Compounds and methods for high-purity chemiluminescent substrates

Abstract

This invention relates to substrate molecules suitable for monitoring enzyme activity by chemiluminescence. These substrates are provided by solid-phase peptide synthesis using aminoluciferin precursors. After solid-phase synthesis, the precursor is converted to the aminoluciferin moiety. The above substrates are highly pure and stable.

Description

[Technical Field]

[0001] This invention relates to substrate molecules suitable for monitoring enzyme activity by chemiluminescence. These substrates are provided by solid-phase peptide synthesis using aminoluciferin precursors. After solid-phase synthesis, the precursor is converted to the aminoluciferin moiety. The substrates are highly pure and stable. [Background technology]

[0002] In recent decades, luminescence imaging has emerged as a powerful tool for monitoring enzyme activity. Generally, luminescence imaging is performed by the oxidation of small molecules, requires no external light source, provides a high signal-to-noise ratio, and typically has a lower background signal than fluorescence. Numerous methods for luminescence imaging have been reported for applications such as the detection of reactive oxygen and nitrogen species, imaging of cancer cells, and in vivo quantification of glucose uptake.

[0003] White and his collaborators were among the first groups to report the use of aminoluciferin (aLuc), a d-luciferin derivative, which can be used in proteolytic assays because this amine can be attached to the C-terminus of peptides (E.H. White et al., J.Am.Chem.Soc. 1966, 88, 2015-2019). The free 6'-amino group of aLuc appears to be important for maintaining its luminescence (similar to the case of the 6'-hydroxyl group in d-luciferin), and modification of this portion usually results in loss of luminescence. These luminescent probes, also known as caged luciferin, can be used in luminescence enzyme assays because they release free aLuc that can be quantified by enzymatic activity. Upon luciferase catalysis, aLuc is oxidized to excited oxyluciferin, which is then relaxed back to the ground state and releases photons. Numerous luminescent probes have been developed using this uncaging principle and are widely used with various enzymes such as hydrolases and proteases; see, for example, International Publication No. 2020 / 079155.

[0004] Within the scope of luminescence imaging applications, there is a need for novel caged luciferins in which the luciferin moiety is cleaved by enzymatic activity to obtain a proportional optical signal. Current preparation methods for luminescent caged peptides mostly rely on solution-phase synthesis. Generally, the C-terminus of a protected peptide is activated with a reagent such as isobutyl chloroformate in the presence of a base such as 4-methylmorpholine (NMM), followed by the addition of the aLuc precursor 6-aminobenzo[d]thiazole-2-carbonitride (6-ABTC, Figure 1A, see D. Sondag et al., ChemBioChem 2022, 23). After this coupling, the protecting group of the peptide's side chain is cleaved, and a final condensation reaction with d-cysteine ​​yields the caged luciferin peptide. [ka]

[0005] The use of solid-phase peptide synthesis (SPPS) for the synthesis of caged luciferin significantly reduces the number of solution-phase synthesis steps, eliminates several purification steps, and enables parallel synthesis. Kovacs et al. (Int. J. Pept. Res. Ther. 2019, 25, 1209-1215) demonstrated that loading the carboxylic acid of aLuc onto a solid support and subsequent peptide chain extension is unsuccessful due to the inherent instability of the thiazoline moiety of aLuc (Figure 1B). The thiazoline ring of luciferin is readily oxidized, which can lead to the formation of dehydrogenated derivatives, which are well-known luciferase inhibitors. Therefore, Kovacs et al. recommended the use of liquid-phase coupling of the peptide to aLuc.

[0006] Improved caged doruciferin is needed. Improved methods for supplying caged doruciferin are needed. Higher purity caged doruciferin is needed. [Overview of the Initiative]

[0007] The inventors have developed a solid-phase peptide synthesis (SPPS) method for synthesizing caged luminescent peptides starting from sidechain anchoring of a side chain containing an amine such as a lysine or ornithine residue (Figure 1C). The amine moiety was guanidylated on a resin to obtain an arginine residue or analog. By introducing an aminoluciferin (aLuc) moiety later, this method can be adapted to SPPS because a more stable 6-ABTC moiety is introduced to the C-terminus of the first residue before the solid-phase reaction. Therefore, the present invention relates to a method for producing peptide conjugates: i) General formula (I): [ka] (wherein n is 1, 2, 3, 4, or 5; p' is a protecting group) The present invention provides a method comprising the steps of: ii) providing a constituent unit; ii) immobilizing the constituent unit on a peptide synthesis resin; and iii) attaching one or more further amino acid residues using conventional solid-phase peptide synthesis to obtain an immobilized peptide conjugate.

[0008] Preferably, the above method further comprises: iv) cleaving the immobilized peptide conjugate from the resin to obtain a free peptide conjugate, and optionally purifying it. Preferably, the above method further comprises: v) contacting the free peptide conjugate with cysteine ​​to obtain a peptide-aminoluciferin conjugate. In a preferred embodiment, step ii) comprises forming an immobilized secondary amine by reductive amination of the free amine of the constituent units onto the aldehyde of the peptide synthesis resin, where the immobilized secondary amine is optionally subsequently protected. Where, in some embodiments, the immobilized secondary amine is guanidylated, and the resulting guanidinium moiety is optionally subsequently protected.

[0009] Compounds of general formula (V) or salts thereof: [ka] (wherein h1 is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; h2 is H, p', C1-4 alkyl, or peptide synthesis resin; n is 1, 2, 3, 4, or 5; R is H, p', X, or polypeptide moiety containing 1 to 10 amino acids; p' is independently a protecting group in each case; X is H, or -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where alkyl and acyl are linear, branched, or cyclic, possibly unsaturated, and possibly substituted with halogens or C1-4 alkoxys.) Also provided. Preferably, R is of the general formula (pep): [ka] (In the formula, sc 1 is preferably an amino acid side chain selected from H or a linear, branched, or cyclic C1-12 alkyl group, where the alkyl is optionally unsaturated and optionally substituted with a halogen, or optionally halogen-substituted -(CH2) 0-4 -Selected from [C5-10 (hetero)aryl]; sc 2 is preferably H, or an amino acid side chain selected from linear, branched, or cyclic C1-12 alkyl groups, where the alkyl is optionally unsaturated and optionally substituted with a halogen, or optionally substituted with a halogen-(CH2) 0-4 -Selected from [C5-10 (hetero)aryl], where sc 2Each of the cases is independently selected; b is -CH2- or absent; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; X is H, or -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where alkyl and acyl are linear, branched, or cyclic, possibly unsaturated, possibly substituted with halogens or C1-4 alkoxys. This is the polypeptide portion. In a preferred embodiment, m is 1. In a preferred embodiment, X is -C(=O)-CH2-O-CH3. In some embodiments, m is 1 or 2, preferably 1;sc 2 If the amino acid moiety adjacent to X is H, then b is -CH2-; if the amino acid moiety adjacent to X is then -CH2-. In some embodiments, h2 is a peptide synthesis resin, preferably an aldehyde-functionalized resin, preferably a highly acid-unstable resin such as (3-formylindolyl)acetamidomethylpolystyrene. Preferably h1 is H, preferably n is 2 or 3, more preferably 3. Preferably h1 is -C(=N-p')-NH-p' or -C(=NH)-NH2, preferably n is 2 or 3, more preferably 2.

