4-TRIFLUOROMETHYLCOUMARIN PEPTIDE DERIVATIVES AND THEIR USE IN PROTEINASE ASSAYS

Abstract: Method for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate.

Description: 4 - TRff LUOROMETHYLCOUMARI PEPTIDE DERIVATIVES AND THEIR USE .IN PROTEINASE ASSAYS BACKGROUND OF THE INVENTION The present invention relates to substrates and methods for determining enzymes. More particularly, the invention relates to qualitative and quantitative methods for determining proteoiytic enzymes. The determination of specific enzymes in biological fluids, such as blood, tissue homogenates, and cytoplasm can be very useful for the diagnosis of certain diseases. The discovery of synthetic substrates for such determinations has resulted In clinical assay procedures having a high degree of specificity, reliability, and sensitivity. Such substrates have been employed for the determination of amylase (Driscoll, R. C, et al. , U.S. Patent ^, 102,7^7) and various proteinases. Synthetic proteinase substrates have generally been amino acid derivatives of aromatic amines. The number and arrangement of amino acids in the peptide moiety determine the enzyme specificity of the substrate and the enzyme activity is measured by the amount of aromatic amine moiety liberated upon hydrolysis of the substrate. Amino acid derivatives of p-nitroaniline have been widely used as synthetic substrates. Erlanger, B. F., U.S. Patent 3,^ 12, 1.50. Other aromatic amines which have been reacted with amino acids or peptides include 2-naphthylamine, Ψ-methoxy-2-naphτhylamine, and 7-amino-^-methylcoumarin. The use of 2-napthylamine and k.-methoxy-2-naphthylamine for the preparation of synthetic substrates and prior art relating thereto are discussed by Smith. R. E., U.S. Patent 3,862,011. Peptide derivatives of 7-amino- f÷-methylcoumarin have recently been reported as fiuorogenic substrates for a number of proteinases. Zimmerman, M ., Yurewicz, E., Patei, G., Anal. Biochem. 70, 258-262 ( 1976) and Zimmerman, M., Quigley, 3. P., Ashe, 3., Dorn, C, Goldfarb, R., Troll, W., Proc. Natl. Acad. Sci., 7 , 750- 753 (1978). " U EA C OMPI WIPO Because the chromophore, p-nitroaniiine, is yellow, enzyme assays employing that chromophore are colorimetric. Fluorescence assays are sometimes preferred over colorimetric assays, because of greater sensi¬ tivity and less background interference. The aromatic amine chromophores heretofore used to prepare synthetic substrates are fluorescent, but their fluorescence generally occurs in the blue region of the spectrum. Such fluorescence is disadvantageous, because it is difficult to measure with inexpensive instruments, and it is similar to fluorescense of other materials present in the analytε, including, in some instances, the intact substrate. These assays are useful for cytological studies for the detection of an enzyme within a single cell. When such cells are viewed under a fluorescence microscope, a blue color is difficult to see or distinguish from the background, but cells emitting light in the yellow region of the spectrum are easily visualized, To overcome these problems, investigators have focused on reac¬ tions involving the enzyme-liberated chromophore to enhance color or fluorescence at a desired wavelength. For instance, aromatic amine chromophores may be reacted with diazoniu salts to form azo dyes which are determined spectrephotometrically. In U.S. Patent Application Serial Number 328, 3 ώ , R. E. -Smith, et al. disclose a reaction of the aromatic amine chromophore with certain aromatic aldehydes to form Shiff base compounds which fluoresce in the yellow-green region of the spectrum. Although such methods have each constituted significant advances over the prior art, there is a need for synthetic substrates for proteinase enzymes which do not fluoresce in the yellow region, but which upon enzyme hydrolysis, release a chromophore which fluoresces strongly in that region of the spectrum. Such substrates would, thus, obviate the need for further reactions involving the liberated chromophore, and the concentra¬ tion of such chromophore could be readily determined