N8-Glycosylated 8-Azapurine and Methylated Purine Nucleobases: Synthesis and Study of Base Pairing Properties

Article abstract of DOI:10.1021/acs.joc.9b01576

In this report, we present the synthesis of N⁸-glycosylated 8-aza-2-methylhypoxanthine and 8-aza-6-thiohypoxanthine 2′-deoxynucleosides as well as methylated 2′-deoxynebularine derivatives. In vitro base pairing properties between each modified and canonical nucleobase were studied. As demonstrated by T_m, incorporation of the modified bases in DNA resulted, with few exceptions, in low stability of duplexes. Modified bases studied in this report are preferentially recognized by T (for N⁸-glycosylated 8-aza-2-methylhypoxanthine and methylated purines) and G (N⁸-glycosylated 8-aza-2-methylhypoxanthine). The base pair formed between N⁸-glycosylated 8-aza-6-thiohypoxanthine and N⁹-glycosylated 2-methyl-6-thiohypoxanthine (X2:X6) showed, to some extent, an orthogonal interaction. Based on T_m studies, the only potential self-pairing system is formed by the N⁸-glycosylated 8-aza-6-thiohypoxanthine nucleoside (X2) but only in the absence of canonical G and T. This study indicated that the canonical thymine base is the preferential base partner of methylated purine bases.

Full text of DOI:10.1021/acs.joc.9b01576

Article
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8
N ‑Glycosylated 8‑Azapurine and Methylated Purine Nucleobases:
Synthesis and Study of Base Pairing Properties
†
†
Piotr Leonczak, Puneet Srivastava, Omprakash Bande, Guy Schepers, Eveline Lescrinier,
and Piet Herdewijn*
KU Leuven, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium
*
S Supporting Information
ABSTRACT: In this report, we present the synthesis of N8-
glycosylated 8-aza-2-methylhypoxanthine and 8-aza-6-thiohypoxan-
thine 2′-deoxynucleosides as well as methylated 2′-deoxynebularine
derivatives. In vitro base pairing properties between each modified
and canonical nucleobase were studied. As demonstrated by Tm,
incorporation of the modified bases in DNA resulted, with few
exceptions, in low stability of duplexes. Modified bases studied in
8
this report are preferentially recognized by T (for N -glycosylated 8-
8
aza-2-methylhypoxanthine and methylated purines) and G (N -
glycosylated 8-aza-2-methylhypoxanthine). The base pair formed
8
9
between N -glycosylated 8-aza-6-thiohypoxanthine and N -glycosy-
lated 2-methyl-6-thiohypoxanthine (X2:X6) showed, to some
extent, an orthogonal interaction. Based on Tm studies, the only
8
potential self-pairing system is formed by the N -glycosylated 8-aza-6-thiohypoxanthine nucleoside (X2) but only in the absence
of canonical G and T. This study indicated that the canonical thymine base is the preferential base partner of methylated purine
bases.
INTRODUCTION
■
The search for new nucleobase pairs is based on the interest in
1
,2
expanding the genetic alphabet for diagnostic reasons, for
3
aptamer selection, or for enlarging the repertoire of amino
4
5
acids that can be introduced in a protein in vitro and in vivo.
In this context, we have observed previously that (a) the
backbone scaffold influences the way modified bases are
6
8
7
recognized in vivo and (b) N -glycosylated and methylated
nucleobases show weak base pairing with the canonical base
8
and are potentially orthogonal. Also, the potential of forming
self-complementary base pairs was investigated. This study has
been extended now with an investigation on the potential base
8
pairing of N -glycosylated nucleosides (derived from 2′-
deoxyinosine, dI) and methylated 2′-deoxynebularine (dN)
nucleosides (Figure 1) in vitro.
9
As nebularine itself forms weak base pairs with natural
1
0−14
bases,
we speculate that methylation of the 2′-
deoxynebularine base would further increase hydrophobicity
and its potential orthogonality (absence of base pairing with
natural bases), making it good candidates as an orthogonal
base. Special attention is given to potential base pairing of the
modified bases with canonical bases (A, G, C, and T), as this
interaction should be avoided when selecting an orthogonal
base pair.
Figure 1. Structure of the nucleosides X1−X6 with modified bases.
7
8
bases. Nucleosides containing N -linked 8-aza-7-deazaade-
2
5
26
nine and its 2-amino analogue were reported as universal
bases, capable of forming base pairs with all canonical DNA
26,27
bases
with a reduction of thermal stability of DNA
8
Analogues of N -glycosylated 8-azapurine nucleosides15−23
duplexes between 4 and 8 °C for a single incorporation. As
demonstrate unexpected pairing properties. They were
7
,24
previously shown to form base pairs with isocytosine
and
Received: June 13, 2019
with 8-substituted (NH , OH) hypoxanthine and guanine
2
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
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8
a
Scheme 1. Synthesis of 2-Methyl-2′-deoxy-N -8-azainosine Phosphoramidite
a
Reagents and conditions: (a) NaNO , NaOH, H SO , 0 to 5 °C, 79%; (b) Na S O , NaHCO , CH CN, H O, 70 °C, 3 h; (c) NaNO , HCl,
2
2
4
2
2
4
3
3
2
2
H SO , 0 °C to rt, overnight, 60% after two steps; (d) DBU, 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose, TMSOTf, toluene, 80 °C, 24 h, 68%; (e)
2
4
NaOCH , CH OH, rt, 1.5 h, 51%; (f) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, pyridine, 0 °C to rt, 3 h, 82%; (g) (i) 1,1′-
3
3
thiocarbonyldiimidazole, CH Cl , rt, 30 h and (ii) Bu SnH, AIBN, toluene, 75 °C, 1.5 h, 58% after two steps; (h) TBAF, THF, rt, 2 h, 98%;
2
2
3
(
i) DMTCl, pyridine, CH Cl , 0 °C to rt, 2 h, 81%; and (j) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, CH Cl , rt, 2 h, 83%.
2
2
2
2
DIPEA: N,N-diisopropylethylamine.
successful in vitro and in vivo base pairing often involve
5 in 68% yield. Deprotection of the hydroxyl groups was done
by sodium methoxide in methanol leading to the nucleoside 6.
Deoxygenation of the 2′ position of the ribose moiety was
28−30
hydrophobic interactions,
we have selected the methy-
lated nebularine analogues (X3−X5) as potential partners for
8
31
the N -glycosylated pairs (X1 and X2). The nucleoside with
carried out in Barton−McCombie reaction conditions. First,
the 2-methyl-6-thiohypoxanthine base (X6) was included
the 5′- and 3′-hydroxyl groups were simultaneously protected
32
because it was previously identified as one of the most
with the tetraisopropyldisilyloxyl group. Then, the nucleoside
8
orthogonal bases in vivo.
7 was converted to the corresponding thiocarbonyl derivative,
which, after radical reduction with Bu SnH in the presence of
3
RESULTS AND DISCUSSION
azobisisobutyronitrile (AIBN), gave the 2′-deoxynucleoside 8.
Removal of the silyl protecting group with TBAF in THF
yielded the nucleoside 9. Subsequent selective protection of 5′-
OH with the 4,4′-dimethoxytrityl group (DMT) and reaction
of protected the nucleoside 10 with 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite using well-established pro-
cedures afforded the phosphoramidite 11 in a good yield.
■
Synthesis of Monomers and Oligonucleotides. The
synthesis of phosphoramidites of X1 (11), X2 (24), and X5
33) started from the heterocycle, which was coupled to the
ribose sugar (for X1 and X2) and 2-deoxyribose sugar (for
X5), respectively. The synthesis of the phosphoramidites of X3
(
(
40) and X4 (46) started from 2′-deoxyadenosine, and
8
Similarly, the synthesis of 2′-deoxy-N -8-aza-6-thioinosine
tetramethyltin was used to introduce the methyl group. Fully
phosphoramidite started from easily available 8-azahypoxan-
protected phosphoramidite of 2-methyl-6-thio-2′-deoxyinosine
7
8
thine (Scheme 2). The BF ·Et O-mediated glycosylation of
X6 was synthesized as previously described.
3
2
8
the compound 13 afforded a mixture of four compounds from
which one compound is predominant (based on the
integration of the doublet signal in the area of anomeric
The synthesis of 2-methyl-2′-deoxy-N -8-azainosine phos-
phoramidite 11 (Scheme 1) started from 8-aza-2-methylpurin-
6
-one (4). Commercially available 4-amino-2-methylpyrimi-
din-6-one (1) was reacted with NaNO under acidic condition
1
protons in the H NMR spectrum). The analytical sample of
2
affording the nitroso derivative 2 in a good yield. Subsequent
reaction of 2 with Na S O yielded crude 4,5-diaminopyr-
the most abundant compound was isolated, and spectral
8
analysis proved it to be the β-anomer of N -8-azainosine 14.
2
2
4
imidine 3, which upon treatment with nitrous acid gave
triazolopyrimidin-6-one 4.
Glycosylation of 4 with commercially available 1,2,3,5-tetra-
O-acetyl-β-D-ribofuranose was carried out in the presence of
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) and trimethylsilyl
trifluoromethanesulfonate (TMSOTf) affording the β-anomer
To avoid tedious separation of the mixture on a larger
synthesis scale, the deprotection of the hydroxyl groups was
performed on the crude mixture using aqueous ammonia and
the resulting mixture containing the nucleoside 15 was reacted
with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane in pyridine.
The compound 16 was readily isolated from this mixture.
B
DOI: 10.1021/acs.joc.9b01576
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8
a
Scheme 2. First Synthetic Pathway to S-Protected 2′-Deoxy-N -8-aza-6-thioinosine Phosphoramidite
a
Reagents and conditions: (a) NaNO , 10% HCl, 0 to 45 °C, 6 h, 72%; (b) 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose, BF -Et O, CH CN, 80 °C, 3 h;
2
3
2
3
(
c) concd aq NH , rt, 5 h; (d) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, pyridine, 0 °C to rt, 2.5 h, 34% after three steps; (e) (i) 1,1′-
3
thiocarbonyldiimidazole, CH Cl , reflux, overnight and (ii) Bu SnH, AIBN, toluene, 95 °C, 5 h, 77% after two steps; (f) P S , pyridine, rt to
2
2
3
2 5
1
05 °C, 6 h, 87%; (g) Et N·3HF, THF, 0 °C to rt, 5 h, 82%; and (h) BrCH CH CN, K CO , DMF, rt.
3
2
2
2
3
8
a
Scheme 3. Synthesis of N -8-Aza-dI Phosphoramidite
a
Reagents and conditions: (a) POCl , 1,2,4-triazole, Et N, CH CN, pyridine, 0 °C to rt, 24 h, 86%; (b) 3-mercaptopropanenitrile (25), DIPEA, rt,
3
3
3
1
.5 h, 96%; (c) Et N·3HF, THF, 0 °C to rt, 5 h, 93%; (d) DMTCl, pyridine, CH Cl , 0 to 35 °C, 17 h, 69%; (e) 2-cyanoethyl-N,N-
3 2 2
diisopropylchlorophosphoramidite, DIPEA, CH Cl , rt, 3 h, 54%; (f) H O, 68 to 100 °C, then 0 °C; and (g) concd NaOH, then H SO .
2
2
2
2
4
31
Radical deoxygenation of the 2′-position of the ribose moiety
afforded the 2′-deoxynucleoside 17 in a good yield. Next, the
6-oxo group of 8-azahypoxanthine moiety was sulfurated using
P S affording the thionucleoside 18. Further, removal of the
2
5
C
DOI: 10.1021/acs.joc.9b01576
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The Journal of Organic Chemistry
Article
a
Scheme 4. Synthesis of 2-Methyl-2′-deoxynebularine Phosphoramidite
a
Reagents and conditions: (a) concd aq NH , 120 °C, sealed flask, overnight, 96%; (b) (C H O) CH, HCOOH, 120 °C, 5.5 h, 72%; (c)
3
2
5
3
Pd(OH) , HCOONH , CH OH, reflux, 2.5 h, 90%; (d) (i) NaH, CH CN, rt; 90 min and (ii) Hoffer’s chlorosugar, rt, 70 min, 62%; (e) NaOCH ,
2
4
3
3
3
CH OH, rt, 2.5 h, then NH Cl, rt, 5 min, 81%; (f) DMTCl, pyridine, CH Cl , 0 °C to rt, 4 h, 66%; and (g) 2-cyanoethyl-N,N-
3
4
2
2
diisopropylchlorophosphoramidite, DIPEA, CH Cl , rt, 3 h, 91%.
2
2
a
Scheme 5. Synthesis of 8-Methyl-2′-deoxynebularine Phosphoramidite
a
Reagents and conditions: (a) TBSCl, pyridine, rt, overnight; (b) (CH ) Sn, Pd(PPh ) dioxane, reflux, overnight, 80% after two steps; (c) TBAF,
3
4
3 4,
THF, overnight; (d) (CH CO) O, pyridine, 0 °C to rt, overnight, 70% after two steps; (e) tert-butyl nitrite, THF, reflux, 2 d, 35%; (f) methanolic
3
2
NH , sealed tube, rt, overnight, 90%; (g) DMTCl, pyridine, rt overnight, 60%; (h) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA,
3
THF, 4 h, 44%.
8
8
silyl protecting group gave the 2′-deoxy-N -8-aza-6-thioinosine
described, this procedure, in the case of the N -8-aza-6-thio-2′-
(
19). As it is apparent that the successful strategy for
incorporating 6-thiopurine nucleosides into oligonucleotide
requires protection of the thiocarbonyl moiety,
to prepare the monomer with the sulfur atom protected with a
-cyanoethyl group. Therefore, the nucleoside 19 was treated
with an excess of 2-bromopropionitrile in DMF in the presence
of K CO . In contrast to literature data where the high yield of
deoxyinosine (19), was unsuccessful in our hands. Therefore,
we turned to another synthesis strategy and used 3-
mercaptopropanenitrile as a synthon for the introduction of
a protected mercapto function into the heterobase moiety via
displacement of the 1,2,4-triazolyl-leaving group (Scheme 3).
This methodology was previously successfully applied for the
synthesis of an electron deficient 4-thiopyrimidine nucleo-
33,34
we decided
2
2
3
33
34
35
sulfur alkylation of 6-thioinosine and 6-thioguanosine is
side. The silyl-protected nucleoside 17 was reacted with
D
DOI: 10.1021/acs.joc.9b01576
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The Journal of Organic Chemistry
Article
a
Scheme 6. Synthesis of 6-Methyl-2′-deoxynebularine Phosphoramidite
a
Reagents and conditions: (a) TMSCl, tert-butyl nitrite, CH Cl , rt, overnight, 52%; (b) (CH ) Sn, Pd(PPh ) , dioxane, reflux, overnight, 77%; (c)
2
2
3
4
3 4
7
N methanolic NH , overnight, sealed tube, 90%; (d) DMTCl, pyridine, 0 °C to rt, overnight, 60%; and (e) 2-cyanoethyl-N,N-
3
diisopropylchlorophosphoramidite, DIPEA, THF, 4 h, 66%.
POCl3 and 1,2,4-triazole to afford the compound 20.
36 was subjected to reductive deamination, which provided
bis-acetyl-protected 8-methyl-2′-deoxynebularine 37 in a
modest 35% yield. The acetyl groups were removed yielding
the compound 38, which was tritylated using DMTCl in
pyridine affording the nucleoside 39 in 55% yield. The 3′-
hydroxyl group was then reacted with 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite to obtain the 8-methyl-2′-
deoxypurine phosphoramidite 40 in 44% yield.
