Total synthesis and stereochemical assignment of micrococcin P1

Full text of DOI:10.1002/anie.200900621

Communications
DOI: 10.1002/anie.200900621
Total Synthesis
Total Synthesis and Stereochemical Assignment of Micrococcin P1**
David Lefranc and Marco A. Ciufolini*
This paper describes the total synthesis of the thiopeptide
antibiotic micrococcin P1 (MP1, 1; Figure 1),[1] thereby estab-
lishing its constitution and its configuration. Compound 1 is
Figure 2. Walker–Lukacs structure of MP1 (3): Z=OH, Z’=H. Syn-
thetic “micrococcin P”[13a,c] (5): Z=H, Z’=OH.
acids. On the basis of these data and of a presumed similarity
with other thiopeptides, in 1977 Walker and Lukacs proposed
structure 3 for MP1 (Figure 2),[11] but without the benefit of
Figure 1. Actual structure of micrococcin P1 (1): R=iPr, R’=H;
Z=OH, Z’=H. Actual structure of micrococcin P2 (2): R=iPr, R’=H;
evidence in support of the alleged topography of the macro-
Z, Z’=O. Bycroft–Gowland structure of MP1 (4): R=H, R’=iPr;
cycle. Shortly thereafter, new chemical evidence induced
Bycroft and Gowland to promulgate the revised structure 4
(Figure 1).[12] The latter authors were unable to assign the
configuration of region b of the molecule, which, correctly,
they left undefined. Moreover, they also left unresolved the
issue of macrocycle topography. Errors possibly present in the
Walker assignment thus propagated to the revised structure,
which nonetheless gained tacit acceptance and gradually
came to be consistently represented with the configuration
shown.
Remarkably, past synthetic work has been unable to
resolve the structural uncertainties surrounding MP1. Indeed,
synthetic epimers of the Walker–Lukacs (see compound 5,
Figure 2)[13a,c] and of the Bycroft–Gowland (see 6, Fig-
ure 1)[13b,d] structures have both been stated to be identical
to the natural product. Not only the two structures are
mutually exclusive: they also possess the (S) configuration,
instead of the secure (R) configuration, at c.[14] Even more
problematic is the fact that synthetic 4 (Figure 1) is not
identical to natural MP1.[15]
Extensive NMR studies ultimately confirmed the
Bycroft–Gowland constitution of MP1,[16] and by default
that of MP2, ruling out the possibility that MP1 may be 3 or 5,
and implying that the difference between 4 and natural MP1
must be purely stereochemical. While spectroscopic methods
failed to unravel the relative configuration of the natural
product, incisive work by Bagley and Merritt[17] led to the
conclusion that MP1 is likely to be 1. Total synthesis now
confirms this surmise.
The retrosynthetic logic that directed the construction of 1
is delineated in Scheme 1. Experience had revealed the
necessity of minimizing chemical operations after macrocycle
formation. Accordingly, MP1 would emerge upon the union
of a pair of suitably COOH- and NH2-protected segments, A
and B. Past experience had also shown that macrocyclization
was facile only if the order of bond formation was (a) first,
then (b). In turn, each segment was accessible by means of the
Z=OH, Z’=H. Synthetic “micrococcin P1”[13b,d] (6): R=H, R’=iPr;
Z=H, Z’=OH.
the major component of “micrococcin P”, a cytotoxic extract
isolated from Bacillus pumilus that consists of a roughly 7:1
mixture of 1 and of the corresponding ketone, 2, which is
termed micrococcin P2 (MP2). MP1 binds tightly to ribo-
somes, thereby disrupting protein synthesis.[2] The compound
thus exerts a potent antibiotic activity toward microorgan-
isms,[3] including the malarial parasite Plasmodium falcipa-
rum.[4]
While MP1[5] is one of the structurally less complex
thiopeptides,[6] its precise structure has remained uncertain
for over 50 years. The constitution of the central pyridine–
thiazole cluster was firmly established by X-ray diffractom-
etry.[7] Important work by Walker and Mijovic ascertained
that the 1-amino-2-propanol segment of 1 (see Figure 2,
region c) has the d-(R) configuration[8] and that MP1
incorporates a l-threonine unit.[9] Walker et al. also advanced
the hypothesis[10] that the valine-derived thiazole in region a
of the molecule had the (R) configuration, thus implying that
the thiazole in question is a formal derivative of d-valine. This
would make MP1 unique among thiopeptide antibiotics, all of
which incorporate thiazole segments derived from l-amino
[*] D. Lefranc, Prof. Dr. M. A. Ciufolini
Department of Chemistry, University of British Columbia
2036 Main Mall, Vancouver, BC V6T 1Z1 (Canada)
Fax: (+1)604-822-2710
E-mail: ciufi@chem.ubc.ca
Homepage: www.chem.ubc.ca/personnel/faculty/ciufolini/
index.shtml
[**] We gratefully acknowledge support for this research from the
University of British Columbia, the Canada Research Chair program,
NSERC, CIHR, and MerckFrosst Canada.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200900621.
