Structural characterization of formaldehyde-induced cross-links between amino acids and deoxynucleosides and their oligomers

Article abstract of DOI:10.1021/ja908282f

Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary genotoxic effect. Although DPCs are biologically important and eight amino acids have been reported to form stable adducts with formaldehyde, the structures of these cross-links have not yet been elucidated. We have characterized formaldehyde-induced cross-links of Lys, Cys, His, and Trp with dG, dA, and dC dT formed no cross-links, nor did Arg, Gln, Tyr, or Asn. Reaction of formaldehyde with Lys and dG gave the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and mass spectrometry established that the cross-links between amino acids and single nucleosides involve a formaldehyde-derived methylene bridge. Lys yielded two additional products with dG in which the linking structure is a 1, N²-fused triazino ring. The Lys cross-linked products were unstable at ambient temperature. Reactions between the reactive N ^a-Boc-protected amino acids and the trinucleotides d(T ¹B₂T₃) where B₂ is the target base G, A, or C and reactions between dG, dA and dC and 8-mer peptides containing a single reactive target residue at position 5 yielded cross-linked products with structures inferred from high resolution mass spectrometry and fragmentation patterns that are consistent with those between N^a-Boc-protected amino acids and single nucleotides rigorously determined by NMR studies. These structures will provide a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in identifying biomarkers for the evaluation of formaldehyde exposure both at the site of contact and at distant sites.

Full text of DOI:10.1021/ja908282f

Published on Web 02/23/2010
Structural Characterization of Formaldehyde-Induced
Cross-Links Between Amino Acids and Deoxynucleosides
and Their Oligomers
Kun Lu,† Wenjie Ye,† Li Zhou,‡ Leonard B. Collins,† Xian Chen,‡ Avram Gold,*,†
Louise M Ball,*,† and James A. Swenberg*,†
Department of EnVironmental Sciences and Engineering and Department of Biochemistry and
Biophysics, UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Received October 6, 2009; E-mail: golda@email.unc.edu; lmball@email.unc.edu; jswenber@email.unc.edu
Abstract: Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary
genotoxic effect. Although DPCs are biologically important and eight amino acids have been reported to
form stable adducts with formaldehyde, the structures of these cross-links have not yet been elucidated.
We have characterized formaldehyde-induced cross-links of Lys, Cys, His, and Trp with dG, dA, and dC.
dT formed no cross-links, nor did Arg, Gln, Tyr, or Asn. Reaction of formaldehyde with Lys and dG gave
the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other
coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and
mass spectrometry established that the cross-links between amino acids and single nucleosides involve a
formaldehyde-derived methylene bridge. Lys yielded two additional products with dG in which the linking
structure is a 1,N2-fused triazino ring. The Lys cross-linked products were unstable at ambient temperature.
Reactions between the reactive NR-Boc-protected amino acids and the trinucleotides d(T1B2T3) where B2
is the target base G, A, or C and reactions between dG, dA and dC and 8-mer peptides containing a single
reactive target residue at position 5 yielded cross-linked products with structures inferred from high resolution
mass spectrometry and fragmentation patterns that are consistent with those between NR-Boc-protected
amino acids and single nucleotides rigorously determined by NMR studies. These structures will provide
a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in
identifying biomarkers for the evaluation of formaldehyde exposure both at the site of contact and at distant
sites.
biological relevance are the induction of DNA adducts,7-14
protein modifications,15,16 interstrand DNA cross-links,17-19 and
Introduction
Formaldehyde is a ubiquitous environmental contaminant,
with human exposures occurring during use in industrial
processes such as the manufacture of resins, particle board,
plywood, leather goods, paper, and pharmaceuticals and through
emission as a vapor over the lifetimes of these products.1
Formaldehyde also occurs endogenously as a normal metabolic
intermediate in human cells as well as under certain pathological
conditions such as oxidative stress. Although formaldehyde is
a known carcinogen and mutagen and has thus evoked serious
health concerns,2-6 its mode of action is not well understood.
Among the interactions of formaldehyde considered to have
DNA-protein cross-links (DPCs),5,6,20,21 with the formation of
DPCs being considered as the primary genotoxic effect follow-
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† Department of Environmental Sciences and Engineering.
‡ Department of Biochemistry and Biophysics.
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J. A. Cancer Res. 1983, 43, 4382–4392.
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Cancer Res. 1996, 56, 1012–1022.
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3388 J. AM. CHEM. SOC. 2010, 132, 3388–3399
10.1021/ja908282f  2010 American Chemical Society

Cross-Linked Amino Acids and Deoxynucleosides
A R T I C L E S
Scheme 1. Formation of Formaldehyde-Induced DPCs Originating from the Initial Attack of Formaldehyde on Protein Residues (A) and from
the Initial Attack of Formaldehyde on DNA (B)
ing exposure to formaldehyde.5,6 The two routes shown in
Scheme 1 may be responsible for the formation of DPCs. In
pathway A, formaldehyde addition at a nucleophilic site on a
protein is followed by cross-linking between the resulting protein
methylol adduct and a nucleophilic site on DNA, whereas in
pathway B, addition at a nucleophilic site on a DNA base is
followed by cross-linking between the DNA methylol adduct
and a protein residue.22 The formation of DPCs is favored by
intimate interactions between DNA and proteins22 and the
lysine-rich DNA-binding histones have been reported to cross-
link with DNA9,20,21 in the presence of formaldehyde.
Key to understanding the toxicity of formaldehyde is the
detection and quantitation of the DPCs as biomarkers for
formaldehyde exposure, both at sites of contact and at sites
removed from contact. Of techniques available for this purpose,
those based on liquid chromatography coupled with mass
spectrometry have shown great promise in recent years.23
Selected ion monitoring (SIM), selected reaction monitoring
(SRM), or multiple reaction monitoring (MRM) provide ideal
sensitivity and specificity for the quantitation of DNA adducts.
To take advantage of these techniques, the molecular structures
of the DPCs must be characterized and standards generated. In
a previous communication, we described a formaldehyde-
derived cross-link formed between the Cys sulfhydryl group of
glutathione and dG and suggested that this reaction may be
relevant to systemic effects of formaldehyde transported as an
S-hydroxymethylene conjugate of glutathione.8 In the present
work, we have established that cross-linking reactions occur
between the amino acids Lys, Cys, His, and Trp and the
nucleosides dG, dA, or dC. We have characterized the nature
of the cross-links formed in the presence of formaldehyde
between the reactive amino acids and trinucleotides d(T1B2T3)
where B2 is the target base G, A, or C. We have also examined
the cross-links formed in the presence of formaldehyde between
dG, dA, and dC and N-terminal protected 8-mer peptides
containing a single target residue at position 5. Finally, we have
determined by NMR and mass spectrometric studies the
structures of the cross-links formed between potentially cross-
linking amino acids and dG, dA, and dC in the presence of
formaldehyde as shown in Chart 1. Such structures are
anticipated to reflect cross-links in vivo in regions of single-
stranded DNA and, implicitly, cross-links formed in double-
stranded DNA.
Experimental Section
Chemicals. Potassium phosphate, ammonium bicarbonate, tri-
fluoroacetic acid, formic acid, acetonitrile, methanol, NR-(tert-
butoxycarbonyl)-L-lysine, tert-butoxycarbonyl-L-cysteine, NR-(tert-
butoxycarbonyl)-L-histidine, NR-(tert-butoxycarbonyl)-L-tryptophan,
NR-(tert-butoxycarbonyl)-L-arginine, NR-(tert-butoxycarbonyl)-L-
asparagine, NR-(tert-butoxycarbonyl)-L-glutamine, NR-(tert-butoxy-
carbonyl)-L-tyrosine, deoxyadenosine, deoxythymidine, deoxycy-
tidine, and deoxyguanosine were purchased from Sigma (St. Louis,
MO). The N-terminal acetylated 8-mer peptides Ac-VEGGRGAA,
Ac-VEGGQGAA, Ac-GEGGCGAA, Ac-GEGGYGAA, Ac-VEG-
GKGAA, Ac-VEGGNGAA, Ac-GEGGHGAA, and Ac-GEGGW-
GAA were synthesized by GenScript Corporation (Piscataway, NJ).
