Avoiding N-nitrosodimethylamine formation in metformin pharmaceuticals by limiting dimethylamine and nitrite.

Since late 2019, concerns regarding trace levels of the probable human carcinogen N-dimethylnitrosamine (NDMA) in Metformin-containing pharmaceuticals have been an issue if they exceeded the maximum allowable intake of 96 ng/day for a medicine with long-term intake. Here, we report results from an extensive analysis of NDMA content along the active pharmaceutical ingredient (API) manufacturing process as well as two different drug product manufacturing processes. Our findings confirm that Metformin API is not a significant source of NDMA found in Metformin pharmaceuticals and that NDMA is created at those steps of the drug product manufacturing that introduce heat and nitrite. We demonstrate that reduction of nitrite from excipients is an effective means to reduce NDMA in the drug product. Limiting residual dimethylamine in the API has proven to be another important factor for NDMA control as dimethylamine leads to formation of NDMA in the drug products. Furthermore, analysis of historical batches of drug products has shown that NDMA may increase during storage, but the levels reached were not shelf-life limiting for the products under study.

Tyrosine O-prenyltransferases TyrPT and SirD displaying similar behavior toward unnatural alkyl or benzyl diphosphate as their natural prenyl donor dimethylallyl diphosphate.

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily are involved in the biosynthesis of secondary metabolites and contribute as modification enzymes significantly to structural diversity of natural products. They show usually broad specificity toward their aromatic substrates with regiospecific prenylations on aromatic rings. However, most members of this superfamily exhibit a high specificity toward their prenyl donors and usually accept exclusively dimethylallyl diphosphate (DMAPP). Recently, several indole prenyltransferases from this family were also demonstrated to accept unnatural DMAPP analogs such as methylallyl, 2-pentenyl and benzyl diphosphate for alkylation, or benzylation of the indole ring. Partial or complete shift of the substitution position was observed for these enzymes. In this study, we report the acceptance of these DMAPP analogs by two tyrosine O-prenyltransferases TyrPT from Aspergillus niger and SirD from Leptosphaeria maculans for alkylation or benzylation of tyrosine and derivatives. NMR and mass spectrometry (MS) analyses of nine isolated enzyme products confirmed the regiospecific O- or N-alkylation or benzylation at position C-4 of the aromatic ring, which is the same prenylation position of these enzymes in the presence of DMAPP.

Regiospecific benzylation of tryptophan and derivatives catalyzed by a fungal dimethylallyl transferase.

A big challenge in organic synthesis is to reach a high regioselectivity. Enzymes catalyze usually highly regiospecific reactions and can function as ideal biocatalysts for such purposes. Some secondary metabolite enzymes can even use distinctly different unnatural substrates and expand therefore their potential usage in chemoenzymatic synthesis. We report here the acceptance of benzyl diphosphate as an alkyl donor by the fungal dimethylallyl transferase FgaPT2 and the regiospecific enzymatic benzylation of tryptophan and several analogues.

Catalytic mechanism of stereospecific formation of cis-configured prenylated pyrroloindoline diketopiperazines by indole prenyltransferases.

Indole prenyltransferases AnaPT, CdpC3PT, and CdpNPT are known to catalyze the formation of prenylated pyrroloindoline diketopiperazines from tryptophan-containing cyclic dipeptides in one-step reactions. In this study, we investigated the different stereoselectivities of these enzymes toward all the stereoisomers of cyclo-Trp-Ala and cyclo-Trp-Pro. The stereoselectivities of AnaPT and CdpC3PT mainly depend on the configuration of the tryptophanyl moiety in the substrates, and they usually introduce the prenyl moiety from the opposite sides. CdpNPT showed lower stereoselectivity, and the structure of the second amino acid moiety in the substrates is important for the stereospecificity in its enzyme catalysis. Moreover, we determined the crystal structure of AnaPT in complex with thiolodiphosphate and compared it with the known structures of CdpNPT. Our results clearly revealed the presence of an indole binding mode that has so far not been characterized.

Breaking cyclic dipeptide prenyltransferase regioselectivity by unnatural alkyl donors.

The behavior of five cyclic dipeptide prenyltransferases, responsible for C2-regular, C2-reverse, or C3-reverse prenylation, was investigated in the presence of the unnatural alkyl donors monomethylallyl and 2-pentenyl diphosphate. Both substrates were well accepted by the tested enzymes. Interestingly, C2-reverse and C3-reverse monoalkylated derivatives were identified as enzyme products in all of the enzyme assays. These findings indicate their similar reaction characteristics in the presence of unnatural alkyl donors.

Expansion of enzymatic Friedel-Crafts alkylation on indoles: acceptance of unnatural ß-unsaturated allyl diphospates by dimethylallyl-tryptophan synthases.

Prenyltransferases of the dimethylallyl-tryptophan synthase (DMATS) superfamily catalyze Friedel-Crafts alkylation with high flexibility for aromatic substrates, but the high specificity for dimethylallyl diphosphate (DMAPP) prohibits their application as biocatalysts. We demonstrate here that at least one methyl group in DMAPP can be deleted or shifted to the δ-position. For acceptance by some DMATS enzymes, however, a double bond must be situated at the ß-position. Furthermore, the alkylation position of an analogue can differ from that of DMAPP.

Structure and catalytic mechanism of a cyclic dipeptide prenyltransferase with broad substrate promiscuity.

Fungal indole prenyltransferases (PTs) typically act on specific substrates, and they are able to prenylate their target compounds with remarkably high regio- and stereoselectivity. Similar to several indole PTs characterized to date, the cyclic dipeptide N-prenyltransferase (CdpNPT) is able to prenylate a range of diverse substrates, thus exhibiting an unusually broad substrate promiscuity. To define the structural basis for this promiscuity, we have determined crystal structures of unliganded CdpNPT and of a ternary complex of CdpNPT bound to (S)-benzodiazepinedione and thiolodiphosphate. Analysis of the structures reveals a limited number of specific interactions with (S)-benzodiazepinedione, which projects into a largely hydrophobic surface. This surface can also accommodate other substrates, explaining the ability of the enzyme to prenylate a range of compounds. The location of the bound substrates suggests a likely reaction mechanism for the conversion of (S)-benzodiazepinedione. Structure-guided mutagenesis experiments confirm that, in addition to (S)-benzodiazepinedione, CdpNPT can also act on (R)-benzodiazepinedione and several cyclic dipeptides, albeit with relaxed specificity. Finally, nuclear magnetic resonance spectroscopy demonstrates that CdpNPT is a C-3 reverse PT that catalyzes the formation of C-3ß prenylated indolines from diketopiperazines of tryptophan-containing cyclic dipeptides.