IL44165A - Method for fluorescently labelling a material with a furanone derivative and products formed by reacting such a material with the furanone derivative - Google Patents

Description

7 UKiisn A method for fluorescentiy labelling a material with a furanone derivative and products formed by reacting such a material with the furanone derivative SPABAMEDICA A..G.

C:- 42345 44165/2 The present invention relates to a method for fluorescently labeling a material selected from a primary amine, an aminoacid, a peptide, a protein, a virus or an unicellular or multicellular organism which method comprises treating said material with a compound of the formula wherein R is lower alkyl, phenyl lower alkyl;, 1¾2 is phenyl or phenyl substituted with one or more halogen, lower alkyl, trifluoromethyl , lower alkoxy, nitro or cya o and is phenyl, naphthyl orNindolyl unsubstituted or substituted with one or more halogen, lower alkyl, trifluoromethyl, lower alkoxy, nitro or cyano, in an aqueous medium. Furthermore the present invention relates to material selected from a primary amine, an aminoacid, a peptide, a protein, a virus or an unicellular or multicellular organism labeled with a compound of formula I . 44165/2 The rapid identification of microorganisms and other pathogenic antigens with the help of fluorescent antibodies is a most important example of the diagnostic utility of fluorophorlc protein conjugates. Existing procedures for fluorescent labeling of proteins, for example, labeling with fluorescein isothlocyanate (FITC) rely upon fluorophors with reactive functionalities which will covalently bind to proteins. However, this methodology is encumbered by serious disadvantages, stemming mainly from the need for extensive purification to remove any excess reagent which would otherwise non-specifically interfere in immunoassays.

It has now been found that the compounds of formula I are non-fluorescent but react with materials selected from a primary amine, an aminoacid, a peptide, a protein, a virus or an unicellular or multicellular organism to produce fluorescent conjugates thus avoiding tedious purification.

In the specification and the appended claims, the term "lower alkyl" shall mean a nomovalent , saturated, 44165/2 straight or branched chain hydrooarbon substituent containing up to and including 8 carbon atoms. Examples of lower alkyl groups are methyl, ethyl, n-propyl, n-butyl, hexyl, octyl, isopropyl, tert. -butyl, and so forth. The term "phenyl lower alkyl" refers to a lower alkyl group as defined above which is attached to a phenyl ring, for example, benzyl, phenyl^ ethyl, phenylpropyl, and so forth. The. term "substituted" as applied to phenyl, naphthyl or indolyl refers to these groups substituted with one or more of the following substituents : halogen (i.e., fluorine, chlorine, bromine or iodine), lower alkyl, trifluoromethyl, lower alkoxy, nitro, and cyano. The term "lower alkoxy" shall mean a group having a lower alkyl residue linked to an ether oxygen and having its valenoe bond from the ether oxygen. Examples of lower alkoxy groups are methoxy, ethoxy,n-propoxy, n-butoxy, isopropoxy, tert . -butoxy, and so forth.

Preferred compounds of formula I are those wherein R.^ is lower alkyl and both R2 and R^ are phenyl. A particularly preferred compound of formula I is the compound wherein R^ is methyl and Rg and R^ are phenyl, i.e., 2-methoxy-2, -diphenyl-3(2 H)furanone.

The compounds of formula I may be prepared by a multi-step synthetic sequence starting from readily available starting materials of formula 44165/2 wherein R and R are as above.

Compounds of formula III wherein and are phenyl or substituted phenyl are generally referred to as benzalacetophenones or substituted benzalacetophenones .

In the first step, the starting material of formula III is epoxidized under basic conditions to afford an epoxy ketone of formula wherein R~ and R_ are as above.

The epoxidation reaction is carried out by treating a compour 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 hydroxide; and alkali metal carbonates, e.g., sodium carbonate and potassium carbonate. Suitable solvents for the epoxidation reaction are alcohols, particularly methanol and ethanol, and aqueous alcohol mixtures. The reaction is generally performed at temperatures from about 10 to about 0°C, most preferably from about 20 to about j30°C.

In the next. step, the compound of formula. IV is 44165/2. ring and afford a diketone of formula wherein 'i and R are as above.

