2′-Deoxyguanosine reacts with a model quinone methide at multiple sites

Article abstract of DOI:10.1021/tx0101043

Quinone methides and related intermediates have been implicated in a range of beneficial and detrimental processes in biology and effectively alkylate a variety of cellular components despite the ubiquitous presence of water. As a prerequisite to understanding the origins of their specificity, the major products generated by DNA and its components with an unsubstituted ortho quinone methide under aqueous conditions were recently characterized [Pande, P., Shearer, J., Yang, J., Greenberg, W. A., and Rokita, S. E. (1999) J. Am. Chem. Soc. 121, 6773-6779]. Investigations currently focus on the complete range of derivatives formed by deoxyguanosine (dG) and guanine residues in duplex DNA through product isolation and structure determination using reversed-phase chromatography and a range of one and two-dimensional NMR techniques. Previous construction of a synthetic standard for dG alkylation is now shown to have yielded the N1-linked adduct rather than the N²-linked adduct. This later adduct has also now been characterized and confirmed to be the major product of reaction between the quinone methide and both duplex DNA and dG under neutral conditions. An N7 adduct of guanine has additionally been identified under these conditions and appears to result from spontaneous deglycosylation of the corresponding N7 adduct of dG. A combination of steric and electronic properties of duplex DNA likely contribute to the enhanced selectivity of the quinone methide for its guanine N² position (7.8:3.2:1.0 for adducts of N²:N7:N1 relative to that of dG (4.7:3.5:1.0 for adducts of N²:NT:N1).

Full text of DOI:10.1021/tx0101043

Chem. Res. Toxicol. 2001, 14, 1345-1351
1345
2′-Deoxygu a n osin e Rea cts w ith a Mod el Qu in on e
Meth id e a t Mu ltip le Sites
Willem F. Veldhuyzen, Yui-Fai Lam, and Steven E. Rokita*
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
Received J une 20, 2001
Quinone methides and related intermediates have been implicated in a range of beneficial
and detrimental processes in biology and effectively alkylate a variety of cellular components
despite the ubiquitous presence of water. As a prerequisite to understanding the origins of
their specificity, the major products generated by DNA and its components with an unsub-
stituted ortho quinone methide under aqueous conditions were recently characterized [Pande,
P., Shearer, J ., Yang, J ., Greenberg, W. A., and Rokita, S. E. (1999) J . Am. Chem. Soc. 121,
6773-6779]. Investigations currently focus on the complete range of derivatives formed by
deoxyguanosine (dG) and guanine residues in duplex DNA through product isolation and
structure determination using reversed-phase chromatography and a range of one and two-
dimensional NMR techniques. Previous construction of a synthetic standard for dG alkylation
is now shown to have yielded the N1-linked adduct rather than the N2-linked adduct. This
later adduct has also now been characterized and confirmed to be the major product of reaction
between the quinone methide and both duplex DNA and dG under neutral conditions. An N7
adduct of guanine has additionally been identified under these conditions and appears to result
from spontaneous deglycosylation of the corresponding N7 adduct of dG. A combination of
steric and electronic properties of duplex DNA likely contribute to the enhanced selectivity of
the quinone methide for its guanine N2 position (7.8:3.2:1.0 for adducts of N2:N7:N1) relative
to that of dG (4.7:3.5:1.0 for adducts of N2:N7:N1).
tive suppression of reaction, electronic contributions by
neighboring bases have the ability to activate certain
sites for reaction (3, 7). Selectivity is also induced by
preferential binding of electrophiles or their precursors
to increase the local concentration of reactants (8). For
example, nitrogen mustards related to chlorambucil
generally react with most all guanine residues through
the major groove (9), but reaction can be limited to a
single region of this groove by conjugating the electro-
phile to a sequencing-directing and triplex-forming de-
oxyoligonucleotide (10). Alternative conjugation to minor
groove-binding agents can redirect or restrict reaction to
A N3 of the minor groove (11).
In tr od u ction
Structural characterization of DNA adducts provides
one of the most useful tools for describing nucleic acid
reactivity and hence contributes to our understanding of
the mechanism by which numerous environmental car-
cinogens and certain anticancer antibiotics function.
Although our ability to predict the target specificity of
many complex intermediates remains limited, some
trends have been established by study of simple electro-
philes (1-5). In general, the sites of modification depend
on the nature of the nucleophile-electrophile pairing and
the accessibility of the target functional group in DNA.
Electrostatic potential calculations suggest that guanine
(G) N7, cytosine (C) N3, and adenine (A) N1 are most
nucleophilic, and other sites with nucleophilic character
include N3 and O6 of G, O2 of C, and N3 of adenine (A)
(6). Relatively soft electrophiles such as dimethyl sulfate
and methyl iodide participate in SN2-like processes and
typically alkylate the most nucleophilic positions such as
dG N7 (1, 3). In contrast, hard electrophiles such as those
formed by N-alkyl-N-nitrosourea participate in SN1-like
processes and often modify the most electronegative
heteroatom, oxygen in dC, dG and the phosphate back-
bone (1-3).
