Oligonucleotide incorporation and base pair stability of 9-deaza-2′-deoxyguanosine, an analogue of 8-oxo-2′-deoxyguanosine

Article abstract of DOI:10.1021/jo1010763

9-Deaza-2′-deoxyguanosine (CdG) is a C-nucleoside and an analogue of the abundant promutagen 8-oxo-2′-deoxyguanosine (OdG). Like 2′-deoxyguanosine (dG), CdG should form a stable base pair with dC, but similar to OdG, CdG contains an N7-hydrogen that should allow it to also form a relatively stable base pair with dA. In order to further investigate the base pairing of CdG, it was incorporated into DNA and paired with either dC or dA. Melting studies revealed CdG:dC base pairs are less stable than dG:dC base pairs, while CdG:dA base pairs are less stable than OdG:dA base pairs. In order to gain a deeper understanding of these results, quantum studies on model structures of nucleoside monomers and base pairs were performed, the results of which indicate that (i) CdG:dC base pairs are likely destabilized relative to dG:dC as a result of structural constraints imposed by the C-nucleotide character of CdG, and (ii) CdG:dA base pairs may be less stable than OdG:dA base pairs, at least in part, because of a third long-range interaction that is possible in OdG:dA but not in CdG:dA.

Full text of DOI:10.1021/jo1010763

pubs.acs.org/joc
Oligonucleotide Incorporation and Base Pair Stability of
9-Deaza-20-deoxyguanosine, an Analogue of 8-Oxo-20-deoxyguanosine
Michelle L. Hamm,*,† Anna J. Parker, Tyler W. E. Steele, Jennifer L. Carman, and
Carol A. Parish*,†
Department of Chemistry, University of Richmond, Gottwald Science Center, Richmond, Virginia 23173.
†Contact M.L.H. for synthesis and melting studies and C.A.P. for quantum studies.
mhamm@richmond.edu; cparish@richmond.edu
Received June 2, 2010
9-Deaza-20-deoxyguanosine (CdG) is a C-nucleoside and an analogue of the abundant promutagen
8-oxo-20-deoxyguanosine (OdG). Like 20-deoxyguanosine (dG), CdG should form a stable base pair
with dC, but similar to OdG, CdG contains an N7-hydrogen that should allow it to also form a
relatively stable base pair with dA. In order to further investigate the base pairing of CdG, it was
incorporated into DNA and paired with either dC or dA. Melting studies revealed CdG:dC base pairs
are less stable than dG:dC base pairs, while CdG:dA base pairs are less stable than OdG:dA base
pairs. In order to gain a deeper understanding of these results, quantum studies on model structures
of nucleoside monomers and base pairs were performed, the results of which indicate that (i) CdG:dC
base pairs are likely destabilized relative to dG:dC as a result of structural constraints imposed by the
C-nucleotide character of CdG, and (ii) CdG:dA base pairs may be less stable than OdG:dA base
pairs, at least in part, because of a third long-range interaction that is possible in OdG:dA but not in
CdG:dA.
Introduction
glycosidic bond conformation of OdG varies depending on
the opposing base. When paired to dC, OdG is in the anti
conformation and the Watson-Crick hydrogen bonding
faces interact.5,6 When base paired to dA however, OdG is
in the syn conformation and uses its Hoogsteen edge to
hydrogen bond with the Watson-Crick face of dA.7,8 Con-
sistent with recent work that indicates the number of hydro-
gen bonds is the best predictor of base pair stability,9 OdG:
dC base pairs, which contain three hydrogen bonds, are more
8-Oxo-20-deoxyguanosine (OdG; Scheme 1) is one of the
most prominent damaged nucleotides in mammalian cells.1
It arises through reaction of 20-deoxyguanosine (dG) with
reactive oxygen species that can be produced by radiation2 or
chemical carcinogens3 and during normal metabolic res-
piration.4 Much research has been focused on OdG, not
only because of its prominence, but also because it is a known
promutagen; OdG can form stable base pairs with both
20-deoxycytosine (dC) and 20-deoxyadenosine (dA; Scheme 2),
thus allowing for the production of dGfT transversions during
replication.
(5) Lipscomb, L.; Peek, M.; Morningstar, M.; Verghis, S.; Miller, E.;
Rich, A.; Essigmann, J.; Williams, L. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
719–723.
(6) Oda, Y.; Uesugi, S.; Ikehara, M.; Nishimura, S.; Kawase, Y.; Ishikawa,
H.; Inoue, H.; Ohtsuka, E. Nucleic Acids Res. 1991, 19, 1407–1412.
(7) McAuley-Hecht, K. E.; Leonard, G. A.; Gibson, N. J.; Thomson,
J. B.; Watson, W. P.; Hunter, W. N.; Brown, T. Biochemistry 1994, 33,
10266–10270.
Crystal structure data from DNA duplexes containing
OdG:dC and OdG:dA base pairs have revealed that the
(1) Kunkel, T. Trends Genet. 1999, 15, 93–94.
(8) Kouchakdjian, M.; Bodepudi, V.; Shibutani, S.; Eisenberg, M.;
Johnson, F.; Grollman, A. P.; Patel, D. J. Biochemistry 1991, 30, 1403–1412.
(9) Geyer, C. R.; Battersby, T. R.; Benner, S. A. Structure 2003, 11, 1485–
1498.
(2) Hutchinson, F. Prog. Nucleic Acid Res. Mol. Biol. 1985, 32, 115–154.
(3) Floyd, R. A. Carcinogenesis 1990, 11, 1447–1450.
(4) Pryor, W. A. Annu. Rev. Physiol. 1986, 48, 657–667.
DOI: 10.1021/jo1010763
r
Published on Web 07/29/2010
J. Org. Chem. 2010, 75, 5661–5669 5661
2010 American Chemical Society

Hamm et al.
JOCArticle
SCHEME 1
SCHEME 3
SCHEME 2
OdG, further insights into the importance of the C8-oxygen
to the promiscuous base pairing of OdG can be obtained.
Results
Oligonucleotide Incorporation of CdG. 9-Deaza-20-deoxy-
guanosine has been incorporated into oligonucleotides pre-
viously. Revankar et al. deoxygenated 9-deazariboguano-
sine at the 20 position before using it to determine that CdG
prevents G-tetrad formation.14 Since publication of that
study, a more efficient route to the CdG monomer has been
published;15 using this route, we were able to generate the
CdG derivative 1 (Scheme 3) in 7 steps and 7% overall yield
starting from commercially available 2-amino-4-hydroxy-6-
methylpyrimidine. Additionally, though Revankar et al. used
an isobutyryl group to protect the exocyclic amine of the base
during DNA synthesis, in our hands the deprotection condi-
tions (29.7% NH4OH, 55 °C, 15 h) required for removal of the
isobutyryl group resulted in roughly 40% R/β epimerization at
C10 of the monomer according to NMR. Thus we instead used
an isopropylphenoxyacetyl (iPrPAc) group, which can be
removed under much milder conditions, to protect the exo-
cyclic amine. Thus nucleoside 1 was treated with isopropyl-
phenoxyacetyl chloride before selective deprotection of the
sugar alcohols with sodium methoxide (Scheme 3). The N1-
benzyl group left over from CdG synthesis was then removed
by pressurized catalytic hydrogenation to yield nucleoside 3.
