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New Bicyclic Nitrogenous Compounds for Enzymology and Pharmacology

Permanent Link: http://ufdc.ufl.edu/UFE0022009/00001

Material Information

Title: New Bicyclic Nitrogenous Compounds for Enzymology and Pharmacology
Physical Description: 1 online resource (259 p.)
Language: english
Creator: Leonik, Fedra
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acetylcholine, amidines, cyclopropanes, glycosidases, nicotinic, receptor, sialyltransferases
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Glycosyltransferases and glycosidases catalyze the transfer of the glycon unit to the acceptor hydroxyl group of carbohydrates and water, respectively. Because glycoconjugates are involved in a variety of metabolic roles, these enzymes play a key role in biological processes such as tumor metastasis, cell-cell development and immune responses. Sialyltransferases (STs) are enzymes that transfer sialic acid from the donor cytidine monophosphate-N-acetylneuraminate (CMP-NeuAc) to the acceptor sugar. STs utilize an SN1-like mechanism in which the leaving group (nucleotide) is mostly dissociated before attack of the incoming nucleophile (oligosaccharide) has started. Previous studies strongly supported a mechanism that involves a transition state (TS) where positive charge is accumulated between the anomeric carbon and oxygen atom of the neuraminate glycon. This study reports the synthesis of diazabicyclic transition state analogs for ST. In the design, a bicyclic system is used to hold the leaving group above the anomeric center mimic, and the amidine functionality will imitate the planarity and positive charge at the transition state. This molecular framework mimics the hypervalent bonding that occurs during a glycosyl transfer reaction. Inhibition experiments were completed with seven-membered ring amidines on different glycosidases and the inhibition constants (Ki) were determined for the most potent inhibitors. The TS analogs tested displayed Ki between 0.15 to 2 mM range. In addition, preliminary inhibition studies were performed on human recombinant alpha(2?6)-sialyltranferase and the Ki of CMP-amidine was estimated to be 50 muM. A second project involved the design and synthesis of agonists to probe the basis for selective activation of neuronal alpha7-type nicotinic acetylcholine receptors (nAChRs). This receptor is a transmembrane ligand gated ion channel, whose three-dimensional structure is not yet known to high resolution. In order to determine the features of the alpha7 nAChR that function as selectivity filters for alpha7 agonist activity, differentially functionalized compounds sharing a common ammonium pharmacophore were synthesized. Among them, derivatives of quinuclidine and quinuclidinone were synthesized which have shown mixed agonist activity.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Fedra Leonik.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Horenstein, Nicole A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022009:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022009/00001

Material Information

Title: New Bicyclic Nitrogenous Compounds for Enzymology and Pharmacology
Physical Description: 1 online resource (259 p.)
Language: english
Creator: Leonik, Fedra
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acetylcholine, amidines, cyclopropanes, glycosidases, nicotinic, receptor, sialyltransferases
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Glycosyltransferases and glycosidases catalyze the transfer of the glycon unit to the acceptor hydroxyl group of carbohydrates and water, respectively. Because glycoconjugates are involved in a variety of metabolic roles, these enzymes play a key role in biological processes such as tumor metastasis, cell-cell development and immune responses. Sialyltransferases (STs) are enzymes that transfer sialic acid from the donor cytidine monophosphate-N-acetylneuraminate (CMP-NeuAc) to the acceptor sugar. STs utilize an SN1-like mechanism in which the leaving group (nucleotide) is mostly dissociated before attack of the incoming nucleophile (oligosaccharide) has started. Previous studies strongly supported a mechanism that involves a transition state (TS) where positive charge is accumulated between the anomeric carbon and oxygen atom of the neuraminate glycon. This study reports the synthesis of diazabicyclic transition state analogs for ST. In the design, a bicyclic system is used to hold the leaving group above the anomeric center mimic, and the amidine functionality will imitate the planarity and positive charge at the transition state. This molecular framework mimics the hypervalent bonding that occurs during a glycosyl transfer reaction. Inhibition experiments were completed with seven-membered ring amidines on different glycosidases and the inhibition constants (Ki) were determined for the most potent inhibitors. The TS analogs tested displayed Ki between 0.15 to 2 mM range. In addition, preliminary inhibition studies were performed on human recombinant alpha(2?6)-sialyltranferase and the Ki of CMP-amidine was estimated to be 50 muM. A second project involved the design and synthesis of agonists to probe the basis for selective activation of neuronal alpha7-type nicotinic acetylcholine receptors (nAChRs). This receptor is a transmembrane ligand gated ion channel, whose three-dimensional structure is not yet known to high resolution. In order to determine the features of the alpha7 nAChR that function as selectivity filters for alpha7 agonist activity, differentially functionalized compounds sharing a common ammonium pharmacophore were synthesized. Among them, derivatives of quinuclidine and quinuclidinone were synthesized which have shown mixed agonist activity.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Fedra Leonik.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Horenstein, Nicole A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022009:00001


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1 NEW BICYCLIC NITROGENOUS CO MPOUNDS FOR ENZYMOLOGY AND PHARMACOLOGY By FEDRA MARINA LEONIK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Fedra Marina Leonik

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3 To the most important people in my life, my husband Daniel, my brother Yuri and my parents Susana and Jorge.

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4 ACKNOWLEDGMENTS My study would not hav e been accomplished wi thout the support and help of many people. To begin with, I would like to specially thank my advisor Dr. Nicole Horenstein for all her scientific guidance and s upport during the course of my studies I also express my gratitude to my doctoral committee members, Dr Ronald Ca stellano, Dr. Tom Lyons, Dr. Linda Bloom and Dr. Roger Papke, for their advice and support. I would like to acknowledge Dr. Ion Ghiviriga for helping with the characterization of the compounds by 2D NMR spectroscopy. I am very grateful to the past and present Dr. Horensteins group members, specially, Jen, Erin, Jeremiah and Jingyi for their friendship and support that made, the day to day in the lab, a better bearable experience. I extend my thanks to my colleagues of the Biochemistry division, in particular, Alonso, Mike, Cory, Kevin, Nancy and Jessica for lending equipment, instructive scientific conversations and sh aring success and frustration. My special acknowledgments go to the financial support from NIH, NSF, Ruegamer Fellowship, College of Liberal Arts and Science Russell Dissertation Fellowship and University of Florida Chemistry Department. I would also like to thank my friends for making my stay in Florida an unforgettable experience. Specially, my gratitude goes to Jo rge, Sarah, Ozge, Giorgos Fabricio and Andrea. We have been, side to side, supporting each other from the very first day. Additionally, I also thank Ece, Giovanni, Laurel, Sophie and Julio. My particular thanks go to the Argentin ean professors, Valeria Kleiman and Adrian Roitberg, for their constant suppor t and guidance on both academic and personal. I have mainly enjoyed our mate and chat afternoons, asados and burako tournament.

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5 My friends from home have also been a valuable support with ceaseless telephone conversations and visits to Gainesville, in pa rticular, Ale, Pablo Mafia, Tamara, Ele, Pablo Engle, Gustavo and Maria Ana. I could not possibly have completed my docto ral studies without my husband Daniel. He has been the foundation of my life and career through his encour agement, patience and love. Special thanks go to my family in law, La ura, Yuji, Fernanda and Marcelo for their unconditional support. Finally, I would like to express my eternal gr atitude to my brother Yuri and my parents Jorge Leonik and Susana Sarabia. Although we are far away from each other, they have always encouraged me to continue this diffi cult journey, by simple believing in me.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 ABREVIATIONS..........................................................................................................................16 ABSTRACT...................................................................................................................................18 CHAP TER 1 INTRODUCTION..................................................................................................................20 Glycoconjugates.....................................................................................................................20 Carbohydrate-Processing Enzymes........................................................................................20 Basic Mechanism................................................................................................................ ....23 Glycosidase Families..............................................................................................................25 Structure..........................................................................................................................26 Inverting and Retaining Mechanisms.............................................................................. 26 Mutagenesis and Labeling Investigations.......................................................................29 Design and Synthesis of Glycosidase Inhibitors .............................................................32 Polyhydroxylated piperidi nes and pyrrolidines ........................................................33 Indolizidine alkaloids............................................................................................... 34 Amino sugars............................................................................................................ 35 Amidines, amidrazones and amidoximes.................................................................35 Imidazoles, tetrazoles and triazole........................................................................... 37 Glycosyltransferases...............................................................................................................40 Structure..........................................................................................................................40 Inverting and Retaining Mechanisms.............................................................................. 42 Sialyltransferases............................................................................................................. 43 Sialic acid................................................................................................................. 44 Structure...................................................................................................................46 Mechanism............................................................................................................... 48 Sialyltransferase inhibitor design............................................................................. 49 Cyclopropane Derivatives......................................................................................................52 Cyclopropanes Biological Implications.......................................................................... 53 Cyclopropane Preparation...............................................................................................55 1,3 cyclopropane bond formation............................................................................. 55 Rearrangement reactions.......................................................................................... 56 Transformation of cyclopropyl derivatives..............................................................57 Combination of C2 and C1 building blocks.............................................................. 58

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7 2 SYNTHESIS OF GLYCOSIDASES AND GLYCOSYLTRANFERASES TRANSITION ST ATE ANALOG S CORE STRUCTURE................................................... 64 Introduction................................................................................................................... ..........64 Results and Discussion......................................................................................................... ..64 Experimental Section........................................................................................................... ...74 3 SYNTHESIS OF DIAZABICYCLIC TRANSI TION STATE ANALOGS.......................... 83 Introduction................................................................................................................... ..........83 Results and Discussions........................................................................................................ ..84 Synthesis of Seven-Membered Ring Amidines............................................................... 84 Synthesis of Five-Membered Ring Amidines Precursor................................................. 91 Synthesis of the Six-Member Ring Amidine Precursor.................................................. 98 Synthesis of the Six-Member Ring Oxazine Precursor ................................................... 99 Synthesis of Seven-Membered Ring Oxazepine...........................................................100 Synthesis of -Function alized Diazoacetate Esters...................................................... 101 Synthesis of TS Nucleotide Analogs............................................................................. 102 Experimental Section........................................................................................................... ...83 4 INHIBITION OF HUMAN SI AL YLTRANFERASE AND GLYCOSIDASES BY DIAZABICYCLIC AMIDINES........................................................................................... 128 Introduction................................................................................................................... ........128 Results and Discussion......................................................................................................... 129 Glycosidases Kinetic Assay.......................................................................................... 129 TS analogs screening..............................................................................................131 TS analogs kinetic characterization........................................................................ 132 (2 6)-Sialyltransferase Inhibition Screen ................................................................. 137 Experimental Section........................................................................................................... .138 5 SYNTHESIS OF QUINUCLIDINE AND QUINUCLIDINONE DE RIVATIVES AS NICOTINIC ACETYLCHOLIN E RECEPTOR AGONISTS............................................. 142 Introduction................................................................................................................... ........142 Acetylcholine Receptor Family..................................................................................... 144 nAChR Structure...........................................................................................................145 Acetylcholine Binding Site............................................................................................ 146 Possible Roles of nAChRs in Human Diseases.............................................................148 nAChR Ligands.............................................................................................................149 Anabaseine and Derivatives.......................................................................................... 152 Results...................................................................................................................................157 Synthesis of Quinuclidine a nd Quinuclidinone Derivatives .........................................157 Electrophysiological Evalua tion of New Com pounds..................................................166 Experimental Section........................................................................................................... .167 6 CONCLUSIONS AND FUTURE WORK ........................................................................... 172

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8 APPENDIX A NMR SPECTRA FROM SYNTHESIZED COMPOUNDS................................................ 175 B KINETIC ASSAY AND ANALYSIS FOR GLYCOSIDASE S WITH DIAZABICYCLIC AMIDINES........................................................................................... 235 Kinetic Screening Graphics..................................................................................................237 Lineweaver-Burke Reverse Plots.........................................................................................242 Michaelis-Menten Curves.....................................................................................................244 Ki Determination with Lineweav er-Burke Plots Infor mation.............................................. 247 LIST OF REFERENCES.............................................................................................................249 BIOGRAPHICAL SKETCH.......................................................................................................259

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9 LIST OF TABLES Table page 2-1 Comparison of the intramolecular cyclopr opanation yield obtained with different catalyst ...............................................................................................................................73 4-1 Inhibition screening data (ni, no inhibiti on at tho se concentra tion of substrate and inhibitor)..........................................................................................................................132 4-2 Inhibition constants (Ki are in mM; because BnCMAM displayed a non-competitive mode of action two Ki were determined)......................................................................... 133

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10 LIST OF FIGURES Figure page 1-1 Schematic pathway of protein glycosylat ion adapted from Stryer, L., Biochemistry, 4th ed...................................................................................................................................22 1-2 Possible configurations in glycosidic bond cleavage......................................................... 23 1-3 Glycosidase catalyzed reaction..........................................................................................25 1-4 Proposed mechanism for inverting glycosidases adapted from the article published by Zechel, D. L. and Withers, S. G.. ..................................................................................27 1-5 Proposed mechanism for retaining glycos idases adapted from the article published by Zechel, D. L. and Withers, S. G.. ..................................................................................28 1-6 N-acteylhexaminidase catalyzed hydrolysis adapted from the article published by Rye, C. S. a nd Withers, S. G.............................................................................................. 29 1-7 1,5-anhydro-D-fructose generation from glycosidic bond hydrolysis. Adapted from the article published by Rye, C. S. and Withers, S. G....................................................... 29 1-8 Conduritol C epoxide....................................................................................................... ..30 1-9 Active site structure of Bacillus circulans xylan ase adapted from the article published by Zechel, D. L. and Withers, S. G................................................................... 31 1-10 D-glucono-1,5-lactone structure........................................................................................ 33 1-11 Polyhydroxylated piperidines and pyrrolidines analogs ....................................................33 1-12 Indolizidine alkaloids.................................................................................................... .....34 1-13 Nectrisine structure...................................................................................................... ......35 1-14 Amidines and amidrazones glycosidases TS analogs........................................................ 36 1-15 Ki comparison of D-glucoazole TS analogs...................................................................... 37 1-16 Tetrahydroxyimidazole derivative..................................................................................... 38 1-17 Proposed glycosidases in-plane or late ral protonation. A) abov e the plane glucoside protonation. B) and C) tetrazole and glucoside protona tion in the plane of the pyranose ring. The figure was adapted from the article published by Heightm an, T. D. and Vasella, A. T...........................................................................................................39 1-18 Glycosyltransferases catalyzed reaction............................................................................ 40

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11 1-19 Glycosyltransferases topological structur e adapted from the article published for Paulson, J. C. and Colley, K. J...........................................................................................41 1-20 Proposed SNi -like mechanism for Neisseria meningitides -galactosyltransferase LgtC adapted from the arti cle published by Lairson, L. L. and Withers, S. G..................43 1-21 Sialyltransferase catalyzed reaction................................................................................... 43 1-22 Sialic acid and N-acetylneuram inic acid structures........................................................... 44 1-23 Inhibitor KI-8110......................................................................................................... ......46 1-24 Cytidine, deoxycytidine and cytosine structures............................................................... 49 1-25 Schimdts ST inhibitors................................................................................................... ..50 1-26 ST transition state analogs s ynthesized by Horensteins group .........................................51 1-27 Proposed diazabicyclic TS analogs.................................................................................... 52 1-28 HPC and PEP structures.....................................................................................................53 1-29 Dihydroxy acid hydrolases enol interm ediates and TS analogs........................................53 1-30 Glutamate receptor (GluR) agonists and i nhib itors adapted from the article published by Salaun, J........................................................................................................................54 1-31 TAM and stilbene-derived compounds.............................................................................. 55 1-32 Cyclopropanation of -unsaturated carbonyl com pound............................................... 56 1-33 Cyclopropanation by nucleophilic su bstitu tion of a Michael acceptor.............................. 56 1-34 Cyclopropanation of 3-butenylstannanes...........................................................................57 1-35 Ring contraction of 2-hydroxycyclobutanone................................................................... 57 1-36 Synthesis of cyclopr opyltrim ethylsilanes..........................................................................57 1-37 Reaction of cyclopropyllithium reagent............................................................................. 58 1-38 Reaction of N-Boc pyrrolinone a nd diphenylsulfonium isopropyl ylide........................... 58 1-39 Simmons-Smith cyclopropanation reactions..................................................................... 59 1-40 Metallocarbene intermediate adapted from Carey, F. A., Advanced organic chemistry ; 4th ed................................................................................................................60

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12 1-41 Cyclopropanation general reacti on adapted from Carey, F. A., Advanced organic chemistry ; 4th ed................................................................................................................60 1-42 Cyclopropanation TS proposed by Casey adapted from the article published by Doyle, M. P........................................................................................................................61 1-43 Cyclopropanation TS adapted from the article published by Doyle, M. P........................ 61 1-44 Cyclopropanation TS proposed by Doyles for carbonyl carbenes adapted from the article published by Doyle, M. P.......................................................................................62 1-45 Rh2(5S-MEPY)4 TS geometry adapted from the article published by Doyle, M. P.......... 63 1-46 The two possible products from the alkene intram olecular cyclopropanation adapted from the article published by Doyle, M. P......................................................................... 63 2-1 Retrosynthetic route for seven-membered diazabicyclic TS analogs................................ 64 2-2 Synthesis of 2-carboxy-4,5-dihydro imidazole.................................................................. 65 2-3 Cyclopropanation using a Michael acceptor...................................................................... 66 2-4 Intermolecular cyclopropana tion of Z -2-butene-1,4-diol.................................................. 66 2-5 Characterization of dioxabicyclo[5.1.0]octane by 2D-NOESY ........................................ 67 2-6 Metal-catalyzed di azoester intram olecula r cycloprapanation............................................ 68 2-7 Synthesis of the Z -bif unctionalized ester 11 ......................................................................68 2-8 Synthesis of diazo ester 8 ...................................................................................................69 2-9 Synthesis of N-trifluoro and FMoc protected glycine ....................................................... 69 2-10 Synthesis of N-trif luoro and FMoc esters .......................................................................... 70 2-11 Improved synthesis of diazoester 8 ....................................................................................72 2-12 Synthesis of Evans ligand................................................................................................ .72 2-13 Lactone 9 2D-NOESY characterization ............................................................................. 73 3-1 Proposed TS analogs with different ring sizes................................................................... 83 3-2 Synthesis of diamine 22 .....................................................................................................84 3-3 Synthesis of hydromethylamidine 23 .................................................................................85 3-4 Oxidation of hydromethy amidine 23 ................................................................................85

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13 3-5 Synthesis of trichloroamidine 28 .......................................................................................87 3-6 Synthesis of chloroamidine 29 ...........................................................................................87 3-7 Synthesis of amidine 31 and diacylated am ine 32 .............................................................88 3-8 2D-NOESY for amidine 31 ...............................................................................................89 3-9 Deprotection of compounds 31 and 23 benzyl groups.......................................................90 3-10 Products of Hofmann and Curtius rearrangements............................................................ 91 3-11 Cis diam ine prepared by Guryn et al. ................................................................................91 3-12 Synthetic route for carbamate 37 .......................................................................................92 3-13 Oxidation of carbamate 38 .................................................................................................93 3-14 Reaction to prep are tran s-aldehyde 41 ...............................................................................93 3-15 NOE interaction for trans-aldehyde 41 ..............................................................................94 3-16 NOE interaction for trans-aldehyde 42 ..............................................................................95 3-17 Synthesis of compound 43 .................................................................................................95 3-18 Synthesis of dicarboxylic acide 44 ....................................................................................96 3-19 Synthesis of diamide 46 .....................................................................................................97 3-20 Synthesis of diamine 53 .....................................................................................................98 3-21 Synthesis to prepare aminoalcohol 54 ...............................................................................99 3-22 Synthesis of oxazepine 58 ................................................................................................100 3-23 Synthesis of functionalized diazoacetates........................................................................ 101 3-24 Nucleoside phosphoramidates......................................................................................... 102 3-25 Phosphotriester approach reaction adapted fr om the article publis hed by Reese, C. B. and Zhang, P. Z................................................................................................................103 3-26 Synthesis of protected nucleoside 68 ...............................................................................104 3-27 Synthesis of 2-chlorophenyl nucleoside 69 .....................................................................104 3-28 Synthesis of CMP-lactone 71...........................................................................................105 3-29 Coupling of amidine 33 by phosphotriester approach ..................................................... 106

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14 3-30 Synthesis of CMP-amidine 73 .........................................................................................106 3-31 Diazabicyclic TS analogs................................................................................................. 107 4-1 Transition State for glucosidase and amidine TS analog................................................. 128 4-2 Library of glycosidases TS analogs................................................................................. 129 4-3 Kinetic assay for glycosidases......................................................................................... 130 4-4 Kinetic assay for -glu at pH 7.5 with all TS analogs .....................................................131 4-5 Inhibition of -galEcoli by TS analog BnCMAM Solid line is reaction without inhibitor; dashed line is enzy matic reaction with BnCMAM.......................................... 134 4-6 Inactivation of -galEcoli by com pound BnCMAM. The -galactosidase was incubated with two chloromet hyl amidine concentrations 0 mM and 0.6 mM, and the reaction started with 0.7 mM of ONPG.............................................................. 135 4-7 Lineweaver-Burke plot for (Z)-(8-( benzyloxym ethyl)-3,5-diazabicyclo[5.1.0]oct-4en-4-yl)methanol (BnHMAM). The concentrations of TS analogs were 4 mM; 2 mM; 0 mM................................................................................................................136 5-1 Acetylcholine, ACh......................................................................................................... .143 5-2 nAChR structure adapted from L odish, H. F. and Darnell, J. E., Molecular cell biology ; 3rd ed. ................................................................................................................ 145 5-3 A model for acetylcholine induced gating of the nAChR receptor.The binding induces a twist on th e subunit that is transmitted to the M2 fragment. The M2 helices assume a new conformation allowi ng the passage of ions. This figure was adapted from the article published by Miyazawa, A., Fujiyoshi, Y. and Unwin, N........147 5-4 nAChR ligands.............................................................................................................. ...150 5-5 Pharmacophore representation adapted from the article published by Barrantes, F. J....151 5-6 Other nAChR agonists.....................................................................................................152 5-7 Nicotine and Anabaseine structures................................................................................. 152 5-8 Anabaseine derivatives.................................................................................................... 153 5-9 Proposed 7-binding pockets that confer selectivity .......................................................155 5-10 Compounds with quinuclidine core that are 7 selective agonists .................................. 155 5-11 Proposed 3-arylidine an d N-alkyl quinuclidines..............................................................156

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15 5-12 Target quinuclidines 7 agonists .....................................................................................157 5-13 Alkylation of quinuclidine hydrochloride........................................................................ 158 5-14 Synthesis of benzylidene quinuclidines...........................................................................159 5-15 NOE enhancements for assignment of olefin geometry of 75a and 75b .........................160 5-16 Methylation of Benzylidene quinuclidines......................................................................161 5-17 Methoxy Benzylidene qui nuclidine dem ethylation......................................................... 162 5-18 Proposed synthesis for BA analog 78 ..............................................................................162 5-19 Synthesis of -pyridyl cyclohexenone 77 .......................................................................163 5-20 Synthesis route A for compound 78 .................................................................................164 5-21 Example of the synthesis of c onjugated dienes through an epoxide ............................... 165 5-22 Proposed Synthetic route B for 3-cyclohenenylpyridine 82 ............................................166 6-1 Antagonist for P2Y1 receptor...........................................................................................174

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16 ABREVIATIONS p-ABSA p-acetam idobenzenesulfonyl azide ACh acetylcholine AChBP acetylcholine binding protein Ac2O acetic anhydride HOAc acetic acid AD Alzheimers BA benzilidene anabaseine Boc t-butyloxycarbonyl BSA bovine serum albumin Bn benzyl CI chemical ionization CMP cytidine 5-monophosphate CDP cytidine 5-diphosphate CTP cytidine 5-triphosphate Cu(acac)2 copper (II) acetylacetonate CuOTf copper triflate DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC N,N-dicyclohexylcarbodiimine DCU dicyclohexylurea DMAP dimethylaminopyridine DME 1,2-dimethoyethane DMF N,N-dimethyl formamide DMSO dimethyl sulfoxide DMTCl 4,4 -Dimethoxytrityl chloride DNA deoxyribonucleic acid DPPA diphenylphosphoryl azide EI electron Ionization ESI electrospray ionization EtOAc ethyl acetate EtOH ethanol FAB fast atom bombardment FMoc 9-fluorenylmethyloxycarbonyl GABA -aminobutyric acid GDP guanosine 5-diphosphate GMP guanosine 5-monophosphate HA hemagglutinin HIV human immunodeficiency virus HPLC high performance liquid chromatography KIE kinetic isotope effect KMnO4 potassium permanganate LacNAc N-acetyl lactosamine LG leaving group LSC liquid scintillation counting MeOH methanol MES 4-morpholineethanesulfonic acid

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17 MS mass spectrometry MsCl methyl sulfonyl chloride MSNT 1-(2-Mesitylenesulfonyl )-3-nitro-1H-1,2,4-triazole nAChR nicotinic acetylcholine receptor NaOEt sodium ethoxide NBS N-bromosuccinimide NeuAc or Neu5Ac N-acetyl neuraminic acid NMO N-methylmorpholine NMR nuclear magnetic resonance NOESY nuclear overhauser enhancement spectroscopy NS nucleotide sugar ONPG o-nitrophenyl -D-galactopyranoside PCC pyridinium chlorochromate Pi inorganic phosphate PIDA iodobenzene diacetate PIFA bis(trifluoroacetoxy)iodobenzene PNP p-nitro phenyl Rh2OAc4 rhodium acetate RNA ribonucleic acid SLeX sialyl lewis ST sialyltransferase tBuOH tert -butanol TBAF tetra-n-butylammonium fluoride TBDMSCl tert -butyldimethylsilyl chloride TBDPSCl tert -butyldiphenylsilyl chloride TFA trifluoro acetic acid TMSI trimethylsilyl iodide THF tetrahydrofuran TLC thin layer chromatography TPAP tetra-n-propylammonium perruthenate TsCl toluensulfonyl chloride TS transition state UDP uracil 5-diphosphate UMP uracil 5-monophosphate UTP uracil 5-triphosphate UV ultraviolet

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18 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NEW BICYCLIC NITROGENOUS CO MPOUNDS FOR ENZYMOLOGY AND PHARMACOLOGY By Fedra Marina Leonik August 2008 Chair: Nicole A. Horenstein Major: Chemistry Glycosyltransferases and glycosidases catal yze the transfer of the glycon unit to the acceptor hydroxyl group of carbohydrates and water, respectively. Because glycoconjugates are involved in a variety of metabolic roles, these enzymes play a ke y role in biological processes such as tumor metastasis, cell-cell development a nd immune responses. Sial yltransferases (STs) are enzymes that transfer sialic acid from the donor cytidine monophosphate-Nacetylneuraminate (CMP-NeuAc) to the acceptor sugar. STs utilize an SN1-like mechanism in which the leaving group (nucleotide) is mostly dissociated before attack of the incoming nucleophile (oligosaccharide) has started. Previous studies strongly supported a mechanism that involves a transition state (TS) where positive charge is accumulated between the anomeric carbon and oxygen atom of the neuraminate gl ycon. This study reports the synthesis of diazabicyclic transition state analogs for ST. In the design, a bicyclic system is used to hold the leaving group above the anomeric center mimic, and the amidine functionality will imitate the planarity and positive charge at the transition state. This molecular framework mimics the hypervalent bonding that occurs during a glycosyl transfer reaction. Inhibi tion experiments were completed with seven-membered ring amidines on different glycosidases and the inhibition constants (Ki) were determined for the most potent inhi bitors. The TS analogs tested displayed

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19 Ki between 0.15 to 2 mM range. In addition, pre liminary inhibition studies were performed on human recombinant (2 6)-sialyltranferase and the Ki of CMP-amidine was estimated to be 50 M. A second project involved the de sign and synthesis of agonists to probe the basis for selective activation of neuronal 7-type nicotinic acetylcholin e receptors (nAChRs). This receptor is a transmembrane ligand gated ion cha nnel, whose three-dimensional structure is not yet known to high resolution. In order to determine the features of the 7 nAChR that function as selectivity filters for 7 agonist activity, differentially functionalized compounds sharing a common ammonium pharmacophore were synthesize d. Among them, derivatives of quinuclidine and quinuclidinone were synthesized whic h have shown mixed agonist activity.

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20 CHAPTER 1 INTRODUCTION Glycoconjugates Oligosacch arides are one of the most important biological polymers in part due to their ability to function in metabolic energy storage (glycogen and starch) and their use in structure (cellulose and chitin).1 They constitute a large portion of the carbon composition in the biosphere, perhaps due to their elevated stabil ity. The glycosidic bond is less susceptible to spontaneous hydrolysis (half life of around 5 million years) than the linkage of amino acids in polypeptides (half life of 460 years) and even DNA nucleotides (half life of 140 thousand years).1 Although they represent the most abundant biopolymeric stru cture, there is still lack of information about their biological roles. Proposing a single comm on hypothesis about an oligosaccharide function is fruitl ess task, not only because of it s extremely complex and varied structure but also because of its diverse biological contex.2 Among their different biological roles, they can a) participate in cell-cell interacti ons; b) modulate protein fo lding and stability; c) act as target or masking for cer tain microorganisms, toxins or binding events; and d) they could serve as structural elements.2 As a consequence of the broad variety of pol ysaccharides, there is a wide assortment of enzymes that are in charge of hydrolyzing and forming these glycosidic bonds. In addition, carbohydrate-processing enzymes are considered among the most cat alytically efficient due to their ability to cleave such a stable bond.1 Carbohydrate-Processing Enzymes The enzym es that are responsible for the glycosidic bond cleavage are divided into two clans, glycosidases, which catal yze the hydrolysis of the glycos idic bond by using only water as the nucleophile and glycosyltransferases, which transfer a sugar unit to another nucleophilic

PAGE 21

21 acceptor.3,4 Moreover, glycosyltransferas es can be divided into two other groups, the transferases that use an oligosaccharide as the donor substr ate and those that utili ze a nucleotide sugar as donor (NS glycosyltransferases).3 Our research only focused on glycosidases and NS glycosyltransferases. As previously mentioned, glycosidases and glycosyltransferases play a key role in biological processes. One example of their metabo lic roles is in protei n glycosylation. Depending on the structural features of the attached oligos accharides, the protein will be trafficked to different intracellular compartments.5 Oligosaccharides could be linked to the protein in two different manners: a) by -N glycosidic bond to an asparagine or b) by a -O-glycosidic bond to serine or threonine.6 Starting at the endoplasmic reticulum, the synthesis of the core oligosaccharide requires the sequential addition of monosaccharide units. This task is performed by specific glycosyltransferases. For example, in the first step of the oligosaccharide building process, a unit of N-acetylglucosamine is added by the enzyme Nacteylglucosaminyltransferase.6,7 Then, several mannose and glucos e units are inserted, but each addition requires the action of unique mannosyl and glucosyltransferases. After the core oligosaccharide is transferred to the protein, th ree units of glucose are trimmed by glucosidases (Figure 1-1).6,7 The glycoprotein processing continues in the Golgi apparatus, where more sugar units are trimmed by glycosidases and more monosaccharide units are added by glycosyltransferases. The final oligosaccharid e composition will depend on the conformation and sequence of the protein that is being processed and the glycos yltransferase composition of the particular Golgi apparatus.6,7 Among other species, di fferent viruses utilize this same pathway to form their viral and cellular surface coats, maki ng the elucidation and complete understanding of the glycosylation process a very attractive t opic for the pharmaceutical industry. In addition,

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22 bacterial wall biosynthesis proceeds through a similar glycosyl tran sfer mechanism. Two compounds that block this oligosaccharide synthesis are tunicamycin and bacitracin.6,7 In particular, bacitracin is actually used as an antibiotic because it can not cross mammalian cell membranes. Figure 1-1. Schematic pathway of protein glycosylation adapted fr om Stryer, L., Biochemistry, 4th ed.8 It has been shown that ca rbohydrate-processing enzymes are also implicated in the development of several types of tumors.9 High levels of glycosidas es in blood are observed in patients with different cancer ous pathologies. Moreover, many tumor cells showed distorted expression of glycosyltranferases, which is ultimately reflected in an abnormal glycosylation pattern.9

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23 Basic Mechanism In general, enzym atic glycoside bond hydrolys is is a nucleophilic substitution at the anomeric carbon of a sugar, which can occur thr ough either retention or inversion (Figure 1-2).4 O OR O OH O O+ OH Retaining Inverting Figure 1-2. Possible configurations in glycosidic bond cleavage Consequently, retaining and inverting en zymes will proceed through two different mechanisms, and it should be pointed out that due to typically rapid mutarotation rates, the product configuration at the anomeric carbon will be rapidly scrambled. The inverting enzymes possess a general acid residue at their active site, which assi sts by protonating the departing aglycon oxygen of the glycosidic bond, and a basi c amino acid that deprotonates the attacking hydroxyl of the acceptor. This process is cons istent with a single-displacement mechanism involving an oxocarbenium ionlike transition state (TS).3,4,10-14 On the other hand, the retaining carbohydrate-processing enzymes are suggested to proceed through a double-displacement mechanism. In this case, one of the active site residues acts as a nucleophile, forming a labile glycosyl-enzyme intermediate, which is immedi ately attacked by the incoming sugar acceptor. The glycosidic oxygen (first step) and the sugar acceptor (second step) are previously activated by another active site residue that operates as an acid/base catalyst. Both steps are considered to proceed through a TS with extensive oxocarbenium ion character.3,4,10-14 X-ray crystallography of proteins covalently labeled with fluorosugar substrate analogs and kinetic characterization of

PAGE 24

24 wild-type and mutant en zymes have been utilized to study these two possible mechanisms.3,13 For example, kinetic isotope effects have served not only to explore the fo rmation of the covalent glycosyl-enzyme intermediate (secondary deut erium isotope effect greater than 1 for deglycosylation step) but also to determine th e oxocarbenium ion character of the TS (inverse isotope effect due to sp2 hybridization of the anomeric carbon).3 In addition, trapping and crystallography experiments with deoxyfluoroglycosides were also highly valuable for the characterization of the covalent intermediate.3,13 Finally, site di rected mutagenesis has been used to identify the residues that may participate as general acid/base catalysts. Changing the critical amino acid required for catalysis will have a strong effect not only on the kinetic parameters but also on the pH profile of the enzyme.3,13 In most cases, asparagine, glutamine and alanine are the substitute residues for glutamic and aspartic acid. They conserve approximately the size of the amino acids that are replacing but lack the possi bility to function as acid or base catalysts.3,13 Non-enzymatic studies of the glycosyl bond hydrolysis have shown that the reaction involved the departure of the aglycon, which is assisted by the oxygen lone pair electrons, followed by the formation of an extremely short lived glycosyl cation.4,15 The stability of this oxocarbenium ion will depend on the solven t and nucleophile used in the reaction.4,15 Based on these studies and other evidence obtained in enzymatic systems, it was proposed that the enzymatic glycosyl hydrolysis occurs as well vi a an oxocarbenium ion TS. One example of this correlation was presented by Bruner and Horens tein with their mechanistic studies on (2 6)sialyltransferases.16 Measurement of beta secondary dideuter ium isotope effects showed that this enzyme TS involves considerable glycosyl catio n formation. Comparison of this result with the one obtained for the acid-catalyzed solvolysis of CMP-N-acetyl neuraminate had established that the sialyltranferase transition state has oxocarbeni um ion character quite similar to the one for

PAGE 25

25 solvent hydrolysis. In addition, the solvolysis results were complemented with an ab-initio computational analysis of the predicted kineti c isotope effects. Both approaches were in agreement with the oxocarbenium ion TS structure.17 Another example on the oxocarbenium ion TS characterization was obtained by W ithers and co workers research on Agrobacterium glucosidase.14,18 This enzyme is a retaining glycosidase and accordingly, it proceeds through a two step mechanism. Another valuable tool us ed to explore details of glycosyl-transferring enzyme mechanisms are TS analogs. Properly designed inhibitors could provide useful information about enzyme-substrate contacts and a good transition state analog would be expected to be a potent inhibitor of the enzyme. Hence, studi es aimed at the synthesis of such compounds are of considerable interest. This aspect of the enzyme mechanism field will be further discussed in the c ourse of this chapter. Glycosidase Families Glycosidases (EC 3.2.1.x) catalyze the hydrolysis of the glycosidic bond that connects a sugar anom eric carbon with a glycan (Figure 1-3). Sugar+ HOH + glycosidase OR HOR Sugar OH Figure 1-3. Glycosidase catalyzed reaction Based on the recently updated intern et database (www.expasy.ch/cgibin/lists?glycosid.txt), glycosidases constitute a clan of around 100 families of more than 1000 enzymes, which are classified by Henris att on the basis of sequence similarities.19,20 Around 150 of these enzymes still remained unclassified and for more than 60 families the three-dimensional structures are known.20