by a fluorometric technique. SUMMARY OF THE INVENTION In accordance with the present invention, there is disclosed a method for determining the presence of an enzyme in an enzyme- containing anaiyte, comprising: (a) contacting the anaiyte with a substrate which can be hydrolyzed by said enzyme to liberate 7-amino- -trifiuoromethylcoumarin, said substrate having the formula wherein R is an amino acid, a peptide, or a derivative thereof, thereby forming an anaiyτe-substrate mixture; (b) incubating the anaiyte-substrate mixture under enzyme hydrolyzing conditions to form an enzyme hydrolyzate; and (c) fluorometrically or spectrophotometrically determining the presence of 7-amino- -trifiuorornethylcoumarin in the enzyme hydrolyzate. DETAILED DESCRIPTION OF THE INVENTION The substrates of the present invention are represented by the formula wherein R may be a single amino acid or a peptide, consisting of two or more amino acids. The terminal amino acid may be reacted with any suitable blocking groups as is well known in the art, such as carbobenzoxy, benzoyl, glutaryl, t-butyloxycarbonyl, and certain d-amino acids, e.g., d-proiine, d-valine, or d-alanine. ι+ - Thus, upon enzymatic hydrolysis, the chromophore, 7-amino -trifluoromεthyicoumarin is released. This chromophore fluoresce strongly in the yellow region of the spectrum when irradiated with ultra violet light, but the intact substrates fluoresce very weakly, if at all, i that region. The fluorescent properties of the substrates and the chromo phore render these compounds particularly useful for the enzyme assays. The presence of the liberated substrate can be qualitatively or quantita tively determined fluorometrically without employing dye-forming or wavelength-shifting reactions. In contrast with prior art substrates which are used either in colorimetric or fiuorometric assays, but not both, the present substrates may be used in both direct colorimetric and fiuorometric assays. The 7-amino-if-trifluoromethylcoumarin chromophore has a yellow color, bu the intact substrates are substantially colorless. Thus, the substrates can be employed in spectrophotometric as well as fiuorometric assays. This property of the substrates makes them particularly valuable for use in enzyme kinetic studies. The number and arrangement of amino acids attached to the chromophore determine the enzyme specificity for the substrate. Any combination of amino acids can be ' employed to obtain the desired specificity. Preferably, the amino acid chain consists of from 1 to about 12 amino acids and, most preferably from 1 to about 6 amino acids. The amino acids are bound together through peptide bonds. Advantageously, the amino acid chain may be terminated with a blocking group. Such a blocking group may be employed during the synthesis of the substrate to prevent reactions with the terminal amino acid, and the blocking group is sometimes employed in substrates to improve enzyme specificity. Such blocking groups are well known in the art as described above. Preferred substrates of the present invention are compounds represented by the above formula wherein R is Cbz-Giy-Gly-Arg-; D-Ala- Leu-Lys-; Cbz-Val-Lys-Lys-Arg- and Leu- (Cbz represents carbobenzoxy and the amino acid abbreviations are generally recognized and accepted in the art). The first substrate is useful for assays for trypsin and urokinase, the second is useful for plasmin assays, the third is useful for the determination of cathepsin B, and the fourth is useful for the determina¬ tion of aminopeptidase M. The substrates may be prepared by acylating 7-amino- -trifluoromethylcoumarin with an appropriate amino acid or peptide. Such acylation may be accomplished by a conventional mixed anhydride reaction. Similarly, amino acids or peptides can be added to substrates having one or more unblocked amino acids. For instance, a urokinase substrate can be prepared by the following reactions scheme: Any desired number and arrangement of amino acids may thus be added onto the chromophore. Blocking groups may be removed, e.g., by hydro- genolysis or treatment with anhydrous hydrogen