Synthesis, structural studies and stability of model cysteine containing DNA-protein cross-links

Article abstract of DOI:10.1039/c7nj00270j

DNA-protein cross-links (DPCs) are bulky, helix-distorting lesions that are formed upon irreversible bonding of proteins to chromosomal DNA in the presence of cross-linking agents. Among a broad range of such agents are α,β-unsaturated carbonyl compounds, which act essentially as bifunctional alkylating agents and form adducts with DNA bases. These adducts can further undergo interactions with other cellular macromolecules leading to the formation of cross-linked products. We synthesized and structurally characterized N-acetylcysteine cross-links formed in the reactions with aldehydic adducts of adenine nucleosides, which possess enol functionality and represent model α,β-unsaturated carbonyl systems. Studies performed by the use of NMR spectroscopy, DFT and ab initio methods established that two of these cross-links exist as rotamers stable at room temperature. Application of Atoms in Molecules Theory enabled hydrogen bonding and other stabilizing interactions within the studied molecules to be estimated. Under physiological conditions the cross-links were found to be relatively stable until N^α-acetyllysine was present in the reaction medium. The presence of this amino acid caused fast transformation of the N-acetylcysteine cross-links into a range of their lysine derivatives. Although instability of the cysteine adduct with acrolein was reported, we showed that the mechanism involved in the gradual decomposition of the N-acetylcysteine cross-links differs from that proposed for acrolein adduct degradation. This demonstrates that in spite of similarities in their structures, numerous α,β-unsaturated carbonyl compounds can interact with nucleophilic biomolecules by different mechanisms leading to structural heterogeneity of the resulting products. Our findings provide an explanation for difficulties in identifying the cysteine containing DPCs in vivo and in vitro, and may be of great importance with respect to detection and isolation of such lesions from biological materials.

Full text of DOI:10.1039/c7nj00270j

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Page 1 of 36
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Synthesis, structural studies and stability of the model cysteine containing
DNAꢀprotein crossꢀlinks
Kinga Salus, Marcin Hoffmann, Tomasz Siodła, Bożena Wyrzykiewicz, Donata Pluskotaꢀ
Karwatka*
Adam Mickiewicz University in Poznań, Faculty of Chemistry, Umultowska 89b,
61ꢀ614 Poznań, Poland
eꢀmail: donatap@amu.edu.pl
Abstract
DNAꢀprotein crossꢀlinks (DPCs) are bulky, helixꢀdistorting lesions that are formed upon
irreversible bonding of proteins to chromosomal DNA in the presence of crossꢀlinking agents. Among
a broad range of the agents there are α,βꢀunsaturated carbonyl compounds which act essentially as
bifunctional alkylating agents and form adducts with the DNA bases. These adducts can further
undergo interactions with other cellular macromolecules leading to the formation of crossꢀlinked
products. We synthesized and structurally characterized Nꢀacetylcysteine crossꢀlinks formed in
the reactions with aldehydic adducts of adenine nucleosides, which possess enol functionality
and represent model α,βꢀunsaturated carbonyl systems. Studies performed by the use of NMR
spectroscopy, DFT and ab initio methods established that two of these crossꢀlinks exist as
rotamers stable at the room temperature. Application of Atoms in Molecules Theory enabled
to estimate hydrogen bonding and other stabilizing interactions within the studied molecules.
Under physiological conditions the crossꢀlinks were found to be relatively stable until Nαꢀ
acetyllysine was present in the reaction medium. Presence of this amino acid caused fast
transformation of the Nꢀacetylcysteine crossꢀlinks into a range of their lysine derivatives.
Although instability of cysteine adduct with acrolein was reported, we showed that
mechanism involved in gradual decomposition of the Nꢀacetylcysteine crossꢀlinks differs
from that proposed for the acrolein adduct degradation. This demonstrates that in spite of
similarities in their structures, numerous α,βꢀunsaturated carbonyl compounds can interact
with nucleophilic biomolecules by different mechanisms leading to the structural
heterogeneity of the resulting products. Our findings provide explanation for difficulties with
identifying the cysteine containing DPCs in vivo and in vitro, and may be of great importance
with respect to detection and isolation of such lesions from biological material.
1

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Introduction
DNAꢀprotein crossꢀlinks (DPCs) are bulky lesions formed when cellular proteins
bound covalently to DNA. These lesions distort DNA helix and can disturb the normal DNAꢀ
protein interactions leading to impairing of the DNAꢀinvolved processes such as transcription,
replication, repair, recombination and chromatin remodeling.1ꢀ3 RNA due to structural
likeness to DNA, is presumed to exhibit similar reactivity towards protein crossꢀlinks
formation. RNAꢀprotein crossꢀlinks can interfere with the processes related to both mRNA
and noncoding RNA.4ꢀ6 Location of crossꢀlinking on the DNA constituent was found to
influence the biological outcomes of DPCs formation.7,8 However the biological
consequences of DPCs formation depend not only on DNA sequence involved in these
lesions, but in a large part, on protein trapped in it. While early reports suggested that only
DNAꢀbinding proteins can undergo crosslinking to DNA, recent studies imply that any
protein can be involved in DPCs formation.9,10
Proteins related to a number of cellular processes were found to form DPCs. Among
them there are DNA replication and repair proteins,3,11 architectural, structural and associated
proteins, cell cycle proteins and chromatin regulators, proteins responsible for cellular
homeostasis and stress response, transcription regulators and RNA splicing
components.3,12,13,14ꢀ17 In the model reactions also lysozyme and serum albumin were shown
to undergo crosslinking to DNA.10,18 Different proteins favor to form DPCs with specified
DNA sequences.2
DPCs can be induced by exposure to a broad range of environmental agents but also
by bifunctional electrophiles of endogenous origin which can react with a nucleophilic site on
both DNA and amino acid side chain. Among such electrophiles, α,βꢀunsaturated carbonyl
compounds and 2ꢀoxoaldehydes such as glyoxal and methylglyoxal are known to induce
DPCs.9,19 Lysine, arginine, cysteine, histidine, and serine were found to form DPCs.1,2,10
Nevertheless, majority of reports refer mainly to the Nꢀterminal and lysine side chain amino
groups and to the guanidine group as protein structure fragments involved in such lesions. The
cysteine residues thiol groups are the most nucleophilic sites in proteins, however contents of
these residues in protein surfaces is much lower than of other amino acids. Moreover cysteine
containing DPCs were reported to be less stable than other types of such lesions.10,11
Therefore studies on cysteine DPCs seem to be challenging.
Bisꢀelectrophiles such as 1,2,3,4ꢀdiepoxybutane, the key carcinogenic metabolite of
1,3ꢀbutadiene and N,Nꢀbisꢀ(2ꢀchloroethyl)ꢀphophorodiamidic acid as well as N,Nꢀbisꢀ(2ꢀ
chloroethyl)amine, the metabolites of cyclophosphamide used to treat some kind of cancers
2

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and autoimmune diseases were shown to induce formation of cysteine containing DPCs.14ꢀ17,
20ꢀ22
Lipid peroxidation product, 4ꢀhydoxyꢀ2ꢀnonenal was demonstrated to modify an
active site’s cysteine residue of protein disulfide isomerase which participates in the
maturation of newly synthesized proteins through promoting correct disulfide formation.23
Another α,βꢀunsaturated aldehyde, acrolein was found to be responsible for
inactivation of protein tyrosine phosphatase 1B by binding covalently to the catalytic cysteine
residue at the enzyme active site.24
Cysteine was also, besides lysine, shown to be involved in the DPCs formed by
methylglyoxal between dG in the substrate DNA and the exonuclease deficient Klenow
fragment of E. coli DNA polymerase I.11 It was found that yield of the cysteine crossꢀlink was
much lower than that of the lysine, however, the chemical nature of the crossꢀlinked
compounds was not determined.11 Glyoxal was also shown to induce formation of this kind of
crossꢀlinks but the aldehyde was found to be a less potent crosslinking agent than
methylglyoxal.11 Although DNAꢀprotein crosslinking activity of methylglyoxal was examine
in the later study 25 only structure of the aldehyde crossꢀlink between dG and Nαꢀacetyllysine
was established.25
DPCs can be potentially used in biochemistry and medicine as biomarkers of
oxidative/carbonyl stress and of chemicals exposure.2 Moreover, controlled formation of
DPCs in vitro is useful tool in proteomics analysis.26,27 Therefore there is a great need for
studies aimed at structure determination, formation mechanisms elucidation and stability
investigation of cysteine containing DPCs.
