Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis

Article abstract of DOI:10.1021/acs.joc.6b02929

Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-

Full text of DOI:10.1021/acs.joc.6b02929

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Note
Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine
Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis
Victor L. Schultz, Xing Zhang, Kathryn Linkens, Jenna Rimel,
Dixy E. Green, Paul L. DeAngelis, and Robert J. Linhardt
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02929 • Publication Date (Web): 27 Jan 2017
Downloaded from http://pubs.acs.org on January 28, 2017
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Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine
Uridine Diphosphate Donors: Chain Terminators in
Glycosaminoglycan Synthesis
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Victor L. Schultz,†# Xing Zhang,†# Kathryn Linkens,† Jenna Rimel,†
Dixy E. Green,‡ 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, Oklahoma Center for Medical Glycobiology, 940 Stanton L. Young Blvd., Oklahoma
City, Oklahoma 73126, United States
# These authors contributed equally to this work
Author to whom correspondence should be addressed at linhar@rpi.edu
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OH
HOOC
O
O
O
HO
O
HO
H
OH
AcHN
H
O
Heparosan oligomer
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9
COOH
O
OH
O
HO
OAc
O
OAc
NHAc
m
HO
F
AcO
AcO
HO
OH
HO
O-UDP
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AcHN
O-UDP
O
HO
HO
m
Chemical and chemoenzymatic synthesis
AcHN
O-UDP
Heparosan synthase 1
OH
O
AcHN
F
HO
HOOC
O
HO
OH
O
HOOC
O
OH
O
H
O
O
HO
O
HO
O
OH
HO
OH
AcHN
O
H
AcHN
O
H
n+m
from n
to n+m
Heparosan polymer
Sequence specific chain termination
ABSTRACT:
Unnatural
uridine
diphosphate
(UDP)ꢀsugar
donors,
UDPꢀ4ꢀdeoxyꢀ4ꢀfluoroꢀNꢀ
acetylglucosamine (4FGlcNAc) and UDPꢀ4ꢀdeoxyꢀ4ꢀfluoroꢀNꢀacetylgalactosamine (4FGalNAc),
were prepared using both chemical and chemoenzymatic syntheses relying on Nꢀ
acetylglucosamineꢀ1ꢀphosphate uridylyltransferase (GlmU). The resulting unnatural UDPꢀsugar
donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various
synthases. UDPꢀ4FGlcNAc was transferred onto an acceptor by Pastuerella multocida
heparosan synthase 1 and subsequently served as a chain terminator.
Sugar nucleotides are the primary building blocks from which most complex carbohydrates
are prepared and, thus, are crucial intermediates in carbohydrate metabolism.1 Uridine
diphosphate (UDP) monosaccharides are among the most common sugar nucleotide donors and
are transferred to glycosyl acceptors by glycosyltransferases or synthases in glycan biosynthetic
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pathways. Both natural and unnatural UDPꢀsugars, can be chemically, enzymatically, or
chemoenzymatically synthesized.2 The synthesis of natural UDPꢀsugars is well established in
literature3, however, the synthesis and application of unnatural UDPꢀsugars, which are otherwise
not synthesized in vivo is not well explored. Such unnatural donors have great potential as
enzymatic substrates in carbohydrate synthesis, as enzyme inhibitors in biochemical studies, as
tools for assay development, and as reagents for the study of glycoconjugate biosynthesis.4
UDPꢀNꢀacetylglucosamine (GlcNAc), UDPꢀNꢀacetylgalactosamine (GalNAc), and UDPꢀ
glucuronic acid (GlcA) are widely studied natural donor substrates involved in
glycosaminoglycan (GAG) synthesis.2,5,6 GAGs are linear polysaccharides composed of
disaccharide repeating units of hexosamine and uronic acid residues that are frequently
sulfated.2,5 Due to the presence of 1→4 linkages in GAGs, particularly those of the heparan
sulfate/heparin family, the C4 position of the UDPꢀdonor offers an interesting target for
modification. By chemically modifying the monosaccharide prior to the addition of a UDP
moiety, the structure of the final polysaccharide chain can be manipulated. Access to the C4
position can be complicated by its low relative reactivity compared to neighboring sites.7 This
and the presence of charged or polar functional groups requires multiple protection/deprotection
steps for C4 modification. Purely chemical approaches for UDPꢀsugar synthesis generally are
associated with low yields due to tedious protection/deprotection strategies, limiting scaleꢀup and
commercialization. A chemoenzymatic approach offers a synthetic route that circumvents many
of these challenges. Chemoenzymatic synthesis of natural UDPꢀsugars has been accomplished
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using
recombinant
Escherichia
coliꢀexpressed
Nꢀacetylglucosamineꢀ1ꢀphosphate
uridylyltransferase (GlmU).8 We hypothesized that UDPꢀdonors chemically modified at the C4
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position could be prepared using GlmU and used to synthesize heparan sulfate/heparin
oligosaccharides with novel structures and also potentially serve as chain terminators.9
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The chemical synthesis of an unnatural UDPꢀGlcNAc donor generally relies on standard
protectionꢀdeprotection chemistry for siteꢀspecific control. Previous work in our laboratory8
reported the chemical synthesis of a series of GlcNRꢀ1ꢀphosphate analogs (R = H2, N2, COR’)
and the recognition of these analogs by GlmU uridylyltransferase. In this study we concluded
that C4 orientation of the hexosamine sugar had little impact on GlmU utilization of an UDPꢀ
HexNR (i.e., UDPꢀGlcNR or UDPꢀGalNR), yet the amide linkage to the Cꢀ2 nitrogen of the
phosphate analogs was crucial for GlmU recognition. While the promiscuity of GlmU has been
notably utilized with 4ꢀOH hexosamine phosphates10,11 subsequent studies, by Wang et al.12,
demonstrated that GlmU was intolerant of any functional group and the C4 position larger than a
hydroxyl functionality. Previous syntheses of 4ꢀfluoroꢀsubstituted sugar nucleotides have been
reported, however these schemes were either purely chemical13ꢀ15 or utilized a lengthy
chemoenzymatic route with relatively low conversion rates by means of UDPꢀGalNAc
pyrophosphorylase.16 The use of fluorinated nucleotides as chain extension terminators has been
well established with galactofuranose derivatives17 while metabolic precursors of fluoroꢀ
substituted sugar nucleotides have been shown to disrupt nucleotide sugar synthesis as the point
of incorporation.18,19 Here we report the first chemoenzymatic synthesis of the 4ꢀfluoro UDPꢀ
GlcNAc/ꢀGalNAc analogs using GlmU in good yields (45%ꢀ50%) and the application of UDPꢀ
4FGlcNAc as a glycosylation terminator in heparan sulfate/heparin synthesis.
