US5493012A
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Ion triggered alkylation of biological targets by silyloxy aromatic agents
Assignee: Research Foundation of the State University of New York
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Filing: 1993-06-02
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Granted: 1996-02-20
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Expires: 2013-06-02
Abstract
Abstract A silyloxy aromatic derivative capable of alkylating a target biological molecule when activated by ionic strength. A sequence directed reagent may be constructed by conjugating a methyl silyloxy aromatic derivative to a hexamethyiamino linker attached to either the 5' or 3' terminus of an oligonucleotide. Annealing this modified fragment of DNA to its complementary sequence allows for target modification subsequent to ionic activation. The product of this reaction is a covalent crosslink between the reagent and target strands resulting from an alkylation of DNA by the activated silyloxy aromatic derivative. In a preferred embodiment, a nitrophenyl or bromo group is attached to a methyl group of the silyloxy aromatic derivative. This reagent may be similarly linked to an oligonucleotide probe. Activation of the alkylating agent by an ionic signal (X) which may naturally occur, or may be introduced into the media containing the target molecule, such as by the introduction of a salt (MX).
Inventors
- 👤 Steven E. Rokita
- 👤 Tianhu Li
- 👤 Qingping Zeng
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 03:37
CPC Classifications
C; C07; C07D; C07D207/00; C07D207/02; C07D207/30; C07D207/34; C07D207/36; C07D207/40; C07D207/404; C07F; C07F7/00; C07F7/02; C07F7/08; C07F7/18; C07F7/1804; C07H; C07H21/00; C07H23/00
Claims Analysis
Total Claims: 54
Claim 1
Independent Claim
236 words
A silyloxy aromatic probe composition having the formula: ##STR30## wherein one of R 1 , R 2 , R 3 , R 4 and R 5 is --OSiR 6 R 7 R 8 ; wherein when R 1 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; when R 2 =--OSiR 6 R 7 R 8 , then R 1 , R 3 and/or R 5 is =--CR 9 R 10 X; when R 3 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; when R 4 =--OSiR 6 R 7 R 8 , then R 1 , R 3 and/or R 5 is =--CR 9 R 10 X; when R 5 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; any remaining R 1 -R 5 substituents that are not --OSiR 6 R 7 R 8 or --CR 9 R 10 X are hydrogen atoms; wherein said R 6 , R 7 , R 8 =alkyl or aromatic groups; wherein R 9 and R 10 is an aliphatic or alkyl group; X=leaving group; L is a linking group for attachment to a probe which may be positioned at any carbon atom of the ring; and Pr is a probe for binding to DNA or RNA.
Claim 2
Dependent Claim
236 words
A silyloxy aromatic probe composition having the formula: ##STR30## wherein one of R 1 , R 2 , R 3 , R 4 and R 5 is --OSiR 6 R 7 R 8 ; wherein when R 1 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; when R 2 =--OSiR 6 R 7 R 8 , then R 1 , R 3 and/or R 5 is =--CR 9 R 10 X; when R 3 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; when R 4 =--OSiR 6 R 7 R 8 , then R 1 , R 3 and/or R 5 is =--CR 9 R 10 X; when R 5 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; any remaining R 1 -R 5 substituents that are not --OSiR 6 R 7 R 8 or --CR 9 R 10 X are hydrogen atoms; wherein said R 6 , R 7 , R 8 =alkyl or aromatic groups; wherein R 9 and R 10 is an aliphatic or alkyl group; X=leaving group; L is a linking group for attachment to a probe which may be positioned at any carbon atom of the ring; and Pr is a probe for binding to DNA or RNA.
Claim 3
Dependent Claim
77 words
A silyloxy aromatic probe according to claim 1, wherein said silyloxy aromatic probe alkylates DNA or RNA in response to ionic activation; and wherein X is selected from the group consisting of Cl, F, I, --OCOR, --OH, --OSO 2 CH 3 , --OSO 2 C 6 H 4 CH 3 --p, --OR, --OCONHR, --OCONHCH 2 CH 2 R, Br, --OC 6 H 5 , --OC 6 H 4 NO 2 , and --SC 6 H 5 .
Claim 4
Dependent Claim
103 words
A silyloxy aromatic probe according to claim 2, wherein L comprises a chain --R 11 --R 12 --R 13 , in which R 11 is selected from the group consisting of --NH----S--, --O--, and --CH 2 --, in which R 12 comprises a stable spacer group between R 11 and R 13 , and in which R 13 is selected from the group consisting of --NH 2 --, --SH, --OH and --COOH; and wherein Pr is a probe that includes a localizing moiety selected from the group consisting of an oligonucleotide, protein, intercalating moiety, or other molecule that localizes to DNA or RNA.
