Accessibility of N-acyl-D-mannosamines to N-acetyl-D-neuraminic acid aldolase

Abstract

N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl-D-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-D-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of D-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl-, and N-phenylacetyl-D-mannosamines may be employed to bioengineer NeuNAc on cells.

Figure 1

UV-visible spetra of the Svennerholm reaction products of: a) NeuNAc, b) NeuNPr, c) NeuNBu and NeuNiBu, d) NeuNPhAc, e) NeuNTFP, f) NeuNPiv, and g) NeuNBz

Figure 2

The reaction kinetics of N-acyl-d-mannosamines (18.2 mM) with pyruvic acid (182 mM) and NeuNAc aldolase (0.9 unit/mL): 37 °C, 0.05 M potassium phosphate buffer (pH 7.4, I = 0.1) containing 7.5% (v/v) dithiothreitol.

Figure 3

The impact of ManNPhAc concentrations (1.8, 3.6, 9.1, 18, 91 and 182 mM) on its reaction with pyruvic acid (182 mM) and NeuNAc aldolase (0.9 unit/mL): 37 °C, 0.05 M potassium phosphate buffer (pH 7.4, I = 0.1) containing 7.5% (v/v) dithiothreitol.

Scheme 1

NeuNAc aldolase-catalyzed reaction

Scheme 2

Preparation of N-acyl-D-mannosamines (2–9)

Scheme 3

Preparation of N-acyl-d-neuraminic acids (11–17)