Tryptase Activity Measurement Substrate

Abstract

An object of the present invention is to provide a method for measuring tryptase activity in a blood sample accurately and rapidly by a convenient operation in order to accurately evaluate the state of a disease whose state involves mast cells. The present invention enables tryptase activity in a blood sample to be directly measured without the pretreatment, such as purification or concentration, of the blood sample, using a substrate for measuring tryptase activity, comprising a tripeptide C-terminally linked through a peptide bond to a dye label, selected from the following formulas (1) to (3): (1) Lys-Ala-Arg-X, (2) Ala-Ala-Arg-X, and (3) Abu-Ala-Arg-X (wherein X represents a dye label whose fluorescence characteristics or color development characteristics change upon the cleavage of the peptide bond with Arg, and Abu represents 2-aminobutyric acid).

Claims

1. A substrate for measuring tryptase activity, comprising a tripeptide C-terminally linked through a peptide bond to a dye label (tripeptide-X), selected from the following formulas (1) to (3):
Lys-Ala-Arg-X,  (1)
Ala-Ala-Arg-X, and  (2)
Abu-Ala-Arg-X,  (3) wherein X represents a dye label whose fluorescence characteristics or color development characteristics change upon the cleavage of the peptide bond with Arg, and Abu represents 2-aminobutyric acid.

2. The substrate for measuring tryptase activity according to claim 1, wherein the substrate for measuring tryptase activity is for directly measuring tryptase activity in a blood sample.

3. The substrate for measuring tryptase activity according to claim 1, wherein a poorly tryptase-digestible water-soluble polymer having a molecular weight of 5,000 or higher is linked to the N terminus of the tripeptide-X.

4. The substrate for measuring tryptase activity according to claim 3, wherein the poorly tryptase-digestible water-soluble polymer is a polyamino acid.

5. The substrate for measuring tryptase activity according to claim 4, wherein the polyamino acid is selected from poly(L-lysine), dendritic poly(L-lysine), poly(D-lysine), dendritic poly(D-lysine), poly(L-ornithine) and poly(D-ornithine).

6. The substrate for measuring tryptase activity according to claim 1, wherein the dye label is a fluorescent dye label selected from an MCA group, an ANS group, a CMCA group, an FMCA group, an AMP group, a rhodamine 110 group, a rhodamine 110 monoamide group, a rhodamine 6G group and a rhodamine B group.

7. A method for measuring tryptase activity in a blood sample, comprising the following steps (A) and (B): (A) contacting the blood sample with the substrate for measuring tryptase activity comprising a tripeptide C-terminally linked through a peptide bond to a dye label (tripeptide-X), selected from the following formulas (1) to (3); and (B) calculating a degree of the tryptase activity in the blood sample by measuring an amount of change in fluorescence characteristics or color development characteristics of a dye label after the step (A):
Lys-Ala-Arg-X,  (1)
Ala-Ala-Arg-X, and  (2)
Abu-Ala-Arg-X  (3) (wherein X represents a dye label whose fluorescence characteristics or color development characteristics change upon the cleavage of the peptide bond with Arg, and Abu represents 2-aminobutyric acid).

8. The method according to claim 7, wherein the blood sample is serum.

9. A kit for measuring tryptase activity in a blood sample, comprising the substrate for measuring tryptase activity comprising a tripeptide C-terminally linked through a peptide bond to a dye label (tripeptide-X), selected from the following formulas (1) to (3):
Lys-Ala-Arg-X,  (1)
Ala-Ala-Arg-X, and  (2)
Abu-Ala-Arg-X,  (3) wherein X represents a dye label whose fluorescence characteristics or color development characteristics change upon the cleavage of the peptide bond with Arg, and Abu represents 2-aminobutyric acid.

10. The kit according to claim 9, wherein the blood sample is serum.

11. The substrate for measuring tryptase activity according to claim 2, wherein a poorly tryptase-digestible water-soluble polymer having a molecular weight of 5,000 or higher is linked to the N terminus of the tripeptide-X.

