3H-1,2-benzodithiol-3-one 1,1-dioxide

[66304-01-6]  · C7H4O3S2  · (MW 200.24)

(reagent extensively used in the efficient conversion of phosphite triesters to phosphorothioate triesters in solid-phase oligonucleotide synthesis)

Alternate Name: Beaucage reagent.

Physical Data: mp 102.5-103 °C.

Solubility: soluble in most organic solvents except nonpolar solvent such as alkanes.

Form Supplied in: white solid.

Analysis of Reagent Purity: 13C NMR, 1H NMR, EI-MS.

Preparative Methods: the reagent is prepared from the oxidation of precursor 3H-1,2-benzodithiol-3-one with trifluoroperoxyacetic acid.

Purification: recrystallization from methylene chloride-hexanes.

Handling, Storage, and Precautions: the reagent can be stored indefinitely as a crystalline material in amber glass bottles at ambient temperature. The reagent decomposes rapidly when in contact with inorganic and organic bases. The reagent will also decompose in solution when exposed to glass and/or metallic surfaces. Disposal the reagent can be safely discarded in a chemical waste container.

Preparation and Handling of 3H-1,2-benzodithiol-3-one 1,1-dioxide

The sulfur-transfer reagent 3H-1,2-benzodithiol-3-one 1,1-dioxide (2, 1)1 is widely used in the preparation of oligonucleoside phosphorothioates, which are especially valuable in the development of therapeutic oligonucleotides against various types of cancer and infectious diseases in humans.2,3

The scope of this report is not to provide an exhaustive list of all oligonucleoside phosphorothioates that have been prepared using 2, but to review those critical issues stemming from the preparation and handling of the reagent along with specific applications of 3H-1,2-benzodithiol-3-one 1,1-dioxide as a sulfur-transfer reagent.

In regard to the preparation of 2, oxidation of 3H-1,2-benzodithiol-3-one (1) to its 1,1-dioxide (1) has initially been performed by treatment with 3-chloroperbenzoic acid.4 While such an oxidation reaction appears satisfactory for the preparation of 2 on a small scale, the generation of hydrophobic 3-chlorobenzoic acid during the course of the reaction complicates the purification of the desired product on large scale.

The search for an oxidizing reagent that would produce hydrophilic side-products has identified trifluoroperoxyacetic acid as a promising oxidizer. Oxidation of 1 with trifluoroperoxyacetic acid is efficient only when the internal temperature of the reaction mixture is maintained at 40-42 °C.1a As the reaction temperature exceeds 42 °C, the yield of 2 decreases presumably as a result of concurrent hydrolysis of the reagent. At a reaction temperature below 37 °C, the oxidation of 1 becomes sluggish and also results in poorer isolated yields of the 1,1-dioxide (2). Thus, when using trifluoroperoxyacetic acid as an oxidizing reagent at 40-42 °C, the preparation of 3H-1,2-benzodithiol-3-one 1,1-dioxide can be scaled up to permit facile isolation of the sulfur-transfer reagent in quantities exceeding 125 g, free from peroxide contaminants, upon precipitation on ice.1c

In addition to 3-chloroperoxybenzoic acid and trifluoroperoxyacetic acid, dimethyldioxirane is also adequate for the conversion of 1 to its 1,1-dioxide.5 The reaction of 3H-1,2-benzodithiol-3-one with 4 equiv of 70-90 mM dimethyldioxirane in acetone at ambient temperature affords a quantitative yield of the corresponding 1,1-dioxide.5 While handling dimethyldioxirane on a large scale may be inconvenient, the production of 3H-1,2-benzodithiol-3-one 1,1-dioxide in high yields under anhydrous conditions further suggests that hydrolysis is likely responsible for the lower yields of 2 obtained under aqueous conditions.