PAGE 26

26 Structure Based on crystallograph ic characterizations, the general amino acid arrangement on the catalytic domain of ma ny glycosidases is ( / )8 or a TIM barrel.10,21 This barrel structure was first found in triose-phosphate isomerase (TIM) but is frequently present is several protein folds.22 The TIM barrel is constituted by eight helices and eight parallel strands. This protein fold has the shape of a toroid in which the strands are located in side the doughnut, with the helices forming the outer surface. In the TIM ba rrel, the secondary structures are connected by loop regions that could differ in length depending on the enzyme family. Among the glycosidases that present the ( / )8 motif are for example, and -amylases, glucanotransferases, xylanase/glucanase from C. fimi and the widely studied -galactosidase from E. coli. Some other families that have a distor ted TIM barrel are endoglucanase E2 and glucanases.10 Due to the diversity of gl ycosidase substrates, there ar e other folds that could be observed such as / barrel, -barrel or -sandwich.10 Usually, glycosidases have one or more catalytic subdomains, one of them utilized for catalysis and the other one for substrate binding.10,23 Active site topologies could be divided in three categorie s: 1) pocket or crater, found in exoglycosidases such as -galactosidases, -glucosidases, sialidases and neuramidases, which hydrolyze sugar units from the non reducing end of glycoconjugates; 2) cleft or groove, found in endoglycosidases such as lysozyme, endocellulase s, chitinases and glucanases, which cleave the internal glycosidic bond within oligosaccharides; and 3) tunnel, observed in cellobiohydrolases when the cleft is covered by some of the protein loops.23 Inverting and Retaining Mechanisms Regarding glycosidase active sites, it was already m entioned that glycosidic bond hydrolysis could be accomplished with either re tention or inversion of the anomeric carbon

PAGE 27

27 configuration. In most glycosidases, the typical active site has an arrangement of two carboxylic amino acids situated on opposite faces of the catalytic pocket.4,10-14 For the inverting glycosidases, they are located around 10.5 apar t from each other, leaving enough space to hold the substrate and a molecule of water.4,10-14 In particular, while one carboxylic acid acts as a general base, helping wate r attack at the anomeric center, the other one proceeds as a general acid facilitating the glycosidic bond hydrolysis (Figure 1-4). O OR O O H O -O H O H O OR O O H O O H O H O O -O O HO OH HOR Figure 1-4. Proposed mechanism for inverting glycosidases adapted from the article published by Zechel, D. L. and Withers, S. G.14 The enzymatic reaction takes place with a single-displacement mechanism having a TS with oxocarbenium ion character. On the other hand, for retaining glycosidases, these carboxylic residues are closer to each ot her (approximately 4.8-5.5 ), which is consistent with a doubledisplacement pathway (Figure 1-5).4,10-14 In the glycosylation (first) step, one carboxyl ic acid acts as a nucleophile forming the enzyme-glycosyl bond, while the other assists th e reaction by protonating the glycosyl oxygen. In the deglycosylating (second) step, the general acid catalytic residue proceeds now as the general base deprotonating a molecule of water that will cleave the covalent enzyme-substrate intermediate.4,10-14 Both steps are known to involve a TS with oxocarbenium ion character. Typically, in retaining glycosidases, these two ca rboxylic acids are identified as glutamate or

PAGE 28

28 aspartate. These amino acids were originally identified in lysozymes, which are the first glycosidases characterize d by X-ray christallography4,23. Nevertheless, with the constant discovering and characterization of new glycosidases families, it was observed that other amino acid such as tyrosine or tryptophan could be critical for enzymatic hydrolysis.10,14 O OR O O H O -O O OR O O H O O O O O O -O H O H O O O O H O O H O OH O HO O -O Figure 1-5. Proposed mechanism for retaining glycosidases adapted from the article published by Zechel, D. L. and Withers, S. G.14 Examples of enzymes that escape from the two types of mechanisms discussed above can also be found. Member of families 18 and 20, N-acetyl-hexosaminidases catalyzed hydrolysis by neighboring group assistance of their substrate Nacetyl group (Figure 1-6). In this case, the acetyl group acts as the nucleophile, forming an oxazoline, with the enzymic carboxylate residue proposed to stabilized the inte rmediate (or its formation).11 Another particular class of glycosidases utilize an elim ination reaction for glycosidic bond hydrolysis, liberating 1,5anhydro-D-fructose instead of glucose (Figure 1-7).11

PAGE 29

29 O OR O O H HN O O O -O H O H HN O O OH O -O HN O Figure 1-6. N-acteylhexaminidase catalyzed hydrolysis adapted from the article published by Rye, C. S. and Withers, S. G.11 Mutagenesis and Labeling Investigations -galactos idase from E. coli a member of family 2 of glycosyl hydrolases, is one of the most studied retaining glycosidas es. Based on its three-dimensiona l structure, the enzyme is a 1023 amino-acid polypeptide formed by four tetramers (465 kDa each).24 Five separate domains and an extended N-terminus constitute the -galactoside monomer. The catalytic domain displays an unconventional TI M barrel where the fifth -helix is missing and the sixth -strand is deformed.24 O O O HO HO HO OH OH HO HO OH O HO HO HO OH O OR HO HO HO OH 1,5-Anhydro-D-fructose (enolform) O HO HO OH O 1,5-Anhydro-D-fructose (ketoform) Figure 1-7. 1,5-anhydro-D-fructose generation from glycosidic bond hydrolysis. Adapted from the article published by Rye, C. S. and Withers, S. G.11

PAGE 30

30 Affinity labeling experiments with conduritol C epoxide (Figure 1-8) initially identified Glu461 as the amino acid responsible of the glycosyl-enzyme intermediate.25 Nevertheless, studies with different -galactosidase mutants of Glu461 di splayed a considerable remnant activity, which was a highly suspicious result for a critical catalytic residue. O HO HO OH OH Figure 1-8. Conduritol C epoxide25 After trapping studies with 2-deoxy-2-fluoro-galactopyranoside and the three dimension structure resolution on this enzyme, Glu537 was assigned as the nucleophilic amino acid instead of Glu461.3,10,24,26 Mutagenic experiments, in which Gl u461 was replaced by Gly, Gln and Asp in conjunction with the crystal structure, reveal ed that this residue was also important for glycosidic bond cleavage due to its role as a general acid/base catalys t. In addition, it was observed that the essential Mg2+ required for the -galactosidase reaction, acts as a pKa modulator for this residue.3,10 Another example where site directed mutagenesis and crystallography have helped identify the amino aci d responsible for catalysis is found in the case of B. circulans xylanase (Bcx). In this case, Glu172 was found to be the acid/base catalyst while Glu78 operates as the nucl eophile (Figure 1-9).3 Interestingly, Tyr69 is located close (~2.95 ) to the enzyme-glycosidic intermed iate. Consequently, it was proposed that this residue has a dual electrostatic interaction with the sugar oxygen and the oxygen of Glu78 which forms the bond with the substrate.13,14 Withers and coworkers suggested th at Tyr69 plays a critical role stabilizing at the same time, the positive ch arge developed on the monosaccharide ring oxygen by electrostatic interac tion with the Tyr oxygen lone pair el ectrons, and the negative charge on

PAGE 31

31 Glu78 by hydrogen bonding during th e deglycosylation step.13,14 This hypothesis was supported by observing a total loss of activity on the Tyr69Phe mutant.27 Moreover, in the Bcx active site cleft, there is another tyrosine assisting Glu172 identified as Tyr80.27 In conjunction with Tyr80, Glu172 activates the water molecu le for the nucleophilic attack. O HO HO HO O O HO HO O O O H O OO H O H H Glu172 Tyr80 Tyr69 Glu78 Figure 1-9. Active site structure of Bacillus circulans xylanase adapted from the article published by Zechel, D. L. and Withers, S. G.13,14 A surprising approach accomplished by muta genesis is the possibility to switch the stereochemical outcome of a glycosidase.13 Due to the close similariti es of the hydrolases TS, an inverting glycosidase could, in principle, be converted into retaining and vice versa.13 For retaining glycosidases, the most extensively ut ilized method is revoking the activity of the enzyme by exchanging the nucleophilic residue for a non-nucleophilic one.3,13,14 An example in support of this hypothesis was obser ved when the nucleophilic Glu358 of -glucosidase from Agrobacterium sp. (Abg) was replaced by an alanine.18 This enzyme was chosen because of its extended substrate specificity. In principle, the Glu358Ala mutant was inactive to perform hydrolysis but, when placed in a medium th at contained good nucleophiles such as azide, formate or acetate, the -glucosidase activity was restored.3,13,14 Thus, the product of this enzymatic reaction was obtained with inversi on stereochemistry. Moreover, Glu358Ala Abg was

PAGE 32

32 able to synthesize a -disaccharide from -glucosyl fluoride without a ny addition of an external nucleophile. In this way, a glycosidase could be turn into a mutant glycosynthase capable of building polysaccharides.3,13,14 In a parallel manner, inverting glycosidases can be transformed into retaining ones by in corporating a nucleophilic amino acid (Glu or His).3,14 The residue needs to be situated in the space typically occu pied by the water molecule. This experiment was successfully carried out by Kuroki et al. on bacteriophage T4 lysozyme.28 This enzyme was also found to be capable of transglycosylation.28 Design and Synthesis of Glycosidase Inhibitors Considerable effort has been dedicated to the design and synthesis of inhibitors of carbohydrate processing enzym es, no t only to elucidate the role of sugar residues in biological systems but also to provide information on enzyme-substrate binding contacts through investigation of structur e-activity relationships. In addition, some glycosidase inhibitors have already been tested as possible therapeutic agents, for instance, against diabetes29 and HIV viral replication.30 Although glycosidases have been the subjec t of intense research, and considerable information is already available on their activ e site structures, inhibitor design remains a complicated task. It is not yet possible to reliab ly design a potent inhibitor by merely inspecting a crystal structure. The work described in this dissertation ta kes a mechanism based approach to inhibitor design, in which our knowledge of the shape and charge of a transition state serves as the template for the design of synthetic targets. A key requirement for an e fficient inhibitor includes high stability and high binding affinity for the en zyme active site. The latter is usually provided by TS analogs.31 Aldolactones and gluconolactones were among the first discovered competitive

PAGE 33

33 glycosidase TS analog inhibito rs; D-glucono-1,5-lactone (Figur e 1-10) was among the most potent.32,33 O HO HO HO HO O D-glucono-1,5-lactone Figure 1-10. D-glucono1,5-lactone structure32,33 The inhibitory potency of these compounds ag ainst numerous glycosidases in part comes from their resemblance to the pyranosyl and furanos yl part of the substrat e sugars. In addition, it was believed that the source of the lactones inhibi ting activity was due to their ability to adopt a half chair conformation simila r to the glucopyranosyl cation.33 In other words, the flattened lactone ring is a geometric analog of a glycosyl tr ansfer transition state. Subsequently, a lot of potent (Ki in the M range) nitrogen-containing glycosidas e inhibitors have been identified, including a) polyhydroxylated pi peridines and pyrrolidines; b) indolizidine alkaloids34; c) amino sugars35,36; d) amidines, amidrazones and amidoximes37-40; and e) imidazoles, tetrazoles and triazole.41-43 These differ from the lactone inhibitors in that the basic ri ng nitrogen can be protonated, providing a mimic of the oxocarbenium ion transition states charge. Polyhydroxylated piperidines and pyrrolidines The first nitrogenous glycosidase inhibito r known was the polyhydroxylated piperidine nojirim ycin (Figure 1-11). NH OH HO HO HO HO NH OH HO HO HO N H CH2OH H OH OH HO Nojirimycin 1-deoxymannojirimycin (DMJ) 1,4-dideoxy-1,4-iminoD-mannitol Figure 1-11. Polyhydroxylated piperidines and pyrrolidines analogs9,44,45

PAGE 34

34 Being a D-glucose analog where the oxygen has been replaced by NH33,45, this compound resulted in a potent and -glucosidase inhibitor. Another piperidine based inhibitor is 1deoxymannonojirimycin (DMJ) (Figure 1-11). This compound is obtained from nojirimycin B (mannojirimycin) by biosynthetic reduction of the anomeric hydroxyl group.46 DMJ blocks the production of high-mannose oligosaccharides to more complex glycans by inhibiting the Golgi -mannosidase (Ki of 750 M).9 Hydroxylated pyrrolidines, whic h mimic the furanosyl moiety, also display inhibition of the corresponding gl ycosidase. An example of this family of compounds is 1,4-dideoxy-1,4-imino-D-mannitol, wh ich turned out to be a good inhibitor for jack bean and human lysosomal -D-mannosidase with Kis of 0.8 M and 13 M, respectively (Figure 1-11).9 Indolizidine alkaloids This kind of glycosidase inhibitor has a fused piperidine and pyrrolidine as a core structure (Figure 1-12). N OH H OH OH Swainsonine N OH H OH HO HO Castanospermine Figure 1-12. Indolizidine alkaloids9,34 In general, target glycosidases are inhibited by these alkaloids because either the piperidine mimics the pyranosyl, or the pyrrolidine mimics the furanosyl moiety of their substrate.9 The interest on indolizidine alkalo ids arose from the study of sw ainsoma toxicosis (an animal neurological disease caused by a pl ant). The toxin, identified as th e alkaloid swainsonine (Figure 1-12), is a very potent lysosomal -mannosidase and Golgi -mannosidase II inhibitor.9 Another common alkaloid is castanospermine (Figure 1-12). In this case, the pipe ridine portion of the

PAGE 35

35 molecule mimics the structure of glucose. C onsequently, this indolizidine acts on lysosomal and -glucosidases (Ki of 0.1 M and Ki of 7 M respectively)9 and endoplasmic reticulum glucosidases I and II.9,34 Both castanospermine and swainson ine were found to be useful in preventing metastasis dissemination in mice. Th ese compounds block the formation of complex oligosaccharides implicated in metastasis, impl ying their potential used in cancer treatment.9 Due to its ability to inhibit carbohydrate-processing glycosidases, castanospermine can influence the infectivity of certain viruses like HIV.9 However, when influenza virus was generated in the presence of this indolizidine, its infective strength remained intact.34 Amino sugars Falling in the category of a mino-pentose, ne ctrisine (Figure 1-13) was a very potent glucosidase and -mannosidase inhibitor with an IC50 of 0.05 M and 6.5 M respectively.35 This inhibitor was first isolated from the fungus Nectria lucida which was found to act as the own fungus inmunomodulator.35 Full synthesis of nectrisine has been reported by Kayakiri et al. from D-glucose35 and by Chen et al. from D-arabinose.47 N HO OH HO Nectrisine Figure 1-13. Nectrisine structure35 Amidines, amidrazones and amidoximes The im portance of this class of compounds arises from their capability of simulating, at the same time, the charge and the planarity of glycosidases oxocarbenium ion-like TS.48 The diaza functional group can be easily pr otonated, allowing equal distri bution of charge within the trigonal planar system. Based on different lite rature reports,39,43,48 not only the charge

PAGE 36

36 distribution, but also the spatial c onfiguration are important issues to take in account in order to produce potential inhibitors that duplicate th e hypervalent transition state geometry. A broad range of amidine-containing com pounds had been synthesized by different research groups.37,48,49 Among the amidines family, D-glucoamidine 1 (Figure 1-14) exhibited a potent inhibitory ac tivity on sweet almond -glucosidases (Ki of 10 M) and jack bean mannosidase (Ki of 9 M).48 N HO HO HO HO NH2 D-glucoamidine1N HO HO HO OH NH N HO HO HO OH NH O HO HO OH OCH3 34N NH 5N HO HO HO OH NH NH2 D-mannoamidrazone2 Figure 1-14. Amidines and amidr azones glycosidases TS analogs37,48,49 Using a group of amidines with a differe nt number of hydroxyl substituents, Bleriot et al. showed the importance of mimicking the backbone of glycosidases sugar substrate. From these studies, the fully hydroxylated amidines 3 and 4 (Figure 1-14) displayed the strongest competitive potency against jack bean -mannosidase and sweet almond -glucosidases, with a Ki of 0.55 M and 5 M, respectively. On the contra ry, the non hydroxylated analog 5 (Figure 114) showed a Ki of only 14 mM for the -mannosidase and 3.7 mM for the -glucosidases.37

PAGE 37

37 With respect to the amidrazones series, Ganem and coworkerss D-mannoamidrazone38,39 2 (Figure 1-14) proved to be one of the most potent inhibitors for -mannosidases, with a Ki of 170 nM. Furthermore, this compound turned out to be a broad spectrum inhibitor because it had also shown activity against -glucosidases, as well as -galactosidases and other mannosidases.38,39 It was suggested that the conformational structure of D-mannoamidrazone is one of the features responsible for its extensive inhi bitory activity. When it is compar ed with the exocyclic carbonyl of aldo and gluconolactones, the amidrazone endoc yclic tautomer seems a better mimic of the half chair conformation adopted by the substrate at the TS.38 Imidazoles, tetrazoles and triazole Tetrazo le and triazoles derivatives43 are aldolactones analogs which have a half chair conformation as a consequence of the fused aromatic ring (Figure 1-15). The similar conformation adopted by aldolactones and the azol e derivatives was actually confirmed by three dimension crystallography.43 N HO HO HO HO N N D-gluco-1,2,3-triazole Ki>8000 M N HO HO HO HO N N N D-glucotetrazole Ki=150 M N HO HO HO HO N N N HO HO HO HO N D-gluco-1,2,4-triazole Ki=19 M D-glucoimidazole Ki=0.1 M Figure 1-15. Ki comparison of D-glucoazole TS analogs43 Tetrazole inhibitory potency was found to be comparable with D-glucono-1,5-lactone. Moreover, based on kinetic and mutageni c studies on different glycosidases,43 tetrazole monosaccharides could be considered a good representation of a TS analog.43 On the other hand, 1,2,3-triazole analogs showed a drastic decrease in activity when compared with the related tetrazoles (Ki > than 8000 M for triazole and 200 M for tetrazole when tested on almond glucosidase).43

PAGE 38

38 The importance of substrate protonation dur ing glycosidase enzymatic reaction was addressed with non annulated imidazole derivatives.42,50 In this case, it was suggested that the proton transfer proceeds from one carboxylic acid to the neighbor through the heterocyclic ring. These kinds of analogs showed modest inhibitory potency, in particular for -glucosidases.42,50 Having these results in mind, several authors re ported the synthesis a nd corresponding kinetic analysis on annulated imidazoles derivatives (Figure 1-15).43 Being part of this category of imidazole analogs, tetrahydropyridoinidazole 6 (Figure 1-16) is one of the most potent glycosidases inhibitors known so far,41 with a Ki of 1.2 nM and 0.11 nM for -glucosidase from almonds and Caldocellum s. respectively. N HO HO HO HO N 5-hydroxymethyl-2-(2-phenylethyl)5,6,7,8-tetrahydroxyimidazole 6 Figure 1-16. Tetrahydro xyimidazole derivative41 This class of compounds was also a valuab le tool for a better characterization and understanding of glycosidases proton transfer. To complete the analysis of enzyme-azole derivatives interaction, 1,2,4triazoles were synthesized.43 A comparison of Ki among the series of annulated azole TS analogs on -glucosidase from almonds is s hown in Figure 1-15. From the difference between tetrazole and 1,2, 3-triazole behavior, and the a ssumption that proton transfer from the enzyme to the substrate is important fo r inhibition, it was proposed that, instead of the generally presumed above-the-anomeric-plane protonation, an in-plane or lateral proton transfer might be occurring (Figure 1-17).43 This theory is also c onsistent with glycosyl bond hydrolysis because the glycosidic oxygen lone pair electrons are oriented one perpendicular to

PAGE 39

39 the other one. Consequently, above-the-plane and in-plane protonation are both perfectly possible. Another result that supported the late ral proton transfer is the correlation of the inhibitory potency and the basicity of the anal ogs. The more basic the he terocycle, the stronger should be its potency, which is observed on the corresponding Ki sequence where tetrazole > 1,2,4-triazole > imidazole.41,43 Additional evidence of latera l protonation was found in docking experiments using te trazole analogs on -galactosidase from E. coli, white clover -glucosidase, Cellulomonas fimi cellulose and 6-phosphogalactosidase from L.lactis .43 For this last enzyme, an X-ray structure in the presence of its substrate also suggested the in-plane proton transfer mechanism was operative.43 Additionally, if the cat alytic carboxylic acid is situated on the same plane of the sugar ring, there is more room for the pseudoaxial departur e of the glycon, which facilitates the hydrolysis. O HO HO HO HO O R O O H O OO HO HO HO HO O R O OO O H N HO HO HO HO O OO O H N N NA BC Figure 1-17. Proposed glycosidases in-plane or lateral protonation. A) ab ove the plane glucoside protonation. B) and C) tetrazole and glucoside protona tion in the plane of the pyranose ring. The figure was adapted from the article published by Heightman, T. D. and Vasella, A. T.43

PAGE 40

40 Glycosyltransferases Com pared with glycosidases, there is much le ss information about NS glycosyltransferases (EC 2.4.x.y). In this case, 78 families have been arranged by sequence homology from which only 20 families have been characterized by X-ray.20,51 Glycosyltransferases catalyze the transfer of a single monosaccharide unit from an activat ed nucleotide donor to the hydroxyl group of an acceptor saccharide (Figure 1-18).52,53 Sugar + HOacceptor O Sugar + glycosyltransferase O P O OH OR acceptor HOP O OH OR Figure 1-18. Glycosyltransf erases catalyzed reaction52 The most common nucleotide sugars are: UDP-glucose (glucosyltransferase), UDPgalactose (galactosyltransferase), UDP-N-acetylglucosamine (N-acetylglucosaminyltransferase), UDP-N-acetyl-galactosamine (N-acetylgalactosaminyl transferase), GDP-mannose (mannosyltransferase), CMP-N-acetylneuramic acid (sialyltransfera se), and GDP-fucose (fucosyltransferase).52,53 Structure Typically, glycosyltransferases are transm embrane glycoproteins localized in the Golgi apparatus.53 Surprisingly, they do not share sequence homology within the family, but they all have the same overall domain structure. In general, glycosyltransferases are characterized by a short N-terminal cytoplasmic tail, a 16-20 amino acid signal-anchor sequence, an extended stem section and a large lumenal C-terminal catalytic domain (Figure 1-19).53 The signal-anchor fragment has the function of not only fixing the enzyme to the membrane, but also giving the catalytic domain a certain direction inside the Golgi.53 The stem domain (35 to 62 residues) may

PAGE 41

41 provide the flexibility needed for the glycosylation of solubl e and membrane proteins during their processing.53 Figure 1-19. Glycosyltransferases topological structure adapted from the article published for Paulson, J. C. and Colley, K. J.53 From the X-ray structures solved to date, it was observed that glycosyltransferases adopt two folds. One of them is the so-called GT-A fold, which was initially discovered in the transferase Bacillus subtilis SpsA, and the other one is the GT-B fold found in phage T4 glucosyltranferase.54-56 The GT-A fold is constituted by two domains that appeared to form a continuous sheet of eight -strands. On the other hand, the GT -B fold exhibits two Rossmann domains separated by a large cleft, frequently observed in nucleotide binding proteins.54 It was shown for both GT-A and GT-B members that while one domain is involved in nucleotide binding, the second is engage d in acceptor binding.54 Interestingly, the GT-A members share the COOH H2NCatalytic Domain Stem Region Signal-Anchor Cytoplasmatic Tail Golgi Lumen Cytoplasm

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42 common feature of containing an M2+ ion (M = metal) which is coordinated by side residues and the nucleotide phosphates. This arrangement usually called DXD motif, is commonly constituted by carboxylic amino acids.54,55 Metal ions are not norma lly found in the crystal structure of GT-B enzymes, which is consistent with the absence of DXD motif within their catalytic active site.54 Inverting and Retaining Mechanisms As for glycosidases, glycosyltranferases coul d be divided into re taining and inverting enzym es, based on the stereochemical outcome of their products. In this case, the inverting transferases have been well ch aracterized but, on the contrary, retaining glycosyltransferase mechanisms are still under debate.54,56 In both cases, it was found that the mechanism involves an oxocarbenium ion TS. For the inverting enzyme s, it is typically proposed that the reaction proceeds through a concerted displacement in whic h the metal cation is used as the general acid catalyst.56 For the retaining glycosyltransferases, no glycosyl-enzyme intermediated has been found. Consequently, the double-displacement mech anism, suggested for glycosidases, seems inconsistent with the experimental results obtain ed for glycosyltransferases. From site directed mutagenesis and 3D structure of Neisseria meningitides -galactosyltranferase LgtC, Withers et al. proposed that the enzymatic reaction occurs by a front side SN2-like attack or SNi-like mechanism, where the acceptor nucleophilic attack and the leaving group departure proceed on the same phase (Figure 1-20).57 Based on this proposed mechanism, the departing aglycon is protonated by the incoming acceptor. Nevertheless, th e feasibility of this r eaction is inconsistent with the low acidity of the acceptor hydroxylic hydrogen. More experimental results will be required for a clearer understa nding of the retaining glycos yltransferases mechanism.

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43 O NH2 O O NH2 O R O H O O NH2 OR' O R HOR' O R' Figure 1-20. Proposed SNi -like mechanism for Neisseria meningitides -galactosyltransferase LgtC adapted from the ar ticle published by Lairson, L. L. and Withers, S. G.56 Sialyltransferases Sialyltran sferases (ST) catalyze the transfer of sialic acid from an activated nucleotide donor cytidine monophosphate-N-acetylneuraminate (CMP-NeuAc) to the hydroxyl group of an acceptor saccharide (Figure 1-21).52 The enzymatic reaction occurs through a nucleop hilic attack with inversion of configuration at the anomeric carbon of sialic acid. The ST family shares the common feature of identifying only -linked CMP-NeuAc as the sial yl moiety donor, but they diverge in their specificity on glycan acceptor and the class of glycosidic bond they form.58,59 As mentioned before, ST are localized in the Golg i apparatus of both microorganism and mammals cell, but they can also be found as the free so luble enzyme in the co lostrum of different animals.59 O HO O CO2 -AcHN O HO HO OH OH OH P O O ON N NH2 O O O OH OH P O O ON N NH2 O O HO AcHN HO HO OH OH OCMP-NeuAc Sialyltransferase ROH -sialoside CMP-NeuAc T S Figure 1-21. Sialyltransferase catalyzed reaction60

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44 Sialic acid Sialic acids are a fam ily of related nine-carbon carboxylated sugars derived from neuramic acid (Figure 1-22). O OH COOH HO H2N HO OH HO Sialicacid (Neuramicacid) O OH COOH HO AcHN HO OH HO N-acetylneuramicacid (Neu5Ac) Figure 1-22. Sialic acid and N-acet ylneuraminic acid structures They could be found at the non-reducing terminal position of sugar chains localized at the surface of microorganism and higher animals cells.61 Differing in the position and characteristic of Oor N-linked substituents, 50 different sialic acid derivatives have been observed so far in nature. However, the most abundant are N -acetylneuraminic acid (Neu5Ac), N glycolylneuraminic acid (Neu5Gc) and N -acetyl-9-O -acetylneuraminic acid (Neu5,9Ac2).61,62 The distribution of sialic acid derivatives in diffe rent organisms is related to the species and the cell function. For example, in humans, the most fr equently found sialic acids are Neu5Ac and the O-acetylated and O-lactylated derivatives.61 The main sialic acid linkages found in glycoproteins, glycolipids or oligosaccharides are: a) 23) or 26) to a galactose, such us Neu5Ac 23)Gal or Neu5Ac 26)Gal; b) 26) to N-acetylgalactosamine or Nacetylglucosamine, such as Neu5Ac 26)GalNAc and Neu5Ac 26)GlcNAc; or c) 28) or 29) to a second sialic acid in Neu5Ac 28)Neu5Ac and Neu5Ac 29)Neu5Ac.58,62 Due to the charge character of sialic acids, they can modulate the el ectrostatic interaction between cells, act as anti-proteolytic agents on glycoproteins, and transport and bind positively charged molecules. Furthermore, the sialic acid chemical properties can be transformed by the inclusion of hydrophobic substituent such us O-acetyl and O-methyl.61

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45 Having a strategic terminal position on glycoc onjugates, sialic acids are involved in a variety of cell-cell recognition processes including cell development and differentiation. Moreover, they have also been implicated in oncogenic transformations, and they could serve as receptors for influenza virus.58,61,62 The influenza virus interacts with the oligosaccharides situated in the outer side of their host cell surfaces. After its attachment to the cell, the virus utilizes a viral lectin (protein that binds oligosaccharides), known as Hemagglutinin (HA), and a viral sialidase to enter and infect the host. While the HA acts as an anchor to the plasma membrane, the sialidase removes the terminal sialic acid of the host oligosaccharide. This mechanism promotes the spreadability of the infl uenza virus because, without the terminal sialic acid, the cell tissue can no longer in teract with other cells and mucu s that protect the respiratory tract.61,63 Other viruses that recognize the host, or use their own sialylated carbohydrates, are HIV and herpes. Sialic acid can also be found as part of the structure of Sialyl Lewis (SLex) oligosaccharides. SLex are ligands for the selectin proteins family which are involved in cell-cell adhesion processes. 58,61,64 The selectins are displayed on the endothelium (Pand E-selectin), platelets (P-selectin) and leukocytes (L-selectin). P-sele ctins assist, for exam ple, the movement of T lymphocytes to infection or inflammation sites. Then, in orde r to start the immune response, the selectin attaches the lymphocytes to the infected tissue, using the lymphocytes SLex oligosaccharide. These SLex structures are also found in tu mor cells, where the expression of sialyltransferases is usually deregulated.58,65 Hypersialylated cell surf aces might camouflage the core oligosaccharide structure a nd keep the cancerous cell away from the immune response. In addition, selectins are generally implicated in metastases because they recognize these SLex oligosaccharides. Consequently, in order to avoid the expansion of the tumor, the patient could

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46 be treated with selectin antibodies, SLex analogs or inhibitors like KI-8110 which prevents the transport of CMP-NeuAc into the Golgi vesicles (Figure 1-23).58,61,66 O CO2Me O O HO AcHN AcO O O N NH O O F OAc AcO KI-8110 Figure 1-23. Inhibitor KI-811066 Finally, some bacteria like E. coli Streptococci and Helicobacter pylori exhibit sialylated carbohydrates receptors on their su rface. Therefore, they can a dhere to the human digestive system and generate gastric inflammation and occasionally cancer. Bact eria adhesion can be revoked by the presence of soluble sialyl contai ning oligosaccharides, which can be found in human and animal milk.61 Structure Although ST shares the common structural features of other glycosyltran sferases, there is a reduced sequence hom ology (only 10 to 12% on thei r catalytic domain) within the family. This small conserved portion of the C-terminus catal ytic domain is usually divided into three sialylmotifs. One of them is the large L-sialyl motif, which consists of a sequence of 48 to 49 amino acids. Site directed mutagenesis using alan ine mutants has suggested that this motif is involved in the donor substrate binding. On the other hand, the short S-sialylmotif contains 23 residues, and it is proposed to bind both acceptor and donor substrate.62,67,68 Both motifs contain a conserved cysteine amino acid and share donor binding participation. Thus, it was proposed that the Land S-sialylmotif are linked by a disulfide bond which contributes to the catalytic

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47 activity of the enzyme. 62,67 There is a third, very short, SV-sialylmotif which is suggested to operate in the stereochemical control of the ST reaction.62 The catalytic role of L-, Sand SV-sialylmotifs was also confirmed by deletion experiments on ST. It was shown that the enzy me conserves its activit y, even after trimming 71 amino acids from the N-terminus. However, C-terminal truncation only delivers inactive enzyme.67 Despite these ST sequence analyses and char acterizations, the first crystal structure of a ST was obtained by Withers group in 2004.69 In this case, the enzyme was crystallized in the presence of free CMP and the substrate anal og CMP-3-fluoro-N-acetyl neuramic acid. The soluble ST CstII was acquired fr om the human mucosal pathogen Campylobacter jejuni by truncating a section of the C-terminus region th at fixes the enzyme to the membrane without disturbing the catalytic activity. CstII presents a tetramer architecture in which each monomer is conformed by 259 amino acids distributed into two domains. The secondary structure of one of these domains is organized in a fold which has four helices and a seven-stranded twisted -sheet.69 While the nucleotide binding region is arranged in a Rossman fold, the pocket for the nucleotide sugar is on the border of the -sheet. Compared with th is first domain (1-154 and 189-259 residues), the second domain is much smalle r, composed only by 33 amino acids. In this case, the ensemble of and structures form a cap that rests over the active site.69 Based on the classification of glycosyltransfer ases, CstII should be considered a member of the GT-A family due to its single Rossman fold. However, this enzyme lacks the DXD and metal cofactor that are generally found on the GT-A transferases.69 The crystal structure also helped to identify so me of the key amino acids that participate in catalysis. Some examples are Tyr156 and Ty r162, which stabilized the nucleotide phosphate departure by hydrogen bond and His188, which mi ght have the role of the general base

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48 catalyst.69 Mutation of all these residues resulted in a complete inactive CstII.69 Unfortunately, CstII sialyltransferase and the mammalian en zymes do not share any significant sequence homology. As a result, any assumptions on mammals ST mechanism or structure, based on CstII 3D analysis, will be pure hypothesis, and await new crystallographic data. Mechanism Based on steady-state kinetics and kinetic is otope effect (KIE) experim ents, the STcatalyzed reaction is suggested to proceed through an SN1-like mechanism with inversion of configuration, in which the leaving group CMP is almost completely dissociated before the incoming nucleophile attacks (sugar-acceptor) (Figure 1-21).16,70 These studies were performed by Horensteins group on (2 6)ST from rat liver, which is one of the best characterized ST. KIE studies on this enzyme, using CMP-NeuA c and UMP-NeuAc, strongly supported an oxocarbenium ion-like transition state where posi tive charge is accumulated at C-1 and O-1, and the carboxylate group approaches coplan arity with the oxocarbenium ion plane.16,70 It was also proposed that the reaction occurs without significant nucleophilic participation from the acceptor substrate side. Using UMP-NeuAc as the subs trate, a bell-shape pH-rate profile of this enzyme was obtained. From this experiment, two ionizable groups, with pKa of 6.2 and 8.9, were observed.70 The pH-rate profile in conjunction with theoretical calculations suggested that the nonbridging oxygen of CMP phosphate group wa s the target of protonation by the ST general-acid catalyst. Finally, KIE and steady state kinetic studies are consistent with a (2 6)ST random sequential mechanism.16 It should also be noted th at there is no evidence for a role for divalent metal cations in sialyltransf erase catalysts which differentiate them from glycosyltransferases that use sugar nu cleotide diphosphates as donor substrates.