36
Subsequent reaction with 3-mercaptopropanenitrile (25)
obtained in a slightly modified procedure as described by
(
37
Gerber and co-workers ) afforded the nucleoside 21 in 96%
yield. Removal of the silyl group with Et N·3HF, selective
3
protection of the 5′-hydroxyl function with DMT, and
phosphitylation of 3′-OH afforded fully protected phosphor-
amidite 24.
The synthesis of 2-methyl-2′-deoxynebularine phosphor-
amidite 33 (Scheme 4) started from 2-methylpurine (29).
Commercially available 5-amino-4,6-dichloro-2-methylpyrimi-
dine (26) was converted to 4,5-diamino-6-chloro-2-methyl-
pyrimidine (27), which, after treatment with triethyl
orthoformate, afforded the compound 28. Subsequent
The synthesis of the 6-methyl-2′-deoxynebularine monomer
46 (Scheme 6) started from the commercially available 2′-
deoxyadenosine. The appropriately protected 2′-deoxyadeno-
41
sine 41 was subjected to dediazotization halogenation to
obtain the 6-chloro-2′-deoxypurine nucleoside 42 in 52% yield.
The product 42 was then methylated with (CH ) Sn and
3
4
38
reductive dechlorination afforded methylated purine 29.
Reaction of 29 with Hoffer’s chlorosugar in the presence of
Pd(PPh ) to obtain acetyl-protected 6-methyl-2′-deoxynebu-
3
4
larine 43 in 77% yield. The acetyl groups were then cleaved by
methanolic ammonia to obtain 6-methyl-2′-deoxynebularine
44 in 90% yield. The 5′-hydroxyl group of 44 was protected
with DMTCl in pyridine to obtain 45 in 60% yield. The 3′-OH
was next reacted with 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite to obtain the 6-methyl-2′-deoxynebularine
phosphoramidite 46 in 66% yield.
7
NaH afforded the nucleoside 30a in 62% yield. The N -isomer,
30b, was formed in minor amounts. Toluoyl-protecting groups
were cleaved from 30a/b by NaOCH in methanol to yield 2′-
3
deoxyribofuranosides 31a/b, the structures of which were
confirmed by 1D/2D NMR analysis. Subsequent protection of
the 5′-hydroxyl function of 31a with DMT followed by
phosphitylation of the 3′-OH afforded phosphoramidite 33 in
a good yield.
The oligonucleotides were synthesized on a DNA
synthesizer using phosphoramidite chemistry. After cleavage
from the support and deprotection, oligomers were purified
using anion-exchange and reversed-phase HPLC and charac-
terized by mass spectrometry (Table 1).
Hybridization Properties of the Modified Oligonu-
cleotides. To investigate the base pairing properties, the
modified nucleosides were incorporated in the antiparallel
duplexes 5′-d(CAC CGX TGC TAC C)-3′ and 3′-d(GTG
GCX ACG ATG G)-5′ (Table 1). The hybridization strength
was studied by matching the oligomers with their comple-
For the synthesis of the 8-methyl-2′-deoxynebularine
monomer 40 (Scheme 5), the commercially available 8-
bromo-2′-deoxyadenosine (34) was converted into the 3′,5′-
bis-TBS (TBS: tert-butyldimethylsilyl)-protected 8-bromoade-
3
9
nosine in quantitative yield. This bis-silyl derivative was then
converted to the 8-methyl derivative via the palladium-
catalyzed reaction, providing the compound 35 in 80% yield
after two steps. The compound 35 was then subjected to
4
0
dediazotization reduction. Extensive decomposition was
observed under the reaction conditions when the silyl-
protected derivative was used. Therefore, the compound 35
was deprotected with TBAF and bis acetylated using acetic
anhydride. The bis-acetyl-protected 8-methyldeoxyadenosine
mentary strands and determining T by temperature-depend-
m
ent UV spectroscopy (Table 2). The T data were determined
m
at 260 nm in a buffer solution containing NaCl (0.1 M),
KH PO (20 mM, pH 7.5), and EDTA (0.1 mM) at a
2
4
36 was obtained in 70% yield after two steps. The compound
concentration of 4 μm for each strand.
E
DOI: 10.1021/acs.joc.9b01576
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Article
Table 1. Chemically Synthesized Oligonucleotides Bearing
Site Specific Modifications Together with Mass
Spectrometry Data
mass calcd mass found
entry
sequence
X
(Da)
(Da)
1
2
3
4
5
6
7
8
9
5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
X1
X1
X2
X2
X3
X3
X4
X4
X5
X5
X6
X6
3893.7
4053.7
3895.6
4055.7
3876.7
4036.7
3876.7
4036.7
3876.7
4036.7
3908.7
4068.7
3893.7
4053.7
3895.7
4055.7
3876.7
4036.7
3876.7
4036.7
3877.0
4036.7
3908.7
4068.7
Figure 2. Potential pairing between X2 and X6 bases.
and G bases is more problematic than with the natural A and C
bases (with the latter bases, base pairing is in most cases weak
giving duplexes of low stability) (Tables 3 and 4).
12
As it was previously presented by Eritja and co-workers,
melting points of duplexes containing 2′-deoxynebularine are
quite low compared to the fully matched canonical duplexes,
except for the nebularine−thymine pair. Also, Rahman and
10
11
12
42
Humayun showed that T was preferably inserted both in
vitro and in vivo opposite 2′-deoxynebularines. We observed
that T is also the preferred partner for the methylated purine
base (X4 > X5 > X3) (Tables 3 and 4). The base pairing of T
with X3 (T:X3) is the least stable among them. Although the
specificity and stability of T:X3 pairing can be rationalized by
formation of one hydrogen bond (Figure 3) between the
Table 2. Hybridization Properties of DNA Duplexes Bearing
Site Specific Modified Base Pairs Expressed by T Values
°C)
m
a
(
12
pyrimidine and purine base, this can also be attributed to
shape recognition of hydrophobic bases as proposed by Kool
43
and co-workers together with a disturbed base pair geometry
and favorable stacking interaction with the neighboring base.
We also observed a sequence specific effect as the ranking of
duplex stability after incorporation of the modified bases is
dependent if the modified bases are inserted in the GXT
sequence or the AXC sequence (Tables 3 and 4). As a last
conclusion from the T studies, X2 might represent the only
potential self-pairing base but only in the absence of G and T,
m
a
The data are the mean value of two measurements. To compare
strength of hybridization, Tm values for unmodified DNA duplexes
together with mismatches (formed between canonical bases) are
given.
as the X2:X2 system gives a lower or comparable T than the
m
X2:G and X2:T systems, respectively.
CONCLUSIONS
Synthetic schemes were developed for the synthesis of
nebularine analogues with a methyl group in the 2-, 6-, or 8-
■
8
The potential base pairing between N -glycosylated bases
X1, X2) and methylated purine bases (X3, X4, and X5), the
(
8
position together with two new N -glycosylated 2′-deoxy-8-
potential base pairing with the canonical bases A, G, C, and T,
and the potential for self-pairing were investigated. The
modified base X6 was involved in this study, because
previously, we demonstrated that it behaves as an orthogonal
base in vivo as it blocks the transliteration of XNA to DNA
aza-(6-thio)inosine analogues. The modified nucleosides were
incorporated in oligonucleotides, and base-pair stability was
investigated. This study revealed that the natural thymine base
is the preferred partner for the methylated purine base and that
8-aza-6-thiohypoxanthine (X2) could form a base pair with 2-
methyl-6-thiohypoxanthine (X6) when the bases are con-
nected to the sugar by N and N , respectively. The 2-
methylpurine base (X5) shows the highest degree of
orthogonality (absence of pairing with other bases). When
looking for orthogonal base pairs with modified nucleosides,
the potential base pairing with the acidic nucleobases (of G
and T) could be more problematic than with the basic
nucleobases (of A and C). It will be important to investigate if
the triphosphates of X2 and X6 could be substrates in a PCR
reaction demonstrating enzyme-catalyzed self-replication.
8
after incorporation of two X6 bases.
With few exceptions, there is a large drop in Tm after
incorporation of the modified bases. When compared with the
canonical base pairs, it makes the methylated purine bases,
studied here, potentially orthogonal, as previously observed for
8
9
8
related methylated nucleobases. The base pair stability
involving methylated bases can be compared with mismatches
between natural bases. The most orthogonal base is X5 as it
gives duplexes with the lowest Tm with all other canonical
bases (except with T). However, the use of such methylated
8
and N -glycosylated modified bases as a partner in a new
artificial base pair is hampered by the preferential recognition
of these modified bases by T (X1, X3, X4, and X5) or by G in
the opposite strand (X1). The only base pair that is somewhat
more stable than those interactions is the X2:X6 pair (Figure
EXPERIMENTAL SECTION
■
1
13
31
1
H, C, and P NMR spectra were recorded on 300 MHz ( H NMR,
13
31
3
00 MHz; C NMR, 75 MHz; P NMR, 121 MHz) or 500 MHz
1
13
1
(
H NMR, 500 MHz; C NMR, 125 MHz) or 600 MHz ( H NMR,
2
).
For designing orthogonal pairs with these modified
nucleobases, the potential hybridization with the natural T
13
6
00 MHz; C NMR, 150 MHz) spectrometers. 2D NMRs (H-
COSY, HSQC, HMBC, ROESY/NOESY) were used for the
assignment of some of the intermediates and final compounds.
F
DOI: 10.1021/acs.joc.9b01576
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The Journal of Organic Chemistry
Article
Table 3. Ranking of the Stability of the Duplexes Bearing X1−X6 Incorporated in the Sequence 5′-GGT AGC AXC GGT G-3′
and X1−X6 or Canonical Nucleotide Incorporated in the Sequence 5′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
5′-CAC CGX TGC TAC C-3′
X1
X2
X3
X4
X5
X6
X6 > T > G > X4 > X3 ≈ A > X2 ≈ X5 > X1 > C
X6 > G > T ≈ X2 > X1 > X4 > A ≈ X3 > X5 > C
T > G > X6 > X1 > X2 > A > X5 > X4 ≈ C ≈ X3
T > X1 > A > X2 ≈ C > G > X6 ≈ X4 ≈ X5 > X3
T > X6 > G > X2 ≈ A ≈ X5 > C ≈ X1 ≈ X3 ≈ X4
X2 > X1 > A > T > X5 > X3 > C > G > X4 > X6
Table 4. Ranking of the Stability of the Duplexes Bearing X1−X6 Incorporated in the Sequence 5′-CAC CGX TGC TAC C-3′
and X1−X6 or Canonical Nucleotide Incorporated in the Sequence 5′-GGT AGC AXC GGT G-3′
5
′-CAC CGX TGC TAC C-3′
5′-GGT AGC AXC GGT G-3′
X1
X2
X3
X4
X5
X6
G > T > X6 > X2 ≈ X4 > X3 > A > X1 > X5 > C
X6 > G > X2 ≈ T > X1 ≈ X3 > X4 ≈ A > X5 > C
T > G > X1 > X2 > X6 > A ≈ X3 > C > X4 ≈ X5
T > X1 > X2 > G > A > C > X3 > X6 ≈ X4 > X5
T > X6 > G > X2 > X1 > X3 > A ≈ C > ≈ X4 = X5
X2 ≈ X1 > A ≈ T > G > X3 > X5 > C > X4 > X6
precipitation started. The mixture was left in a fridge overnight, and
the solid was collected by filtration, washed with a small amount of
ice-cold water, and dried. The filtrate was concentrated, and another
batch of solids was obtained. The crude product was used in the next
step without purification. HRMS-ESI (Q-ToF) (m/z): [M + H]+
calcd for C H N O, 141.0771; found, 141.0778.
5
9
4
4
5
Figure 3. Potential interaction between T and X3 bases.
8-Aza-2-methylhypoxanthine (4). A suspension of the crude
diaminopyrimidine 3 (5.8 g) in 10% aq HCl (87 mL) was cooled in
an ice-water bath. A solution of NaNO (4.89 g, 70.8 mmol) in H O
2
2
Chemical shifts (δ) are reported in parts per million (ppm) relative to
1
(16 mL) was added dropwise, and the resulting mixture was stirred at
room temperature (rt) overnight (with an ice-water bath left until the
ice melted). The solid was collected by filtration, washed with small
amount of ice-cold water, and dried. The filtrate was concentrated in
vacuo. The solid residue was suspended in a small amount of water,
collected by filtration, and dried affording an additional batch of the
crude product. Solids were combined and put on silica gel flash
column chromatography. Elution first with a mixture of 5−20% of
TMS or the residual solvent signal for H, the residual solvent signal
for 1 C (spectra recorded in D O are uncorrected). P spectra were
3
31
2
recorded without using an external reference. Chemical shifts are
described as s (singlet), d (doublet), dd (doublet of doublets), ddd
(
(
doublet of doublet of doublets), t (triplet), dt (doublet of triplets), q
quartet), m (multiplet), appt (apparent triplet), and bs (broad
signal). Coupling constants (J) are reported in hertz. Mass spectra
were acquired on a quadrupole orthogonal acceleration time-of-flight
mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA).
CH OH in CH Cl and next CH Cl /CH OH/AcOH (20:4:1, v/v/
3
2
2
2
2
3
−
1
v) yielded 2.33 g (15.4 mmol, 60% after two steps) of the title
Samples were infused at 3 uL·min , and spectra were obtained in the
1
compound as an off-white solid. H NMR (300 MHz, DMSO-d ): δ
positive (or negative) ionization mode with a resolution of 15,000
6
1
3
1
2
.25 (s, 3H, CH ), 11.18 (bs, 1H, NH). C{ H} NMR (75 MHz,
(
FWHM) using leucine enkephalin as a lock mass. Column silica gel
3
DMSO-d ): δ 21.3 (CH ), 126.8 (C-5), 152.8 (C-4), 158.1 (C-6).
chromatographic separations were carried out by gradient elution with
6
3
+
HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O,
suitable combination of solvents and silica gel (100−200 mesh or
5
6
5
1
52.0567; found, 152.0570.
2
30−400 mesh). Reagents and anhydrous solvents were purchased
8
-Aza-2-methyl-8-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-hy-
from commercial sources and used without further purification unless
otherwise stated. Numbering of (8-aza)purine and derived nucleo-
sides is based on the purine numbering system.
poxanthine (5). To a suspension of 4 (1.82 g, 12.02 mmol) in
anhydrous toluene (120 mL) was added DBU (2 mL, 13.22 mmol).
