Hydrosoluble fluorogenic substrates for plasmin

ANALYTICAL

BIOCHEMISTRY

Hydrosoluble Marzia

Harnois-Pontoni,’

193,

248-255

(1991)

Fluorogenic Substrates for Plasmin Michel

Monsigny,

Dbpartement de Biochimie des Glycoconjugubs et Lectines CNRS, 1 rue Haute, F-45071 Orlbans Cedex 2, France

Received

July

and Roger Endo&nes,

Molbculaire,

9, 1990

New hydrosoluble fluorogenic substrates for plasmin gluconoylpeptidyl-3-amido-S-ethylcarbazole were synthesized. The substitution of the N-terminal end of the peptides by a gluconoyl group prevents the substrates from aminopeptidase degradation and highly increases their hydrosolubility. The substitution of the peptide Cterminal end by a 3-amino-S-ethylcarbazole group leads to substrates suitable for direct fluorometric assay of plasmin present in cell supernatants or in cell lysates. On the basis of the kinetic parameters of the substrate hydrolysis by plasmin, it was found that D amino acids in the P, position decrease systematically the kinetic constants of the substrates. The L configuration of the P, amino acid appears therefore as essential in optimum substrates for plasmin. o 1991 Academic Press,

Mayer’ Centre de Biophysique

Inc.

The role of proteolytic enzymes has been increasingly recognized in a wide variety of biological processes. Peptide derivatives, in which a fluorescent amine is released upon proteolysis, are currently the best tools for the assay of extremely low protease activities. As most of the N-acylated peptidic substrates are sparingly soluble in aqueous solution they have often to be initially dissolved in an organic solvent such as dimethyl sulfoxide and their use in vitro and in vivo is consequently limited. Different strategies have been used to enhance the substrate water solubility: the use of N-terminal free amino acid, preferentially of D configuration in order to avoid aminopeptidase degradation, or the addition of a charged substituent in the N-terminal position. All these procedures call upon the presence of a charge which can strongly impair the binding of the substrate to the enzyme binding site.

r Present address: Roussel-Uclaf, 102 route de Noisy, mainville, France. * To whom correspondence should be addressed.

F-93230

Ro-

On these bases, our interest has been to develop hydrosoluble synthetic fluorogenic substrates that will allow a sensitive detection of proteinase activities in cells as well as in body fluids. Recently we showed that uncharged hydrosolubilizing acylating groups: polyhydroxyalcanoyl groups containing at least two hydroxyl groups were suitable to increase the water solubility of peptide derivatives and to protect them against aminopeptidase degradation (1). This general procedure of hydrosolubilization is applied here to fluorogenic peptidyl substrates specific for plasmin with the additional aim to use these hydrosolubilized peptidic sequences as selective nontoxic carriers of cytotoxic drugs. Plasmin being generated from plasminogen through the action of plasminogen activator, the assay of plasmin is also an indirect assay of plasminogen activator which is relevant to cancer diagnosis. Plasmin splits peptide bonds specifically at the carboxy1 function of a basic amino acid and a large number of tripeptidyl sequence have been investigated or designed as “optimum” substrates (2). A variety of peptidyl substrates was thus hydrosolubilized through Nacylation by a polyhydroxyalcanoyl group, generally a gluconoyl group (G~cA),~ and amidated on the carboxyl function of their lysyl or arginyl C-terminal residue by 3-amino-9-ethylcarbazole (AEC) (3). This fluorescent amine has been selected among several fluorescent detector groups such as 4-methoxy-2-naphthylamine (4), 7-amino-4-methylcoumarin (5), or 7-amino-4-trifluoromethylcoumarin (6) for its spectroscopic properties and for its good coupling yields with amino acids. Furthermore the use of this amine allows quantitative determination of plasmin activity in cell lysates and cell supernatants (7). In this paper the synthesis of hydrosoluble fluoro-

a Abbreviations used: GlcA, gluconic acid (gluconoyl); AEC, amino-9-ethylcarbazole; BOP, benzotriazolyl-N-oxytris(dimethylamino) phosphonium hexafluorophosphate; TMS, tetramethylsilane; DMSO, dimethyl sulfoxide; pNA, p-nitroanilide; TEA, triethylamine.

248 All

Copyright 0 1991 rights of reproduction

3-

0003-2697/91 $3.00 by Academic Press, Inc. in any form reserved.

