α,β-Difluoromethylene deoxynucleoside 5′-triphosphates: A convenient synthesis of useful probes for DNA polymerase β structure and function

Article abstract of DOI:10.1021/ol701755k

αβ-Difluoromethylene deoxynucleoside 5′-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol β Kd_d values for the α, β-CF₂ and unmodified dNTPs, α, β-NH dUTP, and the α, β-CH₂ analogues of dATP and dGTP are discussed in relation to the conformations of α, β-CF₂ dTTP versus α, β-NH dUTP bound into the enzyme active site.2009 American Chemical Society.

Full text of DOI:10.1021/ol701755k

ORGANIC
LETTERS
2009
Vol. 11, No. 9
1883-1886
r,ꢀ-Difluoromethylene Deoxynucleoside
5′-Triphosphates: A Convenient
Synthesis of Useful Probes for DNA
Polymerase ꢀ Structure and Function
Thomas G. Upton,† Boris A. Kashemirov,† Charles E. McKenna,*,† Myron
F. Goodman,† G. K. Surya Prakash,† Roman Kultyshev,† Vinod K. Batra,‡
David D. Shock,‡ Lars C. Pedersen,‡ William A. Beard,‡ and Samuel H. Wilson‡
Departments of Chemistry and Biology, UniVersity of Southern California, Los
Angeles, California 90089, and Laboratory of Structural Biology, NIEHS, National
Institutes of Health, Research Triangle Park, North Carolina 27709
mckenna@usc.edu
Received February 5, 2009
ABSTRACT
r,ꢀ-Difluoromethylene deoxynucleoside 5′-triphosphates (dNTPs, N ) A or C) are advantageously obtained via phosphorylation of corresponding
dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol ꢀ Kd values for the r,ꢀ-CF2 and
unmodified dNTPs, r,ꢀ-NH dUTP, and the r,ꢀ-CH2 analogues of dATP and dGTP are discussed in relation to the conformations of r,ꢀ-CF2
dTTP versus r,ꢀ-NH dUTP bound into the enzyme active site.
In an ongoing multidisciplinary study of structure and function of
DNA polymerase ꢀ, a eukaryotic enzyme primarily involved in
filling short DNA gaps,1 we required a series of R,ꢀ-methylene-
substituted analogues of deoxynucleoside 5′-triphosphates (dNTPs).
When the PR-O-Pꢀ bridging oxygen in a natural mononucleotide
substrate is replaced by an imido (NH)2-4 or methylene (CXY)4,5
group, the P-N or P-C bond should resist cleavage in the
nucleotidyl transfer reaction catalyzed by the enzyme. As a
result, these analogues will remain intact in stable ternary
DNA complexes with the polymerase and therefore should
be useful to probe pre-chemistry enzyme-complex function
and structure, as recently shown with in an X-ray crystal-
lographic study of R,ꢀ-NH dUTP with DNA pol ꢀ.6
Information about such complexes provides a reference point
for theoretical analysis of the chemical mechanism7 for the
complete transfer of a monophosphate nucleoside donor to
the sugar acceptor in the active site. As probes for the
mechanism of polymerase catalysis and its relationship to
polymerase fidelity, R,ꢀ-methylene dNTP analogues permit
exploration of stereoelectronic effects on active site interac-
(3) Ma, Q. F.; Bathurst, I. C.; Barr, P. J.; Kenyon, G. L. J. Med. Chem.
1992, 35, 1938.
† University of Southern California, Los Angeles.
‡ National Institutes of Health.
(1) Beard, W. A.; Wilson, S. H. Chem. ReV. 2006, 106, 361.
(2) Ma, Q. F.; Kenyon, G. L.; Markham, G. D. Biochemistry 1990, 29,
1412.
