α,β-Methylene-2′-deoxynucleoside 5′-triphosphates as noncleavable substrates for DNA polymerases: Isolation, characterization, and stability studies of novel 2′-deoxycyclonucleosides, 3,5′-cyclo-dG, and 2,5′-cyclo-dT

Article abstract of DOI:10.1021/jm800692a

We report synthesis and characterization of a complete set of α,β-methylene-2′-dNTPs (α,β-m-dNTP; N = A, C, T, G, 12-15) in which the α,β-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2′-deoxynucleoside 5′-diphosphate precursors, followed by an enzymatic γ-phosphorylation. All four synthesized α,β-m-dNTPs were found to be potent inhibitors of polymerase β, with K_i values ranging 1-5 μM. During preparation of the dG and dT derivatives of α,β-methylene diphosphate, we also isolated significant amounts of 3,5′-cyclo-dG (16) and 2,5′-cyclo-dT (17), respectively. These novel 2′-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 → C5′ and O2 → C5′, respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5′-cyclo-2′-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.

Full text of DOI:10.1021/jm800692a

6460
J. Med. Chem. 2008, 51, 6460–6470
r,ꢀ-Methylene-2′-deoxynucleoside 5′-Triphosphates as Noncleavable Substrates for DNA
Polymerases: Isolation, Characterization, and Stability Studies of Novel
2′-Deoxycyclonucleosides, 3,5′-Cyclo-dG, and 2,5′-Cyclo-dT
Fengting Liang,|,† Nidhi Jain,|,† Troy Hutchens,† David D. Shock,‡ William A. Beard,‡ Samuel H. Wilson,‡ M. Paul Chiarelli,§
and Bongsup P. Cho*,†
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, UniVersity of Rhode Island, 41 Lower College Road, Kingston,
Rhode Island 02881, Laboratory of Structural Biology, NIEHS, National Institute of Health, DHHS, Research Triangle Park,
North Carolina 27709, Department of Chemistry, Loyola UniVersity, Chicago, Illinois 60626
ReceiVed June 6, 2008
We report synthesis and characterization of a complete set of R,ꢀ-methylene-2′-dNTPs (R,ꢀ-m-dNTP; N )
A, C, T, G, 12-15) in which the R,ꢀ-oxygen linkage of natural dNTP was replaced by a methylene group.
These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails
preparation of 2′-deoxynucleoside 5′-diphosphate precursors, followed by an enzymatic γ-phosphorylation.
All four synthesized R,ꢀ-m-dNTPs were found to be potent inhibitors of polymerase ꢀ, with Ki values
ranging 1-5 µM. During preparation of the dG and dT derivatives of R,ꢀ-methylene diphosphate, we also
isolated significant amounts of 3,5′-cyclo-dG (16) and 2,5′-cyclo-dT (17), respectively. These novel 2′-
deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 f C5′ and O2 f
C5′, respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination.
When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5′-cyclo-2′-
deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.
Introduction
the polymerase ꢀ ternary structures with natural dNTP and
nonhydrolyzable R,ꢀ-methylene-dNTP as incoming substrates
and found negligible difference in active site geometry, permit-
ting facile catalytic metal binding.
Deoxy- and ribonucleoside 5′-triphosphates (dNTPs and
rNTPsa) are essential components in maintaining the integrity
of living cells. Their roles as substrates for DNA or RNA
polymerases as well as for many NTP binding proteins implicate
them in a wide range of potential mechanistic and therapeutic
applications.1,2 The NTP analogues play important roles in
various cellular processes and act as the primary energy sources
for the cell.
NTP derivatives with modifications in the 5′-triphosphate
fragment have been used in studies examining the structures
and mechanisms of NTP binding proteins and enzymes including
polymerases.1,3 In particular, isosteric replacement of the R,ꢀ-
or ꢀ,γ-oxygen linkage in the triphosphate chain with a methylene
or an imido group produces an interesting set of NTPs with
increased stability in the presence of phosphatases.3 Such
modifications also yield nonhydrolyzable and stable dNTP
analogues that are valuable tools for biochemical and structural
studies of reactions in which hydrolysis of the P-O-P linkage
and/or transfer of the phosphate group occur. For example, a
series of ꢀ,γ-methylene and halogenated analogues of dGTP
have recently been used to examine leaving group effects on
polymerase ꢀ catalysis and fidelity.4,5 Batra et al.6,7 compared
As part of our ongoing research program on sequence effects
on arylamine-induced conformational heterogeneity,8 we sought
the isolation of ternary polymerase intermediates involving a
lesion-containing template-primer and dNTP. Strategies to trap
such catalytic complex entities, include utilizing a 3′-dideoxy-
terminated primer (i.e., lacking the attaching O3′)9 or a
nonhydrolyzable dNTP analogue,3,7 in which the R,ꢀ-bridging
oxygen has been replaced with noncleavable isosteres (-CH2-
or -NH-), in order to render the nucleotides inert toward
nucleotidyl transfer. We chose to employ the latter, R,ꢀ-
methylene-dNTP analogues, as they are more synthetically
viable.
Here we describe the preparation of a complete set of R,ꢀ-
methylene-dNTP analogues (N ) A, C, T, G) (12-15, Figure
1a) and a characterization of their polymerase ꢀ binding
properties. Synthetic preparation entails nucleophilic coupling
of methylene-diphosphate with 5′-tosyl nucleosides and a
subsequent enzymatic γ-phosphorylation. All four R,ꢀ-meth-
ylene-dNTPs (12-15) have been found to bind polymerase ꢀ
as efficiently as the natural dNTPs with Ki’s in the ∼1-5 µM
range. We isolated significant amounts of 3,5′-cyclo-2′-deox-
yguanosine (16, cyclo-dG) and 2,5′-cyclo-2′-deoxythymidine
(17, cyclo-dT) (Figure 1b) during preparation of their respective
diphosphate precursors. Here we present a thorough structure
characterization of these novel 2′-deoxycylconucleosides and
their aqueous stabilities.
* To whom correspondence should be addressed. Phone: 401-874-5024.
Fax: 401-874-5766. E-mail: bcho@uri.edu.
†
Department of Biomedical and Pharmaceutical Sciences, College of
Pharmacy, University of Rhode Island.
‡
Laboratory of Structural Biology, NIEHS, National Institute of Health,
DHHS.
§
Department of Chemistry, Loyola University.
These authors contributed equally.
|
a Abbreviations: cyclo-dG, 3,5′-cyclo-2′-deoxyguanosine; cyclo-dT, 2,5′-
cyclo-2′-deoxythymidine; cyclo-dX, 3,5′-cyclo-2′-deoxyxanthosine; dNTP,
2′-deoxyribonucleoside 5′-triphosphates; R,ꢀ-m-dNTP, R,ꢀ-methylene-2′-
deoxyribonucleoside 5′-triphosphates; R,ꢀ-m-dNDP, R,ꢀ-methylene-2′-
deoxyribonucleoside 5′-diphosphates.
