Synthesis of 4-Azido-N-acetylhexosamine Uridine Diphosphate Donors: Clickable Glycosaminoglycans

Article abstract of DOI:10.1021/acs.joc.7b01787

Unnatural chemically modified nucleotide sugars UDP-4-N₃-GlcNAc and UDP-4-N₃-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or

Full text of DOI:10.1021/acs.joc.7b01787

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Note
Synthesis of 4-azido-N-acetylhexosamine uridine
diphosphate donors: Clickable glycosaminoglycans
Xing Zhang, Dixy E. Green, Victor L Schultz, Lei Lin, Xiaorui Han, Ruitong Wang,
Alexander Yaksic, So Young Kim, Paul L. DeAngelis, and Robert J. Linhardt
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01787 • Publication Date (Web): 16 Aug 2017
Downloaded from http://pubs.acs.org on August 17, 2017
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Synthesis of 4-azido-N-acetylhexosamine uridine
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diphosphate donors: Clickable glycosaminoglycans
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Xing Zhang,† Dixy E. Green,‡ Victor L. Schultz,† Lei Lin,† Xiaorui Han,† Ruitong
Wang,† Alexander Yaksic,† So Young Kim,† Paul L. DeAngelis,*‡ and
Robert J. Linhardt*†
†
Department of Chemistry and Chemical Biology, Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180,
United States
‡
Department of Biochemistry and Molecular Biology, University of Oklahoma
Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, Oklahoma
73126, United States
Authors to whom correspondence should be addressed at linhar@rpi.edu and
PaulꢀDeangelis@ouhsc.edu
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Abstract:
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Unnatural chemically modified, nucleotide sugars, UDPꢀ4ꢀN3ꢀGlcNAc and
UDPꢀ4ꢀN3ꢀGalNAc were chemically synthesized for the first time. These unnatural
UDPꢀsugar products were then tested for incorporation into hyaluronan, heparosan or
chondroitin using polysaccharide synthases. The UDPꢀ4ꢀN3ꢀGlcNAc served as a
chain termination substrate for hyaluronan or heparosan synthases; the resulting
4ꢀN3ꢀGlcNAcꢀterminated hyaluronan and heparosan were then successfully
conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is
generally applicable for labeling and detection of suitable GAGs.
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HOOC
OH
O
H
O
O
O
HO
HO
OH
AcHN
O
H
GAG oligomer
n
COOH
OH
O
OBz
O
O
HO
HO
Enzymatic
N3
HO
m
HO
BzO
OBn
incorporation
of unnatural
UDP-sugar
HO
OUDP
OH
AcHN
NHAc
OUDP
m
Chemical synthesis
O
HO
HO
P. multocida
into GAG product
AcHN
OUDP
GAG synthases
OH
O
N
GAGs
N3
HO
N
N
HOOC
AcHN
OH
O
O
O
HO
'CLICK'
Alexa Fluor 488
O
HO
OH
AcHN
O
H
Fluor
from n to n+m
Sequence specific chain termination
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Glycosaminoglycans (GAGs) are
a
family of structurally complex
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heteropolysaccharides composed of repeating disaccharide units containing
hexosamine residue.1 GAGs are prevalent as free glycans or in the core structures of
glycans in glycoproteins and glycolipids, and control a wide range of physiological
and pathological events including cellꢀcell interactions,2 enzyme inhibition,3 cell
proliferation4 and growth factor receptor5 during various metabolic processes.
Heparin/heparan sulfate, hyaluronan and chondroitin/dermatan sulfate are the three
most studied and abundant classes of GAG polymers. Nucleotide sugars are key
intermediates in carbohydrate metabolism and glycoconjugate biosynthesis.6 Uridine
diphosphate (UDP) monosaccharides are common sugar nucleotide donors being
transferred to the nonꢀreducing terminus of carbohydrate chains by glycosylation
reactions with glycosyltransferases in the GAGs biosynthetic pathway.6,7
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GlcNAc and GalNAc are naturally occurring saccharide residues prevalent in
GAGs, Nꢀacetylglucosamine (GlcNAc) found in heparin/ heparan sulfate/hyaluronan
while Nꢀacetylgalactosamine (GalNAc) found in chondroitin/dermatan sulfate, and
playing essential roles in biological processes.6,8 Thus, the corresponding naturally
occurring UDPꢀGlcNAc/GalNAc as well as structural analogues, are of great interest
for enzymatic reactions in carbohydrate synthesis and would be a good approach to
understand the mechanism of GlcNAc/GalNAcꢀrelated pathways.9 Due to its unique
reactivity in biological system, azideꢀderivatized UDPꢀsugar becomes an exciting
target and has been well established.10
The azido moiety is inert to natural processing or reactivity within biological
systems, but can be readily covalently tagged with imaging probes or epitope using an
azideꢀspecific reaction, such as Staudinger ligation11 with phosphines and the [3+2]
cycloaddition with alkynes,12 which permits the detection of specific glycoconjugate
types on cells or in living organisms or the selective capture of glycoproteins from
cell or tissue lysates. At this time, various chemical or enzymatic approaches have
been developed to synthesize azideꢀderivatized UDPꢀsugars. Chen et al.10b reported
a oneꢀpot three enzymes system to produce UDPꢀ6N3GlcNAc; Wang et al.10a
developed a chemoenzymatic approach involved Escherichia coli GlcNAcꢀ1ꢀP
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uridyltransferase (GlmU) catalysis to obtain UDPꢀ6N3GalNAc and UDPꢀGalNAz.
