Plamen P Christov1, Kyu-Jun Son, Carmelo J Rizzo. 1. Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.
Abstract
N(5)-Substituted formamidopyrimidine adducts have been observed from the reaction of dGuo or DNA with aziridine containing electrophiles, including nitrogen mustards. However, the role of substituted Fapy-dGuo adducts in the biological response to nitrogen mustards and related species has not been extensively explored. We have developed chemistry for the site-specific synthesis of oligonucleotides containing an N(5)-nitrogen mustard Fapy-dGuo using the phosphoramidite approach. The lesion was found to be a good substrate for Escherichia coli endonuclease IV and formamidopyrimidine glycosylase.
N(5)-Substituted formamidopyrimidine adducts have been observed from the reaction of dGuo or DNA with aziridine containing electrophiles, including nitrogen mustards. However, the role of substituted Fapy-dGuo adducts in the biological response to nitrogen mustards and related species has not been extensively explored. We have developed chemistry for the site-specific synthesis of oligonucleotides containing an N(5)-nitrogen mustardFapy-dGuo using the phosphoramidite approach. The lesion was found to be a good substrate for Escherichia coliendonuclease IV and formamidopyrimidine glycosylase.
Nitrogen
mustards (bis(2-chloroethyl)amine derivatives; Figure 1) are a family of bifunctional DNA alkylating agents
that were first introduced into a clinical setting in 1942 for the
treatment of non-Hodgkin lymphoma.[1,2] Nitrogen mustards
(NM) are still used in combination with other agents for the treatment
of a variety of cancers.[2] The predominant
site of reaction of nitrogen mustards with DNA is at N7-dGuo. Cationic
N7-dGuo adducts are generally considered to be benign,[3,4] but, like most DNA alkylating agents, an array of minor products
are also formed.[5−7] The cytotoxicity of simple monofunctional alkylating
agents has been ascribed to minor O6-dGuo, O4-dThd, and N3-dAdo adducts.[7−9]
Figure 1
General structure of a nitrogen mustard
(NM) and some of their
dGuo adducts.
The mechanism
of action of nitrogen mustards involves an initial
intramolecular SN2 reaction to form an aziridinium ion,
which is the DNA modifying agent.[10,11] The nucleophilicity
of the N atom can be attenuated by the third substituent, designated
as R in Figure 1. Although the N7-dGuo adduct
is generally the major adduct (N7-NM-dGuo, Figure 1), with the second chloride (X = Cl) partially or fully hydrolyzed
to the corresponding alcohol (X = OH),[12] the bifunctional nature of nitrogen mustards allows them to react
with a second nuclophilic site in DNA to afford intra- and interstrand
cross-links as well as with nucleophilic sites of proteins to form
DNA–protein cross-links.[13−15] This second adduction reaction
presumably involves aziridine formation of the initial N7-adduct.
Cross-links between the N7 positions of dGuo (N7:N7-NM-dGuo), between
the N3-positions of dAdo, and between N7-dGuo and N3-dAdo have been
identified, among others.[16−19] Many crucial cellular processes (i.e., replication
and repair) require the transient separation of the DNA strands. DNA
interstrand cross-links prevent this strand separation and are therefore
predicted to be highly cytotoxic to cells.[20] Indeed, although DNA interstrand cross-links typically account for
only 1–5% to the total adduct burden, they are believed to
be the key lesion in the mechanism of action of nitrogen mustards
and related agents. Interestingly, nitrogen mustards prefer to form
interstrand cross-links in a 5′-GNC-3′ rather than in
a 5′-GC-3′ sequence context.[21−24]General structure of a nitrogen mustard
(NM) and some of their
dGuo adducts.The products from the
reaction of nitrogen mustards with Guo, dGuo,
and DNA have been well-studied. Chetsanga[25] and Hemminki[26−28] have independently characterized the imidazole ring-opening
of the N7-dGuo adduct of a nitrogen mustard to afford the corresponding N5-substituted formamidopyrimidine (Fapy) lesion
(Figure 1). N5-(2-Aminoethyl)-Fapy-dGuo
(AE-Fapy-dGuo) has been observed to be the major product from the
reaction of aziridine with dGuo and DNA.[29,30] A Fapy-dGuo adduct has also been characterized from the reaction
of DNA with acid-activated mitomycin, an aziridine-containing natural
product.[31] However, little attention has
been paid to nitrogen mustardFapy-dGuo adducts (NM-Fapy-dGuo). Previous
studies have shown that Fapy-dGuo adducts have mutagenic potential[32−34] and are substrates for the base excision repair pathway.[35−40] Oligonucleotides containing site-specific NM-Fapy-dGuo lesions will
allow for future replication and repair studies both in vitro and in cells. We have reported the synthesis of oligonucleotides
containing the N5-methyl-Fapy-dGuo lesion
(MeFapy-dGuo) by solid-phase methods.[41] The extension of this chemistry to the synthesis of oligonucleotides
containing the NM-Fapy-dGuo lesion (R = Et) is described herein. We
find that the NM-Fapy-dGuo lesion is a substrate for Escherichia coliendonuclease IV (Endo IV) and formamidopyrimidine
glycosylase (FPG) in vitro.