[0010] Kits of components comprising i) compounds as defined above; and ii) cysteine, preferably D-cysteine ​​are also provided. A method for measuring protease activity is also provided, comprising the steps of: i) providing one or more compounds as defined above; ii) contacting the provided compounds with cysteine ​​to obtain an aminoluciferin-peptide conjugate; iii) contacting the aminoluciferin-peptide conjugate with a protease to obtain free aminoluciferin; and iv) contacting the free aminoluciferin with luciferase to generate a luminescence signal. [Modes for carrying out the invention]

[0011] In the art, the synthesis of caged luminescent peptide substrates remains challenging, particularly when multiple substrates are required. Currently, the most effective methods rely on solution-phase techniques. In this specification, we provide a solid-phase peptide synthesis (SPPS) method for synthesizing C-terminally caged aminoluciferin peptides by side-chain anchoring of residues adjacent to aminoluciferin. The resulting peptides may be, for example, those shown in Examples 1.3.1.1 and 1.3.2.1. Therefore, the present invention relates to a method for producing peptide conjugates: i) General formula (I): [ka] (In the formula, n is 1, 2, 3, 4, or 5; p' is a protecting group. A step of providing a constituent unit; ii) The step of immobilizing the above-mentioned structural units onto a peptide synthesis resin; iii) A step of obtaining an immobilized peptide conjugate by attaching one or more additional amino acid residues using conventional solid-phase peptide synthesis, This provides a method that includes this.

[0012] Such a method is referred to herein as the method according to the present invention. Preferably, the steps are carried out in ascending order of number. This method avoids the formation of dehydrogenated aminoluciferin (aLux), which is a well-known drawback of the well-known SPPS method for aLuc peptide conjugates.

[0013] [Step i - Providing the constituent units of general formula (I)] In step i) of the above method, a constituent unit is provided. The constituent unit has a free primary amine as part of the side chain of an amino acid residue. This amino acid residue is bound to the C-terminus of aminobenzo[d]thiazole-2-carbonitrile (ABTC). This amino acid residue has a protecting group at its N-terminus, which can be conveniently used in SPPS. This amino acid residue is directly adjacent to the resulting aLuc and may be called P1. Similarly, the amino acid residue directly at the N-terminus of P1 may be called P2, the next one may be called P3, and so on.

[0014] n determines the length of the side chain of the P1 amino acid. In preferred embodiments, n is 1, 2, 3, or 4. More preferably, n is 2, 3, or 4. Most preferably, n is 2 or 3 to form ornithine or lysine.

[0015] p' is a protecting group. Preferably, p' is a protecting group suitable for the SPPS program. A person skilled in the art can select a suitable protecting group for p'. The protecting group is preferably a base instability. As used herein, a protecting group has the usual meaning of a group that attaches to a heteroatom in a way that reduces reactivity or protects it from certain conditions. After performing their function, protecting groups can generally be removed again using ordinary chemistry. A person skilled in the art will be familiar with protecting groups and can select a protecting group that is useful for the specific group or atom to be protected, reaction conditions, storage conditions, purification techniques, or available deprotection techniques. One useful reference in this regard is Greene's Protective Groups in Organic Synthesis, Wuts & Greene, DOI:10.1002 / 0470053488.

[0016] In preferred embodiments, p' is 9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), benzyl carbamate (Cbz), acetamide (Ac), trifluoroacetamide, phthalimide, benzylamine (Bn), triphenylmethylamine (Tr, or Trt), or tosylamide (Ts). Preferred embodiments of the base instability of p' are Fmoc, Cbz, Ac, trifluoroacetamide, and phthalimide, more preferably Fmoc or Cbz, most preferably Fmoc. For its reliability in SPPS, p' is most preferably Fmoc.

[0017] The constituent units of general formula (I) can be synthesized by bonding a suitable amino acid with ABTC, preferably 6-ABTC. It is advantageous that p' is already present during this coupling, and that the free amine in general formula (I) is also preferably protected during this coupling. For this protection, the use of a protecting group orthogonal to p', such as Boc, is preferred. Detailed embodiments are shown in Example 2.1.

[0018] In some embodiments, salts of constituent unit (I) are provided. Those skilled in the art will understand that in the case of immobilization on the resin (step ii), the primary amine of P1 should be a free amine. In such cases, this amine should be freed before immobilization. Preferably, the constituent unit of general formula (I) is general formula (II), more preferably general formula (III), and most preferably general formula (IV): [ka] It is a constituent unit.

[0019] [Step II - Immobilization of structural units on peptide synthesis resin] In step ii), the constituent units are immobilized on the peptide synthesis resin. Those skilled in the art can select a suitable resin. This immobilization is conveniently carried out via a free primary amine of the constituent units, which can form a secondary amine that binds the constituent units to the resin. This amine is referred to herein as the immobilization secondary amine. To obtain a free amine after SPPS, the resin is preferably capable of releasing the amine after cleavage. Such a resin may also release a guanidinium moiety, if appropriate. Examples of suitable resins are aldehyde-functionalized resins and 2-chlorotrityl chloride resins. Aldehyde-functionalized resins are preferred, and particularly preferred are highly acid-unstable aldehyde resins, such as (3-formylindolyl)acetamidomethyl polystyrene resins. As used herein, a highly acid-unstable aldehyde resin is a resin that can be cleaved using up to 10% by volume of trifluoroacetic acid (TFA) in water; preferably up to 7% by volume is used, more preferably about 1-6% by volume is used, and most preferably about 2-5% by volume is used. The use of highly acid-unstable aldehyde resins is attractive because it reduces the risk of acid hydrolysis of the nitrile portion present in the constituent units.

[0020] For the immobilization of primary amines on aldehyde resins, the use of reductive amination may be advantageous. In a preferred embodiment, step ii) comprises forming an immobilized secondary amine by reductive amination of the free amine of the constituent units onto the aldehyde of the peptide synthesis resin, the immobilized secondary amine being optionally subsequently protected. Reductive amination is well known in the art. Generally, the primary amine of the constituent units forms an imine with the aldehyde, which is then reduced to form a secondary amine. Reductive amination can be carried out using, for example, NaBH(OAc)3, NaCNBH3, or NaBH4-Ti(OiPr)4, particularly NaBH(OAc)3 in DCE(1,2-dichloroethene) and / or TMOF(trimethyl orthoformate), NaBH3CN in THF(tetrahydrofuran) and / or TMOF and / or AcOH, or NaBH4-Ti(OiPr)4 in THF. Reductive amination is preferably carried out for about 0.5 to 48 hours, preferably about 1 to 36 hours, more preferably about 2 to 30 hours, more preferably about 2.5 to 24 hours, more preferably about 3 to 18 hours, more preferably about 3.5 to 16 hours, and more preferably about 4 to 14 hours. In some embodiments, this is a performer of about 2.5 to 5.5 hours, preferably about 3 to 5 hours. Reductive amination is preferably carried out at a temperature of about 10-40°C, more preferably about 15-30°C, more preferably about 17-25°C, and most preferably about 19-23°C. It is advantageous to carry out reductive amination at room temperature. Good results were obtained when reductive amination was carried out at room temperature for 4 hours using NaCNBH3 in a 1:1 mixture of tetrahydrofuran and trimethyl orthoformate.

[0021] If P1 is intended to ultimately be a residue such as lysine or ornithine, the immobilization amine can be subsequently protected to ensure compatibility with any subsequent SPPS steps. It is advantageous to select an acid-unstable protecting group such as Boc or triphenylmethylamine for this purpose. Methods for introducing such protecting groups are well known to those skilled in the art. Boc can be introduced, for example, conveniently using di-tert-butyl dicarbonate. When introducing such a protecting group, the presence of a base, such as 1 equivalent of DIPEA, may be preferable. Such reactions can be allowed to proceed for 2 to 24 hours, preferably about 8 to 14 hours, for example, overnight.

