Structure-activity relationships of GHRP-6 azapeptide ligands of the CD36 Scavenger Receptor by solid-phase submonomer azapeptide synthesis

Article abstract of DOI:10.1021/ja203007u

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W²]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F⁴]GHRP-6, 2, exhibited a characteristic β-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 μM) affinity for the CD36 scavenger receptor.

Full text of DOI:10.1021/ja203007u

ARTICLE
pubs.acs.org/JACS
StructureÀActivity Relationships of GHRP-6 Azapeptide Ligands of
the CD36 Scavenger Receptor by Solid-Phase Submonomer
Azapeptide Synthesis
David Sabatino,†,§ Caroline Proulx,† Petra Pohankova,‡ Huy Ong,*,‡ and William D. Lubell*,†
†Department of Chemistry and ‡Department of Pharmacology, Universitꢀe de Montrꢀeal, P.O. Box 6128, Downtown Station, Montrꢀeal
QC H3C 3J7, Canada
§Department of Chemistry and Biochemistry, Seton Hall University, 400 South Orange Avenue, South Orange New Jersey 07079,
United States
S Supporting Information
b
ABSTRACT: The cluster of differentiation 36 (CD36) class B scavenger receptor
binds a variety of biologically endogenous ligands in addition to synthetic peptides
(i.e., growth hormone-releasing peptides, GHRPs), which modulate biological
function related to anti-angiogenic and anti-atherosclerotic activities. Affinity
labeling had previously shown that GHRP-6 analogues such as hexarelin,
[2-Me-W2]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor.
Moreover, the azapeptide analogue [aza-F4]GHRP-6, 2, exhibited a characteristic
β-turn conformation as described by CD and NMR spectroscopy and a slightly
higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM,
respectively), suggesting receptor binding was mediated by the conformation
and the aromatic residues of these peptide sequences. Ligand-receptor binding
interactions were thus explored using azapeptides to examine influences of side-
chain diversity and backbone conformation. In particular, considering that
aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to
furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by
submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-
bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover,
certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and
aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues
exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In
covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6
azapeptides retained relatively high (2À27 μM) affinity for the CD36 scavenger receptor.
’ INTRODUCTION
shown improved bioavailability, receptor binding affinity and
selectivity, as well as metabolic stability for potential use in
therapeutic applications.6 For example, in the rare case of
glutamate replacement by aza-glutamate333 (4, Figure 1), the
resulting antigenic hen egg ovalbumin 325À339 azapeptide
exhibited major histocompatibility complex class II (MHC II)
protein binding and partial agonism, demonstrating that conserva-
tive aza-substitution at a T-cell receptor contact site could induce a
minor structural perturbation sufficient to alter T-cell signaling.4c
In spite of a useful example of aza-Glu in studying peptide
recognition,4c as well as successful applications of related aza-
residues (i.e., aza-Gln, aza-Asp, aza-Asn) to furnish enzyme inhi-
bitors, (Figure 1),4,7 aza-amino acids possessing carboxylate side
chains have been rarely used in structureÀactivity relationship
Glutamate residues are often key structural elements for the
biological activity of small molecules and peptides, because the
acidic side chains may interact with target receptors by selective
ionic interactions.1À3 Moreover, constrained glutamate analo-
gues constitute important tools for exploring biological activity at
the molecular level. Such ligands may function as modulators of
receptor activity with enhanced pharmacokinetic properties for
potential therapeutic applications (Figure 1).1À4,7
Azapeptides are a class of conformationally preorganized pep-
tide mimics (peptidomimetics) which have been shown to adopt
β-turn conformations using computation, X-ray and spectro-
scopic analyses.5 Substitution of the R-carbon with nitrogen
rigidifies the peptide conformation because the diacyl hydrazine
and urea moieties of the resulting semicarbazide constrain
respectively the j and ψ dihedral angles of the peptide
backbone.5 In bioactive sequences, aza-amino acid residues have
Received: August 12, 2010
Published: June 21, 2011
r
2011 American Chemical Society
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Figure 1. Bioactive azapeptide analogues bearing aza-residues with carboxylate side chains.
studies. Azapeptides bearing carboxylate side chains may bind by
way of ionic interactions to induce biological activity at target
receptors. However, their application has been particularly ham-
pered due to lack of selectivity when differentiating hydrazine
nitrogen atoms in solution-phase synthesis, which required
separation of isomeric mixtures.4c Moreover, although pyroglu-
tamate analogues of peptides have been shown to improve
bioavailability and stability of active sequences, to the best of
our knowledge, there have been no applications of aza-pyroglu-
tamate analogues.8
The cluster of differentiation 36 (CD36) is a membrane glyco-
protein member of the class B scavenger receptor family which
binds among a variety of endogenous ligands, thrombospondin 1
(TSP1) an extracellular matrix protein and oxidatively modi-
fied low density lipoprotein (oxLDL).9 This receptor ligand
interaction has been found to play a major role in cardiovascular
biology, with inhibitory effects in angiogenesis and in the
development of atherosclerosis in hyperlipidemic states.9,10
Growth hormone-releasing peptides (GHRPs)11 have previously
been reported as the first class of synthetic peptide derivatives,
which serve as ligands of CD36 and exhibit anti-atherosclerotic
activity in a CD36 dependent manner.9c Topographical mapping
of the CD36 receptor by covalent binding using a benzophenone
alanine (Bpa) analogue of hexarelin, (Tyr-Bpa-Ala-His-D-2-
Me-Trp-Ala-Trp-D-Phe-Lys-NH2) has shown that the extracel-
lular binding domain (Asn132-Gln177) encompasses Lys rich
residues,12 and overlaps with the oxLDL binding domain, which
features three conserved Lys residues at position 163, 164 and
166, of which two positively charged Lys residues at positions
164 and 166 directly contribute to binding.13 Considering the
structures of GHRP-6 and the Lys-rich domain of the CD36
receptor, aromatic cation interactions may be hypothesized to
contribute to affinity.14a Recently, [aza-Phe4]GHRP-6 (2) was
shown to exhibit a circular dichroism (CD) spectrum indicative
of a β-turn conformation and to bind discriminately to the CD36
receptor, suggesting that the aromatic character and conforma-
tion of the azapeptide contributed to binding affinity.15a Recog-
nizing that salt bridges have been shown to compete with aro-
matic cation interactions,14bÀd we have explored the influence of
aza-Glu residues on binding affinity in GHRP-6 azapeptide
ligands of the CD36 receptor.
Submonomer solid-phase synthesis was developed to provide
azapeptides without hydrazine chemistry in solution.15,16 Aza-
amino acid residues were built directly onto support-bound pep-
tide by selective alkylation of an aza-Gly residue. This method
consisted of (A) acylation of peptide-bound resin with an
activated benzylidene carbazate, (B) alkylation of the resulting
semicarbazone and (C) deprotection followed by conventional
Fmoc-based SPPS (Scheme 1).15
Novel alkylation chemistry featuring Michael additions of
electron-deficient olefins and conjugate additionÀelimination
of activated allylic acetates is now reported to prepare aza-Glu
azapeptides. Fifteen aza-Glu derivatives were prepared using this
method to systematically modify the Ala3 and Trp4 positions of
GHRP-6. Moreover, novel aza-pyroglutamate analogues were
prepared by replacement of the His1 residue of GHRP-6 with
pyrrazoline amino acids. Structural analyses of the aza-Glu
analogues by CD spectroscopy and [aza-F4]GHRP-6 (2) by
NMR spectroscopy, as well as CD36 receptor binding affinities
were performed to assess relationships between azapeptide
structure and biological activity.
’ RESULTS
Synthesis. Azapeptide synthesis was performed on Rink amide
linker attached to Merrifield resin17 using Fmoc-based SPPS.18
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Scheme 1. Submonomer Solid-Phase Azapeptide Synthesis15
Resin-bound peptides D-Phe-Lys(Boc) 9 and Trp(Boc)-D-Phe-
Lys(Boc) 23 constituted the starting sequences for making
azapeptides with respective aza-modifications at the Trp4 and
Ala3 positions of the GHRP-6 sequence. The aza-Gly residue
was first installed onto supported di- and tripeptides by way
of an activated benzylidene carbazate intermediate 10, which
was generated in situ from benzaldehyde hydrazone and p-
nitrophenylchloroformate.15
Michael Additions. Michael addition reactions were explored
on semicarbazone resins 11 and 24 to generate, respectively, aza-
Glu analogues at positions Trp4 and Ala3, 12À14 and 25À30
(Scheme 2). tert-Butyl acrylate and acrylamide were employed to
install side chains corresponding to aza-Glu and aza-Gln residues.
