Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides

Article abstract of DOI:10.1021/acs.jmedchem.8b01601

In accordance with their common but also divergent physiological actions, human urotensin II (1) and urotensin II-related peptide (2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signaling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2, and fragment U-II_4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, 18 N-methylated UTR ligands were synthesized and their biological profiles evaluated using in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation, indicating relevant hydrogen-bond interactions for the function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.

Full text of DOI:10.1021/acs.jmedchem.8b01601

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Article
Functional Selectivity Revealed by N-Methylation
Scanning of Human Urotensin II and Related Peptides
Francesco Merlino, Etienne Billard, Ali Munaim Yousif, Salvatore Di Maro, Diego Brancaccio, Luigi
Abate, Alfonso Carotenuto, Rosa Bellavita, Roberta d'Emmanuele di Villa Bianca, Paolo Santicioli,
Luciana Marinelli, Ettore Novellino, Terence Hébert, William D. Lubell, David Chatenet, and Paolo Grieco
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01601 • Publication Date (Web): 07 Jan 2019
Downloaded from http://pubs.acs.org on January 8, 2019
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Functional Selectivity Revealed by N-Methylation
Scanning of Human Urotensin II and Related
Peptides
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Francesco Merlino,†,‡ Étienne Billard,∫,‡ Ali M. Yousif, Salvatore Di Maro,
†
†,Ω
Diego
†
†
†
†
Brancaccio, Luigi Abate, Alfonso Carotenuto, Rosa Bellavita, Roberta d’Emmanuele di Villa
†
§
†
†
#
Bianca, Paolo Santicioli, Luciana Marinelli, Ettore Novellino, Terence E. Hébert, William
∏
,∫
,†
D. Lubell, David Chatenet,* and Paolo Grieco*
†Department of Pharmacy, University of Naples ‘Federico II’, via D. Montesano 49, 80131
∫
Naples, Italy; INRS - Institut Armand-Frappier, Groupe de Recherche en Ingénierie des Peptides
et en Pharmacothérapie (GRIPP), Université du Québec, 531 Boulevard des Prairies, Ville de
§
Laval, Québec H7V 1B7, Canada; Department of Pharmacology, Menarini Ricerche, via
∏
Rismondo 12/A, 50131 Florence, Italy; Département de Chimie, Université de Montréal, C.P.
#
6
128, Station Centre-ville, Montréal, Québec H3C 3J7, Canada; Department of Pharmacology
and Therapeutics, McGill University, Montréal, Québec H3A 1A3, Canada.
ABSTRACT
In accordance with their common but also divergent physiological actions, human urotensin II
(hU-II, 1) and urotensin II-related peptide (URP, 2) could stabilize specific urotensin II receptor
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(
UTR) conformations, thereby activating different signalling pathways, a feature referred to as
biased agonism or functional selectivity. Sequential N-methylation of the amides in the
conserved core sequence of 1, 2 and fragment U-II4-11 (3) shed light on structural requirements
involved in their functional selectivity. Thus, eighteen N-methylated UTR ligands were
synthesized and their biological profiles evaluated using both in vitro competition binding
assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological
activity diverged from that of the parent structures contingent on the location of amide
methylation indicating relevant hydrogen-bond interactions for function of the endogenous
peptides. Conformational analysis of selected N-methyl analogs indicated the importance of
specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.
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INTRODUCTION
1
-3
Among the available synthetic strategies to modify peptide structures, N-methylation of amide
bonds has become an attractive tool.4-6 N-Methylation creates tertiary amide bonds that may
exhibit cis-trans isomer equilibrium due to increased steric hindrance with adjacent amino acid
side chains.7,8 Moreover, replacement of the amide hydrogen by a methyl group may disrupt
intra- and inter-molecular hydrogen-bonds, which may be respectively important for stabilization
9
of bioactive conformations and for receptor recognition. Introduction of N-methyl amides may
thus have significant consequences on both pharmacodynamics and pharmacokinetic
properties.6,10
1
1
Considering peptides as relatively flexible and metabolically unstable, susceptibility to
2
,3
proteolytic degradation, poor absorption and short in vivo half-life, all may be considered their
unfavorable pharmacokinetic properties that hinder therapeutic applications.12,13 In this regard,
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N-methylation of amide bonds in peptides may overcome such limitations by improving both
stability and bioavailability, correlated with increased activity and selectivity.4,9,14,15 N-
Methylation of multiple peptide bonds may have complementary effects to improve
bioavailability and target (e.g., receptor subtype) selectivity.15-17 Driven in part by the success of
natural N-methylated peptides, such as the immunosuppressant drug cyclosporine A and the
nematicide omphalotin,18,19 N-methylation has been pursued to improve “drug-like” properties,10
in a wide range of peptides possessing antibiotic,20 anticancer,21 as well as various other
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activities. To our knowledge however, backbone N-methylation has never been investigated as
a tool to reveal functional selectivity that represents the ligand-dependent selectivity for certain
signal transduction pathways relative to the native ligand of the receptor.23
24
The urotensinergic system, consisting of two endogenous ligands, i.e. urotensin II and
urotensin II-related peptide [hU-II (1) and URP (2), Chart 1], and their G protein-coupled
receptor (GPCR) termed UTR, is involved in several patho-physiological conditions in
humans.25,26 Accordingly, several UTR antagonists, such as 1-[2-(4-benzyl-4-hydroxy-piperidin-
1
-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt (ACT-058362, also known as
Palosuran),27
4′-[(1R)-1-[[(6,7-dichloro-3-oxo-2,3-dihydro-4H-1,4-benzoxazin-4-yl)-
acetyl](methyl)amino]-2-(4-morpholinyl)ethyl]-4-biphenylcarboxylicacid
trifluoroacetate
(
GSK1440115),28 and 2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-
dimethoxybenzenesulphonamide hydrochloride (SB-657510),29 have been shown to protect
against renal ischemia,27 asthma,28 liver fibrosis,30 and pulmonary hypertension in rodents.31
However, none of these candidates exhibited efficacy in clinical trials.25
Although studies in vitro using HEK 293 cells have failed to show differences in the
signaling signatures of endogenous UTR ligands, in target tissues, distinct actions of 1 and 2
