Novel chromogenic enzyme substrates

The present invention relates to novel chromogenic substrates for enzymes 
of the type serine proteases (EC 3.4.21). The substrates according to the 
invention are especially suitable for quantitative determination of the 
above-classified enzymes, which split in the peptide chain on the carboxyl 
side of arginine or lysine. Further the substrates can be used for a study 
of reactions in which the said enzymes are formed, inhibited or consumed, 
or for determination of factors influencing or taking part in such a 
reaction. Synthetical substrates for enzyme-determination have great 
advantages as compared to the natural ones, provided that they fulfil 
certain conditions, such as a great sensitivity for and specificity for 
the enzyme, a good solubility in water or the biological test liquid and 
an easy detectibility of some of the splitting products. 
Excellent substrates for the determination of e.g. plasmin, thrombin, 
trypsin, kallikrein and urokinase are inter alia described in the Swedish 
Pat. No. 380.258 and are in principle chromogenic tripeptide derivatives. 
Among the best substrates of this type are those having a benzoylated 
N-terminal end and a chromophoric group coupled to the C-terminal end, 
e.g.: 
EQU Benzoyl-A.sub.1 -A.sub.2 -A.sub.3 -p-nitroanilide I 
wherein A.sub.1, A.sub.2, A.sub.3 are amino acids. 
With specific amino acid sequences it is possible to among the said 
substrates obtain such with a special sensitivity for a certain or certain 
enzymes. Upon enzymatic hydrolysis the substrates form the chromophoric 
product p-nitroaniline which easily can be determined 
spectrophotometrically. These substrates have, however, a delimitation due 
to their relatively low solubility (.ltoreq. 1 mg/ml). A low solubility 
necessitates work very near the saturation limit for the substrates for 
achieving a satisfactory substrate concentration. In enzyme determinations 
in different biological systems it may thus occur that either the 
substrate per se is precipitated or a combination of protein/substrate. 
The said precipitations cause erroneous spectrophotometer readings and 
thus erroneous enzyme determinations. 
The benzoylated enzyme substrates according to type I become considerably 
more soluble if the N-terminal benzoyl group is replaced with H. The now 
free protonized amino group of the amino acid A.sub.1 increases the 
solubility but causes also, however, that the rate with which the enzyme 
splits the substrates decreases (cf. Table II). Further, the substrates 
can now in a biological test solution is a non-desired way be decomposed 
from the N-terminal end by amino peptidases. 
According to the present invention it has quite unexpectedly been found 
that if in a substrate according to formula I, which is satisfactory from 
the activity point of view for a certain enzyme, exchanges the benzoyl 
group to H and simultaneously replaces the hitherto used L-form of the 
amino acid A.sub.1 (L-A.sub.1) with its D-form (D-A.sub.1) the substrate 
so obtained will be very easily soluble as expected, but the activity of 
the substrate in relation to the enzyme does not decrease by the 
introduction of a D-amino acid but is quite surprisingly several times 
better than that of the corresponding substrate with solely L-amino acids 
and often even considerably better than that of the benzoylated good 
starting substrate according to formula I. The N-terminal free D-amino 
acid in the new substrate also prevents a non-desired attack by amino 
peptidases since the said are specific for L-amino acids. 
The novel chromogenic substrates according to the invention are 
characterized by the following general formula: 
EQU H - D-A.sub.1 - A.sub.2 - A.sub.3 - NH - R 
or salts thereof, wherein A.sub.1 and A.sub.2 are chosen among the amino 
acids Gly, Ala, Val, Leu. Ile, Pip, Pro, Aze, A.sub.2 further can be Phe, 
A.sub.3 is chosen among Arg, Lys and Orn, R is chosen among nitrophenyl, 
naphthyl, nitronaphthyl, metoxynaphthyl, quinolyl and nitroquinolyl (as 
regards abbreviations cf. page 4). 
For the synthesis of the novel chromogenic enzyme substrates conventional 
protective groups and coupling methods are used, all of which are 
well-known within the peptide chemistry. 
As the .alpha.-amino protective group it is of advantage to use 
carbobenzoxy or t-butyloxy carbonyl or some group related thereto such as 
for instance p-metoxy, p-nitro or p-metoxyphenylazo-carbobenzoxy. 
