Assay method of enzyme activity

Novel amine compounds of the formula ##STR1## wherein R.sub.1 is lower alkyl and R.sub.2 is substituted methyl amino or D-amino acid residue, or a salt thereof. R.sub.1 can typically be isobutyl or methyl; and R.sub.2 can be methylamino of the formula EQU --NH--CH.sub.2 --R.sub.3 in which R.sub.3 is organic, more particularly methyl, ethyl, n-propyl, isopropyl, n-butyl, amyl, p-hydroxybenzyl, 3,4-di-hydroxybenzyl, 5-imidazolemethyl, 3-imidazolemethyl or phenyl. R.sub.2 can also be D-amino acid residue of the formula ##STR2## in which one of R.sub.4 and R.sub.5 is carboxyl and the other is organic, more particularly methylthioethyl, isobutyl, methyl or phenyl, and C* is a D-asymmetric carbon atom. The compounds are useful in an assay method for leucine and aminopeptidase which comprises reacting a substrate amide as above, or a salt thereof, with a sample for leucine aminopeptidase assay, incubating the reaction product produced thereby, and measuring the thus-consumed oxygen or liberated hydrogen peroxide.

This invention relates to assay methods of enzyme activity, using as a 
substrate a compound of the formula 
##STR3## 
wherein R.sub.1 is lower alkyl and R.sub.2 is substituted methylamino or 
D-amino acid residue, or a salt thereof. 
Leucine aminopeptidase [.alpha.-aminoacyl-peptide hydrolase (cytosol), EC 
3. 4. 11. 1., hereinafter designated LAP; formerly L-leucylpeptide 
hydrolase, EC 3. 4. 1.1] is distributed in tissues and serum, and is known 
to increase under disease conditions. LAP assay is used for the clinical 
diagnosis of various disease and the observation of prognosis. 
The analytical unit of LAP is defined as a G-R unit (Goldbarg Rutenburg). 
The G-R unit=2.72.times.LAP international unit (mU/ml). [Cancer. 11, 283 
(1958)]. 
A number of assay methods for LAP are known. However, each known method has 
disadvantages. Conventional assay methods are the colorimetry of amine 
compounds produced by enzymatic action on a synthetic substrate consisting 
of L-leucine and an amine compound. When a synthetic substrate such as 
L-leucine-p-nitroanilide is incubated with LAP, a wave length of the 
yellow color of p-nitroaniline formed by the enzymatic action is 
overlapped upon colorimetric assay, and moreover the serum composition, 
especially bilirubin pigment, affects the colorimetry. Furthermore, when 
L-leucine-.beta.-naphthyl-amide is used as substrate, the color formed is 
colorimetrically measured, for example, by coupling the formed 
.beta.-naphthylamine with 5-nitro-2-amino-methoxybenzene diazotate; 
coupling the diazotated .beta.-naphthylamine by sodium nitrate with 
N-(1-naphthyl)-ethylenediamine; or condensating the .beta.-naphthylamine 
with p-dimethylaminobenzaldehyde or p-dimethylamino cinnamaldehyde. These 
colorimetric assay methods have a number of disadvantages such as complex 
reaction process and the requirement of strict observance of the operating 
procedures. Moreover the formed .beta.-naphthylamine is high in toxicity 
and is carcinogenic for the bladder. 
Another enzymatic assay method for LAP activity is that in which 
L-leucineamide is used as substrate for LAP and the formed ammonia is 
incubated with .alpha.-ketoglutarate, glutamate dehydrogenase and 
NADH.sub.2 to transform glutamic acid, then the oxidized NADH.sub.2 is 
spectrophotometrically measured. When the substrate L-leucyl-L-alanine is 
used, L-alanine liberated by LAP is incubated with .alpha.-ketoglutarate 
and glutamic-pyruvic transaminase to form pyruvic acid which is converted 
to lactic acid by lactate dehydrogenase, wherein consumed NADH.sub.2 is 
subjected to spectrophotometric assay. 
A further assay method using L-leucine dehydrogenase is known. L-leucine 
liberated from L-leucyl-glycine by LAP is incubated with L-leucine 
dehydrogenase, then NADH.sub.2 is spectrophotometrically measured. 
(Japanese Patent Open No. 54-119290). Furthermore, L-leucinamide is used 
as substrate and the formed L-leucine is incubated with L-amino acid 
oxidase to liberate hydrogen peroxide which is subjected to colorimetric 
assay. [Pharmacia, 14, 872 (1978)]. 
These prior known enzymatic assay methods have a number of disadvantages. 
For example, the enzymatic reaction system is complicated, and bilirubin 
pigment or emulsified serum affects the colorimetric assay. In the assay 
method using L-amino acid oxidase, L-amino acid in the blood inhibits the 
assay of L-leucine liberated by LAP; moreover, the amount of L-amino acid 
in the blood is not constant, which causes difficulties during assay. 
We have found that LAP hydrolyzes the substrates synthesized from 
substituted methylamine, which does not exist in the blood, such as 
benzylamine, tyramine or butylamine, or D-amino acid such as D-methionine 
or D-leucine, and L-leucine or L-alanine to liberate with good yield a 
substituted methylamine such as tyramine or D-amino acid. The liberated 
substituted methylamine or D-amino acid is oxidized by the corresponding 
oxidase and the amount of consumed oxygen or liberated hydrogen peroxide 
is measured, thereby assaying the activity of the LAP. Further, we have 
found a novel useful synthetic substrate amide compound of the formula [I] 
##STR4## 
wherein R.sub.1 and R.sub.2 have the same meanings as hereinbefore. 
