Absolute configurations and stability of cyclic guanosine mono-adducts with glyoxal and methylglyoxal

Article abstract of DOI:10.1002/chir.20875

Glyoxal and methylglyoxal are two endogenous and mutagenic 1,2-dicarbonyl compounds, which can readily form adducts with guanosine. The molecular structures of cyclic guanosine-glyoxal (G-g) and guanosine-methylglyoxal (G-mg) mono-adducts have been extensively studied before. However, diastereoisomers of these adducts have not yet been studied in detail. In this work, one pair of G-g and two pairs of G-mg diastereoisomers were baseline separated by reverse phase HPLC, whose structures were identified as the previously reported cyclic forms, and their absolute configurations were determined by circular dichroism, the octant rule, and molecular modeling. According to the HPLC elution order, configurations of two G-g (as well as trans G-mg) were (6R,7R) and (6S,7S), respectively. Meanwhile, the stability of each isomer in neutral solution was also investigated, which revealed the stability order G-g > cis G-mg > trans G-mg and also indicated distinct transformation processes for different G-mg configurations. Trans G-mg only racemized between each other, while cis G-mg transformed to both cis and trans forms. Different intermediates in the racemization processes were proposed to explain the observations. These results may shed light on further understanding the roles of these two small molecules in mutagenesis. Copyright

Full text of DOI:10.1002/chir.20875

CHIRALITY 23:487–494 (2011)
Absolute Configurations and Stability of Cyclic Guanosine
Mono-adducts with Glyoxal and Methylglyoxal
*
CONGFANG LAI, GUANGXIN LIN, WENYUE WANG, AND HAI LUO
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering,
Peking University, Beijing, 100871, China
ABSTRACT
Glyoxal and methylglyoxal are two endogenous and mutagenic 1,2-dicar-
bonyl compounds, which can readily form adducts with guanosine. The molecular struc-
tures of cyclic guanosine-glyoxal (G-g) and guanosine-methylglyoxal (G-mg) mono-adducts
have been extensively studied before. However, diastereoisomers of these adducts have not
yet been studied in detail. In this work, one pair of G-g and two pairs of G-mg diastereoisom-
ers were baseline separated by reverse phase HPLC, whose structures were identified as
the previously reported cyclic forms, and their absolute configurations were determined by
circular dichroism, the octant rule, and molecular modeling. According to the HPLC elution
order, configurations of two G-g (as well as trans G-mg) were (6R,7R) and (6S,7S), respec-
tively. Meanwhile, the stability of each isomer in neutral solution was also investigated,
which revealed the stability order G-g > cis G-mg > trans G-mg and also indicated distinct
transformation processes for different G-mg configurations. Trans G-mg only racemized
between each other, while cis G-mg transformed to both cis and trans forms. Different inter-
mediates in the racemization processes were proposed to explain the observations. These
results may shed light on further understanding the roles of these two small molecules in
C
VV
mutagenesis. Chirality 23:487–494, 2011.
2011 Wiley-Liss, Inc.
KEY WORDS: glyoxal/methylglyoxal; guanosine adduct; circular dichroism; octant rule;
diastereoisomer; racemization
INTRODUCTION
adduct
3-(b-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-
dihyroxy-imidazo[1,2-a]purine-9-one,7,11,19–25 and a more
stable compound transformed from G-g, named N2-(car-
boxymethyl)-20-deoxyguanosine, was recently found.26
While G-mg was identified as 3-(b-D-erythro-pentofurano-
syl)-5,7-dihydro-6,7-dihyroxy-6-methyl-imidazo[1,2-a]purine-
9-one in the initial reaction21,22,27,28 and then transformed
to N2-(1-carboxylethyl)-20-deoxyguanosine after a longer
incubation time.29,30 The detailed mechanisms for the for-
mation of these open chain products, and particularly their
possible transformation processes from the cyclic adducts
are still far from being clear. Although the molecular struc-
tures of cyclic G-g and G-mg have been reported repeat-
edly, diastereoisomers present in the product19 mixture
have not yet been investigated in detail. Cyclic G-g adducts
Glyoxal and methylglyoxal are two widespread 1,2-dicar-
bonyl compounds which are toxic and mutagenic in mam-
malian cells.1,2 Both of them can be formed endogenously
from a multitude of physiological processes, such as lipid
peroxidation,3 sugar or DNA autoxidation,4,5 and DNA
damage.6 Glyoxal can also be formed from the metabolites
of some carcinogens (e.g., nitrosamines, N-nitrosomorpho-
line, or 2-nitroimidazole7–9) in vivo, which was found to
induce DNA single-strand breaks through the formation of
1,N2-glyoxal-deoxyguanosine adduct.7,10,11 Previous stud-
ies revealed that glyoxal and methylglyoxal had the most
mutagenic activities to S. typhimurium strain TA100
among nine a-dicarbonyl compounds.12 Many data also
showed that accumulation of these compounds in mamma-
lian cells could cause unscheduled DNA synthesis, DNA
single strand breaks13,14 and inhibition to DNA replica-
tion.15 Gene mutations were greatly enhanced by introduc-
tion of glyoxal or methylglyoxal and the main mutation
type was base pair substitution on G:C site.16–18
As DNA adduct formation with glyoxal and methylgly-
oxal changes base pairing and results in gene mutation, it
is highly significant and necessary to study the reactions
and the adduct structures, ideally to have their absolute
configurations. The structures of guanosine-glyoxal (G-g)
and guanosine-methylglyoxal (G-mg) mono-adducts have
been extensively investigated using nuclear magnetic reso-
nance spectroscopy. G-g was confirmed to be a cyclic
C
Additional Supporting Information may be found in the online version of
this article.
