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. Author manuscript; available in PMC: 2017 Feb 24.
Published in final edited form as: J Sulphur Chem. 2016 Feb 24;37(3):307–327. doi: 10.1080/17415993.2015.1137921

S-Ribosylhomocysteine Analogues Modified at the Ribosyl C-4 Position

Christiane Chbib 1, Adam J Sobczak 1, Mukesh Mudgal 1, Cesar Gonzalez 1, Daniel Lumpuy 1, Justyna Nagaj 1, Kamila Stokowa-Soltys 1, Stanislaw F Wnuk 1,*
PMCID: PMC4976779  NIHMSID: NIHMS802520  PMID: 27516805

Abstract

4-C-Alkyl/aryl-S-ribosylhomocysteine (SRH) analogues were prepared by coupling of homocysteine with 4-substituted ribofuranose derivatives. The diastereoselective incorporation of the methyl substituent into the 4 position of the ribose ring was accomplished by addition of methylmagnesium bromide to the protected ribitol-4-ulose yielding the 4-C-methylribitol in 85% yield as single 4R diastereomer. The 4-C hexyl, octyl, vinyl, and aryl ribitols were prepared analogously. Chelation controlled addition of a carbanion to ketones from the (Si-face) was responsible for the observed stereochemical outcome. Oxidation of the primary alcohol of the 4-C ribitols with the catalytic amount of tetrapropylammonium perruthenate in the presence of N-methylmorpholine N-oxide produced 4-C-alkylribono-1,4-lactones in high yields. Mesylation of the latter compounds at the 5-hydroxyl position and treatment with a protected homocysteine thiolate afforded protected 4-C-alkyl/aryl-SRH analogues as the lactones. Reduction with lithium triethylborohydride and successive global deprotections with TFA afforded 4-C-alkyl/aryl SRH analogues. These analogues might impede the S-ribosylhomocysteinase(LuxS)-catalyzed reaction by preventing β-elimination of a homocysteine molecule, and thus depleting the production of quorum sensing signaling molecule AI-2.

Keywords: Homocysteine, LuxS, S-Ribosylhomocysteine, ribonolactone, C-substituted ribose

1. Introduction

Quorum sensing (QS) is a process in which exchange of chemical signals enables bacterial population to take control of crucial functions in united communities for enhancement of symbiosis, virulence, and biofilm formation.[1–4] The interference in this chemical communication among bacteria could results in improving our control of bacterial infection. Numerous small and macromolecules that modulate QS pathways have been designed and synthesized.[5–8] The acetylhomoserine-based isothiocyanate and haloacetamide probes which covalently inhibit bacterial QS[9] and probes for signaling molecules which utilize “click chemistry”[10] have been developed.

The S-ribosylhomocysteinase (LuxS; EC 4.4.1.21) is a key enzyme in the biosynthetic pathway for conversion of S-ribosyl-L-homocysteine (SRH, 1; Figure 1) to homocysteine (Hcy) and 4,5-dihydroxy-2,3-pentadione (DPD), the precursor for the type II autoinducer (AI-2)[11] which mediates the interspecies QS among bacteria (see Figure 2). [12–14] Various SRH analogues has been designed as mechanistic probes and/or inhibitors of LuxS enzyme.[6] Among them, one of the most important are SRH analogues that target mechanistic steps of LuxS catalytic cycle by effecting initial ring opening step (e.g., 1-deoxy-SRH analog 2[15] and [4-aza]-3a[16] or 4-[thio]-SRH analogs 3b[17]; i.e., 1 → intermediate A, Figure 2) or one of the tautomerization/isomerization steps (A → B or B → C). These included substrates lacking enolizable hydroxyl group at C3 (e.g., 4; X = H or OMe),[18] including mechanistically significant C3 halogenated [3-Br or F]-SRH analogues 4.[19] Zhou and coworkers synthesized substrate analogue S-homoribosyl-L-cysteine 5 which was designed to prevent final mechanistic step of LuxS catalytic cycle.[15] Moreover, brominated furanone derivatives were found to modify LuxS selectively leading to the covalent inhibition.[20]

Figure 1.

Figure 1

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Selected LuxS inhibitors.

Figure 2.

Figure 2

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A plausible inhibition of LuxS enzyme by 4-C substituted SRH analogues.

The substitution of the hydrogen at C4 by an alkyl or aryl group in SRH (e.g., 18) should impede the LuxS-catalyzed reaction by preventing β-elimination of a homocysteine molecule (i.e., C → D) since abstraction of the C4-proton by a general base (e.g., Glu158) from the intermediate C, when R = alkyl/aryl, will be disallowed (Figure 2). Consequently, the formation of DPD necessary for the production of AI-2 would be depleted with 4-C-alkyl-SRH analogues.

Since LuxS forms a dimer it is possible that the size and chemical nature of the group incorporated at C4 of ribose ring might also play an additional role in inhibiting dimerization. Recently, the SRH analogous having the sterically-demanding alkyl or aryl group at the Hcy fragment of the SRH have been designed and were attempted to be synthesized.[21] These analogues were thought to be able to bind to one monomer of LuxS protein while blocking the correct association of the second monomer, possibly interfering with dimerization interfaces. [21–23] In theory, for example, the longer the alkyl chain incorporated at C4 position in analogs 18, the more potent inhibition of dimerization of LuxS might be observed since inhibitor can reach both homodimer parts of the protein. The inhibitor might also block one monomer leading to the alteration of the activity and as a consequence conformational changes of the second monomer. Herein, we report synthesis of [4-alkyl/aryl]-SRH analogues which would deplete the production of AI-2 by preventing elimination of Hcy and could also act as dimerization inhibitors.

2. Results and Discussion

The 4-C-alkyl/aryl-S-ribosylhomocysteine analogues were prepared by coupling of the homocysteine with the 4-C-substituted ribofuranose derivatives. The 4-C-substituted riboses can be prepared either by manipulation of natural carbohydrates[24] or chemoenzymatic strategy from non-sugar precursors.[25, 26] From the method available, we chose Maddaford’s method for diastereoselective incorporation of the alkyl substituent into the 4 position of D-ribose ring by addition of Grignard reagents to 4-ulose.[24] Reduction of the protected ribose 6 with NaBH4 provided the acyclic ribitol 7 (Scheme 1). Regioselective silylation of the primary hydroxyl with TBDMSCl and subsequent Dess-Martin or Collins oxidation of the secondary hydroxyl in 8 yielded ketone 9. The overall yield for the conversion of the ribose to the ribitol-4-ulose 9[27] was 75% (5 steps).

Scheme 1.

Scheme 1

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Reagents and conditions: (a) NaBH4/EtOH/H2O; (b) BDMSCI/imidazole/CH2Cl2/16 h; (c) Dess-Martin (3 h) or Collins (1 h) reagents; (d) RMgX/Et2O/−78°C; (e) TBAF/THF; (f) TPAP/NMO/CH2Cl2/6 h; (g) TFA/CH2Cl2/r.t.; and (h) LiEt3BH/CH2Cl2/0°C/0.5 h.

Treatment of ketone 9 with methylmagnesium bromide at −78 °C produced the 4-C-methylribitol 4R-10a in 85% yield as a single isomer after purification by silica gel chromatography. Addition of the hexylmagnesium bromide or octylmagnesium bromide to ketone 9 gave the corresponding 4-C-hexyl and 4-C-octyl ribitols 10b and 10c in 74% and 69% isolated yields, respectively. The 4-C-vinyl ribitol 10d (61%) and 4-C-aryl ribitol 10e (96%) were prepared analogously. The Grignard reagent addition to the ribitol-4-ulose 9, which is an α-alkoxy ketone, is proposed to proceed via a 5-membered ring chelate[28] with the complexation of the metal with the carbonyl group and the α-alkoxy group at C3 allowing transfer of the alkanide carbanion in the anti-fashion (Si-face) to the α-alkoxy group at C3.[24, 29]

Treatment of the 4-C-methylribitol 10a with TBAF (0 °C/30 min) effected desilylation to give ribitol 11a (78%). Analogous deprotection of 10b-e produced the 4-C-substituted ribitols 11b-e (75–87%) with a primary hydroxyl group at C1 and a tertiary hydroxyl group at C3. For the ring closure, we elected the oxidation of the primary alcohol at C1 to the carboxylic acid with the concomitant ring closure to the corresponding ribono-1,4-lactones,[30, 31] since such an approach would provide convenient precursors for the synthesis of both 4-C-substituted SRH lactones[32] and, after reduction, 4-C-alkyl/aryl SRH analogues. Thus, oxidation (6 h) of the 11a with a catalytic amount of tetrapropylammonium perruthenate (TPAP) in the presence of a stoichiometric amount of N-methylmorpholine N-oxide (NMO)[33, 34] gave the corresponding 4-C-methylribono-1,4-lactone 12a (80%). The formation of lactone was supported by disappearance of the signals for H1 and H1′ protons (1H NMR) and the appearance of a peak at 172.1 ppm for the carbonyl carbon at C1 (13C NMR). Oxidation of 4-C-hexyl and 4-C-octyl ribitols 11b or 11c with TPAP/NMO also proceeded smoothly to give the 4-C-hexyl- and 4-C-octylribonolactones 12b and 12c (95%). The longer reaction time (e.g., 14 h), especially with substrates with long alkyl chain (e.g., 11c), led to the formation of byproduct(s) (~30%). The 4-C-vinyl 12d and 4-C-aryl 12e lactones were obtained analogously by oxidation of 11d and 11e, respectively.

