Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Toward the preparation of a directed library of amatoxins containing modified prolines Pellicelli, Jonathan 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-ubc_2006-0609.pdf [ 4.54MB ]
Metadata
JSON: 831-1.0061119.json
JSON-LD: 831-1.0061119-ld.json
RDF/XML (Pretty): 831-1.0061119-rdf.xml
RDF/JSON: 831-1.0061119-rdf.json
Turtle: 831-1.0061119-turtle.txt
N-Triples: 831-1.0061119-rdf-ntriples.txt
Original Record: 831-1.0061119-source.json
Full Text
831-1.0061119-fulltext.txt
Citation
831-1.0061119.ris

Full Text

T O W A R D T H E P R E P A R A T I O N O F A D I R E C T E D L I B R A R Y O F A M A T O X I N S CONTAINING MODIFIED PROLINES by Jonathan Pellicelli B . S c , Universite Laval, 2003 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Chemistry) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A May 2006 © Jonathan Pellicelli 2006 Abstract Amatoxins are bicyclic peptides found in the Amanita mushroom species; these molecules are specific inhibitors of R N A polymerase II. Crystal structure data for the amatoxin-RNA polymerase II complex suggest that the hydrogen bond between Pol II Glu882 and amatoxin hydroxyproline is the key interaction in the binding of amatoxins to Pol II. We decided to synthesize a small library of amatoxins that will investigate this suspected key interaction. The amatoxins in this library contain modified prolines which represent analogue of hydroxyproline. The formation of the library was achieved in three synthetic phases. The first phase of the project was the synthesis of the various proline derivatives chosen to be incorporated into amatoxin peptides. These prolines were fluoroproline, difluoroproline, trifluoromethylproline, cyanoproline, benzylthioproline, azidoproline, hydroxyproline, ketoproline, and thiazolidine acid. The second phase of the project was the synthesis of 3a-hydroxyhexahydropyrroloindoles (Hpi). This molecule is essential for the formation of the tryptathionine bridge (a key feature in the amatoxin molecule) via a Savige-Fontana reaction. The last phase of the project was the formation of linear octapeptides and their subsequent cyclization. Seven linear octapeptides and two monocyclic octapeptides were prepared. Phase 1 Proline derivatives preparation Phase 2 Hpi preparation Fmoc N i O \ / H Phase 3 Peptides preparation N^?~N HN H H j r O j HN o Linear octapeptide on solid support TFA H V 0 ° \_/==\ HN fer y V ^ N H Monocyclic octapeptide H 9 N ^ = 0 nn " \ H I H 2N T - ^ ^ N H Library of amatoxins i i Table of contents Abstract i i Table of contents i i i List of tables v List of figures vi List of schemes vii List of abbreviations viii 1. Introduction 1 1.1 The poisonous amanita mushrooms 1 1.2 Amatoxin and phallotoxins 2 1.3 Phallotoxins target F-actin 4 1.4 Amatoxins target transcription 4 1.5 The importance of hydroxyproline in the inhibition 6 1.6 The aim of the project 8 1.7 The synthesis of amatoxin 11 1.8 The route A synthesis 12 1.9 The Route B synthesis and formation of Hpi 14 1.9.1 Kamenecka and Danishefsky strategy to Hpi 15 1.9.2 Van Vranken strategy to Hpi 16 1.9.3 Ley strategy to Hpi 17 1.10 The Zanotti-Wieland strategy towards amatoxins 18 1.11 Previous advances from our research group 19 1.12 Our goals with the study of amatoxin and Pol II 21 2. Discussion 23 2.1 Preparation of the amino acid derivatives 23 2.1.1 Preparation of Fmoc-4-trifluoromethylproline 23 2.1.2 Preparation of Fmoc-4-fiuoroproline 28 2.1.3 Preparation of Fmoc-4-cyanoproline 29 2.1.4 Preparation of Fmoc-4,4-difluoroproline 30 2.1.5 Preparation of Fmoc-4-ketoproline 31 2.1.6 Preparation of Fmoc-(2S,4S)-4-(benzylfhio)proline 31 2.1.7 Preparation of Fmoc-(R)-thiazolidine-4-carboxylic acid 33 2.1.8 Preparation of Fmoc-4-azidoproline 33 2.2 Preparation of Hpi 35 2.2.1 Preparation of DMDO 35 2.2.2 Autodecomposition of D M D O 36 2.2.3 Preparation of Tr-Trp-Xaa-OMe 38 2.2.4 Preparation of Tr-Hpi-Xaa-OMe 38 2.2.5 Preparation of Tr-Hpi-Gly-OH 41 2.3 Solid phase synthesis of amatoxin derivatives 42 iii 3 . Conclusion 47 3.1 Work completed 47 3.2 Future perspectives 48 4. Experimental Section 49 4.1 General 49 4.1.1 Synthesis 49 4.1.2 Thin layer chromatography 49 4.1.3 Chromatography 50 4.2 Chemical methods 50 4.2.1 Preparation of the various proline derivatives 50 4.2.2 Preparation of the Hpi 70 4.2.3 Preparation of the peptides 81 References 89 Appendices • 91 Appendix 1: Representative HI N M R spectra in deuterated methanol 91 Appendix 2: Representative ESI Mass spectra 95 i v List of tables Table 1: Tr-Hpi-Xaa-OMe dipeptides 40 Table 2: Tr-Hpi-Xaa-OMe dipeptides made by Dr. Jonathan May and Mr. Pierre 41 Table 3: General structure of the linear octapeptide and the different derivatives made 45 Table 4: General structure of the monocycle octapeptide and the different derivatives made 46 List of figures Figure 1: Amanita phalloides 1 Figure 2: Tryptathionine bridge and non ribosomal amino acids in amatoxins 2 Figure 3: Natural amatoxin compounds 3 Figure 4: Natural Phallotoxin compounds 3 Figure 5: Schematic representation of the two essential processes involved in polypeptide synthesis 5 Figure 6: Schematic drawing of Pol II complexed with a-amanitin 6 Figure 7: Structure of a-amanitin and the interaction with the different residues on Pol II 7 Figure 8: Proline ring pucker modes 8 Figure 9: Synthetic proline derivative targets 10 Figure 10: Mechanism of oxidation of a carbonyl with TEMPO and trichloroisocyanuric acid 25 Figure 11: Possible molecules obtained with the SOCb/pyridine elimination reaction and 1H N M R of the compound 6 26 Figure 12: Complexation of molecule 8 to iridium 27 Figure 13: Mechanism of the reaction with DAST 29 Figure 14: Observed NOE effects on compound 41 and 45 35 Figure 15: Autodecomposition of a DMDO solution at -15°C over one month period 37 Figure 16: Autodecomposition mechanism of DMDO 37 Figure 17: Crystal structure of Tr-Hpi-Pro-OMe 40 Figure 18: The different coupling reagents used for the peptide bond formations 44 Figure 19: Nomenclature used for the proline 50 Figure 20: *H N M R of the D M D O solution 70 vi List of schemes Scheme 1: The two routes to the bicyclic amatoxins 11 Scheme 2: Formation of the tryptathionine bridge 12 Scheme 3: Preparation of the phalloidin peptide backbone 13 Scheme 4: Preparation of phalloidin 14 Scheme 5: Fomation of Hpi and reaction with cysteine 15 Scheme 6: Himastatin and synthesis of Hpi 16 Scheme 7: Synthesis of Phakellisatin 17 Scheme 8: Okaramine C and synthesis of Hpi 18 Scheme 9: Zanotti's synthesis of Ile3-Amaninamide 19 Scheme 10: Preparation of Hpi-Glycine by P. Fournier 19 Scheme 11: Preparation of the hexapeptide. Reagents and conditions 20 Scheme 12: Preparation of the amaninamide 21 Scheme 13: Preparation of the amatoxin derivatives 22 Scheme 14: Synthesis of Fmoc-trifluoromethylproline 24 Scheme 15: Synthesis of Fmoc-fluoroproline 28 Scheme 16: Synthesis of Fmoc-4-cyanoproline 30 Scheme 17: Synthesis of Fmoc-4,4-difluoroproline 30 Scheme 18: Synthesis of Fmoc-4-ketoproline 31 Scheme 19: Synthesis of Fmoc-4-(benzylthio)proline 32 Scheme 20: Synthesis of Fmoc-thiazolidine-4-carboxylic acid 33 Scheme 21: The Staudinger reaction 33 Scheme 22: Synthesis of Fmoc-4-azidoproline 34 Scheme 23: Synthesis of cis-Boc-azidoproline 34 Scheme 24: Preparation of D M D O 36 Scheme 25: Synthesis of Tr-Trp-Xaa-OMe 38 Scheme 26: Synthesis of Tr-Hpi-Xaa-OMe 39 Scheme 27: Synthesis of Tr-Hpi-OH 42 Scheme 28: Synthesis of the octapeptide on solid-phase 43 Scheme 29: Synthesis of the bicycle 44 Scheme 30: Iodometric titration of the DMDO solution 70 vii List of abbreviations Asn Asparagine Hpi 3a-Hydroxy-l,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]-indolo-2-carboxylic acid AcOEt Ethyl acetate AcOH Acetic acid ATP Adenosine triphosphate Bn Benzyl Boc t-butyloxycarbonyl Bs Broad singlet C F 3 T M S Trifluoromethyltrimethlsilane CH2CI2 Dichloromethane COSY Correlation spectroscopy CTP Cytidine triphosphate Cys Cysteine d Doublet DAST (Diethylamino)sulfur trifluoride D B U 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC Dicyclohexylcarbodiimide D C U /V,/V'-Dicyclohexylurea dd doublet of doublets DIAD Diisopropyl azodicarboxylate DIEA Diisopropyl ethyl Amine DMDO 3,3-Dimethyldioxirane DMF Af, Af-Dimethylformamide viii DMSO Dimethylsulfoxide D N A Deoxyribonucleic acid dt Doublet of triplets ESI Electro-spray Ionization Et Ethyl E t 2 0 Diethyl ether Fmoc 9-fluorenylmethyloxycarbonyl Fmoc-OSu N-(9-Fluorenylmethoxycarbonyloxy) succinimide Glu Glutamic acid Gly Glycine GTP Guanosine triphosphate H B T U 0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate H C l Hydrochloric acid HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol His Histidine HOBt 1 -Hydroxybenzotriazole HPLC High performance Liquid Chromatography He Isoleucine Ir(Cod)(Py)PCy3 Iridium(I) hexafluorophosphate (l,5-Cyclooctadiene)-(pyridine)-(tricyclohexylphosphine) complex Leu Leucine Lys Lysine m multiplet wCPBA m-Chloroperoxybenzoic acid Me methyl MeOH Methanol MS Mass Spectrometry NMP N-Methyl-2-Pyrrolidone IX N M R Nuclear Magnetic Resonance N O E Nuclear Overhauser enhancement Ns o-Nitrobenzenesulfonyl NTP Nucleoside triphospate Ph phenyl Phe Phenylalanine Pol II R N A Polymerase II ppm part per million Pro Proline j7-TsOH j9-Toluenesulfonic acid PyBOP (Benzotriazol-1 -yloxy)tripyrrolidophosphonium hexafluorophosphate PyBrOP Bromo-tris-pyrrolidino phosphoniumhexafluorophosphate q quartet R f retention factor or ratio to front RNA Ribonucleic acid RT Room temperature s singlet Ser Serine t triplet T B A C N Tetrabutylammonium cyanide • TBAF Tetrabutylammonium fluoride trihydrate tBu ferf-butyl T E A E Tris(aminoethyl)amine TEMPO 2,2,6,6-Tetramethylpiperidine 1-oxyl radical TFA Trifluoroacetic acid THF Tetrahydrofuran THPP Tetrahydropyranyl polystyrene Thr Threonine x TLC Thin-layer chromatograpy Tmse Trimethylsilylethyl Tr trityl, triphenylmethyl Trp Tryptophan UTP Uridine triphosphate Val Valine Z Phenylmethoxycarbonyl 8 chemical shift xi Chapter 1 Introduction 1.1 The poisonous amanita mushrooms Very few wild mushroom species are extremely toxic. The most well known toxic mushroom is Amanita phalloides (Death Cap).1 It is a medium size mushroom (5-12 cm) with white flesh (Figure 1). As it becomes older, it turns greenish and develops a pungent odor. This species is quite common in Europe but not so common in North America. In BC, it can be found underneath beech, sweet chestnut, and hazelnut trees. Death cap is not a native species of British-Columbia, it seems to have been imported from Europe. Amanita virosa is another very toxic mushroom from the same genus. It's been known under the common name of destroying angel. This mushroom is pure white in color and has a sweet silky odor. Unlike Amanita phalloides where at maturity the cap is nearly flat, the cap of Amanita virosa is ovoid. It is quite rare in North America, especially in BC. It is usually found underneath oaks and conifers. Figure 1: Amanita Phalloides.2 1 1.2 Amatoxin and phallotoxins Amatoxins and phallotoxins are the principal toxins in the amanita mushrooms.3 Both families of compounds are bicyclic peptides. Amatoxins are octapeptides, whereas phallotoxins are heptapeptides. These compounds have similar chemical structures; both are N-to-C cyclized peptides that contain an additional transannular tryptathionine bridge between the 2-position on the indole ring of the tryptophan residue and the thiol of the cysteine residue (figure 2a). In natural phallotoxins, this bridge is a simple thioether, whereas in the natural amatoxins the bridge is oxidized to R-sulfoxide. Also these peptides contain a few nonribosomally encoded amino acids. In the case of amatoxin, these are the 4-hydroxyproline, 4,5-dihydroxyisoleucine and 6-hydroxytryptophan (figure 2b). H O HO OH HO 4-hydroxyproline O HO 4,5-dihydroxyisoleucine OH 6-hydroxytryptophane Figure 2: (a) Tryptathionine bridge and (b) non ribosomal amino acids in amatoxins. In the Virosa and Phalloides mushrooms, a large variety of amatoxin and phallotoxin compounds is present. The relative quantity of these toxins varies with the amanita species. The first toxins discovered were oc-amanitin, the neutral and most abundant molecule, and P-amanitin, an acidic compound. Later, y- and s-amanitin, the non-toxic amanullin, amaninamide and their derivatives were discovered.4 Similarly, seven natural phallotoxins were found. The terms amatoxin and phallotoxin describe not only the natural compounds, but also all of the synthetic ones. Figure 3 presents the different natural amatoxin compounds and figure 4 presents the natural phallotoxin compounds. 2 Name R l R2 R3 R4 R5 a-amanitin C H 2 O H OH N H 2 OH OH p-amanitin C H 2 O H OH OH OH OH y-amanitin C H 3 OH N H 2 OH OH s-amanitin C H 3 OH OH OH OH Amanin C H 2 O H OH OH H OH Amaninamide C H 2 O H OH N H 2 H OH Amanullin C H 3 H N H 2 OH OH Amanullinic acid C H 3 H OH OH OH Proamanullin C H 3 H N H 2 OH H Figure 3: Natural amatoxin compounds. Name R l R2 R3 R4 R5 R6 phalloin C H 3 phalloidin C H 3 phallisin C H 3 prophalloin C H 3 phallacin CH(CH 3 ) 2 phallacidin CH(CH 3 ) 2 OH COOH OH phamsadn^OHi^^ C H 3 OH OH C H 3 OH OH C H 3 OH OH C H 3 OH H OH COOH OH C H 3 C H 2 O H C H 2 O H C H 3 C H 3 C H 2 O H CH2OH C H 3 C H 3 C H 2 O H C H 3 C H 3 C H 3 CH2OH Figure 4: Natural Phallotoxin compounds. 3 1.3 Phallotoxins target F-actin Studies of injecting the mushroom extract into mice show the phallotoxin compounds in amanita mushrooms are the first ones to have a toxic effect.3 Their toxicity is due to its strong affinity towards F-actin, a protein involved in muscle contraction.5 When a phallotoxin is bound to F-actin, the depolymerization of F-actin to the G-actin is inhibited. That disturbs the cell and reduces the concentration of G-actin to a very low level. Rapidly, this concentration of G-actin becomes too low and intolerable for the cell. 1.4 Amatoxins target transcription Alpha-amanitin is one of the most toxic natural products known to man. The L D 5 0 in mice is about 0.5 mg/kg of body weight and in human is -0.1 mg/kg.6 A typical mature mushroom of 40-50 g contains 8-12 mg of amatoxins. This quantity can be enough to kill an adult. The inhibitory target of amatoxin is R N A polymerase. There are several kinds of RNA polymerase in the eukaryotic cell: 1. R N A polymerase I, which is located in the nucleoli, a type of organelle, which is responsible for the synthesis of precursors for most of ribosomal RNAs. 2. R N A polymerase II (Pol II), is located in the nucleoplasm and synthesizes mRNA precursors. 3. R N A polymerase III, also located in nucleoplasm synthesizes tRNAs and a variety of cytosolic RNAs. R N A polymerase II is the specific target of amatoxins (Kj of 3 x 10"9 M) . 5 Pol II is one of the main enzymes involved in the transcription, which takes place in every cell whenever information in D N A needs to be transcribed into mRNA (figure 5). After the two strands of the D N A duplex are effectively separated by helicase, one strand of the D N A is used as the template for the synthesis of the R N A from ribonucloside triphosphates (ATP, CTP, GTP, and UTP). The reaction is driven by the release and subsequent hydrolysis of pyrophosphate. 4 Transcription Translation Figure 5: Schematic representation of the two essential processes involved in polypeptide synthesis. The role of R N A polymerase II is clearly shown (Figure taken from Pierre Fournier master degree dissertation).7 R N A polymerase II is a very large enzyme complex (-550 kDa) which contains 12 subunits. The complexity of this enzyme is due to the fact that several constituents need to be brought together to synthesize mRNA. The first crystal structures of Pol II were obtained recently and this was a stepping stone for the study of transcription.9'10 Amanitin is the most potent and specific inhibitor of Pol II." This toxin targets both the transcription initiation and elongation. Bushnell et al. were the first to crystallize the Pol II complexed to a-amanitin. The results were quite relevant for the study of this inhibitory mechanism (figure 6). Alpha-amanitin binds near a bridge helix extending from the cleft between the two biggest Pol II sub-units. The binding site is relatively far from the NTP feeding channel and so the inhibitor doesn't block the NTPs. Since the amatoxin molecule is far from the active site, the binding of NTPs and the formation of phosphodiester is still possible even when Pol II is inhibited. The inhibitory activity of amatoxin comes from the fact that the translocation of the D N A - R N A is almost stopped. Since the D N A cannot move anymore, the transcription is then stopped. To achieve the proper translocation of the D N A strand, 5 the helix bridge has to change position slightly. The inhibition is achieved because the helix bridge cannot move when amanitin is complexed to Pol II. Transcription • Figure 6: Schematic drawing of Pol II complexed with a-amanitin. The main constituents of the transcription process are well shown.1 0 1.5 The importance of hydroxyproline in the inhibition The Pol II-a-amanitin complex crystal structure also showed the principal sites of contact between the enzyme and the inhibitor (figure 7). 