Nucleoside derivatives with photolabile protective groups

Details Nucleoside derivatives with photolabile protective groups Nucleoside derivatives with photolabile protective groups

2002-03-29

2004-06-15

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025300, C536S026100

Reexamination Certificate

active

06750335

ABSTRACT:

TECHNICAL FIELD
The subject matter of the present invention are nucleoside derivatives with photolabile protective groups, processes for preparing the same and the use thereof.
Prior art
Photolabile protective groups for the hydroxy and phosphate functions in nucleosides or nucleotides, respectively, are of particular interest because, for example, they are suitable for light-controlled parallel syntheses of oligonucleotides on a solid carrier (cf. S. P. A. Fodor et al., Science 1991, 251, page 767 et seq.). With their aid, it is possible to produce so-called DNA chips (i.e. carrier platelets on the surface of which many different oligonucleotides are arranged) which, in turn, are required in molecular biology, for example for sequence analyses or expression studies.
According to the prior art, especially the o-nitrobenzyl group and its derivatives have been used as photolabile protective groups in nucleoside and nucleotide chemistry, respectively (cf. V. N. R. Pillai, Org. Photochem. 1987, 9, page 225 et seq., and J. W. Walker et al., J. Am. Chem. Soc. 1988, 110, pages 7170 et seq.) In addition, protective groups of the pyrenyl methoxy carbonyl type have been used (cf. WO 98/39 348). The slow and, in part, incomplete deprotection of the relevant nucleoside or nucleotide derivatives has turned out to be a particular disadvantage of these protective groups. In addition, some undesirable byproducts in the form of toxic nitrosophenyl compounds may result when the o-nitrobenzyl compounds are separated.
The 2-(2-nitrophenyl)ethoxy carbonyl group and the 2-(2-nitrophenyl)ethyl sulfonyl groups and their derivatives have been introduced as additional photolabile protective groups for nucleic acid chemistry (cf. WO 96/18 634 and WO 97/44 345) which may be separated more rapidly and completely when compared to the above-mentioned nitrobenzyl or pyrenyl methoxy carbonyl groups.
One disadvantage of these protective groups turned out to be the deprotection of the relevant nucleosides or nucleotides which is still comparatively slow and incomplete.
SUMMARY OF THE INVENTION
Therefore, it was the objective of the present invention to develop nucleoside derivatives with photolabile protective groups for the 5′-OH function in the sugar portion which does not have the above-mentioned disadvantages of the prior art, but may be deprotected comparatively rapidly, quantitatively and without formation of undesirable byproducts.
This invention achieves this objective by nucleoside derivatives of the general formula (I) according to claim
1
, because it has surprisingly been shown that the protective groups of the invention may be separated much more rapidly and completely than, for example, the o-nitrobenzyl groups. In addition, no major amounts of byproducts have found upon deprotection so far, which was not foreseeable either.
The nucleoside derivatives of the invention have the following general formula (I)
wherein the radicals R
1
and R
2
on the phenyl ring may be defined as follows:
R
1
=H, F, Cl, Br, I, NO
2
R
2
=H, CN,
R
1
and R
2
not being H at the same time
The radical R
3
which is located on the C
2
atom of the o-nitrophenyl ethyl group may either be H, an alkyl radical comprising 1 bis 4 carbon atoms or a phenyl radical. Said alkyl radical may be linear or branched.
The nucleoside portion of the compounds of the invention consists of the customary D-ribofuranose and 2′-deoxyribofuranose units and the pyrimidine (B=cytosine, thymine, uracil) or purine bases (B=adenine, guanine). 2,6-diaminopurine-9-yl, hypoxanthine-9-yl, 5-methylcytosine-1-yl, 5-amino-4-imidazol carboxylic acid amide-1-yl or 5-amino-4-imidazol carboxylic acid amide-3-yl radicals may also be used as bases.
The OH group(s) in the ribofuranoside or 2′-deoxyribofuranose portion may be free or protected as required. In order to protect the 3′ position (R