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Our first objective in accessing the target UDPꢀ4FGlcNAc/ꢀ4FGalNAc donors was to
synthesize the corresponding 4FGlcNAcꢀ/4FGalNAcꢀ1ꢀphosphate analogs. We began the
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synthesis with commercially available
(Scheme 1).
Dꢀglucosamine/Dꢀgalactosamine pentaacetate 1a/1b
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Scheme 1. Synthesis of 4FGalNAcꢀ1ꢀphosphate 9a and 4FGlcNAcꢀ1ꢀphosphate 9b. Although
only one stereochemical series is shown (starting from ꢀglucosamine pentaacetate 1a to
4FGalNAcꢀ1ꢀphosphate 9a), both series were synthesized and the yields for both are provided.
D
o
Refluxing 1 with benzyl alcohol and Yb(OTf)3 in dichloromethane at 60 C provided the βꢀ
benzyl glycoside of
Dꢀglucosamine/galactosamine tetraacetate 2 in good yields (Glc 95% and
Gal 97%) and with excellent selectivity. Zemplen deacetylation reaction with sodium methoxide
in methanol afforded triol 3 in quantitative yield. The Cꢀ4 fluoride functionality was installed by
a selective benzoylation of Cꢀ3 and Cꢀ6 followed by (diethylamino) sulfur trifluoride (DAST)
reaction resulting in a Cꢀ4 configuration inverted intermediate 5a. Using the same approach, the
other corresponding compound 5b was also successfully prepared. Deprotection of the anomeric
hydroxyl followed by phosphorylation with tetrabenzyl pyrophosphate afforded the
phosphorylated intermediate 7 with excellent αꢀselectivity. The targeted substrates 9a/9b were
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obtained in high yields by using deꢀOꢀbenzylation under hydrogenation followed by base
catalyzed deꢀOꢀbenzoylation.
7
8
9
O
OH
O
OH
O
UMP Morpholodate
F
HO
F
HO
NH
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O
O
O
AcHN
Pyridine, Tetrazole
3 days
N
O
AcHN
O P O P O
ONa ONa
O P OH
OH
O
OH OH
4F GlcNAc-UDP 10b, 61%
4F GalNAc-UDP 10a, 55%
4F GlcNAc-1-phosphate 9b
4F GalNAc-1-phosphate 9a
Scheme 2. Chemical synthesis of UDPꢀ4FGalNAc 10a and UDPꢀ4FGlcNAc 10b donors.
With two monophosphate hexosamines in hand, we next focused our attention on the
chemical approach to preparing the UDPꢀ4FGalNAc 10a/UDPꢀ4FGlcNAc 10b donors (Scheme
2). Compounds 9a/9b were converted to their pyridinium salts and then stirred with uridine
monophosphate (UMP)ꢀmorpholidate and tetrazole in pyridine for 3 days to afford the target
unnatural UDPꢀsugars. However, at this stage, the purification became problematic. Neither
size exclusion chromatography (SEC) nor strong anion exchange (SAX) chromatography could
satisfactorily purify the target compounds as numerous organic phosphate salts, contained in the
crude, showed very little difference in molecular weight and polarity. We were able to
circumvent this problem by first converting all the phosphate salts to their sodium form using an
ionꢀexchange resin before subjecting the product to SEC on a 1.2 m BioGel P2 column with
elution by water. Lyophilization afforded the 4FGalNAcꢀ/4FGlcNAcꢀUDP derivatives 10a/10b
in satisfactory isolated yields.
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O
N
OH
O
OH
O
F
HO
GlmU, DTT, UTP
NH
F
HO
O
O
AcHN
O
O
O P O P O
ONa ONa
Tris, MgCl2
pyrophosphatase, 3 h
AcHN
O
O P OH
OH
OH OH
4F GlcNAc-UDP 10b, 45%
4F GalNAc-UDP 10a, 50%
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4F GlcNAc-1-phosphate 9b
4F GalNAc-1-phosphate 9a
Scheme 3. 4FGlcNAcꢀ1ꢀphosphate 9b and 4FGalNAcꢀ1ꢀphosphate 9a as substrates for GlmU.
Next, we examined the ability of GlmU uridylyltransferase to use 4FGlcNAcꢀ/4FGalNAcꢀ1ꢀ
phosphate analogs as substrates. Both 4FGlcNAcꢀ1ꢀphosphate 9b and 4FGalNAcꢀ1ꢀphosphate
9a were accepted by GlmU affording the corresponding UDPꢀsugar nucleotides in satisfactory
yields of 45% and 50%, respectively (Scheme 3).
