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The conversion of saturated fatty acids to high value chiral hydroxy-acids and lactones poses anumber of synthetic challenges:t he activation of unreactive CÀHb onds and the need for regio-and stereoselectivity.H ere the first example of aw ild-type cytochrome P450 monooxygenase (CYP116B46 from Tepidiphilus thermophilus) capable of enantio-and regioselective C5 hydroxylation of decanoic acid 1 to (S)-5hydroxydecanoic acid 2 is reported. Subsequent lactonization yields (S)-d-decalactone 3,ahigh value fragrance compound, with greater than 90 %ee. Docking studies providearationale for the high regio-and enantioselectivity of the reaction.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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The squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC) is a highly efficient enzyme catalyst for stereoselective Brønsted acid catalysis. We engineered AacSHC to catalyze the selective Prins cyclization of citronellal. Four active site variants were identified for the diastereoselective cyclization of (S)‐citronellal to stereoisomers (−)‐iso‐isopulegol, (+)‐isopulegol and (−)‐neo‐isopulegol, respectively. The replacement of active site residues resulted in two triple variants that catalyzed the transformation of (R)‐citronellal to give the isomers (+)‐neo‐isopulegol and (−)‐isopulegol with up to >99 % de, respectively. The newly designed library of functionally diverse active site geometries exhibits high selective control during citronellal cyclization, leading exclusively to a single diastereomer of the desired isopulegol. Whereas the cyclization of citronellal with chemical catalysts was observed to produce the isopulegol isomer with the lowest energy, the reaction with AacSHC variants proceeded with higher product selectivity. The results of this study show that variants of AacSHC are excellent catalysts for the highly selective formation of isopulegol stereoisomers.

Synthesis of Cbz-protected 3-aminopiperidine and 3-aminoazepane using a multi-enzyme cascade consisting of galactose oxidase and imine reductase variants.

A deeper understanding of the >99 % S-selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active-site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)-citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)-citral it remained ≥88 % (S) for all single-residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A-based double-mutant library that enhanced the (E)-citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)-citral to 44 % (S). Formation of (R)-citronellal from an (E/Z)-citral mixture is a desire in industrial (-)-menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.

N-Glycolylneuraminic acid (Neu5Gc) is a common cell-surface ligand in animals which is not biosynthesized in humans, but it can be acquired in human tissue from dietary sources such as red meat. It is important to understand the relevance of this potentially immunogenic glycan on human health, and selective detection methods are needed that can distinguish Neu5Gc from its biosynthetic precursor that is common in humans (i.e., N-acetylneuraminic acid (Neu5Ac)). Here, we demonstrate that Neu5Gc can be selectively oxidized by an engineered variant of galactose oxidase without any reaction toward Neu5Ac. Oxidation of Neu5Gc itself allowed for the full spectroscopic characterization of the aldehyde product. In addition, we show that Neu5Gc is also oxidized when it is part of a typical animal oligosaccharide motif and when it is attached to a protein-linked N-glycan. Oxidation of Neu5Gc introduces bioorthogonal functionality that can be exclusively labeled. We demonstrate that in combination with sialidase-mediated hydrolysis, this twoenzyme system can provide a useful tool for the selective detection of Neu5Gc in complex biological samples such as the biopharmaceutical alpha acid glycoprotein.