Solid-Phase Synthesis of Oligodeoxynucleotides Containing N4-[2-(t-butyldisulfanyl)ethyl]-5-methylcytosine Moieties

An efficient route for the synthesis of the phosphoramidite derivative of 5-methylcytosine bearing a tert-butylsulfanyl group protected thiol is described. This building block is used for the preparation of oligonucleotides carrying a thiol group at the nucleobase at the internal position of a DNA sequence. The resulting thiolated oligonucleotides are useful intermediates to generate oligonucleotide conjugates carrying molecules of interest at internal positions of a DNA sequence.


OPEN ACCESS
We are interested in the incorporation of thiol groups into two-dimensional DNA lattices [17] by introducing a single thiol group per DNA unit. Thiol groups have a strong affinity for gold surfaces and they can be used for the introduction of peptides, proteins and a large number of molecules functionalized with maleimido-or bromo-or iodo-acetyl groups. Previous work has demonstrated that bidimensional DNA arrays carrying N 4 -[2-(t-butyldisulfanyl)ethyl]cytosine residues at the apex of the topological markers can be deposited on gold surfaces [17]. The starting material for the synthesis of the 2'-deoxycytidine phosphoramidite building block carrying the N 4 -(t-butyldisulfanyl)ethyl group was 2'-deoxyuridine [15], which is expensive and the synthetic route is long and tedious. We become interested in optimizing the preparation of a similar building block using thymidine instead of 2'deoxyuridine as starting material. Here, we describe a straightforward synthesis of the 2'-deoxy-N 4 -[2-(t-butyldisulfanyl)ethyl]-5-methylcytidine phosphoramidite building block starting from thymidine. During the use of the new building block a side product coming from the oxidation of the N 4 -(tbutyldisulfanyl)ethyl group was detected. Replacement of iodine solution with a t-butylhydroperoxide one eliminates this side reaction. Finally, the preparation of several fluorescent DNA conjugates using oligonucleotides containing the N 4 -mercaptoethyl-5-methylcytosine is described.
Both DMT-Me C(SS t Bu)-CPG (5) and DMT-C(SS t Bu)-CPG (6) were treated with iodine solution for 5 min and 1 hr and tert-butylhydroperoxide for 3 h. During oligonucleotide synthesis, solid supports are exposed to iodine solution for approx. 1 min per cycle and to tert-butylhydroperoxide for 15 min per cycle. After the treatment with the oxidation solution, the DMT group was removed with trichloroacetic acid and the support treated with ammonia. The resulting nucleosides were analyzed by HPLC. Results are shown in Scheme 2 and Table 1.  Treatment of solid support carrying 5'-DMT-2'-deoxy-N 4 -[2-(t-butyldisulfanyl)ethyl]-5methylcytidine derivative (5) with iodine resulted on the formation of two nucleoside derivatives: the expected 2'-deoxy-N 4 -[2-(t-butyldisulfanyl)ethyl]-5-methylcytidine (7) (retention time 13.6 min, M = 388) and a new nucleoside derivative (retention time 6.2 min, M = 348). HPLC profiles are shown as supplementary material. The new compound was identified as the sulfonic acid derivative 9 resulting from the removal of the t-butylthio group, followed by oxidation of the resulting thiol group to the corresponding sulfonic acid. A 5-min iodine treatment (corresponding to five synthesis cycles) resulted on the formation of 5% of this side compound while 1 h treatment (60 synthesis cycles) resulted on the total conversion of the protected nucleoside to the oxidation product. The use of tertbutylhydroperoxide solution prevented the oxidation reaction (3 h treatment equivalent to 12 synthesis cycles).
On the other hand treatment of solid support carrying 5'-DMT-2'-deoxy-N 4 -[2-(tbutyldisulfanyl)ethyl]cytidine derivative 6, either with iodine or tert-butylhydroperoxide yielded the expected t BuS protected nucleoside as the major compound (> 96%). It has been also described that t BuS protected cysteine is stable to iodine and tert-butylhydroperoxide solutions [21,22]. We hypothesize that the electron donor properties of the methyl group at position 5 is responsible of a slightly higher electron density on the thiol group that may facilitate the oxidation of the sulfur with iodine with subsequent loss of the t BuS group. Although some steric or other effects should be also present as the methyl group at C5 and the sulfur atom are separated by five bonds.
Next we studied the stability of the t BuS protected nucleosides on a dinucleotide sequence (DMT-C Bz p Me C(SS t Bu)-CPG (11) and DMT-C Bz pC(SS t Bu)-CPG (12)) to determine the effect a phosphate group in an environment similar to the one found inside of an oligonucleotide sequence. We used a similar protocol to the one described for the monomer except for the ammonia treatment that was performed at 50 ºC for 6 h (standard conditions for removal of nucleobase protecting groups).  Table 2 shows the results obtained in this study. The formation of the oxidation products were also observed but in a much lower efficiency. For example 1hr treatment of DMT-Cp Me C(SS t Bu)-CPG (11) with iodine solution yielded 19% of side compound ( Table 2). The same treatment on DMT-Me C(SS t Bu)-CPG (5) gave 97% of side product (Table 1). A treatment of 3 h equivalent to 36 synthesis cycles generated a 38% of side compound ( Table 2). As observed in the monomer the side reaction is less efficient with the cytosine derivative than the 5-methylcytosine derivative. A treatment of 3 h of DMT-CpC(SS t Bu)-CPG (12) with iodine solution generated a 12% of side compound instead of 38% that was observed for the support 11 (Table 2). In summary the presence of a nucleotide attached at the 5' position slows the efficiency of the oxidation of t BuS protected nucleoside to sulfonic acid. Table 2. Products resulting from the treatment of DMT-Cp Me C(SS t Bu)-CPG (11) and DMT-CpC(SS t Bu)-CPG (12) with iodine solution.

