JPS6037997A - Measurement of catepsin b in presence of other protein decomposing enzyme and compound useful therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for measuring the activity of cathepsin B in the presence of other proteinaceous Pfl enzymes such as trypsin.
do. The present invention further provides novel substances that are highly specific for cadevcin B. The method of the invention is particularly useful for detecting abnormal concentrations of cathepsin activity in body fluids such as human blood or in body tissues.

Proteinases (protein molecule fi enzymes) are enzymes that digest proteins and polypeptide chains. These enzymes are highly selective for the cleavage site of polypeptide substances.

The activity of this enzyme is precisely regulated in normal biological systems. If this control is impaired, there are serious biological consequences! P
: This leads to.

Many disease states are caused by such disruptions in the activity of proteolytic enzymes [A. J. Barrett, ad. New York 5A-J-Palette and J, MacDonald'
ttVIcDonaldl Press New York. ).

Cathepsin B is normal and zosome (Iysosαηal)
It is cysteine (thiol) flotinase of l.

In recent years, it has been shown that apparent failures in the regulation of cathepsin B activity result in various pathological conditions [A. J. Pallet and J. Macdonald, O.
p, eit, : M, Sandra (5andler
l, -Drugs -1% Uni/(-S5-I Hark-Fres I LIntvessltylPark
Press l, Baltemore].

Several diseases associated with collagen and structural glycoprotein breakdown are associated with elevated cathepsin 8 activity 12 . In particular, the activity and metastasis of cathepsin B
A correlation between TICL and TICL disease has been suggested by several studies, e.g. by assessing the activity of cadevcin 86 or cathepsin B-like substances in the blood of patients with metastatic disease (R. J., Pitras et al. (Pietras) et al., (/97 g l: t7y hot+3-3; R
, J, Beetles etc.t/? 7q1, etc., as described in Cinecol Oncol foncol 1.7: /-/7. ]. Therefore, if there were a method that could accurately and selectively measure the activity of catefcin B in mammalian body fluids, it would be a useful diagnostic aid for detection or monitoring.

Evaluating various enzyme activities 1 is a daily task in clinical laboratories. In a typical evaluation method,
Enzyme-containing zymble f: Involves contact with some synthetic substrate (defined as a substance or compound acted upon by an enzyme) and selective cleavage by the enzyme to a color-changing product as described below. are doing.

-Color) (Colored) or Decolorized) Colorless) Thus, the cathepsin material becomes a peptide or peptide-like compound that synthesizes the product upon cleavage. Evaluation agents used for cathepsin B [A, J, Palette, op, eft, : R, J, Beatrus, O
p, clt, : A, J, Palette (/9 I O
1Blochem-J, 1g7°909-9/l. ] includes peptide substrates incorporating/or more arginyl or polysyl amino acids attached to an enzyme-cleavable indicator group (e.g., Bz-Arg-NA, CB).
Z −Lys −PNP , CBZ −8rg −her
to g-MN, CSZ-81a-8rg-Arg-
AFCV CBZ -Phe -8rg -AMC.

(See abbreviations below). Although the selective cleavage of synthetic standards by cathepsin B is still not fully understood, the nature of their positively charged groups makes these traditionally used substrates susceptible to trypsin-like (also known as serine J) proteases. Thus, evaluation of cathepsin B using such lysyl or arginyl substrates in mammalian body fluids is complicated by the presence of abundant trypsin-like proteases. It is unknown how the activity of cathepsins is highly specified and expressed in the presence of trypsin-like WT.

It is therefore an object of the present invention to provide a substrate (substance) that is highly specific for cathepsin B.

Another object of the present invention is to selectively assess the activity of cathepsin B in the presence of other proteolytic rA'P, such as trypsin. Yet another object of the present invention is to measure the activity of Cadevcin B in body fluids and tissues of mammals. Further objects of the invention will become apparent from the description below.

The present invention relates to cathepsin B in body fluids or tissues of mammals.
A method is provided to test the activity of KX by measuring the rate at which the indicator group fXl is dissociated upon cleavage of a synthetic peptide substrate that is highly selective for cathepsin B. Substrates with suitable indicator groups suitable for the detection of cathepsin B are substances having the following general formula and their salts.

The x-mine in the formula Z-Rt-Rt-X IJe is a group that is cleavable by cathepsin B at the R,-X bond and is detectable upon cleavage. R8 is an amino acid group having an L-structure at the α-position carbon (Ca) of the carbonyl group and having a group that is not positively charged within the pn range of the test conditions, and preferably has a polar side chain. and preferably the side chains therein have 2 to 5 carbon atoms. R1 is a hydrophobic 7-mino P group, preferably phenylalanyl, the carbon at the α-position of the carbonyl group (Ca I has an L-structure, and preferably -CH* -Y I Y is lower alkyl , phenyl, substituted phenyl and indole groups). 2 is an amino blocking group that does not prevent cathepsin B from selectively binding to Ro and R1 groups.

The following abbreviations are used herein:

Amino acids (assumed that all have L'''C unless otherwise noted in percentage) phe -L-phenylalanyl group C1t -L-citrulyl groupAla -L-alanyl group Arg -arginyl group LyS -lysyl group (Not lArg ,,, N 6J-nitroarginyl group (to Tsl8r -N"-) dylarginyl group (i.e. N -p-) luenesulfonylarginyl group) Trp -) ribtohil group Met - methionyl group Asp - aspartyl group (to TF8 Lys -N' -trifluoro-coacetyl lysinyl group (CBZILys -Na -carbobenzyloxylysilyl group Vat -valyl groupLeu -leucyl group 11e -inleucyl group Na1a -naphthylalanyl group amino blocking agent cez -carbobenzyloxy group BZ -benzoyl group t-5ac-t-butyloxycarbonyl group AMC-7-
Amino-hiro-methylcoumarin group 1-lMC-7-hydroxycoumethylcoumarin group AFC-7-amino-coutrifluoromethylcoumarin group (PCT patent W Og Olo, 2,293) 8Q
-4-aminoquinoline group NA -conaphthylamine group MNA -+-methoxy-2-naphthylamine group PNP -varanitrophenyl group PNA -varanitroanilini/group TEA -triethylamine DCC-N,N' -dicyclohexylcarbodiimide DMF -N,N'-dimethylform-11amide T'H
F -tetrahydrofuran DMSO-dimethyl sulfoxide HOAc -acetic acid 1Br/HOAc -HB r -C saturated shitaacetic acid P
d - PEI - palladium polyethyleneimine catalyst other EDTA - ethylenediamine tetrapolymerized dino'thorium salt ole - mmol (1) - mm nrn - nanometer b [α10 (DMFI - 5 at 2S°C in D M F solvent Optical rotation B15-Trls-,2,λ-bis(hydroxymethyl)-λ,2',2"-nitriloethanol HPLC-
Artic [Deerfleld]
High performance liquid chromatography using a column: 'A b X , 250 w, C1g; flow rate:
/ mf, /min: Detector 2 UV311θ, 3/
31.2go and 2S4tnm, fluid compatibilizer, retention time and approximate purity are given in each example.

