Oligonucleotide protecting groups

Details Oligonucleotide protecting groups Oligonucleotide protecting groups

2000-12-22

2003-06-10

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbonate esters

C558S268000, C558S260000, C560S302000, C536S025300

Reexamination Certificate

active

06576783

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to protecting groups, more particularly to compounds useful for protecting amino, amidino, guanidino and hydroxyl groups, as well as methods of use thereof during oligonucleotide synthesis.
BACKGROUND OF THE INVENTION
During oligonucleotide synthesis, convenient amino group protection methodology is important not only for exocyclic amines but also for side chain amino groups (“aminolinkers” or “aminotethers”). These amino group-containing tethers can be conveniently deprotected and used to attach various functionalities to modify the biological or chemical properties of oligonucleotides (e.g., to conjugate groups which can improve uptake of antisense oligonucleotides by living cells); to attach chemical nucleases targeting the pathogenic genes; and to attach reporter groups (such as fluorescein or biotin) which are extensively used in DNA based diagnostics in following cellular trafficking of antisense oligonucleotides (Manoharan, M., in Antisense Research and Applications, S. T. Crooke and B. Lebleu (eds.), CRC Press, Boca Raton, Fla., 1993, 303-349). In spite of their widespread use, the conventional protecting groups used in oligonucleotide chemistry for aminotethers are either too labile during the monomer synthesis (e.g., CF
3
CO—, Fmoc) or somewhat inert, thereby requiring harsh conditions during, for example, oligonucleotide deprotection. The phthalimido group, for example, requires CH
3
NH
2
in addition to the standard ammonium hydroxide conditions. The acid labile MMT (monomethoxytrityl) group is sometimes used, but generally to protect an aminolinker only at the 5′-end of the oligonucleotide.
To overcome these problems the alloc (allyloxycarbonyl) group (Kunz. H. Angew. Chem. 96, 426, 1984; Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem. Soc. 112, 1691, 1990) has been adopted as a protecting group for aminolinkers, as it can be removed using zerovalent palladium (Pd (0)) either in solution phase or in solid phase (Barber-Peoch, I; Manoharan, M.; Cook, P. D. Nucleosides & Nucleotides 16, 1407-1410, 1997; Nelson, P. S.; Muthini, S.; Kent, M. A; Smith T. H.; Nucleosides & Nucleotides 16, 1951-1959, 1997). The chloroformate Cl—(C═O)—O—CH2—CH2—CN also has been used to protect nucleobase amines. Chloroformates, however, are unstable and difficult to use. It would therefore be desirable to provide alternative reagents for protecting amine and other groups during synthesis.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of the formula (I)
wherein
X is aryl or a covalent bond;
Y is aryl or a covalent bond;
R
1
is selected from succinimid-N-yl, phthalimid-N-yl, pyridin-N-yl, 4-nitophenyl, N-imidazol-1-yl, benzotriazol-2-yl, pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl, tetrazol-N-yl and norbornan-N-yl;
R
2
is cyano, nitro or CF
3
;
R
3
and R
4
are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl;
W is C(R
5
) (R
6
) or C(R
7
)═C(R
7
) where each R
5
, R
6
, and R
7
is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl or both R
7
substituents together form an unsaturated aromatic ring; and
n is an integer from 0 to 7.
In another aspect, the invention provide methods for protecting amine, guanidine, amidine or hydroxyl groups comprising reacting a free amine, guanidine, amidine or phosphate with a compound according to the general formula (I).
DETAILED DESCRIPTION OF THE INVENTION
R
1
can be selected from succinimid-N-yl, phthalimid-N-yl, pyridin-N-yl, 4-nitophenyl, -imidazol-1-yl, benzotriazol-2-yl, pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl, tetrazol-N-yl, pyrazol-N-yl and norbornan-N-yl. More preferably, R
1
is succinimid-N-yl, phthalimid-N-yl or pyridin-N-yl, and even more preferably succinimid-N-yl or phthalimid-N-yl. Most preferably, R
