Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo

Article abstract of DOI:10.1002/chem.201802304

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2′-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by T_m measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W?C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).

Full text of DOI:10.1002/chem.201802304

A Journal of
Accepted Article
Title: Methylated nucleobases: Synthesis and evaluation for base
pairing in vitro and in vivo
Authors: Amit M. Jabgunde, Faten Jaziri, Omprakash Bande, Matheus
Froeyen, Mikhail Abramov, Hoai Nguyen, Guy Schepers,
Eveline Lescrinier, Vitor B. Pinheiro, Valérie Pezo, Philippe
Marlière, and Piet Herdewijn
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201802304
Link to VoR: http://dx.doi.org/10.1002/chem.201802304
Supported by

Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
Methylated nucleobases : Synthesis and evaluation for base
pairing in vitro and in vivo
Amit M. Jabgunde, [a] Faten Jaziri, [b] Omprakash Bande, [a] Matheus Froeyen, [a] Mikhail Abramov, [a]
Hoai Nguyen, [a] Guy Schepers, [a] Eveline Lescrinier , Vitor B. Pinheiro, Valérie Pezo, Philippe
[a]
[c]
[b]
[
b]
[a]
Marlière, and Piet Herdewijn*
Abstract: The synthesis, base pairing properties and in vitro
polymerase) and in vivo (E. coli) recognition of 2’-deoxynucleotides
field,[10] and this research has been the subject of many
reviews.[
11]
(
with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-
thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are
Introduction of a methyl group in the 5-position of 2’-deoxyuridine
(giving thymidine) and in the 5-position of 2’-deoxycytidine (giving
5-methyl-2’-deoxycytidine) increases duplex stability of double
stranded nucleic acids due to hydrophobic interactions with the
neighbouring bases.[12] Here we have replaced functionalities of
purine and pyrimidine bases, normally involved in base pairing,
with a methyl group to investigate the influence on duplex stability
and to investigate the potential to involve them in new base
pairing systems. A second small hydrophobic functionality that is
involved in this study is a thiocarbonyl group (replacing a carbonyl
group), for its potential to interact with the methyl group present
in opposite bases in a duplex. By introducing such methyl and
thiocarbonyl group, we will make the major (or minor groove)
more hydrophobic which may not be favourable for duplex
stabilization and could contribute to orthogonality. However, there
are several precedents of the involvement of a hydrophobic
thiocarbonyl group in base pair stabilization and some natural
m
described. As demonstrated by T measurements, the X and Y bases
fail to form a self-complementary base pair. Despite this failure,
enzymatic incorporation experiments show that selected DNA
polymerases recognize the X nucleotide and incorporate this modified
nucleotide versus X in the template. In vivo, X is mainly recognized as
a A/G or C base; Y is recognized as a G or C base and Z is mostly
recognized as T or C. Replacing functional groups in nucleobases
normally involved in W-C recognition (6-carbonyl and 2-amino group
of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality
(absence of base pairing with natural bases).
Introduction
Expanding the genetic alphabet to include an additional base pair
has led to the replication of such a base pair in a bacterial cell,[1]
and in the use of such a base pair to code for unnatural amino
acids.[ Numerous research groups have studied base modified
nucleic acids but few of them have been evaluated in vivo.[2-3]
The candidate base pairs are comprised of reorganized donor–
acceptor hydrogen-bonding groups,[ increased number of
t
RNA contain sulphur modified nucleobases such as 2-thio-U, 4-
thio-U, 2-thio-C.[13] Rappaport has studied the 6-thioguanine (G
S
):
H
[14]
5-methyl-2-pyrimidone (T ) base pair (Figure 1a).
The
2]
laboratory of Switzer has investigated the 2-thio-isoguanine
):5-methyl-isocytosine (iCMe) base pair (Figure 1b) and the
[15]
(
2
(
iG
-thio-isoguanine(iG
Figure 1c). In all these cases the thiocarbonyl group was placed
opposite an NH group or an H-atom and the stability of the base
S
):5-methyl-4-pyrimidone (P)[16] base pair
S
4]
[
5]
[3b, 6]
hydrogen bonds,
size expanded bases,
hydrophobic
2
bases,[ and metal-ion coordinating bases.[8] For example, the
work of Hirao,[9] and Romesberg,[1-2] demonstrates that
hydrophobic interactions may contribute to stable base pairing.
The work of Benner is an example how reorganization of donor-
7]
S
H
S
Me
S
pair (G :T , iG :iC and iG :P) was found to be similar to the
canonical A:T pair.
H
3
C
H
N
O
S
N
N
H
N
N
H
N
N
H
H
O
N
N
CH
3
CH3
dR
N
H
H
O
N
H
accepter interactions may lead to orthogonal base pairing in
vitro.[4c] But many others, likewise, contributed to this research
H
H
N
dR
N
N
N
N
S
N
dR
N
S
dR
N
N
dR
N
N
H
dR
H
H
TH
GS
iGS
iCMe
iGS
P
(
a)
(b)
(c)
[
a]
A. M. Jabgunde, O. Bande, M. Froeyen, M. Abramov, H. Nguyen,
G. Schepers, E. Lescrinier, V. Pezo, P. Marlière, P. Herdewijn
KU Leuven, Rega Institute, Medicinal Chemistry
Herestraat 49 box 1041, 3000 Leuven, Belgium
E-mail: Piet.Herdewijn@kuleuven.be
[
N
H
N
H
CH
N
S
N
H
3
N
S
S
O
H C
N
N
CH
N
3
3
H
3
C
dR
N
O
H
N
N
N
H
N
H
N
dR
N
N
N
dR
N
N
dR
dR
H
H
3
C
N
H
3
C
H
H
N
H
O
[
[
b]
c]
Faten Jaziri, V. Pezo, P. Marlière
dR
Génomique Métabolique, Genoscope, Institut François Jacob, CEA,
CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France
V. B. Pinheiro,
Institute of Structural and Molecular Biology, University College
London, Darwin Building, Gower Street, London
WC1E 6BT, United Kingdom
Z
Y
Y
Y
X
Z
(d)
(e)
(f)
Figure 1. Schematic representation of putative base pairs between (a) 5-
H
S
methyl-2-pyrimidone (T ) with 6-thioguanine (G ) (b) 2-thio-isoguanine (iG
with 5-methyl-isocytosine (iCMe) (c) 2-thio-isoguanine (iG
) with 5-methyl-4-
S
)
S
pyrimidone (P) (d) 6-methyl-4-pyrimidone (Z) with 2-methyl-6-thiopurine (Y) (e)
self-base pair of 2-methyl-6-thiopurine (Y:Y), (f) 2-amino-6-methyl-8-oxo-7,8-
dihydro-purine (X) and 6-methyl-4-pyrimidone (Z)
Supporting information for this article is given via a link at the end of
the document.
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Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
The use of a self-complementary base for the expansion of the
genetic alphabet has the advantage that the potential for
mispairing with the natural bases is reduced (as only one base
has to be considered for mispairing). It is not really a limitation
since the addition of a single self-complementary base to the
genetic alphabet would create 61 new codons. For example
Pochet and Marlière[17] proposed a self-pair using a 2-amino-8-
oxo-7,8-dihydro-purine base, with the potential to form two
hydrogen bonds (Figure 2a). This work is based on the well-
recognized ability of the mutagenic 8-oxoguanine to base pair with
adenine base, the former oriented in the syn conformation. This
Further, we have included in our study the 2-methyl-6-thiopurine
base (Y) to pair with the 6-methyl-4-pyrimidone heterocycle
(Figure 1d) (Z) and with itself (Figure 1e). In addition, potential
pairing of the modified pyrimidone heterocycle (Z) with the
previously mentioned 2-amino-6-methyl-8-oxo-7,8-dihydro-purine
base (X) was investigated (Figure 1f).
There are many precedents in the literature of the stabilization of
macromolecular structure or small molecule interactions by
thiocarbonyl and methyl groups.[21] In addition the higher
polarizability of sulphur over oxygen may contribute to enhanced
stacking interactions.[22]
8
-oxoG (syn):A(anti) base pair was studied by X-ray diffraction
Our investigation started by building a static in silico model to
analyse if X:X interactions and Z:Y interactions are potentially
accessible in a dsDNA context. The models are shown in Figures
and NMR[
18]
H
H
4
and 5. Although distances between functional groups are not
N
N
H
N
H
N
H C
optimal, closer contacts are possible in the pyrimidine:purine
system (Figure 5) than in the purine self-pair system (Figure 4).
The 6-methyl-4-pyrimidone nucleoside was modelled in the trans
configuration, while the presence of the 4-carbonyl group would
suggest that the cis configuration is more stable. The models
demonstrate that, in theory, accommodation of both base pairs in
a dsDNA structure might be possible.
3
H
N
H
N
N
CH3
N
N
O
N
N
H
H
N
O
N
N
dR
dR
N
N
N
O
O
N
N
H
N
H
H
dR
dR
H
X
X
(a)
(b)
Figure 2. Schematic representation of a putative self−complementary base pair
of (a) 2-amino-8-oxo-7,8-dihydro-purine (b) 2-amino-6-methyl-8-oxo-7,8-
dihydro-purine (X).
Replacement of G with 8-oxoG increases thermal stability of
duplex when A is placed in the opposite position.[19] The presence
of the 8-oxo group induces glycosidic bond rotation which led to
8
-oxoG(syn):A(anti) conformation.[18b] When 2-amino-8-oxo-7,8-
dihydro-purine is placed opposite to A, thermal stability of the
duplexes (compared with A:T) is decreased.[20] However, 2-
amino-8-oxo-7,8-dihydro-purine:2-amino-8-oxo-7,8-dihydro-
purine self-pair showed less destabilization. The relative stability
of the 2-amino-8-oxo-7,8-dihydro-purine self-pair could be
explained by the formation of two hydrogen bonds (Figure 2a).
A first example that we investigated is the potential self-
complimentary base pair 2-amino-6-methyl-8-oxo-7,8-dihydro-
purine (X) (Figure 2b) in the syn:anti conformation.
Figure 4. Static model showing potential base:base interactions in a 2-amino -
6-methyl-8-oxo-7,8-dihydro-purine (X) self-pair.
CH3
N
S
N
CH3
N
H
N
N
N
NH
CH3
N
O
O
N
HO
N
NH2
HO
HO
O
O
O
OH
OH
OH
X
Y
Z
Figure 3. 2’-Deoxyribonucleosides, with modified purine (X and Y) and
pyrimidine (Z) bases that have been used in this study. For the sake of simplicity,
we use X, Y and Z as code, as well for the base as for the nucleos(t)ide.
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Chemistry - A European Journal
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FULL PAPER
anomeric mixture of 6 as a white solid. Compound 6 was
converted to its dimethoxytrityl derivative 7 by selective protection
of the primary alcohol with 4,4’-dimethoxytrityl chloride (DMTrCl)
in pyridine. Flash column chromatography was used to separate
the desired β-isomer from the α-isomer and ROESY and HMBC
of 2D NMR was used to prove the correct structure. Trityl
deprotection of compound 7 afforded the nucleoside 8. 2D NMR
analysis of compound 8 confirmed the previous structural
analysis. However, amination of 2-chloro of compound 6 using
ammonia under heating conditions was unsuccessful. Therefore,
we decided to replace first the 2-chloro by a 2-azido group, and
to separate the isomers after introduction of the 2-amino function.
