UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Synthesis on solid phase of a bicyclic octapeptide amatoxin Blanc, Antoine 2009

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
24-ubc_2009_fall_blanc_antoine.pdf [ 1.22MB ]
Metadata
JSON: 24-1.0061354.json
JSON-LD: 24-1.0061354-ld.json
RDF/XML (Pretty): 24-1.0061354-rdf.xml
RDF/JSON: 24-1.0061354-rdf.json
Turtle: 24-1.0061354-turtle.txt
N-Triples: 24-1.0061354-rdf-ntriples.txt
Original Record: 24-1.0061354-source.json
Full Text
24-1.0061354-fulltext.txt
Citation
24-1.0061354.ris

Full Text

SYNTHESIS ON SOLID PHASE OF A BICYCLIC OCTAPEPTIDE AMATOXIN  by Antoine Blanc  B.Sc., Université de Montréal, 1998 M.Sc., Université de Montréal, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2009 © Antoine Blanc, 2009  ii  Abstract The α-amanitin is an excellent template for use in the design of a chemical library on solid phase in order to study the mechanisms involved in the regulation of transcription. Indeed, α-amanitin selectively inhibits with high affinity the RNA Polymerase II. The α-amanitin belongs to the amatoxin family which is characterized by a defined rigid bicyclic structure consisting of a head-to-tail cyclized octapeptide, with a transannular linkage known as a tryptathionine bridge. The latter is made via the Savige-Fontana tryptathionylation of the oxidized tryptophan derivative 3a-hydroxypyrrolo[2,3-b]indoline in neat TFA. Therefore, in order to achieve the synthesis of a α-amanitin-based library on solid phase, the linker must be stable both in TFA and during the peptide synthesis. Such a linker was found in the tartrate-based linker. As proof of this concept, following completion of the linker synthesis on solid phase, the linear octapeptide precursor of Pro2-Glu3-S-deoxo-amaninamide was prepared by standard Fmoc/tert-Bu SPPS. The tryptathionine bridge was achieved by the Savige-Fontana reaction and the second cyclization was performed by a head-to-tail macrolactamization, all on PEGA resin. The bicyclic octapeptide was cleaved off from the linker with the mild sodium periodate oxidant, purified by RP-HPLC and characterized by HRMS (ESI) and UV spectra. OH  Fmoc/tert-Bu SPPS NHFmoc Y  R1  CO2t-Bu O  R5  TFA  HN R6  HN H N Trt  R4 N H O N H  H N O  O  O  R3 N H  S  O  NH NH2  O O  H N R2  O  H N  R5 N H  R6  TrtS O  H N O  R4  N H  O  CO2H Y N R1 H  macrolactamization  HN  R2 HN  Library of amatoxins  Y H N  O O S O N H  R2 N H  R3  R1  R3  Tryptathionine bridge  O  H N  O  H N O  O  R1  Y CO2t-Bu  R6 N H O  N H O HN N H  R4  NH R5 O  iii  Table of Contents Abstract................................................................................................................................. ii Table of Contents ................................................................................................................ iii List of Figures........................................................................................................................v List of Schemes.................................................................................................................... vi List of Abbreviations ........................................................................................................ viii Acknowledgements ........................................................................................................... xiii Chapter 1 ...............................................................................................................................1 Introduction...........................................................................................................................1 1.  Amanitin is a specific RNA Pol II inhibitor ...............................................................1  2.  Amanitin structure activity relationship ....................................................................2  3.  Design of a library to explore the amanitin chemical space......................................6  4.  Library and peptide synthesis on solid phase.............................................................6  5.  Alpha-amanitin analogues synthesis ...........................................................................9 5.1. 5.2.  6.  Synthesis of tryptathionine bridge ......................................................................... 9 Previous syntheses of amanitin analogues........................................................... 10  Linkers – a brief review..............................................................................................12 6.1. Linker classification............................................................................................. 13 6.1.1. Traditional (classical) linker ........................................................................ 14 6.1.2. Traceless linker ............................................................................................ 15 6.1.3. Diversity (multifunctional) linker ................................................................ 16 6.1.4. Safety catch linker........................................................................................ 16 6.2. Cleavage methods ................................................................................................ 17 6.2.1. Electophilic .................................................................................................. 18 6.2.2. Nucleophilic................................................................................................. 19 6.2.3. Photolytic ..................................................................................................... 20 6.2.4. Oxidative...................................................................................................... 21  7.  Promising acid stable linkers .....................................................................................21 7.1. 7.2. 7.3. 7.4.  Silicon-based linkers............................................................................................ 21 Tartrate-based linkers........................................................................................... 25 Anchorage strategies of the first amino acid on the linker .................................. 27 Proposed methodology......................................................................................... 27  Chapter 2 .............................................................................................................................29 Results and discussion ........................................................................................................29 1.  The benzonitrile silyl linker: synthesis and stability test ........................................29  iv 1.1. 1.2. 1.3. 1.4. 2.  The tartrate linker: synthesis and stability test .......................................................39 2.1. 2.2. 2.3. 2.4.  3.  Synthesis of benzonitrile silyl unit in solution..................................................... 30 Benzonitrile silyl linker and spacers loading on solid phase ............................... 30 Synthesis of fluorescent benzonitrile silyl linker on solid phase......................... 33 TFA test stability of the fluorescent silyl-based linker on solid phase ................ 35 Preparation of soluble fluorescent tartrate linker................................................. 40 TFA test stability of the fluorescent soluble tartrate linker ................................. 43 Preparation of tartrate linker on solid phase ........................................................ 45 Assessment of successful tartrate linker synthesis............................................... 47  Preparation of Pro2-Glu3-S-deoxo-amaninamide on solid phase ...........................49 3.1. 3.2. 3.3. 3.4.  Synthesis of linear hexapeptide amatoxin precursor on solid phase.................... 49 TFA test stability of the fluorescent linear heptapeptide on solid phase ............. 50 Synthesis of monocyclic Pro2-Glu3-S-deoxo-amaninamide on solid phase ........ 53 Synthesis of bicyclic Pro2-Glu3-S-deoxo-amaninamide on solid phase .............. 59  Chapter 3 .............................................................................................................................64 Experimental .......................................................................................................................64 1.  General.........................................................................................................................64 1.1. 1.2.  2.  Chemicals............................................................................................................. 64 Analytical methods .............................................................................................. 65  Chemical Methods ......................................................................................................68 2.1. 2.2. 2.3. 2.4. 2.5. 2.6.  Solution phase synthesis ...................................................................................... 68 Solid phase synthesis of the benzonitrile silyl linker........................................... 92 Cleavage of product from benzonitrile silyl linker on resin ................................ 95 Solid phase synthesis of the tartrate linker .......................................................... 96 SPPS on the tartrate linker ................................................................................... 99 Cleavage of product from tartrate-based linker on resin ................................... 105  Chapter 4 ...........................................................................................................................111 Conclusion .........................................................................................................................111 References..........................................................................................................................115 Appendix: Representative 1H-NMR spectra ..................................................................121  v  List of Figures Figure 1: Natural members of the amatoxin family.............................................................. 3 Figure 2: Bushnell’s crystal structure 2a and Lipscomb’s crystal structure. ........................ 5 Figure 3: Traditional (classical) linkers. ............................................................................. 15 Figure 4: Electrophile-labile linkers. .................................................................................. 19 Figure 5: Nucleophile-labile linkers. .................................................................................. 20 Figure 6: Photo-labile linkers.............................................................................................. 20 Figure 7: Oxidant-labile linkers. ......................................................................................... 21 Figure 8: Acid stable silicon-based linkers. ........................................................................ 25 Figure 9: Tartrate-based linker............................................................................................ 26 Figure 10: Acid stable benzonitrile silyl-based linker 60. .................................................. 29 Figure 11: FT-IR spectra of the Merrifield resin (A) and the resin 71 (B). ........................ 32 Figure 12: The fluorescent benzonitrile silyl-based linker 77. ........................................... 33 Figure 13: UV absorption spectra in MeCN of compounds 73 and 74............................... 34 Figure 14: FT-IR of dry resins before or after TFA exposure. ........................................... 36 Figure 15: The two proposed bromobenzonitrile silicon-based isomeres. ......................... 37 Figure 16: The benzonitrile silyl 65 submitted to COSY lrqf............................................. 38 Figure 17: The solid and solution phase tartrate-based linkers........................................... 40 Figure 18: RP-HPLC analysis of the linker 88. .................................................................. 44 Figure 19: MS analysis of the linker 88.............................................................................. 45 Figure 20: LRMS (ESI) of the glyoxamide products 98 and 99......................................... 48 Figure 21: Fluorescent linear heptapeptide 104 on PEGA resin......................................... 51 Figure 22: The eluate (A) and the resin 105 (B) shown under UV lamp............................ 52 Figure 23: UV absorption spectra in MeOH of compounds 106 and 103. ......................... 53 Figure 24: Structure of by-products observed during Hpi synthesis. ................................. 55 Figure 25: RP-HPLC analysis of the monocyclic amatoxin precursors. ............................ 58 Figure 26: MS analysis of the monocyclic amatoxin precursors. ....................................... 59 Figure 27: RP-HPLC analysis of the bicyclic amatoxins 121 and 122............................... 61 Figure 28: UV spectra and MS analysis of the bicyclic amatoxins 121 and 122. .............. 62 Figure 29: Structure of the two bicyclic amatoxin diastereoisomers.................................. 63  vi  List of Schemes Scheme 1: Split-and-pool library synthesis........................................................................... 7 Scheme 2: General principle in solid phase peptide.............................................................. 8 Scheme 3: Savige-Fontana reaction. ..................................................................................... 9 Scheme 4: Trt-Hpi-Gly-OMe 8a/b synthesis. ..................................................................... 10 Scheme 5: Synthesis of L-Pro2-L-Ile3-S-deoxo-amaninamide 12. ...................................... 11 Scheme 6: Head-to-tail macrolactamization. ...................................................................... 12 Scheme 7: Synthesis of amatoxin derivatives on solid phase. ............................................ 13 Scheme 8: Linkers classification. ........................................................................................ 14 Scheme 9: Traceless linkers. ............................................................................................... 16 Scheme 10: Safety catch linkers.......................................................................................... 17 Scheme 11: Electrophilic linker cleavage. .......................................................................... 18 Scheme 12: Silicon bearing linkers used in peptide synthesis. ........................................... 22 Scheme 13: Electrophilic substitution of an aryl silyl 41.................................................... 24 Scheme 14: General hydrophilic tartrate-based linker 49. .................................................. 26 Scheme 15: Proposed synthesis of L-Pro2-L-Glu3-S-deoxo-amaninamide 59. ................... 28 Scheme 16: Synthesis of benzonitrile silyl 65..................................................................... 30 Scheme 17: Synthesis of Merrifield resin hydroquinone spacer 68. ................................... 31 Scheme 18: Synthesis of benzonitrile silyl 70..................................................................... 31 Scheme 19: Synthesis of the benzonitrile silyl-based linker 71 on resin. ........................... 32 Scheme 20: Synthesis of the benzonitrile silyl-based linker 72 on resin. ........................... 33 Scheme 21: Synthesis of Dac-OH 74. ................................................................................. 34 Scheme 22: Synthesis of fluorescent spacer 76c................................................................. 35 Scheme 23: Synthesis of fluorescent silicon-based linker 77 on resin................................ 35 Scheme 24: Metal-halogen exchange of benzonitrile 62 with n-BuLi................................ 39 Scheme 25: Synthesis of the resin spacer mimic 79............................................................ 40 Scheme 26: Synthesis of the soluble Valine linker 81. ....................................................... 41 Scheme 27: Formation of the DBF adduct 82. .................................................................... 41 Scheme 28: Synthesis of protected tartaric acids 84. .......................................................... 42 Scheme 29: Synthesis of the soluble tartrate linker 86........................................................ 42  vii Scheme 30: Synthesis of fluorescent spacer 76c................................................................. 43 Scheme 31: Synthesis of the soluble fluorescent linkers 88 and 88’. ................................. 43 Scheme 32: Preparation of the tartrate linker on amino PEGA resin 93............................. 46 Scheme 33: Preparation of the N-Mmt protected tartrate-based linker 94.......................... 47 Scheme 34: Synthesis of 2-(2,5-dibromobenzamido)acetic acid 96. .................................. 47 Scheme 35: Preparation and isolation of the labeled tartrate linker 98............................... 48 Scheme 36: Preparation of the linear hexapeptide amatoxin precursor 101. ...................... 50 Scheme 37: Synthesis of Fmoc-Lys(Dac)-OH 103. ............................................................ 51 Scheme 38: Preparation and isolation of the labeled heptapeptide 106. ............................. 52 Scheme 39: Synthesis of Trt-Hpi-Gly-OH 9a/b.................................................................. 55 Scheme 40: Preparation of the amatoxin precursor 114 on PEGA resin. ........................... 56 Scheme 41: Isolation of the monocyclic amatoxin 116....................................................... 57 Scheme 42: Preparation of L-Pro2-L-Glu3-S-deoxo-amaninamide 121. ............................. 60  viii  List of Abbreviations Å  Ångström  Abu  L-α-Aminobutyric acid  Ac2O  Acetic anhydride  AcOEt  Ethyl acetate  AcOH  Acetic acid  Ala  Alanine  ANP  3-Amino-3-(2'-nitrophenyl)-2,2- dimethylpropionic acid  Arg  Arginine  Asn  Asparagine  Asp  Aspartic acid  BHA  Benzhydrylamine  Bn  Benzyl  Boc  tert-Butyloxycarbonyl  Br s  Broad singlet  Bz  Benzoyl  Bzl  Benzyl  CD  Circular dichroism  COSY  Correlation spectroscopy  Cys  Cysteine  d  Doublet  Dab  (2S,4)-Diaminobutyric acid  Dac-NHR  7-Diethylamino-coumarin-3-amide derivatives  Dac-OEt  7-Diethylamino-coumarin-3-ethyl ester derivatives  Dac-OH  7-Diethylamino-coumarin-3-carboxylic acid  DBF  Dibenzofulvene  DBU  Diaza(1,3)bicyclo[5.4.0]undecane  dd  Doublet of doublets  DDQ  2,3-Dichloro-5,6-dicyanobenzoquinone  DDQH2  2,3-Dichloro-5,6-dicyano-1,4-hydroquinone  DIAD  Diisopropylazodicarboxylate  DIEA  N,N-Diisopropylethylamine, Hunig's base  DihyIle  (2S,3R,4R)-2-Amino-3-methyl-4,5-dihydroxy-valeric acid (4R,5)-Dihydroxy-L-isoleucine γ,δ-Dihydroxy-L-isoleucine  ix DIPEA  N,N-Diisopropylethylamine, Hunig's base  DMAP  4-Dimethylaminopyridine  DMDO  Dimethyldioxirane  DMF  N,N’-Dimethylformamide  DMP  2,2-Dimethoxypropane  DMS  Dimethylsulfide  DMSO  Dimethylsulfoxide  DNA  Deoxyribonucleic acid  DNP  Dinitrophenylhydrazine  E1  Unimolecualr (rate-limiting step) elimination  E2  Bimolecular (rate-limiting step) elimination  EDC  1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide  EDG  Electron donating group  ESI  Electro-Spray Ionization  Et  Ethyl  Et2O  Diethyl Ether  Et3N  Triethylamine  EtOH  Ethanol  EWG  Electron withdrawing group  Fmoc  9-Fluorenylmethyloxycarbonyl  Fmoc-Lys(Dac)-OH  N-ε-Dac-N-α-Fmoc-L-Lysine  FT-IR  Fourier transformed infra-red  Gln  Glutamine  Glu  Glutamic acid  Gly  Glycine  h  Hour  HBTU  O-Benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluorophosphate  His  Histidine  HOBt  N-Hydroxybenzotriazole  Hpi  3a-Hydroxypyrrolo[2,3-b]indoline  HRMS  High resolution mass spectrometry  HyIle  (2S,3R,4S)-2-Amino-3-methyl-4-hydroxy-valeric acid (4S)-Hydroxy-L-isoleucine γ-Hydroxy-L-isoleucine  Hyp  (4R)-Hydroxy-L-proline trans-4-Hydroxy-L-proline γ-Hydroxy-L-proline  x (2S,4R)-(-)-4-Hydroxy-2-pyrrolinecarboxylic acid HyVal  (2S,3R)-2-amino-4-hydroxy-3-methylbutyric acid (3R)-4-Hydroxy-valine  i  Iso  Ile  L-Isoleucine (2S,3S)-2-amino-3-methylpentanoic acid  i-Pr, iPr, iso-Pr  Iso-propyl  J  Coupling constant  Ki  Inhibitor constant  LAH  Lithium aluminium hydride  LD50  Median lethal dose  Leu  Leucine  LRMS  Low resolution mass spectrometry  LTMP  Lithiated tetramethylpiperidine  Lys  Lysine  m  Multiplet  M  Mass  m/z  Mass-to-charge ratio  MBHA  4-Methylbenzhydrylamine  Me  Methyl  MeCN  Acetonitrile  MeOH  Methanol  Met  Methionine  min  Minute  Mmt  Monomethoxytrityl, methoxytriphenylmethyl, p-anisyldiphenylmethyl  mRNA  Messenger RNA  MS  Mass Spectrometry  MS/MS  Tandem mass spectrometry.  Nbb  3-Nitrobenzamidobenzyl  NBS  N-Bromosuccinimide  n-BuLi  n-Butyllithium  NHS  N-Hydroxysuccinimide  NMM  N-Methylmorpholine  NMP  N-Methylpyrrolidone  NMR  Nuclear Magnetic Resonance  ONB  o-Nitrobenzyl  Orn  Ornithine  xi OSu  Oxysuccinimide  PAAMB  p-acylaminobenzyl  PAM  4-Hydroxymethyl-phenylacetamidomethyl  PEG  Polyethyleneglycol  PEGA  Polyethyleneglycol dimethylacrylamide co-polymer Poly[acryloyl-bis(aminopropyl)polyethylene glycol]  Ph  Phenyl  Phe  Phenylalanine  PMA  Phosphomolybdic acid  PMB  p-Methoxybenzyl  ppm  Part per million  Pr  Propyl  pre  Precursor  Pro  Proline  PTMSEL  (2-Phenyl-2-trimethylsiyl) ethyl linker  PyBOP  (Benzotriazol-1-yloxy)tripyrrolidophosphonium hexafluorophosphate  q  Quartet  RBF  Round-bottom flask  Rf  Retention factor or ratio to front  RNA  Ribonucleic acid (single-stranded)  RNA Pol II  DNA dependent RNA polymerase II  Rpb  RNA polymerase II (B) subunit  RP-HPLC  Reverse phase-high performance liquid chromatography  RT  Room temperature  s  Singlet  SAC  α-Trimethylsilylbenzyl  SAL  Silyl amide linker  SAR  Structure-activity relationship  Ser  Serine  S N1  Unimolecular (rate-limiting step) nucleophilic substitution  S N2  Bimolecular (rate-limiting step) nucleophilic substitution  SPPS  Solid phase peptide synthesis  t  Tert  t  Triplet  TAEA  Tris (2-aminoethyl) amine  TBAF  Tetra-n-butylammonium fluoride t  t-Bu, tert-Bu, Bu  Tert-butyl, tertiary butyl  xii TCA  Trichloroacetic acid  TES  Triethyl silane  TFA  Trifluoroacetic acid  THF  Tetrahydrofuran  Thr  Threonine  TLC  Thin-layer chromatography  TPP  Triphenylphosphine  Trp  Tryptophan  Trt  Trityl, triphenylmethyl  Tyr  Tyrosine  v/v  Volume-to-volume ratio  Val  L-Valine (2S)-2-amino-3-methylbutric acid  w/v  Weight-to-volume ratio  Xaa  Unspecified amino acid  δ  Chemical shift  λ  Wavelength  xiii  Acknowledgements I would like to thank my supervisor Professor David Perrin for his insight and passion for science, which provided inspiration and motivation to better my-self and to complete the thesis project. I am grateful to the service personnel of the NMR Lab and the Mass Spectrometry Lab that helped me with the 2D-NMR experiments and conducted the HRMS (ESI) respectively. I am thankful to Dr. Jonathan May, Dr. Marcel Hollenstein, Dr. Curtis Harwig, Dr. Jason Thomas, Mr. David Dietrich and Ms Irma Hoogendoorn for insightful discussion all along my thesis work and for reading my manuscript. I would like to thank professors Mel Comisarow, Martin E. Tanner and Suzana K. Straus who accepted to be member of my thesis examination committee.  1  Chapter 1 Introduction  1. Amanitin is a specific RNA Pol II inhibitor Alpha-amanitin is a member of the amatoxin bicyclic peptide family found in the highly toxic Amanita phalloides mushroom (death cap mushroom) (Figure 1).1 The oral LD50 of α-amanitin is 0.5 mg per kg of body weight in a mouse. This toxicity is due to an efficient allosteric inhibition of the DNA dependent RNA polymerase II (RNA pol II), in part a result of the high affinity (Ki < 50 nM, varied amongst different eukaryotes).2 The inhibitor constant (Ki) is the in vitro concentration required to produce half maximum inhibition of the RNA polymerase that catalyze the [3H]-UTP incorporation in RNA. Localized in the nucleoplasm, the eukaryotic RNA pol II is a multi-subunit mega Dalton enzyme, which catalyzes the polymerization of the precursor messenger RNA (pre-mRNA) also called heterogeneous nuclear RNA (hnRNA). The mRNA is used as a coding template for the ribosomal synthesis of a majority of functional and structural proteins. When inhibited, the RNA pol II stalls on the DNA and is targeted for proteolysis.3 Subsequently, the α-amanitin is then released to associate with another active polymerase. The overall result is a significant depletion of RNA pol II which in turn compromises cellular homeostasis and leads to cell death. Despite high homologies between the different eukaryotic and prokaryotic RNA polymerases, only the eukaryotic RNA pol II is significantly inhibited, thus indicating the remarkable specificity of the α-amanitin for its target.  2  2. Amanitin structure activity relationship The members of the amatoxin family are produced by the Amanita genus of mushrooms (Figure 1a). They share a general bicyclic structure (24-membered ring) and a common tertiary structure consisting of a head-to-tail cyclized octapeptide with a transannular linkage known as a (R)-S-oxide-tryptathionine bridge 1a (Figure 1b). The bicycle is made of two 18-membered rings at a dihedral angle of 80o (Figure 1c). The indole ring is almost parallel to the hydrophilic ring I (residues 1 to 4). Unnatural amino acid residues, such as (4R)-hydroxy-L-proline 1b (Hyp), 6’-hydroxy-L-tryptophan 1c, (2S,3R,4R)-2-amino-3methyl-4,5-dihydroxy-valeric acid 1d (DihyIle) and (2S,3R,4S)-2-amino-3-methyl-4hydroxy-valeric acid 1e (HyIle) also occur among the amatoxin family members (Figure 1b).  3  a)  R5 3  4  R4 HN  O  O NH  H2 NH C  O  R3  N  S  H N R2 O  O  1  R1  R2  R3  R4  α-amanitin  OH  NH2  OH  OH CH2OH 10-8  β-amanitin  OH  OH  OH  -8 OH CH2OH 10  γ-amanitin  OH  NH2  OH  OH  CH3  10-8  ε-amanitin  OH  OH  OH  OH  CH3  10-8  Amanin  H  OH  OH  OH CH2OH 10-8  Amaninamide  H  NH2  OH  OH CH2OH 10-8  Amanullin  OH  NH2  OH  H  CH3  10-6  Amanullinic acid  OH  OH  OH  H  CH3  10-6  Proamanullin  OH  NH2  H  H  CH3  10-5  O  HN R1  O  2  Name  5  N H  H2 C  NH O  8  6 O  NH  7  O  b)  Ki  HO  O H2N  OH O O S N H  c)  R5  1a  NH  HN  HO  OH N H  CO2H  H2N  1b  Ring I  2b  1c  CO2H  H2N  OH  CO2H  H2N  1d  CO2H  1e Ring II  2b  Figure 1: Natural members of the amatoxin family. a) secondary structure of natural amatoxins with their respective Ki effects on RNA pol II from Drosophilia; b) structure of the (R)-S-oxide-tryptathionine bridge ((R)-sulfoxide) 1a and structures of non ribosomal amino acids (1b – 1e) that are found among the amatoxin family; c) Lipscomb’s crystal structure of ε-amanitin 2b that shares with the amanitin series a very similar tertiary structure. Green and pink represent the amide backbone of ring I and II respectively. Blue, red, yellow and white represent nitrogen, oxygen, sulfur and hydrogen respectively.  