Chromogenic Lactate−Leukocyte Esterase Substrates - American

Jan 1, 1997 - Chromogenic Lactate-Leukocyte Esterase Substrates. Gary M. Johnson* and Robert Schaeper. Diagnostics Division, Bayer Corporation, ...
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Bioconjugate Chem. 1997, 8, 76−80

76

Chromogenic Lactate-Leukocyte Esterase Substrates Gary M. Johnson* and Robert Schaeper Diagnostics Division, Bayer Corporation, Elkhart, Indiana 46515. Received June 24, 1996X

The first successful reported use of lactate-based chromogenic, colorimetric substrates for a serine protease-based enzyme is described. A series of hydroxy-protected 5-phenyl-3-hydroxypyrrolyl L-lactate substrates of the general formula RO-Lac-OPP were prepared and formulated into reagents for the determination of leukocytes in dry phase formats.

INTRODUCTION

The presence of leukocytes in urine is indicative of inflammation or infection. Human leukocyte elastase (HLE) is a serine protease contained within leukocytes, which is associated with physiological processes (1, 2) such as rheumatoid arthritis, adult respiratory distress syndrome, glomerulonephritis, pulmonary emphysema, and phagocytosis. Over the past 20 years, many types of substrates (3-9) have been developed to study the reactivity and specificity of HLE. One substrate (10) has been used to detect HLE in biological fluids as a diagnostic reagent strip in a urinary leukocyte assay (10, 11) known as LEUKOSTIX. The assay operates by the general principle outlined in Scheme 1 using a test strip containing the reagents 3-[(N-tosylalaninyl)oxy]-5-phenylpyrrole (PPTA) and 4-sulfo-1,2-naphthoquinone 1-diazide (DNSA). When a urine sample containing HLE is applied to the strip, the enzyme catalyzes the hydrolysis of PPTA. This releases 3-hydroxy-5-phenylpyrrole (HOPP), which reacts with the diazide to produce a colored azo dye. The concentration of leukocytes in the sample is estimated by comparing the intensity of the final color to a standard color chart or by measuring the reflectance with a clinical reflectance meter such as CLINITEK-10. We were interested in identifying new classes of HLE substrates that yield similar or greater sensitivity toward HLE. We report herein the synthesis and evaluation of a new class of chromogenic, colorimetric esterase substrates, hydroxy-protected 5-phenyl-3-hydroxypyrrolyl L-lactates (12) of the general formula RO-Lac-OPP. These substrates have reactivity and stability comparable to that of the corresponding isostere substrate 5-phenyl3-hydroxypyrrolyl tosyl-L-alaninate (10), Ts-Ala-OPP. CHEMISTRY

The synthesis of 3-(O-tosyl-L-lactoyl)-5-phenylpyrrole (TsO-Lac-OPP, 4a) depicted in Scheme 2 is typical of the sequence used to prepare the lactic acid substrates (12) (RO-Lac-OPP, 4a-e). Tosyl chloride is added to L-lactic acid ethyl ester in the presence of TEA to provide the intermediate O-tosyl ester (1a), in a 91% yield. This intermediate is then hydrolyzed with ethanolic NaOH at 0 °C, which after acidic workup provides the intermediate O-tosyl acid (2a) in a 76% yield. The acid (2a) is then quantitatively converted into the corresponding acid chloride (3a) with neat thionyl chloride. 3a was then added to a chilled solution of 3-hydroxy-5-phenylpyrrole (HOPP) in the presence of pyridine at 0 °C to provide TsO-Lac-OPP (4a) in a 71% yield before purification. X Abstract published in Advance ACS Abstracts, January 1, 1997.

S1043-1802(96)00081-X CCC: $14.00

Table 1 summarizes the synthesized chromogenic lactic acid substrates (4a-e), prepared similarly in like yields. RESULTS AND DISCUSSION