It is of advantage to use protonization, the groups NO.sub.2 or p-toluene 
sulfonyl for protection of the .delta.-guanido group of the arginyl group. 
As protection for the .delta.-amino group in ornithine and for the 
.epsilon.-amino group in lysine it is of advantage to use above all the 
groups carbobenzoxy, t-butyloxy carbonyl or p-toluene sulfonyl. 
As splittable .alpha.-carboxy protective group it is suitable to use 
methyl, ethyl or benzyl ester. 
The coupling between two amino acids or a dipeptide and an amino acid is 
achieved by activation of the .alpha.-carboxy group. The activated 
derivative can either be isolated or generated in situ and can be for 
instance p-nitrophenyl, trichloro phenyl, pentachloro phenyl, N-hydroxy 
succinimide or N-hydroxy benzotriazole ester, symmetric or asymmetric 
anhydride or acid azid. 
The activation to the above-mentioned ester derivative is with advantage 
achieved by the presence of a carbodiimide, e.g. N,N'-dicyclo 
hexylcarbodiimide, which also can serve as activating coupling reagent 
directly between the carboxy and amine components. 
The principle for the substrate synthesis can be stepwise addition of the 
amino acids to the C-terminal arginyl group, which is either from the 
beginning provided with a coupled chromophoric group which then acts as a 
carboxy protective group or provided with a splittable carboxy protective 
group, and the chromophoric group is then coupled to the protected 
tripeptide derivative, or alternatively it is in principle possible to 
choose to synthetize the N-terminal dipeptide fragment per se which 
subsequently is coupled to the arginyl group with or without a 
chromophoric group in principle as discussed above. 
Independent of the principle chosen a purification of the intermediary and 
end products by gel filtration chromatography is suitable since this 
method enables a rapid synthesis work and gives maximal yields.

The invention is described in more detail in the following non-limiting 
specific examples. 
Abbreviations 
Amino acids (if not otherwise stated the L-form is meant): 
______________________________________ 
Arg = Arginine 
Aze = 2-Azetidine carboxylic acid 
Ala = Alanine 
Gly 32 Glycine 
Ile = Isoleucine 
Leu = Leucine 
Lys = Lysine 
Phe = Phenyl alanine 
Pip = Pipecolinic acid 
Pro = Proline 
Val = Valine 
AcOH = Acetic acid 
Bz = Benzoyl 
Cbo-- = Carbobenzoxy-- 
DCCI = Dicyclohexyl carbodiimide 
DMF = Dimethyl formamide 
Et.sub.3 N 
= Triethyl amine 
EtOAc = Ethyl acetate 
GPC = Gel filtration chromatography 
HBT = N-hydroxy benzotriazole 
HMPTA = N,N,N',N',N",N"-hexamethyl phosphoric 
acid triamide 
HONSu = N-hydroxy succinimide 
MeOH = Methanol 
--OpNP = p-nitrophenoxy 
--pNA = p-nitroanilide 
tBoc = t-butyloxy carbonyl 
TFA = Trifluoro acetic acid 
TLC = Thin-layer chromatography 
______________________________________ 
REACTION TYPES USED FOR THE SYNTHESIS 
For synthesis of the novel enzyme substrates enumerated in Table II the 
different reaction steps are performed largely in a similar manner. For 
this reason a general description of the different reaction types is given 
and subsequently, in Table I, a report of intermediary and end products, 
the working-up methods used for different reaction types and certain 
physical data. 
REACTION TYPE 1 
Coupling of the chromophoric group (R) 
20 mmol N.sup..alpha., N.sup.G -protected arginine or N.sup..alpha., 
N.sup..omega. -protected ornithine or lysine or in a corresponding manner 
suitably protected peptide derivative, ground and well-dried, is dissolved 
in 50 ml of dry freshly distilled HMPTA at room temperature, whereupon 20 
mmol Et.sub.3 N and 30 mmol of the chromophoric amine in the form of its 
isocyanate derivative is added under moisture-free conditions and under 
stirring. After one day of reaction time the reaction solution is poured 
down into 0.5 l of 2% sodium bicarbonate solution under stirring. The 
precipitation obtained is removed by filtration and washed well with 
bicarbonate solution, water, 0.5 N hydrochloric acid and water again. From 
the precipitation the desired product is extracted with e.g. methanol, 
certain by-products not being dissolved. The methanol extract can, after 
evaporation, be brought to crystallization from a suitable medium or 
purified by GPC. 