MS - Mlcro Mass VG7 with fast atom bombardment probe and ZAMBLE dissolved in DMSO/Glycerin
Mass spectroscopy using θ′70H instrument 1-1-NMR-d6-DMSO solvent and proton nuclei in Bruker +, 270 MHz instrument using tetramethylsilane as internal standard with chemical shift of θO (a) Exit resonance.Chemical shift, δ, peak splitting: S
Nisingle, d=doublet, 1=)libret, q=quartet, qn:quindent, Se=setstedt,
C - complex multibullet, and number of protons (/ p = -/ protons)
is shown in each example.

MP - melting point (uncorrected, Fitscher-Johns apparatus) HPLCfPrep, l-Altic (Deerfield, 11/,) column I10x, 2SO gate, cig
High performance liquid chromatography using , flow rate, 2 me/min; detector: UV3 0 rta; fluid compatibilizer and retention time are shown in each example.

The peptide substrate compound is defined using the following formula and the following correlation of the structure below for clarity.

The general formula is as follows.

Z-R, -R, -X The structural formula for the above formula can be expressed as follows.

Z Ra RIX In the above formula, Ra is a side chain of the R1 amino acid group, and is a side chain to the R4f'J:Rt amino acid group.

X is R, -, a group that can be cleaved by cathepsin at the bond, and one that can be measured upon cleavage.

Examples of X are AMC, 8FC, and AQ.

Includes HMC, PNP, and PNA.

Salts of the above compounds include any crystalline salts, such as when X is 8Q and this group forms a salt of water-wood chloride.

R1 is the carbon indicated by α1 above and has an L-structure, and is an amino acid group that is not positively charged under the evaluation conditions. For example, the R, amino acid group should not be positively charged within the pH range of about y to about g.

Referring to the above structural formula, R1 is preferably a side chain of R1 having 2 to g atoms. As an example of R9,
When R5 is Ala (Ma-CH++, R8 is (No
When w l Arg, NH1-ICHJs-NHC-NH-Not; when R1 is Cit, I-(Ct], )4-NH-C-CF3.

The structure is necessary for casebutin binding.

Exactly the obi'! The purpose of including l-da-re is to confuse the evaluation or detection of trypsin 4jI! 2) by eliminating the possibility of reaction with enzymes.

R1 is a hydrophobic amino acid group having an L-structure at carbon (Cα), preferably Phe. The l--t'lV structure is required for binding cathepsin B.

The cotv hydrophobic amino acid group also facilitates substrate binding to cathepsin B.

Referring to the above structural formula, R1 is R1's (llIjl
A preferably has the following formula.

-CH! -Y In the above formula, Y is lower alkyl, phenyl, substituted phenyl, or indolyl group. Lower alkyl means 7 to 5 carbon atoms. Examples of R4 are 1" -CR7-Co115 when Rf is Trp, and - when R2 is Leu.
cH2-CH-(CHslt, including -CH-(CHs12 when R3 is Vat).

Z is useful in slightly altering the affinity of the compound for cadepsin BK, although R6 and R7 are not critical. The two groups can be simple amine-binding groups such as CBZXBOC or IkeBz.

It may also be a amino acid group. In any case, these two groups should not prevent cathepsin B from selectively binding to the R and R2 groups.

The chemical structures of the peptides referred to in the present invention and appearing in the Examples are as follows.

i CBZ - Phe - Clt - AMCNll.

, 2. CBZ -Leu -Cit -AMCNt-
lx J, CBZ-11e-Cit-AMCNH.

'A CBZ-Vat-Cit-8MCNH.

& CBZ -Trp -C1t -AMC TOI Ht 4 CBZ -Yen 1e -8la -/WIC'Z C
BZ -Vat -Ala -#VICg,CdayZ-
Trp-Ala-AMC9, csz-Phe-
(Not l rg −/WICH N) (=guNH-Novio, i/, cez-cot tahashi) 8 la −
Phq -Clt -AlvC/, 2. csz-P
he -Met -8MGS"CH3 13, CBZ -Phe -ITsl・-8"g -A
MC/IA CBZ -Phe -(CBZILys
-8 MCI! ;, CBZ -Phe -(1-cho AI I
-ys -NJC/4 CBZ-yen]e -Trp -
AMC/7 CBZ -Na1a -Cit -AMC
pH-l.

〆g, CBZ -Phe -Arg -8FC/9C
BZ -81a -8rg -Arl;i -ΔFC(
) :lO,BZ -Val -Lys -Lys -Ar
g -AFC■ Synthesis of pegtide substrate θ can be carried out by a number of commonly used 4-gtide synthesis methods. Next, three types of synthesis methods will be explained.

Method ADZ The Rx group is coupled to the R,-X group by a DCC1 mixed binocular proboscis or other typical coupling method.

Method B: A complete success! ! Petit ZRz-R,
is coupled with DCC1 mixed anhydride or innocator X using any coupling method that can be used.

Method C: 7% Ri, the Rx group is coupled to 11111, followed by the coupling of the indicator group X using DCC5 mixed anhydride or 110 acceptable coupling methods.

The general methods for most of the compounds mentioned in the implementation sequence and the series to the general methods are as follows.

1''20 In the above, R1 and R2 give 11g of different amines in the general formula, and CBZ for ZIC and X
AMC is used for this. Mixing, and bad water'zu is a type of coupling method.

The details of the reaction/- are as follows.

(1) C8Z-C/reaction: In the case of several cases, CBZ-R induction 1.1 is commercially available, but 11 J) i! In case of J, iif, 'J was produced by the following method. amino l
'ff2R (,25 mmol) and 50 mmol of sodium bicarbonate/θθml in a flask! ; Dissolved in Oml water. Add 1 tablespoon of Carbobenzyloxyflora f-dos (2 mmol) in a vacuum chamber over 7 hours while stirring.

(further 'a 7+'<), 11 @ stirring once, extracted twice with ether, then /θθmA /IvlHa
I lowered it dN inside.

When raw, the product is first precipitated in a semi-solid form and then left to solidify.The product is washed with water, dried and used for one time without further production. 'C0 average) section rate was 53%.

(,21Alv'IC-coupling 2/i'1 mmol of blocked R1-amino7,/3 mg of anhydrous THF and//tatammol of T'E8 were added to a flask with left θme. When the compound dissolved r Su, melt/1y,
The mixture was cooled on ice and 1 mmol of isobutyl chloroformate was added. The solution was stirred on ice for 70 min and then dissolved in 2/l of DMF.
A cooled solution of 1 mmol of AMC was added. Stirring was continued for 7 hours on ice and then in room temperature/overnight.

