IJLSSR, VOLUME 3, ISSUE 4, JULY 2017:1114-1117

Research Article (Open access)

Synthesis of 2,3-O,O-dibenzyl-6-O-tosyl-
Non-L-ascorbic Acid

Pradeep Kumar Swain1* Rama S. Lokhande1, Madhumita Bhattacharjee2
1Department of Chemistry, School of Basic Sciences, Jaipur National University, Jaipur (Rajasthan), India
2Department of Chemistry, Bhavan’s Vivekananda College of Science, Humanities & Commerce, Sainikpuri, Se
Secunderabad, India

*Address for Correspondence: Mr. Pradeep Kumar Swain, R & D Manager, Research and Development,
JAevum Bio Labs Pvt. Ltd, Hyderabad, India

Received: 21 March 2017/Revised: 29 May 2017/Accepted: 25 June 2017

ABSTRACT-- L-Ascorbic acid derivatives was synthesized on treatment with acetone and acetyl chloride afforded 5,6-acetal of L-ascorbic acid then benzylation of C-2 and C-3 hydroxyl groups of the lactone ring was accomplished using K2CO3 and benzyl bromide in DMF, then deblocking of the 5,6-O,O-protected derivative of L-Ascorbic acid with acetic acid and methanol gave 2,3-O,O-dibenzyl-L-Ascorbic acid. Subsequently mono-tosylation at 6 position of 2,3-O, O-dibenzyl-L-Ascorbic acid was carried out with addition of p-toluenetosylchloride (PTSC) in Pyridine and MDC solvent medium gave 2,3-O,O-dibenzyl-6-O-tosyl-L-Ascorbic acid. All the structures were characterized by 1H NMR, 13C NMR and Mass Spectroscopy.

Key-words-L-Ascorbic acid, 5,6-Acetal, Benzylation, Hydrolysis, Tosylation

INTRODUCTION- Vitamin C, or L-Ascorbic acid, is a vital nutrient for humans and has many important functions in the body. L-Ascorbic acid is a white, odorless, crystalline powder. It is freely soluble in water and relatively insoluble in organic solvent. Vitamin C exhibits anti-scorbutic properties, since it contributes to the synthesis of collagen, the main constituent of the protein fibers in human tissue, which is important in maintaining healthy skin elasticity and texture, and also helps maintain the integrity of substances of mese-chymal origin, such as connective tissue, osteoid tissue and dentin.[1-4] L-Ascorbic acid and its derivatives have been found to possess antitumor and antiviral activities[5-7]. Thus L-Ascorbic acid inhibited apoptosis induced by oxidative stress in HL-60 myeloid leukemia cell5. L-Ascorbic acid is highly concentrated in the neurons in the brain, which likely indicates the essential roles in neuronal function and antioxidant protection.[8-9] Recent research on the biochemistry of L-Ascorbic acid has been focused on the transport and accumulation mechanism, by which brain acquires L-Ascorbic acid.

Fig 1: Structure of 2,3-O,O-dibenzyl-6-O-tosyl-L-Ascorbic acid

It has been shown that L-Ascorbic acid and its oxidized form, dehydroascorbic acid (DHA), have distinct transport mechanism, mediated by the Na+-vitamin C transporter SVCT2, and the facilative glucose transporter GLUTI, respectively. [10-13]

L-Ascorbic Acid was purchased from Sigma-Aldrich, USA and other reagents such as Acetyl chloride, Acetone, benzyl bromide, Potassium carbonate, Dimethylformamide, Acetic acid, Methanol etc were purchased from Finar chemicals Ltd, India and p-toluenesulfonylchloride, Pyridine, Di-chloromethane and Diethyl ether were purchased from spectrochem, India. Commercial solvents were used for work up note during synthesis. All the experiments were performed in Aevum Bio Labs Pvt. Ltd, Hyderabad, India. It took six months time period to overcome this Derivative. All analyses were done in Sapala Organics Pvt. Ltd., India.

