Total synthesis and stereochemical reassignment of bisebromoamide

Article abstract of DOI:10.1021/ol101021v

A revised configurational assignment for the thiazoline moiety of the marine peptide bisebromoamide is proposed and validated by total synthesis.

Full text of DOI:10.1021/ol101021v

ORGANIC
LETTERS
2010
Vol. 12, No. 13
3018-3021
Total Synthesis and Stereochemical
Reassignment of Bisebromoamide
Xuguang Gao,† Yuqing Liu,‡ Shuqin Kwong,‡ Zhengshuang Xu,*,†,‡ and
Tao Ye*,†,‡
Laboratory of Chemical Genomics, Peking UniVersity Shenzhen Graduate School,
UniVersity Town of Shenzhen, Xili, Nanshan District, Shenzhen, China, 518055, and
Department of Applied Biology & Chemical Technology, The Hong Kong Polytechnic
UniVersity, Hung Hom, Kowloon, Hong Kong, China
bctaoye@inet.polyu.edu.hk; xuzs@szpku.edu.cn
Received May 4, 2010
ABSTRACT
A revised configurational assignment for the thiazoline moiety of the marine peptide bisebromoamide is proposed and validated by total
synthesis.
Marine cyanobacteria have produced a wide variety of natural
products, many of which have shown striking biological
activity.1 Consequently, they are key synthetic targets in the
quest for new leads in the pharmaceutical industry. One
particular species, Lyngbya, is responsible for the production
of many varied metabolites, the biological activity of which
ranges from anticancer and antiviral to antifungal activity.
Unusual effects include immunosuppression and antifeedant
properties.1d-f These compounds have potential applications
as therapeutic agents, and this has driven our group to
synthesize a range of these metabolites, with the aim of
structure modification for fine-tuning biological activity. We
have been interested for some time in marine secondary
metabolites and view their syntheses as a key route to
structural confirmation, structural modification, and subse-
quent activity control.2 Here we report the first total synthesis
and revised stereochemical assignment of bisebromoamide.
Bisebromoamide is a thiazoline-containing marine peptide
isolated by Suenaga and co-workers3 in 2009 from the marine
cyanobacterium Lyngbya sp. harvested in the Okinawa
(2) (a) Jin, Y.; Liu, Y. Q.; Wang, Z.; Kwong, S.; Xu, Z. S.; Ye, T. Org.
Lett. 2010, 12, 1100–1103. (b) Liang, S.; Xu, Z. S.; Ye, T Chem. Commun.
2010, 46, 153–155. (c) Chen, B.; Dai, L.; Zhang, H.; Tan, W.; Xu, Z. S.;
Ye, T. Chem. Commun. 2010, 46, 574–576. (d) Li, S.; Liang, S.; Tan, W. F.;
Xu, Z. S.; Ye, T. Tetrahedron 2009, 65, 2695–2702. (e) Li, S.; Liang, S.;
Xu, Z. S.; Ye, T. Synlett 2008, 569–574. (f) Ren, Q.; Dai, L.; Zhang, H.;
Tan, W.; Xu, Z. S.; Ye, T. Synlett 2008, 2379–2383. (g) Chen, Z. Y.; Ye,
T. New J. Chem. 2006, 30, 518–520. (h) Pang, H. W.; Xu, Z. S.; Chen,
Z. Y.; Ye, T. Lett. Org. Chem. 2005, 2, 699–702. (i) Pang, H. W.; Xu,
Z. S.; Ye, T. Lett. Org. Chem. 2005, 2, 703–706. (j) Chen, H. L.; Xu, Z. S.;
Ye, T. Tetrahedron 2005, 61, 11132–11140. (k) Chen, Z. Y.; Deng, J. G.;
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† Peking University Shenzhen Graduate School.
‡ The Hong Kong Polytechnic University.
