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Chelation-controlled regioselective alkylation of pyrimidine 2′-deoxynucleosides
Carbohydrate Research 345 (2010) 199–207
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Chelation-controlled regioselective alkylation of pyrimidine 20 -deoxynucleosides R. Lucas a, K. Teste a, R. Zerrouki a,*, Y. Champavier b, M. Guilloton a a b
Laboratoire de Chimie des Substances Naturelles EA1069, Faculté des Sciences et Techniques, 123 Avenue, Albert Thomas, F-87060 Limoges, France Université de Limoges, Service Commun de RMN, Faculté de Pharmacie, 2 rue du Dr Marcland, F-87025 Limoges, France
a r t i c l e
i n f o
Article history: Received 10 September 2009 Received in revised form 23 October 2009 Accepted 27 October 2009 Available online 30 October 2009
a b s t r a c t Protection–deprotection steps, which are usually needed for regioselective alkylation of pyrimidine deoxynucleosides, can be avoided by choosing the appropriate solvent. The combined effects of low dielectric constant and possible sodium chelation by pyrimidine nucleosides may account for the unexpected regioselectivity observed in THF. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Alkylation Nucleosides Chelating agents Regioselectivity
1. Introduction Over the past 50 years, scientists have shown a continuous interest in nucleoside chemistry, most notably in the field of drug synthesis, for example, in the synthesis of anti-HIV drugs (Fig. 1).1 The reactivity of nucleosides is rather complex due to the presence of various nucleophilic sites. Protection/deprotection steps are often necessary to generate nucleoside derivatives. Many applications in bioorganic chemistry concern bioconjugation (e.g., DNA labeling) where purine or pyrimidine bases must be selectively functionalized.2 Another example is presented by the synthesis of oligonucleotide analogues for which functionalization of the 30 O- and 50 -O-position is mandatory. Finally, nucleoside analogue synthesis sometimes requires a regioselective modification of the 30 -O-position (Fig. 1). In fact, modifications of the glycone moiety easily affect the nucleobase unless the nucleoside is adequately protected. Regioselective reactions, should, if possible, be undertaken on the unprotected substrate to avoid this drawback. Functionalization, as well as protection of nucleosides, is frequently accomplished by alkylation. In the case of pyrimidine deoxynucleosides, we are confronted with competition between the 3-N- and 30 -O-nucleophilic positions. The use of a metallic hydride (NaH) in DMF allows the selective alkylation of the 3-N-alkylated.3 Conducted in THF and in the presence of an excess of alkylating agent, this reaction leads to regioselective 30 -O-alkylation.4 This surprising result allowed us to establish the solvent * Corresponding author. Tel.: +33 5 55 45 72 24; fax: +33 5 55 45 72 02. E-mail address: [email protected] (R. Zerrouki). 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.10.021
dependency of high yield (82–99%) in the regioselective 30 -Oalkylation of 50 -O-protected thymidine (Scheme 1).5 In this article, we present a study of 20 -deoxypyrimidine nucleoside alkylation using thymidine (1a), 20 -deoxyuridine (1b), and
Figure 1. Anti-HIV drugs obtained by regioselective modifications.
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Scheme 1. Regioselective alkylation of 50 -O-protected thymidine: (i) NaH (2.5 equiv), THF, RBr (2.5 equiv), ultrasound or MW; (ii) NaH (1.2 equiv), DMF, RBr (1.2 equiv), ultrasound or MW.
2. Results and discussion 2.1. Solvent-dependent regioselectivity
Figure 2. Solvent-dependent regioselective allylation of thymidine.5 *Reactions were carried out with excess sodium hydride/allyl bromide.
20 -deoxycytidine (1c). These experiments were conducted to understand the influence of solvents on regioselectivity, and to master nucleoside alkylation as well as further synthesis of nucleoside analogues.
The regioselective allylation of 50 -O-TBDMS–thymidine (2a) is closely linked to the dielectric constant of the solvent (Fig. 2). Indeed O-allylation is only observed in solvents of low dielectric constant (e 6 10), for example, THF (e = 7.58), excess NaH being required. Under these conditions the best yields are obtained with 2.5 equiv of allyl bromide. In DMF (e = 37), a quasi-stoichiometric amount of both reagents readily leads to N-allylated thymidine. Before clarifying the reactivity of thymidine under such conditions, we repeated these observations with 20 -deoxyuridine (dU). Because the microwave-activated reaction is so fast (completion within 1–3 min), all the following reactions were carried out with ultrasound activation in order to check more easily their evolution as a function of reaction time. Propargyl bromide was used as the alkylating reagent, keeping in mind that the resulting products can be used as ‘click reagents’ in copper-catalyzed azide–alkyne cycloaddition (CuAAC) for further syntheses of oligomers, labeled nucleosides, etc.1,6,7 The corresponding alkylated products are indicated in Scheme 2. The first step was the well-known monoprotection of nucleosides 1a–b to give the corresponding 20 -deoxy-50 -O-tert-butyldimethylsilylnucleosides 2a–b in high yield.8 Then, the 50 -O-protected nucleosides were alkylated using several conditions which
Scheme 2. Synthesis of propargylated 50 -O-protected thymidines: 3a (3-N), 4a (30 -O) and 5a (3-N and 30 -O), and 50 -O-protected 20 -deoxyuridines 3b (3-N), 4b (30 -O), and 5b (3-N and 30 -O). Reagents and conditions: (i) TBDMSCl, DMAP, Py, rt; (ii) NaH, THF or DMF, propargyl bromide, ultrasound.
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207 Table 1 Selected results of propargylation of 50 -O-protected thymidine and 50 -O-protected 20 deoxyuridine Entry
NaH; RBr (equiv)
Solvent
3a (3b)a (%)
4a (4b)a (%)
5a (5b)a (%)
1 2 3 4
2.5 1.2 2.5 1.2
THF THF DMF DMF
0 (0) 0 (0)b 0 (0) 93 (94)
100 (92) 0 (0)b 0 (0) 0 (0)
0 (0) 0 (0)b 90 (89) 0 (0)
a Reaction times allowed virtual reaction completion (checked by TLC); longer reaction times gave no increase in reaction yields. b No evolution was observed even after one day of reaction.
201
are summarized in Table 1. The two solvents used were THF and DMF in order to evaluate their straightforward regioselective effects on thymidine allylation. Firstly, 20 -deoxyuridine behaves essentially in the same way as thymidine whatever the conditions are. 30 -O- and 3-N-propargylation products were obtained in good yields: 4a (30 -O-T) 100%, 4b (30 -O-dU) 92%, 3a (3-N-T) 93%, and 3b (3-N-dU) 94%. The two alkylated compounds 3a and 4a can be differentiated (in the same way as 3b and 4b) by HMBC experiments (Fig. 3). NMR spectra of 3-N-propargylated compounds show a connection between CH2propargyle(a) (4.51–4.55 ppm) and C-2/C-4 (149.6/161.7 ppm) of thymine or 20 -deoxyuridine, whereas for 30 -O-propargylation a
Figure 3. 1H–13C experiments (400 MHz) for analysis of propargylated regioisomers 3a and 4a. Insets show a magnified part of the spectrum containing diagnostic crosspeaks between CH2propargyle(a) and C-2/C-4 for 3a, respectively, C-30 for 4a and between CH(30 ) and Cpropargyle(a) for 4a.
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R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207 Table 2 Selected results of 50 -O-protected thymidine and 20 -deoxyuridine propargylation in the presence of crown ether 15-C-5 Entry
NaH; RBr (equiv)
Solvent
Crown ether (equiv)
3a (3b)a (%)
4a (4b)a (%)
5a (5b)a (%)
1 2 3
2.5 1.2 1.2
THF THF DMF
3.5 1.2 1.2
0 (0) 73 (75) 100 (90)
0 (0) 0 (0) 0 (0)
89 (80) 0 (0) 0 (0)
a Reaction times allowed virtual reaction completion (checked by TLC), longer reaction times gave no increase in reaction yields.
Figure 4. Tentative structures of sodium-enolate complexes accounting for the regioselectivity of O- versus N-alkylation observed in solvents of low dielectric constant in the presence of excess NaH and propargyl bromide.
connectivity between CH(30 ) (4.20–4.25 ppm) of the sugar and CH2propargyle(a) (78.2 ppm) can be observed. If only stoichiometric amounts of NaH and propargyl bromide were used in THF, there was absolutely no alkylation reaction. Furthermore, excess reagent in DMF gave dipropargylated pyrimidine nucleosides in good yields: 89% for 5b and 90% for 5a. Addition of a strong base like NaOH led to similar results. For example, in the presence of excess NaOH and propargyl bromide in THF, the expected compound 4a was isolated in 52% yield. We also tested the effect of Mg2+. After the first activation by ultrasound in THF in the presence of excess NaH, 2.5 equiv of magnesium iodide was added. Then addition of 2.5 equiv of propargyl bromide was followed by a second activation. TLC analysis showed that no reaction took place. A strong interaction between Mg2+ and 30 -O could explain the non-reactivity of this position. The same reaction carried out in DMF with 1.2 equiv of reagents and excess MgI2 allowed selective alkylation of the 3-N position. In other words, when the solvent is THF, the most acidic hydrogen9 (3-NH) must be abstracted by 1 equiv of the 2.5 equiv of sodium hydride before the 30 -OH can react and be further alkylated by propargyl bromide. This means that proton abstraction at the 3-N-position leads to the disappearance of the nucleophilic character of the nitrogen atom, although the reactivity of the 3-N-position is fully restored when THF is replaced by DMF. Since the thymidine/Na+ ion pair created by the consumption of one NaH equivalent must interact more strongly in THF than it does in DMF, we thought that some kind of chelation of sodium by the negatively charged nucleoside could occur in THF. Figure 4 gives a tentative illustration of this hypothesis and suggests that the collapse of 3-N- reactivity could originate from stabilization of the thymine enolate by a sodium ion. 2.2. Effect of crown ether The hypothesis of sodium-chelation-controlled regioselectivity was tested by adding the Na+-selective 15-C-5 crown ether to reaction mixtures. Reactions were carried out in THF or DMF with quasi-stoichiometric or excess amounts of either crown ether or reagents (NaH/propargyl bromide) (Table 2). Excess crown ether, together with excess NaH and propargyl bromide in THF, leads only to dialkylated products 5a and 5b (Table 2, entry 1). The nucleophilic character of the 3-N-position is restored, thanks to sodium trapping by 15-C-5. This result indicates that in the absence of crown ether, thymidine enolate efficiently chelates one sodium ion, thus impairing further alkylation of the 3-N-position. A quasi-stoichiometric amount of crown ether in DMF did not modify the already observed regioselectivity (Table 2, entry 3); however in THF, N-propargylated compounds are actually obtained (Table 2, entry 2).
Taken as a whole, these results are in agreement with the hypothesis of sodium-ion chelation by the pyrimidine nucleoside, which governs the observed regioselectivity and the role of solvent of low dielectric constant in chelate stabilization. Although THF likely stabilizes a sodium-enolate chelate, the exact nature of the complex is presently unknown. We investigated the possible contribution of oxygen in the 50 -position to sodiumion chelation. If the oxygen of the TBDMS-protected 50 -hydroxyl group is involved in this chelation, one may assume that, if unprotected, the 50 -oxygen in the anionic form would interact even more strongly with sodium, thus preventing further alkylation at this position. In order to check this hypothesis, we prepared the 30 -O-protected thymidine and 20 -deoxyuridine (7a and 7b). The starting 20 deoxynucleosides 1a–b were easily transformed into the corresponding 20 -deoxy-30 ,50 -di-(O-tert-butyldimethylsilyl)nucleosides 6a–b in high yield. The second step was the selective removal of the 50 -O-tert-butyldimethylsilyl group according to Oglivie.8 The 30 -O monoprotected 20 -deoxynucleosides when propargylated gave the corresponding compounds 8a–b and 9a–b (Scheme 3). The results show that by using an excess of NaH and propargyl bromide without crown ether in THF we obtain only the 50 -O-alkylated pyrimidine nucleosides in good yield: 8a (50 -O-T) 63%, and 8b (50 -O-dU) 62% (Table 3, entry 1). Thus oxygen in the 50 -position seems not to participate in sodium chelation. With an excess amount of reagents and crown ether, dialkylated compounds are readily obtained (Table 3, entry 2): 9a (3-N-, 50 -O-T) 82%, and 9b (3-N-, 50 -O-dU) 60%. Finally the same reaction without crown ether leads to no reaction at all (Table 3, entry 3), a result reminiscent of reactions with 50 -O-protected deoxynucleosides 2a–b. 2.3. Proposed mechanism in solvents with low dielectric constants The present results permit one to propose a mechanism for the reaction between pyrimidine nucleosides protected in the 50 -position and an alkylating agent, after initial reaction with sodium hydride, in a solvent with a low dielectric constant (Scheme 4). The amount of NaH is the critical parameter which governs alkylation selectivity. Alkylation of 4-N-benzoyl-20 -deoxy-50 -O-tert-butyldimethylsilyl-cytidine: in an attempt to extend this chelation hypothesis to other nucleosides, we investigated the synthesis of the 30 -O-propargylated 4-N-benzoylcytidine protected in the 50 -position (Scheme 5). The first step consisted of the selective benzoylation of the amino group of the heterocycle.10 Compound 10 was then silylated using the literature conditions in a good yield of 84%.11 The impossibility of a chelation (or else chelation with a low energy of coordination) between the base and the sodium ion leads one to conclude that only 1.2 equiv of propargyl bromide and sodium hydride are sufficient to give the expected 30 -O-propargylated 4-Nbenzoylcytidine protected in the 50 -position (Table 4, entry 2).