Formaldehyde 20% in water was purchased from Tousimis (Rock-
ville, MD). 13CH2O was purchased from Cambridge Isotope
Laboratories (Andover, MA). All chemicals were used as received
unless otherwise stated.
Instrumental Methods. NMR Analysis. NMR Spectra were
recorded on a Varian INOVA NMR spectrometer (Varian, Inc.,
1
Palo Alto, CA) at 500 MHz for H NMR and 125 MHz for 13C
spectra with the Varian cold probe.
Liquid Chromatography-Mass Spectrometry (LC-MS).
LC-MS analyses were performed on a LCQ-Deca ion trap mass
spectrometer (Thermo Electron, Waltham, MA), a TSQ Quantum
Ultra triple quadrupole mass spectrometer (Thermo Electron,
Waltham, MA), or a Q-TOF LC/MS (Model 6240, Agilent
Technologies, Santa Clara, CA). The mass spectrometers were
equipped with electrospray ionization (ESI) sources. Analytes were
separated by reverse phase HPLC using a 250 mm × 2.5 mm C18
analytical column from Grace Vydac (Hesperia, CA) eluted at 200
µL/min with a linear gradient from 2% to 60% solvent A in B
over 15 min. Solvent A consisted of 0.1% formic acid in water,
and solvent B was methanol. The ion trap mass spectrometer was
(22) Brodolin, K. Protein-DNA crosslinking with formaldehyde in vitro
In DNA-Protein Interaction; Andrew, T., Malcolm, B., Eds.; Oxford
University Press: Oxford, 2000; pp 141-150.
(23) Koc, H.; Swenberg, J. A. J. Chromatogr. B 2002, 778, 323–343.
9
J. AM. CHEM. SOC. VOL. 132, NO. 10, 2010 3389

A R T I C L E S
Lu et al.
Chart 1. Structures of Cross-Linked Adducts between Amino Acids and Nucleosides Identified in This Studya
a Formaldehyde-derived linkages are shown in red.
operated in full scan as well as dependent scan modes. For the
fragmentation of precursor ions, the normalized collision energy
of the ion trap mass spectrometer varied from 30% to 35%
depending on the adduct. The activation time was set at 30 ms.
The triple quadrupole mass spectrometer was operated in full scan,
parent mode scan, MS/MS scan, SIM, or SRM mode. The collision
energy was set at 17 V for most fragmentation experiments. Q-TOF
high resolution mass spectra were obtained on an Agilent 6250
Accurate Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara,
CA) equipped with a dual spray ESI source. For liquid chroma-
tography, a Hypersil Gold column (Thermo Scientific, Waltham,
MA) (150 mm × 2.1 mm, 3 µm particle size) was used with a
linear gradient from 2% acetonitrile in 0.1% formic acid to 80%
acetonitrile over 15 min, eluted at 200 µL/min. The ESI source
was set as follows: gas temperature, 350 °C; drying gas, 10 L/min;
Vcap, 4000 V; nebulizer, 35 psig; fragmentor, 100 V; skimmer,
65 V. Fourier-transform ion cyclotron resonance mass spectra
(FTICR-MS) (10 scans) were acquired on a hybrid Qe-Fourier
transform ion cyclotron resonance mass spectrometer equipped with
a 12 T actively shielded magnet (Apex Qe-FTICR-MS, 12.0 T AS,
Bruker Daltonics, Billerica, MA) and an Apollo II microelectrospray
(µESI) source. The voltages on the µESI sprayer, interface plate,
heated capillary exit, deflector, ion funnel, and skimmer were set
at 4.2 kV, 3.9 kV, 300 V, 250 V, 175 V, and 30 V, respectively.
The µESI source temperature was maintained at 180 °C. Desol-
vation was carried out by using a nebulization gas flow (2 bar) and
a countercurrent drying gas flow. Before transfer, ion packets were
accumulated inside the collision cell for a duration of 0.02 s. Using
a syringe pump (Cole Parmer, Vernon Hills, IL), sample solutions
were infused with a 250 µL syringe (Hamilton, Reno, NV) at 90
µL/h.
Formation of Formaldehyde-Induced Cross-Links. Typical
reaction mixtures (final volume 50 µL) consisted of amino acid (1
mM) and deoxynucleoside (1 mM) dissolved in potassium phos-
phate buffer (10 mM, pH 7.2) to which formaldehyde was added
to a final concentration of 50 mM. After 48 h, the reaction mixtures
were either separated by reverse phase HPLC or analyzed by
LC-MS. Lys-dG coupling reactions were carried out with 5, 50,
and 100 mM formaldehyde. Reactions using 50 mM formaldehyde
were run for 48, 60, 72, and 84 h. The coupling reaction was
repeated with 50 mM 13C-formaldehyde for 48 h.
Formaldehyde-induced coupling between peptides and deoxy-
nucleosides was accomplished in the same manner in 50 µL reaction
volumes with final concentrations of 50 mM formaldehyde, 1.5 mM
9
3390 J. AM. CHEM. SOC. VOL. 132, NO. 10, 2010

Cross-Linked Amino Acids and Deoxynucleosides
A R T I C L E S
peptide, and 5 mM deoxynucleoside. For coupling between amino
acids and trinucleotides in 50 µL reaction volumes, final concentra-
tions were 50 mM formaldehyde, 5 mM amino acid, and 1 mM
trinucleotide.
NMR (125 MHz, DMSO-d6) δ 172.6 COOH-Cys, 156.7 C6, 155.3
COO-t-Boc, 151.9 C2, 150.0 C4, 136.1 C8, 120.0 C5, 87.8 C4′,
82.9 C1′, 78.2 C-t-Boc, 70.5 C3′, 61.5 C5′, 53.9 CR, 43.2 CH2-
linker, 39.3 C2′, 31.8 Cꢀ, 28.0 CH3-t-Boc.
Cys-CH2-dA. 1H NMR (500 MHz, DMSO-d6) δ 8.42 (bs, 1H,
N6H), 8.37 (s, 1H, H8), 8.26 (s, 1H, H2), 6.35 (Ψt, 1H, H1′, J )
6.8 Hz), 6.01 (d, 1H, NRH-Cys, J ) 5.26 Hz), 4.66-4.77 (m, 1H,
CH2a-linker), 4.50-4.61 (m, 1H, CH2b-linker), 4.40-4.44 (m, 1H,
H3′), 3.85-3.89 (m, 1H, H4′), 3.61 (dd, 1H, H5′, J ) 11.9, 4.3
Hz), 3.66-3.74 (m, 1H, CRH), 3.53 (dd, 1H, H5′′, J ) 11.9, 4.3
Hz), 2.96-3.07 (m, 2H, CꢀH2), 2.66-2.74 (m, 1H, H2′), 2.27 (ddd,
1H, H2′′, J ) 13.1, 6.8, 3.2 Hz), 1.33 (s, 9H, CH3-t-Boc). 13C NMR
(125 MHz, DMSO-d6) δ 172.2 COOH-Cys, 154.6 COO-t-Boc,
153.6 C6, 150.5 C2, 148.5 C4, 139.9 C8, 120.2 C5, 87.3 C4′, 82.7
C1′, 78.0 C-t-Boc, 70.4 C3′, 61.5 C5′, 55.2 CR, 43.0 CH2-linker,
39.3 C2′, 34.7 Cꢀ, 27.8 CH3-t-Boc.
Large scale reactions between amino acids and deoxynucleosides
for structural characterization by NMR were run with 20 mg of
deoxynucleoside and 40 mg of NR-Boc-amino acid in 5 mL of 10
mM potassium phosphate buffer (pH 7.2) and 100 mM formalde-
hyde for 12 h to 1 week, monitored by HPLC until the chromato-
graphic trace remained constant. The reaction mixtures were
separated by semipreparative HPLC, and collected products were
characterized by mass spectrometry and NMR. Exact masses are
given in Table 3. MS/MS data are presented in full as Supporting
Information. H and 13C shifts are shown below. For the Lys-dG
reactions, HPLC fractions were collected on dry ice, lyophilized,
and then stored at -80 °C until analysis.