Triethylamine (460 μL, 3.3 mmol) and N,N-bis(2-chloroethyl)ethylamine hydrochloride
(566 mg, 2.75 mmol) were sequentially added to a stirred solution
of 3 (400 mg, 0.55 mmol) in dry trifluoroethanol (10
mL). The reaction mixture was stirred at ambient temperature for 4
h, after which time it was concentrated to ∼1 mL in
vacuo on a rotary evaporator. Methanolic sodium hydroxide
(0.5 M in 1:3 water/methanol, 10 mL) was added, and the mixture was
stirred for 5 min followed by neutralization by the careful addition
of 20% acetic acid. Water (15 mL) was added, and the mixture extracted
with CH2Cl2 (3 × 20 mL). The combined organic
extracts were dried over anhydrous sodium sulfate, filtered, and evaporated.
Purification of the gummy residue by flash chromatography on silica
afforded 5 as a mixture of isomers (410 mg, 85%). The
eluent was initially 98:1:1 CH2Cl2/CH3OH/pyridine; the CH3OH content was increase by 1% every
column volume (∼200 mL) to a final ratio of 93:6:1. 1HNMR (DMSO-d6) mixture of isomers: δ
8.56 (s, 0.5H, N=CH), 8.52 (s, 0.5H, N=CH), 7.70 (s,
0.5H, CHO), 7.38 (s, 0.5H, CHO), 7.29–7.18 (m, 9H, ArH), 7.05
(d, 0.5H, J = 12.0 Hz, NH), 6.95 (d, 0.5H, J = 12.0 Hz, NH), 6.83–6.79 (m, 4H, ArH), 6.05 (m,
1H, H-1′), 5.12 (d, 1H, J = 6.0 Hz, 3′-OH),
4.36–4.31 (m, 2H, OCHCH2Si), 4.07 (m, 1H, H-3′),
3.76 (m, 1H, H-4′), 3.70 (s, 6H, 2CH3O), 3.60–3.55
(m, 2H, ClCHCH2N), 3.50–3.35 (m, 2H, EtNCHCH2NCHO), 3.07 (s,
3H, CH3-N-CH=), 2.90 (s, 3H, CH3-N-CH=),
3.00–2.98 (m, 2H, H-5′), 2.81–2.60 (m, 4H, ClCH2CHN, EtNCH2CHNCHO), 2.55–2.52 (m, 2H, CHCH3), 1.95–1.87 (m,
2H, H-2′), 0.98 (t, 2H, J = 6.0 Hz, OCH2CHSi), 0.84 (t, 1.5H, J = 6.0 Hz, CH2CH), 0.70 (t, 1.5H, J = 6.0 Hz, CH2CH), 0.07 (s, 9H, Me3-Si).
HRMS (FAB+) m/z calcd for C45H63ClN7O7Si [M + H]+, 876.4252; found, 876.4241.
