Synthesis of CMP-NeuAc

K. Ishige, T. Hamamoto, T. Shiba and T. Noguchi (2001). Novel method for enzymatic synthesis of CMP-NeuAc. Biosci. Biotechnol. Biochem., 65, 1736-1740. 

CMP-N-acetylneuraminic acid (CMP-NeuAc). CMP-N-acetylneuraminic acid has been prepared enzymatically on small scales (> 0.5 mmol) from CTP and NeuAc, under catalysis by CMP-NeuAc synthetase (EC 2.7.7.43) [131l An improvement in this procedure, involving in situ production of CTP from CMP under adenylate kinase and pyruvate kinase catalysis, is suitable for multigram-scale synthesis11321. Adenylate kinase catalyzes the equilibration of CTP and CMP to CDP, which is subsequently phosphorylated by pyruvate kinase to provide CTP. A one-pot synthesis of CMP-NeuAc based on this procedure involves the in situ synthesis of NeuAc from N-acetylmannosamine and pyruvate, catalyzed by sialic acid aldolase (Fig. 11.3-12)[10S1. Chemical syntheses of CMP-NeuAc have also been reported11421. 

Fukumoto S, Miyazaki H, Goto G, Urano T, Furukawa K, Furukawa K (1999) Expression cloning of mouse cDNA of CMP-NeuAc Lactosylceramide a2,3-sialyltransferase, an enzyme that initiates the synthesis of ganglio-sides. J Biol Chem 274 9271-9276... 

Scheme 29 Large-scale synthesis of sialylated lactose using a CMP- ialir acid svnthetase/a2,3-sialyltra enzyme with in situ regeneration of CMP-NeuAc.

CMP-NeuAc. Derivatives of neuraminic acid frequently terminate mammalian glycoconjugates. Activated NeuAc is therefore an especially important target for enzyme-based synthesis. NeuAc is synthesized in vivo by an NeuAc aldolase-catalyzed condensation of N-acetylmannosamine and pyruvate (Scheme 11 above). NeuAc is then coupled directly to CTP by CMP-NeuAc synthase (E.C. 2.7.7.43). We have recently published a multigram preparation of CMP-NeuAc which makes use of both NeuAc aldolase and CMP-NeuAc synthase (Scheme 19) (47). NeuAc aldolase has been cloned and overexpressed, and is commercially available. The final enzyme required, CMP-NeuAc synthase, was isolated from calf brain. 

Synthesis of CMP-NcuAc. CMP-NeuAc can be prepared from CTP and NeuAc using CMP-NeuAc synthase (Equation 10). Several reports on the enzymatic synthesis of nucleotide-sugars have appeared (10) and CMP-NeuAc synthase have been cloned (57). A conveniently obtained immobi-... 

For the synthesis of sialylated oligosaccharides, a fusion enzyme consisting of CMP-NeuAc synthetase and a2,3-sialyltransferase was created (29). The... 

The regeneration system for CMP-NeuAc is more complicated than that for NDP-sugars (Scheme 7) [24]. An additional phosphorylation step must be incorporated, because CMP, a nucleoside monophosphate, is released after reaction with the sialyltransferase. For recycling purposes, nucleoside monophosphate kinase (NMK EC 2.7.4.4) or myokinase (MK EC 2.7.4.3) is added for the conversion of CMP to CDP. In this reaction, the phosphoryl donor is ATP. Subsequently, both CDP and ADP must be re-phosphorylated to CTP and ATP, respectively. Thus, for regeneration of CMP-NeuAc, an additional kinase and two equivalents of PEP are required. The condensation of NeuAc with CTP is catalyzed by CMP-NeuAc synthetase (EC 2.7.7.43). This system was used for the large-scale synthesis of 6 -sialyl-LacNAc(6 -SLN) from LacNAc catalyzed by a2,6-SiaT (EC 2.7.7.43) in 97% yield. 

Scheme 7. Recycling of CMP-NeuAc for the synthesis of 3 -siaIyl-LacNAc. E = a2,3-sialyl-transferase E2 = nucleoside monophosphate kinase E3 = pyruvate kinase E4 = CMP-NeuAc synthetase E5 = pyrophosphatase.

Scheme 10. Recycling of CMP-NeuAc coupled to fran -sialidase activity for the synthesis of novel sialosides.

CMP-NeuAc synthetase (EC 2.7.7.43) to produce CMP-NeuAc. The by-product pyrophosphate (PPi) is hydrolyzed to phosphate (Pi) by inorganic pyrophosphatase (PPase). Sialyla-tion is accomplished with a2,3-sialyltransferase (< 2,3NeuAcT) or a2,6-sialyltransferase (a2,3NeuAcT), respectively. The released CMP is again converted to CDP, to CTP, and finally to CMP-NeuAc. The UDP-Gal and CMP-NeuAc regeneration schemes have been combined in a one-pot reaction and applied to the synthesis of sialyl Lewis X. 

The Schmidt group utilized a sialyl phosphite in a very different synthesis strategy (Scheme 33) [39]. Upon treatment of sialyl donor 54 with cytidine phosphoric acid 103, a phosphite-phosphate exchange reaction occmred to give com-poimd 104 exclusively as the fi isomer. Deacylation by treatment with sodium meth-oxide followed by ester saponihcation through the addition of water provided CMP-NeuAc 88. This method circmnvented the need for an oxidation step or phosphorus deprotection. This method was also applied to the synthesis of another nat-lually occmring CMP-NeuAc derivative 105 [40]. 

Halcomb and Chappell developed a route to CMP-NeuAc 88 that promises to be general for the synthesis of virtually any derivative thereof [44,45]. The route (Scheme 36) utilizes a condensation of sialic acid derivative 99 with the phosphoramidite 112 to afford the phosphite 113 in 62% yield. Oxidation of the phosphite provided the phosphotriester 114 [46], which was taken directly to the next transformation without purification (owing to its instability to chromatography). Deal-lylation of the phosphate gave compound 115 (61% for two steps), which was stable to silica gel chromatography. Compound 115 was deacylated with methoxide, and its methyl ester was subsequently saponified with NaOH to provide CMP-NeuAc 88. The derivatives shown in Scheme 37 were synthesized according to this protocol and were investigated as substrates for sialyltransferases (see below). 

The synthesis of a CMP-NeuAc derivative that was bound to a solid support through the 9-position of the sialic acid has been reported by the Kajihara group [47]. This derivative is quite useful in that it can be utilized to immobilize glycoproteins onto a solid support by transferring the sialic acid to the terminus of the carbohydrate chain of the glycoprotein. 

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