To confirm that no epimerization would occur during depro-
tection of the iPrPAc group, nucleoside 3 was treated with
29.7% NH4OH for 18 h at room temperature. NMR analysis
of the product revealed full deprotection occurred with no
R-anomer present. Finally, in order to prepare CdG for solid
phase synthesis, the 50- and 30-oxygens were protected as a
dimethoxytrityl ether and activated as a phosphoramidite,
respectively, to yield the solid phase synthesis ready nucleotide
4 as a single diastereomer.
stable than OdG:dA mismatches, which contain only two
classical hydrogen bonds.10 Additionally, most likely due
to the abundance and promutagenic character of OdG, cells
have evolved repair enzymes that specifically remove 8-
oxoguanine from OdG:dC base pairs11,12 and adenine from
OdG:dA mismatches.13
The base pairing, replication, and repair of OdG described
above differs significantly from that of dG despite varying
from it at only the N7 and C8 positions. Thus it is possible
that one or both of these sites are key to the distinct bio-
chemical activities of OdG. In order to better address the
individual importance of these two sites, we have set about
creating analogues of OdG that alter either the N7-hydrogen
or C8-oxygen while leaving the other site unaffected. Toward
this goal, we report herein the oligonucleotide incorporation
and base pair stability of the C-nucleotide 9-deaza-20-deoxy-
guanosine (CdG; Scheme 1); by replacing the nitrogen at
position 9 of dG with carbon, CdG eliminates the C8-oxygen
while preserving the N7-hydrogen required for base pairing
to dA. Thus, by comparing the base pairing of CdG to that of
The CdG phosphoramidite 4 was used to synthesize an 11
nucleotide long oligonucleotide with the sequence 50-dC-
CATCXCTACC-30, where X is CdG (5). All standard pro-
cedures were used except that the oligonucleotide was de-
protected with ammonium hydroxide for only 18 h at room
(10) Plum, G. E.; Grollman, A. P.; Johnson, F.; Breslauer, K. J. Biochem-
istry 1995, 34, 16148–16160.
(11) Boiteux, S.; Gajewski, E.; Laval, J.; Dizdaroglu, M. Biochemistry
1992, 31, 106–110.
(12) Bjoras, M.; Luna, L.; Johnson, B.; Hoff, E.; Haug, T.; Rognes, T.;
Seeberg, E. EMBO J. 1997, 16, 6314–6322.
(14) Rao, T. S.; Lewis, A. F.; Durland, R. H.; Revankar, G. R. Tetra-
hedron Lett. 1993, 34, 6709–6712.
(13) Michaels, M. L.; Tchou, J.; Grollman, A. P.; Miller, J. H. Biochem-
istry 1992, 31, 10964–10968.
(15) Gibson, E. S.; Lesiak, K.; Watanabe, K. A.; Gudas, L. J.; Pankiewicz,
K. W. Nucleosides Nucleotides 1999, 18, 363–376.
5662 J. Org. Chem. Vol. 75, No. 16, 2010

Hamm et al.
JOCArticle
destabilization of OdG:dC base pairs relative to dG:dC,
which is likely caused by a steric clash between the C8-
oxygen and ribose ring of OdG. Consistent with this theory,
other nucleosides with steric bulk off C8, including SdG,16
show reduced stability in base pairs with dC. Interestingly,
though CdG contains little steric bulk off C8, it also shows
reduced stability in base pairs with dC as compared to dG.
Also contributing to the relatively similar stability of
OdG:dC and OdG:dA base pairs is the increased stability
of OdG:dA pairs relative to dG:dA pairs. This is most likely
due, at least in part, to the presence of an N7-hydrogen on
OdG. This hydrogen allows OdG to form a base pair with
dA that mimics a natural dT:dA base pair.7 dG lacks the
required N7-hydrogen and thus can only assume mismatches
with dA that do not structurally mimic a natural base
pair.17,18 Though CdG, like OdG and SdG, contains an
N7-hydrogen and CdG:dA base pairs likely adopt a struc-
ture similar to that of OdG:dA and SdG:dA base pairs (vide
infra), they are less stable than either of these pairs.
FIGURE 1. Nuclease digest of oligonucleotide 5 (A) and an
authentic standard of CdG (B).
Computational Study of Monomers and Base Pairs. Since
significant changes in structure and electronics can accom-
pany the change from an N- to a C-nucleotide, a B3LYP/6-
31G* geometry optimization and frequency analysis using
the polarizable continuum model (PCM)19 for water was
undertaken to further investigate the nature of the energetic
differences between dG, CdG, OdG, and SdG both as
monomers and in base pairs. Previous work20 has indicated
that this level of theory yields heavy atom distances that are
TABLE 1. Melting Temperatures (°C) of Duplexes Containing the
Various Base Pairsa
50-dCCATCXCTACC-30
30-dGGTAGYGATGG-50
X = dGb
X = CdG
X = OdGb
X = SdGb
Y = dC
Y = dA
57.5 ( 0.5
43.2 ( 0.2
54.1 ( 0.2
45.2 ( 0.4
52.7 ( 0.5
48.0 ( 0.3
46.6 ( 0.2
47.7 ( 0.3
aConditions: 1 M NaCl, 0.1 mM EDTA, 5 μM duplex, and 100 mM
sodium phosphate, pH 7.0. Average Tm values ( standard deviation
were calculated from three or more melts. bTaken from ref 16.
˚
approximately 0.1 A too short but that interaction energies
are well reproduced. All calculations were performed on
nucleoside structures containing deoxyribose sugars with
and without a CH3-O-CH2- (methoxymethylene) linkage
on the 40 carbon and a CH3-O- (methoxy) linkage on the 30
carbon. These substituents were added to mimic the steric
bulk of the phosphodiester chain in DNA. These structural
variations are shown in the representative model structure in
Figure 2. Base pair interaction energies were determined
using Eint=E(base pair) - E(nucleoside1) - E(nucleoside2)
where a negative Eint indicates a favorable interaction.
Previously, quantum mechanical gas-phase calculations have
been reported on bases20-28 and base pairs.22 In addition,
theoretical results incorporating solvent effects have been
described for monomers22,23,28-31 and base pairs lacking a
ribose ring.22 To our knowledge, the results reported here
temperature. The oligonucleotide was then purified by de-
naturing PAGE and reverse-phase HPLC.
