Synthesis of N2-Deoxyguanosine Modified DNAs and the Studies on Their Translesion Synthesis by the E. coli DNA Polymerase IV

Article abstract of DOI:10.1021/acs.joc.8b02082

We report the synthesis of N²-aryl (benzyl, naphthyl, anthracenyl, and pyrenyl)-deoxyguanosine (dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion synthesis (TLS) across these N²-dG adducts is error free. However, the efficiency of TLS activity decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate TLS. Overall, the results presented here provide insights into the TLS across N²-aryl-dG damaged DNAs by Pol IV.

Full text of DOI:10.1021/acs.joc.8b02082

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Article
Synthesis of N2 -deoxyguanosine modified DNAs and the studies
on their translesion synthesis by the E. coli DNA polymerase IV
Pratibha P. Ghodke, Praneeth Bommisetti, Deepak T Nair, and Pushpangadan Indira Pradeepkumar
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02082 • Publication Date (Web): 10 Jan 2019
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Synthesis of N2-deoxyguanosine modified DNAs and the studies on
their translesion synthesis by the E. coli DNA polymerase IV
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Pratibha P. Ghodke,† Praneeth Bommisetti,† Deepak T. Nair,*, § P. I. Pradeepkumar*, †
†Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India
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§Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-
Gurgaon Expressway, Faridabad-121001, India
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ABSTRACT
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We report the synthesis of N2-aryl (benzyl, naphthyl, anthracenyl and pyrenyl)-deoxyguanosine
(dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer
extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion
synthesis (TLS) across these N2-dG adducts is error free. But the efficiency of TLS activity
decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations
of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino
acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme
and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate
TLS. Overall, the results presented here provide insights into the TLS across N2-aryl-dG
damaged DNAs by Pol IV.
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INTRODUCTION
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Polycyclic aromatic hydrocarbons (PAHs) are fused benzenoid ring containing compounds that
are well known for their carcinogenic and mutagenic properties.1 The exocyclic amino group
of deoxyguanosine (dG) acts as a softer reaction center to form major carcinogenic N2-dG DNA
adducts.2,3 These adducts can lead to genomic alterations via stalling replicative polymerases.
To avoid any detrimental consequence, cells use specialized low-fidelity DNA polymerases
belong to Y-family to bypass the damaged sites. This process called translesion synthesis
(TLS), an evolutionarily conserved DNA damage tolerance pathway, which can be error-free
or error-prone.4 Among various TLS polymerases, Pol IV from E. coli is well known for
accurate and efficient bypass of N2-dG adducts.5–7 Pol IV is an ortholog of human TLS
polymerase, Pol κ.5 A systematic study on the effects of the steric bulkiness of N2-dG adducts
on TLS by E. coli Pol IV is yet to be reported.
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Among the N2-dG DNA adducts, N2-benzyl-dG (N2-Bn-dG, Figure 1) is potentially
formed from three key carcinogens. N-nitroso-N-benzylurea (BnNU) and N-
nitrosobenzylmethylamine (NBnMA) could form N2-Bn-dG adducts via benzyldiazonium ion
8 and methyl hydroxylation9 pathway respectively. Whereas, benzyl halides could directly act
as a benzylating agent.10,11 Among these carcinogens, NBnMA is believed to form N7-benzyl-
dG adduct in in vivo conditions, since the N7-position is one of the major site of DNA
alkylation.11 The 9-(Sulfoxymethyl)anthracene is an electrophilic metabolite of 9-
hydroxymethyl-anthracene, which was alluded to be responsible for the formation of N2-(9-
anthracenylmethyl)-dG adduct (N2-Anth-dG, Figure 1).12 Halomethyl-anthracenes such as
bromo and chloromethyl derivatives have as well been suggested to alkylate the N2-position of
dG.13 1-Methylpyrene, a common environmental pollutant can enzymatically be activated to
1-(hydroxymethyl)pyrene, which in turn can be converted to 1-(sulfoxymethyl)pyrene (SMP).
SMP on reaction with DNA leads to the formation of N2-(1-pyrenylmethyl)-dG adduct (N2-
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Pyre-dG, Figure 1).14,15 Though N2-Bn, N2-Naph, and N2-Anth (Figure 1) adducts are yet to be
detected in cellular DNA, the N2-Pyre-dG adduct was detected in DNA from lung, liver, and
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kidney tissues of mice.14
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O
N
NH
N
N
NH
R
O
O
O
R
=
N2-Benzyl-dG
N2-Bn-dG
N2-(1-naphthylmethyl)-dG N2-(9-anthracenylmethyl)-dG
N2-Naph-dG
N2-Anth-dG
N2-(1-pyrenylmethyl)-dG
N2-Pyre-dG
(
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(
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(
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(
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Figure 1. Structures of N2-dG adducts which originate from polycyclic aromatic hydrocarbons.
To unravel the molecular mechanism of TLS across N2-aryl-dG adducts formed by
non-diol epoxide metabolic pathways by using various biochemical and biophysical methods,
the corresponding modified DNA oligonucleotides are required. Herein we report a short and
reliable synthesis of N2-dG adducts of varying steric bulkiness and the corresponding modified
DNAs. Using primer extension assays, molecular dynamics studies, we were able to gain
insights on the TLS by E. coli Pol IV across N2-(Bn, Naph, Anth, Pyre)-dG damaged templates.
RESULTS AND DISCUSSION
To access various N2-dG damaged DNAs, either phosphoramidite or post-oligomerization
approaches were reported.16–18 Synthesis of N2-Bn-dG adduct was achieved by different
methods such as substitution of C2-trimethanesulfonate group bearing dG by benzylamine,19
aromatic nucleophilic substitution of 2-fluoro-2’-deoxyinosine (2-fluoro-dI),20and reaction of
dG with benzyl bromide,10,21 reduction of N2-benzoyl group using reducing agents,22 reductive
amination of dG with benzaldehyde23 and by Buchwald-Hartwig (B-H) coupling between
TBDMS protected 2-bromo-2’-deoxyinosine (2-bromo-dI) with benzylamine.24 The N2-Naph-
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dG adduct was synthesized using nucleophilic substitution of DMT protected 2-fluoro-dI with
the corresponding amine.47 Synthesis of N2-Anth-dG adduct was achieved using B-H
coupling,24 or by the hydrolysis of 2'-deoxy-4-desmethylwyosine.13 Post-oligomerization
approach was also utilized for the synthesis of N2-Anth-dG adduct employing 2-fluoro-dI
bearing oligonucleotide.23 The N2-Pyre-dG adduct was acessed by fluoro substitution method
using 2-fluoro-dI convertible nucleoside and 1-(aminomethyl)pyrene.25
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Our objective was to develop a robust protocol to synthesize N2-aryl-dG modified
nucleoside and corresponding oligonucleotides (Figure 1) from a common precursor. To
achieve this, first, we attempted various reported methods. However, in our hands, many of
those methods were marred by lack of reproducibility, capricious yields, and poor solubility of
various intermediates/amidites.26 In the present work, we report a reliable and efficient method
for the synthesis of various N2-aryl-dG modified phosphoramidite building blocks employing
B-H coupling as the key step. These modified building blocks were synthesized in the presence
of a versatile para-nitrophenylethyl (NPE) protecting group at O6-position of dG. This route is
more convenient since all the bulky modifications can be synthesized from a common precursor
with good yields.