Embodiments of this synthetic substrate are amide compounds of the formula 
[I] which are synthesized from L-leucine or L-alanine and substituted 
methylamine or D-amino acid, wherein R.sub.1 is lower alkyl such as 
isobutyl or methyl, and R.sub.2 is substituted methylamino of the formula 
[II] 
EQU --NH--CH.sub.2 --R.sub.3 [II] 
wherein R.sub.3 is an organic group, or R.sub.2 is a D-amino acid residue 
of the formula [III] 
##STR5## 
wherein one of R.sub.4 and R.sub.5 is carboxyl, the other is an organic 
residue, and C* is D-asymmetric carbon. Examples of the organic group 
R.sub.3 are methyl, ethyl, n-propyl, isopropyl, n-butyl, amyl, 
p-hydroxybenzyl, 3,4-dihydroxybenzyl, 5-imidazolmethyl, 3-indolmethyl and 
phenyl. Examples of the groups R.sub.4 and R.sub.5 are that one of R.sub.4 
and R.sub.5 is carboxyl and the other is methylthioethyl, isobutyl, methyl 
or phenyl. Examples of substituted methylamine or D-amino acid are, for 
example, ethylamine, n-propylamine, n-butylamine, iso-butylamine, 
n-amylamine, n-hexylamine, tyramine, 3,4-dihydroxyphenylethylamine, 
histamine, tryptamine, benzylamine, D-methionine, D-leucine, D-alanine and 
D-phenylglycine. 
Embodiments of the said substituted amines are compounds consisting of 
L-leucine and substituted methylamine such as L-leucine-benzylamide, 
L-leucine-p-hydroxyphenylethylamide and L-leucine-n-butylamide, a compound 
consisting of L-leucine and D-amino acid such as L-leucyl-D-methionine and 
L-Leucyl-D-leucine, or a compound consisting of L-alanine and substituted 
methylamine such as L-alanine-p-hydroxyphenylethylamide. These substituted 
amines can be used as soluble salts such as hydrochloride, hydrobromide, 
phosphate, formate, acetate, propionate or oxalate. 
An amide compound of the present invention can be produced by a 
conventional peptide synthesis such as protection and removal of the 
protective group and a condensation reaction. 
Examples of the protective group for .alpha.-amino group in L-amino acid, 
such as L-alanine and L-leucine, of the formula [IV] 
##STR6## 
wherein R.sub.1 has the same meanings as hereinbefore, are conventional 
protective groups such as t-butoxycarbonyl, t-amyloxycarbonyl, 
adamantyloxycarbonyl, benzyloxycarbonyl, o-nitrophenylthio or 
nitro-substituted benzyloxycarbonyl. The carboxyl group can be transformed 
to an activated form such as acid azide, mixed anhydride, acid imidazolide 
or activated ester, for example cyanomethyl ester, p-nitrophenyl ester, 
2,4-dinitrophenyl ester, N-hydroxysuccinimide ester or 
N-hydroxyphthalimide ester, or activated by using carbodiimide, 
N,N'-carbonyldiimidazole or an isoxazolium salt such as Woodward reagent. 
These activated forms of the L-amino acid are reacted with a substituted 
methylamine compound or D-amino acid by a condensation reaction such as 
the carbodiimide method, activated ester method or acid anhydride method. 
The reaction is carried out in an inert solvent such as dimethylformamide, 
dimethylacetamide, dimethylsulfoxide or tetrahydrofurane, with an 
equi-molar ratio of the compound, at -30.degree. C. to ambient temperature 
with stirring. The reaction can be terminated after 5 to 50 hours. 
Thereafter, the protective group can be removed. The t-butoxycarbonyl 
group can be removed by trifluoroacetic acid, and the benzyloxycarbonyl 
group is removed by catalytic reduction with palladium-carbon. The product 
can be purified by extraction, washing, chromatography or crystallization. 
The thus-obtained product is transformed to its salt, for example an 
inorganic salt such as hydrochloride, hydrobromide or phosphate, or an 
organic acid salt such as formate, acetate, propionate or oxalate. 
Oxidase corresponding to the substitute methylamine and D-amino acid 
liberated by the action of LAP on the above synthetic substrate of the 
present invention is at least an enzyme which hydrolyzes these substituted 
methylamines or D-amino acids as substrate to consume oxygen and liberate 
hydrogen peroxide in an enzymatic reaction. Examples of the enzyme for 
substituted methylamine are an amine oxidase such as monoamine oxidase, 
diamine oxidase or polyamine oxidase; and the enzyme for D-amino acid is 
D-amino acid oxidase. Monoamine oxidase is an enzyme obtained from porcine 
or bovine serum and Aspergillus niger; tyramine oxidase is an enzyme of 
Sarcina lutea IAM 1099 [Biochem. Biophys. Res. Commn., 27, 350 (1967), 
Methods in Enzymology, 17, 722 (1971)]; and D-amino acid oxidase is an 
enzyme from animal tissues or Trigonopsis variavillis. 
These oxidases may be used in an immobilized form. The immobilized enzyme 
can be assembled in an automatic analyzer, and is used in combination with 
an oxygen electrode or a hydrogen peroxide electrode. The immobilized form 
has advantages for reducing the amount of valuable and expensive enzymes. 
The sensor comprising the combination of the immobilized enzyme of enzyme 
electrode and the above electrodes can be used for rapid and multiple 
measuring without various reagents. The sensor can also be advantageously 
used in colored samples for assaying LAP activity. 
The immobilized enzyme can be prepared by known immobilization techniques, 
for example entrapping with acrylamide, cross-linking with proteins by 
mixing with albumin, entrapping with collagen and fibroin or covalently 
bonding therewith, adsorption or covalent bonding with a porous organic 
polymer or entrapping with photoresist. These immobilized enzymes are 
processed for membrane, fibrous forms, pellets or tubes suitable for 
enzyme electrodes. 