In our previous study,8 we had found that the configuration of the 4ꢀOH group appeared not
to play a critical role in enzyme recognition. This study further confirmed our previous
observation demonstrating that the 4ꢀfluoro substituted substrates were both similarly good
GlmU substrates.
With the chemically and enzymatically synthesized unnatual UDPꢀdonors in hand, we next
tested their utility as substrates in GAG synthesis. Two tests were conducted to determine 4F
GlcNAcꢀ/4FGalNAcꢀUDP were incorporated into a growing GAG chain or served as a chain
extension terminator.20 Coꢀpolymerization assays were conducted using radiolabeled sugar
incorporation to monitor synthaseꢀcatalyzed coꢀpolymerization of UDPꢀ[3H]ꢀGlcA with the
UDPꢀ4FHexNAc donor in the presence of a GAG acceptor. Controls with natural UDPꢀHexNAc
(either UDPꢀGlcNAc or UDPꢀGalNAc) functioned as expected for the GAG synthases,
Pasturella multocida (Pm) hyaluranan synthase HAS, Pm chondroitin synthase (CS), and Pm
heparosan synthase HS 1 or 2.21,22 However, the UDPꢀ4FHexNAc donors showed no signal
above background for the presence of incorporated UDPꢀ4FHexNAc, leading to the conclusion
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that these unnatural sugars once incorporated are not substrates for further GAG chain
elongation. Any inhibitory effect on chain polymerization was approximately the same or less
than shown by UDP alone (~25ꢀ78% at 0.5 mM test range). Since an acceptor with UDPꢀ
4FHexNAc at its nonꢀreducing terminus is missing a C4ꢀhydroxyl required to form the
glycosidic linkage, this was an expected result. It was, however, possible that such an acceptor
could have been extended through the nearby C3ꢀOH instead, as enzyme promiscuity is
unpredictable, but this was not observed.
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3
F
-
4
0
+
5
N
+
6
F
+
2
N
-
Lane
Capping Extension
1
0
-
1. No capping
2. GlcNAc capping
3. 4F-GlcNAc capping
4. No cap, then extension
5. GlcNAc cap, then extension
6. 4F-GlcNAc cap, then
extension
Fig. 1. FACE analysis of dyeꢀtagged heparosan oligosaccharide elongated with authentic or
analog UDPꢀsugar donors by PmHS1.
Termination studies were next performed using synthaseꢀcatalyzed polymerization assays
that normally extends natural GAG chains to ~50ꢀ100 kDa. Parallel reactions were performed in
which UDPꢀ4FHexNAc sugar was added to test if the presence had any effect on GAG chain
length, or if the sugar itself was a synthase inhibitor. The UDPꢀ4FHexNAc substrate were tested
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with each of the Pasteurella GAG synthases that are capable of efficiently polymerizing HA
(PmHAS), heparosan (PmHS1 or 2), or chondroitin (PmCS) chains. Interestingly, the twoꢀstep
terminationꢀtype experiments, reactions with PmHS1 appeared to block chain extension when
the acceptor was preꢀtreated with the UDPꢀ4FGlcNAc analog. Therefore, we did more direct
experiments to verify that the 4FGlcNAc analog sugar from the UDPꢀ4FGlcNAc donor was
transferred onto the nonꢀreducing end of the heparosan acceptor. As observed by fluorophoreꢀ
assisted carbohydrate electrophoresis (FACE) gel (Fig. 1) and by mass spectrometry (Table 1),
new species were observed that had properties consistent with 4FGlcNAc incorporation. While
the 4ꢀfluoro group did block subsequent chain extension, it was not transferred efficiently as
observed by the partial transformation of the starting material.
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Table 1.
Mass spectrometric analysis of heparosan anhydromannitol tetrasaccharide or hexasaccharide acceptors
and PmHS1ꢀcatalyzed extension products. The molecular weight was obtained in negative mode by
matrixꢀassisted laser desorption (MALDI)ꢀtime of flight (ToF)ꢀmass spectrometry (MS). The predicted
masses were observed for the analogꢀterminated products.
Reaction
Heparosan tetrasaccharide
Heparosan hexasaccharide
1097.34
Starting oligosaccharide
+ GlcNAc
718.20
921.30
923.30
1300.42
+ 4FGlcNAc
1302.41
In summary, the successful synthesis of UDPꢀ4FGlcNAc/UDPꢀ4FGalNAc analogs was
completed using both an improved chemical method as well as a new chemoenzymatic approach.
This constitutes the first synthesis of UDPꢀ4FHexNAc analogs by the uridylyltransferase GlmU.
These UDPꢀsugars were then tested for incorporation and termination capabilities using coꢀ
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polymerization assays. While neither analog showed signs of incorporation and extension of
GAGs, reactions with PmHS1 preꢀtreated with UDPꢀ4FGlcNAc resulted in the incorporation of a
single 4FGlcNAc at the nonꢀreducing end of the acceptor acting as a chain terminator. These
results suggest that the unnatural UDPꢀdonor, 4FGlcNAc, can be used to synthesize heparan
sulfate/heparin oligosaccharides having novel functionality and of defined sequence length.
