Structure-functional analysis of interactions of terminal deoxynucleotidyl transferase with new non-nucleoside substrates

Article abstract of DOI:10.1134/S1068162009030091

New non-nucleoside esters of phosphoric acid containing various hydrophobic groups, namely (1) N-(2-tripticencarbonyl)-4-aminobutyl; (2) 5-phenylsubstituted N-(2,4-dinitrophenyl)-4-aminobutyl; (3) N-(4-phenylbenzoyl)- and N-(4-(N-benzylamino)benzoyl)-2-aminoethyl groups, as well as (4) diphenylmethyl and fluorenyl groups were synthesized and studied as substrates of terminal deoxynucleotidyl transferase. With the exception of the two latter derivatives, all the analogues displayed substrate properties and could incorporate into the deoxyoligonucleotide 3′-end. As it was shown in biochemical experiments and by computer modeling, a linker joining the triphosphate and hydrophobic fragments of the molecule was necessary for these compounds to display substrate properties.

Full text of DOI:10.1134/S1068162009030091

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 3, pp. 342–348. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © E.S. Matyugina, L.A. Alexandrova, M.V. Jasco, A.V. Ivanov, I.A. Vasiliev, V.L. Lapteva, A.L. Khandazhinskaya, M.K. Kukhanova, 2009, published in
Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 3, pp. 376–383.
Structure–Functional Analysis of Interactions
of Terminal Deoxynucleotidyl Transferase
with New Non-Nucleoside Substrates
E. S. Matyuginaa, L. A. Alexandrovaa, M. V. Jascoa, A. V. Ivanov, I. A. Vasilieva, V. L. Laptevab,
A. L. Khandazhinskayaa,1, and M. K. Kukhanovaa
a Enhelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia;
b Chemical Department of Moscow State University, Moscow, Russia
Received July 11, 2008; in final form, August 5, 2008
Abstract—New non-nucleoside esters of phosphoric acid containing various hydrophobic groups, namely
(1) N-(2-tripticencarbonyl)-4-aminobutyl; (2) 5-phenylsubstituted N-(2,4-dinitrophenyl)-4-aminobutyl; (3) N-
(4-phenylbenzoyl)- and N-(4-(N-benzylamino)benzoyl)-2-aminoethyl groups, as well as (4) diphenylmethyl
and fluorenyl groups were synthesized and studied as substrates of terminal deoxynucleotidyl transferase. With
the exception of the two latter derivatives, all the analogues displayed substrate properties and could incorporate
into the deoxyoligonucleotide 3'-end. As it was shown in biochemical experiments and by computer modeling,
a linker joining the triphosphate and hydrophobic fragments of the molecule was necessary for these com-
pounds to display substrate properties.
Key words: non-nucleoside substrates, substrate properties, terminal deoxynucleotidyl transferase, triphos-
phate derivatives.
DOI: 10.1134/S1068162009030091
INTRODUCTION
(2,4-dinitrophenyl)-4-aminobutyl derivatives, N-(4-(N-
benzylamino)benzoyl)-2-aminoethyl residues, and the
compounds bearing a fluorenyl fragment or a diphenyl-
ethyl residue mimicking it were used.
Template-independent terminal deoxynucleotidyl
transferase (TdT, EC 2.7.7.31) is the only DNA poly-
merase capable of elongating single-stranded oligonu-
cleotides in the presence of dNTP.2 TdT belongs to the
DNA polymerase X family, which also includes
eukaryotic repair DNA polymerases β, λ, µ, and σ.
Unlike other template-dependent DNA polymerases of
the X family, TdT can utilize substrates of a wide spec-
trum [1–4]. The capacity of the enzyme to utilize both
α-D- and α-L-dNTP provided a suggestion that the
nucleoside fragment did not play an essential role in
enzyme interaction [5]. It was shown earlier that triph-
osphates bearing hydrophobic groups joined with triph-
osphate residues via linkers were effective TdT sub-
strates. The activity of these substrates was dependent
on both the substituent structure and the linker structure
and length [6–8].