REACTION TYPE 2 
Splitting-off a carbobenzoxy protective group (Cbo-) 
10 mmol of the well-dried Cbo-derivative is slurried in 25 ml of dry AcOH 
and 15 ml of 5.6 N HBr in AcOH are added under moisture-free conditions at 
room temperature. After a reaction time of 45-60 min the solution is fed 
drop by drop into 300 ml of dry ether with vivid agitation. The ether 
phase is sucked from the precipitation obtained which is washed with 2-3 
portions of 100 ml of ether. The so obtained hydrobromide of N.sup..alpha. 
-deblocked compound is dried over NaOH-tablets in vacuum at 40.degree. C. 
for 3-16 h. 
REACTION TYPE 3 
Splitting-off of a t-butyloxy carbonyl protective group (tBoc-) 10 mmol of 
the well-dried tBoc-derivative are dissolved in 200 ml of 25% TFA in 
CH.sub.2 Cl.sub.2 under moisture-free conditions at room temperature. 
After a reaction time of 20 min the solution is fed drop by drop into 500 
ml of dry ether. The precipitation obtained is removed by filtration and 
washed freely with ether. The trifluoro acetate of N.sup..alpha. 
-deblocked compound so obtained is dried over NaOH-tablets under vacuum at 
30.degree. C. for 2-3 h. 
REACTION TYPE 4 
Coupling reactions 
Liberation of the .alpha.-amino group 
For acylation of the derivatives obtained in the reaction types 2 or 3 the 
.alpha.-amino group must be present as a free base. The liberation can be 
performed in many different ways. Inter alia it is possible to add one 
equivalent of a dry tertiary amine (e.g. Et.sub.3 N or N-ethyl morpholine) 
to a DMF-solution of the HBr or TFA derivative cooled down to -10.degree. 
C. In cases comprising Et.sub.3 N and HBr derivatives the precipitated 
Et.sub.3 N.HBr is removed by filtration. Alternatively, the HBr or TFA 
derivative may be dissolved in 5% sodium bicarbonate solution from which 
the liberated derivative is extracted by e.g. EtOAc or butanol, whereupon 
the organic phase is dried and evaporated. 
(a) with N.sup..alpha. -protected active ester derivative 
To a solution of 10 mmol peptide or amino acid derivative liberated 
according to the above, in 20-50 ml of freshly distilled DMF 11 mmol of 
N.sup..alpha. -protected p-nitrophenyl or N-hydroxy succinimide ester 
derivative of the amino acid to be coupled on are added at -10.degree. C. 
After a reaction time of 1 h at -10.degree. C., the solution is buffered 
with 5 mmol of tertiary amine and is then allowed to slowly adjust to room 
temperature. The reaction course is suitably followed by TLC-analysis. If 
required further 5 mmol of base is added after a new cooling. When the 
reaction is finished the solution is evaporated on a rotavapor to an oily 
residue which is stirred with a couple of portions of water. The residue 
is purified by GPC or recrystallization. When GPC is used for purification 
of the coupling product and this has an eluation volume which wholly or 
partly coincides with that for the active ester derivative of the coupled 
amino acid the contamination of the coupling product can be avoided if, 
after finished reaction but before the evaporation, unconsumed active 
ester derivative is replaced with an excess (3-5 mmol) of a primary amine, 
e.g. n-butyl amine, during 30 min at room temperature. Thereafter 
working-up is performed as described above. 
(b) with N.sup..alpha. -protected amino acid or peptide and generation of 
active ester in situ. 
To a solution of 10 mmol of the above-mentioned liberated peptide or amino 
acid derivative in 20-50 ml of freshly distilled DMF 11 mmol of 
N.sup..alpha. -protected amino acid or in a corresponding way protected 
peptide derivative with a C-terminal free carboxy group, 11 mmol of HBT or 
HONSu and 11 mmol of DCCI are added at -10.degree. C. After 1-3 h at 
-10.degree. C. the reaction solution is allowed to adjust to room 
temperature. The reaction course is suitably followed by TLC-analysis. 
