H-phosphonates hydroxyl-containing steroids as a reagent for solid-phase synthesis of steroid derivatives of oligonucleotides or their analogs

 

(57) Abstract:

Use: as a reagent for solid-phase synthesis of steroid derivatives of oligonucleotides. The inventive product - H-phosphonate hydroxyl-containing steroids total f-ly 1 , where X is the residue of cholesterol or testosterone. Exit 69 - 89% . table 1.

The invention relates to bio-organic chemistry, namely to new chemical compounds - N-hydroxyl-containing phosphonates steroids of General formula I XO--H where X is the residue of the hydroxyl-containing steroid,

for example:

cholesterol (Chs)

testosterone (TS) which can be used as source reagents for solid-phase synthesis of steroid derivatives of oligonucleotides and their analogs.

Known just one example of solid-phase synthesis cholesterolaemia oligonucleotides based on the use of 2-(cholesteryloxycarbonyl)ethylamine of formula II [1] NH(CH2)2NH-O-cholesterol obtained from cholesterolaemia and Ethylenediamine (output - 76% ). Using this reagent produced a number of oligodeoxyribonucleotides with the rest of cholesterol associated with 31-end mezhnukleotidnyh phosphate. Output cholesterinspiegel of the oligonucleotide - 50% .

Solid phase synthesis of oligonucleotides and their analogues is currently the most widely used method because of its speed and efficiency. Trials of steroid derivatives of oligonucleotides in cell culture systems have shown them to have promise as a means of suppressing the development of viruses. Therefore a very important task is to increase the output steroid derivatives of oligonucleotides and their analogs for solid-phase synthesis of these derivatives.

This task is solved by the fact that, as a reagent for solid-phase synthesis steroidogenesis oligonucleotides and their analogues using new chemical compounds - N-hydroxyl-containing phosphonates steroids of formula 1, which is obtained from the appropriate steroid treatment timidation phosphorus, followed by hydrolysis with water and release by chromatography on silica gel.

The synthesized H-phosphonates hydrox is the breaking of the oligonucleotides of deoxyribo-series the RIBO-some and 21-0-Metallbau-lost, pre-obtained solid phase H-phosphonate method. The obtained results confirm the possibility of solid-phase synthesis of steroid derivatives of oligonucleotides using this reagent, the yield of target products is 80-90% . Conducting the reaction in a solid-phase version greatly simplifies the procedure of excess reagents and can be easily automated.

P R I m e R 1. Synthesis of H-phosphonates hydroxyl-containing steroids.

a) Synthesis of H-phosphonate cholesterol.

The flask with argon 1.2 g (17.5 mmol) of imidazole, previously dried over P2O5at 1-2 mm RT. Art. , dissolved in 60 ml of abs. acetonitrile and cooled with ice. Then added with stirring to 0.35 ml of 5.25 mmol) PCl3and 1.5 ml (18 mmol) of triethylamine. After 15 min added dropwise a solution of pre-dried cholesterol (0.5 g, 1.25 mmol) in 60 ml of abs. chloroform for 30 min, then the cooling is removed and continue stirring for another 2 hours Over the course of the reaction is monitored by thin layer chromatography (TLC) in the system chloroform - ethanol (9: 1). Then the reaction mixture is decomposed by adding 15 ml of water and evaporated to dryness. A mixture of rastaa2SO4, evaporated and chromatographic on silica gel (column 150 ml, a concentration gradient of ethanol (0 -> 50% ) in chloroform containing 1% of triethylamine). The fractions containing the product with Rf= = 0,23 (TLC, chloroform - ethanol 6: 4), evaporated to dryness. Yield 0.6 g (85% ).

The product is homogeneous according to TLC and gives a positive ninhydrin test.

The infrared spectrum, cm-1: 1000, 1060, 1090 (cycle), 1240 (P= O), 1340 (-CH), 1460 (S-CH3), 1600 (C= C), 2400, 2500 (R-OH), 2690 (P-H), 2860, 2950 (CH2-, -CH3WITH-N).

31P-NMR,M. D.(CDCl3): 0,86 (J = 618 Hz)

1H-NMR,M. D.(CDCl3): OF 0.82 (S, 3H, 13-CH3), TO 0.88 (S, H, -(CH3)2), OF 0.95 (S, 3H, 20-CH2), is 1.31 (t, N, CH3CH2-N), from 0.8 to 2.40 (m, 3H, cycle), 3,03 (g, 6H, CH3CH2-N) of 3.94 (m, 3H, 10-CH3), 5,27 (Sm, 1H, 3-H), 6,98 (d, 1H, P-H (J = 618 Hz)), 8,35 (S, 1H, N-H).