Results and Discussion
Preparation of r,ꢀ-Methylene-2′-deoxynucleoside 5′-triph-
osphates (r,ꢀ-m-dNTPs; 12-15). As summarized in Figure 1a,
our synthesis protocol entails two major steps: a nucleophilic
10.1021/jm800692a CCC: $40.75
2008 American Chemical Society
Published on Web 09/24/2008

R,ꢀ-m-dNTP as NoncleaVable Substrates for DNA Polymerases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6461
Figure 1. (a) Synthesis of R,ꢀ-methylene-2′-deoxynucleoside 5′-triphosphates (R,ꢀ-m-dNTP, N ) A, C, T, G) (12-15). (b) Formation of novel
2′-deoxycyclonucleosides (3,5′-cyclo-dG, 16, and 2,5′-cyclo-dT, 17).
coupling of 5′-O-tosyl-2′-deoxynucleoside with methylene-
diphosphate to generate the 2′-deoxynucleoside diphosphate
(dNDP) precursors and a subsequent enzymatic γ-phosphory-
lation. The R,ꢀ-methylene-diphosphate derivatives (R,ꢀ-m-
dNDP; 8-11) were prepared as literature procedures.10-12
Briefly, individual 5′-O-tosylnucleoside was treated with 2 equiv
of tris(tetra-n-butylammonium) hydrogen methylene pyrophos-
phate in dry acetonitrile. The products were exchanged for tetra-
n-butyl ammonium cations to ammonium cations and subjected
to fast performance liquid chromatography (FPLC) with a
cellulose column; elution from the column was monitored by
UV absorption using a diode-array detector. The 1H NMR
spectra are characteristic of coupling diphosphate products such
as R,ꢀ-methylene protons at around 2.0 ppm with a coupling
constant of JP-H ) 19.8 Hz. 31P NMR revealed two distinct
signals at around ∼16 and ∼20 ppm, which corresponds to Pꢀ
and PR, respectively. As expected, an isosteric change from
P-O-P to P-CH2-P resulted in large phosphorus deshieldings
compared to natural dADP (-10 ppm to -15 ppm).1,12
Enzymatic γ-phosphorylation has been shown to be generally
more efficient than the chemical approach for preparation of
R,ꢀ-methylene-dNTPs (12-15). Although pyruvate kinase can
catalyze the phosphorylation of both purine and pyrimidine R,ꢀ-
imido-diphosphate,3c as well as of azole carboxamide ribo-
nucleoside diphosphates,13 the R,ꢀ-methylene dNDP analogues
Figure 2. HPLC chromatograms of a mixture containing R,ꢀ-m-dCDP
(8-11) examined here are poor substrates for pyruvate kinase.
(9) and ATP (a) before and (b) after enzymatic γ-phosphorylation (PK,
Therefore, we employed the substrate nonspecific nucleoside
diphosphate kinase (NDPK).13,14 As shown in Figure 1a, the
reaction entails the incorporation of an ATP regeneration system
to complete an efficient transformation and requires 2 equiv of
phosphoenol pyruvate (PEP) to completely transform ADP to
ATP. Figure 2 shows such a conversion using R,ꢀ-m-dCDP (9)
as a starting material. A complete conversion to R,ꢀ-m-dCTP
(13) was achieved within 4 h as determined by a gradient ion
pair HPLC/UV using program A. Similar conversions were
PEP, ATP) (see Figure 1a). A gradient HPLC solvent program A was
used.
achieved for the remaining cases. The R,ꢀ-methylene-dNTP
products generally exhibited a longer retention time than the
starting 2′-dNDP derivatives. Isolation of the desired R,ꢀ-
methylene-dNTP products was achieved by a three-step process,
which involved (i) addition of 1 equiv of ADP to convert extra
PEP into pyruvate, (ii) boronate affinity gel chromatography to

6462 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Liang et al.
µM) and those reported for commercially available R,ꢀ-imido-
dUTP7 and R,ꢀ-m-dATP.6
Isolation of Novel 2′-Deoxycyclonucleosides. The nucleo-
philic coupling reactions involving the 5′-O-tosyl derivatives
of dA (1) and dC (2) yielded R,ꢀ-m-dADP (8) and R,ꢀ-m-dCDP
(9), respectively, as exclusive products. By contrast, similar
reactions with tosyl-dG (4) and -dT (3) produced two major
fractions in FPLC chromatograms (not shown), with a late
eluting peak corresponding to the desired diphosphates 11 and
10, respectively. The early eluting fractions yielded white fluffy
solids, which showed no signals in the 31P NMR and no R,ꢀ-
methylene signals in the 1H NMR spectra. As elaborated below,
two nonpolar compounds were identified as novel 2′-deoxycy-
clonucleosides: N3,5′-cyclo-dG (cyclo-dG, 16) and O2,5′-cyclo-
dT (cyclo-dT, 17). The proposed structures were confirmed by
comparing the spectroscopic (UV, NMR, MS) data of these
compounds to those of cyclic nucleoside standards prepared by
another method (see below). As shown in Figure 1b, these
results clearly implicated an initial base-catalyzed deprotonation
of the imino proton (N1 and N3 respectively for 4 and 3),
followed by an intramolecular cyclization.
NMR. Figure 4 shows 1H NMR spectra of cyclo-dG and dG
in DMSO-d6. Assignment of the 1H signals was aided by
analyses of COSY, long-range-COSY, and TOCSY spectra
(Figures S13, S15, S16, Supporting Information). Cyclo-dG
showed all of the characteristics of dG except for the imino
proton at N1 (10.63 ppm) and 5′-OH (4.95 ppm). Another
notable difference was significant deshielding of H5′,5′′ with a
large difference in chemical shifts (4.45 and 3.89 ppm) relative
to dG (3.5 ppm).17 By contrast, the H2′,2′′ resonances were
merged at 2.16 ppm. All of the remaining sugar protons were
shifted downfield (∼0.4-0.6 ppm). Interestingly, however, H8
was shielded by about 0.15 ppm. A similar comparison of cyclo-
dT and dT in D2O revealed that all protons, except H1′, were
shifted downfield (Figure 5, also Figures S14, S17, Supporting
Information). As with cyclo-dG, a significant deshielding with
signal split was observed for H5′,5′′ (4.65 and 4.26 ppm) relative
to dT (3.75 ppm). In both cases, H4′ showed the greatest extent
of deshielding (∼0.8 ppm), implicating dramatic change in the
electronic environment of H4′ for the cyclonucleosides.
Figure 3. Inhibition of dCTP incorporation by R,ꢀ-m-dCTP (13). DNA
synthesis was assayed on a single-nucleotide gapped DNA substrate
where the templating nucleotide in the gap was dG. In this assay, the
concentration of dCTP was 20 µM. Dixon analysis for competitive
inhibition indicates that the Ki for 13 is 0.9 µM (solid line).
remove the remaining cis-diol containing ATP from the mixture,
and (iii) final purification by anion exchange with a Q Sepharose
FF anion exchange column.
31P NMR spectra of the final R,ꢀ-methylene dNTP products
clearly showed three well separated signals at around 23, 12,
and -5 ppm for PR, Pꢀ, and Pγ, respectively (Figures S5-S8,
Supporting Information). In all cases, the isosteric methylene
substitution at the R,ꢀ-oxygen linkage resulted in the expected
large deshielding (∼31-32 ppm) of PR and Pꢀ and small
deshielding for Pγ (∼1-3 ppm).1,11 These data indicate that
the structures of the final products were consistent with that
predicted by the spectroscopic data (1H, 31P, HRMS) (Figures
S5-S12, Supporting Information).
Cyclo-dG (16) and cyclo-dT (17) are rigid cage-like mol-
ecules with well-defined syn-glycosidyl conformation, 40.8° (ꢁ
O4′-C1′-N9-C8) and 61.6° (ꢁ O4′-C1′-N1-C6), re-
spectively.18 Such rigidity allows their H1′ and H8 protons to
be in a close spatial proximity, 2.5 and 2.2 Å, respectively
(Figure 6). This is evidenced by observations of strong NOEs
in the NOESY spectrum of cyclo-dG (Figure 7). Such NOEs
are absent in the anti-glycosidic dG and dT (not shown).