Natural products that generate quinone methide or
related intermediates predominantly alkylate the exo-
cyclic amines of dG (N2) and to a lesser extent dA (N6)
(12-17). The origins of this specificity are still under
investigation and may result from selective binding and
orientation as well as optimal pairing of the reactants’
electrophilic and nucleophilic properties. Even simple
quinone methide models exhibit a propensity for reaction
at these exocyclic amines of the purines (18-20). In
contrast, the ring nitrogen N3 of the pyrimidine dC may
be modified instead of its exocyclic N4 position (19, 21).
Reaction is also not limited to these individual sites, and
product profiles can respond to changes in quinone
methide structure and reaction conditions. Loss of the
aziridine ring of mitomycin to form a 2,7-diaminomito-
sene derivative increases formation of a GN7 adduct at
the expense of its N2 adduct (22, 23). Likewise, para-
quinone methides generated from di(tert-butyl)methyl-
phenol derivatives (such as BHTOH) exhibit varying
The intrinsic reactivity of each site within the nucleo-
sides is not the sole variable affecting product distribu-
tion. Both the steric and electronic environments estab-
lished by helical DNA are important determinants in the
specificity of modification as well. While the steric
properties of helical DNA most commonly lead to selec-
* To whom correspondence should be addressed.
10.1021/tx0101043 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/08/2001

1346 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Veldhuyzen et al.
specificities for GN7, N1, and N2 as a function of their
structure and stability (19).
4) to 15% acetonitrile in 26 mM TEAA (pH 4) over 60 min (5
mL/min). The synthesis and isolation was performed 5 times to
obtain sufficient material for spectroscopic characterization (2
mg, 5% yield). 1H NMR (DMF-d7) δ 2.36 (ddd, 1H, J ) 13.0,
6.5, 3.0 Hz), 2.71 (m, 1H), 3.69 (dd, 1H, J ) 12.0, 4.5 Hz), 3.74
(dd, 1H, J ) 12.0, 4.5 Hz), 3.97 (m, 1H), 4.56 (m, 1H), 4.58 (s,
2H), 5.07 (bs, OH), 5.43 (bs, OH), 6.35 (dd, 1H, J ) 7.5, 6.5 Hz),
6.78 (td, 1H, J ) 7.5, 0.5 Hz), 6.94 (dd, 1H, J ) 7.5, 0.5 Hz),
7.12 (td, 1H, J ) 7.5, 1.5 Hz), 7.23 (bs, NH), 7.33 (dd, 1H, J )
7.5, 1.5 Hz), 7.99 (s, 1H).13C NMR (DMF-d7) δ 40.8, 41.0, 63.1,
72.2, 84.2, 88.9, 115.9, 118.2, 119.5, 125.8, 129.1, 130.1, 136.2,
151.3, 154.0, 156.6, 157.7. UV (10 mM potassium phosphate pH
6.5): λmax 256, 280(s) nm. HRMS (FAB, glycerol) m/z 374.1451
(M + H+); calcd for C17H20N5O5 (M + H+): 374.1464.
Our laboratory has developed a series of quinone
methide-forming bioconjugates for sequence specific alkyl-
ation of DNA that are triggered by photochemistry (24),
reduction (24), and fluoride (25), alternatively. A related
model system was concurrently established to identify
the preferred sites of modification on each nucleoside
residue (20, 21). This utilized a simple quinone methide
precursor (QMP) based on a silyl-protected phenol con-
taining an ortho-substituted bromomethyl group (Scheme
1). Upon deprotection with fluoride, bromide is eliminated
N1 Ad d u ct of d G (2). QMP (21) (230 mg, 0.64 mmol) in DMF
(5.0 mL) was combined with dG (130 mg, 0.46 mmol). Reaction
was initiated by addition of aqueous KF (0.90 mL, 2.6 M) and
incubated for 2 h at 37 °C as described previously (20). Adduct
2 was isolated in 18% yield (30 mg) after semipreparative
reversed-phase HPLC using a gradient of 5% acetonitrile in 29
mM TEAA (pH 4) to 25% acetonitrile in 23 mM TEAA (pH 4)
Sch em e 1
1
over 30 min (5 mL/min). H NMR (DMF-d7) δ 2.34 (ddd, 1H, J
) 13.5, 6.0, 2.5 Hz), 2.70 (ddd, 1H, J ) 13.5, 8.0, 5.5 Hz), 3.69
(dd, 1H, J ) 11.5, 4.5 Hz), 3.74 (dd, 1H, J ) 11.5, 4.5 Hz), 3.97
(m, 1H), 4.54 (m, 1H), 5.28 (s, 2H), 6.27 (dd, 1H, J ) 8.0, 6.0
Hz), 6.77 (t, 1H, J ) 7.5 Hz), 6.98 (d, 1H, J ) 7.5 Hz), 7.13 (t,
1H, J ) 7.5 Hz), 7.14 (d, 1H, J ) 7.5 Hz), 7.20 (bs, NH2), 8.05
(s, 1H). 13C NMR (DMF-d7) δ 39.3, 40.7, 63.1, 72.3, 83.8, 89.0,
116.0, 117.2, 120.0, 123.7, 129.0, 129.2, 136.5, 150.2, 154.9,
155.9, 157.8. UV (10 mM potassium phosphate pH 6.5): λmax
257, 275(s) nm.