Recently, we found that aldehydic adducts of adenine nucleosides 28,29 (Fig. 1) exhibit
a potent crossꢀlinking activity.30 We showed that these adducts induce formation of a range of
crossꢀlinks while incubating with Nαꢀ and Nεꢀacetyllysine respectively.30 Enol form
predominates for dialdehyde functionality of the adducts in aqueous solution, therefore these
chemicals represent model systems suitable for studies of α,βꢀunsaturated carbonyl
compounds crossꢀlinking activity. In the present work we used these systems to study their
reactivity toward Nꢀacetylcysteine and we identified a range of crossꢀlinked products. Our
findings provide insight into probable structures and stability of similar modifications
potentially formed in vivo. They also clearly show that difficulties in identification and
structural characterization of cysteine crossꢀlinks may result from their instability.
3

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R1
7
R1
8
8
N
N
O
O
1''
9a
7
1''
N
N
N
N
9b
2
O
OH
5
N
N
3a
N
N
R2
R2
R1 = H, R2 = ribose, M1Gx-A
R1 = CH3, R2 = H, M1MGx-Ade
R1 = CH3, R2 = 2'-deoxyribose, M1MGx-dA
R1 = CH3, R2 = ribose, M1MGx-A
Fig. 1. Structures of the studied adducts: M1GxꢀA, M1MGxꢀAde, M1MGxꢀdA and M1MGxꢀA. 29
4

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Results and discussion
Reactions of M1GxꢀA with Nꢀacetylcysteine.
M1GxꢀA was synthesized as described previously 28, (Scheme 1) and subjected to
reaction with Nꢀacetylcysteine (NAC) in 0.1 M phosphate buffer (PB) solution at 37 and at 50
°C, respectively for 10 days. LCꢀDAD analyses of the reaction mixtures revealed the presence
of one major product named M1GxꢀAꢀNAC (Fig. 2), which achieved maximum concentration
after 36 h of heating at 50 °C.
Scheme 1. Formation of malonaldehydeꢀglyoxal and malonaldehydeꢀmethylglyoxal adducts of adenine nucleosides.
0
5
10
15
[min]
Fig. 2. C18 analytical column LCꢀDAD chromatogram (recorded at λ = 254 nm) of the M1GxꢀA with Nꢀacetylcysteine
reaction mixture held in 0.1 M phosphate buffer solution at 50 °C for 36 h.
Formation of this product was also observed at 37 °C, however, at this temperature the
yield of M1GxꢀAꢀNAC was lower. UV spectrum of this compound exhibited absorption
maxima at λ = 234 and λ = 300 nm, and absorption minimum at λ = 260 nm (Fig. 3).
5

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M1Gx-A-NAC
M1MGx-dA-NAC
M1MGx-Ade-NAC
M1MGx-A-NAC
[nm]
200
400
Fig. 3. UVꢀVis spectra of Nꢀacetylcysteine crossꢀlinks recorded with diode array detector as the compounds eluted
from the column. (For analyses conditions see Experimental section).
Structural data provided by the mass spectrometry (m/z 507 [M + H]+ Supplementary
,
information (SI) Fig. S7) indicated that M1GxꢀAꢀNAC is composed of one Nꢀacetylcysteine
molecule and one M1GxꢀA molecule. Spectrum resulted from the MS/MS analysis, besides
the molecular ion signal, showed two fragment ion signals at m/z 375 and 246 (SI, Fig. S8).
The former signal corresponded to the neutral loss of a ribosyl unit, and the later resulted from
the neutral loss of a ribosyl unit and cleavage of the bond between sulfur atom and βꢀcarbon
atom in Nꢀacetylcysteine residue (Fig. 4 and SI, Fig. S8).
375.0865
246.0445
246.0445
375.0865
202.0719
333.0758
288.0541
507.1296
158.0579
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
[m/z]
Fig. 4. Representative MS/MS trace for the synthesized crossꢀlink; the positive ions MS/MS spectrum of M1GxꢀAꢀ
NAC (collision energy 30 eV).
For M1GxꢀAꢀNAC definitive structural characterization the preparative scale reaction
was performed, the major product was isolated (13 mg, 25% yield) and subjected to
spectroscopic studies. The data obtained from the M1GxꢀAꢀNAC NMR spectra (SI, Fig. S43ꢀ
S48) are presented in Table 1 and in part in Fig.5.
6

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800
700
600
500
400
300
200
100
8
2"
2
5
0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Fig. 5. Representative NMR spectrum of the synthesized crossꢀlink; 1H NMR spectrum of M1GxꢀAꢀNAC (D2O).
Table 1. NMR Data of M1GxꢀAꢀNAC (D2O)
δ(H)
[ppm]
6.21
Multiplicity
d
JH,H [Hz]
5.43
δ(C)
[ppm]
91.29
HMBC
NOESY
HꢀC1’
C2, C3a, C2’, C3’,
HꢀC2, HꢀC2’, HꢀC3’, HꢀC4’
C4’
C1’
C1’, C5’
HꢀC2’
HꢀC3’
4.86
4.50
m
m
76.84
73.25
HꢀC2, HꢀC1’, HꢀC3’, HaꢀC5’
HꢀC2, HꢀC1’, HꢀC2’, HaꢀC5’,
HbꢀC5’
HꢀC4’
HaꢀC5’
4.30
3.88
dd
dd
4.12; 7.34
3.88; 12.72
88.29
64.25
C1’, C2’, C3’, C5’
C3’, C4’
HꢀC1’, HaꢀC5’, HbꢀC5’
HꢀC2’, HꢀC3’, HꢀC4’, Hbꢀ
C5’, HꢀC2
HbꢀC5’
HꢀC2
C3a
HꢀC5
C7
HꢀC8
C9a
C9b
3.95
8.49
dd
s
3.04; 12.73
C3’, C4’
C3a, C9b, C1’
HꢀC3’, HꢀC4’, HaꢀC5’
HꢀC1’, HꢀC2’, HꢀC3’, HaꢀC5’
143.95
141.67
139.51
119.23
137.57
144.45
125.60
127.63
170.62
194,12
57.68
8.63
7.69
s
s
C3a, C7, C9a, C9b
C7, C9a, C1”, C2”
HaꢀCβ
CH3
C1”
HꢀC2”
CHO
HꢀCα
8.66
9.52
4.57
C7, CHO, C1”, Cβ
C7, C1”, C2”
Cβ, COOH, C=O
(Ac)
CHO, HaꢀCβ, HbꢀCβ, HꢀCα
HꢀC2”
HꢀC2”, HaꢀCβ, HbꢀCβ
s
dd
4.33, 7.79
HaꢀCβ
HbꢀCβ
3.42
3.61
dd
dd
7.83, 14.18
4.33, 14.19
40.65
Cα, C2”, COOH
Cα, C2”, COOH
HꢀCα, HbꢀCβ, HꢀC2”
HꢀCα, HaꢀCβ, HꢀC2”
COOH
C=O (Ac)
CH3 (Ac)
178.30
176.58
24.86
2.05
C=O (Ac), Cα, Cβ
1
HꢀC8
The singlet oneꢀproton signal observed, in the H NMR spectrum at 9.52 ppm was
1
assigned to the aldehyde proton based on the chemical Hꢀ13C correlation with the carbon
atom signal at 194.12 ppm. Presence of one aldehyde proton suggested that Nꢀacetylcysteine
was attached to the adduct aldehydic functionality. In the 13C NMR spectrum the signal
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appearing at 119.23 ppm arose from atom not bonded to proton and was assigned to C1”
carbon atom. The structure proposed for M1GxꢀAꢀNAC (Fig. 6) was confirmed by
correlations observed in the HMBC spectrum (Table 1).
