Artificial genetic systems: Exploiting the "aromaticity" formalism to improve the tautomeric ratio for isoguanosine derivatives

Article abstract of DOI:10.1021/jo0497959

The tautomerism of 2′-deoxy-7-deaza-isoguanosine (2) was studied and compared to that of 2′-deoxyisoguanosine (1). The fixed ¹N-methyl (8) and O-methyl (4) derivatives were synthesized to represent the pure extremes of each tautomer. The replac

Full text of DOI:10.1021/jo0497959

Ar tificia l Gen etic System s: Exp loitin g th e
“Ar om a ticity” F or m a lism To Im p r ove th e
Ta u tom er ic Ra tio for Isogu a n osin e
Der iva tives
Theodore A. Martinot and Steven A. Benner*
Department of Chemistry, University of Florida,
P.O. Box 117200, Gainesville, Florida 32611-7200
benner@chem.ufl.edu
F IGURE 1. Isoguanosine (1), a purine (pu), can generate two
possible tautomers, an enol tautomer (giving a puDAD form,
which will pair with T or U) or a keto tautomer (giving a
puDDA form, which will pair with isoC). The extent of this
tautomerism has been reported to be 10:1 in favor of the keto
form.
Received February 4, 2004
Abstr a ct: The tautomerism of 2′-deoxy-7-deaza-isogua-
nosine (2) was studied and compared to that of 2′-deoxy-
1
isoguanosine (1). The fixed N-methyl (8) and O-methyl (4)
derivatives were synthesized to represent the pure extremes
of each tautomer. The replacement of the imidazole ring in
1 with a pyrrole ring in 2 makes the keto form in the latter
more favored by 2 orders of magnitude (KTAUT for 2 ≈ 103,
as opposed to KTAUT for 1 ≈ 10).
presents the donor-acceptor-donor hydrogen bonding
pattern that is complementary to the thymidine and
uridine heterocycles.6 Although the presence of the minor
tautomer does not adversely affect the use of isogua-
nosine in clinical diagnostics, it does inconvenience some
polymerases that prefer to place thymidine (T) and/or
uridine (U), rather than isocytidine (isoC), opposite
isoguanosine in a template.7
It has been a decade since it was shown that the
geometry of the Watson-Crick nucleobase pair can
accommodate as many as 16 nucleobases forming eight
mutually exclusive pairs, to yield an artificially expanded
genetic information system (AEGIS).1 By providing mo-
lecular recognition on demand in aqueous solution,
similar to nucleic acids but with a coding system that is
orthogonal to the system in DNA and RNA, AEGIS today
enables clinical assays such as Bayer’s branched DNA
diagnostics tool that monitors the load of viruses in
patients infected with HIV and hepatitis C viruses.2 Both
assays are FDA-approved and are widely used to provide
personalized patient care in the clinic. Further, AEGIS
components enable an assay for the early detection of the
SARS virus.3 With the emergence of the first six-letter
PCR,4 AEGIS now supports the development of a syn-
thetic biology where higher order processes of living
systems, including reproduction and Darwinian evolu-
tion, are duplicated by artificial, designed chemical
systems.
One issue remaining before a synthetic biology based
on AEGIS is fully implemented arises from the tauto-
meric form displayed by isoguanosine (and 2′-deoxy-
isoguanosine, disoG, 1). In its major keto form (Figure
1), disoG implements a hydrogen bond donor-donor-
acceptor pattern (proceeding from the major to the minor
groove) on a purine heterocycle (Figure 1). A minor enolic
tautomer of disoG is known to be present in aqueous
solution to perhaps 10%,5 however. The enolic tautomer
Given that the keto forms of pyrimidinones normally
dominate over enolic forms, we viewed the enolic tau-
tomer of disoG as an exception in need of explanation.
We reasoned that the enolic form was present in ap-
preciable concentration in disoG because it restores
formal aromaticity to the five-membered imidazole ring,
which must be cross-conjugated in the keto tautomer
(Figure 1). It is well-known that pyrrole is less “aromatic”
than imidazole, with the former being 59% as aromatic
as benzene, the latter being 64% (compared to pyridine
at 86%, thiophene at 66%, and furan at 43%).8 This
suggested that replacement of the imidazole ring of disoG
by a pyrrole ring to give 7-deaza-isoguanine might
generate a purine analogue that would also implement
the puDDA hydrogen bonding pattern (like disoG), but
with less of the enolic tautomer present at equilibrium.
7-Deaza-isoguanine (C7isoG) and its 2′-deoxynucleo-
side analogue (dC7isoG, 2) are both known.9-11 The
crystal structure of dC7isoG12 and its base-pairing ability
have both been studied.13-15 The tautomerism of both
C7isoG and dC7isoG remain undetermined, however.
(6) Robinson, H.; Gao, Y.-G.; Bauer, C.; Roberts, C.; Switzer, C.;
Wang, A. H.-J . Biochemistry 1998, 37, 10897-10905.
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Chemistry, 2nd ed.; Pergamon Press: Oxford, 2000.
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2 1986, 525-530.
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Chim. Acta 1991, 74, 1742-1748.
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K.; Von der Eltz, H.; Batz, H.-G. U.S. Patent 6,211,158 B1, 2001.
(12) Seela, F.; Wei, C.; Reuter, H.; Kastner, G. Acta Crystallogr.
1999, C55, 1335-1337.
(1) Geyer, C. R.; Battersby, T. R.; Benner, S. A. Structure 2003, 11,
1485-1498.
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Hughes, S. H.; Benner, S. A. Nucleic Acids Res. 2004, 32, 728-735.
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(13) Seela, F.; Wei, C. Chem. Commun. 1997, 1869-1870.
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10.1021/jo0497959 CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/01/2004
3972
J . Org. Chem. 2004, 69, 3972-3975