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EXPERIMENTAL SECTION
General Information
Unless noted otherwise, commercially available materials were used without further
purification. All reagents were purchased from commercial corporations. Flash chromatography
(FC) was performed using silica gel (200−300 mesh) according to the standard protocol. All
reactions under standard conditions were monitored by thinꢀlayer chromatography (TLC) on gel
F254 plates. Mass data were acquired by MALDIꢀToFꢀMS or electrospray ionization (ESI)ꢀ
1
highꢀresolution (HR)ꢀMS on an LTQꢀOrbitrap XL FTꢀMS spectrometer. H and 13C NMR
13
spectra were recorded on a 800 MHz (200 MHz for C NMR) spectrometer or 600 MHz (150
MHz for 13C NMR). 19F, 31P NMR spectra were recorded on a 500 MHz NMR spectrometer.
Synthesis of Benzyl 2-acetamido-3,6-di-O-benzoyl-2-deoxy-β-D-gluco-/galacto-pyranoside
(4a/4b)
Commercially available Dꢀglucosamine/galactosamine pentaacetate 1a/1b (1.0 g, 2.6 mmol)
was refluxed with benzyl alcohol (0.85 mL, 8.2 mmol) and Yb(OTf)3 (200 mg, 0.3 mmol) in
o
dichloromethane (20 mL) at 60 C for 3 h. The solution was allowed to cool to room
temperature and the washed three times with deionized water. The organic layer was
concentrated under reduced pressure and the product was recrystallized from methanol and
hexanes to afford 2a/2b as clear crystals in 95% and 97% yields respectively.
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To a solution of 2a/2b (350 mg, 0.8 mmol) in anhydrous MeOH (10 mL) was added sodium
methoxide solution (4.82 mL, 0.5 M in methanol) at room temperature and the resulting solution
left to stir for 2 h. The solution was then neutralized with preꢀwashed and acidified Amberlite
IRꢀ120 Hydrogen ion exchange resin and the reaction mixture filtered through cotton wool to
remove the resin. The eluent was then concentrated under reduced pressure to give the
compound 3a/3b as white solids in quantitative yields. The resulting solids were used in the
following step without further purification.
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Compound 3a/3b (240 mg, 0.77 mmol) was dissolved in pyridine (4 mL) and the solution
o
cooled to ꢀ40 C in a dry ice/ethyl acetate bath. Benzoyl chloride (196 ꢁL, 1.69 mmol) was
added dropwise and the solution was allowed to warm to room temperature overnight. The
pyridine was removed under vacuum and the resulting crude mixture was purified by column
chromatography (hexanes/EtOAc 3:1) to yield compound 4a/4b as white solids (300 mg, 75%
and 307mg, 77% respectively).
Benzyl 2-acetamido-3,6-di-O-benzoyl-2-deoxy-β-D-glucopyranoside (4a)
1H NMR (800 MHz, CDCl3) δ: 8.06 (d, J = 7.8, 2H, HꢀAr), 7.96 (d, J = 7.8, 2H, HꢀAr), 7.56
(t, J = 7.38, 1H, HꢀAr), 7.53 (t, J = 7.38, 1H, HꢀAr), 7.43 (t, J = 7.63, 2H, HꢀAr), 7.37 (t, J = 7.63,
2H, HꢀAr), 7.27ꢀ7.23 (m, 5H, HꢀAr), 5.89 (d, J = 9.28, 1H, Hꢀ1), 5.36 (dd, J = 9.07, 10.45, 1H,
Hꢀ6a), 4.88 (d, J = 12.14, 1H, HꢀOCH2Bn), 4.70 (dd, J = 4.85, 12.14, 1H, Hꢀ3), 4.66ꢀ4.63 (m,
3H, Hꢀ2, Hꢀ5, Hꢀ6b), 4.62 (d, J = 12.14, 1H, H'ꢀOCH2Bn), 3.77ꢀ3.74 (m, 1H, Hꢀ4), 1.77 (s, 3H,
13
HꢀAc); C NMR (200 MHz, CDCl3) δ 170.4, 167.6, 167.1, 137.2, 133.6, 133.4, 130.0, 129.9,
129.6, 129.1, 128.5, 128.5, 128.4, 128.0, 127.9, 99.6, 76.1, 74.3, 70.3, 69.4, 63.7, 54.1, 23.3.
ESIꢀHRMS calcd. for C29H29NO8 ([M + Na] +) 542.1785, found 542.1789
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Benzyl 2-acetamido-3,6-di-O-benzoyl-2-deoxy-β-D-galactopyranoside (4b)
5
6
1
White solid. H NMR (600 MHz, CDCl3) δ 8.14ꢀ8.03 (m, 4H, HꢀAr), 7.66ꢀ7.57 (m, 2H, Hꢀ
7
8
9
Ar), 7.54ꢀ7.43 (m, 4H, HꢀAr), 7.39ꢀ7.29 (m, 5H, HꢀAr), 5.51 (d, J = 8.96, 1H, Hꢀ1), 5.35 (dd, J =
2.81, 11.14, 1H, Hꢀ6a), 4.94 (d, J = 12.14, 1H, HꢀOCH2OBn), 4.74ꢀ4.61 (m, 3H, Hꢀ3, Hꢀ5, H'ꢀ
OCH2OBn), 4.51ꢀ4.44 (m, 1H, Hꢀ2), 4.25 (d, J = 2.47, 1H, Hꢀ6b), 4.01 (t, J = 6.39, 1H, Hꢀ4),
1.87 (s, 3H, HꢀAc); 13C NMR (150 MHz, CDCl3) δ 170.5, 166.5, 166.4, 137.1, 133.7, 133.5,
130.3, 130.1, 129.9, 129.8, 129.3, 128.7, 128.6, 128.3, 128.2, 99.8, 73.4, 72.5, 70.5, 67.1, 63.0,
51.2, 23.5. ESIꢀHRMS calcd. for C29H29NO8 ([M + Na] +) 542.1785, found 542.1760
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Synthesis of Benzyl 2-acetamido-4-fluoro-3,6-di-O-benzoyl-2,4-deoxy-β-D-galacto/gluco-
pyranosides (5a/5b)
Diethylamino sulfur trifluoride (76 µL, 0.57 mmol) was added to a solution of 4a/4b (100
mg, 0.19 mmol) in dry DCM (5 mL) at ꢀ40°C. The mixture was then allowed to warm to room
temperature over 2 h, and quenched by addition with MeOH (several drops) at 0°C. The residue
was diluted with DCM and the solution was washed with water, sat. NaHCO3 aq., brine and then
dried over anhydrous MgSO4. The solvent was removed by evaporation and purified by silica
gel chromatography (hexanes/EtOAc 3:1) to yield 5a/5b as yellow oils (64 mg, 64% for both
compounds).