1 0 There are several interactions between the main chain carbonyls and the enzyme, mainly from the same side of the molecule. The most interesting interaction is the hydrogen bond between glutamate A822 and the hydroxyproline side chain. This interaction is thought to be the key in the amatoxin binding. It has been shown that changing some residues on a-amanitin has only a small effect 19 on the inhibition. Proamanullin is an amatoxin lacking hydroxyl groups on the 4,5-dihydroxyisoleucine and on the hydroxyproline. This compound is 20,000 times less active than a-amanitin. This is likely to be due to the fact that the compound is unable to make a hydrogen bond to Glu A822. The lack of hydroxyl group on the 4,5-dihydroxyisoleucine is without any effect on the inhibition since amanullin, an amatoxin 6 lacking only the 4,5-dihydroxyisoleucine, is four times less active than a-amanitin. Furthermore, it should be noted (figure 7), that the side chains of the 4,5-dihydroxyisoleucine, the tryptophan 6-hydroxyl group and the sulfoxide group don't have any specific interaction with Pol II. Therefore those residues are thought to be relatively unimportant for the inhibition. B743 Gln B765 Pro C H 2 O H A816His H C H C A822 Glu A768 Gin O C 3 H C A760 Gin A766 Gly - O H A688 Gin A726 Arg A767 Gin " C H K 2 . 6 A 3.0AN /3.IA H 5 H 2 O - C -, N H H 4 H - N C -I H 7 C O - C - - N -\ 2.6A A819Gly | 2 H N OC-A1081 Leu • C H 2 H -N H O \S H C O 3.2A,A767 Gin N H ' " C H 3 • 6 / O H H C - — C H A719Val I \ : H 3 C O 3 3.3A - C N-H O • C -7 - H 2 C - - N H A769 Sef A1092 Lys Figure 7: Structure of a-amanitin and the interaction with the different residues on Pol II from Bushnell et al. 10 Besides the H-bond to Glu822, the effects of hydroxyproline on the conformational structure of amanitin, which would in turn affect the interaction with Pol II, are potentially electronic and steric. Often it is difficult to differentiate clearly between these two effects. These are discussed below. 1- The steric effects are mainly caused by the rigid 5 membered ring of the hydroxyproline. Like any 5 membered ring, the proline ring is not flat but puckered. On proline, the pucker mode is determined by the spatial position of the y-carbon, the pucker can be exo or endo13 (Figure 8). The pucker mode is highly dependent on the substituents on the y-carbon. When a bulky group is present in 7 trans fashion as in hydroxyproline (the Y position in scheme 1), the en do form is generally preferred. To illustrate the importance of the pucker of the pyrrolidine ring, it has been shown recently that this characteristic is key to the stability of collagen, a protein which contains a high number of proline residues.14 2- There is a stereoelectronic effect related the the pyrrolidine ring pucker and it is linked to the electronegativity of the ring substituent. A n electronegative substituent on the y-carbon will stabilize the CY-endo pucker mode if the substituent is cis, or CY-exo if the substituent is trans.15 The degree of stabilization correlates with the electronegativity of the substituent. Cy-endo Cy-exo Figure 8: Proline ring pucker modes. 1.6 The aim of the project By studying amatoxin, we would like to achieve three main goals: 1) To study the structure-activity relationship for amatoxin inhibition of Pol II. 2) To build a library of compounds useful for a further screen against a variety of protein targets. 3) To develop robust methods for the formation of bicyclic peptides bearing tryptathionine bridge. We would like to investigate the role of the hydroxyproline residue. This residue may be very important for the interaction of amatoxin with Pol II since hydroxyproline is forming the only H-bond from an amino acid side-chain in amatoxin.10 The modification of the proline residue could greatly influence the binding of amatoxin to Pol II. The synthesis of a series of amatoxins with modified prolines could verify the importance of the hydroxyproline residue in the inhibition. It is possible that we could even get a better inhibition with a synthetic proline than with the natural a-amanitin. Even if we do not 8 observe strong inhibition, this study will permit a preliminary assessment of the structure-activity relationship for amatoxin with regard to the hydroxyproline residue. Nine proline derivatives were chosen for incorporation into amatoxin (figure 9). These prolines are potential isosteres, or at least closely related structural analogs of hydroxyproline, but they have very different steric and electronic properties. We would like to incorporate all these amino acids into amatoxins: 1) Hydroxyproline I is an obvious choice because it is present in the natural amatoxins. It will be used for comparison purposes. The hydroxyl group can act as an H-bond donor and acceptor and it induces ring pucker. 2) Fluoroproline II is a proline bearing a fluorine at the y-position in trans fashion. The Van der Waals radius of fluorine is only 0.015 nm larger than hydrogen and its electronegativity is greater than that of the hydroxyl group.16 Since fluorine is the most electronegative elements, the pyrrolidine ring pucker should be similar to hydroxyproline. Because organofluorides do not usually form hydrogen bonds,17 the importance of the ring pucker in the binding of amatoxins to Pol II will be separated from hydrogen binding effects. 3) Difluoroproline III has similar properties to fluoroproline. Since the radius of fluorine is small, the steric changes should be minimal. 1 8 4) Cyanoproline IV is substituted with the more bulky nitrile group. This can affect the pucker mode of the proline ring. Like hydroxyl group, nitrile groups can form H-bonds, but these will be only acceptors and they are weaker than those of OH. 5) Aminoproline V can also be a strong H-bond donor or acceptor. But, since this substituent is also basic, the protonation of the nitrogen would occur and may ultimately form a salt bridge with Glu822 instead of a hydrogen bond. Salt bridges are usually quite strong interactions, particularly in solvent protected pockets in proteins. 6) Thiazolidinecarboxylic acid VI can be seen as an interesting potential hydrogen bond acceptor. The sulfur atom in the 5 member ring is quite bulky and the ring pucker should be significantly affected. This amino acid has been used before because of its interesting redox properties19 as the thiol atom and the possibility of three oxidation states, it can either be oxidized to sulfoxide or further oxidized to sulfonic acids. 9 7) Ketoproline VII has two lone pairs on the carbonyl group to act as a strong H -bond acceptor. The ring geometry should however be significantly different from 8) Trifiuoromethylproline does not form hydrogen bonds. The substituent is approximately as bulky as the hydroxyl group and is strongly electron withdrawing. The pyrrolidine ring pucker should be affected. Also trifluoromethyl group is quite hydrophobic, that could give rise to interesting properties.20 9) Mercaptoproline VIII presents the SH group which is more bulky and less electronegative than the OH group. Thus the pucker should reflect steric effects and be less C4-endo. Nevertheless, the thiol group can be an H-bond donor. Besides some of the immediate goals for testing sterics and electronics, the thiol functionality could easily be used as a linker arm for the covalent bonding of other molecules, this could lead to interesting applications including the development of prodrugs and photodeprotectable amanitins for intracellular probes of transcription. the other prolines since the gamma carbon is sp2. H HO H H H H H H H H VI IX Vl l VIII Figure 9: Synthetic proline derivative targets 10 1.7 The synthesis of amatoxin To make a library of amatoxins, one needs a reliable synthesis that provides access to such compounds. There are two main difficulties in the amatoxin synthesis. Firstly the non-coding amino acids in amatoxin like 4,5-dihydroisoleucine and hydroxyltryptophan can be quite difficult to synthesize with the right stereochemistry. But it has been shown that these two non-coding amino acids can be replaced by the coding ones with only a very small loss in the activity. The second challenge is the formation of the thioether bond between Cys and Tip which is one of the key steps in the synthesis. Two main strategies related to the cyclizations steps have been used in the synthesis of the amatoxin-phallotoxin compounds. The first strategy starts with the formation of the tryptathionine bridge. The peptide is then cyclized twice to form the two cycles; this is route A shown on scheme 1. The other strategy is the formation of the main peptide chain first, then the tryptathionine bond is formed and finally the last cyclization is achieved, this is route B (scheme l ) . 3 L _ H amatoxin Scheme 1: The two routes to the bicyclic amatoxins. 11 1.8 The route A synthesis Recently, a solid-phase synthesis of phallotoxins has been achieved and it is a good example of the route A . By this method, Anderson et al. synthesized [Ala ]-Phalloidin. A l l the prior syntheses were done using large scale solution-phase synthesis. In this route (A synthesis), the tryptathionine bridge is first formed, then the main chain is formed. The last step in the synthesis is the backbone cyclization. This solid-phase synthesis is based on the intensive use of orthogonal protective groups. The two macrocyclizations were achieved on resin. Since this work is relatively close to what we will present, we will discuss it in depth. The first part of the synthesis is the formation of tryptathionine link. The starting material is a protected cystine molecule which is reacted with thionylchloride to give S-chlorocysteine. The tryptathionine bridge is formed by the reaction of S-chlorocysteine on tryptophane, see scheme 2. o N H - B o c f - B u - O ^ Y " ! ^8 -J> f -Bu -0 N H - B o c O ^ ^ N H - B o c O X X I Scheme 2: Formation of the tryptathionine bridge. Reagents and conditions i) SO2CI2; ii) (o-N0 2Ph)S0 2-Trp-0-Allyl, N a H C 0 3 iii) TFA, (7-Pr)3SiH; iv) Fmoc-Cl, N a 2 C 0 3 . The next part of the reported synthesis is the formation of peptide chain. First, the proline is fixed on the resin, tetrahydropyranyl polystyrene (THPP). After that, alanine was added followed by the addition of intermediate XII (scheme 3). 12 XVI Scheme 3: Preparation of the phalloidin peptide backbone. Reagents and conditions i) Fmoc-c/s-Hyp-O-Allyl, pyridinium /?-toluenesulfonate; ii) Pd(PPh3)4, N,N-dimethylbarbituric acid; iii) //-Ala-O-Tmse , PyBOP, HOBt, DIEA; iv) piperidine in DMF; v) Fmoc-Cys-[.S-(2-((o-N02Ph)S02-Trp-0-Allyl))]-OH XII, PyAOP, HOAt, 2,4,6-collidine. The final part of the synthesis is the addition of the other half amino acid of the toxin and the two successive macrocyclizations, (scheme 4). The whole synthesis is intensively built on the use of orthogonal protective groups and the management of those groups was probably a big challenge for the chemists involved. This preparation of Phalloidin is composed of 18 steps with an overall yield of 1.3 %. 13 o N s - N H ^ ^ O-Tmse O-Allyl C^j/ i) ii) i) iii) i) iii) 9 Fmoc N s - N H ^ A 0 A NH O-Tmse '-[ala7]-phalloidin XX C T H P F D XIX Scheme 4: Preparation of phalloidin. Reagents and conditions i) piperidine in DMF; ii) Fmoc-D-Thr(Od3u)-OH, PyBOP, HOBT, DIEA; iii) Fmoc-Ala-OH, PyBOP, HOBT, DIEA; iv) Pd(PPh 3) 4, W-dimethylbarbituric acid; v) PyAOP, HOAt, DIEA; vi) TBAF; vii) /J-mercaptoethanol, D B U ; viii) diphenylphosphoryl azide, DIEA, ix) TFA/H 2 0 /E t 3 SiH (8:2:10). Anderson and coworkers claimed that they found two compounds after the last cyclization, X X and X X ' . Both compounds found have the same molecular mass but inverse CD spectra. One compound was identified as the natural compound, XX. The other compound the X X ' was thought to be an atropisomer of the natural one which has an inverted shape relative to X X . A similar effect has been observed by Zanotti2 2 and by Pierre Fournier, a previous student in our research group who also worked on an amatoxin derivative.7 1.9 The Route B synthesis and formation of Hpi The synthetic route B was hard to achieve until the discovery of the Savige-Fontana reaction used to make the tryptathionine bridge. Savige was the first one to synthesize L -14 3a-hydroxy-l,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]-indolo-2-carboxylic acid (Hpi) during a study on the oxidation of tryptophan. When reacted with 1 eq. of peroxyacetic acid, tryptophan undergoes an intramolecular cyclization to form Hpi (scheme 5). The usefulness of Hpi was than rapidly established.24 Hpi can react with a thiol in TFA, in particular with the side chain of cysteine, to form a tryptathionine bridge. This reaction can be used as a basis for the synthesis of an amatoxin. Furthermore, Hpi is a structural component of a few natural products and recently a few research groups have been interested in the formation of the Hpi moiety as part of other biologically active compounds. Scheme 5: Fomation of Hpi and reaction with cysteine. Reagents and conditions i) AcOH, CH3CO3H, H 2 0 , 6 °C, 18h; ii) TFA, 2d. 1.9.1 Kamenecka and Danishefsky strategy to Hpi Himastatin is a rather complex molecule containing the Hpi. This natural antibiotic is a dimer of two peptide monocycles, each bearing an Hpi moiety. To construct this moiety, Theodore M . Kamenecka and Samuel J. Danishefsky designed a method to make H p i 2 5 (scheme 6). This method uses Tr-Trp-O-tBu as the starting material and DMDO as the oxidant. The trityl protective group was then removed with acetic acid. These 15 conditions permitted the formation of H-Hpi-OtBu with a reasonable yield over 2 steps (55 %). This was a stereoselective method method leading to just the syn-cis diastereomer (the other diastereomer can be made via more steps and different protecting groups). 1.9.2 Van Vranken strategy to Hpi Recently a research group lead by David L. Van Vranken 2 6 used an interesting way to generate Hpi in the synthesis of phakellistatin 3. This natural product is one of the few monocyclic peptides that contains an Hpi moiety. Initially, the cyclic peptide phakellistatin 13 was made using normal peptide synthesis methods. This compound is the same as phakellistatin 3, but bearing a normal Trp instead of Hpi. The cyclic peptide containing a tryptophan residue was then photooxidized with 0 2 and a sensitizer to give a mixture of phakellistatin and isophakellistatin (scheme 7). The combined yield of 20 % for approximately equal amount of diastereoisomers is the main drawback of the reaction. The poor yield is thought to be caused by the over oxidation of the indole ring. 16 I sophake 11 i statin 3 Scheme 7: Synthesis of Phakellisatin 3. Reagents and conditions: i) O2, hv, sensitizer; ii) Me2S, 20% yield. Dimethylsulfide was used to stop the reaction and to reduce any excess reagent. 1.9.3 Ley strategy to Hpi Recently, Steven V . Ley reported a method to synthesize Hpi with the use of selenium.2 7'2 8 This procedure has been used recently in the synthesis of Okaramine C. The first step involves the selenocyclisation of a protected tryptophane (scheme 8). The success of this reaction depends on the N-protecting group employed. After optimization, the syn-cis selenide product was obtained with a 93 % yield. The selenium atom is then removed by oxidative deselenation in the presence of wet mCPBA. The mechanism of the last reaction is thought to be the following: the selenium atom is oxidized, then the elimination of the phenylseleninic acid occurs and finally water attacks the newly formed carbocation. After the formation of the Hpi moiety, the nitrogen atoms can be selectively deprotected. 17 Scheme 8: Okaramine C and synthesis of Hpi. Reagents and conditions: i) N-(phenylseleno)phthalimide, CH2CI2, pyridinium p-toluene sulfonate, Na2S04, 93%; ii) wet mCPBA 5 eq., K 2 C 0 3 , CH 2 C1 2 , 0-25 °C, quantitative; i i i ) T F A / D C M , 5 minutes, 97%; iv) H 2 , Pd/C, MeOH, 79%. 1.10 The Zanotti-Wieland strategy towards amatoxins Giancarlo Zanotti and Theodor Wieland were the first ones to use a Savige-Fontana reaction in the synthesis of amatoxins.22'29 The first step of the synthesis was the formation of a Boc protected Hpi. A linear octapeptide bearing the natural amino acids and Hpi was then built. For the main chain preparation, the authors used a peptide coupling method called the mixed-anhydride method which involved the use of non symmetrical anhydrides as the activated reactant. The octapeptide was then cyclized by treatment with strong acid to give a monocycle with the tryptathionine bond. Finally the 18 monocycle is reacted with a peptide coupling agent to give the desired bicycle (scheme 9). Scheme 9: Zanotti's synthesis of Ile3-Amaninamide. 