Kinetics of Cross-Link Formation. Solutions were made up
to final concentrations of 4 mM amino acid, 4 mM deoxynucleoside,
and 50 mM formaldehyde in 50 mM potassium phosphate buffer
(pH 7.2). The reaction mixtures were analyzed at 2, 4, 8, 16, 24,
48, and 72 h by HPLC with UV detection at 254 nm. For the Lys-
dG coupling reaction, additional samples were analyzed at 10, 20,
30, and 60 min.
Stability of Cross-Linked Lys-dG Products. To measure the
kinetics of cross-link degradation, a reaction mixture containing
Lys and dG as described above was treated with 50 mM
formaldehyde for 48 h and then separated by HPLC. Fractions
eluting at 17.2 and 26.5 min were collected on dry ice, and 50 µL
aliquots of each fraction were added to 950 µL of 50 mM phosphate
buffer maintained at 37 °C for 1, 5, 10, 20, or 30 min and then
analyzed by HPLC with ESI Q-TOF mass spectrometry.
2-Amino-6-(10-oxo-triazino[1,2-a]purin-7-yl)hexanoic Acid
(TPHA-1). 1H NMR (500 MHz, DMSO-d6) δ 7.91 (s,1H, H2), 7.87
(s, 1H, N5H, J ) 2.1 Hz), 6.74 (d, 1H, Boc-NRH-, J ) 4.7 Hz),
6.09 (dd, 1H, H1′, J ) 7.8, 5.9 Hz), 4.89 (s, 2H, C8H2), 4.33 (m,
1H, H3′), 4.24 (bs, 2H, C6H2), 3.83 (m,1H, H4′), 3.75 (dt, 1, CRH,
J ) 8.1, 8.1, 4.7 Hz), 3.54 (dd, 2H, H5′, J ) 11.7, 4.5 Hz), 3.47
(dd, 2H, H5′′, J ) 11.7, 4.5 Hz), ∼2.47 (CεH2 (overlaps DMSO-
d6)), ∼2.50 (H2′ (overlaps DMSO-d6)), 2.18 (ddd, 1H, H2′′, J )
13.1, 5.9, 3.0 Hz), 1.34-1.48 (m, 2H, CδH2), 1.24-1.37 (m, 2H,
CγH2), 1.44-1.58 (m, 2H, CꢀH2), 1.35 (s, 9H, CH3-Boc). 13C NMR
(125 MHz, DMSO-d6) δ 173.9 COOH, 155.8 C10, 155.2 COO-
Boc, 150.2 C4a, 149.0 C3a, 134.9 C2, 115.5 C10a, 87.3 C4′, 82.1
C1′, 77.4 C-Boc, 70.5 C3′, 61.4 C5′, 60.5 C8, 59.5 C6, 53.8 CR,
49.2 Cε, 39.3 C2′, 30.8 Cꢀ, 27.9 CH3-Boc, 26.6 Cδ, 22.8 Cγ.
2-Amino-6-(5-hydroxymethyl-10-oxo-triazino[1,2-a]purin-
7-yl)hexanoic Acid (TPHA-2). 1H NMR (500 MHz, DMSO-d6) δ
7.96 (s,1H, H2), 6.93-6.99 (m,1H, NRH-Lys), 6.19 (dd, 1H, H1′,
J ) 7.8, 5.9 Hz), 4.99 (d, 2H, N5CH2OH, J ) 1.3 Hz), 4.95 (s,
2H, C8H2), 4.45 (s, 2H, C6H2), 4.35 (m, 1H, H3′), 3.78-3.83 (m,
1H, H4′), 3.77-3.84 (m, 1H, CRH), 3.55 (dd, 1H, H5′, J ) 11.6,
4.8 Hz), 3.49 (dd, 1H, H5, J ) 11.6, 4.7 Hz), 2.58 (ddd, 1H, H2′,
J ) 13.2, 7.7, 5.9 Hz), 2.49 (m, 2H, CεH2), 2.21 (ddd,1H, H2′′, J
) 13.2, 7.8, 3.1 Hz), 1.50-1.69 (m, 2H, CꢀH2), 1.36-1.50 (m,
2H, CδH2), 1.36 (s, 9H, CH3-t-Boc), 1.28-1.34 (m, 2H, CγH2).
13C NMR (125 MHz, DMSO-d6) δ 173.9 COOH-Lys, 156.0 C10,
155.4 COO-t-Boc, 149.4 C4a, 148.2 C3a, 136.0 C2, 115.9 C1a,
87.4 C4′, 82.3 C1′, 77.6 C-t-Boc, 70.7 C3′, 68.5 N5CH2OH, 63.9
C6, 61.5 C5′, 61.2 C8, 53.2 CR, 49.0 Cε, 39.2 C2′, 30.4 Cꢀ, 27.9
CH3-t-Boc, 26.6 Cδ, 22.9 Cγ.
Cys-CH2-dC. 1H NMR (500 MHz, DMSO-d6) δ 8.59 (bs, 1H,
N4H), 7.83 (d, 1H, H6, J ) 7.5 Hz), 6.11 (Ψt, 1H, H1′ J ) 6.9
Hz), 5.99 (d, 1H, NRH-Cys, J ) 4.2 Hz), 5.74 (d, 1H, H5, J ) 7.4
Hz), 4.48 (dd, 1H, CH2a-linker, J ) 13.4, 6.6 Hz), 4.26 (dd, 1H,
CH2b- linker, J ) 13.4 6.5 Hz), 4.20-4.24 (m, 1H, H3′), 3.71-3.74
(m, 1H, H4′), 3.64-3.69 (m, 1H, CRH), 3.53-3.60 (m, 2H, H5′,
H5′′), 2.98 (ddd, 2H, CꢀH2, J ) 42.10, 13.5 4.3 Hz), 2.11 (ddd,
1H, H2′, J ) 12.9, 6.9, 4.4; Hz), 1.93 (td, 1H, H2′′, J ) 12.9, 6.9,
6.4 Hz), 1.35 (s, 9H, CH3-t-Boc). 13C NMR (125 MHz, DMSO-d6)
δ 171.1 COOH-Cys, 162.5 C4, 154.7 C2, 154.3 COO-t-Boc, 140.0
C6, 94.7 C5, 87.1 C4′, 84.5 C1′, 77.2 C-t-Boc, 69.5 C3′, 60.8 C5′,
55.2 CR, 42.3 CH2-linker, 40.1 C2′, 35.1 Cꢀ, 27.8 CH3-t-Boc.
His-CH2-dA. 1H NMR (500 MHz, DMSO-d6) δ 8.83 (bs, 1H,
NRH-His), 8.42 (s, 1H, H8), 8.34 (s, 1H, H2), 7.61 (s, 1H, Hε1-
His), 6.96 (s, 1H, Hδ2-His), 6.4 (bs, 1, N6H), 6.36 (Ψt, 1H, H1′, J
) 6.7 Hz), 5.59 (bs, 2H, CH2-linker), 5.32 (bs, 1H, OH3′), 5.12
(bs, 1H, OH5′), 4.39-4.43 (m, 1H, H3′), 3.84-3.89 (m, 1H, H4′),
3.83-3.87 (m, 1H, CRH), 3.48-3.65 (m, 2H, H5′,H5′′), 2.69-2.81
(m, 2H, CꢀH2-His), 2.70-2.75 (m, 1H, H2′), 2.27 (ddd, 1H, H2′′,
J ) 12.8, 6.7, 2.8 Hz), 1.27 (s, 9H, CH3-t-Boc). 13C NMR (125
MHz, DMSO-d6) δ 173.4 COOH-His, 154.6 COO-t-Boc, 148.7 C4,
140.23 C8, 139.3 C2, 138.2 Cγ-His, 136.1 Cε2-His, 119.5 C5, 115.5
Cδ1-His, 87.8 C4′, 83.7 C1′, 77.1 C-t-Boc, 70.6 C3′, 61.5 C5′, 54.0
CR-His, 49.9 CH2-linker, 39.1 C2′, 30.2 Cꢀ-His, 27.9 CH3-t-Boc.
