NO750485L - - Google Patents

Description

FLUORESCENCE LABELING PROCEDURE.

The present invention relates to a method for fluorescence labeling of a material containing a primary amino group,

and the method consists in treating said material with a compound of the formula

"where means lower alkyl, phenyl-lower-alkyl,

R 2 means phenyl or substituted phenyl, and

R-, is substituted or unsubstituted phenyl, naphthyl or indolyl,

in an aqueous medium at a pH value between approx. 8.0 and 10.5.

The present invention also relates to new compounds which are covered by formula I and which can be used in the above-mentioned method. These compounds have the formula

where R'^ means lower alkyl or phenyl-lower-alkyl,

R'2 means phenyl or substituted phenyl and

R' 3 means substituted or unsubstituted phenyl, naphthyl or indolyl under the proviso that when R' 2 and R' 3 both mean phenyl, then R' means lower alkyl with more than one carbon atom or phenyl-lower alkyl with more than 7 carbon atoms.

The rapid identification of microorganisms and other pathogenic antigens by means of fluorescent antibodies is a very important example of the diagnostic application of fluorophore conjugated proteins. The known procedure for fluorescent labeling of proteins, e.g. labeling with fluorescein isothiocyanate (FITC) is based on fluorophores with reactive functional ability, and which will associate with proteins by means of

covalent bond. However, this methodology is fraught with serious disadvantages, which are mainly due to the need for far-reaching purity, whereby any excess of reagent that would otherwise interfere with immunoassays in a non-specific way is removed.

i It has now been found that the compounds of formula I are non-fluorescent, but that they react with materials containing primary amino groups, thereby resulting in fluorescent conjugated compounds, thereby avoiding time-consuming purification operations.

In the description and in the claims, the term "lower alkyl" shall mean a monovalent, saturated, straight-chain or branched hydrocarbon substituent of up to 8 carbon atoms. Examples of lower alkyl groups are methyl, ethyl, n-propyl, n-butyl, hexyl, octyl, iso-propyl, tert-butyl, etc. The term "phenyl-lower-alkyl" means a lower alkyl group similar to that which is defined above, and which is attached to a phenyl ring, e.g. benzyl, phenylethyl, phenylpropyl, etc. The expression "substituted" in connection with phenyl, naphthyl or indolyl means that these groups are substituted with one or more of the following substituents: halogen (ie fluorine, chlorine, bromine or iodine), lower alkyl, tri fluoromethyl, lower alkoxy, nitro and cyano. The term "lower alkoxy" means a group with a lower alkyl residue which is attached to an ether oxygen, and which has a valence bond from the ether oxygen. Examples of lower alkoxy groups are methoxy, ethoxy, n-propoxy, n-butoxy, isopropoxy, tert-butoxy, etc.

Preferred compounds of formula I are those where R 1 means lower alkyl and both R 2 and R 4 mean phenyl. A particularly preferred compound of formula I is the compound where R^ is methyl and R^ and R^ are phenyl, i.e. 2-methoxy-2,4-diphenyl-

3 (2 H)furanone.

Compounds of formula II can be prepared by means of a multi-step synthesis, whereby one starts from readily available starting materials of formula

where R'2 and R'^ have the meanings indicated above. 1 Compounds of formula III, where R'2 and R'3 mean phenyl or substituted phenyl, will usually be benzalacetophenones or substituted benzalacetophenones. In the first step, the starting material with formula III is epoxidized under alkaline conditions, and this gives an epoxy ketone with formula

where R'2 and R13 have the meaning indicated above. The epoxidation reaction is carried out by treating a compound of formula III with an excess of hydrogen peroxide in the presence of a strong base. Suitable strong bases for the present reaction include alkali metal hydroxides, e.g. sodium hydroxide and potassium hydroxide5 and alkali metal carbonates, e.g. sodium carbonate and potassium carbonate. Suitable solvents for epoxy dering reaction are alcohols, and in particular methanol and ethanol, as well as aqueous alcohol mixtures. The reaction is usually carried out at temperatures from approx. 10 to approx. 40°C, whereby 20 to approx. 30°C is most preferred. In the next step, the compound of formula IV is treated with a strong anhydrous base to cleave the epoxide ring and to to give a diketone of formula

