Synthesis of C6-substituted UDP-GlcNAc derivatives

Article abstract of DOI:10.1016/j.carres.2020.108071

UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The preparation of five UDP-GlcNAc derivatives is reported with 6-deoxy, 6-azido, 6-amino, 6-mercapto, or 6-fluoro substitutions. A concise chemoen

Full text of DOI:10.1016/j.carres.2020.108071

CarbohydrateResearch495(2020)108071
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of C6-substituted UDP-GlcNAc derivatives
Zachary A. Morrison, Mark Nitz∗
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords:
UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The
preparation of five UDP-GlcNAc derivatives is reported with 6-deoxy, 6-azido, 6-amino, 6-mercapto, or 6-fluoro
substitutions. A concise chemoenzymatic synthesis was developed using the kinase NahK (B. longum JCM1217)
and the uridyl transferase GlmU (E. coli K12).
UDP-GlcNAc
Chemoenzymatic synthesis
NahK
GlmU
1. Introduction
The preparation of sugar nucleotides through organic synthesis is
well established, but these procedures are laborious and often give low
N-Acetylglucosamine (GlcNAc) is a ubiquitous amino sugar found in
polysaccharides, glycoproteins and glycolipids. It is a core component
of peptidoglycan, chitin, and many other polysaccharides. GlcNAc is the
key constituent of PNAG, a partially de-N-acetylated β-1,6-linked
homopolymer required for biofilm formation in many gram positive
and negative bacteria [1–10]. Biofilm formation contributes to the
virulence of bacterial pathogens, providing resistance to the host im-
mune response and antibiotics [11–13]. Therefore, the biosynthesis of
PNAG is a potential therapeutic target. In gram negative bacteria in-
cluding E. coli, the glycosyltransferase PgaCD complex polymerizes
uridine 5′-diphosphate-GlcNAc (UDP-GlcNAc) into PNAG [14,15]. De-
spite its key role in bacterial virulence, this enzyme remains relatively
uncharacterized. UDP-GlcNAc substrate analogs provide the opportu-
nity to probe elements of the mechanism of this important complex. To
this end, we report the synthesis of five UDP-GlcNAc analogs 16–20,
carrying deoxy, azido, amino, thiol, or fluoro substitutions at C6
(Fig. 1).
overall yields [21]. For instance, our desired 6-fluoro derivative 20 has
been previously synthesized chemically [22,23]. In the most recent
report,
2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-glucose
was converted to UDP-6-fluoro-GlcNAc in 2% yield over six steps.
Chemoenzymatic synthesis is an attractive alternative, though this re-
quires enzymes that tolerate the modified sugars as substrates. Che-
moenzymatic synthesis of α-D-GlcNAc-1-phosphate (GlcNAc-1P) deri-
vatives has been accomplished using
a GlcNAc-1 kinase from
Bifodobacterium longum JCM1217, NahK [24–26]. These 1-phosphate
sugars can then be uridylated by a suitable uridyl transferase, such as E.
coli GlmU [25–28]. This strategy has been used to prepare various UDP-
GlcNAc analogs, including with deoxy and azido C6-substitutions in
moderate or low yield [24,27]. We hypothesized that other C6-sub-
stituted GlcNAc analogs might also be amenable to this chemoenzy-
matic strategy, including 6-thio, 6-amino, and 6-fluoro derivatives.
2. Results and discussion
These UDP-GlcNAc analogs will also have utility for the preparation
of glycans and glycoconjugates bearing unnatural functionality.
Introducing reactive handles like an azide, a thiol, or an amine can
provide a convenient means of labelling or further derivatizing a glycan
[16–18]. Defined substitutions are also useful for probing structure-
activity relationships or improving the pharmacokinetic properties of
carbohydrates as potential therapeutics [19]. Unnatural oligosacchar-
ides can be chemically synthesized, but the process is encumbered by
protecting group manipulations and challenges with regio- and ste-
reoselectivity. Enzymatic preparation of glycans by Leloir-type glyco-
syltransferases is often preferable [20]. Therefore, convenient routes to
sugar nucleotide analogs are of general interest.
We developed concise synthetic routes for quick access to the re-
quired monosaccharide precursors (Scheme 1). The 6-deoxy and 6-
azido GlcNAc derivatives 5 and 6 have been reported and were pre-
pared using a thiophenyl anomeric protecting group [24]. This ap-
proach takes several synthetic steps [29]. As a more convenient and
higher yielding alternative, we used a N,O-dimethyl hydroxylamine-N-
glycoside which can be installed in a single step without protecting
group manipulations [30]. N-acetyl glucosamine was protected as the
N,O-dimethyl hydroxylamine glycoside under aqueous conditions and
selective tosylation of the C6 hydroxyl in cold pyridine afforded key
intermediate tosylate
1 [31]. The 6-deoxy derivative 2, 6-azido
∗ Corresponding author.
E-mail address: mark.nitz@utoronto.ca (M. Nitz).
https://doi.org/10.1016/j.carres.2020.108071
Received 11 May 2020; Received in revised form 8 June 2020; Accepted 9 June 2020
Availableonline26June2020
0008-6215/©2020PublishedbyElsevierLtd.

Z.A. Morrison and M. Nitz
CarbohydrateResearch495(2020)108071
complete conversion, resulting in the formation of inseparable de-
gradation products. A more practical route was achieved by reducing
the azide prior to uridylation. Catalytic hydrogenation of 6-azido
GlcNAc-1P 12 gave 6-amino GlcNAc-1P 13 without complication.
Though no cationic GlcNAc-1P analogs have been previously reported
as GlmU substrates, we found that compound 13 was smoothly ur-
idylated to UDP-6-amino-GlcNAc 18. By avoiding the liability of the
selective hydrogenation, this route is a safer and more reproducible
approach.
Fig. 1. C6-Substitued UDP-GlcNAc analogs synthesized in this work.
derivative 3, and 6-thioacetyl derivative 4 were prepared divergently
from tosylate 1 by reaction in DMF with NaBH4/LiCl [32], NBu4N3, or
KSAc, respectively. The N,O-dimethyl hydroxylamine glycosides of 2
and 3 were cleaved in 4:1 AcOH/H2O at 60 °C, furnishing 6-deoxy and
6-azido GlcNAc 5 and 6. The 6-thioacetyl derivative 4 was deacetylated
in methanol with K2CO3, then the N,O-dimethyl hydroxylamine gly-
coside was deprotected as above, yielding 6-thio GlcNAc 7.