1.11 Previous advances from our research group In our research group, a master's student, Pierre Fournier, achieved the synthesis of 3 2 7 He -Pro -amaninamide using a modified Hpi dipeptide. The first part of the synthesis was the preparation of an Hpi molecule bearing a glycine methyl ester as a protective group (scheme 10). The use of a glycine as a protective group is an innovation of our research group. The glycine helps to have a better yield and a cleaner reaction. Glycine is also followed by tryptophan in the amatoxin sequence. To verify the generality of the DMDO oxidation, a series of Hpi-amino acid has been prepared and will be discuss later. Trt o HN I II I >r-l^_ Tr Hpi Glycine Scheme 10: Preparation of Hpi-Glycine by P. Fournier. Reagents and conditions: i) LiOH, dioxane/water; ii) DMDO, CH 2C1 2 , -78 °C. .OH O ii) h 8 OH 19 The next part of Fournier's adaptation of Zanotti's approach was the preparation of the hexapeptide with a solution-phase method called the "rapid continuous peptide T A synthesis". In this method amino acids used are activated as acyl fluorides and acyl chlorides (scheme 11). The coupling time is then quite small (10 min). The Fmoc deprotection is achieved with Tris(aminoethyl)amine (TAEA). This peptide coupling method provided fast coupling times and a product in good yield (58% over 5 peptide couplings).7 Scheme 11: Preparation of the hexapeptide. Reagents and conditions: i) CH2CI2, H2C) NaHC0 3 , 15 min, RT; ii) T A E A , 30 min, RT. The final part of Fournier's synthesis was the formation of the octapeptide from Tr-Hpi-Gly-OH and the hexapeptide. This octapeptide is then cyclized twice. A l l the protecting groups and the tryptathionine bond are formed with the help of a strong acid, according to a method from Savige and Fontana.24 The N-terminal is then reacted with the C-terminal in a macrocyclization to give the Pro2-Ile3-S-deoxo-amaninamide using PyBOP as the coupling reagent (scheme 12). Iterative process with every other amino acyl fluoride or chloride according to the sequence. Hexapeptide 20 T K N H o 4 Trityl Hpi glycine (Tr-Hpi-Gly-OH) T r Hexapeptide T K N H 0 = 1 O ^ O / 0 o =  Tr S' i Tr Octapeptide H N b \ H r H?N O O M iii) H H N ' \\ f H H 2 N > - N o N H Pro2-lle3-S-deoxo-amaninamide Scheme 12: Preparation of the amaninamide. Reagents and conditions: i) PyBOP, HOBt, D C M , RT, 15 h, 66 % total; ii) TFA, 3h, 38%; iii) PyBOP, HOBt, D C M , RT, 15h, 48 %. 1.12 Our goals with the study of amatoxin and Pol II We are proposing to prepare a small library of amatoxin compounds. Since our hypothesis is that the hydroxyproline is one of the key amino acids responsible for the inhibition on Pol II by amatoxin, we would like to direct our library toward this amino acid. First, several synthetic proline derivatives will be synthesized. These amino acids are isosteres of hydroxyproline, sharing different steric and electric properties. Then, the amatoxin compounds will be synthesized. We will use the methodology developed by Pierre Fournier;7 a linear peptide bearing the modified proline and Hpi are synthesized. The first macrocyclization is then achieved with a Savige-Fontana reaction23 and the second macrocyclization is then achieved with a peptide coupling reagent. Instead of using a solution-phase synthesis strategy as Fournier did, we would like to use a solid-21 phase synthesis strategy. This methodology would permit an easier purification and synthesis of the library.3 1 Once the library is made, we would like to carry out various biological assays. The first one that we will do is to quantify the potency of inhibition of the various compounds in the library. With the biological results, we would be able to define an accurate structure-function relationship (scheme 13). Preparation of the proline derivatives Scheme 13: Preparation of the amatoxin derivatives . 22 Chapter 2 Discussion The discussion is divided in three parts. The first part is the different preparations of the proline derivatives. The second part explains the formation of Hpi. Finally the third part discusses the solid-phase synthesis of the peptide. 2.1 Preparation of the amino acid derivatives 2.1.1 Preparation of Fmoc-4-trifluoromethylproline The preparation of Fmoc-4-trifluoromethylproline was achieved according to a modified preparation from Del Valle et al32 (scheme 14). The first step of the synthesis was the formation of the methyl ester of hydroxyproline to give compound 2. We chose a straightforward procedure from J. R. Rachele.33 The traditional way to make a methyl ester is to make the acid react with methanol and HCl gas as a catalyst. To avoid the use of gaseous H C l and in order to simplify the purification, the methyl esterification was carried out using 2,2-dimethoxypropane and HCl( a q) and yielded 2. The nitrogen atom on the molecule was then protected with Boc to give 3. The oxidation of 3 was done with trichloroisocyanuric acid and T E M P O 3 4 . This method is very mild and selective. Usually, these types of oxidation are done under the Swern or Dess-Martin conditions, but in this case, oxidation with TEMPO was used for the simple procedure at room temperature (instead of -78°C for Swern). The mechanism of the oxidation reaction is shown on figure 10. 3 5 TEMPO is a stable radical compound and is used to catalyze the oxidation reaction. First TEMPO reacts with a catalytic amount of trichloroisocyanuric acid to form the N-oxoammonium ion, compound a. This ion oxidizes the alcohol giving the hydroxylamine b. To regenerate the catalyst, the hydroxylamine is oxidized back to N-oxoammonium ion with trichlorocyanuric acid. 23 H O H o Boc o i) , / N V A ii) Jl OH < ) X 0 M e C > \ ) M e HO 1 HO HO Boc 0 Boc o c A - A . . . ^ + A - A . . . -A OMe \ / OMe F 3 C -O' A OH 5 Boc Boc 0 - Boc N v . , o ( y j ) _ ^ / N y * C H 2 O H v i i ) N C H 2 O H OMe " F 3 C 6 F 3 C 7 F3CT 8 Boc Q |_| Q F M O CO viii) / N N . A ix) » / N \ A x) r N v ^ F , C ' F 3 C F * C OH 9 r ^ 10 • 11 S c h e m e 14: Synthesis of Fmoc-trifluoromethylproline. Reagents and conditions: i) 2,2-dimethoxypropane, H C l , RT, 14 h, 79%,; ii) (Boc) 20, Et 3 N, CH 2 C1 2 , RT, 18 h, 98 %; iii) Trichloroisocyanuric acid, TEMPO (cat), CH 2 C1 2 , 20 min, 0°C, 76 %; iv) 2 equiv CF 3 TMS, 2.1 equiv TBAF, THF, 0°C^RT, 2 h, 24 %; v) SOCl 2 , pyridine, 20 min, reflux, 93% vi) N a B H 4 , L i C l , THF, Ethanol, RT, 14 h, 62 %; vii) H 2 (1 atm), [Ir(Cod)(Py)PCy3], CH 2 C1 2 , RT, 3 days, 42 %; viii) NaCIO, NaC10 2, TEMPO, MeCN, pH 6.7 N a H 2 P 0 4 buffer, 45°C, 24 h, 88 %; ix) TFA, 2 h, RT; x) Fmoc-OSu, DMF, N a 2 C 0 3 aq, 20 min, RT, 50 % for the last two steps. 24 II R R ' ^ O H i a O C l -O Cat. amount b O H R Figure 10: Mechanism of oxidation of a carbonyl with T E M P O and trichloroisocyanuric acid. Nucleophilic addition with trifluoromethyl anion was performed according to Del Valle et al. Tetrabutylammonium fluoride was added to trifluoromethyl-trimethyl silane. The affinity of silicon for fluoride is quite important. When the trifluoromefhylated silane reacts with the fluoride from T B A F , the silane liberates an equivalent of trifluoromethyl anion. Since the stereochemical outcome is not important for the following steps, it was not studied for product 5.. The dehydration of alcohol 5 was performed differently than that in the original paper. According to Del Valle et al, "reaction of 5 with SOCl 2 /pyridine, para-toluenesulfonic acid/benzene, and P O C I 3 /DBU yielded none of the desired product". This conclusion was unexpected. Elimination with thionyl chloride would shorten the synthesis by 3 steps. In an almost identical paper, Qiu et al.36 tried the same elimination reaction with SOCb/pyridine on the benzyl protected amino acid. Since passing from benzyl ester to methyl should not change the reactivity of the compound, we tried the reaction on our amino acid methyl ester. It worked on the first attempt to give the olefin compound 6. 25 Two compounds can be formed by that elimination; the 3,4 (a) and the 4,5 dehydro proline (b) (figure 11), but the a is the obtained compound 6. Boc ~ KA. Boc p OMe \ \ F 3 C OMe / CHp C H a / / C H 5 ^ X A,' A A A J \ J CHjQMe C H 3 B O C r .1 i - 2.19 J- 0.95 j- 1.00 -600 -500 -400 -300 -200 -100 -0 --100 -200 ppm (f1) 5.0 1 1 1 r 2.0 Figure 11: Possible molecules obtained with the SOCVpyr id ine elimination reaction and ! H N M R of the compound 6. The study of the chemical shift of the CH2 could be a good indication of which elimination took place. The obtained compound has a multiplet of the chemical shift of 4.47-4.27 ppm for the CH2 protons on the pyrrolidine cycle. This is fairly high and implies relatively deshielded protons. If the compound had the b-type of structure, we would expect it to be further upfield (probably below 3 ppm) since there is no adjacent heteroatom. In the case of the structure a, the methylene is right beside a nitrogen atom causing the chemical shift observed. Furthermore, Qiu et al.36 found the same result with a benzyl protected proline. The ester 6 was reduced to give the alcohol 7. We used sodium borohydride and lithium chloride to achieve this reduction. Normally sodium borohydride is known to reduce ketone and aldehyde compounds but in this case the lithium chloride added helped to enhance the reactivity enough for achieving the reduction of an ester. The key to the whole synthesis of 4-trifluoromethylproline is the facial selective hydrogenation with Crabtree catalyst to give 8. The rationale behind this facial selective hydrogenation is that the hydrogen atoms are fixed on the catalyst in trans fashion on the 26 iridium atom. The unsaturated compound then complexes the catalyst by the oxygen atom allowing then the delivery of a facial equivalent of hydrogen (figure 12). Futhermore, the selectivity of the Crabtree catalyst is usually high in this type of hydrogenation (ie. usually more than 99 % for the directed isomer).37 However, the selectivity of this reaction was not a concern because i f isomerization of compound 8 had occurred, the diastereoisomers could have been separated by flash chromatography. Figure 12: Complexation of molecule 8 to iridium. The selectivity of this catalyst is very important, from Del Valle et al.32 it has been shown by 1 9 F N M R of the crude mixtures that the ratio of trans to cis was 158:1. In the original paper, the hydrogenation was done on the 4,5-dehydroproline. As previously stated, we made the 3,4 dehydroproline (product 5). After studying the N M R signature of the resulting compound, we found an identical compound to the one published, compound 8. The only difference was that we noticed the reaction has to be carried out for 3 days instead of 40 minutes as stated in the original paper. The explanation for this is still unclear, further investigation is necessary to find a proper explanation. The regiochemistry of the double bond is different; it's possible that this small change slowed down the hydrogenation reaction a lot. The alcohol 7 is then oxidized to carboxylic acid 8 with a chlorite/hypochlorite mixture according to Del Valle et al. The final step of the synthesis is the swapping of the Boc protecting group for on Fmoc protecting group. The first part consists in the removal of the Boc group on molecule 8 with neat TFA. The second part is the addition of the Fmoc group with Fmoc-OSu, using sodium carbonate as a base, to give the final product 11. This product is ready to use for the solid phase synthesis. + 27 2.1.2 Preparation of Fmoc-4-fiuoroproline The preparation of fmoc-4-fluoromethylproline was achieved according to a preparation by Hodges et al.15 The reaction steps are shown below, scheme 15. Scheme 15: Synthesis of Fmoc-fluoroproline. Reagents and conditions: i) /?-TsOH, BnOH, benzene, reflux, 14 h, 88 %; ii) Boc 2 0, Et 3 N, MeCN, 0 ° C ^ R T , 2 h, 95 %; iii) Triphenylphosphine,/>nitrobenzoic acid, DIAD, THF, RT, 3 h, 38 %; iv) K H C O 3 aq 5%, dioxane, 18 h, RT, 48 %; v) DAST, CH 2 C1 2 , -78°C ^ R T , 20 h, 33 %; vi) H 2 , Pd-C, MeOH, RT, 2 h, 87 %; vii) TFA, 2 h, RT; viii) Fmoc-OSu, DMF, N a 2 C 0 3 aq, 20 min, RT, 13 % for the last two steps. The first step of the synthesis is the benzyl esterification of hydroxyproline to give 12. The product is recovered as the />-toluenesulfonate salt and used without further purification. The product is then Boc protected to give 13. From there on the procedure of Hodges et al. was followed to afford carboxylic acid 17. 28 It is worthwhile talking about the key step of the synthesis, namely the substitution of the hydroxyl group of 15 with fluoride using D A S T as the fluoride source to yield 16. D A S T is a very common reagent in fluorine chemistry. 4 0 The mechanism of the reaction starts by converting the alcohol to a good leaving group (figure 13). This leaving group can displaced with fluoride anion generated by D A S T . Only one inversion has been done on the compound resulting in the trans molecule (ie. SN2). Finally, the protecting groups of 17 were then changed to yield /V-Fmoc-fluoroproline, 18. F Figure 13: Mechanism of the reaction with D A S T . 2.1.3 Preparation of Fmoc-4-cyanoproline cw-Boc-hydroxyproline benzyl ester 15 was used for the preparation of Fmoc-4-cyanoproline 24, (scheme 16). The hydroxyl compound 15 was converted to the tosylated compound 20 which then underwent a SN2 substitution reaction with cyanide to give 21. Tetrabutylammonium cyanide was chosen because it has good solubility in D M F . Initial results showed that the potassium or sodium salt produced a big quantity of the elimination product. Since the starting material is quite expensive, we tried to minimize the elimination reaction by lowering the temperature and by using a very soluble cyanide salt (ie. Tetrabutylammonium cyanide). Finally, after deprotection and reprotection, the product 24 was ready for Fmoc strategy solid phase synthesis. 29 Boc o Boc o Boc n AJ< — A - l AX — \ J OBn { _ / OBn O \ ) B n H0° TsO°' NCT 15 20 21 Boc 0 H 0 F m o c p S OH ^ \ J ' OH ~ yj OH NC* NC N C 22 23 2 4 Scheme 16: Synthesis of Fmoc-4-cyanoproline. Reagents and conditions: i) p-toluenesulfonylchloride, pyridine, 0°C -> RT, 14 h, 21 %; ii) tetrabutylammonium cyanide (TBACN), D M F , 55°C, 15 h, 42 %; iii) H 2 , Pd-C, MeOH, RT, 2 h, 59 %; iv) TFA, 2 h, RT; v) Fmoc-OSu, DMF, N a 2 C 0 3 aq, 20 min, RT, 53 % for the last two steps. 2.1.4 Preparation of Fmoc-4,4-difluoroproline For the preparation of Fmoc-4,4-difluoroproline, DAST was used in a similar fashion as for the preparation of Fmoc-4-fluoroproline 19 (scheme 17). . 0 Boc 0 goc o Boc H 0 13 C f 2 5 F 26 ? ° C .0 H 0 ^ m o c O OH ^ ( 7 X Q H Cj X O H F \ F-"\ F F F „„ F 29 27 28 ^ Scheme 17: Synthesis of Fmoc-4,4-difiuoroproline. Reagents and conditions: i) Trichloroisocyanuric acid, TEMPO (cat), CH 2 C1 2 , 20 min, 0°C, 87 %; ii) DAST, CH 2 C1 2 , - 78°C ^ RT, 15 h, 29 %; iii) H 2 , Pd-C, MeOH, RT, 2 h, 97 %; iv) TFA, 2 h, RT; v) Fmoc-OSu, D M F , N a 2 C 0 3 aq, 20 min, RT, 76 % for the last two steps. Boc-hydroxyproline 13 benzyl ester was oxidized with trichloroisocyanuric acid and TEMPO to give the carbonyl compound ketone 25. This reaction was done earlier with a 30 methyl ester. Instead of the methyl ester, a benzyl ester was used here, because it can be easily removed with hydrogen on Pd/C. The carbonyl compound 25 was reacted with DAST to form the difluoro compound 26. The carbonyl acts as an activating group for DAST and the reaction proceeded faster that the one done on alcohol 15 (scheme 15). Compound 29 is obtained after rearranging the protecting groups as done in the previous syntheses. 2.1.5 Preparation of Fmoc-4-ketoproline Boc o B o c O F m o c 0 fJ 25 0 30 O 31 Scheme 18: Synthesis of Fmoc-4-ketoproline. Reagents and conditions i) H2, Pd-C, MeOH, RT, 2 h, 50 %; ii) TFA, 2 h, RT; iii) Fmoc-OSu, D M F , N a 2 C 0 3 aq, 20 min, RT, 93 % for the last two steps. Compound 25 was first saponified and the Boc-deprotected. The Fmoc protective group is then added to secondary amine to give 31 (scheme 18). 2.1.6 Preparation of Fmoc-(2S,4S)-4-(benzylthio)proline The benzylthioproline is the protected form of the thioproline amino acid. A thioproline would not be suitable for the solid-phase peptide synthesis. Once the benzylthioproline containing peptide is made, we plan to remove the protecting group using a dissolving metal reduction procedure (Na(S) dissolved in NFfjrjiq)). This method is supposed to be mild and the rest of the peptide should not be affected. The Fmoc-4-(benzylthio)proline 36 was achieved according to scheme 19. 31 HO 34 OH Scheme 19: Synthesis of Fmoc-4-(benzylthio)proline. Reagents and conditions i) DIAD, triphenylphosphine, LiBr, THF, 20 h, RT, 78 %; ii) Benzyl mercaptan, sodium ethoxide, ethanol, 15 h, RT, 43 %; iii) L iOH, water, dioxane, 1 h, RT, 44 %; iv) TFA, 2 h; v) Fmoc-OSu, DMF, N a 2 C 0 3 aq, 20 min, RT, 37 % for the last two steps. Boc-hydroxyproline benzyl ester 13 was prepared as previously described and reacted with lithium bromide, triphenylphosphine and DIAD (scheme 19). The gamma carbon stereochemistry of the proline is inverted during the Mitsunobu reaction and the hydroxyl group is substituted with a bromide to give compound 32. This derivative was treated with sodium ethoxide and benzyl mercaptan to produce another inversion product compound 33, which had the desired trans stereochemistry. The reactive solution was kept at room temperature to minimize the elimination product.. The benzyl ester 33 was saponified with lithium hydroxide in dioxane/water. Hydrogenation could not be used in this case because sulfur atoms are well known to poison the hydrogenation catalyst. Also, the molecule contains two benzyl groups, a benzyl ester and a benzyl thioether, both groups could possibly react under hydrogenation conditions. Saponification with a strong base will affect only the benzyl ester and not the benzyl thioether. The usual protection/deprotection reactions were used on 34 to give the final compound 36. 