Polymerase Assays. Mammalian DNA polymerase ꢀ fills
short gaps in DNA during genome maintenance. It has served
as a model enzyme for the study of nucleotidyl transfer and
substrate specificity.15 Strategies to capture catalytic intermedi-
ates for structure determination have utilized a 3′-dideoxy-
terminated primer (without the attaching O3′)9 or a nonhydro-
lyzable dNTP analogues,3,6,7 in which the oxygen bridge
between the R- and ꢀ-phosphates has been replaced with an
imido7 or methylene group,6 rendering the nucleotides inert to
nucleotidyl transfer. A comparison of the resulting ternary
substrates complex structures indicated that structures with the
nonhydrolyzable incoming nucleotides resulted in less active
site distortion and permit catalytic metal binding.7 As expected,
these dNTP analogues are excellent inhibitors of polymerase
ꢀ-dependent DNA synthesis.6,7 Likewise, our R,ꢀ-methylene-
dNTPs (12-15, Figure 1) inhibited DNA synthesis and bound
tightly to polymerase ꢀ (Ki: R,ꢀ-m-dATP, 12, 5.0 µM; R,ꢀ-m-
dCTP, 13, 0.9 µM; R,ꢀ-m-dGTP, 15, 1.0 µM; R,ꢀ-m-dTTP,
14, 0.6 µM). Figure 3 shows inhibition of dCTP incorporation
by R,ꢀ-m-dCTP (13) as an example of the gap-filling assay.
These binding constants are similar to natural dNTPs (2-10
It is also noteworthy that cyclo-dG and cyclo-dT adopted rare
C1′-endo sugar puckering (Figure 6).19,20 This structural feature
distinguishes these cyclonucleosides from dG and dT, which
are known to exist primarily with C2′-endo puckering. This
difference in sugar conformation is clearly indicated in dramatic
changes in chemical shifts (H4′) and the unique coupling
patterns of sugar protons, particularly with respect to H1′ and
H2′,2′′ (Figures 4 and 5). Finally, the rigidity of the cage-like
cyclonucleoside structures places the H5′ (pro-S) and H5′′ (pro-
R) protons in a well-defined electronic environment (Figure 6),
presumably explaining the large differences in chemical shifts
and coupling patterns mentioned above. The pro-S H5′ signal
was assigned tentatively downfield to the pro-R H5” signal
because it is closer to O4′ than the pro-R H5′′. The large
deshielding (∼15 ppm) observed for the C5′ carbon relative to
dT21 in the 13C NMR spectrum (Figure S18, Supporting

R,ꢀ-m-dNTP as NoncleaVable Substrates for DNA Polymerases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6463
1
Figure 4. Comparison of H NMR spectra of (a) dG and (b) cyclo-dG (16) recorded in DMSO-d6.
1
Figure 5. Comparison of H NMR spectra of (a) dT and (b) cyclo-dT (17) recorded in D2O.
However, the UV spectra of dG remained steady across acidic
and neutral pH range but were altered by exposure to alkaline
pHs above 11.9 (Figure S22, Supporting Information). These
results are consistent with the supposition that the N-1 imino
proton appears to be absent in cyclo-dG. The UV spectra of
cyclo-dT changed dramatically with shifting pH through the
range of 1.7-11.9 with a well-defined isosbestic point at 263
nm, possibly implicating multiple hydrolytic species (see below)
(Figure S26, Supporting Information).
CD. The circular dichroism (CD) characteristics of cyclo-
dG and cyclo-dT differed qualitatively from dG and dT,
respectively (Figures S20, S21, S24, Supporting Information).
The syn-conformeric cyclo-dG exhibited well-defined large
negative ellipticities at 214 and 248 nm (Figure S24, Supporting
Information). This behavior precisely complemented that of anti-
conformeric dG, which showed large positive and small negative
ellipticities at 212 and 248 nm, respectively (Figure S20,
Supporting Information).22 The CD spectra of cyclo-dG re-
mained unchanged from pH 5.1 to 11.9 but underwent a
dramatic change when exposed to pHs below 3.4 (Figure S24,
Supporting Information). This sensitivity to acidity suggests that
the protonated base may change conformation. The CD spectra
observed for cyclo-dT were similar to that of dT (Figure S21,
Figure 6. Energy-minimized models (Spartan) of (a) cyclo-dG (16)
and (b) cyclo-dT (17).
Information) can be attributed to the presence of a 2,5′-anhydo
ether linkage in cyclo-dT.
UV. The UV maxima for cyclo-dG and cyclo-dT were shifted
significantly toward shorter wavelengths relative to the diphos-
phate products 11 and 10 (Figure S19, Supporting Information).
A distinct cyclo-dG band at 215 nm most likely reflects the
major change in the electronic configuration of the guanine base.
The UV spectra of cyclo-dG were not affected by exposure to
neutral and basic pHs but were altered by exposure to highly
acidic pHs below 3.4 (Figure S23, Supporting Information).

6464 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Liang et al.
Figure 7. NOESY spectrum of cyclo-dG (16). The cross peaks observed for the H1′ and H8 protons (highlighted in red circles) indicate their
spatial proximity (i.e., syn-glycosidyl conformation).
Supporting Information) but exhibited a strong negative ellip-
ticity at 228 nm.
experiments with the ribose analogue cyclo-G, which produced
a glycolytic product with a half-life of >122 min under similar
conditions.25 The interconversion equilibrium between 16 and
18 at 37 °C appeared to be under kinetic control, affording
quickly the kinetically favored product 18: upon extended
reaction time, however, the reaction preferred the thermody-
namically stable 16. At 100 °C, the reaction was thermodynami-
cally controlled, thus favoring formation of 16.
Stability of Cyclo-dG (16) and Cyclo-dT (17). Cyclo-dG
in a neutral pH solution exhibited virtually no change in CD
after 6 h at 90 °C, whereas cyclo-dT under the same conditions
showed dramatic reduction in absorption (Figures S20, S21,
Supporting Information). These results demonstrate the lesser
stability of cyclo-dT relative to cyclo-dG in aqueous solution,
which motivated us to conduct a systematic hydrolysis study.
To this end, samples of each cyclonucleoside were placed in
an acidic (1.0 M HCl) or basic (1.0 M NaOH) solution. Figures
8 and 9 illustrate the hydrolysis schemes of cyclo-dG and cyclo-
dT, respectively, at specified conditions and their HPLC profiles
as a function of time.
Acid Hydrolysis of Cyclo-dG. After 1 h, a refluxing acid
solution of cyclo-dG produced the hydrolyzed product 18 (RT
) 10.3 min, Figure 8b), which arose from the cleavage of the
glycosidic linkage at N9. Additional heating resulted in reduction
of 18 with a concomitant increase of the starting cyclo-dG. It
was apparent that the glycolytic 18 was formed first and
subsequently converted back to cyclo-dG (16). The latter
deglycolysis process was complete by 6 h and was accompanied
by a small amount of guanine at 4.9 min. Continued heating
(48 h) resulted in complete depurination (not shown), involving
hydrolysis at both positions 9 and 3. Interestingly, acid
hydrolysis at 37 °C yielded an exclusive formation of 18 in
less than 1 h (Figure 8a). Additional incubation at 37 °C for
3 h resulted in a complete deglycosylation (Figure 8b). Note
the similarity of the HPLC profiles between the 3 h reaction at
37 °C and the 6 h reaction at 100 °C. This is in contrast to
The UV spectra of 18 (Figure S28, Supporting Information)
do not differ substantially from those of cyclo-dG (Figure S28,
Supporting Information). Like cyclo-dG, the UV and CD spectra
were particularly sensitive to extreme pHs (Figure S27, Sup-
porting Information). These results collectively indicate that the
overall electronic structures of cyclo-dG remain intact despite
cleavage of the glycosidic bond. ESI-MS of 18 showed (M +
H)+ at 268.1, which is 18 Daltons (H2O) greater than that of
cyclo-dG. The product ion spectrum of m/z 268.1 ion exhibited
fragment ions at 250 and 232.1, corresponding to successive
loss of one and two H2O molecules from the sugar. As expected,
the m/z 152.2 ion corresponds to the protonated guanine formed
by a cleavage of the 3,5′-cyclic linkage. The characteristic
1
patterns of the H1′ and H5′,5′′ protons that existed in the H
NMR of cyclo-dG disappeared. Also, the appearance of the
N9-H and H8 protons as a doublet at 7.27 and 7.85 ppm,
respectively, indicate the prototropic tautomerism exhibited by
N(7)H and N(9)H in 18 (Figures S29, S30, Supporting Informa-
tion). The C1′ signal was shifted downfield (-9.4 ppm) relative
to that of cyclo-dG (89.1 ppm) (Figure S31, Supporting
Information). These results are indicative of a broken glycosidic
linkage and a hydroxyl at C1′.

