N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

Article abstract of DOI:10.1016/j.bmcl.2013.07.036

Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH₂ (DIPP-NH₂) and H-Dmt- TicΨ[CH₂NH]Phe-Phe-NH₂ (DIPP-NH₂[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH₂ and Guan-DIPP-NH₂ in the δ receptor binding site. Lys³-analogues of DIPP-NH₂ and DIPP-NH₂[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.

Full text of DOI:10.1016/j.bmcl.2013.07.036

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5082–5085
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides
results in major changes of the opioid activity profile
Grazyna Weltrowska a, Thi M.-D. Nguyen a, Nga N. Chung a, Brian C. Wilkes a, Peter W. Schiller a,b,
⇑
a Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC H2W 1R7, Canada
b Department of Pharmacology, Université de Montréal, Montreal, QC H3C 3J7, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthe-
sized in an effort to improve their blood–brain barrier permeability. Unexpectedly, N-terminal amidin-
ation significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related d opioid
Received 29 May 2013
Revised 4 July 2013
Accepted 16 July 2013
Available online 23 July 2013
antagonists showed d partial agonist or mixed d partial agonist/
of the mixed agonist/d antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-Tic
Phe-NH2 (DIPP-NH2[ ]) converted them to mixed agonist/d agonists. A docking study revealed distinct
positioning of DIPP-NH2 and Guan-DIPP-NH2 in the d receptor binding site. Lys3-analogues of DIPP-NH2
and DIPP-NH2[ ] (guanidinylated or non-guanidinylated) turned out to be mixed agonists with d
l
partial agonist activity. Guanidinylation
l
W
[CH2NH]Phe-
Keywords:
Peptide synthesis
Guanidinylated opioid peptides
d Opioid antagonists
d Partial opioid agonists
W
l
W
l/j
antagonist-, d partial agonist- or d full agonist activity. Compounds with some of the observed mixed opi-
oid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of
cocaine addiction.
l
l
Opioid agonist/d opioid antagonists
Opioid agonist/d opioid agonists
Ó 2013 Elsevier Ltd. All rights reserved.
The tetrapeptides H-Tyr-Tic-Phe-Phe-OH (TIPP) (1a), H-Dmt-
Tic-Phe-Phe-OH (DIPP) (2a) and H-Tyr-Tic [CH2NH]Phe-Phe-OH
dependence.6 Both d opioid antagonists and mixed
l agonist/d
W
antagonists of the TIPP peptide family proved to be valuable tools
in opioid receptor research and proof-of-concept studies in the opi-
oid field. However, TIPP peptides do not effectively cross the BBB.
Cationization of proteins and peptides by guanidine (Guan)
addition has been shown to improve metabolic stability and BBB
(TIPP[W] (3a) are highly selective d opioid antagonists with low
nanomolar or subnanomolar d opioid receptor binding affinity.1–3
The TIPP-derived tetrapeptide amides H-Dmt-Tic-Phe-Phe-NH2
(DIPP-NH2) (4a) and H-Dmt-Tic
NH2[ ]) (5a) act as agonists at the
antagonists at the d receptor. On the basis of a well established
pharmacological rationale,4,5 compounds with such a mixed
W
[CH2NH]Phe-Phe-NH2 (DIPP-
W
l
opioid receptor and as
permeability.7,8 N-terminal amidination of a
l
-selective dermor-
phin-derived tetrapeptide did not result in a significant change in
l
l
j
receptor binding affinity and high selectivity for
l versus d and
agonist/d antagonist profile are expected to be analgesics with
receptors was retained.9 The resulting compound produced cen-
