# US 5,346,670 A · Phthalocyanine and tetrabenztriazaporphyrin reagents

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- Number: US 5,346,670 A
- Document ID: US5346670A
- Status: Granted
- Kind: A
- Application number: 07/895,601
- Filed: 1992-06-08
- Granted: 1994-09-13
- Earliest claimed: 1986-12-24
- Assignees: British Technology Group USA Inc.
- Inventors: George E. Renzoni; Deborah C. Schindele; Louis J. Theodore; Clifford C. Leznoff; Karen L. Fearon; Barry V. Pepich
- Web page: https://patentgrep.com/patent/US5346670A

## CPC

- G01N 33/533: with fluorescent label
- 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}
- 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
- Y10S 435/808: Optical sensing apparatus
- Y10S 435/968: High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
- Y10S 436/808: Automated or kit
- Y10T 436/13: Tracers or tags
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Red-shifted, water-soluble, fluorescent, monomerically-tetherable derivatives having the formula: ##STR1## wherein, M represents either H.sub.2 or is selected from among the following metals: aluminum, silicon, phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc. Each R.sub.1 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 R.sub.2 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 R.sub.2 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 R.sub.2. Also disclosed are derivatives of the compounds of the above Formula in which 1-4 of the benzo ring(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 R.sub.2 =R.sub.1 and W=-N.sup.+ D.sub.1 D.sub.2 D.sub.3 are also disclosed. Further, compounds containing Tc, Gd, etc. as the metal in the above Formula may be used for imaging or therapy.

## Claims

### Claim 1

A reagent having the formula: ##STR7## wherein M is selected from the group consisting of hydrogen, aluminum, silicon, phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc;
each R.sub.1 is independently --XYW, --O-phenyl, --S-phenyl, --YW, --W, or hydrogen, and wherein at least one R.sub.1 is --XYW, --O-phenyl, or --S-phenyl;
X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium; or X is CR.sub.3 R.sub.4, wherein R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl, or R.sub.3 and R.sub.4 together form a carbonyl oxygen; or X is phenyl;
Y is a linking group between X and W when R.sub.1 is --XYW, or between W and an aromatic ring of said reagent when R.sub.1 is --YW;
W is a water-soluble group;
R.sub.2 is --A or --Y'A, wherein --A is a biological entity and Y' is a linking group to --A; or R.sub.2 is a reactive or activatable group; and
Z is nitrogen.

### Claim 2 (depends on 1)

A reagent according to claim 1, wherein R.sub.1 is --XYW or --YW and wherein Y is a C.sub.1 -C.sub.7 saturated or unsaturated, straight chain, branched, or cyclic hydrocarbon moiety.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

A reagent according to claim 1, wherein W is --OH, --CO.sub.2 H, --OCH.sub.2 CO.sub.2 H, --PO.sub.4.sup..dbd., --PO.sub.3.sup.-, --SO.sub.3.sup.-, --SO.sub.2.sup.-, --SO.sub.2 Cl, --SO.sub.4.sup..dbd., --NH.sub.2, --NHD, --NHD.sub.1 D.sub.2, or --N.sup.+ D.sub.1 D.sub.2 D.sub.3, wherein D, D.sub.1, D.sub.2, and D.sub.3 are independently C.sub.1 -C.sub.10 alkyl, C.sub.6 -C.sub.12 aryl, or C.sub.6 -C.sub.12 aralkyl.

### Claim 5 (depends on 1)

A reagent according to claim 1, wherein R.sub.1 is --XYW or --YW and wherein Y is C.sub.1 -C.sub.3 alkylene and W is sulfonate or carboxylate.

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 5)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 1)

A reagent according to claim 1, comprising
aluminum phthalocyanine 1, 8, 15, 22-tetraglycolic acid, or
aluminum phthalocyanine 2, 9, 16, 23-tetraglycolic acid, linked to R.sub.2.

### Claim 11 (depends on 1)

A reagent according to claim 1, comprising
2, 9, 16, 23 tetraphenoxy aluminum phthalocyanine,
1, 8, 15, 22 tetraphenoxy aluminum phthalocyanine,
2, 9, 16, 22 tetrathiophenyl aluminum phthalocyanine, or
1, 8, 15, 22 tetrathiophenyl aluminum phthalocyanine, linked to R.sub.2.

### Claim 12 (depends on 1)

A reagent according to claim 1, wherein R.sub.2 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.

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 13)

A kit according to claim 13, wherein there are four reagents, and wherein each R.sub.2 group comprises a different nucleotide, deoxynucleotide, or dideoxynucleotide.

### Claim 15 (depends on 10, 14)

A kit according to claim 14, wherein said reagents comprise reagents according to claim 10.

### Claim 16 (depends on 11, 14)

A kit according to claim 14, wherein said reagents comprise reagents according to claim 11.

### Claim 17 (depends on 1)

A kit for sequencing DNA comprising:
each of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTPT as A labeled with a reagent according to claim 1, a sequencing enzyme, a sequencing primer, each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP, and each of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP.

### Claim 18 (depends on 1)

A kit for sequencing DNA comprising:
a reagent according to claim 1 wherein A is DNA sequencing primer, a sequencing enzyme, each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP, and each of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP.

### Claim 19 (depends on 1)

A kit for sequencing DNA comprising:
a plurality of container, each containing a different reagent according to claim 1 attached to a different one of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP as A, a sequencing enzyme, a sequencing primer, each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP, and each of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP.

### Claim 20 (depends on 1)

A kit for sequencing DNA comprising:
a plurality of containers, each containing a different reagent according to claim 1 attached to a sequencing primer as A, a sequencing enzyme, each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP, and each of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP.

### Claim 21

An organometallic reagent useful for magnetic resonance imaging, having the formula: ##STR8## wherein M is a paramagnetic metal or a radioactive metal;
each R.sub.1 is independently --XYW, --O-phenyl, --S-phenyl, --YW, --W, or hydrogen, and wherein at least one R.sub.1 is --XYW, --O-phenyl, or --S-phenyl;
X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium; or X is CR.sub.3 R.sub.4, wherein R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl, or R.sub.3 and R.sub.4 together form a carbonyl oxygen; or X is phenyl;
Y is a linking group between X and W when R.sub.1 is --XYW, or between W and an aromatic ring of said reagent when R.sub.1 is --YW;
W is a water-soluble group;
R.sub.2 is --A or --Y'A, wherein --A is a biological entity and Y' is a linking group to --A; or R.sub.2 is a reactive or activatable group; and
Z is nitrogen or --CR, wherein --R is H, alkyl, aryl, or aralkyl.

### Claim 22 (depends on 21)

An organometallic reagent according to claim 21, wherein M is selected from the group consisting of gadolinium, manganese, and iron.

### Claim 23 (depends on 1)

A fluorescent reagent according to claim 1, wherein R.sub.2 is --A or --Y'A, and wherein the phthalocyanine macrocycle is monomeric.

### Claim 24 (depends on 23)

A fluorescent reagent according to claim 23 having absorbance in both the red and blue portions of the spectrum, wherein the ratio of the relative heights of the maximum red and blue absorbance peaks of the reagent, A(red)/A(blue), is greater than or equal to 1.4.

### Claim 25 (depends on 23)

A fluorescent reagent according to claim 23, wherein M is aluminum, silicon, gallium, germanium, scandium, or tin.