Suitable strong anhydrous bases for this cleavage reaction include alkali metal alkoxides such as sodium methoxide, sodium ethoxide, sodium isopropoxide, potassium tert. -butoxide, and so forth. As suitable solvents for the cleavage reaction, there may be mentioned anhydrous alcohols for example, methanol, ethanol, isopropanol and tert-butanol It is generally preferred to utilize the same alcohol from which the alkoxide base is derived; however, this is not critical, and if a different 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 generally carried out at an elevated temperature, preferably between about 40 and about 100°C, most preferably at the boiling point of the reaction medium.

In the next step, diketone of formula V is converted to an enamine of formula VI wherein R'2 and R'^ are as above, and R ^ and R _ taken independently are each lower alkyl and taken together with the nitrogen atom form a 5- or 6-membered heterocyclic ring having at the most one additional hetero- atom selected from the group consisting of nitrogen and oxygen.

In this reaction, the diketone of formula V is reacted with an amino-methenylating agent to afford the enamine.

Suitable amino methenylating agents include lower alkyl acetals of an Ν,Ν-disubstituted formamide, e.g., dimethyl formamide dimethyl acetalj tris (secondary amino) methanes, e.g., tris (dimethylamino)methane and tris (piperidino) methane } and bis (seoondary amino) lower alkoxy methanes, e.g., bis (dimethyl amino)j -butoxy methane.

The amino as shown in the structural formula for compound VI is introduced from the aminomethenyl-ating agent. Acetals of Ν,Ν-disubstituted formamides have the general formula wherein Rg and R^ are each lower alkyl; tris (secondary amino) methanes have the general formula and bis (secondary amino)lower alkoxymethanes have the general formula wherein RQ is lower alkyl.

Examples of amino moieties - ^ η include those R 5 where R ^ a d R each taken independently are lower alkyl, e.g., dimethylamino and diethylamino, and those where R ^ and R ,. taken together with the nitrogen form a 5- or 6-membered heterocyclic ring, e.g., piperidino, morpholino, pyrrolidino, piperazino, imidazolidino, pyrazolidino, and so forth. Examples of lower alkoxy moieties OR g and OR ^ are methoxy, ethoxy, propoxy, n-butoxy, and so forth.

Examples of lower alkoxy moieties OR g are methoxy, ethoxy, tert-butoxy, and so forth.

This reaction may be carried out in any inert organic solvent. Preferred solvents include formamides, especially dimethylformamide. An excess of aminomethenylatlng agent may also be utilized as solvent. 44165/2 The preparation of the enamine may be effectuated over a temperature range of from about 0 to about 100°C. although a temperature range from about 10 to about 0°C i preferred. A temperature of about room temperature is especially preferred.

In the next step, the enamine of formula VI is converted to the hydroxy furanone of formula wherein R2 and are as above.

This conversion involves a basic aqueous hydrolysis.

Suitable bases for this hydrolysis include alkali metal hydroxides, e.g., sodium hydroxide and potassium hydroxide; and alkali metal carbonates, e.g., sodium carbonate and potassium carbonate. Suitable temperatures for carrying out the above reaction are from about 0 to about 40°C, most preferably about room temperature. After the hydrolysis is complete, the basic solution is neutralized to afford the hydroxy furanone of formula VII.

The hydroxy furanone of formula VII may be then converted to the furanone of formula I by reaction with the appropriate alcohol, i.e. an alcohol of the formula · R^OH. wherein R^ has the meaning given in formula I above. This alcohol may be either a lower alkanol or phenyl lower alkanol, such as methanol, ethanol, benzyl alcohol, phenyl-ethyl alcohol, and so forth. As solvents for this reaction, there may be employed the alcohol itself or a mixture of the alcohol and an inert organic solvent. It is most preferable to carry out this reaction neat in the desired alcohol. The reaction may suitably be carried out at temperatures from about room temperature to about the boiling point of the solvent medium. It is most preferable to carry out the reaction at a temperature between about 40 and about 80°C.

