Chemical and enzymatic synthesis of the alginate sugar nucleotide building block: GDP-D-mannuronic acid

Full text of DOI:10.1016/j.carres.2019.107819

CarbohydrateResearch485(2019)107819
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemical and enzymatic synthesis of the alginate sugar nucleotide building
block: GDP-D-mannuronic acid
Laura Beswick, Sanaz Ahmadipour2, Jonathan P. Dolan1, Martin Rejzek2, Robert A. Field2,
Gavin J. Miller∗
Lennard-Jones Laboratory, School of Chemical and Physical Sciences, Keele University, Keele, Staffordshire, ST5 5BG, UK
A R T I C L E I N F O
Keywords:
Sugar nucleotide
Glycosyl-1-phosphate
Mannuronate
Pyrophosphorylation
Alginate
1. Introduction
As part of a program to investigate the enzymes involved in the
biosynthesis of alginate [4], we were interested to chemically synthe-
sise 1 and deliver an enabling sugar nucleotide tool to support eluci-
dation of the alginate polymerisation process. A chemical synthesis of 1
was recently completed by Codée et al. [5] using PIII-amidite-PV
chemistry to accomplish the key pyrophosporylation step in forming 1.
Herein we report our approach to 1, instead using a PV-PV pyropho-
sphorylation and present the results of evaluating two differentially
protected D-ManA 1-phosphates for coupling. Alongside this we eval-
uated an enzymatic approach to 1 from GDP-D-Man using recombinant
GMD from P. aeruginosa.
Alginate is a heterogenous polysaccharide composed of β-1,4-linked
D-mannuronic acid (M) and its C5 epimer α-L-guluronic acid (G)
(Fig. 1a). Within alginate sub-structure the relative proportions of M
and G units, their homo- or heteropolymeric block-groupings and the
possibility for acetylation at the C2 and/or C3 positions of M residues
produces a structurally diverse biopolymer. This structural micro-
heterogeneity varies depending on the alginate source and the biopo-
lymer is produced by both plants and bacteria. The study of alginate
biochemistry and biosynthesis has largely focused on the bacterial
genera Pseudomonas, owing to the prevalence of the opportunistic
human pathogen Pseudomonas aeruginosa, which causes chronic infec-
tions in cystic fibrosis patients, contributing to a reduction in lung
function and increased mortality rates [1]. Alginate is also an important
industrial biomaterial, currently sourced from marine algae and utilised
as a stabiliser, viscosifier and gelling agent in the food, beverage, paper
and pharmaceutical industries [2].
2. Results and discussion
2.1. Synthesis of D-ManA 1-phosphates
Both enzymatic and chemical approaches to synthesise uronic acid
1-phosphates have been explored [6,7]. From a chemical perspective,
the inclusion of an acidic, charged functional group for pyropho-
sphorylative coupling is challenging and efforts to circumvent this have
involved completing late-stage (post-diphosphate formation) oxidation
to the uronate [8] and protecting the carboxylate [9]. We first sought to
synthesise two differentially protected D-ManA 1-phosphates, 7 and 8,
as we wanted to examine the effect of retaining a protected carboxylate
group (against the free acid form) when completing chemical pyr-
ophosphorylation. Previously it was noted by Linhardt [9] that
Alginate biosynthesis utilises the sugar nucleotide GDP-D-ManA, 1
(Fig. 1b), which is sourced from the cytosolic metabolic pool through a
series of enzymatic transformations starting from fructose 6-phosphate
and ultimately obtained via oxidation of GDP-D-Man to the uronate by
GDP-mannose dehydrogenase (GMD) [3]. Following this, an intricate,
multi-enzyme mediated polymerisation process assembles the β-D-
mannuronate polymer, which is then further modified by epimerisa-
tion, acetylation and truncation before export.
∗ Corresponding author.
E-mail address: g.j.miller@keele.ac.uk (G.J. Miller).
1 Current address: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT, UK.
2 Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
https://doi.org/10.1016/j.carres.2019.107819
Received 2 August 2019; Received in revised form 13 September 2019; Accepted 16 September 2019
Availableonline17September2019
0008-6215/©2019ElsevierLtd.Allrightsreserved.

L. Beswick, et al.
CarbohydrateResearch485(2019)107819
Fig. 1. a) Chemical structure of alginate showing
constituent M/G residues and C2/C3 acetylation for
one M residue, b) GDP-D-ManA 1, the sugar nucleotide
building block of alginate.
Owing to the problems we encountered in accessing 6 via 4 (15%, 2
steps), we explored an alternative route, firstly removing the anomeric
acetate from 3 to give hemi-acetal 5, in 68% yield, followed by con-
version to a trichloroacetimidate donor (88% yield) and immediate
reaction with DBP using TMSOTf as promoter. Although successful, our
attempts at optimisation did not deliver 6 in a yield greater than 49%
(from 5), but did afford the material in 30% overall yield from 3,
double that observed for the route from 4.
We next undertook a two-stage deprotection of 6 to deliver D-ManA
1-phosphates 7 and 8 (Scheme 2). We removed the phosphate benzyl
protecting groups using hydrogenolysis, followed by conversion to a
bis-triethylammonium salt, delivering semi-protected phosphate 7 in
very good yield (71%). The acetate protecting groups of 7 were then
cleaved to give D-ManA 1-phosphate 8. At this juncture we re-visited
the Macdonald phosphorylation and were able to establish conditions
to afford 8 directly from D-mannose 9 (Scheme 2). Following per-
acetylation and anomeric phosphorylation [14], the crude 1-phosphate
could be conveniently oxidised using TEMPO/BAIB or TEMPO/NaOCl
[15] to deliver 8 in 15% yield over three steps. This compares to an
overall yield of 15% over 5 steps for the route to 8 from 2, which whilst
longer, did afford access to the partially protected 1-phosphate 7. With
differentially protected D-ManA 1-phosphates, 7 and 8, in hand we next
evaluated their pyrophosphorylative coupling (with GMP-morpholi-
date) to deliver 1.
