(R)-β,γ-fluoromethylene-dGTP-DNA ternary complex with DNA polymerase β

Article abstract of DOI:10.1021/ja072127v

β,γ-Fluoromethylene analogues of nucleotides are generally considered to be useful mimics of the natural substrates for DNA polymerases, but direct structural evidence defining their active site interactions has not been available. In addition, the effect of introducing a new chiral center (the CHF carbon) has been unexplored. We report here structural studies of the diastereomeric β,γ-CHF analogues (R, 3; S, 4) of dGTP interacting with the active site of DNA pol β, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. The conjugation of dGMP 5′-morpholidate with a tetrabutylammonium salt of (fluoromethylene)bisphosphonic acid (6b, prepared like its difluoro analogue 7b via fluorination of tetraisopropyl methylenebisphosphonate carbanion with Selectfluor) gives a 1:1 mixture of 3 and 4 (by ¹⁹F NMR, pH 10). The β,γ-CF₂ (2) and β,γ-CH₂ (1) dGTP analogues were also synthesized. Crystallization from a solution containing 3 + 4 together with a preformed DNA pol β complex of a 16-mer template DNA and a one-nucleotide gapped primer produced crystals containing the (R)-analogue 3, as shown by the X-ray structure (2.1 A), which revealed a 3.0 A (bonding) distance between a guanidine N of Arg 183 and the CHF fluorine atom. Ligand docking simulations of 3 vs 4 using Autodock 3.0 predicted that both 3 and 4 can adopt an overall orientation closely overlaying that of dGTP itself in the active site; however, a polar C-F··Arg183 bonding interaction is favored only with 3. A similar orientation of one fluorine atom in 2 is observed. The results suggest that introduction of a single fluorine atom at the bridging carbon atom of a β,γ-methylene-dNTP analogue may enable a new, stereospecific interaction within the pre-organized active site complex. Copyright