RESULTS AND DISCUSSION
The structural formulas of the synthesized com-
pounds are shown in the table.
Triphosphate (I) is one of the tripticen derivatives
that have not been studied as TdT substrates. Com-
pounds (II‡) and (IIb) continue the series of earlier-
synthesized substituted N-(2,4-dinitrophenyl)-4-ami-
nobutyl esters of triphosphoric acid: N-(2,4-dinitro-1-
fluorophenyl)-4-aminobutyl, N-(2,4-dinitro-5-imida-
zolylphenyl)-4-aminobutyl, and N-(2,4-dinitrophenyl)-
4-aminobutyl triphosphates [7], which demonstrated
substrate properties towards TdT and inhibited mutant
DNA polymerase λ Y505A [8]. New (II‡) and (IIb)
derivatives contain even more bulky substituents,
namely benzimidazole and benzotriazole residues.
Triphosphates (III‡) and (IIIb) are analogues of the
earlier synthesized 4-(4-phenylbenzoyl)oxybutyl triph-
osphate, an effective TdT termination substrate [8]
which could also be incorporated into the 3'-end of the
oligonucleotide primer in the presence of DNA poly-
merase λ wild and Y505A mutant types, as well as
DNA polymerase β [8]. Incorporation was most effec-
In this work, we describe four types of new non-
nucleoside TdT substrates from calf thymus. They con-
tain hydrophobic groups joined with triphosphate resi-
dues via linkers of different lengths and structures. As
hydrophobic groups, tripticen, 5-phenyl substituted N-
1
Corresponding author; phone: +7 (499) 135-2255, fax: +7(499)
135-1405; e-mail: khandazhinskaya@bk.ru
Abbreviations: CDI, 1,1'-dicarbonyldiimidazole; TdT, terminal
deoxynucleotidyl transferase.
2
342

STRUCTURE–FUNCTIONAL ANALYSIS OF INTERACTIONS
343
O
O
O
P
R
O
P
O
P
O
OH
OH
OH
OH
R
N
N
N
N
N
O
NH(CH2)4-
NH(CH2)4-
CNH(CH2)4-
O2N
NO2
O2N
NO2
(I)
(IIb)
(IIa)
O
O
CNH(CH2)2-
CH2 NH
CNH(CH2)2-
(IIIa)
(IIIb)
CH CH2-
CH CH2-
(IVb)
(IVa)
(I): R = N-(2-tripticencarbonyl)-4-aminobutyl,
(IIa): R = N-[(2,4-dinitro-5-benzimidazolylphenyl)]-4-aminobutyl,
(IIb): R = N-[(2,4-dinitro-5-benzotriazolylphenyl)]-4-aminobutyl,
(IIIa): R = N-(4-phenylbenzoyl)-2-aminoethyl,
(IIIb): R = N-(4-(N-benzylamino)benzoyl)-2-aminoethyl,
(IVa): R = 2,2,-diphenylethyl,
(IVb): R = 9-fluorenylmethyl.
tive against the template adenine in the presence of matic and triphosphate residues enabled us to ascertain
Mg2+ [8]. This is the first example of a substrate that
the impact of these fragments for manifestation of sub-
lacked both a heterocyclic base and a carbohydrate
fragment but could form a pair with the normal base.
strate properties towards TdT.
Triphosphate (I) was synthesized according to
Scheme 1 using the method described in [10]. Coupling
of 2-tripticencarboxylic acid activated with 1,1'-carbo-
nyldiimidazole (CDI) with aminobutanol resulted in
N-(2-tripticencarbonyl)-4-aminobutanol, which was
then phosphorylated with POCl3 in triethyl phosphate.
After isolation on a DEAE cellulose column, the result-
ing monophosphate was transformed into the corre-
sponding imidazolide in the presence of CDI followed
by coupling with tributylammonium pyrophosphate.
