Base pairing involving artificial bases in vitro and in vivo

Article abstract of DOI:10.1039/c5sc03474d

Herein we report the synthesis of N⁸-glycosylated 8-aza-deoxyguanosine (N⁸-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N⁸-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoC^Me), 8-amino-deoxyinosine (8-NH₂-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH₂-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoC^Me:d-isoG artificial genetic system. As demonstrated by T_m measurements, the N⁸-8-aza-dG:d-isoC^Me base pair formed less stable duplexes as the C:G and d-isoC^Me:d-isoG pairs. Incorporation of 8-NH₂-dI versus the N⁸-8-aza-dG nucleoside resulted in a greater reduction in T_m stability, compared to d-isoC^Me:d-isoG. Insertion of the methyl group at the N¹ position of 8-NH₂-dI did not affect duplex stability with N⁸-8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH₂-dI and 8-NH₂-dI) and N⁸-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

Full text of DOI:10.1039/c5sc03474d

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Base pairing involving artificial bases in vitro and in
vivo†
Cite this: Chem. Sci., 2016, 7, 995
Omprakash Bande,‡b Darren Braddick,‡a Stefano Agnello,‡b Miyeon Jang,b
Valerie Pezo,a Guy Schepers,b Jef Rozenski,b Eveline Lescrinier,b Philippe Marlierea
´
`
ab
*
and Piet Herdewijn
Herein we report the synthesis of N8-glycosylated 8-aza-deoxyguanosine (N8-8-aza-dG) and 8-aza-9-
deaza-deoxyguanosine (N8-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-
methyl-isocytosine (d-isoCMe), 8-amino-deoxyinosine (8-NH2-dI), 1-N-methyl-8-amino-deoxyinosine
(1-Me-8-NH2-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-
Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoCMe:d-isoG
artificial genetic system. As demonstrated by Tm measurements, the N8-8-aza-dG:d-isoCMe base pair
formed less stable duplexes as the C:G and d-isoCMe:d-isoG pairs. Incorporation of 8-NH2-dI versus the
N8-8-aza-dG nucleoside resulted in a greater reduction in Tm stability, compared to d-isoCMe:d-isoG.
Insertion of the methyl group at the N1 position of 8-NH2-dI did not affect duplex stability with N8-8-
aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular
interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the
incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The
most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH2-
dI and 8-NH2-dI) and N8-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead
of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-
deoxynucleosides was studied, finding no significant impact in their in vivo assay.
Received 15th September 2015
Accepted 28th October 2015
DOI: 10.1039/c5sc03474d
www.rsc.org/chemicalscience
partners is an N8 substituted purine base and involving
hydrogen-bond formation in the recognition process.
The unnatural isoG:isoCMe base pair9,10 is not adequate to
Introduction
Non-canonical base pairing has been studied by several
research groups,1–6 based on their interest to expand the genetic
alphabet. A major breakthrough in this eld has been reported
by Malyshev et al.,7 showing replication of a synthetic hydro-
phobic base pair (d5SICS–dNAM) in vivo. This base pair system
consists of C-nucleosides that are metabolically not easily
accessible, where recognition is based on hydrophobic inter-
actions, and no hydrogen-bond interactions (such as in
canonical base pairing) are involved. Recently, we have
demonstrated that a change in the backbone (HNA replacing
DNA) could lead to a reduced recognition of non-canonical
bases (i.e. isoCMe and isoG) by natural bases,8 further moving in
the direction of a fully orthogonal information system. Here we
have investigated new base pair systems from which one of the
expand the genetic code and has restricted application due to
facile tautomerization of isoG which enables it to form base
pairs with selected natural nucleobases. Tautomerism leads
to promiscuity and heterocycles (via tautomerism) possibly
leading to pairing with natural bases, should be avoided when
selecting new orthogonal information systems.1 Therefore, we
would like to replace the isoG base in the isoG:isoCMe base pair
by another heterocycle less prone to tautomerization. In search
for new articial genetic systems efforts have been devoted to
the study of C-nucleosides11–13 and nucleosides with an aberrant
glycosidic linkage i.e. with an N8 glycosylated nucleobase,14–17 as
they demonstrate unexpected pairing behavior and some of
them may function as universal bases.18,19 Seela et al., have
demonstrated that altering the glycosylation site of a nucleoside
from N9 to N8 results in a change of the nucleobase recognition
pattern, N8-glycosylated-7-deaza-8-azaguanine behaves like
isoguanine20 and forms base pairs with isoCMe and not with
cytosine. Therefore, we have investigated N8-glycosylated
8-azaguanine21–23 1a together with its N9-deaza-analogue 1b.
Removal of the N9 nitrogen atom (giving 1b) may have
an inuence on the in vitro and in vivo recognition of the
a
`
iSSB – CNRS FRE3561, University of Evry-Val-d'Essonne, 5 rue Henri Desbrueres,
´
ˆ
Genopole Campus 1, Bat. 6, F-91030 Evry Cedex, France
bMedicinal Chemistry, Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, 3000 Leuven, Belgium. E-mail: piet.herdewijn@rega.
kuleuven.be; Tel: +32 16 337387
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5sc03474d
‡ Contributed equally to this article.
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N8-glycosylated purine nucleoside. Potentially, the N8-glycosy-
lated purine nucleoside may be recognized by another purine
nucleoside, when Hoogsteen base pairing is involved. To eval-
uate the potential of involving such Hoogsteen recognition24 in
an unnatural base pair, 8-amino-hypoxanthine25,26 2a was
selected as the pairing partner for the N8-glycosylated bases (N8-
8-aza-dG, N8-8-aza-9-deaza-dG) as shown in Fig. 1. For Hoogs-
teen pairing in a duplex structure, one of the bases should adopt
the ‘syn’ conformation and 8-substituted purines preferentially
adopt this conformation.27,28 The 1-Me-8-NH2-dI nucleobase 2b
was tested in order to exclude Watson–Crick type recognition in
1a/1b:2a base pairing. Changing the 8-amino group in 2a in an
8-hydroxy group will, rst, abolish recognition by 1a and 1b,
which would be further demolished by changing the aad system
of 2 in the dda system of 5 (the dda system is also present
in 1a and 1b) (Fig. 2). Therefore, and to fully understand the
base pairing pattern, we have also synthesized and evaluated
the 7,8-dihydroxy-8-oxonucleosides with the 6-Oxo29 3, 6-Oxo-2-
amino30–34 4 and 6-amino35–38 5 base moieties. Because of the
favored syn conformation of 8-hydroxylated purine, Hoogsteen
base pairing may be preferred over Watson–Crick base pair-
ing.39–41 A conclusion is that Watson–Crick type base pairing as
well as Hoogsteen type base pairing can be considered when
selecting orthogonal base pairs in vitro and in vivo.
In order to determine the orthogonality of the nucleosides
against the biological situation, they were individually analyzed
in vivo in an XNA-dependent DNA synthesis assay. For these
nucleosides the assay studied the pairing properties of each,
with an extension of the study performed for the 7,8-dihydro-8-
oxoguanine and 7,8-dihydro-8-oxoadenine bases in vivo, the
former of which is formed by ROS-mediated damage of DNA.42,43
Both of the 8-oxo-deoxynucleoside bases were analyzed in wild-
type and knockout backgrounds of the important 8-oxo repair
enzyme MutM.44,45 The assay was to determine whether or not
this enzyme in particular was inuencing the base pairing
results determined by tests, and conrm that the non-natural
nucleosides here were not being chemically modied in vivo,
thus affecting the results obtained.
Fig. 2 20-Deoxyribo-nucleosides, with modified purine and pyrimi-
dine bases.
Results and discussion
Synthesis of monomers
As depicted in Scheme 1, the BF3–Et2O-catalysed glycosylation
of commercially available 8-azaguanine (8) with 1,2,3,5-tetra-O-
acetyl-b-D-ribofuranose afforded 9 as the major isomer. The
assignment of N8 glycosidic bond in 9 was made by comparing
spectral data with the known compound 1a in later stage. The
cleavage of the acetyl-protecting group was carried out using
ammonium hydroxide at room temperature for 8 hours,
obtaining compound 10a in 91% yield.46,47 The regioselective
protection of the 30-OH and 50-OH groups with TIPDSiCl2 gave
compound 14a.
Scheme
1
Synthesis of N8-8-aza-G and N8-8-aza-9-deaza-G
nucleoside. Reagents and conditions: (a) BF3–Et2O, 1,2,3,5-tetra-O-
acetyl-b-D-ribofuranose, CH3CN, 75 ꢀC, 3 h, 54%; (b) NH4OH, rt, 8 h,
91%; (c) BSA, DCE, SnCl4, 1,2,3,5-tetra-O-acetyl-b-D-ribofuranose, rt,
Fig. 1 Putative base pair motifs of duplexes with antiparallel strand 36 h, 85%; (d) H2, 10% Pd/C, MeOH, rt, 8 h, 100%; (e) (i) chloro-for-
orientation.
mamidine hydrochloride, DMS, 120 ꢀC, 1 h, (ii) NH4OH, rt, 1 h, 42%.
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For the deoxygenation of the C20-hydroxy group, the Barton– 60% of the protected 8-azido-dI nucleoside 20a. The reduction
McCombie reaction was used. We rst converted the nucleoside of the azide group was carried out using sodium borohydride in
14a into the corresponding thiocarbamate, which, aer radical methanol and further protected with an N,N-dimethylamino
reduction in the presence of tributylstannane and AIBN, gave methylidene residue using dimethylformamide dimethyl acetal
ꢀ
the desired nucleoside 15a (Scheme 2).
in methanol at 70 C for 1 hour. The cleavage of the silyl pro-
Subsequently, the removal of the silyl-protecting group, tecting group was carried out using TBAF in THF at room
carried out using TBAF in THF at room temperature for 4 hours, temperature for 12 hours, to obtain compound 22a to 60% yield.
afforded compound 1a to 94% yield. The spectral and analytical Subsequently, compound 22a was tritylated and phosphity-
data of 1a were found to be in accordance with reported data.21 lated, furnishing the phosphoramidite 24a via 23a. For the
Protection of the amino group of 1a followed by the selective synthesis of the 1-Me-8-NH2-dI building block, the N1 methyl-
protection of the primary alcohol with 4,40-dimethoxytrityl ation of compound 20a was carried out using methyl iodide and
chloride (DMTrCl) in pyridine, provided the protected nucleo- sodium hydride to obtain compound 20b in 88% yield. Then
side 17a,21 which was then converted into phosphoramidite 18a similar to the synthesis of 8-NH2-dI phosphoramidite 24a,
by reaction with bis(diisopropylamino)(2-cyanoethoxy)phos- a series of transformations starting from 20b yielded the cor-
phine in the presence of tetrazole.
responding phosphoramidite 24b.
The synthesis of the N8-8-aza-9-deaza-dG derivative started
Previously,29 8-Oxo-dI 3 was prepared by enzyme-mediated
from the readily available pyrazole 11. Vorbruggen reaction of deamination of 8-Oxo-dA 5. Here, the 8-Oxo-dA50 5 nucleoside
11 with 1,2,3,5-tetra-O-acetyl-b-D-ribofuranose, in the presence was deaminated by diazotization of the 6-NH2 group using
of SnCl4, afforded the desired N1-isomer 12 as the major sodium nitrate in acetic acid and water at room temperature for
product (91% yield).48 The reduction of the nitro group of 12, 12 hours, affording 8-Oxo-dI 3 to 77% yield. Subsequently,
¨
followed by the ring closure with chloroformamidine hydro- compound
3 was tritylated followed by phosphitylation,
chloride and deprotection of acetyl groups in one pot, gave the furnishing the phosphoramidite 26 via 25 (Scheme 4). The
desired compound 10b (Scheme 1). Analogously to what is spectral and analytical data of 26 were found to be in accor-
described for the N8-8-aza-guanine derivatives in Scheme 2, dance with that reported.29 The free amino group of 8-Oxo-dA 5
a series of transformations starting from 10b allowed us to was protected with an N,N-dimethylamino methylidene residue
obtain the deoxy derivative 1b and nally the corresponding using dimethylformamide dimethyl acetal in methanol.
phosphoramidite 18b.
Subsequently, compound 27 was tritylated using DMTrCl in
As depicted in Scheme 3, the synthesis of the phosphor- pyridine and DMAP as a catalyst. Furthermore, phosphitylation
amidite of 8-NH2-dI (2a) started from the silylated deoxyinosine of the DMTr derivative 28, furnished the phosphoramidite 29.
19.49 The compound 19 was treated with tosyl azide and n-
butyllithium in anhydrous THF at ꢁ78 C for 2 hours yielding dimethylamino methylidene residue using dimethylformamide
The amino group of 8-Oxo-dG51 4 was protected with an N,N-
ꢀ
dimethyl acetal in methanol afforded compound 30.