R,ꢀ-m-dNTP as NoncleaVable Substrates for DNA Polymerases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6465
Figure 8. (a) Hydrolysis schemes of 16 and (b) the corresponding HPLC profiles in 1 M HCl, 37 °C, 1 M HCl, 100 °C, and 1 M NaOH, 37 °C
using gradient program B.
Base Hydrolysis of Cyclo-dG. Cyclo-dG (16) was slow to
react in 1.0 M NaOH, but it transformed completely to a 4:1
mixture of products after 41 h at 37 °C (Figure 8b). The major
peak at 10.0 min was identified as N3,5′-cyclo-2′-deoxyxan-
thosine (cyclo-dX, 19), which arose via an oxidative deamination
at C2 position (see below). Holmes and Robins23 reported a
similar deamination reaction on 2′,3′-O-isoprolylidene-3,5′-
anhydroguanosine under similar conditions. The ribose analogue
of 19 has recently been synthesized26 and isolated27 from Eyrus
sp. of marine sponge from the Great Australian Bight.
are consistent with the glycolytic product 20. ESI-MS showed
an (M + H)+ at m/z 269.0, which represents the addition of a
H2O molecule. The base fragment at m/z 153.2 corresponds to
the protonated xanthine base formed by cleavage of the 3,5′-
cyclic linkage of a protonated 19. Both the mass and NMR
spectra are similar to those observed for the glycolytic product
18 isolated from cyclo-dG 16.
Acid Hydrolysis of Cyclo-dT. Acid treatment of cyclo-dT
for 30 min at 37 °C produced a single product 21 at 15.5 min
(Figure 9) with a small amount of thymine at 4.8 min. The
depurination reaction was complete in 6 h. A similar hydrolysis
profile was obtained in a neutral buffer solution. Interestingly,
however, 21 never exceeded that of cyclo-dT (assuming they
have similar molar absorptivity coefficients) throughout the
reaction. It appears that an initial glycolysis was followed
immediately by the cleavage of the 2,5′-anhydro-linkage. Unlike
in the case of cyclo-dG, no recyclization (deglycolysis) of cyclo-
dT was observed. These results indicate a greater fragility of
the 2,5′-O-linkage in acidic conditions. Attempts to isolate the
intermediate 21 in its pure form in order to purse a detailed
characterization were not successful. Nonetheless, the ESI-MS
spectra show an (M + H)+ at 243.1, which is 18 Daltons greater
than starting (pure) cyclo-dT, indicating that there is an addition
of one H2O to the parent compound. The product ion spectrum
of the m/z 243.1 ion exhibited fragment ions at 225.0 and 207.0
for losses of one and two H2O molecules from the sugar. The
base fragment at 127.1 corresponds to the protonated thymine
base. The mass spectrum of 21 is similar to that obtained for
18, thus 21 was assigned as a glycolysis product, as suggested
by their exact masses. The possibility of an initial cleavage of
the 2,5′-O anhydrous linkage, followed by a glycolysis, was
ruled out because no dT was detected in the reaction mixture.
Base Hydrolysis of Cyclo-dT. The base hydrolysis of cyclo-
dT at 37 °C occurred within 30 min to give rise to dT
ESI-MS of the major product showed an (M + H)+ at 251.0,
which is one more Dalton than the starting cyclo-dG. The
product ion spectra show a fragment ion at 233.0 due to loss of
a water molecule involving the 3′-OH and a hydrogen on the
adjacent C2′ (Figure S38, Supporting Information). The UV and
CD spectra of 19 are quite different from those of cyclo-dG,
suggesting that there is a change in the chromophore purine
base (Figures S34, S35, Supporting Information). The 1H NMR
profile of 19 (Figures S34, S35, Supporting Information) was
essentially similar to that of the starting cyclo-dG 16 except
for appearance of a new imide proton at 11.1 ppm. All 1H NMR
signals were assigned and confirmed by COSY/TOCSY spectra.
A strong cross peak between H8 and H1′ in the NOESY
spectrum (Figure S37, Supporting Information) confirmed the
inheritance of the well-defined syn-glycosidic conformation from
cyclo-dG. The 13C NMR of 19 (Figure S36, Supporting
Information) exhibited two distinctive carbonyl signals at 157.9
and 151.7 ppm, which confirms an additional new keto
functionality at C2. The dramatic UV changes that were
observed in both acidic and basic pHs are consistent with the
presence of an imino proton at N1 (Figure S33, Supporting
Information).
Treatment of 19 with 1.0 M HCl (HPLC data not shown)
1
resulted in a single fraction whose ESI-MS and H NMR data

6466 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Liang et al.
Figure 9. (a) Hydrolysis schemes of 17 and (b) the corresponding HPLC profiles in pH 7.0 buffer, 37 °C, 1 M HCl, 37 °C, and 1 M NaOH, 37
°C using gradient program B.
exclusively (Figure 9). This reaction represents an exclusive
cleavage of the 2,5′-anhydrous linkage and no additional
decomposition occurred in 4 h. This was in clear contrast to
cyclo-dG, which underwent an oxidative deamination to 19. The
greater susceptibility to base hydrolysis of cyclo-dT versus
cyclo-dG can be explained by the nature of the 5′-bridge linkage.
That is, the 5′,2-O linkage is more vulnerable to basic hydrolysis
than is the 5′,3-N linkage.
Previous studies on the corresponding ribonucleoside hy-
drolysis showed that the rate of glycosidic hydrolysis in 0.1 N
HCl at 25 °C followed the order 3,5′- , 5′,8-cyclonucleoside
< noncyclic nucleoside.23-25,28 The present findings indicate
that the loss of ring strain during hydrolysis of cyclic nucleosides
does not increase the rate of hydrolysis contrary to what might
have been expected.24,25 Reese et al.28 reported that exclusive
glycosidic hydrolysis of 3,5′-cyclo-guanosine occurred after 27 h
at 37 °C in 0.9 N HCl. That result was based on a loss of starting
material and no product characterization was reported. In the
present study, we found that hydrolysis at the glycosidic bond
was complete in 1 h at 37 °C in 1.0 M HCl. Thus it appears
that the rate of hydrolysis is much greater for 2′-deoxycyclo-
nucleosides.
which may catalyze an intramolecular cyclization with N3 of
guanine and O2 of thymine moiety, respectively. These will be
the subject of future investigations.