where R'2 and R'3 have the meaning indicated above. Suitable, strong, anhydrous bases for this cleavage reaction include alkali metal alkoxides, such as sodium methoxide, sodium ethoxide, sodium isopropoxide, potassium tert.-butoxide, etc. Suitable solvents for the cleavage reaction include anhydrous alcohols, e.g. methanol, ethanol, isopropanol and tert.-butanol. It is usually preferred to use the same alcohol from which the alkoxide base is derived, but this is not critical, and if another alcohol is used as solvent there will be an exchange between the alcohol solvent and the alcohol portion of the alkali metal alkoxide. The cleavage reaction is usually carried out at a higher temperature, preferably between approx. 40 and approx. 100°C, whereby the boiling point of the reaction medium is most preferred. In the next step, the diketone of formula V is converted into an enamine of formula

where R<1>2 and R'^ have the meaning given above, and R^ and R,-, independently of each other, each means lower alkyl, and where together with the nitrogen atom they form a 5- or 6-membered, heterocyclic ring, which in most cases exhibits one additional heteroatom selected from the group consisting of

nitrogen and oxygen.

In this reaction, the diketone of formula V reacts with an amino-methenylating agent to give the enamine.

Suitable amino-methylating agents include lower alkyl acetals of an N,N-disubstituted formamide, e.g. dimethylformamide-dimethylacetal5tris(secondary amino)methanes, e.g. tris(dimethylamino)methane and tris(piperidino)methane^ and bis(secondary amino) lower alkoxymethanes, e.g. bis(dimethylamino)t-butoxymethane.

The amino part

which is shown in the structural formula of compound VI, is introduced from the aminomethenylating agent. Acetals of N,N-disubstituted formamides have the general formula where Rg and R^ each mean lower alkyl$ tris(secondary amino)methanes have the general formula and bis(secondary amino)lower alkoxymethanes have the general formula where RQ means lower alkyl. Examples of amino parts

include those where R^ and R,.

each, independently of the other, means lower alkyl, e.g. dimethylamino and diethylamino, and those where R^ and R^ together with the nitrogen form a 5- or 6-membered, heterocyclic ring, e.g. piperidino, morpholino, pyrrolidino, piperazino, imidazolino, pyrazolidino, etc. Examples of lower alkoxy moieties OR^ and OR^ are methoxy, ethoxy, propoxy, n-butoxy, etc. Examples of lower alkoxy moieties ORg are methoxy, ethoxy, tert. -butoxy etc.

This reaction can be carried out in an inert organic solvent.

Preferred solvents include formamides, and especially dimethylformamide. An excess of aminomethenylating agent can also be used as a solvent.

The production of the enamine can be carried out in a temperature range from approx. 0 to approx. 100°C, whereby a temperature range from approx. 10 to approx. 40°C is preferred. Room temperature is particularly preferred. In the next step, the enamine of formula VI is converted to hydroxy-furanone of formula

where R'^ and R'3 have the meanings indicated above. This conversion involves a basic, aqueous hydrolysis. Suitable bases for this hydrolysis include alkali metal hydroxides, e.g. sodium hydroxide and potassium hydroxide5 and alkali metal carbonates, e.g. sodium carbonate and potassium carbonate. Suitable temperatures for carrying out the above reaction are from approx.

0 to approx. 40°C, with room temperature being the most preferred. After the hydrolysis is complete, the alkaline solution is neutralized, and this gives hydroxyfuranone of formula VII. Compounds of formulas VI and VII, where R'^°9R<l>3 are not both phenyl, are new.

The hydroxyfuranone of formula VII can then be converted into furanone of formula II by reaction with the appropriate alcohol, i.e. an alcohol of formula R'^OH, where R<1>^ has the same meaning as indicated in the compound with formula II above. This alcohol can either be a lower alkanol or phenyl lower alkanol, such as methanol, ethanol, benzyl alcohol, phenylethyl alcohol, etc. As solvents for this reaction, the alcohol itself or a mixture of the alcohol and an inert organic solvent can be used. Most preferred is to carry out the reaction in the desired alcohol. The reaction can conveniently be carried out at temperatures from approx. room temperature to approx. of the solvent

boiling point. It is most preferred to carry out the reaction at a temperature between approx. 40 and approx. 80 oC.

