Concerning the hydrolytic stability of 8-aryl-2′-deoxyguanosine nucleoside adducts: Implications for abasic site formation at physiological pH

Article abstract of DOI:10.1021/jo901080w

(Chemical Equation Presented) Direct addition of aryl radical species to the C⁸-site of 2′-deoxyguanosine (dG) affords C ⁸-aryl-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons (PAHs), and

Full text of DOI:10.1021/jo901080w

pubs.acs.org/joc
Concerning the Hydrolytic Stability of 8-Aryl-20-deoxyguanosine
Nucleoside Adducts: Implications for Abasic Site Formation at
Physiological pH
Katherine M. Schlitt,† Ke-wen M. Sun,† Robert J. Paugh,† Andrea L. Millen,‡
Lex Navarro-Whyte,‡ Stacey D. Wetmore,*,‡ and Richard A. Manderville*,†
†Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and ‡Department of
Chemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4
rmanderv@uoguelph.ca; stacey.wetmore@uleth.ca
Received May 22, 2009
Direct addition of aryl radical species to the C8-site of 20-deoxyguanosine (dG) affords C8-aryl-dG adducts
that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons (PAHs), and certain
phenolic toxins. A common property of C8-arylpurine adduction is the accompaniment of abasic site
formation. To determine how the C8-aryl moiety contributes to sugar loss, UV-vis spectroscopy has been
employed to determine N7 pKa1 values and hydrolysis kinetics, while density functional theory (DFT)
calculations have been utilized to probe the structural features and stability of the C8-aryl-dG adducts
bearing different para and ortho substituents. In all cases, the C8-aryl-dG adducts adopt a syn conformation
containing a strong O -H N3 hydrogen bond with the aryl ring twisted with respect to the nucleobase.
5
0
3 3 3
The adducts undergo N7-protonation with ionization constants and calculated N7 proton affinity (PA)
values similar to those measured for dG. The hydrolysis kinetics shows that C8-aryl-dG nucleoside adducts
are more prone than dG to acid-catalyzed hydrolysis, with those bearing para substituents having k1 values
that are ca. 90- to 200-fold larger than k1 for dG, while the effects for the ortho adducts are only ca. 9- to 60-
fold larger. Changes in the rate of hydrolysis are further explained by calculations showing that glycosidic
bond cleavage in the syn orientation of both neutral and N7-protonated dG has a lower barrier than the anti
orientation, and the bulky (phenyl) group further decreases the barrier. Despite adduct reactivity in acidic
media, all adducts are relatively stable at physiological pH with t1/2 ∼25 days, suggesting that they are
unlikely intermediates leading to abasic site formation at physiological pH. This information has allowed
development of a new rationale for the tendency of abasic site formation to accompany C8-arylpurine
adduction within duplex DNA at neutral pH.
Introduction
adducts.2,3 Modified nucleobases are also used as bioprobes,4
therapeutics,5 or designer molecules such as fleximers.6 Our
DNA modification is an early event in the carcinogenic
process,1 and modified nucleosides serve as biomarkers for
exposure to chemical carcinogens that form covalent DNA
(4) (a) Kool, E. T. Acc. Chem. Res. 2002, 35, 936–943. (b) Lynch, S. R.;
Liu, H. B.; Gao, J. M.; Kool, E. T. J. Am. Chem. Soc. 2006, 128, 14704–
14711. (c) Ogawa, A. K.; Abou-Zied, O. K.; Tsui, V.; Jimenez, R.; Case, D.
A.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 9917–9920.
(5) (a) Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Lancet Oncol.
2002, 3, 415–424. (b) Sigmond, J.; Peters, G. J. Nucleosides Nucleotides
Nucleic Acids 2005, 24, 1997–2022.
(6) (a) Polak, M.; Seley, K. L.; Plavec, J. J. Am. Chem. Soc. 2004, 126,
8156–8166. (b) Bardon, A. B.; Wetmore, S. D. J. Phys. Chem. A 2005, 109,
262–272.
(1) Josephy, P. D.; Mannervik, B. Molecular Toxicology, 2nd ed.; Oxford
University Press, Inc.: New York, 2006; pp 46-82.
(2) Hoffmann, G. R.; Fuchs, R. P. P. Chem. Res. Toxicol. 1997, 10, 347–
359.
(3) Stover, J. S.; Ciobanu, M.; Cliffel, D. E.; Rizzo, C. J. J. Am. Chem.
Soc. 2007, 129, 2074–2081.
DOI: 10.1021/jo901080w
r
Published on Web 07/16/2009
J. Org. Chem. 2009, 74, 5793–5802 5793
2009 American Chemical Society

Schlitt et al.
JOCFeatured Article
interest in modified nucleosides stems from research on DNA
adduction by phenolic toxins that undergo metabolic activa-
tion to form phenoxyl radical intermediates that can attach
covalently to the C8-site of 20-deoxyguanosine (dG).7 Due to
the ambident (O- vs C-attack) reactivity of phenoxyl radicals
both O- and C-linked adducts are produced, as exemplified by
O-PCP-8-dG (Figure 1) derived from pentachlorophenol
(PCP)8 and C-OTA-8-dG derived from the mycotoxin ochra-
toxin A (OTA).9 Other phenolic C-adducts shown in Figure 1
that are likely formed from phenoxyl radical intermediates
include those derived from nickel(II)-salen complexes, such
as the metallopeptide-PNA bioconjugate (C-Ni(II)salen-8-
dG)10 that have been isolated and characterized by the
Burrows laboratory. The hydroquinone adduct C-(3,4-EQ)-
8-dG is formed from reductive activation of 3,4-estrone
quinone (3,4-EQ) that yields semiquinone radical intermedi-
ates.11 These phenolic C-adducts are structurally related to a
familyof C8-aryladducts. Examples include6-BP-8-dG thatis
formed from the polycyclic aromatic hydrocarbon (PAH)
benzo[a]pyrene (BP)12 and N-Ac-ABA-8-dG that is derived
from the powerful PAH mutagen 3-nitrobenzanthrone
(NBA).13 Oxidation of PAHs by CYP peroxidase to yield
PAH radical cations facilitates C8-aryl adduct formation.14
Carcinogenic arylhydrazines that produce aryl radical inter-
mediates also form C8-aryl adducts that contain phenyl (Ph)
groups bearing various para (p) substituents (i.e., X-Ph-8-dG,
X = COOH, CH2OCH3, CH2OH, Figure 1).15
6-BP-8-dG from the enzymatic digest of the precipitated
DNA, while the supernatant contained roughly the same
concentration of the corresponding 6-BP-8-G adduct.12a
Significant levels of abasic site formation has also been
reported for arylhydrazine treatment of DNA.15e Akanni
and Abul-Hajj proposed that C-(3,4-EQ)-8-dG (Figure 1) is
formed as an intermediate prior to the loss of the deoxyribose
sugar to afford C-(3,4-EQ)-8-G.11c Overall, these results
have led to proposals that formation of abasic sites following
C8-aryl adduct formation may contribute to the carcinogeni-
city of PAHs14 and arylhydrazines.15e
For unmodified dG it is well-known that development of a
positive charge at N7, either through protonation16 (pKa for
protonated dG is 2.3417) or alkylation,18 accelerates the rate
of hydrolysis. Acid-catalyzed hydrolysis of dG proceeds via
an A-1 mechanism involving equilibrium protonation (at N7
for the monocation and N3 for the dication), which precedes
the unimolecular rate-limiting cleavage of the C-N bond.16
19
Substitution of dG with electron-withdrawing NO2 and
20
SO2CH3 C8-substituents greatly accelerates hydrolysis,
while electron-donating NH221 and OCH322 C8-substituents
decreases the rate of hydrolysis. Interestingly, bulky elec-
tron-donating arylamino23 and dimethylamino21 C8-substit-
uents accelerate hydrolysis compared to the unmodified base
despite their electron-donating character. This has been
ascribed to release of steric strain upon removal of the
deoxyribose moiety.23,24
A common property of C8-aryl adduction is the accom-
paniment of abasic site formation in studies carried out at
physiological pH. Treatment of DNA with BP in aqueous
media with horseradish peroxidase (HRP)/HOOH afforded
Because the loss of sugar from C8-aryl adducts at neutral
pH was unexpected,12,15 we sought to determine rates of
hydrolysis for the C8-Ph-dG adducts 1-3c (Figure 1) that
bear para (p)-(2a-2e) and ortho (o)-substituents (3a-3c) of
varying electronic and steric properties. These adducts are
readily prepared using palladium-catalyzed Suzuki cross-
coupling reactions25 and include the known arylhydrazine-
derived phenyl adduct 1,15 the isomeric C-phenolic adducts
2a and 3a formed from reactions of DNA with mutagenic
diazoquinones,26 and others serving as structural models for
C8-aryl adducts in general. To determine how the C8-aryl
moiety contributes to sugar loss for adducts 1-3c, we have
measured rates of hydrolysis in aqueous solutions of varying
acidity and employed density functional theory (DFT) cal-
culations to assist interpretation of the kinetic experiments.