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
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Scheme 3. Synthesis of propargylated thymidines: 8a (50 -O-) and 9a (3-N- and 50 -O-), 20 -deoxyuridine 8b (50 -O-) and 9b (3-N- and 50 -O-). Reagents and conditions: (i) TBDMSCl, imidazole, AgNO3, 1:1 THF–DMF, rt; (ii) PPTS, MeOH, rt; (iii) NaH, THF, propargyl bromide, ultrasound.
Table 3 Selected results of propargylation of 30 -O-protected thymidine and 20 -deoxyuridine using crown ether 15-C-5 Entry
NaH; RBr (equiv)
Solvent
Crown ether (equiv)
8a (8b)a (%)
9a (9b)a (%)
1 2 3
2.5 2.5 1.2
THF THF THF
0 3.5 0b
63 (62) 0 (0) 0 (0)b
0 (0) 82 (60) 0 (0)b
a Reaction times allowing virtual reaction completion (checked by TLC), longer reaction times gave no increase in reaction yields. b No evolution was seen even after one day of reaction.
2.4. Effects of other hydrides The mechanism of sodium-chelation-controlled regioselectivity was tested by using two other hydrides LiH and KH. The results are summarized in Table 5. No evolution was observed in the case of LiH in THF, most probably because of a strongly associated pair of intimate ions (Li+ O) in low dielectric solvent. In DMF, compound 3a was isolated in low yield (25%). On the other hand, KH led to the expected product in 50% yield in THF with the K-specific 18-C-6 crown ether. 2.5. Conclusions
When this reaction was conducted in DMF, TLC showed numerous side products along with the initial 20 -deoxycytidine derivative. Purification did not lead to significant results.
We have demonstrated the role of sodium ion in the regioselectivity of the alkylation of pyrimidine nucleosides. The proposed
Scheme 4. A plausible mechanism explaining the role of sodium ion in the regioselectivity of alkylation in a solvent with a low dielectric constant.
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Scheme 5. Propargylation of 4-N- and 50 -O-protected deoxycytidine 11. Reagents and conditions: (i) BzCl, BrSiMe3, Pyr, rt; (ii) TBDMSCl, imidazole, DMF, 0 °C; (iii) NaH, THF, propargyl bromide, ultrasound.
Table 4 Selected results of propargylation ldimethylsilylcytidine in THF
of
4-N-benzoyl-20 -deoxy-50 -O-tert-buty-
Entry
NaH; RBr (equiv)
12a (%)
1 2
2.5 1.2
91 65
a Reaction times allowing virtual reaction completion (checked by TLC), longer reaction times gave no yield increase.
Table 5 Selected results of 50 -O-protected thymidine propargylation in the presence of LiH, KH and corresponding crown ethers 12-C-4 (Li+) and 18-C-6 (K+) Entry
Hydride
NaH; RBr (equiv)
Solvent
Corresponding crown ether (equiv)
3aa (%)
4aa (%)
5aa (%)
1 2 3 4 5 6
LiH KH LiH KH LiH KH
2.5 2.5 2.5 2.5 1.2 1.2
THF THF THF THF DMF DMF
— — 3.5 3.5 — —
0b 0b 0b 0 25 83
0b 0b 0b 0 0 0
0b 0b 0b 50 0 0
a Reaction times allowing virtual reaction completion (checked by TLC), longer reaction times gave no increase in reaction yields. b No evolution was seen even after one day of reaction.
mechanism involves the possible chelation of sodium ion by the 2-enolate form of thymidine and 20 -deoxyuridine. This striking solvent-dependent selectivity is likely to speed up a variety of syntheses involving nucleoside modification and derivatization.
3. Experimental section 3.1. Materials All solvents and chemicals were commercially available, and unless otherwise stated, were used as received. Thymidine, 20 -
deoxyuridine, 20 -deoxycytidine, tert-butylchlorodimethylsilane, and propargyl bromide were purchased from Alfa Aesar. Sodium hydride, silver nitrate, imidazole, and PPTS were purchased from Sigma–Aldrich. Crown ether (15-C-5) was purchased from Acros. All solvents were purchased from Alfa Aesar. Reactions were monitored by thin-layer chromatography (TLC) on precoated 0.2 mm Silica Gel 60 F254 (E. Merck) plates and visualized either with an ultraviolet light source at 254 nm, or by spraying sulfuric acid (6 N) and heating to 200 °C. 1 H NMR (13C NMR) spectra were recorded at 400.13 MHz (100.62 MHz) with a Bruker DPX spectrometer using DMSO-d6. Chemical shifts (d) are expressed in ppm with Me4Si as the internal standard (d0). Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad), coupling constants (Hz), and assignment. Mass spectrometry: Mass spectra were recorded with an R1010 Nermag instrument. 3.2. General procedure For the synthesis of compounds 3a–b, 4a–b, 5a–b, 8a–b, 9a–b, and 12: To 50 -O or 30 -O-tert-butyldimethylsilylnucleoside (6.00 mmol) in dry THF (30 mL) was added 2.5 equiv of NaH (60%, 15.00 mmol), and the mixture was activated by ultrasound activation (20 min). Allyl bromide (2.5 equiv, 15.00 mmol) was then added, and the mixture was activated by ultrasound (from 20 min to 3 h depending on solvent). After workup (NH4Cl–H2O), the product was purified by chromatography (thick layer plates). 3.3. Characterization of compounds Concerning the purity of all the compounds synthesized, no extraneous substances were observed. They were also homogeneous by chromatography. Compound 2a. Rf 0.44 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.31 (br s, 1H, NH), 7.47 (d, 1H, JH6,CH3 0.7 Hz, H6), 1.77 (d, 3H, JCH3,H6 0.7 Hz, CH3); ose: 6.17 (dd, 1H, J 6.3 Hz, J 7.6 Hz, H10 ), 5.26 (s, 1H, OH), 4.20 (m, 1H, H30 ), 3.89 (dd, 1H, J 3.2 Hz, J
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
11.0 Hz, H50 a), 3.81 (m, 1H, H40 ), 3.79 (dd, 1H, J 3.3 Hz, J 11.0 Hz, H50 b), 2.08 (ddd, 1H, J 3.0 Hz, J 6.3 Hz, J 13.2 Hz, H20 a), 2.05 (ddd, 1H, J 5.8 Hz, J 7.6 Hz, J 13.2 Hz, H20 b); TBDMS: 0.88 (s, 9H, C– CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 163.7 (C4), 150.4 (C2), 135.5 (C6), 109.4 (C5), 12.2 (CH3); ose: 86.8 (C40 ), 83.8 (C10 ), 70.5 (C30 ), 63.3 (C50 ), 39.4 (C20 ), TBDMS: 25.8 (C–CH3), 18.0 (C–CH3), 5.4 (Si–CH3); ESIMS: m/z 357.2 (MH+), 380.2 (MNa+). Compound 2b: Rf 0.44 (9:1 CHCl3–EtOH); 1H NMR: uracil: 11.30 (br s, 1H, NH), 7.75 (d, 1H, JH6,H5 8.1 Hz, H6), 5.57 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.14 (t, 1H, J 6.3 Hz, H10 ), 5.29 (br s, 1H, OH), 4.19 (m, 1H, H30 ), 3.82 (m, 1H, H40 ), 3.78 (dd, 1H, J 3.3 Hz, J 11.3 Hz, H50 a), 3.72 (dd, 1H, J 3.5 Hz, J 11.3 Hz, H50 b), 2.15 (ddd, 1H, J 3.6 Hz, J 6.3 Hz, J 13.2 Hz, H20 a), 2.06 (ddd, 1H, J 2.4 Hz, J 6.3 Hz, J 13.2 Hz, H20 b); TBDMS: 0.87 (s, 9H, C–CH3), 0.07 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): uracil: 163.0 (C4), 150.3 (C2), 140.1 (C6), 101.6 (C5); sugar: 86.9 (C40 ), 84.3 (C10 ), 70.1 (C30 ), 63.0 (C50 ), 39.8 (C20 ); TBDMS: 25.8 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.5 (CH3b); ESIMS: m/z 343.0 (MH+), 365.0 (MNa+), 685.0 (M2H+), 706.9 (M2Na+). Compound 3a: (3-N): Rf 0.43 (9:1 CHCl3–EtOH); 1H NMR: thymine: 7.57 (d, 1H, JH6,CH3 0.7 Hz, H6), 1.84 (d, 3H, JCH3,H6 0.7 Hz, CH3); sugar: 6.20 (dd, 1H, J 6.3 Hz, J 7.3 Hz, H10 ), 5.30 (d, 1H, J 4.2 Hz, OH), 4.21 (m, 1H, H30 ), 3.85 (dd, 1H, J 3.0 Hz, J 6.0 Hz, H40 ), 3.80 (dd, 1H, J 3.0 Hz, J 11.4 Hz, H50 a), 3.73 (dd, 1H, J 3.8 Hz, J 11.4 Hz, H50 b), 2.16 (ddd, 1H, J 3.1 Hz, J 6.3 Hz, J 13.3 Hz, H20 a), 2.09 (ddd, 1H, J 6.5 Hz, J 7.3 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.07 (s, 6H, Si–CH3); propargyl: 4.55 (dd, 1H, J 2.4 Hz, J 15.6 Hz, CH2), 4.51 (dd, 1H, J 2.3 Hz, J 15.6 Hz, CH2), 3.10 (br t, 1H, J 2.3 Hz, C–H); 13C NMR (DMSO-d6): thymine: 161.7 (C4), 149.6 (C2), 134.6 (C6), 108.5 (C5), 12.8 (CH3); sugar: 87.0 (C40 ), 85.1 (C10 ), 70.4 (C30 ), 63.2 (C50 ), 39.6 (C20 ); TBDMS: 25.8 (C–CH3), 18.0 (C–CH3), 5.6 (CH3); propargyl: 79.0 (C), 73.0 (CH), 30.0 (CH2); HRESIMS: calcd for C19H30N2O5SiNa [M+Na]+: m/z 417.1816, found: m/z 417.1818. Compound 3b: (3-N): Rf 0.70 (9:1 CHCl3–EtOH); uracil: 7.85 (d, 1H, JH6,H5 8.1 Hz, H6), 5.77 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.17 (t, 1H, J 6.5 Hz, H10 ), 5.33 (d, 1H, J 3.9 Hz, OH), 4.21 (m, 1H, H30 ), 3.86 (br dd, 1H, J 3.2 Hz, J 6.4 Hz, H40 ), 3.80 (dd, 1H, J 3.2 Hz, J 11.4 Hz, H50 a), 3.73 (dd, 1H, J 3.5 Hz, J 11.4 Hz, H50 b), 2.21 (ddd, 1H, J 3.7 Hz, J 6.5 Hz, J 13.3 Hz, H20 a), 2.10 (dt, 1H, J 6.5 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.07 (s, 6H, Si–CH3); propargyl: 4.53 (dd, 1H, J 2.4 Hz, J 17.2 Hz, CH2), 4.48 (dd, 1H, J 2.5 Hz, J 17.2 Hz, CH2), 3.11 (br t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): uracil: 160.9 (C4), 149.7 (C2), 139.1 (C6), 100.6 (C5); sugar: 87.1 (C40 ), 85.5 (C10 ), 70.0 (C30 ), 62.9 (C50 ), 40.0 (C20 ); TBDMS: 25.8 (C–CH3), 18.0 (C–CH3), 5.6 (CH3a), 5.5 (CH3b), propargyl: 78.9 (C), 73.0 (CH), 29.7 (CH2); ESIMS: m/z 381.4 (MH+), 403.4 (MNa+), 419.4 (MK+), 761.5 (M2H+), 783.3 (M2Na+); HRESIMS: calcd for C18H28N2O5SiNa [M+Na]+: m/z 403.5000, found: m/z 403.5011. Compound 4a: (30 -O): Rf 0.53 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.34 (br s, 1H, NH), 7.48 (d, 1H, JH6,CH3 0.8 Hz, H6), 1.78 (d, 3H, JCH3,H6 0.8 Hz, CH3); sugar: 6.10 (dd, 1H, J 5.7 Hz, J 8.7 Hz, H10 ), 4.24 (m, 1H, H30 ), 3.99 (dd, 1H, J 3.7 Hz, J 5.6 Hz, H40 ), 3.79 (dd, 1H, J 4.3 Hz, J 11.2 Hz, H50 a), 3.74 (dd, 1H, J 3.7 Hz, J 11.2 Hz, H50 b), 2.28 (ddd, 1H, J 1.6 Hz, J 5.7 Hz, J 13.7 Hz, H20 a), 2.10 (ddd, 1H, J 6.0 Hz, J 8.7 Hz, J 13.7 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); propargyl: 4.25 (dd, 1H, J 2.3 Hz, J 16.0 Hz, CH2), 4.20 (dd, 1H, J 2.4 Hz, J 16.0 Hz, CH2), 3.47 (br t, 1H, J 2.3 Hz, C–H); 13C NMR (DMSO-d6): thymine: 163.5 (C4), 150.3 (C2), 135.2 (C6), 109.5 (C5), 12.2 (CH3); sugar: 83.8 (C40 ), 83.8 (C10 ), 78.2 (C30 ), 63.1 (C50 ), 35.9 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3); propargyl: 79.9 (C), 77.3 (CH), 55.7 (CH2); HRESIMS: calcd for C19H30N2O5SiNa [M+Na]+: m/z 417.1816, found: m/z 417.1819.
