Structure-activity analysis of the growth hormone secretagogue GHRP-6 by α- And β-Amino γ-lactam positional scanning

Article abstract of DOI:10.1111/j.1747-0285.2009.00913.x

Incorporation of amino lactams into biologically active peptides restricts conformational mobility and may enhance selectivity and increase potency. α- and β-amino γ-lactams (Agl and Bgl), in both S and R configurations, were introduced into the growth hormone secretagogue GHRP-6 using a Fmoc-compatible solid-phase protocol relying on N-alkylation with five- and six-membered cyclic sulfamidates, followed by lactam annulation under microwave heating. Using this protocol in conjunction with IRORI Kan ^TM techniques furnished eleven new GHRP-6 analogs, and their binding affinity IC₅₀ values on both the growth hormone secretagogue receptor 1a (GHS-R1a) and CD36 receptors are herein reported. The results indicate that selectivity towards one receptor or the other can be modulated by lactam substitution, typically at the Ala³ and the d-Phe⁵ positions.

Full text of DOI:10.1111/j.1747-0285.2009.00913.x

ª 2009 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00913.x
Chem Biol Drug Des 2010; 75: 40–50
Research Article
Structure–Activity Analysis of the Growth
Hormone Secretagogue GHRP-6 by a- and
b-Amino c-Lactam Positional Scanning
Nicolas Boutard1, Andrew G. Jamieson1,
Huy Ong2 and William D. Lubell1,*
Received 7 October 2009, revised 23 October 2009 and accepted for
publication 24 October 2009
1Chemistry Department, Universitꢀ de Montrꢀal, C.P. 6128,
Succursale Centre-Ville, Montreal, QC H3C 3J7, Canada
GHRP-6 (HwAWfK, Figure 1) is a synthetic hexapeptide member of
the growth hormone-releasing peptides (GHRPs) family. GHRPs were
derived from Met-enkephalin (1,2) and exert their GH secretagogue
activity by a distinct pathway from that triggered by the growth
hormone-releasing hormone (GHRH) (3). Their GH release-stimulating
action is mediated by their interaction with the growth hormone
secretagogue receptor 1a (GHS-R1a), a G-protein-coupled receptor
located in pituitary and hypothalamic cells (4,5) that binds
ghrelin as its natural ligand (6). GHRP-6 stimulates GH liberation from
somatotrophs in a dose-dependant manner in different species
including humans (2,7) and has clinical perspectives in the treat-
ment of impaired GH secretion-related conditions like obesity
(8), short stature (9), acromegaly (10) or aging (11). In this context,
hexarelin (His-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH2) was developed,
and displayed an increased GH-releasing activity following
intravenous, subcutaneous, intranasal, and oral administration in
humans (12).
2Department of Pharmacology, Faculty of Pharmacy, Universitꢀ de
Montrꢀal, C.P. 6128, Succursale Centre-Ville, Montreal, QC H3C 3J7,
Canada
*Corresponding author: William D. Lubell,
william.lubell@umontreal.ca
Present address: Andrew G. Jamieson, Chemistry Research
Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1
3TA, UK.
Incorporation of amino lactams into biologically
active peptides restricts conformational mobility
and may enhance selectivity and increase potency.
a- and b-amino c-lactams (Agl and Bgl), in both S
and R configurations, were introduced into the
growth hormone secretagogue GHRP-6 using
a
Fmoc-compatible solid-phase protocol relying on
N-alkylation with five- and six-membered cyclic
sulfamidates, followed by lactam annulation under
microwave heating. Using this protocol in con-
junction with IRORI KanTM techniques furnished
eleven new GHRP-6 analogs, and their binding
affinity IC50 values on both the growth hormone
secretagogue receptor 1a (GHS-R1a) and CD36
receptors are herein reported. The results indicate
that selectivity towards one receptor or the other
can be modulated by lactam substitution, typically
at the Ala3 and the D-Phe5 positions.
Another important aspect of GHRPs pharmacological properties is
their multiple effects on the cardiovascular system. For instance,
GHRP-6 analogs are able to exert protective effects on ischemia ⁄
reperfusion injury of isolated rat heart (13) and have also been
shown to display anti-atherosclerotic activity in apolipoprotein
E-deficient mice (14). These effects have recently been shown to be
mediated by their binding to CD36, a multiligand scavenger receptor
expressed in cardiomyocytes and in the microvascular endothelial
cells (15,16). The anti-atherosclerotic properties of GHRPs result from
the overlapping of their binding domain on CD36 with that for
oxidized low density lipoproteins (oxLDL). Therefore, by interacting
with CD36, GHRPs decrease oxidized lipid internalization within
macrophages and the formation of atherosclerotic lesions (17,18).
Furthermore, CD36 features a distinct binding site for thrombos-
pondin and by binding the scavenger receptor, GHRPs may convey
neovascularisation inhibitory effects, suggesting their therapeutic
potential in angiogenesis-related diseases such as age-related
macular degeneration (19).
Key words: CD36, conformational constraint, cyclic sulfamidate,
Freidinger–Veber lactam, GHRP-6, GHS-R1a, lactam, lactam scanning,
peptide, peptidomimetic, solid-phase synthesis
Abbreviations: Agl, a-amino c-lactam; Bgl, b-amino c-lactam;
BSA, N,O-bis(trimethylsilyl)-acetamide; CD36, cluster of differentiation
36; EDTA, ethylenediamine tetraacetate; EGTA, ethyleneglycol tetraace-
tate; FA, formic acid; GH, growth hormone; GHRP, growth hormone-
releasing peptide; GHS-R1a, growth hormone secretagogue receptor 1a;
HBTU, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophos-
phate; RP-HPLC, reverse phase high performance liquid chromatography;
SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;
TES, triethyl silane; PBS, phosphate buffered saline.
Considering that GHRP-6 exhibits dual affinity for both the GHS-R1a
and the CD36 receptors, discovery of analogs selective for one
Editor’s invited manuscript to celebrate the 4th Anniversary of Chemical Biology & Drug Design.
40

Structure-Activity Analysis of the Growth Hormone Secretagogue GHRP-6
(20). Preparation of GHRP-6 analogs bearing b-turn stabilizing pep-
tide mimics may provide information about the structural features
NH2
required to achieve activity and selectivity.
NH
NH
O
O
O
H
H
a-Amino c-lactam (Agl, 1, Figure 2), a member of the so-called Frei-
dinger–Veber lactams, has been successfully utilized to constrain
the backbone conformation of linear peptides to give type II and
type II' b-turn mimics (21–23). b-Amino-c-lactam (Bgl, 2, Figure 2)
has been less frequently used to study peptide structure. Replace-
ment of Asp11 by (S)-Bgl in the fragment [6–13] of human growth
hormone conserved insulin-potentiating activity and provided
extended duration of action (24–27). In addition, Bgl peptide ana-
logs have exhibited CCK-A receptor agonism with high affinity and
selectivity (28), as well as significant enhancement of the binding
of [3H]N-propylnorapomorphine to dopamine receptors (29). Confor-
mational analysis of Bgl peptides by NOESY-NMR studies in DMSO
and molecular dynamics indicated that (S)-Bgl may stabilize a type
II¢-turn-like conformation (27,30,31).