Triphosphate (I) was isolated by ion-exchange chroma-
tography on a DEAE cellulose column in a gradient of
NH4HCO3 and subsequent purification on a LiChroprep
The fourth type of triphosphates, namely com-
pounds (IV‡) and (IVb), can be regarded as analogues
of the earlier prepared N-(9-fluorenylmethoxycarbo-
nyl)-ω-aminoalkyl, N-(9-fluorenylmethoxycarbonyl)-
8-amino-3,6-dioxaoctyl-, and N-[(9-fluorenylmethoxy-
carbonyl)-6-aminohexanoyl]-2-aminoethyl esters [6].
All the earlier synthesized fluorenylmethoxycarbonyl-
bearing triphosphates were substrates of calf thymus
TdT and their substrate properties depended on the
structure and length of the linker joining the 9-fluore-
nylmethoxycarbonyl residue with the triphosphate
fragment, as well as of the nature of the metal activator
[9]. Studies of new compounds (IV‡) and (IVb) lacking
an oxycarbonyl fragment and a linker between the aro- RP-18 column.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

344
MATYUGINA et al.
O
C
O
1) CDI, DMF
2) H2N(CH2)4OH, 15÷
3) NH3/H2O, 15÷
OH
O
CNH(CH2)4OH
POCl3
(EtO)3PO
1) CDI, DMF
2) (Bu3NH)2H2P2O7
O
(I)
CNH(CH2)4O
P
OH
OH
Scheme 1.
(2,4-Dinitrophenyl)-4-aminobutanol derivatives were obtained by phosphorylation by the Ludwig method
synthesized by the reaction of aminobutanol with 1,5- [11] and isolated similarly to triphosphate (I) to give
difluoro-2,4-dinitrobenzene followed by treatment with 25–30% of the product relative to the starting aminob-
a solution of the corresponding heterocycle in DMF utanol.
(Scheme 2) [7]. Triphosphates (II‡) and (IIb) were
F
F
H2N(CH2)4OH
O2N
F
O2N
NH (CH2)4 OH
NO2
NO2
N
N
N
NH
NH
N
N
N
N
N
O2N
NH (CH2)4 OH
NO2
O2N
NH (CH2)4 OH
NO2
1) POCl3, (EtO)3PO
2) (Bu3NH)2H2P2O7
1) POCl3, (EtO)3PO
2) (Bu3NH)2H2P2O7
(IIa)
(IIb)
Scheme 2.
Triphosphates (IIIa) and (IIIb) were prepared from synthesized the corresponding succineimide esters
commercially available 4-phenylbenzoic acid for (IIIa)
or 4-(N-benzylamino)benzoic acid for (IIIb). 4-(N-
Benzylamino)benzoic acid was obtained by the cou-
pling of benzaldehyde and 4-aminobenzoic acid fol-
lowed by reduction with sodium borohydride
(Scheme 3). The attempts to activate the arylcarboxylic
which interacted with aminoethyl phosphate to give tar-
get monophosphates in high yields (75–80%). Mono-
phosphates were activated with CDI and tributylammo-
nium pyrophosphate was added. The resulting triphos-
phates (Scheme 3) were isolated and purified as
acid with CDI (as in Scheme 1) failed. Therefore, we described for the above compounds.
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STRUCTURE–FUNCTIONAL ANALYSIS OF INTERACTIONS
345
O
O
O
NaBH4, 30 min
MeOH
20 min
+
H
H2N
OH
CH
N
OH
O
O
HO
N
O
O
O
NH2CH2CH2OP(O)(OH)2
Et3N, DMF/H2O
CH2 NH
O N
O
CH2 NH
OH
DCC, 15 h
O
O
1) CDI 2) MeOH
OH
3) (Bu3NH)2H2P2O7
(IIIb)
CH2 NH
NH(CH2)2O
P
OH
Sheme 3.