METHODOLOGY- All the Ascorbic acid derivatives were characterized by 1H and 13C NMR and electron impact mass spectra. Melting points of compounds were determined with a Kofler micro hot-stage (Reichert, Wein) were uncorrected. Recoated Merck silica gel 60F-254 plates were used for thin layer chromatography (TLC) and spots were detected under UV light (254 nm). The electron impact mass spectra were recorded with an EXTREL FT MS 2001 instrument with ionizing energy 70 eV. The 1H and 13C NMR spectra were recorded on a Varian Gemini 300 spectrometer, operating at 75.46 MHz for the 13C resonance. The samples were dissolved in DMSO-d6 or CDCl3 chemical shift values are in ppm, referred to TMS. Additional purification of compound (2) by recrystallization from methanol afforded good purity and acetone was dried over calcium chloride and followed by potassium carbonate in reflux condition. DMF was dried over calcium hydride for above 12hrs in reflux condition and MDC was dried over calcium chloride and calcium hydride for about 12hrs. Pyridine was dried by distillation with potassium hydroxide and followed by calcium hydride for couple of Hrs under argon atmosphere. Then Potassium carbonate was dried in oven at 1100C for 6 hrs and p-Toluenesulfonylchloride was purified by diethyl ether.

The 5,6-acetal of L-ascorbic acid (1), benzylation compound of the C-2 and C-3 hydroxyl groups (2) and deblocking compound of the 5,6-O,O-protected derivative (3) were synthesized as described previously [14-18]. Synthesis of 2,3-O,O-dibenzyl-6-O-tosyl-L-Ascorbic acid (4) was synthesized previously14. In this paper it was synthesized with convenient method with good yield (82%) were out-lined in scheme-1.

Fig 2: Root of synthesis of 2,3-O,O-dibenzyl-6-O-tosyl-L-Ascorbic acid

EXPERIMENTAL SECTION- Synthesis of 5,6-O-isopropylidene-L-Ascorbic acid (1) was described as priviously14. MP 198-2020C, MS m/z 215 (MH-).
13C NMR (DMSO-d6) d: C-1(170.329), C-2(152.527), C-3(118.312), C-4(74.390), C-5(73.575), C-6(64.997), C-7(109.149), CH3(25.941-25.537), 1H NMR (DMSO-d6) d: H-4(4.714-4.708,d,1H), H-5(4.282-4.241,dt,1H), H-6(4.118-4.079,t,1H), H-6(3.901-3.864,t,1H), CH3(1.255,s,6H), 2-OH(11.302,s,1H), 3-OH(8.489,s,1H).
D2O Exchange: The peaks at 11.302 and 8.489 were exchanged by D2O.
The 5,6-O-isopropylidene-2,3-O,O-Dibenzyl-L-Ascorbic Acid (2) was synthesized as de¬scribed by Von14.
MP 127-1300C, MS m/z 397 (MH+).13C NMR (CDCl3) d:168.976(C-1),121.045(C-2),156.487(C-3),74.530(C-4), 73.658(C-5),65.153(C-6),110.120(C-7),25.802 & 25.562 (CH3), 73.806 & 73.658(CH2), 135.839-127.664(C6H5). 1H NMR(CDCl3) d:1.409 & 1.366(s,6H,CH3),4.026-3.988 (dd,2H,H-6),4.272-4.231(dt,1H,H-5),4.537-4.530(d,1H, H-4),5.205-5.063(m,4H,CH2Ph),7.401-7.195(m,10H,C6H5).
Synthesis of 2,3-O,O-Dibenzyl-L-Ascorbic acid (3) was synthesized as de¬scribed by Von14. MP 80.2-830C, MS m/z 356.9(MH+). 13C NMR (DMSO-d6) d: 169.437(C-1), 158.166(C-2), 120.691(C-3), 74.670(C-4), 68.792(C-5), 61.687(C-6), 136.292-127.738(C6H5), 73.633, 72.678 (CH2Ph). 1H NMR (DMSO-d6) d: 4.902(1H,s,H-4), 3.733-3.681(1H,q,H-5), 3.493-3.386(2H,m,H-6), 5.270-5.192 (2H,q,OCH2), 4.991-4.927(2H,q,OCH2), 5.165-5.150 (1H,d,5-OH), 4.887-4.873(1H,d,6-OH), 7.429-7.314 (10H,m,C6H5). D2O Exchange: At the region of 5.165-5.150(1H,d,5-OH),4.887- 4.873(1H,d,6-OH) were exchanged by the action of D2O.
Synthesis of 2,3-O,O-Dibenzyl-6-O-Tosyl-L-Ascorbic acid (4) to a solution of 3 (3.56 g, 0.01 mol) in Dry MDC (7.12 ml) and anhyd. Pyridine (14.24 ml) was stirred at 00C under argon atmosphere. p-Toluene sulfonylchloride (1.91g, 0.01mol) dissolved in anhyd. MDC (14.24 ml) was added drop wise to this solution. The stirring of the mixture was continued at RT for 24 Hrs. After completion of re-action, the mixture was diluted with MDC (56.96 ml) and wash with five times of water. Then dried over Na2SO4 and evaporated the solvent completely under reduced pressure. Crude product was crystallized by methanol afforded col-orless crystals of 4 [4.19, 82% yield, MP 110-1110C and MS m/z 511(MH+)]. Reaction was monitored by TLC (Mobile phase: N-Hexane: Ethyl acetate 8:2 Ratio, Rf value 0.8), ) 13C NMR (DMSO-d6) d: 20.97 (CH3), C-6 (66.520), C-5 (70.653), C-4 (75.510), OCH2 (74.250,74.020), C=O (168.967), C-2 (120.930), C-3 (157.038), Ph-Tos (128.380-131.772), Ph-quartC (135.671-136.135), Tos-quartC (131.994-145.167). 1H NMR (DMSO-d6) d: 2.411 (CH3tos, s, 3H), 3.954-3.904 (H-6, q, 2H), 4.171-4.116 (H-5, q, 1H), 4.882 (H-4, s, 1H), 5.789-5.774 (5-OH, d, 1H), 5.244-5.148 (OCH2, q, 2H), 4.971-4.907 (OCH2, q, 2H), 7.824-7.802, 7.493-7.473 (H Ph-Tos, d, 4H), 7.400-7.290 (HBenzyl, m, 10H). D2O Exchange: The peak at 5.789-5.774 was exchanged by D2O.