(1) (a) Jones, A. C.; Gu, L.; Sorrels, C. M.; Sherman, D. H.; Gerwick,
W. H. Curr. Opin. Chem. Biol. 2009, 13, 216–223. (b) Gademann, K.;
Portmann, C. Curr. Org. Chem. 2008, 12, 326–341. (c) Sivonen, K.; Borner,
T. In The cyanobacteria: Molecular Biology, Genomics and EVolution;
Herrero, A., Flores, E., Eds.; Caister Academic Press: Norfolk, U. K., 2008;
pp 159-197. (d) Tan, L. T. Phytochemistry 2007, 68, 954–979. (e) Luesch,
H.; Harrigan, G. G.; Goetz, G.; Horgen, F. D. Curr. Med. Chem. 2002, 9,
1791–1806. (f) Gerwick, W. H.; Tan, L. T.; Sitachitta, N. Alkaloids;
Academic: San Diego, 2001; Vol. 57, pp 75-184. (g) Burja, A. M.; Banaigs,
B.; Abou-Mansour, E.; Burgess, J. G.; Wright, P. C. Tetrahedron 2001,
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10.1021/ol101021v  2010 American Chemical Society
Published on Web 06/08/2010

Prefecture. On the basis of extensive spectroscopic investiga-
tions, chemical degradation, and derivatization studies, planar
structure 1, depicted in Figure 1, was assigned as bisebro-
moamide. In addition to the amino acid L-alanine, bisebro-
moamide contains a significant number of D-amino acids and
N-methylated amino acids along with several other modified
amino acid residues that include D-leucine, N-methyl-3-
bromotyrosine, modified 4-methylproline, a 2-substituted
thiazoline-4-methyl-4-carboxylic acid unit, N-methylphenyl-
alanine, and a rare 2-(1-oxopropyl)pyrrolidine moiety which
has not been previously reported in any natural product.
Furthermore, and to the best of our knowledge, there is no
report on the total synthesis of bisebromoamide.
according to the procedure of Boger8 from D-tyrosine. Ortho-
selective monobromination of 6 was effected with N-
bromosuccinimide and p-toluenesulfonic acid in acetonitrile9
to afford 7 in 87% yield. Coupling of 7 with Boc-L-Ala-OH
was achieved through carboxyl activation with 3-(diethoxy-
phosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT)10 and
provided dipeptide 8 in 77% yield. Saponification of the
methyl ester of 8 gave the corresponding acid 2 in 95% yield.
Scheme 1. Synthesis of Dipeptide 2
The synthesis of fragment 5 commenced with the con-
densation of the known N-methyl L-phenylalanine methyl
ester 911 with Boc-D-leucine (Scheme 2). Bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (BOP-Cl)12 was employed
to effect the condensation process, and dipeptide 10 was
obtained in 86% yield. Amino ethyl ketone 11 was easily
prepared by addition of ethylmagnesium bromide to the Boc-
protected proline-derived Weinreb amide according to pub-
lished procedures.13 Hydrolysis of the methyl ester of 10
followed by a DEPBT-mediated condensation with amine
11 afforded the corresponding tripeptide. Next, cleavage of
the Boc group with trifluoroacetic acid provided fragment 5
as the corresponding ammonium trifluoroacetate salt in 74%
yield (over three steps).
At this juncture, the time had arrived to explore a suitable
strategy for the assembly of fragments and installation of the
sensitive thiazoline heterocycle. In the initial phases of our study,
Cbz-protected 2-methylserine (15) and dipeptide 10 were
employed to examine the feasibility and efficiency of the
important thiazoline-forming reaction from the corresponding
Figure 1. Retrosynthetic analysis of 1.
The principal synthetic challenge associated with the
preparation of bisebromoamide is the efficient formation and
assembly of the sensitive thiazoline heterocycle.4 From the
retrosynthetic perspective, we envisioned that the thiazoline-
ring moiety could arise from either 2-methylserine or
2-methylcysteine derivatives and be installed at a late stage
in the synthesis. Bearing this in mind, we anticipated that 1
might be conveniently constructed by the assembly of four
fragments: dipeptide 2, N-Boc-4-methylproline methyl ester
3, 2-methylserine 4a (or 2-methylcysteine 4b), and tripeptidyl
ketone 5.
While subunits N-Boc-4-methylproline methyl ester 35 and
2-methylserine 4a6 (or 2-methylcysteine 4b7) could be fully
synthesized according to literature procedures, peptide-based
fragments 2 and 5 were unknown and had to be elaborated
from amino acids as shown in Schemes 1 and 2. The
synthesis of fragment 2 (Scheme 1) began with the known
N-methyl D-tyrosine methyl ester 6, which was prepared
(8) Boger, D. L.; Yohannes, D. J. Org. Chem. 1988, 53, 487–499.
(9) Bovonsombat, P.; Khanthapura, P.; Krause, M. M.; Leykajarakul,
J. Tetrahedron Lett. 2008, 49, 7008–7011.
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Org. Lett. 1999, 1, 91–93. (b) Fan, C.-X.; Hao, X.-L.; Ye, Y.-H. Synth.
Commun. 1996, 26, 1455–1457.
