Long wavelength chemically reactive dipyrrometheneboron difluoride dyes and conjugates

• US 5,274,113 A
• Filed: 11/01/1991
• Issued: 12/28/1993
• Est. Priority Date: 11/01/1991
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard P., Kang, Hee C.
• Primary Examiner(s): Berch, Mark L.
• Application Number:
  US07/786,767
• Time in Patent Office: 788 Days
• Field of Search: 548/405, 548/110, 546/13, 544/229
• US Class Current: 548/405
• CPC Class Codes: C07F 5/022 without C-boron linkages
• CPC Class Codes: C07H 13/04 having the esterifying carboxyl radicals attached to acyclic carbon atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

This invention relates to derivatives of dipyrrometheneboron difluoride fluorescent dyes that have an absorption maximum at wavelengths longer than about 525 nm, and are chemically reactive with nucleic acids, proteins, carbohydrates, and other biologically derived or synthetic chemical materials. The dyes generally have the structure: ##STR1## wherein at least one of the substituents R₁ -R₇, is a reactive functional group, and at least one of the substituents R₁ -R₇ contains a bathochromic moiety. The bathochromic moiety is an unsaturated organic group, preferably heteroaryl or alkenyl. The remaining substituents, which may be the same or different, are hydrogen, halogen, alkyl (containing 1-5 carbon atoms), aryl, arylalkyl, or sulfo. The dyes are used to make novel conjugates with members of specific binding pairs that are ligands or receptors.

Claims

1. A compound of the formula:

##STR18## wherein at least one of the substituents R₁ -R₇ ts --L_m G, where G is a reactive functional group that is a carboxylic acid (--(C═

O)OH), a succinimidyl ester (--(C═

O)O(NC₄ O₂ H₄)), an acyl azide (--(C═

O)N₃), a mixed anhydride with ethyl carbonic acid (--(C═

O)O(C═

O)--OC₂ H₅), an acid halide (--(C═

O)X, where X is halide), an acrylamide (--NH(C═

O)CH═

CH₂), an alcohol (--OH), a aldehyde (--CHO), an amine (--NH₂), an azide (--N₃), a haloacetamide (--NH(C═

O)CH₂ X, where X is halide), a halomethyl (--CH₂ X, where X is halide), a sulfonyl halide (--SO₂ X, where X is halide), a hydrazine (--NHNH₂), an isocyanate (--NCO), an isothiocyanate (--NCS), or maleimide group (--NCH₄ H₂ O₂), and --L_m --, where m=0 or 1, is a substituted or unsubstituted alkyl (containing 1-5 carbons) or arylene group, or a linking bathochromic moiety that is an alkenylene group that is an ethenylene, dienylene, or trienylene, or is a heteroarylene; and

wherein the remaining substituents R₁ -R₇, any of which may be the same or different, are hydrogen;

halogen;

alkyl (containing 1-5 carbon atoms);

sulfo;

arylalkyl, the alkyl portion of which contains 1-5 carbon atoms;

or aryl which may be further substituted, one or more times, by alkyl (containing 1-5 carbon atoms), or alkoxy groups wherein the alkyl portions of such groups have less than 5 carbon atoms;

or a separate bathochromic moiety;

or combinations thereof;

such that at least one of the substituents R₁ -R₇ contains a linking bathochromic or contains a separate bathochromic moiety that is attached to the compound by a single covalent bond, where said separate bathochromic moiety is an alkenyl that is an ethenyl, dienyl, or trienyl, or is a heteroaryl group,

where said alkenyl or alkenylene group optionally contains substituents, which may be the same or different, that are hydrogen, halogen, alkyl (containing 1-5 carbon atoms), cyano, methyl or ethyl carboxylate ester, carboxamide (--CONH₂), aryl, or heteroaryl that is optionally substituted by aryl, arylalkyl, alkyl, or alkoxy groups;

the alkyl portions of which have fewer than 5 carbons and are optionally further substituted on the alkyl portion by methyl or ethyl carboxylate ester or carboxamide (CONH₂); and

said heteroarylene or heteroaryl group optionally contains substituents, which may be the same or different, that are aryl, arylalkyl, alkyl, or alkoxy groups;

the alkyl portions of which have fewer than 5 carbons and are optionally further substituted on the alkyl portion by methyl or ethyl carboxylate ester or carboxamide (CONH₂), or heteroaryl that is optionally substituted by aryl, arylalkyl, alkyl, or alkoxy groups;

the alkyl portions of which have fewer than 5 carbons and are optionally further substituted on the alkyl portion by methyl or ethyl carboxylate ester or carboxamide (CONH₂);

said compound having an absorption maximum at a wavelength longer than about 525 nm in methanol and a quantum yield of greater than about 0.45.