3. Conclusion
In conclusion, we achieved a succinct chemical synthesis of 6-deoxy,
6-azido, 6-thio, and 6-fluoro GlcNAc derivatives. Using these pre-
cursors, we prepared five C6-substituted UDP-GlcNAc analogs che-
moenzymatically. Our work extends the substrate scope of NahK,
finding that it tolerates 6-fluoro and 6-thio GlcNAc derivatives. We also
identified 6-fluoro, 6-thio, and 6-amino GlcNAc-1P analogs as novel
GlmU substrates. Evaluation of these UDP-GlcNAc analogs as PgaCD
substrates is currently underway.
6-Fluoro GlcNAc 10 was synthesized from commercially available
benzyl 2-acetamido-2-deoxy-6-O-trityl-α-D-glucopyranoside based on a
previously reported strategy [33]. Known compound benzyl 2-acet-
amido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranoside
8
was deoxy-
fluorinated to give the protected 6-fluoro product 9. This was initially
attempted using XTalFluor-E and Et3N·3HF in DCM [34], but a low
yield of product 9 was obtained even after optimization (~35–50%).
Performing the deoxyfluorination with DAST gave better results (75%).
Catalytic hydrogenation of compound 9 afforded 6-fluoro GlcNAc 10.
The 6-deoxy and 6-azido GlcNAc derivatives 5 and 6 were che-
moenzymatically phosphorylated to their GlcNAc-1P derivatives 11 and
12 as previously described (Scheme 2) [24]. A low yield of 6-azido
GlcNAc-1P 12 was previously reported (34%), presumably due to 6-
azido GlcNAc 6 being a poor substrate. However, by increasing reaction
time and NahK/ATP concentrations, we achieved a high yield (81%) of
product 12. In addition, NahK tolerated the 6-thio and 6-fluoro GlcNAc
derivatives 7 and 10 as substrates, furnishing their respective 1-phos-
phates 14 and 15 in good yields.
4. Experimental section
4.1. General methods
Chemicals and reagents were purchased from Sigma-Aldrich,
Biosynth Carbosynth, or Toronto Research Chemicals and used as
supplied. Moisture-sensitive reactions were performed under Ar(g)
using oven-dried glassware and stir bars. Reaction temperatures are
reported as the temperature of the bath surrounding the vessel.
Reaction monitoring was done by thin layer chromatography, using
silica gel aluminum plates with F254 indicator and visualized by UV
light (254 nm) and chemical staining with p-anisaldehyde. Silica
column chromatography was performed using Silicycle 230–400 mesh
silica gel. Solvents were evaporated under reduced pressure on a rotary
evaporator.
The 6-deoxy and 6-azido GlcNAc-1P derivatives 11 and 12 were
chemoenzymatically uridylated to UDP-GlcNAc analogs 16 and 17
using E. coli GlmU as described previously [27]. As with the NahK re-
action, increasing reaction time and GlmU concentration for the 6-azido
GlcNAc-1P 12 substrate permitted a substantially higher yield (84%) to
be obtained than was originally reported (20%). Chemoenzymatically
prepared UDP-GlcNAc derivatives have generally been purified by
anion exchange chromatography followed by size exclusion chroma-
tography [25–28]. However, we found that silica column chromato-
graphy using an EtOAc/MeOH/H2O solvent system was an effective
alternative, offering high recovery of product and acceptable purity.
The 6-thio and 6-fluoro GlcNAc-1P analogs 14 and 15 were toler-
ated as GlmU substrates, yielding their corresponding UDP-GlcNAc
derivatives 19 and 20. Surprisingly, using TCEP (15 mM, 1.5 equiv) as a
reducing agent for the uridylation of 6-thio GlcNAc-1P 14 resulted in no
product formation, but substituting it with DTT furnished the product
in excellent yield. This suggests that high concentrations of TCEP in-
hibit the enzyme, perhaps by chelating the required Mg2+ cofactor
[35].
1H and 13C nuclear magnetic resonance (NMR) spectra were re-
corded using either a 400 MHz Bruker Advance III or a 500 MHz Agilent
DD2 NMR spectrometer at 296 K. 31P and 19F NMR spectra were
measured on the 400 MHz Bruker Advance III spectrometer. One di-
mensional proton and carbon chemical shifts are reported in parts per
million and referenced to the residual signals of NMR solvents. 1H NMR
was referenced according to CDCl3 = 7.26 ppm, MeOD = 3.31 ppm,
and D2O
=
4.79 ppm. 13C NMR was referenced according
CDCl3 = 77.16 ppm and MeOD = 49.00 ppm. 31P and 19F NMR were
referenced indirectly using the 2H lock frequency of the deuterated
solvent. The following abbreviations were used to show the multi-
plicities: s: singlet, d: doublet, t: triplet, q: quadruplet, dd: doublet of
doublet, ddd: doublet of doublet of doublet, m: multiplet. 1H NMR as-
signments were determined using gCOSY spectra. 13C NMR assignments
were deduced from gHSQC spectra. High resolution mass spectra
(HRMS) were obtained from an Agilent 6538 Q-TOF mass spectrometer
with an electrospray ionization (ESI) ion source or a JEOL AccuTOF
JMST1000-LC mass spectrometer with a Direct Analysis in real Time
(DART) ion source.
UDP-6-amino-GlcNAc 18 has been previously prepared by catalytic
hydrogenation of UDP-6-azido-GlcNAc 17 [18]. We attempted this re-
action but were unable to reproduce the required selectivity. In our
hands, even at short reaction times the catalytic hydrogenation (Pd/C,
1 atm H2, rt, 0.5 h) of compound 17 gave virtually complete reduction
of the uridine moiety. The literature reflects these challenges: though
other selective hydrogenations of azido groups in similar substrates
have been reported [36–38], failed attempts have also been described
[39,40]. The liability of this side reaction is serious, given the difficultly
of separating the over-reduced by-product. We next explored using
triphenylphosphine to directly reduce the azide [41]. Unfortunately,
high temperatures and extended reaction times were required for
4.2. Experimental procedures
4.2.1. N,O-Dimethyl-N-(2-acetamido-2-deoxy-6-O-tosyl-β-D-
glucopyranosyl)hydroxylamine (1)
N,O-Dimethyl-N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)hydro-
xylamine [30] (1.76 g, 6.66 mmol) was dissolved in dry pyridine
(35 mL) under Ar(g). The solution was cooled in an ice/salt bath
(−20 °C). After cooling for 15 min, p-toluenesulfonyl chloride (1.58 g,
1.25 equiv, 8.30 mmol) was added. The solution was stirred overnight
maintained at −20 °C. TLC (5:1 DCM/MeOH) indicated some residual
2

Z.A. Morrison and M. Nitz
CarbohydrateResearch495(2020)108071
Scheme 1. Synthesis of 6-deoxy, 6-azido, 6-thio, and 6-fluoro GlcNAc.
