Key words stereoselective synthesis - heterocycles - medicinal chemistry - nucleosides
The preparation and functionalization of oligonucleotide analogues have attracted
an increasing degree of interest because of the variety of potential applications
of such compounds. Oligonucleotide analogues have been widely used, for example, in
exploring the catalytic mechanisms of enzymes and ribozymes,[
1
] in studying physical and biological properties of nucleic acids and RNA,[
2
] in controlling gene expression,[
3
] in identifying catalytic metal ions,[
4
] and in probing hydrogen-bonding interactions.[
5
] The biological, biochemical, physical, and medicinal importance of oligonucleotide
analogues has fostered the development of various nucleoside modifications.In general,
the two primary strategies that are used to prepare structural modifications of nucleotide
analogues involve modification of the nucleobase or modification of the sugar residue,
respectively. A classical method for modification of the sugar residues involves the
removal or replacement of the oxygen atom in the 3′-position with a larger, more-electropositive,
sulfur atom.[
6
] By this approach, a variety of 3′-S -modified nucleosides containing diverse functionalized bases have been prepared as
building blocks and intermediates for oligonucleotides. These compounds have been
widely used to mimic RNA, to investigate the mechanistic properties of ribozymes,
and to increase the resistance of ribozymes to degradation by nucleases.[
7
] However, 3′-thio nucleoside analogues that contain inosine-derived bases have received
little attention.[
8
]
Recently, Piccirilli and co-workers[
9
] reported the synthesis and biochemical applications of 2′-O -methyl-3′-thioguanosine. The product was incorporated into oligonucleotides to study
the mechanism of a ribozyme reaction in Tetrahymena . Inspired by this design, we developed an efficient method for the synthesis of a
new inosine-containing 2′-deoxy-3′-thionucleoside analogue 6 , together with its phosphorothioamidite 7 , as shown in Figure [1 ]. This product can act as a general base that can pair with any of the four nucleobases[
10
] and it can also match any the four base pairs associated with the hybridization
effect in any position.[
11
] Furthermore, the design of the synthesis of 6 provides an alternative method for the synthesis of nucleoside analogues. We hope
that these products will be useful as intermediates and building blocks for the preparation
of other functionalized nucleosides.
Figure 1 5′-O -{[Bis(4-methoxyphenyl)(phenyl)methyl])-2′-deoxy-3′-thioinosine 6 and its S -phosphorothioamidite 7
In our synthetic route to the required compounds, the preparation of intermediate
3 (Scheme [1 ]) through inversion of the configuration of the hydroxy group at the 3′-position
of the sugar moiety is vital. According to the literature, there are several methods
for realizing a configurational inversion of a 3′-hydroxy group. First, we examined
the approach reported by Challa and Bruice[
12
] for the synthesis of N2
-isobutyryl-2′-deoxy-xylo -guanosine. Treatment 2′-deoxyinosine (1 ) with benzoyl chloride in anhydrous pyridine at room temperature gave 5′-O -benzoyl-2′-deoxyinosine (Scheme [1 ], path a). Treatment of this product with 1.5 equivalents of triflic anhydride in
dichloromethane containing 10% pyridine gave 2′-deoxy-xylo -inosine, which has an inverted configuration at the 3′-position. Although we obtained
a small amount of this required intermediate, we did not take this route any further
because of the low yields and the laborious procedures involved in chromatographic
purifications.
We then tried another synthetic route developed by Piccirilli and coworkers[
9
] (Scheme [1 ], path b). In the presence of 4-(N ,N -dimethylamino)pyridine, the reaction of 5′-O -[tert -butyl(diphenyl)silyl]-2′-deoxyinosine with triflic chloride in dichloromethane at
0 °C gave the 3′-triflate intermediate. Subsequent SN 2 substitution with sodium bromide in acetone resulted in a product with an inverted
configuration at the 3′-position. However, the conversion was very low, even after
prolonged reaction times. This might have been due to the low reactivity and low solubility
of 5′-O -[tert -butyl(diphenyl)silyl]-3′-O -triflyl-2′-deoxy-xylo -inosine in the reaction solvent. Finally we opted to perform the configuration inversion
at C3′ by means of an oxidation–reduction sequence[
13
] (Scheme [1 ], path c).
