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Diastereoselective Synthesis of Glycosyl Phosphates by Using A Angewandte Chemie International Edition:DOI:10.1002/anie.201507710 Phosphorylation German Edition:DOI:10.1002/ange.201507710 DiastereoselectiveSynthesis of Glycosyl Phosphates by Using aPhosphorylase–Phosphatase Combination Catalyst Patricia Wildberger,Martin Pfeiffer,Lothar Brecker,and Bernd Nidetzky* Abstract: Sugar phosphates play an important role in metab- During synthesis of glycosyl phosphates,stereocontrol at olism and signaling,but also as constituents of macromolec- the anomeric center is aproblem that requires special ular structures.Selective phosphorylation of sugars is chemi- attention. Chemical methodologies normally require multiple cally difficult, particularly at the anomeric center.Wereport steps,[4] even if hydroxy-protecting groups on the sugar are phosphatase-catalyzed diastereoselective “anomeric” phos- avoided.[5] Most of these syntheses involve reactions with phorylation of various aldose substrates with a-d-glucose 1- moderate yields and are limited to the formation of only afew phosphate,derived from phosphorylase-catalyzed conversion different glycosyl phosphates.Anumber of glycosyl phos- of sucrose and inorganic phosphate,asthe phosphoryl donor. phates have been obtained effectively from the corresponding Simultaneous and sequential two-step transformations by the b-glycosylsulfonohydrazides,which in turn were derived phosphorylase–phosphatase combination catalyst yielded gly- directly from free sugar hemiacetals.However,diastereose- cosyl phosphates of defined anomeric configuration in yields lectivity was only sufficient to obtain products enriched in the of up to 70%based on the phosphate applied to the reaction. a-anomer.[5] Nucleoside triphosphate (NTP)-dependent sugar An efficient enzyme-assisted purification of the glycosyl 1-kinases,which catalyze phosphorylation of the anomeric phosphate products from reaction mixtures was established. hydroxy group with precise a-selectivity,have proven useful to overcome issues of stereocontrol.[6] However relatively Phosphorylated carbohydrates constitute an important class narrow substrate specificity (e.g. d-Gal,[7a] l-Fuc,[7b] d- of biomolecules.[1] They are intermediates in metabolism and GalNAc/d-GlcNAc,[7c] d-GlcUA,[7d] d-GalUA,[7e] l-Ara[7f]) play central roles in signaling.Macromolecular structures, has essentially restricted the synthetic use of these enzymes including the genome,are also built from sugar phosphates.[1] to the phosphorylation of physiological sugar substrate(s) or Technological uses include applications in the food, cosmetic close structural analogues.Only recently have sugar 1-kinases and detergent sectors.[2,3] Moreover they are used as precur- with more relaxed substrate spectrum been discovered[8] or sors for the synthesis of nucleotide-activated sugars[2d,4a] and alternatively derived through protein engineering.[9] Chen as intermediates in rare sugar synthesis.[2e,f] Therefore,con- and co-workers applied d-GalNAc/d-GlcNAc kinase and d- venient synthetic routes to sugar phosphates is of importance Gal kinase for the synthesis of a-glycosyl phosphates of d- across disciplines,and selective phosphorylation of simple Gal, d-Glc, and d-Man, based on their respective substrate sugar substrates presents astrong option. However,the preferences.Derivatives of the three sugars with the 2-OH inherent structural complexity of sugars makes selective group substituted with H, F, NH2,NHAc,orN3 were also insertion of aphosphoryl group difficult. phosphorylated.[10] Important advances made with “promiscuous” sugar 1- kinases notwithstanding,there still exists gap in the scope of [*] Dr.P.Wildberger,[+] M. Pfeiffer,[+] Prof. Dr.B.Nidetzky sugar substrates that can be phosphorylated by these enzymes Institute of Biotechnology and Biochemical Engineering with good activity.Moreover,itis not economical to use NTP Graz University of Technology phosphoryl donors in stoichiometric amounts,and despite Petersgasse 12, 8010 Graz (Austria) E-mail:[email protected] notable developments in scaling up kinase-catalyzed sugar [11] Prof. Dr.B.Nidetzky phosphorylations to the gram scale, the requirement for acib -Austrian Centre of IndustrialBiotechnology(Austria) their (enzymatic) regeneration adds complexity to the overall [11, 12] Prof. Dr.L.Brecker transformation. Institute of Organic Chemistry,University of Vienna Therefore,analternative method of biocatalytic phos- Währingerstraße 38, 1090 Vienna (Austria) phorylation that combines the key feature of stereoselectivity [+]These authors contributed equally to this work. with the additional advantages of broad substrate acceptance Supportinginformation for this article (including experimental would be very useful, especially when the preparation of