Solid-support based total synthesis and stereochemical correction of brunsvicamide A

Article abstract of DOI:10.1021/ol801064d

(Chemical Equation Presented) A total synthesis of the cyanobacterial metabolite brunsvicamide A and the correction of its originally assigned stereochemistry are reported. Key elements were the construction of a urea building block, peptide elongation on solid phase, and on-resin cyclization of the peptide chain, with good overall yield. Detailed structural investigations uncovered that brunsvicamide A features a previously undetected D-lysine residue in its backbone, setting the foundation for all further investigations in this compound class.

Full text of DOI:10.1021/ol801064d

ORGANIC
LETTERS
2008
Vol. 10, No. 15
3199-3202
Solid-Support Based Total Synthesis
and Stereochemical Correction of
Brunsvicamide A
Thilo Walther,†,‡ Hans-Dieter Arndt,*,†,‡ and Herbert Waldmann*,†,‡
TU Dortmund, Fakulta¨t Chemie, Otto-Hahn-Str. 6, D-44221 Dortmund, Germany, and
Max-Planck-Institut fu¨r Molekulare Physiologie, Otto-Hahn-Str. 11,
D-44227 Dortmund, Germany
hans-dieter.arndt@mpi-dortmund.mpg.de; herbert.waldmann@mpi-dortmund.mpg.de
Received May 8, 2008
ABSTRACT
A total synthesis of the cyanobacterial metabolite brunsvicamide A and the correction of its originally assigned stereochemistry are reported.
Key elements were the construction of a urea building block, peptide elongation on solid phase, and on-resin cyclization of the peptide chain,
with good overall yield. Detailed structural investigations uncovered that brunsvicamide A features a previously undetected D-lysine residue
in its backbone, setting the foundation for all further investigations in this compound class.
Bioactive natural products continue to be a source of
inspiration for chemical biology and medicinal chemistry
research.1 The brunsvicamides (BVA) A-C are a group of
cyclopeptides recently isolated from cyanobacteria (see
Figure 5 for a correct structure) and were shown to inhibit
the tyrosine phosphatase B of Mycobacterium tuberculosis
(MptpB).2 This enzyme is crucial for the host-pathogen
interaction.3 The brunsvicamides belong to a group of
structurally closely related cyclic peptides, the anabaenopep-
tins, which have a urea moiety and N-methylation in common
but differ in amino acid sequence and stereochemistry.
Cyclopeptides are often characterized by increased metabolic
stability, constrained conformation, and pronounced biologi-
cal specificity.4 The N-methylated amide bond is a feature
which also increases conformational rigidity5 and additionally
may lead to improved pharmacological properties.6 The urea
moiety is known as a replacement for the amide bond in
peptidomimetics.7 Besides the brunsvicamides, other mem-
(2) Mueller, D.; Krick, A.; Kehraus, S.; Mehner, C.; Hart, M.; Kuepper,
F. C.; Saxena, K.; Prinz, H.; Schwalbe, H.; Janning, P.; Waldmann, H.;
Koenig, G. M. J. Med. Chem. 2006, 49, 4871–4878.
(3) Singh, R.; Rao, V.; Shakila, H.; Gupta, R.; Khera, A.; Dhar, N.;
Singh, A.; Koul, A.; Singh, Y.; Naseema, M.; Narayanan, P. R.; Parama-
sivan, C. N.; Ramanathan, V. D.; Tyagi, A. K. Mol. Microbiol. 2003, 50,
751–762.
† TU Dortmund, Fakulta¨t Chemie.
‡ Max-Planck-Institut fu¨r Molekulare Physiologie.
(1) (a) Breinbauer, R.; Vetter, I. R.; Waldmann, H. Angew. Chem., Int.
Ed. 2002, 41, 2878–2890. (b) Koch, M. A.; Schuffenhauer, A.; Scheck,
M.; Wetzel, S.; Casaulta, M.; Odermatt, A.; Ertl, P.; Waldmann, H. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 17272–17277. (c) Noeren-Mueller, A.;
Reis-Correa, I., Jr.; Prinz, H.; Rosenbaum, C.; Saxena, K.; Schwalbe, H. J.;
Vestweber, D.; Cagna, G.; Schunk, S.; Schwarz, O.; Schiewe, H.; Wald-
mann, H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10606–10611. (d) Koch,
M. A.; Waldmann, H. Drug DiscoVery Today 2005, 10, 471–483. (e)
Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461–477.
