GB2081257A

GB2081257A - Biologically Interesting Compounds Labeled with Chlorotriazinyl-aminofluorescein

Info

Publication number: GB2081257A
Application number: GB8118754A
Authority: GB
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1980-07-30
Filing date: 1981-06-18
Publication date: 1982-02-17
Also published as: AU554360B2; CA1160626A; IT8123207A0; DE3129705A1; IT1137630B; GB2081257B; FR2487835B1; BE889788A; FR2487835A1; AU7203681A; DE3129705C2; JPS5758695A; SE8104227L

Abstract

Fluorescent derivatives of biologically interesting compounds which permit the detection and determination of said compounds in body fluids such as serum, plasma, saliva and urine, are characterized by the formula: <IMAGE> wherein X is a biologically interesting moiety having a molecular weight of less than 2,000 and having at least one primary or secondary amine or hydroxyl group by which it attaches to a carbon atom of the triazine ring; and Z is 4-aminofluorescein or 5- aminofluorescein attached through the 4-amino or 5-amino, respectively to the 2-position of the triazine ring.

Description

SPECIFICATION Biologically Interesting Compounds Labeled with Dichloritriazinyl-aminofluorescein This disclosure relates generally to fluorescent compounds which have proved particularly useful in a variety of immunofluorescence procedures to detect and determine quantities of small biologically interesting molecules in body fluids. The low molecular weight substances which are desired to be detected or monitored include therapeutically administered drugs, drugs having a known potential for abuse, and various other compounds which are of interest in diagnosing diaease or monitoring thereapy.

While the claimed compounds can be utilized in a variety of immunofluorescence techniques; they are particularly useful in a procedure called fluorescence polarization immunoassay. A fluorescence polarization immunoassay combines the specificity of an immunoassay with the speed and convenience of a homogeneous method. When a fluorescent conjugate is bound to an antibody, linearly polarized light which is used to excite the fluorescent conjugate remains highly polarized upon emission because the fluorophore is constrained from rotating between the time the light is absorbed and emitted. On the other hand, when the fluorescent conjugate is not bound by antibody, its rotation is much faster, molecules are more randomly oriented, and the emitted light is depolarized.

Fluorescence polarization, then, provides a direct measure of bound and free fluorescent conjugate in a competitive binding immunoassay. Fluorescent conjugate competes for antibody binding sites with the compound of interest in the patient sample. The greater the concentration of the compound being assayed, the larger the fraction of fluorescent conjugate unbound by antibody, and therefore, a greater degree of depolarized light is emitted. The precise relationship between the concentration of analyte and polarization is established by generating a standard curve. Calibrators with known amounts of analyte are read and the polarization recorded. When an unknown is read, its concentration is estimated by interpolation between standards.

Fluorescent conjugates of derivatives of low molecular weight compounds are not unique. U.S.

Patent 3,998,943 (1976) specifically describes the preparation of fluorescently labeled insulin using fluorescein isothiocyanate (FITC) and fluorescently-labeied morphine using 4-amino-fluorescein hydrochloride.

The fluorescent derivatives of the present invention are labeled with dichlorotriazinylaminofluorescein which is commonly referred to as DTAF. DTAF is the product of the reaction of aminofluorescein and cyanuric chloride. In an article by Blakeslee, et al, Jounal of Immunological Methods, 13, 305-320, (1976), DTAF was described as an effective reagent for conjugating fluorescein to immunoglobulins (IgG) having molecular weights of at least 160,000.

The present invention discloses and claims the use of DTAF to impart fluorescence to a wide variety of biologically interesting compounds which, when labeled, can be characterized by the formula:

wherein X is a biologically interesting moiety having a molecular weight of less than 2,000 and having at least one reactive substituent selected from the group consisting of a primary or secondary amine or hydroxyl group by which it attaches to a carbon atom of the triazine ring; and Z is selected from the group consisting of 4-amino fluorescein and 5-aminofluorescein attached through the 4-amino or 5amino, respectively to the number 2 carbon of the triazine ring.