HYDROSOLUBLE

FLUOROGENIC

genie substrates of this type is reported with a particular interest on the stereochemistry dependence of the P, residue on the substrate affinity. EXPERIMENTAL

PROCEDURE

Materials Unless otherwise stated, all the amino acids used were of the L configuration. The starting amino acid derivatives were synthesized according to the standard literature procedures. 6-Gluconolactone was from Serva (Heidelberg, FRG). AEC was obtained from Sigma (Saint Louis, MO) and was recrystallized twice from methanollhexane. Benzotriazolyl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) was obtained from Richelieu Biotechnologies (Saint Hyacinthe, Canada). Dimethylformamide and triethylamine were freshly distilled before use on fluoro-2,4dinitrobenzene and benzyloxycarbonylglycyl-p-nitrophenyl ester, respectively. Plasmin from human or porcine blood, thrombin from human plasma, and urokinase from human kidney cells were purchased from Sigma. The substrate H-D-Val-Leu-Lys-pNA was from Bachem (Bubendorf, Switzerland). Analytical thin-layer chromatography (TLC) was carried out with Merck silica gel 60 F-254 precoated glass plates. Spots were visualized by uv, ninhydrin spray, and ammonium heptamolybdate in H,SO, spray or tert-butyl hypochlorite/starch iodide chromatic system. After purification, all the peptides showed a single spot when subjected to TLC using the following solvent systems: A, chloroform/methanol/water (16/6/l); B, chloroform/methanol/water (20/6/l); C, chloroform/ methanol (20/l); D, chloroform/methanol (6/l) (v/v). Methods Melting points were determined on a Leitz melting point apparatus and are uncorrected. Optical rotation measurements were performed on a Perkin-Elmer 141 polarimeter equipped with a thermostat. Infrared spectra were obtained on a Perkin-Elmer 297 spectrophotometer. Ultraviolet and visible absorption spectra were recorded on a Beckman Acta III spectrophotometer. The ‘H (300 MHz) nuclear magnetic resonance spectra were recorded on a Bruker AM-300 spectrometer and are expressed as 6 units (parts per million) relative to tetramethylsilane (TMS) as internal reference in 99.95% Me,SO-d, (C.E.A., Saclay, France) solution. The assignment of the resonance lines was processed by two-dimensional (2-D) correlation spectroscopy (8). Fluorescence was measured using a Fica 55 MK II spectrofluorometer standardized daily with a 0.255 PM quinine sulfate solution in 0.1 N H,SO,. The spectral characteristics of free and N-acylated AEC have been described previously (3). An Hitachi Model 655 A chromatograph

SUBSTRATES FOR PLASMIN

249

was used for analytical HPLC. Preparative-scale HPLC separations were achieved on a Waters 6000 A chromatograph by recycling on a Merck 250 X 10 mm HIBAR stainless steel column with 7 pM RP 18 Lichrosorb packing. Solvents were HPLC grade from BDH Chemicals (Poole, England). Assays The substrates were dissolved, in the absence of DMSO, in a 50 mmol Tris-HCl buffer at pH 8.1 which is the optimal pH for plasmin activity. The kinetic constants were calculated according to the LineweaverBurk method based on initial rate determinations at eight different substrate concentrations in the 20 to 500 pM range, in the presence of 0.05 to 10 pg plasmin/ml depending on the substrate. The amount of AEC released was linear with time. Slopes and intercepts were calculated by a least-squares method. After incubation for 30 min at 37”C, the free AEC released was quantitatively extracted with 1 vol ethyl acetate and assessed spectrofluorometrically as previously described (7). In ethyl acetate, assays were conducted at excitation and emission wavelengths of 370 and 430 nm, respectively. The catalytic constant kcat was calculated by dividing the velocity of the reaction by the molar concentration of the enzyme; the molecular weight of plasmin was taken as 75,400 (9). In order to compare the rates of hydrolysis of 4-nitroanilide and 3-amido-9-ethylcarbazole substrates, both substrates (0.3 mM), dissolved in 50 I'nM Tris-HCl buffer, pH 8.1, were incubated for 5 min at 37°C in the presence of different concentrations of human plasmin. With the chromogenic substrate, the enzymatic reaction was stopped by addition of acetic acid and the increase of absorbance at 410 nm was followed by spectrophotometry. Substrates Synthesis The substrates were synthesized by standard peptide chemistry procedures. In order to investigate the effect of the stereochemistry of the amino acid in the P, position on their affinity for plasmin, the substrates were synthesized by a racemizing fragment coupling procedure in dimethylformamide between a N-gluconoyl dipeptide and lysyl- or arginyl-AEC and the L-L-L and L-D-L diastereoisomers obtained were isolated by preparative-scale HPLC as described above. The following details for the synthesis of the substrate GlcA-Ile-Leu-Lys-AEC are representative of the procedure used. The sequence of reactions leading to this compound is diagrammed in Scheme 1. Boc-Ile-Leu-OBzl. A solution of Tos,H-Leu-OBzl (2.58 g; 6.88 mmol) and of Boc-Ile-OH,DCHA (2.80 g; 6.88 mmol) in ethyl acetate was cooled at -10°C and