However, due to the more challenging modification for C4 position either in chemical
or enzymatic way, very few C4 modified azidoꢀUDP sugars have been reported even
though the 1→4 linkage is particular in GAGs.13
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In our recent work, we reported the first chemoenzymatic synthesis of the
UDPꢀ4ꢀfluoro (F) GlcNAc/ꢀ4ꢀFGalNAc analogs using recombinantly–expressed
Nꢀacetylglucosamineꢀ1ꢀphosphate uridylyltransferase (GlmU) and the application of
UDPꢀ4ꢀFGlcNAc as a glycosylation terminator in the chemoenzymatic synthesis of
heparan sulfate/heparin.14 Here we present the first synthesis of 4ꢀN3 derivatized
GlcNAc and GalNAc UDP sugars, that were tested for their ability to mimic two
natural donor substrates required in GAGs synthesis, resulting in metabolic
conversion to azideꢀfunctionalized GAGs as potential substrates for Huisgen
1,3ꢀdipolar cycloaddition.12 The corresponding unnatural GlcNAcꢀ1ꢀphosphate
substrate specificity of enzyme GlmU was also investigated.
Our first objective was the synthesis of 4ꢀN3ꢀGlcNAcꢀ/4ꢀN3ꢀGalNAcꢀ1ꢀphosphate
analogs. These precursors could then be used as substrates for either chemical or
enzymatic reactions, to access the target UDPꢀ4ꢀN3ꢀGlcNAc or UDPꢀ4ꢀN3ꢀGalNAc
donors. Based on our previous studies,14 we started the synthesis from the known
compounds benzyl 2ꢀacetamidoꢀ2ꢀdeoxyꢀβꢀ ꢀglucoꢀ/galactoꢀpyranoside 1a/1b
D
(Scheme 1)14,15. The placement of the Cꢀ4 azide functionality was initiated through
the reaction of Tf2O in pyridine with 1a/1b to afford the triflates 2a/2b.
Nucleophilic displacement of triflate groups in 2a/2b with sodium azide in
dimethylformamide afforded 4ꢀN3ꢀ GalNAc/4ꢀN3ꢀGlcNAc derivatives 3a/3b in 85ꢀ90%
yields with the inversion of the Cꢀ4 configuration.16 The anomeric benzyl group was
oxidatively removed affording 4a/4b, followed by phosphorylation17 with tetrabenzyl
pyrophosphate to obtain the phosphorylated intermediate 5a/5b in good yield
(80%ꢀ84%) and with excellent αꢀselectivity. Deprotection of benzyl groups was
achieved under hydrogenation to obtain the target benzoateꢀprotected substrates 6a/6b
in good yields (85%ꢀ90%). Hydrogenation needed to be strictly controlled, as longer
reaction times would cause azido reduction.
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Scheme 1. Synthesis of 4ꢀN3ꢀGalNAcꢀ1ꢀphosphate 6a and 4ꢀN3ꢀGlcNAcꢀ1ꢀphosphate 6ba
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aAlthough only one stereochemical series is shown (starting from βꢀ
Dꢀglucopyranoside 1a to 6a),
both series were synthesized and the yields for both are provided.
With both protected 4ꢀN3ꢀNꢀacetylhexosamineꢀ1ꢀphosphates in hand, we next
focused our attention on their deprotection and to determining whether they could be
enzymatically converted with GlmU uridyltransferase to the two unnatural nucleotide
sugar donors.14 Treatment of 6a/6b by sodium methoxide to afforded the deprotected
4ꢀN3ꢀGalNAcꢀ1ꢀphosphate/4ꢀN3ꢀGlcNAcꢀ1ꢀphosphate analogs. Unfortunately, GlmU
recognized neither of the analogs and, thus, no corresponding UDPꢀsugars were
observed by LCꢀMS (Scheme 2A). Wang et al.10a investigated the
UDPꢀGlcNAc/UDPꢀGalNAc substrate specificity of GlmU and suggested that GlmU
was intolerant of any functional group on the C4 position larger than a hydroxyl
functionality.
