Design and Synthesis of Handles for Solid-Phase Peptide Synthesis And

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LSU Doctoral Dissertations Graduate School

2003 Design and synthesis of handles for solid-phase peptide synthesis and convergent peptide synthesis Jose Giraldes Louisiana State University and Agricultural and Mechanical College, [email protected]

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Recommended Citation Giraldes, Jose, "Design and synthesis of handles for solid-phase peptide synthesis and convergent peptide synthesis" (2003). LSU Doctoral Dissertations. 1146. https://digitalcommons.lsu.edu/gradschool_dissertations/1146

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DESIGN AND SYNTHESIS OF HANDLES FOR SOLID-PHASE PEPTIDE SYNTHESIS AND CONVERGENT PEPTIDE SYNTHESIS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Chemistry

by José Giraldés B.S., University of Puerto Rico, 1997 May, 2003

To my family

ACKNOWLEDGMENTS

I would like to thank my major professor Dr. Mark McLaughlin for his

invaluable guidance and support during my stay at LSU. I am very grateful for the

freedom and encouragement he gave me to develop my own ideas. A major amount of

thanks must be given to Dr. Frank Zhou for the magic angle spinning NMR, to Martha

Juban for help with peptide synthesis and purification, to Dr. Frank Fronczek for crystal

structure determinations and to Dr. Tracy McCarley and Ms. Renee Sims for mass specs.

I also greatly appreciate the scientific discussions with many former current students and

post-docs in our laboratory work: Dr. Umut Oguz, Mr. Caleb Clark, Dr. Tanaji Talele,

Dr. Mohanraj Kumar, Dr. Ted Gauthier and Dr. Lars Hammarström.. I would also like to thank my friends for their kindness and support: Mr. Leonardo Baez, Dr. Alfonso Davila,

Dr. Rachel Bolzan and Dr. Robert Koza.

Finally I would like to thank all of my committee members for supervising my work with continuous encouragement: Dr. William Crowe, Dr. Robert Strongin, Dr.

Steven Watkins and Dr. Ding Shih.

iii TABLE OF CONTENTS

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... iii

LIST OF TABLES...... vi

LIST OF FIGURES ...... vii

LIST OF ABBREVIATIONS ...... xi

ABSTRACT...... xv

CHAPTER 1. ACID LABILE LINKERS FOR SOLID PHASE SYNTHESIS OF PEPTIDES...... 1 1.1 Introduction...... 1 1.2 Literature Review...... 7

CHAPTER 2. DESIGN AND SYNTHESIS OF NOVEL FERROCENE LINKER………………………………………………………36 2.1 Introduction...... 36 2.2 Design of the Ferrocene Linker...... 38 2.3 Results and Discussion ...... 39 2.4 Experimental...... 45 2.4.1 4-Ferrocenyl-4-oxobutanoic Acid...... 45 2.4.2 2-Propenyl-4-ferrocenyl-4-oxobutanoate...... 46 2.4.3 Ferrocenyl-1-hydrazide butanoic Acid...... 47 2.4.4 2-Methyl 4-ferrocenyl-4-oxohexanoate ...... 47 2.4.5 Ferrocenyl-1 hydrazide hexanoic Acid ...... 48 2.4.6 Ferrocenyl-1,1'-acid Fluoride...... 48 2.4.7 Methyl ferrocenecarboxylate...... 49 2.4.8 Methyl-1'-formyl-1-ferrocene carboxylateferrocene...... 50 2.4.9 Hept-6-enylamine...... 50 2.4.10 Ferrocenyl-imine-glycine(ethylester), methoxycarbonyl ...... 51 2.4.11 Ferrocene-methylglycine-ethyl ester, methoxycarbonyl ...... 52 2.4.12 Ferrocene-1-carboxylic acid-1`carboxaldehyde...... 52 2.4.13 Ferrocene carboxaldehyde Resin ...... 53 2.4.14 Ferrocenyl heptenoic amine Resin...... 57 2.4.15 Agni[(KLAKKLA)2] (2.24)using ferrocenyl heptenoic amine Resin...... 57 2.4.16 Ferrocene carboxaldehyde-polystyrene Resin...... 58 2.4.17 Allyl-amino ferrocenyl polystyrene Resin ...... 59 2.4.18 Allyl-amino fmoc-phenylalanine ferrocenyl polystyrene Resin ...... 59

iv 2.4.19 Cleavage of (1-allylcarbamoyl-2-phenyl-ethyl)-carbamic acid 9H-fluoren-9-ylmethyl ester from allyl-amino ferrocenyl polystyrene Resin...... 60

CHAPTER 3. CONVERGENT PEPTIDE SYNTHESIS ...... 61 3.1 Introduction...... 61 3.2 Secondary Structure ...... 64 3.3 Solid-Phase Peptide Synthesis Methods ...... 66 3.4 Convergent Peptide Synthesis...... 70 3.4.1 Convergent Solid-Phase Peptide Synthesis(CSPPS) of Protected Peptide Segments...... 71 3.4.2 Chemoselective ligation of Unprotected Peptide Fragments...... 74 3.5 Results and Discussion ...... 77 3.6 Experimental...... 81 3.6.1 Peptide Synthesis ...... 81 3.6.2 Cyh-7...... 84 3.6.3 Cyh-7 MIC Assays...... 85 3.6.4 16-Hydroxypalmitoleic Acid ...... 86 3.6.5 Peptide Synthesis Agni[ Glu(O-All)(KLAKKLA)2heptenoic Acid] ...... 87 3.7 Ring Closing Methatesis Peptide Ligation...... 87 3.7.1 Attempted Peptide Ligation ...... 88 3.7.2 Attempted Peptide Ligation ...... 88

CHAPTER 4 CONCLUSION AND FUTURE STUDIES ...... 89 4.1 Discussion...... 89

REFERENCES...... …90

VITA...... 96

LIST OF TABLES

Table 1.1 Common protecting groups used in SPPS where side chains are cleaved mild to moderate acidic conditions...... 5

Table 3.1 Common coupling reagents used in SPPS ...... 69

Table 3.2 Examples of large synthetic peptides and small proteins synthesized by SPPS...... 70

Table 3.3 Methods for ligating unprotected peptides...... 75

Table 3.4 Minimum inhibitory concentration of Cyh-7 ...... 86

LIST OF FIGURES

Figure 1.1 Schematic representation of a peptide synthesis...... 1

Figure 1.2 Purification of compounds bound to the solid support from those in solution by simple filtration...... 2

Figure 1.3 General scheme for solid-phase peptide synthesis page...... 4

Figure 1.4 Cleavage of peptide from the linker PAL...... 7

Figure 1.5 Cleavage of peptide from BAL(Backbone amide linker) ...... 8

Figure 1.6 Cleavage of peptide from trityl resin ...... 9

Figure 1.7 Cleavage of peptide from the Rink linker...... 10

Figure 1.8 Methoxy substituted benzyl amine linkers ...... 11

Figure 1.9 Peptide cleavage from BDMTA resin ...... 11

Figure 1.10 Cleavage of peptide from BHA resin ...... 12

Figure 1.11 Cleavage of peptide from MBHA resin...... 13

Figure 1.12 Peptide cleavage using Alkoxybenzylamine linker ...... 14

Figure 1.13 Peptide cleavage from CHA and CHE linkers...... 15

Figure 1.14 Peptide cleavage using the semicarbazide linker...... 16

Figure 1.15 4,4'-dimethoxybenzhydryl derived linkers and cleavage conditions ...... 17

Figure 1.16 Peptide cleavage using a dimethoxy diphenyl linker...... 17

Figure 1.17 Peptide cleavage using HMPB linker ...... 18

Figure 1.18 Peptide cleavage using the 2-chlorotrityl linker ...... 19

Figure 1.19 Peptide cleavage from MALDRE linker ...... 19

Figure 1.20 Peptide cleavage from methoxy benzyl amine linker...... 20

Figure 1.21 Modified Benzhydrylamine linker...... 21

vii Figure 1.22 4-Benzyloxytritylamine linker derivatives...... 23

Figure 1.23 Peptide cleavage from SCAL linker ...... 24

Figure 1.24 Use of Boc-DSA--Ala-NH ...... 25

Figure 1.25 Boc-Leu-O-DSB-ß-Ala-NH-linker...... 26

Figure 1.26 Benzyl alcohol linkers ...... 26

Figure 1.27 Peptide cleavage from an acetophenone-based linker ...... 27

Figure 1.28 p-Alkoxybenzyl linker ...... 28

Figure 1.29 Cleavage of peptide mimetic using HMPV linker...... 28

Figure 1.30 Cleave of peptide using caboxybenzaldehyde resin ...... 29

Figure 1.31 Peptide cleavage using arginyl linker ...... 30

Figure 1.32 Peptide cleavage from oxazolidine linker...... 30

Figure 1.33 Peptide cleavage from p-aminoanilide linker ...... 31

Figure 1.34 Peptide cleavage from the silyl phenyl linker...... 32

Figure 1.35 Peptide cleavage using SAL linker...... 32

Figure 1.36 Peptide cleavage from p-Alkoxybenzyl alcohol linker...... 33

Figure 1.37 Peptide cleavage from p- alkoxybenzyl-oxycarbonylhydrazide linker ...... 34

Figure 1.38 Peptide cleavage from alkyloxycarbonylhydrazide linker...... 34

Figure 1.39 Peptide cleavage using imidazole trityl resin ...... 35

Figure 2.1 Benzyl amine linker and ferrocenyl amine linker...... 37

Figure 2.2 Ferrocenyl carbocation formation by acid cleavage ...... 38

Figure 2.3 Ferrocene linker on same and different sites of the cyclopentadienyl ligands...... 38

Figure 2.4 Retrosyntetic analysis of the ferrocene linker...... 39

viii Figure 2.5 Attempted forward synthetic route ...... 39

Figure 2.6 Failed initial attempts to generate the ferrocenyl amine...... 40

Figure 2.7 Ferrocene Friedel-Crafts acylation with methyl adipoyl chloride .....41

Figure 2.8 Synthesis of ferrocene linker and coupling to Clear resin ...... 42

Figure 2.9 Ferrocene 1-carboxylic acid-1`carboxaldehyde synthesis ...... 43

Figure 2.10 Ferrocene linker amino acid coupling ...... 44

Figure 2.11 Ferrocene linker synthesis ...... 45

Figure 2.12 Clear-NH resin magic angle spinning 1H-NMR...... 54

Figure 2.13 Ferrocene carboxaldehyde resin magic angle spinning 1H-NMR ...55

Figure 2.14. Ferrocene linker 1H-NMR ...... 56

Figure 3.1 General amino acid structure...... 63

Figure 3.2 Cylindrical representations of helical secondary structures. Cylinder to the right shows the main hydrogen bonding in an -helix...... 64

Figure 3.3 Parallel and antiparallel -sheet hydrogen bonding...... 65

Figure 3.4 The Fmoc-/Boc-SPPS protecting group strategy...... 67

Figure 3.5. The Benzyl-/Boc-SPPS protecting group strategy...... 68

Figure 3.6 General Scheme for CSPPS. Peptide fragments are coupled to other peptide fragments...... 72

Figure 3.7 Amide backbone protection...... 73

Figure 3.8 Structures of Hmb and Ac-Hmb protecting groups for backbone amides...... 74

Figure 3.9 Trityl protected cysteine ...... 76

Figure 3.10 Structure of Cyh-7 peptide and structures of 1-amino-1-cyclohexane-carboxylic acid (a) and lysine amino acids (b) ...... 77

ix Figure 3.11 Helical wheel motifs showing -helical and 310-helical conformations of Cyh-7 (designed to be an -helix)...... 78

Figure 3.12 Peptide cleavage using a ruthenium catalyst ...... 79

Figure 3.13. a) Peptide ligation using Grubbs-Hoveyda ruthenium catalyst and b) Peptide ligation using Grubbs ruthenium catalyst ...... 80

Figure 3.14 Structure of the reagents used in Cyh-7 synthesis ...... 81

Figure 3.15 Fmoc deprotection ...... 82

Figure 3.16 Fmoc deprotection and amino acid coupling...... 83

LIST OF ABBREVIATIONS

AA C C -disubstituted amino acid

AcCN Acetonitrile

Alloc Allyloxycarbonyl

B Pathlength

BDMTA Benzyloxy dimethoxytrityl amine

BHA Benzhydrylamine

Blys Bis-lysine

Boc tert-Butyloxycarbonyl

Bpoc 2-(p-Biphenyllyl)-2-propyloxycarbonyl

BTC Bis(trichloromethyl) carbonate c Concentration

ECD Electronic circular dichroism cm Centimeter d Doublet

DBU 1,8-Diazobicyclo[4.5.0]undec-7-ene

DCC Dicyclohexylcarbodiimide

DCE 1,2-Dichloroethane

DCM Dichloromethane

DEC Diethylcarbodiimide deg Degree

DIEA Diisopropylethylamine

DIPCDI Diisopropylcarbodiimide

DMAP 4-Dimehtylaminopyridine

DMF N,N-dimethylformamide

DMSO Dimethylsulfoxide

Dmt Dimethoxytrityl

DTH Delayed-type hypersensitivity Dts Dithiasuccinoyl

Et2O Diethyl Ether

EtOAc Ethyl Acetate

Equiv. Equivalents

FAB Fast atom bombardment

FAL Ferrocenyl amine linker

Fmoc 9-Fluorenylmethyloxycarbonyl

Fmoc-Cl 9-Fluorenylmethyloxy acid chloride

GFP Green fluorescent protein h Hour

HATU N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyrindin-1-yl]methylene]-N- methylmethanaminium hexafluorophosphate N-oxide

HBTU O-benzotriazolyl-N,N,N’,N’-tetramethyluronium hexafluorophosphate

HBr Hydrobromic acid

HCl Hydrochloric acid

HF Hydrofluoric acid

HMPV Hydroxymethyl phenyl valeric acid

HOAt 1-Hydroxy-7-azabenzotriazole

xii

HOBt 1-Hydroxybenzotriazole

HPLC High Performance Liquid Chromatography

Lys L-Lysine m Multiplet

M Molar

MALDI Matrix Assisted Laser Desorption Ionization

MHz Megahertz

MIC Minimum Inhibitory Concentration mL Milliliter mM Millimolar mmol Millimole

MS Mass spectrometry

Mtr 4-Methoxy-2, 3, 6 -trimethylbenzenesulfonyl

µM Micromolar

µg Microgram nM Nanomolar

NMR Nuclear Magnetic Resonance

Nsc 2-(4-nitrophenylsulfonyl) ethoxycarbonyl oNBS ortho-Nitrobenzenesulfonyl oNBS-Cl ortho-Nitrobenzenesulfonyl chloride p Para

