Synthesis and in vitro evaluation of substituted aryl- and hetarylmethyl phosphonate and phosphate - UMP derivatives as potential glucosyltransferase inhibitors

Article abstract of DOI:10.1139/v02-119

The enzyme β (1→4)-glucosyltransferase (BGT) catalyses the transfer of glucose from uridine diphospho-glucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) bases in double-stranded DNA. Potential inhibitors of BGT were developed by structure-based design and synthesized. The designed inhibitors 1-6 provide conformational mimicry of the transition state in glucosyltransfer reactions. The key synthetic steps involve a Michaelis-Arbuzov reaction followed by coupling with uridine-5′-morpholidophosphate as activated UMP derivative. 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 the micromolar (μM) range. Useful structure-activity relationships were established which provide guidelines for the design of future generations of inhibitors of BGT.

Full text of DOI:10.1139/v02-119

973
Synthesis and in vitro evaluation of substituted
aryl- and hetarylmethyl phosphonate and
phosphate — UMP derivatives as potential
glucosyltransferase inhibitors
Asish K. Bhattacharya, Florian Stolz, Jürgen Kurzeck, Wolfgang Rüger, and
Richard R. Schmidt
Abstract: The enzyme ꢀ(1J4)-glucosyltransferase (BGT) catalyses the transfer of glucose from uridine diphospho-
glucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) bases in double-stranded DNA. Potential inhibitors of BGT
were developed by structure-based design and synthesized. The designed inhibitors 1–6 provide conformational mim-
icry of the transition state in glucosyltransfer reactions. The key synthetic steps involve a Michaelis–Arbuzov reaction
followed by coupling with uridine-5>-morpholidophosphate as activated UMP derivative. 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 the micromolar ( M) range. Useful structure–activ-
ity relationships were established which provide guidelines for the design of future generations of inhibitors of BGT.
Key words: ꢀ-glucosyltransferase, transition state, enzyme inhibitors, structure-based design, synthesis.
Résumé : La transférase de ꢀ(1J4)-glucosyle (BGT) est un enzyme qui catalyse le transfert du glucose de l’uridine
diphosphoglucose (UDP-Glc) aux bases 5-hydroxyméthylcytosines (5-HMC) de l’ADN à doubles brins. En se basant
sur la structure, on a développé et réalisé la synthèse d’inhibiteurs potentiels de la BGT. Les inhibiteurs proposés (1–6)
fournissent une imitation conformationnelle de l’état de transition des réactions de transfert du glucosyle. Les étapes de
synthèse clés implique une réaction de Michaelis–Arbuzov, suivie d’un couplage avec un 5>-morpholinophosphate
d’uridine sous la forme de dérivé activé UMP. L’activité inhibitrice des composés contre le BGT a été vérifiée in vitro
et on a étudié la cinétique de l’inhibition. On a trouvé que trois des molécules synthétisées sont des inhibiteurs
potentiels du BGT, avec des valeurs de IC50 au niveau micromolaire ( M). On a établi des relations structure–activité
intéressantes; elles fournissent des indications générales pour le développement des futures générations d’inhibiteurs de
BGT.
Mots clés : transférase de ꢀ-glucosyle, état de transition, inhibiteurs d’enzyme, développement sur la base de la struc-
ture, synthèse.
[Traduit par la Rédaction] Bhattacharya et al. 982
Introduction
served in nature (7). What we do know is that there are a
myriad of glycosyltransferases, each with its sugar specific
donor and with acceptor and linkage specificity, which are
responsible for the biosynthesis of these remarkably com-
plex structures. From a medicinal chemist’s standpoint, the
study of these glycosyltransferases has begun to throw new
light onto these areas ranging from bacterial toxicity to
mammalian development. Moreover, the role played by
glycosyltransferases in the etiology of disease, as well as
their potential role as therapeutic targets, are also now being
appreciated (8). Despite tremendous progress made in this
The biosynthesis of complex carbohydrates, oligosaccha-
rides, and other glycosylated cellular metabolites is of major
importance, since these molecules are directly involved in
numerous biological and cellular processes (1). The role of
glycobiology ranges from storage of photosynthetic prod-
ucts, cell–cell recognition and adhesion, as well as glyco-
sylation of proteins (2–6). Thus, in glycobiology, one of the
central questions raised is understanding the fundamental
basis of the incredibly diverse set of glycan structures ob-
Received 4 December 2001. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 7 August 2002.
We dedicate this paper to the memory of Professor Raymond U. Lemieux.
A.K. Bhattacharya, F. Stolz, and R.R. Schmidt.1 Fachbereich Chemie, Universität Konstanz, Fach M725,
D-78457, Konstanz, Germany.
J. Kurzeck and Wolfgang Rüger. Arbeitsgruppe Molekulare Genetik, Fakultät für Biologie, Ruhr Universität, D-44780 Bochum,
Germany.
1Corresponding author (e-mail: Richard.Schmidt@uni-konstanz.de).
Can. J. Chem. 80: 973–982 (2002)
DOI: 10.1139/V02-119
© 2002 NRC Canada

974
Can. J. Chem. Vol. 80, 2002
Fig. 1. Structures of designed inhibitors 1–6.
field, the molecular basis of many processes are still uncer-
tain (9). The glycosyltransferases of the Leloir pathway are
key catalysts for the synthesis of oligosaccharides and
glycoconjugates in vivo (10–14).
O
X
2Na+
NH
Y
ꢀ(1J4)-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-hydroxymethylcytosine
(5-HMC) bases in double-stranded DNA (15). Glucosylation
protects the infecting viral DNA from host restriction en-
zymes (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 modifi-
cation by glucosylation has been found in Trypanosoma
brucei (18) where glucosylation is believed to be involved in
regulating the expression of variant surface glycoproteins,
used by the parasite for protection from immune recognition
(19). BGT is one of two enzymes involved in the gluco-
sylation of T4 phage DNA. In contrast to its counterpart
ꢁ-glucosyltransferase (AGT), which forms ꢁ-glycosidic link-
ages, BGT catalyses the formation of ꢀ-glycosidic linkages,
between the Glc and the modified 5-HMC base. Recognition
specificity resides in the opposite base, namely G, therefore
T4 BGT also glucosylates 5-HMU.2 Regarding this
glucosylation of 5-HMU, it does not take place if A or C or
even an abasic site face 5-HMU on the opposite DNA
strand. Thus, recognition specificity must reside in the
hydroxymethyl group and the opposing G (20, 21). Inhibi-
tion or modulation of this transfer reaction provides an ex-
cellent opportunity for intervention of oligosaccharide
biosynthesis and to obtain a more complete understanding of
the structure–activity relationship of oligosaccharides on a
molecular basis (22). This endeavour is also an interesting
excursion to the basis of carbohydrate–protein recognition,
which has gained seminal contributions by the late Professor
Lemieux (23).
Earlier, we had reported (24) the synthesis of a transition
state based analog of galactosyltransferase which exhibited
high-inhibition properties towards galactosyltransferases.
Encouraged by this, we have recently reported (25) transi-
tion-state-based analogs of sialyltransferases which showed
very high affinity towards the enzyme in the nanomolar
range. However, the aryl analogs mimicking the sugar of
CMP-Neu5Ac turned out to be potent inhibitors exhibiting
very high binding affinity to ꢁ(2J6)-sialyltransferase (26).
Herein we describe the design, synthesis, and in vitro inhib-
iting activities of inhibitors 1–6 (27) (Fig. 1), in order to de-
rive requirements for the structure–activity relationship of
glucosyltransferases as well as to ascertain essential sub-
stituents on aryl and hetaryl moieties.
-
-
O
O
P
O
O
N
O
P
O
O
O
1 X = OH, Y = CH
2 X = H, Y = CH
3 X = OH, Y = N
4 X = H, Y = N
OHOH
O
NHAc
2Na+
-
NH
-
O
O
O
O
O
N
O
P
P
O
O
O
R
5 R = OH
6 R = H
OH OH
(NBS) in the presence of catalytic amounts of dibenzoyl per-
oxide (DBP) in refluxing carbon tetrachloride (Scheme 1).
