RU2131881C1 - Method of synthesis of 3'-phosphate,n,p-nonprotected phosphothioate analogs of oligodeoxyribonucleotides - Google Patents

Abstract

FIELD: bioorganic chemistry, chemistry of nucleic acids. SUBSTANCE: invention relates to a method of synthesis of 3'-phosphate,N,P- -nonprotected phosphothioate analogs of oligodeoxyribonucleotides of the general formula (I)

where B - a residue of thymine, cytosine, adenine or guanine; n = 1-20 that can be used as the parent compounds for synthesis of oligonucleotide reagents for biotechnological aims. Method involves the successive treatment of the parent protected oligonucleotides with iodine solution in an aqueous pyridine followed by treatment with an aqueous ammonia and acetic acid. The parent protected oligonucleotides are 5'-O-dimethoxytrityl,3'-p(S)SMe, N-acyl, P(S)SMe-protected phosphodithioate oligonucleotides of the general formula (III)

where B' - a residue of thymine, N⁴-benzoylcytosine, N⁶-benzoyladenine, N²-isobutyrylguanine; DMTr is 4,4'-dimethoxytrityl; n = 1-20. EFFECT: increased yield of the end product, simplified process. 4 ex

Description

где B - остаток тимина, цитозина, аденина или гуанина;
n = 1-20 (общепринятого, что к олигонуклеотидам относятся полинуклеотидные последовательности длиной цепи от 1 до примерно 20).The invention relates to the field of bioorganic chemistry, and in particular to a method for producing 3'-phosphate, N, P-unprotected phosphothioate oligonucleotides (FTAO) of the general formula I

where B is the residue of thymine, cytosine, adenine or guanine;
n = 1-20 (it is generally accepted that oligonucleotides include polynucleotide sequences with a chain length of from 1 to about 20).

 which can be used as starting compounds for the production of phosphotioate oligonucleotide reagents [1-3] for biotechnological purposes [4-8].

где B' - остаток тимина, N⁴-бензоилцитозина, N⁶-бензоиладенина, N²-изобутирилгуанина;
DMTr - 4,4'-диметокситритил;
n = 1-20,
раствором йода в водном пиридине в течение 20 мин для удаления 3'-p-концевых SMe-защит, затем раствором тиофенола в смеси триэтиламина и диоксана для удаления Me-групп с межнуклеотидных P(O)-SMe остатков (в результате которой образуются межнуклеотидные фосфотиоатные группы) с последующей обработкой водным аммиаком (для удаления N-ацильных защит с гетероциклических оснований нуклеотидов) и уксусной кислотой (для удаления 5'-O-диметокситритильных защитных групп) с выделением целевого продукта ионообменной хроматографией [2,3] (прототип). Недостатком данного способа является низкий выход целевых олигонуклеотидов из-за относительно высокой степени разрыва межнуклеотидных связей при обработке тиофенолом, которая составляет 15% на одну межнуклеотидную связь. В связи с этим выход целевого олигонуклеотида снижается с увеличением длины олигонуклеотидной цепи, который в случае ди-, три- и октануклеотидов составляет 85,71 и 15%, соответственно.A known method of producing 3'-phosphate, N, P-unprotected FTAO of the general formula I by sequential treatment of 5'-O-dimethoxytrityl, 3'-pSMe, N-acyl, P (O) (SMe) -protected phosphothioate oligonucleotides of the general formula II

where B 'is the residue of thymine, N ⁴ -benzoylcytosine, N ⁶ -benzoyladenine, N ² -isobutyryl guanine;
DMTr - 4,4'-dimethoxytrityl;
n = 1-20,
a solution of iodine in aqueous pyridine for 20 min to remove the 3'-p-terminal SMe protections, then a solution of thiophenol in a mixture of triethylamine and dioxane to remove Me groups from internucleotide P (O) -SMe residues (as a result of which internucleotide phosphothioate forms groups) followed by treatment with aqueous ammonia (to remove N-acyl protections from heterocyclic bases of nucleotides) and acetic acid (to remove 5'-O-dimethoxytrityl protective groups) with the isolation of the target product by ion exchange chromatography [2,3] (prototype). The disadvantage of this method is the low yield of the target oligonucleotides due to the relatively high degree of cleavage of internucleotide bonds during treatment with thiophenol, which is 15% per internucleotide bond. In this regard, the yield of the target oligonucleotide decreases with increasing length of the oligonucleotide chain, which in the case of di-, tri- and octanucleotides is 85.71 and 15%, respectively.

