Synthetic Strategies for the Preparation of Affinity Label Dynorphin a (1-11

Home , Deltorphin I, Kelatorphan, Ketazocine, Naloxonazine

AN ABSTRACT OF THE THESIS OF

Leena Leelasvatanakij for the degree of Doctoral of Philosophy in Pharmacy presented on

April 22, 1996. Title: Synthetic Strategies for the Preparation of Affinity Label Dynorphin

A(1-11)NH2 Analogues.

Redacted for Privacy Abstract approved: Jane V. Aldrich

Affinity labels which bind irreversibly to receptors are useful tools to study receptors. Potential affinity label derivatives of dynorphin A based on [D-Prom]Dyn A(1-

11)NH2 were synthesized to use as pharmacological probes for studies of lc opioid receptors.

A reactive functionality (e.g. isothiocyanate or bromoacetamide) was incorporated into either the "message" or "address" sequences of the parent peptide. The peptides were prepared using Fmoc-solid phase synthesis on a polyethylene glycol (PEG)-polystyrene (PS) support with a peptide amide linker (PAL).Orthogonal protection was obtained using the allyloxycarbonyl (Aloc) group which was selectively removed by palladium (0), followed by introducing the reactive functionality.

Preliminary binding assay results indicated that, except for the bromoacetamide derivative [Phe(NHCOCH2Br)4,D-Pro10]Dyn A(1-11)NH2, all peptides bound to lc opioid

receptors with high affinity, with [Lys8,D-Pro10]Dyn A(1-11)NH2 showing the highest affinity for lc opioid receptors. The isothiocyanate derivatives generally showed higher affinity for x opioid receptors than the bromoacetamide derivatives (the bromoacetamide was not introduced into Phe(NH2)').Incorporation of a reactive functional group in the

"message" and "address" sequences of Dyn A yielded peptides with varied selectivity for opioid receptors; for the "message" sequence modifications lc > µ >> 8, and for the "address" sequence modifications lcµ > 8.

The synthesis of [Phe(X)4,D-Pro9Dyn A(1-11)NH2 (where X = -NH2, -N=C=S and

-NHCOCH2Br) on two different resins (PAL-PEG-PS vs. PAL-PS resin) showed that Aloc deprotection rates and the purity of the crude peptide were dependent upon the type of resin used. Removal of Aloc from peptides assembled on the PAL-PEG-PS resin was much faster

(3 h) than was deprotection on the PAL-PS resin (24 h), but the crude peptides from the

PAL-PS resin contained fewer side products than the peptides obtained from the PAL-PEG-

PS.

Radiolabeled affinity label peptides can be useful for studies of the interactions of ligands with opioid receptors. Thus, we developed strategies for the synthesis of a protected precursor for radiolabeling, using [Phe(31,5I-I2,4'-NH2)4,D-Pro9Dyn A(1-11)NH2 as a model.

The protected peptide was assembled on a functionalized NovaSyn TG® resin with a base- labile 4-hydroxymethylbenzoic acid linkage (linker B). Assembly of the peptide on this modified resin proceeded smoothly. The protected peptide was obtained in an excellent yield

(93.1% of the expected peptide substitution). The peptide-resin linkage was cleaved by ammonolysis in isopropanol, with 90% of the peptide cleaved after 1 day. The synthetic

strategies described in this dissertation hopefully will be applicable for other affinity label-

containing peptides. Synthetic Strategies for the Preparation of Affinity Label Dynorphin A(1-11)NH2 Analogues

Leetia Leelasvatanakij

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctoral of Philosophy

APPROVED: Redacted for Privacy

MajProfessor,1-4-4.41-qtriacy

Redacted for Privacy

Dean of College of Pharmacy

Redacted for Privacy

Dean of Gradut School

I understand that my thesis will become part of the permanent collection ofOregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Redacted for Privacy

Leen.al.,eelasvatanaki.i., klthor ACKNOWLEDGEMENT

First my special thanks go to Dr. hine V. Aldrich, my major advisor, who so generously gave her time, advice, creativity, love and patience during these past years.I thank her for her unique wisdom and insight. I also appreciate my committee members,Dr.

Michael Schuyler, Dr. Mark Zabriskie, Dr. Paul Franklin, and Dr. John Gillis, for givingtheir precious time and advice. I would like to acknowledge Dr. Thomas Murray whosupervised the radioligand binding assays and Elizabeth Olenchek who expertlyperformed the

experiments. My thanks also go to Dr. Brian Arbogast for performing thefast atom bombardment experiments and to Barbara Robbins for the amino acid analyses. I would also like to thank the faculty and staff at the College of Pharmacy, Oregon StateUniversity and the School of Pharmacy, University of Maryland at Baltimore; it has been apleasure working with them. I appreciate my colleagues, Dr. Seksiri Arttamangkul, Dr. HeekyungChoi, Dr.

Sandra Vigil-Cruz and Dean Maeda who so generously shared their tremendous talent and wonderful friendship with me. .W very special thanks also go to Dr. Qi Zheng for being

such an excellent co-worker both professionally and personally.

I would like very much to express my heartfelt thanks to my parents for being my

support (pillar) in every way possible.They are truly the inspiration of my life and my

gratitude goes beyond words. I thank my brothers and sisters for encouraging me to stretch

and to grow.I appreciate their love and support, and the beautiful times we havehad

together.My gratitude also goes to the Royal Thai Government for providingfinancial

support, and their staff for all of the titre and energythat they have put into helping my life

run so smoothly during my study in theUnited States. CONTRIBUTION OF AUTHORS

Dr. Jane V. Aldrich provided her advice on the design and analysis of the research and writing of each manuscript. Dr. Thomas Murray and Elizabeth Olenchek were involved in the pharmacological experiments. Dr. Brian Arbogast assisted in the mass spectrometry experiments. TABLE OF CONTENTS

Page

CHAPTER 1 INTRODUCTION 1

1.1 Background 1

1.2 Objectives and rationale 2

CHAPTER 2 LITERATURE REVIEW: OPIOIDS 6

2.1 Opioid peptides 6

2.1.1 Pro-opiomelanocortin (POMC) 9 2.1.2 Proenkephalin A (PROENK) 9 2.1.3 Proenkephalin B or prodynorphin (PRODYN) 10

2.2 Dynorphin A 11

2.2.1Metabolism and enzyme inhibitors 12 2.2.2 Conformational studies 14 2.2.3Structure-activity relationships (SAR) 16

2.3Opioid receptors 18

2.3.1 Opioid receptor types and their ligands 20 2.3.2 Isolation and purification of opioid receptors 28 2.3.3 Molecular biology and cloned opioid receptors 30

2.4. Affinity label-containing opioids 35

2.4.1Non-peptide affinity labels 38 2.4.2Peptide affinity labels 41

CHAPTER 3 LITERATURE REVIEW: SOLID PHASE PEPTIDE SYNTHESIS 42

3.1 Basic synthetic strategies 42

3.2 Protecting groups 44

3.2.1 Mc-amino protecting groups 44 TABLE OF CONTENTS (CONTINUED)

Page

3.2.2 Side chain protecting groups 49

3.3 Insoluble supports 52

3.4 Linkers 55

3.5 Peptide bond formation 60

3.6 Monitoring 66

CHAPTER 4 SYNTHESIS OF POTENTIAL AFFINITY LABEL DERIVATIVES OF DYNORPHIN A(1-11)NH2: MESSAGE SEQUENCE MODIFICATIONS 67

4.1 Abstract 69

4.2 Introduction 70

4.3 Experimental section 75

4.3.1 Materials 75 4.3.2 Methods 76 4.3.3 Receptor binding assays 82

4.4 Results and discussion 84

4.4.1 Amino acid syntheses 84 4.4.2 Peptide syntheses 89 4.4.3 Receptor affinity 92

4.5 Conclusions 95

CHAPTER 5 SYNTHESIS OF POTENTIAL AFFINITY LABEL DERIVATIVES OF DYNORPHIN A(1-11)NH2: ADDRESS SEQUENCE MODIFICATIONS 97

5.1 Abstract 99

5.2 Introduction 100 TABLE OF CONTENTS (CONTINUED)

Page

5.3 Experimental section 103

5.3.1 Materials 103 5.3.2 Methods 103 5.3.3 Receptor binding assays 105

5.4 Results and discussion 105

5.4.1 Peptide syntheses 105 5.4.2 Receptor affinity 106

5.5 Conclusions 111

CHAPTER 6 SOLID PHASE SYNTHESIS OF POTENTIAL AFFINITY LABEL DERIVATIVES OF DYNORPHIN A ASSEMBLED ON PAL-PEG-PS VS PAL-PS RESIN 113

6.1 Abstract 115

6.2 Introduction 116

6.3 Experimental section 120

6.3.1 Materials 120 6.3.2 Methods 121

6.4 Results and discussion 123

6.5 Conclusions 126

CHAPTER 7 SYNTHESIS OF A DYNORPHIN A PRECURSOR FOR RADIOLABELING 131

7.1 Abstract 133

7.2 Introduction 134

7.3 Experimental section 138 TABLE OF CONTENTS (CONTINUED)

Page

7.3.1 Materials 138 7.3.2 Methods 139

7.4 Results and discussion 144

7.4.1 NovaSyn TG® resin functionalization 145 7.4.2 Peptide syntheses 146

7.5 Conclusions 152

CHAPTER 8 CONCLUSIONS 153

BIBLIOGRAPHY 157 LIST OF FIGURES

Figure Page

1.1 [D-Pro 1°]Dyn A(1-11)NH2 and affinity label-containing derivatives 4

2.1 Diagrammatic representation of structures of opioid peptide precursors 8

2.2 Dynorphin sequence and sites for enzymatic degradation 13

2.3 Peptidase inhibitors 14

2.4 Selected opioid receptor ligands 23

2.5 Comparison of the amino acid sequences of 6, lc and u opioid receptors 31

2.6 Schematic diagram of protein structure for, and similarityamong, the three opioid receptors 34

2.7 A schematic illustration of the principle of recognition amplification in the covalent binding of receptor type A by an affinity label containing a group-selective electrophile X 36

2.8 Chemical affinity labeling versus photoaffinity labeling 37

2.9 Affinity label agonists 39

2.10 Affinity label antagonists 40

3.1 Stepwise solid-phase synthesis of linear peptide 43

3.2 "Merrifield" protection scheme for solid-phase synthesis, based on graduated acidolysis 45

3.3 A mild two-dimensional orthogonal protection scheme for solid phase synthesis 47

3.4 Fmoc deprotection 48

3.5 Side chain protecting groups used in Boc SPPS 50 LIST OF FIGURES (CONTINUED)

Figure Page

3.6 Side chain protecting groups used in Fmoc SPPS 51

3.7 Insoluble supports 53

3.8 Resin linkers and handles used in Boc SPPS 56

3.9 Resin linkers and handles used in Fmoc SPPS 58

3.10 Resin linkers and handles used in both Boc and Fmoc SPPS 61

3.11 Peptide bond formation 61

3.12 Active esters of amino acids 63

3.13 Coupling reagents 65

3.14 Ninhydrin test 66

4.1 Synthesis of phenylalanine derivatives 77

4.2 Synthesis of [Phe(X)4,D-Pro10]Dyn A(1-11)NH2 derivatives 79

4.3 Chromatograms of FmocPhe(NH2) (solid line) and FmocPhe(NHAloc) (dotted line.) 85

4.4 Chromatograms of BocPhe(NH2) (solid line) and BocPhe(NHAloc) (dotted line) 86

4.5 Chromatograms of crude peptide lb (top), /c (middle) and ld (bottom) 90

4.6 Chromatograms of crude peptide 2b (top) and 2c (bottom) 91

5.1 Chromatograms of crude peptides lb (top), /c (middle) and ld (bottom) 107

5.2 Chromatograms of crude peptides 2b (top), 2c (middle) and 2d (bottom) 108 LIST OF FIGURES (CONTINUED)

Figure Page

6.1 Deprotection of allyl-base protecting groups 117

6.2 Structures of PAL-PEG-PS (top) and PAL-PS (bottom) resin 117

6.3 Comparison of deprotection rates of Aloe from [Phe(NHAloc)4, D-Pro9Dyn A(1-11)NH-resin synthesized on PAL-PEG-PS (top) vs and PAL-PS (bottom) 125

6.4 Chromatograms of crude peptide [Phe(NH2)4,D-Pro9Dyn A(1-11) NH2 from PAL-PEG-PS (left) and PAL-PS (right) resins 127

6.5 Chromatograms of crude peptide [Phe(N=C=S)4,D-Pro10]Dyn A(1-11) NH2 from PAL-PEG-PS (left) and PAL-PS (right) resins 128

6.6 Chromatograms of crude peptide [Phe(NHCOCH2BW,D-Pro10]Dyn A (1-11)NH2 from PAL-PEG-PS (left) and PAL-PS (right) resins 129

7.1 Synthesis of tritium-labeled peptides bearing a reactive functionality 137

7.2 NovaSyn TG® resin functionalization 140

7.3 Synthesis of dynorphin A precursor for radiolabeling 143

7.4 Chromatograms of Phe(3',5'42,4'-NH2) (dotted line) and FmocPhe(3',5'42, 4'-NH2) (solid line) 148

7.5 Chromatograms of crude protected peptide 1 after ammonolysis; 1 (top), 3 (middle) and 5 (bottom) days 151 LIST OF TABLES

Table Page

2.1 Classification of opioid peptides 7

2.2 Binding profile of dynorphin A(1-13) 12

2.3 Opioid receptors and physiological effects 19

2.4 Distributions of opioid receptors in tissues used in smooth muscle assays 19

2.5 Tentative receptor classification 21

2.6 Pharmacology of the cloned opioid receptors 32

4.1 Analytical data of modified phenylalanine derivatives 87

4.2 Analytical data of potential affinity label derivatives of dynorphin A 93

4.3 Preliminary binding assay data for potential affinity label derivatives of dynorphin A 94

5.1 Analytical data of potential affinity label derivatives of dynorphin A 109

5.2 Preliminary binding assay data for potential affinity label derivatives of dynorphin A 110

6.1 Ratio of swollen/dry volumes for individual polyethylene glycol polystyrene (PEG-PS) graft beads and polystyrene (PS) support in different solvents 119

7.1 Characterization of [Phe(3',5'42,4'-NH2)4,D-Pro19Dyn A(1-11) NH2 before and after ammonolysis 150 ABBREVIATIONS

Aib a-amitioisObutyric acid

Aloc allyioxycarbonyl

Boc t-butyloxycarbonyl

BOP (benzotriazolyloxy) tris (dimethylamino) phosphonium hexafluorophosphate

CTAP D-Phe-Cis-Tyr-D-Trp-Arg-Thr-Peln-Thr-NH2

CTOP D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2

DAMGO Tyr-D-Ala-Gly-MePhe-NH(CH2)20H

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DIC diisopropylcarbodiimide

DIEA 1V,N1-diisopropylethylarnine

DMA N,N-dimethylacetarnide ,

DMF NN-dimethylformamide

DPDPE cyclo[DPen2,D-Pen5]enkephalin

DSLET Tyr-D-Ser-Gly-Phe-Leu-Thr-OH

Dyn A dynorphin A

EDT I,2-ethanedithio!

EKC ethylketocyclazocine

FAB -MS fast :.tour bombardment mass spectrometry

Fmoc 9-fluorenylmethoxycarbonyl

Fmoc-OSu 9-fluorenylmethyl succinimidyl carbonate Fmoc-Cl 9- fluorrNiyitneYhyloxyCalbonyl chloride

GPI gurnea pig ileum

HAPyU (7'-azabenzot riazok -y1)-1,1,3,3-bis(tetramethylene) uronium

HATU 0-(7-azabenzotriazol-1-y1)-1,1,3,3-tetramethyluronium hexafluorophosphate

HOAt 1-hydroxy-7.-azabenzotriazole

HOBt 1 -hy droxybenzoviazole

HPLC high performance liquid chromatography

MBHA 4-methylbenzhydrylamilie

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

MVD mouse vas deferens

Na1Bz0H naiotone benzoylltydrazone

nor-BNI norknaltorpharnine

OPfp pentafitiorophenyl ester

PAL peptide amide linker or 5-(4-aminomethy1-3,5-dimethoxyphenoxy)valeric acid resin

Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

PEG polyethylene glycol

Pen penicillamine

Pmc 2,2,5_7,8-pentamethylchroman-6-sulfonyl

PyAOP 7-azabenzotriazoi-1-34-oxy-trispyrrolidinophosphonium hexafluorophosphate

PyBOP benzotriazol-1-yi-oxy-trispyrrolidino phosphonium hexafluorophosphate TBTU 2-(111-benzOt ia7o1-1-y1)1,1,3,3-tetramethyluronium teti Arkturobor ate t-Bu te;

TFA trifluoi °acetic acid

THE tetrahydrofuran

Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

Tos 4-toluenesulfonyl

U-50,488 (- ) -(.IS, 25)-trans-3,4-dichloro-N-methyl-N42-(1-pyn-olidiny1)- cyclohexylThenzeneacetamide

U-69,593 (5a,711,813)-(-)-N-methyl-N-[7-(1-pyrrolidiny1)-1-oxaspiro- [4,5]dec-8-ylThenzeneacetamide

benzyloxycarbonyl SYNTHETIC STRATEGIES FOR THE PREPARATION OF AFFINITY LABEL DYNORPHIN A(1-11)NH2 ANALOGUES

CHAPTER 1

INTRODUCTION

1.1 Background

Opium from Popover somniferum has been used for controlling pain and diarrhea

since ancient times [Brownstein, 1993]. The active ingredient morphine exerts its actions, including relieving pain, reducing intestinal motility and secretion and altering mood, by binding with opioid receptors [Simon, 1991]. Morphine is clinically usedas an analgesic for severe pain, but long term users can develop tolerance and physical dependence. Thus, researchers have attempted to discover novel nonaddictive analgesics. Studies of opioid receptors have made significant contributions in this search for new analgesics. Three types of opioid receptors, the 11, x and a receptors,were originally proposed by Martin and coworkers [Martin et al., 1976; Gilbert et al., 1976]. Thea receptor was later classified as a non-opioid receptor because its pharmacological effects are not antagonized by naloxone

[Rees and Hunter, 1990; Fries, 1991]. The 8 receptorwas proposed as another type of opioid receptor after the discovery of the enkephalins [Lord et al., 1977]. Today the12, lc and 8 receptors are the generally accepted opioid receptor types.

Characterization of multiple opioid receptors as well as the isolation of opioid peptides has provided a better understanding of the pharmacological effects of opiates and of the physiological functions of endogenous opioid systems.For example x opioid 2

receptors have been characterized by benzomorphan derivatives such as ethylketocyclazocine

(EKC) and bremazocine [Romer et al., 1980], but these ligands can also interact with u opioid receptors [Kosterlitz et al., 1981; Wood et al., 1981; Magnan et al., 1982; Goldstein

1984].The more recently developed ligands, including U50,488, U69,593 and their analogues, show very high lc receptor selectivity [Zimmerman, 1990].

Pharmacological studies of opioid receptors can be facilitated by the availability of affinity label-containing opioids including photoaffinity labels and electrophilic affinity labels (chemical affinity labels). Both types of affinity labels bind with receptors in a two- step process; first binding reversibly followed by covalent bond formation [Takemori and

Portoghese, 1985]. The irreversible binding of affinity labels can be used in the isolation of receptors from cell membranes such as the isothiocyanate derivative of U50,488 which bind irreversibly to lc receptors [de Costa et al., 1989].

1.2 Objectives and rationale

The specific aim of this dissertation was to synthesize and characterize affinity label-containing derivatives of dynorphin A which can be used for x opioid receptor characterization.These analogues are electrophilic affinity labels containing reactive functionalities including isothiocyanate (-N=C=S) and bromoacetamide (-NHCOCH2Br) groups. They will be screened for wash resistant inhibition of binding to cloned opioid receptors.Analogues that bind in a nonequilibrium (irreversible) manner to lc opioid receptors can be used as probes of lc receptor structure and receptor-ligand interaction. The synthetic strategies used to prepare affinity label-containing analogues of dynorphin A 3 described in this dissertation should also be applicable to other affinity label-containing peptides.

K-Opioid receptor characterization is of great interest to many researchers, because these receptors are found in human brain and spinal cord in high concentrations [Pfeiffer et al., 1982; Czlonkowski et al., 1983; Gouarderes et al., 1986]. Unlike morphine, K ligands produce analgesic effects without physical dependence [Mil lan, 1990]. Dynorphin A (Dyn

A), an endogenous opioid peptide derived from prodynorphin [Simmon, 1991], binds to K opioid receptors and is thought to be an endogenous ligand for these receptors [Chavkin et al, 1982]. Therefore, we developed synthetic strategies for the preparation of potential affinity label derivatives of dynorphin A (1-11)NH2. These compounds hopefully will bind selectively and irreversibly to K opioid receptors and can be used as probes for K receptor characterization. In addition, because Dyn A is an endogenous ligand it is important to understand its interactions with opioid receptors and its physiological functions.The truncated peptide, Dyn A(1-13)NH2, has similar activity as the heptadecapeptide in the guinea pig ileum smooth muscle assay [Goldstein et al., 1979]. The C-terminal amide derivative of Dyn A(1-13) was found to have greater metabolic stability than the C-terminal acid [Leslie and Goldstein, 1982]. Gairin [1985] found [D- Pro10]Dyn A(1-11)NH2has higher potency and selectivity (ratio of K. values (K: 8:) = 1: 93: 280) than Dyn A(1-13)NH2.

Therefore, affinity label-containing derivatives of [D-Pro9Dyn A(1-11)NH2 derivatives were prepared (Fig.1.1). The reactive functionalities, isothiocyanate (-N=C=S) and bromoacetamide (-NHCOCH2Br), were incorporated in both the N-terminal "message" and the C-terminal "address" sequences.In the N-terminal sequence, the reactive 4

Dyn A(1-11)NH2

H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-NH2

Affinity label-containing peptides with N-terminal sequence modifications

I. Phel -substituted analogues

H-Phe(X)-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-NH2

2. Phe4-substituted analogues

H-Tyr-Gly-Gly-Phe(X)-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-NH2

Affinity label-containing peptides with C-terminal sequence modifications

1. Phe8-substituted analogues

H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Phe(X)-Arg-D-Pro-Lys-NH2

2. Lys8-substituted analogues

H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Lys(X)-Arg-D-Pro-Lys-NH2

X = -NH2 (or -H for Lys8 series) (control compounds)

= -N=C=S

= -NHCOCH2Br (except for Phe' series)

Fig. 1.1 [D-ProilDyn A(1-11)NR and affinity label-containing derivatives. 5 functionalities were introduced on the p-amino group of phenylalanine in positions 1 and 4

(see chapter 4).In the C-terminal sequence, Iles was replaced by p-amino phenylalanine or

Lys derivatives (see chapter 5).In all cases, the amine compounds without a reactive functionality were prepared as control compounds for the pharmacological assays.

The type of resins used in solid phase synthesis can affect reaction rates and the purity of the final peptides. Therefore we investigated the synthesis of a model peptide,

[Phe(X)4,D-Pro10]Dyn A(1-1 1)NH2 (where X = -NH2, -N=C=S and -NHCOCH2Br) on two different resins (PAL-PEG-PS vs. PAL-PS resin) (see chapter 6), and compared the rates of

Aloc deprotection and purity of the crude peptides obtained from these two resins.

To facilitate the study of receptor-peptide interactions, radiolabeled peptides need to be prepared. We have developed a synthetic strategy for the preparation of radiolabeled Dyn

A analogues containing an affinity label (see chapter 7).The radiolabel and reactive functionalities will be on the same residue so that they will remain associated even after proteolytic digestion of affinity labeled receptors.NovaSyn Tentagel® resin was functionalized with a base labile (NH3/i-PrOH) linkage and used for the preparation of a protected precursor for radiolabeling by replacing Phe by Phe(3,5'- 12,4' -NH2). Following cleavage from the resin this protected peptide amide will be subjected to catalytic hydrogenation prior to introducing a reactive functionality. The procedures developed for this model peptide will be used for the preparation of tritiated affinity label-containing peptides.These peptides will be used as pharmacological tools for lc opioid receptor characterization. 6

CHAPTER 2

LITERATURE REVIEW: OPIOIDS

2.1 Opioid peptides

Opium alkaloids from Popover somniferum, has been used as pain killers since ancient

times. The active ingredient in the opium alkaloids is morphine, which binds to receptors

[Evans et al., 1988] and produces potent analgesia. A search for the endogenous ligands for

opioid receptors resulted in the discovery of the opioid peptides. In 1975, two pentapeptides

with opiate-like activities, methionine enkephalin (Tyr-Gly-Gly-Phe-Met) and leucine

enkephalin (Tyr-Gly-Gly-Phe-Leu) were identified from porcine brain [Hughes et al., 1975].

Since then, several endogenous opioid peptides have been isolated and characterized,

including 13-endorphin (13-End) and the dynorphins (Dyn).

Mammalian endogenous opioid peptides were classified into three groups according

to their precursors, pro-opiomelanocortin (POMC), proenkephalin A (PROENK) and

proenkephalin B or prodynorphin (PRODYN) (Table 2.1) [Negi et al., 1992]. These peptide

precursors are translational products of separate but similar genes [Lutz et al, 1992]. The genes have similarity in the placement and size of their respective introns and exons. This

suggests that the genes for the three precursors are derived from the same ancestoral gene by

gene duplication. POMC, PROENK and PRODYN share commoncharacteristics (Fig. 2.1)

[Simon et al., 1994]; they contain almost the same number of amino acids and their sequence

homology is more than 50 percent Each precursor contains multiple opioid peptide 7

Table 2.1 Classification of opioid peptides.

Precursors Peptides Sequence Pro-opiomelanocortin 13-endorphin YGGFMTSEKSQTPLVTLFKNAIIKN AYKKGE

8-endorphin YGGFMTSEKSQTPLVTLFKNAIIK (P-endorphin 1-27) NAY

y-endorphin YGGFMT SEKSQTPLVTL (P-endorphin 1-17)

a-endorphin YGGFMT SEKSQTPLVT (P-endorphin 1-16)

Proenkephalin A Met-enkephalin YGGFM

Met-enkephalin- YGGFMRF Arg-Phe

Met-enkephalin- YGGFMRFGL Arg-Gly-Leu

Leu-enkephalin YGGFL

Peptide E TGGFMRRVGRPEWWMDYQKRY GFL

Proenkephalin B a-neo-endorphin YGGFLRKYPK (Prodynorphin) P-neo-endorphin YGGFLRKYP

Dynorphin A (1-17)YGGFLRRIRPKLKWDNE

Dynorphin A (1-8) YGGFLRRI

Dynorphin B Y GGFLRRQFKVVT

Dynorphin B TGGFLRRQFKVVTRSQEDPNAYYE (1-29) (Leumorphin) LFDV 8

v.. 5', °,2.' 1 ..t 23 , ..:, ,.., T. Z .-.1 _..j ACTH-11-LPH precursor

Signal cr-MSH (l -MSH peptide N-Terminal peptide ACTH 9 -LPH r, -MSH CLIP y-LPH g-Endorphin

Two OD . a to cm!!.1 ,- J.. °. a° ..J < -J < < < S..4 4. ;A -/' A S. r. ..>.... a..... a, a. U(..) (.1 ...1...1 -.1 -1 -J < -1 III II I_ Preproenkephalin A1.7513 LI Signal Met -enk MetVet-enk -enk Met-enk Met-enk- Met-enk Leu-enk Met-e- nk- peptide Arge-Gly7-Lete Are-Phe'

Peptide Peptide E

a 0: < < A ED < Yiu Preproenkephalin 13

Signal Leu-e- nk Leu-enk Leu-enk Peptide Neo-endorphin Dynorphin Leumorphin

Fig. 2.1 Diagrammatic representation of structures of opioid peptide precursors. MSH and enkephalin (enk) units are shown by solid or shaded boxes. Cysteine and dibasic residue are shown in the upper portion and major peptides derived are shown in the lower portion of each diagram. LPH, lipotrophin; CLIP, corticotrophin-like intermediate lobe peptide.