12. The substrate for measuring tryptase activity according to claim 2, wherein the dye label is a fluorescent dye label selected from an MCA group, an ANS group, a CMCA group, an FMCA group, an AMP group, a rhodamine 110 group, a rhodamine 110 monoamide group, a rhodamine 6G group and a rhodamine B group.

13. The substrate for measuring tryptase activity according to claim 3, wherein the dye label is a fluorescent dye label selected from an MCA group, an ANS group, a CMCA group, an FMCA group, an AMP group, a rhodamine 110 group, a rhodamine 110 monoamide group, a rhodamine 6G group and a rhodamine B group.

14. The substrate for measuring tryptase activity according to claim 4, wherein the dye label is a fluorescent dye label selected from an MCA group, an ANS group, a CMCA group, an FMCA group, an AMP group, a rhodamine 110 group, a rhodamine 110 monoamide group, a rhodamine 6G group and a rhodamine B group.

15. The substrate for measuring tryptase activity according to claim 5, wherein the dye label is a fluorescent dye label selected from an MCA group, an ANS group, a CMCA group, an FMCA group, an AMP group, a rhodamine 110 group, a rhodamine 110 monoamide group, a rhodamine 6G group and a rhodamine B group.

16. The method according to claim 7, wherein a poorly tryptase-digestible water-soluble polymer having a molecular weight of 5,000 or higher is linked to the N terminus of the tripeptide-X.

17. The method according to claim 16, wherein the poorly tryptase-digestible water-soluble polymer is a polyamino acid.

18. The method according to claim 17, wherein the polyamino acid is selected from poly(L-lysine), dendritic poly(L-lysine), poly(D-lysine), dendritic poly(D-lysine), poly(L-ornithine) and poly(D-ornithine).

19. The method according to claim 7, wherein the dye label is a fluorescent dye label selected from an MCA group, an ANS group, a CMCA group, an FMCA group, an AMP group, a rhodamine 110 group, a rhodamine 110 monoamide group, a rhodamine 6G group and a rhodamine B group.

20. The method according to claim 8, wherein a poorly tryptase-digestible water-soluble polymer having a molecular weight of 5,000 or higher is linked to the N terminus of the tripeptide-X.

Description

EXAMPLE 1

1. Screening of Tryptase Substrate

[0045] A total of 17 types of MCA substrates were chemically synthesized by adopting document information on the amino acid sequences of previously reported tripeptide-MCA substrates used for tryptase, and a simplified library approach. .sup.iBoc-Ala-Ala-Arg-MCA having the best responsiveness and physical properties was found for commercially available tryptase separated from the human lung. .sup.iBoc-Ala-Ala-Arg-MCAP_mcs was synthesized by the following procedures.

(1) Under ice cooling, N,N′-dicyclohexylcarbodiimide (DCC) (450 mg, 2.2 mmol) was added to a solution of Fmoc-Arg(Pmc)-OH (manufactured by Watanabe Chemical Industries, Ltd., 4 mmol) in dichloromethane (DCM), and the mixture was stirred for 1 hour. AMC (manufactured by Tokyo Chemical Industry Co., Ltd., 400 mg, 2.2 mmol) was added to the formed symmetric acid anhydride, and the mixture was reacted overnight at room temperature to obtain Fmoc-Arg(Pmc)-MCA.
(2).sup.iBoc-Ala-OH (own made, 1.89 g, 10 mmol) and H-Ala-OBzl_HCl (own made, 2.12 g, 10 mmol) were condensed according to a standard method to prepare .sup.iBoc-Ala-Ala-OBzl. Further, catalytic reduction was performed in methanol using 5% Pd—C to obtain .sup.iBoc-Ala-Ala-OH [yield: 2.08 g (80%)].
(3) Fmoc-Arg(Pmc)-MCA (1.0 mmol) was dissolved in 20% piperidine (5 mL), and the solution was reacted at room temperature for 2 hours. The solvent was distilled off, and the residue was dissolved in N,N′-dimethylformamide (DMF). To this solution, .sup.iBoc-Ala-Ala-OH (260 mg, 1.0 mmol) was added, followed by condensation by the HBTU/HOBt method. The product .sup.iBoc-Ala-Ala-Arg(Pmc)-MCA was purified by silica gel chromatography to obtain 420 mg (50%) of white crystals.
(4).sup.iBoc-Ala-Ala-Arg(Pmc)-MCA (400 mg, 0.45 mmol) was dissolved in trifluoroacetic acid (TFA) (5 mL), and the solution was reacted at room temperature for 2 hours. TFA was distilled off, and diethyl ether was added to the residue to obtain Pmcs salt as a crystalline powder [yield: 365 mg (91%)].

