Structural studies of malonaldehyde-glyoxal and malonaldehyde-methylglyoxal etheno adducts of adenine nucleosides based on spectroscopic methods and DFT-GIAO calculations

Article abstract of DOI:10.1039/c5nj02835c

Etheno adducts are formed in the reactions of DNA bases with chloroacetaldehyde, with lipid peroxidation products, and also with metabolites of vinyl chloride and furan. The presence of such modifications in the genetic material may lead to errors in replication with consequences of mutations and even carcinogenesis. For an understanding of the biological significance of etheno adducts it is important to determine their structures. Structural identification is also essential for using these adducts as inflammatory or cancer biomarkers. This paper reports structural studies on two adducts formed in the reactions of malonaldehyde and glyoxal with adenosine (M₁Gx-A), and malonaldehyde and methylglyoxal with 2′-deoxyadenosine (M₁MGx-dA). NMR spectroscopy and theoretical methods have been used. DFT-GIAO calculations were performed at M06/6-311++G(2df,2pd), B3LYP/6-311++G(2df,2pd) and M06/6-31++G(d,p) levels both in the gas phase and taking into account the effect of solvents (water, methanol and DMSO) using PCM approximation. It has been shown that when M06 or B3LYP functionals with the 6-311++G(2df,2pd) basis set are used, ¹H NMR chemical shifts very close to experimental values are obtained and that the results of GIAO calculations at the M06/6-31++G(d,p) level have a better correlation with measured ¹³C NMR chemical shift values. PCM improves the correlation of results in both cases.

Full text of DOI:10.1039/c5nj02835c

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Structural studies of malonaldehyde–glyoxal and
malonaldehyde–methylglyoxal etheno adducts of
adenine nucleosides based on spectroscopic
methods and DFT-GIAO calculations†
Cite this:DOI: 10.1039/c5nj02835c
Kinga Salus, Marcin Hoffmann,* Bozena Wyrzykiewicz and
˙
Donata Pluskota-Karwatka*
Etheno adducts are formed in the reactions of DNA bases with chloroacetaldehyde, with lipid
peroxidation products, and also with metabolites of vinyl chloride and furan. The presence of such
modifications in the genetic material may lead to errors in replication with consequences of mutations
and even carcinogenesis. For an understanding of the biological significance of etheno adducts it is
important to determine their structures. Structural identification is also essential for using these adducts as
inflammatory or cancer biomarkers. This paper reports structural studies on two adducts formed in the
reactions of malonaldehyde and glyoxal with adenosine (M1Gx-A), and malonaldehyde and methylglyoxal
with 20-deoxyadenosine (M1MGx-dA). NMR spectroscopy and theoretical methods have been used.
DFT-GIAO calculations were performed at M06/6-311++G(2df,2pd), B3LYP/6-311++G(2df,2pd) and
M06/6-31++G(d,p) levels both in the gas phase and taking into account the effect of solvents (water,
methanol and DMSO) using PCM approximation. It has been shown that when M06 or B3LYP functionals
Received (in Montpellier, France)
14th October 2015,
Accepted 26th February 2016
1
with the 6-311++G(2df,2pd) basis set are used, H NMR chemical shifts very close to experimental values are
DOI: 10.1039/c5nj02835c
obtained and that the results of GIAO calculations at the M06/6-31++G(d,p) level have a better correlation
with measured 13C NMR chemical shift values. PCM improves the correlation of results in both cases.
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polymerases27 and taq polymerases in PCR28) and metabolism
of repair enzymes (particularly glycosylases29,30) are impaired.
Introduction
Etheno adducts, 1,N6-etheno-20-deoxyadenosine, 3,N4-etheno- Etheno DNA adducts may lead to conservation of mutations
20-deoxycytidine, 1,N2-etheno-20-deoxyguanosine and N2,3-etheno- and as a consequence to initiation of carcinogenesis or to cell
20-deoxyguanosine, are structural modifications of DNA bases apoptosis.27,31 When etheno RNA adducts are formed, impaired
in which an ethylene unit bridges two nucleophilic sites. cellular processes related to mRNA and epigenetic control may
These derivatives may be formed as a result of interactions occur.7
between the genetic material and lipid peroxidation carbonyl
products.1–16
It is important to determine the structure of etheno adducts
considering their potential use in biochemistry and medicine
Etheno adducts are also formed in the reactions of DNA as biomarkers of oxidative stress, which enhances lipid peroxi-
bases with chloroacetic aldehyde.17 Furthermore, xenobiotics, dation.32–34 Quantitative and qualitative etheno adduct bio-
such as furan,18 tetrahydrofuran19 and vinyl halides,20 generate monitoring may be an indicator of various pathologies, including
etheno adducts after their biotransformation.