The bromides 11 and 12 on Michaelis–Arbuzov reaction
with tris-(trimethylsilyl) phosphite in toluene at 95°C for 4 h
furnished the respective bis-trimethylsilylphosphonates 15
1
and 16 based on the H NMR spectra in quantitative yield.
The bis-trimethylsilylphosphonates are highly labile to mois-
ture and had to be stored under argon. They were used in the
next step without further purification. The deprotection of
the TMS groups and subsequent preparation of sodium salts
were carried out in one pot by treating the phosphonates 15
and 16 with sodium methanolate in methanol at 0°C for
30 min, which after usual work up furnished disodium salts
19 and 20. They were transformed into their corresponding
bis-triethylammonium salts 23 and 24 by ion exchange
(Amberlite IR-120, HNEtꢂ3 form).
The synthesis of mimetics 1–6 was performed following a
recent procedure (29a) which is an improved variant of the
Moffat–Khorana phosphomorpholidate coupling method
(29b). The phosphonophosphates or diphosphates were iso-
1
lated in 42–52% yield. The H NMR and 13C NMR spectra
showed the expected signals and total assignment could be
performed by 2D NMR experiments. The 31P NMR spectra
also showed the characteristic signals associated with the
phosphorous atom. Reaction of bis-triethylammonium salts
23 and 24 with uridine-5>-morpholidophosphate as activated
UMP derivative in the presence of 1H-tetrazole in pyridine
for 3 days at room temperature afforded 1 and 2 as white
powders, respectively, after isolation and purification by
HPLC, ion exchange (Amberlite IR-120 (Na+ form)), and
lyophilization.
In similar fashion, synthesis of 3 and 4 were also carried
out (Scheme 1). Treatment of 9 (30) with NBS in the pres-
ence of catalytic amounts of ꢁ,ꢁ>-azo-isobutyronitrile (AIBN)
in CCl4 and irradiation of the reaction mixture for 8 h with a
Hg lamp furnished 13 whereas bromide 14 (31, 32) was
For the synthesis of sugar mimetics 1–4 the appropriate
phosphonates 15–18 had to be synthesized first. We based
our approach essentially on the Michaelis–Arbuzov reaction
of bromides 11–14 with tris-(trimethylsilyl) phosphite to
generate the corresponding phosphonates 15–18. The re-
quired benzyl bromide derivatives 11 and 12 (28) were pre-
pared from their corresponding methyl precursors 7 and 8,
respectively, by treating them with N-bromosuccinimide
2 A manuscript based on the latest results of recognition specifity of BGT is in preparation by J. Kurzeck and W. Rüger.
© 2002 NRC Canada

Bhattacharya et al.
975
Scheme 1. Reagents and conditions: (i) NBS, cat. DBP, CCl4, reflux, 4 h, 40–45% (for 7 and 8); (ii) NBS, cat. AIBN, CCl4, Hg lamp,
8h, 45% (for 9); (iii) CBr4, PPh3, NEt3, CH2Cl2, 0°C, 1 h, 42% (for 10); (iv) P(OTMS)3, toluene, 95°C, 3.5–4 h, quant.; (v) 0.2 M
NaOMe–MeOH, 0°C, 30 min, Amberlite IR-120 (H+ form); (vi) Amberlite IR-120 (HNEtꢂ3 form), 80–90% (two steps); (vii) UMP-
morpholidate, 1H-tetrazole, py, 3 days, rt, RP-18; (viii) Amberlite IR-120 (Na+ form), 42–52% (two steps).
prepared by treatment of 10 with CBr4–Ph3P–Et3N in
CH2Cl2 at 0°C. Subsequent reactions of bromides 13 and 14
with tris-trimethylsilyl phosphite furnished the correspond-
ing phosphonates 17 and 18 which were transformed into
their bis-triethylammonium salts 25 and 26 in the usual way.
Their reaction with uridine-5>-morpholidophosphate in pres-
ence of 1H-tetrazole (29) in pyridine for 3 days at room tem-
perature furnished 3 and 4 as white powders, respectively,
after isolation and purification by HPLC, ion exchange
(Amberlite IR-120 (Na+ form)) and lyophilization.
Finally, compounds 5 and 6 were synthesized by the reac-
tion of bis-triethylammonium salts 32 and 33 with uridine-
5>-morpholidophosphate as activated UMP derivative in
pyridine catalyzed by 1H-tetrazole (29) for 3 days at room
temperature. Isolation and purification by HPLC followed by
ion exchange [Amberlite IR-120 (Na+ form)] and lyophiliza-
tion afforded 5 and 6 as white powders in 43 and 45% yield,
respectively.
Biological results
The synthesis of compounds 5 and 6 were accomplished
as delineated in Scheme 2. The required alcohol 28 was pre-
pared from the readily available aldehyde 27 (33) in 60%
yield by treating the latter with NaBH4 in KH2PO4 buffer
(34) in dry methanol (Scheme 3). The allyl protected phos-
phates 30 and 31 were synthesized in an one-pot reaction.
The alcohols 28 and 29 (35) were coupled with
bis(allyloxy)(diisopropylamino)phosphine in dry dichloro-
methane–acetonitrile (1:1) activated by 1H-tetrazole and
then the resulting phosphites were oxidized with tert-butyl-
hydroperoxide to phosphates 30 and 31 in 76 and 51% yield,
respectively. The deprotection of allyl protecting groups
were carried out by treating the allylesters 30 and 31 with
dimedone in THF at room temperature catalyzed by tetrakis-
(triphenylphosphine)-palladium. The bis-triethylammonium
salts 32 and 33 were obtained after flash chromatography
(CHCl3–MeOH–H2O, 6:4:1 + 1% Et3N) in 64 and 72%
yield, respectively.
The compounds synthesized were tested for in vitro inhib-
itory activity with ꢀ(1J4)-glucosyltransferase which was
isolated as previously reported (36). The biological studies
reveal a strong structure–activity relationship for the binding
of inhibitors 1–6 to BGT. The IC50 values (concentration of
inhibitor at which 50% activity of the enzyme is observed)
of inhibitors 1–6 were determined and are listed in Table 1.
Inhibitor 2 showed an IC50 value of 1.9 × 10–3 M whereas
compound 1 showed very little inhibition thereby indicating
that the potential inhibitor from the substituted aryl group
should be lipophilic. However, to our surprise compounds 3
and 4 were totally inactive. Acetamidobenzyl derivative 6
showed an IC50 value of 9.6 × 10–5 M (Ki = 3.2 × 10–5 M)
whereas the para-hydroxy-substituted derivative 5 showed
an IC50 value of 7.8 × 10–4 M. Thus, 6 exhibits an affinity to
the enzyme which is comparable to the natural substrate
(UDP-Glc: KM = 3.6 × 10–5 M). X-ray studies with some of
© 2002 NRC Canada

976
Can. J. Chem. Vol. 80, 2002
Scheme 2. Reagents and conditions: (i) (i-Pr2N)P(OAll)2, 1H-
tetrazol, CH2Cl2–CH3CN (1:1), rt, 2.5 h; (ii) t-BuOOH, 0°C,
1.5 h, 76% (for 30), 51% (for 31); (iii) dimedone, Pd(PPh3)4,
THF, Et3N, rt, 1.5 h, 64% (for 32), 72% (for 33); (iv) UMP-
morpholidate, 1H-tetrazol, py, rt, 3 days, RP-18; (v) Amberlite
IR-120 (Na+ form), 43% (for 5), 45% (for 6).
Scheme 3. Reagents and conditions: (i) NaBH4, KH2PO4,
MeOH, 30 min, rt, 60%.