Detritylation of 12a (6 h, rt) with TFA/CH2Cl2 gave 13a (66%). Analogous treatment of the hexyl 12b and octyl 12c with TFA (5 h, rt) gave ribono-1,4-lactones 13b and 13c in 80% and 75% yields, respectively. The longer reaction time should be avoided since formation of byproduct(s) was observed during detritylation of 12b to 13b when reaction was carried out for 16 h.[35] Treatment of the vinyl analogue 12d with TFA (5 h, rt) also gave detritylated ribonolactone 13d but only in 35% yield after purification on silica gel column chromatography. Similarly, 4-C-aryl analogues 12e was converted to 13e (70%).

Careful reduction of the protected 4-C-hexyl ribonolactone 12b with LiEtBH3 (−20 °C, 30 min) gave lactol 14b as an anomeric mixture (α/β, 1:3) in 54% yield after column chromatography. The chemical shifts for the anomeric protons and the magnitude of vicinal 3JH1–H2 coupling constants were diagnostic[36, 37] for the determination of the composition of α/β anomers. The anomeric proton for α-isomer [5.61 ppm (dd, J = 4.2, 11.5 Hz)], appeared as doublet of doublets with splitting to H2 and OH group, while H1 proton for β-anomer appears upfield as doublet [5.15 (d, J = 8.4 Hz)].

Next, the coupling of the selected ribono-1,4-lactones 13 with homocysteine were examined. Thus, treatment of 13b, 13c, or 13e with methylsulfonyl chloride gave the primary 5-O-mesyl derivatives 15b, 15c, and 15e (60–83%). From different approaches[13, 15, 38–40] tested for the nucleophilic displacement of the mesylate in 15 with homocysteine thiolates, we found that reactions with the (N-Boc, COOtBu) protected homocysteine, generated in situ by the reduction of the corresponding homocystine[13] with water extractable tris(2-carboxyethyl)phosphine (TCEP),[18] gave the best results. Thus, treatment of the 15b with homocysteine thiolate (3 equiv) generated from BocNHCH(CHCHSH)CO2t-Bu/LDA afforded protected 4-C-hexyl-SRH lactone 16b (65%, Scheme 2). Analogous coupling of 15c with Hcy gave 16c, contaminated with the protected Hcy substrate (~1:1), which was directly used in the subsequent deprotection step. The 4-C-aryl mesylate 15e was coupled with homocysteine thiolate to give 16e (48%). It is noteworthy to add that displacement of the primary mesylate (or tosylate) from the highly branching ribonolactones 15 having trisubstituted carbon (C4) at the adjacent position need to be carried out with great caution to avoid formation of byproducts. The structure of one such byproduct isolated from the reaction of 15c with Hcy was tentatively assigned as the 2,3-O-isopropylidene-4-C-octyl-D-ribono-1,5-lactone (see note under the experimental procedure for 17c) based on the spectroscopic analysis (1H & 13C NMR and HRMS) and by comparison with the similar ribono-1,5-lactones.[41]

Scheme 2.

Scheme 2

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MsCl/TEA/CH2Cl2; (b) BocNHCH(CH2CH2SH)CO2t-Bu/LDA/DMF; (c) TFA/H2O; and (d) LiEt3BH/THF.

Treatment of 16b with TFA effected global removal of the N-Boc, acetonide and t-butyl ester protecting groups to give 4-C-hexyl-SRH lactone 17b in 55% yield after HPLC purification. Analogous treatment of the crude 16c with TFA and purification of the resulting mixture on Sep-Pak column gave 4-C-octyl-SRH lactone 17c in 21% overall yield from 15c. Deprotection of 16e gave 4-C-(4-methoxyphenyl)-SRH lactone 17e (75%).

Synthesis of the somehow unstable 4-C-alkyl/aryl-SRH derivatives 19 was accomplished by reduction of either protected 16 or deprotected 17 lactones with lithium triethylborohydride. Thus, treatment of 17c with LiEt3BH/THF (2 equiv.) in CH2Cl2 at −20 °C effected reduction of the lactone yielding 4-C-octyl SRH 19c (α/β, ~1:3; 60%). Alternatively, reduction of the protected 4-C-hexyl-SRH lactone 16b with LiEt3BH followed by deprotection of the resulting 18b with TFA and TFA/H2O afforded 4-C-hexyl-SRH 19b (α/β, 1:9; 75%). Similarly, subjection of 16e to the reduction and deprotection sequence afforded 4-C-(4-methoxyphenyl)-SRH 19e (α/β, 1:9; 77%).

3. Conclusion

We have developed synthesis of S-ribosylhomocysteine analogues substituted at the ribosyl C-4 position with alkyl or aryl group. The critical steps in this multistep synthesis starting from ribose were (i) diasteroselective addition of the alkyl/aryl-magnesium bromides to protected ribitol-4-ulose to produce the 4-C-alky/aryl-ribitols in high yields as single 4S diastereomers, (ii) oxidation of the primary alcohol at C1 of the 4-C substituted ribitols with the catalytic amount of tetrapropylammonium perruthenate in the presence of a stoichiometric amount of N-methylmorpholine N-oxide to give 4-C-alkyl/aryl-ribono-1,4-lactones in good yields, (iii) displacement of 5-mesylate with the protected homocysteine thiolate to afford protected 4-C-alkyl/aryl-SRH analogues with a lactone carbonyl at C1 position, and (iv) reduction with lithium triethylborohydride and successive global deprotections with TFA to give 4-C-alkyl/aryl-SRH analogues. Enzymatic and biological properties of these novel analogues of SRH will be published elsewhere.

4. Experimental Section

4.1. General procedures

The 1H (400 or 600 MHz) and 13C (100 MHz) NMR spectra were determined with solutions in CDCl3 unless otherwise noted. Mass spectra (MS) and HRMS were obtained in AP-ESI or TOF-ESI mode. TLC was performed with Merck kieselgel 60-F254 sheets products were detected with 254 nm light or by visualization with Ce(SO4)2/(NH4)6Mo7O24·4H2O/H2SO4/H2O reagent. Merck kieselgel 60 (230–400 mesh) was used for column chromatography. Final products were purified using HPLC [XTerra preparative RP18 OBD column (5μm 19 × 150 mm) with gradient program using CH3CN/H2O as a mobile phase] or Sep-Pak cartridge (C18 classic column) using water and ethanol as eluting system. Reagent grade chemicals were used, and solvents were dried by reflux over and distillation from CaH2 (except for THF/potassium) under argon. The 4-C-substituted SRH analogues need to be handled with care and store in refrigerator (~4 °C) in solid or dried oil state.

4.2. 2,3-O-Isopropylidene-5-O-tritylribitol (7)

NaBH4 (91 mg, 2.4 mmol) was added to a stirred solution of 6[42] (865 mg, 2.0 mmol) in EtOH (20 mL) at 0 °C (ice-bath) under N2 atmosphere. After 1 h, the reaction mixture was partitioned between NaHCO3/H2O and EtOAc. The organic layer was dried over anhydrous MgSO4 and evaporated. The residue was column chromatographed (30% hexane/EtOAc) to give 7[24] (807 mg, 93%): 1H NMR δ 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 2.96 (d, J = 3.6 Hz, 1H, OH), 3.08 (dd, J = 5.0, 8.4 Hz, 1H, H1), 3.34 (dd, J = 6.9, 9.8 Hz, 1H, H5), 3.50 (dd, J = 2.9, 9.8 Hz, 1H, H5′), 3.75–3.81 (m, 1H, H1′), 3.83–3.91 (m, 1H, H4), 4.10–4.17 (m, 1H, H2), 4.33–4.40 (m, 1H, H3), 7.25–7.38 (m, 15H, Ar); MS (ESI+) m/z 457 (M+Na+).

4.3. 1-O-tert-Butyldimethysilyl-2,3-O-isopropylidene-5-O-tritylribitol (8)

TBDMSCl (302 mg, 2.0 mmol) and imidazole (204 mg, 3.0 mmol) were added to a solution of 7 (652 mg, 1.5 mmol) in DMF (10 mL) at room temperature and stirring was continued for 72 h. The volatiles were evaporated and the residue was partitioned between saturated NH4Cl/H2O and EtOAc. The separated organic layer was then washed with NaHCO3/H2O, dried over Mg2SO4, evaporated and the resulting residue was column chromatographed (50% hexane/EtOAc) to give 8[24] (666 mg, 81%) as an amorphous white powder: 1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 3.20 (dd, J = 5.3, 9.7 Hz, 1H, H5), 3.25 (dd, J = 2.8, 9.7 Hz, 1H, H5′), 3.49 (dd, J = 4.1, 10.6 Hz, 1H, H1), 3.68 (dd, J = 8.7, 10.5 Hz, 1H, H1′), 3.79–3.81 (m, 1H, H4), 4.13–4.15 (m, 1H, H2), 4.22 (dd, J = 5.5, 9.2 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); MS (ESI+) m/z 571 (M+Na+).