[0046] In addition to reference to past documents as described above, .sup.iBoc-Y-X-Arg-MCA in which Gly, Ala, Abu, Val, Pro, Phe or hydrophilic Lys having distinctive bulkiness was introduced to positions P.sub.2 and P.sub.3 was synthesized as listed in Table 1 by a peptide library approach, and activity measurement was performed. Abu (2-aminobutyric acid) is a non-protein amino acid, but was added as an optimum peptide sequence search tool.

[0047] The activity measurement was performed by the following method.

(1) A stock of a substrate solution was provided as a 2.0 mM solution in DMSO.
(2) The stock of the substrate solution was diluted 20-fold with 50 mM Tris-HCl (pH 8.0) to prepare a 100 μM solution. 100 μL thereof was added to a polypropylene microtube, further 2 μL of a commercially available tryptase solution (manufactured by Cappel Laboratories, Inc.) was added thereto, and the tube was lightly vortexed for mixing. Immediately thereafter, change in fluorescence intensity at 470 nm was measured at an excitation wavelength of 365 nm using an enzyme activity measurement apparatus (manufactured by Peptide Support Ltd., prototype).

TABLE-US-00001 TABLE 1 Amino acid sequence of fluorescent substrate and comparison of responsiveness to human lung tryptase*.sup.1 .sup.iBoc-P.sub.3-P.sub.2-Arg-MCA*.sup.2 Tryptase activity*.sup.3 .sup.iBoc-Phe-Ser-Arg-MCA 11 .sup.iBoc-Arg-MCA 0 .sup.iBoc-Gly-Gly-Arg-MCA 0 .sup.iBoc-Phe-Gly-Arg-MCA 5 .sup.iBoc-Lys-Gly-Arg-MCA 75 .sup.iBoc-Ala-Ala-Arg-MCA 100 .sup.iBoc-Abu-Ala-Arg-MCA 109 .sup.iBoc-Val-Ala-Arg-MCA 53 .sup.iBoc-Phe-Ala-Arg-MCA 62 .sup.iBoc-Lys-Ala-Arg-MCA 167 .sup.iBoc-Ala-Abu-Arg-MCA 80 .sup.iBoc-Abu-Abu-Arg-MCA 69 .sup.iBoc-Gly-Pro-Arg-MCA 9 Tos-Gly-Pro-Arg-MCA 25 .sup.iBoc-Val-Pro-Arg-MCA 20 .sup.iBoc-Lys-Pro-Arg-MCA 175 .sup.iBoc-Ala-Val-Arg-MCA 50 .sup.iBoc-Ala-Phe-Arg-MCA 1 .sup.iBoc-Ala-Lys-Arg-MCA 11 *.sup.1Information on purchased tryptase. Enzyme reaction conditions. *.sup.2In these compounds, the sulfonic acid (Pmcs, pentamethylchromansulfonic acid) formed a salt as a counter ion with a liberated guanidine group when the Pmc- group, a protective group of the guanidine group of arginine, was removed by trifluoroacetic acid treatment. *.sup.3Relative value with the sensitivity of .sup.iBoc-Ala-Ala-Arg-MCA defined as 100.