CH
3
CH
3
CH
3
H
N
H
N
H
N
N
N
N
O
O
O
HO
O
NH
2
HO
N
N
N
HO
N
N
N
3
HO
N
N
NH
2
O
H
O
O
a
b
c
d
Figure 5. Static model showing potential base:base interactions in the 6-methyl-
α
-
and β-6
4-pyrimidone (Z) and 2-methyl-6-thiopurine (Y) base pair.
α- and β-9
α
-
α- and β-11
-
=
HO
HO
- and β 10
(
β 11 X)
CH
CH
3
CH
3
3
H
N
H
N
H
N
N
N
N
O
O
Result and discussion
N
DMTrO
N
N
N
N
f
DMTrO
O
N
N
N
N
HO
N
N
N
e
O
O
O
α- and β-12
HO
HO
13
O
P
14
The 2-chloro-4-amino-5-nitro-6-methyl pyrimidine
2
was
NC
N
synthesized according to a reported procedure with minor
modifications.[ The nitro group of 2 was reduced using sodium
hydrosulphite to afford 2-chloro-4,5-diamino-6-methyl pyrimidine
23]
Scheme 2. Synthesis of the protected phosphoramidite of 2-amino-6-methyl-8-
oxo-7,8-dihydro-9-(2-deoxy-β-D-erythro-pentofuranos-1-yl)-purine. (a) NH
NH , EtOH, 84 °C, 12 h; (b) NaNO , AcOH, −5 °C - rt, 12 h; (c) H , PtO , MeOH,
55 °C, 45 h, 21% over 3 steps; (d) DMF–DMA, MeOH, rt, 1 h; (e) DMTrCl,
pyridine, 0 °C to rt, 12 h, 58% β anomer; (f) (i-Pr N) POC CN, 1H-tetrazole,
CH Cl , 0 °C to rt, 1 h, 64%.
2
-
3. Reaction of 2-chloro-4,5-diamino-6-methyl- pyrimidine 3 with
1,1’-carbonyldiimidazole in 1,4-dioxane at 110 °C afforded
2
2
2
2
compound 4.
2
2
2 4
H
2
2
CH
3
CH
3
CH
3
CH
3
H
N
O
2
N
ref 23
O
2
2
N
N
a
H
2
2
N
N
b
N
N
N
N
O
N
H
N
Cl
c
Cl
N
Cl
H
N
Cl
H
N
Cl
1
2
3
4
Thus, nucleoside 6 was treated with hydrazine in ethanol to afford
the 2-hydrazino derivative 9 which on further reaction with nitrous
acid at −5 °C, led to formation of 2-azido-6-methyl-8-oxo-7,8-
dihydro-purine nucleoside 10. Reduction of azido group followed
by N,N-dimethylaminomethylidene protection of the resulting
amine function afforded compound 12 which, upon tritylation,
gave a separable mixture of the α,β-anomers 13. The desired β-
anomer was isolated by flash column chromatography and
converted into phosphoramidite 14 by reaction with
bis(diisopropylamino)(2-cyanoethoxy)phosphine in the presence
of tetrazole.
CH
3
CH
3
CH
3
CH
3
H
N
H
N
H
H
N
N
N
N
N
N
O
O
O
O
HO
N
N
Cl
DMTrO
N
N
Cl
HO
N
N
Cl
TolO
N
N
Cl
O
O
O
O
f
e
d
8
HO
7
HO α- and
-
6
OTol α- and β-5
HO
β
Scheme 1. Synthesis of 2-chloro-6-methyl-8-oxo-7,8-dihydro-purine
nucleoside. (a) Na , H O:EtOAc:THF (1:1:3.5), rt, 3h, 39% (b) 1,1'-
2
S
2
O
4
2
carbonyldiimidazole, 1,4-dioxane, 110 °C, 50 min, 86% (c) NaH, 1-chloro-2-
deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranose, 50 °C, 3h, 30%; (d)
NaOMe, MeOH, rt 1h, 95%; (e) DMTrCl, pyridine, 0 °C to rt, 12 h, 58% β isomer;
The triphosphate (of compound β-12) was synthesized according
to the method described by Ludwig[ starting from corresponding
N,N-dimethylaminomethylidene protected nucleoside β-12
24]
2 2
(f) 1.25 M HCl, MeOH:CH Cl (1:1).
(obtained by detritylation of compound 13) (Scheme 3). In this
procedure, regioselective phosphorylation of the 5’-hydroxy group
of the sugar moiety was carried out with phosphoryl oxychloride
in trimethyl phosphate followed by the addition of
tetrabutylammonium pyrophosphate. The reaction product was
deprotected with aqueous ammonia, isolated on Sephadex A25
column and final purification was performed by RP HPLC.
The sodium salt of 4, produced in situ using NaH in acetonitrile
was reacted with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-α-D-
erythro-pentofuranose at 50 °C. The reaction mixture was purified
on silica gel column to afford 2-chloro-6-methyl-8-oxo-7,8-
dihydro-9-(2-deoxy-3,5-di-O-p-toluoyl-D-erythro-pentofuranos-1-
yl)-purine (5) as an inseparable mixture of α and β-anomer in 30%
yield. Treatment of 5 with sodium methoxide in methanol
deprotected the toluoyl groups. The crude product was then
purified by flash column chromatography to yield an inseparable
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Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
CH
3
CH
3
CH
3
H
N
H
N
H
N
phosphoramidite 21 by reaction with bis(diisopropylamino) (2-
N
N
N
O
O
O
cyanoethoxy)phosphine in the presence of tetrazole.
DMTrO
N
N
N
N
HO
N
N
N
N
TPO
N
N
NH
2
O
O
O
R
SH
Cl
N
N
N
N
a
b
N
N
a
c
HO
13
HO β−12
HO
15
TolO
HO
N
N
N
b
CH
3
N
CH3
O
O
Scheme 3. Synthesis of the triphosphate 15 a) 1% TFA on Silica column b) i)
trimethylphosphate, POCl , 0°C, 5 h; ii) Bu N, (NBu HP , 30 min; iii) 25%
NH , 2 h;
N
H
N
CH3
18a; R=Cl
18b;
R=SH
3
3
4
)
3
2 5
O
OTol
OH
Y
3
CN
S
N
CN
CN
S
N
S
N
N
N
N
N
N
N
DMTrO
N
N
CH
3
O
As depicted in scheme 4, glycosylation of commercially available
-methyl-4-pyrimidone with 3,5-bis-(toluoyl)-2-deoxyribosyl
chloride using bis(trimethylsilyl)acetamide and SnCl in dry
HO
DMTrO
CH3
e
N
CH
O
O
3
6
d
f
O
O
P
CN
21
4
OH
OH
N
19
20
acetonitrile afforded a crude mixture of 6-methyl-4-pyrimidone
nucleoside. The cleavage of toluoyl-protecting group was carried
out using 5 M solution of sodium methoxide in methanol to afford
Scheme 5. Synthesis of the protected phosphoramidite of 2-methyl-6-thio-9-
(2-deoxy-β-D-erythro-pentofuranos-1-yl)-purine. (a) NaH, 1-chloro-2-deoxy-3,5-
pure
6-methyl-3-(2-deoxy-β-D-erythro-pentofuranos-1-yl)-4-
pyrimidone Z in 36% yield over two steps. Selective protection of
the primary alcohol with 4,4’-dimethoxytrityl chloride (DMTrCl) in
pyridine gave 16, which was then converted to phosphoramidite
di-O-p-toluoyl-α-D-erythro-pentofuranose, CH
3 2 2 3
CN, rt, 2 h, 76 %; (b) Na S O ,
EtOH-H O, 120 °C, 12 h, 80%. (c) NaOMe, MeOH, 1.5 h, rt, 78%; (d) 3-
2
1
7
by reaction with bis(diisopropylamino)(2-cyanoethoxy)
bromopropionitrile, K
5 %; (f) bis(diisopropylamino)(2-cyanoethoxy)phosphine, tetrazole, CH
C to rt, 2.5 h, 68 %.
2
CO
3
, DMF, rt, 80%; (e) DMTrCl, pyridine, 0 °C to rt, 12 h,
phosphine in the presence of tetrazole.
7
2
Cl , 0
2
°
CH
N
3
CH
N
3
CH
3
N
O
TolO
N
O
O
N
a
TolO
O
b
c
Hybridization properties of XNA modified DNA
HO
O
Cl
OTol
O
N
H
OTol
The oligonucleotides were synthesized by using the
phosphoramidite method and the reagents 14, 17 and 21 on solid
phase employing a DNA synthesizer. The synthesized sequences,
together with the MS data, are shown in Table 1. The
OH
Z
CH
3
CH
N
3
N
O
N
O
DMTrO
N
O
d
oligonucleotides ON 20-23 were used as well for T
in vivo transliteration experiments.
m
studies as for
DMTrO
O
O
O
P
CN
17
OH
N
1
6
Base pairing properties of the X, Y and Z bases were examined
by hybridizing oligomers with their complementary strands and
determining the T
spectroscopy. The T
buffer with KH PO
concentration of 4 µM for each strand. The stability of the
duplexes containing the modified bases (X, Y, Z) was compared
to the stability of the natural DNA duplex (Table 2).
m
of the hybrids by temperature-dependent UV
was determined at 260 nm in NaCl (0.1M)
4
(20 mM, pH 7.5) and EDTA (0.1 mM) at a
Scheme 4. Synthesis of the protected phosphoramidite of 6-methyl-3-(2-deoxy-
-O-DMTr-β-D-erythro-pentofuranos-1-yl)-4-pyrimidone. (a) SnCl BSA,
CH CN, rt, 2 h; (b) NaOMe, MeOH, 1.5 h, rt; (c) DMTrCl, pyridine, 0 °C to rt, 12
h, 69 %; (d) bis-(diisopropylamino)(2-cyanoethoxy)-phosphine, tetrazole,
CH Cl , 0 °C to rt, 2.5 h, 62%.
m
5
4
,
2
3
2
2
The sodium salt of 2-methyl-6-chloropurine, produced in situ
using NaH in acetonitrile, was reacted with l-chloro-2-deoxy-3,5-
di-O-p-toluoyl-α-D-erythro-pentofuranose at room temperature to
afford the desired nucleoside 18a in 76% yield (Scheme 5). The
6-chloro group in 18a was converted into a thiol group by
treatment with sodium thiosulfate. Cleavage of toluoyl protecting
groups by a 5 M solution of sodium methoxide in methanol
(compound Y) followed by reaction with 3-bromopropionitrile in
presence of potassium carbonate gave compound 19. Selective
protection of the primary alcohol with 4,4’-dimethoxytrityl chloride
(DMTrCl) in pyridine gave 20, which was then converted to its
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Chemistry - A European Journal
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FULL PAPER
From this table it is clear that the potential base pairing between
the modified bases X:X, Y:Y, Y:Z and X:Z are very week, if they
exist at all (certainly for Y:Y). For example, the pyrimidone
nucleoside Z is not expected to occur in the conformation as
proposed in Figure 1d. However, the stability Z:X and Z:Y is about
Table 1. DNA sequences with X, Y and Z modified bases that have been
synthesized, together with the MS data.