to yield the ortho-quinone methide intermediate under
mild aqueous conditions. The major product identified
after reaction with DNA was assigned as the N2 adduct
of dG (20), equivalent to that generated by mitomycin
(12, 14) and an anthracycline derivative (13). Interest-
ingly, the selectivity of QMP was not completely reflected
in the intrinsic nature of the nucleosides. Modification
of dC N3 dominated reaction of the nucleosides but was
suppressed by Watson-Crick base pairing in reaction of
duplex DNA (20). The exocyclic amine N2 of dG was least
sensitive to the constraints of duplex DNA and conse-
quently prevailed over the competing sites of reaction.
We have since focused on the minor products of guanine
alkylation in order to place these model studies in context
with the reaction profiles determined for the para-
quinone methide derivatives of BHTOH and mitomycin
that exhibit a range of purine products (15, 16, 19, 22,
23).
Tr is-ter t-bu tyld im eth ylsilyl Der iva tive of th e d G N1
Ad d u ct 2. Adduct 2 (8 mg, 0.02 mmol) and tert-butyldimeth-
ylsilyl chloride (65 mg, 0.43 mmol) were added to a solution of
imidazole (60 mg, 0.88 mmol) in DMF (0.5 mL). The reaction
mixture was stirred at room temperature for 4 h and then
extracted with chloroform, washed with water and brine, dried
(MgSO4), and concentrated under reduced pressure. Purification
by silica gel flash chromatography (6:4 hexanes:ethyl acetate)
yielded the desired product (14 mg, 88%) exhibiting three tert-
1
butyldimethylsilyl groups by H NMR as expected for complete
protection of the three hydroxyl groups. 1H NMR (CD3CN) δ
0.02 (s, 3H), 0.04 (s, 3H), 0.10 (s, 3H), 0.11 (s, 3H), 0.30 (s, 6H),
0.88 (s, 9H), 0.90 (s, 9H), 1.05 (s, 9H), 2.28 (ddd, 1H, J ) 13.5,
6.5, 4.0 Hz), 2.62 (dt, 1H, J ) 13.5, 6.0 Hz), 3.73 (m, 2H), 3.86
(m, 1H), 4.56 (m, 1H), 5.18 (AB quartet, 2H, J AB ) 16.5 Hz),
5.67 (s, NH2), 6.11 (t, 1H, J ) 6.5 Hz), 6.91 (t, 1H, J ) 7.5 Hz),
6.94 (d, 1H, J ) 7.5 Hz), 7.08 (d, 1H, J ) 7.5 Hz), 7.19 (t, 1H,
J ) 7.5 Hz), 7.71 (s, 1H). 13C NMR (CD3CN) δ -5.34, -5.31,
-4.7, -4.5, 18.5, 18.9, 39.9, 40.7, 63.9, 73.1, 83.9, 88.5, 119.9,
123.1, 127.3, 129.0, 129.8, 136.7, 150.1, 153.7, 154.4, 158.1. A
signal corresponding to C5 (∼117.7 ppm) was obscured by the
solvent signal but verified by HMBC (CD3CN). HRMS (FAB,
glycerol) m/z 716.4092 (M + H+); calcd for C35H61N5O5Si3 (M +
H+): 716.4059.
Exp er im en ta l Section
Gen er a l Ma ter ia ls a n d Meth od s. Solvents, chemicals and
reagents of the highest commercial grade were used without
further purification except when noted. All aqueous solutions
were prepared with water purified by a standard filtration
system to yield a resistivity of 18.0 MΩ. Silica gel (230-400
mesh) for column chromatography was purchased from EM
Sciences. 1H and 13C spectra were recorded on an AMX 500
spectrometer (1H, 500.13 MHz; 13C, 125.77 MHz). All NMR
chemical shifts (δ) are reported in parts per million (ppm) and
were determined relative to the standard values for solvent.