[0048] By the screening described above, we were able to obtain .sup.iBoc-Abu-Ala-Arg-MCA and .sup.iBoc-Lys-Ala-Arg-MCA as tripeptide substrates having higher tryptase responsiveness than that of .sup.iBoc-Ala-Ala-Arg-MCA. Also, we were able to confirm that the tryptase substrate .sup.iBoc-Lys-Pro-Arg-MCA having a known tripeptide moiety according to non-patent document 15 have tryptase responsiveness slightly less than two times higher than that of .sup.iBoc-Ala-Ala-Arg-MCA. In the -Arg-Asn-Arg- sequence of the tryptase substrate described in non-patent document 18, Asn at position P.sub.2 also exhibits slightly more than two times the responsiveness of Ala at position P.sub.2, and further, at position P.sub.3, an amino acid having a positively charged side chain has sensitivity about two times better than that of Ala at position P.sub.3. In light of these, the substrates were considered to have responsiveness approximately 5 times higher than that of the -Ala-Ala-Arg- sequence, albeit according to calculation.

[0049] The following findings were obtained as the outcomes of search for suitable amino acid sequences of the tripeptide.

[0050] It is well known as specificity that a proteolytic enzyme (protease) differs in the hydrolysis reaction rate of a peptide bond depending on the structure of the side chain atomic group of an amino acid contained in a protein. Tryptase primarily recognizes Arg having a positively charged side chain and hydrolyzes a peptide bond on the carboxyl group side thereof. The position of an amino acid that is primarily recognized by an enzyme is defined as position P.sub.1, and amino acids toward the N terminus therefrom are designated as positions P.sub.2 and P.sub.3 in order. Even such one amino acid may have not small influence on difference in the rate of enzyme reaction. Amino acids in proteins are broadly divided, according to their side chains, into 4 types, acidic amino acids, basic amino acids, hydrophobic amino acids, and neutral hydrophilic amino acids. These amino acids may be typified by Glu, Arg, Phe, and Ser, respectively. Accordingly, Gly, Ala, Val, Phe, Ser, or Pro was introduced as the amino acid at position P.sub.2 while Glu was excluded therefrom. Likewise, each of the typical amino acids was also introduced to position P.sub.3. Lys was also used at position P.sub.3. Since a number of compounds needed to be synthesized, a method for purifying synthetic intermediates was simplified and a safe .sup.iBoc group was used for trifluoroacetic acid in the removal of a protective group in the final step. After removal of the Pmc protective group on the side chain of Arg with trifluoroacetic acid, the product formed a salt with the side chain of Arg. Therefore, most of .sup.iBoc-tripeptide-MCA compounds were obtained in a favorable powder form.

[0051] As a result of studying the experimental results of Table 1, we were able to summarize features of the amino acid sequences of the substrates for tryptase as the following 5 points. (i) It is evident that Gly at position P.sub.2 is not suitable. (ii) Ala with a methyl group having a small side chain is most suitable for position P.sub.3, and a bulky amino acid such as Val or Phe decreased responsiveness. A large side chain may be difficult for subsite S.sub.2 of the enzyme to accommodate. Pro is an amino acid that also often appears at position P.sub.2 in substrates for other proteases. (iii) Non-protein Abu exhibited slightly lower responsiveness than that of Ala. (iv) At position P.sub.3, as in position P.sub.2, a small side chain of an amino acid such as Ala or Abu is acceptable, and a bulky hydrophobic side chain is difficult to bind. (v) On the other hand, positively charged Lys was found to be accommodated at position P.sub.3. Provided that a natural substrate protein for tryptase will be identified in the future in relation to pathology and the amino acid sequence of its site of action will be determined, Lys will be found at position P.sub.3 thereof.

EXAMPLE 2

[0052] 2. Synthesis of Macromolecular Substrate in which Suc-Ala-Ala-Arg-MCA is Linked to Poorly Tryptase-Digestible Water-Soluble Polymer

[0053] A substrate for measuring tryptase activity linked to poly(L-lysine) as a poorly tryptase-digestible water-soluble polymer was synthesized by the following method.