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have been demonstrated on transcription initiation, astrocyte proliferation, polyphosphoinositide
turnover, ANP release, and myocardial contraction.24,25 Structure-activity relationship (SAR)
studies have demonstrated that the intra-cyclic residues of isoforms of 1 and 2 play critical roles
in both recognition and activation of UTR.25,26 Likewise, SAR studies of both peptides were
usually similar, but, in certain cases, striking differences were noted despite the introduction of
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identical modifications. The N-terminal region of 1 differs across species. On binding, the N-
terminal tail of 1 has been suggested to cause the UTR to undergo a conformational change to
accommodate the Trp-Lys-Tyr triad of the cyclic peptide.32-34 Therefore, differences in the
signaling signatures of 1 and 2 may be due in part to structural variance at their N-terminal
1
regions leading to alternate receptor binding modes. Accordingly, salt bridges between Glu and
Lys196 and between Asp and Arg have been observed in the 1-UTR complex, as supported by
conformational and docking studies.35 Considering that understanding of ligand-receptor
interactions and their consequences on UTR molecular pharmacology is necessary to reveal
divergent signaling pathways (functional selectivity),23,36 probes to dissect the distinct UTR
signaling outcomes in responses to 1 or 2 are essential for assigning specific roles in cell
homeostasis and pathogenesis in UTR-associated diseases.25,37
4
193
Systematic N-methyl scanning of the cyclic peptide sequences of UTR ligands 1-3 may
offer a way to examine the molecular determinants of functional selectivity. The syntheses of N-
methylated derivatives of 1, its paralogue peptide 2 and truncated, equally active analog U-II4-11
(3), all were pursued for subsequent biological evaluations using a combination of radioligand
binding experiments, ex vivo aortic ring contraction assays, and in vitro evaluation of Gq
activation. Finally, conformational analyses were performed on selected analogs. Notably, N-
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methylation of the amides of three residues Phe , Trp and Cys7/10 were identified to affect the
3/6
4/7
biological properties of analogs of 2 in ways that diverged from those exhibited by 1 and 3.
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Chart 1. Chemical structures of hU-II (1), URP (2) and U-II4-11 (3).
RESULTS AND DISCUSSION
Design. Mono N-methylation has been used for years to alter the pharmacological properties of
peptides.4,14 Considering that the N-terminal segments of 1 and 2 likely act as molecular
discriminators for binding and activation processes by way of stabilization of different receptor
conformational subpopulations, these residues were not methylated. Instead, amides of residues
within the common cyclic region present in 1-3 were replaced systematically by their
α
corresponding N -methyl counterpart to provide derivatives 4-21 for head-to-head comparisons
(Table 1).
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Table 1. Peptides 1-3 with N-methylation sites as red-highlighted and N-(methyl)peptide
sequences 4-21.
OH
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O
OH
HN
O
NH
S
R2
S
O
H
N
HN
HN
O
R1
N
H
O
O
NH
NH2
H
N
HO
O
O
O
O
O
NH
H
N
1
: R1
=
H N
N
, R2 = CH CO H
2 2
2
O
OH
2
3
: R1 = H, R2 = CH3
N-methylation sites
: R = H, R2 = CH CO H
1
2
2
Cmpd
Sequence
5
[
(N-Me)Cys ]hU-II (4)
Glu-Thr-Pro-Asp-c[(N-Me)Cys-Phe-Trp-Lys-Tyr-Cys]-Val
Glu-Thr-Pro-Asp-c[Cys-(N-Me)Phe-Trp-Lys-Tyr-Cys]-Val
Glu-Thr-Pro-Asp-c[Cys-Phe-(N-Me)Trp-Lys-Tyr-Cys]-Val
Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-(N-Me)Lys-Tyr-Cys]-Val
Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-(N-Me)Tyr-Cys]-Val
Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-(N-Me)Cys]-Val
Ala-c[(N-Me)Cys-Phe-Trp-Lys-Tyr-Cys]-Val
6
[
(N-Me)Phe ]hU-II (5)
7
[(N-Me)Trp ]hU-II (6)
8
[
(N-Me)Lys ]hU-II (7)
9
[
(N-Me)Tyr ]hU-II (8)
1
0
[
(N-Me)Cys ]hU-II (9)
2
[(N-Me)Cys ]URP (10)
3
[
(N-Me)Phe ]URP (11)
Ala-c[Cys-(N-Me)Phe-Trp-Lys-Tyr-Cys]-Val
4
[
(N-Me)Trp ]URP (12)
Ala-c[Cys-Phe-(N-Me)Trp-Lys-Tyr-Cys]-Val
5
[(N-Me)Lys ]URP (13)
Ala-c[Cys-Phe-Trp-(N-Me)Lys-Tyr-Cys]-Val
6
[
(N-Me)Tyr ]URP (14)
Ala-c[Cys-Phe-Trp-Lys-(N-Me)Tyr-Cys]-Val
7
[
(N-Me)Cys ]URP (15)
Ala-c[Cys-Phe-Trp-Lys-Tyr-(N-Me)Cys]-Val
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[
(N-Me)Cys ]U-II (16)
Asp-c[(N-Me)Cys-Phe-Trp-Lys-Tyr-Cys]-Val
Asp-c[Cys-(N-Me)Phe-Trp-Lys-Tyr-Cys]-Val
Asp-c[Cys-Phe-(N-Me)Trp-Lys-Tyr-Cys]-Val
Asp-c[Cys-Phe-Trp-(N-Me)Lys-Tyr-Cys]-Val
Asp-c[Cys-Phe-Trp-Lys-(N-Me)Tyr-Cys]-Val
Asp-c[Cys-Phe-Trp-Lys-Tyr-(N-Me)Cys]-Val
4
-11
6
[
(N-Me)Phe ]U-II (17)
4
-11
7
[(N-Me)Trp ]U-II (18)
4
-11
1
1
1
1
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[
(N-Me)Lys ]U-II (19)
4-11
9
[
(N-Me)Tyr ]U-II (20)
4
-11
1
0
[
(N-Me)Cys ]U-II (21)
4-11
Chemistry. Peptides 4-21 were produced by solid-phase peptide synthesis (SPPS) using 9-
fluorenylmethoxycarbonyl (Fmoc)-chemistry, as previously reported.38,39 To insert N-methyl
α
α
amide bonds into peptides, commercially available N -Fmoc-N -methyl-α-amino acids can be
employed, although they are relatively expensive. N-Methyl amino acid building blocks have
4
0
41
also been synthesized in solution and by solid-phase methods. A solid-phase N-methylation
procedure was pursued to favor library synthesis of N-methyl-peptides (Scheme S1, see
Supporting Information). The solid-phase protocol was based on reported methods that proved
compatible with most amino acids.4,41,42 Although methylation using DBU as base and dimethyl
4
sulfate as alkylating agent in NMP was typically completed in 5 min, loss of the trityl protection
of the cysteine side chains diminished product quality,40,41 such that a Mitsunobu reaction
procedure was developed to prepare derivatives 4-21.43,44 Coupling to N-methyl amino acids has
been found to be challenging due to steric hindrance. Employment of HATU and HOAt as
coupling reagents in combination with DIEA as base in NMP favored acylation of the N-methyl
45
peptide. Overall, the N-methylation method was effective for synthesizing a library of mono-N-
methyl derivatives of 1-3 (analogs 4-21, Table 1). After cleavage peptides were purified by
reverse-phase (RP) high-pressure liquid chromatography (HPLC). Each peptide (4-21) was
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ascertained to be of >98% purity by analytical RP-HPLC in two solvent systems, and exhibited
the expected molecular ion on analysis by high-resolution mass (HRMS) spectrometry (LTQ
Orbitrap) (Table S1 and Figures S1-10 for the most representative analogues, see Supporting
Information).