250

HARNOIS-PONTONI,

Leu

Ile

Boc --OH

HCI, H --

LYS OBzl

Boc

OBzl

HCI, H

OBzl

GlcA

OBzl

GM

OH

GlcA

MONSIGNY,

i! Boc--LOH 2 BOC--LAEC 2 HCI, H--LAEC z ’ AEC

GICA

AEC

SCHEME I.

Synthesis

of GlcA-Ile-Leu-Lys-AEC.

dicyclohexylcarbodiimide (1.56 g; 7.57 mmol) was added. The reaction mixture was stirred 2 h at -10°C and overnight at room temperature. The DCCU and HCl,DCHA formed were removed by filtration and the filtrate was washed successively with 5% NaHCO,, water, 10% citric acid, and water. The organic phase was dried over Na,SO,, concentrated, and precipitated in hexane to give 2.60 g of a white solid: yield, 88%; mp 99-10l°C; [cy];& -20.7’ (c 1, MeOH); R, 0.80 (C). HCl,H-Ile-Leu-OBzl. A solution of Boc-Ile-LeuOBzl (1.95 g; 4.5 mmol) in 1 N HCl/AcOH (23 ml) was stirred for 20 min at room temperature. The excess of reagent was removed by evaporation under reduced pressure and the residue was triturated several times in hexane. The hygroscopic precipitate was dried to yield 1.5 g (90%): [(Y]& 30.4” (c 1, MeOH); R,O.67 (D); argentimetric titration 99%. GlcA-Ile-Leu-OBzl. To a solution of HCl,H-Ile-LeuOBzl (1.30 g; 3.5 mmol) in freshly distilled DMF (4 ml), neutralized with triethylamine (0.49 ml; 3.5 mmol), 6gluconolactone (1.87 g; 10.5 mmol), and TEA (0.98 ml; 7.0 mmol) were added. The mixture was stirred at 60°C for 24 h. &Gluconolactone (0.62 g; 3.5 mmol) and TEA (0.49 ml; 3.5 mmol) were then added and the mixture was further stirred for 24 h. The mixture was then filtered and the filtrate evaporated under reduced pressure. The residue was purified by preparative chromatography on a silica gel column eluted with the solvent system A: yield, 1.08 g (60%) after precipitation in ether/petroleum ether (l/l (v/v)); mp 115-116°C; [a]& 6.7” (c 1, MeOH); Rf 0.62 (A). GZcA-Ile-Leu-OH. GlcA-Ile-Leu-OBzl (0.97 g; 1.9 mmol) was hydrogenolyzed in the presence of palladium/activated charcoal (10% Pd) (200 mg) in methanol/water (3/l (v/v)) for 4 h. After removal of the catalyst by filtration, the solution was evaporated to dryness and the product was recovered by lyophilization from water in 96% yield (0.76 g): mp 74-76“C; [(Y]& 10.8’ (c 1, MeOH); R, 0.15 (A). Boc-Lys(Z)-AEC. The title compound was synthesized by coupling Boc-Lys(Z)-OH,DCHA (2.70 g; 4.8