32 2.1.7 Preparation of Fmoc-(R)-thiazolidine-4-carboxylic acid O j) H p j n F m o c 0 H S J ^ H OH \ > H 37 38 39 Scheme 20: Synthesis of Fmoc-thiazolidine-4-carboxylic acid. Reagents and conditions i) Formaldehyde .37 % in water, 13 h, RT, 47 %; ii) Fmoc-OSu, DMF, N a 2 C 0 3 aq, 20 min, RT, 60 %. Formaldehyde and cysteine were reacted together in water.41 Beautiful crystals of (R)-thiazolidine-4-carboxylic acid 38 were obtained and used for the next step (scheme 20). An Fmoc protecting group was added to the secondary amine and the Fmoc-(R)-thiazolidine-4-carboxylic acid 39 was used for solid phase synthesis. 2.1.8 Preparation of Fmoc-4-azidoproline The free amino group is not compatible with solid-phase synthesis because during the peptide couplings, this free amine will compete with the main chain N-terminal giving an unwanted product. We decided to retain this amine group as an azide that would serve as an amine equivalent. Thus the normal peptide synthesis was carried out on the azidoproline. Once the azidoproline containing amatoxin is made, deprotection to unmask to the aminoproline should be possible. The Staudinger reaction is a good method to reduce an azide to an amine (scheme 21). Since, a very similar reaction, the Staudinger ligation, is routinely done on peptides,42 the Staudinger reaction should be compatible with amatoxin peptides. P h , , P h R \ i) R i . , P - P h ii) R i ^ N 3 K n W N H 2 R 2 R 2 R 2 Scheme 21: The Staudinger reaction. Reagents i) PPh 3 ii) H2O. 33 The Fmoc-4-azidoproline 44 was achieved according to scheme 22. T s t f H 0 Fmoc iii) i •A O H iv) O H 43 44 Scheme 22: Synthesis of Fmoc-4-azidoproline. Reagents and conditions i) NaN3, DMF, 3 h, 50°C, 95 %; ii) L i O H , MeOH, THF, 2 h, RT, 92 %; iii) TFA, 2 h, RT; iv) Fmoc-OSu, DMF, N a 2 C 0 3 aq, 20 min, RT, 60 % for the last two steps. The previous tosylate compound 20 was first reacted in an SN2 fashion to give only the azido compound 41 (scheme 22). The ester was then saponified to give the acid compound carboxylic acid 42. Since azide compounds can be reduced by catalytic hydrogenation, this could not be used to remove the benzyl ester on compound 41 and thus, saponification conditions had to be used in this synthesis as well. Finally, the Boc protective group was deprotected and reprotected with Fmoc to give the final product 44. We tried also to make the azidoproline 41 using a SN2 on a different leaving group than tosylate. (scheme 23). Scheme 23: Synthesis of cis-Boc-azidoproline. Reagents and conditions i) NaN 3 , DMF, 15 h, 50°C, 47 % for 41 and 2 % for 45. Indeed, cis Boc-4-bromoproline benzyl 32 ester was reacted with sodium azide. The bromide was substituted by azide via an SN2 mechanism (scheme 23). In this case, an inversion of stereochemistry was obtained giving the byproduct 45. We did not perform Br' 34 any further investigations on that direction since cw-azidoproline was not a desired amino acid. The Boc-4-bromoproline benzyl 32 was prepared with a Mitsunobu reaction using LiBr. The stereochemistry at the 4 possition was probably scrambled during this Mitsunobu reaction giving the product 32 with a small quantity of diastereomer contaminating the unwanted cis impurity. Isolated products 41 and 45 are diastereoisomers, which were separated by flash column chromatography. The correct stereochemistry was determined by an NOE experiment (figure 14). On the minor product, we were able to see a very small NOE effect between the proton on the alpha carbon and the proton on the gamma carbon. On the major product, this N O E effect was not observed. Therefore, the minor product is 45, since only this structure allows a NOE between H a and H Y . Here, the NOE experiments proved that our major product 41 is the desired stereodiastereomer. Figure 14: Observed N O E effects on compound 41 and 45. 2.2 Preparation of Hpi 2.2.1 Preparation of D M D O The oxidation of tryptophan is a cornerstone for the whole process of preparing amatoxins and phallotoxins. Prior to this oxidation, D M D O has to be made.43 During the DMDO formation, acetone is oxydized with a very strong oxidant; Oxone™ (scheme 24). The active ingredient in this industrial compound is potassium peroxymonosulfate, KHSO5. This compound is sold as a triple salt with the formula 2KHS05 - K H S 0 4 'K 2 S0 4 . Boc N O E 41 45 35 o i) o—o X 0 K * " O - S - O O H 6 Scheme 24: Preparation of DMDO. Reagents and conditions i) Oxone, NaHC03, water, acetone, reduced pressure, 5-10 °C. The preparation of D M D O involved the oxidation of acetone and then the distillation of D M D O in acetone solution. Great care, i.e. slow addition of oxone and precise control of the reaction temperature, were taken to achieve a high concentration of DMDO. 2.2.2 Autodecomposition of D M D O For a practical purpose, the rate of decomposition of D M D O was studied. The solution of D M D O used in this study was stored in the dark, at -20 °C. The concentration of oxidant was determined by iodometric titrations44 (see experimental section). We found that the D M D O degradation follows a first rate decay with a half-life of 14 days (figure 15). This result was unexpected since these solutions were thought to be quite less stable than what we observed. At first, this decomposition involves the formation of a diradical.4 5 This diradical is transformed into a methyl radical which will propagate. Figure 16 shows the formation of the methyl radical and the formation of the major polymerization products, methyl acetate and l-(acetyloxy)-2-propanone.45 36 The Autodecomposition of a 0.085 M DMDO Solution at -15 °C 0,1 0,09 0,08 0,07 ^ 0,06 E X 0,05 o Q 1 0,04 O 0,03 0,02 y = 0,0862e"t R = 0,9977 10 5 20 Time (day) 25 30 35 Figure 15: Autodecomposition of a DMDO solution at -15°C over one month period. 0-0 A O O _pu„ / \ Acetone X 6 ^ ~ ? H 3 Initiation step C H 3 + o-o ° X ° ' - C H , O ^ Ji ^ Propagation step O Methyl acetate C H , + X 0 C H . + Termination steps 0 0 0 O 1 -(acety loxy)-2-propanone Figure 16: Autodecomposition mechanism of D M D O . 4 5 37 2.2.3 Preparation of Tr-Trp-Xaa-OMe The preparations of the dipeptides that will be used for the D M D O oxidations were done using standard solution phase peptide chemistry. /V-trityl tryptophan was coupled to the desired amino acid methyl ester with DCC and HOBt (scheme 25). Tr t ' NH 2 -Xaa-OMe i) Trt O H N ^ ^ X ,Xaa N H Xaa = Gly 46 = lie 47 = Pro 48 = Phe 49 Scheme 25: Synthesis of Tr-Trp-Xaa-OMe. Reagents and conditions i) DCC, HOBt, Et 3 N, CH 2 C1 2 , 12 h, RT. Where Xaa = Gly, Pro, He, Phe. 2.2.4 Preparation of Tr-Hpi-Xaa-OMe The dipeptides were reacted with DMDO to form the Hpi moiety (scheme 26). Trt o . . . 1 . 11 ^ ^ following page. 38 HN Trt O 49 53a 53b Scheme 26: Synthesis of Tr-Hpi-Xaa-OMe. Reagents and conditions: i) DMDO/Acetone, CH 2 C1 2 , -78°C, ~1 h. Treatment of Tr-Trp-Gly-OMe 46 with D M D O at low temperature afforded clean conversion to Tr-Hpi-Gly-OMe 50(a and b). The yield was 70 % for both diastereomers. A poor diastereoselectivity was observed and a mixture of both syn-cis 50a and anti-cis 50b was recovered (the ratio of syn-cis/anti-cis was approximately 1:1). These molecules were found to run very closely on TLC (ARf < 0.05, AcOEt/Hexane 1:1), therefore the separation of the diastereomers was quite tough. Dr. Jonathan May was able to separate these two constituents by silica gel chromatography and crystallize them to obtain X-ray structures and identify each of the diastereomers.46 To verify the generality of this oxidation reaction with DMDO, we tried the reaction on a variety of other dipeptides as shown in scheme 26. Dipeptides with proline, isoleucine and phenyalanine were prepared. Boc-Trp-Asn(Tr)-OMe was attempted too, but the results were not clear enough to be useful. The other amino acids were prepared by Pierre Fournier and Dr. May and will be briefly discussed for comparison.7 A good conversion was observed on all the studied peptides (43-90 % for two diastereomers). For almost all the conversions, we observed a mixture of the syn-cis and anti-cys isomers (the ratios ranged from only syn-cis to 1:1). The separations of these diastereomers on silica gel chromatography was easier than with Hpi-Gly, probably because of the additional chiral group on the alpha carbon. Hence, all the products were separated without any trouble. With proline, we could see a clear preference for a single diastereomer, syn-cis. We believe that this selectivity is due to some steric preferences in the transition state resulting in only one molecule. Tr-Hpi-Pro-OMe was crystallized by Dr. Jonathan May and it appears that the large group at the C-Terminus favored the less strained syn-cis diastereomer (figure 17). 39 Figure 17: Crystal structure of Tr-Hpi-Pro-OMe, showing the preferred syn-cis geometry. Crystals were grown from AcOEt/hexanes. Using the Rf, lH N M R chemical shift and the crystal structures of Tr-Hpi-Gly-OMe, each diastereomer was identified as syn-cis or the anti-cis. The following tables, 1 and 2, show the result obtained. No. Name Yield (%) Rf N H C O Ng-H H 2 H 3 50a b Tr-Hpi-Gly-OMe 35 35 0.4 0.4 5.54 5.36 5.95 9.21 3.01 4.89 3.82 4.2 2.48,2.28 2.35,1.08 51a b Tr-Hpi-Ile-OMe 29 61 0.5b 0.2b 5.64 5.30 5.74 8.92 3.14 4.74 4.10 4.18 2.28,2.13 2.34,1.45 52a b Tr-Hpi-Pro-OMe 84 0 0.6a 5.49 N / A 3.14 4.31 2.40,2.12 53a b Tr-Hpi-Phe-OMe 11 32 0.5° 0.2C 5.66 5.31 5.62 9.11 2.93 4.77 4.08 4.11 2.18,2.07 2.30,1.90 Table 1: Tr-Hpi-Xaa-OMe dipeptides: isolated yields of cyclization, R f, and characteristic ' H chemical shifts for the syn-cis (a) and anti-cis (b) diastereomers. NB. Rf values are all using hexane/AcOEt (1:1), except for a = (2:1), b = (1:2) and c = C H 2 C l 2 / M e O H (9:1). 40 Name Yield (%) Rf Hga N H C O Tr-Hpi-Ala-OMe 28 0.5 5.52 5.64 25 0.4 5.39 9.12 Tr-Hpi-Asn(Tr)-OMe 59 0.4a 5.54 6.65 38 0.2a 5.35 9.51 Tr-Hpi-Glu(tBu)-OMe 20 0.6 5.53 6.09 25 0.4 5.40 9.18 Tr-Hpi-His(Tr)-OMe 60 0 0.4b 5.42 9.82 Tr-Hpi-Leu-OMe 40 0.6 5.60 5.60 30 0.4 5.35 9.00 Tr-Hpi-Lys(Boc)-OMe 35 0.6 5.55 6.11 40 0.4 5.43 9.12 Tr-Hpi-Ser(tBu)-OMe 42 0.6 5.68 6.04 40 0.3 5.28 9.12 Tr-Hpi-Thr(tBu)-OMe 30 0.6 5.68 5.95 45 0.4 5.43 9.09 Tr-Hpi-Tyr(tBu)-OMe 16 0.6 4.92 5.45 21 0.4 5.29 7.91 Tr-Hpi-Val-OMe 48 0.6 5.62 5.85 44 0.4 5.33 8.97 Table 2: Tr-Hpi-Xaa-OMe dipeptides made by Dr. Jonathan May and Mr. Pierre Fournier46 for sake of comparison purpose and discussion only: isolated yields of cyclization, Rf, and characteristic ' H chemical shifts for the syn-cis (a) and anti-cis (b) diastereomers. N B . Rf values are all using hexane/AcOEt (1:1), except for a = (2:1), b = (3:1). These results allow us to conclude that the synthesis of dipeptides of the structure Tr-Hpi-Xaa-OMe is fairly general. This methodology could not be used on certain amino acids; where Xaa = Trp, Cys(STr), and Met. With these amino acids, the DMDO would oxidize the side chain as well and form unwanted products. 2.2.5 Preparation of Tr -Hpi -Gly-OH The final step for the preparation of an Hpi molecule suitable for solid phase synthesis is the saponification of the methyl ester of Tr-Hpi-Gly-OMe to form the free acid (scheme 27). 41 50a/b 5 4 a / b Scheme 27: Synthesis of Tr-Hpi-OH. Reagents and conditions: i) L i O H , Water/Dioxane, 1 hr, RT, 80 %. The final molecule was quite sensitive to acid. Usually, when a reaction with a base is done, the excess is neutralized with an acid during the work-up. We wanted to avoid this step, so the crude mixture was evaporated and then purified by flash chromatography on silica gel without any sort of work-up. 2.3 Solid phase synthesis of amatoxin derivatives The synthesis of amatoxin derivatives was achieved following a solid-phase strategy (scheme 28). O O Scheme continues on following page. 42 H N ^ O O H N L N H O Scheme 28: Synthesis of the octapeptide on solid-phase. Reagents and conditions: i) Fmoc-Xpro-OH (3 eq.), H B T U , HOBt, DIEA, DMF, RT, 30 min; ii) Piperidine, DMF, RT, 10 min; iii) Fmoc-Asn(Tr)-OH(3 eq.), H B T U or PyBOP, DIEA, D M F , RT, 20 min (condition repeated two times); iv) Fmoc-Cys(Tr)-OH (3 eq.), H B T U , DIEA, DMF, RT, 20 min; v) Fmoc-Gly-OH (3 eq.), HBTU, DIEA, DMF, RT, 20 min; vi) Fmoc-Ile-OH (3 eq.), H B T U , DIEA, D M F , RT, 20 min; vii) Tr-Hpi-Gly-OH (3 eq.), HBTU, DIEA, DMF, RT, 40 min. Where X is CH-SBn, CH-CF 3 , CH-F, C H - N 3 , CH-CN, CH-OtBu, C-F 2 , S, C=0. The 2-Chlorotrityl resin was used for the Fmoc solid phase synthesis. Since the resin is not benzyl alcohol based, the first amino acid is less prone to racemization. Furthermore, the resin is quite acid labile and it is possible to cleave the peptide without removing the trityl protective groups. Since the first amino acid of every single peptide is isoleucine, we used a commercially available preloaded isoleucine 2-chlorotrityl resin. This resin is made from a copolymer of styrene and divinylbenzene. The formation of the linear peptide was performed in an iterative fashion with the succession of Fmoc piperidine deprotection followed by coupling of the next amino acid with HBTU. Proline is a secondary amine and special care need to be taken to have a high yielding coupling with the next amino acid. Thus, coupling with trityl-asparagine ester has to be done two times. Furthermore, on several proline derivatives the addition of trityl asparagine was very difficult, even with a double coupling step. With the peptides containing the difluoroproline, the ketoproline, we had to use longer coupling times, PyBOP instead of H B T U and N M P as solvent instead of DMF to couple the asparagine. These changes to the conditions made these difficult couplings possible. 43 It was also found that a promising coupling reagent could work even better than the previous conditions with PyBOP and NMP. PyBrOP is quite similar to PyBOP but instead of the HOBt moiety there is a bromine atom (figure 18). The activated amino acid formed is then converted to a symmetrical anhydride.47 The thioproline peptide was made with PyBrOP as the coupling agent. O-P-N^J B r - f - V J b—(' PF6 ^ PF6" D F . " N-P F f i / PyBOP PyBrOP HBTU Figure 18: The different coupling reagents used for the peptide bond formations The peptides were treated with TFA on the solid phase to give the monocycle (scheme 29). The monocycles were not cyclized further in this present work. The bicycle molecules are suspected to be extremely toxic, so special care must be taken for for the final cyclizations. W OH ° \ / H2N. JKK. v o T R HN. , i) /=° rL) S. . 0 ^ / \ _ I N ^ L , \ H Q HN HN u \ H . 