Coupling constants (J ) are reported in hertz (Hz). Low and high-
resolution mass spectra were determined with a VG7070E mass
spectrometer. UV absorption spectra were obtained with a
Hewlett-Packard 8453 UV-vis spectrophotometer. Reverse
phase C-18 chromatography was performed on analytical (Vari-
an Microsorb-MV C-18, 300 Å particle size, 250 mm × 4.6 mm)
and semipreparative (Alltech Econosphere C-18, 10 um particle
size, 250 mm × 10 mm) columns using a J asco PU-980 HPLC
N7 Ad d u ct of Gu a n in e (3). QMP (21) (18 mg, 0.060 mmol)
was added to an aqueous solution of dG (5.7 mg, 0.020 mmol),
KF (500 mM) and potassium phosphate (40 mM, pH 7), and
30% DMF (1 mL). The resulting mixture was incubated at 37
°C (24 h) and then directly purified by semipreparative reversed-
phase HPLC using a gradient of 10% acetonitrile in 27 mM
TEAA (pH 4) to 15% acetonitrile in 26 mM TEAA (pH 4) over
60 min (5 mL/min). The synthesis and isolation was performed
nine times to obtain sufficient quantities of the N7 adduct for
1
equipped with
scanning detector.
a
J asco MD-1510 UV-vis multiwavelength
spectroscopic characterization (6 mg, 10% yield). H NMR (0.2
M NaOD) δ 5.39 (s, 2H), 6.49 (td, 1H, J ) 7.5, 0.5 Hz), 6.66 (dd,
1H, J ) 7.5, 0.5 Hz), 6.79 (dd, 1H, J ) 7.5, 1.0 Hz), 7.12 (td,
1H, J ) 7.5, 1.0 Hz), 7.64 (s, 1H). 13C NMR (0.2 M NaOD) δ
46.6, 110.6, 114.1, 119.4, 125.5, 128.8, 129.8, 142.4, 159.5, 161.2,
164.9, 165.7. UV (10 mM potassium phosphate pH 6.5): λmax
280 nm. HRMS (FAB, glycerol) m/z 258.1002 (M + H+); calcd
for C17H20N5O5 (M + H+): 258.0991.
N2 Ad d u ct of d G (1). QMP (21) (12 mg, 0.040 mmol) in DMF
(180 µL) was combined with dG (6 mg, 0.02 mmol). Reaction
was initiated by addition of aqueous KF (420 µL, 2.6 M) and
incubated for 2 h at 37 °C. The N2 adduct was directly purified
by semipreparative reverse-phase HPLC using a gradient of 10%
acetonitrile in 27 mM triethylammonium acetate (TEAA, pH

Quinone Methide Reaction with Guanine
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1347
Ta ble 1. 1H a n d 13C NMR Da ta for th e d G N2 Ad d u ct (1)
a n d th e Gu a n in e N7 Ad d u ct (3)
dG N2 adduct (1)a
dGa
guanine N7 adduct (3)b
position δH (ppm) δC (ppm) δC (ppm) δH (ppm)
δC (ppm)
2
4
5
6
8
154.0
151.3
118.2
157.7
136.2
40.8
156.6
125.8
130.1
119.5
129.1
115.9
154.8
151.8
117.9
157.8
136.2
161.2
159.5
110.6
165.7
142.4
46.6
164.9
125.5
128.8
114.1
129.8
119.4
7.99
4.58
7.64
5.39
-CH2-
10
11
12
13
14
15
7.33
6.78
7.12
6.94
6.79
6.49
7.12
6.66
F igu r e 1. Reversed-phase (C-18) chromatographic analysis of
dG adducts formed from reaction of dG, QMP, and 400 mM KF
in 85% aqueous DMF. For details, see the Experimental Section.
Qu in on e Meth id e Alk yla tion of d G. Reactions were initi-
ated by adding an acetonitrile solution of QMP21 (30 µL) to a
solution of dG and KF in potassium phosphate (pH 7, 70 mL).
The resulting mixture of QMP (25 mM), dG (0.5 mM), KF (500
mM), and buffer (10 mM) was incubated at 37° C (24 h) and
directly separated by analytical reversed-phase HPLC using a
gradient of 3% acetonitrile in 29 mM TEAA (pH 4.0) to 25%
acetonitrile in 21 mM TEAA (pH 4) over 66 min (1 mL/min).
The relative product yields represent averages of three inde-
pendent determinations, and the alkylated products were as-
sumed to have extinction coefficients proportional to their parent
derivatives, dG and guanine, alternatively.
Alk yla tion of Dou ble-Str a n d ed DNA. Reaction of calf
thymus DNA (18 mM in nt, purified by phenol-chloroform
extraction) was performed under identical conditions used for
dG above except the concentration of QMP (21) was increased
to 100 mM. Prior to HPLC analysis, the modified DNA was
digested with alkaline phosphatase and phosphodiesterase as
described previously (20). Products of alkylation were detected
by analytical reversed-phase HPLC using a gradient of 3%
acetonitrile in 29 mM TEAA (pH 4.0) to 25% acetonitrile in 21
mM TEAA (pH 4) over 66 min (1 mL/min). The relative product
yields represent averages of four independent determinations,
and the alkylated products were assumed to have extinction
coefficients proportional to their parent deoxynucleosides and
guanine, alternatively.
a
b
In DMF-d7. In NaOD/D2O.