Source: Imura, H.; Kato, Y.; Nakai, Y.; Nakao, K.; Tanaka, I.; Jingami, H.; Koh, T.; Yoshimasa, T.; Tsukada, T.; Suda, M.; Sakamoto, M.; Norii, N.; Takahashi, H.; Tojo, K.; Sugawara, A. Endogenous Opioids and Related Peptides: from Molecular Biology to

Clinical Medicine../. Endocr.. 1985, 107, 147-157. 9 sequences which are marked by paits of basic amino acids at the C-terminus and the N- teminus contain multiple cysteine residues.

2.1.1Pro-opiomelanocortin (POMC)

Pro-opiomelanocortin (POMC), a translated product of the POMC gene, is a 31 kDa glycoprotein consisting of 267 amino acids. The POMC gene contains 7665 base pairs (bp) including exon 1 (86 bp), exon 2 (152 bp), exon 3 (833 bp), intron A (3708 bp) and intron

B (2886 bp) [Nakanishi, 1979]. The major source of POMC is in the intermediate lobe of the pituitary [Simon, 1991]. POMC has been identified as the precursor of adrenocorticotropic hormone (ACTH), P-lipotrophin (P-LPH), endorphins, y-melanocyte-stimulating hormone

(y-MSH), a-MSH, y-lipotrophin (y-LPH) and the corticotropin-like intermediate lobe peptide (CLIP) [Evans et al., 1988].p-Endorphin (Table 2.1) is the only opioid peptide derived from this precursor which possesses analgesic activity.

2.1.2 Proenkephalin A (PROENK)

Proenkephalin A (PROENK) contains 267 amino acids with multiple copies of enkephalin sequences [Evans et al1988]. The PROENK gene is 5200 by long including exon 1 (70 bp), exon 2 (56 bp), exon 3 (141 bp), exon 4 (980 bp), intron A (87 bp), intron

B (469 bp) and intron C (3400 bp) [Comb et al., 1982]. PROENK identified from bovine adrenal cortex contains a copy of Leu-enkephalin, four copies of Met-enkephalin, two copies of Met-enkephalin-Arg-Phe and one copy of Met-enkephalin-Arg-Phe-Leu [Undenfriend et al., 1984]. Proenkephalin A products (Table 2 1) localize in both central and peripheral 10 tissues [Frederickson et al, 1981; Herbert et al1985]. The processing of PROENK yields smaller opioid peptides by cleavage at all pairs of basic residues and at some single arginine residues. In human adrenal, the processing occurs extensively, whereas in bovine adrenal the processing is less extensive yielding higher molecular weight opioid peptides [Imura et al.,

1985].

2.1.3 Proenkephalin B or prodynorphin (PRODYN)

Proenkephalin B or prodynorphin (PRODYN) contains 254 amino acids.The

PRODYN gene consists of exon 1 (1400 bp), exon 2 (60 bp), exon 3 (145 bp), exon 4 (2200 bp),intron A (1200 bp), intron B (9900 bp) and intron C (1700 b) [Horikawa, 1983].

Prodynorphin gives rise to dynorphins and neo-endorphins. The derived peptides are found in the brain and spinal cord with the highest concentrations in the anterior hypothalamic nuclei. Porcine PRODYN contains three Leu-enkephalin sequences each flanked by pairs of basic amino acids (i.e. Lys-Arg and Arg-Arg) [Kakidani et al., 1982]. When Lys-Arg pairs are the processing signals, prodynorphin will be cleaved into three opioid peptides:13-neo- endorphin [Minomino et al., 1981], dynorphin A [Goldstein et al., 1981] and leumorphin

[Nakao et al., 1983]. Dyn A(1-8) is derived from the cleavage of Dyn A(1-17) between Ilex and Are [Minomino et al., 1980; Seizinger et al., 1981]. A similar cleavage of leumorphin between Thr13 and Arg14 yields dynorphin B (rimorphin) [Fischi et al., 1982; Kilpatrick et al.,

1982]. Opioid peptides derived from prodynorphin (Table 2.1) share a leu-enkephalin, Tyr-

Gly-Gly-Phe-Leu, sequence at the N-terminus and except for dynorphin A(1-8) and I3-neo- endorphin, have selectivity for lc opioid receptors [Kosterlitz et al., 1985; Holt et al., 1983]. 11

Processing patterns of PROENK and PRODYN are different although these precursors are co-localized [Basbaum and Fields, 1984; Evans etal., 1984]. PROENK products from cleavage at basic residue pairs are Leu-enkephalin, Met-enkephalin, Met- enkephalin-Arg-Gly-Leu and Met-enkephalin-Arg-Phe The processing of PRODYN is less

extensive providing opioid peptides with paired basic residues linking C-terminal extensions to the enkephalin sequence. These C-terminal extended enkephalin sequences have higher affinities for x opioid receptors than enkephalins themselves [James et al , 1984].

2.2 Dynorphin A

Dynorphin A (Dyn), a heptadecapeptide derived from prodynorphin, was first identified and isolated from porcine pituitary and duodenum extract [Cox et al., 1975;

Goldstein et al., 1981].It has been postulated to be an endogenous ligand for K opioid receptors [Chavkin et al., 1982]. Dyn is distributed in different organs including the pituitary, brain, hypothalamus [Goldstein et al,1979; Kangawa et al., 1981; Khachaturicn et al.. 1982]

spinal cord, ganglia [Nakao et al., 1981] duodenum [Botticelli et al., 1981] and the adrenal medulla [Lamaire et al., 1983 and 1982]. Dyn A contains a Leu-enkephalin "message" sequence at the N-terminus which accounts for opioid activity, while the "address" sequence at the C-terminus directs the peptide to x opioid receptors (Fig. 2.2) [Chavkin et al, 1982].

The last four amino acid residues (Trp-Asp-Asn-G1n), however,are not necessary for

association of Dyn A with opioid receptors. The binding profile of Dyn A(1-13) to K binding

sites is comparable to that of the heptadecapeptide and also shows affinity for g and 8 opioid receptors (Table 2.2) [Corbett et al., 1982]. The potency of Dyn A(1-13) is 700 times that 12

of Leu-enkephalin and 200 times that of normorphine in guinea pig ileum (GPI) [Corbett et

al., 1982]. The analgesic effects of Dyn are somewhat controversial, with some evidence

showing that it relieves pain from chemical and pressure stimuli but not from heat [Hayes et

al., 1983], and that it is effective only by intraspinai administration [Piercey et al., 1982].

Table 2.2 Binding profile of dynorphin A(1 -13).

IC5o (nM) ic) MVD(8) Rat VD' Rabbit VD2

Dynorphin A(1-13) 0.31 0.33 10,000 2.43

'Low sensitivity to x-ligands. 'Sensitive to lc- but not u- or 6-agonists.

2.2.1 Metabolism and enzyme inhibitors

Like other opioid peptides, dynorphin A is susceptible to enzymatic degradation. The

N-terminal fragment can be inactivated by various peptidases; the Tyr-Gly bond is cleaved by

an aminopeptidase (EC3.4.11), the Gly-Phe bond by either ametalloendopeptidase EC

3.4.24.11 (enkephalinase) or angiotensin converting enzyme (Fig 2.2) [Leslie et al., 1982;

Marks et al., 1980; Benuck et al., 1984]. The C-terminus of both Dyn A(1-17) and Dyn A

(1-13) are cleaved by endopeptidases to provide a shorter biologically active fragment, Dyn

(1-8) [Leslie and Goldstein, 1982]. Dyn (1-8), resulting from cleavage at Iles-Arg9, is less

selective toward x binding sites and is more susceptible to enzymatic inactivation than the

longer parent peptides.Several approaches, including substitution of a D-amino acid, conversion of the terminal COOH to CONH2 [Leslie et al., 1982], conformational constraint 13

and use of enzyme inhibitors in assays, can prevent the metabolic degradation of dynorphin

analogues.

a b d

1 I. Tvr-Glv-Gly-Phe-Leu-Arq-Arq-lle-Arq-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn message sequence address sequence

Fig. 2.2 Dynorphin sequence and sites for enzymatic degradation.aaminopeptidase, b = enkephalinase and c = endopeptidase.

N-Terminal enzymatic inactivation of dynorphin can be suppressed by severalenzyme inhibitors (Fig. 2.3). Bestatin is a specific aminopeptidase inhibitor [Fournie-Zaluskiet al.,

1985], while thiorphan is an enkephalinase inhibitor [Rogues et al., 1980]. Thiorphanitself possesses antinociceptive activity by potentiating the effects of enkephalins released in response to noxious stimuli [Corbett et al., 1982]. The affinity of thiorphan for enkephalinase is independent of its stereochemistry (inhibitory potency (IC50): S, 1.90nM vs R, 1.60 nM).

Aretroisomer of thiorphan is also an effective enkephalinase inhibitor whichinhibits angiotensin converting enzyme (ACE) less than thiorphan. Retro-thiorphan displaysa 100 - fold difference in inhibitory activity by the different isomers (IC50: S, 210nM vs R, 2.3 nM)

[Fournie-Zaluski et al., 1986].Kelatorphan, a hydroxyamido analogue of thiorphan, isa potent enkephalinase and aminopeptidase inhibitor [Fournie-Zaluski et al., 1984]. The /?..S7SS 14 diastereoisomeric mixture was twice as effective as thiorphan against enkephalinase

[Fournie-Zaluski et al.,1984].

H2NCH( CH2Ph )CH(OH)CONHCH(CH2CHMe2)CO2H

Bestatin

PhCH2CH(CH2SH)CONHCH2CO2H

Thiorphan

PhCH2CH(CH2SH)NHCOCH2CO2H

retro-Thiorphan

PhCH2CH(CH2CONHOH)CONHCH(Me)CO2H

Kelatorphan

Fig. 2.3 Peptidase inhibitors.

2.2.2 Conformational studies

Conformation studies of dynorphin A are problematic due to the linear nature of the peptide and the multitude of conformations it can adopt in solution [Maroun et al.,1981;

Zhou et al.,1986;Renugopalakrishman et al.,1988].Data from conformational studies using circular dichroism (CD), nuclear magnetic resonance spectroscopy (NMR including 15

COSY, NOESY, ROESY) and Fourier-transform infrared spectroscopy (FT-IR) techniques are important for revealing possible secondary structures of dynorphin. The results from CD studies of Dyn A(1-13) depended upon the experimental conditions (i.e. temperatures, solvents and additives) [Lancaster et al., 1991; Wu et al., 1986]. In aqueous solution, Dyn

A(1-13) adopted random coil and n-sheet conformations [Vaughn and Taylor, 1989]; when sodium dodecyl sulfate was added, the a-helix content increased [Maroun and Mattice,

1981], whereas in trifluorethanol (TFE) a minimal amount of a-helix was observed

[Lancaster et al., 1991]. Possible conformations of dynorphin were also studied by fluorescence energy transfer between the Tyr' and Trp14 in Dyn A(1-17) in aqueous solution.

There was no energy transfer between these two residues indicating the distance between them was at least 20 A; therefore the N- and C-terminus were not in close proximity

[Schiller, 1983]. Fluorescence energy transfer experiments indicated that the N-terminus of

[Trp4]Dyn A(1-13) was fully extended, whereas in the corresponding [Trp4]Leu- enkephalin analogue, it was folded [Schiller, 1983]. NMR and Raman spectroscopy studies showed dynorphin exists in a mixture of extended (3- pleated sheet and random coil structure in aqueous or methanolic solution [Kallick, 1993; Lancaster et al., 1991; Rapaka et al., 1987].

In a lipid environment, Schwyzer proposed that the N-terminal sequence of dynorphin adopts an a-helix structure when the peptide binds to lc opioid receptors embedded in the hydrophobic membrane [Schwyzer,1986]. 16

2.2.3Structure-activity relationships (SAR)

The heptadecapeptide dynorphin A displays limited selectivity for lc vs pi and 8 receptors. The C-terminal truncated peptide, dynorphin A(1-13), shows a similar receptor selectivity profile to the Dyn A(1-17) [Chavkin et al., 1982]. A truncated sequence Dyn A

(1-11) further shows 3 times higher lc selectivity towards both the pp and the 8 receptors than dynorphin A [Gairin et al., 1985].

Structure-activity relationship studies have indicated which residues are important for the biological activity of the peptide. These residues are normally preserved while other residues can be modified to obtain analogues with greater selectivity and/or stability.

Sequential removal of the C-terminal amino acids of Dyn A(1-13) showed that basic residues

Arg7 and Lys' are essential for high lc selectivity and/or potency [Chavkin and Goldstein,

1981]. Arg7 is important for the interaction with lc opioid receptors while Lys in position 11 is important for potency and lc selectivity. Incorporation of various residues in position 7-15 of Dyn A(1-17) suggests that Prom plays an important role in an amphilic 13-strand structure of Dyn A [Taylor, 1990]. D-Pro substitution at position 10 of Dyn A(1-11) and Dyn A(1-13) also improves lc receptor selectivity and potency [Lemaire et al., 1986; Gairin, et al., 1984 and

1988].D-Amino acid substitution. increases Dyn A(1-13) stability against peptidases

[Kawasaki et al., 1993]. Substitution of hydrophobic residues or D-amino acids for Ile' also enhances lc receptor selectivity [Gairin et al., 1984].

An alanine scan is a standard approach to identify important residues in a peptide.

Introduction of Ala into the Dyn A(1-13) sequence provided structure-activity relationship information for this peptide [Turcotte et al., 1984]. Substitution of Ala into positions 1 and 17

4 dramatically reduced Dyn A(1-13) activities. Thus, Tyr' and Phe 4 are crucial for potency

and receptor affinity of Dyn A(1-13). [A1a3]Dyn A(1-13) retained 35% of the potency in the

GPI and the MVD assay and 12% of binding activity compared to Dyn A(1-13). The linear

peptides [D-AlalDyn A(1-11)NH2 and [Ala 3]Dyn A(1-11)NH2 are very potent for lc opioid

receptors and very selective for x vs g and lc vs 8 receptors [Lung et al., 1995]. Replacement

of Arg6, Arg7, Arg9, and Lys" by Ala decreases the potency in both GPI and binding assays.

[Ala8]Dyn A(1-13), however, showed improved potency in both the MVD and binding

assays [Turcotte et al., 1984]. In rat, [Alas]Dyn A(1-13) was a potent analgesic, while in

guinea pig it was less potent than Dyn A(1-13) itself [Jhamandas et al., 1984]. The D-Ala8

analogue showed only a slight improvement in lc selectivity [Lemaire et al., 1986].

The Dyn A(1-13) analogue DAKLI, [Arg11,Arg13,Gly14]Dyn A(1-13)NH(CH2)5NH2

retains the same lc opioid receptor selectivity as the parent peptide [Goldstein et al., 1988].

DAKLI derivatives containing various labeling groups (e.g.'251, fluorescein or biotin) also

had selectivities comparable to Dyn A(1-13).

Conformationally constrained analogues of Dyn A have also been prepared and

studied for opioid activity and selectivity. A disulfide bridged analogue [D-Cys2,Cys5]Dyn

A(1-13) was the first synthesized cyclic Dyn A analogue and shows greater potency in the

GPI than the parent peptide Dyn A(1-13) [Schiller et al., 1982].This cyclic peptide, however, mainly interacts with 8. opioid receptors instead of with x opioid receptors

[Sherman et al., 1985]. Although introduction of a disulfide bridge between D-Cys8 and D-

Cys13 (or L-Cys13) in Dyn A(1-13)NH2 gives compounds with high affinity forx receptors, these cyclic peptides exhibit low x over m. receptor selectivity [Kawasaki et al., 1990]. The 18 cyclic disulfide-containing analogs cyclo[L-Pen5,Cys11]-, cyc/o[Cys5,Cys10]-, cyclo[Cys5,

Cys9]- and cyc/o[Cys4,Cys9,Arg ]Dyn A(1-11)NH2 possess high x and g opioid receptor affiities for the central receptor (guinea pig brain), but affect only weak potency at peripheral lc and g opioid receptor (guinea pig ileum (GPI)) [Kawasaki et al., 1993]. A lactam analog cyc/o[D-Orn2,Asp1Dyn A(1-8)NH2 shows greater affinity for g. opioid receptors than for

opioid receptors [Schiller et al., 1988]. Several other lactam analogues such as, cyclo[Orn5,

Asp8]-, cyclo[Orn5,Asp10]- and cyclo[Orn5,Asp13]Dyn A(1-13)NH2 are selective for p. opioid receptors and show weak activity in the GPI [Schiller et al., 1988].

2.3 Opioid receptors

Structure-activity relationships suggested that opiate drugs exert their effects through specific receptors. Opioid receptors were first thought to be a single class of homogeneous receptors. Martin and co-workers [Martin 1976; Gilbert 1976], however, proposed the multiple opioid receptor classification, and since then the idea has become generally accepted. In the original classification, three opioid receptor types were proposed which were named after the prototypic drugs used in the studies: mu (g) receptors for morphine, kappa (x) receptors for ketocyclazocine and sigma (a) receptors for N-allylnormetazocine

(SKF10047). These ligands exerted distinct pharmacological effects (Table 2.3) and were unable to replace each other in the suppression of withdrawal symptoms in chronically treated dogs.Sigma (a) receptors are now defined as non-opioid receptors, since pharmacological effects mediated through a receptors are not blocked by naloxone, an opioid antagonist normally used to define opioid receptors [Rees and Hunter, 1990; Fries, 1991]. 19

Delta (8) (from vas deferens) opioid receptors [Lord et al., 1977]were defined after the

discovery of enkephalins. They arepresent in high concentrations in the mouse vas

deferens, but functional 8 receptors are absent from guinea pig ileum (Table 2.4). Mu,kappa

and delta receptors are generally accepted asthe major opioidreceptor types. Two novel

opioid receptors, epsilon (e) and zeta () receptors, have been reported [Wusteret al., 1978;

Zagon et al., 1993].

Table 2.3 Opioid receptors and physiological effects.

mu (p.) kappa (K) delta (8)

analgesia analgesia analgesia constipation sedation hyperthermia respiratory depression diuresis respiratory depression physical dependence

Source: Iyenger, S. and Wood, P., The Opioid receptors. Pasternek, G.W. Ed.,Humana Press, New Jersey, 1988, pp 115-132.

Table 2.4 Distributions of opioid receptors in tissues used in smooth muscleassays.

Tissue 8

Guinea pig ileum (GPI) ++ Hamster vas deferens (HVD) Rabbit vas deferens (LVD) Mouse vas deferens (MVD) ++ Rat vas deferens (RVD) 20

2.3.1 Opioid receptor types and their ligands

2.3.1.1 Mu (p) receptors

Morphine and some opiates have limited selectivity for IA receptors, whereas

DAMGO ([D-Ala2,MePhe4,Gly-ol]enkephalin, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-

Ser-NH2) and DALDA (Tyr-D-Arg-Phe-Lys-NH2, K i6/K 11= 11,400) are highly selective IA agonists [Schiller et al., 1989; Erspamer, 1990]. Radio labeled DAMGO is commonly used for radioligand binding assays foril receptors. The 8 antagonist Tyr-D-Tic-Phe-PheNH2 (Tic

= tetrahydroisoquinoline-3-carboxylic acid) also shows some activity as a µ receptor agonist

[Schiller et al., 1992]. Naloxone and naltrexone are non-selective p-antagonists. Potent 11- antagonists are somatostatin analogues including cyclo [H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-

Cys-Thr-ol] (SMS-201995), cyc/o[H-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2] (CTP), and cyclo[H- D- Phe -Cys- Tyr- D- Trp- Orn -Thr- Pen -Thr -NH2] (CTOP) [Maurer et al., 1982;

Schiller, 1993]. Unlike SMS-201995, CTP and CTOP have high affinity for p.-receptors with reduced affinities for somatostatin receptors [Pe lton et al., 1985, 1986].The heptapeptide cyclo[H -D- Tic -Cys- Tyr- D- Trp- Orn -Thr- Pen -Thr -NH2] (TCTOP, Tic = 1,2,3,4- tetrahydro-isoquinoline-3-carboxylic acid) is the most selective p.-antagonist reported

(IC5PIC50P = 11,400) with a very low affinity for the somatostatin receptors [Kazmierski et al., 1988].

Mu receptors have been classified into two subtypes, and t2 by Pasternak [1994].

Both have some common characteristics including high affinities for morphine and similarities in regional distributions. However, they differ in binding profiles for a number of opioid ligands and opioid peptides (Table 2.5) [Pasternak, 1994]. The postulated pi sites 21

Table 2.5Tentative receptor classification.

Receptor Agonists Antagonists Actions

111 Morphine Naloxonazine Supraspinal analgesia Enkephalin

[12 Morphine CTOP Spinal analgesia Respiratory depression Inhibition of gastrointestinal transit [13 Morphine

K K1 Dynorphin A nor-BNI U-50,488 Spinal analgesia U-69,593 Diuresis Spiradoline K2 Pharmacology unknown NalBzOH Supraspinal analgesia Nalorphine

8 81 Enkephalin; tolerantDALCE Analgesia (spinal of conformations systems are more sensitive than supraspinal ones) 82 Met-enkephalin; nor-BNI not tolerant of conformations

Source: Pasternak, G.W. Basic Pharmacology of Opioids. In The Pharmacologic Basis of Anesthesiology, Bowdel, T.A., Houta, A.; Karasch, E.D., Eds; Churchil Livingstone: New Your, 1994; pp 19-36. 22 bind to morphine and enkephalins with high affinity, whereas the g2 sites have higher affinity for morphine than enkephalins. In addition, naloxonazine and naloxazone are reported to selectively inactivate 111 but not p2 subtypes [Pasternak, 1994]. Mu opioid receptors have been recently cloned [Chen et al., 1993; Wang, et al., 1993; Thompson, et al., 1993], and are a member of the G protein superfamily with high homology with 8 and lc (see section 2.3.3 below).

2.3.1.2 Kappa (x) receptors

Early studies of x opioid receptors were complicated by the lack of x-selective ligands. Benzomorphans (e.g. bremazocine and ethylketocyclazocine) were often used to study lc receptors, but, they have affinity for other opioid receptors as well and are not very selective for lc receptors. The development of the highly selective x-ligand, U-50,488{trans-

3,4-dichloro-N-methyl-N- [2-(1-pyrrolindinyl)cyclohexyl] -benzeneacetamide } [Von Voight- lander et al., 1983] (Fig. 2.4) and the discovery of high levels of lc binding sites in guinea pig ileum [Kosteliz et al., 1981] have greatly facilitated the study of ic-opioid receptors. The endogenous opioid peptide dynorphin A preferentially binds to lc opioid receptors, but it also shows affinity for both p and 8 opioid receptors. [Ala8]Dyn A(1-13) is four times more )(- selective over both the IA- and 8-receptors than Dyn A(1-13), while slight improvements in x-selectivity were found for [Trp8]Dyn A(1-13) and [D-Prow]Dyn A(1-13) [Lemaire et al.,

1986]. The shorter Dyn analogue [D-Pro 1°]Dyn A(1-11) shows an increase in x-selectivity

[Gairin et al., 1984], and 200- to >1,000-fold selectivity for lc over IA receptors was found in the N-monoalkylated [D-Prolc]Dyn A(1-11) derivatives [Choi et al., 1992]. 23

0 Ph` CH2CH3

(CH2)2Ph

Morphine Fentanyl

CH2 CH2

U-50,488 U-69,593

Fig. 2.4 Selected opioid receptor ligands.

Source: McKnight, A.T.; Rees, D.C.; Opioid Receptors and their Lignds. Neurotransmissions 1991, 7, 1-6. 24

CH2CH=CH2 I N N CH2--ci OH

NTI

TENA

nor-BN1

Fig. 2.4 (cont.) Selected opioid receptor ligands. 25

The bivalent ligand TENA is the first selective K opioid receptor antagonist

[Portoghese and Takemori, 1985]. TENA consists of two naltrexone-derived pharmaco- phores connected by a triethylene glycol spacer (Fig. 2.4).Binaltorphimine and nor- binaltorphimine (norBNI) contain a very short and rigid spacer (i.e. a pyrrole ring) are highly potent and selective K opioid receptor antagonists [Portoghese et al., 1987].

At least three K-opioid receptor subtypes, K1, K2 and ic3, have been proposed, K1

Receptors are defined by the highly K selective agonists, U-50,488 and U-69,593 (Fig. 2.4)

[Zukin et al., 1988]. K1-Binding sites bind with high affinity to arylacetamides U-50,488 and

U-69,593, while the K2-sites have lower affinities (Table 2.5) [Zukin et al., 1981]. Further classification of K1-receptors into Klaand Klb [Clark et al., 1989] has been proposed, but, their pharmacology remains unclear. K2-Receptors, which are U-50,488-insensitive, were described in rat brain using 3H-ethylketocyclazocine plus unlabeled IA, 8 and K1 ligands to block these receptors [Zukin et al., 1988]. Another U-50,488-insensitive binding site, the

K3-receptor, was proposed based upon the activity of naloxone benzylhydrazone [Gistrak et al., 1989; Paul et al., 1990]. ic,- Binding sites appear to predominate in calf striatum, where their density is twice that of either IA or 8 receptors [Clark et al., 1989]. K3-Receptors have also been proposed as the iso-receptor of IA receptors [Wollemann et al., 1992; Bardnard and

Simon, 1993]. The cloned K opioid receptors are proposed to be the K1 subtype [Raynor et al., 1994]. 26

2.3.1.3 Delta (6) receptors

Delta receptors were identified after the discovery of enkephalins [Lord et al., 1977].

