DE3208565A1 - Process for the removal of trityl protective groups - Google Patents

Abstract

Trityl protective groups of sensitive organic compounds, in particular nucleosides, oligonucleotides and/or polynucleotides, can be removed rapidly, quantitatively and mildly under absolutely proton-free conditions using halogen compounds of the general formulae RM<II>X, (RO)M<II>X, R2M<III>X, R(RO)M<III>X, (RO)2M<III>X, R3M<IV>X, R2(RO)M<IV>X, R(RO)2M<IV>X, (RO)3M<IV>X, RM<III>X2, (RO)M<III>X2, R2M<IV>X2, R(RO)M<IV>X2, (RO)2M<IV>X2, RM<IV>X3 or (RO)M<IV>X3. In the formulae, R denotes alkyl, cycloalkyl, phenyl or benzyl groups, M<II> denotes tin or zinc, M<III> denotes boron or aluminium, M<IV> denotes titanium or tin and X denotes halogen.

Description

description

The invention relates to a method for splitting off trityl protective groups from sensitive organic compounds. The method of the invention is particular for the detritylation of nucleosides, oligonucleotides and / or polynucleotides suitable, but can also be used for mono-, di- or oligosaccharides, steroids, vitamins and the like, where side reactions occur in the detritylation or are to be feared.

For chemical transformations on nucleosides and nucleotides as well for the synthesis of oligonucleotides is a regioselective protection of the 5'-OH group required (1). For years, mainly derivatives of the trityl group have been used for this purpose used. After completion of the chemical transformations or

The trityl protective group must synthesize oligomers under mild conditions be split off again.

Due to the acid lability of the N-glycosidic bond, in particular of the N-protected purine nucleosides and nucleotides, only the acid-labile ones could so far Derivatives of the trityl group can be used. For syntheses based on the Diester concept was predominantly the 4-monomethoxytrityl group (2,3,4), for syntheses according to the Triester method (5,6,7,8) and phosphite triester method (9) almost exclusively the 4,4'-dimethoxytrityl group used. The latter group in particular is so unstable that that it is partly lost in synthetic operations. To split off the Trityl groups were until recently protonic acids such as 80% aqueous acetic acid, or 1 - 2% benzene or toluenesulphonic acid in chloroform / methanol mixtures used (2 - 9). This occurred when the 4-monomethoxy and 4,4'-dimethoxytrityl groups were split off significant amounts of depurinization, i.e.

Cleavage of the N-glycosidic bond. To avoid this obvious disadvantages has recently been the use of Lewis acids of the ZnBr2 type, SnCl4, All3, TiC14 and BF3 are described (10,11). Such a procedure is also Subject of patent application (12), although the use of ZnBr2 and TiC14 for the cleavage of the chemically similar MEM ethers (13) and BF3 for the cleavage of Trityl ethers (14) has already been described.

A disadvantage when using the above-mentioned Lewis acids their sometimes considerable aggressiveness and their low solubility in non-polar or aprotic solvents. With the most commonly used ZnBr2 is with Nitromethane can only achieve a maximum 0.1 M solution; in methylene chloride it is poorly soluble. This means that especially in the case of syntheses of oligonucleotides on polymeric carriers where high reagent concentrations are absolutely necessary co-solvents such as methanol (15) or even water (16) can be used have to. This of course increases the risk that the hydrobromic acid liberated by hydrolysis in a known manner the N-glycosidic bond of the N-acylated purine nucleosides splits. This has the advantage of the above-mentioned Lewis acids over the direct ones Use of protonic acids is lost again.

On the other hand, the aggressiveness of some of the Lewis acids mentioned by interaction with nucleophilic centers of the heterocyclic bases of the nucleosides lead to side reactions.

De-N-acylation was even observed with the relatively mild ZnBr2 (17).

The disadvantages of the aforementioned methods can be overcome by the method completely avoid the invention with the features specified in the claims.

Under trityl protective groups in the present description are unsubstituted trityl group (C6H5) 3C- and the substituted ones known as protecting groups Derivatives thereof, e.g., 4-monomethoxytrityl and 4,4'-dimethoxytrityl groups to understand.

The alkyl groups R in the general given in the claims Formulas for the halogen compounds can be straight-chain or branched; man can use alkyl groups with up to 10 carbon atoms, e.g. methyl, ethyl, N-propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl groups or the like. Such halogen compounds are partly sterically hindered; the resulting Grading of reactivity can often be exploited in detritylation. Corresponding applies to cycloalkyl groups, in particular polycyclic representatives of the same as norbornyl groups, for aryl groups, especially methyl-substituted phenyl groups, e.g. the mesityl group, as well as for aralkyl groups, e.g. benzyl groups.

Of the cycloalkyl groups, there are in particular the cyclopentyl and cyclohexyl groups preferred.

The halogen compounds to be used according to the invention are relative weak Lewis acids, which are completely in non-polar or aprotic solvents are soluble and their cleavage mechanism with regard to the trityl ether bond is the Precludes the occurrence of protons. High concentrations can therefore be achieved so that the reactions at OOC in a few seconds, even when the right stable trityl ether bond, is quantitatively possible. A quantitative split this bond has so far proved difficult with ZnBr2, since the resulting very reactive Trityl cation considerably favors a reverse reaction to the trityl ether bond.

