Synthesis of a benzene-containing C1-phosphonate analogue of UDP-GlcNAc for the inhibition of O-GlcNAc transferase

Article abstract of DOI:10.1002/bkcs.10609

I report here the design, synthesis, and biological evaluation of a new C1-phosphonate analogue of UDP-GlcNAc as a potential inhibitor of OGT, an enzyme responsible for O-GlcNAc modification. The analogue was designed to mimic the transition state of the

Full text of DOI:10.1002/bkcs.10609

Article
DOI: 10.1002/bkcs.10609
BULLETIN OF THE
J. Im
KOREAN CHEMICAL SOCIETY
Synthesis of a Benzene-containing C1-Phosphonate Analogue of
UDP-GlcNAc for the Inhibition of O-GlcNAc Transferase
*
Jungkyun Im
Department of Nanochemical Engineering, Soonchunhyang University, Asan 336-745, Korea.
*E-mail: jkim5279@sch.ac.kr
Received May 7, 2015, Accepted September 7, 2015, Published online December 17, 2015
I report here the design, synthesis, and biological evaluation of a new C1-phosphonate analogue
of UDP-GlcNAc as a potential inhibitor of OGT, an enzyme responsible for O-GlcNAc modification. The
analogue was designed to mimic the transition state of the natural donor involved in the enzymatic reaction.
However, the analogue showed somehow low activity as an inhibitor of OGT.
Keywords: O-GlcNAc transferase, N-Acetylglucosamine, OGT inhibition, UDP-GlcNAc,
Phosphonate bond
temporarily with a trigonal planar structure. In an
attempt tomimic this transition structure of the carbohy-
drate, the sugar ring was replaced with benzene substi-
tuted by N-acetyl and one hydroxyl group at positions
C2 and C5, respectively. Thereby, such a transition state
analogue could bind effectively to the catalytic region
rather than either the substrate or the product, and be
a strong inhibitor as a result.7
Introduction
Post-translational modification of nuclear and cytoplasmic
proteins with O-linked β-N-acetylglucosamine (O-GlcNAc)
is a ubiquitous process that occurs within all cells of higher
eukaryotes.1 The attachment sites of O-GlcNAc are serine
and threonine residues in proteins, which are sometimes also
O-phosphorylated, making both processes reciprocal.2 Many
proteins, including structural proteins such as nuclear pore
proteins and cytoskeletal proteins, transcription factors such
as the glucose-responsive factor Sp1, and important proces-
sing enzymes such as the proteasome are modified by
O-GlcNAc addition.3 In accord with the abundance of cellular
targets, O-GlcNAc is thought to play an important role in
nuclear transport, cell growth, apoptosis, signaling, and so
on.4 Consistent with its important role, aberrant O-GlcNAc
modification is associated with human diseases including dia-
betes, cancer, and neurodegenerative diseases such as Alzhei-
mer’s disease.5
2. Analogues wherein the glycosidic oxygen of the nucle-
otide diphosphate (NDP) sugar is substituted by a meth-
ylene group to generate a phosphonate bond have
occasionally been used as glycosyltransferase inhibi-
tors.8 Compared to O-glycosides, C-glycosyl phospho-
nate derivatives have been reported to tolerate chemical
degradation and enzyme-catalyzed cleavage well.9 The
phosphonate group in the inhibitor was expected to
favor the recognition by OGT.
By mimicking both the carbohydrate part and diphosphate
linkage of the natural substrate, I synthesized 1 as an inhibitor,
evaluated its inhibitory activity; results are reported below.
Theenzyme thattransfers GlcNAc isO-GlcNAc transferase
(OGT), and the donor substrate is uridine 50-diphosphate
(UDP)-GlcNAc. To further investigate the biological roles
of O-GlcNAc and the structure of OGT at a molecular level,
a potential inhibitor of OGT could be a pertinent molecular
probe. Although many small-molecule inhibitors of OGT
were previously reported, many of them had limitation in inhi-
bition activity, solubility issues, cellular internalization,
in vivo applications, and so on.6 This makes the design of
OGT inhibitors challengeable. Thus, in my efforts to design
a new inhibitor, an analogue of UDP-GlcNAc was chosen
as the synthetic target and the following structural features
were considered.
Results and Discussion
Our retrosynthetic direction is highlighted in Scheme 1.
