Fluoroketones as blocked enzyme inhibitors for use in immunoassays

Abstract: A method for enzyme immunoassay for a ligand suspected to be present in a liquid sample includes signal amplification by use of at least two enzymes and a blocked modulator for one of the enzymes. Ligand present in the liquid binds to an antiligand and an enzyme-labeled tracer. The resulting bound fraction is separated and the enzyme in the tracer removes the blocking group from the blocked modulator. The modulator activates or inhibits a second enzyme which catalyzes the conversion of a substrate to a product. The presence or absence of the ligand in the liquid is indicated by a signal, such as a color change or a rate of color change, associated with the product. The invention includes a new class of enzyme inhibitors and blocked inhibitors and a kit of materials useful for performing the method of the invention.

Description: 1. Field of the Invention . This invention relates to materials for immunoassay in which enhancement of a detectable signal is achieved by inhibition of enzymatic catalysis of an indicator reaction, and more particularly relates to (blocked) fluoroketone inhibitors. 2. Background of the Invention . Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous, requiring a separation of bound tracer from free (unbound) tracer or homogeneous in which a separation step is not required. Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed. Fluoroimmunoassay (FIA) uses fluorochromes as labels and provides direct detection of the label. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium. Enzymes have also been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. When the signal is a color change, detection with the naked eye is limited because the average individual can detect the presence of chromophores only down to about 10⁻⁵ or 10⁻⁶M. EIA sensitivity can often be increased by spectrophotometric techniques: however, these procedures require expensive equipment. In another approach, the sensitivity may be increased by various amplification methods. Single enzyme amplification methods have been disclosed in which ligands present at concentrations of 10⁻⁶ to 10⁻¹⁰ M have been detected. These methods however, have been generally unsatisfactory at ligand concentrations above 10⁻¹¹ M. In cascade amplification procedures, the number of detectable (generally colored) molecules is increased by use of two or more enzymes or enzyme derivatives. U.S. Patent No. 4,463,090 to Harris discloses a cascade amplification immunoassay in which a large molecule activator, such as an enzyme or a proenzyme coupled to a ligand, activates a second enzyme which reacts with a substrate to produce a detectable signal or in turn activates a third enzyme. U.S. Patent No. 4,446,231 to Self discloses a cycling amplification enzyme immunoassay which includes primary and secondary enzyme systems and a modulator for the second enzyme system. The primary system includes a first enzyme coupled to a ligand. In a first embodiment of the Self invention, the first enzyme system acts on a modulator precursor to liberate a modulator. The modulator is a cofactor of the secondary enzyme which activates the second enzyme system to catalyze the reaction of a substrate to a detectable product. During the reaction, the modulator is converted to an inactive form, and cycling is accomplished by a third enzyme which reactivates the modulator. In a second embodiment the modulator is an inhibitor of the secondary system, and is removed by the primary enzyme system whereby the secondary system is activated to act on the substrate and thereby produce the detectable product. Boguslaski et al., U.S. Patent No. 4,492,751 teaches a cycling system in which an enzyme substrate or coenzyme is conjugated to one member of the specifically binding pair. A variety of molecules has been shown to cause specific inactivation of a target enzyme. A subset of inhibitors, termed mechanism-based inhibitors, are substrates for enzymes which react with an enzyme to form a covalent bond. Mechanism-based inhibitors have been reviewed by Walsh (Tetrahedron 38 , 871 (1982). Another subset of inhibitors includes molecules which act as stable transition-state analogs. Gelb et al. have disclosed some fluoroketones as transition-state inhibitors of hydrolytic enzymes in Biochemistry 