Compounds of formula I can be converted ("interconverted"),

ie the alkoxy group OR^ can be replaced by reaction of the compound of formula I with a suitable lower alkanol or phenyl lower alkanol. Thus, e.g. the compound of formula I, where R^ means methyl, is converted into the corresponding compound where R^ means benzyl by heating the first-mentioned compound with an excess of benzyl alcohol.

The "fluorogens" (fluorescence-causing substances) of formula I are relatively insoluble in water and react slowly with water to form decomposition products which are non-fluorescent. The fluorogens of formula I are easily soluble in organic solvents, and are particularly soluble in solvents such as acetone and dichloromethane. As the compounds have low water solubility, and it is desirable to allow these to react with materials occurring in aqueous media either in the form of a solution or a suspension, it is preferred to add the aforementioned compounds either as a solution in an organic solvent, and then preferably in a water-miscible organic solvent, such as acetone, or to carry out the reaction by having the compound of formula I adsorbed on a solid support. This latter technique with adsorption on a solid substrate is particularly preferred when fluorogen reacts with materials that are soluble in the aqueous medium, so that any unreacted fluorogen with formula I, which is adsorbed on the solid substrate, can be removed by

filtration or centrifugation. Suitable solid substrates include neutral, inert materials, such as diatomaceous earth, polysaccharides, etc. A particularly preferred solid substrate is diatomaceous earth. Adsorption of the compound of formula I on the solid support can preferably be carried out using methods known per se, e.g. by suspending the solid support in a solution containing the compound of formula I, e.g. dissolution in acetone or methylene chloride, and evaporation of the solvent from said suspension, followed by careful

! air drying or vacuum drying. It is very beneficial

to produce the solid substrates with a content of from approx. 0.1

to approx. 5.0% by weight compounds ..with formula I, and most preferably with 1 to approx. 2% by weight.'

Fluorogens of formula I react with primary amine-containing materials to form fluorescent products. The types of materials which are allowed to react with the fluorogens of formula I include primary amines, amino acids, peptides, proteins, and especially those with biological characteristics, such as immunoglobulins (antibodies), viruses, single-celled organisms, such as bacteria, protozoa, algae and fungi, as well as multicellular organisms such as helminths,

such as tapeworms. The effect of the reaction of a compound of formula I with one of the aforementioned materials is the introduction of a fluorescent label into said materials. The introduction of the fluorescent label allows rapid identification of such a material using fluorescence technology and in particular fluorescence microscopy. The application of fluorescent labeling is. particularly important when it comes to labeling proteins, and especially those with biological significance, such as e.g. antibodies. A significant advantage of the compounds according to the present invention is that they are not fluorescent in themselves, but that after reaction with materials containing primary amino groups, they produce fluorescent material. Furthermore, after reaction with water, which occurs in the reaction medium,

the compounds of formula I will decompose to materials which are also non-fluorescent. Thus, the purification and isolation of fluorescently labeled materials is considerably simplified, as it is not necessary to separate these materials from any unreacted fluorogen-containing reagents or from their decomposition products, which is the case with previous labeling techniques, such as fluorescein isothio- cyanate.

Preferred materials which can be labeled with the compounds of formula I are indicated in the following Table I:

Table I

■ ■ - ■ v

I. Microorganisms

Bacteria

1. Gram-positive cocci

Streptococci (pyogenes, fecalis and viridans) Staphylococci (aureus and albus) Pneumococci (D. pneumoniae)

2. Gram-negative cocci

Neisseria (gonorrhoeae and meningitidis)

3. Gram-positive aerobic bacilli

Bacillus anthracis

Corynebacterium diphtheriae Erysipelothrix

Listeria monocytogenes

4. Gram-positive anaerobic bacilli

Clostridia (botulinum, perfringens, welchii and tetani) 5. Gram-negative anaerobic bacilli Bacteroides 6. Gram-negative intestinal bacilli Escherichia