Scheme 1
Reaction conditions : (a) BzCl, py; (a) Tf2 O, py, CH2 Cl2 , then H2 O; (b) TBDPSCl, py; (b1 ) TfCl, DMAP, CH2 Cl2 ; (b2 ) NaBr, acetone, reflux; (c) DMTCl, pyridine, ; (c′) 1. Dess–Martin periodinane, CH2 Cl2 ; 2. i -PrOH, NaBH4 , acetone, –45 °C. Dmt = 4,4′-dimethoxytrityl.
The complete preparation began with commercially available 2′-deoxyinosine (1 ). Although oligonucleotide syntheses involving 5′-silyl protecting groups are known,
we choose the bis(4-methoxyphenyl)(phenyl)methyl (4,4′-dimethoxytrityl; Dmt) protecting
group for the 5′-O -hydroxyl group, instead of a tert -butyl(dimethyl)silyl or tert -butyl(diphenyl)silyl group, which would have to have been deprotected by treatment
with tetrabutylammonium fluoride during subsequent steps. Experiments showed protection
with a Dmt group ensured regioselectivity and prevented partial deprotection during
oxidation by Dess–Martin periodinane [1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H )-one]. After reduction by sodium borohydride propan-2-ol, the desired intermediate
product 3 , with the inverted configuration, was obtained in high yield and with high regioselectivity.
Scheme 2 Reaction conditions : (a) DmtCl, py, r.t., 19 h (81.4%); (b) (1) Dess–Martin periodinane, CH2 Cl2 , r.t. 3 h; (2) i -PrOH, NaBH4 , acetone, –45 °C, 13 h (85.2%); (c) MsCl, i -PrOH, r.t., 12 h (44.0%); (d) KSAc, DMF, 60 °C, 24 h (67.4%); (e) LiAlH4 , THF–HOAc, argon, r.t. 3 h (91.5%); (f) i -Pr2 NP(Cl)O(CH2 )2 CN, DIPEA, CH2 Cl2 , r.t., 3 h (95.2%).
Next, we used an O -mesyl group as a leaving group and potassium thioacetate as a nucleophile to realize
the second inversion at the 3′-position (from 4 to 5 ). In this procedure, compound 3 was converted into compound 4 by treatment with mesyl chloride in pyridine. Compound 4 was treated with potassium thioacetate in N ,N -dimethylformamide at 60 °C for 24 hours to give the thioacetate intermediate 5 (Scheme [2 ]).
The target phosphorothioamidite 7 was efficiently obtained in two steps (Scheme [2 ]). First, thioacetate 5 was deprotected by reduction with lithium aluminum hydride to give the sulfanyl derivative
6 in high yield (91.5%). A solution of 6 in dichloromethane was stirred with N,N -diisopropylethylamine and 2-cyanoethyl diisopropylamido-chloridophosphite at room
temperature for three hours to give the target compound 7 in an overall yield of 18.1%.
Compared with literature methods, this synthetic design has advantages of brief reaction
steps, a relatively high overall yield, and regioselectivity in the configuration-inversion
steps. The products should be useful as building blocks and intermediates for the
preparation of new functionalized nucleosides.
All chemicals were obtained commercially and used as received unless otherwise mentioned.
DMF was dried over MgSO4 and distilled. Pyridine was refluxed over KOH and then distilled. CH2 Cl2 was heated with CaH2 for 6 h, decanted, and then distilled. Solvents and liquid reagents were introduced
from oven-dried microsyringes. TLC analyses were carried out on silica gel 60 F254,
and spots were examined under UV radiation. Column chromatography was carried out
on silica gel (200–300 mesh).
1 H and 13 C NMR spectra were recorded on a Bruker AM-500 spectrometer. All mass spectrometric
analyses were performed on a ThermoStar mass spectrometer.
