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(111)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 lc
µ > 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-PEGPS.
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
by
Leetia Leelasvatanakij
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctoral of Philosophy
Completed April 22, 1996
Commencement June 1996
°Copyright by Leena Leelasvatanakij
April 22, 1996
All Rights Reserved
Doctor of Philosophy thesis of Lemt Leeiasvataitakij !.7,resented on April 22 1996
APPROVED:
Redacted for Privacy
Maj
Professor,
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 of Oregon 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 giving their
precious time and advice. I would like to acknowledge Dr. Thomas Murray who supervised
the radioligand binding assays and Elizabeth Olenchek who expertly performed the
experiments. My thanks also go to Dr. Brian Arbogast for performing the fast 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 State University and
the School of Pharmacy, University of Maryland at Baltimore; it has been a pleasure working
with them. I appreciate my colleagues, Dr. Seksiri Arttamangkul, Dr. Heekyung Choi, 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 have had
together.
My gratitude also goes to the Royal Thai Government for providing financial
support, and their staff for all of the titre and energy that they have put into helping my life
run so smoothly during my study in the United 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)
2.1.2 Proenkephalin A (PROENK)
2.1.3 Proenkephalin B or prodynorphin (PRODYN)
2.2 Dynorphin A
2.2.1 Metabolism and enzyme inhibitors
2.2.2 Conformational studies
2.2.3 Structure-activity relationships (SAR)
2.3 Opioid receptors
2.3.1 Opioid receptor types and their ligands
2.3.2 Isolation and purification of opioid receptors
2.3.3 Molecular biology and cloned opioid receptors
2.4. Affinity label-containing opioids
2.4.1 Non-peptide affinity labels
2.4.2 Peptide affinity labels
CHAPTER 3 LITERATURE REVIEW: SOLID PHASE PEPTIDE
SYNTHESIS
9
9
10
11
12
14
16
18
20
28
30
35
38
41
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
4.3.2 Methods
4.3.3 Receptor binding assays
4.4 Results and discussion
4.4.1 Amino acid syntheses
4.4.2 Peptide syntheses
4.4.3 Receptor affinity
4.5 Conclusions
CHAPTER 5 SYNTHESIS OF POTENTIAL AFFINITY LABEL
DERIVATIVES OF DYNORPHIN A(1-11)NH2: ADDRESS SEQUENCE
MODIFICATIONS
5.1 Abstract
5.2 Introduction
75
76
82
84
84
89
92
95
97
99
100
TABLE OF CONTENTS (CONTINUED)
Page
5.3 Experimental section
5.3.1 Materials
5.3.2 Methods
5.3.3 Receptor binding assays
5.4 Results and discussion
5.4.1 Peptide syntheses
5.4.2 Receptor affinity
5.5 Conclusions
CHAPTER 6 SOLID PHASE SYNTHESIS OF POTENTIAL AFFINITY
LABEL DERIVATIVES OF DYNORPHIN A ASSEMBLED ON
PAL-PEG-PS VS PAL-PS RESIN
103
103
103
105
105
105
106
111
113
6.1 Abstract
115
6.2 Introduction
116
6.3 Experimental section
120
6.3.1 Materials
6.3.2 Methods
120
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
7.3.2 Methods
7.4 Results and discussion
7.4.1 NovaSyn TG® resin functionalization
7.4.2 Peptide syntheses
138
139
144
145
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
Schematic diagram of protein structure for, and similarity among,
the three opioid receptors
34
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
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
2.6
2.7
3.3
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
Chromatograms of BocPhe(NH2) (solid line) and BocPhe(NHAloc)
(dotted line)
86
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
Chromatograms of crude peptides 2b (top), 2c (middle) and
2d (bottom)
108
4.4
4.5
5.2
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
Chromatograms of crude peptide [Phe(NH2)4,D-Pro9Dyn A(1-11)
NH2 from PAL-PEG-PS (left) and PAL-PS (right) resins
127
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
Chromatograms of crude peptide [Phe(NHCOCH2BW,D-Pro10]Dyn A
(1-11)NH2 from PAL-PEG-PS (left) and PAL-PS (right) resins
129
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
6.4
6.5
6.6
7.1
151
LIST OF TABLES
Table
Page
2.1
Classification of opioid peptides
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
Ratio of swollen/dry volumes for individual polyethylene glycol
polystyrene (PEG-PS) graft beads and polystyrene (PS) support in
different solvents
119
Characterization of [Phe(3',5'42,4'-NH2)4,D-Pro19Dyn A(1-11)
NH2 before and after ammonolysis
150
6.1
7.1
7
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
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
