Synthesis of oligonucleotides containing 2′-deoxyguanosine adducts of nitropyrenes

Article abstract of DOI:10.1080/15257770902736426

Two different approaches to synthesize oligonucleotides containing the 2′-deoxyguanosine adducts formed by nitropyrenes are described. A direct reaction of an unmodified oligonucleotide with an activated nitropyrene derivative is a convenient biomimetic approach for generating the major adducts in DNA. A total synthetic approach, by contrast, involves several synthetic steps, including Buchwald-Hartwig Pd-catalyzed coupling, but can be used for incorporating both the major and minor adducts in DNA in high yield. Copyright Taylor & Francis Group, LLC.

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Synthesis of Oligonucleotides Containing
2′-Deoxyguanosine Adducts of
Nitropyrenes
Laureen C. Colis a , Debasis Chakraborti a , Pablo Hilario a ,
Christopher McCarty a & Ashis K. Basu a
a Department of Chemistry , University of Connecticut , Storrs,
Connecticut, USA
Published online: 14 Feb 2009.
To cite this article: Laureen C. Colis , Debasis Chakraborti , Pablo Hilario , Christopher
McCarty & Ashis K. Basu (2009) Synthesis of Oligonucleotides Containing 2′-Deoxyguanosine
Adducts of Nitropyrenes, Nucleosides, Nucleotides and Nucleic Acids, 28:2, 67-77, DOI:
10.1080/15257770902736426
To link to this article: http://dx.doi.org/10.1080/15257770902736426
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Nucleosides, Nucleotides and Nucleic Acids, 28:67–77, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770902736426
SYNTHESIS OF OLIGONUCLEOTIDES CONTAINING
2ꢀ-DEOXYGUANOSINE ADDUCTS OF NITROPYRENES
Laureen C. Colis, Debasis Chakraborti, Pablo Hilario, Christopher McCarty,
and Ashis K. Basu
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Two different approaches to synthesize oligonucleotides containing the 2 ꢁ-deoxyguanosine
2
adducts formed by nitropyrenes are described. A direct reaction of an unmodified oligonucleotide
with an activated nitropyrene derivative is a convenient biomimetic approach for generating the
major adducts in DNA. A total synthetic approach, by contrast, involves several synthetic steps,
including Buchwald-Hartwig Pd-catalyzed coupling, but can be used for incorporating both the
major and minor adducts in DNA in high yield.
Keywords Nitropyrene; dinitropyrene; DNA adducts
INTRODUCTION
Nitropyrenes are ubiquitous in the environment and have been detected
in urban air particulates, coal fly ash, and automobile exhaust.[1,2] 1-
Nitropyrene (1-NP) is one of the major mutagenic components of diesel
particulates, whereas the dinitropyrenes (DNPs) (substituted at 1,3-, 1,6-,
and 1,8-positions) are present in much lower concentrations.[3,4] 1-NP
accounts for 25% of the mutagenicity of diesel exhausts. The DNPs, though
present in much smaller quantity in diesel emissions, are much more
mutagenic than 1-NP.[4] All the mono- and dinitropyrenes are tumorigenic
in experimental animals.[5,6] 1,6-DNP is the most potent carcinogen in
this group, whereas 1,8-DNP is the most mutagenic by several assays. The
major DNA adducts formed by 1-NP and the 1,6- or 1,8-DNP both in vitro
and in vivo are the C8 2ꢁ-deoxyguanosine (dG) adducts N -(deoxyguanosin-
Received 11 August 2008; accepted 17 November 2008.
This work was supported by a research grant ES09127 from the National Institute of Environmental
Health Sciences, NIH. CM was supported by a REU grant CHE-0354012 from NSF.
Address correspondence to Ashis K. Basu, Department of Chemistry, University of Connecticut,
Storrs, CT, 06269, USA. E-mail: ashis.basu@uconn.edu
67