0 V - / 5 ^ HN \ NH H,N y " ^ - ^ m X \ / N ^ 0 N H O " o Scheme 29: Synthesis of the bicycle. Reagents and conditions: i) TFA, 4 h, RT; ii) PyBOP, DMF, DIE A , RT, 14 h Where X is CH-CF 3 and CH-F. 44 In total, nine linear peptides were made on solid-phase support (table 3). Two of those were cleaved from the resin with TFA and cyclized to give the monocycle (table 4). These molecules were identified by mass spectroscopy to confirm the structure. A l l the peptides showed the right mass even in the crude mixture. The majority of the peptides was very clean and showed just one peak with the expected mass. The purifications by HPLC of the peptides have not been done. O General structure of the linear octapeptide No Sequence Exact masses g/mol Mass found 54 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-FPro-Ile-OH 1376.6(100.0%) 1377.6 (83.3%) 1378.5 (M+H)+ 1400.5 (M+Na)+ 55 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-CF3Pro-Ile-OH 1426.6(100.0%) 1427.6 (90.3%) 1428.8 (M+H)+, 1450.7 (M+Na)+ 56 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-CNPro-Ile-OH 1383.6(100.0%) 1384.6(90.6%) 1385.9 (M+H)+, 1408.0 (M+Na)+ 57 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-(SBn)Pro-Ile-OH 1480.6 (100.0%) 1481.6(97.6%) 1483.0 (M+H)+ 58 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-N3Pro-Ile-OH 1399.6(100.0%) 1400.6 (90.3%) 1401.9 (M+H)+, 1424.9 (M+Na)+ 59 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-F2Pro-Ile-OH 1394.6 (100.0%) 1395.6 (89.2%) 1396.8 (M+H)+, 1419.8 (M+Na 60 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Hyp(tBu)-Ile-OH 1430.7(100.0%) 1431.7(93.6%) 1431.9 (M+H)+, 1456.0 (M+Na)+ 61 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Ketopro-Ile-OH 1372.6(100.0%) 1373.6(89.2%) 1373.8 (M+H)+ 62 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Thz-Ile-OH 1376.6(100.0%) 1377.6 (88.9%) 1399.7 (M+Na)+, 1421.7 (M-H+2Na)+ Table 3: General structure of the linear octapeptide and the different derivatives made. 45 General structure of the monocyclic octapeptide. No Sequence Exact masses g/mol Mass found 63 NH 2 -T rp-Gly-Ile-Gly-C :ys-Asn-FPro-Ile-OH 874.4(100.0%) 875.4 (47.7%) 875.5 (M+H)+, 897.5 (M+Na)+, 919.5 (M-H+2Na)+ 64 NH 2 -T rp-Gly-Ile-Gly-C :ys-Asn-CF 3Pro-Ile-OH 924.4 (100.0%) 925.4 (48.8%) 925.5 (M+H)+, 947.5 (M+Na)+ Table 4: General structure of the monocycle octapeptide and the different derivatives made. 46 Chapter 3 Conclusion 3.1 Work completed In the course of this work, we made a library of octapeptides to study the inhibition of amatoxin on Pol II. The starting point of this project was the study of the amatoxin-Pol II cocrystal X-ray structure made by Kornberg et al.11 Indeed, from this structure we could conclude that the hydrogen bond between the glutamine residue (Glu882) of Pol II and the hydroxyproline of the amatoxin should be one of the key interactions in the inhibition of Pol II. To assess the extent of interaction we decided to make several mimics of the natural amatoxin. Each of these analogs will comprise a modified proline residue. Once these amatoxin derivatives are synthesized, several biological assays will be carried out, which hopefully should enable a better understanding of the structure-inhibition relationship. The first part of the project was the synthesis of the various proline derivatives which were then to be incorporated into the peptides. The synthesized analogues are fluoroproline, difluoroproline, trifluoromethylproline, cyanoproline, benzylthioproline, azidoproline, hydroxyproline, ketoproline and thiazolidine acid. A l l these derivatives with the exception of thiazolidine acid were synthesized starting from hydroxyproline. Mainly nucleophilic displacements were performed on hydroxyproline to give the desired proline derivatives. A different strategy was used for the thiazolidine acid, which was synthesized from the condensation reaction of formalin and cysteine. A l l the proline derivatives were made as Fmoc-protected amino acids. The second part of the project was the synthesis of Hpi. This molecule is essential for the formation of the tryptathionine bridge of the amatoxins. Hpi was made as the dipeptide (i.e. Hpi-Gly) from Tr-Trp-Gly-OMe. This Hpi dipeptide was found to be necessary to give a clean and high yielding reaction during the formation of Hpi from Trp (the oxidation with DMDO). Several dipeptides were tested in order to verify the generality of this reaction. Once the Hpi was obtained as the methyl ester (Tr-Trp-Gly-47 OMe), a simple saponification of the ester yielded the targeted derivative Tr-Hpi-Gly-OH. The last part of the project was the formation of the octapeptides by standard solid phase synthesis. The different proline derivatives were first coupled on solid-phase. The proper sequence of amino acids were then coupled to the resin after the modified prolines. Finally Tr-Hpi-Gly-OH was coupled to give the linear octapeptides. Two peptides were further cyclized to give monocyclic peptides (the peptides with fiuoroproline and trimethylfluoroproline). 3.2 Future perspectives Work for the completion of the project is already underway. In a first step, the remaining linear peptides need to be cyclized using a Savige-Fontana reaction with TFA. Then all the monocyclic peptides need to be further cyclized using a coupling reagent (PyBOP or HBTU). This second cyclization reaction is still problematic and needs to be optimized. Indeed, when the cyclization was carried out on an octapeptide, two compounds with the expected mass were obtained.7 The chemical nature of these compounds is still elusive. This problem must be resolved prior to the final cyclization of our library peptides. Once all the octapeptides are in the bicyclic form, the amatoxins with the azidoproline and benzylthiolproline will have to be deprotected, to give the amino- and thioprolines respectively. These two deprotection reactions will have to be done carefully and on very small scale since the peptide solid-phase synthesis produces only a small quantity of peptides. The final part of the project and the one that will be the most relevant in the study of Pol II will be the biological assays using the library of amatoxins. The most obvious assay is the inhibitory activity test. Such assays have been done in the past for amatoxins using Drosophilia embryos.48 48 Chapter 4 Experimental Section 4.1 General 4.1.1 Synthesis Ice/water bath (0°C) or a dry ice/acetone bath (-78°C) were used for cold temperature reactions. A mineral oil bath and a temperature controlled hot plate (IKA Ceramag Midi equipped with an IKA ETS-D4 Fuzzy thermometer) were used for controlled temperature reactions. Tetrahydrofuran and tert-butyl alcohol were purchased from E M D and all other solvents were purchased from Fisher. Anhydrous THF was obtained by distillation over sodium with benzophenone as indicator. Anhydrous triethylamine, pyridine and dichloromethane were obtained by distillation over calcium hydride. Methanol (50 mL) was reacted with magnesium turnings to give magnesium methoxide, iodine was used as a catalyst. Anhydrous methanol was obtained by distillation over the magnesium methoxide previously made. A l l the reagent purifications were done according to a literature procedure.49 A l l the amino acids and coupling reagents were purchased form NovaBioChem and all other reagents were purchased from Aldrich Chemicals.The reagents were used as provided by the manufacturer, unless otherwise indicated. The amino acids are L unless otherwise stated. A Biichi rotary evaporator was used for the evaporations under reduced pressure. 4.1.2 Thin layer chromatography Silica gel 60 F254 precoated aluminium plates from E M Science were used for thin layer chromatography (TLC). For acid sensitive compounds, T L C plates pre-treated with a solution of 5 % triethylamine in hexanes were used. The detection on the T L C plates was done with a U V lamp (k = 254 and 365 nm) or with reaction with ninhydrin. The 49 ninhydrin staining was made for a solution containing ninhydrin (150 mg) in n-butanol (50 mL) and acetic acid (1.5 mL). We used silica gel 230-400 mesh from SiliCycle (Quebec) and from E M D for the flash column chromatography purifications. 4.1.3 Chromatography An Agilent HPI 100 Bruker Esquire L C M S was used for all the low-resolution mass spectra. N M R spectra were recorded with deuterated methanol, water, chloroform and dimethylsulfoxide obtained from Cambridge Isotope Laboratory, Inc. Proton ( 'H NMR) spectra were recorded using either a Bruker AV-300 (300 MHz) or a Bruker AV-400 (400 MHz) spectrometer. Carbon nuclear magnetic resonance ( 1 3 C NMR) proton decoupled spectra were recorded using a Bruker AV-400 (100 MHz) spectrometer. Fluorine nuclear magnetic resonance ( l 9 F NMR) proton decoupled spectra were recorded using a Bruker AV-300 (250 MHz) spectrometer and TFA was used as reference (0 ppm). Chemical shifts for all spectra were reported in parts per million (ppm) and, otherwise noted, referenced to the solvent peak. 4.2 Chemical methods 4.2.1 Preparation of the various proline derivatives Note: The nomenclature of the proline carbones was done in a standard fashion with the Greek alphabet (figure 19). H O Figure 19: Nomenclature used for the proline Hydroxyproline methyl ester hydrochloride 1 H O 50 Hydroxyproline (2.26g, 20 mmol) was suspended in 2,2-dimethoxypropane (200 mL). Concentrated hydrochloric acid (20 mL) was added and the mixture was allowed to react at room temperature overnight. The solvent was evaporated to dryness while the temperature of the water bath was kept below 60°C. The residue was dissolved in ethanol, triturated with methanol and the solution was diluted with diethyl ether (500 mL). The precipitate was filtrated, washed with ether, and dried in vacuo. The product was recrystallized from an ether-methanol mixture. White solid, 79 % yield, 2.87 g. * H N M R (D 2 0) 5: 4.77-4.73 (m, merged with solvent peak, 2H, C H a , C H 7 ) , 3.74 (s, 1H, C H O M e ) , 3.46-3.24 (m, 2H, C H 2 5 ) , 2.45-2.35 (m, 1H, C H 2 P ) , 2.25-2.14 (m, 1H, C H 2 P ) . iV-Boc-4(S)-hydroxy-4-trifluoromethylproline methyl ester 5 The preparation of A^-Boc-4(S)-hydroxy-4-trifluoromethylproline methyl ester 5 was done according to the procedure by Del Valle et al.32 in three steps from alcohol 1. Reddish solid, 18 % yield for three steps, 850 mg. ' H N M R (CDC13) 8: 4.52 and 4.43 (2d, J= 9.8 Hz, 1H, C H a , rotamers), 3.78-3.64 (m, 5H, C H 3 o m e , C H 2 5 ) , 2.60-2.48 (m, 1H, C H 2 P ) , 2.22-2.15 (m, 1H, C H 2 P ) , 1.44 and 1.40 (s, 9H, C H 3 b 0 C ) . A7-(2S)-Boc-4-Trifluoromethyl-3,4-dehydroproline methyl ester36 6 C F 3 F 3 C 51 Hydroxyproline (2.26g, 20 mmol) was suspended in 2,2-dimethoxypropane (200 mL). Concentrated hydrochloric acid (20 mL) was added and the mixture was allowed to react at room temperature overnight. The solvent was evaporated to dryness while the temperature of the water bath was kept below 60°C. The residue was dissolved in ethanol, triturated with methanol and the solution was diluted with diethyl ether (500 mL). The precipitate was filtrated, washed with ether, and dried in vacuo. The product was recrystallized from an ether-methanol mixture. White solid, 79 % yield, 2.87 g. *H NMR (D 2 0) 5: 4.77-4.73 (m, merged with solvent peak, 2H, C H a , CH 7 ) , 3.74 (s, 1H, C H O M e ) , 3.46-3.24 (m, 2H, C H 2 5 ) , 2.45-2.35 (m, 1H, C H 2 P ) , 2.25-2.14 (m, 1H, C H 2 P ) . AMBoc-4(S)-hydroxy-4-trifluoromethylproline methyl ester 5 The preparation of iV-Boc-4(S)-hydroxy-4-trifluoromethylproline methyl ester 5 was done according to the procedure by Del Valle et al.32 in three steps from alcohol 1. Reddish solid, 18 % yield for three steps, 850 mg. ' H NMR (CDC13) 5: 4.52 and 4.43 (2d, J= 9.8 Hz, 1H, C H a , rotamers), 3.78-3.64 (m, 5H, C H 3 o m e , C H 2 5 ) , 2.60-2.48 (m, 1H, C H 2 P ) , 2.22-2.15 (m, 1H, C H 2 P ) , 1.44 and 1.40 (s, 9H, C H 3 b 0 C ) . A^-(2S)-Boc-4-Trifluoromethyl-3,4-dehydroproIine methyl ester36 6 C F 3 F 3 C 51 Boc-(4S)-hydroxy-4-trifluoromethyl-L-proline methyl ester 5 (0.72 g, 2.3 mmol) was dissolved in pyridine (29 mL). Thionylchloride (SOCI2) (2.15 mL, 29 mmol) was added. The mixture was heated at reflux for 20 min under N2. 8 mL of water were added to quench the excess SOCI2. The aqueous layer was extracted with ethyl acetate (3 x 15 mL). The combined organic layers were washed once with an 0.1 N HCl solution (30 mL), NaHCC>3 saturated solution (30 mL) and brine (30 mL). The ethyl acetate solution was dried with MgSCU, filtered and evaporated to dryness. The brown product was purified by flash chromatography on silica gel with a mixture of AcOEt/Hexanes 1:30. R f (AcOEt/Hexanes 3:7) 0.74, brown solid, yield 91 %, 0.63 g. *H N M R (CDCI3) 5: 6.24 and 6.20 (s, 1H, C H P , rotamers), 5.22-5.05 (m, 1H, C H a ) , 4.47-4.27 (m, 2H, C H 2 8 ) , 3.74 (s, 3H, C H 3 o m e ) , 1.45 and 1.39 (s, 9H, C H 3 b o c ) . 1 3 C N M R (CDCI3) 5: 169.04 and 168.72 (CO m e t h y l ) , 153.23 and 152.66 (CO b o c ) , 133.16-132.26 (m, CF 3 ) , 122.01 and 119.31 (CH 2 5 ) , 128.16 (CY), 66.42 and 66.15 (CH a ) , 52.53 ( C H 3 o m e ) , 50.86 and 50.70 (CH 2 P ) , 28.04 and 27.42 (CH 3 b o c ) . 7V-Boc-2(S)-hydroxymethyl-4-trifluoromethyl-3,4-pyrroline 7 F 3 C A^-(2S)-Boc-4-Trifluoromethyl-3,4-dehydroproline methyl ester 6 (0.630 g, 2.13 mmol) was dissolved in THF (2.2 mL). Lithium chloride (0.131 g, 3.2 mol), NaBH4 (0.118 g, 3.2 mmol) and ethanol (4.4 mL) were added. The reaction mixture was stirred overnight at room temperature. The pH of the solution was then brought to 4 with a solution of citric acid 5 % in water. The ethanol was evaporated under reduced pressure and the solution was extracted with dichloromethane ( 3 x 1 0 mL). The CH2CI2 solution was then dried with MgSCM, filtered and evaporated to dryness. The product was purified by flash chromatography on silica gel with a gradient of AcOEt/Hexanes ranging from 1:4 to 2:3. 52 R f (AcOEt/Hexanes 1:4) 0.37, colorless oil, 62 % yield, 0.46 g. ! H NMR (CDC13) 8: 6.27-6.28 (m, 1H, CH P ) , 4.82 (s, 1H, OH), 4.45-4.17 (m, 2H, CH 2 OH), 3.83-3.64 (m, 2H, CH 2 8 ) , 1.47 (s, 9H, C H 3 b o c ) . N-Boc-(2S,4R)-trifluoromethylproline 9 The preparation of Boc-trans-trifluoromethylproline 9 was achieved according to a procedure by Del Valle et al.32 starting from A^-Boc-2(S)-hydroxymethyl-4-trifluoromethyl-3,4-pyrroline 7. The N M R of the product agreed with litterature. White solid, 40 % yield, 0.102 g. R f (CH 2C1 2Methanol 9:1) 0.3 ' H NMR (CDCI3) 8: 9.96 (s, br, 1H, COOH), 4.48 and 4.37 (d, J = 4.4 Hz, 1H, CH a ) , 3.82-3.65 (m, 1H, C H 2 5 ) , 3.60-3.45 (m, 1H, CH 2 6 ) , 3.16-2.97 (m, 1H, CH T ), 2.47-2.23 (m, 1H, C H 2 P ) , 1.45 and 1.40 (s, 9H, C H 3 b o c ) . 1 9 F NMR (CDCI3) 5.32 and 5.22 (d, J= 7.7 Hz) Hydroxyproline benzyl ester/Moluenesulfonate 12 Hydroxyproline (5 g, 38 mmol) was suspended into a mixture of benzyl alcohol (16.5 mL) and benzene (33.5 mL) in a Dean-Stark apparatus. /?-Toluenesulfonic acid (8.1 g, 1.1 eq) was added. The mixture was heated at reflux for 14 hours. 100 mL of diethyl ether HO 53 were added and a solid was formed. The solid was filtered off and recrystallized from ethanol and diethyl ether. The product was obtained as the /?-toluenesulfonate salt. R f (methanol/CH2Cl2 1:9) 0.4. White solid, 88% yield, 13.2 g. Boc-hydroxyproline benzyl ester 13 Hydroxyproline benzyl ester p-Toluenesulfonate 12 (3.12 g) was dissolved in acetonitrile (15.6 mL) and triethylamine (1.10 mL) was added. The solution was cooled down to 0°C and di-terr-butyldicarbonate (2.59 g) was added. The reaction was carried on at 0°C for 30 min and then at room temperature until completion as revealed by TLC. The solvent was evaporated to dryness. The solid was dissolved in ethyl acetate (25 mL). The solution was washed with a 0.1 M H C l solution (3 x 25 mL), with a sodium bicarbonate saturated solution (3 x 25 mL) and with brine (3 x 25 mL). The washed solution was dried with M g S 0 4 , filtered and evaporated to dryness. The product was purified with a silica gel plug using a mixture of hexanes, ethyl acetate 1:1 as eluant. R f (AcOEt/Hexanes 1:1) 0.26, colorless oil, 95 % yield, 3.22 g. ! H N M R (CDC13) 6: 7.32 (s, 5H, ArH), 5.25-5.04 (m, 2H, C H 2 b n ) , 4.52-4.38 (m, 2H, C H a , CH y ) , 3.62-3.40 (m, 2H, C H 2 5 ) , 2.30-2.20 (m, 2H, C H 2 P ) , 1.50 and 1.42 (s, 9H, C H 3 b 0 C ) . Boc-(2S,4R)-4-fluoroproline 16 F 54 The preparation of /r<ms-Boc-4-fluoroproline 16 was done according to the procedure by Hodges et al.15 Colorless oil, 9 % yield from Boc-hydroxyproline benzyl ester 13, 0.170 g. lH N M R (CDC13) 8: 9.50 (s, br, 1H, COOH), 5.35-5.10 (m, 1H, CH 1 ) , 4.58-4.32 (m, 1H, C H a ) , 4.00-3.43 (m, 2H, C H 2 6 ) , 2.75-1.85 (m, 2H, C H 2 P ) , 1.49 and 1.44 (s, 9H, C H 3 b o c ) . 1 9 F N M R (CDCI3) 5: -178.5 and -178.8 (decoupled, C 6 F 6 as reference). Benzyl (2S)-N-tert-butoxycarbonyl-4-oxo-prolinate 25 The preparation of Benzyl (2S)-N-tert-Butoxycarbonyl-4-oxo-prolinate 25 was achieved according to a procedure by Del Valle et al.32 starting from Boc-hydroxyproline benzyl ester 13. The oxidation was based on a publication by De Luca et al.34 The N M R agreed with the literature. White product, 87 % yield, 0.860 g. *H N M R (CDCI3) 8: 7.33 (s, br, 5H, ArH), 5.27-5.08 (m, 2H, C H 2 b n ) , 4.84 and 4.72 (d, J = 19.7 Hz, 1H, CH"), 3.89-3.85 (m, 2H, C H 2 8 ) , 2.97-2.85 (m, 1H, C H 2 P ) , 2.57 and 2.42 (s, 1H, C H 2 P ) , 1.45 and 1.35 (s, 9H, C H 3 b o c ) . Boc-(2S)-4,4-difluoroproline benzyl ester50 26 F 55 Benzyl (2S)-N-/-butoxycarbonyl-4-oxo-prolinate 25 (0.500 g, 1.56 mmol) was dissolved in dichloromethane (4 mL). The solution was cooled down to -78 °C in an acetone/dry ice bath under N 2 . (Diethylamino)sulfur trifluoride (DAST, 0.62 mL, 4.68 mmol) was added drop wise. The mixture was then stirred at room temperature until completion (15 hours). The excess reagent was neutralized with a saturated NaHCC>3 solution (10 mL). The mixture was washed with a saturated NaHC03 solution (3 x 5 mL), washed with water (3x5 mL), dried with MgSCM, filtered and evaporated to dryness. The product was purified by flash chromatography on silica gel using a mixture of AcOEt/Hexanes4:l. R f (AcOEt/Hexanes 2:3) 0.6, white solid, 29 % yield, 0.230 g. ! H N M R (CDC13) 6: 7.34 (s, br, 5H, ArH), 5.27-5.08 (m, 2H, C H 2 b n ) , 4.59 and 4.46 (dd, J= 5.8Hz, J= 9.1Hz, 1H, C H a ) , 3.89-3.74 (m, 2H, C H 2 S ) , 2.79-2.56 (m, 1H, C H 2 P ) , 2.50-2.42 (m, 1H, C H 2 P ) , 1.44 and 1.32 (s, 9H, C H 3 b o c ) . E S + / M S : 534.0 (M+Na)+. Boc-(2S)-4,4-difluoroproline 27 Boc-(2S)-4,4-difluoroproline benzyl ester 26 (0.154 g, 0.45 mmol) was dissolved in methanol (2 mL). Palladium on activated charcoal (5 % in Pd, 50 mg) was added carefully to the solution. The reaction mixture was stirred under 1 atm of H 2 for 2 hours. The mixture was filtered over a celite plug and the solvent was evaporated to give the desired product. R f (AcOEt/Hexanes 3:7) 0.1, white solid, 97 % yield, 0.110 g. ' H N M R (CDCI3) 5: 9.02 (s, br, 1H, COOH), 4.63-4.52 and 4.48-4.44 (m, 1H, C H a ) , 3.91-3.71 (m, 2H, C H 2 6 ) , 2.77-2.48 (m, 2H, C H 2 P ) , 1.46 and 1.41 (s, 9H, C H 3 b o c ) . F 56 m-Boc-hydroxyproIine benzyl ester 15 HO° The preparation of c/s-Boc-hydroxyproline benzyl ester 15 was achieved according to Hodges et al.15 starting from Boc-hydroxyproline benzyl ester 13. The N M R agreed with the literature. Rf (AcOEt/Hexanes 1:1), white solid, 24 % yield over the two steps, 630 mg. *H N M R (CDC13) 5: 7.30-7.24 (m, 5H, ArH), 5.24-5.05 (m, 2H, C H 2 b n ) , 4.38-4.25 (m, 2H, C H a , CH y ) , 3.70 (s, 1H, OH), 3.58-3.48 (m, 2H, C H 2 8 ) , 2.31-2.22 (m, 1H, C H 2 P ) , 2.06-2.00 (m, 1H, C H 2 P ) , 1.40 and 1.27 (s, 9H, C H 3 b o c ) . ES + /MS: 344.2 (M+Na)+. (3S,5S)-5-((Benzyloxy)carbonyl)-l-(tert-butoxycarbonyl)pyrrolidin-3-yl 4-methylbenzenesulfonate 20 cw-Boc-hydroxyproline benzyl ester 15 (0.630 g, 1.96 mmol) was dissolved in pyridine (10 mL) and /?