Tetrabutylammonium flouride (1 M in THF,
420 μL, 0.42 mmol) was added to a stirred solution of 6 (250 mg, 0.28 mmol) in dry methylene chloride (15 mL). The
mixture was stirred at room temperature for 4 h. The solvent was removed,
and the residue was purified by flash chromatography on silica to
afford 7 as a mixture of isomers (177 mg, 80%). The eluent
was initially 98:1:1 CH2Cl2/CH3OH/pyridine;
the CH3OH content was increased by 1% every column volume
(∼200 mL) to a final ratio of 95:4:1. 1HNMR (DMSO-d6) mixture of isomers: δ 11.09 (s, 1H,
NH), 8.56 and 8.53 (two s, total 0.5H, N=CH), 8.32 and 8.29
(two s, total 0.5H, N=CH), 7.81 and 7.79 (two s, total 0.5H,
CHO), 7.66 and 7.50 (two s, total 0.5H, CHO), 7.40–7.20 (m,
9.5H, ArH, NH), 6.99 (d, 0.5H, J = 12.0 Hz, NH),
6.90–6.85 (m, 4H, ArH), 6.27 and 6.23 (two m, 1H, H-1′),
5.31 and 5.12 (s, total 1H, 3′-OH), 4.28 (m, 1H, H-3′),
4.10–4.00 (m, 2H, AcOCHCH2N), 4.23 (m, 1H, H-4′), 3.72
(s, 6H, 2CH3O), 3.50–3.35 (m, 2H, EtNCHCH2NCHO),
3.22–3.17 (multiple s, total 6H, 2CH3-N-CH=),
2.98–2.90 (m, 2H, H-5′), 2.60–2.52 (m, 6H, CHCH3,
AcOCH2CHN, EtNCH2CHNCHO), 1.98–1.85 (m, 5H, H-2′, AcO),
1.33–1.22 (multiple t, total 3H, CH2CH). HRMS (FAB+) m/z calcd for C42H54N7O9 [M + H]+, 800.3978; found, 800.3985.
Compound 7 (100 mg, 0.13 mmol)
was dissolved in anhydrous pyridine, evaporated on a rotary evaporator,
and dried overnight under high vacuum. The gummy residue was dissolved
in dry CH2Cl2 (20 mL), and a solution of anhydrous
1H-tetrazole (12.6 mg, 0.18 mmol) was added followed
by 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-phosphorodiamidite
(54.2 mg, 0.18 mmol). The solution was stirred at room temperature
for 3 h. The solvent was removed in vacuo on a rotary
evaporator, and the residue was purified by flash chromatography on
silica to afford 8 as a mixture of isomers (63.0 mg,
50%). The eluent was initially 97:2:1 CH2Cl2/CH3OH/pyridine; the CH3OH content was increased
by 1% every column volume (∼200 mL) to a final ratio of 95:4:1. 1HNMR (acetone-d6) mixture of
isomers: δ 8.34 (s, 0.5H, N=CH), 8.23 (s, 0.5H, N=CH),
7.92 (s, 0.5H, CHO), 7.89 (s, 0.5H, CHO), 7.48–7.30 (m, 9H,
ArH), 6.90–6.85 (m, 4H, ArH), 6.61 (m, 1H, H-1′), 4.84
(m, 1H, H-3′), 4.35–4.17 (m, 3H, H-4′, AcOCHCH2N),
3.78 (s, 6H, 2-CH3O), 3.70–3.63 (m, 2H, POCH2), 3.63–3.43 (m, 4H, isopropyl CH, EtNCHCH2NCHO), 3.10–3.03
(m, 2H, H-5′), 2.91–2.68 (multiple s and m, total 12H,
2CH3-N-CH=, OCH2-CN, AcOCH2CHN, EtNCH2CHNCHO), 2.55–2.52 (m, 2H, CHCH3), 2.03–1.90 (m, 5H, H-2′,
AcO), 1.33–1.21 (multiple t, total 3H, CH2CH), 1.22–1.01
(m, 12H, isopropyl CH3). 31PNMR (acetone-d6, 125 MHz) δ 149.56, 149.25, 149.12,
148.98. HRMS (FAB+) m/z calcd for
C51H71N9O10P [M + H]+, 1000.5056; found, 1000.5079.
Oligonucleotide Synthesis
The oligodeoxynucleotides
were synthesized on a Perseptive Biosystems Model 8909 DNA synthesizer
on a 1 μmol scale using Expedite reagents (Glen Research) with
the standard synthetic protocols for the coupling of unmodified bases.