The reaction mixture was filtered and the THF was removed from the iM solution in a rotary evaporator. The residue is 20-〇H
nα2 and 3θmll with 70% l-1α water/
=2! Add to r me separatory funnel and vigorously: 11
＝It was a long time ago. Third fractionation of acid aqueous solution ri Ia phase k
41 hours out! ε, the product was precipitated into the organic phase. water(
1] was vacucanzed, and the precipitate was washed with ether. The product was dried and used for one serving without further preparation. Average σ month 1 time skewer 7% rl'> -:) lko.

(Removal of unblocking N-carbopene soloking locking structure from Z-RxAMC with hydrogen bromide/ice t:r+
= f1'n rfJ il& (He rll-10A
c) or by catalytic hydrogenation.
and removed it. Two types of samples were used (Ma-t-BOC was used as a substitute for CBZ, and degrocking was performed with formic acid treatment).

HBr/HOAc: into 3θOme flask, 3.
g mmol of Z RI AMC and 3.3 l of 7θml
−HBr/l”0ΔC was added. The sample was dissolved and the solution was stirred for -/S minutes. The reaction mixture is Sθθη
With a needle of te; tfat, the precipitate was dissolved under nitrogen, and the precipitate was poured into ether at 4:0. litsuP+hiddenf
・I was a member. After drying the product, it was used for one serving without further searching for I17. The average yield was 9g hood.

Engineering 1. &/'19 (Hydrogen addition 1 de Sθθml pressure flask into gs mmol Z RI AMC, 4'1
7')"9')') Mu, J?Lyethyleneimine beads (Pd-PET)": and 4θθml of methanol were added. The flask was pressurized with 1 g of hydrogen at 10 ps, heated for 4 hours, and then left for an additional 1 g. Touch i(
The beads were precipitated with alcohol, one bead was soaked with methanol, and the beads were soaked twice. t+￥, ;'′! 'To promote rc/2. Hydrogen monocide salt was prepared by adding 2 portions of Shiome/NHCl water. The solvent was removed by 1 zip, and the residue was redissolved and reevaporated μ times with Oml of denatured alcohol. 11.-AMC was boiled with the product, washed with modified alcohol, dried and used once without making it twice. was 7g%.

Formic acid: 3θθml into flask/,7,'r mmol tBOC-Rt-AMCI! l:,,? θm
Added 97% of e. After the mixture was poured and the proboscis was dissolved, the solution was stirred for 3 hours with a glass spoon. At this mt.
The ether of 25θme and the concentration of aλrJ, (H
αJ, habit was added to 1shot・(zamataruta+'f)).

The white precipitate was filtered and rl, rfi with ether. After drying in vacuo, the product was used 1') without overcoating.

(/,') Ino0 Chidoka Tsuguring:,! ;0mg into the flask 3, ymmol R, -8MCI-IBr (
or Hα)1,)5 mA of anhydrous DMF and 3.2
Millimole of TEA was added. 3λ mmol of CBZ Rz,/θml of anhydrous DM to the flask of λth Sθrrt
F and 3.2 mmol TEA were added. As the compounds dissolved, both solutions were cooled on ice and 22 mmol of isobutyl chloroformate was added to the CBZ-R solution. The reaction mixture was stirred on ice for 70 minutes, and then the cooled R,-AMC solution was added to the CBZ-R reaction mixture. Stirring was continued on ice for 1 hour and then at room temperature for 7 hours. Next, the reaction mixture was added dropwise to 596 tons of an aqueous solution of sodium carbonate (q30) with stirring.

Pass 51 yellow and collect it, wash it with water 3 times and remove it. The average crude yield was .

+n#! : The terminal proboscis was purified by product crystallization and in some cases further by crystal ratio and HPLC. Relying on C13Z R2Rt-AMC (2,9 mmIJ mol), which has a certain amount of production, 3S-
Dissolved in wood vinegar II. Add 5 datqCO to this solution)
Acetonitrile was added and the solution heated to 11 ml. While receiving the heat, g()me of water was added in small portions to the soybean soup. After cooling for 41 hours, the precipitate formed by IE was filtered once and washed with water. When the recrystallized product was dried with nitrogen, the average crystallization yield t, j
gO%. The overall yield and the central point of synthesis are shown in each of the following sections.

Review 1 song Kateph 0 Syn B activation t, indicator ', H
Detection was made by monitoring the dissociation rate of X. The indicator group may be a group whose fluorescence or absorption changes, especially upon cleavage. Any known indicator such as AMC, IMC, FC, Q or U'PNA can be used. - Indicators labeled with radioactive elements can also be used.

The #FI song method of this enzyme, catenosin I3, is
Following the method and general principles I (A, J, Palette, op, clt-; W
, P, Thor? -7 Kusu (Jencks) (/91
．． 6) This is the standard described in Catalysis in Chemistry New York. ). The power of stable heat≠ conditions is based on the ideal enzyme/group ratio (based on the Mlchaells constant factor), which provides accurate speed reports in the shortest experimental time scale. was set using one dose of the enzyme ItlIe).

Catechusin B's plan is to sample Shosae's body fluid (/~Sθ
microliters (μl), the pH range in which Categ/N B is active, the passageway is pH 5-S, preferably iqt.
IJll left 3 was added to an aqueous solution containing ED Dine A and a thiol activator such as cysteine or sothiothreitol (1 monme of raw antioxidant or other suitable reducing agent). After a short activation time, such as 7-2 minutes, methanol, ethanol, acetonitrile or D
The substrate dissolved in a compatible solvent such as MF is vomited in a small volume (2θti l) such that the concentration of the substrate is much higher (preferably at least 100 times) than that of the enzyme.
Te/Jll i k.

Vortex! fVi set temperature, preferably ambient temperature to about 37
The temperature was controlled within a range of ℃. The concentration of the cleaved indicator, t, is measured using well-known techniques such as spectrophotometry, spectrofluorometry, or scintillation counter, or U: if! x can be measured against time by known means of the present invention.

In order to obtain a scale that is comparable to the uncut sample, we created a standard surface of the free indicator I for agricultural deer. S+', fl of the fluorescent indicator can be removed in the following steps 7-11.

Equation: 8.8=C Specifically, in the above equation, A is obtained from sample σ month Q11, day is obtained from the standard curve, and CYi is the result. The word "μM" is micromole (μmol//), L [ml, J is milliliter, ・
And ml is minutes. Calculate other indicators′
The same method can be used for f and 1 history. Based on the information obtained from this dljL, the speed stage expressed by the concentration of free indicator group X and its liquefaction time can be obtained. This rate can be directly related to the rate of cleavage of the radical residue by yxu'C, and thus the enzymatic activity can be estimated.

The present ff5 invention will be explained in more detail in the following implementation sequences, but is not limited to these implementation sequences.

, ili K's synthetic truth, SiMi + (IJ / CBZ -L-P'l]e -L-C1t -AMC, using the contact water of 1st verse 3, 1 person IA, Grotto, King 1 history, And/I Ani 9 / 7meO) HO 8, 7
41m1CQCH婁CN,/'Iθme cl)H
It was synthesized by recrystallizing from scratch as detailed in the above steps. The overall yield was /g.