We also reported the successful GlmU recognition for 4ꢀFꢀ
GlcNAcꢀ1ꢀphosphate /4ꢀFꢀGalNAcꢀ1ꢀphosphate analogs.14 Since the fluorine atom
is considerably smaller and the azido group is considerably larger than a hydroxy
group, our observations of the failure of GlmU to utilize the 4ꢀN3ꢀ and 4ꢀFꢀ
Nꢀactylhexosamineꢀ1ꢀphosphate analogs are consistent with Wang et al.10a
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Scheme 2. GlmU attempt and chemical synthesis of UDPꢀ4ꢀN3ꢀGalNAc and UDPꢀ4ꢀN3ꢀGlcNAc
donors
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Next, we turned our attention to the chemical approach to preparing the unnatural
UDPꢀsugar
donors
(Scheme
2B).
The
protected
4ꢀN3ꢀNꢀacetylhexosamineꢀ1ꢀphosphates 6a/6b were converted to their pyridinium
salts and then stirred with UMPꢀmorpholidate and tetrazole in pyridine for 3 days to
afford the benzoateꢀprotected UDP sugars.18 By treating the reaction crude with
(Na+) resin, all the phosphate salts were converted to their sodium form and purified
on a Biogel P2 column afforded the protected UDP derivatives 8a/8b in modest yields
(45%ꢀ58%). Deprotection of 8a/8b was carried out under standard Zemplén
conditions, affording the corresponding donors UDPꢀ4ꢀN3ꢀGalNAc 7a and
UDPꢀ4ꢀN3ꢀGlcNAc 7b.
The utility of the two chemically synthesized unnatural UDPꢀdonors as substrates
in GAG synthesis was next examined. In the pilot trials, the incorporation of these
new synthetic analogs was attempted using indirect radiochemical sugar assays that
rely on coꢀincorporation of a labeled natural sugar when the analog is used by the
Pasteurella GAG synthases19, but the potential signals were very close to background
levels. Therefore, a direct mass spectrometryꢀbased assay was employed to monitor
the incorporation of any analog directly. Basically, a short GAG acceptor terminating
in GlcA was reacted with the analog and the reaction mixture is analyzed for the
presence of elongated species by MALDIꢀToF MS. A single azidoꢀmonosaccharide
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derived from UDPꢀ4ꢀN3ꢀGlcNAc was incorporated into a growing GAG chain of
heparosan (HEP) with Pasteurella multocida heparosan synthases (PmHS1 or 2) or
hyaluronan (HA) with Pasteurella multocida hyaluronan synthase (PmHAS) based on
the very close agreement between the predicted and the observed masses of the
elongated species (Table 1).
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In parallel tests with UDPꢀ4ꢀN3ꢀGalNAc and the Pasteurella multocida
chondroitin synthase, PmCS, the analog was not incorporated into the chain of the
chondroitin acceptor (not shown). This is somewhat surprising as PmHAS and PmCS
are very similar at the amino acid sequence level (~87% identical) and the tested
reaction conditions were equivalent, but currently there is no structural information
for these two enzymes, thus some differences in their respective active sites seem a
likely explanation for the observed usage of the 4ꢀazido precursors.
Table 1. Mass spectrometric analysis of GAG synthase reaction productsa
Acceptor
HA4b
Donor
none
PmHAS
775.05
PmCS
775.04
978.08
NDe
PmHS1
PmHS2
+ UDPꢀGlcNAc
+ UDPꢀ4ꢀN3ꢀGlcNAc
978.09
1003.12
HEP6c
none
1097.10
1300.10
1325.08
+ UDPꢀGlcNAc
+ UDPꢀ4ꢀN3ꢀGlcNAc
HEP3ꢀBenzd
none
747.28
950.39
NDe
747.33
950.43
975.50
+ UDPꢀGlcNAc
+ UDPꢀ4ꢀN3ꢀGlcNAc
aThe theoretical azide mass addition compared to native sugars is 25.02 Da. bHyaluronan
c
d
tetrasaccharide. Heparosan hexasaccharide. Heparosan trisaccharideꢀbenzaldehyde (aldehyde at
the reducing end). eNo desired product.