где B'-остаток тимина, N⁴-бензоилцитозина, NM⁶-бензоилладенина, N²-изобутирилгуанина;
DMTr - 4,4'-диметокситритил;
n = 1-20,
и обработкой исходных соединений раствором йода в течение 1-2 часов. При этом исключается стадия обработки промежуточной реакционной смеси раствором тиофенола.The objective of the present invention is to increase the yield of the target product and simplify the process. The problem is achieved by using, in contrast to the prototype, as the starting compounds, the well-known [9] 5, -O-dimethoxytrile, 3'-p (S) SMe, N-acyl, P (S) (SMe) -protected phosphodithioate oligonucleotides of the general formula III

where the B'-residue of thymine, N ⁴ -benzoylcytosine, NM ⁶ -benzoyladenine, N ² -isobutyryl guanine;
DMTr - 4,4'-dimethoxytrityl;
n = 1-20,
and treatment of the starting compounds with iodine solution for 1-2 hours. This eliminates the stage of processing the intermediate reaction mixture with a solution of thiophenol.

According to the proposed method, the target 3'-phosphate, N, P-unprotected FTAO of general formula I is obtained by sequential treatment of the known 5'-dimethoxytrityl, 3'-p (S) SMe, N-acyl, P (S) (SMe) -protected phosphodithioate oligonucleotides [9] of the general formula III with a solution of iodine in aqueous pyridine for 1-2 hours to remove the 3'-p-terminal SMe protections (in this case, ³¹ P-NMR spectroscopy showed that internucleotide phosphodithiotriether-OP (S) (SMe) O-groups are retained, while the 3'-terminal p (S) (SMe) group is completely oxidized to phosphate), then with aqueous ammonia solution temporary removal of the SMe group from internucleotide P (S) SMe residues (as a result of which internucleotide phosphothioate groups are formed) and N-acyl protections from heterocyclic nucleotide bases, followed by treatment with acetic acid (to remove 5'-O-dimethoxytrityl protective groups) with the selection of the target product by ion exchange chromatography. The yields of the target di-, tri-, octa- and dodecananucleotides of the general formula I (n = 1, 2, 7 and 11) in this case are 95, 88, 65 and 50%, respectively. Thus, the yields of the target 3'-phosphate, N, P-unprotected FTAO I obtained by the proposed method significantly exceed the yields of the corresponding compounds obtained in a known manner, and this difference in yields increases with the length of the synthesized oligonucleotide chain, which is associated with a relatively low compared with the prototype rupture of internucleotide bonds (in the proposed method, this value does not exceed 5% per internucleotide connection). In addition, in the proposed method there is no stage of processing the intermediate reaction mixture with a solution of thiophenol.

The preparation of the desired 3'-phosphate, N, P-unprotected FTAO I by the proposed method was not obvious, since when treating the starting compounds III, unlike compounds II, with a solution of iodine in aqueous pyridine, the possibility of oxidizing the sulfur atom in internucleotide-OP (S) (SMe) O-phosphodithiothriether groups. In this regard, the procedure for removing SMe protections by the proposed method was specifically investigated. Using ³¹ P-NMR spectroscopy, it was shown that when the starting compounds III were treated with an iodine solution in aqueous pyridine under the above conditions, the internucleotide phosphodithiotriether-OP (S) (SMe) O-groups were not affected, but the 3'-terminal p (S) (SMe) group in this case is oxidized to phosphate. Moreover, when treating the starting compound III with an iodine solution under the conditions described in the prototype (20 min), the 3'-terminal p (S) (SMe) group is oxidized to phosphate by only 40%.

Example 1. Synthesis of 3'-phosphate dithymidyl phosphothioate,
TpsTp (IV).

a) 0.1 mmol (114 mg) of protected DMTTrTp (S) (SMe) Tp (S) (SMe) IVa dimitidylate (compound of general formula III, where B'-thymidine, n = 1), obtained by a known method [9], dissolved in 10 ml of a mixture of pyridine-water (3: 2), add 129 mg of iodine (1 mmol). After 1.5 hours, the reaction mixture was evaporated, dissolved in 10 ml of water, and excess iodine was removed by extraction with ether (3 x 50 ml). The aqueous layers are evaporated. An intermediate compound DMTTrTp (S) (SMe) Tp (IVb) was obtained, which was analyzed by ³¹ P-NMR spectroscopy. In the spectra of ³¹ P-NMR (pyridine-water, 3: 2) found:
1) signals in the range of 93-96 ppm related to the initial internucleotide phosphodithiothriether SMe-phosphodithioate groups (-OP (S) (SMe) O-) [9];
2) the complete disappearance of signals in the region of 72-74 ppm related to the 3'-terminal p (S) (SMe) phosphotidithiophenyl group (-OP (S) (SMe) O ^- ) [10];
3) the appearance of signals in the region of 1.6 ppm related to the unblocked monoester 3'-phosphate group (-OP (O) (O ^- ) O ^- [11].