Synthesis of N2-Bn, N2-Naph, N2-Anth, N2-Pyre-dG modified phosphoramidites 5a-d
The synthetic Scheme 1 was utilized to access N2-modified-dG (N2-Bn, N2-Naph, N2-Anth, N2-
Pyre-dG) phosphoramidite building blocks. Using the protected 2-Br-dI, 1, as a common
precursor,27 the C-N bond formation was carried out with different polycyclic aromatic amines
in presence of Pd(OAc)2, (R)-BINAP, Cs2CO3 in toluene.28 For N2-Bn and N2-Naph-dG
modifications, B-H coupling were carried out using commercially available amine while for
N2-Anth-dG and N2-Pyre-dG modifications, the respective amines were prepared by reported
methods.25,29,30 Deprotection of acyl protecting group was accomplished using 33% MeNH2 in
EtOH (v/v) to obtain N2-modified-dG diols (3a-d). Final modified phosphoramidites 5a-d were
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synthesized by the protection of 5’-OH group using DMT-Cl, followed by phosphitylation
reaction using CEP-Cl, DIPEA in DCM.
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Scheme 1. Synthesis of N2-Bn, N2-Naph, N2-Anth and N2-Pyre-dG modified
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phosphoramidites 5a-d
ONPE
ONPE
N
ONPE
N
N
N
N
N
N
N
N
N
Br
N
NH
N
NH
AcO
AcO
HO
i
ii
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AcO
HO
3a
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3d
57 %
1
2a-d
64 %
54 %
45 %
after two steps
iii
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ONPE
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DMTO
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iv
Solid phase
synthesis
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Modified DNA
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HO
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CN
60 %
60 %
72 %
76 %
5a
61 %
41 %
37 %
40 %
5b
5c
5d
R
=
a
d
b
c
Reagent and conditions: (i) Pd(OAc)2, (R)-BINAP, respective amines, Cs2CO3, toluene, 85 °C; (ii)
33% MeNH2 in EtOH, rt; (iii) DMT-Cl, pyridine, rt; (iv) CEP-Cl, DIPEA, DCM, rt.
Solid phase synthesis of N2-dG modified oligonucleotides
The N2-dG modified phosphoramidite building blocks (5a-5d) were used to synthesize the
desired modified oligonucleotides. The presence of versatile NPE protecting group at O6-
position of dG provided enhanced solubility of respective phosphoramidites during solid phase
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DNA synthesis.39,50,31 We have chosen NPE protecting group because of its chemical inertness
and its stability in the presence of mild acids and bases.32 The N2-aryl-dG damaged DNA
sequences (Table 1) were synthesized using automated DNA synthesizer employing
appropriate CPG solid supports. The 18-mer DNA sequences (Table 1) were utilized for
crystallization with complementary 14-mer primer, incoming nucleotide and E. coli Pol IV.33
These sequences were designed for easy assembling of the three functional complexes of Pol
IV with three different DNA duplexes containing the N2-dG adduct and incoming nucleotide.
The 50-mer N2-dG DNA template sequences were synthesized for the primer extension studies
(Table 1). Deprotection of N2-dG modified DNAs was carried out in four different steps. The
first step involved the selective deprotection of cyanoethyl group using 10% diethylamine in
ACN.34 The deprotection of NPE group was performed using 1 M DBU in ACN.23 The
cleavage of the CPG support and deprotection of the base protecting groups were carried out
separately by treatment of aq. NH3. Modified DNAs were PAGE purified and the integrity of
modified DNAs was confirmed by ESI-MS or MALDI-TOF (Table 1).
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Table 1. N2-dG damaged oligonucleotides and their molecular weights
Code
DNA Sequences (5'-3')
Mol wt. Mol wt.
Error, %
(Calcd.) (Found) (found−calcd.)
D1
D2
D3
D4
TCTAG1GGTCCTAGGACCC
TCTG1GGGTCCTAGGACCC
TCTAGGG1TCCTAGGACCC
5566.7
5581.7
5566.7
15358
5566.0
5581.9
5567.3
15354.8
−0.01
0.004
0.01
TCCTACCGTGCCTACCTGAACAGCTG
GTCACACTG1ATGCCTACGAGTACG
−0.02
D5
D6
D7
D8
TCTAG2GGTCCTAGGACCC
TCTG2GGGTCCTAGGACCC
TCTAGGG2TCCTAGGACCC
5616.8
5632.8
5616.8
5616.8
5635.1
5617.4
0
0.04
0.01
0.007
TCCTACCGTGCCTACCTGAACAGCT
GGTCACACTG2ATGCCTACGAGTACG
15408.1 15409.1
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D9
TCTAG3GGTCCTAGGACCC
5666.9
5682.9
5666.9
5667.6
5682.0
5666.9
0.01
−0.02
0.001
−0.03
D10 TCTG3GGGTCCTAGGACCC
D11 TCTAGGG3TCCTAGGACCC
D12 TCCTACCGTGCCTACCTGAACAGCTG
15458.2 15453.8
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GTCACACTG3ATGCCTACGAGTACG
D13 TCTAG4GGTCCTAGGACCC
D14 TCTG4GGGTCCTAGGACCC
D15 TCTAGGG4TCCTAGGACCC
5690.9
5706.9
5690.9
5690.2
5705.8
5689.9
−0.01
−0.02
−0.02
−0.01
D16 TCCTACCGTGCCTACCTGAACAGCTG
15482.2 15480.9
GTCACACTG4ATGCCTACGAGTACG
Structures of modifications G1 (N2-Bn-dG), G2 (N2-Naph-dG), G3 (N2-Anth-dG) and G4 (N2-
Pyre-dG) are shown in Figure 1. The modified DNAs were characterised by MALDI-TOF in the
positive reflectron/linear mode or negative ion electrospray ionization (ESI) technique
(calculated and found). *Only D2 was characterized by ESI-MS in negative mode.
Primer extension studies using E. Coli DNA PoI IV
Primer extension reactions with E. coli Pol IV were carried out using a primer-template system
as shown in Figure 2A. Reactions were carried out with individual dNTP or with a mixture of
dNTPs employing unmodified and modified DNA templates and were monitored at various
time courses from 30 sec to 2 h (Figure 2 and Figures S1 to S5 of the supporting information).
