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Fmoc Solid-Phase Peptide Synthesis of Human alfa-Calcitonin Gene-Related Peptide and Two Fluorescent Analogs

Authors:
Marina Fuente-Moreno at Parc Sanitari Sant Joan de Déu
Marina Fuente-Moreno
A. Oddo
A. Oddo
A. Oddo
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Majid Sheykhzade at University of Copenhagen
Majid Sheykhzade
D.S. Pickering
D.S. Pickering
D.S. Pickering
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Proceedings of the 24th American Peptide Symposium
Ved Srivastava, Andrei Yudin, and Michal Lebl (Editors)
American Peptide Society, 2015 http://dx.doi.org/10.17952/24APS.2015.264
Fmoc Solid-Phase Peptide Synthesis of Human -Calcitonin
Gene-Related Peptide and Two Fluorescent Analogs
M. Fuente-Moreno1,2, A. Oddo1, M. Sheykhzade1, D.S. Pickering 1,
and P.R. Hansen1
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, DK-2100, Denmark;
2Facultad De Farmacia, Universidad Complutense De Madrid, Madrid, Spain
Introduction
Human -Calcitonin Gene-Related Peptide (h--CGRP) is a naturally occurring 37 amino acid
vasodilatory neuropeptide amide, ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF, with
a disulfide bond between residues 2 and 7. The peptide is found in primary afferent sensory nerves and
is widely distributed throughout the central and peripheral nervous systems in the body [1]. Structure
activity studies of h--CGRP have shown that the middle and C-terminal part of the peptide allow the
formation of the appropriate conformation required for the interaction with the receptor, while the
N-terminus is essential for biological activity and onset of signal [2]. Fluorescent h--CGRP analogs
are useful for investigating the mechanism behind (re)uptake of h--CGRP into the sensory nerve
terminals and monitoring trafficking of CGRP receptors. As part of an ongoing study on the
mechanism of action behind h--CGRP-induced vasodilation, we here present an Fmoc strategy for
the synthesis of [Cys2,7(Acm)] h--CGRP (1), h--CGRP (2), and two fluorescent h--CGRP analogs
labelled with 5-carboxyfluorescein [3] (5CF) at the side-chain of K24. The first analog, [Cys2,7(Acm),
5CFK24] h--CGRP (3) is linear, while the second [5CFK24] h--CGRP (4), contains the native
disulfide bond.
Results and Discussion
The peptides (1) and (2) were
synthesized using standard Fmoc
chemistry on a TentaGel RAM resin
(50 mg, loading 0.24 mmol/g)
(Figure 1). Activation of the Fmoc
amino acids was carried out using
HATU/HOAt/DIEA (4:4:8) [4].
Fig. 1. Strategy for the synthesis of compound (4).
Fmoc-Cys(Acm)-OH was used for residue 2 and 7. Fmoc deprotection was accomplished by treatment
with 20% piperidine in DMF (3x4 min) and final wash with DMF/DCM/DMF (3x/3x/5x). The peptides
were cleaved from the solid support along with the permanent side chain protection groups using
TFA/H2O/TIS (90:2.5:2.5 v/v) for 2 h. The crude peptides were purified by preparative HPLC and
characterized by MALDI-TOF-MS (Figure 2). The peptides (3) and (4) were synthesized as above
with the following modifications: Fmoc-Lys(ivDde)-OH was used at residue 24. Following SPPS, the
ivDde was cleaved by treatment with 2% hydrazine hydrate in DMF (12x5 min). This is significantly
longer than reported in the literature but a cleavage study using the model peptide Boc-A-F-S-
K(ivDde)-S-F-NH-Resin showed that it was necessary. After DMF wash, 5-carboxyfluorescein was
coupled overnight to the side-chain of K24 using HATU/HOAt/DIEA (5:5:10 eq). Following resin
cleavage, disulfide bond formation for compound 2 and 4 was achieved by dissolving the HPLC-
purified and Acm-protected peptides in I2/acetic acid (20mM) and 60 mM HCl [5]. MALDI-TOF-MS
indicated that the reaction was completed after 30 min. Next, 9 vol. eqv. of ice-cold ether was added
and cooled on dry ice for 10-15 min. The suspension was then centrifuged, decanted and purified by
RP-HPLC.
In conclusion, we present an Fmoc strategy for the syntheses of [Cys2,7(Acm)] h--CGRP (1), h--
CGRP (2), and two fluorescent h--CGRP analogs labeled with 5-carboxyfluorescein at the side-chain
of K24. The first analog, [Cys2,7(Acm), 5CFK24] h--CGRP (3) is linear, while the second [5CFK24]
h--CGRP (4), contained the native disulfide bond. However, the compounds were obtained in low
yields. Additional future work will include protocol optimization and performing binding and
functional studies.
Acknowledgments
Birgitte Simonsen is thanked for excellent technical assistance. This work was supported by an ERASMUS grant
to M. Fuente-Moreno.
References
1. Sheykhzade, M., et al. Eur. J. Pharmacol. 667, 375-382 (2011),
http://dx.doi.org/10.1016/j.ejphar.2011.06.031
2. Watkins, H.A., et al. British J. Pharmacol.170, 1308-1322 (2013), http://dx.doi.org/10.1111/bph.12072
3. Fischer, R., et al. Bioconjugate Chem. 14, 653-660 (2003), http://dx.doi.org/10.1021/bc025658b
4. Nielsen, S.L., et al. Protein Sci. 16, 1969-1976 (2007), http://dx.doi.org/10.1110/ps.072966007
5. Zhang, S., et al. Int. J. Pept. Res. Ther. 14, 301-305 (2008), http://dx.doi.org/10.1007/s10989-008-9148-x
Fig. 2. MALDI-TOF-MS of compound 4.
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