Although the reaction yield is generally low, NDP sugars
are mostly built up by coupling between the sugar phosphate
and nucleotide monophosphates activated as morpholidates.10
Because of the commercial availability of uridine 50-
monosphosphomorpholidate (UMP-morpholidate), 8 should
be synthesized as a reaction counterpart. In general, a phos-
phonate can be achieved by the Michaelis–Arbuzov reaction,
which requires an alkyl halide group. In addition, an aromatic
amine can be obtained from aromatic nitro group reduction;
the reduction of ester by metal hydride will provide 8. How-
ever, the reduction should be performed separately because
treatment of an aromatic nitro compound with a metal hydride
could yield an azo compound. Therefore, 8 can be prepared in
1. Our design was based on a proposed model of the tran-
sition state for the OGT reaction (Figure 1). In the SN2-
type transition state, the anomeric carbon of GlcNAc
bears a partial positive charge and becomes sp2-carbon
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Article
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ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
‡
δ
δ
δ
δ
Figure 1. The possible transition state in OGT-mediated transfer of
GlcNAc from UDP-GlcNAc.
Scheme 2. Reagents and conditions: (a) conc. H2SO4, MeOH, reflux,
overnight, 37%; (b) NBS, (BzOꢀ)2, CCl4, reflux, 16 h, 48%;
(c) triethylphosphite, toluene, 115 C, 28 h, 75%; (d) H2 (50 psi),
Pd/C, MeOH, 4 h, 89%; (e) (Ac)2O, pyridine, room temperature,
overnight, 73%; (f ) LiBH4, THF, room temperature, 3 days, 73%.
Catalytic hydrogenation using palladium on carbon reduced
the aromatic nitro group into aniline derivative 6. After acet-
ylation of the amine, the ester group of 7 was reduced by lith-
ium borohydride (LBH) in THF to provide 8 at a yield of 73%
(Scheme 2).
The hydroxyl group of 8 was acetylated initially before the
next step, since deprotection of the diethylphosphonate
always resulted in a very poor yield in the presence of the alco-
hol group. Treatment of 9 with bromotrimethylsilane in
CH2Cl2 followed by deacetylation in a mixture solution
(MeOH/H2O/Et3N) and purification by MPLC on a C8-
reverse phase column afforded fully deprotected phosphonic
acid 10 at 63% yield. Coupling of 10 to UMP-morpholidate
was performed with 1H-tetrazole asa catalyst in dried pyridine
following the reported procedures,10 and as expected, desired
product 1 was obtained as a triethylammonium salt at 60%
yield after sequential purification using MPLC and C18 RP-
HPLC (Scheme 3). Its structure was fully characterized by
Scheme 1. Retrosynthetic overview of 1.
fourstepsfromthekey intermediate 4. Iconsidered 2asuitable
starting material for 4, as 2 is commercially available and
cheap.
Esterification of 2 was carried out in acidic conditions using
methanol as a solvent as well as reagent. Compound 3 was
converted into alkyl bromide 4 in 48% yield with
N-bromosuccinimide (NBS) in the presence of benzoyl perox-
ide in refluxing anhydrous CCl4. Dibromo compound at 34%
yield was a byproduct. The resulting bromide 4 was reacted
with triethylphosphite in toluene and purified to give 5.
1
high-resolution mass spectroscopy, and H, 13C, and 31P
spectra.
Next, the in vitro inhibitory activity of 1 against OGT was
examined. Inhibition of OGT activity was determined by
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BULLETIN OF THE
Article Synthesis of a Benzene-containing C1-Phosphonate Analogue of UDP-GlcNAc KOREAN CHEMICAL SOCIETY
Scheme3. Reagentsandconditions: (a)(Ac)2O, pyridine, roomtemperature, overnight, 83%;(b)TMSBr, CH2Cl2, room temperature, overnight,
MeOH; (c) MeOH–H2O–Et3N (7:3:1), room temperature, overnight, 63%; (d) UMP–morpholidate, 1H-tetrazole, pyridine, room temperature,
7 days, 60%. Purified by RP-HPLC (GRACEVYDAC, C18 column, 0.1M TEAA buffer:CH3CN = 90:10, gradient).
quantitating UDP-GlcNAc incorporation into a protein sub-
strate in the absence or presence of several concentrations
of 1. Purified casein kinase II (CKII) was used as the acceptor
of GlcNAc, because it is a well-known substrate for
O-GlcNAc assays.11 Incubation with OGT was conducted
for a fixed time (typically 1 h), and the mixture was separated
and analyzed by Western blotting using a mouse monoclonal
O-GlcNAc antibody (CTD110.6) (Figure 2).