To further confirm if epimerization occurred during DNA
synthesis or purification, oligonucleotide 5 was not only
characterized by MALDI-TOF but also digested to its
individual nucleosides and analyzed by analytical reverse-
phase HPLC. As seen in Figure 1A, the digest produced only
four main peaks (corresponding to dC, CdG, T, and dA),
suggesting little R-anomer of CdG was present; to further
characterize the CdG peak, it was compared to an authentic
sample of CdG (Figure1B) as well as the R-anomer of CdG
(data not shown).15 The peaks corresponding to CdG from
the digest of 5 and the CdG standard had similar retention
times of 6 min and coeluted, while the R-anomer of CdG had
a retention time of 9 min.
(17) Brown, T.; Hunter, W. N.; Kneale, G.; Kennard, O. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 2402–2406.
Experimental Base Pair Stabilities. Oligonucleotide 5 was
then used to better understand the base pairing of CdG as
compared to dG, OdG, and 8-thio-20-deoxyguanosine (SdG,
which also contains a N7-hydrogen but not a C8-oxygen;
Scheme 1). It was paired to complementary oligonucleotides
(Table 1) and melted under conditions identical to those
previously reported for duplexes containing dG, OdG, and
SdG.16 Since the only difference in the various duplexes
studied was the one X:Y base pair, any difference in melting
temperature is attributable to that particular base pair.
Previous studies have shown that OdG:dC and OdG:dA
base pairs are much more similar in stability than dG:dC and
dG:dA base pairs.10 It is believed this is due, in part, to a
(18) Prive, G. G.; Heinemann, U.; Chandrasegaran, S.; Kan, L. S.;
Kopka, M. L.; Dickerson, R. E. Science 1987, 238, 498–504.
(19) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Chem. Phys. 2002,
117, 43–54.
(20) Hobza, P.; Sponer, J. Chem. Rev. 1999, 99, 3247–3276.
(21) Aida, M.; Nishimura, S. Mutat. Res. 1987, 192, 83–89.
(22) Cysewski, P. J. Chem. Soc., Faraday Trans. 1998, 94, 3117–3125.
(23) Cysewski, P.; Bira, D.; Bialkowski, K. J. Mol. Struct. THEOCHEM
2004, 678, 77–81.
(24) Gu, J. D.; Leszczynski, J. J. Phys. Chem. A 1999, 103, 577–584.
(25) Marian, C. M. J. Phys. Chem. A 2007, 111, 1545–1553.
(26) McConnell, T. L.; Wheaton, C. A.; Hunter, K. C.; Wetmore, S. D. J.
Phys. Chem. A 2005, 109, 6351–6362.
(27) Reynisson,H.;Steenken, S. Phys. Chem. Chem. Phys. 2002, 4, 527–532.
(28) Venkateswarlu, D.; Leszczynski, J. J. Comput.-Aided Mol. Des. 1998,
12, 373–382.
(29) Barbe, S.; Le Bret, M. J. Comput. Chem. 2008, 29, 1353–1363.
(30) Jang, Y. H.; Goddard, W. A.; Noyes, K. T.; Sowers, L. C.; Hwang,
S.; Chung, D. S. Chem. Res. Toxicol. 2002, 15, 1023–1035.
(31) Jang, Y. H.; Goddard, W. A.; Noyes, K. T.; Sowers, L. C.; Hwang,
S.; Chung, D. S. J. Phys. Chem. B 2003, 107, 344–357.
(16) Hamm, M. L.; Cholera, R.; Hoey, C. L.; Gill, T. J. Org. Lett. 2004, 6,
3817–3820.
J. Org. Chem. Vol. 75, No. 16, 2010 5663

Hamm et al.
JOCArticle
the 30 and 40 linkages stabilizes dC containing pairs by
∼5 kcal/mol, while hardly changing the interaction energies
of dA containing base pairs. In order to further understand
the linkage effects, we also determined the structures and
energies for model systems containing only the 30-methoxy or
only the 40-methoxymethylene arms. In all cases, having only
the 30-methoxy linkage does not change the interaction
energies from their values with both arms present. However,
having only the 40-methoxymethylene linkage destabilizes
XdG:dC base pairs by 6-10 kcal/mol as compared to when
both linkers are present. This effect is not seen in the XdG-
(syn):dA structures.
FIGURE 2. Model structure used for B3LYP/6-31G*/PCM(water)
geometry optimizations (dG shown as a representative structure; all
structures are available in the Supporting Information.) The
CH3-O-CH2- and CH3-O- linkages on the 40 and 30 carbons,
respectively, were included to model the steric bulk of the phospho-
diester chain in DNA.
All optimized base pair structures are planar except
SdG:dA and those incorporating CdG, i.e., in each base
pair except these, the bases are aligned in the same plane
(Table 4). For SdG:dA and CdG containing base pairs, one
of the monomers is twisted 3.9-11° relative to the other. For
model compounds containing the 40 CH3-O-CH2- linkage,
the least stable base pair is CdG:dC (-0.83 kcal/mol).
Generally, substitution of the sugar with 30-methoxy and
40-methoxymethylene arms does not significantly affect
intermolecular base pair geometries. For instance, compar-
ing the average intermolecular distances for base pairs with
and without substituents on the sugar, we see that substitu-
contain the largest oligonucleotide/base pair models inves-
tigated to date using quantum mechanical methods and
including the effects of solvent.
Monomers. Starting structures for monomer calculations
began with bases oriented either syn or anti relative to sugars.
All structures oriented the 40 axial CH3-O-CH2- and 30
pseudoequatorial CH3-O- linkages similarly to the phos-
phodiester arms in the experimental crystal structure of
OdG:dC (PDB 183D) or OdG:dA (PDB 178D). Upon geo-
metry optimization, the 40-methoxymethylene and 30-methoxy
linker arms reoriented slightly with all structures displaying a
trans rotamer orientation for both sugar substituents. Mono-
mer energies are shown in Table 2 and indicate that for these
model structures syn and anti OdG conformations are relatively
isoenergetic, whereas SdG prefers syn. Interestingly, for CdG,
the preferred base orientation seems to be strongly dependent
upon the presence or absence of the 40-methoxymethylene arm:
when the linkage is lacking, the anti and syn conformations are
isoenergetic, but when present, the anti conformation is favored
by more than 4 kcal/mol. This effect is negated by the addition
of the 30 pseudoequatorial linkage (vide infra).