NHAc
NHAc
i
CHO
CH2OH
28
27
AcO
AcO
Table 1. IC50 values of transition-state analogues 1–6.a
Inhibitor
IC50 (M)
Ki (competitive) (M)
1
2
3
4
5
6
>5 × 10–3
1.9 (±0.1) × 10–3
>1 × 10–2
>1 × 10–2
7.8 (±0.3) × 10–4
9.6 (±0.4) × 10–5
1.8 (±0.1) × 10–3
3.2 (±0.1) × 10–5b
UDP
aFor details, see the Experimental section.
bKM of UDP-Glc: 3.6 × 10–5 M.
Tetramethylsilane (TMS) or the resonance of the deuterated
solvent was used as an internal standard; solvents: CDCl3 (ꢃ =
7.24), D2O (ꢃ = 4.63), [D6]DMSO (ꢃ = 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-dihy-
droxybenzoic acid (DHB) or 6-aza-2-thiothymine (ATT)
were used as matrices. FAB mass spectra were measured on
a Finnigan MAT312/AMD 5000 spectrometer. Thin layer
chromatography (TLC) was performed on Merck silica gel
plastic plates 60F254 or Merck glass plates RP-18; com-
pounds were visualized by treatment with a solution of
(NH4)6Mo7O24ꢄ4H2O (20 g) and Ce(SO4)2 (0.4 g) in 10%
sulfuric acid (400 mL). Flash chromatography was per-
formed on J.T. Baker silica gel 60 (0.040–0.063 mm) at a
pressure of 0.3 bar. MPLC was performed at a pressure of
5–10 bar; column: silica gel LiChroprep Si 60 (Merck,
15–25 m, 28 × 2.5 cm). Preparative HPLC separations
were performed on an Autochrom system with a Shimadzu
LC8A preparative pump and a Rainin Dynamax UV 1 detector
at 260 nm. The column used was a Lichrosorb RP-18, 7 m,
250 × 16 mm (Knauer, Germany). Mixtures of acetonitrile
and 0.05 M triethylammonium bicarbonate (TEAB) (pH 7.0
to 7.2) were used as the mobile phase.
these compounds bound to BGT will be reported in due
course.
In conclusion, our inhibition studies clearly indicate that:
(i) phosphates 5 and 6 are better inhibitors than phos-
phonates 1–4 in general; (ii) acetamido functionality on the
aryl residue greatly enhances the inhibition property; (iii) in
phosphonates, methyl substitution shows some activity; and
(iv) substituted pyridyl phosphonates 3 and 4 are not active
at all. Our studies have also revealed that benzene rings are
accepted by this glucosyltransferase (and presumably by
carbohydrate processing enzymes in general) as mimics of
carbohydrate residues. Thus, useful structure–activity rela-
tionships and further guidelines for the design of future gen-
erations of inhibitors of BGT are provided.
2-Acetoxymethyl-6-methylpyridine (9)
2-Acetoxymethyl-6-methylpyridine (9) was synthesized
following a procedure by Baker et al. (32). The NMR data
were in good agreement with the reported data (30).
General procedure for the synthesis of compounds (11)
and (12)
To a solution of 7 or 8 (6–9 mmol) in carbon tetrachloride
(CCl4) (20–60 mL), N-bromo succinamide (NBS) (1 equiv)
and a catalytic amount of dibenzoyl peroxide (DBP) were
added and refluxed for 4 h. Evaporation of the solvent and
chromatography (MPLC or flash) of the residue afforded
compounds 11 or 12, respectively.
Experimental section
Solvents were purified according to the standard proce-
dures. NMR measurements were recorded at 22°C on a
Bruker AC250 Cryospec or Bruker DRX 600 spectrometers.
© 2002 NRC Canada

Bhattacharya et al.
977
der argon was used as such in the next step without further
purification.
3-Bromomethylbenzyl acetate (11)
Treatment of 3-methylbenzyl acetate 7 (1.0 g, 6.1 mmol)
according to the general procedure afforded 11 (620 mg,
42%) as a colorless oil after MPLC (toluene). Rf = 0.67 (pe-
troleum ether–ethyl acetate, 19:1). 1H NMR (250 MHz,
CDCl3) ꢃ: 2.11 (s, 3H, COCH3), 4.49 (s, 2H, Ar-CH2Br),
5.10 (s, 2H, Ar-CH2OAc), 7.34–7.38 (m, 4H, Ar-H). MS
(EI) (m/z): 242 (M+). Anal. calcd. for C10H11BrO2 (243.01):
C 49.43, H 4.56; found: C 49.23, H 4.50.
Bis-(trimethylsilyl) 3-(acetoxymethyl)benzylphosphonate (15)
A mixture of 11 (160 mg, 0.67 mmol) and tris-
(trimethylsilyl) phosphite (420 L, 2.0 mmol) in dry toluene
(2 mL) was treated as described in the general procedure to
afford 15 as a colorless oil which was used in the next step
1
without further purification. H NMR (600 MHz, CDCl3) ꢃ:
0.27 (d, J = 1.2 Hz, 18H, 6CH3), 2.08 (s, 3H, COCH3), 3.06
(d, J = 22.3 Hz, 2H, Ar-CH2P), 5.07 (s, 2H, Ar-CH2OAc),
7.22–7.43 (m, 4H, Ar-H). 13C NMR (150.8 MHz, CDCl3) ꢃ:
3-Methylbenzyl bromide (12)
Treatment of m-xylene 8 (1.0 g, 9.4 mmol) according to
the general procedure afforded 12 (780 mg, 45%) as a color-
less oil after flash chromatography (petroleum ether–ethyl
acetate, 19:1). Rf = 0.60 (petroleum ether–ethyl acetate,
19:1). The physical data were in agreement with the reported
(28) data.
0.9, 1.0 (6C, 6CH3), 21.0 (1C, COCH3), 35.5 (d, JC,P
=
156.1 Hz, 1C, Ar-CH2P), 66.1 (1C, Ar-CH2OAc), 126.6,
128.6, 129.7, 130.0, 132.6, 136.1 (6C, Ar), 170.8 (1C,
COCH3). 31P NMR (243 MHz, CDCl3) ꢃ: 21.81. MS (EI)
(m/z): 388 (M+) for C16H29O5PSi2 (388.5).
Bis-(trimethylsilyl) 3-methylbenzylphosphonate (16)
A mixture of 12 (195 mg, 1.06 mmol) and tris-
(trimethylsilyl) phosphite (600 L, 2 mmol) in dry toluene
(2 mL) was treated as described in the general procedure
2-Acetoxymethyl-6-bromomethylpyridine (13)
A mixture of 9 (1.0 g, 6.0 mmol), NBS (1.1 g, 6.1 mmol)
and ꢁ,ꢁ>-azo-isobutyronitrile (AIBN) (100 mg, 0.61 mmol)
in CCl4 (65 mL) was irradiated with a Hg lamp for 8 h.
Flash chromatography (petroleum ether–ethyl acetate, 7:3)
afforded 13 (660 mg, 45%) as a colorless oil. Rf = 0.44 (pe-
troleum ether–ethyl acetate, 3:2). 1H NMR (250 MHz,
CDCl3) ꢃ: 2.17 (s, 3H, COCH3), 4.55 (s, 2H, Ar-CH2Br),
5.23 (s, 2H, Ar-CH2OAc), 7.28 (d, J3,4 = 7.6 Hz, 1H, H-3),
1
above to afford 16 as a colorless oil. H NMR (600 MHz,
CDCl3) ꢃ: 0.30 (d, J = 1.2 Hz, 18H, 6CH3Si), 2.31 (s, 3H,
CH3), 3.02 (d, J = 22.1 Hz, 2H, Ar-CH2P), 7.01–7.30 (m,
4H, Ar-H). 31P NMR (243 MHz, CDCl3) ꢃ: 22.18. MS (EI)
(m/z): 330 (M+) for C14H27O3PSi2 (330.5).