ON Sequence
Mass
Mass
1
2
3
4
5
6
7
8
9
5’-CAC CGX TGC TAC C-3’
3907.7
4067.7
3908.7
4068.7
4012.7
3096.6
3095.6
5472.0
5671.9
5526.0
5686.0
5526.0
5686.0
5580.0
5700.0
5551,9
5605,9
5605,9
5660,0
5552.9
5607.9
5607.9
5662.9
5496.9
5495.9
5495.9
5494.9
5616.9
5575.9
5575.9
5534.9
7059.5
7745.7
3907.7
4067.9
3908.5
4068.7
4012.8
3096.7
3095.6
5472.1
5671.8
5526.2
5685.9
5526.2
5685.9
5580.1
5699.9
5552,2
5606,2
5606,2
5660,3
5552.8
5607.9
5608.0
5663.0
5496.9
5496.0
5496.2
5495.0
5617.0
5576.0
5576.1
5535.2
7058.8
7745.0
the same. The T
when compared with the T
m
of the duplex is decreased with at least 10 °C
of the canonical base pairs. This
5’-GGT AGC AXC GGT G-3’
m
5’-CAC CGY TGC TAC C-3’
could be due to the difficulty of X, Y and Z to adopt a high energy
syn or anti conformation within the duplex or due to steric
hindrance. The Tm of duplexes with a modified pyrimidine,
5’-GGT AGC AYC GGT G-3’
5’-GGT AGC AZC GGT G-3’
m
modified purine base pair (Y:Z and X:Z) is higher than the T of
5’-d GCG CXX GCG C-3’
duplexes with a potential modified self-complementary purine
base pair (X:X and Y:Y), respecting shape-recognition rules, and
the difficulty for the simultaneous orientation of the X and Y
modified purine bases in the syn and anti conformation. Steric
clash between the hydrophobic substituent within the potential
X:X and Y:Y base pair (despite the encouraging modeling data)
may further explain these results.
5’-GXG CAT GCX C-3’
5’-CTA GCX CCG TGC CAT GCA-3’
5’-p- TGC ATG GCA CGG XGC TAG-3’
5’-CTA GCX CXG TGC CAT GCA-3’
5’-p-TGC ATG GCA CXG XGC TAG-3’
5’-CTA GCX XCG TGC CAT GCA-3’
5’-p-TGC ATG GCA CGX XGC TAG-3’
5’-CTA GCX XXG TGC CAT GCA-3’
5’-p TGC ATG GCA CXX XGC TAG-3’
5’-p-CTA GCX CCG TGC CAT GCA-3’
5’-p-CTA GCX CXG TGC CAT GCA-3’
5’-p-CTA GCX XCG TGC CAT GCA-3’
5’-p-CTA GCX XXG TGC CAT GCA-3’
5’-p-CTA GCY CCG TGC CAT GCA-3’
5’-p-CTA GCY CYG TGC CAT GCA-3’
5’-p-CTA GCY YCG TGC CAT GCA-3’
5’-p-CTA GCY YYG TGC CAT GCA-3’
5’-p-CTA GCZ CCG TGC CAT GCA-3’
5’-p-CTA GCZ CZG TGC CAT GCA-3’
5’-p-CTA GCZ ZCG TGC CAT GCA-3’
5’-p-CTA GCZ ZZG TGC CAT GCA-3’
5’-p-TGC ATG GCA CGG ZGC TAG-3’
5’-p-TGC ATG GCA CZG ZGC TAG-3’
5’-p-TGC ATG GCA CGZ ZGC TAG-3’
5’-p-TGC ATG GCA CZZ ZGC TAG-3’
5’-AAC TGX GTC ATA GCT GTT TCC TG-3’
5’-AAC TGX XXG TCA TAG CTG TTT CCT G-3’
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
In order to investigate the selectivity of base pairing, the stability
of duplexes with modified X, Y, Z bases and the four canonical
bases (A, T, G, C) was investigated by T
m
determination (Table
3). In all cases, the potential base interactions are weak,
especially with the Z base. The potential base pairing with the
canonical bases is, in general, somewhat better than the potential
X:X and Y:Y self-pairing. The highest T in this series is found for
m
Y:A, which might be due to syn:anti interactions (Figure 6).
N
H N
2
N
S
N
N
N
NH
dR
N
H
N
dR
CH
3
(
anti)
(syn)
Y
A
Figure 6. Potential base pairing between the 2-methyl-6-thiopurine base (Y)
and adenine (A) in the Y (anti): A (syn) conformation.
As the strength of duplex formation is not only the result of
potential interstrand base:base interactions by H-bonding but also
due to potential base:base interactions by interstrand stacking,
we have evaluated the X:X and the Y:Z system further by
incorporating several modified X, Y and Z bases in a dsDNA,
either separately or consecutively (Table 4). One of the
sequences is phosphorylated at the 5’-position, because this
sequence was also used for insertion in a plasmid for the in vivo
m
Table 2. T values (in °C) of antiparallel stranded DNA oligonucleotide duplexes
A:, 5’-CAC CG1 TGC TAC C-3’ and B:, 3’-GTG GC2 ACG ATG G-5’ containing
A, T, C, G bases and the modified base pairs X:X, Y:Y, Y:Z and X:Z.
experiments. The T
further reduced duplex stability in comparison to dsDNA reference
duplex (T = 72.9 °C).
m
data shows that multiple incorporations
m
Sequence A
Sequence B
Duplex
1
A
T
C
G
X
Y
Y
X
2
T
A
G
C
X
Y
Z
Z
T
m (°C)
57.5
57.3
61.3
59.7
45.2
41.7
47.3
46.6
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Chemistry - A European Journal
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(Table 5). When positioning X at both ends, the oligonucleotide
m
Table 3. T values (in °C) of antiparallel stranded DNA oligonucleotide duplexes
containing A, T, C, G bases and the modified bases X, Y, Z.
remains in a self-complementary duplex just as the natural
sequence, although with considerably reduced T . However,
positioning XX in the middle of the oligonucleotide, force it to
adopt a hairpin conformation. This is in agreement with former
observations that the X:X base pair is not really formed.
m
Sequence
T
m
Sequence
T
m
(°C)
(°C)
5
’-CAC CGX TGC TAC C-3’
’-GTG GCT ACG ATG G-5’
’-GTG GCA ACG ATG G-5’
’-GTG GCG ACG ATG G-5’
’-GTG GCC ACG ATG G-5’
’-GGT AGC AXC GGT G-3’
’-CCA TCG TTG CCA C-5’
’-CCA TCG TAG CCA C-5’
’-CCA TCG TGG CCA C-5’
’-CCA TCG TCG CCA C-5’
5’-CAC CGY TGC TAC C-3’
3’-GTG GCT ACG ATG G-3’
3’-GTG GCA ACG ATG G-3’
3’-GTG GCG ACG ATG G-3’
3’-GTG GCC ACG ATG G-3’
5’-GGT AGC AZC GGT G-3’
3’-CCA TCG TTG CCA C-3’
3’-CCA TCG TAG CCA C-5’
3’-CCA TCG TGG CCA C-5’
3’-CCA TCG TCG CCA C-5’
3
3
3
3
5
3
3
3
3
49.1
48.2
47.7
47.2
50.8
51.1
47.8
44.7
m
Table 5. Concentration dependent T of DNA after positioning X at different
place.
Sequence
2 µM 4 µM 6 µM 8 µM 12 µM Conformation
5’-GCG CXX GCG C-3’ 64.9
5’-GXG CAT GCX C-3’ 27.7
5’-GCG CAT GCG C-3’ 52.6
64.6 64.5 64.1 64.5
29.0 31.3 36.0 37.8
59.6 60.1 62.3 63.8
Hairpin
Duplex
Duplex
49.3
49.0
48.9
49.4
43.7
45.6
48.3
42.0
m
Table 4. T values (in °C) of antiparallel stranded DNA oligonucleotide duplexes
with single and multiple X, Y and Z incorporated.
T
m
T
m
(°C)
Sequence
Sequence
(°C)
5
’-P-TGC ATG GCA CGG XGC
TAG-3’
’- ACG TAC CGT GCC XCG
ATC-5’
5’-p-CTA GCY CCG TGC CAT
GCA-3’
61.7
51.3
54.7
49.0
63.2
3
3’-GAT CGZ GGC ACG
GTA CGT-p-5’
5
’-P-TGC ATG GCA CXG XGC
TAG-3’
’- ACG TAC CGT GXC XCG
ATC-5’
5’-p-CTA GCY CYG TGC CAT
GCA-3’
54.0
55.7
48.8
3
3’-GAT CGZ GZC ACG
GTA CGT-p-5’
5
’-P-TGC ATG GCA CGX XGC
TAG-3’
’- ACG TAC CGT GCX XCG
ATC-5’
5’-p-CTA GCY YCG TGC CAT
GCA-3’
3
3’-GAT CGZ ZGC ACG
GTA CGT-p-5’
Figure 7. CD spectra of oligonucleotide duplexes. The ellipticity is plotted
versus the wavelength for 2-amino-6-methyl-8-oxo-7,8-dihydro purine (dash
dotted green line) and 2-methyl-6-thiopurine (dashed blue line) containing
oligonucleotides along with control sequences with natural base (solid red line).
Sequence: 5’-CACCGX/YTGCTAC-C-3’ and its complement I 5’-GCTAGCAX/Y
CGGTG-3’. In the reference duplex, the base pair in the position of X/Y is A/T.
5
’-P-TGC ATG GCA CXX XGC
TAG-3’
’- ACG TAC CGT GXX XCG
ATC-5’
5’-p-CTA GCY YYG TGC CAT
GCA-3’
3
3’-GAT CGZ ZZC ACG
GTA CGT-p-5’
Finally, we carried out three additional experiments to further
analyze the properties of the 2-amino-6-methyl-8-oxo-7,8-
dihydropurine (X) base: a) the potential of the X base to induce
hairpin formation; b) a CD experiment to compare the shape of
natural dsDNA with the “X incorporated” dsDNA; c) the potential
of the triphosphate of the X nucleoside to function as substrate for
DNA polymerases.
Figure 7 shows the CD spectra of dsDNA with a single X:X and
Y:Y base pair incorporated in the middle of the sequence. All
duplexes have CD spectra indicative of right-handed B-DNA, with
small variation in peak position and ellipticity. The spectra for the
natural DNA sequence exhibit broad negative peak at 252 nm and
positive long wavelength peak at about 272 nm, typical of right
handed B-DNA. The spectra for sequences with 2-amino-6-
methyl-8-oxo-7,8-dihydro-purine X and 2-methyl-6-thiopurine Y
resemble each other but show some deviation in peak position
and ellipticity from the spectra of natural dsDNA.
The 2-amino-6-methyl-8-oxo-7,8-dihydro-purine nucleosides X
was incorporated in the self-complementary 5’-GCG CAT GCG-
C-3’ DNA sequence as well at the second position from both ends
as in the middle positions and the T
m
was measured at different
concentrations ranging from 2 µM to 12 µM in 1N NaCl at 286 nm
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Chemistry - A European Journal
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Figure 8. Polymerase catalysed incorporation of dNTP’s and nucleotide 15 in a DNA primer-template complex.
The spectra shows a more narrow negative peak at 255 nm and
positive peak at 276 nm with reduced height. The introduction of
modified self–complementary base pair with hydrophobic groups
not only destabilizes the DNA duplex but also results in small
conformational changes.
Recognition of modified bases in vivo
In search for new orthogonal base pairs in vivo, it is important to
know “if and how” the modified bases are functioning as template
for canonical bases in vivo in the presence of cellular
polymerases. To test the propagation of the XNA bases in vivo,
the three bases were introduced separately (by chemical
synthesis) in a sequence 5’-CTA GCG CCG TGC CAT GCA-3’
representing the active site of thymidylate synthase (thyA) of E.
coli (coding for Leu – Ala – Pro – Cys – His – Ala). They were
introduced in the GCG codon (coding for Ala) and in the CCG
codon (coding for Pro) (Table 6).