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49 Sialyltransferase inhibitor design The search f or ST inhibitors has been difficult due to intrinsic features of the enzymes such as a complex four-partner transi tion state, weak binding with thei r natural substrates, and limited structural data. Based on inhibitory experiments performed on ST nucleotide analogs, the cytidine portion of the donor substrate seemed to be essential for ST recognition. This hypothesis was demonstrated by the fact that free sialic acid did not show any inhibition on a human serum (2 6)ST catalytic reaction. On the other hand early inhibition studies with different nucleotides placed CMP, CDP and CTP as the best ST competitive inhibitors with Ki of 50 M, 19 M and 16 M, respectively.59,71 Other uridineor adenosine-based nucleotides presented a non-competitive type of inhibition with Ki in the range of 0.2 to 7 mM.71 In addition, it was noticed that the number of phosphate s present in the nucleotide affect ed the inhibitory potency in a sequential manner with CTP > CDP > CMP > cytidine or UTP > UDP > UMP. Comparative studies on cytidine, deoxycytid ine and cytosine (with cytosine being the worst inhibitor) exhibited that, in some way, the ribose moie ty provides favorable en zyme-substrate contacts (Figure 1-24).71 O HO HO OH N N NH2 O O HO OH N N NH2 O N H N NH2 O cytidine 2'-deoxycytidinecytosine Figure 1-24. Cytidine, deoxycytid ine and cytosine structures

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50 Using these early results, seve ral groups have designed ST transition state analogs that contained the cytidine moiety. For example, Schm idt and co-workers synthesized a series of CMP-quinic-acid-based ST anal ogs which displayed a ST inhibitory potency in the M range (Figure 1-25).72,73 O HO HO OH O CO2 -Na+ P O O-Na+ O O OH OH N N O NH2 CMP-quinicacidKi=44 MO O O P O O-Na+ O O OH OH N N O NH2 AcHN 7Ki=2.4 M HO OP O O-Na+ O O OH OH N N O NH2 P O +Na-O OH 8Ki=0.20 MOP O O-Na+ O O OH OH N N O NH2 P O +Na-O OH O HO AcHN O 9Ki=29nM Figure 1-25. Schimdts ST inhibitors60,72-74 Other series of compounds (see 7 in Figure 1-25) had planar ity at the anomeric center mimicry, which incorporated a new TS feature in ST analogs design.60,73,74 This element gives a better representation of the sugar conforma tion during the TS. Moreover, these compounds activity was improved by not only increasing the distance between the anomeric center and the nucleotide but also including an extra negati ve charge into the glycosyl bond mimic (see compounds 8 and 9 in Figure 1-25).60,73,74 From studies on these series of molecules, it was also observed that replacement of the sialic acid portion for an aromatic or alkyl group does not affect the inhibitory activity of the analog (compound 8 in Figure 1-25).60,62 The enzyme might have a

PAGE 51

51 hydrophobic pocket, closed to the catalytic site, th at could help on the enzyme-substrate binding. In accordance with all these studies, th e most potent inhibitor for ST (compound 9) was found to be the molecule that possessed all of the structural elements discussed so far (Figure 1-25).74,75 Previously, our group has proposed and studied a new class of sialyltransferase inhibitors in which a bicyclic system, was used to mimic the TS conformation of these enzyme reactions.76 The shape of these molecules loosely resembled a scorpion, hence the use of scorpio to denote members of this family. In th ese inhibitors, the CMP group attach ed to the bicyclic system was strategically held at a dist ance to simulate a late tr ansition state for bond cleavage.76 The first generation of scorpio inhibitors consisted of an unsaturated bicyclic structure conjugated with a carboxylated group (Figure 1-26). O O OH OH O N N O NH2 P O OOO Ki=10 Mfor (2,3)ST Ki=20 Mfor (2,6)ST O O OH OH O N N O NH2 P O ON H H Ki=5 Mfor (2,3)ST FirstScorpioGeneration SecondScorpioGeneration Figure 1-26. ST transition state analog s synthesized by Horensteins group76,77 Although these TS analogs presented a good inhibitory activity towards both (2 3)ST and (2 6)ST, with a Ki of 10 M and 20 M respectively, they lacked positive charge at the anomeric center mimicry that could simulate the oxocarbenium ion TS. A second generation of scorpio molecules synthesized in our group mimicked the oxocarbenium TS positive charge with an amine functional group. This co mpound displayed a good potency on (2 3)ST with a Ki of 5 M77 (Figure 1-26). Following this structural mimetics approach and trying to improve the

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52 inhibitory potency of the donor derivative, diazabicyclic transition state analogs were synthesized for ST inhibiti on studies (Figure 1-27). OR NH N R' TargetMolecules OR N H N R' Figure 1-27. Proposed diazabicyclic TS analogs Compounds possessing diaza functionality will mi mic the development of positive charge on the anomeric center, allowing eq ual distribution of charge within the trigonal planar system. With the diazabicyclic compounds we will be incor porating certain key structural elements for sialyltransferase recognition: a) geometric requirements, i.e., tr igonal planar center, b) CMP functionality, c) positive charge to mimic oxocarbenium character, and d) proper mimicry of bond distance between the depa rting phosphate and the position analogous to the anomeric carbon. Due to the close similarity between si alyltransferase and gl ycosidase catalytic mechanisms, the diazabicyclic TS analogs will be also suitable for the study and characterization of carbohydrate hydrolyzing enzymes (Chapter 4). Cyclopropane Derivatives Historically, cyclopropane derivatives have caught the attention of different chem istryrelated scientific fields. Th e compounds importance arises not only from their unusual C-C single bonding that resembles a double bond but also from their great angular ring strain (27.5 kcal/mol).78,79 In particular, cyclopropane derivatives ha ve served as unique building blocks for the synthesis of biological related molecules. Either as a natural product or as a synthetic derivative, the cyclopropane ring has been part of molecules that have shown a variety of

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53 biological functions like antiviral, antitumor or antibiotic agents, en zymes inhibitors, insecticides and agonists or antagonists for neural receptors. Consequentl y, cyclopropanoid molecules are useful in the design of novel therapeutic agents and characterization of biological mechanisms. Cyclopropanes Biological Implications An exa mple of a bioactive cyclopropane derivative is 1-hydroxycyclopropanecarboxylic acid phosphate HPC (Figure 1-28). This compo und acts as a competitive inhibitor for the enzymes that utilize phosphoenolpyruvate (PEP ) (Figure 1-28) like PE P carboxylase, enolase and pyruvate kinase. It wa s proposed that HPC potency is related to its geometric and electronic structure similarity to PEP.78 OPO3Na2 COOMe HPC COOMe OPO3Na2 PEP Figure 1-28. HPC and PEP structures78 2,3-dihydroxy acids are used by microorganisms and higher organisms like plants and animals to synthesize amino acids. This synt hesis is regulated by dihydroxy acid hydrolases through enzyme bound enol intermediates (see compound 10 in Figure 1-29). TS analogs for these enzymes could serve as herbicides or antibio tic agents. Via their elec tronic similarity with C-C double bond, cyclopropanoid compounds like 11a-d are known to be good TS analogs for the hydrolases dehydration reaction.78 OH COOH 11a-d R1 R2 OH COOH 10aR1,R2=H bR1,R2=Me cR1=Et,R2=Me dR1=Me,R2=Et R2 R1 Figure 1-29. Dihydroxy acid hydrolases e nol intermediates and TS analogs78

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54 In the mammalian central nervous system, L-glutamic acid works as an excitatory neurotransmitter. In order to investigate the m echanism and function of the glutamate receptors (GluR), different carboxycyclopropy l glycine (CCG) isomers have been prepared (Figure 1-30). In several cases the biological activity of a molecu le is associated with its specific conformation and constraint. These two properties can be achieved by cyclopropane rings, which is demonstrated by the fact that only CCG 12 displayed a selective and potent agonist activity towards GluR.78 HOOC COOH H NH2 HOOC COOH H NH2 FormA FormB COOH H COOH NH2 H GlutamicAcid HOOC H COOH NH2 H CCG12 GluRagonist CCG13 GluRinhibitorH H H COOH NH2 H HOOC CCG14 GluRinhibitorH H H COOH NH2 H COOH CCG15 N-methyl-D-asparticacid receptoragonist Figure 1-30. Glutamate receptor (GluR) agonist s and inhibitors adapted from the article published by Salaun, J.78 As a final example, it wa s reported that dichloro cis diphenylcyclopropanes possess antiestrogenic activity (Figure 1-31). Antiest rogens not only regulate and correct diverse endocrine disorders but also they obstruct the gr owth of estrogen-dependent mammary tumors. Tamoxifen (TAM) is a clinically used antiest rogen drug that operates by competitive inhibition of the estrogen receptor (Figure 1-31). This compound is useful for either treating hormonedependent tumors or preventi ng their appearance in premenopausal women. Stilbene-derived

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55 cyclopropyl compounds showed bett er responses in estrogen-depe ndent and independent breast cancers than the parent TAM.78 Et Me2H2NH2COC Tamoxifen(TAM) Cl Cl R1 R2 R3 Dichlorocisdiphenylcyclopropanes R1-3=H,OH,OMe,OCH2Ph,OCH2CH2NMe2 Figure 1-31. TAM and stilbene-derived compounds78 Cyclopropane Preparation The work described in this dissertation is focused on the synthesis of diazabicyclic TS analogs for ST and glycosidases. Based on the inhibitors design and shap e, an all cis trisubstituted cyclopropane was required as the starting core structure. Three membered carbocycles can be synthesized by se veral different methods. Some of the most relevant reactions that can form the cyclopropyl ring were reviewed by Tsuji and Nishida in 1987.80 The review included: a) 1,3 bond formations; b) rearrangement reactions; c) transformation of cyclopropyl derivatives; and d) combination of two build ing blocks. Among these cyclopropane ring preparation techniques, the combination of two carbon units is the most commonly utilized. Examples of each procedure will be given bellow. 1,3 cyclopropane bond formation The 1,3 bond for mation can be achieved by a simple intramolecular nucleophilic displacement. Because the r eaction proceeds through an SN2 mechanism, inversion of configuration is observed at th e carbon that holds th e leaving group. The reaction of a conjugate

PAGE 56

56 enone to give a -cyclopropyl-unsaturated carbonyl compound is one example (Figure 132).81 O H O H 1)HBr 2)tBuONainalcohol Figure 1-32. Cyclopropanation of -unsaturated carbonyl compound81 Cyclopropyl rings have also been obtained from the nucleophilic s ubstitution of Michael acceptors.82,83 A common intermediate in this reacti on is a carbanion with a good leaving group, usually a halogen, situated at a three carbon distance. In addition, the carbanion is frequently stabilized by an adjacent elec tron-withdrawing group (Figure 1-33). O OMe Cl O OEt MeOOC COOEt MeOOC COOEt and NaOEt Figure 1-33. Cyclopropanation by nucleophi lic substitution of a Michael acceptor82 Finally, a reaction that also falls in this ca tegory of cyclopropanati on is the electrophilic addition to the terminal position of 3butenyl organometallic compounds. Robbins et al. reported the use of 3-butenylstannanes as an ex ample of this reaction (Figure 1-34).84 Rearrangement reactions Substitu ted cyclopropanes can be prepared fr om cyclobutanes by ring contraction. If the cyclobutane is substituted by an electron-donating group a nd a leaving group, the ring contraction is easily accomplished. Usually, this r eaction occurs with inversion of configuration at the carbon containing the leaving group.80 Cyclobutanes that normally undergo this transformation include 2-substituted cyclobutanols and -substituted cyclobutanones. An

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57 example of this method utilized boron trif luoride butyl etherate acidic condition on 2hydroxycyclobutanone (Figure 1-35).85 SnBu3 EX E EX=Cl2,Br2,I2,HgCl2,ArSCl,SO3 Figure 1-34. Cyclopropanati on of 3-butenylstannanes84 O OH O H 1)LiAlH42)BF3.(nBu)2O Figure 1-35. Ring contracti on of 2-hydroxycyclobutanone85 Transformation of cyclopropyl derivatives Substitution transformations on three membered carbocycles are commonly performed via organometallic intermediates. Organometallic cy clopropyl complexes are in general stable and can react avoiding ring opening. This con cept was used in the synthesis of cyclopropyltrimethylsilanes from the 1-bromocyc lopropyltrimethylsilane precursors (Figure 136).86 Si(CH3)3 Br nBuLi -95C THF Si(CH3)3 H Figure 1-36. Synthesis of cyclopropyltrimethylsilanes86 Interestingly, cyclopropyl halides can be eas ily converted into cyclopropyllithiums or Grignard reagents, which could be further utilized in other organic tr ansformations (Figure 137).87

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58 Li OCH3 O O OCH3 Figure 1-37. Reaction of cyclopropyllithium reagent87 Combination of C2 and C1 building blocks One example of this kind of cyclopropanation is the coupling reaction between ylides and compounds that are vulnerable to Michael addi tions. The reaction of the N-Boc pyrrolinone glutamic acid derivative with diphenylsulfonium isopropyl ylide successfully delivered the 3azabicyclo[3.1.0]hexan-2-one in a 79% yield (Figure 1-38).88 N O Boc OTBS N O Boc OTBS Ph2+SC-Me2 79% Figure 1-38. Reaction of N-Boc pyrrolinone and diphenylsulfonium isopropyl ylide88 The preparation of cyclopropanes via carbene intermediates is among the most studied cyclization reaction, not only fo r pure organic synthetic rationale but also for the better understanding of carbene chemistry. An effective transfer of a methylene unit to an alkene is done using the Simmons-Smith reagent.89 A combination of methylene iodide and Zn/Cu couple gives the active iodomethyl zinc i odide alkylating agent. This is a very clean reaction that does not promote alkene isomerizati on or insertion side products frequently generated in carbene additions. The stereochemistry of the Simmons-Sm ith reaction is controlled by steric effects and the coordination of the zinc atom with other heteroatoms and functional groups such as oxygen, nitrogen and -OH.80 A modification of this reaction utilizes diethylzinc and diodomethane as the carbene source.90 Examples of the Simmons-Smith reaction are shown in Figure 1-39.90,91

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59 O O OH CH2I2Cu/Zn O O OH H3CO OH H3CO OH CH2I2Zn(Et)2 Figure 1-39. Simmons-Smith cyclopropanation reactions90,91 For the preparation of acyland alkoxylcyclopropanes, carb enes are generated by thermal or photochemical decomposition of diazo compounds. Normally, the cyclopropanation is performed under dilute conditions. This procedure is utilized to avoid norm al side reactions such as, dimerization of the carbenoid, C-H insertions hydrogen abstraction, 1,3 dipolar additions and Wolff rearrangement.80 Copper, rhodium and palladium salts or complexes with organic ligands are among the most successful catalysts. Among them, Rh(II) carboxylates, having only one coordination site per metal, form stable comp lexes with basic ligand but not with alkenes.92 They are very tolerant to steric factors, which allow th em to participate in cyclizations of disubstituted olefins. On the other hand, Pd derivatives are ve ry suitable for olefin coordination, but they are only effective in monosubstitued double bond cyclopropanations. With respect to copper catalysts, copper triflates (CuOTf) can be complexed with other ligands due to the triflate anions lack of coordination ability. They generall y exhibit intermediate efficiency on the cyclopropanation of alkenes when compared with Rh and Pd derivatives.92 In general, the active species in cyclizations is defined as a metallocarbene intermediate which transfers the carbene unit to the desired mo lecule. The metallocarbene intermediate can be

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60 considered an electrophile in which the carbocation is stabilized by th e metal (Figure 1-40). A summarized representation of the cyclopropanation reaction is shown in Figure 1-41.93 MR2-CN+ N -N2MC+ R R MC R R Figure 1-40. Metallocarbene intermed iate adapted from Carey, F. A., Advanced organic chemistry ; 4th ed.93 LnM MLnCR2 R2CN2N2 R R Figure 1-41. Cyclopropanation general re action adapted from Carey, F. A., Advanced organic chemistry ; 4th ed.93 In the transition state model proposed by Casey94, there is an initial electrostatic interaction between the metallocarbene center and one end of the alkene (CA) (Figure 1-42). Then, the positive charge generated on the other alkene carbon (CB) removes the metal with a simultaneous carbene-CB bond formation which gives ri se to the cis cyclopropane.95 If the size of the alkene substituent groups increase, a metal four-membe r ring intermediate might form, delivering the trans cyclopropane isomer.94,95 This model explained the product stereoselectivity obtained with alkylcarbene-metal complexes, but it did not expl ained the trans favored selectivity when diazo carbonyl compounds were used.

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61 LnM H Z AC H H B C H R Ln M H H R H H Z H H Z R H H LnM Z=carbenefunctionalgroup R=alkenefuctionalgroup Figure 1-42. Cyclopropanation TS proposed by Ca sey adapted from th e article published by Doyle, M. P.94,95 A second model was presented by Doyle et al. in which they propos ed that the initial interaction between the metal-car bene complex and the alkene system is in equilibrium with the free olefin (Figure 1-43). LnM H Z H R H H A LnM H Z H H H R B LnM H Z H R H H LnM H Z R H H H T SB R H H Z LnM H Z H R H H TSA H R H Z Figure 1-43. Cyclopropanation TS adapted from the article published by Doyle, M. P. 95 Then, the parallel position of the double bond relative to the car bene center is established by the electro-donating group R stabilization towa rds the electrophilic center. Consequently, there are two possible TSs (no metallocyclobutan e intermediate) that can account for the two

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62 stereoisomers formed. The TS is then regulated by the initial -metal-carbene steric and electronic interactions. Finally, the ring closure occurs through backside displacement of the metal-ligand complex.95 With Doyles model, it is possible to explain why the trans cyclopropanes are favored with diazo carbonyl carbenes. In this case, the carbonyl group can stabilize the forming electrophilic center with its oxygen lone pair electrons, forcing the olefin position to the one that will give the trans configuration (Figure 1-44).95 LnM H H H H R LnM H R H H H R H H O OEt LnM H H H H R O OEt O OEt O EtO -LnM Figure 1-44. Cyclopropanation TS proposed by Doyl es for carbonyl carbenes adapted from the article published by Doyle, M. P.95 There are two types of diazo cyclopropanations either intermolecular or intramolecular. Usually, the intermolecular cyclization delivers the anti isomer as the favored one due to steric or electronic factors. However, the enantioselectivity of the reaction, in some cases, might be led by a metal catalyst possessing chiral ligands.80,96 Fortunately, intramolecu lar carbenoid reactions can be the solution to this problem. Based on D oyles reports, intramolecular cyclizations only deliver one of the possible three member rings due to geometric constrains.97 The restriction of this kind of reaction is that th e double bond of the substrate has to be in close proximity to the diazo center. Thus, only bicycl o[3.1.0]hexanes and bicyclo[4.1.0] heptanes were successfully obtained. From studies performed on Rh catalyzed cyclizations, the stereoselectivity of the reaction will be dictated by the li gands configuration around the metal, the course of the olefin approach and its orientation towa rds the face of the catalyst. From theoretical calculation on the complex Rh2(5S-MEPY)4, the lower energy TS geometry was found to have the spatial orientation shown in Figure 1-45.

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63 Rh O N N O Rh N O O N CO2Me Rh2(5S-MEPY)4Rh N O N O MeO2C CO2Me R R N O N O MeO2C CO2Me R R H COO R N2CHCOOR Figure 1-45. Rh2(5S-MEPY)4 TS geometry adapted from the article published by Doyle, M. P.95 It was observed that the carbene C-H bond falls between the Rh two lig ands and the olefin approaches the carbenoid center from its less sterically hindered face (Figure 1-46).97 This alkene orientation leads to two possible enantiomers. An example of the two enantiomeric adducts is presented in Figure 1-46.97 RCRh O O N N EtO2C CO2Et H O RA RB RC O Rh O O N N EtO2C CO2Et H O O RA RB RC O RB RA H O RCO RB RA H O AB Figure 1-46. The two possible products from the alkene intramolecular cyclopropanation adapted from the article published by Doyle, M. P.95

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64 CHAPTER 2 SYNTHESIS OF GLYCOSIDASES AND GLYCOSYLTRANFERASES TRANSITION STATE ANALOGS CORE STR UCTURE Introduction As already m entioned in chapter 1, our group has developed a new class of sialyltransferases inhibitors. These compounds [3.1.0] bicyclic systems were used to mimic the apical orientation of a leaving group relative to the oxocarbenium i on plane in the transition state conformation of these enzyme reactions. Following this structural mimetics approach, a family of diazabicyclic compounds will be synthesized tryi ng to improve the inhibitory properties of the compounds known at the moment, and further refi ne our understanding of what constitutes an effective set of features for a good inhibitor. Based on the close si milarity between glycosidases and ST mechanisms and TS, diazabicyclic analogs will be suitable to study both enzymes catalytic reaction. Because the departing l eaving group (LG) mimicry will be achieved by holding the aglycon moiety above the amidine group, one of the synthetic challenges rests in producing the differentia lly functionalized all cis 1,2,3 cyclopropanes that could serve as the starting core structure. A simplifie d retrosynthetic route utilized in the design of TS analogs is shown in Figure 2-1. OR NH NH R' NH2 NH2 RO RO O O OH OH Figure 2-1. Retrosynthetic route for seve n-membered diazabicyclic TS analogs Results and Discussion To have a sense of how to synthesize th e requisite am idine moiety, model compound 2carboxy-4,5-dihydro imidazole 4 was prepared using ethylenedi amine and ethyl glycoloimidate 2 as starting materials (Figure 2-2). Th is method was utilized by Hamilton et al. in the preparation of 2-carboxy-4,5,6,7-tetrahydro-1,3-diazepine.98

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65 HO CN HO NH2 +ClO EtOH/HCl + NH2NH2 N H N OH N H N OH O 76% 30% 15% KMnO41 2 34 Figure 2-2. Synthesis of 2carboxy-4,5-dihydro imidazole Firstly, ethyl glycoloi midate hydrochloride 2 was prepared from the reaction of glycolonitrile 1 with ethanol in acidic media. After that, reaction of the imidate with ethylenediamine afforded 3 as colorless crystals in 30% yield without further purification. Finally, oxidation of hydroxymethyl imidazole was accomplished with potassium permanganate at pH = 11. Centrifugation and decantation of the aqueous solution removed the brown manganese dioxide precipitate. The final pr oduct was purified by Dowex 50 resin in the acid form to give 2-carboxy-4,5-dihydroimidazole 4 as a white solid. It wa s found that this amidine was stable for 3 days in water at pH = 4 which demonstrated the stability of the carboxyamidine functional group. As already described in Chapte r 1, the sialyltransferase donor substrate CMPNeuAc bears a carboxylic acid moiety at the anomer ic carbon. Thus, the rela tive stability of the carboxyamidine is an important pie ce of evidence that the group might be able to be incorporated into TS analog inhibitors. The reaction between a Michae l acceptor and an active methylene compound was used as the preliminary attempt to synthesize a cis cyclopropane dicarboxylic acid (Figure 2-3). Then, methyl acrylate and ethyl chloroacetate were treated with NaOEt at room temperature. The crude reaction mixture resulted in a very complex combination of unreacted starting materials, diastereoisomers and side products Any efforts to purify the mixture by fractional distillation or column chromatography resulted in a group of impure fractions from which it was difficult to determine the presence of the desired products.

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66 O OMe Cl O OEt MeOOC COOEt MeOOC COOEt and NaOEt complexmixture Figure 2-3. Cyclopropanation using a Michael acceptor Intermolecular cyclopropanati on conditions were then utiliz ed as the second approach toward the stereoselective synthesis of trisubstituted cis cyclopropanes. Consequently, the efficacy of metal-catalyzed cyclizations of Z -2-butene-1,4-diol with ethyl diazoacetate were explored. This approach was chosen with foreknowledge that although a preference for exo addition (leading to a trans cyclopropane) might predominate, the poor stereoselectivity of the reaction might yield sufficient endo addition product to allow rapid exploration of downstream synthetic steps. Since allylic alcohol derivatives are known to r eact with carbenoids to form ylids that undergo [2,3]-sigmatropi c rearrangements, the allylic diol was protected to sterically obviate such processes. The requisite Z -2-butene-1,4-diol 5 was transformed to the corresponding acetal form by protecting with 2,2-dimethoxypropane in presence of catalytic amounts of p-toluenesulf onic acid (Figure 2-4).99,100 Fractional distillation at low pressure gave 2,2-dimethyl-1,3-dioxole 6 in a 63% overall yield. OH OH 2,2-dimethoxypropane acetone O O O O EtO O 567 63% Catalyst N2CHCO2Et H H exo Figure 2-4. Intermolecular cycloprop anation of Z-2-butene-1,4-diol99,100

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67 Toward the goal to achieve the desired endotrisubstituted isomer on the cyclopropanation step, the catalyzed reaction of cis -protected diol 6 by rhodium (II) acetate in dichloromethane was first investigated. After tedious purificati on of the cycloaddition reaction mixture between ethyl diazoacetate and 6, only the exo -substituted isomer (8:1 exo : endo ) was isolated in a 24% yield. This preferential stereochemistry of the product was fully confirmed by H1 NMR and 2DNOESY experiments. The NOESY spectrum of 7 exhibited crosspeaks between Hd-Hb, Ha-Hc and Ha-CH3a (Figure 2-5). Following the idea that the favored exo over endo stereochemistry could have been taking place due to a possible coordination between rhodium catalyst and the two oxygens of the allylic protected alcohol, other solvents as THF and benzene were utilized for the cycloaddition. Once again, the results showed that only exo isomer was present in the mixture. Furthermore, despite other atte mpts using catalyst like CuOTf and Cu(acac)2 it was not possible to force the stereochemical outcome of the cycloaddition reaction to yield more endo product. OO CH3a bH3C Hd Hd exo Ha O O Hb Hb Hc Hc Figure 2-5. Characterization of dioxa bicyclo[5.1.0]octane by 2D-NOESY Since bimolecular cyclopropanations were not useful for the problem at hand, the intramolecular cycloaddition of a llyl diazoacetic esters was utili zed as the synthetic solution. Based on previous studies,95,97 intramolecular cyclizations necessarily deliver cyclopropanes with all three substituents in the cis conformation. Because the requisite cyclopropane might also have all three substituents chemoselectiv ely differentiated from each other, the

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68 bicyclo[3.1.0]lactone 9 (Figure 2-6) was chosen to be the ke y core intermediate in the TS analog synthetic route. Consequentl y, corresponding diazoacetic ester 8 was prepared (Figure 2-6). O OR N2 O O OR O Catalyst 89 Figure 2-6. Metal-catalyzed diazoeste r intramolecular cycloprapanation97 The first step on the ester synthe sis involved mono-protection of Z -2-butene-1,4-diol with tert butyldimethysilyl chloride (TBDMSCl). Subse quent reaction of the free hydroxyl group in compound 10 with Boc-protected glycine resulted in the bi functionalized alcohol 11 in 80% yield (Figure 2-7). Treatment of the Boc-protected ester with a solution of trifluoroacetic acid (TFA) in dichloromethane or trimethylsilyl io dide (TMSI) did not result in the desired free amine. Instead, either compound 10 or silyl cleavage of 11 were observed as side products by 1H NMR and TLC. OH OH TBDMSCl OH OTBDMS + DMAP DCC O OTBDMS BocHN OH O NHBoc O 10 11 43%80% O OTBDMS NH2 O TFA/CH2Cl2or TMSI Figure 2-7. Synthesis of the Z -bifunctionalized ester 11

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69 Therefore, the allylic alcohol Z-butene-1,4diol was converted into the less labile monoprotected benzyl alcohol 12 using standard methods (Figure 2-8).101 OH OH OH OBn + DMAP DCC O OBn BocHN OH O NHBoc O 87% O OBn NH2 O Benzylbromide NaOEt/THF O OBn N2 O 15%TFA/CH2Cl2 isoamylNitrite AcOH/CH2Cl242% 20% 60% 12 13 14 8 Figure 2-8. Synthesis of diazo ester 8 Subsequent reaction of 4-benzyloxy diol 12 with Boc-glycine gave the cis alkene 13 in an 87% yield. When the Boc-protected ester was tr eated with 15% TFA in dichloromethane, the reaction delivered the desired glycinate 14 in a 42% yield.102 The benzyl amino alkene 14 was converted to the cis diazoacetic ester 8 by a conventional nitration r eaction using isoamyl nitrite in a 20% yield (Figure 2-8).103 Unfortunately, this synthetic route produced the cis diazo ester in a very low yield. H2N COOH N H COOH O F F F N H COOH O O TFA FMocCl 10%Na2CO370% 79% 15 16 Figure 2-9. Synthesis of N-trifl uoro and FMoc protected glycine

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70 At this stage, because the diazo ester was the starting material for all the different multistep synthetic pathways, the yield obtained in each reaction was particularly important. It was observed that the deprotection of compound 13 with TFA not only was generating the free amine but also was causing the hydrolysis of the ester linkage. The reactivity of other amine protecting groups was tested in order to obtain 14 under reaction conditions that might not affect the ester bond. Then, different N-trifluor o and 9-fluorenylmethyloxycarbonylglycine (FMoc-glycine) moieties were synthesized by literature methods.104,105 These two amino protecting groups are usually used in peptide synthesis and can be removed in mild basic conditions (Figure 2-9). The cis protected diol 12 was coupled to N-trifluoro and FMoc-glycine by the same method utilized for compound 13. The esters N-trifluoro 17 and FMocacetate 18 were obtained in 74% and 38% yield respectively (Figure 2-10). H N O F F F O O O OH O DCC/DMAP H N O O O O DCC/DMAP O N H COOH O F F F 15N H COOH O O 16 74% 38% 17 18 Figure 2-10. Synthesis of Ntrifluoro and FMoc esters

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71 Unfortunately, attempts to deblock the trifluoro amide protecting group with K2CO3 in MeOH resulted, once more, in the hydrolysis of the ester connectivity giving back diol 12. Based on a Carpino et al. report, the FMoc group can be removed by nonhydrolytic conditions for example, when the protected derivative is treated with liquid amm onia for several hours.104 The hydrogen at the position from the carbamate unit of the FMoc conjugated system becomes greatly acidic and can be easily abstracted by we ak bases. This hydrogen acidity is driven by the fact that the conjugated system becomes aromatic after the abstraction of the proton. The same result can be obtained when secondary amines like morpholine or piperidine are utilized as the deprotecting agents. In both cases, dibenzofulve ne is formed as a by-product which could be isolated without difficulty from the reaction mixture. Following Carpinos methodology, cis ester 18 was treated with a piperidine at room temp erature. After the react ion was stirred for 30 minutes, analysis of the crude mixtur e by TLC showed total conversion of 18 again into the starting material 12. This result was also confirmed by H1 NMR. From all these unsuccessful efforts to obtain compound 14, it is believed that the weakness of the ester bond might be related to its proximity to the alkene group. Consequently, a different me thod to synthesize the cis diazo ester 8 had to be pursued. In 2004, Collado et al. presented a new met hodology for the synthesis of these kind of diazo compounds in which th ey are generated from an acetoacetate ester intermediate.106 This synthesis utilized benzyl cis butene-1,4-diol 12 as the starting material. The reaction of its free hydroxyl group with the extremely reactive diketene gave compound 18 in an 61% yield (Figure 2-11). Then, trea tment of the acetoacetate este r with p-ABSA (p-acetamido benzenesulfonyl azide) in Et3N resulted in the desired cis diazoacetic ester. This reaction starts

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72 with a diazo transfer from th e sulfonyl azide. Then, compound 8 is generated by base-induced deacylation of the -diazo acetoacetic ester intermediate. O OBn N2 O OH OBn O OBn O O O O NaOAc/THF 61% 1)p-ABSA CH3CN/Et3N 2)LiOH 79% 12198 Figure 2-11. Improved synthesis of diazoester 8106 A key step in the synthetic design was the intramolecular cyclopropanation of 8 to give the all cis trisubstituted cyclic system. Hence, starting from the cis alkene 8, the resulting fused cyclopropyl lactone 9 will have all its substituents in the cis configuration. The first cyclization reaction was performed utilizi ng the commercially available Rh2OAc4 as the catalyst. The reaction was executed under dilute conditions, adding the diazo compound via a syringe pump over 18 h. In this case, the lactone 9 was obtained in a 32% yield. Due to the poor yield obtained in this reaction, the catalytic efficiency of CuSO4 in benzene was then tested. After refluxing the reaction for 12 h, the cyclic lact one was isolated in 48% yiel d. The last metal catalyst investigated was Cu(I)TfO. This catalyst was ut ilized in presence of the Evans ligand. HO NH2 TBu NC CN N O N O ZnCl2 Cl Evan'sligand Figure 2-12. Synthesis of Evans ligand107 The Evans bis oxazoline ligand was synthesi zed by a literature pr ocedure using L-tertleucinol as starting material (Figure 2-12).107 This reaction conditions were finally chosen for the

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73 cyclization reaction because they afforded the best product yield. A summary of the yields obtained in the intramolecular cycliz ations is presented in Table 2-1. Table 2-1. Comparison of the intramolecular cy clopropanation yield obtained with different catalyst Catalyst Yield Rh2OAc4 32% CuSO4 48% CuTfO / Evans ligand 60% In order to confirm that lactone 9 had the all cis configuration, the molecule was characterized by 2D-NMR spectroscopy. O H Ha O Hb OBn Hc Hd 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm) 2.0 2.5 3.0 3.5 4.0 4.5 F1 Chemical Shift (ppm) 3.7, 4.22 3.42, 4.22 Figure 2-13. Lactone 9 2D-NOESY characterization

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74 Crosspeaks between Ha-Hc (Ha = 4.22 and Hc = 3.42 respectively) and Ha-Hd ( Hd = 3.71) were observed in the NOESY spectrum of 9. This set of hydrogen atoms has a spatial interaction due to the endo configuration of the ring. The NOESY spectrum obtained is shown in Figure 2-13. Experimental Section General me thods. Solvents and reagents were purchased from Aldrich Chemical Company and Acros Organics. The organic solvents were dried overnight over CaH2 or 4 molecular sieves and freshly distilled before use. NMR spectra were obtained using VXR 300, Gemini 300 and 500, or Mercury 300 and 500 MHz spectrometers in appropriate deuterated solvents. Mass spectra were obtained on a Finne gan MAT 95Q spectrometer operated in FAB, CI, ESI or EI modes. Infrared (IR) spect ra were obtained by deposition of CHCl3 solutions on NaCl plates followed by evaporation of the solvent. Ethyl 2-hydroxyacetimidate hydrochloride 2. To a solution of acetylchloride (390 L, 5.51 mmol) in ether (5.0 0 mL), ethanol (640 L) was added with stirring at room temperature, to generate HCl in situ. The mixture was stirred for 3 h and then glycolonitrile 1 (0.50 ml, 5.51 mmol of a 55 wt% solution in H2O) was added at -10 C. The mixture was stirred for 4 h. Then, ether was added and the reaction wa s kept for 24 h at -20 C. Afte r this time, crystals formed which were collected by filtra tion, washed with ether and then dried under vacuum. Compound 2 was obtained as white crysta ls in 76% yield (0.43 g). 1H NMR (D2O) ppm 1.28 (t, 3H), 4.22 (s, 2H), 4.23 (q, 2H), 13CNMR (D2O) ppm 174.6, 62.2, 59.8, 13.4. (4,5-Dihydro-1H-imidazo l-2-yl)-methanol 3. To a solution of ethylenediamine (67 l, 1.1 mmol) at 0 C in absolute ethanol (740 L), ethyl glycoloimidate 2 (130 mg, 1.32 mmol) was added with stirring. The mixture was kept at 0 C for 1 h and then heated up to reflux within 0.5

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75 h. Reflux was maintained until no more ammonia gas evolution was observed (approximately 1.5 h).The hot solution was mixed with alcoholic HCl and then filtered while st ill hot. After leaving the filtrate overnight at -20 C, colorless crystals were collected in 30% yield (0.03 g). 1H NMR (D2O) ppm 3.93 (s, 4H), 4.51 (s, 2H),13C NMR (D2O) ppm 171.7, 55.2, 44.6. 2-Carboxy-4,5-dihydro imidazole 4. After dissolving 3 (35 mg, 0.4 mmol) in water (0.6 mL), adjusting the pH to 11 with 6 N KOH and c ooling the solution to 0 C, small portions of KMnO4 (86 mg, 0.4 mmol) were added with continuous stirring over a 3 h period. The reaction solution was then centrifuged for 15 min at 12000 rpm, the aqueous solution decanted and the brown precipitate MnO2 washed with water (2 x 1 mL). After combining the aqueous solutions, the reaction mixture was concentrated under reduced pressure, and 5 mL of ethanol were added. The white inorganic solid was removed by filtration. The solvent was removed by reduced pressure and the product was purified by ion exchange chromatography. The residue was dissolved in water (1 mL) and applied to Dowex 50 (H+) resin. Product was eluted with 0.5 M aqueous NH4OH to give the carboxy amidine in 15% yield (6.8 mg). 1H NMR (D2O) ppm 3.42 (s, 2H), 3.55 (s, 2H). EI LRMS Calcd for C4H7N2O2 (M + H)+: 115, found: 115. 2,2-Dimethyl-1,3-dioxocyclohept-5-ene 6. A mixture of Z-2-butene-1,4-diol 5 (4.8 mL, 58 mmol), 2,2-dimethoxypropane (7.1 mL, 58 mmo l), acetone (4.2 mL) and benzene (11 mL) with catalytic amount of p-tolu enesulfonic acid (29 mg) were h eated near 65 C for about 1 h. During this time, some methanol, benzene and acetone were distilled off. Dry benzene was added periodically to the reaction flask. The mixt ure was purified by frac tional distillation at 20 mm Hg. Pure product 6 (4.7 g, 63%) was collected from the fraction that distilled in a range of 53-54 C. 1H NMR (C6D6) ppm 1.34 (s, 6H), 4.06 (d, 4H), 5.38 (t, 2H), 13C NMR (C6D6) ppm 129.7, 101.8, 61.2, 24.0. The NMR spectrum was consistent with the literature.100

