β,γ-CHF- and β,γ-CHCl-dGTP diastereomers: Synthesis, discrete 31P NMR signatures, and absolute configurations of new stereochemical probes for DNA polymerases

Article abstract of DOI:10.1021/ja300218x

Deoxynucleoside 5′-triphosphate analogues in which the β,γ-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of β,γ-CXY dNTP diastereomers: (S)- and (R)-β,γ-CHCl-dGTP (12a-1/12a-2) and (S)- and (R)-β,γ-CHF-dGTP (12b-1/12b-2). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,C-dimorpholinamide derivatives 7 of their (R)-mandelic acid monoesters, which provided access to the individual diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by preparative HPLC. Selective acidic hydrolysis of the P-N bond then afforded portal diastereomers, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H₂ (Pd/C) gave the four individual diastereomeric nucleotides 12, which were characterized by ³¹P, ¹H, and ¹⁹F NMR spectroscopy and by mass spectrometry. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH ～10 the diastereomer pairs 12a,b exhibit discrete P_α and P_β³¹P resonances. The more upfield P_α and more downfield P_β resonances (and also the more upfield ¹⁹F NMR resonance in 12b) are assigned to the R configuration at the P_β-CHX-P _γ carbons on the basis of the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polymerase β.

Full text of DOI:10.1021/ja300218x

Communication
pubs.acs.org/JACS
β,γ-CHF- and β,γ-CHCl-dGTP Diastereomers: Synthesis, Discrete
31P NMR Signatures, and Absolute Configurations of New
Stereochemical Probes for DNA Polymerases
Yue Wu,† Valeria M. Zakharova,† Boris A. Kashemirov,† Myron F. Goodman,†,§ Vinod K. Batra,‡
Samuel H. Wilson,‡ and Charles E. McKenna*,†
Departments of †Chemistry and §Biological Sciences, University of Southern California, Los Angeles, California 90089, United States
‡Laboratory of Structural Biology, NIEHS, National Institutes of Health, DHHS, Research Triangle Park, North Carolina 27709,
United States
S
* Supporting Information
ABSTRACT: Deoxynucleoside 5′-triphosphate analogues
in which the β,γ-bridging oxygen has been replaced with a
CXY group are useful chemical probes to investigate DNA
polymerase catalytic and base-selection mechanisms. A
limitation of such probes has been that conventional
Figure 1. Diastereomeric β,γ-CXY analogues of dGTP.
synthetic methods generate a mixture of diastereomers
when the bridging carbon substitution is nonequivalent (X
≠ Y). We report here a general solution to this long-
standing problem with four examples of β,γ-CXY dNTP
diastereomers: (S)- and (R)-β,γ-CHCl-dGTP (12a-1/12a-
2) and (S)- and (R)-β,γ-CHF-dGTP (12b-1/12b-2).
Central to their preparation was conversion of the
prochiral parent bisphosphonic acids to the P,C-
dimorpholinamide derivatives 7 of their (R)-mandelic
acid monoesters, which provided access to the individual
diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by preparative
HPLC. Selective acidic hydrolysis of the P−N bond then
afforded “portal” diastereomers, which were readily
coupled to morpholine-activated dGMP. Removal of the
chiral auxiliary by H2 (Pd/C) gave the four individual
properties that can be tuned by the substituents. The
conventional syntheses of α,β- and β,γ-CXY nucleotide
analogues involve coupling of bisphosphonates with nucleo-
sides and nucleoside monophosphates, respectively.3 The α,β-
and β,γ-analogues where X ≠ Y are therefore obtained as
mixtures of two diastereomers because a new chiral center is
generated at the bisphosphonate bridging carbon (Figure 1).
Several β,γ-CXY nucleotide analogues have been investigated as
enzymatic probes for DNA, viral RNA, or RNA-directed DNA
polymerases,4 but the potential for a stereospecific interaction
of these diastereomeric analogues with the enzyme active site
has received little attention until recently.5
DNA polymerase β (pol β) is a key polymerase in short-
patch base excision repair (BER).6 This is the predominant
diastereomeric nucleotides 12, which were characterized
1
by 31P, H, and 19F NMR spectroscopy and by mass
BER pathway in humans and is crucial for maintaining genome
integrity. Pol β has been an extensively studied model for
spectrometry. After treatment with Chelex-100 to remove
understanding polymerase fidelity.6c,7 In a recent study of the
traces of paramagnetic ions, at pH ∼10 the diastereomer
pairs 12a,b exhibit discrete Pα and Pβ 31P resonances. The
thus-far obscure transition state of pol β, we reported a series of
α,β- and β,γ-CXY dNTP analogues as structural, functional, and
fidelity probes.8 The use of a systematically varied β,γ-CXY
group to probe for a leaving-group effect in the catalytic process
provided evidence for a base-match-dependent chemical
transition step,8b,c consistent with the findings of Lin et al.9
and Tsai et al.10 using different approaches.
