Metabolite Synthesis

Synthesis of O-, N- and acyl-glucuronides


Hypha solves the challenges in synthesising glucuronides using multiple tools from our one-stop metabolite shop. These include microbial biotransformation, mammalian S9 / microsome preparations and our proprietary late-stage chemical synthesis screens, which are scalable to provide gram amounts of O-, acyl, N– and N-carbamoyl glucuronides, as well as other conjugates such as glycosides and sulfates. Diglucuronides and secondary glucuronides can also be produced by these techniques. Microbial biotransformation is particularly suited to producing glucuronides that arise from sequential reactions, such as hydroxylation followed by O-glucuronidation.

We apply the one-stop shop techniques in parallel to determine the most efficient and cost-effective conversion to the glucuronide(s) of interest before proceeding to scale-up. This can determine the optimum system for production of a single glucuronide, or where multiple glucuronides are required, the best system for scale-up of each conjugate. For example, in one client project, screening revealed that 3 of 4 required glucuronides could be made using late-stage chemical synthesis methods, with the other only provided through a liver S9 biotransformation route.

Purification of glucuronides

We are experienced in purifying glucuronides from biological matrices and complex mixtures. This is possible even where small amounts of metabolite are present, such as in one client project where we isolated 0.4 mg of a glucuronide from 24L of urine. The pure material was subsequently used to determine the structure by 2D NMR spectroscopy.

Why is glucuronidation an important route of drug metabolism?

Glucuronidation is the predominant phase 2 metabolic route for a variety of drugs and agrochemicals catalysed by UDP-glucuronosyltransferases (UGTs) into hydrophilic conjugates that are eliminated from the cell by efflux transporters. UGTs are a family of membrane-bound proteins located in the endoplasmic reticulum in the cells of various tissues, with UGT 1A and UGT 2B subfamilies responsible for most drug metabolism.

Although many UGTs are expressed in human liver, some are also expressed extrahepatically and some are only expressed in certain organs (Ge et al., 2016). UGTs catalyse the transfer of glucuronic acid to a variety of O-, N-, C– and S– containing parent substrates and have become increasingly of interest in the elimination of drugs.

Although generally pharmacologically inactive, some acyl glucuronides of carboxylic acid containing drugs can form acyl migration isomers, which result in transacylation and glycation reactions with proteins causing idiosyncratic adverse drug reactions.

Several classes of glucuronides have been shown to interact with CYPs, in particular CYP2C8 due to its distinctive active site (Ma et al., 2017).

Some glucuronides are subject to enterohepatic recycling, such as the O-glucuronide of epacadostat made by one of Hypha’s microbial strains (Boer et al., 2016), where hydrolysis of the glucuronide by gut bacteria can occur. This is followed by the absorption of the drug back into systemic circulation, thereby leading to a prolonged elimination half-life and altered pharmacokinetics and pharmacodynamics. 

Glucuronide synthesis methods infographic

Why access glucuronides?

For pharmacological testing

For definitive structure elucidation

As authentic standards for bioanalysis & quantification

For stability / reactivity testing

For role in any DDIs arising from transporter-mediated effects

Involvement in enterohepatic recirculation


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In recent years, FDA guidance has advised initiating human metabolite profiling earlier in drug development, emphasizing the importance of metabolite identification and quantification to evaluate a drug metabolite’s safety and pharmacological activity. Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator in Phase 2 clinical trials for diabetic nephropathy and heart failure with preserved ejection fraction (HFpEF). During studies on metabolism of praliciguat in preclinical species and in human hepatocytes, a prominent direct O-glucuronide metabolite was detected.

We’ve observed an increase in requests for synthesis of N-glucuronides over the last couple of years. We speculate that this may be due to the increasing use of N-heterocyclic chemistry in the design of new small molecule drugs, and pan company strategies to reduce CYP metabolism. The situation is further complicated by the high interspecies variability in formation of some N-glucuronides, especially aliphatic tertiary amines and aromatic N-heterocycles. UGT1A4 and UGT2B10 are key enzymes responsible for N-glucuronidation reactions in humans, rates of which can be much higher than in other animals. To compound this, synthesis of N-glucuronides is not always straightforward, and can be further muddied by metabolite stability issues, complicating interpretation of data.

The FDA’s 2016 MIST guidance states that phase 2 conjugates are generally pharmacologically inactive, however where a potentially toxic conjugate, such as an acyl glucuronide is formed, additional safety assessments may be needed. Idiosyncratic drug toxicity of carboxylic acid-containing drugs can be caused by the formation of reactive acyl glucuronides,1 which have the ability to directly acylate proteins and undergo intramolecular rearrangement producing reactive aldehydes leading to protein glycation.

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

Hypha Discovery did a fantastic job synthesizing N- and O- glucuronides of our clinical stage drug substance. The project updates were detailed, our questions were answered in a timely manner, and the overall timeline was maintained. Hypha was highly recommended to us and I would not hesitate to recommend them to a colleague.

Joshua Day Ph.D, Director of Chemistry

Crestone, Inc., Colorado, USA

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