Abbreviations: CD, circular dichroism; CE, Cotton effect; G-g, guanosine-
glyoxal mono-adduct; G-mg, guanosine-methylglyoxal mono-adduct;
HPLC, high performance liquid chromatography; MSn, multistage mass
spectrometry; NMR, nuclear magnetic resonance; RSD, relative standard
deviation.
*Correspondence to: Dr. Hai Luo, Beijing National Laboratory for Molecu-
lar Sciences, College of Chemistry and Molecular Engineering, Peking
University, Beijing, 100871, China. E-mail: hluo@pku.edu.cn
Contract grant sponsor: National Natural Science Foundation of China,
Contract grant numbers: 20575006 and 20727002
Received for publication 20 June 2009; Accepted 14 April 2010
DOI: 10.1002/chir.20875
Published online 15 April 2011 in Wiley Online Library
(wileyonlinelibrary.com).
VV
2011 Wiley-Liss, Inc.

488
LAI ET AL.
flow rate 5 units, and capillary temperature 2758C. Helium was the
buffer gas and was used as the collision partner in the collision
induced dissociation (CID) experiments.
are typically a pair of diastereoisomers where the 6- and
7-hydroxyl groups are at transpositions,19,20 though a small
amount of cis-adducts has once been found at hydrolytic
equilibrium conditions.7 Nonetheless, little effort has been
so far attempted to completely separate them and to study
the absolute configuration and stability of each individual
cyclic G-g diastereoisomer. In addition, the cyclic G-mg
adducts had long been expected to have two pairs of dia-
stereoisomers, but only one guanosine mono-adduct could
be detected when human lymphocytes were treated with
methylglyoxal in vitro.27 This result suggests that there
may be significant differences among the four cyclic G-mg
diastereoisomers whose chemical stability and transforma-
tion might be important factors.
Circular dichroism (CD) spectroscopy is a powerful
method for the analysis of chiral compounds in solution.
Configurations of stereoisomers can be obtained by
using the octant rule which was originally proposed by
Moffitt, et al. as a semiempirical theory,31 which was
testified to be very useful in various ketones in particu-
lar with the help of molecular modeling.32,33 As both
G-g and G-mg have new chiral centers close to the car-
bonyl chromophore on a rigid ring, the octant rule is ap-
plicable in elucidation of the absolute configurations
from their CD spectra.
Preparation of Cyclic G-g and G-mg
Guanosine (5.6 mg) suspension in 1 mL of 50 mM sodium phos-
phate buffer (pH 7.4) was incubated with 23 lL glyoxal or 31 lL
methylglyoxal in a water bath at 378C. After 2 h, guanosine was
almost used up in both reaction solutions. Glyoxal containing mixture
was separated by HPLC with gradient 1 at a flow rate of 1 mL/min.
Methylglyoxal containing mixture was separated with gradient 2 at a
flow rate of 0.5 mL/min.
1
For H NMR analysis, scale-up preparations of G-g and G-mg were
taken under the above conditions with 10 times of the reactants’ con-
centrations. Products were separated by semipreparative HPLC at a
flow rate of 2.5 mL/min. Every HPLC fraction of interest was col-
lected and lyophilized by using a Christ Alpha1-4 lyophilizer (Christ
AG, Germany). A Varian 300M Mercury Plus NMR spectrometer
(Chicago, USA) was used to record 1H NMR data of samples in d6-
Me2SO.
CD Analysis of G-g/G-mg Diastereoisomers
Fractions for each G-g (or G-mg) product collected in HPLC (solu-
tion pH 5 6.5) was analyzed by CD immediately. The measurements
were carried out on a Jasco J-810 circular dichroism spectrometer
(Jasco UK, Great Dunmow, Essex, UK) with a 0.2 cm path cell at
room temperature. Each of the CD spectra was the average result of
three scans from 350 to 200 nm at 0.1 nm intervals with a 1 nm slit
width.