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Biological evaluations. The propensity of each analog to bind UTR was initially evaluated using
a competition binding assay. N-Methylated analogs 4-21 were examined on either HEK 293 or
CHO-K1 cells stably expressing the human UTR isoform. The peptides exhibited varying
degrees of binding affinity for the human UTR (Table 2). N-Methylation of the amides at the
Phe , Lys and Tyr9/6 residues of peptides 1-3 resulted in a dramatic loss of binding affinity,
6
/3
8/5
but had no major effect at the Trp7/4 residue. The Phe -Trp -Lys -Tyr segment in the ring of 1
6
7
8
9
has previously been suggested to adopt a turn conformation that is stabilized by hydrogen bonds
6
9
6
9
46
between Phe NH and Tyr CO and between Phe CO and Tyr NH. The loss of affinity
exhibited by 5, 8, 11, 14, 17 and 20 highlighted further the importance of such a hydrogen-bond
network for binding affinity of the parent peptides 1-3, while N-Methylation of the amides of
cysteine residues had a slight effect on binding affinity.
In general, N-methylation diminished binding affinity relative to the parent peptides 1-3.
The decrease in binding affinity on N-methylation was exhibited more profoundly by analogs of
1
(e.g., 4-9) compared to its smaller counterparts 2 (e.g., 10-15) and 3 (e.g., 16-21) (Table 2).
The N-terminal domain of 1 has recently been demonstrated to be involved in specific receptor
interactions that may modulate the receptor binding pocket.35,47 Hence, the decreased tolerance
of N-methylation on longer relative to shorter peptides may reflect a diminution of the ability of
the receptor to accommodate the bulkier peptide tail.
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N-Methyl peptides 4-21 were next tested for ability to induce aortic ring contraction.
Similar potency and efficacy compared to 1 was exhibited in an aortic ring contraction assay by
5
7
10
analogs possessing N-methylation at Cys (e.g., 4), Trp (e.g., 6) and Cys (e.g., 9); however, N-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
8
9
methyl amides at Phe , Lys and Tyr (analogs 5, 7 and 8) decreased significantly contractile
potency (Table 2, Figure 1a), producing weak agonists. Reduced contractile potency of 5 and 8
correlated with their weaker binding affinity and may likely be due to disruption of essential
hydrogen bonds for the active secondary structure. The deleterious effect of N-methylation at
8
Lys (e.g., 7) has not been previously reported and may reflect engagement of the amide NH in a
5
7
hydrogen bond. Although [(N-Me)Cys ]hU-II (4) and [(N-Me)Trp ]hU-II (6) exhibited lower
affinity than 1, they retained similar contractile potency as the parent peptide.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Binding affinity and contractile activity of 1, 2 and 3 and related N-methyl analogs 4-
2
1.