It is well established (13) that hydrolysis of synthetic substrates by the serine protease HLE requires a small alkyl amino acid such as alanine or valine at the P1 site as demonstrated by the substrate 5-phenyl-3-hydroxypyrrolyl tosyl-L-alaninate, Ts-Ala-OPP [the nomenclature of Schecter and Berger (14) is used to designate the individual amino acid residue (P1, P2, P1′, or P2′) of a peptide substrate]. Likewise, diagnostic test strips that detect the presence of HLE contain an alanine subtrate along with a diazonium salt that acts as the colorgenerating system (Scheme 1). The reactivity of these types of dry reagent strips toward an analyte is generally quantified by monitoring the intensity of reflected light at specific wavelengths (i.e. 557 nm for the LEUKOSTIX reagent) and applying the standard Kubelka-Munk equation (28): K/S ) (1 - R)2/ (2R), where K ) the absorption coefficient, S ) the scattering coefficient, and R ) the intensity of reflected light. Use of the equation permits color development to be proportional to analyte concentration. In addition, for the LEUKOSTIX reagent, linear kinetics are also observed over 2 min when the equation is applied. In an average urine specimen, diagnostic reagent strips that contain an alanine substrate detect in 2 min at least 19 ng/mL HLE, which is the trace response with the LEUKOSTIX reagent strip. Lactic acid, is the non-amino acid, oxygen isostere of alanine, and it is well established that changes made to the substrate can dramatically affect the hydrolysis kinetics (15). Published work (1522) on lactate derivatives with orientations at P1 has previously shown that lactates act as inhibitors. Table 2 shows that the direct substitution of the phenylpyrrole ester of alanine with the phenylpyrrole ester of lactic acid produces a highly reactive substrate. As with its alanine parent, an assay incorporating this lactate derivative readily detects 19 ng/mL HLE. Similarly, Table 3 shows that substitutions made to the N terminus also produce reactive substrates. In each case, the assays using the lactate substrates readily detect 19 ng/mL HLE. The substrates listed in Tables 2 and 3 contain an N-tosyl CH3-phenyl-SO2 terminal group, while conventional HLE substrates (13) generally contain amide linkages. Jackson et al. (23) recently confirmed that the N-terminal group dramatically influences reaction kinetics. Substituting a sulfonyl group for the amide group is shown to enhance the hydrolysis kinetics (23). Thus, Table 4 shows a dramatic decrease in hydrolysis when the sulfonyl group is changed to a CdO group. It is known (23, 24) for both the N-tosyl alaninate ester and © 1997 American Chemical Society

Bioconjugate Chem., Vol. 8, No. 1, 1997 77

Chromogenic Lactate−Leukocyte Esterase Substrates Table 1 lactic acid ethyl ester analogsa 1a 1b 1c 1d 1e

L-Ts-OCH(CH3)CO2Et L-nPr-SO2-OCH(CH3)CO2Et L-PhCO-OCH(CH3)CO2Et L-MeSucc-SO2-OCH(CH3)CO2Et L-thiophene-SO2-OCH(CH3)CO2Et

(5-phenyl-3-hydroxypyrrolyl)lactate productsa 4a 4b 4c 4d 4e

Ts-Lac-OPP nPr-SO2-Lac-OPP PhCO-Lac-OPP MeOSucc-SO2-Lac-OPP thiophene-SO2-Lac-OPP

a

Where OPP ) 5-phenyl-3-hydroxypyrrolyl, Lac ) lactate, Ts ) tosyl, PhCO ) benzoyl, nPr ) n-propyl, MeSucc ) methoxysuccinyl, and SO2 ) sulfonyl.

Scheme 1. Reaction Sequence for the Leukocyte Reagent Strip Assay

Scheme 2. Synthetic Sequence for Ts-Lac-OPP (4a)

the N-carbobenzoxyl alaninate ester substrates that the rate-limiting step is the deacylation of the enzyme-acyl intermediate. The dramatic change observed in the hydrolysis rates when the N-terminal CdO group is incorporated suggests that the acyl enzyme deacylates faster with sulfonyl groups. The performance of lactate substrates with different types of N-sulfonyl groups shown in Table 3 is similar to

and at times superior to that of the natural alanine amino acid substrates. Thus, a wide variety of blocking groups can be used, ranging from simple alkyl through aromatic groups. Many functional groups can be envisioned, and this work would anticipate that such substrates would be highly reactive, providing that a sulfonyl group exists between the functional group and the O terminus of the lactate.

78 Bioconjugate Chem., Vol. 8, No. 1, 1997

Johnson and Schaeper

Table 2 response at 120 s

compound

R ) lactate (4a)

R ) alanine

HLEa (ng/mL)

K/S

change

K/S

change

0 19

0.048 0.316

0.268

0.046 0.348

0.302

tosyl-R-OPP

a Results are obtained after immersing the reagent strips in solutions and observing the response at 557 nm after 2 min.

