What We Do

Structure Eludication and NMR

Structure Elucidation by NMR

Hypha are specialists in the structure elucidation of metabolites and other small molecules using NMR spectroscopy. We use cryoprobe technology to gather NMR data on only small amounts of material, and are expert in interpreting spectra obtained from a wide variety of techniques. Our reports detailing the assignment of structures are written to publication standard and are used by clients in submissions to the FDA.

HMBC-spectrum-of-4-hydroxyimidacloprid
The image above shows a portion of the HMBC spectrum of 4-hydroxyimidacloprid and the use of the correlations observed in the structure elucidation of this metabolite.

Structure elucidation by NMR relies on the extensive experience of several of the company’s scientists, developed as natural products chemists. These experiences have been extended through identification of diverse oxidised and conjugated drug metabolites produced by biotransformation and chemical synthesis.

NMR data acquisition is outsourced to partner organisations enabling access to high specification instruments and quantitative NMR purity determination. One of our partners uses a 700 MHz instrument equipped with a 1.7 mm microcryoprobe, enabling acquisition of structure elucidation datasets with as little as 10-30 μg of purified metabolite.

The standard structure elucidation dataset includes 1H, COSY and the heteronuclear shift correlation HSQC and HMBC spectra; NOESY spectra to probe through-space rather than through-bond 1H-1H interactions, if deemed appropriate, and 13C NMR spectra are acquired if needed, provided sufficient material is available. Compounds are anonymised for the data acquisition process and all data are interpreted by scientists at Hypha; access to structural data within the company is restricted for confidentiality purposes.

Much more detail about the use of NMR in metabolite structure elucidation can be found in the following book chapter written by Hypha scientists:

Evans, L., Phipps, R., Shanu-Wilson, J., Steele, J., Wrigley, S., 2020. Chapter 4 Metabolite generation and characterization by NMR. In: Identification and quantification of drugs, metabolites, drug metabolizing enzymes and transporters. Second edition. Eds. Shuguang Ma and Swapan Chowdhury. Elsevier Science. ISBN: 9780128200186

Resources

Explore our library of resources comprising brochures, case studies, posters and publications about the work we do.

It is well known that metabolites of drug compounds may have different efficacy and properties to that of the parent compound. Investigation and production of these metabolites is critical for exploration and understanding of SAR, and to ensure thorough patent coverage. Metabolites can also be sourced to create antibodies used in development of assays for therapeutic drug monitoring.

If clearance mechanisms of the test drug results in sufficient quantities of the major metabolites in biological material such as faeces or urine, purification and subsequent identification of metabolites from such matrices is possible. One such project undertaken at Hypha resulted in tens of milligrams of the R- and S-O-glucuronides of carisbamate, a neuromodulator developed by SK Life Science, which were purified to >95% purity from 150 ml of urine using a three-step purification method.

In Chapter 4 of the book on “Identification and quantification of drugs, metabolites, drug metabolizing enzymes and transporters”, Hypha authors summarise the different methods employed for producing metabolites of drugs, illustrated with representative examples from the literature and work undertaken at Hypha. The chapter also includes a discussion and examples of the use of NMR spectroscopy for structure elucidation of metabolites.

Test tubes with dripper
251A3171-DE01-48EB-AA27-BD82BC5AC10DCreated with sketchtool.

Hypha Discovery has been a valuable metabolite ID partner. Hypha have provided biotransformation, metabolite purification and structure elucidation answers to some of our most challenging metabolism and metabolite ID problems. We really appreciate the breadth of expertise available at Hypha Discovery and will definitely reach out for future work.

Director of DMPK

US Pharma Company

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