Base Pairs. To ascertain the importance of base/backbone
interactions on the stability of base pairs, we also performed
our theoretical study of base pair interactions with and with-
out the 40 CH3-O-CH2- and 30 CH3-O- arms that model
the phosphodiester backbone. By comparison with the crys-
tal structures of duplexes containing OdG:dC5 and OdG:
dA,7 we know that our geometry optimized model structures
are very similar with respect to the nucleoside-base orienta-
tion, but the 40 linker arm is in a very different position than
in the backbone of DNA. This is despite utilizing starting
structures with the correct linker orientation. Geometry
optimization moves the 40 linker arm to ∼180ο orientation,
corresponding to a low energy trans conformation as would
be expected in a steric-free environment (the arm is oriented
at 130ο in the crystal structure). This positions the 40 arm
further away from the base than in the corresponding DNA
structures. The 30 CH3-O- arm in our model compounds
maintains a more similar orientation to the crystal structure
(152ο crystal vs 168ο model). As shown in Table 3, the energy
of interaction is favorable in all cases.
˚
tion increases the average distances by only 0.001-0.016 A.
Discussion
Quantum Results Support the Theory That C8 Steric Bulk
Can Destabilize XdG:dC Base Pairs. CdG is an interesting
nucleoside to study because it lacks significant steric bulk or
hydrogen bonding ability at C8 similar to dG but contains an
N7-hydrogen similar to OdG. Previous experimental work
with dG, OdG, and their analogues16,32 has suggested that
the presence of a large atom off C8 destabilizes base pairs to
dC, perhaps due to a steric clash with the backbone sugar
that destabilizes the anti conformation, leading to a prefer-
ence for a syn conformation. Previous NMR studies support
this theory and indicate OdG,33 and other dG monomers
with large atoms off C8, are primarily in the syn confor-
mation.32,34 The quantum results presented here also indi-
cate that large atoms at C8 might cause a preference for the
syn conformation as evidenced by the anti verses syn pre-
ference for CdG verses SdG when the 40-methoxymethylene
linkage is present (Table 2). It is interesting that the inclusion
of the 30 and 40 arms stabilize XdG:dC pairs, while the
inclusion of only the 40 CH3-O-CH2- linkage destabilizes
XdG:dC base pairs by 6.1-10.2 kcal/mol relative to the
systems with both the 30 and 40 arms present (Table 3). When
the two substituents are present, the stabilizing effect of the 30
arm dominates and even negates the large destabilizing effect
of the 40 linker in CdG:dC. This sensitivity to sugar substitu-
tion is not present in the XdG:dA base pairs, further
suggesting that an unfavorable interaction with the back-
bone is playing a role in base pair stability. A careful analysis
of the energetic components of base pair Eint reveals that the
addition of the 40 arm causes slight destabilization of the
A comparative analysis of the base pair interaction en-
ergies indicates that substitution of the sugar significantly
affects the energetics of dC containing base pairs but has very
little effect on dA containing pairs. A comparison of differ-
ent base pairs with no substituents on the sugar reveals very
similar interaction energies ranging between -6.15 and
-7.54 kcal/mol. However, substitution of the sugar with
(32) Hamm, M. L.; Rajguru, S.; Downs, A. M.; Cholera, R. J. Am. Chem.
Soc. 2005, 127, 12220–12221.
(33) Culp, S. J.; Cho, B. P.; Kadlubar, F. F.; Evans, F. E. Chem. Res.
Toxicol. 1989, 2, 416–422.
(34) Uesugi, S.; Ikehara, M. J. Am. Chem. Soc. 1977, 99, 3250–3253.
5664 J. Org. Chem. Vol. 75, No. 16, 2010

Hamm et al.
JOCArticle
TABLE 2. B3LYP/6-31G* PCM(water) Nucleoside Energiesa
no substituents
40-methoxymethylene and 30-methoxy
40-methoxymethylene
30-methoxy
monomer
E
ΔE
E
ΔE
E
ΔE
E
ΔE
dG(anti)
dG(syn)
-773.8457475
-773.8456820
-757.7951492
-757.7948563
-849.0972735
-849.0969126
-1172.056522
-1172.059257
-1042.197025
-1042.196331
-1026.148490
-1026.148342
-1117.448744
-1117.448158
-1440.406492
-1440.409658
-927.6791134
-927.6784245
-911.6362020
-911.6293070
-1002.930281
-1002.929796
-1325.888170
-1325.891348
-888.363809
-888.364011
-872.313985
-872.313958
-963.615783
-963.615390
-1286.575068
-1286.577567
-0.04
-0.18
-0.23
1.71
-0.43
-0.09
-0.37
1.98
-0.43
-4.32
-0.30
1.99
0.13
-0.02
-0.25
1.57
CdG(anti)
CdG(syn)
OdG(anti)
OdG(syn)
SdG(anti)
SdG(syn)
aAbsolute energies are in atomic units and relative energies are in kcal/mol. Detailed geometries are included in the Supporting Information. Relative
energies (ΔE) were determined as the difference between the anti and syn conformers (anti - syn); a positive difference indicates that the syn is favored.
TABLE 3. B3LYP/6-31G* PCM(water) Base Pair Interaction Energies (Eint, kcal/mol)a
base pair
no substituents
40-methoxymethylene and 30-methoxy
40-methoxymethylene
30-methoxy
dG:dC
-6.75
-6.15
-6.91
-7.05
-6.80
-7.54
-6.60
-11.61
-11.01
-11.76
-11.82
-6.79
-5.48
-0.83
-5.69
-5.71
-6.81
-7.30
-6.31
-11.63
-11.04
-11.82
-11.91
-6.87
CdG:dC
OdG:dC
SdG:dC
CdG:dA
OdG:dA
SdG:dA
-7.27
-7.50
-6.37
-6.62
aDetailed geometries are included in the Supporting Information.
