Macrolactonization of peptide thioesters catalyzed by imidazole and its application in the synthesis of kahalalide B and analogues

Article abstract of DOI:10.1021/ol100596p

The macrolactonization of peptide thioester to yield cyclic depsipeptides was developed using imidazole as a catalyst. This strategy was applied to the synthesis of kahalalide B and its analogues.

Full text of DOI:10.1021/ol100596p

ORGANIC
LETTERS
2010
Vol. 12, No. 10
2250-2253
Macrolactonization of Peptide Thioesters
Catalyzed by Imidazole and Its
Application in the Synthesis of
Kahalalide B and Analogues
Yangmei Li, Marc Giulionatti, and Richard A. Houghten*
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St.
Lucie, Florida 34987 and 3550 General Atomics Court, San Diego, California 92121
houghten@tpims.org
Received March 11, 2010
ABSTRACT
The macrolactonization of peptide thioester to yield cyclic depsipeptides was developed using imidazole as a catalyst. This strategy was
applied to the synthesis of kahalalide B and its analogues.
Bioactive cyclic peptides and depsipeptides that are isolated
from natural sources provide a range of lead structures for
the design of new drugs.1 Kahalalide compounds, A-F, are
cyclic depsipeptides isolated from a sacoglossan mollusk,
Elysia rufescens, and its algal diet Bryopsis sp. They range
from a C31 tetrapeptide to a C75 octapeptide.2 Kahalalide
compounds have similar structures with an endocyclic
depsipeptide bond and fatty acids at the N-termini. Kahala-
lides exhibit a diverse spectrum of biological activities,
including antiviral, antimicrobial, and antitumor activity.3
(1) (a) Xiao, Q.; Pei, D. J. Med. Chem. 2007, 50, 3132. (b) Qin, C.; Bu,
X.; Zhong, X.; Ng, N. L. J.; Guo, Z. J. Comb. Chem. 2004, 6, 398. (c)
Mahlert, C.; Sieber, S. A.; Gru¨newald, J.; Marahiel, M. A. J. Am. Chem.
Soc. 2005, 127, 9571. (d) Liu, S.; Gu, W.; Lo, D.; Ding, X.-Z.; Ujiki, M.;
Adrian, T. E.; Soff, G. A.; Silivermann, R. B. J. Med. Chem. 2005, 48,
3630. (e) Gulavita, N. K.; Wright, A. E.; McCarthy, P. J.; Pomponi, S. A.;
Longley, R. E. J. Nat. Toxins 1996, 5, 225.
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6594.
10.1021/ol100596p  2010 American Chemical Society
Published on Web 04/28/2010