Compounds of formula I may be interconverted, i.e., the alkoxy group OR^ can be changed, by reaction of the compound of formula I with a suitable lower alkanol or phenyl lower alkanol. Thus, for example, the compound of formula I, wherein is methyl, can be converted to the corresponding compound wherein is benzyl by heating the former compound with an excess of benzyl alcohol.

The fluorogens of formula I are relatively insoluble in water and react slowly with water to afford decomposition products which are nonfluorescent. The fluorogens of formula I are readily soluble in organic solvents and are particularly soluble in solvents such as acetone and dichloromethane.

Since the compounds have low water solubility, when it is desired to react them with materials which are present in aqueous media, either as a solution or as a suspension, it 44165/2 solvent, most preferably a water misclble organic solvent such as acetone, or to carry out the reaction by having the compound of formula I adsorbed onto a solid support. This latter technique of adsorption onto a solid support is particularly preferred when reacting the fluorogen with . materials which are soluble in the aqueous medium so that any unreacted fluorogen of formula I adsorbed on the solid support may be removed by filtration or centrifugation.

Suitable solid supports include neutral inert material such as diatomaceous earth, polysaccharides, and so forth. A particularly preferred solid support is diatomaceous earth. Adsorption of the compound of formula I onto the solid support may be performed by methods known per se, for example, by suspending the solid support in a solution containing the compound of formula I, for example, solution in acetone or methylene chloride, and evaporating the solvent from said suspension followed by thorough air drying or drying under vacuum. It is most preferable to prepare solid supports containing from about 0,1 to about 5.0 weight % of compounds of formula I, most preferably from about 1 to about 2 weight %.

Fluorogens of formula I react with materials selected from a primary amine, an aminoacid, a peptide, a protein, a virus or an unicellular or multicellular organism to form fluorescent products. The products obtained with materials with one or more than two carbon atoms are new.

The types of proteins which may be reacted with the V as immunoglobulins (antibodies); viruses; unicellular organisms such as bacteria, protozoa, algae and fungi; and multicellular organisms, for example, helminths, such as tapeworms. The effect of reacting a compound of formula I with one of the aforementioned materials is to introduce a fluorescent label into said material. The introduction of the fluorescent label allows for rapid identification of such material using fluorescent technology, particularly fluorescent microscopy. The use of fluorescent labeling is particularly important in the field of labeling of proteins, particularly those of biological importance such as antibodies. A major advantage of the compounds of the present invention is that they themselves are non-fluorescent, but upon reaction with materials containing primary amino groups, produce fluorescent material. Further, upon reaction with water contained in the reaction medium, the compounds of formula I will decompose into materials which are also non-fluorescent. Thus, the purification and isolation of the fluorescently labeled materials is greatly simplified, since it is not necessary to separate these materials from any unreacted fluorogenic reagents, or decomposition products thereof, as is the case with prior art labeling technique such as fluorescein iso-thiocyanate.