CONCLUSIONS- The present work describes the synthesis of 6-tosylated derivative of 2,3-O,O-dibenzyl-L-Ascorbic acid (3) by using strategy of selective protection and deprotection of 2,3- and 5,6-dihydroxy functional group and subsequent tosylation of 6-hydroxy group. Spectral analysis of 2,3-O,O-Dibenzyl-6-O-Tosyl-L-Ascorbic acid (4) and the structures (1-3) were elucidated by 1H NMR,13C NMR, D2O Exchange and Mass spectroscopy. The aim of re-search is to synthesis and biological screening of new nucleoside analogues of L-Ascorbic acid by using the L-Ascorbic derivatives and pyrimidine derivatives with new selective methods, which will be performed shortly.

ACKNOWLEDGMENT- Support for this study by the Astrel Genome Ltd [Pro-ject No. ASC-01, ASC-02, ASC-03, and ASC-04(MTS)] is gratefully acknowledged. We thank Munisekhar Medasani, Director of Astrel Genome Ltd for providing laboratory and all the chemicals for the synthesis work.

    1. S. Lawson, J. V. Higdon and B. Frei. Vitamin C, 2004; 1-29.
    2. B. V. Nusgens, P. Humbert, A. Rougier, et al. Stimulation of collagen biosynthesis by topically applied vitamin C. European Journal of Dermatology, 2002; 12(4): xxxii-xxxiv.
    3. C. Mitoma and T.E. Smith. Studies on the role of Ascorbic Acid in collagen synthesis. Journal of Biological Chemistry, 1960; 235: 426-428.
    4. W. A. Peck, S. J. Jr. Birge and J. Brandt. Collagen synthesis by isolated bone cells: Stumulation by ascorbic acid in vitro. Biochimica et biophysica acta, 1963; 142(2): 512-525.
    5. Cinatl. J.; Weber, B.; Rabenau, H.; Gumbel, H.O.; Chenot. J. F.; Scholz,M.; Encke, A.; Doerr, H. W. in vitro Inhibition of Human Cytomegalovirus Replication in human Foreskin Fibroblasts and Endothelial cells by Ascorbic Acid-2-phosphare. Antiviral Res. 1995; 27: 405-418.
    6. Bram, S.; Froussard, P.; Guichard, M.; Jasmin, C.; Augery, Y.; Sinoussi-Barre, F.; Wray W. Vitamin C Preferential Toxicity for Malignant Melanoma. Cells. Nature 1980; 248: 629-631.
    7. Grdisa, M.; Kralj. M.; Eckert-Maksic, M.; Maksic, Z. B.; Pavelic, K. 6-Amino-6-Deoxyascorbic Acid Induces Apoptosis in Human Tumor Cells. J. Cancer res. Clin. Oncol, 1995; 121: 98-102.
    8. Witenberg, B.; Kalir. H. H.; Raviv, Z.; kletter. Y.; Kravtsov. V.; Fabian, I. Inhibition by Ascorbic Acid of Apoptosis Induced by Oxidative stress in HL-60 myeloid Leukemia Cells. Biochem. Phamacol, 1999; 5: 823-832.