Klebsiella

Enterobacter

Proteus

Pseudomonas

Salmonella

Shigella

7. Gram-negative nonintestinal bacilli Pasteurella (pestis and tularensis) Hemophilus influenzae

Brucella (melitensis, abortus and suis) Bordetella pertussis

Malleomyces

8. Spirochetes Treponema pallidum Leptospira Borrelia

9. Mycoplasma

10. Mycobacteria

11. Vibrio

12. Actinorayces Protozoa 1. Intestinal Protozoa Amebae 2. Flagellates Trichomonas Leishmania Trypanosomes Toxoplasma

3. Sporozoa

Plasmodia (vivax, falciparum, malariae and ovale)

4. Intestinal nematodes Roundworms Hookworms Whipworms 5. Ve v—nema t ode r Trichinella Filaria (Wuchereria bancroftii) Dracunculus 6. Trematodes Schistosomes Intestinal icterus

Tissue icter

7. Cestodes Tapeworms 8. Toxoplasma (T. gondii)

Mushroom

1. Sporotrichum

2. Cryptococcus

3. Blastomyces

4. Histoplasma

5. Coccidioids

6. Candida Viruses and Richettsia

1. Richettsia

2. Viruses Canine hepatitis Shope papilloma Influenza A & B Honsepest

Herpes simplex Adenoviruses Polyoma

Rous sarcoma Vaccinia Measles Puppies Leukemia

Mumps Newcastle disease

Sendai

"ECHO"

Foot-and-mouth disease Psittacosis

Rabies

Ectromelia

Tree viruses

II. Foreign antigens Polysaccharides Hyaluronidase

Tetanus toxin Egg ovalbumin Serum albumin from sheep Human plasma γ-globulin Human serum albumin

III. Natural antigens 1. Hormones

Pi tui tari a hormones Insulin

Glucagon

Thyroid hormone

Chorionic gonadotrophin Chorionic growth hormone - prolactin

2. Enzymes

Pancreatic chymotrypsinogen Procarboxypeptidase Deoxyribonuclease "Ribonuclease Glyceraldehyde-3-phosphate dehydrogenase Catalase

3. Organ-specific antigens Kidney

Liver

Skin

Heart Gastrointestinal tract

Prostate

Embryonic antigens

Tumor antigens.

4. Connective tissue components

Muscle

The collagen

Amyloid

5. Bl°^cellez5ntigeneri_blood group substances_and_other isoantigens

Platelets

Megakaryocytes

Leukocytes

Erythrocytes

Blood group substances

Forssman antigen

Histocompatible antigens

6. Plasma proteins

Fibrin and fibrinoid

Plasminogen and plasmin

7. Pathological globulins

Myeloma, macroglobulinaemia and dysglobulinaemia proteins

Rheumatoid factor

C-reactive protein

IV Natural antigens

1. Natural gamma globulin

Natural antibodies - nephrotoxic antibodies Complement

2. Auto-antibodies

Antinuclear factor

Thyrojde autoanti drug is

Adrenal autoantibodies Autoantibodies to gastric parietal cells in pernicious anaemia

IN

Anti-coli

Anti-liver

Anti-kidney

Autoantibodies for spermatozoa

Anti-heart

Muscle autoantibodies in myasthenia gravis Autoantibodies for nervous tissue

Autoantibodies against fibrous tissue and vascular components Autoantibodies against platelets and megakaryocytes Antibodies against trophoblasts

3. Artificially named antibodies for: The immunoglobulin classes Igb, IgM, IgA of various species.

Particularly preferred materials which can be labeled with the compounds of formula I are Hymenolepis nana, Escherichia coli, antibodies to pneumococcus type II and gamma globulin.

It has been found that the fluorescent labeling reaction, which uses compounds of formula I, is highly dependent on pH. The marking takes place to a considerable extent between pH values of approx. 8.0 and 10.5. An optimal pH range for the marking is between approx. 9.0 and approx. 9.5. The pH can be regulated using a technique known per se, which also includes the use of a buffer. The use of buffers containing primary amino groups should be avoided.

The degree of labeling of a particular substrate will vary depending on the concentrations of the fluorogen used and on the contact between the fluorogen and the substrate.