The enzymatic incorporation of the modified X nucleotides in DNA
-
was tested using Klenow (exo ) and Pol6G12. The Klenow
fragment was chosen because of its potential relevance for in vivo
work in E. coli. The Pol6G12, an evolved enzyme derived from
the thermophilic archaebacterium Thermococcus gorgonarius,
was chosen because of its potential relevance for in vitro work
(XNA gene synthesis and aptamer). First the triphosphate of the
2-amino-6-methyl-8-oxo-7,8-dihydro-9-(2-deoxy-β-D-erythro-
pentofuranos-1-yl)-purine nucleoside was synthesized (15,
Scheme 3). The incorporation of natural dNTP’s and the modified
nucleotide X was compared using a template with one and three
nucleotides incorporated (see experimental section) and a DNA
primer. Using Klenow polymerase in the absence of Mn2 little X
is incorporated against X in the template (Figure 8). Using Klenow
Table 6 Sequence of the synthesized oligonucleotides representing the active
site of thyA of E. coli indicating the different positions () in which the modified
bases (X, Y, Z) were introduced.
Code
Leu
Ala
Pro
Cys
His
Ala
+
ON 16, 20, 24 5’-p- CTA GC CCG TGC CAT GCA
ON 17, 21, 25 5’-p- CTA GC CG TGC CAT GCA
ON 18, 22, 26 5’-p- CTA GC CG TGC CAT GCA
ON 19, 23, 27 5’-p- CTA GC G TGC CAT GCA
-3’
-3’
-3’
-3’
2+
polymerase in the presence of Mn , the incorporation efficiency
significantly increases (Figure 8). Data are similar for 50 µM and
200 µM of the modified nucleotide and using a template with one
or three modified bases. In Figure 8, only data with one modified
base in the template is shown. Similar results are observed using
2+
Pol6G12. In the presence of Mn , the modified nucleotide X is
incorporated against X in the template, although from previous
experiments we know that X:X does not form a stable base pair,
in vitro. We would also like to exclude that incorporation is due to
a template-independent synthesis. In the presence of Mn2+ no
template-independent synthesis is observed using Klenow
polymerase (Figure 8). However, some template-independent
synthesis is observed using Pol6G12 polymerase (although less
than in the presence of the template).
These oligonucleotides were ligated enzymatically in a gapped
vector, transformed in a strain of E. coli (lacking thyA) and living
colonies were analyzed (ratio between bacterial colony numbers
in ± thymidine (dT) media). Only in case a productive DNA
polymerase ternary complex is obtained with the unnatural
nucleotide and a tolerated amino acid substitution is coded by the
targeted codon, living colonies are obtained (as thyA in an
essential enzyme in E. coli). Sequencing gives us information as
which canonical base the modified bases recognize and which
amino acids are inserted in the active protein. In each case, 20
clones were picked out in the dT deficient media and sequenced.
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Chemistry - A European Journal
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As shown in Figure 9, all three bases are very poorly recognized
in vivo, when compared with the positive control. Even after
introduction of only one base, the efficiency of transliteration of
XNA to DNA is reduced to approximately 10 % in all cases. This
percentage is further reduced between 0 % to 2 % when
introducing two or three modified bases. When bars are not visible
system (the obtained results are steered by the kind of amino
acids that are accepted at a certain position in the protein). In the
Ala codon (GCY), however, this does not make a difference as all
four possible codons (GCA, GCT, GCC, GCG) lead to the
incorporation of Ala at this site of the protein (still a bias may exist
based on the preferential use of E. coli for certain codons).
Finally, the 6-methyl-4-pyrimidone nucleoside Z was analysed in
vivo, which gives a more complicated profile. In the Ala codon
(GCZ), the 6-methyl-4-pyrimidone base is mainly recognized as a
pyrimidine base (75 % as the T giving GCT, and 17 % as C giving
GCC) and as G base to a minor extend (8 % as GCG) (Table 7).
When introduced in the first position of the CCG code (ZCG) we
find equal amounts of the TCG code (Ser) and CCG code (Pro)
which means that 6-methyl-4-pyrimidone behaves as a pyrimidine
base and the Ser substitution is well tolerated in thymidylate
synthase (Table 8). The same amino acids (Ser and Pro) are
coded for when the modified base is introduced at the first and
second position (ZZG). When introduced only at the second
position (CZG), the pyrimidone base is still recognized in majority
as pyrimidine base (CCG), but also as G base in minority (CGG)
coding for Arg. When three modified bases are introduced in a
row (GCZ ZZG), 58 % of Ala-Ser is introduced (GCT TCG) and
42 % of Ala-Pro (17 % GCG CCG, 17 % GCC CCG, 8 % GCT
CCG) (data not shown in Table 8).
(Figure 9), only 1 or 2 colonies could be obtained, except for Y (2-
methyl-6-thiopurine), inserted in GC() C()G, where no colony
was obtained. These modified bases come close to the definition
of orthogonal bases.
2-Amino-6-methyl-8-oxo-7,8-dihydro-purine, when introduced as
third base in the Ala codon GCG (GCX) is recognized as a purine
base (84 % as GCG and 16 % as GCA) (Table 7). Both triplets
code for the amino acid “Ala”. When 2-amino-6-methyl-8-oxo-7,8-
dihydro-purine base is introduced as first or second base in the
Pro codon (XCG and CXG), it is always (100 %) recognized as a
cytosine base (CCG and CCG), coding for Pro (Table 8). This
could be explained by X is recognized by G in a Wobble-
Hoogsteen type association. These data are obtained using ON
1
7 and ON 18 where X is also incorporated in the Ala codon which
might give some bias for base incorporation, especially for XCG
ON 18) It should be mentioned that not only CCG (coding for Pro)
but also TCG (coding for Ser), GCG (coding for Ala) and CGG
coding for Arg) are tolerated at this position of the ThyA gene.
(
(
However, we cannot conclude on the real impact of the individual
amino acid substitutions on the enzyme efficiency. This
observation is of interest as it shows that selection pressure
It is difficult to make general statements about the preferred
recognition of modified purine and modified pyrimidine bases at
this moment. Shape recognition (purine-pyrimidine base pair
formation) is, however, important. The Z base is recognized as a
pyrimidine in 90% of the cases (and only in 8-10% as a purine).
Transversions coming from a purine-purine base pair are
observed in relatively high yield with the X and Y base, probably
because of the syn-anti equilibrium that may convert a purine to a
pyrimidine-like base.
(invariably Ala-Pro is selected) in this case is higher than
structural constrains. The modified base can behave either as a
purine or either as a pyrimidine analogue in vivo dependent on
the place where it is inserted in the gene. Needless to say that
there is no correlation with the results obtained by thermal stability
experiment of the modified oligonucleotides (see previous), where
hybridization of 2-amino-6-methyl-8-oxo-7,8-dihydro-purine
X
with A,G,C,T is very poor, if existing at all, and similar for all 4
canonical bases. Clearly, E.coli DNA polymerases have a key role
in the selection procedure via stabilization of the protein-dsDNA-
dNTP ternary complex.
Table 7 - In vivo recognition of modified bases by canonical bases in the GCG
(Ala) codon (aa: amino acid)
5’-
CTA GC CCG TGC CAT GCA-3’
Leu aa Pro Cys His Ala
The 2-methyl-6-thiopurine base Y, when incorporated in the GCG
(
Ala) codon at the third position (GCY) is recognized almost
exclusively (99 %) as a G (GCG) and in 1 % of the cases as a T
GCT) (Table 7). Both triplets code for Ala. When an extra
aa
X (%)
Y (%)
Z (%)
A
T
Ala
Ala
Ala
Ala
16
0
0
1
0
75
17
8
(
modified base is introduced in the CCG (Pro) codon at the first
position (YCG), it is recognized for 93 % as G base (GCG) coding
for Ala and for 7 % as C base (CCG) coding for Pro (Table 8).
When 2-methyl-6-thiopurine base is incorporated in the second
position of the same codon (CYG) it is recognized as a cytosine
base (CCG) in 100 % of the cases (Table 8). When introduced as
well in the first as the second position (ON 23) of this CCG code
C
G
0
0
84
99
X 2-amino-6-methyl-8-oxo-7,8-dihydro-purine
Y 2-methyl-6-thio-purine
(YYG), the codon only codes for Ala giving GCG as codon, and
Z 6-methyl-4-pyrimidone
not anymore for Pro (data not shown in Table 8). It thus seems
that the presence of one modified base in a codon may influence
the recognition pattern of a second modified base, introduced in
the same codon. Structural constrains seem more challenging
than the selection pressure which leads to the high substitution of
Pro by Ala (93 %). It should be remembered, of course, that only
viable codons can be detected which is a bias of the analytical
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Chemistry - A European Journal
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demonstrated with the T :GS base pair[16] base pairing between
methylated bases or bases with a thiocarbonyl group might easier
occur when a H-atom is present in the opposite base (Figure 1c).
However, the latter bases might not be really orthogonal.
Surprisingly, some selected polymerases are still able to
H
Table 8 - In vivo recognition of modified bases by canonical bases in the GCG
(Pro) codon (aa: amino acid)
5
’-GTA GC CG TGC CAT GCA-3’
5’-CTA GC CG TGC CAT GCA-3’
Leu Ala aa Cys His Ala
CG
Leu Ala aa Cys His Ala
incorporate
the
2-amino-6-methyl-8-oxo-7,8-dihydro-purine
CG
nucleoside opposite itself in a template. In vivo, 2-amino-6-
methyl-8-oxo-7,8-dihydro-purine is mainly recognized as a A/G or
C base. 2-Methyl-6-thio-purine is recognized as a G or C base
aa
X
Y
Z
aa
X (%)
Y (%)
Z (%)
(%)
(%)
(%)
(
and in a minor case as T base). 6-Methyl-4-pyrimodone is in vivo
A
T
Thr
Ser
Pro
Ala
0
0
0
Glu
Leu
Pro
Arg
0
0
0
mostly recognized as T, C but sometimes also as a G base.
Although principles like shape recognition (purine-pyrimidine
base pair formation) plays a role in the recognition process, it is
not the only factor involved. The low efficiency of the
transliteration process of XNA to DNA might implement some
degree of randomness in the nucleotide selection procedure by
the incorporated modified (XNA) nucleotide, when pyrimidine
heterocycles (smaller heterocycles) are involved.
0
0
50
50
0
0
0
0
C
G
100
0
7
100
0
100
0
90
10
93
X 2-amino-6-methyl-8-oxo-7,8-dihydro-purine
Y 2-methyl-6-thio-purine
Z 6-methyl-pyrimidone
Experimental Section
1
1
1
4
2
0
8
6
4
2
0
1H, 13C and P NMR spectra were recorded on 300 MHz ( H NMR, 300
31
1
MHz; 3C NMR, 75 MHz; 31P NMR, 121 MHz) or 500 MHz ( H NMR, 500
1
1
MHz; C NMR, 125 MHz) or 600 MHz ( H NMR, 600 MHz; 13C NMR, 150
MHz) spectrometers. 2D NMRs (H-COSY, HSQC and HMBC) were used
for the assignment of all the intermediates and final compounds. Mass
spectra were acquired on a quadrupole orthogonal acceleration time-of-
flight mass spectrometer. Column chromatographic separations were
carried out by gradient elution with suitable combination of two/three
solvents and silica gel (100–200 mesh or 230–400 mesh). Solvents for
reactions were distilled prior to use: THF and toluene from
13
1
2 2 3 2 3
Na/benzophenone; CH Cl and CH CN from CaH ; Et N and pyridine from
KOH.
