Chemoenzymatic synthesis of ring ¹⁸O-labeled sialic acid

Abstract

Methyl 4,6-O-benzylidene-α-D-glucopyranoside was converted into methyl 2-azido-2-deoxy-4,6-Obenzylidene-α-D-altropyranoside via a synthetic route that incorporated two inversions of configuration. Activation of the C-3 hydroxyl group as a triflate ester followed by an S N 2 reaction with O-18 labeled benzoate gave, after standard functional group manipulations, 2-acetamido-2-deoxy-D-(3-18 O)mannose. Coupling of the labeled N-acetyl-mannosamine with pyruvate was catalyzed by sialic acid aldolase to give ring-oxygen-labeled sialic acid in an overall yield of 11.4% over 10 steps.

Turnover Is Rate-Limited by Deglycosylation for Micromonospora viridifaciens Sialidase-Catalyzed Hydrolyses: Conformational Implications for the Michaelis Complex

“…Neu5Acα2,6GalβFMU ( 1 ) and the necessary seven labeled isotopologues ( 1a − g ) were prepared chemoenzymatically from the β- d -galactopyranoside precursor 6 , ManNAc, and pyruvate (Scheme ). The labeled starting materials for the seven isotopically substituted substrates were (i) pyruvate ( 1a and 1c ); (ii) deuterated sialic acid ( 1e , 1f , and 1g ); (iii) N -acetyl-3- 18 O-mannosamine ( 1b ); and (iv) 18 O- 6 , the synthesis of which is shown in Scheme ( 1d ). Enzyme-mediated synthesis of 1 was used for the current study because, in contrast to most chemical syntheses, formation of the glycal 5-acetamido-2,6-anhydro-3,5-dideoxy- d - glycero - d - galacto -non-2-enonic acid (Neu2en5Ac), a potent inhibitor of sialidases, does not occur during the enzymatic coupling reaction.…”

“…020414nw). Labeled C3-deuterated Neu5Ac and 2-acetamido-2-deoxy- d -(3- 18 O)mannose were synthesized as reported in the literature. The synthesis of 8-fluoro-4-methylumbellyferyl β-galactopyranoside, which upon β-galactosidase-catalyzed hydrolysis gives the fluorescent 8-fluoro-7-hydroxy-4-methylcoumarin (8-fluoro-4-methylumbellyfereone, FMU), is given in the Supporting Information.…”

Transition State Analysis of Vibrio cholerae Sialidase-Catalyzed Hydrolyses of Natural Substrate Analogues

“…16 That is, the necessary isotopologues were synthesized chemoenzymatically starting from pyruvate, ManNAc, and LacβSPh using three enzymes, sialic acid aldolase, CMP-sialic acid synthase, and a sialyltransferase (Scheme 1). 16 The labeled starting materials for the isotopically substituted substrates were (i) (2-13 C)pyruvate for 2a; (ii) (2-13 C)pyruvate and N-acetyl-(3-18 O)mannosamine 20 for 2b; (iii) (2-13 C)pyruvate and phenyl (3′-18 O)-1-thiolactoside, the synthesis of which is shown in Scheme 2, for 2c; (iv) (3-13 C)pyruvate for 2d and 3a; and (v) (3-13 C)pyruvate followed by base-catalyzed deuterium incorporation into the (3-13 C)sialic acid 22 for 2e, 2f, 3b, and 3c. In our hands, the base-catalyzed deuterium exchange onto C3 of (3-13 C)Neu5Ac resulted in a mixture of Neu5Ac isotopologues (3-H 2 , R-3-2 H, S-3-2 H, and 3-2 H 2 ) with the precise ratios dependent on the pH and time that the exchange reaction was allowed to proceed.…”

“…Reactions were stopped when reactions reached approximately 90% completion. ( 18 O 2 )-Benzoic acid and 2-acetamido-2-deoxy- d -(3- 18 O)mannose were synthesized as reported in the literature. Deuterium exchange into (3- 13 C) Neu5Ac, which was made from (3- 13 C)pyruvate using sialic acid aldolase, followed an established protocol .…”

Enzyme‐Catalyzed Aldol Additions

“…NeuA was also used to catalyze the reaction between pyruvate and 2-acetamido-2-deoxy-d-(3-18 O)-mannose to furnish the ring-18 O-labeled sialic acid for investigations of kinetic isotope effects [33].…”