Studies in the total synthesis of himastatin: A revision of the stereochemical assignment

Article abstract of DOI:10.1002/(SICI)1521-3773(19981116)37:21<2993::AID-ANIE2993>3.0.CO;2-I

A stereoisomer of the natural product and not himastatin, an unusual dimeric depsipeptide with promising antibiotic and antitumor properties, was obtained from pyrroloindoline anti-cis-1. This result led to a revision of the proposed stereostructure. The

Full text of DOI:10.1002/(SICI)1521-3773(19981116)37:21<2993::AID-ANIE2993>3.0.CO;2-I

COMMUNICATIONS
the helix was dissolved in sodium acetate buffer (10mm, pH 4) and added
dropwise to a solution of the template in acetate buffer (pH 7.5). The
condensation reaction was complete after 18 h, as monitored by analytical
HPLC.
[23] D. Grell, C. Lehmann, M. Mathieu, G. Tuchscherer, unpublished
results.
[24] J. M. Stewart, J. D. Young, Solid Phase Peptide Synthesis, Pierce
Chemical, Rockford, IL, 1984.
[25] a) P. Dumy, I. M. Eggleston, G. Esposito, S. Nicula, M. Mutter,
Biopolymers 1996, 39, 297; b) G. V. Nikiforovich, C. Lehmann, M.
Mutter, Biopolymers, in press.
[26] J. M. Berg, D. L. Merkle, J. Am. Chem. Soc. 1989, 111, 3759.
[27] I. Bertini, C. Luchinat, Adv. Inorg. Biochem. 1984, 6, 71.
[28] B. Rohwedder, PhD thesis, Lausanne, Switzerland, 1997.
[29] N. P. Pavletich, C. O. Pabo, Science 1991, 252, 809.
[30] H. Rink, Tetrahedron Lett. 1987, 28, 3787.
In the next step, the serine residue in the e position of the lysine residue of
the template was oxidized to the aldehyde (fivefold excess of NaIO4,
5 min), and the peptide purified by RP-HPLC (56%). Subsequently, the
Fmoc protecting group of the aminooxy group at the C-terminal lysine
residue of the helix was removed with 20% piperidine in DMF. After
precipitation with diethyl ether, the crude peptide was dissolved in 10mm
acetate buffer (pH 4); this resulted in immediate formation of the second
oxime bond (>90%). The LIF1 was purified by RP-HPLC and charac-
terized by ESI-MS (m/z: 3788).
[31] M. Mergler, Tetrahedron Lett. 1988, 29, 4005.
Optical absorption spectra of the CoII peptide complex were recorded on a
Beckmann spectrophotometer. Prior to complex formation, the internal
disulfide bond of LIF1 was reduced with 250mm dithiothreitol (DTT) at
room temperature for 10 h. After lyophilization, LIF1 (150mm) was
suspended in Tris ´ HCl buffer (pH 7; degassed with helium) and 150mm
CoCl2 added. The addition of an equimolar amount of ZnCl2 to this
solution readily displaces CoII from the complex.
Studies in the Total Synthesis of Himastatin:
A Revision of the Stereochemical Assignment**
CD spectra were recorded on a Jobin Yvon Marck VI circular dichrometer
in quartz cells (path length 0.1 cm). Prior to recording the spectra the
internal disulfide bond in LIF1 was reduced as described above, LIF1 was
taken up in Tris ´ HCl, and spectra were recorded before and after addition
of equimolar amounts of ZnCl2.
Theodore M. Kamenecka and Samuel J. Danishefsky*
Dedicated to Professor E. J. Corey
The quest for new antibiotic and antitumor agents prompt-
ed scientists at Bristol Myers Squibb to investigate an
actinomycete strain (ATCC 53653) from the state of Hima-
chal Pradesh in India. In doing so, they encountered a new
compound of formula C72H104N14O2, which they named
himastatin.[1] After extensive optimization with the strain,
himastatin could be obtained in scales adequate for sustaining
chemical and biological investigation. While himastatin has
not been developed to the point of clinical trials, its activity
against gram-positive microorganisms and a variety of tumor
probe systems is impressive. Based on spectroscopic inves-
tigations augmented by modest degradative studies, 1 was
Received: April 14, 1998 [Z11719IE]
German version: Angew. Chem. 1998, 110, 3160 ± 3164
Keywords: chemoselectivity
´ protein design ´ protein
mimetics ´ proteins ´ zinc finger
[1] J. S. Richardson, D. C. Richardson, Trends Biochem. Sci. 1989, 14, 304.