After 15 min of stirring at room temperature, β-D-ribofuranose-
4
4
6
-Amino-2-methyl-5-nitrosopyrimidin-4(3H)-one (2). To a
1
,2,3,5-tetraacetate was added (3.44 g, 10.81 mmol) and the resulting
mixture was cooled in an ice-water bath. Then, TMSOTf (4.35 mL,
4.03 mmol) was added dropwise. The cooling bath was removed,
solution of 6-amino-2-methylpyrimidin-4(3H)-one (1) (5 g, 39.96
mmol) in 1 M NaOH (57 mL) was added NaNO (3.03 g, 43.95
2
2
mmol). The resulting mixture was cooled in an ice-water bath, and
then H SO4 (3 mL in 30 mL of H O) was added dropwise,
and the mixture was stirred at 65−80 °C for 24 h. The mixture was
2
2
cooled down to room temperature, and 5% aq NaHCO (60 mL) was
maintaining temperature at 0−5 °C. When addition was completed,
the purple precipitate was collected by filtration, washed with ice-cold
3
added. After stirring for 15 min, layers were separated and the water
phase was extracted with EtOAc (three times). The organic phases
water and Et O, and dried in an oven (65 °C) overnight, affording
2
1
4
.88 g (31.66 mmol, 79%) of product as a blue solid. H NMR (300
were combined and dried over MgSO . The drying agent was
4
MHz, DMSO-d ): δ 2.23 (s, 3H, CH ), 9.06 (bs, 1H, NH), 11.14 (bs,
1
removed by filtration, and filtrate was concentrated in vacuo. The
residue was purified on silica gel column by flash chromatography
6
3
1
3
1
H, NH), 12.33 (bs, 1H, NH). C{ H} NMR (75 MHz, DMSO-d ):
6
δ 21.9 (CH ), 143.7, 148.2, 161.8, 165.8. HRMS-ESI (Q-ToF) (m/
(16−20% of acetone in CH
of product as an off-white foam. H NMR (300 MHz, CDCl
(s, 3H, CH ), 2.15 (s, 6H, 2 × CH ), 2.63 (s, 3H, CH
2 2
Cl ), affording 3.37 g (8.23 mmol, 68%)
3
+
1
z): [M + H] calcd for C H N O , 155.0563; found, 155.0566.
3
): δ 2.13
), 4.23−4.29
5
7
4
2
4
4
5
,6-Diamino-2-methylpyrimidin-4(3H)-one (3). A mixture
3
3
3
of 5-nitroso-pyrimidine 2 (4 g, 25.6 mmol), NaHCO (4.8 g, 57.2
(m, 1H, H-5″), 4.61−4.58 (m, 2H, H-5′, H-4′), 5.83 (t, J = 5.4 Hz,
3
mmol), and Na S O (9.94 g, 57.2 mmol) in a mixture of CH CN
1H, H-3′), 5.95 (dd, J = 5.4, 3.0 Hz, H-2″), 6.40 (d, 1H, J = 3.0 Hz,
2
2
4
3
1
3
1
and H O (400 mL, 1:1 (v/v)) was stirred at 70 °C for 3 h.
H-1′), 11.78 (s, 1H, NH). C{ H} NMR (75 MHz, CDCl ): δ 20.3
2
3
Acetonitrile was removed in vacuo, and the residue was brought to
pH 5 using glacial AcOH. The mixture was concentrated until the
(CH ), 20.4 (CH ), 20.7 (CH ), 22.0 (CH ), 62.9 (C-5′), 70.9 (C-
3′), 74.4 (C-2′), 81.3 (C-4′), 95.3 (C-1′), 130.4 (C-5), 158.0, 158.4,
3
3
3
3
G
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1
58.4 (C-2, C-4, C-6), 169.2 (CO), 169.5 (CO), 170.6 (CO).
membrane pump, and vacuum was released with argon, cycle was
repeated four times), placed in a heating bath, and the reaction
mixture was stirred at 75 °C for 1.5 h. The mixture was cooled down,
and volatiles were removed in vacuo. The crude product was purified
by silica gel flash column chromatography (28−50% of EtOAc in
+
HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O ,
1
6
20
5
8
410.1306; found, 410.1306.
8-Aza-2-methyl-8-(β-D-ribofuranosyl)-hypoxanthine (6). To
a solution of 5 (3.37 g. 8.23 mmol) in anhydrous CH OH (120 mL)
was added commercial solution of sodium methoxide in methanol
3
hexane) affording 1.03 g (2.02 mmol, 58%) of product as an off-white
1
(5.4 M, 4.63 mL, 25 mmol). The resulting mixture was stirred at rt for
foam. H NMR (300 MHz, CDCl ): δ 1.04−1.17 (m, 28H, 4 ×
3
+
1
h. Next, the solution was neutralized using Dowex 50WX8 (H
CH(CH ) ), 2.63 (s, 3H, 2-CH ), 2.63−2.75 (m, 1H, H-2′), 2.91
(dd, J = 13.5, 7.5 Hz, 1H, H-2″), 3.88−4.08 (m, 3H, H-4′, H-5′, H-
5″), 5.23 (m, 1H, H-3′), 6.45 (d, J = 6.9 Hz, 1H, H-1′), 11.97 (s, 1H,
3
2
3
form). The resin was removed by filtration, and the filtrate was
concentrated until dryness. The solid residue was suspended in EtOH
1
3
1
(containing ca. 5% of Et O), and the solid was collected by filtration
NH). C{ H} NMR (75 MHz, CDCl ): δ 12.7, 12.9, 13.4, 13.5, 17.1,
2
3
through a sintered funnel and dried under vacuum, affording 1.19 g
17.3, 17.46, 17.49, 17.59, 17.64 (4 × CH(CH ) ), 22.2 (2-CH ), 40.9
3
2
3
1
(
4.20 mmol, 51%) as an off-white powder. H NMR (500 MHz,
(C-2′), 64.4 (C-5′), 73.0 (C-3′), 86.6 (C-4′), 93.0 (C-1′), 129.8 (C-
5), 157.4, 158.6, 159.2 (C-2, C-4, C-6). HRMS-ESI (Q-ToF) (m/z):
DMSO-d ): δ 2.37 (s, 3H, CH ), 3.47−3.51 (m, 1H, H-5″), 3.59−
6
3
+
3
3
.63 (m, 1H, H-5′), 4.01−4.04 (m, 1H, H-4′), 4.26−4.29 (m, 1H, H-
′), 4.52−4.55 (m, 1H, H-2′), 4.79 (t, J = 5.7 Hz, 1H, 5′-OH), 5.29
[M + H] calcd for C H N O Si , 510.2562; found, 510.2570.
2
2
40
5
5
2
8-Aza-8-(2-deoxy-β-D-ribofuranos-1-yl)-2-methylhypoxan-
thine (9; X1). To the nucleoside 8 (359 mg, 0.70 mmol) dissolved in
anhydrous THF (5 mL) was added solution of TBAF in THF (1 M,
2.11 mL, 2.11 mmol). The resulting mixture was stirred at room
temperature under argon for 2 h. Volatiles were removed in vacuo.
The crude product was purified on silica gel flash column
(
d, J = 5.9 Hz, 1H, 3′-OH), 5.67 (d, J = 5.7 Hz, 1H, 2′-OH), 6.01 (d,
13 1
J = 3.6 Hz, 1H, H-1′), 12.34 (bs, 1H, NH). C{ H} NMR (125
MHz, DMSO-d ): δ 21.6 (CH ), 62.0 (C-5′), 70.7 (C-3′), 74.8 (C-
6
3
2
′), 86.4 (C-4′), 97.3 (C-1′), 130.2 (C-5), 156.7, 157.7, 158.3 (C-2,
+
C-4, C-6). HRMS-ESI (Q-ToF) (m/z): [M + Na] calcd for
C H N O Na, 306.0809; found, 306.0818. UV (H O): λ (nm) =
2
chromatography (0−9% of CH
(0.69 mmol, 98%) of product as an off-white solid. H NMR (300
MHz, DMSO-d ): δ 2.36 (s, 3H, 2-CH
OH in CH Cl ), affording 185 mg
1
0
13
4
5
2
max
3
2 2
3
−1
3
−1
1
74, λ260 (nm), ε = 6.0 × 10 mol ·dm ·cm ; UV (CH OH): λ
3 max
3 3
−1
−1
(
nm) = 273, λ260 (nm), ε = 6.2 × 10 mol ·dm ·cm
6
3
), 2.41−2.50 (m, 1H, H-2″,
.
8
-Aza-2-methyl-8-(3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
overlapped with residual solvent signal), 2.76−2.84 (m, 1H, H-2′),
1
,3-diyl)-β-D-ribofuranosyl)-hypoxanthine (7). The nucleoside 6
3.35−3.41 (m, 1H, H-5″, overlapped with H O signal), 3.50−3.57
2
(
1.19 g, 4.201 mmol) was coevaporated with anhydrous pyridine and
(m, 1H, H-5′), 3.87−3.92 (m, 1H, H-4′), 4.48−4.51 (m, 1H, H-3′),
4.76 (t, J = 5.5 Hz, 1H, 5′-OH), 5.41 (d, J = 4.7 Hz, 1H, 3′-OH), 6.46
dissolved (suspension may be formed) in this solvent (42 mL). The
resulting mixture was cooled in an ice-water bath, and 1,3-dichloro-
1
3
1
(dd, J = 6.5, 4.5 Hz, 1H, H-1′), 12.34 (s, 1H, NH). C{ H} NMR
(75 MHz, DMSO-d ): δ 21.5 (2-CH ), 39.3 (C-2′), 61.9 (C-5′), 70.4
1
,1,3,3-tetraisopropyldisiloxane (1.48 mL, 4.62 mmol) was added
6
3
dropwise. The cooling bath was removed, and the mixture was stirred
(C-3′), 88.7 (C-4′), 93.3 (C-1′), 129.9 (C-5), 156.6 (C-2), 157.5 (C-
+
at room temperature for 3 h. Next, 5% aq NaHCO (55 mL) was
4), 158.1 (C-6). HRMS-ESI (Q-ToF) (m/z): [M + Na] calcd for
3
added, and after 15 min of stirring, the mixture was extracted with
dichloromethane (three times). Organic layers were combined,
C H N O Na, 290.0860; found, 290.0859.
1
0
13
5
4
8-Aza-8-(2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl)-
washed with brine, and dried over MgSO . The drying agent was
2-methylhypoxanthine (10). The nucleoside 9 (180 mg, 0.67
mmol) was coevaporated with anhydrous pyridine (three times) and
next dissolved in pyridine (7 mL). The mixture was cooled in an ice-
water bath, and solution of DMTCl (274 mg; 0.81 mmol) in CH Cl
4
removed by filtration, the filtrate was concentrated in vacuo, and the
residue was coevaporated with toluene (three times). The crude
product was purified by flash column chromatography (hexane/
2
2
EtOAc, 1:1, v/v), affording 1.82 g (3.46 mmol, 82%) of product as an
(3 mL) was added dropwise. The cooling bath was removed, and
resulting mixture was stirred under argon at room temperature for 2 h.
1
off-white foam. H NMR (300 MHz, CDCl ): δ 1.03−1.18 (m, 28H,
3
4
× CH(CH ) ), 2.64 (s, 3H, CH ), 3.34 (s, 1H, 2′-OH), 4.02−4.04
Next, CH OH was added, and after 5 min of stirring, volatiles were
3
2
3
3
(
m, 2H, H-5′, H-5″), 4.19−4.25 (m, 1H, H-4′), 4.57 (d, J = 5.1 Hz,
removed in vacuo. The residue was dissolved in CH Cl and washed
2
2
1
1
1
H, H-2′), 5.13 (dd, J = 7.2, 5.1 Hz, 1H, H-3′), 6.32 (s, 1H, H-1′),
subsequently with 5% aq NaHCO , water, and brine. The organic
3
1
3
1
2.11 (s, 1H, NH). C{ H} NMR (75 MHz, CDCl ): δ 12.7, 12.80,
layer was dried over MgSO . The drying agent was removed by
3
4
3.3, 13.4, 17.2, 17.3, 17.4, 17.51, 17.54 (4 × CH(CH ) ), 22.2 (2-
filtration, and the filtrate was concentrated to dryness and
coevaporated with toluene. The crude product was purified by silica
gel flash column chromatography (0−4% of CH OH in CH Cl with
3
2
CH ), 63.8 (C-5′), 73.6 (C-3′), 76.3 (C-2′), 84.0 (C-4′), 96.9 (C-1′),
3
1
1
30.0 (C-5), 157.7, 158.7, 159.2 (C-2, C-4, C-6). H NMR (300
3
2
2
MHz, DMSO-d ): δ 1.00−1.07 (m, 28H, 4 × CH(CH ) ), 2.37 (s,
0.5 vol % addition of triethylamine), affording 310 mg (0.54 mmol,
6
3 2
1
3
4
3
H, 2-CH ), 3.87−3.97 (m, 2H, H5′, H-5″), 4.06−4.11 (m, 1H, H-
81%) of product as a white foam. H NMR (300 MHz, DMSO-d ): δ
3
6
′), 4.91 (t, J = 4.5 Hz, 1H, H-2′), 4.93 (dd, J = 8.7, 4.5 Hz, 1H, H-
2.37 (s, 3H, 2-CH ), 2.40−2.50 (m, 1H, H-2″, overlapped with
3
′), 5.86 (d, J = 4.5 Hz, 1H, 2′-OH), 6.05 (s, 1H, H-1′), 12.33 (s, 1H,
residual solvent signal), 2.84 (ddd, J = 13.5, 6.6, 2.4 Hz, 1H, H-2′),
3.01 (dd, J = 10.2, 6.6 Hz, 1H, H5″), 3.08 (dd, J = 10.2 Hz, 3.0 Hz,
1H, H5′), 3.70 (s, 3H, OCH ), 3.72 (s, 3H, OCH ), 3.98−4.04 (m,
13
1
NH). C{ H} NMR (75 MHz, DMSO-d ): δ 12.06, 12.10, 12.5,
6
1
2.8, 16.78, 16.84, 16.9, 17.0, 17.13, 17.14, 17.2, 17.3 (4 ×
3
3
CH(CH ) ), 21.5 (2-CH ), 61.4 (C-5′), 71.2 (C-3′), 74.5 (C-2′),
1H, H-4′), 4.55−4.63 (m, 1H, H-3′), 5.45 (d, J = 5.1 Hz, 1H, 3′-
OH), 6.52 (dd, J = 6.9, 2.4 Hz, 1H, H-1′), 7.12−7.28 (m, 13H,
3
2
3
8
1.6 (C-4′), 96.7 (C-1′), 130.2 (C-5), 156.4, 157.7, 158.2 (C-2, C-4,
+
13
1
C-6). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for
DMT), 12.30 (s, 1H, NH). C{ H} NMR (75 MHz, DMSO-d ): δ
6
C H N O Si , 526.2511; found, 526.2514.
21.5 (2-CH ), 45.7 (C-2′), 54.91 (OCH ), 54.94 (OCH ), 63.8 (C-
22
40
5
6
2
3
3
3
8
-Aza-8-(2-deoxy-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
5′), 69.9 (C-3′), 85.2 (DMT), 86.3 (C-4′), 93.0 (C-1′), 112.9
1
,3-diyl)-β-D-ribofuranosyl)-2-methylhypoxanthine (8). The
(
1
DMT), 113.0 (DMT), 126.4 (DMT), 127.6 (DMT), 127.6 (DMT),
29.4 (DMT), 129.7 (DMT), 129.9 (C5), 135.4 (DMT), 135.6
(DMT), 144.8 (DMT), 156.7 (DMT), 157.6 (DMT), 157.8, 157.9,
nucleoside 7 (1.82 g, 3.46 mmol) was dissolved in anhydrous
CH Cl (36 mL), 1,1′-thiocarbonyldiimidazole (0.62 g, 3.46 mmol)
2
2
+
was added, and then resulting mixture was stirred under argon at
room temperature for 30 h. The mixture was washed with H O. The
1
57.9 (C-2, C-4, C-6). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd
2
for C H N O , 570.2347; found, 570.2346.