The bicyclic structure is mandatory for RNA pol II inhibition and can be seen as a structural lock. Bicyclic peptides are often compared to protein loops commonly used as recognition sequences in protein-protein interactions.4 The tryptathionine bridge 1a is a (R)-sulfoxide ether linkage made of the cysteine sulfhydryl group in position 8 (Cys8)  4 covalently bonded to the C-2’ position of the 6’-hydroxy-Trp4 indole ring. The tryptathionine bridge is believed to maintain the three dimensional structure of amatoxins. It may also be involved in water exclusion and hydrophobic interaction with RNA pol II. Interestingly, S-deoxo-α-amanitin with a non-oxidized tryptathionine bridge (S-(2tryptophanyl)cysteine), amaninamide and S-deoxy-amaninamide exhibit the full toxic effect.5 The unnatural (S)-sulfoxide diastereomer of α-amanitin was, however, found to be 8 to 20-fold less active than the naturally occurring (R)-sulfoxide. Recently, Bushnell et al. co-crystallized α-amanitin with the Saccharomyces cerevisiae’s RNA pol II subunit Rpb 1 and Rpb 2 (Figure 2a).6 The two subunits constitute the major part of both the active site and the substrate binding site of the holoenzyme RNA pol II. The crystal structure revealed that the α-amanitin binding site was outside both these sites. Therefore, rather than mimicking a substrate or a transition state at the active site, αamanitin binds to a protein fold leading to the RNA pol II allosteric inhibition. The X-ray crystal structure (resolution of 2.8 Å), also revealed several H-bond exchanges between αamanitin specific residues, its amide backbone and the side chains of Rpb1 (Figure 2a). Importantly, a strong H-bond was observed between the amanitin Hyp2 and Rpb 1’s glutamic acid residue 822 (GluA822). The importance of Hyp2 was supported by previous structure-activity relationship (SAR) experiments and confirmed by a recent cocrystallization.7 The Hyp2 effect likely exceeds that of a single hydrogen bond interaction. The hydroxyl functional group at the C-4’ position of Hyp2, by a combination of steric and electronic effects, might favour a particular proline pucker ring conformation and a β-turn, which are optimized to bind RNA pol II subunits. Data from Bushnell’s crystal 2a supported earlier reports where the α-amanitin active conformation appeared to be stabilized by an intra-molecular H-bond between the βcarbonyl of L-asparagine in position 1 (Asn1) and the amide backbone (Figure 2a).8 The same H-bond was found in β-amanitin between the L-aspartic acid in position 1 (Asp1) and the amide backbone. The importance of the β-carbonyl was supported by a report in which Asn1 or Asp1 substitution to L-α-aminobutyric acid (Abu) led to a significant loss of activity.9  5 a)  GlnA768  HisA816  b)  ArgA726 GlnA767  CH2OH HO  O  O N  N H  O  N H  H O  H O GluA822  O S NH2 H O N O SerA769  N  2a  N H H N  H2 C  O  N H OH Val A719  O H2 N C H O  Hydrophobic pocket  2b  Figure 2: Bushnell’s crystal structure 2a and Lipscomb’s crystal structure. a) Bushnell’s crystal structure 2a of α-amanitin interacting with Rpb1 (A subunit). Red and blue represent H-bond and hydrophobic interactions respectively, between α-amanitin and RNA pol II. Green represents α-amanitin intra-molecular H-bond; b) Lipscomb’s crystal structure 2b of εamanitin. Green, blue, red, yellow and white represent important side chains, nitrogen, oxygen, sulfur and hydrogen respectively.  Bushnell’s crystal 2a revealed that the amide backbone of the α-amanitin’s DihyIle3 was exchanging an H-bond with the RNA pol II subunits (Figure 2a). Substitution of DihyIle3 to HyIle corresponded to the natural γ-amanitin that was similar to α-amanitin, βamanitin and ε-amanitin 2b in their tertiary structures, LD50 and Ki values. However, substitution of HyIle3 to L-isoleucine (Ile) resulted in a total loss of inhibition in whole cell assays, although only an increase of Ki by 4 to 50-fold was measured in vitro.10 Similarly, Ki increased by only 50-fold if the γ-amanitin’s HyIle3 was converted to (4R)-hydroxy-LIle or (2S,3R)-2-amino-4-hydroxy-3-methylbutyric acid (HyVal), but Ki increased by more than 100-fold with substitution to L-allo-Ile ((3R)-methyl), (2S,3S)-2-amino-4-hydroxy-3methylbutyric acid or L-Valine (Val).11 The latter substitutions did not appear to modify the overall rigid bicyclic conformation compared to the natural amanitin series but these substitutions were more likely to induce a local backbone amide distortion in the turn region of Hyp2-Xaa3.12 In conclusion, the 4-hydroxyl is probably mandatory to allow the amanitin translocation through the cellular membrane, and the (3R)-methyl might interact with a hydrophobic region of RNA pol II. An important hydrophobic interaction with RNA pol II was found in the hydrophobic loop conformation made of Gly5-Ile6-Gly7 (part of ring II). For example, αamanitin’s Ki increased by 1000-fold when Ile6 or glycine in position 5 (Gly5) were substituted to L-alanine (Ala).13 The Gly5-Ile6-Gly7 loop conformation may be important to  6 accommodate the insertion of Ile’s side chain into a hydrophobic pocket of RNA pol II. This hydrophobic interaction was supported by Bushnell’s crystal 2a (Figure 2a).  3. Design of a library to explore the amanitin chemical space The mechanisms involved in the allosteric inhibition of RNA pol II by α-amanitin are still under investigation.14 In fact, the pool of unnatural α-amanitin derivatives needed to complete the α-amanitin structure-activity relationship (SAR) information already gathered, is limited. Our aim is to verify the crystal structure data by screening a small chemical library based on the rigid α-amanitin scaffolds against an in vitro RNA pol II assay. Different RNA polymerase species through the prokaryotic and eukaryotic kingdom share important homologies. Therefore, a long range aim is to discover allosteric inhibitors against different types of RNA polymerase involved in bacterial and viral infections by using an α-amanitin bicyclic scaffold-based library.  4. Library and peptide synthesis on solid phase A library is commonly made by combinatorial chemistry since it permits rapid synthesis of a wide range of diverse products. The two common methods for preparing combinatorial libraries are parallel synthesis and split-and-pool synthesis. In parallel synthesis, reactions occur in separate vessels for each combination of compounds. This method can be achieved in solution or in solid phase and products are easily identified since each vessel bears only one type of product. This kind of library synthesis is, however, time consuming and can require both large volumes and high amounts of materials. The number of compounds, therefore, that one can possibly synthesize is limited. The split-and-pool approach is commonly preferred and takes place on the solid phase (Scheme 1). The beads are split proportionally between separate vessels for different reactions. Following reaction completion and appropriate washing, the beads are pooled together into the same vessel, mixed, and split again. The cycle is repeated using the next combination of reactants. This method is advantageous compared to the parallel method  7 because it takes advantage of the solid phase and, with one compound produced per bead, permits the synthesis of a large number of different products in a short time and a condensed space. The solid phase permits the use of excess reagents that are easily washed away by filtration using solvents with a wide range of polarities. Following completion of the synthesis, the product can be cleaved from the solid support and then, recovered in the eluate thus avoiding tedious work-up and purification steps. Solid phase is also used advantageously for a wide range of reaction types, including peptide cyclization performed with a low loading resin and SAR experiments. For example, during SAR experiments, a large number of beads is reversibly and rapidly screened by robots against a wide range of soluble targets.  A A A A  A  B2  A A  A  C1 C1  B2  C1  B3  B1 A  A  B1  B3  B1 B2 B3  A A A  A A A  B1  C2  B2 B3  C2 C2  C3  B1 B2  C3  B3  C3  Scheme 1: Split-and-pool library synthesis. Molecules A anchored to beads react with one type of compound Bi per vessel. Following reaction, all beads are pooled in the same vessel, mixed and split in same proportion into new separated vessels. The process is repeated with the compound Ci.  Therefore, peptide libraries are commonly built by the split-and-pool method based on solid phase peptide synthesis (SPPS) in the C-to-N-terminus synthesis direction (Scheme 2).15 This direction lowers the occurrence of side reactions such as enantiomerization of the α-carbon and diketopiperazine production. Furthermore, the coupling reactions are achieved in about 30 minutes and deprotection occurs in minutes.  8  Pj  P1 Y L  O  H N  Pj  X R1  a  Pj  H N P1  O  O Y L R1  H2N  b  P1  P2 Y L  R1  O  H N  X R2  R2 Pj  c  N H  P2  O  H N  Y L  O R P1 1  d  Pj  H N  Pn+2  Pn+1 Rn+1  O Rn+2  N H  O  O  H N  Y L R1  P1  n+1  Scheme 2: General principle in solid phase peptide. In the C-to-N-terminus synthesis, a linear (L) and a functional group (Y) are attached to a resinous solid phase (bead as a shaded ball) in order to anchor the amino acid to the solid support. a) the peptide synthesis begins by loading the first amino acid from its activated carboxyl (X) with its protected α-amine (Pj) and protected side chain (Pi); b) the α-amine of the anchored residue is deprotected orthogonally to its side chain; c) the next activated amino acid is added; d) “n” number of cycles of deprotection and coupling are repeated until completion of the peptide sequence.  In SPPS, the 9-fluorenylmethyloxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) protecting groups are commonly used for amine protection and belong to the carbamate (urethane)-based protecting group. The benzyl (Bzl) and tert-butyl ester (tert-Bu) are used to protect the C-terminus if the latter is not anchored to the resin. SPPS is currently performed using the protecting group combination of Fmoc and tert-Bu. The protecting group Fmoc is more stable than Boc during SPPS using carbodiimides and onium saltbased coupling reagents. Also, the Fmoc deprotection usually leads to fewer side reactions compared to Boc deprotection that occurs in strong acid. In addition, tert-Bu deprotection is milder than Bzl deprotection. During SPPS, amino acids that have an electrophilic or nucleophilic functional group need to be protected. For example, the carboxamide side chain of asparagine (Asn) can dehydrate to a cyano group (β-cyanoalanine) during activation of the carboxylic acid by the coupling agent.16 Furthermore, the aspartimide by-product can occur during Fmoc deprotection using a strong secondary amine base.17 The sulfhydryl of cysteine (Cys) is a good nucleophile that can compete with the free α-amine in the nucleophilic attack of the activated α-carboxyl. In addition, cysteine is easily oxidized under atmosphere making an  9 intra-molecular or inter-molecular disulfide bond. Thus, the bulky acid labile triphenylmethyl (Trityl (Trt)) group is a common protecting group used for Asn and Cys side chains in SPPS. Furthermore, the Trt protecting group is stable during Fmoc SPPS.  5. Alpha-amanitin analogues synthesis 5.1. Synthesis of tryptathionine bridge  The tryptathionine bridge (S-(2-tryptophanyl)cysteine) synthesis is an important component of the amatoxins synthesis. Because the indole is an electron-rich aromatic heterocycle commonly involved in electrophilic aromatic substitution reactions at its C-3 position, the S-tryptophanylation involving the carbon-sulfur bond formation between the C2 position of the tryptophan indole and the thiol of cysteine is not straightforward. However, Savige and Fontana developed an excellent method to achieve an indole-2-thioether linkage in environments mild enough to preserve the indole and avoid enantiomerization. In this reaction, the Nb-Trt-3a-hydroxypyrrolo[2,3-b]indoline (Trt-Hpi 3) motif is allowed to react with the S-Trt-cysteine 4 for 5 hours in neat trifluoroacetic acid (TFA) where the trityl protecting groups are lost and the tryptathionine bridge 5 is completed (Scheme 3).18  4 HO  3 2  3a 8a  5 6 7  3  N  N H Ph H Na  O  O Nb  Ph Ph  H N S  4  Ph  TFA Ph Ph  5  6  5  4  7  β 3a  3  7a N  H 1  O  α  2  NH2  O  S NH  Scheme 3: Savige-Fontana reaction. In TFA, the reaction affords a tryptathionine bridge (S-(2-tryptophanyl)cysteine) 5.  The Hpi is commonly embedded within a dipeptide sequence. The dipeptide is prepared by a standard peptide coupling to yield the N-α-Trt-L-tryptophan-Xaa-OR (Trt-Trp-XaaOR) followed by oxidation with dimethyldioxirane (DMDO).19 For example, the N-α-TrtL-tryptophanylglycine methyl ester 7 (Trt-Trp-Gly-OMe) oxidation yields a mixture of two Nb-Trt-3a-hydroxypyrrolo[2,3-b]indolylglycine methyl ester diastereoisomeres syn-cis Trt-  10 Hpi-Gly-OMe 8a and anti-cis Trt-Hpi-Gly-OMe 8b (Scheme 4). Stereoselectivity, however, is not of concern here because the stereocenter in position 8a of the pyrroloindole is lost upon the opening of the pyrrolyl cycle during the Savige-Fontana reaction. O O  H N  O CO2Me  N H  CO2Me NHTrt  OH  O  [O]  7  CO2Me HN O  NHTrt  NHTrt N H  H N  N  HO NTrt  N H  N H O H HO N H NTrt  CO2Me  syn-cis, 8a CO2Me  anti-cis, 8b  N H H  Scheme 4: Trt-Hpi-Gly-OMe 8a/b synthesis. One-pot oxidation of the indole double bond of tryptophan 7 followed by intra-molecular attack of the R-NH2 at the indole C-2 position.  May et al. found that the introduction of Hpi in the linear amatoxin precursor was facilitated when an amino acid was used as a protecting group for the carboxyl end of Trp 7 (Hpi precursor) avoiding extensive protecting group manipulations. The trityl lowers the risk of Trp enantiomerization during the coupling to the methyl ester protected amino acid.20 Additionally, the trityl is stable during all reactions before the Savige-Fontana reaction. Several advantages are found in using Hpi: the motif is found in a variety of bioactive natural products and can serve as a template to build a library; it is chemoselectively attacked by cysteine and not by any of the other canonical amino acids; it requires minimal protecting group manipulations; and, it can be prepared from any Trt-TrpXaa-OR sequence. Moreover, the Trt-Hpi-Xaa-OR is stable enough to be purified, saponified, and incorporated into a peptide. 5.2. Previous syntheses of amanitin analogues Based on previous work by Wieland and Zanotti, L-Pro2-L-Ile3-S-deoxo-amaninamide 12 was obtained in our lab by conventional Fmoc/tert-Bu SPPS in the C-to-N terminal direction using the coupling reagent O-benzotriazole-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HBTU), the solvent N,N’-dimethylformamide (DMF) and the base N,N-diisopropylethylamine (DIPEA or DIEA) (Scheme 5). The carboxy-terminus was  11 anchored to an acid labile linker for solid phase synthesis. The linear peptide synthesis ended with compound 10 following incorporation of the Nb-Trt-3a-hydroxypyrrolo [2,3-b] indolylglycine (Trt-Hpi-Gly-OH) 9a/b. A first cyclization was then achieved by the Savige-Fontana reaction simultaneous to the release of product from the solid support. A second cyclization was done in a “ziplike” manner by head-to-tail macrolactamization with the coupling reagent (benzotriazol-1-yloxy)tripyrrolidophosphonium hexafluorophosphate (PyBOP) in solution to produce the bicyclic 12 (Scheme 5).21  a  Ile O Fmoc  HN  CO2H H2N  HN H N Trt  O  b  N  11  O S H N O O  O  H N O  N H  10  H N  O N H  O  O N H  O HN  N OO H  H N  HN  c  O O N  NH  N H  H2N  Ph Ph S H N  Ph  OH  O S H N OO  H2N  O  O N H  O N  O HN Ph Ph Ph O  N H  O O  HN  N OO H N H  O  NH  12  Scheme 5: Synthesis of L-Pro2-L-Ile3-S-deoxo-amaninamide 12. The Ile is substituted for DihyIle: a) standard C-to-N direction Fmoc/tert-Bu SPPS with HBTU; b) tryptathionylation by Savige-Fontana reaction in TFA with simultaneous product release from the solid support; c) macrolactamization in solution using PyBOP, DIEA in DMF.  Head-to-tail macrolactamization as outline in scheme 6, is generally a slow reaction, thus allowing time for side reactions such as enantiomerization at the activated C-terminal residue, hydrolysis of the activated carboxyl, guanylation of the free amine or dimerization.22 Various strategies have been used to reduce these side reactions including, the use of the oxybenzotriazole (OBt)-phosphonium salt coupling reagents to avoid guanylation of free amine and to lower the risk of enantiomerization. Dimerization is usually suppressed under high dilution conditions (10-3 to 10-4 M) in solution or in a low loading resin in solid phase.  12  O H2N  Ri  a OP  Rn+1  O H2N R1  Ri N H  H N O n  O OP Rn+1  b  O R1  HN N H  O n NH Rn+1 O  Scheme 6: Head-to-tail macrolactamization. a) during peptide synthesis, the carboxyl terminus is protected or anchored to a linker on solid support (P); b) the carboxyl terminus is deprotected or released from the solid support during or before macrolactamization.  6. Linkers – a brief review Contrary to the previous amaninamide 12 synthesis in solution, in order to build the library, the α-amanitin derivative should stay anchored by the linker on the solid support during all reactions (Scheme 7). We, therefore, sought a linker stable during the synthesis of the complete α-amanitin derivative. The synthesis includes the Fmoc/tert-Bu SPPS involving strong bases such as piperidine and electrophiles such as coupling reagents, the Savige-Fontana reaction involving strong acid (TFA) and macrolactamization. Additional considerations for linker choice must also be taken in account. In order to not damage the library peptides, the linker must be cleaved and release, in quantitative yield, the product under mild conditions. Moreover, the linker should bring the reaction sites further out from the resin polymer, improve the swelling proprieties of the resin, lower non-specific interactions and disfavour product aggregation. In the present section, to put in context our linker choice for the amatoxin synthesis, we briefly introduce common linkers used in SPPS.  13  NHFmoc Y  R1  OH  a  CO2t-Bu  13 O R5  b  HN R6  R4 N H O N H  H N O  O  O  R3 N H  S  O  O  O  H N R2  O  H N  HN H N Trt  R6  CO2H Y N R1 H  R5 N H  TrtS O  H N O  R4  N H  14  O  R1  c  HN  NH2  15  16  HN R3  R2 N H  R3  R2 NH  O  H N  Y H N  O O S O N H  O  H N O  R1  Y CO2t-Bu  O  R6 N H O  N H O HN N H  NH R5 O  R4  Scheme 7: Synthesis of amatoxin derivatives on solid phase. a) from the functional group (Y) of the linker on solid support 13, Fmoc/tert-Bu SPPS achieves the linear octapeptide 14 with various protected amino acids (Ri = side chain); b) after completion of the octapeptide 14, a first cyclization occurs in strong acid by the Savige-Fontana reaction to achieve 15; c) macrolactamization of the monocyclic octapeptide 15 with an appropriate coupling agent leads to the bicycle 16.  6.1. Linker classification Numerous linker families have been developed and are commonly classified according to the functional group left on the product such as the traditional (classical) linker, the traceless linker, the diversity (multifunctional) linker and the safety-catch linker (Scheme 8).23  14  a)  b) c)  d)  L  X  P  L  X  P  L  X  P  L  X  P  cleavage  X  cleavage  P  P  cleavage Yi  Yi  Aj  Aj Yi  P  P  cleavage  activation L*  X  P  N  P  Scheme 8: Linkers classification. a) in the traditional linker (L), a part of the linker (X) is kept following release of the product (P); b) in the traceless linker (L), the product (P) is anchored by a functional group (X) that, following cleavage, the product is recovered without trace of the linker; c) in the diversity linker (L), a variety of molecules (Yi) are added to the product during the cleavage, or one type of functional group (Y) is created during the cleavage and can be used to link a diversity of molecules in solution (Aj); d) the safety catch linkers (L) can be included in any previous linker family, and is characterized by the requirement of linker activation (L*) by a chemical or physical process before being cleaved, for example, by a nucleophile (N).  6.1.1. Traditional (classical) linker In traditional linkers, the linker part or the functional group that anchors the product to the linker is kept on the product following its release (Scheme 8a). For example, many classical linkers, such as the Wang linker 17a, the Sasrin linker 17b, the Rink linker 18 and the trityl chloride linker 19, take advantage of a benzylic position (Figure 3). These linkers can release alcohols, carboxylic acids, amides and amines by acidolysis. Benzyl ester-based linkers, such as linker 17a, can also be cleaved by hydroxide, alkoxide or amine nucleophilic attack.  15  OMe X2  X  OMe Y  O  17a/b  X1  Y  Y  O  18  19  Figure 3: Traditional (classical) linkers. In the following linkers, R can represent an alkyl chain, a peptide or an aromatic system and the leaving group can be a carboxylic acid (Y = OCOR), an amide (Y = NHCOR), an alcohol (Y = OR) or an amine (Y = NHR) each cleaved by protic acids. 17a: Wang linker (X = H); 17b: Sasrin (X = OMe) linkers; 18: Rink linker; 19: Trityl chloride linker where linker acidolysis efficiency is similar for X1, X2 = H and X1 = H, X2 = Cl but augments in the following order: X1 = Cl, X2 = H; X1, X2 = H; X1 = H, X2 = Me, X1 = H, X2 = OMe when compared with the same leaving group Y.  With classical linkers, the polar group left on the cleaved peptide may modify the activity of the product in biological assays or interfere with the solution phase synthesis at a later step. However, some functional groups, such as the C-terminal carboxamide or the Nalkyl amide left on the product following cleavage, are useful in peptide studies. The Cterminal carboxamide or the N-alkyl amide peptides bring better peptide metabolic stability with higher peptide hydrophobicity, thereby improving the bioactivity and bioavailability of the released peptide. 6.1.2. Traceless linker Originally linkers were included in the traceless family when, after cleavage, the product was recovered without trace of the linker. The family has been extended, however, to include linkers that, upon cleavage, leave aliphatic hydrogen atom, aromatic hydrogen atom or inert residues on the product (Scheme 8b).24 The traceless linker is often made of a labile bond composed of an aryl carbon and an heteroatom that can be cleaved by electrophilic substitution or electrophilic addition as outlined in section 6.2 and scheme 9a. Other cleavage methods have been used such as β-elimination to release peptides made by Boc-SPPS on sulfone linkers (Schemes 9b).25  16 P  a)  L O  b)  N H  X O S O  H  O O  P H  NHBoc P  O  NaOH  HO  NHBoc P  Scheme 9: Traceless linkers. a) the common arene-based traceless linker (L). The product (P) starts with an arene anchored to an heteroatom (i.e. X = B, Si, Ge, N=N, Se, OSO2) that is part of the linker. The linker is substituted for an hydrogen by protogenolytic (H+), hydrogenolytic (H-) or hydridolytic (H•) reaction depending on the arene and X structures; b) the sulfone-based-linker releasing the peptide (P) by βelimination.  6.1.3. Diversity (multifunctional) linker With the development of peptide combinatorial chemistry, a need for linkers with greater functional possibilities led to the development of the diversity or multifunctional linkers (Scheme 8c).26 These linkers can be cleaved by a molecule carrying various functional groups which will stay on the product. Linkers, which upon cleavage leave a reactive group on the product allowing incorporation of diverse substituents to the product, also belong to the diversity linker family as outlined with the tartrate-based linker in section 7.2. Diversity linkers find success in peptide synthesis as they allow the addition of several C-terminal functionalities. They can also be used in peptide cyclization as intermediates for ligation to other peptides, and to develop a combinatorial library with a degenerate C-terminus. 6.1.4. Safety catch linker Before cleavage from the resin in what is usually a late if not the final reaction, the safety catch linkers must be converted to a particular intermediate form (Scheme 8d). Safety catch linkers allow for more flexibility in synthetic methodology as they are stable to a wide range of reactants. One of the earliest safety catch linkers contains a thioether group 20 that, following oxidation to a sulfone 21, releases the carboxylic group upon attack of a suitable nucleophile such as hydroxyl and alkoxides (Scheme 10a). The sulphonamide safety catch linker (Kenner linker) 22, however, with the amide functionality  17 is more stable to acid and base than the thioether (Scheme 10b). Following amide methylation resulting in the linker (Y = CH3 or CH2CN) 23, the product is released from the resin upon attack of a nucleophile.27 The hydrazide (Wieland) linker 24 is frequently used in SPPS and is activated for nucleophilic displacement upon oxidation of the hydrazide to acyl diazene 25 by N-Bromosuccinimide (NBS) (Scheme 10c). O  a)  R  O  [O]  O  S  O S O  20 R  b)  O  O NH S O  R  CH2N2 or ICH2CN  R  O Nu  21 O  NuH  R  O Nu  23 O  O  [O]  O  24  NuH  O  O N S Y O  22  c)  R  N H  H N  R O  NuH  O  25  N  N  R  R  O N 2 Nu  O  Scheme 10: Safety catch linkers. Blue and red represent the leaving groups following cleavage, Nu represents suitable nucleophiles such as hydroxide, alkoxides, amines or thiols and R can be represented by an alkyl chain, an aromatic system or a peptide. a) the thioether linker 20; b) the sulphonamide safety catch linker (Kenner linker) 22 where Y = CH3 or CH2CN; c) the hydrazide linker (Wieland linker) 24.  6.2. Cleavage methods Many types of cleavage methods are possible and, therefore, linker stability can be tailored to suit the particular synthesis conditions.28 Herein, we will introduce the common methods that can be applied to SPPS synthesis of α-amanitin derivatives and libraries of similar compounds. Metal-assisted cleavage methods are not discussed as any trace metals left in the soluble product may lead to artefacts in subsequent SAR experiments. Similarly, because reductive linker cleavage is frequently assisted with metals or can proceed by disulfide bond reduction or desulfurization, this linker type is not included here.  18 6.2.1. Electophilic With electrophilic cleavage, the electrophile (E) is commonly a proton from HF, HBr/TFA or TFA acting on an electron rich system, such as an aromatic system connected to electron donating groups (Scheme 11). This linker type is often used with a good leaving group such as a carboxylic acid, carbamate or β-keto ester. The latter two can further release CO2 to generate an amine or a ketone respectively.  Y A  P  E  E Y  P  B  Scheme 11: Electrophilic linker cleavage. Symbol A represents a methylene group or an electron withdrawing group, B represents an hydrogen, a phenyl, or a methylene silyl, Y represents the protonatable site allowing the product (P) to leave the solid support under electrophilic attack (E+).  Sensitivity to electrophilic cleavage can be fine-tuned by the addition of steric groups, as well as electron donating or withdrawing groups to the pro-carbocation position on the linker. For example, to achieve greater linker stability during Boc deprotection in SPPS, the 4-hydroxymethyl-phenylacetamidomethyl (PAM) 26 linker was developed (Figure 4). The electron withdrawing group (EWG) in the position para to the labile leaving group bond stabilized the linker toward acidolysis (Figure 4). The silyl amide linker (SAL) 27, the benzhydrylamine (BHA) linker 28a and 4-methylbenzhydrylamine (MBHA) linker 28b, each carry an electron donating group (EDG) at the benzylic position (Figure 4). The linker 27 is stable to nucleophiles and is commonly used in Fmoc SPPS.29 The linkers 28a/b are stable during Boc deprotection in SPPS, and allow the release of poorer leaving groups, such as carboxamide, by HF acidolysis (Figure 4).30 In addition, for good leaving groups, these linkers are cleaved under much milder acid conditions.  