AND

MAYER

mmol) and 3-amino-9-ethylcarbazole (1.00 g; 4.8 mmol) in the presence of BOP (2.12 g; 4.8 mmol) in freshly distillated DMF (5 ml). The mixture was stirred in the dark and under nitrogen atmosphere, at room temperature for 6 h. The solution was evaporated to dryness and the residue was dissolved in ethyl acetate and washed successively with 5% NaHCO,, water, 10% citric acid, and water. The organic phase was dried over Na,SO, and concentrated. The residue was precipitated in ether/petroleum ether (l/l (v/v)) and the precipitate was filtered and dried to give 2.2 g of the title compound: yield, 80%; mp 114-115°C; [(Y]& 23.0“ (c 1, MeOH); R, 0.80 (C). HCI,H-Lys(Z)-AEC. A solution of Boc-Lys(Z)-AEC (2.00 g; 3.5 mmol) in 1 N HCl/AcOH (18 ml) was stirred for 20 min at room temperature. The excess of reagent was removed by evaporation under reduced pressure and the residue was dissolved in chloroform and precipitated in dry ether. The precipitate was filtered and dried to give 1.74 g (98% yield); mp 117-119°C; [(Y]& 62.9” (c 1, MeOH); R, 0.09 (C); argentimetric titration 99%. GlcA-Ile-Leu-Lys(Z)-AEC. The title compound was obtained by coupling the two fragments GlcA-Ile-LeuOH (0.76 g; 1.8 mmol) and HCl,H-Lys(Z)-AEC (0.92 g; 1.8 mmol) in distilled DMF (4 ml) in the presence of TEA (0.25 ml; 1.8 mmol), HOBt,H,O (0.27 g; 1.8 mmol), and DCCI (0.41 g; 2.0 mmol). The reaction mixture was stirred 2 h at -10°C and overnight at room temperature. The DCHU and HCl,TEA formed were removed by filtration and the filtrate was evaporated to dryness. The crude product was recrystallized from isopropanol to give 1.4 g (86% yield): mp 208-209’C; R, 0.36 (D). GlcA-Ile-Leu-Lys-AEC,HCl. GlcA-Ile-Leu-Lys(Z)AEC (0.96 g; 1.1 mmol) was dissolved in methanol/ water (3/l (v/v)) (4 ml) and hydrogenolyzed over palladium/activated charcoal (10% Pd) (200 mg) in the presence of 1 N HCl(l.3 ml) for 2 h. After removal of the catalyst by filtration, the filtrate was evaporated to dryness. The residue was taken up with water and lyophilized: yield, 0.80 g (93%); mp llO-112°C; R, 0.15 (D). The 300-MHz NMR spectrum of this compound shows that most of the resonance lines are splitted, indicating that racemization occurred during the fragment coupling step (10). This was also confirmed by analytical reversed-phase HPLC. This compoundwas further purified by preparative-scale HPLC using a recycling procedure as shown on Fig. 1. The NMR resonance lines of both isomers were processed by two-dimensional correlation spectroscopy and are reported in Table 1. In order to provide reference compounds, pure L-L-L and L-D-L stereoisomers were synthesized by a stepwise nonracemizing procedure starting from Nps-Lys(Z)-AEC and adding successively Boc-L-Leu-OH or Boc-D-Leu-OH, and Boc-Ile-OH. The tripeptidyl-AEC was further gluconoylated and the lysine side chain was finally deprotected by catalytic hydrogenolysis.

HYDROSOLUBLE

FLUOROGENIC

SUBSTRATES

FOR

251

PLASMIN

mamide the more sensitive residues are isoleucine, valine, and phenylalanine and that, furthermore, according to Le Nguyen et al. (12) valine or isoleucine as the penultimate residue increases the racemization expected at the activated residue. Physicochemical Properties and Solubility of the N-Gluconoylated Substrates

I 0

I

20

40 Time

60

80

1

l

100

(min)

FIG. 1. Preparative-scale

HPLC separation of the L-L-L and L-D-L diastereoisomers of the substrate GlcA-Ile-Leu-Lys-AEC by recycling procedure. Chromatographic conditions: column HIBAR RP 18, 7 pm, 250 X 10 mm; isocratic elution with a mixture acetonitrile/ methanol/water (3615717 (v/v)); flow rate, 5.0 ml mini; detection at 340 nm.

RESULTS

AND

Melting points, optical rotations and solubilities in 50 mmol Tris-HCl (pH 8.1) buffer of the new substrates synthesized are given in Table 3. In order to study the influence of the hydrophilic N-gluconoyl group on the substrate affinity for plasmin, some substrates with hydrophobic N-acylating groups such as benzyloxycarbony1 or tert-butyloxycarbonyl were also synthesized. In contrast with the aminocoumarin derivatives, all the substrates synthesized are stable in aqueous solution, showing no detectable spontaneous hydrolysis after a 5-h incubation under assay conditions in the absence of enzyme.

