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A Benzotriazole-Based Total Synthesis of Cyclic Heptapeptide Rolloamide B

Permanent Link: http://ufdc.ufl.edu/UFE0045487/00001

Material Information

Title: A Benzotriazole-Based Total Synthesis of Cyclic Heptapeptide Rolloamide B
Physical Description: 1 online resource (46 p.)
Language: english
Creator: Caliskan, Eray
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: benzotriazole -- peptide -- rolloamide
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In the present study, the first total synthesis of Rolloamide B, a cyclic proline-enriched heptapeptide is reported. The work features solution phase benzotriazole-mediated peptide synthesis ligating native amino acids. Cyclic heptapeptide Rolloamide B was achieved on a scale of130 mg in a total of 9 steps ( shortest route, 16 steps for longest route) an overall yield 12.8% (3.6% for shortest route) and an average yield of 79.8%(82.0% average yield of longest route).
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 Eray Caliskan.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045487:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045487/00001

Material Information

Title: A Benzotriazole-Based Total Synthesis of Cyclic Heptapeptide Rolloamide B
Physical Description: 1 online resource (46 p.)
Language: english
Creator: Caliskan, Eray
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: benzotriazole -- peptide -- rolloamide
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In the present study, the first total synthesis of Rolloamide B, a cyclic proline-enriched heptapeptide is reported. The work features solution phase benzotriazole-mediated peptide synthesis ligating native amino acids. Cyclic heptapeptide Rolloamide B was achieved on a scale of130 mg in a total of 9 steps ( shortest route, 16 steps for longest route) an overall yield 12.8% (3.6% for shortest route) and an average yield of 79.8%(82.0% average yield of longest route).
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 Eray Caliskan.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045487:00001


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1 A BENZOTRIAZOLE BASED TOTAL SYNTHESIS OF CYCLIC HEPTAPEPTIDE ROLLOAMIDE B By ERAY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF M ASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2

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3 To my parents Mrs. Gulcan, Mr. Ethem and my siblings Ms. Miray, Mr. T ugay for their endless support

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4 ACKNOWLEDGMENTS I would like to t hank my advisor Alan R. Katritzky for the opportunity, great understanding and support during my research. I thank especially my group members Dr. Mirna El Khatib, Dr. Davit Jishkariani, Mr. Mohamed Elagawany Mr. Mohamed Monem Ibrahim, Dr. Vadim Popov, Ms Emily F. Davis, Dr. C. Dennis Hall, and all other Katritzky group members. I would like to thank my friends in Gainesville, Mr. Huseyin Bahtiyar, Mr. Ersen Gokturk, Mr. Sukru Gulec, Mr. Turgut Sonmez, Mr. Serhat Obuz and my roommate Mr. Caglar Doguer. I thank my committee members, Prof. J. Eric Enholm and Dr. Ion Ghiviriga for their helpful discussion. I also would like to thank my parents and my siblings for their endless support and encourage.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF SCHEMES ................................ ................................ ................................ ........ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 CYCLIC PEPTIDES AND BENZOTRIAZOLE ................................ ......................... 12 1.1 Introduction ................................ ................................ ................................ ....... 12 1.2 Cyclic Peptides ................................ ................................ ................................ 13 1.3 N Substituted Benzotriazoles ................................ ................................ ............ 14 1.4. Acylbenzotriazol es ................................ ................................ ........................... 15 2 TOTAL SYNTHESIS OF CYCLIC HEPTA PEPTIDE ROLLOAMIDE B .................. 17 2.1 Introduction ................................ ................................ ................................ ....... 17 2.2 Synthetic Approach ................................ ................................ ........................... 18 2.2.1 Synthesis of ( S ) ethyl 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((benzoyloxy)methyl) 6,12 di(( S ) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylate (2.3) .... 19 2.2.2 ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((Benzyloxy)carbonyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carbo xylic acid (4a) and ( S ) tert Butyl2 ((( S ) 1 ((( S ) 1 (( S ) 2 (ethoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidine 1 carboxylate (2.4). ............ 21 2.2.3 Synthesis of (6S,9S,12S) 9 ((benzoyloxy)methyl) 6,12 di((S) sec butyl) ................................ ................................ ................................ ............ 22 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oic acid (2.23) ....... 22 2.2.4 Synthesis of ( S ) 1 (( S ) 2 carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminium chloride (2.12a) and ( S ) 1 (( S ) 2 (ethoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminiu m chloride (2.12b). ................................ ................................ ................................ .......... 24 2.2.5 Synthesis of ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((benzoyloxy)methyl) 6,12 di((S) sec butyl) 2, 2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (2.24) ................................ ................................ ................................ ............. 25

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6 2.2.6 Synthesis of 2S,3S) 1 (((S) 3 (benzoyloxy) 1 (((2S,3S) 1 ((S) 2 (((S) 1 (((S) 1 ((S) 2 carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidin 1 yl) 3 methyl 1 oxope ntan 2 yl)amino) 1 oxopropan 2 yl)amino) 3 methyl 1 oxopentan 2 aminium chloride (2.25). ................................ ................................ ................ 25 2. 2.7 Synthesis of cyclic hepta peptide Rolloamide B (2.1) .............................. 26 2.3 Conclusion ................................ ................................ ................................ ........ 27 2.4 Experimental Section ................................ ................................ ........................ 27 2.4.1. General procedure fo r the preparation of N (Z Aminoacyl)benzotriazoles (7a, 9a) ................................ ................................ 27 2.4.2. General procedure for synthesis of N (Boc Aminoacyl)benz otriazoles using DCC (2.14, 2.19). ................................ ....... 29 2.4.3. General Procedure for the preparation of N (Boc aminoacyl)benzotriazoles using BtSO 2 Me ................................ .................... 30 2.4.4. General procedure for the preparation of peptides using benzotriazole methodology (4a, 5, 8a b, 10a, 15a b, 20) .............................. 31 2.4.5. General procedure for the preparation of peptides (2.4b, 2.3) ................ 34 2.4.6. General procedur e for the deprotection of Boc protecting group (2.12a b, 2.21, 2.23b, 2.25) ................................ ................................ ........... 36 2.4.7. General procedure for the macrocyclization of linear heptapeptide (2.26) ................................ ................................ ................................ ............. 38 2.4.8. General procedure for ester hydrolysis of linear heptapeptide (2.24) ..... 39 2.4.9. General procedure for the deprotection of the Cbz and Bz protecting group (2.1) ................................ ................................ .................... 41 3 SUMMARY OF ACHIEVEMENTS ................................ ................................ .......... 42 LIST OF REFERENCES ................................ ................................ ............................... 43 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 46

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7 LIST OF SCHEMES Scheme page 1 1 Benzotriazole 1.5 and typical reactions of N substituted benzotr iazoles ............ 15 1 2 Preparation of N Acylbenzotriazole 1.9 ................................ .............................. 16 2 1 Retrosynthesis of Rolloamide B 2.1 ................................ ................................ .... 19 2 2 Attempted step for deprotection of Cbz group ................................ ................... 20 2 3 Synthesis of linear hepta peptide 2.3 ................................ ................................ .. 20 2 4 Attempted preparation of intermediate 2.4 ................................ ......................... 21 2 5 Retrosynthetic scheme for preparation of intermediate 2.4 ................................ 22 2 6 Synthesis of intermediate 2. 4. ................................ ................................ ............ 22 2 7 Attempted preparation of benzotriazole derivative 2.17 ................................ ...... 23 2 8 Synthesis of intermediate 2.23 ................................ ................................ ........... 23 2 9 Synthesis of 2.12a and 2.12b ................................ ................................ ............ 24 2 10 Synthesis of intermediate 2.24 ................................ ................................ ........... 25 2 11 Synthesis of compound 2.25 ................................ ................................ .............. 25 2 12 Synthesis of Rolloamide B 2.1 ................................ ................................ ............ 26

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8 LIST OF FIGURES Figure page 1 1 Examples of cyclic peptide drugs Vasopressin 1.1 an d Oxytocine 1.2 ............... 14 1 2 1.3 and 1.4 Substituted Benzotriazoles ................................ .............................. 14 2 1 Structure of Rolloamide B 2.1 ................................ ................................ ............. 17