hexafluorophosphate
TBTU
2-(111-benzOt ia7o1-1-y1)1,1,3,3-tetramethyluronium
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
teti Arkturobor ate
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 used as 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]. The a 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 receptor was proposed as another type of opioid
receptor after the discovery of the enkephalins [Lord et al., 1977]. Today the 12, 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 common characteristics (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
Pro-opiomelanocortin
Proenkephalin A
Proenkephalin B
(Prodynorphin)
13-endorphin
Sequence
YGGFMTSEKSQTPLVTLFKNAIIKN
AYKKGE
8-endorphin
(P-endorphin 1-27)
YGGFMTSEKSQTPLVTLFKNAIIK
NAY
y-endorphin
(P-endorphin 1-17)
YGGFMT SEKSQTPLVTL
a-endorphin
(P-endorphin 1-16)
YGGFMT SEKSQTPLVT
Met-enkephalin
YGGFM
Met-enkephalinArg-Phe
YGGFMRF
Met-enkephalinArg-Gly-Leu
YGGFMRFGL
Leu-enkephalin
YGGFL
Peptide E
TGGFMRRVGRPEWWMDYQKRY
GFL
a-neo-endorphin
YGGFLRKYPK
P-neo-endorphin
YGGFLRKYP
Dynorphin A (1-17)
YGGFLRRIRPKLKWDNE
Dynorphin A (1-8)
YGGFLRRI
Dynorphin B
Y GGFLRRQFKVVT
Peptides
TGGFLRRQFKVVTRSQEDPNAYYE
(1-29) (Leumorphin) LFDV
Dynorphin B
8
5',
1
23
,
,..,
Z
v..
°,2.'
..t
..:,
T.
_..j
.-.1
ACTH-11-LPH
precursor
Signal
peptide
cr-MSH
T OD
.
,- J..
..J < -J
wo
1.7513III
Preproenkephalin A
U
..
.4
4.
...1 ...1
-.1
S.
II
(..)
(.1
I_
>. ...
;A-/'
A
a.
....
-1 -J
LI
Met -enk Met
-enk Met-enk
Vet-enk
Signal
peptide
9 -LPH
CLIP
r, -MSH
r.
(l -MSH
ACTH
N-Terminal peptide
y-LPH
a
to
a,
a.
cm
g-Endorphin
!!.1
<
S.
<° <a°
.
<
-1
Met-enk- Met-enk Leu-enk
Arge-Gly7-Lete
Peptide
Met-e- nk-
Are-Phe'
Peptide E
a 0:
A
Yiu
ED
<<
<
Preproenkephalin 13
Signal
Peptide
Leu-e- nk
Neo-endorphin
Leu-enk
Leu-enk
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 Nteminus contain multiple cysteine residues.
2.1.1 Pro-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 al 1988]. 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 al 1985]. 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, TyrGly-Gly-Phe-Leu, sequence at the N-terminus and except for dynorphin A(1-8) and I3-neoendorphin, 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 et al., 1984]. PROENK
products from cleavage at basic residue pairs are Leu-enkephalin, Met-enkephalin, Metenkephalin-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).
ic)
IC5o (nM)
Dynorphin A(1-13)
0.31
MVD(8)
Rat VD'
Rabbit VD2
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 a metalloendopeptidase 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
d
b
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. a
enkephalinase and c = endopeptidase.
aminopeptidase, b =
N-Terminal enzymatic inactivation of dynorphin can be suppressed by several enzyme
inhibitors (Fig. 2.3). Bestatin is a specific aminopeptidase inhibitor [Fournie-Zaluski et al.,
1985], while thiorphan is an enkephalinase inhibitor [Rogues et al., 1980]. Thiorphan itself
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, 1.90 nM vs R, 1.60 nM).
A retro isomer of thiorphan is also an effective enkephalinase inhibitor which inhibits
angiotensin converting enzyme (ACE) less than thiorphan. Retro-thiorphan displays a 100 fold difference in inhibitory activity by the different isomers (IC50: S, 210 nM vs R, 2.3 nM)
[Fournie-Zaluski et al., 1986]. Kelatorphan, a hydroxyamido analogue of thiorphan, is a
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
2.2.3 Structure-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 DCys13 (or L-Cys13) in Dyn A(1-13)NH2 gives compounds with high affinity for x 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 are present 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 opioid receptor types. Two novel
opioid receptors, epsilon (e) and zeta () receptors, have been reported [Wuster et al., 1978;
Zagon et al., 1993].
Table 2.3 Opioid receptors and physiological effects.
mu (p.)
kappa (K)
delta (8)
analgesia
constipation
respiratory depression
physical dependence
analgesia
sedation
diuresis
analgesia
hyperthermia
respiratory depression
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 muscle assays.
Tissue
Guinea pig ileum (GPI)
Hamster vas deferens (HVD)
Rabbit vas deferens (LVD)
Mouse vas deferens (MVD)
Rat vas deferens (RVD)
8
++
++
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-ProSer-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 11antagonists are somatostatin analogues including cyclo [H-D-Phe-Cys-Phe-D-Trp-Lys-ThrCys-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 2.5 Tentative receptor classification.