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Compound 4b: (30 -O): Rf 0.63 (9:1 CHCl3–EtOH); uracil: 11.34 (br s, 1H, NH), 7.74 (d, 1H, JH6,H5 8.1 Hz, H6), 5.61 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.08 (dd, 1H, J 5.9 Hz, J 8.0 Hz, H10 ), 4.24 (m, 3H, H30 and CH2(propargyl)), 4.01 (m, 1H, H40 ), 3.77 (dd, 1H, J 4.1 Hz, J 11.3 Hz, H50 a), 3.73 (dd, 1H, J 3.6 Hz, J 11.3 Hz, H50 b), 2.33 (ddd, 1H, J 2.3 Hz, J 5.9 Hz, J 13.9 Hz, H20 a), 2.12 (ddd, 1H, J 6.2 Hz, J 8.0 Hz, J 13.9 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); propargyl: 4.24 (m, 3H, CH2(propargyl) and H30 ), 3.46 (br t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): uracil: 162.9 (C4), 150.3 (C2), 139.9 (C6), 101.8 (C5); sugar: 84.3 (C10 ), 84.1 (C40 ), 78.1 (C30 ), 63.1 (C50 ), 36.4 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.5 (CH3b), propargyl: 79.9 (C), 77.4 (CH), 55.8 (CH2); ESIMS: m/z 381.4 (MH+), 403.4 (MNa+), 419.4 (MK+), 761.5 (M2H+), 783.3 (M2Na+); HRESIMS: calcd for C18H28N2O5SiNa [M+Na]+: m/z 403.5000, found: m/z 403.5009. Compound 5a: (3-N, 30 -O): Rf 0.65 (98:2 CHCl3–EtOH); 1H NMR: thymine: 7.58 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.85 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.14 (dd, 1H, J 5.8 Hz, J 8.4 Hz, H10 ), 4.26 (m, 1H, H30 ), 4.03 (br dd, 1H, J 3.8 Hz, J 5.8 Hz, H40 ), 3.80 (dd, 1H, J 4.0 Hz, J 11.3 Hz, H50 a), 3.76 (dd, 1H, J 3.8 Hz, J 11.3 Hz, H50 b), 2.36 (ddd, 1H, J 1.8 Hz, J 5.8 Hz, J 13.9 Hz, H20 a), 2.16 (br ddd, 1H, J 6.1 Hz, J 8.4 Hz, J 13.9 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); N-propargyl: 4.55 (dd, 1H, J 2.4 Hz, J 17.1 Hz, CH2), 4.51 (dd, 1H, J 2.5 Hz, J 17.1 Hz, CH2), 3.10 (br t, 1H, J 2.4 Hz, C–H); Opropargyl: 4.24 (br d, 2H, J 2.4 Hz, CH2), 3.47 (br t, J 2.4 Hz, 1H); 13 C NMR (DMSO-d6): 161.6 (C4), 149.6 (C2), 134.4 (C6), 108.6 (C5); sugar: 85.1 (C10 ), 84.1 (C40 ), 78.1 (C30 ), 63.1 (C50 ), 36.1 (C20 ), 12.7 (CH3); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3); Opropargyl: 79.9 (C), 77.3 (CH), 55.7 (CH2); N-propargyl: 78.9 (C), 72.9 (CH), 30.0 (CH2); ESIMS: m/z 433.1 (MH+), 455.3 (MNa+), 471.0 (MK+); HRESIMS: calcd for C22H32N2O5SiNa [M+Na]+: m/z 455.5746, found: m/z 455.5750. Compound 5b: (3-N, 30 -O): Rf 0.85 (9:1 CHCl3–EtOH); uracil: 7.84 (d, 1H, JH6,H5 8.1 Hz, H6), 5.81 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.12 (dd, 1H, J 6.0 Hz, J 7.7 Hz, H10 ), 4.26 (br dd, 1H, J 2.5 Hz, J 5.4 Hz, H30 ), 4.05 (m, 1H, H40 ), 3.80 (dd, 1H, J 4.1 Hz, J 11.3 Hz, H50 a), 3.74 (dd, 1H, J 3.5 Hz, J 11.3 Hz, H50 b), 2.41 (ddd, 1H, J 2.5 Hz, J 6.0 Hz, J 13.8 Hz, H20 a), 2.17 (ddd, 1H, J 6.1 Hz, J 7.7 Hz, J 13.8 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); N-propargyl: 4.53 (dd, 1H, J 2.4 Hz, J 17.0 Hz, 1H, CH2), 4.48 (dd, J 2.5 Hz, J 17.0 Hz, 1H, CH2), 3.12 (br t, 1H, J 2.4 Hz, C–H); O-propargyl: 4.23 (br d, 2H, J 2.0 Hz, CH2), 3.48 (t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): uracil: 160.9 (C4), 149.7 (C2), 139.0 (C6), 100.8 (C5); sugar: 85.6 (C10 ), 84.4 (C40 ), 77.9 (C30 ), 63.0 (C50 ), 36.6 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.5 (CH3b), O-propargyl: 80.0 (C), 77.4 (CH), 55.9 (CH2); N-propargyl: 78.9 (C), 73.0 (CH), 29.7 (CH2); ESIMS: m/z 419.1 (MH+), 441.2 (MNa+), 858.6 (M2Na+); HRESIMS: calcd for C21H30N2O5SiNa [M+Na]+: m/z 441.5480, found: m/z 441.5474. Compound 6a: Rf 0.75 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.32 (br s, 1H, NH), 7.43 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.78 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.15 (dd, 1H, J 6.2 Hz, J 7.7 Hz, H10 ), 4.36 (dt, 1H, J 2.9 Hz, J 5.8 Hz, H30 ), 3.78 (m, 1H, H40 ), 3.76 (dd, 1H, J 4.1 Hz, J 11.0 Hz, H50 a), 3.70 (dd, 1H, J 3.6 Hz, J 11.0 Hz, H50 b), 2.19 (ddd, 1H, J 5.8 Hz, J 7.7 Hz, J 13.4 Hz, H20 a), 2.07 (ddd, 1H, J 2.9 Hz, J 6.2 Hz, J 13.4 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 163.5 (C4), 150.3 (C2), 135.5 (C6), 109.4 (C5), 12.1 (CH3); sugar: 86.6 (C40 ), 83.6 (C10 ), 71.9 (C30 ), 62.6 (C50 ), 39.0 (C20 ), TBDMS: 25.7 (C–CH3a), 25.6 (C–CH3b), 17.9 (C– CH3a), 17.6 (C–CH3b), 5.6 (Si–CH3a), 5.0 (Si–CH3b), 4.9 (Si– CH3c); HRESIMS: calcd for C22H42N2O5Si2Na [M+Na]+: m/z 493.2524, found: m/z 493.2532. Compound 6b: Rf 0.51 (1:1 petroleum ether–EtOAc); 1H NMR: uracil: 11.37 (br s, 1H, NH), 7.74 (d, 1H, JH6,H5 8.1 Hz, H6), 5.62 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.17 (t, 1H, J 6.4 Hz, H10 ), 4.41 (m,
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R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
1H, H30 ), 3.83 (m, 1H, H40 ), 3.81 (dd, 1H, J 4.1 Hz, J 11.0 Hz, H50 a), 3.73 (dd, 1H, J 3.2 Hz, J 11.0 Hz, H50 b), 2.25 (br dd, 1H, J 6.4 Hz, J 13.2 Hz, H20 a), 2.17 (ddd, 1H, J 4.2 Hz, J 6.4 Hz, J 13.2 Hz, H20 b); TBDMS: 0.92 (s, 9H, C–CH3a), 0.91 (s, 9H, C–CH3b), 0.12 (s, 6H, Si– CH3a), 0.11 (s, 6H, Si–CH3b); 13C NMR (DMSO-d6): uracil: 163.0 (C4), 150.3 (C2), 140.1 (C6), 101.7 (C5); sugar: 86.6 (C40 ), 84.0 (C10 ), 71.4 (C30 ), 62.3 (C50 ), 39.5 (C20 ); TBDMS: 25.7 (C–CH3a), 25.6 (C–CH3b), 17.9 (C–CH3a), 17.7 (C–CH3b), 5.6 (Si–CH3a), 5.5 (Si– CH3a), 5.0 (Si–CH3b), 4.8 (Si–CH3c); HRESIMS: calcd for C21H40N2O5Si2Na [M+Na]+: m/z 479.2368, found: m/z 479.2378. Compound 7a: Rf 0.22 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.28 (br s, 1H, NH), 7.66 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.78 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.15 (dd, 1H, J 6.1 Hz, J 7.8 Hz, H10 ), 5.07 (br s, 1H, OH), 4.41 (dt, 1H, J 2.9 Hz, J 5.8 Hz, H30 ), 3.75 (m, 1H, H40 ), 3.58 (br d, 1H, J 11.8 Hz, H50 a), 3.70 (br d, 1H, J 11.8 Hz, H50 b), 2.18 (ddd, 1H, J 5.8 Hz, J 7.8 Hz, J 13.4 Hz, H20 a), 2.07 (ddd, 1H, J 2.9 Hz, J 6.1 Hz, J 13.4 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 163.6 (C4), 150.4 (C2), 135.9 (C6), 109.3 (C5), 12.2 (CH3); sugar: 87.2 (C40 ), 83.6 (C10 ), 72.1 (C30 ), 60.9 (C50 ), 39.5 (C20 ), TBDMS: 25.6 (C–CH3), 17.6 (C–CH3), 4.9 (Si–CH3); HRESIMS: calcd for C16H28N2O5SiNa [M+Na]+: m/z 379.1659, found: m/z 379.1669. Compound 7b: Rf 0.49 (98:2 EtOAc–EtOH); 1H NMR: uracil: 11.29 (br s, 1H, NH), 7.82 (d, 1H, JH6,H5 8.1 Hz, H6), 5.64 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.14 (dd, 1H, J 6.3 Hz, J 7.0 Hz, H10 ), 5.05 (br t, 1H, J 6.6 Hz, OH), 4.41 (m, 1H, H30 ), 3.77 (m, 1H, H40 ), 3.58 (br dd, 1H, J 4.6 Hz, J 12.1 Hz, H50 a), 3.52 (br dd, 1H, J 4.3 Hz, J 12.1 Hz, H50 b), 2.16 (ddd, 1H, J 6.3 Hz, J 7.0 Hz, J 13.2 Hz, H20 a), 2.08 (ddd, 1H, J 3.3 Hz, J 6.3 Hz, J 13.2 Hz, H20 b); TBDMS: 0.87 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): uracil: 163.0 (C4), 150.3 (C2), 140.4 (C6), 101.8 (C5); sugar: 87.3 (C40 ), 83.9 (C10 ), 71.9 (C30 ), 60.8 (C50 ), 39.7 (C20 ); TBDMS: 25.6 (C–CH3), 17.6 (C–CH3a), 5.0 (Si–CH3a), 4.9 (Si–CH3b); HRESIMS: calcd for C15H26N2O5SiNa [M+Na]+: m/z 365.4521, found: m/z 365.4518. Compound 8a: (50 -O): Rf 0.38 (9:1 CHCl3–EtOH); 1H NMR: thymine: 11.24 (br s, 1H, NH), 7.56 (d, 1H, JH6,CH3 0.8 Hz, H6), 1.80 (br s, 3H, CH3); sugar: 6.16 (t, 1H, J 6.3 Hz, J 7.1 Hz, H10 ), 4.39 (dt, 1H, J 3.2 Hz, J 6.3 Hz, H40 ), 4.24 (m, 1H, H30 ), 3.67 (dd, 1H, J 3.4 Hz, J 10.5 Hz, H50 a), 3.60 (dd, 1H, J 4.0 Hz, J 10.5 Hz, H50 b), 2.19 (ddd, 1H, J 6.3 Hz, J 7.1 Hz, J 13.3 Hz, H20 a), 2.06 (ddd, 1H, J 3.2 Hz, J 6.3 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); propargyl: 4.25 (d, 1H, J 2.3 Hz, J 16.0 Hz, CH2), 4.20 (d, 1H, J 2.1 Hz, J 16.0 Hz, CH2), 3.50 (br t, 1H, J 2.2 Hz, C–H); 13 C NMR (DMSO-d6): thymine: 163.7 (C4), 150.4 (C2), 135.8 (C6), 109.6 (C5), 12.3 (CH3); sugar: 85.1 (C40 ), 83.9 (C10 ), 72.3 (C30 ), 69.1 (C50 ), 39.4 (C20 ); TBDMS: 25.7 (C–CH3), 17.7 (C–CH3), 4.9 (CH3a), 4.8 (CH3b); propargyl: 79.9 (C), 77.6 (CH), 57.9 (CH2); HRESIMS: calcd for C19H30N2O5SiNa [M+Na]+: m/z 417.1816, found: m/z 417.1823. Compound 8b: (50 -O): Rf 0.71 (9:1 CHCl3–EtOH); uracil: 11.31 (br s, 1H, NH), 7.69 (d, 1H, JH6,H5 8.1 Hz, H6), 5.62 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.14 (br t, 1H, J 6.5 Hz, H10 ), 4.38 (m, 1H, H30 ), 3.89 (m, 1H, H40 ), 3.64 (dd, 1H, J 3.7 Hz, J 10.6 Hz, H50 a), 3.59 (dd, 1H, J 4.4 Hz, J 10.6 Hz, H50 b), 2.19 (dt, 1H, J 6.5 Hz, J 13.1 Hz, H20 a), 2.09 (ddd, 1H, J 3.9 Hz, J 6.5 Hz, J 13.1 Hz, H20 b); TBDMS: 0.87 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); propargyl: 4.20 (br d, 1H, J 2.1 Hz, CH2), 3.50 (t, 1H, J 2.1 Hz, C–H); 13C NMR (DMSO-d6): uracil: 163.1 (C4), 150.4 (C2), 140.4 (C6), 101.9 (C5); sugar: 85.1 (C40 ), 84.1 (C10 ), 72.1 (C30 ), 68.9 (C50 ), 39.3 (C20 ); TBDMS: 25.7 (C–CH3), 17.7 (C–CH3), 4.9 (CH3a), 4.8 (CH3b), propargyl: 79.9 (C), 77.4 (CH), 55.8 (CH2); HRESIMS: calcd for C18H28N2O5SiNa [M+Na]+: m/z 403.1706, found: m/z 403.1711. Compound 9a: (3-N, 50 -O): Rf 0.73 (9:1 CHCl3–EtOH); 1H NMR: thymine: 7.66 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.87 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.20 (br t, 1H, J 6.6 Hz, H10 ), 4.41 (dt, 1H, J 3.3 Hz, J 6.2 Hz, H30 ), 3.93 (m, 1H, H40 ), 3.69 (dd, 1H, J 4.0 Hz, J