Selective enkephalin derivatives for 6 receptors include the linear analogues DSLET and

DTLET ([D-Ser2,Leu5,Thr6]- and [D-TI112,Leu5,Tlu6]enkephalin) and the cyclic analogues

DPDPE ([D-Pen2,D-Pen5]enkephalin) and its derivatives [Schiller, 1993]. Derivatives of frog skin deltorphins, D-Met-deltorphin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2), [D-Ala2] deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) and [D-Ala2] deltorphin II (Tyr-D-Ala-

Phe-Glu-Val-Val-Gly-NH2), also show high affinity and selectivity for 6 binding sites

[Erspamer, 1992]. The N,N-dialkylated enkephalin derivative ICI 174,864 (N,N-diallyl-Tyr-

Aib-Aib-Phe-Leu) (Aib = aminoisobutyric acid) [Cotton et al., 1984] shows antagonism for oreceptors, whereas TIPP (Tyr-Tic-Phe-Phe-OH) possesses higher potency than ICI 174,864

[Schiller et al., 1992]. Recently developed non-peptide 6-selective agonists are BW373U86

[Chang et al., 1993] and SIOM (7-spiroindino-oxymorphone) [Portoghese et al., 1993], while nonpeptidic 6 antagonists are naltrindole and the naltrindole derivatives naltriben

[Portoghese et al., 1991] and BNTX (7-benzidenenaltrexone) [Portoghese et al., 1992].

Two subtypes of 6 opioid receptors, 81 and 82, have been proposed using agonists

(DPDPE for 61, and [D-Ala2]deltorphin II for 62) [Mattia et al., 1991 and 1992; Jian et al.,

1991], antagonists (BNTX for 6, [Portoghese et al., 1992], and naltriben for 82 [Portoghese et al., 1992]) and affinity labels (DALCE (Tyr -D- Ala -Gly- Phe - Leu- CysOH) for 61 [Bowen et al., 1987], and naltrindole isothiocyanate for 62 [Portoghese et al., 1990]). The cloned 6 receptor has similar a pharmacological profile to 62 sites [Raynor et al., 1994]. 27

2.3.1.4 Epsilon (E) receptors

Epsilon (E) receptors in rat vas deferens (RVD) have been proposed by Schulz [1981] and Gil lan [1981]. Met-enkephalin and Leu-enkephalin have low affinities for these binding sites (IC50> 100 nM), whereas 13-endorphin show higher affinity (IC50 = 74-90 nM) [Wuster et al., 1978]. It was suggested that the 13-endorphin (1-31) sequence was required for binding to E-receptors. In addition, when RVD was made highly tolerant to etorphine, it was only moderately tolerant to I3-endorphin [Wuster et al., 1978]. The high degree of tolerance to etorphine was thought to be due to a small population of 1.t. receptors in the RVD. The presence of E-receptors in other tissues is inconclusive. The benzomorphan binding sites in rat brain may also be the E-receptors [Chang et al., 1984]. Nock and co-workers (1990), however, have reported that the binding sites, previously characterized by them as K2- receptors, may be E-receptors.

2.3.1.5 Zeta (() receptors

Zeta (0 receptors have recently been postulated based on studies in S20Y murine neuroblastoma cells. Some opioids showed growth inhibitory effects in neuroblastomas and in cell cultures derived from neural tumors which have been suggested to be mediated via zeta bindings sites [Zagon et al., 1989]. Zeta (c) receptors have been proposed as opioid growth factor receptors [Zagon et al., 1991 and 1993].Most peptides derived from proenkephalin A and some from prodynorphin have high affinities for zeta receptors, while other alkaloids and selective ligands for 1.1, lc, 8, a and E receptors have low affinity for zeta binding sites [Zagon et al., 1989]. 28

2.3.2 Isolation and purification of opioid receptors

Studies of opioid receptor structure have involved isolation and purification of the receptors. One approach involves a two-step procedure to obtain a specific opioid binding site, first labeling receptors with affinity labels or crosslinking, followed by isolation and purification of the receptor molecules [Simon and Hiller, 1994]. [3H]Etorphine was used to label opioid binding sites from rat brain [Simon et al., 1975], and the labeled complex subsequently solubilized by detergents such as digitonin, lysophosphatidylcholine and {3-

[(3-chloamidopropyl) dimethylamino]-1-propanesulfonatel (CHAPS). CHAPS is a zwitter- ionic derivative of deoxycholate which has an advantage over digitonin in that agonist binding is readily restored by a simple precipitation of the solubilized receptor protein with polyethylene glycol (PEG) [Simon and Gioannini, 1993]. The [3H]etorphine-labeled receptor was unstable under conditions for further purification although it was useful for receptor characterization. The development of FIT (fentanylisothiocyanate) and superFlT (methyl- fentanylisothio-cyanate) have enhanced opioid receptor purification [Simonds et al., 1985].

[3H]FIT was successfully utilized to label opioid receptors on the neuroblastoma glioma hybrid NG108-15 cell line which contains only e opioid receptors. The labeled receptors were solubilized and purified to homogeneity using chromatography on wheat germ agglutinin (WGA)-agarose followed by chromatography on a column of immobilized antibodies to FIT. The isolated receptors were 8 binding sites with a molecular weight of

58 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

An affinity crosslinking technique has been utilized to separate .t- and 8-binding sites using human [125I](3- endorphin ((3-Endh) [Toogood et al., 1986]. Tyr 27 was iodinated instead 29 of Tyr', since the iodination of Tyr' significantly decreases endorphin's binding affinity.

[125I-Tyr 27] -P -Endh bound to II- and 8-receptors with negligible affinity for K-receptors, and thus both and 8-receptors could be studied in the same tissue. The membrane-bound opioid receptors were labeled with [125I]P-Endh and washed to remove free or loosely bound ligands. Then they were crosslinked with bis[2-(succinimidoxycarbonyloxy)ethyl] sulfonate

(BSCOES), which forms linkages between amino groups on the ligand and receptor. The crosslinked membranes were solubilized in SDS, separated by SDS-PAGE and subjected to autoradiography [Howard et al., 1985]. A band of Mr = 65 kDa was present in all tissues containing 11. receptors, while a band of Mr = 53 kDa existed only in 8-receptor containing tissues. To confirm the identity of these bands, crosslinking experiments were performed in the presence of ligands highly selective forµ or 8 receptors [Howard et al., 1986]. The radioactive band at 65 kDa disappeared when the binding of '25I-Tyr22-P-Endh was carried out in the presence of DAMGO (a highly g-selective ligand), while the band at 53 kDa was eliminated when the binding was carried out in the presence of DPDPE (a highly 8-selective ligand).

Kappa-opioid receptors were similarly isolated from guinea pig cerebellum membrane by affinity crosslinking technique using labeled [D-Prow]Dyn A(1-11) [Yao et al., 1989]. Bands at Mr = 55 kDa and K. = 35 kDa were obtained. The 55 kDa band was completely eliminated in the presence of U-50,488 (a highly K-selective ligand), while the

35 kDa band was reduced in intensity but did not completely disappear, even when using higher concentrations of K ligands. The band at 55 kDa was proposed to be high-affinity

K-binding sites, whereas the band at 35 kDa was thought to be low-affinity K-binding sites 30 or a combination of x-binding sites and non-opioid binding sites [Yao et al., 1989]. Itzhak and co-workers [1984] separated K-binding sites from other receptors using a one-step procedure. Guinea pig brain membranes were solubilized with digitonin and layered on top of a sucrose density gradient devoid of sodium and containing only a low concentration of digitonin (0.02%). The mixture was centrifuged, fractions were collected and assayed for binding activities with [3H]bremazocine.Two peaks were observed, the first one corresponded to K-binding sites and the second one was a combination of- and 8- binding sites.

Active kappa receptors had been purified by different techniques. Simon and co- workers [1987] used affinity chromatography on a matrix of DADLE coupled to epoxy- activated Sepharose 4B, followed by gel permeation chromatography on Sepharose 6B to purify x-binding sites from frog brain (Rana esculenta), whereas Mollereau and co-workers

[1988] purified x-receptors from frog brain (Rana ridibunda) using affinity chromatography on a dynorphin-agarose column.

2.3.3 Molecular biology and cloned opioid receptors

The first opioid receptors successfully cloned were 6 receptors from NG 108-15 cells reported in 1992 [Kieffer et al., 1992; Evans et al., 1992]. Since then u, ic and 8-receptors have been cloned from rat, guinea pig, mouse, and human brain [Yasuda et al., 1993; Chen

et al., 1993; Li et al., 1993; Fukuda et al., 1993; Nishi et al 1993; Abood et al., 1994;

Zastawny et al., 1994]. Amino acid sequences of all three cloned receptors are highly homologous (Fig. 2.5), but their pharmacological effects profiles are distinct (Table 2.6) 31

KOPRDI S ARAE LSD PaSAIPIIAZAN OWPGASO mOPRX1 - IDIFRG POP.T.S.SPSAOLLNSSI1APO P-- WAS 0 NOSY SAGIOLSSO ;OMNI DS T CaDllijA0ASS- LIOLSI DONO aBCOLNAT.QLG NOWLCPOS9 P na WPRM(SA,A---.LPLEIJAITALYS LGNV7377FLGJIVRYTXLQKTATNITIFNLALADALAT 100 mOrItX11,LAJ H I KAI IPVIIrTiAaY SV VGNSLVNAVIDAYTKNKTATNITIFNLALADALM 110 rOPRN1 T Ga P...M V TUT I .110.1._2AYWYV.YTKMKTATNITTFNLALADALAT II, no mOPSD1TLPFOS KYLM. WPFGELLCI.SIDYYNNITSIFTLTNNSVDRYIAVCMPVKALDFR 140 mOPRX1 Ta PFOS VYLKN WPFGDVLCKIVISIDISVORYIAVCRPVKALDPR 170 rOPRN1TLPFOSVN C TI CT 179 T244 mOPRD1TP XAK L INICI CVMS D©:. - -VVCHLOFPSa-- TVTKIC214 KOMITPL AKI N C CISAT LGG V EDVDVRECSLAFIDDEY LPNRIC229 rOPRM1 V N ISAILaVaFTEI Y CaS-- Ds.TljTWSMa--T £NLL 235 115 1M6 rflOPRDIVF FAF VPILIITYCYGLMLLRLRSVRLLSGSK EKDRSLRRITRMVLVV GAFV C W AP 270 mOPRX1VFVFAF IPaLIIMVCYMLMILRLKSVR,I,LSGSWEKDRNLRRITEDVLVVVAVFI CWTP 299 rOPRM1 NP/LIITYCY(ILMTT RI KSVPIVILSCSK,',,MRNt RR TTRMVLVVVAVFIVCWTP 29S ITV rOPRD1 WITVOMNRRIPLVVAALIBLCIALGYWNSSLNPVLYAFLDENFKPCFROLR T P 334 em)PRK1 EALGSTSNSTA-ALSRYTFCIALGYTNSALNPVLYAFLDENFKRCFRD FillI341 rOPRM1 I K.LL.ITM-PETFOTVM WHFCI;LGYTNEICILNPVLYAFLGENFKPCF&EFri T T354 froOPRO1 C CPG LA ATTE1RVTACTPSa RPGGGAAA 372 mOPRK1 K MnM R Q TN TVQOPA N R D V GNNXIIIV 310 rOPRM1 SSTS C NST NI-HPTANTVaRTNMOLENLEAETALP 3911

Fig. 2.5 Comparison of the amino acid sequences of 8, lc, and [i opioid receptors. The sequence of mouse 8 (m0PRD1), mouse x (mOPRK 1) and rat µ (rOPRM1) opioid receptors are shown using the single-letter abbreviations for the amino acids. Residues that are identical in two or more of the proteins are boxed. Gaps introduced to generate this alignment are represented by dashes. The seven predicted transmembrane domains are noted (TM1-TM7) and are shown by shading. The potential sites forN-linked glycosylation in the extracellular amino domains of these proteins are: mouse 8 receptor, residues 18 and 33; mouse x receptor, residues 25 and 39; rat u receptor, residues 9, 31, 38, 46 and 53.

Source: Reisine, T.; Bell, G.I. Molecular Biology of Opioid Receptors. TINS 1993, 16, 506-510. 32

Table 2.6 Pharmacology of the cloned opioid receptors.

Ligand K, (iim)

Morphine >1000 163 1.4 Naloxone 95 16 3.9 DAMGO >1000 >1000 0.87 DADLE 2.8 514 6.4 DPDPE 7.6 >1000 >1000 Dynorphin A(1-17) 45 5.48 120 U-50,488 >1000 1.4 >1000 U-69,593 >1000 2.3 >1000

Source: Raynor, K; Kong, H.; Chen, Y.; Yasuda, K.; Yu, L.; Bell, G.I.; Reisine, T. Pharmacological Characterization of the Cloned lc-, 8- and .i-Opioid Receptors. Mol. Pharmacol. 1994, 45, 330-334. 33

[Reisine and Bell, 1993]. The cloned mouse 8 receptor contains 372 amino acidproteins

with 97% identity with rat 8 receptors. Rat11 opioid receptors contain 398 amino acids with

58% and 57% identity and 66% and 68% similarity to thesequences of mouse 8 and ic

receptors, respectively. Rat lc receptors, which consist of 380 amino acids, have 61% amino

acid identity and 70% similarity to thesequence of the mouse 8 receptors. Human [t, 8, and

K-receptors have been recently cloned [Wang et al., 1994; Knapp et al., 1994; Simoninet al.,

1994; Yasuda et al., 1994].

Opioids receptors are guanine nucleotide-binding regulatory proteins(G-proteins)

coupled receptors [Wollemann, 1990]. The receptor protein consists of extracellularregions

(the N-terminus and extracellular loops), seven putative transmembrane (TM)regions, and intracellular regions (the C-terminus and intracellular loops).The highest sequence homology among rat t, rat lc and mouse 8 receptors exist in TM2, TM3 and TM7, and highly preserved sequences are also found in the 2nd and the 3rd intracellular loops (Fig.2.6) [Chen et al., 1993]. The sequences of TM2 and TM3 each contain a conserved Asp residue which may interact with protonated amine groups of opioid ligands. Such an acidic residue has been proposed to mediate sodium inhibition of agonist binding in thea2-adrenergic and somatostatin receptors [Horstman et al., 1990]. The 3rd intracellular loop is required forthe interactions with G-proteins. The 2nd and 3rd extracellular loops and TM1 and TM4-6are less conserved, while the extracellular N-terminus and the intracellular C-terminus havethe greatest diversities. The 1st and 2nd extracellular loops contain Cys residues whichmay be involved in a disulfide linkage.The N-terminal extracellular domain contains several potential sites for N-linked glycosylation, and the intracellular loops and the C-terminus 34

Fig. 2.6 Schematic diagram of protein structure for, and similarity among, the three opioid receptors.

Source: Chen, Y.; Mestek, A.; Liu, J. Molecular Cloning of a Rat lc Opioid Receptor Reveals Sequence Similarities to theµ and 8 Opioid Receptors. Biochem. J. 1993, 295, 625-628. 35

contain several sites for phosphorylation by cAMP-dependent protein kinases and protein kinase C.

2.4 Affinity label-containing opioids

Affinity labels are ligands that have a very high affinity (i.e. low degree of dissociation) for receptors and bind to target sites in a nonequilibrium manner [Takemori and

Portoghese, 1985]. The interactions may or may not involve covalent bonds. Affinity labels that bind covalently to receptors are useful in opioid receptor labeling experiments for differentiating various opioid receptor types and subtypes. Many selective non-peptide and peptide affinity labels, both chemical affinity and photoaffinity labels, have been developed.

Affinity label compounds bind to receptors in a two step recognition process (Fig. 2.7).

Reversible binding in the first step determines receptor affinity, while covalent bond formation in the second step depends on proper orientations of the electrophilegroup and the receptor-based nucleophile. Chemical affinity labels contain an electrophile which interacts with a nucleophile on the receptors. Selectivities of chemical affinity labelsare dependent upon the affinity of the ligand for the receptors, location of the electrophilic group on the ligand, and the chemical selectivity of the electrophile.Photoaffinity labels require a photolysis step (e.g. irradiation at 254 nm) yielding a photoactivated intermediate which could subsequently bind to receptors (Fig. 2.8). Photoaffinity labels have advantages over chemical labels in that they are stable in aqueous solution until activation and they do not require nucleophile group for reaction with the binding sites [Alex et al., 1992 ]. However, photoaffinity labels cannot be used in vivo and photoactivation by short-wavelength 36

RCOOI

RECEPTOR TYPE A

NO REACTION

RECEPTOR TYPE B

Atepsomno NO REACTION

RECFPTOR TYPE C

Fig. 2.7 A schematic illustration of the principle of recognition amplification in the covalent binding of receptor type A by an affinity label containing a group-selective electrophile X. Note that receptor types A-C have similar topographic features that lead to reversible binding (1 °degree recognition). However, the receptor types differ with respect to the reactivity (G' versus G2 in A and B) and location (G1 in A and C) of nucleophiles. Only in A is the nucleophile G' reactive with respect to X and within covalent binding distance(2 °degree recognition).

Source: Takemori, A.E.; Portoghese, P.S. Affinity Labels for Opioid Receptors. Ann. Rev. Pharmacol. Toxicol. 1985, 25, 193-223. 37

Chemical Affinity Labeling

NON-SPECIFIC REACTION

BINDING

SPECIFIC COVALENT ATTACHMENT

Photoaffinity Labeling

BINDING PHOTOLYSIS REACTION /3

Fig. 2.8 Chemical affinity labeling versus photoaffinity labeling.

Source: Nielsen P.E. Photochemical Probes in Biochemistry. J. Biol. Chem. 1962, 237, 3006-3015. 38 ultraviolet light causes losses of opioid receptor-binding capacity [Glasel and Venn, 1981;

Smolarsky and Koshland, 1980].

2.4.1 Non-peptide affinity labels

The first non-peptide potential affinity label agonistswere N-2-bromoalkyl substituted benzomorphans [May et al., 1968]. Their analgesic effects, however, have not been determined. More recently, a number of non-peptide affinity labels for various opioid receptors have been developed [Takemori and Portoghese, 1985; Zimmerman and Leander,

1990]. Selective affinity label agonists include the isothiocyanate derivatives of fentanyl

(FIT and superFIT), U50,488 (UPHIT) and etonitazene (BIT) and the fumaramido derivative of endoethenotetra-hydrooripavine (FAO) (Fig. 2.9) [Rice et al., 1983; Burke et al., 1986].

FIT, superFIT and FAO are highly selective for 8 opioid receptors, whereas BIT is selective for p, opioid receptors. A nitrogen mustard derivative of the opioid antagonist naltrexone,

P-chlornaltrexamine ((3 -CNA) (Fig.2.10), is a non-selective irreversible opioid antagonist which can block all three opioid receptors [Portoghese et al., 1978 and 1979], whereas

13-funaltrexamine (P-FNA) is selective for pt. opioid receptors [Portoghese et al., 1980] and naltrindole isothiocyanate(Fig. 2.10) is an irreversible 8-antagonist [Portoghese et al.,

1990], while the isothiocyanate derivative of U50,488 is an irreversible x-receptor blocker

[de Costa et al., 1989 and 1990]. 39

C61-15, COEt C6H5, COEt \N/ \N/ \ \/-\/CH3 CH2CH2C6H4(p-NCS) CH2CH2C6H4(p-NCS)

FIT SUPERFIT

Eta

NCS

UPHIT BIT

H NHCOC=CCOOMe

HO OMe

FAO

Fig. 2.9 Affinity label agonists. 40

H I NHCOC=CCOOMe HO HO I H

(3 F NA 13CNA

NCS

NT II

Fig. 2.10 Affinity label antagonists. 41 2.4.2 Peptide affinity labels

Reported peptide-based electrophilic affinity labels (chemical affinity labels)are

derivatives of enkephalins and include chloromethyl ketone derivatives DALECK (H-Tyr-D-

Ala-Gly-Phe-Leu-CH2C1) and DAMK (H-Tyr-D-Ala-Gly-MePhe-CH 2C1) [Bowen, et al.,

1987]. DALECK binds preferentially to g-receptors (IC5PIC5OR = 14.3) [Venn and

Barnard, 1981; Sziks et al., 1987; Newman and Barnard, 1984], and DAMK shows higher

selectivity for 1i-receptors (IC50'/IC5og = 40) [Benhye et al, 1987]. DALCE covalently binds to 8-receptors by forming a disulfide bridge between Cys6 and a sulfhydryl groupon the binding site and shows moderate 8-selectivity (IC50 /IC50 g= 0.0745) [Bowen et al, 1987].

Photoaffinity labels are ligands containing a photoreactive moiety suchas an azido or a nitrophenylazido group [Alex et al., 1992 ]. These groups can selectively and covalently label receptors providing high ratios of specific to non-specific bindings.The azide derivatives of DAMGO (H-Tyr-D-Ala-Gly-MePhe(pN3)-Gly-ol), TAPP (H-Tyr-D-Ala-Phe-

Phe(pN3)-NH2) and CTP (H-D-Phe-Cys-Phe(pN3)-D-Trp-Lys-Thr-Lys-Thr-Pen-Thr-NH2) show high 1i-receptor selectivities (IC506/1C50P = 136, 107 and 408, respectively) [Garbay-

Jaureguiberry et al., 1984;Schiller et al.1989; Landis et al., 1989],whereas the incorporation of p-benzoylphenylalanine (Bpa) in a morphinceptin analoggave a compound

(H-Tyr-Pro-BpaNH2) which was less u selective (IC 508/IC 5e = 13.7) [Herblin et al., 1987].

Photoaffinity labels for 8-receptors have been prepared by similar strategies providing the azide derivatives of DTLET (H-Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr-OH, IC 506/IC 5e= 0.0194)

[Garbay-Jaureguiberry et al., 1984] and DPDPE (H-Tyr-D-Pen-Gly-Phe(pN3)-D-Pen-OH,

IC,PIC50[1= 0.00913) [Landis et al., 1989]. 42

CHAPTER 3

LITERATURE REVIEW: SOLID PHASE PEPTIDE SYNTHESIS

3.1 Basic synthetic strategies

Chemical peptide syntheses can be achieved by two strategies,solution

(conventional) and solid phase syntheses.Solution synthesis requires optimization of reaction conditions and purification of intermediates following each step. These can be time-

consuming and result in physical losses during synthesis and purification. In addition, the

unpredictable solubility and crystallization characteristics of intermediates can be problematic.

Solid phase peptide synthesis (SPPS) was first developed by Merrifield [1963].

SPPS differs from solution synthesis in that a peptide chain is attached to an insoluble polymeric support and the anchored peptide is subsequently extended from the C-terminus toward the N-termius by cycles of deprotections and couplings (Fig. 3.1) [Grant, 1992]. The

C-terminal amino acid, containing a "temporary" protecting group (T) at the Na-amine and when needed a "permanent" protecting group on the side chain, is attached to the insoluble polymeric support (R) bearing reactive groups (X) by either an ester linkage for a peptide acid or an amide bond for a peptide amide.In each repetitive cycle, the "temporary" protecting group is removed to free the N"-amine and the next incoming protected amino acid is then coupled to the amine via the activated carboxyl group. Excess soluble reagents are required to drive the reactions to completion and to obtain a homogenous product. At the end of the synthesis surplus amino acids, soluble reagents and side products are removed 43

Functionaluanon

Anchoring Jclile 0

0 lir Deprotection

Repetitive Handle I 0 cycle 0 H70 ,; Coupling

Cleavage

Final deprotecnon

Handle

Fig. 3.1 Stepwise solid-phase synthesis of linear peptide. R, insoluble polymeric support; AA'. AA: NA" amino acid residues numbered starting from C-terminus; T, "temporary" protection; 0, permanent" protection; u, free carboxyl; ED, free amino group.

Source: Fields, G.B.; Tian, Z.; Barany, G. Principles and Solid-Phase Peptide Synthesis. In Synthetic Peptides. A User's Guide. Grant, G.A., Ed.; W.H. Freeman and Company: New York, 1992, pp 77-183. 44

simply by filtration and washing without mechanical losses of the attached peptides. After the peptide chain assembly has been completed, the crude peptide is cleaved from the

support, concurrent with removal of the permanent protecting groups, and purified to give the pure desired peptide. The advantages of SPPS over solution synthesis are simplicity, speed and automation.

3.2 Protecting groups

3.2.1 1V-amino protecting groups

In classical solid phase peptide synthesis, Nu-amine groups are protected by the acid labile Na-tert-butyloxycarbonyl (Boc) group (Fig. 3.2) [Merrifield, 1963]. The Boc groupisintroducedinto amino acids using di-tert-butyldicarbonate or 2-tert- butyloxycarbonyl-oximino-2-phenylacetonitrile (Boc-ON) in aqueous 1,4-dioxane containing

NaOH or triethyl-amine (TEA) [Bodanszky and Bodanszky, 1984]. It is stable to alkali and nucleophiles but readily removed by inorganic and organic acids (e.g. 20-50% trifluoroacetic acid (TFA) in dichloromethane (DCM) or 4 N HC1 in 1,4-dioxane) [Barany and Merrifield, 1979]. A neutralization step using TEA or N,N- diisopropylethylamine

(DIEA) is usually required following Boc deprotection. Coupling amino acids without prior neutralization is feasible in the presence of DIEA or N-methylmorpholine (NMM) (in situ neutralization) [Suzuki, 1975; Schnolzer et al., 1992]. Permanent side chain protecting groups in Boc chemistry are benzyl-type blocking groups. Strong acids either inorganic or organic acids (e.g. liquid anhydrous hydrogen fluoride (HF) at 0°C or trifluoromethane- 45

2,6-C12Bil cH2

PAM Boc c-o O CH2 O C 12 CH3 -=O ±NH-CH-C-NH- CH2NH-6-1C 0 fCH2 CH2CNHCH2< >-0 011 TFA HF

Fig. 3.2 -Merrifield- protection scheme for solid-phase synthesis, based on graduated acidolysis. Temporary N'-amino protection is provided by the Boc group, removed at each step by the moderately strong acid TFA. Permanent Bzl-based and cHex side-chain protecting groups. and the PAM linkage, are then cleaved simultaneously by HF or other strong acids, with a free peptide acid being formed in high yield.

Source: Fields. G.B.; Tian. Z.; Barany, G. Principles and Solid-Phase Peptide Synthesis. In Synthetic Peptides. A User's Guide. Grant, G.A., Ed.; W.H. Freeman and Company: New York. 1992, pp 77-183. 46

sulfonic acid (TFMSA) at 25 °C) in the presence of suitable scavengers are utilized for final

deprotection.