In case of single nucleotide incorporation reactions, we observed a prominent extension
product of 16 nucleotide length, which corresponds to correct base (dCTP) incorporation across
all the N2-dG modified templates (Figure 2B to 2F, lane C). With the use of 4 nM of Pol IV, ~
94% incorporation of the correct base was observed opposite to unmodified dG template at 1.5
h time period (Figure 2B, lane C). For the N2-Bn-dG modification ~ 80% (Figure 2C, lane C),
for the N2-Naph-dG modification ~ 75% (Figure 2D, lane C), for the N2-Anth-dG modification
~ 62% (Figure 2E, lane C), and for the N2-Pyre-dG modification ~ 64% (Figure 2F, lane C)
incorporation products were observed after 1.5 h. These results reveal that the TLS employing
Pol IV is error free irrespective of the steric bulkiness of the N2-dG adducts.5,35 However,
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incorporation efficiency of Pol IV decreases with increasing steric bulkiness from the N2-Bn to
the N2-Pyre-dG adducts. It should be noted that in addition to the correct incorporation, a trace
amount of dTTP misincorporation was also observed opposite to the N2-Bn-dG (~ 10%, Figure
2C, lane T), and the N2-Naph-dG modifications (~ 4%, Figure 2D, lane T).
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Figure 2. PAGE (20%, 7 M urea) of primer extension reactions with single and with the mixture of
dNTPs using unmodified (dG) and modified (N2-dG adducts) templates employing E. coli. Pol IV
(A) Complete sequence of the primer, P (15-mer) and the template, T (50-mer); (B) reactions with
dG template (X = dG) (lanes: P:primer, C:dCTP, A:dATP, G:dGTP, T:dTTP, N:mixture of dNTPs);
(C) reactions with N2-Bn-dG template; (D) reactions with N2-Naph-dG template; (E) reactions with
N2-Anth-dG template; and (F) reactions with N2-Pyre-dG template. All reactions were carried out at
37 °C for 1.5 h time course.
To verify the efficiency of E. coli Pol IV towards full length extension of the primer,
running start experiments were carried out using 15-mer and 11-mer primers with the modified
DNA templates. Running start experiments using 15-mer primer, showed that the DNA Pol IV
is unable to synthesize full length products irrespective of the chemical nature of the damage
(Figures 3B to F). Similar results were obtained in running start experiments with 11-mer
primer (Figures S6 to S7 of the supporting information). As expected, in the case of unmodified
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template, we observed traces of the full-length product. These results are in line with the low
processivity reported for E. coli Pol IV, which is a hallmark of this family of DNA
polymerases.5,36 It should be noted that Pol IV belongs to the Y-family of DNA polymerases
and members of this family are known to bypass DNA lesions and rescue replication stalled at
damaged nucleotides. The majority of Y-family DNA polymerases serve only to extend the
replication fork past the DNA lesion after which they are replaced by replicative DNA
polymerases. 4
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Figure 3. PAGE (20%, 7 M urea) of full-length extension reactions employing Pol IV from E. coli
with all dNTPs. (A) Complete sequence of template, T and primer, P (15-mer); (B) reactions with
dG template (X = dG); (C) reactions with N2-Bn-dG template; (D) reactions with N2-Naph-dG
template; (E) reactions with N2-Anth-dG template; (F) reactions with N2-Pyre-dG template. Lane P,
primer; lanes 1 to 6: primer extension reactions with 250 μM of mixture of dNTPs in different time
course from 30 sec., 30 min., 1, 2, 4 and 8 h; lane C, 50-mer size standard. All the reactions were
carried out at 37 °C.
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Molecular modelling and dynamics studies
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Molecular dynamics (MD) simulations have been carried out to elucidate the factors that
facilitate TLS across N2-dG DNA adducts by E. coli Pol IV. The reported X-ray crystal
structure of furfuryl modified N2-dG DNA adduct in complex with the E.coli Pol IV (PDB ID:
4Q436) was used to prepare the starting structures. The N2-dG adducts, which were used in the
experimental studies (Figure 1) were incorporated into the DNA (Figure S8, Supporting
information). A total of four DNA-Pol IV ternary complexes (enzyme, DNA and incoming
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Figure 4. DNA-Pol IV ternary complex used for MD simulations. The initial structure of N2-Bn
complex prepared from fdG containing crystal structure (PDB ID: 4Q43). The red coloured rectangle
is used to highlight the active site and zoomed view of the active site. Other complexes are shown in
Figure S9, Supporting Information. Cyan, magenta, green and yellow are used for polymerase
associated domain (PAD), finger, palm and thumb domains respectively. DNA backbone is represented
in orange-red colour. The N2-modified residue is highlighted in desire red colour. Carbons atoms are
represented in white (desire red for N2-Bn-dG), nitrogen atoms in blue, oxygen atoms in red, and
magnesium ions in orange colour.
nucleotide components) were prepared and were subjected to 100 ns of unrestrained MD
simulations (Figure 4 and Figure S9, Supporting information). Complexes were generated only
for the insertion stage of dCTP. Trajectory analysis of each of these complexes illuminated the
key interactions between the DNA and Pol IV at the active site.
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Stability of the DNA – Pol IV ternary complexes
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To confirm the adequate sampling and the stability of DNA-Pol IV ternary complexes, root
mean square deviations (RMSD) of backbone atoms of DNA and protein were calculated
separately with respect to the first frame. Protein backbone showed marginal deviations with
an average RMSD value < 1.8 Å for all the complexes (Figure S10, Supporting information).
The DNA backbone showed considerable fluctuations with an average RMSD value > 2.0 Å.
In order to further investigate such large fluctuations in the DNA backbone, root mean square
fluctuation (RMSF) values were calculated for DNA with respect to the first frame. Only a few
nucleotides which don’t interact with protein showed larger fluctuations, whereas the
nucleotides around the active site showed fewer fluctuations (Figure S11, Supporting
information). RMSF values of protein residues showed only nominal fluctuations in all the
complexes (Figure S12, Supporting information). Also, the distance between the α phosphate
of dCTP and the O atom of 3′-OH of the primer terminal dG is < 4.0 Å in all the complexes,
indicative of a productive conformation for the nucleotide incorporation at the insertion stage.
The incoming nucleotide dCTP forms Watson-Crick (W-C) H-bonding with dG at the active
site. These W-C H-bonds were monitored through the course of dynamics and the percentage
occupancy of each of H-bond was calculated (Table 2). The H-bond (a) formed by dG:N2-H ···
O2:dCTP is affected to some extent in all the complexes. The interaction between the
surrounding amino acids (AAs) with the N2-modifications, made the orientation of the N2-
modified dG unfavourable for W-C H-bonding at certain time frames during the dynamics. The
H-bond labelled (a) in Table 2 is most affected in Anth complex in which the H-bond (b) was
also disturbed (Table 2).