starting material after 16 h, and a second portion of p-toluenesulfonyl
chloride (316 mg, 0.25 equiv, 1.66 mmol) was added. The reaction was
stirred at −20 °C for an additional 4 h, then quenched by adding MeOH
(2 mL). The solution was concentrated in vacuo and co-evaporated with
toluene (50 mL) three times, giving a yellow syrup. Silica chromato-
graphy (20:1 CHCl3/MeOH) yielded the product as a white amorphous
solid (1.88 g, 68%). NMR spectra were consistent with the literature
[31]. 1H NMR (400 MHz, MeOD) δ 7.84–7.77 (m, 2H, Ar), 7.47–7.37
(m, 2H, Ar), 4.36 (dd, J = 10.7, 1.8 Hz, 1H, H-6a), 4.16 (dd, J = 10.7,
6.1 Hz, 1H, H-6b), 4.10 (d, J = 9.8 Hz, 1H, H-1), 3.81 (t, J = 9.9 Hz,
1H, H-2), 3.42 (s, 3H, NOCH3), 3.40–3.34 (m, 2H, H-3 and H-5), 3.22
(dd, J = 9.9, 8.7 Hz, 1H, H-4), 2.59 (s, 3H, NCH3), 2.44 (s, 3H, Ts-CH3),
1.96 (s, 3H, COCH3). 13C NMR (101 MHz, MeOD) δ 173.4, 146.3, 134.3,
131.0, 129.1, 93.4, 77.3, 76.6, 71.5, 70.9, 60.3, 53.9, 37.9, 23.0, 21.6.
4.2.2. N,O-Dimethyl-N-(2-acetamido-2,6-dideoxy-β-D-glucopyranosyl)
hydroxylamine (2)
Compound 1 (145 mg, 0.347 mmol) was dissolved in dry DMF
(4 mL) under Ar(g). Sodium borohydride (1.31 g, 100 equiv,
34.7 mmol) and lithium chloride (294 mg, 20 equiv, 6.93 mmol) were
added. The reaction was heated to 40 °C (oil bath) and stirred for 48 h.
The solution was cooled to 0 °C and quenched with acetone, slowly
allowing it to warm up to rt. The solution was diluted with methanol
and filtered through celite. Concentration in vacuo and purification by
silica column chromatography (gradient: 3%–10% MeOH in DCM)
yielded the product as a white solid (73.4 mg, 85%). 1H NMR
(500 MHz, MeOD) δ 4.09 (d, J = 9.8 Hz, 1H, H-1), 3.84 (t, J = 9.9 Hz,
1H, H-2), 3.43 (s, 3H, NOCH3), 3.35 (dd, J = 10.1, 8.9 Hz, 1H, H-3),
3.24 (dq, J = 9.3, 6.1 Hz, 1H, H-5), 3.01 (t, J = 9.1 Hz, 1H, H-4), 2.65
(s, 3H, NCH3), 1.98 (s, 3H, COCH3), 1.29 (d, J = 6.2 Hz, 3H, H-6). 13
C
Scheme 2. Chemoenzymatic synthesis of UDP-GlcNAc analogs. aUsing 1.75 equiv ATP, 2.0 mg/mL Nahk, and a reaction time of 48 h bDTT (40 mM, 1 equiv) was
added to maintain a reducing environment. cUsing 1.5 mg/mL GlmU and a reaction time of 48 h dDTT (15 mM, 1.5 equiv) was added to maintain a reducing
environment.
3

Z.A. Morrison and M. Nitz
CarbohydrateResearch495(2020)108071
NMR (126 MHz, MeOD) δ 173.4, 93.4, 77.41, 77.36, 75.0, 60.3, 54.4,
were consistent with the literature [43]. 1H NMR (400 MHz, MeOD) δ
5.11 (d, J = 3.5 Hz, 0.73H, αH-1), 4.61 (d, J = 8.3 Hz, 0.25H, βH-1),
3.98–3.91 (m, 0.81H, αH-5), 3.86 (dd, J = 10.6, 3.5 Hz, 0.83H, αH-2),
3.68 (dd, J = 10.7, 8.8 Hz, 0.73H, αH-3), 3.63–3.54 (m, 0.47H, βH-2
and -6a), 3.51 (dd, J = 13.1, 2.5 Hz, 0.83H, αH-6a), 3.46–3.32 (m,
2.22H, αH-4,6b, βH-3,4,5,6b), 1.99 (s, 3H, COCH3). 13C NMR
(126 MHz, MeOD) δ 174.2 (βCOCH3), 173.7 (αCOCH3), 97.0 (βC-1),
92.6 (αC-1), 76.6, 75.8, 73.2 (αC-4), 72.8, 72.4 (αC-3), 72.2 (αC-5),
58.8 (βC-2), 55.9 (αC-2), 52.9 (αC-6), 52.8 (βC-6), 22.9 (βCOCH3),
22.6 (αCOCH3).
38.0, 23.0, 18.0. HRMS(ESI) m/z Calcd. for C10H20N2O5Na [M+Na]+
:
271.1264, found 271.1270.
4.2.3. N,O-Dimethyl-N-(2-acetamido-6-azido-2,6-dideoxy-β-D-
glucopyranosyl)hydroxylamine (3)
Compound 1 (398 mg, 0.951 mmol) was dissolved in dry DMF
(4 mL) under Ar(g). Tetrabutylammonium azide (1.08 g, 4 equiv,
3.80 mmol) was added and the reaction stirred for 24 h at 40 °C (oil
bath). The reaction was concentrated to a viscous yellow oil in vacuo.
Silica column chromatography (gradient: 3%–8% MeOH in EtOAc) gave
the product as a white solid (242 mg, 88%). 1H NMR (400 MHz, MeOD)
δ 4.19 (d, J = 9.8 Hz, 1H, H-1), 3.87 (t, J = 9.9 Hz, 1H, H-2),
3.54–3.46 (m, 1H, H-6a), 3.44 (s, 3H, NOCH3), 3.43–3.35 (m, 3H, H-3,
H-4, H-6b), 3.30–3.21 (m, 1H, H-5), 2.69 (s, 3H, NCH3), 1.98 (s, 3H,
COCH3). 13C NMR (101 MHz, MeOD) δ 173.4 (COCH3), 93.7 (C-1), 78.9
(C-4), 77.2 (C-3), 72.7 (C-5), 60.3 (NOCH3), 54.1 (C-2), 52.8 (C-6), 38.0
(NCH3), 23.0 (COCH3). HRMS(ESI) m/z Calcd. for C10H19N5O5Na [M
+Na]+: 312.1278, found 312.1284.