Though the pharmaceutical potential of these natural com-
pounds is high, isolation and purification of larger quantities
of this class of products is difficult. The development of
synthetic strategies for natural compounds and their ana-
logues is essential for the discovery of lead compounds of
this type for use in drug discovery.4
enzymes hydrolyzing the ester bond and the thioester
bond.9 Our recent studies have shown that a cyclic peptide
can be formed through direct aminolysis of a peptide
benzylthioester catalyzed by imidazole.10 The generally
accepted mechanism of the imidazole catalysis involves
the reaction intermediates of acyl imidazole and the acyl
imidazolium cation, which are formed by the direct attack
of imidazole on the carbonyl group. This mechanism can
also be envisioned for the formation of a cyclic depsipep-
tide by macrolactonization. The mild imidazole-catalytic
condition may also eliminate the risk of racemization
during acyl activation. Herein, we report a imidazole-
catalytic approach for the synthesis of cyclic depsipeptides
by macrolactonization of peptide thioesters and the use
of this application in the synthesis of kahalalide B and
its analogues.
The most commonly used methodology for the synthesis
of cyclic depsipeptide involes cyclization of a linear
depsipeptide containing an internal ester bond. The
intramolecular macrolactamization can be carried out
either in solution or on resin.5 Problematically, the
endoester bond may break during the acidic cleavage when
a typical solid-phase synthetic strategy is applied to make
the linear precursor and/or cyclic product. The choice of
resins is thus limited, and some of the widely used resins,
which require strong acidic cleavage conditions, cannot
be successfully used. To overcome this limitation, mac-
rolactonization is a favorable alternative. Since the lac-
tonization is much less efficient than the lactamization
under conventional coupling conditions, synthesis of cyclic
depsipeptides by this strategy is rarely reported and
remains a challenge.6
Biosynthesis of cyclic depsipeptides through peptide
synthetase has been reported by using peptide thioester
as substrate.7 Studies of different peptide synthetase
systems suggest the possibility that a histidine residue
functions as a catalyst of condensation/elongation/cycliza-
tion reaction in the peptide synthesis.8 Imidazole has been
reported as a catalyst mimicking the histidine residues of
The stepwise synthesis of the linear peptides starts with
functionalized mercaptomethylphenyl silica gel 1 as the
“volatilizable” support (Scheme 1).11 PyBOP/DIEA was
used as the protected amino acid activation reagent to
generate resin bound 2. Boc-glycine was coupled on the
resin as the C-terminal residue based on the structure of
kahalalide B. After removal of the Boc group with 55%
TFA, threonine, proline, D-leucine, phenylalanine, D-
serine, tyrosine, and 5-methylhexanoic acid were coupled
stepwise to form the on-resin synthetic precursor of
kahalalide B 3a. The resin-bound 3a was then treated with
anhydrous HF for 2 h at 0 °C. Following evaporation of
the anhydrous HF with a gaseous nitrogen stream, the
unprotected peptide thioester 4a was obtained following
lyophlization.
(3) (a) Rawat, D. S.; Joshi, M. C.; Joshi, P.; Atheaya, H. Anticancer
Agents Med. Chem. 2006, 6, 33. (b) Cruz, L. J.; Luque-Ortega, J. R.; Rivas,
L.; Albericio, F. Mol. Pharm. 2009, 6, 813. (c) Hamann, M. T. Curr. Opin.
Mol. Ther. 2004, 6, 657. (d) Garc´ıa-Rocha, M.; Bonay, P.; Avila, J. Cancer
Lett. 1996, 99, 43. (e) Copp, B. R. Nat. Prod. Rep. 2003, 6, 535.
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Hamann, M. T.; Ganesan, A. J. Med. Chem. 2005, 48, 1330. (c) Jime´nez,
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J. C.; Chavarr´ıa, B.; Lo´pez-Macia`, A.; Royo, M.; Giralt, E.; Albericio, F.
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Giralt, E.; Albericio, F. J. Am. Chem. Soc. 2001, 123, 11398. (e) Gracia,
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C.; Isidro-Llobet, A.; Cruz, L. J.; Acosta, G. A.; Alvarez, M.; Cuevas, C.;
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Waldmann, H. Chem.sEur. J. 2008, 14, 8847. (c) Bowers, A.; West, N.;
Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R. M. J. Am. Chem.
Soc. 2008, 130, 11219. (d) Xie, W.; Ding, D.; Zi, W.; Li, G.; Ma, D. Angew.
Chem., Int. Ed. 2008, 47, 2844. (e) Tan, L.; Ma, D. Angew. Chem., Int. Ed.
2008, 47, 3614. (f) Ruiz-Rodr´ıguez, J.; Spengler, J.; Albericio, F. Angew.
Chem., Int. Ed 2009, 48, 1. (G) Wen, S.; Packham, G.; Ganesan, A J. Org.
Chem. 2008, 73, 9353. (h) Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2,
3743.
The cyclization to the depsipeptide was performed by
macrolactonization in acetonitrile using imidazole as a
catalyst. The effect of imidazole on catalytic esterification
and cyclization was first tested using an N-acetyl pen-
tapeptide thioester Ac-Xxx-Ala-Phe-Tyr-Gly-SCH2Ph, where
Xxx was Ser or Thr. It was found the concentration of
(6) (a) Li, K. W.; Wu, J.; Xing, W.; Simon, J. A. J. Am. Chem. Soc.
1996, 118, 7237. (b) Sarabia, F.; Garc´ıa-Castro, M.; Chammaa, S.
Tetrahedron Lett. 2005, 46, 7695.
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Walsh, C. T.; Sieber, S. A. Biochemistry 2002, 41, 13350. (b) Grnewald,
J.; Sieber, S. A.; Marahiel, M. A. Biochemistry 2004, 43, 2915. (c) Mahlert,
C.; Sieber, S. A.; Grnewald, J.; Marahiel, M. A. J. Am. Chem. Soc. 2005,
127, 9571.
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(b) Bruice, T. C.; Schmir, G. L. J. Am. Chem. Soc. 1958, 80, 148. (c) Bruice,
T. C.; Schmir, G. L. J. Am. Chem. Soc. 1958, 80, 2265. (d) Kirsch, J. F.;
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97, 2651. (c) Sieber, S.; Tao, J.; Walsh, C. T.; Marahiel, M. A. Angew.
Chem., Int. Ed. 2004, 43, 493. (d) Trauger, J. W.; Kohli, R. M.; Mootz,
H. D.; Marahiel, M. A.; Walsh, C. T. Nature 2000, 407, 215. (e) Kohli,
R. M.; Trauger, J. W.; Schwarzer, D.; Marahiel, M. A.; Walsh, C. T.
Biochemistry 2001, 40, 7099. (f) Trauger, J. W.; Kohli, R. M.; Walsh, C. T.
Biochemistry 2001, 40, 7092. (g) Stachelhaus, T.; Mootz, H. D.; Bergendahl,
V.; Marahieli, M. A. J. Bio. Chem. 1998, 273, 22773.
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Org. Lett., Vol. 12, No. 10, 2010
2251