Moreover, acrylonitrile, vinyl phosphonate and vinyl sulfonate
Michael acceptors were employed to examine reaction scope and
expand diversity. The products from addition of the vinyl
phosphonate and sulfonate acceptors were also pursued because
they may be converted to nonhydrolyzable surrogates of phos-
phate and sulfate esters of serine (or cysteine), with interesting
potential for studying inhibition of enzymes that modify such
residues (i.e., kinases, phosphatases and sulfatases).19
diester and monoester. Subsequent peptide elongation by stan-
dard SPPS18 afforded four [aza-Glu4]GHRP-6 (19À22) and
five [aza-Glu3]GHRP-6 (36À40) analogues in acceptable over-
all yields (4À10%) and purities (90À99%) after reverse-phase
HPLC purification (Table 1, entries 4À12).
Conjugate AdditionÀElimination of Activated Allylic
Acetates. Glutamic acid analogues bearing an aromatic alkyli-
dene substituent at the side-chain 4-position have exhibited bio-
logical activity in peptide sequences and small-molecule pepti-
domimetics.20 4-Alkylidene aza-glutamates were thus pursued to
study the influence of the combination of an aromatic and
carboxylate side chain on peptide conformation and affinity to
the CD36 receptor. To procure 4-alkylidene glutamate analo-
gues, the stereoselective alkylation of a glycine Schiff base by
an activated allylic acetate had been previously explored using
chiral phase transfer and transition metal catalysts, as well as
auxiliaries.21 Inspired by these procedures, uninhibited by stereo-
chemical issues, the corresponding aromatic [4-alkylidene aza-
Glu4]GHRP-6 derivatives were pursued by conjugate additionÀ
elimination reactions of activated allylic acetates 48À51 onto
semicarbazone resin 11 (Scheme 4).
Semicarbazone resins 11 and 24 were swollen in THF and
treated with base (potassium tert-butoxide or tert-butylimino-
tri(pyrrolidino)phospharane [BTPP] 300 mol %) and Michael
acceptor (300 mol %). Improved conversions and cleaner reac-
tions were observed with the phosphazene base BTPP (29À
75%) relative to potassium tert-butoxide (21À49%). Diethylvi-
nyl phosphate and phenylvinyl sulfone were observed to be
relatively less reactive Michael acceptors and provided resins 14,
28 and 29 with modest conversions (29À50%) and significant
recovered starting material, as indicated after resin cleavage and
LCMS analyses.
Allylic acetates 48À51 were synthesized in racemic form
by the Baylis-Hillman reaction22 of aromatic aldehydes and
electron-deficient alkenes using trimethylamine as catalyst, fol-
lowed by alcohol acetylation (Scheme 3).23 In our hands, benzal-
dehydes with electron-deficient para-substituents reacted better
than their electron rich counterparts producing allylic alcohols
44, 45 and 47 in 46À65% yields. Alternatively, p-methoxyben-
zaldehyde reacted sluggishly with acrylonitrile giving 25% iso-
lated yield of allylic alcohol 46, after silica gel column chroma-
tography. Allylic acetates 48À51 were prepared in 47À92%
yields from BaylisÀHillman adducts 44À47 by acetylation with
acetyl chloride in a pyridine/dichloromethane solution, followed
by silica gel column chromatography.24
To liberate the amine for further Fmoc-based peptide
synthesis, semicarbazones 12À14 and 25À30 were treated with
NH2OH HCl in pyridine.15 In the case of phosphonates 13 and
Semicarbazone resin 11 was swollen in THF and exposed to
conjugate additionÀelimination reactions using allylic acetates
48À51. Modest conversions (40À55%) were obtained with
3
27, semicarbazone deprotection occurred with some concomi-
tant phosphonate diester solvolysis, giving mixtures of phosphonate
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Scheme 2. Submonomer Solid-Phase Synthesis of [aza-Glu]GHRP-6 Azapeptidesa
a Percents in parentheses refer to crude purities of peptide product as assessed after cleavage of a small aliquot of resin using TFA/H2O/TES, followed by
LCMS analysis using 0À80% MeOH in H2O with 0.1% FA as eluant.
potassium tert-butoxide (300 mol %) as base with allylic acetates
48À51 (300 mol %) as assessed after resin cleavage and LCMS
analysis. Improved conversions (58À74%) were achieved with the
organic soluble, non-ionic Schwesinger base, BTPP (300 mol
%),25 which as a non-nucleophilic alternative, may avoid side
reaction with the electrophile (Scheme 4).21b Diastereomers were
attributedto1:1 mixtures ofolefinisomersE- and Z-52À55, which
were apparent in LCMS analyses as separable peaks having
identical masses (see Supporting Information for LCMS data).
Semicarbazones 52À55 were generally converted to semicar-
bazides using NH2OH HCl in pyridine,15 however, semicarba-
3
zone 53, bearing both electron deficient nitrile and p-nitro-cinnamyl
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Table 1. Characterization Data for Azapeptides
a Crude purity. b Isolated yield calculated from resin loading. c Isolated purity by LCMS at 214 nm. e Retention times using 0À80% MeOH in H2O
(0.1% FA). d Isolated purity by LCMS at 214 nm. f Retention times using 0À80% MeCN in H2O (0.1% FA) as eluants. g Expected mass (observed mass)
as [M + H]+ by LCMS.
moieties, gave no product after repetitive treatments and only
starting material was recovered. The azapeptides 56À58 were
subsequently completed by standard peptide synthesis in accep-
table yields (9À13%) and purities (90À99%) after reverse-phase
HPLC purification (Table 1, entries 13À15).
the tertiary urea. The lack of exchange between the signals of the
vinyl protons at 6.22 and 6.18 ppm during NOESY experiments
confirmed the independence of the olefin isomers (see Support-
ing Information for NMR spectra of 57). Conversely, the 1H and
13C NMR spectra of [aza-Phe4]GHRP-6 (2) at 298 K were
indicative of configurationally pure azapeptide in D2O and
DMSO-d6, as discussed below.
Although azapeptide 57 was observed as a single peak by
1
analytical HPLC, its H and 13C NMR spectra exhibited a
doubling of peaks indicative of a 3:1 E/Z-olefin ratio at 333 K.
At reduced temperatures (318 and 298 K, Figure 2), additional
splitting of signals observed was likely due to isomerization about
Aza-pyroglutamate and Aza-dehydroproline Synthesis.
Under analogous conditions for semicarbazone removal, aza-
Gln analogue 30 was accompanied by intramolecular amidation
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Scheme 3. Preparation of Allylic Acetates via BaylisÀHillman Reaction22
Scheme 4. Submonomer Solid-Phase Synthesis of Aromatic [4-alkylidene aza-Glu4]GHRP-6, 56 and Analogues 57 and 58a
a Crude purities were ascertained from cleavage of resin aliquots using TFA/H2O/TES and LCMS analysis using 0À80% MeOH/H2O with 0.1% FA as eluant.
and converted to a product with mass corresponding to that of the
aza-pyroglutamate, aza-pGlu, 59 (55%, Scheme 5).26 Although
such annulation may be avoided by amide side-chain protec-
tion,27 aza-pyroglutamate64 was pursued insteadasa His1 replace-
ment in the hope of improving pharmacokinetic properties.8
Similarly, alkyl vinyl ketones were used to replace His1 with
aza-dehydroprolines (Scheme 6). Resin-bound aza-Gly hexapeptide
60 was treated with BTPP (300 mol %) andexposedrespectively to
acrylamide, as well as methyl and ethyl vinyl ketones (300 mol %),
to afford alkyl semicarbazones 61À63 in respectable conversions
(60À80%), as assessed by LCMS analysis. Semicarbazone
removal with hydroxylamine was followed by intramolecular
annulation, such that resin cleavage provided N-terminal aza-
pyroglutamate 64 and pyrrazolines 65 and 66 in acceptable
overall yields (8À9%) and purity (90À99%) after reverse-phase
HPLC purification (Table 1, entries 1À3).
CD Spectroscopy. Circular dichroism (CD) spectroscopy
was used to evaluate azapeptide conformation in water
(Figure 3). The CD curve shape of the parent peptide GHRP-
6 is characteristic of a random coil or disordered structure
featuring a negative maximum band at 190 nm, which was
similarly observed for analogues 64-66, possessing N-terminal
heterocycle modifications at His1 (Figures 3a and b).29 Substitu-
tion of aza-Glu analogues at the Ala3 and Trp4 positions of
GHRP-6 caused varying effects on the CD curve shape indicative
of stabilization of secondary structure about the aza-residue
substitution. The CD curve of [aza-Ala3]GHRP-615a also exhibits
a negative maximum band at 190 nm characteristic of a random
coil conformation (Figure 3c).15,29 However, substitution at the
Ala3 position with other aza-amino acid residues in [aza-Glu3]
GHRP-6 analogues 36-40 revealed CD curves which suggested
averaging between random coil29 (more predominant in 39 and
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Figure 2. 1H NMR spectrum of 57 at variable temperatures.