We successfully prepared two azidoꢀtagged GAGs (see the Experimental Section
for details), either heparosan (HEP) or hyaluronan (HA), by employing the
UDPꢀ4ꢀN3ꢀGlcNAc as a chain terminating donor. Thus, these endꢀlabeled GAGs
both possess a 4ꢀN3ꢀGlcNAc residue at their nonꢀreducing end. We next examined
whether these modified chains were ‘Click’ꢀreactive. Reactions between the
4ꢀN3ꢀGlcNAcꢀterminated HEP or HA (~10ꢀ100 kDa) and Alexa Fluor 488 DIBO
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alkyne20 were performed and the products were analyzed by polyacrylamide gel
electrophoresis (PAGE) analysis and fluorescence detection (excitation 488 nm;
emission 494 nm) (Figure 1, lanes 1 and 2). As negative controls, either the starting
material 4ꢀN3ꢀGlcNAcꢀterminated HA or HEP (~10ꢀ100 kDa) without added Alexa
Fluor 488 DIBO alkyne were also loaded on the gel (lane 3 and 4 in Figure 1). The
gel was subjected to electrophoresis for 4 h to run the unreacted, excess Alexa Fluor
488 DIBO alkyne off the gel to reduce the level of residual fluorescent reagent in the
GAG area. All four lanes containing GAG samples were then visualized by Alcian
blue staining; each lane displayed a blueꢀstained band corresponding to the expected
molecular weight and polydispersity of the input GAG samples (Figure 1, left panel).
As expected, only the GAG bands present in lanes 1 and 2 (Figure 1, right panel)
displayed fluorescence, confirming that 4ꢀN3ꢀGlcNAcꢀterminated HEP and HA had
been successfully clickꢀtagged with the alkyne dye.
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Figure 1. PAGE analysis of click reaction between 4ꢀN3ꢀGlcNAcꢀterminated HEP and HA and
Alexa Fluor 488 DIBO alkyne. Products are marked with red arrows. The excess reagent runs as a
smear running faster than the GAGs (denoted with a bracket).
In summary, this is the first synthesis of UDPꢀ4ꢀN3ꢀGlcNAc/UDPꢀ4ꢀN3ꢀGalNAc
analogs reported using an efficient allꢀchemical approach. The final unnatural
UDPꢀsugars were then tested for incorporation into HA, HEP and chondroitin using
polysaccharide synthases and UDPꢀ4ꢀN3ꢀGlcNAc was added to the nonꢀreducing end
of the sugar chain and served as a chain termination substrate in HA and HEP.
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Compared to chemical method to introduce azido groups into polysaccharides, this
enzymatic technique provided a "green" approach to access regioselectively
azideꢀfunctionalized GAGs, which would bring better control of the final structure
and composition of these macromolecules. The resulting 4ꢀN3ꢀGlcNAcꢀterminated
HEP and HA were then conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating
that this technology is generally applicable for labeling and detection of suitable
GAGs.
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EXPERIMENTAL SECTION
General information.
All reagents were purchased from commercial vendors, and unless noted otherwise,
used without further purification. Flash chromatography (FC) was performed using
silica gel (200−300 mesh) according to standard protocols. Reactions were monitored
by thinꢀlayer chromatography (TLC) on silica gel F254 plates. Mass data were
acquired by MALDIꢀToFꢀMS or electrospray ionization (ESI)ꢀhighꢀresolution
1
1
(HR)ꢀMS on an LTQꢀOrbitrap XL FTꢀMS spectrometer. H, 13C, Hꢀ1H COSY and
13
1Hꢀ13C HSQC NMR spectra were recorded on a 800 MHz (200 MHz for C NMR)
spectrometer or 600 MHz (150 MHz for 13C NMR).
Synthesis of benzyl 2ꢀacetamidoꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoylꢀ2,4ꢀdideoxyꢀ
βꢀDꢀgalacto/glucoꢀpyranosides (3a/3b)
The dibenzoylated compound 1a/1b (520 mg, 1.0 mmol, 1.0 equiv) was dissolved in
o
DCM (5 mL) and pyridine (0.2 mL) solvent system. The solution was cooled to 0 C
and trifluoromethane sulfonic anhydride (0.34 mL, 2.0 mmol, 2.0 equiv) was added
dropwise and the resulting mixture was stirred at 0oC for 3 h. The mixture was diluted
with 20 mL EtOAc and washed with H2O and dried over anhydrous Na2SO4. After
filtering, the solvent was removed under vacuum to afford the triflate derivative 2a/2b
as yellow oils, which were used without further purification.
The triflate derivative 2a/2b from above was dissolved in DMF (5 mL) and sodium
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azide (195 mg, 3.0 mmol, 3.0 eq) was added and stirred at room temperature
overnight. The solvent was removed under vacuum and the residue was dissolved in
20 mL EtOAc and washed with H2O. After drying over anhydrous MgSO4, the
solvent was removed and the resulting crude mixture was purified by column
chromatography (Hexanes/EtOAc 1:2) to yield 3a/3b as white solids (348 mg, 64%
and 364 mg, 67% respectively, two steps).