19 O O  O  R  N H  26  O O  N H  27  H N  A Me3Si  R O  O  28a/b  N H  R  Figure 4: Electrophile-labile linkers. In blue the leaving group following electrophilic cleavage and R can be represented by an alkyl chain, an aromatic system or a peptide. In PAM linker 26, an acetamide acts as an EWG para to the labile bond of the leaving group; in SAL 27, the methylene trialkylsilyl acts as EDG; in BHA 28a and MBHA linkers 28b (A = H, Me respectively), the aromatic system acts as an EDG.  6.2.2. Nucleophilic Generally, linkers cleaved by nucleophiles are the methodological mirror image of the electrophilic labile linkers and are commonly built on an electron deficient system. They were developed mainly for Boc SPPS. For example, the Wang resin 29 and linker 30 release peptides with a carboxylic acid, ester or amide C-terminus by the nucleophilic attack of hydroxides, alkoxides, or amines respectively (Figure 5). Other terminal functional groups, such as alcohols released by hydrolysis or aminolysis of linker 31 are accessible (Figure 5). The linker 32 is made from the Wang resin 29 and releases amines by hydrolysis followed by decarboxylation (Figure 5).  20  O  O HN  O R  29  O  30  O  O R O  OR  O  31  32  NHR  Figure 5: Nucleophile-labile linkers. The leaving group expelled following nucleophilic attack is highlighted in blue and R can be represented by an alkyl chain, an aromatic system or a peptide. The Wang resin 29; the glycolamidic ester linker 30; the carboxyl-derivative linker 31; the carbamate linker 32.  6.2.3. Photolytic Photolytic linkers are commonly stable to Boc/Bzl SPPS, Fmoc/tert-Bu SPPS and are slowly cleaved in mild conditions with a wavelength of 350 nm or higher.31 For example, the o-nitrobenzyl (ONB) 33a is found in several photolabile linkers in which the product is usually bonded to the nitrobenzyl by a carboxylic acid, carboxamide or alcohol (Figure 6). The ONB linkers have been up-graded to many derivates such as the 3nitrobenzamidobenzyl  (Nbb)  linker  33b  and  the  3-amino-3-(2'-nitrophenyl)-2,2-  dimethylpropionic acid (ANP) linker 34 (Figure 6). The Nbb linker 33b is stable during the Boc/Bzl SPPS and the peptide is released in better yield compared to the ONB linker 33a by photolysis in trifluoroethanol/CH2Cl2.32 The ANP linkers, such as 34, were shown to be stable to acid and base.33 O NO2 P  R  N H  H N A  O HN  O2N O  33a/b  34  Figure 6: Photo-labile linkers. ONB linker 33a (A = H) and Nbb linker 33b (A = Ph) in which the product (P = OR, OCOR, NHCOR) is released under light (350 nm) exposure; ANP linker 34 allow the release of carboxamide. R can be represented by an alkyl chain or a peptide.  21 6.2.4. Oxidative Oxidative cleavage of linkers can be achieved by treatment of the p-methoxybenzyl (PMB) linker 35 and the acid-stable p-acylaminobenzyl (PAAMB) linker 36 with 2,3dichloro-5,6-dicyanobenzoquinone (DDQ) (Figure 7).34 The PMB linker 35 prepared on the Merrifield resin is the equivalent of the Wang resin. The PMB linker 35 oxidation is used to release alcohols or amines as an alternative to acidolysis in TFA that was found to produce a significant amount of trifluoroacetate esters as by-product during the release of alcohols. Following DDQ treatment, the eluate from the resin containing the product is poured on a mixed-bed ion-exchange scavenger resin to trap the by-product 2,3-dichloro5,6-dicyano-1,4-hydroquinone (DDQH2). Alkene-based linkers such as the linker 37 have been designed to release a C-terminal aldehyde peptide by ozonolysis but peptide epimerization at the carbon next to the aldehyde is common during purification (Figure 7).35 H N O  O  35  H N  XR  O  36  H N  XR  37  R1  R2 O  Figure 7: Oxidant-labile linkers. The product expelled following oxidation and the linker are highlighted in blue and red respectively. R can be represented by an alkyl chain or a peptide. PMB linker 35 (X = NH or O) on Merrifield resin and PAAMB linker 36 (X = O) on ArgoPore resin release an amine (X = NH) or an alcohol (X = O) following DDQ treatment; alkene-based linker 37 on polystyrene resin releases an aldehyde (RCHO) by ozonolysis.  7. Promising acid stable linkers 7.1. Silicon-based linkers Silicon bearing linkers are compatible with a wide range of organic reactions and have been used successfully in Fmoc/tert-Bu SPPS.36 Furthermore, Chenera et al. reported a benzonitrile silyl-based linker stable in TFA, and cleanly cleaved with mild fluoride sources.37 The possibility of using mild fluoride sources is interesting, as it lowers the risk  22 of many side reactions such as aspartimide formation, loss of the N-Fmoc protecting group and peptide epimerization. The stability of Chenera’s linker to TFA as well as to bases convinced us to take advantage of this linker to achieve the synthesis of amatoxins. To put the TFA resistant Chenera’s linker in context, we will briefly discuss common siliconbased linkers. Silicon-based linkers are commonly used as traditional linkers in Fmoc SPPS. For example, the SAL 27 and α-trimethylsilylbenzyl (SAC) linker 39 are often used to release C-terminus carboxamide and carboxylic acid peptides respectively (Scheme 12). SAL 27 is better cleaved by protic acids compared to fluoride sources (Chapter 1, section 6.2.1). SAC linker 39, by releasing a carboxylic acid that is a good leaving group, is cleaved by mild sources of protic acids or fluoride.38 To improve the silicon linker compatibility with Boc/Bzl SPPS, the (2-phenyl-2-trimethylsiyl) ethyl linker (PTMSEL) 40 was made with the leaving group connected away from the linker benzylic position (Scheme 12). This linker 40 was stable during Boc SPPS and Fmoc SPPS while being cleaved with mild fluoride sources such as hydrated tetra-n-butylammonium fluoride (TBAF•3H2O) in CH2Cl2.39 The use of TBAF•3H2O is advantageous because, despite being a strong base in polar aprotic solvents (DMF, THF or NMP), it is a milder base in CH2Cl2. O HN O NH  O  27  R  F-  O R SiMe3  NH  O  O  O H2N  NH  Me3Si  O O  TFA  R  O  O O  NH  CF3CO2SiMe3  SiFMe3 R  39 O NH  O  40  SiMe3 O  FR  O NH  O  O O  SiFMe3 R  O  Scheme 12: Silicon bearing linkers used in peptide synthesis. The linkers SAL 27, SAC linker 39 and PTMSEL 40 represent silicon-based linkers that are frequently used in peptide (R) synthesis. The product expelled following linker cleavage and the soluble silicon by-product are highlighted in blue and red respectively.  23  In contrast with several electrophile labile linkers (Chapter 1, section 6.2.1), the silicon bearing linkers SAL 27, SAC 39 and PTMSEL 40 do not produce an electrophilic carbocation which is left on the resin following linker cleavage. A reactive carbocation could trap nucleophilic functionalities, such as tryptophan residue, included in the leaving group sequence. Instead, the by-product left on the resin is a styrene molecule that is relatively stable during the cleavage of linkers.40 When fluoride is used, the silicon-based linkers are generally cleaved via an E2 type pathway by the fluoride bonding to the silicon in concert with the departure of the leaving group. Under strong acid, however, the electron donating behaviour of the σSiC bond to the β-carbon (β-effect) allows the protonated leaving group connected to the β-carbon to be released by an E1 type process. Thus, silicon-based linkers such as SAL 27, SAC 39 and PTMSEL 40 are likely to be cleaved during the Savige-Fontana reaction, which involves exposure to neat TFA for five hours. For this reason, we did not use these types of silicon-based linkers to complete the synthesis of amatoxins on solid phase. In another type of silicon bearing linker, the silicon is connected directly to an aromatic system. For example, a silyl linker such as the aryl silyl 41 has been used as an acid labile and traceless linker (Scheme 13). When exposed to an electrophile (E+), the aryl silyl 41 undergoes an irreversible regioselective ipso-substitution, called protodesilylation, if the electrophile is a proton. The ipso-substitution is a particular electrophilic aromatic substitution where the electrophile and the silicon share the same ring position in the carbocation intermediate. The first step in the reaction involves the electrophile (E+) attack by the aromatic system, generating a resonance stabilized arenium ion (Wheland intermediate). In the second step, re-aromatization occurs where the weakened σSiC bond is released in the aromatic system during the nucleophilic attack on the silicon (Scheme 13). With the aryl silyl-based linker 41, the nucleophile is generally the electrophile counter ion and not a nucleophilic group found on the released product (P) (Scheme 13). This type of linker is frequently used to introduce bromide and iodide in the aromatic tail of the product during the linker cleavage using Br2 and ICl respectively.41  24  G  R2 R1 Si  +  P  41  E  G  R2 R1 Si E  P  42  Nu  -  G  R2 R1 Si Nu E  43  P  44  Scheme 13: Electrophilic substitution of an aryl silyl 41. The strength of the electrophile needed for the substitution and rate of the reaction can be significantly increased and decreased respectively by using bulky alkyl groups (R1 and R2) on 41. G, P, E+ and Nu- represent an electron withdrawing group or donating group, the product prepared on the linker, the electrophile and the nucleophile respectively. If G = EWG, then the reaction is not favoured and can be slowed down significantly, inversely if G = EDG.  The silicon connected directly to the aromatic system allows control on the acid stability of the silicon-carbon bond. For instance, when an EWG (G) is found meta to the silicon substituent, then the σSiC bond becomes stronger toward electrophiles. The EWG decreases the electronic density at the β-carbon which destabilizes the carbocation intermediate 42 (Scheme 13). Therefore, several strategies have been used to increase the acid stability of the linker carbon-silicon bond. For example, the 1,4-benzodiazepine silicon linker 45 was developed for a library and was stable in 85% TFA (Figure 8). In TFA, the benzodiazepine of linker 45 was protonated turning into an EWG disfavouring the protodesilylation.42 The benzonitrile silyl linker 46 prepared by Chenera et al. was stable to neat TFA, but was mainly designed to improve the electrophilicity of the silicon, allowing the use of milder fluoride sources (Figure 8). A nitrile positioned on the phenyl ring meta to the silicon resulted in the acid stability of linker 46. A similar search led to the 4dimethylsilyl-N-Boc-3-amino-2-chloropyridine linker 47 where only the Boc protecting group was cleaved with 85% TFA (Figure 8).43 Subsequently, a pyridyl propionate system, carrying a beta-silyl group outside the ring 48, was found to be stable in HCl (aq), NaOH (aq), TFA and during Fmoc/tert-Bu SPPS, but was readily cleaved with TBAF (Figure 8).44 Thus, depending on the acid stability of the linker, the acids used for protodesilylation are TFA, HF or HCl while the sources of nucleophilic fluoride are usually CsF or TBAF.45  25  R3 N O  Si  45  R1  N  R2  HN  CN Si  O Cl  Si  R  R1 Ph  Si  N  46  47  OR N  48  Figure 8: Acid stable silicon-based linkers. The synthesis is done from the R position, and the linker is cleaved by fluorodesilylation with the leaving groups coloured in blue and red. R can be represented by an alkyl chain, an aromatic system or a peptide depending on the linker type.  7.2. Tartrate-based linkers In addition to the traceless benzonitrile silicon linker 46, a multifunctional linker 49 developed by Melnyk and Grass-Masse also caught our interest (Scheme 14). The linker 49 was based on a tartrate acetonide unit which was stable during standard Fmoc/tert-Bu SPPS and during deprotection of the tartrate diol with 95% TFA in CH2Cl2.46 This deprotected diol linker was cleaved using mild periodate oxidation. The peptide product was successfully recovered in the eluate even when the peptide contained cysteine and methionine residues. Additionally, a polyethyleneglycol (PEG) unit in the linker was added to contribute to peptide solubility and to lower nonspecific interactions. Moreover, an amide bonded the linker to the resin and the product to the linker, giving more strength to the linker compared to a benzylic ester-based linker. Further functionality could also be introduced to the cleaved product containing a glyoxamide 53 through the use of an hydrazino or aminoxy group to yield the soluble product 54 (Scheme 14).  26  H N  49  a  O O  O O  c  H N  51  O  53  O O  N H  b  O  O  N H  Spacer NH  d  H N  Peptide NHR Spacer NH  O  N H  OH O  R'Y N  g  OH  O  52  Spacer NH  O  Bead Sceening  Peptide NHR  Peptide NHR  O  N H  O  O  O  O O  50  Spacer NH2  N H  O  f  H N  N H  O Peptide NHR Spacer NH  O Peptide NHR Spacer NH  54  Scheme 14: General hydrophilic tartrate-based linker 49. a) Fmoc/tert-Bu SPPS on the linker where R = H or any protecting group or other functionalities; b) beads screening; c) isolation of hits; d) linker deprotection (TFA); f) linker cleavage (HIO4); g) probe addition where Y = N or O and R' = dye or isotope label.  Peptide aldehydes have been prepared by several methods and they are interesting compounds due to the inhibitory properties exhibited toward many classes of proteases.47 In particular, the tartrate linker was used to build a library of protease resistant N-alkylsubstituted glycine peptoid 55 (Figure 9).48 Following periodate cleavage, the product Cterminal aldehyde was linked to a brominated tag yielding 55 that was identified by its characteristic isotope pattern in subsequent mass spectral analysis (Figure 9). This analytical method could be applied to sequence the products from our future library of αamanitin analogues.  Ri N  HN  O N  O OMe  H N O  n  O O  O  55  N H  N  H N  O  Br  O  OMe  Figure 9: Tartrate-based linker. Peptoid (blue) with its C-terminus linked to a hydrophilic spacer (red) where the glyoxamide end have been anchored to an aminoxy isotopic label (green).  27 The stability of tartate linkers in Fmoc/tert-Bu SPPS and TFA and their wide range of application convinced us to incorporate into our methodology a linker analogue to the tartrate linker 49 toward the synthesis of α-amanitin derivatives. 7.3. Anchorage strategies of the first amino acid on the linker Side chain anchorage is a common strategy for head-to-tail macrolactamization on solid support. In this strategy, the product is pulled further from the resin, a technique which favours head-to-tail macrolactamization and the access to soluble targets in library screening. The chosen amino acid to anchor often bears a side chain functionality as is found in Asp, Glu, Asn, glutamine (Gln), lysine (Lys), (2S,4)-diaminobutyric acid (Dab), serine (Ser) or tyrosine (Tyr). Unfortunately, it also limits the chemical space to be explored with the library because one side chain function has already been chosen for anchorage. In our application, side chain anchorage to the linker should have less impact on the amanitin library if a natural amino acid is substituted for DihyIle and used as the anchorage point. The co-crystal structure of RNA pol II with amanitin does not suggest any specific interaction of DihyIle with RNA pol II. As introduced earlier (Chapter 1, section 2), the DihyIle has been substituted to isoleucine with a minimum loss of amatoxin inhibitory activity. Since the DihyIle is not an amino acid naturally encoded by the genome, is not commercially available, and is not easily synthesized in good yield and purity, we proposed its substitution to Glu. The side chain of Glu is not sterically hindered, should contribute to pull out the peptide from the solid support and should therefore not interfere with the natural proprieties of α-amanitin derivatives. Furthermore, the carboxylic side chain permits the anchoring by a stable amide bond to a linker with an amino terminal. 7.4. Proposed methodology As a proof of concept, in the present work we would like to develop a methodology for the synthesis by standard Fmoc/tert-Bu SPPS of Pro2-Glu3-S-deoxo-amaninamide 59 anchored by the Glu side chain to an orthogonal linker 56 (Scheme 15). Pro2-Ile3-S-deoxoamaninamide 12 that is a close analogue of compound 59 had been previously synthesized  28 in our lab and displayed a lower level of toxicity compared to many α-amanitin analogues (Chapter 1, section 5). A similar method will be applied to the synthesis of a library of αamanitin derivatives on solid phase. The library will later be screened in an in vitro RNA pol II binding assay with fluorescent nucleotides followed by an activity assay.49 In our preparation, the α-carboxylic acid of Glu will be protected as a tert-butyl ester. The side chains of Asn and Cys and the secondary amine of Hpi will be protected with the acid labile trityl (Trt) protecting group. The Trt-Hpi-Gly-OH 9a/b will be prepared in solution and coupled to the amino terminus of the linear hexapeptide precursor on solid phase (Chapter 1, section 5). Then the tryptathionine (indole-2-thioether) will be installed on the solid support according to the Savige-Fontana cyclization. Finally, completion of Pro2Glu3-S-deoxo-amaninamide will be achieved by a head-to-tail macrolactamization on beads. Ph  H2N  CO2t-Bu  OH  a  HN H N Trt  O  56  NH Y  Ph H N  O N H  O  H N  O N H  O  57 O  b  O  N HN  OO  O O N H  H2N  S  58  HN  O  N OH N H NH2  O N  O Ph Ph Ph H N Y  HN  c  O  H N  O  O  CO2t-Bu N H  H N  Y  O  HN  Y NH  HO  Ph S H N  O H N  O O O S N H N  NH O  59  OO  O N H  O HN  O O  N H  NH  N H  H2N  Scheme 15: Proposed synthesis of L-Pro2-L-Glu3-S-deoxo-amaninamide 59. The side chain of Glu is used as anchor site to the linker (Y) and as a substitute for the DihyIle. a) standard C-to-N direction Fmoc/tert-Bu SPPS; b) tryptathionylation by Savige-Fontana reaction in TFA; c) macrolactamization using coupling agents as benzotriazole-based phosphonium salt.  29  Chapter 2 Results and discussion  1. The benzonitrile silyl linker: synthesis and stability test Our long-range aim is the preparation of an amatoxin library anchored to a solid support by a linker. The linker needs to be stable during the Fmoc/tert-Bu SPPS of the amatoxin precursor and stable to the 5 hours of exposure to neat TFA required for the formation of the amatoxin tryptathionine bridge (indole-2-thioether). To achieve this goal, we first prepared the electron deficient benzonitrile silyl linker 60 based on Chenera’s paper (Figure 10). Chenera et al. claimed that the linker 60 was stable in neat TFA while the product could be released cleanly from the solid support with TBAF, a mild fluoride source (Chapter 1, section 7.1). O O  N H Si O  N  60  O  O  H N  O  S N H  Figure 10: Acid stable benzonitrile silyl-based linker 60. Shown in blue and red are the linker parts that are cleaved off the resin along with the amatoxin (shown in pink). The green and blue sections represent the key linker parts prepared in solution as adapted from figure 8, linker 46.  30 1.1. Synthesis of benzonitrile silyl unit in solution The first step of the benzonitrile synthesis involved the conversion of 2,5dibromobenzoic acid to the corresponding amide 61 by treatment with oxalyl chloride followed by addition of ammonium hydroxide (Scheme 16). The benzonitrile derivative 62 was  obtained  in  good  yield  by  dehydration  of  the  amide  61  using  chloromethylene(dimethyl)ammonium chloride prepared in situ with DMF and oxalyl chloride in anhydrous acetonitrile (MeCN). The benzonitrile derivative 62 was then converted to the silylated benzonitrile derivative 65 by a metal-halogen exchange followed by trapping the transient lithiated compound 63 with the silyl 64. While the reaction was reported by Chenera et al. (Chapter 1, section 7.1), we did not isolate the intermediate 63. The metal-halogen exchange reaction involved the addition of n-butyllithium (n-BuLi) in one equivalent at -105oC to avoid nucleophilic attack on the nitrile 62 and to lower polymerization induced by the lithiated intermediate 63.  Br  CO2H  a, b  Br  CONH2  c, d  Br  CN  Br  Br  61  e  Li  CN Br  Br  62  63  f Si Cl  Br  Si  CN  Br  64  Br  65  Scheme 16: Synthesis of benzonitrile silyl 65. Reagents and conditions: a) oxalyl chloride, DMF(cat), CH2Cl2, 4 °C to reflux, 4 h; b) NH4OH, EtOAc, 4 oC, 30 min, 78%; c) -15 °C, oxalyl chloride, DMF, MeCN, 10 min; d) pyridine, 0 °C, 45 min, 74%; e) n-BuLi, THF, -105oC, 40 min; f) silyl 64, 2 min at -105oC, -78oC for 45 min, 44%.  1.2. Benzonitrile silyl linker and spacers loading on solid phase In the design of the solid phase linker, a hydroquinone moiety was chosen as a spacer as reported by Chenera et al. (Chapter 1, section 7.1). The hydroquinone was first monoacetylated in acid yielding the acetate 66 (Scheme 17). The protected hydroquinone 66 was anchored to the Merrifield chlorobenzyl via an SN2 reaction in the presence of carbonate followed by deprotection with lithium aluminium hydride (LAH) to afford the hydroquinone 68 (Scheme 17). The presence of the monoacetylated hydroquinone on the  31 resin and its successful deprotection were detected by the presence and disappearance respectively of the carbonyl peak (1760 cm-1) on an FT-IR spectrum.  HO  OH  a  HO  OAc  O  b c  66  OR  67: R = Ac 68: R = H  Scheme 17: Synthesis of Merrifield resin hydroquinone spacer 68. Reagents and conditions: a) AcOH (glacial), 110oC, Ac2O, 3 h, 57%; b) Merrifield chloro resin, DMF, anhydrous K2CO3, 75oC, 2 days; c) THF, 4oC, LAH, 16 h.  In order to couple the benzonitrile silyl 65 on the resin 68 by means of a Mitsunobu reaction, the bromine of benzonitrile silyl 65 needed to be substituted for hydroxyl. This was achieved by an SN2 reaction with the mild nucleophile acetate yielding the acetyl 69 which was converted to the alcohol 70 by acidolysis (Scheme 18). The high yield of the acetyl deprotection suggested that the benzonitrile silyl moiety was stable at low pH.  Br  Si  CN  a  RO  Si  Br  Br  65  CN  b  69: R = Ac 70: R = H  Scheme 18: Synthesis of benzonitrile silyl 70. Reagents and conditions: a) DMF, NaOAc, 55oC 18 h, 81%; b) MeOH, 3N HCl, 0oC to RT, 13 h, 89%.  With the hydroxyl functionality built in, the benzonitrile silyl 70 was loaded on the hydroquinone spacer 68 by a Mitsunobu reaction to yield the resin 71 (Scheme 19). We decided to use the tertiary amine N-methylmorpholine (NMM) in the Mitsunobu reaction according to Chenera et al. (Chapter 1, section 7.1). Richter et al. reported a good yield when using the solvent NMM in the Mitsunobu ether formation of polymer-supported alcohols with soluble phenols.50 They suggested that the weak base NMM (pKa ~ 7.4) enhanced yields by facilitating the deprotonation of the pronucleophile, such as hydroquinone (pKa ~ 11). Lower yields, however, were found when using the solvent  32 NMM in Mitsunobu ether formation of polymer-supported phenols with soluble alcohols.51 On the FT-IR spectrum (KBr pellet) of resin 71, a nitrile peak at 2225 cm-1 confirmed the success of the Mitsunobu reaction outline in scheme 19 and figure 11. A carboxyl peak, however, also appeared at 1735 cm-1 (Figure 11), although its presence may arise from residual phosphine oxide, hydrazinedicarboxylate or by-product 71’ trapped in the resin. The by-product 71’ was reported to form in significant amount when the alcohol was first activated in solution with diisopropylazodicarboxylate (DIAD) and triphenylphosphine (TPP).52 CN  a O  Br i-PrO2C  Si O  OH  O i-PrO2C  68  N Si NH  71’  71  CN Br  Scheme 19: Synthesis of the benzonitrile silyl-based linker 71 on resin. Reagents and conditions: a) benzonitrile silyl 70 activated with NMM, Ph3P, DIAD, 30 min at 4oC, addition of resin 68 and 35oC, 4 days.  Cl  A  CN Si O  Br  B  O  71 1802  1871  1944  2225  1735  Figure 11: FT-IR spectra of the Merrifield resin (A) and the resin 71 (B). The nitrile absorbance appeared at 2225 cm-1.  33 Lastly, the benzoic acid moiety was appended to the resin 71 through a Suzuki coupling of the corresponding boronic acid derivative (Scheme 20). The integrity of the nitrile on linker 72 was confirmed by an FT-IR spectrum. CN Si O  CN  a  Br  Si O  O  71  CO2H  O  72  Scheme 20: Synthesis of the benzonitrile silyl-based linker 72 on resin. Reagents and conditions: a) toluene, 4-boronobenzoic acid, Na2CO3(aq), EtOH, Pd(PPh3)4, 90oC, 18 h.  1.3. Synthesis of fluorescent benzonitrile silyl linker on solid phase In order to verify the linker stability prior to full amatoxin synthesis, we prepared a fluorescent benzonitrile silyl linker 77 (Figure 12). The 2,2’-(ethylenedioxy)-bisethylamine was used as a spacer due to its water-solubility, biocompatibility and its commercial availability. Furthermore, polar spacers are known to lower peptide aggregation and to facilitate the diffusion of polar soluble reagents during peptide synthesis. In addition, they lower the risk of non-specific binding during biological library screening. CN O Si O  O  O  NEt2  HN  77  O  HN  O O  O  Figure 12: The fluorescent benzonitrile silyl-based linker 77. Shown in red is the linker part that is cleaved off the resin along with the coumarin derivative in blue.  Due to its chemical robustness toward a wide range of reactants and its enhanced solubility in numerous solvents, the 7-diethylamino-coumarin-3-carboxyl was chosen as a fluorescent probe. In addition, 7-diethylamino-coumarin-3-carboxylic acid 74 (Dac-OH)  34 and 7-diethylamino-coumarin-3-amide derivatives (Dac-NHR) have UV spectra with λmax ~ 407 nm and 413 nm respectively that are distinct from N-Fmoc protecting groups (λmax ~ 267 nm) UV spectra in 0.1M Tris-HCl buffer at pH = 9.53 In the first step, 7-diethylaminocoumarin-3-ethyl ester 73 (Dac-OEt) was afforded by Knoevenagel condensation of 4diethylaminosalicylaldehyde with diethylmalonate (Scheme 21). The ester 73 was saponified with aqueous sodium hydroxide affording the Dac-OH 74 after acidic crystallization in water (Figure 13). CHO EtO  OH  OEt O  O  O  a  OR  Et2N  NEt2  O  O  73: R = Et 74: R = H  b Scheme 21: Synthesis of Dac-OH 74.  Reagents and conditions: a) piperidine, EtOH, 1 h reflux followed by overnight RT, 59%; b) NaOH(aq), MeOH, 4oC to RT, overnight, 98%.  0.2 Dac-OH  Abs  Dac-OEt  0.15  0.1  0.05  0 220  270  320  λ (nm)  370  420  470  Figure 13: UV absorption spectra in MeCN of compounds 73 and 74. Spectra of Dac-OH 74 is depicted by the blue plot (λmax = 428) and Dac-OEt 73 by the red plot (λmax = 411).  Before coupling Dac-OH 74 to the spacer, the 2,2’-(ethylenedioxy)-bis-ethylamine needed to be monoprotected by treatment with di-tert-butyldicarbonate to achieve the NBoc protected spacer 75 (Scheme 22). The protected spacer 75 was then linked to Dac-OH 74  by  a  standard  1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide  hydrochloride  35 (EDC•HCl) coupling procedure. N-Hydroxybenzotriazole hydrate (HOBt•H2O) was added to the coupling mixture to generate a more stable activated ester intermediate and to avoid guanylation of the primary amine spacer. Finally, the Boc-protecting group was removed using TFA to afford the fluorescent amino spacer 76c in good yield with a UV spectrum similar to Dac-OEt 73.  H2N  O  O  NH2  a  H N  O  O  O  NH2  b  O  HN  O  O  NHR  O  75  O  Et2N  O  76a: R = Boc 76c: R = H  c  Scheme 22: Synthesis of fluorescent spacer 76c. Reagents and conditions: a) CHCl3, (tert-BuOCO)2O, 12 h, 4oC to RT, 97%; b) Dac-OH 74, EDC•HCl, HOBt•H2O, Et3N, DMF, 12 h, 4oC to RT, 67 %; c) TFA, CH2Cl2, 4oC to RT, 4 h, 90%.  