GC● CCG
GC● C●G
GC● ●CG
GC● ●●G
Figure 9. The in vivo propagation of the mosaïc DNA-XNA-DNA
oligonucleotides. 2-amino-6-methyl-8-oxo-7,8-dihydro-purine modified
2-Chloro-4,5-diamino-6-methyl pyrimidine (3). A solution of Na
2 2 4
S O
(
28.67 g, 164.6 mmol) in water (78 mL) was added to the stirred solution
of 2 (10.35 g, 54.9 mmol) in THF (274 mL) and ethyl acetate (78 mL) at
room temperature and the reaction was stirred for 3 hours. Ethyl acetate
oligonucleotides are represented in blue, 2-methyl-6-thio-purine modified
oligonucleotides are represented in red and 6-methyl-4-pyrimidone modified
oligonucleotides are represented in green. Ratios are normalized against
positive control (100%) and correspond to the experimentally derived number
of thymidine-prototrophic colonies (thyA+) from the total number of colonies.
(
2 4
200 mL) was added and extracted. The extract was dried over Na SO
and concentrated in vacuo. The crude mixture was purified by column
chromatography using DCM:MeOH (9:1, v/v) as solvent to afford 3 (3.42
g, 39%). Analytical data of compound 3 was found to be identical to the
reported compound.[25]
2
-Chloro-6-methyl-8-oxo-7,8-dihydro-purine (4).
1,1'-Carbonyl
Conclusions
diimidazole (7.00 g, 43.2 mmol) was added to the solution of 2-chloro- 4,5-
diamine-6-methyl-pyrimidine 3 (3.42 g, 21.6 mmol) in 1,4-dioxane (10 mL)
and stirred at 110 °C under nitrogen for 50 min. Solvents were evaporated
An efficient synthetic scheme was developed for the synthesis of
the protected phosphoramidites of 2’-deoxynucleosides with a 2-
amino-6-methyl-8-oxo-7,8-dihydro-purine (X),
thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety.
These building blocks were successfully used for oligonucleotide
synthesis. These modified bases do not form base pairs with itself
to obtain a yellow oil and CH
vigorously and allowed to settle the precipitate overnight. Solids were
filtered through sintered funnel, washed with CH Cl and dried under
vacuum at 45 °C to afford 4 (3.29, 83%). 1H NMR (300 MHz, DMSO-d
). 13C NMR
δ 12.00 (br s, 1H, NH), 11.39 (br s, 1H, NH), 2.37 (s, 3H, CH
2 2
Cl (150 mL) was added. The flask shaken
a
2-methyl-6-
2
2
6
)
3
(
75 MHz, DMSO-d
120.3 (C5), 18.7 (CH
185.0224; found 185.0219.
6
) δ 153.7 (C6), 151.9 (C8),149.9 (C2), 144.2 (C4),
). HRMS (ESI+): calcd for C H ClN O [M + H]
3 6 5 4
+
(X:X and Y:Y) and with the natural bases (A, G, C, T). Substitution
of a polar functional group (NH , OH) of nucleobases by a methyl
2
group at positions that are normally involved in H-bonding of
canonical bases (the 6-carbonyl group of pyrimidine base, the 2-
amino and 6-carbonyl group of purine base) readily leads to
orthogonality (absence of base pairing with canonical bases). As
2-Chloro-6-methyl-8-oxo-7,8-dihydro-9-(2-deoxy-α/β-D-erythro-
pentofuranos-1-yl)-purine (6). To a suspension of 4 (3.42 g. 18.5 mmol)
in dry CH CN (142 mL) was added sodium hydride (60% in oil, 0.489 g,
3
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Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
2
0.4 mmol) and the mixture was stirred at room temperature under a
crude unstable hydrazine derivative α- and β-9 was used for next reaction
immediately. To an ice-cold solution of crude compound α- and β-9 (3.49
mmol) in acetic acid (23 mL) was added dropwise a solution of sodium
nitrite (53.16 mol) in a least amount of water in an ice bath at −5 °C. The
reaction mixture was allowed to stir at room temperature for 12 hrs and
concentrated in vacuo to afford inseparable anomeric mixture of
nucleoside α- and β-10. To the solution of crude nucleoside α- and β-10
nitrogen atmosphere for 30 min. 1-Chloro-2-deoxy-3,5-di-O-p-toluoyl-D-
erythro-pentofuranose (7.20 g, 18.5 mmol) was added portion wise with
stirring. The reaction mixture was stirred at 50 °C for 3 h and filtered to
remove a small amount of insoluble material. Evaporation of the filtrate
gave an oily residue, which was purified by silica gel column
chromatography using ethyl acetate (35%) in hexane to afford nucleoside
α- and β-5 (3.00 g, 30%). HRMS (ESI+): calcd for C27
H25Cl
1
4
N O
6
[M + H]+
2
(3.49 mmol) in methanol was added PtO (0.71 mmol) and reaction mixture
5
37.1535, found 537.1541.The anomeric mixture (1.50 g, 2.79 mmol) was
was stirred under hydrogen atmosphere (1.38 bar) at 55 °C for 45 hrs. The
reaction mixture was filtered through the celite and concentrated under
vacuo to afford the anomeric mixture of 2-Amino-6-methyl-8-oxo-9-(2’-
deoxy-D-ribofuranosyl)-purine α- and β-11 (X), HRMS (ESI+): calcd for
treated with 5 M solution of sodium methoxide (1.05 mL) for 2 h at room
temperature. The mixture was concentrated and the residue was purified
by flash column chromatography (CH Cl /MeOH 8.5:1.5) to yield the
2 2
anomeric mixture of compound α- and β-6 as a white solid (0.798 mg, 95
%
11 15 5 4
C H N O
[M + H]+ 282.1196; found 282.1199. The crude nucleoside
+
). HRMS (ESI+): calcd for C11
H13Cl
N
1 4
O
4
[M + H] 301.0698, found
α- and β-11 (0.860 mg, 3.06 mmol) was dissolved in methanol (30 mL) and
dimethylformamide dimethyl acetal (15 ml) was added. Reaction mixture
was stirred for 1 hour and concentrated on rotary evaporator to afford
3
01.0707.
dimethylformamidine protected nucleoside diol α- and β-12, HRMS (ESI+):
2
-Chloro-6-methyl-8-oxo-7,8-dihydro-9-(2-deoxy-5-O-dimethoxytrityl-
+
20
calcd for C14H N
6
O
4
[M + H] 337.1618; found 337.1621.
β-D-erythro-pentofuranos-1-yl)-purine (7). Compound α- and β-6 (320
mg, 1.41 mmol) was co-evaporated with dry pyridine twice under argon
atmosphere and then dissolved in dry pyridine (15 mL). 4,4'–
2-N,N-dimethylformamidino-6-methyl-8-oxo-7,8-dihydro-9-(2-deoxy-
5-O-dimethoxy trityl-β-D-erythro-pentofuranos-1-yl)-purine (13).
2 2
Dimethoxytrityl chloride (527 mg, 1.56 mmol) in CH Cl (2 mL) was slowly
added drop wise under argon atmosphere at 0 °C, then the mixture was
stirred at room temperature for 2 h. The reaction was quenched by the
addition of MeOH and the solvents were evaporated. The residue was
Compound α- and β-12 (444 mg, 1.32 mmol) was co-evaporated with dry
pyridine twice under argon atmosphere and then dissolved in dry pyridine
(26 mL). 4,4'–Dimethoxytrityl chloride (447 mg, 1.35 mmol) in CH Cl (2
2 2
dissolved in CH
on Na SO and evaporated under argon atmosphere. The compound 7
was isolated as anomer by column chromatography separation
CH Cl
/MeOH/TEA 97:2:1) as a white foam (372 mg, 58%). 1H and 13
2
Cl
2
and washed with H
2
O, the organic layers were dried
mL) was slowly added drop wise under argon atmosphere at 0 °C, then
the mixture was stirred at room temperature for 3 h. The reaction was
quenched by the addition of MeOH and the solvents were evaporated. The
2
4
β
(
2
2
C
2 2 2
residue was dissolved in CH Cl and washed with H O, the organic layers
NMR Spectrum of compound 7 shows that the compounds contain small
were dried on Na SO and evaporated under argon atmosphere. The
2
4
amounts of trimethylamine and pyridine as compound stabilizer used
compound 13 was isolated as beta anomer by column chromatography
1
during synthesis and purification. These impurities were removed at the
2
(CH Cl
2
/MeOH/Pyridine 96:3:1) as a white solid (360 mg, 43%). H NMR
1
next step of synthesis. H NMR (500 MHz, DMSO-d
6
) δ 11.87 (s, 1H, NH),
(600 MHz, DMSO-d
6
) δ 11.16 (s, 1H, NH), 8.29 (s, 1H, =CH-N), 7.32-7.31
7
4
.38 -7.33 (m, 2H, Ar), 7.22-7.13 (m, 7H, Ar), 6.74 (dd, J = 32.3, 9.0 Hz
H, Ar), 6.18 (dd, J = 7.6, 5.35 Hz, 1H, H-1’), 5.31 (d, J = 3.0 Hz, OH), 4.45
), 3.69 (s, 3H,
), 3.29 (dd, J = 10.1, 7.5 Hz, 1H, H-5a’), 3.11 (dd, J = 10.1, 3.8 Hz,
), 2.19-2.14 (m, 1H,
) δ 148.0, 147.9, 142.2, 140.0, 139.7,
(m, 2H, Ar), 7.20-7.14 (m, 7H, Ar), 6.77-6.70 (m, 4H, Ar), 6.20 (dd, J = 5.2,
7.74 Hz, 1H, H1’), 5.24 (d, J = 4.8 Hz,1H, OH), 4.49-4.45 (m, 1H, H3’),
(
br S, 1H, 3’H), 3.96-3.93 (m, 1H, H-4’), 3.70 (s, 3H, OCH
3
3 3
3.89-3.86 (m, 1H, H4’), 3.70 (s, 3H, OCH ), 3.69 (s, 3H, OCH ), 3.25 (dd,
OCH
3
J = 10.0, 7.5 Hz, 1H, H5a’), 3.09 (dd, J = 10.0, 3.54 Hz, 1H, H5b’), 3.00 (s,
1
2
1
1
H, H-5b’), 2.91-2.85 (m, 1H, H-2a’), 2.41 (s, 3H, CH
b’). 13C NMR (125 MHz, DMSO-d
3
3H, NCH
3
3 3
), 3.01-2.97 (m, 1H, H2a’), 2.96 (s, 3H, NCH ), 2.30 (s, 3H, CH ),
13
6
6
2.11 (ddd, J = 13.2, 8.0, 5.4 Hz 1H, H2b’). C NMR (151 MHz, DMSO-d )
39.6 (C2, C4, C8, C6, Ar), 135.4, 135.1, 126.2, 125.8, 119.8, 119.7,
17.7, 117.6, 116.5, 113.9, 109.37, 102.9, 102.8 (C5, Ar), 75.9 (C4’), 71.1
δ 160.4 (C2), 158.0, 157.9 (OCH
145.2 (C6), 144.1, 135.8, 129.8, 129.6,127.8, 127.7, 126.5 (Ar), 114.9
(C5), 113.0, 112.9 (Ar), 85.5 (C-Ar ), 85.3 (C4’), 80.5 (C1’), 71.2 (C3’), 64.8
(C5’), 55.1, 55.0 (OCH ), 40.3 (C2’), 36.1, 36.4 (NCH ), 19.1 (CH ). HRMS
ESI+) calcd for C35 [M+H]+ 639.2925 found 639.2927.