Agonists
Antagonists
Actions
111
Morphine
Naloxonazine
[12
Enkephalin
Morphine
Supraspinal
analgesia
CTOP
[13
Morphine
Spinal analgesia
Respiratory
depression
Inhibition of
gastrointestinal
transit
Receptor
K
K1
Dynorphin A
U-50,488
U-69,593
Spiradoline
nor-BNI
Pharmacology
unknown
Supraspinal analgesia
K2
NalBzOH
Nalorphine
8
81
Enkephalin; tolerant
of conformations
DALCE
82
Met-enkephalin;
nor-BNI
not tolerant of
conformations
Spinal analgesia
Diuresis
Analgesia (spinal
systems are more
sensitive than
supraspinal ones)
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{trans3,4-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
CI
CI
CH2
U-50,488
CH2
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
N CH2
I
N
OH
HO
NTI
TENA
nor-BN1
Fig. 2.4 (cont.) Selected opioid receptor ligands.
--ci
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 norbinaltorphimine (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 ic 3, 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 K la and K lb [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 K2receptors, 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 (methylfentanylisothio-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 coworkers [1987] used affinity chromatography on a matrix of DADLE coupled to epoxyactivated 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
P
OWPGASO
LSD PaSAIPIIAZAN
0 NOSY
IDIFRG POP.T.S.SPSAOLLNSS
I1APO
P-- WAS
SAGIOLSSO
LIOLSI DONO aBCOLNAT.QLG NOWLCPOS9
CaDllijA0AS Sna
WPRM(SA,A---.LPLEIJA ITALYS
LGNV7377FLGJIVRYTXLQKTATNITIFNLALADALAT 100
KOPRDI
mOPRX1
;OMNI
S ARAE
-
DS
T
KAI IPVI IrTiAaY S
M V TUT
110
VGNSLVNAVIDAYTKNKTATNITIFNLALADALM
.110.1._2AYWYV.YTKMKTATNITTFNLALADALAT II,
no
WPFG ELLCI.SIDYYNNITSIFTLTNNSVDRYIAVCMPVKALDFR 140
mOPSD1TLPFOS K YLM.
mOPRX1 Ta PFOS V YLK N WPFG DVLCKIVISIDISVORYIAVCRPVKALDPR 170
CT
179
rOPRN1TLPFOSVN
C
TI
T244
TVT K IC 214
mOPRD1 TP
XAK L INICI
C VMS
D©:. - -VV C H LOFPSa-KOMI TP L AKI N C
LPN RIC 229
C ISAT LGG
V
EDVDVRE C S LAFIDDEY
£NLL
235
rOPRM1
V
EI Y CaS-- Ds. T ljTWSMa--T
N ISAI LaVaFT
mOrItX11,LAJ H I
rOPRN1 T Ga P ...
rflOPRDI
mOPRX1
VF
VF
V
FAF
FAF
V
I
115
V
I
1M6
PILIITYCYGLMLLRLRSVRLLSGSK EKDRSLRRITRMVLVV GAFV C W AP 270
PaLIIMVCYMLMILRLKSVR,I,LSGSWEKDRNLRRITEDVLVVVAVFI CWTP 299
N P/LIITYCY(ILMTT RI KSVPIVILSCSK,',,MRNt RR TTRMVLVVVAVFIVCWTP 29S
ITV
WIT VOMNRRIPLVVAALIBLCIALGYWNSSLNPVLYAFLDENFKPCFROL R T P 334
FillI 341
em)PRK1
EAL GSTSNSTA-ALSRYTFCIALGYTNSALNPVLYAFLDENFKRCFRD
T
T354
rOPRM1
I K.LL. ITM-PETFOTV
WHFCI;LGYTNEICILNPVLYAFLGENFKPCF&E
M
Fri
372
C PG
froOPRO1 C
LA
ATTE1RVTACTPSa
R PGGGAAA
310
RQ
TN
mOPRK1 K M n
TVQOPA
NRDV
GNNX III V
3911
C
rOPRM1 SSTS
NST
N
TANTVaRTNMOLENLEAETA LP
rOPRM1
rOPRD1
M
I -HP
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 for N-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
Morphine
Naloxone
DAMGO
DADLE
DPDPE
Dynorphin A(1-17)
U-50,488
U-69,593
K, (iim)
>1000
95
>1000
2.8
7.6
45
>1000
>1000
163
16
>1000
514
>1000
5.48
1.4
2.3
1.4
3.9
0.87
6.4
>1000
120
>1000
>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 acid proteins
with 97% identity with rat 8 receptors. Rat 11 opioid receptors contain 398 amino acids with
58% and 57% identity and 66% and 68% similarity to the sequences 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 the sequence of the mouse 8 receptors. Human [t, 8, and
K-receptors have been recently cloned [Wang et al., 1994; Knapp et al., 1994; Simonin et 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 extracellular regions
(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 the a2-adrenergic and
somatostatin receptors [Horstman et al., 1990]. The 3rd intracellular loop is required for the
interactions with G-proteins. The 2nd and 3rd extracellular loops and TM1 and TM4-6 are
less conserved, while the extracellular N-terminus and the intracellular C-terminus have the
greatest diversities. The 1st and 2nd extracellular loops contain Cys residues which may 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
o
p
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 electrophile group and the
receptor-based nucleophile. Chemical affinity labels contain an electrophile which interacts
with a nucleophile on the receptors. Selectivities of chemical affinity labels are 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
GI
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 agonists
were 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,
N
\
N
/
COEt
/-\/CH3
\
CH2CH2C6H4(p-NCS)
CH2CH2C6H4(p-NCS)
SUPERFIT
FIT
Eta
CI
NCS
NCS
UPHIT
BIT
H
NHCOC=CCOOMe
HO
OMe
FAO
Fig. 2.9 Affinity label agonists.