The signals involved in phosphorus-containing part of the molecule in the infrared,31P and1H-NMR spectra correlate well with the literature data [2] . Signals steroid part of the molecules in the spectra of IR and1H-NMR spectrum is also consistent with reference data [3] .

b) Synthesis of H-phosphonate testosterone.

The synthesis are similar to those described above. The yield of 0.49 g (69% ). Rf= 0,29 (TLC, chloroform-ethanol 6: 4)CH2, -CH3).

UV-spectrum (ethanol): max241 nm, lg = = 4,18.

31P-NMR,M. D.(CDCl3): 3,43 m D. (J = 608 Hz).

1H-NMR,M. D.(CDCl3): OF 0.68 (S, 3H, 13-CH3), OF 1.34 (S, 3H, 10-CH3), to 1.21 (t, 9H, CH3-CH2-N), 0,80-2,1 (m, 17H, cycle), 2,94 (g, 6H, CH3-CH2-N), 3,95 (m, 1H, 10-CH3), 5,57 (Sm, 1H, 17-H), of 6.68 (d, 1H, P-H (J = 608 Hz)), 8,11 (S, 1H, N-H).

The data of IR, UV and NMR are consistent with literature data [2-3] .

P R I m m e R 2. The synthesis of steroid derivatives of oligonucleotides.

For the synthesis used by solid state method for the automatic synthesizer "Victoria" [4] protected oligonucleotides in the form of H-phosphonates, connected on 31-end with a polymeric carrier and having on 51-the end free oxygraph.

a) Synthesis of cholesterol derived destimulate

ChSpT(pT)9< / BR>
To the dried sample (8 mg, capacity 70 mol/g at the first nucleotide of the link, i.e., 0.5 mmol), polymer-bound destimulate with H-phosphonate mezhnukleotidnyh links, add 50 l of abs. pyridine, 25 l of abs. acetonitrile, 25 l of 0.1 M solution (25 mmol) of H-phosphonate cholesterol in chloroform and 6 ml (75 mmol) of pivaloate. The mixture was incubated 15 min Emese pyridine - water (98: 2), incubated 30 min and washed with acetone. Then the carrier is treated with 1 ml conc. NH4OH for 1 h, the solution is drained, evaporated to dryness, dissolved in 1 ml of water and applied to a column of ion exchange sorbent. Selected fractions again evaporated, dissolved in water and applied on the column with reversed phase. Conditions of chromatography are shown in table. After separating the fraction containing the target product, evaporated to dryness, dissolved in 100 l of water and added dropwise to 1 ml of 2% aqueous solution of LiClO4in acetone. The precipitation of the lithium salt of the oligonucleotide is separated by centrifugation, washed with acetone and dried under 14-2mm RT. Art. the Output and characteristics of the cholesterol derived destimulate shown in the table.

b) Synthesis of testosterone derived destimulate.

TSpT(pT)9< / BR>
Synthesis and secretion are similar to those described in example 2A. The output and characteristics of the target product are listed in the table.

C) Synthesis of cholesterol derived decorrelate.

ChSpU(pU)9< / BR>
The synthesis are similar to those described in example 2A, the procedure for allocation impose additional step: resulting from the protective groups in the 21-position of the ribose treated with 1 ml of 0.01 M HCl for 2 h at 50aboutC. Then the solution is neutralized conc. NH4OH, evaporated to dryness and dissolved in 1 ml of water. Then put on a column of reversed phase (chromatography conditions shown in the table) and after chromatography produce the target product similar to that described in example 2A. The output and characteristics of the product are shown in table.

g) Synthesis of testosterone derived decorrelate.

TSpU(pU)9< / BR>
Synthesis and secretion are similar to those described in example 2B. The results are shown in the table.

d) Synthesis of cholesterol derived DECA(21-0-methyluridine)

ChSpUm(pUm)9< / BR>
Synthesis and secretion are similar to those described in example 2A. The results are shown in the table.

e) Synthesis of testosterone derived DECA(21-0-methyluridine)

TSpUm(pUm)9< / BR>
Synthesis and secretion are similar to those described in example 2A.

The results are shown in the table. (56) Letsinger, R. L. , Zhang, G. , Sun, D. K. , Ikeuchi , T., P. S. Sarin PNAS, 1989, v. 86, N 17, p. 6553-6556.

P. J. Garegg , Regberg , T., Stawinski, J. Chem. Ser. 1986, v. 26, N 1, p. 59-62.

The Sadtler Standard Spectra. Philadelphia Sadtler research Lab. 1966-1988.

Veniaminova A. G. , Levin Aeriosa steroids of General formula

XO-H

where X is the residue of cholesterol or testosterone,

as a reagent for solid-phase synthesis of steroid derivatives of oligonucleotides or their analogs.

 

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