Somewhat unexpectedly, analogue 1 turned out to be a poor
inhibitor of nucleocytoplasmic OGT (ncOGT) with an IC50 of
more than 5 mM. It may be that employing benzene instead of
a sugar ring to mimic the enzyme-bound transition state was
not sufficient for inhibition. Despite the fact that the formal
charge of the analogue was not altered by the phosphonate
bond, the C(1) O P linkage in the natural substrate could
be very important for the enzyme recognition, because the
phosphonate group may perturb the pKa of the neighboring
phosphate group. Other reported analogues of UDP-GlcNAc
also showed moderate inhibitory activity, which indicates that
the active site of OGT has very little spatial tolerance.12
Figure 2. Inhibition activity test toward OGT by the analogue 1.
(a) Western blot showing the degree of O-GlcNAc modification to
the substrate protein(CKII), and anti CKII, anti FLAG bands for cal-
ibration, (b) the relative intensity of anti O-GlcNAc band in (a).
Conclusion
In conclusion, I designed and synthesized an analogue of
UDP-GlcNAc, 1, to inhibit OGT. The analogue contained a
substituted benzene to mimic the sp2 carbon of the carbohy-
drate ring in the transition state. A phosphonate linkage was
also introduced in the form of an isosteric analogue of
UDP-GlcNAc. Nevertheless, our synthetic analogue had no
distinct activity as an inhibitor of OGT, indicating that the
active site of OGT is very inflexible; even a subtle change
in the analogue was detectable by the OGT assay.
preparation, compounds were dissolved in appropriate sol-
vents such as dichloromethane or chloroform and a thin film
of the solution was spread over a polished NaCl salt plate.
Low-resolution mass spectra were recorded on a micromass
PLATFORM II spectrometer under fast atom bombardment
(FAB) conditions. In case of high-resolution mass spectral
data, HR-FABMS were obtained on a Jeol (Seoul, Korea)
JMS 700 high-resolution mass spectrometer at the Korea
Basic Science Institute (Daegu), and MALDI-HRMS on a
Micromass M@DI at the Biomolecular Diversity Core Facil-
ity (POSTECH). Analytical HPLC was performed on Agilent
(Seoul, Korea) 1100-HPLC ChemStation with an analytical
column Agilent ZORBAX SB-C18 (5 μm, 80 Å,
4.6 × 250 mm), and prep HPLC on Amersham Pharmacia
(Daejeon, Korea)AKTABasic10withasemi-preparative col-
umn GRACEVYDAC C18 (5 μm, 300 Å, 10 × 250 mm).
3-Methyl-4-nitrobenzoic acid methylester (3): To a solu-
tion of 2 (3-methyl-4-nitrobenzoic acid, 4.53 g, 29.98 mmol)
Experimental
General Methods for Synthesis. Column chromatography
was performed on Merck 60 silica gel (70–230 or 230–400
mesh), and MPLC on Fluka (Yongin, Korea) 100 C8-reversed
phase silica gel. NMR spectra were recorded on a Bruker
(MA, USA) ASPECT 300 instrument operating at 300 MHz
for 1H, 75 MHz for 13C and 121 MHz for 31P. IR spectra were
recorded on a BOMEM (Seoul, Korea) FT-IR M100 C15
spectrometer and values are reported in cm−1. In IR sample
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BULLETIN OF THE
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in MeOH (25 mL) was added conc. H2SO4 (97%, 2.66 mL,
48.41 mmol) at room temperature. The turbid reaction mixture
became clear during heating at reflux for overnight and then it
was cooled to room temperature to be a suspension. The pre-
cipitates were filtered and washed with cold MeOH. Recrys-
tallization in MeOH, filtration and drying then gave 3 (1.67
g, 37%) as a yellow crystalline solid. Rf: 0.5 (EtOAc:Hex =
62.70 (d, J = 6.60Hz, OCH2), 125.44 (d, J = 2.85 Hz),
127.87 (d, J = 9.90 Hz), 129.28 (d, J = 3.45 Hz), 134.03 (d,
J = 3.08 Hz), 134.58 (d, J = 5.93 Hz), 151.99 (d, J = 6.38
Hz) [aromatic carbons], 165.13 (C O); 31P NMR
(in CDCl3, δ) 24.38; HR-FABMS [M+H]+ calcd for
C13H19NO7P m/z 332.0899, found 332.0896.
