SAR directed design and synthesis of novel β(1-4)-glucosyltransferase inhibitors and their in vitro inhibition studies

Article abstract of DOI:10.1016/S0968-0896(01)00371-6

This paper describes SAR directed design and synthesis of novel β (1-4)-glucosyltransferase (BGT) inhibitors. The designed inhibitors 1 5 provide conformational mimicry of the transition-state in glucosyltransfer reactions. The compounds were tested for i

Full text of DOI:10.1016/S0968-0896(01)00371-6

Bioorganic & Medicinal Chemistry 10 (2002) 1129–1136
SAR Directed Design and Synthesis of
Novel ꢀ(1-4)-Glucosyltransferase Inhibitors and
Their In Vitro Inhibition Studies
Asish K. Bhattacharya,a Florian Stolz,a Jurgen Kurzeck,b
Wolfgang Rugerb and Richard R. Schmidta,*
aFachbereich Chemie, Universitat Konstanz, Fach M725, D-78457 Konstanz, Germany
¨
bArbeitsgruppe Molekulare Genetik, Fakultat fur Biologie, Ruhr Universitat, D-44780 Bochum, Germany
¨
¨
¨
Received 29 August 2001; accepted 22 October 2001
Abstract—This paper describes SAR directed design and synthesis of novel b(1-4)-glucosyltransferase (BGT) inhibitors. The
designed inhibitors 1–5 provide conformational mimicry of the transition-state in glucosyltransfer reactions. The compounds were
tested for in vitro inhibitory activity against (BGT) and the inhibition kinetics were examined. Three of the designed molecules were
found to be potential inhibitors of BGT having IC50 values in micromolar (mM) range. Useful structure–activity relationships were
established, which provide guidelines for the design of future generations of inhibitors of BGT. # 2002 Elsevier Science Ltd. All
rights reserved.
The specific binding of complex carbohydrates forms
the basis of vital intercellular recognition processes.
Thus, biosynthesis of complex oligosaccharides and
other glycosylated cellular metabolites is of immense
importance, as these molecules are directly involved in
numerous biological and cellular processes.1ꢀ5 Hence, in
glycobiology, one of the central questions raised is
understanding the fundamental basis of the incredibly
diverse set of glycan structures observed in nature.6
There are a myriad of glycosyltransferases, each with its
sugar specific donor and with acceptor and linkage spe-
cificity, which are responsible for the biosynthesis of
these remarkably complex structures. Moreover, the
role played by glycosyltransferases in the etiology of
disease, as well as their potential role as therapeutic
targets, are only now being appreciated.7,8 However, the
molecular basis of many processes are often still uncer-
tain despite tremendous progress made in this field.9
The glycosyltransferases of the Leloir pathway are key
catalysts for the synthesis of oligosaccharides and gly-
coconjugates in vivo.10ꢀ14 These enzymes transfer acti-
vated monosaccharide units in the form of their
nucleotide mono- or diphosphate derivatives to a spe-
cific free hydroxy group of the acceptor molecule. Inhi-
bition or modulation of this transfer reaction provides
an excellent opportunity for intervention of the oligo-
saccharide biosynthesis and to obtain a more complete
understanding of the structure–activity relationship
(SAR) of oligosaccharides on a molecular basis.
b(1-4)-Glucosyltransferase (BGT) is an enzyme encoded
by a number of bacteriophages belonging to the T-even
group. It catalyses the transfer of glucose from uridine
diphosphoglucose (UDP-Glc) to 5-hydroxymethyl-
cytosine (5-HMC) bases in double-stranded DNA.15
Glucosylation protects the infecting viral DNA from
host restriction enzymes.16 In addition to its protective
role, the glucosylation has also been implicated in the
control of phage-specific gene expression by influencing
transcripton in vivo and in vitro.17 Another example of
DNA modification by glucosylation has been found in
Trypanosoma brucei18 where glucosylation is believed to
be involved in regulating the expression of variant sur-
face glycoproteins, used by the parasite for protection
from immune recognition.19 BGT is one of two enzymes
involved in the glucosylation of T4 phage DNA. In
contrast to its counterpart a-glucosyltransferase (AGT),
which forms a-glycosidic linkages, BGT catalyses the
formation of b-glycosidic linkages, between the Glc and
the modified 5-HMC base. The T4 glucosyltransferases
*Corresponding author. Tel.:+49-7531-88-2538; fax:+49-7531-88-
3135; e-mail: richard.schmidt@uni-konstanz.de
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00371-6

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A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
show little DNA sequence specificity but modify only
5-HMC bases, suggesting a mechanism of non-specific
DNA binding combined with specific 5-HMC base
recognition.20,21 Inhibition or modulation of this trans-
fer reaction provides an excellent opportunity for inter-
vention of oligosaccharide biosynthesis and to obtain a
more complete understanding of the SAR of oligo-
saccharides on a molecular basis.22
As part of an ongoing programme in our laboratory for
the development of potential transition-state based gly-
cosyltransferase inhibitors (Scheme 1), we had repor-
ted23 the synthesis of a novel transition-state based
analogue of galactosyltransferase, which exhibited high
inhibition properties (Ki 6.20ꢁ10ꢀ5) towards galacto-
syltransferases. Recently, we have successfully designed
and synthesised24 potential transition-state based ana-
logues of sialyltransferases which showed very high
affinity towards the enzyme in the nanomolar range.
Scheme 1. Postulated transition-state for the sugar transfer catalysed
by BGT.