Sulfurization of tricoordinated phosphorus compounds by treatment with 3H-1,2-benzodithiol-3-one 1,1-dioxide results from the nucleophilic attack of, for example, a phosphite triester on the electrophilic thiosulfonate function of 2 to produce the phosphonium sulfinate intermediate 3 (2). Intramolecular condensation of the sulfinate anion with the carbonyl group of the activated thiol ester function releases the phosphorothioate triester (4) with concomitant formation of 5.1a Because of the intramolecular nature of the sulfur-transfer reaction, sulfurization of phosphite triesters in oligodeoxyribonucleoside phosphorothioate syntheses is rapid and complete within 30 s at 25 °C. Formation of the intermediate 3 is rate-limiting, as the nucleophilicity of phosphite triesters depends on steric and electronic factors. A striking example of such subtle effects relates to the sulfurization of N4-benzoyl-5-o-(4,4-dimethoxytrityl)-2-O-tert-butyldimethylsilyl cytidylyl-(3 -> 5)-N4-benzoyl-2-O-tert-butyldimethylsilylcytidine methylphosphite triester being complete within 30 s upon reaction with 2,6 whereas sulfurization of 5-o-(4,4-dimethoxytrityl)-2-o-methyluridylyl-(3 -> 5)-N4-benzoyl-2-O-methylcytidine(2-cyanoethyl) phosphite triester or its 2-O-tert-butyldimethylsilyl congener under the same conditions reportedly takes 300 s for completion of the reaction.7

The production of 3H-1,2-benzoxathiolan-3-one 1-oxide (5) during the course of the sulfur-transfer reaction effected by 2 appears counterproductive considering that 5 is a potent oxidizing reagent.1a It should, however, be noted that the concentration of 5 generated during sulfurization is at least 50-fold lower than that of 2 at all times. Thus, oxidation of phosphite to phosphate triesters caused by 5 during sulfurization is negligible.

Stability of the sulfur-transfer reagent 2 in solution has been questioned by us1a and others.8-12 The reagent decomposes slowly when in contact with glass and/or metallic surfaces. Consequently, any amber glass containers suitable for storing acetonitrile solutions of 2 must first be completely immersed in concentrated sulfuric acid for at least 15 h before being rinsed with distilled water and dried. The container is then filled to ~20% of its volume with a 10% solution of dichlorodimethylsilane in dichloromethane and vigorously shaken for ~5 min to ensure uniform distribution of the solution all over its internal surface area. The solution is discarded and the siliconized glass container is thoroughly rinsed with 50% aqueous methanol before being dried in an oven at 110 °C for ~1 h. Prior to oligonucleotide synthesis, the siliconized glass container is carefully rinsed with acetonitrile to remove any foreign particulates. A 0.05 M solution of 2 in HPLC grade acetonitrile is then filtered through a 45 mm teflon membrane into the siliconized container and stored under a positive pressure of argon on the DNA/RNA synthesizer. Given the incompatibility of 2 with DNA/RNA synthesis reagents, namely, the aqueous iodine solution, the capping solutions, and ammonium hydroxide, it is critically important that the teflon reagent line be carefully cleaned prior to coupling the siliconized container filled with reagent 2 to the instrument. By fulfilling these precautionary measures, acetonitrile solutions of 2 can be used for months on a DNA/RNA synthesizer without noticeable precipitation.

35S]-3H-1,2-benzodithiol-3-one 1,1-dioxide in the Synthesis of 35S-Labeled Oligonucleoside Phosphorothioates

To facilitate in vivo pharmacokinetic studies of potential therapeutic oligonucleotides such as those DNA, RNA or chimeric RNA-DNA sequences modified with internucleotidic phosphorothioate linkages, mixed phosphodiester-phosphorothioate linkages or mixed phosphorothioate-methyl phosphonate linkages, 35S-labeling of these oligonucleotide analogues is a viable approach. In an attempt to prepare the sulfur-transfer reagent [35S]-2 for this purpose, the synthesis of [35S]-1 is accomplished according to 3.13

