Universal support media for synthesis of oligomeric compounds

Details Universal support media for synthesis of oligomeric compounds Universal support media for synthesis of oligomeric compounds

2002-09-30

2003-11-25

Lambkin, Deborah C. (Department: 1626)

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

Organic compounds

Carbohydrates or derivatives

C536S025300

Reexamination Certificate

active

06653468

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed in one aspect to compounds useful in the preparation of novel universal support media. The universal support media thus prepared are useful in the preparation of oligomeric compounds.
BACKGROUND OF THE INVENTION
Support bound oligonucleotide synthesis relies on sequential addition of nucleotides to one end of a growing chain. Typically, a first nucleoside is attached to an appropriate support medium such as a glass bead support and activated phosphorus compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise to elongate the growing oligonucleotide. When the chain elongation is completed, the oligonucleotide is cleaved from its support and protecting groups are removed. Additional methods for support bound synthesis methods may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. No. 4,725,677 and U.S. Re. Pat. No. 34,069.
In carrying out standard oligonucleotide syntheses, workers minimally need to maintain a supply of eight different nucleoside-loaded supports for DNA and RNA syntheses, each prederivatized with a separate nucleoside corresponding to the 3′ terminus of the desired oligomer (adenosine, guanosine, cytidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine). If a modified nucleoside is desired at the 3′-terminus then additional prederivatized supports are required. Typically, the first nucleoside is covalently bound by a succinate or hydroquinone-O,O′-diacetate linker. Furthermore, certain oligonucleotides with unusual nucleosides are available only as phosphoroamidites but not as supports.
A universal support is a support that may be used as a starting point for oligonucleotide synthesis regardless of the nucleoside species at the 3′ end of the sequence. A universal support has broad application and remedies the aforementioned deficiencies of standard oligonucleotide synthesis procedures because only one support is needed to carry out the oligonucleotide synthesis regardless of what base is desired at the 3′ end. This simplifies the synthetic strategy, reduces the number of required reagents in inventory and reduces the likelihood of errors in parallel synthesis applications.
Some researchers have employed derivatized glass supports with 2′(3′)-O-benzyoluridine 5′-O-succinyl so that the uridine moiety is linked to the glass via a succinate linkage [deBear et al.,
Nucleosides and Nucleotides
6, 821-830 (1987)]. Oligonucleotide synthesis takes place by adding nucleotide monomers to the 2′ or 3′ position of the uridine. Following the synthesis, the newly synthesized oligonucleotide is released from the glass, deprotected and cleaved from the uridinyl terminus in one reaction. Since it is cleaved from the solid support in the cleaving reaction, the uridinyl functionality is no longer available for subsequent oligonucleotide syntheses.
In a similar approach, Crea et al. prepared the dimer 5′-O-p-chlorophenylphospho-2′(3′)-O-acetyluridinyl-[2′-(3′)-3′]-5′-O-dimethoxytritylthymidine p-chlorophenylester and attached the dimer to cellulose via a phosphate linkage. The 5′ position of the thymidine is available for oligonucleotide attachment and synthesis. [Crea et al.,
Nucleic Acids Research
8, 2331 (1980)]. Aqueous concentrated ammonia is used to the release of the synthesized oligonucleotide from the cellulose leaving the uridine portion of the dimer attached to the cellulose. Although Crea et al. utilized the reactive vicinal groups on the uridine as the release site for the oligonucleotide from the uridine the solid support suggested in this reference is not truly a universal solid support because the 3′-terminal oligonucleotide is incorporated in the solid support reagent and a different support is required for oligonucleotides incorporating a different first nucleoside.
Schwartz et al. attached an adapter, 2′-(3′)-O-dimethoxytrityl-3′-(2′)-O-benzoyluridine-5′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, to a thymidine derivatized polystyrene and synthesized an oligonucleotide from the O-dimethoxytrityl position of the uridine [Schwartz et al.,
Tetrahedron Letters
, 36, 1, 27-30, 1995]. While this approach provides a universal solid support for oligonucleotide synthesis, cleavage releases both the adapter and the thymidine from the support and then the synthesized oligonucleotide from the uridine. Thus, thymidine linker must be removed as an impurity and the solid support is unavailable for subsequent reactions.
Some universal supports require cleavage under conditions supplemental to ammonium hydroxide, [Lyttle et al.,
Nucleic Acids Research
, 1996, 24, 14, 2793-2798] making them less useful in many conventional syntheses where ammonium hydroxide is used as cleavage reagent.
The compounds, compositions and processes of the invention provide novel universal support media useful for preparing oligomeric compounds, including oligonucleotides and oligonucleotide mimetics, which may be effectively cleaved without rendering the support media unavailable for subsequent reactions.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the invention is directed to compounds of Formula I:
wherein
X is CH
2
, O, S or NR
3
;
R
3
is alkyl, —C(═O)alkyl or an amino protecting group;
one of R
1
and R
2
is —(L)
n
-sm and the other of R
1
and R
2
is —C(═O)—R
4
or —C(═S)—R
4
;
L is a linking moiety;
n is 0 or 1;
sm is a support medium;
R
4
is —O-alkyl, —N(J
1
)J
2
;
J
1
is H or alkyl;
J
2
is alkyl or a nitrogen-protecting group;
or J
1
and J
2
together with the nitrogen atom they are attached to form a ring structure; and
Z
1
and Z
2
are orthogonal hydroxyl protecting groups.
Preferably, X is O, S or NR
3
. Preferably, R
3
is alkyl or —C(═O)alkyl. More preferably, X is O; and one of R
1
and R
2
is —(L)
n
-sm and the other of R
1
and R
2
is —C(═O)—R
4
. Preferably, L is —C(═O)—. Preferably, R
4
is —N(H)alkyl or N-piperidinyl. More preferably, Z
1
is —C(═O)CH
3
; and Z
2
is dimethoxytrityl.
The support medium may be a controlled pore glass, oxalyl-controlled pore glass, silica-containing particles, polymers of polystyrene, copolymers of polystyrene, copolymers of styrene and divinylbenzene, copolymers of dimethylacrylamide and N,N′-bisacryloylethylenediamine, soluble support medium or PEPS.
Z
1
may be a trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethoxycarbonyl, levulinyl or acetoacetyl groups.
Z
2
may be a 4,4′-dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthen-9-yl (Mox), t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, mesyl, tosyl, 4,4′,4″-tris-(benzyloxy)trityl (TBTr), 4,4′,4″-tris-(4,5-dichlorophthalimido)trityl (CPTr), 4,4′,4″-tris(levulinyloxy)trityl (TLTr); 3-(imidazolylmethyl)-4,4′-dimethoxytrityl (IDTr), 4-decyloxytrityl (C
10
Tr), 4-hexadecyloxytrityl (C
16
Tr), 9-(4-octadecyloxyphenyl)xanthene-9-yl (C
18
Px), 1,1-bis-(4-methoxyphenyl)-1′-pyrenyl methyl (BMPM), p-phenylazophenyloxycarbonyl (PAPoc), 9-fluorenylmethoxycarbonyl (Fmoc), 2,4-dinitrophenylethoxycarbonyl (DNPEoc), 4-(methylthiomethoxy)butyryl (MTMB), 2-(methylthiomethoxymethyl)-ben

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