(11) (a) Van der Auwera, C.; Anteunis, M. J. O. Int. J. Pept. Protein
Res. 1987, 29, 574–88. (b) Adima, A.; Bied, C.; Moreau, J. J. E.; Wong,
M. C. M. Eur. J. Org. Chem. 2004, 2582, 2588. (c) Park, J. D.; Lee, K. J.;
Kim, D. H. Bioorg. Med. Chem. 2001, 9, 237–243.
(4) (a) Wipf, P.; Fritch, P. Tetrahedron Lett. 1994, 35, 5397–5400. (b)
Boden, C. D. J.; Pattenden, G.; Ye, T. Synlett 1995, 417–419.
(5) Murphy, A. C.; Mitova, M. I.; Blunt, J. W.; Munro, M. H. G. J.
Nat. Prod. 2008, 71, 806–809.
(12) (a) Diago-Meseguer, J.; Palomo-Coll, A. L. Synthesis 1980, 547–
551. (b) Cabre, J.; Palomo, A. L. Synthesis 1984, 5, 413–417. (c) Tung,
R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107, 4342–4343.
(13) De Luca, L.; Giacomelli, G.; Taddei, M. J. Org. Chem. 2001, 66,
2534–2537.
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2131–2138.
Org. Lett., Vol. 12, No. 13, 2010
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To our disappointment and surprise, the coupling conditions
failed to produce the desired thioamide 17. To rationalize this
result, we postulate that there exists a severe steric interaction
between the thioacylating agent and the sterically demanding
amine-bearing quaternary center.
Scheme 2. Synthesis of Tripeptide 5
Scheme 4. Synthesis of Proposed Structure (1)
Scheme 3. Attempted Synthesis Toward Thiopeptide 17
Due to the difficulties in forming the thiazoline heterocycle
using cyclodehydration of a ꢀ-hydroxy thioamide-based ap-
proach, we altered our synthetic route by constructing the
thiazoline moiety via cyclocondensation of peptide-derived
nitrile 19 and 2-methylcysteine 4b. Although the revised strategy
was designed to circumvent the problem encountered in the
formation of the thiazoline ring from ꢀ-hydroxy thioamide, this
strategy might have a potential drawback as we do not know
whether the cyclocondensation process and/or the coupling of
thiazoline-containing acid 20 with tripeptide 5 would result in
any epimerization of stereogenic centers presented at two
pyrrolidine moieties. In the event, N-Boc-4-methylproline
methyl ester 3 was converted into the corresponding nitrile 18
according to a published procedure.2f,16 Removal of the Boc
group of 18 followed by a HATU-mediated condensation
with dipeptide 2 furnished the corresponding peptide that
was then resubjected to a TFA-mediated Boc deprotection,
and the resulting amine was acylated with pivaloyl chloride
to afford nitrile 19 in 35% yield (over four steps, Scheme
ꢀ-hydroxy thiopeptide (Scheme 3). Thus, saponification of the
methyl ester of 3 was followed by coupling with diamine 12
via a mixed anhydride to afford an amide which was then
converted into a corresponding thioamide 13 in 55% yield over
three steps. Thioamide 13 was then converted into the corre-
sponding R-amino thionoacid derivative of nitrobenzotriazole
(14) via diazotization as described by Rapoport.14 Treatment
of dipeptide 10 with trifluoroacetic acid effected the removal
of the Boc protecting group to give the corresponding amine
that underwent a HATU15-mediated coupling reaction with Cbz-
protected 2-methylserine (15) to provide 16 in 63% yield.
Hydrogenolysis of the Cbz protecting group of 16 cleanly
afforded the amine, which was immediately treated with excess
thioacylating agent 14 to effect the formation of thioamide 17.
(14) Shalaby, M. A.; Grote, C. W.; Rapoport, H. J. Org. Chem. 1996,
61, 9045–9048.
(16) Gardelli, C.; Nizi, E.; Muraglia, E.; Crescenzi, B.; Ferrara, M.;
Orvieto, F.; Pace, P.; Pescatore, G.; Poma, M.; del Rosario Rico Ferreira,
M.; Scarpelli, R.; Homnick, C. F.; Ikemoto, N.; Alfieri, A.; Verdirame,
M.; Bonelli, F.; Gonzalez Paz, O.; Taliani, M.; Monteagudo, E.; Pesci, S.;
Laufer, R.; Felock, P.; Stillmock, K. A.; Hazuda, D.; Rowley, M.; Summa,
(15) (a) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695–
698. (b) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397–4398. (c)
Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem. 1995, 60, 3561–
3564.
V. J. Med. Chem. 2007, 50, 4953–4975
.