Solid Support Treatment t BuSS-protected / oxidized (-SO 3 ) 11
Iodine solution, 5 min 96 : 4 11 Iodine solution, 1 h 81 : 19 11 Iodine solution, 3 h 62 : 38 12 Iodine solution, 5 min 100 : 0 12 Iodine solution, 1 h 93 : 7 12 Iodine solution, 3 h 88: 12 Next we studied the effect of the iodine or tert-butylhydroperoxide solutions in the purity of a short oligonucleotide containing one modified nucleoside in the middle of the sequence. Oligonucleotide sequence 5'-d(TTCCAXATTACCG)-3' (being X the position of the N 4 -[2-(t-butyldisulfanyl)ethyl]-5methylcytidine derivative) was prepared on 200 nmol scale using three different methods: a) LV200 ® polystyrene support using iodine solution; b) LV200 ® polystyrene support using tertbutylhydroperoxide solution and c) CPG support using iodine solution.  As it can be seen in Table 3, the oxidation reaction is more pronounced on polystyrene support when iodine solution is used obtaining a 59% of the oligonucleotide carrying the sulfonic acid group 18. When using polystyrene and tert-butylhydroperoxide solution as well as CPG and iodine solution only 1% and 6% of side compound 18 was observed. The formation of the side compound when using polystyrene support and iodine solution may be due to an increase of the local concentration of iodine by absorption of iodine on the polystyrene surface.
In order to analyze the stability of the disulfide bond in the presence of tert-butylhydroperoxide , the oligonucleotide attached to polystyrene and oxidized with tert-butylhydroperoxide was treated with the tert-butylhydroperoxide solution for 6 and 12 h more. The resulting crudes were analyzed by HPLC. Figure 1 shows that the disulfide bond is stable to tert-butylhydroperoxide solution at prolonged periods of time as no increase of the peaks around the elution of the oxidation product was observed. Finally, the use of the modified oligonucleotide for the preparation of oligonucleotides carrying fluorescent compounds was demonstrated. To this end oligonucleotide 17 was treated with tris(2carboxyethyl)phosphine to remove the t BuS group and the resulting free thiol oligonucleotide 19 was reacted with three different fluorescent maleimides (Scheme 5). The desired fluorescent oligonucleotides were obtained as a mixture of isomers that were characterized by UV and mass spectrometry analysis. In the case of the fluorescein diacetate 5-maleimide we observed that the acetate groups are partially hydrolyzed in the reaction conditions yielding a complex HPLC profile (see supplementary material). A short treatment with a bicarbonate solution resulted on the total hydrolysis of the acetate groups obtaining a more simple HPLC profile. Coupling efficiency based on the integration of the HPLC peaks was between 80-84% (Table 4).