4.3.3. 2-Acetamido-2,6-dideoxy-6-thio-D-glucopyranoside (7)
N,O-dimethyl hydroxylamine glycoside 4 (25.0 mg, 0.078 mmol)
was dissolved in MeOH (3 mL) and Ar(g) was bubbled through the
solution for 0.5 h. Potassium carbonate (32.2 mg, 3 equiv, 0.233 mmol)
was added and the reaction was stirred for 2 h at rt under Ar(g). Dowex
resin (H+-form, 50WX2, 50–100 mesh) was added until the solution
was no longer basic. It was filtered and concentrated in vacuo. Next, the
N,O-dimethyl hydroxylamine glycoside was hydrolyzed according to
the general procedure. Lyophilization gave the free thiol as an off-white
solid (15.8 mg, 3:1 α/β, 86%). It was used without further purification.
1H NMR (500 MHz, MeOD) δ 5.09 (d, J = 3.5 Hz, 0.74H, αH-1), 4.61
(d, J = 8.4 Hz, 0.25H, βH-1), 3.91–3.80 (m, 1.86H), 3.69 (dd, J = 10.6,
8.9 Hz, 0.84H), 3.60 (dd, J = 10.3, 8.4 Hz, 0.35H, βH-2), 3.47–3.31 (m,
2H), 2.98–2.86 (m, 1.26H), 2.70–2.65 (m, 0.93H), 2.01–1.97 (m, 3H,
COCH3). 13C NMR (126 MHz, MeOD) δ 173.8 (COCH3), 97.0, 92.6,
78.0, 75.7, 74.4, 74.0, 72.8, 72.4, 58.9, 56.0, 27.0, 26.9, 22.9 (COCH3),
22.6 (COCH3). HRMS(ESI) m/z Calcd. for C8H15ClNO5S [M+Cl]-:
272.0365, found 272.0386.
4.2.4. N,O-Dimethyl-N-(2-acetamido-6-S-acetyl-6-thio-2,6-dideoxy-β-D-
glucopyranosyl)hydroxylamine (4)
Compound 1 (100 mg, 0.239 mmol) was dissolved in dry DMF
(2 mL) under Ar(g). Potassium thioacetate (81.9 mg,
3 equiv,
0.717 mmol) was added and the reaction stirred for 16 h at rt. The
solution was concentration in vacuo to an orange residue. It was purified
twice by silica column chromatography (first purification gradient:
2%–8% MeOH in DCM, second purification gradient: 2%–8% MeOH in
EtOAc). The product was obtained as a pale yellow solid (55.9 mg,
73%). 1H NMR (500 MHz, MeOD) δ 4.07 (d, J = 9.8 Hz, 1H, H-1), 3.86
(t, J = 9.9 Hz, 1H, H-2), 3.59 (dd, J = 13.8, 2.7 Hz, 1H, H-6a), 3.42 (s,
3H, NOCH3), 3.41–3.33 (m, 1H, H-3), 3.25 (ddd, J = 9.3, 8.1, 2.8 Hz,
1H, H-5), 3.18 (t, J = 9.1 Hz, 1H, H-4), 3.00 (dd, J = 13.8, 8.1 Hz, 1H,
H-6b), 2.65 (s, 3H, NCH3), 2.33 (s, 3H, SCOCH3), 1.97 (s, 3H, NCOCH3).
13C NMR (126 MHz, MeOD) δ 197.3, 173.4, 93.5, 77.8, 77.2, 74.9, 60.4,
54.1, 38.2, 32.3, 30.4, 23.0. HRMS(ESI) m/z Calcd. for C12H23N2O6S [M
+H]+: 323.1271, found 323.1270.
4.3.4. Benzyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranoside
(8)
A solution of benzyl 2-acetamido-2-deoxy-6-O-trityl-α-D-glucopyr-
anoside (500 mg, 0.903 mmol) in dry DMF (2.5 mL) was chilled to 0 °C
(ice bath) under Ar(g). Sodium hydride (79.5 mg, 2.2 equiv, 1.99 mmol,
60% dispersion in mineral oil) was added and the reaction stirred for
5 min. Benzyl bromide (235 μL, 2.2 equiv, 1.99 mmol) was added
dropwise and the mixture was maintained at 0 °C for 0.5 h. The reaction
was allowed to warm to rt and was stirred for an additional 2 h. The
reaction was cooled to 0 °C and quenched by slow addition of MeOH.
The mixture was diluted into EtOAc and extracted three times with
water then once with brine. The organic layer was concentrated in
vacuo and purified by silica column chromatography (gradient: 0%–5%
EtOAc in DCM). Benzyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-
trityl-α-D-glucopyranoside was obtained as a white solid (554 mg,
84%). NMR spectra were consistent with the literature [44].
4.3. General procedure for N,O-dimethyl hydroxylamine deprotection
A solution of the N,O-dimethyl hydroxylamine glycoside (10 mM) in
4:1 AcOH/H2O was heated to 60 °C (oil bath) and stirred for 4 h. The
reaction was then concentrated in vacuo. The residue was dissolved in
water and lyophilized, giving the product as an off-white solid which
was used without purification or purified by silica column chromato-
graphy.
4.3.1. 2-Acetamido-2,6-dideoxy-D-glucopyranoside (5)
N,O-dimethyl hydroxylamine glycoside 2 (73.0 mg, 0.294 mmol)
was deprotected according to the general procedure. The product was
purified by silica column chromatography (gradient: 5%–10% MeOH in
DCM), yielding a white solid (56.2 mg, 7:4 α/β, 93%). NMR spectra was
consistent with the literature [42]. 1H NMR (500 MHz, MeOD) δ 5.02
(d, J = 3.5 Hz, 0.63H, αH-1), 4.55 (d, J = 8.4 Hz, 0.36H, βH-1),
3.95–3.78 (m, 1.37H, αH-2 and αH-5), 3.65 (dd, J = 10.7, 8.8 Hz,
0.65H, αH-3), 3.59 (dd, J = 10.4, 8.4 Hz, 0.35H, βH-2), 3.45–3.30 (m,
βH-3 and βH-5, 0.60), 3.07–3.00 (m, 1.03H, α+βH-4), 2.00–1.98 (m,
3H, COCH3), 1.29 (d, J = 6.1 Hz, 1.30H, βH-6), 1.23 (d, J = 6.2 Hz,
2.03H, αH-6). 13C NMR (126 MHz, MeOD) δ 174.2, 173.7, 96.8, 92.4,
78.2, 77.5, 75.8, 73.3, 72.4, 68.3, 59.1, 56.2, 22.9, 22.6, 18.25, 18.20.
p-Toluenesulfonic acid monohydrate (264 mg, 2 equiv, 1.39 mmol)
was added to a solution of the above compound (510 mg, 0.695 mmol)
in 2:1 MeOH/DCM (5 mL). The solution immediately became yellow
and was stirred at rt for 2 h. The reaction was quenched by addition of
saturated sodium bicarbonate solution and the mixture was extracted
twice with DCM. The organic layer was washed with brine and con-
centrated in vacuo. Silica column chromatography (gradient: 1%–3%
MeOH in DCM) afforded the product as a white solid (324 mg, 95%).
