THIOPHOSPHORAMIDITES AND THEIR USE IN SYNTHESIZING OLIGONUCLEOTIDE
PHOSPHORODITHIOATE LINKAGES

Xianbin Yang
AM Biotechnologies, 6023 Ave S.
Galveston, Texas 77551

Introduction

Replacing two non-bridging oxygen atoms with sulfur atoms in a DNA phosphodiester linkage creates a phosphorodithioate (PS2) linkage.¹ Like natural DNA, the phosphorodithioate linkage is achiral at phosphorus. Additionally, recent research has demonstrated that this analog is completely resistant to nuclease degradation and forms complexes with DNA and RNA with somewhat reduced stabilities.² Moreover, it has been found that PS2-ODNs bind proteins with a higher affinity than their phosphodiester analogues^2-6 suggesting that PS2-ODNs may have additional utility in the form of sulfur-modified phosphate ester aptamers (thioaptamers)^3,6-8 for therapeutic and diagnostic applications. The biological interest and promise of the PS2-ODNs has spawned a variety of strategies for synthesizing, isolating, characterizing and purifying this compound over the past two decades.⁹ However, the thiophosphoramidite building blocks necessary to synthesize a PS2-ODN have only now become commercially available after recent work at AM Biotechnologies (www.thioaptamer.com). This article highlights a working protocol that enables the widespread use of thiophosphoramidites for the synthesis of PS2-ODNs.

Figure 1: structures of the thiophosphoramdites (thioPA) dA, dC, dG and dT
dA-ThioPA	dC-ThioPA
dG-ThioPA	dT-ThioPA

MATERIALS AND METHODS

Chemicals

Synthesis columns, thiophosphoramidites, and synthesis reagents were obtained from Glen Research, Sterling, VA. Dichloromethane (DCM) anhydrous (#270997), 3Å Molecular Sieve beads (8-12 mesh, #208582), DL-dithiothreitol (#D9779-1G), Ammonium hydroxide (#338818), HPLC grade ethanol (#459828) were from Sigma-Aldrich.

Protocol or guidelines

The structures of thiophosphoramidites (thioPA) are shown in Figure 1. Like normal DNA and RNA phosphoramidites, the dried solid form of the thioPAs is very stable at -20°C for at least one year based on 31P-NMR analysis with no observed reduction in reactivity for synthesizing the PS2 linkage. However, a few simple modifications to standard synthesis protocols are necessary when using the thioPAs.

(1) Unlike normal DNA phosphoramidites, the thioPAs are not completely soluble in anhydrous acetonitrile diluent. Rather, 10% DCM (v/v) in acetonitrile is an ideal diluent for all four of the thioPAs for a final amidite concentration of
0.15 M.

(2) Additionally, while normal DNA phosphoramidites are very stable in anhydrous acetonitrile at room temperature, the thioPAs are somewhat less stable in anhydrous acetonitrile containing 10% DCM; however, the coupling efficiency of all four thioPAs is not reduced after two days in solution at room temperature. Therefore, for best results the thioPA solution should be used within two days.

(3) To avoid the slight chance of some solid precipitant in the reagent bottle, the thioPA bottle on the synthesizer should be replaced with one containing acetonitrile diluent shortly after the completion of the synthesis. Flushing the synthesizer line with acetonitrile is highly recommended.

A Synthesis Cycle for a PS2 linkage

Thiophosphoramidite Activators

Figure 2: Coupling efficiency using tetrazole or DCI as activator.	Figure 3: Sulfurization efficiency comparison using DDTT vs Beaucage Reagent.
Coupling efficiency using Tetrazole or DCI as activator. ThioPA concentration: 0.15 M in acetonitrile containing 10% dichloromethane; the volume of thioPA: 0.125 mL.	Sulfurization efficiency comparison for the synthesis of 5’-GPS2T-3’ by using DDTT vs Beaucage Reagent. dG-thiophosphoramidite concentration: 0.15 M in CH3CN containing 10% DCM; the volume of thioPA: 0.125 mL.

Tetrazole has been commonly used as an activator of choice for phosphoramidite chemistry. However, unpublished studies have shown that tetrazole is not the most efficient activator for the thioPA coupling reaction. It is imperative to use an efficient activator during commercial PS2 production.