PAL Peptide Amide Linker pGlu Pyroglutamyl

xiii

PEG Polyethylene glycol

Pmc 2,2,5,7,8-Pentamethyl-chroman-6-sulphonyl

PS Polystyrene

Psi Pounds per square inch

PyAOP 7-Azabenzotriazole-1-yloxytris(pyrrolindino)phosphonium hexafluorophosphate

RE Electrophile s Singlet

SDS Sodium Dodecyl Sulfate

SPPS Solid-Phase Peptide Synthesis t Triplet tBu tert-Butyl

TEAB Tetraethylammonium bromide

TFA Trifluoroacetic Acid

TFMSA Trifluoromethanesulfonic Acid

TFE Trifluoroethanol

THF Tetrahydrofuran

TMS-Cl Trimethylsilyl chloride

UV Ultraviolet

Vis Visible

Z Benzyloxycarbonyl

Molar absorptivity

Wavelength

xiv

ABSTRACT

The recent popularity of methods for solid-phase peptide synthesis that use

the 9-fluorenylmethyloxycarbonyl (Fmoc) group for N -amino protection has created a

need for compatible anchoring linkages and handles. In an effort to develop mild new

methods for use in solid-phase peptide synthesis (SPPS), a new ferrocene containing

linker or “handle”, the 1’1-ferrocenyl carboxaldehyde handle was designed, synthesized,

characterized and tested. This linker is analogous to those commercially available and

developed by Barany. The ferrocenyl amine linker(FAL) releases C-terminal peptide

amides upon acidolysis. Since the FAL handle is acid labile it is compatible with Fmoc

and N -dithiasuccinoyl (Dts) based chemistries, but not N -tert-butyloxycarbonyl (Boc)

based chemistries. The solid-phase linkage was investigated based on the stability of the

ferrocenium ion. The stability of this ion is greater than that of the benzyl cations that are

used in the handles developed previously.

xv CHAPTER ONE

ACID LABILE LINKERS FOR SOLID PHASE SYNTHESIS OF PEPTIDES

1.1 Introduction

Since the pioneering work of Merrifield in solid phase peptide synthesis1 in the 1960s, several different methods for cleaving peptides have been developed. The cleavage of the peptide from the resin is one of the key steps in solid phase peptide synthesis. One method which imparts flexibility to the cleavage process is to attach a molecule (linker or handle, figure 1.1) to the solid support which facilitates the cleavage of the peptide.

Cleavage Linker Peptide Peptide + Linker

Solid Support

Figure 1.1 Schematic representation of a peptide synthesis on solid phase.

Solid phase chemistry is currently not as developed as solution-phase chemistry, particularly with regard to small organic molecules, but it has some advantages over the solution-phase. First, in solid-phase synthesis, large excesses of reagents can be used to drive reactions to completion; these excess reagents can then be removed at the end of these reactions by filtration and washing. Second, because of easy separation of reagents and products, solid-phase chemistry can be automated more easily than solution

1 chemistry. Separation of compounds bound to the solid support from those in solution is accomplished by simple filtration (figure 1.2).

Excess of Reagents =

Filter

Figure 1.2 Purification of compounds bound to the solid support from those in solution by simple filtration.

The solid support describes the insoluble material that is reversibly bound to the starting reactants. Solid-phase reactions can occur on the surface of the solid particles or inside these particles. There are several types of materials used as solid supports that

2 allow reactions only on the surface, for example, beads made from glass and cellulose fibers2, the reduced surface area in these surface-type solid supports reduces the number of functionalization sites.

Several supports have been developed since the initial introduction of polystyrene cross-linked with 2% divinylbenzene (DVB) by Merrifield. The supports can be chosen depending on the type of reactions and products desired. Different supports may be chosen for different types of chemistries. For example, some supports, such as polyethylene glycol (PEG) grafted resins are suitable for use in polar solvents others such as polystyrene are used with nonpolar solvents. The solid support must have the following characteristics3 for an efficient solid-phase synthesis: 1) physical stability and of the right dimensions to allow for liquid handling and filtration; 2) chemical inertness to all reagents involved in the synthesis; 3) an ability to swell while under reaction conditions to allow permeation of solvents and reagents to the reactive sites within the resin; 4) derivatization with functional groups to allow for the covalent attachment of an appropriate linker or first monomeric unit. In most cases, the linker unit must be cleavable under conditions that allow the isolation of the desired product after synthesis is complete.

Cleavage conditions are dictated by the linker used. The key to successful solid phase synthesis lies in the protection scheme that is used to assure reaction only at the desired position(s). Most monomeric units (X) used in solid-phase synthesis can be expressed with the empirical formula n-X-e, where n is the nucleophilic portion of the residue and e is the electrophilic portion. The first monomeric unit is coupled to the resin at either the nucleophilic or the electrophilic site. However, the portion of the molecule that is not covalently bound to the resin must be protected to avoid subsequent

3 polymerization of excess monomers in solution. Thus, if the electrophilic portion of the first residue is coupled to the resin, the nucleophilic portion must be protected, and vice versa (figure 1.3). The protecting group must be stable to the reaction conditions of each coupling. After coupling is performed, the protecting group is removed to expose a new reactive site and synthesis continues in a repetitive fashion.

Linker

= Solid Support 1st Coupling + = Amino Acid Linker

Deprotection = Side-chain Protection - (Semi-Permanent) 2nd Coupling + = N-terminal Protection (Temporary) Linker

Deprotection -

3rd Coupling + Linker

Deprotection -

4th Coupling + Linker

Deprotection - Cleavage -

Product

4 Figure 1.3 General scheme for solid-phase peptide synthesis.

If several nucleophilic and/or electrophilic groups are present in a monomeric unit, they must be orthogonally protected with groups that vary in reactivity. This allows

4 for deprotection of the portion of the molecule where further reaction is desired to take place in subsequent couplings, while preventing reaction at side-chain functional groups.

SPPS is almost exclusively performed in the C→N direction, with the amino group being the nucleophilic portion and the C-terminus the electrophilic portion. Common protecting groups and the conditions under which they are cleaved are listed in Table 1.1

Table 1.1 Common protecting groups used in SPPS where side chains are cleaved under mild to moderate acidic conditions.

Protecting Group Structure Cleavage Reference

Method

Nα-Protecting

Groups

Fluorenylmethoxy- O Base-catalyzed 5 O carbonyl (Fmoc) (20% Piperidine

in DMF)

Allyloxycarbonyl O Hydrogenolysis 6 O (Alloc) (Pd/C; ethanol)

O 2-(4- O Base catalyzed 7 O2NSCH2CH2 O nitrophenylsulfonyl) O (20% piperidine ethoxycarbonyl in DMF

(Nsc)

Benzothiazole-2- S O Zn-Acetic Acid 8 S N sulfonyl (Bts) O Al-Hg/THF/H2O (table cont’d.)

5 Na2S2O4

5-Methyl-1,3,4- O Zn-Acetic Acid 8 N N S thiadiazole-2- S Al-Hg/THF/H O H3C O 2 sulfonyl (Ths)

Side-Chain

Protecting Groups

Benzyloxycarbonyl O Catalytic 9 O (Z) Hydrogenation

Acidolysis t-Butyl CH3 Acidolysis (TFA) 10,11 H3C CH3

Dimethoxytrityl Acidolysis (Weak 12 H3CO OCH3 (Dmt) Acid)

Acidolysis remains the most popular amongst the synthetic strategies formulated towards the cleavage of a peptide from its protecting groups and resin support. The acidolytic deprotection reaction is a major component in the overall scheme in two widely used stepwise SPPS approaches. The first scheme, the Boc-Bzl strategy adheres to the principle of differential acid lability. The second approach, the Fmoc-tBu strategy, is becoming more widely used because of the huge reactivity difference between acid-labile side-chain protecting groups and the base-mediated cleavage of the Fmoc group.

6 1.2 Literature Review

This review focuses on linkers designed to be highly acid labile. These linkers are predominantly based on the benzylic C-N bond cleavage. Recent advances in linkers have allowed other polar functional groups, such as alcohols and thiols, to be attached to the polymer support. Polar functionalities such as carboxylic acids and amides are released upon cleavage of products from the resin in SPPS. In fact, most of the linkers available for solid support synthesis to date require polar functional groups for binding.

Examples of peptide-resin cleavage are given below for acid labile linkers;

The PAL(peptide amide linker)13 handle (figure 1.4) is suitable for cleavage of complex peptide amides that contain several sensitive side-chain functionalities or arginine residues that contain the blocking groups Mtr or Pmc. The peptide pGlu-Gly-

Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 was prepared in

37% yield.

CH3 a. NHCOOH3C(H2C)4O N Peptide Peptide H CH3

a. TFA/ thiosanisole/1,2-ethanediethol-anisole (90:5:3;2) 3 h at 25 C, to

Resin = aminomethyl polystyrene- 2% divinylbenzene Peptide= Human Gastrin – I (pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-

Gly-Trp-Met-Asp-Phe-NH2) Yield = 68% (cleavage yield), 37% after HPLC Purity = 85% Coupling Method= Fmoc/HOBt/DIPCDI Type of Peptide = Peptide amides Figure 1.4 Cleavage of peptide from the linker PAL.

7 Substituted benzyl amide14 linkers have been compared. The results showed that by adding methoxy groups (figure 1.5) to the benzene and increasing the length of the spacer that separates the electron-donating oxygen para to the anchored amino acid from the electron-withdrawing alkylamide function linking the handle to the support increased the acid liability of the linker. A model peptide (H-Tyr-Gly-Gly-Phe-Met-NH2) was prepared and the cleavage yield was about 80% with 90% purity. This linker is suitable to use with Fmoc or Dts protected amino acids.

OCH3 O TFA

H2C-HN-LeuC(H2C)4O CH2NH-Peptide Peptide Dichloromethane Dimethylsulfide

OCH3

Resin = Aminomethylcopoly(styrene-1%-divinylbenzene) Peptide= analogue methionine-enkephalinamide (H-Tyr-Gly-Gly-Phe-Met-NH2) Purity = 90% Yield = 80% Type of Peptide = amide Coupling Method= Fmoc/DCC/HOBt/DMF

Figure 1.5 Cleavage of peptide from BAL(Backbone amide linker).

The trityl linker15 is widely used to prepare small-protected peptides. It can be used to obtain thiols, alcohols and amines via cleavage. In this example (figure 1.6) The trityl linker was used to synthesize the following peptide: DNP-Pro-Gln-Gly-Ile-Ala-Gly-

Lys(Cum)-Arg-Dah-Acr.

O 0.5% TFA Peptide C(CH2)n O-Peptide DCM 4%MeOH or H2O

Type of Peptide = peptide hydrazides

Coupling method= Fmoc/DIPCDI/HOBt

Figure 1.6 Cleavage of peptide from trityl resin.

The Rink linker16 does not allow the use of HOBt as a coupling catalyst without buffering with DIPEA. A protected peptide[Fmoc-Asp(Obut)-Arg(Mtr)-Gly-Phe-

Tyr(but)-Phe-Ser(but)-Arg(Mtr)-Pro-Ala-Ser(but)-Arg(Mtr)-Val-Ser(But)-

Arg(Mtr)Arg(Mtr)-Ser(But)-Arg(Mtr)-Gly] was prepared and obtained in 23% yield after

HPLC (figure 1.7). N-Substituted hydroxamic peptide acids were synthetized using this linker in yields of 80-90%. This peptides are isolated from microorganisms are known to be potent and selective inhibitors of many metallo proteases 17. A modified Fmoc strategy was used to synthesize this peptide using this linker. A 9-residue peptide consisting of arginine and tryptophan was prepared with high purity and yield.

9 OMe

0.1% TFA in DCM, OMe 2 min, r.t. Peptide

N Peptide

H CO CH3 2

Resin = polystyrene Peptide = Fmoc-Asp(Obut)-Arg(Mtr)-Gly-Phe-Tyr(but)-Phe-Ser(but)-Arg(Mtr)-Pro-Ala- Ser(but)-Arg(Mtr)-Val-Ser(But)-Arg(Mtr)Arg(Mtr)-Ser(But)-Arg(Mtr)-Gly Yield=23 % (after HPLC) Type of Peptide = Peptide esters Coupling method = Fmoc Figure 1.7 Cleavage of peptide from the Rink linker.

Benzyl amine linkers in figure 1.8 were compared by Bertnatowicz18. This study found that linkers c) and d) (figure 1.8) are the most satisfactory for peptide synthesis.

These linkers are well suited for peptide C-terminal amides and are compatible with

Fmoc chemistry. Cleavage is performed with TFA:phenol(95:5) for 2h at room temperature. Yields of crude peptides (Eledoisin and Neuromedin) were isolated in 87-

91 % yield for both linkers.

Benzyloxy dimethoxytrityl amine (BDMTA) resin19 is similar to the trityl resin mentioned earlier but in this case two benzene rings have methoxy groups para to the benzylic carbon attached to the three phenyl groups (figure 1.9). This linker was used to prepare Phe-Thr-Pro-Arg-Leu-NH2 which can be cleaved with dilute TFA. Purity of

90+% (HPLC) and satisfactory yields 80+% based on resin loading were reported.

10 Fmoc-NH Fmoc-NH OCH3 OCH2CO2H

CH3O OCH3 (CH2)2CO2H

a) b)

Fmoc-NH Fmoc-NH OCH3 CH2

OH3C OCH3

OCH3 HO2CCH2O O(CH2)4CO2H C) d)

Figure 1.8 Methoxy substituted benzyl amine linkers.

OCH3

3% TFA CH2O C N Peptide Peptide H

OCH3

Peptide= Phe-Thr-Pro-Arg-Leu-NH2 Yield = 80%+ Purity = 90+% Coupling Method= Fmoc/DCC Type of Peptide = Peptide Amide Figure 1.9 Peptide cleavage from BDMTA resin.

11 Benzhydrylamine(BHA) Resin20 is useful to prepare peptide amides. In this example(figure 1.10) an analogue of arginine vasopressin was prepared.