Table 3 response at 120 s R ) alanine

HLEa compound

R ) lactate (4a,b,d,e)

(ng/mL)

K/S

change

K/S

change

0 19 48 0 19 48 0 19 48 0 19

0.034 0.343 0.822 0.050 0.363 0.881 0.074 0.435 0.976 0.050 0.224

0.309 0.788 0.313 0.831 0.361 0.902 0.174

0.100 0.498 1.049 0.177 0.514 0.916 0.050 0.385

0.398 0.949 0.337 0.739 0.335

tosyl-R-OPP nPr-SO2-R-OPP CH3O-CO(CH2)2SO2-R-OPP thiophene-SO2-R-OPP

a In an average urine, 48 ng/mL HLE produces a small result with the LEUKOSTIX reagent strip. Results are obtained after immersing the reagent strips in solutions and observing the response at 557 nM after 2 min.

Table 4 response at 120 s

compound PhCO-R-OPP

R ) alanine

R ) lactate (4c)

HLEa (ng/mL)

K/S

change

K/S

change

0 19

0.038 0.039

0.001

0.041 0.106

0.065

a

Results are obtained after immersing the reagent strips in solutions and observing the response at 557 nm after 2 min.

In this paper, we have demonstrated that lactate esters can act as substrates for HLE. Other groups (15-22) utilizing different enzymes and different substates indicate that lactate esters either directly or indirectly affect enzymatic reactivity. Much of the previous work focused on designing inhibitors of HLE and other enzymes. In the chymotrypsin work of Ingles (25), he reported a 10fold decrease in the enzymatic rate and a decrease in stereospecificity when phenyl lactate was substituted for phenylalanine. It was suggested that an important hydrogen-bonding interaction has been eliminated. An additional explanation for the apparent change in reactivity was found during the course of our experiments. We observed that, in the presence of 0.25 M NaCl, the reactivity of the lactate derivatives decreases by 33%. Consequently, experimental conditions such as ionic strength can influence the observed reactivity. SUMMARY

In summary, it is well established that simple amino acid compounds can function as colorimetric, chromogenic HLE enzyme substrates. In this manuscript, we have demonstrated that lactate esters of varying length and functional composition act as highly reactive substrates for human leukocyte elastase. MATERIALS AND METHODS

HLE was obtained from Athens Research Technology (Athens, GA). Buffer salts were analytical grade from

Fisher or Aldrich. DMSO and acetone were obtained from J. T. Baker, Inc. (Phillipsburg, NJ), and decanol was from Sigma Chemical Co. (St. Louis, MO). Polyvinylpyrrolidone (PVP) was obtained from Polysciences Inc. (Warrington, PA). Diazonium salts were prepared as previously described (26). IR spectra were recorded on a Perkin-Elmer 710B spectrophotometer. NMR spectra were recorded on a GE 300NB-MHz spectrometer. Mass spectra were determined on a HP5985A instrument by fast atom bombardment (FAB) with direct introduction. Thin layer chromatography was carried out on Merck GF 254 silica plates. Optical rotations were determined on a PE141 polarimeter. C/H/N analyses were done by Galbraith Labs (Knoxville, TN). HLE was dissolved in 50 mM sodium acetate (pH 4.5) buffer, and the concentration of HLE was determined spectrophotometrically according to the method of Baugh and Travis (29). Reagent papers were prepared by impregnating Whatman 3M filter paper with 0.8 M borate/NaOH (pH 8.8) buffer containing 1% PVP K60 and dried in an air foil dryer. The dried paper was subsequently immersed in a solution containing 1.1 mM substrate, 0.7 mM 6-nitro-1,2-naphthoquinone diazide, 1.5% decanol, and 3% DMSO and then dried as above. The reagent paper was made into reagent test strips that consisted of 0.2 in. squares of the final impregnated paper, which was adhered via double-stick adhesive to a polystyrene carrier. The reagent strip was quickly immersed in the testing solution [100 mM NaOAC (pH 5.6), 4 mM benzoic acid, 0.0025% yellow #7, and 0.00002% red #40] that contained 0 or 19 ng/mL HLE. After 2 min, the color intensity was measured with a clinical reflectance meter, the CLINITEK-10. The intensity of the reflected light was quantified by applying the standard Kubulka-Monk equation (28). EXPERIMENTAL PROCEDURES