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Benzyl 2ꢀacetamidoꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoylꢀ2,4ꢀdideoxyꢀβꢀ ꢀ
D
galactopyranosides (3a)
1
White solid. H NMR (600 MHz, CDCl3) δ: 8.13ꢀ8.10 (m, 2H), 8.05ꢀ8.02 (m, 2H),
7.64ꢀ7.58 (m, 2H), 7.53ꢀ7.49 (m, 2H), 7.48ꢀ7.43 (m, 2H), 7.34ꢀ7.29 (m, 3H),
7.29ꢀ7.27 (m, 2H), 5.33 (m, 1H), 4.89 (d, J = 12.50, 1H), 4.72 (dd, J = 12.20, 2.27,
1H), 4.64ꢀ4.58 (m, 2H), 4.56 (d, J = 8.47, 1H), 4.30ꢀ4.25 (m, 1H), 3.88 (dd, J = 10.20,
13
1H), 3.65ꢀ3.62 (m, 1H), 1.84 (s, 3H); C NMR (150 MHz, CDCl3) δ 173.6, 170.1,
169.6, 140.2, 137.2, 136.8, 133.4, 133.3, 133.1, 133.0, 132.0, 131.9, 131.8, 131.7,
131.4, 103.0, 77.6, 75.9, 73.7, 66.8, 64.3, 57.6, 26.6. HRMS (ESIꢀTOF) m/z: [M +
H]+ calcd for C29H29N4O7 545.2031; Found 545.2022.
Benzyl 2ꢀacetamidoꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoylꢀ2,4ꢀdideoxyꢀβꢀDꢀglucopyranosides (3b)
White solid. The NMR and MS data was consistent with the literature.16
Synthesis of 1ꢀphosphoꢀ2ꢀacetamidoꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoylꢀαꢀ ꢀgalacto/gluco
D
ꢀpyranosides (5a/5b)
To a solution of 3a/3b (207 mg, 0.38 mmol) in dry DCM (5 mL) was added FeCl3
(136 mg, 0.84 mmol) under N2 protection. After stirring for 3 h, the reaction was
quenched by the addition of aq. NH4Cl. The aqueous layer was extracted with DCM
(3
× 15 mL). The combined organic phase was washed with brine, dried over
Na2SO4 and concentrated under vacuum. The resulting crude product 4a/4b was
used in the following step without further purification.
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Crude 4a/4b was dissolved in dry THF (5 mL) and the solution cooled to ꢀ78oC. To
the cooled solution, lithium diisopropylamide solution (LDA, 2 M in THF) (0.42 mL,
0.84 mmol) was added dropwise. After 30 minutes, tetrabenzyl pyrophosphate (307
mg, 0.57 mmol) was added to the reaction mixture. The reaction was stirred at ꢀ78oC
for 30 minutes and slowly warmed to 0oC over 3 h. Then the reaction was quenched
by the addition of aq. NH4Cl. The aqueous layers were extracted with EtOAc (3 x 15
mL), and the combined organic phase was washed with brine, dried over Na2SO4 and
concentrated under vacuum. The crude was purified by FC (silica gel,
Hexanes/EtOAc 1:2) to give compounds 5a/5b as yellow oils (187 mg, 69% and 198
mg, 73% respectively, two steps).
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1ꢀphosphoꢀ2ꢀacetamidoꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoylꢀαꢀ ꢀgalactopyranosides (5a)
D
1
Yellow oil. H NMR (800 MHz, CDCl3) δ: 8.06ꢀ8.04 (m, 2H), 7.99ꢀ7.97 (m, 2H),
7.60ꢀ7.57 (m, 1H), 7.54ꢀ7.51 (m, 1H), 7.47ꢀ7.44 (m, 2H), 7.39ꢀ7.36 (m, 2H),
7.35ꢀ7.28 (m, 10H), 5.71 (dd, J = 5.82, 3.29, 1H), 5.60 (d, J = 9.36, 1H), 5.45 (dd, J =
3.29, 11.28, 1H), 5.07ꢀ4.97 (m, 4H), 4.89ꢀ4.84 (m, 1H), 4.45ꢀ4.41 (m, 1H), 4.40ꢀ4.36
13
(m, 1H), 4.35ꢀ4.32 (m, 1H), 4.20ꢀ4.18 (m, 1H), 1.65 (s, 3H); C NMR (200 MHz,
CDCl3) δ 170.2, 166.4, 165.9, 135.4, 135.3, 135.2, 135.1, 134.0, 133.4, 130.2, 129.7,
129.3, 128.9, 128.8, 128.7, 128.7, 128.5, 128.3, 128.2, 128.1, 97.0 (d, J = 6.13), 70.2,
70.0 (d, J = 5.11), 69.9 (d, J = 4.86), 68.8, 62.9, 60.5, 47.9 (d, J = 6.90), 22.9.
HRMS (ESIꢀTOF) m/z: [M + H]+ calcd for C36H36N4O10P 715.2164; Found 715.2153.