bromide in acetic acid, trifluoroacetic acid or other conventional deblocking agents as are known in the art. In the practice of the method of the present invention, an analytε containing, or suspected of containing, an enzyme is contacted with a substrate which can be hydroiyzed by that enzyme. Such analyte is usually a natural biological fluid such as blood, serum, urine, tissue homogenatε, etc., but may also be a synthetic solution used for quality control or as a reference standard. The substrate is generally employed in excεss of the amount which can be completely hydrolyzed by the quantity of enzy presεnt. For instancε, the substrate is prefεrably εmployεd in an amou of from 1 to about 10 times, most preferably from about 1 to about k- tim that amount which can be completely hydrolyzed by the enzyme. The analyte-substrate mixture is incubated under enzym hydrolyzing conditions to form an enzyme hydrolyzate. Such enzym hydroiyzing conditions include conditions of pH and temperature which a conducive to the enzymatic hydrolysis of the substrate. The pH of t analyte-substrate mixture will gεnεraily bε in thε range of the norm physiological εnvironment of the enzyme, and thus may vary from o enzymε to another. Such pH is usually in a rangε of from about to abo 10, and prεfεrably in a range of from about 5 to about 8.5. A pH of about has been employed for urokinase, plasmin, and trypsin assays and a pH about 7.2 has been used for aminopeptidase assays. The pH of t mixture is conveniently controlled by dissolving the analyte and substra in an appropriate buff er, as is well known in the art. A suitable buffer N-tris (hydroxymethyl) methyi-2-aminoethanesulfonic acid (TES). The tempεraturε at which the enzymε hydrolysis is effected is n critical, and may fall within a broad range, provided that the temperatu is high enough to Insure enzyme activity, but not too high to cau dεgradation or other delεtεrious rεactions involving thε substratε, t enzyme, or other components of the mixture. The temperature adva tageously is from about 15°C to about 50°C, prefεrably from about 20° to about ^0°C. Thε fluoromεtric determination of the liberated chromophore m be either a rate detεrmination or an endpoint determination. Ra detεrminations arε preferred, because they are generally more sensiti and precise. In a rate detεrmination, thε fluorεscence of the substrat analytε mixturε may be dεtεrmined promptly aftεr thε analytε is co tactεd with the substrate. In an endpoint detεrmination, thε enzy hydrolysis reaction is allowed to proceed for a predεtεrminεd length time, e.g., from about 5 to about 60 minutes, prefεrably from about 15 about 30 minutes. Such reaction time is selected so that a sufficie quantity of chromophore has been released to provide an acceptable degr of accuracy for the assay. For fiuorometric assays, excitation and emission wavelengths may be selected to conform to existing equipment commonly available in clinical laboratories. Maximum excitation and emission wavelengths for the 7-amino- -trifluoromethylcoumarin chromophore are 365 nm and 495 nm, respεctively. Wavelengths of ttOO nm and 505 nm have beεn employed; and at thεse wavelengths, thε fluorescεnce of thε liberatεd chromophorε is about 700 times greater than an equimolar solution of the substrate, while retaining about 57% of thε maximum fluorεscεncε. The absorbance maximum wavelength for the libεrated chromo- phore is about 370 nm. In spectrophotometric assays, the absorbance easurεmεnts are usually made at about 380 nm to minimize interference by the intact substrate. Those skilled in the art will recognize that the substrates of this invention may be useful in a variety of analytical techniques. For instance, the substrates can be utilized in cytological studies to indicate the presence of certain enzymes in single cells. Other uses of the substrates include their utilization as indicators for various chromatographic or electrophoretic techniques. Enzymes may be isolate by chromatography, e.g., paper chromatography, thin-layer chromatography or column chroma- tography, or by electrophoresis