In summary, we described the preparation of a complete set
of R,ꢀ-methylene dNTPs (12-15, N ) A, C, T, G, Figure 1)
and their substrate binding affinities for polymerase ꢀ. The
synthesis entails a nucleophilic coupling reaction between a 5′-
O-tosyl-2′-deoxynucleoside and methylene diphosphate, fol-
lowed by an enzymatic γ-phosphorylation. In the cases of dG
and dT, significant amounts of cyclo-dG (16) and cyclo-dT (17)
were also isolated. Under acidic conditions, these novel 2′-
deoxycyclonucleosides undergo a glycolysis reaction, followed
by complete depurination. In the case of cyclo-dG, there existed
an equilibrium between glycolysis and deglycolysis prior to a
complete depurination. Under basic conditions, cyclo-dG un-
derwent an oxidative deamination at C2 to give cyclo-dX (19),
while cyclo-dT was transformed to dT via an exclusive cleavage
of the ether linkage between positions 2 and 5′. Gap-filling DNA
synthesis assays showed that all four R,ꢀ-m-dNTPs (12-15)
behave as noncleavable substrates of polymerase ꢀ with binding
affinities (Ki 1-5 µM) similar to natural dNTPs.16
Cyclonucleosides have been synthetic, structural, and biologi-
cal targets for many years.23-27,29 For example, 8,5′-cycload-
enosine is an inhibitor of rhodopsin kinase30 and the 2,5′-
cylclothymidine derivatives have shown antiviral properties.24
The 8,5′-cyclopurine nucleosides such as 8,5′-cyclo-dG and 8,5′-
cyclo-dA have been identified as the major tandem oxidative
DNA damage lesions.31 Rodriguez et al.32 have shown that
lymphoblasts of women with BRCA1 mutations who had been
diagnosed with breast cancer are deficient in the repair of 8,5′-
cyclo-dG and 8-oxo-dG. Virtually nothing is known about the
biological roles of cyclo-dG (16) and cyclo-dT (17) reported in
the present study. These novel cyclo-2′-deoxynuclosides could
be formed because of facile formation of the C5′ radical or ion,33
Experimental Section
All solvents, reagents, and standards were purchased from
Aldrich (Milwaukee, WI) and Fisher Scientific (Pittsburgh, PA)
and used without further purification. Purification of cyclonucleo-
sides and their hydrolysis products were conducted on silica gel
(200-400 mesh) column chromatography (Scientific Adsorbents
Inc., Atlanta, GA). Flash cellulose chromatography was performed
on Whatman CF-11 fibrous cellulose. DOWEX AG-50W column
(H+ form) was purchased from Fisher Scientific. Affi-Gel Boronate
affinity gel, Q sepharose FF anion exchange resin, and associated
columns were purchased from Bio-RAD laboratories, Inc. Nucleo-
side diphosphate kinase (NDPK) and pyruvate kinase were pur-
chased from Sigma-Aldrich.

R,ꢀ-m-dNTP as NoncleaVable Substrates for DNA Polymerases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6467
All NMR (1H, 13C, and 31P) spectra were recorded using an
inverse 5 mm inverse probe on a Bruker DPX400 Avance
spectrometer operating at 400.1, 100.6, and 161.9 MHz, respec-
7.0 Hz, H1′), 4.49 (1H, m, H3′), 3.81 (1H, m, H4′), 3.68 (2H, m,
H5′,H5′′), 2.25 (2H, m, H2′,H2′′), 0.90 (18H, s, 2 t-C4H9), 0.13
(12H, s, 4 CH3)(Figure S1, Supporting Information), LR-MS (ESI)
(M + H)+ 496.34.
1
tively. H and 13C spectra are referenced to TMS. 31P spectra are
referenced using an external 85% aqueous phosphoric acid solution.
The energy-minimized structures of cyclo-dG and cyclo-dT were
obtained using Spartan modeling software (Essential Edition,
Version 3.0.2).
3′-O-(tert-butyldimethylsilyl)-2′-dG (6). Compound 5 (800 mg,
1.6 mmol) was added to a solution of acetic acid, water, and THF
(13:7:3, 160 mL). The mixture was stirred at room temperature
under nitrogen for 18 h. The solvent was reduced to one-fifth of
the original volume under vacuum and lyophilized to dryness.
Elution with methylene chloride:methanol (95:5) on silica yielded
HPLC analyses were carried out on Hitachi EZChrom Elite
HPLC unit with a L2450 diode array detector and a Phenomenex
Luna C18 column (150 cm × 4.5 mm, 5 µm) with flow rate of 1
mL/min. The following two solvent programs were used: (A) a
step gradient involving buffer I (10 mM tetrabutylammonium
hydroxide, 10 mM KH2PO4, 0.25% methanol, pH 7.00) and buffer
II (2.8 mM tetrabutyl ammonium hydroxide, 100 mM KH2PO4,
30% methanol, pH 5.50); 0-15 min 100% I, 15-20 min 0-10%
II, 20-25 min 10-30% II; 25-40 min 30-37% II, 40-55 min
37-45% II, 55-70 min 45-55% II, 70-80 min 55-75% II,
80-90 min 70-100% II; (B) a 30 min isocratic program involving
2% of solvent I (an equal mixture of acetonitrile and 0.10 M
ammonium acetate) and 98% of solvent II (0.10 M ammonium
acetate).
1
380 mg of 6 (63% yield). H NMR (DMSO-d6): δ 10.66 (1H, s,
CONH), 8.00 (1H, s, H8), 7.27 (1H, t, OH), 6.50 (2H, s, NH2),
6.11 (1H, t, J ) 7.0 Hz, H1′), 4.51 (1H, m, H3′), 3.80 (2H, m,
H5′,H5′′), 2.21 (2H, m, H2′,H2′′), 0.92 (s, 9H, t-C4H9), 0.13 (6H,
s, 2 CH3)(Figure S2, Supporting Information), LR-MS (ESI) (M
+ H)+ 382.23.
5′-O-tosyl-3′-O-(tert-butyldimethylsilyl)-2′-dG (7). A solution of
6 (380 mg, 1.0 mmol) in 18 mL of dry pyridine was cooled to 0
°C in an ice-salt mixture. Then 1.0 mmol of p-TsCl in 10 mL of
dry pyridine was added dropwise to the reaction mixture. The
mixture was stirred at 0 °C for 24 h. The reaction was quenched
with ice water, and the mixture was extracted with chloroform.
The organic layer was washed consecutively with saturated NaH-
CO3 and ice-water and dried over anhydrous MgSO4. Silica column
chromatography (methylenechloride:methanol 96:4) gave 340 mg
CD experiments were performed on a Jasco J-810 spectropola-
rimeter equipped with a variable temperature controller. In a typical
run, samples (∼2 ODS) were dissolved in 400 µL of water or a
buffer containing 0.2 M NaCl, 10 mM sodium phosphate, pH )
6.9. Spectra were recorded at 15 °C in a 1 mm path length cell.
The spectropolarimeter was scanned from 200 to 400 nm at a rate
of 50 nm/min. Data points were acquired every 0.2 nm with a 2 s
response time. Spectra were the averages of 10 accumulations and
smoothed using 17-point adaptive smoothing algorithms provided
by Jasco. UV absorption spectra were obtained on a Beckman DU
800 UV/vis spectrophotometer using a 1 cm path length cell.
Samples (∼0.25 ODS) were prepared in 350 µL of a pH 7.0 buffer
solution containing 0.2 M NaCl and 10 mM sodium phosphate.
ESI-MS and MS/MS spectra were acquired with a ThermoFinni-
gan Advantage (San Jose, CA) linear ion trap (LTQ) interfaced to
a Fourier transform mass spectrometer (FTMS) at the Washington
University Mass Spectrometry Research Resource in St. Louis, MO.
Molecular weight and product ion spectra of all the protonated
molecule ions were acquired by direct infusion of low picomolar
amounts of sample into the LTQ. The relative collision energy for
MS/MS experiments was set to 35% of its maximum value for
fragmentation of all the molecule ions in this study. A 70:30 mixture
of methanol and water that was 0.1% in formic acid was used as
the mobile phase. Exact masses of all protonated molecule ions
and product ions formed in the LTQ were acquired in the FTMS.
The capillary temperature was 230 °C and the spray voltage was
4.5 kV. The mass resolution (m/∆m) was 100000 at m/z 400.
Synthesis of 5′-O-Tosylnucleosides. 5′-O-Tosylation of dA, dC,
and dT was carried out in a similar fashion to the literature
procedures10,12,24,34 and is summarized as follows. Briefly, 2′-
deoxynucleoside (2.0 mmol) was treated with a 1.2 equivalent of
p-TsCl in 15 mL dry pyridine at -5 °C. After 16 h at 0 °C, the
reaction was quenched with ice-water and extracted with chloro-
form. Combined chloroform extracts were washed consecutively
with saturated NaHCO3 and water and dried over anhydrous
MgSO4. Flash chromatography on silica (methanol/chloroform)
yielded pure 5′-O-tosyl derivatives (1-3) in 30-40% yields.