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76 4,4-Dimethy-8-ethylformyl-3,5 -dioxa-bicyclo[5.1.0]octane 7. To a solution of 6 (1 g, 8 mmol) in CH2Cl2 (8 mL), Rh2(OAc)4 catalyst (34 mg, 78 mol) was added with stirring. After this mixture has been stirred for 1 h, a hom ogeneous green solution was obtained. Ethyl diazoacetate (0.7 mL, 6.3 mmol) in CH2Cl2 (6.3 mL) was added to th e solution using a syringe pump at a rate of 8 L/h over 24 h. The reaction was mon itored by TLC (10:1 toluene/EtOAc) and stirred for an additional 2 days. After this period solution was filtered through a Celite bed. The filtrate was concentrated under reduced pressure. The mixture was purified by flash chromatography (silica, 100:1 toluene/EtOAc). The exo product was isolated in 25% yield (0.4 g). The same reaction was performed using CuOTf/Evans ligand in CH2Cl2, Rh2(OAc)4 in THF and Rh2(OAc)4 in benzene. The exo product was isolated in 23% (0.4 g), 24% (0.4 g) and 30% yield (0.5 g) respectively. 1H NMR (C6D6) ppm 0.95 (t, 3H), 1.04 (s, 3H), 1.11 (s, 3H), 1.66 (m, 2H), 2.07 (t, 1H), 3.48 (dd, 2H), 3.74 (dd, 2H), 3.96 (q, 2H). ESI HRMS Calcd for C11H18O4Na (M + Na)+ : 237.1097, found: 237.1112. 4-tert-Butyl-dimethyl-silanyloxy)-but-2-en-1-ol 10. To a stirred solution of cis diol 5 (4.2 g, 48 mmol) in DMF (25 mL) were added im idazole (0.7 g, 10 mmol) and TBDMS chloride (1.6 g, 10 mmol) at -10 C. Then, the mixture was stirred at room temperature for 1 h and water was added at 0 C. The reaction mixture was extract ed with ether, washed with brine and dried over Na2SO4. The product was purified by flash ch romatography (silica, 8:1 petroleum ether/EtOAc). Compound 10 was obtained as a colorle ss oil in 43% yield (3.9 g). 1H NMR (CDCl3) ppm 0.05 (s, 6H), 0.9 (s, 9H), 2.22 (bs, 1H), 4.21 (dd, 4H), 5.65 (m, 2H). The NMR spectrum was consistent with the literature.101 4(Z)-4-Benzyloxy-but-2-en-1-ol 12. The title compound was synthesized using the literature procedure.101 Cis diol 5 (56.7 mL, 690 mmol) was added to a stirred suspension of

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77 NaH (60% dispersion in mineral oil, 5.50 g, 138 mmol) in THF (460 mL) which was previously cooled to 0C. Stirring was continued at room temperature for 30 minutes. After this, benzyl bromide (14 mL, 118 mmol) was added. The reaction mixture was left overnight at room temperature until benzyl bromide starting materi al was not observed by TLC. Next, the solvent was evaporated and the residue was dissolved in ether (400 mL). The organic layer was washed with water (3 x 150 mL) and dried with MgSO4. Fractional distillation of the crude product at 5 mm Hg (bp 156-159 C) gave the desired mono protec ted diol in 60% yield (73.7 g). 1H NMR (CDCl3) ppm 2.02 (bs, 2H), 4.06 (d, 2H), 4.13 (d, 2H), s, 2H), 5.75 (m, 2H), 7.32 (m, 5H). The NMR spectrum was consistent with the literature. N-Trifluoroacetylglycine 15.105 Glycine (1.0 g, 13 mmol) in a dry round bottom flask was cooled to 0 C. Freshly distilled triflouro acetic anhydride (TFA) (3.0 ml, 21 mmol) was added slowly, and the reaction was allowed to warm to room temperature. When no more glycine starting material was observed by TLC, TFA was evaporated under reduced pressure. The residue was dissolved in 20 mL of boiling benzene and filtered while still hot. The trifluoroglycine crystallized as a white solid in a 70% yield (1.6 g) (m.p. 114-116 C). The NMR spectra and m.p were consis tent with the literature. 9-fluorenylmethyloxycarbonylglyicine 16. This compound was prepared following the literature procedure.104 A solution of glycine (0.3 g, 4.0 mmol) in 10 mL of 10% aqueous Na2CO3 was cooled on an ice bath. Then, a soluti on of FMocCl (1.1 g, 3.9 mmol) in dioxane was added with stirring. The ice bath was removed and the mixture was stirred for 2 h at room temperature. The reaction was que nched with 200 mL of water a nd extracted with ether. The aqueous phase was acidified with concentrated HCl until a precipitate was observed. This solid was extracted with EtOAc. The product FMoc pr otected glycine was obtai ned in 79% yield (0.9

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78 g) as a white solid with a m.p. of 174-175 C. The NMR spectrum and m.p. were consistent with the literature. General procedure for the preparation of (Z)-4-protected but-2-enyl esters 11, 13, 17 and 18. (Z)-4-protected-but-2-en-1-ol 10 or 12 (10 mmol), dicyclohexylcarbodiimide (DCC) (10.5 mmol) and dimethylaminopyridine (DMAP) ( 0.5 mmol) were dissolved in EtOAc (7 mL). The reaction mixture was cooled to 0 C. Afterwards, N-protected glycine (10 mmol) dissolved in EtOAc (7 mL) was added dropwise to the co ld mixture (half way through the addition, the solution became cloudy white). After stirring r eaction for 5 min at 0 C, the ice bath was removed and the mixture was left at room temper ature for 3 h more. EtOAc was added to dilute the reaction mixture which was then filtered to eliminate dicyclohexylurea DCU. The organic filtrate was washed with 1 N HCl (1 x 40 mL), 10% aqueous NaHCO3 (1 x 50 mL), saturated NaCl (1 x 40 mL) and dried over MgSO4. tert-Butoxycarbonylamino-acetic acid 4-(tert-butyl-dimethyl-silanyloxy)-but-2-enyl ester 11. This compound was prepared fr om N-Boc glycine and compound 10. Purification of the product by column chromatography (silica, 50:1 CHCl3/EtOAc) gave 11 in an 80% yield as a colorless oil. 1H NMR (CDCl3) ppm 0.08 (s, 6H), 0.9 (s, 9H), 1.44 (s, 9H), 3.92 (d, 2H), 4.30 (d, 2H), 4.74 (d, 2H), 5.02 (bs, 1H), 5.56 (m, 2H), 5.76 (m, 2H),13C NMR (CDCl3) (ppm) 170.4, 155.9, 134.7, 123.7, 80.2, 61.4, 59.7, 42.6, 28.5, 26.1,18.5, -5.0. ESI HRMS Calcd for C17H33NO5SiNa (M + Na)+ : 382.2020, found: 382.2033. tert-Butoxycarbonylamino-acetic acid 4-benzyloxy-but-2-enyl ester 13. This compound was prepared from N-Boc glycine and alcohol 12 The product was purified by flash column chromatography (silica, 6:1 hexa ne/EtOAc). The desired ester 13 was obtained in a 87% yield as colorless oil. 1H NMR (CDCl3) ppm s, 9H), 3.89 (d, 2H), 4.12 (d, 2H), s, 2H), 4.68

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79 (d, 2H), 5.03 (bs, 1H), m, 1H), 5.81 (m, 1H), 7.32 (m, 5H),13C NMR (CDCl3) (ppm) 170.6, 156.1, 138.3, 131.8, 128.9, 128.3, 126.5, 80.5, 72.9, 66.1, 61.5, 42.8, 28.8. ESI HRMS Calcd for C18H25NO5Na (M + Na)+ : 358.1625, found: 358.1652. (Z)-4-(benzyloxy)but-2-enyl 2-(2,2, 2-trifluoroacetamido)acetate 17. This compound was prepared from N-trifluoroglycine and alcohol 12. The product was purified by flash column chromatography (silica, 6:1 hexane/EtOAc). The product was obtained as a white solid in a 74% yield. 1H NMR (CDCl3) ppm 4.11 (d, 2H), (d, 2H), 4.53 (s, 2H), 4.77 (d, 2H), 5.72 (m, 1H), 5.86 (m, 1H), 7.34 (m, 5H),13C NMR (CDCl3) (ppm) 168.4, 157.3, 138.4, 128.9, 128.2, 125.8, 73.1, 66.1, 62.3, 41.8. ESI HRMS Calcd for C15H16F3NO4Na (M + Na)+ : 354.0924, found: 354.0947. (Z)-4-(benzyloxy)but-2-enyl 2-(((9H-fluoren-9 -yl)methoxy)carbonylamino)acetate 18. This compound was prepared from FMoc-glycine and alcohol 12. The product was purified by flash column chromatography (silica, 6:1 hexa ne/EtOAc). The product was obtained as a white solid in a 38% yield. 1H NMR (CDCl3) ppm 3.96 (d, 2H), (d, 2H), 4.21 (t, 1H), 4.37 (d, 2H), 4.49 (s, 2H), 4.69 (d, 2H), 5.24 (bs, 1H), 5.68 (m, 1H), 5.84 (m, 1H), 7.37 (m, 9H), 7.58 (d, 2H), 7.74 (d, 2H). ESI HRMS Calcd for C28H27NO5Na (M + Na)+ : 480.1781, found: 480.1819. Amino-acetic acid 4-benzyloxy-but-2-enyl ester 14. Compound 13 (2.0 g, 5.9 mmol) was dissolved in CH2Cl2 (47 mL) at 0 C followed by slow addi tion of TFA (7.1 mL). The mixture was stirred at room temperature for 1 h. After this time, the solvent was evaporated under reduced pressure. The residue wa s dissolved in 100 mL of CH2Cl2 and washed with a saturated solution of NaHCO3 (4 x 50 mL) and saturated solution of NaCl (2 x 50 mL). The organic phase was dried with MgSO4 and evaporated under vacuum. The product was obtained as yellowish oil in a 76% yield (1.1 g) and used in th e following reaction without purification. 1H NMR (CDCl3)

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80 ppm bs, 2H), 3.41 (s, 2H), 4.12 (m, 2H), 4.51 s, 2H), 4.69 (d, 2H), m, 1H), 5.81 (m, 1H), 7.33 (m, 5H),13C NMR (CDCl3) (ppm) 174.5, 138.3, 131.6, 128.9, 128.2, 126.7, 73.0, 66.1, 61.1, 44.4. Diazo-acetic acid 4-benzyloxy-but-2-enyl ester 8. Method A. Glycinate 13 (2.2 g, 9.3 mmol), acetic acid (0.2 mL, 0.3 mmol ) and isoamyl nitrite (1.7 mL, 13 mmol) were dissolved in CH2Cl2 (19 mL) and heated under reflux for 3.5 h. Af terwards, the mixture was diluted with CH2Cl2 (100 mL) and washed with 1M HCl (2 x 50 mL ), deionized water (1 x 50 mL), saturated aqueous NaHCO3 ( 2 x 50 mL), and deionized water (1 x 50 mL). The product was purified by flash chromatography (silica, 10:1 hexane/EtOAc) gi ving 0.5 g of an intense yellow oil in 20% yield. 1H NMR (CDCl3) ppm 4.10 (d, 2H), s, 2H), 4.70 (d, 2H), bs, 1H), 5.70 (m, 1H), 5.78 (m, 1H), 7.31 (m, 5H),13C NMR (CDCl3) (ppm) 167.0, 138.4, 131.4, 128.9, 128.3, 127.0, 72.9, 66.1, 61.0, 46.7. IR: 2116s, 1690.s. EI HRMS Calcd for C13H14N2O3Na (M + Na)+: 269.0897, found: 269.0901. Diazo-acetic acid 4-benzyloxy-but-2-enyl ester 8. Method B. The title compound was synthesized using li terature procedure.106 A solution of acetoacetate 19 (1.7 g, 6.6 mmol) and Et3N (1.2 mL, 8.5 mmol) in anhydrous acetonitrile ( 18 mL) were stirred at room temperature. Then, a solution of p-ABSA (2.1 g, 8.5 mmol) in acetonitrile (18 mL) was added dropwise over a 30 minute period. The reaction mixture was let stir for 3 h and an aqueous solution of 3N LiOH was added. The mixture was left for 27 h. After this time, the mixture was extracted with a mixture of ether/EtOAc 2:1 (3 x 50 mL). The orga nic layers were combined and extracted with brine (100 mL), dried over MgSO4 and evaporated under vacuum. The crude product was purified by flash chromatography (silica, 10:1 petroleum Ether/EtOAc ) to give 1.4 g of 8 as a

PAGE 81

81 yellow oil in a 86% yield. Spectroscopy data were consistent with Method A results and literature information. (Z)-4-(benzyloxy)but-2-enyl 3-oxobutanoate 19. The title compound was synthesized using the literature procedure.106 Cis mono protected diol 12 (2.0 g, 11 mmol) was dissolved in 8 mL of anhydrous THF and NaOAc (56 mg, 0.7 mmol) was added with stirring. A solution of diketene (1.0 mL, 13 mmol) in 4 mL of THF was added dropwise over 1 h to the refluxing mixture. Then, the mixture was refluxed for 1 h more until total consumption of the mono protected diol starting material had occured. The reaction mixtur e was dissolved in ether (40 mL) and washed with brine (60 mL). The organic phase was dried over MgSO4 and evaporated under vacuum. The crude oil was purified by fl ash chromatography (silica, 6:1 petroleum ether/EtOAc) to give 19 in a 60% yield (1.7 g). 1H NMR (CDCl3) ppm 2.25 (s, 3H), 3.44 (s, 2H), d, 2H), 4.51 (s, 2H), 4.68 (d, 2H), 5.71 (m, 1H), 5.82 (m, 1H), 7.33 (m, 5H), 13C NMR (CDCl3) (ppm) 200.7, 167.3, 138.4, 131.9, 128.9, 128.2, 126.4, 73.0, 66.1, 61.6, 50.4, 30.6. Spectroscopy data were consistent with literature information. 6-Benzyloxymethyl-3-oxa-bicyclo[3.1.0]hexan-2-one 9. Diazoester 8 (3.0 g, 12 mmol) was dissolved in dry CH2Cl2 (131 mL) to make a 0.09 M solution of the ester starting material. This solution was added from an addition funnel to a refluxing suspension of CuOTf (61 mg, 0.3 mmol) and Evans ligand (80 mg, 0.3 mmol) in 400 mL of CH2Cl2 over a period of 18 h. When the addition was finished, the so lvent was evaporated under vacuum and the residue was purified by flash chromatography (silica, 5:1 hexane/E tOAc). The bicyclic product was obtained as a colorless oil in 60% yield. The sa me reaction was performed using Rh2(OAc)4 in CH2Cl2 and CuSO4 in benzene. The products were isolat ed in 32% and 48% yield respectively. 1H NMR (CDCl3) ppm 1.83 (m, 1H), dd, 1H), 2.39 (q, 1H), dd, 1H), 3.71 (dd, 1H), 4.22 (d,

PAGE 82

82 1H), 4.41 (dd, 1H), 4.54 (q, 2H), 7.35 (m, 5H), 13C NMR (CDCl3) (ppm) 174.7, 138.2, 128.9, 128.3, 73.8, 66.8, 65.1, 22.9, 21.8. EI HRMS Calcd for C13H14O3 (M)+: 218.0943, found: 218.0944

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83 CHAPTER 3 SYNTHESIS OF DIAZABICYCLIC TRANSITION STATE ANALOGS Introduction Af ter the synthesis of the core all cis trisubstituted cyclopropan e ring, the diaza moiety of the TS analogs needed to be constructed. While the three membered ring gives the desired shape and conformation of the proposed inhibitors, the hetero cyclic portion of the compound plays the role of mimicking the anomeric center of the en zymes sugar-based substrates. In the course of the multistep synthesis, some features of thes e molecules design were modified in order to generate other potential inhibito rs. Accordingly, the resulting expanded set of analogs might provide a greater understanding of glycosidase an d glycosyltransferase cat alytic reactions and the requirements for inhibition of the enzymes. One of the variables was the size of the diaza ring. Consequently, synthetic pathways that l ead to seven-, sixand five-membered rings containing the amidine functionality were designed (Figure 3-1). The size of the heterocyclic ring modifies the position of the LG with respect of the trigonal planar center, and varies the flexibility of the ring; factors which might ha ve an effect on enzyme-TS analog interaction. R1 NH N R1 N H N TargetTSanalogs H H OBn O O R2 R2 allcis cyclopropanelactone R1 NH N H R2 Figure 3-1. Proposed TS analogs with different ring sizes In addition, replacing the ring nitrogen atom (aza) by an oxygen (oxa) will alter the electronic delocalization around the anomeric center mimic, which could be useful for the study of charge-charge interactions between the inhibitor and th e catalytic residues. Finally, preliminary results on the synthesis of diazabicyc lic molecules with func tionalized side chains

PAGE 84

84 were also obtained. Different side chains attached to the bicyclic core will help to better simulate the substrates structure and pr ovide additional binding energy. Results and Discussions As already m entioned in the introduction of this chapter, different synthetic routes will be followed for the preparation of the inhibitors. They all starte d with the cyclopropane lactone 9, whose synthesis was described in Chapter 2. Synthesis of Seven-Membered Ring Amidines In this case, the lactone moiety was opened by LiAlH4 reduction to give the diol 20 in 79% yield. This reaction is shown in Figure 3-2. H H BnO NH2 NH2 H H BnO OH OH 1)MsCl/Et3N 2)NaN3/DMF O O H H BnO LiAlH4/THF H H BnO N3 N3 1)PPh3/CH2Cl22)H2O/ 79%70% 92% 92021 22 Figure 3-2. Synthesis of diamine 22 Activation of compound 20s two primary hydroxyl groups w ith methanesulfonyl chloride (MsCl) and subsequent nucle ophilic displacement with NaN3 in DMF, resulted in the diazide 21. The diamine 22 was obtained in 95% yield from the diazide 21 using mild and selective Staudinger reaction conditions.108 The reduction of the azide functionality proceeds via an iminophosphorane intermediate. Subsequent hydrolys is with water generates the diamine which was found to be an extremely polar compound. Purification of 22 was performed by flash chromatography on silica gel using CH2Cl2:MeOH:NH4OH 1:1:0.01 as the eluent solvent system. Following the Hamilton et al. Methodology (described also in Chapter 2),98 the reaction

PAGE 85

85 of ethyl glycoloimidate and diamine 22 in EtOH, allowed the second ring closure to afford the amidine functional group (Figure 3-3). H H BnO NH2 NH2 H H OBn NH N OH EtOH 70% O OH NH2 +Cl22 23 Figure 3-3. Synthesis of hydromethylamidine 23 The hydroxymethyl amidine 23 was obtained as a white solid in 70% yield. This hydroxylmethyl amidine was envisioned as the precursor to the -carboxy amidine via an oxidation reaction, as precedented in Hamiltons work.98 The carboxy amidine would provide functionality analogous to the carboxylate group that would be found in the oxocarbenium ion derived from N-acetylneuraminic acid (sialic acid). Compound 23 was submitted to different oxidative reaction conditions. The first attempt involved KMnO4 as the oxidating agent, which was successfully utilized previous ly to prepare 2-carboxy imidazole 4 (Chapter 2). H H OBn NH N OH H H O NH N OH O KMnO4 MW=274 H H O NH N OH O O MW=288 OH O MW=122 MW=224 23 24 25 26 m/z91 Figure 3-4. Oxidation of hydromethy amidine 23

PAGE 86

86 After purifying the reaction mixt ure by preparative thin laye r chromatography (TLC), the samples were analyzed by 1H NMR and HPLC/MS spectroscopy. Se veral products were detected by these techniques. Although present as a mi nor component, one of these compounds was identified as the desired carboxy amidine which has a MW of 274 (Figure 3-4). The fragmentation pattern of this peak yielded th e m/z 91 ion which corresponds to the benzyl moiety. Unfortunately, the major impurities present in the sample were benzoic acid with a MW of 122, compound 26 and a non UV-absorbing molecule with MW of 224 which could not be identified. The same alcohol oxidation was tr ied but utilizing ruthenium oxide under the Sharpless conditions.109 In this reaction RuCl3 is oxidized in situ by sodium periodate and then coordinated by acetonitrile. This creates a rapi d and mild oxidizing agent. However, when compound 23 was treated with this reagent, the lo ss of the benzyl protecting group was again observed. The reaction mixture displayed a very similar 1H NMR spectrum to that observed for the KMnO4 reaction. Consequently, the oxidation of the hydroxymethyl group to the aldehyde, instead of the carboxylic acid, wa s investigated. Several reagen ts were employed such as pyridinium chlorochromate (PCC) in CH2Cl2, DMSO and oxalyl chloride under Swern oxidation conditions and Dess-Martin periodinate. In mo st cases, degradation of the substrate was observed. It was noted though, that the poor solubility of 23 in organic solvents may have reduced the efficiency of the oxidation reaction. Numerous authors had reported th e conversion of the trichlorom ethyl functionality into the corresponding ester.110-114 This reaction was performed by eith er basic hydrolysis with NaOH or by treatment with AgNO3 in MeOH. Therefore, the trichloromethyl amidine was synthesized. The corresponding imidate was prepared by adding trichloroacetonitrile in a solution containing K2CO3.115 The reaction of trichloroimidate 27 with diamine 22 afforded compound 28 in 40%

PAGE 87

87 yield (Figure 3-5). Any attempts to convert amidine 28 into the carboxyamidine using the two methods previously described resulted in unrea cted starting material. After these unsuccessful efforts to obtain the carb oxyamidine from compound 23, the synthesis of another amidine substituent synthon was explored. H H BnO NH2 NH2 H H OBn NH N 22 28 Cl H H OBn NH N O Cl Cl Cl CN Cl Cl Cl C HN O K2CO3/MeOH 27 38% Cl Cl MeO 27 /AcOH 40% NaOH or AgNO3/MeOH Figure 3-5. Synthesis of trichloroamidine 28 The methyl phosphonate functionality might be a suitable substitution for the carboxyl acid. Based on Schmidts TS analogs studies on sialyltransferases,60,72-74 the carboxylic residue can be replaced by related f unctional groups. The incorporatio n of a second charge through a phosphonate group on Schmidts anlogs exhibited an increased inhi bitor-enzyme binding affinity (see analog 8 and 9 in Figure 1-25). Following these observations, the phosphonate amidine 30 was pursued (Figure 3-6). 60% H H OBn NH N SO2Cl 2329 P(OMe)3 Cl H H OBn NH N P O OMe OMe 30 H H OBn NH N OH minor component Figure 3-6. Synthesis of chloroamidine 29

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88 In order to synthesize 30 chloromethyl amidine 29 was prepared first. This synthesis was carried out by treating the hydr oxymethyl derivative with th ionyl chloride under reflux conditions.116 The -chlorinated amidine 29 was obtained in 60% yield as a yellow solid after flash chromatography puri fication (Figure 3-6). H H BnO NH2 NH2 88% H H OBn NH N O H NH2 +ClEtOH 2231 H H BnO HN HN O H O H 32 CH(OEt) HCl 40% Figure 3-7. Synthesis of amidine 31 and diacylated amine 32 The reaction of an alkyl halide with a phosphi te ester is known as the Michaelis-Arbuzov reaction which proceeds through an unstable tr ialkoxyphosphonium intermediate. This ion has a great reactivity towards nucleophi lic attack which cau ses the rupture of the C-O bond and the formation of the phosphoryl P=O bond. Thus, a mixture of compound 29 and trimethyl phosphite was stirred under reflux until no more chloro am idine starting material was observed by TLC. The reaction was purified by silica gel flash chromatography using CH2Cl2:MeOH 4:1 as the eluent solvent. Analysis of a promising fracti on by MS spectrometry showed the presence of the desired molecule (MW of 352) and several non identified by-products. Attemps to improve the yield of phosphonate derivative by the modi fied Arbuzov reaction, which utilize KI and

PAGE 89

89 trimethyl phosphite in an acetone-acetonitrile solv ent, reproduced, to some extent, the previous results. After the difficulties meet in the synt hesis of the carboxy or phosphonate amidines, the simpler amidine 31 was prepared. This compound also had the features ambitioned on the TS analogs for the carbohydrate pro cessing enzymes (Figure 3-7). The synthesis of amidine 31 was first carried out using ne at trimethyl orthoformate, and HCl as the catalyst (Figure 3-7). This reaction provided the molecule 32 that had a very similar 1H NMR spectrum when compared with that of the expected compound 31. Analysis of the reaction by MS and 500 MHz NMR resulted in identification of 32 as the diacylated compound. OBn NH N Hb Hb Ha Ha 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm) 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 F1 Chemical Shift (pp m ) 3.78, 4.01 Figure 3-8. 2D-NOESY for amidine 31

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90 When p-toluenesulfonyl acid and one equivale nt of trimethoxy orthof ormate were utilized in this reaction, the desired compound 31 was obtained in 46% yield. Based on these results, it appears that the formation of the diacylated amine with (CH3O)3CH/HCl was driven by the large excess of the orthoformate and th e subsequent hydrolysis by the water present in the system. To improve the yield in the amidine reaction, compound 31 was synthesized by using ethyl formoimidate in EtOH. In this case, the amidine derivative was obtained as a white solid in 88% yield (Figure 3-7). This compounds stereochemis try was analyzed by NOESY 2D NMR in order to confirmed the desired all cis configuration. A crosspeak was observed between Ha ( Ha = 3.78 ppm) and Hb ( Hb = 4.01 ppm), indicated their proximity and was only consistent with an all cis cyclopropane geometry (Figure 3-8). Removal of the benzyl prot ected group of compounds 31 and 23 was performed by conventional ammonium formate hydrogenation, gi ving in both cases quantitative conversion (Figure 3-9). NH N H OH H H OBn NH N Ammonium formate Pd/C NH N OH H H OBn NH N OH OH quantitative 31 23 33 34 Figure 3-9. Deprotec tion of compounds 31 and 23 benzyl groups

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91 Synthesis of Five-Membered Ring Amidines Precursor An i mportant step on the synthe tic route of the five-member ring bicyclic TS analogs was to obtain the amine functionality directly attached to the cyclop ropane ring. Starting from the cyclic lactone, this can be achieved by convert ing the ester into an amide and then, obtain the amine group by Hofmann rearrangement conditions. A nother possibility is to perform a Curtius rearrangement on the carboxylic acid after openi ng the lactone in basic media (Figure 3-10). OBn NH2NH2 OBn O O R1R2 O OH R1R2 O NH2 CurtiusRearrangement R1R2 NH2 HofmannRearrangement OR1 N H N Target R2 Figure 3-10. Products of Hofmann and Curtius rearrangements HOOC COOH -Cl+H3N NH3 +Cl1)SOCl 2)NaN3/ 3)HCl 55% Figure 3-11. Cis diamine prepared by Guryn et al.117

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92 Both reactions are suggested to proceed thr ough a concerted mechanism in which there is a migration of a functional group to an electrondeficient nitrogen atom This migrating group retains the starting stereochem ical configuration. The product of these reactions is the corresponding isocyanate which can be hydrolyzed to the desired amine. There are several examples of these reactio ns in the literature.118-125 One of them was the synthesis of ciscyclopropanediamine by Guryns group utilizing cis -cyclopropanedicarbonyl dichloride (Figure 3-11).117 This was an important piece of evidence that the desired cis diamine synthon might be relatively easy to achieve. Treatment of lactone 9 with a concentrated solution of ammonium hydroxide in MeOH afforded the cyclopropanecarboxamide 35 in a 78% yield. Before converting the amide group into the amine, compound 35s primary hydroxyl group was protec ted with TBDMSCl. From the different Hofmann reaction condition s examined, including Pb(OAc)4 118,126 and Br2 in NaOMe123, the successful method utilized N-bromos uccinimide in MeOH in the presence of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (Figure 3-12). HO O H H OBn H2N TBDMSO O H H OBn H2N TBDMSCl TBDMSO H H OBn HN O O NBS/DBU/MeOH O O H H OBn NH3/MeOH CH2Cl278% 75% 76% 93 53 6 37 Figure 3-12. Synthetic route for carbamate 37 The methyl cyclopropylcarbamate 37 was afforded in 76% yield. At this stage, compound 37s primary hydroxyl group needed to be transformed in a way to obtain the second amino

PAGE 93

93 group directly connected to the cyclopropane ring. Thus, compound 37 was deblocked with tetrabutylammonium fluoride and subjected to different oxidative ag ents (Figure 3-13). HO H H OBn HN O O tBuNF/THF O H H OBn HN O O HO H H OBn HN O O oxidation O H 90% TBDMSO H H OBn HN O O 37 38 Figure 3-13. Oxidation of carbamate 38 Attempted oxidations of alcohol 38 by classical Jones, P CC, tetra-n-propylammonium perruthenate (TPAP)/ N-methylmorpholine (NMO ) and Swern reagents or even the mild RuCl3/NaIO4 could not generate the desired aldehyde or carboxylic acid. In all cases, degradation and spontaneous ri ng opening of the material wa s observed. The carbonyl (electrowithdrawing) group on one side of the cyclopropy l ring could act as a si nk for the nitrogen electron pair, promoting the ring opening. As a resu lt, a new synthetic approach was designed to avoid this problem. In the new pathway, the lact one ring was hydrolyzed in alkaline conditions, followed by esterification with diazomethane/Diazald which gave the hydroxyester 40 in a 76% overall yield. Subsequent oxidation of the free hydroxyl group with TPAP/N MO resulted in the unexpected trans -aldehyde 41 (Figure 3-14). O O H H OBn HO O H H OBn HO 1)2.5NNaOH MeOH 2)HCl CH2N2/ether TPAP/NMO 85% HO O H H OBn O O O OBn O H 93 9 89% 52% 40 41 Figure 3-14. Reaction to prepare trans-aldehyde 41

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94 The 2D-NOESY characteriza tion (Figure 3-15) of compound 41 exhibited crosspeaks between Ha-Hb ( Ha = 9.40 and Hb = 2.46) and Ha-Hc ( Hc = 2.22). Then, Hd ( Hd = 2.55) displayed spatial interaction with the two He ( He = 3.80 and 3.65). From these results, it was believed that the aldehyde was undergoing epimeriz ation due to the exposure to perruthenate basic media. Thus, the oxidation was carried out using the hypervalent iodine (V) of the DessMartin reagent instead. In this occasion, only the cis -aldehyde 42 was produced in a good 82% yield. Once again, the structure of this compound was assigned using 2D-NMR. For the aldehyde hydrogen Ha ( Ha = 9.73), a crosspeak with Hb (Hb = 4.09) was observed. Then, the ester methyl group ( CH3 = 3.71) displayed crosspeaks with Ha and the hydrogens on the phenyl ring ( Ph = 7.33) (Figure 3-16). Hb Hd OBn O Ha Hc O O He He 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm) 2 3 4 5 6 7 8 9 F1 Chemical Shift (pp m ) 2.55, 3.65 2.55, 3.8 2.46, 9.4 2.22, 9. 4 Figure 3-15. NOE interaction for trans-aldehyde 41

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95 O Ha O O O CH3 Hb Hb 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm) 2 4 6 8 10 F1 Chemical Shift (pp m ) 4.09, 9.73 3.71, 9.7 3 3.71, 7.33 Figure 3-16. NOE interaction for trans-aldehyde 42 Further oxidation of compound 42 with NaClO2/H2O2 under buffered conditions,127 gave the carboxylic acid 43 in 87% yield (Figure 3-17). HO O H H OBn O O H H OBn O O HO Dess-Martin 82% O OBn O O H NaClO2/H2O287% 404243 Figure 3-17. Synthesis of compound 43 Then, the free carboxylic group of compound 43 was subjected to the Curtius reaction. Two methods were tested in this case. The fi rst one utilized diphenylphosphoryl azide (DPPA) and triethylamine118 and the second one used ethyl chloroformate and NaN3.125 The acyl azide

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96 that is formed as an intermediate is hydrol yzed with t-BuOH. Neither stepwise procedure afforded the carbamate of interest (Figure 3-18). O H H OBn O O HO 43 BocHN O H H OBn O O H H OBn HO O HO 1)DPPA/Et3Nor ethylchloroformate/NaN32)tBuOH 2.5NNaOH MeOH 80% 44-Cl+H3N-Cl+H3N H H OBn 1)SO2Cl 2)NaN33)HCl Figure 3-18. Synthesis of dicarboxylic acide 44 In order to follow Guryns synthetic procedure117 for the cis-cyclopropanediamine, compound 43 methyl ester was hydrolyzed with aqueous 2.5 N NaOH. The diacid 44 was isolated from the organic extract without fu rther purification in an 80% yield. The free cyclopropyl diacid was sequentially treated with thionyl chloride and a solution of NaN3 in acetone/H2O. After extracting the reaction mixture with ether, the crude mixture was dissolved in toluene and added dropwise to a solution of warmed 10% aqueous HCl. Characterization of the double Curtius reaction by 1H NMR did not reveal any evidence for diamine formation (Figure 3-18). At this point, the synthesis of the cis cyclopropane diamine had turned out to be a very challenging task. Nevertheless, a last synthe tic approach that i nvolved a double Hofmann rearrangement of diamide 46 was pursued. In order to execute this synthesis, compound 43 was esterified with diazomethane which generated the diester 45 in an 88% yield. The diamide 46

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97 was successfully prepared by dissolving compound 45 in a ~16 M solution of NH3/MeOH and letting it react in a pressure bottle for 4 days at 50 C (Figure 3-19).128,129 O H H OBn O O O N H3/ M e O H O H H OBn H2N O H2N 33% O H H OBn O O HO CH2N2/CH2Cl288% O H H OBn H2N O H2N AcHN AcHN H H OBn NBS/DBU/MeOH HN OBn O O 43 45 46 48 47 46 Figure 3-19. Synthesis of diamide 46 Attemps to obtain the dicarbamate 47 by the Hofmann reaction with NBS/DBU, afforded a symmetric product which did not displayed the expected proton NMR chemicals shifts. Later on, this compound was identified by C18 HPLC/ESI-MS to be the succinimide 48 (Figure 3-19). Having in mind that the basic Hofmann reacti on conditions might have promoted the amide intramolecular cyclizatio n, the possibility of performing this reaction under acidic condition was investigated. Usually, iodoorganic reagents such us bis(trifluoroace toxy)iodobenzene known as PIFA or iodobenzene diacetate (PIDA)130-132 are utilized for the rea rrangement of amides under mildly acid conditions. Unfortunately, after treatment of the diamide 46 with these iodo reagents, the synthesis of the diamine of interest co uld not be accomplished. In both cases mostly unreacted starting material and, in minor exte nt, decomposition products were observed by TLC and 1H NMR. Based on the Curtius and Hofmann rearra ngement results, it is believed that the

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98 presence of the bulky CH2OBn might have affected, by steric hindrance, the viability of these double rearrangement reactions. Synthesis of the Six-Member Ring Amidine Precursor The synthetic pathway for the diam ine that gives rise to the six-member ring amidine started with the hydroxyamide 35 (Figure 3-20). HO O H H OBn H2N N3 O H H OBn H2N N3 H H OBn H N O O MsCl/NaN3NBS/DBU/MeOH H2N H H OBn H N O O PPh3/H2O/ H H OBn H N HN O KOH MeOH-H2O 48% 63% 83% 77% 23% H H OBn H2N H2N OR1 Target NH N R2 NH2NH2 35 49 50 51 52 53 Figure 3-20. Synthesis of diamine 53 The convertion of the primary alcohol to the azide group (compound 49) was achieved by the two-step procedure involving MsCl and NaN3. The overall yield was 48% after purification by flash chromatography. Transformation of the amide to give carbamate 50 was done using the Hofmann reaction, in a 63% yiel d. A challenge after synthesizi ng this compound resided in the purification. The desired product co-eluted with an impurity that was only detected when the TLC plate was visualized with the oxidant KMnO4. After this discovery, compound 50 was

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99 successfully purified by flash chromatography usi ng petroleum ether/EtOAc (10:1 ratio) as the eluent. To synthesize the aminocyclopropylcarbamate, triphenylphosphine was employed as the reducing agent for the azide 50. Deprotection of the methyl carbamate group of 51 with ethanolic KOH not only afforded the diamine of in terest, but also the cyclization product 52 (Figure 3-20). The bicyclic urea 52 turned out to be very stable. Only starting material was observed by TLC when 52 was treated with hydrazine in attempts to open the ring. At this stage, it was necessary or either search for different solvents that co uld give a more labile carbamate at the Hofmann rearrangement step or deblock the amide by non al kaline conditions. Unfortunately, the efforts to trap the Hofmann isocyanate us ing other solvents such as H2O and tBuOH failed to give the desired product. In addition, decomposition of the starting material 50 was observed when a mixture of HCl/HOAc, 10% KOH/Me OH or iodotrimethylsilane (TMSI) were tested as carbamate deprotecting agents. In conclusion, improvements on the yield for the carbamate 51 deblocking reaction might be attained using other non-conventional reagents. Synthesis of the Six-Member Ring Oxazine Precursor To take advantage of the synthesis that lead to hydroxym ethyl carbamate 38, the preparation of the corre sponding oxazine was cons idered (Figure 3-21). HO H H OBn HN O O 38 HO H H OBn H2N 10%KOH/MeOH 60% H H OBn O NH H2N Cl Cl Cl H H OBn HONH HN Cl Cl Cl or Cl Cl Cl C HN O 27 54 5556 OR1 Target O N R2 Figure 3-21. Synthesis to prepare aminoalcohol 54

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100 Accordingly, cyclopropylaminoalcohol 54 was obtained by alkaline deprotection of carbamate 38, with a 60% yield after chromatogra phic purification us ing EtOAc/MeOH 10:1. Unfortunately, a preliminary trial to promote the oxazine ring closure with trichloroacetoimidate, finished up in compound 55 or 56 according to the m/z ion of 351 observed by ESI-FTICR mass spectrometry. Both compounds have the same MW and differ from the desired oxazine (MW = 334) in 17 mass units (possible NH3). The 1H NMR was similar to the starting material but with two extra broad singlets which integrated for 2H. This NMR analysis might point to the formation of compound 56 instead of 55. Synthesis of Seven-Membered Ring Oxazepine In this case, the cyclopro pyl carboxamide 35 was used as the starting material. After trying different reaction conditions and reagents for th e amide reduction, the most efficient one turned out to be LiAlH4 in THF in a stoichiometric ratio of 22:1 aluminum w ith respect to the amide. Compound 57 was obtained in a 51% yield after quenc hing the reaction with water, filtration through Celite and purification by fl ash chromatography (Figure 3-22). HO O H H OBn H2N LiAlH4THF 51% HO H H OBn H2N H H OBn O N EtOH O H NH2 +Cl30% 355758 Figure 3-22. Synthesis of oxazepine 58 Oxazepine 58 was obtained in a 30% yield using the same procedure as for compound 31. Although MS analysis indicated the presence of contaminants, the major product of the reaction was oxazepine 58. A more careful purification will be needed in future work with this compound.