Fig. 6. Structures proposed for the crossꢀlinks: M1GxꢀAꢀNAC, M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC.
Głów
prod
M1G
Reactions of M1MGxꢀdA with Nꢀacetylcysteine.
M1MGxꢀdA and NAC were held in 0.1 M and 1 M PB solutions at 37 and 50 °C,
respectively for 14 days. pH of the reaction mixtures was monitored and it was found to be
7.4 for 1 M, and 3 for 0.1 M PB solution. The reaction mixtures chromatograms derived from
LCꢀDAD analyses showed the presence of two major products (M1MGxꢀAdeꢀNAC and
M1MGxꢀdAꢀNAC) in 0.1 M PB solution (Fig. 7) and one major product (M1MGxꢀdAꢀNAC)
in 1 M PB solution (data not shown).
0
5
10
15
[min]
Fig. 7. C18 analytical column LCꢀDAD chromatogram (recorded at 254 nm) of the M1MGxꢀdA with NAC reaction
mixture held in 0.1 M PB solution at 37 °C for 3 h.
The compounds exhibited very similar UV spectra (M1MGxꢀAdeꢀNAC: λmax = 232,
296 nm, λmin = 256 nm; M1MGxꢀdAꢀNAC: λmax = 238, 298 nm, λmin = 260 nm), (Fig. 3). The
mass spectrum of M1MGxꢀAdeꢀNAC displayed the signal at m/z 389 [M + H]+ (SI, Fig. S10),
while the spectrum of M1MGxꢀdAꢀNAC revealed the signal at m/z 505 [M + H]+ (SI, Fig.
S14). Data derived from UV and mass spectra (SI, Fig. S10ꢀS17) of M1MGxꢀAdeꢀNAC and
8

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M1MGxꢀdAꢀNAC indicated that both compounds were structural analogues which differ only
in the presence of 2’ꢀdeoxyribosyl unit. Formation of adenine derivative can be explained by
occurring at low pH hydrolysis of
and in M1MGxꢀdA. Moreover, these data suggested that M1MGxꢀAdeꢀNAC and M1MGxꢀdAꢀ
NAC consisted of one molecule of ꢀacetylcysteine and one molecule of the adduct. The
Nꢀglycosidic bond in both the initially formed crossꢀlink
N
isolated M1MGxꢀAdeꢀNAC (9 mm, 30 % yield), was subjected to spectroscopic studies which
confirmed structure proposed for the compound in the Fig. 6. The HMBC spectrum of
M1MGxꢀAdeꢀNAC exhibited analogous correlations to the HMBC spectrum of M1GxꢀAꢀ
NAC. (For detailed NMR data see SI, Table S1, Fig. S49ꢀS53).
Reaction of M1MGxꢀA with Nꢀacetylcysteine.
Due to M1MGxꢀdA instability under reaction conditions the adenosine analogue of
this adduct (M1MGxꢀA) was used to study its reactivity towards NAC. In the reaction
performed in 0.1 M PB solution at 50 °C one major product (M1MGxꢀAꢀNAC) was formed
(SI, Fig S1). The compound exhibited a very similar UV spectrum (λmax = 238, 298 nm and
λmin = 260 nm), to the UV spectra of M1GxꢀAꢀNAC, M1MGxꢀdAꢀNACand M1MGxꢀAdeꢀ
NAC (Fig 3.). Data derived from the mass spectrum, (m/z 521 [M + H]+, (SI, Fig. S18)),
suggested that the product was structural analogue of compounds formed in the reaction of
M1MGxꢀdA with NAC. However, contrary to the reaction with M1MGxꢀdA, no hydrolysis of
N
ꢀglycosidic bond in the forming product was observed. The spectrum derived from the
M1MGxꢀAꢀNAC MS/MS analysis showed the fragmentation similar to that observed for
M1GxꢀAꢀNAC. Besides molecular ion signal, two fragment ion peaks at m/z 389 and 260
were present in the spectrum (SI, Fig. S19). The former corresponded to the neutral loss of a
ribosyl unit, and the later resulted from the neutral loss of a ribosyl unit and cleavage of the
bond between the sulfur atom and βꢀcarbon atom in the Nꢀacetylcysteine residue.
The preparative scale reaction of M1MGxꢀA with NAC was performed and M1MGxꢀ
AꢀNAC was isolated (22 mg, 28 % yield) for structural studies (Table 2 and SI, Fig. S54ꢀ
S59). Correlations observed in the HMBC spectrum were crucial for M1MGxꢀAꢀNAC
structure determination.
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Table 2. NMR Data of M1MGxꢀAꢀNAC derived from spectra recorded in D2O
δ(H)
[ppm]
δ(C)
[ppm]
Multiplicity
JH,H [Hz]
5.44
HMBC
NOESY
C2, C3a, C2’, C3’,
C4’
HꢀC1’
HꢀC2’
6.20
4.87
d
91.79
76.81
HꢀC2, HꢀC2’, HꢀC3’, HꢀC4’
HꢀC2, HꢀC1’, HꢀC3’, Haꢀ
C5’, HbꢀC5’
dd
5.13; 10.18
C1’
HꢀC2, HꢀC1’, HꢀC2’, Haꢀ
C5’, HbꢀC5’
HꢀC1’, HaꢀC5’, HbꢀC5’
HꢀC2’, HꢀC3’, HꢀC4’, Hbꢀ
C5’, HꢀC2
HꢀC3’
HꢀC4’
HaꢀC5
4.50
4.30
3.90
m
dd
dd
73.31
88.35
64.31
C1’, C5’
C1’, C2’, C3’, C5’
C3’, C4’
3.67; 7.49
4.26; 12.69
HꢀC2’, HꢀC3’, HꢀC4’, Haꢀ
C5’
HꢀC1’, HꢀC2’, HꢀC3’, Haꢀ
C5’
HbꢀC5’
HꢀC2
3.94
8.49
dd
3.06; 12.71
C3’, C4’
s (split)
144.13
C3a, C9b, C1’
C3a
HꢀC5
C7
C8
C9a
C9b
141.98
138.75
115.30
145.10
143.75
124.83
8.55
s (split)
s (split)
C3a, C7, C9a, C9b
C7, C8, C1”, C2”,
CHO
CH3
2.32
15.63
CHO, C2”, HbꢀCβ
C1”
HꢀC2”
CHO
127.27
171.54
193.25
8.74
9.57
s (split)
s
C7, CHO, Cβ
C7, C8, C1”, C2”
HꢀC2”, CH3 (Ac)
HꢀC2”, HaꢀCβ, HbꢀCβ, CH3
(Ac)
HꢀCα, HbꢀCβ, HꢀC2”
HꢀCα, HaꢀCβ, HꢀC2”, CH3
HꢀCα
4.59
dd (split)
4.30; 8.37
57.68
40.37
Cβ, C=O (Ac)
HaꢀCβ
HbꢀCβ
3.41
3.66
dd (split)
dd (split)
8.34; 14.24
4.29; 13.96
Cα, C1”, C2”, COOH
Cα, C1”, C2”, COOH
COOH
C=O (Ac)
CH3 (Ac)
178.29
175.58
24.90
2.08
s (split)
C=O (Ac)
HꢀC2”, CHO, HꢀCα
Mechanism of the Nꢀacetylcysteine crossꢀlinks formation.
