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New Applications for Biocatalysis in Organic Synthesis

Permanent Link: http://ufdc.ufl.edu/UFE0022774/00001

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Title: New Applications for Biocatalysis in Organic Synthesis
Physical Description: 1 online resource (115 p.)
Language: english
Creator: Stowe, Gary
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: biocatalysis, chemistry, organic
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: We synthesized a series of alpha-methyl-beta-ketoesters in two steps. These ketoesters were then reduced to the corresponding alpha-methyl-beta-hydroxyesters by our library of purified ketoreductases from Baker's Yeast and Baker's Yeast whole cells. These steps allowed us to probe the reduction capabilities and specificities of our purified ketoreductases and also demonstrated that they yield far greater enantioselectivities in comparison to whole cells. A phosphonic acid analog of the highly toxic chemical warfare agents Sarin, Soman and VX was synthesized in four steps. This analog was tested as a target against DNA aptamers as part of a program to develop a rapid test for exposure to these nerve agents in the field. Unfortunately, no aptamers bound to the target. We chemoenzymatically synthesized alpha-fluoro-phosphonic acid carbohydrates in seven steps from noncarbohydrate precursors. The stereochemistry of the alpha-fluorine was controlled by selective reduction of an alpha-fluorovinylphosphonate using our library of alkene reductases. The alpha-fluorinated phosphonate alkane reduction product could then be used with aldolase and a suitable acceptor aldehyde to make five and six membered alpha-fluorinated phosphonic acid carbohydrates of defined stereochemistry without the need for protection / deprotection steps.
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 Gary Stowe.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Stewart, Jon D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022774:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022774/00001

Material Information

Title: New Applications for Biocatalysis in Organic Synthesis
Physical Description: 1 online resource (115 p.)
Language: english
Creator: Stowe, Gary
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: biocatalysis, chemistry, organic
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: We synthesized a series of alpha-methyl-beta-ketoesters in two steps. These ketoesters were then reduced to the corresponding alpha-methyl-beta-hydroxyesters by our library of purified ketoreductases from Baker's Yeast and Baker's Yeast whole cells. These steps allowed us to probe the reduction capabilities and specificities of our purified ketoreductases and also demonstrated that they yield far greater enantioselectivities in comparison to whole cells. A phosphonic acid analog of the highly toxic chemical warfare agents Sarin, Soman and VX was synthesized in four steps. This analog was tested as a target against DNA aptamers as part of a program to develop a rapid test for exposure to these nerve agents in the field. Unfortunately, no aptamers bound to the target. We chemoenzymatically synthesized alpha-fluoro-phosphonic acid carbohydrates in seven steps from noncarbohydrate precursors. The stereochemistry of the alpha-fluorine was controlled by selective reduction of an alpha-fluorovinylphosphonate using our library of alkene reductases. The alpha-fluorinated phosphonate alkane reduction product could then be used with aldolase and a suitable acceptor aldehyde to make five and six membered alpha-fluorinated phosphonic acid carbohydrates of defined stereochemistry without the need for protection / deprotection steps.
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 Gary Stowe.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Stewart, Jon D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022774:00001


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NEW APPLICATIONS FOR BIOCATALYSIS IN ORGANIC SYNTHESIS By G. NEIL STOWE 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 1

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2008 G. Neil Stowe 2

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3 ACKNOWLEDGMENTS I would like to thank Dr. Stew art for his steadfast guidance throughout my graduate school career. I would like to thank Dr. Brent Feske fo r teaching me how to function in a bio-organic lab when I started working. Thanks go to (the soon to be Dr.) Jordan Mathias for listening to my unique thoughts on the joys of research and for be ing one of the best friends I have ever had. Thanks go to Despina Bougioukou for being a bette r source of useFULL information than an Encyclopedia. Thanks go to Dimitri Dascier for t eaching me how to behave like a true Parisian. Thanks go to Adam Rothman for informing me of th e true price of a pack of cigarettes. Thanks go to Paige Finkelstein -the kiddofor being my constant source of entertainment for 10 weeks and showing me how to le ss than three chemistry. Thanks go to Richard Farley, Josh Bunger, Jenna Norton, Ashlee Ma ntooth, Lisa Nackers and Janet Cusido and the other members of MUSCO for giving me something fun and competitive to do outside of the lab. Thanks go to Jonathan Sommer for teaching me how to surf and showing me how to stop working and have a good time. Thanks go to Lisa Nackers for smiling and laughing. Heartily.

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 3LIST OF TABLES ...........................................................................................................................6LIST OF FIGURES .........................................................................................................................7ABSTRACT ...................................................................................................................... .............10CHAPTER 1 ENZYMATIC ROUTE FOR THE SYNTHESIS OF CHIRAL BUILDING BLOCKS FROM ALPHA-METHYL -BETA-KETOESTERS ..............................................................11Introduction .................................................................................................................. ...........11Ketoester Synthesis .........................................................................................................12Screening ..................................................................................................................... ....13Results and Discussion ........................................................................................................ ...13Experimental Procedures ....................................................................................................... .15Materials ..................................................................................................................... .....15Synthesis ..................................................................................................................... .....162 SYNTHESIS OF AN APTAMER TARGET FOR RAPID SCREENING OF EXPOSURE TO CHEMICAL WARFARE AGENTS ..........................................................38Introduction .................................................................................................................. ...........38Aptamers for CW Agent Detection .................................................................................38Target Synthesis ..............................................................................................................39Aptamer Selection ...........................................................................................................39Results and Discussion ........................................................................................................ ...40Experimental Procedures ....................................................................................................... .41General Experimental ......................................................................................................41Chemical Synthesis .........................................................................................................41PCR Amplification ..........................................................................................................44Magnetic Nanoparticles ...................................................................................................45Round 1 of SELEX ..........................................................................................................46Rounds 2-5 of SELEX .....................................................................................................47Round 6 of Selex Plus Counterselection Step .................................................................473 CHEMOENZYMATIC ROUTE TO FL UORINATED PHOSPHONIC ACID CARBOHYDRATES OF DEFI NED STEREOCHEMISTRY ..............................................53Introduction .................................................................................................................. ...........53The -Fluorophosphonates ..............................................................................................53Routes to -Monofluorophosphonates ............................................................................53 4

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Methods for Stereochemical Control of -Monofluorophosphonates .............................54The Importance of Stereochemical Control of -Monofluorophosphonates ...................55Aldolases in Synthetic Chemistry ...................................................................................55Selective Reduction of -Fluoro-Ketophosphonates ...................................................56Selective Reduction of -Fluorovinylphosphonates ........................................................57Synthesis of Racemic -Fluorinated Phosphonic Acids ..................................................59Results and Discussion ........................................................................................................ ...61Future Work ............................................................................................................................62Experimental Procedures ....................................................................................................... .63Materials ..................................................................................................................... .....63REFERENCE LIST .....................................................................................................................110BIOGRAPHICAL SKETCH .......................................................................................................115 5

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LIST OF TABLES Table page 1-1 Comparison of -methyl and -chloro-ketoester reductions. .........................................361-2 Comparison of reduction products for -methyl and unsubstituted -ketoesters. .............371-3 Chemical shift difference between ( R ) and (S) MPA esters ( R ,S). ................................372-1 Selection and countersel ection fluorescence data. .............................................................523-1 Fluorination condi tions of carbonate 102. .......................................................................109 6

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LIST OF FIGURES Figure page 1-1 Selective reduction using Bakers yeast whole cells .........................................................251-2 Substrates screened by Kaluzna et al. ................................................................................251-3 Synthetic applications of chiral products from -chloro-ketoester reductions ...............261-4 The -methyl-ketoesters synthesized and possible alcohol products .............................261-5 Previous methods for selective reduction of -methyl-ketoesters ..................................271-6 Synthesis of -methyl-ketoesters ....................................................................................281-7 Cofactor regeneration with G6PDH ...................................................................................281-8 Trifluoroacetyl al cohol derivatives ....................................................................................291-9 Trifluoroacetylation GC values for racemic alcohols ........................................................291-10 Reductions of ketoesters to alcohols with purified enzymes .............................................301-11 Anti 12,13a + syn 12,13a ...................................................................................................311-12 Anti 13a, from enzymatic reduction of ketone 11a ...........................................................321-13 Anti 12 13b + syn 1213b .................................................................................................331-14 Anti 13b from enzymatic reduction of ketone 11b ............................................................341-15 1-D TOCSY NMR of crude ( R ) and (S) MPA esters of anti 13a ......................................351-16 1-D TOCSY NMR of crude ( R ) and (S) MPA esters of anti 13b .....................................351-17 Nomenclature used for assignment of configuration .........................................................352-1 Hydrolysis product of various CW agents .........................................................................492-2 Chemical warfare hydrolysis pr oducts vs. biotinylated analog .........................................492-3 Synthesis of biotinylated CW agent hydrolysis product ....................................................502-4 Template strand, forward and reverse primers ..................................................................512-5 Procedure for SELEX ....................................................................................................... .512-6 Synthesis of acetylated PEG impurity ...............................................................................52 7

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2-7 Direct attachment of methyl phosphonic acid to mnps ....................................................523-1 Phosphate triester, phosphonate and -fluorinated phosphonates ....................................843-2 Phosphate and phosphonic acid p K a2 values ...................................................................843-3 Dihedral angle comparison fo r phosphate and phosphonic acids ......................................843-4 Bisphosphonate route to -fluorophosphonates .................................................................843-5 Savignacs routes to -fluorophosphonates .......................................................................853-6 Fluorination via nucleophili c and electrophilic sources ....................................................863-7 Methods for asymmetric -fluorophosphonate synthesis ..................................................873-8 Glycerol-3-phosphate analogue s synthesized by OHagan ...............................................883-9 OHagans Glycerol-1-phosphate dehydrogease assay .....................................................883-10 The diastereomers of 63 .....................................................................................................883-11 Glucose-6-phosphate analogs synthesized by Berkowitz ..................................................893-12 Oxidation of glucose-6-phos phate to 6-phosphogluconolactone .......................................893-13 Aldol reactions with class I and class II aldolase ..............................................................903-14 Rabbit muscle aldolase catalyzed reaction with nonnatural donor substrate 80 ...............903-15 Phosphonic acid carbohydrates synthesized by Fessner ....................................................913-16 Route to -fluorinated phosphonic acid carbohydrates .....................................................913-17 Fluorinated aldolas e substrate mimics via -ketophosphonates .......................................923-18 Fluorinated acetonide synthesis .........................................................................................9 33-19 Racemic standard, reduction failure and carbonate phosphonate ......................................943-20 Synthesis of carbonates and subsequent difluorination .....................................................943-21 Enone reductase route to -fluorovinylphosphonic acids ..................................................953-22 Synthesis of protected vinyl phosphonate 113 ...................................................................963-23 Dibromofluorophosponate ro ute to vinyl phosphonate 113 ..............................................973-24 Synthesis of vinyl phosphonate 104a ................................................................................97 8

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9 3-25 Synthesis of racemic phosphonate standard ......................................................................973-26 Screening of vinyl -fluorinated phosphonate with en-reductases ....................................983-27 Stoichiometric reduction of vinyl phosphonate with enzyme and NADPH ......................983-28 Fluorine elimination mechanism ........................................................................................983-29 Synthesis of unfluorinated vinyl phosphonate ...................................................................993-30 Inseparable ketal deprotection products .............................................................................993-31 Synthesis of unsaturated phosphonate ...............................................................................993-32 KMnO4 alkene oxidation and phosphonate deprotection ................................................1003-33 RuO4 catalyzed alkene oxidati on and phosphonate deprotection ....................................1003-34 Route to -hydroxyketone via initial phosphonate deprotection .....................................1003-35 Alkene oxidation of phosphonic acid 125 .......................................................................1013-36 Selective diol oxi dation of phosphonic acid 63 ...............................................................1013-37 Synthetic route to diol 63 .................................................................................................1023-38 Reduction of -fluoro-ketophosphonate 130 with purified enzymes ...........................1033-39 Oxidation of diol 63 followed by in situ aldolase reaction ..............................................1033-40 Future route to optically pure -fluorophosphonic acids .................................................1043-41 Future synthetic methods ................................................................................................. 1053-42 Proposed synthesis of optically pure -fluorophosphonate 63b ......................................1063-43 Proposed synthesis of -fluorophosphonic acid 63a .......................................................1063-44 Proposed synthesis of -fluorophosphonic acid carbohydrates .......................................1073-45 Decrease in starting mate rial over a 24 hour period ........................................................108

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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 APPLICATIONS FOR BIOCATALYSIS IN ORGANIC SYNTHESIS By G. Neil Stowe December 2008 Chair: Jon D. Stewart Major: Chemistry We synthesized a series of alpha-methyl-beta-ke toesters in two steps. These ketoesters were then reduced to the co rresponding alpha-methyl-beta-hy droxyesters by our library of purified ketoreductases from Bakers Yeast and Ba kers Yeast whole cells. These steps allowed us to probe the reduction capabilities and specifi cities of our purified ketoreductases and also demonstrated that they yield far greater enan tioselectivities in comparison to whole cells. A phosphonic acid analog of the highly toxic chemical warfare agents Sarin, Soman and VX was synthesized in four steps. This analog wa s tested as a target against DNA aptamers as a part of a program to develop a rapid test for e xposure to these nerve agents in the field. Unfortunately, no aptamers bound to the target. We chemoenzymatically synthesized alpha-f luoro-phosphonic acid carbohydrates in seven steps from noncarbohydrate precursors. The stereochemistry of the alpha-fluorine was controlled by selective reduction of an alpha-fluorovinylphosphonate using our library of alkene reductases. The alpha-fluorinated phosphonate al kane reduction product could then be used with aldolase and a suitable acceptor aldehyde to ma ke five and six membered alpha-fluorinated phosphonic acid carbohydrates of de fined stereochemistry without the need for protection / deprotection steps. 10

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CHAPTER 1 ENZYMATIC ROUTE FOR THE SYNTHESIS OF CHIRAL BUILDING BLOCKS FROM ALPHA-METHYL-BETA-KETOESTERS Introduction Selective reductions of -ketoesters can result in valuab le chiral building blocks for organic synthesis. These reductions, which control the stereochemistry of an alpha substituent in addition to the -alcohol, can be accomplished using Bake rs Yeast whole cells that can be purchased at any supermarket (Figure 1-1).1 However, multiple reductases with potentially different reduction products are present in whole yeast cells; this can significantly decrease the optical purity of the reduced product.2-5 In 2004, Kaluzna et al.5 made a library of 18 purified -ketoreductases from Bakers yeast which were expressed as fusi on proteins with glutathione S -transferase (GST) to allow for simple purification. The library of purified reductase s was screened against a variety of ketoester substrates to yield chiral -substituted-hydroxyesters (Figure 1-2).5 These reductases were overexpressed in E. coli whole cells, allowing for gram scale, selective reductions of -chloro-ketoesters without the need for expensive cofactor regeneration systems. The resulting chiral -chloro-hydroxyesters were then used to efficiently synthesize both anti podes of the Taxol side chain 9a b6 and (-)-Bestatin 107 (Figure 1-3). We wanted to further determine the influence of the substituent at the -position by comparing electronic versus steric environm ents while also expanding our knowledge of substrates for our enzyme library. So, -methyl-ketoesters 11ad were synthesized and screened (Figure 1-4); these -ketoesters are similar in size bu t different electronically to the previously screened -chloro-ketoesters 6ad .8 11

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Previous methods for asymmetric synthesis of -methyl-hydroxyesters employed biocatalytic reduction of -methyl-ketoesters9 in addition to ring opening of optically pure epoxides with Grignard reagents10 and aldol reactions with ch iral oxazoborolidinone reagents11 (Figure 1-5). These approaches were limited: the biocatalytic method offered a small quantity of reductases which gave a limited number of ch iral products while chemical methods required chiral starting materials or reagents. We thought that our lib rary of reductases could prove superior to these methods because we had previously shown with -chloroesters 6ad that multiple chiral products could be made from racemic starting materials. Thus, if the -chlorine of the previously screened -chloro-ketoesters was seen by our enzymes as only a steric entity, then we hypot hesized that substrate acceptance and product stereochemistry should be similar for the -chloro and -methyl compounds. However, if our enzymes could discern between the different electr onic environments of the sterically similar -substituents, then we thought that substrate acceptance and product stereochemistry could differ significantly. We also wanted to compar e stereochemistry of th e alcohol products of nonsubstituted -ketoesters 3ab against the -methyl-ketoesters. Finally, -methyl-ketoesters 11ad were tested for reduction with Bakers yeast whole cells to determine if our purified reductases yield bette r reduction product selectivity in addition to comparison of whole cell substrate acceptance. Ketoester Synthesis The -methyl-ketoesters were made using a two step process. First, ethyl propionate was deprotonated by lithium diisopropylamide (LDA) at -78o C followed by addition of the appropriate aldehyde at the same temperature to yield alcohols 12ad and 13ad .12 The alcohols were oxidized to ketoesters 11ad via standard Swern conditions or PCC (Figure 16).12,13 12