An embodiment of the LAP activity assay is as follows: 
An aliquot portion of synthetic substrate solution is incubated with a LAP 
assay sample such as serum in a buffer. Incubation is carried out at 
37.degree. C. Incubation time need not be limited but is preferably the 
time necessary for liberating substituted amine or D-amino acid from the 
synthetic substrate by LAP. The thus-liberated substituted methylamine or 
D-amino acid is oxidized by the corresponding oxidase to consume oxygen or 
to liberate hydrogen peroxide. The reaction is effected by adding the 
corresponding oxidase solution or contacting the reaction mixture with 
immobilized oxidase at 37.degree. C. The thus-consumed oxygen or liberated 
hydrogen peroxide is preferably measured by an oxygen electrode or a 
hydrogen peroxide electrode. This assay is advantageously performed with 
the enzyme electrode comprising the combination of the immobilized enzyme 
and the electrode. The output is recorded or displayed as electric 
consumption to convert LAP activity. The amount of hydrogen peroxide is 
conventionally measured by a coloration reagent consisting of 
4-amino-antipyrine, phenol and peroxidase, or a luminescent reagent such 
as luminol. 
An embodiment of an LAP activity assay method is a reaction-detection 
method comprising injecting LAP-activity-measuring reagent, substrate 
solution and buffer into an LAP reactor vessel in which the substituted 
methylamine or D-amino acid is liberated from the synthetic substrate, 
oxidizing the thus-formed substituted methylamine or D-amino acid by the 
corresponding oxidase, and detecting the amount of oxygen consumed or 
hydrogen peroxide liberated. 
In the reaction-detector system, the immobilized enzyme column part of 
oxidase and the detection electrode part may preferably be separated, or 
unitarily constructed as an enzyme electrode wherein the immobilized 
enzyme is attached to the detector of the electrode. The assay system can 
be a multiple reactor-detector system in which, for example, sampling is 
taken from multiple LAP reactors to inject the reaction-detector vessel, 
followed by detections and washings. 
In the examples, an assay system using an oxygen electrode is given, by way 
of example. However, in this invention, a hydrogen peroxide electrode can 
also be advantageously used. 
The LAP assay method of the present invention is quite simple, rapid and 
reproducible, and is quite useful for clinical diagnosis.

The abbreviations in the following description have the following 
meansings: 
Boc: t-butoxycarbonyl 
HOSu: N-hydroxysuccinimide 
OSu: N-hydroxysuccinimide ester 
DCC: N,N'-dicyclohexylcarbodiimide 
THF: tetrahydrofuran 
DCU: N,N'-dicyclohexylurea 
DMF: dimethylformamide 
NMM: N-methylmorpholine 
TFA: trifluoroacetic acid 
Z: benzyloxycarbonyl 
The following examples illustrate the present invention but are not to be 
construed as limiting: 
EXAMPLE 1 
L-Leu-NH-CH.sub.2 -C.sub.6 H.sub.5 (L-leucine-benzylamide) 
Boc-Leu-OSu (3.28 g, 10 mM) and benzylamine (1.07 g, 10 mM) dissolved in 
DMF (30 ml) was adjusted to pH 7 by adding NMM, and the mixture was 
stirred overnight at room temperature. DMF was distilled off and the 
residue was dissolved in ethyl acetate. The solution was washed three 
times with 5% w/w sodium bicarbonate solution, twice with 1 N-HCl and 
twice with water, dehydrated with sodium sulfate, then ethyl acetate was 
distilled off. The residue was charged on a column of silica gel (100 g) 
and eluted with benzene-ethyl acetate (1:1) to obtain Boc-Leu-NH-CH.sub.2 
-C.sub.6 H.sub.5 (1.3 g). 
This compound (1.2 g) dissolved in TFA (3 ml) was stirred for 30 minutes. 
After TFA was distilled off, the residue was dissolved in 0.1 N acetic 
acid. The solution was charged on a column of Sephadex LH-20 
(97.0.times.3.0 cm) and fractionated into fractions of 6.3 ml each. 
Fractions Nos. 28-36 were collected and lyophilized to obtain 
L-leucine-benzylamide acetate (900 mg). 
Yield: 32.1% (acetate) 
Molecular formula: C.sub.13 H.sub.20 N.sub.2 O.CH.sub.3 COOH 
Elementary analysis: 
______________________________________ 
C % H % N % 
______________________________________ 
Calculated: 64.26 8.63 9.99 
Found: 64.22 8.68 10.01 
______________________________________ 
Rf value: silica gel plate (n-butanol:acetic acid:water=3:1:1), Rf=0.80 
IR (Nujol): 1690, 1620 cm.sup.-1 (--CO--NH--) 
EXAMPLE 2 
L-Leu-D-Met-OH (L-leucyl-D-methionine) 
D-Met-OH (1.4 g, 10 mM) and sodium bicarbonate (1.68 g, 20 mM) were added 
to a mixture of water (15 ml) and DMF (5 ml). Boc-Leu-OSu (3.28 g, 10 mM) 
in DMF (30 ml) was added thereto and the mixture was stirred overnight at 
room temperature. After cooling to 0.degree. C., the pH was adjusted to pH 
6.5 with 1 N-HCl and the solution was concentrated in vacuo. The residue 
was dissolved in ethyl acetate-1 N-HCl (50 ml-50 ml). The ethyl acetate 
layer was washed with water, dried by adding sodium sulfate and ethyl 
acetate was distilled off. The residue was charged on a column of silica 
gel (100 g) and eluted with benzene-ethyl acetate (1:1) to obtain 
Boc-Leu-D-Met-OH (1.6 g). 
This product (1.5 g) was dissolved in TFA (4 ml) and stirred at room 
temperature for 30 minutes. After TFA was distilled off, the residue was 
dissolved in 0.1 N acetic acid and charged on a column of Sephadex LH-20 
(97.0.times.3.0 cm) to produce fractions of 6.3 ml each. Fractions Nos. 
28-36 were collected and lyophilized to yield L-leucyl-D-methionine 
acetate (1.3 g). 