[0022] If P1 is intended to ultimately become a guanidylated residue such as arginine, the immobilization amine can be guanidylated before any subsequent SPPS step. In preferred embodiments, the immobilization secondary amine is guanidylated, and then the resulting guanidinium moiety is optionally protected. In preferred embodiments, the guanidinium moiety is protected. Guanidylation agents such as N,N'-di-Boc-thiourea or the following 5,6-chlorobenzotriazole agents (N,N'-di-tert-butoxycarbonyl-5-chloro-1H-benzotriazole-1-carboxamidine and N,N'-di-tert-butoxycarbonyl-6-chloro-1H-benzotriazole-1-carboxamidine) can be used. The present invention provides the use of these guanidylation agents in the guanidylation of a secondary amine, preferably a secondary amine to be immobilized on a resin, most preferably an immobilization amine. Preferably, such an agent is in the presence of about 1 equivalent of DIPEA relative to the guanidylating agent. Furthermore, a carbodiimide such as diisopropylmethandiamine (DIPCDI) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), preferably DIPCDI, is preferably present in an amount of 1 equivalent relative to the guanidylating agent. Such a reaction can preferably be carried out for about 4 to 20 hours, more preferably about 8 to 14 hours, for example, overnight. Those skilled in the art will recognize that these agents introduce a protected guanidinium moiety, eliminating the need for further protection. [ka]

[0023] Since the constituent units are immobilized, the reaction can be conveniently washed after completion, and such washing can be carried out using conventional SPPS techniques, for example, by alternating swelling and shrinking washing. Unreacted sites can be capped with acetic anhydride. In a preferred embodiment, after immobilization, the resin is washed with DCM (preferably 3 times) and DMF (preferably 3 times), and unreacted sites are preferably capped by stirring for 10 minutes with a base such as Ac2O (138 μL, 1.46 mmol) and pyridine (118 μL, 1.46 mmol) in a solvent such as DMF. The resin is then washed with a solvent such as DMF (preferably 3 times).

[0024] [Step III - Solid-phase peptide synthesis (SPPS)] After the constituent units are immobilized on the resin, the remainder of the intended peptide can be constructed using another conventional SPPS. For this purpose, p' is generally deprotected and then coupled with a new amino acid residue. This new residue is adequately protected during coupling and can then be deprotected to make the primary amine available. Between steps, the resin can be conveniently washed to remove excess reagent or by-products. In a preferred embodiment, the SPPS follows the Fmoc method. Fmoc can be deprotected using a base, preferably an organic base, preferably a non-nucleophilic base. For example, this can be done using 20% ​​by volume piperidine in DMF, or 3% by volume DBU in DMF, the latter of which has been found to be preferred.

[0025] Preferably, at least one additional amino acid residue is added. Preferably, about 25 or fewer additional amino acid residues are added, more preferably 20 or fewer, more preferably 15 or fewer, and most preferably about 1 to 10 additional amino acid residues.

[0026] In preferred embodiments, the amine at the N-terminal residue is capped. The capping agent is well known in the art and is generally a carboxylic acid without further reactive groups. Good results were obtained using methoxyacetic acid to obtain methoxyacetamide.

[0027] [Step iv - Cleavage of peptide conjugate from resin] For example, maintaining the ABTC-peptide conjugate immobilized on the resin can be useful when long-term storage is anticipated or when further modifications are foreseeable. When the ABTC-peptide conjugate is used, it can be cleaved from the resin using conventional SPPS procedures. Therefore, in some embodiments, the above method: iv) The immobilized peptide conjugate is cleaved from the resin to obtain a free peptide conjugate, and optionally purified. It also includes.

[0028] The free peptide conjugate is as described later in the section on compounds. Cleavage is a standard procedure in SPPS and is preferably carried out using acid treatment, more preferably using TFA treatment. The acid treatment is preferably diluted to avoid the formation of by-products. Dilution can be carried out in any inert aprotic solvent, for example, a solvent known to swell the resin, for example, using dichloromethane (CH2Cl2, DCM). Good purity has been found to be achievable when up to 75% by volume of TFA is used during cleavage. Cleavage may take between about 0.5 and 16 hours, preferably about 1 and 12 hours, more preferably about 1.5 and 8 hours, or about 2 and 6 hours. Cleavage is preferably carried out at a temperature of about 10 to 40°C, more preferably about 15 to 30°C, more preferably about 17 to 25°C, and most preferably about 19 to 23°C. It is advantageous to carry out cleavage at room temperature. Preferably, the immobilized peptide conjugate is cleaved from the resin in 2 hours using 50% by volume of TFA in CH2Cl2.

[0029] If the immobilized peptide conjugate contains certain protecting groups known to generate undesirable byproducts such as reactive carbocations, a scavenger can be used during cleavage. The use of scavengers is well established, and those skilled in the art can design compositions for use in cleavage. For example, thiols such as ethanedithiol (EDT) or silanes such as triisopropylsilane (TIS) can be added. Good results were obtained when 2.5% by volume of TIS and 2.5% by volume of EDT were used to cleave peptides containing a Trt protecting group.

[0030] In some embodiments, the free peptide conjugate is purified after cleavage. Purification can be carried out by any conventional method, for example, by precipitating the cleaved mixture in diethyl ether, preferably about three times. The precipitated product can be concentrated by centrifugation, and then optionally washed and / or concentrated under reduced pressure. In preferred embodiments, the free peptide conjugate is lyophilized after cleavage, more preferably after post-cleavage purification. Further purification can be carried out by chromatographic techniques such as HPLC, preferably preparative or half-cleavage HPLC. A suitable HPLC is RP-HPLC.

[0031] [Step reaction with v-cysteine] The free peptide conjugate or immobilized peptide conjugate contains an ABTC moiety having a nitrile group. This group readily reacts with cysteine ​​to form aLuc. In a preferred embodiment, the above method is: v) A step of contacting a free peptide conjugate with cysteine ​​to obtain a peptide-aminoluciferin conjugate. It also includes.

[0032] Here, the reaction with cysteine ​​takes place after the peptide conjugate has dissociated from the resin. This allows for convenient storage of the peptide conjugate without the risk of aLuc dehydrogenation, as aLuc has not yet been formed. The reaction with cysteine ​​can be performed immediately before the intended use of the caged aLuc conjugate, and can even be performed directly in situ before the assay. In a preferred embodiment, the free peptide conjugate is reacted in situ with cysteine ​​before the addition of the protease.

[0033] In another embodiment, the reaction with cysteine ​​is carried out while the peptide conjugate remains immobilized. This is related to the advantages of SPPS having this final reaction step. In such a case, the above method: v) A step of contacting an immobilized peptide conjugate with cysteine ​​to obtain a peptide-aminoluciferin conjugate. This includes the following. This step is preferably performed between steps iii) and iv). In such cases, the above steps are preferably performed in the order i), ii), iii), v), iv).

[0034] When contacting the free peptide conjugate with cysteine, this is preferably carried out in an aqueous buffer such as 25 mM HEPES, 125 mM NaCl, and 0.5 wt% BSA at pH 7.4. The cysteine ​​is preferably present in excess, such as 10 to 50 equivalents, or 20 to 40 equivalents, or more. The contact can be carried out at room temperature or a slightly higher temperature, for example, 30 to 50°C, preferably 35 to 40°C, for example, about 37°C. The contact is preferably carried out for about 1 to 600 minutes, preferably about 5 to 300 minutes, more preferably about 10 to 180 minutes, more preferably about 15 to 120 minutes, more preferably about 20 to 60 minutes, most preferably about 25 to 45 minutes, for example, about 30 minutes.