Bis-(trimethylsilyl) 2-[6-(acetoxymethyl)pyridyl]methyl-
phosphonate (17)
7.40 (d, J5,4 = 7.6 Hz, 1H, H-5), 7.72 (dd, J4,3 = J4,5
=
7.6 Hz, 1H, H-4). MS (EI) (m/z): 243 (M+). Anal. calcd. for
C9H10NO2Br (244.1): C 44.44, H 4.12, N 5.76; found: C 44.34,
H 4.18, N 5.66.
A mixture of 13 (360 mg, 1.50 mmol) and tris-
(trimethylsilyl) phosphite (950 L, 3.0 mmol) in dry toluene
(2 mL) was treated as described in the general procedure
1
above to afford 17 as a colorless oil. H NMR (600 MHz,
2-Bromomethyl-6-methylpyridine (14)
CDCl3) ꢃ: 0.31 (d, J = 1.2 Hz, 18H, 6CH3Si), 2.12 (s, 3H,
COCH3), 3.31 (d, J = 22.7 Hz, 2H, Ar-CH2P), 5.15 (s, 2H,
Ar-CH2OAc), 7.20 (d, J3,4 = 7.7 Hz, 1H, H-3), 7.31 (d, J5,4
7.7 Hz, 1H, H-5), 7.62 (dd, J4,3 = J4,5 = 7.7 Hz, 1H, H-4).
31P NMR (243 MHz, CDCl3) ꢃ: 12.61. FAB-MS (positive
mode, matrix: CHCl3–NBA–NaI) (m/z): 390 (M + H)+, 412
(M + Na)+ for C15H28NO5PSi2 (389.5).
To a stirred solution of 2-(hydroxymethyl)-6-methylpyri-
dine (1.0 g, 8.1 mmol) in dry CH2Cl2 (80 mL) at 0°C, car-
bon tetrabromide (CBr4) (3.2 g, 9.8 mmol), triphenyl
phosphine (PPh3) (2.6 g, 9.8 mmol), and triethylamine
(Et3N) (1.1 mL, 820 mg, 8.1 mmol) were added succes-
sively. After 1 h when the reaction was complete (TLC), the
reaction mixture was dried in vacuo and purified by flash
chromatography (n-pentane–diethyl ether, 9:1) to afford 14
(630 mg, 42%) as a colorless oil. The physical data were in
full agreement with the reported data (32). Rf = 0.29 (n-
=
Bis-(trimethylsilyl) 2-(6-methylpyridyl)methylphosphonate
(18)
A mixture of 14 (360 mg, 1.50 mmol) and tris-
(trimethylsilyl) phosphite (950 L, 3.0 mmol) in dry toluene
(2 mL) was treated as described in the general procedure
1
pentane–diethyl ether, 4:1). H NMR (250 MHz, CDCl3) ꢃ:
2.56 (s, 3H, CH3), 4.52 (s, 2H, Ar-CH2Br), 7.07 (d, J3,4
=
7.7 Hz, 1H, H-3), 7.25 (d, J5,4 = 7.7 Hz, 1H, H-5), 7.58 (dd,
J4,3 = J4,5 = 7.7 Hz, 1H, H-4). MS (EI) (m/z): 185 (M+), 106
(M – Br)+. Anal. calcd. for C7H8NBr (186.1): C 45.51,
H 4.32, N 7.57; found: C 45.36, H 4.34, N 7.48.
1
above to afford 18 as a colorless oil. H NMR (600 MHz,
CDCl3) ꢃ: 0.32 (d, J = 1.2 Hz, 18H, 6CH3Si), 2.46 (s, 3H,
CH3), 3.24 (d, J = 22.7 Hz, 2H, Ar-CH2P), 6.95 (d, J3,4
7.6 Hz, 1H, H-3), 7.12 (d, J5,4 = 7.6 Hz, 1H, H-5), 7.45 (dd,
J4,3 = J4,5 = 7.6 Hz, 1H, H-4). MS (EI) (m/z): 331 (M+) for
C13H26NO3PSi2 (331.5).
=
General procedure for the synthesis of phosphonates
(15–18)
A mixture of 11, 12, 13, or 14 (1 mmol) and tris-
(trimethylsilyl) phosphite (2 mmol) in dry toluene (2 mL)
was heated under argon to 95°C for 4 h to afford 15, 16, 17,
or 18, respectively, in a quantitative reaction (1H NMR). The
formed trimethylsilyl bromide was distilled off under
slightly reduced pressure. The mixture was concentrated in
vacuo and excess tris-(trimethylsilyl) phosphite was removed
under high vacuum (0.01 mbar). The residue to be kept un-
General procedure for the synthesis of disodium salts
(19–22)
The crude phosphonate ( 0.35g) was disolved in dry
CH2Cl2 (3 mL) under argon and the solution added dropwise
through a syringe to a sodium methoxide solution (30 mL,
0.2 M) at 0°C. After 30 min, small amounts of Amberlite
IR-120 (H+ form) were added to the solution with stirring
till the solution became neutral (pH paper). The solution was
© 2002 NRC Canada

978
Can. J. Chem. Vol. 80, 2002
filtered and the resin was washed once with MeOH (5 mL)
and added to the filtrate. The filtrate was evaporated and
coevaporated with water to furnish a solid. Finally, the solid
obtained was lyophilized with water to afford the disodium
salt as a white powder.
Amberlite IR-120 (HNEtꢂ3-form). The column was washed
thoroughly with H2O and all the fractions collected were
lyophilized to afford 23, 24, 25, or 26 as a colorless oil.
Bis-triethylammonium 3-(hydroxymethyl)benzyl-
phosphonate (23)
Disodium 3-(hydroxymethyl)benzylphosphonate (19)
The phosphonate 15 (270 mg, crude material) was
disolved in dry CH2Cl2 (3 mL) under argon and was treated
with sodium methoxide solution (30 mL, 0.2 M) at 0°C as
per the general procedure described above to yield 19 as a
The disodium salt 19 (250 mg, 1.02 mmol) was treated as
per the general procedure described above to furnish 23 as a
colorless oil (380 mg, 92%). H NMR (600 MHz, D2O) ꢃ:
1
1.19 (t, J = 7.3 Hz, 18H, NCH2CH3), 2.96 (d, J = 20.6 Hz,
2H, Ar-CH2P), (q, J = 7.3 Hz, 12H, NCH2CH3), 4.54 (s, 2H,
Ar-CH2OH), 7.11 (m, 2H, H-4, H-6), 7.17 (s, 1H, H-2), 7.19
(dd, J5,4 = J5,6 = 7.6 Hz, 1H, H-5). 13C NMR (151 MHz,
D2O) ꢃ: 7.7 (1C, NCH2CH3), 35.1 (d, JC,P = 128 Hz, 1C,
Ar-CH2P), 46.2 (1C, NCH2CH3), 63.4 (1C, Ar-CH2OH),
124.7, 128.1, 128.2, 128.4, 135.0, 139.7 (6C, Ar). 31P NMR
(243 MHz, D2O) ꢃ: 19.49 Hz. FAB-MS (negative mode,
1
white powder (166 mg, 98%). H NMR (600 MHz, CDCl3)
ꢃ: 2.72 (d, J = 19.7 Hz, 2H, Ar-CH2P), 4.46 (s, 2H, Ar-
CH2OH), 7.04–7.18 (m, 4H, Ar-H). 13C NMR (151 MHz,
D2O) ꢃ: 36.2 (d, JC,P = 127 Hz, 1C, Ar-CH2P), 63.6 (1C,
Ar-CH2OH), 123.8, 127.9, 128.2, 128.5, 138.0, 139.2 (6C,
Ar). 31P NMR (243 MHz, D2O) ꢃ: 19.01. FAB-MS (negative
mode, matrix: DMSO–H2O–glycerol, 1:1:1) (m/z): 201 [M –
2Na+ + H+]– for C8H9O4PNa2 (246.1).
matrix: MeOH–glycerol, 1:1) (m/z): 201 [M – 2HNEt3ꢂ
+
H+]– for C20H41N2O4P (404.5).
