Tetrabenztriazaporphyrin reagents and kits containing the same

• US 5,135,717 A
• Filed: 08/29/1989
• Issued: 08/04/1992
• Est. Priority Date: 12/24/1986
• Status: Expired due to Term

• Current Assignee: SR One Ltd. (GlaxoSmithKline PLC)
• Original Assignee: British Technology Group Ltd.
• Inventors: Leznoff, Clifford C., Fearon, Karen L., Theodore, Louis J., Renzoni, George E., Schindele, Deborah C., Pepich, Barry V.
• Primary Examiner(s): Hill, Jr., Robert J.
• Application Number:
  US07/398,433
• Time in Patent Office: 1,071 Days
• Field of Search: 128/24.1, 422/61, 436/94, 436/172, 436/501, 436/506, 436/510, 436/512, 436/513, 436/536, 436/546, 436/808, 435/5-7, 435/29, 435/810, 435/7.1, 435/975, 530/345, 536/18.7, 536/27, 540/121, 540/128, 540/129-132, 540/135, 540/139, 540/140
• US Class Current: 422/430
• CPC Class Codes: A61K 41/0071 PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Red-shifted, water-soluble, fluorescent, monomerically-tetherable derivatives having the formula: ##STR1## wherein, M represents either H₂ or is selected from among the following metals: aluminum, silicon, phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc. Each R₁ is independently selected from --XYW, --YW, and --W. X represents either a carbon, or heteroatom selected from among oxygen, nitrogen, sulfur, phosphorus, silicon, and selenium; Y represents a linking group; and W represents a water solubilizing group. The substituent R₂ is selected from among --A, --Y'"'"'A, --XA, and --XY'"'"'A, where A denotes a biological entity such as an antibody, antibody fragment, nucleotide, nucleic acid probe, antigen, oligonucleotide, deoxynucleotide, dideoxynucleotide, avidin, streptavidin or membrane probe, or R₂ is a reactive or activatable group suitable for conjugating to a biological entity. Y'"'"' is a linking group that tethers the biological entity to the phthalocyanine or tetrabenztriazaporphyrin macrocycle. Z is either a nitrogen atom or a carbon substituted with hydrogen, alkyl, aryl, or aralkyl groups. Z may also be attached to R₂. Also disclosed are derivatives of the compounds of the above formula in which 1-4 of the benzo rings(s) contain 1 or 2 N atoms. Methods of sequencing DNA and detecting analytes, including cells, using these derivatives are disclosed, as are kits for carrying out assays for the analytes and flow cytometry. Methods of detecting DNA using cationic compounds of the above formula, wherein R₂ =R₁ and W=--N⁺ D₁ D₂ D₃ are also disclosed. Further, compounds containing Tc, Gd, etc. as the metal in the above formula may be used for imaging or therapy.

Claims

1. A reagent having the formula:

##STR7## wherein M is selected from the group consisting of H₂, aluminium, silicon, phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc;

each R₁ is independently --XYW, --YW, --W, or hydrogen;

X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium;

or X is CR₃ R₄, wherein R₃ and R₄ are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl, or R₃ and R₄ together form a carbonyl oxygen;

or X is phenyl;

Y is a linking group between X and W when R₁ is --XYW, or between W and an aromatic ring of said reagent when R₁ is --YW;

W is a water-soluble group;

R₂ is --A or --Y'"'"'A, wherein --A is a biological entity and Y'"'"' is a linking group to --A;

or R₂ is reactive or activatable group; and

Z is --CR, where --R is H, alkyl, aryl, or aralkyl.

2. A reagent according to claim 1, wherein R₁ is --XYW or --YW and wherein Y is a C₁ -C₇ saturated or unsaturated, straight chain, branched, or cyclic hydrocarbon moiety.

3. A reagent according to claim 1, wherein R₁ is --XYW or --YW and wherein Y is a polyether, polyamine, polyalcohol, sugar, peptide, or nucleotide.