Compounds (IVa) and (IVb) were synthesized from the reason for different substrate activities of com-
the corresponding alcohols (diphenylmethanol for pounds (IIIb), (IVa), and (IVb).
(IVa) and 9-fluorenylmethanol for (IVb)) by treatment
Recently, the crystal structure of the [TdT + ddATP +
with POCl3 in triethyl phosphate followed by the addi-
Co2+] complex was published [12]. The main ddATP
tion of tributylammonium pyrophosphate similar to [6].
binding sites were triphosphate residue, whose position
The target triphosphates were isolated and purified as
was coordinated with two Co2+ atoms, and the base,
described above.
which interacted with the enzyme Trp450. Using the
Alkyl triphosphates (I)–(IV) were studied as TdT
WebLabViewerPro 3.7 program (Accelrys Inc., San
substrates (Fig. 1). 3'-Deoxythymidine triphosphate, an
Diego, California, United States), we fixed the triphos-
effective TdT substrate, served as a control. As is seen
phate residues of compounds (IIIb), (IVa), and (IVb) in
in the figure, the substrate efficacy of triphosphate (IIa)
the position which was occupied by this residue in
bearing a substituted 2,4-dinitrophenyl residue was
ddATP within the [TdT + ddATP + Co2+] complex
lower than that of ddTTP: the 50% primer elongation
(Fig. 2).
indicating the reaction efficacy was not achieved at the
concentration of triphosphate (IIa) of 10 µM, which
exceeded the ddTTP concentration by more than two
orders of magnitude. Similar data were obtained for
triphosphate (IIb) (data not shown).
It is seen in Fig. 2‡ that the N-(4-(N-benzy-
lamino)benzoyl)-2-aminoethyl residue of compound
(IIIb) can also interact with Trp450, although this inter-
action must be weaker than that of the ddATP purine
Compounds (I) and (IIIb) proved to be more effec- base, because only a six-membered purine fragment of
tive TdT termination substrates, although less effective (IIIb) could contact the Trp residue. At the same time,
than reference ddTTP (Fig. 1). Compounds (IVa) and aromatic residues of compounds (IVa) and (IVb)
(IVb) did not display substrate properties towards TdT. (Fig. 2b, c) could not interact with the Trp450 residue
The primer elongation was not observed even at a sub- (probably due to the absence of a linker between the
strate concentration of 50 µM. We attempted to find out triphosphate residue and the hydrophobic fragment of
Primer
K
(d2T)
0.1
(IIa)
(IIIb)
(I)
(IVa)
(IVb)
µM
1
1
2
10 0.4
1
2
10
1
2
10 1 2 10 50 1 2 10 50
Fig. 1. TdT-Catalyzed incorporation of triphosphate derivatives into the primer 3'-end. Compound concentrations are shown in the
figure. Reaction products were analyzed as described in the Experimental section. The left lane indicates the primer position. The
tracks above the primer show the primer elongation form the 3'-end after incorporation of the substrate monophosphate residue.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

346
MATYUGINA et al.
(‡)
(b)
(c)
(IVa)
Co2+
Co2+
(IVb)
(IIIb)
Co2+
Trp450
Trp450
Trp450
Co2+
Co2+
Co2+
Fig. 2. Superposition of compounds (a) (IIIa), (b) (IVa), and (c) (IVb) in the TdT catalytic site. Substrate triphosphate residues
2+
were fixed in the position occupied by the triphosphate residue in ddATP molecule within the crystalline [TdT + ddATP + Co
]
2+
complex [12]. The compounds are in bold. The Trp450 and Co ion positions are shown in the figure.
the molecule). This observation can explain the lack of Shimadzu UV-2401PC spectrophotometer (Japan) (ε is
substrate properties of these compounds.
the molar extinction coefficient).
N-(2-Tripticencarbonyl)-4-aminobutyl triphos-
phate (I): N-(2Tripticencarbonyl)-4-aminobutanol.
