Unambiguous Natural Product ID

Significance of the topic

Identifying exact molecular formulas of natural products is critical for dereplication, avoiding rediscovery of known compounds and accelerating structural elucidation, thus saving time and resources in drug discovery and metabolomics.

Objectives and study overview

This work demonstrates how high-resolution mass spectrometry combined with isotopic fine structure analysis can unambiguously determine the molecular formula of the natural product echinomycin A, illustrating a rapid approach for novel compound discovery and dereplication.

Applied methodology

• Analysis of isotopic fine structures (IFS) exploiting mass defects and natural isotope abundances.
• Comparison of resolved IFS data from a 12T magnetic resonance mass spectrometer (MRMS) versus quadrupole time-of-flight (QTOF) MS.
• Use of SmartFormula to generate candidate formulas under elemental constraints guided by NMR-derived bounds.
• Simulation of isotope patterns with Simulate Pattern to match experimental spectra and discriminate among candidates based on relative peak intensities.

Used instrumentation

• HPLC: Shimadzu LC-20AP, Luna C18 column (250 × 10 mm, 5 μm), gradient 25–50 % MeCN/H₂O with 0.1 % acetic acid.
• MS: Bruker 12T MRMS (ESI positive mode) for high-resolution spectral acquisition; Bruker QTOF MS for comparison.
• Data processing: Bruker Compass DataAnalysis 4.4 with SmartFormula and Simulate Pattern modules.

Main results and discussion

• MRMS data resolved multiple isotopologues ([M+H]+, [M+H+1]+, [M+H+2]+, [M+H+3]+), while QTOF lacked sufficient resolution for IFS.
• SmartFormula generated seven candidate formulas within 2 ppm mass error under constraints C48–C54, S ≤4, N ≤12 based on NMR pre-screening.
• Overlay of simulated IFS patterns for three leading candidates revealed mismatches in sulfur- and nitrogen-derived fine-structure peaks, enabling exclusion of incorrect formulas.
• Matching of experimental and simulated IFS confirmed the formula corresponding to echinomycin A (C51H64N12O2S2), later validated by detailed NMR.

Benefits and practical applications

• Provides rapid, unambiguous molecular formula assignment without isotopic labeling.
• Enhances dereplication workflows in natural product libraries, reducing redundant isolation efforts.
• Supports structural elucidation by supplying definitive elemental composition prior to extensive NMR studies.

Future trends and applications

• Integration of IFS-based formula assignment into automated metabolomics and natural product discovery pipelines.
• Development of machine-learning algorithms to predict IFS patterns and accelerate candidate discrimination.
• Expansion of high-resolution MS instrumentation accessibility, enabling broader adoption in pharmaceutical and environmental analysis.
• Combination with chromatographic and ion mobility separations for multidimensional characterization of complex mixtures.

Conclusion

Isotopic fine structure analysis using high-resolution MRMS, coupled with advanced data-processing tools, offers a powerful, efficient route to unambiguously determine molecular formulas of natural products, streamlining dereplication and structural elucidation tasks in analytical chemistry.

References

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