The coupling of the NM-Fapy-dGuo phosphoramidite was performed off-line
for 30 min as previously described.[41,43]
Enzymatic Digestion
and Analysis of Oligonucleotides
The enzymatic digestion
of oligonucleotides was carried out in a
single step as follows: the oligonucleotide (0.5 A260 units) was dissolved in 70 μL of buffer (pH
7, 10 mM Tris-HCl, 10 mM MgCl2). DNase I (5 units), alkaline
phosphatase (1.7 units), and snake venom phosphodiesterase I, type
II (0.02 units), were added, and the solution was incubated at 37
°C for 1.5 h. HPLC analysis was performed using solvent gradient
1. Conditions for the HPLC and UPLC-MS analyses are provided in the Supporting Information.
5′-GCT AGC-(NM-Fapy-dGuo)-AG
TCC-3′ (9)
Purified by reversed-phase
HPLC using gradient 2 (see Supporting Information for conditions). MALDI-TOF
MS (HPA) m/z calcd for [M –
H]−, 3775.7; found, 3777.2
Purified by reversed-phase HPLC using gradient
3 (see Supporting Information for conditions).
MALDI-TOF MS (HPA) m/z calcd for
[M – H]−, 7406.9; found, 7407.7
5′-ACC
ACG CTA GC-(8-oxo-dGuo)-AGT CCT AAC AAC-3′
This oligonucleotide
was synthesized using commercially available
8-oxo-dGuo phosphoramidite and purified by reversed-phase HPLC using
gradient 4 (see Supporting Information for
conditions). MALDI-TOF MS (HPA) m/z calcd for [M – H]−, 7289.8; found, 7287.0.5′-ACC ACG CTA GC-(MeFapy-dGuo)-AGT CCT AAC AAC-3′
and 5′-ACC ACG CTA GC-(α-dG)-AGT CCT AAC AAC-3′
were prepared previously.[44]
Oligonucleotide
Labeling and Annealing
The oligonucleotides
were labeled using T4 polynucleotide kinase and γ-32P-ATP as previously described.[44]
Time
Course of the Endo IV Incision of the NM-Fapy-dGuo Containing
Duplexes
These experiments were performed as previously described.[44]
Kinetics for the Endo IV Incision of the
NM-Fapy-dGuo Containing
Duplex
These experiments were performed as previously described.[44]
Time Course Incision of the NM-Fapy-dGuo
Containing Duplex by
Endo IV at pH 7.0, 7.5, and 8.0
These experiments were performed
as previously described.[44]
Incision of
the NM-Fapy-dGuo Containing Duplex after Denaturation
and Reannealing
These experiments were performed as previously
described.[44]
Excision of MeFapy-dGuo,
NM-Fapy-dGuo, and 8-Oxo-dGuo Containing
Duplexes by E. coli Formamido-pyrimidine
Glycosylase
The 5′-32P-labeled modified
oligonucleotide (200 nM) was annealed to an equal volume of its complementary
strand (600 nM) in Tris buffer (50 mM). Oligonucleotide duplex (100
nM, 4 μL) was added to the formamidopyrimidine glycosylase (FPG)
reaction buffer (1×: 10 mM Bis-Tris Propane-HCl, 10 mM MgCl2, 1 mM dithiothreitol, 74 μL), followed by addition
of FPG (10 nM). Reactions were incubated at 37 °C. Aliquots (5
μL) were removed at the appropriate times, added to 10 μL
of 95% formamide loading buffer containing xylene cyanol and bromophenol
blue dyes, and heated for 1 min at 90 °C. Aliquots (6 μL)
were separated by electrophoresis on a denaturing gel.
Kinetics for the Excision of MeFapy-dGuo, NM-Fapy-dGuo,
and
8-Oxo-dGuo Containing Duplexes by E. coli Formamidopyrimidine Glycosylase
DNA duplex was formed by
heating 32P-labeled oligonucleotide (100 nM in 50 mM Tris)
and its complementary strand (1.5 equiv) at 95 °C for 5 min and
then slowly cooling to ambient temperature over 1 h. FPG (0.1 nM)
was added to varying concentrations of DNA duplex (5–100 nM
for MeFapy-dGuo, 10–180 nM for NM-Fapy-dGuo, and 1–40
nM for 8-oxo-dGuo in FPG reaction buffer (1×)) to a final volume
50 μL. Reactions were run at 37 °C for 10 min. Aliquots
(5 μL) were taken every 1 min, added to loading buffer (10 μL),
and heated at 90 °C for 1 min. Separation was achieved by PAGE.