24 , 1813 (1985). SUMMARY OF THE INVENTION One aspect of the present invention is a blocked enzyme inhibitor having the formula III: wherein R₃ is H, nitro, alkoxy or halogen; X is O, S or NR₅ wherein R₅ is H or lower alkyl of 1-6 carbon atoms; n is 1-6; A is F or CF 3; and B is a phosphoric acid or salt thereof, a glycosyl group, an amino acid residue covalently conjugated to X through the amino acid carboxyl group, an acyl group of 2-4 carbon atoms, or a peptide of the formula wherein R₆ is (CH₂)₄NH₂. or benzyl R₇ is H or lower alkyl of 1 to 6 carbon atoms, branched or unbranched; R₈ is H, lower alkyl or hydroxy lower alkyl of 1 to 4 carbon atoms, branched or unbranched, CH₂COOH, or (CH₂)₂COOH; R₉ is lower alkyl or lower alkoxy of 1-4 carbon atoms, branced or unbranched, phenyl, or benzyloxy; q is 0-10; and said phosphoric acid or salt has the formula wherein P is bonded to X and n is as described above. Further, the present invention relates to an enzyme inhibitor having the formula III: wherein R₃ is H, nitro, alkoxy or halogen; X is O, S or NR₅ wherein R₅ may be H or lower alkyl of 1-6 carbon atoms; A is F or CF₃; and m is 2-6. The above blocked enzyme inhibitor and enzyme inhibitor can be used in a method for detection of a ligand in a liquid sample, hereinafter referred to as the unknown sample. The unknown sample suspected of containing the ligand is combined with a specific antiligand and a tracer for the ligand. The tracer includes a first enzyme conjugated to either the ligand or to a second antiligand, also specific for the ligand. Conditions conducive to binding between ligand, antiligand and tracer are provided to give a bound phase. After separation of the bound phase from the fluid, the bound phase is contacted in a liquid with a blocked inhibitor and a second enzyme. The first enzyme removes the blocking group to provide an inhibitor for the second enzyme. A substrate for the second enzyme is then added. The substrate is converted by the second enzyme to a product which provides a detectable signal, the conversion of the substrate to the product by the second enzyme being inhibited by the inhibitor. The ligand may be an antigen, a hapten or an antibody. The antigen can be a viral antigen. The method may be carried out by a competitive immunoassay technique, in which case the tracer is the ligand conjugated to the first enzyme, or by a sandwich immunoassay technique, in which case the tracer is a second antiligand conjugated to the first enzyme. The substrate is a chromogen which is convertible by the second enzyme to a product of a different color. Preferably the chromogen is colorless and is converted to a colored product by the second enzyme, the conversion of chromogen to product being inhibited by the inhibitor. In any EIA system which includes unblocking of an enzyme inhibitor, severe constraints are placed on both the inhibitor and the blocked inhibitor if the assay is to achieve maximum sensitivity. The blocked inhibitor in accordance with the present invention is an excellent substrate for the first (unblocking) enzyme, but is essentially unreactive toward the second (color forming) enzyme. Likewise, the unblocked inhibitor is a potent inhibitor of the second enzyme, but has essentially no effect on the first enzyme. Because the reactions of the blocked inhibitor and inhibitor with the first and second enzymes respectively are highly selective, "short circuits" characteristic of prior art cycling EIA systems due to cross reactivities are substantially eliminated. Thus, the invention provides (blocked) enzyme inhibitors which are useful as reagents in assays for ligands present in very low concentrations in a fluid. These reagents make possible naked eye detection and measurement of an assay signal even though the ligand is present in concentrations as low as 10-¹²M and greatly extend the range of ligands which can be detected or determined without expensive or cumbersome equipment. Further, the assay sensitivity, i.e., time required to detect the presence or absence of the ligand, may be reduced by up to 100 fold compared to conventional ElA. Significant savings in cost and space are thereby achieved, enabling assays to be carried out in small clinical laboratories or even in a physician's office. BRIEF DESCRIPTION OF THE DRAWING The Figure depicts the result of a