Compound 30 was tritylated and phosphitylated furnishing the
required phosphoramidite 32 (Scheme 5).
Oligonucleotide synthesis and hybridization properties
The oligonucleotides were prepared by using the common
phosphoramidite method on a solid support employing a DNA
Scheme
2
Synthesis of N8-8-aza-dG and N8-8-aza-9-deaza-dG
nucleoside. Reagents and conditions: (a) TIPDSiCl2, pyridine, 0 ꢀC to rt, Scheme 3 Synthesis of the phosphoramidite of 8-NH2-dI and 1-Me-
8 h, 74–77%; (b) (i) TCDI, CH2–Cl2, rt, 8 h; (ii) AIBN, nBu3SnH, toluene, 8-NH2-dI. Reagents and conditions: (a) TsN3, BuLi, THF, ꢁ78 ꢀC, 2 h,
75 ꢀC, 2 h, 62–68%; (c) TBAF, THF, rt, 4 h, 94–97%; (d) DMF–DMA, 60%; (b) (i) NaBH4, MeOH, 0 ꢀC to rt, 2 h; (ii) DMF–DMA, MeOH, 78 ꢀC, 1
MeOH, 50 ꢀC, 1 h, 93–96%; (e) DMTrCl, pyridine, 0 ꢀC to rt, 55 min, 2 h, h, 70–73%; (c) TBAF, rt, 3 h, 60–73%; (d) (MeO)2TrCl, pyridine, 0 ꢀC to
76–83%; (f) (i-Pr2N)2POCH2CH2CN, 1H-tetrazole, CH2Cl2, 0 ꢀC to rt, rt, 12 h, 89–96%; (e) (i-Pr2N)2POC2H4CN, 1H-tetrazole, CH2Cl2, 0 ꢀC
55 min, 2 h, 69–73%.
to rt, 1 h, 75–77%; (f) Mel, NaH, 0 ꢀC to rt, 12 h, 88%.
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d-isoCMe:d-isoG (Table 1, entry 2). The N8-8-aza-dG:d-isoCMe
base pair gave a less stable duplex than the dC:dG (ꢁ3.1 ꢀC) pair
and d-isoCMe:d-isoG (ꢁ5.4 ꢀC). Incorporation of an 8-NH2-dI 2a
versus an N8-8-aza-dG nucleoside gave a greater decrease in Tm
(ꢁ9.5 ꢀC) than incorporation of d-isoCMe versus N8-8-aza-dG
ꢀ
nucleoside (ꢁ5.4 C), both decreasing on comparison to the d-
isoCMe:d-isoG base pair. Insertion of the methyl group at N1
position of 8-NH2-dI did not affect the duplex stability, which
suggests that the base pairing of 8-NH2-dI with N8-8-aza-dG
takes place through Hoogsteen base pairing (Fig. 1). Surpris-
ingly, 8-Oxo-dI and 8-Oxo-dG also show similar duplex stability,
compared to 8-NH2-dI. As shown in Fig. 3, this could be
explained by Wobble type base pairing in which two hydrogen
bonds are involved. The sudden decrease in Tm of the duplexes
when N8-8-aza-dG is placed opposite to 8-Oxo-dA (ꢁ11.2 ꢀC),
demonstrated that the C6 acceptor (C]O) of 8-Oxo-dI is
necessary for base pairing. The experiment proved that the base
pair stability for N8-8-aza-dG decreases in the order d-isoCMe > 8-
Oxo-dG $ 8-NH2-dI z 1-Me-8-NH2-dI $ 8-Oxo-dI [ 8-Oxo-dA.
Approximately similar results were observed for N8-8-aza-9-
deaza-dG 1b, which indicates that the N9 nitrogen atom does
not play a signicant role in duplex stabilization (Table 2).
In order to investigate the selectivity of base pairing, the
stability of the duplexes with the modied base N8-8-aza-9-
deaza-dG, N8-8-aza-dG, d-isoCMe, 8-NH2-dI, 1-Me-8-NH2-dI, 8-
Oxo-dI, 8-Oxo-dA, and 8-Oxo-dG and the four canonical bases (A,
T, C and G) was investigated by Tm determination (Table 3). This
study is done in function of the potential use of the articial
base pairs in vivo. Among the four inosine derivatives (2a, 2b, 3
and 4) the mismatch discrimination (DTm) is the lowest for 8-
NH2-dI and the highest for 1-Me-8-NH2-dI. For the N8 nucleo-
sides; the N8-8-aza-9-deaza-dG showed better mismatch
discrimination than N8-8-aza-dG.
Scheme 4 Synthesis of the phosphoramidite of 8-Oxo-dI and 8-Oxo-
dA. Reagents and conditions: (a) NaNO2, acetic acid: H2O, 12 h, 77%;
(b) (MeO)2TrCl, pyridine, 0 ꢀC to rt, 12 h, 69–73%; (c) (i-Pr2N)2POC2-
H4CN, 1H-tetrazole, CH2Cl2, 0 ꢀC to rt, 1 h, 77–80%; (d) DMF–DMA,
MeOH, 3 h, 65 ꢀC, 91%.
synthesizer. The base pairing properties of N8-8-aza-9-deaza-dG
and N8-8-aza-dG nucleosides with d-isoCMe,8 8-NH2-dI, 1-Me-8-
NH2-dI, 8-Oxo-dI, 8-Oxo-dA, and 8-Oxo-dG were examined by
hybridizing oligomers with their complementary strands and
determining the Tm of the hybrids by temperature-dependent
UV spectroscopy. In order to investigate the base pairing prop-
erties, the N8-8-aza-dG 1a, N8-8-aza-9-deaza-dG 1b and d-isoG 7
nucleosides were incorporated in the antiparallel duplex 50-
d(GGT AGC AG*C GGT G)-30 replacing the G residue. The 8-NH2-
dI 2a, 1-Me-8-NH2-dI 2b, 8-Oxo-dI 3, 8-Oxo-dA 5, 8-Oxo-dG 4, and
d-isoCMe 6 nucleosides were incorporated in the sequence 30-
d(CCA TCG TC*G CCA C)-50 replacing the C residue. The
strength of hybridization was studied by thermal denaturation
experiments, which were determined at 260 nm in NaCl (0.1 M)
buffer with KH2PO4 (20 mM, pH 7.5) and EDTA (0.1 mM) at
a concentration of 4 mm for each strand. The stability of the
duplexes was compared to the stability of the natural DNA
duplex containing dC:dG (Table 1, entry 1) and unnatural
The selectivity of the 1a:2a base pair is low, as the Tm of the
ꢀ
duplexes containing 1a:2a are only 1.0–3.4 C higher than the
Table 1 Tm values (in ꢀC) of non-self-complementary antiparallel-
stranded oligonucleotide duplexes containing dA, dT, dC, dG, 6 (d-
isoCMe), and 7 (d-isoG)
Entry
Duplex
Tm (ꢀC)
61.3
1
2
50-d(GGT AGC AGC GGT G)-30
30-d(CCA TCG TCG CCA C)-50
50-d(GGT AGC A7C GGT G)-30
30-d(CCA TCG T6G CCA C)-50
63.6
Scheme 5 Synthesis of the phosphoramidite of 8-Oxo-dG. Reagents
and conditions: (a) DMF–DMA, MeOH, 50%; (b) (MeO)2TrCl, pyridine,
0
ꢀC to rt, 12 h, 71%; (c) (i-Pr2N)2POC2H4CN, 1H-tetrazole, CH2Cl2, Fig. 3 Putative base pairs between dG:N8-1-Me-8-NH2-dI and N8-8-
0 ꢀC to rt, 1 h, 81%.
aza-dG:8-Oxo-dI.
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N8-8-aza-dG, N8-8-aza-9-deaza-dG, 8-Oxo-dI, 8-Oxo-dA and
8-Oxo-dG. The DNA backbone represented the 18-base long
coding strand encoding the active site of the essential ThyA
enzyme, whereby the nucleosides indicated replaced DNA
within various codons (Fig. 4).
Table 2 Tm values (in ꢀC) of non-self-complementary antiparallel-
stranded oligonucleotide duplexes containing 1a, 1b {50-d[GGT AGC
A(1a or 1b)C GGT G]-30} and 6, 2a, 2b, 3, 4, 5 {30-d[CCA TCG T(6 or 2a
or 2b or 3 or 4 or 5)G CCA C]-50} and DTm with respect to entry 1
(Table 1)
N8-8-aza-dG
(1a)
N8-8-aza-9-deaza-dG
(1b)
DTm
DTm
XNA-dependent DNA synthesis in vivo
d-isoCMe (6)
8-NH2-dI (2a)
1-Me-8-NH2-dI (2b) 54.0
8-Oxo-dI (3)
8-Oxo-dG (4)
8-Oxo-dA (5)
58.2
54.1
ꢁ3.1 58.3
ꢁ3.0
We investigated the relationship between the nature of the
chemical modications of the 8-derivatives and the response of
the E. coli genetic system to base pair with sufficient recognition
and selectivity to enable survival in vivo. The aim in this study
was to identify which of these modied nucleosides lacked such
recognition, and possessed poor base-pairing ability with the
canonical bases so as to approach orthogonality away from the
biological system.
The xenobiotic nucleosides were tested using the published
and established gapped-vector assay.52,53 In summary, DNA base/
modied nucleoside mosaic oligomers were ligated enzymatically
into a gapped form of the inactive thyA gene, which encodes an
essential section of the ThyA active site. The inactive thyA gene was
located on a plasmid vector (the pAK1/pAK2 heteroduplex)
together with the bla ampicillin resistance gene permitting selec-
tion on two criteria. Once ligated and circularized, the plasmid was
transformed into a strain of E. coli lacking thyA, a lethal growth
phenotype in thymidine (dT) decient media (Fig. 5).
The completion of the thyA gene nalizes the plasmid, which
permits bacteria survival in the dT decient media. The ratio
between bacterial colony numbers in ꢂdT media indicates the
success of the mosaic xenobiotic oligonucleotide to serve as
a template for DNA polymerization. The mosaic oligonucleo-
tides all took the form of the six middle codons that encode the
active site of ThyA around the catalytically essential cysteine
(codon TGC or TGT). The forms of each of the oligomers tested
follow, including the nucleosides and their positions within the
oligomer (Table 4).
ꢁ7.2 54.4
ꢁ7.3 54.0
ꢁ7.9 54.0
ꢁ6.4 55.4
ꢁ14.3 48.6
ꢁ6.9
ꢁ7.3
53.4
54.9
47.0
ꢁ7.3
ꢁ5.9
ꢁ12.7
Table 3 Tm values (in ꢀC) of antiparallel-stranded oligonucleotide
duplexes containing 1a, 1b {50-d[GGT AGC A(1a or 1b)C GGT G]-30} and
2a, 2b, 3, 4, 5 {30-d[CCA TCG T(2a, or 2b or 3 or 4, or 5)G CCA C]-50}
hybridized against complementary oligonucleotides with natural bases
a
A
T
C
G
DTm
N8-8-aza-dG (1a)
N8-8-aza-9-deaza-dG (1b)
8-NH2-dI (2a)
1-Me-8-NH2-dI (2b)
8-Oxo-dI (3)
47.1
46.3
50.7
46.2
55.8
54.7
45.8
53.8
53.0
51.6
43.7
50.3
50.0
54.7
46.9
47.0
53.1
43.1
53.5
57.0
44.5
54.6
55.8
52.9
52.1
47.9
54.9
51.2
7.7
9.5
2.4
9.0
7.9
8-Oxo-dG (4)
8-Oxo-dA (5)
7.0
10.2
a
In this case DTm corresponds to the difference between lowest and
highest Tm (mismatch discrimination).
Tm of the duplexes of 2a and the natural bases (A, T, C and G).
The selectivity of the 1a:2b base pair is much better, due to
a large drop in the Tm of duplexes containing the 2b:A, 2b:T and
2b:C base pairs. However, discrimination with G (2b:G base
pair) stays low. In case of 8-Oxo-dI 3, the stability of the 3:A is
higher than of 3:1a (which is similar than 3:C). Hence the
mismatch discrimination is highest with the G base (3:G). 8-
Oxo-dG (4) shows better base pairing with C (4:C) and with G
(4:C), than 8-Oxo-dI (3:C and 3:G). Base pairing of 8-Oxo-dG with
The thyA plasmid genes from sixteen clones selected on dT
decient media were sequenced for each mosaic oligonucleo-
tide tested (Fig. 6).
canonical bases has been described previously.32 Base pair 1-Me-8-NH2-dI and 8-NH2-dI
stability of 4:A and 4:G is the same as of 4:1a. The base pair
The assay and interpretation of the 8-NH2-dI and 1-Me-8-NH2-dI
stability of 8-Oxo-dA versus canonical bases decreases in the
order of T > G [ A > C. The results with 1b are similar to the
results with 1a, except that 1b:5 is somewhat more stable than
1a:5 and 1b:G is somewhat more stable than 1a:G. The result,
however, could be sequence dependent. Based on the Tm data,
the 1-Me-8-NH2-dI:N8-8-aza-dG (or N8-8-aza-9-deaza-dG) base
pair is the most discriminative versus A, T, and C, except versus
G. However this does not mean that it would be the best
orthogonal base pair in vivo.
and nucleotides in vivo follows (Fig. 7).