    9. Spectro R. Vitamin homeostasis in the central nervous system. N. Eng. J. Med, 1977; 296:1393-1398.
    10. Evance R. M. Currie L. Campbell A. The distribution of as-corbic acid between various cellular components of blood in normal individuals and its relation to the plasma concentration. Br. J. Nutr, 1982; 47:473-482.
    11. Tsukaguchi H, Tokuri T, Mackenzie B, Berger U. V. Chen X. Z. Wang Y. Brubaker R. F. Hediger M. A. A family of mammalian Na+ - dependent L-ascorbic acid transporters. Nature (London), 1999; 399: 70-75.
    12. Vera J. C. Rivas C. I. Fischbarg J. Golde D. W. Mammalian facilitative hexose transporters mediate the transport of de-hydroascorbic acid. Nature (London), 1993; 364:79-82.
    13. Fransson L.A. Mani K. Novel aspects of Vitamin C: How Important is Glypican-1 Recycling. Trends Mol. Med, 2007; 13, 143-149.
    14. Von Dallacker F; Sanders, J, Derivative der L-ascorbinsaure, 1, Darstellung und Eigenschaften der O2, O3-Ethandiyl-und der O2,O3-Dibenzyl-L ascorbinsauren. Chem. Ztg, 1985; 109: 197-202.
    15. Von Dallacker, F; Sanders, J, Derivative der L-ascorbinsaure, 2, Darstellung von Deoxy–L-Ascorbinsauren. Chem. Ztg, 1985; 109: 277-280.
    16. Silvana Raic-Malic, Drazenka Svedruzic, Tatjana Gazivoda, Andrej Marunovic, A antonija Hergold- Brundic, Ante Nagl, Jan Balzarini, Erik De Clercq, and Mladen Mintas. Synthesis and Antitumor Activities of Novel Pyrimidine Derivatives of 2,3-O,O-Dibenzyl-6-deoxy-L-ascorbic Acid and 4,5-Didehydro-5,6-dideoxy-L-ascorbic Acid. J. Med. Chem, 2000; 43: 4806-4811.
    17. Silvana Raic-Malic, antonija Hergold- Brundic, Ante Nagl, Mira Grdisa, Kresimir Pavelic, Erik De Clercq, and Mladen Mintas. Novel Pyrimidine and Purine Derivatives of L-Ascorbic Acid: Synthesis and Biological Evaluation. J. Med. Chem, 1999; 42(14): 2673-2678.
    18. Tatjana Gazivoda, Karlo Wittine, Iva Lovric, Damjan Ma-quc, Janez Plavec, Mario Cetina, Draginja Mrvos-Sermek, Lidija Suman, Marijeta Kralj, Kresimir Pa-velic, Mladen Mintas and Silvana Raic-Malic. Synthesis, structural studies, and cytostatic evaluation of 5,6-di-O-modified L-ascorbic acid derivatives. Carbohydrate Research, 2006; 341(4): 433-442.

      How to cite this article:
      Swain PK, Lokhande RS, Bhattacharjee M: Synthesis of 2,3-O,O-dibenzyl-6-O-tosyl-L-ascorbic Acid. Int. J. Life. Sci. Scienti.
      Res., 2017; 3(4):1114-1117. DOI:10.21276/ijlssr.2017.3.4.2
      Source of Financial Support: Nil, Conflict of interest: Nil