Step 1. To L-lactic acid ethyl ester (5.00 g, 4.81 mL, 42.3 mmol) in CH2Cl2 (50 mL) at 0 °C (ice bath), under argon, was added tosyl chloride (8.1 g, 42.3 mmol) in one portion with stirring. Triethylamine (5.57 g, 7.67 mL, 55 mmol) was added dropwise via syringe, and the reaction mixture was stirred for 7 h at 0 °C. The reaction mixture was then poured over a solution of 1 M aqueous HCl (75 mL) and ice (75 g). After separation, the organic layers were dried over MgSO4, filtered, and concentrated in vacuo to give 10.5 g (91%) of crude product (1a). Step 2. The crude L-tosyl lactate ethyl ester (10.5 g, 38.6 mmol, 1a) in absolute ethanol (12 mL) was added dropwise via addition funnel (1/4 h) to a chilled (0 °C, ice bath) solution of NaOH (1.8 g, 46 mmol) in distilled water (20 mL). After the mixture was stirred for 5 h under argon at 0 °C, the reaction was carefully quenched via dropwise addition of concentrated HCl to pH 2. Solid NaCl was added to saturation, and the aqueous layer was extracted with CH2Cl2 (4 × 50 mL). The organic phase was then dried over MgSO4, filtered, and concentrated in vacuo to give 7.17 g (76%) of crude product (2a). Step 3. The crude L-O-tosyllactic acid (1.50 g, 6.15 mmol, 2a) was placed in a 25 mL round bottom flask under argon equipped with a reflux condenser. Thionyl chloride (10.12 g, 6.2 mL, 85 mmol) was added in one portion and the reaction mixture placed into an oil bath, preheated to 50 °C, and stirred for 2 h. The reaction mixture was then cooled and placed in an ice bath, and ice cold hexane (25 mL) was added. When no solid product was observed, the solution was concentrated in vacuo to give 1.6 g (quantitative) of crude yellow oil (3a).