68
L. C. Colis et al.
SCHEME 1 DNA adducts formed by nitropyrenes.
8-yl)-1-aminopyrene (dG-C8-AP) and N -(deoxyguanosin-8-yl)-1-amino-6(or
8)-nitropyrene (dG-C8-amino-6(or 8)-NP) (Scheme 1).[4] For 1-NP, two N 2
dG adducts have also been isolated in vitro (Scheme 1).[4]
Incorporation of these DNA adducts in desired DNA sequences is an
important step for investigation of mutagenicity, repair, and biophysical
properties of the lesions. In a prior work, we have successfully synthe-
sized oligonucleotides containing dG-C8-AP by allowing the unmodified
oligonucleotides in double stranded form to react with N -hydroxy-1-
aminopyrene.[7] However, we could not detect any N 2 dG adduct by
this biomimetic approach. In recent years Buchwald-Hartwig palladium-
catalyzed amination[8,9] has been extensively employed to synthesize a series
of carcinogen-DNA adducts.[10–14] We and others have used this key step
to generate decent yield of both the major and minor dG adducts of
1-NP.[15,16] In the current work, we have converted these dG adducts into
the corresponding phosphoramidites and incorporated them into DNA. We
determined that the strategy to introduce the major adducts in preformed
oligonucleotides in duplex conditions, as noted in the case of 1-NP,[7] is
also highly efficient for dinitropyrenes. We compared the incorporation of
the nitropyrene and dinitropyrene adducts in a mutagenic hotspot CpG
repetitive sequence d(CGCG∗CG) by both approaches.
RESULTS AND DISCUSSION
Postoligomerization Strategy
A biomimetic approach for a direct reaction with an unmodified
duplex hexamer is shown in Scheme 2, in which the low pH and duplex
nature of the oligonucleotide are the important criteria for a reaction that
occurs almost exclusively with dG. 1-Amino-6-nitropyrene and 1-amino-8-
nitropyrene, synthesized as reported by Purohit and Basu,[17] were oxidized
to the corresponding nitrosonitropyrenes using m-chloroperoxybenzoic
acid. Each nitrosonitropyrene was converted to the N -hydroxy derivative

Nitropyrene-Adducted DNA
69
SCHEME 2 Postoligomerization strategy to introduce DNA adducts.
in situ in the presence of ascorbic acid, which was allowed to react with
the self-complementary oligonucleotide d(CGCGCG) in aqueous DMF at
pH 5.2. DNA adduction was HPLC monitored by removing an aliquot and
injecting it onto a reverse-phase column after the organic reactants were
removed by chloroform extraction. The adducted hexamers eluted more
slowly than the unmodified oligonucleotide, and though three adducted
peaks eluted consistent with the presence of three guanines, one of the
peaks was clearly dominant. Highest yield of the products (∼25%) occurred
at 16 hours when the products were HPLC purified. Figure 1 shows a typical
adduction profile after 16 hours at 260 and 450 nm (unlike unmodified
DNA, the nitro- and/or aminopyrene-containing peaks are detectable at
450 nm). Figure 2 shows the HPLC chromatograms and UV spectra
of the purified adducted hexamers in comparison to their unmodified
counterpart.
Enzymatic digestion followed by HPLC analysis established that the
major adducted hexamers contained the dG-C8-amino-6(or 8)-NP, which
co-eluted with an authentic sample. A portion of the major adducted
hexamer was 32P-radiolabeled and cleaved at the site of the adduct by hot
piperidine, which was compared with Maxam-Gilbert “G” reaction[17] of
the unmodified hexamer on a denaturing polyacrylamide gel. With this
approach, we determined that the major peak in each case contained the
C8 adduct at the second G, where the cleavage occurred. The minor peaks
corresponded to the C8 adduct at two other guanines. It is important
to emphasize that the presence of N 2 guanine adduct, which should be
refractory to the piperidine cleavage, was not detected. Each adducted
hexamer, d(CGCG∗CG), also gave a monoisotopic mass of 2051.6 Da by mass
spectral analysis with electrospray ionization as expected (Table 1).
We believe that in this reaction non-covalent intercalation played an
important role, which was manifested in the most facile reaction at the
duplex middle region of the self-complementary hexamer. As demonstrated

70
L. C. Colis et al.
FIGURE 1 Reverse-phase HPLC profile of crude reaction mixture of d(CGCGCG) with N -hydroxy-1-
amino-6-nitropyrene after stirring for 16 hours at pH 5.2 in DMF: H2O (1:9). The excess organic reagents
were removed by chloroform extraction. The peaks were monitored at 260 nm (A) and 450 nm (B). Peak
1 shows the unreacted hexamer, whereas peaks 2, 3, and 4 show the adducted hexanucleotide peaks. Peak
2 was identified as d(CGCG∗CG) by piperidine cleavage experiment.
FIGURE 2 Reverse-phase HPLC chromatograms (monitored at 260 nm) and UV spectra (shown in
inset) of purified (A) unmodified d(CGCGCG), (B) d(CGCG∗CG) containing dG-C8-amino-6-NP, (C)
d(CGCG∗CG) containing dG-C8-amino-8-NP.