4.4. 1-O-tert-Butyldimethysilyl-2,3-O-isopropylidene-5-O-tritylribitol-4-ulose (9)

Method A

Dess-Martin reagent (2.35 mL of 15 wt% solution/CH2Cl2; 381 mg, 0.9 mmol) was added to a solution of 8 (330 mg, 0.6 mmol) in CH2Cl2 (8 mL) at room temperature and stirred for 3 h. The reaction mixture was partitioned between saturated NaHCO3 (10 mL)/dilute Na2S2O3 (5 mL) and CH2Cl2 (15 mL). The organic layer was dried over anhydrous MgSO4 and evaporated. The residue was column chromatographed (85% hexane/EtOAc) to give 9[24] (300 mg, 91%) as an oil: 1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 3.68 (dd, J = 4.2, 11.2 Hz, 1H, H1), 3.76 (dd, J = 4.0, 11.2 Hz, 1H, H1′), 4.04 (d, J = 17.7 Hz, 1H, H5), 4.20 (d, J = 17.8 Hz, 1H, H5′), 4.51–4.53 (m, 1H, H2), 4.71 (d, J = 7.8 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); MS (ESI+) m/z 569 (M+Na+).

Method B

A freshly prepared solution of Collins reagent [CrO3 (144 mg, 1.44 mmol), pyridine (0.116 mL, 114 mg, 1.44 mmol), and Ac2O (0.272 mL, 294 mg, 2.88 mmol) in CH2Cl2 (2 mL)] was added to a stirred solution of 8 (200 mg, 0.36 mmol) in CH2Cl2 (8 mL) at ambient temperature. The resulting mixture was stirred for 1 h. and was immediately column chromatographed (EtOAc) to give 917 (185 mg, 93%) with spectra properties as above.

4.5. General Procedure for the synthesis of 4-C-substituted ribitols 10

RMgBr reagent (0.75 mmol) was added to a stirred solution of 9 (205 mg, 0.375 mmol) in anhydrous THF (5 mL) at −78 °C under N2 atmosphere. After 15 min, the reaction mixture was allowed to warm up to ambient temperature and was kept stirring for 2 h. The reaction was then quenched by the addition of MeOH (1 mL) and diluted with EtOAc (15 mL). The resulting mixture was washed with 0.1 N HCl and the organic layer dried over anhydrous MgSO4. Volatiles were evaporated and the crude residue was then purified by flash column chromatography (90% hexane/EtOAc).

4.5.1. 1-O-tert-Butyldimethysilyl-2,3-O-isopropylidene-4-C-methyl-5-O-tritylribitol (10a)

Treatment of 9 (205 mg, 0.375 mmol) with MeMgBr (1M/THF, 0.75 mL, 0.75 mmol), using procedure reported in section 4.5 gave 10a[24] (180 mg, 85%) as an clear oil: 1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 1.35 (s, 3H, CH3), 1.40 (s, 6H, 2 × CH3), 3.01 (d, J = 8.7 Hz, 1H, H5), 3.12 (d, J = 8.7 Hz, 1H, H5′), 3.25 (dd, J = 3.8, 10.9 Hz, 1H, H1), 3.70 (dd, J= 3.8, 10.9 Hz, 1H, H1′), 3.90–3.95 (m, 1H, H2), 4.40 (d, J = 5.5 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); HRMS calcd for C34H46O5SiNa+ [M+Na]+ 585.3007, found 585.3009.

4.5.2. 1-O-tert-Butyldimethysilyl-4-C-hexyl-2,3-O-isopropylidene-5-O-tritylribitol (10b)

Treatment of 9 (165 mg, 0.30 mmol) with n-C6H13MgBr (0.8 M/THF; 0.75 mL, 0.6 mmol), using procedure reported in section 4.5 (flash column chromatography; 80% hexane/EtOAc) gave 10b (140 mg, 74%) as a clear oil: 1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 0.89 (t, J = 6.6 Hz, 3H, H6a), 1.30–1.40 (m, 8H, H2a–H5a), 1.40 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 3.06 (d, J = 8.9 Hz, 1H, H5), 3.22 (d, J = 8.9 Hz, 1H, H5′), 3.28 (dd, J = 3.8, 11.1 Hz, 1H, H1), 3.72 (dd, J = 7.6, 11.0 Hz, 1H, H1′), 3.80–3.85 (m, 1H, H2), 4.40 (d, J = 5.2 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); HRMS calcd for C39H56O5SiNa+ [M+Na]+ 655.3789, found 655.3799.

4.5.3. 1-O-tert-Butyldimethysilyl-2,3-O-isopropylidene-4-C-octyl-5-O-tritylribitol (10c)

Treatment of 9 (480 mg, 0.87 mmol) with n-C8H17MgBr (2 M/THF; 0.87 mL, 1.74 mmol), using procedure reported in section 4.5 gave 10c (402 mg, 69%) as a clear oil: 1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 0.89 (t, J = 6.6 Hz, 3H, H8a), 1.28–1.30 (m, 12H, H2a-H7a), 1.38 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 3.05 (d, J = 8.9 Hz, 1H, H5), 3.21 (d, J = 8.9 Hz, 1H, H5′), 3.26 (dd, J = 3.8, 11.0 Hz, 1H, H1), 3.70 (dd, J = 3.9, 11.0 Hz, 1H, H1′), 3.80–3.85 (m, 1H, H2), 4.40 (d, J = 5.2 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); HRMS calcd for C41H60O5SiNa+ [M+Na]+ 683.4102, found 683.4128.

4.5.4. 1-O-tert-Butyldimethysilyl-2,3-O-isopropylidene-5-O-trityl-4-C-vinylribitol (10d)

Treatment of 9 (120 mg, 0.21 mmol) with vinyl-MgBr (1 M/THF; 0.42 mL, 0.42 mmol), using procedure reported in section 4.5 gave 10d (78 mg, 61%) as a clear oil: 1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 2.92 (d, J = 9.0 Hz, 1H, H5), 3.12 (d, J = 9.0 Hz, 1H, H5′), 3.42 (dd, J = 4.5, 10.7 Hz, 1H, H1), 3.80 (dd, J = 8.5, 10.7 Hz, 1H, H1′), 4.00–4.13 (m, 1H, H2), 4.69 (d, J = 6.1 Hz, 1H, H3), 5.16 (dd, J = 1.7, 10.9 Hz, 1H, CH=CHH), 5.40 (dd, J = 1.8, 17.4 Hz, 1H, CH=CHH), 6.20 (dd, J = 10.9, 17.4 Hz, 1H, CH=CHH), 7.25–7.38 (m, 15H, Ar); 13C NMR δ −5.40 (SiMe2), 21.02 & 25.29 (CMe2), 25.77 (SiCMe3), 31.58 (SiCMe3), 60.47 (CPh3), 61.98 (C1), 69.13 (C5), 74.87 (C4), 78.15 (C2), 78.45 (C3), 107.87 (CH=CH2), 114.84 (CMe2), 126.91 & 127.71 & 128.87 & 143.40 (Ar), 146.89 (CH=CH2); HRMS calcd for C35H46O5SiNa+ [M+Na]+ 597.3007; found 597.3006.

4.5.5. 1-O-tert-Butyldimethysilyl-2,3-O-isopropylidene-4-C-4-methoxyphenyl-5-O-tritylribitol (10e)

Treatment of 9 (165 mg, 0.30 mmol) with 4-MeOC6H4MgBr (1 M/THF; 0.60 mL, 0.60 mmol) using procedure reported in section 4.5 gave 10e (190 mg, 96%) as a clear oil:1H NMR δ 0.08 (s, 6H, SiMe2), 0.81 (s, 9H, t-Bu), 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 3.01 (d, J = 9.1 Hz, 1H, H5), 3.15 (d, J = 9.1 Hz, 1H, H5′), 3.41 (dd, J = 4.5, 10.8 Hz, 1H, H1), 3.80 (s, 3H, CH3O), 3.85 (dd, J = 4.5, 10.8 Hz, 1H, H1′), 4.19–4.22 (m, 1H, H2), 5.05 (d, J = 6.4 Hz, 1H, H3), 6.85 (d, J = 6.9 Hz, 2H, Ar), 7.25–7.38 (m, 15H, Ar), 7.61 (d, J = 8.9 Hz, 2H, Ar); HRMS calcd for C40H50O6SiNa+ [M+Na]+ 677.3269, found 677.32567.

4.6. General procedure for desilylation of 4-C-substituted ribitols

TBAF (1 M/THF; 0.4 mL, 0.4 mmol) was added to a stirred solution of 10 (0.33 mmol) in THF (6 mL) at 0 °C (ice-bath). After 1 h, the volatiles were evaporated and the resulting residue was washed with NaHCO3/H2O and extracted with EtOAc. The organic layer was then dried over Mg2SO4 and evaporated to give crude residue which was column chromatographed (80 → 70% hexane/EtOAc).