4-Amino-3-(diethoxy-phosphorylmethyl)-benzoic acid
methylester (6): A mixture of 5 (0.74 mg, 2.24 mmol) and
Pd/C 10% (200 mg) in MeOH (26 mL) was stirred under H2
(50 psi) for 4 h. The reaction mixture was filtered over a short
pad of celite and washed with MeOH. Evaporation gave the
amino compound 6 (0.60 mg, 89%) as a yellow sticky solid.
Rf: 0.3 (EtOAc:Hex = 2:1); IR (film, cm−1) 3356 and 3242
(N H), 2983, 2952 and 2928 ( C H), 1708 (C O), 1606
(N H, bending), 1577 and 1430 (Ar), 1277 (P O), 1025
−1
ꢀ
1:5); m.p. 79.5–80.0 C; IR (film, cm ) 3118 and 3046
( C H), 2959 ( C H), 1732 (C O), 1585 (Ar), 1520
1
(N O), 1429 (Ar), 1344 (N O); H NMR (in CDCl3, δ)
2.63 (s, 3H, CCH3), 3.96 (s, 3H, OCH3), 7.98 (s, 2H), 8.03
(s, 1H) [aromatic protons]; 13C NMR (in CDCl3, δ) 20.30
(CCH3), 52.94 (OCH3), 124.80, 128.28, 133.70, 133.96,
134.23, 152.10 (aromatic carbons), 165.56 (C O); HR-
FABMS [M+H]+ calcd for C9H10NO4 m/z 196.0610, found
196.0608.
1
(P OEt); H NMR (in MeOD, δ) 1.29 (t, 6H, J = 7.04 Hz,
CH2CH3), 3.22 (d, 2H, J = 21.01 Hz, CH2P), 3.85 (s, 3H,
OCH3), 4.04–4.13 (m, 4H, OCH2), 6.77 (d, 1H, J =
8.48Hz), 7.70 (ddd, 1H, J = 8.47 and 2.04 Hz), 7.80 (dd,
1H, J = 2.37Hz) [aromatic protons]; 13C NMR (in MeOD,
δ) 16.76 (d, J = 5.93Hz, CH2CH3), 29.71 (d, J = 138.83 Hz,
CH2P), 64.01 (d, J = 6.98 Hz, OCH2), 116.09 (d, J = 9.38
Hz), 116.29 (d, J = 2.25 Hz), 119.68 (d, J = 2.78 Hz),
131.25 (d, J = 2.85 Hz), 134.94 (d, J = 6.45 Hz), 153.12 (d,
J = 4.80 Hz) [aromatic carbons], 169.04 (C O); 31P NMR
(in MeOD, δ) 28.64; HR-FABMS [M+H]+ calcd for
C13H21NO5P m/z 302.1157, found 302.1158.
3-Bromomethyl-4-nitrobenzoic acid methylester (4): To
a solution of 3 (1.33 g, 7.43 mmol) in anhydrous CCl4 (34 m:)
were added benzoyl peroxide (200 mg, 0.83 mmol) and NBS
(1.98 g, 11.14 mmol) at room temperature. The reaction mix-
turewasheated atrefluxfor16handthenitwascooled toroom
temperature. After filtration and washing with hexane, the fil-
trate was concentrated in vacuo and the obtained residue was
column chromatographed on silica gel to give both 4 (0.98 g,
48%) as a white solid, and a byproduct, 3-dibromomethyl-4-
nitrobenzoic acid methylester (0.89 g, 34%) as a white solid.