[0035] When the immobilized peptide conjugate is brought into contact with cysteine, this is preferably carried out in a solvent suitable for SPPS, such as DMF or NMP. Another characteristic is preferably as described above.

[0036] In this contact, the preferred cysteine ​​is D-cysteine. In this contact, the cysteine ​​is preferably free cysteine ​​without a protecting group.

[0037] [Compound] The method according to the present invention enables the convenient production of peptide-aLuc conjugates and peptide-ABTC conjugates. The latter is a convenient intermediate for peptide-aLuc conjugates and is less prone to dehydrogenation that can occur in aLuc conjugates. Since aLuc can be formed in situ, peptide-ABTC conjugates offer comparable ease of use to aLuc conjugates. Therefore, the present invention relates to compounds of general formula (V) or salts thereof: [ka] (In the formula, h1 is H, p', C1-4alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; h2 is H, p', C1-4 alkyl, or peptide synthesis resin; n is 1, 2, 3, 4, or 5; R is a polypeptide moiety containing H, p', X, or 1 to 10 amino acids; p' is a protecting group in each case independently; X is H, or a -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where the alkyl and acyl are linear, branched, or cyclic, possibly unsaturated, and possibly substituted with a halogen or C1-4 alkoxy. To provide.

[0038] h1 is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; preferably h1 is H, -C(=N-p')-NH-p', or -C(=NH)-NH2; most preferably h1 is H. In some embodiments, h1 is -C(=N-p')-NH-p' or -C(=NH)-NH2. In some embodiments, h1 is H, p', or C1-4 alkyl, preferably H, or p'.

[0039] h2 is H, p', C1-4 alkyl, or peptide synthesis resin; preferably h2 is H or peptide synthesis resin. In preferred embodiments, both h1 and h2 are H. This allows the use of the compounds according to the present invention as constituent units of the methods according to the present invention. In such cases, R is preferably p'. If p' is contained in h1 or h2, it is preferably acid-unstable, and more preferably it is Boc.

[0040] The C1-4 alkyl group is preferably a C1-3 alkyl group, and more preferably a C1-2 alkyl group. Examples of C1-4 alkyl groups are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, tert-butyl, and cyclobutyl.

[0041] Preferred compounds according to the present invention are compounds of general formula (I), more preferably general formula (II), even more preferably general formula (III), and most preferably general formula (IV). n and p' are as defined for compounds of general formula (I). In a further preferred embodiment, the compound is a compound of general formula (VI). Compounds and constituent units, such as those of general formula (I), preferably correspond to the stereochemistry shown in general formula (VI). In other words, the P1 residue is preferably an L-residue. [ka]

[0042] In certain embodiments, h2 is a peptide synthesis resin. In such cases, such a compound is often referred to as a peptide synthesis resin charged with a compound of general formula (V). Since resins generally have multiple charging sites, such resins generally contain multiple compounds of general formula (V). The peptide synthesis resin that can be represented by h2 is preferably as defined herein. In preferred embodiments, h2 is a peptide synthesis resin, preferably an aldehyde-functionalized resin, preferably a highly acid-unstable resin such as (3-formylindolyl)acetamidomethylpolystyrene.

[0043] P1 may preferably be lysine or ornithine. In such cases, h1 is H, and preferably n is 2 or 3, more preferably 3. P1 may preferably be arginine. In such cases, h1 is -C(=N-p')-NH-p' or -C(=NH)-NH2, and preferably n is 2 or 3, more preferably 2.

[0044] R is a polypeptide moiety containing H, p', X, or 1 to 10 amino acids. In preferred embodiments, R is H, p', or X, more preferably H or p'. Such compounds can be advantageously used as constituent units or precursors thereof.

[0045] When R is a polypeptide moiety, the compound may be a substrate for a protease or a resin into which such a substrate is charged. Generally, when R is a polypeptide moiety, one or more SPPS processes are performed on the initial structural unit. In such embodiments, R is a compound of the general formula (pep): [ka] (In the formula, sc 1is an amino acid side chain preferably selected from H, or linear, branched, or cyclic C1-12 alkyl, where the alkyl is optionally unsaturated and optionally substituted with halogen, or optionally substituted with halogen -(CH2) 0-4 - [C5-10(hetero)aryl]; sc 2 is an amino acid side chain preferably selected from H, or linear, branched, or cyclic C1-12 alkyl, where the alkyl is optionally unsaturated and optionally substituted with halogen, or optionally substituted with halogen -(CH2) 0-4 - [C5-10(hetero)aryl], where sc 2 is independently selected in each case; b is -CH2- or is absent; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; X is H, or -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where the alkyl and acyl are linear, branched, or cyclic, optionally unsaturated, and optionally substituted with halogen or C1-4 alkoxy) is the polypeptide moiety of

[0046] sc 1 and sc 2 are amino acid side chains, not optional and preferably substituted; preferably H, C2-5(halo)alkyl-N(h1)2, C1-4(halo)alkyl, 5-9 member (hetero)aryl, C1-2(halo)alkyl-[5-9 member (hetero)aryl], C2-4(halo)alkyl-N(h1)C(N(h1)2)(=Nh1), C1-4(halo)alkyl-C(O)-N(h1)2, or C1-2(halo)alkyl-[3-9 member (hetero)cycloalkyl]; Here, C2-6(halo)alkyl-N(h1)2 is preferably -CH2-CH2-CH2-NH2 or -CH2-CH2-CH2-CH2-NH2; 5-10 membered (hetero)aryl is preferably phenyl; optionally substituted C1-4(halo)alkyl-[5-10 membered (hetero)aryl] is preferably -CH2-phenyl, -CH2-CH2-phenyl, -CH2-imidazolyl, -CH2-CH2-imidazolyl, -CH2-indolyl, -CH2-CH2-indolyl; -CH2-hydroxyphenyl, -CH2-CH2-hydroxyphenyl; optionally substituted C1-6(halo)alkyl is preferably -CH3, -CH(CH3)2, -CH2-CH(CH3)2, -CH(CH3)-CH2-CH3, - CH2-OH, -CH2-SH, -CH2-SeH, -CH2-CH2-CH2-S-CH3, -CH(CH3)-CH2-OH, -CH2-CH2-COOH, or -CH2-COOH, habitually -CH3, -CH(CH3)2, -CH2-CH(CH3)2, -CH(CH3)-CH2-CH3, -CH2-OH, -CH2-SH, -CH2-SeH, -CH2- CH2-CH2-S-CH3 or -CH2(CH3)-CH2-OH; C2-6(halo)alkyl-N(h1)C(N(h1)2)(=Nh1) is preferably -CH2-CH2-CH2-NC(=NH)-NH2; and C1-6(halo)alkyl-C(O)-N(h1)2 is preferably -CH2-CH2-C(O)NH2 or -CH2-C(O)NH2.

[0047] b is either -CH2- or absent; if b is not present in a portion adjacent to X, it is preferably absent. In a preferred embodiment, if b is present in a portion directly adjacent to X, it is -CH2-. This forms a beta-amino acid as the N-terminal residue.

[0048] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. Preferably, it is 0, 1, 2, 3, 4, or 5; more preferably, it is 0, 1, 2, or 3; even more preferably, it is 1, 2, or 3; most preferably, it is 1 or 2; and most preferably, it is 1. This forms a tripeptide that binds to ABTC.