4.6.1. 2,3-O-Isopropylidene-4-C-methyl-5-O-tritylribitol (11a)

Treatment of 10a (189 mg, 0.33 mmol) with TBAF using procedure reported in section 4.6 gave 11a (118 mg, 78%): 1H NMR δ 1.35 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.49 (s, 3H, CH3), 3.05 (d, J = 9.0 Hz, 1H, H5), 3.32 (d, J = 9.0 Hz, 1H, H5′), 3.58 (dd, J = 5.2, 12.0 Hz, 1H, H1), 3.75 (dd, J = 5.5, 12 Hz, 1H, H1′), 4.10–4.20 (m, 1H, H2), 4.30 (d, J = 6.2 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 14.21 (C1a), 25.17 & 27.28 (CMe2), 60.42 (C5), 61.43 (C1), 67.95 (CPh3), 68.38 (C4), 77.61 (C2), 79.10 (C3), 107.67 (CMe2), 127.27 & 127.97 & 128.62 & 143.43 (Ar); HRMS calcd for C28H32O5Na+ [M+Na]+ 471.2142; found 471.2158.

4.6.2. 2,3-O-Isopropylidene-4-C-hexyl-5-O-tritylribitol (11b)

Treatment of 10b (280 mg, 0.44 mmol) with TBAF using procedure reported in section 4.6 gave 11b (145 mg, 87%) as an viscous oil: 1H NMR δ 0.85 (t, J = 6.6 Hz, 3H, H6a), 1.30–1.40 (m, 8H, H2a–H5a), 1.25 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 2.91 (d, J = 9.3 Hz, 1H, H5), 3.20 (d, J = 9.3 Hz, 1H, H5′), 3.30 (dd, J = 5.0, 12.2 Hz, 1H, H1), 3.40 (dd, J = 5.0, 12.0 Hz, 1H, H1′), 3.80 (q, J = 5.8 Hz, 1H, H2), 4.15 (d, J = 5.8 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 14.14 (C6a), 22.61, 23.06, 29.74, 31.83 (C2a–C5a), 25.45 & 27.57 (CMe2), 36.05 (C1a), 61.88 (C1), 64.11 (C5), 74.35 (CPh3), 77.55 (C4), 79.14 (C2), 86.92 (C3), 107.21 (CMe2), 127.31 & 127.96 & 128.65 & 143.37 (Ar); HRMS calcd for C33H42O5Na+ [M+Na]+ 541.2924; found 541.2924.

4.6.3. 2,3-O-Isopropylidene-4-C-octyl-5-O-tritylribitol (11c)

Treatment of 10c (350 mg, 0.52 mmol) with TBAF using procedure reported in section 4.6 gave 11c (200 mg, 84%) as an viscous oil. 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H8a), 1.20–1.30 (m, 12H, H2a-H7a), 1.29 (s, 3H, CH3), 1.40 (s, CH3), 1.50–1.60 (m, 2H, H1a), 2.95 (d, J = 9.4 Hz, 1H, H5), 3.25 (d, J = 9.3 Hz, 1H, H5′), 3.35 (dd, J = 4.92, 12.2 Hz, 1H, H1), 3.45 (dd, J = 5.4, 12.2 Hz, 1H, H1′), 3.87–3.94 (m, 1H, H2), 4.10 (d, J = 5.8 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 14.24 (C8a), 22.79, 23.17, 29.35, 29.68, 30.18, 32.00 (C2a–C7a), 25.54 & 27.65 (CMe2), 36.13 (C1a), 61.94 (C1), 64.27 (C5), 74.41 (CPh3), 77.65 (C4), 79.28 (C2), 87.00 (C3), 107.27 (CMe2), 127.37, 128.03, 128.75 & 143.49 (Ar); HRMS calcd for C35H46O5Na+ [M+Na]+ 569.3237; found 569.3237.

4.6.4. 2,3-O-Isopropylidene-4-C-vinyl-5-O-tritylribitol (11d)

Treatment of 10d (200 mg, 0.34 mmol) with TBAF using procedure reported in section 4.6 gave 11d (124 mg, 77%) as a viscous oil: 1H NMR δ 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 3.38 (d, J = 11.2 Hz, 1H, H5), 3.54 (d, J = 11.2 Hz, 1H, H5′), 3.58 (dd, J = 5.1, 11.3 Hz, 1H, H1), 3.90 (d, J = 8.6, 11.3 Hz, 1H, H1′), 4.25–4.30 (m, 1H, H2), 4.32 (d, J = 6.5 Hz, 1H, H3), 5.20 (dd, J = 1.2, 11.0 Hz, 1H, CH=CHH), 5.40 (dd, J = 1.5, 17.6 Hz, 1H, CH=CHH), 6.20 (dd, J = 11.0, 17.5 Hz, 1H, CH=CHH), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 24.65 & 27.07 (CMe2), 60.29 (CPh3), 60.62 (C1), 68.07 (C5), 77.90 (C2), 78.10 (C4), 82.01 (C3), 115.48 (CMe2), 116.03 (CH=CH2), 126.82 & 127.65 & 128.75 & 143.28 (Ar), 146.88 (CH=CH2); HRMS calcd for C29H32O5Na+ [M+Na]+ 483.2142, found 483.2131.

4.6.5. 2,3-O-Isopropylidene-4-C-4-methoxyphenyl-5-O-tritylribitol (11e)

Treatment of 10e (190 mg, 0.29 mmol) with TBAF using procedure reported in section 4.6 gave 11e (117 mg, 75%) as a viscous oil: 1H NMR δ 1.35 (s, 3H, CH3), 1.37 (s, 3H, CH3), 3.01 (dd, J = 6.0, 11.7 Hz, 1H, H1), 3.03 (d, J = 9.0 Hz, 1H, H5), 3.23 (dd, J = 6.0, 11.7 Hz, 1H, H1′), 3.45 (d, J = 9.0 Hz, 1H, H5′), 3.72 (s, 3H, CH3O), 4.19–4.22 (m, 1H, H2), 4.75 (d, J = 6.6 Hz, 1H, H3), 6.85 (d, J = 6.9 Hz, 2H, Ar), 7.61 (d, J = 8.9 Hz, 2H, Ar), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 24.58 & 27.02 (CMe2), 55.19 (CH3O), 60.04 (CPh3), 61.28 (C1), 69.73 (C5), 78.08 (C2), 79.05 (C3), 82.02 (C4), 113.48 (CMe2), 127.25, 127.37, 127.92, 159.10 (Ar), 128.57 & 129.68 & 132.91 & 146.88 (Ar); HRMS calcd for C34H36O6Na+ [M+Na]+ 563.2404; found 563.2386.

4.7. General procedure for the synthesis of 4-C-substituted ribono-1,4-lactones 12

N-Methylmorpholine N-oxide (NMO; 50 mg, 0.42 mmol), tetrapropylammonium perruthenate (TPAP; 1 mg, 0.002 mmol) and were added to a stirred solution of 11 (0.11 mmol) in CH2Cl2 (3.5 mL) at ambient temperature under N2 atmosphere. After 6 h, the reaction mixture was filtered off and the filtrate was dried over MgSO4 and evaporated. The residue was purified by flash column chromatography (75→50% hexane/EtOAc) to give 12.

Note: Treatment of 11 with NMO and TPAP, as described above, in the presence of 4Å molecular sieves (100 mg) also gave lactones 12 in similar yields.

4.7.1. 2,3-O-Isopropylidene-4-C-methyl-5-O-trityl-D-ribono-1,4-lactone (12a)

Treatment of 11a (51 mg, 0.11 mmol) with NMO/TPAP using procedure reported in section 4.7 gave 12a (40 mg, 80%): 1H NMR δ 1.30 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.40 (s, 3H, CH3), 2.91 (d, J = 10.2 Hz, 1H, H5), 3.50 (d, J = 10.2 Hz, H1, H5′), 4.20 (d, J = 5.6 Hz, 1H, H2), 5.01 (d, J = 5.6 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 16.40 (C1a) 25.90 & 26.78 (CMe2), 66.65 (CPh3), 67.63 (C5), 77.70 (C2), 79.77 (C3), 88.56 (C4), 126.07 (CMe2), 127.46 & 128.25 & 128.67 & 146.87 (Ar), 172.07 (C1); HRMS calcd for C28H28O5Na+ [M+Na]+ 467.1829, found 467.1847.

4.7.2. 2,3-O-Isopropylidene-4-C-hexyl-5-O-trityl-D-ribono-1,4-lactone (12b)

Treatment of 11b (37 mg, 0.09 mmol) with NMO/TPAP using procedure reported in section 4.7 gave 12b (35 mg, 94%): 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.19–1.21 (m, 8H, H2a–H5a), 1.24 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 2.85 (d, J =10.2 Hz, 1H, H5), 3.51 (d, J = 10.2 Hz, 1H, H5′), 4.10 (d, J = 5.6 Hz, 1H, H2), 5.01 (d, J = 5.6 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 13.98 (C6a), 22.45, 23.33, 29.42, 31.30 (C2a–C5a), 25.91 & 26.76 (CMe2), 31.60 (C1a), 66.29 (C5), 77.57 (C3), 80.11 (C2), 87.75 (C4), 88.26 (CPh3), 112.92 (CMe2), 127.41, 128.16, 128.52 & 142.95 (Ar), 174.41 (C1); MS (ESI+) m/z 532 (M+NH4)+.