However, the aryl analogues mimicking the sugar of
CMP-Neu5Ac turned out to be potent inhibitors exhi-
biting very high binding affinity to a(2-6)-sialyltransfer-
ase.25 We have undertaken a programme on the SAR
directed design and synthesis of potential b(1-4)-gluco-
syltransferases. Towards this goal, we have recently
reported26 design and synthesis of several substituted
aryl/hetarylmethyl phosphonate–UMP derivatives A–D
as glucosyltranferase inhibitors (Fig. 1), one of which
showed inhibition properties in the micro molar range.
Encouraged by these results, we have undertaken the
present study in order to evaluate the substitution pat-
tern on the aryl vis a vis their inhibitory activity. The
earlier reported inhibitor B had shown an IC50 value of
1.9ꢁ10ꢀ3 M thereby raising the following questions:
(i) is the methyl substitution on the aryl indispensable or
can it be replaced by other analogous groups and, (ii)
how many fold activity is raised or diminished by the
addition of one more oxygen atom in the chain length?
In order to address to the questions raised above, herein
we describe the design, synthesis and in vitro inhibition
studies of inhibitors 1–5 (Fig. 2).
Figure 1. Reported inhibitors A–D.
For the synthesis of sugar mimetics 1–5 the appropriate
phosphonate 8 and phosphates 9, 15, 16 and 17 had to
be synthesised first. The phosphonate27 8 was synthe-
sised in quantitative yield by treating benzyl bromide
with triallyl phosophite in dry toluene at 95 ꢂC (Scheme
2) whereas phosphates 9,28 15, 16 and 17 were prepared
(Schemes 2 and 3) from their corresponding alcohols 7,
12, 13 and 14 by coupling the latter with bis(allyloxy)
(diisopropylamino)phosphine in dry dichloromethane/
acetonitrile (1:1) activated by 1H-tetrazole and then
oxidising the resulting phosphites to phosphates 9, 15,
16 and 17 with tert-butylhydroperoxide in 82–92%,
respectively, as colorless oils.
The deprotection of allyl groups and subsequent pre-
paration of bis-triethylammonium salts were achieved in
a one-pot procedure. The deprotection of allyl groups
were carried out by treating the allylesters 8, 9, 15, 16
and 17 with dimedone in THF at room temperature
catalysed by tetrakis-(triphenylphosphine) palladium.29
Figure 2. Structures of designed inhibitors 1–5.

A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
1131
Scheme 2. Reagents and conditions: (i) (AllO)3P, toluene, 95 ꢂC, quant (for 8); (ii) (i-Pr2N)P(OAll)2, 1H-tetrazol, CH2Cl2/CH3CN (1:1), rt, 2.5 h;
(iii) t-BuOOH, 0 ꢂ C, 1.5 h, 92% (for 9); (iii) dimedone, Pd(PPh3)4, THF, Et3N, rt, 1.5 h, 50% (for 10), 60% (for 11); (iv) 1H-tetrazol, py, rt, 3 days;
RP-18; (v) Amberlite IR-120 (Na+-form), 88% (for 1), 77% (for 2).
Scheme 3. Reagents and conditions: (i) (i-Pr2N)P(OAll)2, 1H-tetrazol, CH2Cl2/CH3CN (1:1) rt, 2.5 h; (ii) t-BuOOH, 0 ꢂC, 1.5 h, 91% (for 15), 82%
(for 16), 82% (for 17); (iii) dimedone, Pd(PPh3)4, THF, Et3N, rt, 1.5 h, 59% (for 18) 70% (for 19), 44% (for 20); (iv) 1H-tetrazol, py, rt, 3 days; RP-
18; (v) Amberlite IR-120 (Na+-form) 69% (for 3), 22% (for 4), 30% (for 5).

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A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
Table 1. IC50 values of transition state analogues 1–5a
tion-state (Scheme 1), (ii) inactivity shown by
compounds 1 and 2 suggest the necessity of substitution
on the aryl group mimicking the side chain, (iii)
replacement of methyl substitution by methoxy groups
on aryl greatly enhances the activity. Our studies have
revealed useful SARs which provide further guidelines
for the design of future generations of inhibitors of
BGT.
Inhibitors
IC50 [mM]
1
2
3
4
>4
>4
1.60ꢃ0.06
1.58ꢃ0.06
0.71ꢃ0.03
1.80ꢃ0.07
5
UDP
aFor details, see the Experimental.
Experimental
General
After completion of the reaction, NEt3 was added to the
reaction mixture and stirred further for 15 min at room
temperature. bis-Triethylammonium salts 10, 11, 18, 19,
and 20 were obtained after flash chromatography
(CHCl3/MeOH, 6:4+1% NEt3) as colorless oils in 44–
70%, respectively.
Solvents were purified according to the standard proce-
dures. NMR measurements were recorded at 22 ꢂC on a
Bruker AC250 Cryospec or Bruker DRX 600 spectro-
meters. Tetramethyl silane (TMS) or the resonance of
the deuterated solvent was used as an internal standard;
solvents: CDCl3, d 7.24; D2O, d=4.63; DMSO-d6, d
2.49. For 31P NMR, phosphoric acid was used as an
external standard; 31P NMR spectra were broadband
1H-decoupled. MALDI-mass spectra were recorded on
a Kratos Kompact Maldi 1 and 2,5-dihydroxybenzoic
acid (DHB) or 6-aza-2-thiothymine (ATT) were used as
matrices. FAB mass spectra were measured on a Finni-
gan MAT312/AMD 5000 spectrometer. Thin-layer
chromatography was performed on Merck silica gel
plastic plates 60F254 or Merck glass plates RP-18; com-
pounds were visualised by treatment with a solution of
The synthesis of mimetics 1–5 was performed following
a recent procedure30a which is an improved variant of
the Moffat–Khorana phosphomorpholidate coupling
method.30b The phosphonophosphate and diphosphates
1
were isolated in 22–88% yield. The H NMR and 13C
NMR spectra showed the expected signals and total
assignment could be performed by 2D NMR experi-
ments. The 31P NMR spectra also showed the charac-
teristic signals associated with the diphosphate-/
phosphonophosphate-moiety. Reaction of bis-triethy-
lammonium salts 10, 11, 18, 19, and 20 with uridine-
50-morpholidophosphate as activated UMP derivative
in the presence of 1H-tetrazole in pyridine for 3 days at
room temperature afforded 1, 2, 3, 4 and 5 as white
powders, respectively, after isolation and purification by
HPLC, ion exchange [Amberlite IR-120 (Na+ form)]
and lyophilisation.