3
1
32
5
6
water phase was extracted with CH Cl (two times). Organic layers
2
2
(1-(8-Aza-2-methylhypoxanthin-8-yl)-2-deoxy-5-O-dime-
thoxytrityl-β-D-ribofuranos-3-yl)-(2-cyanoethyl)-N,N-diisopro-
pylphosphoramidite (11). The nucleoside 10 (294 mg, 0.52
mmol) was dried overnight. Then, it was dissolved in a mixture of
anhydrous CH Cl and THF (5 mL, 1:1 (v/v)), and DIPEA (270 μL,
were combined and dried over MgSO . The drying agent was
4
removed by filtration, and the filtrate was concentrated in vacuo,
affording yellow foam. The crude intermediate was dissolved under
argon in anhydrous toluene (70 mL). Next, it was added with AIBN
2
2
(
114 mg, 0.69 mmol) followed by Bu SnH (2.3 mL, 8.64 mmol). The
1.55 mmol) was added followed by addition of 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (184 μL, 0.83 mmol). The
3
resulting mixture was degassed (the air was removed using a
H
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1
resulting mixture was stirred under argon at room temperature for 2 h.
mmol) obtained in the previous step. H NMR (500 MHz, DMSO-
The reaction was quenched by addition of 5% aq NaHCO (5 mL).
d ): δ 3.47−3.52 (m, 1H, H5″), 3.59−3.63 (m, 1H, H-5′), 4.02−4.05
3
6
After 10 min of vigorous stirring, the mixture was diluted with CH Cl
(m, 1 H, H-3′), 4.26−4.29 (m, 1H, H-2′), 4.80 (bs, 1H, 5′-OH), 5.31
(bs, 1H, 3′-OH), 5.69 (bs, 1H, 2′-OH), 6.05 (d, J = 4.0 Hz, 1H, H-
2
2
and layers were separated. The organic phase was washed
subsequently with 5% aq NaHCO3 and brine and dried over
1
3
1
1′), 8.16 (s, 1H, H2), 12.47 (bs, 1H, NH). C{ H} NMR (125 MHz,
MgSO . The drying agent was removed by filtration, and the filtrate
DMSO-d ): δ 61.9 (C-5′), 70.6 (C-3′), 74.7 (C-2′), 86.4 (C-4′), 97.3
4
6
was concentrated to dryness. The crude product was purified on silica
gel flash column chromatography (CH Cl /acetone, 4:1 (v/v) with
(C-1′), 131.7 (C-5), 148.8 (C-2), 156.1 (C-4), 157.3 (C-6). HRMS-
+
ESI (Q-ToF) (m/z): [M + Na] calcd for C H N O Na, 292.0653;
2
2
9
11
5
5
0
.5 vol % addition of Et N), affording 330 mg (0.43 mmol, 83%) of
found, 292.0653. UV (H O): λ (nm) = 272, λ260 (nm), ε = 5.5 ×
2 max
3 3
3
3
1
1
−1
−1
product (mixture of two isomers) as a white foam. P{ H} NMR
10 mol ·dm ·cm .
1
(121 MHz, CD Cl ): δ 149.03, 149.14. H NMR (300 MHz,
8-Aza-8-(3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-
D-ribofuranos-1-yl)-hypoxanthine (16). A mixture from the
previous step containing the compound 15 (3.15 g) was coevaporated
with anhydrous pyridine (three times) and next dissolved in the same
solvent (112 mL). The flask was placed in an ice-water bath, and 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (4.1 mL, 12.86 mmol) was
added dropwise. The cooling bath was removed, and mixture was
stirred at room temperature for 2.5 h. The silylating reagent was
2
2
CD Cl ): δ 1.09 (d, 3H, J = 6.8 Hz), 1.16−1.20 (m, 9H), 2.45 (t, 1H,
J = 6.4 Hz), 2.57 (s, 3H), 2.61−2.74 (m, 2H), 3.06−3.15 (m, 1H),
2
2
3
4
.20−3.35 (m, 2H), 3.52−3.70 (m, 3H), 3.73−3.90 (m, 7H), 4.29−
.35 (m, 1H), 4.85−4.97 (m, 1H), 6.56−6.58 (m, 1H), 6.73−6.80
1
3
1
(
m, 4H), 7.13−7.39 (m, 9H), 11.36 (bs, 1H). C{ H} NMR (75
MHz, CD Cl ): δ 20.6, 20.7, 20.8, 20.9, 22.4, 24.7, 24.8, 39.4, 39.5,
2
2
3
6
1
1
1
9.60, 39.64, 43.7, 43.8, 43.91, 43.93, 55.6, 58.8, 58.9, 59.0, 59.1, 64.1,
4.2, 73.4, 73.7, 73.8, 74.1, 86.6, 86.9, 87.0, 87.16, 87.22, 94.39, 94.44,
13.5, 118.0, 118.1, 127.02, 127.05, 128.2, 128.6, 130.40, 130.44,
30.5, 130.6, 136.3, 136.48, 136.51, 145.43, 145.46, 157.54, 157.57,
quenched by adding CH
room temperature, volatiles were removed in vacuo. The residue was
partitioned between CH Cl and saturated aq NaHCO (30 mL).
Layers were separated, and water layer was extracted with CH Cl
(two times). Organic layers were combined and dried over MgSO
OH (30 mL). After stirring for 10 min at
3
2
2
3
+
58.8, 158.9, 158.98, 159.04. HRMS-ESI (Q-ToF) (m/z): [M + H]
2
2
calcd for C H N O P, 770.3425; found, 770.3420.
.
4
4
0
49
7
7
4
6
8
-Azahypoxanthine (13). 5,6-Diaminopyrimidin-4(3H)-one
The drying agent was removed by filtration, the filtrate was
concentrated in vacuo, and the residue was coevaporated with
toluene. The crude product was purified on silica gel flash column
chromatography (hexane/acetone, 3:1 to 2.5:1, v/v) affording 3.96 g
(
12) (4.30 g, 34.09 mmol) dissolved in 10% aq HCl (40 mL) was
cooled in an ice-water bath, and a solution of NaNO (4.02 g, 58.30
2
mmol) in H O (10 mL) was slowly added. The resulting mixture was
2
stirred at 0 °C for 30 min and next for 6 h at 45 °C. Next, it was
cooled in an ice-water bath, the precipitate was collected by filtration,
washed with ice-cold water (until neutral pH of filtrate), and dried in
vacuo, affording 3.39 g (24.73 mmol, 72%) of product as a pale yellow
(7.75 mmol, 34% after three steps) of product as an off-white foam.
1
H NMR (300 MHz, CDCl ): δ 1.06−1.19 (m, 28H, 4 ×
3
CH(CH ) ), 3.35 (s, 1H, 2′-OH), 4.04−4.06 (m, 2H, H-5′, H-5″),
3
2
4.20−4.26 (m, 1H, H-4′), 4.59 (d, J = 5.1 Hz, 1H, H-2′), 5.15 (dd, J
solid. 13C{ H} NMR (300 MHz, DMSO-d ): δ 128.4, 145.0, 155.8,
1
6
= 7.2, 5.4 Hz, 1H, H-3′), 6.34 (s, 1H, H-1′), 8.26 (s, 1H, H-2), 11.78
+
13
1
1
1
56.6. HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O,
(s, 1H, NH). C{ H} NMR (75 MHz, CDCl ): δ 12.8, 13.3, 13.4,
4
4
5
3
38.0410; found, 138.0419.
-Aza-8-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-hypoxanthine
14). A suspension of 8-hypoxanthine (13) (3.34, 24.39 mmol) and
,2,3,5-O-tetraacetyl-β-D-ribofuranose (7.76 g, 22.57 mmol) in
17.10, 17.14, 17.31, 17.38, 17.47, 17.52 (4 × CH(CH ) ), 63.4 (C-
3
2
8
5′), 73.2 (C-3′), 76.2 (C-2′), 83.9 (C-4′), 97.0 (C-1′), 131.8 (C-5),
(
1
1
47.4 (C-2), 158.0, 158.2 (C-4, C-6). HRMS-ESI (Q-ToF) (m/z):
+
[M + H] calcd for C H N O Si , 512.2355; found, 512.2352.
21 38 5 6 2
anhydrous CH CN (95 mL) was stirred at 80 °C for 5 min. Then,
BF -Et O (48%, 3.0 mL, 23.70 mmol) was added, and stirring was
continued at 80 °C for 3 h. Volatiles were removed in vacuo, and the
crude product was purified on silica gel flash column chromatography
8-Aza-8-(2-deoxy-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-β-D-ribofuranosyl)-hypoxanthine (17). The nucleoside
16 (3.96 g, 7.75 mmol) was dissolved in anhydrous CH Cl (76 mL),
3
3
2
2
2
1,1′-thiocarbonyldiimidazole (2.76 g, 15.50 mmol) was added, and
the resulting mixture was stirred under argon at reflux for 24 h. The
mixture was cooled down to rt, and CH Cl was removed in vacuo.
(
(
hexane/EtOAc 2:1 to 1:1) affording 7.05 g of mixture of compounds
difficult to separate on a bigger scale). The small amount of the
2
2
mixture was placed on another silica gel flash column chromatog-
raphy. Elution with a mixture of hexane/EtOAc (3:7, v/v) afforded an
analytical sample (21 mg, 0.053 mmol, as on off-white foam) of the
most abundant compound (highest integration value of anomeric
The crude intermediate was dissolved under argon in anhydrous
toluene (80 mL). Next, it was added with AIBN (0.431 g, 2.62 mmol)
followed by Bu SnH (6.63 mL, 24.60 mmol). The resulting mixture
3
was degassed (the air was removed using membrane pump, and
vacuum was released with argon, cycle was repeated four times) and
placed in a heating bath, and the reaction mixture was stirred at 95 °C
for 5 h. The mixture was cooled down, and volatiles were removed in
vacuo. The crude product was purified by silica gel flash column
chromatography (28−50% of EtOAc in hexane), affording 2.98 g
1
proton on the H NMR spectrum of the above mixture) that was
1
assigned to be the nucleoside 14. H NMR (500 MHz, CDCl ): δ
3
2
.13 (s, 3H, CH ), 2.14 (s, 3H, CH ), 2.15 (s, 3H, CH ), 4.24−4.28
3
3
3
(
m, 1H, H-5″), 4.53−4.56 (m, 2H, H-4′, H-5′), 5.82 (t, J = 5.5 Hz, 1
H, H-3′), 5.96 (dd, J = 5.5, 3.5 Hz, 1H, H-2′), 6.42 (d, J = 3.5 Hz, 1H,
1
3
1
1
H-1′), 8.22 (s, 1H, H-2), 11.31 (bs, 1H, NH). C{ H} NMR (125
MHz, CDCl ): δ 20.5 (CH ), 20.6 (CH ), 20.9 (CH ), 63.0 (C-5′),
(6.01 mmol, 77%) of product as an off-white foam. H NMR (300
3
3
3
3
MHz, CDCl ): δ 0.99−1.19 (m, 28H, 4 × CH(CH ) ), 2.68−2.78
3
3 2
7
1.1 (C-3′), 74.6 (C-2′), 81.6 (C-4′), 95.6 (C-1′), 132.4 (C-5), 147.7
(m, 1H, H-2″), 2.93 (dd, J = 13.5, 7.5 Hz, 1H, H-2′), 3.91−4.09 (m,
3H, H-4′, H-5′, H-5″), 5.22−5.29 (m, 1H, H-3′), 6.49 (d, J = 7.2 Hz,
(
(
C-2), 157.5 (C-4), 157.9 (C-6), 169.4 (CO), 169.7 (CO), 170.9
CO). HRMS-ESI (Q-ToF) (m/z): [M + Na] calcd for
+
13
1
1H, H-1′), 8.24 (s, 1H, H-2), 11.68 (s, 1H, NH). C{ H} NMR (75
C H N O Na, 418.0970; found, 418.0966.
MHz, CDCl ): δ 12.7, 12.9, 13.4, 13.5, 17.1, 17.3, 17.4, 17.5, 17.56,
15
17
5
8
3
4
7
8
-Aza-8-(β-D-ribofuranos-1-yl)-hypoxanthine
(15). The
17.61 (4 × CH(CH ) ), 40.8 (C-2′), 63.9 (C-5′), 72.6 (C-3′), 86.5
3
2
crude product from the previous step (7.02 g) was suspended in a
mixture of H O (18 mL) and concd aq NH solution (29%, 36 mL),
(C-4′), 93.2 (C-1′), 131.5 (C-5), 147.1 (C-2), 157.9, 158.3 (C-4, C-
+
2
3
6). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O Si ,
21 38
5
5
2
and resulting mixture was stirred at room temperature for 5 h.
Volatiles were removed in vacuo, and the crude product was passed
through silica gel column using 5−20% of CH OH in CH Cl as an
496.2406; found, 496.2408.
8-Aza-8-(2-deoxy-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-β-D-ribofuranosyl)-6-thiohypoxanthine (18). The nu-
cleoside 17 (739 mg, 1.49 mmol) was coevaporated with anhydrous
pyridine and next dissolved in this solvent (4 mL). Then, P S (828
3
2
2
eluent. Fractions containing the product were combined and
concentrated, affording 3.16 g of powder containing four compounds
2
5
(
based on the presence of four signals in the anomeric proton region
mg, 3.73 mmol) was added at room temperature, and resulting
1
on the H NMR spectrum). The analytical sample for spectral
characterization (10 mg, 0.038 mmol, 72%) was prepared in an
analogous reaction starting with the pure compound 14 (21 mg, 0.053
mixture was stirred at 105 °C for 6 h. The mixture was cooled to
room temperature, H O (10 mL) was added, and the mixture was
2
stirred at rt for 30 min. Then, it was diluted with CH Cl . Layers were
2
2
I
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
separated. The water layer was extracted with CH Cl (three times).
8-Aza-6-(2-cyanoethyl)mercapto-8-(2-deoxy-3,5-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-β-D-ribofuranosyl)-purine
2
2
Organic phases were combined and dried over MgSO . The drying
4
(
21). The compound 20 (2.00 g, 3.66 mmol) was suspended in
agent was removed by filtration, and the filtrate was concentrated and
coevaporated with toluene. The crude product was purified on silica
gel flash column chromatography (first 0−3% of CH OH in CH Cl
anhydrous CH CN (25 mL), and 3-mercaptopropanenitrile (25; 0.89
3
mL, 10.92 mmol) was added at room temperature followed by
addition of DIPEA (1.70 mL, 9.76 mmol). The mixture was stirred at
room temperature under nitrogen for 1.5 h. Volatiles were removed in
vacuo. The residue was dissolved in CH Cl and washed with 0.5 M
3
2
2
until the product was collected, and next, hexane/EtOAc 2:1 (v/v)
was used to recover the unreacted starting material). The recovered
starting material was reacted and purified in a similar way, affording an
additional batch of the product. The total yield of the target
2
2
aq NaH PO . The water phase was extracted once with CH Cl .