10.6 Hz, H50 a), 3.62 (dd, 1H, J 4.1 Hz, J 10.6 Hz, H50 b), 2.23 (ddd, 1H, J 6.2 Hz, J 6.9 Hz, J 13.3 Hz, H20 a), 2.12 (ddd, 1H, J 3.3 Hz, J 6.4 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); N-propargyl: 4.55 (dd, 1H, J 2.4 Hz, J 16.8 Hz, CH2), 4.51 (dd, 1H, J 2.5 Hz, J 16.8 Hz, CH2), 3.10 (br t, 1H, J 2.4 Hz, C–H); Opropargyl: 4.25 (dd, 1H, J 2.3 Hz, J 15.9 Hz, CH2), 4.21 (dd, 1H, J 2.4 Hz, J 15.9 Hz, CH2), 3.51 (br t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): thymine: 161.7 (C4), 149.7 (C2), 134.9 (C6), 108.7 (C5), 12.9 (CH3); sugar: 85.3 (C10 ), 85.0 (C40 ), 72.2 (C30 ), 68.9 (C50 ), 39.6 (C20 ); TBDMS: 25.6 (C–CH3), 17.6 (C–CH3), 5.0 (CH3a), 4.9 (CH3b); O-propargyl: 79.9 (C), 77.6 (CH), 57.9 (CH2); N-propargyl: 79.0 (C), 73.0 (CH), 30.0 (CH2); HRESIMS: calcd for C22H32N2O5SiNa [M+Na]+: m/z 455.1973, found: m/z 455.1977. Compound 9b: (3-N, 50 -O): Rf 0.73 (9:1 CHCl3–EtOH); 1H NMR: uracil: 7.92 (d, 1H, JH6,H5 8.2 Hz, H6), 5.83 (d, 1H, JH5,H6 8.2 Hz, H5); sugar: 6.17 (br t, 1H, J 6.7 Hz, H10 ), 4.42 (m, 1H, H30 ), 3.81 (m, 1H, H40 ), 3.71 (m, 1H, H50 a), 3.60 (m, 1H, H50 b), 2.21 (ddd, 1H, J 6.1 Hz, J 6.7 Hz, J 13.2 Hz, H20 a), 2.14 (ddd, 1H, J 3.7 Hz, J 6.7 Hz, J 13.2 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); N-propargyl: 4.52 (dd, 1H, J 2.3 Hz, J 16.8 Hz, CH2), 4.51 (dd, 1H, J 2.2 Hz, J 16.8 Hz, CH2), 3.11 (br t, 1H, J 2.4 Hz, C–H); O-propargyl: 4.21 (m, 2H, CH2), 3.51 (t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSOd6): uracil: 160.9 (C4), 149.7 (C2), 139.2 (C6), 100.9 (C5); sugar: 85.5 (C10 ), 85.2 (C40 ), 78.9 (C30 ), 69.8 (C50 ), 39.7 (C20 ); TBDMS: 25.7 (C–CH3), 17.7 (C–CH3), 4.9 (CH3a), 4.8 (CH3b); O-propargyl: 78.9 (C), 77.7 (CH), 57.9 (CH2); N-propargyl: 78.9 (C), 73.0 (CH), 29.8 (CH2); HRESIMS: calcd for C21H30N2O5SiNa [M+Na]+: m/z 441.1816, found: m/z 441.1835. Compound 10: Rf 0.19 (9:1 EtOAc–EtOH); cytidine: 11.05 (s, 1H, NH), 8.40 (d, 1H, J 7.5 Hz, H6), 7.34 (d, J 7.5 Hz, H5); benzoyl: 8.01 (dd, 2H, J 7.3 Hz, J 1.1 Hz, H2–6), 7.62 (tt, 1H, J 7.3 Hz, J 1.1 Hz, H4), 7.52 (br t, 2H, J 7.7 Hz, H3–5); sugar: 6.15 (t, 1H, J 6.3 Hz, H10 ), 5.26 (d, 1H, J 4.1 Hz, 1H, O30 –H), 5.07 (t, 1H, J 5.0 Hz, J 6.0 Hz, O50 –H), 4.25 (m, 1H, H30 ), 3.89 (br dd, 1H, J 3.7 Hz, J 7.3 Hz, H50 b), 3.66 (br dt, 1H, J 4.5 Hz, J 11.8 Hz, H50 a), 3.60 (br dt, 1H, J 4.4 Hz, J 11.8 Hz, H50 b), 2.33 (ddd, 1H, J 3.9 Hz, J 6.3 Hz, J 13.2 Hz, H20 a), 2.07 (dt, 1H, J 6.3 Hz, J 13.2 Hz, H20 b); 13C NMR (DMSO-d6): thymine: 162.8 (C4), 154.2 (C2), 144.8 (C6), 96.0 (C5); benzoyl: 167.4 (CO), 133.1 (C1), 132.6 (C4), 128.3 (C2, C3, C5, C6); sugar: 87.9 (C40 ), 86.1 (C10 ), 69.8 (C30 ), 60.9 (C50 ), 40.2 (C20 ); ESIMS m/z 331.9 (MH+), 353.9 (MNa+), 685.0 (M2Na+). Compound 11: Rf 0.52 (9:1 EtOAc–EtOH); cytidine: 11.22 (s, 1H, NH), 8.31 (br s, 1H, H5), 7.36 (br d, J 6.8 Hz, H5); benzoyl: 8.00 (dd, 2H, J 1.2 Hz, J 8.5 Hz, H2–6), 7.62 (br t, 1H, J 7.5 Hz, H4), 7.51 (br t, 2H, J 7.5 Hz, H3–5); sugar: 6.13 (t, 1H, J 6.1 Hz, H10 ), 5.33 (d, 1H, J 4.4 Hz, 1H, O30 –H), 4.22 (m, 1H, H30 ), 3.93 (br dd, 1H, J 3.1 Hz, J 11.5 Hz, H50 a), 3.87 (dd, 1H, J 3.3 Hz, J 11.5 Hz, H50 b), 3.77 (dd, 1H, J 3.3 Hz, J 11.5 Hz, H50 b), 2.36 (ddd, 1H, J 4.8 Hz, J 6.1 Hz, J 13.3 Hz, H20 a), 2.07 (dt, 1H, J 6.1 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 162.9 (C4), 154.3 (C2), 144.4 (C6), 95.8 (C5); benzoyl: 167.3 (CO), 133.1 (C1), 132.7 (C4), 128.4 (C2, C3, C5, C6); sugar: 87.3 (C40 ), 86.2 (C10 ), 69.6 (C30 ), 62.6 (C50 ), 41.2 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3a), 5.5 (Si–CH3a), 5.6 (Si–CH3b); ESIMS m/z 446.5 (MH+), 468.4 (MNa+), 913.6 (M2Na+); HRESIMS: calcd for C22H31N3O5SiNa [M+Na]+: m/z 468.5733, found: m/z 468.5736. Compound 12: Rf 0.45 (9:1 EtOAc–MeOH); cytidine: 11.26 (s, 1H, NH), 8.28 (br s, 1H, H5), 7.36 (br d, J 7.4 Hz, H5); benzoyl: 8.01 (dd, 2H, J 7.2 Hz, J 1.2 Hz, H2–6), 7.62 (br t, 1H, J 7.4 Hz, H4), 7.51 (br t, 2H, J 7.6 Hz, H3–5); sugar: 6.09 (br t, 1H, J 6.4 Hz, H10 ), 4.27 (m, 1H, H30 ), 4.14 (br dd, J 3.1 Hz, J 6.2 Hz, H40 ), 3.87 (br dd, 1H, J 3.7 Hz, J 11.4 Hz, H50 a), 3.78 (br dt, 1H, J 3.3 Hz, J 11.4 Hz, H50 b), 2.54 (ddd, 1H, J 3.1 Hz, J 6.1 Hz, J 13.3 Hz, H20 a), 2.14 (dt, 1H, J 6.3 Hz, J 13.3 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.10 (s, 6H, Si–CH3a), 0.09 (s, 6H, Si–CH3a); propargyl: 4.23 (br d, 1H, J 2.3 Hz, J 18.0 Hz, CH2), 3.47 (t, 1H, J 2.3 Hz, C–H); 13C NMR (DMSO-d6):
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
thymine: 163.0 (C4), 154.2 (C2), 144.3 (C6), 95.9 (C5); benzoyl: 167.3 (CO), 133.1 (C1), 132.7 (C4), 128.4 (C2, C3, C5, C6); sugar: 86.5 (C10 ), 84.5 (C40 ), 77.7 (C30 ), 62.8 (C50 ), 40.4 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.6 (CH3b); propargyl: 80.0 (C), 77.4 (CH), 55.9 (CH2); ESIMS m/z 484.4 (MH+), 506.4 (MNa+), 989.7 (M2Na+); HRESIMS: calcd for C22H31N3O5SiNa [M+Na]+: m/z 506.6213, found: m/z 506.6221. Acknowledgments Financial support from the MENRT and the ‘Conseil Régional du Limousin’ is gratefully acknowledged. The authors are indebted to Dr. Mircea Darabantu and Dr. Jean Debord for their advices. Supplementary data Supplementary data (spectroscopic data for compounds 2–12) associated with this article can be found, in the online version, at doi:10.1016/j.carres.2009.10.021.
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References 1. (a) Zhang, H.; Yang, B.; Pomerantz, R. J.; Zhang, C.; Arunachalam, S. C.; Gao, L. Nature 2003, 424, 94–98; (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607–3630; (c) Agrofoglio, L.; Suhas, E.; Farese, A.; Condom, R.; Richard Challand, S.; Earl, R. A.; Guedj, R. Tetrahedron 1994, 50, 10611–10670. 2. (a) Weisbrod, S. H.; Marx, A. Chem. Commun. (Cambridge) 2007, 1828–1830; (b) Brazier, J. A.; Shibata, T.; Townsley, J.; Taylor, B. F.; Frary, E.; Williams, N. H.; Williams, D. M. Nucleic Acids Res. 2005, 33, 1362–1371; (c) Sarfati, S. R.; Pochet, S.; Guerreiro, C.; Namane, A.; Huynh-Dinh, T.; Igolen, J. Tetrahedron 1987, 43, 3491–3497. 3. Nervi, C.; Vigna, M. A.; Cavigiolio, G.; Ravera, M.; Osella, D. Inorg. Chim. Acta 2005, 358, 2799–2803. 4. Wu, J. C.; Xi, Z.; Gioeli, C.; Chattopadhyaya, J. Tetrahedron 1991, 47, 2237–2254. 5. Teste, K.; Colombeau, L.; Hadj-Bouazza, A.; Lucas, R.; Zerrouki, R.; Krausz, P.; Champavier, Y. Carbohydr. Res. 2008, 343, 1490–1495. 6. Lucas, R.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y. Tetrahedron 2008, 64, 5467–5471. 7. Weisbrod, S. H.; Marx, A. Chem. Commun. (Cambridge) 2008, 5675–5685. 8. Oglivie, K. K. Can. J. Chem. 1973, 51, 3799–3807. 9. Progress in Inorganic Chemistry; Karlin, K. D., Ed.; Wiley & Sons: New York, 2005. 10. Cui, Z.; Zhang, B. Helv. Chim. Acta 2007, 90, 297–310. 11. Alexander, R. L.; Morris-Natschke, S. L.; Ishaq, K. S.; Fleming, R. A.; Kucera, G. L. J. Med. Chem. 2003, 46, 4205–4208.