30% aqueous DMF. The organic solvent could not be
omitted completely since it was necessary for solubilizing
QMP. Under these conditions with dG, QMP (2 equiv)
and KF (1.8 M), 1 was isolated from a semipreparative
C-18 column in 5% yield with respect to dG and ∼24%
with respect to total dG adducts. Despite this low yield,
sufficient material was collected for complete spectro-
scopic analysis.
The UV absorbance maximum of adduct 1 (256 nm)
exhibited a small 4 nm bathochromic shift relative to dG
(252 nm). Such a shift is consistent with the absorbance
maxima of products measured under neutral aqueous
conditions and formed by alkylation at N2 (256 nm), N1
(256 nm), or N7 (258 nm) (19). In contrast, alkylation at
dG O6 results in a hypsochromic shift (247 nm) (26).
Characterization of adduct 1 by NMR followed an
equivalent approach to that previously published on the
quinone methide adducts of dA and dC (20, 21). Assign-
ment of 1H signals were based on their multiplicities and
chemical shifts and confirmed by 2D COSY analysis. The
13C signals were then identified from their connectivities
to 1H signals observed by HMQC and HMBC experiments
(see Supporting Information) (27, 28). Aromatic carbons
C12-C15 derived from QMP were distinguished by
correlation with their corresponding aromatic hydrogens
as detected by HMQC, and the carbons C10 and C11 were
distinguished by their long range connectivities to aro-
matic hydrogens as detected by HMBC. The purine H8
helped to assign C8 as well as C4 and C5 by direct
(HMQC) and long-range coupling (HMBC), respectively.
The purine C4 was differentiated from C5 by a correlation
between C4 and H1′ of the ribose ring (HMBC). No
geminal or proximal hydrogens are present to discern the
signals for purine C2 and C6. However, based on litera-
ture precedence for 13C NMR assignments of dG and its
derivatives (29-31), C6 was assigned to be more down-
field than C2. Due to the importance of these final signals
in the differentiation of 1 and 2, their assignments were
confirmed experimentally as described below.
Resu lts a n d Discu ssion
Ad d u cts F or m ed by th e Nu cleosid e d G. Prior
analysis of nucleic acid modification by QMP began by
characterizing the major adduct of each residue and
comparing their relative yields using deoxynucleosides
to duplex DNA (20, 21). Additional investigations on the
minor products formed by guanine residues was further
expected to differentiate between reaction specificity
dictated by the electrophile and that dictated by the DNA
target. Our attention consequently turned to such prod-
ucts formed by dG and a model quinone methide conve-
niently generated by QMP and aqueous fluoride. Minor
products were indeed evident by reverse-phase C-18
chromatography (Figure 1), but surprisingly, replacement
of the original column (20) with another provided resolu-
tion of two products 1 and 2 that had previously coeluted
and assigned to the dG N2 adduct (20). Most intriguingly,
the synthetic conditions formerly used to prepare the dG
adduct yielded 2, whereas DNA modification yielded 1
(see below). Structural characterization therefore began
with examination of 1 and, subsequently, 2.
The 13C NMR chemical shifts (Table 1) of adduct 1
relative to dG are most consistent with alkylation at N2
rather than O6, N7, or N1. An O6 product would be
expected to show significant perturbation of its purine
carbon signals C2, C4, C5, C6, and C8 relative to dG
(>2.0 ppm) (32). Similarly, modification at N7 would
result in a downfield shift of C2 and C6 (>3.5 ppm) in
addition to a large upfield shift in C5 (∼9 ppm) relative
to dG (32), and modification at N1 would result an upfield
shift in C4 (∼2-3 ppm) (32). Only alkylation at N2 yields
Isola tion a n d Str u ctu r a l Ch a r a cter iza tion of d G
Ad d u ct 1. Preparative isolation of 1 was initially made
difficult by the great excess (15-fold) of 2 generated under
the original synthetic conditions (85% aqueous DMF)
(20). The formation of 2 was found to be dependent on
DMF concentration and was suppressed 7-fold by use of

1348 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Veldhuyzen et al.
F igu r e 3. 2D COSY (280 K) of dG adduct 1 in DMF-d7.
HMBC data indicating a connectivity between C10 and
the benzylic -CH2- group that was inherent from the QMP
structure. However, the COSY experiment described
above contradicted our earlier assumptions.