4.7.3. 2,3-O-Isopropylidene-4-C-octyl-5-O-trityl-D-ribono-1,4-lactone (12c)

Treatment of 11c (48 mg, 0.08 mmol) with NMO/TPAP using procedure reported in section 4.7 gave 12c (32 mg, 95%): 1H NMR δ 0.80–0.84 (m, 3H, H8a), 1.10–1.20 (s, 12H, H2a-H7a), 1.25 (s, CH3), 1.40 (s, CH3), 1.50–1.60 (m, 2H, H1a), 2.90 (d, J = 10.1 Hz, 1H, H5), 3.55 (d, J = 10.2 Hz, 1H, H5′), 4.10 (d, J = 5.6 Hz, 1H, H2), 5.01 (d, J = 5.6 Hz, 1H, H3), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 14.23 (C8a), 22.77, 23.50, 29.24, 29.29, 30.06, 31.72 (C2a–C7a), 26.06 & 26.92 (CMe2), 31.93 (C1a), 66.39 (C5), 77.57 (C3), 80.11 (C2), 87.94 (C4), 88.38 (CPh3), 113.07 (CMe2), 127.56, 128.31, 128.65, 143.07 (Ar), 174.63 (C1); MS (ESI+) m/z 560 (M+NH4)+.

Note: When oxidation of 11c was carried out overnight instead of 6 h, a second minor product (~30%) was formed in addition to 12c. The minor product had the following ribosyl peaks: 1H NMR δ 3.75 (d, J = 11.6 Hz, 1H), 3.91 (d, J = 11.6 Hz, 1H), 4.60 (d, J = 5.6 Hz, 1H), 4.90 (d, J = 5.6 Hz, 1H).

4.7.4. 2,3-O-Isopropylidene-5-O-trityl-4-C-vinyl-D-ribono-1,4-lactone (12d)

Treatment of 11d (100 mg, 0.21 mmol) with NMO/TPAP using procedure reported in section 4.7 gave 12d (90 mg, 95%): 1H NMR δ 1.22 (s, 3H, CH3), 1.26 (s, 3H, CH3), 2.91 (d, J = 10.2 Hz, 1H, H5), 3.50 (d, J = 10.3 Hz, H1, H5′), 4.20 (d, J = 5.6 Hz, 1H, H2), 5.00 (d, J = 5.6 Hz, 1H, H3), 5.21 (d, J = 11.2 Hz, 1H, CH=CHH), 5.34 (d, J = 17.4 Hz, 1H, CHH), 5.61 (dd, J = 11.2, 17.4 Hz, 1H, CH=CHH), 7.25–7.38 (m, 15 H); 13C NMR δ 25.92 & 26.64 (CMe2), 53.50 (CPh3), 65.50 (C5), 80.01 (C2), 82.03 (C3), 88.87 (C4), 112.03 (CH=CH2), 118.50 (CMe2), 127.29, 127.95, 129.69 & 145.01 (Ar), 146.85 (CH=CH2), 174.14 (C1); HRMS (TOF) m/z calcd for C29H28O5Na+ [M+Na]+ 479.1829; found 479.1829.

4.7.5. 2,3-O-Isopropylidene-4-C-4-methoxyphenyl-5-O-trityl-D-ribono-1,4-lactone (12e)

Treatment of 11e (90 mg, 0.16 mmol) with NMO/TPAP using procedure reported in section 4.7 gave 12e (73 mg, 82%): 1H NMR δ 1.22 (s, 3H, CH3), 1.25 (s, 3H, CH3), 3.25 (d, J = 10.5 Hz, 1H, H5′), 3.35 (d, J = 10.5 Hz, 1H, H5), 3.80 (s, 3H, CH3O), 4.48 (d, J = 5.5 Hz, 1H, H2), 5.20 (d, J = 5.5 Hz, 1H, H3), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.7 Hz, 2H, Ar), 7.25–7.38 (m, 15H, Ar); 13C NMR δ 25.92 & 26.64 (CMe2), 55.18 (CH3O), 62.94 (CPh3), 68.79 (C5), 77.71 (C2), 80.77 (C3), 88.87 (C4), 113.60 (CMe2), 126.9, 127.25, 128.75 & 159.10, (Ar), 128.57, 129.68, 132.91 & 146.40 (Ar), 174.14 (C1); MS (ESI) m/z 554 (M+NH4)+.

4.8. General procedure for detritylation of 4-C-substituted ribono-1,4-lactones

TFA (0.5 mL) and CH2Cl2 (5 mL) were added to a stirred solution of 12 (0.1 mmol) at ambient temperature for 6 h. The volatiles were evaporated and residue co-evaporated with toluene. The oily residue was partitioned between aqueous NaHCO3 and CH2Cl2. The separated organic layer was washed with brine, dried (MgSO4), evaporated and was purified on silica gel column (hexane/EtOAc, 8:2).

4.8.1. 2,3-O-Isopropylidene-4-C-methyl-D-ribono-1,4-lactone (13a)

Treatment of 12a (44 mg, 0.1 mmol) with TFA using procedure reported in section 4.8 gave 13a (13 mg, 66%): 1H NMR δ 1.32 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.42 (s, 3H, CH3), 3.60 (d, J = 11.6 Hz, 1H, H5), 3.71 (d, J = 11.6 Hz, H1, H5′), 4.55 (d, J = 5.6 Hz, 1H, H2), 4.90 (d, J = 5.6 Hz, 1H, H3); 13C NMR δ 16.42 (C1a), 25.81 & 26.76 (CMe2), 67.61 (C5), 77.82 (C2), 80.01 (C3), 86.35 (C4), 113.02 (CMe2), 174.46 (C1); HRMS (TOF) m/z calcd for C9H14O5Na+ [M+Na]+ 225.0733; found 225.0734.

4.8.2. 2,3-O-Isopropylidene-4-C-hexyl-D-ribono-1,4-lactone (13b)

Treatment of 12b (31 mg, 0.06 mmol) with TFA using procedure reported in section 4.8 (flash column chromatography; 80% hexane/EtOAc) gave 13b (12.5 mg, 80%): 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.19–1.21 (m, 8H, H2a–H5a), 1.41 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 3.75 (d, J = 11.8 Hz, 1H, H5), 3.85 (d, J = 11.4 Hz, 1H, H5′), 4.65 (d, J = 5.6 Hz, 1H, H2), 4.96 (d, J = 5.6 Hz, 1H, H3); 13C NMR δ 14.06 (C6a), 22.47, 23.54, 29.42, 31.48 (C2a–C5a), 25.79 & 26.78 (CMe2), 31.91 (C1a), 65.83 (C5), 77.20 (C3), 80.63 (C2), 89.73 (C4), 112.85 (CMe2), 172.67 (C1); HRMS (TOF) m/z calcd for C14H24O5Na+ [M+Na]+ 295.1516, found 295.1516.

4.8.3. 2,3-O-Isopropylidene-4-C-octyl-D-ribono-1,4-lactone (13c)

Treatment of 12c (35 mg, 0.065 mmol) with TFA using procedure reported in section 4.8 gave 13c (16 mg, 75%): 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H8a), 1.30–1.32 (m, 12H, H2a-H7a), 1.40 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 3.75 (d, J = 11.6 Hz, 1H, H5), 3.85 (d, J = 11.6 Hz, 1H, H5′), 4.65 (d, J = 5.6 Hz, 1H, H2), 4.95 (d, J = 5.6 Hz, 1H, H3); 13C NMR δ 14.25 (CH3, C8a), 22.74, 23.64, 29.30, 29.41, 30.21, 31.27 (C2a–C7a), 25.93 & 26.9 (CMe2), 31.98 (C1a), 65.92 (C5), 77.46 (C3), 79.91 (C2), 88.94 (C4), 113.0 (CMe2), 174.98 (C1); HRMS (TOF) m/z calcd for C16H28O5Na+ [M+Na]+ 323.1829; found 323.1915.

4.8.4. 2,3-O-Isopropylidene-4-C-vinyl-D-ribono-1,4-lactone (13d)

Treatment of 12d (30 mg, 0.065 mmol) with TFA using procedure reported in section 4.8 (flash column chromatography; 80% hexane/EtOAc) gave somehow an unstable product 13d (5 mg, 35%): 1H NMR δ 1.22 (s, 3H, CH3), 1.26 (s, 3H, CH3), 3.61 (d, J = 11.7 Hz, 1H, H5), 3.65 (d, J = 11.8 Hz, 1H, H5′), 4.60 (d, J = 5.6 Hz, 1H, H2), 4.82 (d, J = 5.6 Hz, 1H, H3), 5.24 (d, J = 11.3 Hz, 1H, CH=CHH), 5.34 (d, J = 17.5 Hz, 1H, C=CHH), 5.71 (dd, J = 11.2, 17.5 Hz, 1H, CH=CHH); 13C NMR δ 25.87, 26.70 (CMe2), 66.37 (C5), 76.70 (C2), 80.01 (C3), 87.95 (C4), 113.29 (CMe2), 118.21 (CH=CH2), 130.53 (CH=CH2), 174.06 (C1); HRMS (TOF) m/z calcd for C10H14O5Na+ [M+Na]+ 237.0733; found 237.0733.