H2N
N
N
N
NH2
N
N
N
H
H
H
O
O
O
HN
His-D-Trp-Ala-Trp- D-Phe-Lys-NH2
Figure 1: Structure and sequence of GHRP-6.
α-amino γ-lactam = Agl β-amino γ-lactam = Bgl
O
O
R1
R1
N
N
N
R2
R2
N
R1
R1
N
H
H
N
N
R2
R2
N
O
O
H
H
(S)-1
(R)-1
(S)-2
(R)-2
We have recently reported a general protocol for solid-phase syn-
thesis of Agl and Bgl-bridged peptides using, respectively, cyclic sul-
famidate reagents 3 and 4 (Figure 2) (32). Employing this protocol
(Scheme 1), ten lactam-bridged analogs of GHRP-6 were synthesized
(peptides 15–19 using (S)-3 and 20–24 using (S)-4, Table 3) (32).
According to this protocol, standard conditions for solid-phase pep-
tide synthesis with Fmoc protection were employed to synthesize
the peptide chain 6 on Rink amide resin (33,34). When the appro-
priate residue for lactam formation was reached, the N-terminal
Fmoc group was removed, and the free amine was reacted with
N,O-bis(trimethylsilyl)acetamide (BSA) to limit bis-alkylation of the
terminal amine with the respective sulfamidate 3 or 4. Linear alkyl-
ated products were converted to the lactam by microwave-assisted
annulation. Standard peptide synthesis was resumed on amino
lactam peptides 9 and 10. After final deprotection and cleavage
with trifluoroacetic acid (TFA), ether precipitation gave the crude
peptides, which were purified by preparative RP-HPLC to give
the desired products. The binding affinity IC50 values of the GHRP-6
O
O
O
O
OMe
OMe
O
O
S
S
N
CO2Bn
N
CO2Bn
FmocN
FmocN
O
O
Fmoc
Fmoc
O
S
O
O S O
O
O
(S)-3
(R)-3
(S)-4
(R)-4
Figure 2: Three letter codes for amino lactam residues and cyc-
lic sulfamidates 3 and 4.
receptor is of significant interest to probe specific functions. In light
of the large pharmacological profile displayed by CD36 and the fact
that it represents a newly discovered target for GHRPs, selective
binding partners for this receptor are attractive as drug leads, free
of possible unwanted effects associated with action on GHS-R1a.
The bioactive conformation of GHRP-6 as GHS-R1a agonist has
been suggested to adopt a turn motif based on molecular modeling
1) 20% piperidine/DMF
2 X 20 min, RT
1) 20% piperidine/DMF
NHFmoc
2 X 20 min, RT
2) BSA, THF, 16 h, RT
H
H
H
Fmoc
peptide N
Fmoc
Xaa
peptide
N
( )
R
N
Xaa peptide
N
*
2)
Fmoc-Xaa
HBTU, DIEA, DMF
n
H
3)
( )
THF, 24 h
O
S
O
n
7: n = 1, R = CO2Bn
8: n = 0, R = CH2CO2Me
O
5
6
*
N
R
Fmoc
3: n = 1, R = CO2Bn
4: n = 0, R = CH2CO2Me
1) MW, 100 °C
1) 20% piperidine/DMF
X
N
H
2 X 20 min, RT
X
N
DMF or 1% AcOH/DMSO
H
N
Xaa peptide N
*
N
Fmoc peptide Yaa
Xaa peptide
*
Y
FmocHN
Y
H
2)
Fmoc-Yaa
HBTU, DIEA, DMF
2) Boc2O, DIEA, DMF, 1h, RT
3) repeat steps 1) and 2)
9: X = CH2, Y = CO
10: X = CO, Y = CH2
11: X = CH2, Y = CO
12: X = CO, Y = CH2
Yaa, Xaa : amino acid
1) 20% piperidine/DMF
2 X 20 min, RT
X
N
Xaa peptide NH2
*
N
peptide Yaa
Y
H
2) TFA/TES/H2
O
(95 : 2.5 : 2.5, v/v/v)
=
Rink amide resin
H2N
13: X = CH2, Y = CO
14: X = CO, Y = CH2
Scheme 1: General protocol for solid-phase synthesis of Agl and Bgl lactam-bridged peptides.
Chem Biol Drug Des 2010; 75: 40–50
41

Boutard et al.
(S)-amino lactam analogs on both GHS-R1a and CD36 were
determined (32).
strategy to perform a (R)-lactam scan of GHRP-6 using 6-mem-
bered cyclic sulfamidate (R)-3 and 5-membered cyclic sulfamidate
(R)-4. The propylene mesh walled IRORI KanTM reactors allow
for reactions on confined resin beads. They have been used for
the preparation of libraries of discreet compounds in solid-phase
synthesis by a split-and-mix approach (36–40). In the context of
this work, compound tracking was made possible using frequency
tag microchips.
As GHRP-6 contains both L- and D-configured amino acids, a com-
prehensive lactam scan of the later peptide would involve the
use of both (S )- and (R )-configured lactams. We now report the
synthesis of eleven new GHRP-6 lactam analogs employing (R )-Agl
(25–29) and (R )-Bgl residues (30–35) in an efficient split-and-mix
approach using IRORI KanTM techniques and cyclic sulfamidate
reagents (R )-3 and (R )-4. The respective (R )-amino lactam peptides
have been tested for binding affinity with receptors CD36 and
GHS-R1a and compared with their (S)-lactam counterparts in a
summary of the biological data.