Claim 5
Dependent Claim
21 words
A silyloxy aromatic probe according to claim 3, wherein R 6 , R 7 and R 8 comprises a t-butyl moiety.
Claim 6
Dependent Claim
13 words
A silyloxy aromatic probe according to claim 3, wherein Pr is an oligonucleotide.
Claim 7
Dependent Claim
13 words
A silyloxy aromatic probe according to claim 3, wherein Pr is a protein.
Claim 8
Dependent Claim
14 words
A silyloxy aromatic probe according to claim 5, wherein Pr is a DNA strand.
Claim 9
Dependent Claim
20 words
A silyloxy aromatic probe according to claim 3, wherein Pr is an intercalating moiety for intercalating into DNA or RNA.
Claim 10
Dependent Claim
19 words
A silyloxy aromatic probe according to claim 5, wherein said oligonucleotide is linked to L by its 5' terminus.
Claim 11
Dependent Claim
19 words
A silyloxy aromatic probe according to claim 5, wherein the oligonucleotide is linked to L by its 3' terminus.
Claim 12
Dependent Claim
26 words
A silyloxy aromatic probe according to claim 5, further comprising a modified base on said oligonucleotide suitable for linking to R 12 at said modified base.
Claim 13
Dependent Claim
28 words
A silyloxy aromatic probe according to claim 5, further comprising a modified phosphoribose backbone on said oligonucleotide suitable for linking to R 12 at said modified phosphodeoxyribose backbone.
Claim 14
Dependent Claim
93 words
A silyloxy aromatic probe having the formula: ##STR31## wherein R 2 =--OSiR 6 R 7 R 8 , R 3 =--CR 9 R 10 X R 1 ,R 4 and R 5 =hydrogen; said R 6 , R 7 , R 8 =alkyl or aromatic groups; said R 9 and R 10 is an aliphatic or alkyl group, X =leaving group; L is a linking group for attachment to a probe which may be positioned at any carbon atom of the ring; and Pr is a probe for binding to DNA or RNA.
Claim 15
Dependent Claim
93 words
A silyloxy aromatic probe having the formula: ##STR31## wherein R 2 =--OSiR 6 R 7 R 8 , R 3 =--CR 9 R 10 X R 1 ,R 4 and R 5 =hydrogen; said R 6 , R 7 , R 8 =alkyl or aromatic groups; said R 9 and R 10 is an aliphatic or alkyl group, X =leaving group; L is a linking group for attachment to a probe which may be positioned at any carbon atom of the ring; and Pr is a probe for binding to DNA or RNA.
Claim 16
Dependent Claim
105 words
A silyloxy aromatic probe according to claim 13, wherein L comprises a chain --R 11 --R 12 --R 13 --, in which R 11 is selected from the group consisting of --NH--, --S--, --O-- and --CH 2 --, in which R 12 comprises a stable spacer group between R 11 and R 13 , and in which R 13 is selected from the group consisting of --NH 2 --, --SH, --OH and --COOH; and p1 wherein Pr is a probe that includes a localizing moiety, selected from the group consisting of an oligonucleotide, protein, intercalating moiety, or other molecule that localizes to DNA or RNA.
Claim 17
Dependent Claim
24 words
A silyloxy aromatic probe according to claim 14, wherein R 6 and R 7 comprise methyl groups and R 8 comprises a t-butyl moiety.
Claim 18
Dependent Claim
13 words
A silyloxy aromatic probe according to claim 14, wherein Pr is an oligonucleotide.
Claim 19
Dependent Claim
13 words
A silyloxy aromatic probe according to claim 14, wherein Pr is a protein.
Claim 20
Dependent Claim
14 words
A silyloxy aromatic probe according to claim 14, wherein Pr is a DNA strand.
Claim 21
Dependent Claim
20 words
A silyloxy aromatic probe according to claim 14, wherein Pr is an intercalating moiety for intercalating into DNA or RNA.
Claim 22
Dependent Claim
19 words
A silyloxy aromatic probe according to claim 16, wherein said oligonucleotide is linked to L by its 5' terminus.
Claim 23
Dependent Claim
19 words
A silyloxy aromatic probe according to claim 16, wherein said oligonucleotide is linked to L by its 3' terminus.
Claim 24
Dependent Claim
26 words
A silyloxy aromatic probe according to claim 16, further comprising a modified base on said oligonucleotide suitable for linking to R 12 at said modified base.
Claim 25
Dependent Claim
28 words
A silyloxy aromatic probe according to claim 16, further comprising a modified phosphoribose backbone on said oligonucleotide suitable for linking to R 12 at said modified phosphoribose backbone.