Efforts to resolve and reassign the purine carbon
resonances C2 and C6 of 2 were therefore initiated. By
switching solvents from CD3OD to DMF-d7, each 13C
signal became distinct and thus available for direct
characterization. Identification of C2 by correlation to the
protons of its attached -NH2 group were thwarted by their
rapid exchange in DMF. The limited solubility of 2
prevented use of alternative solvents to circumvent this
problem. Instead, the tris-tert-butyldimethylsilyl deriva-
tive of 2 was prepared to enhance its solubility for NMR
studies in CD3CN (Supporting Information). Still, no
correlation between the N2 protons and C2 could be
observed by HMBC despite the relatively sharp signals
of these protons. This result likely reflects the smaller
coupling constant often associated with two vs three bond
heteronuclear systems (34).
The necessary correlations required for unambiguous
assignments were finally obtained by heteronuclear NOE.
Irradiation of the -NH2 protons (5.67 ppm) of the silylated
derivative of 2 yielded an 80% enhancement of its 13C
signal at 154.4 ppm (Supporting Information). In con-
trast, all other 13C signals were unaffected ((5%) by these
conditions. Accordingly, the upfield signal of the silylated
derivative at 154.4 ppm was assigned to C2 and by
default the downfield signal at 158.1 ppm was assigned
to its C6 (Table 2). This in turn indicated that the upfield
signal of the nonsilylated parent 2 was associated with
C2 as typical of other alkylated derivatives of dG (32,
33).
Complete HMBC analysis of 2 in DMF-d7 detected
correlations between the benzylic hydrogens and both
newly assigned purine carbons, C2 and C6, in addition
to those derived from QMP, C10, C11, C12, and C14
(Figure 4). These data are uniquely satisfied by alkylation
of N1 rather than N2 of dG to form adduct 2. The UV
absorbance maximum of 2 (257 nm) is also consistent
with N1 alkylation, but does not differ from that resulting
from N2 alkylation (19). Finally, the downfield shift of
the benzylic hydrogens attached to N1 (5.28 ppm, 2) vs
N2 (4.58 ppm, 1) mimic previous observations for N1 and
N2 adducts of BHTOH (5.06 and 4.31 ppm, respectively)
(19).
F igu r e 2. HMBC of dG adduct 1 in DMF-d7.
little change in the 13C signals of the purine (e0.7 ppm)
(33). Indeed, the chemical shifts of 1 deviate only slightly
from those of dG (e0.7 ppm).
The chemical shift of the benzylic hydrogens are also
very sensitive to the position of their attachment to the
purine, and a value of 4.58 ppm is most reminiscent of
the 4.31 ppm attributed to the equivalent hydrogens of
the N2 adduct of BHTOH and unlike the corresponding
values of N1 (5.06 ppm) and N7 (5.23 ppm) adducts (19).
However, unambiguous assignment of adduct 1 was best
1
determined by the long-range H-13C connectivities ob-
served by HMBC (Figure 2). Such coupling was inherent
for the benzylic hydrogens (-CH2-) and C10, C11, and C12
of the phenolic moiety, but only the N2 derivative would
also exhibit a unique and singular purine coupling to C2.
Alternative alkylation at O6 or N1 would have displayed
a correlation with C6, and alkylation at N7 or N3 would
have displayed correlations to C5 and C8 or C2 and C4,
respectively.
The structure of 1 was further established by observing
the predicted -NH- to -CH2- connectivity using 2D
COSY NMR (DMF-d7). The -NH- proton signals for 1 are
broad at 25° C but were suitably resolved at lower
temperature (7° C) to detect their correlation (Figure 3).
No equivalent connectivity was observed for adduct 2
under identical conditions (Supporting Information).
Initial characterization of the synthetic standard 2 had
also originally led to the same structural assignment as
1 due in part to the uncertainty of the C2 and C6 signals
(20). The COSY experiment strongly suggests that 2 is
not linked to dG through the N2 position.
Str u ctu r a l Ch a r a cter iza tion of d G Ad d u ct 2.
Initial confusion over the assignment of C2 and C6 in 2
resulted from a lack of resolution between the quinone
methide-forming moiety (C10) and a purine carbon in the
13C NMR when CD3OD was used as solvent (20). This
was compounded by ascribing no further consequence to
Isola tion a n d Ch a r a cter iza tion of d G Ad d u ct 3.
The trace quantities of 3 formed under conditions used

Quinone Methide Reaction with Guanine
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1349
Ta ble 2. 1H a n d 13C NMR Da ta for d G N1 Ad d u ct (2) a n d
Its Tr is-ter t-bu tyld im eth ylsilyl Der iva tive
silyl derivative of the
dG N1 adduct (2)a
dG N1 adduct (2)b
position
δH (ppm)
δC (ppm)
δH (ppm)
δC (ppm)
2
4
5
6
8
154.9
150.2
117.2
157.8
136.5
39.3
155.9
123.7
129.2
120.0
129.0
116.0
154.4
150.1
117.7
158.1
136.7
39.9
153.7
127.3
129.0
123.1
129.8
119.9
8.05
5.28
-
7.71
5.18
-CH2-
10
11
12
13
14
15
-
7.14
6.77
7.13
6.98
7.08
6.91
7.19
6.94
a
b
In DMF-d7. In CD3CN.