a measure of cancer risk or an early stage of cancer.32–34
Etheno adducts exhibit promutagenic and procarcinogenic
Glyoxal and methylglyoxal are endogenously formed DNA
properties.21,22 The formation of etheno DNA adducts may damaging agents.35,36 These extremely reactive electrophiles
affect the structure and stability of the double helix through were identified as precursors of advanced glycation end pro-
disturbed base pairing23–26 or else cause changes in glycosidic ducts (AGEs) which comprise a structurally diverse class of DNA
bond conformation in the modified nucleoside.23 As a con- and protein modifications formed in living organisms.37,38
sequence, DNA metabolism (this affects particularly human While protein AGEs are believed to be involved in the pathol-
ogies associated with aging, cancer, Alzheimer’s disease and
diabetes-related complications,39,40 little is known about the
´
Adam Mickiewicz University in Poznan, Faculty of Chemistry, Umultowska 89b,
structural features of this type of DNA lesion. Aldehydes con-
´
61-614 Poznan, Poland. E-mail: mmh@amu.edu.pl, donatap@amu.edu.pl
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nj02835c tinuously generated during a number of cellular processes can
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The largest errors were seen for C7 and C8 atoms of the etheno
ring. Therefore, we performed further thorough spectroscopic
investigation and calculations for alternative structures with
the dialdehyde methyl substituent at C7 (Fig. 2) and on the
basis of the obtained results we found that in both adducts the
dialdehyde methyl group was attached to this carbon atom.
Experimental section
Materials
1,1,3,3-Tetramethoxypropane (TMP), 20-deoxyadenosine mono-
hydrate, adenosine, methylglyoxal solution (40 wt% in H2O),
glyoxal solution (40 wt% in H2O), NH4HCO3, acetonitrile (gradient
grade for chromatography), D2O (containing 0.05% TSP-d4),
CD3OD (containing 0.03% TMS) and DMSO-d6 (containing
0.03% TMS) were purchased from Sigma-Aldrich.
Fig. 1 Structures of M1Gx-A and M1MGx-dA.43
Chromatographic methods
Separation and purification of M1Gx-A and M1MGx-dA were
carried out using an Agilent 1200 Series HPLC system consist-
ing of a binary pump (G1312A), a vacuum degasser (G1379B),
an autosampler (G1329A), a thermostatted column compart-
ment (G1316A), a diode-array detector (UV; G1315B), a fraction
collector (G1364C), and an Agilent ChemStation data handling
program (Agilent Technologies). Isolation was performed under
semi-preparative conditions: 5 mm and 10 ꢀ 250 mm (Hypersil
BDS, Thermo Scientific) reversed-phase C18 column; flow was
set to 3 mL minꢁ1; the thermostat was set to 25 1C; 1.2 mM
NH4HCO3 and ACN were used as eluents.
coexist in biological tissues and can modify biopolymers in a
synergistic manner.41,42
Recently, we synthesized etheno derivatives of adenine
nucleosides formed in reactions with malonaldehyde and
glyoxal (M1Gx-A), and with malonaldehyde and methylglyoxal,
(M1MGx-dA).43 Data resulting from spectroscopic methods
suggested that a dialdehyde methyl moiety was present at
position C8. However, results of our most recent preliminary
studies on M1Gx-A and M1MGx-dA reactivity toward nucleophilic
biomolecules may indicate that the substituent can also be
attached to C7. Therefore, structural studies of both adducts were
undertaken in which computational together with experimental
(NMR spectroscopy) methods were used. Quantum chemical
General procedure for M1Gx-A and M1MGx-dA synthesis
methods provide useful information for determining structures The M1Gx-A and M1MGx-dA adducts were synthesized using
of organic compounds, e.g. by predicting NMR chemical shifts. In a modified procedure developed earlier in our laboratory.43
this study the GIAO method was applied, which is currently the Malonaldehyde was prepared in situ by the acidic hydrolysis of
most widely used method in such investigations.44–58
1,1,3,3-tetramethoxypropane (TMP).59
Calculations of 1H and 13C NMR chemical shifts were performed
Nucleosides (adenosine (170 mg, 0.64 mmol) and 20-deoxy-
for M1Gx-A and M1MGx-dA structures with a dialdehyde methyl adenosine monohydrate (170 mg, 0.63 mmol)) were dissolved
substituent at C843 (Fig. 1). Absolute errors were determined in 15 mL of 0.5 M phosphate buffer at pH = 4.6. Two portions of
by comparison between calculated and experimental values.35 TMP (550 mg, 3.3 mmol) were mixed with 2 mL of 0.1 M HCl
and stirred at 50 1C for 15 min. Glyoxal (2 mL, 17 mmol) and
methylglyoxal (4 mL, 26 mmol) were added separately to the
hydrolyzed TMP. The pH of the resulting solutions was
adjusted to 4.6 with 1 M NaOH. The malonaldehyde–glyoxal
mixture was then added to the solution of adenosine, and the
malonaldehyde–methylglyoxal mixture was combined with
20-deoxyadenosine solution. The reactions were performed at
50 1C for 18 h. The mixtures were then concentrated and passed
through a preparative C18 column. The column was eluted with
H2O (300 mL) and with a gradient of 1% to 10% ACN in H2O.
