Boron NMR as a Method to Screen Natural Product Libraries for B-Containing Compounds

Abstract

Natural products are biologically relevant metabolites exploited for biomedicine and biotechnology. The frequent reisolation of known natural products questions whether existing discovery models are still capable of identifying novel compounds. As innovative NMR-based screening techniques can help overcome these challenges, we applied a phase cycling composite pulse sequence to ¹¹B NMR experiments to enhance their sensitivity to screen libraries for novel boron-containing molecules. Aplasmomycin and autoinducer-2 were detected in crude and enhanced microbial fractions, via their boron signals, as proof of concept. Subsequently, a screen of 21 crude plant and 50 crude marine microbial extracts were chosen at random and analyzed with the optimized ¹¹B experiment for feasibility as a high throughput discovery method. Eight of the plant samples and 13 of the microbial samples were identified as boron-containing, suggesting that there is a higher presence of boron metabolites available from natural sources than previously known due to a lack of appropriate discovery methods. As a result, we believe that this optimized ¹¹B NMR experiment can serve as a robust method for quick and facile discovery of novel boron-containing metabolites from a variety of natural sources.

Figure 1

Examples of boron-containing metabolites.

Figure 2

3.93 mM 5 in CD₃OD, ns = 128, in borosilicate and quartz NMR tubes. The ¹¹B proton decoupled experiment with the standard zgig pulse sequence in both borosilicate (a) and in quartz (b) tubes pick up extraneous probe noise from outside the NMR coil, causing large, asymmetrical peaks, which can overshadow compound peaks. Use of the zgbs pulse sequence with the proton decoupled ¹¹B NMR in quartz tubes (c) is best for suppressing extraneous probe noise.

Figure 3

Structures of additional boronic compounds used to illustrate the effectiveness of the ¹¹B NMR experiment with the applied pulse sequence.

Figure 4

3.22 mM of 6 in CDCl₃ via the standard Bruker ¹¹B decoupling NMR experiment in quartz NMR tubes (blue), ns = 128, yields the boron hump, overshadowing distinctive chemical signals. Application of the zgbs pulse sequence (red), ns = 128, eliminates background resonance noise allowing for clear identification of boron peaks.

Figure 5

¹¹B NMR of 3.22 mM 6 in CDCl₃ via the standard ¹¹B proton decoupled experiment, ns = 128, throughout various stages of purification. Significant reduction of extraneous signals is observed with the pulse sequence resulting in clearly distinguishable boron peaks.

Figure 6

¹¹Bzgbsig proton decoupled NMR of V. harveyi in D₂O, ns = 512. Closer inspection of the peak reveals pentet splitting (insert) from the exchange between D₂O and boron on 3, further proving detection of the molecule.

Figure 7

Percent distribution of boron found in the plant and microbial samples screened using the optimized ¹¹B NMR experiment.