Typically, thiosalicylic acid (6) is heated with 35S-thiobenzoic acid (7) and concentrated sulfuric acid at 50 °C to afford [35S]-3H-1,2-benzodithiol-3-one in 60% yield. Conversion of [35S]-1 to its 1,1-dioxide [35S]-2 is performed according to 1. The radiolabeled sulfur-transfer reagent exhibits a specific activity of 91 mCi/mmol and is isolated in ~30% yield from 6.13

Sulfurization of H-phosphonate and H-phosphonothioate Diesters using 3H-1,2-benzodithiol-3-one 1,1-dioxide

Considering the efficiency with which phosphite triesters are converted to the corresponding phosphorothioate triesters upon reaction with 2, investigations on the use of 3H-1,2-benzodithiol-3-one 1,1-dioxide in the sulfurization of H-phosphonate and H-phosphonothioate diesters were undertaken.14 Conversion of the dinucleoside H-phosphonothioate diester (8) to its dithioated diester (9) is accomplished quantitatively within 30 s when a 0.02 M solution of 2 or 1 in 2% aqueous pyridine is used as a sulfurization reagent.

However, conversion of the dinucleoside H-phosphonate diester 10 to the phosphorothioate analogue 11 effected by a 0.02 M solution of 2 in 2% aqueous pyridine is relatively slow; it reportedly takes ~3 h for a complete reaction.14a While a solution of 2 in 2% aqueous acetonitrile containing triethylamine can completely transform 10 into 11 within 30 s, the use of this sulfurization mixture is incompatible with automated solid-phase oligonucleotide synthesis given the rapid formation of a yellow precipitate caused by triethylamine. In the absence of triethylamine, 10 is not sulfurized under these conditions. Nonetheless, 10 is completely converted to 11 within 20 min when a 0.02 M solution of 1 in 2% aqueous pyridine is used for the sulfurization reaction. Thus, 3H-1,2-benzodithiol-3-one in aqueous pyridine is compatible with automated solid-phase synthesis of both DNA and RNA oligonucleoside phosphorodithioates or phosphorothioates from appropriate H-phosphonate derivatives.14a

3H-1,2-benzodithiol-3-one 1,1-dioxide and other Sulfur- Transfer Reagents

Because of its efficiency and rapid sulfurization kinetics, 3H-1,2-benzodithiol-3-one 1,1-dioxide has served over the years as a reference standard in the evaluation of a number of sulfurizing reagents for the synthesis of oligonucleoside phosphorothioates.9,11,15,16 These include phenylacetyl disulfide,17 tetraethylthiuram disulfide,18 dibenzoyl tetrasulfide,19 bis(O,O-diisopropoxyphosphinothioyl)disulfide,20 benzyltriethylammonium tetrathiomolybdate,12 sulfur-triethylamine,15b bis(ethoxythiocarbonyl)tetrasulfide,10 bis(arylsulfonyl)disulfide,11 pyridinium tetrathionate,11 bis(isopropylsulfonyl) disulfide,11 thiiranes,22 1,2, 4-dithiazoline-3,5-dione,8,21 3-ethoxy-1,2,4-dithiazoline-5-one,8,21 and 3-methyl-1,2,4-dithiazoline-5-one.16

Additional Applications of 3H-1,2-benzodithiol-3-one 1,1-dioxide

In addition to being a potent sulfurizing reagent for phosphite triesters and being particularly useful in the synthesis of phosphorothioated oligonucleotides, 3H-1,2-benzodithiol-3-one 1,1-dioxide has also been used in the preparation of phosphorothioated phospholipid derivatives.23 Also noteworthy is the reaction of 3H-1,2-benzodithiol-3-one 1,1-dioxide with excess thiols to generate unstable hydrosulfides, which have been demonstrated to efficiently cause single strand breaks in duplex DNA.24 Incidentally, the reaction of thiols with 2 is directly analogous to that of phosphite triester with 3H-1,2-benzodithiol-3-one 1,1-dioxide.