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Screening The -methyl-ketoesters 11ad were screened against our library of purified reductases and whole yeast cells. Screenings with pur ified reductases were c onducted in phosphate (KPi) buffer and used NADPH cofactor as the hydride source. The cofactor was regenerated using a glucose-6-phosphate (G6P) / glucose-6-phosphate dehyrogenase (G6PDH) couple (Figure 1-7).5 Whole cell screening of ketoesters 11a d incubated Fleischmans yeast with shaking in KPi buffer with addition of sucrose as needed. Gas chromatography (GC) was used to monitor reaction progress and selectivity. Aliquots of the crude reaction mixture were monitored by ach iral GC in order to follow reaction progress. The stereoselectivity of each reaction was determ ined by chiral GC using trifluoroacetylated derivatives 19ad and 20ad of the possible alcohol products 12ad and 13ad (Figures 1-8, 1-9). Results and Discussion The screenings found that only ketoesters 11a (R = Et) and 11b (R = n-Pr) were substrates for our purified reductases. Both ketoesters were reduced by enzymes encoded by yeast genes YDR541c, YGL039w, YAL060w and YGL157w. Chiral GC analysis of the trifluoroacetylated derivatives found only one of four possible alco hol products was formed from reduction of both ketoesters. NMR analysis of the -methoxy-phenylacetic (MPA) esters of the alcohol showed both reductions yielded -methyl-hydroxy esters with 2( S), 3( S) configurations, corresponding to anti 13a and anti 13b Whole yeast cells also accepted only 11a and 11b as substrates, but gave a 50:50 enantiomeric mixture of anti 12a b or anti 13ab as the major products. (Table 1-1). The -chloro-ketoesters 6ad proved far better substrates for our purified enzymes than the -methyl compounds; both 11a (R = Et, -methyl) and 11b (R = n-Pr, -methyl) were accepted by four purified enzymes while 6a (R = Et, -chloro) and 6b (R = n-Pr, -chloro) were 13

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accepted by 14 and 7 purified enzymes, respectively. However, the major product stereochemistry with respect to the al cohol remained the same for unsubstituted, -chloro and -methyl-ketoesters. Thus, the hydride was delivere d predominately to the same face of the ketone. The -substituent of the major product was also in the same position for both -chloro and -methyl-ketoesters, showing that our purified enzymes preferred to accept the same enantiomer of starting material. (F igure 1-10, Table 1-1, Table 1-2). From a synthetic perspective, our results from reduction of -methyl-ketoesters 11a b with purified enzymes were succes sful since we could produce an optically pure product from racemic starting material. However, our method would prove more versat ile if more than one chiral product could be made. The generalizations made with respect to reduction of unsubstituted, -chloro and methyl-ketoesters with our purified enzymes could be made with whole yeast cells in some cases. For example, -methyl 11a (R = Et) gave a 50 : 50 ratio of anti enantiomeric products in 92% de which was similar to the 38 : 50 anti enantiomeric ratio obtained for -chloro 6a (R = Et). Whole cells were more diastereoselective for reduction of -methyl 11b (R = Pr) than -chloro 6b but 6b still gave a 53 : 36 enantiomeric mixture of anti alcohols as major products. However, these generalizations did not alwa ys hold true. For example, reduction of unsubstituted -ketoesters 3a and 3b was selective, especially for 3b but the hydride was added from the opposite face of the ketone during whol e cell reduction when the alpha substituent was not present. The results of purified enzyme versus whole cell reductions were not surprising since multiple reductases are present in whole cells whic h often give different products. In fact, during this project we discovered fr om whole cell screening of -methyl-ketoesters 11ab that 14

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additional reductases, which could lead to addi tional optically pure produc ts, are present in the yeast genome. These reductases can be is olated and purified via GST fusion protein methodology in the future. However, our current library has proven an excellent method for the production of valuable chiral products from readily or commercially available, racemic starting materials. Experimental Procedures Materials All organic chemicals, G6P and G6PDH were purchased from Sigma-Aldrich and used without further purification. Diis opropylamine and triethylamine (NEt3) were freshly distilled from CaH2 before use. Dimethylsulfoxide (DSMO) was dried overnight over molecular sieves and then freshly distilled before use. n-Butyllithium concentration was determined by titration with 2,5-dimethoxybenzyl alcohol. THF, ether and CH2Cl2 were degassed in 20 L drums and passed through two sequential pur ification columns ( activated alumina) under a positive argon using the GlassContour system (GlassContour, In c.). Thin Layer Chromatography (TLC) was performed on Merck TLC glass sheets with visu alization by UV light or staining using potassium permanganate or vanillin. 1H (300) and 13C NMR (75 MHz) spectra we re recorded on a Varian Mercury 300 spectrometer. Chemical shifts ( ) for 1H and 13C NMR are given in parts per million (ppm) relative to TMS and referenced relative to residual protonated solvent (CHCl3: H 7.27 ppm, C 77.00 ppm or C6D6: H 7.16 ppm, C 128.39). Some carbon signals were isochronous for racemic alcohols and -methyl-ketoesters. Compounds were separated via GC with an HP 5890 Series II Gas Chromatogr aph equipped with an achiral DB-17 column and Chirasil-Dex CB or Chirasil Beta-Dex columns (0.25 mm x 25 m x 0.25 m thickness). GC / MS used an HP 5890 Series II Gas Chromatograph equipped with an achiral DB-17 column (0.25 mm x 25 m x 0.25 m thickness). 15

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GC short runs were 60o C for 2 minutes, 10o C per minute ramp to 180o C followed by 10 minutes at 180o C. GC long runs were 60o C for 2 minutes, 1o C per minute ramp to 150o C, 10o C per minute ramp to 180o C followed by 10 minutes at 180o C. GC / MS runs were 60o C for two minutes, 10o C per minute ramp to 250o C followed by 10 minutes at 250o C. Synthesis Titration of n-Butyllithium. An oven dried round bottom flask was ch arged with 2,5-dimethoxybenzylalcohol (0.225 g / 1.34 mmol) and 8 mL of dry THF. n-Butyllithium (583 L) was then added dropwise at room temperature until a dark brown color pers isted. At this point, ju st over 1 equivalent of nButyllithium had been added, and the concentratio n of the solution was determined to be 2.3 M. General Procedure for synthesis of alcohols 7a d and 8ad A round bottom flask was flame dried while pur ging with Argon, cooled, and charged with dry tetrahydrofuran (THF) (20 mL) and diisopropylamine (3.1 mL 22.1 mmol), then cooled to -78o C. n-Butyllithium ( n-BuLi) (20.1 mL, 22.1 mmol) was then added dropwise at -78o C and the resulting solution stirred at the same temperature for 30 minutes. Ethyl propionate (2.1 mL, 20.3 mmol) in THF (5 mL) was then ad ded dropwise to the so lution of LDA at -78o C and the resulting solution stirred at the same te mperature for one hour. The appropriate aldehyde (22.4 mmol) in THF (5 mL) was then added dropwise at -78o C and the resulting solution stirred at -78o C for 30 minutes before the addition of 8 mL of saturated ammonium chloride. After warming to room temperature the mixture was pour ed into 150 mL of et her and washed with 10 mL of saturated sodium chloride (NaCl). The organic layer was dried with magnesium sulfate (MgSO4) and the solvent removed unde r reduced pressure to give the crude product which was purified by flash chromatography. 16

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Purified by flash chromatography (6:1 Hexane s : EtOAc) to give the pure compound as a slightly yellow oil in 75% yield as a 55 : 45 mixture of diastereomers.10 1H NMR (CDCl3): 0.97 (m, 6H). 1.17 (d, 3H, J = 7.1 Hz ), 1.15 (d, 3H, J = 7.4 Hz ), 1.27 (t, 6H, J = 7.1 Hz), 1.54 (m, 4H), 2.52 (m, 2H), 2.58 (br d, 2H J = 7.0 Hz), 3.58 (m, 1H), 3.81 (m, 1H), 4.17 (q, 4H, J = 7.1 Hz). 13C NMR (CDCl3): 10.01, 10.65, 10.89, 14.44, 14.68, 26.97, 27.83, 44.12, 45.05, 60.77, 60.80, 73.41, 74.92, 176.35, 176.58. FT-IR: (neat) 3500, 2954, 1728, 1182. MS: 145 (M-15, 0.5%), 131 (47.0%), 115 (25.6%), 10 2 (89%), 85 (66%) 74 (100%), 57 (48.3%). OEt CH3 OH O syn 12 13b + anti 12 13b Purified by flash chromatography (8:1 Hexane s : EtOAc) to give the pure compound as a slightly yellow oil in 70% yield as a 53 : 47 mixture of diastereomers.11 1H NMR (CDCl3): 0.92 (t, 6H J = 7.4 Hz), 1.17 (d, 3H, J = 7.4 Hz), 1.23 (d, 3H, J = 7.1 Hz ), 1.27 (t, 6H, J = 7.1 Hz), 1.45 (m, 8H), 2.45 (m, 2H), 2.55 (br d, J = 7.0 Hz), 3.59 (m, 1H), 3.90 (m, 1H), 4.17 (q, 4H, J = 7.1 Hz). 13C NMR (CDCl3): 10.97, 14.20, 14.31, 14.49, 14.62, 18.95, 19.86, 36.22, 37.10, 44.59, 45.56, 60.71, 60.82, 71.64, 73.30, 176.49, 176.76. FT-IR: (neat) 3500, 2960, 1732, 1187. MS: 174 (m/z, 0.2%), 159 (1.9%), 131 (26.2%), 102 (100%), 74 (90.5%), 57 (31.8%). 17

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Purified by flash chromatography (10:1 Hexanes : EtOAc) to give the pure compound as a slightly yellow oil in 60% yield as a 50 : 50 mixture of diastereomers.14 1H NMR (CDCl3): 1.01 (d, 3H, J = 7.1 Hz), 1.14(d, 3H, J = 7.1 Hz), 1.21 (t, 3H, J = 7.2 Hz), 1.25 (t, 3H, J = 7.2 m, Hz), 2.75 (m, 2H), 3.05 (br s, 2H), 4.13 (q, 2H, J = 7.1 Hz), 4.19 (q, 2H, J = 7.1 Hz), 4.65 ( 1H), 5.05 (m, 1H), 7.35 (m, 10H). 13C NMR (CDCl3): 11.08, 14.21, 14.29, 14.63, 46.63, 47.32, 60.87, 60.93, 73.89, 76.49, 126.19, 126.83, 127.61, 128.15, 128.36, 128.60, 141.66, 141.78. FT-IR: (neat) 3450, 3063, 1716. MS: 208 (m/z, 5.3%), 193 (0.6%), 163 (3.7%), 133 (24.7%), 102 (100%), 74 (70.0%), 57 (17.5%). Purified by flash chromatography (10:1 Hexanes : EtOAc) to give the pure compound as a slightly yellow oil in 50% yield as a 51 : 49 mixture of diastereomers. 1H NMR (CDCl3): 1.27 (m, 12H), 2.65 (m, 4H), 2.79 (m, 4H), 3.90 (m, 1H), 4.18 (m13C NMR (CDC ide pwise 2Cl2 (5 mL) at the same temperature. After C 5H), 7.25 m, (10H). l3): 11.20, 14.40, 14.45, 14.62, 40.60, 41.48, 43.82, 44.54, 60.83, 60.93, 72.93, 74.68, 126.79, 128.77, 128.81, 129.53, 129.67, 138.32, 138.40, 176.24. FT-IR: (neat) 3490, 2938, 1731. MS: 204 (M 18, 31.2%), 159 (14.9 %), 131 (100%), 85 (72.6%), 57 (32.0%). General Procedure for the synthesis of ketones 11ad Swern Oxidation: An oven dried round bottom flask was charged with dimethylsulfox (DMSO) (1.27 mL, 17.9 mmol) in CH2Cl2 (20 mL) and cooled to -60o C before the dro addition of oxalyl chloride (1.42 mL, 16.5 mmol) in CH complete addition, the solution was stirred at -60o C for 10 minutes before dropwise addition of alcohol (14.3 mmol) in 5 mL of CH2Cl2 at -60o C. The solution was stirred at -60o 18

PAGE 19

for 20 minutes after complete alcohol additi on and then quenched by dropwise addition of triethylamine (NEt3) at the same temperature. The resu lting solution was allowed to warm to room temperature over a one hour period before the removal of CH2Cl2 under reduced pressure. The resulting residue was dissolved in ether (25 mL), washed with H2O (5 mL) and brine (5 and the organic layer dried with MgSO4. The solvent was removed under reduced pressure to give the crude ketone which was purified by flash chromatography. Pyridinium chlorochromate (PCC) oxidation: A round bottom flask was charged with 50 mL of anhydrous CH2Cl2 and PCC (3.93 g, 18.2 mmol). The alcohol (9.1 mmol) in 5 mL CH2Cl2 was then added dropwise to the solution (The color of the so mL) lution rapidly changed from brigh ck t orange to brown). The solution was then stirred for four hours at room temperature before the addition of 50 mL of anhydrous ether. The supernatant was then separated from the bla precipitate and the precipitate was washed with th ree portions of ether (3 x 10 mL). The solvent was removed under reduced pressure, and th e product purified by flash chromatography. Prepared by Swern oxidation in 70% yield as a colo rless oil after flash chromatography (8 : 1 Hexanes : EtOAc).12 1H NMR (CDCl3): 1.08 (t, 3H J = 7.3 Hz) 1.26 (t, 3H, J = 7.2 Hz), 1.36 (d, 3H, J = 7.2 Hz), 2.58 (m, 2H), 3.57 (q, 1H J = 7.2 Hz), 4.25 (q, 2H, J = 7.2 Hz). 13C NMR (CDCl3): 12.3, 14.1, 35.6, 51.0, 61.3, 169.9, 206.5. FT-IR: (neat) 2970, 1741, 1715. MS: 158 (m/z, 3.6%), 129 (7.9%), 113 (11.7 %), 102 (37.8%), 74 (28.3%), 57 (100%). 19

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Prepared by Swern oxidation in 61 % yield as a colorless oil afte r flash chromatography (8 : 1 Hexanes : EtOAc).15 1H NMR (CDCl3): 0.91 (t, 3H, J = 7.3 Hz), 1.27 (t, 3H J = 7.2 Hz), 1.34 (d, 3H, J = 7.2 Hz), 1.61 (m, 2H), 2.55 (m, 2H), 3.50 (q, 1H J = 7.2 Hz) 4.20 (q, 2H J = 7.0 Hz). 13C NMR (CDCl3): 12.6, 13.9, 16.8, 43.1, 52.6, 52.8, 61.2, 170.5, 205.8. FT-IR: (neat) 2965, 1743, 1716. MS: 172 (m/z, 3.4%), 143 (1.3%), 127 (8.8%), 102 (32.0%), 74 (100%), 57 (12.5%). Prepared by PCC oxidation in 72% yield as a co lorless oi after flash chromatography (12 : 1 Hexanes : EtOAc).16 1H NMR (CDCl3): 1.18 (t, 3H J = 7.2 Hz) 1.50 (d, 3H J = 7.0 Hz), 4.16 (q, 2H J = 7.2 Hz), 4.40 (q, 1H, J = 7.2 Hz), 7.45 (m, 2H), 7.59 (m, 1H), 8.0 (m, 2H). 13C NMR (CDCl3): 13.9, 14.2, 48.6, 61.6, 128.8, 128.9, 133.7, 136.1, 171.1, 196.2. FT-IR: (neat) 2985, 1744, 1684. MS: 206 (m/z, 9.9%), 161 (2.5%), 133 (0.8%), 102 (100%), 77 (36.4%). Prepared by PCC oxidation in 50% yield as a colorless oil after flash chromatography. 17 1H NMR (CDCl3): 1.26 (t, 3H J = 7.2 Hz) 1.32 (d, 3H J = 7.2 Hz), 3.62 (q, 1H J = 7.2 Hz), 3.82 (s, 2H), 4.19 (q, 2H J = 7.2 Hz), 7.25 (m, 5H). 13C NMR (CDCl3): 12.9, 14.2, 48.7, 52.0, 61.5, 127.7, 128.8, 129.7, 133.6, 170.5, 203.4. FT-IR: (neat) 2985, 1730, 1700. MS: 220 (m/z, 19.9%), 174 (6.9%), 129 (44.3%), 102 (16.1%), 91 (100%), 74 (10.4%). General procedure for the synthe sis of optically pure alcohols anti 8a and anti 8b : 20