M1GxꢀAꢀNAC, M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC are structural analogues of
previously identified in our laboratory crossꢀlinks of the aldehydic adenine nucleosides
adducts with lysine derivatives.30 Therefore the mechanism proposed for the lysine crossꢀ
linked products formation is most likely valid also for giving rise to the
Nꢀacetylcysteine
modifications. This mechanism involves Michael addition of the amino acid thiol group to the
adducts α,βꢀunsaturated aldehydic functionality, followed by dehydration (Scheme 2) and is
in agreement with the wellꢀknown rule that for the soft nucleophiles such as thiol groups,
conjugate addition is a typical reaction with α.βꢀunsaturated carbonyls.
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Scheme 2. Proposed mechanism of the Nꢀacetylcysteine crossꢀlinks formation.
Nꢀacetylcysteine crossꢀlinks stability.
To determine stability of the
Nꢀacetylcysteine crossꢀlinks under physiological
conditions pure M1GxꢀAꢀNAC, M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC were dissolved
separately in 0.1 M PB solution (pH 7.4) and stored at 37°C for 30 days. The compounds
stability was monitored by using LC and LCꢀMS systems with diode array detection. M1Gxꢀ
AꢀNAC, M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC were found to be relatively stable under
applied conditions with half life time above 20 days. The crossꢀlinks underwent slow
degradation forming two products (Fig. 8 and SI, Fig. S2, S3).
0
5
10
15
[min]
Fig. 8. C18 analytical column LCꢀDAD chromatogram (recorded at 254 nm) of the 0.1 M PB solution ofM1GxꢀAꢀNAC
held at 37°C for 17 days.
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On the basis of mass and UV spectra, and coꢀelution with the appropriate adducts used
as reference compounds, the products eluted at 8.5 min (Fig. 8), at 2.7 and 6.2 min (SI, Fig.
S2 and S3) were identified as the substrate adducts: M1GxꢀA, M1MGxꢀAde and M1MGxꢀA,
respectively. Compounds having longer retention time (Fig. 8 and SI, Fig. S2 and S3)
exhibited almost identical UV spectra with λmax at 236, 272 nm and λmin at 256 nm (Fig. 9).
Spectrometric studies were performed and the compounds were found to give rise the
protonated molecular ions signals recorded at m/z 361, 243 and 375, respectively (SI, Fig.
S22, S26, S30). The corresponding substrate adducts M1GxꢀA, M1MGxꢀAde and M1MGxꢀA
ions signals were recorded at m/z 362, 244 and 376, respectively (SI, Fig. S20, S24, S28).
Such small differences between the appropriate values were very surprising and prompted us
to perform ESIꢀMS/MS analyses using high collision energy for the degradation product
derived from M1MGxꢀAdeꢀNAC and for M1MGxꢀAde. The resulted spectra showed a range
of signals arisen from similar fragmentation ions (SI Fig. S25 and S27). In the mass spectrum
of M1MGxꢀAde the signal appearing at m/z 226 corresponded to the neutral loss of water
molecule ([M ꢀ H2O + H]+, SI, Fig. S25). In the mass spectrum of the M1MGxꢀAdeꢀNAC
degradation product, the signal recorded at m/z 226 was attributed to the neutral loss of
ammonia molecule ([M ꢀ NH3 + H]+, SI, Fig. S27). Moreover the UV spectra of the
degradation products resembled those of crossꢀlinks arising from reactions of the aldehydic
adducts of adenine nucleosides with Nαꢀacetyllysine 30 (Fig. 9 and 10).
M1Gx-A-NH2
M1MGx-Ade-NH2
M1MGx-A-NH2
[nm]
Nꢀacetylcysteine crossꢀlinks degradation products: M1GxꢀAꢀNH2, M1MGxꢀAdeꢀNH2 and
200
400
Fig. 9. UVꢀVis spectra of
M1MGxꢀAꢀNH2 recorded with diode array detector as the compounds eluted from the column. (For analyses
conditions see Experimental section).
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M1MGx-A-Nα-AcLys
M1MGx-A-(Nα-AcLys)2
[nm]
200
400
Fig. 10. UVꢀVis spectra of products formed in the mixture of M1MGxꢀAꢀNAC with Nαꢀacetyllysine. The spectra were
recorded with diode array detector as the compounds eluted from the column.
In these crossꢀlinks the free amino group of Nαꢀacetyllysine is attached to the adducts
carbonyl carbon atom (Fig. 11 B). Based on all these findings we concluded that the
degradation products of
Fig.11 A.
Nꢀacetylcysteine crossꢀlinks represent structures depicted as shown in
Fig. 11. Proposed structures of M1MGxꢀAdeꢀNAC, M1MGxꢀAꢀNAC and M1MGxꢀAꢀNAC degradation
products (A), and of known Nαꢀacetyllysine crossꢀlinks with the studied adducts21 (B).
Mechanism responsible for these products formation is not clear, however it seems to
be likely that they arise from 1,4ꢀaddition of ammonia to the crossꢀlinks carbon atom Cꢀ2”.
Ammonium bicarbonate solution used for isolation and purification of the crossꢀlinks is a
probable source of ammonia. Although the fractions containing the crossꢀlinks devoted to
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stability studies were evaporated to dryness, the obtained residues might contain ammonia in
amount sufficient for nucleophilic attack. Ammonia could also arise from nonꢀenzymatic
31,32
oxidation of amino acid
studies of the
released from the crossꢀlinks. Results derived from stability
N
ꢀacetylcysteine crossꢀlinks performed in the presence of Nαꢀacetyllysine
provide support for a Michael addition of ammonia being involved in the crossꢀlinks
degradation products formation. These results are also in good agreement with the literature
data suggesting that cysteine containing DPCs are less stable than DPCs in which other amino
acids are involved.10
Nꢀacetylcysteine crossꢀlinks stability in the presence of Nαꢀacetyllysine.
M1GxꢀAꢀNAC and M1MGxꢀAꢀNAC were found to be much more unstable while
incubating at 37 °C with Nαꢀacetyllysine. Half life time of the crossꢀlinks decreased to a few
hours. LCꢀDAD analyses of the reaction mixtures revealed the formation of two products
(Fig. 12 and SI, Fig. S4). On the basis of mass and UV spectra the compounds were identified
as previously characterized in our laboratory 1:2 and 1:1 crossꢀlinked products of M1GxꢀA
and M1MGxꢀA with Nαꢀacetyllysine.30
0
5
10
15
[min]
Fig. 12. C18 analytical column LCꢀDAD chromatogram (recorded at 254 nm) of the M1MGxꢀAꢀNAC mixture with Nα
ꢀ
acetyllysine held in 0.1 M PB solution at 37 °C for 1 day.