Our observations have led to the development of a new
rationale for the noted tendency of abasic site formation to
(7) (a) McLaughlin, C. K.; Lantero, D. R.; Manderville, R. A. J. Phys.
Chem. A. 2006, 110, 6224–6230. (b) Sun, K. M.; McLaughlin, C. K.; Lantero,
D. R.; Manderville, R. A. J. Am. Chem. Soc. 2007, 129, 1894–1895. (c)
Weishar, J. L.; McLaughlin, C. K.; Baker, M.; Gabryelski, W.; Manderville,
R. A. Org. Lett. 2008, 10, 1839–1842.
(8) (a) Dai, J.; Wright, M. W.; Manderville, R. A. Chem. Res. Toxicol.
2003, 16, 817–821. (b) Dai, J.; Sloat, A. L.; Wright, M. W.; Manderville, R. A.
Chem. Res. Toxicol. 2005, 18, 771–779.
(9) (a) Dai, J.; Wright, M. W.; Manderville, R. A. J. Am. Chem. Soc. 2003,
125, 3716–3717. (b) Dai, J.; Park, G.; Perry, J. L.; Il’ichev, Y. V.; Bow, D. A.
J.; Pritchard, J. B.; Pfohl-Leszkowicz, A.; Manderville, R. A.; Simon, J. D.
Acc. Chem. Res. 2004, 37, 874–881. (c) Faucet, V.; Pfohl-Leszkowicz, A.;
Dai, J.; Castegnaro, M.; Manderville, R. A. Chem. Res. Toxicol. 2004, 17,
1289–1296.
(10) (a) Stemmler, A. J.; Burrows, C. J. J. Am. Chem. Soc. 1999, 121,
6956–6957. (b) Kornyushyna, O.; Stemmler, A. J.; Graybosch, D. M.;
Bergenthal, I.; Burrows, C, J. Bioconjugate Chem. 2005, 16, 178–183.
(11) (a) Abul-Hajj, Y. J.; Tabakovic, K.; Tabakovic, I. J. Am. Chem. Soc.
1995, 117, 6144–6145. (b) Akanni, A.; Abul-Hajj, Y. J. Chem. Res. Toxicol.
1997, 10, 760–766. (c) Akanni, A.; Abul-Hajj, Y. J. Chem. Res. Toxicol. 1999,
12, 1247–1253.
(16) (a) Zoltewicz, D.; Clark, D. F.; Sharpless, T. W.; Grahe, G. J. Am.
Chem. Soc. 1970, 92, 1741–1750. (b) Hevesi, L.; Wolfson-Davidson, E.;
Nagy, J. B.; Nagy, O. B.; Bruylants, A. J. Am. Chem. Soc. 1972, 94, 4715–
4720. (c) Zoltewicz, J. A.; Clark, D. F. J. Org. Chem. 1972, 37, 1193–1197.
(17) Da Costa, C. P.; Sigel, H. Inorg. Chem. 2003, 42, 3475–3482.
(18) Gates, K. S.; Nooner, T.; Dutta, S. Chem. Res. Toxicol. 2004, 17,
839–856.
(19) (a) Yermilov, V.; Rubio, J.; Ohshima, H. FEBS Lett. 1995, 376, 207–
210. (b) Tretyakova, N. Y.; Burney, S.; Pamir, B.; Wishnok, J. S.; Dedon, P.
C.; Wogan, G. N.; Tannenbaum, S. R. Mutat. Res. 2000, 447, 287–303.
(12) (a) Rogan, E. G.; Cavalieri, E. L.; Tibbels, S. R.; Cremonesi, P.;
Warner, C. D.; Nagel, D. L.; Tomer, K. B.; Cerny, R. L.; Gross, M. L. J. Am.
Chem. Soc. 1988, 110, 4023–4029. (b) Cavalieri, E. L.; Rogan, E. G.; Li, K.-
M.; Todorovic, R.; Ariese, F.; Jankowiak, R.; Grubor, N.; Small, G. J. Chem.
Res. Toxicol. 2005, 18, 976–983.
(13) Enya, T.; Kawanishi, M.; Suzuki, H.; Matsui, S.; Hisamatsu, Y.
Chem. Res. Toxicol. 1998, 11, 1460–1467.
ꢀ
(14) (a) Cavalieri, E.; Rogan, E. Environ. Health Persp. 1985, 64, 69–84.
(b) Cavalieri, E. L.; Rogan, E. G. Pharmac. Ther. 1992, 55, 183–199. (c)
Chakravarti, D.; Pelling, J. C.; Cavalieri, E. L.; Rogan, E. G. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 10422–10426.
(15) (a) Lawson, T.; Gannett, P. M.; Yau, W.-M.; Dalal, N. S.; Toth, B. J.
Agric. Food Chem. 1995, 43, 2627–2635. (b) Hiramoto, K.; Kaku, M.;
Sueyoshi, A.; Fujise, M.; Kikugawa, K. Chem. Res. Toxicol. 1995, 8, 356–
362. (c) Gannett, P. M.; Lawson, T.; Miller, M.; Thakkar, D. D.; Lord, J. W.;
Yau, W.-M.; Toth, B. Chem.-Biol. Interact 1996, 101, 1–25. (d) Kohda, K.;
Tsunomoto, H.; Kasamatsu, T.; Sawamura, F.; Terashima, I.; Shibutani, S.
Chem. Res. Toxicol. 1997, 10, 1351–1358. (e) Gannett, P. M.; Powell, J. H.;
Rao, R.; Shi, X.; Lawson, T.; Kolar, C.; Toth, B. Chem. Res. Toxicol. 1999,
12, 297–304.
(20) Laayoun, A.; Decout, J.-L.; Lhomme, J. Tetrahedron Lett. 1994, 35,
4989–4990.
(21) Jordan, F.; Niv, H. Nucleic Acids Res. 1982, 10, 4339–4349.