-toluenesulfonylchloride (0.75 g, 3.92 mmol) was added at 0°C. The reactive mixture was kept at 0°C for 1 hour. The solution was then stirred at room temperature for 13 hours (until completion). The mixture was poured into an ice/water mixture (15 mL) and extracted with ether (3x10 mL). The organic layers were combined and washed with water (6 xl5 mL), dried with MgS04, filtered and evaporated on the 57 rotary evaporator. The product was purified on a silica gel flash chromatography column using a mixture of AcOEt/Hexanes 3:2. R f (AcOEt/Hexanes 3:7) 0.30, colorless oil, 21 %, 0.197 mg. ' H N M R (CDC13) 5: 7.69 (d, J= 8.1 Hz, 2H , ArH p " t o 1 ), 7.33-7.28 (m, 7H, ArH), 5.18-4.96 (m, 3H, C H Y , C H 2 b n ) , 4.49-4.46 and 4.38-4.31 (m, 1H, C H a , ) 3.67-3.55 (m, 2H, C H 2 8 ) , 2.43-2.41 (m, 4H, C H 3 t o 1 , C H 2 P ) , 2.34-2.32 (m, 1H, C H 2 P ) , 1.39 and 1.29 (s, 9H, C H 3 b 0 C ) . E S + / M S : 498.1 (M+Na)+, 304.4 (M-OTs) + Boc-(2S,4R)-4-cyanoproline benzyl ester51 21 Compound 20 (270 mg, 0.56 mmol) was dissolved in D M F (9 mL) and tetrabutylammonium cyanide (305 mg, 1.14 mmol) was added. The reaction mixture was stirred until completion (15 hours) at 55°C and then a mixture of ice/water (15 mL) was added. The resulting liquid was extracted with ethyl acetate ( 5 x 5 mL). The organic fractions were combined and washed with water (3 x 20 mL), with brine (3 x 20 mL), dried with MgS04 , filtered and the solvent was evaporated to dryness. The product was purified by flash chromatography on silica gel using a mixture of AcOEt/Hexanes 3:7. R f (AcOEt/Hexanes 1:1) 0.4, white crystalline product, 42 % yield, 0.078 g. J H N M R (CDCI3) 5: 7.34 (s, br, 5H, ArH), 5.24-4.5.07 (m, 2H, C H 2 b n ) , 4.52-4.50 and 4.42-4.39 (m, 1H, C H a , ) 3.92-3.86 (m, 1H, C H 2 5 ) , 3.68-3.60 (m, 1H, C H 2 8 ) , 3.21-3.18 (m, 1H, CYC), 2.52-2.47 (m, 1H, C H 2 P ) , 2.34-2.31 (m, 1H, C H 2 P ) , 1.45 and 1.31 (s, 9H, C H 3 b 0 C ) . E S + / M S : 353.2 (M+Na)+, 304.4(M-CN)+. NC 58 Boc-(2S,4R)-4-cyanoproline 22 Boc-(2S,4R)-4-cyanoproline benzyl ester 21 (70 mg, 0.2 mmol) was dissolved in methanol (2 mL). Palladium on activated charcoal (5 % in Pd, 20 mg.) was added carefully to the solution. The reaction mixture was stirred under 1 atm of H2 for 2 hours. The solution was filtered over a celite plug and the solvent was evaporated. The product was purfied by flash chromatography on silica gel using a mixture of AcOEt/Hexanes White product, 53 % yield, 0.027 mg. l H N M R (CDCI3) 5: 4.25-4.18 (m, 1H, CH",) 3.81-3.76 (m, 1H, C H 2 8 ) , 3.63-3.56 (m, 1H, C H 2 5 ) , 3.16 (q, J= 7.3 Hz, 1H, CH Y), 2.51-2.40 (m, 1H, C H 2 P ) , 2.33-2.27 (m, 1H, C H 2 P ) , 1.45 and 1.42 (s, 9H, C H 3 b o c ) . Boc-(2S,4S)-4-bromoproline benzyl ester 5 2 32 Diisopropyl azodicarboxylate (11.6 mL, 60 mmol) was added drop wise to a solution of triphenylphosphine in THF(15.7 g, 60 mmol in 100 mL of THF) under N 2 . The solution was stirred for 20 min at room temperature. Anhydrous lithium bromide (10.4 g, 120 mmol) and a solution of Boc-hydroxyproline benzyl ester 13 (3.86 g., 12 mmol) in THF (50 mL) were added. The mixture was stirred at room temperature for 20 hours. The reaction solvent was evaporated and the residue was poured into water. The mixture was extracted twice with ether (2 x 30 mL). The organic fractions were combined and washed with brine ( 3 x 80 mL), dried with MgS04, filtered and evaporated in vacuo. The product 3:7. 59 was purified by flash chromatography on silica gel using a mixture of AcOEt/Hexanes 2:8. R f (AcOEt/Hexanes 1:4) 0.3, colorless oil, 78 % yield, 3.57 g. ' H N M R (CDC13) §: 7.34-7.30 (m, 5H, ArH), 5.29-5.06 (m, 2H, C H 2 b n ) , 4.47-4.43 and 4.35-4.31 (m, 1H, C H a , ) 3.30-3.35 (m, 1H, C H 2 5 ) , 4.11-3.97 (m, 1H, C H 2 5 ) , 3.73-3.67 (m, 1H, CH Y ) , 2.86-2.75 (m, 1H, C H 2 P ) , 2.44-2.39 (m, 1H, C H 2 P ) , 1.45 and 1.31 (s, 9H, C H 3 b 0 C ) . E S + / M S : 406.1 and 408.0 (M+Na)+. Boc-(2S,4S)-4-(benzylthio)proline benzyl ester 33 Benzyl mercaptan (0.766 mL, 6.5 mmol) and sodium ethoxide (0.44 g, 6.5 mmol) were dissolved in enough ethanol to give a 5 mL solution. Boc-(2S,4S)-4-bromoproline benzyl ester 32 (1 g, 2.6 mmol) was dissolved in ethanol (5 mL). 4 mL of the thiolate solution was added to the proline derivative solution. The mixture was stirred for one day (15 h) at room temperature. Ethanol was evaporated on the rotary evaporator. The residue was dissolved in a N a H C 0 3 saturated solution (10 mL) and extracted with AcOEt ( 3 x 5 mL). The combined organic were washed with a saturated N a H C 0 3 solution (3 xl5 mL), with brine (3x15 mL), dried with MgS04, filtered and evaporated in vacuo. The product was purified by flash chromatography on silica gel using a mixture of AcOEt/Hexanes 3:7. R f (AcOEt/Hexanes 3:7) 0.55, yellow oil, 43 % yield, 0.480 g. 60 ' H N M R (CDCI3) 5: 7.32-7.23 (m, 10H, ArH), 5.21-5.02 (m, 2H, C H 2 0 b n ) , 4.36-4.31 and 4.28-4.25 (m, 1H, CH",) 3.82-3.77 (m, 1H, CH 7 ) , 3.73 and 3.70 (m, 2H, C H 2 S b n ) , 3.37-3.22 (m, 2H, C H 2 5 ) , 2.15-2.07 (m, 2H, C H 2 P ) , 1.41 and 1.38 (s, 9H, C H 3 b o c ) . ES + /MS: 450.1 (M+Na)+. Boc-(2S,4S)-4-(benzylthio)proline 34 Boc-(2S,4S)-4-(benzylthio)proline benzyl ester 33 (267 mg, 0.59 mmol) was dissolved in dioxane (11 mL). Lithium hydroxide (150 mg, 5.85 mmol) was dissolved in water and then added to the dioxane solution. The reactive mixture was stirred for one hour at room temperature. Dioxane was evaporated and 10 mL of water was added. The solution was washed with ethyl acetate (4x15 mL). The pH of the solution was brought to 1-2 with concentrated hydrochloric acid. The acidified mixture was extracted with AcOEt ( 5 x 5 mL). The organic fractions were combined and washed with brine, dried with MgSCM, filtered and evaporated in vacuo. The product was purified by flash chromatography on silica gel using a mixture of M e O H / C H 2 C l 2 1:19. R f (MeOH/CH 2 Cl 2 3:17) 0.3, white oil, 44 % yield, 86 mg. ' H N M R (CDCI3) 5: 9.52 (s, br, 1H, COOH), 7.29-7.22 (m, 5H, ArH), 4.40-4.38 and 4.33-4.30 (m, 1H, CH",) 3.81-3.74 (m, 2H, C H 2 S b n ) , 3.58-3.54 and 3.40-3.36 (m, 1H, CH 7 ) , 3.30-3.15 (m, 2H, C H 2 5 ) , 2.26-2.14 (m, 1H, C H 2 P ) , 2.07-1.99 (m, 1H, C H 2 P ) , 1.43 and 1.39 ( s ,9H,CH 3 b 0 C ) . ES + /MS: 360.1 (M+Na)+ (R)-Thiazolidine-4-carboxylic acid 4 1 38 'OH 61 Cysteine hydrochloride (15 g) was dissolved in water (50 mL). 12 mL (1.1 eq.) of a formaldehyde solution in water 37 % was added. The mixture was allowed to stand at room temperature for 13 hours. Pyridine (15 mL) was than added to the solution and a cloudy precipitate appeared. After 2 hours, ethanol (25 mL) was added to the mixture. The white solid was filtered and washed 3 times with ethanol. The product was recrystallized from water. White long needles, 47 % yield after the first crop, 5.94 g. ! H N M R (D 2 0) 5: 4.37-4.31 (m, 2H, C H 2 5 ) , 4.25-4.21 (m, 1H, C H a ) , 3.34-3.18 (m, 1H, The /?-toluenesulfonate cw-hydroxyproline benzyl ester, compound 20 (400 mg, 0.84 mmol), was dissolved in D M F (6 mL). Sodium azide (0.382 g, 7 eq.) was added. The mixture was heated to 50°C and allowed to stir to completion (as revealed by TLC). The solution was poured into ice-cold water (20 mL) and extracted with ethyl acetate (3x10 mL). The organic fractions were combined and washed with water (3 x 20 mL), washed with brine (3 x 20 mL), dried with MgS04, filtered and evaporated to dryness. The product was purified by flash chromatography using a mixture of AcOEt/Hexanes 2:8. R f (AcOEt/Hexanes 1:4) 0.2, colorless oil, 95 % yield, 0.276 g. *H N M R (CDC13) 5: 7.33 (s, br, 5H, ArH), 5.25-5.03 (m, 2H, C H 2 b n ) , 4.46 (dd, J= 6.7 Hz, J= 7.6 Hz, 0.5H, CH") 4.35 (t, J= 7.5 Hz, 0.5H, C H a ) , 4.16-4.12 (m, 1H, CH Y), 3.71-3.65 (m, 1H, C H 2 5 ) , 3.59-3.55 (m, 1H, CH 2 5 ) , 2.36-2.25 (m, 1H, C H 2 P ) , 2.18-2.10 (m, 1H, C H 2 P ) , 1.44 and 1.33 (s, 9H, C H 3 b o c ) . E S + / M S : 369.2 (M+Na)+. C H 2 p ) . Boc-(2S,4S)-4-(azido)proline benzyl ester51 41 62 Boc-(2S,4S)-4-(azido)prolineS4 42 Boc-(2S,4S)-4-(azido)proline benzyl ester 42 (276 mg, 0.8 mmol) was dissolved into a mixture of THF:methanol 1:1 (7.14 mL). Lithium hydroxide hydrate (50 mg) dissolved in a minimum amount of water (0.25 mL) was added to the reaction solution. The mixture was stirred for two hours at room temperature. The pH of the solution was then brought to 4 using concentrated HCl . After the addition of water (20 mL) and HCl (5 mL), the solution was extracted with ethyl acetate (4 x 10 mL). The organic fractions were combined, washed with brine (3 x 20 mL), dried with MgSCM, filtered and evaporated to dryness. To remove the benzyl alcohol, further extraction steps were necessary. The product was dissolved in ethyl acetate (20 mL) and extracted with a saturated sodium bicarbonate solution (5x10 mL). The aqueous fractions were combined and acidified carefully to pH = 1 with concentrated HCl . The aqueous layer was extracted with ethyl acetate ( 5 x 1 0 mL). The combined organic fractions were washed with brine (3 x 20 mL), dried with MgSCTt, filtered and evaporated to dryness. Colorless oil, 92 % yield, 0.177 g. l H N M R (CDC13) 5: 7.33 (s, br, 5H, ArH), 5.25-5.03 (m, 2H, C H 2 b n ) , 4.46 (dd, J = 6.7 Hz, J= 7.6 Hz, 0.5H, C H a ) , 4.35 (t, J= 7.5 Hz, 0.5H, C H a ) , 4.16-4.12 (m, 1H, CH Y), 3.71-3.65 (m, 1H, C H 2 8 ) , 3.59-3.55 (m, 1H, CH 2 5 ) , 2.36-2.25 (m, 1H, C H 2 P ) , 2.18-2.10 (m, 1H, C H 2 P ) , 1.44 and 1.33 (s, 9H, C H 3 b o c ) . E S + / M S : 369.2 (M+Na)+. Boc-(2S,4R)-4-(azido)proline 45 63 cw-Boc-4-(azido)proline,45 was synthesized using the same protocol as for compound 41. However, instead of />-toluenesulfonate cw-hydroxyproline benzyl ester 20, the starting material for the reaction was cw-Boc-4-bromoproline benzyl ester 32. The product was recovered as a secondary reaction product. R f (AcOEt/Hexanes 1:4) 0.2, colorless oil, 2 % yield, 0.010 g. *H N M R (CDC13) 5: 7.36 (s, br, 5H, ArH), 5.29-5.08 (m, 2H, C H 2 b n ) , 4.49 (dd, J= 3.4 Hz, J= 8.9 Hz, 0.5H, C H a ) , 4.36 (dd, J = 3.7 Hz, J= 9.0 Hz, 0.5H, C H a ) , 4.16-4.14 (m, IH, CH Y), 4.36 (dd, J = 3.7 Hz, J = 9.0 Hz, 0.5H, C H 2 5 ) , 3.67 (dd, 1H, J = 6.0 Hz, J = II. 5 Hz, 0.5H, C H 2 5 ) , 3.51 and 3.45 (dd, J - 3.7 Hz, J - 11.7 Hz, 1H, C H 2 5 ) 2.50-2.39 (m, 1H, C H 2 P ) , 2.20-2.17 (t, 1H, J=3.8Hz, C H 2 P ) , 1.46 and 1.34 (s, 9H, C H 3 b o c ) . E S + / M S : 369.2 (M+Na)+. Fmoc-(2S)-4-ketoproline 30 (2S)-N-tert-Butoxycarbonyl-4-oxo-prolinate 25 (310 mg) was dissolved in methanol (4 mL). Palladium on activated charcoal (5 % in Pd, 100 mg) was added carefully to the methanol solution. The reaction mixture was stirred under hydrogen atmosphere (1 atm) for 2h. The mixture was then filtered on a celite plug and the solvent was evaporated. The product was purified by flash chromatography on silica gel using a mixture of C H 2 C l 2 / M e O H 4 : l . R f (CH 2 Cl 2 /MeOH 9:1) 0.15, colorless oil, 50 % yield, 0.110 mg. ! H N M R (CDCI3) 5: 4.68 (s br, 1H, C H a ) , 3.86 (s, 1H, C H 2 5 ) , 3.81 (s, 1H, CH 2 8 ) , 2.96-2.80 (m, 1H, C H 2 P ) , 2.70-2.59 (m, 1H, C H 2 P ) , 1.43 (s, 9H, C H 3 b o c ) . 64 General procedure for removal of the Boc protecting groups of amino acids The amino acids (0.44 mmol) were dissolved in trifluoroacetic acid (3 mL). The mixture was stirred for one hour at room temperature. TFA was evaporated on a rotary evaporator and triturated with diethyl ether (5x5 mL). The residues were dried on a high vacuum pump and the resulting products were used without further purification for the addition of Fmoc-protective group. General procedure for the addition of a Fmoc protecting group on amino acids39 The N-deprotected amino acids (4.2 mmol) were dissolved or suspended in 9% sodium carbonate solution (10 mL, 8.4 mmol) and cooled in an ice bath. Fmoc N -hydroxysuccinimide ester (FmocOSu, 1.49g, 4.4 mmol) was dissolved in D M F (8 mL) at 0°C and added to amino acid solution. The heterogeneous mixture was stirred for 20 min. The mixture was then diluted with water (120 mL), extracted with ether (2 x 50 mL) and with ethyl acetate (3 x 50 mL). The aqueous phase was cooled down and acidified to pH 2 using concentrated hydrochloric acid. The aqueous layer and the precipitate were extracted with ethyl acetate ( 6 x 1 5 mL). The extracts were combined and washed with brine (3 x 20 mL), with water (2 x 20 mL), dried with MgS04 and reduced to a small volume in vacuo. A small quantity of petroleum ether was added and a crystalline precipitate was obtained after evaporation to dryness. The product was obtained pure. In case impurities were present, the product was further purified by flash chromatography using M e O H / C H 2 C l 2 3:17 as eluant. Fmoc-(2S,4R)-4-(trifluoromethyl)proline 11 R f (CH 2 Cl 2 /MeOH 9:1) 0.4, white solid, 50 % yield, 0.073 g. 65 ' H N M R (CD3OD) 5: 7.66-7.41 (m, 2H, ArH), 7.61-7.53 (m, 2H, ArH), 7.35 and 7.27 (t, J= 7.4 Hz, 4H, ArH), 4.44- 4.38 (m, 1H, C H a ) , 4.34-4.26 (m, 2H, C H 2 F m o c ) , 4.22-4.13 (m, 1H, C H F m o c ) , 3.78 and 3.66 (dd, J= 8.8 Hz, J= 10.9 Hz, 1H, C H 2 S ) , 3.61-3.55 and 3.47-3.41 (m, 1H, C H 2 5 ) , 3.16 (td, J= 7.9 Hz, J= 16.4 Hz, 1H, CH Y), 2.48-2.17 (m, 1H C H 2 P ) . 1 3 C N M R (CDCI3) 5: 176.59 ( C C O O H ) , 155.32 ( C c a r b a m a t e ) , 143.46 (C a r ) , 141.00 (C a r ) , 127.74 (CH a r ) , 126.98 (CH a r ) , 124.97 (CH a r ) , 119.94 (CH a r ) , 68.41 ( C H 2 F m o c ) , 60.24 (CH a ) , 46.59 (CH 2 8 ) , 45.23 ( C H F m o c ) , 29.60 and 28.77 (CH 2 P ) . Since C C F 3 will give a four peaks and the signal/noise is not important, it was impossible to assign that peak. The peaks were buried under the noise. 1 9 F N M R (CD3OD) 8: 3.68 and 3.63 (decoupled). Fmoc-(2S,4S)-4-(fluoro)proline 19 R f (CH 2 Cl 2 /MeOH 17:3) 0.4, white solid, 12 % yield, 0.034 g. *H N M R (CD3OD) 8: 7.69 (d, J= 5.8 Hz, 2H, ArH), 7.56-7.49 (m, 2H, ArH), 7.31 (t, J= 7.3 Hz, 2H, ArH), 7.25-7.21 (m, 2H, ArH), 5.26-5.24 and 5.14-5.12 (m, 1H, CH Y), 4.46 and 4.38 (t, J = 8.4 Hz, 1H, C H a ) , 4.29-4.19 (m, 2H, C H 2 F m o c ) , 4.14-4.06 (m, 1H, C H F m o c ) , 3.89 and 3.73 (dd, J= 12.8 Hz, J= 21.3 Hz, 1H, C H 2 5 ) , 3.61 and 3.52 (dt, J = 2.7 Hz, J= 12.6 Hz, 1H, C H 2 5 ) 2.70-2.51 (m, 1H, C H 2 P ) , 2.26-2.03 (m, 1H C H 2 P ) . I 3 C N M R (CD3OD) 8: 174.05 and 173.80 ( C C O O H ) , 155.00 (c c a r b a m a , e), 143.62 and 143.65 (C a r ) , 141.04 and 140.93 (C a r ) , 127.35 (CH a r ) , 126.70 (CH a r ) , 124.71 and 124.58 (CH a r ) , 119.50-119.46 (CH a r ) , 92.23 and 90.47 (d, J = 90 Hz (fluorine coupling), CH Y), 67.87 and 67.32 ( C H 2 F m o e ) , 57.51 and 57.15 (CH a ) , 53.33 and 52.70 (CH 2 5 ) , 46.82 and 46.45 ( C H F m o c ) , 37.31 and 36.03 (CH 2 P ) . I 9 F N M R (CD 3 OD) 8: -102.14 and -102.74 (decoupled). Fmoc O F 66 Fmoc-(2S,4S)-4-(cyano)proline 24 Fmoc Q R f (CH 2 Cl 2 /MeOH 17:3) 0.3, white solid, 53 % yield, 0.017 g. ' H N M R (CD 3OD) 5: 7.77 (t,J= 8.4 Hz, 2H, ArH), 7.62-7.59 (m, 2H, ArH), 7.39-7.34 (m, 2H, ArH), 7.32-7.27 (m, 2H, ArH), 4.45 and 4.35 (m, 2H, C H 2 F m o c ) , 4.31 (t,J= 7.0, 1H, C H a ) , 4.27-4.16 (m, H, C H F m o c ) , 3.85 and 3.79 (dd, J = 7.67, J = 10.46 Hz, 1H, C H 2 5 ) , 3.73 and 3.67 (m, 1H, C H 2 5 ) , 3.3-3.57 (m, 1H, CH V), 2.64-2.34 (m, 2H, C H 2 P ) 1 3 C N M R (CD3OD) 5: 169.27 and 169.86 (C C O O H ) , 149.75 (c c a r b a m a t e ), 138.97 and 138.80 (Car), 136.33 (Ca r), 122.65 (CHar), 121.96 (CHar), 119.97 and 119.80 (CHar), 114.65 (CHar), 114.47 (CC N), 63.10 and 62.65 (CH 2 F m o c ) , 53.74 and 53.57 (CHa), 52.73 (CH 2 5 ) , 44.68 (CH F m o c ) , 28.62 and 28.50 (CH2 P), 22.35 and 21.42. ES + /MS: 385.2 (M+Na)+. Fmoc-(2S)-4-difluoroproline 29 Fmoc r, R f (CH 2 Cl 2 /MeOH 17:3) 0.4, colorless oil, 76 % yield, 0.083 g. *H N M R (CD3OD) §: 7.72 (t, J= 6.45 Hz, 2H, ArH), 7.56-7.53 (m, 2H, ArH), 7.33-7.31 (m, 2H, ArH), 7.29-7.26 (m, 2H, ArH), 4.52 and 4.49 (d, J= 4.5, 1H, C H a ) , 4.40-4.30 (m, 2H, C H 2 F m o c ) , 4.19-4.10 (m, 1H, C H F m o c ) , 3.84-3.67 (m, 2H, C H 2 8 ) , 2.85-2.69 (m, 1H, C H 2 P ) , 2.56-2.43 (m, 1H C H 2 P ) . 