The degree of labeling desired for a particular substrate and a particular purpose will naturally vary from time to time. It has been found that a sufficient marking for most purposes will be achieved with a collision time between 2 min. and 2 hours, and then preferably between approx. 5 min. and 30 min.

The fluorescence excitation and emission spectra of the labeled materials will also vary depending on the type of material. As an example, one can mention a typical fluorescence spectrum for a gamma-globulin fraction which is labeled with a compound

with formula I, where one has two excitation maxima at 290 and 390 nm and an emission maximum at 480 nm.

It has further been found that the labeling of living substrates such as bacteria or tapeworms with fluorogens of formula I has little, if any, effect on their viability. Thus, the present procedure provides an efficient method for labeling living organisms. Labeling of biologically important proteins, such as antibodies, has also been found to have little, if any, effect on their biological properties. If e.g. antibodies to pneumococcus type II are fluorescently labeled according to the above technique, the antibody will remain largely unaffected and, more importantly, the specificity of the antibody remains unchanged. In this case, e.g. the antibodies specific to pneumococcus type II, and they do not bind pneumococcus type I.

A further feature of the labeled materials, which are produced according to the above technique, is that they are unusually stable

for extended periods of time even at room temperature and in the presence of. light. Thus, a small breakdown of the fluorescence labeling is obtained, which has been carried out with different substrates including both living and non-living substrates for periods of time as long as a month.

The present invention will be better understood and evaluated with the help of the following examples.

in EXAMPLE 1

To a mechanically stirred mixture of 83.2 g of benzalacetophenone

(0.3 M), 100 ml of methanol and 120 ml of 15% hydrogen peroxide

100 ml of 2 N sodium hydroxide solution was added, while the temperature was kept below 30° by external cooling. After the addition was finished, the mixture was allowed to stand at room temperature for 20 minutes. The crystalline precipitate was filtered off, washed with water and recrystallized from methanol. 59.6 g of 1,3-diphenyl-2,3-epoxy-1-propanone were obtained

m.p. 90°C.

EXAMPLE 2

A hot solution of 30 g of potassium t-butoxide in 500 ml of ethanol was quickly added to a boiling solution of 30 g of 1,3-diphenyl-2,3-epoxy-1-propanone in 500 ml of ethanol. The mixture was kept boiling on a steam bath for 2 minutes. It was then diluted with 3 L of water. The aqueous solution was saturated with carbon dioxide by adding small pieces of ice. The resulting emulsion was extracted with ether. The ether extracts were diluted with benzene, dried over sodium sulphate and evaporated under reduced pressure. The remaining dark oil was distilled under high vacuum to give 19.5 g of 1,3-diphenyl-1,2-propanedione; kp. 136 - 138°/0.1 mm.

EXAMPLE 3

A solution of 44.8 g of 1,3-diphenyl-1,2-propanedione in 90 ml of N,N-dimethylformamide-dimethyl acetal was allowed to stand at room temperature for 2 hours. It then became completely ice/water. The aqueous mixture was extracted three times with ether. The combined extracts were washed with water, diluted with benzene, dried over sodium sulfate and evaporated under reduced pressure. The oily residue was crystallized from ether/petroleum ether to give 40.2 g of the desired product. A second yield of 3.1 g was obtained from the mother liquor by concentration and addition of petroleum ether. A total of 43.3 g of 1-dimethylamino-2,4-di-phenyl-1-butene-3,4-dione was obtained, m.p. 108°C.

EXAMPLE 4

To a solution of 43.3 g of 1-dimethylamino-2,4-diphenyl-1-butene-3,4-dione in 500 ml of ethanol was added 500 ml of 2% aqueous potassium hydroxide. The mixture was stirred at room temperature for 2 h

hours. It was then diluted with 3 1 of water and acidified with 10% hydrochloric acid. The solid 2-hydroxy-2,4-diphenyl-3(2 H )-furanone which precipitated was filtered off by suction. The filter cake was washed with water and dissolved (without further purification)

in 500 ml of methanol. The methanol-containing solution was refluxed for 20 hours, then unconcentrated on a steam bath for approx.