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Chelation-controlled regioselective alkylation of pyrimidine 20 -deoxynucleosides R. Lucas a, K. Teste a, R. Zerrouki a,*, Y. Champavier b, M. Guilloton a a b
Laboratoire de Chimie des Substances Naturelles EA1069, Faculté des Sciences et Techniques, 123 Avenue, Albert Thomas, F-87060 Limoges, France Université de Limoges, Service Commun de RMN, Faculté de Pharmacie, 2 rue du Dr Marcland, F-87025 Limoges, France
a r t i c l e
i n f o
Article history: Received 10 September 2009 Received in revised form 23 October 2009 Accepted 27 October 2009 Available online 30 October 2009
a b s t r a c t Protection–deprotection steps, which are usually needed for regioselective alkylation of pyrimidine deoxynucleosides, can be avoided by choosing the appropriate solvent. The combined effects of low dielectric constant and possible sodium chelation by pyrimidine nucleosides may account for the unexpected regioselectivity observed in THF. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Alkylation Nucleosides Chelating agents Regioselectivity
1. Introduction Over the past 50 years, scientists have shown a continuous interest in nucleoside chemistry, most notably in the field of drug synthesis, for example, in the synthesis of anti-HIV drugs (Fig. 1).1 The reactivity of nucleosides is rather complex due to the presence of various nucleophilic sites. Protection/deprotection steps are often necessary to generate nucleoside derivatives. Many applications in bioorganic chemistry concern bioconjugation (e.g., DNA labeling) where purine or pyrimidine bases must be selectively functionalized.2 Another example is presented by the synthesis of oligonucleotide analogues for which functionalization of the 30 O- and 50 -O-position is mandatory. Finally, nucleoside analogue synthesis sometimes requires a regioselective modification of the 30 -O-position (Fig. 1). In fact, modifications of the glycone moiety easily affect the nucleobase unless the nucleoside is adequately protected. Regioselective reactions, should, if possible, be undertaken on the unprotected substrate to avoid this drawback. Functionalization, as well as protection of nucleosides, is frequently accomplished by alkylation. In the case of pyrimidine deoxynucleosides, we are confronted with competition between the 3-N- and 30 -O-nucleophilic positions. The use of a metallic hydride (NaH) in DMF allows the selective alkylation of the 3-N-alkylated.3 Conducted in THF and in the presence of an excess of alkylating agent, this reaction leads to regioselective 30 -O-alkylation.4 This surprising result allowed us to establish the solvent * Corresponding author. Tel.: +33 5 55 45 72 24; fax: +33 5 55 45 72 02. E-mail address: [email protected] (R. Zerrouki). 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.10.021
dependency of high yield (82–99%) in the regioselective 30 -Oalkylation of 50 -O-protected thymidine (Scheme 1).5 In this article, we present a study of 20 -deoxypyrimidine nucleoside alkylation using thymidine (1a), 20 -deoxyuridine (1b), and
Figure 1. Anti-HIV drugs obtained by regioselective modifications.
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R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
Scheme 1. Regioselective alkylation of 50 -O-protected thymidine: (i) NaH (2.5 equiv), THF, RBr (2.5 equiv), ultrasound or MW; (ii) NaH (1.2 equiv), DMF, RBr (1.2 equiv), ultrasound or MW.
2. Results and discussion 2.1. Solvent-dependent regioselectivity
Figure 2. Solvent-dependent regioselective allylation of thymidine.5 *Reactions were carried out with excess sodium hydride/allyl bromide.
20 -deoxycytidine (1c). These experiments were conducted to understand the influence of solvents on regioselectivity, and to master nucleoside alkylation as well as further synthesis of nucleoside analogues.
The regioselective allylation of 50 -O-TBDMS–thymidine (2a) is closely linked to the dielectric constant of the solvent (Fig. 2). Indeed O-allylation is only observed in solvents of low dielectric constant (e 6 10), for example, THF (e = 7.58), excess NaH being required. Under these conditions the best yields are obtained with 2.5 equiv of allyl bromide. In DMF (e = 37), a quasi-stoichiometric amount of both reagents readily leads to N-allylated thymidine. Before clarifying the reactivity of thymidine under such conditions, we repeated these observations with 20 -deoxyuridine (dU). Because the microwave-activated reaction is so fast (completion within 1–3 min), all the following reactions were carried out with ultrasound activation in order to check more easily their evolution as a function of reaction time. Propargyl bromide was used as the alkylating reagent, keeping in mind that the resulting products can be used as ‘click reagents’ in copper-catalyzed azide–alkyne cycloaddition (CuAAC) for further syntheses of oligomers, labeled nucleosides, etc.1,6,7 The corresponding alkylated products are indicated in Scheme 2. The first step was the well-known monoprotection of nucleosides 1a–b to give the corresponding 20 -deoxy-50 -O-tert-butyldimethylsilylnucleosides 2a–b in high yield.8 Then, the 50 -O-protected nucleosides were alkylated using several conditions which
Scheme 2. Synthesis of propargylated 50 -O-protected thymidines: 3a (3-N), 4a (30 -O) and 5a (3-N and 30 -O), and 50 -O-protected 20 -deoxyuridines 3b (3-N), 4b (30 -O), and 5b (3-N and 30 -O). Reagents and conditions: (i) TBDMSCl, DMAP, Py, rt; (ii) NaH, THF or DMF, propargyl bromide, ultrasound.
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207 Table 1 Selected results of propargylation of 50 -O-protected thymidine and 50 -O-protected 20 deoxyuridine Entry
NaH; RBr (equiv)
Solvent
3a (3b)a (%)
4a (4b)a (%)
5a (5b)a (%)
1 2 3 4
2.5 1.2 2.5 1.2
THF THF DMF DMF
0 (0) 0 (0)b 0 (0) 93 (94)
100 (92) 0 (0)b 0 (0) 0 (0)
0 (0) 0 (0)b 90 (89) 0 (0)
a Reaction times allowed virtual reaction completion (checked by TLC); longer reaction times gave no increase in reaction yields. b No evolution was observed even after one day of reaction.
201
are summarized in Table 1. The two solvents used were THF and DMF in order to evaluate their straightforward regioselective effects on thymidine allylation. Firstly, 20 -deoxyuridine behaves essentially in the same way as thymidine whatever the conditions are. 30 -O- and 3-N-propargylation products were obtained in good yields: 4a (30 -O-T) 100%, 4b (30 -O-dU) 92%, 3a (3-N-T) 93%, and 3b (3-N-dU) 94%. The two alkylated compounds 3a and 4a can be differentiated (in the same way as 3b and 4b) by HMBC experiments (Fig. 3). NMR spectra of 3-N-propargylated compounds show a connection between CH2propargyle(a) (4.51–4.55 ppm) and C-2/C-4 (149.6/161.7 ppm) of thymine or 20 -deoxyuridine, whereas for 30 -O-propargylation a
Figure 3. 1H–13C experiments (400 MHz) for analysis of propargylated regioisomers 3a and 4a. Insets show a magnified part of the spectrum containing diagnostic crosspeaks between CH2propargyle(a) and C-2/C-4 for 3a, respectively, C-30 for 4a and between CH(30 ) and Cpropargyle(a) for 4a.
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R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207 Table 2 Selected results of 50 -O-protected thymidine and 20 -deoxyuridine propargylation in the presence of crown ether 15-C-5 Entry
NaH; RBr (equiv)
Solvent
Crown ether (equiv)
3a (3b)a (%)
4a (4b)a (%)
5a (5b)a (%)
1 2 3
2.5 1.2 1.2
THF THF DMF
3.5 1.2 1.2
0 (0) 73 (75) 100 (90)
0 (0) 0 (0) 0 (0)
89 (80) 0 (0) 0 (0)
a Reaction times allowed virtual reaction completion (checked by TLC), longer reaction times gave no increase in reaction yields.
Figure 4. Tentative structures of sodium-enolate complexes accounting for the regioselectivity of O- versus N-alkylation observed in solvents of low dielectric constant in the presence of excess NaH and propargyl bromide.
connectivity between CH(30 ) (4.20–4.25 ppm) of the sugar and CH2propargyle(a) (78.2 ppm) can be observed. If only stoichiometric amounts of NaH and propargyl bromide were used in THF, there was absolutely no alkylation reaction. Furthermore, excess reagent in DMF gave dipropargylated pyrimidine nucleosides in good yields: 89% for 5b and 90% for 5a. Addition of a strong base like NaOH led to similar results. For example, in the presence of excess NaOH and propargyl bromide in THF, the expected compound 4a was isolated in 52% yield. We also tested the effect of Mg2+. After the first activation by ultrasound in THF in the presence of excess NaH, 2.5 equiv of magnesium iodide was added. Then addition of 2.5 equiv of propargyl bromide was followed by a second activation. TLC analysis showed that no reaction took place. A strong interaction between Mg2+ and 30 -O could explain the non-reactivity of this position. The same reaction carried out in DMF with 1.2 equiv of reagents and excess MgI2 allowed selective alkylation of the 3-N position. In other words, when the solvent is THF, the most acidic hydrogen9 (3-NH) must be abstracted by 1 equiv of the 2.5 equiv of sodium hydride before the 30 -OH can react and be further alkylated by propargyl bromide. This means that proton abstraction at the 3-N-position leads to the disappearance of the nucleophilic character of the nitrogen atom, although the reactivity of the 3-N-position is fully restored when THF is replaced by DMF. Since the thymidine/Na+ ion pair created by the consumption of one NaH equivalent must interact more strongly in THF than it does in DMF, we thought that some kind of chelation of sodium by the negatively charged nucleoside could occur in THF. Figure 4 gives a tentative illustration of this hypothesis and suggests that the collapse of 3-N- reactivity could originate from stabilization of the thymine enolate by a sodium ion. 2.2. Effect of crown ether The hypothesis of sodium-chelation-controlled regioselectivity was tested by adding the Na+-selective 15-C-5 crown ether to reaction mixtures. Reactions were carried out in THF or DMF with quasi-stoichiometric or excess amounts of either crown ether or reagents (NaH/propargyl bromide) (Table 2). Excess crown ether, together with excess NaH and propargyl bromide in THF, leads only to dialkylated products 5a and 5b (Table 2, entry 1). The nucleophilic character of the 3-N-position is restored, thanks to sodium trapping by 15-C-5. This result indicates that in the absence of crown ether, thymidine enolate efficiently chelates one sodium ion, thus impairing further alkylation of the 3-N-position. A quasi-stoichiometric amount of crown ether in DMF did not modify the already observed regioselectivity (Table 2, entry 3); however in THF, N-propargylated compounds are actually obtained (Table 2, entry 2).
Taken as a whole, these results are in agreement with the hypothesis of sodium-ion chelation by the pyrimidine nucleoside, which governs the observed regioselectivity and the role of solvent of low dielectric constant in chelate stabilization. Although THF likely stabilizes a sodium-enolate chelate, the exact nature of the complex is presently unknown. We investigated the possible contribution of oxygen in the 50 -position to sodiumion chelation. If the oxygen of the TBDMS-protected 50 -hydroxyl group is involved in this chelation, one may assume that, if unprotected, the 50 -oxygen in the anionic form would interact even more strongly with sodium, thus preventing further alkylation at this position. In order to check this hypothesis, we prepared the 30 -O-protected thymidine and 20 -deoxyuridine (7a and 7b). The starting 20 deoxynucleosides 1a–b were easily transformed into the corresponding 20 -deoxy-30 ,50 -di-(O-tert-butyldimethylsilyl)nucleosides 6a–b in high yield. The second step was the selective removal of the 50 -O-tert-butyldimethylsilyl group according to Oglivie.8 The 30 -O monoprotected 20 -deoxynucleosides when propargylated gave the corresponding compounds 8a–b and 9a–b (Scheme 3). The results show that by using an excess of NaH and propargyl bromide without crown ether in THF we obtain only the 50 -O-alkylated pyrimidine nucleosides in good yield: 8a (50 -O-T) 63%, and 8b (50 -O-dU) 62% (Table 3, entry 1). Thus oxygen in the 50 -position seems not to participate in sodium chelation. With an excess amount of reagents and crown ether, dialkylated compounds are readily obtained (Table 3, entry 2): 9a (3-N-, 50 -O-T) 82%, and 9b (3-N-, 50 -O-dU) 60%. Finally the same reaction without crown ether leads to no reaction at all (Table 3, entry 3), a result reminiscent of reactions with 50 -O-protected deoxynucleosides 2a–b. 2.3. Proposed mechanism in solvents with low dielectric constants The present results permit one to propose a mechanism for the reaction between pyrimidine nucleosides protected in the 50 -position and an alkylating agent, after initial reaction with sodium hydride, in a solvent with a low dielectric constant (Scheme 4). The amount of NaH is the critical parameter which governs alkylation selectivity. Alkylation of 4-N-benzoyl-20 -deoxy-50 -O-tert-butyldimethylsilyl-cytidine: in an attempt to extend this chelation hypothesis to other nucleosides, we investigated the synthesis of the 30 -O-propargylated 4-N-benzoylcytidine protected in the 50 -position (Scheme 5). The first step consisted of the selective benzoylation of the amino group of the heterocycle.10 Compound 10 was then silylated using the literature conditions in a good yield of 84%.11 The impossibility of a chelation (or else chelation with a low energy of coordination) between the base and the sodium ion leads one to conclude that only 1.2 equiv of propargyl bromide and sodium hydride are sufficient to give the expected 30 -O-propargylated 4-Nbenzoylcytidine protected in the 50 -position (Table 4, entry 2).