An alternative N"-amine protection strategy involves the use of the Na-9-fluorenylmethoxycarbonyl (Fmoc) group (Fig. 3.3). The Fmoc protecting group was developed by

Carpino and Han [1972] to avoid the repetitive treatment of peptides with acids which can

cause loss of peptides from the support and side reactions during the final cleavage. Several reagents including fluorenylmethyl succinimidyl carbonate (Fmoc-OSu) or Fmoc -Cl in a partially aqueous/organic mixture in the presence of base, and [4-(9-fluorenylmethyloxy- carbonyloxy)phenyl]dimethylsulfoniu methyl sulfate (Fmoc-ODSP) in H2O with Na2CO3 or

TEA [Azuse et al., 1989] can be used to introduce an Fmoc group onto the N"-amine. The

Fmoc -Cl reagent, however, can cause formation of the undesired Fmoc dipeptide (2-20%)

[Milton et al., 1987]. The Fmoc group is stable to acids but susceptible to secondary amines such as piperidine [Fields and Fields, 1991] and diethylamine. Piperidine in dimethylformamide (20-55%) is widely used because the reactive dibenzofulvene intermediate generated from proton abstraction at position 9 of the fluorene ring system and 13-elimination is readily trapped by excess secondary amine to form a stable harmless tertiary amine adduct

(Fig. 3.4) [Carpino and Han, 1972]. A non-nucleophilic base 1,8-diazabicyclo[5.4.0]undec-

7-ene (DBU) can be used for removal of Fmoc in continuous flow synthesis. DBU does not form adducts with the dibenzofulvene intermediate, and therefore it must be washed from the peptide resin immediately [Wade et al., 1991]. Permanent side chain protecting groups in Fmoc chemistry are tert-butyl-type blocking groups which can be removed by TFA in the final deprotection. 47

cH3 CH, - C - CH3:err-butyl I Fmoc .113113 CH3 - C CH3 F 0 I 1 0 HMP/PAB C =0 0 6H2 0 CH2 ,H CHx 0- Z -NH- CH- C 0 1 `...../ ._. -NH- CH2 -NH- &I-- C - OICH2 0- CH2CH2 -NH- CH2 i414 6 6 TFA

Fig. 3.3 A mild two-dimensional orthogonal protectionscheme for solid phase synthesis. Temporary Na-amino protection is provided by theFmoc group, removed by the indicated base-catalyzed 13-elimination mechanism.Permanent tBu-base side chain protecting groups and the HMP/PABester linkage are both cleaved by treatment with TFA to yield the free peptide acid. A third dimensionof orthogonality may be added with an acid-stable, photolabile anchoring linkage.

Source: Fields, G.B.; Tian, Z.; Barany, G. Principlesand Solid-Phase Peptide Synthesis. InSynthetic Peptides, A User's Guide.Grant, G.A., Ed.; W.H. Freeman and Company: New York, 1992, pp 77-183. 48

HND

9 -0-C-NHR

0 - 0*--C -NHR CO2+ R-NH2

CH-Ci-iz-ND

Fig. 3.4 Fmoc deprotection.

Source: Bodanszky. M. Peptide Chemistry, Springer-Verlag, Berlin, 1988,p 78. 49

3.2.2 Side chain protecting groups

A key to the success of solid phase peptide synthesis is dependent on the availability

of appropriate side chain protecting groups for different amino acids. Certain side chains

of amino acids such as the E-amino group of Lys, the side chain carboxyls of Asp and Glu

and the mercapto group of Cys require side chain protection. The hydroxylgroups of Ser,

Tyr, and Thr, and the basic groups of Arg and His are usually protected. Choices of side

chain protecting groups should be compatible with the Na-amine protectinggroups (Boc or

Fmoc) and should minimize side reactions which may occur during cycles of deprotection/coupling and during final cleavage of the peptide from the resin.

Benzyl-type and tert-butyl-type derivatives are the two commonly used side chain protecting groups for Boc and Fmoc SPPS, respectively. (Fig. 3.5 and 3.6). The E-amino group of Lys is protected by the 2-chlorobenzyloxycarbonyl (2-C1Z) [Erickson and

Merrifield, 1973] or Fmoc group in Boc-based syntheses and, reciprocally by the Bocgroup for Fmoc chemistry. An allyloxycarbonyl (Aloc) protectinggroup is compatible with both

Boc-and Fmoc-base syntheses. Aloc is insensitive to acids and bases andcan be removed by a palladium (0) catalyst [Lyttle and Hudson, 1992]. The highly basic guanidino side chain of Arg is usually protected by the 4-toluenesulfonyl (Tos) [Tam and Merrifield, 1987] or mesitylene-2-sulfonyl (Mts) [Yajima et al., 1988] in Boc SPPS, and either 4-methoxy-

2 ,3 ,6-trimethylbenzene-sulfonyl (Mtr) [Fujino, 1981], 2,2 ,5 ,7 ,8-pentamethylchroman-6- sulfonyl (Pmc) [Ramage and Green, 1987] or 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl (Pbf) [Carpino, et al., 1993; Fields and Fields, 1993] in Fmoc SPPS. Pbf ismore readily removed than the corresponding Pmc by factors of 1.2-1.4 for TFA/H20 (95/5; 80/20) 50

CH2 CH2

2,6-C12 BzI BzI

O O II II C H20 C cH2oc

2-BrZ 2 -CIZ

CH3 CH3 SO2

C H3

Mts Tos

Fmoc

Fig. 3.5 Side chain protecting groups used in Boc SPPS. 51

CHs CHs 0 CHs C- CHs C-0 IIC

1 I CH3 CH3

t-Bu Boc

CH3O

Tmob

Trt

Mtr

Fig. 3.6 Side chain protecting groups used in Fmoc SPPS. 52

at temperatures between ambient and 37 °C [Carpino et al., 1993]. The side chain carboxyls

of Boc-Asp and Boc-Glu are usually protected by the benzyl ester (OBzl), while tert-butyl

esters are used for Fmoc-Asp and Fmoc-Glu. The aspartamide rearrangement may occur

with Boc- Asp(OBzl) in a susceptible sequence [Bodanszky et al., 1978; Nicolas et al., 1989].

The 2-adamantyl (0-2-Ada) [Okada and Iguchi, 1988], cyclohexyl (Oc Hex) [Tam et al.,

1988] or 9-fluorenylmethyl (OFm) [Albericio et al., 1990] ester are recommended to

minimize this side reaction.

The mercapto group of Boc-Cys canbe protected by 4-methyl-benzyl (Meb)

[Erickson and Merrifield, 1973], and 3-nitro-2-pyridinesulfenyl (Npys) [Rosen et al., 1990].

The acetamidomethyl (Acm) [McDurdy, 1989] and trimethylacetamido-methyl (Tacm) [Kiso et al., 1990] groups can be used for both Boc-Cys and Fmoc-Cys. The side chain hydroxyls of Ser, Tyr and Tyr are often protected as Bzl and t-Bu in Boc and Fmoc chemistry, respectively. The 2,6-dichlorobenzyl (2,6-C12Bz1) [Erickson and Merrifield, 1973b] and 2- bromobenzyloxy-carbonyl (2-BrZ) are preferred to Bzl for Boc-Tyr since the Bzl protecting group can yield in a side product due to the migration of Bzl to the 3-position of the phenol ring.

3.3 Insoluble supports

Polymeric supports of choice must have sufficient mechanical stability and appropriate physicochemical properties which are compatible with solid phase peptide synthesis. Different insoluble supports are compared in Fig. 3.7. The most widely used solid support is a polystyrene polymer cross-linked with 1% 1,3-divinylbenzene (substitution 53 C

--- CH --- ,CH CH2

Polystyrene

ACH2CH20) CH

CH CH2 --CH(CH--C1-42)--

Polyethylene glycol polystyrene

CONAAe2 CONMe2

CH CH2 CH CH2 CH CH2

CONH

(CH2)3

CONH

--CH2 CH--CH2 CH CH, -- S parrow

Fig. 3.7 Insoluble supports.

Source: Fields, G.B.; Tian, Z.; Barany, G. Principles and Solid-Phase Peptide Synthesis. In Synthetic Peptides, A User's Guide. Grant, G.A., Ed.; W.H. Freeman and Company: New York, 1992, pp 77-183. 54 value 0.2-1.0 mmol/g) [Merrifield, 1963]. The resin swells 2.5-6.2 fold in volume in DCM and DMF allowing rapid penetration of solvents and reagents into the resin beads and thus facilitating chemical reactions [Sarin et al., 1980].

Copolymerized dimethylacrylamide, N ,A P -bisacryloethylenediamine, and acryloyl- sarcosine methyl ester resins (commercially known as Pepsyn, substitution value 0.3 mmol/g) [Atherton and Sheppard, 1989] were developed in order to increase the compatibility of the peptide polarity and the insoluble support. Unlike polystyrene resins, polyamide resins also swell sufficiently in polar aprotic solvents.Pepsyn K is an encapsulated polydimethyl-acrylamide resin with a Kieselguhr matrix which provides rigidity to the support (substitution value 0.1-0.2 mmol/g) [Atherton et al., 1981]. Polyhipe is a derivatized polyamide support with high porosity, permeability and physical stability

(substitution value 0.3-1.8 mmol/g) [Small and Sherrington, 1989]. Both Pepsyn K and

Polyhipe withstand high back pressures, and thus they are suitable for continuous flow synthesis. The Sparrow resin is a cross-linked bead of dimethylacrylamide (substitution value 0.65-0.7 mmol/g) which has the advantage of being swollen in aqueous buffers as well as the polar organic solvents used in peptide synthesis [Sparrow et al., 1990; Kanda et al.,

1991]. A polyethylene glycol polystyrene (PEG-PS) graft support (substitution value 0.1-0.4 mmol/g) is a newly developed resin with increased swelling properties [Zalipsky et al.,

1985; Barary et al., 1992].In addition to appreciable swelling in a wide range of solvents the PEG-PS support has improved physical and mechanical properties for both batchwise and continuous-flow SPPS. 55 3.4 Linkers

A key principle in solid phase peptide synthesis is the stepwise chain elongation starting from the C-terminus and proceeding to the N-terminus to avoid racemization. The

C-terminal amino acid is attached to the functionalized resin either directly or by an appropriate linker (handle) [Mitchell et al., 1978]. Most handles contain two functional groups; one has a free or activated carboxyl group which can be coupled to a functionalized amino group on the insoluble support, while the other is a labile protecting group which can be removed to attach the peptide chain. Suitable linkers should provide desired peptide acids or amides upon final cleavage.

Benzyl ester linkers (Fig. 3.8) (e.g. hydroxymethyl resin and 4-(hydroxymethyl) phenylacetic acid (PAM) linker) are commonly utilized for synthesis of peptide acids using

Boc chemistry [Mitchell, 1978; Stewart and Young, 1984].These linkers are cleaved simultaneously with the removal of benzyl-type side chain protecting groups by strong acids

(e.g. anhydrous HF and HBr in TFA). The 4-(2-hydroxyethyl)-3-nitrobenzoic acid (NPE)

[Eritja et al., 1991] and 9-(hydroxymethyl)-2-fluoreneacetic acid (HMFA) [Lui et al., 1990] linkers are used to prepare protected Boc-peptide fragments since they are stable to acids.

Both NPE and HMFA are cleaved by bases, but HMFA can also be cleaved by free Na- amino groups on the peptide resin. Addition of 1-hydroxybenzotriazole (HOBt) during SPPS can prevent premature cleavage of peptides from the HMFA linker [Lui et al., 1990].

Linkers for peptide amides during Boc SPPS are benzhydrylamine derivatives (Fig. 3.8) such as benzhydrylamine (BHA) [Pietta and Marshall, 1970] and 4-methylbenzhydrylamine

(MBHA) [Matsueda and Stewart, 1981]. 56

CICH2

Merrifield resin

HO CH2 CH2COP

PAM resin

HO(CH2)2 COP

02N NPE resin

CH2COP

HOH2C H HMFA resin

BHA resin MBHA resin

Fig. 3.8 Resin linkers and handles used in Boc SPPS. (P = polymeric support) 57

Classical resins for Fmoc chemistry peptide acids are the 4-alkoxybenzyl alcohol resin (Wang resin) and the 4-hydroxymethylphenoxyacetic acid (HMPA) linker (Fig. 3.9)

[Wang, 1973; Sheppard and Williams, 1982; Bernatowicz et al., 1990]. Peptides are cleaved from these resins by 50-100% TFA at 25°C within 1-2 h. Novel linkers (Fig. 3.9) with increased acid liability are 3-methoxy-4-hydroxymethylphenoxyacetic acid [Sheppard and

Williams, 1982], 4-(2',4'-dimethoxyphenylhydroxymethyl)phenoxy (Rink acid) resin [Rink,

1987], 2-methoxy-4-alkoxybenzyl alcohol (SASRIN'; Super Acid Sensitive ResIN)

[Mergler, et al., 1988], 2-chlorotrityl resin [Bar los et al., 1989; Barols et al., 1991] and 5-(4- hydroxymethy1-3,5-dimethoxyphenoxy) valeric acid (HAL; Hypersensitive Acid Labile)

[Albericio and Barany, 1991]. Peptides can be cleaved from these resins under mild acid conditions (1-2% TFA), and thus they are suitable for preparation of protected peptide fragments. Fmoc SPPS peptide amide linkers are based on the benzylhydrylamide structure with the introduction of an electron-donating methoxy groups into the MBHA structure to enhance the linkers' acid liability.These linkers including 5-(4-aminomethy1-3,5- dimethoxyphenoxy) valeric acid (PAL; Peptide Amide Linker) [Albericio and Barany,

1987], 5-(9-aminoxanthen-2-oxy) valeric acid ()CAL) [Sieber, 1987; Bontems et al., 1992],

4-(2',4-dimethoxyphenylamino-methyl)phenoxy resin (Rink amide) [Rink, 1987], 4-(4'- methoxybenzhydryl)phenoxy acetic acid (Dod) [Stuber,etal.,1989], 3-(amino-4- methoxybenzy1)-4,6-dimethoxyphenyl-propionic acid (Breipohl amide linker) [Breipohl, et al., 1989] and 4-succinylamino-2,2',4'-trimethoxy-benzhydrylamine (SAMBHA) [Penke et al., 1988] are sensitive to 50% TFA. 58

HOCH2 OCH2 OCH2 P

Wang resin SASRINTmresin

CH3O

HOCH2 0(cH2)4COp HOCH2 0CH2C0---P

CH3O

HAL resin HMPA linker/ Pac resin

CH3O OCH2 P

Rink acid resin

Fig. 3.9 Resin linkers and handles used in Fmoc SPPS. (P = polymeric support)

Source: (1) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Convergent Solid-Phase Peptide Synthesis. Tetrahedron 1993,49, 11065-11133.

(2) Fields, G.B.; Tian, Z.; Barany, G. Principles and Solid-Phase Peptide Synthesis. In Synthetic Peptide, A User's Guide. Grant, G.A., Ed.; W.H. Freeman and Company: New York,1992, pp 77-183. 59

NHFmoc

0 OCH2 --P O(CH2)4C0 P Sieber amide resin XAL resin

0(CH2)4c0 p

CH3O OCH2 P CH3O Rink amide resin PAL resin

NHCO(CH2)2CONHCHP

OCH3

SAMBHA resin

Fig. 3.9 (cont.) Resin linkers and handles used in Fmoc SPPS. (P = polymeric support) 60

Linkers which are compatible with both Boc and Fmoc SPPS peptide acids are hydroxycrotonyl (HYCRAM') [Kunz and Combo, 1988] and 3-nitro-4-hydroxymethyl benzoic acid (Onb) [Rich and Gurwara, 1975; Kneib-Cordonier et al., 1990] (Fig. 3.10). The

HYCRAMTM linker is cleaved by Pd (0) in presence of weak nucleophiles such as N- methylmorpholine or dimedone, whereas the ONb linker is cleaved photolytically at 350 nm.

For peptide amides, the photolizable 3-nitro-4-aminomethylbenzoic (Nonb) linker (Fig.

3.10) is compatible with both Boc- and Fmoc-based syntheses.

3.5 Peptide bond formation

Coupling ofamino acids to one another requires converting the carboxyl components into acylating agents. This can be achieved by replacement of the OH with an electron-withdrawing group. The electrophilicity of the carbonyl of the amino acid is increased, and therefore, the carbonyl is prone to nucleophilic attack by the amino group of the amino acid to be acylated (Fig. 3.11) [Bodanszky, 1988]. Amino acid chlorides are powerful acylating agents. Conversion of the carboxyl group of the amino acid to the chloride is carried out with phosphorus pentachloride or thionyl chloride. Both reagents give

HC1 as a byproduct, and therefore the use of amino acid chlorides has been limited in SPPS, particularly for Boc chemistry [Grant, 1992]. Acid fluorides prepared by cyanuric fluoride are compatible with both Boc and Fmoc SPPS [Carpino et al., 1990]. Fmoc amino acid chlorides and fluorides are excellent acylating agents for SPPS in the presence of

HOBt/DIEA and DIEA, respectively. Both are useful for either solution or solid phase peptide synthesis providing low levels of racemization [Carpino et al., 1990]. 61

HOCH2 COP

02N Onb resin

H2N(CH2)2 COP

02N

Nonb resin

HOH2C CH= CH CO NH CH2

HYCRAMTM resin

Fig. 3.10 Resin linkers and handles used in both Boc and Fmoc SPPS.

0 II Activation:R COOH R C X r 0 CO -1 O R .0 X RC X RC Coupling: + HX HNH HNH NH R' R' R'

Fig. 3.11 Peptide bond formation.

Source: Bondanszky, M. Peptide Chemistry, Spring-Verlag, Berlin, 1988, p 55. 62

Acylation of amino acids with carboxylic acid anhydrides is the simplest andone of the most efficient methods for peptide bond formation [Merrifield, et al., 1982; Yamashiro,

1987]. The highly reactive preformed symmetrical anhydrides (PSA)are generated in situ from the corresponding Na-protected amino acid (4-12 equiv) plus N, N-diisopropylcarbodiimide (DIC, 1-2 equiv in DCM). PSA's are not widely used for FmocSPPS dueto the wastefulness of the process. Only one amino acid molecule in the anhydride is incorporated into the product, whereas the second amino acid is regenerated but normally not recovered.

Mixed or unsymmetrical anhydrides have been developed in several laboratories [Chen and

Benoiton, 1987], but the inherent ambiguity of synthesis via mixed anhydrides limits their use in SPPS [Barany et al., 1987]. Activation of amino acids to acyl azides has been used in peptide synthesis. Amino acids can either undergo hydrazinolysis of alkyl esters and conversion of the hydrazides to acyl azides with nitrous acid, or direct conversion to acid azides with diphenylphosphoryl azide [Bodanszky, 1988]. An advantage of the acid azide method is that only negligible racemization occurs.

Preactivated amino acids can alternatively be prepared by the formation of active esters. Aryl esters with electron-withdrawing substituents such as 2 -(Ono) and 4-nitrophenyl

(Onp) esters (Fig. 3.12) were used by Bodanszky and co-workers [1973]. Neither Ononor

Onp are popular for SPPS due to their low reactivities even in thepresence of the additive hydroxybenzotriazole (HOBt).Recently developed active esters (Fig. 3.12) including pentafluorophenyl (OPfp) and 3-hydroxy-2,3-dihydro-4-oxobenzotriazine (ODhbt) esters have been successfully used in SPPS. Addition of 1-hydroxybenzotriazole (HOBt) (1-2 equiv) to an OPfp ester increases coupling rates both in Boc and Fmoc SPPS [Atherton et 63

R WNH CH CO -y

Active ester Y

ONp NO2

OPfp

.:,..N OBt 0 N N

kirN OAt 0 im N

Fig. 3.12 Active esters of amino acids. (W = protecting groups)

Source: Fields, G.B.; Tian, Z.; Barany, G. Principles and Solid-Phase Peptide Synthesis. In Synthetic Peptides, A User's Guide. Grant, G.A., Ed.; W.H. Freeman and Company: New York, 1992, pp 77-183. 64 al., 1988; Hudson, 1990]. Boc- and Fmoc-amino acid OBt [Harrison et al., 1989; Fields et al., 1989] and ODhbt [Konig and Geiger, 1970] esters are both highly reactive. Na-

Protected amino acid OBt and ODhbt esters reduce racemization levels during couplings.

Fmoc amino acid ODhbt esters are more stable than OBt esters, whereas the 1-hydroxy-7- azabenzotriazole (OAt) esters are more powerful acylating agents than OBt esters [Carpino,

1993].

Peptide bonds can be formed in situ by using coupling reagents (Fig. 3.13). N,AP-

Dicyclohexylcarbodiimide (DCC) was the first coupling reagent introduced to SPPS [Rich and Singh, 1979; Merrifield et al., 1988]. Unlike DCC, DIC gives a soluble urea byproduct which can be easily washed away during synthesis [Sarantakis et al., 1976]. Addition of

HOBt [Konig and Geiger, 1970; Mojosov et al., 1980] or HOAt [Carpino, 1993] can enhance coupling reactions, suppress racemization and inhibit dehydration of the carboxamide side chains of Asn and Gln to the corresponding nitriles.

New phosphonium and uronium coupling reagents (Fig. 3.13) are highly efficient reagents for SPPS [Fields et al., 1992]. Phosphonium reagents include benzotriazol-1-yl- oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and benzotriazole-1-yl- oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP).BOP generates the carcinogen hexmethylphosphoramide as a byproduct, while PyBOP gives faster reaction rates and less harmful side products [Coste et al., 1990]. Uronium coupling reagents are 2-

(H-benzotriazol-1-y1)-1,1,3 ,3-tetramethyluronium hexafluorophosphate (IIBTU) [Dourtoglou et al., 1978], (0-(7-azabenzotriawl-y1)-1,1,3,3-tetramethyluronium hexafluorophosphate

(HATU) [Carpino, 1993], 2-(benzotriazol-1-y1)-1,1,3,3-tetramethyluronium tetrafluoroborate 65

H3C CH3

N =C =N HCN= C =N CH

H3C CH3 DCC DIC

N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2

N + N + \ BEer 0 P F6 \o

HBTU TBTU

N N N \O \ PF PF6 (CH3)2N P / \mu-4 /P-- (CH3)2N N

BOP PyBOP

Fig. 3.13 Coupling reagents. 66

(TBTU) [Knorr et al..1989], and 2-[2-oxo-1(2H)-pyridy1]-1.1.3.3-bispentamethylene- uronium tetrafluorborate (TOPPipU) [Knorr et al..1990].BOP, HBTU, TBTU and

TOPPipU require a tertiary amine (e.g. NMM or DIEA) for their optimal efficiency

[Gausepohl et al., 1988].Excess amounts of HBTU or TBTU can trap free amino groups giving undesired Schiff base byproducts [Gausepohl et al., 1992].

3.6 Monitoring

SPPS consists of repetitive cycles of deprotection and coupling. Each step has to be driven to completion to obtain a homogenous final product. The ninhydrin test is commonly used to monitor for the presence of unreacted amines after an acylation step [Kaiser et al.,

1970]. This classical color reagent can detect nanomolar quantities of free primary Na- amine which yields the Ruhemann's purple product (Fig. 3.14). The secondary amine of proline gives a yellow color compound which can be monitored at 440 nm.

0 0 C OH 8\ 1,1}- C=N 'CHR c, H2N-C-C-OH -CO2 CH Cr° O H-0

0 9 C + O :CHR ninhvdrin N,C- = C 0

Fig. 3.14 Ninhydrin test.

Source: Bodanszky, M. Peptide Chemistry, Springer-Verlag, Berlin, 1988 p 14. 67

CHAPTER 4

SYNTHESIS OF POTENTIAL AFFINITY LABEL DERIVATIVES OF DYNORPHIN A(1-11)NH2: MESSAGE SEQUENCE MODIFICATIONS

Leena Leelasvatanakij''', Thomas F. Murray', and Jane V. Aldrich'

'College of Pharmacy, Oregon State University 'School of Pharmacy, University of Maryland at Baltimore 68

Abbreviations:

Abbreviations used for amino acids follow the rules of the IUPAC-IUB Joint

Commission of Biochemical Nomenclature in Biochem. J. 1984, 219, 345-373. Amino acids are in the L-configuration except when indicated. Additional abbreviations used are as follows: Aloc, allyloxycarbonyl; Boc, t-butyloxycarbonyl; t-Bu, tert-butyl; DCM, dichloromethane, DIC, N,N'- diisopropylcarbodiimide; DIEA, N,N'-diisopropylethylamine; DMA,

N,N-dimethylacetamide; Dyn A, dynorphin A; DMF, N,N-dimethylformamide; FAB-MS,

fast atom bombardment mass spectrometry; Fmoc, (9-fluorenylmethoxy)carbonyl; HOBt,

1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NMM, N-methylmorpholine; PAL, peptide amide linker, PEG, polyethylene glycol; Pmc, 2,2,5,7,8- pentamethylchroman-6-sulfonyl; PS, polystyrene; TFA, trifluoroaceticacid, THF, tetrahydrofuran; Z, benzyloxycarboxyl. 69

4.1 Abstract

Potentialaffinitylabelderivatives of dynorphin (Dyn) A(1-11)NH2 with modifications in the N-terminal "message" sequence involved incorporation of a reactive functionality (i.e. isothiocyanate and/or bromoacetamide) into the p-amino group of

Phe(NH2) in position 1 or 4 of [D-ProlDyn A(1-11)NH2. Syntheses of these derivatives were performed on a PAL-PEG-PS resin using Fmoc chemistry. An allyloxycarbonyl (Aloc) protecting group was introduced at the p-amino group of Phe(NH2) using allyl chloro-

formate, followed by protecting the Na-amine using Fmoc-Cl.The Aloc group was

selectively removed by Pd(0), followed by incorporation of the reactive functionality.

Except for the isothiocyanate and bromoacetamide derivatives of [Phe(NH2)4,D-Pro10]Dyn

A(1-11)NH2, the peptides were obtained in excellent purity.

Preliminary binding assay results indicated that the control amine compounds

(without a reactive functional group) in both the 1 and 4 positions and the isothiocyanate

derivative of [Phe(NH2)4,D-Pro10]Dyn A(1-11)NH 2 had high affinity for ic opioid receptors.

The bromoacetamide derivative of [Phe(NH2)4,D-Pro10]Dyn A(1-1 1)NH2 and isothiocyanate

of [Phe(NH2)1,D-Pro10]Dyn A(1-11)NH2, however, showed lower affinity for K receptors.

The amine compounds as well as the potential affinity label derivatives of [D-ProllDyn A(1-

11)NH2 had significantly lower affinity for both ii and 8 opioid receptors. These peptides

are being examined for wash-resistant inhibition of binding to opioid receptors. 70

4.2 Introduction

Endogenous mammalian opioid peptides (e.g.enkephalins, dynorphins and endorphins) have been discovered in both the central and peripheral nervous systems [Konig,

1993]. These ligands bind to specific opioid receptors and produce various physiological and pharmacological effects [Simon and Hiller, 1994].The existence of multiple opioid receptors, 11, x and 8 receptors, is well established [Paterson et al., 1984; Simmon, 1991].

Characterization of opioid receptors is important for understanding opiates' functions at a molecular level as well as the physiological and pharmacological roles of opioid receptors.

Highly selective ligands which bind irreversibly to receptors have been valuable tools to study opioid receptor structure and function.These ligands can block certain receptor populations in tissues which contain multiple receptor types, so that the remaining receptor types can be characterized.

Affinity label-containing compounds which covalently bind to receptors differ from reversible ligands [Takemori and Portoghese, 1985]. They are classified into two classes, chemical affinity and photoaffinity labels. The irreversible binding of chemical affinity labels is due to the acylation or alkylation by the electrophilic group on the affinity label of a nucleophilic site on the receptors [Takemori and Portoghese, 1985; Newman, 1991].