3
), 156.9 (C=N), 152.6 (C8), 149.8 (C4),
(
C1’), 61.1 (C3’), 54.6 (C-5’), 45.0, 44.9 (OCH
3
), 26.1 (C2’), 8.9 (CH
[M + H] 603.2004; found 603.2009.
3
).
3
+
HRMS (ESI+): calcd for C32
H31ClN
4
O
6
3
3
3
(
38 6 6
H N O
2
-Chloro-6-methyl-8-oxo-7,8-dihydro-9-(2-deoxy-β-D-erythro-
pentofuranos-1-yl)-purine (8). Compound 7 (0.431 mmol, 0.26 g) was
dissolved in a mixture of CH Cl and MeOH (1:1 v/v, 25 mL), and HCl in
MeOH (0.14 mL of 1.25 mol L solution) was added portion wise. The
reaction was monitored by TLC CH Cl /MeOH(95:5). Once completed, the
2-N, N-dimethylformamidine-6-methyl-8-oxo-9-(2’-deoxy-β-D-erythro -
pentofuranos-1-yl)-purine (β-12): The column of silica gel (10.0 g) was
first prepared in dichloromethane containing 1% pyridine. This column was
overlayed with a small buffer of sand followed by more silica gel (3.0 g) in
dichloromethane containing 1% TFA. The compound 13 (100 mg) was
2
2
–1
2
2
reaction mixture was neutralized with pyridine (1 mL), and the liquid was
concentrated. The resulting oil was purified by column chromatography
2 2
then loaded in 3 mL of CH Cl . The column was eluted with gradient
1
2
CH Cl
2
/MeOH (96:4) to afford 8 (54 mg 70%). H NMR (500 MHz, MeOD-
solvent system CH
2
Cl
2
/MeOH/Pyridine. The compound β-12 was eluted
1
d
3
4
4
) δ 6.30 (dd, J = 7.5, 6.9 Hz, 1H, H1’), 4.59-4.57 (m, 1H Hz, H3’), 3.99-
.96 (m, 1H, H4’), 3.81 (dd, J = 12.1 Hz, 3.8, 1H, H5a’), 3.70 (dd, J = 12.0,
.8 Hz, 1H, H5b’), 2.99 (ddd, J = 13.7, 7.5, 6.1 Hz, 1H, 2a’), 2,46 (s, 3H,
with CH Cl
2
2
/MeOH/Pyridine (96:3:1). H NMR (500 MHz, DMSO-d
6
) δ
11.35 (s, 1H, NH), 8.54 (s, 1H, =CH-N), 6.18 (t, J = 7.4 Hz, 1H, H1’), 5.22
(d, J = 4.0 Hz,1H, OH), 4.89 (t, J = 7.4 Hz, 1H, OH), 4.42 (d, J = 2.7 Hz,
1H, H3’), 3.79 (dd, J = 7.4, 4.65 Hz, 1H, H4’), 3.60 (dt, J = 11.9, 4.0 Hz,
CH
DMSO-d
7.7 (C4’), 82.2 (C1’), 71.7 (C3’), 62.5 (C5’), 36.3 (C2’), 17.5 (CH
3
), 2.20 (ddd, J = 13.5, 6.7, 3.0 Hz, 1H, H-2b’). 13C NMR (125 MHz,
) δ 152.7 (C8), 150.5 (C6), 150.2 (C4), 145.9 (C2), 119.3 (C5),
). HRMS
[M + H] 301.0698; found 301.0707.
6
1H, H5b’), 3.49-3.44 (m, 1H, H5a’), 3.15 (s, 3H, NCH
3.03-2.97 (m, 1H, H2a’), 2.34 (s, 3H, CH ), 2.01 (ddd, J = 13.0, 6.7, 2.9
Hz, 1H, H2b’). 13C NMR (125 MHz, DMSO-d
) δ 158.4 (C=N), 154.3 (C6,
C2), 151.8 (C8), 145.9 (C4), 117.1 (C5), 89.4 (C4’), 83.0 (C1’), 73.1 (C3’),
3 3
), 3.03 (s, 3H, NCH ),
8
3
3
+
(
ESI+): calcd for C11
H
13ClN
O
4 4
6
6
4.2 (C5’), 42.8 (NCH
3
) 37.6 (C2’), 36.8 (NCH
3
), 20.8 (CH
3
). HRMS (ESI+)
2
-N,
N-dimethylformamidine-6-methyl-8-oxo-9-(2’-deoxy-α/β-D-
+
calcd for C14
20
H N
6
O
4
[M+H] 337.1618, found 337.1617.
erythro-pentofuranos-1-yl)-purine 12: To the solution of anomeric
mixture of nucleoside 6 (1.05 gm, 3.49 mmol) in ethanol (10 mL) was
added hydrazine (1.36 mL, 34.92 mmol, 80%). Reaction mixture was
stirred at 84 °C for 12 hrs. The reaction mixture was concentrated and the
3’-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite of 2-N,N-dimethyl
formamimidino-6-methyl-8-oxo-7,8-dihydro-9-(2-deoxy-5-O-
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Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
dimethoxytrityl-β-D-erythro-pentofuranos-1-yl)-purine (14). To
a
DMSO-d
6
) δ 8.41 (s, 1H, H2), 6.19 (s, 1H, H5), 6.16 (d, J = 5.9 Hz,
stirred solution of 13 (350 mg, 1.41 mmol) and
bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.32 mL, 1.32 mmol) in
anhydrous CH Cl (10 mL), under argon atmosphere and at 0 °C, was
1
M
solution of
1H, H1’), 4.31 (t, J = 4.3 Hz, 1H, H4’), 4.25 (d, J = 5.5 Hz, 1H, H3’),
3.49 – 3.36 (m, 2H, H5), 2.61 – 2.53 (m, 1H, H2b’), 2.18 (s, 3H, CH ),
3
1.95 (d, J = 14.2 Hz, 1H, H2a’); 13C NMR (126 MHz, DMSO) δ 163.7
2
2
added 0.45 M solution of 1H-terazole (1.62 mL, 0.728 mmol) dropwise.
After 10 min, the ice bath was removed and reaction mixture was allowed
to stir at room temperature for 45 min. The reaction mixture was diluted
(C4), 160.4 (C6), 148.1 (C2), 111.6 (C5), 90.6 (C4’), 86.8 (C1’), 71.1
(C3’), 62.4 (C5’), 41.0 (C2’), 23.4 (CH
3
); HRMS (ESI+) calcd for
+
C H N O [M+Na] 249.08460, found 249.0853.
10 14 2 4
with CH
dried over Na
purified by flash column chromatography (Hexane/Acetone/TEA, 80:20:1)
to afford amidite 14 (300 mg, 62% yield). 31P NMR (121 MHz, DMSO-d
2
Cl
2
and was washed with 1M TEAB solution. The extracts were
2
SO and concentrated in vacuo. The crude mixture was
4
6
1
-Methyl-3-(2-deoxy-5-O-dimethoxytrityl- β-D-erythro-pentofuranos-
-yl)-4(3H)-pyrimidone (16). Compound Z (320 mg, 1.41 mmol) was co-
6
)
evaporated with dry pyridine (2 x 10 mL) under argon atmosphere and then
dissolved in dry pyridine (15 mL). 4,4'–Dimethoxytrityl chloride (527 mg,
55 8 7 1
δ 147.79, 147.41; HRMS (ESI+) calcd for C44H N O P [M+H]+ 839.4003,
found 839.4009.
1
2 2
.56 mmol) in CH Cl (2 mL) was slowly added drop wise under argon
atmosphere at 0 °C, then the mixture was stirred at room temperature for
2 h. The reaction was quenched by the addition of MeOH and the solvents
2
-Amino-6-methyl-8-oxo-7,8-dihydro 8-9-(2-deoxy-5-O-triphosphate-
β-D-erythro-pento-furanos-1-yl-purine (15). To an ice-cold solution of
protected nucleoside β-12 (30 mg, 0.09 mmol) in trimethyl phosphate
were evaporated. The residue was dissolved in CH
O, the organic layers were dried on Na SO and evaporated under
argon atmosphere. Compound 16 was isolated by column
chromatography (CH Cl /MeOH/TEA 97:2:1) as a white solid (520 mg,
69%). 1H NMR (500 MHz, DMSO-d
) δ 8.40 (s, 1H, H2), 7.38 (d, J = 7.4
Hz, 2H, Ar), 7.33 – 7.19 (m, 7H, Ar), 6.88 (d, J = 8.4 Hz, 4H, Ar), 6.27 –
6.22 (m, 2H, H1’, H5), 5.34 (d, J = 4.6 Hz, 1H, OH), 4.26 (dq, J = 9.0, 4.5
3
Hz, 1H, H3’), 3.99 (dd, J = 8.3, 4.5 Hz, 1H, H4’), 3.74 (s, 6H, 2CH ), 3.24
(dd, J = 10.5, 5.5 Hz, 1H, H5’b), 3.20 (dd, J = 10.5, 3.1 Hz, 1H, H5’a), 2.34
(ddd, J = 13.4, 6.5, 4.5 Hz, 1H, H2b’), 2.22 (dd, J = 13.3, 6.6 Hz, 1H, H2a’),
3 6
2.18 (s, 3H, CH ) δ 163.5 (C4), 159.8,
); 13C NMR (126 MHz, DMSO-d
158.1 (C6, Ar), 147.3 (C2), 144.8, 135.5, 135.4, 129.8, 127.9, 127.7, 126.8
(Ar), 113.3 (Ar), 111.6 (C5), 86.0 (C4’), 85.8 (C), 84.6 C1’), 70.2 (C3’), 63.6
2 2
Cl and washed with
H
2
2
4
(
TMP) (1.0 mL) was added phosphoryl chloride (12 µL, 0.13 mmol.) and
the solution stirred at 0°C for 5 hours. Tributylamine (105 µL, 0.44 mmol)
and tetrabutylammonium pyrophosphate solution (240 mg, 0.27 mmol) in
DMF (1 mL) was added simultaneously, and the solution stirred for a
further 30 minutes. The reaction was then quenched by the addition of 0.5
M triethylammonium bicarbonate (TEAB) buffer (10 mL), and stored at 4°C
overnight. The solvent was evaporated and the residue was treated with
2
2
6
2
5% ammonia (1 mL) for 15 min. The solution was evaporated to dryness
and re-dissolved in water (5 mL) and applied to a Sephadex A25 column
in 0.1 M TEAB buffer. The column was eluted with a linear gradient of 0.1-
1
.0 M TEAB. Appropriate fractions were pooled and evaporated to dryness
to give desired product. The final purification was performed by RP HPLC
(C5’), 55.0 (CH
3
), 40.7 (C2’), 23.1 (CH
3
); HRMS (ESI+) calcd for
+
(
Alltima 5µ C-18 reverse phase column, buffer A, 0.1 M TEAB; buffer B,
31
C H
32
N
O
2 6
[M+Na] 551.2152, found 551.2151.