TABLE 4. B3LYP/6-31G* PCM(water) Base Pair Geometries and Hydrogen Bond Lengthsa
with 40-methoxymethylene linkage
no substituents on sugar
base pair H-bondsc
base pair
base pair geob
base pair H-bondsc
averaged
base pair geob
averaged
dG:dC
planar
twisted
planar
planar
twisted
planar
twisted
1.883
1.930
1.940
1.910
1.901
1.944
1.856
1.904
1.866
1.893
1.916
1.884
1.880
1.906
1.885
1.890
planar
twisted
planar
planar
twisted
planar
planar
1.885
1.884
1.880
1.896
1.869
1.907
1.879
1.926
1.928
1.909
1.901
1.950
1.864
1.907
1.854
1.904
1.856
1.840
1.888
1.905
1.882
1.879
1.910
1.885
1.893
CdG:dC
OdG:dC
SdG:dC
CdG:dA
OdG:dA
SdG:dA
1.896
1.880
1.895
1.867
1.914
1.875
1.913
1.861
1.844
2.911
2.944
2.946
3.001
with 40-methoxymethylene and 30-methoxy linkages
with 30-methoxy linkage
base pair H-bondsc
base pair
base pair geob
base pair H-bondsc
averaged
base pair geob
averaged
dG:dC
planar
twisted
planar
planar
twisted
planar
twisted
1.889
1.882
1.877
1.901
1.872
1.914
1.876
1.925
1.931
1.911
1.902
1.939
1.858
1.908
1.859
1.905
1.861
1.845
1.891
1.906
1.883
1.883
1.905
1.886
1.892
planar
twisted
planar
planar
twisted
planar
twisted
1.885
1.881
1.880
1.895
1.871
1.910
1.882
1.926
1.928
1.909
1.902
1.940
1.862
1.900
1.854
1.906
1.858
1.844
1.888
1.905
1.882
1.880
1.906
1.886
1.891
CdG:dC
OdG:dC
SdG:dC
CdG:dA
OdG:dA
SdG:dA
2.919
2.946
2.948
2.996
aGeometries are included in the Supporting Information. bPlanar = intermolecular torsion angles are (0.5-3.5; twisted = intermolecular torsion
angles are (3.6-11.0. cFor dC containing base pairs, the first, second, and third hydrogen bonded values correspond to XdG(C6carbonyl
O)-dC(C4amine H), XdG(N1H)-dC(N3), and XdG(C2amine H)-dC(C2carbonyl O), and in dA containing base pairs, the first, second, and third
hydrogen bonded values correspond to the XdG(C6carbonyl O)-dA(C6amine H), XdG(N7 H)-dA(N1), and XdG(C8carbonyl O/C8thio S)-dA(C2 H)
interactions, respectively. A minimum for dG:dA was not found at this level of theory presumably due to a lack of hydrogen bond donor-acceptor
functionality. dAverage distance computations did not include the third nonclassical H-bonding distance for OdG:dA and SdG:dA base pairs.
XdG(anti) monomers and, unexpectedly, a slight stabiliza-
tion of the XdG(syn) monomers, resulting in an overall
destabilization of XdG:dC pairs relative to the XdG:dA
pairs (monomer data without arms available in the Support-
ing Information). It should be noted that though all XdG:dC
systems were destabilized by inclusion of the 40 linker, pairs
with large atoms off C8 (as in OdG:dC and SdG:dC) were
not destabilized to a greater extent than those with small
atoms off C8 (as in dG:dC); this is likely due to the differ-
ences in linker arm orientation between our models and the
crystal structure of DNA. It is possible this difference in
configuration limits the destabilizing clash between large
atoms off C8 and the backbone.
Stability of CdG:dC Base Pairs. Interestingly, though CdG
contains only a hydrogen off C8 like dG, CdG:dC base pairs
are less stable than dG:dC base pairs (Tables 1 and 3).
Previous experimental studies on the CdG monomer have
shown some evidence that, like OdG and SdG, it may have
some preference for a syn base conformation despite not
having a large atom off C8.15 Such a preference may explain
the instability of CdG:dC relative to dG:dC; however, the
NMR studies used to determine this finding did not take into
account the known change in sugar pucker present in
C-nucleosides (to increased 20-endo character as indicated
(35) Oleary, D. J.; Kishi, Y. J. Org. Chem. 1994, 59, 6629–6636.
J. Org. Chem. Vol. 75, No. 16, 2010 5665

Hamm et al.
JOCArticle
by an increased J1 -2 ),35 a property that complicates analysis
of the preferred syn/anti base orientation of the CdG mono-
mer and itself may alter base pair stability.36 Our quantum
studies confirm the increased 20-endo character for CdG
relative to the other monomers (as indicated by an increased
H20-C20-C10-H10 torsional angle; data shown in the Sup-
porting Information). However, our theoretical results stand
in contrast to the previous NMR studies and indicate that
either the CdG syn and anti conformations are isoener-
getic or CdG prefers an anti conformation when the 40-
methoxymethylene linkage is present. This finding casts
further doubt on the hypothesis that the instability of
CdG:dC relative to dG:dC is caused by a preference for the
CdG syn monomer conformation.
allows a favorable bifurcating nonclassical hydrogen bond-
ing interaction between a proton on the linker arm, a proton
on the sugar and the O or S off C8 (XdG(anti)) or the N3
(XdG(syn)). This interaction is not possible in dG:dC or
CdG:dC (Figure 3) and is not present in the experimental
crystal structures where instead the O or S off C8 interacts
unfavorably with the 40O in the sugar ring. This indicates
that while our model compounds are, to the best of our
knowledge, the largest base pair structures to be character-
ized using quantum mechanical methods and including the
effects of solvation, they are structurally too small to fully
capture the steric effects present in the oligonucleotides
studied experimentally here.
0
0
It is also noteworthy that our model results suggest that a
C-nucleoside is much less flexible and less able to adjust to
the local environment than an N-nucleoside. The twisted,
nonplanar nature of CdG containing base pairs is one
evidence of this. Structural analysis indicates there is a
pyramidalization at N9 of up to 8° that is not possible in
C-nucleosides where the sp2 hybridized C9 is locked into
a planar geometry (torsional data shown in Supporting
Information). This theoretical observation is difficult to
verify experimentally; to our knowledge no crystal struc-
ture is available containing CdG. However, a comparison
Computational results suggest that on these smaller base
pair models CdG:dC is less stable than dG:dC due to a
number of relatively subtle factors. First, a comparison of
the monomer:monomer dipole interactions with the resul-
tant base pair dipoles indicate that there may be an elec-
trostatic basis for the destabilization: the dipolar orienta-
tion is more complementary/favorable for the other dC
base pairs than for CdG:dC. This is further complicated
by an unusually small dipole moment for the CdG(anti)
monomer (relative to all other monomers including the
CdG(syn) monomer; dipole data shown in the Supporting
Information).
The second factor is that the hydrogen bonding/electro-
static interactions between monomers in the base pair struc-
tures are slightly less favorable for CdG:dC relative to the
other dC base pairs as judged by the hydrogen-bonding
intermonomer distances and perhaps best summarized by
the average of those distances (Table 4). For instance, on
with a crystal structure of formycin (PDB 1T8S),37-39
a
C-nucleoside analogue of adenosine, revealed an average
C9 pyramidalization of only 0.78°, confirming our findings.
Structural data also suggests that the glycosidic bond length
in CdG is less able to adjust to the local environment; for
instance upon addition of the 40 linker arm in N-nucleosides
˚
the base moves 0.010-0.014 A away from the sugar, but is
only able to move 0.002 A away in C-nucleosides.