low propensity to produce analgesic tolerance and physical depen-
trally mediated analgesia with oral and sc administration, indicat-
dence. Indeed, DIPP-NH2[W] given i.c.v. produced a potent analge-
ing its ability to cross the BBB. N-terminally amidinated
sic effect in the rat tail flick test and upon chronic administration
induced less analgesic tolerance than morphine and no physical
endorphin-1 analogues displayed somewhat reduced
binding affinity as compared to their respective non-amidinated
l receptor
parent peptides, were still
hanced BBB permeability.10
Here we describe the syntheses and in vitro opioid activity
profiles of the N-terminally amidinated TIPP antagonists 1a, 2a
and 3a: Guan-Tyr-Tic-Phe-Phe-OH (1), Guan-Dmt-Tic-Phe-Phe-
l receptor-selective and showed en-
Abbreviations: BBB, blood–brain barrier; Boc, tert-butyloxycarbonyl; Cl-HOBt, 6-
chloro-1-hydroxybenzotriazole; DAMGO, H-Tyr-D-Ala-Gly-Phe(NMe)-Gly-ol; DIC,
1,3-diisopropylcarbodiimide; DIPP, H-Dmt-Tic-Phe-Phe-OH; DIPP-NH2, H-Dmt-Tic-
Phe-Phe-NH2; DIPP-NH2[W], H-Dmt-TicW
[CH2NH]Phe-Phe-NH2; Dmt, 20,60-dim-
ethyltyrosine; DSLET, H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH; ES-MS, electrospray mass
spectrometry; GPI, guinea pig ileum; Guan, guanidino; HBTU, 2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HPLC, high performance
liquid chromatography; MVD, mouse vas deferens; Tic, 1,2,3,4-tetrahydroisoquin-
OH (2) and Guan-Tyr-Tic
mixed agonist/d antagonists 4a and 5a: Guan-Dmt-Tic-Phe-
Phe-NH2 (4) and Guan-Dmt-Tic [CH2NH]Phe-Phe-NH2 (5). The
novel Lys3-containing peptides H-Dmt-Tic-Lys-Phe-NH2 (6a) and
H-Dmt-Tic [CH2NH]Lys-Phe-NH2 (7a), and their respective gua-
nidinylated analogues Guan-Dmt-Tic-Lys-Phe-NH2 (6) and Guan-
Dmt-Tic [CH2NH]Lys-Phe-NH2 (7) were also synthesized and
W[CH2NH]Phe-Phe-OH (3), and of the
l
W
oline-3-carboxylic acid; TIPP, H-Tyr-Tic-Phe-Phe-OH; TIPP[
W
], H-Tyr-TicW[CH2-
NH]Phe-Phe-OH; U69,593, (5 ,7
oxaspiro[4.5]dec-8-yl]benzeneacetamide.
a
a,8b-(ꢀ)-N-methyl-N-[7-(1-pyrrolidinyl)-1-
W
⇑
Corresponding author. Tel.: +1 514 987 5576; fax: +1 514 987 5513.
E-mail address: schillp@ircm.qc.ca (P.W. Schiller).
W
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.036

G. Weltrowska et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5082–5085
5083
pharmacologically characterized. Finally, we describe the synthesis
and in vitro opioid activity profile of the guanidinylated dipeptide
opioid d antagonist H-Dmt-Tic-OH11 (8a): Guan-Dmt-Tic-OH (8).
Peptides 1–7 were synthesized by the manual solid-phase
method using a Boc-Phe resin for peptides 1–3 and a p-meth-
ylbenzhydrylamine resin for peptides 4, 5, 6, 6a, 7 and 7a with
Boc-protection and DIC/Cl-HOBt as coupling agents. To introduce
the reduced peptide bond between the Tic2 and Phe3 or Tic2 and
Lys3 residues in peptides 3, 5, 7 and 7a, a reductive alkylation reac-
tion12 between 2-Boc-1,2,3,4-tetrahydroisoquinoline-3-aldehyde13
MVD assay with Ke values in the 0.2–4.8 nM range, the guanidiny-
lated peptides 1, 2 and 3 showed d partial agonist behavior (Ta-
ble 2). For peptide 2 an IC35 of 1.57 nM could be determined
based on 70% maximal inhibition of the electrically evoked con-
tractions of the vas produced by this compound. Peptides 1 and
3 showed lower maximal inhibitions to the extent of 33% and
50%, respectively, which did not permit the determination of accu-
rate IC values.