Photoaffinity labels, however, require a photolysis step (UV irradiation) which prevents their use for in vivo study of opioid receptors [Glasel and Venn, 1981]. Many affinity labels including nonpeptide and peptide-based ligands have been prepared and used for opioid receptor characterizations [Zimmerman and Leander, 1990; Newman, 1991].Selective nonpeptide affinity labels for IA opioid receptors are 13-funaltrexamine ((3-FNA) [Portoghese 71 et al., 1980] and the etonitazene derivative BIT [Rice et al., 1983].The fumaramido derivative of endoethenotetrahydrooripavine (FAO) and the isothiocyanate derivatives of fentanyl FIT and superFlT selectively alkylate 8 receptors [Rice et al., 1983; Burke et al.,

1986]. Alkylating agents for lc receptors are isothiocyanate derivatives of U-50,488

[de Costa et al., 1989; 1990]. Peptide-based affinity labels such as DALECK (H-Tyr-D-Ala-

Gly-Phe-Leu-CH2C1) [Venn and Bernard, 1981], DAMK (H-Tyr-D-Ala-Gly-MePhe-CH gl)

[Benyhe et al., 1987] and DALCE (H-Tyr-D-Ala-Gly-Phe-Leu-Cys0H) [Bowen et al., 1987]

are enkephalin derivatives.

Dynorphin A (Dyn A), a 17-amino acid peptide derived from prodynorphin, is an endogenous ligand for lc opioid receptors [Cox et al.,1975]. The last four amino acids at the

C-terminus (Trp-Asp-Asn-Gln) are not important for the association of Dyn A to opioid receptors, and Dyn A(1-13) accounts for essentially all of the activity of the parent peptide

[Goldstein et al., 1981]. The N-terminal region (Tyr-Gly-Gly-Phe) of Dyn A was proposed as a "message" sequence, while the sequence containing amino acid residues 5-13 was considered an "address" sequence [Chavkin and Goldstein, 1981]. The message sequence, which is also common in other mammalian opioid peptides, is important for opioid activity, whereas the "address" sequence accounts for binding of dynorphin to lc receptors. Dyn

A(1-13)NH2, however, binds with only modest selectivity to K receptors (K, ratios .t: 8: K =

30: 80: 1.0) [Goldstein, 1984]. [D-PronDyn A(1-11)NH2 shows an increase in K selectivity

(K, ratios pt: 8: K = 93: 280: 1.0) [Gairin et al., 1985, 1988]. Thus, [D-ProliDyn A(1-

11)NH2 has been used as a prototype for the synthesis of potential affinity label Dyn A analogues in our laboratory. The peptide structure of Dyn A provides flexibility in the 72

placement of theaffinitylabeling groups. We, therefore, incorporated reactive

functionalities including isothiocyanate (-N=C=S) and bromoacetamide (-NHCOCH2Br) in

both the N- and C-terminus of [D-ProliDyn A(1-11)NH2 to obtain affinity label Dyn A

analogues. The peptides' affinity (IC50 values) for lc, g and 8 opioid receptors were evaluated

by radioligand binding assays using cloned opioid receptors stably expressed in Chinese

hamster ovary (CHO) cells. The peptides' ability to inhibit radioligand binding in a wash-

resistant manner will be determined in the same assays, using concentrations which gave

maximum inhibition of binding under standard conditions.

The synthesis of Dyn A analogues containing affinity labels are described in this

chapter ("message" sequence modifications) and chapter5("address" sequence

modifications). Modifications in the "message" sequence involved Tyr in position 1 and Phe

in position 4. Since opioid peptides share the same "message" sequence, affinity labels with

modifications in this region could provide insight into whether or not this part of the binding

site is similar for opioid receptors. Structure-activity relationship studies indicate that the phenol ring of Tyr' and the aromatic ring of Phe4 in the "message" sequence are critical for the interaction of opioid peptides with opioid receptors [Chavkin and Goldstein, 1981;

Turcotte et al., 1984]. Studies of [Phe(NO)24]Dyn A(1-13) showed that minor modifications

at position 4 of dynorphin A are well tolerated [Schiller et al., 1982]. Thus, we incorporated both isothiocyanate (-N=C=S) and bromoacetamide (-NHCOCH2Br) in the para position of

Phe4. The aromatic ring of non-peptide ligands (e.g. ethylketocyclazocine) mimics the Tyr'

of Dyn A. Therefore, we replaced Tyr' with an isothiocyanate phenylalanine derivative (Phe

(N=C=S)'). The bromoacetamide derivative was not incorporated into position 1 because 73

of its steric hindrance and its lower affinity towards kappa receptors in position 4 of Phe4

comparing to the isothiocyanate derivative. The p-amino (-NH2) compoundswere included

as reversible controls for pharmacological assays. Peptide amides were preferred to peptide

acids because of their improved metabolic stability [Leslie and Goldstein, 1982].

The original approach for preparing the potential affinity label dynorphinA

derivatives by solid phase peptide synthesis was to cleave the protected peptide from the

resin followed by introducing the reactive functionalities in solution [Story andAldrich,

1992].Synthesis of the model peptide Boc-Tyr(t-Bu)-Gly-Gly-Phe-Leu-Arg(Pmc)NH2on

two different commercially available resins, the Rink amide resin (4-(2',4'-dimethoxyphenyl-

Fmoc-amino-methyl phenoxy) and the Pepsyn resin (a polyamide resin with the 4-hydroxymethyl benzoic acid linker) gave unsatisfactory results. Therefore,a modified methylbenz-

hydrylamine (MBHA) resin with a base-labile linker was developed, anda high yield (>80%)

of the model peptide was obtained after ammonolysis in isopropanol. When the modified

MBHA resin was utilized to synthesize the longer protected peptide amides, [Phe(NO2)4,D-

Pro 11DynA( 1 -1 1 )NH2 and [Phe(NHZ)4 ]Dyn A(1 -13 )NH2, however, low recoveries were obtained (25% and 38% yields, respectively).These results may be due to the steric hindrance of the D-amino acid at the C-terminal (i.e. D-Pro'° in the Dyn A(1-11)NH2 derivative) and/or the hydrophobic character of the polystyrene supports.

In this research, we developed an alternative strategy for the synthesis of potential affinity label Dyn A analogues.First, all synthetic steps including incorporation of the reactive functionality were performed on the solid support instead of in solution. Second, an allyl-based protecting group (i.e. allyloxycarbonyl or Aloe) was used on the p-amino 74 group ofp-amino-phenylalanine to obtain orthogonal protection during solid phase synthesis

[Greene and Wut, 1991; Loffet et al., 1992; Lytt le et al., 1992; Kunz et al., 1988; Barany and

Albericio, 1985]. Aloe is stable under acidic and basic conditions, but can be selectively deprotected by palladium (0) [Genet et al., 1993, 1994; Loffet and Zhang, 1993; Kate et al.,

1993; Williams et al., 1991; Dangles et al., 1987; Guibe et al., 1986]. Therefore, itwas suitable for the synthesis of dynorphin A analogues containing affinity labeling groups. The peptides were assembled on the resin and after the Aloe deprotection, the affinity labeling group could then be introduced while other amino acid side chains remained protected.

Lastly, the newly developed polyethylene glycol/polystyrene support witha peptide amide linker (5-(aminomethy1-3,5-dimethyoxy-phenoxy)valeric acid) (PAL-PEG-PS resin),was utilized to enhance solvent compatibilities during peptide synthesis. PAL-PEG-PS resin has proved to offer excellent coupling and deblocking efficiency [Kates et al., 1993; Barany et al., 1993; Auzanneau et al., 1995].Moreover, PAL-PEG-PS resin has high levels of solvation due to the derivatized polyethylene glycol spacer between the functionalgroup on the polystyrene gel bead and the handle or attachment point of the synthetic peptides. It also swells appreciably in a wide range of both organic and inorganic solvents [Auzanneau et al.,

1995] which provides advantages for the synthesis of potential affinity label analogues of

Dyn A. 75

4.3 Experimental section

4.3.1 Materials

The PAL-PEG-PS resin and N,N- diisopropylethylamine (DIEA) were purchased from Perseptive (Bedford, MA). All amino acids except BocPhe(NHAloc) and FmocPhe

(NHAloc) were obtained from Bachem Inc. (Torrance, CA). BocPhe(NHAloc) and FmocPhe

(NHAloc) were synthesized in this laboratory as described below. Other reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI), and all solvents used (HPLC grade) were obtained from Burdick & Jackson (Muskegon, MI).

The phenylalanine derivatives and crude peptides were analyzed on a Beckman model 431 A gradient high performance liquid chromatography (HPLC) system. The HPLC column was a Vydac analytical column (C18, 300 A,5 1.1, 4.6 x 250 mm) equipped with a

Vydac guard cartridge. The samples were eluted using a linear gradient of 0%-75% solvent

B over 50 min with a flow rate of 1.5 mL/min and detected at 214 nm; solvent A was aqueous 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. Preparative reversed phase HPLC was performed on a Rainin gradient HPLC system using a Vydac semipreparative column (C18, 300 A, 10 .t, 21 x 250 mm) equipped with a Vydac guard cartridge. Amino acid analyses were done by the Central Service Laboratory, Center for

Gene Research and Biotechnology, Oregon State University, using standard hydrolysis conditions (6 N HC1 plus 1.0% phenol at 110°C for 20 h). Each amino acid was resolved using a step gradient on a Beckman Spherogel 52C ion exchange column (4.6 x 150 mm) equipped with post-column ninhydrin detection on a Beckman 126A System Gold HPLC 76

amino acid analyzer. Molecular weight of modified amino acids and synthetic peptides were

determined by fast atom bombardment (FAB) mass spectrometry in the positive mode on a

Kratos MS 50RFTC in Department of Agricultural Chemistry at Oregon State University,

Corvallis, OR. Elemental analyses were performed by MWH Laboratories, Phoenix, AZ.

4.3.2 Methods

4.3.2.1 Synthesis of FmocPhe(NHAloc) (Fig.4.1a and Table 4.1)

Allyl chloroformate (1.06 mL, 11 mmol) was added to a solution of p-amino phenylalanine (1.08 g, 11 mmol) in citrate buffer, pH 4.6 (10 mL), and the mixture stirred under pH control (pH = 4.6) [Fahrenhloz and Thierauch, 1980] for 2.5 h. Phe(NHAloc) was collected by filtration and washed with H2O, Et20, EtOH and EtOAc. The white precipitate

was dried in vacuo (1.55 g, 86% yield). Fmoc-Cl (1.53 g, 5.9 mmol) in dioxane (7 mL) was

added to a solution of Phe (NHAloc) (1.55 g, 5.9 mmol) in 10% Na2CO3 /dioxane (1/1, 10

mL) and the mixture was stirred at 0 °C for 1 h and at room temperature for 12 h. The mixture was then poured into ice water (20 mL) and extracted with ether (2 x 20 mL). The aqueous layer was chilled in an ice bath and acidified with HC1 to pH 2, the white precipitate collected by filtration, washed with 0.1 N HC1 H2O and dried in vacuo. The residue was dissolved in EtOAc and the solution, washed with 0.1 N HC1 and H2O, and dried with

MgSO4. Fmoc Phe(NHAloc) was recrystallized from EtOAc/hexane (1/10) (1.50 g, 97% yield). 77

NH2 NHCH2CH=CH2

.11 ly1 c'nloroformate

pH 4.6 CH2 CH2 NH2 CH COOH NH2 CH COOH

Fmoc CI 10% Na2CO3 /dioxane

CH2 FmocNH CH COOH

chloroiorrnate

CH2 CH2 BocNH CH COOH BocNH CH COON

Fig. 4.1 Synthesis of phenylalanine derivatives. 78

4.3.2.2 Synthesis of BocPhe(NHAloc) (Fig.4.1b and Table 4.1)

Allyl chloroformate (1.15 mL, 10.93 mmol)was added to a solution of BocPhe(NH2)

(3.06 g, 10.93 mmol) in methanol/water (1/1, 10 mL). DIEAwas added to maintain the pH of the solution at pH 4-5 and the mixture was stirred overnight [Fahrenhloz andThierauch,

1980]. BocPhe (NHAloc) was collected by filtration, washed with H2O and recrystallized from tetrahydrofuran (THF).

4.3.2.3 Protected [Phe(NHAloc)4,D-Pro"Pyn A(1-11)NH-PAL-PEG-PS resin (protected la-resin) and Phe(NHAloc)1,D-ProwiDyn A(1-11)NH-PAL-PEG-PS resin (protected2a- resin) (Fig. 4.2)

The peptides were synthesized on a Biosearch 9500 automated peptide synthesizer

(Novato, CA) using Fmoc-amino acids, generally according to the standard procedure

[Snyder et al., 1992] except that N,N-dimethylacetamide (DMA)was used in place of dimethylformamide (DMF) as a solvent.Boc-Tyr(t-Bu) and FmocPhe(NHAIoc) were incorporated into the first and fourth positions, respectively, of protected la-resinand

BocPhe(NHAloc) was incorporated into the first position of protected 2a-resin.The protected peptides were assembled on the PAL-PEG-PS resin (1%cross linked polyvinylstyrene, 0.17 mmol/g). The resin was swollen in dichloromethane (DCM) for 20 min and washed with DCM (5x) and DMA/DCM (1/1, 5x). The repetitive cycle for synthesiswas performed by the following steps: (1) removal of the Fmoc protectinggroup using a piperidine/toluene/DMA (30%/35%/35%) mixture; (2) additiona 4-fold excess of Fmoc- amino acid in DMA (0.4 M) which had been activated in-line for 2 min with diisopropylcarbodiimide (DIC) in DCM; (3) mixing for 2 h (completeness of couplingwas monitored 79

t-Bu P Pmc Pmc Pmc Boc I I I I Boc-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Airg-D-Pro-Lys-NH-resin Protected la-resin

Pd(PPh3)4 in 92.5 %DCM/5°/01-10Ac/2.5ToNMM

t-Bu NH2 Pmc Pmc Pmc Boc I I I I I Boc-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-NH-resin Protected lb-resin

Cl2CS (1.5 eq)/DIEA (2.5 eq) or

BrCH2COBr (2 eq)/DIEA (2 eq)

t-Bu X Pmc pale Pmc Boc I Boc-Tyr-Gly-Gly-Phe-Leu-Airg-A I I I rg-Ile-Arg-D-Pro-Lys-NH-resin Protected lc, 1 d-resin modified reagent K I (87.5`)/0TFA /2.5%thioanisole/5%pheno1/5%H20)

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-CONF12 lc, ld

P = -NHCOOCH2CHCH2

X = -N=C=S or -NHCOCH2Br

Fig.4.2. Synthesis of [Phe(X)4,D-Pro10]Dyn A( 1-1 1 )NH, Derivatives. 80

by the ninhydrin test); and (4) washing with DMA/DCM (1/1, 10x). After attachment of the

N-terminal amino acid, the protected peptide-resins were washed with DCM (5x) and dried

in vacuo.

4.3.2.4 Protected [Phe(NH2)4,D-ProlVDyn A(1-11)NH-PAL-PEG-PS resin (protected 1b- resin), [Phe(N112)`,D-Pro10iDyn A(1-1 1)NH-PAL-PEG-PS resin (protected 2b-resin)

The Biosearch 9500 automated peptide synthesizer was used for Aloc deprotection from the peptide-resins. Protected la-resin (0.21 g, 24.4. gmol) and protected 2a-resin

(0.21 g, 25.2 ['mop were separately swollen in DCM for 15 min. A solution of Pd(PPh3)4

(0.14 M, 3 equiv) in 92.5% DCM/5% HOAc/2.5% N-methylmorpholine (NMM)was manually added to the peptide-resins. The reaction was mixing under N2, anda sufficient amount of DCM was added regularly due to the evaporation during mixing. Quantitative

Kaiser test (see chapter 6) was performed after 1 h, 2 h, and 3 h; the reactionwas complete after 3 h.The protected peptide-resins were washed with the following solvents: THE (3 x 2 min), DMF (3 x 2 min), DCM (3 x 2 min), 0.5% DIEA in DCM (3 x 2 min), 0.02 M sodium diethyldithiocarbamate in DMF (3 x 15 min), DMF (5 x 2 min) and DCM (3x 2 min) and then dried in vacuo.

4.3.2.5 Protected [Phe(N=C=S)4,D-Prow]Dyn A(1-1 1)NH-PAL-PEG-PS resin (protected lc-resin) and [Phe(N=C=S)1,D-ProlVDyn A(1-11)NH-PAL-PEG-PS resin (protected 2c- resin)

Protected lb-resin (0.106 g, 21.0 Rmol) and protected 2b-resin (0.208 g, 40.6 !mop were swollen in DCM (3.0 mL) for 15 min, DIEA (2.5 equiv) was added, followed by adding 81 thiophosgene (C12-C=S, 1.5 equiv). The ninhydrin test was monitored until a negative result was obtained (2 h). The resins were filtered, washed with DCM (5 x 2) and dried in vacuo.

4.3.2.6 Protected [Phe(NHCOCH2Br)4,D-PrelDyn A(1-1 1)NH-PAL-PEG-PS resin (protected ld-resin)

Protected lb-resin (0.06 g, 7.57 gmol) was swollen in DCM (3.0 mL) for 15 min and chilled in an ice bath. DIEA (2 equiv) was added to the peptide-resin, followed by adding bromoacetyl bromide (BrCH2COBr, 1.5 equiv). The mixture was stirred at 0°C for

1 h, and at room temperature until the ninhydrin test gave a negative result (24 h). The peptide-resin was filtered, washed with DCM (5 x 2) and dried in vacuo.

4.3.2. 7 Cleavage reactions

Protected peptide-resins la-ld and 2a-2c were treated with 10 mL of modified reagent K (87.5% TFA/2.5% thioanisole/5% phenol /5% H2O) [King et al., 1990] under N2 and protected from light for 4 h at room temperature. The resins were filtered and washed with 2 mL TFA and the filtrates diluted with 10 mL of 10% acetic acid and extracted with ether (3 x 10 mL). The crude peptides were obtained by lyophilization of the aqueous extracts.

4.3.2.8 Purification

All crude peptides were purified on a Rainin preparative gradient HPLC system. The column used was a Vydac semipreparative column with a Vydac guard cartridge. The 82

eluents were monitored at 280 nm using an ISCO UA-5 UV-visible detector. The peptides

were eluted using a linear gradient of 0%-50% solvent B over 50 min with a flow rate of 20

mL/ min; solvent A was 0.1% TFA in H2O and solvent Bwas 0.1% TFA in AcCN. The

homogeneity of the peptides was determined by analytical HPLCon a Vydac analytical

column equipped with a Vydac guard cartridge usinga linear gradient of 0%-75% solvent

B over 50 min with a flow rate of 1.5 mL/min and detection at 214nm. The pure fractions

were collected and lyophilized to obtain the desired peptides. All peptide were > 98% pure

as determined by analytical HPLC.

4.3.3 Receptor binding assays

Radioligand binding assays of the affinity label derivativeswere performed with

Chinese hamster ovary (CHO) cells which express rat clonedlc and ii [Bunzow et al., 1995] and mouse 8 opioid receptors. Cells were harvested 72 h following transfection in 50mM

Tris buffer, pH 7.4,at 4 °C and homogenized using a Dounce homogenizer.The homogenate was then centrifuged at 45,000 x g for 10 min at 4 °C. The pelletwas washed twice by resuspension and recentrifugationas in the previous step.The pellet was resuspended in 50 mM Tris buffer, pH 7.4, at 4 °C to yielda protein concentration of 30-

60 [Lg/mL.Incubations were performed for 90 min at 22 °C with [3H]diprenorphine,

[31-1]DPDPE (cycloP-Pen2,D-Penlenkephalin) and [3H]DAMGO ([D-A1a2,MePhe,Gly-ol] enkephalin) for ic, 8 and p. receptors,respectively. KD values for[3H]diprenorphine,

[3H]DPDPE and [3H]DAMGO were 0.45, 1.76 and 0.49 nM, respectively. Bindingassays were carried out in mixtures containing 100 pg membrane protein, 3 mM Mg', and 83

peptidase inhibitors (10 µM bestatin, 30 µM captopril and 50 µM L-leucyl-L-leucine) ina

final volume of 2 mL 50 mM Tris buffer, pH 7.4, at 22 °C. Nonspecific bindingwas

determined in the presence of 10 [IM unlabeled Dyn A(1-13)NH2, DPDPE and DAMGO, for

8 and [t receptors respectively. The reactions were terminated by rapid filtrationover

Whatman GF/B glass fiber filters using a Brandel M24-R Cell Harvester. The filters had

been presoaked for at least 2 h in 0.5% polyethylenimine to decrease nonspecific binding.

The filter disks were then placed in minivials with 4 mL Cycocint (ICN radiochemicals) and

allowed to elute for at least 6 h before counting in a Beckman LS 6800 scintillation counter.

IC50 values were then derived from nonlinear regression analysis of competition curves.

Potential affinity label derivatives of dynorphin A are being examined for wash- resistant inhibition of binding to opioid receptors.Chinese hamster ovary (CHO) cell membranes expressing lc receptors are incubated in the presence or absence of the dynorphin

analogues for 90 mM at room temperature. The homogenates are then centrifuged at 40,000 x g for 15 mM at 4 °C and the pellet resuspended in 50 mM Tris buffer, pH 7.4, at 4 °C.

The centrifugation and resuspension procedure is repeated four times.After the fifth resuspension (wash) step, the homogenate is recentrifuged and the final pellet resuspended in 50 mM Tris buffer, pH 7.4, at 4 °C as previously described. These final CHO cell membrane homogenates are then subjected to radioligand binding experiments as described above, and the radioligand binding to membranes treated with the dynorphin analogues compared to binding to untreated control membranes. 84 4.4 Results and discussion

4.4.1 Amino acid syntheses

Solid phase synthesis of peptide-based affinity labels requires selective protection of

the side chain to which the affinity label group (reactive functionality) is to be attached. We

designed derivatives in which the reactive functionalities were attached to the anilinegroup

of Phe(NH2)4 or Phe(NH2)' in derivatives of [D-Prol°]Dyn A(1-11)NH2. This anilinegroup

can only be protected by groups which can be introduced using alkyl chloroformates or

anhydrides (i.e.allyloxycarbonyl chloroformate (AlocCl)or (Boc)20) [J. Aldrich unpublished results]. The use of Boc or Fmoc for Na-amine protection and Aloe for the anilinegroup of

Phe(NH2) provided orthogonal protection [Dangles et al., 1987; Guibe et al., 1986; Merzouk et al., 1992; Kates et al., 1994] which was suitable for the synthesis of potential affinity label

Dyn A derivatives. The synthesis of FmocPhe(NHAloc) was performed ina two-step reaction. First, the p-amino group of Phe(NH2) was selectively protected by the Aloegroup, followed by protection of the Mt-amine using Fmoc-Cl. The p-aminogroup can be preferentially acylated by allyl chloroformate in citrate buffer under pH control (pH= 4.6) while the Na-amine remains unreacted (Fig.4.1a) [Fahrenholz et al., 1980]. This selectivity is due to the higher basicity of the Na-amine (pKa= 9.1) compared to the p-amino group

(pKa = 4.3). The final product FmocPhe(NHAloc) was obtained ina high yield (97%, Fig.

4.3 and Table 4.1). The Aloe group was also attached to the aniline amine of BocPhe(NH2) by a one-step acylation reaction (Fig. 4.1b). The reaction was straightforward and the final product BocPhe (NHAloc) was also obtained in a high yield (74.7%, Fig. 4.4 and Table 4.1). 85

1.100.0.

aalia .

f00 .0

401Na. a

30a-a

14100.41,

1.0101.0

-1 a 0 0 0 a 0 N 4 bra 4 4 4 v. manatees

Fig. 4.3 Chromatograms of FmocPhe(NH2) (solid line) and FmocPhe(NHAloc) (dotted line). Column: Vydac (C18). Solvent: A = aqueous 0.1%TFA, B = 0.1%TFA/AcCN. Flow rate: 1.5 mL/min. Gradient: 0-75%B over 50 min. 86

Fig. 4.4 Chromatograms of BocPhe(NH2) (solid line) and BocPhe(NHAloc) (dotted line). For chromatographic conditions see Fig. 4.3. Table 4.1 Analytical data of modified phenylalanine derivatives'.

Phe(NHAloc) FmocPhe(NHAloc) BocPhe(NHAloc) tR (min)2 16.2 36.3 28.0

FAB-MS 265.2 487.4 365.4 (M+H)± (265) (487) (365) [a]D25° 3 +3.71 +12.86 +17.92

MP (°C) 225-227 178-181 138-139

Elemental analysis Formula C13H16N 204 C28H26N 206 C181--124N206 6/0 C 58.9 (59.1) 69.4 (69.1) 59.4 (59.3) % H 6.0 (6.1) 5.3 (5.4) 6.4 (6.6) % N 10.3 (10.6) 5.9 (5.8) 7.7 (7.7)

TLC 0.474 0.904 0.335 0.876 0.567 0.908 'Theoretical values are shown in parentheses. 2For chromatographic conditions see Fig. 4.3. 3c = 0.5, 0.2 and 0.6 in Me0H for Phe(NHAIoc), FmocPhe(NHAIoc) and BocPhe(NHAIoc), respectively. 4So lvent = butanol: acetic acid: water (3:1:1) 5Solvent = chloroform: methanol: acetic acid (77.5:17.5:7.5) 6Solvent = methanol: water: acetic acid (4:1:1) 'Solvent = chloroform: methanol: acetic acid (17:2:1) 'Solvent = butanol: acetic acid: water (4:2:2) 89

4.4.2 Peptide syntheses

The protected peptides la and 2a were assembled on a PAL-PEG-PS resin using

standard Fmoc chemistry.After the peptide chain assembly was complete, the Aloe protecting group was removed by a solution of Pd(PPh3)4 (see experimental section). The

Aloe deprotection reaction monitored by quantitative ninhydrin test was complete within

3 h yielding protected lb- and 2b-resins. The aniline amines of protected lb- and 2b-resins were converted to the isothiocyanate derivatives protected lc- and 2c-resins by reacting with thiophosgene under basic conditions; the reactions gave a negative ninhydrin test after

2 h. The bromoacetamide group was incorporated into the aniline amine of protected lb- resin to give the protected ld-resin using bromoacetyl bromide under basic conditions; the reaction was complete after 24 h. Final cleavage of the peptides from the resins as well as removal of the remaining side chain protecting groups and the N-terminal Boc protecting group were achieved using modified reagent K [King et al., 1990].

All crude peptides were analyzed by analytical HPLC prior to purification. The control compounds lb and 2b were obtained in excellent purity (tR = 17.4 min (Fig. 4.5) and

16.8 min (Fig. 4.6), respectively). The isothiocyanate derivative 2c was also obtained in excellent purity following cleavage from the resin (tR = 22.9 min, Fig. 4.6). Treatment of lb with thiophosgene and bromoacetyl bromide to yield crude peptides /c and Id,respectively, however, resulted in side products (Fig. 4.5).Analyses by reverse phase HPLC showed

70% of the desired peptide /c (tR= 23.9 min) plus the control compound lb (tR = 17.2 min) and other impurities (Fig. 4.5).The bromoacetamide derivative ld and its hydroxyl derivative were eluted with similar retention times (2:1 ratio, tR = 20.7 and 19.8 min, 90

wli.ONYIWO

gaol

Fig. 4.5 Chromatograms of crude peptide lb (top), /c (middle) and ld (bottom). For chromatographic conditions see Fig. 4.3. 91

7. Z

Fig. 4.6 Chromatograms of crude peptide 2b (top) and 2c (bottom). For chromatographic conditions see Fig. 4.3. 92 respectively, Fig. 4.5). All crude peptides along with the reversible controls were purified by preparative reverse phase HPLC. Purified fractions were analyzed by analytical reverse phase HPLC and FAB-MS, and the data (Table 4.2) are consistent with that expected for the desired peptides.