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Table 2: Percentage occupancies of each H-bond at the active site of E. coli Pol IV
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Complex with adduct
a
b
c
N2-Bn-dG
92.08 %
95.26 %
93.01 %
N2-Naph-dG
N2-Anth-dG
N2-Pyre-dG
89.88 %
84.58 %
88.18 %
95.59 %
88.16 %
95.94 %
91.77 %
93.04 %
92.27 %
The dotted lines indicate the H-bond present between the dG and incoming nucleotide dCTP. Red arrow
indicates the position of modification
Clustering analysis
MD snapshots (100 ns) of each of the complex were clustered into 5 ensembles based on
hierarchical agglomerative approach.37 The percentage of each ensemble out of 100 ns of
dynamics is tabulated in Table 3. To identify the differences in the major ensembles,
representative structures of ensemble 1 and ensemble 2 were superimposed for all the
complexes. These representative structures differed primarily in the orientations of terminal
nucleotides of DNA. The similarities in the orientations of the AAs surrounding the
modifications are highlighted in Figure 5. The residues surrounding N2-Anth-dG adduct
showed least differences compared to other adducts. Minor differences were observed in the
orientations of Ser42 and Phe76 residues in the other complexes.5,6
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Table 3. Percentage occupancies of each ensemble out of 100 ns of dynamics.
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Complex with
adduct
Ensemble 1
Ensemble 2
Ensemble 3
N2-Bn-dG
N2-Naph-dG
N2-Anth-dG
N2-Pyre-dG
43.3%
59.9%
56.4%
47.8%
35.4%
23.0%
27.9%
31.8%
14.1%
7.2%
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8.2%
17.8%
Every second frame was considered, and a total of 25000 frames were considered for the
calculation. Clustering was performed using hierarchical agglomerative approach, which was
implemented by CPPTRAJ38 module of AMBER1439
To find out the similarities between the four ternary complexes at the active site, the
representative structures of ensemble 1 of all the complexes were superimposed onto each other
(Figure S14, Supporting information). Orientations of the N2-modified moieties were found to
be strikingly similar to that of the furfuryl adduct in the active site of DNA-Pol IV complex as
revealed by its crystal structure (PDBID: 4Q43). This implies that the Pol IV orients N2-
modified moieties into a particular conformation to facilitate TLS. To validate this, the dihedral
angel labelled ξ (Figure 6) was monitored through the course of dynamics. Bn, Naph and Pyre
complexes appeared to follow a pattern but Anth complex showed slight deviations. This
difference might be a result of the unique position of linkage of the anthracene moiety to the
nucleobase (Figure 1). The average dihedral angle values of 103.18°, 107.58°, 148.13°, 96.31°
were observed for Bn, Naph, Anth and Pyre complexes
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D
C
Figure 5. Superposition of representative structures of ensemble 1 and 2 of N2-dG-modified DNA-Pol
IV ternary complexes. Only residues of interest are highlighted. (A) N2-Bn complex. (B) N2-Naph
complex. (C) N2-Anth complex. (D) N2-Pyre complex. Lime green coloured cartoon representation is
used for the ensemble 1 and light pink coloured cartoon representation is used for the ensemble 2. DNA
backbone is represented in white for both the representative structures. Pale green coloured carbon
framework is used for N2-adducts for ensemble 1 and pale pink coloured carbon framework for
ensemble 2. Nitrogen atoms are represented in blue and oxygen atoms in red. All the hydrogens are
removed for clarity.
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O
N
NH
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C
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O
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
H2C
C(R)
O
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Figure 6: Plot of dihedral Xi (, shown above using red arrow) vs time for all the complexes. Black,
red, blue and pink colours were used to represent N2-Bn, -Naph, -Anth and -Pyre complexes
respectively.
respectively. Based on the above results, few important AA residues were identified and the
trajectory was further analysed to understand the interaction patterns between these adducts
and AAs, and also to probe the nature of the forces responsible for the accommodation of
adducts in the active site.
Key non covalent interactions and interaction energies
Average interaction energies of the N2-dG adducts with the surrounding AA residues were
calculated to identify the nature of interactions in the active site. Since our focus is the modified
nucleobases, only residues in close proximity to these modifications (within 6 Å) were chosen
for the calculations. Interaction energies were calculated using MM/GBSA method
implemented in AmberTools package. The total average energy was calculated by summation
of van der Waals, electrostatic, polar solvation and nonpolar solvation values (Internal = 0 for
reference). The average energy values presented in Table S1, Supporting information helped
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to qualitatively identify the chemical nature of these interactions. The results suggest that Ser42
interacts with the dG adducts primarily through van der Waals interactions.33 Upon visual
inspection of trajectories, Ser42 was found to interact with π cloud of aromatic moiety through
C-H ···π interactions.40 To verify this, the distance between Cβ of Ser42 and the centroid of the
N2-modified aromatic moiety was plotted for all the complexes (Figure S14, Supporting
information). The distances were in the appreciable range i.e. < 4 Å in all the complexes,
favourable for van der Waals interactions. In fact, there is a correlation between this distance
and the dihedral Xi () responsible for the particular orientation of the adducts (Figure S13).
When the distance is between 3-4 Å, the orientation of aromatic moiety favours strong π-cloud
interactions with the Ser42 residue.
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Apart from Ser42, the other major interacting residues displayed similar non-covalent
contacts. Phe76 played a crucial role in the complexes with high steric bulkiness, primarily by
forming T-shaped C-H··· π interactions with the adducts (Figure 5 and 7). The Phe13 also
exhibited C-H ···π interactions. This residue exhibited significant interactions in Anth complex
compared to moderate interactions found in Bn, Anth, and Pyre complexes (Figure 5 and 7).
Similarly, the C-H bond of alpha carbon (Cα) from Gly32 showed C-H··· π interactions in Naph
and Pyre (Figure 7B and 7D) complexes.40 The role of Tyr79 was found to be strategic as it
interacts with Phe13 through parallel displaced stacking and with Phe76 through T-shaped
interactions, which add strong hydrophobicity to the surroundings (Figure 5 and 7). Ile 31 and
Pro73 were also found to form moderate to weak interactions in addition to weak interactions
from Gly33 and Gly74 in all the complexes. The RMSF values for these residues in each of the
complex are reported in Table S2. The RMSF values were mostly < 1 Å, indicating the
importance of these residues in accommodating the adducts.
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D
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Figure 7: van der Waals interactions between AAs of Pol IV and N2-adducts found in DNA-Pol IV
ternary complexes. (A) N2-Bn complex. (B) N2-Naph complex. (C) N2-Anth complex. (D) N2-Pyre
complex. Phe76 interacts with the N2-adduct through T-shaped C-H ··· π interactions in the Bn, Naph,
Anth and Pyre complexes. Carbon atom linked to O-H of Ser42 interacts with π cloud of Naph, Anth
and Pyre moieties. The backbone atom of Gly32 as well interacts with adducts through C-H ··· π
interactions in Naph and Pyre complexes. Phe13 as well forms T-shaped interactions with Anth moiety.
The representative structures of other ensembles of each complex had similar interaction
patterns as discussed above. Overall, the van der Waals interactions are the major forces that
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are responsible for the accommodation of N2-modified moieties in the active site of Pol IV.