Preferred materials which can be labeled with the compounds of formula I are listed in the following table I: Table I I. Microorganisms Bacteria 1. Gram-positive cocci Streptococci (pyogenes, fecalis and viridans) Staphylococci (aureus and albus) Pneumococci (D. pneumoniae) 2. Gram-negative oocci Neisseria (gonorrhoeae and meningitidis) 3. Gram-positive aerobic bacilli Bacillus anthracis Corynebacterium diphtheriae Eryslpelothrix 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 Treponema pallidum Leptospira Borrelia 9. Mycoplasma 0. Mycobacteria 1. Vibrio 2. Actinomyces Protozoa 1. Intestinal Protozoa Amebae 2. Flagellates Trichomonas Leishmania Trypanosomes Toxoplasma 3. Sporozoa Plasmodia (vivax, falciparum, malariae and ovale) 4. Intestinal nematodes Pinworms Hookworms Whip worms 5. Tissue nematodes Trichinella Filaria (Wuchereria bancroftii) Dracunculus 6. Trematodes Schistosomes Intestinal flukes Tissue flukes 7. Cestodes Tapeworms 8. Toxoplasma (T. gondii) Fungi 1. Sporotrichum 2. Cryptococcus j5. Blastomyces 4. Histoplasma 5. Coccidioldes 6. Candida Viruses and Rickettsia 1. Rickettsia 2. Viruses Canine hepatitis Shope papilloma Influenza A & B Fowl plague Herpes simplex Adenoviruses Polyoma Rous sarcoma Vaccinia Poliovirus Measles Canine distemper Leukemia Mumps Newcastle disease Sendai ECHO Foot and mouth disease Psittacosis Rabis Ectromelia Arbor viruses II. Foreign Antigens Polysaccharides Hyaluronidase Tetanus toxin Egg ovalbumin Ovine serum albumin Human plasma 7-globulin Human serum albumin III. Native Antigens ■ 1. Hormones Pituitary hormones Thyroid hormone Chorionic gonadotrophin Chorionic growth hormone - prolactin 2. Enzymes Pancreatic chymotrypslnogen Procarboxypep11dase 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 Collagen Amyloid 5. Blood Cell Antigens, Blood Group Substances and other Isoantigens Platelets Megakaryocytes Leucocytes Erythrocytes Blood group substances Forssman antigen Histocompability antigens 6. Plasma Proteins Fibrin and fibrinoid Plasminogen and plasmin 7. Pathological Globulins Myeloma, macroglubullnaemic and dysglobulinaemia- proteins Rheumatoid factor C-reactive protein IV. Native Antibodies 1. Native gamma globulin Native antibodies - nephrotoxic antibodies Complement 2. Auto antibodies Antinuclear factor Th roid autoantibodies Adrenal autoantibodies Autoantibody to gastric parietal cells in pernicious anemia Ant1-colon Anti-liver • Anti-kidney Autoantibodies to spermatozoa Anti-heart Muscle autoantibodies in myasthenia gravis Autoantibodies to nervous tissue Autoantibodies against fibrous tissue and vascular- components Autoantibodies against platelets and megakaryocytes Antibodies against trophoblasts 3. Induced antibodies to; Immunoglobuline classes Igb, IgM, IgA of different species Particularly preferred materials which oan be labeled with the compounds of formula I are Hymenolepis nana, Escherichia coll, antibodies for pneumococcus type II and gammaglobuline.

It has been found that the fluorescent labeling reaction utilizing compounds of formula I is greatly dependent upon pH. The labeling occurs at a substantial rate between pHs of about 8.0 and 10.5. An optimal pH range for effecting the labeling is between about pH 9.0 and about 9. 5. pH may be controlled by techniques known per se for adjusting pH, including the use of buffers. However, the use of buffers containing free primary amino groups should be avoided.

The extent of labeling a particular substrate will vary depending upon the concentration of fluorogen utilized and the total contact between the fluorogen and the substrate.

The extent of labeling desired for any particular substrate and purpose will of course vary from case to case. It has been found that extensive labeling for most purposes, can be achieved with a contact time between about 2 mins. and 2 hrs., most preferably between about 5 mins. and 30 mins.

The fluorescent excitation and emission spectra for the labeled materials will also vary depending upon the nature of the material. As an example, there may be mentioned a typical fluorescent spectrum for a gamma-globulin fraction labeled with a com ound of formula I in which there are two maximum at 480 nm.

It has further been found that labeling of living substrates such as bacteria or tapeworms with fluorogens of formula I can have little, if any, effect upon their viability. Thus, the present procedure provides an efficient method for labeling living organisms^. It has also been found that labeling of biologically important proteins such as antibodies has little, if any, effect upon their biological properties. For example, if antibodies against pneumococcus Type II are fluorescently labeled in accordance with the above technique, the antibody titer remains largely unaffected and, more importantly, the specificity of the antibody remains unchanged. In this case, for example, the antibodies were still specific for pneumococcus Type II and would not bind with pneumococcus Type I.

One additional feature of the labeled materials prepared by the above technique is that they are unusually stable for long periods of time, even at room temperature and in the presence of light. Thus, there is little destruction of the fluorescent label introduced into a variety of substrates, including both living and nonliving substrates over periods as long as one month.