Binding affinity
Aortic ring contraction
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Peptide
pKia
pEC50b
Emaxc
hU-II (1)
URP (2)
8.11 ± 0.07
8.52 ± 0.11
102 ± 4
116 ± 4
100 ± 4
112 ± 4
96 ± 10
97 ± 10
8.20 ± 0.12
8.34 ± 0.04
8.11 ± 0.06
8.28 ± 0.12
8.01 ± 0.14
6.70 ± 0.30***
8.24 ± 0.42
U-II4-11 (3)
5
[(N-Me)Cys ]hU-II (4)
7.31 ± 0.14**
6.09 ± 0.20***
7.69 ± 0.09*
5.68 ± 0.31***
5.67 ± 0.48***
7.64 ± 0.15*
8.76 ± 0.07*
6.61 ± 0.06***
8.66 ± 0.13*
5.82 ± 0.21***
5.27 ± 0.84***
8.45 ± 0.15
6
[
(N-Me)Phe ]hU-II (5)
7
[
(N-Me)Trp ]hU-II (6)
8
[
(N-Me)Lys ]hU-II (7)
72% @ 10 µM
71% @ 10 µM
7.61 ± 0.10**
9
[(N-Me)Tyr ]hU-II (8)
10
[
(N-Me)Cys ]hU-II (9)
105 ± 3
110 ± 6
2
[
(N-Me)Cys ]URP (10)
8.31 ± 0.19
3
[(N-Me)Phe ]URP (11)
51% @ 10 µM
4
[
(N-Me)Trp ]URP (12)
7.08 ± 0.45***
35 ± 5***
5
[
(N-Me)Lys ]URP (13)
22% @ 10 µM
16% @ 10 µM
8.02 ± 0.49
6
[(N-Me)Tyr ]URP (14)
7
[
(N-Me)Cys ]URP (15)
44 ± 6***
101 ± 8
98 ± 5
5
[
(N-Me)Cys ]U-II (16)
8.53 ± 0.08
8.35 ± 0.15
6.27 ± 0.09***
8.47 ± 0.09
4
-11
6
[
(N-Me)Phe ]U-II (17)
6.64 ± 0.10***
8.76 ± 0.07**
< 5
4-11
7
[(N-Me)Trp ]U-II (18)
105 ± 5
4
-11
8
[
(N-Me)Lys ]U-II (19)
42% @10 µM
4
-11
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9
9
[
(N-Me)Tyr ]U-II (20)
< 5
44% @10 µM
8.28 ± 0.11 95 ± 5
4
-11
1
0
[
(N-Me)Cys ]U-II (21)
8.45 ± 0.05
4
-11
a
b
c
pK : −logK ; pEC : −log EC ; maximum efficacy is expressed as a percentage of the KCl-
i
i
50
50
−
7
induced contraction (40 mM) divided by the tissue-response induced by 1 (10 M). All values
are expressed as mean ± S.E.M. Statistical analysis were performed using unpaired Student’s t
test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, versus values obtained for 1 (4-9), 2 (10-15), or 3 (16-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1). Each replicate (n) was conducted on tissue from different animals or on different cell
passages.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 1. Representative concentration-response curves obtained with rat thoracic aorta rings
after adding cumulative concentrations of a) 1 or related N-methylated analogs 4-9; b) 2 or
related N-methylated analogs 10-15; c) 3 or related N-methylated analogs 16-21. Efficacy is
expressed as a percentage of the KCl-induced contraction (40 mM) divided by the tissue-
response induced by 1 (10−7 M). Each replicate (n) was conducted on tissue from at least 3
different animals. Data represent the mean ± S.E.M.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Analysis of the contractile potential of N-methyl analogs of 2 indicated that the weak
3
5
6
binding affinity of N-Me-Phe (11), N-Me-Lys (13) and N-Me-Tyr (14) correlated with
inability to induce contractile action on rat aortic ring (Table 2, Figure 1b). Although N-
2
methylation at both cysteines of 2 had little impact on binding affinity, N-Me-Cys (10) was a
7
full agonist and N-Me-Cys (15) exhibited partial agonism (E
= 44 ± 6%) in the
max
vasocontractile assay. Previously, inversion of the configuration of the two cysteines in 2 had
differentially affected both binding affinity and biological activity with more pronounced effects
7
2 48
on Cys than Cys .
Evaluation of the contractile action of N-methyl analogs of 3 revealed a similar correlation
6
8
9
between the low binding affinity of the N-methyl Phe , Lys and Tyr analogs (e.g., 17, 19 and
2
0, Table 2) and weak agonist activity. Previously, N-methylation of the Tyr residue in 3 {e.g.,
9
6
[
(N-Me)Tyr ]U-II , 20} and replacement of Phe in 3 with the constrained secondary amino
4-11
acid octahydro-indole-2-carboxylic acid (Oic) were respectively shown to dramatically reduce
4
9
34
peptide potency, and both affinity and contractile activity, consistent with perturbation of
6
9
intra-molecular hydrogen bonds between Phe and Tyr that stabilize the active secondary
4
6
structure. In contrast, respective N-methylation of the amide bonds of Trp and the two Cys
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7
8
9
residues in 16, 18 and 21 led to peptides exhibiting equipotent binding affinity and contractile
activity as 3 (Table 2, Figure 1c).
Comparing the effects of N-methylation on peptides 1-2, certain positions exhibit
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10
divergent activity contingent on the parent structure. For example, N-Me-Cys analogs of 1 and
3
(e.g., 9 and 21) acted as full agonists, but in their counterpart 2 (e.g., 15) N-methylation
resulted in partial agonism (Emax = 44 ± 6%). Such differences in the behavior of the two
2
7
48
cysteines that belong to 2 were also previously observed with [DCys ]URP and [DCys ]URP.
2
Indeed, inversion of configuration of Cys in 2 was less detrimental for the contractile activity
than similar replacement of Cys7,48 which is also illustrated herein comparing the activities
between 10 and 15. As previously demonstrated, the presence of specific interactions between
the N-terminal domain of 1 and 3 and UTR might modulate the topography of the binding pocket
3
5
accommodating the common C-terminal tail of the peptides. Hence, as expected, modifications
1
0
occurring on Cys in 1 and 3 produce distinct outcomes compared to similar modification in 2.
Finally, our recent works suggested that the orientation of Tyr6/9 side chain in 2 and 3 may affect
the contractile activity of the ligand.50-52 It is therefore possible that the N-methylation of Cys7/10
in 1 and 2 may restrict the orientation of the adjacent Tyr residue impacting only on the
contractile profile of 2. Altogether, these results suggest that the conformational requirements of
the Tyr residue may differ between 1, 2 and 3.
Moreover, agonist potency varied with N-methylation of the Phe residue: 5 (pEC 6.70 ±
50
0.30) > 17 (pEC 6.36 ± 0.04) >> 11 (pEC < 5). The Phe amide NH or the conformation of the
5
0
50
phenyl ring appear to be more relevant for the activity of 2 than 1 and 3, therefore suggesting a
subtle but different involvement of this residue in UTR activation. Accordingly, substitution of
the phenylalanine residue with a cyclohexylalanine moiety (Cha) in 3 was reported to generate
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
an inactive analog, i.e. [Cha ]U-II4-11,53 that suppressed the vasoconstrictor effect of 1 on rat
6
3
53
aortic rings. The same modification of 2, i.e. [Cha ]URP, produced a partial agonist. Moreover,
introduction of iodide at the para position of the phenyl ring of Phe in 2 and 3 led to divergent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
6
vasoconstrictor activities: [Phe(pI) ]URP acted as a partial agonist and [Phe(pI) ]U-II behaved
4
-11
5
4
4
193
as a full agonist. As recently reported, interaction between Asp of 1 and 3 with Arg of UTR
may influence the positioning of TM4 and ECL2 of UTR, two domains whose flexibility is
5
4
crucial for receptor activation upon ligand binding. Importantly, these two domains delineate
the binding site of the Phe3/6 residue. Hence, the absence of interaction in the case of 2, in
55
4
which the Asp residue is replaced by an alanine, will therefore create a slightly different binding
pocket that might otherwise accommodate this residue, so explaining the difference of contractile
activity between 2 and 3 Phe-substituted analogues despite retaining similar affinity.54
4
N-Methylation of Trp of 2 in 12 led also to a partial agonist (E
= 35 ± 5*** %) in
max
contrast to agonists 6 and 18. Previously, enantiomeric substitution of DTrp for Trp in both 1 and
7
2
provided [DTrp ]hU-II, which had a 100 fold lower affinity for UTR than 1 and acted as a
3
3
4
partial agonist, and [DTrp ]URP, which exhibited high affinity and completely abolished the rat
48
32,33
aortic ring contraction induced by 1. Accordingly, similar results were obtained elsewhere.