CH2-CH2-CH2OC NH-(Gly)-D-Ala-Arg(Tosyl)-Pro-Cys-Asn-Gln-Phe-Tyr H O-2-Br-CBZ

HF/Anisole(9:1) Peptide

Resin = BHA resin Peptide= arginine vasopressin analogue Yield = 6% Coupling Method= Fmoc/DCC/HOBt Type of Peptide = Peptide Amide Figure 1.10 Cleavage of peptide from BHA resin.

4-Methylbenzhydrylamine (MBHA) linker21 is commonly used in the solid phase synthesis of carboxamides, aldehydes and sulfonamides. This linker is more acid labile than the BHA linker due to the effect of the extra methyl group stabilizing the cation formed upon cleavage. TFMSA and HBr/thioanisole in TFA, HF/anisole can also cleave the MBHA linker22, 23, 24. MBHA resin is the standard resin for the synthesis of peptide carboxamides by the Boc solid phase synthesis methodology 25. The

12 peptide(figure 1.11) was synthesized using amino acid aldehydes and HOBT/Boc couplings. The peptide was cleaved from the resin using HF.

CH3

HF N Peptide Peptide H H

Resin = MBHA polystyrene + Peptide= H3NLeu[CH2N9CH2CH2CH2SH]Ser-Pro-Gly-Lys-

Val[CH2N9CH2CH2CH2SH)]Ala-Pro-Lys-Tyr-NH2 Purity = low levels of side products Yield = 21% (after off-resin disulfide bond cyclization) Type of Peptide = amide Coupling Method= HOBt/Boc/amino acid aldehydes Figure 1.11 Cleavage of peptide from MBHA resin.

Alkoxybenzylamine linker 26 anchors the C-terminal amino acid to the resin by the -nitrogen atom(figure 1.12). This linker is similar to BAL but requires harsher cleavage conditions. This linker is compatible with the alloc-protect/palladium-deprotect chemistry. The linker is also compatible with Boc chemistry and it cleaves from the solid support using standard HF cleavage. The synthesis of stylostatin(a cyclic peptide) with on resin cyclization followed by cleavage and HPLC purification gave yields of 10 % for the monomer, 25.5 % for the dimmer and 1 % for the trimer. Bourne – Smythe also cleaved the peptide, followed by cyclization in solution and the yields, which were

13 dependent on the concentration of DMF, increased for the monomer, were about the same for the dimer and decreased for the trimer.

O Spacer

HF:p-cresol, 10:1 H-Pro-Phe-Asn-Ser-Leu-Ala-IIe-OH

R 39% Yield OH N H-Peptide O Spacer = Gly-Leu-Leu

Resin = Aminomethylpolystyrene Resin Peptide = H-Pro-Phe-Asn-Ser-Leu-Ala-Ile-OH Resin = Benzyl amine resin Yield= 39%(Yields for cyclic peptides range from 10-25%) Type of Peptide = Cyclic amide bond Coupling Method = HBTU/ DIEA/ DMF Figure 1.12 Peptide cleavage using Alkoxybenzylamine linker.

5-{[R,S]-5-[9-Flourenylmethoxy-carbonyl)amino]-10,11- dihydrodibenzo[a,d]cyclohepten-2-yl]oxy}valeric acid (CHA) and 5-{[R,S)-5-[(9- fluorenylmethoxycarbonyl)amino]dibenzo[a,d]cyclohepten-2-yl]oxy} valeric acid (CHE) linkers (figure 1.13) developed by Noda 27 are suitable for peptide amide preparation under mild conditions. The linkers are compatible with Fmoc chemistry. Human Secretin, a 27-residue peptide, was prepared on both linkers and cleaved with TFA-H2O- thioanisole-EMS-EDT-thiophenol (82.5:5:5:3:2.5:2, v/v) at rt for 6 h (Yield 54% for both resins). Although this peptide synthesis gave similar yields, the authors found that CHA

14 was a superior handle for the preparation of peptides, especially peptides that contain acid labile residues such as Trp.

HN Peptide

CHA OC(H2C)4O

Peptide a

HN Peptide

CHE OC(H2C)4O

a = TFA-H2O-thioanisole-EMS-EDT-thiophenol (85:5:5:5:3:2:5:2) v/v ,RT 6 h

Resin = Tanta Gel S NH2 Peptide= Human Secretin Yield = 54%(after HPLC) Purity = 85% Coupling Method= HBTU/HOBt/DIEA Type of Peptide = Peptide amide

Figure 1.13 Peptide cleavage from CHA and CHE linkers.

The semicarbazide linker 28 (figure 1.14) is useful for the synthesis of peptide aldehydes and C-terminal semicarbazones. The method proceeds with no loss of stereochemical integrity. The peptides can be cleaved from the resin using TFA/H2O(9:1) for 1.5 hrs.

15 TFA/H2O(9:1) 1.5 hrs. C O Peptide H2

PeptideHNHN N H O

Peptide= Ac-AAVALLPAVLLALLAPDEVD-H Yield = 50% yield Coupling Method= Fmoc amino acids Type of Peptide = Peptide Aldehyde

Figure 1.14 Peptide cleavage using the semicarbazide linker.

4,4'-dimethoxybenzhydryl derived linkers29 (figure 1.15) are derived from the

4,4’-dimethoxybenzhydryl (Mbh) group used for the protection of amides. The peptide

LHRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) was synthesized in 43% yield after chromatography using linker d in figure 1.15.

[(5-Carboxylatoethyl-2.4-dimethoxyphenyl)-4’-methoxyphenyl]methylamine 29 linker in figure 1.16 is another derivative of 4,4’-dimethoxybenzhydryl(Mbh)-protecting group for amides. Peptide pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-amide was synthesized and cleaved with 95 %TFA, 5 % thioanisole and 5 % ethanedithiol by volume (98 % Yield).

16 O R TFA/thioanisole/ethanedithiol HN C-C-N Peptide 90/5/5 Peptide R1

OOC-H2C-O OCH3 R2

a). R,R1,R2 = H 2 1 b). R, R = H R = CH3 1 2 c). R = H, R ,R = CH3 1 2 d). R = CH2C6H5, R ,R = H 1 2 e). R = (CH3)2CH, R = CH3, R = H

Peptide = LHRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) Yield = 43% Coupling Method= Fmoc/DCC/HOBt Type of Peptide = Peptide Amide

Figure 1.15 4,4'-dimethoxybenzhydryl derived linkers and cleavage conditions.

CH2CH2 HN Peptide 95 %TFA, 5 % thioanisole, 5 % ethanedithiol

H3CO OCH3 Peptide

OCH3

Resin = aminomethylated polystyrene resin Peptide= pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-amide Purity = high Yield = 98% Type of Peptide = amide Coupling Method= Fmoc/HOBt/DIC

Figure 1.16 Peptide cleavage using a dimethoxy diphenyl linker.

17 4-Hydroxymethyl-3-methoxyphenoxybutyric acid (HMPB) 30 is useful to make peptide amides. In this work, a dendrimer was attached to the resin to increase the loading level of the resin. Here the linker is attached after the dendrimer. The results showed that there was only a small increase in substitution. A model peptide consisting of 13 residues was prepared and cleaved with 50% TFA in DCM (44% Yield).

OCH O 3 50% TFA in DCM

Dendrimer CH2-NH-C-(CH2)-O C-O-Peptide Peptide

Resin = Tenta Gel Peptide= 13 residue peptide Yield = 44 % (after HPLC) Type of Peptide = amide

Figure 1.17 Peptide cleavage using HMPB linker.

The 2-chlorotrityl linker 31 is compatible with Fmoc chemistry. This linker is useful when using nucleophilic amino acids such as Trp and Met. The purity when using

Fmoc/Trt amino acids is much higher than when using Fmoc/tBu amino acids.

18 1.1 % TFA/DCM Ser-Peptide Peptide Cl

Peptide= H-Thr-Thr-Trp-Thr-Ser-Met-Ser-Trp-Tyr-OH Purity = 92 % Coupling Method= Fmoc/DCC/HOBt Type of Peptide = Peptide carboxylic acid Figure 1.18 Peptide cleavage using the 2-chlorotrityl linker.

The 2-methoxy-4-benzyloxy-polystyrene aldehyde linker (MALDRE) (figure

1.19) 32 This linker is useful for the synthesis of C-terminal peptide amides. The peptide is cleaved from the resin by treatment with 10-50% trifluoroacetic acid in methylene dichloride for 30 minutes. This linker is compatible with the Fmoc/Boc chemistry. The resin 2-methoxy-4-benzyloxy polystyrene aldehyde linker MALDRE can be prepared from Merrifield chloromethylated polystyrene.

OCH3

CH2O CH2NOCR2 Peptide-CONHR1 R1 R1 = C6H5(CH2)4NH2

Resin = polystyrene

Peptide= Tyr-Gly-Gly-Phe-Leu-NHCH2CH2CH2CH2C6H5 Yield = 44 % after HPLC Type of Peptide = amide Coupling Method= Fmoc/DIC/HOBt Type of Peptide = sec-peptide amides Figure 1.19 Peptide cleavage from MALDRE linker.

19 Benzylamine derivatives in figure 1.20 33 were used to prepare the peptide thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn) was synthesized using both handles. The thymulin-resin-NH-resin were treated with 95 % TFA/thioanisole and the cleavage products were identical and of high purity (90 %).

OCH3

95 % TFA/thioanisole

CH2O R5 Peptide

R6 OCH3

a). p-NH2-resin: R5 =CH2NH2, R6 = H b). o-NH2-resin: R5 = H, R6= CH2NH2

Resin = Chloromethylcopoly(styrene-1 %-divinylbenzene) Peptide= thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn) Purity = high Yield = 50-95 % Type of Peptide = amide Coupling Method= Fmoc/DCC/HOBt/

Figure 1.20 Peptide cleavage from methoxy benzyl amine linker.

20 The benzhydrylamine-derived linker is compatible34 with Fmoc chemistry.

Tetragastrin was prepared and cleaved with 1 M thioanisole /TFA in 41 % yield after

HPLC.

PAM-ILe COCH CH OMe 1 M thioanisole /TFA 2 2 (28 C; 60 min). Peptide CH-NH-Peptide

MeO

Resin = (aminomethyl)polystyrene resin Peptide= tetragastrin Yield = 41 % yield (cleavage yield 60 %, based on Ile in the resin) Coupling Method= Fmoc/DCC/HOBt Type of Peptide = Peptide Amides Figure 1.21 Modified Benzhydrylamine linker.

4-Benzyloxytritylamine linker35 derivatives were substituted with methoxy groups to compare the cleavage of peptides by TFA (figure 1.22). The results showed that the methoxy substituents reduce the cleavage time of the peptide amides from 2h for

BTA to 10 min for 4-benzyloxy-2’,2”,4’,4”-tetramethoxytritylamine (BTEMTA) with 1

% TFA/DCM. This method allows the preparation of protected peptide amides in high yields and purity. Methoxy groups were also introduced to 9-xanthenylamine linker

(XAL). The XAL derivatives, 9-amino-9-(4-methoxyphenyl)-xanthene-3- yloxymethyl(MPXAL) and 9-amino-9-(2,4-dimethoxyphenyl)-xanthene-3- yloxymethyl(DMPXAL), the peptide oxytocin (Fmoc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-

21 Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-OH) in excellent yield and purity. For the linker

MPXAL, the cleavage was achieved in 10 min (80 % yield) and 5 min for DMPXAL (90

% yield) with 1% TFA/DCM.

Safety catch amide linker (SCAL)36 is available in the SO form and the S form.

In the SO form this linker is stable to 100 % TFA, reagent K, 50 % piperidine, HF and

Pd(0) treatment. In the S form is stable to 50 % piperidine but it cleaves with mixture K and 50 % TFA. Synthesis of peptides can be accomplished by Fmoc, Alloc and /or Boc strategy. The SCAL linker was used successfully in the synthesis of a segment related to human calcitonin(figure 1.23). H-Phe-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 was obtained in 95 % purity (HPLC) after cleavage with 1M-Me3SiBr/thioanisole/TFA, for 2h at 0°C. This linker was found to have many advantages for solid-phase chemical ligation. vMIP I, a chemokine and 71 amino acid protein, that contains all 20 natural amino acids was prepared in a three-segment synthesis. Recovered yields were about 10-

15 % for three and four segment ligations.

Benzyl alcohol linkers developed by Sheppard and Williams38 are illustrated in figure 1.26: a) They are is stable to acid and its peptides esters are cleaved by acid or nucleophilic reagents. One of the most common reagents for this is ammonia in the preparation of peptide amides. (b) Benzyl ester-type: acid labile on resin-bound esters are compatible with Boc-benzyl chemistry. The cleavage requires a strong liquid hydrogen fluoride solution. (c) The p-alkoxy derivative has better lability over b and has been used in the Fmoc-t-butyl polyamide method. The peptide ester bond is cleaved by TFA. (d)

These two safety catch linkers37 figures 1.24 and 1.25 are derived from 4- methylsulfinylbenzyl protecting group(Msob) are easily cleaved by mild acid. They are

22 HNH-Fmoc R2 H2NOCH2

O O R3

H2C N C H H XAAL 2 R4 BTA R1=R2=R3=R4=H BMTA R1=OCH3, R2=R3=R4=H BOMTA R1=R4=OCH3, R2=R3=H BTRMTA R1=R2=R4=OCH3, R3=H BTEMTA R1=R2=R3=R4=OCH3

HOH CH3 OCH3

R O O H2N

O(CH2)n N CH H

O OCH2 HXAL n= 1 HXVL n=4 MPXAL R=H DMPXAL R=OCH3

Resin = polystyrene DMPXAL Peptide = oxytocin (Fmoc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu- Gly-OH) Yield= 90% Purity= high Type of Peptide = amide peptide

Figure 1.22 Structures of linkers: 4-benzyloxytritylamine(BTA), 4-benzyloxy-4’- methoxytrytylamine(BMTA), 4-benzyloxy-4’,4”- dimethoxytritylamine(BDMTA), 4-benzyloxy-2’,4’,4”- trimethoxytritylamine(BTRMTA), 4-benzyloxy-2’,2”,4’,4”- tetramethoxytritylamine(BTEMTA), [9-Fmoc-amino-xanthen-4-yl]-acetic acid aminomethyl resin(XAAL), 9-hydroxy-xanthen-3-yloyacetic acid MBHA resin(HXAL), 9-hydroxy-xanthen-3-yloxyvaleric acid MBHA resin (HXBL), 9-amino-9-(4-methoxyphenyl)-xanthene-3-yloxymethyl(MPXAL) and 9-amino-9-(2,4-dimethoxyphenyl)-xanthene-3-yloxymethyl(DMPXAL) polystyrene resin.