Scheme 1. Synthesis of protected D-ManA 1-phosphate 6. a) MeI, K2CO3, DMF,
77% b) NH2NH2. AcOH, DMF, 68% c) TMSOTf, HSPh, DCM, 63% d) i) Cl3CCN,
K2CO3, DCM, 88% ii) HOP(O)(OBn)2, TMSOTf, DCM, 49% e) HOP(O)(OBn)2,
NIS, AgOTf, DCM, 23%.
retaining a methyl ester protecting group (for their synthesis of UDP-L-
IdoA) avoided problems during the pyrophosphorylation coupling re-
action (65% reported yield). However, the synthesis of the related UDP-
D-GlcA reported by Khorana [10] using free D-GlcA 1-phosphate in-
dicated an equally successful approach (66% yield).
Our synthetic route began from mannuronic acid derivative 2, for
which we recently reported a multi-gram scale synthesis [11]. Methy-
lation of 2 was achieved using iodomethane and K2CO3 to give ester 3
in good yield (77%, Scheme 1). We next evaluated MacDonald's con-
ditions (H3PO4(s), high vacuum and 60 °C) [12] to directly form glycosyl
1-phosphates from 2 and 3. Unfortunately, in our hands, 2 and 3 largely
decomposed under the reaction conditions or formed significant
amounts of the C4–C5 elimination product and we instead attempted to
access thioglycoside donor 4, as a means to provide protected man-
nuronate 1-phosphate 6. We found the reaction to form 4 from 3 to be
sluggish and low-yielding, with significant amounts of orthoester for-
mation observed. This was attributed to the disarming nature of the
uronate and use of acetate protecting groups [13]. We were able to
optimise this reaction using TMSOTf as a Lewis acid (BF3 Et2O showed
no reaction) to a yield of 63% (5:1 α/β ratio of 4 with 4:1 α/orthoester,
as judged by 1H NMR) using a reaction temperature of −15 °C for 6 h.
Raising the temperature to 0 °C and extending the reaction time to 32 h
caused a significant reduction in yield (17%, with 20% returned 3), but
did reduce orthoester formation (6:1 α/β ratio of 4 with 33:1 α/or-
thoester). With amounts of pure 4 in hand, following silica gel chro-
matography, we next converted to the protected 1-phosphate 6 using
dibenzyl phosphate (DBP) under standard thioglycoside activation
conditions. This afforded 6, albeit in low yield (23%), but with the
expected 31P NMR resonance for the anomeric phosphate (−3.24 ppm)
and the characteristic doublet of doublets for H1 (3JH1-31P = 6.4 Hz,
3JH1-H2 = 1.9 Hz).
2.2. Chemical synthesis of GDP-D-ManA
In recent years, chemical approaches to synthesise sugar nucleotides
have favoured PV-PV and PV-PIII methods, removing any anomeric in-
tegrity consequences of glycosylating
a nucleoside diphosphate
[16,17]. We selected a PV-PV approach using GMP-morpholidate as the
coupling partner for 7 or 8 and trialled different activators, solvents and
durations, the results of which are summarised in Table 1.
N-Methylimidazole hydrochloride (N-MIC [18], Table 1, entry 1)
has been reported as a superior pyrophosphorylative catalyst to the
traditional use of 1-H-tetrazole. Utilising it here, we were unable to
detect the formation of 1 by TLC (isopropyl alcohol/ammonium hy-
droxide/water, 6:3:1) and observed baseline material after 60h. Re-
peating the reaction (including several co-evaporations with toluene
under N2 prior to reaction) led to similar outcomes and we thus swit-
ched to using 4,5-dicyanoimidazole (DCI, Table 1, entry 2). This reac-
tion proceeded smoothly over 56 h with TLC analysis indicating sig-
nificant consumption of 7 and crude 31P NMR confirming nucleoside
diphosphate formation (δP −11.4, −14.5 ppm). The crude material
isolated was immediately subjected to pyranoside deprotection using
Et3N/MeOH/H2O, followed by strong-anion exchange (SAX) purifica-
tion which delivered 1 but only in very poor yield (< 5%). We
Scheme 2. Synthesis of semi-protected and free D-
ManA 1-phosphates 7 and 8. a) H2, Pd/C, MeOH,
Et3N, 71%; b) Et3N/MeOH/H2O, 2:2:1, IR120 Na+
resin, 96%; c) i) Ac2O, pyridine, DMAP, 93% ii)
H3PO4, then LiOH, 56% iii) TEMPO, BAIB, H2O/
MeCN, 30% or TEMPO, NaOCl, NaOH, H2O/MeCN,
21%.
2

L. Beswick, et al.
CarbohydrateResearch485(2019)107819
Table 1
Evaluation of pyrophosphorylation conditions to synthesise 1.
Entry
1-phosphate
Additive (equiv.)a
Reaction Time (h)
Solvent (conc.)
Yield (%)
Notes
1
2
3
4
5
6
7
7
7
7
7
7
8
8
N-MIC (2.9)
DCI (4.0)
60
DMF (0.06)
DMF (0.05)
DMF (0.05)
Pyr. (0.06)
Pyr. (0.06)
DMF (0.07)
Pyr. (0.04)
0
No rxn.
56
< 5b
< 5b
22b
46b
0
DCI contamination
Reduced DCI
Reduced DCI
Reduced DCI
No rxn.
DCI (1.0)
108
144c
120
40
None then DCI (1.0)
DCI (1.0)
DCI (1.0)
None
144
0
No rxn.
R = Ac, R’ = Me.
a
b
c
Along with 1.5 equiv. GMP-morpholidate and 1.0 equiv. of 1-phosphate.
Following deprotection of the crude coupling reaction (Et3N, MeOH, H2O).
DCI was added after 48 h, as no reaction was indicated to have taken place by TLC.
encountered problems here during SAX purification, namely that the
large amount of DCI used (4.0 equiv.) co-eluted with 1, thus requiring
additional C18 reverse phase purification to remove this impurity
which reduced the overall yield. In order to solve these problematic
final purification(s) we investigated reducing the equivalents of DCI
alongside changing the reaction solvent to pyridine (Table 1, entries 3
and 4). Pleasingly, we were able to improve the yield of 1 to46% using
1.0 equiv. of DCI in pyridine (Table 1 entry 5). We also observed that
the uncatalysed reaction was very slow (no reaction after 48 h), but did
not investigate reducing the amount of DCI further. Using only 1.0
equivalent of DCI we were also able to return to using DMF as solvent,
which improved solubility of the reagents slightly, obtaining similar
results to those using pyridine.
