Combined Use of Subtilisin and N-Acetylneuraminic Acid Aldolase for the Synthesis of a Fluorescent Sialic Acid

Fitz, Wolfgang; Wong, Chi‐Huey

doi:10.1021/jo00105a057

Cited by 35 publications

(13 citation statements)

References 4 publications

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“…The reactions were carried out at 37 °C in a buffer (pH 7.4, I = 0.1) containing 0.05 M potassium phosphate and 7.5% (v/v) dithiothreitol. 23,24 Pyruvic acid was used in large excess (10 equivalents) and its concentration could be considered as a constant during the treatment of reaction kinetics. The sialic acid products of the reactions were assayed with the modified Svennerholm (resorcinol) method.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, NeuNAc aldolase has been used to synthesize a variety of NeuNAc derivatives or analogs by enzymatic method. [19][20][21][22][23][24][25][26][27] To find out the optical substrates for the bioengineering of NeuNAc on tumor cells, and other cells as well, by utilizing the permissibility of NeuNAc aldolase, it is necessary to understand the reactions of various N-acyl-D-mannosamines with the enzyme. For this purpose, the accessibility of a number of N-acyl derivatives of D-mannosamine to NeuNAc aldolase was investigated.…”

Section: Introductionmentioning

confidence: 99%

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

Pan

Ayani

Nadas

et al. 2004

Carbohydrate Research

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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 investigated quantitatively. The N-propionyl-, Nbutanoyl-, 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 Ntrifluoropropionyl 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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Pan

Ayani

Nadas

et al. 2004

Carbohydrate Research

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“…The best utilized member of the pyruvate aldolases, N -acetylneuraminic aldolase (EC 4.1.3.3), has been used extensively to prepare modified sialic acids. − In vivo , NeuAc aldolase catalyzes the reversible condensation of pyruvate and N -acetylmannosamine, to produce N -acetylneuraminic acid. While N -acetylneuraminic aldolase accepts a range of unnatural electrophilic substrates, it does not accept lower-carbon homologues: aldose substrates of less than four carbons are not accepted .…”

Section: Introductionmentioning

confidence: 99%

2-Keto-3-deoxy-6-phosphogluconate Aldolases as Catalysts for Stereocontrolled Carbon−Carbon Bond Formation

et al. 1996

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The pyruvate aldolases use pyruvate as the nucleophilic component in stereoselective aldol condensations, producing a 4-hydroxy-2-ketobutyrate framework. We have examined the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Pseudomonas putida, Escherichia coli, and Zymomonas mobilis for utility as synthetic reagents. Unlike other pyruvate aldolases examined to date, the KDPG aldolases accept short-chain, non-carbohydrate electrophilic aldehydes as substrates, providing a general methodology for the construction of the 4-hydroxy-2-ketobutyrate skeleton. The three aldolases differ markedly with respect to enzyme stability, pH optima, stability in organic cosolvent mixtures, substrate specificity, and diastereoselectivity during aldol condensation. All three enzymes show broad substrate specificity with regard to the electrophilic component. The primary requirements for substrate activity appear to be minimal steric hindrance and the presence of electron-withdrawing substituents at C2. The aldolases from Pseudomonas and Escherichia are also specific for the d-stereochemical configuration at C2, while the enzyme from Zymomonas displays no stereochemical discrimination with regard to the electrophilic substrate. Nucleophiles other than pyruvate are accepted as nucleophilic substrates by all three enzymes, provided the electrophile is sufficiently reactive. In preparative scale reactions with three unnatural electrophiles, the three enzymes show varying degrees of stereochemical fidelity. In most cases, a single diastereomer of the aldol adduct was produced, although in one case, a diastereomeric excess of 50% was observed. In all cases, the diastereoselectivity is exclusively kinetic in origin, despite the reversibility of some reactions. The enzymes are remarkably tolerant of added cosolvent: all three showed >60% of native activity in 30% DMSO and DMF. By appropriate choice of enzyme, the KDPG aldolases offer exceptional utility for stereocontrolled carbon−carbon bond formation under a wide range of experimental conditions.

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“…The first three classes of enzymes have been extensively utilized for the synthesis of a variety of carbohydrate-like products. − The remaining and largest group of aldolases utilize pyruvate or phosphoenolpyruvate as the nucleophile. Two members of this group, N -acetylneuraminic acid aldolase (NeuAc aldolase, EC 4.1.3.3) − and 2-keto-3-deoxyoctulosonate aldolase (KDO aldolase, EC 4.1.2.23), have been used to prepare analogues of their natural substrates. Both enzymes operate under thermodynamic control and frequently yield product mixtures. ,,, In our laboratories, we have utilized 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, EC 4.1.2.14) to prepare a range of structurally varied products. − Unlike other pyruvate aldolases, KDPG aldolase operates exclusively under kinetic control providing stereochemically pure products …”

mentioning

confidence: 99%