Here we report detailed studies of cyclic G-g and G-mg
adducts, using reverse phase HPLC, LC/MSn, NMR, CD
methods, and molecular modeling. The purpose of this
work is to obtain further information of these cyclic mono-
adducts, i.e., the absolute configuration for each diaster-
eoisomer, its stability and racemization at neutral condi-
tion. The detailed information of cyclic guanosine mono-
adducts with glyoxal and methylglyoxal can be very useful
to further understand the roles of these two endogenous
small molecules in biological processes.
Energy Calculation and Conformation Optimization
All the adducts’ conformations were optimized and their internal
energies were calculated by Gaussian 03W software34 using B3LYP/6-
31G(d) basis set. To simplify the calculation, the ribose group of guano-
sine was substituted by a hydrogen atom. Such treatment did not affect
the adduct conformation significantly, nor would it change interpreta-
tion for the major CD absorptions (e.g., ꢀ280 nm for the carbonyl
group) of these adducts. Additionally, the conformation of stereo-selec-
tively synthesized analog, deoxyguanosine-crotonaldehyde mono-
adduct (dG-c),35 was similarly optimized.
MATERIALS AND METHODS
Guanosine, glyoxal aqueous solution (40%) and methylglyoxal
aqueous solution (40%) were purchased from Sigma-Aldrich (St.
Louis, MO). Glyoxal and methylglyoxal have been found to be muta-
genic in bacteria and mammalian cells. Caution should therefore be
exercised in the handling of the compounds. HPLC grade acetonitrile
(ACN) was obtained from Fisher Scientific (Fair Lawn, NJ). All the
other analytical reagents were procured from Beijing Reagent Com-
pany (Beijing, China) and used without further purification.
Stability and Transformation Studies for Cyclic G-g and
G-mg Diastereoisomers
HPLC fraction for each G-g (or G-mg) product was collected (solu-
tion pH 5 6.5) and immediately incubated at 378C water bath for cer-
tain hours, during which aliquots were taken out periodically and ana-
lyzed using LC/UV/MS. The decomposition and transformation prod-
ucts of those diastereoisomers were identified by both retention
times and MSn spectra. The rate constants of the racemization reac-
tions for G-g were calculated from time-dependent changes in the
chromatographic peak areas, and the relative standard deviations
(RSD) were obtained from three individual experiments.
A Thermo Finnigan LCQ Advantage MAX ion trap mass spectrom-
eter (San Jose, CA) was used in conjunction with an HPLC system
which had a build-in variable wavelength UV detector (VWD) and the
wavelength was set at 254 nm for all the LC/UV/MS experiments.
The HPLC system and all the columns used were from Agilent Tech-
nologies (Santa Clara, CA). A ZORBAX Eclipse XDB-C18 column (2.1
3 150 mm, 3.5 lm particle size) was used for LC/MS analysis. A
ZORBAX Eclipse XDB-C18 column (4.6 3 150 mm, 5 lm particle
size) was used for general reaction mixture analysis and a ZORBAX
300SB-C18 column (9.4 3 250 mm, 5 lm particle size) was used for
semipreparation. Two elution gradients were used for separating dif-
ferent products. They are conveniently designated as: gradient 1:
eluting isocratically for 12 min with ACN and H2O (1/99, v/v), gradi-
ent 2: eluting isocratically for 25 min with ACN and H2O (5/95, v/v).
All LC/UV/MS experiments were done with the flow rate of 0.2 mL/
RESULTS AND DISCUSSION
Separation and Identification of Cyclic G-g and G-mg
Diastereoisomers
With optimized HPLC conditions, two G-g products with
almost equal amount were baseline separated (Fig. 1a)
and four G-mg products were also well separated (Fig.
1b). The chromatograms and elution requirement revealed
that these adducts were quite hydrophilic, so it was easy
min, spray voltage 4.5 kV, sheath gas flow rate 24 units, auxiliary gas to overload the reverse phase HPLC column. However,
Chirality DOI 10.1002/chir

DIASTEREOISOMERS OF CYCLIC GUANOSINE MONO-ADDUCTS
489
which could account for the four diastereoisomers (Fig.
1b). Under our experimental conditions, all G-mg can fur-
ther react with another methylglyoxal and form bis-adducts
after 48 h incubation, which is outside the scope of this arti-
cle and is now being investigated in our laboratory.