23 activated by a reductive acidolysis in the final deprotection step. The best protecting groups for the first linker Boc-DSA--Ala-NH-linker (figure 1.24) are Msob or tert-butyl on Asp residues, this is to prevent the formation of succinimide from Asp-containing peptides which can be a side reaction dependent on the amino acid sequence. The peptide

(figure 1.24), Buccalin was synthesized in 40 % overall yield after cleavage with

TFA/DCM.

Boc-Leu-O-DSB-ß-Ala-NH-linker was used to prepare a 17-amino acid residue peptide. γ-Endorphin was synthesized with amino acid derivatives bearing safety catch protecting groups SPPS in which in situ neutralization and BOP activation were employed. The cleavage yield (figure 1.25) was 82 % as determined by amino acid analysis of the hydrolyzate of the resulting resin. The crude peptide was purified to γ- endorphin in 62 % overall yield.

The 2,4-dialkoxy benzyl alcohol derivative polymer bound esters can be cleaved by 1 % TFA in dichloromethane, under controlled conditions t-butyl groups remain in the molecules. Linker (d) has been used with chitosan/chitin as solid supports39.

O 1M-Me3SiBr/thioanisole/TFA Peptide O HN N O H

Peptide NH2 2h at 0°C. H3C CH3 SS O O

Peptide = H-Phe-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 Purity =95 % by HPLC Yield= essentially quantitative cleavage Resin = Benzyl amine resin Coupling Method = Fmoc/Boc

Figure 1.23 Peptide cleavage from SCAL linker.

Peptide-NH OCH3

a Peptide O

S OCH3

CH2-NH-Ala--(CH2)5CH2

a = tetrachlorosilane-m-cresol-thioanisole-ethane-1,2-dithiol/TFA-DCM 25 C 3 h

Resin = alanylaminomethylated polystyrene

Peptide= Buccalin (H-Gly-Met-Asp-Ser-Leu-Ala-Phe-Ser-Gly-Gly-Leu-NH2)

Yield = 40 % (77 % Cleavage Yield)

Coupling Method= Boc/DIPCDI/DMAP

Type of Peptide = Peptide amides

Figure 1.24 Use of Boc-DSA--Ala-NH.

25 O CH3 S a H3C Peptide

CH3

Peptide O-CH-CH2CH2-CO-ß-Ala-NH-CH2

a = tetrachlorosilane-m-cresol-thioanisole-ethane-1,2-dithiol/TFA-DCM 25 C 3 h

Resin = alanylaminomethylated polystyrene Peptide= γ-Endorphin (H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu- Val-Thr-Leu-OH) Yield = 62 % (80 % Cleavage Yield)

Coupling Method= Boc/BOP/iPr2EtN Type of Peptide = Peptide carboxylic acids

Figure 1.25 Boc-Leu-O-DSB-ß-Ala-NH-linker.

CH2OH CH2OH CH2OH CH2OH OMe

CO2H CH2CH2CO2H OCH2CH2CO2H OCH2CO2H a) b) c) d)

Figure 1.26 Benzyl alcohol linkers.

This acetophenone-based linker40 in figure 1.27 linker is compatible with Fmoc chemistry. Peptides can be cleaved with 20 % TFA/DCM. The peptide Fmoc-Glu-His-

Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH was prepared with this linker (64 % yield).

O OPeptide20% TFA/DCM Peptide N O H

Resin = SynPhaseTM PS grafted crowns Peptide = Fmoc-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH Yield = 64 % Purity = 75 % Coupling Method= Fmoc/DIC/HOBt Type of Peptide = Peptide carboxylic acids

Figure 1.27 Peptide cleavage from an acetophenone-based linker.

The p-alkoxybenzyl linker2 was utilized (figure 1.28) to synthesize large numbers of different peptide sequences. Instead of using polystyrene resins or its derivatives cellulose paper discs are used as solid supports for SPPS. Loading of the discs is about

2.5 umol/disc. The peptide chains are linked to the cellulose via a p-alkoxybenzyl ester anchor whis is cleaved by mild treatment with trifloroacetic acid/DCM. This linker is compatible with Fmoc protected amino acids. Cellulose paper is very resistant towards organic solvents but it disintegrates with strong acids. The acid sensitivity prohibits the use t-Boc peptide synthesis but it still allows mild-acid peptide cleavage. The p- alkoxybenzyl alcohol derivative releases ester bound peptides quantitatively by treatment by treatment with 50 % or less trifluoroacetic in DCM. The cleavage cocktail used was

TFA/anisole/dichloromethane (55/5/40 by volume for 2.5 h at RT).

HO-CH2 O-(CH2)5-CH2-O-(Cellulose)-O-Ac

Figure 1.28 p-Alkoxybenzyl linker.

In this example41 the hydroxymethyl phenyl valeric acid(HMPV) linker is used to prepare potential peptide turn mimetics. The peptide in figure 1.29 was cleaved from the resin using 1 % TFA/DCM (68 % Yield). This linker is compatible with Fmoc chemistry and o-NBS protecting groups.

O O O 1% TFA/DCM HMPV O (S) NH2 Peptide N N

tBuO

Peptide

Peptide= Peptide Mimetic Yield = 68 % Coupling Method= Fmoc/HBTU/HOAt/collidine Type of Peptide = Peptide carboxylic acid Figure 1.29 Cleavage of peptide mimetic using HMPV linker.

28 p-Carboxybenzaldehyde42 resin is useful to make peptides such as ocreotide acetate and biotin-ocreotide (figure 1.30). This linker is compatible with Fmoc chemistry.

CH3 O TFA 90%, water,thioanisole, O EDA and Phenol N N Peptide Peptide H O H

Resin = Rink amide Peptide= Ocreotide Yield = 74 %(after HPLC Type of Peptide = amide Coupling Method= Fmoc/HOBt/HBTU/DIEA in NMP

Figure 1.30 Cleave of peptide using caboxybenzaldehyde resin.

Arginyl derived linker43 (figure 1.31)is useful for peptidyl and peptidomimetic argininal derivatives. In this example (figure 1.31) the peptide is cleaved from the resin with TFA, CH2Cl2, H20: 6,3,1.

The oxazolidine linker44 (figure 1.32) allows straight forward and direct attachment of aldehyde functionality to the solid phase. This oxazolidine linker is compatible with

Fmoc peptide synthesis, including TFA treatment, but is cleaved by mild aqueous acid at

60 C.

29 Peptide Peptide N N Alloc m-n N N TFA N N H H OH NH2 O NH2 (CH2)5CONH m - (Ph3P)4Pd(cat), THF, DMSO, 0.5 N HCl, morpholine: 2,2,1,5; n - TFA, CH2Cl2, H2O: 6,3,1

Resin = AM-resin Yield = 55 % Purity = 97 % purity (HPLC) Coupling Method= PyBOP/DIEA/DMF Type of Peptide = Peptide Aldehydes Figure 1.31 Peptide cleavage using arginyl linker.

O CH3 R H AcOH (aq) Spacer Ac-Tyr-Ala-Phe-Val-H N N H O R=Ac-Tyr-Ala-Phe-Val-H

Resin = Synphase crown Peptide= Ac-Tyr-Ala-Phe-Val-H Purity = High Type of Peptide = Peptide Aldehydes Coupling = DIC/HOBt Figure 1.32 Peptide cleavage from oxazolidine linker.

p-Aminoanilide linker45 is useful for preparing p-nitroanilide (PNA) chromogenic substrates to study the kinetics and specificity of proteolytic enzymes. It is compatible with both Boc and Fmoc chemistries. This handle was prepared using

30 hydroxymethyl resin from highly substituted chloromethyl resin. This method works for most amino acids except those containing easily oxidized amino acids like methionine, tryphtophan or cysteine.

O HF Anisole

O N N Peptide Peptide NH2 H H

Resin = Urethane linked p-aminoanilide(UPAA)

Peptide= Succinyl-Ala-Ala-Pro-Arg-PNA

Type of Peptide = paranitroamines, para-nitroanilides (after oxidation with NaBiO3)

Coupling Method= Boc

Figure 1.33 Peptide cleavage from p-aminoanilide linker.

The silyl phenyl linker46 (figure 1.34) for reverse-direction SSPS doesn’t need a spacer group like many other silyl linkers. After the synthesis, the resin can be recycled.

The activation of the amino acids was carried using i-butyl chloroformate/N- methylmorpholine. Other standard conditions such as DCC/DMAP: HBTU/HOBT,

DIPEA; HOBT, DIPCDI were less effective. Cleavage of the free amine leaves behind only CO2 as by-product.

The linker 4-[1-[N-(9-fluorenylmethyloxycarbonyl)-amino]-2-(trimethylsilyl) - ethyl]-pheno-xyacetic acid linker “SAL” 47 takes advantage of organosilicon chemistry.

This linker was designed to undergo deblocking by a β- elimination mechanism under acidic conditions. This linker is useful for peptides with C-terminal tryptophan, which are

31 obtained, only in poor yields using conventional linkers. This linker is also stable to tetrabutylammonium fluoride (TBAF).

Boc N f,g Ph O Si OPeptide H N O N O H O

(Peptide) f : HF, CH3CN; g : (Boc)2O, Et3N

Resin = polystyrene Peptide= above is fine Pro-Phe-Gly-Phe-Phe(O-All) Yield = 54 % Coupling Method= i-butyl chloroformate/N-methylmorpholine Type of Peptide = Allyl or Methyl Ester Figure 1.34 Peptide cleavage from the silyl phenyl linker.

Peptide NH Me Si 3 O NHCH2 Polymer O

TFA

- CF3COO

Me Si + 3 O NHCH2 Polymer Peptide Amide + O

Resin = (aminomethyl)polystyrene resin Peptide= Bz-Ala-Gly Yield = 95 % yield Coupling Method= Fmoc/DIEA/HOBt Type of Peptide = Peptide Amides Figure 1.35 Peptide cleavage using SAL linker.

32 p-Alkoxybenzyl alcohol linker48 is suitable for the preparation of protected peptide fragments. In this example a protected peptide (figure 1.36) was released from resin by 50 % TFA (15 % yield after amino acid deprotection).

Bzl NO2 Bzl Tos

Z-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-OCH2 OCH2

50 % TFA, 30 min

Bzl NO2 Bzl Tos

Z-Asp-Arg-Val-Tyr-Val-His-Pro-Phe XVII

Resin = Merrifield Peptide= Z-Asp(Bzl)-Arg(NO2)-Val-Tyr(Bzl)-Val-His(Tos)-Pro-Phe Yield = 15 % (overall yield after side chain amino acid deprotection) Coupling Method= Bpoc/DCC/pyridine Type of Peptide = Peptide carboxylic acid Figure 1.36 Peptide cleavage from p-Alkoxybenzyl alcohol linker.

The linker (p- alkoxybenzyl-oxycarbonylhydrazide linker) 48.

(figure 1.37) is useful in the preparation of peptide hydrazides. A model peptide Phe-Val-

Ala-Leu-HNNH2 was synthesized and cleaved with 50 % TFA (42 % yield).

33 Phe-Val-Ala-Leu-HNNH-COOCH2 OCH2

50 % TFA, 30 min

Phe-Val-Ala-Leu-HNNH2

Resin = Merrifield Peptide= Phe-Val-Ala-Leu-HNNH2 Yield = 42 % (overall yield) Coupling Method= Bpoc/DCC/pyridine Type of Peptide = Peptide hydrazides Figure 1.37 Peptide cleavage from p- alkoxybenzyl-oxycarbonylhydrazide linker

Alkyloxycarbonylhydrazide linker49 is similar to the one previously reported by

Wang but it has two methyl groups in the position of the carboxylic acid. The addition of these carbonyl groups increases the yield of the peptide synthesis two fold from the previous p- alkoxybenzyl-oxycarbonylhydrazide resin developed by Wang.

H C 3 50% TFA, 30 min

H2CH2COOC-HNNH-Leu-Ala-Val-Phe Phe-Val-Ala-Leu-HNNH

H3C

Resin = styrene- 2% divinylbenzene (200- 400 mesh) Peptide= Phe-Val-Ala-Leu-HNNH2 Yield = 76 % Coupling Method= Bpoc/DCC/pyridine Type of Peptide = Peptide hydrazides Figure11.38 Peptide cleavage from alkyloxycarbonylhydrazide linker.

34 The imidazole trityl resin50 in figure 1.39 was used to prepare head to tail histidine-containing cyclopeptides by a three- dimensional orthogonal solid phase strategy (Fmoc/tBu/allyl). The imidazole ring was anchored to the resin of a peptide.

Cyclization of the linear peptides anchored to the resin was performed with DIPEA and

TBTU.

Ph Ph C Ph N i) Peptide chain elongation (5 cycles) Cyclo(-His-Gly-) N 3 ii) Cyclisation & cleavage (TFA/H O 95:5) Fmoc O 2 N H O

Resin = Fmoc-His(Trt-Resin) Peptide= Cyclo(-His-Gly-)3 Coupling Method= Fmoc/tBu/allyl (HATU/DIPEA) Type of Peptide = cyclopeptides Figure 1.39 Peptide cleavage using imidazole trityl resin.

35 CHAPTER TWO

DESIGN AND SYNTHESIS OF NOVEL FERROCENE LINKER

2.1. Introduction

Despite their usefulness for the chemical synthesis of peptides, strong acids which are often used as deprotecting reagents have led to side reactions and to loss of peptide products usually resulting from modification of peptides by the carbocations generated during the cleavage reaction. That is why there is a need for improvement of linkers.

In solid phase synthesis, synthetic transformations are conducted with one of the reactant molecules attached to an insoluble material referred to as the solid support. It was originally developed for peptide synthesis and then oligonucleotide synthesis.

Linkers are molecules that keep the intermediates in solid-phase synthesis bound to the support. Peptides can be cleaved from the resin easier if they are attached to the resin through a labile linker. Generally a linker is necessary to achieve a good yield in solid phase peptide synthesis. Linkers should enable the easy attachment of the starting material to the support, be stable under a broad variety of reaction conditions, and yet enable selective cleavage at the end of the synthesis, without damage to the product.