(22) Garrett, E. R.; Mehta, P. J. J. Am. Chem. Soc. 1972, 94, 8532–8541.
(23) Novak, M.; Ruenz, M.; Kazerani, S.; Toth, K.; Nguyen, T.-M.;
Heinrich, B. J. Org. Chem. 2002, 67, 2303–2308.
€
(24) Hovinen, J.; Glemarec, C.; Sandstrom, A.; Sund, C.; Chattopad-
hyaya, J. Tetrahedron 1991, 47, 4693–4708.
(25) (a) Western, E. C.; Daft, J. R.; Johnson, E. M. II; Gannett, P. M.;
Shaughnessy, K. H. J. Org. Chem. 2003, 68, 6767–6774. (b) Western, E. C.;
Shaughnessy, K. H. J. Org. Chem. 2005, 70, 6378–6388. (c) Dai, Q.; Xu, D.;
Lim, K.; Harvey, R. G. J. Org. Chem. 2007, 72, 4856–4863.
(26) Kikugawa, K.; Kato, T.; Kojima, K. Mutat. Res. 1992, 268, 65–75.
5794 J. Org. Chem. Vol. 74, No. 16, 2009

Schlitt et al.
JOCFeatured Article
FIGURE 1. Examples of C8-aryl adducts and the structure, atomic numbering, and identification of dihedral angles χ (—(OC10N9C4)) and θ
(—(C11C10C8N9)) for C8-aryl adducts 1-3c.
accompany C8-aryl adduction in duplex DNA at physiolo-
gical pH.
Results and Discussion
Structural Features of C8-Aryl Adducts. Insight into the
structural features of the C8-aryl adducts was obtained from
DFT calculations, as previously presented for the phenolic
adducts 2a (R1 = OH) and 3a (R2 = OH).27 Conformations
for the other adducts were identified by reoptimizing the
previously identified minima for the isomeric phenolic ad-
ducts with the OH substituent replaced with each R1 or R2
group. Representative DFT structures for an anti, syn and
deglyco adduct are shown in Figure 2 for 3a (R2 = OH) and
deglyco3a with N9H. As noted for 2a and 3a,27 anti struc-
FIGURE 2. Global anti,syn and deglyco minima for 3a fully opti-
tures are less stable than syn structures by ∼25 kJ mol-1 for
mized with B3LYP/6-31G(d), highlighting twist angles (θ, deg) and
all C8-aryl-dG adducts 1-3c. This preference is consistent
with the known structural preference of C8-p-NMe2-Ph-
guanosine, which adopts a syn conformation in both the
solid state and solution.28 For 1-3c, all syn minima contain a
hydrogen-bonding interactions (dashed red lines). The syn mini-
.
mum is favored over anti by ∼25 kJ mol-1
For adducts 1-3b, the neutral nucleobase global minima
are planar (θ=0ꢀ), suggesting that the sugar moiety is
inducing the twist within the nucleobase.27 It is also impor-
tant to point out that the barrier to rotation is greater for the
deglyco adducts than the nucleoside adducts, implying that
the sugar also increases conformational flexibility.27 The
lone exception to this trend is adduct 3c (R2=CH3), where
deglyco3c remains significantly twisted (θ=24ꢀ).
Absorbance spectra for adducts 1-3c were initially re-
corded in aqueous pH 4 buffer (0.05 M citrate) at room
temperature. Under these conditions, adducts were relatively
stable, and distinct absorbance changes were noted upon
removal of the deoxyribose sugar moiety. The spectrum
recorded for 3a (Figure 3a, spectrum i) was representative
of spectra for adducts 1, 2a-2c, and 3b, which all showed a
5
3
0
˚
strong O -H N hydrogen bond (1.80-1.96 A), and the
3 3 3
aryl substituent is twisted with respect to the nucleobase,
where the magnitude of the twist angle (θ) depends on steric
considerations and favorable intramolecular interactions.
For example, the global minima for p-adducts 1-2e
are twisted by ∼37ꢀ, while θ increases to 45ꢀ for 3c (R2 =
CH3) and 55ꢀ for 3b (R2 = OCH3) due to steric interactions
between R2 and the nucleobase. In contrast, the 3a (R2 =
OH) global minimum is less twisted (θ = 27ꢀ) than any other
substituent due to O-H N7 hydrogen bonding.27
3 3 3
(27) Millen, A. L.; McLaughlin, C. K.; Sun, K. M.; Manderville, R. A.;
Wetmore, S. D. J. Phys. Chem. A 2008, 112, 3742–3753.
(28) Sessler, J. L.; Sathiosatham, M.; Doerr, K.; Lynch, V.; Abboud, K.
A. Angew. Chem., Int. Ed. 2000, 39, 1300–1303.
J. Org. Chem. Vol. 74, No. 16, 2009 5795

Schlitt et al.
JOCFeatured Article
TABLE 1. UV-vis Parameters and Structural Featuresa for Adducts
1-3c and Their Deglyco Analogues
adduct
1 (R1 = H)
deglyco1b
λ
max, log εc
θ (deg)
C8-C10 (A)
˚
277, 4.33
305, 4.28
277, 4.36
309, 4.35
276, 4.34
310, 4.45
277, 4.38
308, 4.15
311, 4.09
334, 3.92
323, 4.12
350, 4.07
275, 4.10
318, 3.95
278, 4.25
319, 4.04
261, 3.78
285, 3.70
252.7, 4.14d
37.5
0.6
37.0
0.6
37.4
0.6
37.2
0.2
38.0
0.1
38.1
0.5
27.0
0.0
55.3
0.0
1.473
1.464
1.471
1.463
1.471
1.463
1.472
1.463
1.470
1.460
1.471
1.460
1.464
1.455
1.477
1.466
1.477
1.468
2a (R1 = OH)
deglyco2a
2b (R1 = OCH3)
deglyco2b
2c (R1 = CH3)
deglyco2c
2d (R1 = CN)
deglyco2d
2e (R1 = CHO)
deglyco2e
3a (R2 = OH)
deglyco3a
3b (R2 = OCH3)
deglyco3b
3c (R2 = CH3)
deglyco3c
dG
45.3
24.0
aStructures were fully optimized with B3LYP/6-31G(d), and relative
energies were obtained from B3LYP/6-311+G(2df,p) single-point cal-
culations. bCalculations for deglyco adducts with N9H. cDetermined in
0.05 M citrate buffer pH 4, μ = 0.31 M NaCl. dTaken from ref 29.
FIGURE 3. Absorbance spectra for a 50 μM solution of (a) (i) 3a
and (ii) deglyco3a, (b) (i) 2e and (ii) deglyco2e, and (c) (i) 3c and (ii)
deglyco3c in 0.05 M citrate buffer, pH 4, μ = 0.31 M NaCl.
single broad peak at approximately 280 nm that exhibits a
red shift compared to λmax for dG at ∼253 nm.29 The adduct
deglyco3a (Figure 3a, spectrum ii) showed two maxima at
290 and 318 nm, with the 318 nm absorbance being consis-
tent with a planar deglyco3a structure with increased con-
jugation. The p-adducts 2d (R1=CN) and 2e (R1=CHO)
bearing electron-withdrawing groups showed similar fea-
tures. In pH 4 buffer, adduct 2e showed two maxima at
282 and 323 nm (Figure 3b, spectrum i), while deglyco2e
showed a single peak at ∼350 nm (Figure 3b, spectrum ii).