 These data indicate the selective and quantitative release of SMe protections under the given conditions only from the 3'-terminal phosphate group and the formation of compound (IVb).

Next, the reaction mixture containing compound (IVb) is treated with concentrated ammonia for 3 days at 60 ^° C. and an intermediate compound DMTrTpsTp (IVc) is obtained, which is analyzed by ³¹ P-NMR spectroscopy. The absence of signal spectra in the region of 93-96 ppm and the appearance of signals in the region of 55.7 ppm, based on published data [2,3,11], indicates the complete release of the SMe groups of P (S) SMe phosphodithiothriether internucleotide residues (-OP (S) (SMe) O -) in the intermediate compound (IVb) and on the formation of diester phosphothioate internucleotide groups (-OP (S) (O ^- ) O- = ps). These data confirm the structure of the obtained intermediate compound (IVB).

Next, the reaction mixture containing intermediate compound (IVc) is kept in 10 ml of 80% acetic acid for 30 minutes and evaporated on a rotary evaporator (to remove DMTr-protecting groups) to obtain compound (IV). Compound (IV) was isolated by ion-exchange column chromatography (Polysil CA) in a gradient of KH ₂ PO ₄ containing 20% acetonitrile. The desired product is desalted on a reverse phase column, eluting with 50% aqueous acetonitrile and evaporated.

The obtained 0.095 mmol (1605 OE ₂₆₀ ) of the target compound ε ₂₆₀ = 16.8 • 10 ³ l • mol ^-1 . cm ^-1 . Yield 95%.

 The product is homogeneous according to ion-exchange chromatography and in its physicochemical characteristics (ion-exchange and reverse-phase chromatography, UV and NMR spectroscopy) is identical to compound (IV) obtained by the method [2].

 b) The synthesis is carried out similarly to that described in example 1a, while the iodine solution is treated for 1 hour.

Received 0.091 mmol (1522 OE ₂₆₀ ) of the target compound (IV). Yield 91%.

 c) The synthesis is carried out similarly to that described in example 1a, while the iodine solution is treated for 2 hours.

Obtained 0.095 mmol (1596 OE ₂₆₀ ) of the target compound (IV). Yield 95%.

g) synthesis under the conditions given in the prototype [2]
Treatment of 0.1 mmol of protected DMTrTp (S) (SMe) Tp (S) (SMe) (IVa) dithymidylate with iodine solution (for 20 min), ammonia, acetic acid and product isolation by chromatography under the conditions of Example 1a gives 0.032 mmol (540 OE ₂₆₀ ) compounds (IV). Yield 32%.

Analysis of the intermediate reaction mixture (according to ³¹ P-NMR spectroscopy) containing the product (IVb) shows the disappearance of signals in the region of 72-74 ppm related to the 3'-terminal p (S) (SMe) phosphodithio-ester group (-OP (S) (SMe) O ^- ) [10], and the appearance of signals in the 1.6 ppm region related to the unblocked monoester 3'-phosphate group (OP (O) (O ^- ) O ^- [11] If we take for 100% the sum of the signal areas in the region of 72-74 and 1.6 ppm, then the signal area in the region of 1.6 ppm is 40%.

 These data indicate that the release of SMe protections from the 3'-terminal phosphate group in compound (IVa) during treatment with iodine solution for 20 min is only 40%.

Example 2. Synthesis of 3'-phosphate tritimidyl phosphothioate
TpsTpsTp (V).

Treatment of 159 mg (0.1 mmol) of protected tritimidylate DMTrTp (S) (SMe) Tp (S) (SMe) Tp (S) (SMe) (Va) with iodine solution, concentrated ammonia, acetic acid and isolating the product by chromatography under the conditions of the example 1a gives 0.088 mmol (2186 OE ₂₆₀ ) of compound (V) ε ₂₆₀ = 24.9 • 10 ³ L • mol ^-1 • cm ^-1 . Yield 88%.