Analysis of the 1-Me-8-NH2-dI nucleotide in vivo reveals faint
recognition as G and even less as T, with nothing apparent as C
or A. Comparison to the related inosine-derivative 8-NH2-dI,
which lacks the N1 methyl group, shows that recognition is
Recognition of modied bases in vivo
Fig. 4 The general structure of the mosaic test oligos. The 18 bases
derive from the wild-type active site of E. coli thyA. Their positions and
encoded amino acids are shown, including the catalytically essential
Cys residue at 146. The various positions within this sequence
where the modified nucleosides were tested within are bolded and
A range of modied oligonucleosides were chemically synthe-
sized with anking DNA backbones and subsequently analysed
for their biological characteristics in vivo. These oligonucleo-
tides included separately each of 1-Me-8-NH2-dI, 8-NH2-dI, underlined.
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N8-8-aza-dG and N8-8-aza-9-deaza-dG
The assay and interpretation of the two N8 nucleosides, N8-8-
aza-dG and N8-8-aza-9-deaza-dG nucleotides in vivo follow
(Fig. 8 and 9).
Analysis shows that N8-8-aza-dG appears to behave as an A or
C nucleotide, and lacks any recognition as a T or G nucleotide.
Sequencing of positive colonies revealed the presence of
incorporated A's into the XCG codon (changing the amino acid
to Thr from Ala, tolerated to approximately 30% of comparable
wild-type survival through DNA control tests). In the GCX codon
changes were also observed, with mispairing resulting in the
inclusion of silent C and A residues (tolerated to 100% as wild-
type) for an Ala residue in the enzyme primary structure. The N8-
8-aza-dG nucleoside base pairs in the order of T [ G > C ¼ A.
The N8-8-aza-9-deaza-dG nucleotide appears to behave similarly
to the derivative N8-8-aza-dG, with an interpreted base pairing
preferentiality of T [ G ¼ C ¼ A, and similarly a proportion of
changes to A nucleotides as observed through sequencing, also
in the XCG codon (tolerated to Thr).
We hypothesized that the N9 nitrogen atom may assist
Hoogsteen base pairing, although the similar ndings in vivo
between the two N8 nucleosides suggests the N9 nitrogen atom
does not offer a signicant role in recognition, agreeing with the
previously indicated ndings in vitro for this pair with regards
to duplex stabilization. Modications in recognition primarily
occur on the XCG, CXT and GCX codons, but the inuence
remains marginal, as other codons are not affected, indicating
little difference in vivo of the recognition of these two
N8-glycosylated nucleosides. These ndings do not discredit
the in vitro Tm data, where N8-8-aza-dG showed worse mismatch
discrimination than its N9-deaza-analogue. The general appear-
ance of the in vivo results indicate an agreement to the in vitro
ndings, where general mismatch is comparably lower in N8-8-
aza-9-deaza-dG except towards T, which appears easier to pair.
Between the two nucleosides, the N8-8-aza-9-deaza-dG is the
most orthogonal of this pair, and one of the most orthogonal of
all of those tested in this study.
Fig. 5 Selection screen to test DNA oligonucleotides bearing modified
base(s) in templates for DNA synthesis in vivo.
entirely as G (and none with the other canonical bases),
demonstrating that the presence of this 1-Me group is sufficient
to reduce recognition as G but increase slightly recognition as T.
The 8-NH2-dI nucleotide base pairs the canonical bases in
vivo preferentially of the order C \ T ¼ G ¼ A, whereas the
methylated form 1-Me-8-NH2-dI nucleotide appears to base pair
preferentially in the order C [ A > G ¼ T. These ndings agree
with the in vitro Tm data interpretations (Table 3), indicating
perseverance of the recognition patterns between terminal
stabilization in the articial base pairs and enzymatic poly-
merization in vivo. Sequencing analysis was performed on
puried plasmids from positive transformants and in both
experiments revealed only wild-type sequences, indicating no
forced changes of the genetic code by the bases.
8-Oxo-dI, 8-Oxo-dG and 8-Oxo-dA
The assay and interpretation of the 8-Oxo-dI, 8-Oxo-dG and
8-Oxo-dA nucleotides in vivo follow (Fig. 10 and 11).
The response of the 8-Oxo-dI nucleotides reveals the worst in
vivo mismatch discrimination of all of the nucleotides tested
Table 4 Sequence of the oligomers used in the XNA-dependent DNA synthesis experiments
Sequence (50–30)
X
d(P-CTA XCG CCG TGC CAT GCA)
d(P-CTA GCG CCG XGC CAT GCA)
d(P-CTA GCG CCG TGX CAT GCA)
d(P-CTA GCG CCG TGC CXT GCA)
d(P-CTA GCX CCT TGT CAT GCA)
d(P-CTA GCX CXT TGT CAT GCA)
d(P-CTA GCX XCT TGT CAT GCA)
d(P-CTA GCX XXT TGT CAT GCA)
1-Me-8-NH2-dI, 8-NH2-dI, 8-Oxo-dI, N8-8-aza-dG, or N8-8-aza-9-deaza-dG
1-Me-8-NH2-dI, 8-NH2-dI, 8-Oxo-dI, N8-8-aza-dG, or N8-8-aza-9-deaza-dG
1-Me-8-NH2-dI, 8-NH2-dI, 8-Oxo-dI, N8-8-aza-dG, or N8-8-aza-9-deaza-dG
1-Me-8-NH2-dI, 8-NH2-dI, 8-Oxo-dI, N8-8-aza-dG, or N8-8-aza-9-deaza-dG
8-Oxo-dI, N8-8-aza-dG, N8-8-aza-9-deaza-dG, 8-Oxo-dA, or 8-Oxo-dG
8-Oxo-dI, N8-8-aza-dG, N8-8-aza-9-deaza-dG8-Oxo-dA, or 8-Oxo-dG
8-Oxo-dI, N8-8-aza-dG, N8-8-aza-9-deaza-dG, 8-Oxo-dA, or 8-Oxo-dG
8-Oxo-dI, N8-8-aza-dG, 8-Oxo-dA, or 8-Oxo-dG
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Fig. 6 Fate of mosaic oligomers in vivo. The first step in vivo is the polymerisation of the complementary DNA strand followed by canonical
replication. Different possible incorporations of canonical nucleotides facing the base modified DNA nucleoside during the synthesis of the first
DNA strand and the resulting coding strand are presented.
Fig.
9
The in vivo interpretation of the N8-8-aza-9-deaza-dG
nucleotide within the essential thyA gene. The normalized ratio is the
experimentally derived average number of thymidine-prototrophic
colonies (bla+ thyA+) from the average total number of colonies (bla+
thyAꢁ and bla+ thyA+). The modified section of the oligomer sequence
indicates the position of the N8-8-aza-9-deaza-dG nucleotide/s.
Fig. 7 The in vivo interpretation of the 8-NH2-dI nucleotide (top) and
1-Me-8-NH2-dI nucleotide (bottom) within the essential thyA gene.
The normalized ratio is the experimentally derived average number of
thymidine-prototrophic colonies (bla+ thyA+) from the average total
number of colonies (bla+ thyAꢁ and bla+ thyA+). The modified section
of the oligomer sequence indicates the position/s of the 8-NH2-dI or
1-Me-8-NH2-dI nucleotide as appropriate.
widespread changes to the gene. In the XCG codon, over 80% of
the 8-Oxo-dI nucleotides had become T, changing the amino
acid to Ser (tolerated lightly compared to wild-type Ala). Simi-
larly, the codons GCX and (GCX) XCT returned around 15%
each changes to T (silent change). This nucleotide has a base-
pairing preferential order of A ¼ C > G [ T, which support
Hoogsteen recognition in vivo.
The in vitro ndings and hypothesis of 8-Oxo-dI base pairing
stabilities of the order A > C > G agree with the in vivo data. As
with all of the nucleotides tested, the contribution of the oligo
sequence is important as it imposes restrictions on the result
obtained that is not observable in the in vitro work, underlining
the importance of these vital tests in the study of modied
nucleotides.
here. Wide base-pairing capabilities with the canonical bases is
shown by the apparent survival rates recovered through the
tests. As with the previous tests, sequencing was performed of
the now thymidine-prototrophic colonies (bla+ thyA+), showing
The 8-Oxo-dG and 8-Oxo-dA nucleotides show different
patterns of base-pairing discrimination. For 8-Oxo-dG, recog-
nition as an G appears strongest, with weak evidence it behaves
as a T, C or A. Base-pairing preferentiality in vivo appears in the
order of C > T > A [ G, with an aversion to base-pairing G also
shown in the sequencing data across the double (GCX) XCT
codons, which showed changes towards G, T and A in
descending order. The nding of mismatches are largely further
agreed upon by the in vitro dataset, although differences
Fig. 8 The in vivo interpretation of the N8-8-aza-dG nucleotide within
the essential thyA gene. The normalized ratio is the experimentally imposed by the primary sequence structure are observed in
derived average number of thymidine-prototrophic colonies (bla+
these tests.
thyA+) from the average total number of colonies (bla+ thyAꢁ and bla+
The 8-Oxo-dA nucleotide in contrast shows a much cleaner
thyA+). The modified section of the oligomer sequence indicates the
and less promiscuous base-pairing prole than 8-Oxo-dG. The
Chem. Sci., 2016, 7, 995–1010 | 1001
position of the N8-8-aza-dG nucleotide/s.
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Accordingly, both 8-Oxo-dG and 8-Oxo-dA nucleotides were
tested in the same assay in the equivalent strain of E. coli that
had been modied in the MutM locus, deleting the gene. On
following the same experimental procedure it was revealed that
there was in fact no signicant difference between the assay
response in the strain with and the strain without MutM,
indicating that the impact of MutM in this assay format was
negligible. This demonstrated that the known mutagenic base
(8-Oxo-dG) was not being modied in vivo during our assay by
MutM, and therefore not modifying our ndings.
Fig. 10 The in vivo interpretation of the 8-Oxo-dG nucleotide (left)
and 8-Oxo-dA nucleotide (right) within the essential thyA gene. The
normalized ratio is the experimentally derived average number of
thymidine-prototrophic colonies (bla+ thyA+) from the average total
number of colonies (bla+ thyAꢁ and bla+ thyA+). The modified section
of the oligomer sequence indicates the position of the 8-Oxo-dG and
8-Oxo-dA nucleotide/s, for single (left) and double (right) codons for
each.
Conclusions
We have developed an efficient route for the synthesis of
oligonucleotides containing N8-8-aza-dG, N8-8-aza-9-deaza-dG,
8-NH2-dI, 1-Me-8-NH2-dI, 8-Oxo-dI, 8-Oxo-dA, and 8-Oxo-dG
nucleotides on solid phase support by using phosphoramidite
building blocks 18a, 18b, 24a, 24b, 26, 29, and 32 respectively.
The base pairing and mismatch discrimination of N8-8-aza-9-
deaza-dG, N8-8-aza-dG with d-isoCMe, 8-NH2-dI, 1-Me-8-NH2-dI,
8-Oxo-dI, 8-Oxo-dA, and 8-Oxo-dG in duplex DNA with antipar-
allel chain orientation were determined by Tm measurements.
The new base pairs form less stable duplexes compared to the
dC:dG and d-isoCMe:d-isoG pairs. Incorporation of 8-NH2-dI
versus N8-8-aza-dG nucleoside gives a greater decrease in Tm
than of d-isoCMe versus N8-8-aza-dG. Insertion of the methyl
group at N1 position of 8-NH2-dI does not affect duplex stability,
which suggests that H-bonding with N8-8-aza-dG takes place via
Hoogsteen base pairing. As could be expected there is no
straight correlation between duplex stability (Tm measurement)
and base pair recognition in vivo. For example, N8-8-aza-dG is
best recognized by G in vitro (Tm) and by T in vivo (E. coli). This,
of course, reects the contribution of the polymerase in base-
pairing recognition.
in vivo data indicates that this nucleotide is strongly recognized
as an A, affording a base-pairing order of T [ A [ G ¼ C.
Sequencing of the positive clones showed widespread changes
to A, especially within the GCX codon (tolerating the silent
change) and in (GCX) XCT which promoted a change to Thr
(with poor in vivo survival compared to wild-type). As with the
other nucleotides, the base pairing stability determined in vitro
of T > G [ A > C agrees well with the ndings in vivo, possibly
with sequence dependent effects.