Scheme 3. Enzymatic synthesis of 1 from GDP-D-Man. a) NAD+, DTT, MgCl2,
pH 7.4, 70%.
3. Conclusion
We have established chemical (PV-PV) and enzymatic routes to the
alginate sugar nucleotide feedstock GDP-D-ManA. Synthetic access to
partially protected and fully deprotected anomeric 1-phosphates of D-
mannuronic acid enabled their evaluation in pyrophosphorylative
coupling to the target nucleoside diphosphate. Only the partially pro-
tected glycosyl 1-phosphate was effective for this reaction under the
conditions examined. This procedure is complimented by an enzymatic
approach to the same sugar nucleotide using the GDP-D-mannose de-
hydrogenase from P. aeruginosa.
For ManA 1-phosphate 8 we observed no indicative conversion to 1
by TLC (Table 1, entry 6) and we surmised that poor solubility of the
components was hindering the reaction in DMF. Changing solvent to
pyridine (Table 1, entry 7) unfortunately had no positive effect on the
reaction outcome and we concluded that the material was not reacting
under the conditions tried (GMP-morpholidate could still be observed
by crude 31P NMR). In summary, we observed that successful pyr-
ophosphorylative coupling to form 1 could best be achieved using
carboxylate protected mannuronate 1-phosphate 7. The chemical
synthesis route developed here delivers multi-milligram access to 1 in
five steps and 8% overall yield from 2. Whilst more involved than the
direct enzymatic option considered below, this methodology will be
underpinning to the development of analogue syntheses derived from 1,
which is essential to the continued study of sugarnucleotide-mediated
alginate biosynthesis.
4. Experimental section
4.1. General methods and materials
All reagents and solvents which were available commercially were
purchased from Acros, Alfa Aesar, Fisher Scientific, or Sigma Aldrich.
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). 1H NMR spectra were recorded at
400 MHz and 13C spectra at 100 MHz respectively using a Bruker
AVIII400 spectrometer. 1H NMR signals were assigned with the aid of
gDQCOSY. 13C 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 or Mestrenova software. 1H NMR
splitting patterns were assigned as follows: br s (broad singlet), 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
2.3. Enzymatic synthesis of GDP-D-ManA
Within alginate biosynthesis, 1 is produced by dehydrogenative
oxidation of GDP-D-Man by GMD. In order to investigate enzymatic
production of 1 we incubated GDP-D-Man with recombinant GMD from
P. aeruginosa in the presence of NAD+ at room temperature with gentle
shaking. The reaction was monitored by SAX chromatography at dif-
ferent time points. After 21 h, the conversion of GDP-D-Man to 1
reached 70%, using 2 equivalents of NAD+, and enabled the isolation of
mg quantities of the desired material (Scheme 3). After 72 h, complete
consumption of the starting material was evident, following the addi-
tion of four further equivalents of NAD+ (see SI).
3

L. Beswick, et al.
CarbohydrateResearch485(2019)107819
multiple resonances). Reactions were followed by thin layer chroma-
tography (TLC) using Merck silica gel 60F254 analytical plates (alu-
minium support) and were developed using standard visualising agents:
short wave UV radiation (245 nm) and 5% sulfuric acid in methanol/Δ.
Purification via flash column chromatography was conducted using si-
lica gel 60 (0.043–0.063 mm). Optical activities were recorded on au-
tomatic polarimeter Rudolph autopol I or Bellingham and Stanley
ADP430 (concentration in g/100 mL). pH measurements were recorded
using a Hanna® pH 20 m. MS and HRMS (ESI) were obtained on Waters
(Xevo, G2-XS TOF) or Waters Micromass LCT spectrometers using a
methanol mobile phase. High resolution (ESI) spectra were obtained on
a Xevo, G2-XS TOF mass spectrometer. HRMS was obtained using a
lock-mass to adjust the calibrated mass. HPLC was performed on an
Agilent Technologies 1200 series machine, using a Waters Bridge
Reversed-phase prep-C18 column (5 μm OBD, 19 × 100 mm).
MeCN:H2O, 60:40 → 100% was used as a mobile phase and the product
was detected using UV at 254 nm. Purification by C18 chromatography
was conducted using a Thermoscientific ×30 SPE column (HyperSep
C18, 6 mL) eluting with H2O. Purification via strong anion 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 (3.0 mL/min), 0 → 100% 1.0 M (NH4)HCO3 over 33 min or
strong anion-exchange (SAX) HPLC on Poros HQ 50 was performed as
published earlier [19].
(MgSO4), filtered and concentrated under reduced pressure to afford a
pale-yellow oil. Purification by silica column chromatography, eluting
with hexane/ethyl acetate (1/0, 3/1) afforded 4 as an opaque, col-
ourless oil (134 mg, 315 μmol, 63%). Rf 0.30 (hexane/ethyl acetate 3/
1); 1H NMR (400 MHz, CDCl3) δH α-anomer 7.51 (2H, d, J = 6.6 Hz,
Ar–H), 7.30 (3H, m, Ar–H), 5.59 (1H, d, J = 3.8 Hz, H1), 5.48–5.40 (2H,
m, H2, H4), 5.33 (1H, dd, J = 8.4, 3.1 Hz, H3), 4.79 (1H, d, J = 7.8 Hz,
H5), 3.76 (3H, s, C(O)OCH3), 2.11 (3H, s, C(O)CH3), 2.08 (3H, s,
C(O)CH3), 2.02 (3H, s, C(O)CH3); 13C NMR (100 MHz, CDCl3) δC 169.7
(C]O), 169.5 (C]O), 169.3 (C]O), 167.8 (C]O), 132.3 (Ar–C),
131.7 (Ar–C), 129.1 (Ar–C), 128.0 (Ar–C), 84.4 (C1), 71.0 (C5), 69.2
(C2), 68.3 (C3), 67.4 (C4), 52.6 (C(O)OCH3), 20.7 (C(O)CH3), 20.6
(C(O)CH3), (C(O)CH3) 20.5 (C(O)CH3); HRMS [M+NH4]+ calculated
for C19H26O9SN: 444.1323; found: 444.1323.
