Reversible Substrate Anchoring: NC‐SPOS as a Sustainable Approach to Solid‐Supported Organic Synthesis

Porzelle, Achim; Fessner, Wolf‐Dieter

doi:10.1002/ange.200462278

Cited by 3 publications

(3 citation statements)

References 60 publications

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“…Its alkaline pH optimum (pH 8) is a perfect match to the activity profile of the CSS from N. meningitidis as well as to the stability requirements from both the CMP‐activated sialic acid and sialoside products. The lactoside 6 20 was used as an acceptor because it matches the SiaT acceptor specificity for β‐configurated galactosides in combination with a rapid convergent synthetic access, an allylic linker as a potential cleavage site, and a fluorescent acridone tag as an ultrasensitive detection and purification aid 41…”

Section: Resultsmentioning

confidence: 99%

Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

Dong

Kickstein

et al. 2013

Adv Synth Catal

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Sialic acid-containing glycoconjugates at the cell surface are of high importance in carbohydrate-mediated recognition phenomena in physiological and pathological events, as well as in bacterial or viral infection. A key step in the enzymatic synthesis of natural sialoconjugates and functional synthetic analogues is the activation of sialic acids to cytidine 5'-monophosphate (CMP)-sialic acid intermediates catalyzed by CMP-sialic acid synthetase (CSS). Based on our recently developed aligned protein model of substrate binding and a simple colorimetric screening assay, we have engineered the CSS from Neisseria meningitidis by structure-guided site-specific saturation mutagenesis at positions 192/193 to generate enzymes with broadened substrate scope. Top hits, including the F192S/F193Y variant, display an improvement of up to 70-fold catalytic efficiency relative to wild-type CSS for the conversion of sterically demanding N-acyl modified sialic acid analogues, without compromising protein stability. Such significantly enhanced substrate capacity is a major step forward to realizing a generalized chemo-enzymatic strategy for the efficient preparation of neo-sialoconjugate libraries, demonstrated by the highly efficient, regio-and stereospecific synthesis of 2,6-sialyllactose analogues by enzymatic coupling to the highly substrate tolerant a2,6-sialyltransferase from Photobacterium leiognathi JT-SHIZ-145. Our results further document the unusual versatility of the N. meningitidis CSS and engineered variants for a common synthetic approach to sialoconjugates comprising a large diversity of natural and non-natural sialic acid forms without the need for post-synthetic enzymatic modification.

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Section: Resultsmentioning

confidence: 99%

Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

Dong

Kickstein

et al. 2013

Adv Synth Catal

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“…As an acceptor we used β‐lactoside 21 ,34 which matches the SiaT acceptor specificity for LacNAc and lactose in combination with rapid convergent synthetic access, allylic linker as a potential cleavage site, and a fluorescent acridone tag as an ultrasensitive detection and purification aid 42. The resulting neo‐sialoconjugates, structural analogues of 6′‐sialyllactosides, were isolated and characterized by mass spectrometry and NMR analysis to ascertain the regio‐ and stereospecificity of the transfer.…”

Section: Resultsmentioning

confidence: 99%

“…[42] The resulting neo-sialoconjugates, structural analogues of 6'-sialyllactosides, were isolated and characterized by mass spectrometry and NMR analysis to ascertain the regio-and stereospecificity of the transfer.…”

Section: Utilization Of Css Activity For the Synthesis Of Neosialoglymentioning

confidence: 99%

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Dong

Fessner

2011

Adv Synth Catal

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Abstract:We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of cytosine-5'-monophosphate-acetylneuraminate synthetase (CMP-sialate synthetase, CSS) activity based on the pH change of a released proton equivalent upon nucleotide activation of Neu5Ac or related analogues. Using this method, steady-state kinetic data of Neisseria meningitidis CSS were determined for cytosine-5'-triphosphate (CTP), N-acetylneuraminic acid (Neu5Ac) and eleven structural variations of the sialic acid, including backbone truncation, deamination, epimerization, and several N-acyl modifications. These data further demonstrate the unusual versatility of the N. meningitidis CSS for a general access to sialoconjugates containing non-natural sialic acid analogues. Remarkably, the assay allows covering a broad range of substrate parameters that span over more than three orders of magnitude for K M and k cat measurements. With the aid of a structural model built from X-ray crystal structure data, the kinetic data could be used to interpret potential protein contributions in substrate binding of Neu5Ac and its analogues. The Neu5Ac analogues were used for the preparation of neo-sialoconjugate analogues of 2,6-sialyllactose in a one-pot two-enzyme cascade, using N. meningitidis CSS together with the novel a2,6-sialyltransferase (a2,6SiaT) from Photobacterium leiognathi, which proved to be highly effective because of its optimum activity at alkaline pH, appropriately matching the requirements from the overall reaction system.

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Organische Chemie 2005

et al. 2006

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Reversible Substrate Anchoring: NC‐SPOS as a Sustainable Approach to Solid‐Supported Organic Synthesis

Cited by 3 publications

References 60 publications

Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Organische Chemie 2005

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