After finished reaction the solution is poured under stirring down into 
100-300 ml of 5% NaHCO.sub.3 (aq). 
The precipitation obtained is washed with water after filtration or 
decantation. The residue is purified by GPC or recrystallization. 
REACTION TYPE 5 
Splitting-off of all protecting groups and purification and ion exchange 
0.2-1.0 mmol of the protected peptide derivative with the desired 
chromophoric group is deprotected by reaction with 5-20 ml of dry HF in 
the presence of 0.2-1.0 ml of anisole in an apparatus according to 
Sakakibara, intended for this purpose, during 60 min at 0.degree. C. After 
finished reaction and after all of the HF has been distilled the raw 
product is dissolved in 33% aqueous AcOH and purified by GPC. The product 
is isolated by freeze-drying from diluted AcOH and is submitted to ion 
exchange on a column consisting of a weakly basic ion exchange resin 
Sephadex(.RTM.) QAE-25 in the chloride form, swollen in MeOH:water, 95:5, 
with the same medium as dissolution and eluation medium. The pure product 
is freeze-dried from water. 
Gel filtration chromatography 
By GPC of protected peptide or amino acid derivatives, raw products or 
evaporated mother lyes after crystallization a simplified working-up 
procedure and optimal yields are obtained. The substance is then dissolved 
in MeOH and transferred to a column of a suitable size (volume 0.5-7.5 l, 
length 100 cm), packed with Sephadex(.RTM.) LH-20, swollen in MeOH and 
eluated with the same solvent. The eluate is fractionated in suitable 
partial volumes and its UV-absorption (254 nm) is continually determined. 
Product-containing part fractions are checked for purity by TLC and the 
pure ones are combined and evaporated. 
For purification of peptide derivatives after deprotection with HF 
according to 5 above the 30% AcOH (aq) solution of the raw product is 
transferred to a column of a suitable size (volume 0.5-2.0 l, length 60 
cm), packed with Sephadex(.RTM.) G-15, swollen in 30% aqueous AcOH and 
eluated with the same solvent. After proceeding according to the above the 
product-containing pure part fractions are freeze-dried, optionally after 
a partial evaporation on a rotavapor at 25.degree. C. 
Thin-layer chromatography 
For the TLC-analysis preprepared glass plates with "Kiselgel F.sub.254 " 
(Merck) are used as absorption agents. The solvent systems used (volume 
ratios) are: 
______________________________________ 
A: n-butanol: AcOH: water (3:2:1) 
P.sub.1 : 
Chloroform: MeOH (9:1) 
P1/2: Chloroform: MeOH (19:1) 
______________________________________ 
After finished chromatography the plate is studied in UV-light (254 nm) and 
developed with Cl/o-toluidine reagent according to common practice. The 
stated R.sub.f -values are the results from separate chromatographies. 
Determination of serine proteases by chromogenic substrates 
The substrates prepared according to the examples above are used for 
determination of different enzymes according to the procedure outlined 
above. 
The principle for the determination is based on the fact that the splitting 
product formed by enzymatic hydrolysis has a UV-spectrum which is 
essentially different from that of the substrate. Thus, e.g. all p-nitro 
anilide substrates according to the invention have absorption maxima 
around 310 nm with the molar extinction coefficient of about 12000. At 405 
nm the absorption of these substrates has almost completely discontinued. 
p-Nitroaniline which has been split off from the substrate during the 
enzymatic hydrolysis has an absorption maximum at 380 nm and a molar 
extinction coefficient of 13200, which at 405 nm only has decreased to 
9620. By spectrophotometric determination at 405 nm it is thus easy to 
follow the amount of p-nitroaniline formed which is proportional to the 
degree of the enzymatic hydrolysis which in its turn is determined by the 
active amount of enzyme. Table II shows a comparison of relative reaction 
rates between previously known substrates according to the formula I, 
their non-benzoylated forms and substrates according to the invention. 
This table clearly shows the superiority of the substrates according to 
the invention. 
Accordingly, substrates according to the invention are several times better 
than corresponding substrates, with N-terminal L-amino acid and further at 
least as good as the previously known best substrates which are the 
benzoylated substrates according to formula I. Further, the greater 
solubility of the novel substrates (ca 20-300 times greater) is a very 
great advantage for enzyme determinations above all in biological systems, 
in which the poor solubility of previously known substrates caused 
difficult problems, partly due to the fact that substrate saturation could 
not be achieved and partly due to the risk for undesired precipitations. 
