Repetitive Batch Mode Facilitates Enzymatic Synthesis of the Nucleotide Sugars UDP‐Gal, UDP‐GlcNAc, and UDP‐GalNAc on a Multi‐Gram Scale

Abstract

The availability of nucleotide sugars is considered as bottleneck for Leloir‐glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine‐5′‐diphospho‐α‐d‐galactose (UDP‐Gal), Uridine‐5′‐diphospho‐N‐acetylglucosamine (UDP‐GlcNAc), and Uridine‐5′‐diphospho‐N‐acetylgalactosamine (UDP‐GalNAc) in a multi‐gram scale by repetitive batch mode. The authors obtained 12.8 g UDP‐Gal through a high mass based total turnover number (TTNmass) of 494 [gproduct/genzyme] and space‐time‐yield (STY) of 10.7 [g/L*h]. Synthesis of UDP‐GlcNAc in repetitive batch mode gave 11.9 g product with a TTNmass of 522 [gproduct/genzyme] and a STY of 9.9 [g/L*h]. Furthermore, the scale‐up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP‐GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi‐gram scale synthesis of nucleotide sugars by stable enzyme cascades.

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

“…In order to be used in oligosaccharide synthesis mediated by Leloir- glycosyltransferases, these monosaccharides need to be activated first to a high-energy donor form—nucleotide sugars. Production of isotope-labeled nucleotide sugars was carried out as described before, employing cascades of salvage pathway enzymes in a repetitive batch approach (Supplementary Materials, Scheme S1) [39].…”

“…By pre-tempering of all reaction solutions and elongation of the reaction time to one hour, we were able to obtain full substrate conversion (Table 1). Product concentrations were determined by capillary electrophoresis with UV detection (CE-UV) (data not shown) and the product verification was carried out via electrospray ionization mass spectrometry (ESI-MS) as previously reported (Table 1; Supplementary Materials, Figures MS1, and MS2) [32,37,39]. The non-labeled nucleotide sugars utilized in this study were available from our earlier work [39] (Supplementary Materials, Figures MS3, and MS4).…”

“…Product concentrations were determined by capillary electrophoresis with UV detection (CE-UV) (data not shown) and the product verification was carried out via electrospray ionization mass spectrometry (ESI-MS) as previously reported (Table 1; Supplementary Materials, Figures MS1, and MS2) [32,37,39]. The non-labeled nucleotide sugars utilized in this study were available from our earlier work [39] (Supplementary Materials, Figures MS3, and MS4). The synthesized compound 6 (UDP-[ 13 C 6 ]Gal) contains isotopically enriched galactose with a stable 13 C incorporated at each carbon position of the hexose which causes the mentioned mass difference of 6 compared to the natural compound (UDP-Gal).…”

“…Here, we utilize a previously described strategy in a very efficient synthesis set-up for the production of UDP-[ 15 N]GlcNAc and UDP-[ 13 C 6 ]Gal on a 200 and a 400 µmol scales. [39]. As previously demonstrated, nucleotide sugars are readily accessible for Leloir- glycosyltransferases-mediated glycan synthesis without further purification steps [32].…”

“…A chemoenzymatic strategy for the synthesis of UDP-[ 2 H]GlcNAc starting from the sugar-1-phosphate was performed using the uridylyltransferases activity of the bifunctional Escherichia coli ( E.coli ) enzyme GlmU [36]. Recent developments of cascades with salvage pathway enzymes and their use in novel synthesis strategies can provide the nucleotide sugars UDP-Gal and UDP-GlcNAc in a much more efficient way [37,38,39]. Furthermore, recent studies impressively demonstrate the value of defined isotopically labeled OS for in vivo research applications [40].…”