It was suspected that the DNA repair pathways of the host test
strain of E. coli might have been interacting with and perhaps
modifying the 8-oxo modications of these compounds. It is
widely reported that the gene MutM (encoding the MutM DNA
glycosylase) can recognize 8-Oxo-dG and remove it from DNA
towards protection against mutagenic lesions.44,45
From all of the tests in vivo, the compounds belonging to the
family of 8-amino-deoxyinosines (1-Me-8-NH2-dI and 8-NH2-dI)
and N8-8-aza-9-deaza-dG have shown to be the most orthogonal
from the E. coli genetic system and the base-pairing rules of the
canonical bases (A, T, G and C). The 8-aza-deoxyguanosines (N8-
8-aza-dG and N8-8-aza-9-deaza-dG) were recognized largely as
A (base pairing T), although the 9-deaza modications affected
general pairing with canonical bases and afforded more
orthogonality against the unsubstituted N8-8-aza-dG. The 9-
deaza modications did not change base pairing preferentiality,
maintaining the ndings observed in vitro.
In testing the 8-Oxo-dG and 8-Oxo-dA nucleosides, a side test
was performed aer deleting the repair enzyme MutM. This
revealed that at least this enzyme was not modifying the 8-oxo-
deoxynucleosides and therefore not giving an incorrect view
into the orthogonality of the nucleoside (as if it had been cor-
rected to a G through the repair mechanism).
Fig. 11 The in vivo interpretation of the 8-Oxo-dI nucleotide within
the essential thyA gene. The normalized ratio is the experimentally
derived average number of thymidine-prototrophic colonies (bla+
thyA+) from the average total number of colonies (bla+ thyAꢁ and bla+
thyA+). The modified section of the oligomer sequence indicates the
position of the 8-Oxo-dI nucleotide/s, for single (top) and double
(bottom) codons.
Between the in vitro and in vivo tests it appears that a base
pairing of either of the N8-glycosylated nucleosides against 8-
NH2-dI or 1-Me-8-NH2-dI may identify a near orthogonal pair.
Poor responses in vivo for these two pairs of nucleosides suggest
that they are not strongly pairing with the canonical bases. The
biggest problem foreseeable in this pair is the tendency for
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these bases to lack strong discrimination against the canonical
base G, although these pairs would be discriminative against A,
T and C.
5-Amino-2-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-
D-ribofuranosyl]-2H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one
(14a)
To a suspension of the intermediate 10a (3 g, 10.55 mmol), in
anhydrous pyridine (52 mL) under argon atmosphere, 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (3.63 mL, 11.61 mmol)
Experimental section
1H, 13C and 31P NMR spectra were recorded on 300 MHz (1H at 0 ꢀC was added and then the mixture was stirred at room
NMR, 300 MHz; 13C NMR, 75 MHz; 31P NMR, 121 MHz) or 500
temperature for 8 h. The reaction was quenched by the addition
MHz (1H NMR, 500 MHz; 13C NMR, 125 MHz) or 600 MHz (1H
of MeOH. The solvents were evaporated and the product 14a
(4.3 g, 77%) was isolated by ash column chromatography
NMR, 600 MHz; 13C NMR, 150 MHz) spectrometers. 2D NMRs
(H-COSY, HSQC and HMBC) were used for the assignment of all (hexane/EtOAc 1 : 1). 1H NMR (600 MHz, DMSO-d6): d 11.09 (br
the intermediates and nal compounds. Mass spectra were s, 1H, NH), 6.62 (br s, 2H, NH2), 5.89 (s, 1H, H-10), 5.75 (d, J ¼ 4.6
acquired on a quadrupole orthogonal acceleration time-of-ight Hz, 1H, OH), 4.89 (dd, J ¼ 8.5, 4.6 Hz, 1H, H-30), 4.44 (t, J ¼ 3.7
mass spectrometer. Column chromatographic separations were Hz, 1H, H-20), 4.05–3.99 (m, 1H, H-5a0), 3.95–3.79 (m, 2H, H-40,
carried out by gradient elution with suitable combination of H-5b0), 1.12–0.87 [m, 28H, 4 ꢃ CH(CH3)2]; 13C NMR (151 MHz,
DMSO-d6): d 159.7 (C7), 156.8 (C3a), 154.7 (C5), 127.5 (C7a), 96.2
(C10), 81.4 (C40), 74.4 (C30), 71.5 (C20), 61.6 (C50), 17.4, 17.3, 17.2,
17.1, 17.0, 16.96, 16.91, 12.8, 12.5, 12.3, 12.2, 12.1 [CH(CH3)2];
HRMS (ESI+) calcd for C21H38N6NaO6Si2 [M + Na]+ 527.2463,
found 527.2469.
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 Na/benzophenone; CH2Cl2 and CH3CN from
CaH2; Et3N and pyridine from KOH.
5-Amino-2-(2,3,5-O-triacetyl-b-D-ribofuranosyl)-2H-[1,2,3]
triazolo[4,5-d]pyrimidin-7-one (9)
5-Amino-2-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-
deoxy-b-D-ribofuranosyl]-2H-[1,2,3]triazolo[4,5-d]pyrimidin-7-
one (15a)
b-D-Ribofuranose-1,2,3,5-tetraacetate (8.06 g, 23.01 mmol) was
added to a solution of compound 8 (3.50 g, 25.31 mmol) in
acetonitrile (115 mL). The reaction mixture was heated to 75 ꢀC Compound 14a (2 g; 3.80 mmol) was treated with 1,10-thio-
and boron triuoride diethyl etherate (3.03 mL, 24.16 mmol) carbonyldiimidazole (0.676 g; 3.80 mmol) in dry CH2Cl2 (38 mL)
was added. Aer 3 h the volatiles were removed in vacuo and the for 8 h at room temperature under argon atmosphere. The
residue was puried by ash chromatography (hexane/EtOAc solvent was evaporated and the residue was dissolved in CH2Cl2
1
7 : 3) to give the product 9 (5.10 g, 54%). H NMR (300 MHz, (20 mL) and washed with H2O (10 mL). The aqueous layer was
DMSO-d6): d 11.01 (br s, 1H, NH), 6.67 (br s, 2H, NH2), 6.32 (d, J extracted twice with CH2Cl2 (10 mL); the combined organic
¼ 2.8 Hz, 1H, H-10), 5.84–5.75 (m, 1H, H-20), 5.68–5.49 (m, 1H, layers were dried over Na2SO4 and evaporated. The crude
H-30), 4.54–4.33 (m, 2H, H-40, H-5a0), 4.07 (dd, J ¼ 12.1, 4.8 Hz, product obtained as slightly yellow foam, was used without
1H, H-5b0), 2.09 (s, 6H, CH3), 1.98 (s, 3H, CH3); 13C NMR (75 further purication in the next step. The crude product was
MHz, DMSO-d6): d 169.9 (C]O), 169.5 (C]O), 169.2 (C]O), dissolved in dry toluene (76 mL), AIBN (0.125 g; 0.76 mmol) and
159.6 (C7), 156.5 (C3a), 154.6 (C5), 128.0 (C7a), 93.6 (C10), 80.0 Bu3SnH (2.56 mL; 9.50 mmol) were added under argon atmo-
(C40), 73.2 (C30), 70.3 (C20), 62.3 (C50), 20.4, 20.3 (CH3); HRMS sphere and the mixture was degassed with argon for 15 min.
(ESI+) calcd for C15H18N6NaO8 [M + Na]+ 433.1078, found Aerwards, the reaction was heated at 75 C for 2 h. The solu-
ꢀ
433.1076.
tion was evaporated in vacuo, and the product was puried by
ash chromatography (hexane/EtOAc 1 : 1). The compound 15a
was obtained as white solid (1.20 g, 62% over two steps). 1H
NMR (600 MHz, DMSO-d6): d 11.46 (s, 1H, NH), 6.87 (s, 2H,
NH2), 6.30 (d, J ¼ 7.5 Hz, 1H, H-10), 5.12 (m, 1H, H-30), 3.91–3.79
(m, 2H, H-40, H-5a0), 3.72 (dd, J ¼ 12.3, 8.8 Hz, 1H, H-5b0), 2.79
(m, 1H, H-2a0), 2.64–2.50 (m, 1H, H-2b0), [m, 28H, 4 ꢃ
CH(CH3)2]. 13C NMR (151 MHz, DMSO-d6): d 159.6 (C7), 157.0
(C3a), 155.1 (C5), 127.3 (C7a), 91.0 (C10), 84.8 (C40), 72.6 (C30),
63.6 (C50), 40.1 (C20), 17.3, 17.3, 17.2, 17.1, 16.9, 16.83, 16.81,
12.7, 12.5, 12.2, 12.1, 12.0 [CH(CH3)2]. HRMS (ESI+) calcd for
5-Amino-2-(b-D-ribofuranosyl)-2H-[1,2,3]triazolo[4,5-d]
pyrimidin-7-one (10a)46
Concentrated ammonium hydroxide (48 mL) solution was
added to compound 9 (5 g, 12.19 mmol) and the mixture was
stirred for 8 h at room temperature. The aqueous solution
was dried by lyophilization. The residue was puried by ash
column chromatography (CH2Cl2/MeOH 8.5 : 1.5) to yield the
title compound 10a as a white solid (3.17 g, 91%). 1H NMR (300
MHz, DMSO-d6): d 10.96 (br s, 1H, NH), 6.57 (br s, 2H, NH2), 5.87
(d, J ¼ 3.8 Hz, 1H, H-10), 5.60 (br s, 1H, OH), 5.24 (br s, 1H, OH),
4.79–4.77 (m, 1H, OH), 4.49–4.48 (m, 1H, H-20), 4.25–4.21 (m,
1H, H-30), 4.00–3.97 (m, 1H, H-40), 3.60–3.56 (m, 1H, H-5a0),
3.50–3.44 (m, 1H, H-5b0); 13C (75 MHz, DMSO-d6): d 159.6 (C7),
C
21H39N6O5Si2 [M + H]+ 511.2514, found 511.2517.
5-Amino-2-(2-deoxy-b-D-ribofuranosyl)-2H-[1,2,3]triazolo[4,5-
d]pyrimidin-7-one (1a)21
156.8 (C3a), 154.5 (C5), 127.5 (C7a), 96.8 (C10), 86.2 (C40), 74.6 Compound 15a (1.1 g; 2.15 mmol) was dissolved in dry THF
(C30), 70.8 (C20), 62.2 (C50); HRMS (ESI+) calcd for C9H12N6NaO5 (14 mL) and tetrabutylammonium uoride solution 1.0 M in
[M + Na]+ 307.0761, found 307.0772.
THF (6.46 mL, 6.46 mmol) was added. The reaction was stirred
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for 3 h at room temperature. The solvent was evaporated in was added and the reaction was stirred for 30 minutes at room
vacuo, and the crude residue was puried by ash chromatog- temperature. Aerwards, b-D-ribofuranose-1,2,3,5-tetraacetate
raphy (CH2Cl2/MeOH 9 : 1) to yield the product 1a as white solid (3.18 g, 10 mmol) was added into the reaction mixture, followed
(0.560 g, 97%). Spectral and analytical data were in agreement by 1 M solution of stannic chloride in CH2Cl2 (8 mL, 8 mmol)
with previous report.21
and the resulting mixture was stirred at room temperature for
36 h. The reaction solution was quenched with cold saturated
aq. NaHCO3 (20 mL) and extracted with CH2Cl2. The organics
were washed with sat. aq. NaHCO3 (2 ꢃ 15 mL) and brine (2 ꢃ
15 mL), dried over MgSO4, ltered, concentrated, and puried
by ash column chromatography (hexane/EtOAc 7 : 3) to give
compound 12 (3.8 g, 85%) as light yellow oil. 1H NMR (300 MHz,
CDCl3): d 8.47 (s, 1H, H-5), 5.91 (d, J ¼ 2.9, 1H, H-10), 5.70 (dd,
J ¼ 5.2, 2.9 Hz, 1H, H-20), 5.45 (dd, J ¼ 6.2, 5.2 Hz, 1H, H-30),
4.47–4.22 (m, 4H, H-40, H-5a0, OCH2), 4.25 (dd, J ¼ 12.5, 3.6 Hz,
1H, H-5b0), 2.15 (s, 6H, CH3), 2.07 (s, 3H, CH3), 1.38 (t, J ¼ 7.1
Hz, 3H, CH3); 13C (75 MHz, CDCl3): d 170.2, 169.2, 169.0 (C]O),
159.8 (C]O), 140.1 (C4), 134.2 (C3), 129.3 (C5), 92.5 (C10), 80.6
(C40), 74.2 (C30), 69.5 (C20), 62.5 (C50), 62.0 (CH2O), 20.5, 20.2,
13.8 (CH3); HRMS (ESI+) calcd for C17H21N3O11 [M + Na]+
466.1068, found 466.1063.