DISCUSSION

HPLC and NMR Analysis of Racemization upon Peptide Fragment Coupling We have taken advantage of the possibility of racemization of the C-terminal residue of the carboxylic fragment during fragment condensation to obtain at one and the same time both L-L-L and L-D-L diastereoisomers. Quantitation and sequence dependence of racemization during peptide synthesis has been reviewed by Benoiton (11). The amount of each isomer in the substrates synthesized was determined by analytical reversed-phase HPLC and by high-field proton NMR; both methods were used without further chemical treatment of the compound. By HPLC, as little as 0.1% of the contaminating isomer can be detected, while NMR can detect about 1% of the other isomer in a 1-mg sample (Fig. 2). The different chemical shifts of the NH protons of the L-L-L and L-D-L diastereoisomers of the substrate GlcA-Ile-Leu-Lys-AEC provide a convenient method for assessing racemization during fragment coupling synthesis. All the NH resonance lines of the L-D-L isomer were shifted downfield with regard to those of the L-L-L isomer as can be seen on Fig. 2 and in Table 1. In the present case, change in the configuration of the substrate P, residue induces a conformational modification altering the spatial relationship between most of its protons and the ability to modify the orientation of the substrate in the active site of plasmin. The amounts of L-D-L diastereoisomers formed during the fragment coupling synthesis of the different substrates are reported in Table 2. These results agree with the fact that in polar solvents such as dimethylfor-

B

I I

I

I

8X

,

a2 PPM

FIG. 2.

NMR spectra at 300 MHz of the amido protons of the optically pure L-L-L and L-D-L diastereoisomers of GlcA-Be-Leu-Lys-OH, 5 X 10m3 M in Me,SO-d, at 300 K. (A) Overlapping of the spectra of L-L-L (-) and L-D-L (- - -) pure isomers. (B) Spectrum of a mixture of 95% L-L-L and 5% L-D-L isomers.

HARNOIS-PONTONI,

252 TABLE

MONSIGNY,

1

Proton Magnetic Resonance Data of the L-L-L and L-D-L Isomers of the Substrate GlcA-Ile-Leu-Lys-AEC (5 X 10e3 M) in Me,SO-ds at 300 K from Tetramethylsilane (TMS = 0.00 rwm) Chemical shifts kwm) Protons NH CH-4 a-NH a-NH CH-5 CH-2 CH-1 a-NH CH-8 CH-7 CH-6 OH-2 OH-5 OH-4 OH-3 CH, (u-CH a-CH a-CH CH-2 CH-3 CH-6 CH-4 CH-5 &Hz P-CH, /3-CH &CH, @-CH -y-CH r-CH, CH, -VW &CH, r’-CH, &CH,

AEC AEC LYS Leu AEC AEC AEC Ile AEC AEC AEC GlcA GlcA GlcA GlcA AEC Leu LYS Ile GlcA GlcA GlcA GlcA GlcA LYS LYS Ile LYS Leu Leu LYS AEC Ile Leu Ile Ile

L-L-L

9.85 8.46 7.98 8.04 8.03 7.64 7.54 7.60 7.57 7.43 7.16 5.48 4.64 4.64 4.64 4.41 4.42 4.39 4.19 4.12 3.95 3.56 3.56 3.56 2.77 1.77 1.77 1.58 1.50 1.50 1.39 1.27 1.04 0.87 0.81 0.81

L-D-L

9.86 8.42 8.29 8.24 8.03 7.63 7.66 7.60 7.57 7.43 7.16 5.59 4.56 4.56 4.45 4.40 4.30 4.28 4.40 4.15 3.92 3.47 3.47 3.47 2.75 1.84 1.84 1.56 1.52 1.52 1.45 1.28 0.92 0.73 0.84 0.84