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2′-deoxyinosine (2)[
14
]
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2′-deoxyinosine (2)[
14
]
A stirred soln of 2′-deoxyinosine (1 ; 0.252 g, 1.0 mmol) in anhyd pyridine (10 mL) was treated with DMTCl (0.406 g, 1.2
mmol). After 19 h, MeOH (2 mL) was added and stirring was continued for 5 min. The
mixture was then transferred into CH2 Cl2 (20 mL) and the soln was washed successively with H2 O (2 × 20 mL) and brine (2 × 20 mL) then dried (Na2 SO4 ), filtered, and concentrated under vacuum. The resulting yellow oil was purified
by chromatography [silica gel, CH2 Cl2 –EtOH (20:1)] to give a white solid; yield: 0.451 (81.4%).
1 H NMR (400 MHz, DMSO-d
6 ): δ = 12.37 (s, 1 H), 8.19 (s, 1 H), 7.99 (s, 1 H), 7.34–6.77 (m, 13 H), 6.34 (dd,
J
1 = 4.0 Hz, J
2 = 12 Hz, 1 H), 5.38 (d, J = 4 Hz, 1 H), 4.42 (m, 1 H), 3.97 (dd, J
1 = 4 Hz, J
2 = 12 Hz, 1 H), 3.72 (s, 6 H), 3.15 (dd, J
1 = 4.0 Hz, J
2 = 12 Hz, 2 H), 2.77 (m, 1 H), 2.34 (m, 1 H).
13 C NMR (100 MHz, DMSO-d
6 ): δ = 157.94, 156.62, 147.90, 145.50, 144.75, 138.73, 135.47, 129.60, 127.70, 127.59,
126.62, 124.53, 113.01, 85.80, 85.35, 83.49, 70.41, 63.88, 56.06, 54.95.
MS (ESI): m/z [M + H]+ calcd for C31 H31 N4 O6
: 555.2; found: 555.3.
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2′-deoxy-xylo -inosine (3)[
15
]
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2′-deoxy-xylo -inosine (3)[
15
]
Product 2 (0.554 g, 1.0 mmol) was added to a chilled stirred soln of Dess–Martin periodinane
(0.640 g, 1.5 mmol) in anhyd CH2 Cl2 (8 mL) at 0 °C, and the soln was stirred at 0 °C for 1 h and then at r.t. for 4 h.
i -PrOH (8 mL) was added and the resulting white slurry was cooled to –45 °C. After
20 min, freshly powdered NaBH4 (76 mg, 2.0 mmol) was added and the mixture was stirred for 12 h at –45 °C. Acetone
(8 mL) was added and the mixture was allowed to warm to r.t., diluted with CH2 Cl2 (20 mL), and washed sequentially with aq NaHCO3 (2 × 20 mL), H2 O (2 × 20 mL), and brine (2 × 20 mL). The organic layer was separated, dried (Na2 SO4 ), filtered, and concentrated under vacuum. The residue was purified by chromatography
[silica gel, CH2 Cl2 –EtOH (15:1)] to give a white foamy solid; yield: 0.472 g (85.2%).
1 H NMR (400 MHz, DMSO-d
6 : δ = 12.39 (s, 1 H), 8.14 (s, 1 H), 8.09 (d, J = 4 Hz, 1 H), 7.41–6.77 (m, 13 H), 6.33 (dd, J
1 = 4.0 Hz, J
2 = 12 Hz, 1 H), 5.42 (d, J = 4 Hz, 1 H), 4.33 (m, 1 H), 4.21 (dd, J
1 = 4 Hz, J
2 = 12 Hz, 1 H), 3.73 (s, 3 H), 3.72 (s, 3 H), 3.18 (dd, J
1 = 4.0 Hz, J
2 = 12 Hz, 2 H), 2.72 (m, 1 H), 2.26 (m, 1 H).
13 C NMR (100 MHz, DMSO-d
6) : δ = 157.96, 156.60, 147.73, 145.76, 144.98, 138.78, 135.68, 135.54, 129.71, 127.68,
126.56, 124.02, 113.01, 85.42, 83.93, 83.01, 69.28, 63.14, 54.94, 40.82.
MS (ESI): m/z [M + H]+ calcd for C31 H31 N4 O6 : 555.2; found: 555.3.