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101 Synthesis of -Functionaliz ed Diazoacetate Esters One strategy to produce inhibitors with side chains attached to the cyclopropyl ring fusion carbons would be to use alpha-substituted diazo esters in the in itial intermolecular cyclopropanation reaction. Introduction of th e side chain to the acetoacetate ester 19 is outlined below (Figure 3-23). Compound 19 was treated with NaH followed by a solution of either allyl or benzyl bromide in dimethoxyethan e (DME) to give alkylation products 59 and 60 in 73% and 61% yields, respectively (Figure 3-23). Subsequent diazo transfer reaction based on Collados procedure106 produced the corresponding -functionalized diazoesters of interest without any difficulties. These two compounds are ready to be subjected to cyclopropanation reaction conditions with the purpose of generating 63 and 64. These side chains could be further modified to prepare other TS analogs (Figure 3-23). For example, a variety of functionalizations could be performed on the allyl group of 64 including dihydroxylati ons and hydroxylation. CH3CN/Et3N p-ABSA CH3CN/Et3N p-ABSA O OBn O O NaH/vinylbromide THF 73% NaH/benzylbromide THF 61% 60% 43% O O OBn O O OBn 19 59 60 61 62 63 64 O OBn O O O OBn O O O OBn N2 O O OBn N2 O Figure 3-23. Synthesis of f unctionalized diazoacetates

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102 Synthesis of TS Nucleotide Analogs For the sialyltransferase TS analogs, the final step on the synthetic route involved conjugation of the m olecule 33 with cytidine monophosphate (CMP). In order to accomplish this coupling, different methodol ogies can be followed. The synthesis of oligoand poly-nucleotides is one of the most chal lenging areas in the nucleic acids world due to the high number of possible side reactions that their chemistry involves. Among the difficulties that are needed to overcome, a matter of essential importance is the choice of the appropriate protecting groups not only for the nucleosides but also, for the internucleotide linkage. These groups should be able to be removed under conditions that will not affect other groups present in the rest of th e molecule. Several methods have been developed in order to prepare oligonucleotides.133 One of them is the called phosphoramidite approach. This method was introduced by Beaucage and Caru thers, in 1981, with the highly reactive intermediate phosphoramidite (Figure 3-24).134 O PO Base O P NR2 R1O P=protectinggroup R1=Meor-CH2CH2CN R2=MeorMe2CH2 Figure 3-24. Nucleoside phosphoramidates These nucleoside phosphoramidates are successfully utilized in solid phase synthesis of dinucleoside phosphates. The disadvantage of th is approach, when applied in liquid phase synthesis, is the high moisture sensitivity of the reagents, which makes their handling extremely difficult. Another method that is widely used and was utilized in this thesis research is the phosphotriester approach.133,135 A summary of the reagents a nd procedure applied in this synthesis is indicated in Figure 3-25.

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103 O Base O O P O ArO OO Base HO O O Base O O P O ArO O O Base O O Base O O P O -O O O Base O O2S N NO2 H N OH MSNT/pyridine 1)NBO tetramethylguanidine 2)NH3(c) MSNT NBO N N NO2 Figure 3-25. Phosphotriester approach reaction adap ted from the article p ublished by Reese, C. B. and Zhang, P. Z.135 With the idea of applying the phosphotriester ap proach in the synthesis of ST inhibitors, the triacetylated cytidine 68 was prepared as one of the build ing blocks (Figure 3-26). This compound was obtained by two different literature methods. The first one (Method A) involved the preparation of the acet yl dimethoxytrityl cytidine 66.136 This compound was converted to the desired product by a modified protocol in which the nucleoside secondary hydroxyl groups are acetylated and the 5 alcohol is deprotected in a one pot reaction. Compound 68 was obtained in 30% overall yield. Following the two steps Halcomb et al. Procedure,137 the triacetyl cytidine was achieved in a 25% total yield (Method B). In this case, t-but yldiphenylchlorosilane (TBD PSCl) is utilized as the 5 protecting reagent. Although, Method B afford ed a slightly lower yi eld than Method A, the reaction was cleaner, which facilitated purification.

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104 O HO HO OH N N O NH2 1)Ac2O/DMF 2)DMTCl/pyridine 1)TBDPSCl/imidazole 2)Ac2O/pyridine O O O O HO OH N N O NHAc O HO AcO OAc N N O NHAc O Si O AcO OAc N N O NHAc MethodA MethodB nBu4NF AcOH 25%overall 1)Ac2O/pyridine 2)80%AcOH 30%overall 64 66 67 68 Figure 3-26. Synthesis of protected nucleoside 68 O P O O OCl H+N O HO AcO OAc N N O NHAc 68 O AcO OAc N N O NHAc 69 O P O Cl Cl Cl triazole 51% Figure 3-27. Synthesis of 2-chlorophenyl nucleoside 69 Compound 69 was prepared in 51% yield using Reese 2-chlorophenyl dichlorophosphate reagent (Figure 3-27).138,139 This phosphoester was used in the next reaction without further purification. The function of the 2-chorophenyl substituent is to protect the phosphate linkage from any imminent hydrolysis. A model coupling reaction between 69 and deprotected lactone 70 was successfully achieved utiliz ing 1-(mesityl-2-sulfonyl)-3-nitro-1 H -1,2,4-triazole also

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105 known as MSNT (Figure 3-28).138,139 When the same coupling technique was tried on amidine 33, the reaction did not affo rded the desired compound 72. The analysis of the complex crude sample by HPLC/ESI-MS did not even detected 72 as a minor component (Figure 3-29). The last methodology employed to couple the amidine 33 with the cytidine nucleotide was the Khorana and coworkers phosphodiester approach.140,141 In this case, the phosphodiester linkage is left completely unprotected. Either DC C or sulfonyl chlorides can be utilized as the coupling reagent. In order to follow this approach, the phosphorylation of amidine 33s primary hydroxyl group was first attempted. Several phos phorylating reagents were tried, such as tetrachloropyrophosphate (P2O3Cl4), POCl3/PO(OEt)3,142 dibenzylchloro phosphate/pyridine143,144 (prepared from dibenzylphosphite) w ith subsequent benzyl remova l, but the presence of high amounts of inorganic phosphate salts made the detection and the purification of the phosphoramidine impossible. O P O O OCl H+N O AcO OAc N N O NHAc 69 O OBn O O OH O H2/Pd/C CH2Cl2quantitative 970 O OH O 69 O P O O O Cl O AcO OAc N N O NHAc 71 O O MSNT pyridine 32% Figure 3-28. Synthesis of CMP-lactone 71

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106 O P O O OCl H+N O AcO OAc N N O NHAc 69 OH 33 O P O O O Cl O AcO OAc N N O NHAc 72 MSNT pyridine HNN HN N Figure 3-29. Coupling of amidine 33 by phosphotriester approach Finally, the inverse reaction, in which the cy tidine was the phosphate carrier, became the solution to the synthetic challenge. Tr eatment of a suspension of amidine 33 and cytidine-5monophosphate in pyridine with DCC,140 resulted in the desired CMP-amidine 73 (Figure 3-30). Two difficulties found for this reaction were the low yield and tedious purification, which was carried out by C18 HPLC chromatography. After three consequtive HP LC purifications, the CMP-amidine was obtained in a 0.3% yield in 95% purity. O P HO O OH O HO OH N N O NH2 OH 33 DCC pyridine HNN O P O O OH O HO OH N N O NH2 HNN 73 Figure 3-30. Synthesis of CMP-amidine 73 In summary, the synthesis of the sevenmembered ring diazabicyclic TS analogs was fruitfully accomplished in a relatively high yield. Consequently, compounds 23, 29, 31, 33, 34 and 73 will be tested for inhibition activity on di fferent glycosidases and human recombinant (2 6)ST. The set of compounds that will be eval uated as inhibitors is shown in Figure 3-31.

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107 O P O O OH O HO OH N N O NH2 HNN 73 H H OBn NH N 29 Cl 23 H H OBn NH N OH H H OBn NH N 31 H H OH NH N OH H H OH NH N 33 34 Figure 3-31. Diazabicyclic TS analogs Experimental Section General Methods. Solvents and reagents were purchased from Aldrich Chem ical Company and Acros Organics. The organic solvents were dried overnight over CaH2 or 4 molecular sieves and freshly distilled before use. NMR spectra were obtained using VXR 300, Gemini 300 and 500, or Mercury 300 MHz spect rophotometers in appropriate deuterated solvents. Mass spectra were obtained on a Finni ngan MAT 95Q spectrometer operated in FAB, EI, CI or ESI modes. Infrared (IR) spect ra were obtained by deposition of CHCl3 solutions on NaCl plates followed by evaporation of the solvent. The HPLC column utilized was a Phenomenex C18 Synergi 10 Hydro-RP 80 (250 x 15 mm). ((1R,2S,3S)-3-(benzyloxymethyl)cyclop ropane-1,2-diyl)dimethanol 20. Lactone 9 (1.0 g, 4.6 mmol) was added dropwise to a suspension of LiAlH4 (1.6 g, 41 mmol) in dry THF (46 mL) at 0 C. After 3 h, total consumption of lactone starting material was observed by TLC and

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108 the reaction was quenched with a saturated aqueous solution of NH4Cl. The reaction mixture was diluted with more THF and filtered through Celite. The organic layer was dried with MgSO4. After evaporation of the solv ent, the reaction mixture was purified by flash chromatography (silica, 2:1 petroleum ether/EtOAc). Th e yield of the reaction was 74% (0.8 g). 1H NMR (CDCl3) ppm 1.49 (m, 3H), 2.55 (bs, 2H), d, 2H), 3.70 (dd, 2H), 3.78 (dd, 2H), 4.54 (s, 2H), 7.34 (m, 5H), 13C NMR (CDCl3) (ppm) 138.0, 128.9, 128.3, 73.6, 66.6, 59.2, 22.2, 19.5. EI HRMS Calcd for C13H19O3 (M + H)+ : 223.1334, found: 223.1330. ((((1S,2R,3S)-2,3-bis(azidomethyl)cycl opropyl)methoxy)methyl)benzene 21. A mixture of diol 20 (0.4 g, 1.7 mmol) and Et3N (0.6 ml, 5.1 mmol) in CH2Cl2 (57 mL) was cooled to 0 C. Then, freshly distilled methanesulfonyl chloride was added dropwise (0.4 ml, 5.1 mmol) and the resulting mixture was stirred at the same temper ature for 20 min. After that time, all the diol starting material was consumed. The organic phas e was washed with water and brine and dried over MgSO4. The crude dimesylated product was dissolved in DMF (35 mL) and NaN3 was added. The reaction was stirred at 60 C for 4 h and the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc ( 150 mL) and washed with water (3 x 100 mL). The organic layer was dried with MgSO4 and the solvent evaporated under vacuum. The product was purified by flash chromatography (silica, 20:1 petroleum ethe r/EtOAc) giving 0.3 g of 21 as a white solid in a 73% yield. 1H NMR (CDCl3) ppm 1.48 (m, 2H), 1.55 (m, 1H), (dd, 2H), 3.39 (dd, 2H), 3.56 (d, 2H), 4.50 (s, 2H), 7.32 (m, 5H). IR (NaCl) 2096 cm-1. EI HRMS Calcd for C13H17ON6 (M + H)+ : 273.1464, found: 273.1388. ((1R,2S,3S)-3-(benzyloxymethyl)cyclop ropane-1,2-diyl)dimethanamine 22. Triphenylphosphine (0.74 g, 2.84 mmol) was added to a solution of diazide 21 (0.19 g, 0.71 mmol) in dry CH2Cl2 (7.10 mL). The mixture was stirred at room temperature under argon for 24

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109 h. Then, water (0.38 mL) was added and the mixture refluxed for 3 h. After the solvent was evaporated, the mixture was purified by column (silica, CH2Cl2:MeOH 1:1 then CH2Cl2:MeOH:NH4OH 1:1:0.01). The desired cycloproane diamine 22 was obtained as a white solid in 95% yield (0.15 g). 1H NMR (CDCl3) ppm 1.17 (m, 2H), 1.30 (m, 1H), 1.81 (bs, 4H), 2.76 (d, 4H), 3.56 (d, 2H), 4.50 (s, 2H), 7.31 (m, 5H), 13C NMR (CDCl3) (ppm) 138.6, 128.9, 128.2, 128.1, 73.5, 66.8, 37.9, 22.9, 18.5. EI HRMS Calcd for C13H21ON2 (M + H)+ : 221.1654, found: 221.1654. (Z)-(8-(benzyloxymethyl)-3,5-diazabicyclo[5.1.0]oct-4-en-4-yl)methanol 23. Diamine 22 (0.5 g, 2.5 mmol) was dissolved in 13 mL of EtOH. Then, ethyl 2-hydroxyacetimidate hydrochloride 2 (0.3 g, 2.5 mmol) was added and the reac tion mixture was stirred under reflux for 12 h. After the solvent was evaporated und er vacuum, the residue was purified by flash chromatography (silica, CH2Cl2/MeOH 15:1) to give the hydroxymethyl amidine (0.5 g) as a white solid in a 70% yield. 1H NMR (CDCl3) ppm 1.42 (m, 1H), 1.76 (m, 2H), 3.57 (dd, 2H), 3.77 (d, 2H), 3.95 (dd, 2H), 4.19 (s, 2H), 4.53 (s, 2H), 7.33 (m, 5H), 13C NMR (CDCl3) (ppm) 166.9, 138.5, 128.5, 128.0, 127.9, 72.9, 65.4, 59.1, 40.3, 19.4, 17.6. ESI-FTICR (+) Calcd for C15H21O2N2 (M + H)+: 261.1598, found: 261.1575. Methyl 2,2,2-trichloroacetimidate 27. The title compound was synthesized using the literature procedure.115 To a suspension of K2CO3 (0.3 g, 1.8 mmol) in anhydrous methanol (13 mL), trichloroacetonitrile (25 mL 0.3 mol) was added slowly with stirring. Inmediately after the addition, the reaction mixture was distilled and the fraction that boiled around 151 C was collected. The product was obtained as a clear li quid in a 38% yield (20 g). IR (NaCl) 1666 cm-1, 1H NMR (CDCl3) ppm 3.94 (s, 3H), (bs, 1H), 13C NMR (CDCl3) (ppm) 163.9, 91.5, 56.7. ESI-FTICR (+) Calcd for C3H5Cl3NO (M + H)+: 176, found: 176.

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110 (Z)-8-(benzyloxymethyl)-4-(t richloromethyl)-3,5-diazabicyclo[5.1.0]oct-3-ene 28. Trichloroacetoimidate 27 (0.11 g, 0.62 mmol) was added slowly to a suspension of diamine 22 (0.12 g, 0.56 mmol) in 1.86 mL of glacial acetic acid previously cooled to 0 C. The ice bath was taken away and the reaction was stirred at room temperature until no more diamine 22 was observed by TLC. The mixture was diluted with a solution of 5% aqueous Na2CO3 (50 mL) and extracted with CH2Cl2 (3 x 30 mL). The organic layer was dried with MgSO4 and the solvent evaporated under vacuum. The product was pur ified by flash chromatography (silica, CH2Cl2/MeOH 46:1) to give the trichloroamidine (78 mg) as a clear oil in a 40% yield. 1H NMR (CDCl3) ppm 1.18 (m, 1H), 1.63 (m, 2H), 3.62 (m 6H), 4.46 (s, 2H), 7.27 (m, 5H), 13C NMR (CDCl3) (ppm) 152.4, 138.2, 128.7, 128.3, 128.1, 128.0, 96.6, 73.2, 66.1, 42.2, 18.0, 17.9. ESIFTICR (+) Calcd for C15H18ON2Cl3 (M + H)+ : 347.0479, found: 347.0477. (Z)-8-(benzyloxymethyl)-4-(chloromethy l)-3,5-diazabicyclo[5.1.0]oct-3-ene 29. Hydroxymethyl amidine 23 (0.4 g, 1.5 mmol) was added slowly to 8.0 mL of freshly distilled thionyl chloride. After the suspension was heated under reflux for 10 minutes, the amidine started to dissolve; thus the r eaction was left for 2 h more. Th e unreacted thionyl chloride was evaporated under vacuum. The brown residue wa s purified by flash chromatography (silica, CH2Cl2/MeOH 20:1) to give the chloromethyl amidine 29 as a yellow solid in a 60% yield (0.3 g). 1H NMR (CDCl3) ppm 1.25 (m, 1H), 1.41 (m, 1H), 1.78 (m, 1H), 3.56 (dd, 2H), 3.75 (d, 2H), 4.01 (dd, 2H), 4.24 (s, 2H), 4.52 (s, 2H), 7.32 (m, 5H), 13C NMR (CDCl3) (ppm) 162.7, 138.4, 129.1, 128.4, 127.9, 127.8, 72.8, 62.3, 40.8, 40.5, 18.9, 17.1. ESI-FTICR (+) Calcd for C15H20ON2Cl (M + H)+ : 279.1259, found: 279.1256. (Z)-8-(benzyloxymethyl)-3,5-dia zabicyclo[5.1.0]oct-3-ene 31. Diamine 22 (0.5 g, 2.3 mmol) was dissolved in 20 mL of EtOH. Th en, ethyl formimidate hydrochloride (0.3 g, 2.3

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111 mmol) was added and the reaction mixture was st irred under reflux for 10 h. After the solvent was evaporated under vacuum, the residue was purified by flash chromatography (silica, CH2Cl2/MeOH 10:1) to give the amidine 31 as a white solid in a 88% yield (0.5 g). 1H NMR (CD3OD) ppm 1.37 (m, 1H), 1.73 (m, 2H), 3.46 (dd, 2H), 3.70 (d, 2H), 3.93 (dd, 2H), 4.48 (s, 2H), 7.28 (m, 5H), 7.49 (s, 1H), 13C NMR (CD3OD) (ppm) 154.4, 138.5, 128.4, 127.9, 127.8, 72.8, 65.4, 40.6, 19.7, 17.7. ESI-FTICR (+) Calcd for C14H19ON2 (M + H)+ : 231.1489, found: 231.1489. General procedure for the benzyl depr otection of cyclic amidines 33 and 34. The amidine (1 mmol) is dissolved in 5 mL of fres hly distilled MeOH. Amm onium formate (9 mmol) and 0.4 g of 10% Pd/C were added to the solutio n of amidine. The mixture was refluxed until no more amidine was observed. The black suspensi on was filtered through Celite. The celite bed was washed five times with MeOH to maximize product recovery. The solvent was evaporated under vacuum. All the products were used without further purification. (Z)-3,5-diazabicyclo[5.1.0] oct-4-en-8-ylmethanol 33. This compound was obtained as previously described as a yellow solid in a 100% yield. 1H NMR (CD3OD) ppm 1.29 (m, 1H), 1.70 (m, 2H), 3.48 (dd, 2H), 3.72 (d, 2H), 3.95 (dd, 2H), 7.49 (s, 1H), 13C NMR (CD3OD) (ppm) 154.4, 56.9, 40.5, 22.1, 17.6. ESI-FTICR (+) Calcd for C7H13ON2 (M + H)+ : 141.1022, found: 141.1024. (Z)-3,5-diazabicyclo[5.1.0]oct-4 -ene-4,8-diyldimethanol 34. This compound was obtained as previously described as a white solid in a 100% yield. 1H NMR (CD3OD) ppm 1.24 (m, 1H),1.65 (m, 2H), 3.52 (dd, 2H), 3.74 (d, 2H), 3.86 (dd, 2H), 4.14 (s, 2H), 13C NMR (CD3OD) (ppm) 166.7, 59.0, 56.9, 40.1, 21.7, 17.5. ESI-FTICR (+) Calcd for C8H15O2N2 (M + H)+ : 171.1128, found: 171.1138.

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112 (1R,2S,3R)-2-(benzyloxymethyl)-3-(hydr oxymethyl)cyclopropanecarboxamide 35. Lactone 9 (0.5 g, 2.3 mmol) was dissolved in a saturated solution of NH3 gas in MeOH (23 mL). The reaction mixture was stirred for 48 h. The crude amide was purified by flash chromatography (silica, 1:5 petr oleum ether/EtOAc) giving a colo rless oil in 78% yield (0.4 g). 1H NMR (CDCl3) ppm 1.67 (m, 2H), 1.81 (m, 1H), 3.18 (bs, 1H), 3.68 (m, 2H), 3.94 (m, 2H), 4.51 (s, 2H), 5.61 (bs, 1H), 6.19 (bs, 1H), 7.32 (m, 5H),13C NMR (CDCl3) (ppm) 173.2, 138.1, 128.9, 128.3, 73.7, 66.2, 58.7, 24.3, 23.8, 22.2. EI HRMS Calcd for C13H18O3N (M + H)+ : 236.1287, found: 236.1287. (1S,2S,3R)-2-(benzyloxyme thyl)-3-((tert-butyldime thylsilyloxy)methyl) cyclopropanecarboxamide 36. Hydroxymethylamide 35 (0.6 g, 2.5 mmol) and imidazole (0.2 g, 2.8 mmol) were dissolved in dry CH2Cl2 (17 mL). Then, TBDMSCl (0.4 g, 2.8 mmol) in CH2Cl2 (10 mL) was added dropwise to the previous solution. After addition of this reagent, the reaction turned cloudy. The mixture was stirred at room temperature for 12 h. The organic phase was washed with saturated NaHCO3 and brine. The crude pr oduct was purified by flash chromatography (silica, 1:1 petr oleum ether/EtOAc) to give 36 in a 75% yield (0.7 g). 1H NMR (CDCl3) ppm 0.06 (s, 3H), 0.06 (s, 3H), 0.07 (s, 3H), 0.89 (s, 9H), 1.58 (m, 2H), 1.84 (t, 1H), 3.72 (dd, 1H), 3.85 (dd, 2H), 3.98 (dd, 1H), 4.52 (dd, 2H), 5.24 (bs, 1H), 6.68 (bs, 1H), 7.33 (m, 5H),13C NMR (CDCl3) (ppm) 174.3, 138.1, 128.9, 128.3, 73.6, 66.8, 59.8, 26.4, 23.9, 21.3, 18.7. EI HRMS Calcd for C19H32O3NSi (M + H)+ : 350.2151, found: 350.2139. Methyl(1S,2S,3R)-2-(benzyloxymethyl)-3-( (tert-butyldimethyl silyloxy)methyl) cyclopropylcarbamate 37. A solution of amide 36 (0.8 g, 2.2 mmol), DBU (9.9 L, 6.6 mmol) and NBS (0.4 g, 2.2 mmol) in MeOH (22 mL) were heated to reflux for 15 min. Then, another equal amount of NBS (0.4 g, 2.2 mmol) was adde d and the reflux was continued for 10 min

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113 more. The solvent was evaporated under vaccum and the residue dissolved in EtOAc. The solution was washed with 0.01 M HCl and saturated solution of NaHCO3. The organic layer was dried over MgSO4 and evaporated to give a yellow oil. The crude product was purified by flash chromatography (silica, 7:1 petroleu m ether/EtOAc) to give 0.6 g of 37 in a 76% yield. 1H NMR (CDCl3) ppm 0.02 (s, 6H), 0.83 (s, 9H), 1.38 (m, 2H), 2.76 (t, 1H), 3.55 (dd, 1H), 3.62 (s, 3H), 3.70 (dd, 2H), 3.80 (dd, 1H), 4.45 (s, 2H), 5.37 (bs, 1H), 7.28 (m, 5H),13C NMR (CDCl3) (ppm) 158.6, 138.6, 128.8, 128.2, 73.4, 66.1, 59.4, 52.7, 31.1, 26.3, 22.3, 20.6, 18.6. EI HRMS Calcd for C20H33O4NSiNa (M + Na)+ : 402.2071, found: 402.2063. Methyl (1R,2S,3R)-2-(benzyloxymethyl)-3-(h ydroxymethyl)cyclopropylcarbamate 38. A solution of cyclopropylcarbamate 37 (0.7 g, 2.0 mmol) in dry THF (50 mL) was treated with a 1 M solution of tetrabuthylammmonium fluor ide in THF (3.9 mL, 3.9 mmol). The reaction mixture was stirred at room temperature for 2 h until no more carbamate was observed by TLC. After this time, the solvent was evaporated und er vaccum and the residue dissolved in EtOAc. The organic phase was washed with sa turated saturated solution of NaHCO3 and dried over MgSO4. The colorless oil was purified by flash chromatography (silica, 3:2 petroleum ether/EtOAc) to give 38 in a 90% yield (0.5 g). 1H NMR (CDCl3) ppm 1.49 (m, 2H), 2.76 (t, 1H), 3.47 (m, 3H), 3.69 (m and s, 5H), 4.50 (dd, 2H), 5.30 (bs, 1H), 7.34 (m, 5H),13C NMR (CDCl3) (ppm) 159.9, 138.1, 129.0, 128.3, 128.2, 73.5, 65.6, 57.8, 53.0, 30.2, 24.4, 19.4. EI HRMS Calcd for C14H19O4NNa (M + Na)+ : 288.1206, found: 288.1204. 2-(benzyloxymethyl)-3-(hydroxymethy l)cyclopropanecarboxylic acid 39. To a solution of the lactone 9 (0.2 g, 0.9 mmol) in methanol (1.8 mL) was added an aqueous solution of 2.5 N NaOH (1.8 mL). The mixture was stirred at room temperature for 3 h. Then, the solvent was evaporated under vacuum and the solid dissolve d in water (30 mL). The basic aqueous phase

PAGE 114

114 was extracted with ether (3 x 30 mL) and afterwards it was acidifie d with HCl to reach pH of 1. Finally, the acidic aqueous la yer was extracted with CH2Cl2 (4 x 30 mL). The organic phase obtained from extraction of the acidifi ed aqueous phase was dried over MgSO4 and evaporated under vacuum. The product was obtained as a white solid (0.2 g) in a 85% yield. 1H NMR (CDCl3) ppm 1.84 (m, 2H), (m, 1H), 3.92 (m, 4H), 4.51 (s, 2H), 7.34 (m, 5H), 13C NMR (CDCl3) (ppm) 176.8, 138.0, 128.9, 128.3, 73.7, 64.8, 57.5, 26.6, 23.9, 21.4. EI HRMS Calcd for C13H16O4 (M + 2Na)+ : 281.0760, found: 281.0773. Methyl 2-(benzyloxymethyl)-3-(hydr oxymethyl)cyclopropanecarboxylate 40. The cyclopropyl carboxylic acid 39 (0.4 g, 1.9 mmol) was dissolved in an Erlenmeyer flask with a mixture of diethyl ether/CH2Cl2 (15 mL/5 mL) and cooled on an ice bath. Diazomethane was generated from Diazald (p-toluensulphonylmethylni trosamide). It was assumed that 1 g of Diazald generated 3 mmol of diazomethane (CH2N2). In a filter flask that did not have any noticeable scratches on the walls, Diazald (2 g) was dissolved in EtOH (10 mL) and the mixture was stirred under flowing N2 at 0 C. This flask was connected to the reaction mixture Erlenmeyer by latex hoses. Then, a solution of 5 N NaOH was added drop wise from a septum with a plastic syringed until the Diazald was di ssolved. The solution of NaOH was added until yellow color of CH2N2 persisted in the reaction mi xture. After addition of CH2N2, the mixture was stirred for 1 h more and quenched with 3 drops of glacial acetic acid. The solvent was evaporated under vacuum and the residue purifie d by flash chromatography (silica, petroleum ether/EtOAc 3:1). The ester was obtained as a clear oil in a 89% yield (0.4 g). 1H NMR (CDCl3) ppm 1.82 (m, 2H), (m, 1H), 2.49 (bs, 1H), 3.65 (s, 3H), 3.82 (m, 2H), 4.02 (m, 2H), 4.54 (s, 2H), 7.34 (m, 5H), 13C NMR (CDCl3) (ppm) 172.1, 138.0, 128.8, 128.2, 73.6, 64.8, 57.5, 52.1, 25.9, 23.1, 21.4. ESI-MS (+) Calcd for C14H18O4 (M + Na)+ : 273.1, found: 273.1.

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115 (1R,2R,3R)-methyl 2-(benzyloxymethyl) -3-formylcyclopropanecarboxylate 41. Molecular sieves 4 () (93 mg) were added to a solution of ester 40 (50 mg, 0.2 mmol) in dry CH2Cl2 (2.3 mL). This suspension was stirred at room temperature under argon for 5 minutes. Then, tetra-n-propylammonium perruthenate (TPAP) (2.0 mg, 5.1 mol) and Nmethylmorpholine N-oxide (NMO) (37 mg, 0.3 mmol) were added to the reaction mixture which was left stirring for 18 h. After filtering the so lution through Celite, the solvent was evaporated under vacuum. The product was purified by fl ash chromatography (silica, petroleum ether/EtOAc 10:1). The trans cyclopropyl aldehyde 41 (26 mg) was obtained in a 52% yield as a clear oil. 1H NMR (CDCl3) ppm 2.16 (m, 1H), (m, 1H), 2.55 (m, 1H), 3.65 (m, 1H), 3.66 (s, 2H), 3.75 (m, 1H), 4.45 (s, 2H), 7.27 (m, 5H), 9.36 (d, 1H), 13C NMR (CDCl3) (ppm) 197.8, 170.1, 138.2, 128.8, 128.1, 128.0, 73.4, 66.4, 52.6, 34.9, 28.5, 26.4. CI-MS (+) Calcd for C14H17O4 (M + H)+ : 249.1127, found: 249.1120. (1R,2R,3S)-methyl 2-(benzyloxymethyl )-3-formylcyclopropanecarboxylate 42. DessMartin periodinane reagent (0.5 g, 1.2 mm ol) was suspended in 3 mL of dry CH2Cl2. A solution of the ester 40 (0.2 g, 0.6 mmol) in dry CH2Cl2 (3 mL) was added drop wise to this suspension. The mixture was stirred for 45 minutes and quenched with 6 mL of aqueous 5% NaHCO3 containing 98 mg of Na2S2O3. The stirring continued for 10 more minutes and the aqueous mixture was extracted with CH2Cl2 (3 x 30 mL). The organic layer was dried over MgSO4 and evaporated under vacuum. The all cis cyclopropyl aldehyde 42 was purified by flash chromatography (silica, petroleum ether/EtOAc 10:1) and obtained as a clear oil in a 82% yield (0.1 g). 1H NMR (CDCl3) ppm 2.16 (m, 2H), (m, 1H), 3.71 (s, 3H), 4.09 (m, 2H), 4.53 (s, 2H), 7.33 (m, 5H), 9.73 (d, 1H), 13C NMR (CDCl3) (ppm) 198.9, 170.3, 138.1, 128.8, 128.2,

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116 128.1, 73.5, 63.5, 52.7, 32.5, 27.2, 27.0. CI-MS (+) Calcd for C14H17O4 (M + H)+ : 249.1127, found: 249.1125. 2-(benzyloxymethyl)-3-(methoxycarbony l)cyclopropanecarboxylic acid 43. To a solution of the cis aldehyde 42 (0.5 g, 2.0 mmol) in acetonitrile (20 mL) was added 30% H2O2 (0.5 mL, 4.8 mmol) and an aqueous solution of 0.65 M NaH2PO4 (8 mL). After this mixture was cooled to 0 C, a 0.1 M solution of NaClO2 (29 mL, 2.9 mmol) was added dropwise over 1.5 h. The reaction was stirred for 2 h more at 0 C and then Na2SO3 (0.1 g, 0.8 mmol) was added to quench the excess of NaClO2. The mixture was acidified to pH around 2 with 1M HCl. The aqueous phase was extracted with EtOAc (3 x 40 mL). The organic layer was dried with MgSO4 and the solvent evaporated under vacuum. The product was purified by flash chromatography (silica, petroleum ether/EtOAc/HOAc 2:1:0.005). The acid 43 was obtained in a 87% yield (0.5 g) as a clear oil. 1H NMR (CDCl3) ppm 1.95 (m, 1H), (d, 2H), 3.67 (s, 3H), 3.97 (d, 2H), 4.50 (s, 2H), 7.30 (m, 5H), 13C NMR (CDCl3) (ppm) 173.6, 169.9, 138.3, 128.7, 128.1, 128.0, 73.5, 64.8, 52.7, 24.6, 24.4. CI-MS (+) Calcd for C14H17O5 (M + H)+ : 265.1076, found: 265.1057. 3-(benzyloxymethyl)cyclopropan e-1,2-dicarboxylic acid 44. This diacid was obtained in a similar manner as for acid 39. The acid (0.1 g, 0.4 mmol) was di ssolved in MeOH (0.7 mL). A solution of 2.5 N NaOH (1 mL) wa s added to the previous solution and the mixture stirred for 4 h at room temperature. After this time, the solv ent was evaporated and th e residue was dissolved in water (3 mL). This solution was acidified to pH of around 1 and extracted with EtOAc (3 x 30 mL). The organic layer was dried over MgSO4 and the solvent evaporated under vacuum. The product was obtained in an 80% yi eld (80 mg) as a white solid and was considered pure enough to be used in the following step. 1H NMR (CD3OD) ppm 1.77 (m, 1H), (d, 2H), 3.93 (d,

PAGE 117

117 2H), 4.40 (s, 2H), 7.23 (m, 5H), 13C NMR (CD3OD) (ppm) 172.6, 139.7, 129.3, 128.8, 128.7, 74.0, 66.3, 25.0, 24.7. ESI-MS (+) Calcd for C13H15O5 (M + H)+: 251.0919, found: 251.0.952. Dimethyl 3-(benzyloxymethyl)cy clopropane-1,2-dicarboxylate 45. This diester was obtained in a similar manner as for ester 40. Compound 44 (0.3 g, 1.0 mmol) was dissolved in diethyl ether/CH2Cl2 (13 mL/8 mL) and cooled on ice. Diazomethane was freshly generated from Diazald (1.5 g, 4.0 mmol) which was suspended in 8 mL of EtOH. The mixture was stirred on ice for 1 h after yellow color of CH2N2 persisted in the reaction. Gl acial acetic acid was used to quench the reaction. After the solvent was evaporated, the product was purified by flash chromatography (silica, petroleu m ether/EtOAc 9:1). The diester 45 (0.2 g) was obtained in a 88% yield as a clear oil. 1H NMR (CDCl3) ppm 1.88 (m, 1H), (d, 2H), 3.70 (s, 6H), 4.02 (d, 2H), 4.51 (s, 2H), 7.33 (m, 5H), 13C NMR (CDCl3) (ppm) 169.5, 138.7, 128.7, 128.0, 127.9, 73.4, 65.2, 52.4, 24.2, 24.1. CI-MS (+) Calcd for C15H19O5 (M + H)+ : 279.1248, found: 279.1250. 3-(benzyloxymethyl)cyclopr opane-1,2-dicarboxamide 46. The diester cyclopropyl 45 (82 mg, 0.3 mmol) was dissolved in a ~16 M so lution of ammonia in MeOH (4 mL). This solution was made by bubbling NH3 gas into MeOH, previously cooled to -15 C with an ice-salt bath. The reaction was performed in a pressure bottle and stirred for 4 days at 50 C. After this time, the solvent was evaporated and the residue was purified by flash chromatography (silica, EtOAc/MeOH 5:1). The diamide was obtained in a 33% yield (25 mg) as a white solid. 1H NMR (CD3OD) ppm 1.79 (m, 1H), (d, 2H), 3.93 (d, 2H), 4.44 (s, 2H), 7.25 (m, 5H), 13C NMR (CD3OD) (ppm) 172.9, 128.3, 127.9, 127.7, 73.0, 65.3, 25.1, 22.9. CI-MS (+) Calcd for C13H17O3N2Na (M + Na)+ : 271.1053, found: 271.1067.