For 3c, bearing the o-CH3 substituent (Figure 3b, spectrum
i), a single peak at ∼261 nm was observed of weaker intensity
and blue-shifted compared to all other adduct absorbances.
The deglyco3c adduct (Figure 3b, spectrum ii) showed a
broad absorbance with λmax=285 nm, which was also
significantly blue-shifted compared to the absorbance for
the other deglycosylated adducts. The UV-vis parameters
and structural features of adducts 1-3c are summarized in
Table 1.
FIGURE 4. (a) Overlay spectra for 2a as a function of pH at 20 ꢀC.
(b) Initial absorbance at 320 nm vs pH for 2a.
the spectrophotometric procedure at 20 ꢀC, as outlined
previously for C8-(arylamino)-dG adducts.23 Figure 4a
shows overlay absorbance spectra for adduct 2a (R1=
OH) as a function of pH at 20 ꢀC, while shown in
Figure 4b is a plot of initial absorbance at 320 nm vs pH
from the absorbance data shown in Figure 4a. The spectro-
photometrically determined pKa1 for 2a (2.43 ( 0.12) is in the
range expected for pKa1 of dG; for example, a recent pKa1
Proton Affinity. Ionization constants for the C8-aryl-dG
adducts in the low pH region (1-4) were determined using
(29) Onidas, D.; Markovitsi, D.; Marguet, S.; Sharonov, A.; Gustavsson,
T. J. Phys. Chem. B 2002, 106, 11367–11374.
5796 J. Org. Chem. Vol. 74, No. 16, 2009

Schlitt et al.
JOCFeatured Article
TABLE 2. Gas-Phase Proton Affinities (PA), Ionization Constants, and Structural Featuresa for Adducts 1-3c Protonated at N7 (N7H+
)
adduct
PAb
pKa
θ (N7H+
)
Δθ g
C8-C10 (N7H+
)
Δ C8-C10
h
1 (R1 = H)
230.0
231.7
233.1
231.8
222.7
2.41 ( 0.04d
2.43 ( 0.12d
2.48 ( 0.06d
2.41 ( 0.12d
2.00e
42.3
42.8
42.1
40.7
42.0
42.3
28.3
28.8
68.2
4.8
5.8
4.7
3.5
4.0
4.2
1.3
-26.5
22.9
1.462
1.454
1.453
1.459
1.464
1.464
1.457
1.456
1.471
-0.011
-0.017
-0.018
-0.013
-0.006
-0.007
-0.007
-0.021
-0.006
2a (R1 = OH)
2b (R1 = OCH3)
2c (R1 = CH3)
2d (R1 = CN)
2e (R1 = CHO)
3a (R2 = OH)
3b (R2 = OCH3)
3c (R2 = CH3)
dG
225.2
226.9
2.12e
2.21 ( 0.17d
2.57 ( 0.26d
2.40 ( 0.22d
2.34f
234.1
231.7
232.0 (234.4)c
aStructures were fully optimized with B3LYP/6-31G(d) (relative energies from B3LYP/6-311+G(2df,p) single-point calculations). bN7 proton
affinity (PA) in the gas phase calculated as the negative of the enthalpy change for protonation (kcal mol-1). cExperimental PA for dG taken from ref 30.
dObtained from spectrophotometric titration at 20 ꢀC. eEstimated from gas-phase N7 PA and experimentally determined pKa values. fTaken from ref 17.
gΔθ = θ(N7H+) - θ(neutral adduct). hΔC8-C10 = C8-C10 (N7-H+) - C8-C10 (neutral adduct).
value for N7-protonated dG is 2.34.17 The pKa1 value for 2a
was similar to the values determined for the other p-sub-
stituted adducts 1-2c, which all exhibited a red shift upon
protonation and gave a narrow pKa1 range (2.41-2.48), as
reported in Table 2. Variation in pKa1 was observed for o-
substituted analogues 3a (R2 = OH) and 3b (R2 = OCH3).
Compared to the p-substituted analogues, 3a exhibited a
decrease in pKa1 (2.21 ( 0.17), while 3b showed an increase
(2.57 ( 0.26).
Also reported in Table 2 are the calculated gas-phase
proton affinity (PA) values for the most basic N7-site of
adducts 1-3c. For 1, the N7 PA is 230.0 kcal mol-1, which
is similar to the calculated N7 PA for dG (232 kcal mol-1) as
well as the experimental PA for dG (234.4 kcal mol-1).30 The
N7 PA values for 1-3c lie within a narrow range (222.7-234.1
kcal mol-1) with the lowest value for 2d (R1=CN) and
the highest value for 3b (R2=OCH3). A plot of N7 PA vs
spectrophotometrically determined pKa1 values affords a
straight line, from which pKa1 values for 2d and 2e were
estimated. Even though the data reported in Table 2 support
protonation at N7, it has been speculated that C8-substituents
may differentially stabilize protonation at N3 rather than
N7.23 However, this seems unlikely given that N7-protonation
is much more favorable energetically than N3-protonation for
FIGURE 5. Hammett plot for adducts 1-2e using σp vs log k/ko in
0.1 M HCl at 37.2 ꢀC.
tions involving the phenolic group in both the neutral and
protonated adduct. The relatively low pKa1 value for 3a
suggests preferential hydrogen bonding in the neutral adduct,
with a concomitant decrease in N7 PA.30 In contrast, adduct
3b (R2=OCH3) becomes much less twisted (by 27.2ꢀ) upon
N7-protonation and has the same degree of twist as neutral 3a
due to N7-H O-CH3 hydrogen-bonding. This preferen-
dG (PA for N3 of dG is 220.1 kcal mol-1 31
and that
)
3 3 3
tial stabilization of the N7-protonated adduct for 3b is also
reflected in the relatively large decrease in the C8-C10 bond
length and the increase in pKa1. For 3c (R2=CH3), the N7-
protonated adduct is significantly more twisted (22.9ꢀ) than
the neutral species due to increased sterics. Overall, the
calculations and spectrophotometrically determined pKa1
values support N7-protonation for C8-aryl-dG adducts 1-3c.
Rates of Hydrolysis. First-order rate constants were de-
termined spectrophotometrically by monitoring formation
of the deglycosylated product at its absorbance maximum.
Apparent first-order rate constants, k (min-1), and half-
lives, t1/2 (min), were first determined for the hydrolysis of
adducts 1-3c in 0.1 M HCl at 37.2 ꢀC and 0.05 M pH 4
citrate buffer at 48.4 ꢀC, with ionic strength maintained at
μ = 0.31 M NaCl. These particular conditions were chosen
in order to draw direct comparison to hydrolysis rates of
dG.16b Figure 5 shows a plot of log k/ko versus Hammett σp
for hydrolysis of p-adducts 1-2e in 0.1 M HCl at 37.2 ꢀC. A
linear correlation with a small positive slope (F) of 0.543 was
observed, implying an increase in rate by electron-withdrawing
groups and that the hydrolysis is less sensitive than benzoic
acid dissociation to the electronic nature of the p-substituent.
protonation of 8-oxoguanine occurs at N3 with pKa1 of 0.22.32
Table 2 also reports changes in the twist (θ) angle and C8-
C
10 bond length upon N7-protonation. For the p-substituted
adducts 1-2e, the optimized protonated structures were not
greatly altered from the neutral structures. The global mini-
5
0
ma are in the same syn conformation with a strong O -
H
N3 hydrogen bond, and the anti structures are higher in
3 3 3
energy (by ∼19 kJ mol-1). The N7-protonated p-adducts are
more twisted than the neutral species (by about 3-6ꢀ) due to
increased steric interactions between the aryl ring and the
protonated nucleobase. Despite the increase in twist angle
(θ), the N7-protonated nucleosides have shorter C8-C10
bonds, especially when R1 is electron-donating (OH, OCH3).
