Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis , MN 55455-0431, USA and ¹ Department of Chemistry, Louisiana State University, Baton Rouge , LA 70803, USA

Received July 12, 1996 ; Revised and Accepted July 28, 1996

ABSTRACT

We recently reported on the use of 1,2,4-dithiazolidine- 3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents for the preparation of phosphorothioate-containing oligodeoxyribonucleotides [Xu et al . (1996) Nucleic Acids Res. , 24, 1602-1607]. One challenge in automated solid-phase synthesis of phosphorothioate-containing RNA is to develop sulfurization reagents that are effective in the presence of bulky 2 ' -OH protecting groups. The present study demonstrates that EDITH is exceedingly effective at low concentrations (0.05 M) and short reaction times (2 min) for the automated synthesis of oligoribonucleotides.

Phosphorothioate-containing nucleic acids have found widespread use in molecular biology ( 2 , 3 ). The increased resistance to nuclease digestion that is displayed by sulfur-containing backbone analogues has prompted the consideration of these molecules for medical purposes. At least one phosphorothioate antisense DNA oligonucleotide is in phase II clinical trials ( 4 ). Antisense RNAs are potentially valuable therapeutic agents as well ( 5 ). Backbone modifications, including phosphorothioate substitutions, are also being explored as an approach for increasing the nuclease resistance, and thus enhancing the therapeutic potential, of ribozymes ( 6 ).

The introduction of phosphorothioates into RNA can be achieved in several ways ( 7 - 9 ). For oligoribonucleotides that are accessible by automated, solid-phase phosphoramidite chemistry, sulfur can be introduced at any internucleotidic linkage with 3 H -1,2-benzodithiol-3-one l, l-dioxide, the Beaucage reagent ( 8 ). Alternatively, solid-phase synthesis with H-phosphonate chemistry can introduce phosphorothioates into oligoribonucleotides at all linkages ( 9 ). One challenge in automated RNA synthesis is to find reagents that are effective in the presence of the bulky 2'- O -silyl protecting group. This paper describes the use of 3-ethoxy-1,2,4-dithiazoline-5-one (`EDITH') to create phosphorothioate-containing oligoribonucleotides using phosphoramidite chemistry, with superior results over existing reagents.

Oligoribonucleotide hexamers containing a phosphorothioate diester at every position were prepared with EDITH, the Beaucage reagent, or 1,2,4-dithiazolidine-3,5-dione (DtsNH) by carrying out the sulfurization reaction after each coupling cycle during automated chain assembly. Materials and Methods were as described elsewhere ( 1 , 10 ), with the following additions. Separations of crude oligoribonucleotides that were fully sulfurized were achieved by ion-exchange high performance liquid chromatography using a Dionex NucleoPac PA-100 column (0.4 * 25 cm) heated to 60^oC to minimize secondary structural effects ( 11 , 12 ). RNA synthesis was accomplished essentially as described ( 13 ), except that the activator 5-ethylthio-1H-tetrazole from American International Chemical, Inc. (Natick, MA) was used for the coupling reactions ( 14 ). The capping step in the synthesis cycle was performed after the sulfurization reaction ( 15 ). The remaining steps in the synthesis cycle were performed according to standard Pharmacia Gene Assembler protocols, and all oligoribonucleotides were synthesized trityl-off. Cleavage of oligoribonucleotides from the polymer support and cleavage of side-chain protecting groups were as previously described ( 13 ). The 2'-silyl protecting groups were removed by incubation at 25^oC for 24 h in neat triethylamine trihydrofluoride (Aldrich), and oligoribonucleotides were recovered by n -butanol precipitation ( 16 ). DtsNH and the Beaucage reagent appear to be less effective than EDITH at sulfurizing an RNA hexamer, as revealed by ³¹ P NMR analysis (Table 1 ). These results were confirmed by anion exchange chromatography (data not shown), wherein the amount of product containing a singly oxygenated species can be quantified ( 1 , 12 ). Reaction with EDITH resulted in only 5.5% of this undesired product, confirming a >99% sulfurization efficiency at each step. Higher levels of approximately 13 and 21% of the singly oxygenated species, were obtained using the Beaucage reagent and DtsNH, respectively. These latter numbers correspond to approximately 98 and 96% sulfurization efficiency.