The kinetics parameters were calculated using KaleidaGraph (v. 4.5,
Synergy Software). Reactions were carried out in duplicate.
Results
and Discussion
Phosphoramidite Synthesis
The synthesis
of the NM-Fapy-dGuophosphoramidite reagent is shown in Scheme 1. O6-(2-Trimethylsilyethyl)-dGuo (1) was prepared in three steps according to a literature procedure[42] and was further protected at the 5′-O- and N2-positions to afford 3 in 43% overall yield from dGuo. Reaction of 3 with bis(2-chloroethyl)ethylamine in trifluoroethanol presumably
gives the cationic N7-adduct (4),[18,26,28,45] which was
not isolated. Rather, the reaction mixture was briefly treated with
methanolic NaOH followed by careful neutralization with 20% acetic
acid to afford protected NM-Fapy-dGuo intermediate 5.
Competing alkylation at O6 was observed
if this position was not protected. The second chloride, which surprisingly
survived the hydroxide treatment, was displaced by cesium acetate,
and the O6-protecting group was removed
by fluoride ion. Phosphitylation of the 3′-hydroxyl group provided
the desired phosphoramidite reagent (8) as a mixture
of isomers. The synthesis required nine steps from dGuo and proceeded
in ∼11% overall yield.
Scheme 1
Synthesis of the NM-Fapy-dGuo Phosphoramidite
Phosphoramidite 8 was used to synthesize 12- and 24-mer
oligonucleotides containing
the NM-Fapy-dGuo lesion shown in Table 1. An
off-line, manual coupling protocol was used to incorporate the modified
nucleotide.[41,43] Fapy-dGuo lesions with a free
5′ hydroxyl group can rearrange to the pyranose form.[31,46] Therefore, the critical step of the oligonucleotide synthesis was
the detritylation of the NM-Fapy-dGuo nucleotide. We previously observed
that standard DNA synthesis protocols resulted in significant rearrangement
to the pyranose form of the MeFapy-dGuo adducts. However, shortening
the deprotection cycle minimized the furanose to pyranose rearrangement.[41] Oligonucleotides containing the NM-Fapy-dGuo
adduct were synthesized using a “short” deprotection
cycle. Two products were identified as having the correct mass in
a ∼1:2.6 ratio (see Figure S10 of the Supporting
Information). The major product (later eluting) was assumed
to be the furanose form based on our previous work with the MeFapy-dGuo
adduct in the same sequence. The two products were observed in a ∼1:1
ratio under a long deprotection cycle, which is consistent with the
assignment because the long deprotection should favor the isomerization
to the pyranose form.
Table 1
Sequences and Mass
Spectrometric Characterization
of the Oligonucleotides Containing the NM-Fapy-dGuo Lesion
m/z
oligonucleotide
found
calcd
9
5′-GCTAGC-(NM-Fapy-dGuo)-AGTCC-3′
3777.2
3775.7
10
5′-ACCACGCTAGC-(NM-Fapy-dGuo)-AGTCCTAACAAC-3′
7407.7
7406.9
The NM-Fapy-dGuo containing 12-mer oligonucleotide
was enzymatically
digested to the individual nucleosides. The unmodified nucleosides
were readily observed by HPLC analysis in the expected ratio (Figure 2A),[47] but NM-Fapy-dGuo
was not detected. Substituted Fapy-dGuo nucleosides exist as multiple,
slowly interconverting species, which may include the furanose and
pyranose forms of the deoxyribose, α- and β-anomers, cis
and trans geometric isomers of the formamide, and possibly atropisomers.[31,46,48−53] The slow interconversion of the Fapy-isomers often results in poor
HPLC chromatographic behavior and diminished detection.[54] Therefore, the enzymatic digestion reaction
was examined by UPLC-MSn. The extracted ion chromatograms
are shown in Figure 2B, in which the neutral
loss of the deoxyribose (−116 Da) was monitored. A mass consistent
with NM-Fapy-dGuo (400 → 285) was observed along with the unmodified
nucleosides. As anticipated, NM-Fapy-dGuo was observed as multiple
broad peaks, reflecting its conformational heterogeneity. The enzymatic
digestion reaction was also subjected to acid hydrolysis and UPLC-MS
analysis (Figure 2C). A broad peak with a mass
consistent with the NM-Fapy-Gua base (m/z 285.2) was observed. The broad nature of this peak likely reflects
geometric isomers of the formamide group; the geometric isomers of
the MeFapy-Gua base could be resolved into two separate peaks by HPLC.