4. A reagent according to claim 1, wherein W is --OH, --CO₂ H, --OCH₂ CO₂ H, --PO₄⁼, --PO₃^-, --SO₃^-, --SO₂^-, --SO₂ Cl, --SO₄⁼, --NH₂, --NHD, --NHD₁ D₂, or --N⁺ D₁ D₂ D₃, wherein D, D₁, D₂, and D₃ are independently C₁ -C₁₀ alkyl, C₆ -C₁₂ aryl, or C₆ -C₁₂ aralkyl.

5. A reagent according to claim 1, wherein R₁ is --XYW or --YW and wherein Y is C₁ -C₃ alkylene and W is sulfonate or carboxylate.

6. A reagent according to claim 5, wherein --XYW is substituted at 1-4 of the 1, 8, 15, and 22 positions on the macrocycle.

7. A reagent according to claim 5, wherein --XYW is substituted at 1-4 of the 2, 9, 16, and 23 positions on the macrocycle.

8. A reagent according to claim 1, wherein M is aluminum, and at least one R₁ is --XYW, wherein X is O or S, and W is sulfonate or sulfonyl chloride.

9. A reagent according to claim 8, wherein Y is phenyl.

10. A reagent according to claim 1, comprising9, 16, 23 tetraphenoxy aluminum 20-phenyl tetrabenztriazaporphyrin,8, 15, 22 tetraphenoxy aluminum 20-phenyl tetrabenztriazaporphyrin, or8, 15, 22 tetrathiophenyl aluminum 20-phenyl tetrabenztriazaporphyrin, linked to R₂.

11. A reagent according to claim 1, wherein R₂ is --A or --Y'"'"'A, and wherein A is a nucleic acid selected from the group consisting of ATP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP, dUTP, ddATP, ddCTP, ddGTP, ddTTP, ddUTP, and derivatives thereof, oligonucleotides, and polynucleotides.

12. A kit for sequencing DNA, comprising:

a plurality of containers, each containing a different reagent according to claim 1,

wherein the different reagents are characterized by having a common excitation wavelength and different wavelengths of maximum emission.

13. A kit according to claim 12, wherein there are four reagents, and wherein each R₂ group comprises a different nucleotide, deoxynucleotide, or dideoxynucleotide.

14. A reagent according to claim 1, wherein M is aluminum, each R₁ is sulfonate, and Z is C-phenyl, attached to R₂.

15. A reagent having the formula:

##STR8## wherein M is selected from the group consisting of H₂, aluminum, silicon, phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc;

each R₁ is independently --XYW, --YW, --W, or hydrogen;

X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium;

or X is CR₃ R₄, wherein R₃ and R₄ are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl, or R₃ and R₄ together form a carbonyl oxygen;

or X is phenyl;

Y is a linking group between X and W when R₁ is --XYW, or between W and an aromatic ring of said reagent when R₁ is --YW;

W is a water-soluble group;

R₂ is --A or --Y'"'"'A, wherein --A is a biological entity and Y'"'"' is a linking group to --A;

Z is nitrogen or --CR, wherein --R is H, alkyl, aryl, or aralkyl; and

wherein at least one of the benzo rings a, b, c, and d depicted in the formula above is a pyridine or pyrazine ring.

16. A reagent having the formula:

##STR9## wherein M is selected from the group consisting of H₂, aluminum, silicon, phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc;

each R₁ is independently --XYW, --YW, --W, or hydrogen;

X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium;

or X is CR₃ R₄, wherein R₃ and R₄ are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl, or R₃ and R₄ together form a carbonyl oxygen;

or X is phenyl;

Y is a linking group between X and W when R₁ is --XYW, or between W and an aromatic ring of said reagent when R₁ is --YW;

W is a water-soluble group; and

Z is --C--R₂, wherein R₂ is --A is or --Y'"'"'A, wherein is --A is a biological entity and Y'"'"' is a linking group to --A;

or R₂ is a reactive or activatable group suitable for conjugating to a biological entity without substantially impairing the fluorescence of the reagent or the biological activity of the biological reagent.