(4) Barrie, S. E.; Paine, R. M.; Stock, J. A.; Vincent, R. B.; Harrap,
K. R. AdV. Exp. Med. Biol. 1984, 165, 371.
(5) Wang, G.; Boyle, N.; Chen, F.; Rajappan, V.; Fagan, P.; Brooks,
J. L.; Hurd, T.; Leeds, J. M.; Rajwanshi, V. K.; Jin, Y.; Prhavc, M.; Bruice,
T. W.; Cook, P. D. J. Med. Chem. 2004, 47, 6902.
10.1021/ol701755k CCC: $40.75
Published on Web 04/07/2009
 2009 American Chemical Society

tions, by making appropriate substitutions X,Y on the
adjacent PR CXY bridging carbon. The largest obtainable
electron-withdrawing effect with minimal steric perturbation
can be achieved using X,Y ) F, resulting in analogues in
which the bisphosphonate group is expected to be less basic
than the pyrophosphate moiety in the natural dNTPs.8,9
In this Letter, we describe the first synthesis of R,ꢀ-CF2
dCTP 6, using a modified chemical-enzymological approach
that also can be applied to synthesis of R,ꢀ-CF2 dATP 3,
affording these compounds in sufficient purity to virtually
eliminate detectable contaminating substrate activity in
polymerase inhibition kinetics assays. DNA pol ꢀ Kd values
were determined for these analogues and compared to Kd
values for R,ꢀ-CF2 dTTP 9, the natural substrates dATP,
dCTP and dTTP, R,ꢀ-NH dUTP 10, and the dATP and dGTP
R,ꢀ-CH2 analogues (11, 12). We also describe the ternary
complex of 9 bound with template-primer DNA into the
active site of DNA polymerase ꢀ, as determined by X-ray
crystallography, and discuss its structural implications.
An obvious route to R,ꢀ-methylene-dNTP analogues entails
coupling a particular methylenebis(phosphonate) derivative to the
target nucleoside, followed by phosphorylation10 to add the terminal
γ-phosphate. Several R,ꢀ-CXY NDPs (X,Y ) H or F) were
previously synthesized via tosylation of the 5′-OH in the protected
ribonucleoside with tosyl chloride and (dimethylamino)pyridine,
followed by deprotection and displacement of the tosyl moiety with
the appropriate tris(tetrabutylammonium)11 bisphosphonate. A
similar approach was used to prepare an R,ꢀ-CF2 dGDP (N2-(p-
N-n-butylphenyl) derivative),12 dATP and dTTP,13 and related
nucleotide analogues.14 In a synthesis of AZT 5′-carbonylbis(pho-
sphonate), trimethyl (diazomethylene)bis(phosphonate) was coupled
to AZT under Mitsunobu conditions, but subsequent deprotection
(demethylation) at phosphorus was required.15 Direct 5′-coupling
of 2-6 equiv of methylenebis(phosphonic dichloride) with un-
protected nucleosides also has been demonstrated.16
the R,ꢀ-methylene dNTP analogue product. Carbonyldiimidazole
(CDI) activation18 of the dGDP derivative referred to above has
been utilized as an alternative route.12 We found phosphorylation
of our dNDP substrates using CDI to be problematic because of
the reactivity of the imidazolate, which tended to give unwanted
nucleoside phosphorylation at the 3′-OH and other side products.
As an alternative to syntheses of R,ꢀ-imido dNTPs using
CDI to activate the NDP analogue for coupling with
tributylammonium phosphate,3,19 Kenyon proposed enzy-
matic phosphorylation by either creatine kinase (CK)2,19 or
pyruvate kinase (PK) and phosphoenolpyruvate (PEP).19
However, inadequate CK and PK activity in attempted PEP
phosphorylation of our dNDP intermediates led us to consider
adaptation of a method for phosphorylation of azole car-
boxamide riboxynucleoside 5′-diphosphates (NDPs) by ∼
stoichiometric ATP and nucleoside diphosphate kinase
(NDPK)20a previously applied to synthesis of 11,20b in which
PEP and PK are included to recycle the ADP produced in
the phosphoryl transfer and drive the reaction to completion.
Synthesis of r,ꢀ-dNTPs. We first converted dA or N4-
benzoyl-dC 7 to the corresponding 5′-tosylates 1 or 4, respectively
(see Scheme 1), by reaction with tosyl chloride in pyridine
(75-80%).13
Scheme 1.