The reaction mechanism involved in the process of the invention can be seen from FIG. B means a base like thymine, uracil, 5-bromouracil, 5-methyluracil, adenine, guanine or cytosine, the latter also in N-protected form. The groups R are as defined in the claims. The groups R 'can be hydrogen, a 3'-O-protecting group, phosphoryl groups, oligonucleotidyl groups, or the like be.

When adding the halogen compound, which is in hexane, methylene chloride, Chloroform, benzene, toluene or the like may be dissolved to the tritylated Compound occurs the yellow color of the trityl cation.This is due to the reaction of the resulting TrCl e.g. with excess R2A1X to Tr R2AlCl29.

This provides a very good response indicator.

In the immediately following work-up by extraction with aqueous, excess sodium bicarbonate, ammonium acetate, triethylammonium bicarbonate or the like, the alcoholic fenction is released at the same time quantitative trapping of hydrohalic acids by neutralization. The nucleotides are therefore during the entire detritylation process either in a non-polar or aprotic environment, or when shaken out the pH of the organic phase with the buffer systems mentioned does not reach 7 fallen below. This means that a detritylation under very precisely defined, very gentle conditions is possible.

The detritylation reaction of the invention also proceeds with the very stable trityl protective group at OOC in a few seconds. Yet can with these short reaction times even at room temperature or higher temperatures to be worked.

Solvents which can be used in the process of the invention can be hexane, Benzene, toluene, chloroform, methylene chloride, nitromethane, acetonitrile and the like, Mixtures of the solvents mentioned can also be used. It's an essential one Advantage of the method of the invention that solvents can be used which in no case can give rise to the formation of protonic acids. For the same Basically, with the exclusion of moisture and with absolutely dry solvents and reagents are worked.

The use of the halogen compounds according to the drive before the invention for the cleavage of the trityl ether bond in nucleosides, nucleotides, oligonucleotides and polynucleotides are based on the use of protonic acids or Lewis acids Far superior to the state of the art, since the reaction is extremely fast below absolute exclusion of protons can be carried out. This becomes the destructive Effect that can emanate from protonic acids is completely prevented. Especially for the synthesis of oligonucleotides and polynucleotides can be carried out by the method of Invention, the unsubstituted trityl group was also used as a protective group for the first time which was previously considered to be too stable for these syntheses.

The method of the invention can be used in chemical transformations of Nucleosides and nucleotides, as well as for the synthesis of oligo- and polynucleotides the ribo and deoxyribo series can be used. Here it is beneficial for the Applicability of the procedure is irrelevant whether after the above-mentioned diester, tri-ester or phosphite triester method is synthesized. The method is also advantageous applicable for the synthesis of at the base (e.g. by incorporation of deoxyuridine, 5-bromodeoxyuridine), on sugar (e.g. by incorporating arabinonucleosides) or in the internucleotide bond (e.g. synthesis of oligophosphonates) modified oligo- or polynucleotides.

The 0 and N-acyl groups and O-silyl groups can be used as well as aryl or alkyl protecting groups for permanent or temporary protection of the phosphoric acid function.

The method of the invention is particularly advantageously applicable to the synthesis of nucleosides, nucleotides, oligonucleotides and polynucleotides using organic (18) or inorganic polymeric carriers (9,19,20,21). This is especially true because vn both non-polar as well as polar, aprotic Solvent very high concentrations of the detritylating halogen compound can be used. Here, however, it is important that after thorough washing e.g. of the excess R AlX the dialkylaluminum-2 bond to the 5'-O of the nucleosides, Nucleotides and oligo- or polynucleotides by treatment with n-butanol / pyridine mixtures is split.

For the advantageous applicability of the method of the invention is it is irrelevant whether the anchoring is to be synthesized on the polymeric carrier Oligo- or polynucleotide linkage via an ester linkage or via another linkage he follows.

The method of the invention is described below on the basis of an operating procedure and the preferred examples given in Table 1 are explained in more detail.

Working procedure for detritylation with dialkyl aluminum chlorides.

The 5'-O-tritylated nucleosides, nucleotides, oligonucleotides or Polynucleotides are azeotroped with pyridine (distilled over tosyl chloride and KOH) dried; Traces of pyridine are removed by repeated evaporation with toluene / chloroform (3: 7, V / V) removed in a high vacuum.

The residue is dissolved in distilled and dried methylene chloride recorded. 10-15 equivalents are added to the clear solution with magnetic stirring added to the dialkyl aluminum chloride solution. The detritylation is by short Appearance of a yellow color. Reactions with diethylaluminum chloride were after 1 - 3 minutes, that with diisobutylaluminum chloride completed in 5 - 10 minutes. The excess reagent is removed by the dropwise addition of 1 M aqueous NaHCO3 solution decomposes at 0oC. The organic phase is separated off and the aqueous phase which has precipitated out Aluminum compounds heterogeneous phase washed thoroughly with chloroform. The United organic phases are washed with saturated NaCl solution and dried azeotropically. After dissolving in a little methylene chloride, either direct precipitation occurs with Petroleum ether (bp 60-700C) or purification by column chromatography on silica gel. The control of the reaction by thin-layer chromatography on silica gel shows the quantitative Detritylation on; after column chromatography between 70 and 85% of the pure detritylated compounds obtained.