a- methods;enzyme production and purification;assays;enzymatic glycosyl phosphates as commodity chemicals is considered. conversions;and product analysis, purification and identification)is Theuse of aphosphoryl-group donor more convenient than available on the WWW under http://dx.doi.org/10.1002/anie. NTP could further improve the procedure.Weherein 201507710. describe aphosphorylase–phosphatase combination catalyst 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. for the anomeric-center phosphorylation of aldose sugars KGaA. This is an open access article under the terms of the Creative from inorganic phosphate (Scheme 1). Commons AttributionNon-CommercialNoDerivs License, which permitsuse and distribution in any medium, provided the original Theoverall two-step conversion proceeded via the work is properly cited, the use is non-commercial and no modifica- activated phosphoryl donor a-d-glucose 1-phosphate (aGlc tions or adaptations are made. 1-P), which was derived from sucrose and phosphate in Angew.Chem. Int.Ed. 2015, 54,15867 –15871 2015 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA,Weinheim 15867 Angewandte. Communications Scheme 2. Kinetic scheme of AGP-catalyzed transphosphorylation. Ais the sugar acceptor and Pi is phosphate. Scheme 1. Diastereoselective synthesis of glycosyl phosphates by dephosphorylation by the sugar acceptor;low hydrolysis of asucrose phosphorylase (SPase) and glucose 1-phosphatase (AGP) the phosphoenzyme intermediate;and low hydrolysis of the combination catalyst. synthesis product.[16] 1 Purified AGP(0.2 UmLÀ )was applied to the phosphor- athermodynamically favored reaction catalyzed by sucrose ylation (378C; pH 7.0) of different monosaccharides phosphorylase (from Leuconostoc mesenteroides;SPase; (200 mm; d-Man, d-Gal, d-GlcNAc, d-GlcNH2, d-GalNH2, EC 2.4.1.7).[13] aGlc 1-P was then utilized directly in aselec- d-GlcUA, l-Fuc, d-Xyl, d-Ara, and l-Ara) from aGlc 1-P tive transphosphorylation, in which the sugar acceptor was (20 mm). Xylitol (Xol) was also used. Reaction time courses reacted in the presence of asuitable phosphomonoester are shown in Figure S2 in the Supporting Information. Except hydrolase (glucose 1-phosphatase (AGP);EC3.1.3.10). The for d-GlcUAand d-GalNH2,which were not accepted by large number of phosphatases encoded by the genome and AGP, all other compounds were active.Extensive 1D and 2D the often strongly overlapping substrate specificities of these NMR analyses (1H, 31P) conducted directly on the reaction enzymes[14] makes the dedicated selection of candidate mixtures and on recovered compounds revealed the identity biocatalysts achallenging task. of the phosphoryl transfer products (Figures S3–S7, Expedient synthesis of several aldohexose phosphates by Tables S2,S3). AGPphosphorylated d-Man, d-GlcNAc, d- phosphatase-catalyzed transphosphorylationfrom pyrophos- Gal, and l-Fuc at the anomeric hydroxy group,and it did so phate has been reported.[15] However,the phosphorylation with absolute axial selectivity to yield the corresponding a-d- was always at C6 and never at the anomeric center. aGlc 1-P or b-l-glycosyl phosphate products (Figures S3–S6). Interest- [16] is hydrolyzed by various phosphatases but until recently, its ingly, d-GlcNH2 was phosphorylated primarily at the 6-OH use as aphosphoryl donor for enzymatic synthesis was not (Figure S7). Xol, d-Xyl, d-Ara, and l-Ara were also phos- considered. We speculated that phosphatases active in the phorylated. cleavage of aGlc 1-P might be also efficient and specific in Theefficiency with which each sugar acceptor was synthesizing phosphomonoesters with the anomeric sugar phosphorylated was evaluated by using the rate ratio of hydroxy group.Three sugar phosphate phosphatases from aGlc 1-P consumption (rS)and phosphate release (rP)asthe Escherichia coli (AGP,HAD4, HAD13;see the Supporting key parameter (Scheme 2). Under hydrolysis conditions, rS/rP Information) that have been reported to use aGlc 1-P as their has avalue of unity.Phosphoryl transfer to the acceptor is preferred substrate for hydrolysis[14,17] were tested for phos- phorylation of different sugars (see below,100–200 mm)inthe presence of aGlc 1-P (20 mm). Reactions were followed by monitoring aGlc 1-P consumption, phosphate release,and formation of the phosphorylated product(s) (see Methods in the Supporting Information). HAD4 and HAD13 converted aGlc 1-P exclusively through hydrolysis and thus proved useless for synthesis.Bycontrast, AGPwas active with various acceptors (e.g. d-Man) and utilized asubstantial portion ( 50%) of the offered donor substrate for sugar phosphate synthesis.Preliminary product analysis of the AGP reaction with d-Man revealed that the phosphorylation product was not d-mannose 6-phosphate (Man 6-P). This indicates that AGPmight
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