Release, "2.2'/-, 230℃.Q.(+)MFl
=-no magazine 77°.

HPLCC70%CHgOH/3θ%1hO)9. Am
1r1973%.

``Land'' μdan: δ/, 30-1! ;! r, c, 2p:
Al! ; Shio-1g / #Cr 21) lδ, 1θ
t 5r3p: δ, 2.7S, t, /l); δ, 2.
ri0-3. /θe c+Jp; δ'12g 'A4
'0. c, / p: δlAhiro θ-4I43/, c
, /p; δ'A9'l, s, , 2p; δ3'1
3. s.

=2p+δmuθθ,t,/piδl>28. s,
/p; δZ/θ−””e Cr / (:) p i
δ7. dagld, /p:δ7. S-/, d, /ρ;
δ273. d, /p; δ7IO, d, /piδg,
33. d, /p: δ/θ! ;/, s, /p.

MS Parent molecule ion = 4/1 Elemental analyzer JTia (as G55HssNsOy) C1,'
A! f9% H left 7S% N 7777% Actual 011j value Cl,'Ag/CHH&77CHN/it, 1% Implementation [flCoC3Z-1--Leu-L-Cl -AMC gold,
Previous: 1 self-produced 1 J, f, using catalytic hydrogen addition blocking and ly AT/7 HOAc, 36 m
1 of C, HsCN, and 136 ml of HzO, and synthesized as detailed in steps 1 to 1 above. The overall yield was 79%.

~1P=/half 9EC%reli. (DMF)=-λ left 7
P1-I P L C1 (4/,, S-%CHs CN
/ks%HmO) 5! g min 99.9%,
1-NMR: δ θυ, = 2d, 6pi δ
/41. G, t, J p; δl! ;4' 777
, c, 1Ipiδ-1-, :19. s, ,? p
:a, 2. gb −J, / (7, c* 2peδ
'A10. q, jp; δlA'13. q, jp
;a ri 0 beg s, , 2p; Jj:! ko + stλρ
; δ left 9g, t, jp; δA, 27. s, jp
;+52. J, ll-733,c, 7p; δ27
,2゜d,jp; δ77g, d, / p rδg,
/6. d, jp; δ/θ'Ily, s, jp.

MS parent molecule ion = k g O elemental content υt ;;h1. Value (as Cgo 1.-+xyNsOy)
C4,2,/Otsuchi H & Mudachi N/2θgchi actual measurement value C62,3? %Hmu5θchiN/2.06% Example 3 CBZ-L-+1e-L-C1t-AMCi, r71
J I + own process 30J catalytic hydrogenation process 1 history, and lI at D, 20 ml HOA c x
CHsCN of 4' me was recrystallized from 3/1 mg of HO and synthesized as detailed above (Example/~Q). The overall yield was /ochi.

M scale = 2'l0-2'l2°C, deaf. (DMF)=-puff 6°, )(PLC(jθ%CHsCN/!;θ%Hg0
) Z/rnln, 9/8chi, 'H-NMR: δθ7
3-θ9J, c, 4p; a lOθ-1gθ,
c, 7p; δyuku θt st , jp; a, 2. gb-
,3,/,2. c.

=2p; δ3.9, t, /p+δ4/, 413. q
, jp: δ left θy, s.

, 2p; δbuda 3. S, -2p iδ left 99. t
, jp;δmuλ7. S.

jp;δZ 2! r Z Q 2p c-A p ;
' 7 'I 9 r d, / p r δZ? 2
．． d, / p rδ7.7g, s, jp; 11g
2θ, d, jp; a/θ gu A, s, jp,,, one sacrifice” - Book molecular ion = sgθ Elemental analyzer 3'? :ffK (as Cso Hst Ns Oy) G /, λ, /6chi ■ Muda 3% NZ, 2.0g% -4 Measured value 06 Yu 2 Hirochim 36g6 N /, 2. /4% Example 1 CBZ-L-Val-L-Cl t-AMC was subjected to the J-catalyst hydrogenated grotzking of the above-mentioned process 4 and 3,
and/waata'), 2'I mg)-10Ac,
7! ; - CHsCN, /2gme of H,0 was recrystallized and synthesized as detailed above. The overall one-to-one rate was l ochi.

艮且=2'l弘-J7℃，falll. (DτφF)=-22
36°, HPLC (35% CHsCN/4j%HmO)
/ 7 / min, 93. g%, “H-NMR
: δθ7 j 70 # C, A p i δ1yo
-is lI.

c,, 2p; δi 5 lI-/, g j -c r
2 ptδlza3-2. /θ, C1/piδ2daθ,
s, 3p; 82. g 3-J, / 2. c, e2p
iδ, 3.9'l, t, jp; δ44413+
ql' prδfu0. ! ;, s, λp: δoichi, s
, 2p; δ left 9g, t, jp; δ Otsu, -g, s,
, / p ; δZ-202'l J* C,j p
iδz+l/l-'z+7. c,, 2p; δ772,
d, ll); δ777. d, /p +δg/9
．． d, jp: δlO, Hirosu, s, jp.

MS Parent molecule ion = S Otsu 6 Elemental analysis calculation value (as C 鵞eH*sNs Or) c bisg
% Hmu 21chiN 723g% Actual θsu 1st Cl, ig Otsuchi Hmu e27% N /,), 11guti Example 5 CBZ -L-Trp -L -Cit -AMC, =iJ 6self Using catalytic hydrogen modification Grocking in step 3, HOAc of 1/6 HOAc per lI, 37
ml of C, HsCN.

The compound was synthesized by recrystallization from 6 liters of HmO as detailed in the above steps. The overall yield was /7.

MP=233-. 23gC1(su.(DMF)=-ko3
7S0, HPLC, (l15%Cl-1sCN15j [
HsO)=7. min, left 6th, 'HNMR: δ1
30 1 to 'l, c, :2p:'i! i'l-/8
”*Cl e2p ; δ, 2.4'0.s, 31
) iδQg, 2-. 3. Ty, o.

c, 4piδri, j3-'A! ;'l, c, JpiJ 4. s, Jp; δ tag, /, s, = 2p; δ left wawa t, /pzδ mukoku, s, /pi δ47! i,t
, /p;δZ(75,t, / p iδ'Z / 0
7'A3.

cp&p: δ'730. d, /p; δZ
/)! i − d r / pr δ7.7 (1°d,
/p; δ7gO, d, /p; δg, J3. d, /p
iδ/a daso, s, /piδ/θ79. s, /p,
-μj-base molecule ion = 633 Elemental analysis calculation i+@, (as Css H2S N@C)y)
C+14'/CH]" Left uncle J N Z2. gg% lol measurement direct C6 116 1-1,! r, 41 g% N 72.9 pieces Example 6 CBZ-L-Phe-L-81a-ΔMC, l]! f
If using catalytic hydrogenation blocking at 3 p/l and HOAc at 27 m
The compound was recrystallized from CH3CN%29- of HaO and synthesized as described above. The overall yield was 3/ti.