31
0
.1 M TEAB, 50% MeCN. 0% to 100% buffer B over 60 min). P NMR
(
(
C
2
D O, δ, ppm): -6.38 (1P-γ, d, J 20 Hz), -11.91 (1P-α, d, J 20 Hz), -22.64
The 3’-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite of 6-methyl-
1P-β, t,
J
20 Hz). ESIHRMS found: (M-H)– 520.0041. calcd for
3
4
’-(2-deoxy-5-O-dimethoxytrityl-β-D-erythro-pentofuranos-1-yl)-
(3H)-pyrimidone (17). To a stirred solution of 16 (350 mg, 1.41 mmol)
–
11
H
18
N
O
5 13
3
P : (M-H) 520.0041.
and 1 M solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.32
mL, 1.32 mmol) in anhydrous CH Cl (10 mL), under argon atmosphere
and at 0 °C, was added 0.45 M solution of 1H-terazole (1.62 mL, 0.728
mmol) dropwise. After 10 min, the ice bath was removed and reaction
mixture was allowed to stir at room temperature for 45 min. The reaction
6
-Methyl-3-(2-deoxy-β-D-erythro-pentofuranos-1-yl)-4(3H)-
pyrimidone (Z). To a stirred solution of 6-methyl-4-pyrimidone (637 mg,
.79 mmol) in acetonitrile (25 mL) at room temperature under argon was
2
2
5
added bis(trimethylsilyl)acetamide (1.18 g, 5.79 mmol). After stirring at
ambient temperature for 40 min, 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-D-
mixture was diluted with CH
The extracts were dried over Na
2
Cl
2
and was washed with 1M TEAB solution.
SO and concentrated in vacuo. The
erythro-pentofuranose (1.5 g, 3.86 mmol) and dropwise SnCl
.86 mmol) was added. After 1 h, complete dissolution of the 2-
deoxyribofuranoside had occurred. To the reaction was added EtOAc, and
the resulting solution was successively extracted with saturated NaHCO
and brine. The organics were dried over anhydrous Na SO , and solvents
4
(3.87 mL,
2
4
3
crude mixture was purified by flash column chromatography
(Hexane/Acetone/TEA, 80:20:1) to afford amidite 17 (300 mg, 62% yield).
3
1
3
P NMR (121 MHz, DMSO-d
6
) δ 147.75, 147.37; HRMS (ESI+) calcd for
+
2
4
C
40
H
49
N
4 7
O P [M+Na] 751.3231, found 751.3222.
were removed in vacuo. Purification via flash column chromatography on
silica gel (30-50% ethyl acetate in hexanes) afforded a mixture of two
anomers. The anomeric mixture was treated with 5 M solution of sodium
methoxide (0.650 mL) for 2 h at room temperature. The mixture was
concentrated and the residue was purified by flash column
2
-Methyl-6-chloro-9-(2-deoxy-3,5-di-O-p-toluoyl-β-D-erythro-
pentofuranos-1-yl)-purine (18a). A mixture of 6-chloro-2-methylpurine
520 mg, 3.09 mmol) and 60% sodium hydride (164 mg, 4.11 mmol) in
[26]
(
anhydrous acetonitrile (15 mL) was stirred at ambient temperature for 30
minutes under a nitrogen atmosphere. The 1-chloro-2-deoxy-3,5-di-O-p-
toluoyl-D-erythro-pentofuranose (800 mg, 2.06 mmol) was added
portionwise, and stirred for 2 hours. The reaction mixture was diluted with
ethylacetate and washed with water. The organics were dried over
chromatography (CH
2
Cl
2
/MeOH 8.5:1.5) to yield the title compound Z as a
) δ 8.63 (s,
H, H2), 6.25 – 6.11 (m, 2H, H5,H1’), 5.27 (d, J = 4.2 Hz, 1H, OH),
.07 (t, J = 5.1 Hz, 1H, OH), 4.27 (dq, J = 7.5, 3.8 Hz, 1H, H3’), 3.87
white solid (315 mg, 36 %). 1H NMR (600 MHz, DMSO-d
6
1
5
(
q, J = 3.6 Hz, 1H, H4’), 3.64 (ddd, J = 11.9, 5.0, 3.7 Hz, 1H, H5b’),
2 4
anhydrous Na SO , and solvents were removed in vacuo. Purification via
3
3
.57 (ddd, J = 11.9, 4.7, 4.1 Hz, 1H, H5a’), 2.29 (ddd, J = 13.3, 6.2,
.8 Hz, 1H, H2b’), 2.19 (s, 3H, CH
), 2.12 – 2.08 (m, 1H, H2a’); 13C
) δ l163.4 (C4), 159.9 (C6), 147.7 (C2),
11.3 (C5), 88.0 (C4’), 84.4 (C1’), 70.1 (C3’), 60.9 (C5’), 41.1 (C2’),
3.1(CH ); HRMS (ESI+) calcd for C10
[M+Na]+ 249.0846,
column chromatography on silica gel (30% ethyl acetate in hexanes)
afforded the desired product 18a as a white solid compound (815 mg,
3
NMR (151 MHz, DMSO-d
1
2
6
6%). 1H NMR (600 MHz, CDCl
H, Ar-H), 7.54 (d, J = 8.0 Hz, 2H, Ar-H), 7.29-7.25 (m, 2H, Ar-H), 7.20-
.16 (m, 2H, Ar-H), 6.64 (d, J = 6.6 Hz, 1H, H1’), 5.71 (d, J = 6.4 Hz, 1H,
7
2
7
3
) δ 8.41(s, 1H, H8), 7.96 (d, J = 8.0 Hz,
3
14 2 4
H N O
found 249.0847.
H3’), 4.91 (br S, 1H, H4’), 4.66-4.59 (m, 2H, H5’), 3.20 (d, J = 15.0 Hz,
H, H2b’), 3.04 (dt, J = 15.0, 6.6 Hz, 1H, H2a’), 2.72 (s, 3H, CH ), 2.43 (s,
3H, CH ), 2.39 (s, 3H, CH ) δ 166.1, 165.7
); 13C NMR (151 MHz, CDCl
(C=O), 162.5 (C2), 151.5 (C4), 150.4 (C6), 144.7, 144.3 (Ar), 142.5 (C8),
1
3
Further elution with (CH
2
Cl
2
/MeOH 8:2) afforded alpha isomer of
3
3
3
1
compound
Z as a white solid (157 mg, 18 %). H NMR (500 MHz,
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Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
1
(
29.8, 129.6, 129.3, 129.3 (Ar, C5), 126.6, 125.7 (Ar), 86.3 (C1’), 84.7
C4’), 74.8 (C3’), 63.9 (C5’), 38.3 (C2’), 25.6 (CH ), 21.7 (CH ), 21.6 (CH );
[M+Na] 543.1405, found 543.1409
7.13 (m, 7H, ArH), 6.79 – 6.72 (m, 4H, ArH), 6.43 (t, J = 6.4 Hz, 1H, H1’),
5.39 (d, J = 4.6 Hz, 1H, OH), 4.49-4.45 (m, 1H, H3’), 4.01 (dt, J = 6.8, 4.0
3
3
3
+
HRMS (ESI+) calcd for C27
H25ClN
4
O
5
Hz, 1H, H4’), 3.71 (s, 3H, OCH
H, SCH ), 3.26 (dd, J = 10.1, 6.7 Hz, 1H, H5b’), 3.16 (dd, J = 10.1, 3.8
Hz, 1H, H5a’), 3.04 (t, J = 6.7 Hz, 2H, CH CN), 2.88 (dt, J = 12.8, 6.2 Hz,
H, H2b’), 2.50 (s, 3H, CH
), 2.35-2.37 (m, 1H, H2a’); 13C NMR (151 MHz,
DMSO-d ) δ 160.7 (C2), 158.1, 158.0, 157.4 (C6, Ar), 148.9 (C4), 144.9
Ar), 143.2 (C8), 135.6, 135.5 (Ar), 129.7, 129.6, 129.5, 127.7, 127.6,
3 3
), 3.70 (s, 3H, OCH ), 3.60 (t, J = 6.8 Hz,
2
2
2
2
-Methyl-6-sulfhydryl-9-(2-deoxy-3,5-di-O-p-toluoyl-β-D-erythro-
1
3
pentofuranos-1-yl)-purine (18b): To a solution of compound 18a (750
mg, 1.4 mmol) in 1:1-ethanol and water (40 mL), sodium thiosulfate (1.14
g, 7.2 mmol) was added and heated at 120 °C in a seal tube for 12 hours.
The reaction mixture was concentrated and diluted with ethyl acetate and
washed with water. The organics were dried over anhydrous Na SO , and
2 4
solvents were removed in vacuo. Purification via column chromatography
6
(
1
26.6 (Ar, C5), 119.3 (CN), 113.1, 113.0 (Ar), 86.2 (C4’), 85.4 (C), 83.9
), 38.4 (C2’), 25.6 (CH ),
). HRMS (ESI+) calcd for C35
S [M+H]+
38.2431, found 638.2446.
(
C1’), 70.8 (C3’), 64.2 (C5’), 55.1, 55.0 (OCH
3
3
2
6
3.7 (CH
2
), 18.0 (CH
2
35 5 5
H N O
on silica gel (50% ethyl acetate in hexanes) afforded the desired product
1
1
d
2
8b as a white solid compound (600 mg, 80%). H NMR (600 MHz, DMSO-
6
) δ 8.41 (s, 1H, H8), 7.93 (d, J =8.2 Hz, 2H, Ar-H), 7.81 (d, J =8.2 Hz,
The 3’-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite of 2-methyl-
6-cyanoethylthio-9-((2-deoxy-5-O-dimethoxytrityl-β-D-erythro-
pentofuranos-1-yl)-purine (21). The reaction of compound 20 (250 mg,
0.39 mmol) and 1 M bis(diisopropylamino)(2-cyanoethoxy)phosphine (784
μl, 0.78 mmol) and 0.45 M 1H-terazole (958 μl, 0.431 mmol) in anhydrous
H, Ar-H), 7.36 (d, J =8.2 Hz, 2H, Ar-H), 7.29 (d, J =8.2 Hz, 2H, Ar-H), 6.46
(
t, J =7.1 Hz, 1H, H1’), 5.80-5.76 (m, 1H, H3’), 4.64 (dd, J = 10.8, 4.2 Hz,
H5b’), 4.58-4.52 (m, 2H, H5a’, H4’), 3.21-3.16 (m, 1H, H2b’), 2.77 (ddd, J
=
3
9.3, 6.7, 2.9 Hz, 1H, H2a’), 2.47 (s, 3H, CH
3 3
), 2.39 (s, 3H, CH ), 2.36 (s,
H, CH ) δ 176.8 (C6), 165.5, 165.3
3
); 13C NMR (151 MHz, DMSO-d
6
CH
by flash column chromatography (CH
amidite 21 (223 mg, 68% yield); 31P NMR (202 MHz, DMSO-d
2
Cl
2
(8 mL), was carried out as described for compound 21 and purified
Cl /MeOH/TEA 98:1:1) afforded
) δ 147.86,
PS [M+H]+ 838.3509, found
(
1
(
(
C=O), 155.4 (C2), 144.2, 144.1, 143.9 (Ar-C, C4), 140.9 (C8), 133.9 (C5),
29.5, 129.4, , 129.3, 126.6, 126.5 (Ar-C), 83.8 (C1’), 81.8 (C4’), 74.8
C3’), 63.9 (C5’), 35.8 (C2’), 21.3 (CH ), 21.2 (CH ), 21.0 (CH ); HRMS
S [M+H] 519.1696, found 519.1700.
2
2
6
3
3
3
147.22; HRMS (ESI+) calcd for C44
52 7 6
H N O
+
26 4 5
ESI+) calcd for C27H N O
838.3516.