40
H
I
NHCOC=CCOOMe
HO
I
HO
H
13CNA
(3 F NA
NCS
HO
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 group on 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 such as 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 analog gave 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
Jclile
0
0 lir
Handle
0
I
H70
0
,;
Anchoring
Deprotection
Repetitive
cycle
Coupling
Cleavage
Final deprotecnon
Handle
Fig. 3.1 Stepwise solid-phase synthesis of linear peptide. R, insoluble polymeric support;
NA" amino acid residues numbered starting from C-terminus; T,
"temporary" protection; 0, permanent" protection; u, free carboxyl; ED, free amino
AA'. AA:
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
group is introduced into amino acids using di-tert-butyl dicarbonate or 2-tertbutyloxycarbonyl-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
Boc
c-o
O
CH3
CH2
-=O ±NH-CH-C-NH- CH2
TFA
PAM
O
C 12
NH-6-1C 0
f
CH2
CH2CNHCH2< >-0
0
11
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-fluorenyl-
methoxycarbonyl (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-fluorenylmethyloxycarbonyloxy)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]undec7-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
Fmoc
I
.113
113
CH3 - C CH3
I
1
0
,H
1
i414
0
0
F
HMP/PAB
C =0
6H2
CHx`...../
0- Z -NHCH- C -NH- CH2
._.
6
0
CH2
-NH- &I-- C - OICH2
6
0- CH2
0
CH2
-NH- CH2
TFA
Fig. 3.3 A mild two-dimensional orthogonal protection scheme for solid phase synthesis.
Temporary Na-amino protection is provided by the Fmoc group, removed by the
indicated base-catalyzed 13-elimination mechanism. Permanent tBu-base side chain
protecting groups and the HMP/PAB ester linkage are both cleaved by treatment with
TFA to yield the free peptide acid. A third dimension of orthogonality may be added
with an acid-stable, photolabile anchoring linkage.
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.
48
H
ND
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 hydroxyl groups 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 protecting groups (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 Boc group
for Fmoc chemistry. An allyloxycarbonyl (Aloc) protecting group is compatible with both
Boc-and Fmoc-base syntheses. Aloc is insensitive to acids and bases and can 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-methoxy2 ,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 is more
readily removed than the corresponding Pmc by factors of 1.2-1.4 for TFA/H20 (95/5; 80/20)
50
CI
CH2
CH2
CI
2,6-C12 BzI
BzI
O
O
cH2oc
II
II
C H20 C
CI
2 -CIZ
2-BrZ
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-0 C
CHs C-
II
I
1
CH3
CH3
Boc
t-Bu
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 can be 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 2bromobenzyloxy-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 Naamino 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
COP
HO(CH2)2
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, et al., 1989], 3-(amino-4methoxybenzy1)-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
0CH2C0---P
HOCH2
CH3O
HMPA linker/ Pac resin
HAL 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
O(CH2)4C0 P
OCH2 --P
Sieber amide resin
XAL resin
0(CH2)4c0 p
CH3O
OCH2 P
CH3O
PAL resin
Rink amide resin
NHCO(CH2)2CONHCHP
OCH3
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 Nmethylmorpholine 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 of amino 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
COP
HOCH2
02N
Onb resin
COP
H2N(CH2)2
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
RCX
II
Activation: R COOH
0
Coupling:
r
CO
R .0 X
RC X
HNH
HNH
R'
R'
-1
O
RC
NH
+ HX
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 and one 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-diisopropylcarbo-
diimide (DIC, 1-2 equiv in DCM). PSA's are not widely used for FmocSPPS due to 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 Ono nor
Onp are popular for SPPS due to their low reactivities even in the presence 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
OBt
OAt
0
.:,..N
N
0 imkir
N
N
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-7azabenzotriazole (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,APDicyclohexylcarbodiimide (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-yloxy-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
CH3
H3C
HCN= C =N
N =C =N
H3C
DCC
CH
CH3
DIC
N(CH3)2
N(CH3)2
N(CH3)2
\
N+
0
P F6
HBTU
N(CH3)2
\o
N+
BEer
TBTU
N
N
N
\O
(CH3)2N P
(CH3)2N
PF6
/ \mu-4
BOP
Fig. 3.13 Coupling reagents.