Benzyl 2-acetamido-4-fluoro-3,6-di-O-benzoyl-2,4-deoxy-β-D-galactopyranoside (5a)
1
Yellow oil. H NMR (600 MHz, CDCl3) δ: 8.06 (d, J = 7.90, 4H), 7.59 (t, J = 7.50, 2H), 7.48ꢀ
7.43 (m, 4H), 7.34ꢀ7.29 (m, 5H), 5.55 (ddd, J = 2.48, 11.24, 27.30, 1H), 5.45 (d, J = 8.83, 1H),
5.01 (dd, J = 2.48, 50.51, 1H), 4.94 (d, J = 12.09, 1H), 4.90 (d, J = 8.46, 1H), 4.70ꢀ4.64 (m, 2H),
4.56 (dd, J = 6.79, 11.24, 1H), 4.26ꢀ4.20 (m, 1H), 4.06 (dt, J = 6.74, 26.40, 1H), 1.85 (s, 3H); 13C
NMR (150 MHz, CDCl3) δ 173.8, 169.5, 169.4, 140.3, 137.0, 136.8, 133.4, 133.2, 133.1, 132.8,
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132.3, 131.9, 131.8, 131.6, 131.4, 102.5, 89.2 (d, J = 186.67), 74.5 (d, J = 19.57), 74.1, 74.0,
65.4, 55.2, 26.8. ESIꢀHRMS calcd. for C29H28FNO7 ([M + H] +) 522.1923, found 522.1910;
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Benzyl 2-acetamido-4-fluoro-3,6-di-O-benzoyl-2,4-deoxy-β-D-glucopyranoside (5b)
Yellow oil. 1H NMR (600 MHz, CDCl3) δ: 8.10ꢀ8.02 (m, 4H), 7.64ꢀ7.55 (m, 2H), 7.50ꢀ7.43 (m,
4H), 7.35ꢀ7.28 (m, 5H), 5.80ꢀ5.75 (br, 1H), 5.42 (d, J = 9.03, 1H), 5.00ꢀ4.93 (m, 2H), 4.74ꢀ4.62
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(m, 2H), 4.49ꢀ4.39 (m, 2H), 4.30ꢀ4.29 (m, 1H), 3.97ꢀ3.92 (m, 1H), 1.85 (s, 3H); C NMR (150
MHz, CDCl3) δ 173.7, 169.4, 169.2, 140.3, 137.0, 136.8, 133.4, 133.2, 132.9, 132.3, 131.9,
131.9, 131.6, 131.5, 102.95, 90.4 (d, J = 188.90), 75.0 (d, J = 22.21), 74.2, 74.0, 66.3, 57.7, 26.9.
ESIꢀHRMS calcd. for C29H28FNO7 ([M + H] +) 522.1923, found 522.1902;
Synthesis of 1-phospho-2-acetamido-4-fluoro-α-D-galacto-/gluco-pyranosides (7a/7b)
To a solution of 5a/5b (200 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 6a/6b was used in the following step without further purification. Crude
◦
6a/6b was dissolved in dry THF (5 mL) and the solution cooled to ꢀ78 C. To the cooled
solution, lithium diisopropylamide solution (LDA, 2M in THF) (0.42 mL, 0.84 mmol) was added
dropwise (CAUTION: this is a pyrophoric reagent and care must be taken in undertaking this
reaction). After 30 min, tetrabenzyl pyrophosphate (307 mg, 0.57 mmol) was added to the
◦
◦
reaction mixture. The reaction was stirred at ꢀ78 C for 30 min and slowly warmed to 0 C over
3h. Then the reaction was quenched by the addition of aq. NH4Cl. The aqueous layer was
extracted with EtOAc (3 × 15 mL), and the combined organic phase was washed with brine,
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dried over Na2SO4 and concentrated under vacuum. The crude was purified by FC (silica gel,
Hexanes/EtOAc 3:1) to give compounds 7a/7b as yellow oils (191 mg, 72% and 204 mg, 77%
respectively).