The fractions containing products were combined respectively,
concentrated and subjected to further isolation using a semi-
preparative C18 column. M1Gx-A isolation was carried out in a
gradient of 0% to 0.7% ACN for 19 min, and then of 0.7% to
15% ACN for 4 min. For the purification of M1MGx-dA the column
Fig. 2 Alternative structures for M1Gx-A and M1MGx-dA.
was eluted with a gradient of 0% to 0.7% ACN for 21 min,
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and then of 0.7% to 20% ACN for 5 min. The solutions Results and discussion
containing pure adducts were combined, evaporated to dryness
and lyophilized. The resulting M1Gx-A (13 mg, yield: 3.6%) and
M1MGx-dA (24 mg, yield: 6.7%) were characterized by NMR.
Synthesis of malonaldehyde–glyoxal and malonaldehyde–
methylglyoxal etheno adducts of adenine nucleosides
M1Gx-A was formed in the reaction of malonaldehyde and glyoxal
with adenosine, while M1MGx-dA was prepared in the reaction
of malonaldehyde and methylglyoxal with 20-deoxyadenosine43
(Fig. 3). The optimization of the synthesis conditions resulted
in the product yields of 3.6% and 6.7%, respectively.
NMR spectroscopy
Samples were dissolved in D2O (internal standard: 3-(trimethyl-
silyl)propionic-2,2,3,3-d4 acid sodium salt, TSP-d4), CD3OD or
DMSO-d6 (internal standard: tetramethylsilane, TMS). The
experiments were performed at the temperature of 298 K. All
spectra were acquired on a Bruker Avance III DRX system,
operating at frequencies of 600.3 MHz (1H) and 150.9 MHz
(13C). The spectrometer was equipped with a 5 mm triple-
resonance inverse probe head [1H/31P/BB]. High-power 1H,
13C p/2 pulses of 9.00 and 15.00 ms, respectively, were used.
1D and 2D homo- and heteronuclear correlation experiments
were carried out using pulse sequences from the Bruker pulse
program library.
Mechanism for the formation of the studied adducts
M1Gx-A and M1MGx-dA belong to the group of conjugate
adducts – the nucleoside modifications that comprise units
derived from the condensation products of carbonyl compounds.
The presence of malonaldehyde in the reaction mixtures results
in the formation of dimers and trimers of this aldehyde, but also
of conjugates with glyoxal and methylglyoxal, respectively. Multi-
meric adducts of malonaldehyde were proposed to be formed by
reactions of this aldehyde oligomers with the DNA bases.61 An
analogous mechanism was suggested for the formation of the
nucleoside–malonaldehyde–acetaldehyde conjugate adducts62,63
and a similar mechanism is most likely valid also for the formation
of M1Gx-A and M1MGx-dA. We proposed that these adducts are
derived from the reactions of adenine nucleosides with the initially
formed malonaldehyde–glyoxal and malonaldehyde–methylglyoxal
conjugates, respectively. The reaction is thought to take place
through a two-step addition, however the mechanism for this
addition is yet to be clarified in further kinetics studies, which
is beyond the scope of the current paper.
Calculation methods
The initial structures of M1Gx-A, M1MGx-dA and NMR standards:
tetramethylsilane (TMS) and 3-(trimethylsilyl)-2,20,3,30-tetra-
deuteropropionic acid (TSP-d4) were generated based on aver-
age bond lengths and valence angles; torsional angles for
rotatable bonds were sampled randomly. All initial conformers
of M1Gx-A (11 of enol and 11 of dialdehyde forms), M1MGx-dA
(11 of enol and 13 of dialdehyde forms), TMS and TSP
(1 of anion and 7 neutral forms) were optimized at the
M06/6-31G(d) level.