General
All reagents were purchased from Aldrich, Sigma or Fluka (Sigma-Aldrich Química S.A., Spain) and were used without further purification. Phosphoramidites and ancillary reagents used during oligonucleotide synthesis were from Applied Biosystems (PE Biosystems Hispania S.A., Spain). Flash column chromatography was carried out on silica gel SDS 0.063-0.2 mm/70-230 mesh. NMR spectra were recorded on a Varian Mercury 400 spectrometer operating at 400 MHz ( 1 H) and 100 MHz ( 13 C). Chemical shifts are reported in ppm relative to the singlet at δ = 7.24 ppm of CHCl 3 for 1 H-NMR and to the centre line of the triplet at δ = 77.0 ppm of CDCl 3 for 13 C-NMR. J values are given in Hz. 31 P-NMR spectra were recorded on a Varian Inova 300 spectrometer. HPLC separations were performed using a Waters HPLC system. MALDI mass spectrometry was recorded on a Fisons VG Tofspec spectrometer and ESI mass spectra on a Fisons VG Platform II spectrometer. Oligonucleotide sequences were prepared using solid phase methodology. The syntheses of oligonucleotides were carried out on an Applied Biosystems Model 3400 DNA synthesizer. Details of the synthesis of 2aminoethyl-tert-butyl disulfide (1) [15] and DMT-T (2) are described as supplementary material. HPLC profiles are shown as supplementary material.

Synthesis of 2'-deoxy-5'-O-(4,4'-dimethoxytriphenylmethyl)-N 4 -(tert-butyldithio-ethyl)-5-methylcytidine (3)
DMT-T (2, 1.25 g, 2.30 mmol) was dried by evaporation of anhydrous ACN under reduced pressure, and the residue was dissolved in anhydrous DMF (30 mL) under argon. Hexamethyldisilazane (0.96 mL 4.6 mmol) was added with a syringe to the solution with exclusion of moisture. After 30 min of magnetic stirring at room temperature, the reaction was complete as judged by TLC (ethyl acetate, R f = 0.61). Then, the solution was evaporated under reduced pressure. The residue was dissolved in toluene (4 × 10 mL) and concentrated to dryness. 1,2,4-Triazole (1.67 g, 24.15 mmol) was dried by evaporation of anhydrous ACN under reduced pressure, and the residue was dissolved in anhydrous ACN (40 mL) under argon. Triethylamine (3.7 mL, 26.45 mmol) was added and the solution was cooled on ice. Then, phosphorus oxychloride (0.53 mL, 5.75 mmol) was added with a syringe to the solution with exclusion of moisture. After 30 min of magnetic stirring at T = 0 ºC, the protected nucleoside dissolved in anhydrous ACN (40 mL) was added dropwise to the solution under argon. The reaction mixture was stirred at room temperature. After 6 hours, the reaction was complete as judged by TLC (ethyl acetate, R f = 0.39). The solvent was removed under reduced pressure and the residue dissolved in DCM (50 mL). The solution was washed with saturated aqueous NaCl (50 mL). After drying the organic phase with Na 2 SO 4 , the solvent was evaporated under reduced pressure. The residue was dissolved in dry DMF (20 mL) under argon. Compound 1 (0.57 g, 3.45 mmol) and DBU (0.52 mL, 3.45 mmol) were added to the solution. The reaction mixture was stirred overnight at room temperature. DBU was added (0.34 mL, 2.30 mmol) and the reaction mixture was stirred at room temperature. After 6 hours, the solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (100 mL). The solution was washed with saturated aqueous sodium chloride (100 mL). After drying the organic phase with sodium sulphate, the solvent was evaporated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and purified by chromatography on silica gel. The column was packed with silica gel using a 1% triethylamine solution in ethyl acetate. The product was eluted with a gradient of methanol from 0 to 5% in ethyl acetate. The pure compound was obtained as pale yellow foam (0.85 g, 53%). TLC (3% methanol in ethyl acetate) R f = 0. 25