\
PF
/P-N
PyBOP
66
(TBTU) [Knorr et al.. 1989], and 2-[2-oxo-1(2H)-pyridy1]-1.1.3.3-bispentamethyleneuronium 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 Naamine 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.
8\ 1,1
0
c,
OH
CH
0
C
}-
H2N-C-C-OH
C=N
-CO2
Cr°
H-0
O
0
ninhvdrin
'CHR
9
C
N,C- =
+
O :CHR
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, 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-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NMM, N-methyl-
morpholine; PAL, peptide amide linker, PEG, polyethylene glycol; Pmc, 2,2,5,7,8pentamethylchroman-6-sulfonyl; PS, polystyrene; TFA, trifluoroacetic acid, THF,
tetrahydrofuran; Z, benzyloxycarboxyl.
69
4.1 Abstract
Potential affinity label derivatives 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(NH 2)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-AlaGly-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(111)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 the affinity labeling 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 chapter
5
("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) compounds were 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 dynorphin A
derivatives by solid phase peptide synthesis was to cleave the protected peptide from the
resin followed by introducing the reactive functionalities in solution [Story and Aldrich,
1992]. Synthesis of the model peptide Boc-Tyr(t-Bu)-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 on
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-hydroxy-
methyl benzoic acid linker) gave unsatisfactory results. Therefore, a modified methylbenzhydrylamine (MBHA) resin with a base-labile linker was developed, and a 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,DPro 11DynA( 1 -1 1 )NH2 and [Phe(NHZ)4 ]Dyn A(1 -13 )NH 2, 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, it was
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 with a 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 functional group 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
a)
NHCH2CH=CH2
NH2
.11 ly1 c'nloroformate
pH 4.6
CH2
CH2
NH2 CH COOH
NH2 CH COOH
Fmoc CI
10% Na2CO3 /dioxane
CH2
FmocNH CH COOH
b)
chloroiorrnate
CH2
BocNH CH COOH
Fig. 4.1 Synthesis of phenylalanine derivatives.
CH2
BocNH CH COON
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). DIEA was added to maintain the pH
of the solution at pH 4-5 and the mixture was stirred overnight [Fahrenhloz and Thierauch,
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 (protected 2aresin) (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-resin and
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 polyvinyl-
styrene, 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 synthesis was
performed by the following steps: (1) removal of the Fmoc protecting group using a
piperidine/toluene/DMA (30%/35%/35%) mixture; (2) addition a 4-fold excess of Fmocamino 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 coupling was monitored
79
t-Bu
P
I
Pmc Pmc
I
I
Pmc
Boc
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
I
I
Pmc Pmc
I
Pmc
I
Boc
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
I
Pmc
Boc
Boc-Tyr-Gly-Gly-Phe-Leu-Airg-A I
rg-Ile-Arg-D-Pro-Lys-NH-resin
I
I
Protected lc, 1 d-resin
I
modified reagent K
(87.5`)/0TFA /2.5%thioanisole/5%pheno1/5%H20)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-CON F12
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 1bresin), [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, and a 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 reaction was 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 (3 x 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 2cresin)
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 B was 0.1% TFA in AcCN. The
homogeneity of the peptides was determined by analytical HPLC on a Vydac analytical
column equipped with a Vydac guard cartridge using a linear gradient of 0%-75% solvent
B over 50 min with a flow rate of 1.5 mL/min and detection at 214 nm. 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 derivatives were performed with
Chinese hamster ovary (CHO) cells which express rat cloned lc and ii [Bunzow et al., 1995]
and mouse 8 opioid receptors. Cells were harvested 72 h following transfection in 50 mM
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 pellet was washed
twice by resuspension and recentrifugation as in the previous step. The pellet was
resuspended in 50 mM Tris buffer, pH 7.4, at 4 °C to yield a protein concentration of 3060 [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. Binding assays
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) in a
final volume of 2 mL 50 mM Tris buffer, pH 7.4, at 22 °C. Nonspecific binding was
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 filtration over
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 aniline group
of Phe(NH2)4 or Phe(NH2)' in derivatives of [D-Prol°]Dyn A(1-11)NH2. This aniline group
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 aniline group 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 in a two-step
reaction. First, the p-amino group of Phe(NH2) was selectively protected by the Aloe group,
followed by protection of the Mt-amine using Fmoc-Cl. The p-amino group 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 in a 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
N
manatees
a
4
-1
0
bra
0
4
0
a
4
0
4
v.
