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Date: 30-03-15 12:53:37
Pages: 6
A. El-Faham, F. Albericio et al.
Z-Phg-Pro-NH2: A linear gradient of 25–50% CH3CN (0.1%
FULL PAPER
was used for data processing. LCMS was carried out with a
Shimadzu 2020 UFLC-MS (Kyoto, Japan) instrument using a TFA)/H2O (0.1% TFA) over 15 min was used, with a flow rate
YMCTriartC18 (5 μm, 4.6ϫ150 mm) column, and data processing of 1.0 mLmin–1, detection at 220 nm, and a Phenomex C18 (3 μm,
was carried out using the LabSolution software. Buffer A: H2O
(with 0.1% formic acid); and buffer B: CH3CN (with 0.1% formic
acid). High-resolution mass spectrometric data was obtained using
4.6ϫ50 mm) column. tR(ll)
tR(Z-Phg-OH) = 8.8 min.
= 6.4 min, tR(dl) = 6.8 min,
Z-Phe-Val-Pro-NH2: A linear gradient of 30–60% CH3CN (0.1%
TFA)/H2O (0.1% TFA) over 15 min was used, with a flow rate
of 1.0 mLmin–1, detection at 220 nm, and a Phenomex C18 (3 μm,
4.6ϫ50 mm) column. tR(lll) = 5.8 min, tR(ldl) = 6.9 min,
tR(Z-Phe-Val-OH) = 7.7 min.
a
Bruker micrOTOF-Q II instrument (Bremen, Germany)
operating at room temperature and a sample concentration of
approximately 1 ppm.
Synthesis of K-Oxyma-B (9; Potassium Salt of Oxyma-B): 1,3-Di-
methylbarbituric acid (15.6 g, 0.1 mol) was dissolved in water
(60 mL) containing KOH (8.4 g, 0.15 mol). Methanol (10 mL) was
added to the resulting clear solution, then sodium nitrite (7.6 g,
0.11 mol) was added, and the resulting mixture was stirred for
10 min. The mixture was cooled to 0 °C, then acetic acid (18 g,
0.3 mol) was added dropwise, and the mixture was kept at that
temperature for 2 h. The resulting precipitate was collected by fil-
tration, washed with a mixture of water and methanol (1:1) and,
after that, with water. The product was recrystallized from a mix-
ture of water and methanol (2:1) to give K-Oxyma-B (9) (16.7 g,
Acknowledgments
This work was funded in part by the National Research Founda-
tion (NRF) of the University of KwaZulu-Natal (South Africa)
and the Secretaria Nacional de Educación Superior, Ciencia, Tec-
nología e Innovación (SENESCYT), Ecuador, further by the
Centro de Investigación Científica
y Tecnológica (CICYT)
(CTQ2012-30930), the Generalitat de Catalunya (2014 SGR 137),
and the Institute for Research in Biomedicine, Barcelona (IRB Bar-
celona, Spain). Additionally, the authors thank the Deanship of
Scientific Research at King Saud University for partially funding
this work through research group No. RGP-234 (Saudi Arabia).
Finally, the authors thank Dr Karine Salim (Pcas) for the generous
gift of ChemMatrix resin, and Yoav Luxembourg (Luxembourg
Bio Technologies Ltd) for his continuous support of this study.
1
75.0%) as yellow-orange crystals, m.p. 228 °C (decomposition). H
NMR (400 MHz, [D6]DMSO): δ = 3.05 (s, 3 H, CH3), 3.21 (s, 3
H, CH3) ppm. 13C NMR ([D6]DMSO): δ = 26.8, 27.7, 141.2, 151.2,
151.4, 161.3 ppm. HRMS (ESI): calcd. for C6H6N3O4 [M]Ϫ
–
184.0358; found 184.0367.
Solid-Phase Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH2: The synthe-
sis was carried out in a plastic syringe, which was attached to a
vacuum manifold so as to effect rapid removal of reagents and
solvent. The Fmoc-RinkAmide-AM-PS resin (0.6 mmolg–1,
[1] F. Albericio, Curr. Opin. Chem. Biol. 2004, 8, 211–221.