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9 LIST OF ABBREVIATIONS Anal. Analytical Aq. Aqueos BtH Benzotriazole BtSOBt 1, 1' sulfinylbis(1H benzo[d][1,2,3]triazole) Boc Ter butoxycarbonyl group o C Celsius degree Cbz (benzyloxy)carbonyl group Calcd. Calculated CDCl 3 Deuterated chlorof orm CH 2 Cl 2 Methylene chloride CH 3 CN Acetonitrile d doublet DCC Dicyclohexylcarbodiimide DCM Methylene chloride dd Doublet of doublet DIPEA Diisopropylethylamine DMSO d 6 Deuterated dimethyl sulfoxide DMF Dimethyl formamide e. g. Exempli grata et al And others Et 3 N Triethylamine EtOAc Ethyl acetate FDPP Pentafluorophenyl diphenylphosphinate g Gram

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10 h Hour HCl Hydrochloric acid Hz Hertz J Coupling constant K 2 CO 3 Potassium carbonate LiOH Lithium hydroxide m Multiplet MeCN Acetonitrile mL Mililiter mmol Mil imol MgSO 4 Magnesium sulfate mp Melting point N Normal Na 2 CO 3 Sodium carbonate Na 2 SO 4 Sodium sulfate NMR Nuclear magnetic resonance Ph Phenyl Pka Logarithmic measure of the acid dissociation constant q Quartet rt room temperature s Singlet t Triplet THF T etrahydrofuran W Watt Chemical shift

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A BENZOTRIAZOLE BASED TOTAL SY N THESIS O F CYCLIC HEPTAPEPTIDE ROLL O AMIDE B By Eray May 2013 Chair: Alan R. Katritzky Major: Chemistry In the present study, t he first total synthesis of Rolloamide B, a cyclic proline enriched heptapeptide is reported. The work features solution phase benzotriazole mediated peptide synthesis ligating native amino acids. Cyclic heptapeptide Rolloamide B was achieved on a scale of 130 mg in a total of 9 steps ( shortest route, 16 steps for longest route) an overall yield 12.8% (3.6% for shortest route) and an average yield of 79.8% (82.0% average yield of longest route).

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12 CHAPTER 1 CYCLIC PEPTIDES AND BENZOTRIAZOLE 1.1 Introduction Peptides a group of compounds consisting of two or more amino acids linked by peptide bond, are abundantly present in living organisms. Thousands of peptides have been isolated from animals, plants and microorganisms. Based on their chemical structure, peptides can be divided into linear and cyclic peptides. Studies have shown that most peptides isolated from plants are cyclic p eptides and is they exhibit more potent biological activity, possibly due to the stable configuration provided by their cyclic structure. Pharmacological studies have proved that many peptides, including those isolated from plants, have potential antitumor effects. These peptides have a number of advantages over other chemical reagents including their low molecular weight, relatively simple structure, lower antigenicity and fewer adverse actions, easy absorption and various routes of administration [06AJTM1 ]. Peptides display a wide variety of biological functions and many have marked physiological properties. For example, they function as structural molecules in tissues, as enzymes, antibodies, neurotransmitters, and hormones that control many physiological processes ranging from gastric acid segreg ation and carbohydrate metabolism to growth. The toxic components of snake and spider venoms are usually peptide in nature, as a consequence of the sequence of amino acids in the peptide or protein [ 02 MNP]. Today, peptides have attracted considerable attention as therapeutics due to their roles as mediators of key biological functions together with their low toxicity and high specificity. In 2010, four of the 60 peptide drugs on the market, reached global

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13 sales ove r US$ 1 billion while over 500 pepti des are in clinical development [12CEJ11536, 10DDT40] 1.2 Cyclic Peptides Cyclic peptides and their derivatives continue to hold the attention of synthetic chemists and biologists alike. Apart from the occurrence of a v ariety of naturally occurring bioactive metabolites, cyclic peptides are often more stable in vivo than their linear counterparts and therefore often represent promising drug candidates. Another feature that contributes to the appeal of cyclic peptides is their reduced conformational mobility which allows them to be used in the study and mimicry of protein folding and to present diverse functionality in a defined and predictable manner [01JCSPT1 471]. Their t opology enhances their activity and their physico chemical stability provides a mechanism to resist digestion by exo and endo peptidase [11NC509] making them attractive targets for drug discovery and biomedical research [03JPS472, 01JCSPT1 471]. Examples of cyclic peptide drugs include natural antibiotic vancomycin, hormone oxytocine, neuropeptide vasopressin and antibiotics cyclosporine and tyrocidine A [10ACIE8625, 11ACIE12240, 12CEJ2409, 10CEJ5528, 12ACIE4637]. Cyclic peptides are also important targets in peptide synthesis for their interesting biolo gical properties. Constraining highly flexible linear peptides by cyclization is one of the most widely used approaches to define the bioactive conformation of peptides. In addition, cyclic peptides often have increased receptor affinity/selectivity and me tabolic stability over their linear counterparts [99TL8197].

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14 Vasopressin 1.1 Oxytocine 1.2 Figure 1 1. Examples of cyclic peptide drugs Vasopressin 1.1 and Oxytocine 1.2 1.3 N Substituted B enzotriazoles Benzotriazole is a synthetic auxiliary which offers many advantages. It is an inexpensive, stable compound. It is soluable in ethanol, benzene, toluene, chloroform, and DMF; sparingly soluable in water but highly soluable in basic solution be cause it is an acidic of appreciable strength with acid pKa = 8.2. Figure 1 2. 1.3 and 1.4 Substituted Benzotriazoles To be useful as a synthetic auxiliary, a group must be display several characteristics. First, it must be easy to remove at the end of the synthetic sequence (it is an added advantage if it can be recovered and used again) second, it must be able to be introduced readily at the end of sequence. Third, it should be stable during various synthetic operations, and if possible, exert an activating influence on other parts of molecule. Benzotriazole displa ys all of these characteristics [98CR409]

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15 Scheme 1 1. Benzotriazole 1.5 and typical reactions of N substituted benzotriazoles N Substituted derivatives of benzotriazole have some interesting properties. Benzotriazole possesses both electron donor and electron ac ceptor properties and, attached to a benzotriazole nitrogen can ionize in two ways, either to form the benzotriazole anion and an immon ium, oxonium, or thioni um cation, or to ioniz e off the heteroatom substituent [98CR409, 00JOC8210] 1.4. Acylbenzotriazoles N (Protected aminoacyl) benzotriazoles 1.9 are efficient intermediates for N and O aminoacylation. These intermediates allow rapid preparation of peptides in high yields and purity under mild reaction conditions with full retention of the original chirality. The developed methodology allows simple solution and solid phase preparative techniques to generate complex peptides and peptide conjugates [09S2392, 11CR6577]. Two me thods have been developed recently to prepare a wide range of N acylbenzotriazoles starting directly from carboxylic acid the first method uses

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16 Et 3 N, carboxyl acids are converted into the desired acylbenzotriazoles, possibly through intermediate formation of the mixed carboxylic sulfonic anhydride and benzotriazole anion, which is then acylated by the mixed anhydride [00JOC8210]. The second method involves treatment of a carboxyl acid with BtSOBt prepared in situ from thionyl chloride and an excess of benzotriazole (Scheme 1.2B) [03S2795]. Scheme 1 2. Preparation of N Acylbenzotriazole 1.9 The two methods allow the preparation of a wide range of N acylbenzotriazoles. The c arboxylic acid starting materials include alkyl aryl and, a w ide range of heterocyclic acids [09S2392, 11CR6577]

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17 CHAPTER 2 TOTAL SYNTHESIS OF CYCLIC HEPTA PEPTIDE ROLLOAMIDE B 2.1 Introduction Marine sponges continue to be a source of secondary metabolites with unusual chemical diversity and remarkable biological activity. Numerous peptides have been isolated from marine sponges and, have attracted considerable attention because of their unique structural framework, r ich physiochemical properties, and potential as important drug candidates [05BMNP]. A noteworthy class of marine cyclopeptides is represented by proline rich compounds, usually containing seven or eight amino acid residues. The role of proline in these mol ecules, which often occur as complex mixtures of structurally related derivatives, has been associated with control of conformation of the molecule in solution because of restricted dihedral angle of proline [97JACS6962, 09JNP1555]. 2.1 Figure 2 1 Structure of Rolloamide B 2.1 Rolloamide B 2.1 is a cyclic, proline enriched heptapeptide derived from a marine sponge. Marine sponges are a rich source of natural products and non ribosomal cyclic peptides with seven to ten amin o acid residues derived from secondary sponge

