Structure Elucidation

Overviewtambromycin

Structure elucidation of natural products and unexpected reaction products are routinely carried out in IMSERC. In ~90% pure mixtures, compound structure can be determined with as little as 1 mg material, although ~100 mg of material is ideal for most projects. Structure elucidation projects can be carried out on a fee for service basis or in collaboration with research groups. The Kelleher group example to the right was determined using mass spectrometry and NMR spectroscopy. Only ~1 mg of sample was used for the NMR experiments. The full details of this project are published in ACS Central Science (DOI: 10.1021/acscentsci.5b00331). Fees for structure elucidation projects are highly dependent on the complexity of the project, so communication with IMSERC staff through out projects is important. Frequently, it will be less expensive to engage IMSERC staff as these scientists are experts in experiment design and data interpretation. Fees for common services can be found here.

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Process Flow (Click on each step for more details)

  1. Isolation and Purification
  2. Initial Characterization
  3. Dereplication
  4. Structure Elucidation
  5. Validation

Detailed Process Flow

  1. Isolation and Purification
    •       This step is typically carried out in the research labs and involves production of new compounds and purification by liquid-liquid extraction or solid phase extraction to isolate compounds.
    •       Further purification can be completed using sublimation, crystallization or preparative HPLC.
    •       Goal should be to isolate ~ 100 mg material if multiple crystallization strategies will be used, although as little as 5 mg can be used for crystallization and less for NMR/MS based elucidation projects. For more information on crystallizing compounds, click here.

  2. Initial Characterization
    •       These steps can typically be completed in less than 2 hours of staff time at which point, the complexity of the problem can be determined.
    •       Obtain FTIR spectrum to determine functional groups and likely solvents.
    •       Obtain accurate mass data to determine molecular formula if possible.
    •       Obtain 1D proton, carbon and HSQC data.

  3. Dereplication

    CSD Refcode TUWMOP – the 800,000th entry added to the CSD

    •       The goal of this process is to compare the “new” compound to existing published structures and relies heavily on the use software and databases IMSERC has purchased for this activity.
    •       X-Ray Crystallography: If single crystals that are 50-100 μm on two sides can be produced, a unit cell can be determined and compared to those loaded in the Cambridge Structure Database which currently has over 850,000 small molecule and metal organic structure and is growing by > 50,000 structures each year. For details on dereplication by X-Ray crystallography, click here. If initial data yields good quality data, the crystallographer will automatically collect a full data set.
    •       Mass Spectrometry: With as little as 1 ug of sample, MS analysis using accurate mass and fragmentation can be used to both determine molecular formulae of organic compounds and possible structures. For samples that are amenable for analysis by GCMS, IMSERC provides access to the NIST version 2.0 library which has 1 mega-compounds. For LCMS samples, possible known structures can be found using ACD/Spectrus and the ChemSpider database which contains over 20 mega-compounds. Even when there is insufficient data to identify compounds, fragmentation patterns provide critical data on likely functional groups. For details on dereplication by MS, click here.
    •      NMR Spectrometry: When a single formula or several formulae are provided, ChemSpider can be used to further narrow structure based on proton and carbon NMR shifts. For more information on dereplication by NMR, click here.

  4. Structure Elucidation
    •       Single crystal X-Ray diffraction is the gold standard for structure elucidation and if high quality crystals can be obtained, is the best method to use. To learn more about submitting samples for X-Ray analysis, click here.
    •       In cases where crystals cannot be formed or when there is insufficient material, molecular structure elucidation can be performed using mass spectrometry and NMR data. IMSERC can collaborate with you to determine compound structure using ACD Elucidator software. Examples of compounds solved using this method can be found at http://www.acdlabs.com/comm/elucidation/past_elucidations.php.

  5. Validation
    •       Crystallographically derived solutions typically do not require additional validation.
    •       NMR/MS based structures can be published, but important structures will eventually need to be validated through a total synthesis.

 

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