Total synthesis of 2'-O-methyl-β-L-arabinosyluridine and reassignment the nucleoside from Penicillium sp. as 2'-O-methyl-β-L-uridine

In order to validate the structure of a rarely reported naturally occurring nucleoside isolated from the broth of Penicillium sp. (NO. 64), practical syntheses of 2'-O-methylβ-L-arabinosyluridine, 2'-O-methyl-α-L-arabinosyluridine, and 2'-O-methyl-β-L-uridine were accomplished. Comparing their nuclear magnetic resonance (NMR) spectra and physical data, its structure was reassigned as 2'-O-methyl-β-L-uridine instead of former reported 2'-O-methyl-β-L-arabinosyluridine.


Introduction
Naturally occurring nucleosides have played extraordinarily important role for discovering new pharmaceutical and chemical identities [1][2][3].In the past century, large amount nucleosides have been isolated and identified from various natural resources, such as plants, microorganisms, and recent marine origins.Reprehensive examples are antiviral drug Vidarabine (Ara-A) and Cytarabine (Ara-C) from sponges, which are still prescribed in clinical practice nowadays [4].
Examination the chemical structures of all reported naturally occurring nucleosides showed that almost all of them are D-nucleosides [5].Actually, D-nucleosides are also the main components of DNA and RNA.But in recent years, L-nucleosides have attracted tremendous interests of medicinal chemists, because of their excellent absorption, distribution, metabolism, and excretion (ADME) properties [6].Since then, amount of nucleosides with the unnatural β-L-configuration have been synthesized and their biological activities were evaluated [7].Many have been found to possess very potent antiviral activities [8].The most noble compound is Lamivudine (3TC, Scheme 1), which is the first-line drug for treating HBV at the moment [9].
In 2017, identification of 2'-O-methyl-β-L-arabinosyluridine (compounds 1, Figure 1) from the broth of Penicillium sp.(NO.64) was reported by Guo et al [10].We immediately realized that discovering L-nucleoside from nature was a breakthrough, which might change our acknowledge about nucleoside's natural occurrence.In addition, as we all know, L-arabinopyranoside and L-arabinofuranoside are widely presented in nature, especially presence as the component of bacterial cell wall [11].
However, to the best our knowledge, there is very rare example of L-nucleoside reported as natural product.Because only small amount of nucleoside 1 was isolated, its biological activity was not reported by the authors.As our continuous study on total synthesis of naturally occurring nucleosides, we started to carry out its synthesis and investigate its biological activities [12][13][14][15][16][17].From the synthetic point view, synthesis of β-L-arabinosyluridine could be carried out in two different approaches shown in Figure 2. The most straightforward approach is selective methylation of β-L-arabinosyluridine, which can be synthesized from 2,3,5tri-O-benzyl-α-L-arabinofuranosyl chloride and uracil (Route A, Figure 2).But, the glycosylation always affords a mixture of anomers and their separation is notorious.In addition, this approach needs tedious protection and deprotection manipulations.Thus, the overall efficiency is low.The other convenient approach is synthesis nucleoside 1 using Vorbrüggen glycosylation of corresponding properly protected 2-O-methyl-Larabinofuranose with uracil directly (Route B, Figure 2).However, because of 2-Omethyl group lacking of neighboring participation effect, an anomeric mixture of nucleosides 1 and 2 will be formed inevitably.Considering that the corresponding αconfiguration nucleoside was also not reported before and the α-configuration nucleosides were attracted much interests in recent years, we decided to use the second approach to synthesize 1 and 2 at the same time [18][19][20][21].
Encouragingly, a single crystal of L-arabinofuranose 6 suitable for X-ray crystallography was obtained and its structure was ambiguously confirmed (Figure 3) [24].Next, methylation of 2-OH of compound 6 was investigated.Literature survey revealed that CH3I/Ag2O and trimethylsilyl diazomethane (TMSCHN2)/HBF4 were frequently used for the O-methylation of alcohols [25,26].At first, the method of CH3I/Ag2O in DMF was applied.Because Ag2O are weak base, transesterification of benzoyl to 2-OH occurred and a complicated mixtures was obtained [27].Therefore, the method of TMSCHN2/HBF4 was further employed.In our preliminary experiment, L-arabinose methyl ether 7 was successfully obtained but in low yield accompanied by unreacted starting material.After extensive optimization of solvents, reaction temperature, equivalence ratios of TMSCHN2 and HBF4, it was found that the best condition was using of 1.0 equiv.1,3,5-tri-O-benzoyl-α-L-arabinofuranose 6, 2.0 equiv. of TMSCHN2, and 1.0 equiv. of HBF4 in DCM at room temperature.It afforded methyl ether 7 in 70% yield accompanied with 20% unreacted starting material arabinose 6.
Subsequent, vorbrüggen glycosylation of uracil with methyl ether 7 was performed in MeCN/BSA/TMSOTf.It afforded the β-L-arabinosyluridine 8 and α-L-arabinosyluridine 9 (approximation ratio of 3:1) as expected in 74% yield.Careful recrystallization of the mixed isomers can give part of nucleoside 8 as a white solid.The remaining residue containing the mixed isomers 8 and 9 was difficult to separate using silica gel chromatography.Thus, the mixture was subjected to a saturated solution of ammonia in methanol directly to afforded nucleoside 1 and 2, which can be separated successfully by HPLC.Discrimination of the α-anomer and β-anomer was done based on 3 JH1',H2' coupling constant.The resonances for the anomeric hydrogens of α/β-Larabinosyluridine appeared as a doublet ( 3 JH1',H2' = 2.9 Hz) at δ 5.81 ppm and a doublet ( 3 JH1',H2' = 5.6 Hz) at δ 6.13 ppm, respectively [28].It is noteworthy that the small value of 3 JH1',H2' is a characteristic trans-relationship between H1' and H2' in ribose nucleoside [29].1).This inconsistent means that the nucleoside structure from the broth of Penicillium sp.
Revisiting the two-dimensional NMR data, we supposed that the uridine might be 2'-  Company, China).NMR spectra were recorded on a Bruker AV400 spectrometer, and chemical shifts (δ) are reported in ppm. 1 H NMR and 13 C NMR spectra were calibrated with TMS as an internal standard, and coupling constants (J) are reported in Hz.The ESI-HRMS were obtained on a Bruker Dalton microTOFQ II spectrometer in positive ion mode.Melting points were measured on an electrothermal apparatus uncorrected.
Optical rotation was measured on a Rudolph Autopol IV at a wavelength of 589 nm.