DTAF can be readily prepared according to the method adopted by Blakeslee, et al (supra). DTAF Isomer I is prepared from 5-amino fluorescein; Isomer II is derived from 4-amino fluorescein.

The conjugation of either isomer of DTAF to compounds of interest can generally be accomplished by dissolving equal molar amounts of DTAF and a compound having a reactive amine (primary or secondary) or a hydroxyl group in an appropriate solvent such as water, methanol, dimethylformamide or dimethylsulfoxide. If the reactive amine is a salt, a suitable base is added to the reaction mixture to form the free base of the reactive amine. After completion of the reaction, the conjugates can be purified by using either thin-layer or column chromatography.

The biologically interesting compounds employed in the claimed invention must have at least one primary or secondary amino group or hydroxyl group by which it can react with and attach to a carbon atom of the triazine ring. The reactive group may be inherent in the biologically active compounds as it is in the quinidine, procainamide, thyroxine and the aminoglycoside antibiotics, or it may be introduced into the compound by derivatization. Examples of biological compounds that require the formation of amino derivatives before DTAF conjugates can be prepared include theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide, digoxin, chloramphenicol, aspirin, acetaminophen, carbamazepine, desimprmine, and nortriptyline.

Structures illustrating the most preferred compounds of the disclosed invention include the following: 8-AMINOMETHYLTHEOPHYLINE-DTAF

2-ETHYL-5-AMINOPENTANOIC ACID-DTAF

and 2-PROPYL-5-AMINOPENTANOIC ACID-DTAF

The following examples will demonstrate the preparation of specific DTAF conjugates within the scope of the claimed invention. Note that in the following examples, DTAF may be either isomer unless specifically stated.

Example I Gentamicin-DTAF Gentamicin sulfate (200mg) was dissolved in 1 ml of distilled water. The pH was adjusted to 9.0 with approximately 0.8ml of 1 .OM sodium hydroxide. DTAF (20mg) was dissolved in 1.5my of methanol and added dropwise to the gentamicin solution with stirring and allowed to react for one hour. The reaction mixture was added to a DEAE cellulose medium mesh column and the resulting gentamicin-DTAF eluted with 0.1 M phosphate buffer at pH 8.0.

Example II Tobramycin-DTAF Tobramycin (250mg) was dissolved in 2ml of carbonate buffer, 0.1 M, pH 9.0. DTAF (20mg) was dissolved in 1 ml of methanol and added to 1 ml of the tobramycin solution. After about five minutes, the reaction mixture was purified by application to a 20ml DEAE cellulose column and equilibrated with 0.1 M phosphate buffer at pH 8.0. The reaction product was eluted with the same buffer.

Example III Amikacin-DTAF Amikacin (24mg) was dissolved in 0.2ml of water, and 4.5mg of DTAF were suspended in 0.2ml of methanol. The methanolic suspension of DTAF was added to the amikacin solution with stirring. The small particles of DTAF rapidly dissolved, so the reaction mixture was not stirred. After thirty minutes the reaction mixture was applied to a 17ml column of DEAE cellulose equilibrated with pH 8.1 phosphate buffer, 0.1 M. The reaction product was eluted with the same buffer.

Example IV Desethyl-N-acetyl-procaina mide-DTAF Paraacetamidobenzoic acid (1.799) and 1.159 of N-hydroxysuccinimide were dissolved in 15ml of pyridine. Then, 2.3g N,N'-dicyclohexylcarbodiimide was added and dissolved. The reaction mixture was chilled in a refrigerator for two hours and then filtered. Crystals were rinsed with about 2ml of acetone. N-ethylethylenediamine (0.88g) was added to the combined pyridine-acetone filtrate. The mixture was stirred for two hours and then chilled in a refrigerator for about twenty-four hours. The resulting crystals were filtered and rinsed with acetone. The crystals (2.09) were dissolved in 50ml of distilled water, and the solution adjusted to pH 10 with 6N sodium hydroxide solution. A white precipitate was filtered off and dried in a dessicator.The DTAF conjugate was formed by dissolving equal molar amounts of desethyl-N-acetylprocainamide and DTAF in methanol. The reaction was completed in about ten minutes. Purification was performed by silica gel thin layer chromatography, using a developing solvent of chloroform/acetone (1 :1).