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
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Scheme 3. Synthesis of propargylated thymidines: 8a (50 -O-) and 9a (3-N- and 50 -O-), 20 -deoxyuridine 8b (50 -O-) and 9b (3-N- and 50 -O-). Reagents and conditions: (i) TBDMSCl, imidazole, AgNO3, 1:1 THF–DMF, rt; (ii) PPTS, MeOH, rt; (iii) NaH, THF, propargyl bromide, ultrasound.
Table 3 Selected results of propargylation of 30 -O-protected thymidine and 20 -deoxyuridine using crown ether 15-C-5 Entry
NaH; RBr (equiv)
Solvent
Crown ether (equiv)
8a (8b)a (%)
9a (9b)a (%)
1 2 3
2.5 2.5 1.2
THF THF THF
0 3.5 0b
63 (62) 0 (0) 0 (0)b
0 (0) 82 (60) 0 (0)b
a Reaction times allowing virtual reaction completion (checked by TLC), longer reaction times gave no increase in reaction yields. b No evolution was seen even after one day of reaction.
2.4. Effects of other hydrides The mechanism of sodium-chelation-controlled regioselectivity was tested by using two other hydrides LiH and KH. The results are summarized in Table 5. No evolution was observed in the case of LiH in THF, most probably because of a strongly associated pair of intimate ions (Li+ O) in low dielectric solvent. In DMF, compound 3a was isolated in low yield (25%). On the other hand, KH led to the expected product in 50% yield in THF with the K-specific 18-C-6 crown ether. 2.5. Conclusions
When this reaction was conducted in DMF, TLC showed numerous side products along with the initial 20 -deoxycytidine derivative. Purification did not lead to significant results.
We have demonstrated the role of sodium ion in the regioselectivity of the alkylation of pyrimidine nucleosides. The proposed
Scheme 4. A plausible mechanism explaining the role of sodium ion in the regioselectivity of alkylation in a solvent with a low dielectric constant.
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R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
Scheme 5. Propargylation of 4-N- and 50 -O-protected deoxycytidine 11. Reagents and conditions: (i) BzCl, BrSiMe3, Pyr, rt; (ii) TBDMSCl, imidazole, DMF, 0 °C; (iii) NaH, THF, propargyl bromide, ultrasound.
Table 4 Selected results of propargylation ldimethylsilylcytidine in THF
of
4-N-benzoyl-20 -deoxy-50 -O-tert-buty-
Entry
NaH; RBr (equiv)
12a (%)
1 2
2.5 1.2
91 65
a Reaction times allowing virtual reaction completion (checked by TLC), longer reaction times gave no yield increase.
Table 5 Selected results of 50 -O-protected thymidine propargylation in the presence of LiH, KH and corresponding crown ethers 12-C-4 (Li+) and 18-C-6 (K+) Entry
Hydride
NaH; RBr (equiv)
Solvent
Corresponding crown ether (equiv)
3aa (%)
4aa (%)
5aa (%)
1 2 3 4 5 6
LiH KH LiH KH LiH KH
2.5 2.5 2.5 2.5 1.2 1.2
THF THF THF THF DMF DMF
— — 3.5 3.5 — —
0b 0b 0b 0 25 83
0b 0b 0b 0 0 0
0b 0b 0b 50 0 0
a Reaction times allowing virtual reaction completion (checked by TLC), longer reaction times gave no increase in reaction yields. b No evolution was seen even after one day of reaction.
mechanism involves the possible chelation of sodium ion by the 2-enolate form of thymidine and 20 -deoxyuridine. This striking solvent-dependent selectivity is likely to speed up a variety of syntheses involving nucleoside modification and derivatization.
3. Experimental section 3.1. Materials All solvents and chemicals were commercially available, and unless otherwise stated, were used as received. Thymidine, 20 -
deoxyuridine, 20 -deoxycytidine, tert-butylchlorodimethylsilane, and propargyl bromide were purchased from Alfa Aesar. Sodium hydride, silver nitrate, imidazole, and PPTS were purchased from Sigma–Aldrich. Crown ether (15-C-5) was purchased from Acros. All solvents were purchased from Alfa Aesar. Reactions were monitored by thin-layer chromatography (TLC) on precoated 0.2 mm Silica Gel 60 F254 (E. Merck) plates and visualized either with an ultraviolet light source at 254 nm, or by spraying sulfuric acid (6 N) and heating to 200 °C. 1 H NMR (13C NMR) spectra were recorded at 400.13 MHz (100.62 MHz) with a Bruker DPX spectrometer using DMSO-d6. Chemical shifts (d) are expressed in ppm with Me4Si as the internal standard (d0). Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad), coupling constants (Hz), and assignment. Mass spectrometry: Mass spectra were recorded with an R1010 Nermag instrument. 3.2. General procedure For the synthesis of compounds 3a–b, 4a–b, 5a–b, 8a–b, 9a–b, and 12: To 50 -O or 30 -O-tert-butyldimethylsilylnucleoside (6.00 mmol) in dry THF (30 mL) was added 2.5 equiv of NaH (60%, 15.00 mmol), and the mixture was activated by ultrasound activation (20 min). Allyl bromide (2.5 equiv, 15.00 mmol) was then added, and the mixture was activated by ultrasound (from 20 min to 3 h depending on solvent). After workup (NH4Cl–H2O), the product was purified by chromatography (thick layer plates). 3.3. Characterization of compounds Concerning the purity of all the compounds synthesized, no extraneous substances were observed. They were also homogeneous by chromatography. Compound 2a. Rf 0.44 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.31 (br s, 1H, NH), 7.47 (d, 1H, JH6,CH3 0.7 Hz, H6), 1.77 (d, 3H, JCH3,H6 0.7 Hz, CH3); ose: 6.17 (dd, 1H, J 6.3 Hz, J 7.6 Hz, H10 ), 5.26 (s, 1H, OH), 4.20 (m, 1H, H30 ), 3.89 (dd, 1H, J 3.2 Hz, J
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
11.0 Hz, H50 a), 3.81 (m, 1H, H40 ), 3.79 (dd, 1H, J 3.3 Hz, J 11.0 Hz, H50 b), 2.08 (ddd, 1H, J 3.0 Hz, J 6.3 Hz, J 13.2 Hz, H20 a), 2.05 (ddd, 1H, J 5.8 Hz, J 7.6 Hz, J 13.2 Hz, H20 b); TBDMS: 0.88 (s, 9H, C– CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 163.7 (C4), 150.4 (C2), 135.5 (C6), 109.4 (C5), 12.2 (CH3); ose: 86.8 (C40 ), 83.8 (C10 ), 70.5 (C30 ), 63.3 (C50 ), 39.4 (C20 ), TBDMS: 25.8 (C–CH3), 18.0 (C–CH3), 5.4 (Si–CH3); ESIMS: m/z 357.2 (MH+), 380.2 (MNa+). Compound 2b: Rf 0.44 (9:1 CHCl3–EtOH); 1H NMR: uracil: 11.30 (br s, 1H, NH), 7.75 (d, 1H, JH6,H5 8.1 Hz, H6), 5.57 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.14 (t, 1H, J 6.3 Hz, H10 ), 5.29 (br s, 1H, OH), 4.19 (m, 1H, H30 ), 3.82 (m, 1H, H40 ), 3.78 (dd, 1H, J 3.3 Hz, J 11.3 Hz, H50 a), 3.72 (dd, 1H, J 3.5 Hz, J 11.3 Hz, H50 b), 2.15 (ddd, 1H, J 3.6 Hz, J 6.3 Hz, J 13.2 Hz, H20 a), 2.06 (ddd, 1H, J 2.4 Hz, J 6.3 Hz, J 13.2 Hz, H20 b); TBDMS: 0.87 (s, 9H, C–CH3), 0.07 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): uracil: 163.0 (C4), 150.3 (C2), 140.1 (C6), 101.6 (C5); sugar: 86.9 (C40 ), 84.3 (C10 ), 70.1 (C30 ), 63.0 (C50 ), 39.8 (C20 ); TBDMS: 25.8 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.5 (CH3b); ESIMS: m/z 343.0 (MH+), 365.0 (MNa+), 685.0 (M2H+), 706.9 (M2Na+). Compound 3a: (3-N): Rf 0.43 (9:1 CHCl3–EtOH); 1H NMR: thymine: 7.57 (d, 1H, JH6,CH3 0.7 Hz, H6), 1.84 (d, 3H, JCH3,H6 0.7 Hz, CH3); sugar: 6.20 (dd, 1H, J 6.3 Hz, J 7.3 Hz, H10 ), 5.30 (d, 1H, J 4.2 Hz, OH), 4.21 (m, 1H, H30 ), 3.85 (dd, 1H, J 3.0 Hz, J 6.0 Hz, H40 ), 3.80 (dd, 1H, J 3.0 Hz, J 11.4 Hz, H50 a), 3.73 (dd, 1H, J 3.8 Hz, J 11.4 Hz, H50 b), 2.16 (ddd, 1H, J 3.1 Hz, J 6.3 Hz, J 13.3 Hz, H20 a), 2.09 (ddd, 1H, J 6.5 Hz, J 7.3 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.07 (s, 6H, Si–CH3); propargyl: 4.55 (dd, 1H, J 2.4 Hz, J 15.6 Hz, CH2), 4.51 (dd, 1H, J 2.3 Hz, J 15.6 Hz, CH2), 3.10 (br t, 1H, J 2.3 Hz, C–H); 13C NMR (DMSO-d6): thymine: 161.7 (C4), 149.6 (C2), 134.6 (C6), 108.5 (C5), 12.8 (CH3); sugar: 87.0 (C40 ), 85.1 (C10 ), 70.4 (C30 ), 63.2 (C50 ), 39.6 (C20 ); TBDMS: 25.8 (C–CH3), 18.0 (C–CH3), 5.6 (CH3); propargyl: 79.0 (C), 73.0 (CH), 30.0 (CH2); HRESIMS: calcd for C19H30N2O5SiNa [M+Na]+: m/z 417.1816, found: m/z 417.1818. Compound 3b: (3-N): Rf 0.70 (9:1 CHCl3–EtOH); uracil: 7.85 (d, 1H, JH6,H5 8.1 Hz, H6), 5.77 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.17 (t, 1H, J 6.5 Hz, H10 ), 5.33 (d, 1H, J 3.9 Hz, OH), 4.21 (m, 1H, H30 ), 3.86 (br dd, 1H, J 3.2 Hz, J 6.4 Hz, H40 ), 3.80 (dd, 1H, J 3.2 Hz, J 11.4 Hz, H50 a), 3.73 (dd, 1H, J 3.5 Hz, J 11.4 Hz, H50 b), 2.21 (ddd, 1H, J 3.7 Hz, J 6.5 Hz, J 13.3 Hz, H20 a), 2.10 (dt, 1H, J 6.5 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.07 (s, 6H, Si–CH3); propargyl: 4.53 (dd, 1H, J 2.4 Hz, J 17.2 Hz, CH2), 4.48 (dd, 1H, J 2.5 Hz, J 17.2 Hz, CH2), 3.11 (br t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): uracil: 160.9 (C4), 149.7 (C2), 139.1 (C6), 100.6 (C5); sugar: 87.1 (C40 ), 85.5 (C10 ), 70.0 (C30 ), 62.9 (C50 ), 40.0 (C20 ); TBDMS: 25.8 (C–CH3), 18.0 (C–CH3), 5.6 (CH3a), 5.5 (CH3b), propargyl: 78.9 (C), 73.0 (CH), 29.7 (CH2); ESIMS: m/z 381.4 (MH+), 403.4 (MNa+), 419.4 (MK+), 761.5 (M2H+), 783.3 (M2Na+); HRESIMS: calcd for C18H28N2O5SiNa [M+Na]+: m/z 403.5000, found: m/z 403.5011. Compound 4a: (30 -O): Rf 0.53 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.34 (br s, 1H, NH), 7.48 (d, 1H, JH6,CH3 0.8 Hz, H6), 1.78 (d, 3H, JCH3,H6 0.8 Hz, CH3); sugar: 6.10 (dd, 1H, J 5.7 Hz, J 8.7 Hz, H10 ), 4.24 (m, 1H, H30 ), 3.99 (dd, 1H, J 3.7 Hz, J 5.6 Hz, H40 ), 3.79 (dd, 1H, J 4.3 Hz, J 11.2 Hz, H50 a), 3.74 (dd, 1H, J 3.7 Hz, J 11.2 Hz, H50 b), 2.28 (ddd, 1H, J 1.6 Hz, J 5.7 Hz, J 13.7 Hz, H20 a), 2.10 (ddd, 1H, J 6.0 Hz, J 8.7 Hz, J 13.7 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); propargyl: 4.25 (dd, 1H, J 2.3 Hz, J 16.0 Hz, CH2), 4.20 (dd, 1H, J 2.4 Hz, J 16.0 Hz, CH2), 3.47 (br t, 1H, J 2.3 Hz, C–H); 13C NMR (DMSO-d6): thymine: 163.5 (C4), 150.3 (C2), 135.2 (C6), 109.5 (C5), 12.2 (CH3); sugar: 83.8 (C40 ), 83.8 (C10 ), 78.2 (C30 ), 63.1 (C50 ), 35.9 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3); propargyl: 79.9 (C), 77.3 (CH), 55.7 (CH2); HRESIMS: calcd for C19H30N2O5SiNa [M+Na]+: m/z 417.1816, found: m/z 417.1819.