AND MAYER

strongly hydrophobic fluorescent detector (AEC) does not facilitate the hydrosolubilization of these substrates but it mimics aromatic drugs of prodrugs. It can be seen in Table 3 that compounds 9 and 10 are almost insoluble in aqueous medium. The use of a Ngluconoyl group increases 20-fold the solubility of this sequence in water. However, it is not enough to obtain substrate concentrations consistant with the substrate K, value. Five percent DMSO is still necessary with the substrate GlcA-Ile-L-Phe-Lys-AEC but this must be compared to more than 50% DMSO for the N-Boc and N-Z analogues (substrate 8 compared to 9 and 10, respectively). The hydrosolubilizing effect of the N-gluconoyl group is still more obvious for compound 24 which is 1900 times more soluble in water than the N-Z analogue (substrate 25). The stereochemistry of the substrates also has a noticeable effect upon their solubilities. The L-D-L isomer is generally three to five times more soluble in water than the L-L-L isomer. Enzymatic Hydrolysis of the Hydrosoluble Substrates Conventionally (16), the amino acid sequence of proteinases cleavage site is denoted as H,N-P,-P,-P,-P,Pi-Pk-COOH where the bond cleaved is between amino acids P, and PI. Plasmin splits peptide bonds specifically after a basic amino acid. Effect of the nature of the N-terminal residue. Table 4 summarizes the K,,, and k,, values which characterize substrate affinity and reaction velocity respectively, as well as the proteolytic constant k,,IK,,, and the solubility/K,,, ratio. As already shown, it appears that plasmin favors lysine substrates at P, position. It has also been observed (17) that tripeptidyl substrates with a benzyloxycarbonyl or a benzyl N-protecting group are usually better substrates than their analogues with a N-succinyl

Note. s, singlet; d, doublet; t, triplet; m, multiplet.

On the basis of structure-activity relationship data for plasmin substrates, the sequence H-D-Ile-Phe-LyspNA has been predicted by regression analysis of kinetic parameters (2) as 40 times better than H-D-ValLeu-Lys-pNA (S 2251) (13), which is currently used. This was recently confirmed experimentally (14,15), although data on the water solubility of this compound were not given. In these sequences four of the most hydrophobic amino acids are found. This prompted us to synthesize such highly hydrophobic sequence, analogous to these potentially best substrates, and to try to hydrosolubilize them by means of N-acylation with a gluconoyl group (1). In the present work, the use of a

TABLE2 Extent of Racemization, Expressed as the Percentage of and L-L-L Diastereoisomers” upon Fragment Condensation in Dimethylformamide L-D-L

Peptides GlcA-Ile-Leu-Arg-AEC GlcA-Ile-Leu-Lys-AEC GlcA-Ile-Phe-Arg-AEC GlcA-Ile-Phe-Lys-AEC GlcA-Val-Leu-Arg-AEC GlcA-Val-Leu-Lys-AEC GlcA-Val-Phe-Arg-AEC GlcA-Val-Phe-Lys-AEC a Numbers in parentheses Tables 3 and 4.

L-D-L

44 63 54 35 32 47 39 39

(1) (31 (5) (7) (12) (14) (17) (20)

L-L-L

66 (2) 37 (41

46 (6) 65 (8) 68 (13) 53 (15) 61 (18) Sl(21)

are related to the compounds listed in

HYDROSOLIJBLE

FLUOROGENIC

SUBSTRATES TABLE

Characteristics No.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 a The very ’ Solubilities

FOR

3

of the Fluorogenic Peptidic Substrates

Substrates

low solubility of these compounds precludes their were determined in 50 mM Tris-HCl, pH 8.1.

Synthesized l&‘& (c 1)

mp (“Cl

GlcA-Ile-n-Leu-Arg-AEC GlcA-Ile-L-Leu-Arg-AEC GlcA-Ile-D-Leu-Lys-AEC GlcA-Ile-L-Leu-Lys-AEC GlcA-Ile-D-Phe-Arg-AEC GlcA-Ile-L-Phe-Arg-AEC GlcA-Ile-D-Phe-Lys-AEC GlcA-Ile-L-Phe-Lys-AEC Boc-Ile-L-Phe-Lys-AEC” Z-Ile-L-Phe-Lys-AEC” GlcA-Gly-Ile-L-Phe-Lys-AEC GlcA-Val-D-Leu-Arg-AEC GlcA-Val-L-Leu-Arg-AEC GlcA-Val-D-Leu-Lys-AEC GlcA-Val-L-Leu-Lys-AEC Boc-Val-L-Leu-Lys-AEC GlcA-Val-D-Phe-Arg-AEC GlcA-Val-L-Phe-Arg-AEC GlcA-Gly-Val-L-Phe-Arg-AEC GlcA-Val-D-Phe-Lys-AEC GlcA-Val-L-Phe-Lys-AEC GlcA-Gly-Val-L-Phe-Lys-AEC GlcA-Val-Leu-Gly-Arg-AEC GlcA-Gly-L-Pro-Lys-AEC Z-Gly-L-Pro-Lys-AEC