˚
˚
average, the CdG:dC monomers are 0.023-0.036 A further
Thus, the quantum studies indicate the lower stability of
CdG:dC base pairs as compared to dG:dC base pairs is likely
due to the C-nucleoside character of CdG and the various
structural and electronic changes that result. These findings
are also consistent with previous work that shows oligo-
nucleotide duplexes containing a C-glycoside usually have
melting temperatures that are a few degrees lower than an
analogous duplex with an N-glycoside.9
Stability of CdG:dA Base Pairs. Though CdG contains an
N7-hydrogen like OdG and SdG, which should allow it to
form relatively stable base pairs to dA, duplexes containing a
CdG:dA base pair are less stable than duplexes containing an
OdG:dA or SdG:dA base pair (Table 1). This experimentally
observed difference in base pair stability may be due to a
number of different factors. First, the pKa at the N7 of CdG
is likely much higher in value than that at the N7 of OdG or
SdG. Pyrrole (a model compound for the five-membered
ring in the base of CdG) has a pKa value of 17.5,40 much
higher than that of 2-hydroxyimidazole or 2-mercaptoimidazole
(comparable analogues for OdG and SdG, respectively),
which have pKa values of 12.8 and 11.3, respectively (see
Supporting Information). It is known that the strength of a
hydrogen bond increases as the pKa values for the acceptor
apart than all other dC containing base pairs. Third, all other
monomers form optimally oriented, planar base pair struc-
tures (Figure 3) except for those with CdG likely due to the
C-nucleoside nature of the base (vide infra). The most
significant CdG:dC destabilization (of ∼10 kcal/mol relative
to the model with both arms) occurs in the presence of the
40-methoxymethylene arm. An analysis of the orientation of
the plane of the base relative to the pseudoplane of the sugar
reveals that in all of the 40-methoxymethylene containing
systems except CdG:dC, the free monomer orientation is
preserved (to within 1-9°) in the optimized base pair struc-
ture. However, the base in the CdG(anti) monomer (orien-
tation = 67.7°) must torque 20° in the glycosidic torsional
angle (O40-C10-C9-C8) in order to base pair with dC
(orientation of CdG base relative to sugar in base pair =
47.7°). Thus it appears that whereas all the other monomers
are “pre-organized” for bonding with their corresponding
base pair, CdG(anti) must undergo a significant structural
rearrangement in order to pair with dC. This builds an
inherent strain into the system, resulting in a much less
favorable interaction. In this case, the addition of the 40
linker arm increases the separation between monomers by
˚
0.011 A and changes significantly the orientation of the base
to the sugar.
Finally, in our model systems, the reorientation of the
40-methoxymethylene arm upon geometry optimization
(37) Giranda, V. L.; Berman, H. M.; Schramm, V. L. Biochemistry 1988,
27, 5813–5818.
(38) Prusiner, P.; Brennan, T.; Sundaral., M Biochemistry 1973, 12, 1196–
1202.
(39) Zhang, Y.; Cottet, S. E.; Ealick, S. E. Structure 2004, 12, 1383–1394.
(40) Archeson, R. M. An Introduction to the Chemistry of Heterocyclic
Compounds, 3rd ed.; John Wiley & Sons, Inc.: New York, 1976.
(36) Williams, A. A.; Darwanto, A.; Theruvathu, J. A.; Burdzy, A.;
Neidigh, J. W.; Sowers, L. C. Biochemistry 2009, 48, 11994–12004.
5666 J. Org. Chem. Vol. 75, No. 16, 2010

Hamm et al.
JOCArticle
FIGURE 3. B3LYP/6-31G*/PCM(water) optimized base pair structures. For clarity, only model compounds are shown containing 40
methoxymethylene arms. All other structures may be found in the Supporting Information.
and donor atoms become more similar.41,42 Since the N1
position of dA (the acceptor) has a pKa value of 3.5, the
N7-hydrogen (the donor) pKa values for OdG and SdG
would be closer than that for CdG, possibly allowing for
the formation of a stronger hydrogen bond and stabilizing
OdG:dA and SdG:dA base pairs overall.
The instability of CdG:dA base pairs as compared to
OdG:dA and SdG:dA base pairs may also be due to a lack
of steric bulk at the C8 position of CdG. As explained above,
while dG prefers an anti conformation about the glycosidic
bond, OdG, SdG, and other dG analogues with large atoms
at C8 prefer a syn base conformation. While the preferred
base orientation of CdG is controversial, it is possible that, as
compared to OdG and SdG, CdG may be less likely to adopt
the syn conformation needed for base pairing to dA.
between the number of hydrogen bonds formed and base pair
stability confirms the results reported previously by Geyer et al.9
Monomer Conformational Preferences Are a Strong Pre-
dictor of Base Pair Stabilities. When combining the experi-
mental dC and dA base pairing data, we find a trend where
CdG forms much more stable base pairs to dC, OdG forms
somewhat more stable base pairs to dC, and SdG forms
slightly more stable base pairs to dA (Table 1). Though we
realize many factors can lead to the observed stability
differences in the corresponding duplexes, it is interesting
to note that the monomeric conformational preferences of
CdG, OdG, and SdG determined computationally are con-
sistent with these findings. With the 40-methoxymethylene
arm present, the CdG monomer has a relatively large ener-
getic preference for the anti conformation (Table 2), and
experimentally we see a relatively large preference for base
pairing to dC where the anti conformation is required. The
syn conformation is more energetically stable than the anti
conformation in the SdG monomer, and experimentally
SdG:dA containing duplexes, where the syn conformation
of SdG is required, are more stable than SdG:dC containing
duplexes. As a note, dG was not addressed in this analysis
since it cannot form similar base pairs to dA due to a lack of a
N7-hydrogen (a geometry optimization starting with a planar
intermonomer arrangement led to a relatively high energy
structure containing one hydrogen bond and a twisted (50°)
orientation between nucleosides).
Final Note. Differences between the theoretically and
experimentally determined stabilities of the various base
pairs may be due to very small differences in individual base
pair interactions that become amplified by interaction with
flanking nucleotides when incorporated into the oligomers,
an effect too large for current quantum mechanical methods.
The model structures employed here also appear to be too
small and flexible to capture the true nature of the duplexes
containing damaged DNA base pairs. For instance, contri-
butions from base-stacking are not included, and thus addi-
tional effects from structural differences in the base pairs
Finally, though computational results suggest that for
these model structures, CdG:dA, OdG:dA, and SdG:dA are
isoenergetic with interaction energies ranging from -6.20 to
-7.54 kcal/mol (Table 3), we do note that the average inter-
monomer distances in OdG:dA and SdG:dA are similar and
˚
∼0.02 A shorter than in CdG:dA. We also see the potential for a
˚
third, albeit relatively long (∼2.9 A) electrostatic/nonclassical
hydrogen bonding interaction43,44 present in OdG:dA and SdG:
dA that is not possible in CdG:dA (Figure 3). Natural bond
orbital analysis and Wiberg Bond Indices (Supporting
Information) determined for OdG:dA agree with the structural
data and suggest the possibility of a third, albeit weak, long-
range interaction. The similarities for OdG:dA and SdG:dA in
base pair interaction energy and structure is in accord with
previous results suggesting that sulfur is close to oxygen in its
hydrogen bond acceptor behavior.45 The strong correlation
(41) Chen, J. G.; McAllister, M. A.; Lee, J. K.; Houk, K. N. J. Org. Chem.