SCHEME 1
SCHEME 2
To test our hypothesis, the O-methyl-7-deaza-isogua-
nine derivative was prepared by the procedure of Davoll.16
Reaction with 1-(R)-chloro-3,5-di-O-(p-toluoyl)-2-deoxy-D-
ribose17 gave the 3′,5′-ditoluoyl-protected 2′-deoxyriboside
of 2-methoxy-6-amino-7-deaza-purine (3). Seela’s proce-
dure9 for the removal of the 2-methoxy group (HCl, 1,4-
dioxane, 100 °C) did not work well in our hands (Scheme
1). We therefore used thiophenol in ethylene glycol18 to
generate the di-O-(p-toluoyl)-derivative of 2 (5). This
compound was converted to the 2′-deoxynucleoside using
NaOMe in MeOH to afford 2, which had spectroscopic
properties identical to those reported for this compound
by Seela et al.9 The structure of this compound was
further confirmed by treatment with diazomethane,
which generated 4′. The structure of 4′ was identical in
all aspects to that of 4. The N-methyl derivative was
prepared by protection of the exocyclic amino group as
the diisobutylformamidine19,20 (generating 6), reaction
with CH3I (to give 7), and immediate deprotection by
treatment with methanolic NH3/NaOMe, affording 8.
1
N-Methylation at the N position was confirmed by long-
range (HMBC21) and short-range (HMQC22) 1H-13C NMR
correlation spectroscopy.
The pKa of the nucleoside analogue (2) was determined
by spectrophotometric titration (pH 2.8 to 13.7)23 at 240-
350 nm to give values for pK1 and pK2 as 4.3 ((0.1) and
9.9 ((0.2), respectively (Supporting Information). These
are comparable to the pKa values of natural nucleobases
and ensure that the C7isoG heterocycle is largely un-
charged in neutral water.
Four tautomeric forms are worthy of consideration for
7-deaza-isoguanine (Scheme 2). On the basis of literature
precedent, as well as precedent in other heterocycles, the
imino form was considered to be the least likely.5,24-26
(19) Zemlicka, J .; Holy, A. Collect. Czech. Chem. Commun. 1967,
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(16) Davoll, J . J . Chem. Soc. 1960, 131-138.
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463.
J . Org. Chem, Vol. 69, No. 11, 2004 3973