Nitropyrene-Adducted DNA
71
TABLE 1 ESI-MS characterization of the d(CGCG∗CG)
Calculated
(M-H) m/z
(Da)
Found (M-H)
m/z (Da)
Chemical
1,6-DNP
Adduct
Synthetic approach
dG-C8-amino-6-NP
Direct reaction with
N -hydroxy-1-amino-6-
nitropyrene
Direct reaction with
N -hydroxy-1-amino-8-
nitropyrene
Direct reaction with
N -hydroxy-1-aminopyrene
Total synthesis
2051.61
2051.61
2006.60
2051.39
2051.52
2006.45
1,8-DNP
1-NP
dG-C8-amino-8-NP
dG-C8-AP
1-NP
1-NP
dG-C8-AP
2006.60
2006.60
2006.42
2006.50
dG-N 2–8-AP
Total synthesis
with structurally similar DNA adducts,[18] it is likely that the initial N7
guanine adduct subsequently rearranged to the C8 guanine adduct.
Total Synthesis by Using a Phosphoramidite Reagent
The Buchwald-Hartwig C-N bond formation strategy[8,9] has been
successfully applied by many researchers to synthesize various DNA
adducts.[10–14] For the synthesis of the C8 dG adduct, we followed the
synthetic strategy of Gillet and Scha¨rer[15] to convert dG (4) to dG-C8-AP
(7) in eight steps (Scheme 3). In our hands, the yield in each step, including
the Pd2(dba)3 catalyzed coupling, was similar to the published procedure.
The C8 adduct 7 was converted to the protected phosphoramidite monomer
in three steps (Scheme 3).
Similarly, a protected monomer (9) was subjected to Buchwald-Hartwig
amination and the N 2 dG adduct derivative (10) was isolated in high yield
as we reported in our communication Chakraborti et al.[16] (Scheme 4).
Following removal of the hydroxyl protecting groups, the adduct derivative
11 was converted to fully protected adduct monomer 12 in good yield.
The protected monomers 8 and 12 were used to synthesize modified
hexamers, d(CGCG∗CG), by standard protocol in an automated DNA syn-
thesizer, except that the coupling time for these monomers was increased.
Deprotection of the oligonucleotides was carried out with concentrated
NH4OH in the presence of β-mercaptoethanol. For the dG-C8-AP, the final
product d(CGCG∗CG) was the major peak in reverse-phase HPLC, which
gave a monoisotopic mass of 2006.42 Da (theoretical mass 2006.60 Da)
(Table 1). For the N 2 adduct, on the other hand, the isolated product was
the precursor of the desired product, which was treated with palladium
black to reduce the 8-nitro group and to deprotect the benzyl protecting
group at the O6 position. The final product was isolated by reverse-phase
HPLC. As shown in Figure 3, the reduced product eluted approximately

72
L. C. Colis et al.
SCHEME 3 Synthesis of fully protected dG-C8-AP.
SCHEME 4 Synthesis of a fully protected monomer that can be converted to the N 2-dG adduct after
oligonucleotide synthesis.

Nitropyrene-Adducted DNA
73
FIGURE 3 Reverse-phase HPLC chromatograms (monitored at 260 nm): Before (solid line) and after
(dotted line) Pd black/cyclohexadiene reduction of d(CGCG∗CG) synthesized with monomer 12.
4 minutes earlier. ESI-MS analysis of the reduced product at 2006.50 Da
was in excellent agreement with the theoretical monoisotopic mass of
2006.60 (Table 1). Indeed, as shown in Table 1, both the postoligomer-
ization strategy as well as total synthesis generated the hexamers that gave
monoisotopic mass by electrospray ionization MS consistent with theoretical
calculations.
In conclusion, using two different strategies, we synthesized the DNA
adducts formed by 1-NP, 1,6-DNP, and 1,8-DNP, three carcinogenic nitropy-
renes, in a mutagenic hotspot sequence, d(CGCG∗CG). The biomimetic
approach of the reaction between the unmodified oligonucleotide and
the N -hydroxypyrene derivative worked well for preparing the major DNA
adducts, whereas a more involved total synthesis approach enabled us to
synthesize both the major and minor adducts.
MATERIALS AND METHODS
General Procedures
All starting materials, reagents and solvents were of commercial grade
and used as such unless otherwise specified. Anhydrous solvents were
purchased from Aldrich (St. Louis, MO, USA) and THF was dried by
standard methods. NMR (1H and 13C) spectra were recorded on a Bruker
(Coventry, UK) AC-400 spectrometer. Samples prepared for NMR analysis
were dissolved in either CDCl3 or DMSO-d6. Chemical shifts are reported in
δ ppm relative to TMS in the proton spectra and to the deuterated solvent
in the carbon spectra. LCMS (electrospray ionization) was recorded on a
Micromass Quattro II (Manchester, UK) triple quadrupole mass spectrom-
eter in acetonitrile mobile phase and the cone voltage set to 30 V. High
resolution MS spectras were obtained either from Nebraska Center for Mass
Spectrometry, University of Nebraska, Lincoln or Yale Cancer Center Mass