Elucidation of Configurations of Cyclic G-g and G-mg
Diastereoisomers
All of the six adducts (two G-g and four G-mg) clearly
showed Cotton effect (CE) around 280 nm in the CD anal-
ysis (see Fig. 2). G-g 1 and 2 showed mostly overlapping
curves except exhibiting opposite signals from 260 to 300
nm, which strongly confirmed the enantiotropy in these
two G-g adducts. G-mg adducts exhibited similar CD
behaviors. Considering the area of each G-mg peak in the
chromatogram (Fig. 1b), i.e., the concentration of the col-
lected fraction, and its corresponding absorption intensity
Fig. 1. HPLC chromatograms of (a) guanosine-glyoxal and (b) guano-
sine-methylglyoxal reaction mixtures. 1 and 2 in (a) stand for the two G-g
diastereoisomers, while 1, 2, 3, and 4 in (b) represent the four G-mg dia-
stereoisomers. Structures of G-g and G-mg are shown as insets and the
newly formed chiral carbons are indicated by asterisks.
the optimized HPLC methods could be used to desalt for
on-line LC-MS analysis of these adducts.
LC-MSn (n 5 1–5) experiments suggested that two G-g
adducts had identical fragmentation pattern (data not
shown), and four G-mg also showed almost the same prod-
uct ion spectra, except their MS3 spectra (Fig. S1). These
results indicated that the molecular structures of two G-g
or four G-mg were the same, but the configurations of
newly formed chiral carbons differentiated the adducts in
chromatography.
Because of complete separation, each adduct could be
1
analyzed by H NMR spectrometry with little interference
from isomeric impurities. However, such interference is in-
evitable because of the stability and racemization of these
diastereoisomers (vide infra). The two G-g diastereoisom-
1
ers had almost the same H NMR chemical shifts as the
reported results7,19 [Table S1 in supporting information
(SI)], which suggested that G-g was formed as the previ-
ously determined cyclic structure with the two hydroxyl
groups at trans positions (the inset in Fig. 1a). Little G-g
cis adduct,7 G-g carboxyl containing adduct or G-2g bis
adduct26 was observed under this condition. Almost identi-
cal 1H NMR chemical shifts for four G-mg were also
observed, suggesting the cyclic structure (Table S2 in SI)
with two hydroxyl groups at both trans and cis positions,
Fig. 2. CD spectra of (a) G-g 1 and G-g 2, (b) G-mg 1 and G-mg 3, (c)
G-mg 2 and G-mg 4.
Chirality DOI 10.1002/chir

490
LAI ET AL.
that internal energies for cis G-mg are indeed more stable
with 15.8 kJ/mol lower in energy than trans pair (Table S3
in SI). The typical value of OꢁꢁHꢁꢁO hydrogen bond is 21
kJ/mol,37 therefore, the additional stabilization in cis
adducts might be from intramolecular hydrogen bonding
between the neighboring OH groups. Thus, as G-g diaster-
eoisomers, the absolute configurations of trans G-mg 1
and 3 can be identified as (6R, 7R) and (6S, 7S), respec-
tively, but that of cis G-mg cannot be unambiguously deter-
mined with the present data (Fig. S2 in SI). The above con-
figuration assignments for G-g and trans G-mg diaster-
eoisomers can be further justified by using analog
compounds with known absolute configurations. For exam-
ple, the deoxyguanosine-crotonaldehyde mono-adducts
(dG-c) stereo-selectively synthesized by Harris and co-
workers35 have similar structures with G-g and G-mg.
Their CD observations are in very good agreement with
what we predict for dG-c using the octant rule (Figs. S3
and S4, in SI), which is a convincing proof that the classic
octant rule can be used to determine the absolute configu-
rations for this type of compounds.
The reaction of 20-deoxyguanosine with glyoxal and
methylglyoxal were also studied in our lab. The HPLC
trace of dG-g (dG-mg) was quite comparable with that of G-
g (G-mg). Additionally, LC-MSn (n 5 2–5) spectra of dG-g
(dG-mg), which corresponded to the CID results on parent
ion after loss of the deoxyribose, were identical with the
corresponding data of G-g (G-mg) (data not shown). There-
fore, the conclusion for guanosine adducts may also be ap-
plicable for the corresponding deoxyguanosine adducts.