[2] J. W. Bryson, S. F. Betz, H. S. Lu, D. J. Suich, H. X. Zhou, K. T. OꢁNeil,
W. F. DeGrado, Science 1995, 270, 935.
[3] a) S. Vuilleumier, M. Mutter, Angew. Chem. 1989, 101, 653; Angew.
Chem. Int. Ed. Engl. 1989, 28, 535; b) M. Mutter, G. Tuchscherer, Cell.
Mol. Life Sci. 1997, 53, 851.
[4] S. Kamtekar, J. M. Schiffer, H. Xiong, J. M. Babik, H. M. Hecht,
Science 1993, 262, 1680.
[5] S. Olofsson, L. Baltzer, Fold. Design 1996, 1, 347.
[6] M. D. Struthers, R. P. Cheng, B. Imperiali, Science 1996, 271, 342.
[7] R. T. Sauer, Fold. Design 1996, 1, R27.
[8] B. Nolting, R. Golbik, A. R. Fersht, Proc. Natl. Acad. Sci. USA 1995,
92, 10668.
[9] E. Shakhnovich, V. Abkevich, O. Ptitsyn, Nature 1996, 379, 96.
[10] a) M. Mutter, G. Tuchscherer, Makromol. Chem. Rapid Commun.
1988, 9, 437; b) G. Tuchscherer, C. Servis, G. Corradin, U. Blum, J.
Rivier, M. Mutter, Protein Sci. 1992, 1, 1377.
[11] a) M. Mutter, G. Tuchscherer, C. Miller, K.-H. Altmann, R. I. Carey,
D. F. Wyss, A. M. Labhardt, J. E. Rivier, J. Am. Chem. Soc. 1992, 114,
1463; b) G. Tuchscherer, L. Scheibler, P. Dumy, M. Mutter, Biopolym.
Pept. Sci. 1998, 47, 63.
[12] P. Dumy, I. M. Eggleston, S. E. Cervigni, U. Sila, X. Sun, M. Mutter,
Tetrahedron Lett. 1995, 36, 1255.
[13] M. Mutter, P. Dumy, P. Garrouste, C. Lehmann, M. Mathieu, C.
Peggion, S. Peluso, A. Razaname, G. Tuchscherer, Angew. Chem.
1996, 108, 1587; Angew. Chem. Int. Ed. Engl. 1996, 35, 1482.
[14] K. Rose, J. Am. Chem. Soc. 1994, 116, 30.
[15] a) G. Tuchscherer, Tetrahedron Lett. 1993, 34, 8419; O. Nyanguile, M.
Mutter, G. Tuchscherer, Lett. Pept. Sci. 1994, 1, 9.
[*] Prof. S. J. Danishefsky,[‡] T. M. Kamenecka
Laboratory for Bioorganic Chemistry
Sloan-Kettering Institute for Cancer Research
1275 York Avenue, Box 106, New York, NY 10021 (USA)
Fax: (‡1)212-772-8691
E-mail: c-kandell@ski.mskcc.org
‡
[ ] Further address:
Department of Chemistry, Columbia University
Havemeyer Hall, New York, NY 10027 (USA)
[**] This work was supported by the National Institutes of Health (grant
nos. CA 28824 and HL 25848 (S.J.D) and CA-08748 (SKI Core
Grant)). T.M.K. gratefully acknowledges the NIH for postdoctoral
fellowship support (grant no. AI09355). We thank Bristol Myers
Squibb for providing us with an authentic sample of himastatin. We
thank Dr. George Sukenick and the NMR Core Facility Laboratory,
Sloan-Kettering Institute for Cancer Research, for mass spectral and
NMR analyses.
[16] P. E. Dawson, S. B. H. Kent, J. Am. Chem. Soc. 1993, 115, 7263.