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A round bottom flask was charged with NADP+ (7 mg), glucose-6-phosphate (86 mg), rified reduc t Glucose-6-phosphate dehydrogenase (50 g), -methyl-ketoester (95 L) and pu tase (2.2 mL) in 6 mL of KPi buffer. The flask was then incubated with gentle shaking a 30 oC for 36 hours. The reaction mixture was then extracted with ether (3 x 15 mL), the combined organics were dried with MgSO4, and the solvent removed un der reduced pressure to give the crude product as an oil. Purification by flash chromatography (12 : 1 Hexanes : EtOAc) gave the pure product as a slightly yellow oil in 80% yield.18,19 1H NMR (CDCl3): J = 7.4 Hz), 1.20 ( Cl3): 0.96 (t, 3H d, 3H, J = 7.1 Hz), 1.26 (t, 3H J = 7.1 Hz), 1.51 (m, 2H), 2.50 (dq, 1H J1 = 7.0 Hz, J2 = 7.0 Hz), 2.65 (br d, 1H, J = 7.0 Hz), 3.58 (m, 1H), 4.17 (q, 2H J = 7.1 Hz). 13C NMR (CD 10.02, 14.38, 14.53, 27.77, 45.00, 60.73, 74.85, 176.30. FT-IR: (neat) 3500, 2969, 1730, 1185. [ ]D = +3o (C = 0.3, CHCl3). MS was the same as 1213a. Purification by flash chromatography (12 : 1 Hexanes : EtOAc) gave the pure product as a slightly yellow oil in 80% yield.11 1H NMR (CDCl3): 0.90 (t, 3H J = 7.1 Hz), 1.20 (d, 3H, J = 7.1 Hz), 1.24 (t, 3H, J = 7.1 Hz), 1.50 (m, 4H), 2.45 (dq, 1H, J1 = 7.0 Hz, J2 = 7.0 Hz), 2.59 (br d, 1H, J = 7.0 Hz), 3.59 (m, 1H), 4.15 (q, 2H, J = 7.1 Hz). 13C NMR (CDCl3): 14.17, 14.37, 14.44, 18.93, 37.05, 45.48, 60.71, 73.25, 176.31. FT-IR: (neat) 3500, 2964, 1733, 1186. [ ]D = +4o (C = 0.2, CHCl3). MS was the same as 1213b 21

PAGE 22

General Procedure for MPA al cohol derivitization: An NMR tube was charged with one equivalent of alcohol, CDCl3 (1 mL), and one : 1), followed by addition of 1.5 equivalents of dic ( S) for both alcohols.20,21 We determined relative configuration for both comp Pr, Ph, Bn), H3 is shifted ture data ( 3.58 observed, lit. value: 3.58 ( anti ), 3.84 ( syn )) as ical shift for H3 ( 3.59 from purified enzyme reduction vs. 3.59 and 3.90 from nonse the ed t equivalent of a mixture of ( R) -MPA and ( S)-MPA (2 yclohexylcarbodiimide (D CC) and 0.5 equivalents of N,N-dimethylaminopyridine (DMAP).8 The crude reaction was monitored by 1H and 1D TOCSY NMR. The chemical shift differences, listed as the change in chemical shift between (R ) and (S) esters ( R ,S), are summarized in Table 3. The chemical shift differences between ( R ) and (S) MPA esters allowed us to assign absolute stereochemistry ounds based on vicinal coupling constants (3JHH) for the H2 signal (7.0.5 for anti and 2.5-3.0 for syn )22 and comparison of the H3 chemical shift for anti and syn isomers with literature data. For these -hydroxyketones (R = Me, Et, iupfield for anti isomers.11,14,19,23 For -methyl-hydroxyester 13a (R = Et), 3JHH of 7.0 Hz for H2 in addition to an H3 chemical shift identical with litera19 allowed us to conclude anti relative stereochemistry, assi gning the full conf iguration 2( S) 3( S ). For -methyl-hydroxyester 13b (R = n-Pr), 3JHH of 7.0 Hz for H2 in addition to an upfield chem lective aldol reaction) allowed us to conclude anti relative stereochemistry, assigning full configuration as 2( S), 3( S). This assignment does not agree with Hena et al., who publish chemical shifts of H3 for anti and syn isomers as 3.7 and 3.5, respectively.11 However, Hena e al. did not report any coupling constants for H2 to support their assignment. Thus, we believe the 22

PAGE 23

configuration was not assigned correctly since other -methyl-hydroxyesters in this series (R = Me, Et, i -Pr, Ph, Bn) assigned the chemical shift of H3 for anti compounds upfield from syn And, our coupling constants support anti relative stereochemistry, while Hena et al. did not report this data. General procedure for reductions of -methyl-ketoesters using Bakers Yeast whole cells: Fleishmans yeast (10 g) was added to a solution of 45 mL of tap water in a baffled 0o C for en at hed ere Erlenmeyer flask followed by the addition of 15 g of sucrose. The flask was then shaken at 3 one hour followed by the addition of 100 L of substrate. The reaction was then shak 30o C for 5 days with addition of sucrose as needed. Aliquots of the reaction mixture were analyzed via chiral and achiral GC. After five days, the reaction mixture was filtered over Celite and the aqueous layer extracted with EtOAc (3 x 75 mL). The combined organics were was with brine (1 x 8 mL), dried with MgSO4 and the solvent removed under reduced pressure to give the crude compound as a yellow oil which was purified by filtration through a short plug of silica to give the pure compound. For each com pound, the major products using a long run w one of two possible peaks on ach iral GC and two of four po ssible peaks on chiral GC. Purified by a short flash chromatography colu mn to give a slightly yellow oil in 60% yield.24 Alcohols were analyzed as trifluoroacetyla ted derivatives anti -20a and syn 1920a. The p t 19 roducts from NaBH4 reduction were separated using an achiral DB-17 column (peaks a 11.14 and 12.50 min, long run) and a chiral Dex column (peaks at 26.39, 27.66, 28.23 and 29.08 min, long run). Trifluoroacetylated derivatives of the reaction products showed peaks at 11.14 23

PAGE 24

and 12.50 min (long run) (1 : 21.5, 91% de, l ong run) on achiral GC and 27.70, 28.38 and 29.11 min (11.0 : 1.0 : 11.0, 0% ee, long run) on ch iral GC. The majo r products are the anti enantiomers since the peak at 27.70 min corresponds to the produc t obtained from purified enzyme reduction. Our results al so agree with the literature. For this compound, alcohols anti 12 13b and syn 1213b were not derivatized. The products from NaBH4 reduction were separated using an achiral DB-17 column (peaks at 22.51 and 24.14 min, long run) and a chiral Dex column (peaks at 53.56, 53.91, 54.58 and 54.90 min, long run). After reaction, produc t appeared on achiral GC at 22.41 min (>99% de, long run) and on chiral GC at 53.66 and 54.32 (1.0 : 1.0, 0% ee, long run). These are the anti enantiomers since the peak at 53.66 min corresponds to th e alcohol from purified enzyme reduction. 24

PAGE 25

Figure 1-1. Selective reduction us ing Bakers yeast whole cells Figure 1-2. Substrates sc reened by Kaluzna et al. 25

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Figure 1-3. Synthetic applications of chiral products from -chloro-ketoester reductions Figure 1-4. The -methyl-ketoesters synthesized a nd possible alcohol products 26

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Figure 1-5. Previous methods for selective reduction of -methyl-ketoesters 27

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Figure 1-6. Synthesis of -methyl-ketoesters Figure 1-7. Cofactor regeneration with G6PDH 28

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Figure 1-8. Trifluoroacet yl alcohol derivatives Figure 1-9. Trifluoroa cetylation GC values for racemic alcohols 29

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Figure 1-10. Reductions of ketoesters to alcohols with purified enzymes 30

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Figure 1-11. Anti 12,13a + syn 12,13a 31

PAGE 32

Figure 1-12. Anti 13a, from enzymatic reduction of ketone 11a 32

PAGE 33

Figure 1-13. Anti 12 13b + syn 1213b 33

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Figure 1-14. Anti 13b from enzymatic reduction of ketone 11b 34

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Figure 1-15. 1-D TOCSY NMR of crude ( R ) and (S) MPA esters of anti 13a Figure 1-16. 1-D TOCSY NMR of crude ( R ) and (S) MPA esters of anti 13b Figure 1-17. Nomenclature used for assignment of configuration 35

PAGE 36

Table 1-1. Comparison of -methyl and -chloro-ketoester reductions Yeast GenePlasmid anti 12a : syn 12a : anti 7a : syn 7a : anti 12b : syn 12b : anti 7b : syn 7b : anti 13a : syn 13a anti8a : syn8a :anti 13b : syn 13b anti8b : syn8b : YJR096wpIK9--------YDL124wpIK8--<1 : 19 : 7 : 73----YBR149wpIK12--<1 : >99 : <1 : <1----YOR120wpIK30--<1 : >99 : <1 : <1----YHR104wpIK29--------YDR368wpIK4--<1 : >99 : <1 : <1----YGL185cpAKS1--<1 : 40 : <1 : 60----YNL274cpIK13--<1 : 40 : <1 : 60----YPL275wpIK18--------YPL113cpIK15--22 : 78 : <1 : <1----YLR070cpIK23--------YAL060wpTM3<1 : <1 : >99 : <1<1 : <1 : 95 : 5 <1 : <1 : >99 : <1<1 : <1 : 98 : 2 YGL157wpIK7<1 : <1 : >99 : <1<1 : <1 : >99 : <1<1 : <1 : >99 : <1<1 : <1 : >99 : <1 YDR541cpIK5<1 : <1 : >99 : <1 ---<1 : <1 : >99 : <1--YGL039wpIK6<1 : <1 : >99 : <1<1 : <1 : 90 : 10<1 : <1 : >99 : <1<1 : <1 : 97 : 3 YNL331cpIK11--<1 : <1 : <1 : >99--<1 : <1 : <1 : >99 YCR107wpIK10--<1 : <1 : <1 : >99--<1 : <1 : <1 : >99 YOL151wpIK3--------Yeast Cells---50 : <1 : 50 : <150 : 7 : 38 : 550 : <1 : 50 : <112 : 4 : 77 : 7 a <20% conversion after 24 hours8 36

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Table 1-2. Comparison of reduction products for -methyl and unsubstituted -ketoesters Yeast GenePlasmid anti 12a : syn 12a : 4a : 5a anti 12b : syn 12b : 4b : 5b anti 13a : syn 13a anti 13b : syn 13b YJR096wpIK9--------YDL124wpIK8--------YBR149wpIK12--------YOR120wpIK30--<1 : >99 --<1 : >99 YHR104wpIK29--------YDR368wpIK4--<1 : >99 --<1 : >99 YGL185cpAKS1--------YNL274cpIK13--------YPL275wpIK18--------YPL113cpIK15--<1 : >99 --16 : 84 YLR070cpIK23--------YAL060wpTM3<1 : <1 : >99 : <1<1 : >99 <1 : <1 : >99 : <1<1 : >99 YGL157wpIK7<1 : <1 : >99 : <1<1 : >99 <1 : <1 : >99 : <1<1 : >99 YDR541cpIK5<1 : <1 : >99 : <1<1 : >99 <1 : <1 : >99 : <1<1 : >99 YGL039wpIK6<1 : <1 : >99 : <1<1 : >99 <1 : <1 : >99 : <1<1 : >99 YNL331cpIK11------23 : 77 YCR107wpIK10--38 : 62 ----YOL151wpIK3--<1 : >99 --<1 : >99 Yeast Cells---50 : <1 : 50 : <170 : 30 50 : <1 : 50 : <1>99 : <1 a <20% conversion after 24 hours1,5 bArbitrary assignment, syn product absolute configuration was not assigned Table 1-3. Chemical shift difference between ( R ) and (S) MPA esters ( R ,S) CompounddH2'dH1'dH1a'dH2dHMedH3dH4adH4bdH5adH5bdHAlcohol Conf anti 13a nma0.280.240.100.170-0.1-0.13nabnab-0.31( S) anti13b nm 0.210.260.110.190-0.1-0.13-0.36-0.36-0.21( S) aNot measured bNot applicable 37

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CHAPTER 2 SYNTHESIS OF AN APTAMER TARGET FOR RAPID SCREENING OF EXPOSURE TO CHEMICAL WARFARE AGENTS Introduction Chemical warfare (CW) began during World War I with the deployment of toxic gases such as phosgene, chlorine and dich loroethylsulfide (mustard gas).25 More recently, CW was used by terrorists during the release of Sa rin gas in Matsumoto City and Tokyo, Japan.26 CW agents are toxic at low levels: patients admitte d to the Nippon Medical Center after exposure to Sarin gas in the Tokyo incident had ingested es timated levels of 0.13 0.25 mg for a comatose patient (who never recovered) to 16-32 g for patients who were sick but fully recovered.27 The risk of serious injury, or death, from exposur e to CW agents continues to the present day. Rapid, accurate detection of CW agents is of vital importance in order to minimize their potentially devastating effects. Detection of exposure to CW agents relies upon detection of their degradation products since the intact agent is rapidly hydrolyzed in vivo (Figure 2-1).28 Current methods for CW agent detection include GC / MS28, HPLC / MS29 or assays detecting inhibition of acetylcholine esterase (AChE).30 Both of these techniques have disadvantages: GC / MS28 or HPLC / MS29 require at least 24 hours after samp le acquisition, while AChE inhibition assays will not identify specific CW agents30 and suffer from false positive results due to AChE inhibition from unrelated chemicals. Clearly, a need persists for rapid and accurate detection of CW agents. Aptamers for CW Agent Detection Aptamers are single stranded (ss)DNA or RNA oligonucleotides that can bind to targets due to their tertiary structure.31 Aptamers are selected using an in vitro process called SELEX ( S ystematic E volution of L igands by EX ponential enrichment) which begins with a large random pool of oligonucleotides and, through repeated r ounds of selection against a target, identifies a 38

PAGE 39

small number of oligonucle otides that tightly bind.32-34 Aptamers can bind tightly to a wide range of targets ranging in size from live cancer cells ( Kd = 0.80 nM)35 to ethanolamine ( Kd = 6 nM).36,37 We set out to isolate DNA aptamers that could bind to 18, a biotinylated analog of CW agent hydrolysis product 17 (Figure 2-2). These aptamers coul d, in principle, be isolated since phosphonic acid 17 (n = 2) was detected at approximately 100 nM in the urine of a Japanese man one day after Sarin exposu re in Matsumoto City.38 Similar levels of 17 (135 25 nM) were found in the serum of victims 1.5 hours after hosp ital admittance in Sarin attacks in Tokyo and Matsumoto City.29 These people became ill, but their exposure was not fatal. We chose to biotinylate our analog to allow for easy separation of binding aptamers in solution using streptavidin coated magnetic nanoparticles.39 After isolation of the DNA aptamers, a rapid colorimetric test for nerve ag ent exposure in the fiel d would be developed by ADA Technologies using thei r proprietary technology. Target Synthesis Biotinylated phosphonic acid 18 was chemically synthesized in four steps starting from phosphonic dichloride 19 The dichloride was converted to the mixed phosphonate ester 20 by sequential treatment with benzyl alcohol and propylene glycol.40 Ester 20 was then oxidized to carboxylic acid 21 using TEMPO41 followed by attachment of polyethylene glycol (PEG) amine 22 with polymer bound EDC42 to form phosphonate 23. After this step, some of amine 22 remained as an inseparable impurity. The benzyl ester was removed using H2 / Pd43 to give phosphonic acid 18 plus amine impurity 22 (Figure 2-3). Aptamer Selection We used a 76 nucleotide ssDNA template with a 40 nucleotide random sequence surrounded by forward and reverse priming regi ons. The primer sequences were chosen 39