There are different types of linkers that meet these conflicting requirements to some extents. Linkers can be prepared so they yield amides, carboxylic acids or sulfonamides among others. The aim of this study was to design new solid support linkers for peptides that are commonly found in medicinally important agents.

A series of linkers have been developed which yield amides upon cleavage

[figure 2.1 a)]. The most common strategies used to date for the release of amides from insoluble supports are: (a) cleavage of a benzylic C-N bond of resin-bound N-alkyl-N- benzyl amides, (b) nucleophilic cleavage of resin bound acylating agents with amines and

36 (c) acylation/debenzylation of resin-bound N,N-dibenzylamines 51. Incorporation for the first time of a ferrocenyl group into a solid-phase acid labile linker (figure 2.1 b) was studied.

spacer + R H R a). Peptide N Peptide N H O O

Linker

spacer

+ Fe H R b). R Peptide N Peptide N H O O

Linker

Figure 2.1 a). Benzyl amine linker52, b). ferrocenyl amine linker.

Little is known about ferrocene adducts being incorporated into solid-phase peptide synthesis. In an attempt to increase the efficiency in our solid phase peptide synthesis a new type of linker was designed. This linker consists of a ferrocenyl moiety instead of the benzyl group used in most of the acid labile linkers developed up to date.

Based on the ferrocenium carbocation stability,53,54 it was considered important to test the hypothesis that a peptide cleavage would occur under very mild acidic conditions in

37 which a ferrocenium carbocation and a terminal amide group form at the point of cleavage. The cleavage occurs at the ferrocenyl amino bond (figure 2.2).

HN Peptide cleavage H+ + O

+ H2N Peptide Fe Fe

Figure 2.2 Ferrocenyl carbocation formation by acid cleavage.

2.2. Design of the Ferrocene Linker

Different synthetic routes were explored to obtain a molecule that contains a ferrocenyl amino bond and a carbon chain that will allow attachment of the linker to the solid support at any position within the linker. Two possibilities were explored (figure

2.3).

NH2 NH O 2 X n Fe Fe

a) b)

Figure 2.3 a) Ferrocene linker attached to resin and site of cleavage on the same site; b) Ferrocene linker attached to the resin and cleavage site on different sites of the cyclopentadienyl ligands.

In the first case a) both the ferrocenyl amino and carbon chain that attaches to the resin are on the same ferrocenyl carbon, and in b) the carbon chain that connects the linker to the resin is on different cyclopentadienyl ligands.

38 2.3 Results and Discussion

Figure 2.4 illustrates a retrosynthetic route toward the target molecule for the linker of the type a. This route involves Friedel Crafts acylation and reduction of the ferrocenyl ketone to the amine.

NH2 O O O

X X O O n n Fe Fe Fe + X X n

Figure 2.4 Retrosyntetic analysis of the ferrocene linker.

The first attempted forward synthetic route to the linker is outlined in figure 2.5.

The initial step was a Friedel Crafts acylation in which 4-ferrocenyl-4-oxobutanoic acid was obtained

O NH OOO 2 OH OH

O O Fe Fe Fe AlCl3

2.2 2.1 K2CO3 / Acetone

O O O O O Fe Fe

2.3 2.4

Figure 2.5 Attempted forward synthetic route.

39 Attempts to reduce the ketone formed the lactone instead. To prevent the lactone formation, the preparation of the tert-butyl ester was unsuccessfully. Failed attempts to reduce this ketone are given in figure 2.6.

NH2 R OH R O HO Fe NH O Fe R O Fe

NaBH3CN, MeOH CH3COONH4 NaBH4, MeOH

. NH2OH HCl Ethanol, Pyridine N3 O NH2 R R R BF3,NaN3 i O Ti(O Pr)4,NH4Cl,NEt3 Fe O O Fe Fe

Na(OAc) BH 1.NaBH4,TMS-Cl Formamide 3 2.NaN Benzyamine 3 OH-CHO NH3.OH-CHO

N3 R NCHO HN R R O Fe O O Fe Fe R = allyl

Figure 2.6 Failed initial attempts to generate the ferrocenyl amine.

It is known 5-membered rings are usually formed easily due to entropy factors followed by 6- and 3-membered rings. To prevent the lactone (ring) formation, an increase in the carbon chain to 6 carbons was performed. Now the lactone formation would be less favorable because 7-membered ring closures are less favorable. To prepare the compound with 6 carbons between the linker and the resin the original synthesis was modified. The ferrocene Friedel Crafts acylation was easily done with methyl adipoyl chloride(figure 2.7).

40 O O O O

Cl (CH2)4 OMe (CH2)4 OMe

FeFe FeFe AlCl3 DCM 2.1 2.5

Figure 2.7 Ferrocene Friedel-Crafts acylation with methyl adipoyl chloride.

But it was found that the methyl ester of this compound has low solubility in organic solvents and the deprotected acid has low solubility in aqueous and polar solvents, which made the reduction of the ketone to the amine difficult. At this point the synthesis of linker type a) was abandoned and it was decided to make a linker of the type b) (figure 2.3).

Two linkers were successfully synthesized each has the ferrocenyl amine bond and the carbon chain that attaches to the resin on different cyclopentadienyl rings of the ferrocene. The synthesis of the first linker is summarized in figure 2.8. The synthetic steps involved Friedel-Crafts acylation with chloromethyl methyl ether and then coupling to the solid support. The solid support in this case is Clear Resin in which most of the core CLEAR structure is composed of ethylene glycol (-CH2-CH2-O-)n units. Then reductive amination on solid support using sodium cyanoborohydride and heptenyl amine was done.

To make the ferrocene 1-carboxylic acid-1`carboxaldehyde, there was no need to protect the carboxylic acid during the Friedel-Crafts acylation if the equivalents of aluminum chloride were doubled (figure 2.9).

H 11 N H O 2. H O 9 Fe 2. Fe 2.7 Fe O N H O O N H HO t 3 and coupling to Clear resin. 2 l B 3

Cl C H 2 H M Al I/HO C -OC / 2 x F DC 2 PCD CH 2 NH DM DI Cl CN 4 H H O B Na Fe Fe 7 2.6 2. O O Synthesis of ferrocene linker H O HO HO + 9 Fe 2. 2 Figure 2.8 NH 2.8 O N H

O 2 H / H O Na H H O O 13 Fe 2. 7 Fe 2. O O O carboxaldehyde synthesis. Me HO 3 3 l 3 C 3 l CH OCH M O DCM or AlCl DC CH- 2 x A 2 CH- 2 Cl Cl Ferrocene 1-carboxylic acid-1` 2 Fe 1 6 Fe 2. 2. O O HO O Figure 2.9 Me

43 After the synthesis and characterization of the linker it was tested in a peptide synthesis using Fmoc amino acids (figure 2.10).

N N H R Fe R O O Fe HATU N N H 2.11 H 2.12

H+

R= Peptide or Amino Acid R

Figure 2.10 Ferrocene linker amino acid coupling.

The cleavage yield for peptides or just one amino acid was <5 %. When this linker was designed it was beleived that ferrocene was going to provide enough stability to the carbocation formed regardless of the functional groups attached to the ferrocene.

The amide that holds the ferrocene to the solid support is an electron-withdrawing group.

Since this group can lower the carbocation stability it was a good idea to replace this group with an electro-donating group.

The synthesis of the new linker with an electron-donating group is summarized in figure 2.11. The synthesis of the linker was carried out in only 3 steps. The synthesis started with the Merrifield resin functionalized with triphenyl phosphine 2.13 and ferrocene dicarboxaldehyde 2.15. The reaction gave the desired product, confirmed by solid state NMR and later by Fmoc cleavage of the amine attached to the linker. The reductive amination with allyl amine and sodium cyanoborohydride gave the linker 2.16.

A model three-residue peptide(Ala-Gly-Phe-Fmoc) was synthesized and it was

44 characterized by FAB. A big increase in the cleavage product (40 %) was observed compared to the previous linker (>5 %).

O O H H + - PPh3 Cl + Fe O Fe 2.13 H 2.14 2.15

H NaBH4CN NH Fe Fe DCM, DMF

2.15 2.16

Figure 2.11 Ferrocene linker synthesis.

2.4 Experimental

2.4.1 4-Ferrocenyl-4-oxobutanoic Acid (2.2).

OOO O OH

Fe Fe O

2.1 2.2

To a solution of ferrocene (1.86 g, 0.01 mol) and succinic anhydride (1.0 g, 0.01 mol) in 1,2-dichloroethane (50 mL), anhydrous aluminum chloride (2.67 g, 0.02 mol) was added and refluxed for 12 h. After cooling the solution, water (50 mL) and concentrated HCl (50 mL) were added to the solution, and the organic layer was separated. The organic layer was washed with concentrated sodium carbonate solution with a final pH of 10. The aqueous layer was separated and 6N HCl was added to the

45 solution with a final pH of 3. The aqueous layer was extracted with ether (50 mL × 3).

The organic layers were combined and washed with water (50 mL x 2), followed by

1 drying with Na2SO4. The solvent was removed in vacuo. Yield 1.87 g (65 %). H-NMR

(250 MHz; CDCl3) 11.352 (s, 1H), 4.885 (s, 2H), 4.415 (s, 4H), 4.215 (s, 4H), 3.625 (t,

2H), 2.598 (t, 2H) ppm. 13C-NMR (250 MHz; CDCl3) 202.012, 177.496, 79.781, 77.982,

77.673, 76.972, 66.689, 32.661, 29.766 ppm.

2.4.2 2-Propenyl-4-ferrocenyl-4-oxobutanoate (2.3).

O O OH 1.CsCO3 / MeOH O O Fe O Fe 2. Br

CCl3 2.2 2.3

To a solution of 4-ferrocenyl-4-oxobutanoic acid (0.95 g, 3.3 mmol) in CHCl3,

20% aqueous solution of Cs2CO3 (~ 25 mL) was added to a pH of 10. The mixture was evaporated in vacuo. The residue was stirred with allyl bromide (0.48 g, 4.0 mmol) in

CHCl3 for 6 h. The mixture was washed with water (3 x 40 mL), and the organic layer was dried over Na2SO4. The solvent was removed in vacuo. The ferrocene derivative was recrystallized by dissolving the crude material in a hot ethanol solution, followed by slow cooling to room temperature. Yield 0.75 g (70 %). 1H-NMR (250 MHz; CDCl3) 5.995

(m, 1H), 5.325 (q, 2H), 4.853 (s, 2H), 4.525 (s, 4H), 4.215 (s, 5H), 3.090 (t, 2H), 2.661(t,

13 2H) ppm. C-NMR (250 MHz; CDCl3) 28.242, 34.561, 65.742, 69.590, 72.639, 76.978,

77.698, 78.774, 118.657, 132.607, 173.191, 202.473 ppm. MS, m/e (M+) 326.

46 2.4.3 Ferrocenyl-1-hydrazide butanoic Acid (2.17).

O S HN O O N O p-toluenesulfonhydrazide O O Fe Benzene Fe O Reflux 12h

2.1 2.17

To a solution of 2-propenyl-4-ferrocenyl-4-oxobutanoate (0.53 g, 0.003 mol) in benzene (40 mL) p-toluenesulfonhydrazide (0.56 g, 0.003 mol) was added and refluxed for 12h. The product was present in the reaction as indicated by MALDI. However it was not isolated in a sufficient quantity to continue with the synthesis. MALDI (Matrix

Assisted Laser Desorption Ionization) (M+H)+ 497.10.

2.4.4 2-Methyl 4-ferrocenyl-4-oxohexanoate (2.5).

O O O O

Cl (CH2)4 OMe (CH2)4 OMe FeFe Fe AlCl3 DCM 2.1 2.5

To a solution of ferrocene (5.30 g, 0.0285 mol) and methyl adipoyl chloride (7.67 g, 0.043 mol) in CH2Cl2 (50 mL), anhydrous AlCl3 (3.8 g, 0.02 mol) was added and refluxed for 12 h. After cooling the solution, ice-cold water (50 mL) was added to the solution, and then the organic layer was separated. The organic layer was washed with a

5 % solution of NaHCO3. The solvent was removed in vacuo. The product was purified by flash chromatography starting with a hexane: ethyl acetate 4:1 mixture increasing the concentration of ethyl acetate gradually. Yield 1.87 g (80 %). MS, m/e 328, MALDI,

47 329.07. 1H-NMR 4.390-4.001 (m, 9H), 3.60 (s 3H), 2.741-1.242 (m, 8H) ppm. (250

MHz; C6D6). 13C-NMR (250 MHZ; CDCl3) 206.132, 174.046, 76.083, 72.847, 70.381,

69.920, 52.044, 39.420, 33.854, 25.096, 24.824 ppm.

2.4.5 Ferrocenyl-1 hydrazide hexanoic Acid (2.18).

O S O HN O O p-toluenesulfonhydrazide N O (CH2)4 OMe

FeFe Benzene (CH2)4 OMe

Reflux 12h FeFe

2.5 2.18

To a solution of 2-methyl 4-ferrocenyl-4-oxohexanoate (0.53 g, 0.003 mol) in benzene (40 mL) p-toluenesulfonhydrazide (0.56 g, 0.003 mol) was added and the mixture was allowed to reflux overnight. The product was present in the reaction as indicated by MALDI. However it was not isolated in a sufficient quantity to continue with the synthesis. MALDI (M+H)+ 497.10.

2.4.6 Ferrocenyl-1,1'-acid Fluoride(2.19)

F N N

F N F O Fe O Fe Piridine, CHCl2 HO F 2.6 2.19

To a solution of ferrocene carboxylic acid55 (1.01 g, 3.7 mmol) in CH2Cl2 (30 mL) under argon was added pyridine (1 mL, 12.4 mmol). The suspension was cooled to 0

48 °C. Cyanuric fluoride was added (2.60 mL, 20 mmol), and the contents were stirred for

1h to provide an orange solution. Crushed ice/water (30 mL) was added, the contents were filtered, and the organic layer was separated and washed with cold water. The organic layer was then dried with CaCl2, filtered, and concentrated in vacuo. The solid was dissolved in acetone, precipitated with cold water, filtered, and dried in vacuo. Yield

1 0.89g (88 %), mp 166.5-167 °C. H-NMR (250MHz;CDCl3) 4.898 (s, 1H), 4.5873 (s,

1H), 4.5800 (s, 1H), 4.3148 (s, 1H) ppm, as reported in the literature.