Individual examples can be found in Table 1 below.

The abbreviations used are explained in (4) and (15).

The conversion of the detritylation determined by thin-layer chromatography was 100% in each example; CH2Cl2 was used as solvent.

The publications mentioned in the description result from the following list.

1) News Chem.Tech.Lab. 27, 694 (1979) 2) 3. Mol. Biol. 72, 209 (1972) 3) Z. Physiol. Chem. 356, 1585 (1975) 4) Liebigs Ann. Chem. 1978, 839-1006 5) J. Org. Chem. 38, 245 (1973) 6) Nucleic Acids Res. 6, 1371 (1979) 7) Tetrahedron Letters 22, 3209 (1981) 8) Tetrahedron Letters 28, 2449 (1978) 9) Tetrahedron Letters 21, 719 (1980) 10) Tetrahedron Letters 21, 2683 (1980) 11) Tetrahedron Letters 21, 3243 (1980) 12) EU-OS 00 35 255 13) Tetrahedron Letters, 11, 809 (1976) 14) Angew.Ch§Wlnt.Ed. 18, 148 (1979) 15) Nucleic Acids Symposium Ser.No. 7: 39 16 (1980) Nucleic Acids Res. 9, 2807 (1981) 17) Tetrahedron Letters, 22, 3761 (1981) 18) Chemical Reviews, 77, 183 (1977) 19) Tetrahedron Letters 16, 1527 (1972) 20) Tetrahedron Letters, 21, 4159 (1980) 21) Tetrahedron Letters, 22, 4177 (1981) 22) Tetrahedron 37, 363 (1981) Table 1: Detritylation with diethyl aluminum chloride (A) and diisobituyl aluminum chloride (B) product solvent equivalents of time yield according to per mmol (ml) reagent () (min) chromatography in% (Tr) Td (25) 10 (B) 10 (Tr) tl4Cd (30) 10 (B) 10 (Tr) bz6Ad (39) 15 (B) 10 (Tr) bz6Ad (tBuBz) (39) 15 (B) 10 (Tr) an4Cd (tBuBz) (30) 15 (B) 10 (Tr) ib²Gdp (ClPh) (50) 20 (B) 10 (Tr) Td (20) 15 (A) 2 (Tr) tl4Cd (30) 10 (A) 2 (Tr) ib²Cd (100) 10 (A) 2 (Tr) bz6Ad (tBuBuz) (60) 15-20 (A) 3 85 (Tr) an4Cd (tBuBz) (60) 10 (A) 3 85 (Tr) td (tBuPh2Si) (50) 10 (A) 3 80 (Tr) Tdp (ClPh, TCE) (30) 10 (A) 3 70 (Tr) bz6Adp (ClPh) tl4Cd (45) 10 (A) 3 (Tr) Tdp (ClPh) tl4Cd (50) 10 (A) 3 (Tr) ib²Gdp (ClPh) bz6Ad (100) 10 (A) 3 L e r s e i t e

Claims (5)

Process for splitting off trityl protective groups. Claims 1. Process for splitting off triyl protective groups from sensitive organic compounds, especially nucleosides, oligonucleotides and / or polynucleotides with Lewis acids in a solvent, characterized in that that the Lewis acid is a halogen compound of the general formulas RMIIX (RO) MIIX R2MIIIX R (RO) MIIIX (RO) 2MIIIX R3MIVX R2 (RO) MIVX R (RO) 2MIVX (RO) 3MIVX RMIIIX (RO) M X2 R2MIVX2 R (RO) MIVX2 (RO) 2MIVX2 RMIVX3 or (Ro) X3 in which R is straight-chain or branched, saturated or unsaturated alkyl groups with up to 10 carbon atoms, saturated or unsaturated, optionally alkyl-substituted cycloalkyl groups with up to 10 carbon atoms or optionally alkyl-substituted phenyl or benzyl groups are, where two groups R can optionally form an alkylene group, MII Tin or zinc, MIII boron or aluminum, MIV titanium or tin and X fluorine, chlorine, Used to mean bromine or iodine. 2. The method according to claim 1, characterized in that the halogen compound i-Bu2AlCl or Et2AlCl is used. 3. The method according to claim 1 or 2, characterized in that one a non-polar or aprotic solvent is used. 4. The method according to one (of claims 1 - 3, characterized in that that the halogen compound, based on the tritylated compound, in 10 -15 - a fold excess. 5. The method according to any one of claims 1 - 4, characterized in that that for the detritylation of nucleosides, nucleotides and oligo- and polynucleotides these are convalently bound to inorganic or organic carriers.