ꢀ
Rf: 0.4 (EtOAc:Hex = 1:10); m.p. 117.0–117.5 C; IR (film,
cm−1) 3124 and 3053 ( C-H), 3002 and 2952 ( C H),
1722 (C O), 1586 (Ar), 1529 (N O), 1442 (Ar), 1356
4-Acetylamino-3-(diethoxy-phosphorylmethyl)-
benzoic acid methyl ester (7): To a solution of 6 (0.45 mg,
1.50 mmol) in dried pyridine (10 mL) was added acetic anhy-
dride(0.60mL, 6.39mmol)atroomtemperature. Afterstirring
for overnight, the reaction mixture was diluted and neutralized
by EtOAc and aq. HCl (1 N), respectively. Organic layer was
washed by HCl (1 N) twice, aq. NaHCO3, and brine succes-
sively, and then dried with MgSO4. Filtration and condensa-
tion of the organic layer in vacuo gave a crude product,
which was purified by column chromatography on silica gel
to yield 7 (0.37 mg, 73%) as colorless oil. Rf: 0.5 (EtOAc:
Hex = 2:1); IR (film, cm−1) 3251 (N H), 3109 ( C H),
2982, 2953 and 2924 ( C H), 1716 (C O, ester), 1668
(C O, amide), 1590 and 1436 (Ar), 1285 (P O), 1024
1
(N O), 1277 (CH2 Br, bending), 624 (C Br); H NMR
(in CDCl3, δ) 3.99 (s, 3H, OCH3), 4.83 (s, 2H, CH2Br),
8.06 (d, 1H, J = 8.43 Hz), 8.13 (dd, 1H, J = 8.49 and 1.77
Hz), 8.24 (d, 1H, J = 1.59Hz) [aromatic protons]; 13C NMR
(in CDCl3, δ) 28.09 (CBr), 53.17 (OCH3), 125.80, 130.83,
133.28, 133.95, 134.80, 150.72 (aromatic carbons), 164.85
(C O); HR-FABMS [M+H]+ calcd for C9H9BrNO4 m/z
273.9715, found 273.9711.
3-(Diethoxy-phosphorylmethyl)-4-nitrobenzoic
acid
methylester (5): To a solution of 4 (0.85 g, 3.11 mmol) in tol-
uene (40 mL) was added triethylphosphite (2.40 mL,
13.72 mmol) at room temperature. The reaction mixture was
ꢀ
1
heated at 115 C for 28 h and then it was cooled to room tem-
(P OEt); H NMR (in CDCl3, δ) 1.25 (t, 6H, J = 7.02Hz,
perature. The solution was concentrated in vacuo by rotꢀary
evaporation and kugelrohr bulb-to-bulb distillation at 70 C.
The product was purified by column chromatography on silica
gel to give 5 (0.77 g, 75.10%) as a yellow oil. Rf: 0.5 (EtOAc:
Hex = 2:1); IR (film, cm−1) 3111 and 3044 ( C H), 2956 and
2908 ( C H), 1729 (C O), 1586 (Ar), 1533 (N O), 1438
CH2CH3), 2.23 (s, 3H, C OCH3), 3.19 (d, 2H, J = 21.12
Hz, CH2P), 3.90 (s, 3H, OCH3), 3.95–4.08 (m, 4H, OCH2),
7.86 (s, 1H), 7.90 (d, 1H, J = 8.52 Hz), 8.01 (d, 1H, J =
8.55 Hz) [aromatic protons]; 13C NMR (in CDCl3, δ) 16.48
(d, J = 5.70 Hz, CH2CH3), 24.55 (OCH3), 31.52 (d, J =
137.18 Hz), 52.28 (OCH3), 63.28 (d, J = 6.98Hz, OCH2),
122.79 (d, J = 9.15 Hz), 124.46 (d, J = 7.35 Hz), 141.57 (d,
J = 4.50 Hz) [aromatic carbons], 166.58 (NC O), 169.37
(OC O); 31P NMR (in CDCl3, δ) 28.64; HR-FABMS [M
+H]+ calcd for C15H23NO6P m/z 344.1263, found 344.1267.