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Thus, the union of 10 and 12 could be achieved only through
the use of a heterogeneous catalytic system comprising
powdered Li2CO3 in EtOAc (Scheme 3).[18] The resultant 13
was converted into 14, and the latter was then advanced to the
complete pyridine core of MP1, 21.
Parallel work reached 27 through the sequence outlined in
Scheme 4. Owing to the propensity of valine-derived thiazole
Scheme 1. Retrosynthetic disconnection of micrococcin P1 (1) into
fragments C–H. Pg=protecting group.
fusion of a triad of appropriately protected subunits: C–E for
A; F–H for B.
Building blocks 8, 10, and 11 were thus prepared from the
known 7[5a,15] and 9[18] as previously described (Scheme 2).[15]
A challenging aspect of the synthesis of 1 was the assembly of
the central pyridine–thiazole cluster, an objective that is best
attained through a Hantsch-type pyridine construction pro-
ceeding through the merger of 10 with 12.[19] The proclivity of
12 to undergo base-promoted polymerization precluded the
implementation of traditional procedures for the initial
Michael reaction leading to intermediate 13 (Scheme 3).
Scheme 3. Construction of the pyridine–thiazole cluster of MP1. a) 10,
cat. Li2CO3, EtOAc, 92%; b) NH4OAc, EtOH then DDQ, toluene, 97%;
c) LiOH, H2O, THF; d) Boc2O, DMAP, Et3N, DCM; e) 8, BOP-Cl, Et3N,
CH3CN, 77% over 3 steps (c–e); f) MsCl, Et3N, then DBU, DCM;
g) TBAF, THF; h) Dess–Martin periodinane, NaHCO3, DCM, 88% over
three steps (f–h); i) NaClO2, 2-methyl-2-butene, NaH2PO4, THF, H2O,
84%. DDQ=2,3-dichloro-5,6-dicyano-1,4-benzoquinone, DCM=di-
chloromethane, BOP-Cl=bis(2-oxo-3-oxazolindinyl)phosphinic chlo-
ride, MsCl=mesityl chloride, DBU=1,8-diazabicyclo[5.4.0]undec-7-
ene, TBAF=tetrabutylammonium fluoride.
22[20] and of its derivatives to racemize/epimerize,[21] the
stereochemical integrity of each intermediate in this sequence
was ascertained by 13C and 19F NMR scrutiny of Mosher
derivatives. No racemization/epimerization occurred during
subsequent transformations. This was also apparent from the
1H NMR spectra of intermediates 23, 25, and 26, wherein a
single diastereomer was discernible.
The final sequence of the synthesis (Scheme 5) com-
menced with the coupling of 21 and 27 to furnish 28, which
subsequently underwent deblocking and macrocyclization
(reaction with DPPA).[22] This produced compound 1 con-
taminated with a byproduct of unknown structure and with
similar chromatographic characteristics. This contaminant
appeared to be present also in an aged sample of natural
Scheme 2. Synthesis of fragments 8, 10, and 11. a) DCC, (R)-isoalani-
nol, CH2Cl2, RT, overnight; b) Ac2O, DMAP, pyridine, 2 h, 85% a–b;
c) 4n HCl in dioxane, 20 min, then addition of H2O, 15 min, 100%;
d) 3 equiv of 2-(lithiomethyl)-4-(tert-butyldimethylsilyloxy)methylthia-
zole, THF, ꢀ788C, 81%; e) Boc2O, Et3N, DMAP, 99% (crude);
f) LiOH, 50% aq. THF, then acidification to pH 3 with NaH2PO4 sol.,
95% (crude). TBS=tert-butyldimethylsilyl, DCC=N,N’-dicyclohexylcar-
bodiimide, DMAP=4-dimethylaminopyridine, Boc=tert-butoxycar-
bonyl.