typical assay using the (blocked) enhyme inhibitors of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is satisfied by embodiments in many different forms, there is described in detail preferred embodiments of the invention, with the understanding that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the embodiments described. The scope of the invention will be measured by the appended claims and their equivalents. The sandwich assay can be described with reference to the assay flow sheet below to provide a general understanding of the assay components and their interaction, after which each component will be discussed in detail. In the above flow sheet, the following definitions apply, wherein a colon indicates an immunological binding, a hyphen indicates a chemical bond or a physical attachment, such as absorption, a solid arrow indicates a chemical conversation and a dotted arrow indicates inhibition of a reaction or an assay component. AL - antiligand L - ligand T - tracer E₁ - first enzyme E₂ - second enzyme B- I - blocked inhibitor I - inhibitor for enzyme B - blocking group for inhibitor Sub - substrate P - product It is seen from the flow sheet that conditions conducive to binding of ligand to antiligand and tracer are provided, followed by a separation step and appropriate wash steps. A second enzyme and a blocked inhibitor consisting of an inhibtor and a blocking group are added, and the blocking group is removed by the first enzyme component of the tracer. A substrate for the second enzyme is added, and the conversion of the substrate to a detectable product by the second enzyme is inhibited by the inhibitor liberated from the blocked inhibtor by the first enzyme. The level of product is inversely proportional to the level of inhibitor, and thus to the ligand. The actual signal measured may be a color associated with the indicator reaction, as, for example, the color of the product or rate of formation thereof, or the color of the substrate or the rate of disappearance thereof. Turning now to a detailed description of the assay components, any enzyme which can remove a blocking group from a blocked inhibitor may be used as the first enzyme. Suitable first enzymes are generally hydrolases, such as phosphatases, peptidases, esterases and glycosidases. Exemplary of, but not limited to, suitable first enzymes are trypsin, thrombin, mammalian liver esterase, acetylcholinesterase, β-galactosidase, or most preferably, alkaline phosphatase. The inhibitor component should inhibit the activity of the second enzyme while having substantially no effect on the first enzyme. Thus, the nature of the second enzyme and its substrate will be discussed prior to further description of the blocked inhibitor and the inhibitor. In the assay the second enzyme is generally a hydrolase which converts the substrate to the product. Suitable hydrolases are, for example, phosphatases, peptidases such as trypsin, chymotrypsin and pepsin, or preferably esterases such as acetyl cholinesterase (AChE) and butyl cholinesterase. The most preferred second enzyme is a carboxyesterase, such as rabbit liver esterase (RLE). The substrate may be any substance containing a group which can be cleaved by the second enzyme to provide a product detectable by a signal associated with color. Thus, the signal detected is the develpment or disappearance of a color, or a change from one color to another. It is preferred that the substrate be colorless until cleaved by the second enzyme to give a colored product. Suitable substrates are indoxyl esters and, preferably, esters of nitrophenols, such as ortho and para nitrophenyl acetates or butyrates. These substrates are colorless until cleavage of the acetyl or butyryl groups by carboxyesterase occurs to give colored nitrophenols. Thus, when the substrate is an ester of a nitrophenol, the signal which is measured is inhibition of color formation. It is evident that the blocked inhibitors may also be used in a fluorescence immunoassay. In such assay, the second enzyme may convert a nonfluorogenic substrate to a fluorogenic product wherein the signal measured is modulation of fluorescence. For this assay, it is preferred that the second enzyme be an esterase. As mentioned above, the first enzyme component of the tracer cleaves the blocking group from the blocked inhibitor to provide the