Diethyl-(2-acetylamino-5-hydroxymethyl-benzyl)-
1
(Ar), 1357 (N O), 1271 (P O), 1024 (P OEt); H NMR
(in CDCl3, δ) 1.25 (t, 6H, J = 7.08 Hz, CH2CH3), 3.72 (d,
2H, J = 22.56 Hz, CH2P), 3.97 (s, 3H, OCH3), 3.99–4.10
(m, 4H, OCH2), 7.98 (d, 1H, J = 8.49 Hz), 8.06 (ddd, 1H,
J = 8.49 and 1.98 Hz), 8.13 (dd, 1H, J = 2.04 Hz) [aromatic
protons]; 13C NMR (in CDCl3, δ) 16.44 (d, J = 5.93Hz,
phosphonate (8): A mixture of 7 (0.36 mg, 1.03 mmol) and
LiBH4 (4 mL, 2.0 M in THF) was stirred for 3 days at room
CH2CH3), 30.70 (d, J = 136.51 Hz, CH2P), 52.97 (OCH3),
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BULLETIN OF THE
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temperature. To the reaction mixture was dropwise added
aq. NH4Cl at 0 C until bubble formation ceased. The result-
excess reagent were evaporated twice with toluene, and the
residue was dissolved in MeOH (2 mL), and treated with
few drops of H2O at room temperature. After stirring for
10 h, the solution was condensed to dryness twice with tolu-
ene. To the resulting residue was added a mixture solution,
MeOH–H2O–Et3N (2 m:, 7:3:1), and the reaction mixture
was stirred for overnight at room temperature. After concen-
tration to dryness, the residue was purified by using MPLC
on reverse phase C8 silica gel (0.1 M triethylammonium ace-
tate [TEAA, pH = 7.01]:CH3CN = 100:0 to 90:10) to give 10
(40.50 mg, 63%) as a white sticky solid. 1H NMR (in D2O, δ)
ꢀ
ing suspension was diluted with EtOAc and then washed with
aq. NH4Cl and brine. The organic layer was dried with
MgSO4, filtered and condensed in vacuo to give the crude
product, which was purified by column chromatography on
silica gel to yield 8 (0.24 mg, 73%) as colorless oil. Rf: 0.5
(CH2Cl2:MeOH = 10:1); IR (film, cm−1) 3297 (O H, broad),
3120 ( C H), 2983, 2925 and 2854 ( C H), 1673 (C O),
1595 and 1442 (Ar), 1282 (P O), 1025 (P OEt); 1H NMR
(in CDCl3, δ) 1.24 (t, 6H, J = 7.03 Hz, CH2CH3), 2.20 (s,
3H, C OCH3), 3.13 (d, 2H, J = 21.15 Hz, CH2P),
3.93–4.08 (m, 4H, OCH2), 4.62 (s, 2H, CH2OH), 7.17 (s,
1H), 7.24 (d, 1H, J = 8.20 Hz), 7.73 (d, 1H, J = 8.22Hz) [aro-
matic protons]; 13C NMR (in CDCl3, δ) 16.50 (d, J = 5.78Hz,
1.16 (t, 9H, J = 7.31 Hz, CH2CH3), 2.11 (s, 3H, C OCH3),
2.92 (d, 2H, J = 20.43 Hz, CH2P), 3.08 (q, 6H, J = 7.30Hz,
NCH2), 4.51 (s, 2H, OCH2), 7.18 (d, 1H, J = 7.96 Hz), 7.23
(s, 1H), 7.29 (d, 1H, J = 8.08 Hz) [aromatic protons]; 13C
NMR (in D2O, δ) 7.75 (NCH2CH3), 22.05 (C OCH3),
31.90 (d, J = 126.68 Hz, CH2P), 46.20 (NCH2), 62.99
(OCH2), 125.53 (d, J = 3.15Hz), 126.11 (d, J = 2.63 Hz),
129.73 (d, J = 5.63 Hz), 129.96 (d, J = 9.00 Hz), 133.52 (d,
J = 5.85 Hz), 138.42 (d, J = 2.93 Hz) [aromatic carbons],
172.90 (C O); 31P NMR (in D2O, δ) 21.53; MALDI-HRMS
[M+Na]+ calcd for C10H14NO5PNa m/z 282.0507, found
282.0566; Analytical HPLC (ZORBAX SB-C18): Rt =
10.21 min (flow rate = 1 mL/min, UV 220 nm, 0.1 M
TEAA:CH3CN = 90:10, gradient).
OCH2CH3), 24.33 (C OCH3), 31.37 (d, J = 137.03 Hz,
CH2P), 63.13 (d, J = 6.98 Hz, OCH2CH3), 64.71 (CH2OH),
123.76 (d, J = 9.45 Hz), 125.92 (d, J = 3.23 Hz), 126.82 (d,
J = 3.68 Hz), 129.95 (d, J = 7.05 Hz), 126.23 (d, J = 5.03
Hz), 138.42 (d, J = 2.70 Hz) [aromatic carbons], 169.41
(C O); 31P NMR (in CDCl3, δ) 29.14; HR-FABMS [M
+H]+ calcd for C14H23NO5P m/z 316.1314, found 316.1312.