Reactions between a,b-unsaturated aldehydes and 20-deoxy-
guanosine are supposed to take place through Michael addition
occurring by N2 or N1 of the nucleoside at the b-carbon atom
followed by nucleophilic attack of N1 or N2 of dG at the carbon
atom of the aldehyde group.64 The presence of a substituent
at the b-carbon atom can preclude initial attack by N1.64
Reactions of a,b-unsaturated aldehydes with 20-deoxyadenosine
are suggested to occur by the addition of N6 of dA to the carbonyl
carbon atom with subsequent ring closure by attack of N1 at C2
of the carbonyl compound.2,5
The lowest-energy conformers were selected for further
research (one each for the enol and dialdehyde form for each
adduct) and for the NMR standards. NMR shielding constants
were calculated for the structures using GIAO at the following
levels: M06/6-311++G(2df,2pd), B3LYP/6-311++G(2df,2pd) and
M06/6-31++G(d,p). The calculations were performed for the
compounds in the gas phase and also taking into account
the effect of solvents (water, methanol and DMSO) using the
polarizable continuum model (PCM). Geometry was optimized
and GIAO calculations were performed using Gaussian 09.60
1
Calculations of H and 13C NMR chemical shifts (scalc [ppm])
were based on the isotropic magnetic shield tensor (IMS)
[ppm]. scalc values [ppm] were derived as usual55 by subtracting
IMS values for the test structures from the IMS value of the
NMR standard (TSP for calculations in the gas phase and water
or TMS for calculations in the gas phase, methanol and DMSO),
that is: scalc = IMSref ꢁ IMScalc
.
The NMR chemical shifts calculated using quantum chemical
methods were compared with the experimental values. Compar-
isons for the enol and dialdehyde forms of the studied compounds
were made for each solvent. Absolute errors of NMR chemical
shifts and mean absolute errors (MAEs) were calculated. The
experimental and calculated results were also compared using
linear regression.
Energy barriers for proton transfer in the b-dicarbonyl
moieties in M1Gx-A and M1MGx-dA were calculated using the
M06/6-31++G(d,p)//M06/6-31G(d) level.
Fig. 3 Formation of M1Gx-A and M1MGx-dA.
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Due to the distinctive structure of malonaldehyde–glyoxal and
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malonaldehyde–methylglyoxal conjugates all plausible routes
leading to the formation of M1Gx-A and M1MGx-dA should be
considered. One mechanism that can be proposed, involves
Michael-type addiction of N6 of the adenine nucleosides to the
b-carbon atom followed by addition of the nucleoside N1 to the
carbonyl group derived from glyoxal or methylglyoxal, respec-
tively (ESI,† Fig. S1A). An alternative mechanistic explanation for
M1Gx-A and M1MGx-dA formation is based on 1,4-addition of N1
to the conjugate and subsequent attack of N6 at the appropriate
carbonyl carbon atom (ESI,† Fig. S1B). Preference of the first
route would result in the formation of adducts with the dialde-
hyde methyl substituent at C8, while the second mechanism
would give rise to compounds having this substituent at C7.
Discussion on the M1Gx-A and M1MGx-dA formation is
additionally complicated by the fact that in both plausible
mechanisms the opposite order of the addition reactions could
not be ruled out (ESI,† Fig. S1C and D, respectively).
Fig. 4 Major correlations in the HMBC spectrum (D2O) (A) and in the
NOESY spectrum (DMSO-d6) (B) of M1Gx-A.
the NOESY spectrum of the adduct showed correlation between
the aldehyde protons and the H-C5 proton (Fig. 4B). Therefore, the
dialdehyde methyl substituent was assumed to be at position C7.
This was additionally confirmed by the lack in the NOESY
spectrum of M1Gx-A of correlation between the H-C5 proton
and proton of the etheno ring. This correlation should be strong
if the dialdehyde methyl group was bonded to the C8.
In the etheno ring of M1MGx-dA neither C7 nor C8 is bonded
to the proton and correlation observed in the HMBC spectrum
between the H-C5 proton signal and one of these carbon atom
signals is not sufficient to distinguish one of these atoms from
the other (Table 2 and Fig. 5A). Therefore, correlations observed
in the NOESY spectrum between the H-C5 proton and the
aldehyde protons were crucial for the assignment of the signal
at d 117.65 ppm (Table 2 and Fig. 5B) to the C7 carbon atom
bearing the dialdehyde methyl group.
Based on the presence of respective correlations in the NOESY
spectra, and also on the lack thereof, the structures of other
substituted etheno adducts were determined.9,65 The position of
the 2-oxobutyl group in the etheno ring of the 20-deoxyadenosine
derivative formed in a reaction between the nucleoside and 4-oxo-
2-hexanal9 as well as the position of the methyl group in etheno
adenosine derivatives was determined.65 The 4-oxobutyl substi-
tuent at position C7 was additionally confirmed by correlation
between protons of the methylene group of the substituents
bonded to the etheno ring and the adenosine N1 atom seen in
the 1H–15N HMBC spectrum.9
Determination of the dialdehyde methyl substituent position in
M1Gx-A and M1MGx-dA on the basis of spectroscopic analysis
1H NMR, 13C NMR and correlation spectra (COSY, HSQC, HMBC
and NOESY) were recorded for the M1Gx-A and M1MGx-dA
adducts.