1ꢀphosphoꢀ2ꢀacetamidoꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoylꢀαꢀ ꢀglucopyranosides (5b)
D
1
Yellow oil. H NMR (600 MHz, CDCl3) δ: 8.07ꢀ8.04 (m, 2H), 8.04ꢀ8.02 (m, 2H),
7.61ꢀ7.57 (m, 2H), 7.48ꢀ7.44 (m, 4H), 7.41ꢀ7.33 (m, 10H), 5.85 (d, J = 9.60, 1H),
5.73 (dd, J = 3.20, 5.98, 1H), 5.42 (dd, J = 9.28, 10.88, 1H), 5.15ꢀ5.03 (m, 4H),
4.53ꢀ4.46 (m, 2H), 4.41 (dd, J = 2.03, 12.27, 1H), 3.94ꢀ3.87 (m, 2H), 1.63 (s, 3H);13C
NMR (150 MHz, CDCl3) δ 173.7, 170.0, 169.4, 138.7, 138.6, 138.5, 138.4, 137.2,
136.7, 133.3, 133.1, 132.8, 132.3, 132.2, 132.1, 132.0, 131.9, 131.8, 131.5, 99.8 (d, J
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= 5.73), 74.8, 73.7, 73.4 (d, J = 4.46), 73.3 (d, J = 4.71), 65.9, 63.5, 55.4 (d, J = 7.50),
26.1.
HRMS (ESIꢀTOF) m/z: [M + H]+ calcd for C36H36N4O10P 715.2164; Found 715.2147.
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Synthesis of disodium uridine 5'ꢀ(2ꢀacetamidoꢀ2,4ꢀdideoxyꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀ
benzoylꢀαꢀ ꢀgalacto/glucoꢀpyranosyl) diphosphate (8a/8b)
D
The protected phosphate 5a/5b (10 mg, 0.013 mmol) was dissolved in MeOH (2
mL). Pd/C (10 wt% on activated carbon) (6 mg) was added and the mixture was
stirred under a hydrogen atmosphere for 2 h. The palladium was then filtered off
and the solvent removed in vacuo to produce crude 6a/6b in 90% and 85% yield,
respectively, which was used in the next step without further purification.
A solution of monoꢀphosphate 6a/6b (8 mg, 0.013 mmol) in MeOH (2 mL) was
treated with Et3N (9 µL, 0.07 mmol), and then concentrated to yield crude
bis(triethylammonium) phosphate. Without purification, this crude material was
repeatedly coꢀevaporated with dry pyridine (3
5′ꢀmonophosphomorpholidate 4ꢀmorpholineꢀN,N′ꢀdicyclohexylcarboxamidine salt (14
mg, 0.02 mmol) was coꢀevaporated with pyridine (3 3 mL) in a separate vessel, then
transferred in 2 mL pyridine via a cannula into the reaction flask. The combined
reagents were coꢀevaporated with pyridine (2 2 mL), then 1Hꢀtetrazole (4 mg, 0.05
×
3
mL). Uridine
×
×
mmol) in pyridine (2 mL),was added, and the reaction mixture stirred at rt for 3 d.
The reaction mixture was then concentrated in vacuo and was converted into Na+
form by passing through a Dowex (Na+) column. The resulting fraction was
concentrated in vacuum and the residue was loaded onto a Bio Gel P2 column (1
×
120 cm) eluted with H2O. Fractions were collected, and those containing the product
as determined by MS were combined and freezeꢀdried to afford 8a/8b as white
powders (5 mg, 45% and 7 mg, 58% respectively).
Disodium uridine 5'ꢀ(2ꢀacetamidoꢀ2,4ꢀdideoxyꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoyl
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5
ꢀαꢀ ꢀgalactopyranosyl) diphosphate (8a)
D
1
White solid. H NMR (600 MHz, D2O) δ: 7.98ꢀ7.90 (m, 3H), 7.86 (d, J = 8.15, 1H),
7.81 (d, J = 8.15, 1H), 7.63ꢀ7.54 (m, 3H), 7.48ꢀ7.41 (m, 3H), 5.87ꢀ5.81 (m, 1H),
5.76ꢀ5.74 (m, 1H), 5.65 (d, J = 8.22, 1H), 5.61ꢀ5.58 (m, 1H), 5.45ꢀ5.41 (m, 1H),
4.53ꢀ4.43 (m, 2H), 4.30ꢀ4.19 (m, 3H), 4.18ꢀ4.12 (m, 2H), 4.00ꢀ3.89 (m, 2H), 1.85 (s,
3H); 13C NMR (150 MHz, D2O) δ 178.2, 170.7, 169.7, 169.4, 155.1, 145.2, 144.9,
144.6, 137.7, 137.3, 133.0, 132.9, 132.2, 132.1, 106.1, 105.2, 97.8, 91.6, 87.1, 86.5,
77.6, 77.2, 75.1, 73.3, 73.1, 72.3, 71.4, 67.0, 63.8, 25.4. HRMS (ESIꢀTOF) m/z: [M ꢀ
H]ꢀ calcd for C31H33N6O18P2 839.1332; Found 839.1340.
