C O M M U N I C A T I O N S
Figure 1. Biosynthesis of rifamycin B. RifA is primed with AHBA, derived from amino-DHS by action of AHBA synthase (RifK). The loading module
consists of an adenylation domain (A) for AHBA activation and a peptidyl carrier protein (PCP). Subsequently, 10 cycles of C2 elongation are carried out
by RifA-RifE. Each cycle plus reductive modifications is catalyzed by one module composed of a â-keto synthase domain (KS), an acyl transferase domain
(AT), an acyl carrier protein (ACP), and optionally a keto reductase domain (KR) or a dehydratase domain (DH, inactive in module 1). Premature chain
release after module 3 of RifA gives rise to the shunt product P8/1-OG (or the derived lactone 2). Macrolactamization of the fully assembled polyketide
chain is catalyzed by amide synthetase RifF leading to proansamycin X, which undergoes post-PKS modifications finally yielding rifamycin B.
Scheme 2 In Vivo Transformations
Supporting Information Available: Synthetic procedures, protein
expression, and purification conditions, in vitro labeling procedure,
characterization data, LC-MS traces, KR sequence alignment, and
modeling results. This material is available free of charge via the
References
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nonnaturally configured substrates.12 Therefore, substrate 12 likely
contains the natural configuration of intermediates in the rifamycin
PKS pathway. Additionally, the keto reductase domain of module
1 can be expected to be D-selective based on sequence analysis,13
thus establishing that, for example, C7 in lactone 2 is S-configured
(as in 11 and 12). The P8/1-OG-derived lactone 2 shows a large
1H NMR coupling constant (>9 Hz) for this benzylic proton.4a
Computational minimization of conformational energies for the syn-
and anti-diastereomers of lactone 2 led to a dihedral angle of
173.86° for the anti-2, which is in agreement with the observed
coupling constant (Karplus calculation: 9.1 Hz; measured: 9.0 Hz
in d6-DMSO). This additional evidence underscores that anti-
diketide 12 contains the natural configuration of biosynthetic
intermediates in the rifamycin PKS pathway.
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HMPA can be replaced by DMPU (50% yield after chromatography).
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Acknowledgment. This research was supported by a grant from
the NIH (AI38947 to C.K. and GM072293 to N.A.S.). I.V.H. is a
recipient of a fellowship from the Deutsche Forschungsgemeinschaft
and M.A.R. of a Stanford Research Fellowship. We thank the
Stanford MS facility and Dr. L. O. Haustedt and Dr. M. Sukopp
for support with MacroModel. We also thank Dr. H. G. Floss for
providing strain HGF003.
(11) Danada, H.; Hansen, M. M.; Heathcock, C. H. J. Org. Chem. 1990, 55,
173. The anti/syn-diastereoselectivity was at best 3.5:1.0.
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Biol. Chem. 2003, 278, 42020 and references therein.
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