The present invention may be more fully understood and appreciated by reference to the following specific Example 1 To a mechanically stirred mixture of 83· 2 g benzal-acetophenone (0.3 )# 1000 ml methanol and 120 ml 15$ hydrogen peroxide was added 100 ml 2N sodium hydroxide solution, while maintaining the temperature below 30° by external cooling. After completed addition, the mixture was left standing at room temperature for 20 min. The crystalline precipitate was filtered off, washed with water and recrystallized from methanol. There were obtained 59.6 g l, 3-diphenyl-2,3-epoxy-l-propanone; m.p. 90°C.

Example 2 To a boiling solution of 30 g l,3-diphenyl-2, 3-epoxy-l-propanone in 500 ml ethanol was rapidly added a hot solution of 30 g potassium t-butoxide in 500 ml ethanol.

The mixture was kept boiling on a steambath for 2 minutes. It was then diluted with 3 1 · water. The aqueous solution was saturated with carbon dioxide by the addition of small pieces of dry ice. The resulting emulsion was extracted with ether. The ether extracts were diluted with benzene, dried over sodium sulfate and evaporated under reduced pressure. The remaining dark oil was distilled in high vacuum to afford 19.5 g l, 3-diphenyl-l, 2-propanedione; b.p. 136-138°/ 0.1 mm.

Example 3 A solution of 44.8 g 1 , 3-diphenyl-l, 2-propanedione in 90 ml Ν,Ν-dimethylformamide dimethyl acetal was allowed to stand at room temperature for 2 hours. It was then poured into 1 1. ice/water. The aqueous mixture was extracted three times with ether. The oombined 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 crop of 3.1 g was obtained from the mother liquor upon concentration and addition of petroleum ether. In total .3 g l-dimethylamino-2, 4-di-phenyl-l-butene-3, 4-dione; m-p. 108°C were obtained.

Example 4 To a solution of 4 . g l-dimethylamino-2, 4.-diphenyl- 1-butene-3, 4-dione in 500 ml ethanol was added 500 ml 2% aqueous potassium hydroxide. The mixture was stirred at room temperature for 2 hours. It was then diluted with 3 1 · water and acidified with 10$ hydrochloric acid. The solid 2-hydroxy-2,4-diphenyI-3 (2 H)-furanone, which precipitated, was filtered off with suction. The filter-cake was washed with water, and dissolved (without further purification) in 500 ml methanol. The methanolic solution was refluxed for 20 hrs., then concentrated on the steambath to approximately ' # This was further purified by reorystallizing twioe from methanol, yielding 25· 5 g of the desired material; m.p. 93-95°C. The mother liquors were combined and evaporated. The residue was redissolved in chloroform and filtered through 200 g silica gel. The eluate was evaporated and the residue was recrystallized from ethanol, giving an additional 5.8 g product; m.p. 93-95°C. In total 31.3 8 2-methoxy-2,4-diphenyl-3 (2 H)-furanone, m.p. 93-95°C are obtained.

Example 5 Following the procedures of examples 1-4, the following compounds, including the respective intermediates in their preparation, were prepared: 2-ethoxy-2,4-diphenyl-3 (2 H)-furanone, m.p. 87°: 2-Benzyloxy-2,4-diphenyl-3 (2 H)-furanone, m.p. l40°C: 2-methoxy-2-phenyl-4-(4-nitrophenyl) -3- (2 H)-furanone, m.p. 115-117°C.

Example 6 Following the procedure of examples 1-4, there may be prepared the following oompounds: 2-methoxy-2-phenyl-4- (2-naphthyl) -3 (2 H)-furanone; 2-methoxy-2- (4-chlorophenyl) -4-phenyl-3 (2 H)-furanone; 2-ethoxy-2- (2,4-dimethoxyphenyl) -4- (3-indolyl) -3 (2 H) -furanone; furanone.

Example 7 Mouse tapeworms (Hymenolepls nana) In 9 ml of aqueous buffer, pH 9. 5* were stained for 10 minutes at 22-26°C by adding 1 ml of an acetone solution (of various concentrations) of 2-methoxy-2, -diphenyl-3 (2 H)-furanone. Excess dye was removed by washing the worms in BME (eagle's basal medium) culture medium. The intensity of fluorescence, judged on an arbitrary scale from no staining (0) to maximum staining (1 .00) , was determined using an American Optical Fluorescent microscope at 1 hr., 22 hr. and 8 days after staining. These results are presented below. Both gross morphology and activity of the worms appeared to be unaffected by the staining procedure.