(4) Li, P.; Roller, P. P.; Xu, J. Curr. Org. Chem. 2002, 6, 411–440.
(5) Vitoux, B.; Aubry, A.; Cung, M. T.; Marraud, M. Int. J. Pept. Protein
Res 1986, 27, 617–32.
(6) Biron, E.; Chatterjee, J.; Kessler, H. J. Pept. Sci. 2006, 12, 213–
219.
(7) Burgess, K.; Linthicum, D. S.; Shin, H. Angew. Chem., Int. Ed. Engl.
1995, 34, 907–909.
10.1021/ol801064d CCC: $40.75
Published on Web 07/01/2008
2008 American Chemical Society

bers of this class have been reported. Many anabaenopeptins
feature a D-lysine, an unusual homotyrosine, and were shown
to inhibit the human carboxypeptidase A.8 The amino acid
sequence of monzamide A and B includes the very rare allo-
isoleucine and D-valine or D-isoleucine.9 Schizopeptin 79110
was shown to inhibit trypsin, and the latest addition to the
anabaenopeptin family was psymbamide.11
L-Lys(Fmoc)OAll to form the orthogonally protected14 urea
building block. Column chromatography removed residual
4-nitrophenol and was directly followed by tert-butyl ester
cleavage to yield acid 4.
Solid phase peptide synthesis (SPPS) was used to extend
the peptide chain. Treatment of 2-chlorotrityl chloride resin
with the purified urea building block 4, followed by capping
with methanol, gave access to the solid-support bound urea
12 (Scheme 2). To obtain sufficient loadings, it was necessary
1
The sequence of brunsvicamide A features Lys-2Val-3
Leu-4NMeTrp-5Phe and forms a 19-membered cyclopeptide
ring via the lysine side chain ε-NH2. A urea function with
an additional Ile residue is attached to the R-amino group
of the N-terminal Lys. All amino acid residues of brunsvica-
mide A were initially reported to be L-configured. The
inhibition of MptpB, which could be used to develop agents
targeting tuberculosis, prompted us to develop a synthetic
route for creating a library of BVA and related natural
products for the purpose of structural confirmation and further
biological evaluation.
Scheme 2
.
Synthesis of the Reported Brunsvicamide A
Structure
Here, we report the first total synthesis of BVA A which
led to the correction of the previously reported stereochem-
istry. We planned to create the urea moiety at an early
synthetic stage, to elongate and then close the peptide ring
on solid support. The synthesis of the urea began with the
reaction of isoleucine tert-butylester (2) and 4-nitrophenyl
chloroformate (1) to give 4-nitrophenyl carbamate 3 which
can be purified by column chromatography and stored for
months (Scheme 1).12 Upon addition of Hu¨nig’s base to the
Scheme 1. Synthesis of Urea Building Blocks (4-11)
to increase the reaction time to 16 h. This led to a loading
of 0.6 mmol/g (UV-based Fmoc determination).15 Alternative
attempts of assembling the urea directly from solid-support
attached amino acids16 gave inferior results in our hands.
The Fmoc groups were removed with DMF/piperidine
(4:1, v/v, 15 min). Peptide couplings were performed using
carbamate 3, intense yellow coloration indicated the in situ
formation of the isocyanate,13 which was reacted with
(12) Boeijen, A.; Liskamp, R. M. J. Eur. J. Org. Chem. 1999, 2127–
2135.
(8) Harada, K.; Fujii, K.; Shimada, T.; Suzuki, M.; Sano, H.; Adachi,
K.; Carmichael, W. W. Tetrahedron Lett. 1995, 36, 1511–1514.
(9) Schmidt, E. W.; Harper, M. K.; Faulkner, D. J. J. Nat. Prod. 1997,
60, 779–782.
(13) Kruijtzer, J. A. W.; Lefeber, D. J.; Liskamp, R. M. J. Tetrahedron
Lett. 1997, 38, 5335–5338.
(14) Schelhaas, M.; Waldmann, H. Angew. Chem., Int. Ed. Engl. 1996,
35, 2056–2083.
