a
Table 2: Pharmacology of commercial and synthesized SHU9119 and MTII at the mouse melanocortin receptors.
Peptide
mMC1R
mMC3R
mMC4R
mMC5R
EC50 (nM)
0.23±0.03
NDP-MSH
SHU9119
0.04±0.01
0.98±0.17
0.47±0.21
Antagonist
0.18±0.07
Partial agonist
Partial agonist
(
Commercial)
PA
2
= 8.8, K
i
= 1.6 nM
PA
2
= 9.3, K
i
= 0.5 nM
SHU9119 (SRT5-134)
1.43±0.33
0.06±0.02
0.05±0.01
Partial agonist
PA = 8.5, K
Antagonist
PA = 9.1, K
Partial agonist
0.14±0.05
2
i
= 3.2 nM
2
i
= 0.79 nM
MTII
0.18±0.04
0.15±0.04
0.15±0.06
0.14±0.02
(
Commercial)
MTII
SRT5-148)
0.12±0.02
(
a
The indicated errors represents the standard error of the mean determined from at least three independent experiments. * The pA2 values were calculated by a Schild analysis.
In conclusion, an efficient microwave-assisted synthetic
References and notes
strategy for the syntheses of side-chain to side-chain lactam-
bridged cyclic peptides on a solid support without aspartimide
formation is reported. This synthetic route makes the purification
of these peptides efficient and the synthesis amenable to rapid
SAR compound generation and purification. Utilization of
microwave irradiation in each step of the peptide synthesis,
which include i) deprotection of Fmoc group, ii) coupling of
amino acid, iii) selective deprotection of alloc and allyl
protecting groups, iv) formation of lactam cyclic-bridge, v) N-
terminal acetylation, and vi) cleavage of peptide from resin,
reduces the reaction times at each step. The current method also
permits the syntheses of these peptides efficiently in an open
reaction vessel without need for complete absence of oxygen.
The methodology presented here could be automated and applied
to the synthesis of other side-chain to side-chain lactam-bridge
cyclic peptides, decreasing the synthetic times and improving the
synthetic efficiency.
1
.
Rezai, T.; Yu, B.; Millhauser, G. L.; Jacobson, M. P.; Lokey, R. S.
J. Am. Chem. Soc. 2006, 128, 2510-1.
March, D. R.; Abbenante, G.; Bergman, D. A.; Brinkworth, R. I.;
Wickramasinghe, W.; Begun, J.; Martin, J. L.; Fairlie, D. P. J. Am.
Chem. Soc. 1996, 118, 3375-3379.
2.
3
4
5
.
.
.
Gentilucci, L.; De Marco, R.; Cerisoli, L. Curr. Pharm. Des. 2010,
1
6, 3185-3203.
Murage, E. N.; Gao, G. Z.; Bisello, A.; Ahn, J. M. J. Med. Chem.
2010, 53, 6412-6420.
Park, B. W.; Zhang, H. T.; Wu, C. J.; Berezov, A.; Zhang, X.;
Dua, R.; Wang, Q.; Kao, G.; O'Rourke, D. M.; Greene, M. I.;
Murali, R. Nat. Biotechnol. 2000, 18, 194-198.
Colgrave, M. L.; Korsinczky, M. J. L.; Clark, R. J.; Foley, F.;
Craik, D. J. Biopolymers 2010, 94, 665-672.
Athanassiou, Z.; Dias, R. L. A.; Moehle, K.; Dobson, N.; Varani,
G.; Robinson, J. A. J. Am. Chem. Soc. 2004, 126, 6906-6913.
Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz,
P. G. J. Am. Chem. Soc. 1991, 113, 9391-9392.
6
.
7.
8
9
.
.
Felix, A. M.; Heimer, E. P.; Wang, C. T.; Lambros, T. J.;
Fournier, A.; Mowles, T. F.; Maines, S.; Campbell, R. M.;
Wegrzynski, B. B.; Toome, V.; Fry, D.; Madison, V. S. Int. J.
Pept. Protein Res. 1988, 32, 441-454.
Acknowledgments
This work has been supported by NIH Grants R01DK091906
1
0. Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.;
Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J.
Science 2004, 305, 1466-1470.
(C. H.-L.).
Supplementary Material
11. Taylor, J. W. Biopolymers 2002, 66, 49-75.
1
2. Al-Obeidi, F.; Castrucci, A. M.; Hadley, M. E.; Hruby, V. J. J.
Med. Chem. 1989, 32, 2555-61.
Detailed procedure for the syntheses of peptides, analytical
HPLC chromatograms, analytical data of peptides and
AlphaScreen cAMP assay are given in the supporting
1
3. Hruby, V. J.; Lu, D.; Sharma, S. D.; Castrucci, A. L.; Kesterson,
R. A.; Al-Obeidi, F. A.; Hadley, M. E.; Cone, R. D. J. Med. Chem.
®
1
995, 38, 3454-61.
information.
14. Al-Obeidi, F.; Hadley, M. E.; Pettitt, M. B.; Hruby, V. J. J. Am.
Chem. Soc. 1989, 111, 3413-3416.
Abbreviations: MWA-SPPS, microwave-assisted solid phase
1
5. Haskell-Luevano, C.; Lim, S.; Yuan, W.; Cone, R. D.; Hruby, V.
peptide
synthesis;
MBHA,
methylbenzhydryl;
MCR,
J. Peptides 2000, 21, 49-57.
16. Grieco, P.; Gitu, P. M.; Hruby, V. J. J. Pept. Res. 2001, 57, 250-6.
melanocortin receptors; NDP, [Nle4, DPhe7]-αMSH; MTII,
melanotan-II; RP-HPLC, reverse phase high performance liquid
chromatography;
triisopropylsilane, H
N,N,N′,N′-tetramethyluronium hexafluoro-phosphate; DIEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
DCM, dichloromethane; HATU, O-(7-azabenzotrizol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluoro-phosphate; PyBOP,
1
7. Grieco, P.; Han, G.; Weinberg, D.; Van der Ploeg, L. H.; Hruby,
V. J. Biochem. Biophys. Res. Commun. 2002, 292, 1075-80.
8. Grieco, P.; Balse-Srinivasan, P.; Han, G.; Weinberg, D.; MacNeil,
T.; Van der Ploeg, L. H.; Hruby, V. J. J. Pept. Res. 2003, 62, 199-
206.
TFA,
trifluoroacetic
acid;
TIS,
1
2
O, water; HBTU, O-(benzotriazol-1-yl)-
19. Vaz, E.; Dames, S. A.; Geyer, M.; Brunsveld, L. Org. Biomol.
Chem. 2012, 10, 1365-73.
2
0. Guo, H.; Gallazzi, F.; Miao, Y. Bioconjugate Chem. 2012, 23,
341-8.
1
(benzotriazol-1-yloxy)tripyrrolidinophospho-nium hexafluoro-
2
1. Yang, B.; Li, X.; Zhang, C.; Yan, S.; Wei, W.; Wang, X.; Deng,
X.; Qian, H.; Lin, H.; Huang, W. Org. Biomol. Chem. 2015, 13,
4551-61.
phosphate; HOBt, N-hydoxybenzotriazole; TES, triethylsilane;
NMP, N-methyl-2-pyrrolidone.
2
2
2. Lidstrom, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron
2
001, 57, 9225-9283.
3. Pedersen, S. L.; Tofteng, A. P.; Malik, L.; Jensen, K. J. Chem.
Soc. Rev. 2012, 41, 1826-44.