Trp-CH2-dG. 1H NMR (500 MHz, DMSO-d6) δ 7.90 (s, 1H,
H8), 7.06 (d, 1H, Hε3-Trp, J ) 7.14 Hz), 6.91-6.98 (m, 1H, Hη2-
Trp), 6.50-6.58 (m, 2H, Hꢁ2, Hꢁ3-Trp), 6.14 (ψt, 1H, H1′, J ) 6.8
Hz), 6.24-6.06 (m, 1H, N2H), 5.30 (bs, 2H, CH2-linker), 4.36-4.32
(m, 1H, H3′), 4.29-4.21 (m, 1H, CHR), 3.79-3.83 (m, 1H, H4′),
3.68-3.66 (m, 1H, CꢀH2a), 3.58-3.44 (m, 3H, CꢀH2b, + H5′, H5′′),
2.51-2.61 (overlapping DMSO-d6), 2.17-2.25 (m, 1H, H2′′), 1.40
(s, 4H, CH3-t-Boc1), 1.34 (s, 5H, CH3-t-Boc2). 13C NMR (125
MHz, DMSO-d6) δ 156.3 C6, 152.8 CO2H, 150.0 C4, 150.0 Cδ2,
135.5 C8, 129.6 Cε2, 128.1 Cη2, 122.8 Cε3, 116.9 Cꢁ3, 116.8 C5,
108.4 Cꢁ2, 87.5 C4′, 82.8 C1′, 79.8 CH2-linker, 79.0 C-t-Boc, 70.8
C3′, 61.5 C5′, 56.4 CR, 45.3 Cꢀ, 39.3 C2′, 27.9 CH3-t-Boc.
Results and Discussion
Eight amino acids previously reported to form stable adducts
with formaldehyde15 were investigated (as NR-Boc derivatives)
in coupling reactions with all four nucleosides to determine
which would be of interest for characterization of reactions with
oligonucleotides and oligopeptides. No cross-links could be
detected with Arg, Gln, Tyr, or Asn, and consistent with
previous studies,10-13 the endocyclic nitrogen of dT did not form
a coupling product with any of the amino acids. Figure 1 shows
the differences in extent and rate of formation; reactions between
Lys and dG, and Cys and dG are essentially complete after 24 h,
whereas product increased over 72 h for the less reactive
combinations Cys and dA, Cys and dC, His and dA, and Trp
and dG.
Cys-CH2-dG. 1H NMR (500 MHz, DMSO-d6) δ 12.73 (s, 1H,
COOH-Cys), 10.82 (s, 1H, N1H-dG), 7.99 (s, 1, H8), 7.11 (bs,
1H, N2H-dG), 6.99-7.08 (m, 1H, NRH-Cys), 6.16 (ψt, 1H, H1′, J
) 6.8 Hz), 4.48-4.57 (m, 2H, CH2-linker, J ) 10 Hz), 4.35 (td, 1,
H3′, J ) 3.2, 3.2, 6.1 Hz), 4.06-4.12 (m, 1H, CRH-Cys), 3.78-3.82
(m, 1H, H4), 3.55 (dd, 1H, H5′, J ) 11.6, 4.8), 3.48 (dd, 1H, H5′′,
J ) 11.62, 4.8 Hz), 3.03 (dd, 1H, CꢀH2a, J ) 13.5, 4.47 Hz), 2.84
(dd, 1H, CꢀH2b, J ) 13.5, 9.19 Hz), 2.62-2.65 (m, 1H, H2′), 2.21
(ddd, 1H, H2′′, J ) 13.1, 6.8, 3.2 Hz), 1.36 (s, 9H, CH3-t-Boc. 13
C
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A R T I C L E S
Lu et al.
Table 1. Exact Masses for Reaction Products of Trinucleotides and NR-Boc-Protected Amino Acids Cross-Linked by Formaldehyde,
Determined by Negative Ion ESI-QTOF-MS
cross-links
exptl mass ([M - H]-)
calcd mass ([M - H]-)
composition
∆ (ppm)
-
T(G-CH2-Lys)T
T(TPHA-1)T
T(TPHA-2)T
T(G-CH2-Trp)T
T(G-CH2-Cys)T
T(A-CH2-His)T
T(A-CH2-Cys)T
T(C-CH2-Cys)T
1132.3360
1144.3373
1174.3494
1190.3264
1107.2526
1125.3048
1091.2556
1067.2464
1132.3395
1144.3395
1174.3501
1190.3239
1107.2537
1125.3085
1091.2588
1067.2476
C42H60N11O22P2-
C43H60N11O22P2-
C44H62N11O23P2-
C47H58N11O22P2
C39H53N10O22P2S-
-3.1
-1.9
-0.6
2.2
-1.0
-3.3
-2.9
-1.1
-
C42H55N12O21P2
C39H53N10O21P2S-
C38H53N8O22P2S-
The high amount of cross-links formed by Lys is of particular
interestbecausethisresidueisinvolvedinextensiveDNA-protein
contacts and may therefore be considered highly likely to form
cross-links in vivo. The second most abundant cross-link,
between Cys and dG, may also have relevance for the active
site of alkylguanine alkyltransferases24 where a Cys residue can
come into proximity with a formaldehyde adduct of guanine.
Trinucleotides Cross-Linked to Nr-Boc-Protected Amino
Acids. Since dT did not form adducts, we selected trinucleotides
having G, A, or C flanked by T as targets for studies of
trinucleotide-amino acid cross-linking. Elemental compositions
of the deprotonated molecules [M - H]- of cross-linked
products of the trinucleotides and NR-Boc-protected amino acids
were determined by high resolution ESI-QTOF mass spectrom-
etry and are given in Table 1. All trinucleotides yielded
deprotonated molecules with summed masses expected for the
trinucleotide + NR-Boc-protected amino acid + 12 mass units,
consistent with formation of a methylene link between the
trinucleotide and amino acid. The reaction of TGT with Lys
and formaldehyde yielded two additional products: [T(TPHA-
1)T], having a composition expected for the formation of two
methylene linkages and [T(TPHA-2)T], having a composition
expected for two methylene linkages and a hydroxymethylene
adduct.
intramolecular condensation of terminal amino nitrogens from
two peptide residues with formaldehyde as well as from glycine-
formaldehyde condensation with the guanidino moiety of Arg.15
Biological significance of these products is unlikely because of
the lower concentrations of formaldehyde in vivo.
Fragmentation of the reaction products between trinucleotides
and amino acids was investigated by high resolution QTOF MS/
MS to confirm that the target base in the second position was
indeed the site of the cross-linking reaction. The high resolution
data provide elemental compositions to support structural
assignments of product ions. The product ion nomenclature
applied for describing backbone fragmentation patterns of the
trinucleotides follows the widely used convention given in Chart
2.
MS/MS spectra, acquired on an ion trap mass spectrometer,
have been reported for all 64 possible unmodified trinucle-
otides.25 While extensive sequential decomposition would be
more likely in the ion trap than in the QTOF used in our work,
the fragmentation patterns observed for the modified trinucle-
otides show reaction sequences similar to those reported
previously.25 Without exception, the cross-linked trinucleotides
formed singly charged anions. The MS/MS spectra of the cross-
linked trinucleotides were characterized by initial cleavage of
the coupling linkage, with the resulting product ions undergoing
backbone fragmentation. With the exception of TAT cross-
linked with His, none of the ions containing an intact base-
methylene-amino acid linkage was the source of backbone
fragmentations. Full MS-MS spectra and assignments of product
ions for all cross-linked trinucleotides are presented as Sup-
porting Information (Figures S1-S8).
As discussed below, NMR analysis of the products with
multiple-methylene linkages establishes T(TPHA-1)T as T(10-
oxo-triazino[1,2-a]purin-7-yl)T-substituted 2-aminohexanoic acid
and T(TPHA-2)T as the corresponding N5-hydroxymethyl
adduct. Formation of triazinane rings has precedent in the
TGT Derivatives. In addition to the w1 ion (dT-5′-P-; m/z
-
321), a major ion in the MS/MS of all of the TGT derivatives
was observed at nominal mass m/z 866 [(TGT - H + 12)-].