253

PLASMIN

110-111 135-136 120-121 134-135 108-109 115-116 130-131

202-203 227-230 233-236 220-221 114-115 132-133 1044105 123-124 132-133 125-126 140-141 162-164 122-123 195-196 180-185 159-160 181-183 141-143

-27.3 (MeOH) -19.2

(MeOH) -28.4 (MeOH) -34.4 (MeOH) -2.8 (DMF) -4.0 (DMF) -4.7 (DMF) -1.6 (DMF) -2.1 (DMF) - 19.7 (DMSO) -23.0 (MeOH) -39.2 (MeOH) -13.0 (MeOH) -20.8 (MeOH) -30.6 (MeOH) -53.3 (MeOH) -43.5 (MeOH) -18.0 (MeOH) ~21.6 (MeOH) -39.5 (MeOH) -42.0 (MeOH) -5.0 (DMSO) -25.0 (MeOH) -76.1 (MeOH) -26.9 (MeOH)

Solubilityb (mM

at 20’c)

33 7.0 40 17

5.0 4.1 1.0 0.8

0.04 0.03 5.0 94 61 80

68 2.5 65 17

3.6 64 10

4.2 67 380 0.2

use as substrates,

or a free N-terminal L-amino acid. This is still illustrated here for substrates 24 and 25. The contribution of the hydrosolubilizing N-gluconoyl group in the P, position results in a lowering of the catalytic constant of these water-soluble substrates when compared to the rather insoluble N-Z analogue, with a good affinity of the substrate being retained. In order to space this hydrophilic group from the proteolytic cleavage site further, substrates with a glycyl residue in position P, and the gluconoyl group in P, position were synthesized. The addition of this glycyl residue lowers the water solubility of the substrate and decreases its affinity for plasmin but increases its reaction velocity. This can be seen when comparing substrates 21 and 18 with 22 and 19, respectively. Effect of a D amino acid in position P2. As shown in Table 4, the stereochemistry of the amino acid in the P, position markedly affects the kinetic constants of the substrates. The L-L-L substrates are 4- to lo-fold better ligands than their L-D-L analogues and their hydrolysis rate is also slightly higher, suggesting a more favorable interaction of the L-L-L diastereoisomers with the active site H of the enzyme. The L configuration of the amino acid at the P, position is therefore essential for suitable orientation of the substrate in the active site of plasmin.

Comparison of the kinetic constants of substrates 4 and 15,8 and 21,2 and 13, and 6 and 18 shows that the S, hydrophobic crevice of plasmin exhibits a distinct preference for isoleucine upon valine which has only a slightly less bulky side chain. The same preference was observed for the corresponding L-D-L isomers (3 and 14,7 and 20, or 1 and 12). This corroborates the predominant role of the P, residue in serine protease substrates (18). Furthermore, when P, is an isoleucine residue the S, hydrophobic crevice seems to prefer phenylalanine versus leucine, while it is the opposite when P, is a valine residue. This is also true with the L-D-L isomers. Examination of the proteolytic constants shows that only four substrates emerge from the others: Z-GlyPro-Lys-AEC (substrate 25) with a weakly hydrophobic peptidic sequence but a N-Z terminal hydrophobic end, and substrates 4,8, and 15 with a strongly hydrophobic peptidic sequence and a hydrophilic N-gluconoyl terminal group. While the kJK,,, values of these substrates are lower than the values found for human plasmin with substrates such as H-D-Val-Leu-Lys-pNA, HD-Ile-Leu-Lys-pNA, or H-D-Ile-Phe-Lys-pNA (14) it must be noticed that fluorogenic substrates are much more sensitive than chromogenic substrates.

254

HARNOIS-PONTONI,

MONSIGNY, TABLE

Kinetic Constants

of the Hydrosoluble

Substrates

AND

MAYER

4

Measured in 50 mmol Tris-HCI KflI

No.