Considering the divergent behavior of Trp analogs of 1 and 2, allosteric modulators were
developed by substitution of alternative residues at this particular residue in 2 and 3.39,56,57 To
resume, N-methyl substitution of residues Phe , Trp and Cys7/10 resulted in divergent effects
3
/6
4/7
in peptides 1-3 highlighting the relevance of their backbone amides for affinity and activity.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Representative concentration-response curves of G protein activation for peptides, 5,
q
6
, 9, 11, 12, 15, 17, 18, 21 and 1, 2, 3 using a BRET-based biosensor. Efficacy is expressed as a
−
5
percentage of the activation induced by 2 (10 M). Each replicate (n) was conducted on at least
3 different cellular passages. Data represent the mean ± S.E.M.
Table 3. G activation of 1, 2, 3 and selected analogs 5, 6, 9, 11, 12, 15, 17, 18, and 21.
q
G activation
q
Peptide
pEC50a
Emaxb
100 ± 3
100 ± 4
98 ± 3
91 ± 7
95 ± 5
95 ± 5
93 ± 5
94 ± 5
URP (2)
U-II4-11 (3)
hU-II (1)
8.57 ± 0.08
8.73 ± 0.14
8.52 ± 0.09
3
[
(N-Me)Phe ]URP (11)
6.98 ± 0.17***
7.73 ± 0.13**
7.65 ± 0.05***
7.86 ± 0.15***
8.44 ± 0.17
6
[(N-Me)Phe ]U-II (17)
4
-11
6
[
(N-Me)Phe ]hU-II (5)
4
[
(N-Me)Trp ]URP (12)
7
[
(N-Me)Trp ]U-II (18)
4
-11
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
[
(N-Me)Trp ]hU-II (6)
8.25 ± 0.08
8.28 ± 0.10
91 ± 3
91 ± 3
89 ± 4
101 ± 5
7
[
(N-Me)Cys ]URP (15)
1
0
[(N-Me)Cys ]U-II (21)
8.33 ± 0.15
8.57 ± 0.05
4
-11
1
0
[
(N-Me)Cys ]hU-II (9)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
pEC : –log EC ; Maximum efficacy is expressed as a percentage of the amplitude of the
5
0
50
–5
activation induced by 2 (10 M). All values are expressed as mean ± S.E.M. Statistical analysis
was performed using an unpaired Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, versus
values obtained for non-N-methylated-peptide (either 1, 2 or 3); # P ≤ 0.05, comparing the
isoforms sharing the same modification. Each replicate (n) was conducted on different cellular
passage. N.S.: non significant.
58
Activation of G was associated with aortic contraction mediated by 1 and 2. The influence of
q
N-methylation at the Phe , Trp and Cys7/10 residues on G activation was thus investigated in
3
/6
4/7
q
a comparison of the nine N-methyl analogs (5, 6, 9, 11, 12, 15, 17, 18, and 21) to peptides 1-3.
The latter three reference peptides 1-3 activated G with similar potency and efficacy (Table 3,
q
Figure 2). N-Methylation had a limited influence that usually reduced agonist potency to activate
G . At the Phe residue, this resulted in weak agonists, but 11 appeared to be significantly weaker
q
than 5 and 17, which was consistent with their affinities and contractile activities. N-Methylation
of Trp in 2 led to a significantly weaker agonist (12) compared to 6 and 18, which appeared to be
equally active as their parent peptides, consistent with their aortic activities. N-Methylation of
7
Cys impacted however the contractile activity of 2 without affecting 1 and 3, which activated G
q
with the same potency as their parent peptides. In contrast to analogs of 1, 9 and 21, an analog of
2
, 15, may activate another pathway involved in aortic contraction, perhaps the small GTPase
5
9
RhoA through G activation contributing to aortic ring contraction mediated by 1. Although
12
activation of G was not examined, the present results suggest that 15 and potentially the partial
1
2
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4
5
6
7
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9
contractile agonist 12 behave as biased agonists. For reference, biased agonists have previously
3
6
50,54
been produced on substitution of Phe and Tyr of 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Conformational analysis by NMR spectroscopy. Previously, NMR spectroscopy supported
6
9
with computational analysis demonstrated that the amide NH and CO moieties of Phe and Tyr
engaged in two hydrogen bonds that stabilized a β-hairpin conformation in peptides 1-
6
9
3
.46,47,51,52,61,62 Consistently, N-methylation of the Phe and Tyr amides reduced binding affinity
and activity in the different analogs. In contrast, spectroscopic analysis of peptides 1-3 has not
implicated the Lys amide in intramolecular hydrogen bonding,46,47 yet N-Me-Lys derivatives 7,
13 and 19, all lost affinity and potency. To examine the latter effect of N-methylation, [(N-
8
46
Me)Lys ]U-II (19) was investigated by NMR spectroscopy in SDS micelles in solution. The
4
-11
7
improved affinity and activity of [(N-Me)Trp ]U-II
(18) relative to 3 also required a
-11
4
conformational analysis. Considering that N-methylation of the Trp and Lys amides had limited
influence on binding of peptides 1-3, and that the three latter peptides adopted similar
conformations at their cyclic regions in earlier NMR studies,35,46 the results of the spectroscopic
analyses of N-methyl peptides 18 and 19 may be used to interpret the effects of similar
modifications on their respective 1 and 2 homologues (e.g., 6, 12, and 7, 13). Peptides 18 and 19
4
6
and their parent 3, here reported for comparison, exhibited similar NMR spectra (Tables S2-S4,
see Supporting Information), which indicate the presence of a β-hairpin structure centered on the
7
8
Trp and Lys residues. Mainly, medium range NOE signals demonstrated the presence of this
structural feature (Tables S5-S6, see Supporting Information). The NMR spectrum of [(N-
7
Me)Trp ]U-II (18) differed from that of 3 by intense downfield shifts of the Hα resonances of
4
-11
residues 4-6 and 9-11, which flank the central turn (Figure S11, see Supporting Information), and
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7
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
indicated that methylation at Trp caused hairpin stabilization. Significant up-field shifts of the
8
7
NH and Hγ resonances of Lys and the Hα signal of Trp , due to the ring current of the indole
7
moiety, indicated stabilization of the trans χ -dihedral angle orientation of the Trp side chain. In
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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1
2
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4
5
6
7
8
9
0
8
7
8
contrast, in the spectrum of [(N-Me)Lys ]U-II
(19), the Hα resonances of Trp and Lys
-11
4
exhibited large downfield shifts compared to those of random coil peptides indicative of a
6
3
conformational change at the turn region (Figure S11). Computational analysis using molecular
dynamics (MD) and NMR-derived constraints (Tables S5-S6, see Supporting Information)
provided an ensemble of conformations for each peptide, from which the ten lowest energy
conformers were grouped together (Figure 3).