74
L. C. Colis et al.
Spectrometry Source. Thin-layer chromatography (TLC) was performed
on silica gel sheets containing fluorescent indicator. Components were
visualized by UV light. Chromatographic separations were carried out on
silica gel 60 µm (230–400 mesh). Preparative TLC was performed on
fluorescent silica gel glass backed plates (250 µm). All experiments dealing
with moisture and air-sensitive compounds were conducted under dry
nitrogen. All new products showed a single spot on TLC analysis, after
purification.
Synthesis of O6-benzyl-8-bromo-3ꢁ,5ꢁ-O-bis(tert-butyldimethylsilyl)-N2-
dimethoxytrityl-2ꢁ-deoxyguanosine (5), 8-(N 2-aminopyrene)-O6-benzyl-3ꢁ,5ꢁ-
O-bis(tert-butyldimethylsilyl)-2ꢁ-deoxyguanosine (6), and N -(deoxyguano-
sine-8-yl)-1-aminopyrene (7) essentially followed a method reported by
Gillet and Scha¨rer.[15]
2,8-Diisobutyryl-8-(1-aminopyrenyl)- 5ꢁ-O-(4,4ꢁdimethoxytrityl)-3ꢁ-O-[N,
Nꢁ-diisopropylamino(2-cyanoethoxy)phosphinyl] -2ꢁ-deoxyguanosine (8).
Compound 7 (60 mg, 0.2 mmol) was twice dried with pyridine and sus-
pended in 2 mL of dry pyridine to which a large excess of chlorotrimethyl-
silane (0.2 mL, ∼2 mmol) was added. The mixture was cooled for 30
minutes in an ice-water bath. Isobutyric anhydride (0.2 mL, ∼1 mmol)
was added, and the mixture was left standing at ambient temperature for
2 hours. The mixture was cooled again in an ice-cold water bath and
quenched with water. A solution of ammonium hydroxide (1 mL) was added
and the solvent was removed by evaporation. The residue was dissolved
in 2 mL water and extracted with diethyl ether (5 mL). The precipitate
appearing in the aqueous phase was collected, dried twice with pyridine,
and dissolved in 0.5 mL dry pyridine. Two equivalent 4,4ꢁ-dimethoxytrityl
chloride (143 mg, 0.4 mmol) and DMAP (0.01 mmol) were added and the
solution was stirred for 2 hours at room temperature when TLC monitoring
showed that the reaction was complete. A saturated NaHCO3 solution was
added and the solution was extracted three times with methylene chloride.
The combined organic layers were dried over Mg2SO4 and the solvent
was removed in vacuo. Purification of the residue by flash chromatog-
raphy on silica gel with 1% methanol in methylene chloride containing
0.5% triethylamine gave the desired 2,8-diisobutyryl-5ꢁ-O-DMT-protected
product.
The 2,8-diisobutyryl-8-(1-aminopyrenyl)-5ꢁ-dimethoxytrityl-2ꢁ-deoxygua-
nosine (95 mg, 0.1 mmol) was coevaporated with dry toluene (2 ×10 mL),
dissolved in dry methylene chloride (5 mL), and treated with triethylamine
(0.1 mL) followed by 2-cyanoethyl-N,N -diisopropylchlorophosphoramidite
(0.035 mL, 0.15 mmol). The reaction mixture was stirred at room tem-
perature for 1 hour under nitrogen and then diluted with methylene
chloride solution containing 1% TEA (25 mL). The mixture was washed
with aqueous saturated NaHCO3 solution, dried over Na2SO4, filtered,

