Biomimetic formation of gramicidin S by dimerization-cyclization of pentapeptide precursor on solid support

Article abstract of DOI:10.1016/j.tetlet.2006.09.146

The biomimetic formation of gramicidin S, cyclo(-d-Phe-Pro-Val-Orn-Leu-)₂, by the dimerization and cyclization of pentapeptide precursor without the protection of δ-amino group of the Orn residue was examined on a solid support. The cyclization of H-d-Phe-Pro-Val-Orn-Leu-oxime on a resin with an oxime group of 0.62 mmol/g in 1,4-dioxane directly gave gramicidin S in a 50% yield. The dimerization-cyclization mode on the solid support was similar to that of the biosynthesis of gramicidin S on an enzyme.

Full text of DOI:10.1016/j.tetlet.2006.09.146

Tetrahedron Letters 47 (2006) 8475–8478
Biomimetic formation of gramicidin S by
dimerization–cyclization of pentapeptide precursor on solid support
Makoto Tamaki,* Kenji Honda, Sho Kikuchi and Rie Ishii
Department of Chemistry, Faculty of Science, Toho University, Miyama, Funabashi, Chiba 274-8510, Japan
Received 22 August 2006; revised 27 September 2006; accepted 28 September 2006
Available online 17 October 2006
Abstract—The biomimetic formation of gramicidin S, cyclo(-D-Phe-Pro-Val-Orn-Leu-)2, by the dimerization and cyclization of penta-
peptide precursor without the protection of d-amino group of the Orn residue was examined on a solid support. The cyclization of
H-D-Phe-Pro-Val-Orn-Leu-oxime on a resin with an oxime group of 0.62 mmol/g in 1,4-dioxane directly gave gramicidin S in a 50%
yield. The dimerization–cyclization mode on the solid support was similar to that of the biosynthesis of gramicidin S on an enzyme.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Gramicidin S (GS),1,2 cyclo(-D-Phe-Pro-Val-Orn-Leu-)2,
is an antibiotic cyclodecapeptide isolated from Bacillus
brevis ATCC9999. In Bacillus brevis, GS is produced
via the dimerization and cyclization of a pentapeptide
fragment (-D-Phe-Pro-Val-Orn-Leu-) having an Leu resi-
due at the C-terminus on GS synthetase.3
the Orn residue on a solid support. However, the linear
decapeptide (-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-
Orn-Leu-) as a precursor peptide was used. Hence the
mode of cyclization in the reported synthetic methods
is significantly different from that of the biosynthesis
of GS in Bacillus brevis.
In 1957, the synthesis of GS by the cyclization of the
pentapeptide precursor (H-Val-Orn(Tos)-Leu-D-Phe-
Pro-p-nitrophenyl ester) was reported by Schwyzer and
Sieber.4 Since then, various analogous of GS have been
synthesized by the dimerization and cyclization of penta-
peptide precursors having a D-Phe, Pro, Val, Orn(Z),
and Leu residue at the C-terminus.5 In these cycliza-
tions, p-nitrophenyl ester, N-hydroxysuccinimide ester,
and azide methods are most often used for the cycliza-
tion of precursor peptides.5 However, these methods
are multistep processes suffering from low yields and
racemization.6 On the other hand, in the preparations
of GS and its analogous using solid-phase peptide syn-
thesis, many difficult challenges lie in the C-terminus
activation and cyclization of the linear precursor on a
solid support.7 Recently, biomimetic syntheses of GS
and its derivatives on a solid support have been reported
by some research groups.8 In these reported synthetic
methods, the cyclizations of precursor peptides were car-
ried out without the protection of the d-amino group of
In this study, we would like to report the novel bio-
mimetic synthesis of GS via dimerization and cyclization
of pentapeptide precursor (-D-Phe-Pro-Val-Orn-Leu-)
without the protection of the d-amino group of the
Orn residue on a solid support.
Using Boc-solid phase peptide synthesis, a fully depro-
tected linear peptide oxime, H-D-Phe-Pro-Val-Orn-
Leu-oxime (1) mimicking the biosynthetic precursor of
GS was synthesized on a resin9 (loading of oxime group:
0.48 mmol/g resins). The formation of the cyclic pep-
tide by the cyclization-cleavage of oxime 1 on the resin
was performed in several solvents (1,4-dioxane, CCl4,
benzene, CH2Cl2, pyridine, ethanol, and DMF) with
2 equiv of N,N-diisopropylethylamine and AcOH for
1 day at 25 ꢁC (concentration of peptide in solution;
3 · 10À3 M).10 The yields and contents of GS and
semi-GS were determined by HPLC analysis (Table 1).
The cyclization of oxime 1 on the resin in CCl4 and benz-
ene mainly gave GS (cyclic dimer) in 15% and 19%
yields, respectively, and the production of semi-GS (cyc-
lic monomer) was not detected by HPLC analysis. On
the other hand, the cyclizations of oxime 1 on the resin
in other solvents afforded semi-GS and GS. The cycliza-
tion in 1,4-dioxane gave the best yield among several
Keywords: Gramicidin S; Biomimetic synthesis; Dimerization–cycliza-
tion on solid phase; Oxime resin; Reaction mechanism.
*
Corresponding author. Tel./fax: +81 47 472 4323; e-mail: tamaki@
chem.sci.toho-u.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.09.146