1
of 7 (64%). H NMR (DMSO-d6): δ 10.68 (1H, s, CONH), 7.83
(1H, s, H8), 7.75 (2H, d, J ) 8.0, tosyl), 7.41 (2H, d, J ) 8.0 Hz,
tosyl), 6.48 (2H, s, NH2), 6.09 (1H, t, J ) 7.0 Hz, H1′), 4.44 (1H,
m, H3′), 4.17 (1H, m, H4′), 3.8 (2H, m, H5′,H5′′), 2.29 (2H, m,
H2′,H2′′), 1.28 (3H, s, tosyl-CH3) 0.84 (s, 9H, t-C4H9), 0.05 (6H,
s, 2 CH3)(Figure S3, Supporting Information), LR-MS (ESI) (M
+ H)+ 536.29.
5′-O-Tosyl-2′-dG (4). Aqueous hydrofluoric acid (15 mL, 2%)
was added to 15 mL of acetonitrile containing 7 (340 mg, 0.64
mmol). After stirring for 24 h at room temperature, a homogeneous
solution was neutralized with excess solid ammonium bicarbonate.
The acetonitrile was removed under vacuum, and the water was
removed by lyophilization, leaving a white solid. The solid was
extracted with 9:1 chloroform:methanol and the extract was eluted
on silica gel with with dichloromethane:methanol (90:10) and
yielded 160 mg of 4 (70%). 1HNMR (DMSO-d6): δ 10.62 (1H, s,
CONH), 8.64 (1H, s, OH), 8.03 (1H, s, H8), 7.77 (2H, d, tosyl),
7.32 (2H, d, tosyl), 6.41 (2H, s, NH2), 6.01 (1H, s, H1′), 4.25 (1H,
m, H3′), 4.14-3.84 (3H, m, H4′, H5′,H5′′), 2.25 (2H, m, H2′,H2′′),
1.16 (3H, s, Ar-CH3)(Figure S4, Supporting Information). LR-MS
(ESI) (M + H)+ 421.99.
Synthesis of r,ꢀ-Methylene 2′-Deoxynucleoside 5′-Diphosphate
(r,ꢀ-m-dNDP:). r,ꢀ-Methylene 2′-Deoxyadenosine 5′-Diphosphate
(8). Freshly prepared tris(tetra-n-butylammonium) hydrogen me-
thylene pyrophosphate (472 mg, 0.53 mmol) was added to
compound 1 (140 mg, 0.35 mmol) in 0.2 mL of acetonitrile. This
viscous solution was allowed to stir under N2 at room temperature
for 48 h. The solution was diluted with water (10 mL). The tetra-
n-butylammonium cation was exchanged for an ammonium cation
by passing the solution through a DOWEX AG-50W column
(NH4+) form with 2 column volumes of deionized water. The eluent
was lyophilized and the solid was extracted with acetonitrile/100
mM NH4HCO3/NH4OH (7:3:2). The extract was loaded onto CF-
11 cellulose column (5 cm × 23 cm) connected to a FPLC and
eluted with the same solution. The elution was monitored using
UV detector set at 230 nm. The major UV absorbing fractions were
pooled, and the acetonitrile was removed by rotary evaporation.
The resulting aqueous solution was lyophilized to give 90 mg of 8
(63%) as a white fluffy solid. HPLC RT 79.4 min (program A). 1H
NMR (D2O) δ 8.39 (1H, s, H8), 8.13 (1H, dd, J ) 6.6 Hz, H2),
6.37 (1H, t, J) 6.6 Hz, H1′), 4.65 (1H, m, H3′), 4.16 (1H, m, H4′)
3.99 (2H, m, H5′,H5′′), 2.72-2.48 (2H, m, H2′,H2′′) 2.04 (2H, t,
J ) 20.3 Hz, PR-CΗ2-Pꢀ). 31P NMR (D2O) δ 19.70 (m, PR), 16.13
(m, Pꢀ). LR-MS (ESI) (M + H)+ 410.29.
A direct 5′-O-tosylation of dG was problematic due to its low
solubility in pyridine. Instead, 5′-tosyl-2′-deoxyguanosine (4) was
obtained by the three-step indirect procedure shown in Figure 1.
5′,3′-O-(bis-tert-butyldimethylsilyl)-2′-dG (5). A mixture of dG
(500 mg, 1.86 mmol), TBDMSCl (670 mg, 4.46 mmol), and
imidazole (316 mg, 4.65 mmol) was stirred in 3 mL of dry DMF
at room temperature under nitrogen for 24 h. The solvent was
removed under reduced pressure, and water was added to the
reaction mixture. The precipitated solid was filtered and dried under
vacuum. Recrystallization from 95% ethanol gave 5 as shiny white
crystals (800 mg, 87% yield). 1H NMR (DMSO-d6): δ 10.62 (1H,
s, CONH), 7.90 (1H, s, H8), 6.49 (2H, s, NH2), 6.11 (1H, t, J )
r,ꢀ-Methylene 2′-Deoxycytidine 5′-Diphosphate (9). Compound
9 was prepared in a similar manner from 2 (134 mg, 0.35 mmol)

6468 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Liang et al.
and tris(tetra-n-butylammonium) hydrogen methylene pyrophos-
phate (472 mg, 0.53 mmol). Compound 9 (74 mg (55%) was
obtained as a white fluffy solid. HPLC RT 59.7 min (program A).
1H NMR (D2O) δ 7.85 (1H, d, J ) 7.8 Hz, H6), 6.19 (1H, t, J )
6.5 Hz, H1′), 5.98 (1H, d, J ) 7.6 Hz, H5), 4.47 (1H, m, H3′),
4.03 (1H, m, H4′), 3.99 (2H, m, H5′,H5′′), 2.25-2.35 (2H, m,
H2′,H2′′), 1.99 (2H, t, J ) 19.6 Hz, PR-CΗ2-Pꢀ). 31P NMR (D2O)
δ 19.92 (m, PR), 16.54 (m, Pꢀ). LR-MS (ESI) (M + H)+ 386.29.
r,ꢀ-Methylene 2′-Deoxyguanosine 5′-Diphosphate (11) and 3,5′-
cyclo-2′-Deoxyguanosine (16). The coupling reaction between 4 (147
mg, 0.35 mmol) and tris(tetra-n-butylammonium) hydrogen methy-
lene pyrophosphate (472 mg, 0.53 mmol) gave two main fractions
with an approximately equal intensity after an FPLC. An early
eluting major fraction was collected and purified by HPLC to afford
3,5′-cyclo-2′-deoxyguanosine (cyclo-dG, 16) (14 mg, 16%) as a
Final purification of the triphosphate product was performed on
FPLC with a Q Sepharose FF anion exchange column (2.5 cm ×
-
20 cm, HCO3 form) by a linear gradient elution with NH4HCO3
(from 0.05 to 0.5 M, pH 7.8) at a flow rate of 2 mL/min monitored
at 230 nm. The desired fractions were pooled and lyophilized to
+
give a white fluffy solid 12 (25 mg, NH4 salt, 52%). HPLC RT
1
89.3 min (program A). H NMR (D2O) δ 8.37 (1H, s, H8), 8.10
(1H, dd, J ) 6.6 Hz, H2), 6.35 (1H, t, J ) 6.6 Hz, H1′), 4.70 (1H,
m, H3′), 4.13 (1H, s, H4′), 3.97 (2H, m, H5′), 2.71-2.44 (1H, m,
H2′, H2′′), 2.20 (2H, t, J ) 20.3 Hz, PR-CΗ2-Pꢀ). 31P NMR (D2O)
δ 23.33 (m, PR), 12.42 (m, Pꢀ) -4.82 (d, JP-P ) 24.7 Hz, Pγ).