Scheme 5. Synthesis of aza-pGlu 59
40) and polyproline type II,31 (more predominant in 36) or
β-turn30 (more predominant in 37 and 38) conformations
(Figure 3d). Previous studies of GHRP-6 analogues by CD
spectroscopy have shown that replacement of Trp4 by an aza-
Phe4 residue led to a conformational change in water from a
curve shape characteristic of a random coil to one indicative of a
β-turn (Figure 3e).15a,28,30 The CD signatures for aza-residue
modifications at Trp4, possessing [aza-Glu4]GHRP-6 derivatives
19-22, as well as, aromatic 4-alkylidene aza-Glu4 residues, 56-58,
all were indicative of β-turn conformers characterized by nega-
tive maximum around 200 and 230 nm and a positive maximum
near 215 nm, albeit 19 and 21 may have significant populations
of random coil (Figure 3f).30 Therefore, aza-residue replace-
ments at Trp4 appear to stabilize a β-turn conformation within
the D-Trp-Ala-Trp-D-Phe region of GHRP-6 with significant
tolerance of side-chain diversity.
NMR Spectroscopy. A deeper study of the azapeptide con-
formation of [aza-Phe4]GHRP-6 (2) has begun using NMR
spectroscopy to add additional support for the β-turn structure
observed by CD spectroscopy. Two-dimensional COSY,
TOCSY and HMQC experiments were performed to facilitate
1
chemical shift assignments of the H and 13C NMR spectra in
DMSO-d6 at 298 K (see Experimental Section and Supporting
Information for details). The 1H NMR spectrum of 2 in D2O was
also used to assign NH signals. Complete structural assignments
have, however, been hindered by overlapping of signals and
residual H2O and DMSO solvent peaks which masked the
methylene signals of the D-Phe and D-Trp residues. Nevertheless,
chemical shift values of the amide protons were employed to
determine intramolecular hydrogen bonding in 2. Chemical
shifts for solvent-exposed amide protons and those involved in
intramolecular hydrogen bonds are respectively observed
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Scheme 6. Submonomer Solid-Phase Synthesis of Aza-pyr-
oglutamate and Pyrrazoline Azapeptidesa
amide signals indicated values for solvent-exposed NHs (Δδ/
ΔT = À4, À8, and À11 ppb/K, respectively). In the two-dimen-
sional ROESY experiment (700 ms), a number of interactions
between sequential side chain residues suggested folding about a
preorganized turn conformation (see Supporting Information).
The amide temperature dependencies and long-range side chain
interactions were consistent with [aza-Phe4]GHRP-6 adopting a
turn geometry in which the D-Phe residue NH is engaged in an
intramolecular hydrogen bond. Although the correlation of the
CD and NMR spectral data for [aza-Phe4]GHRP-6 in aqueous
solution supports a β-turn, the relevance for such a conformation
when 2 is bound to the receptor remains to be elucidated.
CD36 Binding. The affinities (EC50 values) of azapeptides
containing potentially ionic side chains were evaluated in com-
petition binding studies with the GHRP-6 prototype ligand,
hexarelin (His-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH2, Table 2,
entry 1). In this assay, [aza-Phe4]GHRP-6 (2, Table 2, entry 8)
exhibited slightly enhanced affinity relative to hexarelin (1.34 μM
vs. 2.27 μM, respectively). Moreover, several [azaGlu]GHRP-6
analogues also maintained low micromolar affinity for the CD36
receptor (Table 2).
At the Ala3 position, [aza-Ala3]GHRP-6 (3) exhibited a 3-fold
loss in binding affinity relative to hexarelin (6.9 μM vs 2.3 μM,
Table 2, entries 1 and 2). The [aza-Glu3]GHRP-6 analogues
36À38 and 40 (Table 2, entries 3À5 and 7) maintained
relatively high affinity (2.4À27 μM) for the CD36 receptor.
In particular, the [aza-(cyanoethyl)Gly3]GHRP-6 analogue had
affinity similar to that of hexarelin (2.4 μM vs 2.3 μM, Table 2,
entries 4 and 1).
At the Trp4 position, relative to [aza-Phe4]GHRP-6 (2, 1.34 Â
10À6 M), none of the novel analogues exhibited improved
affinity. For example, 8- and 13-fold lower CD36 binding
affinities were respectively observed for [aza-Glu4]GHRP-6
(19, 11 μM) and [4-alkylidene aza-Glu4]GHRP-6 (56, 18 μM)
(Table 2, entries 8, 9 and 13). On the other hand, relative
to the non-ionic aliphatic sequences, [azaLeu4]GHRP-616a (67,
2.9 mM) and [(2-cyano-p-methoxycinnamyl)Gly4]GHRP-6
(57, 3.5 mM), [aza-Glu4]GHRP-6 analogues 19 and 56 exhibited
>150-fold higher affinity for the CD36 receptor, illustrating that
the ionic carboxylate enhanced affinity relative to the simple
hydrophobic aliphatic counterparts.
’ DISCUSSION AND CONCLUSION
a Percents in parentheses refer to crude purity ascertained after resin
cleavage using TFA/H2O/TES, by LCMS analyses using 0À80%
MeOH in H2O with 0.1% FA as eluant.
Azapeptide side-chain diversity has been expanded by employ-
ing Michael acceptors and allylic acetates in conjugate addi-
tionÀelimation reactions to a semicarbazone-derived aza-Gly
using submonomer solid-phase azapeptide synthesis. Fifteen new
aza-analogues of the GHRP-6 hexapeptide have been synthesized
in acceptable isolated yields (4À13%) and purities (>90%) after
HPLC purification. The phosphazene base, BTPP, proved more
effective than potassium tert-butoxide in the Michael addition
and conjugate additionÀelimination reactions onto the semi-
carbazone. Michael adducts with reactive side chain functional
groups were also found to undergo intramolecular annulation
during semicarbazone deprotection to afford novel N-terminal
pyrrazoline analogues.
between 4 and 6 ppm and between 7 and 9 ppm in model
azapeptides dissolved in CDCl3 at 298 K.35 The backbone amide
protons of the Ala, D-Trp, residues (8.7, and 8.2 ppm, re-
spectively) of 2, were shifted downfield from the remaining
amide signals (7.6À7.3 ppm) in DMSO-d6 at 298 K. The
chemical shifts and temperature dependencies for the amide
protons were measured in DMSO-d6 from 283 to 383 K to
distinguish H-bonding of the amide signals from those exposed
to solvent. Specifically, the chemical shifts of intramolecular
H-bonding amide protons show less temperature dependence
(Δδ/ΔT e 4 ppb/K) than those exposed to solvent ((6 ppb/K
e Δδ/ΔT e 10 ppb/K).36 In the case of azapeptide 2, the D-Phe
NH signal (7.3 ppm) exhibited a small temperature dependence
(Δδ/ΔT = À1 ppb/K) consistent with intramolecular hydrogen
bonding; on the other hand, examination of the Lys, Ala and D-Trp
Ligands of the CD36 receptor have been specifically pursued
in our program to develop treatments for angiogenesis-related
diseases, such as age-related macular degeneration and diabetic
retinopathy, as well as atherosclerosis.9À11 Our synthetic meth-
odology has provided rapid access to a small library of side-chain
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Figure 3. Circular dichroism spectra for GHRP-6 azapeptides (20 μM) in water.