2
4
2
2
1
Organic extracts were combined and dried over MgSO . The drying
compound was 661 mg (1.29 mmol, 87%). H NMR (300 MHz,
4
agent was removed by filtration, and the filtrate was concentrated in
vacuo. The crude product was purified by silica gel flash column
chromatography (0−2% of CH OH in CH Cl ), affording 1.99 g
CDCl ): δ 1.03−1.15 (m, 28H, 4 × CH(CH ) ), 2.68−2.75 (m, 1H,
3
3 2
H-2″), 2.88 (dd, J = 13.5, 7.5 Hz, 1H, H-2′), 3.94−4.06 (m, 3H, H-4′,
H-5′, H-5″), 5.19−5.28 (m, 1H, H-3′), 6.51 (d, J = 6.9 Hz, 1H, H-1′),
3
2
2
1
1
3
1
(3.52 mmol, 96%). H NMR (300 MHz, CDCl ): δ 0.97−1.27 (m,
3
8
.23 (s, 1H, H-2), 11.95 (s, 1H, NH). C{ H} NMR (75 MHz,
2
2
8H, 4 × CH(CH ) ), 2.72−2.82 (ddd, J = 13.6, 9.4, 7.3 Hz, 1H, H-
3
2
CDCl ): δ 12.9, 13.0, 13.5, 13.6, 17.3, 17.4, 17.59, 17.65, 17.76, 17.81
3
″), 2.90 (dd, J = 13.4, 7.4 Hz, 1H, H-2′), 3.00 (t, J = 6.9 Hz, 2H,
(
9
4 × CH(CH ) ), 41.0 (C-2′), 64.2 (C-5′), 72.8 (C-3′), 86.7 (C-4′),
3
2
CH CN), 3.67 (t, J = 6.9 Hz, 2H, SCH ), 3.95−4.02 (m, 2H, H-5′,
2
2
3.6 (C-1′), 139.7 (C-5), 146.2 (C2), 151.9 (C-4), 180.0 (C-6).
+
H-5″), 4.04−4.10 (m, 1H, H-4′), 5.28−.36 (m, 1H, H-3′), 6.59 (d, J
HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O Si S,
2
1
38
5
4
2
13 1
=
6.9 Hz, 1H, H-1′), 8.93 (s, 1H, H-2). C{ H} NMR (75 MHz,
5
12.2177; found, 512.2180.
-Aza-8-(2-deoxy-β-D-ribofuranos-1-yl)-6-thiohypoxanthine
19; X2). The nucleoside 18 (661 mg, 1.29 mmol) was dried in a
CDCl ): δ 12.6−17.6 (4 × CH(CH ) ), 18.2 (CH CN), 24.8
3
3
2
2
8
(
1
SCH ), 41.0 (C-2′), 64.1 (C-5′), 72.7 (C-3′), 86.4 (C-4′), 93.7 (C-
(
2
′), 117.8 (CN), 133.9 (C-5), 155.0 (C-2), 155.9 (C-4), 164.3 (C-6).
vacuum overnight. Next, it was dissolved in anhydrous THF (15 mL)
+
HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O Si S,
24
41
6
4
2
and cooled in an ice-water bath, and Et N·3HF (956 μL, 5.87 mmol)
3
5
65.2443; found, 565.2457.
was added dropwise. The cooling bath was removed, and the mixture
was stirred at rt for 5 h. Volatiles were removed in vacuo. The product
was purified on silica gel flash column chromatography (5−6% of
CH OH in CH Cl ), affording 284 mg (1.06 mmol, 82%) of product
8
-Aza-6-(2-cyanoethyl)mercapto-8-(2-deoxy-β-D-ribofura-
nos-1-yl)-purine (22). The compound 21 (1.99 g, 3.52 mmol) was
dried under vacuum overnight. Next, it was dissolved in anhydrous
3
2
2
THF (33 mL) and cooled in an ice-water bath, and Et N·3HF (2.61
1
3
as a yellow solid. H NMR (600 MHz, DMSO-d ): δ 2.47 (ddd, J =
6
mL, 16 mmol) was added dropwise. The cooling bath was removed,
and mixture was stirred under nitrogen at rt for 5 h. Volatiles were
removed in vacuo. The product was purified silica gel flash column
chromatography (0−3% of CH OH in CH Cl ). Fractions containing
1
2
6
1
3.8, 6.9, 5.8 Hz, 1H, H-2″), 2.84 (ddd, J = 13.8, 6.5, 4.1 Hz, 1H, H-
′), 3.39−3.43 (m, 1H, H-5′), 3.54−3.57 (m, 1H, H-5″), 3.93 (dt, J =
.1, 4.8 Hz, 1H, H-4′), 4.50−4.54 (m, 1H, H-3′), 4.75 (t, J = 5.5 Hz,
H, 5′-OH), 5.42 (d, J = 4.8 Hz, 1H, 3′-OH), 6.52 (dd, J = 6.9, 4.1
3
2
2
the target compound were combined and evaporated, affording 0.84 g
of product. The fraction containing contaminated product was
repurified on silica gel column using acetone/CH Cl (1:4, v/v),
1
3
1
Hz, 1H, H-1′), 8.21 (s, 1H, H-2). C{ H} NMR (125 MHz, DMSO-
d ): δ 39.1−40.0 (C-2′, overlapped with residual solvent signal), 61.9
6
2
2
(
C-5′), 70.4 (C-3′), 88.9 (C-4′), 93.9 (C-1′), 138.9 (C-5), 147.2 (C-
affording additional 0.22 g of product. In total, 1.06 g (3.29 mmol,
+
2), 151.7 (C-4), 179.6 (C-6). HRMS-ESI (Q-ToF) (m/z): [M + H]
calcd for C H N O S, 270.0655; found, 270.0663. UV (H O): λ
max
1
9
3%) of compound 22 as a white foam was obtained. H NMR (600
9
12
5
3
2
MHz, DMSO-d ): δ 2.50−2.56 (m, 1H, H-2′ overlapped with residual
3
−1
3
−1
6
(
nm) = 347, λ260 (nm), ε = 2.2 × 10 mol ·dm ·cm ; UV
3 3
solvent signal), 2.90 (ddd, J = 13.8, 6.4, 3.8 Hz, 1H, H-2′), 3.10 (t, J =
−1
(CH OH): λ (nm) = 348, λ260 (nm), ε = 2.6 × 10 mol ·dm ·
3
max
6
.7 Hz, 2H, CH CN), 3.43−3.47 (m, 1H, H-5′), 3.57−3.61 (m, 1H,
−
1
2
cm .
-Aza-8-(2-deoxy-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
,3-diyl)-β-D-ribofuranosyl)-6-(1,2,4-triazol-1-yl)-purine (20).
,2,4-Triazole (7.66 g, 110.9 mmol) was dried overnight at 0.2
mbar. Then, it was dissolved in anhydrous CH CN (36 mL), Et N
H-5′), 3.71 (t, J = 6.7 Hz, 2H, SCH ), 3.96−3.98 (m, 1H, H-4′),
4
=
2
8
.56−4.60 (m, 1H, H-3′), 4.74 (t, J = 5.7 Hz, 1H, 5′-OH), 5.45 (d, J
1
1
4.8 Hz, 1H, 3′-OH), 6.68 (dd, J = 7.0, 3.8 Hz, 1H, H-1′), 8.99 (s,
13
1
1
2
H, H-2). C{ H} NMR (125 MHz, DMSO-d ): δ 17.5 (CH CN),
6 2
3
3
4.2 (SCH ), 39.8 (C-2′), 61.8 (C-5′), 70.3 (C-3′), 89.2 (C-4′), 94.7
2
(15.4 mL, 110.5 mmol) was added, and the mixture was cooled in an
(
C-1′), 119.1 (CN), 133.2 (C-5), 155.0 (C-2), 155.3 (C-4), 164.0
ice bath. Next, POCl (2.97 mL, 31.8 mmol) was added dropwise.
C-6). HRMS-ESI (Q-ToF) (m/z): [M + H]+ calcd for
3
(
Suspension was stirred at 0 °C for 10 min, and solution of the
C H N O S, 323.0921; found, 323.0924.
1
2
15
6
3
nucleoside 17 (2.1 g, 4.24 mmol) in a mixture of CH CN and
3
8
-Aza-6-(2-cyanoethyl)mercapto-8-(2-deoxy-5-O-dimethox-
pyridine (50 mL, 1:1 (v/v)) was added. The mixture was stirred at rt
ytrityl-β-D-ribofuranos-1-yl)-purine (23). The nucleoside 22
for 24 h. Then, H O (3.2 mL) was added, and resulting mixture was
2
(0.84 g, 2.61 mmol) was coevaporated with anhydrous pyridine
stirred for 30 min. Volatiles were removed in vacuo. The residue was
(
three times). Then, it was dissolved in anhydrous pyridine (30 mL),
partitioned between saturated NaHCO (115 mL) and EtOAc. Layers
3
and the resulting solution was cooled in an ice-water bath. Next,
were separated, and the aqueous phase was extracted with EtOAc
DMTCl (1.06 g, 3.13 mmol) in CH Cl (3 mL) was added under N .
2
2
2
(
two times). Organic extracts were combined and dried over MgSO4.
The cooling bath was removed, and the mixture was stirred at rt
The drying agent was removed by filtration, the filtrate was
concentrated to dryness, and the residue was coevaporated with
toluene. The product was purified using silica gel flash column
overnight and next at 35 °C for 5 h. The reaction was quenched with
CH OH. Volatiles were removed in vacuo. The residue was
3
partitioned between CH Cl and 5% aq NaHCO . The water phase
2
2
3
chromatography (hexane/EtOAc, 3:1 to 2:1, v/v), affording 2.00 g
was extracted with CH Cl . Combined organic fractions were washed
2 2
1
(
3.66 mmol, 86%) as a white foam. H NMR (300 MHz, CDCl ): δ
with H O and brine and dried over MgSO . The drying agent was
2 4
3
0
7
4
.99−1.26 (m, 28H, 4 × CH(CH ) ), 2.73−2.83 (ddd, J = 13.7, 9.3,
removed by filtration, and the filtrate was concentrated and
coevaporated with toluene. The crude product was purified by silica
gel flash column chromatography (acetone/CH Cl , 5:95 (v/v), to
3
2
.4 Hz, 1H, H-2″), 2.92−2.99 (dd, J = 13.6, 7.4 Hz, 1H, H-2′), 4.03−
.15 (m, 3H, H-4′, H-5′, H-5″), 5.34−5.41 (m, 1H, H-3′), 6.76 (d, J
2
2
=
6.9 Hz, 1H, H-1′), 8.36 (s, 1H, H-3-imidazole), 9.15 (s, 1H, H-2),
remove yellow contamination then 5% of CH OH in CH Cl , all
3 2 2
1
3
1
9
1
4
.56 (s, 1H, H-5-imidazole). C{ H} NMR (75 MHz, CDCl ): δ
eluent systems contained 0.5 vol % Et N), affording 1.13 g (1.81
3
3
1
2.8, 12.9, 13.3, 13.4, 17.1, 17.3, 17.47, 17.53, 17.6 (4 × CH(CH ) ),
mmol, 69%) of product as a white foam. H NMR (300 MHz,
3
2
1.5 (C-2′), 64.3 (C-5′), 73.0 (C-3′), 86.9 (C-4′), 94.6 (C-1′), 125.7
CDCl ): δ 2.45 (bs, 1H, 3′-OH), 2.57−2.66 (m, 1H, H-2″), 2.94 (t, J
3
(C-5), 144.7 (C-5-imidazole), 148.5 (C-6), 155.2 (C-3-imidazole),
= 7.1 Hz, 2H, CH CN), 3.01 (ddd, J = 13.7, 6.6, 3.0 Hz, 1H, H-2′),
2
+
1
55.7 (C-2), 161.1 (C-4). HRMS-ESI (Q-ToF) (m/z): [M + H]
3.33 (dd, J = 10.1, 5.3 Hz, 1H, H-5″), 3.41 (dd, J = 10.1, 5.7 Hz, 1H,
H-5′), 3.61 (t, J = 7.1 Hz, 2H, SCH ), 3.75 (s, 3H, OCH ), 3.76 (s,
calcd for C H N O Si , 547.2627; found, 547.2636.
23
39
8
4
2
2
3
J
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
3
H, OCH ), 4.20 (q, J = 5.4 Hz, 1H, H-4′), 4.89−4.95 (m, 1H, H-3′),
heated to 45 °C and held at 45−47 °C for 45 min. The heating was
removed, and the flask with the mixture was placed in an ice-salt bath.
After cooling to 20 °C, aq H SO (6.0 M, 5 mL) was slowly added
3
6
(
.64 (dd, J = 6.9, 3.0 Hz, 1H, H-1′), 6.71−7.36 (m, 13H, DMT), 8.89
s, 1H, H-2). 13C{ H} NMR (75 MHz, CDCl ): δ 18.3 (CH CN),
1
3
2
2
4
2
4.9 (SCH ), 40.3 (C-2′), 55.3 (2 × OCH ), 64.2 (C-5′), 72.5 (C-
until pH 6, keeping the internal temperature between 20−25 °C.
2
3
3
′), 86.6 (DMT), 87.1 (C-4′), 94.9 (C-1′), 113.3 (DMT), 117.8
Next, Et O (7.1 mL) was added, the mixture was stirred for 1 min,
2
(
1
(
CN), 126.9 (DMT), 127.9 (DMT), 128.2 (DMT), 130.1 (DMT),
30.2 (DMT), 134.0 (C-5), 135.95 (DMT), 135.99 (DMT), 144.8
DMT), 155.1 (C-2), 155.9 (C-4), 158.6 (DMT), 164.4 (C-6).
and layers were left to separate. The ether layer was transferred (using
a syringe and a needle) to the Erlenmeyer flask containing MgSO4
(ca. 5.5 g). The process was repeated four times using the same
+
HRMS-ESI (Q-ToF) (m/z): [M + Na] calcd for C H N O SNa,
amount of Et O each time. The organic layer was transferred to the
3
3
32
6
5
2
647.2047; found, 647.2045.
round-bottom flask, concentrated (35 °C, 200−300 mbar, and then
(
1-(8-Aza-6-(2-cyanoethyl)mercaptopurin-8-yl)-2-deoxy-5-
50 mbar) until half of the beginning volume, and dried over MgSO
4
O-dimethoxytrityl-β-D-ribofuranos-3-yl)-(2-cyanoethyl)-N,N-
diisopropylphosphoramidite (24). The compound 23 (1.13 g,
(ca. 2 g). After removal of the drying agent, solution was concentrated
and transferred to a 25 mL round-bottom flask. The product was
distilled through a short-path vacuum distillation apparatus (8 mbar,
1
.81 mmol) was dried at high vacuum at room temperature overnight.
Next, it was dissolved under argon in CH Cl (15 mL). Then, DIPEA
2
2
5
7−59 °C) with a receiving flask placed in a dry ice to the level of the
(
0.95 mL, 5.43 mmol) was added followed by addition of 2-
ground glass joint, affording 0.9 mL (11.05 mmol, 35%) of the
cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.65 mL, 2.89
mmol). The resulting mixture was stirred at room temperature
under argon for 3 h. The reaction was quenched with of 5% aq
product as transparent colorless liquid with very unpleasant odor. The
product was kept tightly closed at −30 °C and used within a few days.