Yield: 40.4% 
Molecular formula: C.sub.11 H.sub.22 N.sub.2 SO.sub.3.CH.sub.3 COOH 
Elementary analysis: 
______________________________________ 
C % H % N % S % 
______________________________________ 
Calculated: 48.43 8.13 8.69 9.94 
Found: 48.40 8.15 8.65 9.95 
______________________________________ 
Rf value: silica gel plate (n-butanol:acetic acid:water=3:1:1), Rf=0.50 
IR (Nujol): 1687, 1620 cm.sup.-1 (--CO--NH--) 
EXAMPLE 3 
L-Leuc-NH-CH.sub.2 -CH.sub.2 -C.sub.6 H.sub.5 -OH 
(L-leucine-p-hydroxyphenylethylamide) 
Boc-Leu-OSu (3.28 g, 10 mM) and tyramine (1.37 g, 10 mM) dissolved in DMF 
(30 ml) were adjusted to pH 7 with NMM at 0.degree. C. and the mixture was 
stirred overnight at room temperature. After DMF was distilled off, the 
residue was dissolved in ethyl acetate (50 ml), washed three times with 5% 
w/w sodium bicarbonate solution, twice with 1 N-HCl and twice with water, 
and then dried by adding sodium sulfate. After the ethyl acetate was 
distilled off, the residue was charged on a column of silica-gel (100 g), 
and eluted with benzene-ethyl acetate (1:1) to obtain Boc-Leu-NH-CH.sub.2 
-CH.sub.2 -C.sub.6 H.sub.4 -OH (1.58 g). 
This compound (1.4 g) was dissolved in TFA (5 ml), stirred for 30 minutes 
at room temperature and TFA was distilled off. The residue dissolved in 
0.1 N acetic acid was charged on a column of Sephadex LH-20 
(97.0.times.3.0 cm) to produce fractions of 6.3 ml each. Fractions Nos. 
30-38 were collected and lyophilized to yield 
L-leucine-p-hydroxyphenylethylamide acetate (1.25 g). 
Yield: 40.3% (acetate) 
Molecular formula: C.sub.14 H.sub.22 N.sub.2 O.sub.2.CH.sub.3 COOH 
Elementary analysis: 
______________________________________ 
C % H % N % 
______________________________________ 
Calculated: 61.92 8.44 9.03 
Found: 61.90 8.45 9.02 
______________________________________ 
Rf value: silica gel plate (n-butanol:acetic acid:water=3:1:1), Rf=0.75 
IR (Nujol): 1685, 1615 cm.sup.-1 (--CO--NH--) 
EXAMPLE 4 
L-Leu-NH-(CH.sub.2).sub.3 -CH.sub.3 (L-leucine-n-butylamide): 
Boc-Leu-OH (2.5 g, 10 mM), n-butylamine (1 ml, 1 mM) and 
1-hydroxybenztriazole (2.7 g) were dissolved in THF (25 ml) and cooled in 
an ice bath. Water-soluble carbodiimide (1.83 ml, 1 mM) was added thereto 
and the mixture was stirred overnight. After THF was distilled off, 
residue was dissolved in ethyl acetate (50 ml), which was washed three 
times with 5% w/w sodium bicarbonate solution, twice with 1 N-HCl and 
twice with water, then dried with sodium sulfate and ethyl acetate was 
distilled off. The residue was charged on a column of silica gel (100 g) 
and eluted with benzene-ethyl acetate (1:1) to obtain 
Boc-Leu-NH-(CH.sub.2).sub.3 (1.4 g). 
This compound (1.2 g) was dissolved in TFA (3 ml), stirred for 30 minutes 
at room temperature then TFA was distilled off. The residue dissolved in 
0.1 N acetic acid was charged on a column of Sephadex LH-20 
(97.0.times.3.0 cm) and fractionated to produce fractions of 6.3 ml each. 
Fractions Nos. 30-35 were collected and lyophilized to obtain 
L-leucine-n-butylamide acetate (800 mg). 
Yield: 32.5% (acetate) 
Molecular formula: C.sub.10 H.sub.22 N.sub.2 O.CH.sub.3 COOH 
Elementary analysis: 
______________________________________ 
C % H % N % 
______________________________________ 
Calculated: 58.5 10.6 11.4 
Found: 58.4 10.58 11.4 
______________________________________ 
Rf value: silica gel plate (n-butanol:acetic acid:water=3:1:1), Rf=0.52 
IR (Nujol): 1690, 1620 cm.sup.-1 (--CO--NH--) 
EXAMPLE 5 
L-leucine-p-hydroxyphenylethylamide 
Boc-Leu-OH.H.sub.2 O (49.9 g, 0.2 M) and HOSu (23.0 g, 0.2 M) were 
dissolved in THF (300 ml) in a 1 lit. round flask. DCC (41.3 g, 0.2 M) in 
THF (200 ml) solution was added dropwise at -10.degree. C. for about 10 
minutes and the mixture was stirred at room temperature overnight. 
Precipitated DCU was filtered off and the filtrate was concentrated. The 
residue was dissolved in ethyl acetate (300 ml), washed with 1 N-HCl, NaCl 
solution and a small amount of water and dried with anhydrous sodium 
sulfate. After removal of the drying agent, the solution was concentrated, 
and n-hexane was added thereto to obtain Boc-Leu-OSu as a colorless powder 
(m.p. 106.degree.-110.degree. C., 59.11 g, yield: 90%). 
To tyramine (1.51 g, 11 mM) dissolved in DMF (15 ml) was added Boc-Leu-OSu 
(3.28 g, 10 mM) and DMF (5 ml), neutralized by adding NMM, and stirred 
overnight. DMF was distilled off in vacuo and ethyl acetate (100 ml) was 
added to the residue, which was then washed with 5% w/w sodium bicarbonate 
solution, NaCl solution, 1 N-HCl, NaCl solution and a small amount of 
water, and dried with anhydrous sodium sulfate. The drying agent was 
removed, and the solution was concentrated. Then n-hexane was added to the 
residue to obtain a precipitated powder (3.01 g). After drying, the powder 
was dissolved in dioxane (5 ml), and then 4.3 N-HCl/dioxane (dioxane 
absorbed with anhydrous hydrogen chloride) (5 ml) was added at 5.degree. 