Table 2. EC50 Binding Values for the CD36 Receptor
entry
azapeptide sequence (#)
EC50 binding (Â 10À6 M)
1
His-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH2 (1)
2.27 ( 0.32
6.89 ( 0.21
26.9 ( 0.15
2.36 ( 0.47
27.0 ( 0.70
1210 ( 1.23
5.38 ( 0.19
1.34 ( 0.22
11.1 ( 0.20
2890 ( 1.56
6610 ( 1.38
4500 ( 0.25
18.4 ( 0.37
3530 ( 1.01
2
His-D-Trp-aza-Ala-Trp-D-Phe-Lys-NH2 (3)
3
His-D-Trp-aza-Glu-Trp-D-Phe-Lys-NH2 (36)
4
His-D-Trp-aza-(cyanoethyl)Gly-Trp-D-Phe-Lys-NH2 (37)
His-D-Trp-aza-(ethyl methylphosphoryl)Gly-Trp-D-Phe-Lys-NH2 (38)
His-D-Trp-aza-(ethyl dimethylphosphoryl)Gly-Trp-D-Phe-Lys-NH2 (39)
His-D-Trp-aza-(ethyl diethylphosphoryl)Gly-Trp-D-Phe-Lys-NH2 (40)
His-D-Trp-Ala-aza-Phe-D-Phe-Lys-NH2 (2)
5
6
7
8
9
His-D-Trp-Ala-aza-Glu-D-Phe-Lys-NH2 (19)
10
11
12
13
14
His-D-Trp-Ala-aza-Leu-D-Phe-Lys-NH2 (67)
His-D-Trp-Ala-aza-(ethyl methylphosphoryl)Gly-D-Phe-Lys-NH2 (20)
His-D-Trp-Ala-aza-(ethyl diethylphosphoryl)Gly-D-Phe-Lys-NH2 (22)
His-D-Trp-Ala-aza-(E/Z)-(2-carboxy-p-nitrocinnamyl)Gly-D-Phe-Lys-NH2 (56)
His-D-Trp-Ala-aza-(E/Z)-(2-cyano-p-methoxycinnamyl)Gly-D-Phe-Lys-NH2 (57)
diverse GHRP-6 azapeptides for evaluating structureÀactivity
relationships with the CD36 scavenger receptor. According to
the CD spectra curve shapes, NMR and binding data, the
presence of an aza-amino acid residue can influence the con-
formation and affinity of the GHRP-6 analogues contingent on
its location in the sequence and side chain. The two aza-
analogues exhibiting highest affinity possessed curve shapes
indicative of β-turn geometry: [aza-(cyanoethyl)Gly3]- and
[aza-Phe4]GHRP-6 (37 and 2); both had affinities (2.4 and
1.3 μM) similar to that of hexarelin (2.3 μM). Although a
putative turn geometry in [aza-Phe4]GHRP-6 was also sup-
ported by NMR spectroscopy which suggested a fold about the
aza-residue, the relevance of the turn structure obtained in
solution to that of the bound conformation remains to be eluci-
dated. The loss of affinity on respective substitutions at positions
3 and 4 using the described aza-amino acid residues may be due
to various factors, including incorrect backbone geometry and
side-chain interactions. For example, although [aza-(ethyl
methylphosphoryl)Gly4]- and [aza-(ethyl diethylphosphoryl)
Gly4]GHRP-6 (20 and 22) exhibited curve shapes indicative of
β-turn geometry, neither possessed significant affinity for the
CD36 receptor. The aza-glutamate analogues, [aza-Glu4]- and
[aza-(E/Z)-(2-carboxy-p-nitrocinnamyl)Gly4]GHRP-6 (19 and
56), featuring ionic side chains at the Trp4 position, both
exhibited interesting affinity (18 and 27 μM) for the CD36
receptor, >150-fold superior to their counterparts possessing
aliphatic side chains: [aza-Leu4]- and [aza-(E/Z)-(2-cyano-p-
methoxycinnamyl)Gly4]GHRP (67 and 57). The manner by
which such ionic side chains improve affinity relative to aliphatic
counterparts remains a matter of debate; however, their potential
to form salt bridges with conserved lysine residue side chains
within the CD36 receptor emerges as an interesting hypothesis
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Journal of the American Chemical Society
ARTICLE
for future ligand design. In conclusion, the submonomer synth-
esis method offers a powerful means for making azapeptides for
studying structureÀactivity relationships with target receptors.
Employing this method, we are now studying the anti-angiogenic
and anti-atherosclerotic effects of such ligands in pursuit of their
full therapeutic potential.
2-[Hydroxyl(4-nitrophenyl)methyl]-tert-butylacrylate (47).
47was preparedasdescribedfor44above andisolatedbysilicagel column
chromatography using 4:1 Hexane/EtOAc, Rf (2:1 Hexane/EtOAc): 0.7,
pale-yellow oil. Yield (890 mg, 47%);1H NMR(400 MHz, CDCl3) δ 1.43
(s, 9H) 3.53 (d, J = 8 Hz, 1H), 5.58 (d, J = 8 Hz, 1H), 5.77 (s, 1H), 6.31 (s,
1H), 7.58 (d, J = 12 Hz, 2H), 8.21 (t, J = 4 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ 27.6, 72.6, 82.0, 123.2, 126.2, 126.9, 141.9, 147.0, 148.7, 164.9;
HRMS Calcd m/z for C14H17NO5Na [M + Na]+ 302.0999, found
302.1007.
’ EXPERIMENTAL SECTION
2-[Acetyloxy(phenyl)methyl]acrylonitrile (48).
A stirred
General Methods. Polystyrene Rink Amide resin (0.67 mmol/g)
was purchased from Advanced Chemtech, and the manufacturer’s
reported loading of the resin was used in the calculation of the yields
of the final products. Benzaldehyde, p-nitrobenzaldehyde, p-methoxy-
benzaldehyde, acetyl chloride, tert-butyl acrylate, acrylonitrile, phenylvi-
nyl sulfate, diethylvinyl phosphate, dimethylvinyl phosphate, acrylamide,
methylvinyl ketone, ethylvinyl ketone, hydrazine hydrate, p-nitrophenyl
chloroformate, potassium tert-butoxide, tert-butylimino-tri(pyrrolidino)
phosphorane (BTPP), hydroxylamine hydrochloride, pyridine, formic
acid (FA), and N,N-diisopropylethylamine (DIEA) were all purchased
from Aldrich and used without further purification. Thin-layer chroma-
tography (TLC) was performed on silica gel 60 F254 plates from Merck.
Commercially available Fmoc amino acids, Fmoc-His(Trt), Fmoc-D-
Trp(Boc), Fmoc-Ala, Fmoc-Trp(Boc), Fmoc-D-Phe, and FmocLys-
(Boc) and coupling reagents such as HBTU and diisopropylcarbodii-
mide (DIC) were purchased from GL Biochem and used as received. All
solvents were obtained from VWR International. Anhydrous solvents
[N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and dichlor-
omethane (DCM)] were obtained by passage through solvent filtration
system (Glass-Contour, Irvine, CA).
solution of alcohol 44 (0.5 g, 3.2 mmol) in pyridine (0.4 mL, 4.9 mmol)
and dichloromethane (3.5 mL) was treated with acetyl chloride (0.3 mL,
4.2 mmol) dropwise over 5 min at room temperature (20 °C). The clear
solution was stirred until TLC indicated complete product formation
[(2:1 Hexane/EtOAc) Rf (48): 0.5]. The crude was diluted in dichlor-
omethane (10 mL) and washed with water (10 mL) and brine (10 mL).
The organic phase was separated and dried over anhydrous magnesium
sulfate, and the extracts were evaporated in vacuo. The crude product
was purified by column chromatography using 2:1 Hexane/EtOAc to
afford acetate 48 as a yellow oil. Yield (660 mg, 61%); 1H NMR (400
MHz, CDCl3) δ 2.17 (s, 3H), 6.02 (d, J = 4 Hz, 1H), 6.07 (d, J = 4 Hz,
1H), 6.35 (s, 1H), 7.39À7.42 (m, 5H). 13C NMR (100 MHz, CDCl3) δ
20.5, 74.0, 115.9, 122.8, 126.6, 128.6, 128.9, 131.8, 135.3, 168.9.
HRMS Calcd m/z for C12H11NO2Na [M + Na]+ 224.0682, found
224.0688.
2-[Acetyloxy(4-nitrophenyl)methyl]acrylonitrile (49). 49
was prepared as described for 48 above and isolated by silica gel
column chromatography using 2:1 Hexane/EtOAc, Rf (2:1 Hexane/
1
EtOAc) 0.5, orange oil. Yield (300 mg, 47%); H NMR (400 MHz,
2-[Hydroxyl(phenyl)methyl]acrylonitrile (44). A stirred solu-
tion of benzaldehyde (0.5 g, 4.7 mmol) and 25 wt % aqueous
trimethylamine (1.5 mL, 5.9 mmol) in methanol (2.5 mL) was treated
with acrylonitrile (0.94 mL, 14.2 mmol) dropwise over the span of 5 min
at room temperature (20 °C). The clear solution was stirred until TLC
indicated formation of a more polar product without further consump-
tion of starting material [(2:1 Hexane/EtOAc), Rf (benzaldehyde): 0.7
and Rf (44): 0.4]. The crude was diluted in chloroform (20 mL) and
treated with water (5 mL). The organic phase was separated, and the
aqueous phase was extracted with chloroform (2 Â 10 mL). The
combined organic extracts were dried over anhydrous magnesium
sulfate and evaporated in vacuo. The crude product was purified by
column chromatography using 2:1 Hexane/EtOAc to afford the pure
adduct (44) as a colorless oil. Yield (486 mg, 65%), 1H NMR (400 MHz,
CDCl3) δ 3.14 (d, J = 4 Hz, 1H), 5.25 (d, J = 4 Hz, 1H), 6.0 (s, 1H), 6.08
(s, 1H), 7.37À7.43 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 73.68,
116.7, 125.8, 126.2, 128.6, 129.7, 138.8. HRMS Calcd m/z for
C10H10NO [M + H]+ 160.0757, found 160.0758.