Synthesis of 2'-deoxy-5'-O-(4,4'-dimethoxytriphenylmethyl)-N 4 -(2-aminoethyldi-thioethyl)-5methylcytidine-3-O-(2-cyanoethyl-N,N'-diisopropylphosphoramidite) (5)
Compound 3 (200 mg, 0.22 mmol) was dried by evaporation of anhydrous ACN in vacuo, and the residue was dissolved in anhydrous DCM (10 mL) under argon. N,N'-diisopropylethylamine (154 µL, 0.88 mmol) was added with exclusion of moisture. The solution was cooled on ice and 2-cyanoethoxy-N,N'-diisopropylaminochlorophosphine (73 µL, 0.33 mmol) was added dropwise with a syringe. Afterward, the solution was stirred at room temperature for 1 h. After this time, TLC (1% cyclohexane in ethyl acetate) showed the presence of the starting compound. As a consequence more 2cyanoethoxy-N,N-diisopropylaminochlorophosphine (26 μL, 0.11 mmol) once the mixture was cooled on ice. After the addition the reaction mixture was allowed to stir at room temperature for 1 h. The solvent was removed in vacuo and the residue was dissolved in dichloromethane (20 mL). The solution was washed with 5% aqueous sodium hydrogen carbonate (20 mL) and with saturated aqueous sodium chloride (20 mL). After drying the organic phase with sodium sulphate, the solvent was evaporated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate/cyclohexane 2:1 and purified by chromatography on silica gel. The column was packed with silica gel using a 5% triethylamine solution in ethyl acetate/cyclohexane 2:1. The product was eluted with ethyl acetate/cyclohexane 2:1. The pure compound was obtained as pale yellow foam (220 mg, 86%). TLC (1% cyclohexane in ethyl acetate) R f = 0.39 and 0.26. 1

Study of the stability of DMT-Me C(SS t Bu)-CPG under oxidative conditions and comparison with DMT-C(SS t Bu)-CPG
The solid support functionalized with the cytidine derivative, 5'-DMT-N 4 -(2aminoethyldithioethyl)cytidine (DMT-C(SS t Bu)-CPG) (6) was synthesized as described in [15]. Five mg of each solid support (DMT-C(SS t Bu)-CPG) (6) and (DMT-Me C(SS t Bu)-CPG) (5) were treated with oxidation solutions (iodine or t-butylhydroperoxide). Iodine solution was the same solution used in synthesis of oligonucleotides (0.02 M iodine in water/ pyridine/ tetrahydrofuran). The 10% t-butylhydroperoxide solution was prepared freshly by mixing 86.4 mL of acetonitrile and 14.3 mL of commercially available 70% t-butylhydroperoxide solution in water. At different time intervals the solution were filtered and the resulting solid support washed with ACN. The resulting supports were treated with a solution of trichloroacetic acid 3% in DCM for 5 min to remove the DMT group and with aqueous concentrated ammonia for 30 min to cleave the product from the resin. The mixture was filtered and the ammonia solution was concentrated to dryness. The resulting products were analyzed by HPLC (Column: X-Bridge TM OST C 18 (2.5 μm; 4.6 × 50 mm); solvent A: 100 mM triethylammonium acetate (pH = 7) and solvent B: 70% ACN in 100 mM triethylammonium acetate (pH = 7). Flow rate: 1 mL/min. Conditions: 2.5 min 100% A, then 5.5 min linear gradient from 0-12.5% B, then 12 min linear gradient from 12-100% B, and mass spectrometry. HPLC profiles are shown as supplementary material.