48
6
-Chloro-2-methylpyrimidine-4,5-diamine (27). The com-
NaHCO (7.5 mL). After 10 min of vigorous stirring, the mixture was
diluted with CH Cl . Layers were separated, and aqueous phase was
extracted with CH Cl (one time). Organic layers were combined,
and washed subsequently with 5% aq NaHCO and brine, and dried
3
pound 26 (10.5 g, 58.98 mmol) in concd aq NH (30 mL) was stirred
3
2
2
in a sealed flask at 120 °C overnight. Next, the mixture was cooled in
an ice-water bath. The solid was collected by filtration, washed with a
small amount of ice-cold water, and dried, affording the product as a
yellow precipitate (8.6 g). The filtrate was concentrated and subjected
2
2
3
over MgSO . The drying agent was removed by filtration, and the
4
filtrate was concentrated to dryness. The residue was purified by silica
gel flash column chromatography (4−5% of acetone in CH Cl with 1
vol % Et N), affording 0.80 g (0.97 mmol, 54%) of product (mixture
of two isomers) as a white foam. P{ H} NMR (121 MHz, CD Cl ):
δ 148.94, 149.03. H NMR (300 MHz, CD Cl ): δ 1.10 (d, 3H, J =
6
6
1
4
7
to silica gel chromatographic purification (5−10% of CH OH in
3
2
2
CH Cl ), affording additional 0.35 g of product. Total yield: 8.95 g
2
2
3
1
3
1
1
(56.44 mmol, 96%). H NMR (300 MHz, DMSO-d
CH ), 4.72 (bs, 2H, 5-NH ), 6.65 (bs, 2H, 4-NH ). C{ H} NMR
75 MHz, DMSO-d ): δ 24.0 (CH ), 120.3 (C-5), 138.0 (C-6), 153.8
6
): δ 2.19 (s, 3H,
2
2
13
1
1
3
2
2
2
2
(
(
6
3
.8 Hz), 1.17−1.20 (m, 9H), 2.46 (t, 1H, J = 6.4 Hz), 2.62 (t, 1H, J =
.3 Hz), 2.67−2.80 (m, 1H), 2.96 (t, 2H, J = 7.0 Hz), 3.08−3.17 (m,
H), 3.26−3.37 (m, 2H), 3.52−3.70 (m, 5H), 3.74−3.90 (m, 7H),
.36−4.43 (m, 1H), 4.86−4.99 (m, 1H), 6.69−6.76 (m, 5H), 7.11−
+
C-4), 154.1 (C-2). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for
C H ClN , 159.0432; found, 159.0435.
5
8
4
4
9
6
-Chloro-2-methyl-9H-purine (28). The compound 27 (5.01
1
3
1
g, 31.59 mmol), triethylorthoformate (57 mL), and formic acid (1.25
mL) were stirred at 120 °C for 5.5 h. The mixture was cooled to room
temperature, and the precipitate was collected by filtration, washed
.37 (m, 9H), 8.89 and 8.90 (2 × s, 1H). C{ H} NMR (75 MHz,
CD Cl ): δ 18.6, 20.6, 20.7, 20.8, 20.9, 24.8, 24.9, 25.4, 39.9, 40.0,
2
2
4
7
1
1
1
0.09, 40.13, 43.8, 43.9, 55.6, 58.8, 58.9, 59.1, 59.2, 64.2, 64.4, 73.4,
3.6, 73.8, 74.1, 86.6, 87.3, 87.4, 87.5, 87.6, 95.4, 95.5, 113.4, 118.0,
18.1, 118.3, 127.01, 127.03, 128.1, 128.5, 128.6, 130.43, 130.46,
30.49, 134.4, 136.3, 145.35, 145.38, 155.4, 156.4, 158.98, 159.01,
64.7. HRMS-ESI (Q-ToF) (m/z): [M + H]+ calcd for
subsequently with hexane and Et O, and dried in vacuo. The crude
2
product was purified by silica gel flash column chromatography (5%
of CH OH in CH Cl ), affording 3.86 g (22.90 mmol, 72%) of
3
2
2
1
product as an off-white solid. H NMR (300 MHz, CD OD): δ 2.70
s, 3H, CH ), 4.98 (bs, 1H, NH), 8.45 (s, 1H, H-8). C{ H} NMR
3
1
3
1
(
C H N O PS, 825.3306; found, 825.3323.
3
4
2
3
50
8
6
3
6,37
(75 MHz, CD OD): δ 25.2 (CH ), 128.1 (C-5), 146.4 (C-8), 149.4
-Mercaptopropanenitrile (25).
The compound 25 was
3
3
1
37
(C-6), 156.0 (C-5), 163.5 (C-2). H NMR (300 MHz, DMSO-d ): δ
6
synthesized according to a slightly modified reported procedure. In
a two-necked round-bottom flask were placed thiourea (3.83 g, 50.38
mmol), 3-bromopropionitrile (3.10 mL, 37.32 mmol), and H O (2.5
mL). In the first neck, it was installed with a condenser with an
attached balloon with N , and in the second neck, it was equipped
1
3
1
2
.6 (s, 3H, CH ), 8.6 (s, 1H, H-8), 13.6 (bs, 1H, NH). C{ H} NMR
3
(
(
75 MHz, DMSO-d ): δ 24.9 (CH ), 127.3 (C-5), 145.3 (C-8), 147.4
6 3
2
C-6), 154.4 (C-5), 160.9 (C-2). HRMS-ESI (Q-ToF) (m/z): [M +
+
H] calcd for C H ClN , 169.0275; found, 169.0278.
6
6
4
2
5
0
2
-Methyl-9H-purine (29). To a suspension of chloropurine 28
with a balloon with N . The reaction mixture was placed in a heating
2
(
3.86 g, 22.90 mmol) and Pd(OH) (20% on carbon, 3.86 g) in
bath (room temperature), stirred using a magnetic stirrer, and heated
to 68 °C (during ∼20 min). The mixture was kept stirring at this
temperature for 1 h. Then, the temperature of the heating bath was
increased to 100 °C (during ∼15 min), and stirring was continued at
this temperature for 2 h. The heating bath was replaced with a cooling
bath (ice-water). Stirring stopped as the product started to solidify.
After 30 min of cooling, it was added with cold acetone (12 mL, kept
2
anhydrous CH OH (150 mL) was added ammonium formate (4.43 g,
3
7
0.29 mmol). The resulting mixture was stirred at reflux for 2.5 h.
Then, the mixture was filtered through Celite. Celite was washed with
CH OH. The filtrate was concentrated to dryness. The crude product
was purified using silica gel flash column chromatography (10−15%
of CH OH in CH Cl ), affording 2.77 g (20.65 mmol, 90%) of
product as a white powder. H NMR (300 MHz, DMSO-d
(s, 3H, CH ), 8.49 (s, 1H, H-6), 8.98 (s, 1H, H-8), 13.21 (bs, 1H,
3
3
2
2
1
5
h at −30 °C) and the solid was carefully crushed using a spatula.
): δ 2.66
6
The content of the flask was stirred for 5 min. The solid was collected
by filtration, subsequently washed with 24 mL of cold acetone and 12
mL of cold Et O, and dried in vacuo for 18 h, affording 6.6 g (31.42
3
+
NH). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C
H N ,
7 4
6
135.0665; found, 135.0667.
2
mmol, 84%) of 2-(2-cyanoethyl)isothiouronium bromide as a white
9-(2-Deoxy-3,5-di-O-toluoyl-β-D-ribofuranosyl)-2-methyl-
1
solid. H NMR (300 MHz, D O): δ 3.04 (t, J = 6.6 Hz, 2H, CH CN),
purine (30a). To a suspension of the compound 29 (1.30 g, 9.69
2
2
1
3
1
3
.49 (t, J = 6.6 Hz, 2H, SCH2). C{ H} NMR (75 MHz, D O): δ
mmol) in anhydrous CH CN (75 mL) was added NaH (60%
3
2
1
7.9 (SCH ), 26.2 (CH CN), 118.8 (CN), 169.7 (C-uronium).
In a three-necked round-bottom flask equipped with a
dispersion in mineral oil, 465 mg, 11.63 mmol), and the resulting
mixture was stirred under nitrogen at rt for 90 min. Next, Hoffer’s
sugar (3.96 g, 10.18 mmol) was added, and stirring was continued for
70 min. The mixture was filtered through Celite. The filtrate was
concentrated and put on silica gel column. Elution with a mixture of
AcOEt/hexane (3:7, v/v) afforded the product as a pale brown foam
(2.95 g, 6.06 mmol, 63%). Further, elution afforded 7-(2-deoxy-3,5-
di-O-toluoyl-β-D-ribofuranosyl)-2-methylpurine 30b.
2
2
thermometer, a septum, and a needle connected to a N source
2
were placed thiouronium salt (6.60 g, 31.41 mmol) and water (6.6
mL). The mixture was purged with nitrogen while stirring for 15 min.
Then, a concentrated NaOH solution (11.25 M, 5.3 mL) was slowly
added under a nitrogen atmosphere, keeping internal temperature
below 25 °C. After the addition was completed, the mixture was
K
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1
3
0a: H NMR (300 MHz, CDCl ): δ 2.40 (s, 3H, CH -tol), 2.45
product was purified by silica gel flash column chromatography (2−
3% of CH OH in CH Cl ), affording the product as a pale-yellow
3
3
(
=
4
1
4
s, 3H, CH -tol), 2.83−2.89 (m, 4H, H2′, 2-CH ), 3.10−3.20 (ddd, J
3
3
3
2
2
1
14.3, 8.1, 6.3 Hz, 1H, H-2″), 4.65−4.71 (m, 2H, H-4′, H-5′), 4.76−
foam (1.77 g, 3.21 mmol, 66%). H NMR (300 MHz, CDCl ): δ
3
.82 (dd, J = 13.1, 5.6 Hz, 1H, H-5″), 5.83−5.86 (dt, J = 6.1, 2.15 Hz,
H, H-3′), 6.58−6.63 (dd, J = 8.1, 6.0 Hz, 1H, H-1′), 7.20−7.31 (m,
H, H-Ph), 7.87−8.00 (m, 4H, H-Ph), 8.18 (s, 1H, H8), 9.03 (s, 1H,
2.51−2.59 (m, 1H, H-2″), 2.71 (s, 3H, CH ), 2.84 (dt, J = 13.5, 6.4
3
Hz, 1H, H-2′), 3.06 (bs, 1H, 3′-OH), 3.36−3.42 (dd, J = 10.2, 4.8 Hz,
1H, H-5″), 3.43−3.48 (dd, J = 10.2, 4.5 Hz, 1H, H-5′), 3.76 (s, 6H, 2
1
3
1
H-6). C{ H} NMR (75 Hz, CDCl ): δ 21.77 (CH ), 21.83 (CH ),
× OCH ), 4.17−4.19 (m, 1H, H-4′). 4.70 (bs, 1H, H-3′), 6.52 (t, J =
3
3
3
3
2
(
1
1
6.0 (2-CH), 37.9 (C-2′), 64.1 (C-5′), 75.2 (C-3′), 83.1 (C-4′), 84.7
6.4 Hz, 1H, H-1′), 6.76−7.40 (m, 13H, DMT), 8.15 (s, 1H, H-8),
1
3
1
C-1′), 126.6, 126.8, 129.4, 129.8, 129.9, 132.7 (C-5), 142.8 (C-8),
9.00 (s, 1H, H-6). C{ H} NMR (75 MHz, CDCl ): δ 26.0 (CH ),
3 3
44.3, 144.7, 148.8 (C-6), 151.6 (C-4), 162.8 (C-2), 166.1 (CO),
40.2 (C-2′), 55.3 (2 × OCH ), 64.0 (C-5′), 72.7 (C-3′), 84.2 (C-1′),
3
+
66.3 (CO). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for
86.4 (C-4′), 86.8 (DMT), 113.3 (DMT), 127.1 (DMT), 128.0
(DMT), 128.2 (DMT), 130.1 (DMT), 132.6 (C-5), 135.7 (DMT),
135.7 (DMT), 143.2 (C-8), 144.6 (DMT), 148.5 (C-6), 151.5 (C-4),
158.7 (DMT), 162.5 (C-2). HRMS-ESI (Q-ToF) (m/z): [M + H]+
calcd for C H N O , 553.2445; found, 553.2450.
C H N O , 487.1976; found, 487.1976.
2
7
3
27
4
5
1
0b: H NMR (300 MHz, CDCl ): δ 2.39 (s, 3H, CH -tol), 2.45
3
3
(
s, 3H, CH -tol), 2.88 (s, 3H, 2-CH ), 2.89−2.92 (m, 2H, H-2′, H-
3
3
2
6
7
″), 4.67−4.72 (m, 3H, H-4′, H-5′, H-5″), 5.71−5.74 (m, 1H, H-3′),
3
2
33
4
5
.58−6.43 (t, J = 6.8 Hz, 1H, H-1′), 7.18−7.29 (m, 4H, H-Ph), 7.78−
(2-Deoxy-5-O-dimethoxytrityl-1-(2-methylpurin-9-yl)-β-D-ri-
bofuranos-3-yl)-(2-cyanoethyl-N,N-diisopropyl)-
phosphoramidite (33). The compound 32 (500 mg, 0.90 mmol)
was dried at high vacuum at rt. Next, it was dissolved in anhydrous
1
3
1
.97 (m, 4H, H-Ph), 8.38 (s, 1H, H-8), 8.99 (s, 1H, H-6). C{ H}
NMR (75 Hz, CDCl ): δ 21.72 (CH ), 21.79 (CH ), 26.0 (2-CH ),
3
3
3
3
3
(
1
8.6 (C-2′), 63.8 (C-5′), 74.6 (C-3′), 83.2 (C-4′), 86.4 (C-1′), 122.3
C-5), 126.3, 126.4, 129.4, 129.6, 129.9, 140.8 (C-6), 144.4, 144.8,
45.2 (C-8), 162.1, 163.3 (C-2, C-4), 165.9 (CO), 166.1 (CO).
HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O ,
87.1976; found 487.1975.
CH Cl (9 mL). DIPEA (473 μL, 2.71 mmol) was added and
2 2
followed by addition of 2-cyanoethyl-N,N-diisopropylchlorophos-
phoramidite (323 μL, 1.45 mmol). The resulting mixture was stirred
under argon at rt for 3 h. The reaction was quenched with 4.5 mL of
+
2
7
27
4
5
4
9
-(2-Deoxy-β-D-ribofuranos-1-yl)-2-methylpurine (31a; X5).
5% aq NaHCO
diluted with CH
was extracted with CH
combined, washed subsequently with 5% aq NaHCO
and dried over MgSO . The drying agent was filtered-off, and the
filtrate was concentrated until dryness. The product was purified by
silica gel flash column chromatography. Elution with CH Cl /acetone
2:1 (v/v, with 0.5 vol % addition of Et N) afforded the mixture (703
3
. After 10 min of vigorous stirring, the mixture was
Cl . Layers were separated, and the aqueous phase
Cl (one time). Organic layers were
and brine,
To a solution of the nucleoside 30a (2.96 g, 6.09 mmol) in anhydrous
2
2
CH OH (90 mL) was added solution of NaOCH in CH OH (5.4 M,
2
2
3
3
3
2
72 μL, 1.47 mmol), and resulting mixture was stirred at rt for 2.5 h.