[17] C. F. Liu, J. P. Tam, Proc. Natl. Acad. Sci. USA 1994, 91, 6584.
[18] S. F. Michael, V. J. Kilfoil, M. H. Schmidt, B. T. Amann, J. M. Berg,
Proc. Natl. Acad. Sci. USA 1992, 89, 4796.
[19] E. Rebar, C. O. Pabo, Science 1994, 263, 671.
[20] J. Miller, A. D. McLachlan, A. Klug, EMBO J. 1985, 4, 1609.
[21] R. S. Brown, C. Sanders, P. Argos, FEBS Lett. 1985, 186, 271.
[22] P. R. Gerber, K. Müller, J. Comput. Aided Mol. Des. 1995, 9, 251.
Angew. Chem. Int. Ed. 1998, 37, No. 21
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
1433-7851/98/3721-2993 $ 17.50+.50/0
2993

COMMUNICATIONS
advanced as the structure of himastatin. A cleavage product
of himastatin retaining only a d-valinol moiety from the
peptidal domain was accordingly formulated as 2.
Our interest in himastatin first arose from the intrinsic
challenge it posed to chemical synthesis. Solutions to prob-
lems of this scope often carry with them learning opportu-
nities which transcend the confines of the particular problem
under inquiry. Moreover, we had a focusing biological goal in
mind. We hoped to evaluate representative ªmonomersº (see
compound 17) to ascertain whether the ªdimericº motif is
constructed by the microorganism in response to an identi-
fiable biological advantage.
Scheme 2. Synthesis of 11. a) 1. Al(Hg), THF; 2. CbzCl, pyridine, CH2Cl2,
80%; 3. TBSCl, DBU, CH3CN, 508C, 90%; b) ICl, CH2Cl2, 2,6-di-tert-
butylpyridine, 90%; c) Sn2Me6, THF, [Pd(PPh3)4], 608C, 85%;
d) [Pd2dba3], AsPh3, 8, 458C, DMF, 50±70%; e) 1. TBAF; 2. TESCl,
TEA; 3. H2, Pd/C, EtOAc; 4. FMOC-HOSu, pyridine; 5. TESOTf, 2,6-
lutidine; 6. allyl alcohol, EDCI, DMAP; 7. piperidine, CH3CN, 30%
(7 steps).
Cbz ˆ benzyloxycarbonyl,
dba ˆ 1,5-dibenzylideneacetone,
DBU ˆ 1,8-diazabicyclo[5.4.0]undec-7-ene, EDCIˆ 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide, FMOC ˆ (9H-fluoren-9-ylmethoxy)carbonyl,
HOSu ˆ N-hydroxysuccinimide, TBAF ˆ tetrabutylammonium fluoride,
TBS ˆ tert-butyldimethylsilyl, TES ˆ triethylsilyl, Tf ˆ trifluoromethylsul-
fonyl.
One of the many provocative structural features of 1 is the
2,3,3a,8a-hexahydropyrrolo[2,3-b]indole moiety bearing
a
potentially eliminable hydroxyl group in position 3a. Even if
attention is confined to the system bearing a cis fusion of the
five-membered rings, two possibilities for the relationship of
the tryptophan carboxy equivalent at C2 to the junction
present themselves. Given the structure 1 proposed for
himastatin, we focused on the subtype with an anti relation-
ship of this carboxy center.
The possibility, in principle, of oxidative cycloaromatization
of various tryptophan derivatives (see compound 4) had been
known since the pioneering work of Witkop et al.[2] For the
purpose of our synthesis, the use of an Nb-anthracenosulfonyl
protecting group on the tert-butyl ester of tryptophan (3,
Scheme 1)[3] proved to be of immense advantage.[4] Conver-
sion of 3 into 4 was readily achieved, as was the conversion of
4 into 5. For purposes of additional verification, the trans-
formation of 5 to 6 was also accomplished. The latter
correlates with a compound previously assigned to be in the
anti-cis series.[5]
protecting groups chosen, iodination at C5 of the indoline
occurred with ICl (!8). The stannyl indole 9 was prepared
from 8. Palladium-mediated coupling of 8 and 9 gave rise to
10. Following extensive trial and error probes, it was found
that the arrangement of protecting groups in 11, which was
synthesized as shown, would allow us to advance toward 1
with viable prospects for eventual deprotection. We were
attracted to the possibility of twofold leveraging of the
subsequent interpolation by conducting it simultaneously in
both sectors of the dimer.