IRORI KanTM experimental procedures for (R)-
Agl and (R)-Bgl GHRP-6 analogs preparation
Ten IRORI KanTM macroreactors containing frequency tag micro-
chips with designated serial numbers were each charged with
Rink amide resin SS (70–90 mesh, 300 mg, 0.7 mmol ⁄ g,
0.2 mmol). The Kan reactors were sealed, and peptide synthesis
was preformed by placing the Kan reactors in a 250 mL glass
bottle containing a stirrer bar. After each coupling step, the
Materials and Methods
General methods
Sulfamidates (R)-3 and (R)-4 were synthesized, respectively, from
D-methionine and D-aspartic acid as previously described (32). Rink
amide resin SS (70–90 mesh, 0.7 mmol ⁄ g loading) was purchased
from Advanced ChemtechTM (Louisville, Kentucky). Fmoc amino acids
were purchased from Novabiochemꢀ (EMD Bioscience Inc., San
Diego, CA, USA) or GL Biochem Ltd. (Shangai, China). O-Benzotriazole-
N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) was
purchased from GL Biochem (Shangai, China). Anhydrous tetrahydro-
furane (THF) and dimethyl formamide (DMF) were obtained from a
Seca Solvent Filtration System (GlassContourTM Laguna Beach,
California). Diisopropylethylamine (DIEA; from Aldrichꢀ, Saint-Louis,
MO, USA) was distilled first from ninhydrin, then from calcium
hydride. Peptide couplings and Fmoc removal were qualitatively moni-
tored by Kaiser test (35). Reactions requiring anhydrous conditions
were performed under an atmosphere of dry argon. Microwave-
assisted reactions were preformed in a Biotage Initiator microwave
apparatus (Biotage AB, Uppsala, Sweden) in Biotage 5 mL V-shaped
glass reaction vessels containing a 10-mm triangular magnetic stirrer
bar and sealed with a septum cap.a Alkylation and lactam formation
reactions were monitored by LC-MS (UV: k = 214 nm, 0–60% MeCN
in H2O, 0.1% formic acid (FA) in 4 min, 0.5 mL ⁄ min, GeminiTM 5u C18
110A, 50 · 4.6 mm, 5 lm). Analytical RP-HPLC analyses of crude and
purified peptides were performed on a GeminiTM 5u C18 110A column
(150 mm · 4.6 mm, 5 lm, column A; Phenomenexꢀ Inc., Torrance,
CA, USA) or a SynergiTM 4u Polar RP80A column (150 mm · 4.6 mm,
4 lm, column B; Phenomenexꢀ Inc.) with a flow rate of 0.5 mL ⁄ min
using a linear gradient of acetonitrile [0.1% formic acid (FA)] in water
(0.1% FA). Retention times (tR) from analytical RP-HPLC are reported
in minutes; UV absorption was recorded at k = 214 nm. Peptides
were purified with a GeminiTM 5u C18 110A column (250 mm · 21.2
mm, 5 lm, column C; Phenomenexꢀ Inc.) or a SynergiTM 4u Polar
RP80A Axial Packed column (100 mm · 21.2 mm, 4 lm, column D;
Phenomenexꢀ Inc.) using a specified linear gradient of acetonitrile
(0.1% FA), in water (0.1% FA), with a flow rate of 10.6 mL ⁄ min.
appropriate Kan was identified by scanning the microchip serial
number using the SYNTHESIS MANAGER
TM
softwarec, removed from
the batch reaction vessel and transferred to a syringe tube for
silylation and alkylation. Because of the incompatibility of the
frequency tag and microwave conditions and to improve stirring,
the resin was temporarily removed from the IRORI KanTM, trans-
ferred to a microwave reaction vessel for the microwave-assisted
annulation for the specified time. The resin was then washed
back into the IRORI KanTM along with its corresponding frequency
tag. The Kan containing the supported lactam-bridged peptide was
recombined with the batch of Kan reactors and peptide synthesis
was continued. When the scan was complete, the terminal Fmoc
groups were removed, the IRORI KansTM were then identified and
separated, and the crude peptides were cleaved off the resin
and purified by preparative RP-HPLC to give the desired peptides
25–35 as white fluffy solids (Table 2).
Resin swelling, Fmoc deprotection and
washings
The ten Kan reactors were placed in a 250 mL bottle and sus-
pended in DMF (100 mL) for 0.5 h in order for the resin to swell.
The solvent was decanted, and the Kan reactors were suspended in
a freshly prepared 20% piperidine in DMF solution (150 mL) and
stirred gently for 20 min. The solution was decanted and the opera-
tion was repeated. For washings, the Kan reactors were sequen-
tially stirred for 5 min in DMF (3 · 150 mL), MeOH (3 · 150 mL)
and dichloromethane (DCM, 3 150 mL). Residual solvent was
removed from the Kan reactors by centrifugation.
Amino acid coupling
Resin in Kan reactors was swollen as described above. In a 250 mL
round-bottom flask, a solution of N-(Fmoc)amino acid (3 equiv.),
HBTU (3 equiv.), and DIEA (6 equiv.) in DMF (150 mL for ten Kan
reactors, 0.04 M in amino acid) was prepared, stirred for 10 min
and then added to the bottle containing Kan reactors filled with
swollen resin. The bottle was tightly sealed with a screw cap, and
the reaction mixture was gently stirred at room temperature for
1 h, in the cases of Fmoc-Lys(Boc) and Fmoc-Ala couplings, and for
Combinatorial strategy using IRORI KanTM
reactors
With the goal to accelerate the process of lactam scanning,
IRORI KanTM reactorsb were utilized in a combinatorial synthesis
42
Chem Biol Drug Des 2010; 75: 40–50

Structure-Activity Analysis of the Growth Hormone Secretagogue GHRP-6
Table 1: Optimization of the alkylation reaction of peptidyl resin 36 with cyclic sulfamidate (R)-4
Alkylation
temperature
Entry
BSA (time)
(R)-4 (time)
DIEA (equiv.)
37:38:39a
1 (init.)
5 equiv. (12 h)
10 equiv. (48 h)
10 equiv. (2 h)
10 equiv. (1 h)
10 equiv. (1 h)
10 equiv. (1 h)
5 equiv. (1 h)
5 equiv. (1 h)
5 equiv. (1 h)
RT
_
_
_
_
_
_
53:47:0
52:48:0
55:45:0
23:77:0
49:51:0
57:43:0
80:20:0
52:27:21
2
3
4
5
6
7
8
5 equiv. (1 h)b
60 ꢁC lW
60 ꢁC lW
RT
60 ꢁC lW
60 ꢁC lW
60 ꢁC lW
60 ꢁC lW
5 equiv. (1 h)b
3 equiv. (1 h)
3 equiv. (1 h)b
_
_
_
1 equiv.
1.2 equiv.
lW, microwave irradiation; DIEA, diisopropylethylamine.
aRatio determined by LC-MS analysis (UV: k = 214 nm, 0–60% MeCN in H2O, 0.1% FA over 4 min, 0.5 mL ⁄ min, GeminiTM 5u C18 110A, 50 mm · 4.60 mm).
bResin silylation performed in the microwave reaction vessel, washings with dry THF, and supernatant removal carried out by syringe under argon before
alkylation.
Table 2: Yields and purities of (R)-lactam-bridged GHRP-6 peptides 25–35
HRMS
Crude
Peptidea
nꢁ
purity %b
Purity %c
Yield %d
m ⁄ z (calcd.)
m ⁄ z (obsd.)
(R)-Agl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
His-(R)-Agl-Ala-Trp-D-Phe-Lys-NH2
His-D-Trp-(R)-Agl-Trp-D-Phe-Lys-NH2
His-D-Trp-Ala-(R)-Agl-D-Phe-Lys-NH2
His-D-Trp-Ala-Trp-(R)-Agl-Lys-NH2
(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
Ac(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
His-(R)-Bgl-Ala-Trp-D-Phe-Lys-NH2
His-D-Trp-(R)-Bgl-Trp-D-Phe-Lys-NH2
His-D-Trp-Ala-(R)-Bgl-D-Phe-Lys-NH2
His-D-Trp-Ala-Trp-(R)-Bgl-Lys-NH2
25
26
27
28
29
30
31
32
33
34
35
19
45
30
61
55
30
20
31
22
40
60
>93
>99
>98
>99
>99
>97
>99
>99
>99
>99
>99
14
12
4
11
7
5
7
8
8
819.4301
770.4096
443.2296
770.4096
809.4205
819.4301
861.4406
385.7085
443.2296
770.4096
809.4205
819.4308
770.4097
443.2301e
770.4097
809.4199
819.4307
861.4413
385.7083e
443.2301
770.4095
809.4198
5
3
HRMS, high resolution mass spectrometry.
aBold lettering indicates lactam residues.
bRP-HPLC purity at 214 nm of the crude peptide.
cRP-HPLC purity at 214 nm of the purified peptide.
dYields after purification by RP-HPLC are based on Fmoc loading test for Rink resin.
eThe dicharged cation [M+2H]2+ was observed, theoretical mass was calculated accordingly.