Scheme 1
.
Synthesis of Kahalalide B and Its Analogues through
Scheme 2
.
Mechanism of Imidazole Catalysis of
Macrolactonization
Macrolactonization of Peptide Thioester Catalyzed by Imidazole
Because there are also hydroxyl groups on the residue of
serine or threonine at position aa1 and tyrosine at position
aa5, the cyclic product identified in LC-MS analysis could
be either the expected kahalalide B or the esterification
product of C-terminal thioester by the hydroxyl groups of
these residues. To distinguish these possible products,
semipreparative reverse-phase HPLC was used to purify the
cyclic product. The purified product was analyzed by NMR
spectroscopy and found to be identical to the natural
kahalalide B reported earlier.12 In addition, linear peptides
CH3CO-Tyr-Ala-Phe-Tyr-Gly-SCH2Ph, CH3CO-Ser-Gly-
SCH2Ph, and CH3CO-Thr-Gly-SCH2Ph were also synthe-
sized, and cyclizations were attempted under the same
conditions as the cyclization of kahalalide B. No cyclization
products were detected in the three cases even after reacting
for 3 days.
imidazole significantly accelerated the macrolactonization.
Macrolactonization by the hydroxyl on the serine residue
was complete after a 24 h reaction at room temperature
gave a yield over 95% when the concentration of
imidazole was 1.5 M, but failed even after reacting for
10 days when the concentration was 0.15 M. The effect
of the concentration of imidazole on macrolactonization
by peptide threonine had a similar result but a lower yield
of 70% after reacting for 24 h. The need for a high
concentration of imidazole likely suggests that the forma-
tion of the imidazolyl intermediate was rate-limiting
(Scheme 2). Increasing the concentration of imidazole thus
drives the formation of the imidazolyl intermediate. The
formation of kahalalide B (5a) was tested by dissolving
4a to a concentration of 1 mM in 1.5 M imidazole in
acetonitrile at room temperature for 24 h. A portion of
the reaction mixture was examined by LC-MS. It was
found that the cyclic depsipeptide of kahalalide B was
quantitatively formed. When compared to the reported
strategy of PyBOP-DIEA activation for macrolactoniza-
tion, the yield of cyclization of the same compound is
much higher using the present experimental approach
(reported, 28%; current, quantitative).12 To accecelarate
the cyclization, microwave reaction was tested under the
same concentrations but at 65 °C in a separate experiment.
It was found that the cyclization was complete within 2 h
with a quantitative conversion.
Analogues of kahalalide B (5b-e) were synthesized by
selectively changing residues at positions aa1, aa2, aa3, aa4,
and aa5. Residues of glycine and proline were reserved in
the cyclic product. The yield and purity of kahalalide B and
its analogues are shown in Table 1.
Table 1. Kahalalide B and Analogues Synthesized by
Macrolactonization
linear peptide thioesters
yield yield MW MW
entry (%)a (%)b (calcd)c (obsd)d (%)e (calcd)c (obsd)d
cyclic products
yield MW MW
a
b
c
d
e
90
92
90
94
90
42
40
57
51
46
1002.5 1002.5
1004.5 1004.5
98
90
85
80
95
878.5
880.4
806.4
836.4
892.5
878.5
880.4
806.4
836.4
892.5
930.4
960.5
930.4
960.5
1016.5 1016.5
a Yield is based on the weight of crude product and the amount of resin
used. b Yield is based on the weight of purified product (purity >95%) and
d
the amount of resin used. c Caclulated from [M + H]. [M + H]+ by
ESI-MS. e Yield is based on the weights of purified cyclic products and
their purified linear peptide thioesters.
In summary, we present here a novel method for the
synthesis of cyclic depsipeptides by macrolactonization of
`
(12) Lo´pez-Macia`, A.; Jime´nez, J. C.; Royo, M.; Giralt, E.; Albericio,
F. Tetrahedron Lett. 2000, 41, 9765.
2252
Org. Lett., Vol. 12, No. 10, 2010

peptide thioesters. The esterifaction/cycliczation was greatly
faciliated with catalysis by imidazole. Kahalalide B and its
analogues were obtained in high yields and purities by this
method. This strategy is promising for the synthesis of other
natural cyclic depsipeptides.
ence Foundation (RAH CHE 0455072), 1P41GM079590,
1P41GM081261, and U54HG03916-MLSCN.
Supporting Information Available: Experimental detail,
1
compound characterization, copies of H NMR, 13C NMR
spectra, HRMS,and LC-MS for cyclic compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. This work was supported by the State
of Florida, Executive Officer of the Governor’s Office of
Tourism, Trade and Economic Development, National Sci-
OL100596P
Org. Lett., Vol. 12, No. 10, 2010
2253