Diethyl-(2-acetylamino-5-acetoxymethyl-benzyl)-
phosphonate (9): To a solution of 8 (0.15 mg, 0.46 mmol) in
dried pyridine (4mL) was added acetic anhydride (0.20 mL,
2.13 mmol) at room temperature. After stirring for overnight,
thereaction mixture wasdiluted and neutralized byEtOAc and
aq. HCl (1 N), respectively. Organic layer was washed by HCl
(1 N)two times, aq. NaHCO3, and brine successively, andthen
dried with MgSO4. Filtration and condensation of the organic
layer in vacuo gave the crude product, which was purified by
column chromatography on silica gel to yield 9 (137 mg, 83%)
as colorless oil. Rf: 0.7 (EtOAc:MeOH = 20:1); IR (film, cm−1)
3263 (N H), 3116 ( C H), 2984 and 2932 ( C H), 1739
(C O, ester), 1694 (C O, amide), 1596 and 1443 (Ar), 1282
(P O), 1229 (OC O), 1026 (P OEt); 1H NMR (in CDCl3, δ)
Uridine 50-{[(2-acetylamino-5-hydroxymethyl-benzyl)-
phosphono]phosphate}, bistriethylammonium salt (1): A
mixture of 10 (9.8 mg, 27.19 μmol) and 4-morpholine-N,N0-
dicylcohexylcarboxamidinium uridine 50-monophosphomor-
pholidate (33.70 mg, 49.08 μmol) was coevaporated thrice
with dried pyridine. To the residue were added dried pyridine
(0.4ml)and1H-tetrazole (0.1 ml, ~0.45MinCH3CN), andthe
resulting solution was stirred for 7 days at room temperature.
The solution was treated with few drops of H2O and concen-
trated in vacuo several times with toluene. The residue was
purified sequentially by using MPLC and RP-HPLC
(GRACEVYDAC, C18) (0.1 M TEAA buffer; gradient,
0–10% CH3CN over 30 min) to give 1 (13.0 mg, 60%) as a
1.24 (t, 6H, J = 7.00 Hz, CH2CH3), 2.08 (s, 3H, OC OCH3),
2.21 (s, 3H, NC OCH3), 3.14 (d, 2H, J = 21.14 Hz, CH2P),
3.97–4.06 (m, 4H, OCH2CH3), 5.05 (s, 2H, CH2OC O), 7.15
(s, 1H), 7.29 (d, 1H, J = 6.70 Hz), 7.80 (d, 1H, J = 8.23 Hz)
[aromatic protons], 9.54 (s, 1H, NH); 13C NMR (in CDCl3, δ)
16.44 (d, J = 5.85Hz, CH2CH3), 21.13 (OC OCH3), 24.36
1
white sticky solid. H NMR (in D2O, δ) 1.26 (t, 18H, J =
7.32 Hz, NCH2CH3), 2.20 (s, 3H, C OCH3), 3.13–3.22 (m,
14H, NCH2 and CH2P), 3.94–4.24 (m, 5H, CH2CHCHCH)
4.24 (s, 2H, CH2OH), 5.83 (d, 1H, J = 8.15 Hz, C OCH),
5.87 (d, 1H, J = 4.74 Hz, NCsp3H), 7.24 (d, 1H, J = 8.05
Hz), 7.32 (s, 1H), 7.36 (d, 1H, J = 7.42 Hz) [aromatic protons],
7.82 (d, 1H, J = 8.12 Hz, NCsp2H); 13C NMR (in D2O, δ) 7.74
(CH2CH3), 22.11 (C OCH3), 31.43 (d, J = 132.90 Hz,
CH2P), 46.18 (NCH2), 62.97 (CH2OH), 63.98 (d, J = 5.70
Hz, POCH2), 68.73, 73.27, 82.45 (d, J = 9.00 Hz), 88.18 (fur-
anose carbons), 101.97 (C OCsp2), 125.57 (d, J = 3.45 Hz),
126.10 (d, J = 2.48 Hz), 129.03 (d, J = 9.45 Hz), 129.89 (d, J =
5.63 Hz), 133.55 (d, J = 6.34 Hz), 138.33 (d, J = 3.08 Hz) [aro-
matic carbons], 140.91 (NCsp2H), 151.85 (NHC ON),
161.59 (Csp2HC O), 172.90 (C OCH3); 31P NMR
(in D2O, δ) −10.01 (d, 1P, J = 26.98 Hz, OPO), 14.03 (d,
1P, J = 26.98 Hz, CP); MALDI-HRMS [M+Na]+ calcd for
(NC OCH3), 31.40 (d, J = 137.02 Hz, CH2P), 63.13 (d,
J = 6.98 Hz, OCH2CH3), 65.81 (CH2OC O), 123.64 (d,
J = 9.45 Hz), 125.81 (d, J = 2.15 Hz), 128.37 (d, J = 3.60
Hz), 131.47 (d, J = 7.05 Hz), 133.02 (d, J = 2.78 Hz),
137.09 (d, J = 5.03 Hz) [aromatic carbons], 169.25
(NC O), 170.90 (OC O); 31P NMR (in CDCl3, δ) 28.94;
HR-FABMS [M+H]+ calcd for C16H25NO6P m/z 358.1420,
found 358.1418.
