Solid-phase total synthesis and structure proof of callipeltin B

Article abstract of DOI:10.1021/ja0666250

The cytotoxic, cyclic heptadepsipeptide, natural product callipeltin B was synthesized on a solid-phase support in 15% overall yield. Comparison of the ¹H NMR spectra of three synthetic isomers with those of callipeltin B confirmed the configurational reassignment of its threonine residues as d-allothreonine and the assignment of the configuration of its β-methoxytyrosine residue as (2R,3R). Copyright

Full text of DOI:10.1021/ja0666250

Published on Web 11/09/2006
Solid-Phase Total Synthesis and Structure Proof of Callipeltin B
Ravi Krishnamoorthy, Leslie D. Vazquez-Serrano,† Jeffrey A. Turk,† Jennifer A. Kowalski,†
Alan G. Benson,† Nneka T. Breaux,† and Mark A. Lipton*
Department of Chemistry and Cancer Center, Purdue UniVersity, 560 OVal DriVe,
West Lafayette, Indiana 47907-2084
Received September 13, 2006; E-mail: lipton@purdue.edu
The cyclic depsipeptide callipeltin B (1) was isolated from the
Lithistid sponge callipelta sp. and structurally characterized by
Minale and co-workers in 1996.1 Along with the related cyclic
depsipeptide callipeltin A,2 1 possesses a 22-membered macrolac-
tone composed of the unique, non-proteinogenic amino acids
â-methoxytyrosine (â-MeOTyr) and (3S,4R)-3,4-dimethyl-L-pyro-
glutamic acid (DiMePyroGlu) as well as several D- and N-
methylated amino acids. Both callipeltins A and B show broad-
spectrum cytotoxicity against a number of tumor cell lines, including
several drug-resistant cell lines,1 and callipeltin A shows potent
anti-HIV activity.2
Although the initially published structure of callipeltin B (1a)
has never been revised, revisions to the structure of the closely
related cyclic depsipeptide callipeltin A have strongly implied a
need for a structural revision of 1. Specifically, D’Auria and co-
workers, after isolating and characterizing two smaller, linear
peptidic fragments of callipeltin A that they termed callipeltins D
and E, suggested that one of the residues initially identified as
L-threonine should be reassigned as D-allothreonine.3 Such a change
thus implied that the structure of callipeltin B should be reassigned
as 1b, in which the configuration of C-21 is R.
implied that the structure of callipeltin B should be further revised
to 1c, in which the configuration of C-18 has been changed, and
those of C-1′ and C-6 are assigned as R. Herein we report the
synthesis of 1a-c and the confirmation of 1c as the structure of
1
callipeltin B by H NMR correlation.
A solid-phase synthetic strategy was chosen to expedite the
synthesis of 1, its analogues, and related depsipeptides. To permit
macrocyclization on a solid support, it was decided to anchor the
side chain of the N-methylglutamine residue to a peptide amide
synthesis resin, thereby leaving available both N- and C-termini
for macrocyclization by amide bond formation. The Tentagel-based
TG Sieber amide resin7 (2) was chosen for the relatively low
amounts of TFA (1-5% in CH2Cl2) needed for efficient cleavage
of the peptide, thereby minimizing the possibility of acid-catalyzed
â-MeOTyr decomposition during cleavage from the resin. A
standard Fmoc-based, C-to-N peptide synthesis approach thus
required the use of allyl-based protecting groups for N- and
C-termini immediately prior to macrocyclization. All side-chain
protecting groups were selected for their ability to be removed either
during the mildly acidic cleavage from the Sieber resin or a
subsequent catalytic hydrogenation.
The initial plan to close the macrocycle at the MeGln/â-MeOTyr
peptide bond, permitting a C-to-N synthesis from the MeGln
residue, had to be abandoned because of spontaneous diketopip-
erazine formation upon deprotection of the Leu-MeGln dipeptide;
instead, it was decided to close the macrocycle at the â-MeOTyr/
N-methylalanine peptide bond, despite the known problem of slower
acylation of secondary amines. Additionally, an initial plan to install
the N-terminal DiMePyroGlu after macrocyclization was revised
because of rapid O-to-N transacylation upon deprotection of the
N-terminal Fmoc group. As a result, it was decided to install the
DiMePyroGlu residue prior to forming the ester bond to prevent
transacylation.
All of the constituent residues needed to make 1a-c were either
commercially available or had been previously synthesized. In
addition to D’Auria’s synthesis of â-MeOTyr,5 several others had
been published;8 several different syntheses of DiMePyroGlu had
also been reported.9 The syntheses of 1a-c began with the
deprotection and acylation of 2 (Scheme 1) with the cyclic
anhydride of Fmoc-N-methylglutamic acid (3),10 followed by
activation of the resin-bound acid with HATU and coupling with
(2R,3R)-O-benzyl-â-methoxytyrosine allyl ester (4)8c to afford a
resin-bound dipeptide. All deprotection and coupling reactions were
monitored by removal of an aliquot of 1-2 mg of resin, cleavage
of the peptide from the resin, and analysis of the crude cleavage
More recently, Bifulco and D’Auria have published computa-
tional studies that suggest that both residues in callipeltin A initially
assigned as L-threonine by Minale and co-workers are actually
D-allothreonine.4 At the same time, the configuration of the
â-MeOTyr residue of callipeltin A, which could not be determined
in Minale’s original structural study, was determined to be (2R,3R)
by D’Auria and co-workers, using a combination of synthesis and
degradation studies.5 Both of these structural reassignments were