This ion corresponds in composition to the Schiff base derivative
of the trinucleotide at G, which is possible only at the exocyclic
N2 and confirms that the formaldehyde-induced cross-linking
reactions of TGT involve the target G. The non sequence ion
from loss of neutral TH from the Schiff base adduct of
the trinucleotide is also common to all the MS/MS spectra
of cross-linked TGT products. In the MS/MS spectrum of
T(G-CH2-Lys)T, bonds on either side of the methylene linkage
cleave, leading to a prominent ion at m/z 874 resulting from
the loss of the Schiff base adduct of Lys in addition to the ion
at m/z 886. Backbone cleavages of the product ions at m/z 874
-
and 886 give rise to parallel series of sequence ions w2-, x2
,
y2-, and z2- separated by 12 mass units, as expected for source
ions TGT- and its Schiff base adduct at G. The MS/MS of
Figure 1. Formation of cross-links between 4 mM amino acid and 4 mM
nucleoside treated with 50 mM formaldehyde: (, sum of Lys products; 0,
Cys-CH2-dG; 2, Cys-CH2-dA; ×, Cys-CH2-dC; 4, His-CH2-dA;
b, Trp-CH2-dG. Relative yields are based on integration of HPLC peak
areas in UV traces recorded at 254 nm.
(24) Daniels, D. S.; Woo, T. T.; Luu, K. X.; Noll, D. M.; Clarke, N. D.;
Pegg, A. E.; Tainer, J. A. Nat. Struct. Mol. Biol. 2004, 11, 714–720.
(25) Vrkic, A. K.; O’Hair, R. A. J.; Foote, S. Aust. J. Chem. 2000, 53,
307–319.
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A R T I C L E S
Chart 2
cross-link between dA and Cys discussed below. Although
product ions in MS/MS spectrum of T(C-CH2-Cys)T (Figure
S8) were in low abundance, the exact mass of the base peak
lies within an acceptable tolerance (4.1 ppm) for a composition
corresponding to the Schiff base adduct of trinucleotide which
is consistent with a methylene bridge between the C-N4 and
-
-
the Cys sulfhydryl group. Sequence ions w2 , x2 , and w1- were
also present at the expected nominal masses, in addition to the
nonsequence ion (M - H - TH)-.
Peptides Cross-Linked to Deoxynucleosides. We reacted dG,
dA, and dC with formaldehyde and N-terminal acetylated 8-mers
in which position 5 contained one of the four residues that had
been established as targets for cross-linking in the screening
reactions. The exact masses of the protonated molecules are
given in Table 2. As in the case of the trinucleotides, all of the
8-mers yielded singly charged ions with compositions corre-
sponding to formation of a methylene cross-link, and the Lys-
containing 8-mers yielded two additional products consistent
with formation of the tricyclic cross-linked structures TPHA-1
and TPHA-2. In accord with a report on the formation of
formaldehyde adducts of 7-mer and 9-mer peptides under
conditions similar to ours,15 we found no evidence of
peptide-peptide cross-linking by LC-MS analysis over the
range of mass-to-charge ratios expected for cross-linked peptides.
By MS/MS (Figures S9-S16 in Supporting Information), the
protonated molecules undergo initial fragmentation of the
glycosidic bond or the bridging structure, and the resulting
product ions undergo backbone fragmentations similar to those
observed for unmodified peptides, giving predominantly yn, an
and bn ions.26 Analysis of MS/MS spectra of the protonated
molecules definitively established the site of formaldehyde-
induced cross-linking for all of the peptides with the exception
of the His-containing 8-mer cross-linked with dA.
Backbone fragmentations in the MS/MS spectra of the Lys-
and Trp-containing 8-mers with a single methylene link to dG
(Figures S9 and S10) are derived from the intermediate product
ions in which the cross-link has broken to yield a peptide
sequence containing the Schiff base of Lys (Scheme 2) or the
2-methylene indole derivative of Trp. Figure 3 illustrates the
MS/MS of the singly bridged Lys-containing octapeptide.
The mass difference between the b5 and b4 ions corresponds
to the Schiff base adduct of the target Lys (or 2-methylene-
substituted Trp) at position 5, which establishes the point of
attachment of the methylene bridge at the predicted target
residues. The modification of the Lys and Trp residues is further
confirmed by the identification of y4 ions, and the determination
that the mass differences between (b5 + y4) and the intermediate
product ions yielding the backbone fragmentation series (m/z
742 for the Lys 8-mer and 758 for the Trp 8-mer) correspond
to the mass of the modified residue. The product ions represent-
ing loss of the Schiff base adduct of dG from the cross-linked
peptides establishes the exocyclic N2 of dG as the second point
of attachment of the methylene bridge, as discussed above for
the cross-linked trinucleotides.
T(G-CH2-Lys)T in Figure 2 is illustrative of the data obtained
from QTOF analysis.
The triazino ring of T(TPHA-1)T fragments to yield the
product ions TGT- and Schiff base adduct of the trinucleotide
from which are derived the same parallel series of backbone
fragmentations observed in the MS/MS spectrum of the singly
bridged TGT-Lys product (Figure S2; formation of sodium
adducts of the major ions in the MS/MS spectrum of T(TPHA-
1)T should be noted). In the MS/MS of T(TPHA-2)T (Figure
S3), the cross-linking structure fragments sequentially to give
prominent ions corresponding to [M - H - hydroxymethyl-
ene]- (nominal m/z 1144) and [M - H - NR-Boc - hydroxym-
ethylene]- (nominal m/z 1044) in addition to the Schiff base
adduct of TGT at m/z 866, which is progenitor of the sole series
-
of observed sequence ions w2-, x2-, y2-, and z2
.
The MS/MS spectrum of T(G-CH2-Cys)T (Figure S4)
shows a single fragmentation pathway consistent with loss of
Cys to give the Schiff base adduct of TGT at m/z 866 and
-
-
formation of the sequence ions w2-, x2 y2-, and z2 from the
expected backbone fragmentations. The MS/MS spectrum of
T(G-CH2-Trp)T (Figure S5) yielded product ions in low
abundance. The Schiff base adduct of TGT and backbone
-
-
cleavage ions w2 , x2- y2 , and z2- were present at the expected
nominal mass-to-charge ratios; however, the accuracy of mass
measurements was low.
TAT and TCT Derivatives. Loss of NR-Boc (m/z 1025) and
(NR-Boc + His) (m/z 870) were prominent ions in the MS/MS
spectrum of T(A-CH2-His)T (Figure S6). The ion at m/z 1025
gave the non sequence ion (M - H - TH)- and sequence ions
-
-
w2 and x2 from backbone fragmentation in which the cross-
link is intact (the only instance of backbone fragmentations with
an intact cross-link). The Schiff base derivative from loss of
(NR-Boc + His) (m/z 870) yielded the parallel series of sequence
-
ions w2 and x2-, fixing the point of cross-link attachment at
exocyclic N6 of adenine. The MS/MS spectrum of the depro-
tonated molecule T(A-CH2-Cys)T (Figure S7) features (y2 -
B2)-, w1- and T- (base peak) ions along with a prominent ion
at nominal mass m/z 545. However, no ion containing the Schiff
base adduct of A was detected. The nominal mass m/z 545
corresponds to a 2′,3′-dideoxy-2′,3′-dehydro-AMP or 2′-deoxy-
adenosine-5′-phosphenate linked through a methylene bridge
to NR-Boc-His. The exact mass differs by +10.8 ppm from that
calculated for the composition of the proposed structures, within
sufficient tolerance to support the methylene bridge between
Cys and the target N6 of A. However, a backbone fragmentation
pathway yielding ions of either of the proposed structures was
not reported for any of the 64 possible unmodified trinucleotides
or observed for any of the other cross-linked trinucleotides in
this study. Thus, support for the proposed structure of the cross-
link relies on the NMR studies of the formaldehyde-derived
In the MS/MS spectrum of the Cys-containing 8-mer cross-
linked to dC (Figure S11), a series of low-abundance bn ions
originates from the intermediate product ion (m/z 697) after loss
of the deoxyribose from the protonated molecule. The difference
in mass between the b5 and b4 ions of this bn series corresponds
to the mass of the Cys-CH2-C unit, confirming that the cross-
link is attached to the peptide at Cys. Additional support for
(26) Paizs, B.; Suhai, S. J. Am. Soc. Mass Spectrom. 2004, 15, 103–113.
