17 research outputs found
Mapping Functional Substrate–Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs
We report high-resolution
crystal structures of DNA polymerase
(pol) β in ternary complex with a panel of incoming dNTPs carrying
acidity-modified 5′-triphosphate groups. These novel dNTP analogues
have a variety of halomethylene substitutions replacing the bridging
oxygen between Pβ and Pγ of the incoming dNTP, whereas
other analogues have alkaline substitutions at the bridging oxygen.
Use of these analogues allows the first systematic comparison of effects
of 5′-triphosphate acidity modification on active site structures
and the rate constant of DNA synthesis. These ternary complex structures
with incoming dATP, dTTP, and dCTP analogues reveal the enzyme’s
active site is not grossly altered by the acidity modifications of
the triphosphate group, yet with analogues of all three incoming dNTP
bases, subtle structural differences are apparent in interactions
around the nascent base pair and at the guanidinium groups of active
site arginine residues. These results are important for understanding
how acidity modification of the incoming dNTP’s 5′-triphosphate
can influence DNA polymerase activity and the significance of interactions
at arginines 183 and 149 in the active site
Two Scaffolds from Two Flips: (α,β)/(β,γ) CH<sub>2</sub>/NH “Met-Im” Analogues of dTTP
Novel
α,β-CH<sub>2</sub> and β,γ-NH (<b>1a</b>) or α,β-NH and β,γ-CH<sub>2</sub> (<b>1b</b>) “Met-Im” dTTPs were synthesized
via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate
synthons (<b>4a</b>, <b>4b</b>), formed via a base-induced
[1,3]-rearrangement of precursors (<b>3a</b>, <b>3b</b>) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization
during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu
conjugation with dT was avoided by microwave conditions. <b>1a</b> was 9-fold more potent in inhibiting DNA polymerase β, attributed
to an NH-group interaction with R183 in the active site
Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid
It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF2-substituted nucleotide analogs—one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have “correct” polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF2-substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol β) binding pocket by obtaining a 2.5 Å resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg2+ interaction over Watson–Crick hydrogen bonding was found and discussed
Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State
We examine the DNA
polymerase β (pol β) transition
state (TS) from a leaving group pre-steady-state kinetics perspective
by measuring the rate of incorporation of dNTPs and corresponding
novel β,γ-CXY-dNTP analogues, including individual β,γ-CHF
and -CHCl diastereomers with defined stereochemistry at the bridging
carbon, during the formation of right (R) and wrong (W) base pairs.
Brønsted plots of log <i>k</i><sub>pol</sub> versus
p<i>K</i><sub>a4</sub> of the leaving group bisphosphonic
acids are used to interrogate the effects of the base identity, the
dNTP analogue leaving group basicity, and the precise configuration
of the C-X atom in <i>R</i> and <i>S</i> stereoisomers
on the rate-determining step (<i>k</i><sub>pol</sub>). The
dNTP analogues provide a range of leaving group basicity and steric
properties by virtue of monohalogen, dihalogen, or methyl substitution
at the carbon atom bridging the β,γ-bisphosphonate that
mimics the natural pyrophosphate leaving group in dNTPs. Brønsted
plot relationships with negative slopes are revealed by the data,
as was found for the dGTP and dTTP analogues, consistent with a bond-breaking
component to the TS energy. However, greater multiplicity was shown
in the linear free energy relationship, revealing an unexpected dependence
on the nucleotide base for both A and C. Strong base-dependent perturbations
that modulate TS relative to ground-state energies are likely to arise
from electrostatic effects on catalysis in the pol active site. Deviations
from a uniform linear Brønsted plot relationship are discussed
in terms of insights gained from structural features of the prechemistry
DNA polymerase active site