 The product is homogeneous according to ion exchange chromatography and in its physicochemical characteristics (ion exchange and reverse phase chromatography, UV and NMR spectroscopy) is identical to compound (V) obtained by the method [2].

 Example 3. Synthesis of 3'-phosphate deoxyoctanucleotide phosphotioate CpsApsTpsTpsGpsTpsGpsAp (VI).

Treatment of 41.4 mg (0.01 mmol) of protected octanucleotide phosphodithioate DMTrC ^bz p (S) (SMe) A ^bz p (S) (SMe) Tp (S) (SMe) Tp (S) (SMe) G ^ib p ( S) (SMe) Tp (S) (SMe) G ^ib p (S) (SMe) A ^bz p (S) (SMe) (VIa) iodine solution, concentrated ammonia, acetic acid and product isolation by chromatography under the conditions of Example 1a, gives 0.0065 mmol (519 OE ₂₆₀ ) of compound (VI) ε ₂₆₀ = 79.5 • 10 ³ L • mol ^-1 • cm ^-1 . Yield 65%.

 The product is homogeneous according to the data of ion exchange chromatography and in its physicochemical characteristics (ion exchange and reverse phase chromatography, UV and NMR spectroscopy) is identical to compound (VI) obtained by the method [2].

 Example 4. Synthesis of 3'-phosphate deoxydecane nucleotide phosphotioate.

 GpsCpsApsTpsCpsApsApsGpsCpsApsGpsCp (VII).

Treatment of 58 mg (0.01 mmol) of the triethylammonium salt of the protected deoxydecodanucleotide phosphodithioate DMTrG ^ib p (S) (SMe) C ^bz p (S) (SMe) A ^bz p (S) (SMe) Tp (S) (SMe) C ^bz p (S) (SMe) A ^bz p (S) (SMe) A ^bz p (S) (SMe) G ^ib p (S) (SMe) C ^bz p (S) (SMe) A ^bz p (S) ( SMe) G ^ib p (S) (SMe) C ^bz p (S) (SMe) (VIIa) with iodine solution, concentrated ammonia, acetic acid and product isolation by chromatography under the conditions of Example 1a gives 0.005 mmol (586 OE ₂₆₀ ) of compound (VII ) Yield 50%.

The product is homogeneous according to ion-exchange chromatography, has a longer retention time compared to the control deoxydecodanucleotide GpCpApTpCpApApGpCpApGpCp, which does not contain internucleotide phosphothioate groups. In reverse phase chromatography, the resulting product (VII) is eluted as a mixture of diastereomers, the retention time of which is longer than the retention time of the control deoxydecane nucleotide GpCpApTpCpApApGpCpApGpCp. UV spectra of the obtained compound (VII) in water: λ _min = 234 nm, λ _max = 268 nm, ε ₂₆₀ = 117.3 • 10 ³ L • mol ^-1 • cm ^-1 - are identical with the spectra of the control deoxydecane nucleotide GpCpApTpCpApApGpCpApGpCp. In the ³¹ P-NMR spectra (30% CH ₃ CN in H ₂ O), signals corresponding to the signals of internucleotide phosphothioate groups (ps) (δ = 55.2 - 56.2 ppm) and 3'-terminal phosphate (δ = -0.2 ppm) in the ratio of 10.7: 1 (theoretically 11: 1).

фThe sum of the data presented confirms the structure of the obtained compound (VII)

f

Claims (1)

The method of obtaining 3'-phosphate, N, P-unprotected phosphothioate analogues of oligonucleotides of the General formula I

where B is the residue of thymine, cytosine, adenine or guanine;
n = 1 - 20,
by sequentially treating the starting protected oligonucleotides with a solution of iodine in aqueous pyridine and then with aqueous ammonia and acetic acid, characterized in that 5'-O-dimethoxytrityl, 3'-P (S) SMe, N-acyl is used as the starting protected oligonucleotides, P (S) SMe-protected phosphodithioate oligonucleotides of the general formula III

where B 'is the residue of thymine, N ⁴ -benzoylcytosine, N ⁶ -benzoyladenine, N ² -isobutyryl guanine;
DMTr - 4,4'-dimethoxytrityl;
n = 1 - 20,
and treatment with iodine solution is carried out for 1 to 2 hours