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A R T I C L E S
Lu et al.
Figure 2. ESI-QTOF-MS/MS of the deprotonated molecule T(G-CH2-Lys)T: (green) ions containing Schiff base guanine; (red) ions containing unmodified
guanine; (black) ions that do not contain guanine.
Table 2. Exact Masses of Protonated 8-Mers Cross-Linked to Deoxynucleosides by Formaldehyde Determined by Positive Ion
ESI-QTOF-MS
cross-links
exptl mass ([MH]+)
calcd mass ([MH]+)
composition
∆ (ppm)
+
+
+
+
acetyl-VEGG(K-CH2-dG)GAA
acetyl-VEGG(TPHA-1) GAA
acetyl-VEGG(TPHA-2)GAA
acetyl-GEGG(W-CH2-dG)GAA
acetyl-GEGG(C-CH2-dG)GAA
acetyl-VEGG(H-CH2-dA)GAA
acetyl-GEGG(C-CH2-dC)GAA
acetyl-GEGG(C-CH2-dA)GAA
1009.4687
1021.4702
1051.4805
1025.4048
942.3372
1002.4383
902.3304
926.3427
1009.4697
1021.4697
1051.4803
1025.4071
942.3370
1002.4388
902.3309
926.3421
C41H65N14O16
C42H65N14O16
C43H67N14O17
C43H57N14O16
-1.0
0.5
0.2
-2.2
0.2
-0.5
-0.6
0.6
C35H52N13O16S+
+
C41H60N15O15
C34H52N11O16S+
C35H52N13O15S+
Scheme 2. Fragmentation of AcVEGG(K-CH2-dG)GAA
the cross-link to Cys is a second series of low abundance b5,
b6, and b7 ions having compositions consistent with backbone
fragmentation of the product ion from cleavage of the C-S bond
of methylene bridge which transforms Cys to an R-amidoacrylic
acid residue. As required by this scheme, the difference in mass
between the b5 (m/z 412) and b4 (m/z 343), which does not
contain a modified residue, is 69 mass units, corresponding to
R-amidoacrylic acid at position 5. Major backbone fragmentation
series for the Cys-containing 8-mers cross-linked with dA
(Figure S12) and dG (Figure S13) originate from the product
ions subsequent to the loss of the Schiff base derivatives of the
nucleosides and therefore are not informative with respect to
the site of attachment of the methylene bridge to the peptide.
However, the bn series from the intermediate product ions
containing R-amidoacrylic acid at position 5 are present in the
MS/MS spectra of both cross-linked 8-mers, fixing the meth-
ylene bridge attachment at Cys.
The MS/MS spectrum of the Lys-containing 8-mer linked
by two methylene groups through formation of a fused triazino
ring (Figure S14) is consistent with initial fragmentation of the
triazino ring followed by proton transfer to give Schiff base
adducts of both dG and the 8-mer (nominal mass, m/z 742)
according to Scheme 3, with the charge residing predominantly
on the 8-mer fragment. The Schiff base adduct of dG loses
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3394 J. AM. CHEM. SOC. VOL. 132, NO. 10, 2010

Cross-Linked Amino Acids and Deoxynucleosides
A R T I C L E S
Figure 3. ESI-QTOF-MS/MS spectrum of the protonated molecule acetyl-VEGG(K-CH2-dG)GAA: (green) ions containing Schiff base derivative of Lys;
(red) ions containing unmodified Lys; (black) ions that do not contain residue 5.
Scheme 3. Fragmentation of AcVEGG(TPHA-1)GAA
deoxyribose to give an ion at m/z 164 in low abundance. The
absence of an ion corresponding to [MH - dG]+ (nominal mass
m/z 730) accompanied by product ions from its backbone
fragmentation, such as observed above for a single cross-linking
methylene, supports the triazino structure. The difference in mass
between b5 and b4 ions corresponds to the Schiff base adduct
at the terminal -NH2 of the Lys residue as required by Scheme
3.
N2-Schiff base adduct of G and at m/z 178 compatible with the
protonated tricyclic base 1,N2-ethano-G.
In the MS/MS spectrum of the His-containing 8-mer cross-
linked to dA, a single series of backbone fragmentations arises
from an intermediate product ion via loss of the Schiff base
adduct of dA. This fragmentation pattern is not informative
regarding the site of methylene attachment to the 8-mer, and
the structure of the cross-link is inferred from the structures
determined by NMR studies for cross-linked monomeric units
dA-CH2-His.
Nr-Boc-Protected Amino Acids Cross-Linked to Deoxy-
nucleosides. Formaldehyde-induced coupling reactions between
the NR-Boc-protected amino acids and nucleoside monomers
were investigated on a scale that allowed detailed structural
determination of the linkages by NMR spectrometry. Table 3
gives the exact masses and corresponding elemental composi-
tions of the cross-linked products, and MS/MS spectra are given
in Figures S17-S24 in Supporting Information. LC-MS
analysis revealed no cross-links formed between deoxynucleo-
The MS/MS spectrum of the product the containing the
tricyclic (TPHA-2) cross-linking structure with an N5 hy-
droxymethyl-substituted triazinopurine (Figure S15) is domi-
nated by two series of ions derived from peptide backbone
fragmentations of the intermediate product ions shown in
Scheme 4. The y4, b4, and b5 ions can be identified for both
series, and the compositions establish that the terminal -NH2 of
Lys has been incorporated into the linking structure. The
sequential fragmentations of the protonated molecule that lead
to the intermediate products in Scheme 4 are suggested by the
presence of product ions both at m/z 164 for the protonated
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A R T I C L E S
Lu et al.
Scheme 4. Fragmentation of AcVEGG(TPHA-2)GAA
When 13C-formaldehyde was used in the coupling reaction, the
protonated molecules are observed at m/z 527, 540, and 571,
confirming formaldehyde as the origin of the methylene linkers
and the hydroxymethylene group.
Table 3. Exact Masses of Formaldehyde-Induced
Deoxynucleoside-Amino Acid Cross-Links by FTICR-MS
cross-links
exptl mass ([MH]+) calcd mass ([MH]+)
composition
∆ (ppm)
Lys-CH2-dG
TPHA-1
TPHA-2
Trp-CH2-dG
Cys-CH2-dG
His-CH2-dA
Cys-CH2-dA
Cys-CH2-dC
526.2622
538.2623
568.2726
584.2465
501.1763
519.2312
485.1813
461.1701
526.2619
538.2619
568.2725
584.2463
501.1762
519.2310
485.1813
461.1700
C22H35N7O8
C23H35N7O8
C24H37N7O9
C27H33N7O8
C19H28N6O8S
C22H30N8O7
C19H28N6O7S
C19H28N6O8S
0.6
0.7
0.2
0.3
0.2
0.4
0
Well-resolved peaks were collected by semipreparative HPLC
(Figure 4) and characterized by NMR (Figures S26-S31 in
Supporting Information). 1H NMR spectra of the fractions
collected at 17.2 and 26.5 min indicated they were dG-
containing mixtures, suggesting postcolumn decomposition.