To investigate the mode of the cyclization of H-D-Phe-
8476
M. Tamaki et al. / Tetrahedron Letters 47 (2006) 8475–8478
Table 1. Solvent effect on the cyclization of H-D-Phe-Pro-Val-Orn-
Leu-oxime (1) on resin
Pro-Val-Orn-Leu-oxime (1) on the resin, the cyclizations
of several derivatives 2–4, Z-D-Phe-Pro-Val-Orn-Leu-
oxime (2), H-D-Phe-Pro-Val-Orn(Z)-Leu-oxime (3),
and H-(D-Phe-Pro-Val-Orn-Leu-)2-oxime (4), were per-
formed on the resin with oxime groups of 0.68 mmol/g
at 25 ꢁC for 1 day using 1,4-dioxane as a solvent.
Entry
Solvent
Ratio of
cyclic products
semi-GS:GS
Total yield (%) of
semi-GS and GS
1
2
3
4
5
6
7
CCl4
0:100
0:100
18:82
20:80
20:80
25:75
18:82
15
19
49
24
43
32
11
Benzene
1,4-Dioxane
CH2Cl2
Pyridine
DMF
The main products of each cyclization were confirmed
on HPLC analysis by direct comparison with authentic
samples.5e–g In the cyclization of oxime 2, cyclic dimer,
Ethanol
5f,h
Z-D-Phe-Pro-Val-Orn-Leu
Leu-Orn-Val-Pro-D-Phe-Z
in a 56% yield, but the produce of cyclic mono-
,
produced
5f,h
mer,
,
having
a
highly
Z-D-Phe-Pro-Val-Orn-Leu
solvents tried. The yields of semi-GS and GS were 9%
and 40%, respectively (Table 1). Thus, the polarities of
solvents significantly affect the cyclization mode of
oxime 1 on the resin.
strained nine-membered ring, could not be found on
HPLC. In addition, the cyclization of oxime 3 gave
diZ-GS (the benzyloxycarbonyl derivative of GS)5e
and Z-semi-GS5e in 53% and ꢀ3%, respectively. These
results suggested that in the intramolecular cyclization
of H-D-Phe-Pro-Val-Orn-Leu-oxime (1) on the resin,
the C-terminal oxime reacts very slowly with the a-ami-
no group of the D-Phe residue to give the semi-GS, but
does not react with the d-amino group of the Orn resi-
due under the condition used.
The cyclizations were carried out in different amounts of
1,4-dioxane (peptide concentration: 3 · 10À3 M and
0.6 · 10À3 M) at 25 ꢁC for 1 day. The amounts of reac-
tion solvents slightly affect the total yield and the ratios
of GS and semi-GS. This result indicates that the three
steps of dimerization, cyclization, and cleavage of pre-
cursor pentapeptide occur on the solid support.
Next, the cyclization of oxime
tions similar to those in the case of the reaction of
oxime on the resin gave GS and
in 56%
4 under condi-
Next, the effect of concentration of oxime 1 on the resin
for the formation of GS was examined on resins with
oxime of 0.48, 0.68, and 0.88 mmol/g. The ratios of
GS and semi-GS depended directly on the concentration
of oxime 1 on the resin. However, the use of the resin
with 0.88 mmol/g gave very low yields (7.5%) of cyclic
peptides. On the other hand, the use of the resin with
0.68 mmol/g gave GS and semi-GS with the best yield
among three resins tried in this study (Fig. 1). The yields
of semi-GS and GS were 7% and 50%, respectively.
Thus, the peptide concentration on the resin signifi-
cantly affects the yield of GS.
1
5f
H-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu
and 15% yields, respectively. These results indicated that
in the cyclization of decapeptide precursor on resin,
the C-terminal oxime reacts with both the a-amino
group of D-Phe residue and the d-amino group of
the Orn residue near the N-terminal. On the
other hand, the cyclization of oxime 1 on the resin
gave GS in
H-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu
a
50% yield, but the produce of
5f be slightly
found on HPLC (Fig. 1). These results indicated that in
the synthetic process of GS from H-D-Phe-Pro-Val-Orn-
Leu-oxime (1) on the resin, the formation of decapeptide
precursor, H-(D-Phe-Pro-Val-Orn-Leu-)2-oxime, as an
intermediate is for a moment.
On the basis of the above results, the mode of biomi-
metic synthesis of GS from H-D-Phe-Pro-Val-Orn-Leu-
oxime (1) on the resin is proposed as follows. When
GS is synthesized by the dimerization–cyclization of
H-D-Phe-Pro-Val-Orn-Leu-oxime (1) on the resin, the
two pentapeptide-oximes (1) on the solid support take
an anti-parallel type of conformation that brings the
N- and C-terminals into close proximity (Fig. 2). Next,
in an intermolecular reaction, the decapeptide-oxime
was formed by the coupling of the two H-D-Phe-Pro-
Val-Orn-Leu-oximes (1) on the resin and then cyclized
to give GS with a great facility. Finally, the head-to-tail
cyclization of the decapeptide precursor directly sepa-
rates GS from the solid support.
In summary, the present protocol provides a practical
method for the biomimetic synthesis of GS and its anal-
ogous from their fully deprotected pentapeptide precur-
sors oxime on a resin. It is interesting that both in
Figure 1. HPLC profile of crude product of the cyclization of peptide-
oxime 1 on resin with oxime group of 0.68 mmol/g in 1,4-dioxane. a = GS,
b = semi-GS, c =
.
H-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu

M. Tamaki et al. / Tetrahedron Letters 47 (2006) 8475–8478
8477
Figure 2. Biomimetic formation of gramicidin S by the dimerization–cyclization of H-D-Phe-Pro-Val-Orn-Leu-oxime (1) on resin.
Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D. Chem.
Rev. 1997, 97, 2661–2674.
4. Schwyzer, R.; Sieber, P. Helv. Chem. Acta. 1958, 41, 2186–
2189.
biological synthesis and in chemical synthesis on a solid
phase, the formations of the cyclic peptides are subjected
to a similar regiospecific control, although the condi-
tions of their cyclizations are different from each other.
This novel dimerization–cyclization method may be
applicable to the synthesis of other natural cyclic
peptides and its derivatives with a C2 symmetry.
5. (a) Izumiya, N.; Kato, T.; Aoyagi, H.; Waki, M.; Kondo,
M. Synthetic Aspects of Biologically Active Cyclic Peptide-
Gramicidin S and Tyrocidines; Kodansha Ltd. and Wiley:
Tokyo, 1979; (b) Minemutsu, Y.; Waki, M.; Suwa, K.;
Kato, T.; Izumiya, N. Tetrahedron Lett. 1980, 21, 2179–
2180; (c) Imazu, S.; Shimohigashi, Y.; Kodama, H.;
Sakaguchi, K.; Waki, M.; Kato, T.; Izumiya, N. Int. J.
Pept. Protein Res. 1988, 32, 298–306; (d) Tamaki, M.;
Takimoto, M.; Hayashi, M.; Muramatsu, I. Bull. Chem.
Soc. Jpn. 1989, 62, 594–596; (e) Tamaki, M.; Akabori, S.;
Muramatsu, I. Bull. Chem. Soc. Jpn. 1993, 66, 3113–3115;
(f) Tamaki, M.; Akabori, S.; Muramatsu, I. J. Am. Chem.
Soc. 1993, 115, 10492–10496; (g) Tamaki, M.; Komiya,
M.; Akabori, S.; Muramatsu, I. J. Chem. Soc., Perkin
Trans. 1 1997, 14, 2045–2050; (h) Tamaki, M.; Ishi, R.;
Kikuchi, S.; Watanabe, E. J. Antibiotics 2005, 58, 293–
295.
Acknowledgments
We are grateful to the staff of the Research Laboratories
of Asahi Chemical Industry Company (Ohito, Shizuoka,
Japan) for elemental analysis.
Supplementary data
Details of synthesis and cyclization of penta- and deca-
peptide-oxime on resin (1–4); HPLC profiles of the
crude products of biomimetic cyclization of penta- and
decapeptide-oxime on resin (1–4); CD, NMR and LR-
Mass (FAB) spectra of GS synthesized in preparative
scale on resin (13 pages). Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2006.09.146.
6. (a) Izumiya, N.; Kato, T.; Waki, M. Biopolymers 1981, 20,
1785–1791; (b) Ji, A.-Xm.; Bodansky, M. Int. J. Pept.
Protein Res. 1983, 22, 590–596.
7. (a) Klosteremyer, H. Chem. Ber. 1968, 101, 2823–2831; (b)
Losse, G.; Neubert, K. Tetrahedron Lett. 1970, 15, 1267–
1270; (c) Ohno, M.; Kuromizu, K.; Ogawa, H.; Izumiya,
N. J. Am. Chem. Soc. 1971, 93, 5251–5254; (d) Sato, K.;
Abe, H.; Kato, T.; Izumiya, N. Bull. Chem. Soc. Jpn. 1977,
50, 1999–2004; (e) Aimoto, S. Bull. Chem. Soc. Jpn. 1988,
¨
61, 2220–2222; (f) Osapy, G.; Profit, A.; Taylor, J. W.
Tetrahedron Lett. 1990, 31, 6121–6124; (g) Xu, M.;
Nishino, N.; Mihara, H.; Fujimoto, T.; Izumiya, N.
Chem. Lett. 1992, 191; (h) Wishart, D. S.; Kondejewski, L.
H.; Semchuk, P. D.; Hodges, R. S. Lett. Pep. Sci. 1996, 3,
References and notes
1. (a) Dubos, R. J. J. Exp. Med. 1939, 70, 1–10; (b) Gause,
G. F.; Brazhnikova, M. Nature 1944, 154, 703; (c)
Battersby, A. R.; Craig, L. C. J. Am. Chem. Soc. 1951,
73, 1887–1888.
2. Amino acid residues with no prefix have the L-configura-
tion. The abbreviations for amino acids and peptides are
in accordance with the rules of the IUPAC-IBU Nomen-
clature of Organic Chemistry; Rigaudy, J., Klesney, S. P.,
Eds.; Pergamon: Oxford, 1979.
3. (a) Kra¨tzschmar, J.; Krause, M.; Maraheil, M. A. J.
Bacteriol. 1989, 171, 5422–5429; (b) Kleinkauf, H.; von
Do¨hren, H. Eur. J. Biochem. 1990, 192, 1–15; (c) Turgay,
K.; Krause, M.; Marahaeil, M. A. Mol. Micriobiol. 1992,
6, 529–546; (d) Kleinkauf, H.; von Do¨hren, H. Eur. J.
Biochem. 1996, 236, 335–351; (e) von Do¨hren; Keller, U.;
Vater, J.; Zocher, R. Chem. Rev. 1997, 97, 2675–2706; (f)
53; (i) Andreu, D.; Ruiz, S.; Carreno, C.; Alsina, J.;
˜
´
Albericio, F.; Jimenez, J. A.; Figuera, N.; Herranz, R.;
´
´
´
Garcia-Lopez, M. T.; Gonzalez-Muniz, R. J. Am. Chem.
Soc. 1997, 119, 10579–10586; (j) Arai, T.; Maruo, N.;
Sumida, Y.; Korosue, C.; Nishino, N. Chem. Commun.
1999, 16, 1503–1504; (k) Qin, C.; Bu, X.; Wu, X.; Guo, Z.
J. Comb. Chem. 2003, 5, 353; (l) Bu, X.; Wu, X.; Wong, K.
M.; Ng, N. L.; Mak, C. K.; Qin, C.; Guo, Z. J. Org. Chem.
2004, 69, 2681–2685.
8. (a) Trauger, J. W.; Kohi, R. M.; Mootz, H. D.; Marahiel,
M. A.; Walsh, C. T. Nature 2000, 407, 215–218; (b)
Trauger, J. W.; Kohi, R. M.; Walsh, C. T. Biochemistry
2001, 40, 7092–7098; (c) Kohi, R. M.; Trauger, J. W.;
Schwazer, D.; Marahiel, M. A.; Walsh, C. T. Biochemistry
2001, 40, 7099–7108; (d) Kohi, R. M.; Walsh, C. T.;