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-3′-O -mesyl-2′-deoxyinosine (4)[
16
]
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-3′-O -mesyl-2′-deoxyinosine (4)[
16
]
A soln of MsCl (77 μL, 1.0 mmol) in pyridine (2 mL) was added dropwise to a stirred
soln of compound 3 (0.277 g, 0.5 mmol) in anhyd pyridine (4 mL) at 0 °C. The cooling bath was removed
and the mixture was stirred for 12 h at r.t. The mixture was then poured into ice–water
and stirred for another 10 min. The resulting mixture was diluted with CH2 Cl2 (20 mL) and the organic layer was separated, washed with aq NaHCO3 (2 × 20 mL), H2 O (2 × 20 mL), and brine (2 × 20 mL) then dried (Na2 SO4 ), filtered, and concentrated under vacuum. The crude product was purified by chromatography
[silica gel, CH2 Cl2 –EtOH (20:1)] to give a white foamy solid; yield: 0.139 g (44.0%).
1 H NMR (400 MHz, CDCl3 ): δ = 11.91 (s, 1 H), 8.02 (s, 1 H), 7.99 (s, 1 H), 7.44–6.82 (m, 13 H), 6.43 (m,
1 H), 5.46 (m, 1 H), 4.37 (m, 1 H), 3.80 (s, 6 H), 3.66 (dd, J
1 = 4.0 Hz, J
2 = 8 Hz, 1 H), 3.36 (dd, J
1 = 4.0 Hz, J
2 = 8 Hz, 1 H), 2.93 (m, 2 H), 2.74 (s, 3 H).
13 C NMR (100 MHz, CDCl3 ): δ = 153.44, 153.24, 142.34, 140.10, 139.41, 135.22, 130.63, 130.52, 124.79, 122.86,
122.62, 121.61, 119.99, 107.91, 81.36, 79.11, 78.85, 65.71, 57.10, 49.97, 35.83, 24.47.
MS (ESI): m/z [M + H]+ calcd for C32 H33 N4 O8 S: 633.2; found: 633.3.
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-3′-thioinosyl 3′-S -Acetate (5)[
16
]
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-3′-thioinosyl 3′-S -Acetate (5)[
16
]
KSAc (70.3 mg, 0.617 mmol) was added to a colorless soln of compound 4 (0.130 g, 0.21 mmol) in anhyd DMF (8 mL), and the mixture was stirred at 60 °C for
24 h under argon, The mixture was then transferred into CH2 Cl2 (10 mL) and the soln with washed with aq NaHCO3 (2 × 20 mL), H2 O (2 × 20 mL), and brine (2 × 20 mL). The organic layer was separated, dried (Na2 SO4 ), filtered, concentrated, and co-evaporated with toluene to remove the DMF. The residual
oil was purified by chromatography [silica gel, CH2 Cl2 –EtOH (15:1)] to give a white foamy solid; yield: 86.6 mg (67.4%).
1 H NMR (400 MHz, CDCl3 ): δ =12.13 (s, 1 H), 8.07 (s, 1 H), 7.99 (s, 1 H), 7.44–6.77 (m, 13 H), 6.33 (dd,
J
1 = 4.0 Hz, J
2 = 8 Hz, 1 H), 4.27 (m, 1 H), 4.17 (dd, J
1 = 4 Hz, J
2 = 8 Hz, 1 H), 3.78 (s, 6 H), 3.40 (d, J = 4.0 Hz, 2 H), 3.03 (m, 1 H), 2.56 (m, 1 H), 2.34 (s, 3 H).
13 C NMR (100 MHz, CDCl3 ): δ = 194.04, 159.21, 158.53, 148.42, 144.99, 144.39, 139.48, 138.39, 135.57, 130.09,
129.15, 128.15, 127.84, 126.93, 125.14, 113.17, 86.64, 84.48, 84.02, 63.29, 55.25,
40.80, 39.61, 30.62.
MS (ESI): m/z [M + H]+ calcd for C33 H33 N4 O6 S: 613.2; found: 613.3.