33
Cai et al. examined the reactivity of acrolein towards model peptides containing cysteine,
lysine and histidine, and found that in the initial step the reaction pathway involves a Michael
addition of the cysteine thiol group. Due to the loss of electron donation from the double C=C
bond, the aldehyde group in the resulted adduct is more electrophilic than in acrolein and
reacts with amino group forming Schiff base adduct. This adduct can undergo rearrangement
through the intermediate enamine to more stable Schiff base adduct which contains C=C bond
conjugated with the imine group (Scheme 3). 33
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Scheme 3. Formation of adducts in reaction of model peptides with acrolein. 33
This mechanism cannot be involved in transformation of M1GxꢀAꢀNAC and M1MGxꢀAꢀNAC
into the Nαꢀacetyllysine crossꢀlinked products. The
ꢀacetylcysteine derivatives possess
N
substituted α,βꢀunsaturated aldehydic functionality therefore are unable to form enamine
intermediate. In the reactions of M1GxꢀA and M1MGxꢀA with Nαꢀacetyllysine, apart from
crossꢀlinks resulted from Michael addition also imineꢀenamine adducts containing two amino
acid residues were formed.30 These adducts arose from 1,2ꢀaddition of the Nαꢀacetyllysine
amino group to the initially formed Michael addition products, the enamine crossꢀlinks,
followed by dehydration.30 The imineꢀenamine adducts are stabilized by hydrogen bond
between the imine nitrogen atom and proton of the enamine NꢀH group.30 Such stabilization
would not be possible in the products resulted from 1,2ꢀaddition of Nαꢀacetyllysine to M1Gxꢀ
AꢀNAC and M1MGxꢀAꢀNAC and consequently formation of such compounds was not
observed.
In the light of these data we proposed that mechanistic explanation for the transformation of
M1GxꢀAꢀNAC and M1MGxꢀAꢀNAC into 1:1 crossꢀlinked products of M1GxꢀA and M1MGxꢀ
A with Nαꢀacetyllysine is based on a Michael addition of Nαꢀacetyllysine to the
acetylcysteine crossꢀlinks in conjunction with ꢀacetylcysteine elimination (Scheme 4).
Formation of 1:2 crossꢀlinks occurs as it was previously described.30
Nꢀ
N
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Scheme 4. Mechanism proposed for transformation of the cysteine crossꢀlinks into their Nαꢀacetyllysine derivatives.
To provide additional support for the proposed mechanism, studies on M1GxꢀA and M1MGxꢀ
A reactivity towards the N
ꢀacetylcysteine and Nαꢀacetyllysine mixture were performed.
M1GxꢀA and M1MGxꢀA reactivity towards the mixture of Nꢀacetylcysteine and Nαꢀ
acetyllysine.
M1GxꢀA and M1MGxꢀA were separately subjected to simultaneous reactions with
Nꢀ
acetylcysteine and Nαꢀacetyllysine. LCꢀDAD and LCꢀMS analyses revealed that M1GxꢀAꢀ
NAC and M1MGxꢀAꢀNAC were initially formed crossꢀlinked products (SI, Fig. S5, S6).
Prolonged reactions led to gradual decline in the amounts of
N
ꢀacetylcysteine crossꢀlinks and
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to the formation of Nαꢀacetyllysine derivatives which became the main products of the
reactions.
For the purpose of estimating difference in energy between the Nꢀacetylcysteine and Nαꢀ
acetyllysine crossꢀlinks, quantumꢀchemical investigations using isodesmic model systems
were performed. The obtained results showed that energy of compound formed by the amino
group addition is lower than that of product arising from the thiol group addition (Fig. 13). ꢀE
calculated at M06/6ꢀ311++G(2df,2pd) and M06/augꢀccꢀpVDZ levels was equal 4.4 and 5.5
kcal/mol, respectively indicating that, in accordance with the experimental data, Nαꢀ
acetyllysine crossꢀlinks are more stable than their Nꢀacetylcysteine derivatives.
Fig. 13. Isodesmic models for crossꢀlinks: product resulted from the thiol group addition (A), compound arisen from
the amino group addition (B). (Calculations performed at M06/6ꢀ311++G(2df,2pd) level).
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VariableꢀTemperature 1H NMR studies on M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC.
1H NMR spectra of M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC recorded at room
temperature exhibited doubling of signals (SI, Fig. S49, S54). This phenomenon was also
observed in the 13C NMR spectra of both crossꢀlinks (SI, Fig. S50, S55). However not all of
the carbons and protons signals were duplicated. A very intriguing was fact that no
duplication of signals was seen in the spectra of M1GxꢀAꢀNAC (SI, Fig. S43, S44). The three
crossꢀlinked products differ only in the substituent at C8; proton attached to C8 in M1GxꢀAꢀ
NAC is replaced in M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC with a methyl group. Presence
of this group was supposed to cause hindered rotation over the C7ꢀC1” bond resulted in
existing of two rotational conformers (rotamers). To confirm this hypothesis, variableꢀ
temperature 1H NMR studies together with computational investigations were performed.
1
The pattern of signals duplication in the H NMR spectrum of M1MGxꢀAdeꢀNAC
(D2O) suggested that at 297 K the crossꢀlink existed as two rotamers (A and B) separated by a
rotational energy barrier around the C7ꢀC1” bond sufficiently high to prevent these
conformers fast interconversion at room temperature. Both rotamers were detected by the use
of signals at 1.96 ppm assigned to methyl protons of the acetyl group as a marker (Fig. 14).
373 K
372 K
370 K
342 K
297 K
3.0
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2.0
1.9
1
Fig. 14. Fragments of M1MGxꢀAdeꢀNAC H NMR spectra (D2O) recorded at 297.2 K (1), 342.6 K (2), 370.4 K (3),
371.5 K (4) and 372.6 K (5). The split acetyl proton signals at 1.96 coalesced to singlet at Tc = 371.5 K, methyl protons
signals at 2.23 ppm coalesced at lower temperature.
At 297 K the signals appeared as a split singlet due to existence of the two rotamers in
the ratio of 1.44 : 1.56. Concentration of both conformers was PA = 0.48 and PB = 0.52. The
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frequency difference, ꢁν, between the methyl proton signals was 8.42 Hz. This value
increased to 8.52 Hz when the sample was cooled to 286 K, but reheating to the room
temperature resulted in the return to the initial ꢁν. As the temperature increased the signals
for the two rotamers were still detected, although within particular pairs, signals broadened
and moved closer together. The two signals coalesced to a single one which appeared at 2.63
ppm in the spectrum measured at 371.5 K (Tc) (Fig. 14). The other pairs of signals coalesced
at lower temperature.
The Gibbs free energy of activation for interconversion between two rotamers was
calculated on the basis of NMR studies using the Earing’s equations modified by Shananꢀ
Atidi and BarꢀEli 34:
ꢁGǂA = 4.57Tc {10.62 + log[X/2π(1 ꢀ ꢁP)] + log(Tc/ꢁν)}
ꢁGǂB = 4.57Tc {10.62 + log[X/2π(1 + ꢁP)] + log(Tc/ꢁν)}
X was obtained by use of equation 34:
PA – PB = ꢁP = [(X2 – 2)/3]3/2*1/X
For ꢁP = 0.04 the X value is equal 1.636.
The free energy of rotamer A activation was ꢁGǂA = 19.97 kcal/mol, and of rotamer B
ꢁGǂB = 19.85 kcal/mol.
The high temperature caused partial decomposition of the studied compound and
formation of degradation products being a source of additional signals in the 1H NMR spectra.
Analogous variableꢀtemperature 1H NMR studies were also performed for M1MGxꢀAꢀ
NAC (D2O). The acetyl methyl proton signals which appeared at 1.96 ppm in the spectrum
recorded at 279 K were again used as a marker. Two rotational conformers of the crossꢀlink
existed nearly in the same ratio as rotamers of the adenosine analogue (1.43 : 1.57) (SI, Fig.