(2-Acetylamino-5-hydroxymethyl-benzyl)-phospho-
nate, monotriethylammonium salt (10): To a solution of
9 (63.60 mg, 0.18 mmol) in CH2Cl2 (1 mL) was added bromo-
trimethylsilane (1 mL, 7.50 mmol) at room temperature, and
the solution was stirred for overnight. The solvent and the
Bull. Korean Chem. Soc. 2016, Vol. 37, 7–12
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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BULLETIN OF THE
Article Synthesis of a Benzene-containing C1-Phosphonate Analogue of UDP-GlcNAc KOREAN CHEMICAL SOCIETY
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C19H25N3O13P2Nam/z588.0760, found588.0779;Analytical
HPLC (ZORBAX SB-C18): Rt = 2.18 min (flow rate = 1 mL/
min, UV 220 nm, 0.1 M TEAA:CH3CN = 70:30, isocratic).
In Vitro OGT Inhibition Assay. Transfection, expression,
and purification of human ncOGT were similarly performed
as previously described.11,12(d),13 Briefly, human full-length
ncOGT was cloned into the p3XFLAG-CMV-7.1 expression
vector (Sigma, Yongin, Korea). The vector encoding FLAG-
tagged ncOGT was transfected using Tranfectin (Bio-Rad,
Seoul, Korea). FLAG-tagged human ncOGT was expressed
in 293T cells and immunoprecipitated using FLAG/agarose
beads (F2426; Sigma). Purified CKII protein (2.2 μg, GlcNAc
acceptor; New England BioLabs P6010, MA, USA), ncOGT
binding beads (20 μL), UDP-GlcNAc (20 μM; Sigma), and
synthesized O-GlcNAc inhibitor were combined in assay
buffer (25 mM Hepes pH 7.0, 1 mM EDTA, 10 mM MgCl2,
ꢀ
40 μL). The reaction mixture was incubated at 37 C and
mixed gently every 10 min. After 1 h, the reaction
was quenched by adding SDS sample buffer (0.0642 M
Tris pH 6.8, 10% glycerol, 2% SDS, 0.002% BPB, 5%
β-mercaptoethanol). The solution was centrifuged, and the
supernatant was collected for SDS–PAGE. Proteins were
transferred from the SDS gel to a cassette that contained nitro-
cellulose membrane (RPN303D; Hybond ECL; Amersham
Biosciences, NJ, USA). Western blotting was then performed
with casein kinase II antibody (Sc-12738; Santa Cruz Biotech-
nology, Inc., TX, USA), O-GlcNAc monoclonal antibody
(CTD110.6; Sigma), and anti-FLAG M2 monoclonal
antibody (F3165; Sigma). For visualization, West-oneTM solu-
tion (iNtRON Biotechnology, Seongnam-si, Korea) was
sprayed over the membrane and images were analyzed by
LAS-4000 (GE Healthcare, Seoul, Korea). The density of each
band was calculated using the Multi-gauge V3.1 program
(GE Healthcare, Seoul, Korea). The relative density of each
band was considered the amount of O-GlcNAc modification
of CKIIα by ncOGT incubation. Densities of CKIIα and FLAG
bands were used as standards for relative density calculations.
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Acknowledgments. I would like to thank Dr. Sung-Kee
Chung (honorary professor, Postech) for his interest and
advices. I am thankful with much gratitude for Prof. Jin
Won Cho at Yonsei university for carrying out the inhibition
assays and the Western blot. This work was supported by the
Soonchunhyang University Research Fund (No. 20140000)
and partly supported by the Industry technology R&D pro-
gram of MOTIE/KEIT (10051080, Development of mechan-
ical UI device core technology for small and medium-sized
flexible display).
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Bull. Korean Chem. Soc. 2016, Vol. 37, 7–12
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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