Fig. 3. Optimized G-g (6R,7R) conformation (a) and its projections in
the octant coordinates (b and c). Note that the nodal surface A is the plane
of the paper in b and it dissects at the middle of the carbonyl double
bond. The CE signs due to the main perturbers are indicated. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Stability and Racemization Studies of Cyclic G-g and
G-mg Diastereoisomers
Each diastereoisomer in G-g pair was found to transform
to the other one when it was incubated in an almost neu-
in the CD spectra (Figs. 2b and 2c), one can easily con- tral solution at 378C. LC/UV/MS results showed that G-g
clude that G-mg 1 and 3 are a pair of diastereoisomers 1 and 2 transformed to each other at almost equal rates
while G-mg 2 and 4 are the other pair.
with little decomposition. After 45 h, the transformation
Further determination of their absolute configurations is process seemed to reach a steady state where the amount
facilitated by molecular modeling and the classic octant of G-g 1 and G-g 2 were almost equal, no matter starting
rule31,36 in CD spectroscopy. Take trans G-g (6R,7R) as an with G-g 1 or 2 (see Fig. 4). Assuming no decomposition
example. Figure 3b clearly shows that all perturbers at C-6 occurred in the first 10 h, we obtained the rate constants
and C-7 are in the right octants. Further seen from the of this racemization for G-g 1 and G-g 2 to be 0.045 h21
negative Y axis in the octant coordinate (Fig. 3c), the (RSD 5 10%) and 0.044 h21 (RSD 5 10%), respectively
hydrogen atom of OH-7 is in the upper front octant, which (see the supporting information for details).
gives a positive CE contribution, while the oxygen atom is
In comparison with G-g, the racemization of G-mg iso-
just cross the nodal surface A, exhibiting no CE; the H-7 is mers was much more complicated under the same condi-
in the lower back octant, though it is close to the nodal tions (see Fig. 5). The trans pair G-mg 1 and 3 quickly
surface A, still giving a positive CE contribution. OH-6 and decomposed to the reactants and also underwent racemi-
H-6 are distant from the carbonyl group and they are in zation between each other in the first incubation hour. Af-
the lower and upper back octant, respectively. Their addi- ter 6 h, the amount ratio of G-mg 1/G-mg 3 reached about
tive CE contributions should be slightly positive as OH 1:1 (no matter starting with G-mg 1 or 3). In contrast, the
group is larger. Taking these together, G-g (6R, 7R) transformations of cis isomers (G-mg 2 and 4) occurred
should show a positive CD signal around 280 nm which is not only between each other, but also to the trans pair.
observed for G-g 1. Similarly, G-g 2 can be assigned with Except to the transformed G-mg and decomposition prod-
absolute configuration of (6S, 7S) (Fig. S2 in SI).
uct guanosine, the LC/UV/MS traces only showed
Our stability and racemization studies suggest that one another minor product (Fig. 5, indicated by an asterisk)
pair of G-mg (G-mg 2 and 4) are more stable than the with a molecular weight of 313. Its quantity increased
other pair (G-mg 1 and 3) under the same conditions steadily during the incubation process. It might be a deg-
(vide infra). Calculations by Gaussian software also reveal radation product whose identity and formation mechanism
Chirality DOI 10.1002/chir

DIASTEREOISOMERS OF CYCLIC GUANOSINE MONO-ADDUCTS
491
of the 6-h and the 24-h incubation mixtures, the G-mg
transformation was believed to reach its equilibrium in the
first 6 h. Compared with the behavior of G-g isomers,
decomposition was the main reaction channel for G-mg.
Cis isomers(G-mg 2 and 4)behaved more stable than the
trans pair (G-mg 1 and 3), and could generate trans struc-
tures through racemization. Meanwhile, the trans isomers,
just like the G-g pair, only underwent racemization (except
some unknown side reactions) between each other even
in prolonged 24-h incubation.
There were some pH-dependent studies on the stability
of G-g adducts in the previous reports. For example, Sha-
piro38 and Montoya39 independently revealed that cyclic
G-g was more stable in acidic solution, while Loeppky7
suggested that the equilibrium constant for dG-g dissocia-
tion decreases with increasing pH due to the formation of
an unknown complex between dG-g and hydroxyl anion.