The gel Sephadex(.RTM.) G-15 used for the gel filtration is a crosslinked 
dextran gel. The gel Sephadex(.RTM.) LH-20 is a hydroxypropylated 
crosslinked dextran gel. The ion exchanger Sephadex(.RTM.) QAE-25 used is 
a crosslinked dextran gel with diethyl-(2-hydroxy-propyl)-amino-ethyl as 
functional group. These gels are from Pharmacia Fine Chemicals, Uppsala, 
Sweden. 
Table I 
__________________________________________________________________________ 
Syn- 
the- 
sis 
acc. Cl.sup.- 
to 
content 
reac. 
Yield 
Working- TLC found 
Product No. Starting material 
type 
(%) up [.alpha.] .sub.D.sup.24.sps 
p.x (R.sub.f) 
theor. 
__________________________________________________________________________ 
Cbo-Arg(NO.sub.2)-pNA 
I Cbo-Arg(NO.sub.2)-OH 
p-NO.sub.2 -phenylisocyanate 
1 63 GPC +20.5(D) 
P.sub.1 (0.34) 
tBoc-Lys(Cbo)-pNA 
II tBoc-Lys(Cbo)-pNA 
1 67 Cryst. 
-8.5(M) 
P.sub.1 (0.72) 
p-NO.sub.2 -phenylisocyanate 
(EtOH) 
Cbo-Pro-Arg(NO.sub.2)-pNA 
III I, Cbo-Pro-OpNP 
2, 4a 
96 GPC -33.0(D) 
P.sub.1 (0.28) 
Cbo-D-Val-Pro-Arg(NOhd 2)-pNA 
IV III. Cbo-D-Val-OpNP 
2, 4a 
75 GPC +26.2(D) 
P.sub.1 (0.38) 
Cbo-Pip-Arg(NO.sub.2)-pNA 
V I, Cbo-Pip-OpNP 
2, 4a 
86 GPC -26.2(D) 
P.sub.1 (0.30) 
Cbo-Val-Pip-Arg(NO.sub.2)-pNA 
VI V, Cbo-D-Val-OpNP 
2, 4a 
23 GPC -24.0(D) 
P.sub.1 (0.40) 
Cbo-Leu-Arg(NO.sub.2)-pNA 
VII I, Cbo-Leu-OpNP 
2, 4a 
85 GPC +3.7(D) 
P.sub.1 (0.38) 
-33.4(M) 
Boc-D-Ile-Leu-Arg(NO.sub.2)-pNA 
VIII VII, tBoc-D-Ile-OH 
2, 4b 
94 GPC -2.5(M) 
P.sub.1 (0.38) 
Cbo-D-Val-Leu-Arg(NO.sub.2)-pNA 
IX VII Cbo-D-Val-OpNP 
2, 4a 
64 Cryst. 
+1.7(D) 
P.sub.1 (0.42) 
(MeOH) 
tBoc-Leu-Lys(Cbo)-pNA 
X II, tBoc-Leu-OpnP 
3, 4A 
74 GPC -4.7(D) 
P1/2(0.62) 
tBoc-D-Ile-Leu-Lys(Cbo)-pNA 
XI X, tBoc-D-Ile-OH 
3, 4b 
77 GPC+ -27.1(M) 
P.sub.1 (0.70) 
+Cryst. 