The fluorescent probe 76c was loaded on the Merrifield resin 72 with a standard EDC•HCl/HOBt•H2O coupling procedure (Scheme 23). The integrity of the product 77 was confirmed by the absorption of the nitrile functionality in the FT-IR spectra. CN Si O  CO2H  O CN  72  O  a  Si O  77  O  O  NEt2  HN O  HN  O O  O  Scheme 23: Synthesis of fluorescent silicon-based linker 77 on resin. Reagents and conditions: a) DMF, HOBt•H2O, EDC•HCl, Et3N, fluorescent spacer 76c, RT, 16 h.  1.4. TFA test stability of the fluorescent silyl-based linker on solid phase The resin 77 carrying the fluorescent linker was treated with an excess of neat TFA for 5 hours at room temperature. The fluorescence under UV lamp was significantly reduced suggesting that the linker had degraded in these conditions. Likewise, the nitrile  36 absorbance band (2228-2223 cm-1) in the FT-IR spectra (KBr pellet) of resin 72 was slightly reduced relative to the resin absorbance band (1949 to 1727 cm-1) following exposure to an excess of TFA for 5 hours (Figure 14b). Surprisingly, the TFA exposed resin 77 was still weakly fluorescent following treatment with TBAF. We were expecting the fluoride from TBAF to cleave all silicon-based linkers from the resin. Similarly, when the TFA exposed resin 72 was further exposed to TBAF for 4 hours, the nitrile peak in the FT-IR spectra (KBr pellet) was still apparent (Figure 14c).  1869  1783  1950  1873  1942  2228  2228 1734  1803 1871  1944  2223  (b)  1735  1783  1727  (a)  (c)  Figure 14: FT-IR of dry resins before or after TFA exposure. a) resin 72; b) resin 72 exposed to TFA for 5 h; c) resin 72 exposed to TFA for 5 h followed by TBAF for 4 h.  Unfortunately, we were unable to purify and characterize the products found in the eluate of the TFA treated resins 72 and 77 after or prior the TBAF treatment. However, the results from the TFA test were suggestive of a lack of stability of the silicon linkers 72 and 77 to acidic media. The residual fluorescence following the resin 77 TFA treatments was likely due to nitrile by-products anchored or trapped in the resin without the silicon functionality. This inherent lack of stability in neat TFA could be attributed to an incorrect synthesis that led to the regioisomer 3-cyano-4-(bromomethyldimethylsilyl)-bromobenzene 65’ instead of 2-cyano-4-(bromomethyldimethylsilyl)-bromobenzene 65 (Figure 15a). Even though the 1H-NMR spectra of benzonitrile silyl 65 was identical to the reported spectrum  37 in the literature (Figure 15b), we expected the H-5 of the benzonitrile silyl 65 to be upfield to H-6 due to the electro-donating nature of the silicon atom. However, the H-5 chemical shift was found between H-3 and H-6. The H-5 was assigned to the doublet of doublets due to its typical scalar coupling constant (J) for the ortho (J3H-5;H-6 ~ 8) and meta (J4H-5;H-3 ~ 2) positions. 7'  7  a) 9  Si  1  4  8  Br  b)  2' 3' CN  3 2 CN  Br  Br  4'  1'  5 6  6'  65  65’  Br  8'  Si  9'  5'  H-6 or H-5’  H-3 or H-2’ H-5 or H-6’  Figure 15: The two proposed bromobenzonitrile silicon-based isomeres. a) structure of compounds 65 and 65’; b) the aromatic portion of the 1H-NMR spectra of compound 65 with the following chemical shift assignment: H-3 (d, 7.86-7.85), H-5 (dd, 7.76-7.73 ppm), H-6 (d, 7.53-7.51 ppm).  In a COSY experiment optimized for long-range coupling, the signal cross peak intensity corresponding to H-9 (s, 0.52 ppm) and H-6 (d, 7.53-7.51 ppm) increased with decreasing delay time (d6) (Figure 16). The opposite behaviour was observed for the cross peak signal of H-9 with H-5 (dd, 7.76-7.73 ppm). These results suggested a coupling constant J6H-6,H-9 value higher than J5H-5,H-9 in the structure of benzonitrile silyl 65 which is unusual. However, if the compound submitted to the NMR experiments corresponded to  38 the structure 65’, then the doublet of doublet associated to H-6’ gave a chemical shift relative to H-5’ and a long range coupling constant with H-9’ in the expected range.  a)  b) 1/d6 = 1 Hz  J5H-5, H-9  J6H-6, H-9  1/d6 = 3 Hz J6H-6, H-9  Figure 16: The benzonitrile silyl 65 submitted to COSY lrqf. a) the delay time d6 used for evolution of the long range coupling constant in the COSY lrqf pulse program; b) comparison of the cross peak region of H-9 and aromatic protons H-6 and H-5 under different delay time d6 where the threshold peak intensity is 0.2% for the first level and 95% for last level.  The preferential stabilization of aryl-lithium 63’ compared to 63 by the electronwithdrawing nitrile moiety may lead to the undesired derivative 65’ following addition of the bromomethylchlorodimethylsilane 64 (Scheme 24).54 However, we did not isolate the intermediates 63 or 63’ as the reaction was achieved in one pot from the starting material 62.  39  Br NC  Br  62  n-BuLi  Li  -105oC  NC  Br  63’  Br  64  NC  Li  Si  Cl  Br Br  -78oC  Br  Si NC  63  Br  65’  NC  Si  65  Br  Scheme 24: Metal-halogen exchange of benzonitrile 62 with n-BuLi. Compound 62 was mixed with n-BuLi followed by addition of chlorosilane 64 leading to two possible regioisomers 65 and 65’.  The electron-withdrawing effect of the nitrile contributed to the benzonitrile pKa value of 38.1 when the benzonitrile was mixed with the lithiated tetramethylpiperidine (LTMP) in THF at -78oC.55 Under these conditions, 36% of the benzonitrile was ortho-lithiated after 12 minutes from the addition of LTMP, longer use led to benzonitrile decomposition. However, the o-lithiobenzonitrile formation was completed in 5 minutes when the obromobenzonitrile was exposed to n-BuLi in THF at -100oC and the o-lithiobenzonitrile was found to be stable for hours.56 In conclusion, we cannot rule out that the benzonitrile silyl 65’ instead of 65 was incorporated leading to the linker acid sensitivity in neat TFA.  2. The tartrate linker: synthesis and stability test Following the disappointing results of the benzonitrile silyl linker 72, two papers from Zuckermann and Gras-Masse caught our interest (Chapter 1, section 7.2). They reported a tartrate linker stable in 95% TFA during SPPS on a hydrophilic resin such as the poly[acryloyl-bis(aminopropyl)polyethylene glycol] resin (PEGA resin). In addition, the chemical nature of the synthesized peptides was unaltered by the periodate treatment necessary for cleavage of the product off the resin. Thus, we made use of the methodologies developed by Zuckermann and Gras-Masse to synthesize the tartrate linker 78 on a PEGA resin (Figure 17). In addition, a soluble linker bearing a fluorophore 88 was prepared to test the synthetic methodology and to test the stability of the linker to acidic media.  40  H N  O O  O n  O  N H  O  H N  O  O  N H  NEt2 O  O O  O  O  O O  78 H N  NHCOR  O  O  N H  88  O  H N  H N  O  O  O O  O  O  N H  Figure 17: The solid and solution phase tartrate-based linkers. Red depicts the product that is cleaved off the linker after the acidic diol deprotection and oxidative cleavage with NaIO4.  2.1. Preparation of soluble fluorescent tartrate linker As a mimic of the PEGA resin spacer, we envisioned using the soluble spacer 2,2’(ethylenedioxy)-bis-(ethylamine). The spacer was mono-protected with isobutyric anhydride leading to the amphiphilic 2-[2-(2-{isobutyramide}-ethoxy)-ethoxy]-ethylamine 79 in good yield (Scheme 25).57  O H2N  O  O  NH2  a  N H  O  O  NH2  79  Scheme 25: Synthesis of the resin spacer mimic 79. Reagents and conditions: a) isobutyric anhydride, CH2Cl2, -78oC, 1 h 30 min, warmed-up to RT, overnight, 77%.  Since the projected linker 78 contains a valine residue between the PEG linker and the cleavable diol, N-α-Fmoc-L-Valine (Fmoc-Val-OH) was coupled to the amine 79 under standard conditions (Scheme 26). HOBt•H2O was added to minimize side reactions which could occur due to the slow rate of the coupling reaction caused by the valine steric hindrance. Following removal of the Fmoc protecting group from compound 80 using tris (2-aminoethyl) amine (TAEA), the amphiphilic amine 81 was obtained in reasonable yield (Scheme 26).  41 H N O  O  O  NH2  H N  a  O O  O  79  b  O  NHR  N H  80: R = Fmoc 81: R = H  Scheme 26: Synthesis of the soluble Valine linker 81. Reagents and conditions: a) CH2Cl2, HOBt•H2O, Fmoc-Val-OH, EDC•HCl, Et3N, 4oC to RT, overnight, 95%; b) TAEA, CH2Cl2, 1 h, RT, 73%.  Excess of TAEA was used as a base because a significant amount of dibenzofulvene (DBF) adduct 82 was contaminating the product when excess of piperidine or diethylamine was used (Scheme 27).58  CO2(g)  H N  O O  Base CONHR  H2N  HN CONHR  82  CONHR  Scheme 27: Formation of the DBF adduct 82. Compound 82 appears following elimination of the N-Fmoc protecting group with a non-sterically hindered base.  Prior to incorporing the key diol unit into the soluble linker system, the diol was protected using dimethoxypropane (DMP) affording the diethyl-(2S,3S)-O-isopropylideneD-tartrate 83a (Scheme 28). Trace amounts of the asymmetric ester 83b were observed and caused by trans-esterification with MeOH generated in the process. Both diesters 83a/b were monosaponified using one equivalent of KOH in ethanol yielding the tartaric acid 84 in good yields.59 Due to predominant saponification on both tartrate carboxyl ends, lower yields were obtained using aqueous LiOH, NaOH or KOH in THF or dioxane at various experimental conditions.60  42  O  OH O EtO  OEt O  a  O O  R'O O RO  OH  b  83a: R, R’ = Et, Et; 83b: R, R’ = Me, Et 84: R, R’ = Et, H  Scheme 28: Synthesis of protected tartaric acids 84. Reagents and conditions: a) i) cyclohexane, dimethoxypropane, p-TsOH•H2O, reflux in Dean– Stark, 3 h, ii) RT, Na2CO3, 1 h, 85% of 83a with trace amounts of 83b; b) KOH, EtOH, 4oC to RT, 98% of 84.  The protected diol tartaric acid 84 was incorporated into the soluble linker precursor 81 by applying a standard EDC•HCl/HOBt•H2O coupling procedure (Scheme 29). The ester 85 was saponified using KOH in dry ethanol to give the soluble linker precursor 86 in good yield.  O N H  O  O  H N  NH2  a  O N H  O  81  b  O  O  O  H N O  N H  O  OR O  O  85: R = Et 86: R = H  Scheme 29: Synthesis of the soluble tartrate linker 86. Reagents and conditions: a) monoester tartaric acid 84, HOBt•H2O, EDC•HCl, Et3N, CH2Cl2, 4oC to RT, overnight, 75%; b) EtOH, KOH, 4oC to RT, 2 h, 91%.  Prior to the fluorescent probe incorporation in the soluble linker 86, the fluorescent probe 74 was first coupled to the spacer 2,2’-(ethylenedioxy)-bis-ethylamine 87 mono protected with monomethoxytrityl (Mmt) (Scheme 30). Herein, the Mmt was chosen as the spacer protecting group because it was used in the corresponding solid phase linker synthesis. The Mmt protected spacer 87 was then deprotected in TFA but the resulting purified fluorescent spacer 76c was unstable as a free base and was, therefore, stored as an ammonium chloride salt.  43 OMe O H2N  O  a  NH2  Ph  O  Ph N H  2  NH2 2  b  N H  87  O  O  Et2N  O  NHR 2  76b: R = Mmt 76c: R = H  c  Scheme 30: Synthesis of fluorescent spacer 76c. Reagents and conditions: a) anhydrous K2CO3, CH2Cl2, Mmt-Cl, 4oC to RT, 4 h, 90%; b) Dac-OH 74, EDC•HCl, HOBt•H2O, Et3N, CH2Cl2, overnight, 4oC to RT, 94%; c) TFA, TES, CH2Cl2, RT, 2 h, 80%.  To complete the soluble fluorescent linker synthesis, the fluorescent spacer 76c was coupled to the soluble tartaric acid linker precursor 86 by means of a standard EDC•HCl/HOBt•H2O coupling procedure to yield the soluble fluorescent linker 88 with trace amount of the deprotected diol 88’ (Scheme 31).  O N H  O N H  O N H  O  O  O  86  O  O  H N  O  N H  O  O  O  N H  O  H N  O O  O  O  N H  N H O  O  NEt2  O  O  O  a  O  88 H N  OH O  O  H N  O  O  H N  O O  O  OH  N H O  O  NEt2  OH  88’ Scheme 31: Synthesis of the soluble fluorescent linkers 88 and 88’. Reagents and conditions: a) fluorescent spacer 76c, HOBt•H2O, EDC•HCl, Et3N, CH2Cl2, 4oC to RT, overnight, 62% of 88 with trace amount of 88’.  2.2. TFA test stability of the fluorescent soluble tartrate linker To test the stability of the linker to acidic treatment, the fluorescent soluble tartrate linker 88 (0.064 g) was dissolved in an excess of TFA (10 mL) for 6 hours at room  44 temperature. After removal of the TFA under reduced pressure, the fluorescent mixture was analyzed by RP-HPLC (Figure 18). Four main peaks were observed (λ = 410 nm, Dac), compared to the RP-HPLC chromatogram of the starting material 88 and further analyzed by HRMS (ESI) (Figure 19). The first peak A grew from 4% to 24% (peak area) which was shown by ESI-MS to be composed of the diol deprotected derivative 88’ contaminated with trace amounts of the starting material 88. The second peak B was lowered from 78% to 57% (peak area) which was shown to be composed of starting material 88. The two last peaks C+D were collected in the same fraction and their area sum did not change significantly (from 17% to 19%). The resulting fraction C+D was shown to be composed of the starting material 88 or an isomer with the same m/z and was not further investigated. This experiment suggested that the tartrate linker chosen was stable to acidic treatment since no apparent cleavage was observed other than the diol protecting group.  Before TFA treatment  B  A  C+D  After TFA treatment  Figure 18: RP-HPLC analysis of the linker 88. RP-HPLC (see experimental section) chromatograms of the linker 88 before and after the acidic treatment (TFA): peak A (36.2 min) and peak B (37.1 min) were each collected in a single fraction, denoted fraction A and B respectively, peak C (37.6 min) and D (37.9 min) were both collected both in the same fraction, denoted fraction C+D.  45  O H N  O O H N  m/z  H N O  O  2  N H  O  2  OH  88’ from A [M+H]+ = 823.4448 O  O  H N  O H N  O 2  m/z  O  O  N H  NEt2  O  OH  O  H N  O  H N  O  O  O  NEt2  H N 2  O  O  88 from B, C+D [M+H]+ = 863.4761  Figure 19: MS analysis of the linker 88. HRMS (ESI) of parent ion [M+H]+ corresponding to compound 88’ found in fraction A and to compound 88 found in fraction B and C+D. Under each compound structure the calculated parent ion [M+H]+ is given.  2.3. Preparation of tartrate linker on solid phase Next, we built the tartrate linker on PEGA resin. This resin was chosen because it swells in a wide range of solvents (toluene to water) allowing many different synthetic reactions to occur with a low level of product aggregation.61 In addition, the porous, gellike phase of the amphiphilic PEGA resin allows for efficient diffusion of bio-molecules up to 35 kD.62 Therefore, the PEGA resin is an excellent support for the synthesis and biological screening of a peptides library with a low level of non-specific protein interactions. Furthermore, the low loading (0.4 mmol/g) resin gives a pseudodilution effect favouring macrolactamization over dimerization. The linker synthesis started with the acylation of the PEGA resin primary amine 89 by Fmoc-Valine-OH using the standard HBTU/HOBt•H2O coupling procedure (Scheme 32). The coupling afforded resin 90, with loading of 0.35 mmol/g of resin. The extend of loading was estimated by the amount of dibenzofulvene (DBF) produced following Fmoc deprotection.63 Valine was used because it was reported by Gras-Masse et al. to stabilize the linker in subsequent reactions (Chapter 1, section 7.2). The Fmoc group was removed from the resin 90 by treatment with 20% piperidine to yield resin 91 (Scheme 32). Due to  46 steric hindrance, the acylation of the primary amine’s valine on resin 91 by the monoester tartaric acid 84 was expected to proceed more slowly. The slower the coupling reaction, the higher the risk of primary amine guanylation by coupling agents such as carbodiimides or uronium/guanidinum salts. Thus, to avoid guanylation of the free primary amine of resin 91, the coupling reaction was carried out with the phosphonium salts PyBOP (Scheme 32).64 The nucleophilic base 4-dimethylaminopyridine (DMAP) was preferred as an additive to the bulkier and less basic HOBt.65 Following completion of the coupling reaction, the tartrate esters derivative 92 was saponified with KOH to the tartrate acid 93 (Scheme 32).  NH2  H N  a  NHR  c  O  O  89  b  90: R = Fmoc 91: R = H  O  H N  d  N H  O  O  OR' O  92: R’ = Et 93: R’ = H  Scheme 32: Preparation of the tartrate linker on amino PEGA resin 93. Reagents and conditions: a) Fmoc-Val-OH, HOBt•H2O, HBTU, DIEA, DMF, 1h, RT, coupling repeated for 2h; b) 20% piperidine in DMF, 5 min, RT, deprotection repeated for 10 min; c) monoester tartaric acid 84, PyBOP, DMAP, DIEA, DMF, 1h, RT, coupling repeated for 2 h; d) KOH in 2:1 MeOH/H2O, 18 h, RT.  To complete the tartrate-based linker synthesis on PEGA resin, the resin 93 was coupled to the N-Mmt protected amino spacer 87 using PyBOP overnight according to the Paulick and Zuckermann methodology introduced in Chapter 1, section 7.2 (Scheme 33). Despite PyBOP having a half life of 5 hours with concomitant pyrrolidide release caused by PyBOP degradation, the coupling was successful in our case.66 The N-Mmt protected tartrate-based linker 94 was stored dry at -20oC for months without problem. When the linker on PEGA resin 94 was needed for peptide synthesis, the Mmt protecting group was cleaved with trichloroacetic acid (TCA) in MeCN and the resulting linker was used the same day.  47  O  H N O  N H  93  O  OH  b  H N  O  O  O O  N H  94  H N  O  O O  O  O NHMmt  Scheme 33: Preparation of the N-Mmt protected tartrate-based linker 94. Reagents and conditions: a) N-Mmt protected amino spacer 87, PyBOP, HOBt•H2O, DIEA, DMF, 2 h, RT, coupling repeated for 12 h.  2.4. Assessment of successful tartrate linker synthesis In order to verify the successful synthesis of the linker 94 on PEGA resin, we appended a dibromobenzoic acid functionality at the N-terminus of the linker arm. The presence of the two bromines was sought to give a characteristic triplet isotopic mass in HRMS (ESI) due to the natural  79  Br and  81  Br distribution of 50.7% and 49.3% respectively. Therefore,  the triplet signature was a quick and unambiguous way to identify small amounts of cleaved products from the resin. To this end, the 2,5-dibromobenzoic acid was first coupled to  glycine  methyl  ester  hydrochloride  (H-Gly-OMe•HCl)  under  the  standard  EDC•HCl/HOBt•H2O coupling procedure to give the methyl ester derivative 95 (Scheme 34). The latter was then saponified to the 2-(2,5-dibromobenzamido)acetic acid 96. O Br  O OH  a  Br  N H  Br  Br  b  OR O  95: R = Me 96: R = H  Scheme 34: Synthesis of 2-(2,5-dibromobenzamido)acetic acid 96. Reagents and conditions: a) HOBt•H2O, EDC•HCl, H-Gly-OMe•HCl, Et3N, DMF, overnight, 4oC to RT, 91%; b) KOH, 1:1 MeOH/H2O, reflux, 1 hour, 93%.  The N-Mmt protected tartrate-based linker 94 was deprotected in TCA, washed with 1% DIEA and coupled to the acetic acid derivative 96 with HBTU/HOBt•H2O (Scheme 35). The diol of the tartrate-based linker 97 was deprotected in TFA and the product was cleaved from the resin with sodium metaperiodate (NaIO4) to afford the soluble glyoxamide product 98 (Scheme 35). The presence of the glyoxamide derivative 98 was  48 shown by LRMS (ESI) to be present in the same methanol mixture as methyl ketal 99 (Figure 20).  O  H N  O H N O  OMe  O  a, b, c  O  H N  O H N  Ph  N H  94  O  N H  Ph  O  O  O  O  N H  97  O  N H  Br H N  O  Br  O  O  O  d, e O  H N  O O  O  N H  O  98  Br H N  Br  O  Scheme 35: Preparation and isolation of the labeled tartrate linker 98. Reagents and conditions: a) 4% (w/v) TCA in MeCN, 30 min, RT, the deprotection repeated two more times; b) 1% DIEA in DMF washed; c) carboxylic acid 96, HOBt•H2O, HBTU, DIEA, DMF, 1 h, RT, the coupling repeated for 2 h; d) 95:2.5:2.5 TFA/H2O/anisole, 2 h, RT; e) NaIO4, 5:1 H2O/AcOH, 5 min, RT, 16%. O MeO OH  N H  O  O  O  99  O O  H N  N H  O  O  98  O  Br  O  Br  N H Br  H N O  N H Br  Figure 20: LRMS (ESI) of the glyoxamide products 98 and 99. The parent ion [M+Na]+ of m/z = 575.9, 577.9, 579.8 corresponded to compound 99; the parent ion [M+Na]+ of m/z = 543.8, 545.8, 547.7 corresponded to compound 98.  49  3. Preparation of Pro2-Glu3-S-deoxo-amaninamide on solid phase One of our long range goals is the synthesis of an amatoxin library and its screening against RNA pol II. The library will be used to broaden our knowledge of the outstanding α-amanitin allosteric inhibition on the eukaryotic RNA pol II. First, as a proof of principle, we decided to prepare the bicyclic Pro2-Glu3-S-deoxo-amaninamide 59 that has a low level toxicity using the N-Mmt protected tartrate-based linker 94 on PEGA resin. 3.1. Synthesis of linear hexapeptide amatoxin precursor on solid phase After deprotection of the N-Mmt protected tartrate-based linker 94 with TCA, the free primary amine of the spacer was acylated by N-α-Fmoc-L-Glu-OtBu (Fmoc-Glu-OtBu) using the system HBTU/HOBt•H2O to afford the resin 100 (Scheme 36). The estimated loading was found to be 0.32 mmol per gram of resin. The same method was kept throughout the synthesis of the linear hexapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 101 (Scheme 36). In order to ensure good coupling yields and to avoid the use of capping agents, each coupling reaction was carried out twice. To avoid side reactions associated with activated amino acids such as epimerization, we did not use a preactivation step.67  50  O  NHTrt TrtS HO2C  NHFmoc HO2C  NHFmoc HO2C  O NHFmoc HO2C  NHFmoc HO2C  OH  NFmoc t-BuO2C  NHFmoc  a, b, c d, e  CO2t-Bu  FmocHN CO2t-Bu  d, e  O C N H  O C NH  O C N H  d, e FmocHN  O C NH  O H C N  O C NH  O C N H  d, e FmocHN  FmocHN  O H C N  O C N H  O C N H  O C NH  O C N H  O  H N  H N  O  H N  O  H N  O  H N  H N  O  O  H N  N H  O  N H  N H  O  O H N O H N O  O  O  N H  O  H N O  O  O O  N H  Scheme 36: Preparation of the linear hexapeptide amatoxin precursor 101.  100  H N  O  O  2 O  O  O O  2 O  H N  O  O  2 O H N  N H  O  2 O H N  O  O  2 O  O CO2t-Bu  O H C N  O  O CO2t-Bu  O C N H  H N  O CO2t-Bu  O C N H  H N 2 O  O CO2t-Bu  FmocHN  O  O  d, e FmocNH  H N  H N O  101  Reagents and conditions. a) N-Mmt protected tartrate-based linker 94, 4% (w/v) TCA in MeCN, 30 min, RT, the deprotection repeated two more times; b) 1% DIEA in DMF washed; c) Fmoc-GluOtBu, HOBt•H2O, HBTU, DIEA, DMF, 1 h, RT, the coupling repeated for 1 h 30 min; d) 20% piperidine in DMF, RT, 5 min, deprotection repeated for 10 min; e) Fmoc-Xaa-OH, HBTU, HOBt•H2O, DIEA, DMF, RT, 1 h, the coupling repeated for 1 h 30 min.  3.2. TFA test stability of the fluorescent linear heptapeptide on solid phase To test the stability of the linker on solid phase to acidic treatment and to optimize the oxidative cleavage of the linker, the linear hexapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 101 was made fluorescent as shown in compound 104 (Figure 21).  51 O  O  NEt2  H N  H2N  O  O  HN  O HS O HN  N H  H N  H N  N O O O  O  HN  O  2 O  O  O  O  H N  O O  N H  H N O  OH  104  NH2  Figure 21: Fluorescent linear heptapeptide 104 on PEGA resin. Shown in red is the linker part that is cleaved off the resin along with the coumarin derivative (in blue) and the peptide (in green).  To be consistent with our peptide synthesis methodology, we preferred to incorporate a fluorescent amino acid instead of only Dac-OH 74. The lysine was chosen for its side chain amine which allowed standard coupling to the coumarin carboxylic acid 74. However, to avoid unnecessary protection steps, we first prepared the N-hydroxysuccinimide (NHS) ester of coumarin (Dac-NHS) 102 from Dac-OH 74 using EDC•HCl as the coupling agent (Scheme 37).68 Without further purification, the N-α-Fmoc-L-Lysine (Fmoc-Lys-OH) was acylated by the crude product Dac-NHS 102 to afford the N-ε-Dac-N-α-Fmoc-L-Lys-OH (Fmoc-Lys(Dac)-OH) 103 in 71% yield. O O  O  a  O  OH Et2N  O  74  O  N  Et2N  O  O  102  O  CO2H  O  b  N H Et2N  O  NHFmoc  O  103  Scheme 37: Synthesis of Fmoc-Lys(Dac)-OH 103. Reagents and conditions: a) NHS, EDC•HCl, DMF, 48 h, 4oC to RT, used without purification; b) Fmoc-Lys-OH, DMF, overnight, 4oC to RT, 71%.  The fluorescence-labeled N-protected lysine 103 was coupled to the hexapeptide on resin 101 by standard HBTU/HOBt•H2O activation followed by Fmoc deprotection to afford the fluorescent resin 104 (Scheme 38). After exposing resin 104 for 30 minutes to neat TFA, the resin was still strongly fluorescent and only weak fluorescence could be  52 detected in the eluate (Figure 22). A similar result was obtained when the resin was exposed two additional times for 1 hour each and once for 2 hours (Figure 22). Following TFA treatment, the resulting fluorescence-labeled heptapeptide 105 was cleaved off from the resin with NaIO4 to achieve the soluble glyoxamide product 106 (Scheme 38).  CO2t-Bu FmocHN  O H C N  O C NH TrtS  O C N H TrtHN  O C N  O  a, b, c  C N OH  N H Et2N  O  N H O  TrtS  O C N H TrtHN  O C N  O  C N OH  O C N H (CH2)4  O C N H  e  H CO N HS  H C N O H2N  O C N H (CH2)4  O C N H  H CO N HS  H C N O H2N  O  H N O  H N  O  H N  O  O  N H  O  2 O  H N O  O C N H  CO2H  H N  O  O  H N  OH  2 O  O  OH  N H  H N O  105 C N O  O  O  N H  104 CO N  O  O  O  2 O  O  CO2H  N H O  d  CO N H  O H3N O  Et2N  (CH2)4  O H C N  H N  101  O H3N O  Et2N  O C N H  O  O  CO2t-Bu H2N O  H N  CN OH  H N O  O  H N  O  2 O  106  Scheme 38: Preparation and isolation of the labeled heptapeptide 106. Reagents and conditions: a) 20% piperidine in DMF, RT, 1 x 5 min, deprotection repeated for 10 min; b) Fmoc-Lys(Dac)-OH 103, HBTU, HOBt•H2O, DIEA, DMF, RT, 4 h; c) 20% piperidine in DMF, RT, 1 x 5 min, deprotection repeated for 10 min; d) 95:2.5:2.5 TFA/H2O/anisole, 5h 30 min, RT; e) NaIO4, 5 to 6 min, RT, 5:1 H2O/AcOH.  A  TFA: 30’ 1h30 3h30  B  5h30  5h30  Figure 22: The eluate (A) and the resin 105 (B) shown under UV lamp. The eluate (A) was collected from resin 104 exposed to 95:2.5:2.5 TFA/H2O/anisole at RT for varying times.  53 Due to the small amount of starting material, the high hydrophilicity of the linker and the electrophilic glyoxamide, the desired product was difficult to purify from the eluate 106. Furthermore, the ethanolamine used to quench the periodate was contaminating the product even after flash chromatography. However, when the eluate 106 was loaded on different TLC slides with the same solvent system, presence of a single product spot with the same Rf was revealed under UV (Dac fluorescence), ninhydrin (revealing a free primary  amine),  bromocresol  green  (revealing  a  free  carboxylic  acid)  and  dinitrophenylhydrazine (revealing a free aldehyde). Moreover, the UV spectra of the cleaved product 106 showed absorption maxima at 423 nm consistent with the presence of the Dac derivative (Figure 23). Overall, this qualitative assay showed that the tartrate linker on PEGA resin was stable in TFA and the peptide product could be cleaved off the solid support under mild conditions. Thus, we continued with the synthesis of Pro2-Glu3-Sdeoxo-amaninamide.  1.2 Abs 1 0.8  A  0.6  B  0.4 0.2 0 240  290  340  λ (nm)  390  440  490  Figure 23: UV absorption spectra in MeOH of compounds 106 and 103. Spectra of compound 106 is depicted by the blue plot (λmax = 423) and Fmoc-Lys(Dac)-OH 103 by the red plot (λmax = 419). Inset: in the right-upper corner shown under UV lamp, the eluate (A) containing compound 106 collected by filtration from the fluorescence-labeled heptapeptide resin 105 (B) poured in a mildly acidic NaIO4 aqueous solution.  