(1) Fmoc-Arg(Pmc)-MCA (own made, 1.1 mmol) was dissolved in 20% piperidine in DMF (10 mL) and reacted for 90 minutes. DMF was distilled off to obtain free amine in a white solid form. This solid was dissolved in DMF (5 mL). To the solution, Boc-Ala-Ala-OH (own made, 1.2 mmol) was added, followed by condensation by the HBTU/HOBt method. After reaction for 4 hours, Boc-Ala-Ala-Arg(Pmc)-MCA was isolated and purified by silica gel chromatography [yield: 505 mg (62%)].
(2) Boc-Ala-Ala-Arg(Pmc)-MCA (440 mg) was dissolved in TFA (5 mL) and reacted for 10 minutes. TFA was rapidly distilled off, and the residue was crystalized with diethyl ether.
(3) TFA H-Ala-Ala-Arg(Pmc)-MCA (390 mg) was dissolved in DMF (5 mL). Under ice cooling, NEt.sub.3 (2.2 eq.) and succinic anhydride (1.2 eq.) were added to the solution, and the mixture was reacted overnight at room temperature. Formed Suc-Ala-Ala-Arg(Pmc)-MCA was extracted and isolated, further dissolved in TFA (3 mL), and reacted for 2 hours to remove the Pmc group.
(4) Poly(L-lysine) hydrochloride having a molecular weight of 8,000 or higher (manufactured by Peptide Institute, Inc., Lot. 670515, 165 mg, 1 mmol) was dissolved in water (5 mL). To the solution, Suc-Ala-Ala-Arg-MCA_Pmcs (164 mg, 0.2 mmol) was added, and the hydrochloride was neutralized with NaHCO.sub.3 (21 mg, 0.5 mmol). Further, pH was kept at 8 to 9 by the addition of N-hydroxysuccinimide (HOSu, 23 mg, 0.2 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide_HCl (EDC, MW 191.7, 96 mg, 0.5 mmol). Since the reaction solution foamed, ethanol (1 mL) was added as an antifoaming agent 5 hours later. Reaction was performed for 2 days.
(5) The reaction solution was concentrated by the addition of acetic acid in a small amount. When ethanol was added to the residual syrup, it was solidified. On the next day, the solvent was removed by decantation, and the residue was dissolved again in distilled water. The solution was concentrated with ethanol as an antifoaming agent, and ethanol was added to the residual syrup to obtain a white solid. 220 mg of Suc-Ala-Ala-Arg-MCA-linked poly(L-lysine) was obtained by vacuum drying.
(6) 3.81 mg of Suc-Ala-Ala-Arg-MCA-linked poly(L-lysine) was dissolved in 50 mL of distilled water. When an absorption spectrum was measured, absorbance at 280 nm was 0.55.

[0054] Also, a substrate for measuring tryptase activity linked to poly(D-lysine) as a poorly tryptase-digestible water-soluble polymer was synthesized by the following method.

(7) Tripeptide-MCA was condensed with poly(D-lysine) by the same procedures as in the steps (1) to (6) except that poly(D-lysine) hydrobromide having a molecular weight of 4,000 to 15,000 (manufactured by Sigma-Aldrich Co. LLC, Lot. SLBZ6409, 42 mg, 0.2 mmol) and Suc-Ala-Ala-Arg-MCA_Pmcs (33 mg, 0.04 mmol) were used in the step (4). 38 mg of the title compound was obtained.

[0055] Dendritic poly(lysine) was considered as a macromolecule suitable for suppressing the access of MCA substrates to blood coagulation system protease such as thrombin trapped by α2 macroglobulin tetramer. Accordingly, dendritic poly(L-lysine) and dendritic poly(D-lysine) were prepared by the following method.

(8) Two molecules of di-Boc-L-lysine were condensed with hexamethylenediamine. After removal of the Boc groups with TFA, four molecules of di-Boc-L-lysine were condensed with four liberated amino groups (second-generation K6B8 wherein K represents the number of L-lysine molecules, and B represents the number of Boc groups). This operation was repeated to expand the dendrite to third-generation K14B16, fourth-generation K30B32, and fifth-generation K62B64 (Chem. Commun., 1999, 2057-2058 (1999)). K14B16, K30B32, and K62B64 were treated with TFA to obtain TFA salts of K14A16 (wherein A represents a free amino group (TFA salt) after deprotection), K30A32, and K62A64 as white powders (Table 2).
(9) TFA salts of k14A16 (wherein k represents the number of D-lysine molecules), k30A32, and k62A64 were obtained as white powders by the same procedures as above except that di-Boc-D-lysine was used instead of di-Boc-L-lysine in the step (8) (Table 2).