Chromogenic Lactate−Leukocyte Esterase Substrates

Step 4. To a chilled solution (0 °C, ice bath) of 5-phenyl-3-hydroxypyrrole (986 mg, 6.20 mmol) in CH2Cl2 (30 mL) under argon was added pyridine (1.5 mL, 18.6 mmol) dropwise via syringe, followed immediately by rapid dropwise addition of a solution of L-O-tosyllactic acid chloride (1.60 g, 6.11 mmol, 3a) in CH2Cl2 (5.0 mL). The residual contents were then added dropwise with an additional 5.0 mL of CH2Cl2. The reaction mixture was stirred for 1 h at 0 °C, allowed to come to room temperature over approximately 1 h further, and then stirred overnight at room temperature (16 h). The reaction mixture was then extracted with aqueous 1 M HCl (2 × 25 mL), and the combined aqueous phases were back-extracted with CH2Cl2 (25 mL). The combined organic layers were then extracted with saturated aqueous bicarbonate (2 × 25 mL). After back-extraction of the basic aqueous phase, the combined organic layers were treated with norite carbon and MgSO4, filtered, and concentrated in vacuo. The resulting oil was dissolved in hexane/EtOAc (1:1, 25 mL), treated with norite carbon once more, filtered, and concentrated in vacuo to give 1.7 g (71%) of solid crude product (4a). This material was triturated with warm hexane (10 mL) followed by hexane/EtOAc (4:1, 2 × 8 mL), and decanted, and the resultant solid was dried under vacuum to provide 1.04 g of a pink solid. This material was dissolved in hexane/EtOAc (1:1), treated with norite carbon, filtered, and concentrated in vacuo at room temperature to give 932 mg (unoptimized yield) of creme product (4a). Product 4a: 1H NMR (300 MHz, CDCl3, ppm) 1.65 (d, 3H), 2.42 (s, 3H), 5.12 (qu, 1H), 6.26 (dd, 1H), 6.84 (dd, 1H), 7.18-7.5 (m, 7H), 7.84 (d, 2H), 8.15 (brs, 1H); 13C NMR (75 MHz, CDCl3, ppm) 18.51 (methyl), 21.66 (methyl), 73.96 (CH, lactate), 93.39 (CH, pyrrole), 107.89 (CH, pyrrole), 123.84 (CH, aromatic), 126.85 (CH, aromatic), 128.12 (CH, aromatic), 128.97 (CH, aromatic), 129.87 (CH, aromatic), 166.68 (CdO); EI/MS (18 EV, DIP) 385 (M+, 21.8), 227 (2.3), 199 (22.4), 158 (67.5), 155 (BASE), 91 (41.7); IR: (CDCl3, cm-1) 3450, 3022, 1770, 1600, 1570, 1550, 1514, 1450, 1370, 1260, 1240; optical rotation (λ ) 578 nm) 10 mg sample, 1 mL of MeOH, room temperature, 1 cm cell, -64.8°. Anal. Calcd: C, 62.39; H, 4.97; N, 3.63; S, 8.33. Found: C, 62.44; H, 5.03; N, 3.57; S, 8.65. Product 4b: 1H NMR (300 MHz, CDCl3, ppm) 1.10 (t, 3H), 1.75 (d, 3H), 2.20 (m, 2H), 3.30 (m, 2H), 5.35 (qu, 1H), 6.41 (dd, 1H), 6.95 (dd, 1H), 7.20-7.60 (m, 5H), 8.30 (brs, 1H); 13C NMR (75 MHz, CDCl3, ppm) 12.87 (methyl), 17.23 (methylene), 18.59 (methyl), 53.64 (methylene), 73.49 (CH, lactate), 98.32 (CH, pyrrole), 107.94 (CH, pyrrole), 123.86 (CH, aromatic), 126.90 (CH, aromatic), 128.96 (CH, aromatic), 166.5 (CdO); FAB/MS 337.2 (M+, 55), 159 (BASE); IR: (CDCl3, cm-1) 3400, 3010, 2980, 1768, 1573, 1512, 1454, 1400, 1360, 1254, 1169; optical rotation (λ ) 578 nm) 13 mg sample, 1 mL of MeOH, room temperature, 1 cm cell, -48.5°. Anal. Calcd: C, 56.97; H, 5.64; N, 4.15; S, 9.49. Found: C, 57.06; H, 5.75; N, 3.87; S, 9.91. Product 4c: 1H NMR (300 MHz, CDCl3, ppm) 1.75 (d, 3H), 5.55 (qu, 1H), 6.40 (dd, 1H), 6.95 (dd, 1H), 7.15-7.7 (m, 8H), 8.1 (d, 2H), 8.2 (brs, 1H); 13C NMR (75 MHz, CDCl3, ppm) 17.13 (methyl), 69.08 (CH, lactate), 98.57 (CH, pyrrole), 108.03 (CH, pyrrole), 123.82 (CH, aromatic), 126.68 (CH, aromatic), 128.41 (CH, aromatic), 128.89 (CH, aromatic), 129.91 (CH, aromatic), 133.33 (CH, aromatic), 165.98 (CdO), 166.48 (CdO); EI/MS (18 EV, DIP) 335 (M+, 3.1), 177 (42.8), 149 (22.2), 105 (BASE), 77 (0.7); IR: (CDCl3, cm-1) 3400, 3000, 1763, 1723, 1606, 1585, 1573, 1512, 1452, 1318, 1177; optical