3.3. Synthesis of monocyclic Pro2-Glu3-S-deoxo-amaninamide on solid phase  54 With the Fmoc protected linear hexapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 101 in hand, we continued the synthesis of amatoxin on the solid support by incorporating the Trt-Hpi-Gly-OH 9a/b (Chapter 1, sections 5.1 and 7.3). The synthesis of Hpi 9 was initiated by the protection of L-tryptophan yielding the amine and carboxyl trityl protected compound 107 (Scheme 39). The detritylation of the carboxyl was done by reflux in methanol to give the N-α-Trt-L-tryptophan 108 (Trt-Trp-OH) that was then amidated by glycine methyl ester hydrochloride (H-Gly-OMe•HCl) using the coupling agent EDC•HCl with HOBt•H2O as the additive to yield the Trt-Trp-Gly-OMe 7 (Scheme 39). The trityl group was reported to be stable to HOBt (pKa ~ 5) in DMF for at least two hours.69 The resulting Trt-Trp-Gly-OMe 7 was then oxidized to a 10:8 diastereoisomeric mixture of TrtHpi-Gly-OMe syn-cis 8a and anti-cis 8b using the mild volatile oxidant DMDO (Scheme 39). Caution must be applied in this oxidation since a slightly elevated temperature or excess DMDO may lead to side reactions such as DMDO decomposition, N-oxidation, oxindolylalanine 111 production caused by nucleophilic attack of water on Hpi with isomerisation of 2-hydroxytryptophan, loss of trityl, and/or Hpi dehydration, which gives compound 112 (Figure 24).70 Nevertheless, due to the starting material co-eluting with the product during purification by flash chromatography, a small DMDO excess was used to consume the starting material at the expense of minimal by-product formation. The resulting Trt-Hpi-Gly-OMe syn-cis 8a and anti-cis 8b were difficult to separate by standard flash chromatography in good purity and thus, the mixture was used for the amatoxin synthesis following its saponification to 9a/b with lithium hydroxide (Scheme 39).  55  CO2R''  CO2R'' O  OH  O  a  NH2  O  OR'  c  NHR  N H  O  OMe  d  O  NHTrt  HO  NH NTrt H NH  O  HO  NH NTrt H NH  N H  N H  b  H N  107: R, R’ = Trt 108: R, R’ = Trt, H  7  8a: R’’ = Me e 9a: R’’ = H  e  8b: R’’ = Me 9b: R’’ = H  Scheme 39: Synthesis of Trt-Hpi-Gly-OH 9a/b. Reagents and conditions: a) TrtCl, 2:1 CHCl3/DMF, Et3N, 4oC to RT, overnight; b) MeOH reflux, 5h then Et3N in Et2O, 79%; c) H-Gly-OMe•HCl, EDC•HCl, HOBt•H2O, Et3N, DMF, RT, overnight, 79 %; d) DMDO, acetone, CH2Cl2, -78oC, ~ 1 hour, 55%; e) LiOH, 2:1 THF/H2O, RT, 2 h, 85%. O HO NH2  N H  109  N H  O NH  110  O O N H  O  NH2  111  N H  NH  112  Figure 24: Structure of by-products observed during Hpi synthesis. Structure of Trp motif 109 (λmax = 280 nm), its oxidized derivatives Hpi motif 110 (λmax = 300 nm), the by-products oxindolylalanine motif 111 (λmax = 250 nm, small peak at 290 nm) and 2,3dihydropyrrolo[2,3-b]indole motif 112 (λmax = 310 nm, shoulder at 290 nm). These by-products were observed during the oxidation reaction or Savige-Fontana reaction in our lab.  The Fmoc protected linear hexapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 101 was deprotected and acylated with Trt-Hpi-Gly-OH 9a/b in a standard HBTU/HOBt•H2O solid phase peptide synthesis procedure to yield the linear resin-bound octapeptide 113 (Scheme 40). The resulting linear resin 113 was subjected to a SavigeFontana reaction (TFA, 5 hours) to yield a side chain-to-side chain cyclic peptide 114 with the tryptathionine linkage (S-(2-tryptophanyl)cysteine) in place of the cysteine and Hpiresidue (Scheme 40).  56  FmocHN  O H C N  O H C N  O C N H TrtHN  TrtS  101  CO2t-Bu  O C N H  O C N  N N H H Trt  O C N H  O H C N  O C N H TrtHN  TrtS  NH3  114  O  c  O N  NH  O NH H N  O C N H  O C N  O  113  H N  O  O  2 O  N H  O  H N O  a, b  O  O H C N  O  O  HO O H C N  H N  S O O  HN NH  O  CO2t-Bu  H N  O  O  CO2H  N H O O  H N 2 O  H N O  O  H N 2 O  O  O  N H  O  O  H N O  O O  N H  H N O  NH2  N H  Scheme 40: Preparation of the amatoxin precursor 114 on PEGA resin. Reagents and conditions: a) 20% piperidine in DMF, RT, 5 min, deprotection repeated for 10 min; b) Trt-Hpi-Gly-OH•Et3N 9a/b, HBTU, HOBt•H2O, DIEA, DMF, RT, 1 h, the coupling repeated for 1 h 30 min; c) neat TFA excess, 5 h, RT.  After the Savige-Fontana reaction, the tartrate diol of resin 114 was deprotected under acidic conditions to yield the monocyclic Pro2-Glu3-S-deoxo-amaninamide 115 (Scheme 41). Triethyl silane (TES) was found to be more efficient than anisole to quench the carbocations and to protect nucleophilic residues such as Asn, Cys and Trp. Compound 115 was oxidatively cleaved from the resin to yield the soluble glyoxamide monocyclic Pro2-Glu3-S-deoxo-amaninamide 116 and Pro2-Glu3-S-oxo-amaninamide 117 (Scheme 41).  57 O NH3  S O O  NH H N O  N  NH  O  CO2H  N H O O  HN  O  H N  O  O  O  2 O  O  H N  N H  O  114  NH2  O  NH  H N  a  N H O NH3 NH H N O  N  NH  O  S O O  CO2H  N H O O  HN  O  2 O  O OH  N H  H N O  115 b  N H O NH3 NH H N  N  NH  O  O  OH  H N  O  NH2  O  NH  H N  S O O  HN NH  N H  CO2H  N H O O  O  O  116  2 O  O  NH3  N  NH  O  O  O  H N  NH2  O  NH H N  H N  S O O O  HN NH  CO2H  N H O O  O  H N  O  O  NH2  H N  O  2 O  117  N H  Scheme 41: Isolation of the monocyclic amatoxin 116. Reagents and conditions: a) 95:2.5:2.5 TFA/TES/H2O, 2 h, RT; b) NaIO4, 6 min, RT, 5:1 H2O/AcOH, product 116 in 6% yield, product 117 in 3% yield.  During linker cleavage, dimethylsulfide (DMS) was preferred over the use of ethanolamine or ethylene glycol for the quenching of excess of NaIO4 and yielding DMSO with iodine. The crude peptidic glyoxamide mixture of 116 and 117 (~ 20 mg) recovered from the oxidative cleavage was first loaded on a Sep-Pak C18 column mainly to eliminate salts  and  dimethylsulfoxide  (DMSO).  The  characteristic  tryptathionine  (S-(2-  tryptophanyl)cysteine) UV absorbance (λmax = 289 nm) was used to detect the product 116 in the eluate that was mainly contaminated with iodine and the sulfoxide compound 117. About 10 mg of the product mixture 116 and 117 was loaded on a RP-HPLC from which was isolated 1 mg of the soluble glyoxamide monocyclic Pro2-Glu3-Sulfur-oxoamaninamide 117 (Peak M[O]) and 2 mg of the non-oxidized monocyclic thioether 116 (Peak M) (Figure 25). Sulfur oxidation, however, was of little concern since cleavage of  58 the peptides of the library will occur after the biological assays. The products were characterized by HRMS (ESI) and the monocycles 116 and 117 were found to be in equilibrium with their respective imine 118 and 119 (Figure 26). Spontaneous ketal and imine formation was previously reported with glyoxamide peptides.71 M  M[o]  Figure 25: RP-HPLC analysis of the monocyclic amatoxin precursors. RP-HPLC (see experimental section) elution profile of the cleaved product from resin 115 following C18 Sep-Pak gel chromatography. Peak M[O] (21.0 min) and peak M (23.4 min) were each collected in a single fraction, denoted fractions M[O] and M respectively.  59  O O  O O  N NH H  HN  O O  O  HN  O  NH2 O  OO O S  NH  N H N  NH O  HN O  O  HN  N NH H  O N  O  OO O S  NH O OH  O  O  HN  OH  O O  O  HN  O  NH2 O  O O S  O  119 from M[O] calculated m/z = 1055.4262 found m/z = 1055.4141  O  N NH H  N HN  H2N  H2N  O  O  O  O O  117 from M[O] calculated m/z = 1073.4368 found m/z = 1073.4331  O  NH  N H  HN  HN  O O  HN  NH  N H  O  HN NH  N  O O O  N NH H  HN O  O N  O  O O S  NH  N H  O  HN NH  HN  HN  N  O O  OH  H2N  116 from M calculated m/z = 1057.4419 found m/z = 1057.4388  O O  O  HN O  OH  H2N  118 from M calculated m/z = 1039.4313 found m/z = 1039.4271  Figure 26: MS analysis of the monocyclic amatoxin precursors. Structure of compounds 116 and 118 found in fraction M, compounds 117 and 119 found in fraction M[O]. Under each compound structure the parent ion [M-H]- found by HRMS (ESI) and calculated are given.  3.4. Synthesis of bicyclic Pro2-Glu3-S-deoxo-amaninamide on solid phase Head-to-tail macrolactamization of the monocycle 114 completed the solid phase synthesis of Pro2-Glu3-S-deoxo-amaninamide. We opted for PyBOP as the coupling agent since it had been used successfully in macrolactamization of the soluble monocyclic Pro2Ile3-S-deoxo-amaninamide (Chapter 1, section 5.3). Furthermore, phosphonium salt-based coupling reagents reduce the risk of both epimerization and guanylation during macrolactamization (Chapter 1, section 5.2). Thus, the monocyclic derivative 114 was macrolactamized on solid phase with PyBOP (2 eq.), HOBt•H2O (2 eq.) and DIEA (3 eq.) in DMF (Scheme 42). Due to the short half-life of PyBOP, the resin 114 was exposed to the  60 coupling mixture successively 3 hours for each equivalent. Because the amine and carboxyl were already deprotonated by DIEA washes, only three equivalents of base (DIEA) were used to lower the risk of epimerization.72 Finally, the Pro2-Glu3-S-deoxo-amaninamide 120 bound to the amino PEGA resin by the tartrate linker was oxidatively cleaved. As explained below, we believed that the crude product recovered from the resin eluate was composed of a mixture of L-Pro2-L-Glu3-S-deoxo-amaninamide 121 and L-Pro2-D-Glu3-Sdeoxo-amaninamide 122 (Scheme 42).  O NH3  HN  S O O  NH H N O  O  NH  HN HN  O  S  O HN O NH HN O  d  N O  NH  H N  O NH O  O  122  O  2 O  O N H  O  H N O  114  N NH  O  S  O  NH  H N  O  O HN O  NH  NH HN  H2N  O  O  H2N  HN  O  H N  O  O  HN  H N  NH2  O  NH  N H  a, b, c  N H O O  N  NH  O  CO2H  O  2 O  O  O  OH  O OH  N H  H N O  120  O  O  H2N O  O  H N  O  H N O  N NH  HN HN  O  S  O HN O NH HN  O  NH  H N  O NH O  O  O  O  H N O  121  O  Scheme 42: Preparation of L-Pro2-L-Glu3-S-deoxo-amaninamide 121. Reagents and conditions: a) 5% DIEA in DMF; b) PyBOP, HOBt•H2O, DIEA, DMF, 3 h, RT, the coupling repeated three other times; c) 95:2.5:2.5 TFA/TES/H2O, 2 h, RT; d) NaIO4, 5 min, RT, 5:1 H2O/AcOH, ~ 10% in total yield including the epimerized D-Glu3 derivative 122.  The crude product resulting from the NaIO4 treatment of resin 120 was desalted with a Sep-Pak C18 column and purified by RP-HPLC (Figure 27). The major products eluting from the RP-HPLC were found in two fractions called A and B in a ratio of 1:2.  61 Both fractions were shown by HRMS (ESI) to be composed of a product with a mass-tocharge ratio corresponding to compound 121 and 122 (Figure 28). Furthermore, both fractions were contaminated with trace amounts of a product of mass-to-charge ratio corresponding to the sulfoxide analogue of compound 121 and 122. As previously, the tryptathionine (S-(2-tryptophanyl)cysteine) UV spectrum helped in the identification of Pro2-Glu3-S-deoxo-amaninamide in the fractions collected (Figure 28). The UV spectrum of both fractions displayed the absorbance maximum at 289 nm with a shoulder on either side that matches the known UV spectrum of the tryptathionine moiety.73 a) B A  b) A’  B’  Figure 27: RP-HPLC analysis of the bicyclic amatoxins 121 and 122. a) RP-HPLC (see experimental section) elution profile of the cleaved product from resin 120 following C18 Sep-Pak gel chromatography where peak A (12 min) and peak B (13 min) were collected in separate fractions denoted A and B, respectively; b) RP-HPLC (see experimental section) elution profile of fraction A and B respectively where both afforded a single major peak A’ (11 min) and B’ (12 min) respectively.  62  a)  0.8 Abs 0.7  Fraction A Fraction B  0.6 0.5 0.4 0.3 0.2 0.1 0 200  225  250  275  300  325  350  375  400  λ (nm)  b)  O H2N N  O  NH  HN HN  O  S  NH  H N  O  OHN O NH  O  NH HN  O  O  H N  O  2 O  O  O  121 and 122 from A and B calculated m/z = 1039.4313  m/z from A  m/z from B  Figure 28: UV spectra and MS analysis of the bicyclic amatoxins 121 and 122. a) UV absorption spectra in 0.06:2:8 TFA/MeCN/water of fraction A (in blue) and B (in red); b) HRMS (ESI) of parent ion [M-H]- from fraction A and B. Under the corresponding compound structure the calculated parent ion [M-H]- is given.  Based on a report of May et al., we believed that epimerization occurred at the glutamate residue during the macrolactamization of compound 114 on solid phase.74 May et al. reported a similar epimerization took place in solution phase head-to-tail  63 macrolactamization of the monocyclic Pro2-Ile3-S-deoxo-amaninamide 11 with PyBOP in DMF (Chapter 1, section 5.3). A 1:1 mixture of the diastereoisomers L-Pro2-L-Ile3-Sdeoxo-amaninamide 12 and L-Pro2-D-allo-Ile3-S-deoxo-amaninamide 12’ was isolated and characterized (Figure 29). The product 12’ was faster to elute from RP-HPLC compared to compound 12, thus suggesting that fraction A corresponded to compound 122 and fraction B to compound 121. May et al. described a βII-turn that was found in ring I (residue 1-4) of the epimerized bicyclic amatoxin 12’ causing a variation in the CD spectrum compared to the bicyclic 12 which displayed a βI-turn in ring I. The epimerized bicyclic amatoxin 12’ ring II (residue 4-8) equilibrated between a βI-turn and a βΙI-turn, whereas only the βΙIturn was observed in the NMR structure of 12 and α-amanitin. The βΙI-turn in ring II is thought to be stabilized by the amide side chain of Asn and to play a role in the amatoxins’ affinities to RNA pol II.75 However, the inhibitory activity of the bicyclic compounds 12 and 12’ on RNA pol II was weak due to the absence of the hydroxyproline residue. O  O  H2N O  7 HN  8  NH  HN  S  O  NH  12  HN  5 O  O  2 O  4  8  NH  O  OHN O  6  H2N  1 N  O  NH  HN  3 NH  1  7 HN  O  S  NH  12’  O  2 NH  O  OHN O  6  N  NH  HN  5  3  4 O  O  Figure 29: Structure of the two bicyclic amatoxin diastereoisomers. The compounds 12 and 12’ were purified from the RP-HPLC following macrolactamization in solution. The Ile3 included in ring I (residue 1-4) is shown in red and ring II (residue 4-8) is shown in green.  64  Chapter 3 Experimental  1. General 1.1. Chemicals All reactions were performed under argon or nitrogen atmosphere in flame-dried glassware and dried solvent at room temperature, unless otherwise stated. Controlled temperature reactions were performed using a mineral oil bath and a temperature controlled hot plate (IKA Ceramag Midi equipped with an IKA ETS-D4 Fuzzy thermometer). Temperature reactions below room temperature were performed in an ice/water bath (4°C), dry ice/ethylene glycol (-15oC), dry ice/acetone bath (-78°C) or Et2O in liquid nitrogen (116°C). Solvents were evaporated under reduced pressure using a Büchi rotary evaporator. Lyophilisation of water, water/MeCN mixture or DMSO solutions proceeded by sublimation at 200 to 300 mTorr after freezing the solution in a dry ice/acetone bath. A brine solution refers to a saturated NaCl (aq) solution. The silicon-based linker synthesis was carried out on a Merrifield chloro resin (chloromethyl-copoly(styrene-1%)-divinylbenzene, 100-200 mesh, loading of 1.00-1.60 mmole/g, Novabiochem (Germany)). The synthesis of the tartrate-based linker and amatoxin was performed on an amino PEGA resin 89 (poly[acryloyl-bis(aminopropyl) polyethylene glycol, 50-100 mesh, loading of 0.4 mmol/g, Novabiochem (Germany)). Anhydrous solvents were achieved by distillation under nitrogen atmosphere. Ethers were distilled from sodium in the presence of benzophenone as indicator. Et3N, pyridine and CH2Cl2 were distilled over calcium hydride. Methanol was distilled from magnesium.  65 DMF and ethanol were dried over 4Å molecular sieves (three times) under argon atmosphere. Anhydrous MeCN was purchased from Sigma-Aldrich Chemicals. Reagents were purified according to literature procedures.76 Unless otherwise indicated, all reagents and solvents were purchased from NovaBioChem, Sigma-Aldrich Chemicals or Fisher. All products were stored at –20oC under argon. Dimethyldioxirane (DMDO) O O  A three-neck round-bottom flask (RBF, 2 L) fitted to a three-way claisen adapter connected to a collecting RBF (250 mL) and mounted with a Dewar condenser was filled with dry ice/acetone. A small amount of anhydrous K2CO3 was added to the collecting RBF and immersed in a dry ice/acetone bath. The three-neck RBF was filled with NaHCO3 (100 g, 1190 mmol), distilled water (300 mL), acetone (300 mL, 4080 mmol) and cooled to 4oC. After addition of Oxone® (5:3:2:2 2KHSO5/KHSO4/K2SO4) in five portions of 40 g at 3 minute intervals, the mixture was kept at 4oC, and was distilled from 80 to 60 mmHg. Then, the distillate (~80 mL) was filtered against anhydrous K2CO3 to yield DMDO as a clear yellow solution in acetone. The product was stored under 4 Å molecular sieves at 20oC for no more than two days. The DMDO concentration in acetone was approximated according to Adam et al. and typical yield of ~1% was obtained at ~0.06 M in acetone.77 1  H NMR (300 MHz, CDCl3): δ = 1.60 (s) 1.2. Analytical methods Thin-layer chromatography (TLC): TLC was done using silica gel 60 F254 precoated aluminium plates (EM Science). TLC  analyte migration is reported as Rf that refers to a ratio-to-solvent front measured on plates. Detection of TLC spots were effected by different methods using UV lamp at 254 and 365 nm; iodine crystal mixed in silica; p-anisaldehyde; cerium sulphate; potassium permanganate; cerium molybdate (Hanessian’s stain); phosphomolybdic acid (PMA),  66 ninhydrin; dinitrophenylhydrazine (DNP); bromocresol green; ferric chloride; morin hydrate according to literature procedures.78 Solid phase: Colorimetric resin analysis was performed by a Kaiser or Green Malachite test according to literature procedures.79 Resin loading with Fmoc protected residue was determined by Fmoc cleavage.80 For example, following PEGA resin 89 loaded with Fmoc-Val-OH, the resulting resin 90 (~0.090 g MeOH wet resin, 0.012 g dry resin) was dried overnight under vacuum against anhydrous CaCl2 of 4-20 Mesh. The dried resin was precisely weighed and then swollen in 1 mL of DMF for 30 min. To the resuspended resin was added diaza (1,3) bicycle [5.4.0] undecane (DBU) (20 μL) and the resulting mixture was shaken for 30 min. Then, MeCN (4 mL) was added to the mixture from which 1 mL was taken and added to MeCN (9 mL) in a separate vial. A solution of 2:8 DBU/DMF was diluted in the same way and used as a blank. The absorbance value was measured at 304 nm where DBF has ε = 7624 M-1cm-1. Chromatography: Flash column chromatography purification was performed with silica gel 60 (230-400 mesh, Silicycle, Quebec).81 Reverse phase column chromatography was performed on Sep-Pak C18 gel (Waters, Delaware) using Zeba Desalt polypropylene spin column (5 mL format) from PierceTM. Pressure was applied manually using a syringe. Linear gradient was achieved with 1 mL/min flow rate and by combining Buffer A’ and B’ as the mobile phase. Buffer A’ was 0.065% TFA in water and Buffer B’ was 0.05% TFA in MeCN. The product elution was monitored by UV spectra. RP-HPLC purification was performed using an Agilent 1100 HPLC, equipped with a photodiode array detector. Two reverse phase columns were utilized for RP-HPLC purification at 50oC: (1) A Phenomenex Jupiter C18 (150 x 1.5 mm, internal diameter of 8.5 μm); (2) A Phenomenex Jupiter C18 (250 x 4.6 mm, internal diameter of 10 μm). The sample dissolved in a mixture of 8:2 to 7:3 of Buffer A/B was filtered through a PVDF membrane filter (0.2 μm, Pall Life Science) before injection into the RP-HPLC system. Linear gradient was achieved with a 1 mL/min flow rate and by combining Buffer A and B as the mobile phase. Buffer A was 0.1% TFA in water and Buffer B was 0.05% TFA in  67 MeCN. Three different wavelengths were applied for the detection of products (λ = 229, 292 and 410 nm). Spectra analysis: Low-resolution mass spectra (LRMS) in electrospray ionization (ESI) mode were determined on a Bruker Esquire spectrometer. High-resolution mass spectra (HRMS) in ESI mode were recorded on a Micromass LCT time-of-flight spectrometer. Nuclear magnetic resonance spectra were recorded in deuterated solvents (chloroform, dichloromethane, methanol or acetone) supplied by Cambridge Isotope Laboratory, Inc. Proton (1H NMR) and carbon (13C NMR) nuclear magnetic resonance spectra were recorded using a Bruker AV-300 (300 MHz) spectrometer with exception of the long-range COSY (COSY lrqf pulse program) which was performed using a Bruker AV-600 (600 MHz). Chemical shifts for all spectra were reported in parts per million and referenced to the solvent peak. Fourier Transform-Infra Red spectra were determined on a Bomen Michelson spectrometer MB-100 using KBr pellets. UV spectra were recorded on a Beckman Coulter DU800 spectrophotometer.  68  2. Chemical Methods 2.1. Solution phase synthesis N-α-Trt-L-Tryptophanylglycine methyl ester 7 H N  O O NH  Ph Ph  O  NH Ph  To a solution of Trt-Trp-OH•Et3N 108 (6.00 g, 11 mmol) in DMF (100 mL) at 4oC was added HOBt•H2O (1.48 g, 11 mmol), EDC•HCl (2.10 g, 11 mmol) and H-Gly-OMe•HCl (1.25 g, 10 mmol). The mixture was stirred for 5 min followed by addition of Et3N (3 mL, 20 mmol) at 4oC. After being stirred overnight at room temperature, the solvent was removed under reduced pressure and the crude product was resuspended in EtOAc (275 mL). The organic layer was washed with saturated NH4Cl(aq) (3 X 150 mL), saturated NaHCO3(aq) (3 X 150 mL) and brine (3 X 150 mL), dried under anhydrous MgSO4, and the solvent was removed under reduced pressure. The resulting brown solid was recrystallized twice in EtOH/MeOH/H2O and the residual product was purified by flash chromatography (Silica, 1:1 Hexanes/EtOAc) to afford product 7 (4.50 g, 79%) as a white solid. Rf = 0.4 (Hexanes/EtOAc 1:1) 1  H NMR (300 MHz, CD2Cl2): δ = 2.43-2.36 (dd, J = 7 Hz; 6 Hz, 1H, β-CHTrp), 2.79 (br s,  1H, NH), 3.15-3.08 (dd, J = 7 Hz; 6 Hz, 1H, β-CHTrp), 3.52-3.46 (m, 2H, CH2Gly), 3.713.63 (m, 4H, OCH3, α-CHTrp), 6.92 (br s, 1H, NH), 7.38-6.93 (m, 19H, ArH), 7.48-7.45 (d, J = 7 Hz, 1H, ArH), 8.19 (br s, 1H, NH). 13  C NMR (100 MHz, CD2Cl2): δ = 29.9 (CH2), 41.1 (CH2), 52.1 (CH3), 57.8 (CH), 71.7  (C), 110.8 (C), 111.1 (CH), 119.4 (CH), 119.5 (CH), 122.3 (CH), 124.0 (CH), 126.7 (CH), 128.0 (CH), 128.9 (CH), 129.7 (CH),136.5 (C), 145.9 (C), 170.2 (C), 174.9 (C). HRMS (ESI) m/z: calculated for C33H31N3O3 [M-H]-: 516.2293, found: 516.2303.  69  Nb-Trt-3a-Hydroxypyrrolo[2,3-b]indolylglycine methyl ester 8a (syn-cis) and 8b (anticis) 5 6 7  4  5  OH 3 8a  2  3a  N N H H Ph  6  O 1  α  HN Ph O Ph  7  O  8a  4  OH 3 8a  2  3a  N N H H Ph  O 1  α  HN Ph O Ph  O  8b  To a solution of Trt-Trp-Gly-OMe 7 (0.50 g, 1 mmol) in CH2Cl2 (100 mL) at -78oC was added by portion a solution of ~ 0.06 M DMDO (9 X 2 mL, 5 min intervals) in acetone kept at -78 °C. The resulting yellow mixture was concentrated to dryness under reduced pressure at room temperature to yield a brown solid. The same procedure was repeated using Trt-Trp-Gly-OMe 7 (4 X 0.40 g, 1 X 0.20 g) in CH2Cl2 (100 mL). The crude products were pooled and purified by flash chromatography (Silica, 0.1:4:6 Et3N/Hexanes/EtOAc) to afford a diastereoisomeric solid mixture (1.32 g, 55%) of syn-cis 8a and anti-cis 8b in a ratio of ~10:8. Note: The characterisation of these products was achieved with the help of May et al. (Chapter 2, section 3.3). Rf = 0.3 (Et3N/Hexanes/EtOAc 0.1:5:5) 1  H NMR (300 MHz, CDCl3): δ = 1.16-1.06 (dd, J = 10 Hz; 14 Hz, 1H, CH-3 (8b)), 2.30-  2.25 (d, J = 14 Hz, 1H, CH-3 (8a)), 2.39-2.34 (d, J = 14 Hz, 1H, CH-3 (8b)), 2.54-2.47 (dd, J = 14 Hz; 9 Hz, 1H, CH-3 (8a)), 3.07-3.06 (d, J = 4 Hz, 1H, NH (8a)), 3.21-3.14 (dd, J = 3 Hz; 18 Hz, 1H, α-CH (8b)), 3.64-3.60 (m, 2H, α-CH (8a)), 3.72 (s, 3H, OCH3 (8a)), 3.87 (s, 3H, OCH3 (8b)), 3.90-3.87 (d, J = 9 Hz, 1H, CH-2 (8a)), 4.19-4.07 (m, 2H, CH-2 (8b), α-CH (8b)), 4.88-4.86 (d, J = 5 Hz, 1H, NH (8b)), 5.33 (s, 1H, OH (8b)), 5.40-5.38 (d, J = 5 Hz, 1H, CH-8a (8b)), 5.54 (s, 1H, OH (8a)), 5.59-5.58 (d, J = 4 Hz, 1H, CH-8a (8a)), 5.94-5.90 (br s, 1H, NHCO (8a)), 6.38-6.35 (d, J = 8 Hz, 1H, CH-7 (8a)), 6.68-6.65 (d, J = 8 Hz, 1H, CH-7 (8b)), 6.81-6.73 (m, 2H, CH-5 (8a), CH-5 (8b)), 7.15- 7.04 (m, 3H, CH-6 (8a), CH-6 (8b), CH-4 (8b)), 7.40-7.25 (m, 19H, ArHTrt (8a), ArHTrt (8b), CH-4  70 (8a)), 7.55-7.53 (d, J = 7 Hz, 6H, ArHTrt (8a)), 7.74-7.72 (d, J = 7 Hz, 6H, ArHTrt (8b)), 9.19 (br s, 1H, NHCO (8b)). HRMS (ESI) m/z: calculated for C33H31N3O4 [M+Na]+: 556.2207, found: 556.2213. Nb-Trt-3a-Hydroxypyrrolo[2,3-b]indolylglycine 9a (syn-cis) and 9b (anti-cis) 5 6 7  4  5  OH 3 8a  3a  N N H H Ph  2  O 1  OH 3  6  α  HN Ph O Ph  HNEt3+ O  9a  4  7  8a  3a  N N H H Ph  2  O 1  α  HN HNEt3+ OPh O Ph  9b  To a solution of Trt-Hpi-Gly-OMe 8a/b (1.32 g, 2 mmol) in THF (82 mL) at 4oC was added a solution of LiOH(aq) (41 mL, 0.3 M). After the mixture was stirred at room temperature for 2 hours, the reaction mixture was evaporated to dryness under reduced pressure. The crude product was purified by a flash chromatography (Silica, 0.1:0.5:9.4 Et3N/MetOH/CH2Cl2) to afford a diastereoisomeric salt mixture (1.31 g, 85%) of syn-cis 9a and anti-cis 9b in a ratio of ~1:2. Note: The characterisation of these products was achieved with the help of May et al. (Chapter 2, section 3.3). Rf = 0.1 (Et3N/MetOH/CH2Cl2 0.1:0.5:9.4) 1  H NMR (300 MHz, CDCl3): δ = 1.05-0.98 (dd, 1H, J = 10 Hz, 13 Hz, CH-3 (9b)), 1.34-  1.29 (t, J = 7 Hz, 9H, NCH2CH3), 2.27-2.23 (d, J = 13 Hz, 1H, CH-3 (9b)), 2.43-2.38 (d, 1H, J = 14 Hz, CH-3 (9a)), 2.50-2.46 (m, 1H, CH-3 (9a)), 3.09-2.99 (m, 7H, NCH2CH3, αCH (9b)), 3.46-3.42 (m, 2H, α-CH (9a)), 3.56-3.48 (m, 1H, α-CH (9b)), 3.97-3.89 (m, 2H, CH-2 (9a), CH-2 (9b)), 4.06-4.03 (d, 1H, J = 10 Hz, NH (9b)), 5.34 (s, 1H, CH-8a (9b)), 5.57 (s, 1H, CH-8a (9a)), 6.21 (br s, 1H, NHCO (9a)), 6.36-6.34 (d, J = 8 Hz, 1H, CH-7 (9a)), 6.60-6.58 (d, J = 8 Hz, 1H, CH-7 (9b)), 6.75-6.58 (m, 2H, CH-5 (9a), CH-5 (9b)), 7.09-7.01 (m, 3H, CH-6 (9a), CH-6 (9b), CH-4 (9b)), 7.37-7.22 (m, 19H, ArHTrt (9a), ArHTrt (9b), CH-4 (9a)), 7.56-7.54 (d, J = 7 Hz, 6H, ArHTrt (9a)), 7.77-7.75 (d, J = 7 Hz, 6H, ArHTrt (9b)), 9.14-9.13 (br s, 1H, NHCO (9b)). HRMS (ESI) m/z: calculated for C32H29N3O4 [M-H]-: 518.2085, found: 518.2079.  71  2,5-Dibromobenzamide 61 Br H2N O  Br  To a solution of 2,5-dibromobenzoic acid (5.04 g, 18 mmol) in CH2Cl2 (32 mL) at 4°C was added dropwise oxalyl chloride (3 mL, 32 mmol) followed by DMF (11 μL). The reaction was heated to reflux for 5 hours, and the solvent with residual oxalyl chloride was removed under reduced pressure to afford 2,5-dibromobenzoyl chloride as a brown oil. The acyl chloride was resuspended in EtOAc (28 mL) and added dropwise to an ice-cold solution of EtOAc (143 mL) containing concentrated aqueous NH4OH (30 mL). Following addition, the mixture was stirred cold for 30 min and the layers were separated. The organic layer was washed with saturated NaHCO3(aq) (3 X 60 mL), water (3 X 60 mL) and brine (3 X 60 mL), dried over anhydrous MgSO4, and concentrated to dryness. The resulting whiteyellow solid was recrystallized from a mixture of 3:97 MeOH/CH2Cl2 to afford the compound 61 (3.92 g, 78%) as a white crystal. mp: 180-181oC Rf = 0.4 (MeOH/CH2Cl2 5:95) 1  H NMR (300 MHz, CDCl3): δ = 6.05 (br, 2H, NH), 7.43-7.40 (dd, J = 2 Hz; 8 Hz, 1H,  ArH-4), 7.49-7.46 (d, J = 8 Hz, 1H, ArH-3), 7.78-7.77 (d, J = 2 Hz, 1H, ArH-6). 13  C NMR (100 MHz, acetone-d6): δ = 118.01 (C), 120.78 (C), 131.60 (CH), 133.70 (CH),  135.02 (CH), 141.19 (C), 167.48 (C). HRMS (ESI) m/z: calculated for C7H5Br2NO [M-H]-: 275.8660, found: 275.8664. 2,5-Dibromobenzonitrile 62 Br Br  N  Oxalyl chloride (3 mL, 33 mmol) was added over 10 min to a solution of DMF (2 mL) in MeCN (71 mL) at -15°C. The resulting chloromethylene(dimethyl)ammonium chloride  72 white suspension was stirred for 5 min at -15°C. An ice-cold solution of benzamide 61 (4.00 g, 14 mmol) in DMF (21 mL) was added slowly, the resulting mixture was stirred for 30 min at -15°C and then pyridine (5 mL, 64 mmol) was added over 1 min. After the reaction mixture was stirred for 45 min at 4°C, saturated NH4Cl  (aq)  (145 mL) was added  and the product was extracted with 1:1 EtOAc/Hexanes (5 X 50 mL). The organic layer was washed with saturated NH4Cl(aq) (3 X 100 mL), water (3 X 100 mL) and brine (3 X 60 mL), dried over anhydrous MgSO4, and concentrated to dryness. The resulting white solid was recrystallized from Et2O to afford the compound 62 (2.77 g, 74%) as a white crystal. mp: 144-145oC Rf = 0.4 (EtOAc/Hexanes 1:9) 1  H NMR (300 MHz, CDCl3): δ = 7.63-7.56 (m, 2H, ArH-3, ArH-4), 7.81-7.80 (d, J = 2 Hz,  1H, ArH-6). 