inhibitor of the second enzyme, and another aspect of the invention is a new class of enzyme inhibitors and blocked enzyme inhibitors of the general formula III, set forth below, wherein the nature of group B, as described later, determines whether the compound is an inhibitor or a blocked inhibitor: In formula III, R₃ may be H, nitro, alkoxy or halogen; X may be O, S or NR₅ wherein R₅ may be H or lower alkyl of 1-6 carbon atoms; n may be 1-6; A may be F or CF₃; and B may be H, a phosphoric acid or salt, a glycosyl group, an amino acid residue, such as a lysine or arginine residue covalently conjugated to X through the amino acid carboxyl group, an acyl group of 2-4 carbon atoms such as an acetyl or butyryl group, or a peptide of the formula wherein R₆ is (CH₂)₄NH₂. or benzyl R₇ may be H or lower alkyl of 1 to 6 carbon atoms, branched or unbranched; R₈ may be H, lower alkyl or hydroxy-lower alkyl of 1 to 4 carbon atoms, branched or unbranched, CH₂COOH or (CH₂)₂COOH; R₉ may be lower alkyl or lower alkoxy of 1 to 4 carbon atoms, branched or unbranched, phenyl, or benzyloxy; and q may be 0-10. When B is H, formula III represents enzyme inhibitors. When B is any group other than H, formulas I to IV represent blocked enzyme inhibitors. When B is a phosphoric acid or salt thereof, it is intended that B have the formula wherein P is bonded to X and n may be as described above. The preferred blocked inhibitor is having the formula: wherein n is 1-3; and B is a glycosyl group or a phosphoric acid or salt thereof of the structure and the preferred inhibitor is having the formua: wherein m is 2-3. The inhibitor and blocked inhibitor in accordance with formula III may be synthesized by any sequence of conventional chemical reactions as may be envisioned by one skilled in the art. Suitable and convenient methods are given in the Examples, below. The following list of effective enzyme inhibitors is intended to be exemplary only. EXPERIMENTAL Routine Analytical Techniques - Flash Silica gel chromatography was performed on ICN silica gel 32-63 mesh at 0.21-0.48 bar (3-7 psi). Analytical TLC was performed on 0.25 mm 5 x 20 cm aluminum-backed silica gel plates from EM Scientific. Preparative TLC was performed on 2.0 mm 20 x 20 cm glass-back silica gel plates from EM Scientific. Melting points were performed on a Thomas Hoover capillary melting point apparatus and are uncorrected. NMR spectra were recorded on an IBM WP-200SY spectrophotometer and chemical shifts are reported in ppm relative to tetramethylsilane. HPLC was performed on a Waters 510 two pump system with UV detection using one of two solvent systems on a Brownlee AX- 300 7 x 250 mm column; System A) initial hold for 5 minutes at 30mM NH₄OAc pH 6.5 followed by a linear gradient to 2.0 M NH₄OAc over a 30 minute period followed by a hold at 1.0 M NH₄OAc for 5 minutes. System B) used an isocratic buffer system of 30mM NH₄OAc pH 6.5 for 40 minutes. Flow rates were 1.0 ml/min. Gas Chromatography was performed on a H.P. 5840A Gas Chromatograph equipped with a FID and an automatic injector using a 30 M DB-l Megabore® column purchased from J&W Scientific, Inc. GC conditions were as follows; A three minute hold at 100°C followed by a 10°C/min gradient to 250°C followed by a 3.0 minute hold at 250°C at 16.0 ml/min flow rate. Inhibition constants were measured in 50 mM Tris pH = 8.0. Enzyme and inhibitor were incubated at ambient temperature for 20 minutes. Substrate for the enzyme was then added and the rate of hydrolysis was followed spectrophotometrically. The substrate for PLE and RLE was o-nitro-phenylbutrate and for AChE was acetyl thiocholine and Ellman's reagent. EXAMPLE I Diammonium [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate A. Ethyl 3-(4-methoxyphenyl)-2-(1-oxo-2,2,2-trifluoroethyl)propionate A 1l four neck round bottom flask, fitted with reflux condenser, dropping funnel, argon inlet, and magnetic stirrer was charged with 7.17g (0.149mol) of a 50% oil dispersion of sodium hydride and 300 ml of dry ethyl ether. Nine ml of absolute ethanol was slowly added to the stirring solution. After the evolution of hydrogen stopped, a mixture of 25g(0.136mol) of ethyl 4,4,4-trifluoromethyl acetoacetate and 21.3g(0.136mol) of 4-methoxybenzyl chloride was added over a 1 hour period. The mixture was then heated at reflux overnight. The reaction mixture