Figure 2
(A) HPLC
(A254) and (B) UPLC-SRM-MS2 analysis of the enzymatic digestion of NM-Fapy-dGuo containing
12-mer oligonucleotide (9). (C) UPLC-MS analysis of the
NM-Fapy-Gua after acid hydrolysis of the enzymatic digestion reaction
of oligonucleotide (9). The reconstructed ion chromatogram
of NM-Fapy-Gua ([M + H]+m/z 285.2) is shown at the top of this panel, and the CID fragmentation
of the parent ion is shown at the bottom. See the Supporting Information for the conditions for each analysis.
(A) HPLC
(A254) and (B) UPLC-SRM-MS2 analysis of the enzymatic digestion of NM-Fapy-dGuo containing
12-mer oligonucleotide (9). (C) UPLC-MS analysis of the
NM-Fapy-Gua after acid hydrolysis of the enzymatic digestion reaction
of oligonucleotide (9). The reconstructed ion chromatogram
of NM-Fapy-Gua ([M + H]+m/z 285.2) is shown at the top of this panel, and the CID fragmentation
of the parent ion is shown at the bottom. See the Supporting Information for the conditions for each analysis.Duplexes containing the MeFapy-dGuo
and N5-aflatoxin B1-Fapy-dGuo
(AFB1-Fapy-dGuo)
lesions showed biphasic thermal melting profiles.[41,55] It was hypothesized that the two phases represented the α-
and β-anomers of the Fapy-dGuo adduct. Attempts to characterize
the thermal melting profile of the NM-Fapy-dGuo containing 12-mer
gave inconsistent results in our hands. We cannot offer an explanation
for this observation.
Incision of the NM-Fapy-dGuo Duplex with E. coli Endonuclease IV (Endo IV)
An unusual
property of Fapy lesions
is that they can isomerize to the unnatural α-anomer. Endo IV
incises the 5′-phosphodiester bond of an abasic site in duplex
DNA[56−58] and has been shown to incise α-nucleotides.
Endo IV has been used previously to approximate the α/β
ratio of Fapy-dGuo, Fapy-dAdo, and MeFapy-dGuo in duplex DNA by the
selective incision of the α-anomer.[44,59−61] We applied this assay to the NM-Fapy-dGuo containing
24-mer to determine the α/β ratio of the lesion. The modified
24-mer (10) was 5′-32P-labeled, annealed
to its complement, and incubated with Endo IV (Figure 3). Approximately 50% of the duplex was incised after a 40
min incubation period at 37 °C, affording a product consistent
with phosphodiester hydrolysis at the 5′-side of NM-Fapy-dGuo.
The reaction mixture, containing the remaining 50% of the duplex as
the β-anomer, was denatured by heating at 90 °C and slowly
cooling to reanneal the duplex. This process should re-equilibrate
the NM-Fapy-dGuo lesion to its original mixture of anomers.[44,61] Additional Endo IV was added, and after 40 min, gel analysis indicated
that the total level of incision was ∼75%; the 25% increase
represents 50% of the duplex remaining after the first incision reaction.
The reaction mixture was denatured and reannealed again, and a third
portion of Endo IV was added. The third cycle resulted in an additional
∼12% of the incised product (∼87% total), which represents
approximately half of the duplex remaining after the second Endo IV
treatment (25%). The results are consistent with a 1:1 mixture of α-
and β-anomers after initial annealing of the duplex.
Figure 3
(Top) Gel electrophoretic
analysis of the incision of the NM-Fapy-dGuo
containing 24-mer (10) duplex with E.
coli Endo IV. The right lane contains a standard of
the 5′-32P-ACCACGCTAGC-3′ incision product.
(Bottom) Percentage of incision product after denaturation–reannealing
and additional Endo IV (average of two experiments).
(Top) Gel electrophoretic
analysis of the incision of the NM-Fapy-dGuo
containing 24-mer (10) duplex with E.
coli Endo IV. The right lane contains a standard of
the 5′-32P-ACCACGCTAGC-3′ incision product.