1 3 C N M R (CD3OD) 5: 172.23 ( C C O O H ) , 154.49 ( C c a r b a m a t e ) , 143.46 (C a r ) , 141.12 (C a r ) , 128.35 (t, J = 250 Hz, C 5 ) 127.36 (CH a r ) , 126.71 (CH a r ) , 124.54 (CH a r ) , 119.46 (CH a r ) , 67.80 and 67.53 ( C H 2 F m o c ) , 56.82 (CH a ) , 52.77 (q, J= 29.1 Hz, C H 2 5 ) , 46.80 ( C H F m o c ) , 35.44 (CH 2 P ) . F 67 " F N M R (CD3OD) 5: -101.78 (d, J= 154 Hz), -101.60 (d,J= 154 Hz), -103.41 (d, J = 184 Hz), 104.23 (d, J= 184 Hz) (decoupled, CFC1 3 as reference). Fmoc-(2S)-4-ketoproline 31 R f (CH 2 Cl 2 /MeOH 17:3) 0.45, brownish oil, 93% yield, 0.157 g. *H N M R (CD3OD) 5: 7.75 (d,J= 6.7 Hz, 2H, ArH), 7.63-7.56 (m, 2H, ArH), 7.34 (t, J = 7.4 Hz, 2H, ArH), 7.29-7.27 (m, 2H, ArH), 4.76 and 4.65 (d, J= 9.7 Hz, 1H, CH"), 4.42-4.35 (m, 2H, C H 2 F m o c ) , 4.32-4.28 (m, 1H, C H F m o c ) , 4.26-4.14 (m, 2H, C H 2 5 ) , 3.14-3.00 (m, 1H C H 2 P ) . Fmoc-(2S,4S)-4-(benzylthio)proline 36 Fmoc n R f (CH 2 Cl 2 /MeOH 19:1) 0.4, colorless oil, 37 % yield, 0.041 g. *H N M R (CD3OD) 5: 7.76 (t, J= 8.8 Hz, 2H, ArH), 7.57-7.53 (m, 2H, ArH), 7.37-7.18 (m, 9H, ArH), ), 5.26-5.24 and 5.14-5.12 (m, 1H, CH Y ) , 4.37-4.27 (m, 3H, C H 2 F m o ° , CH"), 4.19 and 4.13 (t, J = 6.6 Hz, 1H, C H F m o c ) , 3.77-3.71( m, 2H, C H 2 S B n ) , 3.71 and 3.61 (dd,, J= 6.5 Hz, J= 10.5 Hz, 1H, CH 2 S ) , 3.46-3.30 and 3.21-3.17 (m, 1H, CH 2 Y ) , 3.27-3.25 (m, 1H, CH Y ) , 2.22-2.08 (m, 2H C H 2 P ) . 1 3 C N M R (CD3OD) 5: 174.01 ( C C O O H ) , 154.84 and 154.68 ( C c a r b a m a t e ) , 143.77 and 143.55 (C a r ) , 141.12 and 140.96 (C a r ) , 138.22 and 138.13 (C a r ) , 128.40 (CH a r ) , 128.11 (CH a r ) , 127.33 (CH a r ) , 126.71 (CH a r ) , 124.67 (CH a r ) , 124.60 (CH a r ) , 119.47 (CH a r ) , 67.66 and 67.22 ( C H 2 F m o c ) , 58.42 and 58.27 (CH a ) , 52.52-52.11 (CH 2 S ) , 46.86 ( C H F m o c ) , 40.45 and 39.61 (CHY), 36.99 and 35.94 (CH 2 P ) , 35.38 and 35.33 ( C H 2 C H 2 B n ) . 68 Fmoc-(R)-4-thiazoIidinecarboxylic acid 39 Fmoc Q Rf (CH 2 Cl 2 /MeOH 17:3) 0.5, white solid, 60 % yield, 0.80 g. *H N M R (CD 3 OD) 5: 7.76 (d, J= 5.3 Hz, 2H, ArH), 7.61-7.56 (m, 2H, ArH), 7.36 (d, J = 3.4 Hz, J= 7.4 Hz, ArH), 7.30-7.26 (m, 2H, ArH), 4.77 and 4.70 (d, J= 4.0 Hz), 1H, C H a ) , 4.60-4.57 and 4.47-4.40 (m, 2H, C H 2 F m o c ) , 4.40-4.37 (m, 2H, C H F m o c , C H 2 5 ) , 4.24 and 4.17 (t, J= 6.4 Hz, 1H, C H 2 5 ) , 3.36-3.17 (m, 2H, C H 2 P ) . l 3 C N M R (CD3OD) 5: 154.33 and 154.17 ( C c a r b a m a t e ) , 143.65 and 143.48 (C a r ) , 141.08 and 141.00 (C a r ) , 127.35 (CH a r ) , 126.71 (CH a r ) , 124.58 (CH a r ) , 119.45 (CH a r ) , 67.82 and 67.63 ( C H 2 F m o c ) , 61.77 and 61.12 (CH a ) , 48.41 (CH 2 S ) , 46.82 ( C H F m o c ) , 33.66 and 32.43 (CH 2 P ) . Fmoc-(2S,4S)-4-(azido)proline 44 R f (CH 2 Cl 2 /MeOH 17:3) 0.4, white solid, 60 % yield, 0.170 g. ' H N M R (CD3OD) 5: 7.81 (d, J= 7.0 Hz, 2H, ArH), 7.64 (dt, J= 2.8 Hz, J= 8.0 Hz, 2H, ArH), 7.40 (dt, J= 7.3 Hz, J= 3.4 Hz, 2H, ArH), 7.35-7.30 (m, 2H, ArH), 4.48-4.18 (m, 5H, C H a , C H 2 F m o c , C H F m o c , CH y ) , 3.70-3.71 (m, 1.5H, C H 2 S ) , 3.53-3.49 (m, 0.5H, C H 2 8 ) 2.51-2.38 (m, 1H, C H 2 P ) , 2.31-2.18 (m, 1H C H 2 P ) . 1 3 C N M R (CD3OD) 5: 173.83 and 173.60 ( C C 0 0 H ) , 154.84 and 154.78 ( C c a r b a m a t e ) , 143.73 and 143.45 (C a r ) , 141.07 and 140.96 (C a r ) , 127.35 (CH a r ) , 126.69 (CH a r ) , 124.68 and 124.60 (CH a r ) , 119.49-119.45 (CH a r ) , 67.77 and 67.32 ( C H 2 F m o c ) , 59.50 and 58.73 (CH a ) , 57.68 and 57.3 (CH 2 Y ), 51.50-51.16 (CH 2 5 ) , 46.87 and 46.78 ( C H F m o c ) , 35.90 and 34.87 (CH 2 P ) . Fmoc O 69 4.2.2 Preparation of the Hpi D M D O preparation and titration The preparation of D M D O in acetone was carried out using Oxone®, following the procedure of Adam et al43 Standardization of the solution was done with two quantitative methods. The first one was done by taking a ' H N M R of the D M D O solution. The concentration was calculated from the comparison of the height of the methyl proton signal of the dioxirane with the C satellite peak. Every satellite peak should be 0.5 % of the main acetone peak44 (figure 20). Satellite peaks LJ / Acetone solvent peak 1 DMDO peak Water peak 2.00 1.50 ppm (f1) Figure 20: ' H N M R of the D M D O solution. We can clearly see the satellite peaks. The second method is an iodometric titration (scheme 30). The D M D O solution (1 mL) was added to acetic acid/acetone mixture 3:2 (2mL). A saturated solution of KI (2 mL) was added and some small pieces of dry ice were added to remove the dissolved oxygen from the solution. The mixture was stored in the dark at room temperature for 10 min. The sample was brought to 10 mL and aliquots (1 mL) were titrated against an aqueous sodium thiosulfate solution of 0.001 N . When the yellow colored solution started to fade, a starch solution (5 drops) was added to give a sharper turning point. The solution was then titrated until the disappearance of the iodine-starch blue-grey complex. i) 2 H + + 2 I" + D M D O -> Acetone + I 2 (aq) + H 2 0 ii) 2 S 20 3" 2 + I 2 ( a q) 2 r + S 40 6" 2 Scheme 30: Iodometric titration of the DMDO solution, i) D M D O and iodide reaction to form iodine; ii) Titration of the iodine by thiosulfate. 70 AVTrityl-tryptophan Trityl chloride (24. lg , 86.2 mmol, 2.2 eq.) was dissolved a chloroform/DMF mixture (2:1, 250 mL) and tryptophane (8.1 g, 39.17 mmol, leq.) was then added. The resulting slurry was stirred for l h at room temperature until most of the tryptophan had dissolved. Whilst checking that the temperature was constant, triethylamine (22 mL) was added slowly. After 5 hours of stirring, the ditritylated product was observed on T L C (Rf 0.3 in Hexanes/AcOEt 4:1). Ethanol (500 mL) was then added and the reactive mixture was stirred for 5h at 50°C. Diethyl ether (400 mL) was added and the mixture was washed with a 5 % aqueous solution of citric acid (3 x 150 mL) and with brine (3 x 150 mL). The organic phase was dried with MgSC^, filtered and evaporated to 200 mL. Triethylamine (4 mL) was added and the mixture was left to stand overnight. The formed solid was filtered, resuspended in ether (250 mL) and washed with a 5 % citric acid in water until the entire solid dissolved. The ether solution was washed with brine (2 x 150 mL), dried with MgS04, filtered and evaporated to dryness. The product was recovered as a light brown solid. R f (Hexanes/AcOEt 1:1) 0.4, light brown solid, 68 % yield, 26 g. R f (CH 2 Cl 2 /MeOH 95:5) 0.2. MS (ESI) m/z: calculated for C 3 o H 2 7 N 2 0 2 [M+H]+: 447.19, found: 447.32. 71 General method for preparation of Tr-Trp-Xaa-OMe In a round bottom flask, AVtrityl-tryptophan (6.59 g, 12 mmol) was dissolved in dry CH2CI2 (62 mL). The amino acid (13.2 mmol) methyl ester denoted Xaa-OMe, HOBt (2.02 g, 13.2 mmol) and triethylamine (1.8 mL, 13.2 mmol) were added. The reactive mixture was cooled down to 0 °C. DCC (2.72 g, 13.2 mmol) was then added and the solution was left to react at 0 °C for 30 minutes. The reaction was continued overnight at room temperature until completion. The solvent was evaporated to dryness. The residue was dissolved in AcOEt and filtered to remove the dicyclohexaneurea (DCU) formed. This was repeated on the solid obtained. The filtrate was combined and reduced to 100 mL. The solution was then washed with a 5 % citric acid solution (3 x 70 mL), with a saturated solution of sodium bicarbonate (3 x 70 mL) and with a brine solution (3 x 70 mL). The organic phase was dried with MgS04, filtered and evaporated to dryness. The solid obtained was then purified by flash chromatography on silica gel to give a pure product. Nb-Trityl-tryptophanylglycine methyl ester 46 R f (Hexanes/AcOEt 1:1) 0.4, 80 %. ! H N M R (CDCb) 5: 8.03 (s, 1H, NH), 7.55 (d, J= 8.3 Hz, 1H, ArH), 7.30-7.00 (m, 18H, ArH), 3.63 (s, 3H, C H 3 0 M e ) , 3.45 (m, 3H, C H a t r p , C H 2 a g l y ) , 3.12 (dd, J = 6.2, 14.6 Hz, 1H, C H 2 p t r p ) , 2.83 (d, J= 5.0 Hz, 1H, NH), 2.55 (dd, J= 5.5, 14.6 Hz, 1H, C H 2 p t r p ) . Trt O 72 Nb-Trityl-tryptophanylisoleucine methyl ester 47 HN R f (Hexanes/AcOEt 1:1) 0.5, 60 % yield. *H N M R (CDC13): 5 7.98 (s, 1H, NH), 7.48 (d, J= 7.4 Hz, 1H, ArH), 7.41-7.12 (m, 14H, ArH), 7.03 (t, J= 3.67 Hz, A r H , 1H), 6.84 (s, 1H, NH), 4.05 (dd, J = 7.4 Hz, J= 4.6 Hz, 1H, C H a p h e ) , 3.67 (s, 3H, C H 3 O M e ) , 3.61 (t, J= 5.4 Hz, 1H, C H a t r p , ) , 3.14 (dd, J= 4.8 Hz, J= 14.0 Hz, 1H, C H 2 p t r p ) , 2.29-2.18 (m, 1H, C H 2 p t r p ) , 1.64-1.41 (m, C H p i l e ) , 1.03-0.87 (m, 2H, C H 3 y l i l e ) , 0.80 (t, J= 7.3 Hz, 3H, C H 3 5 1 i l e ) , 0.461 (d, J = 7.3 Hz, 3H, C H 2 Y 2 t r p ) . Nb-Trityl-tryptophanylproline methyl ester 48 Trt O V - 0 . . .1 . II - N R f (Hexanes/AcOEt 1:1) 0.3, 90 % yield. Nb-Trityl-tryptophylphenylalanine methyl ester 49 HN 73 R f (Hexanes/AcOEt 0.35:0.64) 0.25, 39 % yield. ' H N M R (CDC13) 8: 8.02 (s, 1H, NH), 7.56 (d, J= 8.1 Hz, 1H, ArH), 7.53 (d, J= 9.2 Hz, 1H, ArH) 7.50-7.35 (m, 14H, ArH), 6.70 (d, J= 2.2 Hz, 1H, ArH), 6.31 (d, J = 7.1 Hz, 2H, ArH) 4.74-4.64 (m, 1H, C H a p h e ) 3.67 (s, 3H, C H 3 0 M e ) , 3.57-3.49 (m, 1H, CH a t r p , ) , 3.15 (dd, 1H, J= 3.9 Hz, J= 14.1 Hz, 1H, C H 2 p p h e ) , 2.92 (dd, J= 13.6 Hz, J = 3.8 Hz, , 1H, C H 2 p p h e ) , 2.38 (d, J= 6.8Hz, 1H, NH) 2.07 (dd, J= 6.2 Hz, J= 13.7 Hz, 1H, C H 2 p t r p ) 1.65 (dd, J= 5.4 Hz, J= 14.1 Hz, 1H, C H 2 p t r p ) . General method for preparation of Hpi derivatives Tr-Trp-Xaa-OMe (0.10 mmol) was dissolved in dry CH 2 C1 2 (1 mL) at -78°C. A DMDO solution in acetone was added (0.11 mmol, 1.1 eq.). The reactive mixture was stirred for 15 min. The solvent was evaporated to dryness under reduced pressure. The water bath was kept at room temperature. The crude reaction product was purified by flash chromatography using NEt3-pretreated silica gel and using hexanes/AcOEt/NEt3 80:20:1 to 60:40:1 as eluent gradients. A fast-running product a (syn-cis) and a slow running product b (anti-cis) were obtained. AT-Trityl-3a-hydroxy-pyrrolo[2,3-6]indolyl-glycine methyl ester (syn-cis) (Tr-Hpi-Gly-OMe) 50a O Note: The characterisation of this product was mostly achieved by Dr. Jonathan May. R f (Hexane/AcOEt 1:1) 0.4, white solid, 35% yield, 1.86 g. 74 ' H N M R (CDCI3) 5: 7.50 (d, 6H, J= 6.8 Hz, A r H T r ) , 7.48-7.18 (m, 10H, ArH), 7.07 (t, 1H, J= 7.6 Hz, CH 6 ) , 6.73 (t, 1H, ./= 7.6 Hz, CH 5 ) , 6.38 (d, 1H, J= 7.8 Hz, CH 7 ) , 5.95 (dd, 1H, J= 5.0, 4.9 Hz, NHCO), 5.54 (d, 1H, J = 3.6 Hz, C H 8 a ) , 5.49 (s, 1H, OH), 3.81 (d, 1H, J= 9.3 Hz, CH 2 ) , 3.65 (s, 3H, C H 3 O M e ) , 3.59 (dd, 1H, J= 18.5, 5.0 Hz, C H a ) , 3.52 (dd, 1H, J = 18.5, 4.9 Hz, C H a ) , 3.01 (d, 1H, J= 3.6 Hz, NH), 2.48 (dd, 1H, J = 13.9, 9.3 Hz, CH 3 ) , 2.28 (d, 1H, J= 13.9 Hz, CH 3 ) . 1 3 C N M R (CDCI3) 5: 176.62 ( C C O N H ) , 169.82 ( C c o ) , 146.96 (C 7 a ) , 143.78 (C T r ) , 131.11 (CH T r ) , 130.16 (C 3 b ) , 128.84 (CH 6), 127.54 (CH T r ) , 127.06 (CH T r ) , 122.74 (CH 4), 118.81 (CH 5 ), 110.04 (CH 7 ), 92.31 (CH 8 a ) , 86.83 (C 3 a ) , 75.61 (C T r ) , 64.59 (CH 2), 52.39 ( C H 3 O M e ) , 42.84 (CH 2 3 ) , 40.97 (CH 2 a ) . E S + / M S : 556.2 (M+Na)+. H R M S (ES +) for C 3 3 H 3 , N 3 0 4 (M+H)+: calculated 534.2393, found 534.2390. UV/vis (MeOH): 239 nm ( l . S x l o W m o l ' 1 ) , 297 nm (0.3x1 o W m o l " 1 ) . 7Vl-Trityl-3o-hydroxy-pyrrolo[2,3-6]indolyl-glycine methyl ester (anti-cis) (Tr-Hpi-Gly-OMe) 50b O Note: The characterisation of this product was mostly achieved by Dr. Jonathan May. R f (Hexane/AcOEt 1:1) 0.4, white solid, 35% yield, 1.86 g. J H N M R (CDCI3) 5: 9.25-9.19 (br, 1H, NHCO), 7.71 (d, 6H, J= 7.6 Hz, A r H T r ) , 7.48-7.25 (m, 9H, ArH), 7.13-6.98 (m, 2H, CH 6 ' 4 ) , 6.73 (t, 1H, J= 7.4 Hz, CH 5 ) , 6.62 (t, 1H, J = 7.4 Hz, CH 7 ) , 5.36 (d, 1H, J= 5.5 Hz, C H 8 a ) , 4.89 (d, 1H, J= 5.5 Hz, NH), 4.25-4.19 (m, 2H, C H 2 , C H a ) , 3.80 (s, 3H, C H 3 O M e ) , 3.18 (dd, 1H, J= 18.3, 3.2 Hz, C H a ) , 2.35 (d, 1H, J= 13.4 Hz, CH 3 ) , 1.08 (dd, 1H, J= 13.4, 9.8 Hz, CH 3 ) . 75 1 3 C N M R (CDCI3) 5: 174.26 ( C C O N H ) , 169.82 ( C c o ) , 148.50 (C 7 a ) , 144.78 (C T r ) , 130.11 (C 3 b ) , 129.29 (CH T r ) , 128.81 (CH 6), 128.25 (CH T r ) , 126.83 (CH T r ) , 124.54 (CH 4), 120.69 (CH 5 ), 110.32 (CH 7 ) , 90.65 (C 3 a ) , 89.62 (CH 8 a ) , 78.55 (C T r ) , 66.70 (CH 2 ), 52.17 ( C H 3 O M e ) , 44.89 (CH 2 3 ) , 41.25 (CH 2 a ) . E S + / M S : 556.1 (M+Na)+. H R M S (ES+) for C 3 3 H 3 1 N 3 O 4 (M+H)+: calculated 534.2393, found 534.2396. UV/vis (MeOH): 235 nm (1.8xl06cm2mor'), 292 nm (0.2x10'Wmol"1). For the ease of the synthesis, the last two compounds were only separated from each other for the full characterization. For the subsequent synthesis, the Hpi-glycine was used as a mixture of diastereoisomers. Tr-Hpi-Ile-OMe 51a R f (CH 2 Cl 2 /MeOH 9:1) 0.5. White solid, 29% yield, 0.048 g. *H N M R (CDCI3) 5: 7.53 (d, 6H, J= 7.0 Hz, A r H T r ) , 7.33-7.24 (m, 10H, ArH), 7.02 (t, 1H, J= 7.8 Hz, CH 6 ) , 6.71 (t, 1H, J= 7.3 Hz, CH 5 ) , 6.31 (d, 1H, J= 7.8 Hz, CH 7 ) , 5.83 (s, 1H, OH), 5.74 (d, 1H, J= 1A Hz, NHCO), 5.64 (d, 1H, J= 3.7 Hz, C H 8 a ) , 4.10 (d, \U,J= 9.1 Hz, CH 2 ) , 3.68 (s, 3H, C H O M e ) , 4.09 (d, 1H, J= 8.7 Hz, C H a ) , 2.93 (d, 1H, J = 3.8 Hz, NH), 3.14 (d, 1H, J= 3.7 Hz, NH), 2.28 (dd, 1H, J = 13.7 , 9.1 Hz, CH 3 ) , 2.13 (d, 1H, J= 13.7 Hz, CH 3 ) , 1.50-1.37 (m, 1H, CH P ) , 1.02-0.84 (m, 1H, CH Y ) , 0.84 (t, 3H, J= 7.0 Hz, CH 8 ) , 0.52 (d, 3H, J= 7.0 Hz, C H Y ) . 1 3 C N M R (CDCI3) 5: 176.23 ( C C O N H ) , 171.35 ( C c o ) , 146.98 (C 7 a ) , 144.12 (C T r ) , 131.16 (C 3 b ) , 130.94 (CH T r ) , 128.75 (CH 6 ), 127.06 (CH T r ) , 126.96 (CH T r ) , 122.62 (CH 4), 76 118.74 (CH 5 ), 110.03 (CH 7 ) , 92.55 (CH 8 a ) , 86.94 (C 3 a ) , 75.50 (C T r ) , 65.05 (CH 2), 56.53 (CH a ) , 51.94 ( C H 3 O M e ) , 41.92 (CH 2 3 ) , 37.23 (CH 3 P ) , 25.74 (CH 3 Y ), 14.58 (CH 3 Y), 11.63 (CH 8). E S + / M S : 612.2 (M+Na)+. Tr-Hpi-Ile-OMe 51b R f (CH 2 Cl 2 /MeOH 9:1) 0.2. White solid, 61% yield, 0.100 g lH N M R (CDC13) 5: 8.92 (d, 1H, J= 8.6 Hz, NHCO), 7.68 (d, 6H, J= 7.8 Hz, A r H T r ) , 7.34-7.02 (m, 11H, A r H , C H 4 , CH 6 ) , 6.69 (t, 1H, J= 7.4 Hz, CH 5 ) , 6.60 (d, 1H, J= 8.2 Hz, CH 7 ) , 5.30 (d, 1H J= 5.0 Hz, C H 8 a ) , 4.74 (d, 1H, J= 5.0 Hz, NH), 4.38 (dd, 1H, J = 8.6, 5.1 Hz, C H a ) , 4.18 (d, 1H, J= 10.6 Hz, CH 2 ) , 3.77 (s, 3H, C H O M e ) , 2.34 (d, 1H, J = 14.1 Hz, CH 3 ) , 1.45 (dd, 1H, J= 14.1 Hz, 10.6 Hz CH 3 ) , 1.18-1.10 (m, 3H, C H P , C H 2 Y ) (t, 3H, J= 7.3 Hz, C H 3 8 ) , 0.63 (d, 3H, J= 7.3 Hz, CH 3 Y ' ) . 1 3 C N M R (CDCI3) 5: 174.38 ( C C O N H ) , 172.20 ( C c o ) , 148.198 (C 7 a ) , 144.90 (C T r ) , 131.46 (C 3 b ) , 129.70 (CH 6 ), 129.29 (CH T r ) , 128.24 (CH T r ) , 126.80 (CH T r ) , 124.35 (CH 4), 120.78 (CH 5 ) , 111.05 (CH 7), 91.15 (CH 8 a ) , 90.95 (C 3 a ) , 78.77 (C T r ) , 67.29 (CH 2), 56.40 ( C H 3 O M e ) , 51.83 (CH a ) , 45.92 (CH 2 3 ) , 33.85 (CH 3 P ) , 24.86 (CH 2 Y ), 14.71 (CH 3 Y ') , 11.52(CH 3 5). E S + / M S : 612.2 (M+Na)+. 77 Tr-Hpi-Pro-OMe 52a R f (Hexane/AcOEt 1:1) 0.4. White solid, 84% yield, 87 mg. ' H N M R (CDC13) 5: 7.60 (d, 6H, J= 6.8 Hz, A r H T r ) , 7.30-7.23 (m, 10H, ArH), 7.00 (t, 1H, J= 7.8 Hz, CH 6 ) , 6.78 (t, 1H, J= 7.8 Hz, CH 5 ) , 6.31 (d, 1H, J= 7.8 Hz, CH 7 ) , 5.88 (s, 1H, OH), 5.49 (d, 1H, J= 3.6 Hz, C H 8 a ) , 4.31 (d, 1H, J= 9.0 Hz, CH 2 ) , 4.12-4.08 (m, 1H, C H a ) , 3.71 (s, 3H, C H O M e ) , 3.25-3.18 (m, 1H, CH 8 ) , 3.14 (d, 1H, J = 3.6 Hz, NH), 2.78-2.70 (m, 1H, CH 8 ) , 2.40 (dd, m,J= 13.4 Hz, 9.0 Hz, CH 3 ) , 2.12 (d, 1H, J= 13.4 Hz, CH 3 ) , 1.89-1.81 (m, 2H, CH P ) , 1.67-1.53 (m, 2H, CH Y ) . 1 3 C N M R (CDCI3) 5: 176.66 ( C C O N H ) , 172.00 ( C c o ) , 147.29 (C 7 a ) , 144.17 (C T r ) , 131.22 (CH T r ) , 128.64 (C 3 b ) , 127.84 (CH 6 ), 127.22 (CH T r ) , 126.77 (CH T r ) , 122.71 (CH 4), 118.47 (CH 5 ), 109.77 (CH 7 ), 91.58 (CH 8 a ) , 87.08 (C 3 a ) , 74.93 (C T r ) , 61.38 (CH 2), 58.84 (CH a ) , 52.23 ( C H 3 0 M e ) , 41.93 (CH 2 8 ) , 33.84 (CH 2 3 ) , 28.37 (CH 3 P ) , 24.56 (CH 3 Y). E S + / M S : 596.2 (M+Na)+. Tr-Hpi-Phe-OMe 53a 78 R f (Hexane/AcOEt 3:1) 0.5. White solid, 11% yield, 0.018 g. lH N M R (CDC13) 5: 7.51 (d, 6H, J - 8.2 Hz, A r H T r ) , 7.29-7.21 (m, 13H, ArH), 7.02 (t, 1H, J = 7.6 Hz, CH 6 ) , 6.72 (m, 3H, A r H , CH 5 ) , 6.31 (d, 1H, J= 7.8 Hz, CH 7 ) , 5.88 (s, 1H, OH), 5.66 (d, 1H, J= 3.8 Hz, C H 8 a ) , 5.62 (d, \U,J= 6.8 Hz, NHCO), 4.08 (d, 1H, J = 9.1 Hz, CH 2 ) , 3.64 (s, 3H, C H O M e ) , 3.44-3.38 (m, 1H, CH") 2.93 (d, 1H, J = 3.8 Hz, NH), 2.83 (d, 1H, C H P ) , 2.75-2.69 (m, 1H, CH P ) , 2.18 (dd, 1H, J= 15.7, 9.1 Hz, CH 3 ) , 2.07 (d, 1H, J= 15.7 Hz, CH 3 ) . 1 3 C N M R (CDCI3) 5: 176.70 ( C C O N H ) , 171.43 ( C c o ) , 146.91 (C 7 a ) , 144.20 (C T r ) , 135.00 (C A r ) , 130.88 (CH T r ) , 128.91 (CH A r ) , 128.74 (C 3 b ) , 128.53 (CH 6 ) , 127.61 (CH T r ) , 127.09 (CH A r ) , 126.92 (CH T r ) , 122.61 (CH 4), 118.78 (CH 5 ), 110.59 (CH 7), 92.67 (CH 8 a ) , 86.95 (C 3 a ) , 75.75 (C T r ) , 64.97 (CH 2), 52.97 ( C H 3 O M e ) , 52.20 (CH a ) , 41.94 (CH 2 3 ) , 37.17 (CH 3 P ) . E S + / M S : 646.1 (M+Na)+. Tr-Hpi-Phe-OMe 53b R f (Hexane/AcOEt 3:1) 0.2. White solid, 32% yield, 0.052 g. *H N M R (CDCI3) 5: 9.11 (d, 1H, J= 7.3 Hz, NHCO), 7.63 (d, 7H, J= 7.62 Hz, A r H T r ) , 7.34-7.02 (m, 15H, ArH), 6.74 (t, 1H, J= 7.4 Hz, CH 5 ) , 6.69 (d, 1H, J = 8.2 Hz, CH 7 ) , 5.31 (d, 1H, J= 5.1 Hz, C H 8 a ) , 4.77 (d, 1H, J = 5.1 Hz, NH), 4.45-4.38 (m, 1H, CH"), 4.11 (d, 1H, J= 7.7 Hz, CH 2 ) , 3.61 (s, 3H, C H O M e ) , 2.59-2.53 (m, 1H, C H P ) 2.30 (d, 1H, J= 13.5 Hz, CH 3 ) , 2.06-1.99 (m, 1H, CH P ) , 1.90 (dd, 1H, J= 13.5, 7.7 Hz CH 3 ) . 