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Communications
the molecule (see Scheme 2). In any event, purification of
synthetic 1 was accomplished by HPLC. Purified 1 was
chromatographically (HPLC, TLC) indistinguishable from
25
authentic micrococcin P1, and its optical rotation {½aꢁD
=
+ 688 (90% aq. EtOH, c = 0.45 gcmꢀ3; lit. ½aꢁD21 = + 63.78 (c =
1.19 gcmꢀ3, 90% aq. EtOH)[24]} and 1H and 13C NMR spectra
are coincident with those of authentic MP1. This established
the identity of 1 to the natural product.
In summary, chemical synthesis has now settled the
structural ambiguities that have surrounded micrococcin P1
during more than fifty years since its discovery. The methods
detailed herein are applicable to a number of other syntheti-
cally appealing thiopeptide antibiotics, and developments in
this domain will be the subject of future reports.
Received: February 2, 2009
Published online: April 30, 2009
Scheme 4. Synthesis of segment 27 of MP1. a) 11, HOBt, DCC,
Keywords: antibiotics · heterocycles · micrococcin P1 ·
thiopeptides
.
CH2Cl2, 84%; b) 4m HCl in dioxane, 100%; c) 7, DCC, CH2Cl2, 81%;
d) MsCl, Et3N, CH2Cl2, then DBU, 93%; e) 4n HCl in dioxane, then
H2O, THF, 100%. HOBt=1-hydroxy-1H-benzotriazole.
[1] Isolation from Bacillus pumilus: A. T. Fuller, Nature 1955, 175,
722. Micrococcin P is very similar, possibly identical, to an
antibiotic isolated from a Micrococcus species and named
“micrococcin” by: T. L. Su, Br. J. Exp. Path. 1948, 29, 473. No
structural work on the Su micrococcin appears to have been
described in the literature.
[2] a) G. Rosendahl, S. Douthwaite, Nucleic Acids Res. 1994, 22, 357;
b) E. Cundliffe, C. J. Thompson, Eur. J. Biochem. 1981, 118, 47;
recent work: c) J. M. Harms, D. N. Wilson, F. Schluenzen, S. R.
Connell, T. Stachelhaus, Z. Zaborowska, C. M. T. Spahn, P.
Fucini, Mol. Cell 2008, 30, 26 – 38.
[3] Review: S. Pestka in Antibiotics, Vol. 3 (Eds.: J. W. Corcoran,
F. E. Hahn) Springer, New York, 1975, p. 480 – 486.
[4] M. J. Rogers, E. Cundliffe, T. F. McCutchan, Antimicrob. Agents
Chemother. 1998, 42, 715 – 716.
[5] Synthetic studies: a) Y. Nakamura, C.-G. Shin, K. Umemura, J.
Yoshimura, Chem. Lett. 1992, 1005 – 1008; b) K. Okumura, M.
Shigekuni, Y. Nakamura, C.-G. Shin, Chem. Lett. 1996, 1025 –
1026; c) Y. Yonezawa, A. Konn, C.-G. Shin, Heterocycles 2004,
63, 2735 – 2746; d) E. A. Merritt, M. C. Bagley, Synlett 2007,
954 – 958.
[6] Reviews on thiopeptide antibiotics: a) M. C. Bagley, J. W. Dale,
E. A. Merritt, X. Xiong, Chem. Rev. 2005, 105, 685 – 714;
b) R. A. Hughes, C. J. Moody, Angew. Chem. 2007, 119, 8076 –
8101; Angew. Chem. Int. Ed. 2007, 46, 7930 – 7954. Key synthetic
work in this area: c) R. A. Hughes, S. P. Thompson, L. Alcaraz,
C. J. Moody, J. Am. Chem. Soc. 2005, 127, 15644 – 15651; d) M. C.
Bagley, K. E. Bashford, C. L. Hesketh, C. J. Moody, J. Am.
Chem. Soc. 2000, 122, 3301 – 3313; e) C. J. Moody, R. A. Hughes,
S. P. Thompson, L. Alcaraz, Chem. Commun. 2002, 1760 – 1761;
f) K. C. Nicolaou, M. Nevalainen, B. S. Safina, M. Zak, S. Bulat,
Angew. Chem. 2002, 114, 2021 – 2025; Angew. Chem. Int. Ed.
2002, 41, 1941 – 1945; g) K. C. Nicolaou, B. S. Safina, M. Zak,
S. H. Lee, M. Nevalainen, M. Bella, A. A. Estrada, C. Funke,
F. J. Zecri, S. Bulat, J. Am. Chem. Soc. 2005, 127, 11159 – 11175;
h) O. Delgado, H. M. Muller, T. Bach, Chem. Eur. J. 2008, 14,
2322 – 2339; i) H. M. Mꢀller, O. Delgado, T. Bach, Angew. Chem.