[2] S.-Y. Han, Y.-A. Kim, Tetrahedron 2004, 60, 2447–2467.
[3] C. A. G. N. Montalbetti, V. Falque, Tetrahedron 2005, 61,
10827–10852.
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[6] J. C. Sheehan, G. P. Hess, J. Am. Chem. Soc. 1955, 77, 1067–
1068.
[7] J. P. Adams, C. M. Alder, I. Andrews, A. M. Bullion, M.
Campbell-Crawford, M. G. Darcy, J. D. Hayler, R. K. Hender-
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H. F. Sneddon, M. D. Walker, Green Chem. 2013, 15, 1542–
1549.
100 mg)
or
Fmoc-RinkAmide-AM-ChemMatrix
resin
(0.52 mmolg–1, 50 mg) was washed with DMF, CH2Cl2, and DMF
(2ϫ 10 mL each), and was then treated with piperidine (20% in
DMF; 10 mL) for 10 min. The resin was then washed with DMF,
CH2Cl2, and DMF (2ϫ 10 mL each), and then it was acylated with
a solution of Fmoc-Leu-OH (3 equiv.), the corresponding additive
(3 equiv.), and the corresponding carbodiimide (3 equiv.) in the
minimum amount of solvent (DMF, THF, or MeCN). After the
peptide coupling, the resin was washed with DMF, and was then
deblocked by treatment with piperidine (20% in DMF) for 7 min.
The resin was washed with DMF, CH2Cl2, and DMF (2ϫ 10 mL
each). Then coupling with the next amino acid and deblocking were
carried out as described above, and repeated to obtain the penta-
peptide. The peptide was cleaved from the resin by treatment with
TFA/H2O (9:1) at room temperature for 2 h. The TFA was removed
under a stream of nitrogen, and the crude peptide was purified by
washing with cold Et2O (3ϫ 10 mL), and then lyophilized. The
ratio of the penta- and tetrapeptides was determined by HPLC
analysis using a Phenomex C18 (3 μm, 4.6ϫ50 mm) column, with
a linear gradient of 20–40% of CH3CN (0.1% TFA)/H2O (0.1%
TFA) over 15 min, flow rate: 1.0 mLmin–1, detection at 220 nm.
The tR values for the pentapepide and des-Aib were 6.68 and
6.78 min, respectively. LC–MS showed the expected mass for the
pentapeptide at m/z = 611.0, and also for des-Aib at m/z = 526.
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26, 2525–2528.
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Chemistry of Functional Groups, John Wiley & Sons Ltd, Ho-
boken, NJ, USA, 2009.
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Chem. Soc., Chem. Commun. 1994, 201–203.
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Albericio, Chem. Eur. J. 2009, 15, 9394–9403.
[15] R. Subiros-Funosas, S. N. Khattab, L. Nieto-Rodriguez, A. El-
Faham, F. Albericio, Aldrichim. Acta 2013, 46, 21–40.
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General Method for the Racemization Experiments: Z-Phg-OH or
Z-Phe-Val-OH (0.125 mmol), H-Pro-NH2 (0.125 mmol), and the
corresponding additive (0.125 mmol) were dissolved in DMF
(2 mL), and the solution was cooled in an ice bath, and treated
with carbodiimide (DIC or EDC·HCl, 0.125 mmol). The mixture
was stirred at 0 °C for 1 h, and then at room temperature overnight.
Then, an aliquot (10 μL) of the solution was taken, and was diluted
to 1 mL with a mixture of CH3CN/H2O (1:2), and 5 μL of this
solution was injected into a reverse-phase HPLC apparatus.
[18] A. El-Faham, F. Albericio, Org. Lett. 2007, 9, 4475–4477.
[19] T. Bruckdorfer, O. Marder, F. Albericio, Curr. Pharm. Biotech-
nol. 2004, 5, 29–43.
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