2
-Methyl-6-sulfhydryl-9-(2-deoxy-β-D-erythro-pentofuranos-1-yl)-
Oligonucleotide synthesis.
purine (Y). 5 M solution of sodim methoxide (0.25 mL) solution was added
to compound 18b (650 mg, 1.25 mmol) and the mixture was stirred for 1.5
h at room temperature. The reaction mixture was concentrated and the
Oligonucleotide assembly was performed with an Expedite DNA
synthesizer (Applied Biosystems) by using the phosphoramidite approach.
The oligomers were deprotected and cleaved from the solid support by
treatment with aqueous ammonia (30%) for 1/2 h at 25 °C and 1 h at 55 °C.
After gel filtration on a NAP-25 column (Sephadex G25-DNA grade from
GE Healthcare) with water as eluent, the crude mixture was analyzed by
using a Mono-Q HR 5/5 anion exchange column, after which purification
was achieved by using a Mono-Q HR 10/100 GL column (Pharmacia) with
residue was purified by flash column chromatography (CH
to yield the title compound Y as a white solid (276 mg, 78%). H NMR (600
MHz, DMSO-d ) δ 8.41(s, 1H, H8), 6.28 (dd, J = 7.1, 4.0 Hz, 1H, H1’), 5.32
d, J = 4.0 Hz, 1H, OH), 4.98 (t, J = 5.5 Hz, 1H, OH), 4.40-4.35 (m, 1H,
H3’), 3.87- 3.83 (m, 1H, H4’), 3.59 (dt, J = 9.9, 4.8 Hz, 1H, H5a’), 3.53-3.48
2 2
Cl /MeOH 9:1)
1
6
(
(
9
1
(
C
m, 1H, H5b’), 2.63- 2.56 (m, 1H, H2b’), 2.49 (s, 3H, CH
.9, 6.1, 3.3 Hz, 1H, H2a’); 13C NMR (151 MHz, DMSO-d
55.3 (C2), 144.2 (C4), 140.7 (C8), 133.6 (C5), 88.1 (C1’), 83.5 (C4’), 70.7
); HRMS (ESI+) calcd for
S [M+H] 283.0859, found 283.0863.
3
), 2.28 (ddd, J =
6
) δ 176.6 (C6),
the following gradient system: Buffer A = 10 mM NaClO
HCl in 15% CH CN, pH = 7,4. Buffer B = 600 mM NaClO
HCl in 15% CH
4
with 20 mM Tris-
with 20 mM Tris-
CN, pH=7,4. The low-pressure liquid chromatography
3
4
C3’), 61.6 (C5’), 48.7 (C2’), 21.0 (CH
3
3
+
11 14 4 3
H N O
system consisted of a HITACHI Primaide organizer with a HITACHI
Primaide 1410 UV detector and with a HITACHI Primaide 1110 pump and
a Mono-Q HR 10/100 GL column (Pharmacia). The product-containing
fraction was desalted on a NAP-25 column and lyophilised, followed by
analysis by mass spectrometry.
2
-Methyl-6-cyanoethylthio-9-(2-deoxy-β-D-erythro-pentofuranos-1-
yl)-purine (19). To a solution of dry compound Z (25 mg, 0.88 mmol) in
dry DMF (6 mL) was added powdered anhydrous potassium carbonate
(
474 mg, 3.54 mmol). This was followed by the addition of 3-
bromopropionitrile (367 mg, 2.66 mmol) via a dry syringe. After stirring at
room temperature for 12 hours the DMF was removed by coevaporation
with xylenes and the residue was purified by flash column chromatography
UV melting experiments.
Oligomers were dissolved in a buffer solution containing NaCl (0.1 M),
(
CH
mg, 80%). 1H NMR (500 MHz, DMSO-d
.1 Hz, 1H, H1’), 5.34 (d, J = 3.9 Hz, 1H, OH), 5.05 (t, J = 5.6 Hz, 1H,
OH), 4.43 (br s, 1H, H3’), 3.89 (d, J = 2.7 Hz, 1H, H4’), 3.64-3.51 (m, 4H,
SCH , H5’), 3.06-3.02 (t, J = 6.6 Hz, 2H, CH ), 2.75-2.68 ( m, 1H, H2a’),
.64 (s, 3H, CH
), 2.34-2.30 (m, 1H, H2b’); 13C NMR (126 MHz, DMSO-
) δ 160.7, 157.5 (C2, C6), 148.9 (C4), 142.8 (C8), 129.3 (C5), 119.3
),
S [M+H]+
2
Cl
2
/MeOH 95:5) to yield the title compound 19 as a white solid (240
KH
determined by measuring the absorbance in Milli-Q water at 260 nm at
0°C. The following extinction coefficients were used: dA, ε=15.060; dC,
2 4
PO (0.02 M, pH 7,5) and EDTA (0.1 mM). The concentration was
6
) δ 8.60 (s, 1H, H8), 6.41 (t, J =
7
8
ε=7.100; dG, ε=12.180; dT, ε=8.560; X, ε= 2089; Y, ε = 903; Z, ε = 2919.
Extinction coefficient have been determined at 260 nm, which is not the
maximum of absorption, but the wavelength at which the concentration of
the oligonucleotide is determined. The concentration for each strand was
2
2
2
d
(
2
3
3
6
CN), 88.1 (C4’), 83.8 (C1’), 70.8 (C3’), 61.7 (C5’), 39.5 (C2’), 25.6 (CH
3
3.6 (CH
4
µM in all experiments. Melting curves were determined with a Varian
2 2 17 5 3
S), 18.0 (CH CN); HRMS (ESI+) calcd for C14H N O
Cary 100 BIO spectrophotometer. Cuvettes were maintained at constant
temperature by water circulation through the cuvette holder. The
temperature of the solution was measured with a thermistor that was
directly immersed in the cuvette. Temperature control and data acquisition
were carried out automatically with an IBM-compatible computer by using
Cary WinUV thermal application software. A quick heating and cooling
cycle was carried out to allow proper annealing of both strands. The
samples were then heated from 10 to 80 °C at a rate of 0.2 °C min-1, and
were cooled again at the same speed. Melting temperatures were
36.1124, found 336.1134.
2
-Methyl-6-cyanoethylthio-9-(2-deoxy-5-O-dimethoxytrityl-β-D-
erythro-pentofuranos-1-yl)-purine (20). The reaction of compound 19
220 mg, 0.62 mmol) in dry pyridine (13 mL) and 4,4'–dimethoxytrityl
chloride (233 mg, 0.69 mmol) was carried out as described for compound
(
1
9
6 and purified by flash column chromatography (CH
7:2:1), affording compound 20 as a white solid (261 mg, 75%); H NMR
2
Cl
2
/MeOH/TEA
1
(
6
600 MHz, DMSO-d ) δ 8.50 (s, 1H, H8), 7.31-7.29 (m, 2H, ArH), 7.24 –
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
determined by plotting the first derivative of the absorbance as a function
of temperature; data plotted were the average of two runs. Up and down
curves showed identical Tm values.
and cooled as before. Ligation was performed by adding 1 mM ATP and 5
U T4 DNA ligase (NEB) to the samples before overnight incubation at 16
°C. Ligated mixtures were dialyzed as before, and transformed by
electroporation into fresh electro-competent E. coli K12 strain (ΔthyA:aad).
Incubation of the electroporated bacteria was performed at 37 °C for 1
hour, before plating 100 μL of a serial dilution of the suspension onto
Muller-Hinton (MH) media containing 100 μg mL-1 ampicillin (spreading the
Circular dichroism materials and methods.
For CD analysis, 4 µM of modified duplex [5’-d (CAC CGX/Y TGC TAC C)-
1
00 and 10-1 dilutions) and onto the same media supplemented additionally
3
’ and 5’-d (GGT AGC AX/Y C GGT G)-3’)] and natural duplex [5’-d (CAC
CGA TGC TAC C)-3’ and 5’-d (GGT AGC ATC GGT G)-3’) ] were dissolved
in buffer (0.1 M NaCl, 20 mM KH PO and 0.1 mM EDTA) at pH 7. CD
with 0.3 mM thymidine (10 and 10-4 dilutions).
-3
2
4
experiments were performed with Jasco J-1500 spectropolarimeter
equipped with a Julabo F12 Peltier thermoelectric temperature control unit.
Spectra were recorded at 25 °C using a 1 mm path length quartz cuvette
with scanning from 320 to 200 nm. Three scans were averaged with a
bandwidth of 1 nm and a 0.5 s averaging time at a rate of 50 nm/min.
Acknowledgements
We thank Luc Baudemprez for technical assistance and Chantal
Biernaux for editorial help. The research leading to these results
has received funding from the European Research Council under
the European Union's Seventh Framework Programme
Primer extension experiments by DNA polymerases.
(
FP7/2007-2013)/ERC Grant agreement no. ERC-2012-
The incorporation of natural dNTPs and the 2-amino-6-Methyl-8-oxo-7,8-
dihydro-purine nucleoside triphosphate was carried out using a Cy5
fluorescence labeled primer and a template containing one or three 2-
amino-6-methyl-8-oxo-7,8-dihydro-purine nucleosides, incorporated (ON
ADG_20120216/320683, from FWO Vlaanderen, Belgium
(G078014N) and from the KU Leuven Research Council
(OT/1414/128). Mass spectrometry was made possible by the
support of the Hercules Foundation of the Flemish Government
2
8, 29). The primer 5’Cy5-CAGGAAACAGCTATGAC-3’ was annealed
with one of the templates 5’-AACTGXGTCATAGCTGTTTCCTG-3’ or 5’-
AACTGXXXGTCATAGCTGTTTCCTG-3’ in a 1:2 molar ratio by heating
the mixture at 95 °C for 5 minutes followed by slow cooling to room
temperature. A mesophilic DNA polymerase: Klenow fragment exo- (New
England Biolabs) and a thermostable polymerase Pol6G12[27] were used.
The final DNA polymerization mixtures each contained 125 nM primer-
template duplex, reaction buffer (NEBuffer 2 was used for Klenow
fragment exo- and ThermoPol buffer was used for Pol6G12) and 50 µM of
the natural dNTPs or 50 µM or 200 µM of 2-amino-6-Methyl-8-oxo-7,8-
dihydro-purine nucleoside triphosphate. Final concentration in a reaction
of Klenow fragment exo- and Pol6G12 were 0.0017 U/µl and 0.0186 U/µl,
(
grant 20100225-7).
Keywords: methylated nucleobases • self-pairing • duplex
stability • polymerases • transliteration
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10.1002/chem.201802304
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Chemistry - A European Journal
10.1002/chem.201802304
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Methylation
of
nucleobases
at
[a]
[b]
A. M. Jabgunde, F. Jaziri, O.
positions normally involved in W-C
recognition of canonical bases readily
leads to orthogonality.
[a]
[a]
[a]
Bande, M. Froeyen, M. Abramov, H.
[
a]
[a]
Nguyen, G. Schepers, E.
Lescrinier,[ V. B. Pinheiro, V. Pezo,
a]
[c]
[b]
P. Marlière,[ and P. Herdewijn*
b]
[a]
CH
3
S
N
CH
3
Page No. – Page No.
H
N
N
N
N
N
NH
CH3
O
Methylated nucleobases: Synthesis
and evaluation for base pairing in
vitro and in vivo
N
N
NH2
N
O
Rd
Rd
dR
This article is protected by copyright. All rights reserved.