4.4. Methyl 2,3,4-tri-O-acetyl-α-D-mannopyranosyluronate (5)
To a stirred solution of 3 (100 mg, 0.27 mmol, 1.0 equiv.) in an-
hydrous DMF (2 mL) was added hydrazine acetate (38 mg, 0.41 mmol,
1.5 equiv.). The solution was stirred at room temperature for 3 h,
whereupon TLC analysis (hexane/ethyl acetate, 1/1) indicated com-
plete conversion of starting material to a lower Rf spot. The solvent was
removed in vacuo and the residue dissolved in ethyl acetate (10 mL).
The organic layer was washed with distilled water (10 mL) and the
aqueous layer re-extracted with ethyl acetate (10 mL). The combined
organic layers were washed with distilled water (20 mL), dried
(MgSO4), filtered and concentrated in vacuo. Purification by silica gel
column chromatography eluting with hexane/ethyl acetate (3/1, 1/1)
afforded 5 as a colourless oil (95% α-anomer, 58 mg, 0.17 mmol, 64%).
Rf 0.47 (hexane/ethyl acetate, 1/1); 1H NMR (400 MHz, CDCl3) δH α-
anomer 5.45 (1H, d, J = 3.2 Hz, H3), 5.41 (1H, d, J = 9.0 Hz, H4), 5.34
(1H, d, J = 2.5 Hz, H1), 5.26–5.25 (1H, m, H2), 4.58 (1H, d, J = 8.9 Hz,
H5), 2.15 (3H, s, C(O)OCH3), 2.07 (3H, s, C(O)OCH3), 2.02 (3H, s, C(O)
OCH3); 13C NMR (100 MHz, CDCl3) δC 170.2 (C]O), 169.7 (C]O),
168.6 (C]O), 162.8 (C]O), 92.1 (C1), 69.7 (C2), 69.6 (C5), 68.3 (C3),
67.2 (C4), 54.7 (C(O)2CH3), 20.9 (C(O)CH3), 20.7 (C(O)CH3), 20.6
(C(O)CH3); HRMS [M+NH4]+ calculated C13H22O10N: 352.1283;
found: 352.1246.
4.2. Methyl (1,2,3,4-tetra-O-acetyl-β-D-mannopyranosyl)uronate (3)
To a stirred solution of 1,2,3,4-tetra-O-acetyl-β-D-mannuronic acid 2
[11] (600 mg, 1.70 mmol, 1.0 equiv.) in anhydrous dimethylformamide
(8 mL) was added methyl iodide (250 μL, 4.02 mmol, 2.4 equiv.) and
K2CO3 (156 mg, 1.13 mmol, 1.5 equiv.). The solution was stirred at
room temperature for 72 h, whereupon TLC analysis (hexane/ethyl
acetate, 3/1) indicated complete conversion of starting material to a
higher Rf spot. The reaction was quenched with methanol (5 mL), ethyl
acetate (25 mL) was added and the solution washed with H2O (15 mL).
The aqueous layer was extracted with ethyl acetate (25 mL), the com-
bined organic layers washed with water (15 mL) and brine (15 mL),
dried (MgSO4), filtered and concentrated in vacuo. The resultant yellow
solid was triturated with methanol to afford 2 as a white solid (480 mg,
4.5. Methyl 2,3,4-tri-O-acetyl-α-D-mannopyranosyluronate dibenzyl 1-
26
1.3 mmol, 77%). Rf 0.23 (hexane/ethyl acetate, 3/1); [α]
= −16.0
D
phosphate (6)
(c = 0.5, CHCl3); 1H NMR (300 MHz, CDCl3) δH 5.91 (1H, d,
J = 1.3 Hz, H1), 5.50 (1H, dd, J = 3.5, 1.2 Hz, H2), 5.42 (1H, t,
J = 9.4 Hz, H4), 5.19 (1H, dd, J = 9.6, 3.2 Hz, H3), 4.15 (1H, d,
J = 9.4 Hz, H5), 3.74 (3H, s, C(O)OCH3), 2.21 (3H, s, C(O)CH3), 2.12
(3H, s, C(O)CH3), 2.07 (3H, s, C(O)CH3), 2.03 (3H, s, C(O)CH3); 13C
NMR (100 MHz, CDCl3) δC 170.6 (C]O), 170.0 (2 x C]O), 168.8 (C]
O), 167.2 (C]O), 90.1 (C1), 73.6 (C5), 70.0 (C3), 67.8 (C2), 66.7 (C4),
53.3 (CO2CH3), 21.1 (C(O)CH3), 21.1 (C(O)CH3), 21.0 (C(O)CH3), 20.9
(C(O)CH3); HRMS [M+NH4]+ calculated for C15H24O11N: 394.1344;
found: 394.1337.
From 5: To a stirred solution of 5 (550 mg, 1.65 mmol, 1.0 equiv.)
and oven dried anhydrous K2CO3 (360 mg, 2.64 mmol, 1.6 equiv.) in
anhydrous dichloromethane (5.5 mL) was added trichloroacetonitrile
(0.37 mL, 4.62 mmol, 2.8 equiv.). The solution was stirred at room
temperature for 24 h, whereupon TLC analysis (hexane/ethyl acetate,
1/1) indicated conversion of the starting material to a higher Rf spot.
The dark brown solution was filtered through Celite®, washing with
dichloromethane and concentrated in vacuo to afford the tri-
chloroacetimidate as a pale brown oil (774 mg, 1.62 mmol, 88%). This
crude material (774 mg, 1.62 mmol, 1.0 equiv.) was dissolved in an-
hydrous dichloromethane (15 mL), powdered 4 Å molecular sieves were
added and the suspension stirred for 2 h. Dibenzyl phosphate (770 mg,
2.75 mmol, 1.7 equiv.) was then added and stirring continued for
30 min. The solution was then cooled to −10 °C and TMSOTf (0.15 mL,
810 μmol, 0.5 equiv.) added dropwise. The solution was warmed slowly
to room temperature over 1 h. TLC analysis (hexane/ethyl acetate, 1/1)
indicated complete conversion of starting material to a lower Rf spot.