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Disodium uridine 5'ꢀ(2ꢀacetamidoꢀ2,4ꢀdideoxyꢀ4ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzoyl
ꢀαꢀD
ꢀglucopyranosyl) diphosphate (8b)
1
White solid. H NMR (600 MHz, D2O) δ: 7.99ꢀ7.91 (m, 4H), 7.84ꢀ7.80 (m, 2H),
7.64ꢀ7.60 (m, 2H), 7.48ꢀ7.44 (m, 3H), 5.85ꢀ5.81 (m, 1H), 5.78 (d, J = 3.05, 1H), 5.73
(d, J = 8.08, 1H), 5.59 (dd, J = 3.12, 6.74, 1H), 5.44 (m, 1H), 4.63ꢀ4.53 (m, 2H), 4.45
(dt, J = 3.05, 10.49, 1H), 4.31ꢀ4.27 (m, 1H), 4.24ꢀ4.18 (m, 3H), 4.17ꢀ4.11 (m, 2H),
13
1.78 (s, 3H); C NMR (150 MHz, D2O) δ 177.8, 171.4, 171.0, 169.2, 154.8, 144.9,
144.7, 137.7, 137.5, 133.0, 132.8, 132.2, 132.1, 132.0, 106.0, 105.5, 97.7, 92.4, 86.1,
77.4, 77.1, 76.1, 72.9, 72.8, 72.6, 68.4, 66.8, 63.1, 55.2, 25.0. HRMS (ESIꢀTOF) m/z:
[M ꢀ H]ꢀ calcd for C31H33N6O18P2 839.1332; Found 839.1335.
Synthesis
of
disodium
uridine
5'ꢀ(2ꢀacetamidoꢀ2,4ꢀdideoxyꢀ4ꢀazidoꢀαꢀ ꢀ
D
galacto/glucoꢀpyranosyl) diphosphate (7a/7b)
To a solution of 8a/8b (6 mg, 0.007 mmol) in anhydrous MeOH (1 mL) was added
Sodium methoxide solution (20 µL, 5.5 M in methanol) and the resulting solution left
to stir overnight. The solution was then neutralized with preꢀwashed and acidified
Amberlyte IRꢀ120 Hydrogen ion exchange resin and the reaction mixture filtered
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through cotton wool to remove the resin. The reaction mixture was concentrated
5
6
under reduced pressure and the residue was loaded onto a Bio Gel P2 column (1
× 75
7
8
cm) and eluted with H2O to give 7a/7b as white solids. (2 mg, 55% and 3 mg, 60%
9
respectively).
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Disodium
uridine
5'ꢀ(2ꢀacetamidoꢀ2,4ꢀdideoxyꢀ4ꢀazidoꢀαꢀ ꢀgalactopyranosyl)
D
diphosphate (7a)
1
White solid. H NMR (600 MHz, D2O) δ: 7.82 (d, J = 8.06, 1H, uridineꢀH''ꢀ6),
5.86ꢀ5.82 (m, 2H, uridineꢀH''ꢀ5, ribꢀH'ꢀ1), 5.38ꢀ5.35 (m, 1H, Hꢀ1), 4.26ꢀ4.20 (m, 2H,
ribꢀH'ꢀ2, ribꢀH'ꢀ5a), 4.17ꢀ4.08 (m, 4H, Hꢀ2, Hꢀ5, ribꢀH'ꢀ3, ribꢀH'ꢀ4 ), 4.07ꢀ4.00 (m, 2H,
Hꢀ3, ribꢀH'ꢀ5b ), 3.67ꢀ3.63(m, 1H, Hꢀ6a), 3.62ꢀ3.56 (m, 2H, Hꢀ4, Hꢀ6b), 1.95 (s, 3H);
13C NMR (150 MHz, D2O) δ 185.8, 172.9, 171.7, 151.9, 141.7, 102.6, 94.6, 88.5, 83.1,
73.6, 70.5, 69.5, 68.0, 65.1, 62.8, 60.7, 21.9.
HRMS (ESIꢀTOF) m/z: [M ꢀ H]ꢀ calcd for C17H25N6O16P2 631.0808; Found 631.0808.