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118 2-(azidomethyl)-3-(benzyloxymethyl)cyclopropanecarboxamide 49. The hydroxylamide 35 (50 mg, 0.2 mmol) and triethylamine (44 l, 0.3 mmol) were dissolved in dry CH2Cl2 (2.6 mL) and cooled to 0 C. Methanesulfonyl chloride (25 l, 0.3 mmol) was adde d dropwise to this mixture. The reaction was stirred at the sa me temperature for 30 minutes and more CH2Cl2 was added (10 mL). The organic layer was washed with H2O and brine. The solvent was evaporated under vacuum and the crude mixture dissolved in dry DMF (11 mL). Then, sodium azide (27 mg, 0.4 mmol) was added and the reaction stirred for 22 h at r oom temperature. Finally, DMF was evaporated under high vacuum. The residue was dissolved in CH2Cl2 and washed with H2O and brine. The organic layer was dried with MgSO4 and the solvent evap orated under vacuum. The product 49 was purified by flash chromatography (sil ica, petroleum ether/EtOAc 4:1 then 1:1). The amide was obtained in a 48% yield (25 mg) as a white so lid. IR (NaCl) 2102, 1667 cm1, 1H NMR (CDCl3) ppm 1.65 (m, 1H), (m, 2H), 3.63 (dd, 1H), 3.79 (m, 2H), 3.91 (m, 1H), 4.49 (s, 2H), 5.69 (bs, 1H), 6.02 (bs, 1H), 7.31 (m, 5H), 13C NMR (CDCl3) (ppm) 172.4, 138.4, 128.8, 128.2, 128.1, 73.6, 65.0, 46.5, 23.0, 22.3, 22.1. ESI-FTICR (+) Calcd for C13H17O2N4 (M + H)+ : 261.1346, found: 261.1340. Methyl 2-(azidomethyl)-3-(benzyloxymethyl)cyclopropylcarbamate 50. This compound was synthesized by the same procedure as 37. In this case, the amide (0.2 g, 0.8 mmol), NBS (0.1 g, 0.8 mmol) and DBU (0.4 L, 2.3 mmol) were dissolved in 8.3 mL of MeOH. The time and workup of the reaction were followed as described above for compound 37. The product was purified by flash chromatography (silica, pe troleum ether/EtOAc 10:1). The carbamate 50 (0.2 g) was obtained in a 63% yield as colorless oil. IR (NaCl) 2102, 1731 cm-1, 1H NMR (CDCl3) ppm 1.46 (m, 2H), (t, 1H), 3.33 (dd, 1H), 3.52 (t, 2H), 3.67 (s, 3H), 3.74 (dd, 1H), 4.50 (s, 2H), 5.18 (bs, 1H), 7.33 (m, 5H), 13C NMR (CDCl3) (ppm) 158.4, 138.0, 128.9, 128.3, 128.1,

PAGE 119

119 73.5, 65.8, 52.8, 47.1, 30.9, 20.1, 19.4. ESI-FTICR (+) Calcd for C14H18O3N4Na (M + Na)+ : 313.1271, found: 313.1264. Methyl 2-(aminomethyl)-3-(benzyloxymethyl)cyclopropylcarbamate 51. This compound was obtained by the same procedure as 22. In this case, methyl carbamate (0.1 g, 0.4 mmol) was dissolved in 5.5 mL of CH2Cl2 and PPh3 (0.2 g, 0.7 mmol) was added. After stirring the reaction for 22 h, 0.1 mL of H2O were added. The mixture was purified by flash chromatography (silica, CH2Cl2: MeOH: NH4OH 20:1:0.2). The desired amine 51 was obtained as a white solid in 83% yield (88 mg). 1H NMR (CDCl3) ppm 1.23 (m, 1H), 1.43 (m, 1H), 2.55 (bs, 2H), 2.75 (t, 1H), 2.88 (m, 2H), 3.49 (t, 2H), 3.64 (s, 3H), 4.47 (s, 2H), 5.42 (bs, 1H), 7.31 (m, 5H), 13C NMR (CDCl3) (ppm) 159.0, 138.2, 128.9, 128.2, 128.1, 73.4, 65.8, 52.7, 36.8, 30.4, 23.7, 19.0. CI-HRMS (+) Calcd for C14H21O3N2 (M + H)+ : 265.1552, found: 265.1582. 7-(benzyloxymethyl)-2,4-diazabicyclo[4.1.0]heptan-3-one 52. Compound 51 (84 mg, 0.3 mmol) was dissolved in a so lution of 10% KOH in MeOH-H2O (13 mL). The mixture was stirred under reflux for 4 h. The solvent was ev aporated under vacuum and the residue was dissolved in CH2Cl2 and washed with water. The organic layer was dried with MgSO4 and the solvent was evaporated under reduced pressu re. The crude product was purified by flash chromatography (silica, CH2Cl2/MeOH 7:1 then CH2Cl2/MeOH/NH4OH 7:1:0.25) to give 54 mg of the bicyclic product 52 as a white solid in a 77% yield and the diamine 53 in a 23% yield (14 mg) as a clear oil. For diazabicyclo 52 : 1H NMR (CDCl3) ppm 1.23 (m, 1H), 1.49 (m, 1H), 2.93 (t, 1H), 3.36 (d, 1H), 3.57 (m, 3H), 4.49 (dd, 2H), 4.83 (bs, 1H), 5.17 (bs, 1H), 7.32 (m, 5H), 13C NMR (CDCl3) (ppm) 156.9, 138.4, 128.8, 128.2, 128.1, 73.7, 65.0, 37.2, 30.6, 22.1, 9.1. ESI-FTICR (+) Calcd for C13H17O2N2 (M + H)+ : 233.1285, found: 233.1284. For diamine

PAGE 120

120 53: 1H NMR (CDCl3) ppm 1.00 (m, 1H), 1.15 (m, 1H), 1.99 (bs, 4H), 2.60 (t, 1H), 2.92 (d, 2H), 3.70 (d, 2H), 4.51 (s, 2H), 7.32 (m, 5H). ((1R,2R,3S)-2-amino-3-(benzyloxymethyl)cyclopropyl)methanol 54. The cyclopropylcarbamate 38 (0.2 g, 0.8 mmol) was dissolved in a solution of 10% KOH in MeOHH2O (7.5 mL). The mixture was stirred under refl ux for 4 h. The solvent was evaporated under vacuum and the residue was dissolved in CH2Cl2 and washed with water. The organic layer was dried with MgSO4 and the solvent was evaporated unde r reduced pressure. The crude product was purified by flash chromatography (sili ca, 15:1 EtOAc/MeOH) to give 99 mg of 54 in a 60% yield. 1H NMR (CDCl3) ppm 1.26 (m, 2H), 1.78 (bs, 3H), 2.71 (m, 1H), 3.87 (dd, 4H), 4.56 (s, 2H), 7.35 (m, 5H). EI HRMS Calcd for C12H18O2N (M + H)+ : 208.1332, found: 208.1332. ((1S,2R,3R)-2-(aminomethyl)-3-(benzyl oxymethyl)cyclopropyl)methanol 57. A suspension of LiAlH4 (0.2 g, 5.5 mmol) in dry THF (3 mL ) was stirred at 0 C under argon. Then, amide 35 (60 mg, 0.3 mmol) in dry THF (3 mL ) was added dropwise. The reaction mixture was refluxed for 5 h. After that time, th e reaction was diluted with more THF (15 mL) and water was added slowly, with caution (0.3 mL). The mixture was stirred at room temperature for 1 h and filtered through Celite. The organic solvent was evaporated under reduce pressure. The crude product was purified by flash chromatography (silica, 1:1 CH2Cl2:MeOH then 1:1:0.01 CH2Cl2:MeOH:NH4OH) to give 56 in a 51% yield (34 mg). 1H NMR (CDCl3) ppm 1.25 (m, 1H), (m, 2H), 2.53 (t, 1H), 2.86 (bs, 3H ), 3.16 (dd, 1H), 3.56 (m, 3H), 3.78 (dd, 1H), 4.50 (s, 2H), 7.33 (m, 5H), 13C NMR (CDCl3) (ppm) 138.3, 128.8, 128.1, 73.3, 66.6, 58.8, 37.3, 21.7, 19.9. EI HRMS Calcd for C13H20O2N (M + H)+ : 222.1489, found: 222.1482. (Z)-8-(benzyloxymethyl)-3-oxa-5azabicyclo[5.1.0]oct-4-ene 58. The hydroxylamine 57 (50 mg, 0.2 mmol) and ethyl formimidate (24 m g, 0.2 mmol) were dissolved in 2 mL of EtOH

PAGE 121

121 and heated to reflux for 10 h. The material resulting from evaporat ion of the solvent was purified by flash chromatography (silica, CH2Cl2/MeOH 12:1 5:1) to give 14 mg of the oxazepine in a 30% yield. 1H NMR (CD3OD) ppm 1.20 (m, 1H), 1.31 (m, 2H), 3.45 (m, 6H), 4.41 (s, 2H), 7.32 (m, 5H), 7.80 (s, 1H), 13C NMR (CDCl3) (ppm) 154.2, 138.3, 128.4, 128.0, 127.8, 72.9, 65.8, 57.4, 37.8, 21.1, 18.7, 17.2. DIP-CI-MS (+) Calcd for C14H18ON2 (M + H)+ : 232.1338, found: 232.1339. (Z)-4-(benzyloxy)but-2-enyl 2-benzyl-3-oxobutanoate 59. To a suspension of NaH (60% in oil) (16 mg, 0.4 mmol) in dry dimethoxyeth ane (DME) (0.5 mL) wa s added a solution of acetoacetate (0.1 g, 0.4 mmol) in DME. The mixtur e was stirred at room temperature for 30 minutes under argon. After this time, benzyl bromide (91 l, 0.8 mmol) was added and the mixture was left to react for 18 h. The solvent was evaporated under vacuum and the residue was dissolved in diethyl ether (20 mL). The mixtur e was extracted with water (3 x 20 mL). The organic phase was washed with sa turated NaCl and dried over MgSO4 and the ether was removed under reduced pressure. The crude product was purified by flash chromatography (silica, petroleum ether/AcEtO 20:1 to15:1 to 8:1) to give th e functionalized acetoacetate 59 as a clear oil in a 73% yield (0.1 g). 1H NMR (CDCl3) ppm 2.17 (s, 3H), 3.17 (d, 2H), 3.78 (t, 1H), 4.09 (d, 2H), 4.50 (s, 2H), 4.66 (d, 2H), 5.61 (m, 1H), 5.79 (m, 1H), 7.33 (m, 10H). (Z)-4-(benzyloxy)but-2-enyl 2-acetylpent-4-enoate 60. This compound was obtained by the same procedure as 59 In this case, allyl bomide (66 l, 0.8 mmol) was used as alkylating agent. The amounts of the other r eagents were kept the same as 59. The crude product was purified by flash chromatography (silica, petroleum ether/AcEtO 20:1 then 8:1) to give the allyl functionalized acetoacetate 60 as a clear oil in a 61% yield (0.2 g). 1H NMR (CDCl3) ppm 2.22

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122 (s, 3H), 2.59 (t, 2H), 3.53 (t, 1H), 4.13 (d, 2H), 4.51 (s, 2H), 4.71 (d, 2H), 5.06 (m, 2H), 5.71 (m, 3H), 7.34 (m, 5H). (Z)-4-(benzyloxy)but-2-enyl 2-diazo-3-phenylpropanoate 61. The title compound was synthesized using the procedure described for 8. A solution of benzyl acetoacetate (90 mg, 0.3 mmol) and Et3N (47 l, 0.3 mmol) in anhydrous acetonitrile (0.7 mL) were stirred at room temperature. Then a solution of p-ABSA (81 mg, 0.3 mmol) in acetonitrile (0.7 mL) was added dropwise over a 30 minutes period. After 3 h, an aqueous solution of 3 N LiOH (0.3 mL) was added. The work up of the mixture was the same as compound 8. The crude product was purified by flash chromatography (silica, petroleum ether/EtOAc 20:1) to gi ve 53 mg of benzyl azide as a yellow oil in a 53% yield. 1H NMR (CDCl3) ppm 3.62 (s, 2H), d, 2H), 4.51 (s, 2H), d, 2H), 5.72 (m, 1H), 5.80 (m, 1H), 7.33 (m, 10H),13C NMR (CDCl3) (ppm) 167.3, 138.3, 137.4, 131.2, 129.2, 128.8, 128.7, 128.1, 128.1, 127.5, 127.1 72.8, 66.0, 61.0, 29.7. ESIFTICR Calcd for C20H20N2O3Na (M + Na)+ : 359.1366, found: 359.1367. (Z)-4-(benzyloxy)but-2-enyl 2-diazopent-4-enoate 62. This compound was prepared from allyl functionalized acetoa cetate (63 mg, 0.2 mmol) using the same procedure used for synthesis of compounds 8 and 61. The crude product was purified by flash chromatography (silica, petroleum ether/EtOAc 20:1) to give ally l azide as a yellow oil in a 32% yield (18 mg). 1H NMR (CDCl3) ppm 3.04 (d, 2H), d, 2H), 4.51 (s, 2H), d, 2H), 5.12 (m, 2H), 5.81 (m, 3H), 7.33 (m, 5H). ESI-FTICR Calcd for C16H18N2O3Na (M + Na)+ : 309.1210, found: 309.1210. Method A. N4-Acetyl-5-O-(4,4-d imethoxytrityl)cytidine 66. This compound was synthesized by literature procedure.136 Cytidine 65 (5.0 g, 21 mmol) was suspended in dry DMF (42 mL). Freshly fractionally distilled Ac2O (2.1 mL, 23 mmol) was added to this suspension.

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123 After the mixture was stirred at room temperat ure for 14 h, the organic solvent was removed by high vacuum. The residue was dissolved in py ridine (37 mL) and DMTCl (7.7 g, 23 mmol) was added slowly to that solution. The reaction was s tirred at room temperature for 1 h and diluted with CH2Cl2 (150 mL). The organic phase was extracte d with water (3 x 100 mL) and saturated NaHCO3 (3 x 100 mL), dried with MgSO4 and the solvent evaporated under vacuum. The product was purified by recrystallizati on with MeOH. The trityl cytidine 66 was obtained as a white solid in 44% yield (5.4 g) after two recr ystallizations. 1H NMR (CDCl3) ppm 2.23 (s, 3H), 3.48 (dd, 2H), 3.72 (bs, 1H), 3.79 (s, 6H), 4.39 (m, 3H), 5.83 (bs, 1H), 5.89 (d, 2H), 6.84 (m, 4H), 7.26 (m, 11H), 8.25 (d, 1H), 9.16 (bs, 1H). The NMR spectrum was consistent with the literature. 2,3-O,N4-Triacetyl Cytidine 68. Trityl N-acetyl cytidin e (5.3 g, 9.1 mmol) was dissolved in dry pyridine ( 18 mL) and treated with Ac2O (3.0 mL, 32 mmol). After the mixture was stirred for 22 h, the solvent was evapor ated under vacuum. The reaction was quenched slowly with saturated NaHCO3 and, as soon as the evolution of gas ceased, it was extracted with CH2Cl2 (3 x 100 mL). The organic layer was dried with MgSO4 and the solvent evaporated under vacuum. The product was pure enough by TLC (CH2Cl2/MeOH 9:1) to proceed with second step. The fully protected cytidine (6.7 g, 10 mmol) was tr eated with 90% solution of glacial acetic acid in water (55 mL) and let react at room temp erature for 2 h. Then, the reaction was slowly neutralized with 5 N NaOH and extracted with CH2Cl2 (3 x 200 mL). The organic layer was dried with MgSO4 and the solvent evaporated under v acuum. The product was purified by flash chromatography (silica, CH2Cl2/ MeOH 25:1) to give 2.3 g of the triacetyl cytidine 68 as a white solid in 68% yield for two steps. 1H NMR (DMSO) ppm 2.03 (s, 3H), 2.07 (s, 3H), 2.09 (s,

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124 3H), 3.33 (s, 1H), 3.66 (dd, 2H), 4.18 (s, 1H), 5. 37 (m, 2H), 6.01 (d, 1H), 7.22 (d, 1H), 8.31 (d, 1H), 10.95 (s, 1H). The NMR spectrum wa s consistent with the literature. Method B. 2,3-O,N4-Triacetyl-5-tert-Buyldi phenylsilyl Cytidine 67. This compound was synthetized by literature procedure.137 Cytidine 65 (2.0 g, 8.2 mmol) was dissolved in DMF (34 mL) and imidazole (1.2 g, 18 mmol) and te rt-Butyldiphenylchloros ilane (TBDPSCl) (2.6 mL, 9.9 mmol) were added. The reaction was stir red at room temperature for 20 h. Then, the reaction was diluted with pyridine (8 mL) and treated with Ac2O (4.0 mL, 43 mmol). After stirring for 12 h, the mixture was dissolved in EtOAc (100 mL) and washed with water (3 x 100 mL), saturated NaHCO3 (3 x 100 mL) and brine (3 x 100 mL). The organic layer was dried with MgSO4 and the solvent evaporated under v acuum. The product was purified by flash chromatography (silica, EtOAc/petroleum ether 2:1 to 9:1) to give 1.7 g of the protected cytidine 67 as a white solid in a 35% yield. 1H NMR (CDCl3) ppm 1.10 (s, 9H), 2.04 (s, 3H), 2.06 (s, 3H), 2.26 (s, 3H), 3.80 (dd, 1H), 4.08 (m, 1H), 4.20 (dd, 1H), 5.43 (m, 2H), 6.29 (d, 1H), 7.23 (d, 1H), 7.43 (m, 6H), 7.64 (m, 4H), 8.16 (d, 1H), 10.48 (bs, 1H). The NMR spectrum was consistent with the literature. 2,3-O,N4-Triacetyl Cytidine 68. This compound was synthesized by the literature procedure.137 To a solution of fully protected cyti dine (1.8 g, 3.0 mmol) in dry THF (4.6 mL) was added glacial acetic acid (0.3 mL 4.5 mmol) and a 1 M solution of n-Bu4NF in THF (15 mL, 15 mmol). After stirring the mixture 19 h at room temperature, 1 mL of acetic acid was added. The solvent was evaporated under vacuum. The product was purified by flash chromatography (silica, CH2Cl2/ MeOH 25:1) to give 8.0 g of the triacetylated cytidine 68 as a white solid in a 72% yield. The spectral data wa s consistent with literature and previously reported NMR.137

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125 Triethylammonium (5-(4-acetamido2-oxopyrimidin-1(2H )-yl)-3,4-diacetoxy tetrahydrofuran-2-yl)methyl 2-chlorophenyl phosphate 69. To a solution of 2-chlorophenyl dichlorophosphate (220 L, 1.36 mmol) in anhydrous aceto nitrile (2.80 mL) was added Et3N (376 L, 2.71 mmol) and 1,2,4-triazole (0.24 g, 3.51 mmol). A white precipitate formed immediately and the mixture was stirred for 30 mi nutes at room temperature. A solution of triacetyl cytidine 68 (0.20 g, 0.54 mmol) in dry pyridine (2 .70 mL) was added to the previous suspension. After stirring the so lution for another 30 minutes, th e reaction was quenched with a combination of Et3N (0.38 mL), water (0.1 mL) and pyridine (1 mL). This mixture was stirred for 15 minutes, diluted with 50 mL of saturated NaHCO3 and extracted with CHCl3 (8 x 20 mL). The organic layer was washed with more saturated NaHCO3, dried with MgSO4 and the solvent evaporated under vacuum. The residue was dissolved in a minimum amount of CHCl3 and added dropwise to 50 mL of petroleum ether. The product 69 was collected by gravity filtration as a yellow powder in a 51% yield (0.18 g). The chlo rophenyl triacetylated cytidine obtained was sufficiently pure enough to be us ed without further purification. 1H NMR (DMSO) ppm 1.92 (s, 3H), 1.95 (s, 3H), 2.12 (s 3H), 4.23 (m, 3H), 5.32 (m, 2H), 6.16 (d, 1H), 7.23 (m, 5H), 8.23 (d, 1H), 11.42 (s, 1H), 31P NMR (CDCl3) (ppm) -5.46. C18 HPLC/UV ESI-MS (-) Calcd for C21H22O11N3ClP (M H): 558, found: 558. 6-(hydroxymethyl)-3-oxabic yclo[3.1.0]hexan-2-one 70. Lactone 9 (0.2 g, 0.9 mmol) was dissolved in dry CH2Cl2 (35 mL). Then, 30 mg of 10% Pd activated with carbon were added. The reaction was stirred for 5 h at room temperature under H2 (g) atmosphere. Then, the black suspension was filtered through Celite. The ce lite bed was washed five times with CH2Cl2 to maximize recovery of the product. The solvent was evaporated under vacuum. The product was obtained as a clear oil in 100% yield and was used without further purification. 1H NMR (CDCl3)

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126 ppm 1.85 (m, 1H), 1.95 (bs, 1H), 2.37 (dd, 1H), 2.45 (dd, 1H), 3.69 (dd, 1H), 3.84 (dd, 1H), 4.32 (dd, 1H), 4.46 (dd, 1H), 13C NMR (CDCl3) (ppm) 175.1, 66.7, 57.4, 23.8, 22.7. 2-(4-acetamido-2-oxopyrimidin-1(2H )-yl)-5-(((2-chlorophenoxy)((2-oxo-3oxabicyclo[3.1.0]hexan-6-yl)methoxy)phosphoryloxy)methyl)tetrahydrofuran-3,4-diyl diacetate 71. A solution of lactone 70 (21 mg, 0.2 mmol) in dry pyridine (2.6 mL) was treated with 2-chlorophenyl phosphate cytidine (84 mg, 0.1 mmol) and 1-mesitylene-sulphonyl-3-nitro1,2,4-triazole (MSNT) (0.3 g, 0.8 mmol). The reaction was stirred at room temperature until no more lactone starting material was observed by TLC (2 h). The mixture was diluted with a saturated solution of NaHCO3 (20 mL) and extracted into CHCl3 (8 x 20 mL). The organic layer was dried with MgSO4 and the solvent evaporated under vacuum. The product was purified by flash chromatography (silica, CH2Cl2/ MeOH 50:1 then 25:1) to give 43 mg of the product 71 as a clear oil in a 32% yield. 1H NMR (DMSO) ppm 1.97 (s, 1H), 2.07 (s, 6H), 2.23 (s, 3H), 2.44 (m, 2H), 4.39 (m, 7H), 5.41 (dd, 2H), 6.12 (dd, 1H), 7.31 (m, 5H), 7.88 (m, 1H), 9.68 (bs, 1H), 31P NMR (CDCl3) (ppm) -5.22, -5.56, -5.65, -5.97. ESI-TOF-MS (+) Calcd for C27H30O13N3ClP (M + H)+ : 670.1199, found: 670.1177. (Z)-3,5-diazabicyclo[5.1.0]oct-4 -en-8-ylmethyl (5-(4-amino-2-oxopyrimidin-1(2H)-yl)3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen phosphate 73. Amidine 33 (0.1 g, 0.8 mmol) was suspended in a few milliliters of dr y pyridine and evaporated under high vacuum. Then, a solution of cytidine-5-monophosphate ( 0.3 g, 0.9 mmol) in 1 mL of pyridine was mixed with the amidine and again evaporated under va cuum. The white residue was suspended in 6.5 mL of dry pyridine and DCC ( 0.8 g, 3.8 mmol) was added. After th e mixture was stirred for six days, water was added and the mixture was stirred for 1.5 h more and filtered. The solvents were evaporated and the crude r eaction was partitioned in H2O/CHCl3. The organic phase was

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127 discarded. Finally, the aqueous phase was concentr ated and the resulting sample was purified by HPLC. The solvent utilized was a mixture 95% of H2O/acetonitrile with a flow rate of 5.5 mL/min and the detector set it at 270 nm. Unde r this eluent solvent system, the CMP-amidine exhibit a retention time of 8.1 minutes. The desi red amidine was obtained in a 0.3% yield as a white solid with a 95% purity. 1H NMR (D2O) ppm 1.44 (m, 1H), 1.77 (m, 2H), 3.56 (dt, 2H), 4.05 (m, 13H), 5.94 (d, 1H), 6.08 (d, 1H), 7.45 (s, 1H), 7.31 (m, 5H), 7.86 (d, 1H), 31P NMR (D2O) (ppm) 1.06. C18 HPLC/UV ESI-MS (+) Calcd for C16H25O8N5P (M + H)+ : 446.4, found: 446.1.

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128 CHAPTER 4 INHIBITION OF HUMAN SI AL YLTRANFERASE AND GLYCOSIDASES BY DIAZABICYCLIC AMIDINES Introduction The design and synthesis of enzym es specifi c and potent enzyme inhibitors is of considerable interest due to their possible applications in several research areas. For example, mechanism-based analogs are valuable tools in the examination of an enzyme catalyzed mechanism. In addition, they could serve to identi fy which amino acids could be involved in the substrate binding and recognition. Th e only disadvantage of this ki nd of inhibitor resides in the difficulty of preparing the synt hetic products. In general, the ideal compound should display high binding affinity to the active site of the protein. This feature is frequently found in TS analogs with competitive binding. From diverse kinetic experiments, the best TS analog inhibitors interact with the enzymes 107 to 1015 times tighter than the corres ponding substrate does in the ground state.145 Compounds that possess the aza functionality ar e found among the most potent inhibitors for glycosidases and glycosyltransferases (see Chapter 1 for further discussion on these derivatives). One of the reasons is their ability to bind to enzyme active site by charge-charge interaction, as well as, by hydrogen bonding (Figure 4-1). O HO HO OH O OH OO H ANO2 OO N H H N H O H AFigure 4-1. Transition State for gl ucosidase and amidine TS analog

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129 With the intention to explore the general utility of the bicyclic amidines as mimics of oxocarbenium ion-like transition states, a small fa mily of bicyclic amidines which differed in their substitution and mimicry of a leavi ng group (LG) moiety was tested on several glycosidases. In addition, CMP-amidine 73 was tested on human recombinant (2 6)sialyltranferase ( (2 6)-ST). Results and Discussion Glycosidases Kinetic Assay The seven-m embered ring amidines utilized for glycosidases scr eening are shown in Figure 4-2. They are divided in two classes. While the amidines that have a OBn group (with Bn being benzyl) as the LG mimicry were meant to simulate the aglycon portion of the substrate, the ones that have hydroxyl group might mimic the attacking water molecule. OBn N NH H OBn N NH OH OBn N NH Cl OH N NH H OH N NH OH BnAM OHHMAM OHAM BnCMAM BnHMAM Figure 4-2. Library of gl ycosidases TS analogs For the inhibitors kinetic characterizati on, a continuous assay with a chromogenic substrate was utilized. A re presentation of this assa y is shown in Figure 4-3.

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130 Figure 4-3. Kinetic assay for glycosidases The commercially available oor p-nitrophenyl or -glycopyranosides were incubated with the inhibitor at 37 C in a buffer of 50 mM Na-citra te-phosphate. After the mixture equilibrated to temperature, the appropriate amount of enzyme was added. The glycosidase hydrolyses the nitrophenol moiety which develops a yellow color in the solution. The continuous increase in the reaction absorbance is followe d at 400 nm by a UV/VIS spectrophotometer. The velocity of the enzymatic reaction can be calcula ted from the absorbance vs time profile and the extinction coefficient of the nitrophenol, which was determined independantly. The inhibitor screening was performed at pH of 7.5 and 6. Any va riations in the kinetic parameters with pH might provide a preliminary clue into the charge state of the inhibito r or inhibitor/enzyme complex.

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131 TS analogs screening The glycosidases utilized in these experim ents were -galactosidase from green coffee beans ( -gal), -galactosidase from Escherichia coli ( -galEcoli), -galactosidase from Aspergillus orizae ( -galAsp), -glucosidase from Saccharomyces cerevisiae ( -glu) and glucosidase from sweet almonds ( -glu) which are all retaining enzymes. 0 0.01 0.02 0.03 0.04 0.05 012345678910 Time (min) mol product PNPbglu BnAM BnCMAM BnHMAM OHAM OHHMAM Figure 4-4. Kinetic assay for -glu at pH 7.5 with all TS analogs The kinetic screenings were accomplished under in itial velocity conditions. In all cases the concentration of substrate and i nhibitor were set close to the Km of the corresponding glycosidase. The reported Km data in the literature were the following: a) -gal, 0.25 mM (pH 6.5),146 b) -glu 0.3 mM(pH 7.0),146 c) -galAsp, 0.72 mM (pH 4.5),147 d) -galEcoli, 0.65 mM (pH 7.0),146 and e) -glu,1.3 mM(pH 5.0).146 Consequently, the concentration of substrate and inhibitors utilized were 0.25 mM for -gal, 0.3 mM for -glu, 1 mM for -galAsp and galEcoli and 2 mM for -glu. The inhibitor screening with di fferent glycosidases is reported in Table 4-1.

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132 Table 4-1. Inhibition screening da ta (ni, no inhibition at those concentration of substrate and inhibitor) Compound -gal -glu -glaAps -galEcoli -glu pH 7.5 pH 6 pH 7.5 pH 6 pH 7.5 pH 6 pH 7.5 pH 6 pH 7.5 pH 6 BnAM 2% ni ni ni 16% 16% 10% ni 83% 65% OHAM ni 4% 2% ni 11% ni 3% ni 45% 24% BnHMAM 2% ni ni 3% 40% 16% 10% 13% 87% 71% OHHMAM ni 3% ni ni 34% ni 10% 5% 46% 26% BnCMAM ni ni ni ni 13% ni 51% 12% 46% 29% The inhibition is presented as a percentage inhibition when the initial velocity of the reaction without inhibitors is compared with the enzymatic reaction in the presence of inhibitor. An example of the appearance of the velocity profile when inhibitors are tested against -glu is presented in Figure 4-4 (P NPglu is p-nitrophenyl-glucopyranoside). The full set of kinetic inhibition assay plots can be found in Appendix B. Based on th e kinetic screening of the different inhibitors, neither -gal nor -glu showed a significant decr ease on activity at either pH 6 or 7.5. On the other hand, all three -glycosidases tested showed selective responses to these molecules. TS analogs kinetic characterization The kinetic param eters (Km, Vmax and Ki) for the most potent amidine inhibitors were determined by full steady stat e kinetic analysis. While Km and Vmax were obtained by the corresponding Linewearve-Burke plots, the Ki data were estimated by fitting the MichaelisMenten curves with Excel (a complete illustra tion of Michelis-Mente n equations, LineweaverBurke plots and Michaelis-Menten curves are enclosed in Appendix B). The Km and Vmax determined were the following: a) for -galAsp, 1.5 mM and 1.9 molprod/min.mgenz ; b) for galEcoli, 0.08 mM and 446 molprod/min.mgenz; c) for -glu, 3.8 mM and 25 molprod/min.mgenz. A summary of the Ki obtained with the TS analog s is shown in Table 4-2.

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133 Table 4-2. Inhibition constants (Ki are in mM; because BnCMAM displayed a non-competitive mode of action two Ki were determined) Compound -galAsp -galEcoli -glu pH 7.5 pH 7.5 pH 7.5 BnAM --------0.27 OHAM ------------BnHMAM 0.76 ----0.15 OHHMAM 1.55 --------BnCMAM ----0.80 and 0.56 ----For -galAsp only the hydroxylmethyl amidines (BnHMAM and OHHMAM) displayed some inhibitory activity. This result implies that CH2OH functionality, next to the anomeric center mimic, might be forming a hydrogen bond to the active site nucleophilic carboxylate.10-14 In addition, the amidine BnHMAM, which has the benzyl group as the mimic of the departing aglycon, was the most potent inhibitor for this -galactosidase. Consequently, this amidines higher inhibitory potency might be explained by a favorable hydrophobic interaction of its benzyl group with the aglycon binding portion of the enzyme active site. None of the molecules tested on -galAps at pH 6 showed any significant in hibition, which suggest s that there is a catalytic active residue that need s to be deprotonated in order to stabilize the transition state Titration of either BnAM or BnHMAM failed to reveal a pKa up to pH 10, indicating that the pH sensitivity of the inhibition data reflects an ionization on the enzyme. A completely different inhibitory profile was observed with -galEcoli. The only modest inhibitor was molecule BnCMAM that contains a chloromethyl functional group. Particularly, this molecule presented a mixed or non-competiti ve type of profile based on the LineweaverBurke plot (see appendix B). In addition, a slight curvature in the initi al velocity data was observed, suggesting a time dependa nt inactivation mechanisms might be operative (Figure 4-5).

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134 0 1000 2000 3000 4000 5000 0100200300400500600 Time (sec) molprod/mgenz Figure 4-5. Inhibition of -galEcoli by TS analog BnCMAM. Solid line is reaction without inhibitor; dashed line is en zymatic reaction with BnCMAM. Because it was suspected that an attack to the chloromethyl center was occurring, thus, forming a covalent inhibitor-enzyme intermedia te, an inactivation assay was performed on the galEcoli. This kind of nucleophilic attack from the enzyme was previously observed with glycosides that included reactive functional groups like triazenes and fluor ine near the anomeric center.148,149 The methyl triazene compounds acted as affinity labels generating a carbonium ion that irreversible inhibited -galEcoli.148 Alternatively, 2-deoxy-2-fl uoro glycosyl molecules showed a decreased in the deglycosylation rate reaction and subse quent accumulation of glycosyl enzyme intermediate.149 To test these hypotheses, -galEcoli was incubated with the TS analog before adding the substrate. This experiment showed a curvature on the slope vs. time plot which suggested that the molecule not only inhibits the enzyme but al so inactivates it (Figure 4-6).

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135 0 1 2 3 4 5 6 7 8 0510152025 Time (h)v ( molprod/mgenz/h) Figure 4-6. Inactivation of -galEcoli by compound BnCMAM. The -galactosidase was incubated with two chloromet hyl amidine concentrations 0 mM and 0.6 mM, and the reaction started with 0.7 mM of ONPG. Analysis of this data did not show pse udo first order kinetics which implied that a complex mechanism of inactivation is taking place. Specifically, after an initial, rapid inactivation phase, a slower rate of inactivation was observed, s till in progress after 25 hours. This interaction needs to be further examined, and appears consistent with the chloromethyl group functioning as an alkylating agent. Similar to the -galAsp enzyme, -galEcolis best inhibiti on was observed at pH 7.5, which it is expected in an enzyme that has a cat alytic amino acid side chain that needs to be deprotonated before the reaction occurs. In this thesis research, -galactosidases belonging to two different organisms ( A. oryzae and E. coli ) were studied. Surprisingly, they showed a different inhibitory profile when tested with this series of TS analogs. This result mi ght suggest that these enzymes do not present a parallel arrangement of catalytic residues in their active sites.