C. thereto, and the mixture was stirred for 2 hours at room temperature. 
Hydrogen chloride and dioxane were distilled off in vacuo. Then n-hexane 
was added to the oily residue at 0.degree. C. to obtain the precipitate. 
The precipitate was dried in vacuo to yield 
L-leucine-p-hydroxyphenylethylamide.HCl powder (2.23 g). 
Yield: 78.0% (from Boc-Leu-OSu) 
Molecular formula: C.sub.14 H.sub.22 N.sub.2 O.sub.2.HCl 
Elementary analysis: 
______________________________________ 
C % H % N % 
______________________________________ 
Calculated: 58.63 8.08 9.77 
Found: 58.35 8.23 9.50 
______________________________________ 
m.p.: 125.degree.-130.degree. C. 
IR spectrum: FIG. 8 
TLC: 
silica gel plate (merck, No. 5715) 
developer: (n-butanol:pyridine:glacial acetic acid:water=15:10:3:12) 
Rf=0.70 
EXAMPLE 6 
L-leucine-p-hydroxyphenylethylamide 
Ethyl chloroformate (0.94 ml, 10 mM) was added to Z-Leu-OH (2.7 g, 10 mM) 
and NMM (1.0 g, 10 mM) dissolved in THF (30 ml) at -20.degree. C., and 
stirred at -15.degree. C. for 2 minutes. Tyramine (1.4 g, 10 mM) in DMF 
(20 ml) was added thereto and the mixture was stirred at room temperature 
overnight. The solvent was removed in vacuo, ethyl acetate (100 ml) was 
added thereto, and the mixture was washed with 10% citrate solution, 5% 
w/w aqueous sodium bicarbonate and water, and dried with anhydrous sodium 
sulfate. After removal of the drying agent, n-hexane was added to the oily 
residue. The precipitate was recrystallized from ethyl acetate-n-hexane to 
yield a white powder (3.0 g, 78%). This powder was dissolved in 50% v/v 
aqueous ethanol (600 ml), 5% w/w palladium carbon (600 ml) and aqueous 
N-HCl (5 ml) were added, and hydrogen gas was introduced with stirring at 
room temperature. After the generation of carbon dioxide gas stopped, the 
catalyst was removed and the filtrate was concentrated. The colorless oily 
product was dried in vacuo to yield 
L-leucine-p-hydroxyphenylethylamide.HCl (1.8 g, 62.7%). 
EXAMPLE 7 
L-leucine-p-hydroxyphenylethylamide 
Boc-Leu-OSu (3.28 g, 10 mM) and tyramine (1.37 g, 10 mM) were dissolved in 
DMF (30 ml), adjusted to pH 7 by adding NMM and the mixture was stirred at 
room temperature overnight. After DMF was removed, the residue was 
dissolved in ethyl acetate (50 ml) and washed three times with aqueous 5% 
w/w sodium bicarbonate, twice with 1 N-HCl, twice with water and sodium 
sulfate. Ethyl acetate was removed and the residue was charged on a column 
of silica gel (100 g), then eluted with benzene-ethyl acetate (1:1) to 
obtain Boc-leu-NHCH.sub.2 -CH.sub.2 -C.sub.6 H.sub.4 -OH (1.58 g). This 
compound (1.4 g) was dissolved in trifluoroacetic acid (5 ml), stirred for 
30 minutes at room temperature and the trifluoroacetic acid was removed. 
The residue was dissolved in 0.1 N acetic acid and charged on a column of 
Sephadex LH-20 (97.0.times.3.0 cm), then fractionated to fractions of 6.3 
ml each. Fractions Nos. 30-38 were collected and lyophilized to yield 
L-leucine-p-hydroxyphenylethylamide acetate [1.25 g, yield: 40.3%, 
molecular formula: C.sub.14 H.sub.22 N.sub.2 O.sub.2.CH.sub.3 COOH, TLC: 
(silica gel plate, n-butanol:acetic acid:water=3:1:1), Rf=0.75]. 
EXAMPLE 8 
L-alanine-p-hydroxyphenylethylamide 
Boc-Ala-OH (37.8 g, 0.2 M) and HOSu (23.0 g, 0.2 M) were dissolved in THF 
(300 ml) in a 1 lit. round flask. DCC (41.3 g, 0.2 M) in THF (200 ml) 
solution was added dropwise at -10.degree. C. for about 10 minutes and the 
mixture was stirred at room temperature overnight. Precipitated DCU was 
filtered off and the filtrate was concentrated. The residue was dissolved 
in ethyl acetate (300 ml), washed with 1 N-HCl, NaCl solution and a small 
amount of water and dried with anhydrous sodium sulfate. After removal of 
the drying agent, the solution was concentrated, and n-hexane was added 
thereto to obtain a colorless powder (52.7 gm, m.p. 
140.degree.-143.degree. C., yield: 91%). 
Boc-Ala-OSu (2.86 g, 10 mM) and DMF (5 ml) were added to tyramine (1.51 g, 
11 mM) dissolved in DMF (20 ml); and after neutralizing by adding NMM, the 
mixture was stirred overnight. DMF was distilled off in vacuo and ethyl 
acetate (100 ml) was added to the residue, which was then washed with 5% 
w/w sodium bicarbonate solution, NaCl solution, 1 N-HCl, NaCl solution and 
a small amount of water, and dried with anhydrous sodium sulfate. The 
drying agent was removed, and the solution was concentrated. n-Hexane was 
added to the oily residue to obtain a powder (2.55 g). 