2-(2-Deoxy-b-D-ribofuranosyl)-5-(dimethylaminomethylidene)-
2H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (16a)21
To a solution of compound 1a (0.50 g, 1.86 mmol) in MeOH
(12 mL) was added dimethylformamide dimethyl acetal (499 mL,
3.73 mmol), and the mixture was heated at 50 C for 1 h. The
reaction was cooled to room temperature and concentrated
under reduced pressure. The crude residue was puried by ash
column chromatography (CH2Cl2/MeOH 95 : 5) to yield the
compound 16a as a white solid (0.56 g, 93%). Spectral and
analytical data were in agreement with previous report.21
ꢀ
2-(5-O-Dimethoxytrityl-2-deoxy-b-D-ribofuranosyl)-5-
(dimethylaminomethylidene)-2H-[1,2,3]triazolo[4,5-d]
pyrimidin-7-one (17a)21
The compound 16a (0.50 g, 1.55 mmol) was co-evaporated with
dry pyridine twice under argon atmosphere and then dissolved
in dry pyridine (15 mL). 4,40-Dimethoxytrityl chloride (0.524 g,
1.55 mmol) in CH2Cl2 (1.5 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
dissolved in CH2Cl2 and washed with H2O, the organic layers
were dried on Na2SO4 and evaporated under argon atmosphere.
The compound 17a was isolated by column chromatography
(CH2Cl2/MeOH/TEA 97 : 2 : 1) as a white solid (0.81 g, 83%). Spec-
tral and analytical data were in agreement with previous report.21
Ethyl 4-amino-1-(2,3,5-O-triacetyl-b-D-ribofuranosyl)-1H-
pyrazole-3 (13)
To a solution of compound 12 (3.7 g, 8.35 mmol) in MeOH (41
mL) was added 10% Pd/C (88 mg), and the mixture was stirred
at room temperature for 8 h under a hydrogen atmosphere
(1 atm). The catalyst was removed by ltration over celite. The
ltrate was concentrated and compound 13 (3.45 g, 100%) was
1
used as such in the next step. H NMR (300 MHz, DMSO-d6):
d 7.38 (s, 1H, H-5), 5.99 (d, J ¼ 4.2 Hz, 1H, H-10), 5.70 (dd, J ¼ 5.2,
2.9 Hz, 1H, H-20), 5.45 (dd, J ¼ 6.2, 5.2 Hz, 1H, H-30), 4.47–4.22
(m, 4H, H-40, H-5a0, OCH2), 4.25 (dd, J ¼ 3.6, 12.5 Hz, 1H, H-5b0),
2.15 (s, 6H, CH3), 2.07 (s, 3H, CH3), 1.28 (t, J ¼ 7.1 Hz, 3H, CH3);
13C (75 MHz, DMSO-d6): d 170.1, 169.5, 169.3, 162.8 (C]O),
135.7 (C4), 130.6 (C3), 116.2 (C5), 90.7 (C10), 79.6 (C40), 72.9
(C30), 70.4 (C20), 62.8 (C50), 59.8 (CH2O), 20.5, 20.3, 14.3 (CH3);
HRMS (ESI+) calcd for C17H23N3O9 [M + Na]+ 436.1326, found
436.1331.
2-(5-O-Dimethoxytrityl-2-deoxy-b-D-ribofuranosyl)-5-
(dimethylaminomethylidene)-2H-[1,2,3]triazolo[4,5-d]
pyrimidin-7-one-4-(2-cyanoethyl-N,N-diisopropyl)
phosphoramidite (18a)
To a stirred solution of 17a (0.320 g, 0.511 mmol) and 1 M
solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine
(767 mL, 0.767 mmol) in anhydrous CH2Cl2 (4 mL), under argon
5-Amino-2-(b-D-ribofuranosyl)-2H-pyrazolo[4,3-d]pyrimidin-7-
one (10b)
ꢀ
atmosphere and at 0 C, was added 0.45 M solution of 1H-ter-
A mixture of 13 (1.2 g, 2.9 mmol), chloroformamidine hydro-
azole (1.13 mL, 0.562 mmol) dropwise. Aer 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 with
CH2Cl2 and was washed with 1 M TEAB solution. The extracts
were dried over Na2SO4 and concentrated in vacuo. The crude
mixture was puried by ash column chromatography (hexane/
acetone/TEA, 65 : 34 : 1) to afford amidite 18a (0.310 g, 73%
yield). 31P NMR (121 MHz, CDCl3) d 149.3, 149.2. HRMS (ESI+)
calcd for C42H53N9O7P [M + H]+ 826.3799, found 826.3818.3
chloride (0.84 g, 7.26 mmol) and dimethylsulfone (1.37 g, 14.5
mmol), was heated for 1 hour at 120 C in an open ask with
ꢀ
magnetic stirring. Aer the mixture was cooled to room
temperature, H2O (5 mL) was added. The solution was then
neutralized with NH4OH, stirred for 1 h and then the aqueous
solution was dried by lyophilization. The crude residue was
puried by ash column chromatography (CH2Cl2/MeOH 8 : 2)
1
to yield the compound 10b as a white solid (0.35 g, 42%). H
NMR (300 MHz, DMSO-d6): d 10.95 (br s, 1H, NH), 8.00 (s, 1H, H-
9), 6.07 (br s, 2H, NH2), 5.72 (d, J ¼ 4.1 Hz, 1H, H-10), 5.54 (d, J ¼
5.0 Hz, 1H, OH), 5.19 (m, 1H, OH), 4.98 (t, J ¼ 5.8 Hz, 1H, OH),
4.31–4.29 (m, 1H, H-20), 4.14–4.09 (m, 1H, H-30), 3.95–3.91 (m,
Ethyl 1-(2,3,5-O-triacetyl-b-D-ribofuranosyl)-4-nitro-1H-
pyrazole-3-carboxylate (12)
To the compound 11 (1.85 g, 10 mmol) in 66 mL of 1,2-dichlo- 1H, H-40), 3.65–3.60 (m, 1H, H-5a0), 3.54–3.33 (m, 1H, H-5b0); 13C
roethane, N,O-bis(trimethylsilyl) acetamide (4.9 mL, 20 mmol) (75 MHz, DMSO-d6): d 157.3 (C7), 151.2 (C5), 139.3 (C3a), 133.4
1004 | Chem. Sci., 2016, 7, 995–1010
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(C7a), 119.5 (C3), 94.9 (C10), 85.4 (C40), 75.1 (C30), 70.3 (C20), 61.4 (C3a), 133.1 (C7a), 119.3 (C3), 90.6 (C10), 88.1 (C40), 70.5 (C30),
(C50); HRMS (ESI+) calcd for C10H13N5O5 [M + Na]+ 306.0809, 61.8 (C50), 40.0 (C20); HRMS (ESI+) calcd for C10H14N5O4 [M +
found 306.0810.
H]+ 268.1040, found 268.1055.
5-Amino-2-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-
D-ribofuranosyl]-2H-pyrazolo[4,3-d]pyrimidin-7-one (14b)
2-(2-Deoxy-b-D-ribofuranosyl)-5-(dimethylaminomethylidene)-
2H-pyrazolo[4,3-d]pyrimidin-7-one (16b)
Reaction of compound 10b (0.8 g, 2.82 mmol), in anhydrous Reaction of compound 1b (0.430 g, 1.61 mmol) with dime-
pyridine (14 mL) under argon atmosphere with 1,3-dichloro- thylformamide dimethyl acetal (430 mL, 3.22 mmol) in MeOH
1,1,3,3-tetraisopropyldisiloxane (970 mL, 3.11 mmol), was (10 mL) was carried out as described for compound 16a and
carried out as described for compound 14a and puried by ash puried by ash column chromatography (CH2Cl2/MeOH
column chromatography (CH2Cl2/MeOH 95 : 5) affording 95 : 5) to yield the compound 16b as a white solid (0.50 g, 96%).
compound 14b as white solid (1.1 g, 74%) by 1H NMR (300 MHz, 1H NMR (500 MHz, DMSO-d6): d 11.11 (br s, 1H, NH), 8.54 (s,
DMSO-d6): d 10.60 (br s, 1H, NH), 7.83 (s, 1H, H-9), 5.96 (br s, 1H, N]CH), 8.16 (s, 1H, H-9), 6.19 (t, J ¼ 6.1 Hz, 1H, H-10), 5.32
2H, NH2), 5.79 (s, 1H, H-10), 5.69 (d, J ¼ 4.4 Hz, 1H, OH), 4.59 (d, J ¼ 4.3 Hz, OH), 4.92 (t, J ¼ 5.4 Hz, 1H, OH), 4.38 (m, 1H, H-
(dd, J ¼ 8.2, 4.6 Hz, 1H, H-30), 4.29 (t, J ¼ 4.4 Hz, 1H, H-20), 4.03– 30), 3.87–3.85 (m, 1H, H-40), 3.62–3.51 (m, 1H, H-5a0), 3.49–3.40
3.98 (m, 2H, H-40, H-5a0), 3.90–3.87 (m, 1H, H-5b0), 1.05–0.94 [m, (m, 1H, H-5b0), 3.12 (s, 3H, NCH3), 3.00 (s, 3H, NCH3), 2.62–2.57
28H, 4 ꢃ CH(CH3)2]; 13C (75 MHz, DMSO-d6): d 156.9 (C7), 150.9 (m, 1H, H-2a0), 2.34–2.29 (m, 1H, H-2b0); 13C NMR (125 MHz,
(C5), 139.1 (C3a), 133.9 (C7a), 119.8 (C3), 94.0 (C10), 80.9 (C40), DMSO-d6): d 158.0 (N]CH), 157.4 (C7), 155.1 (C5), 138.3 (C3a),
75.8 (C20), 70.1 (C30), 60.3 (C50), 17.3, 17.2, 17.1, 17.1, 16.9, 16.8, 134.6 (C7a), 121.0 (C3), 90.7 (C10), 88.2 (C40), 70.4 (C30), 61.7
13.0, 12.8, 12.7, 12.4, 12.3, 12.2 [CH(CH3)2]; HRMS (ESI+) calcd (C50), 40.4 (NCH3), 39.8 (C20), 34.5 (NCH3); HRMS (ESI+) calcd
for C22H40N5O6Si2 [M + H]+ 526.2511, found 526.2507.
for C13H19N6O4 [M + H]+ 323.1462, found 323.1455.
5-Amino-2-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-
deoxy-b-D-ribofuranosyl]-2H-pyrazolo[4,3-d]pyrimidin-7-one
(15b)
2-(5-O-Dimethoxytrityl-2-deoxy-b-D-ribofuranosyl)-5-
(dimethylaminomethylidene)-2H-pyrazolo[4,3-d]pyrimidin-7-
one (17b)
Compound 14b (1.2 g; 2.28 mmol) was treated with 1,10-thio- The reaction of compound 16b (0.450 g, 1.4 mmol) in dry
carbonyldiimidazole (0.406 g; 2.28 mmol) in dry CH2Cl2 (22 mL) pyridine (14 mL) and 4,40-dimethoxytrityl chloride (0.473 g, 1.4
for 8 h at 35 ꢀC then with AIBN (75 mg; 0.456 mmol) and mmol) was carried out as described for compound 17a and
Bu3SnH (1.54 mL; 5.70 mmol) in dry toluene (45 mL), was puried by ash column chromatography (CH2Cl2/MeOH/TEA
carried out as described for compound 15a and puried by ash 97 : 2 : 1), affording compound 17b as a white solid (0.665 g,
column chromatography (1% to 3% MeOH in CH2Cl2). The 76%). 1H NMR (500 MHz, DMSO-d6): d 11.20 (br s, 1H, NH), 8.53
compound 15b was obtained as white powder (0.790 g, 68% over (s, 1H, N]CH), 8.12 (s, 1H, H-9), 7.35–7.08 (m, 9H, ArH), 6.80–
two steps). 1H NMR (500 MHz, DMSO-d6): d 10.83 (br s, 1H, NH), 6.73 (m, 4H, ArH), 6.26 (dd, J ¼ 6.7, 3.8 Hz, 1H, H-10), 5.35 (s, 1H,
7.82 (s, 1H, H-9), 6.16 (d, J ¼ 7.3 Hz, 1H, H-10), 6.13 (s, 2H, NH2), OH), 4.46–4.43 (m, 1H, H-30), 4.00–3.84 (m, 1H, H-40), 3.71 (s,
4.97 (m, 1H, H-30), 3.85–3.79 (m, 2H, H-40, H-5a0), 3.74 (dd, J ¼ 3H, OCH3), 3.69 (s, 3H, OCH3), 3.13 (s, 3H, NCH3), 3.10–3.04 (m,
12.4, 8.3 Hz, 1H, H-5b0), 2.74–2.70 (m, 1H, H-2a0), 2.50–2.45 (m, 2H, H-5a0,b0), 3.01 (s, 3H, NCH3), 2.85–2.67 (m, 1H, H-2a0), 2.34–
1H, H-2b0), 1.05–0.94 [m, 28H, 4 ꢃ CH(CH3)2]; 13C NMR (125 2.30 (m, 1H, H-2b0); 13C NMR (125 MHz, DMSO-d6): d 158.0 (N]
MHz, DMSO-d6): d 157.0 (C7), 151.0 (C5), 139.2 (C3a), 133.6 CH), 157.9, 157.5, 157.4 (Ar, C7), 155.2, 155.1 (Ar, C5), 144.9,
(C7a), 119.7 (C3), 88.6 (C10), 84.8 (C40), 72.1 (C30), 63.3 (C50), 40.1 135.7, 135.6, 135.5, 134.7, 134.6 (C7a, C3a, Ar), 129.8, 129.5,
(C20), 17.4, 17.3, 17.2, 17.1, 17.0, 16.9, 16.8, 13.1, 12.8, 12.5, 12.3, 127.7, 126.5, 121.4, 113.1, 91.1 (Ar), 89.9 (C10), 85.9 (C40), 85.3
12.0 [CH(CH3)2]; HRMS (ESI+) calcd for C22H40N5O5Si2 [M + H]+ (CPh3), 70.3 (C30), 63.9 (C50), 55.0 (2 ꢃ OCH3), 40.5 (NCH3), 40.0
510.2562, found 510.2561.