NMR spectra were consistent with the literature [45]. 1H NMR
(400 MHz, CDCl3) δ 7.33–7.17 (m, 15H, Ar), 5.24 (d, J = 9.3 Hz, 1H,
NH), 4.90–4.69 (m, 3H, H-1 and OCH2Ph), 4.68–4.51 (m, 3H, OCH2Ph),
4.35 (d, J = 11.8 Hz, 1H, OCH2Ph), 4.15 (td, J = 9.7, 3.7 Hz, 1H, H-2),
3.80–3.55 (m, 5H, H-3,4,5, 6a+b), 1.88 (dd, J = 7.6, 5.3 Hz, 1H, OH),
1.72 (s, 3H, COCH3). 13C NMR (101 MHz, CDCl3) δ 169.9 (COCH3),
138.5, 138.0, 137.2, 128.67, 128.66, 128.6, 128.3, 128.24, 128.23,
128.11, 128.08, 127.9, 97.3 (C-1), 80.2, 78.3, 75.3, 75.0, 71.9, 69.8,
61.8, 52.8, 23.5 (COCH3).
4.3.2. 2-Acetamido-6-azido-2,6-dideoxy-D-glucopyranoside (6)
N,O-dimethyl hydroxylamine glycoside 3 (178 mg, 0.615 mmol)
was deprotected according to the general procedure. The product was
purified by silica column chromatography (gradient: 8%–15% MeOH in
CHCl3), yielding a white solid (138 mg, 3:1 α/β, 91%). NMR spectra
4

Z.A. Morrison and M. Nitz
CarbohydrateResearch495(2020)108071
4.3.5. Benzyl
2-acetamido-3,4-di-O-benzyl-2,6-dideoxy-6-fluoro-α-D-
1H, H-5), 3.95 (ddd, J = 10.4, 3.4, 2.2 Hz, 1H, H-2), 3.75 (dd,
J = 10.5, 9.1 Hz, 1H, H-3), 3.24 (t, J = 9.5 Hz, 1H, H-4), 2.06 (s, 3H,
COCH3), 1.29 (d, J = 6.3 Hz, 3H, H-6). 31P NMR (162 MHz, D2O) δ
1.77. 13C NMR (126 MHz, D2O) δ 174.6, 92.5 (d, J = 5.6 Hz), 75.7,
71.2, 68.0, 54.3 (d, J = 7.5 Hz), 22.0, 16.8. MS(ESI) m/z Calcd. for
C8H15NO8P [M-H]-: 284.05, found 284.05.
glucopyranoside (9)
A solution of compound 8 (230 mg, 0.468 mmol) in dry DCM (6 mL)
chilled to 0 °C was added dropwise to neat DAST (310 μL, 5 equiv,
2.34 mmol) in a plastic reaction vessel maintained at 0 °C (ice bath)
under Ar(g). The ice bath was allowed to slowly warm up to rt and the
reaction was stirred for 12 h. The solution was cooled again to 0 °C and
quenched by the addition of saturated sodium bicarbonate solution.
When the bubbling stopped the mixture was extracted twice with DCM.
The organic layer was concentrated in vacuo and purified by silica
column chromatography (gradient: 0%–10% EtOAc in DCM). The
product was afforded as a white solid (174 mg, 75%). NMR spectra
were consistent with the literature [46]. 1H NMR (500 MHz, CDCl3) δ
7.45–7.19 (m, 15H, Ar), 5.30 (d, J = 9.4 Hz, 1H, NH), 4.92–4.84 (m,
3H, H-1 and OCH2Ph), 4.71–4.44 (m, 6H, H-6a+b and OCH2Ph), 4.28
(ddd, J = 10.4, 9.4, 3.7 Hz, 1H, H-2), 3.88–3.63 (m, 3H, H-3,4, and 5),
1.81 (s, 3H, COCH3). 19F NMR (377 MHz, CDCl3) δ −233.22 (td,
J = 47.7, 28.2 Hz). 13C NMR (126 MHz, CDCl3) δ 169.8, 138.4, 137.8,
137.1, 128.7, 128.6, 128.29, 128.26, 128.24, 128.19, 128.17, 128.0,
97.4 (C-1), 81.9 (d, J = 173.2 Hz, C-6), 80.3, 77.6 (d, J = 5.8 Hz, C-4),
75.4, 75.08, 75.07, 70.8 (d, J = 18.4 Hz, C-5), 70.0, 52.6, 23.5. HRMS
(ESI) m/z Calcd. for C29H33FNO5 [M+H]+: 494.2337, found 494.2331.
4.4.2. Ammonium 2-acetamido-6-azido-2,6-dideoxy-α-D-glucopyranosyl-1-
phosphate (12)
Compound 6 (43.5 mg, 0.177 mmol) was phosphorylated according
to the general procedure with several modifications to enhance yield:
the NahK concentration was increased to 2.0 mg/mL, more ATP was
used (1.75 equiv, 70 mM), and the reaction time was increased to 48 h.
The product was obtained as a white solid (51.6 mg, 81%). NMR
spectra were consistent with the literature [24]. 1H NMR (400 MHz,
D2O) δ 5.41 (dd, J = 7.3, 3.3 Hz, 1H, H-1), 4.03 (ddd, J = 10.0, 4.4,
2.7 Hz, 1H, H-5), 3.97 (ddd, J = 10.6, 3.5, 2.4 Hz, 1H, H-2), 3.80 (dd,
J = 10.5, 8.9 Hz, 1H, H-3), 3.74 (dd, J = 13.6, 2.7 Hz, 1H, H-6a), 3.65
(dd, J = 4.4, 1.8 Hz, 1H, H-6b), 3.61–3.52 (m, 1H, H-4), 2.07 (d,
J = 0.9 Hz, 3H, COCH3). 31P NMR (162 MHz, D2O) δ −0.59. 13C NMR
(126 MHz, D2O) δ 174.6 (COCH3), 93.1 (d, J = 6.0 Hz, C-1), 71.0 (C-5),
70.6 (C-3), 70.3 (C-4), 53.8 (d, J = 8.0 Hz, C-2), 50.6 (C-6), 21.9
(COCH3). HRMS(ESI) m/z Calcd. for C8H14N4O8P [M-H]-: 325.0555,
found 325.0558.