Guanidinylation of the mixed
l agonist/d antagonist H-Dmt-
Tic-Phe-Phe-NH2 (4a) had unexpected effects on the in vitro opioid
and the
a-amino group of the resin-bound H-Phe-Phe- or H-Lys-
activity profile. While Guan-Dmt-Tic-Phe-Phe-NH2 (4) retained
Phe dipeptide segments was performed. Amidination on the resin
was performed using the reagent N,N0-bis-(2-chloro-benzyloxycar-
bonyl)-1H-1-pyrazole-1-carboxamidine.14 Peptides were cleaved
from the resin by HF/anisole treatment. The guanidinylated dipep-
tide Guan-Dmt-Tic-OH (8) was prepared in solution by coupling
Boc-Dmt-OH with H-Tic-OMe using HBTU as coupling agent. The
reagent 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine15 was
used for N-terminal guanidinylation. Subsequent NaOH hydrolysis
of the methyl ester and Boc deprotection with TFA afforded the tar-
get product. Crude products were purified by reversed-phase HPLC
and their purity (>98%) and structural identity were established by
TLC, analytical HPLC and ES-MS.
very high d and l
receptor binding affinities (Kid = 0.146 nM,
l
Ki = 0.518 nM), it showed significant
j
receptor binding affinity
affinity of the non-guanid-
lM). Surprisingly, this compound turned
j
(Ki = 35 nM), in contrast to the low
j
j
inylated peptide (Ki > 1
out to be a potent d full agonist in the MVD assay (IC50 = 1.72 nM).
The effect was naloxone-reversible (Ke = 0.308 0.57 nM), indicat-
ing that it was mediated by opioid receptors. As expected on the
basis of its high
l receptor binding affinity, this compound also
showed high agonist potency in the GPI assay (IC50 = 8.09 nM).
l
Guan-Dmt-Tic-Phe-Phe-NH2 thus represents a potent, balanced
l
agonist/d agonist.
A study of flexible docking of compounds 4a and 4 to the d opi-
Binding affinities (Ki values) for
l
and d opioid receptors were
oid receptor was performed using Mosberg’s models of the recep-
determined by displacing, respectively, [3H]DAMGO and [3H]DSLET
tor in the inactive and activated state.17 The mixed
l
agonist/d
from rat brain membrane binding sites, and
j
opioid receptor
antagonist 4a and the mixed l agonist/d agonist 4 were docked
binding affinities were measured by displacement of [3H]U69,593
from guinea pig brain membrane binding sites, as described.16 Opi-
oid agonist potencies (IC50 values) or antagonist activities (Ke val-
ues) were determined in the mouse vas deferens (MVD) assay (d
to the inactive and the activated form of the d receptor, respec-
tively (Fig. 1). In general, a comparison of the ligand–receptor
interactions of 4a bound to the inactive receptor form with those
of 4 bound to the activated form revealed that most of the interac-
tions involved the same lipophilic receptor residues, including
Tyr129, Phe133, Val217, Phe218, Ile277, Val281, Leu200, Trp284, Leu299
and Met199. Both the N-terminal amino group of 4a and the N-ter-
minal guanidino group of 4 were engaged in an electrostatic inter-
action (salt bridges) with Asp128 in the third transmembrane helix
of the receptor. However, due to the steric bulk of the guanidino
group, peptide 4 was shifted relative to the position of peptide
4a (average rms deviation = 1.06 Å). This resulted in somewhat dif-
ferent interactions with corresponding receptor residues which in
some cases also have different side chain orientations between the
two receptor forms. These distinct receptor interactions may ex-
plain the d antagonist versus d agonist behavior of compounds 4a
and 4.