F igu r e 5. HMBC of guanine N7 adduct 3 in 0.2 M NaOD/D2O.
1
for all subsequent characterization. The H and 13C NMR
signals of 3 were first assigned as described for the dG
N2 adduct 1 (Table 1). However, the chemical shifts of
C4 and C5 were not distinguishable by HMBC in this
case due to the absence of the deoxyribose H1′ that would
otherwise provide a unique coupling to C4. Assignment
of the downfield signal (159.5 ppm) as C4 and the
remaining signal (110.6 ppm) as C5 was instead based
on extensive literature precedence for guanine N7 ad-
ducts (36-39). Similarly, C6 and C2 that also lack
discernible couplings were again assigned by literature
precedence (36-39) and are consistent with hetero-
nuclear NOE data for the dG N1 adduct 2 in which C6
is downfield relative to C2.
Long-range correlations were observed by HMBC
between the benzylic hydrogens (-CH2-) and C10, C11,
C12 and, most importanly, purine C5 and C8 (Figure 5).
Only alkylation of N7 would generate this pattern, and
neither alkylation of O6, N1, N2, or N3 would exhibit
comparable coupling to C8. The facile deglycosylation
leading to 3 is again expected for N7 alkylation.
Ch a r a cter iza tion of d G Ad d u ct 4. An additional
adduct 4 was observed by HPLC after reaction of QMP
and dG (Figure 1). This material was also isolated by
semipreparative HPLC. During subsequent removal of
solvent by lyophilization, 85% of 4 decomposed to 3 as
indicated by reanalysis using HPLC. Examination of the
resulting mixture by 1H NMR (DMF-d7) verified this
decomposition and indicated an equivalent 85% loss of
the deoxyribose hydrogens. Thus, 4 appears to be the
initial N7 adduct of dG that spontaneously converts to
the guanine derivative 3. The UV spectrum of 4 observed
during HPLC elution exhibited an absorbance maximum
at 260 nm (pH 4), closely corresponding to that of N7
methyl dG (257 nm, pH 3; 258 nm, pH 7) (40). Further
characterization of 4 was prevented by its extreme
lability.
F igu r e 4. HMBC of dG adduct 2 in DMF-d7.
to prepare 1 (30% aqueous DMF) or 2 (85% aqueous
DMF) were insufficient for structural characterization.
However, generation of 3 was enhanced by maintaining
neutral pH with potassium phosphate in 30% DMF. This
afforded an isolated yield of 10% for 3 relative to dG and
accounted for almost 30% of all purine alkylation under
these conditions. Preliminary examination of 3 by 1H
NMR immediately suggested that deglycosylation had
occurred since no signals for the deoxyribose group were
evident. Mass spectral analysis (FAB) further confirmed
this observation. Such a process is typical for intermedi-
ates containing an N7 alkyl substituent (8, 19, 35). The
UV absorbance maximum of 3 (280 nm) is also equivalent
to that observed for a depurinated N7 adduct formed by
the related BHTOH (λmax ) 282 nm) (19).
Alkylation of N7 was formally established by the same
one- and two-dimensional NMR techniques used success-
fully to solve the structures of 1 and 2 above. The poor
solubility of 3 and incomplete resolution of its 13C NMR
signals precluded extensive use of DMF-d7. An NMR
solvent of 0.2 M NaOD in D2O was found to be suitable

1350 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Veldhuyzen et al.
quinone methides (43-46), QMP produces an electro-
philic intermediate that is not completely quenched
under aqueous conditions and is instead capable of
forming adducts with the cyclic and exocyclic nitrogens.
The product profile is not dependent on the intrinsic
nucleophilicity of each site and may instead be related
to the thermodynamics more than the kinetics of reaction,
a topic under current investigation.
Alk yla tion P r ofile of d G in Du p lex DNA. Product
determination was reinvestigated for duplex DNA since
our original analysis had unwittingly measured the
combined yields of adducts 1 and 2 (20). An overall
suppression of quinone methide reactivity was again
expected and observed as a result of the reduced acces-
sibility of nucleophiles in duplex DNA relative to its
constituent deoxynucleosides. Modification of dG N2 still
dominated the profile of DNA, and the relative reactivity
of dC and dA was similar to that reported previously
(Figure 6B) (20).