2.4.7 Methyl ferrocenecarboxylate (2.12).

MeOH O Fe O Fe DMAP,Pyridine F MeO 2.12 2.19

To a solution of (0.2 mmol) of ferrocenyl-1,1-acid fluoride and (0.2 mmol) 4-

(dimethylamino)pyridine in dry CH2Cl2 was added (0.2 mmol) of methanol. The mixture was stirred at room temperature for 12 h and purified by recrystallization ether: hexane

10:1 mixture. 1H-NMR (250 MHz; C6D6) 4.814 (s, 1H), 4.805 (s, 1H), 4.794 (s, 1H),

4.406 (s, 1H), 4.397 (s, 1H), 4.386 (s, 1H), 4.206 (s, 1H), 3.8059 (s, 1H) ppm as reported in the literature.55 13C-NMR(250 MHz; C6D6) (data not found in literature) 172.606,

73.020, 72.551, 71.613, 71.063, 66.422, 52.273, ppm.

49 2.4.8 Methyl-1'-formyl-1-ferrocene carboxylateferrocene (2.13).

O Cl2CH-OCH3 H AlCl3 Fe O O Fe CH2Cl2 MeO MeO 2.12 2.13

To an ice-cold mechanically-stirred CH2Cl2 solution (100 mL) of methyl ferrocenecarboxylate (11.5 g, 0.047 mol) and dichloromethyl methyl ether (5.4 g, 0.047 mol), was added AlCl3 (12 g, 0.188 mol) in small portions using a solid addition funnel.

The reaction was maintained at 0ºC for 0.5 h. Water (100 mL) was added in small portions to quench the reaction. The product was extracted with CH2Cl2. The organic phase was dried over NaSO4, and the crude product was chromatographed over silica with ethyl acetate: hexane, in a 1:2 mixture. Yield 10.35 g (87 %). The product was obtained as a red solid, mp 83-84°C, 1H-NMR (250 MHz; C6D6) 3.80 (s, 3H), 4.45 (t,

2H), 4.58 (t, 2H), 4.80 (t, 2H), 4.88 (t, 2H) 9.95 (s, 1H) ppm. 13C-NMR(250 MHz; C6D6)

(data not found in literature) 193.155, 172.183, 75.047, 74.717, 73.434, 72.699, 72.084,

52.475 ppm

2.4.9 Hept-6-enylamine (2.10).

LiAlH4 NC H2N 2.10

To a solution of 6-cyano-hex-1-ene (15 g, 0.1374 mol) in tetrahydrofuran under argon at 0L C was added dropwise a 1.0 M solution of LiAlH4 in ethyl ether (137 ml,

0.1374 mol). The mixture was refluxed overnight at 70 L C and then quenched at 0L C

50 with water (12 ml), 15 % NaOH (12 ml) and water (24 ml). The mixture was filtered and the organic layer was separated. A 1M solution of NaOH was added to the aqueous layer and extracted with dichloromethane until the extracts gave a negative ninhydrin test. The organic layers were combined and evaporated in vacuo. The oily product was distilled under vacuum (60 mm of mercury) and 1ml fractions were collected. Purity was confirmed by TLC with methanol: ethyl acetate: triethylamine (10:10:1) followed by nynhydrin test. The product was dried with NaOH pellets (1 pellet / ml of amine), which gave pure (by NMR) product 10g (65 % yield).

2.4.10 Ferrocenyl-imine-glycine(ethylester), methoxycarbonyl (2.21).

HO O N O H Ethyl ester glycine H O Fe O Fe MeO CHCl3 Reflux MeO 2.13 2.21

To a solution of glycine ethyl ester hydrochloride (2 mmol) and triethylamine (2 mmol) in dry CHCl3 was added (2.13) (2 mmol) and refluxed for 10h. The solvent was removed in vacuo. The imine was obtained as a yellow solid. The purity was checked by

1H-NMR. The organic phase was dried and evaporated to afford ferrocenyl imine methyl amino acid in 90% yield. 1H-NMR (250 MHz; C6D6) 7.855 (s, 1H), 4.299-4.031 (m,

11H), 3.646 (s, 3H), 1.431 (m, 3H), 1.097 (m, 2H) ppm. 13C-NMR confirms the presence

51 of the product. 13C-NMR (250 MHz; C6D6) 173.352, 172.588, 165.272, 73.752, 73.294,

72.645, 71.568, 63.292, 61.779, 52.319, 15.261 ppm.

2.4.11 Ferrocene-methylglycine-ethyl ester, methoxycarbonyl(2.22).

HO HO N O NaBH4 HN O H MeOH O Fe O Fe MeO MeO

2.21 2.22

To a solution of the imine in dry CH3OH, 4 equiv. of solid NaBH4 were added in small portions at 0 °C. After the mixture was stirred for 30 min, 20 mL water was added and the organic phase extracted with CHCl3 (3 x 40 mL). The solvent was removed in vacuo to afford the pure ferrocene methyl amino acid. Yield (~ 95 %). 1H-NMR (250

MHz; C6D6) 4.4-3.32 (m, 15H) 1.79-1.02 (m, 4H) ppm.

2.4.12 Ferrocene-1-carboxylic acid-1`carboxaldehyde (2.7).

O Cl CH-OCH 2 3 H AlCl3 Fe O O Fe DCM HO HO

2.6 2.7

To a solution of ferrocene carboxylic acid (10 g, 0.04 mol) and aluminum chloride (22 g, 0.16 mol) in dichloromethane (250 mL) dichloromethyl methyl ether (5.0 g, 0.04 mol) was added and stirred at 0L for 0.5 hours. Water (150 mL) and 2N HCl (150 mL) were added to the solution, and the organic layer was separated. The organic layer

52 was washed with concentrated sodium carbonate solution with a final pH of 10. The aqueous layer was separated and 6N HCl was added to the solution with a final pH of 3.

The aqueous layer was extracted with dichloromethane (100 mL × 3). The organic layers were combined and washed with water (50 mL x 2), followed by drying with Na2SO4.

The solvent was removed in vacuo. The compound was purified with hexane/ethyl acetate (3:1). Yield 3 g (20%). The compound was characterized by NMR (figure 2.14).

2.4.13 Ferrocene carboxaldehyde Resin (2.9).

O O

H H DIPCDI/HOBt NH2 Fe + O O Fe DMF/CH2Cl2 HO N H 2.8 2.9

A glass peptide reaction vessel equipped with a fritted glass was siliconized with

Surfasil (Pierce) prior to the reaction. Clear-NH resin (1.7 g 0.66 meq) (Peptides

International) resin was swelled in the reaction vessel for 1hr with dichloromethane

(NMR in figure 2.12). To a mixture of 20 ml of dichloromethane and Clear-NH resin ferrocene 1-carboxylic acid-1`carboxaldehyde (1.11 g, 5.30 mmol) diisopropylcarbodiimide (0.66 g, 5.30 mmol), HOBt (0.71 g, 5.30 mmol) and 20 ml of dimethylformamide were added. The mixture was stirred with a nitrogen flow for 20 hrs

53 followed by rinsing of the resin with DCM (30 ml x 3), DMF (30 ml x 3), DCM. (30 ml x

3) and diethyl ether (30 ml). The compound was characterized by NMR (figure 2.13).

Figure 2.12 Clear-NH resin magic angle spinning 1H-NMR(CDCl3, 400 MHz).

Figure 2.13 Ferrocene carboxaldehyde resin 2.9 1H-NMR(CDCl3, 400 MHz).

Figure 2.14. Ferrocene linker. 1H-NMR(CDCl3, 250 MHz).

56 2.4.14 Ferrocenyl heptenoic amine Resin (2.11).

NaBH4CN N H H O Fe O Fe NH2 N N H H

2.9 2.11

A glass peptide reaction vessel equipped with a fritted glass was siliconized with Surfasil

(Pierce) prior to the reaction. Clear ferrocene carboxaldehyde resin was swelled in the reaction vessel for 1hr with dichloromethane. To a mixture of 20 mL of dichloromethane and Clear ferrocene carboxaldehyde resin hept-6-enylamine (0.75 g, 6.63 mmol) was added and stirred for 10 min. A solution of sodium cyanoborohydride(0.41g, 6.63 mmol) in 20 ml of dimethylformamide was added and stirred with a nitrogen flow overnight followed by rinsing of the resin with DCM (30 mL x 3), DMF (30 mL x 3), DCM. (30 mL x 3) and diethyl ether (30 mL).

2.4.15 Agni[(KLAKKLA)2] (2.24)using ferrocenyl heptenoic amine Resin.

Peptide Synthesis N H N O Fe OPFe eptide N H N H O 2.11 2.23 88% TFA, phenol triethyl silane, H2O HN Peptide ~2% Yield O 2.24

57 The peptide was synthesized using the Fmoc protecting group, HOBt/TBTU coupling reagents and HATU/DIEA was used for the first coupling. Lysine side chains were protected with Cbz. The peptide was cleaved from the resin using a solution

(prepared fresh) composed of 88 % (10 mL) TFA, 5 % (0.5 mL) phenol, 2 % (0.2 mL) triisopropylsiliane, and 5 % (0.5 mL) dd water. The TFA solution was purged with argon.

This cleavage solution was added to the resin/peptide under argon with exclusion of light and occasional shaking. After 2 hours the peptide/TFA solution was filtered and diluted with 100 mL of cold 20 % acetic acid. This solution was extracted with cold diethyl ether

(50 mL x 4). The peptide was lyophilized two days and purified by HPLC and characterized by mass spectrometry. The crude product was purified by preparative reverse-phase HPLC on a Waters 15 µm Deltapak C4 column using a water (0.05 %

TFA) and acetonitrile (0.05 % TFA) gradient system and the absorption was monitored at

222 nm. Peptide purity was verified on a Vydac 5 µm C18 column using the same conditions (5 % yield calculated from resin substitution level). Matrix assisted laser desorption ionization (MALDI) mass spectrometry was used to verify peptide mass.

Agni 2534 calculated observed 2552 mass (M+H)+.

2.4.16 Ferrocene carboxaldehyde-polystyrene Resin (2.15).

O O

H H + - PPh3 Cl + Fe O Fe

2.13 H

2.14 2.15

58 A solution of ferrocene dicarboxaldehyde 2.14 (0.06 g, 0.23 mmol) and benzyl- triphenylmethylphosphonium chloride resin 2.13 (0.33 g, 0.23 mol) in dichloromethane

(30 mL) was refluxed for 10 min. DBU(1,8-diazabicyclo[5.4.0]undec-7-ene) (1mL) was added and reflux was continued for 30 min. The mixture then was rinsed in a filter with

DCM(3 x 40 mL), DMF(2 x 30 mL) and DCM ( 3x 40 mL).

2.4.17 Allyl-amino ferrocenyl polystyrene Resin (2.16).

H NaBH4CN NH

Fe Fe DCM, DMF

2.15 2.16

To a solution of ferrocene carboxaldehyde polystyrene resin and allyl amine

(0.13g, 2.3 mmol) in DCM (20 mL) a solution of sodium cyanoborohydride (0.22g, 0.34 mmol) in DMF was added and stirred overnight. The mixture then was rinsed in a filter with DCM(3 x 40 mL), DMF(3 x 40 mL) and DCM (3x 40 mL). The product was characterized by solid-phase NMR and the aldehyde proton was identified at 9.5351ppm

(s, 1H).

2.4.18 Allyl-amino fmoc-phenylalanine ferrocenyl polystyrene Resin (2.25).

O O NH HN O HATU, DIEA, DMF N Fe Fe

2.16 2.25

59 Allyl-amino ferrocenyl polystyrene resin was swelled in 20 mL of DCM. Fmoc- phenylalanine (1.54 g, 4 mmol) HATU (1.52 g, 4 mmol) and DIEA (1.55 g, 12 mmol) were dissolved in DMF and added to the resin and the mixture was stirred overnight.

2.4.19 Cleavage of (1-allylcarbamoyl-2-phenyl-ethyl)-carbamic acid 9H-fluoren-9- ylmethyl ester (2.26) from allyl-amino ferrocenyl polystyrene Resin.

O O HN O HN 5% TFA/DCM , Et3SiH

O O HN O 40% Yield by weight after column N

Fe O O HN O 1 % TFA/DCM , Et SiH 3 HN

2.25

20% Yield by weight after column 2.26

The resin was swelled in DCM for an hour. In two separate cleavage studies 5 % and 1 % TFA solutions in DCM with 5 % triethylsilane were added to the resin. The mixtures were shaken slowly for 4 hours. The product[(1-allylcarbamoyl-2-phenyl- ethyl)-carbamic acid 9H-fluoren-9-ylmethyl ester] TLC showed small impurities. The resin amino acid mixture was filtered using glass wool and the product was dried in vacuo. The product was washed with water (2 x 20 ml). The product was purified using preparative TLC (2% methanol/chloroform) to give yields 40 % and 20 %, respectively.

60 CHAPTER THREE

CONVERGENT PEPTIDE SYNTHESIS

3.1 Introduction

Peptides are endogenous, polycationic molecules that have broad microbiocidal activity against various bacteria and fungi. Water-membrane soluble proteins and peptides are used in the defense and offense systems of all organisms, including plants and humans. 56-60 Peptides serve as nonspecific defense system that complements the highly specific cell mediated immune system. Many bacteria are now resistant to the conventional antibiotics like penicillin 61,62. This has motivated the investigation of new antimicrobial agents with different mechanisms of action.