receptor-representative) or in the guinea pig ileum (GPI) assay (
and receptor-representative) using previously described
protocols.16
l
j
Guanidinylation of the d antagonist TIPP (1a) resulted in a com-
pound (1) with d receptor binding affinity (Kid = 2.29 nM) similar to
that of the TIPP parent (Kid = 1.22 nM) (Table 1). In comparison
with the d antagonists DIPP (2a) and TIPP[
guanidinylated peptides Guan-Dmt-Tic-Phe-Phe-OH (2) and
Guan-Tyr-Tic [CH2NH]Phe-Phe-OH (3) retained similar subn-
anomolar
receptor binding affinities (Kid = 0.146 nM and
W] (3a), the respective
W
d
0.968 nM, respectively). Like their parent peptides, the guanidiny-
lated peptides 1, 2 and 3 showed high d receptor binding selectivity
l
with weak binding affinities for
l
opioid receptors (Ki = 126–
j
875 nM) and very weak affinity for
j
receptors (Ki > 2000 nM).
Compared to guanidinylated peptide 4, the pseudopeptide
Whereas peptides 1a, 2a and 3a were potent d antagonists in the
Guan-Dmt-Tic
W
[CH2NH]Phe-Phe-NH2 (5) showed a similar opioid
Table 1
Receptor binding affinities of TIPP- and TIPP-NH2 analoguesa
Kid (nM)
Ki (nM)
Ki (nM)
Selectivity ratio (d/l/j)
l
j
Compound
1
1a
2
2a
3
3a
4
4a
5
5a
6
6a
7
7a
8
Guan-Tyr-Tic-Phe-Phe-OH
H-Tyr-Tic-Phe-Phe-OHb
Guan-Dmt-Tic-Phe-Phe-OH
H-Dmt-Tic-Phe-Phe-OHb
2.29 0.51
1.22 0.07
875 21
1720 50
126 10
141 25
704 82
3230 440
0.518 0.047
1.19 0.11
1.02 0.19
0.943 0.052
0.354 0.005
19.4 2.2
>5000
>1000
2260 10
>1000
>10,000
>1000
35.8 3.0
>1000
40.1 10.1
>1000
1.69 0.09
2.23 0.17
24.7 1.1
2.06 0.59
>10,000
>1000
1/382/>2180
1/1410/>820
1/863/15500
1/569/>4030
1/727/>10300
1/10500/>3250
1/4/245
1/10/>8470
1/1/51
1/2/>2240
1/0.2/0.8
1/63/7
1/10/13
1/3/0.8
1/6/>3760
1/829/>714
0.146 0.007
0.248 0.025
0.968 0.011
0.308 0.060
0.146 0.041
0.118 0.016
0.789 0.141
0.447 0.007
2.20 0.38
0.306 0.061
1.83 0.41
2.73 0.80
2.66 0.10
1.64 0.07
Guan-Tyr-Tic
W
[CH2NH]Phe-Phe-OH
H-Tyr-Tic
W
[CH2NH]Phe-Phe-OHb
Guan-Dmt-Tic-Phe-Phe-NH2
b
H-Dmt-Tic-Phe-Phe-NH2
Guan-Dmt-Tic
W
[CH2NH]Phe-Phe-NH2
b
H-Dmt-Tic [CH2NH]Phe-Phe-NH2
W
Guan-Dmt-Tic-Lys-Phe-NH2
H-Dmt-Tic-Lys-Phe-NH2
Guan-Dmt-Tic
W
[CH2NH]Lys-Phe-NH2
18.1 3.1
9.11 0.97
15.0 1.3
H-Dmt-Tic [CH2NH]Lys-Phe-NH2
W
Guan-Dmt-Tic-OH
8a
H-Dmt-Tic-OHb
1360 160
a
Mean of 3 determinations SEM.