1 2 3 4 5 6 7 8

11 12 13 14 15 16 17

18 19 20 21 22 23 24 25

Substrates GlcA-Ile-D-Leu-Arg-AEC GlcA-Ile-L-Leu-Arg-AEC GlcA-Ile-D-Leu-Lys-AEC GlcA-Ile-L-Leu-Lys-AEC GlcA-Ile-D-Phe-Arg-AEC GlcA-Ile-L-Phe-Arg-AEC GlcA-Ile-D-Phe-Lys-AEC GlcA-Ile-L-Phe-Lys-AEC GlcA-Gly-Ile-L-Phe-Lys-AEC GlcA-Val-D-Leu-Arg-AEC GlcA-Val-L-Leu-Arg-AEC GlcA-Val-D-Leu-Lys-AEC GlcA-Val-L-Leu-Lys-AEC Boc-Val-L-Leu-Lys-AEC GlcA-Val-D-Phe-Arg-AEC GlcA-Val-L-Phe-Arg-AEC GlcA-Gly-Val-L-Phe-Arg-AEC GlcA-Val-D-Phe-Lys-AEC GlcA-Val-L-Phe-Lys-AEC GlcA-Gly-Val-L-Phe-Lys-AEC GlcA-Val-Leu-Gly-Arg-AEC GlcA-Gly-L-Pro-Lys-AEC Z-Gly-L-Pro-Lys-AEC

1.40 0.40 2.40 0.35 2.50 0.29 1.70 0.18 0.74 1.11 0.30 2.00 0.20 0.68 3.30 0.33 0.72 1.90 0.21 0.71 0.70 0.20 0.16

In order to assessthe sensitivity of the assay using gluconoylated substrates bearing a 3-amino-g-ethylcarbazole fluorogenic reporter with regards to the commonly used 4-nitroanilide substrates, the initial rates of hydrolysis of both GlcA-Val-Leu-Lys-AEC and H-DVal-Leu-Lys-pNA by various concentrations of human plasmin were determined. The minimal concentration of human plasmin allowing quantitative measurements was about 8 ng/mL (100 PM) and 100 rig/ml with the hydrosoluble fluorogenic substrate and with the chromogenic substrate, respectively. Taking into consideration that an optimal hydrolytic sensitivity is obtained when the substrate concentration is at least 10 times higher than the K, value, the best substrates should have a solubility/K, ratio above 10 in order to maintain a saturation of the enzyme binding site above 90%. As usual a good substrate must have a maximal Iz,,lK,. It appears therefore that the best substrates of the series presented here are GlcA-Ile-LeuLys-AEC and GlcA-Val-Leu-Lys-AEC. To optimally detect plasmin activity, these two substrates should be used at about a 3 mM concentration; at this concentration the peptides are readily hydrosoluble. For the choice of a suitable synthetic substrate both its sensitivity and its specificity have to be considered. The susceptibility of the best substrates for plasmin GlcA-Ile-Phe-Lys-AEC, GlcA-Val-Leu-Lys-AEC,

8.1,

at 37°C

LlKm (M-l

(mM)

Sensitivity

Buffer, pH

0.083 0.150 0.160 0.350 0.150 0.145 0.130 0.206 0.079 0.055 0.083 0.100 0.190 0.130 0.052 0.072 0.115 0.072 0.124 0.203 0.126 0.020 0.270

SolubilitylK,

S-l)

59 375 67 1000 60 500 76 1144 107 50 277 50 950 192 16 218 160 38 590 286 180 100 1690

23 17.5 19 43 2 14 0.6 4.4 7 65 156 25 85 4 20 39 5 25 48 6 96 1900 1

GlcA-Ile-Leu-Lys-AEC, and GlcA-Gly-Pro-Lys-AEC was therefore tested with other enzymes such as thrombin and urokinase at pH 8.1. Under the experimental conditions used, no hydrolysis of these substrates was detected with enzyme concentrations up to 50 pug/ml as expected (15). Hydrosolubilization of highly hydrophobic peptidic sequence by a iV-gluconoyl or more generally by a Npolyhydroxyalcanoyl moiety allows one to obtain potent hydrosoluble fluorogenic substrates of plasmin. This general hydrosolubilizing procedure furthermore prevents the substrates from aminopeptidases degradation. The use of N-gluconoyl acylated hydrophobic peptides as nontoxic carriers of cytotoxic drugs will give rise to hydrosoluble selective prodrugs usable in vitro and in Go. Further work along this line is in progress in our laboratory. ACKNOWLEDGMENT We thank spectroscopy.

Mrs.

Anita

Caille

for

her

skillful

assistance

in NMR

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