Activators that are commercially available include 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), 4,5-dicyanoimidazole (DCI), and 5-(bis-3.5-trifluoromethylphenyl)-1H-tetrazole (Activator-42). The coupling efficiency of all of these activators during PS2 synthesis was compared on an Expedite 8909 DNA synthesizer. Significant differences were observed, which suggests that the thioPA activation mechanism differs from that of the normal phosphoramidite. Therefore, a practical protocol for use on an Expedite 8909 DNA synthesizer was developed for the commonly used tetrazole activator; however, another protocol for the DCI activator, which is more efficient for PS2 linkage synthesis, was also developed.

(a) Use of Tetrazole as a thioPA Activator

(http://www.glenresearch.com/Technical/Expedite_8909_PS2_Tetrazole_Protocol.PDF)

A dimer model sequence, 5’-XPS2T-3’, was used to study the thioPA coupling efficiency. Tetrazole was used as the activator and DDTT as a sulfurizing reagent. The experiments demonstrated that to achieve > 93% coupling yield for all of the thioPAs, a coupling time of 9 minutes was required. Even the dC-thioamidite exhibited a very slow rate (Figure 2) of PS2 linkage formation using tetrazole.

(b) Use of DCI as a thioPA Activator

(http://www.glenresearch.com/Technical/Expedite_8909_PS2_DCI_Protocol.PDF)

DCI is emerging as an alternative activator to tetrazole. DCI is soluble in acetonitrile up to 1.1 M at room temperature and experiments with thioPAs have shown that DCI decreases the time required to achieve up to 96% coupling yield by a factor of three as compared with tetrazole (Figure 2).

Sulfurizing Reagents in the Synthesis of PS2 Linkage

Beaucage Reagent is a very popular sulfurizing agent for the synthesis of phosphoromonothioate linkages using normal phosphoramidites. When Beaucage Reagent was used with the thioPAs to synthesize PS2 linkages, it was observed that the by-product formed in the sulfurization reaction oxidizes the thiophosphite triester. This oxidation leads to phosphoromonothioate by-products, thus lowering the desired product yield and complicating the purification of the desired product. In addition, Beaucage Reagent has a tendency to precipitate from solution and clog the solvent and reagent transfer lines of a DNA synthesizer. DDTT is another sulfurizing agent primarily used to synthesize RNA phosphoromonothioates; however, it can be used to synthesize DNA phosphoromonothioates as well. Comparing DDTT efficiency to Beaucage Reagent in sulfurizing PS2 linkages, it was found that DDTT has slightly better sulfurizing reactivity than Beaucage Reagent (Figure 3); however, importantly, DDTT reduces the formation of phosphoromonothioate linkages during the synthesis of PS2 linkages thus increasing product yield.

Cleavage from the Support and Removal of Base, Phosphate and Thiophosphate Protecting Groups

Upon completion of the automated synthesis, the support was removed from the synthesizer and dried with argon. The support was transferred into a 4 mL sealable vial where 1 mL of concentrated ammonia:ethanol (3:1, V:V) mix containing 20 mM DTT was added to the vial. The vial was sealed and incubated at 55 °C for 15-16 h. After the vial was removed from the oven and cooled to room temperature, the solution was transferred to a larger vial and 4~5 mL of distilled water was added. Solvents were removed by lyophilization.

Example

The above protocol was used to synthesize a model sequence: 5’-TPS2TPS2TPS2TPS2TPS2T-3’. The 31P-NMR spectra are provided in Figure 4.

Figure 4: 31P-NMR spectra of 5’-TPS2TPS2TPS2TPS2TPS2T-3’.

31P-NMR spectra of 5’-TPS2TPS2TPS2TPS2TPS2T-3’. T-thiophosphoramidite concentration: 0.15M ; T-thiophosphoramidite volume: 0.125ml ; sulfurization reagent : DDTT ; coupling time : 3 minutes ; Activator: (A) Tetrazole and (B) DCI.

Synthesis of Chimeric DNA Containing PS2 Linkage(s)

It should be noted that the Expedite 8909 oxidizing time as well as the oxidizing reagent volumes found in the manufacturer’s protocol are not sufficient to synthesize chimeric DNA containing PS2 linkage(s). The protocols referenced above correct these problems.

ABI Synthesis Cycle
Glen Research has also developed a PS2 synthesis cycle for the ABI394 DNA synthesizer using regular tetrazole or 0.25M DCI activators.
(http://www.glenresearch.com/Technical/ABI_394_PS2_Protocol.PDF)

References

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