Data taken from Ref. 3.
b

5084
G. Weltrowska et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5082–5085
Table 2
Opioid activities of TIPP- and TIPP-NH2 analogues
Compound
MVD
GPI
a
a
a
Ke (nM)
IC50 (nM)
P.A. (33%)c
IC50 (nM)
1
1a
2
2a
3
3a
4
4a
5
5a
6
6a
7
Guan-Tyr-Tic-Phe-Phe-OH
H-Tyr-Tic-Phe-Phe-OHb
Guan-Dmt-Tic-Phe-Phe-OH
H-Dmt-Tic-Phe-Phe-OHb
Inactive
4.80 0.20
Inactive
1050 150
Inactive
Inactive
Inactive
d
1.57 0.16 (IC35
P.A. (50%)c
)
0.196 0.008
2.89 0.14
Guan-Tyr-Tic
W
[CH2NH]Phe-Phe-OH
H-Tyr-Tic
W
[CH2NH]Phe-Phe-OHb
Guan-Dmt-Tic-Phe-Phe-NH2
1.72 0.19
8.09 0.53
18.2 1.8
1.22 0.18
7.71 0.31
0.289 0.033
16.5 1.9
9.46 2.11
5.69 0.90
P.A. (50%)c
Inactive
b
H-Dmt-Tic-Phe-Phe-NH2
0.209 0.037
0.537 0.026
2.18 0.30
Guan-Dmt-Tic
W
[CH2NH]Phe-Phe-NH2
0.750 0.069
4.69 0.94
b
H-Dmt-Tic [CH2NH]Phe-Phe-NH2
W
Guan-Dmt-Tic-Lys-Phe-NH2
H-Dmt-Tic-Lys-Phe-NH2
d
d
Guan-Dmt-Tic
W
[CH2NH]Lys-Phe-NH2
7.35 1.99 (IC35
23.2 2.66 (IC35
P.A. (50%)c
)
)
7a
8
8a
H-Dmt-Tic [CH2NH]Lys-Phe-NH2
W
Guan-Dmt-Tic-OH
H-Dmt-Tic-OHb
6.55 0.27
a
b
c
Mean of 3 determinations SEM.
Data taken from Ref. 3.
Partial agonist (% of maximal inhibition of contractions).
Partial agonist (IC35).
d
Figure 1. d Receptor docking studies. H-Dmt-Tic-Phe-Phe-NH2 (4a) in green bound to the d receptor in the inactive state (key residues depicted in white and Asp128 in red),
and Guan-Dmt-Tic-Phe-Phe-NH2 (4) in magenta bound to the d receptor in the activated state (key residues depicted in yellow and Asp128 in orange).
receptor binding profile with high
ties and moderate receptor binding affinity. In the GPI assay it
was a seven fold more potent opioid agonist (IC50 = 1.22 nM)
than its non-guanidinylated parent (5a) and, as was the case with
4, it behaved as a d full agonist in the MVD assay with subnanom-
olar potency (IC50 = 0.750 nM).