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18 metabolites show biological activity [05CSR255, 11NC509, 10JNP738, 10JNP650]. Their reduced zwitterionic character provides more lipophilicity and thus increases membrane permeability, compared to linear analog s [10JNP738]. The two prolines present in 2.1 render this natural product intriguing. Proline units reduce the backbone flexibility of cyclic peptide raising affinity and selectivity for protein binding [10JNP738]. In addition, proline facilitates the form ation of turns [06ACIE1780, 03JACS1221] and increases structural diversity as a result of cis/trans isomerization across the Xaa Pro peptide bond [06ACIE1780, 12ACIE3708]. In addition, Rolloamide B 2.1 possesses a hydroxy amino acid moiety in its serine unit, which is particularly attractive due to its solubilizing effect. Hydroxy amino acid units, Ser/Thr, are present in numerous biological by active natural products possessing a wide range of activity as antibiotics and immunosuppressants, e.g. vancomyci n, and echinocandin D [97JACS11734, 06JOC7106]. 2.2 Synthetic Approach We attempted the synthesis of Rolloamide B 2.1 (Figure 2 1) by generation of lactone 2.2 by the reaction of the free OH group in Ser with the carboxylic group. Deprotection, gives a m ore conformationally flexible peptide bringing the free N terminus in close proximity to the C terminius thus facilita ting an O to N acyl transfer affording the cyclic native peptide.

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19 Scheme 2 1 Retrosynthesis of Rolloamide B 2.1 2.2.1 Synthesis of ( S ) ethyl 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((benzoyloxy)methyl) 6,12 di(( S ) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylprop anoyl)pyrrolidine 2 carboxylate (2.3) With both the tetrapeptide 2.4 and tripeptide 2.23 in hand, the second synthesis subgoal was fragment ligation of 2.4 with 2.23 to prepare the requisite linear heptapeptide 2.3 However, the anticipated Cbz deprotecti on of 2.4a,b with hydrogen and catalytic amount of Pd/C consistently failed to give intermediates 2.5a,b (Scheme 2 2).

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20 Scheme 2 2 Attempted step for deprotection of Cbz group However, removal of the Boc protecting gro up removal of 2.4c gave 2.5c which coupled with fragment 2.23 to give linear heptapeptide 2.3 in 81% yield. Scheme 2 3 Synthesis of linear hepta peptide 2.3

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21 2.2.2 ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((Benzyloxy)carbonyl)pyrrolid ine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (4a) and ( S ) tert Butyl2 ((( S ) 1 ((( S ) 1 (( S ) 2 (ethoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidine 1 carb oxylate (2. 4 ). The initial synthesis of 2.4 began by the linear coupling of amino acid residues starting from Z L Pro OH 2.6a using benz otriazole methodology (Scheme 2 4). Although the linear tripeptide 2.10a was prepared succesfully, all attempts to prepa re tri peptide Bt 2.11a failed. Thus, a different strategy for the synthesis of 2.4 was devised. Scheme 2 4 Attempted preparation of intermediate 2.4 Alternatively, intermediate 2.4 was synthesized by ligating two dipep ti de fragments together (Scheme 2 5). Starting material 2.9a or 2.8a,b synthesized according to Scheme 2.3, fulfil to be one of the dipeptide fragments.

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22 Scheme 2 5 Retrosynthetic scheme for preparation of intermediate 2 .4 The addition of 2.9 to 2.12a in MeCN and three equivalents of DIPEA at 78 o C gave the desired intermediate 2.4a (75%) as a crystalline solid (m.p. 56 o C) (Scheme 2 6). Direct esterification of tetrapeptide 2.4a failed to give 2.4b but, the reaction o f 2.12b with 2.9a,b gave desired 2.4b,c Intermediate 2.4c was also prepared by the direct coupling of 2.12b and 2.8b both prepared using benzotriazole activation Scheme 2 6 Synthesis of intermediate 2. 4. 2.2.3 Synt hesis of (6S,9S,12S) 9 ((benzoyloxy)methyl) 6,12 di((S) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oic acid (2.23) The synthesis of intermediate 2.23 started from commercially available Boc L Ser OH 2.16 (Scheme 2 7). However, preparation of the benzotriazole derivative of tert

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23 butyl (S) (1 (1H benzo[d][1,2,3]triazol 1 yl) 3 hydroxy 1 oxopropan 2 yl)carbamate 2.17 was unsuccessful. Scheme 2 7 Attempted preparation of benzotriazole derivative 2 .17 The benzotriazole derivative 2.19 from commercially available 2.18 was coupled with L Ile to give dipeptide 2.20 Removal of the Boc group gave 2.21 which 2.22 reacted under microwave irradiation to afford intermediate 2.23 (Scheme 2 8) Scheme 2 8 Synthesis of intermediate 2.23

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24 2.2.4 Synthesis of ( S ) 1 (( S ) 2 carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminium chloride (2.12a) and ( S ) 1 (( S ) 2 (ethoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminium chlo ride (2.12b). Starting material 2.12a was prepared as a model reaction to see if the preparation of the tetrapeptide fragment 2.4 was feasible. However, for preparation of the linear heptapeptide 2.3 ( via coupling of 2.4 with 2.23 ) the protected carboxylic moiety 2.12b was needed. Activating the carboxylic group with benzotriazole using DCC in methylene chloride at room temperature gave Boc L Phe Bt 2.14 in 88% yield. Coupling with free L Pro OH 2.15a and L Pro OEt 2.15b gave compounds 2.12a and 2.12b after Boc deprotection using HCl dioxane solution (Scheme 2 9). Scheme 2 9 Synthesis of Synthesis of ( S ) 1 (( S ) 2 Carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminium chloride 2.12a and ( S ) 1 (( S ) 2 (ethoxycarbonyl)pyrrolid in 1 yl) 1 oxo 3 phenylpropan 2 aminium chloride 2.12b

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25 2.2.5 Synthesis of ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((benzoyloxy)methyl) 6,12 di((S) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (2.24) Orthogonal protection/deprotection strategy was proceeded selective re moval of the ester function in 2.3 with LiOH gave 2.24 Scheme 2.10 Synt hesis of ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((b enzoyloxy)methyl) 6,12 di((S) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (2. 24) 2.2.6 Synthesis of 2S,3S) 1 (((S) 3 (benzoyloxy) 1 (((2S,3S) 1 ((S) 2 (((S) 1 (((S) 1 ((S) 2 carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidin 1 yl) 3 methyl 1 oxopentan 2 yl)amino) 1 oxopropan 2 y l)amino) 3 methyl 1 oxopentan 2 aminium chloride (2.25). Scheme 2 11 Synthesis of compound 2.25

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26 Peptide 2.24 was treated with HCl dioxane solution to remove the Boc group to afford 2.25 prior to macrocyclization. 2. 2.7 Synthesis of cyclic hepta peptide Rolloamide B (2.1) Key intermediate 2.25 was then cyclized with FDPP peptide coupling reagent in the presence of DIPEA to give macrocycle intermediate 2.26 in 74% yield. Removal of the benzyl protecting group gave the desired macrocycle peptide natural product, Rolloamide B ( 1 ) in 80 % an average yield. Scheme 2 12 Synthesis of Rolloamide B 2.1

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27 2.3 Conclusion The first total synthesis of cyclic heptapeptide Rolloamide B was achieved on a scale of 130 mg in a total of 9 steps (shortest route, 16 steps for longest route) an overall yield 12.8% (3.6% for shortest route) and an average yield of 79.8% (82.0% average yield of longest route). 2.4 Experimental Section Melting points were dete rmined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. NMR spectra were recorded with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. Data are reported as follows: chemical shift, multiplicity (s= sin glet, d = doublet, t = triplet, q = quartet, br s = broad singlet, m = multiplet), coupling constant ( J values) expresses in Hz. Reaction progress was monitored by thin layer chromatography (TLC) and visualized by UV light. Elemental analyses were performe d on a Carlo Erba EA 1108 instrument. 2.4.1. General procedure for the p reparation of N (Z Aminoacyl)benzotriazoles (7a, 9a) Thionyl chloride (0.6 mL, 8.00 mmol, 1.2 equiv) was added to a solution of 1 H benzotriazole (3.17 g, 26.67 mmol, 4 equiv) in methylene chloride (15 mL) to give a clear yellow solution that was stirred for 15 min a t room temperature. The amino acid (6.67 mmol, 1 equiv) was then added to give a suspension which was stirred at room