The peak eluting at 17.2 min contained dG/Lys/TPHA-1 in
1:1:1 ratio, whereas the peak at 26.5 min contained a 1:1.4:7
mixture of dG/TPHA-1/TPHA-2 based on signals in the H1′
region of the NMR spectra. In the early eluting fraction, Lys
and dG were readily identified by comparison of well resolved
signals in critical regions of the spectrum with authentic
standards, and in the late-eluting fraction, signals of TPHA-1
and TPHA-2 were readily distinguished because of the differ-
ence in signal intensities and peak integrals. The composition
of the fractions may be explained by co-chromatography of
TPHA-1 and Lys-CH2-dG at 17.2 min and sequential uncou-
pling of formaldehyde units in both fractions. The absence of
signals in either fraction attributable to singly bridged
Lys-CH2-dG suggests that Lys-CH2-dG degrades signifi-
cantly faster than the tricyclic adducts. The appearance of dG
and TPHA-1 in the 26.5 min fraction suggests that both TPHA-1
0.2
sides or between amino acids. As observed for the trinucleotide
and 8-mer, coupling products of Lys with dG incorporated one
or two methylene groups or two methylenes with a hydroxym-
ethyl adduct. All of the Lys coupling products were labile and
were isolated and stored at subambient temperature. The lability
of the Lys coupling products is consistent with reports that
formaldehyde-induced DPCs involving the Lys- and Arg-rich
major histones are hydrolytically unstable.15,20 The structures
of formaldehyde-induced cross-links formed between the amino
acids and nucleosides are given in Chart 1.
Lys Cross-Links with dG. In the presence of formaldehyde,
Lys and dG formed three coupling products with the structures
given in Chart 1. All three structures were detected in coupling
reactions run at the lowest formaldehyde concentration. The
proportion of tricyclic and N-hydroxymethyl-substituted tricyclic
nucleosides increased with increasing formaldehyde concentra-
tion and reaction time (confirmed with 13C-formaldehyde),
consistent with progressive incorporation of formaldehyde
molecules into the initial coupling product (Figure S25). As
described above, the products were all labile at ambient
temperature and required care in isolation and characterization.
The tricyclic structures TPHA-1 and TPHA-2 were definitively
established by mass spectrometry and NMR. Lys-CH2-dG was
too labile for characterization by NMR, and its assignment as
a product of cross-linking is based on indirect evidence from
the analyses below.
1
and TPHA-2 are unstable. The mixtures observed in the H
NMR spectra are consistent with the degradation at 37 °C of
the 17.2 and 26.5 min fractions monitored by mass spectrometry
(Figure S32). Uncoupling is supported additionally by a signal
Exact mass measurements by ESI-MS in the positive ion
mode of major ions at m/z 526, 538, and 568 in the coupling
reaction mixture were compatible with the protonated molecules
Lys-CH2-dG, TPHA-1 and TPHA-2, respectively (Table 3).
Figure 4. Trace (254 nm) of semipreparative HPLC of the reaction mixture
from the formaldehyde-induced coupling of Lys and dG. The peaks at 17.2
and 26.5 min were characterized by NMR. Peaks at 7.3 and 10.2 min are
dG and N2-hydroxymethyl-dG, respectively.
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A R T I C L E S
1
Figure 5. HPLC peak at 17.2 min containing TPHA-1: expansion of the HMBC spectrum between 2.0 and 5.2 ppm on the H-axis and 15 and 166 ppm
1
on the 13C-axis. Key signals are identified on the marginal H and 13C traces. Unsuppressed 1-bond couplings are indicated by brackets.
1
Figure 6. HPLC peak at 26.5 min containing TPHA-2: expansion of the HMBC spectrum between 2.0 and 5.2 ppm on the H-axis and 40 and 160 ppm
1
on the 13C-axis. Key signals are identified on the marginal H and 13C traces. Unsuppressed 1-bond couplings are indicated by brackets.
1
in the H NMR spectra of both early and late-eluting fractions
attributable to formaldehyde or formaldehyde hydrate (Figures
S26, S29) in accord with reported reversibility of formaldehyde-
induced DPCs.20 The formaldehyde/formaldehyde hydrate as-
signment is based on the appearance of a proton singlet below
8 ppm having unsuppressed 1-bond coupling with a carbon
signal at 164.4 ppm that has no connectivity with any component
in the HMBC spectra of the mixtures (Figures S26, S29).
In each fraction, the NMR signals of the components of the
mixtures could be resolved, allowing the structures of TPHA-1
and TPHA-2 to be unambiguously assigned. Critical in estab-
lishing the triazine ring is the presence of two formaldehyde-
derived methylenes and identification of the connectivities
between the methylene groups, guanine, and Lys moieties.
Figures 5 and 6 show expansions of the HMBC spectra that
are key to establishing the fused triazine linkages. In the HMBC
spectrum of TPHA-1 (Figure 5), a broad two-proton singlet at
4.89 with three-bond coupling to a 13C signal at 59.5 ppm and
3
a second two-proton singlet at 4.24 ppm with JC-H coupling
to a 13C signal at 60.5 ppm are assigned to methylene groups at
positions 8 and 6, respectively, of the triazino[1,2-a]purine
framework. Unsuppressed one-bond couplings, confirmed by
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A R T I C L E S
Lu et al.
the corresponding C/H cross peaks in the HSQC spectrum
(Figure S27) are observed for both methylene signals and allow
assignment of the methylene carbon shifts. The protons attached
to the methylene carbon assigned to C8 show the expected
connectivities within the tricyclic framework between C4a at
150.2 ppm and C10 at 155.8 ppm, while the methylene protons
at position 6 couples only with the carbon at C4a. Attachment
of the hexanoic acid moiety at N7 is confirmed by cross peaks
between the methylene protons at positions 6 and 8 and a carbon
signal at 49.2 ppm, which can be assigned to hexanoic acid Cε
by virtue of unsuppressed one-bond coupling in the HMBC
spectrum and the corresponding C/H cross peak in the HSQC
spectrum. The complementary three-bond coupling between the
methylene protons attached to Cε (overlapping with the DMSO
signal and sugar H2′′ signals at ∼2.5 ppm) and C6 and C8 is
also observed.
By a similar analysis of the HMBC spectrum in Figure 6,
connectivities can be identified to confirm the triazino-
[1,2a]purine framework of TPHA-2, the major component of
the late-eluting peak. The critical cross peaks are between C8H2
(4.95 ppm) and C6 (63.9 ppm), C4a (149.4 ppm), C10 (156.0
ppm), and Cε (49.0 ppm); between C6H2 at 4.45 ppm and C8
(61.2 ppm), C4a (149.4 ppm), and Cε (49.0 ppm); and between
CεH2 (2.5 ppm, overlapping with DMSO) and C6 and C8. A
methylene proton signal at 4.99 ppm can be assigned to the
hydroxymethylene group with the position of attachment fixed
at N5 by virtue of cross peaks with C6 and C4a. In the 1H NMR
spectrum (Figure S29), a singlet, assignable to formaldehyde
by the rationale described above, strongly supports the conclu-
sion that the presence of THPA-1 in the late-eluting fraction
results from the hydrolytic elimination of the N5-hydroxym-
ethylene substituent of TPHA-2. The dynamic nature of
formaldehyde-induced Lys-dG cross-links (Scheme S1) is
supported by the lability of the products and the presence of
formaldehyde in the NMR spectra of TPHA-1 and TPHA-2,
which indicate that the condensations are reversible.
identification of Cys-NRH as one of two incompletely resolved
proton signals at ∼7.10 ppm based on cross peaks with Cys CR
(53.9 ppm) and Cꢀ (31.8 ppm).
Cys formed stable cross-links with dA and dC in minor
amounts. By high resolution mass spectrometry, both products
had empirical compositions consistent with a methylene bridge
linking the nucleoside and Cys. Both products were character-
ized by NMR (Figures S35-S38 in Supporting Information).
Critical C/H connectivities in the HMBC spectra (Figures S35,
S37) were observed between the proton signals of the bridging
methylene and a carbon signal of the base, and between the
bridging methylene and Cys ꢀCH2. In the case of Cys-CH2-dA,
attachment to dA at the exocyclic amino group was established
by coupling between the diastereotopic methylene proton
multiplets of the bridging groups centered at 4.56 and 4.73 ppm
and C6 of dA at 153.6 ppm (Figure S35). Attachment to the
Cys sulfhydryl group was established by coupling between the
diastereotopic methylene bridge protons and the Cꢀ signal of
Cys at 34.7 ppm. Identification of a one-proton N6H signal
confirmed the substitution at the exocyclic amino group of dA,
while presence of the Boc-NRH proton signal ruled out attach-
ment at the R-amino group of Cys. The cross-link between dC
and Cys was similarly determined to be between the exocyclic
amino group of dC and the Cys sulfhydryl because of the
observed coupling between the bridging methylene protons at
4.48 and 4.26 ppm and C4 of dC at 162.5 ppm and between
the bridging methylene protons and Cꢀ of Cys at 35.1 ppm
(Figure S37). As in the case of Cys-CH2-dA, the position of
the cross-link determined from the HMBC spectrum is supported
by one-proton signals attributable to N4H of dC and Boc-NRH.