S40). The frequency difference, ꢁν, between the marker signals was 9.48 Hz. The two signals
coalesced to a single peak at 2.67 ppm in the spectrum recorded for the overheated sample at
1
375 K (Tc). At Tc the coalescence of split signals was observed for the entire H NMR
spectrum, although the other pairs of signals coalesced at lower temperature (SI, Fig. S40).
The Gibbs free energy of activation for the rotamer A was 19.95 kcal/mol, and for the
rotamer B was 20.01 kcal/mol. Almost identical values were in the good agreement with the
observation that in the 1H NMR spectrum the paired signals appeared in a nearly to 1:1 ratio.
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Quantumꢀchemical studies on the M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC rotamers
interconversion.
The aim of the quantum chemical investigations was to examine hindered rotation
around the C7ꢀC1” bond in M1MGxꢀAꢀNAC and M1MGxꢀAdeꢀNAC. Rotation around this
bond is associated with changes in the dihedral angle N6ꢀC7ꢀC1”ꢀC2” values and induce the
molecules helicity. P helicity corresponds to positive values of this angle, and M helicity to
the angle of negative values (Fig. 15). TS_0 describes transition state structure with the
dihedral angle N6ꢀC7ꢀC1”ꢀC2” of about 0° (Fig. 16).
Fig. 15. The geometry of conformers in energy minima: s-cis/M, s-cis/P, s-trans/M and s-trans/P.
Fig. 16. The synperiplanar (TS_0) and antiperiplanar (TS_180) geometry of the transition states with appropriate
conformation of aldehyde group: s-cis/TS_0, s-cis/TS_180, s-trans/TS_0 and s-trans/TS_180
.
In this structure atoms within the N6ꢀC7ꢀC1”ꢀC2” fragment adopt synperiplanar
geometry. In the transition state structure with the dihedral angle of about 180° (TS_180)
atoms within the N6ꢀC7ꢀC1”ꢀC2” fragment adopt antiperiplanar geometry (Fig. 16). The
studied molecules differ also in conformation of the aldehyde group. This group exists in s-cis
conformation when the dihedral angle OꢀCHOꢀC1”ꢀC2” adopts the value of about 0°, and
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possess s-trans conformation for this angle of about 180 ° (Fig. 15 and 16). The rotation of
aldehyde group occurs through transition states structures with the dihedral angle OꢀCHOꢀ
C1”ꢀC2” value of about 90° (TS_90) and ꢀ90° (TS_ꢀ90).
Quantumꢀchemical studies in vacuo at the M06/6ꢀ31G(d) level of theory showed that
relative energies of s-trans conformers of M1MGxꢀAdeꢀNAC were generally slightly higher
(about 1ꢀ4 kcal/mol), than energies of appropriate s-cis conformers (Chart 1). Only for N6ꢀ
C7ꢀC1”ꢀC2” dihedral angle values from ꢀ150° till ꢀ170° the s-trans conformers were
energetically favorable (Chart 1).
14
12
10
8
6
4
2
0
-180 -150 -120
-90
-60
Dihedral angle N6-C7-C1"-C2" value
s-cis s-trans
-30
0
30
60
90
120
150
180
Chart 1. The relative energy of s-cis and s-trans conformers of M1MGxꢀAdeꢀNAC dependent on the N6ꢀC7ꢀC1”ꢀC2”
dihedral angle value calculated at M06/6ꢀ31G(d) level of theory in vacuo.
Two energy minima were found for the s-cis conformers; for structure with the N6ꢀ
C7ꢀC1”ꢀC2” dihedral angle value of ꢀ120.6° (minimum s-cis/M) and also of 130.8° (minimum
s-cis/P) (Chart 1, Table 3, Fig. 17).
Table 3. Comparison of the relative energies (ꢁ
E) and Gibbs free energies (ꢁG) of s-cis and s-trans conformers of
M1MGxꢀAdeꢀNAC calculated at M06/6ꢀ31G(d) level of theory in vacuo and using PCM for water
In vacuo
PCM (water)
Dihedral
angle*
ꢁE
ꢁG
Dihedral
angle*
ꢁE
ꢁG
[kcal/mol] [kcal/mol]
[kcal/mol] [kcal/mol]
s-cis
s-cis
/
M
M
ꢀ120.6º
130.8º
5.0º
0
0
ꢀ116.7º
129.0º
0.6º
0
0
/
0.85
9.08
10.07
1.41
3.00
12.72
10.50
1.08
0.66
9.43
10.02
0.97
1.58
12.78
10.53
0.28
9.70
11.45
1.12
0.97
14.04
11.45
s-cis/TS_0
11.89
12.47
2.87
s-cis/TS _180
177.3º
ꢀ134.7º
130.0º
3.4º
ꢀ176.7º
ꢀ130.2º
128.9º
ꢀ1.1º
s-trans
/M
s-trans
/
P
2.94
s-trans/TS_0
14.45
11.73
s-trans/TS _180
179.2º
179.0º
* N6ꢀC7ꢀC1”ꢀC2” dihedral angle value
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Fig. 17. Energy minima structures of M1MGxꢀAdeꢀNAC and energy barriers of their interconversion (calculated at
M06/6ꢀ31G(d) level of theory in vacuo, see SI, Fig. S42 for transition state structures).
Difference in the relative energy between these two minima was very small (0.85
kcal/mol). This fact was in good agreement with experimental data which indicated that two
rotamers of M1MGxꢀAdeꢀNAC existed in almost equal amounts. The energetically preferred
transition state structure was found for s-cis conformer with the N6ꢀC7ꢀC1”ꢀC2” dihedral
angle value of about 0
E values of other transition state structures were slightly higher (up to 12.72 kcal/mol, Table
3). Application of thermal correction led to obtaining the transition state structures with the
Gibbs free energies ( G) from the range of 11.73ꢀ14.45 kcal/mol (Table 3). To take into
° (s-cis/TS_0, Table 3). It was characterized by ꢀE equal 9.08 kcal/mol.
ꢀ
ꢀ
account the effect of solvent (water), the calculations were performed with the use of the
polarizable continuum model (PCM). The obtained results were very similar to those received
for the molecules in vacuo (Table 3). ꢀE values calculated using the M06/6ꢀ31G(d) method
did not differ significantly from the values obtained with larger basis sets (M06/6ꢀ31++G(d,p)
and B3LYP/6ꢀ31++G(d,p)) (Table 4). Application of the atomꢀatom dispersion correction
(ωB97XꢀD) led to slightly higher ꢀE values comparing to those obtained with the use of
standard hybrid functionals (Table 4). The best correlation between the experimentally
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obtained rotational energy barrier and calculated
perturbation method.
ꢀE values was only achieved using MP2
Table 4. The relative energy values (ꢁE) of s-cis and s-trans conformers of M1MGxꢀAdeꢀNAC calculated using M06/6ꢀ
31++G(d,p), B3LYP/6ꢀ31++G(d,p), ωB97XꢀD/6ꢀ31G(d) and MP2/6ꢀ31G(d) in vacuo. For comparison the ꢁE values
calculated at initial M06/6ꢀ31G(d) level were given
ωB97XꢀD/
6ꢀ31G(d)
Dihedral
angle*
M06/
6ꢀ31G(d)
M06/
B3LYP/
MP2/
6ꢀ31G(d)
6ꢀ31++G(d,p) 6ꢀ31++G(d,p)
0.00
1.22
s-cis/M
s-cis/M
ꢀ120.6º
130.8º
5.0º
0.00
0.85
0.00
1.26
0.00
0.07
0.27
1.83
10.68
11.87
1.06
s-cis/TS_0
s-cis/TS _180
s-trans/M
9.08
9.29
9.24
15.22
17.73
0.00
177.3º
ꢀ134.7º
130.0º
3.4º
10.07
1.41
10.73
1.52
10.32
1.34
3.05
s-trans/P
3.00
3.23
2.73
2.08
13.82
11.32
s-trans/TS_0
s-trans/TS _180
12.72
10.50
12.32
11.00
13.16
10.73
16.13
14.29
179.2º
* N6ꢀC7ꢀC1”ꢀC2” dihedral angle value
Quantumꢀchemical studies of rotation around the C7ꢀC1” bond in M1MGxꢀAꢀNAC
gave very similar results to those described for M1MGxꢀAdeꢀNAC indicating that the
presence of the ribosyl unit had no significant impact on the values of energy barriers between
the M1MGxꢀAꢀNAC conformers, nor on the ꢀE values of conformers representing structures
of transition states and minima found in the curves of relative energies as function of the N6ꢀ
C7ꢀC1”ꢀC2” dihedral angle values (SI, Chart S2, Tables S3, S4, Fig. S41).