.
(NH4)6Mo7O24 4H2O (20 g) and Ce(SO4)2 (0.4 g) in
10% sulfuric acid (400 mL). Flash chromatography was
performed on J.T. Baker silica gel 60 (0.040–0.063 mm)
at a pressure of 0.3 bar. Preparative HPLC separations
were performed on an Autochrom System with a Shi-
madzu LC8A preparative pump and a Rainin Dynamax
UV 1 Detector at 260 nm. The column used was a
Lichrosorb RP-18, 7 mm, 250ꢁ16 mm (Knauer, Ger-
many). Mixtures of acetonitrile and 0.05 M triethyl-
ammonium bicarbonate (TEAB) (pH 7.0–7.2) were used
as the mobile phase. Elemental analysis were carried out
at the Microanalysis Unit, Fachbereich Chemie, Uni-
versitat Konstanz, Konstanz.
Biological Results
The compounds synthesised were tested for in vitro
inhibitory activity with b(1-4)-glucosyltransferase,
which was isolated as previously reported.31 The bio-
logical studies reveal strong SAR for the binding of
inhibitors 1–5 to BGT. The IC50 values (concentration
of inhibitor at which 50% activity of the enzyme is
observed) of inhibitors 1–5 were determined and are
summarised in Table 1. Inhibitor 3 showed an IC50
value of 1.60ꢁ10ꢀ3 M whereas compounds 1 and 2
showed no inhibition (>4ꢁ10ꢀ3 M) thereby implying
that the methyl substitution on the the aryl group is
mandatory in order to develop potential inhibitors from
this series. Diphosphate 3 showed slightly better inhibi-
tion than its phosphonophosphate analogue B (IC50
1.9ꢁ10ꢀ3 M). However, better inhibition was shown by
dimethoxy substituted compound 5 (IC50 7.1ꢁ10ꢀ4 M)
followed by compound 4 (IC50 1.58ꢁ10ꢀ3 M). X-ray
studies with some of these compounds bound to BGT
will be reported in due course.
Diallyl benzylphosphonate (8). A mixture of 6 (1.0 g,
5.88 mmol) and triallyl phosphite (1.01 mL, 5.88 mmol)
in dry toluene (5 mL) was heated under argon to 95 ꢂC
for 4 h to afford 827 in quantitative yield. The formed
allyl bromide was distilled off under slightly reduced
pressure. The residue was used as such in the next step
without further purification. Rf=0.14 (pentane/diethyl
ether, 1:1); 1H NMR (600 MHz, CDCl3): d 3.20 (d,
J=20.2 Hz, 2H, CH2–Ar), 4.41–4.58 (m, 4H,
CH2¼CH–CH2), 5.18–5.39 (m, 4H, CH2¼CH–CH2),
5.83–5.95 (m, 2H, CH2¼CH–CH2), 7.25–7.31 (m, 5H,
Ar–H); 31P (243 MHz, CDCl3): d 28.39 (s, 1P); MS (EI):
m/z 252 [M]+ for C13H17O3P (252.3).
General procedure for the synthesis of (9), (15), (16) and
(17). To a solution of 7, 12, 13 or 14 (1.4–4.6 mmol) in
a mixture of dry CH2Cl2 and dry CH3CN (each 10 mL)
under argon, vacuum dried 1H-tetrazole (2.5–12.5
mmol) was added at room temperature. bis(Allyloxy)
In conclusion, our inhibition studies clearly indicate
that: (i) introduction of an extra oxygen atom in the
chain length appreciably increases the inhibition prop-
erties as could be concluded from the postulated transi-

A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
1133
(diisopropylamino)phosphine (1.5–5.7 mmol) was
added dropwise to the reaction mixture through a syr-
inge under argon. After stirring for 2.5 h at rt, the
reaction mixture was cooled to 0 ꢂC and 5.5 M tert-
BuOOH solution in nonane (1.5–5.7 mmol) was added
dropwise and on completion of the addition, reaction
mixture was further stirred for 0.5–1.5 h at rt. After
completion of the reaction (TLC), Na2S2O3 solution
(50 mL) was poured into the reaction mixture. The
organic phase was washed successively with NaCl
solution, NaHCO3 solution and once again with NaCl
solution (each 50 mL). The aqueous solution was
extracted with CH2Cl2 (2ꢁ25 mL) and added to the
organic phase. The pooled organic phase was dried
(MgSO4) and evaporated to dryness. The residue after
flash chromatography afforded 9, 15, 16 or 17 as a col-
orless oil.
workup afforded a residue which on flash chromato-
graphy (toluene/acetone, 9:1) afforded 16 as a colorless
oil (552 mg, 82%); Rf=0.22 (toluene/acetone, 9:1); H
NMR (250 MHz, CDCl3): d 3.81 (s, 3H, p-OCH3), 3.84
(s, 6H, m-OCH3), 4.47–4.53 (m, 4H, CH2¼CH–CH2),
4.99 (d, JC,P=8.5 Hz, 2H, CH2–Ar), 5.20–5.36 (m, 4H,
CH2¼CH–CH2), 5.82–5.97 (m, 2H, CH2¼CH–CH2),
6.59 (s, 2H, Ar–H); C16H23O7P (358.3) calcd C 53.63, H
6.47; found C 53.43, H 6.66.