The residues Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76 and Tyr79 were found
to form a hydrophobic pocket6 into which the adducts are accommodated and our current MD
results support the same. The role of Ser42 was elucidated before6,41 and our results from the
MD simulations underscore the importance of this residue in stabilizing non-polar adducts
through van der Waals interactions. General interaction patterns from Phe13, Phe76, Tyr79,
and Gly32 were also identified. In summary, our MD studies provide insights on the molecular
interactions that are responsible for the accommodation of N2-dG adducts and their bypass by
E. coli Pol IV, which is line with the results emerged from the experiments.
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SUMMARY AND CONCLUSIONS
In conclusion, we have developed a robust and reliable protocol for the synthesis of N2-dG
modified DNAs. These modified DNAs were utilized to investigate the effect of steric
bulkiness of the N2-dG adducts on the TLS by Pol IV from E. coli. The Pol IV was found to be
efficient in bypassing these adducts with high fidelity but with low processivity. Molecular
modelling and dynamics studies were also carried out incorporating these N2-dG modified
DNAs at the active site of E. coli. Pol IV. The results revealed a common mechanism used by
the enzyme that involves using its hydrophobic pocket at the active site to accommodate these
modifications resulting in the efficient bypass of adducts. Further structural studies to validate
these observations are currently being pursed.
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EXPERIMENTAL SECTION
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All chemicals and dry solvents were obtained from commercial sources and used without any
further purification. Toluene, ACN, DCM, Et3N, DIPEA, dioxane and pyridine were dried
using calcium hydride. Thin layer chromatography (TLC) was performed on silica gel plates
pre-coated with fluorescent indicator with visualization by UV light (at 260 nm) or by dipping
into a solution of 5% conc. H2SO4 in EtOH (v/v) and heating. Silica gel (100−200 mesh) was
used for column chromatography. 1H NMR (500 or 400 MHz), 13C NMR (125 or 100 MHz),
31P NMR (202 or 162 MHz) were recorded on 500 MHz or 400 MHz instruments. Chemical
shifts in parts per million (δ) are reported downfield from TMS (0 ppm) and referenced to the
TMS signal or residual proton signal of the deuterated solvent as follows: CD3OD (3.31 ppm)
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for H NMR spectra, and CDCl3 (77.2 ppm) or CD3OD (49.1 ppm) for C NMR spectra.
Multiplicities of 1H NMR spin couplings are reported as s for singlet, br s for broad singlet, d
for doublet, t for triplet, br t for broad triplet, dd for doublet of doublet, ddd for doublet of
doublet of doublets, quint for quintet or m for multiplet and overlapping spin systems. Values
for apparent coupling constants (J) are reported in Hz. High resolution mass spectra (HRMS)
were obtained from Q-TOF analyzer in positive ion electrospray ionization (ESI) mode. All
the modified DNA sequences were synthesized using an automated solid phase synthesizer.
Mass spectra of DNA oligonucleotides were obtained by MALDI (in positive linear or
reflectron mode) or ESI (in negative mode). E. coli Pol IV was expressed and purified in
house.41
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General procedure for the Buchwald-Hartwig coupling and deacetylation
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In a screw capped tube, Pd(OAc)2 (0.1 equiv), (R)-BINAP (0.3 equiv) were taken and flushed
with N2 gas followed by addition of dry toluene (9.9 mL/mmol). Reaction mixture was stirred
at room temperature for 5 min. Cs2CO3 (1.4 equiv), respective amines (1.1 equiv) and bromo
nucleoside 1 (1 equiv) were added in the sequential manner. Further, the tube was purged with
N2 gas, sealed with a teflon-lined cap and heated in an oil bath at 85 °C. After completion of
reaction, the reaction mixture was passed through a celite pad and washed with EtOAc (250
mL). The filtrate was concentrated to give a crude B-H coupled products. Further, the crude
compound was subjected directly to deacetylation using 33% MeNH2 in EtOH (v/v, 22
mL/mmol) and stirred at room temperature. After completion of the reaction, the mixture was
concentrated under reduced pressure. The oily residue was purified by silica gel column
chromatography to obtain deprotected nucleosides.
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N2-Benzyl-O6-(2-(4-nitrophenyl)ethyl)-2'-deoxyguanosine (3a)
Pd(OAc)2 (5.94 mg, 0.02 mmol), (R)-BINAP (50 mg, 0.07 mmol), dry toluene (3 mL), Cs2CO3
(131 mg, 0.37 mmol), benzylamine (0.03 mL, 0.29 mmol) and bromo nucleoside 1 (150 mg,
0.26 mmol) were used and heated for 18 h to give a brown crude compound 2a (156 mg, 0.26
mmol), which was further dissolved in 33% MeNH2 in EtOH (5 mL) and stirred at room
temperature for 3 h. Silica gel column chromatography (4% MeOH in DCM) yielded slightly
brownish solid 3a (76 mg, 57% after two steps). Rf = 0.47 (10% MeOH in DCM); mp: 60−63
°C; 1H NMR (400 MHz, CDCl3):δ 8.12–8.07 (m, 2H), 7.61 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H),
7.31–7.21 (m, 5H), 6.19 (dd, J = 9.4, 5.6 Hz, 1H), 5.58 (br t, 1H), 4.70 (d, J = 5 Hz, 1H), 4.64
(t, J = 6.9 Hz, 2H), 4.60 (dd, J = 5.9, 2.4 Hz, 2H), 4.15 (s, 1H), 3.92 (dd, J = 12.6, 1.8 Hz, 1H),
3.71 (d, J = 11.3 Hz, 1H), 3.16 (t, J = 6.7 Hz, 2H), 2.97 (ddd, J = 14.2, 9.1, 5.1 Hz, 1H), 2.23
13
(dd, J = 12.6, 5.6 Hz, 1H); C{1H} NMR (100 MHz, CDCl3):δ 160.1, 158.5, 152.8, 146.9,
145.9, 139.3, 139.1, 130, 128.7, 127.3, 127.3, 123.8, 116.5, 89.1, 87.3, 73.3, 66.1, 63.5, 46.1,
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40.2, 35.1; HRMS (ESI): Calcd for C25H27N6O6, [M + H]+ 507.1992; found, [M + H]+ 507.1980
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(∆m −0.0012, error −2.4 ppm).
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The Pd(OAc)2 (11 mg, 0.04 mmol), (R)-BINAP (88 mg, 0.14 mmol), dry toluene (7 mL),
Cs2CO3 (214 mg, 0.65 mmol), 1-naphthylmethylamine (0.07 mL, 0.51 mmol) and bromo
nucleoside 1 (269 mg, 0.47 mmol) were used and heated for 21 h to give a dark brown crude
compound 2b (260 mg, 0.40 mmol), which was further dissolved in 33% MeNH2 in EtOH (9
mL) and stirred at room temperature for 2.5 h. Silica gel column chromatography (4% MeOH
in DCM) yielded yellow solid 3b (171 mg, 64%, after two steps). Rf = 0.49 (10% MeOH in
DCM); mp: 67−70 °C; 1H NMR (400 MHz, CDCl3):δ 8.09–8.02 (m, 1H), 7.97 (d, J = 8.3 Hz,
2H), 7.92–7.85 (m, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H), 7.57–7.36 (m, 6H), 7.16 (br s,
1H), 6.18 (dd, J = 9.2, 5.6 Hz, 1H), 5.51 (br s, 1H), 5.14–4.99 (m, 2H), 4.67 (d, J = 5.3 Hz,
1H), 4.60 (t, J = 7.0 Hz, 2H), 4.33 (s, 1H), 4.13 (s, 1H), 3.90 (d, J = 12 Hz, 1H), 3.68 (dd, J =
12.5, 1.8 Hz, 1H),3.07 (t, J = 6.7 Hz, 2H), 3.00 (ddd, J = 14, 9.5, 5.5 Hz, 1H), 2.22 (dd, J =
13.4, 5.6 Hz, 1H); 13C{1H} NMR (100 MHz, CDCl3):δ 161, 158.4, 153, 146.8, 145.6, 139.2,
134.2, 133.9, 131.3, 129.8, 129, 128.1, 126.4, 126, 125.5, 125.1, 123.7, 123.1, 116.4, 89, 87,
73, 66.1, 63.3, 44, 40.1, 35; HRMS (ESI): Calcd for C29H29N6O6, [M + H]+ 557.2149; found,
[M + H]+ 557.2156 (∆m +0.0007, error +1.3 ppm).