The selectivity within dG residues of DNA could now
also be determined, and the relative yield of the N2
adduct 1 was found to increase (7.8:3.2:1.0 for adducts
of N2:N7:N1 in duplex DNA) over that formed by the
deoxynucleoside. The diminished reactivity of dG N1 is
consistent with its central position within the Watson-
Crick hydrogen-bond array of helical DNA and far
removed from the solvent accessible surface. In contrast,
the N7 and N2 groups maintain exposure on the surface
of the major and minor grooves, respectively (6). Acces-
sibility alone cannot control reaction since the preference
for alkylation of N2 was enhanced from deoxynucleoside
to DNA. The highly ordered structure of duplex DNA also
effects the electrostatic potential of its components (6),
and reaction at dG N2 in particular may be assisted by
the ability of the O2 group of the complementary dC to
act as a general base (47). The hydrophobic nature of the
minor groove may additionally promote reaction by
increasing the local concentration of hydrophobic reac-
tants such as QMP (20).
Dominant formation of the N2 adduct of dG (1) with
QMP has now been confirmed under conditions that
resolve numerous products of dG and DNA alkylation.
Reassignment of the NMR spectra of 1 was made possible
by COSY and heteronuclear NOE experiments as well
as by changing NMR solvents. In the future, related
structures may be implied merely by the diagnostic
chemical shift of their benzylic hydrogens since adducts
formed with both ortho- and para-quinone methide
intermediates exhibit equivalent trends (19). While the
variable reactivity of dG N1 may be rationalized by its
protonation state in part, the remaining processes con-
trolling the specificity between conjugated electrophiles
such as quinone methides and other nucleophiles of DNA
may now be examined. Optimal pairing of nucleophilic
and electrophilic properties likely represent only one
determinant, and product stability and kinetics may play
a substantial but as yet unappreciated role (16).
F igu r e 6. Reversed-phase chromatographic analysis of nucleo-
side adducts formed from reaction of QMP with (A) dG (0.5 mM)
and (B) calf thymus DNA (18 mM nucleotides) in potassium
phosphate (10 mM, pH 7) and 30% CH3CN. Reactions with calf
thymus DNA were digested with alkaline phosphatase and
phosphodiesterase prior to HPLC analysis (20).
Alk yla tion P r ofile of d G. Identification of the ad-
ducts (1-4) was a prerequisite for returning to questions
on the specificity of guanine alkylation at the deoxy-
nucleoside and duplex DNA levels. Previous analytical
conditions had focused on only the major adduct and had
not resolved products 1 and 2 (20). The improved HPLC
resolution illustrated in Figure 1 allowed for a more
complete analysis of the derivatives formed by QMP and
for direct comparison to the product profile formed by its
more stable and sterically hindered para analogues (such
as BHTOH) published earlier (19). Most dramatically, the
dG N1 adduct 2 formed more readily than the N2 (1) or
N7 (3, 4) adducts by g15-fold in unbuffered 85% aqueous
DMF as indicated above. The N2 rather than N1 adducts
of dG are customarily produced in greatest yield under
neutral pH with quinone methide intermediates (12, 13,
19, 41). Thus, efficient formation of the N1 adduct 2 in
this example is most likely due to deprotonation of N1
(pKa ) 9.2) (40) since the unbuffered conditions used to
form 2 become mildly basic upon addition of potassium
fluoride. Interestingly, the behavior of the dG anion is
very much influenced by its electrophilic partner and
reaction conditions. Deprotonation of dG appears to
promote alkylation of its N2 position by benzyl bromide
under polar protic conditions, although the specificity
shifts to the N1 position with a polar aprotic solvent
(42).
Yield of the N1 adduct 2 decreased significantly below
that of the common N2 adduct 1 when the reactivity of
QMP was examined with dG under neutral conditions
equivalent to those used for the DNA analysis below (pH
7, 30% aqueous CH3CN). Quantitative analysis of the
products by HPLC indicated a ratio of 4.7:3.5:1.0 for the
products N2 (1):N7 (3 + 4):N1 (2) (Figure 6A). Interest-
ingly, this ratio does not significantly differ from that
measured for the more stable para-quinone methide
generated by BHTOH (5.5:2.8:1.0) (19). Despite the
highly reactive nature predicted for unsubstituted ortho-
Ack n ow led gm en t. This work was supported in part
by the National Institutes of Health (CA81571). We
thank Ms. Carolyn Ladd for the mass spectral analyses
and Dr. Gene Mazzola for technical advice and as-
sistance.
Su p p or tin g In for m a tion Ava ila ble: Full HMQC and
HMBC of the dG N2 (1), dG N1 (2), and guanine N7 (3) adducts.

Quinone Methide Reaction with Guanine
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1351
(23) Maliepaard, M., de Mol, N. J ., Tomasz, M., Gargiulo, D., J anssen,
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(24) Chatterjee, M., and Rokita, S. E. (1996) Inducible Alkylation of
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Full HMBC and heteronuclear NOE data of the tris-tert-
butyldimethylsilyl derivative of the dG N1 adduct (2). Full 2D
COSY of the dG N2 (1) and dG N1 (2) adducts. This material is
available free of charge via the Internet at http://pubs.acs.org.
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