The light orange reaction mixture was quenched by addition of Et3N
(until pH = 7) and filtered over Celite™ washing with dichloromethane
(20 mL). The organic layer was then washed with saturated aqueous
NaHCO3 solution (25 mL), distilled water (25 mL), brine (25 mL), dried
(MgSO4), concentrated in vacuo to give a yellow oil. This crude material
was purified by silica gel column chromatography eluting with toluene/
acetone (10/1, 7/1, 3/1) to afford 6 as a colourless oil (223 mg,
4.3. Methyl phenyl-2,3,4-tri-O-acetyl-1-thio-α/β-D-
mannopyranosyluronate (4)
Uronate 3 (200 mg, 500 μmol, 1.0 equiv.) and powdered 4 Å mo-
lecular sieves were dissolved in anhydrous dichloromethane (3 mL) and
stirred under N2 atmosphere for 12 h. Thiophenol (82 μL, 820 μmol. 1.5
equiv.) was added, the solution cooled to −15 °C and TMSOTf
(0.30 mL, 1.62 mmol, 3 equiv.) was added dropwise. The reaction
mixture was stirred at − 15 °C for 6 h, whereupon TLC analysis
(hexane/ethyl acetate 3/1) showed complete conversion of the starting
material to a higher Rf spot. The yellow reaction mixture was quenched
through the addition of triethylamine until pH = 7, filtered over
Celite™ and diluted with dichloromethane (25 mL). The organic layer
was washed with distilled water (15 mL) and brine (15 mL), dried
4

L. Beswick, et al.
CarbohydrateResearch485(2019)107819
0.38 mmol, 34%). Rf 0.30 (hexane/ethyl acetate, 1/1); 1H NMR
(400 MHz, CDCl3) δH 7.35–7.34 (8H, m, ArH), 7.19–7.17 (2H, m, ArH),
5.70 (1H, dd, J = 6.4, 1.9 Hz, H1), 5.41–5.31 (2H, m, H3, H4), 5.24 (1H,
d, J = 2.1 Hz, H2), 5.09 (4H, m, CH2Ph), 4.39 (1H, d, J = 8.8 Hz, H5),
3.69 (3H, s, C(O)OCH3), 2.13 (3H, s, C(O)CH3), 2.05 (3H, s, C(O)CH3),
2.01 (3H, s, C(O)CH3); 13C NMR (100 MHz, CDCl3) δC 169.6 (C]O),
169.5 (C]O), 169.5 (C]O), 167.2 (C]O), 129.1 (Ar–C), 128.7 (Ar–C),
128.7 (Ar–C), 128.2 (Ar–C), 128.1 (Ar–C), 94.7 (C1), 70.8 (C5), 70.1
(CH2Ph), 69.9 (CH2Ph), 68.2 (C2), 67.6 (C3), 66.4 (C4), 52.8
(C(O)2CH3), 21.5 (C(O)CH3), 20.7 (C(O)CH3), 20.6 (C(O)CH3); 31P
NMR (161 MHz, CDCl3) δP −3.20 (1 P, s); HRMS [M+H]+ calculated
for C27H32O13P: 595.5158; found: 595.1586. These data were in good
agreement with literature values [5].
(Dowex® 50 W-X4 Na+ form, 200–400 mesh) eluting with water. The
sugar containing fractions were pooled and freezedried to afford 8 as a
fluffy cream solid (53 mg, 0.19 mmol, 96%). Rf 0.33 (acetonitrile/water
26
with 4 drops NH4OH, 2/1); [α]
= + 22.22 (c = 0.45, H2O); 1H
D
NMR (400 MHz, D2O) δH 5.28 (1H, d, JH-P = 8.6 Hz, H1), 4.02 (1H, d,
J = 10.0 Hz, H5), 3.89–3.83 (2H, m, H2, H3), 3.75–3.65 (1H, m, H4);
13C NMR (100 MHz, D2O) δC 177.4 (C]O), 95.2 (C1), 72.9 (C4), 71.0
(C2), 70.0 (C3), 69.0 (C5); 31P NMR (161 MHz, D2O) δP 1.35 (s); HRMS
[M-H]− calculated for C6H11O10P: 273.0012; found: 273.0013.
From 9: D-mannose (5.00 g, 30.0 mmol, 1.0 equiv.) and DMAP
(61 mg, 0.5 mmol, 0.02 equiv.) were dissolved in anhydrous pyridine
(70 mL) and cooled to 0 °C. Acetic anhydride (18.0 mL, 190 mmol, 6.8
equiv.) was added dropwise and reaction warmed to room temperature
and stirred for 71 h. After this time the solution was poured onto iced
water (100 mL) and stirred vigorously for 1 h, whereupon the majority
of the solvent was removed in vacuo and the water extracted with ethyl
acetate (2 × 50 mL). The combined organic extracts were washed with
water (50 mL), saturated aqueous NaHCO3 solution (3 × 50 mL), brine
(2 × 50 mL), dried (MgSO4), and concentrated in vacuo to give a yellow
oil. This crude material was purified by silica gel column chromato-
graphy eluting with EtOAc/hexane (2/1) to afford (1,2,3,4,6)-penta-O-
acetyl-D-mannose as a colourless syrup (9.80 g, 25.1 mmol, 90%). This
material (9.80 g, 25.1 mmol, 1.0 equiv.) and phosphoric acid (14.1 g,
144.0 mmol, 5.7 equiv.) were dissolved in anhydrous THF (20 mL) and
the solvent removed in vacuo. The resulting syrup was stirred at room
temperature under high vacuum for 1 h (0.35 kPa). The temperature
was ramped to 60 °C over a period of 30 min and stirred for a further 2 h
under vacuum (0.35 kPa). The reaction was cooled to room tempera-
ture, THF (20 mL) was added and the solution further cooled to 0 °C.
The reaction was then quenched using 25% NH4OH solution (12 mL),
the resulting precipitate filtered off and washed with ice-cold THF
(10 mL). To the filtrate was added LiOH (3.08 g, 128 mmol, 5.1 equiv.)
in H2O (5 mL) and the solution stirred at room temperature overnight.