(C20), 39.8 (NCH3); HRMS (ESI+) calcd for C34H37N6O6 [M + H]+
625.2768, found 625.2748.
5-Amino-2-(2-deoxy-b-D-ribofuranosyl)-2H-pyrazolo[4,3-d]
pyrimidin-7-one (1b)
2-(5-O-Dimethoxytrityl-2-deoxy-b-D-ribofuranosyl)-5-
(dimethylaminomethylidene)-2H-pyrazolo[4,3-d]pyrimidin-7-
one-3-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (18b)
Reaction of compound 15b (0.8 g; 1.57 mmol) with tetrabuty-
lammonium uoride in THF (4.71 mL, 4.71 mmol) in dry THF
(9.4 mL) and was carried out as described for compound 1a and The reaction of compound 17b (0.250 g, 0.40 mmol) and 1 M
puried by ash column chromatography (CH2Cl2/MeOH 8 : 2) bis(diisopropylamino)(2-cyanoethoxy)phosphine (600 mL, 0.60
1
to yield the product 1b as white solid (0.395 g, 94%). H NMR mmol) and 0.45 M 1H-terazole (880 mL, 0.440 mmol) in anhy-
(300 MHz, DMSO-d6): d 10.38 (br s, 1H, NH), 7.95 (s, 1H, H-9), drous CH2Cl2 (4 mL), was carried out as described for
6.14 (t, J ¼ 4.1 Hz, 1H, H-10), 5.94 (br s, 2H, NH2), 5.28 (d, J ¼ 4.3 compound 18a and puried by ash column chromatography
Hz, 1H, OH), 4.87 (t, 1H, J ¼ 5.5 Hz, OH), 4.37–4.34 (m, 1H, H- (hexane/acetone/TEA, 55 : 44 : 1) afforded amidite 18b (0.230 g,
30), 3.86–3.82 (m, 1H, H-40), 3.57–3.50 (m, 1H, H-5a0), 3.46–3.40 69% yield). 31P NMR (121 MHz, CDCl3) d 149.1, 148.8. HRMS
(m, 1H, H-5b0), 2.62–2.54 (m, 1H, H-2a0), 2.32–2.28 (m, 1H, H- (ESI+) calcd for C43H53N8O7P [M + H]+ 825.3847, found
2b0); 13C (75 MHz, DMSO-d6): d 157.3 (C7), 150.9 (C5), 139.2 825.3849.
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8-Azido-30-50-O-di(tertbutyldimethylsilyl)-20-deoxyinosine
(20a)
solution was stirred overnight at room temperature. Aer
removing volatiles, the crude residue was puried by ash
column chromatography (CH2Cl2/MeOH 20/1) to yield
compound 22a (0.19 g, 60%) as a white solid. 1H NMR (300
MHz, DMSO-d6): d 8.56 (s, 1H, N]CH), 7.82 (s, 1H, H-2), 6.50 (t,
J ¼ 6.4 Hz, 1H, H-10), 4.41–4.42 (m, 1H, H-30), 3.82 (br s, 1H, H-
40), 3.64 (dd, J ¼ 11.8, 4.2 Hz, 1H, H-5a0), 3.48 (dd, J ¼ 11.7, 4.3
Hz, 1H, H-5b0), 3.14 (s, 3H, NCH3), 3.02 (s, 4H, NCH3, H-2a0),
1.98–2.05 (m, 1H, H-2b0); 13C-NMR (75 MHz, DMSO-d6): d 159.0,
156.8, 153.0, 147.3, 145.1 (C6, C]N, C2, C8, C4), 122.3 (C5),
87.7, 82.8 (C10, C40), 71.6 (C30), 62.5 (C50), 40.2 (NCH3), 37.4
(C20), 34.2 (NCH3). HRMS: calcd for C13H19N6O4 [M + H]+
323.1462, found 323.1474.
To a solution of compound 19 (1.0 g, 2.08 mmol) in dry THF
ꢀ
(40 mL) under argon atmosphere at ꢁ78 C was added n-BuLi
(2.5 M solution in hexane, 4.2 mL, 10.4 mmol) and the solution
was stirred 1 h at ꢁ78 ꢀC. p-Toluenesulfonyl azide (0.1 M in
toluene, 13.7 mL, 6.24 mmol) was added dꢀrop wise and the
reaction mixture was stirred for 2 h at ꢁ78 C and quenched
with NH4Cl. Aer removing volatiles, the residue was extracted
with CH2Cl2. The combined organic layers were washed with
water, brine and dried over Na2SO4 and the solvent was removed
under reduced pressure and the residue was puried by ash
column chromatography (CH2Cl2/MeOH 50/1) to yield
compound 20a as light yellow solid (0.65 g, 60%). 1H NMR (300
MHz, CDCl3): d 13.5 (s, 1H, NH), 8.02 (s, 1H, H-2), 6.20 (t, J ¼ 6.9
Hz, 1H, H-10), 4.69–4.74 (m, 1H, H-30), 3.89–3.92 (m, 1H, H-40),
3.82 (dd, J ¼ 10.8, 6.5 Hz, 1H, H-5a0), 3.69 (dd, J ¼ 10.8, 4.7 Hz,
1H, H-5b0), 3.25 (quint, J ¼ 6.8 Hz, 1H, H-2a0), 2.14–2.21 (m, 1H,
H-2b0), 0.92 (s, 9H, CCH3), 0.86 (s, 9H, CCH3), 0.12 (s, 6H, SiCH3),
0.019 (s, 3H, SiCH3), ꢁ0.006 (s, 3H, SiCH3); 13C-NMR (75 MHz,
CDCl3): d 158.4 (C6), 149.3, 145.7, 143.7 (C4, C8, C2), 123.1 (C5),
87.9, 83.7 (C40, C10), 72.3 (C30), 62.8 (C50), 37.4 (C20), 26.0, 25.9
(CH3), 18.5, 18.1 (CCH3), ꢁ4.5, ꢁ4.6, ꢁ5.2, ꢁ5.3 (SiCH3). HRMS:
calcd for C22H40N7O4Si2 [M + H]+ 522.2675, found 522.2682.
8-Amino-N8-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-20-deoxyinosine (23a)
Reaction of compound 22a (0.16 g, 0.50 mmol) with 4,40-dime-
thoxytrityl chloride (0.168 g, 0.50 mmol) in dry pyridine/DMF
(16 mL, 7/1 v/v) was carried out as described for compound 17a
and puried by ash column chromatography (CH2Cl2/MeOH/
TEA 80/2/1) to yield compound 23a (0.3 g, 96%) as white solid.
1H NMR (500 MHz, DMSO-d6): d 8.53 (s, 1H, N]CH), 7.93 (s, 1H,
H-2), 7.27 (d, J ¼ 7.3 Hz, 2H, ArH), 7.30–7.27 (m, 8H, ArH), 6.85–
6.74 (m, 4H, ArH), 6.52 (t, J ¼ 7.2 Hz, 1H, H-10), 5.25 (d, J ¼ 5.25
Hz, OH), 4.47–4.43 (m, 1H, H-30), 3.97–3.70 (m, 1H, H-40), 3.72
(s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.451–3.26 (m, 2H, H-H-50),
3.14–3.09 (m, 4H, CH3, H-2a0), 2.94 (s, 3H, NCH3), 2.14–2.06 (m,
1H, H-2b0). 13C NMR (125 MHz, DMSO-d6) d 158.3, 157.4, 156.0,
154.9 (Ar, C6, C]N, C2, C8, C4), 147.0, 145.5, 136.2, 136.1,
130.1, 130.0, 128.0, 127.9, 126.8 (Ar), 122.0 (C5), 113.3, 113.3
(Ar), 86.0, 85.6 [C10, C(CH3)3], 82.5 (C40), 71.8 (C30), 64.9 (C50),
55.4 (OCH3), 36.9, 34.7, 33.7 (2NCH3, C20). HRMS: calcd for
8-Amino-N8-(dimethylaminomethylidene)-30-50-O-
di(tertbutyldimethylsilyl)-20-deoxyinosine (21a)
To a pre-cooled solution of compound 20a (600 mg, 1.15 mmol)
in MeOH (6 mL) was added NaBH4 (0.218 g, 5.75 mmol) portion
wise at 0 ꢀC. The reaction mixture was allowed to warm to room
temperature and stirred for 2 h. Aer the completion of the
reaction, excess NaBH4 was carefully quenched with saturated
NH4Cl (2 mL) and the solvents were removed under reduced
pressure. The crude product was used without further puri-
cation for the next step. The solution of crude product in N,N-
dimethylforamide-dimethylacetal (5 mL) was heated at 70 ꢀC for
1 h. Aer removing solvent, the crude residue was puried by
ash column chromatography (CH2Cl2/MeOH 30/1) to yield
compound 21a (448 mg, 70%) as light yellow solid. 1H NMR (300
MHz, CDCl3): d 13.4 (s, 1H, NH), 8.71 (s, 1H, N]CH), 7.75 (s,
1H, H-2), 6.64 (t, J ¼ 7.3 Hz, 1H, H-10), 4.71–4.72 (m, 1H, H-30),
3.83–3.92 (m, 2H, H-40, H-5a0), 3.68 (dd, J ¼ 4.2, 9.7 Hz, 1H, H-
5b0), 3.39 (quint, J ¼ 5.9 Hz, 1H, H-2a0), 2.08–2.15 (m, 1H, H-2b0),
0.91 (s, 9H, CCH3), 0.86 (s, 9H, CCH3), 0.10 (s, 6H, SiCH3), ꢁ0.01
(s, 3H, SiCH3), ꢁ0.02 (s, 3H, SiCH3); 13C-NMR (75 MHz, CDCl3):
d 158.7, 157.6, 155.8, 148.7, 141.6 (C6, C]N, C2, C8, C4), 123.1
(C5), 87.4, 83.1 (C10, C40), 73.2 (C30), 63.4 (C50), 40.9 (NCH3), 36.9
(C20), 34.8 (NCH3), 26.0, 25.9 (CH3), 18.5, 18.1 (CCH3), ꢁ4.5,
ꢁ5.1, ꢁ5.2 (SiCH3). HRMS: calcd for C25H47N6O4Si2 [M + H]+
551.3191, found 551.3198.
C
34H37N6O6 [M + H]+ 625.2769, found 625.2770.
8-Amino-N8-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-20-deoxyinosine-30-(2-cyanoethyl-N,N-
diisopropyl)-phosphoramidite (24a)
Reaction of compound 23a (0.2 g, 0.32 mmol) with 1 M bis
(diisopropylamino)(2-cyanoethoxy)phosphine (1 M in CH3CN,
0.96 mL, 0.96 mmol) and 1H-tetrazole (0.45 M in CH3CN, 1.56
mL, 0.70 mmol) in anhydrous CH2Cl2 (10 mL), was carried out
as described for compound 18a and puried by ash column
chromatography (CH2Cl2/MeOH/TEA 80/2/1) to yield compound
24a (0.20 g, 75%) as white solid. 31P NMR (121 MHz, CDCl3)
d 148.5, 148.0. HRMS: calcd for C13H19N6O4 [M + H]+ 323.1462,
found 323.1474.