These peptides are mobilized shortly after microbial infection, and act rapidly to neutralize a broad range of microbes. This rapid response is important because activation of pathogen specific immune responses occur slowly relative to the potential kinetics of microbial proliferation. Many of these antibacterial peptides are helix-forming peptides, which are amphipathic (with polar and nonpolar groups on opposite faces of the helix) a structural feature believed to be important in their function as antimicrobial agents. Most of these peptides are linear with a potential to form amphipathic -helical or -sheet structures, whereas others are cyclic due to the presence of one or more disulfide bonds or thioethers 63-67. Despite many similar sructural features among antimicrobial peptides, their spectrum of activity differs significantly and they can be classified into several groups: (1) peptides such as cecropins, 68,69 isolated from the cecropia moth that are toxic to microorganisms but not to normal mammalian cells and which are active mainly on Gram-positive bacteria. Others are active on both Gram-positive and Gram-

61 negative bacteria, e.g.,magainins 70 and dermaseptins, both isolated from the skin of frogs. (2) Peptides that are toxic to both microorganisms and mammalian cells, such as the bee venom melittin, and the Moses Sole fish venom pardaxin.71,72

Antimicrobial peptides represent a good alternative to traditional antibiotics because most of them kill bacteria by physical disruption of cell membranes, which may prevent microorganisms from developing resistance against these agents. The discovery of thousands of antimicrobial peptides with variable lengths and sequences suggests a general mechanism for killing bacteria rather than a specific mechanism that requires preferred active structures. Antimicrobial peptides are considered promising drug candidates because of their potential to overcome bacterial resistance73-76. They are ancient components of all species of life, and their expression pathways in all organisms, including insects and plants, are conserved. Conventional antibiotics penetrate the cell wall and act on specific targets. In these circumstances, the bacterial morphology is preserved and the bacteria can develop resistance. In contrast, most antibacterial peptides disrupt and permeate the target cell membrane and inflict irreversible damage. The exact mechanism of antimicrobial action is not known but there are two proposed mechanisms; the raft or carpet model77 and the barrel-stave or pore model78. The raft model does not require any specific structure or sequence and starts with the binding of the peptide to the surface of the membrane79. The membrane is disrupted after a threshold peptide concentration has been reached. The peptide first binds to the phospholipid headgroups.

The peptide helix assembles on the membrane of a bacterium so that the positively charged molecules can interact with the negatively charged phospholipid groups of the membrane79. The peptide inserts partially into the membrane so that the hydrophobic

62 residues interact with the hydrophobic core of the membrane and finally destruction of the membrane occurs by disruption of the bilayer curvature. In the pore model, amphipathic -helices form bundles in the cell, in which their hydrophobic surfaces interact with the lipid core of the membrane79. In this orientation, the hydrophilic surfaces of the helices form a water filled pore. Membrane binding of the peptide is favored by hydrophobic interactions. It seems that when there is an appropriate balance between hydrophobicity and a net positive charge, the peptides destroy the susceptible cell79.

Amino acids are the basic components of peptides. There are twenty naturally occurring -amino acids and hundreds of non-natural amino acids that have the general structure shown below (figure 3.1). Amino acids are also present in proteins but the term

“protein” is usually used for those polypeptides that occur in nature and have definite three-dimensional structures under physiological conditions.80 This dissertation focuses on peptides.

H2NCHC OH

Figure 3.1 General -amino acid structure.

The distinguishing feature in the amino acids is the structure of the R group located at the -carbon. Some unnatural amino acids are routinely included in many peptide sequences. Side chains that do not occur in biological systems distinguish

63 unnatural amino acids from their natural counterparts. The R groups affect the peptide secondary structure.

3.2 Secondary Structure

Peptides can adopt defined 3-D structures. The -helix and the 310-helix are two of the most common helices in antimicrobial peptides (figure 3.2). Helices can be characterized by the number of residues (amino acids) per turn and the hydrogen-bonding pattern. In the -helix, there are 3.6 residues per turn of the helix. In a 310-helix, there are

3 residues per turn. A combination of hydrogen bonding and hydrophobic, electrostatic and steric interactions stabilize the helix. The -helix is by far the most common; the 310- helix makes up less than 10% of all peptide structures.

Figure 3.2 Cylindrical representations of helical secondary structures. Cylinder to the right shows the main hydrogen bonding in an -helix.

64 -sheets form as a result of either inter or intramolecular hydrogen bonds between peptide segments. Hydrophobic interactions further stabilize the -sheet. Secondary structures that are not included above are classified as random coil. The random coil has no regular, repeating structure in significant stretches of the peptide chain. 81 The -sheet results from two or more -strands forming interchain hydrogen bonds. Two types of - sheet are known: parallel and anti-parallel (figure 3.3). 82

O O O H N H N H N H N O

O O O N H N H N H N H O

O O O H N H N H N H N O

O O O N H N H N H N H O

O O O H N H N H N H N O

Parallel Antiparallel

Figure 3.3 Parallel and antiparallel -sheet hydrogen bonding.

65 3.3 Solid-Phase Peptide Synthesis Methods

One of the difficulties associated with solid-phase peptide chemistry is prevention of reaction on functionalized side chains of amino acids such as lysine, glutamic acid and cysteine. In order to prevent these reactions from taking place, side-chain protecting groups must be used. These groups must be stable to the coupling conditions, stable under conditions that cleave the active terminus for continued growth, but labile under conditions which cleave the product from the resin, allowing for isolation of the fully deprotected product. Thus, the N-terminal and side-chain protecting groups must be orthogonal to each other in a solid-phase coupling scheme. Several coupling schemes have been developed which match linker, N-terminal protection and side-chain protection in an orthogonal manner. Some of the most commonly used include the Boc-benzyl 83

84 strategy (figure 3.4) 85 and the Fmoc-Boc strategy 86,87 (figure 3.5).85

One aspect of solid phase synthesis that has seen tremendous development is in the field of coupling reagents. Traditionally, peptide coupling under solid phase conditions can be realized in two ways: 1) A reactive electrophilic derivative of the amino acid, such as an acid halide88 or an N-carboxyanhydride (NCA) 89 can be synthesized, isolated and then allowed to react with the nucleophilic portion of the resin- bound residue. These active species are easy to synthesize and relatively stable, allowing for facile characterization. In addition, the simplicity of by-products released from such couplings, such as a carbon dioxide, chloride or fluoride ion, allow for simple purification after coupling has occurred. Alternately, a coupling reagent may be added to the reaction mixture, to generate a reactive electrophilic derivative in situ. These coupling reagents 90 include carbodiimides91,92 such as DCC, DEC, and DIPCDI, HOBt 93 and HOAt 94,95

66 and tertiary ammonium salts of triazole N-oxides, such as HBTU 96and HATU 95 (table

3.1), in addition to several others.

gy. te

ting group stra

SPPS prote -

-/Boc

Fmoc

The

Figure 3.4

N H

O gy . te

M Z r PA

2-B ting group stra c

HF Br O O

O O SPPS prote O C - 2 H N H O yl-/Boc nz HF H N Be

O O The O C

2 H N H A

TF zl O Figure 3.5. 3 O

OB CH Boc

C C 3 3 H H

68 The advantage of these types of coupling is the relative ease of handling of unactivated species, which are introduced into the reaction along with the coupling reagent. In addition, in situ activation allows for the preparation of highly activated species that are often not isolable.

Table 3.1. Common coupling reagents used in SPPS.

Coupling Reagent Structure Reference

DCC 91 92 NCN , N,N'-Dicyclohexyl- carbodiimide

DEC H3CH2CNCNCH2CH3 91, 92 N,N'-Diethyl- carbodiimide

DIPCDI H3C CH3 91, 92 NCN N,N'-Diisopropyl- H C CH carbodiimide 3 3

HOBt N 93 1-Hydroxybenzotriazole N N OH

HOAt N 94,95 7-Aza-1-hydroxybenzo- N triazole N N OH HBTU 96 (H C) NN(CH) N-[(1-H-Benzotriazol-1-yl)- 3 2 3 2 (dimethylamino)methylene]- N N-methylmethanaminium N hexafluorophosphate N- N oxide O

(table cont’d.)

69 HATU 95 (H C) NN(CH) N-[(1-H-7-Azabenzotriazol- 3 2 3 2 1-yl)-(dimethylamino)- N methylene]-N- N methylmethanaminium N N hexafluorophosphate O

3.4 Convergent Peptide Synthesis

Efforts have been devoted to increasing coupling yields such as the improvement of protecting groups, solid supports, coupling reagents, linkers and automation of SPPS.

As a result of these improvements, several impressive linear solid-phase syntheses of peptides have been reported using the SPPS linear approach (table 3.2).

Table 3.2. Examples of large synthetic peptides and small proteins synthesized by SPPS.

Number of Name Ref.

Residues

48 Insulin-fragments 97,98

99 HIV- protease 99

126 Cardiodilatin 100

188 Growth hormone 101

analog

70 Successful peptide assembly is still hampered by problems such as poor solvation of the growing peptide chain during solid phase synthesis as well as limited solubility of fully protected peptide fragments in the solution approach, often leading to incomplete coupling steps102. It was believed that these problems were mainly caused by interactions between the peptide and the resin support,103 but recent work,104 shows that these undesirable physicochemical problems originate from intermolecular aggregation of the protected peptide chains and/or the formation of secondary structures, most notably of -sheets. These hydrogen bonds reduce solvation of the peptide, which decreases the access to the incoming activated amino acid resulting in low coupling.

Hydrogen bonding is partially reversible which results in a series of deleted sequences making the separation and purification of the peptide difficult.

To overcome these difficulties, different convergent strategies have been devised, especially for homogeneous large molecules. Some of these methods involve protected peptide segments, which is generally referred as convergent solid-phase peptide synthesis(CSPPS) and the second method, which involves unprotected peptide segments, which is referred as chemo-selective ligation.

3.4.1 Convergent Solid-Phase Peptide Synthesis(CSPPS) of Protected Peptide Segments

The CSPPS method was developed for the preparation of difficult and complex peptides and small proteins. In the step-by-step peptide chain elongation, the resin-bound

C-terminal amino acid is reacted sequentially with suitably protected and activated amino acids. In this procedure, the peptide is elongated towards the N-terminal direction (figure

3.6). Elongation performed from the N- to the C-terminus can be problematic because the

C-terminal amino acid needs to be activated, which leads to its racemization. In CSPPS

71 method, the possibility for racemization is minimized by reducing the number of individual activation steps. In this method, protected peptide fragments rather than the free peptide can be cleaved from the solid support after synthesis. The protected peptide fragments are then purified by chromatography before coupling on a solid support thus spanning the entire peptide sequence, either on the solid support or in solution.

fragment1 fragment1 + fragment2

fragment 1 fragment2 fragment1 fragment2 + fragment3

fragment1 fragment2 fragment3 fragment1 fragment2 fragment3

Figure 3.6 General Scheme for CSPPS. Peptide fragments are coupled to other peptide fragments.

In CSPPS, all reactive functional groups except the ones required for coupling are protected. These protecting groups must be stable to the cleavage conditions necessary to prepare each fragment.

Protected segment solubility is one of the problems of CSPPS. Selecting compatible protecting groups for the individual segment usually can increase segment solubility. Usually peptide segments no longer than 15 residues are used in CSPPS.

Longer segments usually give incomplete couplings. Successful segment couplings can take days 105, 106. Sometimes amide backbone protection is required to increase solubility. Amide backbone protection of every 4-6 residues can be sufficient in many cases. This amide backbone protection prevents hydrogen bonding thus eliminating

72 unpredictable changes in conformation like -sheet-like and amide backbone hydrogen bonding. Figure 3.7 illustrates the amide backbone protection method.

Protected backbone amide

O RRO O H N N N N H R O n O

Free backbone amide

R= amino acid side chain

Figure 3.7 Amide backbone protection.

The TFA-labile N-(2-hydroxy-4 -methoxybenzyl) (Hmb) protecting group and its reversibly acid-stable counterpart, N-(2-acetoxy-4 -methoxybenzyl) (AcHmb) (figure

73 3.8) are widely used in amide backbone protection. Literature reports demonstrate that the use of this type of amide backbone protection eliminates problems in syntheses increasing segment solubility, and insures excellent peptide-resin solvation.

R O R O

N N

O CH2 O CH2

CH3CO-O HO

OCH3 OCH3

a) b)

Figure 3.8 a) Structures of Hmb and, b) Ac-Hmb protecting groups for backbone amides.

3.4.2 Chemoselective ligation of unprotected peptide fragments

Chemoselective ligation is similar to convergent synthesis in that large peptide fragments are coupled to other peptide fragments but the difference between the two techniques is that that in chemoselective ligation, the synthesis occurs with unprotected peptide fragments. This type of synthesis is carried in aqueous solution and is based on the selective formation of particular covalent bonds in the presence of the unprotected peptide side-chain functionalities. This type of approach has some benefits: by coupling small to medium sized peptides, the purification can be greatly simplified; and by using unprotected peptides, the problems of poor fragment solubility normally are eliminated.

Chemoselective ligation was by introduced by Liu and Tam and others 107 108.

There are three general methods (table 3.3) for the preparation of large peptides using unprotected segments. The first method is based on the thiol chemistry method,

74 which includes thioalkylation, thiol addition, and thiol disulfide exchange. The second method is based on carbonyl chemistry and involes an addition-elimination reaction between a weak base and an aldehyde. The third method uses enzymes in reverse proteolysis.

Table 3.3. Methods for ligating unprotected peptides.

Methods Reaction Remarks

1 2 1 R -SH + X-CH2-CO-R R -S-CH2-CO-R2 X= Cl or Br Thiol chemistry - Thioalkylation R1-SH + R’-S-S-R2 R1-S-S-R2 R’= aromatic - Thiol-disulfide exchange

O 1 2 1 2 R C H + NH2-X-R R -CH=N-X-R X = O or N

Weak base-aldehyde O HX X 1 2 1 R C H + H2N R R N R2 X = O or S

O 1 2 R -CO-NHNH-CO-NHNH2 + R C H R1 = peptide R1-CO-NHNH-CO-NHN=CH-R3

1 2 1 Reverse proteolysis R -CO-OH + NH2NH-CO-R R = NHNH2

R1-CONHNH-R2

75 Thiol chemistry exploits the reaction of sulfhydryls in the alkylation with - halocarbonyls, addition to conjugated olefins, and sulfur-sulfur exchange with disulfides.

Thioalkylation is very popular in protein chemistry as a way to attach ligands and peptides 109 because of the easy access of thiol groups in proteins 110. The ease of adding haloacetyl(-halocarbonyl) groups to peptides during stepwise solid-phase synthesis makes this type of chemistry convenient. Alkyl thiols are usually used in the form of cysteine, which can be coupled to a peptide using Fmoc chemistry. The sulfhydryl group is usually protected with the trityl protecting group(figure 3.9).

H2N

Figure 3.9 Trityl protected cysteine.

The haloacetyl residue can be attached to the N-terminus or the side chain of lysine positioned anywhere in the sequence 111. The haloacetyl moiety is stable to HF cleavage conditions in the absence of thiol scavengers. The reagent of choice is usually bromoacetyl because it is more reactive than the corresponding chloro analog, especially in aqueous media. The iodo analog is used rarely because of instability to cleavage

76 conditions in solid phase synthesis. Synthetic peptides with N -bromoacetyl and C- terminal cysteine are usually oligmerized at pH 7-8, to prevent formation of disulfide bonds.