In a parallel series of investigations, the ester tripeptide 12,
bearing a free carboxyl group in the terminal d-threonine
residue, was synthesized (Scheme 3).[7] Coupling of 11 with 12
under the conditions shown gave rise to 13 after reprotec-
tion.[8] Cleavage of the carboxyl and amino protecting groups
led to 14, the substrate for the critical macroloactamization.
Cyclization smoothly led to 15, which on deprotection
afforded fully synthetic 1.
1
At this point it was clear that the H NMR spectrum of 1
synthesized in our laboratory did not correspond with that
recorded for naturally derived himastatin.[1c] Particularly
striking were the two upfield signals at d ˆ 0.55 and 0.35 for
1 corresponding to the valine isopropyl group. No such upfield
1
resonances had been observed in the H NMR spectrum of
himastatin.
The strategy used to reach dimer 1 was applied to synthesize
monomeric 17 (Scheme 4). For this goal, we utilized 16, which
is readily derived from 5, and the previously described 12. The
synthesis proceeded in much the same manner as for 1. Once
again, the 1H NMR spectrum of 17 contained upfield peaks at
d ˆ 0.55 and 0.35 having no counterpart in that of himasta-
tin.[9] That this discrepancy was rooted in the pyrrolindoline
sector of the molecule was shown when we obtained
compounds 18 and 19. The recorded chemical shifts of the
Scheme 1. Synthesis of 6. a) NBS, TEA, CH2Cl2, 80%; b) 1. DMDO,
CH2Cl2, 788C; 2. NaBH4, MeOH, 75% (2 steps); c) 1. TFA; 2. CH2N2;
3. Al(Hg); 4. ClCO2Me, pyridine, 50% (4 steps). anth ˆ anthracen-9-yl,
DMDO ˆ 3,3-dimethyldioxirane, NBS ˆ N-bromosuccinimide.
The use of a Stille coupling[6] was envisioned for joining the
two indolyl moieties through a carbon ± carbon bond. In
practice compound 5 lent itself to conversion into 7 through a
three-step sequence (Scheme 2). With the particular set of
2994
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
1433-7851/98/3721-2994 $ 17.50+.50/0
Angew. Chem. Int. Ed. 1998, 37, No. 21

COMMUNICATIONS
centers are reversed (S,S rather than R,R). The structure of
himastatin is thus assigned as 20, and that of its valinol
degradation product as 21. In the newly proposed structure of
himastatin, the components in the depsipeptide domain are
presented in alternating d and l configurations.[10] These
various proposals were validated when a total synthesis of
himastatin was realized.[11]
Received: June 5, 1998 [Z11949IE]
German version: Angew. Chem. 1998, 110, 3164 ± 3166
Keywords: alkaloids ´ antibiotics ´ himastatin ´ structure
elucidation ´ total synthesis
[1] a) K. S. Lam, G. A. Hesler, J. M. Mattel, S. W. Mamber, S. Forenza, J.
Antibiot. 1990, 43, 956; b) J. E. Leet, D. R. Schroeder, B. S. Krishnan,
J. A. Matson, J. Antibiot. 1990, 961; c) J. E. Leet, D. R. Schroeder, J.
Golik, J. A. Matson, T. W. Doyle, K. S. Lam, S. E. Hill, M. S. Lee, J. L.
Whitney, B. S. Krishnan, J. Antibiot. 1996, 49, 299.
[2] M. Ohno, T. F. Spande, B. Witkop, J. Am. Chem. Soc. 1968, 90, 6521.
[3] Prepared from d-tryptophan: 1) anthSO2Cl, TEA, THF, H2O;
2) N,N'-diisopropyl-O-tert-butylisourea, CH2Cl2 (65% yield overall).
[4] a) The anthracene sulfonamide was readily cleaved with Al(Hg) at
room temperature. b) The use of the tert-butyl ester afforded a much
higher anti:syn stereoselectivity (>15:1) than the corresponding
methyl ester (3:1).