Analysis of the stability of DMT-Cp( Me C(SS t Bu))-CPG and DMT-Cp(C(SS t Bu))-CPG solid supports to oxidative conditions
DMT-dC Bz phosphoramidite was incorporated in the solid supports 5 and 6 previously synthesized in a DNA synthesizer (Applied Biosystems 3400) on 1 μmol scale using commercially available chemicals. The synthesis was carried out using the standard phosphite triester methodology. For the oxidation step was used a solution of tert-butyl hydroperoxide 10% in ACN instead of the commercially available solution of iodine 0.02 M. In both cases the synthesis was carried out without the removal of the 5'-DMT group.
The analysis of the stability of the thiolated nucleosides under oxidation conditions were studied as described for supports 5 and 6. Five mg of each solid support were treated with iodine solution. At different time intervals the solution were filtered and the resulting solid support washed with acetonitrile. The resulting supports were treated with a solution of trichloroacetic acid 3% in DCM for 5 min to remove the DMT group and with aqueous concentrated ammonia at 55 ºC for 6 h to cleave the products from the support and to remove the benzoyl group. The mixtures were filtered and the ammonia solutions were concentrated to dryness. The resulting products were characterized by HPLC (Column: X-Bridge TM OST C 18 (2.5 μm, 4.6 × 50 mm); solvent A: 100 mM triethylammonium acetate (pH = 7) and solvent B: 70% ACN in 100 mM triethylammonium acetate (pH = 7).
The protecting group for dC and dA was the benzoyl (Bz) group. The isobutyryl ( i bu) group was used for protection of dG. In all cases, at the end of the synthesis the DMT group was removed. The coupling efficiency for the modified phosphoramidite was between 95-99% (as measured by the absorbance of the DMT group). After the assembly of the sequence the solid supports were treated with concentrated aqueous ammonia for 6 h at 55 ºC. Under these conditions, all base and phosphate groups were also removed. The mixtures were filtered and the ammonia solutions were concentrated to dryness. The resulting crudes were analyzed by HPLC (Column: X-Bridge TM OST C 18 (2.5 μm; 4.6 × 50 mm); solvent A: 5% ACN in 100 mM triethylammonium acetate (pH = 7) and solvent B: 70% ACN in 100 mM triethylammonium acetate (pH = 7). Flow rate: 1 mL/min. Conditions: 10 min linear gradient from 0-30%B. The different peaks observed in the HPLC profiles were analyzed by mass spectrometry. HPLC profiles are shown as supplementary material.

Conjugation with tetramethylrhodamine-5-maleimide
The HPLC analysis of the crude revealed the presence of four bands between t R = 7.6 min and t R = 8.3 min. Only the band with t R = 7.9 min showed an UV spectra consistent with an oligonucleotide (λ max = 262 nm) with rhodamine (λ max = 554 nm). The other three peaks showed λ max around 556 nm without the maxima at 260 nm, indicating that they are tetramethylrhodamine-5-maleimide derivatives that could not be removed with a NAP-5 column. This is due to the positive charge of tetramethylrhodamine that interacts with the negative charge of the phosphate bonds. The conjugate was characterized as well by EM (MALDI-TOF). The coupling efficiency was determined from the HPLC profile (~80%). Compound 21: t R = 7.9 min, ESI-MS m/z (negative mode [M-H] -) calc for C 156 H 189 N 46 O 82 P 12 S 4424.20, found 4422.58.

Conclusions
The excellent molecular recognition properties of synthetic oligonucleotides are being exploited for the rational construction of 2-D DNA lattices with well defined structures [23,24]. A step ahead is to incorporate chemical groups at specific sites of 2-D DNA lattices to direct the deposition of specific molecules or nanomaterials [25] or to direct deposition to surfaces [17]. In this article we have described the synthesis of the 2'-deoxy-N 4 -[2-(t-butyldisulfanyl)ethyl]-5-methylcytidine phosphoramidite building block. This compound has been developed for the specific introduction of thiol groups at the nucleobase. During the use of the new building block a side reaction was observed. This side reaction has not been observed previously yielding a non reactive sulfonic acid. The side reaction is mostly observed in the 5-methylcytosine derivative and using iodine solution for the oxidation of phosphites on polystyrene supports. Replacement of iodine solution with a solution of tbutylhydroperoxide eliminates this side reaction. Several examples of the use of oligonucleotides containing N 4 -mercaptoethyl-5-methylcytosine for the preparation of fluorescent DNA conjugates are described.