8478
M. Tamaki et al. / Tetrahedron Letters 47 (2006) 8475–8478
Burkart, M. D. Nature 2002, 418, 658–661; (e) Bu, X.; Wu,
E. T. J. Org. Chem. 1983, 48, 678–685; (d) Nakagawa, S.
H.; Kaiser, E. T. J. Am. Chem. Soc. 1985, 107, 7087–7092;
(e) Mihara, H.; Kaiser, E. T. Science 1988, 242, 925–927;
(f) Sasaki, T.; Findeis, M. A.; Kaiser, E. T. J. Org. Chem.
1991, 56, 3159–3168.
X.; Bu, X.; Xie, G.; Guo, Z. Org. Lett. 2002, 4, 2893–2895;
(f) Wu, X.; Bu, X.; Wong, K. M.; Yan, W.; Guo, Z. Org.
Lett. 2003, 5, 1749–1752; (g) Lin, H.; Walsh, C. T. J. Am.
Chem. Soc. 2004, 126, 13998–14003.
9. (a) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1980, 45,
1295–1300; (b) DeGrado, W. F.; Kaiser, E. T. J. Org.
Chem. 1982, 47, 3258–3261; (c) Nakagawa, S. H.; Kaiser,
10. (a) Gisin, B. F.; Merrifield, R. B. J. Am. Chem. Soc. 1972,
¨
94, 3102–3106; (b) Osapay, G.; Taylor, J. W. J. Am. Chem.
Soc. 1990, 112, 6046–6051.