The o-substituted adducts 3a-c behave differently upon N7-
protonation. Adduct 3a (R2 = OH) shows only a small 1.3ꢀ
increase in θ due to O-H N7 hydrogen-bonding interac-
3 3 3
(30) Greco, F.; Liguori, A.; Sindona, G.; Uccella, N. J. Am. Chem. Soc.
1990, 112, 9092–9096.
(31) Xia, F.; Xie, H.; Cao, Z. Internat. J. Quantum Chem. 2008, 108, 57–
65.
(32) Jang, Y. H.; Goddard, W. A. III; Noyes, K. T.; Sowers, L. C.;
Hwang, S.; Chung, D. S. Chem. Res. Toxicol. 2002, 15, 1023–1035.
J. Org. Chem. Vol. 74, No. 16, 2009 5797

Schlitt et al.
JOCFeatured Article
TABLE 3. Summary of First-Order Rate Constants, k (min-1), and Half-Lives, t1/2 (min), for Hydrolysis of Adducts 1-3c in HCl and pH 4 Buffer at 37
and 48 ꢀC
adduct
kobs(HCl), t1/2
kobs/kobs (dG)
kobs (pH 4), t1/2
k1 (pH 4)d
k1/k1 (dG)
Δθe
ΔC8-C10
a
c
f
1 (R1 = H)
0.790 ( 0.008, 0.877
0.478 ( 0.009, 1.45
0.611 ( 0.006, 1.13
0.651 ( 0.009, 1.06
1.78 ( 0.02, 0.389
1.41 ( 0.01, 0.491
0.282 ( 0.001, 2.46
0.291 ( 0.003, 2.38
0.196 ( 0.002, 3.45
0.0391, 17.7b
20.2
12.2
15.6
16.6
45.5
36.1
7.2
(2.61 ( 0.08) ꢀ 10-2, 26.6
(3.1 ( 0.1) ꢀ 10-2, 22
(2.95 ( 0.09) ꢀ 10-2, 23.5
(2.84 ( 0.08) ꢀ 10-2, 24.4
(2.3 ( 0.1) ꢀ 10-2, 30
(2.38 ( 0.07) ꢀ 10-2, 29.1
(1.06 ( 0.02) ꢀ 10-2, 65.4
(7.4 ( 0.2) ꢀ 10-3, 94
(2.6 ( 0.2) ꢀ 10-3, 270
2.44 ꢀ 10-4, 2840b
1.015
1.152
0.977
1.105
2.300
1.805
0.654
0.199
0.103
0.011
92.3
104.7
88.8
100.5
209.1
164.1
59.5
-42.3
-42.6
-42.0
-39.8
-42.0
-42.1
-28.3
-28.8
-39.2
0.002
0.009
0.010
2a (R1 = OH)
2b (R1 = OCH3)
2c (R1 = CH3)
2d (R1 = CN)
2e (R1 = CHO)
3a (R2 = OH)
3b (R2 = OCH3)
3c (R2 = CH3)
dG
0.004
-0.004
-0.004
-0.002
0.010
7.4
5.0
18.1
9.4
-0.003
aDetermined in 0.1 M HCl at 37.2 ꢀC. bTaken from ref 16b. cDetermined in 0.05 M citrate buffer pH 4, μ=0.31 M NaCl at 48.4 ꢀC. dk1
≈
kobsKa1/[Hþ]. eΔθ = θ (N7Hþ adduct) - θ (neutral nucleobase with N7H). fΔ C8-C10 = C8-C10 (N7Hþ adduct) - C8-C10 (neutral nucleobase).
For comparison to dG, the C8-aryl adduct 8-Ph-dG (1),
which bears no substituent, underwent deglycosylation ∼20
times faster than dG in 0.1 M HCl. This behavior was
contrasted by adducts 3a-c with o-substituents that under-
went hydrolysis at a significantly slower rate than their p-
substituent counterparts in 0.1 M HCl. In particular, 8-o-
CH3Ph-dG (3c) underwent hydrolysis ∼3 times slower than
8-p-CH3Ph-dG (2c) in 0.1 M HCl and only showed a 5-fold
increase in rate compared to dG.
For the rate data obtained at pH 4, the pKa1 values
reported in Table 2 were used to estimate k1 for the rate-
limiting C-N bond cleavage from the N7-protonated ad-
ducts 1-3c. As reported previously for acid-catalyzed hy-
drolysis of dG-16 and C8-substituted dG analogues,23 both
the monoprotonated and diprotonated substrates are subject
to rate-limiting C-N bond cleavage. The pKa2 for the
diprotonated substrate is ca. -2.5,16c and the C8-substituent
is expected to have little effect on Ka2.23 At pH 4, where
involvement of the diprotonated species can be ignored, the
rate expression simplifies to kobs = kHaH, where kH ≈ k1/Ka1.
Thus, at pH 4, k1 ≈ kobsKa1/[H+], and k1 values for 1-3c are
given in Table 3. The C8-aryl substituent clearly increases the
magnitude of k1 relative to k1 for dG. The effects for the p-
adducts 1-2e are ca. 90- to 200-fold larger than k1 for dG,
while the effects for the o-adducts 3a-c are only ca. 9- to 60-
fold larger. For comparison, C8-arylamino substituents in-
crease k1 by ca. 3- to 15-fold,23 which is similar to the rate
enhancement exerted by the least reactive C8-aryl-dG adduct
3c (R2=CH3).
FIGURE 6. Structural changes in the nucleobase upon deglycosy-
lation for 2b, 3b, and 3c.
C8-C10 bond length also shortens, suggesting preferential
stabilization of the neutral nucleobase compared to the
protonated nucleoside. This phenomenon would also be
expected to increase the rate of hydrolysis.
For o-adducts 3a and 3b, the decrease in twist angle upon
sugar removal to form the planar nucleobase is only ca. 28ꢀ
because the N7-protonated nucleoside is relatively planar
due to hydrogen-bonding interactions between the R2 sub-
stituent and N7Hþ. Thus, the relief in strain for 3a and 3b
upon sugar removal is not as great as it is for 1-2e, and
hence, the rate of hydrolysis is diminished. Differentiation
between 3a and 3b is apparent from the C8-C10 bond length
changes, where the bond becomes shorter for 3a and longer
for 3b. This suggests preferential stabilization of the neutral
nucleobase for 3a, and hence, its rate of hydrolysis is greater
than the rate for 3b. For 3c, which bears the o-CH3 sub-
stituent, the change in twist of 39ꢀ suggests considerable
relief of strain. However, in this case, unlike the other C8-
aryl-G adducts, the neutral nucleobase is not planar, but has
a twist angle θ of 29ꢀ (Figure 6). This suggests that the
nucleobase is also sterically hindered and that the relief of
strain for 3c upon sugar removal is diminished due to
hindrance in the free nucleobase.