N2-(9-Anthracenylmethyl)-O6-(2-(4-nitrophenyl)ethyl)-2'-deoxyguanosine (3c)
Pd(OAc)2 (3.9 mg, 0.01 mmol), (R)-BINAP (33 mg, 0.05 mmol), dry toluene (2 mL), Cs2CO3
(87 mg, 0.24 mmol), 9-(aminomethyl)-anthracene29 (41 mg, 0.19 mmol), and bromo nucleoside
1 (100 mg, 0.17 mmol) were used and heated for 8 h to give a dark brown crude compound 2c
(127 mg, 0.18 mmol), which was further dissolved in 33% MeNH2 in EtOH (3.7 mL) and
stirred at room temperature for 3 h. Column chromatography (3% MeOH in DCM) yielded
slightly greenish compound 3c (59 mg, 54% after two steps). Rf = 0.50 (10% MeOH in DCM);
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mp: 83−86 °C; 1H NMR (400 MHz, CDCl3):δ 8.47 (s, 1H), 8.32–8.27 (m, 2H), 8.12 (d, J = 8.5
Hz, 2H), 8.03 (dd, J = 8.5, 1.0 Hz, 2H), 7.64 (s, 1H), 7.54–7.42 (m, 6H), 6.18 (br t, 1H), 5.57–
5.47 (m, 2H), 5.18 (t, J = 4.4 Hz, 1H), 4.90–4.71 (m, 2H), 4.63 (br s, 1H), 4.07 (br s, 1H), 3.88–
3.77 (m, 1H), 3.63–3.53 (m, 1H), 3.29 (br s, 2H), 3.12–2.92 (m, 1H), 2.25–2.13 (m, 1H);
13C{1H} NMR (100 MHz, CDCl3):δ 161.1, 158.3, 153.1, 146.9, 145.9, 139.2, 131.6, 130.6,
130, 129.3, 128.7, 128.2, 126.6, 125.2, 124.1, 123.8, 116.5, 88.8, 87, 73, 66.3, 63.2, 40.1, 38.9,
35.3; HRMS (ESI): Calcd for C33H31N6O6, [M + H]+ 607.2300; found, [M + H]+ 607.2297 (Δm
−0.0003, error −0.5 ppm).
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N2-(1-Pyrenylmethyl)-O6-(2-(4-nitrophenyl)ethyl)-2'-deoxyguanosine (3d)25
Pd(OAc)2 (16 mg, 0.07 mmol), (R)-BINAP (132 mg, 0.21 mmol), dry toluene (7 mL), Cs2CO3
(323 mg, 0.99 mmol), 1-(aminomethyl)pyrene30 (182 mg, 0.78 mmol), and bromo nucleoside
1 (403 mg, 0.71 mmol) were used and heated in oil bath for 21 h at 85 °C to get crude compound
2d (547 mg, 0.76 mmol), which was further dissolved in 33% MeNH2 in EtOH (15 mL) and
stirred at room temperature for 1 h 15 min. Column chromatography on a silica gel (2.5%
MeOH in DCM) yielded yellow compound 3d (202 mg, 45% after two steps). Rf = 0.49 (10%
MeOH in DCM); mp: 75−78 °C; 1H NMR (400 MHz, MeOH-d4 + CDCl3):δ 8.24 (d, J = 9.1
Hz, 1H), 8.16–8.09 (m, 2H), 8.08–7.89 (m, 7H), 7.72 (s, 1H), 7.66 (br s, 2H), 6.86 (br s, 1H),
6.22 (dd, J = 8.9, 5.9 Hz, 1H), 5.23 (s, 2H), 4.55 (d, J = 5.4 Hz, 1H), 4.38 (t, J = 6.6 Hz, 2H),
4.09 (d, J = 1.3 Hz, 1H), 3.88 (d, J = 12.4 Hz, 1H), 3.73–3.66 (m, 2H), 3.58–3.49 (m, 1H), 2.88
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(ddd, J = 14.5, 9.1, 5.6 Hz, 1H), 2.30–2.20 (m, 1H); C{1H} NMR (100 MHz, MeOH-d4 +
CDCl3):δ 160.9, 158.5, 152.9, 146.4, 145.5, 139.1, 132.4, 131.3, 130.7, 129.4, 128.4, 127.9,
127.4, 127.2, 126.2, 125.4, 125.2, 124.9, 124.8, 123.4, 122.5, 115.9, 88.8, 87, 72.5, 65.9, 63.2,
44, 40.1, 34.7; HRMS (ESI): Calcd for C35H31N6O6, [M + H]+ 631.2300; found, [M + H]+
631.2293 (∆m −0.0007, error −1 ppm).
General procedure for the DMT-protection
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The N2-modified-dG nucleosides (1 equiv) was co-evaporated with dry pyridine (5 mL) and
dissolved in the same solvent (10 mL/mmol). To this, DMT-Cl (1.22 equiv) was added and
stirred at room temperature. Further, the reaction mixture was diluted with DCM (100 mL) and
washed with saturated NaHCO3 (40 mL) and water (2 × 50 mL). DCM layer was dried over
Na2SO4 and evaporated. The crude DMT-protected compound was purified by silica gel
column chromatography (using appropriate solvents along with 2% Et3N) to afford pure
compound.