Figure 3. Superposition of the ten lowest energy conformers of a) 18 (PDB code: 6HVB) and b)
1
9 (PDB code: 6HVC). Structures were superimposed using the backbone heavy atoms of
residues 5-10. Heavy atoms are shown with different colors (carbon, green or cyan; nitrogen,
blue; oxygen, red; sulfur, yellow). Only polar hydrogen atoms are shown for clarity. A star
indicates the N-methylated residues.
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A well-defined β-hairpin structure was observed for peptide 18 having a rms deviation of
the cyclic backbone heavy atoms equal to 0.10 Å (Figure 3a). The ensemble of NMR-derived
conformers of peptide 18 was significantly better defined than that previously obtained for 3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
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4
4
4
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5
5
5
5
5
5
5
5
6
0
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6
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6
7
8
9
0
rmsd 0.22 Å).46 The slight yet significant improvement of both the affinity and activity of
(
peptide 18 compared to 3 may tentatively be attributed to stabilization of this conformation.
4
A similar stabilization of the β-hairpin conformer for [(N-Me)Trp ]URP (12) may account
for the improved binding affinity compared to 2. The reduced potency and efficacy of 12 may be
7
due to the loss of flexibility of the Trp side chain, which is forced into a trans conformation to
avoid a steric clash with the N-methyl amide. UTR activation by 2 may thus require a post-
7
binding movement of the Trp side chain, which is inhibited in 12. Considering both [(N-
7
7
Me)Trp ]hU-II (6) and [(N-Me)Trp ]U-II
(18) exhibited increased binding affinity and
-11
4
7
potency compared to their parent peptides 1 and 3, adjustment of the Trp side chain may not be
necessary for their activation of the UTR, in contrast to 2. Accordingly, constraint of the Trp side
chain to a gauche-(+) or trans orientation by cyclization onto the nitrogen of the C-terminal Lys5
in a 4-amino-1,2,4,5-tetrahydroindolo[2,3-c]-azepin-3-one residue (Aia) has provided
4
47
[
Aia ]URP, which exhibited partial agonism in the aortic ring contraction assay.
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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3
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0
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0
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0
1
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3
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Superposition of the lowest energy conformers of 18 and 19. Structures were
superimposed using the backbone heavy atoms of residues 5-10. Heavy atoms are shown with
different colors (carbon, green or cyan; nitrogen, blue; oxygen, red; sulfur, yellow). Only polar
hydrogen atoms are shown for clarity.
8
The β-hairpin structure typical of UTR ligands was retained in [(N-Me)Lys ]U-II4-11 (19,
7
9
Figure 3b); however significant differences in the orientation of the side chains of Trp and Tyr
were observed in comparison with 3 and 18 (Figure 4), due likely to steric clashes between the
7
N-methyl amide and the proximal indole and phenol side chains. Although the Trp side chain χ
1
9
angle was oriented trans in both 3 and 19, the χ angle rotated 60° to −127° in 19. The Tyr side
2
chain χ angle rotated from a gauche-(−) (in 3 and 18) to a trans conformation in 19. Moreover,
1
8
the Lys side chain, which is relatively flexible in peptides 3, 18 and 19, rotated from a preferred
gauche-(+) χ angle in 3 and 18 to a preferred gauche-(−) orientation in 19 (Figure 4), placing
1
the positively charged nitrogen in a different position. The trans orientation of the χ angle of
1
9
Tyr shown by 19 has been also observed in a few analogs of 3 and suggested to promote
antagonism.52,61,63 Low efficacy of analogs 9, 15, and 21 is in accordance with this suggestion. In
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contrast, the observed Trp orientation around χ angle in 19 is unprecedented in our studies and
2
may account in part for the drop in affinity and potency of the N-Me-Lys8/5 derivatives 7, 13 and
19. In contrast, N-methylation of the corresponding lysine residue in octreotide analogs has been
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
well tolerated in certain somatostatin receptors binding ligands that engage sst and sst5
2
subtypes.5,64 Such a structural requirement may account in part for the reciprocal activity of
ligands of UTR vs somatostatin receptors.
CONCLUSIONS
N-Methylation of peptide backbone amide bonds is commonly used to improve “drug-like”
properties of peptides. A new application of this technique has been demonstrated by the N-
methylation of amides at the cyclic core regions of the two endogenous peptide ligands of UTR.