The observed decrease in the rate of hydrolysis for the o-
adducts 3a-c compared to the p-adducts 1-2e was some-
what surprising given that a bulky substituent at C8 is
expected to increase the rate of hydrolysis due to the relief
of strain in the activated complex in an A-1 mechanism.24
However, a rationale for the decrease in rates of hydrolysis
for 3a-c relative to 1-2e is provided by considering the
changes in the twist angle (θ) and C8-C10 bond length
(Table 3) when going from the N7-protonated nucleoside
adduct to the neutral nucleobase with N7H that lacks the
sugar moiety (Figure 6). It is important to point out that the
twist angle (θ) for the N7H nucleobase intermediate differs
slightly from the N9H nucleobase shown in Figure 2 with
θ values given in Table 1 for the deglyco adducts. For the
p-adducts 1-2e, a decrease in twist angle of ca. 40ꢀ is
observed upon removal of the sugar group from the twisted
N7-protonated species to form the planar nucleobase. For
2d and 2e, which bear electron-withdrawing groups, the
5798 J. Org. Chem. Vol. 74, No. 16, 2009

Schlitt et al.
JOCFeatured Article
TABLE 4. Summary of First-Order Rate Constants, k (min-1), and Half-Lives, t1/2 (min), for Hydrolysis of Adducts 1, 2a, 2e, and 3c at 37 ꢀC
adduct
a
b
b
b
pH 1 k, t1/2
pH 2 k, t1/2
pH 3 k, t1/2
pH 4 k, t1/2
pH 7 k, t1/2 (days)c
1
1.36 ( 0.08, 0.510
0.95 ( 0.02, 0.73
4.6 ( 0.1, 0.15
0.300 ( 0.006, 2.31
0.217 ( 0.004, 3.19
0.504 ( 0.007, 1.36
0.047 ( 0.002, 15
0.0591 ( 0.0005, 11.7
0.0424 ( 0.0005, 16.3
0.046 ( 0.003, 15
0.0041 ( 0.0001, 170
0.0055 ( 0.0001, 125
0.0043 ( 0.0001, 160
0.0004 ( 0.0001, 1730
1.90 ꢀ 10-5, 25
3.80 ꢀ 10-5, 13
4.06 ꢀ 10-6, 118
5.20 ꢀ 10-7, 924
2a
2e
3c
0.37 ( 0.03, 1.9
0.0054 ( 0.0005, 130
aDetermined in 0.05 M phosphate buffer, μ = 0.31 M NaCl. bDetermined in 0.05 M citrate buffer, μ = 0.31 M NaCl. cEstimated rate data based on
first-order dependence on Hþ activity.
C8-Ph group is still observed (green vs orange line in Figure 7).
Interestingly, N7-protonation has a larger (80 kJ mol-1
,
purple vs red line) effect on the barrier for dG deglycosyla-
tion than it does for C8-Ph-dG (50 kJ mol-1, orange vs light
blue line). This diminishes the role of the C8-phenyl sub-
stituent in acidic media where N7 is protonated, although
the calculations correctly predict that N7Hþ 1 still has the
lowest barrier for C-N cleavage overall (by ∼20 kJ mol-1).
This role of pH on the relative rates of deglycosylation
of modified dG bases vs natural dG is noted in the rate
data presented in Table 3 where kobs(1)/kobsdG is 20 in
0.1 M HCl and ∼100 at pH 4. The same phenomenon was
observed for the hydrolysis of C8-arylamine adducts that
exhibited considerably higher rates of hydrolysis than dG
at pH ∼3-7 but became comparable (within a factor of 5)
at pH <2.23
Hydrolysis at Physiological pH. Hydrolysis kinetics be-
tween pH 1-4 for C8-aryl-dG adducts 1, 2a, 2e, and 3c at
37 ꢀC yielded straight lines, as observed for dG16,23 and other
purine nucleosides.33 This has been interpreted to mean that
k1/Ka1 ≈ k2/Ka216,23,33 and that the hydrolysis reaction shows a
continuous first-order dependence on Hþ activity.16 This
permitted extrapolation of the rate data to pH 7 for an estimate
of hydrolysis rates at physiological pH (Table 4). For C8-Ph-
dG (1), a first-order rate constant of 1.90 ꢀ 10-5 min-1, with
t1/2 = 25 days, was determined at pH 7. For comparison, a rate
for spontaneous loss of purines from duplex DNA at pH 7.4 at
37 ꢀC is ∼3 ꢀ 10-11 s-1 (t1/2 = 730 years).34 Thus, while C8-
aryl-dG adducts are significantly more reactive than dG
toward hydrolysis, they are reasonably stable at physiological
pH and should be even more stable within duplex DNA where
purines are more resistant to hydrolysis.18 The hydrolysis
kinetics for C8-aryl-dG adducts 1-3c suggests that these
adducts are unlikely to be intermediates prior to loss of the
deoxyribose sugar and that an alternative explanation ac-
counts for the accompaniment of abasic site formation during
C8-aryl-purine formation at physiological pH.
FIGURE 7. Deglycosylation profile calculated with constrained
PCM-B3LYP/6-31G(d) optimizations for anti (ꢀ , purple) and
syn (4, green) neutral dG, anti (0, red) and syn (), dark blue)
N7Hþ dG, and neutral (O, orange) and N7Hþ (*, light blue) syn-1.
The effects of the structural changes in the modified base
on the rates of deglycosylation can be further broken down
and analyzed through consideration of experimentally de-
termined activation parameters and DFT calculations of the
energetic effects of extending the glycosyl bond (Figure 7).
For dG, previously reported activation parameters deter-
mined in 0.1 M HCl are as follows: ΔHq = 22.5 kcal mol-1
(94.2 kJ mol-1); ΔSq = 4.3 cal mol-1 K-1; and ΔGq = 21.2
kcal mol-1 (88.7 kJ mol-1).16b For comparison, the corre-
sponding parameters for C8-Ph-dG (1) (ΔHq = 19.7 kcal
mol-1 (82.5 kJ mol-1); ΔSq = 4.5 cal mol-1 K-1; ΔGq = 18.4
kcal mol-1 (77.0 kJ mol-1) were determined in 0.1 M HCl at
six different temperatures ranging from 25 to 60 ꢀC to allow
generation of an Eyring plot (data not shown). This indicates
that attachment of the C8-phenyl substituent lowers the
Rationale for Abasic Site Formation. Insight into the cause
of abasic site formation during C8-aryl-purine adduction is
€
provided by recent studies from the Kenttamaa laboratory
C-N cleavage barrier by ∼12 kJ mol-1
.
on reactions of phenyl radicals toward nucleic acids in the
gas phase.35 These studies employ mass spectrometry
coupled with laser-induced acoustic desorption (LIAD) to
examine the reactivity of phenyl radicals with dinucleoside
phosphates in the gas phase. Both H-atom abstraction from
the sugar moiety and direct radical addition to the C8-site of
Dissection of the structural features that account for this
decrease in barrier height is provided by inspection of the
calculated deglycosylation profile for dG vs 1 shown in
Figure 7. By estimating the barriers from the energetic
plateaus, it can be seen that although natural dG prefers
the anti conformation over syn by ∼11 kJ mol-1, the barrier
to deglycosylation of the syn conformer (∼175 kJ mol-1) is
∼20 kJ mol-1 less than the corresponding barrier for anti
(∼195 kJ mol-1). Therefore, the syn orientation induced by
the bulky C8-substituent may contribute to the acceleration
of the hydrolysis rates of C8-aryl adducts. Nevertheless,
when the barrier of neutral syn dG is compared to neutral
C8-Ph-dG (1), a large effect (∼ 40 kJ mol-1) of the bulky
€
(33) Oivanen, M.; Lonnberg, H.; Zhou, X.-X.; Chattopadhyaya, J.