PAGE 40

according to Stoltenburg.36 The forward primer contained a fluorescent (FAM) tag at its 5 end, while the reverse primer had a biotin tag at the 5 position (Figure 2-4). The fluorescent tag allowed for detection of the DNA and biotin allowed for purification and separation of double stranded DNA using streptavidin co ated magnetic nanoparticles. For the initial selection round, biotinylated target 18 was mixed with streptavidin (SA) coated magnetic nanoparticles (mnps) a nd allowed to stand overnight at 4o C before addition of the template strand. The resulting solution was th en allowed to incubate at room temperature with gentle shaking for 30 minutes followe d by removal of the nonbinding oligonucleotide containing supernatant from the mnps. Bound DNA was eluted from the target by denaturation and the resulting ssDNA was amplified by PCR to give double stranded dsDNA containing a fluorescent tag and biotinylated tag on opposite strands. The ds DNA was then allowed to stand overnight with streptavidin coated mnps; ssD NA containing the fluorescent tag was eluted by denaturation to give the starting material for the second selection round. Selection rounds 2-5 followed the same procedure as the first round (Figure 2-5). Selection round 6 employed a counterselecti on step to determine if DNA was binding the mnps or PEG impurity 22 instead of target 18. The counterselection st ep used acetylated amine 23 as a mimic for PEG impurity 22 (Figure 2-6). The step was performed by binding acetylated amine 23 to streptavidin coated mnps as descri bed previously followed by incubation with isolated DNA from the selection process. As shown in Table 3, counterselection after round 6 resulted in a large decrease in fluorescence of the isolated DNA. Additionally, no DNA was isolated after PCR amplification. Results and Discussion A steady increase in fluorescence to blank s hould be observed after the initial selection round if DNA is tightly binding si nce larger quantities are recove red. We did not observe this 40

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steady increase, allowing us to conclude th at DNA was not specifica lly binding to target 18, but instead to the polyethyl ene glycol impurity. Generation of the PEG impurity could be a voided by attachment of the methyl phosphonic acid directly to the mnps, as employed by Strehlitz et. al. for their preparation of ethanolamine modified mnps.36 This process would requ ire incubation of phosphonic acid 24 with commercially available tosyl activated mnps (F igure 2-7). These phosphonic acid modified mnps could then be used for SELEX without any major modifications to our current procedure. Finally, the similarity of the methyl phosphonic acid and phosphate esters in the DNA backbone should be noted. This could prevent D NA from binding tightly using either procedure since it is possible that DNA can not tell the difference between itself and the target. If this similarity is too great, then the detection of hydrolysis pr oducts of CW agents with DNA aptamers is not possible. Experimental Procedures General Experimental All chemicals and dNTPs were purchased from Sigma Aldrich or Fish er and used without further purification. Solvents were purified as de scribed in the previous procedure. Biotinylated amine 22 was purchased from Pierce. Taq polymerase was purchased from New England Biolabs. All DNA was synthesized by IDT DNA Technologies. Fluores cence was measured using a Tecan Safire microplate read er with 384 well Corning plates. Chemical Synthesis 41

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An oven dried flask was equipped with a stir bar and charged with 20 mL of CH2Cl2, methyl phosphonic dichloride (8.8 mmol, 0.8 mL ) and triethylamine (17.6 mmol, 2.45 mL). Benzyl alcohol (8.8 mmol, 0.91 mL) was then adde d dropwise at room temperature so that a gentle reflux was maintained. The solution was stirred at room temperat ure for two hours before dropwise addition of propylene glycol (8.8 mmol, 0.64 mL) at room temperature. The reaction was then stirred overnight at room temperatur e. Triethylammonium salts were removed by filtration, the solvent was removed and the product purified by flash chromatography (EtOAc) to give the product (1.61 g, 75 % yield) as a colorless oil. 1H NMR: (CDCl3) 1.49 (d, 3H, J = 17.6 Hz), 1.82 (m, 2H), 3.72 (t, 2H, J = 5.7), 4.07 (m, 1H), 4.21 (m, 1H), 5.08 (d, 2H, J = 9.1 Hz), 7.4 (m, 5H); 31P NMR: (CDCl3) 33.82. Aqueous 15 % NaHCO3 (3 mL) was added to a solution of 20 (1.19 mmol, 0.29 g) in 25 mL of acetone and the solution was cooled to 0o C. Sodium bromide (0.24 mmol, 0.024 g) and TEMPO (0.024 mmol, 0.004 g) were then added, followed by the addition of trichloroisocyanuric acid (2.38 mmol, 0.55 g) over a 20 minute period. The reaction was then stirred at room temperature for two hours before the addition of 1.5 mL of isopropanol. The mixture was filtered over Celite, concentrated under vacuum, and 8 mL of saturated Na2CO3 was added. The aqueous phase was washed with EtOA c (2 x 20 mL), then acidified with 1 M HCl and extracted again with EtOAc (3 x 20 mL). The combined organic layers from the second extraction were dried with MgSO4 and concentrated under vacuum to yield the product as a semicrystalline solid (crude yield 0.26 g, 85 %) which was used without pur ification in the next 42

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step. 1H NMR: (CDCl3) 1.50 (d, 3H, J = 17.8 Hz), 2.68 (t, 2H, J = 6.12 Hz), 4.26 (m, 2H), 2.08 (dd, 2H, J = 3.9, 9 Hz), 7.36 (m, 5H). 31P NMR: (CDCl3) 36.87. 23 HN NH S O O 4N H O O 2 H N O O P O BnO Carboxylic acid 21 (0.06 mmol, 16 mg) and PEG bioti nylated linker (0. 06 mmol, 25 mg) were combined in an oven dried flask that contained 2 mL of CH2Cl2 and 2 mL of DMF. Polymer bound EDC (100 mg) was then added and the reaction was stirred overnight at room temperature. The solvents we re then evaporated and the cr ude product was purified by flash chromatography (5 : 1 EtOAc : MeOH) to give th e product (crude yield 0.025 g, 65 % yield) plus some of the PEG biotinylated li nker as an inseparable impurity. 1H NMR: (CD3OD) 1.26 (m, 3H), 1.44 (m, 2H), 1.52 (d, 3H, J = 15 Hz), 1.64 (m, 4H), 2.22 (t, 2H, J = 7.4 Hz), 2.56 (t, 2H, J = 6.0 Hz), 2.70 (m, 1H), 2.93 (m, dd, J = 5.1, 12.7 Hz), 3.20 (m, 2H), 3.37 (m, 10H), 3.53 (m, 5H), 3.62 (m, 11H), 4.28 (m, 3H), 4.5 (dd, 1H, J = 4.5, 8.1 Hz), 5.07 (dd, 2H J = 2.3, 8.5 Hz), 7.4 (m, 6H); 31P NMR: (CD3OD) 33.8. Biotinylated phosphonate 23 (0.025 g, 0.039 mmol) was added to a flask containing 15 mL of MeOH. A spatula tip full of Pd on Carbon wa s then added to the flask and the mixture was hydrogenated at room temperature under a balloon of hydrogen overnight. The mixture was then 43

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filtered over Celite and MeOH wa s removed under reduced pressure. The product was then lyophilized to give yellow cr ystals (0.022 g, 94 % yield). 1H NMR: (D2O) 1.10 (m, 6H), 1.30 (s, 1H), 1.50 (m, 5H), 1.75 (m, 2H), 1.95 (s, 1H), 2.15 (m, 1H), 2.42 (t, 2H, J = 5.7 Hz), 2.68 (s, 1H), 2.84 (s, 1H), 3.2 (m, 9H), 3.52 (m, 6H), 4.57 (s, 1H). 31P NMR: (D2O) 28.20. (+) ESI-MS: 584 (M + 16, impurity from oxidation of biotin sulfur to sulfoxide), 568 (m/z for biotinylated methyl phosphonic acid 18, most abundant compound), 418 (m/z for biotin linker impurity 22). PEG biotinylated linker (12 mg, 0.028 mmol), acetic anhydride (5.3 L, 0.056 mmol) and N,N-dimethylaminopyridine (1 small crystal) we re combined in an oven dried flask that contained 6 mL of CH2Cl2 at room temperature and stirred ov ernight at room temperature. The solvent was removed under reduced pressure and the resulting solid purified by flash chromatography (5 : 1 EtOAc : MeOH) to give 23 (6.4 mg, 50 % yield). 1H NMR: (CDCl3) 1.45 (m, 2H), 1.70 (m, 4 H), 1.95 (s, 3H), 2.25 (t, 2H, J = 6.9 Hz), 2.75 (m, 1H), 2.92 (dd, 1H, J = 5.1, 12.9 Hz), 3.15 (m, 1H), 3.44 (m, 4H), 3.57 (t, 4H, J = 5.1 Hz), 3.64 (s, 6H), 4.33 (m, 1H), 4.51 (m, 1H), 5.50 (s, 1H), 6.49 (s, 1H), 6.60 (s, 1H), 6.76 (s, 1H). PCR Amplification The reactants for each PCR round were 0.2 mM dNTPs, 1 M forward primer, 1 M reverse primer, 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl pH 8.8, 10 mM MgSO4, 0.1% Triton X-100. PCR conditions were 5 minutes at 94oC and 30 cycles of 1 min at 94oC, 1 min at 47oC, 1 min at 72oC, then 10 minutes at 72oC after the last cycle. 20 units of Taq polymerase (5 44

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U / L) were used for a 100 L PCR; 10 units were initially added via hot start methodology and 10 units were added after 15 cycles. Electrophoresis on a 2% agarose gel with Hae III phix174 markers were used to determine if the correct size of DNA was produced. Magnetic Nanoparticles Magnetic nanoparticles were synthe sized by Joshua Smith at the Un iversity of Florida. An aqueous mechanically stirre d solution of ammonium hydroxi de (2.5%), ferric chloride hexahydrate (0.5 M), ferrous chlo ride tetrahydrate (0.25 M) and HCl (0.33 M) was stirred for 10 minutes at 350 RPM. The iron ox ide nanoparticles were then wash ed with water three times and ethanol once. The MNPs were dispersed in an ethanol solution containing ~1.2% ammonium hydroxide at a final concentration of ~7.5 mg / mL to a final volume of ~6 mL. The magnetite core particles were coated with silica by adding 200 L tetraethylorthosilicate (TEOS) to the ethanolic solution and sonicating for 90 minutes at room temperature. Additional TEOS (10 L) was introduced and sonication was continued at room temperature for an additional 90 minutes to post-co at the nanoparticles. The sample was washed three times with ethanol. To introduce surface carboxyl groups, 80 L of carboxy-silane (N-(trimethoxysilylpropyl)ethylened iamine, triacetic acid trisodi um salt, 45% in water) was added to 1 mL of silica-coated magnetic na noparticles (10 mg / mL suspension in 10 mM phosphate-buffered saline, pH 7.4) and the reaction was vortexed for 4 hours at room temperature. The particles were then washed three times with 10 mM phosphate-buffered saline and stored at room temperature. To prepare for streptavidin coupling, a 1 mg sample of carboxyl-modified magnetic nanoparticles was washed three times with 250 L aliquots of 0.5 mM MES, pH 5.0. Protein immobilization was carried out by adding 50 L of a 20 mg / mL EDC solution to the washed 45

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nanoparticles in 250 L of 0.5 mM MES, pH, 5.0 and inc ubating at room temperature for 15 minutes before 100-300 g of streptavidin was added to th e reaction mixture. The suspension was incubated for 2-4 h with continuous vortexing. The streptavidin-coated nanoparticles were magnetically extracted and washed three times with 500 L aliquots of 20 mM Tris-Cl, 5 mM MgCl2, pH 8, then resuspended at a final concentr ation of 2 mg / mL in the same buffer and stored at 4oC. Round 1 of SELEX A 500 L eppendorf reaction tu be containing 3 x 10-18 mole of target biotinylated phosphonic acid (30 L from a 1 x 10-12 M stock solution), 20 L of streptavidin coated magnetic nanoparticles and 380 L of buffer was allowed to stand overnight at 4oC. 3 nanomoles of the template sequence was then added (70 L from a 1 g / L stock solution) and the reaction was allowed to incubate at room te mperature for 30 minutes with gentle shaking. (Before addition, DNA was denatured and renatured by heating to 80oC for 10 min and then cooling on ice for 10 minutes). The eppendorf tube was then attached to a magnet and allowed to stand for 5 minutes. The supernatant was removed, 200 L of fresh buffer was added, the tube was gently vortexed, attached to a magnet and allowed to stand for 5 minutes before removing the supernatant. This procedure was performed three total times. The DNA bound to the phosphonic aci d was eluted by adding 100 L of fresh binding buffer to the eppendorf tube, attaching the tube to a magnet, binding for 5 minutes and then heating to 94oC with shaking for 8 minutes followed by immediate removal of the supernatant while the solution was still hot. This procedure was performed two total times. After elution, DNA was precipitated with EtOH and 4 M NaCl (EtOH = 2.5 x total volume buffer used, 4 M NaCl = 0.025 x total volume buffer used), a nd allowed to stand overnight at -20oC. The solution 46

PAGE 47

was then microfuged for 20 minutes and the supern atant was removed to give the eluted DNA. The eluted DNA was PCR amplifie d using the conditions listed in the general experimental. After amplification, the ds DNA was purif ied and separated into ss DNA by adding 20 L of MNPs to the PCR vial and allowing to stand at 0oC overnight. The tube was then attached to a magnet, allowed to stand for 5 minut es and the supernatant removed. 100 L of fresh buffer was added, the tube was vortexed, attached to a magnet and allowed to stand for five minutes followed by removal of the supernatant. This procedure was performed three total times. After washing was complete, 100 L of fresh buffer was added, the t ube was attached to a magnet, bound for 5 minutes, heated to 94o C for 8 minutes while bound to a magnet with shaking and the supernatant was removed while still hot. This pr ocedure was performed two total times to give the purifed ss FAM labelled DNA in 200 total L of buffer. The presence of DNA was monitored by fluoresence and by visualization of the amplified DNA after gel electrophoresis. Rounds 2-5 of SELEX For subsequent selex rounds the entire P CR amplified and purified ss FAM labelled DNA from the previous round was used. For example, selex for round 2 contained 200 L of purified DNA solution, 250 L of buffer and 30 L of the phosphonic acid from a 1 x 10-12 M stock solution and 20 L of magnetic nano particles. Round 6 of Selex Plus Counterselection Step Round 6 introduced a counterse lection step to ensure that DNA was binding to the phosphonic acid and not to the magnetic nanoparticles or biotinylated polye thylene glycol linker impurity 1. The selex procedure was carri ed out as usual to give 200 L of solution after elution from the phosphonic acid. To this 200 L solution was added 50 L of a solution which contained 20 L of magnetic nanoparticles and 30 L of a 1 x 10-12 M solution of acetylated PEG linker (The acetylated PEG linker and ma gnetic nanoparticles were allowed to bind 47

PAGE 48

overnight at 0oC before addition). The entire solution was shaken gently for 30 minutes, the tube was attached to a magnet a nd the supernatant removed. 100 L of buffer was then added, the tube was gently vortexed, attached to a magnet and the supernatant was removed. The recovered solution was precipitated and subjected to PCR amp lification conditions as described previously. 48

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Figure 2-1. Hydrolysis pr oduct of various CW agents Figure 2-2. Chemical warfare hydrolysis products vs. biotinylated analog 49

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Figure 2-3. Synthesis of biotinyl ated CW agent hydrolysis product 50

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Template Strand 5-ATA CCA GCT TAT TCA ATT N40 AGA TAG TAA GTG CAA TCT-3 Forward Primer 5-FAM-ATA CCA GCT TAT TCA ATT-3 Reverse Primer 5-BIOTIN-AGA TTG CAC TTA CTA TCT-3 Figure 2-4. Template strand, forward and reverse primers Figure 2-5. Procedure for SELEX 51

