Ion Mobility Separation Of Marine Natural Products: Characellides from the Deep-sea Sponge Characella pachastrelloides

Importance of the Topic

A deep-sea sponge, Characella pachastrelloides, produces unique glycolipopeptides called characellides that show promising bioactivity. Structural characterization of these compounds is challenging due to their low abundance and conformational similarity. Ion mobility spectrometry (IMS) offers an orthogonal separation dimension beyond chromatography and mass measurement, enabling differentiation of isomeric natural products when conventional nuclear magnetic resonance is limited.

Objectives and Overview of the Study

This study aimed to evaluate the capability of high-definition ion mobility spectrometry (HDMSE) and cyclic IMS to separate and characterize characellide isomers. Major goals included:

• Isolation of characellides A through D from C. pachastrelloides extract
• Separation of conformational isomers using multiple IMS passes
• Determination of collision cross-section (CCS) values for structural insight
• Use of feature-based molecular networking to discover additional related analogs

Methodology and Instrumentation

Sample Preparation and Chromatography

• Collection of sponge biomass from 809 m depth off southwest Ireland
• Powdered biomass extracted with MeOH:CH2Cl2 (1:1), fractionated via RP-C18 VLC
• LC separation on ACQUITY Premier HSS T3 column (2.1×100 mm, 1.8 µm) with water/ACN + 0.1% formic acid gradient

Ion Mobility and Mass Spectrometry

• Waters SELECT SERIES cyclic IMS system with ESI+ source (2 kV capillary voltage)
• Single- and multi-pass IMS (1 to 6 passes) achieving up to ~159 resolving power (Ω/ΔΩ)
• MS acquisition range m/z 50–1200 at ~60 000 FWHM resolution
• AllCCS software for CCS prediction and comparison

Main Results and Discussion

IMS Separation of Isomeric Characellides

• Characellides A and B (C41H66N6O14, m/z 867.468) were baseline separated after multiple IMS passes; observed CCS values 289.5 Å2 and 286.8 Å2
• Characellides C and D (C40H64N6O14, m/z 853.451) resolved with six IMS passes, CCS values around 286.8 Å2 and 284.4 Å2

Molecular Networking and Discovery of New Analogs

• Feature-based molecular networking on the GNPS platform revealed additional peaks at m/z 849–881
• Six new characellide-related compounds were annotated by MS/MS and CCS matching

Benefits and Practical Applications

IMS delivers rapid separation of conformational isomers that are otherwise inseparable by conventional LC or too dilute for NMR. Determined CCS values provide structural dimensions that aid annotation of unknown marine natural products. Combining IMS with molecular networking accelerates discovery of novel bioactive compounds in complex extracts.

Future Trends and Opportunities

The integration of high-resolution IMS with advanced data analysis will expand collision cross-section databases and improve structural predictions. Coupling IMS with machine learning and enhanced fragmentation workflows promises automated isomer classification. These developments will strengthen marine drug discovery and complex mixture analysis in environmental and pharmaceutical research.

Conclusion

This work demonstrates that cyclic IMS with multiple passes effectively separates isomeric characellides and uncovers new analogs from deep-sea sponges. Measured CCS values complement MS/MS and support structural assignments when NMR is impractical. IMS-based workflows offer a powerful tool for characterizing low-abundance natural products.

References

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