Then, solid NH Cl (82 mg, 1.53 mmol) was added, and stirring was
3
4
4
continued for 5 min. The mixture was deposited on silica gel and
subjected to flash column chromatography. Elution with a mixture of
CH Cl /CH OH (95:5−93:7−9:1, v/v) afforded the product as an
2
2
2
2
3
3
1
off-white foam (1.23 g, 4.92 mmol, 81%). H NMR (600 MHz,
mg) of the product (as a mixture of two isomers) contaminated with a
DMSO-d ): δ 2.33 (ddd, J = 13.2, 6.6, 3.6 Hz, 1H, H-2″), 2.69 (s, 3H,
hydrolyzed phosphorylating reagent (2-cyanoethyl-N,N-diisopropyl-
6
3
1
1
CH ), 2.75 (ddd, J = 13.2, 7.8, 6.0 Hz, 1H, H-2′), 3.53−3.56 (m, 1H,
amine-H-phosphonate; P{ H} NMR [121 MHz, acetone-d ]: δ
6
3
3
1
1
H-5″), 3.62−3.65 (m, 1H, H-5′), 3.89 (m, 1H, H-4′), 4.43−4.46 (m,
13.48 ppm). Based on the P{ H} NMR spectrum, the purity of the
product was 88.5% (wt) (91% yield), and it was used in
oligonucleotide synthesis without further purification P{ H} NMR
(121 MHz, acetone-d ): δ 148.2, 148.4. P{ H} NMR (121 MHz,
CD Cl ): δ 148.70 (bs). H NMR (300 MHz, CD Cl ): δ 1.13 (d,
2 2
1
3
(
3
(
H, H-3′), 5.06 (t, J = 5.4 Hz, 1H, 5′-OH), 5.36 (d, J = 4.2 Hz, 1H,
3
1
1
′-OH), 6.46 (dd, J = 7.8, 6.6 Hz, 1H, H-1′), 8.70 (s, 1H, H-8), 9.03
s, 1H, H-6). 13C{ H} NMR (125 MHz, DMSO-d ): δ 25.5 (CH ),
1
31
1
6
3
6
1
9.3 (C-2′), 61.7 (C-5′), 70.8 (C-3′), 83.5 (C-1′), 88.0 (C-4′), 132.1
2
2
C-5), 144.6 (C-8), 148.0 (C-6), 151.2 (C-4), 161.2 (C-2). HRMS-
3H, J = 6.8 Hz), 1.18−1.21 (m, 9H), 2.48−2.52 (m, 1H), 2.56−2.71
(m, 5H), 2.92−3.01 (m, 1H), 3.34−3.41 (m, 2H), 3.59−3.91 (m,
10H), 4.26−4.33 (m, 1H), 4.77−4.85 (m, 1H), 6.47 (t, 1H, J = 6.6
Hz), 6.73−6.79 (m, 4H), 7.19−7.30 (m, 7H), 7.37−7.42 (m, 2H),
+
ESI (Q-ToF) (m/z): [M + H] calcd for C H N O , 251.1138;
1
1
15
4
3
found, 251.1135. UV (H O): λ (nm) = 268, λ260 (nm), ε = 6.2 ×
2
max
3
−1
3
−1
1
6
0 mol ·dm ·cm ; UV (CH OH): λ (nm) = 268, λ260 (nm), ε =
3 max
3 3
−1
−1
13
1
.0 × 10 mol ·dm ·cm .
-(2-Deoxy-β-D-ribofuranos-1-yl)-2-methylpurine (31b).
The compound 31b (27 mg, 0.108 mmol, 61%) was obtained from
8.15−8.16 (2 × s, 1H), 8.95 (s, 1H). C{ H} NMR (75 MHz,
7
CD Cl ): δ 19.6, 19.65, 19.67. 19.8, 23.7, 23.8, 25.0, 29.9, 38.2, 38.3,
2
2
38.4, 42.6, 42.7, 54.5, 57.6, 57.8, 57.9, 58.0, 63.0, 63.2, 72.8, 73.1,
73.3, 73.6, 83.7, 83.8, 85.0, 85.1, 85.2, 85.3, 85.7, 112.4, 116.5, 117.0,
117.1, 126.1, 126.2, 127.1, 127.41, 127.45, 129.35, 129.39, 129.42,
3
3
0b (65 mg, 0.176 mmol) by following the procedure described for
1a. H NMR (600 MHz, DMSO-d ): δ 2.33 (ddd, J = 13.2, 6.06, 3.6
1
6
Hz, 1H, H-2′), 2.67 (s, 3H, 2-CH ), 2.58 (ddd, J = 13.2, 7.8, 6.0 Hz,
132.1, 135.1, 142.8, 144.2, 147.6, 150.8, 158.0, 161.49, 161.53.HRMS-
3
+
1
3
H, H-2″), 3.54−3.61 (m, 1H, H-5″), 3.62−3.65 (m, 1H, H-5′),
.90−3.92 (m,1H, H-4′), 4.40−4.41 (m, 1H, H-3′), 5.10 (bs, 1H, 5′-
ESI (Q-ToF) (m/z): [M + H] calcd for C41
H
N
50
O
6
P, 753.3524;
6
found, 753.3528.
OH), 5.45 (bs, 1H, 3′-OH), 6.41 (dd, J = 7.8, 6.0 Hz, 1H, H-1′), 8.82
6-Amino-9-(3,5-di-O-(tert-butyldimethylsilyl)-2-deoxy-β-D-
ribofuranosyl)-8-methylpurine (35). Commercially available 8-
bromodeoxyadenosine 34 (5.0 g, 15 mmol) was suspended in
anhydrous pyridine (50 mL), TBSCl (6.8 g, 45 mmol) was added, and
the reaction was stirred overnight. The reaction was quenched with
1
3
1
(
6
1
s, 1H, H-8), 9.22 (s, 1H, H-6). C{ H} NMR (125 MHz, DMSO-
d ): δ 25.6 (2-CH ), 40.0 (C-2′), 61.2 (C-5′), 70.4 (C-3′), 85.9 (C-
3
′), 88.0 (C-4′), 122.0 (C-5), 141.9 (C-6), 147.4 (C-8), 161.2, 161.3
+
(
C-2, C-4). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for
C H N O , 251.1138; found, 251.1145. UV (H O): λ (nm) =
saturated aq NaHCO
were combined, dried over MgSO
4
3
and extracted with CH
2 2
Cl . The organic layers
1
1
15
4
3
2
max
3
−1
3
−1
2
71, λ260 (nm), ε = 4.4 × 10 mol ·dm ·cm .
9
methylpurine (32). The nucleoside 31a (1.21 g, 4.82 mmol) was
coevaporated with anhydrous pyridine (three times). Then, it was
dissolved in anhydrous pyridine (48 mL) and cooled in an ice-water
bath. Next, DMTCl (1.96 g, 5.78 mmol) in CH Cl (9 mL) was
, and evaporated. The residue was
39
-(2-Deoxy-5-O-dimethoxytrityl-β-D-ribofuranos-1-yl)-2-
purified to get 8-bromo-5′,3′-bis-TBDMS-deoxyadenosine in a
quantitative yield. In the next reaction, 10 g (17.8 mmol) of bis-
TBS-protected 8-bromodeoxyadenosine was dissolved in anhydrous
dioxane (50 mL). Then, (CH ) Sn (10 mL,72 mmol) and Pd (PPh )
3 4
3
4
(2.0 g, 1.78 mmol) were added, and the mixture was refluxed for 6−8
h. After completion of the reaction, as shown by TLC, the solvent was
evaporated and the residue was purified by silica gel chromatography
2
2
added under N . The cooling bath was removed, and the mixture was
stirred under N at rt for 4 h. The reaction was quenched with
2
2
1
saturated aq NaHCO3 (25 mL). The mixture was diluted with
CH Cl , and layers were separated. The water phase was extracted
(EtOAc/heptane) to obtain the target compound 35 (6.0 g, 80%). H
NMR (300 MHz, CDCl ) δ −0.04 (s, 3H), 0.00 (s, 3H), 0.14 (s,
2
2
3
with CH Cl (two times). Combined organic fractions were dried
over MgSO . The drying agent was removed by filtration, and the
filtrate was concentrated and coevaporated with toluene. The crude
6H), 1.48 (s, 9H), 1.57 (s, 9H), 2.23 (ddd, J = 13.1, 6.6, 3.0 Hz, 1H,
H-2″), 3.67 (m, 2H, H-2′, H-5′′), 3.93 (m, 2H, H-5′, H-4′), 4.87 (m,
2
2
4
1
3
1
1H, H-3′), 6.34 (appt, J = 6.72 Hz, 1H), 8.24 (s, 1H, H-2). C{ H}
L
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
NMR (75 MHz, CDCl ): δ −4.6, −4.5, 15.3 (C-8 methyl), 25.9, 37.6
(2-Deoxy-5-O-dimetoxytrityl-1-(8-methylpurin-9-yl)-β-D-ri-
bofuranos-3-yl)-(2-cyanoethyl)-N,N-diisopropylphosphorami-
dite (40). The compound 39 (0.5 g, 0.9 mmol) was dissolved in
anhydrous THF and cooled in an ice bath. DIPEA (0.8 mL, 4.5
mmol) was added followed by addition of 2-cyanoethyl-N,N-
diisopropyl chlorophosphoramidite (0.3 mL, 0.99 mmol). The
reaction was kept at 0 °C for 30 min and then at rt for 2 h.
Additional 1.1 equiv of phosphitylation reagent was added, and
reaction again left for 2 h. After completion, the reaction was
quenched with methanol and the solvent evaporated. The residue was
3
(
C-2′), 62.8 (C-5′), 72.3 (C-3′), 84.3 (C-4′), 87.5 (C-1′), 118.9 (C-
5
(
), 150.6 (C-4), 151.2 (C-2), 151.9 (C-6), 154.4 (C-8). HRMS-ESI
Q-ToF) (m/z): [M + H]+ calcd for C H N O Si , 494.2977;
2
3
44
5
3
2
found, 494.2976.
6
-Amino-9-(3,5-di-O-acetyl-2-deoxy-β-D-ribofuranosyl)-8-
methylpurine (36). The compound 35 was deprotected with TBAF
and purified. The residue was dissolved in pyridine and acetylated
using acetic anhydride. The target compound 36 was obtained in 70%
1
yield. H NMR (300 MHz, CDCl ): δ 2.04 (s, 3H), 2.14 (s, 3H), 2.39
3
(
(
ddd, J = 14.0, 6.6, 2.7 Hz, 1H, H-2″), 2.66 (s, 3H, C-8 Methyl), 3.73
purified by chromatography (acetone/heptane + 2% Et N) to obtain
3
3
1
1
ddd, J = 14.0, 8.2, 7.7 Hz, 1H, H-2′), 4.24−4.32 (m, 2H, H-4′, H-
the product as a mixture of two isomers (0.29 g, 44%). P{ H} NMR
1
5
″), 4.47 (dd, J = 11.0, 4.46 Hz, 1H, H-5′), 5.57 (m, 1H, H-3′), 6.29
(121 MHz, CDCl ): δ 149.16, 149.33. H NMR (300 MHz, CDCl ):
3
3
1
3
1
(
dd, J = 7.9, 6.5 Hz, 1H, H-1′), 8.28 (s, 1H, H-2); C{ H} NMR (75
δ 1.13−1.37 (m, 14H), 2.50 (m, 1H), 2.63 (m, 1H), 2.74 (s, 3H),
3.27 (m, 1H), 3.38 (m, 1H), 3.34−3.62 (m, 4H), 3.77 (s, 6H), 4.26
(m, 1H), 4.97 (m, 1H), 6.32 (m, 1H), 6.76 (m, 4H), 7.16−7.36 (m,
9H), 8.67 (s, 0.5H), 8.69 (s, 0.5H), 8.94 (s, 0.5H), 8.95 (s, 1H).
MHz, CDCl ): δ 15.1 (methyl-C-8), 20.8 (acetyl), 21.1(acetyl), 34.8
3
(
C-2′), 63.7 (C-5′), 74.9 (C-3′), 82.4 (C-4′), 84.5 (C-1′), 118.9 (C-
5
), 149.8 (C-4), 151.2 (C-2), 152.2 (C-6), 154.5 (C-8), 170.4 (CO-
+
13
1
acetyl), 170.7 (CO-acetyl). HRMS-ESI (Q-ToF) (m/z): [M + H]
C{ H} NMR (75 MHz, CDCl ): δ 15.4, 20.2, 20.3, 20.4, 20.5, 20.6,
3
calcd for C H N O , 350.1458; found, 350.1458.
23.0, 23.1, 23.2, 23.3, 24.6, 24.7, 24.8, 36.5, 36.6, 36.7, 36.8, 43.4,
43.6, 45.5, 45.6, 55.3, 58.3, 58.4, 58.6, 58.8, 63.4, 63.6, 73.5, 73.7,
74.3, 74.5, 84.5, 84.6, 85.5, 85.6, 85.7, 85.8, 86.4, 126.8, 126.9, 127.8,
128.3, 128.4, 130.2, 133.8, 136.0, 136.1, 144.8, 146.9, 147.0, 151.6,
151.7, 152.5, 152.6, 155.5, 158.6 HRMS-ESI (Q-ToF) (m/z): [M +
15
20
5
5
9-(3,5-di-O-Acetyl-2-deoxy-β-D-ribofuranosyl)-8-methylpur-
ine (37). The compound 36 (6.0 g, 17 mmol) was dissolved in dry
THF and placed under reflux. Neat tert-butyl nitrite (50 mL, 343
mmol) was added in three portions and refluxed for 2 days, and after
cooling, the reaction was quenched with saturated aq NaHCO3.
CH Cl was added, and the organic layer washed with saturated aq
+
Na] calcd for C H N O PNa, 775.3343; found, 775.3357.
41
49
6
6
2
2
6-Chloro-9-(3,5-di-O-acetyl-2-deoxy-β-D-ribofuranosyl)-pu-
rine (42). Acetyl protected deoxyadenosine 41 (5.0 g, 14.9 mmol)
was dissolved in anhydrous CH Cl (100 mL) at 0 °C. To this was
NaHCO . The CH Cl layer was dried and evaporated, and the
3
2
2
residue chromatographed over silica (CH Cl /CH OH) to afford the
2
2
3
2
2
1
product (1.9 g, 35%). H NMR (300 MHz, CDCl ): δ 2.02 (s, 3H),
added trimethylsilyl chloride (4.0 mL, 30.0 mmol) followed by tert-
butyl nitrite (9.0 mL, 75 mmol). The solution was left to warm to
room temperature and stirred at rt overnight. After the completion of
the reaction, as shown by TLC, the reaction was quenched with
saturated aq NaHCO and extracted with CH Cl . The organic layers
3
2
3
4
.15 (s, 3H), 2.45 (ddd, J = 14.2, 6.1, 2.2 Hz, 1H, H-2″), 2.75 (s, 3H),
.81 (ddd, J = 14.2, 7.5, 7.1 Hz, 1H, H-2′), 4.33 (m, 2H, H-5″, H-4′),
.49 (m, 1H, H-5′), 5.60 (m, 1H, H-3′), 6.34 (appt, J = 6.87 Hz, 1H,
1
3
1
H-1′), 8.90 (s, 1H, H-2), 8.99 (s, 1H, H-6). C{ H} NMR (75 MHz,
3
2
2
CDCl3): δ 15.4 (C-8-methyl), 20.8 (CH -acetyl), 21.1 (CH -acetyl),
were combined, dried over Na SO , and evaporated. The residue was
3
3
2
4
3
4.7 (C-2′), 63.6 (C-5′), 74.7 (C-3′), 82.5 (C-4′), 84.6 (C-1′), 133.9
purified by silica gel column chromatography (acetone/heptane) to
1
(
(
C-5), 147.2 (C-6), 151.9 (C-2), 152.5 (C-4), 155.0 (C-8), 170.4
CO-acetyl), 170.7 (CO-acetyl). HRMS-ESI (Q-ToF) (m/z): [M +
obtain the product (2.74 g, 52%). H NMR (300 MHz, CDCl ): δ
3
2.09 (s, 3H), 2.15 (s, 3H), 2.70 (ddd, J = 14.1, 6.0, 2.73 Hz, 1H, H-
2″), 2.97 (ddd, J = 14.1, 7.7, 6.4 Hz, 1H, H-2′), 4.39 (m, 3H, H-4′, H-
5′, H-5″), 5.46 (m, 1H, H-3′), 6.50 (dd, J = 7.5, 6.0 Hz, 1H, H-1′),
+
H] calcd for C H N O , 335.1350; found, 335.1351.