MP=, 23.2-, :13ac, (a) o (D
M E ) =23. e / 0, IPLc (j,
t % Cl-15 CN/43%Hz o) A, g
min, 7g. 2%, 'H-NMR: δ736.
d, 3piδ−,41θ, s, jp+δ, 11711
．． t, /p: 229g -, 3, /3. c, /
p ; δ1A (13, c.

/p;JIAII4. C, /p; δ4491I, S
, =2p; δe2g, s.

/p; δ7/2 7+'0. c, /(7F)iδ7
．． lI'l-7,,5A, c.

2p; δZ7g, d, /pi δ77g, d, /pi
δg, gθ, d.

/p;δ/θlI3. s, /p% MS Parent molecule ion = 5.2g Elemental analysis calculation 1st shift (as CIO821N308) C1, g, 3
0chi H left 5guchi N 7.91. Actual measured value C g, 37% H left g% N 796%, 4 Example 7 CBZ -L-Va l -L -A Ia -AMC
Before? Using the JjG catalyst block of the r self-representation J, and the HO of /θQ trl per /y
Ac, CHmCN of /θ'l me.

9, which was recrystallized from 67 ml of T (20) and prepared as detailed in steps 1 to 9, with an overall yield of 3/3.
It was Chi.

MP=K0-24s℃, [α) (DMF)=-3'
A! ;10, □ □υ HPLC (l/-5%Cl-l5 CN155%Hg0
) A&rnln, 9 g 7%, 1 Steller NM
I'(: δ and 1g, 2-09 Otsu, C2ro p
; δ 733. d.

jp; 'Xg g-co, θ7. c, /p;δko,v0. s, 3p:δ, 3.7 flood, t, /pi“4
ri'l'l, c, / I) iδ left (75, s, cop: δlz2g, s, /p; δ7-2 A -7"
i'=2. c, 3 p; δ2 da S-752, c
,,2p iδ7.73. d, /l); δz7 Otsu, d,
/p;ag,,23. d, /p;δ/ (2J
9+ s, / 1) skACJ Takeo + 4-Heeno Lacquer Saw〃2n Elemental Analyzer - Cicada: Value (as Cps l-129Ns Os)C
6 left 12chi H670% N, g, 7bchi actual measurement I direct C6fu! i 7 % Hm 0.2 inches N g, 79 width Example Z CB Z -L -T r p-L -AA! a-
AMC was used for catalytic hydrogenation blocking of 3 times a day, and 33 mg/l)
-10 Ac, 7 ml CHsCN, 41 mg H
! O was also recrystallized and synthesized as described in Steps 7 to 1 above. The overall yield was fi/7%.

MP=, 2 < Lee-233℃, (su (OM Elmi-
749°, HPLC (4'5% CHI CN/!;!r
%H*O) /JJn+in, 93.4%,”H-N
MR: δ13da, d, Jpiδλ, tlO,s, 3
p ; δ, 2. f41. c, /p: 23.0g
-3,,22,c,/p ;δri3, se.

/p; a'A4'3. t, /p; '4
'r''?A;, S# -2prδ1x26.s.

/p;δ/, 95. t, /p; δ/Zos, t, /
piδZ/θ−′7. 'IO, C, gr) + δ7 to θ, d, /p; δZ 44, d, / p; δ7
7 ='lr d −/p; δ'27g, d,
/p iδg, 36, dt / p iδ/θ, J
g, s, /p; δ/θ79. s, /p.

-1 [Σ iσ tree molecule ion = 567 Elemental analysis 81', iT 1ift (Csz Hso Ni
(as Os) C1,7,g3% To1 Left 34t% R 9. g9% >'% +llI firj, C1,g, 0 0%
H, Da/1.2% N 9ri 6 Fu Dami JJI! I13 Riwa CBZ-L-Phe-L-(Now)Arg-AMC was added to the previous m1. L1γ3 contact ARM water latency slippage I
One 7g pg ml! , of 10Ac
,/4 ml CHsCN.

/3 tni of H, O was re-bonded and synthesized in 1j11 km/~ as described in detail. The overall yield was 20%.

IVI P = / ! θ-76θ℃ (dec), [c
rll,COMF)=-3,gOo,)(PLC(5A
%OHmcN/'13HzO) ggm group kH back,”
H-NMR: δi'A-i9. c, q−p iδ
w2.4', s, J r) iδ near -3,7,
c, 2p; δJ, / 9. c,,2p;δri,
Jll, c.

/ p iδlA'I 5 t Cv / p rδ'
A9! ;,s,=2p iδ&-g,s.

/p; δγ/ OZ Jll-2r C, / 3p *
δZ<27. d, -2'p: δ274', d, /
p; δ7'7g, d, /p; δg,, J11/!
, d, /p: δ10. 9. s, /p.

MS Mother wood molecule ion = Elemental analyzer 1? Direct (as Css Hss Ni Os) C1
,0,27 Chito 1 , Yo 3 Otsu Osamu N /lA de/Chimi 1HIIl Direct ci, o, o 3% H left 4t/% N /! r, 0 Tachi-s, Example/θ CBZ-L-8la-L-Phe-L-Cl-8MC, CBZ-L-Pbe-L-C1t-
AMC(-)4ji:li+FIJ/), T1';
I red-J2) 1% H day r as in 3
The reaction product was synthesized by grogging with /HOAc and then pegtide-caggling the reaction product with CBZ-L-AA'a as in the above factory. The final product is /3'I ml l-10Ac%41 per /I
'1 ml of Ct-hCN-27, recrystallized from 3 mA of H*O and further 1-IPLC (Prep.

'l 5% Cl-1scNi3j%Hx Ol-0
(to m1) was prepared and used as II chord analysis j'll. The overall composition ratio was 0%.

M P = ,2,? Otsu-JθC1, @Yu. ([JI
vlF)=-J9wa10.11PLc(4θ%CHsC
N/'10%t(+Oj lh3 min, ?=2.7
%, '11-NMR: δ l/'I, d, jp;
δ12g-1! ;'l, c.

, 2p:δi3′l 7g3. c, 2p: δ2.4'2
．． s, Jp: δ, 27 J −3, / 'I,
c, 9'l);δ'A O,2,t, /p
iδ ri gp, q.

/p; 6 people 5, c, /p, δ 0θtq+λp; δ left 3. s.