and the appropriate substrate may be applied to the chromatographic or electrophoretic medium to indicate the location or intensity of the enzyme spot, band, or zone. Thus, there has beεn discoverεd a sensitive and reliable method and novel substrates for the determination! of proteinase enzymεs. The invention is further illustrated by the following examples which are not intended to be limiting. EXAMPLE 1 This examplε describes a procedure for preparing a substrate of the formula OMPI whεrεin Cbz is carbobεnzoxy, and is applicablε to the preparation of any of the substrates of the presεnt invεntion by selεction of the proper reac- tants. Cbz-argininε, 1.7g, was dissolved in 10 ml of dry dimεthylforma- midε, the solution was cooled in an ice-acεtonε bath, and 0:75 ml of isoamylchloroformate was addεd. Thε mixture was stirred for threε hours at -15°C. 7-amino- -trifluoromεthylcoumarin, 1.1 g, was addεd and stirring was continuεd for anothεr 20 hours whilε the bath was allowed to warm to room temperaturε. The solvents were removed by vacuum distillation at 5 mm Hg pressure at room temperature, and the residuε was driεd overnight undεr 10 μ of Hg pressure at room temperaturε. Thε crudε reaction mixture was purified by high pεrformancε liquid chromatography using a silica gεl column and 10% mεthanol in methyiεnεdichloride as the εluant, thus yiεlding a product of thε formula: That product, 555 mg, was dissolvεd in a 5 ml of 32% HBr in acεtic acid. Aftεr 30 minutεs at room temperaturε, thε orangε solution was pourεd into 80 ml of εthεr. Thε mixture was centrifuged and the precipitate was washed twice with ethεr and dried ovεrnight. This procεdurε is effective for removing the carbobenzoxy blocking group. The resulting product, 0.98g, was dissolved in 5.0 ml of dry dimethylformamide, and this solution was combined with the mixed anhydride preparεd from 660 mg of Cbz-Gly-Gly in 5.0 ml of driεd dimethylformamide at -15 C (mixed anhydride prepared by reacting Cbz-Gly-Gly with isobutylchioro- formate in the presεncε of N-mεthylmorpholine in DMF solvent). The mixture was stirred overnight as the tempεrature was allowεd to reach room temperaturε. Thε solvεnts wεrε rεmovεd by vacuum distillation at 5 mm Hg prεssurε at room temperature, and the rεsiduε was dried overnight at room temperaturε at 30 μ Hg prεssurε. Thε product was purifiεd by twice subjecting it to high pressurε liquid chromatography on a silica gel column using 20% ethanol in methylenedichloride. The nuclear magnetic resonance spectrum of thε product was consistent with the assigned structure. The optical rotation of the product (195 mg/10 ml methanol) (α) n - 6Λ°. The elemental analysis for carbon, hydrogen, and nitrogen was also consistent with the assigned structure. EXAMPLE II A series of experiments was conducted to demonstrate the method of the present invention. Solutions of each of the enzymes, urokinase, plasmin, aminopeptidase M, and trypsin wεrε prεpared at various concentrations within the ranges indicated in Table I. For trypsin and urokinase assays, the substrate cbz-gly-gly-arg-7-amino- -trifluoromethyi- coumarin was used as the substrate. For plasmin assays, d-ala-leu-lys- 7-amino- ~trifluoromεthylcoumarin was used, and for aminopeptidase M assays, leu-7-amino-Ψ-trifluoromethylcoumarin was used. Dimethylfσrma- 2 mide solutions of the substrates (10 millimolar for aminopeptidase M - assays and 20 millimolar for trypsin, urokinase, and plasmin assays) were prepared. To conduct an assay, 50 ι l of substrate solution was added to 900 μ l of buffer (0.05 M TES, pH 8, for urokinase and plasmin; 0.05 M TE5, pH 7.2, for aminopeptidase M; 0.5 M TES, pH 8.0 for trypsin) in a cuvette. To this solution, 50 μl of enzyme solution was added and the temperature was controlled at 25 C. Fluorescence was recorded for five minutes or more on a recording spectrofluorometer using an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The rate of increasε of fluorescence was linear over the enzyme concentration ranges indicated in Table I, and the rate of increase of fluorescence was found to be directly