[0049] X is H, or a -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where the alkyl and acyl are linear, branched, or cyclic, optionally unsaturated, and optionally substituted with halogens or C1-4 alkoxys. If X is optionally substituted, it is substituted with methoxy. The C1-20 in the alkyl or acyl as defined with respect to X is preferably C1-12, more preferably C1-8, and even more preferably C1-6. With respect to C1-6 acyl, this is preferably C2-6 acyl. Examples of C1-6 alkyl moieties are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, and hexyl. An example of an acyl moiety is an alkyl moiety having an oxo substituent on a carbon atom directly adjacent to the nitrogen to which X is bonded. It is highly preferable that X is -C(=O)-CH2-O-CH3.

[0050] In a preferred embodiment, m is 1 or 2, preferably 1; sc 2 b is H when it is an amino acid adjacent to X; b is -CH2- when it is an amino acid adjacent to X.

[0051] The present invention provides a kit of components comprising the compound defined above and cysteine. Preferably, the cysteine ​​is D-cysteine. Such a kit is useful for performing a luciferase assay. The kit may further comprise the luciferase enzyme. The kit may further comprise ATP, or MgCl2, or instructions for use.

[0052] Method for determining protease activity The above compounds can be appropriately used in assays to determine protease activity. The target protease may be, for example, a blood coagulation factor. Preferably: i) A step of providing one or more compounds according to the present invention; ii) The step of contacting the provided compound with cysteine ​​to obtain an aminoluciferin-peptide conjugate; iii) The step of contacting the above aminoluciferin-peptide conjugate with a protease to obtain free aminoluciferin; iv) A step of contacting the above-mentioned free aminoluciferin with luciferase to generate a luminescence signal, A method for determining protease activity including [specific enzymes].

[0053] The characteristics and definitions are preferably as defined above. For example, a 6-ABTC-binding peptide can be incubated with D-cysteine ​​in a buffer at 37°C for 30 minutes to obtain each aLuc derivative, after which protease, ATP, MgCl2, and luciferase can be added. The luminescence can then be recorded at 37°C for 30 minutes, and the area under the curve (AUC) can be calculated. The AUC is, for example:

number

[0054] Luciferase is a general term for a type of oxidase that produces (bio)luminescence using luciferin as a substrate. Luciferase does not require an external light source, but it does require luciferin and O2, and often ATP as well. Mg 2+It is known that this increases the luminescence yield of some luciferases. Luciferases and their assays are well known in the art, and the conditions suitable for their activity are also well known. Suitable luciferases are Enhanced Beetle luciferase (ELuc), Click Beetle green luciferase (CBG), R. ohbai luciferase (RoLuc), Firefly luciferase (FLuc), Red Firefly luciferase (RedF), P. hirtus red luciferase (RedLuc), Nanoluciferase (NLuc), Renilla luciferase (Renilla), Metridia luciferase (MetLuc), Lucia luciferase (Lucia), Gaussia luciferase (GLuc), or Green Renilla luciferase (GrRenilla), most preferably having NLuc activity. Many luciferases are commercially available from manufacturers such as Promega and Sigma.

[0055] [General definition] In preferred embodiments, the compounds and compositions according to the present invention are for use in the methods according to the present invention or for use according to the present invention. Unless otherwise specified, each of the embodiments distinguished herein can be combined with one another.

[0056] Wherever parameters of a substance are discussed in the context of the present invention, unless otherwise specified, it is assumed that such parameters are determined, measured, or revealed under physiological conditions. Physiological conditions are well known to those skilled in the art and include aqueous solvent systems, atmospheric pressure, pH values ​​between 6 and 8, temperatures ranging from room temperature to about 37°C (about 20°C to about 40°C), and appropriate concentrations of buffer salts or other components. It is understood that electric charge is often associated with equilibrium. A portion described as carrying or having a certain charge is a portion in which the state of having or carrying such a charge is more frequently observed than the state of not having or carrying such a charge. Thus, as will be understood by those skilled in the art, atoms indicated in this disclosure as being charged may not withstand electricity under certain conditions, and neutral portions may be charged under certain conditions.

[0057] In the context of the present invention, a decrease or increase in the parameter being evaluated means a change of at least 5% in the value corresponding to that parameter. More preferably, a decrease or increase in the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In the latter case, the value may no longer be detectable as associated with the parameter.

[0058] In this document and its claims, the verb “including” and its inflections are used in a non-restrictive sense, meaning that the items following the word are included, but not that items not specifically mentioned are excluded. “Hemostasis” and “Haemostasis” can be used synonymously in this specification. Furthermore, unless the context explicitly requires the existence of one and only one element, a reference to an element with the indefinite article “a” or “an” does not preclude the possibility that there may be more than one of that element. Thus, the indefinite article “a” or “an” usually means “at least one.” When used in relation to a number, the words “about” or “approximately” preferably mean that the value can be a specific value that is 1% greater or less than that value. Furthermore, the verb “consisting of” can be replaced with “essentially consisting of,” meaning that the compositions of the present invention may contain additional components other than the particularly specified components, and such additional components do not alter the properties specific to the present invention.

[0059] Whenever a structural formula or chemical name is understood by those skilled in the art to have a chiral center, chirality has not yet been indicated, and for each chiral center, individual references are made for all three: racemic mixtures (with any enantiomer excess), pure R enantiomers, and pure S enantiomers. Whenever a molecular fragment, often called a part, is shown, a dotted or dashed line indicates which bond connects it to the whole molecule; an asterisk (*) indicates where the shown part connects to the rest of the molecule. This asterisk does not indicate an atom, nor does the bond where the dotted or dashed line intersects convey any information about which atom is on the non-partial side of the bond. All of this is well known and common practice in the art.

[0060] All patents and references cited herein are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0061] [Figure 1A] A conventional solution-phase method for the synthesis of aminoluciferin peptides by C-terminal activation of a protected peptide, followed by deprotection of the side chain, and concentration of cysteine ​​in solution. [Figure 1B] A well-known method using SPPS (Kovacs et al., Int.J.Pept.Res.Ther. 2019, 25, 1209-1215). The loading of aminoluciferin onto a solid support yields a dehydrogenation product (thiazole, indicated by a thick, curved arrow), which does not provide the appropriate activity in the assay. [Figure 1C] According to the present invention, side chain anchoring of a first amino acid on a resin is obtained, followed by conventional SPPS chemistry and then a condensation reaction with cysteine. This yields an aminoluciferin-caged peptide with good activity in assays, while simultaneously avoiding the formation of dehydrogenated products. [Figure 2] Assay plan using aLuc precursors: i. βA-XR / βA-XK substrate (1.0 equivalent), D-cysteine ​​(1.5 equivalents), buffer (25 mM HEPES, 125 mM NaCl, 0.5% BSA, pH=7.4), 37°C, 30 min. ii. Protease (thrombin / FXa, final concentration 10 nM), ATP (1.5 equivalents), MgCl2 (10 equivalents), Quantilum® recombinant firefly luciferase (final concentration 10 μM, Promega). [Figure 3] Total luminescence of candidate substrates (βA-XR / βA-XK) using thrombin and factor Xa. Library screening of peptide libraries using the substrates (677 μM) methoxyacetamide-βA-XR-6ABTC and methoxyacetamide-βA-XK-6ABTC after in-situ condensation with cysteine. The proteolytic activity of the substrates against either FXa or thrombin was determined, and the ratio was plotted against the total luminescence of either thrombin or factor Xa (ratio 1:1:y=x). [Examples]