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136 0 3 6 9 00.511.52 1/[S]( mM-1)1/v (mgenz/min/ molprod) Figure 4-7. Lineweaver-Burke plot for (Z)(8-(benzyloxymethyl)-3,5-di azabicyclo[5.1.0]oct-4en-4-yl)methanol (BnHMAM). The concentrations of TS analogs were 4 mM; 2 mM; 0 mM. Finally, -glu was the most susceptible and the less selective enzyme to this set of TS analogs. All compounds showed a degree of inhi bitory action, with BnAM and BnHMAm being the most potent inhibitors. The Lineweaver-Burke plot for -glu against hydroxymethyl amidine BnHMAM is showed in Figure 4-7. These compounds shared the common feature of a benzyl group which implies that the enzyme active sites aglycon region may also recognize hydrophobic residues. From the comparison of kinetic data for BnAM and BnHM AM, it is established that including a hydroxyl group only improved the potency of the amidine TS analog by a factor of 2. When performing the screening assays on -glu at pH 6, it was observed that the inhibition only decreased 20% relative to the pH 7.5 data. This result was different from wh at was observed with the other enzymes, where the potency of the amidines at pH 7.5 was at least 30% higher than for pH 6. This effect is consistent with a -glu pH-independent inhibition m ode of action against some TS

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137 analogs, also observed by other groups.37,39,49 With respect to the rela tive contributions of a hydrophobic aglycon mimic, or the hydrophilic h ydroxymethyl group to the potency of the inhibitors, it can be concluded that the hydrophobic aglycon mi mic group was more effective than the hydroxymethyl group. (2 6)-Sialyltransferase Inhibition Screen A point assay was utilized to perform the kinetic characterization of compound 73 as a ST inhibitor. The glycosyl donor substrate, cytidine monophosphate-N-acetylneuraminate (CMPNeuAc) was 14C radiolabeled at the N-acetyl group. Upon transfer of the NeuAc group to the acceptor saccharide N-acetyl lactosamine (LacNAc), the resulting 14C-labeled trisaccharide product was quantified with liquid scintillation counting (LSC) by passing time point aliquots through a Dowex-1 (Pi form) column, which retain ed unreacted starting material. The inhibition assay was carried out under initial velocity c onditions utilizing a 200 M concentration of CMPamidine 73, two different concentrations of CMP-NeuAc (43 M and 100 M) and the LacNAc concentration held at 4.6 mM. A control runs were also performed using the same concentrations of CMP-NeuAc and LacNAc but without the inhib itor. All the reaction mi xtures were incubated at 30 C for a short period of time before a dding the enzyme. After in itiating the reaction by addition of enzyme, time point aliquots at 5 and 10 minutes were applied to Dowex 1 (Pi) mini columns. The estimation of the Ki was obtained by plotting the slope of the Lineawear-Burke plots againts the [I] and getting the x-axis intercept of these plot after linear regressions. Assuming competitive inhibition, CMP-amidine 73 exhibited a Ki of ~ 50 M. Clearly the analysis is preliminary with so few da ta collected, but it is clear that compound 73 is indeed a reasonable micromolar inhibitor. The inhibition constant estimated for compound 73 is comparable with the one exhibited by (2 6)-ST from human serum in the presence of free

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138 CMP (Ki of 50 M).71 This result confirms the fact that the cytidine portion of the donor is required for enzyme recognition. On the other han d, the amidine fraction did not seem to have any significant contribution to bi nding. In addition, the pr evious generations of scorpio inhibitors obtained in our lab displayed 10 times lower Ki when compared with amidine 73. Although those molecules were tested on the rat (2 3)and (2 6)-ST, it can be inferred that the inhibitory activity of the TS analog might have been affected by the greater size of the seven-membered ring amidine. These suggestions must be taken w ith caution, given the preliminary nature of the data. A more complete characteriza tion of the inhibition kinetics of 73 and its selectivity profile for other sialyltransferases are required. Experimental Section Solvents an d reagents were purchased from Aldrich Chemical Company and Acros Organics. Glycosidase enzymes and nitrophenyl glycosid e substrates were purchased from Sigma. UV/visible spectra were obtaine d in Beckman DU 640 and Agilent 8453 spectrophotometers. An Isotemp 210 from Fisher Scientific was used to maintain constant temperature. Inhibition Studies on Glycosidases Initial velocities for enzyme catalyzed reactions were dete rmined spectrophotometrically based on the appearance of nitrophenol produced by hydrolysis of the corresponding nitrophenyl hexopyranoside. The kinetic assa ys were performed at 37 C at pH 6.0 and pH 7.5 using 50 mM Na-citrate-phosphate buffers with the exception of the -galactosidase from E. coli, whose buffer contained also 10 mM MgCl2. The glycosidases used were -galactosidase from green coffee beans, -galactosidase from Escherichia coli -galactosidase from Aspergillus orizae glucosidase from Saccharomyces cerevisiae and -glucosidase from sweet almonds. The substrate used for each enzyme were the following: p-nitrophenyl-galactopyranoside PNPgal

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139 ( -galactosidase), p-nitrophenyl-glucopyranoside PNP glu ( -glucosidase), p-nitrophenylglucopyranoside PNP glu ( -glucosidase) and o-nitrophenyl-galactopyranoside ONPG ( galactosidase). The concentrations of substrat e and TS analog used we re the following: 0.25 mM for -galactosidase from green coffee beans, 0.3 mM for -glucosidase from baker yeast, 1 mM for -galactosidase from A. oryzae and -galactosidase from E. coli and 2 mM for -glucosidase from almonds. The enzymatic reaction were init iated by the addition of the following units and volumes of enzymes: 0.2 U of -galactosidase ( Aspergillus orizae) in 5 L for both pHs; 0.2 U of -galactosidase in 10 L at pH 7.5 and 0.02 U in 15 L at pH 6.0; 0.1 U of -glucosidase in 5 uL at pH 7.5 and 0.2 U in 10 uL at pH 6.0; 0.3 U of -galactosidase ( E.coli ) in 5 L at pH 7.5 and 0.5 U in 10 L at pH 6.0; 0.1 U of -glucosidase in 5 L at pH 7.5 and 0.01 U in 5 L at pH 6.0. For the inhibition screening assays, the substrate concentration used was around Km and the amount of enzyme was adjusted to obtain less th an 10% hydrolysis of the substrate over 5 or 10 minutes time courses. In all cases, 1 mL reactio n mixtures were incubated at 37 C for 3 minutes before enzyme addition. The increase of nitr ophenol absorbtion was mon itored at 400 nm after adding the enzyme. The reactions were followed for 5 to 10 minutes to obtain initial velocity data. The kinetic parameters (Km, Vmax, Ki) were determined using 5 to 8 substrate concentrations between 0.05 mM and 30 mM, and 3 different inhibitors concentrations between 0.3 mM and 4 mM. Velocity data were obtained from the slope of the plot mol product vs. time (see Appendix B). The Km and Vmax were calculated by fittin g the corresponding MichaelisMenten curves and double reciprocal plots (Linew eaver-Burke reverse plots) (see Appendix B). The inhibition constants were obtained by fitting the different Michaelis-Menten curves with and without inhibitor with Excel. These constants were compared with the values obtained by

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140 plotting the slope of the Lineawea r-Burke plots againts the [I] and getting the x-axis intercept of these plot after linear regressions (Appendix B). The extinction coefficient for oand p-nitro phenol were calculated by the Beer-Lambert Law in 50 mM Na-citrate-phosphate buffer usi ng known concentrations of the phenol. For pnitrophenol, was 15059 and 2983 cm-1M-1 at pH 7.5 and 6 respectively. For o-nitro phenol, was 2558 and 1159 cm-1M-1 at pH 7.5 and 6 respectively. Inactivation of -galactosidase from E.coli These experiments were carried out by incuba ting 1.5 U of enzyme in the presence of 0.6 mM (Z)-8-(benzyloxymethyl)-4-(chloromethyl)3,5-diazabicyclo[5.1.0]oc t-3-ene (BnCMAM) on an ice bath during a period of 25 h. In parallel to this mixture, another 1.5 U of enzyme were incubated without inhibitor under the same temp erature and time conditions. The buffer used was 50 mM Na-citrate-phosphat e containing 10 mM MgCl2 at pH 7.5. A solution of 0.7 mM ONPG (~ 10 Km) was equilibrated at 37 C for 3 minutes. At appropriate intervals of time, 10 L aliquots (0.05 U of enzyme) from the vial of enzyme with or wit hout inhibitor were added to the solution of the substrate. The final volume fo r each reaction mixture was 1 mL. The hydrolysis of o-nitrophenyl-galactopyranoside was monitored at 400 nm. Inhibition Studies on (2 6)-Sialyltransferase The (2 6)-ST kinetic assays were performed at 30 C using a 50 mM MES (pH = 7.2) buffer containing 0.05% Triton CF-54 and 0.1mg/mL BSA. The enzyme was a recombinant, human (2 6)-ST (Calbiochem), lacking the N-termin al membrane spanning region which was expressed in S. frugiperda insect cells using a baculovirus expression system. The concentrations of substrate and TS analog used were 43 M and 100 M of CMP-NeuAc, 4.6 mM LacNAc and 200 M of CMP-amidine 73. The concentrations of CMP-NeuAc were

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141 obtained by mixing 4 L of a stock 273 M solution of 14C-CMP-NeuAc (specific activity of 2.65x10-8 mol/cpm) and the appropriate amount of a 720 M solution of cold CMP-NeuAc. The enzymatic reactions were initiated by the addition of 1 L of enzyme (1.43 mU). In all cases, the 210 L reaction mixtures were incubated at 30 C for approximately 2 minutes before enzyme addition. Two 100 L aliquots were taken at 5 and 10 minutes and quenched in 500 L of ice-cold 5 mM phosphate buffer (pH of 6.8). Then, 580 L of the quenched mixtures were applied to the Dowex 1 (Pi) columns and eluted with 3.42 mL of 5 mM phosphate buffer. After collecting the columns eluate, the vials were fi lled with 10 mL of Scintisafe30 LSC fluid. The radioactivity of each vial was counted for 3 minutes. The Dowex-1 (Pi) resin was prepared from the Clform by washing it first with 2 L of deionized water and 500 mL of EtOH. Then, the resin was treated with 1 L of 4 M NaOH. The OHresin was washed with deionized water until the pH was approximately 7. The OHresin was converted into the Pi by adding 1 L of a 4 M solution of 85% aqueous H3PO4. Finally, the Pi resin was extensively washed with deionized water until pH reached neutrality again.

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142 CHAPTER 5 SYNTHESIS OF QUINUCLIDINE AND QUINUCLIDINONE DE RIVATIVES AS NICOTINIC ACETYLCHOLINE RECEPTOR AGONISTS Introduction In this chapter a study involvi ng the synthesis of potential agonists for the nicotinic acety lcholine receptor (nAChR) is outlined. The goa l of this work is to use these compounds to help define, and identify what constitutes the st ructural requirements for agonism of the human 7 receptor subtype. The following section prov ides background information to develop the ideas behind the design of the target quinuclidine compounds. Neurons communicate intracellularly with each other through synapse, by passing an electric signal (action potential) or chemical signal. The electric potential across the plasma membrane is regulated prim arily by opening and closing K+, Na+, Cland Ca2+ ion channels. On the other hand, because the gap (s ynaptic cleft) between neurons sometimes is too large for a direct transmission, the communication between th ese cells is mediated by neurotransmitters. These molecules travel across the gap to the corresponding receptor where they bind and trigger an action potential on the other neuron. If the neuron is conn ected with a muscle cell, the signal transmission may induce muscle c ontraction. If the posts ynaptic cell is part of glandular tissue, the action potential may induce a hormone secretion.150 One big family of synaptic receptor is the ionophores. This type of receptor molecule allows the diffusion of ions through the cell membrane. There are two classes of ionophores: carriers, which bind the ion on one side of the cel l membrane and release it on the other side, and ion channels, which have the capability of opening a pore through which ions can go through.8,150 The specificity of carriers is much broader than for the ion channe l. In contrast, the transport of current through an ion channel is fast er than the carriers. Th e ion selective channels open and close for a very short period of time in response to different cellular signals. This gated

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143 mode of action is regulated by the binding of a variety of ligands located inside or outside the neuron or, by changes of the electrical potential across the plasma membrane. Consequently, the channels that are controlled by the interacti on with a chemical transmitter are known as ligandgated channels. On the other hand, ion channels th at respond to an electri cal gradient across the membrane are called voltage-gated channels.8,63,150 One example of a voltage-gated channel is the Na+ ion channel located in the membrane of both neurons and mu scles. When the outside of the cell becomes negative (depolarization), positively charge voltage-sensing helixes of this channel change position, allowing a flux of Na+ through the membrane. The channel closes after a short period of time when the interior of the cell becomes negative again. At this stage, the helixes return to the relaxed position and a channel-inactivating segment blocks the gate on the interior making the channel moment arily inactive. This guarantees that the action potential only moves in one direction.150 A large family of ligand-gated channels re spond to the binding of acetylcholine (Ach). Biosynthesized from acetyl coenzyme A and cho line, ACh (Figure 5-1) is accumulated next to the neuron presynaptic plasma membrane. The releas e of this neurotransm itter to the synaptic cleft is regulated by voltage-gated Ca2+ ion channels. After traveli ng across the synapse, ACh binds to the acetylcholine receptor (AChR) located in the postsynaptic neuron. This interaction triggers a conformational change of the receptor which allows the entrance of alkali ions like Na+ and K+; thus effecting transfer of the el ectrical signal from cell to cell.150 O N+ O Figure 5-1. Acetylcholine, ACh

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144 Acetylcholine Receptor Family The acety lcholine ligand-gated ch annels are called cholinergi c receptors and are divided into two major types. The ones that are sensitiv e to the mushroom alkaloid muscarine are the muscarinic acetylcholine receptors (mAChRs).151,152 Some of these receptors are responsible for the entrance of K+ in heart muscle, activation of phospho lipases and inhibition of adenylyl cyclase. The other subfamily of acetylcholine re ceptors are the nicotinic acetylcholine receptors (nAChRs).151,152 These ligand-gated channels are so name d because they are susceptible to the alkaloid nicotine. Among the functions of this receptor, it was found that nAChRs are found at brain synapses and neuromuscular junctions. Be ing one of the first ion channels to be characterized and purified, they ha ve served as a starting point fo r the study of the structure and mechanism of other receptors. Their importance also arises due to the fact that these ligand-gated channels are involved in severa l human neuronal diseases like Alzheimers, schizophrenia and Tourettes syndrome (characteri zed by the development of a vari ety of vocal and motor tics).151 The nAChR belong to the super family of i ontropic receptors that includes glycine, serotonin, and GABA receptors,152 and they have been extensively studied due to their ready isolation from the electric organ of the Torpedo California (electric ray).150,153,154 Moreover, the nAChR are strongly inhibited by tw o snake neurotoxins such as -bungarotoxin and cobratoxin which facilitated their tagging and purification.150 The muscle-type nAChR is composed of five different subunits, or and 2 being the domains the binding sites for the acetylcholine neurotransmitter. Neuronal nA ChRs are formed by an arrangement of and subunits ( 2 through 6, 2 through 4) if they are heteromeric, or only subunits ( 7 through 10) if they are homomeric.154,155

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145 nAChR Structure The nAChR is a pentam eric, integral membrane protein with a subunit molecular weight of approximately 250-270 kDa.156 The amino acid sequence among subunits is considerably conserved, and they share a similar transmembr ane topology: 1) a glycosylated N-terminal extracellular domain involved in agonist bindi ng; 2) three short transmembrane fragments (M1, M2 and M3) connected to a fourth piece by an in tracellular loop; and 3) a short extracellular Cteminal domain. It is believed that each M2 fragment folds into an -helix followed by a loop. The five M2s (one from each subunit) are arranged to form the actual pore of the channel.156 By electron microscopy it was possible to measure an outer pore diameter of approximately 25 ( -helix) that becomes ~ 7 in the interior of the channel (lower loop, Figure 5-2).156 This narrow part of the ion channel provides selectivity towards th e charge and size of the ions. Figure 5-2. nAChR structure adapted from Lodish, H. F. and Darnell, J. E., Molecular cell biology ; 3rd ed.150 60 20 25 7 Neurotransmitter binding site Gate M2 helix

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146 Site directed mutagenesis and affinity labe ling experiments support that the ion pore is organized by rings of amino acids which contribute to ion conductance.154,157 First, the channel screens for cations with a ring of negatively charge d residues like glutamate and aspartate, also found at the end of the channel. By studies wi th chloripromazin, it was found that the channel has two rings of serines and th reonines in the middle region.154 Finally, there are three more rings of leucines and valines that impart hydrophobic ity to the pore. These residues may undergo an allosteric conformational change after acetylcholine binding, which make them twist and move away from the channel, allowing the passage of the cations.158 Moreover, it was found that nAChRs permeability to positively charged ions in creases when Leu251 is mutated to serine or threonine in nAChRs expressed in Xenopus oocytes.157,159,160 Acetylcholine Binding Site Acetylcho line binds at the interface of the subunit and an adjacent subunit. Based on the stoichiometry of the channel, two molecules of neurotransmitter bind per oligomer (except for homopentamers like 7 that carry five acetylcholine-binding sites).156 These two ACh binding sites do not neighbor each ot her and, even though both -subunits are usually encoded by a single gene, they do not show exactly the same binding affinity for snake toxins.154 The binding process is considered cooperative because the ch annel interaction with the first ACh increases the binding of the seco nd. Acetylcholine-binding pockets ar e localized on the large N-teminus domain of the channel (Figure 5-2). It wa s observed that the bi nding site region is asymmetrically distributed, with a principal section on the subunit and a complementary section on the neighbor subunits.156 Studies on the electric orga n and muscle nAChR revealed that the principal section of the binding site is constituted by a disulfide bridge of Cys192 and Cys193 and aromatic amino acids like Tyr93, Trp149 and Tyr190.156,161 The complementary

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147 section is constituted by residues including tryptophan, tyrosine and arginine.156,161 From these experiments, it is observed that the nAChR cont ains a congregation of electron-rich aromatic residues on the ACh-binding site which are believed to stabilize the AC h quaternary ammonium positive charge. Specifically, an X-ray study of acetylcholinesterase from Torpedo californica suggested that the most potent cationinteraction is given through the van der Waals contact between ACh quaternary ammonium and Trp84.156 Figure 5-3. A model for acetylcholine induced gating of the nAChR receptor.The binding induces a twist on the subunit that is transmitted to the M2 fragment. The M2 helices assume a new conformation allowi ng the passage of ions. This figure was adapted from the article published by Miyazawa, A., Fujiyoshi, Y. and Unwin, N.158 More information about the acetylcholine binding site was obtained by electron microscopy of the electric organ membranes of the Torpedo electric ray and the snail acetylcholine binding protein (AChBP).161 AChBP is a homopentameric protein, isolated from freshwater snail, and has a 26% homology with the N-terminus of the neuronal 7 nAChR. When the microscopy electron images of the nA ChR from the electric ray were fitted by the AChBP -sheet portion, it was observed that ACh binding induces a rotation of the subunits S S S S M2 fragments

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148 barrels.158 When this movement is transmitted to the M2 transmembrane fragment, the hydrophobic interaction among helices is disrupted and the channel becomes permeable to ions (Figure 5-3).158 Despite the structural information brie fly reviewed here, to our knowledge there does not yet exist even a single high resolution cr ystal structure of a pentameric nAChR suitable for the design of new agonists. Thus, as will be developed later in this chapter, structurally defined changes in known agonists, obtained through chemical synthesis, will be used to further probe the basis for agonist binding and the selectivity of agonis ts for one type of nAChR over another. Possible Roles of nAChRs in Human Diseases It is known that nAChR are involved in num erous inherited and ac quired hum an diseases. Among the most common illnesses, myasthenia gravis (MG) is an autoantibody-mediated disorder in which the nicotinic receptor is the target of the antibodies.151 The clinical manifestation is severe muscle weakness whic h is a consequence of reduced neuromuscular transmission. The diminution of postsynaptic currents is a result of the decrease in the number of nAChR arising from the autoimmune body response.151 One of the treatments for MG includes immunosupresive drugs like oral antigens, but due to their lack of sel ectivity, they displayed generalized immunosuppression. Anot her choice for patients who have MG is plasma exchange, but there is a high risk of contracting transmitted diseases.151 A decrease in the number of nAChR is also ob served in neurodegenerative diseases such as Alzheimers (AD) and Parkinsons.151,162 In the case of Alzheimers, the pathology is showed by the lost of memory. For Parkinsons disease, the patient usually presents motor dysfunction and cognitive disorders. A major dimunition of nAChRs was observed in th e cerebral cortex and hippocampus.162 Different techniques, such as western-blot, immunohistochemical analysis or/and radiolabeled ligand binding, have helped to identify the primary nAChR subtypes

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149 compromised in these diseases. Based on these anal yses, the most affected nicotinic proteins are 7 and 4 2 subtypes.162 Unfortunately, the exact mechanism that accounts for this loss still remains unclear. Until today, the main treatmen t utilized for AD is the administration of acetylcholinesterase inhibitors. These agents prev ent the hydrolysis of ACh, resulting in a higher level of the neurotransmitter at the postsynaptic neuron terminal, which c ould compensate for the loss of brain cells. But their action was shown to be only temporary, losing their therapeutic effect at later stages on the disease. The discovery of agonists selective for 7 and 4 2 receptors has become one of th e current challenges for the scientific community. Nowadays, GTS-21 (Figure 5-8) is under cl inical trial for AD disease.162,163 Another typical neurological disease is schizophrenia.151 Once more, the pathology affects primarily the 7-nAChR and in lesser extent the 4 2 subtypes. Some typical manifestations of this disease include auditory gating insuffi ciency and unpredictable eyes movements. Schizophrenics are not able to filter repeated auditory stimu li in the brain which could be reflected in their lack of concentration and hypervigilance. Nicotine helps to normalize the auditory response by stimulating the hippocampus cholinergic system. As a result, schizophrenic patients have a higher tendency to smoke that a nor mal person because they utilize the cigarettes nicotine as a self-medication. nAChR Ligands Several potent agonists and antagonists for the nA ChR have been found so far, among them, -bungarotoxin ( -Btx) from krait Bungarus multicinctus is an antagonist that binds tightly to muscle nAChR and 7-nAChRs.151 This 76 amino acid peptide has been used for labeling experiments and purif ication of the receptor.

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150 N N N H N O Nicotine Cytisine H N N Cl Epibatidine N O O N Strychnine NN S O Trimethaphan Agonists Antagonists N+ O H3CO OH NH+ OH OCH3 Tubocuranine Figure 5-4. nAChR ligands151 Other examples are the curare alkaloids such as, tubocurarine and toxi ferine (Figure 5-4). These compounds are nAChR antagonists and are known to be used as muscle relaxants.151 Moreover, epibatidine an agonist alkaloid extracted from the Ecuadorian poison frog Epipedobates tricolor has served to induce analgesia a nd has a high affinity for different neuronal nAChRs (Figure 5-4).151,164 Another class of al kaloids that have high affinity for the 4 2-nAChR but low affinity for the 7 is nicotine and cytisine (Figure 5-4). With the help of radioligand binding and the numer ous nAChR agonists and antagoni sts discovered, it is possible to define a pharmacophore for these ion ch annels. A pharmacophore is the minimal 3D arrangement of essential functional groups necessary to be recognized by the receptor.151,165 These functional groups could be a particularly defined point (an at om) or a point of interaction

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151 on the molecule, for example, a hydrogen-bond d onor or acceptor. A number of groups have proposed different pharmacophore models, one of the first ones was presented by Barlow and colleagues where they considered that an onium feature is re quired and a flat hydrophobic area, at 4.5-6.5 from the onium point is necessary for activity.166 After studying several nicotine analogs, it became evident that the position of the pyridine nitrogen atom was contributing in some way to receptor binding.167 However, there are examples in the literature of compounds that still retain receptor a ffinity after the pyridine nitrogen is substituted by a carbon.167 The Beers-Reich/Sheridan models suggested that the nAChR pharmacophore is defined by 3 points within the molecule.168 By using different nicotinic a gonist and antagonists like cytisine, strychnine, nicotine and trimetha phan (Figure 5-4), the first point A, is given by the coulombic interaction between the quaternary (charged) nitrogen of the molecules and the receptor (Figure 5-5). The second point, B, will be the -bonded electronegative atom that provides a hydrogen bond interaction. The distance between point A a nd B is at approximately 5.9 base on the Beer-Reich model or 4.8 based on Sheridan mode l. Finally, point C is identified as the other end of the local dipole moment set up by point B (for example C will be the carbon of a carbonyl group, Figure 5-5).151,165,167,168 Figure 5-5. Pharmacophore represen tation adapted from the article published by Barrantes, F. J.151 B C A 4.8-5.9 4.0 1.2 N+

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152 However, it was observed by comparison of se veral nAChR agonists and antagonist that there is not a unique way of bindi ng. Consequently, it became apparent that systematic structural changes on the molecules tested did not always re sult in the expected pa rallel affinity response on the receptor.167 Moreover, it was found that cholin e and tetramethylammonium are also efficient agonists for 7 receptors which reduces the minimum pharmacophore structure to a quaternary nitrogen (Figure 5-6).169 Based on these observations, it can be concluded that the point of hydrogen bond interaction is not a requisite for nAChR activ ation but it may be seen as a center that could provide binding affinity.167 HO N+ N+ Choline Tetramethylammonium Figure 5-6. Other nAChR agonists169 Anabaseine and Derivatives Anabaseine, a paralyzing toxin found in certain m arine worms and ants, is a heterocyclic molecule related to nicotine (Figure 5-7).170 Nicotine N NH+ N N H+ Anabaseine Figure 5-7. Nicotine and Anabaseine structures Even though this compound displayed activity on several nAChR, it has a potent efficacy and affinity as agonist upon 7 nAChRs (Ki of 58 nM and EC50 6.7 M for rat 7) compared with nicotine (Ki of 400 nM and EC50 47 M for rat 7).171 Although, nicotine and anabaseine share the common feature of havi ng the positively charged ring n itrogen, these molecules differ

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153 in the relative orientation of the pyridyl ring wi th respect to the ring bearing the charge. While the two rings of anabaseine are coplanar, because of the conjugation of the imine functionality with the pyridyl ring, the saturated ring of nicotine is twiste d approximately 90 with respect to the aromatic ring (Figure 5-7). This structural di fference may be reflected in their different mode of action against the nAChR subtypes.171 After discovering that neuronal 7-nAChR may be involved in neurodegenerative diseases like Alzheimers,162 the study of the possible therapeutic app lications of anabas eine attracted the interest of numerous research groups. Several anabaseine deriva tives have been synthesized and tested in order to identify nAChR subtype sel ectivity and reduce the t oxicity of the parent compound.172-175 One of these derivatives was 3-(2,4dimethoxy)benzylidene-anabaseine (GTS21) (Figure 5-8). N N Anabaseine N N OCH3 OCH3 N N OH OCH3 N N OCH3 G T S-21 4-OH-GS T -21 DM A C Figure 5-8. Anabaseine derivatives

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154 Although this compound showed part ial agonist selectivity for 7-nAChR, improving memory related behavior in primates and rode nt species, it was much less efficient on human 7 receptor when it was compared with the parent agonist anabaseine.163 The receptors dissimilar response to these two compounds ma y be a result of a specific in teraction with the benzylidene ring.175 Moreover, GTS-21 showed a 4 2 antagonist behavior when tested on rat receptors,172 which could be a demonstration of the grea ter tolerance of agonist structure of the 7 towards activation when compared with 4 2. A different explanation to this antagonist action towards the receptor could be th rough channel blockade. When molecules of GTS-21 open the channel, additional binding of this compound to th e receptor could cause the obstruction.172 Another benzylidene anabaseine (BA), 4-OH-GST-21, displa yed around 10 fold greater efficacy against both rat and human 7 receptor than for the nAChR constituted with subunits (Figure 58).163,176 Interestingly, 4OH-GST-21 is one of the main metabolites of GST-21 and is the first 7-nAChR agonist to exhibit cytoprotective activity on human cells.163 Another anabaseine derivative, 3-(4-dimethylamino) cinnamylidine (DMAC) (Figure 5-8), that has been studied by De Fiebre et al. showed very potent selectiv e agonist activit y of the rat brain 7-nAChR.172 This compound bound to the receptor with a Ki of 33 nM exceeding the potency of GTS-21 on this subtype of receptor.172 Further, DMAC displayed an extremely poor agonist action not only on 4 2 but also on 4 4, 2 2, and 3 2.172 BA derivatives are considered to have two elements for receptor activation, the anabaseine center, which gives the nAChR recognition site to the molecules, and the benzylidene or cinnamylidene center, which might provide the selectivity for 7 subtype.

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155 largehydrophobic pocket smallhydrophobicpocket NH+ N N+ BA N,N-dimethyl piperidine Figure 5-9. Proposed 7-binding pockets that confer selectivity Nevertheless, in addition to this hydrophobic st ructural motif that imparts selectivity for the 7-nAChR, it has been found that the simple N, N-dimethyl piperidine is also selective towards this neuronal receptor.177 Based on this experimental resu lt, it was believed that another distinct hydrophobic pocket may be present nearby the quaternary nitrogen (Figure 5-9). In the past few years, new kinds of nACh R agonists have been synthesized where a quinuclidine core structure is used for receptor activation. Am ong these compounds, the spirooxazolidinone (AR-R17779) (Figur e 5-10) turned out to be the first full and most selective agonist reported for rat 7-nAChR.178 This agonist displayed a great selectivity for rat 7nAChR (Ki of 92 nM) expressed on Xenopus oocytes but low when compared with 4 2 nicotinic receptor (Ki of 16000 nM).178 It has been noted though, that small structural changes on AR-R17779 provoked a drastic decrease of the 7 receptor affinity.178 N NH O O A R R 17779 N HN O Cl PNU-282987 N N TC-1698 Figure 5-10. Compounds with quinuclidine core that are 7 selective agonists

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156 Other two members of the quinuclidine nAChR-agonists family are TC-1698179 and the 4chlorobenzamidine PNU-282987180 (Figure 5-10). From the PNU-282987 studies, it could be concluded that para-substituted benzamidines were the most potent, and the ortho substitution the worst.180 It was also noticed that only the analogues that containe d small substituents (chloro, fluoro, methoxy) on the para positio n were among the most potent.180 A similar response for the 7-nAChR was found when TC-1698 was tested by Bencherif et al. .179 This molecule displayed very low or no agonist effect when it was applied on the -subunit-containing nAChR 4 2, 3 2, and 1 1 nicotinicsubtypes.179 In order to find a better 7-nAChR agonist candidate, the quinuclidine and quinuclidinone core structures were utilized as a starting material for a new class of 7 analogs. With these kind of compounds it is possible not only to search fo r a novel therapeutic agent, but also to study the hypothesis of the dual hy drophobic motif present on 7 subtype of nicotinic receptors (Figure 511). This will be performed by incorporating aryl groups that will look like the BA 7-nAChR series or alkyl functionality that will resemble dialkyl piperidines. Something important to notice is that these compounds will not have the pyr idyl H-bond acceptor present in anabaseine derivatives, a feature already noted as lacking in some 7 selective agonists. largehydrophobic pocket smallhydrophobicpocket Ar NH+ N+ Figure 5-11. Proposed 3-arylidin e and N-alkyl quinuclidines

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157 Results and Discussions Synthesis of Quinuclidine and Quinuclidinone Derivatives Com pounds 74 and 75 were synthesized and tested to st udy their selectivity as agonists of neuronal 7 and 4 2 nAChRs (Figure 5-12). To the best of our knowledge, surprisingly little has been reported regarding one step 3-benzylidene quinuc lidine syntheses,181 though olefination via organometallic additions to 3-quinuclidinone and dehydra tive eliminations are known.182 We envisioned a straightforward synthesis of the 3-benzylidine analogs of quinuclidine via Wittigtype olefinations and report the results of these studies. N H+ N+ R R 75 aR=H b R =OMe 74 aR=Me b R =Et Figure 5-12. Target quinuclidines 7 agonists Alkylation of quinuclidine hydrochloride with methyl or ethyl iodide in methanolic solution in the presence of K2CO3 afforded N-methyl and N-ethyl quinuclidines 74a and 74b in 99% and 90% yields, resp ectively (Figure 5-13).183,184 This method to quaternize amines is extensively used due to its sel ectivity, mild reaction conditions and high yields obtai ned. The desired compounds were obtained from the organic filtrate without needing further purification.

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158 An initial attempt to synthesize 3-benzylidene quinuclidine 75a utilized the Wittig185,186 reaction between 3-quinuclidinone hydrochloride and the ylid derived from treatment of triphenyl benzyl phosphonium iodide with n-BuLi Firstly, two equivalents of n-BuLi, one to deprotonate the amine and the second to form the ylid, were used, but base on 1H NMR spectroscopy no formation of the double bond was observed and quinuclidone was left unreacted. Then, the same reaction was performe d on the free base quinuclidone. Unfortunately, only an 8% yield of an E/Z mixture of 75a was obtained, with ~30% co nversion of the starting 3quinuclidinone based on 1H-NMR analysis of the crude reaction mixture. Based on this result, it was considered that the acidity of the 2-posi tion in 3-quinuclidinone required a less basic olefination reagent. N H+ N I CH3I/MeOH KHCO3N CH3CH2I/MeOH KHCO3 Cl-74a 74b 99% 90% I Figure 5-13. Alkylation of quinuclidine hydrochloride Benzyl phosphonates were utilized in the Wads worth-Emmons reaction to provide more satisfactory yields.187 In this case, diethyl benzyl phosphonate for Z/E-75a and diethyl-4methoxy benzyl phosphonate for Z/E75b were used as olefination reagents (Figure 5-14). The best solvent for this reaction was found to be 1,2-dimethoxy ethane (DME).188 Chromatographic

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159 separation of the geometric isomers proved to be difficult, not only because the two isomers had very similar Rf but also, because other amine containing side products were coeluting with the desired compounds. A variety of bi nary and ternary systems failed to provide a clean separation in a single step. After two successive chromatographic steps on silica in CHCl3/MeOH/Et3N mixtures, the two isomers were obtained in pure form, providing 75a in 46% yield184 (Z:E ratio, 2:1). Compound 75b was obtained in 23% yield with a Z:E ratio of 7:1 after chromatographic purification.184 N O NaH/DME N H N H NaH/DME and P O O O 23% 75a N H OMe N H MeO and 75b MeO P O O O 46% Figure 5-14. Synthesis of benzylidene quinuclidines The olefin geometry for each isomer was una mbiguously established based on analysis of NOESY spectra and analysis of chemical shifts (Figure 5-15). The NOESY spectrum for Z75b revealed crosspeaks for interactions between H2-H6, and H4-H5. The E-isomer of 75b presented a complementary set of data, with crossp eaks corresponding to interactions between H2-H5 and

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160 H4-H6. The NOESY spectrum of Z75a displayed crosspeaks for interactions between H2-H6, and H4-H5 as was observed for Z75b Finally, characteristic chemi cal shifts were identified for H2 and H4 depending on the isomer in question. Thus, for the Eisomers of 75a,b the chemical shift of H4 was found downfield relative to the shift for H4 of the Z-isomers, while in the case of the Z-isomers, H2 was shifted downfield relative to the chemical shift of H2 in the E-isomers. These effects may be attributed to deshielding from the phenyl ring.184 N N H5 H2 H4 H6 OMe H5 H4 H6 H2 N H5 H2 H4 H6 E-75b Z-75b E-75aMeO Figure 5-15. NOE enhancements for assignment of olefin geometry of 75a and 75b Finally, both benzyl quinuclidones were converted to their corre sponding hydrochloride salts in order to improve their so lubility in aqueous solution to facilitate their subsequent testing with nicotinic receptors. With the purpose of further investigate the hypothesis of the dual hydrophobic pocket present in neuronal nAChR, Z and E75a were N-methylated utiliz ing the method previously

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161 described for the quinucli dines. The Z-N-methyl-75a was obtained in a 62% yield while the E isomer was isolated in a 94% yield (Figure 5-16). N+IN+IE-N-methyl-75a Z-N-methyl-75a N N CH3I/MeOH KHCO3CH3I/MeOH KHCO394% 64% Figure 5-16. Methylation of Benzylidene quinuclidines Furthermore, OH-Z75b was synthesized by dealkylation reaction of Z75b This reaction could have been done by treating the methoxybe nzylidene quinuclidone with trimethylsilyl iodide (TMSI).189 In our case, TMSI was prepared from trimethylsilyl chloride and KI. This crude mixture was added to Z-75b but after leaving the reaction for 72 h at room temperature only unreacted Z75b was observed by 1H NMR. Another conventional method to cleave the ether functionality is by using BBr3 190-192 as a Lewis acid and performing the reaction at -60 C under nitrogen atmosphere. Unluckily, 1H NMR and TLC characterizations of the crude mixture revealed the presence of a very complex mixtur e of products, leading to the abandonment of this synthetic route.