Carbonyldiimidazole (123 mg, 0.75 mmol) was added
to a solution of 2-tripticencarboxylic acid (149 mg,
0.5 mmol) (obtained as described in [13]) in DMF
(3 ml), and the mixture was added in 10 min to a solu-
tion of 4-aminobutanol (270 mg, 3 mmol) in DMF
(5 ml). The mixture was stirred for 20 h at room tem-
perature, 24% aqueous ammonia (5 ml) was added, and
the mixture was kept for 20 h. The solvents were evap-
orated in a vacuum, and a 1 : 1 mixture of 1 M HCl and
chloroform (25 ml) was added. The aqueous phase was
washed with chloroform (3 × 10 ml), and the chloro-
form fraction was dried with Na2SO4 and evaporated.
The residue was dissolved in chloroform (2 ml), loaded
on a silica gel column (2 ×18 cm), and eluted in a gra-
To summarize, new nonnucleotide TdT substrates of
four types, namely esters of triphosphoric acid, were
synthesized. It was shown that the presence of a linker
between the triphosphate residue and the aromatic part
of the molecule was necessary for the manifestation of
substrate properties.
EXPERIMENTAL
4-Aminobutanol, triethyl phosphate, 1,1'-carbonyl-
diimidazole,
N-hydroxysuccineimide,
dioxane,
N-methylimidazole, benzaldehyde, N,N'-dicyclohexyl-
carbodiimide, sodium borohydride, and acetonitrile
were from Fluka (Switzerland); 2-aminoethyl phos-
phate, phosphorus oxychloride, DMF, and 4-phenyl-
benzoic acid were from Aldrich (United States); tribu-
tylammonium pyrophosphate was from Sigma (United
States). [γ-32P]ATP (specific activity 6000 Ci/mM) was
from Isotop (Russia). For enzymatic reactions, sodium
cacodylate, 1,4-dithiothreitol, EDTA, acrylamide, for-
mamide, and calf thymus TdT were from Amersham
(Great Britain). The 18-mer deoxyoligonucleotide
primer (5')CCGTCAATTCCTGTAGTC was obtained
from Litex (Russia). Adsorption column chromatogra-
phy was carried out on LiChroprep RP-18 (25–40 µm)
and Kieselgel (63–100 µm) (Merck) columns. Ion-
exchange column chromatography was performed on a
dient of ethanol in chloroform (0
5%). The frac-
tions containing the target compound were evaporated
in a vacuum to give 147 mg (78%) of the product. UV
1
(ethanol, λmax, nm): 277 (ε 1570). H NMR (CDCl3):
7.38, 7.29, 6.99 (11 H, three m, aryl), 6.27 (1 H, br. s,
NH), 5.45, 5.44 (2 H, two s, bridges), 3.69 (2 H, m,
CH2O), 3.43 (2 H, m, CH2N), 1.53 (5 H, m (CH2)2 +
OH).
N-(2-Tripticencarbonyl)-4-aminobutyl triphos-
phate (I) was obtained in a yield of 23% from N-(2-
tripticencarbonyl)-4-aminobutanol as described in [10].
1
UV (H2O, pH 6, λmax, nm): 276 (ε 1450). H NMR
Dowex-50 WX8 (Fluka, Switzerland) in the NH+4 form
(D2O): 7.62, 7.37, 6.96 (11 H, three m, aryl), 5.66, 5.64
(2 H, two s, bridges), 3.98 (2 H, m, CH2O), 3.33 (2 H,
m, CH2N), 1.53 (4 H, m (CH2)2). 31P NMR (D2O): –10.07
(1 P, d, J 18.5, Pγ), –10.34 (1 P, d, J 20.3, Pα), –21.94 (1 P,
dd, Pβ).