was then cooled, extracted with water, 1N HCl, dried (anhydrous MgSO₄), and solvent removed under reduced pressure. The crude reaction mixture, 33.5g, was chromatographed on a 60 x 300 mm silica gel column with a 1:3 ethyl acetate:hexane mixture. Similar fractions were combined and gave 9.0g (27%) of the desired product as an oil. nmr(CDCl₃) - δ 2.12(m,3H), 2.67(m,2H), 3.85(m,3H), 3.90(s,3H), 7.24(q,4H). B. 1,1,1-Trifluoro-4-(4-hydroxyphenyl)-2-butanone A 100ml round bottom flask, fitted with reflux condenser, magnetic stirrer and argon inlet was charged with 2.05g(6.7mmol) of ethyl 3-(4-methoxyphenyl)-2-(1-oxo-2,2,2-trifluoroethyl)propionate, 20 ml of 31% HBr in AcOH, and 10 ml of water. This mixture was heated overnight at 120°C, concentrated under reduced pressure and partitioned between dichloromethane and water. The organic-layer was extracted with aqueous bisulfite, saturated sodium bicarbonate, dried (anhydrous MgSO₄), and the solvent removed under reduced pressure. The crude reaction mixture was chromatographed on a 50 x 300 mm silica gel column with 1:1 ethyl acetate:hexane. Similar fractions were combined and solvent was removed under reduced pressure to yield 600mg(41%) as a clear oil. nmr(CDCl₃) - δ 2.95(m,4H), 4.90(bs,1H), 6.93(dd,4H)J=4,60Hz. C. Diethyl [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate A 10 ml one neck round bottom flask, fitted with argon inlet and magnetic stirrer was placed in an ice bath and charged with 400mg(1.8mmol) of 1,1,1-trifluoro-4-(4-hydroxyphenyl)- 2-butanone, 400mg(2.4mmol) of diethyl chlorophosphate, 0.15 ml of dry pyridine and 5ml of dichloromethane at 5°C. The mixture was stirred overnight at ambient temperature, filtered to remove pyridine HCl, extracted with 0.2N HCl, extracted with water, and dried (anhydrous MgSO₄). Solvent removal under reduced pressure gave a crude yield of 600mg of a brown oil. Preparative TLC using 1:1 ethyl acetate:hexane gave 600mg(92%) of a clear oil. nmr(CDCl₃) - δ 1.50(m,6H), 3.0(m,4H), 4.20(m,4H), 7.15(s,4H). D. Diammonium [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate A 25 ml one neck round bottom flask, fitted with argon inlet and magnetic stirrer was charged with 5.0 ml of dichloromethane, 140mg(0.4mmol) of diethyl [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate and 2.0 ml of bromotrimethylsilane. After stirring this mixture for 3 hours at ambient temperature, 10 ml of methanol was added and the volatile materials were removed under reduced pressure. The residue was dissolved in water and the pH adjusted to 7.3 with 1.0 N NaOH. The aqueous solution was extracted with ethyl ether and lyophilized to give 190 mg of a white solid. This material was dissolved in 10 ml of 30mM NH₄OAc buffer and purified by HPLC using system A. Yield of the product after lyophilization was 50mg (37%). mp 235 - 240° C. nmr(D₂0) -δ 1.90(m,2H), 2.56(m,2H), 4.65(s,DOH), 6.88(dd,4H)J = 6, 82 Hz. EXAMPLE II Half Sandwich Assay for Hexon Antigen from Adenovirus General Procedure: Two-fold serial dilutions of purified Adenovirus hexon protein were coated onto a polyvinyl microtiter plate in pH 9.5 carbonate buffer (10mM) for a period of 1-18 hours. The plate was rinsed several times with a solution consisting of 0.05% polyoxyethylenesorbitan monolaurate in phosphate buffered saline. The immunological reaction was performed using an antibody (mouse-anti-hexon IgG) conjugated to alkaline phosphatase diluted to an appropriate level in 10% culture medium (containing fetal calf serum). After incubation for 1 hour, and a standard rinse procedure, a mixture of blocked inhibitor (10⁻³ - 10⁻⁴M) and RLE (10⁻⁸ - 10⁻⁹M) was added, and the plate was preincubated for 10 to 60 minutes. A chromogenic substrate (i.e. indoxyl butyrate or o-nitrophenyl butyrate) was added to the mixture. Color development in control wells having no antigen typically occured within 15 minutes, and could be stabilized for a longer period of time by the addition of excess free modulator. Wells which were coated with dilutions containing 1 ng/ml of hexon protein gave a positive response with this system. EXAMPLE III Sandwich Assay for Adenovirus A polyvinyl chloride microtiter plate was coated with anit-adenovirus antibody by incubating the plate for 1 hour at 37°C in a 1:900 dilution of 4.5mg/ml stock solution of the antibody in a 10mM carbonate 20mM