confirmed in our laboratory by the synthesis and spectral correlation
of two diastereomers of callipeltin E.6 Taken together, these studies
† Present addresses: (L.D.V.S.) Owens Corning Composite Solutions, Granville,
OH 43023; (J.A.T.) International Flavors and Fragrances, Union Beach, NJ
07735; (J.A.K.) Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT 06877;
(A.G.B.) Mallinckrodt, St. Louis, MO 63134; (N.T.B.) Dow Agrosciences,
Indianapolis, IN 46268.
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10.1021/ja0666250 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
exchangeable amide protons in CD3OD.12 On the other hand, the
1H NMR spectra of 1a and 1b showed considerable differences
from those of callipeltin B; in fact, the spectra of 1a and 1b showed
evidence of conformational heterogeneity at ambient temperature.
On the basis of these comparisons, we now conclude that the correct
structure of callipeltin B is 1c. Current studies are underway to
synthesize analogues of 1c and several related cyclic depsipeptides
using the methodology described herein.
Scheme 1. Solid-Phase Synthesis of 1c
In summary, the synthesis of the cyclic depsipeptide natural
product, callipeltin B, has been accomplished, thereby providing
confirmation of the recent configurational reassignment of the
previously assigned L-threonine residues as D-allothreonine and the
recent configurational assignment of the â-methoxytyrosine residue
as (2R,3R). This synthesis illustrates the facility with which even
complex natural products can be synthesized on solid supports. In
addition, the expeditious solid-phase synthesis of 1 opens the door
for the rapid synthesis of analogues to explore structure-function
relationships and for the synthesis of other, more complex, cyclic
depsipeptides.
Acknowledgment. We thank Prof. Maria Valeria D’Auria and
Dr. Kirk Gustafson for supplying spectral information and for
helpful discussions. We also thank Selc¸uk C¸ alimsiz for his
assistance and are grateful to both him and Minoo Sedighi for their
synthesis of dimethylpyroglutamic acid for this project. We
gratefully acknowledge the National Institutes of Health (AI-50888)
for financial support of this work.
mixture by reverse-phase HPLC (RP-HPLC) and MALDI-MS. It
was found that use of HATU with no added HOAt afforded
complete consumption of starting material with no epimerization
evident by HPLC analysis. Sequential installation of Fmoc-Leu,
Fmoc-D-Arg(NO2), Fmoc-D-aThr(THP), and Fmoc-D-aThr using
coupling conditions optimized for each step proceeded without
incident to afford the resin-bound hexapeptide 5 in >95% purity
as determined by HPLC analysis. For the syntheses of 1a and 1b,
protected L-threonine was substituted in this sequence for the
second, or both, D-allothreonine residues. Additionally, in the
syntheses of 1a and 1b, Fmoc-D-Arg(Z,Z) was substituted for Fmoc-
D-Arg(NO2).
Supporting Information Available: Complete experimental details
and spectroscopic details for 1 and the novel protected amino acids
1
used in its synthesis; a tabulated comparison of H NMR spectra of 1
and callipeltin B and reproduced spectra of both. This information is
available free of charge via the Internet at http://pubs.acs.org.
References
Deprotection of the N-terminal Fmoc afforded a â-amino alcohol
that could be selectively N-acylated with the N-hydroxysuccinimide
ester of DiMePyroGlu (6). Esterification of the hydroxyl with alloc-
N-methylalanine using the sulfonyl nitrotriazole reagent MSNT11
in conjunction with N-methylimidazole afforded a resin-bound,
protected heptadepsipeptide in high purity. Palladium-catalyzed
deprotection of N- and C-termini followed by macrolactamization
using PyAOP to minimize epimerization of the activated â-meth-
oxytyrosine afforded the resin-bound macrocycle in roughly 80%
purity as judged by HPLC analysis of the crude deprotection
mixture. Removal of the cyclized product from the resin with 2%
TFA/CH2Cl2 and complete deprotection by catalytic transfer
hydrogenation afforded 1c, after purification by RP-HPLC, in 15%
overall yield. The isomeric depsipeptides 1a and 1b were obtained
in 15% and 14% overall yields, respectively.
(1) D’Auria, M. V.; Zampella, A.; Gomez-Paloma, L.; Minale, L. Tetrahedron
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(10) The regioselectivity of opening of 3 was established by cleavage of the
acylation product from the resin and HPLC correlation with authentic
samples of Fmoc-NR-methylglutamine and the C-terminal carboxamide
of Fmoc-NR-methylglutamic acid. Exclusive attack on the less hindered
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(12) The only differences outside of the amide proton region noted in the
comparison were three singlets present in the 1H NMR of the natural
product (at δ ∼2.08, 2.1, and 2.2) that were not found in the spectrum of
1c, nor were they included in the tabulated data in ref 1; additionally, a
Because of the unavailability of a sample of natural callipeltin
B, the correlation of synthetic 1a-c with callipeltin B involved
the comparison of spectroscopic data. Comparison of the 1H NMR
spectra of 1c with those of callipeltin B showed no significant
differences between the two, apart from the absence of several
1
singlet at δ 2.85 was found in the H NMR of 1c that didn’t correspond
to any resonance in callipeltin B and might possibly have resulted from
a small amount of DMF present in the sample.
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