and a DEAE-Toyopearl (Toyosoda, Japan). TLC was
performed on a Kieselgel 60 F254 (Merck, Germany);
the eluting system was a 6 : 4 : 1 mixture of dioxane–
water–25% NH3. NMR spectra were registered on an
AMX III-400 spectrometer (Bruker) with a working
frequency of 400 MHz for 1H NMR (chemical shifts are
given in δ in ppm relative to the internal standard of
sodium 3-trimethylsilyl-1-propanesulfonate (DSS); J
N-[(2,4-Dinitro-5-benzimidazolylphenyl)]-4-
aminobutyl triphosphate (IIa). N-[(2,4-Dinitro-5-
benzimidazolylphenyl)]-4-aminobutanol. Benzimida-
zole (73 mg, 0.62 mmol) and triethylamine (139 µl,
are given in Hz) and 162 MHz for 31P NMR (with 1 mmol) were added to a solution of N-[5-(2,4-dinitro-
hydrogen–phosphorus spin–spin decoupling; the exter- 1-fluorophenyl)-4-aminobutanol (100 mg, 0.31 mmol)
nal standard was 85% phosphoric acid; chemical shifts in acetonitrile (3 ml). The reaction mixture was
are given in δ in ppm). UV spectra were registered on a refluxed for 2 h, evaporated, and the product was iso-
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STRUCTURE–FUNCTIONAL ANALYSIS OF INTERACTIONS
347
lated by chromatography on a silica gel column (2 × NH4HCO3 (0
20%). The target fractions were evap-
20 cm) eluted with a 95 : 5 mixture of chloroforom–
orated, dissolved in water (3 ml), and lyophilized to
methanol in a yield of 70%.
give 41 mg (23%) of the product. UV (H2O, pH, 6, λmax
,
1
nm): 274 (ε 24000). H NMR (D2O): 7.72, 7.53, 7.44
N-[(2,4-Dinitro-5-benzimidazolylphenyl)]-4-
aminobutyl triphosphate (IIa). Phosphorus oxychlo-
ride (12 µl, 0.13 mmol) was added to a solution of N-[5-
(2,4-dinitro-1-benzimidazolylphenyl)-4-aminobutanol
(35 mg, 0.1 mmol) in triethyl phosphate (2 ml). The
reaction mixture was stirred for 3 h, and a solution of
tributylammonium pyrophosphate (91 mg, 0.2 mmol)
in DM (1 ml) was added. The mixture was kept for 1 h
(9 H, three m, aryl), 4.02 (2 H, m, CH2O), 3.68 ((2 H, m,
CH2N). 31P NMR (D2O): 1.73.
N-(4-Phenylbenzoyl)-2-aminoethyl triphosphate
(IIIa) was obtained from the corresponding phosphate
by the method described in [10] in a yield of 23%
(8.5 mg). UV (H2O, pH 6, λmax, nm): 274 (ε 24000);
1H NMR (D2O): 7.73, 7.54 and 7.45 (9 H, three m, aryl),
at room temperature, loaded on a Dowex-50 (NH+4 )
column (2 × 4 cm), and eluted with water. The eluate
was evaporated and chromatographed on a LiChroprep
RP-18 column (2 × 18 cm) eluted with water to give
30% of the product. UV (H2O, pH 6, λmax, nm): 242
31
4.12 (2 H, m, CH2O), 3.78(2 H, m, CH2N). P NMR
(D2O): –7.77 (1 P, d, J 17.5, Pγ), –10.35 (1 P, d, J 19.3,
Pα), –21.79 (1 P, dd, Pβ).
N-(4-(N-Benzylamino)benzoyl)-2-aminoethyl
triphosphate (IIIb) (Scheme 3). 4-(N-ben-
zylimino)benzoic acid. Benzaldehyde (886 µl, 8 mmol)
was added to a solution of 4-aminobenzoic acid
(548 mg, 4 mmol) in ethanol (3 ml) and the mixture
was kept for 20 h at +4°ë. The residue was filtered and
washed with cold methanol to give 540 mg (60%) of the
product. UV (H2O, pH 6, λmax): 254 (ε 22700).
1H NMR (DMSO-d6): 12.22 (1 H, COOH), 7.98, 7.65,
7.33, 6.55 (9 H, four m, aryl), 5.81 (1 H, c, CH=N).