Disodium 3-methylbenzylphosphonate (20)
Bis-triethylammonium 3-methylbenzylphosphonate (24)
The phosphonate 16 (374 mg, crude material) was dis-
solved in dry CH2Cl2 (3 mL) under argon and was treated
with sodium methoxide solution (30 mL, 0.2 M) at 0°C as
per the general procedure described above to yield 20 as a
The disodium salt 20 (100 mg, 0.43 mmol) was treated as
per the general procedure described above to furnish 24 as a
1
colorless oil (156 mg, 92%). H NMR (600 MHz, D2O) ꢃ:
1.09 (t, J = 7.3 Hz, 18H, NCH2CH3), 2.14 (s, 3H, CH3), 2.82
(d, J = 20.7 Hz, 2H, Ar-CH2P), 3.01 (q, J = 7.3 Hz, 12H,
NCH2CH3), 6.80–7.12 (m, 4H, Ar-H). FAB-MS (negative
mode, matrix: MeOH–glycerol, 1:1) (m/z): 185 [M –
2HNEtꢂ3 + H+]– for C20H41N2O3P (388.5).
1
white powder (250 mg, 96%). H NMR (600 MHz, CDCl3)
ꢃ: 2.17 (s, 3H, CH3) 2.67 (d, J = 19.8 Hz, 2H, Ar-CH2P),
6.97 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.05 (d, J5,6 = 7.8 Hz, 1H,
H-6), 7.10 (s, 1H, H-2), 7.13 (dd, J5,4 = J5,6 = 7.8 Hz, 1H,
H-5). 31P NMR (243 MHz, D2O) ꢃ: 19.02. FAB-MS
(negative mode, matrix: H2O–glycerol, 1:1) (m/z): 185 [M –
2Na+ + H+]– for C8H9O3PNa2 (230.1).
Bis-triethylammonium 2-[6-(hydroxymethyl)pyridyl]methyl-
phosphonate (25)
The disodium salt 21 (320 mg, 1.30 mmol) was treated as
per the general procedure described above to furnish 25 as a
colorless oil (478 mg, 92%). H NMR (250 MHz, D2O) ꢃ:
Disodium 2-[6-(hydroxymethyl)pyridyl]methylphosphonate
(21)
1
The phosphonate 17 (580 mg, crude material) was dissolved
in dry CH2Cl2 (3 mL) under argon and was treated with so-
dium methoxide solution (30 mL, 0.2 M) at 0°C as per the
general procedure described above to yield 21 as a white
powder (320 mg, 86%). 1H NMR (250 MHz, CDCl3) ꢃ: 2.97
(d, J = 20.7 Hz, 2H, Ar-CH2P), 4.96 (s, 2H, Ar-CH2OH),
7.12 (d, J3,4 = 7.8 Hz, 1H, H-3), 7.18 (d, J5,4 = 7.8 Hz, 1H,
H-5), 7.59 (dd, J4,3 = J4,5 = 7.8 Hz, 1H, H-4). FAB-MS (pos-
itive mode, matrix: H2O – CH3CN – 0.1% TFA, 1:1:1) (m/z):
248 [M + 2Na+ + H]+ for C7H8NO4PNa2 (247.1).
1.06 (t, J = 7.3 Hz, 18H, NCH2CH3), 2.95 (q, J = 7.3 Hz,
12H, NCH2CH3), 3.09 (d, J = 21.7 Hz, 2H, Ar-CH2P), 5.04
(s, 2H, Ar-CH2OH), 7.31 (m, 2H, H-3, H-5), 7.79 (dd, J4,3
=
J4,5 = 7.7 Hz, 1H, H-4). FAB-MS (positive mode, matrix:
H2O – CH3CN – 0.1% TFA (1:1:1), glycerol) (m/z): 248
[M – HNEtꢂ3 + 2Na+ + H+]+ for C19H40N3O4P (405.5).
Bis-triethylammonium 2-(6-methylpyridyl)methylphos-
phonate (26)
The disodium salt 22 (428 mg, 1.85 mmol) was treated as
per the general procedure described above to furnish 23 as a
1
Disodium 2-(6-methylpyridyl)methylphosphonate (22)
The crude phosphonate 18 (1.11 g) was dissolved in dry
CH2Cl2 (3 mL) under argon and was treated with sodium
methoxide solution (30 mL, 0.2 M) at 0°C as per the general
procedure described above to yield 22 as a white powder
colorless oil (605 mg, 84%). H NMR (250 MHz, D2O) ꢃ:
1.09 (t, J = 7.3 Hz, 18H, NCH2CH3), 2.56 (s, 3H, CH3), 3.02
(q, J = 7.3 Hz, 12H, NCH2CH3), 3.24 (d, J = 20.8 Hz, 2H,
Ar-CH2P), 7.59 (d, J3,4 = 7.9 Hz, 1H, H-3), 7.62 (d, J5,4
=
7.9 Hz, 1H, H-5), 8.23 (dd, J4,3 = J4,5 = 7.9 Hz, 1H, H-4).
31P NMR (243 MHz, D2O) ꢃ: 13.80 Hz. FAB-MS (positive
mode, matrix: CH3CN – 0.1% TFA (1:1), glycerol) (m/z):
188 [M – 2NEtꢂ3 + H+]– for C19H40N3O3P (389.5).
1
(693 mg, 90%). H NMR (250 MHz, CDCl3) ꢃ: 2.41 (s, 3H,
CH3), 2.95 (d, J = 19.6 Hz, 2H, Ar-CH2P), 7.15 (d, J3,4
=
7.8 Hz, 1H, H-3), 7.28 (d, J5,4 = 7.8 Hz, 1H, H-5), 7.74 (dd,
J4,3 = J4,5 = 7.8 Hz, 1H, H-4). FAB-MS (negative mode,
matrix: H2O–glycerol, 1:1) (m/z): 186 [M – 2Na+ + H+]–,
208 [M + Na+]– for C7H8NO3PNa2 (231.1).
General procedure for the synthesis of (1–6)
A mixture of 23, 24, 25, 26, 31, or 32 (0.13–0.25 mmol)
and 4-morpholine-N,N>-dicyclohexylcarboxamidinium uridine
5>-monophosphomorpholidate (0.17–0.27 mmol) was evapo-
rated with dry pyridine (3 × 1.5 mL). To this mixture,
vacuum-dried 1H-tetrazole (0.29–1.10 mmol) and dry pyridine
(1 mL) were added under argon and the solution was stirred
General procedure for the synthesis of bis-triethyl-
ammonium salts (23–26)
The disodium salts of 19, 20, 21, or 22 were dissolved in
H2O and passed through a column (10 × 2.5 cm) containing
© 2002 NRC Canada

Bhattacharya et al.
979
at room temperature (rt) for 3 days. The reaction mixture
was evaporated and coevaporated with water (2 × 1 mL).
The residue was purified by preparative HPLC (0.05 M
13C NMR (151 MHz, D2O) ꢃ: 19.89 (1C, CH3), 35.0 (d,
JC,P = 135 Hz, 1C, C-1>>), 63.9 (1C, C-5>), 68.8 (1C, C-3>),
73.2 (1C, C-2>), 82.5 (1C, C-4>), 8.0 (1C, C-1>), 102.0 (1C,
C-5), 126.2, 127.8, 129.91, 129.95, 133.9, 137.8 (6C, Ar),
140.9 (1C, C-6), 151.3, 165.8 (2C, C-2, C-4). 31P NMR
(243 MHz, D2O) ꢃ: –10.3 (d, J = 27.7 Hz, P(O)O3), 15.1 (d,
J = 27.7 MHz, CP(O)O2). MS (MALDI, negative mode, ma-
trix: ATT, H2O) (m/z): 491.6 [M – 2Na+ + H+]– for
C17H20N2O11P2Na2 (536.28).
triethylammonium hydrogencarbonate buffer
+
4ꢅ8%
MeCN, flow rate: 10–18 mL min–1). The fractions contain-
ing product were pooled and evaporated. The residue was
dissolved in water (2 mL) and passed through a column
(2.5 × 12 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 dissolved in
a 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 lyophilized. After
lyophilization, 1, 2, 3, 4, 5, or 6 was obtained as a white
powder in 42–52% yield.
