Human carbonic anhydrase B (HCAB), prepared by a new affinity chromatography procedure, was carboxymethylated exclusively at NT of its active-site histidine-200 using 90% [1-13C]bromoacetate. The 13C nuclear magnetic resonance signal of the covalently attached carboxylate was easily detected over the natural abundance background due to the other carbonyl and carboxyl carbons of this 29 000 molecular weight zinc metalloenzyme. Its chemical shift proved very sensitive to the presence of inhibitors in the active site and to variations in pH. Two perturbing groups with pKa values of 6.0 and 9.2 were assigned to the modified histidine-200 itself and the zinc-bound water ligand, respectively, making use of 13C NMR titration data on Nr- and Nr-carboxymethyl-L-histidine model compounds. The results rule out histidine-200 as the critical group whose ionization controls the catalytic activity. They also strongly suggest an interaction of the carboxylate of the carboxymethyl group with either the zinc or its water ligand around pH 8, possibly explaining the basis for the major differences between HCAB and CmHCAB.
The regioselective gold‐catalysed hydration of propargylic alcohols to β‐hydroxy ketones can be achieved by diverting the gold‐catalysed Meyer–Schuster rearrangement through the addition of a protic additive with a pK
a of 7–9 such as p‐nitrophenol, boric acid or a boronic acid. This provides an interesting alternative to an aldol reaction when combined with the straightforward addition of an alkyne to an aldehyde or ketone. The gold‐catalysed reaction of an electron‐deficient, sterically hindered propargylic alcohol with a boronic acid led to the formation of an unusually stable cyclic boron enolate.
Understanding the factors that influence N → S acyl transfer in native peptide sequences, and discovery of new reagents that facilitate it, will be key to expanding its scope and applicability. Here, through a study of short model peptides in thioester formation and cyclisation reactions, we demonstrate that a wider variety of Xaa-Cys motifs than originally envisaged are capable of undergoing efficient N → S acyl transfer. We present data for the relative rates of thioester formation and cyclisation for a representative set of amino acids, and show how this expanded scope can be applied to the production of the natural protease inhibitor Sunflower Trypsin Inhibitor-1 (SFTI-1).
In this paper we describe the use of a chiral aldehyde derived from lactate esters for determining the enantiopurity of primary amines, via the formation of diastereomeric imines. The method was shown to be suitable for reproducibly determining the enantiopurity of a diverse set of chiral amines. Both enantiomers of the aldehyde can be prepared in two steps from commercially available materials.
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