180 min in the cases of Fmoc-D-Phe, Fmoc-Trp(Boc), Fmoc-D-Trp(Boc),
and Fmoc-His(Trt) couplings. The resin in Kan reactors was then
washed as described above.
was added to the Kan in the tube, which was tightly sealed with
stoppers and parafilm. After shaking for 24 h, the solution was
removed by suction, and the Kan was washed with dry THF
(3 · 7 mL, under argon), MeOH (3 · 7 mL), and DCM (3 · 7 mL)
and dried in vacuo.
Silylation and alkylation
For lactam synthesis, the appropriate Kan was taken out of the
batch and transferred to a 12 mL plastic syringe tube. The resin
was swollen in DMF (7 mL) for 0.5 h and treated twice with a
freshly prepared 20% piperidine in DMF solution (7 mL), for
20 min. It was then washed sequentially with DMF (3 · 7 mL),
MeOH (3 · 7 mL), and DCM (3 · 7 mL) and dried in vacuo for at
least 180 min. The syringe tube containing the Kan was flushed
with argon, and the resin was swollen in dry THF (7 mL) under
argon, treated with BSA (N,O-bis(trimethylsilyl)-acetamide, 5 equiv.),
and the tube was tightly sealed with stoppers and parafilm and
shaken for 16 h. The solution was then filtered off under argon. A
solution of sulfamidate (R)-3 or (R)-4 (5 equiv.) in dry THF (7 mL)
Microwave-assisted annulation
The Kan was opened, the resin was separated from the correspond-
ing radio frequency tag and equally split into two 5 mL V-shaped
microwave glass vessels fitted with a triangular stirring bar. After
DMF (2 mL) addition, both vessels were sealed and heated under
microwave irradiation at 100 ꢁC (pressure 1 bar) for 75 min or as
specified. The two batches of resin were then combined back in an
IRORI Kan with their radio frequency tag and transferred to a
12 mL plastic filtration tube with polyethylene frit, where they were
washed sequentially with DMF (3 · 7 mL), MeOH (3 · 7 mL), and
DCM (3 · 7 mL) and dried in vacuo.
Chem Biol Drug Des 2010; 75: 40–50
43

Boutard et al.
Table 3: IC50 Binding values for GHS-R1a and CD36 receptors for Agl and Bgl GHRP-6 analogs
Compound
Peptide
IC50 binding GHS-R1a
IC50 binding CD36
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
GHRP-6
Hexarelin
15a
25
His-D-Trp-Ala-Trp-D-Phe-Lys-NH2
6.08 · 10)9
M
His-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH2
(S)-Agl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
(R)-Agl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
(S)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
Ac(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2
His-(S)-Agl-Ala-Trp-D-Phe-Lys-NH2
His-(R)-Agl-Ala-Trp-D-Phe-Lys-NH2
His-(S)-Bgl-Ala-Trp-D-Phe-Lys-NH2
His-(R)-Bgl-Ala-Trp-D-Phe-Lys-NH2
His-D-Trp-(S)-Agl-Trp-D-Phe-Lys-NH2
His-D-Trp-(R)-Agl-Trp-D-Phe-Lys-NH2
His-D-Trp-(S)-Bgl-Trp-D-Phe-Lys-NH2
His-D-Trp-(R)-Bgl-Trp-D-Phe-Lys-NH2
His-D-Trp-Ala-(S)-Agl-D-Phe-Lys-NH2
His-D-Trp-Ala-(R)-Agl-D-Phe-Lys-NH2
His-D-Trp-Ala-(S)-Bgl-D-Phe-Lys-NH2
His-D-Trp-Ala-(R)-Bgl-D-Phe-Lys-NH2
His-D-Trp-Ala-Trp-(S)-Agl-Lys-NH2
His-D-Trp-Ala-Trp-(R)-Agl-Lys-NH2
His-D-Trp-Ala-Trp-(S)-Bgl-Lys-NH2
His-D-Trp-Ala-Trp-(R)-Bgl-Lys-NH2
3.33 · 10)6
1.45 · 10)5
M
M
4.09 · 10)8
3.46 · 10)6
3.71 · 10)7
1.01 · 10)6
2.75 · 10)6
2.86 · 10)5
M
M
M
M
M
M
>>10)5
>>10)5
M
20a
30
M
1.37 · 10)5
M
M
31
>>10)5
M
16a
26
1.34 · 10)5
>>10)5
>>10)5
>>10)5
M
M
M
>>10)5
>>10)5
>>10)5
M
M
M
21a
32
17a
27
2.39 · 10)6
2.82 · 10)6
6.54 · 10)7
3.39 · 10)6
M
M
M
M
7.45 · 10)6
M
M
>>10)5
>>10)5
M
22a
33
M
2.11 · 10)5
18a
28
>>10)5
>>10)5
M
>>10)5
>>10)5
M
M
M
23a
34
>> 10)5
>> 10)5
M
M
>> 10)5
>> 10)5
M
M
19a
29
3.10 · 10)6
M
M
2.14 · 10)5
M
>>10)5
M
>>10)5
M
24a
35
2.45 · 10)5
2.65 · 10)5
9.40 · 10)5
M
M
>>10)5
M
aReference (32).
Optimized alkylation and lactamization
Peptide cleavage
conditions for (R)-Bgl peptides 32 and 33 (vide
infra)
Each Kan was shaken in a separate 12 mL syringe tube contain-
ing TFA ⁄ H2O ⁄ TES (7 mL, 95 ⁄ 2.5 ⁄ 2.5, v ⁄ v ⁄ v) for 2 h. The liquid
was filtered off, the resin was washed with TFA (2 · 8 mL), and
the combined filtrate was concentrated in vacuo. The resulting
residue was dissolved in a minimum volume of TFA (ꢀ1 mL),
transferred to a centrifuge tube and precipitated by the addition
of ice-cold diethyl ether (40 mL). The suspension was then cen-
trifuged, and the diethyl ether was carefully decanted from the
tube. The precipitate was resuspended in cold diethyl ether, and
the process was repeated twice. The resulting white solid was
dissolved in water (10 mL) and freeze-dried to give a white fluffy
solid that was purified by preparative RP-HPLC using the
specified conditions.