4.4.3 Receptor affinity

Potential affinity label Dyn A analogues lc, ld and 2c and the reversible control peptides lb and 2b were evaluated for their binding affinity using CHO cells expressing cloned lc, u and 8 opioid receptors. Preliminary binding assay data are shown in Table 4.3.

Incorporation of affinity labeling groups in the "message" sequence of dynorphin A(1-

11)NH2 resulted in different binding affinities for the three opioid receptors. The control peptides lb and 2b and the isothiocyanate lc derivative exhibited high affinities (IC50 = 2.34-

5.75 nM), whereas 2c showed a somewhat lower affinity (IC50 = 14.1 nM) for lc receptors.

The isothiocyanate analogue lc of [Phe(X)4,D-Pro10]Dyn A(1-11)NH2 showed greater

affinity for lc receptors than the bromoacetamide analogue ld (IC50 = 5.57 vs 27.4 nM). The affinity of these analogues for lc receptors is 1/20 to 1/110 that of the parent peptide [D-

Pro' Dyn A(1-11). In the [Phe(X)4,D -Pro' Dyn A(1-11)NH2series, the control peptide lb and the isothiocyanate lc surprisingly tended to have comparable affinities for each opioid receptor type, while distinct affinities were found in the [Phe(X)1,D-Pro9Dyn A(1-11)NH2

series. The control peptide 2b had 7-fold higher selectivity for lc receptors than the isothiocyanate 2c. Incorporating either isothiocyanate or bromoacetamide into the p-amino group of Phe in position 1 or 4 caused dramatic decreases in affinity for both p. and 8 opioid Table 4.2 Analytical data of potential affinity label derivatives of dynorphin A.

Compounds tR (min)1 FAB-MS2 Amino acid analysis (M+H)± Tyr Gly Phe Leu Arg Ile D-Pro Lys Phe(NH2) (1) (2) (1) (1) (3) (1) (1) (1) (1)

[Phe(X)4,D-Pro10]Dyn A(1-11)NH2 lb, X= NH2 17.4 1376.9 0.97 1.93 1.04 3.07 0.90 0.92 1.05 1.12 (1377) /c, X= N=C=S 23.9 1418.9 (1419) Id, X= NHCOCH2Br 20.7 1498.5 (1499)

[Phe(X)1,D-Pro10]Dyn A(1-11)NH2 2b,X=NH2 16.8 1361.0 _3 2.00 1.01 1.02 3.05 0.92 0.98 1.01 (1361) 2c, X= N=C=S 22.9 1403.0 (1303)

'For chromatographic conditions see Fig. 4.3 'Theoretical values are shown in parentheses. 3[Phe(X)1,D-Pro10]Dyn A(1-11)NH2 series contain no Tyr. Table 4.3 Preliminary binding assay data for potential affinity label derivatives of dynorphin A'.

Compound name IC50 (nM) ±SE IC50 ratio

lc 11 8 K/µ /8

[p-Phe(X)4, D-Pro' lDyn A(1-11)NH2 lb,X= NH2 5.75 ± 2.95 146 ± 16 481 1/28/84 /c, X = N=C=S 5.57 ± 0.97 328 ± 58 315 1/48/57 Id, X = NHCOCH2Br 27.4 ± 5.9 173 ± 5.5 1,650 1/6/60

[p- Phe(X)', D-ProliDyn A(1-11)NH2 2b,X= NH2 2.34 ± 0.31 78.9 572 ± 4.7 1/34/244 2c,X= N=C=S 14.1 ± 1.8 92.5 ± 10.5 1,933 1/6/37

[D-Pro ilDyn A(1-11) 0.25 ± 0.03 1.04 ± 0.15 5.52 ± 0.07 1/4/22

'Values without SE are from one experiment. 95 receptors, resulting in IC50 values greater than 75 nM. For the [Phe(X)4,D-Pro10]Dyn A(1-

11)NH2series, the isothiocyanate /c showed better lc selectivity (x/µ /S ratio = 1/48/57) than the bromoacetamide ld (x/g/8 ratio = 1/6/60 ).For the [Phe(X)1,D-Pro' iDyn A(1-11)NH2 series, the control compound 2b unexpectedly had greater x selectivity (x/µ /S ratio=

1/34/244) than the isothiocyanate 2c (x/pt/8 ratio = 1/6/37).

4.5 Conclusions

Potential affinity label derivatives of Dyn A(1-11)NH2 assembled on PAL-PEG-PS resin have been successfully prepared using standard Fmoc chemistry and the Aloe protecting group.Removal of the Aloe group from the protected peptide-resins was complete in 3 h. Conversion of the protected [Phe(NH2)4,D-Pro10]Dyn A(1-11)NH-resin and the protected [Phe(NH2)1,D-Pro10]Dyn A(1-11)NH-resin to their isothiocyanate derivatives was complete in 3 h, whereas conversion to their bromoacetamide derivatives took 24 h to go to completion. The control compounds [Phe(NH2)4,D-Pro10]Dyn A(1-11)NH 2 and [Phe

(NH2)', D-ProllDyn A(1-11)NH2 and the isothiocyanate [Phe (N=C=S)I,D-ProllDyn A

(1-11)NH2 were obtained in excellent purity. However, the isothiocyanate [Phe(N=C=S)4,

D-ProlDyn A(1-11)NH2 and the bromoacetamide [Phe(NHCOCH2Br)4,D-Pro9Dyn A(1-

11)NH2, contained side products, namely the control compound [Phe(NH2)4,D-Pro10]Dyn

A(1-11)NH2 and the hydroxyl side product [Phe(NHCOCH2OH)4,D-Pro10]Dyn A(1-11)NH2, respectively.

Preliminary binding assay data showed that the control amine compounds and the isothiocyanate [Phe(N=C=S)4,D-Pro10]Dyn A(1-11)NH2 had high affinity for x opioid 96 receptors, whereas the bromoacetamide [Phe(NHCOCH2BW,D-Pro10]Dyn A(1-1 1)NH2 and isothiocyanate [Phe(N=C=S)1,D-Pro9Dyn A(1-1 1)NH2 exhibited lower affinity for lc opioid receptors. All peptides had markedly decreased affinity for both u and 8 opioid receptors.

Thus, they showed higher selectivity for lc opioid receptor than the parent peptide [D-

Pro9Dyn A(1-1 1)NH2. Additional pharmacological experiments with these compoundsare in progress. 97

CHAPTER 5

SYNTHESIS OF POTENTIAL AFFINITY LABEL DERIVATIVES OF DYNORPHIN A(1-11)NH2: ADDRESS SEQUENCE MODIFICATIONS

Leena Leelasvatanakij 'a, Thomas F. Murray' and Jane V. Aldrich'

'College of Pharmacy, Oregon State University 'School of Pharmacy, University of Maryland at Baltimore 98

Abbreviations:

Abbreviations used for amino acids follow the rules of the IUPAC-IUB Joint

Commission of Biochemical Nomenclature in Biochem. J. 1984, 219, 345-373. Amino acids

are in the L-configuration except when indicated. Additional abbreviations used are as

follows: Aloe, allyloxycarbonyl; Boc, t-butyloxycarbonyl; t-Bu, tert-butyl; DCM, dichloro-

methane, DIC, N,N'-diisopropylcarbodiimide; DIEA, N,N'-diisopropylethylamine; DMA,

N,N- dimethylacetamide; Dyn A, dynorphin A; DMF, N,N- dimethylformamide; FAB-MS,

fast atom bombardment mass spectrometry; Fmoc, (9-fluorenylmethoxy)carbonyl; HOBt,

1-hydroxy-benzotriazole; HPLC, high performance liquid chromatography; NMM, N-

methylmorpholine; PAL, peptide amide linker, PEG, polyethylene glycol; Pmc, 2,2,5,7,8-

pentamethylchroman-6-sulfonyl; PS, polystyrene; TFA, trifluoroaceticacid, THF,

tetrahydrofuran; Z, benzyloxy-carbonyl. 99

5.1 Abstract

The "address" sequence of dynorphin (Dyn) A is important for directing the peptide to kappa opioid receptors. We replaced Ile of [D-Pro9Dyn A(1-11)NH2 with either isothiocyanate or bromoacetamide derivatives of para-substituted phenylalanine or lysine.

Syntheses of affinity label derivatives of [D-ProlDyn A(1-11)NH2 were achieved by the strategy described in chapter 4. Crude peptides in the [Lys(X)8,D-Pro10]Dyn A(1-11)NH2 series were obtained in high purity, whereas those from [Phe(X)8,D-Pro10]Dyn A(1-11)NH2 series contained minor side products (where X = NH2 (for amine compounds) or a reactive functional group (e.g.-N=C=S or -NHCOCH2Br)).

Preliminary binding assay data revealed that the amine compounds (without a reactive functional group) as well as the affinity label derivatives of [D-Prolc]Dyn A(1-

11)NH2 had high affinity for both lc and p, opioid receptors (IC50 = 0.16-1.54 and 0.77-2.58 nM, respectively). Among these peptides, the amine compound [Lyss,D-Pro9Dyn A(1-

11)NH2 exhibited the highest affinity for lc opioid receptors (IC50 = 0.16 nM) with modest selectivity (IC50 ratio (x/µ /8) = 1/4/31). Affinity of all compounds for 8 opioid receptors (IC

50 = 4.92-13.9 nM) were lower than affinity for lc and p, opioid receptors. 100 5.2 Introduction

The concept of multiple opioid receptors which was first proposed in 1965

[Portoghese, 1965], is supported by many in vitro and in vivo studies [Lord et al., 1977;

Martin, 1976]. Opioid receptors were initially designated as 1.1, ic and a receptors by the

prototype ligands used in the study (morphine, ketazocine and N-allylnormetazocine (SKF

10,047), respectively) [Gilbert and Martin, 1976]. The a receptors areno longer considered

as opioid receptors since their effects are not reversed by naloxone [Fries, 1991]. An

additional type of opioid receptor, the 8 receptors, was identified after isolation of the

enkephalins [Hughes et al., 1975; Lord et al., 1977]. In vitro studies showed that the guinea-

pig ileum (GPI) is rich in j.t and ic receptors, whereas 8 receptors are predominant in the

mouse vas deferens (MVD) [Takemori, 1976; Ward et al., 1985].

Irreversible ligands specific for a certain receptor type are useful tools to elucidate

the involvement of different receptor types in physiological and pharmacologicalresponses.

Affinity label derivatives including chemical affinity labels (electrophilic ligands) and

photoaffinity labels require two recognition steps for covalent bonding; 1) primary recognition which is a reflection of the reversible affinity of the ligand for thereceptor, and

2) secondary recognition which involves the reaction of the electrophilic center witha proximal nucleophile [Portoghese and Takemori, 1985]. Photoaffinity labelsare not useful for in vivo studies since UV iradiation is required to convert these ligands to highly reactive intermediates. Electrophilic affinity labels), however, can be used for both in vitro and in vivo studies. Alkylating groups have been introduced into opiates with the epoxymorphinan structure by converting an amino derivative to the corresponding isothiocyanateor 101 bromoacetamido derivatives [Rice et al., 1983]. Examples of compounds containing an isothiocyanate group are 6a- and 6P-isothiocyanato-4,5-epoxymorphinans [Sayre et al.,

1983], fentanyl isothiocyanates derivatives FIT, SUPERFIT, naltrindole and BIT [Klee et al., 1982; Rothman et al., 1989], N-alkyl -6,14-endo-ethanotetrahydrooripavine-derived isothiocyanates [Jacobson et al., 1983] and derivatives of U-50,488 [de Costa et al., 1989;

1990]. Studies of K receptors were initially difficult due to the lack of selective ligands.

Benzomorphan derivatives such as ethylketocyclazocine (EKC) and bremazocine were often used for K receptor characterization, but these analogs also show affinity for IA receptors

[Paterson et al., 1984]. The benzacetamide U-50,488 was the first non-peptide x-selective ligand [Szumuszkovicz and Voigtlander, 1982], and many derivatives of this ligand have been prepared [Zimmerman and Leander, 1990].U-50,488 and U-69,593 are utilized to distinguish two populations of lc binding sites in rat brain, a U-69,593- (or U-50,488-) sensitive K1 subtype and a U-69,593- (or U-50,488-) insensitive K2 subtype [Zukin et al.,

1988]. Isothiocyanate derivatives of U-50,488 selectively and irreversibly block binding sites which recognize [3H]U- 69,593 but not those which bind r H]bremazocine [de Costa,

1989; 1990].

Research in our laboratory focuses on opioid peptide analogues as tools to characterize multiple types of opioid receptors. In the present study, we are interested in preparing affinity labels for lc opioid receptors based on dynorphin A (Dyn A). These analogues can be used as probes of lc receptor structure and function. Dyn A is a 17-amino acid endogenous opioid peptide for lc receptors [Chavkin et al., 1982], and thus it is important to understand its interaction with opioid receptors and physiological functions. 102

Dyn A, however, displays limited selectivity for x versus IA and 6 receptors [Gairinet al.,

1984; Corbett et al., 1982]. The C-terminally truncated peptide Dyn A(1-13) shows similar

receptor selectivity profile to Dyn A(1-17) [Lemaire et al., 1986], whereas [D-Pro 11Dyn

A(1-11) exhibits a high binding affinity (K1 = 0.032 nM) against thex ligand [3H]

bremazocine. Therefore, we prepared affinity label derivatives of Dyn A using [D- Pro10]Dyn

A(1-11) as the parent compound. We incorporated a reactive functionality (e.g. isothio-

cyanate (-N=C=S) or bromoacetamide (-NHCOCH2Br)) into either the N-terminal "message"

(see chapter 4) or the C-terminal "address" (Leu-Arg-Arg-Ile-Arg-D-Pro-Lys)sequence.

Modifications in the C-terminus of [D-Prol°]Dyn A(1-11)are described in this chapter. The "address" sequence of Dyn A plays an important role in directing the peptide to the kappa opioid receptors. Structure-activity relationship studies indicated that the basic residues Are and Lys" are the most important for potency and kappa receptor selectivity

[Chavkin and Goldstein, 1981; Turcotte et al., 1984; Snyder et al., 1992] The positive charges of these residues may form two electrostatic interactions with negative chargeson the kappa binding site, which would increase bindingenergy and stabilize the bioactive conformation [Hruby and Gehrig, 1989]. Replacements of the basic amino acids in position

7 and 11 cause a larger decrease in opioid activity in the GPI than do substitution in positions

8 and 10 [Turcotte, 1984]. Substitution of Ilex by Ala or D-Ala enhances kappa binding affinity [Lemaire et al., 1986]. Since structural changes in the position 8are well tolerated, we selected the nonbasic residue Ile as a site for introducing a reactive functionality in our studies. Ilea was replaced with either isothiocyanate or bromoacetamide derivatives ofpara- substituted phenylalanine or lysine. The amine containing peptides, [Phe(NH2)8,D-Pro 103

Dyn A(1-11)NH2and [Lys8,D-Pro10] Dyn A(1-11)NH2 were included as reversible controls

in the pharmacological assays. The synthetic strategy which includes the use of the allyl-

oxycarbonyl (Aloc) protecting group and the novel polyethylene glycol (PAL-PEG-PS) resin

were described in detail in chapter 4.

5.3 Experimental section

5.3.1 Materials

The reagents and instrumentation used were those described previously (see chapter

4) except for FmocLys(Aloc), which was purchased from Perseptives (Bedford, MA).

5.3.2 Methods

5.3.2.1 Protected [Phe(NHAloc)8,D-Pro10]Dyn A(1-11)NH-PAL-PEG-PS resin (protected la-resin) and [Lys(NIL4loc)8,D-Pro10 ]Dyn A(1-11)NH-PAL-PEG-PS resin (protected 2a- resin), protected [Phe(N112)8,D-Pro10JDyn A(1-11)NH-PAL-PEG-PS resin (protected lb- resin) and [Lys(NHZD-Prow]Dyn A(1-11)NH-PAL-PEG-PS resin (protected 2b-resin)

The protected peptides la and 2a were assembled on PAL-PEG-PS resin (1% cross linked polyvinyl-styrene, 0.17 mmollg, 1.0 g) using standard Fmoc chemistry procedures as described in chapter 4. Either FmocPhe(NHAloc) or FmocLys(Aloc) were incorporated into position 8 where the reactive functionality was to be introduced into the peptide chain. Other side chain functionalities and the N-terminal amine were protected with TFA-labile protecting groups as described in chapter 4. Protected peptide la (0.14 g, 17 umol) and 2a 104

(0.75 g, 89.8 mop were then treated with Pd(PPh3)4 for 3 h and 2 h, respectively, as

described in chapter 4, providing protected peptide lb- and 2b-resins.

5.3.2.2 Protected [Phe(N=C=S)8,D-Prow] Dyn A(1-11)NH-PAL-PEG-PS resin (protected lc-resin) and [Lys(N=C=S)8,D-ProwiDyn A(1-11)NH-PAL-PEG-PS resin (protected 2c- resin), protected [Phe(NHCOCH2Br)8,D-Pro10JDyn A(1-11)NH-PAL-PEG-PS resin (protected ld-resin) and [Lys(NHCOCH2Br)8,D-Pro10]Dyn A(1-11)NH-PAL-PEG-PS resin (protected 2d-resin)

Protected peptide-resin lb (0.11 g, 12.9 mol) and 2b (0.28 g, 38.0 mop were

swollen in DCM (3.0 mL) for 15 min, DIEA (2.5 equiv) was added, followed by adding

thiophosgene (C12-C=S, 1.5 equiv). The ninhydrin test was monitored until a negative result

was obtained (2 h). The products, protected peptide /c- and 2c-resins, were filtered, washed

with DCM (5 x 2) and dried in vacuo.

Protected peptide-resin lb (0.08 g, 9.89 mop and 2b (0.17 g, 23.0 mop were

swollen in DCM (3.0 mL) for 15 min and chilled in an ice bath. DIEA (2 equiv) was added

to the peptide-resin, followed by adding bromoacetyl bromide (BrCH2COBr, 1.5 equiv). The

mixture was stirred at 0°C for 1 h, and at room temperature until the negative ninhydrin test was obtained (24 h and 18 h for ld and 2d, respectively). The peptide-resins ld and 2d were

filtered, washed with DCM (5 x 2) and dried in vacuo.

5.3.2.3 Cleavage reactions and purification

Protected peptide-resins la-ld and 2a-2d were treated with 10 mL of modified reagent K as described in chapter 4. Following cleavage of the peptides, the peptides were purified on a Rainin preparative gradient HPLC system and purity of peptides (> 98%) was 105

verified by analytical HPLC as described in chapter 4. The purified peptides were sumbitted

for fast atom bombardment mass spectrometry.

5.3.3 Receptor binding assays

Opioid receptor binding studies were performed in Chinese hamster ovary (CHO)

cells which express cloned II, S and lc opioid receptors. Wash-resistant inhibitions of binding

CHO cell membranes will also be determined. The procedures for both assays are described

in detail in chapter 4.

5.4 Results and discussion

5.4.1 Peptide syntheses

The protected peptides la and 2a were assembled on PAL-PEG-PS resin using

standard Fmoc chemistry followed by removal of the Aloc protecting group usinga Pd (0)

catalyst [Kates et al., 1993] using the procedures previously described (see chapter 4). The

Aloc deprotections of protected la- and 2a-resin were complete in 3 h and 2 h, respectively.

Isothiocyanate and bromoacetamide reactive functionalities were subsequently introduced into protected lb- and 2b-resin using thiophosgene and bromoacetyl bromide, respectively, under basic conditions.Conversion of the amines protected lb- and 2b-resin to the isothiocyanates protected lc- and 2c-resin was complete in 2 h, as indicated by quantitative ninhydrin test. The bromoacetamide analogues protected ld- and 2d-resin, however, took

24 h and 18 h, respectively, for the reactions to go to completion. The N-terminal Boc 106

protecting group and side chain protecting groups were removed and the peptide amides then

liberated from the resins using modified reagent K (ethanedithiol was eliminated to prevent

reactions with the alkylating group, see chapter 4).In all cases, crude peptides in the

[Lys(X)8,D-Pro10]Dyn A(1-11)NH2 series were obtained in excellent purity following

cleavage from the resin ( Fig. 5.2). Crude peptides in [Phe(X)8,D-Pro10]Dyn A(1-11)NH2

series, however, contained some minor side products ( Fig. 5.1). Our results indicate that the

incorporation of isothiocyanate or bromoacetamide functional groups into [Lys(X)8,D-

Pro10]Dyn A(1-11)NH2 was faster and cleaner than into [Phe(X)8,D-Pro' lDyn A(1-11)NH2.

After purification by preparative reverse phase HPLC, all peptides were characterized by analytical reverse phase HPLC and FAB-MS (Table 5.1) and the parent peptides lb and 2b by amino acid analysis.

5.9.2 Receptor affinity

The purified peptides were evaluated for their binding affinity to lc, p, and 8 opioid receptors using competition binding assays. The affinities for x, ti and 8 were determined with cloned receptors expressed in Chinese hamster ovary (CHO) cell using radioligands

[3H]diprenorphine, [3H]DAMGO and [31-I]DPDPE, respectively, and IC50 values (Table 5.2) were calculated from nonlinear regression analysis of competition curves.

Binding affinity of potential affinity label derivatives of dynorphin A for opioid receptors varied. Preliminary data show that all peptides had high affinity for both lc and opioid receptors (IC50 = 0.16-2.89 nM) compared to the parent peptide [D-ProllDyn A(1-11)

(IC50 = 0.25 and 1.04 nM for lc andµ opioid receptors, respectively). The isothiocyanate 107

.11.

Fig. 5.1 Chromatograms of crude peptides lb (top), /c (middle) and Id (bottom). Column: Vydac (C18). Solvent: A = aqueous 0.1% TFA, B= acetonitrile containing 0.1% TFA. Flow rate: 1.5 mL/min. Gradient: 0- 75% B over 50 min. 108

Fig. 5.2 Chromatograms of crude peptides 2b (top), 2c (middle) and 2d (bottom). For chromatographic conditions see Fig 5.1. Table 5.1 Analytical data of potential affinity label derivatives of dynorphin A.

Compounds tR (min)' FAB-MS2 Amino acid analysis (M+H)* Tyr Gly Phe Leu Arg D-Pro Lys' Phe(NH2) (1) (2) (1) (1) (3) (1) (1) (1)

[Phe(X)8,D-Pro10]Dyn A(1-11)NH2 1 b,X= NH2 19.9 1410.8 1.07 2.19 1.12 1.09 2.93 0.76 0.82 1.05 (1411) /c, X= N=C=S 22.5 1454.0 (1454) Id, X= NHCOCH2Br 22.0 1532.8 (1533) [Lys(X)8,D-Pro10]Dyn A(1-11)NH2 2b,= - 19.0 1377.0 0.93 1.92 1.041.04 3.10 0.99 1.98 (1377) 2c, X= N=C=S 19.1 1419.0 (1419) 2d, X= NHCOCH2Br 20.9 1498.8 (1499) 'For chromatographic conditions see Fig. 5.1. 2Theoretical values are shown in parentheses. 3[Lys(X)8D-Pro10]Dyn A(1-11)NH2 contains two Lys residues. Table 5.2 Preliminary binding assay data for potential affinity label derivatives of dynorphin A'.

Compound name IC50 (nM) ±SE IC50 ratio

K 11 43 K/µ /8

[p-Phe(X)8, D-ProllDyn A(1-11)NH2 16,X =NH2 1.54 ± 0.15 1.06 ± 0.29 6.83 ± 2.1 1.5/1/9 lc, X = N=C=S 0.49 ± 0.09 1.71 ± 0.27 13.9 1/4t25 ld, X = NHCOCH2Br 1.42 ± 0.06 2.89 ± 0.31 8.14 ± 0.44 1/2/6

[Lys(X)8, D-ProllDyn A(1-11)NH2 2b,X= - 0.16 ± 0.03 0.71 + 0.13 4.92 + 0.36 1/4/31 2c, X = N=C=S 0.54 ± 0.03 1.04 ± 0.20 5.24 ± 0.42 1/2/10 2d, X = NHCOCH2Br 0.68 ± 0.10 1.16 ± 0.03 6.07 ± 0.16 1/2/9

[D-Pro9Dyn A(1-11) 0.25 ± 0.03 1.04 ± 0.15 5.52 ± 0.07 1/4/22

'Values without SE are from one experiment. 111

derivative of [Phe(X)8,D-Pro10]Dyn A(1-11)NH2(/c) bound to -lc andµ opioid receptors with

greater affinity than the bromoacetamide derivative (1d) (IC50 = 0.49 and 1.71 vs 1.42 and

2.89 nM, respectively). For [Lys(X)8,D-Pro10]Dyn A(1-11)NH2 series, both the isothiocyanate (2c) and bromoacetamide (2d) derivatives had comparable affinities for x and IA opioid receptors. Unexpectedly, the amine control peptide 2b had the greatest affinity for lc binding sites (IC50 = 0.16 nM). Potential affinity label derivatives of dynorphin A also bound to 8 opioid receptors with modest affinity (IC50 = 4.92-13.9 nM).

5.5 Conclusions

Isothiocyanate and bromoacetamide derivatives of [Phe(X)8,D-Pro10]Dyn A(1-

11)NH2 and [Lys(X)8,D-Pro10]Dyn A(1-11)NH2 (where X = NH2 (for amine compounds)or a reactive functional group (e.g.-N=C=S or -NHCOCH2Br)) have been prepared using the synthetic strategy described in chapter 4. Removal of Aloc group from the protected [Lys-

(NHAloc)8,D-ProliDyn A(1-11)NH-resin was faster than from the protected [Phe (NH-

Aloc)8,D-ProllDyn A(1-11)NH-resin (2 h vs 3 h).Conversion of the protected [Phe-

(NH2)8,D-ProliDyn A(1-11)NH-resin and the protected [Lys 8,D-Pro 10 ]Dyn A(1-11)NH- resin to their isothiocyanate derivatives was complete in 2 h. Whereas, conversion of the protected [Lysg,D-ProliDyn A(1-11)NH-resin to its bromoacetamide derivative tooka shorter reaction time than that of the protected [Phe(NH2)8,D-Pro10]Dyn A(1-11)NH-resin

(18 h vs 24 h).Crude peptides in the [Lys(X)8,D-Pro10]Dyn A(1-11)NI-12 series were obtained in high purity, while crude peptides in the [Phe(X)8,D-Prol° ]Dyn A(1-11)NH2 series contained minor impurities which could be separated by reverse phase HPLC. 112

Preliminary binding assay data showed that the control compounds and affinity label

derivatives of [D-ProliDyn A(1-11)NH2 had affinity for all three opioid receptors (c, ii and

6). Affinity for 8 opioid receptors (IC50 = 4.92 - 13.9 nM), however, was slightly lower than

for K (IC50 = 0.16 - 1.42 nM) and I.L (IC50 = 1.06 - 2.89 nM) opioid receptors. Except for

[Phe(NH2)8,D-Pro10]Dyn A(1-11)NH2, all peptides possessed higher affinity for ic opioid

receptors than for ji opioid receptors.The highest affinity for lc opioid receptors was

surprisingly found in the control compound [Lys8,D-Pro10]Dyn A(1-11)NH2 (IC50 = 0.16

nM). Kappa selectivity of this peptide was comparable to the parent peptide [D- Pro10] Dyn

A(1-11)NH2 (IC50 ratio (K/g/8) = 1/4/31 vs 1/4/22). In both [Phe(X)8,D-Pro10]Dyn A(1-

11)NH2 and [Lys(X)8,D-Pro10]Dyn A(1-11)NH 2series, the isothiocyanate derivatives showed

slightly higher affinity for K opioid receptors than the bromoacetamide derivatives.