HRMS (ESI): calcd, 488.0137 (M - H)-; found, 488.0143 (M -
H)-.
r,ꢀ-methylene-dCTP (13). Compound 13 was prepared in a
similar manner to 12 from 9 (30 mg, 0.08 mmol) to yield 18 mg
(NH4+ salt, 49%) of 13 as a white fluffy solid. HPLC RT 69.1 min.
1H NMR (D2O) δ 7.83 (1H, d, J ) 7.8 Hz, H6), 6.18 (1H, m, J )
6.1 Hz, H1′), 5.96 (1H, d, J ) 7.8, H5) 4.48 (1H, m, H3′), 4.04
(1H, m, H4′), 3.96 (2H, m, H5′,H5′′), 2.15-2.32 (2H, m, H2′,H2′′),
2.24 (2H, t, J ) 20.4 Hz, PR-CΗ2-Pꢀ). 31P NMR (D2O) δ 23.29
(m, PR), 12.27 (m, Pꢀ), -4.87 (d, JP-P ) 25.8 Hz, Pγ). HRMS
(ESI): calcd, 464.0025 (M - H)-; found, 464.0032 (M - H)-.
r,ꢀ-Methylene-dTTP (14). Compound 14 was prepared from 10
1
white fluffy solid. 16: H NMR (DMSO-d6) δ 7.64 (1H, s, H8),
7.04 (2H, bs, NH2), 6.50 (1H, dd, J ) 5.2 Hz, H1′), 5.53, (1H, bs,
3′-OH), 4.54 (1H, m, H4′), 4.50 (1H, d, J ) 13.6 Hz, H5′), 4.40
(1H, m, H3′), 3.84 (1H, d, J ) 13.6 Hz, H5′′), 2.16 (2H, m,
H2′,H2′′). 13C NMR (D2O): 89.15 (C1′), 84.39 (C4′), 70.50 (C3′),
53.80 (C5′), 42.97 (C2′). LR-MS (ESI) [M + H]+ 250.13. HRMS
(ESI): calcd, 250.0935 (M + H)+; found, 250.0925 (M + H)+.
The late eluting fractions were identified as the desired diphosphate
product 11 (44 mg, 30%) as a white fluffy solid. 11: HPLC RT
+
(25 mg 0.06 mmol) as described above to yieled 13 mg (NH4
1
salt, 45%) of 14 as a white fluffy solid. HPLC RT 82.5 min (program
A). 1H NMR (D2O) δ 7.72 (1H, s, H6), 6.31 (1H, dd, J ) 6.8 Hz,
H1′), 4.61 (1H, m, H3′), 4.2 - 4.1 (3H, m, H4′, H5′,H5′′), 2.37
(2H, m, H2′, H2′′), 2.34 (2H, t, J ) 20.4 Hz, PR-CΗ2-Pꢀ), 1.90
(3H, s, CH3). 31P NMR (D2O) δ 21.82 (m, PR), 11.42 (m, Pꢀ),
-6.04 (d, JP-P ) 16.7 Hz, Pγ). HRMS (ESI): calcd, 479.0022 (M
- H)-; found, 479.0028 (M - H)-.
61.8 min (program A). H NMR (D2O) δ 8.28 (1H, s, H8), 6.32
(1H, t, J ) 6.9 Hz, H1′), 4.72 (1H, m, H3′), 4.25 (1H, m, H4′),
4.10 (2H, m, H5′), 2.80-2.52 (2H, m, H2′,H2′); 2.13 (2H, t, J )
19.8 Hz, PR-CΗ2-Pꢀ). 31P NMR (D2O) δ 19.47 (m, PR), 16.08
(m, Pꢀ). LR-MS (ESI) (M + H)+ 425.83.
r,ꢀ-methylene 2′-deoxythymidine 5′-diphosphate (10) and 2,5′-
cyclo-2′-Deoxythymidine (17). The usual coupling reaction of 3 (100
mg, 0.25 mmol) with tris(tetra-n-butylammonium) hydrogen me-
thylene pyrophosphate (340 mg, 0.38 mmol) gave two main
fractions of equal abundance by FPLC. An early eluting major
fraction was collected and purified by HPLC to give 2,5′-cyclo-dT
r,ꢀ-methylene-dGTP (15). Compound 15 was prepared from 11
+
(30 mg 0.08 mmol) to give 21 mg (NH4 salt, 52%) of 15 as a
white fluffy solid. 15: HPLC RT 69.4 min. 1H NMR (D2O) δ 8.47
(1H, s, H8), 6.31 (1H, t, J ) 6.5 Hz, H1′), 4.84 (1H, m, H3′), 4.23
(1H, m, H4′), 4.15 (2H, m, H5′,H5′′), 2.85-2.50 (2H, m, H2′,H2′′),
2.32 (2H, t, J ) 19.8 Hz, PR-CH2-Pꢀ). 31P NMR (D2O) δ 22.19
(m, PR), 11.37 (m, Pꢀ), -6.07 (t, JP-P ) 23.8 Hz, Pγ). HRMS
(ESI): calcd, 504.0087 (M - H)-; found 504.0092 (M - H)-.
Alternative Synthesis of Cyclo-dG (16) and Cyclo-dT (17).
Cyclo-dG (16) was prepared according to a modified literature
procedure.35,36 A solution of DIAD (690 mg, 3 mmol) in dioxane
(1.0 mL) was added dropwise to a suspension of dG (268 mg, 1.0
mmol) and triphenyl phosphine (786 mg, 3.0 mmol) in dioxane
(2.0 mL) at 70 °C. dG was solubilized as DIAD was added. The
resulting homogeneous solution was stirred at 70 °C for 3 h and
then allowed to cool to ambient temperature. The resulting
precipitate was filtered and washed with dioxane. Purification by
silica gel column chromatography (ethyl acetate:methanol 60:40)
gave 16 (80 mg, 30%). The chromatographic (TLC, HPLC) and
spectroscopic (1H NMR, UV, CD, ESI-HRMS) properties of this
sample match to those obtained from the coupling reaction between
5′-O-tosyl-2′-dG 4 and methylene diphosphate described above.
Cyclo-dT (17) was prepared according to a general cyclization
procedure.24 To a solution of 3 (1.13 g, 2.54 mmol) in acetonitrile
(70 mL) was added DBU (0.7 g, 4.6 mmol). The reaction mixture
was refluxed for 1 h and was monitored for starting material by
TLC (CH2Cl2-CH3OH (80:20)). After 90 min, TLC indicated the
reaction was complete. The hot solution was immediately filtered
through a celite pad, and the filtrate was evaporated to dryness.
The residue was purified by silica gel column chromatography
(CH2Cl2-CH3OH (80:20)) to give 285 mg (50%) of pure 17. The
chromatographic (TLC, HPLC) and spectroscopic (1H NMR, UV,
CD, ESI-HRMS) properties of this sample match to those obtained
from the coupling reaction between 3 and methylene diphosphate
described above.
1
17 (cyclo-dT; 167 mg, 31%). H NMR (D2O) δ 7.75 (1H, s, H6),
6.1 (1H, dd, J ) 8.0, 1.8 Hz, H1′), 4.82 (1H, m, H4′), 4.62 (1H, d,
J ) 12.9 Hz, H5′), 4.55 (1H, m, H3′), 4.26 (1H, d, J ) 12.9 Hz,
H5′′), 2.67 (1H, m, H2′), 2.49 (1H, m, H2′′), 1.92 (3H, s, CH3).