4.8.5. 2,3-O-Isopropylidene-4-C-4-methoxyphenyl-D-ribono-1,4-lactone (13e)

Treatment of 12e (70 mg, 0.13 mmol) with TFA using procedure reported in section 4.8 (flash column chromatography; 80% hexane/EtOAc) gave 13e (23 mg, 70%): 1H NMR δ 1.22 (s, 3H, CH3), 1.25 (s, 3H, CH3), 3.80 (s, 3H, CH3O), 3.85 (d, J = 12.3 Hz, 1H, H5), 3.95 (d, J = 12.5 Hz, 1H, H5′), 4.95 (d, J = 5.4 Hz, 1H, H2), 5.15 (d, J = 5.3 Hz, 1H, H3), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.0 Hz, 2H, Ar); 13C NMR δ 25.83 & 26.65 (CMe2), 55.25 (CH3O), 68.50 (C5), 77.41 (C2), 80.57 (C3), 90.12 (C4), 112.93 (CMe2), 113.76, 126.17, 126.89, 159.33 (Ar); 174.86 (C1); HRMS (TOF) m/z calcd for C15H18O6Na+ [M+Na]+ 317.0996; found 317.0982.

4.9. 2,3-O-Isopropylidene-4-C-hexyl-5-O-trityl-D-ribofuranose (14b)

LiEt3BH (1M/THF, 0.18 mL, 0.18 mmol) was added dropwise to a solution of 12b (36 mg, 0.07 mmol) in CH2Cl2 (0.5 mL) and the resulting mixture was stirred for 30 min at −20 °C under N2 atmosphere. The reaction was quenched with MeOH and the volatiles were evaporated. The residue was partitioned between CH2Cl2 and NaHCO3, washed with brine and dried with anhydrous MgSO4. The resulting oil was column chromatographed (75:15, hexane/EtOAc) to give 14b (α/β; 1:3; 20 mg, 54%). The major β-anomer had: 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.33 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 3.16 (d, J = 10.1 Hz, 1H, H5), 3.32 (d, J = 10.1 Hz, 1H, H5′), 3.74 (d, J = 8.8 Hz, OH), 4.50 (d, J = 6.0 Hz, 1H, H3), 4.75 (d, J = 6.0 Hz, 1H, H2), 5.15 (d, J = 8.4 Hz, 1H, H1). 7.25–7.38 (m, 15H, Ar). The minor α-anomer had: 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.33 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 2.94 (d, J = 9.9 Hz, 1H, H5), 3.25 (d, J = 9.9 Hz, 1H, H5′), 3.86 (d, J = 11.5 Hz, OH), 4.20 (d, J = 6.1 Hz, 1H, H3), 4.62 (dd, J = 4.2, 6.1 Hz, 1H, H2), 5.61 (dd, J = 4.2, 11.5 Hz, 1H, H1), 7.25–7.38 (m, 15H, Ar). 13C NMR for the mixture of α/β-anomers: δ 14.01 (C6a), 23.61 & 23.94 (CMe2), 24.57 & 24.98 (CMe2), 25.99, 26.22, 29.65, 31.51 (C2a–C5a), 31.83 (C1a), 67.33 & 68.18 (C5), 80.01 & 82.78 (C2), 83.09 & 83.82 (C3), 88.05 & 88.12 (C4), 96.55 & 102.85 (C1), 112.06 & 112.32 (CMe2), 127.22, 127.45, 127.97, 128.05, 128.65, 128.76, 142.90, 143.49 (Ar); HRMS (TOF-ESI) m/z calcd for C33H40O5Na+ [M+Na]+ 539.2768; found 539.2789.

4.10. General procedure for the synthesis of 5-O-mesyl-4-C-substituted ribono-1,4-lactones 15

TEA (48 μL, 34 mg, 0.33 mmol) and MsCl (11.4 μL, 19.5 mg, 0.15 mmol) were added to a stirred solution of 13 (0.1 mmol) in dry CH2Cl2 (2 mL) at 0 °C (ice-bath) under N2 atmosphere. After 1 h, the reaction mixture was partitioned between CH2Cl2 and diluted HCl. The separated organic layer was washed with aqueous solution of NaHCO3, brine and dried over anhydrous MgSO4. Volatiles were evaporated and the residue was purified on silica column chromatography (hexane/EtOAc, 6:4).

4.10.1. 2,3-O-Isopropylidene-4-C-hexyl-5-O-mesyl-D-ribono-1,4-lactone (15b)

Treatment of 13b (27 mg, 0.1 mmol) with MsCl using procedure reported in section 4.10 gave 15b (21 mg, 60%): 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.33 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 2.99 (s, 3H, Ms), 4.20 (d, J = 11.0 Hz, 1H, H5), 4.32 (d, J = 11.0 Hz, 1H, H5′), 4.60 (d, J = 5.7 Hz, 1H, H2), 4.88 (d, J = 5.7 Hz, 1H, H3); 13C NMR δ 14.03 (CH3, C6a), 22.46, 23.36, 29.55, 31.38 (C2a–C5a), 25.78 & 26.69 (CMe2), 31.45 (C1a), 37.62 (Ms), 71.80 (C5), 76.70 (C3), 78.91 (C2), 85.77 (C4), 113.79 (CMe2), 173.21 (C1); HRMS (TOF-ESI) m/z calcd for C15H26O7SNa+ [M+Na]+ 373.1291; found 373.1307.

4.10.2. 2,3-O-Isopropylidene-5-O-mesyl-4-C-octyl-D-ribono-1,4-lactone (15c)

Treatment of 13c (32 mg, 0.1 mmol) with MsCl using procedure reported in section 4.10 gave 15c (19 mg, 68%): 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H8a), 1.20–1.28 (m, 12H, H2a-H7a), 1.35 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 3.05 (s, 3H, Ms), 4.20 (d, J = 10.9 Hz, 1H, H5), 4.32 (d, J = 10.9 Hz, 1H, H5′), 4.60 (d, J = 5.6 Hz, 1H, H2), 4.84 (d, J = 5.6 Hz, 1H, H3); 13C NMR δ 14.24 (CH3, C8a), 22.78, 23.52, 29.22, 29.30, 30.02, 31.58 (C2a–C7a), 25.92 & 26.82 (CMe2), 31.94 (C1a), 37.76 (Ms), 71.93 (C5), 76.88 (C3), 79.05 (C2), 85.91 (C4), 113.92 (CMe2), 173.34 (C1); MS (ESI) m/z 401 (M+Na)+.

4.10.3. 2,3-O-Isopropylidene-5-O-mesyl-4-C-4-methoxyphenyl-D-ribono-1,4-lactone (15e)

Treatment of 13e (24 mg, 0.08 mmol) with MsCl using procedure reported in section 4.10 gave 15e (25 mg, 83%): 1H NMR δ 1.35 (s, 3H, CH3), 1.41 (s, 3H, CH3), 3.0 (s, 3H, Ms), 3.80 (s, 3H, CH3O), 4.20 (d, J = 11.2 Hz, 1H, H5), 4.55 (d, J = 11.2 Hz, 1H, H5′), 5.01 (d, J = 5.5 Hz, 1H, H2), 5.15 (d, J = 5.5 Hz, 1H, H3), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.0 Hz, 2H, Ar); 13C NMR δ 25.90 & 26.58 (CMe2), 44.10 (Ms), 55.33 (CH3O), 73.26 (C5), 77.35 (C2), 79.71 (C3), 87.04 (C4), 113.79 (CMe2), 114.10, 124.91, 127.00, 159.95 (Ar); 173.01 (C1); HRMS (TOF-ESI) m/z calcd for C16H20O8SNa+ [M+Na]+ 395.0771; found 395.0793.

4.11. General procedure for the synthesis of 4-C-substituted S-ribosylhomocyteine lactones 16

Step a

H2O (0.24 mL) and tris(2-carboxyethyl)phosphine hydrochloride (88 mg, 0.31 mmol) were added to a stirred solution of N,N′-di(tert-butoxycarbonyl)-L-homocystine di(tert-butyl) ester[13] (160 mg, 0.28 mmol) in anhydrous DMF (2.4 mL) at ambient temperature under Ar atmosphere. After 20 h, the reaction mixture was partitioned between EtOAc, and saturated NaHCO3/H2O. The aqueous layer was extracted with EtOAc, and the combined organic layer was washed with brine, dried (Na2SO4), and concentrated to give N-tert-butoxycarbonyl-L-homocysteine tert-butyl ester (159 mg, 99%) as colorless oil of sufficient purity to be directly used in next step.

Step b

LDA (2M/THF and heptanes, 48 μl, 0.96 mmol) was added slowly to a stirred solution of the freshly prepared protected L-homocysteine (29 mg, 0.1 mmol; from step a) in anhydrous DMF 1.5 ml under Ar atmosphere at 0 °C (ice bath). After 30 min, solution of 15 (0.035 mmol) in DMF (1 mL) was added by a syringe and the mixture was left stirring for 15 min at 0 °C then at room temperature for 24 hours. The reaction was quenched with NH4Cl and the volatiles were evaporated under high vacuum. The residue was partitioned between EtOAc and NaHCO3, washed with brine and dried over anhydrous MgSO4. Volatiles were evaporated and the resulting oil was column chromatographed (hexane/EtOAc, 8:2).