Additional pharmacological experiments of these compounds are in progress. 113

CHAPTER 6

SOLID PHASE SYNTHESIS OF POTENTIAL AFFINITY LABEL DERIVATIVES OF DYNORPHIN A ASSEMBLED ON PAL-PEG-PS VS PAL-PS RESINS

Leena Leelasvatanakij''' and Jane V. Aldrich'

'College of Pharmacy, Oregon State University 'School of Pharmacy, University of Maryland at Baltimore 114

Abbreviations:

Abbreviations used for amino acids follow the rules of the IUPAC-IUB Joint

Commission of Biochemical Nomenclature in Biochem. J. 1984, 219, 345-373. Amino acids are in the L-configuration except when indicated. Additional abbreviations used are as follows: Aloc, allyloxycarbonyl; Boc, t-butyloxycarbonyl; t-Bu, tent- butyl; DCM, dichloromethane, DIC, N,N'- diisopropylcarbodiimide; DIEA, N,N'- diisopropylethylamine; DMA,

N,N-dimethylacetamide; Dyn A, dynorphin A; DMF, N,N-dimethylformamide; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, (9-fluorenylmethoxy)carbonyl; HOBt,

1-hydroxy-benzotriazole; HPLC, high performance liquid chromatography; NMM, N- methylmorpholine; PAL, peptide amide linker, PEG, polyethylene glycol; Pmc, 2,2,5,7,8- pentamethylchroman-6-sulfonyl; PS, polystyrene; TFA, trifluoroaceticacid, THF, tetrahydrofuran; Z, benzyloxy-carbonyl. 115

6.1 Abstract

The synthesis of affinity label-containing dynorphin A (Dyn A) analogues in our

laboratory generally involves modification of a substituted phenylalanine residue at different positions. An appropriate protecting group is required for an amine functionality on the para position of Phe. Allyloxycarbonyl (Aloc) was used to provide orthogonal protection which was removed by Pd(PPh3)4 in DCM/5% AcOH/2.5% N-methylmorpholine (NMM). Rates

of Aloe deprotection were affected by the resin type used for peptide assembly; the complete

removal of Aloe was achieved in 3 h and 24 h for PAL-PEG-PS and PAL-PS resins, respectively. The reactive functionalities isothiocyanate (-N=C=S) and bromoacetamide

(-NHCOCH2Br) were introduced into the Aloe deprotected peptide-resins. Analyaes by

HPLC revealed the crude peptides assembled on the PAL-PS resin contained less impurities than those on the PAL-PEG-PS resin. 116

6.2 Introduction

One research project in our laboratory involves the preparation of electrophilic affinity labels for kappa opioid receptors based on dynorphin A. Affinity label-containing compounds can bind covalently to receptors and thus differ from reversible ligands. The irreversible binding of electrophilic affinity labels is due to the acylation or alkylation by the electrophilic group on the affinity label to a nucleophilic site on the receptors. This irreversible binding can increase the ligand's selectivity for one receptor type over another

[Takemori et al 1985]. Therefore, affinity labels can be utilized to differentiate receptor types and even receptor subtypes. Dyn A analogues containing an affinity label are being synthesized to use as tools to study kappa opioid receptor structure and function. The reactive functionalities generally are being introduced at the p-amino group of a phenylalanine residue, for example at position 4 to give [Phe(X)4,D-Pro10]Dyn A(1-11)NH2, and include isothiocyanate (X = -N=C=S) and bromoacetamide (X = -NHCOCH2Br) derivatives.

Allyl-based protecting groups have been used extensively in organic synthesis and recently have been applied to peptide and DNA syntheses [Lytt le and Hudson, 1992].

Deprotection of allyl derivatives occurs smoothly and quantitatively with palladium (0) (Fig.

6.1). The mild conditions to deprotect allyl functionalities are compatible with classical Boc

(butyloxycarbony1)/Bz1(benzyloxycarbonyl) and Fmoc ( 9- fluorenylmethyloxycarbonyl) /t -Bu

(t-butyl) chemistries [Albericio et al., 1992; Kates et al., 1994], and provide orthogonal protection schemes for the construction of complex peptides. An allyl-based protecting group such as allyloxycarbonyl or Aloe is easy to introduce using the readily available and 117

RCH,CH=CH,

ti -Pd. RCH=CHCH2Nu t Pd °H RCHCH=CH, Pcii. O2CCH,

Fig. 6.1 Deprotection of allyl-based protectinggroups.

HANDLE PEG PS

CH .,O FmocNHCH2

CH 0

Fig. 6.2 Structures of PAL-PEG-PS (top) and PAL-PS (bottom) resin. 118 inexpensive allyl chloroformate. Deprotection of Aloe can also be achieved using only a catalytic amount of catalyst (e.g. 125-250 mg of Pd(0) per 0.1 mmol peptide-resin) and the reaction tolerates a wide range of solvents (e.g. DCM and DMF).Thus, allyl-based protecting groups (e.g. Aloc) are suitable for use in the synthesis of dynorphin A analogues containing reactive functional groups which require different levels of protection. We used an Aloc protecting group for the amino acid residue where a reactive functionality was incorporated.The peptides were assembled on the resin and an affinity label group introduced after Aloc deprotection while the other amino acid side chains and N-terminus remained protected.

A novel polyethylene glycol-polystyrene resin with a peptide amide linker (PAL-

PEG-PS) offers excellent coupling and deblocking efficiency [Kates et al., 1994; Barany et al., 1993; Auzanneau et al., 1995]. It has a high level of solvation due to the derivatized polyethylene glycol spacer (Fig. 6.2) between the functional group on the polystyrene gel bead and the handle or attachment point of the synthetic peptide. It also swells appreciably in a wide range of both organic and inorganic solvents (Table 6.1) enhancing rapid diffusion of reagents throughout the polymer network. Thus, the PAL-PEG-PS resin may be suitable for the synthesis of affinity label derivatives of Dyn A.

Previous studies (see chapter 4 and 5), however, showed that the crude peptides assembled on PAL-PEG-PS resin contained the desired peptides but that some impurities were also often present. Therefore, we synthesized potential affinity label Dyn A analogues, for example [Phe(X)4,D-Pro9Dyn A(1-11)NH2 (where X = -N=C=S or -NHCOCH2Br), on a polystyrene resin, PAL (Peptide Amide Linker, 5-( 4- Fmoc- aminomethyl -3,5- dimethoxy- 119

Table 6.1 Ratio of swollen/dry volumes for individual polyethylene glycol polystyrene (PEG-PS) graft beads and polystyrene (PS) support in different solvents.

Solvent PEG-PS PS

DCM 6.9 5.8 DMF 4.8 3.6 AcCN 3.5 1.4 TFE 5.5 1.3 EtOAc 3.7 3.7 THE 4.5 5.7 EtOH 2.3 1.2 H2O 1.7 1.0

Source: Sarin, V.K; Kent, S.B.H.; Merrifield, R.B. Properties of Swollen Polymer Network: Solvation and Swelling of Peptide-Containing Resins in Solid-Phase Peptide Synthesis. J. Am.Chem.Soc. 1980, 102, 5463-5470. 120

phenoxy) valeric acid-MBHA (methylbenzylhydrylamine))-PS resin.PAL-PS resin is

normally used in our laboratory to obtain peptide amides, but preliminary studies using the

qualitative ninhydrin test indicated that the Aloc deprotection of the protected peptide-PAL-

PS resin required a longer reaction time (more than 12 h) than deprotection on the PAL-

PEG-PS resin. In this study, we compared the effects of using PAL-PEG-PS vs PAL-PS

resin on the Aloc deprotection rates and the purity of the final potential affinity label Dyn A

analogues. The results from this study could be useful to improve the synthesis of potential

affinity label derivatives of Dyn A as well as potential affinity label derivatives of other peptides.

6.3 Experimental section

6.3.1 Materials

The reagents and instruments used were those described previously (see chapters 4

and 5) with the following modification: PAL-PS resins (1% cross linked polyvinylstyrene,

0.38 mmol/g) was purchased from Perseptives (Bedford, MA). 121

6.3.2 Methods

6.3.2.1 Protected 1Phe(NHAloc)4,D-Pro10iDyn A(1-11)NH-PAL-PEG-PS resin (protected la-resin) and Phe(NHAloc)4 D-ProlVDyn A(1-11)NH-PAL-PS resin (protected lb-resin)

Protected la- and lb-resin were assembled on the PAL-PEG-PS resin (1% cross linked polyvinylstyrene, 0.17 mmol/g) and PAL-PS resin (1% cross linked polyvinylstyrene,

0.38 mmol/g), respectively, using the standard procedure described in chapter 4.

6.3.2.2 Time course studies of Aloc deprotection

A Biosearch 9500 automated peptide synthesizer was used for the Aloc deprotection.

A time course study of Aloc deprotection was performed using 20 mg aliquots of protected la- and lb-resin and the following times: 0.5, 2 and 3 h for protected la-resin, and 2, 6, 12 and 24 h for protected lb-resin. The peptide-resin was swollen in DCM for 15 min and a solution of Pd(PPh3)4, (0.14 M, 3 equiv) in 92.5% DCM/5% AcOH/2.5% NMM (N- methylmorpholine) was manually added to the reaction vessel [Kates et al., 1993]. The reaction was mixed under N2 and a sufficient amount of DCM was added occasionally due to evaporation during mixing. A freshly prepared palladium solution was added after prolonged mixing times (more than 6 h). Each aliquot of peptide-resin was washed with the following solvents: THE (3 x 2 min), DMF (3 x 2 min), DCM (3 x 2 min), 0.5% DIEA in

DCM (3 x 2 min), 0.02 M sodium diethyldithiocarbamate in DMF (3 x 15 min) and DCM

(3 x 2 min) [Kates et al., 1993]. The peptide-resin was dried in vacuo and the completeness of Aloc deprotection was determined by quantitative ninhydrin test [Stewart et al., 1984]. 122

The amount of amine remaining was calculated based on the absorbance at 570 nm using the following formula:

amine (gmol/g) = A x V x 106 E570 X Wt

A = absorbance at 570 nm

V = volume of final solution (mL)

E570 = molar absorptivity at 570 nm (1.5 x 104 M-1 cm-')

Wt = weight of peptide-resin used (mg)

6.3.2.3 [Phe(NH2)4,D-Pro10JDyn A(1-11)NH-PAL-PEG-PS resin (protected 2a-resin) and [Phe (NH2)4,D-Pro10]Dyn A(1-11)NH-PAL-PS resin (protected 2b-resin)

The time course study of Aloc deprotection described in the previous section indicated that the Aloc deprotection of the peptide assembled on the PAL-PEG-PS resin was complete in 3 h, while the reaction on PAL-PS resin required in 24 h. This data and the conditions described above were used on a larger scale Aloc deprotection of protected la- and lb-resin (0.5 and 0.45 g, respectively) to yield protected 2a- and 2b-resin, respectively.

6.3.2.4 Protected [Phe(N=C=S)4,D-Pro10]Dyn A(1-11)NH-PAL-PEG-PS resin (protected 3a-resin), [Phe(N=C=S)4,D-Pro10JDyn A(1-11)NH-PAL-PS resin (protected 3b-resin), [Phe(NHCOCH2Br)4,D-Pro10]Dyn A(1-11)NH-PAL-PEG-PS resin (protected 4a-resin) and [Phe(NHCOCH2Br)4,D-Pro10] Dyn A(1-11)NH-PAL-PS resin (protected 4b-resin)

The reactive functionality groups isothiocyanate (- N =C =S) and bromoacetamide

(-NHCOCH2Br) were introduced into the protected peptide-resins la and lb as described in chapter 4 to give the protected peptide-resins 3a, 3b, 4a and 4b.The products were 123

subsequently cleaved by modified reagent K (87.5% TFA/2.5% thioanisole/5% phenol/ 5%

H2O) [King et al., 1990] for 4 h and after filtering diluted with 10% acetic acid and extracted with ether (see chapter 4). Crude peptides 3a, 3b, 4a and 4bwere analyzed by HPLC.

6.4 Results and discussion

The synthesis of potential affinity label derivatives of Dyn A described previously in chapter 4 and 5 were achieved by utilizing the allyl/Fmoc/tBu protection scheme. The

Aloe group provides orthogonal protection which can be selectively removed by Pd(0).

Removal of Aloc followed by incorporation of a reactive functionalitygroup could be achieved on a PAL-PEG-PS resin. Polyethylene glycol comprises 20-70 percent weight of the resin which makes the resin compatible with both organic and inorganic solvents [Kates et al., 1993]. Analyses by reverse phase HPLC, however, indicated that the crude products often contained some impurities in addition to the desired peptides. This promptedus to synthesize the potential affinity label derivatives of Dyn A ona different resin, PAL-PS resin, and to directly compare the Aloc deprotection rates and purity of the crude peptides on the two resins. Therefore, [Phe(NHAloc)4,D-Pro10 ]Dyn A(1-11)NH2 was assembled on the two resins, PAL-PEG-PS and PAL-PS resins. The reactive functionalgroups were introduced after removal of the Aloe group.

Deprotection of the Aloe group can be achieved using palladium (0) [Kates et al.,

1993; Baeza et al, 1992]. The deprotection procedure, however, can be complicated bya number of factors including the insolubility of palladium, requirement for an inert atmosphere and the formation of side products (e.g. metal ions and allylamine) [Genet et al., 124

1993; 1994]. A nucleophile (e.g. morpholine [Kunz, 1987; Kunz et al., 1984 and 1988],

formic acid [Minami et al., 1985; Hayakawa et al., 1990], potassium 2-ethylhexanoate

[Jeffery et al., 1982], dimedone [Guibe et al., 1981] or tributyltin hydride [Guibe et al., 1989;

Dangles et al., 1987; Boullanger et al., 1986]) is required as an allylic scavenger to minimize

the competitive formation of allylamine; palladium catalyzes the transfer of the allyl unit to

these nucleophiles [Loffet et al., 1993] (Fig 6.1). The optimum conditions used in our study

were Pd(PPh3)4 in 92.5% DCM/5% AcOH/2.5% NMM; DCM was used instead of

chloroform [Kates et al., 1993] to avoid hepatic toxicities of the latter solvent. Organic acid

(i.e. AcOH) was preferred to mineral acid (i.e. HCl) to prevent a heterogenous solvent mixture. Metal ions and side products were removed by washing with THF, DMF, DCM,

5% DIEA in DCM, 0.02 M sodium diethyldithiocarbamate in DMF, DMF and DCM; sodium

diethyldithiocarbamate was required as a palladium scavenger [Kates et al., 1993].

Aloe deprotection rates of protected [Phe(NHAloc)4,D-Pro10]Dyn A(1-11)NH2

assembled on PAL-PEG-PS and PAL-PS resins are compared in Fig.6.3.Quantitative

ninhydrin test was performed to measure amine levels. Quantitative ninhydrin data indicate that Aloc deprotection on the PAL-PEG-PS resin is significantly faster than on the PAL-PS resin. After 2 h, 73.8% and 11.1% of the expected maximum amine were obtained on PAL-

PEG-PS and PAL-PS resins, respectively. The removal of Aloc was complete in 3 h on the

PAL-PEG-PS resin, whereas a longer reaction time (24 h) was required for the PAL-PS

resin.The polyethylene glycol spacer in PAL-PEG-PS resin probably enhances Aloc

deprotection rates by allowing complete solvation of the reactive sites which resulted in a more efficient reaction.The protected peptides 2a and 2b on the resin were used as 125

Deprotection of Aloc on PAL-PEG-PS resin

time (h)

Deprotection of Aloc on PAL-PS resin

100

.04 60

de 40

6 20

0 0 2 6 12 24 time (h)

Fig. 6.3 Comparison of deprotection rates of Aloc from [Phe(NHAloc)4, D-Pro 9Dyn A(1- 1 1)NH-resin synthesized on PAL-PEG-PS (top) vs PAL-PS (bottom) resins. 126

precursors for the preparation of affinity label-containing DynAanalogues 3a, 3b, 4a and

4b. Crude peptides were obtained after the cleavage reactions and analyzed by reverse phase

HPLC.The amine peptides 2a and 2b assembled onPAL-PEG-PSandPAL-PSresin,

respectively, were obtained in excellent purity (> 90%, Fig 6.4). The potential affinity label

derivatives DynAassembled onPAL-PEG-PS,however, contained more side products than

those assembled on thePAL-PSresin. The isothiocyanate 3a contained approximately 30%

side products in addition to the desired peptide, whereas the isothiocyanate 3bwas the only

major product (>88%) assembled on thePAL-PSresin (Fig 6.5). Reverse phaseHPLC

analysis of the bromoacetamide derivatives assembled onPAL-PEG-PSandPAL-PSresin

showed two major peptide products in approximately 2:1 and 1:3 ratios, respectively (Fig.

6.6). The less polar peptide (tR = 20.8 min) had a molecular ion (M+1 = 1498.5) which

corresponded with the bromoacetamide derivative Dyn A, while the more polar peptide

(tR = 19.7 min) had a molecular ion (M+1 = 1435.5) which was identifiedasthe

hydroxylated peptide [Phe(NHCOCH2OH)4]Dyn A(1-11)NH2. Therefore, although the Aloe

deprotection was faster on thePAL-PEG-PSresin (complete reaction within 3 h), the crude potential DynAaffinity label derivatives 3a and 4a contained more impurities compared to the crude peptides 3b and 4b assembled on thePAL-PSresin.

6.5 Conclusions

The allyloxycarbonyl(Aloc)group provides orthogonal protection of the para-amine on phenylalanine which can be selectively removed by palladium (0) without affecting the

N-terminal Fmoc group or acid-labile side chain protecting groups. Deprotection rates of the TN

11111.

111111.

PAL-PEG-PS (2a) PAL-PS (2b)

Fig. 6.4 Chromatograms of crude peptide [Phe(NH2)4,D-Pro10]DynA(1-11)NH, from PAL- PEG-PS (left) and PAL-PS (right) resins. Column: Vydac (Cis).Solvent: A = aqueous 0.1% TFA, B= acetonitrile with 0 1% TFA. Flow rate: 1.5 mL/min. Gradient: 0- 75% B over 50 min. ; ; Wleme009

PAL-PEG-PS (3a) PAL-PS (3b)

Fig. 6.5 Chromatograms of crude peptide [Phe(N=C=S)4,D-Pro10]Dyn A(1-11)NH2from PAL-PEG-PS (left) and PAL-PS (right) resins. For chromatographic conditions see Fig. 6.4.

00 1114. 080.0

7141

7.0..

'WS

'WO

SW.

S0111.

1.0

;--

PAL-PEG-PS (4a) PAL-PS (4b)

Fig. 6.6 Chromatograms of crude peptide [Phe(NHCOCH2Br)4,D-Pro10JDynA(1-1 1)NH2 from PAL-PEG-PS (left) and PAL-PS (right) resins. For chromatographic conditionsee Fig. 6.4. 130

Aloc group while the peptides are attached to the resin are dependent upon the resin used.

Our study shows that the PAL-PEG-PS resin facilitates the Aloe removal from protected

[Phe(NHA1oc)4,D-Pro9Dyn A(1-11)NH2, presumably due to the greater accessibility of the catalyst reagent compared to the PAL-PS resin. The crude potential Dyn A affinity label derivatives obtained from the PAL-PS resin, however, contained fewer side products than the peptides from the PAL-PEG-PS resin. 131

CHAPTER 7

SYNTHESIS OF A DYNORPHIN A PRECURSOR FOR RADIOLABELING

Leena Leelasvatanakij''' and Jane V. Aldrich'

'College of Pharmacy, Oregon State University 'School of Pharmacy, University of Maryland at Baltimore 132

Abbreviations:

Abbreviations used for amino acids follow the rules of the IUPAC-IUB Joint

Commission of Biochemical Nomenclature in Biochem. 1 1984, 219, 345-373. Amino acids are in the L-configuration except when indicated. Additional abbreviations used are as follows: Boc, t-butyloxycarbonyl; t-Bu, tent- butyl; DCM, dichloromethane, DIC, N,N'- diisopropylcarbodiimide;DIEA, N,N'-diisopropylethylamine; DMA, N,N-dimethylacetamide; Dyn A, dynorphin A; DMF, NN-dimethylformamide; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, (9-fluorenylmethoxy)carbonyl; HOBt,1- hydroxybenzotriazole;HPLC,highperformanceliquidchromatography;OPfp, pentafluorophenyl ester; PAL, peptide amide linker; Pmc, 2,2,5,7,8-pentamethylchroman-6- sulfonyl; TFA, trifluoroacetic acid. 133

7.1 Abstract

Protected [Phe(3',5'42,4'-NH2)4,D-Pro10]Dyn A(1-11)NH2, 1, was synthesized by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting

groups. The NovaSyn TG® resin was functionalized to bear a base-labile 4-hydroxymethylbenzoic acid linkage (substitution value 0.19 mmol /g) and used for the synthesis of the protected peptide 1. The peptide-resin linkage was cleaved by ammonolysis in isopropanol

without removing other side chain protecting groups. Different ammonolysis times affected the recovery and purity of the protected peptide 1. The longest reaction time (5 days) gave the peptide with the highest purity (74.7%), but the lowest recovery (31.0%). Catalytic hydrogenation (or tritiation) of this peptide will yield the desired precursor for introduction of the affinity label group. 134 7.2 Introduction

Opiate drugs and opioid substances produce various pharmacological and

physiological effects through a heterogenous opioid system. Both multiple opioid receptors

(i.e. p,, 8 and lc opioid receptors) [Martin et al., 1976; Lord, 1977] and numerous endogenous

opioid peptides (e.g. enkephalins, endorphins and dynorphins) [Holt et al., 1983] have been

identified.Research in the opioid area involves the development of ligands with high

selectivity for a particular receptor. Such ligands are useful as potential therapeutic agents

and as tools for characterization of opioid drug-receptor interactions. Studies ofan opioid receptor type can be greatly facilitated by using selective ligands.

Affinity labels are classified into electrophilic affinity labels and photoaffinity labels.

Both are useful because of their complimentary applications. Electrophilic affinity labelscan be utilized in vivo for pharmacological studies, while photoaffinity labelsare reversible until after photoactivation occurs [Glasel and Venn, 1981]. Non-peptide affinity labels have been used to irreversibly block selected receptor populations in tissues suchas the guinea pig ileum and brain membranes which contain multiple receptor types,so that the remaining receptors can be studied in isolation [Rothman et al., 1990]. For example, BIT (2-(p- ethoxybenzy1)-1-diethylaminoethy1-5-isothioicyanatobenzimidazole) selectively alkylates

opioid receptors [Rice et al., 1983], whereas FIT (N-phenyl-N-[1-(2-(p-isothiocyanato) phenylethyl)-4-piperidinyl]propanamide) and superFlT selectively acylate 8 opioid receptors

[Burke et al., 1983, 1986]. p-FNA (P-funaltrexamine) binds reversibly to bothp, and ic receptors, but only reacts irreversibly with p receptor. [Takemori and Portoghese, 1985].

Peptide-based affinity labels (e.g. DALECK (Tyr-D-Ala-Gly-Phe-LeuCH2C1), DALCE (Tyr- 135

D- Ala- Gly- Phe- leu- CysOH) and DAMK (Tyr-D-Ala-Gly-MePheCH2C1)) have also been

prepared and used as biochemical tools to study IA and 6 opioid receptors [Venn and Barnard,

1981; Szucs et al., 1987; Benhyhe et al., 1987; Bowen et al., 1987].

Affinity labels need to be labeled (i.e. with a radiolabel) for maximum utility. A

radioligand should have very high affinity for the binding site to be labeled, be highly

selective for that site and have high enough specific radioactivity to be usableat a low

concentration in order to maximize its binding at the preferred site relative to thatat the next-

preferred site [Goldstein et al., 1988].Radioisotopes with a high-energy mode of decay

such as 3H and 1251 are normally incorporated into affinity label-containing ligands.

We are interested in studying lc opioid receptor structure and function, since selective agonists for lc receptor produce analgesia without the side effects associated with morphine

or other IA receptor-selective analgesic agents [Milian, 1990; Rees, 1992]. Dyn A is a 17-

amino acid endogenous peptide possessing high affinity andsome selectivity for lc opioid receptors [Chavkin et al., 1982]. The [D-Pro9Dyn A(1-11) analog, however, shows greater selectivity for K receptors than the heptadecapeptide itself [Gairin et al., 1985; 1989]. The specific goal in this study was to synthesize potential affinity labels forK opioid receptors using [D-Pro'°]Dyn A(1-11) as a prototype. The peptide structure offers flexibility inthe design of affinity labels and allows incorporation of other useful functionalities(e.g. radiolabel and/or biotin). Our goal is to identify modified Dyn A analogues which will retain high lc receptor affinity, exhibit ic receptor specificity and achieve irreversible attachment to ic opioid receptors.Peptides which show wash-resistant inhibition of binding to cloned wild type opioid receptors will subsequently be labeled using a radiolabel. The 136

labeled peptides will be reacted with cloned opioid receptors followed by isolation of the receptor complexes. The point of attachment of the affinity label can then be determined after proteolytic digestion of the receptor.