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162 N HO N MeO N H+Cl40% OH-Z-75b Figure 5-17. Methoxy Benzylidene quinuclidine demethylation The desired OH-Z75b was finally obtained when Z75b was heated at 190 C in the presence of pyridinium hydrochloride for 2 h.193,194 After purifying the amine by flash column chromatography, OH-Z75b was acquired in 40% yield (Figur e 5-17). Unfortunately, neither Z or E-N-methyl75a nor OH-Z75b were tested for 7 and 4 2 nAChRs possible agonist activity. In both cases, the quanti ties and purity in hand were unsatis factory to perform a reliable binding response experiment on the nicotinic ion channels. N O I N B OH OH BAanalog 78 Figure 5-18. Proposed synthesis for BA analog 78 With the observation that BA compounds can show considerable potency and efficacy for 7 activation, we considered th e possibility that perhaps th e hydrophobic group and its relative position in the tricyclic molecule might be suffici ent in itself to produ ce receptor activation. Hence, compound 78 (Figure 5-18) was identified as a synt hetic target. It lacks the ammonium

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163 pharmacophore, but it otherwise identical to BA. In our attempts to synthesize this deaza BA analog (Figure 5-18), 2-iodocyclohex-2-enone was required as a precursor for the subsequent Suzuki coupling with pyridine boronic acid. This cyclohexenone was prepared by -iodination of 2-cyclohexanone following Johnsons method but us ing DMAP as the base catalyst (Figure 519).195,196 The -iodoketone 76 was obtained in a 69% yield af ter chromatographic purification. The signals observed in the 1H NMR were consistent with the literature.197 O I N B OH OH O I2/pyridine-CCl4DMAP O N Pd(Ph3)4DME 69%44% 7677 Figure 5-19. Synthesis of -pyridyl cyclohexenone 77 Afterward, -pyridyl cyclohexenone 77 was synthesized by the cross-coupling reaction known as Suzuki-Miyaura.198 This reaction is one of the most used methods for carbon-carbon bond formation at sp2 centers. The reaction proceeds through c onjugation of a haloalkene or haloalkynes with an arylboronic ac id. Palladium catalysts are known to give the best results in this kind of reactions. Among th em, the tertiary phosphines ligands are the most popular,199 but recently, Felpins research group observed that he terogeneous catalysis with Pd(0)/C could also produce coupling products in very good yields.200 Initially, the heteroge neous Pd(0)/C Suzuki coupling was chosen for the synthesis of 77. The previously prepared 2-iodocyclohex-2-enone and pyridine-3-boronic acid were utilized as vi nyl halogen and aryl sources. In this case, the formation of the product was almost undetectable by 1H NMR. Under otherwise identical reaction conditions, Pd(0)/C was replaced with Pd(Ph3)4. Once again, the coupling reaction did not produce the desired product, leav ing a large amount of unreacted -iodoketone. In our case, the conditions that gave the best results were when Pd(Ph3)4 was first complexed with the

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164 iodonone for 10 minutes, followed by the sequen tial addition of pyrid ine boronic acid and aqueous Na2CO3.199 The product 77 was obtained as yellowish crystals in 44% yield. O N 77 N 78 P O O O NaH/DME PhCH2P(Ph)3 +Cl-/n-BuLi Br Mg/ether Figure 5-20. Synthesis route A for compound 78 The first attempt in the synthesis of 78 utilized Wadsworth-Emmons reaction conditions already used for 75a and 75b (Synthesis route A (Figure 5-20 )). The 2-pyridylcyclohexenone 77 was treated with diethyl benz yl phosphonate using NaH as the base. Analysis of the crude mixture by 1H NMR did not show evidence of eith er new double bond formation or pyridine hydrogen peaks. In a second effort to make this compound, triphenyl benzyl phosphonium chloride was used as the Wittig reagent precursor but again, the reaction did not result in the desired product (Figure 5-20). Th e corresponding benzyl bromide Grignard reagent was prepared in a final attempt to produce this compound via th e Synthetic route A. From the chromatographic

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165 purification of the crude mi xture a promising fraction was obtained. Unfortunately, the compound of interest was not present in the fraction mixture by analysis of the 1H NMR. One hypothesis for why this scheme was troublesome would be that deprotonation of the basic cyclohexenone -hydrogens was occurring with use of th e relatively basic benzylidenating reagents discussed above. Thus, a new synthe tic route was pursued. Based on the literature, conjugated dienes have been prep ared by the reaction of an epoxi de and the appropriate aldehyde or ketone in presence of tributylphosphine (Figure 5-21).201 O O H Bu3P Figure 5-21. Example of the synthesis of conjugated dienes through an epoxide In order to test this possibility, 3-cyclohexenylpyridine 82 was chosen as the epoxide precursor (Synthesis route B (Figure 5-22)). Unfortunately, efforts thus far at synthesizing 82 have been unsuccessful. First, 3-bromopyridine 79 was metallated with n-BuLi, and used for a carbonyl addition reaction with cyclohexanone. Despite spectroscopic evidence for the cyclohexanol intermediate in the crude product, attempts to dehydrate the crude mixture were unfruitful. Finally, it was believed that the Suzuki coupling be tween pyridine-3-boronic acid 80 and 1-chlorocyclohexene 81 could be the solution to the problem in hand (Figure 5-22). Unfortunately, this vinyl halide is not commerciall y available and attempts to synthesize this compound were unsuccesful. A thir d possibility, as yet untest ed might be to deoxygenate compound 77, above, via reduction of the corresponding thioketal.

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166 N N Br O N O N B OH OH Br n-BuLi Pd(Ph3)4/DME O 79 80 81 82 Figure 5-22. Proposed Synthetic ro ute B for 3-cyclohenenylpyridine 82 Electrophysiological Evalua tion of Ne w Compounds Xenopus oocytes expressing mRNAs corresponding to 7, 3 4, or 4 2 subunits of nAChRs177 were used by Dr. Roger Papkes group (UF Department of Pharmacology) to determine agonism of compounds 74a,b and 75a,b The reference agonist was acetylcholine, applied at 300 M, which produces a maximal channel curre nt at this concentration. It was found that E75a and E75b were 7 selective part ial agonists (Emax ~ 40%) with respective EC50s of 1.5 and 1.3 M. By comparison, 4OH-GTS-21 has an EC50 of approximately 5 M for human 7 and an Emax of about 40% the maximum response produced by the full agonist ACh. Compound 74a was an 7-selective full agonist with an EC50 of 40 M, and the data for 74b suggested it was a weak 7 selective agonist, but receptor-independent currents were observed upon application of the compound to oocytes making detailed interpretation of this

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167 compounds activity difficult. Compounds E75a,b and 74a,b were tested in co-application experiments to see if they antagonized the activation of 3 4 or 4 2 receptors by ACh. When tested at a concentration of 10 M, co-applied with ACh, E75b produced a transient inhibition of the 3 4 receptor of approximately 50%, but otherw ise there appeared to be no significant antagonist actions for the quinuclidine compounds at that concentration. In summary, the results of these experiment s are consistent with the proposed model for selectivity of 7 agonists, showing that selectivity and activity may be obtained with molecules possessing a charged nitrogen and suitable hyd rophobic residue. It was believe the EC50 values indicate that the binding site for the aryl group is tolerant of substituti on and therefore amenable to further development of agonists. Further details of the biologica l activity of these and other quinuclidine compounds will be reported elsewhere. Experimental Section General Methods. Solvents and reagents were purchased from Aldrich Chemical Company and Acros Organics. The organic solvents were dried overnight over CaH2 or 4 molecular sieves and freshly distilled before use. NMR spectra were obtained using VXR 300, Gemini 300 and 500, or Mercury 300 and 500 MHz spectrophotometers in appropriate deuterated solvents. Mass spectra were obtained on a Finningan MAT 95Q spectrometer operated in FAB, CI, ESI or EI modes. 1-methyl-1-azoniabicyclo[2.2.2]octane iodide 74a.183 A mixture of methyl iodide (0.54 mL), KHCO3 (0.34 g) and quinuclidine hydrochloride (50 mg) was stirred in methanol at room temperature for 12 h. Then, solv ent was evaporated and CHCl3 was added and stirred overnight. The mixture was filtered and solvent evaporated under vacuum giving methyl quinuclidine as a white solid in 99% yield (85 mg). The solid started to decompose at temperatures greater than

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168 230 C. 1H NMR (D2O) ppm 1.96 (m, 6H), 2.17 (m, 1H), 2.90 (s, 3H), 3.37 (t, 6H),13C NMR (D2O) (ppm) 57.4, 52.3, 23.9, 19.1. EI Calcd for C8H16IN (M-I)+: 126.1277, found: 126.1286. 1-ethyl-1-azoniabicyclo[2.2.2]octane iodide 74b.183 This compound was synthesized by the same procedure explained above using 0.55 mL of ethyl iodide. The reaction gave the ethyl quinuclidine as a white solid in a 90% yield (81 mg). The solid star ted to decompose at temperatures greater than 210 C. 1H NMR (D2O) ppm 1.30 (t, 3H), 1.99 (m, 6H), 2.20 (m, 1H), 3.21 (q, 2H), 3.38 (t, 6H),13C NMR (D2O) (ppm) 60.0, 54.5, 23.8, 19.5, 7.6. ESI-FT-ICR Calcd for C9H18IN (2M+I)+: 407.1918, found: 407.1921. 3-benzylidene-1-azoniabicyclo[2.2.2]octane chloride 75a To a suspension of NaH (60% in mineral oil, 0.23 g, 5.8 mmol) in 1,2-dimet hoxy ethane (DME) (6.4 mL) was added dropwise a solution of diethyl benzyl phosphonate (1.3 g, 5.8 mmol) in DME (2 mL) at room temperature under argon. After this addition, a solution of quinuclidone (0.41 g, 2.54 mmol) in DME (1.78 mL) was injected slowly. The reaction mixture was refluxed for 1.5 h. Then, the mixture was quenched carefully with water (30 mL) and DME evaporated under re duced pressure. The aqueous phase was extracted with CH2Cl2 (3 x 30 mL) and the organic layer dried over MgSO4 and evaporated under vacuum. The crude oil was purified by flash chromatography on silica gel using CH2Cl2: MeOH: Et3N 20:1:0.1 as eluent system to give the Z isomer in a 30% yield (0.15 g) and E isomer in a 16% yield (81 mg). The hydrochloride salts of these isomers were obtained by adding ether-HCl to a solution of the bases in a mixture of CH2Cl2/ether. Both isomers were obt ained as white solids, which decomposed at temperatures above 200 C. The free base quinuclidone used in the pr eceeding syntheses was obtained by treating quinuclidone hydrochloride with a 2 M aqueous solution of K2CO3 (20 mL). Then, this solution

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169 was extracted three times with ether (30 mL), dried over MgSO4 and, the solvent was evaporated under reduced pressure. For the Z isomer: 1H NMR (CDCl3) ppm 2.06 (td, 4H), 2.78 (m, 1H), 3.28 (m, 2H), 3.33 (m, 2H), 4.15 (s, 2H), 6.45 (t, 1H), 7.17-7.37 (m, 5H), 13C NMR (CDCl3) (ppm) 145.3, 137.7, 130.2, 128.8, 128.7, 126.5, 120.5, 56.9, 48.1, 27.5, 26.1. ESI-FT-ICR Calcd for C14H18N (M)+: 200.1439, found: 200.1423. For the E isomer: 1H NMR (CDCl3) ppm 2.06 (td, 4H), 3.39 (m, 5H), 4.05 (s, 2H), 6.48 (t, 1H), 7.19-7.36 (m, 5H),13C NMR (CDCl3) (ppm) 144.5, 137.6, 128.9, 128.7, 126.7, 122.0, 56.0, 47.9, 34.1, 28.1. ESI-FT-ICR Calcd for C14H18N (M)+: 200.1439, found: 200.1423. 3-(4-methoxybenzylidene)-1-azoniabicyclo[2.2.2]octane chloride 75b. These compounds were synthesized using the same pro cedure explained above. A suspension of NaH (60% in mineral oil, 0.87 g, 22 mmol) in DME (24 mL) was stirred at room temperature under argon. A solution of diethyl -methoxy benzylphosphonate (3.6 mL, 21 mmol) in DME (7 mL) was added dropwise to that solu tion. After this, a solution of quinuclidone as a free base (1.1 g, 9.0 mmol) in DME (6 mL) was added dropwise. The reaction mixture was refluxed for 1.5 h and quenched with water. The DME was evaporated under vacuum and the residue dissolved with dichloromethane and washed with water. Then, the organic layer was washed with brine and dried over MgSO4.The mixture was purified by flas h chromatography (silica gel, CH2Cl2:MeOH 35 :1). The two isomers where isolated as free ba ses giving the Zisomer in 20% yield (0.41 g) and the Eisomer in 3% yield (61 mg). For the Z isomer: 1H NMR (CDCl3) ppm 2.10 (td, 4H), 2.80 (m, 1H), 3.31 (dt, 2H), 3.39 (dt, 2H), 3.83 (s, 3H), 4.19 (s, 2H), 6.42 (t, 1H), 6.88 (d, 2H), 7.08 (d, 2H),13C NMR (CDCl3)

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170 (ppm) 159.3, 131.1, 130.0, 128.2, 125.2, 114.6, 55.7, 54.4, 47.1, 32.2, 25.3. EI Calcd for C15H19NO (M)+: 229.1467, found: 229.1475. For the E isomer: 1H NMR (CDCl3) ppm 2.04 (td, 4H), 3.35 (m, 5H), 3.82 (s, 3H), 3.99 (s, 2H), 6.40 (t, 1H), 6.91 (d, 2H), 7.11 (d, 2H),13C NMR (CDCl3) (ppm) 159.3, 131.1, 129.9, 127.9, 125.5, 114.4, 55.7, 54.7, 47.0, 24.7, 24.2. EI Calcd for C15H19NO (M)+: 229.1467, found: 229.1475. 3-benzylidene-1-methyl-1-azoniabicyc lo[2.2.2]octane iodide (N-methyl-75a). A mixture of methyl iodide (16 mmol), KHCO3 (10 mmol) and Z or E-benzylidene quinuclidones (1 mmol) were stirred in methanol at room temperature for 12 h. Then, the solvent was evaporated and CHCl3 was added and stirred overnight. The mixture was filtered and solvent evaporated under vacuum. The Z isomer was obtained in a 64% yiel d (80 mg) and the E in a 94% yield (26 mg). For the Z isomer: 1H NMR (D2O) ppm 2.17 (m, 4H), 2.91 (m, 1H), 3.06 (s, 3H), 3.48 (m, 4H), 4.45 (s, 2H), 6.61 (s, 1H), 7.28-7.44 (m, 5H). For the E isomer: 1H NMR (D2O) ppm 2.14 (m, 4H), 3.11 (s, 3H), 3.39 (m, 1H), 3.58 (m, 4H), 4.27 (s, 2H), 6.55 (s, 1H), 7.30-7.49 (m, 5H). (Z)-4-(quinuclidin-3-ylidenemethyl)phenol (OH-Z-75b). Pyridinium hydrochloride (0.13 g, 0.88 mmol) and Z75b (29.8 mg, 0.13 mmol) were heated at 190 C under argon atmosphere for 2 h. After the crude mixture was pour ed into an ice-cooled saturated solution of sodium chloride, the reaction was neutralized w ith a solution of 1 M ammonium hydroxide to pH 7-8 and extracted into CH2Cl2 (3 x 10 mL). The organic phase was dried over Na2SO4 and the solvent evaporated under vac uum. The crude product was purified by flash chromatography (silica gel, CH2Cl2:MeOH 9:1). The product was isolated as the free base (11 mg) giving the OHZ75b in a 40% yield.1H NMR (CDCl3) ppm 1.94 (m, 4H), 2.59 (m, 1H), 3.11 (m, 4H), 4.03 (s,

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171 2H), 6.29 (s, 1H), 6.76 (d, 2H), 7.04 (d, 2H). EI Calcd for C14H17NO (M)+: 215.1310, found: 215.1292. 2-iodocyclohex-2-enone 76.195,196 2-cyclohexe-1-one (2.1 mL, 21 mmol) was dissolved in a mixture of 1:1 pyridine/CCl4 (100 mL) and the solution was cool ed to 0 C. Then, a catalytic amount of DMAP (51 mg, 0.42 mmol) and iodine (13 g, 52 mmol) in pyridine/CCl4 (1:1, 60 mL) were added. After stirring the mixture at room temperature for 24 h, the reaction was quenched with 50 mL of 20% aqueous Na2S2O3 solution. The aqueous phase was extracted with ether (3 x 100 mL). The combined organic layers were dried over MgSO4 and the solvent evaporated under vacuum. The crude was purified by flash chroma tography (silica gel, petroleum ether:EtOAc 25 :1). The -iodoketone (3.2 g, 69% yield) was obtained as a yellow oil.1H NMR (CDCl3) ppm 2.08 (m, 2H), 2.42 (q, 2H), 2.66 (t, 2H), 7.76 (t, 1H). The 1H NMR was consistent with the literature.197 2-(pyridin-3-yl)cyclohex-2-enone 77. Iodoenone (2 g, 9 mmol) was added to a suspension of Pd(Ph3)4 (0.31 g, 0.27 mmol) in dimethoxyethane (DME) (27 mL). After stirring this mixture for 10 min at rt, pyridine boronic acid (1.65 g, 13.5 mmol) dissolved in a minimum amount of EtOH followed by a 2 M aqueous solution of Na2CO3 (2.2 mL, 4.4 mmol) were added. The reaction was heated under reflux for 6 h. The cr ude mixture was diluted with 5% aqueous Na2CO3 and extracted four times with EtOAc. The co llected organic extracts were dried with MgSO4 and concentrated under reduced pressure. Purification of 77 by flash chromatography (silica gel, petroleum ether:EtOAc 2:1) gave 2-pyridylcyclohexenone (0.7 g, 44% yield) as yellowish crystals.1H NMR (CDCl3) ppm 2.13 (m, 2H), 2.59 (m, 4H), 7.11 (t, 1H), 7.27 (m, 1H), 7.98 (dt, 1H), 8.52 (d, 2H). 13C NMR (CDCl3) (ppm) 197.7, 149.5, 149.4, 148.9, 137.6, 136.6, 132.5, 123.0, 39.1, 26.9, 23.1. EI Calcd for C11H11NO (M)+: 173.0549, found: 173.0564.

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172 CHAPTER 6 CONCLUSIONS AND FUTURE WORK This dissertation work has resu lted in the synthesis of a new class of TS analogs for glycosidases and glycosyltransferases where the apical geom etry of the leaving group is held over the plane of atoms that present the oxocarbenium charge mimicry, using an all cis trisubstituted cyclopropyl ring fuse d to a second amidine containing ring The synthesis of the seven-membered ring di aza analogs was successfully accomplished in a relatively good overall yield following a short mu ltistep pathway. The design of the synthetic route also allows the preparation of amidines w ith a variety of side groups attached. As already demonstrated in this work, this can be accomplished using the corresponding imidate derivative in the last step of the synthesis. Other amidines differing in the nucleophile moiety could then be tested for glycosidases inhibi tory activity. Moreover, the porti on of the analogs that mimic the departing leaving group might as well be m odified by changing the substituent of the cyclopropane bridging oxygen. Alte rations of these two features may help to improve the molecules inhibitory potency a nd/or to explore new enzyme-TS analogs binding contacts. This synthetic pathway might be suit able to obtain inhibitors for other enzymes. For example, substitution of the amidines benzyl group w ith a small oligosaccharide might convert the compound into a TS analog for endoglycosidases wh ich have several pockets for binding a long polysaccharide chain. Although the amidines lacked th e hydroxyl groups present in the sugars backbone that are known to facilitate favorable enzyme-substr ate interaction, they still displayed Ki values on the high M range. Based on Bleriot et al.37 results, the incorpora tion of hydroxyl groups on glycosidases TS analogs increase d the inhibitory power of the molecule. T hus, the incorporation

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173 of hydroxyl groups on future amidine designs should also be investigated in order to increase the binding interactions of these compounds. With the exception of the chloromethyl amidine 29 (BnCMAM), all the diaza compounds presented a competitive mode of inhibition wh ich was expected for a TS analog. Amidine 23 (BnHMAM) was the best inhibitor from the library of analogs with a Ki of 150 M against glucosidase from sweet almonds. Consequently, BnHMAM binds almost 25 times tighter to the enzyme than the substrate PNP glu. This result may be an indication that the BnHMAM 23 shape resembles, in some way, the geometry of glycosidases TS. Only BnCMAM displayed a mixed or non-competitive mode of inhibition. Th is unpredicted result may be explained by a possible interaction of BnCMAM with other sites in the enzyme Further kinetic experiments will be necessary in order to clarify this hypothesis. Moreover, it might be concluded that the analogs were selective for the -glycosidases because none of the glycosidases were significantly inhibited. The significance of th is observation remains to be explored. For the series of (2 6)-ST TS analogs, the final step on these multi-step synthetic route involved conjugation of the molecules with CM P. Analysis of the coupling reaction of compound 33 and CMP by mass spectrometry and NMR proved the presence of CMP-amidine 73. The desired compound was obtained with 95 % purity after purification by reverse phase HPLC. The estimated Ki for CMP-amidine 73 on human recombinant (2 6)-ST was ~50 M. This TS analogs inhibitory activity turned out to be comparable with the one displayed by free CMP and higher than those observed for previous generation scorpio in hibitors. The modest activity might be a reflection of the size of the amidine ring which could situate the amidine center in a less favorable position for binding with respect to the leaving group mimic.

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174 The all cis trisubstituted cyclopropan es, synthesized during the course of this study, may also find use in other applications. For example, they could serve as chiral ligand frameworks for asymmetric catalysis that could provide enantiomerically enriched comp ounds. In addition, they could be utilized as building bl ocks for targeting other protei ns such as G-protein-coupled receptors (P2Y).202 An example of a known antagonist for th is receptor is shown in Figure 6-1. OPO3 -2 OPO3 -2 N N N N NH Cl Figure 6-1. Antagonist for P2Y1 receptor202 Finally, for the study of 7 nAChR agonists, quinuclidine and quinuclidinone derivatives were synthesized. By their agonis t activity profiles, they supporte d the requirement for a charged nitrogen center but als o, they supported the hypothesis that se lectivity may be achieved with hydrophobic moieties having two very di fferent sizes and spatial rela tionship with respect to the ammonium center. An ongoing work involves synt hesis of functionalized tropanes to further investigate the selectivity filter of the 7 nAChR.

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175 APPENDIX A NMR SPECTRA FROM SYNTHESIZED COMPOUNDS The NMR spectra of the synthesized com pounds are shown in this Appendix. While the structure and number of the co mpound is shown in the spectra, the name of the molecule is described at the bottom of the page.

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176 Figure A-1. (4,5-Dihydro-1H -imidazol-2-yl)-methanol

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177 Figure A-2. 2,2-Dimethyl-1,3-dioxocyclohept-5-ene

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178 Figure A-3. 4,4-Dimethy-8-ethylfor myl-3,5-dioxa-bicyclo[5.1.0]octane

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179 Figure A-4. Diazo-acetic acid 4-benzyloxy-but-2-enyl ester

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180 Figure A-5. 6-Benzyloxymethyl-3oxa-bicyclo[3.1.0]hexan-2-one

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181 Figure A-6. tert-Butoxycarbonylamino-ace tic acid 4-benzyloxy-but-2-enyl ester

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182 Figure A-7. Amino-acetic acid 4-benzyloxy-but-2-enyl ester

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183 Figure A-8. (Z)-4-(benzyloxy)but-2-enyl 2-(2,2,2-trifluoroa cetamido)acetate

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184 Figure A-9. (Z)-4-(benzyloxy)but-2-enyl 2-(((9Hfluoren-9-yl)methoxy)carbonylamino)acetate

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185 Figure A-10. (Z)-4-(benzyloxy)but-2-enyl 3-oxobutanoate

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186 Figure A-11. ((1R,2S,3s)-3-(benzyloxymethy l)cyclopropane-1,2-diyl)dimethanol

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187 Figure A-12. ((((1s,2R,3S)-2,3-bis(azidomethy l)cyclopropyl)methoxy)methyl)benzene

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188 Figure A-13. ((1R,2S,3s)-3-(benzyloxymethyl) cyclopropane-1,2-diyl)dimethanamine

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189 Figure A-14. (Z)-(8-(benzyloxymethyl)-3,5-d iazabicyclo[5.1.0]oct-4-en-4-yl)methanol

PAGE 190

190 Figure A-15. (Z)-8-(benzyloxymethyl)-4-(trichlor omethyl)-3,5-diazabi cyclo[5.1.0]oct-3-ene

PAGE 191

191 Figure A-16. (Z)-8-(benzyloxymethyl)-4-(chlo romethyl)-3,5-diazabicyclo[5.1.0]oct-3-ene

PAGE 192

192 Figure A-17. (Z)-8-(benzyloxymethyl)3,5-diazabicyclo[5.1.0]oct-3-ene

PAGE 193

193 Figure A-18. (Z)-3,5-diazabicyclo[5.1.0]oct-4-en-8-ylmethanol

PAGE 194

194 Figure A-19. (Z)-3,5-diazabicyclo[ 5.1.0]oct-4-ene-4,8-diyldimethanol

PAGE 195

195 Figure A-20. (1R,2S,3R)-2-(benzy loxymethyl)-3-(hydroxymethyl )cyclopropanecarboxamide

PAGE 196

196 Figure A-21. (1S,2S,3R)-2-(benzyloxymethy l)-3-((tert-butyldimethylsilyloxy )methyl) cyclopropanecarboxamide

PAGE 197

197 Figure A-22. Methyl(1S,2S,3R)-2-(benzyloxymethyl)-3-((tertbutyldimethylsilyloxy)methyl ) cyclopropylcarbamate

PAGE 198

198 Figure A-23. Methyl (1R,2S,3R)-2-(benzyloxymeth yl)-3-(hydroxymethyl)cyclopropylcarbamate

PAGE 199

199 Figure A-24. 2-(benzyloxymethyl)-3-(hydr oxymethyl)cyclopropanecarboxylic acid

PAGE 200

200 Figure A-25. Methyl 2-(benzyloxymethyl)-3 -(hydroxymethyl)cyclopropanecarboxylate

PAGE 201

201 Figure A-26. (1R,2R,3R)-methyl 2-(benzyloxy methyl)-3-formylcyclopropanecarboxylate

PAGE 202

202 Figure A-27. (1R,2R,3S)-methyl 2-(benzyloxy methyl)-3-formylcyclopropanecarboxylate

PAGE 203

203 Figure A-28. 2-(benzyloxymethyl)-3-(methoxy carbonyl)cyclopropanecarboxylic acid

PAGE 204

204 Figure A-29. 3-(benzyloxymethyl)cyclopropane-1,2-dicarboxylic acid

PAGE 205

205 Figure A-30. Dimethyl 3-(benzyloxymet hyl)cyclopropane-1,2-dicarboxylate

PAGE 206

206 Figure A-31. 3-(benzyloxymethyl) cyclopropane-1,2-dicarboxamide

PAGE 207

207 Figure A-32. 2-(azidomethyl)-3-(benzyloxymethyl)cyclopropanecarboxamide

PAGE 208

208 Figure A-33. Methyl 2-(azidomethyl)-3-(ben zyloxymethyl)cyclopropylcarbamate

PAGE 209

209 Figure A-34. Methyl 2-(aminomethyl)-3-(benzyloxymethyl)cyclopropylcarbamate

PAGE 210

210 Figure A-35. 7-(benzyloxymethyl)-2,4-diazabicyclo[4.1.0]heptan-3-one

PAGE 211

211 Figure A-36. (1R,2S,3S)-2-(ami nomethyl)-3-(benzyloxymethyl)cyclopropanamine

PAGE 212

212 Figure A-37. ((1R,2R,3S)-2-amino-3-(benzy loxymethyl)cyclopropyl)methanol

PAGE 213

213 Figure A-38. ((1S,2R,3R)-2-(aminomethyl)-3-( benzyloxymethyl)cyclopropyl)methanol

PAGE 214

214 Figure A-39. (Z)-8-(benzyloxymethyl)-3 -oxa-5-azabicyclo[5.1.0]oct-4-ene

PAGE 215

215 Figure A-40. (Z)-4-(benzyloxy)but-2-enyl 2-benzyl-3-oxobutanoate

PAGE 216

216 Figure A-41. (Z)-4-(benzyloxy)but-2-enyl 2-acetylpent-4-enoate

PAGE 217

217 Figure A-42. (Z)-4-(benzyloxy)but-2-enyl 2-diazo-3-phenylpropanoate

PAGE 218

218 Figure A-43. (Z)-4-(benzyloxy)but-2-enyl 2-diazopent-4-enoate

PAGE 219

219 Figure A-44. 2,3-O,N4-Triacetyl Cytidine

PAGE 220

220 Figure A-45. Triethylammonium (5-(4-ace tamido-2-oxopyrimidin-1(2 H)-yl)-3,4-diacetoxy tetrah ydrofuran-2-yl)methyl 2chlorophenyl phosphate

PAGE 221

221 Figure A-46. 6-(hydroxymethyl)-3 -oxabicyclo[3.1.0]hexan-2-one

PAGE 222

222 Figure A-47. 2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)5-(((2-chlorophenoxy)((2-oxo-3-oxabicyclo[3.1.0]hexan-6yl)methoxy)phosphoryloxy)methyl)tetra hydrofuran-3,4-diyl diacetate

PAGE 223

223 Figure A-48. (Z)-3,5-diazabicyclo[5.1.0]oct-4-en-8-ylme thyl (5-(4-amino-2-oxopyr imidin-1(2H)-yl)-3,4dihydroxytetrahydrofuran-2-yl)methyl hydrogen phosphate

PAGE 224

224 Figure A-49. 1-methyl-1-azoniabicyclo[2.2.2]octane iodide

PAGE 225

225 Figure A-50. 1-ethyl-1-azoniabicyclo[2.2.2]octane iodide

PAGE 226

226 Figure A-51. E-3-benzylidene-1-azon iabicyclo[2.2.2]octane chloride

PAGE 227

227 Figure A-52. Z-3-benzylidene-1-azon iabicyclo[2.2.2]octane chloride

PAGE 228

228 Figure A-53. E-3-(4-methoxybenzylidene)-1-a zoniabicyclo[2.2.2]octane chloride

PAGE 229

229 Figure A-54. Z-3-(4-methoxybenzylidene)-1 -azoniabicyclo[2.2.2]octane chloride

PAGE 230

230 Figure A-55. E-3-benzylidene-1-methyl-1 -azoniabicyclo[2.2.2]octane iodide

PAGE 231

231 Figure A-56. Z-3-benzylidene-1-methyl-1 -azoniabicyclo[2.2.2]octane iodide

PAGE 232

232 Figure A-57. (Z)-4-(quinuclidin-3-ylidenemethyl)phenol

PAGE 233

233 Figure A-58. 2-iodocyclohex-2-enone

PAGE 234

234 Figure A-59. 2-(pyridin-3-yl)cyclohex-2-enone

PAGE 235

235 APPENDIX B KINETIC ASSAY AND ANALYSIS FOR GL YCOSIDASE S WITH DIAZABICYCLIC AMIDINES The inhibitors screening gra phics are displayed bellow. Assuming competitive and mixed or non-competitive inhibition, th e models utilize to analyze the data were the following6: Competitive inhibition ][ ]][[ EI IE Ki ][][][][ ESEIEET ][ ][ ][ S ESK EM i MKS IESK EI ][ ]][[ ][ 1 ][ 1 ][ ][][i M TK I S K ESE ][ ][ 1 ][][ ][ S K I K SE ESi M T ][ ][ 1 ][][ ][2 20S K I K SEk ESkvi M T TEkv ][2 max ][ ][ 1 ][max 0SK K I Sv vM i 0v and maxv are initial velocity and ma ximal velocity respectively

PAGE 236

236 For the Lineweaver-Burke revers e plots, the competitive inhi bition reciprocal ecuation is: max max 01 ][ 1][ 1 1 vSv K K I vM i Mixed or non-competitive inhibition ][ ]][[ EI IE Ki ][ ]][[ ESI IES Ki ][][][][][ ESIESEIEET i i TK I ES K I EE'][ 1][ ][ 1][][ If ][ ][ ][ S ESK EM then i M i TK I S K K I ESE'][ 1 ][ ][ 1][][ If ][20ESkv and TEkv ][2 max then, ][ ][ 1 ][ 1 ][' max 0S K I K K I Sv vi M i For the Lineweaver-Burke reverse plots, the mixed or non-competitive inhibition reciprocal ecuation is: max max 01][ 1 ][ 1][ 1 1 vK I Sv K K I vi M i

PAGE 237

237 Kinetic Screening Graphics 0 0.004 0.008 0.012 0.016 0.02 012345678910 Time (min) mol product PNPgal BnAM BnHMAM BnCMAM OHHMAM OHAM Figure B-1. Kinetic assay of -gal at pH 7.5 0 0.02 0.04 0.06 0.08 0.1 0246810 Time (min) mol product PNPgal BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-2. Kinetic assay for -gal at pH 6

PAGE 238

238 0 0.02 0.04 0.06 0.08 00.511.522.533.544.55 Time (min) mol product PNPaglu BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-3. Kinetic assay for -glu at pH 7.5 0 0.05 0.1 0.15 0.2 0.25 00.511.522.533.544.55 Time (min) mol product PNPaglu BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-4. Kinetic assay for -glu at pH 6

PAGE 239

239 0 0.05 0.1 0.15 0.2 0.25 0.3 012345678910 Time (min) mol product ONPG BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-5. Kinetic assay for -galAsp at pH 7.5 0 0.2 0.4 0.6 0.8 01234567 Time (min) mol product ONPG BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-6. Kinetic assay for -galAsp at pH 6

PAGE 240

240 0 0.04 0.08 0.12 0.16 012345678910 Time (min) mol product ONPG BnAM BnCMAM BnHMAM OHAM OHHMAM Figure B-7. Kinetic Assay of -galEcoli at pH 7.5 0 0.05 0.1 0.15 0.2 0.25 012345678910 Time (min) mol product ONPG BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-8. Kinetic assay for -galEcoli at pH 6

PAGE 241

241 0 0.01 0.02 0.03 0.04 0.05 012345678910 Time (min) mol product PNPbglu BnAM BnCMAM BnHMAM OHAM OHHMAM Figure B-9. Kinetic assay for -glu at pH 7.5 0 0.04 0.08 0.12 0.16 0246810 Time (min) mol product PNPbglu BnAM BnHMAM BnCMAM OHAM OHHMAM Figure B-10. Kinetic assay for -glu at pH 6

PAGE 242

242 Lineweaver-Burke Reverse Plots 0 3 6 9 00.20.40.60.811.21.41.61.82 1/[S]( mM-1)1/v (mgenz/min/ molprod) Figure B-11. Lineweav er-Burke plot of -glu with BnHMAM. The concentrations of TS analogs were 4 mM; 2 mM; 0 mM. 0 1 2 3 4 5 00.20.40.60.811.21.41.61.82 1/[S] (mM-1)1/v (mgenz/min/ molprod) Figure B-12. Lineweav er-Burke plot of -glu with BnAM. The concentrations of TS analogs were 4 mM; 2 mM; 0 mM.

PAGE 243

243 0 0.002 0.004 0.006 0.008 0.01 0.012 02468101214161820 1/[S] (mM-1)1/v (mgenz/min/ molprod) Figure B-13. Linweaver-Burke plot of -galEcoli with BnCMAM. The concentrations of TS analogs were 0.6 mM; 0.3 mM; 0 mM. 0 2 4 6 8 10 12 14 16 00.511.522.533.544.55 1/[S] (mM-1)1/v (mgenz/min/ molprod) Figure B-14. Linweaver-Burke plot of -galAsp with BnHMAM. The concentrations of TS analogs were 3 mM; 1 mM; 0 mM.

PAGE 244

244 0 2 4 6 8 10 12 00.511.522.533.544.55 1/[S] (mM-1)1/v (mgenz/min/ molprod) Figure B-15. Linweaver-Burke plot of -galAsp with OHHMAM. The concentrations of TS analogs were 3 mM; 1 mM; 0 mM. Michaelis-Menten Curves 0 5 10 15 20 25 30 051015202530 [S] (mM)v ( molprod/min/mgenz) Figure B-16. Michaelis-Menten curves of -glu with BnHMAM. The concentrations of TS analogs were 4 mM; 2 mM; 0 mM.

PAGE 245

245 0 5 10 15 20 25 30 051015202530 [S] (mM)v ( molprod/min/mgenz) Figure B-17. Michaelis-Menten curves of -glu with BnAM. The concentrations of TS analogs were 4 mM; 2 mM; 0 mM. 0 100 200 300 400 500 00.40.81.21.62 [S] (mM)v ( molprod/min/mgenz) Figure B-18. Michaelis-Menten curves of -galEcoli with BnCMAM. The concentrations of TS analogs were 0.6 mM; 0.3 mM; 0 mM.

PAGE 246

246 0 0.4 0.8 1.2 1.6 2 012345678910 [S] (mM)v ( molprod/min/mgenz) Figure B-19. Michaelis-Menten curves of -galAsp with BnHMAM. The concentrations of TS analogs were 3 mM; 1 mM; 0 mM. 0 0.4 0.8 1.2 1.6 2 012345678910 [S] (mM)v ( molprod/min/mgenz) Figure B-20. Michaelis-Menten curves of -galAsp with OHHMAM. The concentrations of TS analogs were 3 mM; 1 mM; 0 mM.

PAGE 247

247 Ki Determination with Lineweaver-Burke Plots Information 0 0.5 1 1.5 2 2.5 3 00.511.522.53[I] (mM)slope ( molprod. mMONPG/mgenz/min) Figure B-21. Ki determination of -galAsp with BnHMAM 0 0.5 1 1.5 2 2.5 00.511.522.53[I] (mM)slope ( molprod.mMONPG/mgenz/min) Figure B-22. Ki determination of -galAsp with OHHMAM

PAGE 248

248 0 0.5 1 1.5 2 2.5 00.511.522.533.54[I] (mM)slope ( molprod.mMPNPbglu/mgenz/min) Figure B-23. Ki determination of -glu with BnAM 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 00.511.522.533.54[I] (mM)slope ( molprod.mMPNPbglu/mgenz/min) Figure B-24. Ki determination of -glu with BnHMAM

PAGE 249

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259 BIOGRAPHICAL SKETCH Fedra Marina Leonik, daughter of Jorge P. Leonik and Susana E. Sarabia, was born in Buenos Aires, Argentina, on May 20, 1978. She gr aduated with a Bachelor of Science degree in chem istry from the School of Science (Facultad de Ciencias Exactas y Naturales), University of Buenos Aires (UBA), Argentina, where she studied the variation in digestibility and dispersibility of milk proteins during storage, under the supervision of Dr. Maria Susana Vigo. She moved to Gainesville, Florida, on Augus t 2002 to pursue her PhD in chemistry at the University of Florida. In May 2003, she married Da niel G. Kuroda in Buenos Aires, Argentina. She received her PhD in chemistry in August, 2008, under the guidance of Dr. Nicole A. Horenstein. After completion of her studies, sh e and her husband will carry on their carreers in Pennsylvania.