6 R / S -deutero- α - d -mannopyranoside 1-phosphate

: 6 R / S -deutero- α - d -mannopyranoside 1-phosphate was synthesised from a C6 aldehydic mannose thioglycoside donor in four steps. Using NaBD 4 as the reductant, isotopic enrichment at C6 was achieved and the resultant C6-deuterated material was converted through to the glycosyl 1-phosphate using a protection / glycosylation / deprotection sequence. The product was fully characterised by 1 H, 13 C, 31 P and 2D NMR, alongside MS analysis.


Introduction
Glycosyl 1-phosphates are key intermediates in carbohydrate primary metabolism and are utilised by microorganisms to form polyphosphate architectures that constitute keys parts of their extracellular capsule and cell walls [1][2][3][4][5]. They serve as precursors to sugar-nucleotides [6,7], the sugar-donor components utilised by glycosyltransferases in the assembly of oligosaccharides and glycans and have played a key role in the development of glycosylated natural-product-based therapeutics [8]. Additionally, glycosyl 1-phosphates have been used as substrates for glycoside phosphorylases (a rapidly expanding family of CAZy enzymes) for the synthesis of oligosaccharide targets [9,10] and also play important technological roles in the food and detergent sectors [11][12][13][14].
A chemical synthesis approach to these target molecules allows modification of the native structure, rendering a capability to then interrogate the biosynthetic enzymes/processes that utilise them. This is typified by regio-and stereoselective deuteration, which has proven the underpinning for the elucidation of biosynthetic mechanisms involving carbohydrates and has also been used to confirm assignments of their NMR and mass spectra [15,16]. Carbohydrates diastereoselectively deuterated at C6 have provided important chemical tools [17], illustrated for mannose by a study reported by Tanner concerning the synthesis of ADP-[6 -2 H]-D,D-Hep for elucidating the mechanism of ADP-l-glycero-d-manno-heptose 6-epimerase [18]. As part of wider project concerning the chemical synthesis of modified mannose 1-phosphates and derived sugar-nucleotides [19], we required access to the title compound to establish a proof of concept methodology for incorporating C6 deuterium. Herein we provide our record of its synthesis and full characterisation from S-phenyl thioglycoside C6 aldehyde (1).

Results
Our synthetic route began from d-mannose which was appropriately transformed into C6-aldehyde thioglycoside (1) using established procedures [19]. We next completed a reduction of (1) with NaBD 4 to deliver (2) in 65% yield (Scheme 1). 1 H and 13 C NMR analyses of (2) were unable to unambiguously distinguish the diastereomeric ratio of the product mixture, nor were the diastereoisomers separable by TLC. We thus completed a small-scale synthesis of the 1,6-anhydro derivative of (2), using NBS to activate the thioglycoside and close the C6-OH onto the anomeric centre. 2D-HSQC data for this compound showed the expected correlation between H 6 and C 6 for both diastereoisomers, with a 1 J 13 C-2 H coupling of 23.1 Hz. 1 H NMR data provided a clear resolution of the diastereomeric anhydro-sugar mixture at H 6 and an expected almost equal product ratio from the NaBD 4 reduction (0.7:0.6, endo/exo) which could be extrapolated back to give the indicative diastereomeric ratio at C6 for (2) [0.7/0.6, S/R].
We next completed our route to (4) containing a C6-deuterium. Accordingly, alcohol (2) was protected at C6 with a benzyl group in good yield (84%) and the required 1-phosphate installed (in protected form) using dibenzylphosphate (DBP) as the acceptor under thioglycoside activation conditions (NIS/AgOTf) in satisfactory 51% yield to deliver (3). 1 H and 31 P NMR for (3) confirmed the presence of an anomeric phosphate with the characteristic doublet of doublets observed for H 1 coupling to H 2 and 31 P ( 3 J H1-H2 = 1.9 Hz, 3 J H1-P = 6.1 Hz). Finally, a global hydrogenolysis using H 2 with Pd/C and Pd(OH) 2 /C was completed, providing the title compound in a moderate 61% yield. Analytical data collected for (4) supported the structural assignment and gave an indicative level of purity. Copies of NMR, and MS data are included in Supplementary Materials. diastereoisomers separable by TLC. We thus completed a small-scale synthesis of the 1,6-anhydro derivative of (2), using NBS to activate the thioglycoside and close the C6-OH onto the anomeric centre. 2D-HSQC data for this compound showed the expected correlation between H6 and C6 for both diastereoisomers, with a 1 J 13 C-2 H coupling of 23.1 Hz. 1 H NMR data provided a clear resolution of the diastereomeric anhydro-sugar mixture at H6 and an expected almost equal product ratio from the NaBD4 reduction (0.7:0.6, endo/exo) which could be extrapolated back to give the indicative diastereomeric ratio at C6 for (2) [0.7/0.6, S/R]. We next completed our route to (4) containing a C6-deuterium. Accordingly, alcohol (2) was protected at C6 with a benzyl group in good yield (84%) and the required 1-phosphate installed (in protected form) using dibenzylphosphate (DBP) as the acceptor under thioglycoside activation conditions (NIS/AgOTf) in satisfactory 51% yield to deliver (3). 1 H and 31 P NMR for (3) confirmed the presence of an anomeric phosphate with the characteristic doublet of doublets observed for H1 coupling to H2 and 31 P ( 3 JH1-H2 = 1.9 Hz, 3 JH1-P = 6.1 Hz). Finally, a global hydrogenolysis using H2 with Pd/C and Pd(OH)2/C was completed, providing the title compound in a moderate 61% yield. Analytical data collected for (4) supported the structural assignment and gave an indicative level of purity. Copies of NMR, and MS data are included in Supplementary Materials. Scheme 1. Synthesis of 6R/S-deutero-α-D-mannopyranoside 1-phosphate (4) from C6-aldehyde (1).