1
(ε 6300), 365 (ε 7000). H NMR (D2O): 9.31 (1 H, s,
H3), 8.48 (1 H, br. s, benzimidazole), 7.88, 7.46 and
7.42 (4 H, three m, benzimidazole), 7.31 (1 H, s, H6),
3.99 (2 H, m, CH2é), 3.56 (2 H, t, J 6.8, CH2N), 1.84–
1.75 (4 H, m, (CH2)2). 31P NMR (D2é): –10.25 (1 P, d, J
19.3, Pγ), −10.37 (1 P, d, J 20.3, Pα), –22.68 (1 P, dd, Pβ).
N-[(2,4-Dinitro-5-benzotriazolylphenyl)]-4-ami-
nobutyl triphosphate (IIb) was obtained in a yield of
25% similar to triphosphate (IIa) using benzotriazole.
UV (H2O, pH 6, λmax, nm): 255 (ε 7000), 369 (ε 8000).
1H NMR (D2O): 9.31 (1 H, s, H3), 8.18, 7.72 and 7.61
(4 H, three m, benzotriazole), 7.41 (1 H, s, H6), 4.00
(2 H, m, CH2é), 3.56 (2 H, t, J 6.8, CH2N), 1.85–1.75
(4 H, m, (CH2)2). 31P NMR (D2é): –9.13 (1 P, d, J 19.3, Pγ),
−10.12 (1 P, d, J 20.3, Pα), –21.77 (1P, dd, Pβ).
4-(N-Benzylamino)benzoic acid. Sodium borohy-
dride (152 mg, 4 mmol) was added to a solution of
4-(N-benzylimino)benzoic acid (540 mg, 4 mmol) in
ethanol (10 ml) under vigorous stirring. The reaction
mixture was stirred for 1 h at room temperature and the
solvents were evaporated in a vacuum. A 1 : 1 water–
chloroform mixture was added to the residue, the aque-
ous phase was washed with chloroform (3 × 10 ml),
acidified with 1 M HCl to pH 1.5, and the target acid
was extracted with chloroform (3 × 10 ml). The extracts
were dried with Na2SO4 and evaporated to give 452 mg
(83%) of the product. UV (H2O, pH 6, λmax, nm): 286
N-(4-Phenylbenzoyl)-2-aminoethyl triphosphate
(III‡). N-Hydroxysuccineimide ester of p-phenylben-
zoic acid. Dicyclohexylcarbodiimide (227 mg,
1.1 mmol) was added to a solution of p-phenylbenzoic
acid (198 mg, 1 mmol) and N-hydroxysuccineimide
(127 mg, 1 mmol) in absolute dioxane (5 ml) and the
mixture was stirred for 17 h at 20°ë. The reaction mix-
ture was filtered and the filtrate was evaporated. The
residue was washed with petroleum, dissolved in a 2 : 1
ethyl acetate–hexane mixture, and loaded onto a silica
gel column (2 × 18 cm). The mixture was eluted in a
1
(ε 12100). H NMR (DMSO-d6): 11.9 (1 H, COOH),
7.65, 7.30, 6.98, and 6.59 (9 H, four m, aryl), 4.34 (2 H,
m, CH2-N).
N-Hydroxysuccineimide ester of 4-(N-benzy-
lamino)benzoic
acid.
N-Hydroxysuccineimide
(253 mg, 2.2 mmol) was added to a solution of 4-ben-
zylaminobenzoic acid (452 mg, 2 mmol) in dioxane
(10 ml). After it was dissolved, DCC (474 mg,
2.3 mmol) was added and the reaction mixture was
stirred for 20 h at room temperature. Ethyl acetate (5
ml) was added and the mixture was kept for 1 h at room
temperature. The precipitate was filtered and the sol-
vents were evaporated in a vacuum. The residue was
dissolved in a 2 : 1 ethyl acetate–hexane mixture and
loaded onto a silica gel column (2 ×18 cm). The product
was eluted in a gradient of ethyl acetate concentration
gradient of ethyl acetate in hexane (30
80%) and
the target fractions were evaporated in a vacuum to give
215 mg (73%) of the product. 1H MMR (CDCl3): 7.95,
7.66, 7.42 (9 H, three m, aryl), 2.83 (4 H, m, (CH2)2).