(10) Reshef, V.; Carmeli, S. J. Nat. Prod. 2002, 65, 1187–1189.
(11) Robinson, S. J.; Tenney, K.; Yee, D. F.; Martinez, L.; Media, J. E.;
Valeriote, F. A.; Van Soest, R. W. M.; Crews, P. J. Nat. Prod. 2007, 70,
1002–1009.
(15) Coin, I.; Beyermann, M.; Bienert, M. Nat. Protoc. 2007, 2, 3247–
3256.
(16) Marsh, I. R.; Bradley, M.; Teague, S. J. J. Org. Chem. 1997, 62,
6199–6203.
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Org. Lett., Vol. 10, No. 15, 2008

either 3 equiv of the Fmoc-amino acid/DIC/HOBt (2 h, DMF,
rt) or 3 equiv of Fmoc-Leu-OH/HATU/HOAt with 6 equiv
of DIPEA (2 h, DMF, rt) for the coupling to the NMe-Trp.
In each case, complete conversion was evident from negative
Kaiser or chloranil tests.17 Cleavage of the allylester 13 with
Pd(PPh3)4 proceeded smoothly when phenylsulfinic acid
sodium salt was used as a scavenger.14,18 Deblocking of the
N-terminal Fmoc group was followed by macrolactam
formation on resin using DIC/HOBT in DMF. The crude
product 14 was released with TFA and purified by prepara-
tive reversed phase HPLC, using MeOH/water (65:35) as
eluent.
In this direct approach, 14 was obtained under pseudodi-
lution conditions on solid support with an overall yield of
11% based on the loading of urea 12. Using other reagent
combinations for the macrocyclization (e.g., HATU/HOAt,
PyBrop/DIPEA, DIC/C6F5OH) did not provide better results.
Alternatively, the peptide precursor was released from the
resin, and then macrocyclization was attempted in solution
under pseudohigh dilution conditions (syringe pump). How-
ever, this strategy was more laborious and did not improve
overall efficiency and was therefore abandoned.
Much to our surprise, the NMR spectra of the such
obtained compound 14 were similar, but not identical, to the
originally reported data for the natural product.2 An HPLC
coinjection of synthetic 14 and natural BVA revealed that
they were not identical (Figure 1). However, the MS-MS
fragmentation patterns were identical with the patterns of
the natural product. This finding indicated that the structural
differences should reside in the stereochemistry of the
residues. We compared the recorded NMR spectra with the
reported data and found major differences for the signals
belonging to the L-lysine and L-isoleucine (Figure 2 and
Supporting Information).
Figure 1. HPLC chromatograms showing coinjections of synthetic
and natural brunsvicamides. (A) Authentic sample of BVA A. (B)
14 with L-Lys as initially reported. (C) 18 with D-Lys. (D)
Coinjection of authentic sample and 18. (E) Coinjection of authentic
sample, 18, and 14.
Since many of the anabaenopeptins contain a D-lysine, and
the monzamides feature the very uncommon allo-isoleucine,
we decided to synthesize a collection of all eight possible
stereochemical combinations of lysine and isoleucine in
brunsvicamide A (Figure 3). The required tert-butyl esters
of D-allo-Ile-OH, D-Ile-OH, and L-allo-Ile-OH, respectively,
were readily obtained with tert-butyl isourea.19 The 4-ni-
trophenyl carbamates 3b-d were prepared and each reacted
with L- and D-Lys(Fmoc)OAll to give the ureas 5-11
(Scheme 1). Eight portions of 2-chlorotrityl resin were loaded
with the individual stereoisomers. Peptide chain elongation,
allyl ester cleavage, and ring closure were performed as
before and simplified by the successful application of IRORI
MacroKans and RFID technology.20 The compounds 15-21
were obtained smoothly in good and reproducible overall
yield (13-18%, Figure 3).
1
Figure 2. H spectra showing characteristic signals of BVA (500
MHz, MeOH-d4). (A) 14 with L-Lys as initially reported.2 (B) 18
with D-Lys (pH ) 4). (C) 18 (pH ) 10).
that the compound 18 had NMR spectra with only minor
differences from those reported earlier, whereas 21 showed
major differences to the reported data. After consideration
of the original data in conjunction with our synthetic efforts,
we decided to remeasure the samples for compound 18 at
different concentrations and pH values, both of which were
not clear from the isolation procedure.