79 1 3 C N M R (CDCI3) 5: 173.85 ( C C O N H ) , 172.21 ( C c o ) , 148.34 (C 7 a ) , 145.76 (C A r ) , 144.77 (C T r ) , 136.41 (C 3 b ) , 131.37 (CH A r ) , 130.06 (CH T r ) , 129.70 (CH 6 ), 129.29 (CH T r ) , 128.24 (CH A r ) , 126.80 (CH T r ) , 124.55 (CH 4), 120.88 (CH 5 ) , 110.94 (CH 7), 90.87 (CH 8 a ) , 90.32 (C 3 a ) , 78.67 (C T r ) , 66.95 (CH 2), 53.49 ( C H 3 O M e ) , 51.61 (CH a ) , 37.98 (CH 2 3 ) , 33.85 (CH 3 P ) . Nl-Trityl-3a-hydroxy-pyrrolo[2,3-b]indolyl-glycine triethylammonium salt 54 b Tr-Hpi-Gly-OMe 50 (mixture of diastereomers) (0.13 g, 0.2 mmol) was dissolved in dioxane/water 2:1 (15 mL). L i O H (0.05 g, lOeq.) was added to the solution. The reaction mixture was stirred at room temperature, the completion of the reaction was followed by TLC (CH2Cl2/MeOH 9:1). The solvent was evaporated to dryness. The crude mixture was purified by by flash chromatography on silica gel, using CH2Cl 2/MeOH/NEt3 (90:10:1) as eluant. The product was obtained as the triethylamine salt of the mixture of the two diastereomers, 0.13g, (85%). Comprehensive characterization of the final product was avoided since the product was very acid sensitive. R f (CH 2 Cl 2 /MeOH 9:1) 0.2. Obtained as a mixture of diastereomers, but ' H N M R was possible to be assigned separately: Note: The characterisation of this product was achieved by Dr. Jonathan May 80 54a *H N M R (d 4-MeOH) 5: 9.59-9.53 (br, 1H, NH), 7.51 (d, 6H, J= 7.8 Hz, A r H T r ) , 7.27-7.11 (m, 10H, ArH), 7.01-6.45 (m, 2H, ArH 5 ' 6 ) , 6.25 (t, 1H, J = 7.9 Hz, CH 7 ) , 5.49 (s, 1H, C H 8 a ) , 4.21 (d, 1H, J= 9.5 Hz, CH 2 ) , 3.14-3.09 (m, 2H, C H 2 a ) , 2.25 (dd, 1H, J = 13.8, 9.5 Hz, CH 3 ) ,2.18(d, 1H,J= 13.8 Hz, CH 3 ) . 54b ! H N M R (d 4-MeOH) 5: 9.55-9.48 (br, 1H, NH), 7.75 (d, 6H, J= 7.8 Hz, A r H T r ) , 7.40-7.25 (m, 10H, ArH), 7.01-6.45 (m, 3H, ArH 5 ' 6 ' 7 ) , 5.26 (s, 1H, C H 8 a ) , 3.98 (d, 1H, J = 10.1 Hz, CH 2 ) , 3.77 (d, 1H, J= 17.6 Hz, C H a ) , 2.81 (d, 1H, J= 17.6 Hz, C H a ) , 2.07 (d, 1H, J= 13.0 Hz, CH 3 ) , 1.21 (dd, 1H, J= 13.0, 10.1 Hz, CH 3 ) . 54a/b: mixture of diastereomers 1 3 C N M R (d 4-MeOH) 5: 180.00, 175.97 ( C C O N H ) , 175.74 ( C c o ) , 156.27, 151.32 (C 7 a ) , 146.42, 145.41 (C T r ) , 130.89 (CH T r ) , 129.88 (CH T r ) , 130.31 (C 3 b ) , 129.35 (CH 6 ), 129.17 (CH T r ) , 128.79 (CH T r ) , 125.06 (CH 4 ), 119.95, 119.76 (CH 5 ), 111.03 (CH 7 ), 94.82, 93.56 (CH 8 a ) , 90.73, 90.19 (C 3 a ) , 80.08 (C T r ) , 67.95, 65.98 (CH 2 ), 56.35, 54.86 (CH 2 a ) , 46.55 (NEt3), 44.78, 44.36 (CH 2 3 ) , 7.75 (NEt 3). ES7MS: 518.4 (M-H)". H R M S (ES") for C 3 2 H 2 8 N 3 0 4 (M-H)": calculated 518.2080, found 518.2062. 4.2.3 Preparation of the peptides Solid phase synthesis The solid phase synthesis of the octapeptide was carried out on 2-chlorotrityl chloride resin functionalized with isoleucine (0.54 mmole/g) from Novabiochem (Germany). The protocol is made for 100 mg of resin. 81 1- Preparation of the resin The correct amount of resin was weighed out (75 mg) and put in a solid-phase reaction vessel. CH2CI2 (2mL) was added and the vessel was agitated for 1 hour to swell the resin. CH2CI2 was removed and DMF (2 mL) was added and the vessel was agitated for 3 hours. The first coupling was carried out without deprotection since the resin was not protected. 2- Fmoc deprotection The resin was suspended in a 20% piperidine solution in D M F (2 mL) and agitated for 10 min. The reactive solution was removed and the resin was washed 5 times with DMF ( 5 x 2 mL). 3- Coupling of a proline derivative amino acid The proline derivative amino acid (4 eq.) was weighed out into an Eppendorf tube and H B T U (3 eq.) and HOBt (3 eq.) were added. The solids were dissolved in DMF and DIEA (55 uL) was added. The solution was poured in the reaction vessel and the suspension was agitated for 40 min. The coupling mixture was removed and the resin was washed with D M F ( 5 x 2 mL). 4- Coupling of trityl asparagine For the peptides 54(-F), 55(-CF3), 56(-CN), 57(-SBn), 58(-N3) Fmoc trityl asparagine (6 eq.) was weighed out in an Eppendorf tube and HBTU (6 eq.) was added. The solids were dissolved in DMF and DIEA (55 uL) was added. The solution was poured in the reaction vessel and the suspension was agitated for 20 min. The coupling mixture was evacuated and the resin was washed with D M F ( 5 x 2 mL). This coupling was repeated with the same conditions for a second time. 82 For the peptides 59(-F2), 60(-OtBu), 61 (=0) Fmoc trityl asparagine (6 eq.) was weighed out in an Eppendorf tube. PyBOP (6 eq.) and HOBt (6 eq.) were added. The solids were dissolved in N M P and DIEA (55 pL) was added. The solution was poured in the reaction vessel and the suspension was agitated for 2 h. The coupling mixture was removed and the resin was washed with DMF ( 5 x 2 mL). This coupling was repeated with the same conditions for a second time. For the peptide 62(thz) Fmoc trityl asparagine (6 eq.) was weighed out in an Eppendorf tube. PyBrOP (6 eq.) was added. The solids were dissolved in N M P and DIEA (55 uL) was added. The solution was poured in the reaction vessel and the suspension was agitated for 2 h. The coupling mixture was removed and the resin was washed with D M F ( 5 x 2 mL). This coupling was repeated with the same conditions for a second time. Note: For peptide 62; Asn, Cys, Gly and He were coupled with PyBrOP instead of HBTU. 5- Coupling of the natural amino acids The amino acid (6 eq.) was weighted out in an Eppendorf tube and H B T U (6 eq.) was added. The solids were dissolved in DMF and DIEA (55 uL) was added. The solution was poured in the reaction vessel and the suspension was agitated for 20 min. The coupling mixture was removed and the resin was washed with D M F ( 5 x 2 mL). 6- Fmoc-(Tr)Hpi-gly coupling Fmoc-(Tr)Hpi-Gly (4 eq.) was weighted out into a Eppendorf tube. H B T U (4 eq.) and HOBt (4 eq.) were added. The solids were dissolved in D M F and DIEA (55 uL) was 83 added. The solution was poured in the reaction vessel and the suspension was agitated for 30 min. The coupling mixture was removed and the resin was washed 5 times with DMF ( 5 x 2 mL). 7- Preparation of a sample for the mass spectrum analysis A very small quantity of the resin (« 1-2 mg of wet resin) was put in a small screw cap vial. CH 2 C1 2 (500 pL) and HF1P (l,l,l,3,3,3-Hexafiuoro-2-propanol) (100 uL) were added. The vial was agitated for 5 min. The reactive mixture was evaporated to dryness and further dried on the high vacuum. Methanol (1 mL) was added, and the solution was centrifuged to aid separation of the solution from the resin beads. The solution was transferred into a mass spectrum vial for MS analysis. Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-FPro-Ile-OH 54 C 7 7H 8 5 FNioOiiS Exact Mass: 1376.6 g/mol E S + / M S : 1378.5 (M+H)+, 1400.5 (M+Na)+ Note: For these molecules, the mass spectrum molecular peak is often off by one unit. Since these molecules are fairly big, on most of them, the isotopic peak is as important as the molecular peak. 84 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-CF 3Pro-Ile-OH 55 C 7 8H g 5 F3N 1 0 OiiS Exact Mass: 1426.6 g/mol. ES + /MS: 1428.8 (M+H) +, 1450.7 (M+Na)+. Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-CNPro-Ile-OH 56 CygHgsNiiOiiS Exact Mass: 1383.6 g/mol, 51 mg on resin. ES + /MS: 1385.9 (M+H) +, 1408.0 (M+Na)+. Hpi-Gly-Ile-GIy-Cys(Tr)-Asn(Tr)-(SBn)Pro-Ile-OH 57 C84H92N10O11S2 Exact Mass: 1480.6 g/mol, 38 mg on resin. ES + /MS: 1483.0 (M+H) +. Hpi-Gly-Ile-GIy-Cys(Tr)-Asn(Tr)-N3Pro-Ile-OH 58 C77H85N13O11S Exact Mass: 1399.6 g/mol, 88 mg on resin. ES + /MS: 1401.9 (M+H)+, 1424.9 (M+Na)+. Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-F 2Pro-Ile-OH 59 C77H84F2N10O11S Exact Mass: 1394.6 g/mol, 38 mg on resin. ES + /MS: 1396.8 (M+H)+, 1419.8 (M+Na)+. Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Hyp(tBu)-Ile-OH 60 Trv C81H94N10O12S Exact Mass: 1430.7 g/mol, 36 mg on resin. ES + /MS: 1431.9 (M+H)+, 1456.0 (M+Na)+. Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Ketopro-Ile-OH 61 C77H.84N10O12S Exact Mass: 1372.6 g/mol, 34 mg on resin. ES+/MS: 1373.8 (M+H) +. Hpi-GIy-Ile-Gly-Cys(Tr)-Asn(Tr)-Thz-Ile-OH 62 C76H84N10O11S2 Exact Mass: 1376.6 g/mol, 31 mg on resin. ES+/MS: 1399.7 (M+Na)+, 1421.7 (M-H+2Na)+. Cleavage from the resin and first cyclization The resin was dissolved in TFA (10 mL) and stirred for 5 hours at room. Trifluoroacetic acid was evaporated and the residue was triturated 5 times with ether (5 x 5 mL). The residue was dissolved in methanol (5 mL). Diethyl ether was slowly added to the solution until no more precipitate is forming. The residue was filtered and washed with Et 2 0. 87 Monocyclic octapeptide with fluoroproline 63 C 3 9 H 5 5 F N 1 0 O 1 0 S Exact Mass: 874.4 g/mol, 10 mg. ES + /MS: 875.5 (M+H) +, 897.5 (M+Na)+, 919.5 (M-H+2Na)+. Monocyclic octapeptide with trifluoromethylproline 64 C40H55F3N10O10S Exact Mass: 924.4 g/mol, 11 mg. ES + /MS: 925.5 (M+H)+, 947.5 (M+Na)+. 88 References (1) Gibson, I.; Gibson, E . http://www, pfc. forestry, ca/biodiver•sity/matchmaker-/index_e. html. (2) Ho, S.; O'Ryan, K . http://www.anhg.zov.au/poison-plants/AB-poison.html 2004. (3) Wieland, T. Peptides of Poisonous Amanita Mushrooms; Springer-Verlag: New York, 1986. (4) Wieland, T.; Faulstich, H . Experientia 1991, 47, 1186-1193. (5) Voet, D . ; Voet, J. G . Biochemistry second edition N e w York, 1995. (6) Shoham, G . ; Ress, D . C. ; Lipscomb, W . N . ; Zanotti, G . ; Wieland, T. J. Am. Chem. Soc. 1984, 4606-4615. (7) Fournier, P. Master thesis, U B C , 2003. (8) Cramer, P. Curr. Opin. Genet. Dev. 2004,14, 218-226. (9) Armache, K . - J . ; Kettenberger, H . ; Cramer, P. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6964-6968. (10) Bushnell, D . A . ; Kornberg, R. D. Proc. Natl. Acad. Sci. U. S. A. 2003,100, 6969-6973. (11) Bushnell, D . A . ; Cramer, P.; Kornberg, R. D . Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 1218-1222. (12) Zanotti, G . ; Mohringer, C ; Wieland, T. Int. J. Pept. Protein Res. 1987, 30, 450-459. (13) Koskinen, A . M . P.; Helaja, J.; Kumpulainen, E . T. T.; Koivisto, J.; Mansikkamaki, H . ; Rissanen, K . J. Org. Chem. 2005, 70, 6447-6453. (14) Jenkins, C. L . ; L i n , G . ; Duo, J.; Rapolu, D. ; Guzei, I. A . ; Raines, R. T.; Krow, G. R. J. Org. Chem. 2004, 69, 8565-8573. (15) Hodges, J. A . ; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 9262-9263. (16) Pinto, D . ; Sarocchi-Landousy, M . - T . ; Guschlbaeur, W . Nucleic Acids Res. 1979, 6. (17) Holmgren, S. K . ; Taylor, K . M . ; Bretscher, L . E . ; Raines, R. T. Nature 1998, 392, 666. (18) Shirota, F . N . ; Nagasawa, H . T.; Elberling, J. A . J. Med. Chem. 1977, 20, 1176-1181. (19) Fitzpatrick, P. F. ; Massey, V . J. Biol. Chem. 1982, 257, 1166-1171. (20) Niemz, A . ; Tirrell , D . A . J. Am. Chem. Soc. 2001,123, 7407-7413. (21) Anderson, M . O.; Shelat, A . A . ; Guy, R. K . J. Org. Chem. 2005, 70, 4578-4584. (22) Zanotti, G . ; Birr , C ; Wieland, T. Int. J. Pept. Protein Res. 1981, 18, 162-168. (23) Savige, W . E . Aust. J. Chem. 1975, 28, 2275-2287. (24) Savige, W . E . ; Fontana, A . Chem. Commun. 1976, 600-601. (25) Kamenecka, T. M . ; Danishefsky, S. J. Chem.-Eur. J. 2001, 7, 41-63. (26) Greenman, K . L . ; Hach, D . M . ; Van Vranken, D. L . Org. Lett. 2004, 6, 1713-1716. (27) Ley, S. V . ; Cleator, E . ; Hewitt, P. R. Org. Biomol. Chem. 2003,1, 3492-3494. (28) Hewitt, P. R.; Cleator, E . ; Ley, S. V . Org. Biomol. Chem. 2004, 2, 2415-2417. (29) Zanotti, G . ; Birr , C ; Wieland, T. Int. J. Pept. Protein Res. 1978,12, 204-216. (30) Beyermann, M . ; Bienert, M . ; Niedrich, H . ; Carpino, L . A . ; Sadat-Aalaee, D . J. Org. Chem. 1990, 55, 721-728. 89 (31) Aimoto, S. Curr. Org. Chem. 2001, 5, 45-87. (32) Del Val le , J. R.; Goodman, M . Angew. Chem. 2002,114, 1670-1672. (33) Rachele, J. R. J. Org. Chem. 1963, 28, 2898-2898. (34) De Luca, L . ; Giacomelli , G ; Porcheddu, A . Org. Lett. 2001, 3, 3041-3043. (35) De Luca, L . ; Giacomelli , G . ; Masala, S.; Porcheddu, A . J. Org. Chem. 2003, 68, 4999-5001. (36) Qiu, X . - l . ; Qing, F . - l . J. Org. Chem. 2002, 67, 7162-7164. (37) Crabtree, R. H . ; Davis, M . W. J. Org. Chem. 1986, 57, 2655-2651. (38) Bodanszky, M . ; Bodanszky, A . The Practice of Peptide Synthesis; Springer-Verlag: Berlin, 1984. (39) Lapatsanis, L . ; Mi l ias , G . ; Froussios, K . ; Kolovos, M . Synthesis 1983, 8, 671-673. (40) Middleton, W . J. J. Org. Chem. 1975, 40, 574-578. (41) Ratner, S.; Clarke, H . T. J. Am. Chem. Soc. 1937, 59, 200-204. (42) Nilsson, B . L . ; Kiessling, L . L . ; Raines, R. T. Org. Lett. 2001, 3, 9-12. (43) Waldemar, A . ; Bialas, J.; Hadjiarapoglou, L . Chem. Ber. 1991,124, 2377. (44) Waldemar, A . ; Chan, Y . - Y . ; Cremer, D. ; Gauss, J.; Scheutzow, D. ; Schindler, M . J. Org. Chem. 1987, 52, 2800-2803. (45) Singh, M . ; Murray, R. W. J. Org. Chem. 1992, 57, 4263-4270. (46) May, J. P.; Fournier, P.; Pellicelli , J.; Patrick, B . O.; Perrin, D . M . J. Org. Chem. 2005, 70, 8424-8430. (47) Coste, J.; Frerot, E . ; Jouin, P. J. Org. Chem. 1994, 59, 2437-2446. (48) Zanotti, G . ; Petersen, G . ; Wieland, T. Int. J. Pept. Protein Res. 1992, 40, 551-558. (49) Perrin, D . D . ; Armarego, W . L . F. Purification of Laboratory Chemicals, 3rd edition; Pegamon: Oxford, 1988. (50) Demange, L . ; Menez, A . ; Dugave, C. Tetrahedron Lett. 1998, 39, 1169-1172. (51) Webb, T. R.; Eigenbrot, C. J. Org. Chem. 1991, 56, 3009-3016. (52) Tamaki, M . ; Han, G . ; Hruby, V . J. J. Org. Chem. 2001, 66, 1038-1042. (53) Eswarakrishnan, V . ; Field, L . J. Org. Chem. 1981, 46, 4182-4187. (54) Wennemers, H . ; Conza, M . ; Nold , M . ; Krattiger, P. Chem.-Eur. J. 2001, 7, 3342-3347. 90 Appendix 2: Representative ESI Mass spectra 1- Linear octapeptides Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-FPro-Ile-OH 54 Peak ID Mass Found 1 1: ICO :MS ES+ 1428.8 4.le+006 c n , 7->fi A 1071 6 1412.7 f 7 - 3 5 9 5 . 3 7 0 9 . 2 ' 2 | - r | 7 4 8 ; 4 8 7 6 ; ^ 1 0 . 6 ^ 0 5 3 , 5 ^ I 1 1 1 5 | 3 ; 6 1 2 3 2 . 6 1 ^ ^ ^ 600.00 800.00 1000.00 1200.00 Nfrt alamonto mora frttmA 8 i 1450.7 / 1451.9 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-CF3Pro-Ile-OH 55 Peak ID Mass Found 1 1: 10 0-. 4 507.3 / 595.3 708.2 :MS ES+ 1428.8 4.1e+00€ noe, A 1071 6 1412.7 7 2 ? - 4 . 7 4 8 . 4 876.4 „ , „ „ , \ ° 1153. 6 1 2 3 2 . 6 1 2 6 7 • 7 ^ '910.6 1053.5^ 14S0.7 / 1451.9 600.00 M A H lomonfc i t i a r a fr\imH 800.00 1000.00 1200.00 1400.00 — m/z Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-CNPro-Ile-OH 56 1262.9 525.4 " 5 - 6 705.7 8 2 1 " 8 865.9 , . • . , > , ; ^ • ,1 . . — 988.7 1 1 2 5 . 9 1 1 6 * - 9 1385.9 1:MS ES+ 6 . 2 » * 0 0 5 1286.9 V .Li.,-.!.-..*..,. 1450.8 1452.8 600.00 800.00 1000.00 1200.00 1400.00 n/z Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-(SBn)Pro-Ile-OH 57 1: 100 I A 1:MS 601.5 « 4 5 - 7 7 7 e . 0 . 8 ° 5 - 7 8 6 5 . 9 » 0 9 - 9 M 7 . 9 ' 1 3 6 ; f " | ^ 2 3 . 8 1469,9 1483.0_g.ls+006 J1484.0 J1485.1 600.00 800.00 1000.00 m/z 1200.00 1400.00 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-N3Pro-Ile-OH 58 95 1: 100-624.3 721 .5780 .7 884.7 921.7 1 0 5 5 . 8 1 1 2 6 ' 9 H B O . 8 1 1:M3 ES+ 1401.9 5.2e+006 1403.0 1372.9 . . V • m/z Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-F2Pro-Ile-OH 59 Peak ID Mass Found 1 1: 100-, 507.3 :MS ES+ 1428.8 4.1e+006 595.3 708.2 726 .4 , , , , , „ 1071.6 , , „ , 1412.7 , , ,748.4876.4 Q , „ K , „ , . „ , . , , 1153. 6 1 2 3 2 . 6 * 2 6 • 7 ^ \ 910.6 1053.5_ 1450.7 1451.9 600.00 800.00 1000.00 1200.00 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Hyp(tBu)-Ile-OH 60 2:MS ES+ 1432.9 9.1a+006 567.4 683.4 721-4 7 5 5 - 4 , 8 1 2 . 5 926.1 994.4 1 1 0 9 ^ H 5 7 . 9 . 8 „ 1 4 1 5 - » T • • • • '-'T ' '—1—i—•—•—•—•—i——r •> -•—r-1—•—*—'—i——^—n—•—>—1—' 1 * 600.00 No elements were found. 1000.00 1200.00 1400.00 J1434.0 ,,1456.0 Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Ketopro-Ile-OH 61 100-, 613.4 550.4 | . <j41-6 7 1 4 - 3 8 2 5 . 4 881.7 9 8 8 . 8 1067.7 1284.8 1069.8 1244.7 . . . , • , . . ^ 2:MS ES+ 1.6e+006 600.00 1000.00 1200.00 1427.8 m/z Hpi-Gly-Ile-Gly-Cys(Tr)-Asn(Tr)-Thz-Ile-OH 62 100 658. 4 680. 4 . , , , i-{!.,..,-„—^. 600.00 800.00 868.8 » » - « 0 3 8 . 3 1 0 8 2 _ 6 1182.7 3:MS ES+ 1399.7 1.2e+006 ,1421.7 1000.00 1383.7 \ J1423.8 1465.6 1200.00 1400.00 L i .I486 96 2- Monocycle octapeptides Monocyclic fluoroproline octapeptide 63 Peak ID Mass Found ~ j 1 \ J 1: -JJ * 1:MS BS+ 100-, 875.5 897.5 ^ 2.6.+007 ^ 1 5 ' 2 629.3 657.2 761. 3,783. 3 | 919.5 f 9 20 .6 1053.5 1 1 9 1 5 1 3 2 ? 4 1368.1 1463.4 *J—r-l—i—i—•—r—S—i—i—'. -i—<—<—. fS..,.....r , ^ m / z 600.00 800.00 1000.00 1200.00 1400.00 Monocyclic trifluoromethylproline octapeptide 64 i •; Sample 1 Instrument Method^ :\MassLynx\OALogin\OAMethods\MEOH_FIA_ES_2.olp 669.3 925.5 811.4,833.4 7 4 8 ; 3 If,-, 947.5 1:MS ES+ 2.6a+007 948.7 V ^,964.8 1083 .5 1 1 6 9 . 7 " °7 - 6 1 2 7 9 . 9 1 4 1 1 , 1 4 3 5 . 9 m/ z 800.00 1000.00 97 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0061119/manifest

Comment

Related Items