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Figure 2-6. Synthesis of acetylated PEG impurity Figure 2-7. Direct atta chment of methyl phos phonic acid to mnps Table 2-1. Selection and count erselection fluorescence data Round Ratio of Fluorescence to Blank 1 320 2 11 3 267 4 11 5 21 6 173 6 + Counterselection 14 52

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CHAPTER 3 CHEMOENZYMATIC ROUTE TO FL UORINATED PHOSPHONIC ACID CARBOHYDRATES OF DEFI NED STEREOCHEMISTRY Introduction The -Fluorophosphonates Phosphate esters and anhydrides are widespread in biological systems. These compounds interact with enzyme binding sites through el ectrostatic, hydrogen bonding and dipole-dipole interactions.24 The frequent presence of phosphate es ters/anhydrides has fueled research to synthesize molecules that contain these useful functional groups. Unfortunately, phosphate esters/anhydrides are susceptible to hydrolysis in vivo by phosphatases, limiting their potential as successful drugs. Phosphonates, which differ from phosphates by methylene replacement of one bridging oxygen, have been envisioned as nonhydrolyzable mimics of phosphate esters/anhydrides. Blackburn et al.44 proposed that -fluorinated phosphonates might serve as superior phosphate analogs because of increased met hylene polarity (Figure 3-1). -Fluorinated phosphonic acids can also be considered as isopolar mi mics of phosphates due to the reduced p K a of the free hydroxyl groups (Figure 3-2).45 Other reasons postulated for the superiority of fluorinated phosphonates include increased br idging atom dihedral angle46 and the possibility for C-FH-X hydrogen bonding (Figure 3-3).47,48 Routes to -Monofluorophosphonates -Fluorophosphonates are made using vari ous techniques. Blackburn et al.49 and Prestwich et al.50 used the Horner-Wadsworth-Emmons (HWE) reaction of fluorinated bisphosphonate 36 to build vinyl -fluorophosphonates 37. The vinyl phosphonates can be converted to alkyl -fluorophosphonates by reduction with Pd / C at atmospheric pressure (Figure 3-4).50 53

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HWE,51 Peterson52 and alkylation53 reactions with dibromofluorophosphonate 40 were used by Savignac to make vinyl and alkyl -fluorophosphonates. Phosphonate 40 is the Arbuzov product of triethyl phosphite and tribromofluoromethane.54 Savignacs strategy generally relied upon double lithium halogen exchange using n-BuLi with in situ trimethylsilyl (TMS) protection/stabilization of the resulting lithiated anion before reaction with an aldehyde or alkyl halide (Figure 3-5). Electrophilic fluorination using N-fluorobenzenesulfonimide (NFSI) 52 or Selectfluor 53 has been used for the selective fluorin ation of phosphonates, phosphonoacetates and sulfonophosphonates. NFSI and Selectfluor55 are stable, solid, easily handled sources of fluorine that can be used without any special precau tions or special trai ning. McKenna et. al56 used Selectfluor to build -fluorophosphonoacetates while Wnuk et. al57 used Selectfluor to make fluorosulfones. The choice of counterion (Na+ or K+) can be important when optimizing electrophilic fluorinations. Finally, (diethylamino) su lfur trifluoride (DAST) 54 has been employed for the nucleophilic displacement of -hydroxyphosphonates by fluorine. DAST was used by Prestwich et al.58 for their synthesis of analogs of lysophospha tidic acid. DAST is th ermally unstable, so reactions must be conducted at room temperature or below (Figure 3-6).59 Methods for Stereochemical Control of -Monofluorophosphonates Optically pure -fluorophosphonates are currently made using chiral Lewis Acid or organometallic complexes. Joergensen et al.60 used chiral Lewis acid complex 57 to fluorinate -substited -ketophosphonates with good enantiomeric excess while Sodeoka et al.61 used chiral Pd complex 60 to carry out enantioselective fluorinati on (Figure 3-7). However, neither method was reported to sel ectively fluorinate -unsubstituted phosphonates. 54

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The Importance of Stereochemical Control of -Monofluorophosphonates OHagan and coworkers synthesized sn -glycerol-3-phosphate (G3P) analogs 6164 (Figure 3-8) and assayed them for activity against glycerol-1-phosphate de hydrogenase (G1DH) by monitoring the rate of NADH formation (Figure 3-9).46 OHagan found that -monofluorinated phosphonic acid 63 was a substrate for the enzyme. In fact, both methylene 62 and monofluoro 63 were better than the natural substrate, 61. OHagan also examined the diastereomers of 63 to see if G1DH preferred one, and di scovered that one was consumed 20% faster (Figure 3-10). Unfortunate ly, he was not able to determin e the configuration of the better substrate or detect any accumulation of -hydroxyketone product 65. Berkowitz et al.62 constructed phosphonate analogs 6669 of glucose-6-phosphate (G6P) (Figure 3-11).63 Berkowitz tested these G6P analogs as substrates for glucose-6-phosphate dehydrogenase (G6PDH) (Figure 3-12), and f ound that the analogs with the highest ( 68) and lowest ( 67 ) Km varied only by the stereochemistry of the -fluorine. In fact, an order of magnitude separated the binding of the tw o compounds. Clearly, further research on stereochemical control of -fluorinated phosphonates was warr anted from the findings of OHagan and Berkowitz. Aldolases in Synthetic Chemistry Aldolases are enzymes that selectively catalyze the aldol r eaction between a donor substrate and acceptor aldehyde to create two new stereocenters. Aldol ases are classified according to their reaction mechanism: class I use a lysine residue in the active site to create a donor substrate enamine which then attack s an acceptor aldehyde, class II use Zn2+ to stabilize a donor enolate within the active site before attack on the acceptor aldehyde (Figure 3-13). The donor substrate for most class I and class II aldolases is dihydroxyacetone phosphate (DHAP), 72. 55

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Aldolases have been studied as tools for asymmetric orga nic synthesis using non-natural phosphonic acid donor substrate 80. Stribling63 discovered 80 could substitute for natural phosphate substrate 72 in the fructose 1,6-bisphosphate al dolase (FruA) catalyzed aldol reaction with D-glyceraldehyde 3-phosphate 81 to make phosphonic acid carbohydrate 82 (Figure 3-14). Fessner et al.64 synthesized phosphonic acid sugars 83 and 84 using substrate 80 with aldolases from class I and class II, allowing him to make different diastereomeric products from the same starting materials (Figure 3-15). Thus we reasoned that optic ally pure fluorinated DHAP mimics 85ab could be used with aldolase to synthsize optically active fluorinated phosphonic acid carbohydrat es (Figure 3-16). Selective Reduction of -Fluoro-Ketophosphonates Our initial route to optic ally pure DHAP mimics 85ab relied upon selective reduction of -fluoro-ketophosphonate 88. The corresponding alcohol 89 would then be deoxygenated using standard Barton-McCombie conditions, followed by acetonide deprotection and selective oxidation of the secondary alc ohol to give phosphonate ester 92 Deprotection of the phosphonate ester would give phosphonic acid DHAP mimics 85ab (Figure 3-17). -Fluoro-ketophosphonate 88 was made in two steps via reaction of dimethyl methylphosphonate with acetonide methyl ester 9565 followed by electrophilic fluorination of ketophosphonate 97 with Selectfluor.57 Methyl ester 95 was made by selective protection of Dmannitol66 followed by NaIO4 cleavage of diol 9465 and immediate oxidation of the resulting aldehyde to the methyl ester using Br2 / NaHCO3 / MeOH. Fluorination of 97 gave low yields of monofluorinated compound in addition to a difficult chromatographic separation of difluorinated and monofluorinated pro ducts (Figure 3-18). The -fluoro-ketophosphonate 88 was then reduced by NaBH4 to give racemic standard 98. Compound 88 was then screened agains t our library of purified -ketoreductases from 56

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Bakers Yeast using the same conditions as listed in Figure 1-7. The reactions were monitored with gas chromatography. Unfortunately, none of the reductases accepted the ketone. We next attempted to modify the acetonide protecting group of -fluoro-ketophosphonate 88 with the less sterically demanding carbonate 99 (Figure 3-19). Since quantities sufficient for further synthesis of -fluoro-ketophosphonate 88 were difficult to obtain it was decided not to in stall the carbonate by acetonide deprotection / carbonate reprotection of 88 to give carbonate 100 Instead, acetonide deprotection of ketophosphonate 97, which was available on a multigram scale, using Dowex H+ resin67 to give diol 101 followed by carbonate formation with triphosgene68 to access carbonate 102 on a gram scale. Unfortunately, all attempts to fluorinate 102 yielded only difluorinated product 103 and recovered starting material (Figure 3-20 and Table 3-1). The synthetic route to -fluorinated DHAP mimics of defined stereo chemistry via reduction of an -fluoro -ketophosphonate followed by Barton-McCombie deoxygenation of the resulting alcohol was not further pursued. Selective Reduction of -Fluorovinylphosphonates We next hypothesized that DHAP mimics 85ab could be synthesized by selective reduction of -fluorovinylphosphonates 104a-b using our library of e none reductases cloned and purified by Despina Bougioukou (Figure 3-21).5,69 Our route to -fluorovinylphosphonates used Horner-Wadsworth-Emmons (HWE) methodology to install the fluorin ated alkene. The initial synthesis of 104ab used the HWE reaction of fluorinated bisphosphonate 108 with protected aldehyde 112 to make the E isomer of protected phosphonate 113 as the sole product.49 The fluorinated bisphosphonate 108 was constructed in two steps by intial synthesis of methylene bisphosphonate 107 from diethyl methylphosphonate 106 and diethyl chlorophosphate70 followed by electrophilic fluo rination with Selectfluor.71 The protected aldehyde was made by ketalation of 1,3-dihydroxyacetone 109 followed by mono acetyl 57

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protection of the 1,3-diol with Pseudomonas fluorescens Lipase72 and subsequent Swern oxidation to aldehyde 112 (Figure 3-22). This synthetic route proved laborious due to difficulty purifying fluorinated bisphosphonate 108 via flash chromatography or fractional distillation. Additionally, it was found that the HWE reaction of crude 108 with aldehyde 112 gave an inseparable mixture of products. However, HWE reaction of the in situ generated lithiated 108a gave vinyl fluorinated phosphonate 113 in one step starting from -diethyl-dibromofluoromethyl phosphonate 40 (Figure 3-23).51 The dibromofluoromethyl phosphonate was gene rated by the Arbuzov reaction of triethyl phosphite and tribromofluoromethane.54 This reaction sequence allowed us to completely circumvent the purification problems associated with -fluorobisphosphonate 108. After considerable experimentation, it was found that deacetylation of 113 with Amberlyst A-26 resin72 followed by ketal deprotection with Montmorillonite clay73 afforded -fluorinated vinyl phosphonate 104a in decent yield (Figure 3-24). Racemic standard 115, the reduction product of -fluorinated vinylphosponate 104a b was prepared before screening vinyl phosphonate 104a against our library of purified en-reductases to allow for reaction monitoring by gas chromat ography. Synthesis of the racemic standard began with reduction of -fluorinated phosphonate 113 with PdOH / C followed by acetyl deprotection using Amberlyst A-26 and deketalation with Montmor illonite clay (Figure 3-25). This route proved cumbersome due to diffi culty separating the reduction products of 113, but served to make sufficient quantities of crude 115 for GC analysis. -Fluorinated phosphonate 104a was then screened against our library of en-reductases using a glucose-6-phosphate / glucose-6-phosphate dehyrogenase cofactor regeneration system; 58

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the screenings revealed that starting material was consumed, but no product could be detected by GC (Figure 3-26). In order to identify the reaction product(s), we ran small scale enzymatic reactions in D2O with stoichiometric quantities of NADPH. The 19F NMR after four hours showed complete consumption of starting material with the production of a single peak corresponding in chemical shift to inorganic fluoride (Figure 3-27). Presumabely, the fluorine was eliminated during reduction of the -fluorovinylphosphonate via the mechanism in Figure 3-28 to give unfluorinated vinyl phosphonate 119 To support this mechanism, we synthesized vinyl phosphonate 119 via the same reaction sequence used to make fluorinated vinyl phosphonate 104a. Thus, bisphosphonate 107 was condensed with aldehyde 112 to give protected vinyl phosphonate 113. Acetyl deprotection with Amberlyst A26 followed by deketalation with montmorillonite clay gave 119 (Figure 3-29). Unfluorinated vinyl phosphonate 119 was not observed as a product of the enzymatic reduction of fluorinated vinyl phosphonate 104a with stoichiometric quantities of NADPH (reaction monitored by 31P NMR), but 119 was detected as an inseparable by-product from the HWE chemical synthesis of racemic fluorinated phosphonate 115 during ketal deprotection (Figure 3-30). Synthesis of Racemic -Fluorinated Phosphonic Acids We decided to optimize the synthesis of racemic -fluorinated phosphonate 115 before further investigation on the construction of -fluorinated phosphonic acids of defined stereochemistry 85ab to determine if the fluorinated phos phonic acids are aldol ase substrates. Our HWE route to 115 was not sufficient for this purpose, but recent literature reported that terminal alkenes can be converted to -hydroxyketones in one step using Ruthenium or KMnO4 oxidants. 59

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Thus, unsaturated phosphonate 122 was made via double lithium halogen exchange / in situ trimethylsilyl (TMS) protec tion of dibromofluorophosphonate 40 followed by nucleophilic attack on allyl iodide to give th e TMS protected unsaturated phosphonate.53 This product was not isolated, but instead deprot ected in the same pot using lit hium ethoxide to give pure 122 after flash chromatography puri fication (Figure 3-31). Unfortunately, oxidation of 122 with KMnO4 yielded a mixture of -hydroxyketone 115 plus unfluorinated vinyl phosphonate 119 in roughly a 1 : 1 ratio. Phosphonate deprotection of this mixture led to multiple unidentified phosphorus containing products (F igure 56). Oxidation with RuO4 gave no defluorination, but in stead yielded carboxylic acid 123 in low yield (Figure 3-32). We then hypothesized that deprot ection of unsaturated phosphonate 122 followed by alkene oxidation could le ad to racemic phosphonic acid 124 (Figure 3-33). Phosphonate 122 was smoothly deprotected with TMSBr followed by hydrolysis of the TMS protected phosphonic acid with water to give phosphonic acid 125.73 However, KMnO4 alkene oxidation was not successful and RuO4 oxidation gave carboxylic acid 126 as the only reaction product (Figure 3-34). Our results with alkene oxida tion revealed that this was not a method for synthesis of racemic -fluorophosphonic acids. Our next route to racemic -fluorophosphonic acids sought to selectivly oxidize the secondary alcohol of diol 120 to -hydroxyketone 118 using glycerol-1-phosphate dehydrogenase (G1DH) (Figure 3-35). For th is procedure we pla nned to use NADH oxidase from Lactobacillus sanfranciscensis for cofactor regeneration. Our route to diol 63 started from the NaIO4 cleavage of acetonide protected D-mannitol 94 followed by immediate reduction of the result ing aldehyde to acetoni de protected alcohol 60

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127.76 This alcohol was c onverted to triflate 12877 and then condensed with phosphonate 40 to give acetonide protected phosphonate 129 in decent yield.46 Both protecting groups of 129 were removed by initial treatment of the neat phosph onate with TMSBr followed by addition of water for silyl ether cleavage and acetonide deprotection to give diol 63 as a 0.8 : 1 diastereomeric mixture as determine by 19F NMR (Figure 3-36).46 Phosphonic acid 63 (6 mg / 0.53 mol) was combined with G1DH (80 Units), NADH oxidase from L. sanfranciscensis (80 U) and NAD+ (50 mg / 0.08 mmol) in 50 mM phosphate buffered D2O and stirred at room temperature for 24 hours. The diastereomeric ratio changed during this time period from 0.8 : 1 to 0.4 : 1, indi cating that starting material was consumed. We are currently unsure if product was formed, but this data will be obtained by monitoring the reaction by 13C NMR to observe ketone formation or by mass spectrometry to observe molecular weight change. Results and Discussion While logical, our routes to -fluorinated DHAP mimics of defined stereochemistry were not successful. We initially believed that our li brary of purified reductases from Bakers yeast could accept acetonide protected -fluoro-ketophosphonate 88 due to our previous success with sterically similar furan derivative 130 (Figure 3-37). Unfortuna tely, acetonide protected phosphonate 88 proved too large for our enzymes, and in stallation of a less sterically demanding protecting group resulted in difluorinated products. Our enone reductase route for asymmetric synthesis of -fluorinated DHAP mimics via vinyl -fluorophosphonates was not viable due to the unexpected elimination of fluorine after enzymatic reduction. Chemical synthesis of phosphonate 115 or phosphonic acid 65 as racemic mixtures via olefin oxidation also did not produce the pure compound due to fluorine 61