Disodium
uridine
5'ꢀ(2ꢀacetamidoꢀ2,4ꢀdideoxyꢀ4ꢀazidoꢀαꢀ ꢀglucopyranosyl)
D
diphosphate (7b)
1
White solid. H NMR (600 MHz, D2O) δ: 7.86 (d, J = 7.95, 1H, uridineꢀH''ꢀ6),
5.88ꢀ5.84 (m, 2H, uridineꢀH''ꢀ5, ribꢀH'ꢀ1), 5.42 (dd, J = 3.18, 7.32, 1H, Hꢀ1),
4.27ꢀ4.22 (m, 2H, ribꢀH'ꢀ2, ribꢀH'ꢀ5a), 4.19ꢀ4.13 (m, 2H, ribꢀH'ꢀ3, ribꢀH'ꢀ4), 4.10ꢀ4.06
(m, 1H, ribꢀH'ꢀ5b), 3.95(dt, J = 2.84, 10.70, 1H, Hꢀ2), 3.79ꢀ3.66 (m, 4H, Hꢀ3, Hꢀ5,
13
Hꢀ6a, Hꢀ6b), 3.48 (m, 1H, Hꢀ4), 1.97 (s, 3H, HꢀNAc); C NMR (150 MHz, D2O) δ
178.5, 169.9, 145.1, 106.0, 98.0, 92.3, 86.5, 77.2, 74.9, 73.7, 72.7, 68.2, 64.9, 63.8,
56.9, 25.6. HRMS (ESIꢀTOF) m/z: [M ꢀ H]ꢀ calcd for C17H25N6O16P2 631.0808;
Found 631.0809.
GAG synthase assays
Either azidoꢀcontaining UDPꢀhexosamine analog or authentic UDPꢀhexosamine (0.5
mM) was incubated in a reaction (25 ꢁl) containing 50 mM Tris, pH 7.2, 1 mM
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MnCl2, radiolabeled UDPꢀ[3H]GlcA (0.05 mM; 0.1 ꢁCi; Perkin Elmer), and 25 ꢁg of
purified recombinant enzyme (PmHS1 or PmHS2; PmHAS or PmCS) (as well as 1 M
ethylene glycol for PmHAS and PmCS) at 30°C for 5.25 h. The reaction mixtures
were quenched with sodium dodecyl sulfate (2% final conc) and analyzed by
descending paper chromatography (overnight in 65:35 ethanol/1 M ammonium
acetate buffer, Whatman 3MM paper). As a negative control for assay background, a
reaction with no UDPꢀhexosamine was tested in parallel (GAG chain polymerization
can only occur when UDPꢀGlcA and a functional UDPꢀhexosamine are present
simultaneously).
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Mass Spectroscopy
HA21a or Heparosan oligosaccharides21b (0.1ꢀ1 ꢁg) were reacted with a 2ꢀ10ꢀfold
molar excess of either UDPꢀGlcNAc, UDPꢀGalNAc, the azidoꢀcontaining analog, or
no analog in a buffer containing 50 mM Tris, pH 7.2, 1 mM MnCl2, and purified
recombinant synthase (5ꢀ10 ꢁg) for 16 h at 30 °C. The reactions were assessed by
MALDIꢀToFꢀMS, with the matrix 6ꢀazaꢀ2ꢀ thiothymine at a concentration of 5 mg/mL
in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA). HA oligosaccharides were
employed as mass calibrants.
Azido tagged GAG preparations
GAGs (HA, 68 kDa or HEP, 40.1 kDa) were azido endꢀlabeled at the nonꢀreducing
terminus using the donor sugar UDPꢀ4ꢀN3ꢀGlcNAc in a reaction buffer containing 50
mM HEPES, pH 7.2, 1 mM MnCl2, and 1 ꢁg/ꢁl purified enzyme (PmHAS or
PmHS1).
Click chemistry
The 4ꢀN3ꢀGlcNAcꢀterminated HEP or HA polymers (30 ꢁg) were mixed with a
10ꢀfold excess Alexa Fluor 488 DIBO alkyne in 15 ꢁL deionized water and incubated
at room temperature in the dark for 5 h. The crude reaction was then directly
subjected to PAGE analysis.
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Polyacrylamide gel electrophoresis (PAGE)
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Phenol red dye was added to the sample for visualization of the ion front during
electrophoresis. A 10ꢀꢁg aliquot of each sample was analyzed on a 4ꢀ15%
MiniꢀProtean® TGX™ Precast Gel ran in TrisꢀGlycine buffer (at constant 30 mA for
4 h). The gel was first visualized by UV fluorescence and followed by Alcian Blue
staining (0.5% w/v Alcian blue dye and 2% v/v aqueous acetic acid) and destained in
water.22
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS publications
website at DOI:
HRMS, 1H, 13C, COSY and HSQC NMR spectra of the components (PDF)
AUTHOR INFORMATION
Corresponding Author
linhar@rpi.edu and PaulꢀDeangelis@ouhsc.edu
Notes
The authors declare no completing financial interest.
ACKNOWLEDGMENT
The authors are grateful for funding from the National Institutes of Health in the form
of grants HL62244 and HL094463.
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