Example V N-p-acetamidobenzoyl Ethylene Diamine-DTAF The procedure of example IV was repeated using 0.9g of ethylenediamine in place of Nethylethylenediamine. The reaction mixture was stirred for one hour, and chilled for one and one-half hours. The precipitate was filtered and rinsed with acetone. The DTAF conjugate was prepared the same as in Example IV, but methanol was used as the developing solvent.

Example VI N-p-acetamidobenzoyl-N'-ethyl-N'-amino-acetyl Ethylene Diam ine-DTAF Desethyl-N-acetyl procainamide (1.259) from example IV and 0.89 of chloroacetyl chloride were dissolved in 25ml of acetone and refluxed for two hours. After refluxing, the mixture was filtered and the filtrate evaporated to dryness. The yellow residue from the filtrate and 0.75g of sodium iodide were dissolved in 20ml of acetone and refluxed for one hour. The solution was filtered and the filtrate evaporated to dryness. The red residue was dissolved in 20ml of methanol. Concentrated ammonium hydroxide (20ml) was added to the solution, which was refluxed for one and one-half hours. After cooling, the mixture was extracted twice with 20ml of chloroform.The combined extracts were dried over sodium sulfate, filtered, and evaporated. The DTAF conjugate was prepared the same as in example IV and purified by thin-layer chromatography (chloroform/acetone (1 :)).

Example VII Amino-primidone-DTAF Primidone (1.1 g) was dissolved in 1 Oml of concentrated sulfuric acid. Another solution of 1 ml concentrated nitric acid and 2ml of concentrated sulfuric acid was added to the reaction mixture slowly without cooling. The mixture was then shaken at room temperature for forty-five minutes, and the reaction mixture was poured over 50ml ice and crystals of para-nitro-primidone were filtered and rinsed with water. The crystals (1.1 7g), melting point 225 0--2280C, were dissolved in 200ml of hot ethanol. Iron powder (1.5g) and 100ml of water were added. The mixture was heated to boiling, 2ml of concentrated hydrochloric acid were added, and the mixture was refluxed for two hours. The hot mixture was filtered and the filtrate evaporated to dryness. The residue was dissolved in 100% ethanol and precipitated out by adding diethyl ether. The precipitate was filtered to give 0.8g of brown hydroscopic crystals. The DTAF conjugate was formed by dissolving 5mg of DTAF and 5mg of the brown crystals in 0.5ml of methanol. The reaction was complete in 10 minutes and the conjugate was purified by silica gel thin-layer chromatography using chloroform/methanol (3:1) as the developing solvent. Final purification was performed by thin-layer chromatography using chloroform/methanol (2:1).