However, we here aimed to follow the transformation reac-
tions of cyclic G-g and G-mg mainly at close to neutral con-
dition, expecting to study the transformation behaviors
between various isomers in a pseudo physiological envi-
ronment. Comparing the stability results of G-g and G-mg,
one could conclude that G-mg was less stable than G-g in
a neutral solution, which was in agreement with the previ-
ous report that G-mg had a smaller formation constant
than G-g under neutral condition.39
The obviously different racemization behaviors for cis
and trans isomers in an almost neutral solution suggest
different mechanisms for these mono-adduct transforma-
tion. The simple dissociation/reassociation model of a re-
versible formation of the hemiaminal link cannot explain
the difference. We hypothesized that the distinct behav-
iors between cis and trans adducts might be due to their
different dehydrolyzed intermediates. In G-g, G-mg 1 and
3, the OH-6, and OH-7 groups are at trans positions. Dur-
ing the incubation, these isomers may experience an ep-
oxy intermediate structure with the loss of a H2O molecule
via SN2 mechanism in the slightly acidic solution. When
another H2O attacks C-6 or C-7, because of the spatial hin-
drance, it could only attack from the back of the epoxy
structure, which results in the reformation of two transpo-
sitioned hydroxyl groups (Fig. 6a). For G-mg 2 and 4,
however, the intermediate structure after loss of a H2O
molecule might be planar at the enol segment. Another
H2O can freely attack C-6 or C-7 from both sides of the pla-
nar segment and consequently to form trans and cis struc-
tures (Fig. 6b). Different dissociation patterns for trans
and cis G-mg were observed in LC-MS3 analysis. The two
pairs of G-mg gave two different MS3 spectra under the
same CID conditions (Fig. S1, in SI), where relative inten-
sity of the daughter ion [M-H2O]1 was about two times
higher for cis G-mg than trans one, suggesting the former
ion is more stable than the later. In general consideration,
an enol structure in a partial aromatic system could be
more stable than an epoxy ion structure. Though there
were no such intermediates directly detected in the incu-
bation solution, the observed intrinsic discrepancies of
trans and cis G-mg supported this proposal to a large
extent.
Fig. 4. Chromatograms of (a) G-g 1 and (b) G-g 2 at 0 and 45 h of the
incubation clearly indicate the transformation. The integration curves that
are used to calculate the transformation rate constants are presented in
the Supporting Information.
are not yet clear. The relative amounts of G-mg transfor-
mation products are summarized in Table 1. The large
deviations observed are mostly due to the decomposition
reaction, though it is not surprise to see smaller relative
standard deviations for the most abundant peaks. As there
was no significant difference between the chromatograms
Chirality DOI 10.1002/chir

492
LAI ET AL.
Fig. 5. HPLC studies of the transformation from (a) G-mg 1, (b) G-mg 2, (c) G-mg 3 and (d) G-mg 4. The sign of ; points to the initial compound
which decomposes and/or transforms into the other structures which are indicated by : during the incubation time. Unidentified products are indicated
by asterisks. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
intermediates for the racemization processes were proposed
to explain the observations. As guanosine residue is the hot
CONCLUSIONS
spot in DNA adduct formation with methylglyoxal, these
Cyclic guanosine mono-adducts with glyoxal and methyl-
results may shed light on the possible involvement of differ-
glyoxal were studied in detail, particularly with respect to
ent types of G-mg adducts in mutagenesis.
their absolute configurations and transformation characteris-
tics under nearly neutral conditions. Under the experimental
conditions, these mono-adducts had the following stability
order: G-g > cis G-mg > trans G-mg. Cis G-mg was further
found to have different racemization behavior from that of
SURPPORTING INFORMATION
The supporting information includes: LC-MSn (n 5 1–5)
1
spectra of G-mg, H NMR data and Gaussian calculations
trans G-mg, and mechanistic pathways involving different of G-g and G-mg diastereoisomers, CD responses of G-g,
TABLE 1. Relative amounts of transformation products from four G-mg isomers in a weak acidic solution (pH 5 6.5)a
Transformation products
Transformation
starting from
Guanosine
G-mg 1
G-mg 2
G-mg 3
G-mg 4
G-mg 1
G-mg 2
G-mg 3
G-mg 4
78.4 (10.9)
37.0 (16.5)
83.9 (10.0)
29.8 (20.3)
11.6 (5.8)
0.5 (0.8)
7.1 (4.4)
0.9 (1.1)
N/A
58.5 (16.6)
N/A
10.0 (5.1)
0.3 (0.6)
9.0 (5.6)
0.8 (1.3)
N/A
3.7 (1.3)
N/A
5.5 (2.1)
63.0 (20.1)
N/A stands for ‘‘none of this product being observed in HPLC.’’
aThe incubation time is 6 h and the temperature is 378C. Values in the table are average percentages of the peak areas of the transformation products
with respect to that of all the five compounds detected in HPLC. Values in the parentheses are the standard deviations of 3 independent experiments.
Chirality DOI 10.1002/chir

DIASTEREOISOMERS OF CYCLIC GUANOSINE MONO-ADDUCTS
493
10. Loeppky RN, Fuchs A, Janzowski C, Humberd C, Goelzer P, Schnei-
der H, Eisenbrand G. Probing the mechanism of the carcinogenic acti-
vation of N-nitrosodiethanolamine with deuterium isotope effects: in
vivo induction of DNA single-strand breaks and related in vitro assays.
Chem Res Toxicol 1998;11:1556–1566.