2p; δ left 7wo, t, /p; δ m2gHs, / p i
δ70g-7,4tθ, C1/θp; δZ'l 5
# d-/l) #' Z 53- d p/
p iδ77 l/-, d, / p iδ'7.79
．． d, /p; δ29θ, d, /p; δL
village, d, /p; δ/θlIg, S, /l)%MS 1σ
Body molecule ion = A & 5 Elemental analyzer n1 direct (as Oss day HN60s) cb3. Is
Person in charge]] Left g 7 Chi Ni, 2. ．． 27% Actual measurement 1st shift CA3.10~Htaγ / ChiNi, 2.73%, Chisel hm+Tari / / CBZ-D-Ala-L-Phe-L-C (t -AM
C, 1cBl-D in the final peptide power tunneling
Implemented using -A/a and L-Phe-L -CI t-AMC! ;'l/t). Final generation 1'! IJf, 7g/(l ml of H)
To 〇8 * j b ml of CH3Cl'J, / 7
J m”, recrystallized from θ month 120 and further HPLC (
Prep, , '15% 011gCNi 3
5% HxO, θmin,)
! ,,I, ;Analysis/11. The overall yield was Vi3.

MP=/7der 796℃, palanquin. (DIv'lF)=
7/ 7g 0.1-IPLC, (1,0% CH3CN
/lIO%H,O) A3rnln, g g, /!
%, 'H-NMR: δ(2pgp d-jp 12
1.2g-136,c.

, 2p: δl 561 g g , c, -2p; δQ
'l /, s, j p iδ, :) -7g -3,
20, c, lp; δ4', oj, t, / p i
a'A'14', c.

lp;δ+C1/p;δl/, light+q+-21)?
δFens, s.

e2p; δ1x00. t, lp:aA, 2g, s
, /piδxto-'23g, c, /θp:δ7
．． '14. d, / p r '75'7', d
, / p 1Z73. d, lp; δ7. gt),
da/p+δg,/9. d, / p iδg
, 24t, d, lp; δ/θ32. s,lp
, y±'#, water molecule ion - gs elemental analyzer'f (itge (as C36H2ON60s) Cl,
, 3.7! i% 1” left 89% N/, 2. ．． 27chi actual measurement C Otsu 297chi H left q/chi N/, 2. ．． 2θ Chi Example /, 2 CBZ-L-Phe-L-Met-”AMC, HBr/HOAc unblocking of previous n self-engineering 3, total blocking and / eQ >) or Jml! HOAc, /θr
M's CHsCN%4'Go's H! Recrystallize from 0 and further t
-IPLC (Prep, .

60%CHs CN/'IO%H20,/5 r
It was diluted and used for enzyme evaluation. The overall yield was e/6.

MP=μm-109°C, Deaf D(DMF)=-3,79°
, HPLC, (4'5%CHsCN/&shiochiH*O)
/2λmin, δ3.2%,”) l-NMR:
δ1lf3-2.20. c, , 2p iδμ, θ7
．． S.

3 p iδyu J5 Qb JC, δ2 p; δλ
,'1.2. s, 31): δ-17 Otsu 1 tt
, 2p; δ'A (1g, c, lp; δda 5 J+ C
1/ p; δ'A9 Otsu, s,, 2piδmu2g, s,
lp; δZh 73'7゜c, /θp; δ7.3
θ, d, -2p rδZ 7'l, d, / p
rδ77g.

d, lp; δg, 3 Otsu, d, /p; δ/θg gr Sl
'l'%MS Parent molecule ion = 3gg Elemental analysis aFJLi direct (as Cs* Ham NsO@S)C
0% l from A] Left 66% N 'Z15chi S Yo14% Actual value C6 Left 7 Ochi H! 177% N 7θ Wachi S Left Gu S Tibu S1 No 1 iii 14;su/3 C8Z-L-Phe-L-(Ts)Arg-AMC,
α-t-BOC is used as a substitute for the α-cBZ conductor in the step λ.
(Ts) Starting from 8rg, it was synthesized as detailed in steps λ to 1 above. The α-t-soc group was removed as described above in a yield of 7% formic acid. Total 14 [
The composition ratio of this material is UICHPL.
C(Prep, 78%CHsOH/25%H)
mIn,) was purified.

-MP=//g-/, 2jU, (so +7-00, H
P L c, (4-!;%CHsCN/3..ff%H
z O) / 13 min, 909%, '-H-N
MR: δ/29-1311', c, 2p;ai-!
;g IgA.

c,,2p;δ,2. ．． 2g, s, 3p; δYui(θt
se3p+', 2.741:, t, lp iδyu9
e2-Yuko Oy Cm 31) + 'Sa Ku4r3
2. c, ,2p; a'Ari'p se =21
); δA, 2gHs, / pi δlh4'J 7
//, c, 3p: δ7 / / -2'I'l,
c', /'I p + δ7 A: l, d, 2
p rδ773e d, / p; a77g, s
, / p ; δg, j 41 p ce / p +
δ10.3.2. ．． S, 'I) iMS: Mother wood molecule ion = 767 Elemental analysis calculation 1 shift (C40Ham N5OsS at °) C6λ
, 63% H left S2 chi N /θ96 chi S 447 g chi actual measurement value C6λ, O1/, q6 H tall 6 chi N /θri θ chi S IAgu7chi Example/1I CBZ-L-Phe-L-( C[3Z)Lys-AMC
, instead of the α-CBZ flange conductor in the step λ, α-t-B
Starting from OC(CBZ)Lys, it was synthesized in a very detailed manner. The above α-t-BOC group was removed with formic acid in a yield of 3 kg as described above. Recrystallization was performed using HOA cl of 7g/λθme,
! r b mg CHsCN, /27-HgO
It was done from

The overall yield was 3 units.

MP=/7θ-/73C, gL. (DMF) = - λ
g0, HPLC, (11,3%CHsCN/33%Hm
O) 3mIn at 7, 72.5-1, =H-NlvlR:
δ1.2θ−1jlI, c, lIp *δi! r
'l-71j2. c, ,2p;δyukθ,s,3
p; δ276, t, Jp; 2.2. gg-3,θri e Cp 2p+δ'1.2g-'Allza,c,,2
p; δ'A q dz 'I ρ*δA, 27. s, /
p: a7/θ-7,11,,3,c.

/ 7 p + δ2 ta9 z t −/ p ; δ
773. d, /p; δ7.7'7゜s, /p; δg
,,29. d, /p; δ/θQ7. s, / l
)%MS: Parent molecule ion-779 Elemental analysis calculation directly (as C41Ha2N40g) C115/ChiH left g9 ChiN 7.79% Real (0 (1 value C6g', Yu3chiHyoq/ChiN7Otsu9 19 Li /, 3- CBZ-L-Phe-L- (TFA) Lys-AMC was added using catalytic hydrogen (Oka blocking) in +if notation factory 3, and -CHs CN to I
I+ was crystallized and combined with C as described in detail in the above steps. The overall yield was excellent.

MP=2θg-,2,10℃, [σ](1)MF)=
g/30, - -0 HPLC, (1%Cl-1++CN/, t511-h
O) / left g min, θ+S, 1 H-NMR 2 δl, 2θ-l 1'j1 c, = 2p: δ l
'13-i6/, c, = 2p; δ16/-113, c
,,2p iδ,2. 'J2. s.