Tetrahedron 1987, 43, 1133–1140.
(34) Lindahl, T.; Karlstrom, O. Biochemistry 1973, 12, 5151–5154.
(35) (a) Ramirez-Arizmendi, L. E.; Heidbrink, J. L.; Guler, L. P.;
€
Kenttamaa, H. I. J. Am. Chem. Soc. 2003, 125, 2272–2281. (b) Liu, J.;
Petzold, C. J.; Ramirez-Arizmendi, L. E.; Perez, J.; Kenttamaa, H. I. J. Am.
€
Chem. Soc. 2005, 127, 12758–12759. (c) Yang, L.; Nash, J. J.; Yurkovich, M.
J.; Jin, Z.; Vinueza, N. R.; Kentt€amaa, H. I. Org. Lett. 2008, 10, 1889–1892.
J. Org. Chem. Vol. 74, No. 16, 2009 5799

Schlitt et al.
JOCFeatured Article
SCHEME 1. Proposed Pathways for the Degradation of the Nucleoside Radical Intermediate Following Phenyl Radical Attachment at
C8-dG
purine bases are observed,35 as noted for the reactivity of
phenyl radicals toward DNA in solution.15,36 Interestingly,
the radical addition reaction at C8 of purines is always
followed by C-N bond cleavage with formation of the
nucleobase lacking the sugar moiety.35 Thus, as outlined in
Scheme 1, direct phenyl radical attachment at C8-dG gen-
erates the resonance-stabilized radical nucleoside intermedi-
ate. In path A, the radical intermediate undergoes homolytic
C-N bond cleavage to eliminate the free base, as observed in
the gas phase for addition of the phenyl radical to C8 of
purines.35 H-atom abstraction to form the stable nucleoside
adduct in path B would be a competitive process to path A.
In the gas phase, path B cannot compete with path A and so
only homolytic C-N bond cleavage is observed.35 However,
path B would be expected to compete with path A in water.
As a model for path B, it is known that the resonance-
stabilized cyclohexadienyl radical undergoes very rapid H-
atom transfer with molecular oxygen with a second order
rate of 1.64 ꢀ 109 M-1 s-1 in benzene and that this reaction
shows essentially no dependence on solvent polarizability,
polarity and ability to hydrogen bond.36 Assuming an oxy-
gen concentration of 1.2 mM for an O2-saturated solution,37
a first-order rate of ∼2 ꢀ 106 s-1 for path B can be estimated.
This suggests a similar rate for the homolytic C-N bond
cleavage in path A given that significant levels of abasic site
formation accompany C8-aryl-purine adduction;12,15the
C10-radical would be expected to form the oxidized abasic
site 2-deoxyribonolactone in the presence of O2 and water.38
Thus, oxygen is likely to promote C8-dG adduct formation in
solution by providing a rapid and productive path for
aromatization of the nucleoside radical intermediate.39 Ex-
periments are currently underway to test this hypothesis.
Conclusions
The current study has allowed us to conclude the following:
(1) C8-aryl-dG nucleoside adducts adopt a syn conformation
containing a strong O -H N3 hydrogen bond with the aryl
5
0
3 3 3
ring twisted with respect to the nucleobase. In general, removal
of the deoxyribose sugar moiety affords a planar nucleobase
adduct that exhibits a red shift compared to λmax for the
nucleoside adduct. These adducts undergo protonation at N7
with ionization constants and calculated N7 proton affinity
(PA) values that are similar to those measured for dG. (2) C8-
aryl-dG nucleoside adducts are more prone than dG to acid-
catalyzed hydrolysis. C8-aryl-dG adducts 1-2e that bear para
substituents have k1 values that are ca. 90- to 200-fold larger
than k1 for dG, while the effects for the ortho adducts 3a-c are
only ca. 9- to 60-fold larger. Relief of steric strain upon
removal of the deoxyribose sugar moiety provides a rationale
for this relative reactivity. Calculated deglycosylation path-
ways show that protonation at N7, the bulky substituent, and
the syn orientation all contribute to the increase in the rate of
C-N glycosidic bond cleavage. (3) Despite the enhanced
reactivity of these bases in acid compared to dG, they are
relatively stable at physiological pH with t1/2 ∼25 days. Given
that abasic site formation is known to accompany C8-aryl-dG
adduct formation, our results suggest that these nucleoside
adducts are unlikely intermediates leading to abasic site for-
mation at physiological pH. Instead, a resonance-stabilized,
radical nucleoside intermediate that forms upon direct radical
attachment at the C8-site of purines, and is a known precursor
to C8-aryl-dG adduction following H-atom abstraction, is the
more likely intermediate leading to the elimination of the
nucleobase by homolytic C-N bond cleavage.
(36) Maillard, B.; Ingold, K. U.; Scaiano, J. C. J. Am. Chem. Soc. 1983,
105, 5095–5099.
(37) Linke, W. F. Solubilities, Inorganic and Metalo Organic Compounds:
A Compilation of Solubility Data from the Periodocal Literature; Van
Nostrand: Princeton, 1958-1965; p 1228.
(38) (a) Chatgilialoglu, C.; Ferreri, C.; Bazzanini, R.; Guerra, M.; Choi,
S.-Y.; Emanuel, C. J.; Horner, J. H.; Newcomb, M. J. Am. Chem. Soc. 2000,
122, 9525–9533. (b) Huang, H.; Greenberg, M. M. J. Org. Chem. 2008, 73,
2695–2703.
(39) Curran, D. P.; Keller, A. I. J. Am. Chem. Soc. 2006, 128, 13706–
13707.
Experimental Section
Suzuki Coupling of 8-Br-dG with Boronic Acids. These
reactions were conducted according to the literature25 and are
briefly described here. Palladium acetate (2.2 mg, 0.01 mmol),
5800 J. Org. Chem. Vol. 74, No. 16, 2009

Schlitt et al.
JOCFeatured Article
tris(2-sulfophenyl)phosphine trisodium salt (TPPTS) (14.8 mg,
0.025 mmol), sodium carbonate (80 mg, 0.75 mmol), 8-Br-dG
(0.375 mmol), and the appropriate boronic acid (0.45 mmol)
were added to deoxygenated 2:1 water:acetonitrile (3.5 mL) and
heated to 80 ꢀC for 4 h under an argon balloon. The reaction was
diluted with ca. 20 mL of water and the pH adjusted to 6-7 with
1 M HCl (aq). The mixture was allowed to cool to 0 ꢀC for
several hours before the product was recovered by vacuum
filtration. Adducts 1,25a 2a-e,25a,7b,7c and 3a,b7a were prepared
as described and NMR spectra were identical to previously
published spectra provided in Supporting Information.7,25a The
corresponding guanine analogues of adducts 1-3c were pre-
pared, as previously outlined,7a,27 by placing the adduct in ca.
20 mL of 10% formic acid under heat (75 ꢀC) for 1 h. After
cooling, the reaction mixtures were brought to pH 6 with 1 M
NaOH, and products were recovered by crystallization and
filtering from the aqueous media. The deglycosylated adducts
were used as standards for spectroscopic measurements.