Claim 26
Dependent Claim
18 words
A silyloxy aromatic probe recited in claim 15, wherein X comprises --OC 6 H 4 NO 4 .
Claim 27
Dependent Claim
115 words
A process for alkylating DNA or RNA, comprising steps of: a) providing a probe for binding to DNA or RNA; b) providing a silyloxy benzene derivative for linking to the probe; c) linking the probe to the silyloxy benzene derivative to form a targeted alkylating agent; d) introducing the targeted alkylating agent to an in vitro system containing DNA or RNA, whereby the probe associates with the DNA or RNA, localizing the linked silyloxy aromatic derivative near the DNA or RNA; and e) activating the targeted alkylating agent, thereby causing covalent binding between the linked aromatic derivative and the DNA or RNA proximal to the association site of the probe with the DNA or RNA.
Claim 28
Dependent Claim
115 words
A process for alkylating DNA or RNA, comprising steps of: a) providing a probe for binding to DNA or RNA; b) providing a silyloxy benzene derivative for linking to the probe; c) linking the probe to the silyloxy benzene derivative to form a targeted alkylating agent; d) introducing the targeted alkylating agent to an in vitro system containing DNA or RNA, whereby the probe associates with the DNA or RNA, localizing the linked silyloxy aromatic derivative near the DNA or RNA; and e) activating the targeted alkylating agent, thereby causing covalent binding between the linked aromatic derivative and the DNA or RNA proximal to the association site of the probe with the DNA or RNA.
Claim 29
Dependent Claim
244 words
A process as recited in claim wherein the targeted alkylating agent has the molecular formula: ##STR32## wherein one of R 1 , R 2 , R 3 , R 4 and R 5 is --OSiR 6 R 7 R 8 ; wherein when R 1 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; when R 2 =--OSiR 6 R 7 R 8 , then R 1 , R 3 and/or R 5 is =--CR 9 R 10 X; when R 3 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; when R 4 =--OSiR 6 R 7 R 8 , then R 1 , R 3 and/or R 5 is =--CR 9 R 10 X; when R 5 =--OSiR 6 R 7 R 8 , then R 2 and/or R 4 is =--CR 9 R 10 X; wherein any remaining R 1 -R 5 substituents that are not --OSiR 6 R 7 R 8 or --CR 9 R 10 X are hydrogen atoms; wherein said R 6 , R 7 , R 8 =alkyl or aromatic groups; said R 9 and R 10 is an aliphatic or alkyl group; X=leaving group; L is a linking group for attachment to a probe which may be positioned at any carbon atom of the ring; and Pr is a probe for binding to DNA or RNA.
Claim 30
Dependent Claim
39 words
A process as recited in claim 26, wherein said linking step (c) further comprises adapting the silyloxy aromatic derivative by the addition of an acidic linking group suitably modified for linking the silyloxy aromatic derivative to the probe molecule.
Claim 31
Dependent Claim
35 words
A process as recited in claim 27, wherein said probe is an oligonucleotide, and further comprising the step of: suitably modifying a base of said oligonucleotide probe for linking to L at the modified base.
Claim 32
Dependent Claim
37 words
A process as recited in claim 27, wherein said probe is an oligonucleotide, and further comprising the step of: suitably modifying a phosphoribose backbone of said oligonucleotide probe for linking to L at the modified phosphoribose backbone.
Claim 33
Dependent Claim
18 words
A process as recited in claim 27, wherein said activating step comprises introduction of an ionic activating signal.
Claim 34
Dependent Claim
27 words
A process as recited in claim 27, wherein said adapting step further includes brominating the X group, and said activating step comprises activation with an ionic signal.
Claim 35
Dependent Claim
69 words
A process recited in claim 31, wherein said brominating step is followed by substituting the bromine by molecules selected from the group consisting of Cl, F, I, --OCOR, OH, --OSO 2 CH 3 , --OSO 2 C 6 H 4 CH 3 --p, --OR, --OCONHR, --OCONHCH 2 CH 2 R, Br, --OC 6 H 5 , --OC 6 H 4 NO 2 , and --SC 6 H 5 .
Claim 36
Dependent Claim
103 words
A process as recited in claim 32, wherein L comprises a chain --R 11 R 12 --R 13 --, in which R 11 is selected from the group consisting of --NH--, --S--, --O--, and --CH 2 --, in which R 12 comprises a stable spacer group between R 11 and R 13 , and in which R 13 is selected from the group consisting of --NH 2 , --SH, --OH and --COOH; and wherein Pr is a probe that includes a localizing moiety selected from the group consisting of an oligonucleotide, protein, intercalating moiety, or other molecule that localizes to DNA or RNA.