Bioconjugate Chem., Vol. 8, No. 1, 1997 79

rotation (λ ) 578 nm) 12 mg sample, 1 mL of MeOH, room temperature, 1 cm cell, +13.3°. Anal. Calcd: C, 71.64; H, 5.07; N, 4.18. Found: C, 71.92; H, 5.22; N, 4.28. Product 4d: 1H NMR (300 MHz, CDCl3, ppm) 1.78 (d, 3H), 1.98 (t, 2H), 2.62 (t, 2H), 2.75 (s, 3H), 5.37 (qu, 1H), 6.42 (brs, 1H), 6.98 (brs, 1H), 7.2-7.55 (m, 5H), 8.2 (brs, 1H); 13C NMR (75 MHz, CDCl3, ppm) 18.52 (methyl), 28.42 (methylene), 47.29 (methylene), 52.4 (methyl), 74.14 (CH, lactate), 98.37 (CH, pyrrole), 107.93 (CH, pyrrole), 123.89 (CH, aromatic), 126.94 (CH, aromatic), 128.98 (CH, aromatic); FAB/MS 381 (M+, 63), 186 (8.5), 159 (BASE), 91 (12), 55 (24.5); IR: (CDCl3, cm-1) 3450, 3000, 1751, 1741, 1572, 1513, 1453, 1439, 1359, 1319, 1255; optical rotation (λ ) 578 nm) 7 mg sample, 1 mL of MeOH, room temperature, 1 cm cell, -27.1°. Anal. Calcd: C, 53.54; H, 4.99; N, 3.67; S, 8.39. Found: C, 53.05; H, 5.07; N, 3.41; S, 7.75. Product 4e: 1H NMR (300 MHz, CDCl3, ppm) 1.70 (d, 3H), 5.19 (qu, 1H), 6.32 (dd, 1H), 6.85 (dd, 1H), 7.12 (t, 1H), 7.20-7.50 (m, 5H), 7.70 (dd, 1H), 7.79 (dd, 1H), 8.19 (brs, 1H); 13C NMR (75 MHz, CDCl3, ppm) 18.44 (methyl), 74.91 (CH, lactate), 98.34 (CH, pyrrole), 107.90 (CH, pyrrole), 123.85 (CH, aromatic), 126.85 (CH, aromatic), 127.54 (CH, thiophene), 128.95 (CH, aromatic), 134.09 (CH, thiophene), 134.71 (CH, thiophene); EI/MS (18 EV, DIP) 377 (M+, 63.1), 236 (4.4), 191 (13.8), 164 (4.9), 159 (BASE), 147 (66.1), 99 (8.2), 83 (1.1); IR (CDCl3, cm-1) 3450, 3050, 2940, 1760, 1573, 1509, 1453, 1404, 1378; optical rotation (λ ) 578 nm) 12 mg sample, 1 mL of MeOH, room temperature, 1 cm cell, -34.3°. Anal. Calcd: C, 54.1; H, 3.98; N, 3.71; S, 16.98. Found: C, 53.4; H, 4.16; N, 3.69; S, 16.81. LITERATURE CITED (1) Powers, J. C., Kam, C. M., Hou, H., Oleksyszyn, J., and Meyer, E. F. (1992) in Biochemistry of Pulmonary Emphysema (C. Grassi, J. Travis, L. Casali, and M. Luisetti, Eds.) pp 123141, Springer-Verlag, London. (2) Bieth, J. G. (1989) Human Neutrophil Elastase. In Elastin and Elastases, Vol. II, pp 23-31, CRC Press, Inc., Boca Raton, FL. (3) Ashe, B. M., and Zimmerman, M. (1980) Fluorogenic substrates for human leukocycte and porcine pancreatic elastase. J. Appl. Biochem., 445-447. (4) Powers, J. C., Harper, J. W., Cook, R. R., Roberts, C. J., and McLaughlin, B. J. (1984) Active Site Mapping of Serine Proteases Using Tripeptide Thiobenzyl Ester Substrates. Biochemistry 23, 2995-3002. (5) Nakajima, K., Powers, J. C., Ashe, B. M., and Zimmerman, M. (1979) Mapping the Extended Substrate Binding Site of Cathepsin G and Human Leukocyte Elastase. Studies with Peptide Substrates related to the alpha-1-Proteinae Inhibitor. J. Biol. Chem. 254, 4027-4032. (6) Castillo, M. J., Nakajima, X., Zimmerman, M., and Powers, J. C. (1979) Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal. Biochem. 99, 53-64. (7) Stein, R. L., Viscarello, B. R., and Wildonger, R. A. (1984) Catalysis by human leukocycte elastase. Rate limiting deacylation for specific p-nitroanilides and amides. J. Am. Chem. Soc. 106, 796-798. (8) Stein, R. L., Strimpler, A. M., Hori, H., and Powers, J. C. (1987) Catalysis by Human Leukocyte Elastase: Mechanistic Insights into Specificity Requirement. Biochemistry 26, 1301-1305. (9) Stein, R. L., Strimpler, A. M., Hori, H., and Powers, J. C. (1987) Catalysis by Human Leukocyte Elastase: Proton Inventory as a Mechanistic Probe. Biochemistry 26, 13051314. (10) Corey, P. F., Skjold, A. L., Pendergrass, J. H., and Stover, L. (1987) Composition and Test Device for Determining the