8-(200-Tolyl)-20-deoxyguanosine (3c). Starting from 8-Br-dG
(0.504 g, 1.46 mmol), 2-tolylboronic acid (0.594 g, 4.37 mmol),
Pd(OAc)2 (32.8 mg, 0.146 mmol), TPPTS (0.144 g, 0.244 mmol),
and Na2CO3 (0.312 g, 2.92 mmol), adduct 3c was obtained as an
off-white solid (65.7 mg, 13%) following purification by flash
chromatography on silica (1:10 MeOH/CH2Cl2): 1H NMR
(DMSO-d6) (300 MHz) δ 11.4 (s, 1H), 7.42-7.30 (m, 4H),
6.46 (s, 2H), 5.71 (m, 1H), 5.66 (brs, 1H), 5.10 (brs, 1H), 4.20
(m, 1H), 3.72 (m, 1H), 3.50-3.43 (m, 2H), 2.96 (m, 1H), 2.16 (s,
3H), 1.93 (m, 1H); 13C NMR (DMSO-d6) (75.5 MHz) δ 159.0,
154.8, 151.1, 145.5, 138.0, 130.6, 130.3, 130.2, 129.6, 125.6,
117.0, 87.8, 84.7, 71.3, 62.2, 37.1, 19.4; HRMS calcd for
C17H18N5O4 [M - H]- 356.1359, found 356.1346.
Spectroscopic Measurements. Stock solutions of adducts were
made in DMSO to a concentration of 4 mM. Spectroscopic
solutions were prepared with 25 μL of stock solution and 1.975 mL
of 0.1 M HCl, 0.05 M phosphate buffer (pH 1), or 0.05 M
citrate buffer (pH 2, 3 and 4), with ionic strength (μ) maintained
using 0.31 M NaCl. Absorbance scans were taken at 25 ꢀC for
spectroscopic solutions in 0.1 M HCl and 0.05 M phosphate
buffer (pH 1) and at 37 ꢀC for spectroscopic solutions in 0.05 M
citrate buffer (pH 2, 3, and 4). Spectral measurements were
observed from 220 to 400 nm, with a measurement obtained
every 5 to 10 min. Spectroscopic measurements were made until
deglycosylation was observed, and the absorbance maximum for
the deglycosylated product was obtained.
until a plateau in the graphical fit could be observed. The first-order
rate constants for the hydrolysis of adducts were obtained from
plots of log [adduct] versus time and were linear to 4 half-lives.
DFT Calculations. The B3LYP/6-311þG(2df,p)//B3LYP/6-
31G(d) potential energy surfaces of deglyco1-3c and their N7
protonated counterparts were considered as a function of θ, the
dihedral angle that controls the relative orientation of the
phenyl and nucleobase rings (Figure 1). For 1-3c, the sugar
puckering and the orientation of the nucleobase about the
glycosidic bond (defined by the dihedral angle χ, Figure 1) must
be considered in addition to rotation about θ in the correspond-
ing adducts 1-3c. Previously,27 systematic B3LYP/6-31G(d)
potential energy surface scans of 2a and 3a were performed
where the dihedral angles referred to as χ and θ (Figure 1) were
constrained in 10ꢀ increments from 0 to 360ꢀ. Preliminary
conformational searches were performed using Monte Carlo
with MMFF as implemented in the Spartan40 software package
to identify the preferred sugar conformation.27 The ten lowest
energy structures identified from these scans were subsequently
optimized to a minimum with B3LYP/6-31G(d). For both 2a
and 3a,27 the lowest energy conformer involves C20-endo sugar
puckering, which is the puckering present in B-DNA. Addition-
ally, the C50 hydroxyl group is directed toward the nucleobase.27
Finally, the C30 hydroxyl group is directed toward C20
(—(HC30OH) is approximately -60ꢀ). To more accurately deter-
mine the geometries and relative energies of the minima and
transition states identified from the potential energy surface
scans for 2a and 3a, full optimizations (i.e., all constraints
released) were subsequently performed.27
All minima identified for 2a and 3a were subsequently reopti-
mized with different R1 and R2 substituents to generate all adducts
1-3c. Additional Monte Carlo calculations were performed on
these adductstoensure that lower energy minima werenot missed.
Subsequently, each minimum structure was protonated at N7 and
reoptimized to study the effects of an acidic environment on the
adduct structure. The proton affinities were calculated in the gas
phase as the negative of the enthalpy change,30 for the reaction
with the global minimum of the N7-protonated species as the
product and the global minimum of the neutral species as the
reactant. Higher level (B3LYP/6-311þG(2df,p)) single-point cal-
culations as well as B3LYP/6-31G(d) frequency calculations were
performed in the gas phase on all fully optimized structures with
zero-point vibrational energies included.
Deglycosylation scans were performed using constrained
PCM-B3LYP/6-31G(d) optimizations in the solvent (water)
phase. In these calculations, only the C-N bond length was con-
pKa Determination. N7 pKa values for C8-Ph-dG adducts were
determined spectrophotometrically at 20 ꢀC. Solutions were pre-
pared using 25 μL of 4 mM stock solution of the adduct in DMSO
and 1.975 mL of 0.05 M phosphate buffer (pH 1 and 1.5), 0.05 M
citrate buffer (pH 2, 2.5, 3, 3.5, 4, 4.5, 5), and 0.05 M MOPS buffer
(pH 6 and 7). Spectral measurements were observed from 220 to
400 nm, with absorbance measurements obtained at pH values 1-
7 and the initial absorbance recorded at 280 or 320 nm. pKa values
were obtained with the following equation: pKa = pH þ log [(A -
AM)/(AI - A)] with A representing the initial absorbance at 280 or
320 nm, AM representing absorption of the neutral species (pH 7),
and AI representing absorption of the protonated species (pH 1).
pKa values were determined at the pH values between 1.5 and 6,
and the average was taken.
Kinetic Measurements. The first-order kinetics of hydrolysis
was obtained spectrophotometrically at 37.2 or 48.4 ꢀC. Solutions
were prepared using 25 μL of 4 mM stock solution of the adduct in
DMSO and 1.975 mL of 0.1 M HCl, 0.05 M phosphate buffer (pH 1)
or 0.05 M citrate buffer (pH 2, 3, and 4). Kinetic runs were carried
out in parallel using the multicell changer, with 6 kinetic runs
obtained for each adduct in each set of conditions. The appearance
of the deglycosylated product was monitored at its absorbance
maximum, which was determined separately via a UV-vis absor-
bance scan as detailed above. Data points were recorded every 0.1 s
˚
strained, where it was gradually increased in 0.1 A increments
˚
and optimized at each step from 1.4 to 3.4 A.
All B3LYP calculations were performed using Gaussian 03.41
Acknowledgment. Support for this research was provided
by the Natural Sciences and Engineering Research Council
(40) Spartan 002 Wavefunction, Inc. Irvine, CA.
(41) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci,
B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada,
M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.;
Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A.
D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari,
K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.;
Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komar-
omi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.;
Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revisions C.02 and
D.01; Gaussian, Inc.: Wallingford, CT, 2004.
J. Org. Chem. Vol. 74, No. 16, 2009 5801

Schlitt et al.
JOCFeatured Article
(NSERC) of Canada, the Canada Research Chair program,
the Canada Foundation for Innovation, and the Ontario
Innovation Trust Fund. R.A.M. thanks Ms. Karen Shipley
for carrying out preliminary experiments. A.L.M. thanks
NSERC and Alberta Ingenuity for student scholarships.
Supporting Information Available: General experimental
procedures, 1H and 13C NMR spectra of adduct 3c, and Cartesian
coordinates for N7Hþ of adducts 2b, 3b, and 2c. This material
is available free of charge via the Internet at http://pubs.
acs.org.
5802 J. Org. Chem. Vol. 74, No. 16, 2009