Synthesis and examination of the biological profiles of N-methyl-peptides 4-21 of the
endogenous peptides 1 and 2 has proven effective to dissect their shared conformer from the
different conformational requirements responsible for their distinct activities. Notably, N-
methylation at the Lys and Tyr residues resulted in a loss of agonist activity of both natural
ligands 1 and 2. The N-methylation of the Phe residue of 2 (e.g., 11) reduced the ability to induce
aortic contraction and to activate G more significantly compared to the same modification of 1
q
and 3 (e.g., 5 and 17, respectively). N-Methylation of the Trp residue of 2 (e.g., 12) enhanced
binding affinity, but drastically reduced contractile efficacy and G activation potency, in
q
contrast to the same modification on 1 and 3 (e.g., 6 and 18, respectively). N-Methylation of the
Cys7/10 (e.g., 9, 15 and 21) had different effects on vasocontractile action without influencing
binding affinity of 1-3. Conformational analysis of select analogs (e.g., 18 and 19) revealed
different conformational requirements that may explain the functional selectivity observed
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
between peptide hormones 1 and 2. Finally, considering the interest in the urotensinergic system
has been renewed in light of the discovery of biased agonism at UTR and an emerging role in
inflammatory disease and cancer, our study focused on the N-methylation scanning
of 1, 2 and 3 peptide sequences offers, for the first time in this context, tools to discern the
specific molecular determinants involved in such functional selectivity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
EXPERIMENTAL SECTION
Materials and general methods. The amino acids Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-
Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Glu(OtBu)-OH, all were
purchased from GL Biochem Ltd (Shangai, China). Coupling reagents used, that are N,N,N’,N’-
tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate
(HBTU),
(HATU),
O-(7-
azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
1-
hydroxybenzotriazole (HOBt), and 1-hydroxyazabenzotriazole (HOAt), all were also obtained
commercially from GL Biochem Ltd (Shangai, China). The Wang resin (0.64 mmol/g as
loading) was purchased from Chem-Impex, and the manufacturer’s reported loading of the resin
was used in the calculation of final product yields. N,N-diisopropylethylamine (DIEA), acetic
anhydride, 4-(dimethylamino)pyridine (DMAP), orto-nitrobenzenesulfonyl (o-NBS) chloride,
2
,4,6-collidine, DIAD, Ph P, 2-mercaptoethanol, anisole, dimethyl sulfoxide (DMSO), all were
3
purchased from Sigma-Aldrich/Merck and used as received. Piperidine, trifluoroacetic acid
TFA) and DBU, were purchased from Iris Biotech GmbH. Solvents for peptide synthesis such
(
as N,N-dimethylformamide (DMF), dichloromethane (DCM), NMP, diethyl ether (Et O), and for
2
HPLC analyses and purifications such as water, MeOH and acetonitrile (MeCN), were reagent
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8
9
grade acquired from commercial sources (Merck/Sigma-Aldrich and VWR) and used without
further purification. Anhydrous solvents [DCM, DMF and tetrahydrofuran (THF)] were obtained
by Merck/Sigma Aldrich. LC-MS analyses were performed on a LC-MS instrument from
Agilent Technologies equipped with an analytical C18 column (Phenomenex Kinetex, 5 μm, 150
mm × 4.6 mm, 100 Å) and 6110 Quadrupole, in positive electrospray ionization (ESI) mode, to
confirm N-methylation step and difficult couplings had achieved >90% conversion. High-
resolution mass (HRMS) measurements were recorded on a LTQ Orbitrap mass spectrometer in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
positive ESI mode, and proton adducts [M + H] were used for empirical formula confirmation.
Purification of compounds 4-21 was performed on a semi-preparative reverse-phase C18
column (Phenomenex Kinetex, 5 μm, 150 × 21.2 mm, 100 Å) using specific linear gradients of
MeCN (0.1% TFA) in water (0.1% TFA) with a flow rate of 10 mL/min (from 10 to 90% over
4
0 min) and UV detection at 220 nm. Final products were obtained by freeze-drying the
appropriate fractions after removal of MeCN under reduced pressure by rotary evaporation. The
purity of compounds 4-21 was ascertained using analytical HPLC analyses, which were
performed on a reverse-phase C18 column (Phenomenex Kinetex, 5 μm, 150 mm × 4.6 mm, 100
Å) with a flow rate of 1 mL/min using a gradient of MeCN (0.1% TFA) or MeOH (0.1% TFA)
in water (0.1% TFA), and UV detection at 220 and 254 nm. All compounds examined for
biological activity were purified to >98%, and the correct molecular ions were confirmed by
HRMS (Table S1, see Supporting Information).
Synthesis of protected linear sequences. The synthesis of N-methylated analogs 4-21 was
performed manually in a stepwise fashion by SPPS,38,39,65 using Fmoc/tBu orthogonal protection.
Wang resin (0.2 mmol) was placed into a 10-mL plastic syringe tube equipped with Teflon™
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
filter, stopper, and stopcock, pre-swollen in DMF on an automated shaker at rt for 30 min,
treated with the first amino acid, Fmoc-Val-OH (4 equiv), HBTU (4 equiv), HOBt (4 equiv),
DIEA (8 equiv) and a catalytic amount of DMAP (0.15 equiv), to facilitate ester formation, all
suspended in DMF, and shaken for 3 h, at rt. After the reaction was ascertained to have gone to
completion, the resin was washed with DMF (2 mL × 3) and DCM (2 mL × 3). To avoid
potential parallel synthesis of side products, any remaining reactive resin sites were end capped
by treatment with acetic anhydride (2 equiv), DIEA (4 equiv) and DMAP (0.4 equiv) in DCM,
and the resulting mixture was agitated for 16 h at rt. The resin was washed with DMF (2 mL × 3)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
α
and DCM (2 mL × 3). The N -Fmoc protecting group was removed from the Val residue by
treatment with 20:80 piperidine/DMF (v/v) (2 mL × 5 min, 2 mL × 25 min). The resin was
washed with DMF (2 mL × 3) and DCM (2 mL × 3). The protected amino acids (4 equiv) needed
for the construction of required peptide sequence [Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Ala-
38
OH] were then coupled according to SPPS protocols, using HBTU (4 equiv) and HOBt (3
equiv) in the presence of DIEA (8 equiv) as base, in DMF for 2 h, at rt. Accordingly, Fmoc
protection was removed after amino acid couplings as described above. The resins were washed
after each coupling and Fmoc group removal step with DMF (2 mL × 3) and DCM (2 mL × 3).