1
Diallyl 3,5-dimethoxybenzyl phosphate (17). A mixture
of 14 (240 mg, 1.45 mmol), 1H-tetrazole (200 mg, 2.86
mmol), bis(allyloxy)(diisopropylamino)phosphine (389
mL, 1.45 mmol) in dry CH2Cl2/CH3CN (10 mL each)
was treated as per the general procedure described
above. After 2.5 h, 5.5 M tert-BuOOH solution in non-
ane (264 mL, 1.45 mmol) was added dropwise and after
workup afforded a residue which on flash chromato-
graphy (toluene/acetone, 6:1) afforded 17 as a colorless
Diallyl benzylphosphate (9). A mixture of 7 (500 mg,
4.63 mmol), 1H-tetrazole (875 mg, 12.5 mmol),
bis(allyloxy) (diisopropylamino)phosphine (1.5 mL, 5.74
mmol) in dry CH2Cl2/CH3CN was treated as per the
general procedure described above. After 2.5 h, 5.5 M
tert-BuOOH solution in nonane (1.04 mL, 5.74 mmol)
was added dropwise and after workup afforded a resi-
due which on flash chromatography (pentane/diethyl
ether, 1:1) afforded 928 as a colorless oil (1.14 g, 92%).
Rf=0.11 (pentane/diethyl ether, 1:1); 1H NMR
(600 MHz, CDCl3): d 4.51 (m, 4H, CH2¼CH–CH2),
5.09 (d, J=8.2 Hz, 2H, CH2–Ar), 5.24–5.34 (m, 4H,
CH2¼CH–CH2), 5.88–5.92 (m, 2H, CH2¼CH–CH2),
7.33–7.39 (m, 5H, Ar–H); 31P NMR (243 MHz, D2O): d
0.34 (s, 1P); MS (EI): m/z 268 [M+] for C13H17O4P
(268.25).
1
oil (390 mg, 82%); Rf=0.32 (toluene/acetone, 4:1); H
NMR (250 MHz, CDCl3): d 3.76 (s, 6H, OCH3), 4.47–
4.55 (m, 4H, CH2¼CH–CH2), 4.99 (d, J=8.0 Hz, 2H,
CH2–Ar), 5.19–5.36 (m, 4H, CH2¼CH–CH2), 5.82–5.98
(m, 2H, CH2¼CH–CH2), 6.39 (t, J=2.3 Hz, 1H, 4-H),
6.50 (d, J=2.3 Hz, 2H, 2-H, 6-H); MS (MALDI, posi-
tive mode, matrix: DHB, dioxane): m/z 351
[M+Na+]+, 367 [M+K+]+ C15H21O6P (328.3) calcd
C 54.87, H 6.45; found C 54.85, H 6.46.
General procedure for the synthesis of bis-triethylammo-
nium salts (10), (11), (18), (19) and (20). To a solution
of 8, 9, 15, 16 or 17 (0.5–1.4 mmol) in dry THF (6–30 mL),
dimedone (2.7–7.0 mmol) and tetrakis-(triphenylphos-
phine)-palladium (0.11–0.24 mmol) was added and stirred
at room temperature under argon protected from light.
After 1.5 h when the TLC showed no starting material,
NEt3 (0.15 mL) was added and further stirred for 15 min
at room temperature. The reaction mixture was evapo-
rated and the residue was purified over flash chromato-
graphy to furnish 10, 11, 18, 19 or 20 in 44–70%.
Diallyl 3-methylbenzylphosphate (15). A mixture of 12
(400 mg, 3.28 mmol), 1H-tetrazole (620 mg, 8.86 mmol),
bis(allyloxy)(diisopropylamino)phosphine (1.12 mL,
4.29 mmol) in dry CH2Cl2/CH3CN (10 mL each) was
treated as per the general procedure described above.
After 2.5 h, 5.5 M tert-BuOOH solution in nonane (782
mL, 4.30 mmol) was added dropwise and after work up
afforded a residue which on flash chromatography
(pentane/diethyl ether, 1:1) afforded 15 as a colorless oil
(841 mg, 91%). Rf=0.12 (pentane/diethyl ether, 1:1);
1H NMR (600 MHz, CDCl3): d 2.36 (s, 3H, CH3), 4.52
(m, 4H, CH2¼CH–CH2), 5.05 (d, J=8.0 Hz, 2H, CH2–
Ar), 5.23–5.35 (m, 4H, CH2¼CH–CH2), 5.89–5.93 (m,
2H, CH2¼CH–CH2), 7.14–7.27 (m, 4H, Ar–H); 13C
NMR (151 MHz, CDCl3, assignment by HMQC): d
21.27 (1C, CH3), 68.01 (2C, CH2¼CH–CH2), 69.27 (1C,
CH2–Ar), 118.17 (2C, CH2¼CH–CH2), 124.93, 128.42,
128.59, 129.21 (4C, Ar), 132.39 (2C, CH2¼CH–CH2);
31P NMR (243 MHz, CDCl3): d 0.29 (s, 1P); MS (EI):
m/z 282 [M+] for C14H19O4P (282.28).
