D-luciferin derivatives, their application and processes for the detection of ligands marked with an enzyme in the determination of biochemical substances

• US 5,098,828 A
• Filed: 08/19/1987
• Issued: 03/24/1992
• Est. Priority Date: 10/24/1985
• Status: Expired due to Fees

• Current Assignee: Reinhard Geiger, Werner Miska
• Original Assignee: Reinhard Geiger, Werner Miska
• Inventors: Geiger, Reinhard, Miska, Werner
• Primary Examiner(s): Ceperley, Mary E.
• Application Number:
  US07/064,256
• Time in Patent Office: 1,679 Days
• Field of Search: 435/7, 435/8, 435/14, 435/15, 435/18, 435/19, 435/21-24, 435/6, 435/7.72, 548/114, 548/178, 530/802, 530/300, 536/4.1, 536/17.1, 536/17.3, 536/17.4, 536/27, 536/29
• US Class Current: 435/7.72
• CPC Class Codes: C07D 277/64 with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
• CPC Class Codes: C07F 9/65583 each of the hetero rings containing nitrogen as ring hetero atom
• CPC Class Codes: C07H 17/00 Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
• 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: C07K 5/0821 with the first amino acid being heterocyclic, e.g. His, Pro, Trp
• CPC Class Codes: C07K 7/18 Kallidins; Bradykinins; Related peptides
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Abstract

The object of the invention consists of D-luciferin derivatives of General Formula (I) ##STR1## wherein R¹ =OH, alkoxy, alkenyloxy, amino acid, NH₂ or oligopeptide and

R² =H; H₂ PO₃ --; HSO₃ --; alkyl or alkenyl, optionally substituted by phenyl; aryl; ##STR2## with R³ =alkyl or alkenyl, optionally substituted by phenyl; mono- or disaccharide or nucleotide.

An object of the invention is further the use of said luciferin derivatives for the detection of ligands in the determination of biochemical substances, in particular in enzyme immuno assays, in blot processes and in nucleic acid hybridization. These ligands are attached to an enzyme (enzyme conjugate). In the process, for example an antigen, a hapten or an antibody is conjugated with an enzyme. The enzyme conjugate is capable of releasing luciferin from the luciferin derivative. The luciferin released is reacted with luciferase. The amount of light emitted in the process is determined. From the data obtained in this manner, the quantity of the substances to be determined may be derived.

Claims

1. D-luciferin derivatives which are enzyme substrates, which release luciferin upon reaction with enzyme and which have a formula (I):

##STR35## and wherein said luciferin derivative is D-luciferyl-L-N-alpha-arginine (R¹ is L-H-alpha-arginin, R² is H);

D-luciferyl-L-phenylalanin (R¹ is --NH--CH(CH₂ C₆ H₅)--COOH, R² is H);

D-luciferyl-L-methionine (R¹ is --NH--CH((CH₂ SCH₃)--COOH, R² is H);

D-luciferyl-L-glycine (R¹ is --NH--CH₂ COOH, R² is H);

D-luciferin-O-sulfate (R¹ is OH, R² is --SO₂ --OH);

D-luciferin-O-phosphate (R¹ is --OH, R² is --P(O)--(OH)₂);

D-luciferin-O-(C¹)glucose(R¹ is OH and R² is glucose);

or

D-luciferin-O-(C¹)galactose (R¹ is OH and R² is galactose).

2. A luciferin derivative according to claim 1 of formula (I), which is

D-luciferyl-L-N-alpha-arginine (R¹ is L-H-alpha-arginin, R² is H);

D-luciferyl-L-phenylalanin (R¹ is --NH--CH(CH₂ C₆ H₅)--COOH, R² is H);

D-luciferyl-L-methionine (R¹ is --NH--CH((CH₂ SCH₃)--COOH, R² is H);

D-luciferyl-L-glycine (R¹ is --NH--CH₂ COOH, R² is H);

D-luciferin-O-sulfate (R¹ is OH, R² is --SO₂ --OH);

or

D-luciferin-O-phosphate (R¹ is --OH, R² is --P(O)--(OH)₂).

3. A luciferin derivative according to claim 1 of formula (I), which is

D-luciferin-O-(C¹)glucose (R¹ is OH and R² is glucose);

or

D-luciferin-O-(C¹)galactose (R¹ is OH and R² is galactose).

4. In a chemical test wherein a ligand conjugated to an enzyme (enzyme-ligand conjugate) is reacted with its biochemical co-reactant to produce a combination (enzyme-ligand+coreactant) and wherein the enzyme-ligand conjugate, which is either contained in said combination or not, is detected by its reaction with a substrate for such enzyme whereby a measurable species is released and wherein the improvement comprises using as an enzyme substrate a compound which releases luciferin and has a formula (I):

##STR36## wherein R¹ is a hydroxy or amino group;

a linear or branched C₁ -C₂₀ alkoxy or C₂ -C₂₀ alkenyloxy group;

an L-amino acid radical bound by the alpha-amino group;

or an oligopeptide radical with up to 10 L-amino acid units, bound by the α

-amino group of the terminal amino acid unit and R² is hydrogen atom;

a H₂ PO₃ -- or HSO₃ group;

a linear or branched C₁ -C₂₀ alkyl or C₂ -C₂₀ alkenyl group, optionally substituted by one or several phenyl radicals;

an aryl group with 6 to 18 carbon atoms;

a group of the formula (II);

##STR37## wherein R³ is a linear or branched C₁ -C₂₀ alkyl or a C₂ -C₂₀ alkenyl group optionally substituted by a phenyl radical;

or a C₆ -C₁₈ aryl group;

a naturally occurring nucleotide radical with 1 to 3 phosphate groups attached by means of the phosphate group or groups;

or a glucosidically attached mono- or disaccharide;

which comprises reacting the enzyme conjugate with said luciferin derivative to release luciferin;

the released luciferin reacts with the enzyme luciferase of the fire fly Photinus pyralis and the amount of light emitted is measured quantitatively and the quantity of the substance to be detected is obtained from the data.

5. A process according to claim 4 for the detection of antigens, haptens or antibodies marked with an enzyme in immunoassays, which comprises releasing luciferin by means of the enzyme conjugate from a luciferin derivative of formula (I):

##STR38## wherein R¹ is a hydroxy or amino group;

a linear or branched C₁ -C₂₀ alkoxy or C₂ -C₂₀ alkenyloxy group;

an L-amino acid radical bound by the α

-amino group;

or an oligopeptide radical with up to 10 L-amino acid units, bound by the α

-amino group of the terminal amino acid unit and R² is hydrogen atom;

a H₂ PO₃ -- or HSO₃ group;

a linear or branched C₁ -C₂₀ alkyl or C₂ -C₂₀ alkenyl group, optionally substituted by one or several phenyl radicals;

an aryl group with 6 to 18 C atoms;

a group of the formula (II);

##STR39## wherein R³ is a linear or branched C₁ -C₂₀ alkyl or a C₂ -C₂₀ alkenyl group optionally substituted by a phenyl group;

or a C₆ -C₁₈ aryl group;

a naturally occurring nucleotide radical with 1 to 3 phosphate groups attached by means of the phosphate group or groups;

or a glucosidically attached mono- or disaccharide;

which comprises reacting the enzyme conjugate with said luciferin derivative to release luciferin;

such that the luciferin released is reacted with the enzyme luciferase of the fire fly Photinus pyralis, the amount of light emitted determined quantitatively and the quantity of the antigen, antibody or hapten to be detected is determined from the obtained data.