Synthesis of R,ꢀ-Methylene dNTP Analoguesa
Chemical phosphorylation of R,ꢀ-CH2 dADP and dTDP has
been carried out via the p-nitrobenzyl-phosphoromorpholidates.13
Unfortunately, subsequent deprotection by hydrogenolysis of the
p-nitrobenzyl group restricts the pyrimidine substrate, because the
cytosine ring of dC is prone to reduction under these conditions.17
Side products from the phosphorylation step typically contaminate
a 1, 2, and 3: base ) adenine. 4: base ) N4-Bz-cytosine. 5 and 6: base )
cytosine. Note: before tosylation, dC was converted to N4-Bz-deoxycytidine 7 with
benzoic anhydride, DIEA in pyridine, microwave irradiation (2 min, 300 W). Prior
to the enzymatic phosphorylation, the N4-protected dCDP analogue 8 obtained
directly from 4 was debenzoylated to 5 using methanolic ammonia.
(6) Batra, V. K.; Beard, W. A.; Shock, D. D.; Krahn, J. M.; Pedersen,
L. C.; Wilson, S. H. Structure 2006, 14, 757
.
The NH2 group of dC was protected via facile microwave-
induced reaction with benzoic anhydride and diisopropylethylamine
(DIEA) in pyridine,21 to give 7 (76%). When purifying 4 from
the reaction mixture, we initially used conventional extraction with
aq NaHCO3 (pH ∼8.3) but obtained low isolated yields (∼20%).
The pKa of the N4-proton in 4 was estimated (ACDLabs pKaDB
8.01 software program) to be ∼8. Accordingly, we changed to a
slightly acidic aqueous workup (citric acid buffer, pH ∼4.5), which
increased the isolated yield to ∼70%.
(7) (a) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.;
Martinek, V.; Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.;
McKenna, C. E.; Florian, J.; Warshel, A.; Goodman, M. F. Biochemistry
2007, 46, 461. (b) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna,
J.; Oertell, K.; Beard, W. A.; Wilson, S. H.; Florian, J.; Warshel, A.;
McKenna, C. E.; Goodman, M. F. Biochemistry 2008, 47, 870
(8) McKenna, C. E.; Shen, P.-D. J. Org. Chem. 1981, 46, 4573
(9) Blackburn, G. M.; England, D. A.; Kolkmann, F. J. Chem. Soc.,
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(10) McKenna, C. E.; Kashemirov, B.; Blazewska, K. M. Phosphoric
Acids and DeriVatiVes; Science of Synthesis, Houben-Weyl Methods of
Molecular Transformations; Mathey, F., Ed.; Georg Thieme Verlag:
Stuttgart, 2009; Vol. 42, pp 779-921.
The purified tosylates were converted to the dNDP R,ꢀ-
difluoromethylene analogues 2 or 5 via condensation with the
tris(tetrabutylammonium) salt13 of difluoromethylenebis(phospho-
nic acid) (DFBP). The N4-benzoyl dCDP analogue 8 obtained as
the direct tosylation product from 4 was then deprotected in
methanolic ammonia to give 5 quantitatively, which was purified
by preparative ion-exchange HPLC. DFBP was obtained by
(11) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J. Org.
Chem. 1987, 52, 1794.
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2002, 30, 383.
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Org. Lett., Vol. 11, No. 9, 2009

bromotrimethylsilane dealkylation22 of tetra-isopropyl (difluoro)-
methylenebis(phosphonate),8 synthesized by fluorination of the
carbanion of tetra-isopropyl methylenebis(phosphonate).7,23
Phosphorylation to the dNTP analogues 3 or 6 was cleanly
achieved using NDPK and a catalytic amount of ATP, regener-
ated with 2.5 equiv of PEP with PK in 50 mM HEPES buffer
(75-90% by HPLC and 31P NMR). Importantly, the latter
modification renders unnecessary the use of an affinity column20
to purify the product from excess ATP.