Example VIII 2-Ethyl-amino-pentanoic Acid-DTAF Delta-5-valerolactam (7.5g) was dissolved in 60ml of dry tetrahydrofuran, under a dry nitrogen atmosphere and n-butyllithium (1.6M, 90ml) in hexane were added dropwise to the reaction flask and chilled with a dry iceacetone bath. After all the n-butyllithium was added, the reaction mixture was stirred at room temperature for one hour, refluxed for thirty minutes, and cooled to room temperature (still under dry nitrogen atmosphere). 1-Bromoethane (8.09) was slowly added to the reaction flask while the flask was chilled in an ice bath. The mixture was then stirred for sixteen hours at room temperature and then 100ml of water was added slowly. This mixture was stirred at room temperature for thirty minutes and the organic layer separated.The aqueous layer was extracted with 50ml of diethyl ether and the organic layers combined and dried over sodium sulfate. The solvent was evaporated to give a dark oil, which crystallized on standing, and was recrystallized from petroleum ether to give 3.89 of product. This product (2.8g) was refluxed in 25ml of 6N hydrochloric acid for six hours. The water was evaporated to give a dark, thick oil. The DTAF conjugate was formed by dissolving equimolar amounts of 2-ethyl-5-amino-pentanoic acid and DTAF in methanol. The reaction was completed in about ten minutes. The product was purified by silica gel thin-layer chromatography with chloroform/methanol (3:1) as the developing solvent.

Example X D-Thyroxine-DTAF DTAF (5mg) was dissolved in 0.5ml of methanol. Smg of D-thyroxine was added and then dimethylsulfoxide was added dropwise until a clear solution was formed. Two drops of triethylamine were added and a conjugate formed after about sixteen hours. The product was purified by silica gel thin-layer chromatography using chloroform/methanol (3:1) as the developing solvent.

Example XI L-Thyroxine-DTAF This conjugate was prepared following the procedure of Example X, but substituting L-thyroxine.

Example XII 3,3',5-TrHiodo-L-thyronine-DTAF This conjugate was prepared following the procedure of Example X, but substituting 3,3',5triiodo-L-thyronine.

Example XIII 5-Amino-dibenzocycloheptane-DTAF The following were dissolved in 50ml of methanol: 8.00gel of ammonium acetate, 630mg of sodium cyanoborohydride, and 2.10g of 10,11-dihydro-5H-dibenzo [a,d] cycloheptent-5-one.The solution was refluxed for twenty-four hours and then evaporated to dryness. A tan residue remained, which was dissolved in 25ml of 2N hydrochloric acid and extracted twice with 25ml dichloromethane.

6N sodium hydroxide was added to the aqueous phase until the pH reached 14. A brown oil began to form and the solution was chilled in a freezer for 1 6 hours. All water was evaporated and the residue was taken up in methanol and filtered. The filtrate was evaporated to give a white residue. The DTAF conjugate was formed by dissolving equimolar amounts of DTAF and the white residue in methanol.

The product was purified by silica gel thin-layer chromatography using chloroform/acetone (1 :1) as developing solvent.

Example XIV Dibenzosuberone Hydrazone-DTAF 10,1 -Dihydro-5H-dibenzo [a,d] cyclohepten-5-one (10g) and 18g of dimethylhydrazine were refluxed for twenty-four hours in 100% ethanol. 100ml of distilled water were added and the yellow solution was extracted with diethyl ether until extracts were colorless. The combined ether extracts were washed with 25ml of 2N hydrochloric acid. The organic phase was then dried over sodium sulfate and evaporated. The residue was a thick orange oil, dibenzosuberone dimethylhydrazone. This product (2.0g) was refluxed for twelve hours with 39 of hydrazine in 1 Oml of 100% ethanol. The reaction mixture was poured over 1 Oml of ice water then extracted twice with 25ml of diethyl ether.The combined ether extracts were dried over sodium sulfate and evaporated to dryness to give a yellow oil, dibenzosuberone hydrazone. The DTAF conjugate was prepared the same as in Example XIII, but using chloroform/methanol (3:1) as the developing solvent.

Example XV 5-(y-Aminopropylidene)-5H-dibenzo[a,d]-1 0,11 -dihydrocycloheptene-DTAF 5-(y-Bromopropylidene)5H-dibenzo [a,d]-lO,l 1 -dihydrocycloheptene and its precursor 5 cyciopropyl-5-hydroxy-5H-dibenzo [a,d]- 10.11 -dihydrocycloheptene were prepared by the procedure described in Jounal Organic Chemistry, Vol. 27, pages 4134-4137 (1962) by R. D. Hoffsomer, D.