Full text of DOI:10.1021/ja072127v

Published on Web 11/22/2007
(R)-â,γ-Fluoromethylene-dGTP-DNA Ternary Complex with DNA Polymerase â
Charles E. McKenna,*, Boris A. Kashemirov, Thomas G. Upton, Vinod K. Batra,
†
†
†
‡
†
‡
‡
‡
Myron F. Goodman, 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, DHHS,
Research Triangle Park, North Carolina 27709
Received March 26, 2007; E-mail: mckenna@usc.edu
DNA polymerases are crucial for maintaining the fidelity of
genetic information encoded into DNA, and failure to repair aberrant
bases in damaged DNA strands is notoriously implicated in
oncogenesis. During base excision repair (BER),1 DNA poly-
merase â (pol â) inserts the correct deoxynucleoside triphosphate
Chart 1. â,γ-CXY Analogues of dGTP (Charges Omitted)
,2
(dNTP), replacing an excised damaged or mismatched nucleoside
residue, with release of pyrophosphate. The smallest eukaryotic
cellular DNA polymerase, pol â is an error-prone enzyme that is
tightly down-regulated in normal somatic cells, but often overex-
pressed in human tumors, and has been the subject of extensive
2
3
studies examining its roles in BER and cancer.
Probes of molecular interactions with nucleic acid polymerases
can be created by designed modifications in the structures of natural
dNTPs. Although such analogues may be usefully modified in their
nucleoside moieties to create active site probes, as shown for
(
2
R/S)-â,γ-CHF-dGTP (3/4) (and also the corresponding CH and
CF
2
analogues) were prepared by DCC-mediated conjugation of
the morpholidate8 of dGMP (5) in anhydrous DMSO with the
tributylammonium salt of the appropriate methylenebisphosphonic
acid, followed by two-stage preparative HPLC to achieve a high
degree of purity with respect to contaminating nucleotides. The
,9
4
example by the recent work of Kool, changes to the triphosphate
group are of particular interest because this group is the locus of
chemical transformation catalyzed in the polymerase active site.
Replacement of the PR-O-Pâ bridging oxygen by a carbon atom
1
0,11
(
mono- and difluoromethylene)bisphosphonic acids
were prepared using the more convenient Selectfluor (8) in place
of perchloryl fluoride to synthesize the intermediate fluorinated
esters (6a, 7a)
(6b, 7b)
12
(
CXY) will prevent hydrolysis, whereas a Pâ-CXY-Pγ modifica-
10
tion will alter the leaving group properties, depending on the nature
of substituents X and Y, while conferring resistance to dephos-
5
10,11
from tetraisopropyl methylenebisphosphonate.
phorylation. The introduction of these substituents potentially may
also enable entirely new bonding (or repulsive) active site interac-
tions, not present with the natural nucleoside triphosphate, and thus
could inform inhibitor design seeking to exploit pol â as a drug
target.
In consistency with an early study of the mixed (R/S)-â,γ-CHF
analogues of ATP, at pH 10 or higher, 3 and 4 could be
13
individually detected and quantified (1:1) by their overlapping but
19
resolvable F NMR resonances, exhibited as a pair of ddd with δ
-218.61 and -218.77 ppm.
Recently, a series of unfluorinated (X,Y ) H, 1) and fluorinated
For protein crystallography, human pol â was overexpressed in
(
X,Y ) F, 2; X,Y ) H,F, 3 (R) or F,H 4 (S)) â,γ-methylene-
E. coli and purified as described previously.14 The double-stranded
DNA substrate consisted of a 16-mer template (5′-CCGACCGCG-
CATCAGC-3′), a complementary 9-mer primer (5′-GCTGATGCG-
bisphosphonate analogues of dGTP (Chart 1) and related com-
pounds were used to examine leaving group effects on pol â
catalysis and fidelity.6 These analogues are substrates of the
3′), and a 5-mer downstream oligonucleotide (5′-pGTCGG-3′), thus
polymerase, but release an X,Y-methylenebisphosphonate in place
creating a two-nucleotide gap with annealed primer. Addition of
ddCTP terminates the primer and creates a one-nucleotide gap. The
1:1 mixture of 3 and 4 was added to a solution of the preformed
protein-DNA complex.
of the natural pyrophosphate leaving group. Several â,γ-CXY dNTP
analogues were previously investigated as inhibitors of different
DNA polymerases and viral reverse transcriptases,7 but the
structures of the putative complexes formed during turnover were
not determined. In previous studies, the CHX analogues (or more
generally, CXY analogues where X * Y) have been used as the
diastereomer mixtures always obtained synthetically when the
substituted methylenebisphosphonate is coupled to a dNMP, and
their potential for a stereospecific interaction with the polymerase
active site has received little or no attention.
In this Communication, we report X-ray crystallographic studies
and computer docking simulations of terminal ddNMP primer-
template pol â complexes with dGTP â,γ-fluoromethylenebis-
phosphonate analogues 3 and 4, leading to the first demonstration
of stereospecificity in dNTP â,γ-CHX analogue-enzyme complex
formation.
The crystal structure of the resulting complex was resolved at
2.1 Å. The analogue was found in the enzyme active site position
normally occupied by dGTP, and its configuration overall was a
good match for that of the natural substrate. However, despite both
CHF stereoisomers being present at very similar concentrations in
the crystallizing mixture, electron density was only observed for a
single fluorine atom in the complex, corresponding to the (R)-CHF
stereoisomer (3) (Figure 1). In contrast, in a comparable crystal-
lography experiment, the corresponding â,γ-CHCl analogue stereo-
isomers populated the DNA pol active site about equally (Pedersen,
L. C., personal communication). Exclusion of the (S)-â,γ-CHF
analogue is not the result of an unattainable binding conformation,
because the crystal structure of the corresponding CF
complex superimposes well with that of the (R)-stereoisomer.
2
-analogue
†
University of Southern California.
National Institutes of Health, DHHS.
‡
6
15412
9
J. AM. CHEM. SOC. 2007, 129, 15412-15413
10.1021/ja072127v CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Note Added in Proof: A PDB search for ligand C-F
interactions with the guanidinium group of Arg has documented
a number of examples underscoring the fluorophilic character
of the Arg side chain: M u¨ ller, K.; Faeh, C.; Diederich, F.
Science 2007, 317, 1881-1886.
Supporting Information Available: Synthesis and characterization
data for 1-7; crystallographic data for the complex of 3 with DNA
pol â (PDB ID, 2PXI); computer docking results. This material is
available free of charge via the Internet at http://pubs.acs.org.
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2
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