Initial DNA polymerase inhibition studies on R,ꢀ-CF2 dCTP, 6
at 1 mM showed no detectable background DNA synthesis with
0.5 nM enzyme, and only a trace with 50 nM enzyme, representing
<0.01% of the inhibitor (Figure 1A). Comparable absence of
Purification of r,ꢀ-dNTPs. To obtain product free of
detectable nucleotide contaminants, we found that separa-
tion on DEAE Sephadex or Dowex3,5,9,12-14,24-30 or
single-pass preparative HPLC using a C-18 or ion-
exchange3,5,9,12-14,24-31 column was not sufficient.
However, dual-HPLC (ion exchange, then C-18) provided
1
highly purified products based on analytical HPLC and H,
31P, and 19F NMR analysis (Supporting Information).
This overall synthesis/purification route has the advantage of
being applicable to both purine and pyrimidine examples, including
the previously unavailable R,ꢀ-CF2 dCTP analogue 6. The reactions
are relatively clean (particularly compared to standard CDI phos-
phorylation), do not require protection/deprotection chemistry in
the phosphate moieties, eliminate the problem of excess ATP in
the enzymatic phosphorylation, and after the dual-HPLC purifica-
tion provide exceptionally pure analogues suitable for polymerase
inhibition studies (see below). The dual-HPLC purification protocol
was also effective in reducing impurities in R,ꢀ-CF2 dTTP 9
prepared by the p-nitrobenzyl-phosphomorpholidate method (see
below).
Figure 1. Gapped DNA synthesis assay with DNA pol ꢀ for dNTPs
(dCTP, dATP, dTTP) and R,ꢀ-CXY analogues. Primer (n) extension was
assayed in the presence of low (L, 0.5 nM) or high (H, 50 nM) pol ꢀ and
1 mM analogue for 5 or 10 min, respectively. (A) dCTP versus 6. (B) 3
versus 11, 9, dATP, and dTTP. The mobility of the extension product (n
+ 1) with dCTP, dATP, or dTTP serves as a reference, where most of
the primer (200 nM) is extended after a 21 min incubation with 50 nM
enzyme and 100 µM dNTP.
background synthesis was evident in the assay using R,ꢀ-CF2
dATP, 3 (Figure 1B). For comparison, with a preparation of
inhibitor 9 obtained via the p-nitrobenzyl phosphoromorpholidate
method, at 50 nM pol ꢀ most of the primer strand in the assay has
been extended, with some incorporation still detectable using the
lower concentration of enzyme (Figure 1B). After dual-HPLC
purification, this artifact was no longer observable with 0.5 nM
enzyme, although incorporation at 50 nM enzyme was more
evident than for 6 and 3 (Supporting Information).
Inhibition of natural nucleotide insertion by the different R,ꢀ-
modified dNTP analogues was investigated at various concentra-
tions of inhibitor (I) and subsaturating dNTP substrate (S). Steady-
state kinetic parameters (km, Km) were determined by fitting the
rate data to the Michaelis equation, and the inhibitor constant Ki
()Kd) was determined by fitting the inhibition data to the equation
for competitive inhibition (Supporting Information). Both fits used
nonlinear regression methods. Equilibrium dissociation constants
(Kd) for the natural dNTPs were taken from previously published
data [dATP;33 dCTP;34 dTTP35]. The Ki’s for 10, 11, and 12 were
reported previously.6,36
DNA Synthesis Assays. DNA synthesis was assayed on four
single-nucleotide gapped DNA substrates where the templating
base in the gap was complementary to the incoming dNTP. The
DNA sequence was as described previously32 with the core
sequence identical to that used for crystallization.
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Org. Lett., Vol. 11, No. 9, 2009
1885

dehydrogenase, the Km of the R-CF2 analogue was an order of
magnitude greater than the Km of glucose 6-phosphate, but that
of the R-CH2 derivative was also higher (4-fold).38
Crystallization and Analysis of r,ꢀ-Modified dNTP
Analogues Complexed with DNA Primer and DNA pol
ꢀ. The crystal structure of the ternary complex of pol ꢀ with
the incoming analogue 9 opposite dA represents the precatalytic
state of the nucleotidyl transfer reaction for correct incorporation,
containing all atoms required for catalysis including the two
catalytic metal ions. As expected, the substitution of the CF2
for the bridging oxygen prevented dissociation of the pyro-
phosphate leaving group, trapping the complex. All correlated
atoms in the active site of this structure superimpose well with
previously determined ternary complex structures of pol ꢀ where
the reaction was trapped by deletion of the nucleophilic 3′-OH
on the primer terminus39 or by using a nitrogen in place of the
R,ꢀ bridging oxygen1,4,6 (Figure 3).