bis-Triethylammonium benzylphosphonate (10). A mix-
ture of 8 (150 mg, 0.60 mmol), dimedone (420 mg, 3.0
mmol) and tetrakis-(triphenylphosphine)-palladium
(139 mg, 0.12 mmol) in dry THF (6 mL) was treated as
per the general procedure described above. After evap-
oration of the solvent, the residue was purified over
flash chromatography (CHCl3/MeOH, 6:4+1% NEt3)
to afford 10 as a colorless oil (110 mg, 50%); Rf=0.29
1
(CHCl3/MeOH, 6:4+1% NEt3); H NMR (600 MHz,
CDCl3): d 1.08 (t, J=7.3 Hz, 18H, NCH2CH3), 2.79 (q,
J=7.3 Hz, 12H, NCH2CH3), 2.92 (d, J=21.5 Hz, CH2–
Ar), 7.20–7.40 (m, 5H, Ar–H); 13C NMR (151 MHz,
CDCl3): d 8.30 (6C, NCH2CH3), 35.90 (d, J=134 Hz,
CH2P), 45.28 (6C, NCH2CH3), 125.63, 127.92, 130.03,
130,07, 135.20, 135.26 (6C, Ar); 31P NMR (243 MHz,
CDCl3): d 21.7 (s, 1P); MS (FAB, positive mode,
matrix: NBA/THF): m/z 274 [MꢀHNEt+3 +2H+]+,
375 [M+H+]+ for C19H39N2O3P (374.5).
Diallyl 3,4,5-trimethoxybenzyl phosphate (16). A mix-
ture of 13 (369 mg, 1.87 mmol), 1H-tetrazole (280 mg,
4.0 mmol), bis(allyloxy)(diisopropylamino)phosphine
(740 mL, 2.80 mmol) in dry CH2Cl2/CH3CN (10 mL
each) was treated as per the general procedure described
above. After 2.5 h, 5.5 M tert-BuOOH solution in non-
ane (545 mL, 3.0 mmol) was added dropwise and after
bis-Triethylammonium benzylphosphate (11). A mixture
of 9 (250 mg, 0.93 mmol), dimedone (651 mg, 4.65
mmol) and tetrakis-(triphenylphosphine)-palladium

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A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
(219 mg, 0.19 mmol) in dry THF (10 mL) was treated as
per the general procedure described above. After evap-
oration of the solvent, the residue was purified over
flash chromatography (CHCl3/MeOH, 6:4+1% NEt3)
to afford 11 as a colorless oil (216 mg, 60%); Rf=0.18
(CHCl3/MeOH/H2O, 8:4:0.5+1% NEt3); 1H NMR
(250 MHz, D2O): d 1.06 (t, J=7.3Hz, 18H, NCH2CH3),
2.95 (q, J=7.3 Hz, 12H, NCH2CH3), 3.62 (s, 6H,
OCH3), 4.64 (d, overlapping with D2O peak, CH2–Ar),
6.28 (t, J=2.3 Hz, 1H, 4-H), 6.48 (d, J=2.3 Hz, 2H, 2-
H, 6-H); MS (MALDI, negative mode, matrix: ATT,
H2O): m/z 247 [Mꢀ2HNEt+3 +H+]ꢀ for C21H43N2O6P
(450.6).
1
(CHCl3/MeOH, 6:4 +1% NEt3); H NMR (600 MHz,
CDCl3): d 1.24 (t, J=7.3 Hz, 18H, NCH2CH3), 2.96 (q,
J=7.3 Hz, 12H, NCH2CH3), 4.98 (d, J=6.2 Hz, 2H,
CH2–Ar), 7.20–7.40 (m, 5H, Ar–H); 13C NMR
(151 MHz, CDCl3): d 8.50 (6C, NCH2CH3), 45.30 (6C,
NCH2CH3), 66.67 (d, JC,P=17.4 Hz, 1C, CH2-Ar),
127.01–139.26 (6C, Ar); 31P NMR (243 MHz, CDCl3): d
3.11 (s, 1P); MS (FAB, positive mode, matrix: NBA/
THF): m/z 290 [M–HNEt+3 +2H+]+, 391 [M+H+]+
for C19H39N2O4P (390.5).
General procedure for the synthesis of (1), (2), (3), (4)
and (5). A mixture of 10, 11, 18, 19 or 20 (0.13–0.33
mmol) and 4 - morpholine - N,N0 - dicyclohexylcarbox -
amidinium uridine 50-monophosphomorpholidate (0.16–
0.36 mmol) was evaporated with dry pyridine (3ꢁ1.5
mL). To this mixture, vacuum dried 1H-tetrazole (0.39–
1.0 mmol) and dry pyridine (1–4 mL) were added under
argon and the solution was stirred at rt for 3 days. The
reaction mixture was evaporated and co-evaporated
with water (2ꢁ1 mL). The residue was purified by pre-
parative HPLC (0.05 M triethylammonium hydro-
gencarbonate buffer+7.5–10% CH3CN, flow rate: 10–
15 mL/min). The fractions containing product were
pooled and evaporated. The residue was dissolved in
water (2 mL) and passed through a column (2.5ꢁ12 cm)
of Amberlite IR-120 (Na+ form). The column was
washed with water (50 mL) and the collected fractions
were evaporated. The residue thus obtained was dis-
solved in few drops of water and then precipitated with
EtOH (5 mL). The suspended solution was centrifuged
and the supernatant was carefully decanted to afford a
white solid which was dissolved in water and lyophil-
ised. After lyophilisation, 1, 2, 3, 4 or 5 was obtained as
a white powder in 22–88% yield.
bis-Triethylammonium 3-methylbenzyl phosphate (18). A
mixture of 15 (150 mg, 0.53 mmol), dimedone (371 mg,
2.65 mmol) and tetrakis-(triphenylphosphine)-palla-
dium (122 mg, 0.11 mmol) in dry THF (10 mL) was
treated as per the general procedure described above.