This latter powder was dissolved in dioxane (5 ml), and 4.3 N-HCl/dioxane 
solution (5 ml) was added thereto at 5.degree. C., and then the mixture 
was stirred at room temperature for 2 hours. Hydrogen chloride and dioxane 
were distilled off in vacuo. n-Hexane was added to the oily residue at 
0.degree. C. to obtain a precipitate. The precipitate was dried in vacuo 
to yield a powder of L-alanine-p-hydroxyphenylethylamide.HCl (1.81 g). 
Yield: 74.0% (from Boc-Ala-OSu) 
Molecular formula: C.sub.11 H.sub.16 N.sub.2 O.sub.2.HCl 
Elementary analysis: 
______________________________________ 
C % H % N % 
______________________________________ 
Calculated: 53.99 6.95 11.45 
Found: 53.78 7.11 11.23 
______________________________________ 
m.p.: 105.degree.-110.degree. C. 
IR spectrum: FIG. 9 
TLC: 
silica gel plate (Merck, No. 5715) 
developer (n-butanol:pyridine:glacial acetic acid:water=15:10:3:12) 
Rf=0.64 
EXAMPLE 9 
L-alanine-p-hydroxyphenylethylamide 
Ethyl chloroformate (0.94 ml) was added to Z-Ala-OH (2.2 g, 10 mM) and NMM 
(1.0 g, 10 mM) in THF at -20.degree. C. and the mixture was stirred at 
-15.degree. C. for 2 minutes. Tyramine (1.4 g, 10 mM) in DMF (20 ml) was 
added thereto and the mixture was stirred at room temperature overnight. 
THF was distilled off and ethyl acetate (100 ml) was added. The solution 
was washed with 10% w/w aqueous citrate, 5% w/w aqueous sodium bicarbonate 
and a small amount of water and dried with anhydrous sodium sulfate. After 
the drying agent was removed, n-hexane was added to the oily residue 
obtained from the concentrated solution. The precipitate was 
recrystallized from ethyl acetate-n-hexane to obtain a white powder. This 
powder was dissolved in 50% aqueous ethanol (600 ml), 5% palladium carbon 
(600 mg) and N-HCl (5 ml) were added, and hydrogen gas was introduced with 
stirring at room temperature. After the generation of carbon dioxide has 
terminated, the catalyst was removed and the filtrate was concentrated and 
dried in vacuo to yield L-alanine-p-hydroxyphenylethylamide.HCl (1.1 g, 
46.1%). 
EXAMPLE 10 
LAP activity assay using L-leucine-benzylamide and amine oxidase 
A phosphate buffer solution (pH 7.0, 1 ml) of L-leucine-benzylamide (50 mM) 
was introduced into a reaction vessel provided with an oxygen electrode. 
LAP sample (50 .mu.l, 50 U/ml, 100 U/ml, 150 U/ml, 200 U/ml and 250 U/ml) 
(Boehringer G.m.b.H.) was added thereto, and the mixture was incubated at 
37.degree. C. for 15 minutes with stirring. Amine oxidase (2 U/ml, Miles 
Lab., 50 .mu.l) was added and the mixture was incubated for one minute at 
37.degree. C. The benzylamine formed by the LAP was oxidized and the 
amount of consumed oxygen gas measured with an oxygen electrode as an 
electric current variation. 
The result is shown in FIG. 1 in which the variation of electric current is 
in proportion to LAP activity. LAP activity can thus be determined by 
measuring electric current variation. 
EXAMPLE 11 
LAP activity assay using L-leucine-p-hydroxyphenylethylamide and tyramine 
oxidase 
The substrate solution and enzyme in Example 10 were replaced by a 0.1 M 
phosphate buffer solution (pH 7.0, 1 ml) of 
L-leucine-p-hydroxyphenylethylamide (50 mM) and tyramine oxidase (3 U/ml, 
produced by Sarcina lutea IAM 1099, 50 .mu.l), and the process was carried 
out the same as in Example 10 for LAP activity assay. The result is shown 
in FIG. 2, which shows good results. 
EXAMPLE 12 
LAP activity assay using L-leucyl-D-methionine and D-amino acid oxidase 
The substrate solution and enzyme of Example 10 were replaced by a 0.1 M 
phosphate buffer solution (pH 7.0, 1 ml) of L-leucyl-D-methionine (50 mM) 
and D-amino acid oxidase (11.5 U/ml, Boehinger G.m.b.H., 10 .mu.l), and 
the process was carried out the same as in Example 10 for LAP activity 
assay. The result is shown in FIG. 3. 
EXAMPLE 13 
LAP activity assay using L-leucine-n-butylamide and amine oxidase 
FIG. 4 illustrates the flow diagram for a LAP activity assay system. 
The LAP activity assay sample added through sample injector 1 and substrate 
solution 2 are introduced into LAP reactor vessel 3. The sample is 
introduced by using a micropipette or an autosampler and the substrate 
solution is added by constant volume pump 4. After incubation, the 
reaction mixture is transferred to immobilized enzyme column 5 wherein 
simultaneously the buffer solution in buffer vessel 6 is supplied through 
constant volume pump 7. 
The solution passed through the immobilized enzyme column is transferred 
into flow cell 9 wherein an electrode 8 such as an oxygen electrode or a 
hydrogen peroxide electrode for measuring consumed oxygen or liberated 
hydrogen peroxide by enzymatic action is provided. These are kept at 
constant temperature in constant temperature vessel 10. The electric 
variation detected by electrode 8 is recorded in recorder 12, digital 
meter 13 or digital recorder 14 through amplifier 11). 