(Bottom) Percentage of incision product after denaturation–reannealing
and additional Endo IV (average of two experiments).We previously observed that the initial α/β-anomeric
ratio of the MeFapy-lesion slowly equilibrated over time.[44] The labeled NM-Fapy-dGuo containing 24-mer duplex
was incubated at 37 °C and pH 7.5. Aliquots were removed after
0.5 h and then approximately every 24 h and were subjected to Endo
IV treatment. The level of incision steadily decreased over time to
about 14% after 5 days (Figure 4). The anomerization
was also examined at pH 7.0 and 8.0 and was surprisingly insensitive
to pH over this range; this is in contrast to that of the MeFapy-dGuo
lesion, which anomerized more slowly at pH 7.5 and not at all at pH
8.0.
Figure 4
Time course for Endo IV incision of the NM-Fapy-dGuo containing
24-mer (10). Endo IV was omitted in the control lane
(C). The day 0 time point was after an incubation time of 30 min.
Time course for Endo IV incision of the NM-Fapy-dGuo containing
24-mer (10). Endo IV was omitted in the control lane
(C). The day 0 time point was after an incubation time of 30 min.The catalytic efficiency (kcat/Km, Table 2) for the Endo
IV incision of the NM-Fapy-dGuo containing 24-mer was 0.16 nM–1 min–1 (kcat = 5.4 ± 0.5 min–1, Km= 33 ± 6.8 nM). The incision efficiency was approximately
half of that of the MeFapy-dGuo lesion and ∼4.3-fold lower
than α-dGuo in the same sequence context (Table 2).[44] The kcat values for the three substrates were similar, and differences
in their incision efficiency were largely due to Km. An active site pocket of Endo IV is hypothesized to
accommodate α-nucleotides.[62] The
affinity for our three substrates decreases with increasing steric
bulk, perhaps reflecting the ability of the active site pocket to
accommodate the substrate. We previously reported that the very bulky
AFB1-Fapy-dGuo adduct is a poor substrate for Endo IV.
Table 2
Steady Kinetic Parameter for the Incision
of the NM-Fapy-dGuo, MeFapy-dGuo, and α-dGuo Containing 24-mer
Duplexes by E. coli Endo IV
Excision of the NM-Fapy-dGuo Lesion of E. coli Formamidopyrimidine Glycosylase (FPG)
Fapy lesions are
substrates for the base excision repair pathway and are excised by
FPG in bacteria and by OGG1, NEIL1, and NTH1 in eukaryotes.[35−40] Less is known about the excision of N5-substituted Fapy-dGuo lesions. The MeFapy-dGuo, ethyl-Fapy-dGuo,
(2-hydroxyethyl)-Fapy-dGuo, (pyridyloxobutyl)-Fapy-dGuo, and Fapy-dGuo
lesions derived from a phosphoramide and sulfur mustard have been
reported to be substrates for FPG,[25,52,63−66] whereas AFB1-Fapy-dGuo is not a substrate.[66] A ring-opened oxidation product of dThd is also
a substrate for FPG.[67] MeFapy-dGuo is a
substrate for human[68] and yeast[69] OGG1, hNEIL1,[70] and
yeast,[71] mouse,[72] and human[70,73] NTH1, but it is a poor substrate
for hNEIL2,[70] mouseNEIL3,[74] and E. coli Endo III and
Endo VIII[70,72,75] when paired
with dCyd. Excision by NEIL3 improved markedly in single-stranded
DNA.[74] There is indirect evidence that
FPG and hOGG1 will excise the AE-Fapy-dGuo lesion. The overexpression
of FPG or hOGG1 was up to 100-fold protective of cells treated with
thioTEPA or aziridine, suggesting a role for the AE-Fapy-dGuo lesion
in the cytotoxic mechanism of these agents.[76−79] FPG expression also provided
a 10- and 2-fold protective effect against 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU) and the nitrogen mustardmafosfamide,[76,80] respectively, suggesting that the corresponding N5-substituted Fapy-dGuo lesions are part of the mechanism
of action of these agents as well. The lower protective effect against
BCNU and mafosfamide is probably reflective of the greater role of
interstrand cross-links in their cytotoxicity.The excision
of MeFapy-dGuo and NM-Fapy-dGuo opposite dCyd from duplex DNA by E. coli FPG was examined and compared to that of
8-oxo-dGuo; all three lesions were incorporated into the same 24-mer
sequence. Asagoshi et al. previously reported the excision of MeFapy-dGuo
and 8-oxo-dGuo from a 25-mer duplex in a 5′-TXG-3′ sequence by FPG and hOGG1.[68] Both glycosylases had nearly identical catalytic efficiencies for
the two substrates opposite dCyd, although the activity for FPG was
significantly higher than that of hOGG1. We found that the MeFapy-dGuo
and NM-Fapy-dGuo lesions were good substrates for E.