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Dibenzyl 2-acetamido-4-fluoro-3,6-di-O-benzoyl-2,4-deoxy-α-D-galactopyranosyl
phosphate (7a)
Yellow oil. 1H NMR (600 MHz, CDCl3) δ: 8.08ꢀ7.97 (m, 4H), 7.62ꢀ7.51 (m, 2H), 7.49ꢀ7.42 (m,
2H), 7.41ꢀ7.23 (m, 12H), 5.82ꢀ5.76 (m, 1H), 5.66ꢀ5.60 (m, 1H), 5.35ꢀ5.24 (m, 1H), 5.12ꢀ4.96 (m,
5H), 4.93ꢀ4.86 (m, 1H), 4.52ꢀ4.41 (m, 2H), 4.38ꢀ4.28 (m, 1H), 1.67 (s, 3H); 13C NMR (150
MHz, CDCl3) δ 173.6, 169.8, 169.2, 138.7, 138.5, 137.1, 136.7, 133.4, 133.1, 132.7, 132.3,
132.1, 132.1, 132.0, 131.8, 131.5, 100.4 (d, J = 6.04), 89.4 (d, J = 189.64), 73.4, 73.3, 72.7, 72.6,
+
71.9, 71.8, 65.2, 65.2, 50.8, 26.2. ESIꢀHRMS calcd. for C36H36FNO10P ([M + H] ) 692.2055,
found 692.2041;
Dibenzyl 2-acetamido-4-fluoro-3,6-di-O-benzoyl-2,4-deoxy-α-D-glucopyranosyl
phosphate (7b)
Yellow oil. 1H NMR (600 MHz, CDCl3) δ: 8.09ꢀ8.01 (m, 4H), 7.96ꢀ7.94 (m, 2H), 7.91ꢀ7.88 (m,
1H), 7.84ꢀ7.80 (m, 2H), 7.63ꢀ7.56 (m, 2H), 7.52ꢀ7.43 (m, 9H), 5.93ꢀ5.89 (m, 2H), 5.67ꢀ5.64 (m,
1H), 5.49 (dd, J = 3.16, 11.46, 1H), 5.15ꢀ5.03 (m, 4H), 4.97ꢀ4.91 (m, 1H), 4.54ꢀ4.49 (m, 1H),
4.46ꢀ4.41 (m, 1H), 4.34ꢀ4.29 (m, 1H), 1.66 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 173.7, 169.7,
169.5, 138.8, 138.7, 137.0, 136.9, 136.7, 136.6, 133.3, 133.2, 133.1, 132.7, 132.4, 132.2, 132.2,
132.1, 132.0, 131.9, 131.8, 131.7, 100.5 (d, J = 6.04), 89.5 (d, J = 181.56), 73.4, 73.3, 72.6, 71.6,
+
71.2, 65.3, 51.6, 26.2. ESIꢀHRMS calcd. for C36H36FNO10P ([M + H] ) 692.2055, found
692.2041;
Phosphate 2-acetamido-4-fluoro-α-D-galacto-/gluco-pyranoside (9a/9b)
The protected phosphate (25 mg, 0.036 mmol) was dissolved in MeOH (2 mL). Pd/C (10
wt% on activated carbon) (3 mg) was added and the mixture was stirred under a hydrogen
atmosphere for 2 h. The palladium was filtered off and the solvent removed to yield a crude
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8a/8b, which was used in the next step without further purification. To a solution of crude 8a/8b
in anhydrous MeOH (3 mL) was added sodium methoxide solution (20 µL, 5.5 M in methanol)
at 0 oC and the resulting solution was left to stir for 2 h. The solution was then neutralized with
preꢀwashed and acidified Amberlyte IRꢀ120 Hydrogen ion exchange resin and the reaction
mixture filtered through cotton wool to remove the resin. The eluent was then concentrated
under reduced pressure to give the corresponding 4ꢀF phosphoꢀsugar analog 9a/9b as white
solids (10 mg, 93% and 9 mg, 84% respectively).
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Phosphate 2-acetamido-4-fluoro-α-D-galactopyranoside (9a)
1
White solid. H NMR (800 MHz, D2O) δ 5.25 (d, J = 3.8, 1H, Hꢀ1), 4.32 (dt, J = 9.12,
50.87, 1H, Hꢀ4), 4.01ꢀ3.93 (m, 2H, Hꢀ3, Hꢀ5), 3.89ꢀ3.84 (m, 1H, Hꢀ2), 3.77ꢀ3.73 (m, 1H, Hꢀ6a),
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3.71ꢀ3.66 (m, 1H, Hꢀ6b), 1.93 (s, 3H, HꢀAc); C NMR (200 MHz, D2O) δ 174.5, 92.8 (d, J =
31
5.0), 89.3 (d, J = 181.57), 69.7 (d, J = 23.82), 69.4 (d, J = 17.53), 59.8, 53.5, 22.0; P NMR
(202.5 MHz, D2O) δ ꢀ0.5; 19F NMR (470 MHz, D2O) δ ꢀ198.0 (dd, J = 14.1, 51.7). (ESIꢀHRMS
calcd. for C8H15FNO8P ([M ꢀ H] ꢀ) 302.0447, found 302.0435
Phosphate 2-acetamido-4-fluoro-α-D-glucopyranoside (9b)
White solid. 1H NMR (800 MHz, D2O) δ: 5.31 (s, 1H, Hꢀ1), 4.34 (dt, J = 9.48, 50.44, 1H, Hꢀ
4), 4.00ꢀ3.93 (m, 2H, Hꢀ3, Hꢀ5), 3.93ꢀ3.86 (m, 1H, Hꢀ2), 3.76ꢀ3.68 (m, 2H, Hꢀ6a, Hꢀ6b), 1.93 (s,
3H, HꢀAc); 13C NMR (200 MHz, D2O) δ 174.7, 93.2 (d, J = 5.3), 89.0 (d, J = 179.91), 70.2 (d, J
19
= 24.67), 68.8 (d, J = 19.51), 59.7, 53.3, 21.9; 31P NMR (202.5 MHz, D2O) δ ꢀ1.7; F NMR
ꢀ
(470 MHz, D2O) δ ꢀ198.2 (dd, J = 14.1, 51.7). ESIꢀHRMS calcd. for C8H15FNO8P ([M ꢀ H] )
302.0447, found 302.0440
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Chemical synthesis of disodium uridine 5'-(2-acetamido-2,4-dideoxy-4-fluoro-α-D galacto-/
gluco-pyranosyl) diphosphate (10a/10b)
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A solution of monoꢀphosphate 9a/9b (4 mg, 0.013 mmol) in MeOH (2 mL) was treated with
Et3N (9 µL, 0.07 mmol), and then concentrated under vacuum to yield crude
bis(triethylammonium) phosphate. Without purification, this crude material was coꢀevaporated
with dry pyridine (3 x 3 mL). Uridine 5′ꢀmonophosphomorpholidate 4ꢀmorpholineꢀN, N′ꢀ
dicyclohexylcarboxamidine salt (14 mg, 0.02 mmol) was coꢀevaporated with pyridine (3 x 3
mL), then transferred via a cannula into the reaction flask. The combined reagents were coꢀ
evaporated with pyridine (2 x 2 mL) then pyridine (2 mL), 1Hꢀtetrazole (4 mg, 0.05 mmol) were
added and the reaction mixture stirred at room temperature for 3 d. The reaction mixture was
then concentrated under vacuum and was converted into Na+ form by passing through a Dowex
(Na+) column. The resulting fraction was concentrated under vacuum and the residue was loaded
onto a BioꢀGel P2 column (1 × 120 cm) and eluted with H2O. Fractions were collected, and those
containing the product as determined by MS were combined and freezeꢀdried to afford 4Fꢀ
GlcNAc/GalNAcꢀUDP as white powders (5 mg, 61%).