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-3′-thioinosine (6)[
16
]
5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-3′-thioinosine (6)[
16
]
A suspension of LiAH4 (15.2 mg, 0.4 mmol) in anhyd THF (5 mL) was cooled to 0 °C and then a soln of compound
5 (61.2 mg, 0.1 mmol) in THF (5 mL) was added dropwise under argon. The mixture was
stirred for 4 h at r.t. then treated with 1 M aq HOAc (2 × 20 mL). CH2 Cl2 (10 mL) was added and the organic layer was separated, washed with brine (2 × 20
mL), dried (Na2 SO4 ), filtered, and concentrated under vacuum. The residue was purified by chromatography
[silica gel, CH2 Cl2 –EtOH (12:1)] to give a white foamy solid; yield: 52.2 mg (91.5%).
1 H NMR (400 MHz, CDCl3 ): δ = 12.62 (s, 1 H), 8.10 (s, 1 H), 8.04 (s, 1 H), 7.43–6.80 (m, 13 H), 6.32 (dd,
J
1 = 4.0 Hz, J
2 = 8 Hz, 1 H), 4.00 (m, 1 H), 3.78 (s, 6 H), 3.75–3.70 (m, 1 H), 3.53 (dd, J
1 = 4.0 Hz, J
2 = 8 Hz, 1 H), 3.42 (dd, J
1 = 4.0 Hz, J
2 = 8 Hz, 1 H), 2.93 (m, 1 H), 2.49 (m, 1 H), 1.65 (d, J = 8 Hz, 1 H).
13 C NMR (100 MHz, CDCl3 ): δ = 158.21, 157.53, 147.16, 143.99, 143.38, 137.59, 134.52, 129.01, 127.07, 126.93,
125.94, 124.21, 112.21, 87.39, 85.61, 83.15, 60.75, 54.22, 41.68, 34.01.
MS (ESI): m/z [M + H]+ calcd for C31 H30 N4 O5 S: 571.2; found: 571.3.
S -[5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-3′-thioinosinyl] O -(2-Cyanoethyl) Diisopropylamidothiophosphite (7)
S -[5′-O -[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-3′-thioinosinyl] O -(2-Cyanoethyl) Diisopropylamidothiophosphite (7)
DIPEA (68.4 μL, 0.40 mmol) and i -Pr2 NP(Cl)(CH2 )2 CN (31.8 μL, 0.14 mmol) were added to a stirred soln of compound 6 (40 mg, 0.070 mmol) in anhyd CH2 Cl2 (5 mL), and the mixture was stirred at r.t. for 3 h. The mixture was then diluted
with CH2 Cl2 (10 mL) and washed successively with H2 O (2 × 20 mL) and brine (2 × 20 mL) then dried (Na2 SO4 ), filtered, and concentrated under vacuum. The residue was purified by chromatography
(silica gel, 0.5% Et3 N in 5% EtOH–CH2 Cl2 ) to give a white solid; yield: 51.3 mg (95.2% yield).
1 H NMR (400 MHz, CDCl3 ): δ = 13.17 (s, 1 H), 8.15–8.06 (s, 2 H), 7.40–6.76 (m, 13 H), 6.39 – 6.29 (m, 1
H), 4.54–4.44 (m, 1 H), 4.24–4.17 (m, 1 H), 3.76 (s, 6 H), 3.66–3.52 (m, 4 H), 3.48–3.36
(m, 2 H), 3.04–2.95 (m, 1 H), 2.75–2.66 (m, 1 H), 2.60–2.55 (m, 1 H), 2.45–2.41 (m,
1 H), 1.28–1.02 (m, 12 H).
13 C NMR (100 MHz, CDCl3 ): δ = 158.74, 158.54, 148.60, 145.28, 144.47, 138.37, 135.61, 130.06, 128.14, 127.87,
126.94, 125.13, 117.48, 113.15, 86.49, 84.57, 73.47, 63.33, 55.35, 55.25, 45.92, 43.27,
29.69, 24.62, 20.19.
31 P NMR (162 MHz, CDCl3 ): δ = 165.42, 165.05.
HRMS: m/z [M]+ calculated for C40 H47 N6 O6 PS: 770.3015; found: 770.3038.