[0062] [Example 1 - Materials and Method] [1.1 Overview] The NMR spectra were recorded using a Bruker Avance III 400 MHz or Bruker 500 MHz spectrometer, and the compounds were identified as follows: 1 1H NMR, 13 The classifications were determined using 13C NMR, COSY, HSQCED, and HMBC spectra. Chemical shifts were reported in parts per million (ppm) relative to a baseline (CDCl3: 1 H: 7.26 ppm. and 13 C 77.16ppm.;CD3OD: 1 H: 3.31 ppm. and 13 C 49.00 ppm; D2O: 1 H: 4.79 ppm; (CD3) 2SO: 1 H: 2.50 ppm. and 13(C 39.52 ppm). NMR data are presented as follows: chemical shift, multiplicity (s=singleline, bs=broad singleline, d=doubleline, t=tripline, dd=double doubleline, ddd=double double doubleline, dtd=double triple doubleline, h=septatyl, m=multiline and / or multiple resonances), and binding constant J in units of Hz. Peptides were synthesized on resin coupling using Fmoc solid-phase peptide (SPPS) chemistry, and the deprotection step was monitored using the Kaiser test. Mass spectra were recorded on a JEOL AccuTOF CS JMS-T100CS (ESI) mass spectrometer. Automated flash column chromatography was performed on a Biotage Isolera Spektra One using 4-50 g of SNAP or Silicone cartridges (Biotage, 30-100 μm, 60 angstroms). Preparative HPLC was performed on a Phenomenex® Gemini-NX 3u C18 110A reverse-phase column (150 × 21.2 mm) using gradient elution at a constant flow rate of 10 mL / min at 30°C. MiliQ (0.1% TFA) and CH3CN (0.1% TFA) were used as solvents. The pure fractions containing the product were combined and freeze-dried overnight to obtain the target compound. The reaction was carried out under positive pressure with Ar. / N2 flow and a protective atmosphere using flame-dried glassware. Adenosine 5'-triphosphate (ATP), bovine serum albumin (BSA), 4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES), magnesium chloride hexahydrate (MgCl2), sodium chloride (NaCl), and sodium hydroxide (NaOH) were obtained from Merck KGaA (Germany). D-cysteine ​​was obtained from Fluorochem (UK). Factor Xa and thrombin were obtained from Haematologic Technologies (USA). Quantilum® recombinant firefly (Photinus pyralis) luciferase was obtained from Promega (Leiden, the Netherlands).I obtained a Half-area white Greiner Bio-One 96-well plate (#675075) from VWR (Amsterdam, Netherlands).

[0063] [1.2 Synthesis of constituent units] [1.2.1 Synthesis of 6-aminobenzo[d]thiazole-2-carbonitrile (6-ABTC)] [Scheme S1.2.1 - Synthesis of 6-ABTC from 2-chlorobenzo[d]thiazole] [ka] [ka] 2-chloro-6-nitrobenzo[d]thiazole (8): 2-chlorobenzo[d]thiazole (15.00 g, 88.43 mmol) was added in several portions to concentrated H2SO4 (90 mL) on an ice bath. KNO3 (9.834 g, 97.27 mmol) was added in several portions, and the reaction mixture was stirred at 0°C for 30 minutes. The reaction mixture was warmed to room temperature and stirred at this temperature for 18 hours. The mixture was poured onto ice water (300 mL), and the formed precipitate was collected by filtration. The crude product was washed with ice water and NaHCO3 aqueous solution until the acid was removed. The product was dried overnight under reduced pressure and recrystallized from EtOH (650 mL) to obtain 8 (16.81 g, 89%) as an off-white solid. TLC (siRNA / n-heptane, 1:4 v / v):R f = 0.60. 1 H NMR(500MHz,CDCl3)δ 8.75(d,J=2.3Hz,1H),8.38(dd,J=9.0,2.3Hz,1H),8.07(d,J=9.0Hz,1H). 13 C NMR(126MHz,CDCl3)δ 158.9,154.9,136.6,123.4,122.3,117.8.

[0064] [ka] 6-Nitrobenzo[d]thiazole-2-Carbonitrile(9):2-chloro-6-nitrobenzo[d]-thiazole (8, 10.78 g, 50.23 mmol) was dissolved in ACN (1000 mL), and triethylenediamine (845.2 mg, 7.53 mmol) was added. NaCN (2.78 g, 56.83 mmol) was dissolved in water (100 mL) and added dropwise to the stirred reaction mixture. After 24 hours, the reaction mixture was quenched with an aqueous solution of iron(III) chloride hexahydrate (0.3 M, 50 mL) and diluted with water (350 mL). The reaction mixture was extracted with RINKAN (3 × 400 mL), and the combined organic layer was washed with brine (100 mL), dried over MgSO4, and concentrated under reduced pressure. The crude product was charged onto a silica plug, flushed with CHCl3 (2000 mL), concentrated, and dried under high vacuum to obtain 9 (8.30 g, 81%) as a yellow solid. TLC(CHCl3):R f = 0.33. 1 H NMR(500MHz,CDCl3)δ 8.95(d,J=2.2Hz,1H),8.52(dd,J=9.1,2.2Hz,1H),8.38(d,J=9.1Hz,1H). 13 C NMR(126MHz,CDCl3)δ 155.4,147.4,141.9,135.7,126.2,123.2,118.6,112.1.

[0065] [ka] 6-aminobenzo[d]thiazole-2-carbonitrile (1): 6-nitrobenzo[d]thiazole-2-carbonitrile (9, 7.70 g, 37.53 mmol) was suspended in AcOH (700 mL). Iron dust (104.80 g, 1.88 mmol) was added, and the reaction mixture was stirred for 24 hours. The reaction mixture was diluted with water (1400 mL) and filtered over Celite. The aqueous solution was extracted with RINKAN (3 × 700 mL) and washed with brine (400 mL). The crude product was charged onto a silica plug, flushed with CHCl3 (2000 mL), concentrated, and dried under high vacuum to obtain 1 (3.45 g, 53%) as a yellow solid. TLC (DCM, 1:1 v / v):R f = 0.26. 1 H NMR(500MHz,CDCl3)δ 7.95(d,J=8.9Hz,1H),7.08(d,J=2.2Hz,1H),6.95(dd,J=8.9,2.3Hz,1H). 13 ¹³C NMR (126 MHz, CDCl3): δ 147.6, 145.5, 138.0, 131.0, 125.9, 117.6, 103.8, 77.2. HRMS (m / z): Calculated for C8H5N3S [M+H] + : 176.0282, measured value 176.0294.