4
position), the following known phosphite amide groups have turned out to be effective, for example
wherein the R
7
groups may be the same or different and represent linear or branched alkyl radicals having 1 to 4 carbon atoms. Preferably they are ethyl or isopropyl radicals.
In position 2′ of the ribofuranoside portion (position R
5
), a free or protected OH group may be present in addition to the hydrogen or halogen atom (especially F, Cl, Br). In that case, any protective group customary in nucleotide chemistry (R
6
) may be used. In particular, use may be made of the customary alkyl, alkenyl, acetal or silyl ether protective groups for oxygen atoms (X=O). R
5
may also be an S-alkyl group (X=S, R
6
=alkyl). O-methyl or O-ethyl radicals are preferred examples for O-alkyl protective groups, O-allyl radicals for O-alkenyl protective groups, O-tetrahyropyranyl or O-methoxytetrahydropyranyl radicals for O-acetal protective groups and O-t-butyldimethylsilyl radicals for O-silylether protective groups.
In accordance with a preferred embodiment, the pyrimidine or purine bases having primary amino functions (e.g. adenine, cytosine and guanine) may also have permanent protective groups, preferably on a carbonyl basis. For this purpose, especially phenoxy acetyl or dimethyl formamidino radicals are preferred, because they may be used for all of the three cited bases. In addition, there are special protective groups which are introduced only with certain bases. In case of adenine, for example, these are benzoyl or p-nitrophenyl ethoxy carbonyl (p-NPEOC) radicals. In addition to the p-NPEOC radicals, isobutyroyl or p-nitrophenyl ethyl (p-NPE) protective groups may be introduced for guanine (for the O-6 function). Finally, benzoyl or isobutyroyl are suitable protective groups for cytosine in addition to p-NPEOC radicals.
Preparation of the nucleoside derivatives is carried out in at least two stages. In the first stage, an alcohol of the general formula (II)
wherein R
1
, R
2
and R
3
are as defined above is reacted with a phosgene derivative, preferably in a non-plar organic solvent at temperatures between −20 and +25° C. In addition to phosgene, diphosgene (chloroformic acid trichloromethyl ester) or triphosgene (bistrichloromethyl carbonate) may be used as the phosgene derivative.
The alcohol component is known in most cases or may be prepared analogously by known methods. In stage (a), toluene or THF is preferably used as the non-polar organic solvent. Even though the reaction components may be used in an almost stoichiometric ratio, the phosgene derivative is preferably used in a clear excess based on the alcohol component. The concentration of the alcohol component may also be varied within wide limits, but it has turned out to be particularly advantageous to adjust this concentration to 0. 1 to 10.0 mmol per 10 ml of solvent.
This reaction (duration of the reaction about 1 to 2 hours), yields 95% or more of the relevant chlorocarbonic acid esters of the general formula (IV) which are of high purity:
The pertinent products are preferably worked up by first distilling off any excess phosgene derivative and the solvent under vacuum. In stage (b), the chlorocarbonic acid ester (IV) may then be reacted without further work-up with the nucleosides of the general formula (III)
wherein R
4
, R
5
and B are as defined above.
The reaction is preferably carried out in a solvent mixture consisting of dichloromethane and a polar organic solvent, optionally in the presence of a base, at temperatures between −60 and +25° C. DMF or pyridine is preferably used as the polar organic solvent, no additional base being required when pyridine is used. However, if a solvent mixture of dichloromethane/DMF is used, it is recommended to add a base such as pyridine, triethyl amine or ethyl diisopropyl amine in order to scavenge the protons released during the reaction. The mixing ratio of dichloromethane to pyridine or DMF is not critical, but it is preferred to use 1 to 3 parts by vol. of dichloromethane per part by vol. of pyridine or DMF, respectively.
In a preferred embodiment, the relevan

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