(MeOH) 
Cbo-D-Val-Leu-Lys(Cbo)-pNA 
XII X, Cbo-D-Val-OpNP 
3, 4a 
90 Cryst. +17.1(D) 
P1/2(0.68) 
(EtOH) 
H-D-Val-Pro-Arg-pNA . 2 HCl 
XIII IV (1.46 mmol) 
5 87 GPC -117(A) 
A(0.38) 
12.1(12.6) 
H-D-Val-Pip-Arg-pNA . 2 HCl 
XIV VI (0.20 mmol) 
5 85 GPC -81.5(A) 
A(0.40) 
11.7(12.2) 
H-D-Ile-Leu-Arg-pNA . 2 HCl 
XV VIII (0.38 mmol) 
5 75 GPC -58.2(A) 
A(0.46) 
12.4(11.9) 
H-D-Val-Leu-ARg-pNA . 2 HCl 
XVI IX (0.28 mmol) 
5 75 GPC -63.7(A) 
A(0.46) 
11.7(12.1) 
H-D-Ile-Leu-Lys-pNA . 2 HCl 
XVII XI (0.18 mmol) 
5 72 GPC -66.6(A) 
A(0.44) 
13.0(12.5) 
H-D-Val-Leu-Lys-pNA . 2 HCl 
XVIII XII (0.27 mmol) 
5 87 GPC -67.3(A) 
A(0.42) 
12.5(12.9) 
Cbo-Ala-Arg(NO.sub.2)-pNA 
XIX I, Cbo-Ala-OpNP 
2, 4a 
76 Cryst. 
+2.0(D) 
P.sub.1 (0.34) 
MeOH 
Cbo-D-Ala-Ala-Arg(NO.sub.2)-pNA 
XX XIX, Cbo-D-Ala-OpNP 
2, 4a 
80 GPC P.sub.1 
Cbo-Gly-Arg(NO.sub.2)-pNA 
XXI I, Cbo-Gly-OpNP 
2, 4a 
89 Cryst. 
-35.0(M) 
P.sub.1 (0.12) 
MeOH 
Cbo-D-Leu-Gly-Arg(NO.sub.2)-pNA 
XXII XXI, Cbo-D-Leu-OpNP 
2, 4a 
78 GPC P.sub.1 (0.29) 
Cbo-Ile-Arg(NO.sub.2)-pNA 
XXIII I, Cbo-Ile-OpNP 
2, 4a 
75 GPC +2.8(D) 
P.sub.1 (0.50) 
Cbo-D-Leu-Ile-Arg(NO.sub.2)-pNA 
XXIV XXIII, Cbo-D-Leu-OpNP 
2. 4a 
81 GPC P.sub.1 (0.53) 
Cbo-Val-Arg(NO.sub.2)-pNA 
XXV I, Cbo-Val-OpNP 
2, 4a 
84 Cryst. 
+4.9(D) 
P.sub.1 (0.40) 
MeOH 
Cbo-D-Leu-Val-Arg(NO.sub.2)-pNA 
XXVI XXV, Cbo-D-Leu-OpNP 
2, 4a 
79 GPC P.sub.1 (0.65) 
Cbo-Aze-Arg(NO.sub.2)-pNA 
XXVII I, Cbo-Aze-OpNP 
2, 4a 
69 GPC P.sub.1 (0.47) 
Cbo-D-Val-Aze-Arg(NO.sub.2)-pNA 
XXVIII 
XXVII, Cbo-D-Val-OpNP 
2, 4a 
73 GPC P.sub.1 (0.55) 
Cbo-Phe-Arg(NO.sub.2)-pNA 
XXIX I, Cbo-Phe-OpNP 
2, 4a 
80 GPC +5.7(D) 
P.sub.1 (0.32) 
Cbo-D-Pro-Phe-Arg(NO.sub.2)-pNA 
XXX XXIX, Cbo-D-Pro-OH 
2, 4b 
61 GPC P.sub.1 (0.36) 
Cbo-D-Pip-Phe-Arg(NO.sub.2)-pNA 
XXXI XXIX, Cbo-D-Pip-OpNP 
2, 4a 
57 GPC P.sub.1 (0.38) 
Cbo-D-Leu-Leu-Arg(NO.sub.2)-pNA 
XXXII VII, Cbo-D-Leu-OpNP 
2, 4a 
91 GPC P.sub.1 (0.37) 
Boc-Phe-Lys(Cbo)-pNA 
XXXIII 
II, Boc-Phe-OpNP 
3, 4a 
65 Cryst. P.sub.1 (0.78) 
EtOAc 
Cbo-D-Pro-Phe-Lys(Cbo)-pNA 
XXXIV XXXIII, Cbo-D-Pro-OH 
3, 4b 
62 Cryst. P.sub.1 (0.78) 
MeOH 
H-D-Ala-Ala-Arg-pNA . 2HCl 
XXXV XX (0.5 mmol) 
5 87 GPC -42.0(A) 
A(0.44) 
13.2(13.8) 
H-D-Leu-Gly-Arg-pNA . 2HCl 
XXXVI XXII (0.3 mmol) 
5 79 GPC -51.0(M) 
A(0.44) 
13.0(13.1) 
H-D-Leu-Ile-Arg-pNA . 2HCl 