3.5 Results and Discussion

The peptide in this study(Cyh-7) is comprised of 57% of an ,-disubstituted amino acid. The ,-disubstituted amino acid increases the stability of the helix112.

This peptide was designed be to helical and it can form an amphipathic -helix (figure

3.10) or an amphipathic 310-helix. The peptide synthesis of Cyh-7 was successful and the biological activity of this peptide was significant. At this point chemical ligation was considered a good method to combine monomeric units to make a larger peptide.

O O

H N C

(H2C)5 Lys-Cyh-Cyh-Lys-Cyh-Cyh-Lys-NH (CH2)5 NH2 O

H NH2 OH O H2N

O NH2 a. b.

Figure 3.10 Structure of Cyh-7 peptide and structures of 1-amino-1-cyclohexane- carboxylic acid (a) and lysine amino acids (b).

-Helical wheel 310-helical wheel

Figure 3.11 Helical wheel motifs showing -helical and 310-helical conformations of Cyh-7 (designed to be an -helix).

The coupling yield for each step in the synthesis of Cyh-7 was lower than 85 % and in most cases, just 70 %, so if a longer sequence was needed a more efficient route other than stepwise synthesis was necessary in order to synthesize the peptide. Thus several synthetic convergent routes were deviced to make this peptide.

The first synthetic strategy was cross metathesis. To allow this, a new linker(16- hydroxypalmitoleic acid) was synthesized in just one step from aleuritic acid. The double bond in the molecule, in theory, will cleave the peptide from the resin using a ruthenium catalyst. Figure 3.12 shows the strategy proposed.

O n2 Ruthenium Catalyst n2 OOPeptide n O Peptide 1 H2CCH2

n1=7 n2=6

n2 O Peptide oligomer Ruthenium Catalyst

Figure 3.12 Peptide cleavage using a ruthenium catalyst.

Attempts to form an ester bond between the terminal alcohol of the linker and the first residue of the peptide failed. This is probably due to solubility problems. Other methods to make shorter linkers with a double bond in the center were in theory easy, but costly compared to our next strategy. In this strategy, the double bond is at the side chain of a commercially available amino acid and the cleavage of the peptide from the resin is performed with commercially available resins with acid labile linkers. Two residues at the C-Terminus and N-terminus of the model peptide (KLAKKLA)2 were added. The first residue, the side chain of aspartyl allyl ester, bears a double bond that, when reacted with the ruthenium catalyst and the terminal residue bearing a double(figure 3.13) bond yields a cyclic peptide. The ring closing metathesis was attempted in several ways, but the desired product it was not obtained.

a).

O H O O O N TFA H O H O N Peptide N Ruthenium Catalyst N H H2N Peptide H2N Peptide

CHCl2,Reflux OO OO OO

+ Peptide= Agni =(ALKKALKA) 2 Helical Oligomers Peptide Lysines are protectde with Cbz NH = PAL-PEG-PS

H2,Pd,AcOH 12 h Side Chain depro- Ruthenium Catalyst tection

iPr Cl- Cl- O N O H O Ru 2- N 4+ N H2N Peptide 2-

+ Helical Oligomers

b).

O H O O O N TFA H O H O N Peptide N Ruthenium Catalyst N H H2N Peptide H2N Peptide

CHCl2,Reflux OO OO OO

+ Peptide= Agne=(ALKKALKA) 2 Helical Oligomers Peptide Lysines are protectde with Cbz NH = PAL-PEG-PS

H2,Pd 12h Ruthenium Catalyst = P

-Cl O H O N 4+ Ru 2- H2N Peptide CH -Cl OO P + Helical Oligomers Bis(tricyclohexylphosphine)isopentenylidene ruthenium dichloride

Figure 3.13. a) Peptide ligation using Grubbs-Hoveyda ruthenium catalyst and b) Peptide ligation using Grubbs ruthenium catalyst.

80 3.6 Experimental

3.6.1 Peptide Synthesis

The solid phase peptide synthesis was carried out using Fmoc amino acid fluorides (figure 3.14), which have been shown to be efficient, rapid-acting coupling reagents.

H3CO O O H2 C OC

Fmoc N C O (CH2)4 H H N 2 H H

H3CO Structure of the PAL Support Structure of Fmoc a. b.

O H Fmoc N(CH) 2 4 BOC NH O

F Fmoc N O F H

O Fmoc-Lys-F-Boc Fmoc-1-Amino-1-cyclohexane-carboxyl fluoride c. d.

Figure 3.14 Structure of the reagents used in Cyh-7 synthesis.

81 General peptide synthesis procedure steps:

1. Deprotection (figure 3.15).

The Fmoc protection of the support-based amino acid is removed with a solution containing a mild base (20 % Piperidine/DMF/2 % DBU) to liberate the amino group for coupling. The spacer Fmoc-6-aminohexanoic acid is used to reduce the steric interaction between the peptide chain and the resin. The peptide synthesis was carried out on the resin PAL-PEG-PS (peptide amide linker-polyethylene glycol-polystyrene).

Spacer Fmoc N H O

20% Piperidine/DMF/DBU

Spacer H2N

Figure 3.15 Fmoc deprotection.

82 2. Activation and Coupling (figure 3.16). The carbonyl fluoride group of the next amino acid is coupled with the deprotected amino group of the previous amino acid by formation of an amide bond. Excess reagents and high concentration are used to drive reactions as close to completion as possible.

O H Fmoc N F

Collidine +

Spacer H2N

O R1 H Fmoc N Spacer N H O R2

Figure 3.16 Fmoc deprotection and amino acid coupling.

83 3. The deprotection and coupling steps are repeated until the peptide is fully assembled.

4. The spacer Fmoc-6-aminohexanoic acid is coupled to the terminal lysine and the amino group of the spacer is Fmoc deprotected and is acetylated using Ac2O and pyridine.

5. Cleavage of peptide from resin. The resin/peptide was rinsed with DCM and dried for a minimum of 4 h under vacuum. The cleavage solution was prepared fresh. It is composed of 88 % (10 mL) TFA, 5 % (0.5 mL) phenol, 2 % (0.2 mL) triisopropylsiliane, and 5 % (0.5 mL) dd water. The TFA solution was purged with argon. This cleavage solution was added to the resin/peptide under argon with exclusion of light and occasional shaking. After 2 hours the peptide/TFA solution was filtered and diluted with

100 mL of cold 20 % acetic acid. This solution was extracted with cold diethyl ether (50 mL x 4). The peptide was freeze dried for two days and purified by HPLC and characterized by mass spectrometry.

3.6.2 Cyh-7

Cyh-7 was synthesized by manually coupling aminohexanoic acid onto a PAL-

PEG-PS solid support. The couplings were done by reacting 8 equivalents of the Fmoc- acid fluoride, 3 equivalents of DIEA and the resin in methylene chloride. The reagents were allowed to react until an acceptable yield was determined by quantitative Fmoc tests. In some cases, the resin was rinsed with 3 x 20 mL portions of methylene chloride and treated with fresh reactants until >70 % coupling yields were obtained. A solution of

20% piperidine/2 % 1,8-diazobicyclo [4.5.0]undec-7-ene (DBU) in DMF was used for

Fmoc removal. The spacer, Fmoc-6-aminohexanoic acid, is coupled to the terminal lysine and the amino group of the spacer is Fmoc deprotected and is acetylated using Ac2O and pyridine. The resin/peptide was rinsed with DCM and dried for a minimum of 4 h under

84 vacuum. The peptide was cleaved from the resin using a solution (prepared fresh) composed of 88 % (10 mL) TFA, 5 % (0.5 mL) phenol, 2 % (0.2 mL) triisopropylsiliane, and 5 % (0.5 mL) dd water. 113 The TFA solution was purged with argon. This cleavage solution was added to the resin/peptide under argon with exclusion of light and occasional shaking. After 2 hours the peptide/TFA solution was filtered and diluted with

100 mL of cold 20 % acetic acid. 113 This solution was extracted with cold diethyl ether

TFA) and acetonitrile (0.05 % TFA) gradient system. The gradient was run from 15 % to

45 % organic and the absorption was monitored at 222 nm. Peptide purity was verified on a Vydac 5 µm C18 column using the same conditions. Matrix assisted laser desorption ionization (MALDI) mass spectrometry was used to verify peptide mass. Cyh-7 1192

(M+H)+.

3.6.3 Cyh-7 MIC Assays

E. coli American type culture collection (ATCC) 25922 and S. aureus ATCC 25922 were used as representative Gram-negative and Gram-positive bacteria in the MIC (minimum inhibitory concentration) assays. The bacterial cultures were grown in nutrient broth to midlog phase and standardized using McFarland standard before dilution. A 512 µg/mL peptide stock solution was prepared and 1:2 serial dilutions were prepared and added to the culture media to give final peptide concentrations of 256 µg/mL and less.

Fifty microliters of cells (5 X 104) and 50 µL of the peptide solution were added to a sterile well and the MIC was determined by the lowest concentration that inhibited cell growth. The inhibition of cell growth was indicated by the absence of turbidity, after

85 four hours. Turbidity in the wells was visualized manually. The MIC values are reported as the median value for at least three experiments.

Table 3.4 Minimum inhibitory concentration of Cyh-7.

Concentration g/mL. 256 128 64 32 16 8 4 2 Inhibition Against E. coli. High High High High High Moderate Low Low Inhibition Against S. aureus. High High High High Moderate Low Low Low

3.6.4 16-Hydroxypalmitoleic Acid.

O OH n2 O H n2 n =7 OOH H 1 n1 OOHn2=6 OH n1

Triethyl orthoformate (8.5 ml, 50.1 mmol), benzoic acid 0.25 g and aleuritic acid

(5 g, 16.45 mmol were heated gently (~110L C) for 1 hr until the ethanol evaporated. The temperature was then slowly raised to 170L C for 4 hours. To this mixture, a 1 N solution of NaOH was added at room temperature and stirred overnight. HCl 2N was added until a pH of 10. The mixture was extracted with dichloromethane (3x 100 ml). The organic layers were combined, dried with sodium sulfate and dried in vacuo. The product was very pure by TLC but it can be purified using column chromatography (8 % methanol in chloroform).

86 3.6.5 Peptide Synthesis Agni[ Glu(O-All)(KLAKKLA)2heptenoic Acid]

The peptide was synthesized using the Fmoc protecting group, HOBt/TBTU coupling.

Lysine side chains were protected with Cbz. The peptide was cleaved from the resin using a solution (prepared fresh) composed of 76 % (18 mL) TFA, 17 %(4 mL) dichloromethane 17 % (0.5 mL) phenol, 2 % (0.5 mL) triisopropylsiliane, and 2 % (0.5 mL) dd water. The TFA solution was purged with argon. This cleavage solution was added to the resin/peptide under argon with occasional shaking. After 3 hours the peptide/TFA solution was filtered and diluted with 100 mL of cold 20 % acetic acid. The mixture was lyophilized two days.

The peptide was dissolved in dichloromethane and it was washed with water. The organic layer was concentrated in vacuo. TLC 1:9 methanol/chloroform showed a major spot with minor impurities. Matrix assisted laser desorption ionization (MALDI) mass spectrometry was used to verify peptide mass. 2533 (M+H)+.

3.7 Ring Closing Methatesis Peptide Ligation

The ruthenium catalyst was purchased from Senn chemicals [cas#194659-03-5].

Fmoc glutamic acid allyl ester was purchased from Neosystem. Heptenoic acid was synthesized as in the ferrocene linker I.

The peptide Agni was synthesized using Fmoc amino acids. To a segment of the peptide Agni earlier reported by this group were added two residues.

The C-terminus residue glutamic acid with the side chain as an allyl ester, which can undergo metathesis with other residues and heptenoic acid was used to undergo metathesis at the N-terminus residue.

87 3.7.1 Attempted Peptide Ligation.

To a solution of 0.0339mg (0.0073mmol) of the peptide in dichloromethane was added in 5 % by mol portions of the Grubbs-Hoveyda catalyst until the total amount was

40 % by mol. The reaction was monitored by TLC. MALDI was used to verify the appearance of peptide oligomers, but only masses below 1100 were observed.

3.7.2 Attempted Peptide Ligation

To a solution of 0.2500 mg (.0050 mmol) of the peptide in dichloromethane was added in 5 % by mol portions of the new Grubbs catalyst until the total amount was 40 % by mol. The reaction was monitored by TLC. MALDI was used to verify the appearance of peptide oligomers, but only masses below 1100 were observed.

CHAPTER FOUR

SUMMARY AND FUTURE STUDIES

4.1 Discussion

In Chapter 2 of this dissertation, a novel ferrocene linker was prepared. The effects of the electron-withdrawing groups on the cleavage rates of molecules from the ferrocenyl-amine position were demonstrated. The ferrocenyl cation was stabilized with electron-donating groups to increase the cleavage rates. It was found that by having a vinylic bond instead of a carboxyl amide group directly attached to the cyclopentadienyl ring increases the acid lability of the linker. This suggests that other groups may be attached to the ferrocene in order to make linkers with different degrees of acid lability. These changes can be beneficial when different protection schemes are needed. In Chapter 3, ways to make antimicrobial helical peptides incorporating natural and unnatural amino acids were explored. A synthetic antimicrobial peptide Cyh-7 was synthesized and tested for biological activity, which proved to be significant. Attempts to make a longer homogeneous sequences using cross metathesis failed. Possible causes for this could be undesired conformations of the peptide in solution, which makes them inaccessible to react with the catalyst. A study of the peptide conformation in suitable solvents for the catalysis may provide the necessary information to make large peptides in a convergent way.

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95 Vita

José Giraldés was born on March 25, 1974, in Hato Rey, Puerto Rico. He received his bachelor of science degree (American Chemical Society certified) in chemistry from the University of Puerto Rico , San Juan, in 1997. He decided to pursue his interests in organic synthesis by seeking a doctorate degree in organic chemistry at Louisiana State

University from June 1998 to present, where he studied under the direction of Dr. Mark

L. McLaughlin. Currently he is a candidate for the degree of Doctor of Philosophy in the

Department of Chemistry and has accepted a postdoctoral fellowship at the University of

South Florida, H. Lee Moffitt Cancer Center.