4.3.6. 2-Acetamido-2,6-dideoxy-6-fluoro-D-glucopyranoside (10)
Compound 9 (139 mg, 0.282 mmol) was dissolved in AcOH (8 mL)
with Pd/C (250 mg, 10 wt %). The flask was purged with N2(g) and
then with H2(g). After 48 h, the mixture was filtered through celite. The
solution was concentrated to a residue in vacuo, dissolved in water and
lyophilized. The product was obtained as a white solid (60.2 mg, 4:1 α/
β, 96%). NMR spectra were consistent with the literature [46]. 1H NMR
(500 MHz, MeOD) δ 5.10 (d, J = 3.4 Hz, 0.76H, αH-1), 4.70–4.48 (m,
1.98H, H-6a+b, βH-1), 3.93 (dddd, J = 27.2, 10.2, 4.3, 1.7 Hz, 0.80H,
αH-5), 3.84 (dd, J = 10.7, 3.5 Hz, 0.79H, αH-2), 3.71 (dd, J = 10.6,
8.8 Hz, 0.76H, αH-3), 3.67–3.49 (m, 0.28H), 3.49–3.34 (m, 1.27H, αH-
4), 1.99 (s, 3H, COCH3). 19F NMR (377 MHz, MeOD) δ −235.5 (td,
J = 47.7, 23.9 Hz, βF), −236.40 (td, J = 47.9, 27.3 Hz, αF). 13C NMR
(126 MHz, MeOD) δ 174.2, 173.7, 97.0 (βC-1), 92.6 (αC-1), 84.3, 84.1,
82.9, 72.6, 72.0, 71.9, 71.3, 58.8, 55.8, 22.64, 22.58. HRMS(DART) m/
z Calcd. for C8H15FNO5 [M+H]+: 224.0929, found 224.0933.
4.4.3. Ammonium 2-acetamido-6-amino-2,6-dideoxy-α-D-glucopyranosyl-
1-phosphate (13)
Pd/C (5.0 mg, 10 wt %) was added to a solution of compound 12
(5.0 mg, 13.9 μmol) in H2O (1 mL). The vial was purged with N2(g) and
then the atmosphere was replaced with H2(g). The reaction was stirred
for 5 h at rt. The mixture was filtered through celite and lyophilized,
giving the product as a white solid (4.4 mg, 95%). 1H NMR (400 MHz,
D2O) δ 5.39 (dd, J = 7.1, 3.4 Hz, 1H, H-1), 4.15 (td, J = 10.0, 2.8 Hz,
1H, H-5), 3.99 (dt, J = 10.5, 2.8 Hz, 1H, H-2), 3.80 (t, J = 9.8 Hz, 1H,
H-3), 3.49 (dd, J = 13.2, 2.8 Hz, 1H, H-6a), 3.42 (t, J = 9.5 Hz, 1H, H-
4), 3.12 (dd, J = 13.2, 9.9 Hz, 1H, H-6b), 2.07 (s, 3H, COCH3). 31P NMR
(162 MHz, D2O) δ 1.27. 13C NMR (126 MHz, D2O) δ 174.6 (COCH3),
92.6 (d, J = 5.8 Hz, C-1), 71.8 (C-4), 71.0 (C-3), 68.2 (C-5), 53.8 (d,
J = 7.8 Hz, C-2), 40.4 (C-6), 22.0 (COCH3). HRMS(ESI) m/z Calcd. for
C8H16N2O8P [M-H]-: 299.0650, found 299.0651.
4.4. General procedure for chemoenzymatic phosphorylation using NahK
4.4.4. Ammonium 2-acetamido-2,6-dideoxy-6-thio-α-D-glucopyranosyl-1-
phosphate (14)
The phosphorylation reactions were performed using the conditions
reported by Cai et al. [24]. The solutions contained GlcNAc analog
(40 mM), ATP (1.25 equiv, 50 mM), MgCl2 (10 mM), 100 mM Tris-HCl
buffer (pH 9), and Nahk (1.5 mg/mL). The reactions were incubated for
24 h at 37 °C. Protein was removed by boiling the sample for 1 min
followed by centrifugation. The clarified solution was lyophilized and
purified by silica column chromatography. The sample was dry loaded
on silica by sonicating a suspension of the material and silica in MeOH
and concentrating the solution in vacuo. A 1.5 × 25 cm column of silica
gel was used with a step gradient of methanol (5 mM NH4HCO3) in
DCM: 30%, 50%, 70%, 80%, 90%, and 100%. Typically, the product co-
eluted with Tris and a small quantity of glycerol from the enzyme stock
solution. The Tris was removed by passing an aqueous solution of the
material through a plug of Dowex resin (H+-form, 50WX2, 50–100
mesh). The solution was neutralized with ammonium bicarbonate and
lyophilized. The glycerol impurity (~0.0–0.3 equiv) was carried for-
ward to the next step.
Compound 7 (15.0 mg, 63.2 μmol) was phosphorylated according to
the general procedure, adding DTT (40 mM, 1 equiv) to maintain a
reducing environment. The product was obtained as a white solid
(14.5 mg, 65%). 1H NMR (500 MHz, D2O) δ 5.43 (dd, J = 7.3, 3.4 Hz,
1H, H-1), 4.06–3.92 (m, 2H, H-2 and H-5), 3.80 (dd, J = 10.6, 9.1 Hz,
1H, H-3), 3.61 (dd, J = 9.8, 9.1 Hz, 1H, H-4), 3.00 (dd, J = 14.4,
3.0 Hz, 1H, H-6a), 2.84 (dd, J = 14.4, 5.7 Hz, 1H, H-6b), 2.06 (s, 3H,
COCH3). 31P NMR (162 MHz, D2O) δ −1.55. 13C NMR (126 MHz, D2O)
δ 174.5 (COCH3), 93.1 (d, J = 5.9 Hz, C-1), 71.6 (C-3), 71.3 (C-4), 70.6
(C-5), 53.9 (d, J = 7.9 Hz, C-2), 24.9 (C-6), 21.9 (COCH3). HRMS(ESI)
m/z Calcd. for C8H15NO8PS [M-H]-: 316.0261, found 316.0256.