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N2-Benzyl-O6-(2-(4-nitrophenyl)ethyl)-5'-(4,4’-dimethoxytrityl)-2'-deoxyguanosine (4a)
Compound 3a (251 mg, 0.49 mmol) in dry pyridine (5 mL), DMT-Cl (332 mg, 0.99 mmol)
were stirred for 24 h. Column chromatography (90% DCM in pet. ether + 2% Et3N) yielded
compound 4a as a pale yellow solid (237 mg, 60%). Rf = 0.55 (2% MeOH in DCM + 2% Et3N);
mp: 71−74 °C; 1H NMR (400 MHz, CDCl3):δ 8.13–8.09 (m, 2H), 7.68 (s, 1H), 7.39 (d, J = 7.3
Hz, 4H), 7.31–7.26 (m, 8H), 7.25–7.14 (m, 4H), 6.78 (d, J = 8.0 Hz, 4H), 6.27 (t, J = 6.7 Hz,
1H), 5.17 (t, J = 5.5 Hz, 1H), 4.66 (t, J = 6.8 Hz, 2H), 4.63–4.58 (m, 1H), 4.53 (t, J = 5.8 Hz,
2H), 4.09–4.04 (m, 1H), 3.76 (s, 6H), 3.38 (dd, J = 9.8, 4.8 Hz, 1H), 3.34 (dd, J = 10.1, 5.4 Hz,
1H), 3.20 (t, J = 6.8 Hz, 2H), 2.87–2.81 (m, 1H), 2.38 (ddd, J = 13.2, 6.2, 4 Hz, 1H); 13C{1H}
NMR (100 MHz, CDCl3):δ 160.6, 158.8, 158.7, 153.8, 146.9, 146.1, 144.7, 139.6, 137.9, 135.8,
130.1, 130, 128.6, 128.2, 128, 127.4, 127.2, 127, 123.8, 115.5, 113.3, 86.6, 85.9, 84, 72.8, 66,
64.1, 55.3, 46.9, 39.5, 35.3. HRMS (ESI): Calcd for C46H45N6O8, [M + H]+ 809.3299; found,
[M + H]+ 809.3329 (∆m +0.003, error +3.7 ppm).
N2-(1-Naphthylmethyl)-O6-(2-(4-nitrophenyl)ethyl)-5'-(4,4’-dimethoxytrityl)-2'-
deoxyguanosine (4b)
Compound 3b (171 mg, 0.30 mmol) in dry pyridine (3 mL), DMT-Cl (126 mg, 0.37 mmol)
were stirred for 10 h. Column chromatography (1% MeOH in DCM + 2% Et3N) yielded
compound 4b as a yellow solid (154 mg, 60%). Rf = 0.50 (2% MeOH in DCM + 2% Et3N);
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mp: 85−88 °C; 1H NMR (400 MHz, CDCl3):δ 8.08–7.98 (m, 3H), 7.91–7.86 (m, 1H), 7.77 (d,
J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.55–7.46 (m, 2H), 7.44–7.35 (m, 4H), 7.30–7.26 (m, 5H), 7.26–
7.13 (m, 4H), 6.80–6.72 (m, 4H), 6.29 (t, J = 6.6 Hz, 1H), 5.24–5.17 (m, 1H), 5.06–4.88 (m,
2H), 4.63 (t, J = 6.9 Hz, 2H), 4.56 (br s, 1H), 4.08–4.01 (m, 1H), 3.74 (d, J = 1.3 Hz, 6H),
3.68–3.63 (m, 1H), 3.39 (dd, J = 10, 5 Hz, 1H), 3.33 (dd, J = 9.8, 4.8 Hz, 1H), 3.14 (br s, 2H),
2.85–2.74 (m, 1H), 2.38 (ddd, J = 13.5, 6.5, 4.5 Hz, 1H); 13C{1H} NMR (100 MHz, CDCl3):δ
160.7, 158.7, 158.6, 146.8, 146, 144.7, 137.9, 135.8, 134.9, 134.5, 133.9, 131.4, 130.1, 129.8,
129, 128.2, 128.1, 128, 127, 126.5, 126, 125.6, 123.8, 123.4, 115.7, 113.3, 86.6, 85.8, 84, 72.7,
66, 64.1, 55.3, 44.1, 39.6, 35.2; HRMS (ESI): Calcd for C50H47N6O8, [M + H]+ 859.3455;
found, [M + H]+ 859.3478 (∆m +0.0023, error +2.7 ppm).
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N2-(9-Anthracenylmethyl)-O6-(2-(4-nitrophenyl)ethyl)-5'-(4,4’-dimethoxytrityl)-2'-
deoxyguanosine (4c)
Compound 3c (162 mg, 0.26 mmol) in dry pyridine (3 mL), DMT-Cl (180 mg, 0.53 mmol)
were stirred for 10 h. Column chromatography (1% MeOH in DCM + 2% Et3N) yielded
compound 4c as a slightly greenish solid (177 mg, 72%). Rf = 0.50 (2% MeOH in DCM + 2%
Et3N); mp: 86−89 °C; 1H NMR (500 MHz, CDCl3):δ 8.46 (s, 1H), 8.29–8.24 (m, 2H), 8.08 (d,
J = 7.9 Hz, 2H), 8.05–8.00 (m, 2H), 7.75 (s, 1H), 7.50–7.36 (m, 8H), 7.28 (dd, J = 5.8, 2.7 Hz,
3H), 7.20–7.15 (m, 3H), 7.12–7.08 (m, 1H), 6.73 (d, J = 7.3 Hz, 4H), 6.46–6.28 (m, 1H), 5.51–
5.36 (m, 2H), 5.14 (t, J= 4.0 Hz, 1H), 4.74–4.59 (m, 2H), 4.09 (br s, 1H), 3.70 (d, J = 1.5 Hz,
6H), 3.46–3.40 (m, 1H), 3.38–3.30 (m, 1H), 3.26–3.15 (br s, 2H), 2.91–2.85 (m, 1H), 2.52–
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2.44 (m, 1H); C{1H} NMR (125 MHz, CDCl3):δ 160.7, 158.6, 153.9, 146.8, 146.1, 144.6,
137.7, 135.8, 135.7, 131.6, 130.5, 130.1, 130, 130, 129.4, 129.3, 129.1, 128.3, 128.2, 128, 127,
126.5, 125.2, 124.2, 123.8, 115.5, 113.2, 86.6, 85.9, 83.9, 72.8, 66, 64.1, 55.3, 45.9, 38.7, 35.3;
HRMS (ESI): Calcd for C54H49N6O8, [M + H]+ 909.3606; found, [M + H]+ 909.3608 (∆m
+0.0002, error +0.1 ppm).