Claim 37
Dependent Claim
101 words
A silyloxy aromatic compound, comprising a substituted benzene ring, wherein a position on the benzene ring is occupied by an --OSiR 6 R 7 R 8 group and at least one position on the benzene ring in conjugation with the --OSiR 6 R 7 R 8 group is occupied by a --CR 9 R 10 X group, in which R 6 , R 7 , and R 8 are selected from the group consisting of alkyl and aromatic groups; R 9 and R 10 are selected from the group consisting of alkyl and aromatic groups; and X is a leaving group.
Claim 38
Dependent Claim
101 words
A silyloxy aromatic compound, comprising a substituted benzene ring, wherein a position on the benzene ring is occupied by an --OSiR 6 R 7 R 8 group and at least one position on the benzene ring in conjugation with the --OSiR 6 R 7 R 8 group is occupied by a --CR 9 R 10 X group, in which R 6 , R 7 , and R 8 are selected from the group consisting of alkyl and aromatic groups; R 9 and R 10 are selected from the group consisting of alkyl and aromatic groups; and X is a leaving group.
Claim 39
Dependent Claim
31 words
The silyloxy aromatic compound of claim 34, wherein two positions on the benzene ring in conjugation with the --OSiR 6 R7R 8 are occupied by --CR 9 R 10 X group.
Claim 40
Dependent Claim
35 words
The silyloxy aromatic compound of claim 34, wherein a heteroatom is present within the benzene ring without destroying the conjugation between the --OSiR 6 R 7 R 8 and --CR 9 R 10 X groups.
Claim 41
Dependent Claim
20 words
The siloxy aromatic compound of claim 36, wherein said heteroatom is selected from the group consisting of O and N.
Claim 42
Dependent Claim
25 words
The silyloxy aromatic compound according to claim 34, wherein said R 6 and R 7 comprise methyl groups and R 8 comprises a t-butyl moiety.
Claim 43
Dependent Claim
62 words
The silyloxy aromatic compound according to claim 34, wherein --X is selected from the group consisting of F, Cl, Br, I, --OCOR, OH, --OSO 2 CH 3 , --OSO 2 C 6 H 4 CH 3 --p, --OR, --OCONHR, --OCONHCH 2 CH 2 R, --OC 6 H 5 , --OC 6 H 4 NO 2 , and --SC 6 H 5 .
Claim 44
Dependent Claim
25 words
The silyloxy aromatic compound according to claim 34, wherein X is selected from the group consisting of OC 6 H 4 NO 2 and Br.
Claim 45
Dependent Claim
22 words
The silyloxy aromatic compound according to claim 34, wherein the silyloxy aromatic compound alkylates DNA or RNA in response to ionic activation.
Claim 46
Dependent Claim
49 words
The silyloxy aromatic compound according to claim 34, wherein a position on the benzene ring is occupied by a --L--Pr group, in which L is a linking group for attachment of the aromatic ring system to a probe; and Pr is a probe for binding to DNA or RNA.
Claim 47
Dependent Claim
68 words
The silyloxy aromatic compound according to claim 42, wherein L comprises a chain --R 11 --R 12 --R 13 --, in which R 11 is selected from the group consisting of --NH--, --S--, --O--, and --CH 2 --; R 12 comprises a stable spacer group between R 11 and R 13 ; R 13 is selected from the group consisting of --NH 2 , --SH--, --OH, and --COOH.
Claim 48
Dependent Claim
38 words
The silyloxy aromatic compound according to claim 42, wherein Pr is a probe that includes a localizing moiety selected from the group consisting of an oligonucleotide, protein, intercalating moiety, or other molecule that localizes to DNA or RNA.
Claim 49
Dependent Claim
18 words
The silyloxy aromatic compound according to claim 44, wherein the localizing moiety non-specifically localizes to DNA or RNA.
Claim 50
Dependent Claim
18 words
The silyloxy aromatic compound according to claim 44, wherein the localizing moiety specifically localizes to DNA or RNA.
Claim 51
Dependent Claim
15 words
The silyloxy aromatic compound according to claim 44, wherein the localizing moiety is an oligonucleotide.
Claim 52
Dependent Claim
15 words
The silyloxy aromatic compound according to claim 47, wherein the localizing moiety is a protein.
Claim 53
Dependent Claim
16 words
The silyloxy aromatic compound according to claim 44, wherein the localizing moiety is a DNA strand.
Claim 54
Dependent Claim
22 words
The silyloxy aromatic compound according to claim 44, wherein the localizing moiety is an intercalating moiety for intercalating into DNA or RNA.
References Cited (Backward Citations)
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