proportional to enzyme concentration. Table I Substrate C=7-amino- -trifluoro- Linear Detεction Enzyme methylcoumarin Range Limit Trypsin Cbz-Gly-Gly-Arg-C 0.08-25ng/ml 0.08ng/mi Urokinase Cbz-Gly-Gly-Arg-C 0.75-50 I.U./ml 0.75 l.U./ l Plasmin d-Ala-Lεu-Lys-C 0.0006-0.06 0.0006 CTA/ml CTA/ml Aminopεptidasε M Lεu-C 6.0-600ng/ml 6.0ng/ml EXAMPLE III A patient serum may be assayed for thε εnzyme cathepsin B by the following procedurε. A 2 millimolar solution of the substrate Cbz-Val- Lys-Lys-Arg-7-amino-4-trifIuoromεthylcoumarin in dry dimεthylformam- ide was prepared. This substrate solution, .50 μ l, was added to 900 μ l of .05 M sodium cacodylate buffer (pH 5.6-6.2) in a cuvette. To this solution, 50 μ l of 1 : 10 diluted patient serum was added and the tempεrature was controlled at 25 C. Fluorescencε was mεasurεd as described in Example II and the rate of increase of fluorescence was compared to a standard calibration curve to determine enzymε concεntration. ~

Claims: CLAIMS 1. A method for determining the presence of an enzyme in an enzyme-containing analyte, comprising: (a) contacting the analyte with a substrate which can be hydrolyzed by said enzyme to liberate 7-amino- -trifluoromethylcoumarin, said substrate having the formula wherein R is an amino acid, a peptide, or a derivative thereof, thereby forming an analyte-substrate mixture, (b) incubating the analyte-substrate mixture under enzyme hydrolyzing conditions to form an enzyme hydrolyzate; and (c) fluorometrically or spectrophotometrically determining the presence of 7~amino- -trifluoromethylcoumarin in the enzyme hydrolyzate. 2. The method of claim 1, wherein R is an amino acid chain comprising from 1 to about 12 amino acids. 3. The method of claim 1, wherein R is an amino acid chain comprising from 1 to about 6 amino acids. . The method of claim 1, wtierein R is gly-gly-arg-. 5. Thε method of claim 1, wherεin R is d-ala-leu-lys-. , V— . ff- f GΓ. ' .PI l? o 6. The method of claim 1, wherein R is leu-. 7. The method of claim 1, wherεin R is Cbz-Val-Lys-Lys-Arg- 8. The mεthod of claim 2, 3, 4, 5, 6, or 7 whεrein a blocking group is bonded to the terminal amino acid. 9. The method of claim 8, wherεin said blocking group is carbobenzoxy, bεnzoyl, glutaryl, t-butyloxycarbonyl, d-prolinε, d-valinε, or d-alaninε. 10. Thε mεthod of claim 1, whεrein said enzymε-hydroiyzing conditions inciudε a pH of from about 4 to about 10 and a tεmperature of from about 15°C to about 50°C. 11. The method of claim 1, wherεin said εnzymε-hydrolyzing conditions include a pH of from about 5 to about 8.5 and a tempεraturε of from about 20°C to about 40°C. 12. The method of claim 10, wherεin thε dεtεrmination is a fluoromεtric rate dεtεrmination and employs an excitation wavelεngth of about 400 nm and an εmission wavelength of about 505 nm. 13. The method of claim 10, wherein the detεrmination is spεctrophotomεtric and absorbancε measurεments are made at about 380 nm. 14. The method of claim 10, 1 1, 12, or 13,' wherεin thε analyte is a biological fluid and the enzyme is a proteinase enzyme. 15. Thε method of claim 14, wherein the biological fluid is the cytoplasm within a single cell. 16. A compound of the formula wherein R is an amino acid, a peptide, or a derivative thereof. 17. The compound of claim 16, wherein R is an amino acid chain comprising from 1 to about 6 amino acids. 18. The compound of claim 16, wherein R is an amino acid chain comprising from 1 to-about 6 amino acids. 19. The compound of claim 16, wherein R is gly-gly-arg- 20. The compound of claim 16, wherein R is d-ala-leu-lys-. 21. The compound of claim 16, wherein R is leu-. 22. The compound of claim 16, wherein R is Cbz-Val-Lys-Lys- Arg-. 23. The compound of claim 17, 18, 19, 20, or 21, wherein a blocking group is bonded to the terminal amino acid. 24. The compound of claim 23, wherein said blocking group is carbobenzoxy, benzoyi, glutaryl, t-butyioxycarbonyl, d-proline, d-valine, or d-alanine. OMPI

Inventors: BISSELL E, SMITH R
Applicants: BISSELL E, SMITH R
Application year: 1980
Main classification: C12Q1
IPC codes: C07D311, C07K5, C12Q1, G01N33