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28 temperature for the reported time. The suspension was filtered, the filtrate evaporated, the residue dissolved in EtOAc and the solution w as washed with a saturated solution of sodium carbonate. The organic portion was dried over anhyd Na 2 SO 4 filtered, and dried to give the corresponding N (Z aminoacyl)benzotriazole (2.7a, 2.9a) ( S ) Benzyl 2 (1H benzo[d][1,2,3]triazole 1 carbonyl)pyrrolidine 1 carboxylate (2.7a). Stirred for 12 h at room temperature. Yellow oil (77%). 1 H NMR (CDCl 3 ): 8.23 (d, J = 12 Hz, 1H), 8.11 (d, J = 12.9 Hz, 1H), 7.62 (t, J = 12.6 Hz, 1H), 7.48 (t, J = 11.4 Hz, 1H), 7.40 7.17 (m, 10H), 7.01 6.95 (m, 2H), 5.29 4.91 (m, 3H), 4.51 4.42 (m, 1H), 3.81 3.45 (m, 3H), 2.21 1.93 (m, 7H); 13 C NMR (CDCl 3 ): 176.2, 175.6, 171.5, 171.0, 155.7, 155.2, 154.8, 154.3, 145.8, 138.4 136.4, 136.3, 135.7, 131.2, 131.0, 131.7, 130.6, 128.5, 128.5, 128.3, 128.1, 128.0, 127.8, 127.8, 127.5, 127.4, 126.5, 126.4, 126.1, 120.2, 120.1, 114.8, 114.3, 114.3, 67.4, 60.0, 59.3, 59.3, 58.9, 47.4, 47.0, 46.6, 31.5, 30.9, 30.7, 29.7, 24.4, 24.3, 23 .7, 23.4. [06S411]. (S) Benzyl 2 (((S) 1 (1H benzo[d][1,2,3]triazol 1 yl) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidine 1 carboxylate (2.9a). Stirred for 4 h at 0 o C. Yellow solid (70%); m.p. 74 o C. Converted to compound 2.10a after checking NMR; 1 H NM R (CDCl 3 ): 8.30 8.21 (m, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.70 7.62 (m, 1H), 7.55 7.48 (m, 1H), 7.40 7.10 (m, 6H), 5.99 5.94 (m, 1H), 5.23 5.13 (m, 2H),4.61 4.30 (m, 1H), 3.68 3.30 (m, 2H), 2.40 1.40 (m, 7H), 1.18 0.70 (m, 6H); 13 C NMR (CDCl 3 ): rotamers: 175.5, 172.4, 172.0, 146.1, 136.2, 131.3, 130.7, 128.7, 128.3, 128.0, 126.5, 126.4, 126.5, 120.4, 114.6, 67.7, 60.7, 60.6, 52.1, 47.3, 47.2, 41.6, 41.2, 31.3, 29.0, 28.5, 25.6, 25.5, 25.1, 24.8, 23.4, 23.1, 22.0, 21.9, 21.6. ; Anal. Calcd for C 2 5 H 31 N 5 O 5: C, 62.35 ; H, 6. 49 ; N 14. 54 Found: C, 6 2.73 ; H, 6. 62 ; N 13. 98

PAGE 29

29 2.4.2. General procedure for s ynthesis of N (Boc Aminoacyl)benzotriazoles using DCC (2.14, 2.19). Boc protected amino acid (0.03 mol) was added to a solution of DCC (1 equiv) in methylene chloride (10 15 mL) under an atmosphere of nitrogen. After 30 minutes, BtH (1 equiv) was added and this was stirred for 12 h. The suspension was filtered on a bed of silica and celite, the filtrate evaporated, and the residue dissolved in EtOAc, then filtered on a bed of silica and celite and washed with a solution of saturated sodium carbonate, then with wa ter and brine. The organic portion was dried over anhyd Na 2 SO 4 filtered on a bed of silica, and dried to give the corresponding N (Boc aminoacyl) benzotriazole. 1 H NMR and mp of Boc L Phe Bt 14 matched that reported in the literature [04S1806]. ( S ) tert Butyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)carbamate (2.14). White solid (88%). m.p. 114.0 116.0 o C; 1 H NMR and 13 C NMR matched that reported in literature [04S1806]. ( S ) tert Butyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 3 (benzyloxy) 1 oxopropan 2 yl)carbamate (2.19). Colorless oil (75%). Converted to compound 2.20 after checking NMR; 1 H NMR (CDCl 3 ): 8.29 (d J = 8.1 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.18 7.05 (m, 5H), 5.85 5.73 (m, 2H), 4.51 (d, J = 12.3 Hz, 1H), 4.40 (d, J = 12.3 Hz, 1H), 4.20 (dd, J = 9.5, 3.2 Hz, 1H), 3.97 (dd, J = 9.9, 3.0 Hz, 1H), 1. 48 (s, 9H); 13 C NMR (CDCl 3 ): 169.6, 155.6, 146.0, 137.1, 131.3, 130.8,

PAGE 30

30 128.4, 127.9, 127.7, 126.6, 120.4, 114.6, 80.6, 73.3, 70.0, 55.3, 28.5; Anal. Calcd for C 21 H 24 N 4 O 4 : C, 63.62; H, 6.10; N 14.13. Found: C, 63.83; H, 6.49; N 13.68. 2.4.3. General Procedure for the p reparation of N (Boc a minoacyl)benzotriazoles using BtSO 2 Me A mixture of Boc protected amino acid (4.646 mmol), 1 (methylsulfonyl) 1H benzo[d][1,2,3]triazole (0.9 g, 4.646 mmol) and TEA (0.7 g, 6.969 mmol) in DMF was heated by microwave at 50 C, 50 W for 1 h. The r eaction mixture was poured onto cold Na 2 CO 3 solution and extracted with ethyl acetate. The organic layer was extracted twice with water and brine. The organic layer was dried over Na 2 SO 4 filtered and filtrate evaporated to give the corresponding N (Boc aminoacyl) benzotriazole. ( S ) tert Butyl 2 (1H benzo[d][1,2,3]triazole 1 carbonyl)pyrrolidine 1 carboxylate (2.7b). Colorless oil (81%). Converted to compound 2.8b after checking NMR. 1 H NMR (CDCl 3 ): 8.34 8.27 (m, 1H), 8.18 8.10 (m, 1H), 7.74 7.62 (m, 1H ), 7.59 7.48 (m, 1H), 5.84 5.76 (m, 1H), 3.80 3.63 (m, 2H), 2.65 2.52 (m, 1H), 2.23 2.02 (m, 3H), 1.49 (s, 4H), 1.21 (s, 5H); 13 C NMR (CDCl 3 ): 172.3, 171.6, 154.5, 153.6, 146.0, 131.4, 131.2, 130.8, 130.5, 126.5, 126.3, 125.5, 120.4, 120.2, 114.7, 114.4, 80.4, 66.0, 59.7, 59.4, 47.2,, 46.9, 31.6, 30.8, 28.6, 28.2, 24.7, 24.1, 15.4, 14.4.