The assignment of the appropriate signals from 13C NMR
spectra to the etheno ring carbon atoms C7 and C8 was
essential for the determination of the dialdehyde methyl group
position. The assignment of the signal observed in the 13C NMR
spectrum of M1Gx-A at d 133.32 ppm (Table 1) to the carbon
atom C8 was based on the one-bond correlation (HSQC) with
the proton signal at d 7.49 ppm (Table 1) and long-range
correlations (HMBC) between this proton signal and carbon
signals derived from C7, C100, CHO and C9a (Fig. 4A). The
assignment of the signal at d 121.55 ppm to the carbon atom C7
was achieved on the basis of correlation observed in the HMBC
spectrum between this signal and the H-C5 proton signal
(Table 1 and Fig. 4A), while no correlation were seen between
the signal assigned to C8 and H-C5 signals (Fig. 4A). Furthermore,
Structural determination of M1Gx-A and M1MGx-dA based on
comparison between calculated and experimental chemical
shift values
Table 1 NMR data of M1Gx-A (D2O). (NMR data derived from spectra
recorded in CD3OD and DMSO-d6 and NOESY data – Tables S2–S4 in the ESI)
Most of the M1Gx-A and M1MGx-dA conformers selected for
GIAO calculations (Fig. 6) had syn conformation, even though it is
generally assumed that the anti conformation prevails in DNA, in
nucleosides and nucleotides. However, results of spectroscopic
studies combined with calculations indicated that adenine
nucleosides take both conformations at a similar level in
solution.66 Furthermore, results based on quantum chemical
calculations suggest that syn conformers of nucleoside deriva-
tives are more stable in aqueous media.67 Etheno adducts
reveal a tendency for syn conformation in double-strand DNA
that facilitates the formation of Hoogsteen base pairs.23,68
Furthermore, etheno adducts in the syn conformation are more
susceptible to DNA repair processes.69
d(H)
JH,H
d(C)
[ppm] HMBC
[ppm] Multiplicity [Hz]
2 CHO 8.98
s
193.21 C7
110.58
C100
C3a
H-C2
H-C5
H-C7
C8
142.85
8.46
8.60
s
s
143.77 C3a, C9b, C10
139.89 C3a, C9a, C9b, C7
121.55
7.49
s
133.32 C7, C100, CHO, C9a
141.56
C9a
C9b
125.13
H-C10 6.21
H-C20 4.90
d
t
5.43
5.43
4.18; 5.10
4.18; 7.21
91.90 C2, C3a, C20, C30, C40
76.74 C10, C40
73.28 C10, C40, C50
88.31 C20, C30, C10, C50
H-C30 4.49 dd
H-C40 4.30 dd
H-C50a 3.94 dd
H-C50b 3.88 dd
3.02; 12.76 64.25 C30, C40
GIAO calculations were carried out at the M06/6-311++G(2df,2pd),
B3LYP/6-311++G(2df,2pd) and M06/6-31++G(d,p) levels. TMS and a
4.25; 12.76
C30, C40
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Table 2 NMR data of M1MGx-dA (D2O). (NMR data derived from spectra in CD3OD and DMSO-d6 – Tables S6 and S7 in the ESI)
d(H) [ppm] Multiplicity JH,H [Hz]
d(C) [ppm] HMBC
NOESY
2 CHO 9.02
s
193.25
110.14
142.01
143.71
139.08
117.65
14.77
141.70
142.12
124.05
87.82
C100, C7
CH3, H-C5
C100
C3a
H-C2
H-C5
C7
CH3
C8
8.44
8.54
s
s
C3a, C9b, C10
C3a, C9a, C9b, C7
H-C10, H-C20a, H-C30, H-C50a, H-C50b
CHO
2.32
s
C7, C8, C100, CHO
CHO
C9a
C9b
H-C10
6.57
t
m
ddd
dt
td
dd
dd
6.80
C2, C3a, C2, C30, C40, C50 H-C2, H-C20a, H-C20b, H-C40, H-C50a, H-C50b
H-C20a 2.92
H-C20b 2.63
6.80, 14.02
3.92, 6.43, 14.02
3.73, 6.43
3.59, 7.21
3.59, 12.51
4.87; 12.51
41.92
C10, C30, C40
C30, C40
H-C10, H-C20b, H-C30, H-C2
H-C10, H-C20b, H-C30, H-C40
H-C30
H-C40
4.68
4.18
73.94
90.24
64.43
C10, C40, C50
C10, C20, C30
C30, C40
H-C2, H-C20a, H-C20b, H-C40, H-C50a, H-C50b
H-C10, HC20a, H-C30, H-C50a, H-C50b
C-H10, H-C20b, H-C30, H-C40, H-C2
C-H10, H-C20b, H-C30, H-C40, H-C2
H-C50a 3.83
H-C50b 3.79
C30, C40
performed for the enol forms shows that they are closer to the
experimental data. The calculated values with the best correla-
tion with the spectroscopic data are shown in Tables 3 and 4,
and also S26 and S27 in the ESI† (remaining data in ESI,†
Tables S9–S28).