Hydrogen bond formation in M1MGxꢀAdeꢀNAC and M1MGxꢀAꢀNAC based on Atoms
in Molecules theory.
35
The Bader’s Atoms in Molecules (AIM) theory was applied in order to investigate
the intramolecular hydrogen bond formation (or other stabilizing interactions) which may
stabilize rotamers structures and inhibit their fast interconversion at room temperature.
The topological parameters, such as the electron density in bond critical points
(BCPs), ρBCP, its Laplacian, ∇ ,
2ρBCP and density of the total electron energy in BCP, HBCP
correlate well with the Hꢀbond energy, so they well describe the Hꢀbond strength, but may be
applied rather for samples of related compounds.36 For the particular case of OꢀH···O
intermolecular hydrogen bonds in water and methanol clusters, a reasonably good linear
correlation between the charge density at the BCPs and the strength of the HꢀBonds was
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reported.37ꢀ39 Thus, analysis of such parameters may be useful for the estimation of the
relative strength of hydrogen bonding or other stabilizing interactions.40ꢀ43
The analysis of BCP also provides information on the nature of interatomic
interaction.44 For shared interactions like covalent and polarized bonds, the Laplacian of
electron density is negative because there is concentration of electron density within the atomꢀ
atom region. For the interactions between closedꢀshell systems like van der Waals
interactions, ionic ones, and hydrogen bonds, there is the depletion of electron charge within
the atomꢀatom region, and hence the Laplacian is positive. Thus, the sign of the Laplacian
may indicate the kind of interaction. There is a very interesting situation in the case of
hydrogen bonding where usually the Laplacian is positive; however, for very strong hydrogen
bonds like those in H5O2+ or (FHF)ꢀ, the Laplacians are negative, respectively, for both H···O
or H···F contacts. This is the strong evidence for the covalent character of hydrogen
bonding.45
Rozas et al. have classified hydrogen bonds on the basis of
Weak and medium in strength hydrogen bonds show both positive
For strong Hꢀbonds,
2ρBCP is positive and HBCP is negative. For very strong hydrogen bonds,
2ρBCP and consequently HBCP values are negative. Cremer and Kraka involved the energetic
∇
2ρBCP and HBCP values46.
2ρBCP and HBCP values.
∇
∇
∇
topological parameters into the classification of interactions much earlier.47
To sum up, for samples of related compounds the stronger interactions should exhibit
greater value of the electron density in BCP (
ρBCP), greater absolute value of the electron
density’s Laplacian (
∇
2ρBCP) and lower value of the total electron energy in BCP (HBCP).
The mentioned above topological parameters for hydrogen bonds and other stabilizing
interactions in investigated structures are collected in Table 5. Only interactions which need
to be rearranged in order to initiate the rotation P→M (or reverse) were considered as the ones
which may inhibit such interconversion.
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Table 5. Bonding Characteristics: Electron Density,
Density, HBCP. All Values in Atomic Units
ρ
BCP ; Laplacian of the Electron Densities, ∇
2ρBCP ; and Energy
∇
2ρBCP
Structure
Rotamer
Complex
HBCP
ρBCP
s-cis/Mꢀ50
C5ꢀH ··· O=Cacetyl
+0.017
+0.053
+0.032
+0.044
+0.041
+0.047
+0.048
+0.042
+0.047
+0.034
+0.028
+0.034
+0.042
+0.045
+0.042
+0.050
+0.052
+0.042
+0.0002
s-cis/Mꢀ120
s-cis/P
C5ꢀH ··· O=Cacetyl
C8ꢀCH3 ··· O=Cacetyl
C5ꢀH ··· O=Caldehyde
C5ꢀH ··· O=Cacetyl
C5ꢀH ··· O=Caldehyde
C8ꢀCH3 ··· O=Cacetyl
C5ꢀH ··· O=Cacetyl
C5ꢀH ··· C2’’
+0.008
+0.014
+0.010
+0.013
+0.012
+0.014
+0.013
+0.009
+0.008
+0.008
+0.011
+0.014
+0.010
+0.014
+0.013
+0.014
+0.0013
+0.0003
+0.0017
+0.0010
+0.0015
+0.0002
+0.0011
+0.0018
+0.0006
+0.0011
+0.0012
+0.0003
+0.0017
+0.0009
+0.0015
+0.0001
s-trans/M
s-trans/P
s-cis/Mꢀ50
N4 ··· O=Cacetyl
C2’ꢀOH ··· O=Cacetyl
C5ꢀH ··· O=Cacetyl
C8ꢀCH3 ··· O=Cacetyl
C5 ··· O=Caldehyde
C5ꢀH ··· O=Cacetyl
C5ꢀH ··· O=Caldehyde
C8ꢀCH3 ··· O=Cacetyl
s-cis/Mꢀ120
s-cis/P
s-trans/M
s-trans/P
The considered topological parameters of electron density have always positive values. This
indicates that there is no strong interactions which may inhibit rotation and fast
interconversion between rotamers P and M at room temperature. However, the most stable
conformers s-cis/M can exist as the two stable equivalent forms (having almost the same
energy): one with the dihedral angle N6ꢀC7ꢀC1”ꢀC2” of about ꢀ50° (s-cis/Mꢀ50); and other one
with the angle of about ꢀ120° (s-cis/Mꢀ120) (both of them are having the M helicity). Within
the form having the dihedral angle N6ꢀC7ꢀC1”ꢀC2” of about ꢀ50° in compound M1MGxꢀAꢀ
NAC (containig sugar moiety) there are 4 possible interactions recognized by the AIM
analysis (Fig. 18).
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A
B
Fig. 18. The bond paths (black lines), bond critical points (small green dots), ring critical points (small purple dots)
and interatomic surfaces (grey lines) for rotamer s-cis/Mꢀ50 of compound M1MGxꢀAdeꢀNAC (A) and M1MGxꢀAꢀNAC
(B).
Probably, all 4 of them simultaneously are capable to inhibit, to some extent, rotation M→P.
What is interesting, in the analogous case of compound M1MGxꢀAdeꢀNAC, the one without
sugar substituent, such rotamer has only one (C5ꢀH ··· O=Cacetyl) of the four interactions
observed in compound containing the sugar moiety. This shows the role of the sugar
substitution in the stabilization of rotamer s-cis/M, where the sugar moiety is involved in
interaction (C2’ꢀOH ··· O=Cacetyl) (Fig. 18). Moreover, the one interaction in rotamer s-cis/Mꢀ
of compound without the sugar substituent, seems to be the strongest one: having the
50
highest value of electron density (+0.017), the highest absolute value of Laplacian (+0.053)
and the lowest value of total electron energy (+0.0002).
In the case of rotamers s-cis/P there is only one interaction (C8ꢀCH3 ··· O=Cacetyl
)
considered as the one which may inhibit such interconversion. Further, conformers having the
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aldehyde moiety in s-trans configuration (s-trans/M and s-trans/P) are stabilized by the two
interactions each (Table 5).