4.11.1. S-(2,3-O-Isopropylidene-4-C-hexyl-D-ribono-1,4-lactone-5-yl)-N-tert-butoxycarbonyl-L-homocysteine tert-butyl ester (16b)

Treatment of 15b (11 mg, 0.031 mmol) with homocysteinate salt using procedure reported in section 4.11 gave 16b (18 mg, 65%): 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.31 (s, 3H, CH3), 1.39 & 1.41 (2 × s, 2 × 9H, 2 × t-Bu), 1.42 (s, 3H, CH3), 1.50–1.60 (m, 2H, H1a), 1.75–1.85 (m, 1H, H8), 1.95–2.05 (m, 1H, H8′), 2.46–2.54 (m, 2H, H7,7′), 2.72 (d, J = 14.7 Hz, 1H, H5), 2.80 (d, J = 14.7 Hz, 1H, H5′), 4.20–4.25 (m, 1H, H9), 4.40 (d, J = 5.9 Hz, 1H, H3), 5.00 (br. d, J = 7.8 Hz, 1H, NH), 5.10 (d, J = 5.9 Hz, 1H, H2); 13C NMR δ 14.01 (C6a), 22.5 & 22.7 (CMe2), 23.45, 25.66, 26.56, 30.67 (C2a–C5a), 27.9 (C7), 28.08 (t-Bu), 28.32 (t-Bu), 34.34 (C1a), 35.8 (C8), 39.86 (C5), 53.6 (C9), 77.0 (C2), 77.20 (C3), 80.59 (t-Bu), 82.41 (t-Bu), 88.98 (C4), 113.30 (CMe2), 156.1 (Boc), 171.3 & 174.0 (C1 & C10); HRMS (TOF-ESI) calcd for C27H48NO8S+ [M+H]+ 546.3095; found 546.3104.

4.11.2. S-(2,3-O-Isopropylidene-4-C-4-methoxyphenyl-D-ribono-1,4-lactone-5-yl)-N-tert-butoxycarbonyl-L-homocysteine tert-butyl ester (16e)

Treatment of 15e (22 mg, 0.07 mmol) with homocysteinate salt using procedure reported in section 4.11 gave 16e (20 mg, 48%): 1H NMR δ 1.35 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.39 &1.41 (2 × s, 2 × 9H, 2 × t-Bu), 1.75–1.89 (m, 1H, H8), 1.95–2.05 (m, 1H, H8′), 2.52–2.68 (m, 2H, H7,7′), 2.85 (d, J = 14.8 Hz, 1H, H5), 3.20 (d, J = 15.0 Hz, 1H, H5′), 3.80 (s, 3H, OCH3), 4.20–4.25 (m, 1H, H9), 4.80 (d, J = 5.8 Hz, 1H, H3), 5.01 (br. d, J = 8.1 Hz, 1H, NH), 5.30 (d, J = 5.8 Hz, 1H, H2), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.0 Hz, 2H, Ar); 13C NMR δ 25.9 & 26.5 (CMe2), 28.1 (t-Bu), 28.4 (t-Bu), 28.9 (C7), 31.0 (C8), 43.9 (C5), 53.1 (C9), 55.4 (OCH3), 77.16 (C2), 77.37 (C3), 81.6 (t-Bu), 82.6 (t-Bu), 90.2 (C4), 113.5 (CMe2), 113.7, 126.7, 130.1, 156.2 (Ar), 155.8 (Boc), 171.2 & 172.0 (C1 & C10); HRMS (TOF-ESI) calcd for C28H41NO9SNa+ [M+Na]+ 590.2394; found 590.2378.

4.12. General procedure for deprotection of 4-C-substituted S-ribosylhomocyteine lactones

Compound 16 (0.03 mmol) was stirred in TFA (2 mL) at 0 °C for 1 h and then at ambient temperature for 3 h. H2O (0.1 mL) was then added and stirring was continued for an additional 1 h. Volatiles were evaporated in vacuum below 30 °C and the residue was coevaporated with MeCN (2 × 0.5 mL). The crude product was redissolved in deionized water (2.5 mL) and washed with CHCl3 (2 × 1 mL). The aqueous layer was evaporated in vacuum below 30 °C.

4.12.1. S-(4-C-Hexyl-D-ribono-1,4-lactone-5-yl)-L-homocysteine (17b)

Treatment of 16b (17 mg, 0.03 mmol) with TFA using procedure reported in section 4.12 gave 17b (7 mg, 60%). This product was additionally purified by HPLC (CH3CN/H2O, 15:85; tR = 21.0 min) to give 6.5 mg (55%) of 17b 1H NMR (D2O) δ 0.82 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.45–1.50 (m, 2H, H1a), 1.87–2.00 (m, 1H, H8), 2.05–2.12 (m, 1H, H8′), 2.45–2.55 (m, 2H, H7,7′), 2.82 (d, J = 13.6 Hz, 1H, H5), 2.87 (d, J = 13.6 Hz, 1H, H5′), 4.20 (d, J = 5.4 Hz, 1H, H3), 4.21–4.23 (m, 1H, H9), 4.72 (d, J = 5.4 Hz, 1H, H2); 13C NMR (D2O) δ 15.01 (C6a), 23.20, 23.56, 23.90, 29.20 (C2a–C5a), 27.3 (C7), 29.73 (C8), 32.10 (C1a), 41.99 (C5), 52.4 (C9), 72.55 (C2), 78.50 (C3), 88.50 (C4), 172.3 & 173.5 (C1 & C10); HRMS calcd for C15H27NO6S+ [M+Na]+ 372.1451; found 372.1469.

4.12.2. S-(4-C-Octyl-D-ribono-1,4-lactone-5-yl)-L-homocysteine (17c)

Step a. Treatment of 15c (24 mg, 0.063 mmol) with homocysteinate lithium salt using procedure reported in section 4.11 gave 16c contaminated with protected homocysteine (~1:1, 40 mg). Compound 16c had: 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H8a), 1.20–1.25 (m, 12H, H2a-H7a), 1.26 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.39 & 1.41 (2 × s, 2 × 9H, 2 × t-Bu), 1.50–1.60 (m, 2H, H1a), 1.75–1.89 (m, 1H, H8), 1.9–2.0 (m, 1H, H8′), 2.48–2.54 (m, 2H, H7,7′), 2.80–2.83 (m, 2H, H5,5′), 4.20–4.26 (m, 1H, H9), 4.40 (d, J = 5.9 Hz, 1H, H3), 5.05 (br. d, J = 7.6 Hz, 1H, NH), 5.15 (d, J = 5.9 Hz, 1H, H2). Step b. Treatment of the crude 16c (~1:1, 40 mg; Step a) with TFA (2 mL), using procedure reported in section 4.12, gave an oily residue that was partitioned between water and CHCl3. The aqueous layer was evaporated in vacuum below 30 0C and the residue (20 mg) was divided into two portions. Each portion of crude 17c was dissolved in deionized water/MeCN (2.5 mL, 19:1, v/v) and was injected into the Sep-Pak cartridge (C18 classic column). The columns were eluted with deionized water (5 mL), a second portion of deionized water (5 mL) and ethanol (5 mL). The combined water eluents contained mainly Hcy (TLC and 1H NMR) while the combined ethanol eluents were evaporated in vacuum to give 17c (5 mg, 21% from 15c): 1H NMR (MeOH-d4) δ 0.82 (t, J = 6.6 Hz, 3H, H8a), 1.20–1.28 (m, 12H, H2a-H7a), 1.50–1.60 (m, 2H, H1a), 1.90–2.00 (m, 1H, H8), 2.05–2.12 (m, 1H, H8′), 2.55–2.65 (m, 2H, H7,7′), 2.80 (d, J = 13.8 Hz, 1H, H5), 2.87 (d, J = 13.9 Hz, 1H, H5′), 4.20 (d, J = 5.4 Hz, 1H, H3), 4.19–4.21 (m, 1H, H9), 4.75 (d, J = 5.4 Hz, 1H, H2); 13C NMR (MeOH-d4) δ 15.01 (C8a), 23.00, 23.50, 23.85, 29.00, 30.67, 30.51 (C2a–C7a), 27.40 (C7), 29.7 (C8), 32.07 (C1a), 39.86 (C5), 52.21 (C9), 71.54 (C2), 77.20 (C3), 84.59 (C4), 172.21 & 173.52 (C1 & C10); HRMS calcd for C17H31NNaO6S+ [M+Na]+ 400.1764; found 400.1783.

Note. Varying on reaction conditions different quantities of 2,3-O-isopropylidene-4-C-octyl-D-ribono-1,5-lactone were isolated during the column chromatography of the crude reaction mixture from step a: 1H NMR δ 0.88 (t, J = 6.6 Hz, 3H, H8a), 1.25–1.32 (m, 12H, H2a-H7a), 1.40 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.62–1.70 (m, 2H, H1a), 3.86 (m, 2H, H5,5′), 4.60 (d, J = 5.7 Hz, 1H, H2), 4.85 (d, J = 5.7 Hz, 1H, H3); 13C NMR δ 14.23 (CH3, C8a), 22.76, 22.87, 29.29, 29.48, 29.91, 31.92 (C2a–C7a), 25.93 & 26.9 (CMe2), 35.47 (C1a), 63.45 (C5), 76.55 (C3), 80.07 (C2), 87.05 (C4), 114.57 (CMe2), 173.25 (C1); HRMS (TOF) m/z calcd for C16H28O5Na+ [M+Na]+ 323.1798; found 323.1805.