Taub, and N. L. Wendler. Procedure (b) for preparation of end product, 5-(y-bromopropylidene)-5H dibenzo [a,d]-10,11 -dihydrocycloheptene, was followed substituting the bromopropylidene compound for the chloropropylidene compound. The DTAF conjugate was formed by dissolving equimolar amounts of DTAF and the amine in methanol. An access amount of triethylamine was added and the reaction was completed in thirty minutes. The reaction product was purified by silica gel thin-layer chromatography using chloroform/methanol (2:1) as the developing solvent.

Example XVI N-Aminoacetyli m inostilbene-DTAF Iminodibenzyl (6.0g) was dissolved in 30ml of chloroform. Chloroacetyl chloride (6my) was added and the mixture was refluxed for forty-five minutes. Water (60ml) was added and the mixture was stirred for thirty minutes at room temperature. The chloroform layer was separated and dried over sodium sulfate and evaporated to dryness. The residue was dissolved in 25ml of acetone and a solution of 4.5g sodium iodide dissolved in 25ml of acetone was added. This mixture was refluxed for thirty minutes, 100ml of water was added, and the reaction product was extracted twice with 50ml of chloroform and evaporated to dryness. The residue was dissolved in 40ml of methanol. Concentrated ammonium hydroxide (60ml) was added and the mixture was refluxed for one hour.The solution was evaporated to dryness and the residue was taken up in 100ml of chloroform and washed twice with 30ml of 2N hydrochloric acid. The organic phase was dried over sodium sulfate and evaporated to dryness. The DTAF conjugate was prepared by dissolving 5mg of DTAF and 5mg of the amine in 0.5ml of methanol. Conjugate formed in ten minutes and was purified the same as Example Xlil.

Example XVII N-Aminoacetyidesipramine-DTAF Desipramine hydrochloride (1.33g) and 0.809 of chloracetyl chloride was dissolved in 25ml chloroform. This was refluxed for two hours, then the chloroform was evaporated. The residue was dissolved in 25ml of acetone. Sodium iodide (0.759) was added, and the solution was refluxed for thirty minutes. The solution was filtered and the precipitated salt was rinsed with acetone. The acetone filtrate was evaporated and the residue was taken up in 20ml of methanol. Concentrated ammonium hydroxide (20ml) was added and the solution was refluxed for one hour. The reaction mixture was extracted three times with 25ml of chloroform and combined extracts were dried over sodium sulfate, filtered and evaporated.The conjugate was prepared by dissolving 5mg each of DTAF and the amine in 0.5ml of methanol. About five drops of dimethylsulfoxide were added to dissolve the precipitate. The reaction was completed in ten minutes and purified by using the procedure of Example XIII using chloroform/methanol (3:1) as the developing solvent.

Example XVIII 8-Aminomethyl-theophylline-DTAF DTAF (1 Omg) and 8-aminomethyl-theophylline (5mg) were dissolved in 0.5ml of dimethylsulfoxide. After five minutes the reaction was complete and the conjugate was purified by silica gel thin-layer chromatography, using chloroform/acetone (1 :1) as the developing solvent.

Example IXX 8-Aminoethyl-theophylline-DTAF Procedure same as Example XVII I substituting 8-aminoethyltheophylline for the methyl derivative.

Example XX Quinidine-DTAF DTAF (5mg) and anhydrous quinidine (5mg) were dissolved in 0.5ml of dimethylformamide. After sixteen hours the reaction was complete and the conjugate was purified by silica gel thin-layer chromatography, using chloroform/methanol (3:1) as the developing solvent.

As mentioned above, the fluorescently labeled tracers prepared according to this invention can be used in a variety of immunoassay procedures. The following assays are offered to demonstrate the suitability of the representative sampling of these tracers in fluoroscence polarization assays.