4.4.5. Ammonium 2-acetamido-2,6-dideoxy-6-fluoro-α-D-glucopyranosyl-
1-phosphate (15)
Compound 10 (25.4 mg, 0.114 mmol) was phosphorylated ac-
cording to the general procedure, affording the product as a white solid
(28.5 mg, 74%). 1H NMR (400 MHz, MeOD) δ 5.46 (dd, J = 6.9, 3.3 Hz,
1H, H-1), 4.77–4.48 (m, 2H, H-6a+b), 4.02–3.87 (m, 2H, H-2 and -5),
3.72 (ddd, J = 10.3, 8.9, 1.0 Hz, 1H, H-3), 3.54–3.47 (m, 1H, H-4),
2.01 (s, 3H, COCH3). 19F NMR (377 MHz, MeOD) δ −238.5 (td,
J = 48.3, 29.6 Hz). 31P NMR (162 MHz, MeOD) δ −1.07. 13C NMR
(126 MHz, D2O) δ 174.6 (COCH3), 92.9 (d, J = 5.7 Hz, C-1), 82.1 (d,
J = 167.5 Hz, C-6), 71.1 (C-3), 71.0 (d, J = 17.1 Hz, C-5), 68.7 (d,
4.4.1. Ammonium 2-acetamido-2,6-dideoxy-α-D-glucopyranosyl-1-phosphate
(11)
Compound 5 (17.8 mg, 86.7 μmol) was phosphorylated according to
the general procedure, yielding the product as a white solid (22.9 mg,
83%). NMR spectra were consistent with the literature [24]. 1H NMR
(500 MHz, D2O) δ 5.33 (dd, J = 7.2, 3.4 Hz, 1H, H-1), 4.03–3.97 (m,
5

Z.A. Morrison and M. Nitz
CarbohydrateResearch495(2020)108071
J = 6.7 Hz, C-4), 53.9 (d, J = 7.7 Hz, C-2), 22.0 (COCH3). HRMS(ESI)
m/z Calcd. for C8H14FNO8P [M-H]-: 302.0447, found 302.0451.
chromatography, using 5:2:1 EtOAc/MeOH/H2O (5 mM NH4HCO3) for
the first column and 4:2:1 for the second. The product was obtained as a
white solid (5.8 mg, 69%). NMR spectra were consistent with the lit-
erature [18]. 1H NMR (400 MHz, D2O) δ 7.95 (d, J = 8.1 Hz, 1H, uracil
H-6), 6.03–5.93 (m, 2H, uracil H-5 and ribose H-1), 5.56 (dd, J = 7.2,
3.4 Hz, 1H, H-1), 4.44–4.34 (m, 2H, ribose H-2 and 3), 4.33–4.21 (m,
3H, ribose H-4 and 5a+b), 4.20–4.12 (m, 1H, H-5), 4.06 (dt, J = 10.5,
3.2 Hz, 1H, H-2), 3.83 (dd, J = 10.6, 9.0 Hz, 1H, H-3), 3.55–3.41 (m,
2H, H-6a and H-4), 3.16 (dd, J = 13.3, 9.7 Hz, 1H, H-6b), 2.09 (s, 3H,
COCH3). 31P NMR (162 MHz, D2O) δ −11.41 (d, J = 21.3 Hz), −13.28
(d, J = 21.3 Hz). 13C NMR (126 MHz, D2O) δ 174.7 (COCH3), 166.4
(uracil C-4), 151.9 (uracil C-2), 141.6 (uracil C-6), 102.5 (uracil C-5),
94.0 (d, J = 6.5 Hz, C-1), 88.5 (ribose C-1), 83.0 (d, J = 9.1 Hz, ribose
C-4), 73.6 (ribose C-2), 71.5 (C-4), 70.4 (C-3), 69.5 (ribose C-3), 69.1
(C-5), 65.0 (d, J = 5.6 Hz, ribose C-5), 53.4 (d, J = 8.4 Hz, C-2), 40.4
(C-6), 21.9 (COCH3). HRMS(ESI) m/z Calcd. for C17H27N4O16P2 [M-H]-:
605.0903, found 605.0901.
4.5. General procedure for chemoenzymatic uridylation using GlmU
The uridylation reactions were performed using the conditions re-
ported by Guan et al. [27]. The solutions contained GlcNAc-1P analog
(10 mM), UTP (1.5 equiv, 15 mM), MgCl2 (10 mM), 100 mM Tris-HCl
buffer (pH 7.5), GlmU (1.0 mg/mL), and inorganic pyrophosphatase
(PPA, 1 U/mL) from C. cerevisiae. The reactions were incubated for
24 h at 37 °C. Protein was removed by adding one volume of acetoni-
trile to the reaction and incubating for 0.5 h at rt, followed by cen-
trifugation. The clarified solution was lyophilized and purified by silica
column chromatography. The sample was dry loaded on silica by so-
nicating a suspension of the material and silica in MeOH and con-
centrating the solution in vacuo. A 1.5 × 25 cm column of silica gel was
used with solvent system: 6:2:1 EtOAc/MeOH/H2O (5 mM NH4HCO3).
Fractions containing product were concentrated in vacuo and lyophi-
lized. Two rounds of chromatography were usually required to achieve
high purity.
4.5.4. Uridine 5′-diphospho-2-acetamido-2,6-dideoxy-6-thio-α-D-glucopyranose
diammonium salt (19)
Compound 14 (6.3 mg, 17.9 μmol) was uridylated according to the
general procedure, adding DTT (15 mM, 1.5 equiv) to maintain a re-
ducing environment. The product was obtained as a white solid
(11.0 mg, 93%). 1H NMR (400 MHz, D2O) δ 7.95 (d, J = 8.2 Hz, 1H,
uracil H-6), 6.02–5.91 (m, 2H, uracil H-5 and ribose H-1), 5.52 (dd,
J = 7.3, 3.4 Hz, 1H, H-1), 4.43–4.32 (m, 2H, ribose H-2 and 3),
4.32–4.14 (m, 3H, ribose H-4 and 5a+b), 4.09–3.96 (m, 2H, H-2 and H-
5), 3.81 (dd, J = 10.5, 9.0 Hz, 1H, H-3), 3.67–3.58 (m, 1H, H-4), 2.99
(dd, J = 14.4, 3.0 Hz, 1H, H-6a), 2.84 (dd, J = 14.4, 5.3 Hz, 1H, H-6b),
2.08 (s, 3H, COCH3). 31P NMR (162 MHz, D2O) δ −11.52 (d,
J = 19.9 Hz), −13.29 (d, J = 19.6 Hz). 13C NMR (101 MHz, D2O) δ
174.7 (COCH3), 166.5 (uracil C-4), 151.9 (uracil C-2), 141.6 (uracil C-
4), 102.6 (uracil C-5), 94.4 (d, J = 6.5 Hz, C-1), 88.7 (ribose C-1), 83.0
(d, J = 9.2 Hz, ribose C-4), 73.8 (ribose C-2), 72.3 (C-5), 71.2 (C-4),
70.7 (C-3), 69.6 (ribose C-3), 65.0 (d, J = 5.1 Hz, ribose C-5), 53.7 (d,
J = 8.6 Hz, C-2), 25.0 (C-6), 22.1 (COCH3). HRMS(ESI) m/z Calcd. for
4.5.1. Uridine 5′-diphospho-2-acetamido-2,6-dideoxy-α-D-glucopyranose
diammonium salt (16)
Compound 11 (11.1 mg, 34.8 μmol) was uridylated according to the
general procedure, affording the product as a white solid (18.6 mg,
86%). NMR spectra were consistent with the literature [27]. 1H NMR
(400 MHz, D2O) δ 7.97 (d, J = 8.1 Hz, 1H, uracil H-6), 6.06–5.88 (m,
2H, uracil H-5 and ribose H-1), 5.49 (dd, J = 7.2, 3.3 Hz, 1H, H-1),
4.43–4.35 (m, 2H, ribose H-2 and 3), 4.33–4.17 (m, 3H, ribose H-4 and
5
a+b), 4.09–3.96 (m, 2H, H-2 and 5), 3.77 (dd, J = 10.5, 9.1 Hz, 1H, H-
3), 3.28 (t, J = 9.5 Hz, 1H, H-4), 2.09 (s, 3H, COCH3), 1.32 (d,
J = 6.3 Hz, 3H, H-6). 31P NMR (162 MHz, D2O) δ −11.49 (d,
J = 19.9 Hz), −13.24 (d, J = 19.6 Hz). 13C NMR (126 MHz, D2O) δ
174.6 (COCH3), 166.2 (uracil C-4), 151.7 (uracil C-2), 141.6 (uracil C-
6), 102.6 (uracil C-5), 94.2 (d, J = 6.4 Hz, C-1), 88.5 (ribose C-1), 83.0
(d, J = 9.1 Hz, ribose C-4), 75.2 (C-4), 73.7 (ribose C-2), 70.6 (C-3),
69.5 (ribose C-3), 69.1 (C-5), 64.9 (d, J = 5.4 Hz, ribose C-5), 53.8 (d,
J = 8.8 Hz, C-2), 22.0 (COCH3), 16.7 (C-6). HRMS(ESI) m/z Calcd. for
C17H26N3O16P2 [M-H]-: 590.0794, found 590.0784.