The reaction was then neutralised using IR120H+ ion exchange resin
and filtered through a Whatman® GF/A glass microfibre filter. The
solvent was evaporated in vacuo and the residue treated with MeOH
(30 mL). The resulting suspension was centrifuged at 4000 rpm for
5 min., the supernatant removed, the pellets washed with ice cold
MeOH and then dried in vacuo to give α-D-mannose-1-phosphate
(3.74 g, 14.5 mmol, 56%) as a white amorphous solid. 1H NMR
(400 MHz, CDCl3) δH 5.23 (1H, d, J = 8.6 Hz, H1), 3.82–3.87 (2H, m,
H2, H3), 3.75–3.82 (2H, m, H5, H6b), 3.63 (1H, dd, J = 11.7, 6.1 Hz,
From 4: Uronate 4 (180 mg, 0.4 mmol, 1.0 equiv.) and powdered
4 Å molecular sieves were dissolved in anhydrous dichloromethane
(5 mL) and stirred under a N2 atmosphere at RT for 12 h. Dibenzyl
phosphate (198 mg, 0.7 mmol, 1.7 equiv.) was added and stirred for
30 min. N-iodosuccinimide (0.14 g, 0.6 mmol, 1.5 equiv.) and silver
trifluoromethanesulfonate (33 mg, 0.1 mmol, 0.3 equiv.) were added at
−30 °C and the temperature was raised to −10 °C over 40 min. TLC
analysis (hexane/ethyl acetate, 1/1) indicated complete conversion of
the starting material to a higher Rf spot. The dark red reaction mixture
was quenched through the addition of triethylamine until pH = 7, fil-
tered through Celite™ and diluted with dichloromethane (25 mL). The
organic layer was washed with saturated aqueous Na2S2O3 solution
(15 mL), saturated aqueous sodium hydrogen carbonate solution
(15 mL), distilled water (15 mL) and brine (15 mL). The organic layer
was dried (MgSO4), filtered and concentrated under reduced pressure to
afford a dark orange oil. Purification by silica gel column chromato-
graphy, eluting with hexane/ethyl acetate (3/1, 2/1, 1/1), afforded 6 as
a colourless oil (61 mg, 100 μmol, 23%).
4.6. Methyl 2,3,4-tri-O-acetyl-α-D-mannopyranosyluronate 1-phosphate
(bis-triethylammonium salt) (7)
A suspension of 6 (200 mg, 0.34 mmol, 1.0 equiv.) and 10% Pd/C
(15 mg, 0.14 mmol, 0.2 eq. per Bn) in anhydrous methanol (5 mL) was
stirred under an atmosphere of hydrogen (1 atm, balloon) at room
temperature for 5 h. TLC analysis (hexane/ethyl acetate, 1/2) showed
complete conversion of starting material to a lower Rf spot. The reac-
tion mixture was filtered through Celite®, washing with methanol and
the filtrate treated with Et3N (95 μL, 0.68 mmol, 2.0 equiv.) followed by
solvent removal in vacuo to afford 7 as a white solid (148 mg,
H
6a), 3.50 (1H, apt, J = 9.7 Hz, H4); 13C NMR (100 MHz, CDCl3) δC
0.24 mmol, 71%). Rf 0.45 (ethyl acetate/methanol/water, 5/3/1);
94.1 (d, J1,P = 4.4 Hz, C1), 72.0 (C5), 70.2 (d, J2,P = 7.3 Hz, C2), 69.3
(C3), 66.2 (C4), 60.4 (C6); 31P NMR (161 MHz, D2O) δP 1.79 (d,
JP,1 = 7.8 Hz); HRMS [M+Li]+ calculated for C6H13O9PLi: 267.0457;
found: 267.0469.
26
[α]
= + 16.05 (c = 0.3, MeOH); 1H NMR (400 MHz, CDCl3) δH
D
5.62 (1H, d, J = 7.0 Hz, H1), 5.47 (1H, dd, J = 9.9, 3.3 Hz, H3),
5.37–5.30 (2H, m, H2, H4), 4.70 (1H, d, J = 10.0 Hz, H5), 3.70 (3H, s,
CO2CH3), 2.93 (12H, q, J = 6.6 Hz, [CH3CH2]3NH+), 2.13 (3H, s,
C(O)CH3), 2.03 (3H, s, C(O)CH3), 1.96 (3H, s, C(O)CH3), 1.25 (18H, t,
J = 6.9 Hz, [CH3CH2]3NH+); 13C NMR (100 MHz, CDCl3) δC 169.9 (2 x
C]O), 169.6 (C]O), 168.6 (C]O), 93.6 (C1), 69.6 (C2), 69.5 (C5),
68.6 (C3), 67.1 (C4), 52.5 (CO2CH3), 45.6 (N(CH2CH3)3), 20.9
(C(O)CH3), 20.7 (C(O)CH3), 20.6 (C(O)CH3), 9.2 (N(CH2CH3)3); 31P
NMR (160 MHz, CDCl3) δP −0.90 (s); HRMS [M+H]+ calculated for
4.7.1. Oxidation of mannose 1-phosphate
4.7.1.1. Secondary oxidant NaOCl. α-D-mannose-1-phosphate (89 mg,
0.33 mmol, 1.0 equiv.) and TEMPO (9 mg, 0.06 mmol, 0.2 equiv.) were
dissolved in a mixture of H2O and MeCN (2 mL, 1:1, v/v) at 0 °C. To this
solution, aqueous 1 M NaOH was added to pH 9. NaOCl solution (1 mL,
available chlorine 10%) was then added slowly to the rapidly stirring
solution. The pH was maintained at 9 by adding 1 M NaOH several
times over the course of the reaction. After 2 h, the reaction mixture
was concentrated and MeOH (5 mL) was added to the resultant residue
causing a precipitate to form. This suspension was centrifuged at
4000 rpm for 5 min., the supernatant removed, the pellet washed
(once with MeOH and twice with MeCN) and dried in vacuo to give 8
(23 mg, 0.08 mmol, 21%) as a white amorphous solid.
C
13H19O13P: 413.0951; found: 413.0945.