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Compound 4b: (30 -O): Rf 0.63 (9:1 CHCl3–EtOH); uracil: 11.34 (br s, 1H, NH), 7.74 (d, 1H, JH6,H5 8.1 Hz, H6), 5.61 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.08 (dd, 1H, J 5.9 Hz, J 8.0 Hz, H10 ), 4.24 (m, 3H, H30 and CH2(propargyl)), 4.01 (m, 1H, H40 ), 3.77 (dd, 1H, J 4.1 Hz, J 11.3 Hz, H50 a), 3.73 (dd, 1H, J 3.6 Hz, J 11.3 Hz, H50 b), 2.33 (ddd, 1H, J 2.3 Hz, J 5.9 Hz, J 13.9 Hz, H20 a), 2.12 (ddd, 1H, J 6.2 Hz, J 8.0 Hz, J 13.9 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); propargyl: 4.24 (m, 3H, CH2(propargyl) and H30 ), 3.46 (br t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): uracil: 162.9 (C4), 150.3 (C2), 139.9 (C6), 101.8 (C5); sugar: 84.3 (C10 ), 84.1 (C40 ), 78.1 (C30 ), 63.1 (C50 ), 36.4 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.5 (CH3b), propargyl: 79.9 (C), 77.4 (CH), 55.8 (CH2); ESIMS: m/z 381.4 (MH+), 403.4 (MNa+), 419.4 (MK+), 761.5 (M2H+), 783.3 (M2Na+); HRESIMS: calcd for C18H28N2O5SiNa [M+Na]+: m/z 403.5000, found: m/z 403.5009. Compound 5a: (3-N, 30 -O): Rf 0.65 (98:2 CHCl3–EtOH); 1H NMR: thymine: 7.58 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.85 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.14 (dd, 1H, J 5.8 Hz, J 8.4 Hz, H10 ), 4.26 (m, 1H, H30 ), 4.03 (br dd, 1H, J 3.8 Hz, J 5.8 Hz, H40 ), 3.80 (dd, 1H, J 4.0 Hz, J 11.3 Hz, H50 a), 3.76 (dd, 1H, J 3.8 Hz, J 11.3 Hz, H50 b), 2.36 (ddd, 1H, J 1.8 Hz, J 5.8 Hz, J 13.9 Hz, H20 a), 2.16 (br ddd, 1H, J 6.1 Hz, J 8.4 Hz, J 13.9 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); N-propargyl: 4.55 (dd, 1H, J 2.4 Hz, J 17.1 Hz, CH2), 4.51 (dd, 1H, J 2.5 Hz, J 17.1 Hz, CH2), 3.10 (br t, 1H, J 2.4 Hz, C–H); Opropargyl: 4.24 (br d, 2H, J 2.4 Hz, CH2), 3.47 (br t, J 2.4 Hz, 1H); 13 C NMR (DMSO-d6): 161.6 (C4), 149.6 (C2), 134.4 (C6), 108.6 (C5); sugar: 85.1 (C10 ), 84.1 (C40 ), 78.1 (C30 ), 63.1 (C50 ), 36.1 (C20 ), 12.7 (CH3); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3); Opropargyl: 79.9 (C), 77.3 (CH), 55.7 (CH2); N-propargyl: 78.9 (C), 72.9 (CH), 30.0 (CH2); ESIMS: m/z 433.1 (MH+), 455.3 (MNa+), 471.0 (MK+); HRESIMS: calcd for C22H32N2O5SiNa [M+Na]+: m/z 455.5746, found: m/z 455.5750. Compound 5b: (3-N, 30 -O): Rf 0.85 (9:1 CHCl3–EtOH); uracil: 7.84 (d, 1H, JH6,H5 8.1 Hz, H6), 5.81 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.12 (dd, 1H, J 6.0 Hz, J 7.7 Hz, H10 ), 4.26 (br dd, 1H, J 2.5 Hz, J 5.4 Hz, H30 ), 4.05 (m, 1H, H40 ), 3.80 (dd, 1H, J 4.1 Hz, J 11.3 Hz, H50 a), 3.74 (dd, 1H, J 3.5 Hz, J 11.3 Hz, H50 b), 2.41 (ddd, 1H, J 2.5 Hz, J 6.0 Hz, J 13.8 Hz, H20 a), 2.17 (ddd, 1H, J 6.1 Hz, J 7.7 Hz, J 13.8 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); N-propargyl: 4.53 (dd, 1H, J 2.4 Hz, J 17.0 Hz, 1H, CH2), 4.48 (dd, J 2.5 Hz, J 17.0 Hz, 1H, CH2), 3.12 (br t, 1H, J 2.4 Hz, C–H); O-propargyl: 4.23 (br d, 2H, J 2.0 Hz, CH2), 3.48 (t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): uracil: 160.9 (C4), 149.7 (C2), 139.0 (C6), 100.8 (C5); sugar: 85.6 (C10 ), 84.4 (C40 ), 77.9 (C30 ), 63.0 (C50 ), 36.6 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.5 (CH3b), O-propargyl: 80.0 (C), 77.4 (CH), 55.9 (CH2); N-propargyl: 78.9 (C), 73.0 (CH), 29.7 (CH2); ESIMS: m/z 419.1 (MH+), 441.2 (MNa+), 858.6 (M2Na+); HRESIMS: calcd for C21H30N2O5SiNa [M+Na]+: m/z 441.5480, found: m/z 441.5474. Compound 6a: Rf 0.75 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.32 (br s, 1H, NH), 7.43 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.78 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.15 (dd, 1H, J 6.2 Hz, J 7.7 Hz, H10 ), 4.36 (dt, 1H, J 2.9 Hz, J 5.8 Hz, H30 ), 3.78 (m, 1H, H40 ), 3.76 (dd, 1H, J 4.1 Hz, J 11.0 Hz, H50 a), 3.70 (dd, 1H, J 3.6 Hz, J 11.0 Hz, H50 b), 2.19 (ddd, 1H, J 5.8 Hz, J 7.7 Hz, J 13.4 Hz, H20 a), 2.07 (ddd, 1H, J 2.9 Hz, J 6.2 Hz, J 13.4 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 163.5 (C4), 150.3 (C2), 135.5 (C6), 109.4 (C5), 12.1 (CH3); sugar: 86.6 (C40 ), 83.6 (C10 ), 71.9 (C30 ), 62.6 (C50 ), 39.0 (C20 ), TBDMS: 25.7 (C–CH3a), 25.6 (C–CH3b), 17.9 (C– CH3a), 17.6 (C–CH3b), 5.6 (Si–CH3a), 5.0 (Si–CH3b), 4.9 (Si– CH3c); HRESIMS: calcd for C22H42N2O5Si2Na [M+Na]+: m/z 493.2524, found: m/z 493.2532. Compound 6b: Rf 0.51 (1:1 petroleum ether–EtOAc); 1H NMR: uracil: 11.37 (br s, 1H, NH), 7.74 (d, 1H, JH6,H5 8.1 Hz, H6), 5.62 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.17 (t, 1H, J 6.4 Hz, H10 ), 4.41 (m,
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1H, H30 ), 3.83 (m, 1H, H40 ), 3.81 (dd, 1H, J 4.1 Hz, J 11.0 Hz, H50 a), 3.73 (dd, 1H, J 3.2 Hz, J 11.0 Hz, H50 b), 2.25 (br dd, 1H, J 6.4 Hz, J 13.2 Hz, H20 a), 2.17 (ddd, 1H, J 4.2 Hz, J 6.4 Hz, J 13.2 Hz, H20 b); TBDMS: 0.92 (s, 9H, C–CH3a), 0.91 (s, 9H, C–CH3b), 0.12 (s, 6H, Si– CH3a), 0.11 (s, 6H, Si–CH3b); 13C NMR (DMSO-d6): uracil: 163.0 (C4), 150.3 (C2), 140.1 (C6), 101.7 (C5); sugar: 86.6 (C40 ), 84.0 (C10 ), 71.4 (C30 ), 62.3 (C50 ), 39.5 (C20 ); TBDMS: 25.7 (C–CH3a), 25.6 (C–CH3b), 17.9 (C–CH3a), 17.7 (C–CH3b), 5.6 (Si–CH3a), 5.5 (Si– CH3a), 5.0 (Si–CH3b), 4.8 (Si–CH3c); HRESIMS: calcd for C21H40N2O5Si2Na [M+Na]+: m/z 479.2368, found: m/z 479.2378. Compound 7a: Rf 0.22 (95:5 CHCl3–EtOH); 1H NMR: thymine: 11.28 (br s, 1H, NH), 7.66 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.78 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.15 (dd, 1H, J 6.1 Hz, J 7.8 Hz, H10 ), 5.07 (br s, 1H, OH), 4.41 (dt, 1H, J 2.9 Hz, J 5.8 Hz, H30 ), 3.75 (m, 1H, H40 ), 3.58 (br d, 1H, J 11.8 Hz, H50 a), 3.70 (br d, 1H, J 11.8 Hz, H50 b), 2.18 (ddd, 1H, J 5.8 Hz, J 7.8 Hz, J 13.4 Hz, H20 a), 2.07 (ddd, 1H, J 2.9 Hz, J 6.1 Hz, J 13.4 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 163.6 (C4), 150.4 (C2), 135.9 (C6), 109.3 (C5), 12.2 (CH3); sugar: 87.2 (C40 ), 83.6 (C10 ), 72.1 (C30 ), 60.9 (C50 ), 39.5 (C20 ), TBDMS: 25.6 (C–CH3), 17.6 (C–CH3), 4.9 (Si–CH3); HRESIMS: calcd for C16H28N2O5SiNa [M+Na]+: m/z 379.1659, found: m/z 379.1669. Compound 7b: Rf 0.49 (98:2 EtOAc–EtOH); 1H NMR: uracil: 11.29 (br s, 1H, NH), 7.82 (d, 1H, JH6,H5 8.1 Hz, H6), 5.64 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.14 (dd, 1H, J 6.3 Hz, J 7.0 Hz, H10 ), 5.05 (br t, 1H, J 6.6 Hz, OH), 4.41 (m, 1H, H30 ), 3.77 (m, 1H, H40 ), 3.58 (br dd, 1H, J 4.6 Hz, J 12.1 Hz, H50 a), 3.52 (br dd, 1H, J 4.3 Hz, J 12.1 Hz, H50 b), 2.16 (ddd, 1H, J 6.3 Hz, J 7.0 Hz, J 13.2 Hz, H20 a), 2.08 (ddd, 1H, J 3.3 Hz, J 6.3 Hz, J 13.2 Hz, H20 b); TBDMS: 0.87 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): uracil: 163.0 (C4), 150.3 (C2), 140.4 (C6), 101.8 (C5); sugar: 87.3 (C40 ), 83.9 (C10 ), 71.9 (C30 ), 60.8 (C50 ), 39.7 (C20 ); TBDMS: 25.6 (C–CH3), 17.6 (C–CH3a), 5.0 (Si–CH3a), 4.9 (Si–CH3b); HRESIMS: calcd for C15H26N2O5SiNa [M+Na]+: m/z 365.4521, found: m/z 365.4518. Compound 8a: (50 -O): Rf 0.38 (9:1 CHCl3–EtOH); 1H NMR: thymine: 11.24 (br s, 1H, NH), 7.56 (d, 1H, JH6,CH3 0.8 Hz, H6), 1.80 (br s, 3H, CH3); sugar: 6.16 (t, 1H, J 6.3 Hz, J 7.1 Hz, H10 ), 4.39 (dt, 1H, J 3.2 Hz, J 6.3 Hz, H40 ), 4.24 (m, 1H, H30 ), 3.67 (dd, 1H, J 3.4 Hz, J 10.5 Hz, H50 a), 3.60 (dd, 1H, J 4.0 Hz, J 10.5 