In the above flow diagram, the following were used: 
Substrate solution: 0.1 M phosphate buffer (pH 7.0) of 
L-leucine-n-butylamide (50 mM); LAP activity assay sample: LAP solutions 
of various concentrations (50 U/ml, 100 U/ml, 150 U/ml, 200 U/ml and 250 
U/ml); immobilized enzyme column: 2.8.times.30 mm containing 100 mg of 
immobilized amine oxidase (15 U/g carrier), covalently bonded with amine 
oxidase and porous polymer (carrier: polyacrylnitrile, cross-linking 
reagent: glutaraldehyde, British Patent No. 2,015,001); and flow-cell: 0.1 
ml inner volume, provided with an oxygen electrode. 
A LAP sample (10 .mu.l) was added to the substrate solution (50 .mu.l) and 
incubated at 37.degree. C. for 15 minutes. 0.1 M phosphate buffer (pH 7.0) 
flowed at a rate of 1 ml/min. into immobilized enzyme column 5 from buffer 
vessel 6. After the amount of dissolved oxygen detected by oxygen 
electrode 8 became constant in flow cell 9, the incubation mixture (10 
.mu.l) was transferred into the immobilized enzyme column 5. The 
n-butylamine formed by the LAP was oxidized by the immobilized enzyme and 
the amount of dissolved oxygen consumed in the enzymatic reaction was 
measured by oxygen electrode 8 in flow cell 9 as an electric current 
variation which was recorded through amplifier 11. 
The result is shown in FIG. 5 wherein good results were obtained which are 
advantageous for automatic analysis. 
EXAMPLE 14 
LAP activity assay using L-leucyl-D-methionine and D-amino acide oxidase 
LAP samples (50 U/ml, 100 U/ml, 150 U/ml, 200 U/ml and 250 U/ml) (10 .mu.l) 
were added to 0.1 M phosphate buffer solutions (pH 7.0, 50 .mu.l) of 
L-leucyl-D-methionine (50 mM), and incubated at 37.degree. C. for 15 
minutes. The LAP activity was measured by the system shown in FIG. 6. In 
FIG. 6, 15 indicates an injector for the LAP incubation mixture, 16 
indicates a 0.1 M phosphate buffer vessel, 17 indicates a constant volume 
pump which transfers the buffer at a flow rate 1 ml/min., and 18 indicates 
the flow cell. The flow cell consists of a reactor-detector provided with 
oxygen electrode 20 having an immobilized enzyme membrane 19 of D-amino 
acid oxidase (carrier: polyacrylnitrile membrane containing an amino 
group; cross-linking reagent: glutaraldehyde, British Patent No. 
2,015,001) (30 U/g carrier, diameter 5 mm, 0.8 mg, D-amino acid oxidase 
activity 2.4 mU). Electric current variation detected by the oxygen 
electrode was recorded in recorder 22 through amplifier 21. Numeral 23 
indicates the exhaust outlet. 
In the above apparatus, 0.1 M phosphate buffer (pH 7.0) flowed at 1 
ml/min., and after dissolved oxygen has stabilized, the above LAP 
incubation mixture (10 .mu.l) was injected through injector 15. 
D-methionine liberated by LAP action was oxidized by the enzymatic action 
of immobilized enzyme membrane 19 to consume dissolved oxygen which was 
detected by oxygen electrode 20 and the electric current variation was 
recorded. 
The result is shown in FIG. 7, wherein good results were obtained for the 
automatic assay system. 
Furthermore, LAP activity was continuously assayed 100 times using LAP 
activity assay samples (100 U/ml and 200 U/ml). After each respective 
assay, the amount of dissolved oxygen had immediately (one minute after 
finishing the detection) shown the original stable value and each of the 
100 assays revealed the same good results and reproducibility. 
EXAMPLE 15 
Substrate specificity on various synthetic substrates by LAP and 
aminopeptidase 
Synthetic substrate of the following were used: L-leucinamide (50 mM), 
L-leucine-p-nitroanilide (50 mM), L-leucine-.beta.-naphthylamide 
(saturated solution), L-leucine-n-butylamide (50 mM) and 
L-leucine-p-hydroxyphenylethylamide (50 mM). 
A 0.1 M phosphate buffer solution (pH 7.0, 10 .mu.l) containing LAP (300 
U/ml) was added to a 0.1 M phosphate buffer solution (pH 7.0, 50 .mu.l) of 
the above substrate, and the mixture was incubated at 37.degree. C. for 15 
minutes. 
Also aminopeptidase (20 U/ml, 10 .mu.l, Boehringer G.m.b.H.) was used under 
the same conditions. 
The L-leucine liberated by the LAP and aminopeptidase was measured by the 
system shown in FIG. 4 as specified in Example 13, except that L-amino 
acid oxidase was used in place of amine oxidase. A 0.1 M phosphate buffer 
(pH 7.0) was fed at 1 ml/min. by the constant volume pump; and after 
dissolved oxygen in the flow cell reached the stable state, the above 
incubation mixture (10 .mu.l) was injected. A column of immobilized 
L-amino acid oxidase fibers (28 U/g-carrier, 100 mg, 2.8.times.30 mm) was 
used. Consumed oxygen corresponding to the formation of L-leucine by 
L-amino acid oxidase was detected by the oxygen electrode in the flow 
cell, which was amplified to change the electric current variation; and 
the action of LAP and aminopeptidase on various substrates was thus 
assayed. The results are shown in Table 1. 
TABLE 1 
______________________________________ 
LAP Aminopeptidase 
______________________________________ 
L-leucinamide +++ +++ 
L-leucine-p-nitroanilide 
+ +++ 
L-leucine-.beta.-naphthylamide 
+ +++ 
L-leucine-n-butylamide 
+++ + 
L-leucine-p-hydroxy- 
phenylethylamide +++ +.+-. 