8-Azido-30-50-O-di(tertbutyldimethylsilyl)-N1-methyl-20-
deoxyinosine (20b)
To a solution of sodium hydride (344 mg, 8.62 mmol) in dry THF
(30 mL), compound 20a (3.0 g, 5.75 mmol) in THF (50 mL) was
ꢀ
added drop wise at 0 C and stirred for 15 min. Methyl iodide
8-Amino-N8-(dimethylaminomethylidene)-20-deoxyinosine
(22a)
(0.72 mL, 11.50 mmol) was added slowly to the reaction mixture
at 0 ꢀC. The reaction mixture was stirred at room temperature for
To a solution of compound 21a (0.55 g, 1.0 mmol) in THF (10 12 h and then NH4Cl (5 mL) was added to the reaction mixture to
mL) was added TBAF (1 M in THF, 2.2 mL, 2.2 mmol) and the quench the excess sodium hydride and the reaction mixture was
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8-Amino-N8-(dimethylaminomethylidene)-50-O-
evaporated to dryness. The residue obtained was extracted with
CH2Cl2 (2 ꢃ 100 mL), washed with water (2 ꢃ 15 mL), brine (10
mL) and dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure and the residue was puried by
ash column chromatography (n-hexane/ethyl acetate 7/3) to
yield compound 20b as a yellow solid (2.71 g, 88%). 1H NMR (500
MHz, CDCl3) d 7.87 (s, 1H, H-2), 6.16 (t, J ¼ 6.9 Hz, 1H, H-10),
4.74–4.69 (m, 1H, H-30), 3.90–3.86 (m, 1H, H-40), 3.79 (dd, J ¼
10.9, 6.4 Hz, 1H, H-5b0), 3.68 (dd, J ¼ 10.9, 4.6 Hz, 1H, H-5a0), 3.63
(s, 3H, NCH3), 3.18 (pent, J ¼ 6.9 Hz, 1H, H-2b0), 2.15 (ddd, J ¼
10.8, 6.9, 3.9 Hz, 1H, H-2a0), 0.92 (s, 9H, CCH3), 0.85 (s, 9H,
CCH3), 0.11 (s, 6H, SiCH3), 0.02 (s, 3H, SiCH3), ꢁ0.01 (s, 3H,
SiCH3); 13C NMR (125 MHz, CDCl3) d 156.1 (C6), 147.7 (C4), 146.0
(C2), 145.1 (C8), 122.9 (C5), 87.6 (C40), 83.4 (C10), 72.1 (C30), 62.7
(C50), 37.3 (C20), 34.4 (NCH3), 26.0, 25.9 (CCH3), 18.4, 18.1 (CH3C),
ꢁ4.5, ꢁ4.6, ꢁ5.2, ꢁ5.3 (CH3Si); HRMS (ESI+): calcd for
C23H41N7O4Si2 [M + H]+ 536.2831; found 536.2832.
dimethoxytrityl-N1-methyl-20-deoxyinosine (23b)
Reaction of 22b (420 mg, 1.25 mmol) with 4,40-dimethoxytrityl
chloride (465 mg, 1.37 mmol) in dry pyridine (45 mL), in dry
CH2Cl2 (5 mL) was carried out as described for compound 17a
and puried by ash column chromatography (CH2Cl2/MeOH/
TEA 80/2/1) to yield compound 23b (710 mg, 89%) as white
solid. 1H NMR (500 MHz, CDCl3) d 8.68 (s, 1H, N]CH), 7.45 (s,
1H, H-2), 7.41 (d, J ¼ 7.2 Hz, 2H, Ar), 7.31–7.15 (m, 7H, Ar), 6.78–
6.73 (m, 4H, Ar), 6.66 (t, J ¼ 6.9 Hz, 1H, H-10), 4.85–4.80 (m, 1H,
H-30), 4.11–4.06 (m, 1H, H-40), 3.77 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3), 3.54 (s, 3H, NCH3), 3.52–3.45 (m, 1H, H-5b0), 3.32–3.25
(m, 2H, H-2b0, H-5b0), 3.07 (m, 3H, NCH3), 3.02 (m, 3H, NCH3),
2.28 (ddd, J ¼ 12.3, 6.9, 4.5 Hz, 1H, H-2a0); 13C NMR (125 MHz,
CDCl3) d 158.5 (Ar), 157.5 (N]C), 156.7 (C6), 155.4 (C8), 146.9
(C4), 145.0 (Ar), 143.7 (C2), 136.3, 136.1, 130.2, 128.2, 127.8,
126.7 (Ar), 122.8 (C5), 113.1 (Ar), 86.3 (CPh3), 85.5 (C40), 82.6
(C10), 73.6 (C30), 64.6 (C50), 55.3 (OCH3), 40.9 (NCH3), 37.2 (C20),
34.8, 34.1 (NCH3); HRMS (ESI+): calcd for C35H38N6O6 [M + H]+
639.2925; found 639.2926.
8-Amino-N8-(dimethylaminomethylidene)-30-50-O-
di(tertbutyldimethylsilyl)-N1-methyl-20-deoxy-inosine (21b)
The reaction of compound 20b (2.5 g, 4.67 mmol) with NaBH4
(882 mg, 23.33 mmol) in MeOH (26 mL) and then with N,N-
dimethylformamide-dimethylacetal (15 mL) was carried out as
described for compound 21a, and puried by ash column
chromatography (CH2Cl2/MeOH 15/1) to yield yellow solid (1.92
8-Amino-N8-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-N1-methyl-20-deoxyinosine-30-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite (24b)
The reaction of compound 23b with 1 M bis(diisopropyla-
mino)(2-cyanoethoxy)phosphine (1.18 mL, 1.40 mmol) and 0.45
M 1H-tetrazole (1.45 mL, 0.77 mmol) in CH2Cl2 (25 mL) was
carried out as described for compound 18a and puried by ash
column chromatography (CH2Cl2/MeOH/TEA 40/1/1) to yield
compound 24b (0.380 g, 77%) as white solid. 31P NMR (202 MHz,
CDCl3) d 148.6, 148.2. HRMS (ESI+): calcd for C44H55N8O7P [M +
H]+ 839.4003; found 839.4014.
1
g, 73%). H NMR (500 MHz, DMSO-d6) d 8.56 (s, 1H, CH]N),
8.18 (s, 1H, H-2), 6.47 (t, J ¼ 7.0 Hz, 1H, H-10), 4.71 (dt, J ¼ 6.7,
3.6 Hz, 1H, H-30), 3.80 (dd, J ¼ 10.4, 6.7 Hz, 1H, H-5b0), 3.77–3.72
(m, 1H, H-40), 3.59 (dd, J ¼ 10.4, 4.9, 1H, H-5a0), 3.47 (s, 3H,
NCH3), 3.28–3.21 (m, 1H, H-2b0), 3.15 (s, 3H, NCH3), 3.02 (s, 3H,
NCH3), 2.09 (ddd, J ¼ 10.9, 7.0, 3.6 Hz, 1H, H-2a0), 0.90 (s, 9H,
CCH3) 0.83 (s, 9H, CCH3), 0.11 (s, 6H, SiCH3), ꢁ0.01 (s, 3H,
SiCH3), ꢁ0.03 (s, 3H, SiCH3). 13C NMR (125 MHz, DMSO-d6)
d 157.1 (C]N), 155.7 (C6), 154.6 (C4), 146.8 (C8), 145.5 (C2),
121.7 (C5), 86.3 (C40), 81.9 (C10), 72.5 (C30), 62.8 (C50), 40.2
(NCH3), 36.2 (C20), 34.3 (NCH3), 33.4 (NCH3), 25.7 (CH3), 18.0
(CCH3), 17.7 (CCH3), ꢁ4.6 (SiCH3), ꢁ4.7 (SiCH3). HRMS (ESI+):
calcd for C23H43N5O4Si2 [M + H]+ 565.3348; found 565.3358.
7,8-Dihydro-8-oxo-20-deoxyinosine (3)29
A solution of NaNO2 (516 mg, 7.48 mmol) in (2 mL) of water was
added to a stirred solution of 1 g (3.74 mmol) of compound 5 in
50 mL of 95% aqueous acetic acid. The reaction mixture was
stirred overnight. The solvent was removed under reduced
pressure and the oily residue was puried by ash column
chromatography (CH2CI2/MeOH, 4 : 1) to give 3 (780 mg, 77%)
as a white solid. Spectral and analytical data were in agreement
with previous report.29
8-Amino-N8-(dimethylaminomethylidene)-N1-methyl-20-
deoxyinosine (22b)
The reaction of compound 21b (565 mg, 1.0 mmol) with TBAF
(1 M in THF, 2.2 mL, 2.2 mmol) in THF (10 mL) was carried out
as described for compound 22a and puried by ash column
chromatography (CH2CI2/MeOH, 9 : 1) to give 22b (246 mg,
73%) as a pale yellow solid. 1H NMR (500 MHz, DMSO-d6) d 8.56
(s, 1H, CH]N), 8.21 (s, 1H, H-2), 6.49 (t, J ¼ 6.4 Hz, 1H, H-10),
5.25 (s, 1H, OH), 4.96 (s, 1H, OH), 4.43 (br s, 1H, H-30), 3.81 (br s,
1H, H-40), 3.64 (dd, J ¼ 11.4, 4.6 Hz, 1H, H-5b0), 3.47 (br s, 4H,
NCH3, H-5a0), 3.14 (s, 3H, NCH3), 3.09–2.90 (m, 4H, NCH3, H-
2b0), 2.05 (ddd, J ¼ 8.8, 6.4, 2.0 Hz, 1H, H-2a0); 13C NMR (125
MHz, DMSO-d6) d 157.1 (C]N), 155.5 (C6), 154.4 (C8), 146.5
(C4), 145.6 (C2), 121.7 (C5), 87.5 (C40), 82.5 (C10), 71.3 (C30), 62.3
(C50), 40.3 (NCH3), 37.1 (NCH3), 34.4 (C20), 33.4 (NCH3); HRMS
7,8-Dihydro-50-O-dimethoxytrityl-8-oxo-20-deoxyinosine (25)29
Reaction of compound 3 (350 mg, 1.35 mmol) with 4,40-dime-
thoxytrityl chloride (486 mg, 1.44 mmol) in dry pyridine (70 mL)
was carried out as described for compound 17a and puried by
ash column chromatography (CH2CI2/MeOH/TEA, 95 : 4 : 1) to
give 25 (510 mg, 69%) as a white foam. Spectral and analytical
data were in agreement with previous report.29
7,8-Dihydro-50-O-dimethoxytrityl-8-oxo-20-deoxyinosine-30-
(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (26)29
(ESI+): calcd for C14H20N6O4 [M + Na]+ 359.1438; found The reaction of compound 25 (400 mg, 0.70 mmol) with 1 M
359.1444.
bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.40 mL, 1.40
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mmol) and 0.45 M 1H-tetrazole (1.7 mL, 0.77 mmol) in CH2Cl2 (ESI+): calcd for C43H53N8O7P [M + H]+ 825.3847; found
(25 mL) was carried out as described for compound 18a and 825.3842.
puried by ash column chromatography (n-hexane/acetone/
TEA, 39 : 60 : 1) to give 26 (430 mg, 80%) as a white foam.
Spectral and analytical data were in agreement with previous
report.29
7,8-Dihydro-N2-(dimethylaminomethylidene)-8-oxo-20-
deoxyguanosine (30)
To a suspension of compound 4 (1 g, 3.53 mmol) in MeOH
7,8-Dihydro-N6-(dimethylaminomethylidene)-8-oxo-20-
deoxyadenosine (27)
(25 mL), N,N-dimethylformamide dimethyl acetal (4.7 mL,
35.3 mmol) was added. The reaction mixture was stirred at 25 ꢀC
for 12 h. The resulting precipitate was isolated, and was washed
with CH2Cl2 to give 30 (600 mg, 50%) as a white solid. 1H NMR
(600 MHz, DMSO-d6) d 8.46 (s, 1H, N]CH), 6.10 (t, J ¼ 7.3 Hz,
1H, H-10), 5.16 (s, 1H, OH), 4.75 (s, 1H, OH), 4.39–4.33 (m, 1H,
H-30), 3.76–3.71 (m, 1H, H-40), 3.56 (dd, J ¼ 11.5, 4.6 Hz, 1H, H-
5b0), 3.44 (dd, J ¼ 11.5, 4.2 Hz, 1H, H-5a0), 3.12 (s, 3H, NCH3),
3.00 (s, 3H, NCH3), 2.99–2.91 (m, 1H, H-2b0), 1.94 (ddd, J ¼ 9.6,
6.7, 2.7 Hz, 1H, H-2a0); 13C NMR (150 MHz, DMSO-d6) d 157.8
(C]N), 156.4 (C6), 152.2 (C8), 151.7 (C2), 145.6 (C4), 102.2 (C5),
87.3 (C40), 81.1 (C10), 71.2 (C20), 62.3 (C50), 40.7 (NCH3), 36.0
(C20), 34.6 (NCH3); HRMS (ESI+): calcd for C13H18N6O5 [M + Na]+
361.1231; found 361.1227.