X-ray crystallographic studies of ternary complexes formed
from diastereomeric β,γ-CXY dGTP analogue mixtures
incubated with binary DNA-pol β complex crystals have
revealed the presence of only one diastereomer in the active site
for monofluorinated analogues (X = H, Cl, Me; Y = F),
associated with an interaction between the F atom and
Arg183.5,11 Other β,γ-monohalogenated (X = H; Y = Cl, Br),
monomethylated (X = H, Y = Me), and heterodihalogenated
more upfield Pα and more downfield Pβ resonances (and
also the more upfield 19F NMR resonance in 12b) are
assigned to the R configuration at the Pβ-CHX-Pγ carbons
on the basis of the absolute configurations of the individual
diastereomers as determined from the X-ray crystallo-
graphic structures of their ternary complexes with DNA
and polymerase β.
ucleotide bisphosphonate analogues in which a pyro-
Nphosphate bridging oxygen is replaced by a methylene
carbon were first described by Myers.1 Subsequently, it was
suggested that the bisphosphonate moiety more closely mimics
pyrophosphate when the bridging carbon is fluorinated.2 Pα-
CXY-Pβ substitution blocks nucleotidyl transfer catalyzed by
polymerases, whereas Pβ-CXY-Pγ substitution (Figure 1)
produces a dNTP substrate analogue with leaving-group
Received: January 9, 2012
Published: March 7, 2012
© 2012 American Chemical Society
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(X = Cl, Y = Br) analogues populated the active site evenly in
the crystal complex.5 These observations provided a strong
impetus to obtain the individual diastereomers.
and then esterified with (R)-(−)-methyl mandelate using
Mitsunobu coupling, giving esters 4 with inversion at the chiral
center of the auxiliary.20 These mixtures of diastereomers were
subjected to selective silyldemethylation with BTMS21 in
anhydrous CH3CN followed by methanolysis to afford the
(S)-mandelyl bisphosphonates 5a,b as diastereomer pairs.
Nucleotide-analogue stereoisomers resulting from replacing a
nonbridging Pα or Pβ oxygen with B, S, or Se have been isolated
by HPLC, or, in the case of ATPαS and ATPβS, by selective
enzymatic depletion.12 In contrast, separation of diastereomers
where a bridging oxygen is replaced by a CXY group has
remained a long-standing challenge. The diastereomers of α,β-
CMe(N3)-dATP were recently prepared via the corresponding
dADP isomers, which could be separated by preparative
reversed-phase C18 HPLC.13 However, efforts to separate the
α,β-CH(N3) stereoisomers were unsuccessful.13 (R/S)-β,γ-
CH(N3)-dGTP and (R/S)-β,γ-CMe(N3)-dGTP also proved
refractory to this method,13 suggesting that both the substituent
size and the distance of the CXY group from the chiral ribose
affect separation. It seemed apparent that preparation of the
elusive individual β,γ-CXY stereoisomers, particularly the highly
desirable monohalo derivatives,5,14 required a new approach,
ideally one achieving stereochemical separation at the
bisphosphonate level.
Here we report the synthesis, analytically discrete 31P NMR
signatures, and absolute configuration assignments of all four
diastereomers of β,γ-CHX-dGTP (X = F, Cl), based on fixing
the chirality at the bridging carbon of the prochiral α-halo
bisphosphonate prior to conjugation using a novel chiral
auxiliary strategy. After separation, the intermediate is
conjugated conventionally with the targeted dNMP. As the
incorporated nucleoside suffices to maintain the bisphospho-
nate stereochemistry, the chiral auxiliary can then be removed
reductively under mild, nonracemizing conditions.
Figure 2. 31P NMR spectra (202 MHz, D2O) of (a) a diastereomeric
mixture of 7a-1/7a-2 at pH 9.8, (b) diastereomer 7a-1 at pH 9.8, and
(c) diastereomer 7a-2 at pH 10.0.
Scheme 1. Synthesis of Chiral Bisphosphonate Synthons 7
Attempts to separate the stereoisomers of 5 by RP-C18
HPLC were not successful. However, after facile (pH 8)
hydrolysis of the carboxylate methyl ester (which proceeded
without loss of stereochemistry at the benzyl carbon), the
resulting mandelic acid bisphosphonate esters 6a-1/6a-2 could
be separated by preparative RP-C18 HPLC under isocratic
conditions. Unfortunately, the α-fluoro bisphosphonate dia-
stereomers 6b-1/6b-2 could not be resolved by this method,
even on analytical scale. To explore the effect of masking both
the carboxylic and phosphonic acid groups on improving the
chromatographic separability, we converted the mixtures of
diastereomers of 6 to the corresponding P,C-dimorpholin-
amides 7, which gratifyingly made possible the facile preparative
isolation of all four diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by
preparative HPLC. The 31P NMR spectra of 7a before and after
separation are shown in Figure 2. The relatively more
downfield chemical shift in each diastereomer corresponds to
1
the benzyl ester phosphonate P nucleus, based on the H−31P
gradient heteronuclear multiple bond correlation between the
downfield phosphorus peak and the benzyl proton.