N-(4-Phenylbenzoyl)-2-aminoethyl phosphate. A
solution of N-hydroxysuccineimide ester of 4-phenyl-
benzoic acid (148 mg, 0.5 mmol) in DMF (6 ml), trieth-
ylamine (210 µl, 1.5 mmol), and methylimidazole
(47 µl, 0.55 mmol) were added to a solution of amino-
ethyl phosphate (72 mg, 0.5 mmol) in water (2 ml), and
the reaction mixture was stirred for 3 h at room temper-
ature. Organic solvents were evaporated in a vacuum,
the residue was dissolved in water (4 ml), and loaded
onto a reverse-phase LiChroprep RP-18 column (2 ×
18 cm) eluted in a linear gradient of ethanol in 0.01 M
in hexane (30
80%). The target fractions were
evaporated in a vacuum to give 505 mg (78%). UV
1
(EtOH, pH 6, λmax, nm): 289 (ε 12200). H NMR
(CDCl3): 7.89, 7.35, 7.27, 6.59 (9H, four m, aryl), 4.39
(2 H, m, CH2-N), 2.83 (4 H, m, (CH2)2).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

348
MATYUGINA et al.
N-(4-(N-Benzylamino)benzoyl)-2-aminoethyl
ACKNOWLEDGMENTS
phosphate. A solution of N-hydroxysuccineimide ester
of 4-(N-benzylamino)benzoic acid (162 mg, 0.5 mmol)
in DMF (4 ml), triethylamine (210 µl, 1.5 mmol), and
methylimidazole (47 µl, 0.55 mmol) were added to a
solution of aminoethyl phosphate (212 mg, 1.5 mmol)
in water (1 ml) and the reaction mixture was stirred for
3 h at room temperature. Organic solvents were evapo-
rated in a vacuum, and the residue was dissolved in
water (2 ml) and centrifuged. The solution was evapo-
rated up to a volume of 2 ml, loaded on a reverse-phase
LiChroprep RP-18 column (2 × 18 cm), and eluted with
0.01 M NH4HCO3. The target fractions were evapo-
rated, and the residue was dissolved in water (3 ml) and
lyophilized to give 45 mg (23%) of the product. UV
(H2O, pH 6, λmax, nm): 290 (ε 11500). 1H NMR (D2O):
7.65, 7.43, 7.37 and 6.83 (9 H, four m, aryl), 4.43 (2 H,
m, CH2-aryl), 4.13 (2 H, m, CH2O), 3.64 (2 H, m,
CH2N). 31P NMR (D2O): –1.3.
2,2-Diphenylethyl triphosphate (IVa) was
obtained by treatment of diphenylmethanol with POCl3
in triethyl phosphate followed by the addition of tribu-
tylammonium pyrophosphate similar to [6] to give 16%
of the product. UV (H2O, pH 6, λmax, nm): 258
(ε 540).1H NMR (D2O): 7.39 (8H, m, Ph), 7.27 (2H, m,
Ph), 4.55 (2 H, m, CH2é), 3.56 (1 H, t, J 6.6, CHCH2).
31P NMR (D2é): –9.25 (1 P, d, J 19.3, Pγ), –10.69 (1 P,
d, J 20.3, Pα), –22.43 (1 P, dd, Pβ).
The work was supported by the Russian Foundation
for Basic Research, project no. 06-04-48248, and Fede-
ral Program: Studies and Design in Foreground Direc-
tions of Development of Research and Technical Com-
plex of Russia (State contract no. 02.512.11.2237).
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009