The difference in topological parameter’s values for corresponding interactions in
compounds M1MGxꢀAdeꢀNAC (without sugar) and M1MGxꢀAꢀNAC (with sugar) allows us
to investigate the impact of the sugar moiety on the strength of considered interactions. All
considered interactions seem to be equal or stronger for the case of compound M1MGxꢀAꢀ
NAC containing the sugar moiety, which indicates that this compound should be less labile in
the considered rotation P→M or reverse (assuming the same energy barrier). However,
relatively low differences in the discussed numerical values do not allow us to make clear
statements.
Conclusions
We studied reactivity of the aldehydic adenine nucleosides adducts towards Nꢀ
acetylcysteine and provided structural characterization of crossꢀlinks that could be induced by
DNA adducts containing carbonyl moieties, in reactions with the cysteine residues of
proteins.
Bifunctional carbonyl compounds of endogenous origin are considered, to be responsible for
different types of crossꢀlinks formation.48 Such carbonyls are known to form adducts with
nucleophilic biomolecules and these adducts can further undergo interactions with other
cellular macromolecules giving rise to crossꢀlinked products.49,50 Our investigations show that
such mechanism can be, at least in part, valid also for the cysteine containing crossꢀlinks
formation. It is wellꢀknown that cysteine residues are involved in the catalytic activity of
enzymes. Moreover the cysteine thiol groups take part in disulfide bonds formation which
plays important role in the protein structure stabilization. Therefore structural modifications
of such sites can have broad functional implications. As strong nucleophilic centers, the
cysteine thiol groups are very probable target for electrophilic attack that leads to DPCs
formation. Despite their ubiquitous presence in living cells, structural features of DPCs, and
mechanisms of their formation still remain to be explored. The literature reports on cysteine
containing DPCs synthesis and structural determination do not refer to stability of such
lesions. Our results show that stability studies seem to be crucial for establishing biological
importance of cysteine containing DPCs and for clarifying role that the amino acid plays in
their formation. Our results also provide insight into the chemical nature of the crossꢀlinks
possibly induced by α,βꢀunsaturated carbonyl compounds between adenine residues of DNA
and cysteine residues of proteins. The highest yield of the Nꢀacetylcysteine crossꢀlinks
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formation was observed in reactions performed at pH about 3. This fact does not exclude
possibility of such crossꢀlinks formation in vivo because they were found to be formed, (in
smaller amount), also under physiological conditions. Apart from that different cellular
compartments are characterized by individual environment including pH. It is worth noticing
that our studies provide first discussion on acidity/neutrality of the cysteine containing
reaction medium, and pH was showed to be a key factor for formation of Nꢀacetylcysteine
crossꢀlinks.
Mechanism proposed for Nꢀacetylcysteine crossꢀlinks degradation and transformation gives
clear explanation for difficulties with detection and isolation of such lesions. Fast
transformation of the cysteine crossꢀlinks occurring in the presence of the lysine was probably
the reason for much lower yield of the methylglyoxal induced crossꢀlinks between cysteine
residues of DNA Klenow fragment Polymerase and guanine residue of the substrate DNA
comparing to analogous crossꢀlinks involving the lysine residues.25 Difficulties with detection
of cysteine containing crossꢀlinks do not rule out the high probability of such lesions
formation in vivo.
Experimental
Materials
1,1,3,3ꢀTetramethoxypropane (TMP), 2’ꢀdeoxyadenosine monohydrate, adenosine,
adenine, methylglyoxal solution (40 wt. % in H2O), glyoxal solution (40 wt. % in H2O), Nꢀ
acetylcysteine, Nαꢀacetyllysine, NH4HCO3, ACN (gradient grade for chromatography), D2O
(containing 0.05 % TSPꢀd4) were purchased from SigmaꢀAldrich.
Chromatographic methods
Progress of the reactions was monitored by LCꢀDAD and the analyses were performed
on an Agilent 1100 series liquid chromatographic system consisting of a binary pump
(G1312A), autosampler (G1313A), vacuum degasser (G1379A), diodeꢀarray detector (UV;
G1315B), a 5 ꢂm, 4.6 x 150 mm reversedꢀphase C18 anal. column (Hypersil BDS, Thermo
Scientific) and Agilent ChemStation data handling program (Agilent Technologies). The
column was eluted isocratically for 5 min with 0.01 M phosphate buffer, pH 7.1, and then
with a gradient from 0 % to 20 % ACN in 20 min at a flow rate of 1.5 ml/min.
Separation and purification of the synthesized compounds was carried out using an
Agilent 1200 Series HPLC system consisting of a binary pump (G1312A), vacuum degasser
(G1379B), autosampler (G1329A), thermostated column compartment (G 1316A), diodeꢀ
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array detector (UV; G1315B), fraction collector (G1364C), and Agilent ChemStation data
handling program (Agilent Technologies). 1.2 mM NH4HCO3 and ACN were used as eluents.
The flow was set to 3 mL/min. Isolation of M1GxꢀA, M1MGxꢀA and M1MGxꢀdA was
performed using a semiprep. 5 ꢂm, 10 x 250 mm (Hypersil BDS, Thermo Scientific)
reversedꢀphase C18 column thermostated at 25 °C. Isolation of M1GxꢀAꢀNAC, M1MGxꢀAꢀ
NAC, M1MGxꢀdA and M1MGxꢀAdeꢀNAC was carried out by the use of a semiprep. 5 ꢂm,
10 x 250 mm (Hypersil GOLD, Thermo Scientific) reversedꢀphase C18 column thermostated
at 20 °C.
ESIꢀLC/MS and ESIꢀLC/MS/MS analyses
The ESIꢀLC/MS and ESIꢀLC/MS/MS analyses were performed with Agilent 1200 LC
system with Agilent QꢀTOF 6540 spectrometer with DUAL AJS ESI source. Ionization was
carried out using nitrogen as nebulizing/drying gas (8 L/min, 300 °C, 35 psi) and sheath gas
(11 L/min, 325 °C). The VCap was set to 3500 V, the fragmentor was set to 100 V. Ion
polarity mode and collision energy values for MS/MS experiments were chosen individually
and given with the appropriate spectra. The analyzed compounds were introduced trough the
LC system using a reversed phase analytical column (5 ꢂm, 4.6 mm x 150 mm, Thermo
Scientific) eluted isocratically for 3 min with water, and then with a gradient from 0 % to 20
% of ACN for 20 min with a flow rate of 0.5 min/min. Thermostat was set to 25 °C.
General procedure for M1GxꢀA and M1MGxꢀdA and M1MGxꢀA synthesis
M1GxꢀA, M1MGxꢀdA, and M1MGxꢀA were synthesized by using a modified
procedure developed earlier in our laboratory.28 Malonaldehyde was prepared in situ by the
acidic hydrolysis of 1,1,3,3ꢀtetramethoxypropane (TMP).51 Syntheses of the adducts were
accomplished according to the methods published previously 29.
Studies on the M1GxꢀA and M1MGxꢀdA and M1MGxꢀA reactivity towards Nꢀ
acetylcysteine (analyticalꢀscale reactions)
Reaction of M1GxꢀA with Nꢀacetylcysteine
Nꢀacetylcysteine (3.6 mg, 22.6 ꢂmol) was dissolved in the solution of M1GxꢀA (0.8
mg, 2.26 ꢂmol) in 0.5 mL of 0.1 M phosphate buffer (pH 7.4). The mixture was divided into
two parts and incubated at 37 °C and 50 °C, respectively. Progress of the reactions was
monitored by LCꢀDAD.
29