Formation of the target nucleotides was carried out by
conjugation with a 5′-activated dGMP (Scheme 2). The isolated
7a or 7b stereoisomer was first exchanged on a Dowex H+
column, and the pH of the eluate was adjusted to 1 (1 M HCl)
to complete the hydrolysis of the P−N bond, giving the
“portal” monoesters 10. Each of these diastereomers was
coupled with dGMP-morpholidate (9)5 [prepared by N,N-
dicyclohexylcarbodiimide coupling of dGMP (8) with morpho-
line]22 by stirring in anhydrous DMSO for 3 days to afford the
Synthesis of the chiral bisphosphonate synthons 7a-1/7a-2
and 7b-1/7b-2 is outlined in Scheme 1. The readily available α-
halo bisphosphonic acid 1a15 or 1b2a,5a,16 was heated at reflux
with trimethylorthoformate to afford the corresponding
tetramethyl ester 2a17 or 2b.18 Monodemethylation using 1
equiv of NaI in acetone19 afforded the racemic trimethyl esters
3, which were converted to their acid forms on Dowex (H+)
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Scheme 2. Synthesis of Target Diastereomeric Nucleotides
Table 1. Relative 31P and 19F NMR Chemical Shifts and
Absolute Configurations of β,γ-CHX-dGTP Diastereomer
a
Pairs in D2O
Compound
31P NMR Pα 31P NMR Pβ 19F NMR CHX A.C.
12a-1 (CHCl)
12a-2 (CHCl)
12b-1 (CHF)
12b-2 (CHF)
D
U
D
U
U
D
U
D
N/A
N/A
D
S
R
S
U
R
a
Abbreviations: A.C., absolute configuration; D, downfield; U, upfield.
NMR data: 12a-1/12a-2: Pα, Δδ = 5.4 Hz; Pβ, Δδ = 8.5 Hz (202
MHz, pH 10.2). 12b-1/12b-2: Pα, Δδ = 2.4 Hz; Pβ, Δδ = 5.3 Hz (162
MHz, pH 10.5); 19F, Δδ = 22.6 Hz (376 MHz, pH 10.5). Δδ values
were measured from the NMR spectra of the artificial mixtures.
diastereomers were derived by simulation5b but could not be
assigned to a specific configuration at the β,γ-bridging carbon.
The absolute configuration at the chiral CHF carbon of 12b-2
is found to be R by X-ray crystallographic analysis of its ternary
complex with DNA and pol β (Figure 3; PDB entry 4DO9),
allowing the assignment of the more upfield 19F resonance to
this diastereomer (Table 1). It was possible to obtain the
absolute configurations of the other three diastereomers (12b-
1, 12a-1, and 12a-2) similarly (PDB entries 4DOA, 4DOC, and
4DOB, respectively).
nucleoside triphosphate analogues 11. These were purified by
strong anion exchange HPLC and obtained as the triethyl-
ammonium salts. Removal of the chiral morpholinamide
auxiliary by hydrogenolysis over 10 wt% Pd/C in 0.1 M
triethylammonium bicarbonate/MeOH (1:1, pH 8) gave the
deprotected individual β,γ-CHCl-dGTP (12a-1/12a-2) and
β,γ-CHF-dGTP (12b-1/12b-2) diastereomers, which were
then purified by RP-C18 HPLC and obtained as the
triethylammonium salts.
The 19F NMR spectrum of the 12b-1/12b-2 mixture as
obtained by conventional synthesis was previously reported to
display nonoverlapping peaks for the two diastereomers.5a,b
The chemical shifts and correct coupling constants of the two
Figure 4. 31P NMR spectra (202 MHz, D2O) of Pα in 12a: (a)
Artificial mixture of 12a-1/12a-2 at pH 10.2. 12a-1 was added in
excess, demonstrating that the 12a-2 signal is more upfield (U in Table
1) by Δδ = 5.4 Hz (0.027 ppm). (b) Diastereomer 12a-1 at pH 10.6.
(c) Diastereomer 12a-2 at pH 10.3.