Concentration mg/ml Time post staining 1 hr. 22 hrs. 8 days 0.0 0.06 0. 06 0. 06 0. 5 0. 37 0.37 0. 50 0. 1 0.67 0. 67 - 0. 2 0.75 0.75 O. 67 Example 8 Escherichia coli was suspended in 9 ml borate buffered saline solution, pH 9. 5» and was stained for 30-60 mins. 4 (of various concentrations) of 2-methoxy-2,4-diphenyl-;5(2 H)-furanone. Excess dye was removed by centrifugation, the bacteria were resuspended in, saline, stored at 4°C and samples were examined for fluorescence at 24 hr., 8 days and 16 days post staining. Cell viability was estimated at 22 hrs. post staining by a standard plate count technique. Staining intensity was judged on an arbitrary scale from no staining (0) to maximum staining (1.00). The results are presented below: Cone, mg/ml Time post staining Cell viability 1 day 8 days l6 days 0.0 0.00 0.00 0.00 2 x 108 0.05 0.67 0.67 0.67 - 0.1 1.00 0.87 0.87 7 x 106 0.2 1.00 1.00 0.87 ,5 x 105 Example 9 Specific Type II rabbit antipneumococcal serum was diluted in buffer, pH 9.0, and reacted for 10 minutes at room temperature with celite (dlatomaceous earth) containing 1% or 2% w/w of 2-methoxy-2,4-diphenyl-5(2 H)-furanone. The celite was then removed by filtration or centrifugation and the labeled serum stored at 4°C. Saline suspension of Type II pneumococci were placed on microscope slides. The bacteria, after air drying, were fixed to the slides by heat, and stained with labeled Type II antiserum for 10-30 washing the slides with saline and fluorescence was determined under direct oil immersion. Staining intensity was measured at 10, 25 and 31 days post labeling, and was judged on an arbitrary scale from no staining (0) to maximum staining (l.OO) The results are presented below: Time post Dilution of labeled serum labeling (days) 1:10 1:100 Staining Intensity 0 0.87 10 0.75 1.00 25 0.75 31 0.87 1.00 No fluorescence was seen when Type I pneumococci were used as antigen, thus indicating that immunological specificity was retained.

The effect of labeling on antibody titer was determined by standard immunological tests. The results are presented below. The titer is expressed as the reciprocal of the last serum dilution giving a positive reaction.

Immunologic Test Titer labeled/serum unlabeled/serum Quellung Reaction 100 80 Latex slide agglutination 400 320-640 Fluorescent microscope: 14 days post labeling 640-1280 0 1 days post labeling 400-800 0 Example 10 50 mg of 7-globulin (horse, ^> 98$ pure) was dissolved in 500 ml of 0.05 M buffers of varying pH. 200 mg celite containing 2% w/w of 2-methoxy-2, 4-diphenyl-3 (2 H)-furanone (prepared by treating 10 g celite with a solution of 200 mg of the furanone in acetone and evaporating to dryness) was added. After 15 mins. of stirring magnetically at room temperature, the mixture was filtered through a fine funnel, the filtrate was frozen in dry ice-acetone and stored in a freezer. Fluorescence was measured after centrifugation. Relative fluorescence is given in arbitrary units. pH rel. fluorescence 8.00 4 8.35 9.5 8.50 16 8.83 3 9.00 46 9.31 75 9.50 80.5 9.75 84 10.06 89 Identical results were obtained after allowing the solutions to stand for 3 days at room temperature.