2-[Hydroxyl(4-nitrophenyl)methyl]acrylonitrile (45). 45
was prepared as described for 44 above and isolated by silica gel column
chromatography using 2:1 Hexane/EtOAc, Rf (2:1 Hexane/EtOAc) 0.2,
pale-yellow oil. Yield (620 mg, 46%); 1H NMR (400 MHz, CDCl3) δ
3.60 (br., 1H), 5.44 (s, 1H), 6.09 (s, 1H), 6.19 (s, 1H), 7.59 (d, J = 12 Hz,
2H), 8.20 (t, J = 4 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 72.73,
116.1, 123.6, 124.9, 127.1, 131.2, 146.0, 147.5; HRMS Calcd m/z for
C10H9N2O3 [M + H]+ 205.0608, found 205.0613.
CDCl3) δ 2.22 (s, 3H), 6.17 (d, J = 12 Hz, 2H), 6.42 (s, 1H), 7.61
(d, J = 8 Hz, 2H), 8.26 (d, J = 8 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ 20.5, 73.1, 115.2, 121.6, 123.8, 127.5, 133.0, 142.2, 147.9. 168.7.
HRMS Calcd m/z for C12H9N2O4 [M À H]À 245.0567, found 245.057.
2-[Acetyloxy(4-methoxyphenyl)methyl]acrylonitrile (50).
50 was prepared as described for 48 above and isolated by silica gel
column chromatography using 2:1 Hexane/EtOAc, Rf (2:1 Hexane/
1
EtOAc) 0.5, yellow oil. Yield (198 mg, 64%); H NMR (400 MHz,
CDCl3) δ 2.16 (s, 3H), 3.82 (s, 3H), 6.00 (s, 1H), 6.06 (s, 1H), 6.30 (s,
1H), 6.93 (t, J = 4 Hz, 2H), 7.34 (d, J = 8 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 20.6, 55.0, 73.7, 113.9, 116.0, 123.0, 127.3, 128.2, 131.2, 159.9,
168.9. HRMS Calcd m/z for C13H13NO3Na [M + Na]+ 254.0788,
found 254.0791.
2-[Acetyloxy(4-nitrophenyl)methyl]-tert-butylacrylate (51).
51 was prepared as described for 48 above and isolated by silica gel column
chromatography using 2:1 Hexane/EtOAc, Rf (1:1 Hexane/EtOAc) 0.8,
pale-yellow oil. Yield (876 mg, 92%); 1H NMR (400 MHz, CDCl3) δ 1.40
(s, 9H), 2.14 (s, 3H), 5.85 (s, 1H), 6.39 (s, 1H), 6.69 (s, 1H), 7.57 (d, J = 8
Hz, 2H), 8.21 (d, J = 8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 20.60,
27.54, 71.89, 81.63, 123.2, 125.5, 128.2, 139.6, 145.0, 147.3, 163.2, 168.8.
HRMS Calcd m/z for C16H19NO6Na [M + Na]+ 344.11046, found
344.10993.
Fmoc-based SPPS: Fmoc Deprotection and Peptide Cou-
plings. Peptide syntheses were performed under standard conditions18
on an automated shaker using Polystyrene Rink Amide resin (0.67
mmol/g). Couplings of amino acids (3 equiv) were performed in DMF
using HBTU (3 equiv) as coupling reagent and DIEA (6 equiv) for 3 h.
Fmoc deprotections were performed by treating the resin with 20%
piperidine in DMF for 30 min. Resin was washed after each coupling and
deprotection step sequentially with DMF (3 Â 10 mL), MeOH (3 Â
10 mL), THF (3 Â 10 mL), and DCM (3 Â 10 mL). The purity of di-[D-
Phe-Lys-NH2], tri-[Trp-D-Phe-Lys-NH2], and penta-[D-Trp-Ala-Trp-D-
Phe-Lys-NH2] peptide fragments was ascertained by LCMS analysis
after cleavage and deprotection of a small aliquot of resin.
2-[Hydroxyl(4-methoxyphenyl)methyl]acrylonitrile (46).
46 was prepared as described for 44 above and isolated by silica gel
column chromatography using 2:1 Hexane/EtOAc, Rf (2:1 Hexane/
EtOAc): 0.2, colorless oil. Yield (344 mg, 25%); 1H NMR (400 MHz,
CDCl3) δ 3.30 (d, J = 4 Hz, 1H), 3.80 (s, 3H), 5.18 (s, 1H), 5.97 (s, 1H),
6.06 (s, 1H), 6.90 (dd, J = 4, 6 Hz, 2H), 7.27 (d, J = 8 Hz, 2H); 13C NMR
(100 MHz, CDCl3) δ 55.0, 73.2, 113.9, 114.1, 116.8, 126.1, 127.6, 128.1,
129.2, 131.1, 159.5; HRMS Calcd m/z for C11H12NO2 [M + H]+
190.0863, found 190.0856
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Journal of the American Chemical Society
ARTICLE
Azapeptide couplings were performed according to the symmetric
anhydride method.32 Fmoc-amino acids (10 equiv) were activated with
diisopropylcarbodiimide (5 equiv) in dry DCM at 0 °C for 20 min. The
suspension was concentrated in vacuo, dissolved in DMF, and added to
the semicarbazide resin. The reaction was continued for 24 h at room
temperature, and the resin was filtered and washed under vacuum with
DMF (3 Â 10 mL), MeOH (3 Â 10 mL), THF (3 Â 10 mL), and DCM
(3 Â 10 mL). The extent of reaction was monitored by subjecting an
aliquot (3 mg) of resin to Fmoc deprotection (20% piperidine/DMF,
0.3 mL, 30 min) followed by the cleavage conditions [0.3 mL, TFA/
TES/H2O (95:2.5:2.5, v/v/v)], and the crude was analyzed by LCMS.
The target sequences were completed according to the conventional
Fmoc-based SPPS.18
Cleavage Test of Resin-Bound Peptide. A sample of peptide-
bound resin (3À5 mg) was treated with a freshly made solution of TFA/
H2O/TES (95:2.5:2.5, v/v/v, 0.3 mL) for 30 min at room temperature.
The cleavage mixture was filtered and then concentrated, and the crude
peptide was precipitated with cold ether (1.5 mL). Crude peptide
samples were agitated on a vortex shaker, and spun in a centrifuge.
Decantation of the supernatant left a pellet, which was dissolved in 10%
MeOH/H2O (1 mg/mL) and subjected to LCMS analysis.
Representative Protocol for aza-Gly-Peptide Synthesis:
Preparation of Semicarbazone-Protected aza-Gly-Peptide
Resin 11. To a stirred solution of EtOH (3 mL) and hydrazine
hydrate (120 μL, 3.7 mmol) at 0 °C was added benzaldehyde (190 μL,
1.9 mmol) dropwise. Complete formation of phenyl hydrazone was
usually observed after 15 min by TLC, [(2:1 Hexane/EtOAc), Rf
(benzaldehyde) 0.7 and Rf (benzaldehyde hydrazone) 0.6]. The mix-
ture was poured directly into H2O (5 mL) and extracted with DCM
(3 Â 5 mL). The organic phase was separated, dried with MgSO4, and
concentrated in vacuo to yield the benzaldehyde hydrazone as a yellow-
tinged oil that was employed without further purification.
Benzaldehyde hydrazone (230 mg, 1.9 mmol, 10 equiv) in DCM
(2 mL) was added dropwise over 15 min to a solution of p-nitrophenyl
chloroformate (240 mg, 1.2 mmol, 6 equiv) in DCM (2 mL) at 0 °C.
The reaction mixture was stirred at room temperature under argon for
an additional 1.5 h, and treated with DIEA (420 μL, 2.4 mmol, 12 equiv)
dropwise over 20 min at 0 °C, when TLC [(2:1 Hexane/EtOAc), Rf:
0.75] indicated complete conversion to the activated methylidene
carbazate intermediate. The suspension was quickly transferred to
the resin (250 mg, 0.19 mmol). The resin suspension was agitated on
an automated shaker for 16 h at room temperature, filtered, washed
under vacuum with DMF (3 Â 10 mL), MeOH (3 Â 10 mL), THF
(3 Â 10 mL), and DCM (3 Â 10 mL). The extent of reaction conversion
was monitored on an aliquot (3 mg) of resin which was subjected
to 0.3 mL of TFA/TES/H2O (95:2.5:2.5, v/v/v) for resin cleavage and
the crude was analyzed by LCMS.
THF (2 Â 10 mL), and DCM (2 Â 10 mL) and dried under vacuum.
The extent of reaction conversion was monitored on an aliquot (3 mg)
of resin which was subjected to 0.3 mL of TFA/TES/H2O (95:2.5:2.5,
v/v/v) for resin cleavage, and the crude was analyzed by LCMS.