Synthesis of 2-O-trifluoromethylsulfonyl-1,3,5-tri-O-benzoyl-α-L-ribofuranose (5)
A mixture of 1,3,5-tri-O-benzoyl-α-L-ribofuranose 4 (4.62 g, 10 mmol, 1 eq.) and anhydrous pyridine (11.7 mL, 14.6 mmol, 14.6 eq.) in anhydrous DCM (150 mL) was stirred under ice bath for 10 min.Trifluoromethanesulfonic anhydride (12 mmol, 2 mL) was then added dropwise with vigorous stirring.The obtained reaction mixture was stirred for another 1 hr at 0 °C and then stirred at room temperature for 3 hrs.Then, the reaction was quenched by the addition of 200 mL of ice water.The aqueous solution was extracted with DCM (3×200 mL) and the combined extract was washed with a saturated solution of sodium carbonate (3×200 mL) and brine (3×200 mL), respectively.The organic phase was dried with anhydrous MgSO4.After filtered and evaporated under reduced pressure, the obtained syrup was purified by column chromatography on silica gel to give 5 as a white solid (5.34 g, 90%).

Synthesis of 2'-O-methyl-3',5'-di-O-benzoyl-β-L-arabinosyluridine (8)
To a solution of uracil (0.27 g, 2.4 mmol) in dry MeCN (10 mL) was added BSA (1.95 g, 2.4 mL, 9.6 mmol) and stirred under nitrogen for 1 h at room temperature.After addition of 7 (0.95 g, 2 mmol), TMSOTf (1.78 g, 1.5 mL, 8 mmol) was added to the mixture at ice bath.The mixture was stirred for 15 min before heating to 80 °C for 12 hrs.After cooling, the reaction mixture was poured into water (30 mL) and extracted with EtOAc (3 × 30 mL).The combined extract was washed third with a saturated solution of sodium carbonate (3 × 30 mL) and brine (3 × 30 mL), respectively.The organic phase was dried with anhydrous MgSO4, filtered and evaporated under reduced pressure.The residue was purified by column chromatography on silica gel to give crude products.The crude products were recrystallized from a mixture of EA and PE to give a white solid 8 (0.51 g, 55%).The filtrated stock solution containing isomers of nucleoside 9 was evaporated under reduced pressure and used directly without further purification.

Synthesis of 2'-O-methyl-α-L-arabinosyluridine (2)
The nucleoside 2 was synthesized as described for 1 starting from crude nucleoside 9.The reaction mixture was concentrated to dryness and the residue was purified by column chromatography on silica gel to give a crude product.The nucleoside 2 (0.04 g, 14%) was obtained by preparative HPLC purification from crude product.

Synthesis of 2'-O-methyl-β-L-uridine (3)
2,2′-Anhydrouridine 12 (0.57g, 2.5 mmol) was added to a freshly prepared solution of 12% magnesium methoxide (7 mL, 9.8 mmol) in anhydrous methanol.The reaction mixture was refluxed under nitrogen.Once TLC monitoring showed disapperance of the starting material, the reaction was quenched with sat.NH4Cl.After cooling, the mixture was concentrated to dryness and the residue was purified by column chromatography on silica gel to give nucleoside 3 as a white solid (0.48 g, 75%).

Figure 1 .
Figure 1.Chemical structures of Lamivudine and the synthesized L-nucleosides in