PAGE 31

31 2.4.4. Gener al procedure for the preparation of peptides using benzotriazole m ethodology ( 4a, 5, 8a b, 10a, 15a b, 20 ) N (Pg Aminoacyl)benzotriazoles (1.0 mmol) in MeCN (5 mL) was added dropwise to a solution of free amino acid (2 equiv; for AA OEt, 1 equiv should be used) and Et 3 N (or DIPEA, as noted in paper)(2.5 3.5 equiv) in MeCN/H 2 O (9:1, 15 mL) at the temperature reported for each reaction and stirred until all the N (Pg aminoacyl)be nzotriazoles were consumed. Acetonitrile was evaporated the residue dissolved in EtOAc (50 mL) and washed with 3N HCl (5 x 50 mL). The organic portion was dried over anhyd. NaSO 4 filtered and concentrated to give the desired p eptide fragment No further purification was required in all cases. ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((Benzyloxy)carbonyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (2.4a). Stirred for 18 h at 78 o C rt. Colorless oil (75%). 1 H NMR (CDCl 3 ): 7.40 7.25 (m, 10H), 5.20 5.14 (m, 2H), 4.30 3.35 (m, 3H), 2.20 2.05 (m, 2H), 2.00 1.80 (m, 4H), 1.80 1.40 (m, 4H), 1.30 1.00 (m, 6H), 0.95 0.85 (m, 6H); 13 C NMR (CDCl 3 ): 176.3, 172.9, 130.1, 129.1, 128.7, 128.5, 126.6, 115.6, 68.1, 50.1, 41.6, 34.3, 30 .3, 26.1, 25.4, 23.4, 22.5, 14.7. Anal. Calcd for C 33 H 42 N 4 O 7 : C, 66.07; H, 6.70; N, 6.63. Found: C, 65.33; H, 6.98; N, 9.23.

PAGE 32

32 ( S ) 2 (( S ) 1 ((Benzyloxy)carbonyl)pyrrolidine 2 carboxamido) 4 methylpentanoic acid (2.8a). Yellow oil (69%). 1 H NMR (CDCl 3 ): 7 .52 7.32 (m, 5H), 5.17 (br s, 2H), 4.53 (br s, 1H), 4.39 (br s, 1H), 3.58 3.38 (m, 2H), 2.00 1.81 (m, 2H), 1.73 1.46 (m. 3H), 0.92 0.88 (m, 6H). 13 C NMR (CDCl 3 ): 176.6, 176.3, 175.4, 172.1, 156.5, 156.0, 136.3, 135.9, 129.5, 128.7, 128.6, 128.3, 128.1, 1 27.9, 127.2, 127.1, 67.2, 66.0, 60.7, 56.3, 54.8, 51.1, 41.1, 38.5, 37.9, 24.9, 21.9, 20.9, 15.2, 14.3. [61JOC2136]. ( S ) 2 (( S ) 1 ( tert Butoxycarbonyl)pyrrolidine 2 carboxamido) 4 methylpentanoic acid (2.8b). White sticky solid (73%). 1 H NMR (CDCl 3 ): 9 .45 (br s, 1H), 7.44 (br s, 1H), 6.82 (br s, 1H), 4.59 (br s, 1H), 4.32 (br s, 1H), 3.52 3.33 (m, 2H), 2.32 2.18 (m, 1H), 1.94 1.84 (m, 2H), 1.73 1.57 (m, 4H), 1.45 (s, 9H), 0.93 (s, 6H); ; 13 C NMR (CDCl 3 ): 176.1, 173.2, 172.6, 156.0, 155.5, 81.5, 80.8, 61.2, 59.8, 59.1, 50.9, 47.2, 41.3, 31.3, 28.5, 28.4, 25.0, 23.8, 23.1, 21.9. [89DANSSSR364]. ( S ) 2 (( S ) 2 (( S ) 1 ((b enzyloxy)carbonyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoic acid (2.10a). Colorless oil (81%). 1 H NMR (CDCl 3 ): 8. 10 (br s, 1H), 7.34 7.14 (m, 10H), 5.30 5.02 (m, 3H), 4.40 4.18 (m, 4H), 2.20 2.17 (m, 2H), 2.00 1.88 (m, 4H), 1.60 1.26 (m, 5H), 0.98 0.82 (m, 6H); 13 C NMR (CDCl 3 ): 136.4, 129.7, 129.6, 128.8, 128.7, 128.4, 128.1, 127.1, 68.0, 67.8, 67.2, 59.2, 47.2, 4 6.7, 39.1, 30.8, 30.2, 29.9, 29.2, 25.1, 25.0, 24.5, 24.2, 23.7, 23.2, 23.0, 22.0, 21.9, 14.3. [90EJMC171]. ( S ) 1 (( S ) 2 (( tert Butoxycarbonyl)amino) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (2.15a). Colorless oil (85%). 1 H NMR (CDCl 3 ): 9.13 (br s 1H), 7.42 7.18 (m, 5H), 5.76 5.73 (m, 1H), 4.67 4.64 (m, 1H), 4.55 4.51 (m, 1H), 3.65 3.62 (1H), 3.18 2.90 (m, 3H), 2.15 2.02 (m, 3H), 1.91 1.21 (m, 9H); 13 C NMR (CDCl 3 ): 176.0,

PAGE 33

33 174.6, 172.1, 155.6, 136.3, 129.8, 128.5, 126.9, 80.0, 59. 3, 53.6, 47.3, 38.9, 28.8, 28.5, 24.9, 22.2, 21.0. [90EJMC171]. ( S ) Ethyl 1 (( S ) 2 (( tert butoxycarbonyl)amino) 3 phenylpropanoyl)pyrrolidine 2 carboxylate (2.15b). Stirred for 16 h at 40 o C rt. Colorless oil (90%). 1 H NMR (CDCl 3 ): 7.30 7.15 (m, 5H), 5.40 5.37 (m, 1 H), 4.22 4.18 (m, 1H), 3.71 3.53 (m, 1H), 2.85 3.2 (m, 2H), 1.80 2.00 (m, 2H), 1.40 1.22 (m, 12 H); 13 C NMR (CDCl 3 ): rotamers 171.9, 170.9, 155.4, 136.5, 129.8, 129.5, 128.6, 128.4, 127.0, 126.8, 79.7,61.9, 61.3, 59.4, 59.2, 54.0, 53.4,52.3, 47.0,39.1, 30.7, 29.2, 28.4, 25.0,22.3, 20.9, 14.3 Anal. Calcd for C 21 H 30 N 2 O 5 : C, 64.60; H, 7.74; N 7.17. Found: C, 64.29; H, 8.04; N 6.95. (2 S ,3 S ) 2 (( S ) 3 (Benzyloxy) 2 ((tert butoxycarbonyl)amino)propanamido) 3 methylpentanoic acid (2.20). Sticky solid/gel (80%). Converted to compound 21 after checking NMR; 1 H NMR (CDCl 3 ): 11.15 (br s, 1H), 7.34 7.24 (m, 6H), 5.70 (br s, 1H), 4.61 (dd, J = 8.5, 4.4 Hz, 1H), 4.51 (s, 2H), 4.40 (br s, 1H), 3.87 (dd, J = 9.3, 3.9 Hz, 1H), 3.60 (dd, J = 9.3, 6.6 Hz, 1H), 1.97 1.86 (m, 1H), 1.43 (s, 9H), 1.27 1.02 (m, 2H), 0.90 0.82 (m, 6H); ); 13 C NMR (CDCl 3 ): 174.7, 170.9, 155.7, 137.3, 128.4, 127.8, 127.6, 80.4, 73.4, 69.8, 56.7, 53.9, 37.7, 28.2, 24.8, 15.5, 15.4, 11.6 ; Anal. Calcd for C 21 H 32 N 2 O 6 : C, 61.68; H, 8.16; N, 6.88. Found: C, 61.75; H, 7.90; N, 6.86. (6 S ,9 S ,12 S ) 9 ((Ben zoyloxy)methyl) 6,12 di(( S ) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oic acid (2.5). Required microwave irradiation at 50 W, 50 o C for 1.5 h. White solid (65%). mp 75.0 77.0 o C; 1 H NMR (CDCl 3 ): 7.39 7.20 (m, 5H), 5.38 (br s, 1H), 4.75 4.68 (m, 1H), 4.57 4.53 (m, 3H), 4.14 4.06 (m, 1H), 3.87 3.82 (m, 1H), 3.61 3.54 (m, 1H), 1.97 1.81 (m, 2H), 1.42 (s,