Benzaldehyde semicarbazone-(glutamyl)-D-Phe-Lys-NH2
(12): LCMS (0À80% MeOH, with 0.1% FA, 10 min) R.T. = 5.8 min;
LCMS (ESI) calcd for C26H36N6O5 [M + 2H]+, 513.0 found m/e 513.2.
Benzaldehyde semicarbazone-(ethyl dimethylphosphate)-
D-Phe-Lys-NH2 (13): LCMS (0À80% MeOH, with 0.1% FA, 10 min)
R.T. = 5.8 min; LCMS (ESI) calcd for C27H41N6O6P [M + 2H]+, 577.0
found m/e 577.2.
Benzaldehyde semicarbazone-(ethyl diethylphosphate)-
D-Phe-Lys-NH2 (14): LCMS (0À80% MeOH, with 0.1% FA, 10 min)
R.T. = 5.9 min; LCMS (ESI) calcd for C29H45N6O6P [M + 2H]+, 605.0
found m/e 605.2.
Benzaldehyde semicarbazone-(E/Z)-(2-cyanocinnamyl)-
D-Phe-Lys-NH2 (52): LCMS (0À80% MeOH, with 0.1% FA, 10
min) R.T. = 6.2 min; LCMS (ESI) calcd for C33H39N7O3 [M + 2H]+,
581.7 found m/e 582.3.
Benzaldehyde semicarbazone-(E/Z)-(2-cyano-p-nitrocin-
namyl)-D-Phe-Lys-NH2 (53): LCMS (0À80% MeOH, with 0.1%
FA, 10 min) R.T. = 6.3 min; LCMS (ESI) calcd for C33H38N8O5 [M +
2H]+, 626.7 found m/e 627.3.
Benzaldehyde
semicarbazone-(E/Z)-(2-cyano-p-meth-
oxycinnamyl)-D-Phe-Lys-NH2 (54): LCMS (0À80% MeOH, with
0.1% FA, 10 min) R.T. = 6.3 min; LCMS (ESI) calcd for C34H41N7O4
[M + 2H]+, 611.7 found m/e 612.2.
Benzaldehyde semicarbazone-(E/Z)-(2-carboxy-p-nitro-
cinnamyl)-D-Phe-Lys-NH2 (55): LCMS (0À80% MeOH, with
0.1% FA, 10 min) R.T. = 6.4 min; LCMS (ESI) calcd for C33H39-
N7O7 [M + 2H]+, 645.7 found m/e 646.2.
Benzaldehyde semicarbazone-(glutamyl)-Trp-D-Phe-Lys-
NH2 (25): LCMS (0À80% MeOH, with 0.1% FA, 10 min) R.T. =
6.5 min; LCMS (ESI) calcd for C37H46N8O6 [M + 2H]+, 698.8 found
m/e 699.3.
Benzaldehyde semicarbazone-(cyanoethyl)-Trp-D-Phe-Lys-
NH2 (26): LCMS (0À80% MeOH, with 0.1% FA, 10 min) R.T. =
5.4 min; LCMS (ESI) calcd for C37H45N9O4 [M + 2H]+, 679.8 found
m/e 680.3.
Benzaldehyde semicarbazone-(ethyl dimethylphosphate)-
Trp-D-Phe-Lys-NH2 (27): LCMS (0À80% MeOH, with 0.1% FA, 10
min) R.T. = 6.2 min; LCMS (ESI) calcd for C38H51N8O7P [M + 2H]+,
762.8 found m/e 763.2.
Benzaldehyde semicarbazone-(ethyl diethylphosphate)-
Trp-D-Phe-Lys-NH2 (28): LCMS (0À80% MeOH, with 0.1% FA, 10
min) R.T. = 6.7 min; LCMS (ESI) calcd for C40H55N8O7P [M + 2H]+,
790.9 found m/e 791.3.
Benzaldehyde semicarbazone-(ethylphenylsulfonyl)-Trp-
D-Phe-Lys-NH2 (29): LCMS (0À80% MeOH, with 0.1% FA, 10 min)
R.T. = 6.3 min; LCMS (ESI) calcd for C42H50N8O6S [M + 2H]+, 794.9
found m/e 795.3.
Benzaldehyde semicarbazone-(glutaminyl)-Trp-D-Phe-
Lys-NH2 (30): LCMS (0À80% MeOH, with 0.1% FA, 10 min)
R.T. = 6.2 min; LCMS (ESI) calcd for C37H47N9O5 [M + 2H]+, 697.8
found m/e 698.3.
Benzaldehyde semicarbazone-D-Phe-Lys-NH2 (11): LCMS
(0À80% MeOH, with 0.1% FA, 10 min) R.T. = 5.9 min; LCMS (ESI)
calcd for C23H32N6O3 [M + 2H]+, 440.5 found m/e 441.3.
Benzaldehyde semicarbazone-Trp-D-Phe-Lys-NH2 (24):
LCMS (0À80% MeOH, with 0.1% FA, 10 min) R.T. = 6.1 min; LCMS
(ESI) calcd for C34H42N8O4 [M + 2H]+, 627.1 found m/e 627.3.
Benzaldehyde semicarbazone-D-Trp-Ala-Trp-D-Phe-Lys-
NH2 (60): LCMS (0À80% MeOH, with 0.1% FA, 10 min) R.T. =
6.2 min; LCMS (ESI) calcd for C48H57N11O6 [M + 2H]+, 884.1 found
m/e 884.4.
Representative Alkylation of aza-Gly, Preparation of Resin
(12À14 and 52À55). To the swollen semicarbazone peptide bound
resin 11 (0.05 g, 33 μmol) in THF (1 mL), potassium tert-butoxide
(12 mg, 0.1 mmol, 3 equiv) or BTPP (30 μL, 0.1 mmol, 3 equiv)
was added followed by Michael acceptor or allylic acetate (48À51)
(0.1 mmol, 3 equiv). After agitation at room temperature for 16 h, the
resin was filtered, washed with DMF (2 Â 10 mL), MeOH (2 Â 10 mL),
Benzaldehyde semicarbazone-(glutaminyl)-D-Trp-Ala-
Trp-D-Phe-Lys-NH2 (61): LCMS (0À80% MeOH, with 0.1%
FA, 20 min) R.T. = 14.5 min; LCMS (ESI) calcd for C51H62N12O7
[M + 2H]+, 955.1 found m/e 955.3.
Benzaldehyde semicarbazone-(ethyl methylketone)-D-
Trp-Ala-Trp-D-Phe-Lys-NH2 (62): LCMS (0À80% MeOH, with
0.1% FA, 10 min) R.T. = 6.5 min; LCMS (ESI) calcd for C52H63N11O7
[M + 2H]+, 954.1 found m/e 954.4.
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Journal of the American Chemical Society
ARTICLE
Benzaldehyde semicarbazone-(ethyl ethylketone)-D-Trp-
Ala-Trp-D-Phe-Lys-NH2 (63): LCMS (0À80% MeOH, with 0.1%
FA, 10 min) R.T. = 6.5 min; LCMS (ESI) calcd for C53H65N11O7 [M +
2H]+, 968.1 found m/e 968.4.
28.2, 27.1, 22.6, 17.1. LCMS (0À40% MeOH, 20 min) R.T. = 16.3 min;
(0À40% MeCN, 20 min) R.T. = 13.3 min; LCMS (ESI) calcd for
C43H55N12O6 [M + H]+, 835.4 found m/z 835.2; HRMS Calcd m/z for
C43H55N12O6 [M + H]+ 835.4362, found 835.43494.
Representative Protocol for Semicarbazone Removal,
Preparation of Aza-pGlu-Trp-D-Phe-Lys-Peptide Resin 59.
Resin-bound semicarbazone 30 (0.05 g, 33 μmol) was treated with a
His-D-Trp-Ala-aza-Glu-D-Phe-Lys-NH2 (19): Yield (1.4 mg,
6%), LCMS (0À80% MeOH, 20 min) R.T. = 9.56 min; (0À80%
MeCN, 20 min) R.T. = 8.60 min; LCMS (ESI) calcd for C39H51N12O8
[M + H]+, 816.0 found m/z 816.4; HRMS Calcd m/z for C39H50N12O8
[M + H]+ 815.3947, found 815.3947.
solution of 1.5 M NH2OH HCl in pyridine (2.5 mL) and heated with
3
ultrasound at 60 °C for 12 h. The resin was filtered and washed under
vacuum with 10% DIEA/DMF (3 Â 10 mL), DMF (3 Â 10 mL),
MeOH (3 Â 10 mL), THF (3 Â 10 mL), and DCM (3 Â 10 mL). The
extent of reaction conversion was monitored on an aliquot (3 mg) of
resin, which was subjected to 0.3 mL of TFA/TES/H2O (95:2.5:2.5,
v/v/v) for resin cleavage, and the crude was analyzed by LCMS.