To a suspension of compound 5 (1 g, 3.74 mmol) in MeOH (35
mL), N,N-dimethylformamide dimethyl acetal (2.5 mL, 18.7
mmol) was added. The reaction mixture was stirred at 65 ꢀC for
3 h. The reaction mixture was evaporated to dryness and puri-
ed by ash column chromatography (CH2Cl2/MeOH : TEA,
1
90 : 9 : 1) to give 27 (1.1 g, 91%) as a white solid. H NMR (500
MHz, DMSO-d6) d 8.72 (s, 1H, HC]N), 8.19 (s, 1H, H-2), 6.17
(dd, J ¼ 8.2, 6.5 Hz, 1H, H-10), 5.20 (s, 1H, OH), 5.08 (s, 1H, OH),
4.42–4.38 (m, 1H, H-30), 3.83–3.79 (m, 1H, H-40), 3.62 (dd, J ¼
11.7, 4.5 Hz, 1H, H-5b0), 3.47 (br s, 1H, H-5a0), 3.14 (s, 1H,
NCH3), 3.10 (s, 1H, NCH3), 3.10–2.95 (m, 1H, H-2b0), 2.01 (ddd, J
¼ 9.2, 6.5, 2.6 Hz, H-2a0); 13C NMR (125 MHz, DMSO-d6) d 156.0
(C]N), 152.2 (C8), 149.6 (C2), 148.9 (C6), 148.3 (C4), 111.7 (C5),
87.5 (C40), 81.4 (C10), 71.4 (C30), 62.4 (C50), 40.4 (NCH3), 36.0
(C20), 34.3 (NCH3); HRMS (ESI+): calcd for C13H18N6O4 [M + H]+
323.1462; found 323.1455.
7,8-Dihydro-N2-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-8-oxo-20-deoxy guanosine (31)
The reaction of compound 30 (480 mg, 1.42 mmol) with 4,40-
dimethoxytrityl chloride (528 mg, 1.56 mmol) in dry pyridine
(60 mL) was carried out as described for compound 17a and
puried by ash column chromatography (CH2CI2/MeOH/TEA,
7,8-Dihydro-N6-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-8-oxo-20-deoxy adenosine (28)
The reaction of compound 27 (600 mg, 1.86 mmol) with 4,40-
dimethoxytrityl chloride (693 mg, 2.05 mmol) in dry pyridine (60
mL), was carried out as described for compound 17a and
puried by ash column chromatography (CH2CI2/MeOH/TEA,
95 : 4 : 1) to give 28 (850 mg, 73%) as a white foam. 1H NMR (600
MHz, DMSO-d6) d 8.70 (s, 1H, HC]N), 8.04 (s, 1H, H-2), 7.36–
7.34 (m, 2H, Ar), 7.24–7.14 (m, 7H, Ar), 6.71–6.81 (m, 4H, Ar),
6.17 (dd, J ¼ 7.2, 6.2 Hz, 1H, H-10), 4.52 (br s, 1H, H-30), 3.94–3.88
(m, 1H, H-40), 3.71 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.20–3.05
(m, 9H, 2 ꢃ NCH3, H-5a0,b0, H-2b0), 2.09 (ddd, J ¼ 12.7, 7.2, 4.8
Hz, 1H, H-2a0). 13C NMR (150 MHz, DMSO-d6) d 157.9, 157.8,
155.8, 152.3, 149.7, 148.5, 145.1 (C2, C4, C8, C6, C]N, Ar),
135.8, 135.6, 129.7, 129.5, 128.9, 127.6, 127.6, 127.41, 126.4,
113.0, 112.9, 112.7 (Ar), 111.7 (C5), 85.2 (CPh3), 85.1 (C40), 80.7
(C10), 71.0 (C30), 64.2 (C50), 55.0, 54.9 (OCH3), 40.4 (NCH3), 35.6
(C20), 34.3 (NCH3); HRMS (ESI+): calcd for C34H36N6O6 [M + H]+
625.2768; found 625.2776.
1
97 : 2 : 1) to give 31 (650 mg, 71%) as a white foam. H NMR
(600 MHz, DMSO-d6) d 11.53 (s, 1H, NH), 10.82 (s, 1H, NH), 8.33
(s, 1H, C]N), 7.36–7.31 (m, 2H, Ar), 7.25–7.15 (m, 7H, Ar), 6.72–
6.82 (m, 4H, Ar), 6.13 (dd, J ¼ 7.9, 5.0 Hz, 1H, H-10), 5.24 (d, J ¼
4.9 Hz, 1H, OH), 4.46 (quin, J ¼ 4.9 Hz, 1H, H-30), 3.81–3.85 (m,
1H, H-40), 3.72, 3.71 (OCH3), 3.20 (dd, J ¼ 9.9, 7.5 Hz, 1H, H-5b0),
3.07 (dd, J ¼ 9.9, 3.3 Hz, 1H, H-5a0), 3.02, 2.99 (NCH3), 2.90–2.96
(m, 1H, H-2b0), 2.09 (ddd, J ¼ 13.5, 7.9, 5.0 Hz, 1H, H-2a0); 13C
NMR (150 MHz, DMSO-d6) d 157.9, 157.8, 157.4, 156.0, 152.1,
151.6, 145.5, 145.1 (C6, C2, C]N, C8, C4, Ar), 135.8, 135.7,
129.6, 129.5, 127.7, 127.6, 126.4, 113.0, 112.9 (Ar), 112.7 (C5),
102.2 (CPh3), 85.1 (C40), 80.3 (C10), 70.9 (C30), 64.5 (C50), 54.9,
54.9 (OCH3), 40.6 (NCH3), 36.4 (C20), 34.6 (NCH3); HRMS (ESI+):
calcd for C34H36N6O7 [M + Na]+ 663.2537; found 663.2537.
7,8-Dihydro-N2-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-8-oxo-20-deoxyguanosine-30-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (32)
7,8-Dihydro-N6-(dimethylaminomethylidene)-50-O-
dimethoxytrityl-8-oxo-20-deoxyadenosine-30-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (29)
The reaction of compound 31 (440 mg, 0.68 mmol), 1 M
bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.37 mL, 1.37
The reaction of compound 28 (900 mg, 1.44 mmol), 1 M mmol) and 0.45 M solution of 1H-tetrazole (1.7 mL, 0.75 mmol)
bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.88 mL, 2.88 in CH2Cl2 (20 mL) was carried out as described for compound
mmol) and 0.45 M solution of 1H-tetrazole (3.5 mL, 1.58 mmol) 18a and puried by ash column chromatography (n-hexane/
in CH2Cl2 (30 mL) was carried out as described for compound acetone/TEA, 49 : 50 : 1) to give 32 (470 mg, 81%) as a white
18a and puried by ash column chromatography (n-hexane/ foam. 31P NMR (202 MHz, DMSO-d6) d 147.8, 147.6. HRMS
acetone/TEA, 49 : 50 : 1) to give 29 (920 mg, 77%) as a white (ESI+): calcd for C43H53N8O8P [M + Na]+ 863.3616; found
foam. 31P NMR (202 MHz, DMSO-d6) d 147.4, 146.8. HRMS 863.3620.
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ambient temperature over 2 h, before water dialysis on 0.05 mM
nitrocellulose lters (Millipore) for 30 min. The oligomers
(0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA)
and negative d(CTAGCGCCG/CATGCA) oligomer controls,
were added separately to the dialyzed heteroduplex mixture
diluted into 1ꢃ DNA ligase T4 reaction buffer (NEB) for 20 mL
per sample. The mixture was denatured at 85 ꢀC as before.
Ligation was performed by adding 5 U T4 DNA ligase (NEB)
supplemented with 1 mM ATP to the samples before overnight
incubation (16 ꢀC). Ligated mixtures were dialyzed as before,
and transformed by electroporation into electro-competent E.
coli K12 (DthyA:aadA). Incubation of the electroporated bacteria
Oligonucleotide synthesis
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 2 h. Aer gel ltration on a NAP-25 column (Sephadex G25-
DNA grade; Pharmacia) with water as eluent, the crude mixture
was analysed by using a Mono-Q HR 5/5 anion exchange
column, aer which purication was achieved by using a Mono-
Q HR 10/100 GL column (Pharmacia) with the following
gradient system: A ¼ 10 mM NaClO4 in 15% CH3CN, pH 7.4, B ¼
600 mM NaClO4 in 15% CH3CN, pH 7.4. The low-pressure liquid
chromatography system consisted of a Merck-Hitachi L-6200A
intelligent pump, a Mono-Q HR 10/100 GL column (Pharmacia),
an Uvicord SII 2138 UV detector (Pharmacia-LKB) and
a recorder. The product-containing fraction was desalted on
a NAP-25 column and lyophilised. Analysis by mass spectrom-
etry followed.
ꢀ
was made at 37 C for 1 hour, before plating 100 mL of a serial
dilution of the suspension onto Muller-Hinton (MH) media
containing 100 mg mLꢁ1 ampicillin (spreading the 100 and 10ꢁ1
dilutions) and onto the same media supplemented additionally
with 0.3 mM thymidine (10ꢁ3 and 10ꢁ4 dilutions).
In vivo assay of control DNA-oligonucleotides
Control DNA oligomers were designed by modifying the 18-long
standard oligomer to incorporate single changes at the codons
tested. All DNA oligomers were ordered at Eurons MWG and
dissolved in MilliQ water to a concentration of 100 mM before
dilution ten-fold for testing. Assay of these oligos followed that
of the XNA-oligonucleotides, following the same protocol as
above.
UV melting experiments
Oligomers were dissolved in a buffer solution containing NaCl
(0.1 M), potassium phosphate (0.02 M, pH 7.5) and EDTA (0.1
mM). The concentration was determined by measuring the
ꢀ
absorbance in Milli-Q water at 260 nm at 80 C. The following
extinction coefficients were used: dA, 3 ¼ 15 000; dT, 3 ¼ 8500;
C, 3 ¼ 7100; dG, 3 ¼ 12 100; N8-8-aza-dG, 3 ¼ 2200; N8-8-aza-9-
deaza-dG, 3 ¼ 2500; 8-NH2-dI, 3 ¼ 6800; 1-Me-8-NH2-dI, 3 ¼
7600; d-isoCMe, 3 ¼ 6300; d-isoG, 3 ¼ 4600; 8-Oxo-dI, 3 ¼ 7500; 8-
Oxo-dA, 3 ¼ 11 100; 8-Oxo-dG, 3 ¼ 5900. The concentration for
each strand was 4 mM in all experiments. Melting curves were
determined with a Varian 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 acqui-
sition were carried out automatically with an IBM-compatible
computer by using Cary WinUV thermal application soware. A
quick heating and cooling cycle was carried out to allow proper
annealing of both strands. The samples were then heated from
Data analysis and sequencing
As before,8 the total numbers of thymidine-prototrophic colo-
nies and ampicillin resistant colonies were taken from the plate
counts, and the ratio of the former over the latter was calcu-
lated. Positive thymidine-prototrophic colonies were restreaked
twice to MH ampicillin, before growth in 2 mL liquid cultures of
the same medium. Plasmids were recovered by Miniprep (Qia-
gen) and the plasmids sequenced using an oligo for pAK1
d(AACAGTGGCGCGCCTGG).
Generation of the DMutM strain
The MutM gene was deleted from the E. coli K12 strain
(DthyA:aadA) through pKD46 meditation recombination, using
a deletion construct containing anking regions of the gene
around an apramycin resistance cassette (aac(3)IV). Recombi-
nation was made by the published procedure.54 Deletion was
conrmed by external primers through colony-PCR.
10 to 80 C at a rate of 0.2 C minꢁ1, and were cooled again at
the same speed. Melting temperatures were determined by
plotting the rst 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.
ꢀ
ꢀ
In vivo assay of XNA-oligonucleotides
Acknowledgements
The oligomers were dissolved in MilliQ water to 100 mM. Before
assay, they were diluted ten-fold in the same medium. Testing We thank Luc Baudemprez for technical assistance and
was performed with a gapped heteroduplex vector produced Chantal Biernaux for editorial help. The research leading to
through the enzymatic digestion and PCR assisted denaturation these results has received funding from the European Research
and hybridization of the pAK1 and pAK2 plasmids.45 A mix of Council under the European Union's Seventh Framework Pro-
equimolar (25 ng each) puried pAK1 (NheI and NsiI cut) and gramme (FP7/2007-2013)/ERC Grant agreement no. ERC-2012-
puried pAK2 (two-fold EcoRI cut and dephosphorylated) was ADG_20120216/320683, from FWO Vlaanderen, Belgium
diluted into 10 mM Tris–HCl (pH 7.5) with 100 mM NaCl. The (G078014N) and from the KU Leuven Research Council (OT/
mixture was denatured at 95 ꢀC for 5 min before cooling to 1414/128). Mass spectrometry was made possible by the support
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