coli FPG when paired with dCyd (Figure 5). Our kcat and Km values for the MeFapy-dGuo and 8-oxo-dGuo substrates
are in reasonable agreement with those previously reported (Table 3);[68] however, we found
8-oxo-dGuo to be a better substrate for FPG by ∼2-fold. The
excision of NM-Fapy-dGuo was 2.5 and 4.6 times less efficient than
that for MeFapy-dGuo and 8-oxo-dGuo, respectively. The excision efficiencies
are largely reflective of differences in the Km values, which roughly correlate with the steric demands of
the modification. Consistent with our observation, the Km for the excision of N5-ethyl-Fapy-dG
was reported to be 7-fold higher than that for MeFapy-dGuo.[52] However, the apparent KD’s of nonhydrolyzable cyclopentane analogues of Fapy-dGuo
and N5-benzyl-Fapy-dGuo with FPG were
nearly identical.[81]
Figure 5
Gel analysis of the excision
of 8-oxo-dGuo, MeFapy-dGuo, and NM-Fapy-dGuo
containing duplexes by E. coli formamidopyrimidine
glycosylase (FPG).
Table 3
Steady
Kinetic Parameter for the Excision
of the NM-Fapy-dGuo, MeFapy-dGuo, and 8-Oxo-dGuo Containing Duplexes
by E. coli FPG
NM-Fapy-dGuo
MeFapy-dGuo
8-oxo-dGuo
kcat (min–1)
7.6 ± 0.9
9.5 ± 0.4
4.6 ± 0.3
Km (nM)
60 ± 18
29 ± 3.8
7.7 ± 1.4
kcat/Km (nM–1 min–1)
0.13
0.33
0.60
Gel analysis of the excision
of 8-oxo-dGuo, MeFapy-dGuo, and NM-Fapy-dGuo
containing duplexes by E. coliformamidopyrimidine
glycosylase (FPG).
Conclusions
MeFapy-dGuo
and AFB1-Fapy-dGuo have been reported to
be persistent lesions from exposure to methylating agents and aflatoxin
B1, respectively.[82,83] This observation suggests
that N5-substituted Fapy-dGuo lesions
play a role in the carcinogenicity of DNA alkylating agents and secondary
tumor development from chemotherapeutic agents such as temozolomide,
thioTEPA, BCNU, and nitrogen mustards.We have site-specifically
incorporated an N5-nitrogen mustard Fapy-dGuo
lesion into oligonucleotides using
the phosphoramidite approach. A DNA duplex containing the NM-Fapy-dGuo
lesion is a good substrate for E. coli Endo IV and FPG when paired with dCyd. The Endo IV incision indicates
that NM-Fapy-dGuo exists as a ∼50:50 α/β-anomeric
ratio after annealing, but it will slowly equilibrate to a ∼14:86
ratio. The NM-Fapy-dGuo lesion will be initially formed as the natural
β-anomer in cellular DNA and will slowly equilibrate to a mixture
of anomers over time. Therefore, the α-anomer will be a relevant
lesion if NM-Fapy-dGuo persists. The efficient excision of NM-Fapy-dGuo
from duplex DNA suggests that FPG could be used as part of an enrichment
protocol for the mass spectrometric detection of NM-Fapy-Gua from
treated cells, animals, or clinical samples. Oligonucleotides containing
NM-Fapy-dGuo will also be useful in repair and replication studies in vitro and in cells. Our work compliments previously reported
work in which a nitrogen mustard interstrand cross-link model was
engineered into oligonucleotides.[84] It
should also be noted that the parent Fapy-dGuo lesion derived from
oxidative damage has previously been incorporated into oligonucleotides.[85−87]
Figure 1
Scheme 1
Figure 2
Figure 3
Figure 4
Figure 5