Nitropyrene-Adducted DNA
75
concentrated under reduced pressure, and the residue was purified by flash
chromatography on silica, eluting with 1% methanol in methylene chloride
containing 0.5% pyridine, and provided 8 (62 mg, 55% yield) as a pale
yellow solid.
1H NMR (DMSO-d6): δ 8.76 (s, 1H, NH), 8.68–8.01 (m, 9H; pyrene),
7.85–7.20 (m, 9H), 6.38 (d, J = 6 Hz, 4H), 6.31 (t, J = 6.5 Hz, 1H; H-1ꢁ),
4.32 (m, 1H; H-3ꢁ), 4.15 (m, 2H, OCH2), 3.85 (m, 1H; H-4ꢁ), 3.75 (s,
6H), 3.62 (m, 1H), 3.51 (m, 2H; H-5ꢁ), 3.42 (m, 2H), 2.86 (t, J = 6 Hz,
2H), 2.58 (m, 1H; H-2ꢁβ), 2.42 (m, 2H), 2.20-2.32 (m, 1H; H-2ꢁα),1.32-1.01
(m, 24H). 13C NMR: δ 158.56, 130.99, 130.13, 129.97, 129.92, 128.34,
128.15, 127.82, 126.91, 125.97, 116.84, 113.04, 112.87, 85.79, 77.24, 76.99,
76.73, 58.14, 58.09, 55.17, 45.32, 45.27, 43.12, 29.67, 24.57, 22.96, 22.95,
22.88, 22.86, 22.62, 20.41, 20.11, 20.05, 18.61, 18.43, 14.08. 31P-NMR: 148.5
(diastereomeric pair).
HRMS: 1124.0627 (M—H) (Calcd. C64H69N8O9P minus H: 1124.2607)
2- (8-nitro-1-pyrenyl) -O6-benzyl- 3ꢁ, 5ꢁ-bis-O- (tert-butyldimethylsilyl) -2ꢁ-
deoxyguanosine (10): Compound 9 (0.1 g, 0.17 mmol), 1-bromo-8-
nitropyrene (70 mg, 0.21 mmol), Cs2CO3 (80 mg, 0.23 mmol), Pd(OAc)2
(4 mg, 0.01mmol) and BINAP (80 mg, 0.12 mmol) were weighed out in a
vial and flushed with argon. Anhydrous toluene (1.5 mL) was added and
the flask was refilled with argon before it was sealed. The mixture was
allowed to stir on a sand bath at 80◦C for 8–16 hours. It was subsequently
filtered through a celite pad, concentrated under reduced pressure, and
chromatographed with 8% ethyl acetate in hexanes to provide the coupled
product 10 in 88% yield.
1H NMR (CDCl3): δ 8.98 (d, J = 9.6 Hz, 1H), 8.83 (d, J = 8.6 Hz, 1H),
8.69 (d, J = 8.6 Hz, 1H), 8.52 (d, J = 9.6 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H),
8.22 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 8.06 (s, 1H), 8.03 (d, J =
8.9 Hz, 1H), 7.79 (s, 1H; H-8), 7.41 (m, 2H; phenyl), 7.29 (m, 3H; phenyl),
6.44 (t, J = 6.5 Hz, 1H; H-1ꢁ), 5.60 (dd, J = 12.3, 14.0 Hz, 2H; CH2Ph), 4.58
(m, 1H; H-3ꢁ), 4.02(q, J = 3.4 Hz, 1H; H-4ꢁ), 3.80 (dq, J = 6.2, 12.3 Hz, 2H;
H-5ꢁ), 2.58 (m, 1H; H-2ꢁβ), 2.42 (m, 1H; H-2ꢁα), 0.09 (s, 3H; Si-Me), 0.094 (s,
3H; Si-Me), 0.93 (s, 9H; Me3C), 0.92 (s, 9H; Me3C), 0.11 (s, 3H; Si-Me), 0.10
(s, 3H; Si-Me). 13C NMR: δ 160.7, 155.7, 153.2, 142.0, 138.6, 136.3, 135.9,
135.6, 130.9, 128.4, 128.3, 128.0, 126.9, 125.2, 124.0, 123.1, 121.6, 121.0,
120.9, 117.2, 87.8, 83.9, 76.8, 72.0, 68.4, 62.9, 41.4, 29.99, 25.8, 18.4, 18.0,
-4.65, -4.7.
HRFAB MS: 831.3748 (M+ +H) (Calcd. C45H55N6O6Si2: 831.3722).
2-(8-nitro-1-pyrenyl)-O6-benzyl-2ꢁ-deoxyguanosine (11): To a stirred so-
lution of 10 (100 mg, 0.12 mmol) in THF (3 mL) was added tetrabuty-
lammonium fluoride (0.36 mmol, 1.0 M solution in THF). The mixture
was stirred at room temperature for 1.5 hours, after which the solvent
was removed under reduced pressure. The crude product was purified by