After Fmoc deprotection, as described above, the resin was taken
out of its Kan, separated from the corresponding radio frequency tag
microchip and split into two 5 mL microwave glass vessels. A
solution of cyclic sulfamidate (R)-4 (5 equiv.) and DIEA (1 equiv.) in
THF (2 mL) was added, the reactors were sealed and irradiated under
microwave at 60 ꢁC for 1.5 h. The two batches of resin were then
combined into a 12 mL syringe tube, washed with DMF (3 · 7 mL),
MeOH (3 · 7 mL), DCM (3 · 7 mL), and with three cycles comprising
washing with 2% AcOH in DMSO (7 mL) for 30 min and washing
with MeCN (7 mL) for 5 min. The resin was split back into two 5 mL
V-shaped microwave glass vessels fitted with a triangular stirring
bar, treated with a DMSO ⁄ H2O ⁄ AcOH 75:23:2 mixture (2 mL), and
the vessels were sealed and irradiated under microwave at 100 ꢁC
for 3 h. The resin was then washed from the microwave vessels into
a 12 mL plastic filtration tube fitted with a polyethylene frit and
washed sequentially with DMF (3 · 7 mL), MeOH (3 · 7 mL), and
DCM (3 · 7 mL) and dried in vacuo.
(R)-Agl-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (25)
(R)-Agl-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (25) was prepared using the
split-and-mix process as described above in IRORI KansTM. Micro-
wave-assisted annulation gave the desired lactam-bridged peptide
TFA salt (97 mg) in 19% crude purity as determined by analytical
RP-HPLC (column A, 2–40% MeCN in H2O, 0.1% FA, 60 min
gradient). Final lactam peptide purification was carried out by
preparative RP-HPLC (column C, 2–40% MeCN in H2O, 0.1% FA,
60 min gradient) to give the desired FA salt product (8 mg, 14%)
as a white fluffy solid. The purified product was analyzed by
analytical RP-HPLC (column A) using both MeCN tR 11.39
(0–60% MeCN in H2O, 0.1% FA, 30 min gradient) and MeOH tR
14.50 (0–60% MeOH in H2O, 0.1% FA, 30 min gradient) and
revealed >93% purity. HRMS calcd. m ⁄ z for C44H55O6N10 [M+H]+
819.4301, found 819.4308.
Resin capping and peptide completion
The resin in a 12 mL plastic filtration tube was swollen in DMF,
suspended in a solution of di-tert-butyl dicarbonate (5 equiv.) in
DMF (7 mL), treated with DIEA (10 equiv.), shaken for 1 h, filtered
and washed sequentially with DMF (3 · 7 mL), MeOH (3 · 7 mL)
and DCM (3 · 7 mL). The resin was then transferred back to a Kan
with the corresponding frequency tag microchip. The peptide syn-
thesis was then completed, and the N-terminal Fmoc group
removed as described above.
44
Chem Biol Drug Des 2010; 75: 40–50

Structure-Activity Analysis of the Growth Hormone Secretagogue GHRP-6
His-(R)-Agl-Ala-Trp-D-Phe-Lys-NH2 (26)
8.83 (0–60% MeCN in H2O, 0.1% FA, 30 min gradient) and MeOH
tR 10.21 (0–60% MeOH in H2O, 0.1% FA, 30 min gradient) and
revealed >99% purity. HRMS calcd. m ⁄ z for C41H53O6N12 [M+H]+
809.4205, found 809.4199.
His-(R)-Agl-Ala-Trp-D-Phe-Lys-NH2 (26) was prepared as described
above in an IRORI KanTM. Microwave-assisted annulation gave the
desired lactam-bridged peptide TFA salt (178 mg) in 45% crude
purity as determined by analytical RP-HPLC (column A, 0–20%
MeCN in H2O, 0.1% FA, 20 min gradient). Final purification was
carried out by preparative RP-HPLC (column C, 0–20% MeCN in
H2O, 0.1% FA, 25 min gradient) to give the desired FA salt product
(19 mg, 12%) as a white fluffy solid. The purified product was
analyzed by analytical RP-HPLC (column A) using both MeCN tR
8.80 (0–60% MeCN in H2O, 0.1% FA, 30 min gradient) and MeOH
tR 10.03 (0–60% MeOH in H2O, 0.1% FA, 30 min gradient) and
revealed >99% purity. HRMS calcd. m ⁄ z for C39H52O6N11 [M+H]+
770.4096, found 770.4097.
(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (30)
(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (30) was prepared in an IRORI
KanTM as described above. Microwave-assisted annulation over
180 min gave the desired lactam peptide TFA salt 30 (78 mg) in
30% crude purity as determined by analytical RP-HPLC (column
A, 0–30% MeCN in H2O, 0.1% FA, 20 min gradient). After final
Fmoc deprotection, and before cleavage, half of the resin was
kept for preparation of compound 31 (vide infra). Purification
was carried out by preparative RP-HPLC (column C, 10–25%
MeCN in H2O, 0.1% FA, 50 min gradient, flow rate: 15 mL ⁄ min)
to give the desired FA salt 30 (4.8 mg, 5%) as a white
fluffy solid. The purified product was analyzed by analytical RP-
HPLC (column A) using both MeCN tR 15.50 (0–60% MeCN in
H2O, 0.1% FA, 25 min gradient) and MeOH tR 13.50 (0–60%
MeOH in H2O, 0.1% FA, 25 min gradient) and revealed >97%
purity. HRMS calcd. m ⁄ z for C44H55O6N10 [M+H]+ 819.4301, found
819.4307.
His-D-Trp-(R)-Agl-Trp-D-Phe-Lys-NH2 (27)
His-D-Trp-(R)-Agl-Trp-D-Phe-Lys-NH2 (27) was prepared as described
above in an IRORI KanTM. Microwave-assisted annulation gave the
desired lactam-bridged peptide TFA salt (144 mg) in 30% crude
purity as determined by analytical RP-HPLC (column A, 0–40%
MeCN in H2O, 0.1% FA, 15 min gradient). Purification was carried
out by preparative RP-HPLC (column C, 0–40% MeCN in H2O,
0.1% FA, 60 min gradient) to give the desired FA salt product
(7 mg, 4%) as a white fluffy solid. The purified product was
analyzed by analytical RP-HPLC (column A) using both MeCN tR
10.60 (0–60% MeCN in H2O, 0.1% FA, 30 min gradient) and
MeOH tR 13.77 (0–60% MeOH in H2O, 0.1% FA, 15 min gradient)
and revealed >98% purity. HRMS calcd. m ⁄ z for C47H58O6N12
[M+2H]2+ 443.2296, found 443.2301.
Ac(R)-Bgl-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (31)
H2N-(R)-Bgl-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(Boc)-NH-Rink (prepared
in the synthesis of lactam peptide 30) was acetylated using a
solution of acetic anhydride (5 equiv.) and DIEA (5 equiv.) in
DMF (4 mL) over 1.5 h. After washing, cleavage, and precipita-
tion as described above, the desired lactam peptide TFA salt
(68 mg) was obtained in 20% crude purity as determined by
analytical RP-HPLC (column A, 0–30% MeCN in H2O, 0.1% FA,
20 min gradient). Purification was carried out by preparative
RP-HPLC (column C, 20–40% MeCN in H2O, 0.1% FA, 40 min
gradient, flow rate: 15 mL ⁄ min) to give the desired FA salt
(7 mg, 7%) as a white fluffy solid. The purified product was
analyzed by analytical RP-HPLC (column A) using both MeCN tR
14.42 (0–80% MeCN in H2O, 0.1% FA, 25 min gradient) and
MeOH tR 19.42 (0–90% MeOH in H2O, 0.1% FA, 25 min gradi-
ent) and revealed >99% purity. HRMS calcd. m ⁄ z for
C46H57O7N10 [M+H]+ 861.4406, found 861.4413.