PAGE 34

34 9H), 1.30 1.04 (m, 4H), 0.93 0.82 (m, 12H); 13 C NMR (CDCl 3 ): 174.4, 172.4, 170.3, 156.2, 137.4, 128.6, 128.0, 80.3, 73.6, 69. 6, 59.5, 57.2, 52.6, 37.5, 28.4, 25.1, 24.8, 15.7, 15.6, 11.7, 11.5; Anal. Calcd for C 27 H 43 N 3 O 7 : C, 62.17; H, 8.31; N 8.06. Found: C, 62.17; H, 8.61; N 7.83. 2.4.5. General procedure for the preparation of p eptides (2.4b, 2.3) A solution of the amino aci d with free carboxyl group (3 mmol) in dry THF (10 mL) under argon was cooled to 15 C in an ice bath with stirring. N Methylmorpholine (0.33 g, 3.2 mmol), followed by isobutyl chloroformate (0.45 g, 3.2 mmol) were added. After 4 min, a solution of the amino acid hydrochloride salt (1.5 mmol) and N methylmorpholine (0.7 g, 1.6 mmol) i n DMF (5 mL) was added. The ice bath was removed after 5 min, and the solution was allowed to stir for 12 h at room temperature. The solution was concentrated under vacuum and the residue was dissolved in ethyl acetate (30 mL) and water (5 mL). The organic phase was washed successively with saturated Na2CO3 (2 15 mL), water (10 mL), 2N HCl (15 mL), and water (10 mL). The solution was dried over MgSO4, filtered, and then concentrated under vacuum. The peptide was recrystallized from ethyl acetate hexanes to give the desired peptide. ( S ) tert Butyl 2 ((( S ) 1 ((( S ) 1 (( S ) 2 (ethoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidine 1 carboxylate (2.4b). White solid (83%). mp 81.0 83.0 o C; 1 H NMR (CD 3 OD): 7.34 7.19 (m, 5H), 4.43 4.36 (m, 2H), 4.21 4.13 (m, 3H), 3.75 3.67 (m, 1H), 3.52 3.34 (m, 3H),

PAGE 35

35 3.12 (dd, J = 14.1, 6.3 Hz, 1H), 2.91 (dd, J = 14.1, 7.5 Hz, 1H), 2.24 2.13 (m, 1H), 2.00 1.79 (m, 6H), 1.76 1.60 (m, 2H), 1.52 1.35 (m, 12H), 1.26 ( t, J = 7.2 Hz, 3H), 0.96 0.87 (m, 6H); 13 C NMR (CD 3 OD): 175.5, 175.3, 174.4, 173.4, 172.8, 172.1, 171.9, 156.5, 156.1, 138.1, 137.7, 130.7, 130.6, 129.8, 129.6, 128.3, 127.9, 81.4, 62.3, 61.3, 60.8, 60.7, 54.0, 54.0, 53.1, 48.0, 47.5, 42.4, 38.7, 32.6, 3 0.2, 28.8, 25.9, 25.9, 24.7, 23.6, 22.2, 14.7; Anal. Calcd for C 32 H 48 N 4 O 7 : C, 63.98; H, 8.05; N 9.33. Found: C, 63.68; H, 8.32; N 8.64. S ) Ethyl 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((benzoyloxy)methyl) 6,12 di(( S ) sec butyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylate (2.3). White solid (81%). mp 74.0 76.0 o C; 1 H NMR (CDCl 3 ): 7.25 7.09 (m, 10H), 4.85 (br s, 1H), 4.72 4.63 (m, 1H), 4.55 4.32 (m, 4H), 4.22 (br s, 1H), 4.14 4.00 (m, 2H), 3.90 3.72 (m, 3H), 3.70 3.20 (m, 5H), 3.10 2.95 (m, 1H), 2.89 2.79 (m, 1H), 2.16 1.75 (m, 9H), 1.60 1.41 (m, 4H), 1.34 (s, 9H), 1.19 (t, J = 7. 1 Hz, 3H), 1.13 1.04 (m, 4H), 0.88 0.70 (m, 18H); 13 C NMR (CDCl 3 ): 174.3, 173.6, 171.9, 171.7, 170.1, 169.9, 169.6, 155.8, 137.4, 136.2, 129.7, 129.3, 128.6, 128.3, 127.8, 126.8, 79.8, 73.5, 73.4, 73.3, 71.8, 69.8, 69.6, 61.1, 59.1, 59.0, 52.7, 52.0, 47 .0, 38.4, 37.5, 29.0, 28.3, 27.9, 26.2, 24.8, 24.7, 23.0, 22.1, 21.7, 19.0, 15.6, 15.5, 14.4, 14.2, 11.8, 11.7, 11.5; Anal. Calcd for C 54 H 83 N 7 O 12 : C, 63.44; H, 8.18; N 9.15. Found: C, 63.19; H, 8.63; N 9.12.

PAGE 36

36 2.4.6. General p rocedure for the deprotection of Boc protecting group (2.12a b, 2.21, 2.23b, 2.25) Boc Protected amino acid (1.0 mmol) was dissolved in HCl dioxane (4.0 M HCl in dioxane, 15 mL) and stirred for 1 h. Solvent was evaporated, ether was added to the residue and the suspension was sti rred for 2h. Filtration (when sticky solid resulted, decantation of ether several times was performed instead) gave the required amino acid/peptide as a hydrochloride salt. ( S ) 1 (( S ) 2 Carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminium chloride (2.12a ). White solid (95%). m.p. 119 o C; 1 H NMR (CD 3 OD): mixture of rotamers 7.31 7.13 (m, 5H), 4.42 4.20 (m, 2H), 3.22 3.09 (m, 2H), 3.02 2.85 (m, 2H), 2.22 2.02 (m, 1H), 1.98 1.62 (m, 2H), 1.60 1.40 (m, 1H); 13 C NMR (CD 3 OD): 175.1, 168.9, 135.7, 131.5, 131.0, 130.6, 130.5, 129.3, 68.5, 61.2, 54.8, 48.1, 38 .2, 30.5, 26.3. Anal. Calcd for C 28 H 40 Cl 2 N 4 O 7 : C, 54.63; H, 6.55; N 9.10. Found: C, 54.19; H, 6.68; N 8.76. ( S ) 1 (( S ) 2 (E thoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 aminium chloride (2.12b). White solid (97%). m.p.114 o C; 1 H NMR (CDCl 3 ): mixt ure of rotamers 7.36 7.10 (m, 5H), 4.42 4.25 (m, 1H), 4.20 4.00 (m, 2H), 3.55 (s, 2H), 3.25 3.00 (m, 2H), 3.05 2.90 (m, 2H), 2.01 2.20 (m, 1H), 1.84 1.72 (m, 3H), 1.22 1.16 (m, 3H); 13 C NMR (CDCl 3 ): 174.5, 168.4, 135.2, 130.9, 130.6,129.7, 130.1, 130.0, 128.8,

PAGE 37

37 68.1, 64.0, 62.3, 60.8, 54.4, 54.2, 53.0, 48.1, 40.0, 37.8, 31.5, 30.0, 25.9, 23.5, 14.5 [50BJ196]. ( S ) 3 (Benzoyloxy) 1 (((1 S ,2 S ) 1 carboxy 2 methylbutyl)amino) 1 oxopropan 2 aminium chloride (2.21). Gel (95%); 1 H NMR (CD 3 OD): 7.39 7.28 (m, 5H), 4.61 (s, 2H), 4.44 (d, J = 5.3 Hz, 1H), 4.21 (dd, J = 6.6, 3.9 Hz, 1H), 3.92 (dd, J = 10.5, 3.9 Hz, 1H), 3.80 (dd, J = 10.5, 6.6 Hz, 1H), 1.98 1.89 (m, 1H), 1.57 1.44 (m, 1H), 1.34 1.20 (m, 1H), 0.99 0.90 (m, 6H); 13 C NMR (CD 3 OD): 174.1, 168.0, 138.7, 1 29.6, 129.2, 129.1, 74.7, 69.4, 58.6, 54.6, 38.5, 26.3, 16.2, 12.1; Anal. Calcd for C 32 H 52 Cl 2 N 9 O 4 : C, 54.31; H, 7.41; N 7.92. Found: C, 54.05; H, 7.28; N 7.88. ( S ) 2 ((( S ) 1 ((( S ) 1 (( S ) 2 (Ethoxycarbonyl)pyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidin 1 ium chloride (2.23b). White solid (87%). m.p. 93.0 95.0 o C; 1 H NMR (CD 3 OD): 8.38 8.28 (m, 1H), 7.18 7.05 (m, 5H), 4.32 4.22 (m, 2H), 4.17 4.13 (m, 1H), 4.06 3.99 (m, 2H), 3.64 3.55 (m, 1H), 3.31 3.19 (m, 3H ), 2.96 (dd, J = 14.1, 5.7 Hz, 1H), 2.80 2.73 (m, 1H), 2.33 2.20 (m, 1H), 2.11 1.76 (m, 7H), 1.57 1.35 (m, 4H), 1.11 (t, J = 7.2 Hz, 3H), 0.81 0.73 (m, 6H); 13 C NMR (CD 3 OD): 173.9, 173.5, 172.0, 169.8, 169.7, 138.1, 137.7, 130.8, 130.6, 129.8, 129.6, 128 .4, 127.9, 62.5, 62.4, 61.0, 60.8, 60.7, 53.9, 53.8, 53.7, 48.3, 47.6, 47.5, 41.9, 41.5, 38.6, 32.0, 31.2, 30.2, 26.0, 25.1, 23.5, 22.1, 14.6; Anal. Calcd for C 27 H 43 ClN 4 O 6 : C, 58.42; H, 7.81; N 10.09. Found: C, 58.61; H, 7.98; N 9.54.