It was found that at pH ) 10 all the signals now matched
those initially observed for the isolated material (Figures 2
and 4). In the HPLC coinjection, compound 18 and the
HPLC coinjection experiments narrowed the selection of
possible candidates to compounds 18 and 21. We then found
(17) (a) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595–8. (b) Christensen, T. Acta Chem. Scand. B 1979,
33, 763–6.
(18) (a) Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. Engl. 1984,
23, 71–72. (b) Honda, M.; Morita, H.; Nagakura, I. J. Org. Chem. 1997,
62, 8932–8936. (c) Coulson, D. R. Inorg. Synth. 1990, 28, 107–9
(19) Mathias, L. J. Synthesis 1979, 561–76.
(20) http://www.irori.com/.
.
Org. Lett., Vol. 10, No. 15, 2008
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Figure 5. Brunsvicamide A with its correct stereochemistry.
The very efficient 12-step sequence gave access to a
collection of eight brunsvicamide A stereoisomers in 11-18%
overall yield and allowed the identification of the correct
stereochemistry of the brunsvicamides which feature a
previously undetected D-lysine residue.
These studies highlight the vital role that chemical
synthesis continues to play in confirming the structures of
promising natural products of low abundance.22,23 Efforts
directed toward the synthesis of other members of the
anabaenopeptin family of natural products and studies of their
biological activity are underway.
Figure 3. Structures of synthesized stereochemical variants of
brunsvicamide A.
natural product were indistinguishable as well (Figure 1).
This led us to the conclusion that the Lys residue in
brunsvicamide A must be D-configured and by inference
should also be assumed D for the other brunsvicamides. This
finding is consistent with the occurrence of D-Lys in all other
Acknowledgment. Funding by the Fonds der Chemischen
Industrie, the Max-Planck-Society (to H.W.), and the Deut-
sche Forschungsgemeinschaft (Emmy-Noether young inves-
tigator grant to H.-D.A.) is highly appreciated. This work
was supported in part by the EC and the state of Nordrhein-
Westfalen (ZACG Dortmund). T.W. acknowledges the
IMPRS Chemical Biology for financial support. We thank
Prof. Dr. G. Ko¨nig (U Bonn) and C. Mehner (U Bonn) for
providing authentic samples of brunsvicamides A and B and
for helpful discussions.
Note Added after ASAP Publication. This paper was
published ASAP on July 1, 2008. A change was made to
Scheme 1. The revised paper was reposted on July 3, 2008.
Figure 4.
Chemical shift differences of 13C signals reported for
isolated BVA A and synthetic material (125 MHz, MeOH-d4). (A)
14 with L-Lys as initially reported. (B) 18 with D-Lys (pH ) 4).
(C) 18 (pH ) 10).
Supporting Information Available: Experimental details,
characterization of the compounds discussed, detailed NMR
characterization of compounds 14 and 18, HPLC traces, and
1H and 13C NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
anabaenopeptins reported to date which are produced from
cyanobacteria.21
OL801064D
(22) Nicolaou, K. C.; Snyder, S. A. Angew. Chem., Int. Ed. 2005, 44,
In summary, a total synthesis of brunsvicamide A (18,
Figure 5) has been accomplished on solid support from a
urea building block which is easily varied. The solid phase
synthesis entailed peptide chain elongation, allyl ester
cleavage, and a practical on-resin cyclization.
1012–1044
.
(23) A referee pointed out that amino acid stereochemistry determination
by chiral GC (as reported in ref 2) is typically reliable. We agree, but note
that depending on the amount and quality of the isolated materials
misassignments can occur, especially with noncanonical linkages (e.g., urea)
or less common stereoisomers (e.g., allo). For a related case see: (a)
Zampella, A.; Randazzo, A.; Borbone, N.; Luciani, S.; Trevisis, L.; Debitus,
C.; D’Auria, M. V. Tetrahedron Lett. 2002, 43, 6163–6166. For a general
account on the technology, see: (b) Fountoulakis, M.; Lahm, H. W.
(21) Welker, M.; von Doehren, H. FEMS Microbiol. ReV. 2006, 30, 530–
563.
J. Chromatogr. A. 1998, 826, 109–134.
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