4.7. α-D-mannopyranuronic acid 1-phosphate (disodium salt) (8)
From 7: To a stirred solution of 7 (130 mg, 0.21 mmol, 1.0 equiv.) in
methanol/water (3 mL/1.5 mL) was added triethylamine (3 mL). The
solution was stirred for 18 h at room temperature, whereupon TLC
analysis (acetonitrile/water with 4 drops of NH4OH, 2/1) indicated
conversion of starting material to a lower Rf spot. The solution was
concentrated in vacuo (water bath temperature not exceeding 30 °C) to
afford a yellow residue. This was passed down an ion-exchange column
4.7.1.2. Secondary oxidant BAIB. α-D-mannose-1-phosphate (505 mg,
1.85 mmol, 1.0 equiv.), TEMPO (46 mg, 0.3 mmol, 0.15 equiv.) and
bis(acetoxy)iodobenzene (1.29 g, 3.99 mmol, 2.2 equiv.) were dissolved
5

L. Beswick, et al.
CarbohydrateResearch485(2019)107819
in a mixture of H2O/MeCN (8 mL, 1:1, v/v). The reaction was stirred at
room temperature for 24 h, concentrated in vacuo and MeOH (10 mL)
added to the resultant residue, causing a precipitate to form. This
suspension was centrifuged at 4000 rpm for 5 min., the supernatant
removed, the pellet washed (once with MeOH and once with MeCN)
and dried in vacuo to give crude 8. This material was dissolved in H2O
and passed through a Sephadex® G25 gel filtration column. The sugar-
sequential evaporations from methanol (2 × 25 mL) and the solid was
lyophilized to afford the title compound as a white solid (161 mg,
0.37 mmol, 30%). Rf 0.52 (isopropyl alcohol/NH4OH/water, 7/1/2);
26
[α]
= − 16.0 (c = 0.5, H2O); 1H NMR (400 MHz, D2O) δH 7.93 (1H,
D
s, H8), 5.79 (1H, d, J = 5.0 Hz, H1’), 4.67 (1H, t, J = 5.1 Hz, H2’), 4.41
(1H, t, J = 4.8 Hz, H3’), 4.20 (1H, br. s, H4’), 3.95–3.88 (2H, m, H5’),
3.47 (4H, t, J = 4.5 Hz, 2 × CH2 morpholine), 2.85–2.82 (4H, m,
2 × CH2 morpholine); 13C NMR (101 MHz, D2O) δC 159.5 (guanine C),
154.5 (guanine C), 151.7 (guanine C), 137.2 (C8), 116.4 (guanine C),
87.3 (C1’), 83.7 (C4’), 83.7 (C1’), 73.7 (C2’), 70.4 (C3’), 66.9 (CH2 mor-
pholine), 64.1 (C5’), 44.7 (CH2 morpholine); 31P NMR (161 MHz, D2O)
δP −7.46 (1s); HRMS [M−H]− calculated for C4H20N6O8P: 431.1080;
found: 431.1082.
+
containing fractions were pooled, treated with an NH4 ion exchange
resin, filtered and freeze-dried to yield 8 (182 mg, 0.67 mmol, 30%) as
needle-like crystals. See above for analytical data for 8.
4.8. Guanosine-5′-phosphoromorpholidate
Method
A
(Khorana [20]): Dowex® 50 W-X8 resin (H+ form,
17 × 700 mm column) was exchanged to its morpholine form by pas-
sing a 10% aqueous morpholine solution through the column (200 mL).
Exchange was indicated through a basic pH of the eluate (pH 10.87).
Guanosine 5′-monophosphate disodium salt (Na2GMP) (407 mg,
1.0 mmol, 1.0 equiv.) in distilled water (50 mL) was then applied to the
column and eluates containing sodium morpholine-GMP were con-
centrated to a final volume of 10 mL. Morpholine (210 μL, 2.4 mmol,
2.4 equiv.) and t-butanol (10 mL) were added and the solution was
4.9. General procedure for sugar nucleotide synthesis
Glycosyl 1-phosphate and GMP-morpholidate were exchanged to
their bis-triethylammonium salt forms prior to reaction and lyophilized.
Glycosyl 1-phosphate (bis-triethylammonium salt, 1.0 equiv.), GMP-
morpholidate (bis-triethylammonium salt, 1.5 equiv.) and activator
were each co-evaporated with toluene or pyridine (3 × 2 mL) and then
dissolved in DMF or pyridine, respectively. The reaction mixture was
stirred at room temperature and conversion monitored by TLC analysis
(isopropyl alcohol/NH4OH/water, 6/3/1). The reaction mixture was
concentrated under reduced pressure (water bath temperature not ex-
ceeding 30 °C) and dried under high vacuum before analysis by crude
1H and 31P NMR to confirm presence of the NDP-sugar.
heated to 100 °C at reflux, whilst
a solution of dicyclohex-
ylcarbodiimide (825 mg, 4.0 mmol, 4.0 equiv.) in t-butanol (15 mL) was
added dropwise over 2 h. The solution was heated at reflux for a further
3 h, whereupon TLC analysis (isopropyl alcohol/NH4OH/water, 7/1/2)
showed two new spots (Rf = 0.52 + 0.93). The yellow solution was
cooled to room temperature and left for 72 h whereupon a white
crystalline by-product (dicyclohexylurea) had formed. The suspension
was filtered, concentrated in vacuo and the remaining aqueous phase
extracted with diethyl ether (2 × 20 mL). The combined aqueous
phases were concentrated in vacuo then purified by Sephadex® G25
column chromatography, eluting with a linear gradient of triethy-
lammonium bicarbonate (0.005–0.5 M). Fractions containing the pro-
duct were collected and concentrated under reduced pressure. Residual
bicarbonate was removed by sequential evaporations from methanol
(2 × 25 mL). The residue was dissolved in methanol (10 mL), 4-mor-
pholine-N,N′-dicyclohexylcarboxamidine (600 mg, 2.0 mmol, 2.0
equiv.) was added then the solution concentrated under reduced pres-
sure. The residue was dissolved in methanol (5 mL) and diethyl ether
(25 mL) was added to form a white precipitate. The liquid was decanted
and the precipitate was washed with diethyl ether (2 × 10 mL), re-
dissolved in water and lyophilized to afford the title compound as a
cream solid (198 mg, 0.27 mmol, 27%). Rf 0.52 (isopropyl alcohol/
ammonium hydroxide/water, 7/1/2); 31P NMR(161 MHz, D2O) δP
−7.41 (s).