After evaporation of the solvent, the residue was pur-
ified over flash chromatography (CHCl3/MeOH,
6:4+1% NEt3) to afford 18 as a colorless oil (126 mg,
59%); Rf=0.21 (CHCl3/MeOH, 6:4+1% NEt3); 1H
NMR (600 MHz, CDCl3): d 1.25 (t, J=7.3 Hz, 18H,
NCH2CH3), 2.29 (s, 3H, CH3), 2.98 (q, J=7.3 Hz, 12H,
NCH2CH3), 4.95 (d, J=6.0Hz, 2H, CH2–Ar), 7.01–7.22
(m, 4H, Ar–H); 13C NMR (151 MHz, CDCl3): d 8.47
(6C, NCH2CH3), 21.30 (1C, CH3), 45.27 (6C,
NCH2CH3), 66.66 (d, JC,P=4.5 Hz, 1C, CH2–Ar),
124.49, 127.72, 127.83, 128.20, 137.46, 139.02 (6C, Ar);
31P NMR (243 MHz, CDCl3): d 3.10 (s, 1P); MS (FAB,
positive mode, matrix: CH3CN/glycerol/0.1% TFA,
1:1:1): m/z 304 [M–HNEt+3 +2H+]+, 405 [M+H+]+
for C20H41N2O4P (404.53).
Disodium uridine 50-phosphono benzylphosphate (1). A
mixture of 10 (53 mg, 0.14 mmol) and 4-morpholine-
N,N0-dicyclohexylcarboxamidinium uridine 50-mono-
phospho-morpholidate (158 mg, 0.23 mmol) and 1H-
tetrazole (27 mg, 0.39 mmol) in dry pyridine (1 mL) was
treated as per general procedure described above. After
3 days, product was purified by HPLC (0.05 M triethyl-
ammonium hydrogencarbonate buffer+8% CH3CN,
flow rate: 10 mL/min, tR 11.1 min). Compound 1 was
obtained as a white powder (36 mg, 88%) after ion-
exchange and precipitation. 1H NMR (600MHz, D2O): d
3.04 (d, J=21.4 Hz, 2H, 1a00-H, 1b00-H), 3.93 (m, 1H, 5a0-
H), 3.97 (m, 2H, 30-H, 5b0-H), 4.04 (m, 1H, 40-H), 4.11 (m,
bis-Triethylammonium 3,4,5-trimethoxybenzyl phosphate
(19). A mixture of 16 (500 mg, 1.40 mmol), dimedone
(980 mg, 7.0 mmol) and tetrakis-(triphenylphosphine)-
palladium (176 mg, 0.15 mmol) in dry THF (30 mL)
was treated as per the general procedure described
above. After evaporation of the solvent, the residue was
purified over flash chromatography (CHCl3/MeOH/
H2O, 8:4:0.5+1% NEt3) afford 19 as a colorless oil (470
mg, 70%). Rf=0.22 (CHCl3/MeOH/H2O, 8:4:0.5+1%
1
NEt3); H NMR (250 MHz, D2O): d 1.05 (t, J=7.3 Hz,
18H, NCH2CH3), 3.00 (q, J=7.3 Hz, 12H, NCH2CH3),
3.59 (s, 3H, p-OCH3), 3.70 (s, 6H, m-OCH3), 4.64 (d,
overlap with HDO, 2H, CH2–Ar), 6.67 (s, 2H, Ar–H);
MS (MALDI, negative mode, matrix: ATT, H2O): m/z
277 [Mꢀ2HNEt+3 +H+]ꢀ for C22H45N2O7P (480.6).
1H, 20-H), 5.73 (d, J5,6=7.8 Hz, 1H, 5-H), 5.81 (d,
0
0
0
J1 ,2 =4.6 Hz, 1H, 1 -H), 7.11–7.23 (m, 5H, Ar–H), 7.68
(d, J6,5=7.8 Hz, 1H, 6-H); 31P NMR (243 MHz, D2O): d
ꢀ9.95 (d, J=27.2 Hz, P(O)O3), 15.23 (d, J=27.2 Hz,
CP(O)O2); MS (FAB, positive mode, matrix: CH3CN/
glycerol/0.1% TFA, 1:1:1): m/z 523 [M+H+]+, 545
[M+Na+]+ for C16H18N2O11P2Na2 (522.25).