We designed a potential peptide affinity label with a radiolabel andan affinity labeling group incorporated in the same residue, so that the radiolabel will remain associated with the affinity label even after proteolytic digestion of affinity labeled receptors. Labeling methods for peptides depend upon the label incorporated (radiolabel and/or other label), sensitive groups on the peptide and the length of the peptide. This chapter describes the synthesis of a Dyn A precursor for radiolabeling. Since further modifications of the peptide is planned after assembly of the peptide chain, it is necessary to isolate the peptide in its protected form.Solid phase synthesis of the protected peptide requires an appropriate peptide-resin linkage and cleavage conditions which permit the peptide to be cleaved without removing the side-chain protecting groups. Thus, we functionalized the NovaSyn TG® resin and incorporated linker B (hydroxymethylbenzoic acid pentafluorophenyl ester)as a base- labile linkage. The NovaSyn TG® resin contains a polyethylene glycol polystyrene (PEG-

PS) support which provides advantages over the commonly used polystyrene support. The

PEG-PS support swells in a wide range of solvents and has greater solvation capacity than the PS support (chapter 6). The 9-fluorenylmethoxycarbonyl group (Fmoc) was utilizedas the amine protecting group since it provides the flexibility of an orthogonal protection strategy and permits the use of mild acid for the final deprotection of side-chain protecting groups. We incorporated FmocPhe(3',5'42,4'-NH2) in position 4 of [D-PronDyn A(1-11)

NH2 peptide. After assembly of the peptide chain, the protected peptide was cleaved from 137

the functionalized NovaSyn TG® resin by ammonolysis: subsequentcatalytic hydrogenation

will yield protected peptide precursor of the affinity label. Theconditions used for catalytic

hydrogenation reaction will be optimized for small scale synthesisto give the unlabeled

peptides. and similar conditions can later be appliedto catalytic tritiation which will be

performed by New England Nuclear. Following tritiation of the protectedpeptide, the

affinity labeling group (e.g. isothiocyanateor bromoacetamide) can be attached to the p-

amino group of the Phe(31,5'-3H2,4'-NH2) and the peptide deprotected(Fig. 7.1).

TFA Final peptide scavenger

Fig. 7.1 Synthesis of tritium-labeled peptides bearinga reactive functionality. 138

7.3 Experimental section

7.3.1 Materials

The NovaSyn TG® resin was purchased from Calbiochem-Novabiochem Corp. (La

Jolla, CA). Linker B-OPfp ester (hydroxymethylbenzoic acid-OPfp) and N,N-diisopropylethylamine (DIEA) were purchased from Perseptives (Bedford, MA). FmocNle and

FmocLys(Boc)NCA (N-carboxyanhydride) amino acids were obtained from BioResearch

(San Diego, CA). Phe(3',5'42,4'-NH2) was from Bachem Bioscience (King of Prussia, PA), and it was converted to FmocPhe(31,5'I2,4' -NH2) in this laboratory as described below. All other amino acids were obtained from Bachem Inc. (Torrance, CA). Reagents were obtained from Aldrich (Milwaukee, WI), and all solvents used (HPLC grade) were obtained from

Burdick & Jackson (Muskegon, MI).

The peptides were synthesized on a Biosearch 9500 automated peptide synthesizer

(Novato, CA). FmocPhe(3',5'I2,4' -NH2) and crude peptides were analyzed on a Beckman

System Gold high performance liquid chromatography (HPLC) system consisting of a model

126 solvent module, model 168 detector and model 507 autosampler. The HPLC column was a Vydac analytical column (C18, 300 A , 5 11, 4.6 x 250 mm) equipped with a Vydac guard cartridge. The samples were eluted using a linear gradient of 0%-75% solvent B over

50 min with a flow rate of 1.5 mL/min (except where indicated) and detected at 214 nm; solvent A was aqueous 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA.

Preparative reversed phase HPLC was performed on a Rainin gradient HPLC system using a Vydac semipreparative column (C18, 300 A, 10 ii, 21 x 250 mm) equipped with a Vydac 139

guard cartridge. The amino acid analysis was done by the Protein and Nucleic Acid Facility,

Beckman Center, Stanford University. The samples were hydrolyzed for 24 h at 110 °C with 6 N HC1 plus 1% phenol (peptide samples) or 1/1 propionic acid/conc HC1 (peptide- resin samples) and analyzed on a Beckman Model 6300 amino acid analyzer using ninhydrin

detection.Molecular weights of modified amino acids and synthetic peptides were

determined by fast atom bombardment (FAB) mass spectrometry in the positive mode on a

Kratos MS 50RFTC in the Department of Agricultural Chemistry at Oregon State University,

Corvallis, OR. Elemental analyses were performed by MWH Laboratories, Phoenix, AZ.

7.3.2 Methods

7.3.2.1 NovaSyn TG* resin functionalization (Fig. 7.2)

NovaSyn TG® resin (1.01 g, reported substitution value: 0.29 mmol/g) was swollen

with DCM (20 mL) for 15 min and then washed with DCM/DMA (1:1) (3 x 20 mL). The

resin was neutralized with a 10% DIEA in DCM (3 x 15 mL) and washed with DCM (6 x

20 mL) and DCM/DMA (1:1) (3 x 20 mL).FmocNle was incorporated as an internal standard.FmocNle (0.51 g, 1.4 mmol) and HOBt (0.22 g, 1.4 mmol) in DMA (3 6 mL) were coupled to the resin with 3.2 mL of DIC (1.4 mmol) in DCM. The ninhydrin test was negative (yellow) after 2 h. An aliquot of peptide-resin was taken for Fmoc analysis (calcd.

substitution value: 0.22 mmol/g). The Fmoc protecting group was removed using piperidine/toluene/DMA (30%135%135%),and the resin then washed with DCM/DMA and

DCM (3 x 20 mL each). A positive blue color was obtained from the ninhydrin test. 4- 140

NovaSyn TG Resin

10% DIEA

FmocNle

DIC/HOBt

FmocNle - NovaSyn TG Resin

piperidine/toluene/DMA (30%/35%/35%)

Nle - NovaSyn TG Resin

1)1 HOCH2 COPfp / HOAt

HOCH2 Nle - NovaSyn TG Resin

FmocLys(Boc)-NCA

1-acetylimidazole (capping)

FmocLys(Boc) C Nle - NovaSyn TG Resin

Fig.7.2 NovaSyn TG®resin functionalization. 141

Hydroxymethylbenzoic acid pentafluorophenyl ester (linker B, 0.37g, 1.2 mmol, 4-fold

excess) was coupled to the resin with HOAt (0.16 g, 1.2 mmol) in DCM (5 mL), and the

resin was washed with DCM/DMA (1:1) and DCM (5x 20 mL each). The coupling,

monitored by the ninhydrin test, was complete after 5 h.FmocLys(Boc)-NCA (0.43 g, 0.92

mmol, 3-fold excess) in a mixture of toluene (4.37 mL) and NMM (9.26 µL, 84.3 umol )was

coupled to the resin, and the resin then washed with toluene (5x 10 mL) and Me0H (3 x 10

mL). Fmoc analysis after 1, 3 and 4 h showed substitution values of 0.22, 0.28 and 0.28

mmol/g, respectively. The resin was treated with 0.30 M 1-acetylimidazole in DCM (20mL) for 30 min, washed with DCM/ DMA (1:1) and DCM (3x 20 mL each) and then dried in vacuo. Quantitative amino acid analysis after 4 h coupling showed a substitution of 0.19 and

0.16 mmol/g of Nle and Lys, respectively.

7.3.2.2 Synthesis of FmocPhe(3',5'-I2,4'-NH)

Fmoc-Cl (0.38 g, 1.46 mmol) in dioxane (3 mL) was added toa solution of Phe(3',51-

I24'-NH2) (0.64 g, 1.47 mmol) in 10% Na 2CO3 /dioxane (1/1), 5 mL) and the mixturewas stirred at 0 °C for 1 h and at room temperature for 12 h. The mixturewas then poured into ice water (20 mL) and extracted with ether (2 x 20 mL). Theaqueous layer was chilled in a ice bath and acidified with 1 N HC1 to pH 2, the white precipitate collected by filtration, washed with 0.1 N HC1, H2O and dried in vacuo. The residuewas dissolved in EtOAc and the solution washed with 0.1N HC1 and H2O, and dried with MgSO4. Fmoc Phe(3',5'42,4'-

NH2) was recrystallized from THE (0.90 g, 93.7% yield): mp 132-134°C; TLC (silica gel,

DCM/Me0H, 1:1) Rf = 0.62; FAB-MS m/z 655 (M+1); [a]E, +27.6 (c 0.4, DCM). 142

Anal. calcd for C24H2012N2041.5THF: C 47.24, H 4.2, N 3.67%. Found: C 47.64, H 3.97, N

3.73%.

7.3.2.3 Synthesis of protected [Phe(3',5'-12,41-NH2)4,D-Pro wiDyn A(1-11)NH-functionalized NovaSyn TG® resin (1) (Fig. 7.3)

The potential affinity label precursor protected [Phe(3',5'42,4'-NH 2)4,D-ProilDyn

A(1-11)NH2, 1, was assembled on the functionalized NovaSyn TG® resin (0.80 g,

substitution value from quantitative amino acid analysis: 0.16 rnmol/g). All amino acids

used were Fmoc-amino acids, except for BocTyr(t-Bu) which was incorporated at the N- terminus. Side chain protecting groups used were Pmc for Arg, t-Bu for Tyr and Boc for

Lys. The resin was swollen in DCM/CMA for 15 min before beginning the synthesis. The

solid-phase synthesis cycle was started by washing the resin with 4 x 10 mL DCM/DMA and

removing the Fmoc protecting group from the C-terminal amino acid using a piperidine/

toluene/DMA (30%/35%/35%) solution. Fmoc-amino acids (4.0-fold excess in DMA) were

coupled to the peptide with an equimolar amount of HOBt and an equal volume of 0.40 M

DIC in DCM for 2 h, followed by washes with DCM/DMA (4 x 5 mL). After coupling the

N-terminal amino acid, the peptide-resin was washed with DCM/DMA (10 x 5 mL), DCM

(5 x 10 mL) and Me0H (4 x 5 mL) and dried in vacuo. The protected peptide-resin was

submitted for quantitative amino acid analysis (%peptide found compared to Nle internal

standard: 93.0%). 143

Boc FmocLys-linker B-Nle-NovaSyn TG resin

piperidine/toluene/DMA (30%/35%/35%) 1 deprotection/ coupling cycles Fmoc-amino acids DIC/HOBt

t-Bu CH2 PmcPmcPic Boc I I I I I I Boc-Tyr-Gly-Gly-NH-CH-CO-Leu-Arg-Arg-Ile-Arg-D-pro-Lys-linker B-Nle-NovaSyn TG resin

NH2 NH3/i-PrOH

t-Bu CH2 PmcPmc Pic Boc

I I I I Boc-Tyr-Gly-Gly-NH-CH-co-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-CONFI2

Fig. 7.3 Synthesis of dynorphin A precursor for radiolabeling. 144

7.3.2.4 Ammonolysis of protected peptide-resin 1

Aliquots of protected peptide-resin 1 (0.61, 0.52 and 0.95 g) were treated with ammonia in i-PrOH in a heavy-walled pressure bottle (250 mL, 6' long, 2.5' outer diameter) for 1, 3 and 5 days, respectively. At the end of the reaction time, the resins were filtered and washed with i-PrOH (3 x 10 mL). The i-PrOH filtrates were combined and evaporated to yield the crude protected peptide amide. The crude protected peptides were characterized by an analytical RP-HPLC column (C18, 300 A, 5 .t, 4.6 x 250 mm), where solvent A was aqueous 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. A linear gradient of 55 to 100% acetonitrile over 30 min was used at a flow rate of 1.5 mL/min and peptides were detected at 214 nm. Aliquots of the resins left after ammonolysis were sent for amino acid analysis to determine percentages of cleavage. The crude protected peptide amide after

5 days ammonolysis was purified on a preparative RP-HPLC column (C18, 300 A, 5 22 x

250 mm) using the conditions described above except that the wavelength monitored was

280 nm. The purified peptide was analyzed by analytical RP-HPLC (tR = 22.7 min) and

FAB-MS ((M+1) = 2683).

7.4 Results and discussion

The dynorphin A precursor for labeling was prepared by solid phase peptide synthesis

(SPPS) (Fig.7.3) and the protected peptide cleaved from the resin in preparation for further modification in solution. Thus Na-amino and side chain protecting groups and a peptide- resin linkage of differing labilities were needed. We utilized the base-labile 9-fluorenylmethyloxycarbonyl (Fmoc) group to protect the N"-amine, and acid-labile tert-butyl (t-Bu) 145 type groups to protect the side chains of amino acids. A resin with a linker that yields protected peptide amides was preferred, since the amide derivatives of dynorphin A possess higher chemical and metabolic stability [Leslie and Goldstein, 1982] than the acid derivatives. Commercially available resins such as the PAL® (peptide amide linker) resin, an MBHA (4-methylbenzhydrylamine) resin with a 5-(4-aminomethy1-3,5-dimethoxyphenoxy)valeric acid linker [Albericio et al., 1990], and the Samba resin (4-succinylamino-

2,T,4'-trimethoxyphenyl-propionic acid) [Penke et al., 1989] yield peptide amides upon treatment with acids. Final cleavage requires concentrations of trifluoroacetic acid (TFA) which also remove the t-butyl-type side chain protecting groups used in Fmoc syntheses.

We, therefore, developed a base-labile resin that yields protected peptide amides following ammonolysis.

7.4.1 NovaSyn TG® resin functionalization

The NovaSyn TG® resin was functionalized to incorporate the base-labile 4-hydroxymethylbenzoic acid linkage (Fig. 7.2). The NovaSyn TG® resin contains a polyethylene glycol spacer which enhances its compatibility with various solvents. First, FmocNle was incorporated as an internal standard using FmocNle, DIC and HOBt; after removal of the

Fmoc group by piperidine, 4-hydroxymethylbenzoic acid (linker B) was incorporated by its pentafluoro-phenyl ester and HOAt. The functionalized NovaSyn TG® resin was coupled to the intramolecularly activated FmocLys(Boc)-NCA, and subsequently capped with 1- acetyl-imidazole to acylate any unreacted sites. Coupling of FmocNle to the resin, as monitored by a negative result from the ninhydrin test, was complete within 2 h, and the 146 substitution value calculated from the Fmoc analysis was comparable to the reported value

(0.22 vs 0.29 mmol/g, respectively). Coupling of 4-hydroxymethylbenzoic acid (linker B) to the Nle-NovaSyn TG® resin was complete after 5 h. The resin was then esterified with

FmocLys(Boc)-NCA. Urethane-protected amino acid N-carboxy anhydrides (UNCAs) are very reactive amino acid derivatives, and thus are efficient reagents for peptide synthesis

[Wakselman et al., 1994]. They are soluble in various solvents and can be used at high concentrations for difficult coupling reactions.UNCAs are activated internal mixed anhydrides which form peptide bonds directly, quickly and cleanly with carbon dioxide as the only byproduct [Fuller et al., 1990]. Our study shows that the FmocLys(Boc)-NCA was successfully esterified to the functionalized resin after 3 h; the completeness of the reaction was monitored by quantitative Fmoc analysis at 1, 3 and 4 h coupling and showed loadings of 0.22, 0.28 and 0.28 mmol/g, respectively. Quantitative amino acid analysis after Fmoc removal showed a substitution of 0.19 and 0.16 mmol/g resin for Nle and Lys, respectively.

7.4.2 Peptide syntheses

The synthesis of dynorphin A analogues in this model study involves modifications of phenylalanine in position 4 of [D-PronDyn A(1-1 OM-12.The affinity label (e.g. isothiocyanate and bromoacetamide) and the radiolabel (e.g. tritium) group will be incorporated on the same residue. Tritiation of the model peptide will be accomplished by incorporation of Phe(3',5'42,4'-NH2) into the peptide (Fig. 7.1), followed by catalytic tritiation of a protected peptide precursor. We prepared FmocPhe(3',5'I2,4' -NH2) from

Phe(3',5'42,4'-NH2) using Fmoc-Cl under basic conditions. The product was obtained in a 147 good yield (93.7%) after recrystallization from THE (Fig. 7.4). Because of the bulk of the

3',5'-bisiodine functionality, the p-amino group was left unprotected during the assembly of the peptide chain.

The functionalized NovaSyn TO® resin described in the previous sectionwas used for solid phase peptide chain assembly of the model dynorphin A precursor for labeling. The

Fmoc protecting group was removed by piperidine/toluene/DMA (30%/35%/35%) and the next Fmoc-amino acid was coupled to the growing peptide chain using DIC and HOBt in

DCM/DMA (1/1). Completeness of individual coupling reactions was monitored by the ninhydrin test. Coupling of each amino acid was achieved after 2 h, except for the D-Pro'° which required a recoupling. After peptide chain assembly, the peptide on the resinwas submitted for amino acid analysis. The results shows a substitution value of 0.12 mmol/g,

93.1% of the expected value based on the internal standard, and the expected ratios of individual amino acids.

Protected peptide 1 was cleaved from the functionalized NovaSyn TO® resin by ammonolysis using isopropanol instead of methanol to avoid the methyl ester side product.

Previous studies [Story and Aldrich, 1992] in our laboratory indicated that the yield and purity and of the crude peptide Boc-Tyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 from ammonolysis depended upon reaction times. A negligible recovery of the peptide was obtained after 4 h ammonolysis, but the yield was increased to 83% when the reaction time was extended to

4 days. A 2.5" x 6", 250 mL heavy-walled pressure bottle was found to be optimum in these studies. In the current study, we compared reaction times of 1, 3 and 5 days for ammonolysis of protected peptide 1 using the same dimension bottle with a teflon lined cap. Cleavage of 148

a. - O a

T 1

Fig. 7.4 Chromatograms of Phe(31,51-I,,4' -NHS) (dotted line) and FmocPhe(3',5' -I 2,4'-NH2) (solid line). Column: Vydac (C18). Solvent: A = aqueous 0.1%TFA, B = 0.1%TFA/AcCN. Flow rate: 1.5 mL/min. Gradient: 0-75%B over 50 min. 149 the peptide was determined by amino acid analysis by comparison of the amino acid content of peptide vs the internal standard Nle on the resin (Table 7.1). The majority of the protected peptide / was cleaved from the resin after 1 day ammonolysis (87.4% cleavage), while the recovery of peptide for different reaction times varied (%recovery: 54.8, 67.0 and 31.0%, respectively). The crude protected peptide after ammonolysis showed a major peak by analytical RP-HPLC (tR = 22.7 min, Fig. 7.5) plus several side products. The longer reaction time (5 days) gave the crude protected peptide of the highest purity (74.7%) with an acceptable recovery (31.0%). The crude protected peptide with lower purities, 27.6 and

22.9%, was obtained after 1 and 3 days ammonolysis (Fig. 7.5), respectively. The crude protected peptide after 5 days ammonolysis was purified by reverse phase preparative HPLC and further characterized by analytical revese phase HPLC and FAB-MS. The pure peptide obtained showed a single peak (96%) by reverse phase HPLC and the expected mass by

FAB-MS ((M+H)+ = 2683).

The ultimate goal of our study is to prepare dynorphin A analogues containing a radiolabel and an affinity labeling group on the same residue. These labeled peptides, hopefully, will assist in the isolation and characterization of lc opioid receptors.The procedures described in this chapter will be used as guidelines for the synthesis of such analogues. The optimum conditions will be determined for the catalytic hydrogenation and subsequently applied to the catalytic tritiation (to be performed at New England Nuclear).

An affinity labeling group (e.g. isothiocyanate or bromoacetamide) will then be incorporated into the p-amino group of the Phe(31,51-3H2,4'-NH2), followed by deprotection of the peptide.

The purified tritiated affinity label peptides will be characterized by analytical RP-HPLC. Table 7.1 Characterization of [Phe(31,5'-12,4'-NH2,)4, D-ProlDyn A(1-11)NH2 before and after ammonolysis'.

Amino acid analysis Peptide Nle internal % Peptide % Cleavage IT2 G F L R I P K substitution standard on resin (% Recovery) (1) (2) (1) (1) (3) (1) (1) (1) (mmol/g) (mmol/g)

Before 1.53 0.88 0.97 3.35 0.98 0.96 1.33 0.12 0.13 93.1 cleavage

After cleavage 1 d 0.02 0.17 11.7 88.3 (54.8) 3 d 0.01 0.21 3.3 96.7 (67.0) 5 d 0.01 0.36 4.7 95.3 (31.0)

'Theoretical values are shown in parentheses. 2Tyr was destroyed during hydrolysis and was excluded from the calculation. 151

.1 v

Fig. 7.5 Chromatograms of crude protected peptide 1 after ammonolysis; 1 (top), 3 (middle) and 5 (bottom) days. Column: Vydac (C18).Solvent: A = aqueous 0.1%TFA, B = 0.1%TFA/AcCN. Flow rate: 1.5 mL/min. Gradient: 55-100% B over 30 min. 152

7.5. Conclusions

The Fmoc protection strategy provides orthogonality for the synthesis of protected peptide amides. A proper peptide-resin linkage which permits cleavage without removing the side chain protecting groups is required. The NovaSyn TG® resin containing a base- labile 4-hydroxymethylbenzoic acid linkage was successfully developed in our laboratory and used for peptide synthesis. The resin gave the expected model dynorphin A precursor

1, [Phe(3',5'42,4'-NH2)4,D-Pro10]Dyn A(1-11)NH2, needed for labeling with 93.1% of the expected peptide substitution, calculated from amino acid analysis of peptide-resin. The protected peptide 1 was liberated from the resin by ammonolysis in isopropanol, and greater than 85% of peptide was successfully cleaved from the resin. The longest reaction time (5 days) gave the crude peptide with the highest purity (74.7%) compared to those from 1

(27.6%) or 3 (22.9%) days ammonolysis. 153

CHAPTER 8

CONCLUSIONS

Potential affinity label derivatives of dynorphin A basedon [D-Prom]Dyn A(1-

11)NH2 were synthesized for use as pharmacological probes forlc opioid receptors. A

reactive functionality (isothiocyanate or bromoacetamide)was incorporated into the N-

terminal "message" or C-terminal "address " sequences of the parent peptide [D-Prom]Dyn

A(1-11)NH2. Modifications in the "message" sequence (chapter 4) involved the replacements of tyrosine in position 1 with the isothiocyanate derivative of phenylalanine, and phenylalanine in position 4 with the isothiocyanateor bromoacetamide derivatives of p-aminophenylalanine.Structural changes in the "address" sequence (chapter 5)were performed at isoleucine in position 8; Ile' was replaced with either the isothiocyanateor bromoacetamide derivatives of p-aminophenylalanine or lysine. The peptides containingan amine (i.e. without a reactive functional group)were also prepared and used as reversible controls for the pharmacological assays.

We have developed synthetic strategies for the preparation of these potential affinity label derivatives of dynorphin A.Initially, all protected peptides were assembledon a polyethylene glycol (PEG)-polystyrene (PS) support witha peptide amide linker (PAL) using standard Fmoc chemistry. The allyloxycarbonyl (Aloc)group was introduced into the side chain of the amino acid residue where a reactive functionalgroup was to be incorporated in order to provide orthogonal protection. The Aloc groupcan be selectively removed by palladium (0) in DCM/5% AcOH/2.5% N-methylmorpholine, and the reactive functional 154 group then introduced. Finally, peptides were liberated from the support and other side chain protecting groups were removed simultaneously using modified reagent K (87.5% TFA/5% phenol /5% H20/2.5% thioanisole); ethanedithiol was excluded from the cleavage cocktail to prevent reaction with the alkylating group. Reverse phase HPLC analysis of the crude peptides after cleavage indicatedthe purity of the peptides.Crude peptides from the

[Phe(X)I,D-Prom]Dyn A(1-11)NH2 series, where X = -NH2 (amine compound) or -N=C=S

(for the potential affinity label derivative), and [Lys(X)8,D-Pro10]Dyn A(1-11)NH2 series, where X = -H (amine compound), or -N=C=S and -NHCOCH2Br (for potential affinity label derivatives), were obtained in excellent purity. Affinity label derivatives from the other series, namely [Phe(X)4,D-Pro Dyn A(1-11)NH2] and [Phe(X)8,D-Pro 11Dyn A(1-11)NH2 where X = -N=C=S and = -NHCOCH2Br, however, contained side products, including the starting amine and other side products, (e.g. the hydroxyl derivative (-NHCOCH2OH)) as well.

The peptides' affinities (IC50 values) for different opioid receptors were determined in radioligand binding assays using cloned opioid receptors stably expressed in Chinese hamster ovary cells. Preliminary binding assay data (Table 4.3 and 5.2) indicated that, except for the bromoacetamide derivative of [Phe(NH2)11,D-Pro10]Dyn A(1-11)NIi,all peptides had high affinity for lc opioid receptorsSurprisingly, [Lys8,D-Pro10]Dyn A(1-

11)NH2 exhibited the highest affinity for lc opioid receptor(IC= 0.16 nM) with comparable lc selectivity to the parent peptide. The isothiocyanate derivatives generally showed higher affinity for lc opioid receptors than did the bromoacetamide derivatives (the bromoacetamide group was not introduced into Phe'). Incorporation of a reactive functional 155 group in the N-terminal "message" and C-terminal "address" sequences of Dyn A yielded peptides with varying selectivity for opioid receptors:lc > µ >> S (for the "message" sequence modifications) andxµ > 8 (for the "address" sequence modifications).

Since some of the potential affinity label derivatives of Dyn A assembled on PAL-

PEG-PS resin contained side products, we investigated a different resin, PAL-PS resin, using

[Phe(X)4,D-Pro10]Dyn A(1-11)NH2 as a model (chapter 6). Rates of Aloc deprotection and purity of crude peptides from the two resins were compared. Aloe deprotection was greatly enhanced on the polyethylene glycol polystyrene support; removal of Aloc from peptides assembled on this support was complete within 3 h compared to 24 h from peptides assembled on a polystyrene support. The polyethylene glycol spacer in the PAL-PEG-PS resin may play an important role in enhancing Aloe deprotection rates by allowing complete

solvation of the reactive sites which would result in a more efficient reaction. Crude peptides

assembled on both resins were characterized by reverse phase HPLC. The amine compounds

assembled on either the PAL-PEG-PS or the PAL-PS resin both showed excellent purity,

but the potential affinity label derivatives assembled on the PAL-PEG-PS resin contained more side products than those synthesized on the PAL-PS resin.

The last part of this research focused on the synthesis of a dynorphin A precursor for radiolabeling using protected [Phe(31,5'42,4'-NH2,D-Pro10iDyn A(1-11)NH 2 as a prototype.

NovaSyn TG® resin was functionalized by incorporating a base-labile 4-hydroxymethyl-

benzoic acid linkage (linker B) (substitution value 0.19 mmol/g) and used for the synthesis.

The protected peptide was obtained with 93.1% of the expected peptide substitution,

calculated from comparison of amino acid analysis of the peptide on the resin vs. an internal 156 standard Nle. Almost 90% of the protected peptide was liberated from the resin after 1 day ammonolysis in isopropanol. An extended reaction time (5 day), however, gave the product with higher purity. This peptide can then be subjected to catalytic tritiation and the affinity label group then introduced. This strategy allows us to introduce a radiolabel and affinity labeling group on the same residue. The radiolabel will then remain associated with the affinity label even after proteolytic digestion of affinity labeled receptors. The radiolabel affinity label peptides will hopefully be useful for studies of interactions of peptide ligands with x opioid receptors.

Additional pharmacological assays will be performed to identifyaffinity label derivatives of dynorphin A which bind irreversibly to opioid receptors. The synthetic strategies described in the previous section will then be applied to these lead peptides, optimum conditions will then be determined for catalytic tritiation (to be performed at New

England Nuclear), and a reactive functionality (e.g. isothiocyanate or bromoacetamide) will be incorporated into the tritiated protected peptide. Deprotection will then give the desired peptides containing both an affinity label and tritium label which can be used in future pharmacological studies. 157

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