350 ml. Crystalline product was obtained on cooling. This was further purified by recrystallization twice from methanol, yielding 25.5 g of the desired material 5 m.p. 93 - 95°C. The mother liquors were combined and evaporated. The rest were redeemed

in chloroform and filtered through 200 g of silica gel. The eluate was evaporated and the residue was recrystallized from ethanol, yielding 5.8 g of additional product m.p. 93 - 95°C. A total of 31.3 g of 2-methoxy-2,4-diphenyl-3(2H)-furanone was obtained, m.p. 93 - 95°C.

EXAMPLE 5

By following the procedures from examples 1-4 they became

following compounds, including respective intermediates, prepared.

2-ethoxy-2,4-diphenyl-3 (2 H )-furanone, m.p. 87°C$

2-benzyloxy-2,4-diphenyl-3(2H)-furanone, m.p. 140°C$ 2-methoxy-2-phenyl-4-(4-nitrophenyl)-3-(2 H )-furanone, m.p. 115 - 117°C.

EXAMPLE 6

By following the procedures from examples 1-4, the following compounds can be prepared: 2-methoxy-2-phenyl-4-(2-naphthyl)-3(2H)-furanone5

2-Methoxy-2-(4-chlorophenyl)-4-phenyl-3 (2H)-furanone$

2-ethoxy-2-(2,4-dimethoxyphenyl)-4-(3-indolyl)-3(2H)-furanone-, 2-methoxy-2-phenyl-4-(2-trifluoromethylphenyl)-3(2 H)-furanone.

EXAMPLE 7

Mouse tapeworms (Hymenolepis nana) in 9 ml aqueous buffer,

pH 9.5, was stained for 10 minutes at 22 - 26°C by adding 1 ml of an acetone solution (with different concentrations)

of 2-methoxy-2,4-diphenyl-3(2H)-furanone. Excess of color

in substance was removed by washing the worms in BME (boar's basal medium) culture medium. The intensity of fluorescence, which was graded on an approximate scale, from none (0) to maximum staining (1.00), was determined using an American optical fluorescence microscope at 1 hour, 22 hours and 8 days after staining. These results are shown below. Both morphology and activity of the worms appear to remain unaffected by the staining procedure.

EXAMPLE 8

Escherichia coli was suspended in 9 ml of borate-buffered saline, pH 9.5, and was stained for 30 - 60 minutes at room temperature by the addition of 1 ml of an acetone solution (various concentrations) of 2-methoxy-2,4- diphenyl-3-(2H)-furanone. Excess dye was removed by centrifugation, the bacteria were resuspended in saline, stored at 4°C,

and the sample was examined for fluorescence at 24 hours, 8 days and 16 days after staining. Cell viability was assessed 22 hours after staining using a standard plate-counting technique. Staining intensity was assessed using a graded scale, ranging from no staining (0) to maximum staining (1.00). The results are shown below:

EXAMPLE 9

Specific type II rabbit antipneumococcal serum was diluted in buffer, pH 9.0, and allowed to react for 10 minutes at room temperature with celite (diatomaceous earth) containing 1 or 2% by weight 2-methoxy-2,4-diphenyl-3 (2 H)-furanone. The celite was then removed by filtration or centrifugation, and the labeled serum was stored at 4°C. The salt suspension of type II pneumococci was placed on microscopic slides. After air drying, the bacteria were fixed to the slides by heating, and they were stained with labeled type II antiserum for 10 - 30 minutes at room temperature. Excess anti-serum was removed by washing the slides with saline, and fluorescence was determined under direct oil immersion. The color intensity was measured 10, 25 and 31 days after labeling and was graded using a discretionary scale, which covers the range of no coloration (0) to maximum coloration (1.00). The results are shown below.

No fluorescence could be observed when type I pneumococci were

used as antigen, and this indicated that the immunological specificity was retained.

Effect of labeling of antibody titers was determined by standard immunological samples. The results are shown below. The titer is expressed as the reciprocal of the last serum dilution that gives a positive reaction.