Jpiδ, 2.7! ;-3,0otr Cg 2p *δ
, :/,7g,c,λp;δlA2g,se,/p;δ
'A4'g, q, /p ;a'A9g, s, J
p: a 4.2g, s, / p; δ7/4t-
7.39. c, /θp; δ79/&-2! ;'
l v c * −2p rδ7.74. d, /p
;δ7.7g, d, /p:δg, J/, d, /
p; δwag θ, c, /p; δ/θ months”-Sr/l
'xMS Parent molecule ion = 6g/ Elemental analyzer - Itch 1 direct (as Csg HsIIN40y F3)
C6/7 Chi H left/g% N g, 23% F g, 37 Chi actual measurement C/, 2.2. ff% H 3g % N g, 70% F g, 01. % Mosuichi 1kari/Otsu CBZ-L-Phe-L-Trp-AMC, pre-construction (
Part 3 of Presentation II! using hydrogenated grogging and/or
HOAc of yatap//rrt,//ml
l's CHsCN, '1me's H! 0 to 4'+ crystals were synthesized as detailed in the above steps.

The overall yield was 3/T.

MP=/4 left -767°C. (Silence F) Scream S1gri0.
1-IPLC, C1Lt5%CHsCN/33'1rh
hO) / MUλmln, RI9RIW, 'II-NMR:
δko,gθtS+jp*δλ741. , t, , 2p
; δ2, g 3-3.30. c, 2p; δ1A
33. qnt /p, δ1A76゜q+/l): δriq7. s, , 2p; δ O - 1g, s, / p
;δμ97゜t, /p;δ707. t, /p
; δ712-73g, c, /Jp; δ'l'1g, d
, / p + δ7.63. d, /p;J27J d,
/p; δ776, S, /I); δg, 3g, d,
/p; 2 for δ/θ. s.

l +, ψ λ l/IQ/ + 7+MS Parent molecule ion-OtlI3 Elemental content tlr 1st row: J'i I direct (as Css Ha<N406)
C71θ/VH left 33% N g12 coefficient actual measurement value C7i, 2. /chih leftguθchiNghaI 夷〃(haexample/7 CBZ-L-Nalla-L-CIt-AMC, front 7
1 self-produced 1 3 catalytic hydrogenation using a 7J log locking box, HOAc of 6pθ-, g lx me
C)-1sC:N, was recrystallized from HzO of λme and attached to the former 1st layer and 2nd layer as yP yB L. The overall yield was t/3.

!ヱ=, 2.2S9 to 7℃, gate. (D:ψF) =-
,,2θ3°,-I-IPLC,C40%Cl-1*c
N/%O%l1-1tO), 0mln, 999%.

"l-l-1-N: δ130-13A, c, 2p; δ
l, fear-1g3*Co, 2p; δ-Z,'l/
7. s, j p +6.2. g 17-3. ．． 2
2. c.

lIp; δri3g-ri5'/, c, e2p; δl/
,S,,zp;δ””'r ”e 2p;δl>00.
t, /p; δlh2g, s, /p pδ7θ7'
Z4! 5'p e, / =2 p rδ7.7tl
, d, /p; δγ7mu d, /p; δ792- d
, / p iδg, , 22. d, /p ;δzaj
4' t d , / p ; δ/θ30. s, /p
sMS parent molecule ion-Hiroshi Ototo elemental analysis d value (as C5yHsyNsOr)Cot'A,S
-9chiH left 75chiN /l'l/chimi translation 11 , 1 pig liver and 1 neoplastic using the vomit salting out method.
It was prepared from 11 livers of human + t4J. Evaluation is pH! r
. Thiol activity ratio? 7 stain (10 mM) was used as ill for one period, and the buffer solution containing this activation/TJ was prepared fresh at 7°ζ every day. A small tear containing 6 fluffs of cuffs, and 5 g of thickened sand at 37°C in the tappet of an SX Saho fluorometer.
The mixture was mixed with 5 μl of a fresh buffer solution containing a diol activator, and incubated at 37° C. for 2 minutes. θ(to θθ5+n
M~, 1 tremor of DMF substrate in the range of 2mM, θ
It was added with mixing. increase in fluorescence, )l?
-Kin x Lumar- (Perkin Elmer) L
S-5'y) Light quenching using current trr for 37 minutes
The temperature was monitored hourly.

The excitation wavelength for AMC is 3Sθμm, its emission wavelength is l16θn m, -t and the emission wavelength for AFC is t'j: 3g 5 n (in 11, (bsu, its meaning) ゛The 0 wavelength was getta θnm.+
3 h・, t: U' 3 vyrn,
The filter response was / and the scale factor of 7 was set to θ~10. Substrate Zanol/~/7 and commercially available Ql$CBZ-pHQ-8rq-AFC (Example/g), CBZ-Ala-Arg-Arp・-A
F C (琵h'lU row/9) and BZ-Va/J-L
ys -Lys-Arg-AFC (actual 1lii column, 2o
) The results of the enzymatic reaction are listed in the t4C/Table.

From the above data, this 'f@Ming group -R/~17
(pH-i table) is f4'j / 1Q base 4/za ~
λ0A Rirui combines with X and cures the positively charged fortune in the ljR1 position.It is currently used for R Guchido-no-like.Compared to Buchige, it is a highly selective song and impression II. It is clear that -C has -C. Trift-dried enzymes are commonly found in human 11'IL fluid, so such enzymes can be used to measure catezin B.
J)j,! f. Motoyaku's pegtide substrate avoids or reduces the problem of interference caused by tryfusin-like enzymes, and inhibits the sensitivity to cadezocin B. 1 to 6θ to 3Sθ times the sensitivity IW of 20 pegtides
1 is in dispute.

In Mizumoto/iJ's other Kotobuki 1 child, Katezushin 8's life 1
By contacting the prepared body fluids of 4 cows, 4 sucklings, and 1 cutler body fluid with a suitable sample of the present invention, it is possible to make a definite or semi-definite, ++llI >, L result. . For example, it'll selectively smear cysts, tissue or organ samples in a clinical setting such as 1!・1].
The method includes chromatographic analysis of cells in which the cells are brought into contact with the aforementioned substrates or reagents. Another suitable clinical analysis is to use cellulosic materials or non-woven materials, or one or more of them, such as by encasing them in a relatively soft inert carrier.
You can also go with Satomoe/j Death Strig. When the lean pull is brought into contact with the test strip, the sample is absorbed by the test strip and the color change is observed for a comparatively quick analysis.