Fig. 4.3 Chromatograms of FmocPhe(NH2) (solid line) and FmocPhe(NHAloc) (dotted
line). Column: Vydac (C 18 ). 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
1
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
(265)
487.4
(487)
365.4
(365)
[a]D25°
+3.71
+12.86
+17.92
(M+H)±
3
MP (°C)
225-227
Elemental
analysis
Formula
C13H16N 204
C28H26N 206
C181--124N
0.474
0.876
0.904
0.567
0.335
0.908
6/0 C
%H
%N
TLC
58.9 (59.1)
6.0 (6.1)
10.3 (10.6)
178-181
69.4 (69.1)
5.3 (5.4)
5.9 (5.8)
138-139
206
59.4 (59.3)
6.4 (6.6)
7.7 (7.7)
'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 lbresin 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
r
wli.ONYIWO
gaol
11
Fig. 4.5 Chromatograms of crude peptide lb (top), /c (middle) and ld (bottom). For
chromatographic conditions see Fig. 4.3.
91
7.
Z
a
a
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(111)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 [DPro' 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
(M+H)±
Tyr
(1)
Gly
(2)
1376.9
(1377)
1418.9
(1419)
1498.5
(1499)
0.97
1.93
1361.0
(1361)
1403.0
(1303)
_3
Amino acid analysis
Phe
Leu Arg Ile
(1)
(1)
(3)
(1)
D-Pro Lys Phe(NH2)
(1)
(1)
(1)
[Phe(X)4,D-Pro10]Dyn A(1-11)NH2
lb, X= NH2
17.4
/c, X= N=C=S
23.9
Id, X= NHCOCH2Br
20.7
1.04
3.07
0.90
0.92
1.05
1.02
3.05
0.92
0.98
1.01
[Phe(X)1,D-Pro10]Dyn A(1-11)NH2
2b,X=NH2
16.8
2c, X= N=C=S
22.9
'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.
2.00
1.01
1.12
Table 4.3 Preliminary binding assay data for potential affinity label derivatives of dynorphin A'.
Compound name
IC50 (nM) ±SE
lc
[p-Phe(X)4, D-Pro' lDyn A(1-11)NH2
lb,X= NH2
/c, X = N=C=S
Id, X = NHCOCH2Br
[p- Phe(X)', D-ProliDyn A(1-11)NH2
2b,X= NH2
2c,X= N=C=S
[D-Pro ilDyn A(1-11)
'Values without SE are from one experiment.
5.75 ± 2.95
5.57 ± 0.97
IC50 ratio
8
11
27.4 ± 5.9
146 ± 16
328 ± 58
173 ± 5.5
2.34 ± 0.31
14.1 ± 1.8
78.9
92.5 ± 10.5
0.25 ± 0.03
1.04 ± 0.15
K/µ /8
481
315
1,650
572 ± 4.7
1,933
5.52 ± 0.07
1/28/84
1/48/57
1/6/60
1/34/244
1/6/37
1/4/22
95
receptors, resulting in IC50 values greater than 75 nM. For the [Phe(X)4,D-Pro10]Dyn A(111)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)NH 2 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 [DPro9Dyn A(1-1 1)NH2. Additional pharmacological experiments with these compounds are
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, Nmethylmorpholine; PAL, peptide amide linker, PEG, polyethylene glycol; Pmc, 2,2,5,7,8-
pentamethylchroman-6-sulfonyl; PS, polystyrene; TFA, trifluoroacetic acid, 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 isothio-
cyanate 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(111)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(111)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 are no 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 pharmacological responses.
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 the receptor, and
2) secondary recognition which involves the reaction of the electrophilic center with a
proximal nucleophile [Portoghese and Takemori, 1985]. Photoaffinity labels are 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 isothiocyanate or
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 [Gairin et 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 the x 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. isothiocyanate (-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 charges on
the kappa binding site, which would increase binding energy 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 8 are 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 of para-
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 allyloxycarbonyl (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 2aresin), protected [Phe(N112)8,D-Pro10JDyn A(1-11)NH-PAL-PEG-PS resin (protected lbresin) 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 using a 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,DPro10]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
[Phe(X)8,D-Pro10]Dyn A(1-11)NH2
1 b,X= NH2
tR (min)'
19.9
/c, X= N=C=S
22.5
Id, X= NHCOCH2Br
22.0
[Lys(X)8,D-Pro10]Dyn A(1-11)NH2
2b,= -
19.0
2c, X= N=C=S
19.1
2d, X= NHCOCH2Br
20.9
'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.