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elimination. But, these methods gave us valuab le insight about the sensitive nature of these compounds; any future syntheses must avoid the facile loss of fluorine. Our current route for the synthesis of -fluorinated DHAP mimics as a racemic mixture appears promising. Additional data, obtained from 13C NMR or mass spectrometry, must be collected to ensure -hydroxyketone formation from oxidation of diol 63, since we currently are unsure of the fate of consumed starting materi al. If the oxidation is successful, then the fluorinated DHAP mimic will not be isolated, but in stead reacted in situ with aldolase to form diastereomeric mixtures of -fluorophosphonic acid carbohydrates The in situ reaction of hydroxyketone 65 should minimize defluorination since the enolizable ketone will be converted to the lactol by aldol ase (Figure 3-38). Future Work Future work will chemoenzymatically synthesize optically pure -fluorophosphonic acids 85ab from optically pure -hydroxyphosphonates 133ab (Figure 3-39). Alphahydroxyphosphonates 133ab have been made previously from ( S)-malic acid 134 by Prestwich et al. as diastereomeric mixture 135.58 We plan to separate the diastereomers via lipase catalyzed kinetic resolution as shown by Backvall et al. for -hydroxyphosphonate 136 (Figure 3-40).78 Nucleophilic displacement of ki netically resolved alcohol 132a with diethylaminosulfur trifluoride (DAST) will give optically pure -fluorophosphonate 140b .62 Or, the alcohol will be converted to the triflate followed by nucle ophilic displacement w ith cesium fluoride79 or tetrabutylammonium fluoride (TBAF)80 to give 140b Deprotection of 140b as previously described will give -fluorophosphonic acid 63b (Figure 3-41). Diastereomer 63a will be obtained by removal of the acteyl group under neutral conditions81 followed by nucleophilic fluorination and deprotection (Figure 3-42). Optically 62

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pure -flurophosphonic acids 63ab will then be selectively oxidi zed and reacted in situ with aldolase to make -fluorinated phosphonic acid carbohydrat es of defined stereochemistry. Experimental Procedures Materials All organic chemicals were purchased from Sigma or Fisher and used without further purification. THF was distilled from Sodium metal. Flash chromatography was monitored by TLC with staining of plates by KMnO4. D-mannitol (28 g / 0.15 mol), dry DMSO ( 30 mL), 2,2-dimethoxypropane (46 mL / 0.38 mmol), and p-TsOH (0.5 g / 2.63 mmol) were added to an oven dried flask and stirred under Argon overnight. The solution was then poured into 3% NaHCO3 (150 mL) and the resulting aqueous solution extracted with CH2Cl2 (3 x 100 mL). The combin ed organic layers were washed with water (2 x 20 mL) and brine (1 x 20 mL), dried over MgSO4, and the solvent removed under reduced pressure. The resul ting product was placed under high vacuum for 1.5 hours to remove residual solvent and the crude product recrystallized from heptane : CHCl3 (10 : 1 volume / volume based on crude mass of product) to give 22 g of the pure product as white needles (56% yield).82 Melting point 118 120o C; 1H NMR: (CDCl3) 1.36 (s, 6H), 1.43 (s, 6H), 2.80 (m, 2H), 3.75 (m, 2H), 3.8-4.3 (m, 6H); 13C NMR: (CDCl3) 109.3, 75.8, 70.9, 66.7, 26.7, 25.2. 63

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Acetonide 94 (6.07 g / 23.15 mmol) was added to 150 mL of CH2Cl2 followed by addition of NaIO4 (9.89 g / 46.28 mmol) and then 4 mL of aqueous saturated NaHCO3. The resulting mixture was mechanically stirred at room temper ature for 3 hours before the addition of 20 g of MgSO4, then stirred at room temperature for another 15 minutes. The solution was then filtered, and the solvent removed under redu ced pressure to give the crude aldehyde that was dissolved into 100 mL of 9 : 1 MeOH : H2O which also contained 14.58 g / 173.55 mmol of NaHCO3. Br2 (4.8 mL / 93.78 mmol) was then added dropwise to the solution and the resu lting orange mixture was stirred overnight at room temperature. Sa turated aqueous sodium bisulfite was then added dropwise to the solution until th e orange color disappeared. Methanol was removed under reduced pressure, and the remaining aqueous phase was extracted with CH2Cl2 to yield the crude product which was purified by flash chromatography to give the pure produc t as a colorless oil (4.81 g, 65% yield from 94).65 1H NMR: (CDCl3) 1.42 (s, 3H), 1.45 (s, 3H), 3.80 (s, 3H), 4.12 (m, 1H), 4.23 (m, 1H), 4.61 (m, 1H). 13C NMR: (CDCl3) 25.66, 26.00, 52.52, 67.40, 74.20, 111.50, 177.78. A round bottom flask equipped with an addition funnel was flam e dried while purging with Argon before the addition of 25 mL of THF and then cooled to -10o C. n -butyllithium (13.34 mL 64

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/ 33.34 mmol from a 2.5 M solution) was then ad ded and the mixture was cooled to -78o C. Dimethyl methylphosphonate 96 (3.58 mL / 31.75 mmol) in 7 mL of THF was then added dropwise to the solution of n-BuLi at -78o C and the resulting white slurry was stirred at -78o C for 30 minutes. The slurry was then transferred vi a cannula to a separate flask containing methyl ester 95 (4.37 mL / 30.16 mmol) in 15 mL of THF at -78o C. The resulting solution was stirred at -78o C for one hour before allowing to warm to room temperature overnight. Saturated ammonium chloride (10mL) was added dropwise at room temperature to quench the reaction and THF was removed under reduced pressure. The resulting aqueous laye r was extracted with EtOAc (3 x 20 mL), the combined or ganic layers were dried with MgSO4 and the solvent removed under reduced pressure to give the cr ude product as an oil which was purified by flash chromatography (3:1 Hex : EtOAc to 3 : 1 EtOAc : Hex) to give the pure product as a colorless oil. (6.89 g, 86% yield).83 1H NMR: (CDCl3) 1.37 (s, 3H), 1.46 s (3H), 3.16 (m, 1H), 3.49 (m, 1H), 3.77 (d, 2H, J = 3.6 Hz), 4.35 (m, 4H), 4.55 (m, 1H). 13C NMR: (CDCl3) 25.14, 26.21, 35.81, 37.55, 53.31 (m), 66.18, 80.32, 111.43, 202.39. 31P NMR: (CDCl3) 23.26. A round bottom flask equipped w ith an addition funnel was ch arged with NaH (0.15 g / 3.7 mmol of a 30% dispersion) and the NaH was wash ed two times with 5 mL of pentane before addition of 15 mL of THF. The re sulting solution was cooled to 0o C and phosphonate 97 (850 L / 3.7 mmol) dissolved in 5 mL THF was added dropwise to the flask at 0o C. The resulting solution was stirred at 0o C for 30 minutes and room temperat ure for one hour before recooling to 0o C. This solution was then transferred via cannula to a suspension of Selectfluor (1.31 g / 65

PAGE 66

3.7 mmol) in dry CH3CN at 0o C and the resulting solution stirred at 0o C for one hour before warming to room temperature overnight. The reaction was quenched with saturated ammonium chloride (7 mL), THF and acetonitrile were re moved under reduced pressure and the resulting aqueous layer was extracted with EtOAc (3 x 10 mL ). The combined organics were dried with MgSO4 and the solvent removed under reduced pressu re to give the crude product as an oil. Purification via flash chromatography (5 : 1 Hex : EtOAc to 1:1 Hex : EtOAc) gave the pure product as a slightly yellow oil. 1H NMR: (CDCl3) 1.46 (s, 3H), 1.51 (s, 3H), 3.80 (m, 4H), 4.25 (m, 2H), 4.9 (m, 1H), 5.4 (dd, J = 47.21, 60.39 Hz), 5.75 (dd, J = 47.5, 47.5 Hz). 19F NMR: (CDCl3) -215.5 (dd, J = 70.24, 70.24 Hz), -216.0 (dd, J = 69.03, 70.24 Hz). O O P O OMe OMe F OH 98 A round bottom flas k containing NaBH4 (0.017 g / 0.44 mmol) in MeOH (4 mL) was cooled to 0o C before the addition of ketone 88 (0.12 g / 0.44 mmol). The solution was stirred at 0o C for one hour and at room temperature for 4 hours before the reaction was quenched with saturated aqueous ammonium chloride (1 mL). MeOH was then removed under reduced pressure and the resulting a queous layer extracted with CH2Cl2 (3 x 7 mL). The combined organics were dried with MgSO4, the solvent evaporated under reduced pressure and the crude oil purified by flash chromatography (1 : 1 Hexane s : EtOAc to 4 : 1 Hexanes : EtOAc) to give the product as a colorless oil. 1H NMR: CDCl3 1.38 (m, 6H), 3.80 (m, 10H), 4.70 (m,1H). 13C NMR: (CDCl3) 25.16, 25.20, 25.23, 26.17, 26.41, 26.70, 26.91, 65.42, 65.80, 66.36, 67.00, 68.71, 69.01, 69.74, 69.98, 69.74, 69.98, 70.87, 71.10, 71.87, 72.13, 73.73, 73.79, 73.88, 74.54, 74.69, 75.26, 75.46, 75.15, 75.61, 75.66, 84.94, 86.14, 87.18, 87.41, 88.37, 88.64, 89.65, 90.87. 66

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31P NMR: (CDCl3) 18.89, 19.27, 19.50, 19.90, 20.18, 20.32, 20.90. 19F NMR: (CDCl3) 213.4, dddd ( J = 46.02 Hz), -215.4, dddd ( J = 44.81, 46.02, 46.02, 44.81 Hz), -223.4 dddd (J = 46.02Hz), -230.7 dddd ( J = 44.81, 46.24, 44.81, 44.81 Hz). Acetonide protected phosphonate 97 (0.41 g / 1.63 mmol) was di ssolved in 7 mL of MeOH at room temperature before the addition of 0.6 g of Dowex 50WX8-100 resin at room temperature. The resin was then filtered and washed multiple times with acetone to remove the diol product. Acetone and methanol were rem oved under reduced pressure and the resulting oil / water mixture was azeotroped several times with CH2Cl2 to remove the residual water. The resulting oil was then purified by flash chromatography (EtOAc) to give the pure product as a slightly yellow o il (72 % / 0.25 g). 1H NMR: (CDCl3) 3.35 (d, 2H J = 3.8 Hz), 3.80, (m, 6H), 3.92 (m, 1H), 4.05 (m, 1H), 4.27 (m, 1H). 13C NMR: (CDCl3) 36.00, 37.76, 48.31, 48.61, 50.48, 62.68, 77.89. 31P NMR: (CDCl3) 23.74. A flask was charged with diol 101 (0.44 g / 2.09 mmol) and pyridine (0.51 mL / 6.27 mmol) in CH2Cl2 (15 mL) and then cooled to -40o C under Argon. Triphosgene (0.62 g / 2.09 mmol) dissolved in CH2Cl2 (5 mL) was then added dropwise at the same temperature. The solution was allowed to warm slowly to room temperature over two hours and then stirred for an additional two hours at room temperature. The resulting solution was washed with 10 % HCl 67

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(10 mL), saturated NaHCO3 (10 mL) and brine (10 mL), dried with MgSO4, and the solvent removed under reduced pressure to give the cr ude product as an oil. The crude product was purified by flash chromatography (2 : 1 Hexanes : EtOAc) to give the pu re product as a yellow oil. 1H NMR: (CDCl3) 3.42 (m, 2H), 3.82 (m, 6H), 4.64 (m 2H), 5.32 (m, 1H). 31P NMR: (CDCl3) 21.18. OH O O HO 110 Dihydroxyacetone dimer (7.5 g / 41.6 mmol), tr imethylorthoformate (9 mL / 82.2 mmol), and p-TsOH (0.03 g / 0.16 mmol) and anhydrous Me OH (100 mL) were combined in a flask and stirred under Argon overnight at room temperatur e. Amberlyst A26 resin (0.28 g) was then added at room temperature and the solution was stirred at room temperature for 15 minutes. The solution was then filtered and the solvent was removed under redu ced pressure to give a white solid (5.37 g / 95 % yield) which was pure enough fo r further use. The solid could be purified by flash chromatography (1 : 1 Hexanes : EtOAc) if desired.72 1H NMR: (CDCl3) 3.25 (s, 6H), 3.70 (s, 4H). MS: 105 (M 31, 4.5%), 77 (100%), 55 (35.4%). Protected diol 110 (3.75 g / 27.6 mmol), Pseudomonas fluorescens lipase (0.33 g), vinyl acetate (40 mL) and diisopropyl ether (20 mL) we re stirred under Argon overnight at room temperature. The lipase was removed by filtration over Celite and the solvents removed under reduced pressure. The resulting oil was purified by flash chromatography(5 : 1 Hexanes : EtOAc to 3 : 1 Hexanes : EtOAc) to give the pure co mpound as a colorless oi l (2.95 g / 60% yield).72 1H NMR: (CDCl3) 2.18 (s, 3H), 3.25 (s, 6H), 3.60 (s, 2H), 4.20 (s, 2H). 13C NMR: (CDCl3) 68

PAGE 69

20.47, 48.06, 59.80, 99.81, 170.86. FT-IR: (neat) 3463.7, 295.1, 1711.7. MS: 147 (100%), 129 (1.7%), 105 (62.9%), 87 (28.5%), 73 (35.2%). An oven dried round bottom flask was charged with DMSO (257 L / 3.54 mmol) in 15 mL of dry CH2Cl2 and cooled to -60o C under Argon. Oxalyl chloride (280 L / 3.27 mmol) was then added dropwise at -60o C and the resulting solution stir red at this temperature for 10 minutes. Acetylated alcohol 103 (450 L / 2.83 mmol) was then added dropwise to the solution at -60o C followed by stirring at -60o C for 20 minutes before quenc hing via dropwise addition of NEt3 (1.3 mL) at -60o C. The solution was stirred at -60o C for 15 minutes before allowing to warm to room temperature over 45 minutes. Solvents were then removed under reduced pressure and the resulting residue di ssolved into half saturated NaHCO3 (6 mL) and extraction of the aqueous layer with ether (3 x 10 mL). The combined organics were washed with brine (5 mL), dried with MgSO4, and the solvents removed under redu ced pressure to give the crude compound (0.42 g / 85% crude yield) as a slightly yellow oil which was used without further purification. 1H NMR: (CDCl3) 2.05 (s, 3H), 3.30 (s, 6H), 4.25 (s, 2H), 9.45 (s, 1H). 13C NMR: (CDCl3) 20.80, 50.16, 61.11, 66.59, 170.1, 204.12. FT-IR: (neat) 2950.02, 1750.32, 1172.08. MS: 147 (100%), 133 (0.9%), 115 ( 5.6%), 103 (56.9%), 73 (31.3%). A flame dried round bottom flask was charge d with diisopropylamine (8.85 mL / 63.14 mmol) and THF (15 mL), then cooled to -78o C before the dropwise addition of n-butyllithium 69