4.10. General procedure for sugar nucleotide deprotection
The crude reaction mixture was suspended in a mixture of MeOH
and H2O (1:1) and Et3N was added until pH = 9. The reaction mixture
was stirred for 24 h at room temperature or until TLC analysis (iso-
propyl alcohol/NH4OH/water, 6/3/1) indicated complete conversion
of starting material to a lower Rf value spot. The reaction mixture was
concentrated under reduced pressure (water bath temperature not ex-
ceeding 30 °C) to give a dark yellow residue. This was dissolved in H2O
and, for entries using DCI, passed down a Thermoscientific ×30 SPE
column (HyperSep C18, 6 mL), eluting with H2O to remove DCI. The
resulting aliquots were purified by SAX chromatography as described in
General Methods.
Table 1, Entry 5: 7 (22 mg, 53 μmol, 1.0 equiv.), GMP-morpholidate
(36 mg, 84 μmol, 1.5 equiv.) and DCI (6 mg, 53 μmol, 1.0 equiv.) were
dissolved in pyridine (1 mL) and stirred for 120 h. Following depro-
tection/purification, as described in 4.9 & 4.10, afforded 1 as a white
powder (15 mg, 24 μmol, 46%).
Method B (Mukaiyama [21]): Dowex® 50 W-X8 resin (H+ form,
17 × 700 mm column) was exchanged to its morpholine form by pas-
sing a 10% aqueous morpholine solution through the column (200 mL).
Exchange was indicated through a basic pH of the eluate (pH 10.87).
Guanosine 5′-monophosphate disodium salt (Na2GMP) (500 mg,
1.23 mmol, 1.0 equiv.) in distilled water (50 mL) was applied to the
column and eluates containing sodium morpholine-GMP were con-
centrated in vacuo to afford a cream solid. To a solution of morpholine-
GMP in dimethyl sulfoxide (10 mL) was added morpholine (0.58 mL,
6.64 mmol, 5.4 equiv.) to form an opaque white solution. After stirring
for 5 min. at room temperature, dipyridyl disulfide (0.89 g, 4.06 mmol,
3.3 equiv.) was added slowly to the solution, followed by triphenyl
phosphine (1.06 g, 4.06 mmol, 3.3 equiv.). The resultant bright yellow
solution was stirred for 4 h at room temperature and a solution of so-
dium iodide (0.1 M in acetone) was then added until a precipitate
formed. This was collected by filtration, dissolved in distilled water and
purified by Sephadex® G25 column chromatography, eluting with a
Table 1, Entry 4: 7 (44 mg, 0.10 mmol, 1.0 equiv.) and GMP-mor-
pholidate (73 mg, 0.17 mmol, 1.6 equiv.) were dissolved in pyridine
(1.5 mL) and stirred for 48 h. DCI (11 mg, 93 μmol, 1.0 equiv.) was
added and the reaction mixture was stirred for a further 96 h. Following
deprotection/purification, as described in 4.9 & 4.10, afforded 1 as a
white powder (14 mg, 22 μmol, 22%).
4.11. Guanosine 5’-(α-D-mannopyranuronic diphosphate) (1)
Rf 0.19 (isopropyl alcohol/NH4OH/water, 6/3/1); 1H NMR
(600 MHz; D2O) δ 7.96 (1 H, s, H8G), 5.79 (1 H, d, J = 6.1 Hz, H1Rib),
5.39 (1 H, dd, J = 8.0, 1.7 Hz, H1Man), 4.64 (1 H, hidden, H2Rib), 4.35
(1 H, dd, J = 4.8, 3.3 Hz, H3Rib), 4.34–4.29 (1 H, m, H4Rib), 4.18 (2 H,
dd, J = 5.2, 3.5 Hz, H5Rib), 3.95 (1 H, d, J = 10.0 Hz, H5Man), 3.88 (1 H,
dd, J = 2.2, 3.3 Hz, H2Man), 3.78 (1 H, dd, J = 9.6, 3.3 Hz, H3Man), 3.64
(1 H, t, J = 9.7 Hz, H4Man); δP (101 MHz D2O) δ −11.2, −13.7; HRMS
[M-H]- calculated for C16H22N5O17P2: 618.0491; found: 618.0484.
linear
gradient
of
triethylammonium
bicarbonate
(TEAB)
(0.005–0.5 M). Fractions containing the product were collected and
concentrated under reduced pressure. Residual TEAB was removed by
6

L. Beswick, et al.
CarbohydrateResearch485(2019)107819
4.12. Guanosine 5’-(α-D-mannopyranuronic diphosphate) (1)
doi.org/10.1016/j.carres.2019.107819.
GDP-α-D-mannose (1.6 mg, 2.5 μmol) and NAD+ (3.5 mg,
5.25 μmol) were dissolved in buffer (0.9 mL, 200 mM sodium phos-
phate, pH 7.4, 1 mM DTT, 0.5 mM MgCl2) and GMD (0.8 mg/mL final
concentration) was added to give a total volume of 1.0 ml. The mixture
was incubated at room temperature with gentle shaking whilst being
monitored by SAX on a Poros HQ 50 column. Samples (10 μL) were
taken at time points, mixed with methanol (10 μL), vortexed and cen-
trifuged to remove precipitated protein. The supernatant (10 μL) was
analysed by SAX. After 21 h the conversion of GDP-α-D-mannose to 1
reached 70%. The enzymatic transformation was stopped by addition of
methanol (1 mL) and the mixture vortexed for 1 min. and centrifuged.
The supernatant was filtered through a syringe disc filter (0.45 μm,
PTFE) and the resulting crude product purified by SAX. Fractions
containing 1 were pooled and freeze-dried to give the title compound as
a bisammonium salt (1.2 mg, 2.0 μmol, 70%). See analytical data above
for 1.
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Supplementary data to this article can be found online at https://
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