[0066] [1.2.2 Composition of constituent units of general formula (I)] [ka] Fmoc-Orn(Boc)-6ABTC(4):6-aminobenzo[d]thiazole-2-carbonitrile (1,300 mg, 1.71 mmol) was dissolved in 6 mL of dry pyridine in a flame-dried flask. Trichlorophosphan (78 μL, 890 μmol) was added dropwise, and the reaction mixture was stirred for 1.5 hours. Fmoc-Orn(Boc)-OH (778 mg, 1.71 mmol) was added to 3 mL of dry pyridine, and the reaction mixture was stirred at 40°C for 3 hours. The reaction mixture was cooled to room temperature, diluted with HCl (150 mL), and washed with 150 mL of 10% citric acid aqueous solution. The aqueous phase was re-extracted with HCl (75 mL). The combined organic layers were washed with 10% citric acid aqueous solution (100 mL), saturated NH4Cl aqueous solution (2 × 100 mL), and brine (100 mL). The combined organic layer was dried over MgSO4, concentrated under reduced pressure, and purified by silica gel column chromatography (20 → 80% toluene / n-heptane) to obtain Fmoc-Orn(Boc)-6ABTC (4, 1.03 g, 98%) as a yellow solid. TLC (toluene / n-heptane, 1:1 v / v):R f = 0.69. 1 H NMR(500MHz,CDCl3)δ 9.26(s,1H),8.66(d,J=2.1Hz,1H),8.09(d,J=8.9Hz,1H),7.76(d,J=7.6Hz,2H),7.60( t,J=7.2Hz,2H),7.57-7.53(m,1H),7.39(t,J=7.5Hz,2H),7.30(t,J=5.5Hz,2H),5.76- 5.71(m,1H),4.87-4.83(m,1H),4.68-4.64(m,1H),4.42(d,J=7.1Hz,2H),4.22(t,J=7. 0Hz,1H),3.63-3.50(m,1H),3.16-3.06(m,1H),2.04(s,2H),1.67(s,2H),1.45(s,9H). 13 C NMR(126MHz,CDCl3)δ 171.3,157.6,148.7,143.7,141.4,136.9,127.9,127.2,125.3,125.2,121.0,120.1,120.1,113.2,111.5,47.3,28.5.

[0067] [ka] Fmoc-Orn-6ABTC(6):Fmoc-Orn(Boc)-6ABTC (4,785,1.28 mmol) was dissolved in formic acid (10 mL) and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the product was freeze-dried overnight to obtain the formate of Fmoc-Orn-6ABTC (6,733 mg, quantified) as a yellow solid. 1 H NMR(400MHz,CD3OD)δ 8.64(d,J=2.1Hz,1H),8.12(d,J=9.0Hz,1H),7.78(d,J=7.6Hz,2H),7.70(dd,J=9.0,2.1Hz,1H),7.66(t,J=7.5Hz,2H),7.37(t,J=7.5Hz, 2H),7.33-7.24(m,2H),4.42(qd,J=10.6,6.7Hz,2H),4.35-4.31(m,1H),4.21(t,J=6.7Hz,1H),2.97(t,J=7.3Hz,2H),1.88-1.73(m,3H). 13 C NMR(101MHz,CD3OD)δ 177.3,172.9,169.9,158.3,149.9,145.1,142.2,140.4,138.0,136.8,128.8,128.1 ,125.9,122.3,120.9,114.0,112.9,67.9,56.5,39.7,30.1,24.2,22.1.HRMS(m / z):C 28 H 25 [M+H] calculated for N5O3S + : 512.1756, measured value 512.1752.

[0068] [ka] Fmoc-Lys(Boc)-6ABTC(5):6-aminobenzo[d]thiazole-2-carbonitrile (1,501 mg, 2.86 mmol) was dissolved in 10 mL of dry pyridine in a flame-dried flask. Trichlorophosphan (130 μL, 1.49 μmol) was added dropwise, and the reaction mixture was stirred for 1.5 hours. Fmoc-Lys(Boc)-OH (1340 mg, 2.86 mmol) was added to 5 mL of dry pyridine, and the reaction mixture was stirred at 40°C for 3 hours. The reaction mixture was cooled to room temperature, diluted with 150 mL of ethyl acetate, and washed with 150 mL of 10% citric acid aqueous solution. The aqueous phase was re-extracted with 75 mL of ethyl acetate. The combined organic layers were washed with 100 mL of 10% citric acid aqueous solution, 2 × 100 mL of saturated NH4Cl aqueous solution, and 100 mL of brine. The combined organic layer was dried over MgSO4, concentrated under reduced pressure, and purified by silica gel column chromatography (0 → 70% toluene / n-heptane) to obtain Fmoc-Lys(Boc)-6ABTC (5, 1.38 g, 77%) as a yellow solid. TLC (toluene / n-heptane, 9:1 v / v):R f = 0.83. 1 H NMR(500MHz,CD3OD)δ 8.65-8.60(m,1H),8.08(dd,J=9.2,4.3Hz,1H),7.79-7.74(m,2H),7.69-7.63(m,3H),7.39-7.34(m,2H),7.32-7.25(m,2H),4.40-4.36 (m,2H),4.28-4.24(m,1H),4.22-4.18(m,1H),3.07-3.00(m,2H),1.89-1.82(m,1H),1.80-1.71(m,1H),1.55-1.47(m,4H),1.39(s,9H). 13 C NMR(126MHz,CDCl3)δ 173.7,158.6,149.8,145.2,145.1,142.5,140.5,138.0,136.7,128.7,128.1,126.2,1 25.9,122.2,120.9,114.0,112.8,79.8,67.9,57.2,48.4,38.4,32.9,30.6,28.7,24.2.

[0069] [ka] Fmoc-Lys-6ABTC(7):Fmoc-Lys(Boc)-6ABTC (5, 1.38 g, 2.21 mmol) was dissolved in formic acid (10 mL) and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the product was freeze-dried overnight to obtain the formate of Fmoc-Lys-6ABTC (7, 1260 mg, quantified) as a yellow solid. 1 H NMR(400MHz,CD3OD)δ 8.61(d,J=2.1Hz,1H),8.07(d,J=9.0Hz,1H),8.28(s,2H),7.75(d,J=7.5Hz ,2H),7.70-7.60(m,3H),7.35(t,J=7.5Hz,2H),7.26(t,J=7.5Hz,2H),4.40 (dd,J=6.7,3.4Hz,2H),4.28(dd,J=8.9,5.3Hz,1H),4.19(t,J=6.7Hz,1H), 2.93-2.88(m,2H),1.94-1.83(m,1H),1.83-1.62(m,3H),1.58-1.40(m,2H). 13 C NMR(101MHz,CD3OD)δ 172.0,157.2,148.5,143.7,141.2,139.1,136.6,135.4,127.4,126.7,124.8,124.6 ,120.9,119.6,112.7,111.6,66.5,55.6,47.0,39.1,31.3,26.8,22.5.HRMS(m / z):C 29 H 27 [M+H] calculated for N5O3S + : 526.1912, measured value 526.1909.

[0070] [1.3 Solid-phase peptide synthesis] [1.3.1 General Procedure A] A typical 100 mg resin (3-formylindolyl)acetamidomethyl resin (100 mg, 0.073 mmol depending on the amount used) was suspended in dry THF:TMOF (1:1, 2 mL) in a flame-dried flask. Fmoc-Orn-6ABTC (6, 82 mg, 0.146 mmol) was added, and the suspension was stirred at room temperature for 4 hours. NaBH3CN (9 mg, 0.146 mmol) and AcOH (15 μL, 0.256 mmol) in THF (2 mL) were added, and the suspension was stirred for 2 hours. The resin was washed with THF (3 times), DCM (3 times), MeOH (3 times), and Et2O (3 times), and dried under high vacuum for 1 hour. The resin was swollen in dry DCM for 20 minutes. N,N'-di-Boc-thiourea (40 mg, 0.146 mmol), DIPEA (25 μL, 0.146 mmol), and diisopropylmethandiimine (23 μL, 0.146 mmol) were added, and the reaction mixture was stirred overnight. The resin was washed with DCM (3 times) and DMF (3 times), and the unreacted sites were capped by stirring for 10 minutes with Ac2O (138 μL, 1.46 mmol) and pyridine (118 μL, 1.46 mmol) in DMF. The resin was washed with DMF (3 times). Subsequently, this resin was used in a standard SPPS using standard Fmoc chemistry. Fmoc amino acid coupling was performed using diisopropylmethandiimine (3.30 equivalents) and hydroxybenzotriazole (3.6 equivalents) in DMF. After each coupling, the resin was washed with DMF (3 times), and the Fmoc group was removed using 3% DBU (v / v) in DMF. Before the final cleavage of the peptide, the resin was washed with DMF (3 times), DCM (3 times), MeOH (3 times), and Et2O (3 times). The peptide was cleaved from the resin for 2 hours using 1:1 TFA:DCM (2.5% TIS and EDT were added for Trt-containing sequences), concentrated under reduced pressure, freeze-dried overnight, and purified by RP-HPLC in half to obtain the target peptide.