13  C NMR (100 MHz, CDCl3): δ = 115.97 (C), 117.75 (C), 121.40 (C), 124.22 (C), 134.67  (CH), 136.87 (CH), 137.26 (CH). HRMS (EI) m/z: calculated for C7H3Br2N [M]+: 258.86322, found: 258.86322. 2-Cyano-4-(bromomethyldimethylsilyl)-bromobenzene 65 Br Br Si  N  The n-BuLi stock solution (~1.6 M in Hexanes) was first titrated against diphenylacetic acid in dry THF82. Then, to a solution of benzonitrile 62 (3.48 g, 13 mmol) in tetrahydrofuran (128 mL) at -105°C was added dropwise the n-BuLi stock solution (9 mL, 13 mmol) over 30 minutes and the resulting mixture was stirred for 10 min. To the lithiated intermediate 63 was added dropwise bromomethylchlorodimethylsilane 64 (2.5 mL, 17 mmol) over 2 min and the solution was stirred at -78oC for 45 min. The reactive mixture was quenched with ice water (60 mL) and the product was extracted with 1:1 Et2O/Hexanes (4 X 80 mL). The organic layer volume was reduced to 100 mL by evaporation under reduced pressure. The resulting organic layer was washed with water (3 X 70 mL), brine (3 X 70 mL), dried over anhydrous MgSO4, and concentrated to dryness.  73 The crude product was purified by flash chromatography (silica, 3:97 EtOAc/Hexanes) to afford compound 65 (1.97 g, 44%) as a yellow oil. Rf = 0.3 (EtOAc/Hexanes 1:9) 1  H NMR (300 MHz, CDCl3): δ = 0.60 (s, 6H, SiCH3), 2.85 (s, 2H, SiCH2), 7.53-7.51 (d, J  = 8 Hz, 1H, ArH-6), 7.76-7.73 (dd, J = 2 Hz; 8 Hz, 1H, ArH-5), 7.86-7.85 (d, J = 2 Hz, 1H, ArH-3). 13  C NMR (100 MHz, CDCl3): δ = -3.98 (CH3), 14.94 (CH2), 118.44 (C), 119.03 (C),  124.47 (C), 136.22 (CH), 137.27 (CH), 137.43 (CH), 139.53 (C). HRMS (EI) m/z: calculated for C10H11Br2NSi [M]+: 330.90275, found: 330.90275. Hydroquinone monoacetate 66 OH  O O  To a solution of hydroquinone (4.00 g, 36 mmol) in glacial acetic acid (18 mL) at 110oC was added acetic anhydride (2 mL, 18 mmol) dropwise over 1 hour. The mixture was stirred for 2 hours, and concentrated to dryness under reduced pressure. The crude product was resuspended in toluene (50 mL) at 4oC to precipitate-out the hydroquinone overnight. The organic layer was filtered and the solvent was removed under reduced pressure to yield a white solid contaminated with the by-product diacetate hydroquinone. The by-product was recrystallized in water and the product was extracted from the aqueous layer with Et2O (5 X 20 mL). The organic layer was washed with brine (3 X 75 mL), dried over anhydrous MgSO4, and concentrated to dryness. The product was recrystallized in petroleum ether to afford compound 66 (1.57 g, 57%) as a white crystal with a trace amount of the diacetate derivative. mp: 57-59oC Rf = 0.2 (EtOAc/Hexanes 25:75)  74 1  H NMR (300 MHz, CDCl3): δ = 2.31 (s, 3H, CH3), 4.25 (s, 1H, OH), 6.79-6.76 (d, J = 9  Hz, 2H, ArH), 6.95-6.92 (d, J = 9 Hz, 2H, ArH). 13  C NMR (100 MHz, CDCl3): δ = 21.26 (CH3), 116.22 (CH), 122.55 (CH), 144.17 (C),  153.63 (C), 170.66 (C). HRMS (ESI) m/z: calculated for C8H8O3 [M+Na]+: 175.0366, found:175.0371. 2-Cyano-4-(acetomethyldimethylsilyl)-bromobenzene 69 N O Br  Si  O  To a solution of benzonitrile silane 65 (1.97 g, 6 mmol) in DMF (20 mL) was added sodium acetate (1.45 g, 18 mmol) and the mixture was warmed to 55oC overnight. After cooling to room temperature, the mixture was poured into ice water (40 mL) and the product was extracted with 1:1 EtO2/Hexanes (5 X 40 mL). The organic layer volume was reduced to 100 mL by evaporation under reduced pressure. The resulting organic layer was washed with water (3 X 60 mL), brine (3 X 60 mL), dried over anhydrous MgSO4. Evaporation of solvents under reduced pressure followed flash chromatography (silica, 1:9 EtOAc/Hexanes) afforded compound 69 (1.50 g, 81%) as a yellow oil. Rf = 0.2 (EtOAc/Hexanes 1:9) 1  H NMR (300 MHz, CDCl3): δ = 0.52 (s, 6H, SiCH3), 2.04 (s, 3H, COCH3), 4.12 (s, 2H,  SiCH2), 7.51-7.48 (d, J = 8 Hz, 1H, ArH-3), 7.74-7.71 (dd, J = 2 Hz; 8 Hz, 1H, ArH-4), 7.85 (d, J = 2 Hz, 1H, ArH-6). 13  C NMR (100 MHz, CDCl3): δ = -4.65 (CH3), 20.84 (CH3), 55.80 (CH2), 118.44 (C),  119.36 (C), 124.29 (C), 135.23 (CH), 136.18 (CH), 136.59 (CH), 140.14 (C), 171.73 (C). HRMS (ESI) m/z: calculated for C12H14BrNO2Si [M+Na]+: 333.9869, found: 333.9875.  75 2-Cyano-4-(hydroxymethyldimethylsilyl)-bromobenzene 70 N OH Br  Si  To a solution of benzonitrile silane 69 (1.51 g, 5 mmol) in MeOH (40 mL) at 4°C was added slowly HCl(aq) (8 mL, 3N), then the mixture was stirred overnight at room temperature. Following pH neutralization with NaHCO3, the mixture was filtered and the solvent was removed under reduced pressure. The resulting oil was resuspended in EtOAc (80 mL) and the organic layer was washed with water (1 X 50 mL) and brine (3 X 50 mL), dried over anhydrous MgSO4, and concentrated to dryness. The crude product was purified by flash chromatography (silica, 3:7 EtOAc/Hexanes) to afford 70 (1.16 g, 89%) as a yellow oil. Rf = 0.2 (EtOAc/Hexanes 3:7) 1  H NMR (300 MHz, CDCl3): δ = 0.50 (s, 6H, SiCH3), 3.79 (s, 2H, SiCH2), 7.60-7.58 (d, J  = 8 Hz, 1H, ArH-3), 7.74-7.71 (dd, J = 2 Hz; 8 Hz, 1H, ArH-4), 7.84 (d, J = 2 Hz, 1H, ArH-6). 13  C NMR (100 MHz, CDCl3): δ = -4.61 (CH3), 54.24 (CH2), 118.71 (C), 119.20 (C),  124.05 (C), 135.23 (CH), 136.06 (CH), 136.98 (CH), 141.01 (C). HRMS (ESI) m/z: calculated for C10H12BrNOSi [M+Na]+: 291.9764, found: 291.9763. 7-Diethylamino-coumarin-3-ethyl ester (Dac-OEt) 73 6 7 N  5  8  4 3  O 1  O 2 O  O  To a mixture of diethylmalonate (4 mL, 30 mmol) and 4-diethylaminosalicylaldehyde (3.88 g, 20 mmol) in EtOH (40 mL) was added piperidine (1.5 mL, 15 mmol) and the mixture was refluxed for 1 hour and stirred at room temperature overnight. Following completion of reaction, the solvent was removed under reduced pressure and the crude product was resuspended in EtOAc (100 mL). The organic layer was washed with 1:9 (w/v) citric  76 acid/water (3 X 75 mL), brine (3 X 75 mL), dried over anhydrous MgSO4, and concentrated to dryness. The oily crude product was purified by flash chromatography (silica, 2:8 EtOAc/Hexanes) to afford 73 (3.38 g, 59%) as a yellow solid. Rf = 0.3 (EtOAc/Hexanes 1:1) 1  H NMR (300 MHz, CDCl3): δ = 1.25-1.20 (t, J = 7 Hz, 6H, NCH2CH3), 1.41-1.36 (t, J = 7  Hz, 3H, OCH2CH3), 3.47-3.40 (q, J = 7 Hz, 4H, NCH2CH3), 4.40-4.33 (q, J = 7 Hz, 2H, OCH2CH), 6.46 (d, J = 2 Hz, 1H, ArH-8), 6.61-6.58 (dd, J = 2 Hz; 9 Hz, 1H, ArH-6), 7.36-7.33 (d, J = 9 Hz, 1H, ArH-5), 8.42 (s, 1H, CH-4). HRMS (ESI) m/z: calculated for C16H19NO4 [M+Na]+: 312.1206, found: 312.1219. UV spectra (MeCN) λmax = 253 nm (carboxyl), 411 nm (Dac). 7-Diethylamino-coumarin-3-carboxylic acid (Dac-OH) 74 O OH N  O  O  To a solution of Dac-OEt 73 (3.38 g, 12 mmol) in MeOH (30 mL) at 4oC was added a solution of NaOH(aq) (30 mL, 0.5 M). After the mixture was stirred overnight at room temperature, the solvent was removed under reduced pressure. The crude salt was dissolved in an aqueous solution of 2 M HCl to reach a pH ~2 and the product was left crystallized overnight at 4oC to yield the orange solid compound 74 (2.98 g, 98%) with a trace amount of the methyl ester derivative. Rf = 0.2 (EtOAc) 1  H NMR (300 MHz, CDCl3): δ = 1.29-1.24 (t, J = 7 Hz, 6H, NCH2CH3), 3.53-3.45 (q, J =  7 Hz, 4H, NCH2CH3), 6.54-6.53 (d, J = 2 Hz, 1H, ArH-8), 6.73-6.69 (dd, J = 2 Hz; 9 Hz, 1H, ArH-6), 7.47-7.44 (d, J = 9 Hz, 1H, ArH-5), 8.66 (s, 1H, CH-4). 13  C NMR (100 MHz, CDCl3): δ = 12.5 (CH3), 45.5 (CH2), 97.1 (CH), 105.8 (C), 108.8  (C), 111.1 (CH), 132.1 (CH), 150.5 (CH), 153.9 (C), 158.2 (C), 164.6 (C), 165.7 (C). HRMS (ESI) m/z: calculated for C14H15NO4 [M-H]-: 260.0928, found: 260.0921.  77 UV spectra (MeCN) λmax = 262 nm (carboxyl), 428 nm (Dac). 2-[2-(2-{tert-Butoxycarbamate}-ethoxy)-ethoxy]-ethylamine 75 O  H2N  H N  O  O O  A solution of di-tert-butyldicarbonate (2.18 g, 10 mmol) in CH3Cl (60 mL) was added dropwise to a solution of 2,2'-(ethylenedioxy)-bis-(ethylamine) (15 mL, 100 mmol) in CH3Cl (100 mL) at 4oC. After having been stirred overnight at room temperature, the solvent was removed under reduced pressure to yield thick oil that was taken up in CH2Cl2 (100 mL). The organic layer was washed with brine (3 X 50 mL), dried over anhydrous MgSO4, and concentrated to dryness to afford product 75 (2.42 g, 97%) as a thick clear oil. 1  H NMR (300 MHz, CDCl3): δ = 1.43 (s, 9H, CH3tert-Bu), 2.89-2.85 (t, J = 5 Hz, 2H,  CH2NH2), 3.33-3.30 (m, 2H, CH2NH), 3.55-3.49 (m, 4H, CH2CO), 3.60 (s, 4H, OCH2CH2O), 5.14 (br s, 1H, NHCO). LRMS (ESI) m/z: calculated for C11H24N2O4 [M+H]+: 249.2, found: 249.4. Dac-N-(2-[2-(2-{tert-butoxycarbamate}-ethoxy)-ethoxy])-ethylamide 76a O N H N  O  O  O  O  H N  O O  To a mixture of Dac-OH 74 (1.40 g, 5 mmol), HOBt•H2O (1.44g, 10 mmol), EDC•HCl (2.05g, 10 mmol) in DMF (78 mL) at 4oC was added a solution of Boc protected spacer 75 (1.33 g, 5 mmol) in DMF (10 mL). Following addition of Et3N (1.5 mL, 10 mmol), the mixture was stirred for 30 min at 4oC and overnight at room temperature. The solvent was removed under reduced pressure to yield yellow oil that was resuspended in EtOAc (150 mL). The organic layer was washed with 2:8 (w/v) citric acid/water (3 X 100 mL), saturated NaHCO3(aq) (3 X 100 mL), brine (3 X100 mL), dried over anhydrous MgSO4, and concentrated to dryness. The crude oil was purified by flash chromatography (Silica, EtOAc) to yield product 76a (1.77 g, 67%) as a yellow oil with blue fluorescence.  78  Rf = 0.2 (EtOAc) 1  H NMR (300 MHz, CDCl3): δ = 1.25-1.18 (t, J = 7 Hz, 6H, NCH2CH3), 1.40 (s, 9H,  CH3tert-Bu), 3.33-3.30 (t, J = 5 Hz, 2H, CH2NH), 3.45-3.38 (q, J = 7 Hz, 4H, NCH2CH3), 3.56-3.52 (m, 4H, CH2CO), 3.64-3.62 (m, 8H, OCH2CH2O, CH2NH), 5.21 (s, 1H, NH), 6.46-6.45 (d, J = 2 Hz, 1H, ArH-8), 6.63-6.59 (dd, J = 2 Hz; 9 Hz, 1H, ArH-6), 7.40-7.37 (d, J = 9 Hz, 1H, ArH-5), 8.66 (s, 1H, CH-4), 9.02 (s, 1H, NH). LRMS (ESI) m/z: calculated for C25H37N3O7 [M+Na]+: 514.2, found: 514.2. Dac-N-(2’-[2’-(2’-{monomethoxytrityl-amino}-ethoxy)-ethoxy])-ethylamide 76b N  O  O  O H N O  O  O  N H  To a solution of Dac-OH 74 (0.57 g, 2 mmol) in CH2Cl2 (14 mL) at 4oC was added HOBt•H2O (0.29 g, 2 mmol), EDC•HCl (0.42 g, 2 mmol) and the mixture was stirred for 10 min. To the resulting mixture was added a solution of Mmt protected spacer 87 (0.61g, 1 mmol) in CH2Cl2 (2 mL), followed by addition of Et3N (0.3 mL, 2 mmol) at 4oC. After having been stirred at room temperature overnight, the solvent was removed under reduced pressure and the crude product was dissolved in EtOAc (100 mL). The organic layer was washed with saturated NH4Cl(aq) (3 X 50 mL), saturated NaHCO3(aq) (3 X 50 mL), brine (3 X 50 mL), dried over anhydrous MgSO4, and concentrated to dryness. The crude oil was purified by flash chromatography (Silica, 1:9 EtOAc/Et2O) to afford product 76b (0.90 g, 94%) as a yellow oil with blue fluorescence. Rf = 0.3 (EtOAc/Et2O 2:8) 1  H NMR (300 MHz, CD2Cl2): δ = 1.26-1.22 (t, J = 7 Hz, 6H, NCH2CH3), 2.14 (s, 1H,  NH), 2.34-2.31 (t, J = 5 Hz, 2H, CH2NH), 3.50-3.43 (q, J = 7 Hz, 4H, NCH2CH3), 3.643.55 (m, 10H, CH2NH, CH2CO, OCH2CH2O ), 3.77 (s, 3H, OCH3), 6.50-6.49 (d, J = 2 Hz, 1H, ArH-8), 6.70-6.66 (dd, J = 2 Hz; 9 Hz, 1H, ArH-6), 6.84-6.81 (d, J = 9 Hz, 2H,  79 ArHMmt), 7.31-7.16 (m, 6H, ArHMmt), 7.51-7.38 (m, 7H, ArHMmt, ArH-5), 8.65 (s, 1H, CH4), 8.89 (s, 1H, NH). 13  C NMR (100 MHz, CD2Cl2): δ = 12.3 (CH3), 39.5 (CH2), 43.3 (CH2), 45.1 (CH2), 55.2  (CH3), 69.9 (CH2), 70.4 (CH2), 70.6 (CH2), 71.3 (CH2), 96.5 (CH), 108.4 (C), 110.0 (CH), 110.5 (C), 113.1 (CH), 126.2 (CH), 127.8 (CH), 128.7 (CH), 129.9 (CH), 131.1 (CH), 138.4 (C), 146.7 (C), 147.8 (CH), 152.7 (C), 157.8 (C), 162.6 (C), 163.0 (C). HRMS (ESI) m/z: calculated for C40H45N3O6 [M+H]+: 664.3381, found: 664.3372. UV spectra (MeCN) λmax = 234 nm, 413 nm 2-[2-(2-Dac-N-ethoxy)-ethoxy]-ethylamine 76c O N H N  O  O  O  NH2  O  Method A: To a solution of compound 76a (1.78 g, 4 mmol) in CH2Cl2 (70 mL) at 4oC was slowly added TFA (8 mL, 108 mmol). After having been stirred for 4 hours at room temperature, the solution was concentrated to dryness under reduced pressure to afford the crude product 76c (1.64 g, 90%) as an ammonium trifluoroacetate salt. Method B: Compound 76b (0.87 g, 1 mmol) was dissolved in a mixture of 50 mL of 0.5:2:97.5 TES/TFA/CH2Cl2 (50 mL) at 4oC. After being stirred for 2 hours at room temperature, the solution was concentrated to dryness under reduced pressure. The crude oil was purified by flash chromatography (Silica, 0.1:1:8.9 NH4OH/EtOH/CH2Cl2) to yield product 76c (0.41 g, 80%) as a yellow clear thick oil. The product was stored as an ammonium chloride salt but the product was contaminated with trace of ethanol. Rf = 0.2 (NH4OH/EtOH/CH2Cl2 0.1:1:8.9) 1  H NMR (300 MHz, CD2Cl2): δ = 1.26-1.21 (t, J = 7 Hz, 6H, NCH2CH3), 2.84-2.81 (t, J =  5 Hz, 2H, CH2NH2), 3.76-3.27 (m, 14H, NCH2CH3, CH2CO, CH2NH, OCH2CH2O), 6.53-  80 6.52 (d, J = 2 Hz, 1H, ArH-8), 6.70-6.67 (dd, J = 2 Hz; 9 Hz, 1H, ArH-6), 7.48-7.45 (d, J = 9 Hz, 1H, ArH-5), 8.68 (s, 1H, CH-4), 8.90 (s, 1H, NH). 13  C NMR (100 MHz, CD2Cl2): δ = 12.3 (CH3), 39.5 (CH2), 42.0 (CH2), 45.1 (CH2), 69.9  (CH2), 70.4 (CH2), 70.6 (CH2), 73.5 (CH2), 96.5 (CH), 108.3 (C), 109.9 (CH), 110.0 (C), 131.1 (CH), 147.8 (CH), 152.7 (C), 157.8 (C), 162.6 (C), 163.0 (C). HRMS (ESI) m/z: calculated for C20H29N3O5 [M+H]+: 392.2180, found: 392.2187. 2-[2-(2-{Isobutyramide}-ethoxy)-ethoxy]-ethylamine 79 O H2N  O  O  NH  To a solution of 2,2'-(ethylenedioxy)-bis-(ethylamine) (8 mL, 55 mmol) in CH2Cl2 (200 mL) at -78oC was added by canulation over 1 hour a solution of isobutyric anhydride (2 mL, 14 mmol) in CH2Cl2 (250 mL) kept at 4oC. After being stirred overnight at room temperature, the solvent was evaporated under reduced pressure to afford a clear oil that was purified by flash chromatography (Silica, 0.1:2:9.7 NH4OH/EtOH/CH2Cl2) to afford product 79 (2.31 g, 77%) as a clear yellow oil. Rf = 0.3 (NH4OH/EtOH/CH2Cl2 0.1:2:9.7). 1  H NMR (300 MHz, CDCl3): δ = 1.08-1.06 (d, J = 7 Hz, 6H, CH3i-Pr), 2.36-2.26 (sixtuplet,  J = 7 Hz, 1H, CHi-Pr), 2.83-2.80 (t, J = 5 Hz, 2H, CH2NH2), 3.42-3.35 (m, 2H, CH2NH), 3.51-3.45 (m, 4H, CH2CO), 3.56 (s, 4H, OCH2CH2O), 6.31 (br s, 1H, NHCO). 13  C NMR (100 MHz, CDCl3): δ = 19.7 (CH3), 35.5 (CH), 39.1 (CH2), 41.8 (CH2), 70.0  (CH2), 70.2 (CH2), 70.3 (CH2), 73.4 (CH2), 117.2 (C). HRMS (ESI) m/z: calculated for C10H22N2O3 [M+Na]+: 241.1523, found: 241.1534.  81 N-α-Fmoc-L-valine-N-{2-[2-(2-{isobutyramide}-ethoxy)-ethoxy]-ethylamide} 80 O HN  O O  O  N H  H N  O O  To a solution of N-α-Fmoc-L-Val-CO2H (4.48 g, 13 mmol) in CH2Cl2 (100 mL) at 4oC was added HOBt•H2O (1.79 g, 13 mmol), and EDC•HCl (5.01 g, 13 mmol) and the mixture was stirred for 10 min. To the resulting mixture was added the amino spacer 79 (2.31 g, 13 mmol) followed by addition of Et3N (2 mL, 13 mmol) at 4oC and stirred overnight at room temperature. The reactive mixture was quenched in saturated NH4Cl(aq) (200 mL) and the product was extracted with EtOAc (6 X 50 mL). The organic layer was washed with 1 M HCl(aq) (3 X 150 mL), saturated NaHCO3(aq) (3 X 150 mL), brine (3 X 150 mL), dried over anhydrous MgSO4, and concentrated to dryness under reduced pressure. The crude product was purified by flash chromatography (Silica, 0.5:9.5 EtOH/EtOAc) to afford product 80 (5.43 g, 95%) as a white solid. Rf = 0.14 (EtOH/EtOAc 0.5:9.5) 1  H NMR (300 MHz, CDCl3): δ = 0.94 (br s, 6H, β-CH3Val), 1.12-1.10 (d, J = 7 Hz, 6H,  CH3i-Pr), 2.11-2.09 (m, 1H, β-CHVal), 2.35-2.31 (m, 1H, CHi-Pr), 3.48-3.41 (m, 12H, HNCH2CH2CO, OCH2CH2O), 3.96 (m, 1H, α-CHVal), 4.43-4.17 (m, 3H, CHFmoc, CH2Fmoc ), 5.64-5.61 (d, J = 9 Hz, 1H, NH), 6.12 (s, 1H, NH), 6.58 (s, 1H, NH), 7.41-7.26 (m, 4H, ArH), 7.58-7.56 (d, J = 7 Hz, 2H, ArH), 7.76-7.73 (d, J = 7 Hz, 2H, ArH). 13  C NMR (100 MHz, CDCl3): δ = 18.2 (CH3), 19.4 (CH3), 19.7 (CH3), 31.4 (CH), 35.7  (CH), 39.2 (CH2), 39.4 (CH2), 47.3 (CH), 60.8 (CH), 67.2 (CH2), 69.8 (CH2), 70.1 (CH2), 70.3 (CH2), 70.5 (CH2), 120.1 (CH), 125.2 (CH), 127.2 (CH), 127.9 (CH), 141.4 (C), 143.9 (C), 156.6 (C), 171.4 (C), 177.3 (C). HRMS (ESI) m/z: calculated for C30H41N3O6 [M+Na]+: 562.2888, found: 562.2880.  82 L-Valine-N-{2-[2-(2-{isobutyramide}-ethoxy)-ethoxy]-ethylamide} 81 O HN  O O  O  N H  NH2  To a solution of N-α-Fmoc-L-valine derivative 80 (1.40 g, 2 mmol) in CH2Cl2 (26 mL) was added TAEA (19 mL, 130 mmol) and the mixture was stirred for 60 min. Following completion of the reaction, the mixture was diluted in CH2Cl2 (180 mL) and the organic layer was washed with saturated NaHCO3(aq) (3 x 50 mL), brine (50 mL), dried over anhydrous MgSO4, and concentrated to dryness under reduced pressure. The crude oil was purified by flash chromatography (Silica, 0.5:0.5:9 H2O/AcOH/2-propanol) to afford the product 81 as an ammonium acetate salt which was diluted in HCl(aq) (120 mL, 0.1M). The acetic acid was extracted with CH2Cl2 (5 x 25 mL) and the aqueous layer was concentrated to dryness under reduce pressure to afford product 81 (0.67 g, 73%) as a white ammonium chloride salt. Rf = 0.3 (H2O/ AcOH/ 2-propanol 0.5:0.5:9) 1  H NMR (300 MHz, CDCl3): δ = 0.82 (d, J = 7 Hz, 3H, β-CH3Val), 0.98 (d, J = 7 Hz, 3H,  β-CH3Val), 1.16-1.14 (d, J = 7 Hz, 6H, CH3i-Pr), 1.25 (br s, 1H, NH2), 2.38-2.27 (m, 2H, CHi-Pr, β-CHVal), 3.23 (br s, 1H, α-CHVal), 3.47-3.44 (m, 4H, CH2NH), 3.56-3.54 (m, 4H, CH2CO), 3.61 (s, 4H, OCH2CH2O), 6.03 (br s, 1H, NH), 7.52 (br s, 1H, NH). HRMS (ESI) m/z: calculated for C15H31N3O4 [M+Na]+: 340.2207, found: 340.2212. Diethyl ester-(2S,3S)-O-isopropylidene-D-tartrate 83a O  O O O  O O  To a solution of D-(-)diethyltartrate (4 mL, 24 mmol) in cyclohexane (21 mL) was added 2,2-dimethoxypropane (24 mL, 192 mmol) and p-toluenesulfonic acid hydrate (0.046 g, 0.2 mmol). The mixture was heated to reflux in a flask equipped with a Dean–Stark apparatus for 3 hours followed by addition of anhydrous Na2CO3 (0.04 g, 0.3 mmol) at room  83 temperature. After being stirred for 1 hour, the salt was removed by filtration and the mixture was concentrated to dryness under reduced pressure. The crude oil was taken up in Et2O (100 mL) and the organic layer was washed with saturated K2CO3(aq) (3 x 75 mL), water (75 mL), brine (3 x 75 mL), dried over anhydrous MgSO4, and concentrated to dryness. The resulting oil was distilled to yield a mixture of diethyl ester 83a with a small amount of methyl ethyl diester 83b. The mixture was purified by flash chromatography (Silica, 3:7 Et2O/Hexanes) to afford diethyl ester 83a (5.05 g, 85%) contaminated with a trace amount of methyl ethyl diester 83b. Rf = 0.2 (Et2O/Hexanes 3:7) 1  H NMR (300 MHz, CDCl3): δ = 1.27-1.23 (t, J = 7 Hz, 6H, OCH2CH3), 1.43 (s, 6H,  C(CH3)2), 4.25-4.17 (q, J = 7 Hz, 4H, OCH2CH3), 4.70 (s, 2H, CH). 13  C NMR (100 MHz, CDCl3): δ = 14.2 (CH3), 26.5 (CH3), 61.9 (CH2), 77.1 (CH), 113.5  (C), 169.7 (C). HRMS (ESI) m/z: calculated for C9H14O6 [M+Na]+: 269.0996, found: 269.1001. Ethyl ester-(2S,3S)-O-isopropylidene-D-tartaric acid 84 O  O O O  HO O  To a solution of diester 83a/b (0.97 g, 4 mmol) in EtOH (6 ml) at 4oC was added KOH (0.22 g, 4 mmol) in EtOH (7 mL). The mixture was stirred at room temperature for 2 hours, the solvent was removed under reduced pressure and the product was stored as a white potassium tartrate salt. Just before being used, the crude salt was purify by flash chromatography (Silica, 0.3:1:4:5 AcOH/Acetone/n-Heptane/EtOAc) to afford the ethyl ester tartaric acid 84 (0.84 g, 98%) as a colourless clear oil. All AcOH was removed under high vacuum because the product 84 was used for amidation. Rf = 0.3 (AcOH/Acetone/n-Heptane/EtOAc 0.3:1:4:5)  84 1  H NMR (300 MHz, CDCl3): δ = 1.33-1.29 (t, J = 7 Hz, 3H, OCH2CH3), 1.48 (s, 3H,  C(CH3)2), 1.50 (s, 3H, C(CH3)2), 4.32-4.25 (q, J = 7 Hz, 2H, OCH2CH3), 4.79-4.77 (d, J = 5 Hz, 1H, CH), 4.87-4.85 (d, J = 5 Hz, 1H, CH), 7.6 (br s, 1H, OH). 13  C NMR (100 MHz, CDCl3): δ = 14.2 (CH3), 26.5 (CH3), 62.4 (CH2), 76.5 (CH), 77.3  (CH), 114.3 (C), 169.8 (C), 174.5 (C). HRMS (ESI) m/z: calculated for C9H14O6 [M-H]-: 217.0718, found: 217.0717. N-α- (1-Ethyl ester-2,3-O-isopropylidene-D-tartramide)-L-Valine-N-{2-[2-(2{isobutyramide}-ethoxy)-ethoxy]-ethylamide} 85 O HN  O O  O  N H  O  H N O  O  O OEt  To a solution of ethyl ester tartaric acid 84 (0.62 g, 3 mmol) in CH2Cl2 (19 mL) at 4oC was added HOBt•H2O (0.38 g, 3 mmol), EDC•HCl (1.08 g, 3 mmol) and the mixture was stirred for 10 min. To the resulting mixture was added the Valine derivative 81 (0.67 g, 2 mmol) followed by addition of Et3N (0.4 mL, 3 mmol) at 4oC. After having been stirred overnight at room temperature, the solvent was removed under reduced pressure and the crude product was resuspended in EtOAc (100 mL). The organic layer was washed with saturated NH4Cl(aq) (3 X 50 mL), saturated NaHCO3(aq) (3 X 50 mL), brine (3 X 50 mL), dried under anhydrous MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (Silica, 1:9 EtOH/EtOAc) to yield product 85 (0.74 g, 75%) as thick yellow oil. Rf = 0.3 (EtOH/EtOAc 1:9) 1  H NMR (300 MHz, CDCl3): δ = 0.95-0.91 (m, 6H, β-CH3Val), 1.13-1.11 (d, J = 7 Hz, 6H,  CH3i-Pr), 1.31-1.27 (t, J = 7 Hz, 3H, OCH2CH3), 1.45 (s, 3H, C(CH3)2), 1.50 (s, 3H, C(CH3)2), 2.17-2.03 (m, 1H, β-CHVal), 2.40-2.28 (m, 1H, CHi-Pr), 3.46-3.38 (m, 4H, CH2NH), 3.56-3.51 (m, 4H, CH2CO), 3.58 (s, 4H, OCH2CH2O), 4.29-4.18 (m, 3H, OCH2CH3, α-CHVal), 4.68-4.66 (d, J = 5 Hz, 1H, CH), 4.77-4.75 (d, J = 5 Hz, 1H, CH), 6.22 (br s, 1H, NH), 6.64 (br s, 1H, NH), 7.14-7.11 (d, J = 9 Hz, 1H, NH).  85 13  C NMR (100 MHz, CDCl3): δ = 14.3 (CH3), 18.3 (CH3), 19.4 (CH3), 19.8 (CH3), 26.4  (CH3), 26.9 (CH3), 31.3 (CH), 35.7 (CH), 39.2 (CH2), 39.4 (CH2), 58.5 (CH), 62.2 (CH2), 69.7 (CH2), 70.2 (CH2), 70.3 (CH2), 70.5 (CH2), 78.0 (CH), 113.6 (C), 170.0 (C), 170.1 (C), 170.5 (C), 177.4 (C). HRMS (ESI) m/z: calculated for C24H43N3O9 [M+H]+: 518.3072, found:518.3078. N-α- (1-Carboxylic-2,3-O-isopropylidene-D-tartramide)-L-Valine-N-{2-[2-(2{isobutyramide}-ethoxy)-ethoxy]-ethylamide} 86 O HN  O O  O  N H  O  H N O  O  O OH  To a solution of KOH (0.10 g, 2 mmol) in EtOH (18 mL) was added the soluble tartrate linker precursor 85 (0.74 g, 1.4 mmol). After being stirred for 2 hours, the solvent was removed under reduced pressure and the product was stored as a white potassium tartrate salt. Just before being used, the crude salt was purified by flash chromatography (Silica, 1:2:1:1:5 AcOH/2-propanol/Acetone/n-Heptane/EtOAc) to afford product 86 (0.64 g, 91%) as a white foamy solid contaminated with a trace amount of AcOH and the deprotected diol derivative. Rf = 0.2 (AcOH/2-propanol/Acetone/n-Heptane/EtOAc 1:2:1:1:5) 1  H NMR (300 MHz, CDCl3): δ = 0.98-0.93 (m, 6H, β-CH3Val), 1.17-1.15 (d, J = 7 Hz, 6H,  CH3i-Pr), 1.50 (s, 6H, C(CH3)2), 2.16-2.09 (m, 1H, β-CHVal), 2.44-2.39 (m, 1H, CHi-Pr), 3.48-3.46 (m, 4H, CH2NH), 3.76 (s, 0.7H, OH), 3.62-3.58 (m, 8H, CH2CO, OCH2CH2O), 4.32-4.27 (m, 1H, α-CHVal), 4.47-4.44 (d, J = 5 Hz, 1H, CH), 4.55-4.53 (d, J = 5 Hz, 1H, CH), 6.42 (br s, 1H, NH), 7.46-7.44 (d, J = 9 Hz, 1H, NH), 7.58 (br s, 1H, NH). 13  C NMR (100 MHz, CDCl3): δ = 18.2 (CH3), 19.3 (CH3), 19.8 (CH3), 26.1 (CH3), 26.3  (CH3), 31.3 (CH), 35.6 (CH), 39.1 (CH2), 39.4 (CH2), 58.2 (CH), 63.8 (CH2), 69.7 (CH2), 70.2 (CH2), 70.3 (CH2), 78.6 (CH), 112.3 (C), 171.0 (C), 171.6 (C), 174.7 (C), 177.8 (C). HRMS (ESI) m/z: calculated for C22H39N3O9 [M+Na]+: 512.2579, found: 512.2579.  86 2-[2-(2-{Monomethoxytrityl-amino}-ethoxy)-ethoxy]-ethylamine 87  HN  O  O O H2N  To a mixture of anhydrous K2CO3 (1.12 g, 8 mmol) and 2,2'-(ethylenedioxy)-bis(ethylamine) (6.00 g, 40 mmol) in CH2Cl2 (250 mL) at 4oC was added slowly a solution of p-anisylchlorodiphenyl methane (2.62 g, 8 mmol) in CH2Cl2 (100 mL). After the mixture was stirred at room temperature for 4 hours, K2CO3 was filtered out and the solvent was evaporated under reduced pressure. The residual oil was resuspended in CH2Cl2 (200 mL), washed with saturated K2CO3(aq) (2 X 100 mL), dried over anhydrous MgSO4, and concentrated to dryness. The crude oil was purified by flash chromatography (Silica, 0.1:0.5:9.4 NH4OH/EtOH/CH2Cl2) to afford product 87 (3.21 g, 90%) as a clear lightly yellow thick oil. Rf = 0.15 (NH4OH/EtOH/CH2Cl2 0.1:0.5:9.4) 1  H NMR (300 MHz, CDCl3): δ = 2.37-2.34 (t, J = 5 Hz, 2H, CH2NH2), 2.82 (m, 2H,  CH2NH), 3.62-3.46 (m, 8H, CH2CO, OCH2CH2O), 3.77 (s, 3H, OCH3), 6.81-6.78 (d, J = 9 Hz, 2H, ArHMmt), 7.28-7.14 (m, 6H, ArHMmt), 7.39-7.36 (d, J = 9 Hz, 2H, ArHMmt), 7.487.45 (d, J = 7 Hz, 4H, ArHMmt). 13  C NMR (100 MHz, CD2Cl2): δ = 41.9 (CH2), 43.2 (CH2), 55.3 (CH3), 70.3 (CH2), 70.4  (CH2), 71.4 (CH2), 73.6 (CH2), 113.2 (CH), 126.3 (CH), 127.9 (CH), 128.8 (CH), 130.0 (CH), 138.4 (C), 146.6 (C), 158.0 (C). HRMS (ESI) m/z: calculated for C26H32N2O3 [M+Na]+: 443.2305, found: 443.2299.  87  Soluble fluorescent tartrate-based linker 88 O  O O HN  O O  O  N H  O  H N  H N  O O  O  O  NEt2  O  O  N H  To the fluorescent spacer 76c (0.05 g, 0.1 mmol) in CH2Cl2 (2 mL) at 4oC was added the soluble tartaric linker precursor 86 (0.11 g, 0.2 mmol), HOBt•H2O (0.02g, 0.2 mmol), EDC•HCl (0.03 g, 0.2 mmol). The mixture was stirred for 5 min followed by addition of Et3N (0.03 mL, 0.2 mmol) at 4oC. After having been stirred overnight at room temperature, the solvent was removed under reduced pressure and the crude product was resuspended in EtOAc (50 mL). The organic layer washed with saturated NH4Cl(aq) (3 X 25 mL), saturated NaHCO3(aq) (3 X 25 mL), brine (3 X 25 mL), dried under anhydrous MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (Silica, 0.9:9.1 EtOH/DCM) to afford product 88 (0.06 g, 62%) as a yellow solid with a trace amount of the diol deprotected derivative 88’. The product 88 was further analysed by RP-HPLC (column (1)) as follows. The product 88 was diluted to 1 mg/mL in 1:1 MeCN/H2O and 1 μL of the solution was loaded on RPHPLC. The elution was carried out as follows with buffer B given as a percentage of total mobile phase composition: 5% buffer B over 5 min; 5% to 34.2% over 25 min; 34.2% to 100% over 5 min, 100% over 5 min and 100% to 5% over 5 min. Four peaks were detected (410 nm), collected and analyzed by HRMS (ESI). The eluates were denoted as follows: peak A (36.2 min, 4% area) corresponding to the diol deprotected derivative 88’; peak B (37.1 min, 78% area) corresponding to the product 88; peak C (37.6 min, 8% area) and peak D (37.9 min, 9% area). The peaks C and D were collected in the same fraction which was shown to be composed of the protected diol 88. The compound 88 (0.075 mmol, 0.064 g) was dissolved in neat TFA (134.62 mmol, 10 mL) and mixed for 6 hours. The crude product was concentrated to dryness under reduced pressure, diluted in 1:1 MeCN/H2O and loaded on RP-HPLC (column (1)). The elution was carried out as described above affording peaks with the same retention time but with  88 different areas such as, 24% area for peak A, 57% area for peak B, 6% area for peak C and 13% area for peak D. Product 88 (peak B, RP-HPLC): Rf = 0.2 (EtOH/DCM 0.9:9.1) 1  H NMR (300 MHz, CD2Cl2): δ = 0.97-0.91 (m, 6H, β-CH3Val), 1.12-1.10 (d, J = 7 Hz, 6H,  CH3i-Pr), 1.26-1.21 (t, J = 7 Hz, 6H, NCH2CH3), 1.48 (s, 6H, C(CH3)2), 2.24-1.93 (m, 1H, β-CHVal), 2.44-2.29 (m, 1H, CHi-Pr), 3.64-3.38 (m, 28H, NCH2CH3, OCH2CH2O, HNCH2CH2CO), 4.29-4.24 (m, 1H, α-CHVal), 4.57-4.55 (d, J = 7 Hz, 1H, CH), 4.61-4.59 (d, J = 7 Hz, 1H, CH), 6.24 (br s, 1H, NH), 6.55-6.54 (d, J = 2 Hz, 1H, ArH-8), 6.73-6.69 (dd, J = 2 Hz; 9 Hz, 1H, ArH-6), 6.92 (br s, 1H, NH), 7.36 (br s, 2H, NH), 7.49-7.46 (d, J = 9 Hz, 1H, ArH-5), 8.67 (s, 1H, CH-4), 8.95 (br s, 1H, NH). 