His Cross-Link with dA. A low yield of stable cross-linked
product was isolated from the reaction of His and dA with
formaldehyde, incorporating one methylene linkage identified
by accurate mass measurement. Despite the absence of C,H
cross-peaks between the linker methylene and His, attachment
at Nε2 could readily be established by the presence of imidazole
ring C-H proton signals Hδ2 at 6.96 and Hꢀ1 at 7.61 ppm, which
were definitively assigned by unsuppressed one-bond coupling
to carbon signals at 115.5 and 136.1 ppm, respectively, in the
HMBC spectrum (Figure S39). Site of attachment to dA rests
on the presence of a signal having an integral value of one
proton assigned to N6H. When the cross-link is derived from
13C-formaldehyde, a broad two-proton signal at 5.59 ppm in
Cys Cross-Links with dG, dA, and dC. A single product was
isolated from the cross-linking of Cys and dG with formalde-
hyde. In contrast to the cross-linked products of Lys, the product
with Cys was stable and readily isolated and purified. The exact
mass corresponded in elemental composition to addition of one
methylene group. ESI-MS/MS of the protonated molecule
(Figure S20) yielded product ions from loss of deoxyribose and
t-Boc. The base peak of the MS/MS spectrum appears at m/z
164 (Figure S20), in accord with fragmentation to a Schiff base
derivative of guanine, which indicates that the methylene cross-
link bridges N2 of guanine and the Cys sulfhydryl group.
NMR spectrometry (Figures S33, 34) confirmed guanine N2
and Cys SH as the points of attachment of the methylene cross-
link. The carbon and proton signals of formaldehyde-derived
methylene cross-link are assigned from the HMBC spectrum
on the basis of unsuppressed one-bond coupling between the
carbon signal at 43.2 ppm and a 2-proton methylene multiplet
centered at 4.52 ppm. Attachment at the Cys sulfhydryl is then
fixed by coupling between the diastereotopic CꢀH2 protons of
Cys at 2.84 and 3.03 ppm and the formaldehyde-derived
methylene bridging carbon, while attachment at guanine N2 is
fixed by coupling between the bridging methylene protons and
C2 of guanine at 151.9 pm. The presence of the guanine imino
1
the H NMR spectrum is replaced by a two-proton doublet
centered at 5.59 ppm, 1JC-H ) 153.2 Hz (Figure S40), and can
be assigned to the formaldehyde-derived methylene bridge. This
signal has the expected NOESY interactions with the imidazole
protons in the ROESY spectrum (Figure S41).
Trp Cross-Link with dG. The Trp coupling product incor-
porated one methylene cross-link determined by accurate mass
measurement. The site of attachment to Trp can be definitively
fixed at C2 of the indole ring and is analogous to the reported
formation of tetrahydro-ꢀ-carboline from Trp via intramolecular
incorporation of a formaldehyde-derived methylene when the
R-amino group is unprotected.27,28 The proton signal of the
formaldehyde-derived methylene forming the cross-link to dG
is identified by a broad two-proton resonance at 5.30 ppm in
1
the H NMR spectrum attached to a carbon having a signal at
79.8 ppm in the HSQC spectrum (Figure S42), which is also
1
N1H at 10.82 ppm in the H NMR spectrum, unambiguously
(27) Tammler, U.; Quillan, J. M.; Lehmann, J.; Sadee, W.; Kassack, M. U.
Eur. J. Med. Chem. 2003, 38, 481–493.
assigned from coupling with guanine C5 at 120 ppm in the
HMBC spectrum (Figure S33), rules out attachment of the cross-
link at guanine N1. Attachment to Cys at NR is ruled out by
(28) Zhao, M.; Bi, L.; Bi, W.; Wang, C.; Yang, Z.; Ju, J.; Peng, S. Bioorg.
Med. Chem. 2006, 14, 4761–4774.
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Cross-Linked Amino Acids and Deoxynucleosides
A R T I C L E S
detected by unsuppressed one-bond coupling (1JC-H ) 164.4
Hz; Figure S43) in the HMBC spectrum. Attachment of the
methylene at indole C2 is supported by the detection of indole
benzo-ring carbons as the only indole carbons having attached
protons in the HSQC spectrum and by NOESY interactions
between the bridge methylene and Trp CꢀH2 proton signals in
the ROESY spectrum (Figure S44), which would not be
observed if attachment of the methylene were at the indole
nitrogen. While the proton-bearing carbons of the Trp indole
ring were readily identified in the HSQC spectrum, overlapping
of the quaternary carbon signals of both indole and dG moieties
precludes establishing connectivity between the methylene
bridge and dG from the HMBC spectrum. Attachment of the
methylene bridge at the exocyclic amino group is inferred from
the absence of a 2-proton signal assignable to an unsubstituted
exocyclic amino group of guanine and the fragmentation of the
protonated molecule to yield the Schiff base of dG as the base
peak in the MS/MS spectrum (Figure S24).
levels tested (5 mM). The three Lys cross-linked products were
labile in solution, and the cross-link formed by a single
methylene bridge was too unstable for characterization by NMR
spectrometry. The lability of the Lys-dG cross-links supports
the reported reversibility of formaldehyde-induced cross-links
formed in vivo between histones and DNA.20 The cross-links
Cys-CH2-dG, Cys-CH2-dA, and Cys-CH2-dC were stable
and readily isolated and characterized. The cross-links charac-
terized in this work will contribute to a better understanding of
DPC formation induced by formaldehyde and of the mode of
action of this known human carcinogen. The stable structures
identified in this study have potential as biomarkers for the
occurrence of DPCs following formaldehyde exposure. Fur-
thermore, by using [13CD2]-formaldehyde, specific exposure-
related exogenous cross-links can be differentiated from en-
dogenous cross-links. Such sophisticated methods will be
necessary to carefully examine formaldehyde adducts formed
at the site of contact versus adduction at distant sites to
determine the plausibility of inhaled formaldehyde as a causative
agent for leukemia.29
Conclusion
We have investigated the formation of formaldehyde-induced
cross-links between nucleosides and amino acids and provide
the first rigorous structural characterizations of DNA-protein
cross-links induced by coupling with formaldehyde. Eight cross-
linked structures have been characterized. In five of these
structures, the cross-linking arose via a methylene bridge
connecting the exocyclic amino group of a nucleoside to a
nucleophilic nitrogen or sulfur of an amino acid side chain. In
the case of Trp, the points of attachment were the N2 exocyclic
amino group of dG and C2 of the indole ring. Lys, in addition
to forming an expected dG-N2-CH2-Nε-Lys cross-link, formed
two tricyclic adducts with dG, a 10-oxo-triazino[1,2-a]purinyl
derivative incorporating two formaldehyde molecules and a
5-hydroxymethyl-10-oxo-triazino[1,2-a]purinyl adduct incor-
porating three formaldehyde molecules. While the formation
of the tricyclic adducts was favored at formaldehyde levels of
50 and 100 mM, all three adducts were identified at the lowest
Acknowledgment. This work was supported in part by NIH
grants P30-ES10126 and P42-ES05948 and a grant from the
Formaldehyde Council, Inc.
Supporting Information Available: Complete HMBC and
ROESY NMR spectra of NR-Boc-amino acid-deoxynucleoside
cross-links. Full HSQC spectra of TPHA-1, TPHA-2, and
Trp-CH2-dG. Complete QTOF-MS/MS spectra of NR-Boc-
amino acid-trinucleotide cross-links and complete QTOF-MS/
MS spectra of N-terminal acetylated octapeptide-deoxynucle-
oside cross-links. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA908282F
(29) Zhang, L.; Steinmaus, C.; Eastmond, D. A.; Xin, X. K.; Smith, M. T.
Mutat. Res. 2009, 681, 150–168.
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