Tyr
(1)
Gly
(2)
Amino acid analysis
D-Pro Lys'
Phe
Leu Arg
(1)
(1)
(3)
(1)
(1)
1410.8 1.07
(1411)
1454.0
(1454)
1532.8
(1533)
2.19
1.12
1.09
2.93
0.76
0.82
1377.0 0.93
(1377)
1419.0
(1419)
1498.8
(1499)
1.92
1.04
1.04
3.10
0.99
1.98
FAB-MS2
(M+H)*
1.05
Phe(NH2)
(1)
Table 5.2 Preliminary binding assay data for potential affinity label derivatives of dynorphin A'.
Compound name
IC50 (nM) ±SE
K
[p-Phe(X)8, D-ProllDyn A(1-11)NH2
16,X =NH2
lc, X = N=C=S
ld, X = NHCOCH2Br
[Lys(X)8, D-ProllDyn A(1-11)NH2
2b,X=
-
2c, X = N=C=S
2d, X = NHCOCH2Br
[D-Pro9Dyn A(1-11)
'Values without SE are from one experiment.
11
IC50 ratio
43
K/µ /8
1.54 ± 0.15
0.49 ± 0.09
1.42 ± 0.06
1.06 ± 0.29
1.71 ± 0.27
2.89 ± 0.31
6.83 ± 2.1
13.9
8.14 ± 0.44
1.5/1/9
1/4t25
1/2/6
0.16 ± 0.03
0.54 ± 0.03
0.68 ± 0.10
0.71 + 0.13
1.04 ± 0.20
1.16 ± 0.03
4.92 + 0.36
5.24 ± 0.42
6.07 ± 0.16
1/4/31
1/2/10
1/2/9
1.04 ± 0.15
5.52 ± 0.07
1/4/22
0.25 ± 0.03
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(111)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 took a
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(111)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, 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, Nmethylmorpholine; PAL, peptide amide linker, PEG, polyethylene glycol; Pmc, 2,2,5,7,8-
pentamethylchroman-6-sulfonyl; PS, polystyrene; TFA, trifluoroacetic acid, 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
RCHCH=CH,
-Pd.
RCH=CHCH2Nu t Pd °H
Pcii.
O2CCH,
Fig. 6.1 Deprotection of allyl-based protecting groups.
xH
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 (PALPEG-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
DMF
AcCN
TFE
EtOAc
5.8
3.6
1.4
EtOH
6.9
4.8
3.5
5.5
3.7
4.5
2.3
H2O
1.7
THE
1.3
3.7
5.7
1.2
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 PALPEG-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 (Nmethylmorpholine) 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 laand 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 4b were 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 functionality group 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 prompted us to
synthesize the potential affinity label derivatives of Dyn A on a 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 functional groups 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 by a
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
so
.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(11 1)NH-resin synthesized on PAL-PEG-PS (top) vs PAL-PS (bottom) resins.
126
precursors for the preparation of affinity label-containing Dyn A analogues 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 on PAL-PEG-PS and PAL-PS resin,
respectively, were obtained in excellent purity (> 90%, Fig 6.4). The potential affinity label
derivatives Dyn A assembled on PAL-PEG-PS, however, contained more side products than
those assembled on the PAL-PS resin. The isothiocyanate 3a contained approximately 30%
side products in addition to the desired peptide, whereas the isothiocyanate 3b was the only
major product (>88%) assembled on the PAL-PS resin (Fig 6.5). Reverse phase HPLC
analysis of the bromoacetamide derivatives assembled on PAL-PEG-PS and PAL-PS resin
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 identified as the
hydroxylated peptide [Phe(NHCOCH2OH)4]Dyn A(1-11)NH2. Therefore, although the Aloe
deprotection was faster on the PAL-PEG-PS resin (complete reaction within 3 h), the crude
potential Dyn A affinity label derivatives 3a and 4a contained more impurities compared
to the crude peptides 3b and 4b assembled on the PAL-PS resin.
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
NG
IN
IN
11111.
N
111111.