______________________________________ 
As shown in Table 1, previously known L-leucinamide, 
N-L-leucyl-p-nitroanilide and L-leucine-.beta.-naphthylamide were 
hydrolyzed not only by LAP but also by aminopeptidase, and hence these 
substrates are less preferable for LAP-activity assay in serum because of 
containing LAP and aminopeptidase. On the contrary, since 
L-leucine-n-butylamide and L-leucine-p-hydroxyphenylethylamide of the 
present invention were hydrolyzed by LAP and almost not hydrolyzed by 
aminopeptidase, these substrates are preferable for serum LAP assay. 
EXAMPLE 16 
LAP activity assay using L-leucine-p-hydroxyphenylethylamide and amine 
oxidase 
A substrate solution (50 mM) was prepared by dissolving 
L-leucine-p-hydroxyphenylethylamide hydrochloride obtained as in Example 5 
in 0.1 M phosphate buffer (pH 7.0). The following solution [I] was 
prepared. 
______________________________________ 
0.2 M Tris-HCl buffer (pH 8.0) 
0.1 ml 
0.3% 4-aminoantipyrine 0.05 ml 
0.2% phenol 0.05 ml 
0.5 mg/ml peroxidase (Sigma Chem. 
Co. Type-I) 0.05 ml 
0.1 M magnesium chloride 0.025 ml 
50 mM substrate solution (L-leucine- 
p-hydroxyphenylethylamide) 
0.025 ml 
distilled water 0.1 ml 
Solution [I] Total 0.4 ml 
______________________________________ 
A reaction composition solution [II] (0.45 ml) was prepared by adding amine 
oxidase (5 U/ml, Miles Lab., 50 .mu.l) in the above solution [I] (0.4 ml). 
Normal rat serum*, parenchymal liver disease model rat serum** and 
extrahepatic cholestasis liver model rat serum***, each respectively 50 
.mu.l, were added to the reaction composition solution [II] (0.45 ml), and 
the mixture was incubated at 37.degree. C. for 30 minutes and 
spectrophotometrically measured at 480 nm. 
FNT *serum of Wister rat, male, weight about 250 g. 
FNT **50% carbon tetrachloride-olive oil (V/V) (1 ml/kg/day) was subcutaneously 
injected twice in a week for 4 weeks into backside of Wister rat, male, 
weight about 250 g. Serum was collected after 48 hours from the final 
administration. 
FNT ***Wister rat, male, weight about 250 g was etherized, and the common bile 
duct was ligated. Serum was collected after 48 hours from ligation. 
Serum of known LAP activity (Seraclea N, 122 G-R units, Nihon Shoji Co.) 
was treated as the same way hereinabove and the LAP activity calculated. 
The result is shown in Table 2. 
In comparison with the above assay method, a known LAP activity assay 
method (Wako Pure Chem. Co., Code 274-41901, substrate: 
L-leucine-p-diethylaminoanilide) was applied to the same serum sample. The 
result is shown in Table 2. 
TABLE 2 
______________________________________ 
Substrate 
L-leucine-p-hydroxy- 
L-leucine-p-diethyl- 
phenylethylamide 
aminoanilide 
Serum sample 
(G-R unit) (G-R unit) 
______________________________________ 
normal rat 331 148 
parenchymal liver 
disease model rat 
2486 237 
extrahepatic 
cholestasis liver 
model rat 379 241 
______________________________________ 
As shown in the table, L-leucine-p-hydroxyphenylethylamide of the present 
invention can be used as an advantageous substrate for the diagnosis of 
parenchymal liver disease. 
EXAMPLE 17 
LAP assay using L-alanine-p-hydroxyphenylethylamide and amine oxidase 
L-alanine-p-hydroxyphenylethylamide hydrochloride obtained as in Example 8 
was dissolved in 0.1 M phosphate buffer (pH 7.0) to prepare a substrate 
solution (50 mM). The substrate solution (0.025 ml) was used in place of 
the substrate solution of Example 16 and solution [I] and reaction 
composition solution [II] were prepared in the same way as in Example 16. 
LAP activity was assayed the same as in Example 16. The result is shown in 
Table 3. 
TABLE 3 
______________________________________ 
Substrate 
L-alanine-p-hydroxyphenylethylamide 
Serum (G-R unit) 
______________________________________ 
normal rat 117 
parenchymal liver disease 
model rat 165 
extrahepatic cholestasis 
liver model rat 
219 
______________________________________ 
As shown in the table, L-alanine-p-hydroxyphenylethylamide of the present 
invention is a preferable synthetic substrate for diagnosis of 
extrahepatic cholestasis of the liver. 
EXAMPLE 18 
______________________________________ 
0.2 M Tris-HCl buffer (pH 8.0) 
0.1 ml 
0.3% 4-aminoantipyridine 
0.05 ml 
0.2% phenol 0.05 ml 
0.05% peroxidase (Sigma Chem. 
Co. Type II) 0.05 ml 
0.1 M magnesium chloride 
0.025 ml 
50 mM L-leucine-p-hydroxy- 
phenylethylamide 0.025 ml 
amine oxidase (7.6 U/ml) 
0.01 ml 
distilled water 0.14 ml 
Total 0.45 ml 
______________________________________ 
A standard serum [Wako Pure Chem. Co., Standard LAP assay serum, LAPC-Test 
Wako (L-leucine-p-diethylaminoanilide substrate method), 50 .mu.l] was 
added to the above reaction composition solution and the mixture was 
incubated at 37.degree. C. for 30 minutes. Ethanol (2.5 ml) was added to 
stop the reaction. A sedimented substance was separated and the 
supernatant solution was colorimetrically measured at 480 nm. The standard 
curve is shown in FIG. 10. A linear relation between LAP activity and 
absorption at 480 nm was observed. The method can be used as a 
quantitative assay method of serum LAP activity using an assay kit. 
Serum LAP activity of human patients was assayed by the above standard 
curve. A correlation coefficient .gamma.=0.984 [sample n=24] and a 
regression equation Y=1.024X-2.898, which are good results, were observed. 
The correlation curve is shown in FIG. 11.