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Disodium uridine 5'-(2-acetamido-2,4-dideoxy-4-fluoro-α-D galactopyranosyl) diphosphate
(10a)
White solid. 1H NMR (800 MHz, D2O) δ: 7.83 (d, J = 7.72, 1H, uridineꢀH''ꢀ6), 5.87ꢀ5.81 (m,
2H, uridineꢀH''ꢀ5, ribꢀH'ꢀ1), 5.39 (br, 1H, Hꢀ1), 4.35 (dt, J = 8.93, 50.75, 1H, Hꢀ4), 4.26ꢀ4.20 (m,
2H, ribꢀH'ꢀ2, ribꢀH'ꢀ5a), 4.18ꢀ4.10 (m, 2H, ribꢀH'ꢀ3, ribꢀH'ꢀ4), 4.09ꢀ4.03 (m, 1H, ribꢀH'ꢀ5b), 4.01ꢀ
3.91 (m, 3H, Hꢀ2, Hꢀ3, Hꢀ5), 3.75 (d, J = 12.12, 1H, Hꢀ6a), 3.68 (d, J = 12.12, 1H, Hꢀ6b), 1.95
(s, 3H, HꢀAc); 13C NMR (200 MHz, D2O) δ 174.7, 166.4, 151.7, 141.5, 102.6, 94.1, 88.7 (d, J =
179.60), 88.5, 83.1 (d, J = 9.50), 73.8, 70.4 (d, J = 25.0), 69.5, 69.2 (d, J = 18.56), 64.9 (d, J =
5.14), 59.6, 53.2, 22.0; 31P NMR (202.5 MHz, D2O) δ ꢀ11.5, ꢀ13.3; 19F NMR (470 MHz, D2O) δ
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ꢀ163.4 (dd, J = 14.1, 51.7). ESIꢀHRMS calcd. for C17H24FN3O16P2Na2 ([M ꢀ 2NaꢀH] )
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608.0700, found 608. 0720
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Disodium uridine 5'-(2-acetamido-2,4-dideoxy-4-fluoro-α-D glucopyranosyl) diphosphate
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(10b)
White solid. 1H NMR (800 MHz, D2O) δ: 7.87 (d, J = 8.15, 1H, uridineꢀH''ꢀ6), 5.90ꢀ5.85 (m,
2H, uridineꢀH''ꢀ5, ribꢀH'ꢀ1), 5.43ꢀ5.41 (m, 1H, Hꢀ1), 4.39 (dt, J = 9.49, 50.95, Hꢀ4), 4.29ꢀ4.25
(m, 2H, ribꢀH'ꢀ2, ribꢀH'ꢀ5a), 4.21ꢀ4.18 (m, 1H, ribꢀH'ꢀ3,), 4.18ꢀ4.14 (m, 1H, ribꢀH'ꢀ4), 4.12ꢀ4.08
(m, 1H, ribꢀH'ꢀ5b), 4.04ꢀ3.95 (m, 3H, Hꢀ2, Hꢀ3, Hꢀ5), 3.81ꢀ3.76 (m, 1H, Hꢀ6a), 3.74ꢀ3.70 (m, 1H,
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Hꢀ6b), 1.99 (s, 3H, HꢀAc); C NMR (200 MHz, D2O) δ 174.8, 166.2, 151.8, 141.6, 102.6, 94.2
(d, J = 5.85), 88.8 (d, J = 182.54), 88.5, 83.1 (d, J = 9.25), 73.8, 70.4 (d, J = 24.58), 69.5, 69.2
(d, J = 18.56), 65.0, 59.6, 53.2, 22.0; P NMR (202.5 MHz, D2O) δ ꢀ11.5, ꢀ13.3; 19F NMR (470
31
ꢀ
MHz, D2O) δ ꢀ198.4. ESIꢀHRMS calcd. for C17H24FN3O16P2Na2 ([M ꢀ 2NaꢀH] ) 608.0700,
found 608. 0714
General Procedure for the Enzymatic Preparation of the UDP-Sugar Nucleotides.