The ability to detect discrete 31P resonances for non-F-
containing analogues such as 12a-1/12a-2 would render them
more generally useful as stereoprobes. At pH ∼10 and after
removal of traces of paramagnetic metal ions by passage
through Chelex-100, the diastereomeric Pα and Pβ resonances
of 12a-1/12a-2 and 12b-1/12b-2 proved to be observable at
162 and 202 MHz (0.30 Hz/point digital resolution). As shown
in Figure 4, a 2:1 mixture of 12a-1/12a-2 exhibits a 31P Δδ of
Figure 3. Detailed view of the incoming nucleotide (R)-β,γ-CHF-
dGTP (12b-2) in the active site of the X-ray crystal structure of its
ternary complex with pol β and DNA (PDB entry 4DO9). The
Arg183, Asp190, and Asp192 side chains in the enzyme active site are
shown, along with the nucleotide-binding magnesium and a water
molecule. The interatomic distance between the F atom and Nη2 of
Arg183 is 3.11 Å.
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Am. Chem. Soc. 2007, 129, 15412. (b) Batra, V. K.; Pedersen, L. C.;
Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.;
Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617.
(c) It should be noted that the formation of both fluoromethylene
diastereomers in a synthesis of β,γ-CHF ATP and GTP was discussed
by Blackburn et al., who reported that the individual isomers could not
be distinguished by 19F (94 MHz) or 31P (40.5, 162 MHz) NMR:
Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc., Perkin
Trans. 1 1984, 1119.
(6) (a) Barnes, D. E.; Lindahl, T. Annu. Rev. Genet. 2004, 38, 445.
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Biochemistry 2001, 40, 5368.
5.4 Hz for Pα at 202 MHz, leading to the assignment of the
more downfield signal to the S isomer, 12a-1. The Pβ
resonances, which are separated by 8.5 Hz under the same
conditions, show the reverse relationship (Table 1).
In conclusion, the first examples of individual β,γ-CXY-
dNTP diastereomers have been successfully prepared, and their
absolute configurations have been correlated with discrete
features of their 31P and 19F NMR spectra. The synthetic
strategy developed, based on constructing a “portal” chiral
bisphosphonate synthon, should be adaptable to the synthesis
of cognate nucleotide bisphosphonate diastereomers. The
availability of the individual diastereomers of 12a,b now
makes possible kinetic analysis of their binding and turnover
interactions with pol β and other polymerases.
(8) (a) 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. Org. Lett. 2009, 11, 1883.
(b) 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. (c) 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.
(d) Chamberlain, B. T.; Batra, V. K.; Beard, W. A.; Kadina, A. P.;
Shock, D. D.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.;
Wilson, S. H. ChemBioChem 2012, 13, 258.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
mckenna@usc.edu
■
Notes
(9) (a) Lin, P.; Pedersen, L. C.; Batra, V. K.; Beard, W. A.; Wilson, S.
H.; Pedersen, L. G. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13294.
(b) Lin, P.; Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.;
Pedersen, L. G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5670.
(10) (a) Bakhtina, M.; Roettger, M. P.; Tsai, M. D. Biochemistry
2009, 48, 3197. (b) Roettger, M. P.; Bakhtina, M.; Tsai, M. D.
Biochemistry 2008, 47, 9718.
(11) Hardegger, L. A.; Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert, R.;
Stihle, M.; Gsell, B.; Thoma, R.; Diez, J.; Benz, J.; Plancher, J.-M.;
Hartmann, G.; Banner, D. W.; Haap, W.; Diederich, F. Angew. Chem.,
Int. Ed. 2011, 50, 314.
(12) (a) Kowalska, J.; Zuberek, J.; Darzynkiewicz, Z. M.;
Lukaszewicz, M.; Darzynkiewicz, E.; Jemielity, J. Collect. Symp. Ser.
2008, 10, 383. (b) Kowalska, J.; Lewdorowicz, M.; Zuberek, J.;
Grudzien-Nogalska, E.; Bojarska, E.; Stepinski, J.; Rhoads, R. E.;
Darzynkiewicz, E.; Davis, R. E.; Jemielity, J. RNA 2008, 14, 1119.
(c) Kowalska, J.; Lukaszewicz, M.; Zuberek, J.; Darzynkiewicz, E.;
Jemielity, J. ChemBioChem 2009, 10, 2469. (d) Lin, J. L.; Shaw, B. R.
Chem. Commun. 2000, 2115. (e) Lin, J. L.; Porter, K. W.; Shaw, B. R.
Nucleosides, Nucleotides Nucleic Acids 2001, 20, 1019. (f) Eckstein, F.;
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by NIH Grant 5-U19-CA105010
and in part by Research Project Z01-ES050158 (S.H.W.),
Intramural Research Program of the National Institutes of
Health, National Institute of Environmental Health Sciences.
We thank Inah Kang for assistance in preparing the manuscript.
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