His-D-Trp-Ala-(R)-Agl-D-Phe-Lys-NH2 (28)
His-D-Trp-Ala-(R)-Agl-D-Phe-Lys-NH2 (28) was prepared as described
above in an IRORI KanTM. Microwave-assisted annulation gave the
desired lactam-bridged peptide TFA salt (132 mg) in 61% crude
purity as determined by analytical RP-HPLC (column A, 0–20%
MeCN in H2O, 0.1% FA, 35 min gradient). Purification was carried
out by preparative RP-HPLC (column C, 0–20% MeCN in H2O,
0.1% FA, 20 min gradient) to give the desired FA salt product
(17 mg, 11%) as a white fluffy solid. The purified product was
analyzed by analytical RP-HPLC (column A) using both MeCN tR
8.68 (0–60% MeCN in H2O, 0.1% FA, 30 min gradient) and MeOH
tR 9.77 (0–60% MeOH in H2O, 0.1% FA, 30 min gradient) and
revealed >99% purity. HRMS calcd. m ⁄ z for C39H52O6N11 [M+H]+
770.4096, found 770.4097.
His-(R)-Bgl-Ala-Trp-D-Phe-Lys-NH2 (32)
His-(R)-Bgl-Ala-Trp-D-Phe-Lys-NH2 (32) was prepared in an IRORI
KanTM using the optimized alkylation and lactamization conditions
(vide infra), to give the desired lactam peptide TFA salt (112 mg) in
31% crude purity as determined by analytical RP-HPLC (column B,
0–80% MeCN in H2O, 0.1% FA, 30 min gradient). Purification was
carried out by preparative RP-HPLC (column D, 10–40% MeCN in
H2O, 0.1% FA, 30 min gradient) to give the desired FA salt product
(16 mg, 8%) as a white fluffy solid. The purified product was ana-
lyzed by analytical RP-HPLC (column B) using both MeCN tR 14.26
(0–60% MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR
15.91 (0–60% MeOH in H2O, 0.1% FA, 30 min gradient) and
revealed >99% purity. HRMS calcd. m ⁄ z for C39H53O6N11 [M+2H]2+
385.7085, found 385.7083.
His-D-Trp-Ala-Trp-(R)-Agl-Lys-NH2 (29)
His-D-Trp-Ala-Trp-(R)-Agl-Lys-NH2 (29) was prepared as described
above in an IRORI KanTM. Microwave-assisted annulation over
90 min gave the desired lactam-bridged peptide TFA salt (101 mg)
in 55% crude purity as determined by analytical RP-HPLC (column
A, 0–20% MeCN in H2O, 0.1% FA, 15 min gradient). Purification
was carried out by preparative RP-HPLC (column C, 0–20% MeCN
in H2O, 0.1% FA, 20 min gradient) to give the desired FA salt prod-
uct (11 mg, 7%) as a white fluffy solid. The purified product was
analyzed by analytical RP-HPLC (column A) using both MeCN tR
Chem Biol Drug Des 2010; 75: 40–50
45

Boutard et al.
His-D-Trp-(R)-Bgl-Trp-D-Phe-Lys-NH2 (33)
pH to 7.1 (HBSS) by alternating one drop ⁄ two air bubbles. The
transfection mix reaction was incubated at RT for 30 min. After the
incubation period, 1 mL of the mix was added to each plate and
distributed evenly for incubation. The media was replaced with
standard DMEM-high glucose media for another 24 h, and cells
were collected for membrane preparation.
His-D-Trp-(R)-Bgl-Trp-D-Phe-Lys-NH2 (33) was prepared in an IRORI
KanTM using the optimized alkylation and lactamization conditions
(vide infra), to give the desired lactam peptide TFA salt (120 mg) in
22% crude purity as determined by analytical RP-HPLC (column B,
15–40% MeCN in H2O, 0.1% FA, 30 min gradient). Purification was
carried out by preparative RP-HPLC (column D, 8–30% MeCN in
H2O, 0.1% FA, 30 min gradient) to give the desired FA salt (14 mg,
8%) as a white fluffy solid. The purified product was analyzed by
analytical RP-HPLC (column A) using both MeCN tR 17.57 (0–60%
MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR 21.38
(0–80% MeOH in H2O, 0.1% FA, 25 min gradient) and revealed
>99% purity. HRMS calcd. m ⁄ z for C47H58O6N12 [M+2H]2+ 443.2296,
found 443.2301.
Membrane preparation
The experiment was carried out at 4 ꢁC unless specified. Cells
were washed two times with PBS and with the homogenization
buffer (HB) consisting of 50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA,
30 lg ⁄ mL bacitracin at pH 7.3 and were scraped in Eppendorf
tubes. Cells were lyzed with two cycles of freeze ⁄ thawing using
liquid nitrogen and were then centrifuged at 4 ꢁC for 20 min at
10 000 · g to collect the membranes. The membranes were resus-
pended in a small volume of HB and aliquoted for storage at
)80 ꢁC.
His-D-Trp-Ala-(R)-Bgl-D-Phe-Lys-NH2 (34)
His-D-Trp-Ala-(R)-Bgl-D-Phe-Lys-NH2 (34) was prepared in an IRORI
KanTM as described above. Microwave-assisted annulation over
180 min gave the desired lactam peptide TFA salt (180 mg) in 40%
crude purity as determined by analytical RP-HPLC (column A,
0–30% MeCN in H2O, 0.1% FA, 25 min gradient). Purification was
carried out by preparative RP-HPLC (column C, 10–25% MeCN in
H2O, 0.1% FA, 20 min gradient) to give the desired FA salt product
(9.6 mg, 5%) as a white fluffy solid. The purified product was
analyzed by analytical RP-HPLC (column A) using both MeCN tR
12.13 (0–60% MeCN in H2O, 0.1% FA, 20 min gradient) and MeOH
tR 14.32 (0–60% MeOH in H2O, 0.1% FA, 20 min gradient) and
revealed >99% purity. HRMS calcd. m ⁄ z for C39H52O6N11 [M+H]+
770.4096, found 770.4095.