ethylenediaminetetraacetic acid (EDTA) buffer. The plate was washed 3 times with a wash buffer solution consisting of 0.5% casein in 10mM TRIS and 154mM NaCl at pH 7.6. Adenovirus infected HeLa cells diluted to ca 1 x 10⁵ plaque forming units (PFU)/ml in pH 7.4 phosphate buffered saline containing 0.05% polyoxyethylenesorbitan monolaurate, 0.1mg/ml gentamicin, 0.5% phenol red, 0.1% bovine serum albumin, 10mM EDTA and 100 mM ethylene bis(oxyethylenenitrilo) tetraacetic acid were serially diluted in 2-fold steps across the plate. The plate was incubated as above to bind the cells to the antibody, washed with wash buffer, incubated with a 0.005mg/ml tracer solution of anti-adenovirus conjugated to alkaline phosphatase in wash buffer containing 0.5% gelatin and 10% inactivated fetal calf serum. Excess tracer solution was decanted and the plate was washed 3 times with TRIS buffer (without casein). A mixture of a 1 x 10⁻⁴M solution of the blocked inhibitor from Example I and 5 x 10⁻⁹M RLE in 50 mM TRIS buffer, pH 8.5, was added and the plate was incubated at room temperature for 10 minutes. o-Nitrophenyl butyrate (1mM) was then added to each well of the plate. Control wells having no antigen typically showed color detectable with the naked eye in ca 15 minutes, and addition of free inhibitor (product B from Example I) to the control wells inhibited color formation for longer periods. Test wells having antigen dilutions remained colorless for periods of time in excess of 15 minutes. The Figure shows the relationship of color formation to antigen concentration as determined with a Beckman DU7 spectrophotometer 20 minutes after chromogen addition. It is seen that the optical density (OD) of the solution in the control wells is substantially constant at about 0.24, and that the OD of the solutions in the test wells is inversely proportional to antigen concentration and approaches the control value at very low antigen concentrations. In summary, the invention provides a method for detection or determination of a ligand present in a liquid sample at very low levels. After binding the ligand, antiligand and tracer, the bound phase is separated and contacted with a second enzyme and a blocked inhibitor whereby a first enzyme component of the tracer removes the blocking group to provide a inhibitor for the second enzyme. The resulting inhibitor affects conversion of a substrate to a product by the second enzyme leading to a detectable signal. Detection of the signal establishes the presence or absence of the ligand in the sample. By measuring the magnitude of the signal, the concentration of the ligand may be determined. The inhibitor and the second enzyme provide two amplification stages whereby the signal is amplified by 10⁶ fold or more, enabling naked eye detection of the signal in a time up to 100 fold less than by conventional EIA.

Claims: A blocked enzyme inhibitor having the formula III: wherein R₃ is H, nitro, alkoxy or halogen; X is O, S or NR₅ wherein R₅ is H or lower alkyl of 1-6 carbon atoms; n is 1-6; A is F or CF 3; and B is a phosphoric acid or salt thereof, a glycosyl group, an amino acid residue covalently conjugated to X through the amino acid carboxyl group, an acyl group of 2-4 carbon atoms, or a peptide of the formula wherein R₆ is (CH₂)₄NH₂. or benzyl R₇ is H or lower alkyl of 1 to 6 carbon atoms, branched or unbranched; R₈ is H, lower alkyl or hydroxy lower alkyl of 1 to 4 carbon atoms, branched or unbranched, CH₂COOH, or (CH₂)₂COOH; R₉ is lower alkyl or lower alkoxy of 1-4 carbon atoms, branced or unbranched, phenyl, or benzyloxy; q is 0-10; and said phosphoric acid or salt has the formula wherein P is bonded to X and n is as described above. A blocked enzyme inhibitor of claim 1 having the formula: wherein n is 1-3; and B is a glycosyl group or a phosphoric acid or salt thereof of the structure An enzyme inhibitor having the formula III: wherein R₃ is H, nitro, alkoxy or halogen; X is O, S or NR₅ wherein R₅ may be H or lower alkyl of 1-6 carbon atoms; A is F or CF₃; and m is 2-6. An enzyme inhibitor of claim 3 having the formula: wherein m is 2-3.

Inventors: MIZE PATRICK D, O'CONNELL JAMES P, MIZE, PATRICK D., O'CONNELL, JAMES P.
Applicants: BECTON DICKINSON CO, BECTON DICKINSON AND COMPANY
Application year: 1987
Main classification: C07C49
IPC codes: C07C49, C07F9, C07C45, G01N33, C07C225