76
L. C. Colis et al.
chromatography on silica, with 4% methanol in chloroform. The product
11 (69 mg, 95% yield) was isolated as pale yellow oil.
1H NMR (CDCl3): δ10.11 (s, 1H, NH), 8.73–8.16 2.29 (m, 9H; H-8 and
pyrene), 7.34 (m, 2H; phenyl), 7.29 (m, 3H; phenyl), 6.33 (t, J = 6.5 Hz,
1H; H-1ꢁ), 5.48 (s, 2H; CH2Ph), 5.26 (m, 1H, CH-OH), 4.86 (m, 1H, CH2-
OH), 4.32 (m, 1H; H-3ꢁ), 3.83 (m, 1H; H-4ꢁ), 3.47 (m, 2H; H-5ꢁ), 2.68 (m,
1H; H-2ꢁβ), 2.29 (m, 1H; H-2ꢁα). 13C NMR: δ 169.9, 150.6, 150.4, 137.2,
132.9, 131.9, 131.8, 128.0, 127.9, 127.4, 125.9, 125.8, 125.7, 125.6, 125.5,
125.4, 125.1, 123.1, 122.0, 119.5, 119.4., 88.5, 83.4, 71.4, 62.5, 62.4, 61.3,
55.8, 49.47, 24.5, 23.4.
HRES MS: 625.1819 (M+Na) (Calcd. C33H26N6O6Na: 625.1812).
N2-(8-nitro-1-pyrenyl)-O6-benzyl- 5ꢁ-O-(4,4ꢁdimethoxytrityl)-3ꢁ-O-[N,Nꢁ-
diisopropylamino(2-cyanoethoxy)phosphinyl]-2ꢁ-deoxyguanosine (12): The
method was nearly identical to the synthesis of 8.
1H NMR (DMSO- d6): δ 10.12 (s, 1H, NH), 8.73–8.16 (m, 9H; H-8 and
pyrene), 7.35 (m, 2H; phenyl), 7.28 (m, 3H, phenyl), 6.90–6.85 (m, 9H),
6.38 (d, J = 6 Hz, 4H), 6.31 (t, J = 6.5 Hz, 1H; H-1ꢁ), 5.52 (s, 2H; CH2Ph),
4.32 (m, 1H; H-3ꢁ), 4.15 (m, 2H, OCH2), 3.85 (m, 1H; H-4ꢁ), 3.51 (m, 2H;
H-5ꢁ), 3.46 (m, 2H), 3.35 (s, 6H), 2.86 (t, J = 6 Hz, 2H), 2.68 (m, 1H; H-2ꢁβ),
2.34 (m, 1H; H-2ꢁα), 1.05 (dd, 12H). 31P-NMR: 147.5 (diastereomeric pair).
HRMS: 1103.50 (M—H) (Calcd. C63H61N8O9P minus H: 1103.4221).
Oligonucleotide synthesis: Phosphoramidite 8 and 12 were used to
synthesize modified oligonucleotides in automated synthesizer as per the
manufacturer’s instructions, except a prolonged coupling time of 20 min-
utes was used for these monomers. Deprotection of the oligonucleotides was
carried out with concentrated ammonium hydroxide for 20 hours at 55◦C in
the presence of 0.25 M β-mercaptoethanol. After drying to remove excess
ammonia, the reaction mixture was desalted on a Sep-PAK C18 cartridge
and purified by reverse-phase HPLC.
Nitroreduction and O6-benzyl deprotection: The modified oligonu-
cleotide was suspended in a solution of 35% formamide, 35% ethanol,
10% ethyl acetate, 20% cyclohexadiene, and 0.2 mg palladium black. The
reaction mixture was sonicated for one hour and then stirred at room
temperature overnight. The reduced product was purified by reverse-phase
HPLC on a C18 Phenomenex Ultracarb column using a gradient of 0.1 M
ammonium acetate/1% acetonitrile (pH = 6.9) and 80% acetonitrile/ 20%
water over 50 minutes. The purified product was characterized using ESI
mass spectrometry.
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