2. A compound, as claimed in claim 1, wherein G is a carboxylic acid, a succinimidyl ester, an acyl azide, an anhydride, an acid halide, a sulfonyl halide, an isocyanate, or an isothiocyanate.

3. A compound, as claimed in claim 1, wherein G is a haloacetamide, a maleimide, or an alkyl halide.

4. A compound, as claimed in claim 1, wherein L is an unsaturated organic group that is a substituted or unsubstituted phenylene, ethenylene, dienylene, trienylene, or heteroarylene.

5. A compound, as claimed in claim 1, wherein at least one of R_1-7 is L_m G, wherein L is a straight chain unsubstituted alkyl group containing 1-5 carbons and G is a carboxylic acid, a succinimidyl ester, an acyl azide, an anhydride, an acid halide, a sulfonyl halide, an isocyanate, or an isothiocyanate;

and one of R_1-7 is a heteroaryl group that is pyrrole, thiophene, furan, oxazole, isoxazole, oxadiazole, imidazole, benzoxazole, benzothiazole, benzimidazole, benzofuran, or indole, that is optionally substituted or unsubstituted, or

one of R_1-7 is an alkenyl group that is ethenyl, butadienyl or hexatrienyl, that is optionally substituted or unsubstituted; and

the remaining substituents of R_1- R₇, which may be the same or different, are hydrogen or alkyl, or sulfo.

6. A compound, as claimed in claim 1, substituted by 1-4 separate bathochromic moieties wherein at least one separate bathochromic moiety is a heteroaryl group that is a pyrrole, thiophene, furan, oxazole, isoxazole, oxadiazole, imidazole, benzoxazole, benzothiazole, benzimidazole, benzofuran, or indole, that is optionally substituted.

7. A compound, as claimed in claim 5, wherein R₁ is L_m G, wherein G is a carboxylic acid and R₇ is a heteroaryl group or an alkenyl group.

8. A compound, as claimed in claim 1, substituted by 1-4 separate bathochromic moieties wherein at least one separate. bathochromic moiety is an alkenyl group that is ethenyl, butadienyl or hexatrienyl, that optionally is substituted

9. A compound, as claimed in claim 1, wherein L is a straight chain unsubstituted alkyl group containing 1-5 carbons and G is a succinimidyl ester (--COO(NC₄ O₂ H₄)).

10. A compound, as claimed in claim 1, wherein R₂ or R₆, or R₂ and R₆ are sulfo.

11. A compound, as claimed in claim 1, wherein R₁ is L_m G that optionally contains a linking bathochromic moiety;

R₇ is a separate bathochromic moiety;

R₅ is optionally a separate bathochromic moiety; and

the remaining substituents of R₁ -R₇, which may be the same or different, are hydrogen, alkyl, or sulfo.

12. A compound, as claimed in claim 11, wherein R₁ is L_m G, wherein L is a straight chain unsubstituted alkyl group containing 1-5 carbons and G is a succinimidyl ester (--(C═

O)O(NC₄ O₂ H₄));

R₇ is a heteroaryl group that is a pyrrole, thiophene, furan, oxazole, isoxazole, oxadiazole, imidazole, benzoxazole, benzothiazole, benzimidazole, benzofuran, or indole, that is optionally substituted one or more times by aryl, arylalkyl, heteroaryl, alkyl, or alkoxy groups, or combinations thereof, the alkyl portions of which have fewer than 5 carbons;

any substituent is optionally further substituted by an ester or amide;

or

R₇ is an alkenyl group that is ethenyl, butadienyl or hexatrienyl, that optionally is substituted;

R₅ is optionally a separate bathochromic moiety;

R₂ and R₆, which may be the same or different, are hydrogen, alkyl, or sulfo;

and the remaining substituents of R₁ -R₇, which may be the same or different, are hydrogen or alkyl.