Disodium uridine 5>-phosphono {2-[6-(hydroxymethyl)py-
ridyl]methyl}phosphate (3)
A mixture of 25 (100 mg, 0.25 mmol) and 4-morpholine-
N,N>-dicyclohexylcarboxamidinium uridine 5>-monophos-
phomorpholidate (227 mg, 0.33 mmol) and 1H-tetrazole
(45 mg, 0.56 mmol) in dry pyridine (1 mL) was treated as
per the general procedure described above. After 3 days, the
product was purified by HPLC (0.05 M triethylammonium
Disodium uridine 5>-phosphono [3-(hydroxymethyl)ben-
zyl]phosphate (1)
hydrogencarbonate buffer
+
5% MeCN, flow rate:
10 mL min–1, tR 29.5 min). Compound 3 was obtained as a
A mixture of 23 (86 mg, 0.21 mmol) and 4-morpholine-
N,N>-dicyclohexylcarboxamidinium uridine 5>-monophos-
phomorpholidate (187 mg, 0.27 mmol) and 1H-tetrazole
(32 mg, 0.46 mmol) in dry pyridine (1 mL) was treated as
per the general procedure described above. After 3 days, the
product was purified by HPLC (0.05 M triethylammonium
white powder (59 mg, 42%) after ion-exchange and precipi-
1
tation. H NMR (600 MHz, D2O) ꢃ: 3.28 (d, J = 21.8 Hz,
2H, H-1a>>, H-1b>>), 3.98 (m, 1H, H-5a>), 4.03 (m, 1H,
H-5b>), 4.11 (m, 1H, H-4>), 4.13 (dd, J3>>,2>> = J3>>,4>> = 4.9 Hz,
1H, H-3>), 4.20 (dd, J2>,3> = J2>,1> = 5.0 Hz, 1H, H-2>), 4.59 (s,
2H, Ar-CH2OH), 5.74 (d, J5,6 = 7.8 Hz, 1H, H-5), 5.87 (d,
1H, J1>,2> = 5.0 Hz, H-1>), 7.24 (d, J5 >>,6 >> = 7.6 Hz, 1H, H-
hydrogencarbonate buffer
+
6% MeCN, flow rate:
18 mL min–1, tR 14.0 min). Compound 1 was obtained as a
white powder (62 mg, 52%) after ion exchange and precipi-
5>>), 7.30 (d, J7 >>,6 >> = 7.6 Hz, 1H, H-7>>), 7.67 (dd, J6 >>,5 >>
=
1
tation. H NMR (600 MHz, D2O) ꢃ: 3.10 (d, J = 21.4 Hz,
J6 >>,7 >> = 7.6 Hz, 1H, H-6>>), 7.72 (d, J6,5 = 7.8 Hz, 1H,
H-6). 31P NMR (243 MHz, D2O) ꢃ: –10.00 (d, J = 27.6 Hz,
P(O)O3), 12.20 (d, J = 27.6 MHz, CP(O)O2). FAB-MS (pos-
itive mode, matrix: H2O – CH3CN – 0.1% TFA (1:1:1),
glycerol) (m/z): 532 [M – Na+ + 2H+]+ for C16H19N3O12P2Na2
(553.27).
H 2H, -1a>>, H-1b>>), 3.96 (m, 1H, H-5a>), 4.02 (m, 1H, H-5b>),
4.12 (m, 2H, H-3>, H-4>), 4.20 (dd, J2>,3> = J2>,1> = 4.9 Hz, 1H,
H-2>), 4.53 (s, 2H, -CH2OH), 5.77 (d, J5,6 = 8.0 Hz, 1H,
H-5), 5.86 (d, J1>,2> = 4.9 Hz, 1H, H-1>), 7.14–7.25 (m, 4H,
H-3>>, H-5>>, H-6>>, H-7>>), 7.75 (d, J6,5 = 8.0 Hz, 1H, H-6).
13C NMR (151 MHz, D2O, assignment by HMQC) ꢃ: 35.06
(d, JC,P = 133.5 Hz, 1C, C-1>>), 63.4 (1C, Ar-CH2OH), 64.0
(1C, C-5>), 68.9 (1C, C-3>), 73.2 (1C, C-2>), 82.4 (1C,
C-4>), 88.1 (1C, C-1>), 102.2 (1C, C-5), 124.7, 128.2, 128.3,
128.6, 134.4, 139.7 (6C, Ar), 140.6 (1C, C-6). 31P NMR
(243 MHz, D2O) ꢃ: –9.95 (d, J = 27.6 Hz, P(O)O3), 15.04
(d, J = 27.6 MHz, CP(O)O2). MS (MALDI, negative mode,
matrix: ATT, H2O) (m/z): 507.5 [M – 2Na+ + H+]– for
C17H20N2O12P2Na2 (552.28).
Disodium uridine 5>-phosphono [2-(6-methylpyri-
dyl)methyl]phosphate (4)
A mixture of 26 (70 mg, 0.18 mmol) and 4-morpholine-
N,N>-dicyclohexylcarboxamidinium uridine 5>-monophos-
phomorpholidate (124 mg, 0.18 mmol) and 1H-tetrazole
(22 mg, 0.31 mmol) in dry pyridine (1 mL) was treated as
per the general procedure described above. After 3 days, the
product was purified by HPLC (0.05 M triethylammonium
hydrogencarbonate buffer
+
4% MeCN, flow rate:
Disodium uridine 5>-phosphono (3-methylbenzyl)phos-
phate (2)
10 mL min–1, tR 14.13 min). Compound 4 was obtained as a
white powder (58.5 mg, 42%) after ion exchange and precip-
itation. 1H NMR (600 MHz, D2O) ꢃ: 2.46 (s, 3H, CH3), 3.00
(d, J = 19.6 MHz, 2H, H-1a>>, H-1b>>), 4.06 (m, 1H, H-5a>),
4.13 (m, 2H, H-5b>, H-4>), 4.21 (m, 2H, H-3>, H-2>), 5.80
(d, J5,6 = 8.1 Hz, 1H, H-5), 5.82 (d, J1>,2 = 4.2 Hz, 1H, H-
1> ), 7.20 (d, J5 >>,6 >> = 7.7 Hz, 1H, H-5>>), 7.33 (d, 1H, J7 >>,6 >>
= 7.8 Hz, H-7>>), 7.79 (m, 2H, H-6>>, H-6). 31P NMR
(243 MHz, D2O) ꢃ: –9.63 (bd, P(O)O3), 17.14 (bd, CP(O)O2).
FAB-MS (positive mode, matrix: H2O – CH3CN – 0.1%
TFA (1:1:1), glycerol) (m/z): 516 [M – Na+ + 2H+]+ for
C16H19N3O11P2Na2 (537.27).
A mixture of 24 (50 mg, 0.13 mmol) and 4-morpholine-
N,N>-dicyclohexylcarboxamidinium uridine 5>-monophos-
phomorpholidate (116 mg, 0.17 mmol) and 1H-tetrazole
(20 mg, 0.29 mmol) in dry pyridine (1 mL) was treated as
per the general procedure described above. After 3 days, the
product was purified by HPLC (0.05 M triethylammonium
hydrogencarbonate buffer
+
8% MeCN, flow rate:
18 mL min–1, tR 27.8 min). Compound 2 was obtained as a
white powder (29 mg, 42%) after ion exchange and precipi-
1
tation. H NMR (600 MHz, D2O) ꢃ: 2.22 (s, 3H, CH3), 3.05
(d, J = 21.4 MHz, 2H, H-1a>>, H-1b>>), 3.95 (m, 1H, H-5a>),
4.04 (m, 1H, H-5b>), 4.11 (m, 2H, H-3>, H-4>), 4.21 (dd,
J2>,3> = J2>,1> = 4.9 Hz, 1H, H-2>), 5.78 (d, 1H, J5,6 = 8.1 Hz,
H-5), 5.85 (d, J1>,2> = 4.9 Hz, 1H, H-1>), 6.99–7.14 (m, 4H,
H-3>>, H-5>>, H-6>>, H-7>>), 7.78 (d, J6,5 = 8.1 Hz, 1H, H-6).