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deoxyguanosine (4d)
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Compound 3d (151 mg, 0.24 mmol) in dry pyridine (3 mL), DMT-Cl (102 mg, 0.30 mmol)
were stirred for 12 h. Column chromatography (1% MeOH in DCM + 2% Et3N) yielded
compound 4d as a pale yellow solid (178 mg, 76%). Rf = 0.55 (2% MeOH in DCM + 2% Et3N);
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mp: 77−80 °C; H NMR (400 MHz, CDCl3):δ 8.25 (d, J = 9.3 Hz, 1H), 8.22–8.16 (m, 2H),
8.10–7.86 (m, 7H), 7.71 (s, 1H), 7.42–7.36 (m, 2H), 7.32–7.26 (m, 5H), 7.25–7.10 (m, 5H),
6.78–6.72 (m, 4H), 6.30 (t, J = 6.5 Hz, 1H), 5.37–5.32 (m, 1H), 5.27–5.13 (m, 2H), 4.64–4.50
(m, 3H), 4.09–4.03 (m, 1H), 3.70 (d, J = 2.5 Hz, 6H), 3.39 (dd, J = 9.9, 4.6 Hz, 1H), 3.35 (dd,
J = 10, 5.2 Hz, 1H), 3.04 (br s, 2H), 2.87–2.77 (m, 1H), 2.37 (ddd, J = 13.4, 6.4, 4.0 Hz, 1H);
13C{1H} NMR (100 MHz, CDCl3):δ 160.7, 158.7, 158.6, 153.9, 146.6, 145.9, 144.7, 137.9,
135.8, 132.4, 131.4, 130.8, 130.1, 129.7, 128.2, 128, 127.5, 127.4, 127, 126.2, 125.5, 125.3,
125, 124.9, 124.8, 123.6, 122.8, 115.7, 113.3, 86.6, 85.9, 84, 72.8, 65.9, 64.1, 55.3, 44.3, 39.6,
35.1; HRMS (ESI): Calcd for C56H48N6NaO8, [M + Na]+ 955.3426; found, [M + Na]+ 955.3431
(∆m +0.0005, error +0.6 ppm).
General procedure for the phosphitylation
DMT protected nucleoside (1 equiv) was dissolved in DCM (10 mL/mmol) followed by
addition of DIPEA (8 equiv) and CEP-Cl (2 equiv). Reaction mixture stirred at room
temperature under N2 atmosphere. After completion, the reaction was quenched by addition of
trace amount of MeOH and stirred for 5 to 10 min. Further, the reaction mixture was diluted
with DCM (100 mL) and washed with NaHCO3 (3 × 20 mL), dried over Na2SO4, and
evaporated under reduced pressure. Crude compound was purified by silica gel column
chromatography (using appropriate solvents along with 2% Et3N) to obtain phosphitylated
compound.
N2-Benzyl-O6-(2-(4-nitrophenyl)ethyl)5'-O-(4,4’-dimethoxytrityl)-3'-(2-cyanoethyl
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diisopropylphosphoramidite)-2'-deoxyguanosine (5a)
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Nucleoside 4a (150 mg, 0.18 mmol), DCM (2 mL), DIPEA (0.25 mL, 1.48 mmol) and CEP-
Cl (127 mg, 0.54 mmol) were used and stirred for 2 h. Further, MeOH (0.5 mL) was added and
stirred for additional 10 min. Column chromatography (35% DCM in pet. ether + 2% Et3N)
yielded compound 5a as a pale yellow solid (111 mg, 61%). Rf = 0.65 (90% DCM in pet. ether
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+ 2% Et3N); mp: 58−61 °C; P NMR (162 MHz, CDCl3):δ 148.81, 148.63; HRMS (ESI):
Calcd for C55H62N8O9P, [M + H]+ 1009.4377; found, [M + H]+ 1009.4348 (∆m −0.0029, error
−3 ppm).
N2-(1-Naphthylmethyl)-O6-(2-(4-nitrophenyl)ethyl)5'-O-(4,4’-dimethoxytrityl)-3'-(2-
cyanoethyldiisopropylphosphoramidite)-2'-deoxyguanosine (5b)
The tritylated nucleoside 4b (150 mg, 0.17 mmol), DCM (2 mL), DIPEA (0.25 mL, 1.36 mmol)
and CEP-Cl (83 mg, 0.34 mmol) were used and stirred for 2 h 15 min. Further, MeOH (0.5
mL) was added and stirred for additional 10 min. Column chromatography (35% DCM in pet.
ether + 2% Et3N) yielded compound 5b as a pale yellow solid (74 mg, 41%). Rf= 0.65 (75%
DCM in pet. ether + 2% Et3N); mp: 70−73 °C; 31P NMR (162 MHz, CDCl3):δ 148.72, 148.57;
HRMS (ESI): Calcd for C59H64N8O9P, [M + H]+ 1059.4528; found, [M + H]+ 1059.4529 (∆m
+0.00009, error +0.1 ppm).
N2-(9-Anthracenylmethyl)-O6-(2-(4-nitrophenyl)ethyl)5'-O-(4,4’-dimethoxytrityl)-3'-(2-
cyanoethyldiisopropylphosphoramidite)-2'-deoxyguanosine (5c)
Nucleoside 4c (170 mg, 0.18 mmol), DCM (2 mL), DIPEA (0.26 mL, 1.49 mmol) and CEP-
Cl (88 mg, 0.37 mmol) were used and stirred for 1 h. Further, MeOH (0.5 mL) was added and
stirred for additional 10 min. Column chromatography (40% DCM in pet. ether + 2% Et3N)
yielded compound 5c as a slightly greenish solid (75 mg, 37%). Rf = 0.60 (75% DCM in pet.
ether + 2% Et3N); mp: 77−80 °C; 31P NMR (202 MHz, CDCl3):δ 148.85, 148.72; HRMS (ESI):
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Calcd for C63H66N8O9P [M + H]+1109.4685; found, [M + H]+1109.4686 (∆m +0.00009, error
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cyanoethyldiisopropylphosphoramidite)-2'-deoxyguanosine (5d)
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Cl (100 mg, 0.42 mmol) were used and stirred for 1 h 15 min. Further, MeOH (0.5 mL) was
added and stirred for additional 10 min. Column chromatography (45% DCM in pet. ether +
2% Et3N) yielded compound 5d as a pale yellow solid (93 mg, 40%). Rf = 0.66 (75% DCM in
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pet. ether + 2% Et3N); mp: 80−83 °C; P NMR (202 MHz, CDCl3):δ 148.72; HRMS (ESI):
Calcd for C65H66N8O9P, [M + H]+ 1133.4685; found, [M + H]+1133.4690 (∆m +0.0005, error
+0.4 ppm).
Solid phase synthesis, deprotection and purification of N2-dG modified oligonucleotides
All DNA sequences (Table 1) were synthesized on 1 µmol scale using appropriate controlled
pore glass (CPG) solid supports. Unmodified and modified phosphoramidite building blocks
were dried under P2O5 for 24 h, and then dissolved in anhydrous ACN and stored at 4 °C under
molecular sieves (4 Å) for 10 h. Concentration used for the unmodified phosphoramidite was
0.067 M and for modified phosphoramidite was 0.1 M. Coupling time used for unmodified
phosphoramidite was 2 min. and for the N2-dG modified phosphoramidites was 6 min. After
solid phase synthesis, deprotection of N2-Bn, N2-Naph, N2-Anth and N2-Pyre-dG modified
oligos were carried out in four different steps. Initially, the deprotection of cyanoethyl group
of the modified DNA sequences were carried out using 10% diethylamine in ACN (800 μl, v/v)
for 5 min. Supernatant layer was collected and solid support was washed with ACN (2 × 400
μl). The CPG beads was air dried (3 h) and treated with 1 M DBU in ACN (1 mL, v/v) for 1 h
at room temperature to remove NPE group.23 After 1 h, the supernatant was removed and the
CPG beads were washed with MeOH (2 × 1 mL) and ACN (3 × 1 mL). Furthermore, the
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