Structure elucidation of uniformly ¹³C labeled small molecule natural products

Abstract

Utilization of 2 H, 13 C, and 15 N isotopically labeled proteins and peptides is now routine in biomolecular NMR investigations. The wide-spread availability of inexpensive, uniformly 13 C enriched glucose now makes it possible to isolate uniformly 13 C labeled natural products from microbial fermentation. We now wish to describe an approach for the rapid structural characterization of uniformly 13 C labeled natural products that avoids the pitfalls of relying on parameters typically employed in biomolecular NMR studies.

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research

“…Finally, Gross and coworkers conducted labeling experiments employing doubly 13 C-labeled acetate and confirmed in this way the structure by the determination and localization of intact acetate units via measurement of J C,C 37. Similarly, Reibarkh et al have emphasized the utility of uniform 13 C labeling of microbial NPs, which becomes feasible via the availability of uniformly 13 C labeled glucose 52…”

Tryptorubin A: A Polycyclic Peptide from a Fungus-Derived Streptomycete

“…A 13 C- 13 C COSY of 13 C-labeled tryptorubin A ( 1 ) provided the carbon–carbon connectivity for most of the structure, including the tryptophan and tyrosine. 11 …”

“…In order to confirm the carbon connectivity in these two amino acids, fermentation of strain CLI2509 in ISP2 medium containing 13 C-labeled glucose produced 13 C-labeled tryptorubin A ( 1 ). A 13 C- 13 C COSY of 13 C-labeled tryptorubin A ( 1 ) provided the carbon–carbon connectivity for most of the structure, including the tryptophan and tyrosine …”

“…A 13 C- 13 C COSY of 13 C-labeled tryptorubin A (1) provided the carbon−carbon connectivity for most of the structure, including the tryptophan and tyrosine. 11 To confirm the location of the hydroxyl groups, tryptorubin A (1) was acetylated (see Supporting Information). Acetylation of tryptorubin A (1) resulted in a product with three acetyl groups, but NMR analysis did not allow the unequivocal location of the acetyl groups to be determined.…”

Epipyrone A, a Broad-Spectrum Antifungal Compound Produced by Epicoccum nigrum ICMP 19927

“…While the majority of 1 H and 13 C NMR chemical shifts observed for compound 1 (Figure 1) were similar to those reported for synthetic epipyrone A (compound 2, Figure 1) [22], resonances associated with the pyrone ring were noticeably different ( Table 3). To facilitate complete assignment of the carbon skeleton of the molecule, the fungus was cultured on minimal media supplemented with [U-13 C]glucose [23] Purification of the disalt using RP-8 column chromatography again afforded 1 (Figure 1), this time highly enriched with 13 C. Connectivity of the contiguous carbon backbone of compound 1 was determined by analysis of a magnitude mode 13 C- 13 C COSY (Bruker pulse sequence cosydcqf) data set [24]. Tracing the carbon-carbon connectivity, derived from the neighbouring carbon atom correlations observed in the 13 C-13 C COSY NMR experiment and a 1 H- 13 C HMBC NMR experiment ( Figure S7), afforded unambiguous assignment the backbone of the molecule.…”