4.12.3. S-(4-C-4-Methoxyphenyl-D-ribono-1,4-lactone-5-yl)-L-homocysteine (17e)

Treatment of 16e (11.4 mg, 0.02 mmol) with TFA (1 mL) using procedure reported in section 4.12 gave 17e (5.6 mg, 75%): 1H NMR (MeOH-d4) δ 1.80–1.83 (m, 1H, H8), 1.90–1.92 (m, 1H, H8′), 2.50–2.65 (m, 2H, H7,7′), 2.85 (d, J = 14.8 Hz, 1H, H5), 3.20 (d, J = 15.1 Hz, 1H, H5′), 3.80 (s, 3H, CH3O), 4.22–4.27 (m, 1H, H9), 4.60 (d, J = 5.8 Hz, 1H, H3), 4.90 (d, J = 5.8 Hz, 1H, H2), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.0 Hz, 2H, Ar); 13C NMR (MeOH-d4) δ 27.33 (C7), 29.73 (C8), 41.37 (C5), 52.50 (C9), 55.33 (CH3O), 74.40 (C2), 78.20 (C3), 85.40 (C4), 117.20, 125.81, 127.00, 162.28 (Ar), 172.31, 173.49 (C1 & C10); HRMS calcd for C16H21NO7SNa+ [M+Na]+ 394.0931; found 394.0908.

4.13. General procedure for the reduction of lactones. Synthesis of 4-C-substituted S-ribosylhomocyteines 19

LiEt3BH (1M/THF, 0.045 mL, 0.045 mmol) was added dropwise to a solution of 0.02 mmol of 16 in CH2Cl2 (1 mL) or 17 in THF/CH2Cl2 (1:1; 1 mL) and the resulting mixture was stirred at −20 °C for 30 min under N2 atmosphere. MeOH (0.5 mL) was then added slowly to quench the reaction and volatiles were evaporated in vacuum below 25 °C. The residue for the protected products 18 was partitioned between CH2Cl2/NaHCO3, washed with brine, dried (MgSO4) and was column chromatographed (75:25, hexane/EtOAc); whereas the residue for the deprotected product 19 was redissolved in deionized H2O/MeOH (4:1, 2.5 mL) and washed with CHCl3 (2 × 1 mL) and then the aqueous layer was evaporated in vacuum below 30 °C. The 4-C-substituted SRH analogues 19 are somehow unstable and need to be manipulated with care but are stable when stored as powder or well-dried syrup in refrigerator at 4 °C for a month.

4.13.1 S-(5-Deoxy-4-C-hexyl-D-ribofuranos-5-yl)-L-homocysteine (19b)

Step a. Treatment of 16b (10.9 mg, 0.02 mmol) with LiEt3BH, using procedure reported in section 4.13, gave 2,3-O-isopropylidene-5-[(tert-butoxycarbonyl)-L-homocysteine tert-butyl ester]-4-C-hexyl-D-ribofuranose 18b (α/β, ~1:9, 9.5 mg, 90%). The major β anomer had: 1H NMR δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.35 & 1.41 (2 × s, 2 × 9H, 2 × t-Bu), 1.35 (s, 6H, 2 × CH3), 1.50–1.60 (m, 2H, H1a), 1.91–2.02 (m, 2H, H8,8′), 2.48–2.58 (m, 2H, H7,7′), 2.60 (d, J = 12.8 Hz, 1H, H5), 2.94 (d, J = 12.6 Hz, 1H, H5′), 4.19–4.21 (m, 1H, H9), 4.30 (d, J = 5.9 Hz, 1H, H3), 4.92 (d, J = 5.9 Hz, 1H, H2), 5.19–5.21 (m, 1H, NH), 5.35 (s, 1H, H1). The minor α anomer had a peak for H1 at δ 5.47 (d, J = 3.9 Hz). Step b. Treatment of 18b (α/β, ~1:9; 9.5 mg, 0.02 mmol) with in TFA (1 mL) using procedure reported in section 4.12 gave 19b (α/β, ~1:9; 4 mg, 75%). The major β anomer had: 1H NMR (D2O) δ 0.80 (t, J = 6.6 Hz, 3H, H6a), 1.20–1.28 (m, 8H, H2a–H5a), 1.50–1.60 (m, 2H, H1a), 1.91–2.01 (m, 2H, H8,8′), 2.48–2.58 (m, 2H, H7,7′), 2.63 (d, J = 12.8 Hz, 1H, H5), 2.94 (d, J = 12.6 Hz, 1H, H5′), 4.12 (t, J = 5.9 Hz, 1H, H2), 4.19–4.21 (m, 1H, H9), 4.20 (d, J = 5.9 Hz, 1H, H3), 5.33 (s, 1H, H1); MS (ESI−) m/z 350 (MH−). HRMS calcd for C15H29NO6SNa+ [M+Na]+ 374.1608, found 374.1617.

4.13.2. S-(5-Deoxy-4-C-octyl-D-ribofuranos-5-yl)-L-homocysteine (19c)

Treatment of 17c (6 mg, 0.01 mmol) with LiEt3BH (0.03 mL), using procedure reported in section 4.13, gave 19c (α/β, ~1:3; 4 mg, 60%). The major β anomer had: 1H NMR (MeOH-d4) δ 0.80 (t, J = 6.6 Hz, 3H, H8a), 1.21–1.32 (m, 12H, H2a-H7a), 1.50–1.60 (m, 2H, H1a), 1.9–2.0 (m, 2H, H8,8′), 2.48–2.58 (m, 2H, H7,7′), 2.79 (d, J = 12.8 Hz, 1H, H5), 2.90 (d, J = 12.6 Hz, 1H, H5′), 4.15 (t, J = 5.9 Hz, 1H, H2), 4.19–4.21 (m, 1H, H9), 4.20 (d, J = 5.9 Hz, 1H, H3), 5.19–5.21 (m, 1H, NH), 5.39 (s, 1H, H1), [the minor α anomer had a peak for H1 at δ 5.44 (d, J = 3.5 Hz)]; 13C NMR (MeOH-d4) δ 15.01 (C8a), 23.00, 23.50, 23.85, 29.00, 30.67, 30.51 (C2a–C7a), 27.3 (C7), 29.6 (C8), 32.07 (C1a), 41.99 (C5), 50.51 (C9), 69.77 (C3), 72.09 (C2), 87.16 (C4), 99.90 (C1), 172.62 (C10); HRMS calcd for C17H33NO6SNa+ [M+Na]+ 402.1921, found 402.1933.

4.13.3 S-(5-Deoxy-4-C-4-methoxyphenyl-D-ribofuranos-5-yl)-L-homocysteine (19e)

Step a. Treatment of 16e (11.3 mg, 0.02 mmol) with LiEt3BH, using procedure reported in section 4.13, gave 2,3-O-isopropylidene-5-[(tert-butoxycarbonyl)-L-homocysteine tert-butyl ester]-4-C-4-methoxyphenyl-D-ribofuranose 18e (α/β, ~1:9; 7.8 mg, 68%). The major β-anomer had: 1H NMR δ 1.35 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.39 &1.41 (2 × s, 2 × 9H, t-Bu), 1.75–1.89 (m, 1H, H8), 1.95–2.05 (m, 1H, H8′), 2.48–2.54 (m, 2H, H7,7′), 3.01 (d, J = 14.8 Hz, 1H, H5), 3.20 (d, J = 15.1 Hz, 1H, H5′), 3.80 (s, 3H, CH3O), 4.20–4.25 (m, 1H, H9), 4.65 (d, J = 5.8 Hz, 1H, H3), 4.85 (d, J = 5.8 Hz, 1H, H2), 5.01 (d, J = 8.1 Hz 1H, NH), 5.40 (s, 1H, H1), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.0 Hz, 2H, Ar), [the minor α-anomer had a peak for H1 at δ 5.60 (d, J = 3.8 Hz)]; HRMS calcd for C28H43NO9SNa+ [M+Na]+ 592.2551, found 592.2522. Step b. Treatment of 18e (α/β, ~1:9; 7.6 mg, 0.013 mmol) with in TFA (1 mL), using procedure reported in section 4.12, gave 19e (α/β, ~1:9; 3.7 mg, 77%). The major isomer had: 1H NMR (MeOH-d4) δ 1.90–2.00 (m, 2H, H8,8′), 2.48–2.58 (m, 2H, H7,7′), 3.01 (d, J = 14.8 Hz, 1H, H5), 3.20 (d, J = 15.1 Hz, 1H, H5′), 3.80 (s, 3H, CH3O), 4.15 (d, J = 5.8 Hz, 1H, H2), 4.20–4.25 (m, 1H, H9), 4.40 (d, J = 5.8 Hz, 1H, H3), 5.45 (s, H1, 1H), 6.82 (d, J = 8.8 Hz, 2H, Ar), 7.12 (d, J = 9.0 Hz, 2H, Ar); 13C NMR (MeOH-d4) δ 27.51 (C7), 29.70 (C8), 52.52 (C9), 55.33 (CH3O), 42.20 (C5), 72.61 (C3),74.40 (C2), 85.40 (C4), 101.10 (C1), 117.20, 125.81, 127.00, 162.28 (Ar), 172.3 (C10). HRMS calcd for C16H23NO7SNa+ [M+Na]+ 396.1087, found 396.1062.

Acknowledgments

This work was partially supported by NIGMS/NCI (1SC1CA138176). We thank the NIH MARC U*STAR (GM083688) program for the fellowship to DL and University of Wroclaw and EU social funds for supporting KSS and JN summer internship training at FIU.

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