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38 2 S ,3 S ) 1 ((( S ) 3 (Ben zoyloxy) 1 (((2 S ,3 S ) 1 (( S ) 2 ((( S ) 1 ((( S ) 1 (( S ) 2 carboxypyrrolidin 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 4 methyl 1 oxopentan 2 yl)carbamoyl)pyrrolidin 1 yl) 3 methyl 1 oxopentan 2 yl)amino) 1 oxopropan 2 yl)amino) 3 methyl 1 oxopentan 2 aminium chlor ide (2.25). White solid (91%). m.p. 115.0 117.0 o C; 1 H NMR (C D3OD ): 7.36 7.19 (m, 10H), 4.87 4.79 (m, 2H), 4.58 4.54 (m, 2H), 4.46 4.25 (m, 3H), 3.87 3.70 (m, 5H), 3.58 3.36 (m, 3H), 3.16 3.10 (m, 1H), 2.97 2.88 (m, 1H), 2.23 2.14 (m, 1H), 1.98 1.81 (m, 8H), 1.69 1.49 (m, 4H), 1.43 1.22 (m,2H), 1.05 (dd, J = 6.9, 2.8 Hz, 2H), 0.98 0.87 (m, 18H); 13 C NMR (C D3OD ): 175.2, 174.7, 174.3, 174.2, 171.9, 171.5, 169.5, 139.1, 138.1, 130.8, 129.8, 129.5, 129.1, 128.9, 127.8, 74.5, 72.8, 71.5, 71.0, 60.7, 60.6, 5 9.0, 58.5, 56.0, 54.9, 54.5, 53.8, 53.2, 48.0, 42.0, 42.1, 38.7, 38.5, 38.1, 32.0, 31.0, 30.2, 29.3, 27.4, 26.2, 25.9, 25.5, 24.6, 23.6, 22.3, 19.5, 16.1, 15.2, 12.3, 12.0, 11.8; HRMS (+ESI) m/z for C 47 H 70 N 7 O 9 [M + H] + calcd. 879.5230, found 879.5057. 2.4. 7. General procedure for the macrocyclization of linear h eptapeptide (2.26) A modified literature procedure [04JOC8804] was used ; FDPP (63 mg, 0.164 mmol, 1.5 equiv) was added to a solution of the linear heptapeptide 2.25 (100 mg, 0.110 mmol) in acetonitrile (20 mL, 0.005 M) under anhydrous conditions. DIPEA (51 mg,

PAGE 39

39 0.394 mmol, 3.6 equiv) was added to this solution. The reaction mixture was then stirred at rt overnight, then the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (30 mL) and washed with Na 2 CO 3 solution (2x 15 mL). The organic layer was dried over Na 2 SO 4 filtered and concentrated under vacuum to yield cyclized heptapeptide 2.26 ((6 S ,9 S ,12 S ,14a S ,20 S ,23 S ,25a S ) 20 benzyl 6,1 2 di(( S ) sec butyl) 23 isobutyl 5,8,11,14,19,22,25 heptaoxotetracosahydro 1H dipyrrolo[1,2 a:1',2' j][1,4,7,10,13,16,19]heptaazacyclohenicosin 9 yl)methyl benzoate (2.26). White solid (74%). m.p. 122 124 o C; 1 H NMR (C D3OD ): 7.44 7.16 (m, 10H), 4.75 4.63 (m, 1H), 4.56 4.46 (m, 2H), 4.39 4.16 (m, 3H), 3.80 3.63 (m, 6H), 3.59 3.38 (m, 3H), 3.21 3.06 (m, 1H), 3.01 2.84 (m, 1H), 2.30 2.12 (m, 1H), 2.05 1.76 (m, 8H), 1.68 1.55 (m, 2H), 1.53 1.42 (m, 2H), 1.40 1.28 (m, 3H), 1.02 0.81 (m, 19H); 13 C NMR (C D3OD ): 175.2, 175.2, 174.3, 173.7, 172.1, 139.3, 138.3, 130.7, 129.9, 129.8, 129.6, 129.5, 129.0, 128.9, 128.8, 127.9, 74.4, 72.9, 71.1, 61.7, 61.0, 55.8, 53.3, 43.8, 42.2, 41.7, 38.4, 32.8, 31.5, 30.5, 29.4, 26.2, 25.9, 24.7, 23.6, 22.3, 22.0, 19.6, 18.2, 16.2 13.3, 12.1, 11.9. HRMS m/z for C 47 H 65 N 7 O 9 [M+H] + calcd. 858.5124, found 858.5115. 2.4.8. General procedure for ester hydrolysis of linear h eptapeptide (2.24)

PAGE 40

40 A modified literature method was used. To a solution of Boc Heptapeptide ethyl ester 2.3 (400 mg, 0.398 mmol), in methanol (15 mL), THF (9 mL) was added LiOH (84 mg, 1.992 mmol) dissolved in water (3 mL). The mixture was allowed to stir at room temperature (25 C) over 2h. Solvent was evaporated under reduced press ure, the residue dissolved in water and acidified with 2N HCl, extracted with ethyl acetate. The organic layer was dried over anhyd Na2SO4 and concentrated to obtain 2.24. ( S ) 1 (( S ) 2 (( S ) 2 (( S ) 1 ((6 S ,9 S ,12 S ) 9 ((Benzoyloxy)methyl) 6,12 di((S) sec butyl ) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oyl)pyrrolidine 2 carboxamido) 4 methylpentanamido) 3 phenylpropanoyl)pyrrolidine 2 carboxylic acid (2.24). White solid (75%); m.p. 98 99 C; 1 H NMR (CDCl 3 ): 9.72 (br s, 1H), 7.28 7.10 (m, 10H) 4.86 (br s, 1H), 4.67 4.64 (m, 1H), 4.51 4.41 (m, 4H), 4.23 (br s, 1H), 3.80 3.72 (m, 3H), 3.59 3.34 (m, 4H), 3.10 2.98 (m, 2H), 2.85 (dd, J = 13.3, 5.5 Hz, 1H), 2.08 2.01 (m, 1H), 1.93 1.75 (m, 8H), 1.52 1.40 (m, 4H), 1.34 (s, 9H), 1.11 0.92 (m, 4H), 0. 87 0.73 (m, 18H); 13 C NMR (CDCl 3 ): 175.1, 174.6, 174.2, 173.7, 172.3, 170.2, 169.4, 156.5, 156.0, 137.4, 135.9, 129.9, 129.6, 128.5, 127.9, 127.9, 127.0, 80.1, 73.5, 73.4, 71.9, 69.6, 60.5, 59.4, 57.0, 55.8, 52.7, 52.6, 47.4, 41.5, 38.3, 38.0, 37.5, 28.7 28.4, 28.1, 26.2, 25.0, 24.8, 23.1, 21.9, 21.0, 19.1, 15.7, 15.6, 14.5, 11.9, 11.7, 11.5; Anal. Calcd for C 52 H 81 N 7 O 13 : C, 61.70; H, 8.07; N 9.69. Found: C, 61.57; H, 8.29; N 8.49.