TABLE-US-00002 TABLE 2 The numbers of lysine molecules, the numbers of free amino groups, molecular weights as TEA salt, synthesis yields and yield percentages of dendritic poly(L-lysine) and poly(D- lysine) in each generation The number Synthesis of free Molecular yield and amino weight (+ yield Generation Abbreviation groups*.sup.) TFA salt) percentage 3G K14A16 16 1908 + 1824 908 mg (86%) 4G K30A32 32 3956 + 3648 762 mg (77%) 5G K62A64 64 8052 + 7296 341 mg (75%) 3G k14A16 16 1908 + 1824 1.85 g (83%) 4G k30A32 32 3956 + 3648 1.13 g (80%) 5G k62A64 64 8052 + 7296 962 mg (75%) *.sup.)k is an abbreviation of D-lysine.

[0056] The obtained dendritic poly(lysine) was linked to Suc-Ala-Ala-Arg-MCA by the following method.

(10) The dendritic poly(D-lysine) 5Gk62A64 (316 mg) was dissolved in water (10 mL). To the solution, Suc-Ala-Ala-Arg-MCA_Pmcs (330 mg, 0.4 mmol) was added, and the trifluoroacetate was neutralized with NaHCO.sub.3 (43 mg, 1 mmol). Further, N-hydroxysuccinimide (HOSu, 50 mg, 0.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide_HCl (EDC, 200 mg, 1 mmol) were added thereto, followed by reaction as described in the step (4). 343 mg of 5Gk62A64-Suc-Ala-Ala-Arg-MCA acetate was obtained.

EXAMPLE 3

3. Activity Measurement of Tryptase in Serum Using Substrate of Present Invention

[0057] Suc-Ala-Ala-Arg-MCA-linked poly(L-lysine) prepared in Example 2 was used to test whether to be able to specifically measure tryptase activity in serum.

(1) 20.0 mg of Suc-Ala-Ala-Arg-MCA-linked poly(L-lysine) was dissolved in 50 mL of 50 mM Tris-HCl (pH 8.0).
(2) 2 μL of commercially available human serum (manufactured by BioWest) was mixed with 2 μL of PBS(−) (pH 7.4) (control) or 2 μL of 10 μM nafamostat (tryptase inhibitor, manufactured by Tokyo Chemical Industry Co., Ltd.) or 2 μL of 10 U/mL hirudin (thrombin inhibitor, manufactured by Sigma-Aldrich Co., LLC). After a lapse of 10 minutes at room temperature, each mixed solution was added to 100 μL of the solution prepared in the step (1). Immediately thereafter, enzyme activity was measured.

[0058] The results are shown in the following Table 3.

TABLE-US-00003 TABLE 3 Comparison of human serum enzyme activity by addition of various inhibitors Reaction solution Enzyme activity *.sup.1 Serum + PBS (−) 100 Serum + nafamostat 0 Serum + hirudin 83 *.sup.1 Relative value with the enzyme activity of serum + PBS(−) defined as 100

[0059] As seen from Table 3, the enzyme activity in serum detected with the macromolecular substrate of the present invention was completely inhibited by the tryptase inhibitor nafamostat, but not by the thrombin inhibitor hirudin. These results indicated that the enzyme activity in serum detected with the macromolecular substrate of the present invention was ascribable to tryptase and the tryptase activity can be specifically measured even in a serum sample containing a blood coagulation system enzyme such as thrombin in a state trapped by α2 macroglobulin tetramer.

INDUSTRIAL APPLICABILITY

[0060] The present invention enables tryptase activity in a blood sample to be directly measured without the pretreatment, such as purification or concentration, of the blood sample. Hence, mast cell activation syndrome, which has been determined so far only from the abundance in blood of a tryptase protein-related antigen, can be determined from tryptase activity which more accurately reflects the disease state. Therefore, the present invention has high industrial applicability in the medical field.