Hz, H50 b), 2.19 (ddd, 1H, J 6.3 Hz, J 7.1 Hz, J 13.3 Hz, H20 a), 2.06 (ddd, 1H, J 3.2 Hz, J 6.3 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); propargyl: 4.25 (d, 1H, J 2.3 Hz, J 16.0 Hz, CH2), 4.20 (d, 1H, J 2.1 Hz, J 16.0 Hz, CH2), 3.50 (br t, 1H, J 2.2 Hz, C–H); 13 C NMR (DMSO-d6): thymine: 163.7 (C4), 150.4 (C2), 135.8 (C6), 109.6 (C5), 12.3 (CH3); sugar: 85.1 (C40 ), 83.9 (C10 ), 72.3 (C30 ), 69.1 (C50 ), 39.4 (C20 ); TBDMS: 25.7 (C–CH3), 17.7 (C–CH3), 4.9 (CH3a), 4.8 (CH3b); propargyl: 79.9 (C), 77.6 (CH), 57.9 (CH2); HRESIMS: calcd for C19H30N2O5SiNa [M+Na]+: m/z 417.1816, found: m/z 417.1823. Compound 8b: (50 -O): Rf 0.71 (9:1 CHCl3–EtOH); uracil: 11.31 (br s, 1H, NH), 7.69 (d, 1H, JH6,H5 8.1 Hz, H6), 5.62 (d, 1H, JH5,H6 8.1 Hz, H5); sugar: 6.14 (br t, 1H, J 6.5 Hz, H10 ), 4.38 (m, 1H, H30 ), 3.89 (m, 1H, H40 ), 3.64 (dd, 1H, J 3.7 Hz, J 10.6 Hz, H50 a), 3.59 (dd, 1H, J 4.4 Hz, J 10.6 Hz, H50 b), 2.19 (dt, 1H, J 6.5 Hz, J 13.1 Hz, H20 a), 2.09 (ddd, 1H, J 3.9 Hz, J 6.5 Hz, J 13.1 Hz, H20 b); TBDMS: 0.87 (s, 9H, C–CH3), 0.08 (s, 6H, Si–CH3); propargyl: 4.20 (br d, 1H, J 2.1 Hz, CH2), 3.50 (t, 1H, J 2.1 Hz, C–H); 13C NMR (DMSO-d6): uracil: 163.1 (C4), 150.4 (C2), 140.4 (C6), 101.9 (C5); sugar: 85.1 (C40 ), 84.1 (C10 ), 72.1 (C30 ), 68.9 (C50 ), 39.3 (C20 ); TBDMS: 25.7 (C–CH3), 17.7 (C–CH3), 4.9 (CH3a), 4.8 (CH3b), propargyl: 79.9 (C), 77.4 (CH), 55.8 (CH2); HRESIMS: calcd for C18H28N2O5SiNa [M+Na]+: m/z 403.1706, found: m/z 403.1711. Compound 9a: (3-N, 50 -O): Rf 0.73 (9:1 CHCl3–EtOH); 1H NMR: thymine: 7.66 (d, 1H, JH6,CH3 0.9 Hz, H6), 1.87 (d, 3H, JCH3,H6 0.9 Hz, CH3); sugar: 6.20 (br t, 1H, J 6.6 Hz, H10 ), 4.41 (dt, 1H, J 3.3 Hz, J 6.2 Hz, H30 ), 3.93 (m, 1H, H40 ), 3.69 (dd, 1H, J 4.0 Hz, J
10.6 Hz, H50 a), 3.62 (dd, 1H, J 4.1 Hz, J 10.6 Hz, H50 b), 2.23 (ddd, 1H, J 6.2 Hz, J 6.9 Hz, J 13.3 Hz, H20 a), 2.12 (ddd, 1H, J 3.3 Hz, J 6.4 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); N-propargyl: 4.55 (dd, 1H, J 2.4 Hz, J 16.8 Hz, CH2), 4.51 (dd, 1H, J 2.5 Hz, J 16.8 Hz, CH2), 3.10 (br t, 1H, J 2.4 Hz, C–H); Opropargyl: 4.25 (dd, 1H, J 2.3 Hz, J 15.9 Hz, CH2), 4.21 (dd, 1H, J 2.4 Hz, J 15.9 Hz, CH2), 3.51 (br t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSO-d6): thymine: 161.7 (C4), 149.7 (C2), 134.9 (C6), 108.7 (C5), 12.9 (CH3); sugar: 85.3 (C10 ), 85.0 (C40 ), 72.2 (C30 ), 68.9 (C50 ), 39.6 (C20 ); TBDMS: 25.6 (C–CH3), 17.6 (C–CH3), 5.0 (CH3a), 4.9 (CH3b); O-propargyl: 79.9 (C), 77.6 (CH), 57.9 (CH2); N-propargyl: 79.0 (C), 73.0 (CH), 30.0 (CH2); HRESIMS: calcd for C22H32N2O5SiNa [M+Na]+: m/z 455.1973, found: m/z 455.1977. Compound 9b: (3-N, 50 -O): Rf 0.73 (9:1 CHCl3–EtOH); 1H NMR: uracil: 7.92 (d, 1H, JH6,H5 8.2 Hz, H6), 5.83 (d, 1H, JH5,H6 8.2 Hz, H5); sugar: 6.17 (br t, 1H, J 6.7 Hz, H10 ), 4.42 (m, 1H, H30 ), 3.81 (m, 1H, H40 ), 3.71 (m, 1H, H50 a), 3.60 (m, 1H, H50 b), 2.21 (ddd, 1H, J 6.1 Hz, J 6.7 Hz, J 13.2 Hz, H20 a), 2.14 (ddd, 1H, J 3.7 Hz, J 6.7 Hz, J 13.2 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); N-propargyl: 4.52 (dd, 1H, J 2.3 Hz, J 16.8 Hz, CH2), 4.51 (dd, 1H, J 2.2 Hz, J 16.8 Hz, CH2), 3.11 (br t, 1H, J 2.4 Hz, C–H); O-propargyl: 4.21 (m, 2H, CH2), 3.51 (t, 1H, J 2.4 Hz, C–H); 13C NMR (DMSOd6): uracil: 160.9 (C4), 149.7 (C2), 139.2 (C6), 100.9 (C5); sugar: 85.5 (C10 ), 85.2 (C40 ), 78.9 (C30 ), 69.8 (C50 ), 39.7 (C20 ); TBDMS: 25.7 (C–CH3), 17.7 (C–CH3), 4.9 (CH3a), 4.8 (CH3b); O-propargyl: 78.9 (C), 77.7 (CH), 57.9 (CH2); N-propargyl: 78.9 (C), 73.0 (CH), 29.8 (CH2); HRESIMS: calcd for C21H30N2O5SiNa [M+Na]+: m/z 441.1816, found: m/z 441.1835. Compound 10: Rf 0.19 (9:1 EtOAc–EtOH); cytidine: 11.05 (s, 1H, NH), 8.40 (d, 1H, J 7.5 Hz, H6), 7.34 (d, J 7.5 Hz, H5); benzoyl: 8.01 (dd, 2H, J 7.3 Hz, J 1.1 Hz, H2–6), 7.62 (tt, 1H, J 7.3 Hz, J 1.1 Hz, H4), 7.52 (br t, 2H, J 7.7 Hz, H3–5); sugar: 6.15 (t, 1H, J 6.3 Hz, H10 ), 5.26 (d, 1H, J 4.1 Hz, 1H, O30 –H), 5.07 (t, 1H, J 5.0 Hz, J 6.0 Hz, O50 –H), 4.25 (m, 1H, H30 ), 3.89 (br dd, 1H, J 3.7 Hz, J 7.3 Hz, H50 b), 3.66 (br dt, 1H, J 4.5 Hz, J 11.8 Hz, H50 a), 3.60 (br dt, 1H, J 4.4 Hz, J 11.8 Hz, H50 b), 2.33 (ddd, 1H, J 3.9 Hz, J 6.3 Hz, J 13.2 Hz, H20 a), 2.07 (dt, 1H, J 6.3 Hz, J 13.2 Hz, H20 b); 13C NMR (DMSO-d6): thymine: 162.8 (C4), 154.2 (C2), 144.8 (C6), 96.0 (C5); benzoyl: 167.4 (CO), 133.1 (C1), 132.6 (C4), 128.3 (C2, C3, C5, C6); sugar: 87.9 (C40 ), 86.1 (C10 ), 69.8 (C30 ), 60.9 (C50 ), 40.2 (C20 ); ESIMS m/z 331.9 (MH+), 353.9 (MNa+), 685.0 (M2Na+). Compound 11: Rf 0.52 (9:1 EtOAc–EtOH); cytidine: 11.22 (s, 1H, NH), 8.31 (br s, 1H, H5), 7.36 (br d, J 6.8 Hz, H5); benzoyl: 8.00 (dd, 2H, J 1.2 Hz, J 8.5 Hz, H2–6), 7.62 (br t, 1H, J 7.5 Hz, H4), 7.51 (br t, 2H, J 7.5 Hz, H3–5); sugar: 6.13 (t, 1H, J 6.1 Hz, H10 ), 5.33 (d, 1H, J 4.4 Hz, 1H, O30 –H), 4.22 (m, 1H, H30 ), 3.93 (br dd, 1H, J 3.1 Hz, J 11.5 Hz, H50 a), 3.87 (dd, 1H, J 3.3 Hz, J 11.5 Hz, H50 b), 3.77 (dd, 1H, J 3.3 Hz, J 11.5 Hz, H50 b), 2.36 (ddd, 1H, J 4.8 Hz, J 6.1 Hz, J 13.3 Hz, H20 a), 2.07 (dt, 1H, J 6.1 Hz, J 13.3 Hz, H20 b); TBDMS: 0.88 (s, 9H, C–CH3), 0.09 (s, 6H, Si–CH3); 13C NMR (DMSO-d6): thymine: 162.9 (C4), 154.3 (C2), 144.4 (C6), 95.8 (C5); benzoyl: 167.3 (CO), 133.1 (C1), 132.7 (C4), 128.4 (C2, C3, C5, C6); sugar: 87.3 (C40 ), 86.2 (C10 ), 69.6 (C30 ), 62.6 (C50 ), 41.2 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3a), 5.5 (Si–CH3a), 5.6 (Si–CH3b); ESIMS m/z 446.5 (MH+), 468.4 (MNa+), 913.6 (M2Na+); HRESIMS: calcd for C22H31N3O5SiNa [M+Na]+: m/z 468.5733, found: m/z 468.5736. Compound 12: Rf 0.45 (9:1 EtOAc–MeOH); cytidine: 11.26 (s, 1H, NH), 8.28 (br s, 1H, H5), 7.36 (br d, J 7.4 Hz, H5); benzoyl: 8.01 (dd, 2H, J 7.2 Hz, J 1.2 Hz, H2–6), 7.62 (br t, 1H, J 7.4 Hz, H4), 7.51 (br t, 2H, J 7.6 Hz, H3–5); sugar: 6.09 (br t, 1H, J 6.4 Hz, H10 ), 4.27 (m, 1H, H30 ), 4.14 (br dd, J 3.1 Hz, J 6.2 Hz, H40 ), 3.87 (br dd, 1H, J 3.7 Hz, J 11.4 Hz, H50 a), 3.78 (br dt, 1H, J 3.3 Hz, J 11.4 Hz, H50 b), 2.54 (ddd, 1H, J 3.1 Hz, J 6.1 Hz, J 13.3 Hz, H20 a), 2.14 (dt, 1H, J 6.3 Hz, J 13.3 Hz, H20 b); TBDMS: 0.89 (s, 9H, C–CH3), 0.10 (s, 6H, Si–CH3a), 0.09 (s, 6H, Si–CH3a); propargyl: 4.23 (br d, 1H, J 2.3 Hz, J 18.0 Hz, CH2), 3.47 (t, 1H, J 2.3 Hz, C–H); 13C NMR (DMSO-d6):
R. Lucas et al. / Carbohydrate Research 345 (2010) 199–207
thymine: 163.0 (C4), 154.2 (C2), 144.3 (C6), 95.9 (C5); benzoyl: 167.3 (CO), 133.1 (C1), 132.7 (C4), 128.4 (C2, C3, C5, C6); sugar: 86.5 (C10 ), 84.5 (C40 ), 77.7 (C30 ), 62.8 (C50 ), 40.4 (C20 ); TBDMS: 25.7 (C–CH3), 17.9 (C–CH3), 5.6 (CH3a), 5.6 (CH3b); propargyl: 80.0 (C), 77.4 (CH), 55.9 (CH2); ESIMS m/z 484.4 (MH+), 506.4 (MNa+), 989.7 (M2Na+); HRESIMS: calcd for C22H31N3O5SiNa [M+Na]+: m/z 506.6213, found: m/z 506.6221. Acknowledgments Financial support from the MENRT and the ‘Conseil Régional du Limousin’ is gratefully acknowledged. The authors are indebted to Dr. Mircea Darabantu and Dr. Jean Debord for their advices. Supplementary data Supplementary data (spectroscopic data for compounds 2–12) associated with this article can be found, in the online version, at doi:10.1016/j.carres.2009.10.021.
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