PAGE 70

(60.2 mL / 66.2 mmol) at the same temperature. The anion was allowed to stir at -78o C for 30 minutes before the dropwise addition of diet hylmethyl phosphonate (4.5 mL / 30.8 mmol) in 5 mL of THF at -78o C. The resulting solution was stirred at -78o C for 30 minutes before the dropwise addition of diethyl ch lorophosphate (4.7 mL / 32.3 mmo l) in 5 mL of THF. The resulting solution was stirred for 30 minutes at -78o C before slowly allowing to warm to -35o C over a period of 2 hours. The solution was then cooled to -50o C and quenched by the dropwise addition of 3M HCl (22 mL). The resulting vi scous mixture was allowed to warm to room temperature and THF was removed by rotary ev aporation. The resulting aqueous layer was extracted with CH2Cl2 (3 x 20 mL) and the combined organics were dried with MgSO4 and the solvent removed under reduced pressure. The resulting oil was dissolved into 60 mL of anhydrous ether and the triethylammonium salts removed by filtration. The ether was removed to give the crude product as a yellow oil. Th e product was purified by vacuum distillation (165o C bath temperature / 125o C apparatus temperature / 0.05 mm Hg) to give the pure compound as a slightly yellow oil (8.0 g / 90% yield).70 1H NMR: (CDCl3) 1.30 (t, 12H, J = 7.0 Hz), 2.4 (t, 2H J = 21.0 Hz), 4.05 (m, 8H). 13C NMR: (CDCl3) 16.42, 23.61, 25.42, 27.24, 62.72. 31P NMR: (CDCl3) 20.51. IR: (neat) 2985.30, 1480.10, 1024.76. MS: 288 (m/z, 10.2%), 261 (40.8%), 205 (2.0%), 177 (0.6 %), 159 (100%), 125 (65.7%). Potassium hydride (0.29 g / 2.21 mmol of a 30% dispersion in mineral oil) was washed twice with pentane before the addition of 7 mL of THF. The resulting suspension was then cooled to 0o C before the dropwise addition of bisphosphonate 107 (0.5 mL / 2.01 mmol) in 5 mL of THF. The resulting solutio n was stirred for 30 minutes at 0o C and one hour at room 70

PAGE 71

temperature before recooling to 0o C. The anion was then transf erred via cannula to a solution of Selectfluor (0.71 g / 2.01 mmol) in CH3CN (20 mL). The resulting solution was then allowed to warm to room temperature overnight before the addition of saturated NH4Cl (5 mL). THF and acetonitrile were then removed under reduced pressure and the remaining aqueous layer was extracted with ether (3 x 15 mL). The combined organics were dried with MgSO4 and the solvent removed under reduced pressure to give the crude compound as an oil. Purification by flash chromatography (4.5 : 1 Hexanes : EtOAc to 2: 1 EtOAc : Hex) gave the pure compound as a colorless oil ( 1.62 g / 50% yield).82,84 CAUTION: If this proce dure is used the purification requires a much longer time than for standard organic compounds. 1H NMR: (CDCl3) 1.20 (t, 12H J = 7.1 Hz), 4.05 (m, 8H), 4.82 (dt, 1H J = 13.4, 45.9 Hz). 13C NMR: (CDCl3) 16.15, 64.81 (d, J = 30.72 Hz), 84.05 (dt J = 156.1, 312.7 Hz). 31P NMR: (CDCl3) 11.05 (d, J = 64.09 Hz). 19F NMR: (CDCl3) 312.6 (dt J = 62.3, 45.9 Hz). FT-IR: (neat) 2984.95, 1260.04, 1026.07. MS: 306 (m/z, 7.1%), 279 (41.4%), 22 3 (61.4%), 194 (80.8%), 177 (100%), 143 (50.6%). Tribromofluoromethane (1.35 mL / 13.8 mmol ), triethyl phosphite (2.36 mL / 13.8 mmol) and THF (12 mL) were combined in a round bottom flask and heated to 50o C and stirred overnight at this temperature. The reaction wa s then cooled, the solven t removed under reduced pressure and the resulting oil purified by flash chromatography (12 : 1 Hexanes : EtOAc) to give the pure product as a slightly yellow oil (4.30 g / 95% yield). 1H NMR: (CDCl3) 1.42 (t, 6H J = 7.2 Hz), 4.39 (m, 4H). 13C NMR: (CDCl3) 16.60 (d, J = 10 Hz), 66.99 (d, J = 7.0 Hz). 31P NMR: (CDCl3) 2.55 (d, J = 76.9 Hz). 19F NMR: (CDCl3) 76.50 (d, J = 76.8 Hz). IR: (neat) 71

PAGE 72

2984.95, 1274.84, 1018.98. MS: 327 (m/z, 0.1%), 299 (1.1%), 247 (2.6%), 191 (9.2%), 137 (100%), 109 (90.8%). From purified fluorinated bisphosphonate 108: An oven dried round bottom flask was charged with fluorinated bisphophonate 108 (100 L / 0.38 mmol) in 6 mL of THF and cooled to -78o C before the dropwise addition of n-BuLi (345 L / 0.38 mmol from a 1.1 M solution). The resulting solution was stirred at -78o C for 10 minutes before the addition of crude aldehyde 112 (84 L / 0.52 mmol) at the same temperature. The solution was then allowed to stir at -78o C for 1.5 hour before slowly allowing to warm to room temperature overnight. Water (3 mL) was then added, THF was removed under reduced pressure and the resulting aqueous solution was extracted with ether (3 x 8 mL). The comb ined organic layers were dried with MgSO4 and the solvent removed under reduced pressure to give the crude product as a yellow oil which was purified by flash chromatography (3 : 1 Hexanes : EtOAc to 1.5 : 1 Hexanes : EtOAc) to give the pure product as a slightly yellow oil (0.088 g / 70% yield). 1H NMR: (CDCl3) 1.40 (t, 6H, J = 7.0 Hz), 2.05 (s, 3H), 3.25 (s, 6H), 4.15 (m, 4H), 4.25 (s, 2H), 5.85 (dd, J = 9.24, 41.91 Hz, 1H). 13C NMR: (CDCl3) 16.14, 16.22, 20.66, 49.05, 62.80, 63.37 (d, J = 5.7 Hz), 99.65 (d, J = 14.6 Hz), 122.3 (d, J = 27.5 Hz), 152.1 (dd, J = 231.6, 288.6 Hz), 170.0. 31P NMR: (CDCl3) 4.7 (d, J = 100.72). 19F NMR: -120.4 (dd, J = 41.52, 99.65). FT-IR: (neat) 2985.09, 1751.85, 1020.20. MS: 297 (M 31, 7.1%), 277 (3.1%), 235 (48%), 207 (15.5%), 99 (68.8%). From -dibromofluorophosphonate 40: A flame dried round bottom flask was charged with THF (7 mL) and n-BuLi (325 L / 0.52 mmol from a 1.6 M solution) and cooled to -78o C 72

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followed by dropwise addition of phosphonate 40 (100 L / 0.52 mmol) at -78o C (the solution turned red / brown at this point). Th e resulting solution was stirred at -78o C for one hour before dropwise addition of aldehyde 112 at the same temperature. The solution was then stirred at -78o C for one hour before allowing to warm to room temperature overnight. Water (5 mL) was then added, THF was removed under reduced pressure and the aqueous layer extracted with ether (3 x 10 mL). The combined organics were dried with MgSO4 and the solvent removed under reduced pressure to give the crude product as a brown oil which was purified by flash chromatography using the same solvent system as listed above to give the product as a slightly yellow oil (55.4 mg / 65% yield). Spectral data we re also the same as listed above. A round bottom flask contai ning acetylated alcohol 113 (0.65 g / 1.98 mmol) and Amberlys A26 resin in 8 mL Me OH was stirred overnight at room temperature. The resin was removed by filtration then washed with acetone. The resulting solution was concentrated under reduced pressure and the crude oil purified by flash chromatogra phy (2 : 1 Hexanes : EtOAc to 1.5 : 1 EtOAc to Hexanes) to give the pure produ ct as a pale yellow oi l (0.38 g / 75% yield). 1H NMR: (CDCl3) 1.40 (t, 6 H, J = 7.1 Hz), 3.15 (s, 6H), 3.78 (s, 2H), 4.20 (m, 4H), 5.85 (dd, J = 9.4, 42.51 Hz, 1H). 13C NMR: (CDCl3) 18.36, 49.40, 63.37, 63.66 (d, J = 5.4 Hz), 101.47(d, J = 14.3 Hz), 123.12 (d, J = 27.5 Hz), 152.34 (dd, J = 287.1, 233.0 Hz). 31P NMR (CDCl3) 4.8 (d, J = 102.54). 19F NMR (CDCl3) -120.85 (dd, J = 41.44, 101.73 Hz). IR (neat) v 3418.12, 2985.48, 1019.28. MS 255 (M 31, 66.4%), 235 (46.3%), 207 (14.0%), 179 (23.2%), 167 (17.3%), 117 (30.2%), 99 (100%). 73

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P O OEt OEt F 104a HO O A round bottom flask containing alcohol 113 (0.55 g / 1.93 mmol) and Montmorillonite clay (1 g) in 10 mL of CH2Cl2 was stirred at room temperature overnight. The solution was then filtered over Celite, the solvent was removed under reduced pressure and the resulting oil purified by flash chromatography (1 : 1 Hexanes : Et OAc to 3 : 1 EtOAc to Hexanes) to give the pure product as a slightly ye llow oil (0.26 g / 65% yield).85 1H NMR: (CDCl3) 1.40 (t, 6H, J = 7.0 Hz), 4.20 (m, 4H), 4.58 (s, 2H), 6.35 (dd, J = 8.2, 42.51 Hz). 13C NMR: (CDCl3) 16.54, 64.55 (d, J = 5.8 Hz), 69.85, 116.85 (d, J = 25.9 Hz), 161.26 (dd, J = 307.2 Hz, 225.6 Hz), 196.10. 31P NMR: (CDCl3) 2.70 (d, J = 100.11 Hz). 19F NMR: (CDCl3) -101.6 (dd, J = 43.6, 101.73 Hz). FT-IR: (neat) 3418, 2897, 1699, 1642, 1019. A round bottom flask was charge d with fluorinated alkene 113 (0.50 g / 1.52 mmol), EtOAc (8 mL) and a spatula tip of PdOH / C be fore hydrogenation overnight at 1 atm at room temperature. The suspension was then filtered over Celite and the solvents removed under reduced pressure to give the crude product as a slightly yellow oil. The crude product was purified by flash chromatography (6 : 1 Hex : Et OAc to 2 : 1 Hex : EtOAc) to give the pure product as a slightly yellow oil ( 0.15 g / 30% yield). 1H NMR: (CDCl3) 1.32 (t, 6H, J = 7.2 Hz), 2.05 (s, 3H), 2.29 (m 2H), 3.05 (s, 6H), 3.40 (s, 2H), 4.10 (m 4H), 4.87 (m, 1H). 31P NMR: (CDCl3) 19.02 (d, J = 75.69 Hz). 19F NMR: (CDCl3) -208.3 (m). 74

PAGE 75

P O OEt OEt F 117 O O HO A round bottom flask was charged with reduced phosphonate 116 (0.15 g / 0.45 mmol), Dowex A26 resin (0.10 g) and Methanol (6 mL) before stirring overnight at room temperature. The solution was then filtered, and the filtrered resin washed several times with acetone. Solvents were then removed under reduced pressu re to give the crude compound as a slightly yellow oil. The crude product was purified by fl ash chromatography (2 : 1 Hex : EtOAc to 2 : 1 EtOAc : Hex) to give the pure product as a slig htly yellow oil (0.097 g / 75% yield). 1H NMR: (CDCl3) 1.40 (t, 6H, J = 7.1 Hz), 2.20 (m, 2H), 3.10 (s, 3H), 3.40 (s, 2H), 4.10 (m, 4H), 4.79 (m, 1H). A round bottom flask was charged with alcohol 117 (0.097 g / 0.34 mmol), montmorillonite clay (0.10 g) and CH2Cl2 (5 mL) before stirring at room temperature overnight. The suspension was then filtered over Celite and th e solvents removed under reduced pressure to give the crude product as a yellow oil which contained the desired -fluorophosphonate as the major product plus defluorinated vinyl phosphonate 119 as an inseparable byproduct (crude yield 80 % / 0.065 g). 1H NMR: (CDCl3) 1.25 (m, 6H), 3.05 (m, 2H), 4.12 (m, 4H), 4.20 (s, 2H), 5.31 (m, 1H), 6.90 (m, 2H, this signal is from the defluorinated vinyl phosphona te side product). 31P NMR: (CDCl3) 17.35 (d, J = 73.25 Hz), 15.35 (s, this signal is from the defluorinated vinyl phosphonate side product). 19F NMR: (CDCl3) 208.13 (m). 75

PAGE 76

An oven dried round bottom flask was charged with bisphosphonate 107 (646 L / 2.60 mmol) in THF (10 mL) and cooled to -78o C under Argon. n -Butyllithium ( 1.04 mL from a 2.5 M solution in hexanes) was then added dropwise at -78o C and the resulting solution stirred at the same temperature for 1 hour. Aldehyde 112 (569 L / 3.56 mmol) was then added dropwise at 78o C and the resulting solution stirred at the same temperature before allowing to warm overnight. Water (3 mL) was then added, and THF was removed under reduced pressure. The remaining aqueous layer was extracted with ether (3 x 10 mL), the combined organics were washed with brine (4 mL), dried with MgSO4 and the solvent removed un der reduced pressure to give the crude product as a slightly yellow oi l. The crude product was purified by flash chromatography (4 : 1 Hex : EtOAc) to give th e pure product as a colo rless oil (0.67 g / 82% yield). 1H NMR: (CDCl3) 1.30 (t, 6H, J = 7.0 Hz), 2.05 (s, 3H), 3.20 (s, 6H), 4.05 (m, 4H), 4.08 (s, 2H), 6.15 (dd, 1H, J = 20.3, 17.5 Hz), 6.50 (dd, 1H, J = 22.53, 17.3 Hz). 31P NMR: (CDCl3) 18.18. P O OEt OEt 121 O O HO A round bottom flask was charged with vinyl phosphonate 120 (0.67 g / 2.13 mmol), A26 resin (0.70 g) and methanol (8 mL) and then sti rred at room temperatur e overnight. The product was then filtered, the resin was washed several tim es with acetone and the solvents evaporated to give the crude product as a slightly yellow oi l. The crude product was purified by flash 76

PAGE 77

chromatography (1 : 1 Hex : EtOAc) to give the pure product as a slight ly yellow oil (0.44 g / 78 % yield). 1H NMR: (CDCl3) 1.21 (t, 6H J = 7.1 Hz), 3.05 (s, 6H), 3.50 (s, 2H), 3.98 (m, 4H), 6.25 (m, 2H). 31P NMR: (CDCl3) 18.71. P O OEt OEt 119 HO O A round bottom flask was charged with alcohol 121 (0.44 g / 1.66 mmol), montmorillonite clay (0.55 g) and CH2Cl2 (7 mL) before stirring overnight at room temperature. The suspension was then filtered over Celite a nd the solvent removed under reduced pressure to give the crude product as a yellow oil. The crude product was purified by flash chromatography (1 : 1 Hex : EtOAc) to give the pure product as a sli ghtly yellow oil ( 0.33 g / 90% yield). 1H NMR: (CDCl3) 1.25 (t, 6H J = 7.1 Hz), 4.05 (m, 4H), 4.42 (s, 2H), 6.80 (m, 2H). 31P NMR: (CDCl3) 15.44 (s). A three neck round bottom flask equipped with a pressure equalizing dropping funnel was flame dried while purging with Ar gon before charging the flask with n-Butyllithium (6.9 mL / 17.1 mmol from a 2.5 M solution in hexanes) in THF (20 mL). The dropping funnel was charged with a mixture of dibromofluorophosphonate 40 (1.5 mL / 7.83 mmol) and trimethylsilyl chloride (993 L / 7.83 mmol) in THF (10 mL). The flask was then cooled to -78o C and the contents of the funnel were added dropwise at th is temperature. The resulting brown solution was stirred at -78o C for 10 minutes before the dropw ise addition of allyl iodide (927 L / 10.2 77