C
17H26N3O16P2S [M-H]-: 622.0514, found 622.0517.
4.5.5. Uridine
5′-diphospho-2-acetamido-2,6-dideoxy-6-fluoro-α-D-
4.5.2. Uridine
5′-diphospho-2-acetamido-6-azido-2,6-dideoxy-α-D-
glucopyranose diammonium salt (20)
glucopyranose diammonium salt (17)
Compound 15 (5.5 mg, 16.3 μmol) was uridylated according to the
general procedure, affording the product as a white solid (10.0 mg,
95%). NMR spectra were consistent with the literature [23]. 1H NMR
(400 MHz, D2O) δ 7.95 (d, J = 8.1 Hz, 1H, uracil H-6), 6.09–5.87 (m,
2H, uracil H-5 and ribose H-1), 5.56 (dd, J = 7.3, 3.3 Hz, 1H, H-1),
4.90–4.61 (m, 2H obscured by HOD, H-6a+b), 4.42–4.33 (m, 2H, ribose
H-2 and 3), 4.32–4.17 (m, 3H, ribose H-4 and 5a+b), 4.13–3.98 (m, 2H,
H-5 and 2), 3.85 (t, J = 9.8 Hz, 1H, H-3), 3.71–3.64 (m, 1H, H-4), 2.09
(s, 3H, COCH3). 19F NMR (377 MHz, D2O δ −236.4 (td, J = 47.2,
30.0 Hz). 31P NMR (162 MHz, D2O) δ −11.52 (d, J = 19.9 Hz),
−13.34 (d, J = 19.8 Hz). 13C NMR (126 MHz, D2O) δ 174.6 (COCH3),
166.6 (uracil C-4), 152.0 (uracil C-2), 141.5 (uracil C-6), 102.6 (uracil
C-5), 94.3 (d, J = 6.5 Hz, C-1), 88.5 (ribose C-1), 83.0 (d, J = 9.1 Hz,
ribose C-4), 81.8 (d, J = 168.4 Hz, C-6), 73.7 (ribose C-2), 71.8 (d,
J = 17.3 Hz, C-5), 70.6 (C-3), 69.5 (ribose C-3), 68.4 (d, J = 6.7 Hz, C-
4), 64.9 (d, J = 5.6 Hz, ribose C-5), 53.5 (d, J = 8.7 Hz, C-2), 22.0
(COCH3). HRMS(ESI) m/z Calcd. for C17H25FN3O16P2 [M-H]-:
608.0700, found 608.0691.
Compound 12 (10.5 mg, 29.1 μmol) was uridylated according to the
general procedure with several modifications to enhance yield: the
GlmU concentration was increased to 1.5 mg/mL and the reaction time
was increased to 48 h. The product was obtained as a white solid
(16.4 mg, 84%). NMR spectra were consistent with the literature [27].
1H NMR (400 MHz, D2O) δ 7.96 (d, J = 8.1 Hz, 1H, uracil H-6),
6.04–5.92 (m, 2H, uracil H-5 and ribose H-1), 5.53 (dd, J = 7.2, 3.3 Hz,
1H, H-1), 4.45–4.32 (m, 2H, ribose H-2 and 3), 4.33–4.17 (m, 3H, ri-
bose H-4 and 5a+b), 4.12–3.94 (m, 2H, H-2 and H-5), 3.81 (dd,
J = 10.5, 9.1 Hz, 1H, H-3), 3.74 (dd, J = 13.6, 2.6 Hz, 1H, H-6a),
3.68–3.53 (m, 2H, H-6b and H-4), 2.08 (s, 3H, COCH3). 31P NMR
(162 MHz, D2O) δ −11.48 (d, J = 19.8 Hz), −13.31 (d, J = 19.6 Hz).
13C NMR (101 MHz, D2O) δ 174.7 (COCH3), 166.4 (uracil C-4), 151.9
(uracil C-2), 141.6 (uracil C-6), 102.6 (uracil C-5), 94.4 (d, J = 6.5 Hz,
C-1), 88.6 (ribose C-1), 83.1 (d, J = 9.2 Hz, ribose C-4), 73.8 (ribose C-
2), 71.7 (C-5), 70.7 (C-3), 70.1 (C-4), 69.6 (ribose C-3), 65.0 (d,
J = 5.5 Hz, ribose C-5), 53.6 (d, J = 8.5 Hz, C-2), 50.6 (C-6), 22.1
(COCH3). HRMS(ESI) m/z Calcd. for C17H25N6O16P2 [M-H]-: 631.0808,
found 631.0796.
Declaration of competing interest
4.5.3. Uridine
5′-diphospho-2-acetamido-6-amino-2,6-dideoxy-α-D-
glucopyranose diammonium salt (18)
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Compound 13 (4.4 mg, 13.2 μmol) was uridylated according to the
general procedure. The product was purified twice by silica column
6

Z.A. Morrison and M. Nitz
CarbohydrateResearch495(2020)108071
Acknowledgements
subsequent glycan-profiling and visualization via staudinger ligation, Nat. Protoc. 2
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