l
and d receptor binding affini-
In comparison with peptide 4a, its Lys3-analogue, H-Dmt-Tic-
Lys-Phe-NH2 (6a) showed similar subnanomolar d receptor binding
j
l
affinity, 16-fold lower
receptor affinity. In the functional assays 6a displayed a mixed
l
receptor affinity and >1000-fold higher
j
l
agonist/d antagonist profile similar to that of 4a. The fact that de-
spite the reduced receptor binding affinity of compound 6a, its
l

G. Weltrowska et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5082–5085
5085
agonist potency in the GPI assay is similar to that of 4a can be ex-
plained with its high receptor binding affinity. In addition to
receptors the GPI also contains receptors and their activation
by 6a compensated for the reduced receptor affinity of 6a as
orientations of lipophilic residues of the binding site seen in the
activated form of the receptor and may explain the conversion
from d antagonism to d agonism. Peptides 4 and 5 with a mixed
j
l
j
l
l agonist/d agonist profile are of interest because there is strong
compared to 4a. Interestingly, the guanidinylated analogue of 6a,
Guan-Dmt-Tic-Lys-Phe-NH2 (6) showed seven fold lower d recep-
evidence to indicate that compounds with this profile may be
effective for the treatment of pain with reduced side effects.18
tor affinity and 55-fold higher
l
receptor affinity than 6a and com-
receptor affinity of
The Lys3-analogues of DIPP-NH2 and DIPP-NH2[
W
] (guanidinylated
parable receptor affinity. The subnanomolar
j
l
or non-guanidinylated) are essentially mixed
l
/j
agonists with a d
6 explains its extraordinary agonist potency (IC50 = 0.289 nM) in
the GPI assay. As was the case with guanidinylated peptides 4
and 5, compound 6 was a d full agonist in the MVD assay. Its some-
what lower d agonist potency (IC50 = 4.69 nM) is in agreement with
antagonist, d full agonist or d partial agonist component. Com-
pounds with mixed l/j opioid receptor agonist activity have ther-
apeutic potential for treatment of cocaine abuse.19
l
its relatively lower d receptor binding affinity (Ki = 2.20 nM).
Acknowledgments
The receptor binding profile of the pseudopeptide H-Dmt-
Tic
W
[CH2NH]Lys-Phe-NH2 (7a) was similar to that of 6a, except
The work was supported by grants from the Canadian Institutes
of Health Research (MOP-89716) and the U.S. National Institutes of
Health (DA004443).
for its about nine fold lower d receptor affinity. Compound 7a
was a full agonist in the GPI assay with three fold higher potency
than 6a, but unlike 6a it behaved as a d partial agonist in the
MVD assay. In comparison with 7a, its guanidinylated derivative
(7) showed a similar opioid receptor binding profile, comparable
agonist activity in the GPI assay and similar d partial agonist activ-
ity in the MVD assay.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 07.
036.
Guanidinylation of the dipeptide d antagonist H-Dmt-Tic-OH11
(8a) had quite a drastic effect on the opioid activity profile. While
Guan-Dmt-Tic-OH (8) had d receptor binding affinity similar to
that of 8a, its
and it showed partial agonist activity in both the MVD assay and
the GPI assay. Thus, Guan-Dmt-Tic-OH is a partial agonist/d par-
tial agonist with low nanomolar binding affinity for and d recep-
tors and with no affinity for receptors at concentrations up to
10 M.
In conclusion, N-terminal amidination of various peptides of the
References and notes
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showed similar or enhanced
l and d receptor binding affinities as
compared to their non-guanidinylated parent peptides. In the case
of compounds 4 and 5 guanidinylation resulted in a significant
enhancement of
j receptor binding affinity. Amidination also pro-
duced unexpected effects on the efficacy of the compounds, pri-
marily at the d receptor. The guanidinylated derivatives of the d
antagonists 1a, 2a, 3a and 8a turned out to be d partial agonists
with subnanomolar or low nanomolar d receptor binding affinity.
An interesting case is the dipeptide Guan-Dmt-Tic-OH (8) which
retained high d receptor affinity, showed greatly enhanced
tor binding affinity as compared to H-Dmt-Tic-OH (8a) and turned
out to be a mixed partial agonist/d partial agonist. Also unex-
pected was the conversion of the mixed agonist/d antagonists
4a and 5a to mixed agonist/d full agonists (compounds 4 and
l recep-
l
l
l
5). In the case of peptides 4a and 4 the results of a d receptor dock-
ing study indicated a slightly shifted position of receptor-bound 4
relative to that of receptor-bound 4a as a consequence of the in-
creased steric bulk of the guanidino group as compared to the ami-
no group in the electrostatic interaction with the Asp128 residue of
the receptor. This results in the stabilization of side chain
19. Mello, N. K.; Negus, S. S. Ann. N.Y. Acad. Sci. 2000, 909, 104.