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Org. Lett., Vol. 12, No. 13, 2010

4). This set the stage for the cyclocondensation. Gratifyingly,
condensation reaction between the nitrile 19 and the (S)-2-
methylcysteine (S-4b) in a sealed tube at 90 °C led to the
key intermediate 20 as a single isomer in 55% yield. Finally,
carboxyl activation of 20 with a HATU/collidine system and
coupling with fragment 5 produced the proposed structure
(1) for bisebromoamide in 30% yield. Unfortunately, neither
1H nor 13C NMR spectra for 1 were identical with those of
the natural product. In addition, the optical rotation was of
equal magnitude but of opposite sign [natural bisebromoa-
mide [R]D22 +17.8 (c 1.00, CHCl3); synthetic sample [R]D
20
- 19.0 (c 0.31, CHCl3)]. Taken together, these data suggested
that the reported structure (1) must be incorrect.
Figure 2. Effect of compounds 1 and epi-1 on cancer cell and
hepatic stellate cell proliferation.
A thorough examination of 1H and 13C NMR spectra of 1
and comparison of reported spectra for natural bisebromoa-
mide revealed that the discrepancies of chemical shifts are
The initial cytotoxicity evaluation of 1 and epi-1 was
performed across a panel of three cancer cell lines of different
histological origins (HeLa, cervix; HT29, colon; and HCT-116,
colon), and cell proliferation was measured by MTS assay. The
IC50 values of 1 were 43.1 nM (Hela), 62.9 nM (HT29), and
134.2 nM (HCT116), and the IC50 values of epi-1 were 48.8
nM (Hela), 55.1 nM (HT29), and 115 nM (HCT116). These
data indicated that both 1 and epi-1 inhibited the proliferation
of cancer cells at comparable levels of potency, with IC50 values
slightly higher than these reported for natural bisebromoamide.
These results led us to believe that stereochemistry at the
thiazoline moiety of bisebromoamide may not be an important
factor for its bioactivities. We also expanded the scope of our
tests by screening the effect of 1 and epi-1 on hepatic stellate
cells (LX-2 and HSCT6) (Figure 2). The IC50 values of 1 were
42.3 nM (LX-2) and 53.4 nM (HSCT6), which were also
comparable to the IC50 values of epi-1 [45.4 nM (LX-2) and
46.4 nM (HSCT6)].
1
mostly located in the thiazoline ring region, particularly H
NMR chemical shifts of the CH2 at C-5 of the thiazoline
ring and the 13C NMR chemical shifts at carbons at and
adjacent to the thiazoline ring. The actual structure of
bisebromoamide appeared to be epimeric to the proposed
structure at one or more stereocenters in or adjacent to the
thiazoline ring system.
Scheme 5. Synthesis of Revised Structure (epi-1)
In summary, the first total synthesis of bisebromoamide
was completed, leading to a revision of the reported
stereochemistry from structure 1 to epi-1.
Acknowledgment. We acknowledge financial support from
the Hong Kong Research Grants Council (project nos.
PolyU5407/06M and PolyU5638/07M) and financial support
from the Shenzhen Bureau of Science, Technology & Informa-
tion (JC200903160367A, ZD200806180051A), Shenzhen Foun-
dation for R&D (SY200806300179A, JC200903160372A), and
Nanshan Science & Technology (NANKEYUAN2009083,
NANKEPING2007005).
On the basis of these chemical shift variations, we hypothesized
that the stereogenic center at the thiazoline ring of the originally
proposed bisebromoamide structure might require revision. We
therefore elected to synthesize the epimer (with R-configuration
of the stereogenic center at the thiazoline ring) of the proposed
structure 1. As shown in Scheme 5, we synthesized epi-1 following
the same synthesis as for 1, but incorporating (R)-2-methylcysteine
(R-4b) to the thiazoline moiety. This was readily achieved, and
epi-20 was incorporated into the final synthesis as previously
performed to afford epi-1 in 51% yield. To our delight, the synthetic
bisebromoamide’s (epi-1, [R]D20 + 15.9 (c 0.48, CHCl3)) spectral
data (1H and 13C NMR) are identical to those of the natural
bisebromoamide.
Note Added after ASAP Publication. Scheme 4 con-
tained an error in the version published ASAP June 8, 2010;
the correct version reposted June 11, 2010.
Supporting Information Available: Full details for
experimental procedures for compounds 1, epi-1, 2, 5, 7, 8,
1
10, 11, 13, 14, 16, 19, 20, epi-20, and S1-S3 and H and
13C NMR spectra for compounds 1, epi-1, 7, 8, 10, 13, 16,
19, 20, epi-20, and S1-S3. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL101021V
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