2-Acetamido-5-acetoxy-benzaldehyde (27)
2-Acetamido-5-acetoxy-benzaldehyde (27) was synthesized
as previously described (33).
© 2002 NRC Canada

980
Can. J. Chem. Vol. 80, 2002
residue was purified over flash chromatography (CHCl3–
MeOH–H2O, 6:4:1 + 1% Et3N) and afforded a mixture of 32
and 5-O-acetylated product which was dissolved in MeOH
(50 mL). To this solution, Et3N (4 mL) was added and
stirred at 45°C for 20 h. The reaction mixture was evapo-
rated in vacuum and lyophilized in water. After lyophiliza-
tion, compound 32 was obtained as a colorless oil (140 mg,
64%). Rf = 0.3 (CHCl3–MeOH–H2O, 6:4:1). 1H NMR
(250 MHz, D2O) ꢃ: 1.08 (t, J = 7.4 Hz, 18H, NCH2CH3),
2.01 (s, 3H, COCH3), 3.00 (q, J = 7.4 Hz, 12H, NCH2CH3),
4.59 (d, J = 6.4 Hz, 2H, CH2Ar), 6.67 (dd, J4,3 = 8.6 Hz,
J4,6 = 2.9 Hz, 1H, H-4), 6.87 (d, J6,4 = 2.9 Hz, 1H, H-6),
7.04 (d, J3,4 = 8.6 Hz, 1H, H-3). MS (MALDI, positive
4-Acetamido-3-hydroxymethylphenyl acetate (28)
To a solution of 27 (220 mg, 1.0 mmol) in dry methanol
(25 mL), potassium dihydrogen phosphate (1.36 g, 10 mmol)
was added. The reaction mixture was cooled to 0°C and then
NaBH4 (94 mg, 2.5 mmol) was added under argon and fur-
ther stirred for 30 min. Then cold water (20 mL) was poured
into the reaction mixture and the solution was acidified to
pH 5 with 5% HCl solution. The methanol was distilled off
and the aqueous phase was extracted with CH2Cl2 (5 ×
80 mL). The organic phase was washed with water and dried
over MgSO4. Evaporation and flash chromatography (tolu-
ene–acetone, 3:1) afforded 28 (130 mg, 60%) as a white
solid. Rf = 0.45 (toluene–acetone, 1:1), mp 140–142°C.
1H NMR (250 MHz, [D6]DMSO) ꢃ: 2.03 (s, 3H, NHCOCH3),
2.25 (s, 3H, OCOCH3), 4.45 (d, J = 5.6 Hz, C 2H, H2-Ar),
5.31 (t, J = 5.7 Hz, 1H, CH2OH, exchanged with D2O), 6.95
mode, matrix: DHB) (m/z): 284 [M – 2HEt3N+ + 2H+
+
Na+]+ for C21H41N3O6P (463.5).
(dd, J2,6 = 2.7 Hz, J5,6 = 8.6 Hz, 1H, H-6), 7.15 (d, J2,6
=
Disodium (2-acetamido-5-hydroxy)benzyl-uridine-5 >-
2.7 Hz, 1H, H-2), 7.41 (d, J5,6 = 8.6 Hz, 1H, H-5), 9.29
(s, 1H, NH). Anal. calcd. for C11H13NO4 (223.2): C 59.19,
H 5.87; found: C 58.90; H 5.71.
diphosphate (5)
A mixture of 32 (32 mg, 0.069 mmol) and 4-morpholine-
N,N>-dicyclohexylcarboxamidinium uridine 5>-monophos-
phomorpholidate (106 mg, 0.15 mmol) and 1H-tetrazole
(24 mg, 0.34 mmol) in dry pyridine (1 mL) was treated as
per the general procedure described above. After 3 days, the
product was purified by HPLC (0.05 M triethylammonium
(2-Acetamido-5-acetoxy-benzyl) diallyl phosphate (30)
To a solution of 28 (160 mg, 0.717 mmol) in a mixture of
dry CH2Cl2 and dry MeCN (each 10 mL) under argon, vac-
uum-dried 1H-tetrazole (135 mg, 1.94 mmol) was added at
room temperature. Bis(allyloxy)(diisopropylamino)phosphine
(250 L, 0.95 mmol) was added dropwise to the reaction
mixture through a syringe under argon. After stirring at
room temperature for 2.5 h, the reaction mixture was cooled
to 0°C and 5.5 M t-BuOOH solution in Nonan (195 L,
1.07 mmol) was added dropwise and after completion of the
addition, the reaction mixture was further stirred for 1.5 h at
room temperature. After completion of the reaction (TLC),
Na2S2O3 solution (50 mL) was poured into the reaction mix-
ture. The organic phase was washed successively with NaCl
solution, NaHCO3 solution, and once again with NaCl solu-
tion (each 50 mL). The aqueous solution was extracted with
CH2Cl2 (2 × 25 mL) and added to the organic phase. The
pooled organic phase was dried (MgSO4) and evaporated to
dryness. The residue after flash chromatography (toluene–
acetone, 4:1) afforded 30 as a colorless oil (210 mg, 76%).
Rf = 0.52 (toluene–acetone, 1:1). 1H NMR (250 MHz,
CDCl3) ꢃ: 2.24 (s, 3H, NHCOCH3), 2.30 (s, 3H,OCOCH3),
4.49 (m, 4H, CH2=CH-CH2), 5.03 (d, J = 11.2 Hz, 2H,
CH2Ph), 5.22–5.37 (m, 4H, CH2=CH-CH2), 5.81–5.95 (m,
2H, CH2=CH-CH2), 7.12 (d, J6,4 = 1.8 Hz, 1H, H-6), 7.14
hydrogencarbonate buffer
+
5% MeCN, flow rate:
16 mL min–1, tR 8.5 min). Compound 5 was obtained as a
white powder (18.3 mg, 43%) after ion exchange and precip-
1
itation. H NMR (250 MHz, D2O) ꢃ: 2.00 (s, 3H, COCH3),
3.95–4.02 (m, 5H, H-2>, H-3>, H-4>, H-5a>, H-5b>), 4.69 (d,
2H, CH2Ar), 5.56 (d, J = 8.1 Hz, 1H, 5-H), 5.66 (d, J =
2.9 Hz, 1H, H-1>), 6.61 (dd, J4,3 = 8.6 Hz, J4,6 = 2.8 Hz, 1H,
H-4>>), 6.80 (d, J6,4 = 2.8 Hz, 1H, H-6>>), 6.93 (d, J3,4
=
8.6 Hz, 1H, H-3>>), 7.62 (d, J = 8.1 Hz, 1H, H-6). 31P NMR
(243 MHz, D2O) ꢃ: –12.2 (bd, 2P). MS (MALDI, negative
mode, matrix: ATT, H2O) (m/z): 566 [M – 2Na+ + H+]– for
C18H21N3O14P2 Na2 (611.4).
2-Acetamidobenzyl alcohol (29)
2-Acetamidobenzyl alcohol (29) was synthesized from
2-aminobenzyl alcohol by following the procedure reported
by Söderbaum and Widman (35a) in 95% yield. All the
physical data were in agreement with the reported (35b)
data.