Moreover, the progression of the coupling and Fmoc-deprotection reactions was monitored by
the observation of colorimetric tests such as the Kaiser and p-chloranil tests for primary and
6
6
secondary amines, respectively. Ambiguous couplings were confirmed by LC-MS monitoring.
N-Methylation procedure and coupling on secondary amines. N-Methylation was performed
after coupling and Fmoc-deprotection of the corresponding amino acid in three steps.4,16,41 First,
the amino group was converted to an o-nitrobenzene sulfonamide by reaction with o-NBS
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chloride (5 equiv) and 2,4,6-collidine (10 equiv) in NMP for 15 min at rt. The N-methylation was
performed under Mitsunobu conditions:43,44 triphenylphosphine (10 equiv), DIAD (5 equiv) and
MeOH (5 equiv), together with the resin, all were suspended in anhydrous THF (4 mL) and
shaken at rt for 10 min. Finally, the o-NBS group was removed by treatment with 2-
mercaptoethanol (10 equiv) and DBU (5 equiv) in NMP for two cycles of 5 min each. This
deprotection procedure yielded the corresponding secondary amine, which was detected by the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
6
observation of both Kaiser and p-chloranil colorimetric tests. Due to the steric hindrance of N-
methyl amino acids, the next coupling employed HATU (3 equiv) and HOAt (3 equiv) in the
presence of DIEA (6 equiv), dissolved in a small volume of NMP (2-3 mL). The reaction was
carried out at rt for 16 h and complete coupling was confirmed by LC-MS analysis of the residue
after an aliquot of resin (5 mg) was treated with a cleavage cocktail of 95:2.5:2.5 TFA/TIS/H O
2
(v/v/v) at rt for 1 h, filtered, and evaporated.
Peptide cleavage. After Fmoc group removal as described above, the resin was washed with
DMF (2 mL × 3) and DCM (2 mL × 3) and dried in vacuo. Then, peptide was released from the
solid support, protecting groups were removed, and, simultaneously, the disulfide bridge was
formed, by treating the resin with a solution of 89:10:1 TFA/DMSO/anisole (v/v/v) at rt for 3 h.
The resin was filtered and washed with a small amount of fresh TFA. The combined filtrate was
precipitated in chilled diethyl ether, stored at −6 °C for 15 min, and centrifuged to yield crude
peptide as pale yellow pellet, which was purified by HPLC to afford the respective peptide (4-
21) as white powder.
5
[
(N-Me)Cys ]hU-II (4) - purity >98%; t = 15.5 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 24.7 min
2
R
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
6
5
88 13 18 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
402.5812, found 1402.5681.
6
[
(N-Me)Phe ]hU-II (5) - purity >98%; t = 15.2 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.5 min
2 R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
6
5
88 13 18 2
1
402.5812, found 1402.5831.
7
[
(N-Me)Trp ]hU-II (6) - purity >98%; t = 16.4 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 24.3 min
2 R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
6
5
88 13 18 2
1
402.5812, found 1402.5983.
8
[
(N-Me)Lys ]hU-II (7) - purity >98%; t = 14.8 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.2 min
2 R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
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+
[M + H]+
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S
88 13 18 2
6
5
1
402.5812, found 1402.5934.
9
[(N-Me)Tyr ]hU-II (8) - purity >98%; t = 15.0 min [analytical HPLC/Gradient#1,
R
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(
0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.2 min
2 R
[
analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
6
5
88 13 18 2
1
402.5812, found 1402.5861.
1
0
[(N-Me)Cys ]hU-II (9) - purity >98%; t = 15.8 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(
0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 23.3 min
2
R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
6
5
88 13 18 2
1
402.5812, found 1402.5928.
2
[
(N-Me)Cys ]URP (10) - purity >98%; t = 15.7 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(
0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 23.1 min
2
R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
5
0
67 10 10 2
1031.4478, found 1031.4493.
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Journal of Medicinal Chemistry
Page 28 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
[
(N-Me)Phe ]URP (11) - purity >98%; t = 15.2 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.4 min
2
R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
50 67 10 10
2
1
031.4483, found 1031.4470.
4
[
(N-Me)Trp ]URP (12) - purity >98%; t = 16.8 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 24.8 min
2
R
[
analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
5
0
67 10 10 2
1
031.4483, found 1031.4496.
5
[
(N-Me)Lys ]URP (13) - purity >98%; t = 14.8 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(
0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.4 min
2 R
[
analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
5
0
67 10 10 2
1
031.4483, found 1031.4509.
6
[
(N-Me)Tyr ]URP (14) - purity >98%; t = 14.9 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
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Page 29 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.0 min
2
R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
[M + H]+
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S
67 10 10 2
5
0
1
031.4483, found 1031.4505.
7
[
(N-Me)Cys ]URP (15) - purity >98%; t = 16.0 min [analytical HPLC/Gradient#1,
R
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 23.5 min
2 R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
+
[M + H]+
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S
67 10 10 2
5
0
1
031.4483, found 1031.4506.
5
[(N-Me)Cys ]U-II
(16) - purity >98%; t = 15.4 min [analytical HPLC/Gradient#1,
R
4
-11
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 22.6 min
2 R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
and gradient 10-90% MeOH (0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0
2
mL/min]; HRMS (ESI) m/z calculated for molecular formula C H N O S [M + H]+
+
5
1
67 10 12 2
1
075.4381, found 1075.4438.
6
[(N-Me)Phe ]U-II
(17) - purity >98%; t = 15.2 min [analytical HPLC/Gradient#1,
R
4
-11
isocratic 10% MeCN (0.1% TFA) in H O (0.1% TFA) over 5 min, and gradient 10-90% MeCN
2
(0.1% TFA) in H O (0.1% TFA) over 20 min, flow rate of 1.0 mL/min], and t = 21.3 min
2
R
[analytical HPLC/Gradient#2, isocratic 10% MeOH (0.1% TFA) in H O (0.1% TFA) over 5 min,
2
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