13  C NMR (100 MHz, CDCl3): δ = 12.6 (CH3), 18.1 (CH3), 19.5 (CH3), 19.8 (CH3), 26.2  (CH3), 26.4 (CH3), 30.8 (CH), 35.6 (CH), 39.2 (CH2), 39.3 (CH2), 39.6 (CH2), 41.2 (CH), 45.2 (CH2), 58.7 (CH), 69.7 (CH2), 69.8 (CH2), 70.0 (CH2), 70.1 (CH2), 70.3 (CH2), 70.4 (CH2), 70.6 (CH2), 77.8 (CH), 96.7 (CH), 108.5 (C), 110.1 (CH), 110.3 (C), 112.7 (C), 131.3 (CH), 148.2 (CH), 152.7 (C), 157.8 (C), 162.8 (C), 163.5 (C), 169.9 (C), 170.1 (C), 170.8 (C), 177.4 (C). HRMS (ESI) m/z: calculated for C42H66N6O13 [M+Na]+: 885.4580, found: 885.4574. HRMS (ESI) m/z: calculated for C42H66N6O13 [M+H]+: 863.4761, found: 863.4844. UV spectra (MeCN) λmax = 258 nm (carboxyl), 414 nm (Dac). Product 88’ (peak A, RP-HPLC): Rf = 0.03 (0.9:9.1 EtOH/DCM) HRMS (ESI) m/z: calculated for C39H62N6O13 [M+H]+: 823.4448, found: 823.4460. UV spectra (MeCN) λmax = 262 nm (carboxyl), 420 nm (Dac).  89 Methyl 2-(2,5-dibromobenzamido)acetate 95 O Br  O  N H  O  Br  To a solution of 2,5-dibromobenzoic acid (1.00 g, 3 mmol) in DMF (36 mL) at 4oC was dissolved HOBt•H2O (0.96 g, 7 mmol), EDC•HCl (1.37 g, 7 mmol) and H-Gly-OMe•HCl (0.45 g, 3 mmol). The mixture was stirred for 10 min followed by addition of Et3N (1.5 mL, 11 mmol) at 4oC. After having been stirred overnight at room temperature, the mixture was quenched in 1:9 (w/v) citric acid/water (150 mL) and the product was extracted with EtOAc (5 X 50 mL). The organic layer was washed with 1:9 (w/v) citric acid/water (3 X 100 mL), saturated NaHCO3(aq) (3 X 100 mL), brine (3 X 100 mL), dried under anhydrous MgSO4, and the solvent was removed under reduced pressure. The resulting white solid was purified by flash chromatography (Silica, 2:3 Hexanes/Et2O) to afford product 95 (1.15 g, 91%) as a white solid. Rf = 0.3 (Hexanes/Et2O 2:3) 1  H NMR (300 MHz, CDCl3): δ = 3.81 (s, 3H, OCH3), 4.26-4.25 (d, J = 6 Hz, 2H, CH2),  7.72-7.42 (m, 3H, ArH), 8.10 (s, 1H, NH). LRMS (ESI) m/z: calculated for C10H9Br2NO3 [M+Na]+: 371.9, 373.9. 375.9, found: 371.8, 373.8, 375.8. 2-(2,5-Dibromobenzamido)acetic acid 96 Br H N Br  O OH  O  To a solution of KOH (0.68g, 12 mmol) in 1:1 MeOH/water (67 mL) was added methyl 2(2,5-dibromobenzamido) acetate 95 (1.15 g, 3 mmol) and the mixture was heated to reflux for 1 hour. The solvent was removed under reduced pressure and the resulting salt was purified by flash chromatography (Silica, 0.1:1:8.9 AcOH/2-propanol/CH2Cl2) followed by  90 triturating in 2:8 Et2O/Hexanes to afford product 96 (1.02 g, 93%) as a white solid. AcOH was removed under high vacuum because the product 96 was used for amidation. Rf = 0.3 (AcOH/2-propanol/CH2Cl2 0.1:1:8.9) 1  H NMR (300 MHz, Acetone (D3)): δ = 4.16-4.14 (d, J = 6 Hz, 2H, CH2), 7.64-7.51 (m,  3H, ArH), 7.92 (s, 1H, NH) 13  C NMR (100 MHz, Acetone (D3)): δ = 40.8 (CH2), 118.4 (C), 120.7 (C), 131.9 (CH),  134.0 (CH), 135.1 (CH), 140.5 (C), 166.1 (C), 170.1 (C). HRMS (ESI) m/z: calculated for C9H7Br2NO3 [M-H]-: 333.87, 335.87, 337.87, found: 333.8714, 335.8705, 337.8697. N-ε-Dac-N-α-Fmoc-L-lysine 103 O  O OH HN  HN O  O O  O N  To a mixture of N-hydroxysuccinimide (0.07 g, 0.6 mmol) and EDC•HCl (0.11 g, 0.6 mmol) in DMF (2 mL) at 4oC was added a solution of Dac-OH 74 (0.10 g, 0.4 mmol) in DMF (3 mL). After being stirred at room temperature for 48 hours, the mixture was diluted in ethyl acetate (100 mL). The organic layer was washed with brine (3 X 100 mL), dried under anhydrous MgSO4, and the solvent was removed under reduced pressure to yield Dac-NHS 102 as an orange solid which was taken forward in the synthesis without further purification. To a solution of Dac-NHS 102 (0.14 g, 0.4 mmol) in DMF (20 mL) at 4oC was added a solution of N-α-Fmoc-L-Lys-OH (2.92 g, 8 mmol) in DMF (60 mL). After being stirred at room temperature overnight, the solvent was evaporated under reduced pressure and the crude product was dissolved in EtOAc (100 mL). The organic layer was washed with 2M HCl(aq) (3 X 100 mL), dried under MgSO4, and the solvent was removed under reduced pressure to yield a yellow solid. The crude product was purified by flash chromatography  91 (Silica, 0.1:0.7:9.2 AcOH/2-propanol/DCM) to afford product 103 (0.17 g, 71%) as a yellow foam. Rf = 0.2 (AcOH/EtOH/DCM 0.1:0.5:9.4) 1  H NMR (300 MHz, CD2Cl2): δ = 1.21-1.17 (t, J = 7 Hz, 6H, NCH2CH3), 1.95-1.50 (m,  6H, CH2Lys), 3.54-3.40 (m, 6H, NCH2CH3, CH2Lys), 4.36-4.16 (m, 4H, CH2Fmoc, α-CHLys, CHFmoc), 6.00 (br s, NH), 6.42 (s, 1H, ArH-8), 6.55 (br s, 1H, ArH-6), 7.36-7.27 (m, 5H, ArH-5, ArHFmoc), 7.62-7.57 (m, 2H, ArHFmoc), 7.72 (br s, 2H, ArHFmoc), 8.68 (s, 1H, CH4), 9.02 (br s, 1H, NH). 13  C NMR (100 MHz, CD2Cl2): δ = 12.3 (CH3), 22.2 (CH2), 29.2 (CH2), 31.1 (CH2), 38.7  (CH2), 45.1 (CH2), 47.3 (CH), 67.0 (CH2), 96.4 (CH), 108.4 (C), 109.6 (C), 110.1 (CH), 119.9 (CH), 125.3 (CH), 127.1 (CH), 127.7 (CH), 131.2 (CH), 141.3 (C), 144.0 (C), 148.4 (CH), 152.9 (C), 156.4 (C), 157.8 (C), 162.7 (C), 164.1 (C), 174.1 (C). HRMS (ESI) m/z: calculated for C35H37N3O7 [M-H]-: 610.2559, found: 610.2563. U.V. spectra (MeOH) λmax = 261 nm (N-α-Fmoc group), 419 nm (Dac). N-α-Trt-L-Tryptophan triethylammonium salt 108 H N O O NHEt3+ NH  To a solution of trityl chloride (30.03 g, 108 mmol) in 2:1 CHCl3/DMF (300 mL) at 4oC was added L-tryptophan (10 g, 49 mmol) by portion and the mixture was stirred for 2 hours. Following addition of Et3N (27 mL, 196 mmol) over 20 min at 4oC, the mixture was stirred overnight at room temperature. To the resulting yellow solution was added MeOH (300 mL) and the mixture was refluxed for 5 hours. The resulting brown mixture was cooled to room temperature, divided into three fractions (200 mL) and the product was extracted with Et2O (150 mL). Each organic layer was washed with 1:9 (w/v) citric acid/water (3 X 100 mL), brine (3 X 100 mL) and dried over anhydrous MgSO4. The  92 product was precipitated overnight at 4oC by adding Et3N (2 ml) to each organic layer. The resulting salt was filtered out, washed with Et2O and dried under reduced pressure to yield product 108 (21 g, 79%) as a beige triethylammonium salt. Rf = 0.3 (Hexanes/EtOAc 2:8) 1  H NMR (300 MHz, CD2Cl2): δ = 1.04-1.00 (t, J = 7 Hz, 9H, NCH2CH3), 2.75-2.65 (m,  7H, NCH2CH3, β-CHTrp), 3.13-3.07 (dd, J = 6 Hz; 7 Hz, 1H, β-CHTrp), 3.64-3.60 (t, J = 6 Hz, 1H, α-CHTrp), 7.40-7.05 (m, 19H, ArH), 7.54-7.51 (d, J = 7 Hz, 1H, ArH), 8.20 (br s, 1H, NH). HRMS (ESI) m/z: calculated for C30H26N2O2 [M-H]-: 445.1922, found: 445.1919. 2.2. Solid phase synthesis of the benzonitrile silyl linker Hydroquinone acetate resin 67 O O  O  To the Merrifield chloro resin (2.00 g, 2 mmol) swollen and resuspended in DMF (20 mL) was added hydroquinone monoacetate 66 (1.53 g, 9 mmol) and anhydrous K2CO3 (1.37 g, 10 mmol). After having been gently stirred at 75oC for 48 hours, the mixture was diluted with EtOH (9 ml) and quenched with addition of 2:1 EtOH/3 M HCl(aq) (20 mL). The resin was filtered with EtOH (5 X 20 mL), THF (5 X 20 mL), MeOH (5 X 20 mL), water (5 X 20 mL), MeOH (5 X 20 mL) and dried under reduced pressure to afford resin 67. FT-IR (KBr): 1760 cm-1 (carbonyl). Hydroquinone resin 68 O  OH  To the monoacetate hydroquinone resin 67 (2.11 g) swollen and resuspended in THF (17 mL) at 4oC was added a solution of LiAlH4 (7 mL, 1 M in THF). After having been gently stirred for 16 hours at room temperature, the mixture was quenched at 4oC with the  93 addition of EtOAc (10 mL) over 30 min, followed by addition of methanol (10 mL). The mixture was stirred at room temperature for one hour and the resin was filtered with THF (5 X 30 mL), MeOH (5 X 30 mL), 1:1 MeOH/3 M HCl (5 X 30 mL), water (5 X 30 mL), MeOH (5 X 30 mL), and dried under reduced pressure to afford resin 68. FT-IR (KBr): disappearance of 1760 cm-1. Benzonitrile silyl resin 71 Br Si  N  O  O  To a solution of benzonitrile silyl 70 (1.16 g, 4 mmol) in NMM (6 mL) at 4oC was added Ph3P (1.13 g, 4 mmol) and DIAD (0.8 mL, 4 mmol). After being stirred for 30 min, hydroquinone resin 68 (1.32 g) was added to the mixture, warmed to 35oC and gently stirred for 4 days. The resin was filtered with THF (5 X 30 mL), CH2Cl2 (5 X 30 mL), 8:2 CH2Cl2/MeOH (5 X 30 mL), 1:1 CH2Cl2/MeOH (5 X 30 mL), 2:8 CH2Cl2/MeOH (5 X 30 mL), MeOH, water (5 X 30 mL), MeOH (5 X 30 mL), and dried under reduced pressure to afford resin 71. FT-IR (KBr): 2225 cm-1 (nitrile). Benzoic benzonitrile silyl resin 72 O OH  Si O  N  O  To the resin 71 (1.57 g) swollen and resuspended in toluene (15 mL) was added 4boronobenzoic acid (0.65 g, 4 mmol), Na2CO3(aq) (2 mL, 2 M), EtOH (2 mL) and Pd(Ph3P)4 (0.18 g, 0.15 mmol). After being gently stirred at 90oC for 18 hours, the resin was filtered  94 with EtOH (5 X 30 mL), 1:1 EtOH/water (5 X 30 mL), EtOH (5 X 30 mL), toluene (5 X 30 mL), THF (5 X 30 mL), 1:1 MeOH/CH2Cl2 (5 X 30 mL), MeOH (5 X 30 mL) and dried under reduced pressure to afford resin 72. A sample of resin 72 was exposed to an excess of TFA for 5 hours, filtered and dried under reduced pressure. FT-IR (KBr): 2223 cm-1 (nitrile), 1727 cm-1 (carbonyl). Fluorescent benzonitrile silyl resin 77 O  O N H Si O  O  O  O  N  H N O  N  O  To the resin 72 (0.30 g) swollen and resuspended in DMF (8 mL) was added a mixture of HOBt•H2O (0.07 g, 0.5 mmol), EDC•HCl (0.09 g, 0.5 mmol), Et3N (0.06 mL, 0.5 mmol) and fluorescent spacer 76c (0.18 g, 0.5 mmol) in DMF (3 mL). After being gently stirred for 16 hours, the resin was filtered with DMF (5 X 10 mL), THF (5 X 10 mL), EtOH (5 X 10 mL), MeOH (5 X 10 mL), 1:1 MeOH/CH2Cl2 (5 X 10 mL), CH2Cl2 (5 X 10 mL) and dried under reduced pressure to afford fluorescent resin 77. A sample of resin 77 was exposed to an excess of TFA for 5 hours, filtered, dried under reduced pressure and exposed under UV lamp.  95 2.3. Cleavage of product from benzonitrile silyl linker on resin Cleavage of product from resin 72 with TBAF-TFA O OH  N  To the resin 72 (0.02 g) swollen and resuspended in CH2Cl2 (2 mL) at 4oC was added a solution of TBAF (2 mL, 1M in THF) with or without TFA (2 mL). After having been gently stirred for 4 hours at room temperature, the mixture was drained out of the resin by filtration. More products were recovered by filtering the resin with MeOH (5 X 10 mL), 1:1 MeOH/CH2Cl2 (5 X 10 mL) and CH2Cl2 (5 X 10 mL). All eluates were pooled into a same fraction and solvents were evaporated under reduced pressure but we were not able to isolate the product. Cleavage of product from resin 77 with TBAF-TFA O  O N H  O  O  O  N  H N O  N  To the resin 77 (0.02 g) swollen and resuspended in CH2Cl2 (2 mL) at 4oC was added a solution of TBAF (2 mL, 1M in THF) with or without TFA (2 mL). After having been gently stirred for 4 hours at room temperature, the mixture was drained out of the resin by filtration. More products were recovered by filtering the resin with MeOH (5 X 10 mL), 1:1 MeOH/CH2Cl2 (5 X 10 mL) and CH2Cl2 (5 X 10 mL). All eluates were pooled into a same fraction and solvents were evaporated under reduced pressure but we were not able to isolate the product.  96 2.4. Solid phase synthesis of the tartrate linker Reactions were carried under atmosphere at room temperature by shaking in Zeba Desalt polypropylene spin column (5 mL format) from PierceTM. The amino PEGA resin 89 was handled in swollen state and high vacuum was avoided due to the fragile resin structure when dried. The general procedure below describes a typical synthesis that starts with the amino PEGA resin 89 (0.40 g, dry resin). Resin Swelling: When the amino PEGA resin 89 was stored dry or the solvent system A needed to be changed for solvent B, then the resin was filtered with CH2Cl2 (50 mL) and solvent B (50 mL). The resin was shaken in solvent B (4 mL) for 10 min, filtered and the procedure was repeated three more time with fresh solvent B. Reaction: After resin washing or swelling, the reactive mixture in solvent B was added to the amino PEGA resin derivative and shaken for the indicated time. Washing: After completion of reaction, the amino PEGA resin derivative was filtered with solvent B (50 mL), shaken in solvent B (4 mL) for 3 min and filtered. The shaking procedure was repeated once for 3 min and twice for 5 min, each time with fresh solvent B. A resin sample was filtered with CH2Cl2, EtOH and submitted to Kaiser test or Green Malachite test. Storage: The amino PEGA resin derivative was filtered with CH2Cl2 (50 mL), dried under air filtration or kept wet in EtOH and stored at -20oC.  97  Fmoc-Val-PEGA resin 90  O  H N  O  N H  O  After being washed and swollen in DMF, a mixture of N-α-Fmoc-L-Val-OH (0.27 g, 0.8 mmol), HOBt•H2O (0.11 g, 0.8 mmol), HBTU (0.30 g, 0.8 mmol) and DIEA (0.27 mL, 1.6 mmol) in DMF (4 mL) was added to the amino PEGA resin 89 (0.40 g). The mixture was gently shaken for 1 hour and drained from the resin. After having been filtered with DMF (10 mL), a mixture of N-α-Fmoc-(L)Val-OH (0.13 g, 0.4 mmol), HOBt•H2O (0.05 g, 0.4 mmol), HBTU (0.15 g, 0.4 mmol), DIEA (0.13 mL, 8 mmol) in DMF (4 mL) was poured on the resin and gently shaken for 2 hours. The resin was washed with DMF to afford Fmoc-Val-PEGA resin 90. The loading was found to be 0.35 mmol per gram of dry resin. Tartrate-Val-PEGA resin 93  O KO  O  O  H N O  O N H  After being washed and swollen in DMF, a solution of 20% piperidine in DMF (4 mL) was added to the Fmoc-Val-PEGA resin 90 (0.40 g). The mixture was gently shaken for 5 min, the solution was drained from the resin and the procedure was repeated for 10 min. The resin was washed in DMF to afford the Fmoc-deprotected Val-PEGA resin 91. To the drained resin 91 (0.40 g) was added a mixture of tartaric acid derivative 84 (0.13 g, 0.6 mmol), PyBOP (0.33 g, 0.6 mmol), DMAP (0.008 g, 0.06 mmol), and DIEA (0.16 mL, 0.9 mmol) in DMF (4 mL). The mixture was gently shaken for 1 hour and the solution was drained from the resin. After having been filtered with DMF (10 mL), a fresh activated tartrate mixture was poured on the resin and gently shaken for 2 hours. The resin was washed in DMF to afford the ethyl ester tartrate-Val-PEGA resin 92.  98 The resin 92 (0.40 g) was filtered with MeOH (50 mL) and swollen in 2:1 MeOH/water (4 mL). To the drained resin was added a solution of KOH (0.06 g, 1 mmol) in 2:1 MeOH/water (3.5 mL) and the mixture was gently shaken for 18 hours. The resin was washed with 2:1 MeOH/water, MeOH, 1:1 MeOH/CH2Cl2 and filtered with CH2Cl2 (50 mL) to afford the Tartrate-Val-PEGA resin 93. N-Mmt-PEG-Tartrate-Val-PEGA resin 94 Ph  Ph N H  O  O O  MeO  O O  NH O  NH  H N  O  After being washed and swollen in DMF, a mixture of N-Mmt protected spacer 87 (0.34 g, 0.8 mmol), PyBOP (0.42 g, 0.8 mmol), HOBt•H2O (0.11 g, 0.8 mmol) and DIEA (0.28 mL, 1.6 mmol) in DMF (4 mL) was added to the tartrate-Val-PEGA resin 93 (0.40 g). The mixture was gently shaken for 2 hours and the solution was drained from the resin. After being filtered with DMF (10 mL), a mixture of N-Mmt protected spacer 87 (0.21 g, 0.5 mmol), PyBOP (0.26 g, 0.5 mmol), HOBt•H2O (0.07 g, 0.5 mmol) and DIEA (0.18 mL, 1 mmol) in DMF (4 mL) was poured on the resin. The mixture was gently shaken overnight and the resin was washed to afford the N-Mmt-PEG-Tartrate-Val-PEGA resin 94. 2-(2,5-dibromobenzamido) acetate labeled tartrate linker 97 O  O  O  O  H N Br  N H  O  O  NH  O  O NH  H N O  Br  After having been washed and swollen in MeCN, a solution of 2% (w/v) TCA in MeCN (5 mL) was added to the N-Mmt-PEG-Tartrate-Val-PEGA resin 94 (0.20 g) making the solution yellow due to Mmt acidolysis. The yellow mixture was gently shaken for 30 min,  99 the solution was drained from the resin and the procedure was repeated twice with fresh TCA solution. To the drained resin was added 2% (w/v) TCA in MeCN, then the resin was gently shaken for 3 min and filtered. The procedure was repeated with fresh TCA solution until the soluble fraction was colourless. Then, the resin was filtered with MeCN (25 mL), DMF (25 mL), 1% DIEA in DMF (25 mL) and washed in DMF. To the resulting drained NH2-PEG-Tartrate-Val-PEGA resin was added a mixture acetic acid derivative 96 (0.12 g, 0.4 mmol), HOBt•H2O (0.05 g, 0.4 mmol), HBTU (0.14 g, 0.4 mmol), DIEA (0.16 mmol, 0.9 mmol) in DMF (5 mL). The mixture was gently shaken for 1 hour and the solution was drained from the resin. After being filtered with DMF (10 mL), a fresh activated acetic acid derivative 96 mixture was poured on the resin, the mixture was gently shaken for 2 hours and the resin was washed to afford the 2-(2,5dibromobenzamido) acetate labeled tartrate linker 97. 2.5. SPPS on the tartrate linker Reactions were carried out under atmosphere at room temperature by shaking in Zeba Desalt polypropylene spin column (5 mL or 10 mL format) from PierceTM. The PEGA resin was handled in a swollen state and high vacuum was avoided due to the fragile resin structure when dried. Following coupling reaction and deprotection reaction, a resin sample was submitted to Kaiser test. In addition, Green Malchite test was performed following the Savige-Fontana reaction and the macrolactamization. The general procedure below describes a typical SPPS that starts with the deprotection of N-Mmt-PEG-Tartrate-ValPEGA resin 94 (0.46 g, dry resin). Deprotection of the N-Mmt-PEG-Tartrate-Val-PEGA resin 94: The resin 94 was filtered with CH2Cl2 (50 mL), MeCN (100 mL) and shaken in MeCN (4 mL) for 10 min. The shaking procedure was repeated three more time with fresh MeCN. To the drained resin was added a solution of 5% (w/v) TCA in MeCN (5 mL) making the solution yellow due to Mmt acidolysis. The yellow mixture was gently shaken for 30 min, the solution was drained from the resin and the shaking procedure was repeated twice with fresh TCA solution. To the drained resin was added a fresh TCA solution, the resin was  100 then shaken for 3 min and filtered. The procedure was repeated with fresh TCA solution until the soluble fraction turned colourless. Then, the resin was filtered with MeCN (50 mL), DMF (50 mL), and shaken in DMF (4 mL) for 5 min. The resin was filtered and the shaking procedure was repeated three times, each with fresh DMF. The resin was filtered with 1% DIEA in DMF (50 mL) and shaken in DMF (4 mL) for 3 min. The resin was filtered and the shaking procedure was repeated two times, each with fresh DMF. The resin was used directly in the coupling reaction. Resin Swelling: If the Fmoc-protected resin was stored dry, the resin was shaken in solvent (4 mL) for 10 min and filtered. The shaking procedure was repeated three more time with fresh solvent. Fmoc deprotection: A solution of 20% piperidine in DMF (5 mL) was added to the Fmoc protected residue anchored to the PEGA resin via the tartrate linker. The mixture was gently shaken for 5 min, the solution was drained from the resin and the procedure was repeated for 10 min with fresh piperidine solution. The resin was filtered with DMF (50 mL), shaken in DMF (4 mL) for 3 min. The resin was filtered and the shaking procedure was repeated once for 3 min and twice for 5 min each with fresh DMF. Peptide coupling: A mixture of N-α-Fmoc-L-Xaa-OH (0.8 mmol), HOBt•H2O (0.11 g, 0.8 mmol), HBTU (0.30 g, 0.8 mmol), DIEA (0.34 mL, 2 mmol) in DMF (4 mL) was added to the Fmoc deprotected residue anchored to the PEGA resin via the tartrate linker without preactivation to avoid side reactions associated with an activated amino acid. The mixture was gently shaken for 1 hour, the solution was drained from the resin. The resin was filtered with DMF (10 mL) and a fresh activated Fmoc protected amino acid mixture was poured on the resin. The mixture was gently shaken for 1 hour 30 min, the solution was drained from the resin and filtered with DMF (50 mL). Then, the resin was shaken in DMF (4 mL) for 3 min, filtered and the shaking procedure was repeated once for 3 min and twice for 5 min each with fresh DMF. After loading the first amino acid on the linker PEGA resin, the overall peptide sequence was assembled in a linear fashion from the C-terminus to the Nterminus by repetitive cycles of Fmoc deprotection and amino acid coupling reactions. Storage:  101 The Fmoc protected peptide anchored to the PEGA resin via the tartrate linker was filtered with CH2Cl2 (100 mL), dried under air filtration or kept wet in EtOH and stored at -20oC. Fmoc-Glu-ΟtBu-PEG-Tartrate-Val-PEGA resin 100 O O O  O  O  O  H N  O  N H  O  NH  O H N  NH  O  O  O  N-Mmt-PEG-Tartrate-Val-PEGA resin 94 (0.46 g) was deprotected, washed and coupled to N-α-Fmoc-L-Glu-OtBu (0.34 g, 0.8 mmol), all in DMF, as described in the general procedure above. The loading was found to be 0.32 mmol per gram of dry resin 100. Fmoc-Ile-Gly-Cys-Asn-Pro-Glu-OtBu-Tartrate-Val-PEGA resin 101 O  O  O O O  H N  FmocHN  H N  O O N H Ph Ph  S Ph  N  O  HN  O O HN  Ph  Ph  Ph  O  O N H  O  NH O  NH HN O  O  Fmoc-Glu-ΟtBu-PEG-Tartrate-Val-PEGA resin 100 (0.46 g) was deprotected, washed, and coupled to N-α-Fmoc-L-Xaa-OH, all in DMF, as described in the general procedure above. The coupling sequence used was: N-α-Fmoc-Pro-OH (0.27 g, 0.8 mmol), N-α-Fmoc-N-βTrt-Asn-OH (0.47 g, 0.8 mmol), N-α-Fmoc-S-Trt-Cys-OH (0.46 g, 0.8 mmol), N-α-FmocGly-OH (0.23 g, 0.8 mmol), N-α-Fmoc-Ile-OH (0.28 g, 0.8 mmol) to afford the linear hexapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 101 anchored to PEGA resin by the tartrate linker.  102  Dac-Lys-Ile-Gly-Cys-Asn-Pro-Glu-OtBu-Tartrate-Val-PEGA resin 104 O  O  O O O  O  O  N O  HN NH2  O N H  H N  H N  O O N H Ph Ph 2  S Ph  N  O  HN  O O HN  Ph  Ph  Ph  O  O N H  O  NH O  NH HN O  O  3  The linear hexapeptide Pro -Glu -S-deoxo-amaninamide precursor 101 (0.02 g) was deprotected, washed, and swollen, all in DMF as described in the general procedure above. To the resin 101 was added a mixture of Dac-Fmoc-Lys-OH 103 (0.006 g, 0.009 mmol), HOBt•H2O (0.001 g, 0.009 mmol), HBTU (0.003 g, 0.009 mmol), DIEA (0.003 mL, 0.02 mmol) in DMF (0.5 mL). The mixture was shaken for 4 hours and the resin was washed, all in DMF, as described in the general procedure above. However, additional washing was done using 1:1 DMF/CH2Cl2, CH2Cl2, 1:1 MeOH/CH2Cl2 and MeOH. The resulting Fmoc protected resin was swollen in DMF and poured in a solution of 20% piperidine in DMF (0.6 mL). The mixture was gently shaken for 5 min, the solution was drained from the resin and the shaking procedure was repeated three other times with fresh piperidine solution. The resin was filtered with DMF (5 mL), CH2Cl2 (5 mL), shaken in DMF (1 mL) for 2 min and filtered. The shaking procedure was repeated four more times, each with fresh DMF. The same shaking procedure (1 mL, 4 X 2 min) was repeated using the following: 1:1 DMF/CH2Cl2 and CH2Cl2 to afford the fluorescent linear heptapeptide 104 anchored to PEGA resin by the tartrate linker that was exposed to an excess of TFA for varying times and analyzed under UV lamp.  103  Trt-Hpi-Gly-Ile-Gly-Cys-Asn-Pro-Glu-OtBu-Tartrate-Val-PEGA resin 113 O  O  O NH Ph  O  Ph  N  Ph O  HO  H N  N H Ph  N H  O  H N  O O  Ph  S Ph  N  O  HN  Ph  Ph  Ph  O  NH HN O  N H  HN  O O  O  NH O  O  O  The linear hexapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 101 (0.46 g) was deprotected, washed, swollen, and coupled to Trt-Hpi-Gly-OH•Et3N 9a/b (0.49 g, 0.8 mmol), all in DMF, as described in the general procedure above. The product of coupling yielded the linear octapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 113 anchored to PEGA resin by the tartrate linker. Monocyclic Pro2-Glu3-S-deoxo-amaninamide on Tartrate-Val-PEGA resin 114 O  O O  NH3 O  N NH H  HN  CF3 HO  O O S  O  O N H  NH O  HN NH  N  O O O  O  O HN  O  N H  HN O  O  OH  N H  OH  H2N  The linear octapeptide Pro2-Glu3-S-deoxo-amaninamide precursor 113 (0.46 g) was filtered with CH2Cl2 (100 mL) and transferred wet to a Zeba Desalt polypropylene spin column (10 mL format) from PierceTM. The resin 113 was filtered with TFA (40 mL) and poured in TFA (13 mL). The solution turned yellow as soon as it was exposed to TFA due to Trt acidolysis. The yellow mixture was gently shaken for 5 hours, the solution was flushed from the resin and the resin was filtered with TFA (40 mL) and 95:2.5:2.5 TFA/TES/water (40 mL). The resin was shaken in 95:2.5:2.5 TFA/TES/water (12 mL) for 5 min, filtered and the procedure was repeated with a fresh TFA/TES/water mixture until the solution  104 turned colourless. The resin was shaken in 9:1 AcOH/water (12 mL) for 3 min, filtered and the shaking procedure was repeated three more times, each with fresh 9:1 AcOH/water. The same shaking procedure (12 mL, 4 X 3 min) was repeated using the following: 1:5 AcOH/water, MeCN and DMF. The resin was filtered with DMF (100 mL), CH2Cl2 (200 mL), dried under air filtration and stored at -20oC to afford monocyclic Pro2-Glu3-S-deoxoamaninamide 114 anchored to PEGA resin by the tartrate linker. Pro2-Glu3-S-deoxo-amaninamide on Tartrate-Val-PEGA resin 120 HO O NH O  O OO NH2  HN  HN O O  HN  OH  O  NH O  NH S  O  O N H  N H  HN  N O  O  O  HN HN  O  N H  The monocyclic Pro2-Glu3-S-deoxo-amaninamide 114 precursor (0.46 g) was washed and swollen, all in DMF, according to general procedure but additional washing was done with 1% DIEA in DMF. To the resin 114 was added a mixture of PyBOP (0.16 g, 0.3 mmol), HOBt•H2O (0.04 g, 0.3 mmol), DIEA (0.11 mL, 0.6 mmol) in DMF (12 mL). The mixture was shaken for 3 hours, drained from the resin and filtered with DMF (25 mL). Macrolactamization was repeated three more times with fresh PyBOP mixture. The resin was drained from mixture, filtered with DMF (300 mL), shaken (5 X 5 min) with fresh DMF (12 mL), filtered with CH2Cl2 (500 mL), dried under air filtration and stored at -20oC to afford Pro2-Glu3-S-deoxo-amaninamide 120 anchored to PEGA resin by the tartrate linker.  105  2.6. Cleavage of product from tartrate-based linker on resin Linker cleavage was carried out under atmosphere at room temperature by shaking in Zeba Desalt polypropylene spin column (5 mL or 10 mL format) from PierceTM. Herein, the typical experiment involved a peptide prepared from the N-Mmt-PEG-Tartrate-Val-PEGA resin 94. Tartrate Diol deprotection: For example, 0.2 g of dry resin was filtered with CH2Cl2 (50 mL), 95:2.5:2.5 TFA/water/TES (25 mL) and poured in 95:2.5:2.5 TFA/water/TES (5 mL). The mixture was gently shaken for 2 hours, the solution was flushed from resin. Then, the resin was shaken in 95:2.5:2.5 TFA/water/TES (4 mL) for 5 min, filtered and the procedure was repeated twice with fresh mixture. The same shaking procedure (4 mL, 3 X 5 min) was repeated using the following: 1:1 AcOH/CH2Cl2, AcOH, 1:1 AcOH/water and water. Tartrate oxidative cleavage: The wet acetonide deprotected resin was shaken in 5:1 water/AcOH (4 mL) for 10 min, filtered and the shaking procedure was repeated three more time with fresh 5:1 water/AcOH mixture. The resin was poured in mixture of NaIO4 (2.5 mL, 0.12 M) in 5:1 water/AcOH and shaken for 6 min. The soluble fraction was collected by filtration to which was added DMS (0.76 mL, 10 mmol). The reduction of NaIO4 by DMS produced DMSO that could carry products through gloves and skin. Thus, caution was taken during the handling of eluates. The resin was filtered with different solvent systems with polarities corresponding to the expected cleaved product. All fractions collected from the resin were pooled together. The elution of the products was followed by TLC and/or UV spectroscopy.  