In summary, we have synthesized a series of R,ꢀ-CF2
dNTP analogues. Two of these, 3 and 6, were synthesized
by an advantageous chemical-enzymatic method that was
used to obtain the previously unavailable dCTP analogue,
6.40 The isolation method provides the inhibitors essentially
free of contaminating “dNTP” activity. Kd values for these
and comparable analogues, combined with an X-ray crystal-
lographic study of 9 bound into a pol ꢀ ternary complex,
indicate that CXY modification in the PR-O-Pꢀ bridging
position of dNTPs where X,Y ) F or H can modulate
binding properties over a wide range of Kd while substantially
retaining a dNTP-like conformation.
Figure 2. DNA pol ꢀ dissociation constants (Kd) for three natural dNTPs
(dATP, dCTP, dTTP), for R,ꢀ-NH dUTP (10), the R,ꢀ-CH2 analogues of
dATP and dGTP (11, 12), and the R,ꢀ-CF2 analogues of dATP, dCTP,
and dTTP (3, 6, 9). Kd values determined by single-turnover analysis.
Acknowledgment. We thank Dr. Ron New (Mass Spectrom-
etry Facility, University of California, Riverside) for assistance in
obtaining high resolution mass spectra of nucleotide analogues,
Ms. Keriann Oertell (University of Southern California) for
verifying the DNA synthesis background activity in 9 after dual-
HPLC purification, and Prof. George L. Kenyon (University of
Michigan) for helpful discussions. This research was supported by
the Intramural Research Program of the NIH, National Institute of
Environmental Health Sciences and by NIH program project grant
5-U19-CA105010.
Figure 3. Superposition of the active site of the ternary substrate
complex of pol ꢀ with incoming 9, R,ꢀ-CF2-dTTP (gray carbons), and
the corresponding ternary complex with 10, R,ꢀ-NH-dUTP (yellow
atoms; pdb ID 2FMS). Active site carboxylates (D190, D192, D256)
that coordinate two magnesium ions are shown, as well as the 3′-
OH of the primer terminus (P10). The fluorine atoms of 9 are cyan.
Electron density for the 2Fo-Fc simulated annealing map (blue) is
contoured to 5σ illustrating the high quality of the electron density.
Supporting Information Available: Detailed synthetic
procedures, compound characterization data including NMR
and HRMS spectra, details of enzyme studies, crystal-
lographic methods and statistics. This material is available
free of charge via the Internet at http://pubs.acs.org.
an order of magnitude lower than those of the natural nucle-
otides. Interestingly, the -NH- and -CH2- analogue exhibited
the tightest binding (∼10-fold tighter than natural dNTPs),
despite polarity differences between the R,ꢀ-NH and R,ꢀ-CH2
groups. This pattern contrasts with the results obtained by
O’Hagan for NADH-dependent glycerol 3-phosphate dehydro-
genase with -CH2- and -CF2- phosphonate analogues of sn-
glycerol-3-phosphate,37 which respectively had the same and
3.5-fold greater Km values than the natural substate. In a study
by Berkowitz on substrate mimics for glucose 6-phosphate
OL701755K
(38) Berkowitz, D. B.; Bose, M.; Pfannenstiel, T. J.; Doukov, T. J. Org.
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(40) Preliminary accounts of this work: Upton, T. G.; Kashemirov, B. A.;
McKenna, C. E.; Goodman, M. F.; Prakash, G. K. S.; Kultyshev, R.; Batra,
V. K.; Shock, D. D.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. No.
P148, XVIIth International Conference on Phosphorus Chemistry, Xiamen,
China, April 15-19, 2007. Upton, T.; Osuna, J.; Kashemirov, B. A.;
McKenna, C. E. ORGN-278, 235th National Meeting of the American
Chemical Society, New Orleans, April 6-10, 2008.
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