PAGE 78

mmol) in THF (7 mL) at the same temperature. The resulting solutio n was stirred at -78o C for 45 minutes, then allowed to warm to 0o C on an ice bath. A solution of lithium ethoxide (prepared by adding 0.3 g of lithium wire to et hanol at room temperature) was then added dropwise at 0o C and the resulting solution was allowed to stir at the same temperature for 1 hour before pouring into 15 mL of 2 M HCl. THF was removed under reduced pressure, and the remaining aqueous solution was extracted with ether (3 x 15 mL). The combined organic phases were washed with a freshly prepared solution of saturated sodium bisulfite (3 x 7 mL) and brine (1 x 7 mL), dried with MgSO4 and the solvent removed under reduced pressure to give the crude product as a dark yellow oil. The crude produc t was purified by flash ch romatography (12 : 1 Hex : EtOAc to 8 : 1 Hex : EtOAc) to give the pu re product as a slightly yellow oil (1.40 g / 85% yield).86 1H NMR: (CDCl3) 1.25 (t, 6H J = 7.05 Hz), 2.6 (m, 2H), 4.10 (m, 4H), 4.72 (m, 1H), 5.14 (m, 2H). 13C NMR: 15.42, 34.05, 62.31 (m), 67.46, 67.52, 112.48, 131.12. 31P NMR: (CDCl3) 17.8 (d, J = 74.26 Hz). 19F NMR: (CDCl3) -200.67 (m). A round bottom flask was charged with NaHCO3 (0.37 g / 4.5 mmol) followed by addition of a 0.1 M aqueous solution of RuCl3 (139 L) then CH3CN (8.4 mL) and EtOAc (8.4 mL). Oxone was then added in one portion to the brown suspension to give a bright yellow suspension (gas was evolved at this point). The reaction mixture was then cooled to 0o C in an ice bath and unsaturated phosphonate 122 (0.30 g / 1.39 mmol) was added in one portion. The mixture was stirred at 0o C for one hour before dilution with EtOAc (20 mL), the mixture was then filtered over Celite followed by washing of the solution w ith brine (1 x 5 mL). The organic phase was dried with MgSO4 and the solvent removed under reduced pr essure to give the crude product as a 78

PAGE 79

yellow oil. Purification by flas h chromatography (3 : 1 EtOAc : Hex) gave the product as a colorless oil (0.11 g / 34% yield). 1H NMR (CDCl3) 1.25 (t, 6H, J = 7.1 Hz), 2.92 (m, 2H), 4.05 (m, 4H), 5.25 (m, 1H). 31P NMR: (CDCl3) 17.82 (d, J = 74.47 Hz). 19F NMR: (CDCl3) -203.45 (m). A round bottom flask was charge d with unsaturated phosphonate 122 (0.10 g / 0.48 mmol) and then placed under vacuum for four hours. Th e flask was then sealed with a rubber septa and placed under Argon and TMSBr (218 L / 1.61 mmol) was added dropwise to the neat phosphonate at room temperature. The rubber se pta was removed and quickly replaced with a yellow Caplugs stopper and the reaction was stirred at room temperature overnight. Volatile materials were then removed (fir st on the rotovap, then on hi gh vacuum for four hours) and water (1.5 mL) was added to the resulting oil and the solution stirred overnight at room temperature. (The solution instantly became turbid before slowly clearing overnight). Water was then removed by freeze drying to yield the phosphoni c acid product as a colorless, viscous oil which was used without further purification in the next step (0.07 g / 95% yield). 1H NMR: (D2O) 2.25 (m, 2H), 4.60 (m, 1H), 4.91 (m, 2H), 5.62 (m, 1H). 13C NMR: (D2O) 40.15, 62.45 (m), 112.76, 132.04. 31P NMR: (D2O) 16.34 (d, J = 74.49 Hz). 19F NMR: (D2O) 200.48 (m). 79

PAGE 80

P O OH OH F 126 HO O This compound was prepared in the same way as 123 in 25%yield. 1H NMR: (D2O) 2.45 (m, 2H), 4.62 (m, 1H). 13C NMR: (D2O) 39.90 (d, J = 21.7 Hz), 91.05 (dd, J = 169.7, 153.1 Hz), 180.11. 31P NMR: (D2O) 14.55 (d, J = 70.11 Hz). 19F NMR: (D2O) -204.75 (m). Protected D-mannitol was cleaved as de scribed for the synthesis of ester 95 The resulting aldehyde (5.16 g / 39.7 mmol) was dissolved in EtOH (45 mL) and the solution cooled to 0o C before the slow addition of NaBH4 over a period of five minutes. The solution was then allowed to warm to room temperature and stirred at r oom temperature for two hours. The reaction was then quenched by the addition of 3 M HCl until the pH was neutral. EtOH was then removed under reduced pressure and the aque ous phase was extracted with CH2Cl2 (4 x 40 mL). The combined organic phases were dried with MgSO4 and the solvent removed under reduced pressure to give the crude produc t as a colorless oil. The cr ude product was purified by flash chromatography (10 : 1 Hex : EtOAc) to give th e pure product as a colorless oil (4.0 g / 76% yield).87 1H NMR (CDCl3) 1.37 (s, 3H) 1.42 (s, 3H), 3.58 (m, 1H), 3.74 (m, 1H), 3.80 (m, 1H), 4.05 (m, 1H), 4.24 (m, 1H). 13C NMR (CDCl3) 25.6, 26.8, 62.5, 65.0, 77.1, 108.4. 80

PAGE 81

A solution of alcohol 127 (750 L / 6.30 mmol) and pyridine (627 L / 7.75 mmol) in CH2Cl2 (15 mL) was cooled to -60o C before the addition of tr iflic anhydride at the same temperature. The resulting solution was stirred at -60o C for 30 minutes before allowing to warm to room temperature over one hour. The organic la yer was washed with brine (1 x 6 mL), dried with MgSO4, and concentrated under reduced pressure to give a dark brown / purple oil which was used immediately in the next step without further purification.77 A round bottom flask equipped w ith a pressure equalizing dr opping funnel was flame dried while purging with Argon before charging with n -butyllithium (2.71 mL / 10.6 mmol of a 2.0 M solution in hexanes) in THF ( 15 mL). Dibromofluorophosphonate 40 (469 L / 2.45 mmol) and TMSCl (2.45 mmol / 310 L) in THF (10 mL) were added to the dropping funnel. The flask was cooled to -78o C before the dropwise addition of the c ontents of the dropping funnel at the same temperature. The resulting dark yello w / brown solution was stirred at -78o C for 15 minutes before the addition of triflate 128 (0.65 g / 2.45 mmol) in THF (8 mL) at the same temperature. This solution was stirred at -78o C for one hour before warming to 0o C followed by the dropwise addition of a solution of lithium ethoxide (prepare d by adding 0.2 g of lithium wire to 12 mL of ethanol) and the resulting solution allowed to stir at 0o C for one hour before the solution was poured into 7 mL of saturated NH4Cl. THF was removed under reduced pressure and the aqueous layer extracted with EtOAc (3 x 20 mL). The combined organics were dried with MgSO4 and the solvent removed under reduced pre ssure to give the crude product which was purified by flash chromatography (8 : 1 Hex : Et OAc to 2 : 1 Hex : EtOAc) to give the pure 81

PAGE 82

product as a slightly ye llow oil (0.49 g / 70%).46 1H NMR: (CDCl3) 1.25 (m, 12H), 2.10 (m, 2H), 3.48 (m, 1H), 4.10 (m, 6H), 4.80 (m, 1H). 13C NMR: (CDCl3) 16.5 (d, J = 3.2 Hz), 25.6, 26.8 27.0, 33.8 (d, J = 19.75 Hz), 34.95, 62.9, 63.1 (d, J = 6.60 Hz), 63.5 (m), 68.8, 69.4, 71.7 (d, J = 14.89 Hz), 72.5 (d, J = 11.74 Hz), 83.8 (d, J = 17.75 Hz), 86.1 (dd J = 7.73, 18.32 Hz), 88.4 (d, J = 17.46 Hz), 109.26. 31P NMR: (CDCl3) 18.67 (d J = 74.07 Hz), 18.35 (d J = 75.30 Hz). 19F NMR (CDCl3): -207.9 (m), -212.3 (m). A round bottom flask charged with protected phosphonate 129 (100 L / 0.39 mmol) was dried under high vacuum for four hours before sealing with a rubber septa followed by the dropwise addition of TMSBr (302 L / 2.30 mmol) to the neat compound at room temperature. The rubber septa was immediately replaced with a yellow caplugs stopper, and the resulting solution stirred at room temperature for four hours. Volatile materials we re then removed; first on the rotovap and then on high vacuum for three hours. Water (1 mL) was then added, and the resulting solution was stirred overn ight at room temperature. The resulting solution was then loaded onto a column of Dowex IX-8 resin (HCO3 form, prepared by washing the resin sequentially with di H2O, 1 M NaHCO3 and diH2O until the pH was neutral) and eluted with 100 mM NH4HCO3. The eluant was then freeze dried to give the ammonium salt as a white powder. The free phosphonic acid was obtained by several cycles of freeze drying. 1H NMR: (D2O) 1.65 (m, 2H), 3.25 (m, 2H), 3.65 (m, 1H), 4.65 (m, 1H). 13C NMR: (D2O) 34.35 (d, J = 19.5 Hz), 64.74, 65.60, 68.10 (dd, J = 2.9, 11.2 Hz), 69.80 (dd, J = 2.3, 11.5 Hz), 89.45 (dd, J = 82

PAGE 83

83 169.8, 133.45 Hz), 91.23 (dd, J = 169.5, 154.3 Hz). 31P NMR: (D2O) 16.8 ( d, J = 71.4 Hz), 17.0 ( d, J = 70.11 Hz). 19F NMR: (D2O) -203.75 (m), -207.8 (m). A solution of diol (32 mol / 6 mg), NAD+ (50 mg / 80 mol), glycerol-1-phosphate dehydrogenase (80 U), and oxidase from L Sansfrancinsens (80 U) in deuterated KPi was gently stirred for 24 hours at room temperature. The reaction was monitored by 19F NMR. After 24 hours, integration between the two diastereomers changed from 0.8 : 1 to 0.4 : 1 (Figure 3-45).

PAGE 84

Figure 3-1. Phosphate triester, phosphonate and -fluorinated phosphonates Figure 3-2. Phosphate and phosphonic acid p K a2 values O P O OH OP O OH OP O OH OF P O OH OR R R R F F 118o112o113o116o33 34 35 36 Figure 3-3. Dihedral angle compar ison for phosphate and phosphonic acids P O P O O-iPr O-iPr iPr-O iPr-O F 1)LDA/THF/-78oC 2)Benzaldehyde 46% P O O-iPr O-iPr F 37 38 Figure 3-4. Bisphosphonate route to -fluorophosphonates 84

PAGE 85

Figure 3-5. Savignacs routes to -fluorophosphonates 85

PAGE 86

Figure 3-6. Fluorination via nucle ophilic and elec trophilic sources 86

PAGE 87

Figure 3-7. Methods for asymmetric -fluorophosphonate synthesis 87

PAGE 88

Figure 3-8. Glycerol-3-phosphate analogues synthesized by OHagan Substrate K m 61 X = O 0.20 mM 62 X = CH2 0.18 mM 63 X = CHF 0.17 mM 64 X = CF2 0.73 mM Figure 3-9. OHagans Glycerol -1-phosphate dehydrogease assay Figure 3-10. The diastereomers of 63 88

PAGE 89

Figure 3-11. Glucose-6-phosphate analogs synthesized by Berkowitz Figure 3-12. Oxidation of glucose6-phosphate to 6-phos phogluconolactone 89

PAGE 90

Figure 3-13. Aldol reactions w ith class I and class II aldolase Figure 3-14. Rabbit muscle aldolase catalyzed reaction with nonnatural donor substrate 80 90

PAGE 91

Figure 3-15. Phosphonic acid carbo hydrates synthesized by Fessner Figure 3-16. Route to -fluorinated phosphonic acid carbohydrates 91

PAGE 92

Figure 3-17. Fluorinated al dolase substrate mimics via -ketophosphonates 92

PAGE 93

O O P O OMe OMe P O OMe OMe O O O OCH3 2)Br2/MeOH/NaHCO3/ H2O 60% 1)NaIO4/NaHCO3/H2O CH2OH H HO H HO OH H OH H CH2OH O O /DMSO/ p -TsOH H O H HO OH H O H O O 53% 1) n -BuLi/THF/-78oC 2) 88 /THF/-78oC 90% 1)NaH/THF/0oCtor.t. 2)Selectfluor/CH3CN 20% O O P O OMe OMe F O O 93 94 95 96 97 97 88 Figure 3-18. Fluorinated acetonide synthesis 93

PAGE 94

O O P O OMe OMe F OH Reductase([H]) 88 89 O O P O OMe OMe F OH NaBH4/MeOH 88 98 O O P O OMe OMe O 99 O F [H]/Reductase O O P O OMe OMe OH 100 O F Figure 3-19. Racemic standard, redu ction failure and carbonate phosphonate Figure 3-20. Synthesis of carbonate s and subsequent difluorination 94

PAGE 95

Figure 3-21. Enone reductase route to -fluorovinylphosphonic acids 95

PAGE 96

Figure 3-22. Synthesis of protected vinyl phosphonate 113 96

PAGE 97

Figure 3-23. Dibromofluorophosponate route to vinyl phosphonate 113 P O OEt OEt F 113 O O AcO AmberlystA-26 P O OEt OEt F 114 O O HO MontmorilloniteClay MeOH P O OEt OEt F 104a HO O 75% CH2Cl260% Figure 3-24. Synthesis of vinyl phosphonate 104a Figure 3-25. Synthesis of racemic phosphonate standard 97

PAGE 98

Figure 3-26. Screening of vinyl -fluorinated phosphonate with en-reductases Figure 3-27. Stoichiometric reduction of vinyl phosphonate with enzyme and NADPH Figure 3-28. Fluorine elimination mechanism 98

PAGE 99

P P O OEt OEt 107 O EtO EtO + H 112 O O AcO O n -BuLi/THF/-78oC P O OEt OEt 120 O O AcO 120 AmberlystA-26 P O OEt OEt 121 O O HO MontmorilloniteClay MeOH P O OEt OEt 119 HO O 82% 78% CH2Cl290% Figure 3-29. Synthesis of unf luorinated vinyl phosphonate Figure 3-30. Inseparable ketal deprotection products Figure 3-31. Synthesis of unsaturated phosphonate 99

PAGE 100

Figure 3-32. KMnO4 alkene oxidation and phosphonate deprotection Figure 3-33. RuO4 catalyzed alkene oxidati on and phosphonate deprotection Figure 3-34. Route to -hydroxyketone via initial phosphonate deprotection 100

PAGE 101

Figure 3-35. Alkene oxidation of phosphonic acid 125 Figure 3-36. Selective diol oxidation of phosphonic acid 63 101

PAGE 102

Figure 3-37. Synthetic route to diol 63 102

PAGE 103

Figure 3-38. Reduction of -fluoro-ketophosphonate 130 with purified enzymes Figure 3-39. Oxidation of diol 63 followed by in situ aldolase reaction 103

PAGE 104

O O P O OEt OEt 132a OH P O OH OH 85b F O HO steps O O P O OEt OEt 132b OH P O OH OH 85a FO HO steps Figure 3-40. Future route to optically pure -fluorophosphonic acids 104

PAGE 105

Figure 3-41. Future synthetic methods 105

PAGE 106

Figure 3-42. Proposed synt hesis of optically pure -fluorophosphonate 63b O O P O OEt OEt 139 OAc O O P O OEt OEt 132b OH [ t -Bu2SnOH(Cl)]2MeOH P O OH OH 63a F steps HO OH Figure 3-43. Proposed synthesis of -fluorophosphonic acid 63a 106

PAGE 107

Figure 3-44. Proposed synthesis of -fluorophosphonic ac id carbohydrates 107

PAGE 108

Figure 3-45. Decrease in starting material over a 24 hour period 108

PAGE 109

Table 3-1. Fluorination conditions of carbonate 102 Base / Solvent / Temperature F + Agent / Solvent / Temperature NaH / THF / 0o C Selectfluor / THF / 0o C NaH / THF / 0o C Selectfluor / CH3CN / 0o C NaH / CH3CN / 0o C Selectfluor / CH3CN / 0o C NaH / CH3CN / -10o C 2 equiv. Selectfluor / CH3CN / -10o C LDA / THF / -78o C to -10o C Selectfluor / THF / -10o C LDA / THF / -78o C to -10o C Selectfluor / CH3CN / -10o C KH / THF / -10o C Selectfluor / CH3CN / -10o C NaH / THF / -10o C NFSI / THF / -10o C LDA / THF / -78oC to -10o C NFSI / THF / -10o C KH / THF / -10o C NFSI / THF / -10o C 109

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115 BIOGRAPHICAL SKETCH Neil Stowe was born in Atlanta, GA. He went Delta State University in Cleveland, MS where he received his B.S. in Chemistry in 2003. He came to the University of Florida in Fall, 2003. Upon completion of his Ph. D. he plans to work at the Scri pps Research Institute in San Diego, California.