80 Bioconjugate Chem., Vol. 8, No. 1, 1997 Presence of Leukocytes, Esterase and Protease in a Test Sample, U.S. Patent 4,657,855. (11) Skjold, A. C., and Hugel, H. (1987) Composition and Test Device for Determining the Presence of Leukocytes Containing a Zwitterion Coupling Reagent, U.S. Patent 4,637,979. (12) Johnson, G. M., and Schaeper, R. J. (1995 and 1996) Test Device for Determining the Presence of Leukocyte Cells, Esterase or Protese in a Test Sample, U.S. Patent 5,464,739; U.S. Patent 5,512,450. (13) Bieth, J. G. (1989) Mechanism of Action of Elastases. In Elastin and Elastases, Vol. II, pp 23-31, CRC Press, Inc., Boca Raton, FL. (14) Schechter, I., and Berger, A. (1967) On the size of the active site of proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157-162. (15) Dorn, C. P., and Yang, S. S. (1977) Peptide Carbazates, U.S. Patent 4,064,236. (16) Dorn, C. P., Zimmerman, M., Yang, S. S., Yurewicz, E. C., Ashe, B. M., Frankshun, R., and Jones, H. (1977) Proteinase Inhibitors 1. Inhibitors of Elastase. J. Med. Chem. 20, 14641468. (17) Digenis, G., Agha, B. J., Tsuji, K., Kato, M., and Shinogi, M. (1986) Peptidyl Carbamates Incorporating Amino Acid Isosteres as Novel Elastase Inhibitors. J. Med. Chem. 29, 1468-1476. (18) Krantz, A., Spencer, R. W., Tam, T. F., Liak, T. J., Copp, L. J., Thomas, E. M., and Rafferty, S. P. (1990) Design and Synthesis of 4H-3,1-Benzoxazin-4-ones as Potent Alternate Substrate Inhibitors of HLE. J. Med. Chem. 33, 464-479. (19) Umezawa, H., Takeuchi, T., Aoyagi, T., Morishima, H., and Takamatru, A. (1977) N-Lactoyl Peptides, JP 52,057,121.

Johnson and Schaeper (20) Kellog, R. M., and Moorlag, H. (1990) Pig Liver Esterase Catalyzed Hydrolyses of Racemic Alpha-substituted Alphahydroxy Esters. J. Org. Chem. 55, 5878-5881. (21) Kellog, R. M., and Moorlag, H. (1991) Pig Liver Esterase Catalyzed Hydrolyses of Alpha-substituted Alpha-hydroxy Esters: The Influence of Substituents. Tetrahedron: Asymmetry 2, 705-720. (22) Kraicsovits, F., and Otvos, L. Effect of the Alcohol Moiety upon Reactivity and Stereospecificity in Alpha-Chymotrypsincatalyzrd Ester Hydrolysis. Symp. Pap.sIUPAC Int. Symp. Chem. Nat. Prod., 11th, 37-40. (23) Jackson, D. S., Brown, A. D., Schaeper, R. J., and Powers, J. C. (1995) A Kinetic Study of the Hydrolysis of the N-Tosylalanine Ester of 3-Hydroxy-5-phenylpyrrole and Related Compounds by Human Leukocyte Elastase. Arch. Biochem. Biophys. 323, 108-114. (24) Stein, R. L. (1983) Catalysis by human Leukocyte Elastase: Substrate Structural Dependence of Rate-Limiting Protolytic Catalysis and Operation of the Charge Relay System. J. Am. Chem. Soc. 105, 5111-5116. (25) Ingles, D. W., and Knowles, J. R. (1968) The Stereospecificity of Alpha-Chymotripysin. Biochem. J. 108, 561-569. (26) Putter, R. (1965) In Methoden der Organischen Chemie (E. Muller, Ed.) pp 89-92, George Thiem Verlag, Stuttgart. (27) Baugh, R. J., and Travis, J. (1976) Human Leukocyte Granule Elastase: Rapid Isolation and Characterization. Biochemistry 15, 836-841. (28) Kortum, G. (1976) Reflectance Spectroscopy: Principles, Methods, & Applications, Springer-Verlag, New York.

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