Similar to R2 values, the analysis of mean absolute errors
(MAEs) for 1H NMR chemical shifts for M1Gx-A (Chart 1A) shows
that error values are typically higher for the dialdehyde form.
Computational results for the enol form of the adduct are much
Fig. 5 Major correlations in the HMBC spectrum (D2O) (A) and in the
NOESY spectrum (DMSO-d6) (B) of M1MGx-dA.
neutral and anionic TSP form were standards in NMR chemical more similar to the experimental data other than results for the
shift calculations. The calculations were performed for in vacuo spectrum with DMSO-d6 as solvent. When a model for solvent
molecules and using the PCM to take into account the effect of was included in calculations, lower MAE values were obtained.
solvents (water, methanol and DMSO).
The lowest MAE values (approx. 0.2 ppm, Chart 1A) were found
Computational results were compared with the experimental with M06 and B3LYP functionals and the 6-311++G(2df,2pd)
data. The comparison was based on the following parameters: basis set for the enol form of the adduct when the results were
absolute errors, mean absolute errors (MAEs) and coefficients compared to the NMR spectra measured using D2O and CD3OD
of determination R2. Furthermore, the solvent effects on the as solvents.
correlation between experimental and theoretical results were
determined.
The comparison of experimental and calculated 13C NMR
chemical shifts for M1Gx-A (Chart 1B) showed that mean
The analysis of calculated 13C NMR chemical shifts for the absolute errors were higher for the dialdehyde form, similar
1
dialdehyde forms of M1Gx-A and M1MGx-dA showed that the to the H NMR chemical shifts. The best correlation with the
largest absolute errors occurred for the C100 atoms (up to 60 ppm) experimental data derived from NMR spectra recorded using
and C8 and CHO (up to 20 ppm) (see ESI,† Tables S9–S12 and D2O, CD3OD and DMSO-d6 as solvents was seen for the
S17–S20). The largest errors for the enol forms were seen for the calculations based on the M06/6-31++G(d,p) level for the enol
CHO, C10 and C2 atoms (up to 20 ppm) in M1Gx-A and CHO, form of M1Gx-A with solvent effects included (MAE = 3 ppm,
C8 (up to 20 ppm) in M1MGx-dA (see ESI,† Tables S13–S16 Chart 1B).
1
and S21–S24). The largest absolute errors for 1H NMR chemical
The comparison of MAE values for H NMR chemical shift
shifts for the dialdehyde and enol forms of M1Gx-A and M1MGx-dA calculations for M1MGx-dA shows significant separation of
occurred for the aldehyde protons (2 ppm) (see ESI,† Tables S9–S24). results for the dialdehyde and the enol form (Chart 1C).
The R2 coefficients determined for calculations of 13C NMR Calculated results for the enol form are more similar to the
chemical shifts for M1Gx-A and M1MGx-dA were 0.83–0.89 and spectroscopic data. Calculations that include solvent effects
0.91–0.93, respectively, for the dialdehyde forms and 0.98–0.99 show even better correlation with the experimental data than
and 40.99, respectively, for the enol forms of both compounds. those based on the same methods for the compound in the gas
The R2 coefficients determined for calculations of 1H NMR phase. Similar to M1Gx-A, the lowest MAE values (approx. 0.2 ppm,
chemical shifts for M1Gx-A and M1MGx-dA were 0.97–0.98 and Chart 1C) were obtained using M06 and B3LYP functionals and
0.93–0.97, respectively, for the dialdehyde forms and 0.96–0.99 the 6-311++G(2df,2dp) basis set including PCM for the solvent
and 0.98–0.99, respectively, for the enol forms (see ESI,† when the results were compared to the NMR spectra recorded
Tables S9–S24). Comparison of R2 values for the calculations using D2O and CD3OD. Slightly higher MAEs were obtained
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Table 3 Comparison of NMR experimental data for M1Gx-A (D2O) with
data calculated for the enol form of M1Gx-A using the B3LYP/6-311++G
(2df,2pd) method, PCM and a neutral form of TSP as the NMR standard
d
exp(H)
d
exp(C)
d
calc(H)
|D|d(H)
[ppm]
d
calc(C)
|D|d(C)
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
2 CHO
C100
C3a
H-C2
H-C5
C7
8.98
193.21
110.58
142.85
143.77
139.89
121.55
133.32
141.56
125.13
91.9
76.74
73.28
88.31
64.25
8.78
0.20
193.81
112.67
145.38
149.65
140.47
125.60
142.53
150.69
134.36
100.73
80.05
0.60
2.09
2.53
5.88
0.58
4.05
9.21
9.13
9.23
8.83
3.31
7.52
8.11
5.50
8.46
8.6
8.03
8.92
0.43
0.32
C8
C9a
C9b
7.49
7.66
0.17
H-C10
H-C20a
H-C30
H-C40
H-C50a
H-C50b
6.21
4.9
4.49
4.3
3.94
3.88
5.98
4.88
4.63
4.38
3.81
4.07
MAE
R2
0.23
0.02
0.14
0.08
0.13
0.19
0.19
0.9877
80.80
96.42
69.75
MAE
5.47
0.9922
R2
Table
4
Comparison of NMR experimental data for M1MGx-dA