GHS-R1a receptor binding assay
The competitive binding assay consists of 200 lL HB, 100 lL 125I-
Ghrelin (40,000 cpm), 100 lL competitive ligand (from 10)12 to
10)5 M), and 100 lL of GHS-R1a transiently transfected in LLC-PK1
cells as source of binding sites (10 lg protein ⁄ tube). The non-
specific binding was determined by excess of competitive ligand
at 10)5 M. The reaction was carried out at RT for 1 h. After the
incubation period, the separation of bound from free fraction was
performed by filtration over a GF ⁄ C filter presoaked with 0.5%
polyethyleneimine, and the filters were washed with 4 mL of HB
consisting of 50 mM Tris, 10 mM MgCl2, 2.5 mM EDTA, and 3 mL of
wash buffer consisting of 50 mM Tris–10 mM MgCl2, 20 mM EDTA,
0.015% Triton X100 (pH 7.3) and were then collected for radio-
activity counting using a gamma scintillation counter (LKB Wallac
1277, Turku Finland).
His-D-Trp-Ala-Trp-(R)-Bgl-Lys-NH2 (35)
His-D-Trp-Ala-Trp-(R)-Bgl-Lys-NH2 (35) was prepared in an IRORI
KanTM as described above. Microwave-assisted annulation over
180 min gave the desired lactam peptide TFA salt (67 mg) in 60%
crude purity as determined by analytical RP-HPLC (column A, 0–30%
MeCN in H2O, 0.1% FA, 25 min gradient). Purification was carried
out by preparative RP-HPLC (column C, 0–30% MeCN in H2O, 0.1%
FA, 25 min gradient) to give the desired FA salt (5.4 mg, 3%) as a
white fluffy solid. The purified product was analyzed by analytical
RP-HPLC (column A) using both MeCN tR 12.05 (0–60% MeCN in
H2O, 0.1% FA, 20 min gradient) and MeOH tR 14.11 (0–60% MeOH
in H2O, 0.1% FA, 20 min gradient) and revealed >99% purity. HRMS
calcd. m ⁄ z for C41H53O6N12 [M+H]+ 809.4205, found 809.4198.
Membrane preparation for CD36
Animal use was in accordance with the Institutional Animal Ethics
Committee and the Canadian Council on Animal Care guidelines for
the use of experimental animals. Sprague Dawley (275–350 g) rats
were anaesthetized with sodium pentobarbital, and their hearts
were promptly removed in ice-cold saline, and the cardiac mem-
branes were prepared according to Harigaya and Schwartz (41).
Competitive covalent CD36 binding assay using
photoactivatable [125I]-Tyr-Bpa-Ala-Hexarelin as
radioligand
Membrane preparation for GHS-R1a receptor
Transfection LLC-PK1 cells were seeded at 1.5 · 106 cells ⁄ 10-cm
Petri dishes and grown for 24 h in DMEM high-glucose (4.5 g ⁄ L)
with 10% fetal bovine serum supplemented with penicillin
(10 000 Units ⁄ mL) and streptomycin (10 000 lg ⁄ mL), and cultured
at 37 ꢁC, with 5% CO2. Four to 5 h after replacement of the med-
ium, transfection was performed using Ca3(PO4)2 calcium phosphate.
The DNA solution consisted of 40 lg of DNA in a volume of
500 lL in which 500 lL of 2 mM Tris–HCl pH 8.0, 0.2 mM EDTA pH
8.0 containing 500 mM CaCl2 was added to a final volume of 1 mL,
followed by 1 mL of 50 mM Hepes, 280 mM NaCl, 1.5 mM Na2HPO4
The radioiodination procedure of the photoactivatable ligand and
the receptor binding assays were performed as previously described
by Ong et al. (42). Briefly, the rat cardiac membranes (200 lg) as
source of CD36 were incubated in the dark, in 525 lL 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 with increasing concentrations of com-
petitive ligands (ranging from 0.1 to 50 lM). Non-specific binding
46
Chem Biol Drug Des 2010; 75: 40–50

Structure-Activity Analysis of the Growth Hormone Secretagogue GHRP-6
was defined as binding not displaced by 50 lM peptide. After an
stoichiometry of BSA and (R)-4, the temperature, the reaction time,
and the use of base as additive. Alkylation at the Ala residue of
GHRP-6 with (R)-4 was used for the optimization experiments
(peptide 36, Scheme 2; Table 1).
incubation period of 60 min at 22 ꢁC, membranes were submitted
to UV irradiation at 365 nm for 15 min at 4 ꢁC. After centrifugation
at 12 000 g for 15 min, the pellets were resuspended in 100 lL 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 being subjected 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 phos-
phor intensifying screen (Amersham Biosciences), and analysed by
using a Typhoon PhosphorImager (Amersham Biosciences) and IMAGE-
QUANT 5.0 softwared to establish competition curves. Protein bands
corresponding to the specifically labeled protein of 87 kDa were
quantified by densitometry analysis.
The silylation time could be reduced to 1 h (entry 2) and the alky-
lation time to 1 h (entry 3) by performing the alkylation at 60 ꢁC
under microwave irradiation without affecting the conversion to
secondary amine 37. Conversion to the latter was however halved
by conducting the reaction at room temperature (entry 4). The sily-
lation step could be removed without any significant change in
the ratio of non-alkylated and mono alkylated product 38:37
(around 1:1) without bis-alkylation product 39 (entry 6). We con-
sidered that the sulfamic acid, formed by the opening of the cyc-
lic sulfamidate, protonated the terminal primary amines preventing
alkylation leading to only 50% conversion. Employment of DIEA
(1 equiv., entry 7) as base confirmed our hypothesis and gave
80% conversion to 37. However, increasing the amount of base
to 1.2 equiv. gave rise to a noticeable amount of bis-alkylation
product 39 (21%, entry 8), indicating that excess base should be
avoided. In these studies, ACS grade THF worked as effectively
as dry THF.
Results and discussion
Optimized protocol
Using our standard protocol, lactam peptides 25–31 and 34–35
have been prepared in moderate to good crude purity and isolated
in acceptable yields (Table 2). However, the syntheses of peptides
32 and 33 were problematic, giving product in low crude purity
(ꢁ5%), both from IRORI KanTM and syringe tubes. This led us to
retool our method for difficult Bgl synthesis cases, beginning by
monitoring the alkylation step protocol using LC-MS. Considering
that quantification depended on the presence of a chromophore on
starting material and product, direct cleavage of small resin sample
was deemed biased, because it afforded alkylation product bearing
a UV active Fmoc group and starting peptide without Fmoc group.
A Fmoc reprotection step was thus performed before cleavage
by treating the resin aliquot with a solution of N-(9-fluorenyl-
methoxycarbonyloxy) succinimide (Fmoc-OSu) and DIEA (Scheme 2).