1998, 63, 4611–4619.
(42) Shan, S. O.; Herschlag, D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
14474–14479.
(43) Costa, M. T. C. M. J. Mol. Struct. THEOCHEM 2005, 729, 47–52.
(44) Khan, A. J. Phys. Chem. B 2000, 104, 11268–11274.
(45) Wierzejewska, M.; Saldyka, M. Chem. Phys. Lett. 2004, 391, 143–
147.
J. Org. Chem. Vol. 75, No. 16, 2010 5667

Hamm et al.
JOCArticle
may not be fully accounted for. For example, the twist
between the two bases observed computationally for CdG:
dC, CdG:dA, and SdG:dA (Table 4) could weaken stacking
interactions with the adjacent base pairs, thus helping to
account for the relatively lower melting temperatures ob-
served experimentally with these base pairs (Table 1). Also,
the overall structure of DNA positions the backbone arm
more rigidly near the base, and this likely prevents small
changes in the base-sugar orientation that are possible in
our model systems. Hybrid (QM/MM) and molecular dyna-
mics (MD) analyses are currently underway to probe these
effects and will be reported elsewhere. It is also possible that
the experimental effects are not captured with the approach
taken here but necessitate a determination of the free energy
including the entropy. It is not currently possible to perform
a multiconformer entropic analysis with quantum mecha-
nical methods on model systems of this size while including
the effects of solvent.
1H NMR (DMSO-d6) δ: 11.50 (s, 1H), 11.04 (s, 1H), 8.40 (s, 1H),
7.21 (s, 1H), 5.22 (m, 1H), 5.17 (dd, 1H), 4.92 (m, 1H), 4.25 (m,
1H), 3.79 (m, 1H), 3.54 (m, 1H), 3.47 (m, 1H), 3.09 (s, 3H), 2.99
(m, 3H), 2.32 (dt, 1H), 1.90 (dd, 1H). 13C NMR (DMSO-d6)
δ:157.4, 155.3, 154.6, 143.6, 126.0, 116.3, 116.1, 87.9, 73.8, 73.7,
63.6, 42.5, 40.8, 34.8. HR-ESI (M þ Hþ) for C29H33N4O6:
expected 533.2395, found 533.2389.
2-N-Isopropylphenoxyacetal-9-deaza-20-deoxyguanosine (3).
Compound 2 (220 mg, 0.41 mmol) in 150 mL of methanol was
added to 75 mg of palladium hydroxide. The solution was
shaken for 15 h under 40 psi of hydrogen at room temperature,
filtered over Celite, concentrated, and purified by silica gel
chromatography using 4% methanol in chloroform to yield
1
120 mg (0.27 mmol; 66%) of 3 as a white powder. H NMR
(DMSO-d6) δ: 11.96 (b, 1H), 11.6 (b, 2H), 7.37 (s, 1H), 7.17 (d,
2H), 6.88 (d, 2H), 5.20 (dd, 1H), 4.95 (m, 1H), 4.79 (s, 2H), 4.20
(m, 1H), 3.71 (m, 1H), 3.44 (m, 2H), 2.83 (m, 1H), 2.14 (m, 1H),
2.00 (m, 1H), 1.17 (d, 6H). 13C NMR (DMSO-d6) δ: 171.5,
156.4, 153.0, 145.1, 142.5, 141.6, 127.7, 126.4, 117.4, 115.5,
114.9, 73.0, 87.8, 72.3, 67.2, 63.1, 41.9, 33.1, 24.5. HR-ESI
(M þ Hþ) for C22H27N4O6: expected 443.1925, found 443.1940.
2-N-Isopropylphenoxyacetal-50-O-dimethoxytrityl-9-deaza-20-
deoxyguanosine. Compound 3 (120 mg, 0.27 mmol) was coeva-
porated three times with pyridine to remove any associated water
before addition of 137 mg (0.40 mmol) of dimethoxytrityl
chloride and 2 mg (0.016 mmol) of 2-(dimethylamino)pyridine.
The flask was covered with argon, and 4 mL of anhydrous
pyridine and was added. After 1.5 h of stirring at room tempera-
ture, the reaction mixture was concentrated, and the resulting oil
was purified by silica gel chromatography using 1-2% methanol
in chloroformto yield 160mg of 2-N-isopropylphenoxyacetal-50-
O-dimethoxytrityl-9-deaza-20-deoxyguanosine (0.21 mmol; 80%)
as a white foam. 1H NMR (CDCl3) δ: 11.83 (b, 1H), 10.89 (b, 1H),
9.15 (b, 1H) 7.4-7.1 (14H), 6.88 (d, 2H), 6.78 (d, 2H), 5.45 (dd,
1H), 4.60 (s, 2H), 4.50 (m, 1H), 4.11 (m, 1H), 3.75 (s, 6H), 3.36 (m,
1H), 3.18 (m, 1H), 2.90 (m, 1H), 2.31 (m, 1H), 1.89 (m, 1H), 1.24
(d, 6H). 13C NMR (CDCl3) δ: 169.7, 158.5, 154.8, 153.2, 149.8,
144.9, 143.4, 142.6, 136.11, 136.08, 130.1, 128.2, 127.80, 127.75,
126.8, 126.4, 123.8, 117.5, 116.0, 114.8, 113.1, 86.2, 86.0, 74.8, 72.1,
67.2, 64.8, 55.2, 41.0, 33.3, 29.7, 24.1. HR-ESI (M þ Hþ) for
C43H45N4O8: expected 745.3232, found 745.3263.
2-N-Isopropylphenoxyacetal-50-O-dimethoxytrityl-9-deaza-
20-deoxyguanosin-30-yl β-Cyanoethyl-N,N-diisopropylphosphor-
amidite (4). A 180 mg portion of 2-N-isopropylphenoxyacetal-
50-O-dimethoxytrityl-9-deaza-20-deoxyguanosine (0.24 mmol)
was coevaporated three times with pyridine to remove any
associated water before addition of 41 mg (0.35 mmol) of 4,5-
dicyanoimidazole (Chemgenes). The flask was covered with
argon, and 5 mL of anhydrous methylene chloride and 113 μL
(0.35 mmol) of 2-cyanoethyl-N,N,N,N-tetraisopropyl-phos-
phane (Chemgenes) were added. The reaction was stirred for
40 min at room temperature before addition of 15 mL of
ethylacetate. The solution was washed twice with 10 mL of
saturated NaHCO3, dried with Na2SO4, and concentrated. The
resulting foam was purified by silica gel chromatography using
15-25% acetone and 0.1% triethylamine in methylene chloride
to yield 110 mg of 4 (0.114 mmol; 47%) as a white foam. 31P
NMR (CDCl3) δ: 148.0. HR-MS (M þ Naþ) for C52H61N6O9P-
Na: expected 967.4129, found 967.4125.