EXAMPLE 10'

50 mg of γ-globulin (horse, ^> 98% purity) was dissolved in 500 ml of 0.05 M buffer of different pH. 200 mg of celite, containing 2% by weight of 2-methoxy-2,4-diphenyl-3(2H)-furanone (prepared by treating 10 g of celite with a solution of 200 mg of furanone in acetone and evaporating to dryness) was added. After 15 minutes of magnetic stirring at room temperature, the mixture was filtered through a fine funnel, and the filtrate was frozen in dry-ice-acetone and stored in a freezer. The fluorescence was measured after centrifugation. Relative fluorescence is given in relative units.

Similar results were obtained after the solution was allowed to stand for 3 days at room temperature.

EXAMPLE 1 11

20 ml of 1% solutions of γ-globulin (horse ^> 98%) in buffer

I (either pH 9.00 or 9.50) was labeled by treatment with 200 mg of celite containing 2% by weight 2-methoxy-2,4-diphenyl-3(2H)-furanone at room temperature for 10 minutes. The solutions were immediately neutralized to pH 7.00 with 1 N HCl, centrifuged and the supernatant was stored at room temperature. Relative fluorescence was measured (activation 390 nm, emission 484 nm) at various time intervals, and judged by reasonable units.

Claims (19)

1. Method for fluorescent labeling of material containing a primary amino group, characterized by treating said materials with a compound of formula wherein means lower alkyl or phenyl-lower-alkyl, R^ phenyl or substituted phenyl and R^ substituted or unsubstituted phenyl, naphthyl or indolyl-, in an aqueous medium at a pH between approx. 8.0 and 10.5. 2. Method according to claim 1, characterized in that the pH is between 9.0 and 9.5. 3. Method according to claim 1 or 2, characterized in that the material which is labeled is a protein. 4. Method according to claim 3, characterized in that the protein is an antibody. 5. Method according to claim 1 or 2, characterized in that the material. which is marked, is a unicellular or multicellular organism. 6. Procedure according to claim 4 or 5, characterized in that the material to be marked is I Hymenolepis nana. 7. Method according to claim 1 or 2, characterized in that the material to be labeled is Escherischia coli. 8. Method according to claim 1 or 2, characterized in that the material to be labeled is an antibody for pneumococcus type II. 9. Method according to claim 1 or 2, characterized in that the material to be labeled is gamma globulin. 10. Method according to claims 1-9, characterized in that the compound of formula I is adsorbed on an inert, solid substrate. 11. Method according to claim 10, characterized in that the solid substrate is diatomaceous earth. 12. Method according to one of claims 1-11, characterized in that the compound of formula I is 2-methoxy-2,4-diphenyl-3(2H)-furanone. 13. Method according to one of claims 1-11, characterized in that the compound with formula I is 2-benzyloxy-2,4-diphenyl-3-(2H)-furanone. 14. Method according to one of claims 1-11, characterized in that the material containing a primary amino group is treated with a compound of the formula wherein R<1>^ means lower alkyl or phenyl-lower-alkyl, R'2 means phenyl or substituted phenyl and R'2 is substituted or unsubstituted phenyl, naphthyl or indolyl under the proviso that when R12 and R'3 are both phenyl then R'^ is lower alkyl with more than one carbon atom or phenyl-lower alkyl with more than 7 carbon atoms. 15. Means for carrying out the method according to claims 1-14, characterized in that it is a compound of formula wherein R'^ means lower alkyl or phenyl-lower-alkyl, R<1>2 means phenyl or substituted phenyl and R'2 means substituted or unsubstituted phenyl, naphthyl or indolyl under the proviso that when R'2 and R'3 both mean phenyl then R'^ is lower alkyl with more than one carbon atom or phenyl-lower alkyl of more than 7 carbon atoms. j 16. A compound according to claim 15, character cert in that R <1>^ means lower alkyl.. 17. A compound according to claim 15 or 16, characterized in that R'2 and both mean phenyl. 18. A compound according to claim 15, characterized in that the compound is 2-ethoxy-2,4-diphenyl-3(2H)-furanone. 19. A compound according to claim 15, characterized in that the compound is 2-methoxy-2-phenyl-4-(4-nitrophenyl)-3(2H)-furanone.