bis-Triethylammonium 3,5-dimethoxybenzyl phosphate
(20). A mixture of 17 (350 mg, 1.07 mmol), dimedone
(750 mg, 5.36 mmol) and tetrakis-(triphenylphosphine)-
palladium (138 mg, 0.12 mmol) in dry THF (20 mL)
was treated as per the general procedure described
above. After evaporation of the solvent, the residue was
purified over flash chromatography (CHCl3/MeOH/
H2O, 8:4:0.5+1% NEt3). The product obtained after
flash chromatography was lyophilised with water to
afford 20 as a colorless powder (210 mg, 44%). Rf 0.29
Disodium benzyl uridine 50-diphosphate (2). A mixture of
11 (107 mg, 0.27 mmol) and 4-morpholine-N,N0-dicy-
clohexylcarboxamidinium uridine 50-monophospho-
morpholidate (261 mg, 0.38 mmol) and 1H-tetrazole (46
mg, 0.66 mmol) in dry pyridine (1 mL) was treated as
per general procedure described above. After 3 days,
product was purified by HPLC (0.05 M triethylammo-
nium hydrogencarbonate buffer+8% CH3CN, flow

A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
1135
rate: 10 mL/min, tR 13.8 min). Compound 2 was
obtained as a white powder (64 mg, 77%) after ion-
400-C), 140.72 (1C, 6-C), 150.86 (2C, 300-C, 500-C), 151.84,
165.23 (2C, 2-C, 4-C); 31P NMR (243 MHz, D2O): d
ꢀ10.20, ꢀ10.02 (2 bd, 2P(O)O3); MS (MALDI, negative
mode, matrix: ATT, H2O): m/z 583.4 [Mꢀ2Na+H+]ꢀ
for C19H24N2O15P2 Na2 (628.4).
1
exchange and precipitation. H NMR (600 MHz, D2O):
d 4.02 (m, 1H, 5a0-H), 4.07 (m, 1H, 20-H), 4.09 (m, 2H,
40-H, 5b0-H), 4.13 (m, 1H, 30-H), 4.86 (d, J=6.3 Hz, 2H,
1a00-H, 1b00-H), 5.640 (d, J5,6=7.9 Hz, 1H, 5-H), 5.79 (d,
J1 ,2 =4.6 Hz, 1H, 1 -H), 7.21–7.29 (m, 5H, Ar–H), 7.65
(d, J6,5=7.9 Hz, 1H, 6-H); 13C NMR (151 MHz, D2O,
assignment by HMQC): d 64.8 (1C, 50-C), 68.5 (1C,
CH2–Ar), 69.4 (1C, 30-C), 74.3 (1C, 20-C), 83.3 (1C, 40-C),
89.3 (1C, 10-C), 103.3 (1C, 5-C), 141.2 (1C, 6-C); 31P
NMR (243 MHz, D2O): d ꢀ10.21, ꢀ9.84 (2d, 3JP,P=21.6
Hz, 2P(O)O3); MS (FAB, positive mode, matrix:
CH3CN/glycerol/0.1% TFA, 1:1:1): m/z 539 [M+H+]+,
561 [M+Na+]+ for C16H18N2O12P2Na2 (538.25).
Disodium 3,5-dimethoxybenzyl uridine-50-diphosphate
(5). A mixture of 20 (60 mg, 0.13 mmol) and 4-mor-
pholine-N,N0-dicyclohexylcarboxamidinium uridine 50-
monophosphomorpholidate (110 mg, 0.16 mmol) and
1H-tetrazole (27 mg, 0.39 mmol) in dry pyridine (2 mL)
was treated as per general procedure described above.
After 3 days, product was purified by HPLC [0.05 M
triethylammonium hydrogencarbonate buffer+10%
CH3CN, flow rate: 15 mL/min, tR 17.0 min]. Compound
5 was obtained as a white powder (23 mg, 30%) after
ion-exchange and precipitation. 1H NMR (600 MHz,
D2O): d 3.68 (s, 6H, OCH3), 4.05 (m, 2H, 5a0-H, 20-H),
4.10 (m, 1H, 40-H), 4.16 (m, 2H, 30-H, 5b0-H), 4.81 (d,
J=5.6 Hz, 2H, CH2–Ar), 5.54 (d, J5,6=8.1 Hz, 1H, 5-
0
0
Disodium (3-methylbenzyl) uridine 50-diphosphate (3). A
mixture of 18 (55 mg, 0.14 mmol) and 4-morpholine-
N,N0-dicyclohexylcarboxamidinium uridine 50-mono-
phospho-morpholidate (158 mg, 0.23 mmol) and 1H-
tetrazole (27 mg, 0.39 mmol) in dry pyridine (1 mL) was
treated as per general procedure described above. After
3 days, product was purified by HPLC [0.05 M triethy-
lammonium hydrogencarbonate buffer+10% CH3CN,
flow rate: 10 mL/min, tR 16.5 min]. Compound 3 was
obtained as a white powder (52 mg, 69%) after ion-
exchange [Amberlite IR-120 (Na+ form)] and pre-
0
00
0
0
H), 5.71 (d, J1 ,2 =4.1 Hz, 1H, 1 -H), 6.31 (s, 1H, 4 -H),
6.49 (s, 2H, 200-H, 600-H), 7.65 (d, J6,5=8.1 Hz, 1H, 6-H);
13C NMR (151 MHz, D2O, assignment by HMQC): d
54.84 (2C, OCH3), 63.90 (1C, 50-C), 66.67 (1C, CH2–
Ar), 68.42 (1C, 30-C), 73.48 (1C, 20-C), 82.14 (1C, 40-C),
88.17 (1C, 10-C), 98.78 (400-C), 101.60 (1C, 5-C), 104.73
(2C, 200-C, 600-C), 139.66 (1C, 100-C), 140.57 (1C, 6-C),
150.87 (1C, 2-C or 4-C), 159.61 (2C, 300-C, 500-C), 165.36
(1C, 2-C or 4-C); 31P NMR (243 MHz, D2O): d
ꢀ12.85,ꢀ12.51 (2 d, J=20.9 Hz, 2P(O)O3) MS
(MALDI, negative mode, matrix: ATT, H2O): m/z 553
[Mꢀ2Na+H+]ꢀ for C18H22N2O14P2 Na2 (598.4).