1
is succinimid-N-yl.
R
2
can be cyano, nitro or CF
3
. In a preferred embodiment, R
2
is cyano while X and Y are both a covalent bond. In another preferred embodiment R
2
is nitro while one of X and Y is aryl while the other is a covalent bond. In another embodiment R2 is cyano while Y is aryl (e.g., 1,4-phenylene) and X is a covalent bond.
R
3
, R
4
, R
5
, R
6
, and R
7
each be independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl.
As used herein, the term “alkyl” includes but is not limited to straight chain, branch chain, and cyclic unsaturated hydrocarbon groups having 1 to about 10 (preferably 1 to about 4) carbon atoms. “Alkenyl” includes but is not limited to straight chain, branch chain, and cyclic saturated hydrocarbon groups having 2 to about 10 carbon atoms. “Alkynyl” includes but is not limited to hydrocarbon groups having 2 to about 10 carbon atoms and a carbon—carbon triple bond. By “cycloalkyl” is meant mono- or bicyclic rings of 3 to 10 members optionally unsaturated and optionaly attached to the carbon atom from which R
3
to R
7
depend by an alkyl chain thereby forming a “cycloalkyl” group. When any of R
3
through R
7
is alkenyl or alkynyl, the unsaturated bond or bonds preferably are spatially removed from the carbon atom from they depend. In other words, the unsaturated bond is preferably not adjacent to said carbon atom.
“Aryl” is used herein interchangeably with “aromatic”, and includes optionally substituted mono-, bi- and tricyclic, 5 to 14 membered rings incorporating carbon atoms exclusively or incorporating one or more heteroatoms such as N, O and S (as well as SO and SO
2
), thereby forming a heteroaryl group. Prefered aryl groups include phenyl, naphthyl, pyridyl, quinolinyl, isoquinolinyl and naphthyridyl.
In preferred embodiments, R
3
through R
7
are independently H, alkyl or aryl. In a particularly prefered embodiment R
3
, R
4
and R
5
are H and R
6
is phenyl (preferably while n is 1). In another particularly prefered embodiment, both R
3
and R
4
are H and both R
5
and R
6
are methyl (preferably while n is 1). In another preferred embodiment, each of R
3
through R
6
are H (preferably while n is 1).
In other embodiments in which W includes olefinic moieties, R
2
preferably is cyano or nitro. When W is olefinic, adjacent R
7
substituents together may form an aromatic ring. Prefered aromatic (i.e., aryl) rings formed in this respect include benzene, naphthalene, pyridine and more preferably benzene.
X and Y preferably are both covalent bonds. In an alternate embodiment, one of X and Y is aryl (e.g., 1,4-phenylene) wherein X and CR
5
R
6
are para to one another.
Compounds of the invention can be prepared according established organic synthetic techniques. In a particular general method, compounds are prepared by reacting an alcohol of formula (II) under suitable conditions (e.g. in acetonitrile/dichloromethane in the presence of pyridine) with a dicarbonate of formula (III), wherein X, Y, n and R
1
through R
7
are as previously defined. See Scheme 1 below. The alcohol (II) and dicarbonate (III) are either commercially available or themselves are prepared from commercially available reagents according to established synthetic techniques.
Alternatively, compounds of formula (I) can be prepared by reacting a chloroformate of formula (IV) under suitable conditions with an alcohol of formula (V). See Scheme 2 below.
Again, chloroformate (IV) and alcohol (V) are either commercially available or themselves may be prepared from commercially available reagents using established organic synthetic techniques.
The invention also provides methods for protecting amine, guanidine, amidine or hydroxyl groups comprising reacting a free reactive amine, guanidine, amidine, or hydroxyl with a compound according to the general formula (I). Said amine, guanidine, amidine or hydroxyl group may be incorporated on or within various chemical molecular entities requiring protection, including but not limited to ami

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