(DMSO-d6) with data calculated for the enol form of M1MGx-dA using
the M06/6-31++G(d,p) method, PCM and TMS as the standard
d
exp(H)
d
exp(C)
d
calc(H)
|D|d(H)
[ppm]
d
calc(C)
|D|d(C)
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
2 CHO
C100
C3a
H-C2
H-C5
C7
CH3
C8
C9a
C9b
8.91
183.64
106.56
137.86
139.56
136.1
115.37
14.16
138.42
138.68
122.33
83.87
9.03
0.12
182.54
101.19
137.88
140.00
135.56
113.88
9.92
145.70
141.07
125.20
86.64
1.10
5.37
0.02
0.44
0.54
1.49
4.24
7.28
2.39
2.87
2.77
1.59
8.49
8.33
8.13
8.88
0.36
0.55
2.18
2.61
0.43
H-C10
H-C20a
H-C20b
H-C30
H-C40
H-C50a
H-C50b
6.47
2.74
2.36
4.44
3.9
3.63
3.54
6.34
3.49
2.26
4.20
4.28
3.99
3.76
MAE
R2
0.13
0.75
0.10
0.24
0.38
0.36
0.22
0.33
0.9819
39.71
38.12
70.68
87.9
61.65
75.13
89.28
64.31
4.45
1.38
2.66
MAE
2.57
0.9951
R2
solvent that yields the best match between the experimental
and the computational results (MAE = 2.57 ppm) (Chart 1D).
The largest differences between the experimental and the
computational results concerned the b-dicarbonyl moiety in
both adducts. The computational results for the dialdehyde
Fig. 6 Lowest-energy conformers of M1Gx-A ((A) – dialdehyde form;
(B) – enol form) and of M1MGx-dA ((C) – dialdehyde form; (D) – enol form).
when the calculated results based on the above parameters forms significantly differed from experimental data especially
were compared with the experimental results with DMSO-d6 as for the shielding constant of the C100 atom. The computational
solvent (Chart 1C).
results for the enol forms were much closer to the experimental
The comparison of MAE values for the 13C NMR chemical data. Furthermore, one signal with integration 2 from the alde-
1
shifts calculated and measured for the M1MGx-dA adduct hyde protons was seen in the H NMR spectra and one signal
shows clear separation of results for the dialdehyde and the from the aldehyde carbon atoms in the 13C NMR spectra. This
enol form (Chart 1D). The chart shows that calculations for the proves that the experimental NMR chemical shifts within those
enol form at the M06/6-31++G(d,p) level including PCM for parts of the adducts were averaged. This most likely results from
the solvent provided results closest to the experimental data the enol form being dominant for the compounds and more
(MAE of 2.5–3.5 ppm). The comparison shows that DMSO is the rapid conversions between the two possible enol forms than the
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relaxation of excited hydrogen and carbon nuclei.70–72 The calcu-
lated energy barriers for proton transfer in the b-dicarbonyl
moieties are 1.8 kcal molꢁ1 for M1Gx-A and 1.5 kcal molꢁ1 for
M1MGx-dA. Literature reports are available that confirm that the
energy barriers for proton transfer in similar systems are low.73,74
The energy barrier for proton transfer in malonaldehyde calcu-
lated using ab initio methods is similar to the values obtained for
M1Gx-A and M1MGx-dA,73 while the results of semiempirical
methods seem to be very high.75 The results of the crystallo-
graphic investigation of compounds with a b-dicarbonyl system
also confirm the averaged position of the hydrogen atom between
the two oxygen atoms.76
It is noted that the experimental results (D2O) were obtained
with TSP-d4 as the NMR standard. The standard was also used
in chemical shift calculations. TSP may have a neutral or an
anionic form. Both structures were optimized and subjected
to GIAO calculations using the same methods as the adducts
studied. Subsequently, IMS values for both the neutral and
anionic TSP form were used to determine NMR chemical shifts of
the adducts. Better correlation with the experimental data was
seen with the anionic TSP form as the NMR standard in calcula-
tions for 13C NMR chemical shifts. The neutral form of the
standard, in turn, provided better correlation with spectroscopic
data for 1H NMR chemical shifts. More thorough investigation
would be needed to determine the actual TSP form present in the
studied solutions, and it goes beyond the scope of this project. It is
noted, however, that TSP was used in literature reports for NMR
experiments whose results were compared to computational
results with TMS as the reference.77,78 This may be due to the fact
that NMR chemical shifts for both TMS and TSP are by definition
dH = 0 ppm and dC = 0 ppm. Experiments prove, however, that
minor differences occur between chemical shifts of the standards,
1
being approx. 0.1 ppm for H NMR and approx. 0.2 ppm for 13C
NMR depending on the solvent.79–82 The conformers of TSP in the
neutral and anionic form used in GIAO calculations with the
calculated IMS values are listed in ESI,† Fig. S2, S3 and Table S8.