[5] M. Nakagawa, S. Kato, S. Kataoka, S. Kodato, H. Watanabe, H.
Okajima, T. Hino, B. Witkop, Chem. Pharm. Bull. 1981, 29, 1013.
[6] J. K. Stille, Angew. Chem. 1986, 98, 504; Angew. Chem. Int. Ed. Engl.
1986, 25, 508.
[7] Compound 12 was synthesized similarly to 20, which is described in
ref. [11].
[8] J. M. Humphrey, A. R. Chamberlin, Chem. Rev. 1997, 97, 2243.
[9] The 1H NMR spectra of synthetic 1 and 17 are virtually identical
upfield of 6.0 ppm.
Scheme 3. Synthesis of ªepi-himastatinº (1). a) 1. HATU, HOAt, collidine,
CH2Cl2,
108C ! RT, 65%; 2. TESOTf, 2,6-lutidine, CH2Cl2, 63%;
b) 1. [Pd(PPh3)4], PhSiH3, THF; 2. H2, Pd/C, EtOAc, 40%; c) HOAt,
HATU, iPr2NEt, DMF; d) TBAF, THF, HOAc, 25% (from 14). HATU ˆ
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-
methylmethaneaminium hexafluorophosphate, HOAt ˆ 1-hydroxy-7-
aza-benzotriazole.
[10] See M. R. Ghadiri, R. J. Granja, R. A. Milligan, D. E. McRee, N.
Khazanovich, Nature 1993, 366, 324.
[11] T. M. Kamenecka, S. J. Danishefsky, Angew. Chem. 1998, 110, 3166 ±
3168; Angew. Chem. Int. Ed. 1998, 37, 2995 ± 2998.
Total Synthesis of Himastatin: Confirmation of
the Revised Stereostructure**
Scheme 4. Synthesis of the ªepi-himastatinº monomer (17).
Theodore M. Kamenecka and Samuel J. Danishefsky*
isopropyl methyl groups of the synthetic syn-cis monomer 19
(d ˆ 0.92 and 0.88)[7] were in much closer harmony with the
data (d ˆ 0.94 and 0.89)[1c] of the degradation product of
himastatin (hitherto formulated as 2) than were those of the
synthetic anti-cis structure 18 (d ˆ 0.78 and 0.62).
Dedicated to Professor E. J. Corey
In the preceding communication[1] we have provided the
background of the himastatin problem and the findings that
necessitated a revision in the assignment of the configuration
[*] Prof. S. J. Danishefsky,[‡] T. M. Kamenecka
Laboratory for Bioorganic Chemistry
Sloan-Kettering Institute for Cancer Research
1275 York Avenue, Box 106, New York, NY 10021 (USA)
Fax: (‡1)212-772-8691
E-mail: c-kandell@ski.mskcc.org
‡
[ ] Further address:
Therefore, we postulated that the relationship of the cis
junction and the C2-carboxamido group in naturally occurring
himastatin is syn rather than anti, as previously proposed.
Given the chiroptical data accumulated in the Bristol Myers
Squibbs investigation,[1c] we further favored a revision from a
d-tryptophan matrix to the l-tryptophan series. Thus, the
absolute stereochemistry of the pyrrolindoline junction re-
mains unchanged from the previous assignment. However, the
stereochemical assignment of the tricyclic carboxamido
Department of Chemistry, Columbia University
Havemeyer Hall, New York, NY 10027 (USA)
[**] This work was supported by the National Institutes of Health (grant
nos. CA 28824 and HL 25848 (S.J.D) and CA-08748 (SKI Core
Grant)). T.M.K. gratefully acknowledges the NIH for postdoctoral
fellowship support (grant no. AI09355). We thank Bristol Myers
Squibb for providing us with an authentic sample of himastatin. We
thank Dr. George Sukenick and the NMR Core Facility Laboratory,
Sloan-Kettering Institute for Cancer Research, for mass spectral and
NMR analyses.
Angew. Chem. Int. Ed. 1998, 37, No. 21
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
1433-7851/98/3721-2995 $ 17.50+.50/0
2995