13C NMR (DMSO-d6) δ 171.90 (C4), 158.09 (C2), 141.42 (C6),
118.69 (C5), 94.64 (C1′), 86.34 (C4′), 75.36 (C5′), 71.96 (C3′),
41.95 (C2′), 12.36 (CH3). LR-MS (ESI) (M + H)+ 225.07. HRMS
(ESI): calcd, 225.0870 (M + H)+; found, 225.0859 (M + H)+.
The late eluting fractions were identified as the desired diphosphate
product 10 (30 mg, 30%) as a white fluffy solid. HPLC RT 45.4
1
min (program). H NMR (D2O) δ 7.60 (1H, s, H6), 6.19 (1H, dd,
J ) 6.7 Hz, H1′), 4.48 (1H, m, H3′), 4.03 (1H, m, H4′), 3.98 (2H,
m, H5′,H5′′), 2.15-2.45 (2H, m, H2′,H2′′), 2.03 (2H, t, J ) 19.8
Hz, PR-CΗ2-Pꢀ), 1.78 (3H, s, CH3). 31P NMR (D2O) δ 19.90 (m,
PR), 16.45 (m, Pꢀ). LR-MS (ESI) (M + H)+ 400.99.
Synthesis of r,ꢀ-Methylene 2′-Deoxynucleoside 5′-Triphos-
phate (r,ꢀ-m-dNTP; 12-15). r,ꢀ-methylene-dATP (12). A mixture
of 8 (40 mg, 0.1 mmol), ATP (55 mg, 0.1 mmol), and phosphoenol
pyruvate (PEP, 38 mg, 0.2 mmol) were placed in a 10 mL solution
of triethanolamine (83 mM), MgCl2 (17 mM), KCl (67 mM). The
pH of the mixture was adjusted to 7.6 with 6.0 M NaOH.
Nucleoside diphosphate kinase (NDPK) (200 unit) and pyruvate
kinase (400 unit) were added to this solution, and the mixture was
incubated at 37 °C for 4 h. The reaction progress was monitored
by HPLC (program A) until γ-phosphorylation was complete. Then
ADP (45 mg, 0.1 mmol) was added to remove the excess PEP in
the reaction mixture. After 1 h, the mixture was filtered through a
0.2 µm syringe disk and lyophilized. The residual solid on the disk
was dissolved in 10 mL of 1 M NH4HCO3 and adjusted to pH 8.5
with concentrated NH4OH. The mixture was then applied to a Bio-
Rad boronate affinity gel column (2.5 cm × 5.5 cm, in 1 M
NH4HCO3, pH 8.5). The column was eluted with the same buffer
at a flow of 1 mL/min and the fractions containing the first two-
column volumes were pooled and lyophilized. Excess NH4HCO3
was removed by repeated lyophilization from deionized water after
adjustment to pH 7.2 with CO2.
Stability Studies of 2′-Cyclonucleosides. General Procedure.
Cyclonucleosides (0.2 mmol) were placed in 5.0 mL of either 1.0
M HCl or 1.0 M NaOH for acid and base hydrolysis, respectively.
Reaction progress at either 37 or 100 °C was monitored by HPLC/
UV using gradient program B. After appropriate time, the reaction

R,ꢀ-m-dNTP as NoncleaVable Substrates for DNA Polymerases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6469
mixture was neutralized, lyophilized, and purified by silica column
chromatography.
determined using a reaction mixture containing 50 mM Tris-HCl,
pH 7.4, 20 mM MgCl2, 200 nM single-nucleotide gapped DNA, at
various dNTP concentrations. Reactions were initiated with 0.5 nM
enzyme at room temperature and stopped with EDTA mixed with
formamide dye. The substrates/products were separated on 15%
denaturing polyacrylamide gels and quantified in the dried gels by
phosphorimagery. Steady-state kinetic parameters (kcat, Km) were
determined by fitting the rate data to the Michaelis equation.
Inhibition of dNTP insertion by various R,ꢀ-methylene-dNTPs was
determined at different concentrations of inhibitor (I) and subsatu-
rating dNTP concentration (S). The inhibitor equilibrium binding
constant, Ki, was determined by fitting the inhibition data to eq 1
for competitive inhibition by nonlinear regression methods.
Acid Hydrolysis of Cyclo-dG (16). Complete disappearance of
16 (RT ) 5.0 min) occurred after 1 h at 37 °C with an exclusive
formation of a new peak at 10.5 min (Figure 8b). The product was
identified as the glycosidic cleavage product 18. 1H NMR (DMSO-
d6): δ 7.85 (1H, d, J ) 8.0 Hz, H8), 7.27 (1H, d, J ) 8.0 Hz, NH),
6.81 (2H, s, NH2), 6.22 (1H, bs, OH), 5.43 (1H, t, H1′), 4.41-4.33
(1H, m, H3′), 4.25-4.04 (2H, m, H5′, H4′), 4.01-3.93 (1H, m,
H5′′), 2.33 (1H, m, H2′), 2.05 (1H, m, H2′′). 13C NMR (DMSO-
d6): δ 140.19 (C8), 98.54 (C1′), 82.96 (C4′), 71.81 (C3′), 48.26
(C5′), 40.59 (C2′). HRMS (ESI): calcd, 268.1045 (M + H)+; found,
268.1039 (M + H)+. Continued incubation at 37 °C triggered a
reverse cyclization reaction (e.g., deglycosylation). After 3 h, entire
18 was reverted back to the starting cyclo-dG along with trace of
the free base guanine (Figure 8b). The formation of cyclo-dG and
guanine was confirmed by cochromatography with standards on
HPLC. When the above reaction was carried out at 100 °C, a similar
HPLC profile was obtained after 6 h (Figure 8b). Prolonged reaction
time (48 h) at 100 °C resulted in a complete depurination.
Base Hydrolysis of Cyclo-dG. No decomposition of cyclo-dG
16 (RT 4.9 min, Figure 8b) occurred in 1.0 M NaOH after stirring
for 1 h at 37 °C. A longer reaction time (18 h), however, led to
formation of one major product at 10.0 min. The base hydrolysis
completed after 41 h. An acid workup, followed by a silica
chromatography (ethyl acetate:methanol 30:70%), yielded 3,5′-
cyclo-2′-deoxyxanthosine 19 (cyclo-dX) as a main product. A polar
minor product at 4.0 min decomposed on passing through a reverse
phase column. 19: 1H NMR (DMSO-d6): δ 11.11 (1Η, s, CONH),
7.71 (1H, s, H8), 6.47 (1H, m, H1′), 5.46 (1H, bs, 3′-OH), 4.65
(1H, J )16 Hz, H5′), 4.60 (1H, m, H3′), 4.36 (1H, m, H4′), 3.65
(1H, m, J ) 16.0 Hz, H5′′), 2.21 (2H, m, H2′,H2′′). 13C NMR
(DMSO-d6): δ 157.91 (C6), 151.78 (C2), 141.58 (C8), 118.56 (C5),
88.43 (C1′), 86.08 (C4′), 70.37 (C3′), 51.59 (C5′), 44.79 (C2′).
HRMS (ESI): calcd, 251.0780 (M + H)+; found, 251.0775 (M +
H)+.
(kcat × S)
kobs
)
(1)
I
S + Km 1 +
(
(
K ))
i
Acknowledgment. This research was supported by the NIH
grant CA098296 and the Intramural Research Program of the
NIH, NIEHS, in association with the NIH grant 1U19CA105010.
This research was also made possible in part by the use of the
INBRE Research Core Facility, which is supported by the
NCRR/NIH (P20 RR016457). Mass spectrometry was provided
by Washington University Mass Spectrometry Resource with
support from the NCRR/NIH (P41RR0954).
Supporting Information Available: 1H (1D, NOESY, TOCSY),
13C, and 31P NMR spectra; ESI-LQT-HRMS spectra; UV and CD
Spectra. This material is available free of charge via the Internet at
http://pubs.acs.org.
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