All Examples followed the same basic protocol: 1) A small voluem of standard or test serum is delivered into a test tube and diluted with buffer; 2) A small volume of concentrated fluorescent tracer containing surfactant is then added to each tube; 3) Finally, a volume of diluted antiserum is added; and 4) The reaction mixture is incubated at room temperature.

Example XXI Valproic Acid Assay 2-Ethyl-5-.amino-pentanoic Acid DTAF Conjugate Materials Required: 1) Buffer: 0.1M phosphate, pH 7.5, containing 0.01% (w/v) sodium azide and 0.01% (w/v) bovine.

gamma globulin (BGG).

2) Tracer: 2-ethyl-5-amino-pentanoic acid DTAF conjugate 50x 1 0-9M in 0.1 M tris hydrochloride buffer, pH 7.8, containing 0.1% (w/v) sodium dodecyl sulfate, 0.01% (w/v) bovine gamma globulin, and 0.01% (w/v) sodium azide.

3) Antibody: Sheep antiserum to valproic acid diluted to 1 to 3.75 in buffer.

4) Standards or unknowns: Human serum (or other biological fluid) containing valproic acid in the concentration range 0 to 150 g ug/ml.

5) Fluorescence polarimeter: Instrument capable of reading the polarization of fluorescense of a 1 xl 0-9M fluorescein solution to + 0.001 polarization unit.

Protocol: 1) 0.75 of standard or unknown sample placed in a 12x75nm disposable culture tube (cuvette). This is accomplished by pipetting 20,u1 of standard or unknown into a predilution container followed by 500y1 of buffer. Next, 20y1 of diluted sample is pipetted into the 1 2x75 culture tube followed by 400y1 of buffer.

2) 40y1 of tracer and 800 l of buffer are added to the cuvette.

3) 40y1 of antiserum and 800,u1 of buffer are added to the cuvette. The contents of the cuvette are mixed and incubated for approximately 15 minutes at room temperature.

4) The fluorescence polarization is read. Typical results are presented in Table Table I ValproicAcid Conc. (gjml) Polarization 0 0.217 12.5 0.186 25 0.165 50 0.132 100 0.099 150 0.081 The polarization changes in a regular manner as the concentration of valproic acid is varied allowing the construction of a standard curve. Unknown samples are treated in an identical manner; from the polarization of fluorescence of the unknown sample, the concentration of valproic acid in the unknown sample may be determined by reference to the standard curve.

Example XXII Gentamicin Assay Materials: 1) Buffer: (See valproic acid assay).

2) Tracer: Gentamicin-DTAF at 1 00nM in a trihydrochloride buffer pH 7.5 containing 0.125% sodium dodecyl sulfate, 0.01% sodium azide, and 0.01% bovine gamma globulin.

3) Antibody: Rabbit or sheep antisera to gentamicin diluted appropriately in buffer.

4) Standards or unknowns: Human serum (or other biological fluid) containing gentamicin.

5) Fluorescence polarimeter: (See valproic acid assay).

Protocol: 1) 1.8ul of standard or unknown sample is placed in a 12x75nM disposable culture tube (cuvette). This is done by pipetting 20 l of sample followed by 200,u1 of buffer. Next 20 l of diluted sample is pipetted into the curvette followed by 200ul of buffer.

2) 40 l of tracer and 1000ul of buffer are added to the cuvette.

3) 40y1 of antibody and 1 OOOyi of buffer are added, the contents of the cuvette are mixed and incubated for approximately fifteen minutes at room temperature.

4) The fluorescence polarization is read following the incubation. Typical results are presented in Table II.

Table II Genatmicin Concentration f g/miJ Polarization 0 0.178 0.5 0.158 1.0 0.140 2.0 0.115 4.0 0.090 8.0 0.074 The polarization changes in a regular manner allowing construction of a standard curve. Unknown samples are tested in an identical manner, and the gentamicin content is determined by reference to the standard curve. The utility of the gentamicin-DTAF tracer for determining the concentration of entamicin in biological samples is thereby illustrated.