1
cipitation. H NMR (600 MHz, D2O): d 2.18 (s, 3H,
CH3), 4.01 (m, 2H, 5a0-H, 20-H), 4.07 (m, 2H, 5b0-H, 40-
H), 4.10 (m, 1H, 30-H), 4.82 (d, J=6.2 Hz, 2H, CH2–
Ar), 5.59 (d, J=7.7 Hz, 1H, 5-H), 5.79 (d, J=4.6 Hz,
1H, 10-H), 7.03–7.15 (m, 4H, Ar–H), 7.57 (d, J=7.7 Hz,
1H, 6-H); 13C NMR (151 MHz, D2O, assignment by
HMQC)): d 20.10 (1C, CH3), 64.11 (1C, 50-C), 67.28
(1C, CH2–Ar), 68.69 (1C, 30-C), 73.36 (1C, 20-C), 81.87
(1C, 40-C), 88.20 (1C, 10-C), 102.40 (1C, 5-C), 123.97,
127.53, 128.01, 128.19 (4C, Ar), 139.57 (1C, 6-C); 31P
NMR (243 MHz, D2O): d ꢀ9.76, ꢀ9.41 (2d, J=21.8 Hz,
2P(O)O3); MS (FAB, positive mode, matrix: CH3CN/
glycerol/0.1% TFA, 1:1:1): m/z 553 [M+H+]+, 575
[M+Na+]+ for C17H20N2O12P2Na2 (552.28).
Inhibition studies of ꢀ-glucosyltransferase of phage T4.
The activity of b-glucosyltransferase was monitored by
the transfer of 14C-glucose from the substrate 14C-
UDPG to 5-hydroxymethylcytosine (HMC) of unglu-
cosylated T4*DNA. T4*DNA was obtained from pro-
geny phage propagating in a UDPGꢀ Escherichia coli K
host strain (W4597). The reaction mixture used to
determine the IC50 in the inhibition studies, contained in
a final volume of 100 mL, 100 mM Tris–HCl, pH 7.9, 9
mg of T4*-DNA, 25 mM MgC12, 200 mM UDPG
(Sigma), 5 mL of 14C-UDPG (NEN, 330 mCi/mmol or
1.85 MBq, delivered in a total volume of 2 mL), 0.5mg
BGT and different inhibitors, with concentrations vary-
ing between 0.5 and 2.5 mM. After 15 min of incubation
at 30 ꢂC, the reaction mixtures were transferred onto
DEAE filter disks (Whatman DE81). The filters were
washed three times with 5 mL of a buffer containing
0.5 M NaH2PO4, pH 7.0, dried and counted in a Beck-
man scintillation counter, model LS 6000 TA.
Disodium 3,4,5-trimethoxybenzyl uridine 50-diphosphate
(4). A mixture of 19 (160 mg, 0.33 mmol) and 4-mor-
pholine-N,N0-dicyclohexylcarboxamidinium uridine 50-
monophosphomorpholidate (250 mg, 0.36 mmol) and
1H-tetrazole (70 mg, 1.0 mmol) in dry pyridine (4 mL)
was treated as per general procedure described above.
After 3 days, product was purified by HPLC [0.05 M
triethylammonium hydrogencarbonate buffer+7.5%
CH3CN, flow rate: 15 ml/min, tR 12.7 min]. Compound
4 was obtained as a white powder (47 mg, 22%) after
ion-exchange and precipitation. 1H NMR (600 MHz,
D2O): d 3.62 (s, 3H, p-OCH3), 3.73 (s, 6H, m-OCH3),
4.02 (m, 5a0-H), 4.09 (m, 2H, 20-H, 40-H), 4.15 (m, 2H,
Km and Vmax values were determined in essentially the
same buffer and reaction volume, however, the con-
centrations of the substrate mixture varied between 1.6
and 200 mM and the inhibitors were added at constant
concentrations of 0.5, 1, and 2 mM, respectively. The
different substrate concentrations were obtained by the
appropriate dilution of a mixture containing UDPG
and 14C-UDPG in a ratio of 27.5:1. Radioactivity
transferred from the substrate to the T4*DNA, again
was measured as described above.
30-H, 5b0-H), 4.80 (d, J=4.9 Hz, 2H, CH2–Ar), 5.59 (d,
0
0
0
J5,6=8.1 Hz, 1H, 5-H), 5.73 (d, J1 ,2 =4.6 Hz, 1H, 1 -
H), 6.65 (s, 2H, Ar–H), 7.67 (d, J6,5=8.1 Hz, 1H, 6-H);
13C NMR (151 MHz, D2O, assignment by HMQC): d
55.47 (2C, m-OCH3), 60.20 (1C, p-OCH3), 64.11 (1C, 50-
C), 67.00 (1C, CH2–Ar), 68.72 (1C, 30-C), 73.39 (1C, 20-
C), 82.35 (1C, 40-C), 87.98 (1C, 10-C), 101.79 (1C, 5-C),
104.12 (2C, 200-C, 600-C), 133.57 (1C, 100-C), 135.36 (1C,

1136
A. K. Bhattacharya et al. / Bioorg. Med. Chem. 10 (2002) 1129–1136
13. (a) Palcic, M. M.; Heerze, L. D.; Srivastava, O. P.;
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One of the authors (A.K.B.) is grateful to the Deutscher
Akademischer Austauschdienst (DAAD) for a research
fellowship. This work was supported by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemi-
schen Industrie. We are grateful to Ms. Anke Friemel
and Mr. Klaus Hagele, Fachbereich Chemie, Uni-
versitat Konstanz, Konstanz for recording NMR
(600 MHz) and FAB-MS, respectively.
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