Aza-pGlu-Trp-D-Phe-Lys-peptide resin 59: LCMS (0À80%
MeOH, with 0.1% FA, 10 min) R.T. = 5.7 min; LCMS (ESI) calcd for
C30H38N8O5 [M]+, 590.7 found m/e 591.2.
His-D-Trp-Ala-aza-(ethyl
methylphosphoryl)Gly-D-Phe-
Lys-NH2 (20): Yield (1 mg, 5%), LCMS (0À80% MeOH, 20 min)
R.T. = 11.5 min; (0À80% MeCN, 20 min) R.T. = 9.46 min; LCMS
(ESI) calcd for C39H56N12O9P [M + 2H]+, 868.1 found m/z 868.2;
HRMS Calcd m/z for C39H56N12O9P [M + H]+ 867.4025, found
867.4016.
His-D-Trp-Ala-aza-(ethyl dimethylphosphoryl)Gly-D-Phe-
Lys-NH2 (21): Yield (0.4 mg, 4%), LCMS (0À80% MeOH, 20 min)
R.T. = 9.58 min; (0À80% MeCN, 20 min) R.T. = 8.65 min; LCMS
(ESI) calcd for C40H58N12O9P [M + H]+, 881.1 found m/z 881.4;
HRMS Calcd m/z for C40H58N12O9PNa [M + H]+ 881.4181, found
881.4180.
Deprotection and Cleavage of Azapeptide from the Resin.
The Rink resin-bound peptide was deprotected and cleaved from the
support using a freshly made solution of TFA/H2O/TES (95:2.5:2.5,
v/v/v, 20 mL/g of peptide resin) at room temperature for 2 h. The resin
was filtered and rinsed with 1 mL of TFA. The filtrate and rinses were
concentrated under a flow of Ar(g) until a crude oil persisted, from which
a precipitate was obtained by addition of cold ether (10À15 mL). After
centrifugation (12 000 rpm for 10 min.), the supernatant was removed,
and the crude peptide was taken up in aqueous methanol (10% v/v) and
freeze-dried to a white solid prior to analysis.
His-D-Trp-Ala-aza-(ethyl
diethylphosphoryl)Gly-D-Phe-
Lys-NH2 (22): Yield (1 mg, 5%), LCMS (0À80% MeOH, 20 min)
R.T. = 10.9 min; (0À80% MeCN, 20 min) R.T. = 9.12 min; LCMS
(ESI) calcd for C42H62N12O9P [M + H]+, 909.0 found m/z 909.4;
HRMS Calcd m/z for C42H62N12O9P [M + H]+ 909.4494, found
909.4484.
His-D-Trp-Ala-aza-(E/Z)-(2-carboxy-p-nitrocinnamyl)Gly-
D-Phe-Lys-NH2 (56): Yield (1.5 mg, 9%), LCMS (0À80% MeOH, 20
min) R.T. = 14.3 min; (0À80% MeCN, 20 min) R.T. = 10.3 min; LCMS
(ESI) calcd for C46H56N13O10 [M + H]+, 950.0 found m/z 950.2;
HRMS Calcd m/z for C46H56N13O10 [M + H]+ 950.4268, found
950.4302.
Analysis and Purification of Azapeptides. Analyses and
characterization of crude azapeptides were performed on either an
Agilent Technologies 1100 series LCMS instrument with ESI ion-
source, single quadropole mass detection and positive mode ionization
or a ThermoFinnigan LCQ Advantage MS with ESI ion-source, ion-trap
mass detection, positive mode ionization and equipped with a Gilson LC
322 pump containing autosampler and injector. Azapeptide samples
were dissolved in 10% H2O in methanol. The LCMS analyses were
performed on a Gemini C18 reverse-phase column (150 mm  4.60 mm,
5 μm), using binary solvent system consisting of 0.1% formic acid in
H2O, and 0.1% formic acid in methanol at a flow rate of 0.5 mL/min and
UV detection at 214 nm. Linear gradients of the mobile phase (0.1%
formic acid in methanol, 0À80% over 15 min) were used for analyses of
crude peptides.
Purification of peptides was conducted on a Waters PrepLC instru-
ment equipped with a reverse-phase Gemini C18 column (250 mm Â
21.2 mm, 5 μm), using binary solvent system consisting of 0.1%
formic acid in H2O, and 0.1% formic acid in methanol at a flow rate
of 10 mL/min and UV detection at 214 nm. Linear gradients of the
mobile phase (0.1% formic acid in methanol, 0À80% in 20 min) were
used for purifications of peptides. Fractions containing pure azapeptide
were combined, freeze-dried and lyophilized to a white powder. Purified
azapeptide samples were analyzed for purity by LCMS with a Gemini
C18 reverse-phase column (150 Â 4.60 mm, 5 μm), with a flow rate of
0.5 mL/min using a 0À80% gradient from water (0.1% FA) to CH3CN
(0.1% FA) or to MeOH (0.1% FA).
His-D-Trp-Ala-azaPhe-D-Phe-Lys-NH2 (2): Yield (1.2 mg, 5%),
1H NMR (700 MHz, DMSO-d6) δ 1.11 (5H, d, J = 7.49 Hz), 1.44 (3H,
br s), 1.67 (1H, br s), 2.51À2.52 (1H, m), 2.69À2.71 (3H, m), 2.77 (1H,
dd, J = 4.8 10 Hz), 2.96À3.01 (3H, m), 3.11À3.19 (1H, m), 3.52À3.54
(1H, m), 3.99 (1H, m), 4.06 (1H, br s), 4.30 (1H, m), 4.62 (1H, br s),
6.79 (1H, s), 6.94 (1H, t, J = 15 Hz), 7.05 (1H, t, J = 15 Hz), 7.11 (2H, s),
7.17À7.36 (12H, m), 7.42 (1H, s), 7.53 (1H, d, J = 8.1 Hz), 7.57 (1H, s),
8.23 (1H, s), 8.38 (1H, s), 8.72 (1H, s), 10.8 (1H, s). 13C NMR (125
MHz, DMSO-d6) δ 174.2, 173.8, 172.6, 172.2, 164.5, 158.2, 157.6,
137.7, 136.4, 135.5, 129.7, 128.8, 128.7, 128.1, 127.8, 127.6, 126.7, 124.1,
121.3, 118.8, 118.6, 111.7, 110.1, 54.9, 53.6, 52.6, 51.5, 40.3, 32.1, 31.1,
His-D-Trp-Ala-aza-(E/Z)-(2-cyano-p-methoxycinnamyl)-
Gly-D-Phe-Lys-NH2 (57): Yield (1.7 mg, 12%), 1H NMR (700 MHz,
D2O, T = 318 K) δ 1.08 (d, J = 4 Hz, 0.65H), 1.10 (m, 2H), 1.16 (d, J =
7 Hz, 3H), 1.61 (m, 3.6H), 1.79 (m, 2H), 2.81 (d, J = 7 Hz, 2H), 2.90 (td,
J = 2, 7 Hz, 0.70H), 2.98 (td, J = 2, 7 Hz, 2H), 3.15 (d, J = 7.7 Hz, 2H),
3.84 (bs, 1H), 3.88 (s, 3H), 3.89 (s, 0.85H), 3.91 (bs, 1H), 4.13 (q, J = 7
Hz, 1H), 4.18 (dd, J = 5, 10 Hz, 1H), 4.30 (t, J = 7 Hz, 1H), 4.33 (d, J = 15
Hz, 1H), 4.48 (d, J = 15 Hz, 1H), 4.54 (t, J = 7.7 Hz 1H), 6.68 (s, 1H),
7.08 (d, J = 5.6 Hz, 2H), 7.22 (bs, 2H), 7.27 (bs, 2H), 7.31 (d, J = 5 Hz,
2H), 7.33 (d, J = 7.7 Hz, 3H), 7.56 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 7.7 Hz,
1H), 7.63 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 9 Hz, 2H) . 13C NMR
(125 MHz, D2O) δ 15.58 (16.04), 22.02 (22.24), 26.27 (26.41), 27.12
(27.33), 30.21, 30.74 (31.14), 37.22, 39.21 (39.27), 40.52, 49.25
(50.27), 51.99, 53.41, 53.68, 54.20, 54.75, 54.88, 55.65, 56.61, 102.2,
108.9, 112.0, 114.4, 114.6 (114.5), 118.4, 119.3 (119.4), 122.1, 124.5,
126.0, 126.9, 127.3, 128.8 (128.9), 129.4, 130.4, 130.9 (131.0), 135.9,
136.3 (136.5), 148.3, 157.6, 161.2, 171.1, 174.2, 174.4, 175.7, 176.6.
LCMS (0À80% MeOH, 20 min) R.T. = 11.8 min; (0À80% MeCN,
20 min) R.T. = 9.33 min; LCMS (ESI) calcd for C47H58N13O7 [M +
H]+, 916.0 found m/z 916.4; HRMS Calcd m/z for C44H58N13O7 [M +
H]+ 916.4577, found 916.4601.