Oligoribonucleotide 19- or 20mers containing a phosphorothioate diester at a single position or at every position were also prepared. Figure 1 shows a direct comparison of the anion exchange HPLC results obtained with the sequence S-U ₁₉ A prepared with either EDITH (Fig. 1 A) or the Beaucage reagent (Fig. 1 B). The major peaks (I) correspond to the fully sulfurized product and the small shoulder peaks (II) were confirmed to be the singly-oxygenated species by co-injection of the analogous 20mer prepared with a single phosphodiester linkage (data not shown). Once again, the amount of singly oxygenated species (5.6%) indicates that >99% sulfurization efficiency was obtained at each step using EDITH. The amount of singly oxygenated species obtained using the Beaucage reagent (27.5%) suggests that the sulfur transfer efficiency was only 98% at each step, very similar to the RNA hexamer result described above. These results were confirmed by ³¹ P NMR spectroscopy.

Table 1 . Sulfurization of RNA oligonucleotides by various reagents

Sulfurizing	Conc. (M) /	RNA	Distribution (%) b
reagent	time (min) a	sequence	P=O	P=S
DtsNH	0.20/5.0	S-U 5 A	4	96
EDITH	0.05/2.0	S-U 5 A	N.D.	>99
Beaucage	0.05/2.0	S-U 5 A	1	99
EDITH	0.05/2.0	S-U 19 A	N.D.	>99
Beaucage	0.05/2.0	S-U 19 A	2	98
EDITH	0.05/2.0	i	N.D.	>99
Beaucage	0.05/2.0	i	4	96
EDITH	0.05/2.0	ii	95	5
Beaucage	0.05/2.0	ii	96	4

S-U ₅ A: 5'-U _s U _s U _s U _s U _s A-3' S-U ₁₉ A: 5'-U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s U _s A-3' i: 5'-A _s A _s U _s C _s C _s U _s C _s U _s C _s U _s C _s G _s C _s C _s G _s A _s C _s C _s A-3' ii: 5'-AAUCCUCUCUCGCC _s GACCA-3' ^a The sulfurizing reagent solution (1.25 ml/cycle) was recycled through the column at a flow rate of 2.5 ml/min. ^{b 31} P nuclear magnetic resonance (NMR) spectroscopy was used to analyze phosphorothioate-containing oligoribonucleotides: fully deprotected material from 1 [mu]mol scale syntheses was dissolved in 0.25 ml of DEPC-treated H ₂ O, and spectra were recorded on a Varian VXR 300 MHz NMR spectrometer, and referenced to external 85% phosphoric acid. The relative amounts of P=S ([delta] ~58 p.p.m.) and P=O ([delta] ~0 p.p.m.) were determined by integration of the ³¹ P NMR spectra. N.D., not detected.

Figure 1 . Both anion-exchange HPLC and ³¹ P NMR (insets) of the oligoribonucleotide S-U ₁₉ A containing multiple phosphorothioate linkages. A two-eluent system was used: (A) 20 mM LiClO ₄ /20 mM NaOAc-HOAc, pH 6.5, CH ₃ CN (10:1); (B) 0.8 M LiClO ₄ /20 mM NaOAc-HOAc, pH 6.5, CH ₃ CN (10:1). Elution was at 1.2 ml/min by a gradient starting from 100% eluent A increased over 18 min by 2.8%/min of eluent B. Phosphorothioate linkages were established by 2 min reactions with 0.05 M sulfurizing reagent in CH ₃ CN. Sulfurization was performed with EDITH ( A ) or with the Beaucage reagent ( B ).

A mixed 19mer oligoribonucleotide containing all four bases and phosphorothioates at every position (Table 1 , sequence i) or at a single position (Table 1 , sequence ii) was synthesized using both EDITH and the Beaucage reagent. In all cases, use of EDITH resulted in nearly quantitative sulfur transfer. The Beaucage reagent performed similarly to EDITH in the case of the singly-sulfurized sequence (Table 1 ). However, only 96% sulfurization per step was obtained using the Beaucage reagent in the synthesis of the multiply-sulfurized RNA 19mer. Polyacrylamide gel analysis of the mixed sequences was also carried out (data not shown), and the results confirm that comparable yields of full-length products were obtained with EDITH and the Beaucage reagent.

In summary, we have previously shown that the new sulfurizing agent EDITH offers several advantages over existing reagents in the automated synthesis of phosphorothioate-containing DNA oligonucleotides ( 1 ). The reagent is relatively easy and inexpensive to prepare ( 10 ), and is stable in solution ( 1 ). The present work demonstrates that EDITH is also a highly effective sulfurization agent for incorporating single or multiple phosphorothioate linkages into the very sterically demanding 2'- O -silyl protected RNA oligomers.

ACKNOWLEDGEMENTS

We thank Mr Lin Chen for preparing some of the DtsNH and EDITH used in this work. Financial support from NIH grants GM 28934, 42722 and 43552 to GB; GM 49928 to KMF; and grants LEQSF(RF/1993-1996)-RD-A-42 and NSF CHE9500992 to RPH are gratefully acknowledged.

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