Example XXIII N-Acetyl Procainamide Assay Materials required: 1) Buffer: (See valproic acid assay).

2) Tracer: Desethyl-N-acetyl procainamide-DTAF conjugate at a concentration of 50x 10-9M in a 5.75% (w/v) solution of sodium toluene sulfonate.

3) Antiserum: Rabbit antiserum to N-acetyl-procainamide diluted one to six in buffer.

4) Standards or unknowns: Human serum (or other biological fluid).

5) Fluorescence polarimeter: (See valproic acid assay).

Protocol: 1) 0.4881 of standard or unknown in placed in a cuvette by pipetting 1 oil of sample into a predilution container and mixing with 200,us of buffer. Tenyl of diluted sample is next pipetted into the cuvette followed by 200ul of buffer.

2) 4081 of tracer and 1000yl of buffer are added to the cuvette.

3) 4081 of antiserum and 1000yl of buffer are next added to the cuvette. The contents of the cuvette are mixed and incubated at room temperature for approximately 1 5 minuteqat room temperature.

4) The fluorescence polarization is read following the 15-minute incubation period. Typical results for N-acetyl-procainamide are presented in Table Ill.

Table Ill N-Ace tyl Procainamide (ug/mli Polarization 0 0.239 1 0.218 2 0.209 4 0.190 8 0.173 16 0.158 A standard curve can be constructed from the data in Table III. Unknown samples treated identically to the standards can be quantitated by reference to the standard curve, thereby illustrating the usefuiness of the des-ethyl-N-acetyl procainamide-DTAF conjugate for the determination of Nacetyl procainamide in biological fluids.

Claims

1. A fluorescent compound of the formula:

wherein X is a biologically interesting moiety having a molecular weight of less than 2000 and a reactive substituent selected from the group consisting of a primary or secondary amine or hydroxyl group by which it attaches to a carbon atom of the triazine ring; and Z is selected from the group consisting of 4-aminofluorescein and 5-aminofluorescein attached through the 4-amino or 5-amino, respectively to the number 2 carbon of the triazine ring.

2. The compound according to Claim 1 wherein X is an aminoglycoside antibiotic.

3. The compound according to Claim 2 wherein the aminoglycoside is gentamicin.

4. The compound according to Claim 2 wherein the aminoglycoside is tubramycin.

5. The compound according to Claim 2 wherein the aminoglycoside is amikacin.

6. The compound according to Claim 1 wherein X is amino-phenobarbital.

7. The compound according to Claim 1 wherein X is desethyl-N-acetylprocainamide.

8. The compound according to Claim 1 wherein X is N-para-acetamidobenzoyl ethylenediamine.

9. The compound according to Claim 1 wherein X is N-acetamidobenzoyl-Nt-ethyl-N'-amino acetyl ethylenediamine.

10. The compound according to Claim 1 wherein X is para-aminoprimidone.

11. The compound according to Claim 1 wherein X is 2-ethyl-5-aminopentanoic acid.

1 2. The compound according to Claim 1 wherein X is D-thyroxine.

13. The compound according to Claim 1 wherein X is L-tyroxine.

14. The compound according to Claim 1 wherein X is 2-propyl-5-aminopentanoic acid.

1 5. The compound according to Claim 1 wherein X is 3,3',5-triiodo-L-thyronine.

1 6. The compound according to Claim 1 wherein X is 2-(2-aminoethyl)-5,5-diphenyl hydantoin.

1 7. The compound according to Claim 1 wherein X is 8-aminomethyl theophylline.

18. The compound according to Claim 1 wherein X is 8-(2-aminoethyl) theophylline.

19. The compound according to Claim 1 wherein X is quinidine.

20. A fluorescent compound as claimed in Claim 1 and according to any one of the Examples herein.