106  Cleavage of product 98 from resin 97 with NaIO4 O  O  O  H N Br  N H  O  O  O NH  Br  The resin 97 (~0.2 g) was deprotected and the product cleaved as described in the general procedure above using the following: Zeba Desalt polypropylene spin column (5 mL format) from PierceTM, 95:2.5:2.5 TFA/water/anisole (6 mL) as deprotection mixture, NaIO4 (3 mL, 0.1 M) in 5:1 water/AcOH as cleavage mixture and DMS (0.9 mL) as quenching mixture. Following cleavage, the resin was filtered with 5:1 water/AcOH (10 mL) and 3:2:1 tert-BuOH/AcOH/water. Then, the resin was shaken in 1:1 tertBuOH/MeOH (5 mL) for 3 min, filtered and the procedure was repeated twice with fresh 1:1 tert-BuOH/MeOH mixture. The same shaking procedure (5 mL, 3 X 5 min) was repeated using 1:1 MeOH/CH2Cl2. Water (100 mL) was added to the pooled eluate fraction which was then extracted with CH2Cl2 (5 X 20 ml). The crude product however was found in the organic and aqueous layers after having been concentrated to dryness under reduced pressure. The crude product was purified by flash chromatography (Silica, 1:1:8 AcOH/2propanol/CH2Cl2) to afford product 98 (0.005 g, 16%) as a mixture of glyoxamide, hemiacetal and methanol acetal. Rf = 0.2 (AcOH/2-propanol/CH2Cl2 1:1:8) LRMS (ESI) m/z: calculated for C17H21Br2N3O6 [M+Na]+: 544.0, 546.0, 548.0, found: 543.8, 545.8, 547.7.  107  Cleavage of product from resin 104 with NaIO4 O O  O  O  N  O  O  HN O O  NH3  O N H  H N  O O  H N  O O  N H HS  N  O  O  O  N H  HN O  O NH  OH  H2N  The resin 104 (~0.07 g) was deprotected and the product cleaved as described in the general procedure above using the following: DNA synthesizer reactor (1 mL format), 95:2.5:2.5 TFA/water/anisole (0.6 mL) as deprotection mixture, NaIO4 (1 mL, 0.18 M) in 5:1 water/AcOH as the cleavage mixture and ethanolamine (0.05 mL) as the quenching mixture. Following cleavage, the resin was filtered with 5:1 water/AcOH (2 mL), and shaken in 5:1 water/AcOH (1 mL) for 5 min, filtered. The shaking procedure was repeated twice with fresh 5:1 water/AcOH mixture. The eluate was concentrated to dryness under reduced pressure. Despite attempting different purification methods, we were not able to isolated the product 106 in quantity for clear characterisation. However, TLC analysis confirmed the presence of a product spot from the resin eluate bearing Dac residue, free amine, free carboxylic acid and free aldehyde. Moreover, the UV spectra of resin eluate showed absorption maxima at 431 nm consistent with the presence of the Dac derivative. Rf = 0.3 (H2O/ AcOH/ 2-propanol 1:1:2) U.V. spectra (MeOH) λmax = 423 nm (Dac)  108 Cleavage of monocyclic Pro2-Glu3-S-deoxo-amaninamide from resin 114 O O  N NH H  HN  NH2  O O S  O  O N H  NH O  HN NH  N  O O O  O  HN O  O  O  N H OH  H2N  The resin 114 (~0.1 g) was deprotected and the product cleaved as described in the general procedure above using the following: Zeba Desalt polypropylene spin column (5 mL format) from PierceTM, 95:2.5:2.5 TFA/water/TES (3.6 mL) as the deprotection mixture, NaIO4 (2.4 mL, 0.1 M) in 5:1 water/AcOH as cleavage mixture and DMS (0.6 mL) as the quenching mixture. Following cleavage, the resin was filtered with 5:1 H2O/AcOH (20 mL) and 3:2:1 tert-BuOH/AcOH/water (20 mL). Then, the resin was shaken with 3:2:1 tert-BuOH/AcOH/water (5 mL) for 3 min, filtered and the procedure was repeated three more times with fresh 3:2:1 tert-BuOH/AcOH/water mixture. The same shaking procedure (5 mL, 4 X 5 min) was repeated using 4:1 tert-BuOH/water, 1:1 tert-BuOH/CH2Cl2, CH2Cl2. The pooled fractions were evaporated to dryness under reduced pressure. The crude solid was triturated in CH2Cl2 and solvent was removed under reduced pressure. The crude product (~20 mg) was solubilised in mixture of 8:2 Buffer A’/B’ as described in section 1.2 (Chapter 3). The mixture was loaded on Sep-Pak C18 column, washed with 8:2 Buffer A’/B’ and the product eluted in 7:3 Buffer A’/B’ mainly contaminated with iodine. Following lyophilisation, a yellow solid (~10 mg) was recovered, dissolved in 8:2 Buffer A/B (10 mL), filtered and loaded on RP-HPLC (column (1), Chapter 3, section 1.2). Elution of the injected material on RP-HPLC was carried out under the following program: 5% Buffer B over 5 minutes, linear gradient of 5% to 34% Buffer B over 25 min, linear gradient of 34% to 100% Buffer B over 5 min, 100% Buffer B over 5 min, linear gradient of 100% to 5% Buffer B over 5 min. Two peaks were collected, which were denoted M[O] (1 mg, 3% yield, eluting at 21 min) and M (2 mg, 6% yield, eluting at 23 min). Following lyophilisation, the white solid M[O] displayed two major mass spectra peaks corresponding to the oxidized tryptathionine bridge (sulfoxide, oxo-S-(2-tryptophanyl)cysteine) denoted  109 117 contaminated with the cyclic glyoxylic imine denoted 119. The white solids M displayed two major mass spectra peaks corresponding to the deoxo tryptathionine bridge (S-(2-tryptophanyl)cysteine) denoted 116 contaminated with the cyclic glyoxylic imine denoted 118 and trace amount of compound 117. Product 116 (peak M, RP-HPLC): HRMS (ESI) m/z: calculated for C46H66N12O15S [M-H]-: 1057.4419, found: 1057.4413. Product 117 (peak M[O], RP-HPLC): HRMS (ESI) m/z: calculated for C46H66N12O16S [M-H]-: 1073.4368, found: 1073.4331. Product 118 (peak M, RP-HPLC): HRMS (ESI) m/z: calculated for C46H64N12O14S [M-H]-: 1039.4313, found: 1039.4271. Product 119 (peak M[O], RP-HPLC): HRMS (ESI) m/z: calculated for C46H64N12O15S [M-H]-: 1055.4262, found: 1055.4141. Cleavage of Pro2-Glu3-S-deoxo-amaninamide from resin 120 O  O NH O  O OO NH2  NH O  NH S  O HN  HN O O  O  N H  HN  N O  O  HN HN  O  N H  The resin 120 (~0.2 g) was deprotected and the product cleaved as described in the general procedure above. Following NaIO4 exposure, the resin was filtered with 5:1 water/AcOH (10 mL) and 4:6 Buffer A’/B’ (10 mL) as described in section 1.2, Chapter 3. The resin was shaken in 4:6 Buffer A’/B’ (4 mL) for 5 min. The shaking procedure was repeated at least 10 times until the product was not detected in the eluate. Following solvent evaporation, the crude product was solubilised in a minimum amount of 9:1 Buffer A’/B’. The mixture was loaded on Sep-Pak C18 column, washed with 8:2 Buffer A’/B’ and the  110 product eluted in 6:4 Buffer A’/B’ mainly contaminated with iodine. Following lyophilisation, a yellow solid was recovered, dissolved in 7:3 Buffer A/B, filtered and loaded on RP-HPLC (column (1), Chapter 3, section 1.2). Elution of injected material on RP-HPLC was carried out using 10% buffer B over 2 min, followed by a linear gradient of 10% to 50% buffer B over 18 min, followed by a linear gradient of 50% to 100% buffer B over 5 min, kept to 100% buffer B for 3 min and lowered to 10% buffer B over 2 min. Two peaks were collected at 12 min and 13 min, which were denoted peak A and peak B respectively. The peak A and B were collected in fraction denoted A and B respectively found in a ratio of 1:2. To asses the purity of the collected fraction A and B, in each case a sample (5 μL) was loaded on RP-HPLC (column (2)) with the same program and one major peak at 11 min was found from fraction A and one major peak at 12 min for fraction B. Following lyophilisation, the resulting white solid from both fractions displayed the same major mass spectra peaks corresponding to Pro2-Glu3-S-deoxo-amaninamide bearing a PEG glyoxamide end with trace amounts of the corresponding oxidized tryptathionine bridge (sulfoxide) isomer. Both fractions displayed a UV spectrum corresponding to the known UV spectrum of S-deoxo-tryptathionine bridge (S-(2-tryptophanyl)cysteine). Based on a previous report (Chapter 2, section 3.4), fraction A and B were believed to be composed of glyoxamide L-Pro2-D-Glu3-S-deoxo-amaninamide 122 (0.002 mmol, 3.5% yield) and glyoxamide L-Pro2-L-Glu3-S-deoxo-amaninamide 121 (0.004 mmol, 7% yield) respectively. Quantification of product was determined in 0.06:2:8 TFA/MeCN/H2O according to the Beer-Lambert law. As a first approximation, the extinction coefficient (10 755 M-1cm-1) caused by the S-(2-tryptophanyl)cysteine in the cyclic (L-Cys-Gly-Gly-Gly2-mercapto-L-Trp-Gly-Gly-Gly) cyclic (1 to 5) sulfide determined at 289 nm in MeOH was used (Chapter 2, section 3.4). Product 121 (peak B, RP-HPLC): HRMS (ESI) m/z: calculated for C46H64N12O14S [M-H]-: 1039.4313, found: 1039.4310. UV spectra (0.06:2:8 TFA/MeCN/H2O) λmax = 289 nm (S-(2-tryptophanyl)cysteine) Product 122 (peak A, RP-HPLC): HRMS (ESI) m/z: calculated for C46H64N12O14S [M-H]-: 1039.4313, found: 1039.4305. UV spectra (0.06:2:8 TFA/MeCN/H2O) λmax = 289 nm (S-(2-tryptophanyl)cysteine)  111  Chapter 4 Conclusion  The mechanisms involved in the allosteric inhibition of RNA pol II by α-amanitin are of significant interest to us and others. Toward these goals, our interest focuses on the chemical basis for the interactions between amanitin and RNA pol II. In order to complete the α-amanitin structure-activity relationship information already gathered, we would like to prepare an amatoxin library anchored to a solid support by a linker, and screen the resulting library against an in vitro RNA pol II assay. To achieve an amatoxin library on solid support, the linker needs to be stable during the Fmoc/tert-Bu SPPS of the amatoxin and stable to the 5 hours of exposure to neat TFA required for the formation of the tryptathionine bridge (S-(2-tryptophanyl)cysteine). Based on Chenera’s paper reporting a silicon-based linker stable in neat TFA, we first prepared the electron deficient benzonitrile silyl linker 72. Following incorporation of the fluorescent spacer 76c in the benzonitrile silyl linker 72, the resulting product 77 was exposed to neat TFA (Chapter 2, section 1.4). Under UV lamp, the bead fluorescence was significantly reduced suggesting that the linker had degraded in neat TFA. Unfortunately, we were unable to purify and characterize the products found in the eluate of the TFA treated resins 72 and 77 after or prior to the TBAF treatment. However, the results from the TFA test suggested a lack of stability of the silicon-based linkers to acidic media. From the long range COSY experiment (Chapter 2, section 1.4), we concluded that the benzonitrile silyl 65’ rather than the desired compound 65 was incorporated leading to the linker degradation in neat TFA. Although Chenera’s protocol was followed we contend that the difference in results that we obtained compared to those reported in the original work may explain the disappointing results we obtained. If indeed silyl 65’ had been prepared in lieu  112 of 65, Chenera did not report on this possibility and in light of the considerable amount of time already invested in a linker that might have been a regioisomer of the desired compound, no further attempts were made to fully address the variance in regiochemistry that we obtained compared to that in Chenera’s published report, which may account for the stability they observed. A multifunctional linker 49 developed by Melnyk and Grass-Masse was reported to be stable during standard Fmoc/tert-Bu SPPS and to 95% TFA exposure in CH2Cl2. Before preparing an analogue of linker 49, acid stability of the tartrate-based linker was confirmed by exposing the soluble fluorescent linker mimic 88 to neat TFA. The only side-reaction observed was the loss of diol protecting group in 20% of the linkers exposed to TFA (Chapter 2, section 2.2). The diol linker deprotection was not of concern because it occurred only just before the macrolactamization, which is the last step in the amatoxin synthesis. Thus, we decided to carry on the tartrate-based linker 94 syntheses on PEGA resin. The successful preparation of the tartrate-base linker was shown by LRMS (ESI) when the linker 97 coupled to a dibromobenzoic acid was cleanly recovered as compound 98 in the eluate following the oxidative linker cleavage (Chapter 2, section 2.4). With the tartrate-based linker in hand, we prepared Pro2-Glu3-S-deoxoamaninamide 120 on solid phase from linker 94 as follows. Previously, we showed that the linear fluorescent amatoxin precursor 104 built on linker 94 was stable in neat TFA (Chapter 2, section 3.2). Then, the monocyclic L-Pro2-L-Glu3-S-deoxo-amaninamide 114 was prepared from the linear hexapeptide 101 by a clean Savige-Fontana reaction on solid phase (Chapter 2, section 3.3). Subsequently, the monocycle 114 was macrolactamized using PyBOP as the coupling agent. This resulted in the bicycle 120 that, following the linker cleavage with NaIO4, was recovered into the soluble fraction as products 121 and 122 along with trace amount of sulfoxide by-products (Chapter 2, section 3.4). Products 121 and 122 had RP-HPLC retention times with UV spectrum, and HRMS (ESI) m/z that were expected for Pro2-Glu3-S-deoxo-amaninamide. Based on a report of May et al., we believe that the two products are the two diastereoisomers 121 and 122 resulting from an epimerization occurring at the glutamate residue during the macrolactamization of compound 114. The Pro2-Glu3-S-deoxo-amaninamide synthesis on PEGA resin is currently  113 being scaled-up to afford enough product for further characterizations by CD spectra, NMR spectra and MS experiments. Some drawbacks are found in our methodology that needs to be fine-tuned. Our amatoxin synthesis led to the production of two diastereoisomers, required the use of Glu3 that lacks a β-methyl, and oxidized a small proportion of tryptathionine. The diastereoisomere, with the (R)-configuration at its α-carbon found on the same bead than its stereoisomer counterpart, could lead to a significant increase in the product’s Ki toward RNA pol II. However, Dr Jonathan May, a previous post-doctoral researcher in our lab, enhanced  the  monocyclic  Pro2-Ile3-S-deoxo-amaninamide  11  macrolactamization  enantioselectivity in solution with the addition of 10% water to the DMF mixture (unpublished results). Despite a decreased yield, from 39.3% to 23.2%, the ratio of bicyclic derivatives 12 (L-Ile3) to 12’ (D-allo-Ile3) was improved from 1:1 to 25:1. The present methodology is based on a report claiming that Hpi needed to be incorporated last, during the conventional amatoxin synthesis from C-to-N-terminus (Chapter 1, section 5.2). Thus, we have used the residue in position 3 as the anchoring site, but with an absence of a βmethyl, the product’s Ki might increase (Chapter 1, section 2). However, May et al. have developed a new methodology for the Pro2-Ile3-S-deoxo-amaninamide 12 synthesis with the incorporation of NH2-[syn-cis]-Ile-Hpi-Gly-OH in the middle of the linear peptide sequence during the SPPS.83 This method is of interest as it enables the synthesis to begin at any location on the amatoxin sequence. The sequence could be chosen to avoid macrolactamization at a sterically constrained amino acid since they are prone to enantiomerization. In addition, this gives more freedom in the choice of residue, that is to be anchored to the linker during the library synthesis. Consequently, Gly5 or Gly7 appears as an interesting anchoring site to the solid phase. Unfortunately, the conformation of the amatoxin’s ring II composed of the Gly5-Ile6-Gly7 seems to accommodate the insertion of Ile’s side chain into a hydrophobic pocket of RNA pol II. Thus using any of these residues in the loop might lower the resulting amatoxin’s inhibitory activity. On the other hand, only the Asn1’s β-carbonyl was reported to be important to the amatoxin activity. Therefore, a long aliphatic side chain with a β-ketone could replace Asn1 and be used as an anchoring site to the solid support. The tryptathionine oxidation during the tartrate-based linker cleavage is not of concern as it will occur following the library screening on beads. But, the  114 (S)-S-oxo-amatoxin derivatives that are cleaved from the tartrate-based linker might loose their inhibitory activity during a subsequent screening against an RNA pol II assay in solution. However, current experiments show that only a small proportion of amatoxins are oxidized during the linker cleavage with NaIO4. In addition, the oxidized amatoxins can be separated in good part from the S-deoxo-amatoxins by RP-HPLC. To the best of our knowledge, the current work represents a novel methodology to achieve the synthesis of Pro2-Glu3-S-deoxo-amaninamide on solid phase. Furthermore, the presence of a glyoxamide end in the cleaved product allows incorporation of additional functionalities such as probes that are useful in product characterization or bio-assay. Notably, the present methodology could be expanded to the synthesis of other tryptathionine analogues such as the phallotoxins. Our next goal is to build an amatoxin library to better understand the critical structure-activity relationship between α-amanitin and RNA pol II. Hopefully, the library will also lead to the discovery of bicyclic peptides showing an higher affinity and/or specificity for different RNA polymerase types compared to α-amanitin. Finally, these molecules could find application in the study of cellular pathways and diseases in which the RNA pol II is involved.  115  References (1)  Wieland, T. Peptides of Poisonous Amanita Mushrooms; Springer-Verlag: NewYork, 1986.  (2)  Wieland, T. and Faulstich H. Experientia 1991, 47, 1186.  (3)  Lee, K. B.; Wang, D.; Lippard, S. J.; Sharp, P. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4239.  (4)  Katsara, M.; Tselios, T.; Deraos, S.; Deraos, G.; Matsoukas, M. T.; Lazoura, E.; John Matsoukas, J.; Apostolopoulos, V. Curr. Med. Chem. 2006, 13, 2221.  (5)  Wieland, T.; Gotzendorfer, C.; Zanotti, G.; Vaisius, A. C. Eur. J. Biochem. 1981, 117, 161; Zanotti, G., Birr, C., Wieland, T. Int. J. Pept. Protein Res. 1981, 18, 162.  (6)  Bushnell, D. A.; Cramer, P.; Kornberg, R. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1218.  (7)  Brueckner, F.; Cramer, P. Nature Struct. Mol. Biol. 2008, 15, 811; Brueckner, F.; Armache, K. J.; Cheung, A.; Damsma, G. E.; Kettenberger, H.; Lehmann, E.; Sydow J.; Cramer P. Acta Crystallographica 2009, D65, 112.  (8)  Shoham, G.; Rees, D. C.; Lipscomb, W. N.; Wieland, T. J. Am. Chem. Soc. 1989,111, 4791.  (9)  Zanotti, G.; Wieland, T.; D’Auria, G.; Paolillo, L.; Trivellone, E. Int. J. Pept. Protein Res. 1990, 35, 263.  (10)  Shoham, G.; Rees, D. C.; Lipscomb, W. N.; Zanotti, G.; Wieland, T. J. Am. Chem. Soc. 1984, 106, 4606; Zanotti, G.; Wieland, T.; Benedetti, E.; Blasio, B. D.; Pavone, V.; Pedone, C. Int. J. Pept. Protein Res. 1989, 34, 222; Zanotti, G.; Möhringer, C.; Wieland, T. Int. J. Pept. Protein Res. 1987, 30, 450.  (11)  Zanotti, G.; Petersen, G.; Wieland, T. Int. J. Pept. Protein Res. 1992, 40, 551.  (12)  Schmitt, W.; Zanotti, G.; Wieland, T.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 4380.  (13)  Shoham, G.; Rees, D. C.; Lipscomb, W. N.; Zanotti, G.; Wieland, T. Protein Sci. 1994, 3, 750; Zanotti, G.; D’Auria, G.; Paolillo, L.; Trivellone, E. Biochim.  116  Biophys. Acta 1986, 870, 454; Zanotti, G.; D’Auria, G.; Paolillo, L.; Trivellone, E. Int. J. Pept. Protein Res. 1988, 32, 9. (14)  Kaplan, C. D.; Larsson, K. M.; Kornberg, R. D. Mol. Cell 2008, 30, 547; Kornberg, R. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12955; Svetlov, V.; Nudler, E. Nature Struct. Mol. Biol. 2008, 15, 777; Brueckner, F.; Cramer, P. Nature Struct. Mol. Biol. 2008, 15, 811.  (15)  Coin, I.; Beyermann, M.; Bienert, M. Nature Protocols 2007, 2, 3247; Amblard, M.; Fehrentz, J. A.; Martinez, J.; Subra, G. Molecular Biotechnology 2006, 33, 239.  (16)  Paul, R.; Kende, A. S. J. Am. Chem. Soc. 1964, 86, 4162; Gausepohl, H.; Kraft, M.; Frank, R. W. Int. J. Peptide Protein Res. 1989, 34, 287.  (17)  Capasso, S.; Balboni, G.; Di Cerbo, P. Pept. Sci. (Biopolymers) 2000, 53, 213.  (18)  May, J. P.; Perrin, D. M. Pept. Sci. (Biopolymers) 2007, 88, 714.  (19)  May, J. P.; Fournier, P.; Pellicelli, J.; Patrick, B. O.; Perrin, D. M. J. Org. Chem. 2005, 70, 8424.  (20)  Barlos, K., Papaioannou, D., Patrianakou, S. and Tsegenidis,T., Liebigs Ann. Chem. 1986, 1950.  (21)  Teixido, M.; Altamura, M.; Quartara, L.; Giolitti, A.; Maggi, C. A.; Giralt, E.; Albericio, F. J. Comb. Chem. 2003, 5, 760.  (22)  Karskela, T.; Virta, P.; Lönnberg, H. Curr. Org. Synth. 2006, 3, 283.  (23)  Comely, A. C.; Gibson, S. E. Angew. Chem. Int. Ed. 2001, 40, 1012.  (24)  Blaney, P.; Grigg, R.; Sridharan, V. Chem. Rev. 2002, 102, 2607.  (25)  Katti, S.B.; Misra, P. K.; Haq, W.; Mathur, K. B. J. Chem. Soc., Chem. Commun. 1992, 843.  (26)  Scott, P. J. H.; Steel, P. G. Eur. J. Chem. Org. 2006, 10, 2251.  (27)  Heidler, P.; Link, A. Bioorg. Med. Chem. 2005, 13, 585.  (28)  Guillier, F.; Orain, D.; Bradley M. Chem. Rev. 2000, 100, 2091.  (29)  Chao, H. G.; Bernatowicz, M. S.; Matsueda, G. R. J. Org. Chem. 1993, 58, 2640.  (30)  Pietta, P. G.; Marshall, G. R. J. Chem. Soc., Chem. Commun. 1970, 650; Hruby, V. J.; Upson, D. A.; Agarwal, N. S. J. Org. Chem. 1977, 42, 3552; Akaji, K.; Yoshida, M.; Tatsumi, T.; Kimura, T.; Fujiwara, Y.; Kiso, Y. J. Chem. Soc., Chem. Commun.  117  1990, 288; Nefzi, A.; Ostresh, J. M.; Meyer, J.-P.; Houghten, R. A. Tetrahedron Lett. 1997, 38, 931. (31)  Bochet, C. G. J. Chem. Soc., Perkin Trans. I 2002, 125; Bley, F.; Schaper, K.; Gomer, H. Photochemistry and Photobiology 2008, 84, 162.  (32)  Nicolas, E.; Clemente, J.; Ferrer, T.; Albericio, F.; Giralt, E. Tetrahedron 1997, 53, 3179.  (33)  Sternson, S. M.; Schreiber, S. L. Tetrahedron Lett. 1998, 39, 7451.  (34)  Deegan, T. L.; Gooding, O. W.; Baudart, S.; Porco, J. A., Jr. Tetrahedron Lett. 1997, 38, 4973; Fukase, K.; Egusa, K.; Nakai, Y.; Kusumoto, S. Mol. Div. 1996, 2, 182; Fukase, K.; Nakai, Y.; Egusa, K.; Porco, J. A., Jr.; Kusumoto, S. Synlett 1999, 1074.  (35)  Paris, M.; Heitz, A.; Guerlavais, V.; Cristau, M.; Fehrentz, J. A.; Martinez, J. Tetrahedron Lett. 1998, 39, 7287 ; Hall, B. J.; Sutherland, J. D. Tetrahedron Lett. 1998, 39, 6593.  (36)  Lee, Y.; Silverman, R. B. Tetrahedron 2001, 57, 5339.  (37)  Chenera, B.; Finkelstein, J. A.; Veber, D. F. J. Am. Chem. Soc. 1995, 117, 11999.  (38)  Chao, H. G.; Bernatowicz, M. S.; Reiss, P. D.; Klimas, C. E.; Matsueda, G. R. J. Am. Chem. Soc. 1994,116, 1746.  (39)  Wagner, M.; Kunz, H. Angew. Chem. Int. Ed. 2002, 41, 317; Wagner, M.; Dziadek, S.; Kunz, H. Chem. Eur. J. 2003, 9, 6018.  (40)  Chao, H. G.; Bernatowicz, M. S.; Matsueda, G. R. J. Org. Chem. 1993, 58, 2640.  (41)  Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 303; Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 3743.  (42)  Plunkett, M. J.; Ellman, J. A. J. Org. Chem. 1995, 60, 6006.  (43)  Woolard, F. X.; Paetsch, J.; Ellman, J. A. J. Org. Chem. 1997, 62, 6102.  (44)  Ramage, R.; Andrews, M. J. I.; Raphyb, J.; Wanga, P. Tetrahedron Lett. 2004, 45, 2403.  (45)  Allen, J. M.; Aprahamian, S. L.; Sans, E. A.; Shechter, H. J. Org. Chem. 2002, 67, 3561.  118  (46)  Melnyk, O.; Fruchart, J. S.; Grandjean, C.; Gras-Masse, H. J. Org. Chem. 2001, 66, 4153.  (47)  Melnyk, O.; Fehrentz, J.A.; Martinez, J.; Gras-Masse, H. Pept. Sci. (Biopolymers) 2000, 55, 165.  (48)  Paulick, M. G.; Hart, K. M.; Brinner, K. M.; T jandra, M.; Charych, D. H.; Zuckermann, R. N. J. Comb. Chem. 2006, 8, 417.  (49)  Halling P. J. Biochem. Soc. Trans. 2006, 34, 309; Kress, J.; Zanaletti, R.; Amour, A.; Ladlow, M.; Frey, J. G.; Bradley, M. Chem. Eur. J. 2002, 8, 3769; Kuhlman, P.; Duff, H. L.; Galant, A. Analytical Biochemistry 2004, 324, 183.  (50)  Richter, L. S.; Gadek, T. R. Tetrahedron Lett. 1994, 35, 4705.  (51)  Krchnak, V.; Flegelova, Z.; Weichsel, A. S.; Lebl, M. Tetrahedron Lett. 1995, 36, 6193.  (52)  Ma, X.; Shi, R.; Zhang, B.; Yan, B. J. Comb. Chem. 2009, 11, 438.  (53)  Berthelot, T.; Talbot, J. C.; Lain, G.; Deleris, G.; Latxague, L. J. Pept. Sci. 2005, 11, 153.  (54)  Parham, W. E.; Piccirilli, R. M. J. Org. Chem. 1977, 42, 257.  (55)  Fraser, R. R.; Bresse, M.; Mansour, T. S. J. Am. Chem. Soc. 1983, 105, 7790.  (56)  Parham, W. E.; Jones, L. D. J. Org. Chem., 1976, 41, 1187; Caron, S.; Do, N. M. Synlett 2004, 1440.  (57)  Jacobson, A. R.; Makris, A. N.; Sayre, L. M. J. Org. Chem. 1987, 52, 2592.  (58)  Carpino, L. A.; Ghassemi, S.; Ionescu, D.; Ismail, M.; Sadat-Aalaee, D.; Truran, G. A.; Mansour, E. M. E.; Siwruk, G. A.; Eynon, J. S.; Morgan, B. Organic Process Research & Development 2003, 7, 28; Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J. Org. Chem. 1990, 55, 1673.  (59)  Yeager, A. R.; Finney, N. S. Bioorg. Med. Chem. 2004, 12, 6451; Gogoi, S.; Argade, N. P. Tetrahedron 2004, 60, 9093.  (60)  Niwayama, S.; Rimkus, A. Bull. Chem. Soc. Jpn. 2005, 78, 498; Niwayama, S. J. Org. Chem. 2000, 65, 5834.  (61)  Fruchart, J. S.; Gras-Masse, H.; Melnyk, O. Tetrahedron Lett. 1999, 40, 6225.  119  (62)  Conza, M.; Wennemers, H. J. Chem. Soc., Chem. Commun. 2003, 866; Renil, M.; Ferreras, M.; Delaisse, J. M.; Foged, N. T.; Meldal, M. J. Pept. Sci. 1998, 4, 195.  (63)  Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2002, 9, 203.  (64)  Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem. 1998, 63, 9678.  (65)  Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M. N.; Jouin, P. Tetrahedron 1991, 47, 259.  (66)  Alsina, J.; Barany, G.; Albericio, F.; Kates, S. A. Lett. Pept. Sci. 1999, 6, 243.  (67)  Han, Y.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307.  (68)  Berthelot, T.; Lain, G.; Latxague, L.; Deleris, G. Journal of Fluorescence 2004, 14, 671.  (69)  De la Torre, B. G.; Marcos, M. A.; Eritja, R.; Albericio, F. Lett. Pept. Sci. 2002, 8, 331.  (70)  Savige, W. E.; Fontana, Int. J. Pept. Protein Res. 1980, 15, 102.  (71)  Dubs, P.; Bourel-Bonnet, L.; Subra, G.; Blanpain, A.; Melnyk, O.; Pinel, A. M.; Gras-Masse, H.; Martinez, J. J. Comb. Chem. 2007, 9, 973.  (72)  Davies, J. S. J. Pept. Sci. 2003, 9, 471.  (73)  Zanotti, G.; Birr, C.; Wieland, T. Int. J. Pept. Protein Res. 1978, 12, 204.  (74)  May, J. P.; Fournier, P.; Patrick, B. O.; Perrin, D. M. Chem. Eur. J. 2008, 14, 3410.  (75)  Schmitt, W.; Zanotti, G.; Wieland, T.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 4380.  (76)  Armarego, W. L. F.; Perrin, D. D. Purification of Laboratory Chemicals; Elsevier, 4th Edition, 1997.  (77)  Adam, W.; Chan, Y. Y.; Cremer, D.; Gauss, J.; Scheutzow, D.; Schindler, M. J. Org. Chem. 1987, 52, 2800.  (78)  Sherman, J.; Fried, B. Handbook of Thin-Layer Chromatography; Eds. Marcel Dekker: New York, 1991.  (79)  Gaggini, F.; Porcheddu, A.; Reginato, G.; Rodriquez, M.; Taddei, M. J. Comb. Chem. 2004, 6, 805.  120  (80)  Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2002, 9, 203; Varady, L.; Rajur, S. B.; Nicewonger, R. B.; Guo, M.; Ditto, L. J. Chromatography A, 2000, 869, 171.  (81)  Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.  (82)  Kofron, W. G.; Baclawski, L. M. J. Org. Chem. 1976, 41,1879.  (83)  May, J. P.; Perrin, D. M. Chem. Eur. J. 2008, 14, 3404.  121  Appendix Representative 1H-NMR spectra Br Br  N  62  122  Br Br Si  N  65  123  OH  O O  66  124  N OH Br  Si  70  125  O OH O  N  74  O  126  O N H N  O  O  O  H N  O O  O  76a  127  N  O  O  O H N  O  O  76b  O  N H  128  O N H N  O  O  76c  O  O  NH2  129  O H2N  O  O  79  NH  130  O HN  O O  O  N H  80  H N  O O  131  O  O O O  HO O  84  132  O HN  O O  O  N H  85  O  H N O  O  O OEt  133  HN O O H2N  87  O  134  O  O O HN  O O  O  N H  O  H N  H N  O O  O  O  88  N H  O  O  NEt2  135  O Br  N H Br  95  O O  136  Br H N Br  O  96  O OH  137  O  O OH HN  HN O  O O  O N  103  

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.24.1-0061354/manifest

Comment

Related Items