F IGURE 2. UV spectra of compounds 2, 4, and 8 in water. (Inset) UV spectra of the same compounds, in 99:1 1,4-dioxane/water.
This was confirmed by NMR: the -NH2 group was easily
identified in the NMR by integration and D2O exchange.
The UV spectra of 2′-deoxy-7-deaza-isoguanosine (2)
in aqueous solution resembles closely that of the N-
methylated derivative (8) and not the O-methylated
derivative (4) (Figure 2). This similarity suggests but does
not prove that the keto tautomer predominates, following
the same logic used by Sepiol et al. to infer that the keto
tautomer predominates for isoguanosine.5
The multiwavelength analysis described by Dewar and
Urch27 was used to suggest that, at most, one additional
tautomer was present as a major contributor to the
mixture (Supporting Information).
Information), chosen to maximize the difference between
the keto and enol forms, was chosen as a metric for the
tautomeric equilibrium constant in dioxane/water mix-
tures in varying proportions. This was plotted against
the Dimroth ET(30) value, which provides a measure of
the local dielectric constant.29-33
Even qualitatively, disoG and dC7isoG behave differ-
ently in these experiments. The UV spectrum of disoG
changes well before the water is completely removed. In
contrast, the UV spectrum of dC7isoG is not identical to
that of the O-methylated derivative even at the highest
concentrations of dioxane tested. Further, the UV spec-
trum of disoG continues to change even in mixtures
approaching pure water; this suggests that the conver-
sion of the enolic tautomer to the keto tautomer of disoG
is not complete even in pure water (the conclusion of
Sepiol et al.5). With dC7isoG, however, the UV spectrum
ceases to be solvent-dependent when the fraction of water
is greater than 50%. These results suggest that our
hypothesis at outset had manipulative value: the keto
tautomer of dC7isoG is more stable relative to its enolic
tautomer than the keto tautomer of disoG is relative to
its enolic tautomer.
The procedure of Voegel et al.28 was then used to
estimate a value of the keto:enol tautomers in pure water
for dC7isoG. This procedure exploits the fact that in
nonpolar solvents the UV spectra for both disoG and
C7isoG shift from their form in pure water (which
resembles the N-methylated derivatives) to a form that
resembles the UV spectra of the O-methylated deriva-
tives. This is interpreted as evidence that the tautomeric
equilibrium shifts from one favoring the keto tautomer
in pure water to one favoring the enol tautomer in pure
dioxane.
We then estimated the KTAUT () [keto]/[enol]) for
dC7isoG using the method of Voegel et al.28 Here, the
fraction of enol form was estimated over a range of ET-
(30) values where the KTAUT ≈ 1, regions where an
To obtain quantitative data, the ratio of the extinction
coefficients at two wavelengths (λ ) 296 and 255 nm for
disoG and λ ) 305 and 254 nm for dC7isoG; Supporting
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Liebigs Ann. Chem. 1963, 661, 1-37.
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3974 J . Org. Chem., Vol. 69, No. 11, 2004

F IGURE 3. Plot of log([enol]/[keto]) versus ET(30) for dC7isoG (2) and disoG (1) in mixtures of dioxane (ET(30) ) 36.0) and water
(ET(30) ) 63.1).
estimate could be made with some reliability. This is the
region where ET(30) ≈ 40-50. The log fraction of enol
was plotted against ET(30) (Figure 3), and the line was
extrapolated to the ET(30) of pure water (63.1).31 This
generated a value of KTAUT ≈ 103. Recalculating the KTAUT
for isoG gave a value of ≈10, consistent with that
previously reported. This can be compared with the value
for the natural guanosine nucleoside (KTAUT ≈ 104-105).34
With this result, we have now fixed each of the
problems identified in AEGIS components exploiting the
full range of hydrogen bonding patterns. Adjusting the
electron-withdrawing groups has generated a pyDDA
derivative that is free of epimerization.35 Adjusting side
chains of the pyAAD system has managed deamination
issues.36-40 Adjusting the ring nitrogens has corrected the
problematic pKa values of bases of acidic purines.41-44 In
the work presented here, adjustment of the aromaticity
of rings has resolved a tautomerism issue.45 Thus, with
this paper, we have the ingredients for a fully functional
12-letter genetic alphabet that meets the specifications
met by natural nucleic acids.
Ack n ow led gm en t. The authors are grateful to the
National Institutes of Health (GM54048) and the Uni-
versity of Florida (fellowship for T.M.) for funding.
Skillful assistance of Sonia K. Reback in the preparation
of starting materials is appreciated.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures, characterization data, HPLC traces, and experimental
details for the pKa measurements and tautomerization data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
J O0497959
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(45) It is clear that the ET(30) value at the interior of the DNA helix
is not known; our current work is looking into whether the behavior
of dC7isoG in water will correlate to its behavior in DNA.
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J . Org. Chem, Vol. 69, No. 11, 2004 3975