Biocatalysis

Primary amination of C(sp3)–H bonds

J. Am. Chem. Soc. https://doi.org/10.1021/jacs.0c03428 (2020)

Primary amines play an important role in the biological, chemical and pharmaceutical world. Interestingly, the direct primary amination of C(sp3)–H bonds remained unknown — both in nature and synthetic chemistry.

Now, Zhi-Jun Jia, Shilong Gao and Frances H. Arnold report C(sp3)–H primary aminases as new-to-nature enzymes that can do exactly that. Specifically, the engineered enzymes allow the selective primary amination of tertiary, secondary and primary C–H bonds at benzylic and allylic positions with high regio- and enantioselectivity. A readily available hydroxylamine ester is used as the nitrogen source.

The C(sp3)–H primary aminases are based on cytochrome P450 from Bacillus megaterium, which performs C(sp3)–H hydroxylation of fatty acids in nature. The rationale was to identify P450 variants that can use an unprotected iron nitrenoid species for the desired amination reaction.

First, benzylic C–H bonds were targeted for functionalization. A previously engineered P450 variant produced the wanted primary benzylic amine, but with a very low total turnover number (TTN). Based on structural data, residues for site-saturation mutagenesis were chosen and some rounds of engineering and screening afforded a variant that performed the reaction in an efficient and enantioselective manner. This enzyme allowed the primary C–H amination of several substrates with primary, secondary and tertiary benzylic C–H bonds, and was therefore termed a benzylic C–H primary aminase.

Due to the sensitivity of olefin groups, the selective primary amination of allylic C–H bonds provides an extra challenge compared to benzylic C–H bonds. An intermediate variant from previous screens was taken and successfully evolved into an allylic C–H primary aminase. This enzyme was shown to be capable of selectively functionalizing primary, secondary and tertiary allylic C–H bonds with up to 3930 TTN and 94% enantiomeric excess.

These primary aminases offer new opportunities to introduce amine groups into organic molecules in a stepwise and atom-economic fashion and could be useful for strategies to reformulate the nitrogen metabolism.

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Correspondence to Jan-Stefan Völler.

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Völler, JS. Primary amination of C(sp3)–H bonds. Nat Catal 3, 473 (2020). https://doi.org/10.1038/s41929-020-0477-2

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