Claims (1)

[Scope of Claims] (1) A method for selectively evaluating the activity of cathepsin B in a substance that may contain trypsin and trypsin-like enzymes; Z-R, -R, -X group JJIL or an acid salt thereof (wherein X is an indicator group that is released by cathepsin B upon cleavage of the R, -X bond and can be detected upon cleavage; R. is an amino acid group that has a structure at the carbon α position relative to the carbonyl group and is not positively charged within the pn range to be evaluated; R1 is the carbon α position relative to the carbonyl group. The above is a hydrophobic amino acid group having an L-structure, and 2 is an amino blocking group that does not prevent cathepsin B from selectively binding to R1 and R1 groups.)
and the mixture has a p range in which cathepsin B is active.
and the mixture contains the above substrate at a concentration sufficient to detect the cleaved X-group. and measuring the rate of cleavage of the X-group from the substrate. C2X is a cleavable group whose fluorescent emission changes upon cleavage fs (method according to item C. (♂ X is a cleavable group whose visible spectrum or ultraviolet spectrum changes upon cleavage) Claim No. (
The method described in Section C. (4'lR+ is a citrullyl group. Method according to claim (C) (5) The method according to claim (1), where R+ is a nitroarginyl group. The method according to claim (1), where Rt is a phenylalanyl group.
The method described in Section 1. (g) Rt is a tryptohyl group (method described in claim 1); (91Rt is inleucyl group) claim (c); (101R, is valyl) Claim No. (//
The method described in Section 1. (//IR, is a phenylalanyl group, and R is a citrulyl group. , and R
. The method according to claim @(1), wherein is a nitroarginyl group. ! /, 7) The method according to claim @(1), wherein R is a citrulyl group, and R7 is a valyl group. i/4tIR, is a citrulyl group, and R1 is a tryptohyl group. The method according to claim (1). (8) A method suitable for f'F #i; (8) Sample f of the above substance l1gI;
-rt. The substrate of -X or their acid salts (in the formula It is an amino acid group that has an L-structure on the carbon and is not charged within the range of pu to be evaluated: Rt is a hydrophobic amino acid group that has an L-structure on the carbon located at the α position with respect to the carbonyl group. amine P group, and Z is cathepsin B is R, and R1
It is an amino blocking group that does not prevent selective binding to the group. ), and the contact with the substrate is carried out under conditions in which cathepsin B is active, at a concentration in which the substrate is present at a concentration substantially greater than the concentration of cathepsin B. and (bJ) The above detection method comprising the step of measuring Xf generated by cleavage from a substrate. The method according to (1) Claims in which X is a cleavable group whose visible spectrum or ultraviolet spectrum changes upon cleavage.
The method according to No. ll (/61J7i. 1/9) R is a citrullyl group.
! The method described in concave item +/A1. The method according to claim (16), wherein t:vlR, is a nitroarginyl group. [,2/l R, Ka, %i' which is a phenylalanyl group
f The method according to claim (76). The method according to claim (/6), wherein , 221R1 is a leucyl group. L: 4'3 + R, is a tryptohyl group lR#″r
The method according to claim (16). Claim 11, in which u/JR is an isoleucyl group
The method described in paragraph 1 (m). Claim No. (/) in which L7jlR, is a valyl group
The method described in section 6). t, 2Alr+, but 7E; lualanyl group and J1R+
The method according to item (/6), wherein is a citrullyl group. ψ) The method according to claim 1, wherein R is a phenylalanyl group and RI is a nitroarginyl group. The method according to claim (/6), wherein IJIR is a citrulyl group and R2 is a valyl group. (, 291R+ is a citrulyl group, 69, and R7 is tryptohyl). (Claim 301R, is a citrulyl group, and R1 is an inleucyl group) The method according to item (16). (3) Compounds of formula 1 Z'''Rz -Rt -X and their acid salts, where X is an indicator group suitable for cleavage from R5. , Ri: Rx is an amino acid group which has Li structure on the carbon at the α position of the carbonyl group and is not positively charged; R2 is an amino acid group at the carbon at the α position of the carbonyl group.
2 is a hydrophobic amino acid group having the following structure; and 2 is an amino blocking group). (,?,21 The compound according to claim (3/), wherein X is a υIJ cleavable group whose fluorescence changes upon cleavage. +3.?) Claim i, which is a cleavable group whose spectrum or ultraviolet spectrum changes? /1. The compound according to item (31) (Claim v in which 341Rt is a phenylalanyl group) (The compound according to item 3/+) (Claim v in which 341Rt is a phenylalanyl group) (,
Compound described in Section 7/1. The compound according to claim 3/1, wherein t3glRt is a tryptohyl group. (The compound according to claim 3, wherein 391Rt is an inleucyl group. , Claim No. 3 which is a valyl group
/) Method described in section. (4/J R,, phenylalanyl group 6'), R
The compound according to claim (3/), wherein + is a citrullyl group. (4t, 1. I R, Ka, , y enylalanyl group with 6
R+ is a nitroarginyl group? iF A compound according to claim (3/). (lA31R+ is a citrulyl group and R2 is) (
The compound according to claim (3/), which is a lyle group. The compound according to claim (3/), wherein f IRI is a citrulyl group and R2 is a l-IJ buthiryl group. (The compound according to claim (3/), wherein VIR+ is a citrulyl group and R2 is an isoleucyl group. (76) Compounds of the following formula and their acid salts. Rs is a side chain of an amino acid group that has an L-structure and is not positively charged at the carbon at the α position of the carbonyl group, and R3 has 2 to 5 atoms; R4 is at the carbon at the α position of the carbonyl atom. is a side chain of a hydrophobic amino acid group having an L-structure in the vertical atom of , and has the formula -c+-+ of 841
t-"v'ty is selected from the group consisting of lower alkyl, phenyl, substituted phenyloyothintole groups; X is an indicator group suitable for cleavage from the described compound; and 2 is The compound according to claim 6, which is a chlorine-blocking group. A compound according to Claim No. 6. A compound according to Claim No. 6, wherein (/Lt?JR, is -CHz -C6He. (SI) R4 is , -CHt -CH-(CHs lt
The compound according to claim 6, which is The compound according to claim 6, which is The compound according to claim 6, which is (531R4 is -CH-CHv-CHs. The compound according to scope item (q6). (531R4 is -CH2-CsHe, R3 is
1 -CHt -CHt -CHt -N1-(-C-NH
The compound according to claim 6, which is y. (,ffj)R, is -CHt-C6He, and R
5 is NH 1 -CHz -CHt -CHt -NH-C-NH
The compound according to claim (6), which is -N Os. (561R, is -CHs -CHs -CHt -N
H-C-NHt8, and R4 is C)(-CH, the compound according to claim I CHx Disciple (da 6). -CH2-NH-C-NH2r6
A compound according to claim 1. (If IR8 in S is C H1 -CH!-CH! -CHx-NH-C-NH2, and R4 is CH-CH, -CH, then the range of Hll is allowed in the (da 6) term. The compound according to Claim 1'16), wherein #l X is a cleavable group whose fluorescence changes upon cleavage. Compounds described in Section 1, “Spectrum Range @ (G6),” which are cleavable groups whose spectra change.