Carbapenem Biosynthesis: Confirmation of Stereochemical Assignments and the Role of CarC in the Ring Stereoinversion Process from L-Proline Anthony Stapon, Rongfeng Li, and Craig A. Townsend* Contribution from the Department of Chemistry, The Johns Hopkins UniVersity, 3400 North Charles Street, Baltimore, Maryland 21218 Received January 20, 2003; E-mail: ctownsend@jhu.edu Abstract: (5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem -lactam antibiotics. It co-occurs with two saturated (3S,5S)- and (3S,5R)-carbapenam carboxylic acids. Confusion persists in the literature about the signs of rotation and absolute configurations of these compounds that is resolved in this paper. (3S,5S)-Carbapenam carboxylic acid was prepared from L-pyroglutamic acid to unambiguously establish its absolute configuration as identical to the natural product isolated from Serratia marcescens and from overexpression of the biosynthetic genes carAB in Escherichia coli. L-Proline labeled with deuterium or tritium at the diastereotopic C-5 methylene loci was shown to incorporate one label at the bridgehead of (3S,5S)-carbapenam carboxylic acid, but not into the “inverted” (3S,5R)-carbapenam carboxylic acid or the final carbapenem product. CarC, the third enzyme of the biosynthetic pathway required to synthesize the carbapenem, was demonstrated in cell-free studies to be dependent on R-ketoglutarate and ascorbate in keeping with weak sequence identities with other non-heme iron, R-ketoglutarate-dependent oxygenases. CarC mediated the stereoinversion of synthetic (3S,5S)-carbapenam carboxylic acid to the (5R)-carbapenem as judged by bioassay. These findings suggest that L-proline is desaturated to pyrroline-5-carboxylic acid prior to uptake into the biosynthetic pathway. The loss of the bridgehead hydrogen from the (3S,5S)-carbapenam during the ring inversion process to form the epimeric (3S,5R)-carbapenam and desaturation to the (5R)-carbapenem are proposed to be coupled by CarC to the reduction of dioxygen to drive the formation of these higher energy products, an unprecedented reaction for this enzyme class. Carbapenem-3-carboxylic acid (4) is the simplest of over 50 naturally occurring carbapenem -lactam antibiotics. 1 Members of this family and their derivatives are clinically important for their potent, broad-spectrum antibacterial activity and their resistance to -lactamases. 2 Early experiments established that the -lactam carbons of 4 are derived from acetate 3 and the fused pyrrolidine carboxylic acid arises ultimately from glutamate. 3 Assembly of its primary metabolic precursors into the carbap- enem nucleus takes place through the action of just three enzymes, CarA, B, and C (Scheme 1). 4 This efficient biosyn- thetic process is accompanied by a remarkable stereochemical inversion at C-5 mediated by CarC. 4 While the absolute configurations of 2, 3, and 4 were in part misassigned initially, 5 stereochemical correlations by Bycroft corrected these to those shown in Scheme 1. 6 A chemical logic can be discerned in this pathway where a natural L-amino acid is utilized in the formation of 1 by CarB and cyclized to carbapenam 2 by CarA. CarC then acts to invert the absolute configuration of the bridgehead to 5R essential to the ultimate biological activity of the antibiotic and to subtly raise the strain energy of the bicyclic system by placing the C-3 carboxyl endo. 4 Unfortunately, the amended stereochemical assignments of Bycroft, which we relied upon in the interpretation of the biochemical experiments summarized in Scheme 1, 4 have been called into question by Ogasawara et al., 7 and are further clouded by reversal of the absolute stereochemistry of 2 to (3R,5R) in a 1998 review. 8 We describe in this paper a third stereochemical correlation in which 2 as its p-nitrobenzyl (PNB) ester was prepared by total synthesis from L-pyroglutamic acid and compared to the same product isolated from wild-type Serratia marcescens and a recombinant Escherichia coli bearing carAB in an overexpression vector. These findings unambiguously confirm the corrected absolute configurations made for 2, 3, and 4 by Bycroft as depicted in Scheme 1. 4 In addition, we address the issue of the cryptic stereochemical inversion at C-5 that occurs in the CarC-catalyzed transformation (1) Parker, W. L.; Rathnum, M. L.; Wells, J. S. J.; Trejo, W. H.; Principe, P. A.; Sykes, R. B. J. Antibiot. 1982, 35, 653-660. (2) Bradley, J. S.; Garau, J.; Lode, H.; Rolston, K. V. I.; Wilson, S. E.; Quinn, J. P. Int. J. Antimicrob. Agents 1999, 11, 93-100. (3) Bycroft, B. W.; Maslen, C. J. Antibiot. 1988, 41, 1231-1242. (4) Li, R.; Stapon, A.; Blanchfield, J. T.; Townsend, C. A. J. Am. Chem. Soc. 2000, 122, 9296-9297. (5) Bycroft, B. W.; Maslen, C.; Box, S. J.; Brown, A. G.; Tyler, J. W. J. Chem. Soc., Chem. Commun. 1987, 1623-1625. (6) Bycroft, B. W.; Chhabra, S. R. J. Chem. Soc., Chem. Commun. 1989, 1989, 423-425. (7) Tanaka, H.; Sakagami, H.; Ogasawara, K. Tetrahedron Lett. 2002, 43, 93- 96. (8) McGowan, S. J.; Bycroft, B. W.; Salmond, G. P. C. Trends Microbiol. 1998, 6, 203-208. Published on Web 06/18/2003 8486 9 J. AM. CHEM. SOC. 2003, 125, 8486-8493 10.1021/ja034248a CCC: $25.00 © 2003 American Chemical Society