15
19
4
5
9-(2-Deoxy-β-D-ribofuranos-1-yl)-8-methylpurine (38; X3).
1
3
1
The compound 37 (1.5 g, 2.9 mmol) was treated with 7 N methanolic
ammonia (50 mL) in a sealed tube at rt overnight. The solvent was
evaporated, and the residue chromatographed over silica (CH Cl /
CH OH) to obtain the product 38 (0.67 g, 90%). H NMR (300
MHz, DMSO-d + D O): δ 2.59 (ddd, J = 13.1, 6.7, 3.5 Hz, 1H, H-
8.32 (s, 1H, H-8), 8.77 (s, 1H, H-2). C{ H} NMR (75 MHz,
CDCl ): δ 20.8 (acetyl), 20.9 (acetyl), 37.8 (C-2′), 63.7 (C-5′), 74.4
3
2
2
(C-3′), 83.0 (C-1′), 85.3 (C-4′), 132.5 (C-5), 143.4 (C-8), 151.3 (C-
1
3
6), 151.6 (C-4), 152.2 (C-2), 170.3 (CO-acetyl), 170.4 (CO-acetyl).
+
6
2
HRMS-ESI (Q-ToF) (m/z): [M + Na] calcd for C H ClN O Na,
14 15
4
5
2
(
″), 3.04 (s, 3H), 3.18 (ddd, J = 13.1, 7.0, 6.8 Hz, 1H, H-2′), 3.46
dd, J = 11.7, 5.5 Hz, 1H, H-5″), 3.64 (dd, J = 11.8, 4.7 Hz, 1H, H-
′), 6.62 (m, 1H), 3.86 (m, 1H, H-4′), 4.50 (m, 1H, H-3′), 6.36
377.0623; found, 377.0626.
9-(3,5-Di-O-acetyl-2-deoxy-β-D-ribofuranosyl)-6-methylpur-
ine (43). The compound 42 (2.0 g, 5.6 mmol) was dissolved in
5
1
3
1
(appt, J = 6.87 Hz), 8.82 (s, 1H, H-2), 8.97 (s, 1H, H-6). C{ H}
anhydrous dioxane (50 mL). To this were added (CH
) Sn (3.1 mL,
3 4
NMR (75 MHz, DMSO-d + D O): δ 15.3 (methyl-C-8), 37.5 (C-
22.0 mmol) and Pd(PPh (0.64 g, 0.56 mmol), and the mixture was
)
3 4
6
2
2
′), 62.0 (C-5′), 71.1 (C-3′), 84.5 (C-4′), 87.8 (C-1′), 133.6 (C-5),
refluxed for 6−8 h. After the completion of the reaction, the solvent
1
46.6 (C-6), 151.4 (C-2), 152.2 (C-4), 156.4 (C-8). HRMS-ESI (Q-
was evaporated and the residue purified by silica gel column
+
ToF) (m/z): [M + H] calcd for C H N O , 251.1138; found,
chromatography (acetone/heptane) to obtain the product (1.4 g,
1
1
15
4
3
3
1
2
51.1139. UV (CH OH): λ (nm) = 261, λ260 (nm), ε = 7.0 × 10
77%). H NMR (300 MHz, CDCl ): δ 2.09 (s, 3H), 2.14 (s, 3H),
3
max
3
−
1
3
−1
mol ·dm ·cm .
-(2-Deoxy-5-O-dimetoxytrityl-β-D-ribofuranos-1-yl)-8-
2.64 (ddd, J = 13.9, 5.9, 2.5 Hz, 1H, H-2″), 2.87 (s, 3H), 3.02 (ddd, J
= 13.9, 8.0, 7.6 Hz, 1H, H-2′), 4.38 (m, 3H, H5′, H-5″, H-4′), 5.46
(m, 1H, H-3′), 6.49 (dd, J = 6.0 Hz, 1H, H-1′), 8.21 (s, 1H, H-8),
9
methylpurine (39). The compound 38 (0.5 g, 1.9 mmol) was
coevaporated and dissolved in anhydrous pyridine. DMTCl (1.0 g, 2.8
mmol) was added, and the reaction was warmed to rt overnight. The
reaction was quenched with saturated aq NaHCO and extracted with
CH Cl . The organic layers were combined, dried, and evaporated.
1
3
1
8.85 (s, 1H, H-2). C{ H} NMR (75 MHz, CDCl ): δ 19.64
3
(methyl-C-6), 20.8 (acetyl), 21.0 (acetyl), 37.6 (C-2′), 63.8 (C-5′),
74.6 (C-3′), 82.8 (C-1′), 84.8 (C-4′), 133.8 (C-5), 141.9 (C-8), 150.2
(C-4), 152.5 (C-2), 159.9 (C-6), 170.3 (CO-acetyl), 170.4 (CO-
acetyl). HRMS-ESI (Q-ToF) (m/z): [M + H]+ calcd for
C H N O , 335.1350; found, 335.1350.
3
2
2
The residue was purified by silica gel column chromatography
acetone/heptane + 2% pyridine) to yield the product 39 (0.55 g,
(
1
5
19
4
5
1
5
3
3
6
8
4
1
5%). H NMR (300 MHz, CDCl ): δ 2.30 (m, 1H, H-2″), 2.70 (s,
9-(3,5-Di-O-acetyl-2-deoxy-β-D-ribofuranosyl)-6-methylpur-
3
H) 3.10 (m, 3H, H-2′, H-5′, H-5′′), 3.70 (s, 3H, OCH ), 3.71 (s,
ine (44; X4). The compound 43 (1.2 g, 3.6 mmol) was dissolved in 7
N methanolic ammonia (50 mL) in a sealed tube and stirred at rt
overnight. After complete deprotection of the acetyl groups, as shown
by TLC, the solvent was evaporated and the residue purified by silica
gel column chromatography (CH Cl /CH OH) to obtain the
3
H, OCH ), 3.99 (m, 1H, H-4′), 4.69 (m, 1H, H-3′), 6.38 (appt, J =
3
.81 Hz, 1H), 6.73 (m, 4H), 7.08−7.22 (m, 9H), 8.71 (s, 1H, H-2),
1
3
1
.97 (s, 1H, H-6). C{ H} NMR (75 MHz, CDCl ): δ 14.9, 36.7,
3
5.7, 54.9, 63.4, 70.4, 83.9, 85.1, 112.9, 113.0, 126.5, 127.5, 127.6,
2
2
3
1
29.6, 133.3, 135.5, 144.8, 146.2, 151.0, 151.7, 156.0, 157.8, 157.9.
product (0.8 g, 90%). H NMR (300 MHz, DMSO-d ): δ 2.38
6
+
HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for C H N O ,
5
(ddd, J = 13.9, 6.6, 3.6 Hz, 1H, H-2″), 2.50 (s, 3H), 2.66 (ddd, J =
3
2
33
4
5
53.2445; found, 553.2464.
14.0, 7.2, 6.7 Hz, 1H, H-2′), 3.59 (dd, J = 12.3, 4.5 Hz, 1H, H-5″),
M
DOI: 10.1021/acs.joc.9b01576
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
8
.66 (dd, J = 12.3, 3.5 Hz, 1H, H-5′), 3.96 (m, 1H, H-4′), 4.47 (m,
determined for the ribonucleoside analogue 6), N -8-aza-6-thio-dI
3
3
H, H-3′), 6.37 (appt, J = 6.75 Hz, 1H, H-1′), 8.49 (s, 1H, H-8), 8.60
(X2), ε = 2.2 × 10 ; 8-Me-dN (X3), ε = 7.0 × 10 ; 6-Me-dN (X4), ε
3 3 8
s, 1H, H-2). 13C{ H} NMR (75 MHz, DMSO-d ): δ 18.9 (C-6-
1
= 6.5 × 10 ; 2-Me-dN (X5), ε = 6.2 × 10 ; 2-Me-6-thio-dI (X6) , ε =
6
3
0.9 × 10 . Extinction coefficients given above have been determined at
260 nm. Melting curves were determined with a Varian Cary 100 BIO
spectrophotometer using Cary WinUV thermal application software.
A quick heating and cooling cycle were carried out to allow proper
annealing of both strands. The samples were then heated from 10 to
+
), 158.1 (C-6). HRMS-ESI (Q-ToF) (m/z): [M + H] calcd for
1
1
15
4
3
3
max
3
−1
3
−1
65, λ260 (nm), ε = 6.5 × 10 mol ·dm ·cm .
−
1
9
-(2-Deoxy-5-O-dimetoxytrityl-β-D-ribofuranos-1-yl)-6-
80 °C at a rate of 0.2 °C·min and were cooled again at the same
speed. Melting temperatures were 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.
3
2
2
The organic layers were combined, dried, and evaporated. The residue
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.9b01576.
■
was purified by silica gel column chromatography (acetone/heptane
*
S
1
+
2% pyridine) to obtain the product (1.0 g, 60%). H NMR (300
MHz; CDCl ) δ 2.53 (ddd, J = 13.5, 6.2, 4.0 Hz, 1H, H-2″), 2.84 (s,
3
3
H), 2.85−2.92 (m, 1H, H-2′), 3.40 (dd, J = 12.0, 4.63, 2H, H-5′5″),
3
.77 (s, 6H, OCH ), 4.16 (m, 1H, H-4′), 4.71 (m, 1H, H-3′), 6.49
NMR and HRMS spectra of synthesized compounds
3
(
appt, J = 6.51 Hz, 1H, H-1′), 6.78 (m, 4H), 7.19−7.40 (m, 9H), 8.17
(PDF)
13 1
(s, 1H, H-8), 8.75 (s, 1H, H-2). C{ H} NMR (75 MHz, CDCl ): δ
3
1
1
9.6, 40.2, 55.3, 63.9, 72.8, 84.6, 86.3, 113.4, 127.2, 128.0, 128.2,
AUTHOR INFORMATION
Corresponding Author
*E-mail: piet.herdewijn@kuleuven.be.
ORCID
Piet Herdewijn: 0000-0003-3589-8503
Author Contributions
P.L. and P.S. contributed equally to this work.
30.1, 133.8, 135.8, 142.3, 144.6, 150.1, 152.3, 158.8, 159.5. HRMS-
■
+
ESI (Q-ToF) (m/z): [M + H] calcd for C H N O , 553.2445;
3
2
33
4
5
found 553.2453.
′-O-Dimethoxytrityl-6-methyl-2′-deoxynebularine-3′-O-
2-cyanoethyl-N,N-diisopropyl)phosphoramidite (46). The
5
(
compound 45 (1.0 g,1.8 mmol) was dissolved in anhydrous THF
and cooled in an ice bath. DIPEA (1.5 mL, 9.0 mmol) was added
followed by the addition of 2-cyanoethyl-N,N-diisopropyl chloro-
phosphoramidite (0.6 mL, 2.7 mmol). The reaction was kept at 0 °C
for 30 min and then at room temperature for 4 h. After completion,
the reaction was quenched with methanol and the solvent evaporated.
The residue was purified by chromatography (acetone/heptane + 2%
†
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Et N) to obtain the product as a mixture of two isomers (0.9 g, 66%).
■
3
31
1
1
P{ H} NMR (121 MHz, CDCl ): δ 149.47, 149.34. H NMR (300
We thank Luc Baudemprez for technical assistance. We wish to
thank Prof. Jef Rozenski for conducting mass spectrometry
measurements. Mass spectrometry was made possible by the
support of the Hercules Foundation of the Flemish Govern-
ment (grant 20100225−7). We are indebted to FWO
Vlaanderen, Belgium, (grant G078014 N) for financial support.
3
MHz, CDCl ): δ 1.11−1.29 (m, 14H), 2.47 (m, 1H), 2.62 (m, 2H),
3
2
3
4
0
2
3
6
8
1
.85 (s, 3H), 1.95 (m, 1.5H), 3.35 (m, 2.5H), 3.58−3.72 (m, 4H),
.77 (s, 6H), 4.32 (m, 1H), 4.80 (m, 1H), 6.49 (m, 1H),6.78 (m,
H), 7.21−7.40 (m, 10H), 8.20 (s, 0.5H), 8.22 (s, 0.5H), 8.76 (s,
1
3
1
.5H), 8.77 (s, 0.5H). C{ H} NMR (75 MHz, CDCl ): δ 19.6, 20.2,
3
0.3, 20.3, 20.4, 20.5, 20.6, 23.0, 23.1, 24.5, 24.6, 24.7, 24.8, 39.4,
9.5, 43.3, 43.4, 43.5, 43.6, 55.3, 55.4, 58.3, 58.4, 58.6, 58.7, 63.5,
3.7, 73.7, 73.9, 74.3, 75.5, 84.8, 85.3, 85.4, 85.9, 86.0, 86.1, 86.2,
6.6, 113.3, 113.4, 127.0, 127.1, 127.9, 128.0, 128.3, 128.4, 130.1,
REFERENCES
■
(1) Benner, S. A. Understanding Nucleic Acids using Synthetic
Chemistry. Acc. Chem. Res. 2004, 37, 784−797.
30.2, 135.8, 135.9, 142.4, 144.7, 152.3, 158.7. HRMS-ESI (Q-ToF)
+
(m/z): [M + H] calcd for C H N O P, 753.3524; found, 753.3543.
(2) Kimoto, M.; Cox, R. S., III; Hirao, I. Unnatural Base Pair
Systems for Sensing and Diagnostic Applications. Expert Rev. Mol.
Diagn 2011, 11, 321−331.
4
1
50
6
6
Oligonucleotide Synthesis. Oligonucleotides were synthesized
on an Expedite DNA synthesizer (Applied Biosystems) by using the
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Pac
Ac
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2
the solid support and deprotected by treatment with aqueous
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1
00 GL column, Pharmacia) using following conditions: flow rate = 2
−
1
mL·min , solvent A = 100 mM NaCl, pH 12; solvent B = 900 mM
NaCl, pH 12. The fraction containing the product was desalted on a
NAP-25 column, lyophilized, and repurified on RP-HPLC (C18
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3
(
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min . Oligomers were characterized by mass spectrometry.
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solution containing NaCl (0.1 M), KH PO (0.02 M, pH 7.5), and
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2
4
EDTA (0.1 μM). The concentration for each strand was 4 μM in all
Base Pairing Involving Artificial Bases In Vitro and In Vivo. Chem. Sci.
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−
1
3
−1
experiments. The following extinction coefficients (mol ·dm ·cm )
3
3
3
were used: dA, ε = 15.1 × 10 ; dC, ε = 7.1 × 10 ; dG, ε = 12.2 × 10 ;
dT, ε = 8.6 × 10 ; N -8-aza-2-Me-dI (X1), ε = 6.0 × 10 (value
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8
3
N
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