Example 11 20 ml of \ solutions of 7-globulin (horse, > 98$) In buffer, either pH 9.00 or 9.50, were labeled by treatment with 200 mg of celite containing 2% w/w of 2-methoxy-2, 4-diphenyl-3(2 H)-furanone at room temperature for 10 mins. The solutions were neutralized immediately to pH 7.00 with 1 N HC1, centrifuged, and the supernatant was stored at room temperature. Relative fluorescence was measured (activation 390 nm , emission 484 nm) at various intervals, and is given arbitrary units. pH 9.00 Days post labeling Rel. fluorescence 0 56.Ο 1 56.0 2 55.5 . 4 56.0 6 57.0 8 11 55.0 pH 9.5Ο Days post labeling Rel. fluorescence 0 89.5 1 89.0 2 88.0 4 88.0 6 89.0 8 11 86.0

Claims (24)

44165/2 CLAIMS :

1. A method for fluorescently labeling a material selected from a primary amine, an aminoacid, a peptide, a protein, a virus or an unicellular or multicellular organism, which method comprises treating said material with a compound of the formula wherein is lower alkyl, phenyl lower alkyl;, R2 is phenyl or phenyl substituted with one or more halogen, lower alkyl, trifluoromethyl , lower alkoxy, nitro or cyano and is phenyl, naphthyl or indolyl unsubstituted or substituted with one or more halogen, lower alkyl, trifluoromethyl, lower alkoxy, nitro or cyano, in an aqueous medium at a pH between about 8.0 and 10.5.

2. A method according to Claim 1 wherein the pH i between 9.0 and 9.5. 44165/2

3. A method according to Claim 1 or Claim 2 wherein the material being labeled is a protein.

4. A method according to Claim 3 wherein the protein is an antibody.

5. A method according to Claim 1 or Claim 2 wherein the material being labeled is unicellular or multicellular organism.

6. A method according to Claim 1 or Claim 2 wherein the material to be labeled is Hymenolepis nana.

7. A method according to Claim 1 or Claim 2 wherein the material to be labeled is Escherichia coll.

8. A method according to Claim 1 or Claim 2 wherein the material to be labeled is an antibody for pneumococcus Type II.

9. A method according to Claim 1 or Claim 2 wherein the material to be labeled is gammaglobuline.

10. A method according to any one of Claims 1 to 9 wherein the compound of formula I is 2-methoxy-2,4-diphenyl-3 (2H) -furanone.

11. A method according to any one of Claims 1 to 9 wherein the compound of formula I is 2-benzyloxy-2,4-diphenyl-3- (2H) -furanone. 44165/2

12.. A method according to any one of Claims 1 to i! wherein the compound of formula I is adsorbed onto an inert solid support.

13. ' A method according to Claim 12 wherein said solid support is diatomaceous earth.

14. Product formed by reacting a material selected from a primary amine, an aminoacid, a peptide, a protein, a virus or an unicellular or multicellular organism having one or more than two carbon atoms with a. compound of the formula wherein is lower alkyl or phenyl lower- alkyl, R2 is phenyl or phenyl substituted with one or more halogen, lower alkyl, trifluoromethyl , lower alkoxy, nitro or cyano and is phenyl, naphthyl or indolyl unsubstituted with one or more halogen, lower alkyl, trifluoromethyl , lower alkoxy, nitro or cyano.

15. Product according to Claim 14 wherein the substituents and in the compound of formula I are both phenyl. 44165/2

16. Product according to Claim 14 or Claim 15 wherein the compound of formula Z is 2-methoxy~2,4~ dipheny1-3 (2Π) -furanone.

17. « Product according to Claim 14 or Claim 15 wherein the compound of formula I is 2-benzyloxy-2,4-dipheny1-3 (2H)-fur none.

18. Product according to any one of Claims 14 to 17 wherein the primary amino group containing material is a protein.

19. Product according to Claim 18 wherein the protein is an antibody.

20. Product according to any one of Claims 14 to 17 wherein the primary amino group containing material is a unicellular or multicellular organism.

21. Product according to any one of Claims 14 to 17 wherein the primary amino group containing material is Hymenolepis nana.

22. Product according to any one of Claims 14 to 17 wherein the primary amino group containing material is Escherichia coli. 44165/2

23. Product according to any one of Claims 14 to 17 Wherein the primar amino group containing material is an antibod for pneumococcus type XI.

24. Product according to any one of Claims 14 to 17 wherein the primary amino group containing material is gananaglobuline.