2-Acetamidobenzyl diallyl phosphate (31)
To a solution of 29 (190 mg, 1.15 mmol) in a mixture of
dry CH2Cl2 and dry MeCN (each 12.5 mL) under argon,
vacuum dried 1H-tetrazole (220 mg, 3.14 mmol) was added
at room temperature. Bis(allyloxy)(diisopropylamino)phos-
phine (557 L, 2.18 mmol) was added dropwise to the reac-
tion mixture through a syringe under argon. After stirring for
2.5 h at rt, the reaction mixture was cooled to 0°C, and
5.5 M t-BuOOH solution in Nonan (400 L, 2.2 mmol) was
added dropwise and on completion of the addition, the reac-
tion mixture was further stirred for 1.5 h at rt. After comple-
tion 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 aque-
ous solution was extracted with CH2Cl2 (2 × 25 mL) and
added to the organic phase. The pooled organic phase was
(dd, J4,3 = 9.5 Hz, J4,6 = 1.8 Hz, 1H, H-4), 8.03 (d, J3,4
=
9.5 Hz, 1H, H-3), 9.16 (bs, 1H, NH). MS (MALDI, positive
mode, matrix: DHB, THF): 406.5 [M + Na]+. Anal. calcd.
for C17H22NO7P (383.3): C 53.27, H 5.78, N 3.65; found:
C 53.16, H 5.87, N 3.45.
Bis-triethylammonium (2-acetamido-5-hydroxy)benzyl
phosphate (32)
To a solution of 30 (180 mg, 0.47 mmol) in dry THF
(6 mL), dimedone (330 mg, 2.35 mmol) and tetrakis-
(triphenylphosphine)-palladium (100 mg, 0.087 mmol) was
added and stirred at room temperature protected from light.
After 1.5 h when the TLC showed no starting material, Et3N
(0.25 mL) was added and further stirred for 20 min at room
temperature. The reaction mixture was evaporated and the
© 2002 NRC Canada

Bhattacharya et al.
981
dried (MgSO4) and evaporated to dryness. The residue after
flash chromatography (toluene–acetone, 3:1) afforded 31 as
a colorless oil (190 mg, 51%). Rf = 0.5 (toluene–acetone,
[M – H+]–, 572 [M – Na+]–, 550 [M – 2Na+ + H+]– for
C18H21N3O13P2Na2 (595.4).
1
1:1). H NMR (250 MHz, CDCl3) ꢃ: 2.24 (s, 3H, COCH3),
Inhibition studies of ꢀ-glucosyltransferase of phage T4
The activity of ꢀ-glucosyltransferase was monitored by
the transfer of 14C-glucose from the substrate 14C-UDPGlc
to 5-hydroxymethylcytosine (HMC) of unglucosylated
T4*DNA. T4*DNA was obtained from progeny phage prop-
agating in a UDPGlc– Escherichia coli K host strain
(W4597). The reaction mixture used to determine the IC50 in
the inhibition studies, contained 100 mM Tris–HCl, pH 7.9,
4.48 (m, 4H, CH2=CH-CH2), 5.07 (d, J = 11.0 Hz, 2H, CH2-
Ar), 5.22–5.35 (m, 4H, CH2=CH-CH2), 5.80–5.96 (m, 2H,
CH2=CH-CH2), 7.14 (dd, J5,6 = 7.5 Hz, J5,4 = 7.4 Hz, 1H,
H-5), 7.34 (d, J6,5 = 7.5 Hz, 1H, H-6), 7.41 (dd, J4,3
=
7.5 Hz, J4,5 = 7.4 Hz, 1H, H-4), 8.01 (d, J3,4 = 7.5 Hz, 1H,
H-3), 9.16 (bs, 1H, NH). MS (MALDI, positive mode, ma-
trix: DHB, THF) (m/z): 348.5 [M + Na+]+, 364.6 [M + K+]+.
Anal. calcd. for C15H20NO5P (325.3): C 55.38, H 6.20;
found: C 55.11, H 6.27.
9
g of T4*DNA, 25 mM MgC12, 200 M UDPGlc
(Sigma), 5 L of 14C-UDPGlc (NEN, 330 mCi mmol–1 or
1.85 MBq, delivered in a total volume of 2 mL), 0.5 g BGT
and different inhibitors, with concentrations varying between
0.5 and 2.5 mM in a total assay volume of 100 L. After
15 min of incubation at 30°C, the reaction mixtures were
transferred onto DEAE filter disks (Whatman DE81). The
filters were washed three times with 5 mL of a buffer con-
taining 0.5 M NaH2PO4, pH 7.0, dried and counted in a
Beckman scintillation counter, model LS 6000 TA.
Bis-triethylammonium-2-acetamidobenzyl phosphate (33)
To a solution of 31 (130 mg, 0.40 mmol) in dry THF
(6 mL), dimedone (320 mg, 2.28 mmol) was added and
stirred at room temperature protected from light. After 1.5 h
when the TLC showed no starting material, Et3N (0.15 mL)
was added and further stirred for 15 min at room tempera-
ture. The reaction mixture was evaporated and the residue
was purified over flash chromatography (CHCl3–MeOH–
H2O, 6:4:1 + 1% Et3N). The product obtained after flash
chromatography was lyophilized with water to afford 33 as a
colorless powder (128 mg, 72%). Rf 0.37 (CHCl3–MeOH–
H2O, 6:4:1). 1H NMR (250 MHz, [D6]DMSO) ꢃ: 1.09 (t, J =
7.3 Hz, 18H, NCH2CH3), 2.07 (s, 3H, COCH3), 2.89 (q, J =
7.3 Hz, 12H, NCH2CH3), 4.64 (d, J = 9.0 Hz, 2H, CH2Ar),
6.99 (dd, J5,4 = J5,6 = 8.0 Hz, 1H, H-5), 7.24–7.27 (m, 2H,
H-4, H-6), 8.00 (d, J = 8.0 Hz, 1H, H-3), 11.26 (bs, 1H,
Km and Vmax values were determined in essentially the
same buffer and reaction volume, however, the concentra-
tions of the substrate mixture varied between 1.6 and
200 M and the inhibitors were added at constant concentra-
tions of 0.5, 1, and 2 mM, respectively. The different sub-
strate concentrations were obtained by the appropriate
dilution of a mix containing UDPGlc and 14C-UDPGlc in a
ratio of 27.5:1. Radioactivity transferred from the substrate
to the T4*DNA, again was measured as described above.
1
NH). H NMR (250 MHz, D2O) ꢃ: 1.09 (t, J = 7.3 Hz, 18H,
Acknowledgments
NCH2CH3), 2.04 (s, 3H, COCH3), 3.01 (q, J = 7.3 Hz, 12H,
NCH2CH3), 4.73 (d, overlapping with D2O peak, CH2Ar),
7.19–7.25 (m, 3H, H-4, H-5, H-6), 7.37 (d, J = 7.3 Hz, 1H,
H-3). MS (MALDI, positive-mode, matrix: DHB, H2O)
(m/z): 268.6 [M – 2HNEtꢂ3 + 2H+ + Na+]+, 284.5 [M –
2HNEtꢂ3 + 2H+ + K+]+ for C21H41N3O5P (447.53).
This work was supported by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen
Industrie. A.K.B. is grateful to the Deutscher Akademischer
Austauschdienst (DAAD) for a research fellowship. We are
grateful to Ms. Anke Friemel and Mr. Klaus Hägele,
Fachbereich Chemie, Universität Konstanz, Konstanz for re-
cording NMR (600 MHz) and FAB-MS, respectively.
Disodium (2-acetamidobenzyl)-uridine-5>-diphosphate (6)
A mixture of 33 (100 mg, 0.22 mmol) and 4-morpholine-
N,N>-dicyclohexylcarboxamidinium uridine 5>-monophos-
phomorpholidate (160 mg, 0.23 mmol) and 1H-tetrazole
(77 mg, 1.1 mmol) in dry pyridine (1 mL) was treated as per
the general procedure described above. After 3 days, the
product was purified by HPLC (0.05 M triethylammonium
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