A
reaction
mixture
containing
the
monoꢀphosphate,
glucosamineꢀ1ꢀphosphate
acetyltransferase/Nꢀacetyl glucosamineꢀ1ꢀphosphate uridylyltransferase (GlmU), TrisꢀHCl (46
mM, pH 7.0), MgCl2 (5 mM), dithiothreitol (200 ꢁM), UTP (2.5 mM), and inorganic
pyrophosphatase (0.012 units/ꢁL), from SigmaꢀAldrich, was incubated at 30 °C for 3 h. For
GalNAc substrates, a second portion of GlmU was added to the reaction mixture on the second
day. The reaction was monitored through MS. Then three reaction volumes of ethanol was
added to quench the reaction and stored overnight at −20 °C. Centrifugation at 3000 rcf for 30
min afforded the crude product in the supernatant which was then concentrated and further
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purified on a BioGel (BioꢀRad) P2 column (1 × 100 cm) and eluted with H2O. Fractions were
collected, and those containing the product as determined by UV absorbance at 262 nm were
combined and freezeꢀdried to afford the pure product. Characterization data were consistent with
chemically synthesized products.
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Expression of GlmU Enzyme.
The procedures for the expression and purification of GlmU were described previously. Briefly,
GlmU was cloned into a PET 21b vector (Novagen) to form a Cꢀterminal (His)6ꢀtagged fusion
protein. The expression was carried out in BL21 star cells (Invitrogen). The protein was purified
by a NiꢀSepharose 6 Fast Flow column (GE Health) following a standard procedure.
Polymerization Assays.
To assess if the analogs would serve as inhibitors or terminators of the GAG synthases, various
protocols were used to detect if the reagents could slow polymerization rates or block elongation
of nascent chains. For the kinetic experiments, each of the fluorineꢀcontaining UDPꢀhexosamine
analogs (0.5 mM) was coꢀincubated in a reaction containing radiolabeled UDPꢀ[3H]GlcA (0.05ꢀ
0.5 mM; 0.1 µCi; PerkinꢀElmer), with or without the addition of the authentic UDPꢀ
hexosamine. Reactions (25 µl) also contained 50 mM Tris, pH 7.2, and 1 mM MnCl2 with 0.35ꢀ
25 µg 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 10 minꢀ24 h. In other control reactions,
UDP, the byꢀproduct of GAG polymerization and a part of the analog structure, was employed to
assess for any inhibitorꢀlike effects. The reaction mixtures were quenched with sodium dodecyl
sulfate detergent (2% final conc) and analyzed by descending paper chromatography (overnight
in 65:35 ethanol/1 M ammonium acetate buffer, Whatmann 3MM paper). As a negative control
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for assay background, a reaction with no UDPꢀhexosamine was tested in parallel (GAG chain
polymerization can only occur when UDPꢀGlcUA and a functional UDPꢀhexosamine are present
simultaneously).
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To assess the analogs’ termination functionality (i.e. stop GAG chain growth), a twoꢀstep
assay was used with unlabeled UDPꢀsugars and enzyme (6ꢀ10 µg). First, a quasiꢀmonodisperse
hyaluronan (for PmHAS and PmCS) or heparosan (PmHS1) polysaccharide acceptor (each ~12
kDa) was capped by reacting it with an excess (1.1 to 10 molar excess over acceptor) of the
various UDPꢀhexosamine analogs (or no analog) for 2 h at 30°C. Second, both the authentic
UDPꢀsugars (8 mM each UDPꢀGlcA and the natural UDPꢀhexosamine) were added and the
reactions were continued overnight. If the acceptor had been capped with an unextendable 4ꢀF
group, then the polysaccharide acceptor’s size would not increase. The polysaccharide products
were analyzed on agarose gels with StainsꢀAll staining.23
FACE Electrophoresis.
To confirm that the analogs were indeed being added to the GAG acceptor, a heparosan
trisaccharide was labeled with AMAC (2ꢀaminoacridone) to facilitate fluorescent visualization of
the extended oligosaccharides and polysaccharides.24 First, the AMACꢀlabeled oligosaccharide
was capped by reacting it with a 10ꢀfold molar excess of the either the fluorineꢀcontaining analog
or UDPꢀGlcNAc over acceptor or no analog in a buffer containing 50 mM Hepes, pH 7.2, 1 mM
MnCl2, and purified recombinant PmHS1 (5 µg) for 9 h at 30°C. Second, both of the authentic
UDPꢀsugars required for polymerization were added (final 1.6 mM each) to a portion of these
reactions and the incubation was continued overnight. The products of each step were analyzed
on a 21% 1X TAE polyacrylamide gel run in TrisꢀTricine buffer and visualized by UV
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fluorescence.
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Mass Spectroscopy.
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Heparosan oligosaccharides (mixture of 4 and 6 sugars long; 1 µg) were reacted with a 10ꢀfold
molar excess of either UDPꢀGlcNAc, the fluorineꢀcontaining analog, or no analog in a buffer
containing 50 mM Tris, pH 7.2, 1 mM MnCl2, and purified recombinant PmHS1 or PmHS2 (5
µg) for 5.5 h at 30°C. The reactions were assessed by MALDIꢀToFꢀMS, with the matrix 6ꢀazaꢀ
2ꢀthiothymine at a concentration of 5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA)
(REF 3). HA oligosaccharides were employed as mass calibrants (Hyalose, LLC, Oklahoma
City, OK).
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Supporting Information Available: H, C, F and P NMR spectra of the components are
available free of charge via the Internet at http://pubs.acs.org
ACKNOWLEDGEMENTS
The authors are grateful for funding from the National Institutes of Health in the form of grants
HL62244 and HL094463
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