2007, 119, 4855 – 4858; Angew. Chem. Int. Ed. 2007, 46, 4771 –
4774.
Scheme 5. Total synthesis of micrococcin P1. a) BOP-Cl, Et3N, 27,
MeCN, 73%; b) LiOH, THF/H2O (1:1); c) 4n HCl in dioxane;
d) DPPA, Et3N, DMF, 24 h, 41% over 3 steps (b–d). DPPA=diphenyl-
phosphoryl azide.
micrococcin P1.[23] At this time, we believe that the unknown
material is likely to be a product of dehydration of the
threonine-derived thiazole segment comprising region b of
[7] M. N. G. James, K. J. Watson, J. Chem. Soc. C 1966, 1361 – 1371.
[8] M. P. V. Mijovic, J. Walker, J. Chem. Soc. 1960, 909 – 916. The
side-chain amino alcohol is levorotatory. This segment was
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originally assigned as alaninol, but it was later ascertained to be
isoalaninol. In either case, the levo isomer is the one of d-(R)
configuration: W. Klyne, J. Buckingham, Atlas of Stereochemis-
try: Absolute Configurations of Organic Molecules, 2nd ed.,
Oxford University Press, New York, 1978.
mixture of MP1 and MP2, but rather a mixture of Walker–
Lukacs MP1, Bycroft–Gowland MP1, plus the corresponding C2
epimers, plus the Walker–Lukacs and the Bycroft–Gowland
MP2s. Our NMR studies (Ref. [16]) excluded this possibility.
[15] M. A. Ciufolini, Y.-C. Shen, Org. Lett. 1999, 1, 1843 – 1846.
[16] B. Fenet, F. Pierre, E. Cundliffe, M. A. Ciufolini, Tetrahedron
Lett. 2002, 43, 2367 – 2370.
[17] a) M. C. Bagley, E. A. Merritt, J. Antibiot. 2004, 57, 829 – 831;
b) E. A. Merritt, M. C. Bagley, Synlett 2007, 954.
[18] M. A. Ciufolini, Y. C. Shen, J. Org. Chem. 1997, 62, 3804 – 3805.
[19] The preparation of this material is provided in the Supporting
Information.
[9] P. Brookes, A. T. Fuller, J. Walker, J. Chem. Soc. 1957, 689 – 699.
[10] The evidence that led to the assignment of this configuration as
(R), implying that the thiazole in question derived from d-valine,
is tenuous: B. M. Dean, M. P. V. Mijovic, J. Walker, J. Chem. Soc.
1961, 3394 – 3400.
[11] J. Walker, A. Olesker, L. Valente, R. Rabanal, G. Lukacs, J.
Chem. Soc. Chem. Commun. 1977, 706 – 708.
[12] B. W. Bycroft, M. S. Gowland, J. Chem. Soc. Chem. Commun.
1978, 256 – 258.
[20] Prepared by the method of: A. I. Meyers, E. Aguilar, Tetrahe-
dron Lett. 1994, 35, 2473 – 2476.
[13] a) C.-G. Shin, K. Okamura, M. Shigekuni, Y. Nakamura, Chem.
Lett. 1998, 139 – 140; b) K. Okamura, A. Ito, D. Yoshioka, C.-G.
Shin, Heterocycles 1998, 48, 1319 – 1324; c) K. Okumura, Y.
Nakamura, C.-G. Shin, Bull. Chem. Soc. Jpn. 1999, 72, 1561 –
1569; d) K. Okumura, T. Suzuki, Y. Nakamura, C.-G. Shin, Bull.
Chem. Soc. Jpn. 1999, 72, 2483 – 2490.
[21] B. M. Dean, M. P. V. Mijovic, J. Walker, J. Chem. Soc. 1961,
3394 – 3400.
[22] For a recent review see: H. Liang, Synlett 2008, 2554 – 2555.
[23] A sample of natural MP1 was kindly provided by Prof. E.
Cundliffe, University of Leicester.
[24] E. P. Abraham, N. G. Heatley, A. Brooks, T. Fuller, J. Walker,
Nature 1956, 178, 44 – 45.
[14] The alleged identity of both 4 and 5 to the natural product may
be tenable in the event that “micrococcin P” were not just a
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