11. Chung FL, Hecht SS. Formation of the cyclic 1,N2-glyoxal-deoxygua-
nosine adduct upon reaction of N-nitroso-2-hydroxymorpholine with
deoxyguanosine. Carcinogenesis 1985;6:1671–1673.
12. Dorado L, Montoya MR, Mellado JMR. A contribution to the study of
the structure-mutagenicity relationship for alpha-dicarbonyl com-
pounds using the Ames test. Mutat Res 1992;269:301–306.
13. Kielhorn J, Pohlenz-Michel C, Schmidt S, Mangelsdorf I. Glyoxal. In:
Concise international chemical assessment document 57. Geneva:
World Health Organization Press; 2004.
14. Kalapos MP. Methylglyoxal toxicity in mammals. Toxicol Lett
1994;73:3–24.
15. Klamerth OL. Influence of glyoxal on cell function. Biochim Biophys
Acta 1968;155:271–279.
16. Murata-Kamiya N, Kamiya H, Kaji H, Kasai H. Glyoxal, a major prod-
uct of DNA oxidation, induces mutations at G:C sites on a shuttle vec-
tor plasmid replicated in mammalian cells. Nucleic Acids Res
1997;25:1897–1902.
17. Murata-Kamiya N, Kaji H, Kasai H. Deficient nucleotide excision
repair increases base-pair substitutions but decreases TGGC frame-
shifts induced by methylglyoxal in Escherichia coli. Mutat Res Genet
Toxicol Environ Mutagen 1999;442:19–28.
18. Marnett LJ, Basu AK, O’Hara SM. The role of cyclic nucleic acid
adducts in the mutational specificity of malondialdehyde and beta-sub-
stituted acroleins in Salmonella. IARC Sci Publ 1986;70:175–183.
19. Olsen R, Molander P, Ovrebo S, Ellingsen DG, Thorud S, Thomassen
Y, Lundanes E, Greibrokk T, Backman J, Sjoholm R, Kronberg L.
Reaction of glyoxal with 20-deoxyguanosine, 20-deoxyadenosine, 20-
deoxycytidine, cytidine, thymidine, and calf thymus DNA: identifica-
tion of DNA adducts. Chem Res Toxicol 2005;18:730–739.
Fig. 6. Possible mechanisms for the racemization of G-g and G-mg.
Trans (a) and cis (b) structures have different reaction pathways.
20. Awada M, Dedon PC. Formation of the 1,N2-glyoxal adduct of deoxy-
guanosine by phosphoglycolaldehyde, a product of 30-deoxyribose oxi-
dation in DNA. Chem Res Toxicol 2001;14:1247–1253.
G-mg, dG-c diastereoisomers and their structure projec-
tions in the octant coordinates, calculation of the racemiza-
tion rate constants of cyclic G-g adducts.
21. Shapiro R, Hachmann J. The reaction of guanine derivatives with 1,2-
dicarbonyl compounds. Biochemistry 1966;5:2799–2807.
22. Shapiro R, Cohen BI, Shiuey SJ, Maurer H. On the reaction of gua-
nine with glyoxal, pyruvaldehyde, and kethoxal, and the structure of
the acylguanines. A new synthesis of N2-alkylguanines. Biochemistry
1969;8:238–245.
LITERATURE CITED
1. Kalapos MP. Methylglyoxal in living organisms chemistry, biochemistry,
toxicology and biological implications. Toxicol Lett 1999;110:145–175.
23. Ruohola AM, Koissi N, Andersson S, Lepisto I, Neuvonen K, Mikkola
S, Lonnberg H. Reactions of 9-substituted guanines with bromomalon-
dialdehyde in aqueous solution predominantly yield glyoxal-derived
adducts. Org Biomol Chem 2004;2:1943–1950.
2. Schauenstein E, Esterbauer H, Zollner H. Aldehydes in biological sys-
tems. Their natural occurrence and biological activities. Great Britain:
Pion Ltd; 1977.205p.
24. Czarnik AW, Leonard NJ. Unequivocal assignment of the skeletal struc-
ture of the guanine-glyoxal adduct. J Org Chem 1980;45:3514–3517.
3. Loidl-Stahlhofen A, Spiteller G. Alpha-hydroxyaldehydes, products of
lipid peroxidation. Biochim Biophys Acta 1994;1211:156–160.
25. Shapiro R, Cohen BI, Clagett DC. Specific acylation of the guanine
residues of ribonucleic acid. J Biol Chem 1970;245:2633–2639.
4. Wells-Knecht KJ, Zyzak DV, Litchfield JE, Thorpe SR, Baynes JW.
Mechanism of autoxidative glycosylation: identification of glyoxal and
arabinose as intermediates in the autoxidative modification of proteins
by glucose. Biochemistry 1995;34:3702–3709.