General
All reagents and solvents which were available commercially were purchased from Acros (Geel, Belgium), Alfa Aesar (Heysham, UK), Fisher Scientific (Geel, Belgium), or Sigma Aldrich (Gillingham, UK). All reactions in non-aqueous solvents were conducted in oven dried glassware under a nitrogen atmosphere with a magnetic stirring device. Solvents were purified by passing through activated alumina columns and used directly from a Pure Solv-MD solvent purification system and were transferred under nitrogen. Reactions requiring low temperatures used the following cooling baths: −30 °C (dry ice/acetone), −15 °C (NaCl/ice/water) and 0 °C (ice/water). Infrared spectra were recorded neat on a Perkin Elmer Spectrum 100 FT-IR spectrometer; selected absorbtion frequencies (νmax) are reported in cm −1 . 1 H NMR spectra were recorded at 400 MHz and 13 C spectra at 100 MHz, respectively, using a Bruker AVIII400 spectrometer. 1 H NMR signals were assigned with the aid of gDQCOSY. 13 C NMR signals were assigned with the aid of gHSQCAD. Coupling constants are reported in Hertz. Chemical shifts (δ, in ppm) are standardised against the deuterated solvent peak. NMR data were analysed using Nucleomatica iNMR software. 1 H NMR splitting patterns were assigned as follows: s (singlet), d (doublet), app. t (apparent triplet), t (triplet), dd (doublet of doublets), ddd (doublet of doublet of doublets), or m (multiplet and/or multiple Scheme 1. Synthesis of 6R/S-deutero-α-d-mannopyranoside 1-phosphate (4) from C6-aldehyde (1).

General
All reagents and solvents which were available commercially were purchased from Acros (Geel, Belgium), Alfa Aesar (Heysham, UK), Fisher Scientific (Geel, Belgium), or Sigma Aldrich (Gillingham, UK). All reactions in non-aqueous solvents were conducted in oven dried glassware under a nitrogen atmosphere with a magnetic stirring device. Solvents were purified by passing through activated alumina columns and used directly from a Pure Solv-MD solvent purification system and were transferred under nitrogen. Reactions requiring low temperatures used the following cooling baths: −30 • C (dry ice/acetone), −15 • C (NaCl/ice/water) and 0 • C (ice/water). Infra-red spectra were recorded neat on a Perkin Elmer Spectrum 100 FT-IR spectrometer; selected absorbtion frequencies (ν max ) are reported in cm −1 . 1 H NMR spectra were recorded at 400 MHz and 13 C spectra at 100 MHz, respectively, using a Bruker AVIII400 spectrometer. 1 H NMR signals were assigned with the aid of gDQCOSY. 13 C NMR signals were assigned with the aid of gHSQCAD. Coupling constants are reported in Hertz.
Chemical shifts (δ, in ppm) are standardised against the deuterated solvent peak. NMR data were analysed using Nucleomatica iNMR software. 1 H NMR splitting patterns were assigned as follows: s (singlet), d (doublet), app. t (apparent triplet), t (triplet), dd (doublet of doublets), ddd (doublet of doublet of doublets), or m (multiplet and/or multiple resonances). For 13 C NMR data quaternary carbons are indicated as C q . Reactions were followed by thin layer chromatography (TLC) using Merck silica gel 60F254 analytical plates (aluminium support) and were developed using standard visualising agents: Short wave UV radiation (245 nm) and 5% sulfuric acid in methanol/∆. Purification via flash column chromatography was conducted using silica gel 60 (0.043-0.063 mm). MS and HRMS (ESI) were obtained on a Waters (Xevo, G2-XS TOF) spectrometers using a methanol mobile phase. Purification via ion exchange chromatography was conducted on Bio-Rad Biologic LP system using a Bio-Scale Mini UNOsphere Q (strong anion exchange) cartridge (5 mL): flow rate (1.5 mL/min), 0→90% 1.0 M (NH 4 )HCO 3 over 28 min.