Conclusions
It is now commonly agreed that exocyclic etheno adducts together
with other oxidative DNA damage could play an important role in
the multistage process of carcinogenesis. The use of such adducts
as biomarkers offers a promising tool in studies on cancer
etiology and prevention, particularly for human neoplasms in
which the causative factors and mechanisms are still poorly
understood.83 Therefore detailed knowledge of DNA adduct
structure is extremely important.
Experimental methods (NMR spectroscopy) together with
quantum chemical calculations were applied for the structural
studies of substituted malonaldehyde–glyoxal and malonaldehyde–
methylglyoxal etheno adducts of adenine nucleosides (M1Gx-A and
M1MGx-dA, respectively). It was found on the basis of the
results that in both adducts the dialdehyde methyl substituent
was attached to C7 of the etheno rings, in contrast to the
earlier report.43 Our results as well as those derived from the
Chart 1 Comparison of MAE values for chemical shift calculations:
(A) 1H NMR, M1GxA; (B) 13C NMR, M1Gx-A; (C) 1H NMR, M1MGx-dA;
(D) 13C NMR, M1MGx-dA; (1) M06/6-311++G(2df,2pd), PCM; (2) M06/
6-311++G(2df,2pd), in vacuo; (3) M06/6-31++G(d,p) PCM; (4) M06/
6-31++G(d,p), in vacuo; (5) B3LYP/6-311++G(2df,2pd) PCM; (6) B3LYP/
6-311++G(2df,2pd), in vacuo.
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literature9,65 clearly indicate that correlation spectra, especially
NOESY, are necessary for the determination of the substitution
position in the etheno rings of the adducts among spectro-
scopic techniques. However NOE data are not always pivotal.2
Our studies demonstrate that quantum chemical calculations
provide valuable information that helps in solving the struc-
tural problems associated with the presence of the substituent
at C7 or C8 in substituted etheno adducts.
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optimum computational method for the compounds was
1
selected. The experimental H NMR chemical shift values had
the highest correlation with computational results for M06 or
B3LYP functionals with the 6-311++G(2df,2pd) basis set. However,
13C NMR chemical shift calculations had the highest correlation
with GIAO results at the M06/6-31++G(d,p) level. PCM improved
the correlation of results in both cases.
9 K. Kawai, P.-H. Chou, T. Matsuda, M. Inoue, K. Aaltonen,
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adduct structures were closer to the experimental data. There-
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compounds studied. Furthermore, the averaging of 1H and
13C NMR chemical shifts indicated rapid proton transfer between
both enol forms of the compounds. This resulted from a low
energy barrier for proton transfer in the b-dicarbonyl system. The
calculated energy barrier for proton transfer is 1.8 kcal molꢁ1 for
M1Gx-A and 1.5 kcal molꢁ1 for M1MGx-dA.
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List of abbreviations
MA
Malonaldehyde
MAE
M1Gx
Mean absolute error
Conjugate of one molecule of malonaldeyde and
one molecule of glyoxal
M1MGx
M1Gx-A
Conjugate of one molecule of malonaldeyde and
one molecule of methylglyoxal
7-(Diformylmethyl)-3-(b-D-
ribofuranosyl)imidazo[2,1-i]purine
M1MGx-dA 7-(Diformylmethyl)-8-methyl-3-(20-deoxy-b-D-
ribofuranosyl)imidazo[2,1-i]purine
TMP
TMS
1,1,3,3-Tetramethoxypropane
Tetramethylsilane
20 F. P. Guengerich and V. D. Raney, J. Am. Chem. Soc., 1992,
114, 1074–1080.
TSP-d4
3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid
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Acknowledgements
This research was supported in part by PL-Grid Infrastructure
(http://www.plgrid.pl/en).
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