1. (a) Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan, J. B.; Beaucage, S. L., J. Org. Chem. 1990, 55, 4693. (b) Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L., J. Am. Chem. Soc. 1990, 112, 1253. (c) Regan, J. B.; Phillips, L. R.; Beaucage S. L., Org. Prep. Proc. Int. 1992, 24, 488.
2. Crooke, S. T.; Bennett, C. F., Annu. Rev. Pharmacol. Toxicol. 1996, 36, 107.
3. Beaucage, S. L.; Iyer R. P., Tetrahedron 1993, 49, 6123.
4. Hortmann, A. G.; Aron, A. J.; Bhattacharya A. K., J. Org. Chem. 1978, 43, 3374.
5. Marchán, V.; Gibert, M.; Messeguer, A.; Pedroso, E.; Grandas, A., Synthesis 1999, 43.
6. Morvan, F.; Rayner, B.; Imbach, J.-L., Tetrahedron Lett. 1990, 31, 7149.
7. DiRenzo, A.; Grimm, S.; Levy, K.; Haeberli, P.; Maloney, L.; Usman, N.; Wincott, F., Nucleosides Nucleotides 1996, 15, 1687.
8. Xu, Q.; Musier-Forsyth, K.; Hammer, R. P.; Barany, G., Nucleic Acids Res. 1996, 24, 3643.
9. Zhang, Z.; Nichols, A.; Tang, J. X.; Alsbeti, M.; Tang, J. Y., Nucleosides Nucleotides 1997, 16, 1585.
10. Zhang, Z.; Nichols, A.; Alsbeti, M.; Tang, J. X.; Tang, J. Y., Tetrahedron Lett. 1998, 39, 2467.
11. Efimov, V. A.; Kalinkina, A. L.; Chakhmakhcheva, O. G.; Hill, T. S.; Jayaraman, K., Nucleic Acids Res. 1995, 23, 4029.
12. Rao, M. V.; Macfarlane, K., Tetrahedron Lett. 1994, 35, 6741.
13. Iyer, R. P.; Tan, W.; Yu, D.; Agrawal, S., Tetrahedron Lett. 1994, 35, 9521.
14. (a) Stawinski, J.; Thelin, M., J. Org. Chem. 1991, 56, 5169. (b) Stawinski, J.; Thelin, M.; von Stedingk, E., Nucleosides Nucleotides 1991, 10, 517.
15. (a) Wyrzykiewicz, T. K.; Ravikumar, V. T., Bioorg. Med. Chem. Lett. 1994, 4, 1519. (b) Cheruvallath, Z. S.; Cole, D. L.; Ravikumar, V. T., Nucleosides Nucleotides 1996, 15, 1441.
16. Zhang, Z. D.; Nichols, A.; Tang, J. X.; Han, Y. X.; Tang, J. Y., Tetrahedron Lett. 1999, 40, 2095.
17. Kamer, P. C. J.; Roelen, H. C. P. F.; van den Elst, H.; van der Marel, G. A.; van Boom, J. H., Tetrahedron Lett. 1989, 30, 6757.
18. Vu, H.; Hirschbein, B. L., Tetrahedron Lett. 1991, 32, 3005.
19. Rao, M. V.; Reese, C. B.; Zhengyun, Z., Tetrahedron Lett. 1992, 33, 4839.
20. Stec, W. J.; Uznanski, B.; Wilk, A.; Hirschbein, B. L.; Fearon, K. L.; Bergot, B. J., Tetrahedron Lett. 1993, 34, 5317.
21. Xu, Q.; Barany, G.; Hammer, R. P.; Musier-Forsyth, K., Nucleic Acids Res. 1996, 24, 3643.
22. Arterburn, J. B.; Perry M. C., Tetrahedron Lett. 1997, 38, 7701.
23. Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W., J. Org. Chem. 1994, 59, 4805.
24. Breydo, L.; Gates, K. S., Bioorg. Med. Chem. Lett. 2000, 10, 885.

Serge L. Beaucage

Food and Drug Administration, Bethesda, MD, USA



Copyright 1995-2000 by John Wiley & Sons, Ltd. All rights reserved.