His-D-Trp-Ala-aza-(E/Z)-(2-cyano-cinnamyl)Gly-D-Phe-Lys-
NH2 (58): Yield (1.6 mg, 13%), LCMS (0À80% MeOH, 20 min) R.T. =
11.4 min; (0À80% MeCN, 20 min) R.T. = 9.20 min; LCMS (ESI) calcd
for C44H56N13O6 [M + H]+, 886.0 found m/z 886.4; HRMS Calcd m/z
for C46H56N13O6 [M + H]+ 886.4471, found 886.44845.
His-D-Trp-aza-Glu-Trp-D-Phe-Lys-NH2 (36): Yield (2.7 mg,
10%), LCMS (0À80% MeOH, 20 min) R.T. = 11.3 min; (0À80%
MeCN, 20 min) R.T. = 8.96 min; LCMS (ESI) calcd for C47H58N13O8
[M + H]+, 932.0 found m/z 932.4; HRMS Calcd m/z for C47H58N13O8
[M + H]+ 932.4526, found 932.4542.
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Journal of the American Chemical Society
ARTICLE
His-D-Trp-aza-(cyanoethyl)Gly-Trp-D-Phe-Lys-NH2 (37): Yield
(1.2 mg, 5%), LCMS (0À80% MeOH, 20 min) R.T. = 10.7 min; (0À80%
MeCN, 20 min) R.T. = 8.85 min; LCMS (ESI) calcd for C47H57N14O6
[M + H]+, 913.0 found m/z 913.4; HRMS Calcd m/z for C47H57N14O6
[M + H]+ 913.4580, found 913.4573.
His-D-Trp-aza-(ethyl methylphosphoryl)Gly-Trp-D-Phe-Lys-
NH2 (38): Yield (1 mg, 6%), LCMS (0À80% MeOH, 20 min) R.T. =
13.7 min; (0À80% MeCN, 20 min) R.T. = 10.1 min; LCMS (ESI) calcd
for C47H61N13O9P [M + H]+, 982.1 found m/z 982.4; HRMS Calcd
m/z for C47H61N13O9P [M + H]+ 982.4447, found 982.4455.
His-D-Trp-aza-(ethyl dimethylphosphoryl)Gly-Trp-D-Phe-
Lys-NH2 (39): Yield (1.2 mg, 4%), LCMS (0À80% MeOH, 20 min)
R.T. = 11.2 min; (0À80% MeCN, 20 min) R.T. = 8.71 min; LCMS
(ESI) calcd for C48H63N13O9P [M + H]+, 997.1 found m/z 997.4;
HRMS Calcd m/z for C50H62N13O9PNa [M + Na]+ 1018.4423, found
1018.4407.
His-D-Trp-aza-(ethyl diethylphosphoryl)Gly-Trp-D-Phe-Lys-
NH2 (40): Yield (1 mg, 4%), LCMS (0À80% MeOH, 20 min) R.T. =
12.2 min; (0À80% MeCN, 20 min) R.T. = 9.31 min; LCMS (ESI) calcd
for C50H67N13O9P [M + H]+, 1024.1 found m/z 1024.4; HRMS Calcd
m/z for C50H67N13O9P [M + H]+ 1024.4917, found 1024.4918.
aza-pGlu-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (64): Yield (2.0 mg,
9%), LCMS (0À80% MeOH, 20 min) R.T. = 16.7 min; (0À80%
MeCN, 20 min) R.T. = 12.0 min; LCMS (ESI) calcd for C44H54N11O7
[M + H]+, 848.0 found m/z 848.4; HRMS Calcd m/z for C44H54N11O7
[M + H]+ 848.4202, found 848.4209.
2-Methyl-pyrrazoline-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (65):
Yield (2.6 mg, 8%), LCMS (0À80% MeOH, 20 min) R.T. = 18.8 min;
(0À80% MeCN, 20 min) R.T. = 14.0 min; LCMS (ESI) calcd for
C45H56N11O6 [M + H]+, 846.0 found m/z 846.4; HRMS Calcd m/z
for C45H56N11O6 [M + H]+ 846.4410, found 846.4417.
Competitive Covalent CD36 Binding Assay Using Photo-
activatable [125I]-Tyr-Bpa-Ala-Hexarelin as Radioligand. The
radioiodination procedure of the photoactivatable ligand and the
receptor binding assays were performed as previously described by
Ong et al.34 Briefly, the rat cardiac membranes (200 μg) as source of
CD36 were incubated in the darkness, in 525 μL of 50 mM Tris-HCl pH
7.4 containing 2 mM EGTA (Buffer A) in the presence of a fixed
concentration of [125I]-Tyr-Bpa-Ala-Hexarelin (750 000 cpm) in Buffer
B (50 mM Tris-HCl pH 7.4 containing 2 mM EGTA and 0.05%
Bacitracin) and of increasing concentrations of competitive ligands
(ranging from 0.1 to 50 μM). Nonspecific binding was defined as
binding not displaced by 50 μM peptide. After an incubation period of
60 min at 22 °C, membranes were submitted to UV irradiation with UV
at 365 nm for 15 min at 4 °C. After centrifugation at 12 000 rpm for
15 min, the pellets were resuspended in 100 μL of sample buffer
consisting of 62 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 15%
2-mercapto-ethanol, and 0.05% bromophenol blue and boiled for 5 min
prior to electrophoresis on 7.5% SDS-PAGE. The SDS/PAGE gels were
fixed, colored in Coomassie Brilliant Blue R-250, dried, exposed to a
storage phosphor intensifying screen (Amersham Biosciences), and
analyzed by using a Typhoon PhosphorImager (Amersham Biosciences)
and ImageQuant 5.0 software to establish competition curves. Protein
bands corresponding to the specifically labeled protein of 87 kDa were
quantified by densitometry analysis.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details including
b
analytical data, NMR spectra, LCMS chromatograms, competi-
tive covalent binding curves for CD36 receptor binding affinities
(EC50). This material is available free of charge via the Internet at
http://pubs.acs.org.
2-Ethyl-pyrrazoline-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (66): Yield
(2.8 mg, 8%), LCMS (0À80% MeOH, 20 min) R.T. = 19.4 min; (0À80%
MeCN, 20 min) R.T. = 14.4 min; LCMS (ESI) calcd for C46H58N11O6
[M + H]+, 860.0 found m/z 860.4; HRMS Calcd m/z for C46H58N11O6
[M + H]+ 860.4566, found 860.4566.
Mass Spectrometry. Accurate mass measurements were per-
formed on a LC-MSD-TOF instrument from Agilent technologies
in positive electrospray mode. Either protonated molecular ions
[M + H]+ or sodium adducts [M + Na]+ were used for empirical for-
mula confirmation.
’ AUTHOR INFORMATION
Corresponding Author
huy.ong@umontreal.ca; william.lubell@umontreal.ca
’ ACKNOWLEDGMENT
Financial Support from the Natural Sciences and Engineering
Research Council (NSERC) of Canada, le Fond Quꢀebꢀecois de
la Recherche sur la Nature et les Technologies (FQRNT) and
the CIHR Team Grant Program (Funding No: CTP79848) in
G-Protein Coupled Receptor Allosteric Regulation (CTIGAR)
is gratefully acknowledged. We thank Dr. Alexandra F€urt€os
and Marie-Christine Tang for assistance in LCMS and HRMS
analyses. We also thank Dr. Minh Tan Phan Viet, Cedric Malveau
and Sylvie Bilodeau for assistance with NMR analyses of azape-
ptides 2 and 57.
CD Spectroscopy. All CD spectra were recorded on a Chirascan
CD Spectrometer (Applied Photophysics, Leatherhead, United King-
dom) using a 1.0 cm path length quartz cell containing 20 μM of peptide
dissolved in Milli-Q water. The experimental settings were: 1 nm,
bandwidth; 0.5 nm, step size; 3 s, sampling time.
NMR Spectroscopy. 1H and 13C NMR spectra were measured on a
Bruker NMR spectrometer (400 or 700 MHz) with samples (2À5 mM)
dissolved in CDCl3, DMSO-d6 and D2O (99.9%) and referenced to
H2O (4.79 ppm), DMSO (2.5 and 39.5 ppm) or CHCl3 (7.26 and 77.8
ppm). Coupling constants, J values were measured in hertz (Hz) and
chemical shift values in parts per million (ppm). For variable-tempera-
ture experiments, the sample was allowed to equilibrate within the
probe, 5À10 min before the data were collected over a temperature
range of 283À338 K. Two-dimensional COSY, HMQC, TOCSY (80 ms
mixing time), NOESY and ROESY (150, 300, 500, and 700 ms mixing
time) spectra were acquired at 298 K.
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