26. Pluskota-Karwatka D, Pawlowicz AJ, Tomas M, Kronberg L. Forma-
tion of adducts in the reaction of glyoxal with 20-deoxyguanosine and
with calf thymus DNA. Bioorg Chem 2008;36:57–64.
5. Glomb MA, Monnier VM. Mechanism of protein modification by gly-
oxal and glycolaldehyde, reactive intermediates of the Maillard reac-
tion. J Biol Chem 1995;270:10017–10026.
27. Vaca CE, Fang JL, Conradi M, Hou SM. Development of a P-32 Postlab-
eling method for the analysis of 20-deoxyguanosine-30-monophosphate
and DNA-adducts of methylglyoxal. Carcinogenesis 1994;15:1887–1894.
6. Murata-Kamiya N, Kamiya H, Iwamoto N, Kasai H. Formation of a mu-
tagen, glyoxal, from DNA treated with oxygen free radicals. Carcino-
genesis 1995;16:2251–2253.
28. Vaca CE, Nilsson JA, Fang JL, Grafstrom RC. Formation of DNA
adducts in human buccal epithelial cells exposed to acetaldehyde and
methylglyoxal in vitro. Chem Biol Interact 1998;108:197–208.
7. Loeppky RN, Cui W, Goelzer P, Park M, Ye Q. Glyoxal-guanine DNA
adducts: detection, stability and formation in vivo from nitrosamines.
IARC Sci Publ 1999;150:155–168.
29. Papoulis A, Alabed Y, Bucala R. Identification of N-2-(1-carboxyethyl)-
guanine (Ceg) as a guanine advanced glycosylation end-product. Bio-
chemistry 1995;34:648–655.
8. Chung FL, Hecht SS, Palladino G. Formation of cyclic nucleic acid
adducts from some simple alpha, beta-unsaturated carbonyl com-
pounds and cyclic nitrosamines. IARC Sci Publ 1986;70:207–225.
30. Frischmann M, Bidmon C, Angerer J, Pischetsrieder M. Identification of
DNA adducts of methylglyoxal. Chem Res Toxicol 2005;18:1586–1592.
9. Whitmore GF, Varghese AJ, Gulyas S. Reaction of 2-nitroimidazole
metabolites with guanine and possible biological consequences. IARC
Sci Publ 1986;70:185–196.
31. Moffitt W, Woodward RB, Moscowitz A, Klyne W, Djerassi C. Struc-
ture and the optical rotaory dispersion of saturate ketones. J Am
Chem Soc 1961;83:4013–4018.
Chirality DOI 10.1002/chir

494
LAI ET AL.
32. Gergely A, Zsila F, Horvath P, Szasz G. Determination of absolute con-
figuration of ketamine enantiomers by HPLC-CD-UV technique. Chir-
ality 1999;11:741–744.
Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL,
Fox DJ, Keith T, Laham A, Peng CY, Nanayakkara A, Challacombe
M, Gill PMW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople JA.
Gaussian 03, Revision B. 05; Gaussian Inc.: Pittsburgh, PA, 2003.
33. Butkus E, Berg U, Zilinskas A, Kubilius R, Stoncius S. Enantiomer
separation and absolute configuration of densely functionalized 2-oxa-
tricyclo[4.3.1.0(3,8)]decanes by CD spectroscopy and chemical corre-
lation. Tetrahedron: Asymmetry 2000;11:3053–3057.
35. Brock AK, Kozekov ID, Rizzo CJ, Harris TM. Coupling products of
nucleosides with the glyoxal adduct of deoxyguanosine. Chem Res
Toxicol 2004;17:1047–1056.
34. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheese-
man JR, Montgomery JA, Vreven T, Kudin KN, Burant JC, Millam
JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani
G, Rega N, Petersson GA, 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 JE, Hratchian HP, Cross JB, Bakken V,
Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin
AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth
GA, Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels
AD, Strain MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K,
Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J,
36. Lightner DA. Determination of absolute configuration by CD. Applica-
tions of the octant rule and the exciton chirality rule. In: Purdie N,
Brittain HG, editors. Analytical applications of circular dichroism,
Chapter 5. Amsterdam: Elsevier; 1994. p129–204.
37. Emsley J. Very strong hydrogen bonds. Chem Soc Rev 1980;9:91–124.
38. Shapiro R, Sodum RS, Everett DW, Kundu SK. Reactions of nucleo-
sides with glyoxal and acrolein. IARC Sci Publ 1986;70:165–173.
39. Montoya MR, Mellado JMR. Addition of alpha-dicarbonyl compounds
to guanine and guanosine. Gazz Chim Ital 1995;125:309–313.
Chirality DOI 10.1002/chir