Conclusion
CdG was incorporated into oligonucleotides, and its base
pairing, as compared to dG, OdG, and SdG, was studied
through both experimental and computational analysis.
These findings shed additional light on the reasons for the
promiscuous base pairing, and thus promutegenic character,
of OdG and are consistent with current theories that suggest
the steric bulk of the C8-oxygen of OdG contributes to the
destabilization of OdG:dC base pairs. Additionally, the
results suggest OdG:dA base pairs are stabilized not only
by the presence of an N7-hydrogen but possibly also by the
presence of a third, albeit long-range, electrostatic interac-
tion between the C8-oxygen of OdG and the C2-hydrogen
of dA.
Experimental Section
General Methods. Methylene chloride used in chromatogra-
phy was passed through alumina (Active Basic, Activity I), and
Dowex (50 ꢀ 4-400) was washed with methanol prior to use.
MALDI-TOF and HR-ESI analyses were performed at the
University of California-Riverside Mass Spectrometry Facility.
Preparative and analytical HPLC were performed using a
semiprep C18 column (10 ꢀ 250 mm) run at 3 mL/min and an
analytical C18 column (4.6 ꢀ 250 mm) run at 1 mL/min, res-
pectively. HPLC solvents A and B were 0.1 M triethylammo-
nium acetate (TEAAC) pH 7 and acetonitrile, respectively.
˚
Merck silica gel, 200-400 mesh, 60 A was used for column
chromatography.
1-N-Benzyl-2-N-isopropylphenoxyacetal-9-deaza-20-deoxygua-
nosine (2). A 340 mg (0.52 mmol) portion of 115 was coevaporated
three times with pyridine to remove any associated water before
being covered with argon and put on ice. Dry pyridine (5 mL) was
added before 108 μL (0.57 mmol) of isopropylphenoxyacetyl
chloride was added dropwise over 5 min while stirring. The
reaction was then removed from the ice and stirred for 1.5 h
before an additional 50 μL of isopropylphenoxyacetyl chloride
was added, and the reaction stirred for 30 min longer at room
temperature. The solution was concentration in vacuo and used
directly in the next reaction without purification. To the resulting
oil, 12 mL of 65/35 pyridine/methanol and 440 μL of 25% sodium
methoxide in methanol were added. The reaction was stirred for
1.5 h, quenched with Dowex, filtered, concentrated, and purified
by silica gel chromatography using 3-5% methanol in chloro-
form to yield 220 mg (0.41 mmol; 79%) of 2 as a yellow foam.
Oligonucleotide Synthesis. All standard DNA was purchased
from IDT Technologies. Synthesis of 5 was performed at the
University of Virginia Biomolecular Research Facility using all
standard procedures. It was then deprotected and cleaved from
the column using 29.7% ammonium hydroxide incubated at
room temperature for 18 h. MALDI-TOF for 5 (MHþ): ex-
pected 3236, found 3236.
DNA Purification. All oligonucleotides were purified by 20%
denaturing PAGE before UV visualization. The slowest running
5668 J. Org. Chem. Vol. 75, No. 16, 2010

Hamm et al.
JOCArticle
band was excised and soaked twice in water, in the dark, for 24 h.
The resulting solutions were then concentrated, combined by
resuspension in 1 mL of water, and filtered. The oligonucleo-
tides were then further purified by preparative HPLC using a
linear gradient of 5-20% solvent B in A over 30 min.
Nuclease Digestion. Compound 5 (0.2 OD260) was incubated
for 16 h at 37 °C with 12 μg units snake venom phosphodiesterase
(Crotalus adamanteus), 2 units bacterial alkaline phosphatase,
32 mM Tris pH 7.5, and 15 mM MgCl2 in a final volume of
80 μL. When the reaction was complete, 10 μL of 3 M sodium
acetate pH 7 and 250 μL of ethanol were added, and the solution
incubated for 30 min at -78 °C before centrifugation for 20 min
at 12,000 rpm. The supernatant was removed, and the remaining
solid was dried in vacuo and resuspended in 150 μL of water
before analysis by analytical HPLC using a linear gradient of
5-6.5% B in A over 20 min.
rate of 0.5 °C/min. The resulting melting curves were analyzed
by least-squares fitting to ΔH° and Tm for a two state stable
transition.
Computational Details. Geometry optimizations of base pairs
and the corresponding monomers were performed using the
B3LYP density functional with the 6-31G* basis set as imple-
mented in the Gaussian0346 program. Solvent effects were
included at every step of the geometry optimization using the
polarizable continuum model (PCM)19 for water. This approach
has been shown to adequately describe similar systems.29 Har-
monic frequency analysis was used to confirm all structures as
minima on the B3LYP/6-31G* (PCM) potential energy sur-
faces. All calculations were performed on nucleoside structures
containing deoxyribose sugars with and without 40 CH3-O-
CH2- and 30 CH3-O- linkages intended to mimic the steric
bulk of the phosphodiester chain as shown in the representative
dG structure in Figure 2. Base pair interaction energies were
determined using the formula Eint = E(base pair) - E(nucleo-
side 1) - E(nucleoside 2) where a negative Eint indicates a favo-
rable interaction. Interaction energies were not corrected for
zero point vibration.
Melting Studies. Each oligonucleotide at 5 μM, 1 M NaCl, 0.1
mM EDTA, and 100 mM phosphate buffer pH 7 in a total
volume of 1 mL was heated for 5 min at 90 °C. The solution was
then allowed to cool at room temperature for at least 30 min and
at 4 °C for at least 30 min. The absorbance at 260 nm was
monitored from 20-80 °C with the temperature increased at a
Acknowledgment. The authors thank Bojan Dragulev and
ꢀ
(46) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.;
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Rene Kanters for assistance with DNA synthesis and pKa
determination, respectively. The work was supported by the
NSF CAREER (Grant CHE-0239664), NSF RUI (Grant
CHE-0809462), and Henry Dreyfus Teacher Scholar Award
(M.L.H. and C.A.P.) programs, as well as the HHMI
Foundation and the Floyd D. and Elisabeth S. Gottwald
Endowment. Support is also acknowledged from the Donors
of the American Chemical Society Petroleum Research Fund.
1
Supporting Information Available: pKa determination; H
and 13C NMR; computational results (geometries, energies,
dipoles). This material is available free of charge via the Internet
at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 16, 2010 5669