XXXVII 
XXIV (0.35 mmol) 
5 75 GPC -64.3(A) 
A(0.46) 
12.3(11.9) 
H-D-Leu-Val-Arg-pNA . 2HCl 
XXXVIII 
XXVI (0.43 mmol) 
5 80 GPC -53.0(A) 
A(0.46) 
11.6(12.1) 
H-D-Val-Aze-Arg-pNA . 2HCl 
XXXIX XXVIII (0.25 mmol) 
5 72 GPC -133(A) 
A(0.42) 
12.2(12.9) 
H-D-Pro-Phe-Arg-pNA . 2HCl 
XL XXX (0.4 mmol) 
5 70 GPC -1.0(A) 
A(0.40) 
10.9(11.6) 
H-D-Pip-Phe-Arg-pNA . 2HCl 
XLI XXXI (0.3 mmol) 
5 65 GPC -32.1(A) 
A(0.44) 
11.0(11.3) 
H-D-Leu-Leu-Arg-pNA . 2HCl 
XLII XXXII (0.3 mmol) 
5 72 GPC -47.0(A) 
A(0.47) 
11.6(12.0) 
H-D-Pro-Phe-Lys-pNA . 2HCl 
XLIII XXXIV (0.4 mmol) 
5 63 GPC -6.0(M) 
A(0.50) 
12.1(12.1) 
__________________________________________________________________________ 
.sup.x Data for the determination of [.alpha.].sub.D.sup.24 : 
C = 0.5-1.0; solvent: 
(D) = DMF, 
(M) = MeOH, 
(A) = 50% AcOH (aq) 
Table II 
______________________________________ 
Solu- 
bility 
mg/ml Rel Reaction rates 
Substrate buffer.sup.x 
T Try Pl Kal UK 
______________________________________ 
Bz-Val-Pro-Arg-pNA 
0.3 6 60 15 
H-Val-Pro-Arg-pNA 
40 6 55 15 
H-D-Val-Pro-Arg-pNA 
(XIII) 100 80 100 95 
Bz-Val-Pip-Arg-pNA 4 45 30 
H-Val-Pip-Arg-pNA 4 35 45 
H-D-Val-Pip-Arg-pNA 
(XIV) 100 70 100 
Bz-Val-Leu-Arg-pNA 
0.2 100 
H-Val-Leu-Arg-pNA 50 
H-D-Val-Leu-Arg-pNA 
XVI 600 100 
Bz-Val-Leu-Lys-pNA 
0.5 100 
H-Val-Leu-Lys-pNA 25 
H-D-Val-Leu-Lys-pNA 
(XVIII) &gt;100 100 
Bz-Ile-Leu-Arg-pNA 
0.2 55 100 
H-Ile-Leu-Arg-pNA 10 20 
H-D-Ile-Leu-Arg-pNA 
(XV) 4 75 100 
Bz-Ile-Leu-Lys-pNA 
1 130 
H-Ile-Leu-Lys-pNA 35 
H-D-Ile-Leu-Lys-pNA 
(XVII) 20 100 
______________________________________ 
.sup.x Buffer = Tris, pH 8.2, I 0.15 
In the table above the relative reaction rates for the different substrates 
are stated in relation to a reference substrate chosen for each enzyme. 
Symbols, reference substrates and their sensitivity for the respective 
enzymes are according to the following: 
______________________________________ 
Sensitivity (stated amount 
Ref. of enzyme gives the activity 
Enzyme (symbol) 
substrate No. 
0.1 nkat .DELTA.OD/min = 0.0254 
______________________________________ 
Thrombin (T) 
XIV 0.06 INH 
Trypsin (Try) 
XIII 0.03 .mu.g (Novo) 
Plasmin (Pl) 
XVIII 0.01 CU 
Kallikrein (Kal) 
XVI 0.2 Be 
Urokinase (UK) 
XIV 40 Ploug E 
______________________________________