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41 2.4.9. General p rocedure for the deprotection of the Cbz and Bz protect ing group (2.1) The amino acid/peptide (1.0 mmol) was dissolved in anhydrous MeOH (30 mL) and stirred under an atmosphere of hydrogen in the presence of a catalytic amount of Pd/C for 48 h. Filtration through a bed of celite and evaporation afforded the desired amino acid/peptide. (6 S ,9 S ,12 S ,14a S ,20 S ,23 S ,25a S ) 20 Benzyl 6,12 di(( S ) sec butyl) 9 (hydroxymethyl) 23 isobutylhexadecahydro 1H dipyrrolo[1,2 a:1',2' j][1,4,7,10,13,16,19]heptaazacyclohenicosine 5,8,11,14,19,22,25(25a H ) heptaone (2.1). NMR and op t ical rotation matched reported [09JNP1253]

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42 CHAPTER 3 SUMMARY OF ACHIEVEMENTS In the present of study, cyclic hepta peptide Rolloamide B was synthesized by N (p rotected aminoacyl) benzotriazole methodology and macrocyclization. The deprotection method ology was applied to remove Boc Cbz, and Bz groups for several intermadiates. Natural product Rollamide B was achieved in a total 9 steps for shortest route 16 steps for longest route and an overall yield of 13 % and an average yi eld for each step of 80 %.

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43 LIST OF REFERENCES The reference citation system employed throughout this thesis is from Katritzky, A.R.; Rees, C.W.; Scriven, E.) Each time a reference is cited, a number lett er code is designated to the corresponding reference with the first two or four of the and the page number in the end. Additional notes to this reference system are as fo llow: 1. each reference code is followed by conventional literature citation in ACS style. 2. Journals which are published in more than ine part including in abbreviation cited the appropriate part. 3. Less commonly used books and journals are still abbreviated as using initials of the journal name. 4. Patents are given by their application number. 5. The list of the references arranges according to the designated code in the order of (i) year, (ii) journal/book in alphabetical order, (iii) part number or volume number if it is included in the code, and (iv) page number. [50BJ196] Harris J.I.; Work, T.S. Biochem. J ., 1950 46, 196 [61JOC2136] Cash, W.D. J. Org. Chem. 1961 26, 2136. [89DANSSSR] Mazurov, A. A.; Kabanov V. M. ; Andronati, S.A. Doklady Akademii Nauk SSSR 1989, 306, 364. [90EJMC171] Salvadori, S.; Marastoni, M.; Balboni, G.; Borea P.; Tomatis, R. Euro. J. Med. Chem., 1990 25, 171. [ 97JACS11734] Kimura, T.; Vassilev, V. P.; Shen, G. J.; Wong, C. H. Journal of the American Chemical Socie ty 1997 119 11734. [97JACS6962] Delbert, L. H.; Giovanni, L.C.; Georg e, R. P.; Jayaram K. S J. Am. Chem. Soc. 1997 119, 6962 [98CR409] Katritzky, A R.; Lan, X ; Yang, J Z.; Denisko, O V. Chemical Reviews, 1998, 409.

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44 [99TL8197] Yang, L ; Morriello, G Tetrahedron Letters 40 1999 8197 8200 [99JACS9790] Meutermans, W D. F.; Golding, S W.; Bourne, G T.; Miranda, L P.; Dooley, M J.; Alewood, P l F.; Smythe, M L. Journal of the American Chemical Society, 1999 9790. [00J OC8210] Katritzky, A R.; He, H ; Suzuki, K Journal of Organic Chemistry, 2000 8210. [01JCSPT, 471] Lambert, J.N.; Mitchell, J.P.; Roberts, K.D. J. Chem. Soc ., Perkin Trans.1 2001 471. [02MNP] tic 2002 [03JPS471] Davies, J. S. Journal of Peptide Science 2003 9 471 [03S2795] Katritzky, A R.; Zhang, Y ; Singh, S K. Synthesis, 2003 2795. [03JACS1221] Zhang, W. J.; Berglund, A.; Kao, J. L. F.; Couty, J. P.; Gershengor n, M. C.; Marshall, G. R. Journal of the American Chemical Society 2003 125 1221. [04S1806] Katritzky, A. R .; Shestopalov A. A.; Suzuki, K. Synthesis 2004 1806 [05CSR355] Keyzers, R. A.; Davies Coleman, M. T. Chemical Society Reviews, 2005 34 355. [05BMNP] Springer, 2005 [06ACIE1780] Shi, T.; Spain, S. M.; Rabenstein, D. L. Angewandte Chemie, International Edition 2006 45 1780. [06S411] Katritzky, A.R.; An grish P.; Suzuki, K. Synthesis 2006, 411 [06JOC7106] Crich, D ; Banerjee, A Journal of Organic Chemistry, 2006 7106. [06AJTM1] Ma, X.; Wu, C.; Wang, W.; Li, X. Asian Journal of Traditional Medicines, 2006 1. [08TL4674] Lecaillon, J.; Gilles, P .; Subra, G.; Martinez, J.; Amblard, M. Tetrahedron Letters 2008 49 4674. [09JNP1555] Vera, B.; Vicente J.; A. 2009 1555.

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45 [09S2392] Katritzky, A.R.; Angrish, P.; Todadze, E. Synlett 2009 2392. [10DDT40] Vlieghe, P.; Liso wski, V.; Martinez, J.; Khrestchatisky, M. Drug discovery today 2010 15 40. [10ACIE8625] Jebrail, M. J.; Ng, A. H. C.; Rai, V.; Hili, R.; Yudin, A. K.; Wheeler, A. R. Angewandte Chemie, International Edition 2010 49 8625. [10CEJ5528] Yo o, B.; Shin, S. B. Y.; Huang, M. L.; Kirshenbaum, K. Chemistry -A European Journal 2010 16 5528. [10JNP650] Zhang, H. J.; Yi, Y. H.; Yang, G. J.; Hu, M. Y.; Cao, G. D.; Yang, F.; Lin, H. W. Journal of Natural Products 2010 73 650. [10JNP738] Cychon, C.; Kock, M. Journal of Natural Products 2010 73 738. [11NC509] White, C. J.; Yudin, A. K. Nature Chemistry 2011 3 509 [11ACIE12240] Gnanaprakasam, B.; Balaraman, E.; Ben David, Y.; Milstein, D. Angewandte Chemie, International Edi tion 2011 50 12240. [11CR6577] El Faham, A.; Albericio, F. Chemical Review 2011 111, 6557. [11MCC1087] El Khatib, M.; Jauregui, L.; Tala, S. R.; Khelashvili, L.; Katritzky, A. R. MedChemComm 2011 2 1087. [12CEJ11536] Masurier, N.; Zajde l, P.; Verdie, P.; Pawlowski, M.; Amblard, M.; Martinez, J.; Subra, G. Chemistry -A European Journal 2012 18 11536. [12CEJ2409] Marti Centelles, V.; Burguete, M. I.; Luis, S. V. Chemistry A European Journal 2012 18 2409. [12ACIE4637] Maxwell D. C.; Sandrine V. Angewandte Chemie, International Edition, 2012 4637. [12ACIE3708] Tischler, M.; Nasu, D.; Empting, M.; Schmelz, S.; Heinz, D. W.; Rottmann, P.; Kolmar, H.; Buntkowsky, G.; Tietze, D.; Avrutina, O. Angewandte Chemie, International Edition 2012 51 3708. [12OBC4836] El Khatib, M.; Elagawany, M.; Jabeen, F.; Todadze, E.; Bol'shakov, O.; Oliferenko, A.; Khelashvili, L.; El Feky, S. A.; Asiri, A.; Katritzky, A. R. Organic & Biomolecular Chemistry 2012 10 4836.

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46 BIOGRAPHICAL SKETCH Er ay Caliskan was born and raised in Elazig, Turkey. He received his Bachelor degr ee from Firat University in 2009 In 2011, he was admitted by University of Florida as a graduate student and joined the research group of Professor A.R. Katritzky at Departmen t of Chemistry. His research focuses on the total synthesis natural products and peptide ligation in synthetic organic chemistry. He received his master degree in chemistry from the University of Florida in the spring of 2013.