PAL-PEG-PS (2a)
PAL-PS (2b)
Fig. 6.4 Chromatograms of crude peptide [Phe(NH2)4,D-Pro10]Dyn A(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)NH2 from 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-Pro10JDyn A(1-1 1)NH2 from PAL-PEG-PS (left) and PAL-PS (right)
resins. For chromatographic condition see 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-dimethyl-
acetamide; Dyn A, dynorphin A; DMF, NN-dimethylformamide; FAB-MS, fast atom
bombardment mass spectrometry; Fmoc, (9-fluorenylmethoxy)carbonyl; HOBt,
hydroxybenzotriazole;
HPLC,
high performance
liquid chromatography;
1-
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)NH 2, 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-hydroxymethyl-
benzoic 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 of an 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 labels can
be utilized in vivo for pharmacological studies, while photoaffinity labels are reversible until
after photoactivation occurs [Glasel and Venn, 1981]. Non-peptide affinity labels have been
used to irreversibly block selected receptor populations in tissues such as 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-(pethoxybenzy1)-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 both p, 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 usable at a low
concentration in order to maximize its binding at the preferred site relative to that at 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 17amino acid endogenous peptide possessing high affinity and some 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 for K opioid receptors
using [D-Pro'°]Dyn A(1-11) as a prototype. The peptide structure offers flexibility in the
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 and an 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 baselabile linkage. The NovaSyn TG® resin contains a polyethylene glycol polystyrene (PEGPS) 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 utilized as
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: subsequent catalytic hydrogenation
will yield protected peptide precursor of the affinity label. The conditions used for catalytic
hydrogenation reaction will be optimized for small scale synthesis to give the unlabeled
peptides. and similar conditions can later be applied to catalytic tritiation which will be
performed by New England Nuclear. Following tritiation of the protected peptide, the
affinity labeling group (e.g. isothiocyanate or bromoacetamide) can be attached to the pamino 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 bearing a 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-diisopropyl-
ethylamine (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 (peptideresin 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
HOCH2
1)1
COPfp / HOAt
0
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, 0.37 g, 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 (5 x 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 (5 x 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 (20 mL)
for 30 min, washed with DCM/ DMA (1:1) and DCM (3 x 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 to a solution of Phe(3',51-
I24'-NH2) (0.64 g, 1.47 mmol) in 10% Na 2CO3 /dioxane (1/1), 5 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
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 residue was 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
Fmoc-amino acids
DIC/HOBt
t-Bu
CH2
PmcPmc
I
I
I
Pic
I
I
deprotection/
coupling cycles
Boc
I
Boc-Tyr-Gly-Gly-NH-CH-CO-Leu-Arg-Arg-Ile-Arg-D-pro-Lys-linker B-Nle-NovaSyn TG resin
NH2
t-Bu
I
CH2
NH3/i-PrOH
PmcPmc Pic
Boc
I
Boc-Tyr-Gly-Gly-NH-CH-co-Leu-Arg-Arg-Ile-Arg-D-Pro-Lys-CONFI2
I
I
1
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-hydroxy-
methylbenzoic 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 section was 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 resin was
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'.
IT2
(1)
Before
cleavage
G
(2)
Amino acid analysis
F
L
R
I
(1)
(1)
(3)
(1)
P
(1)
K
(1)
1.53
0.88
0.96
1.33
0.97
3.35
0.98
Peptide
substitution
(mmol/g)
Nle internal
standard
(mmol/g)
% Peptide
on resin
0.12
0.13
93.1
0.02
0.01
0.01
0.17
11.7
3.3
% Cleavage
(% Recovery)
After
cleavage
1d
3d
5d
'Theoretical values are shown in parentheses.
2Tyr was destroyed during hydrolysis and was excluded from the calculation.
0.21
0.36
4.7
88.3 (54.8)
96.7 (67.0)
95.3 (31.0)
151
.1
v
LI
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 baselabile 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 based on [D-Prom]Dyn A(111)NH2 were synthesized for use as pharmacological probes for lc opioid receptors. A
reactive functionality (isothiocyanate or bromoacetamide) was incorporated into the Nterminal "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 isothiocyanate or 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 isothiocyanate or
bromoacetamide derivatives of p-aminophenylalanine or lysine. The peptides containing an
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 assembled on a
polyethylene glycol (PEG)-polystyrene (PS) support with a 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 functional group was to be incorporated in
order to provide orthogonal protection. The Aloc group can 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 indicated the 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 receptors Surprisingly, [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) andx
µ > 8 (for the "address" sequence modifications).
Since some of the potential affinity label derivatives of Dyn A assembled on PALPEG-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-hydroxymethylbenzoic 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 identify affinity 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
BIBLIOGRAPHY
Abood, M.E.; Noel, M.A.; Farnsworth, J.S.; Tao, Q. Molecular Cloning and Expression of
a delta-Opioid Receptor from Rat Brain. J. Neurosci. Res. 1994, 37, 714-719.
Albericio, F.; Nicolas, E.; Rizo, J.; Ruiz-Gayo, M.; Pedroso, E.; Giralt, E. Convenient
Synthesis of Fluorenylmethyl-Based Side Chain Derivative of Glutamic and
Aspartic Acids, Lysine and Cysteine. Synthesis 1990, 119-122.
Albericio, F.; Barany, G. Hypersensitive Acid-labile (HAL) Tris(alkoxy)benzyl Ester
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