Optimization of the alkylation conditions involved varying the
The alkylated peptidyl resin from conditions described in entry 7 of
Table 2 was subjected to lactam formation in 1% AcOH ⁄ DMSO at
100 ꢁC under microwave for 3 h (32). LC-MS analysis of a cleaved
sample indicated the presence of the expected lactam compound
along with the corresponding Fmoc-deprotected lactam, accounting,
respectively, for 75% and 16% of the mixture. A plausible explana-
tion would be the presence of DIEA trapped on the resin as an
ammonium sulfamate salt. Thermal decomposition of the sulfamate
during microwave irradiation would liberate free DIEA accountable
for Fmoc deprotection at 100 ꢁC. Improvements in lactam formation
yields were obtained by washing the resin beforehand with three
Fmoc-Ala-Trp-D-Phe-Lys-NH2
1) BSAa
MeO2C
2) (R)-4a
38
H
N-Ala-Trp-D-Phe-Lys-NH2
+
Ala-Trp(Boc)-D-Phe-Lys(Boc)-NH-
FmocHN
NHFmoc
3) FmocOSu, DIEAb
36
MeO2C
MeO2C
4) TFA cleavage c
37
N-Ala-Trp-D-Phe-Lys-NH2
NHFmoc
39
a
b
Scheme 2: Alkylation reaction of peptide 36 with cyclic sulfamidate (R)-4. see table 2 for condition; with N-(9-fluorenyl-methoxycarbo-
c
nyloxy)succinimide (Fmoc-OSu, 0.04M in DCM, 1 mL), DIEA (3 drops), 1 h, RT; TFA/H2O/TES 95/2.5/2.5, 2h, RT.
O
MeO2C
1) 20% piperidine/DMF
H
H
H
H
Fmoc Xaa peptide N
Xaa peptide
N
N
N
Xaa peptide N
FmocHN
FmocHN
MW, 100 °C, 3 h
DMSO:H2O:AcOH
75:23:2
2) (R)-4, 5 eq.
1 eq. DIEA, THF
60 °C μW, 1 h
6
8
10
3) 2% AcOH/DMSO
and MeCN washings
1) 20% piperidine/DMF
2) Fmoc-Yaa-OH
O
HBTU, DIEA, DMF
3) repeat steps 1) and 2)
N Xaa peptide NH2
peptide Yaa
N
H
4) 20% piperidine/DMF
5) TFA/TES/H2O
(95 : 2.5 : 2.5, v/v/v)
14
Scheme 3: Optimized protocol for the synthesis of Bgl-peptides.
Chem Biol Drug Des 2010; 75: 40–50
47

Boutard et al.
cycles of 2% AcOH in DMSO (7 mL) before lactamization, and by
performing the microwave irradiation at 100 ꢁC for 3h in a DMSO ⁄
H2O ⁄ AcOH 75:23:2 mixture. Addition of water to the lactamization
mixture proved to enhance conversion to the lactam compounds by
5–10%.
hypothesis of a b-turn about residues 2–4 of GHRP-6, which may
account for binding and differentiating the CD36 receptor from the
GHS-1Ra receptor (45).
Conclusion
Using this optimized protocol (Scheme 3) gave peptides 32 and 33,
respectively, in 35% and 21% crude purity; both being isolated in
8% yield after purification on preparative RP-HPLC.
A (R)-Agl and (R)-Bgl positional scan of GHRP-6 was efficiently
accomplished using IRORI KanTM techniques and cyclic sulfami-
dates (R)-3 and (R)-4 to produce eleven new GHRP-6 analogs in
good overall yields and crude purities. Improvements in the syn-
thesis of Bgl were made by microwave heating in the presence
of a base during the alkylation step and introduction of a mild
acid wash before microwave-assisted annulation in a DMSO ⁄
H2O ⁄ AcOH 75:23:2. The new compounds were evaluated for their
binding affinity towards the GHS-R1a and the CD36 receptors.
Results emphasized the importance of Trp residues for affinity
especially with the ghrelin receptor. The study yielded compounds
selective for the CD36 receptor relative to GHS-R1a, typically
with substitution of Ala3 and D-Phe5 with both Agl and Bgl. In
the structure–affinity relationship contingent on stereochemistry
and sequence, changes in selectivity were observed using Agl
and Bgl residues, suggesting that they may induce complemen-
tary turn structures. Agl and Bgl lactam scanning has thus iden-
tified new leads for selective GHRP-6 mimics as well as a
better understanding of the importance of side chains and con-
formation on affinity and selectivity.
Biology
The affinity of Agl- and Bgl-containing GHRP-6 analogs 25–35 for
the ghrelin receptor was assessed in a competition-binding study
using radiolabelled ghrelin [1–28] as radioligand and a GHS-R1a
transfected cell membrane as source of binding site (43). The affin-
ity of the GHRP-6 amino lactam peptides for the receptor CD36 has
been assessed by covalent competition binding study using a radio-
labelled photoactivatable hexarelin derivative as radioligand and rat
cardiac membranes as source of CD36 receptor as previously
reported (15). GHRP-6 lactam analogs were used as competitive
ligands in the range concentrations from 10)12 to 10)6 M and IC50
values of competition binding curves were calculated by iterative
nonlinear curve-fitting programe.
Lactam analogs generally lost affinity for both the GHS-R1a and
CD36 receptors. The (S)-Agl and (R)-Bgl peptides exhibited gener-
ally better affinity for the CD36 receptor than their enantiomeric
counterparts. In the case of the GHS-R1a receptor, the (S)-lactam
peptide displayed better affinities.
Acknowledgments
At His1, the (S)-Agl and (S)-Bgl peptides 15 and 20 (entries 3 and
5) displayed a 10- to 100-fold loss of affinity towards the ghrelin
receptor (32), as observed with previous analogs featuring substitu-
tions at this position (44); however, (R)-Agl1, (R)-Bgl1, and Ac(R)-
Bgl1 peptides (peptides 25, 30 and 31) caused a drop of affinity
of 2 to 6 · 103 for the GHS-R1a receptor. (S)-Agl1 and (R)-Bgl1 pep-
tides 15 and 30 conserved some affinity for the CD36 receptor.
The authors acknowledge financial support from the Natural
Sciences and Engineering Research Council of Canada (NSERC),
Universitꢀ de Montrꢀal, Canadian Institutes of Health Research
(CIP-79 848) CIHR-Team in GPCR allosteric regulation (CTiGAR), the
Fonds Quebecois de la Recherche sur la Nature et les Technolo-
gies (FQRNT), The Ministre du Developpement Economique, de
l'Innovation et de l'Exportation du Quebec, and equipment made
possible from the Canadian Foundation for Innovation. We are
grateful to Petra Pohankova for performing binding measurements
on GHS-R1a and CD36 receptors, to Dalbir Sekhon for his assis-
tance in peptide purifications, and to Marie-Christine Tang and
Karine Venne from the Universitꢀ de Montrꢀal Mass spectrometry
facility for MS analyses.
Replacement of the Trp residues by Agl and Bgl of both configura-
tions was particularly detrimental to the binding affinity at the
GHS-R1a receptor (entries 8–11 and 16–19) likely because of the
importance of the indolic side chains for interaction with the ghrelin
receptor. The same trend was observed for binding to CD36 recep-
tor with the exception of (S)-Agl2 16 (entries 8) that exhibited a
fourfold reduced affinity compared with hexarelin.
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the CD36, receptor compared with hexarelin for 19 and 24. The
residue at position 5 could play a role for selectivity towards CD36.
(R)-Bgl peptide 35 lost affinity for both receptors.
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Notes
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php.
TM
cSYNTHESIS MANAGER Nexus Biosystems, Inc., Poway, CA, USA.
dIMAGEQUANT 5.0, GE Healthcare, Piscataway, NJ, USA.
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50
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