[0071] A typical 10 g of resin (10.00 g, 7.50 mmol depending on usage) was suspended in dry THF:TMOF (1:1, 150 mL) in a flame-dried flask. Fmoc-Orn-6ABTC (6, 3.46 g, 6.75 mmol) was added, and the suspension was stirred on a rotary evaporator at room temperature for 4 hours. NaBH3CN (848 mg, 13.5 mmol) and AcOH (1.5 mL, 26.2 mmol) in THF (5 mL) were added, and the suspension was stirred for 2 hours. After washing the resin with DMF (3 times) and DCM (3 times), 10 (2.82 g, 6.75 mmol) and DIPEA (3.26 mL, 18.7 mmol) in DMF (100 mL) were added, and the mixture was stirred for 18 hours. Subsequently, the resin was washed with DMF (3 times), DCM (3 times), and Et2O (3 times). The resin was dried under high vacuum and used in a standard SPPS. Multiple batches were prepared with final usage amounts between 0.20 and 0.22 mmol / g (Fmoc quantification at 301 nm).

[0072] [1.3.1.1 Peptide conjugates containing arginine as P1] [ka] Methoxyacetamide-βAVR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.14(d,J=9.0Hz,1H),7.76(dd,J=9.0,2.1Hz,1H),4.58(dd,J=9.1,4.9Hz,1H),4.10(d,J=7.3Hz,1H),3.86(s,2H),3.49-3.43(m,2H), 3.37(s,3H),3.25(q,J=6.6Hz,2H),2.52(t,J=6.8Hz,2H),2.12-1.96(m,3H),1.90-1.83(m,1H),1.81-1.68(m,3H),1.05-0.95(m,6H). 13C NMR(126MHz,CD3OD)δ 174.4,174.1,172.4,172.2,158.6,149.9,140.4,138.1,136.9,126.0,122.2,114.0, 112.8,72.6,61.1,59.5,54.8,48.4,42.0,36.4,31.4,30.1,26.4,19.6.HRMS(m / z):C 25 H 35 [M+H] calculated for N9O5S + : 574.2560, measured value 574.2557.

[0073] [ka] Methoxyacetamide-β-AGR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.70(d,J=2.0Hz,1H),8.15(d,J=9.0Hz,1H),7.81(dd,J=9.0,2.1Hz,1H),4.61(dd,J=9.0,5.0Hz,1H),3.89(d,J=5.8Hz,2H),3.85 (s,2H),3.56-3.52(m,2H),3.35(s,3H),3.24(td,J=7.0,3.7Hz,2H),2.52(t,J=6.6Hz,2H),1.89-1.80(m,2H),1.76-1.66(m,2H). 13 C NMR(126MHz,CD3OD)δ 174.9,172.4,172.0,158.6,150.0,140.4,138.0,137.0,126.0,122.4,114.0, 113.0,72.6,59.5,54.8,44.0,42.0,36.5,36.4,30.1,26.3,20.7.HRMS(m / z):C 22 H 29 [M+H] calculated for N9O5S + : 532.2090, measured value 532.2113.

[0074] [ka] メトキシアセトアミド-βACR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.70(d,J=2.0Hz,1H),8.14(d,J=9.0Hz,1H),7.78(dd,J=9.0,2.1Hz,1H),4.59(dd,J=9.3, 4.9Hz,1H),4.45(dd,J=7.0,5.5Hz,1H),3.86(d,J=1.4Hz,2H),3.54(dt,J=11.0,6.7Hz,2H) ,3.37(s,3H),3.27-3.22(m,2H),2.93(dd,J=13.9,5.6Hz,1H),2.86(dd,J=13.9,7.0Hz,1H) ,2.53(t,J=6.7Hz,2H),2.07-2.01(m,1H),1.86(dp,J=14.0,4.7Hz,1H),1.81-1.68(m,2H). 13 C NMR(126MHz,CD3OD)δ 174.5,172.8,172.6,172.2,158.6,150.0,140.3,138.0,136.9,126.0,122.3,114 .0,112.9,72.6,59.5,57.8,55.0,41.9,36.6,36.4,30.0,26.4,26.4.HRMS(m / z):C 23 H 31 Calculation of N9O5S2 cases[M+H] + :578.1967, measured value 578.1975.

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[0077] [ka] Methoxyacetamide-βATR-6ABTC: 1 1H NMR (500MHz, CD3OD) δ 8.70(d,J=2.1Hz,1H),8.14(d,J=9.0Hz,1H),7.78(dd,J=9.0,2.2Hz,1H),4.6 2(dd,J=9.4,4.7Hz,1H),4.33(d,J=4.5Hz,1H),4.23-4.17(m,1H),3.86(s,2H) ),3.56-3.51(m,2H),3.37(s,3H),3.25(q,J=6.7Hz,2H),2.58-2.54(m,2H),2 .10-2.02(m,1H),1.89-1.82(m,1H),1.79-1.70(m,2H),1.24(d,J=6.4Hz,3H). 13 C NMR(126MHz,CD3OD)δ 174.5,172.9,172.6,172.3,158.5,150.0,140.3,138.0,136.9,126.0,122.3,114.0, 112.9,72.6,68.2,60.7,59.5,54.8,41.9,36.5,36.5,30.0,26.3,20.0.HRMS(m / z):C 24 H 33 For N9O6S, the calculated [M+H] was 576.2352, and the measured value was 576.2357.

[0078] [ka] Methoxyacetamide-βASR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.69(d,J=2.1Hz,1H),8.14(d,J=9.0Hz,1H),7.80(dd,J=9.0,2.1Hz,1H),4.62(dd,J=9. 7,4.5Hz,1H),4.39(dd,J=6.3,5.5Hz,1H),3.91(dd,J=10.7,5.5Hz,1H),3.86-3.85(m,2H) ),3.81(dd,J=10.7,6.3Hz,1H),3.56-3.51(m,2H),3.37(s,3H),3.25(td,J=6.9,3.8Hz,2 H),2.53(dt,J=6.6,3.2Hz,2H),2.16-2.09(m,1H),1.88-1.82(m,1H),1.80-1.70(m,2H). 13 C NMR(126MHz,CD3OD)δ 174.5,173.3,172.6,172.3,158.0,150.0,140.3,138.0,137.0,126.0,122.4,114 .0,113.0,72.6,62.8,59.5,57.4,54.8,41.9,36.6,36.4,29.7,26.4.HRMS(m / z):C 23 H 31 [M+H] calculated for N9O6S + : 562.2196, measured value 562.2206.

[0079] [ka] Methoxyacetamide-βAER-6ABTC: 1H NMR(500MHz,CD3OD)δ 8.70(d,J=2.1Hz,1H),8.14(d,J=9.0Hz,1H),7.78(dd,J=9.0,2.1Hz,1H),4.58(dd,J =9.1,4.9Hz,1H),4.32(dd,J=8.4,5.8Hz,1H),3.86(d,J=0.8Hz,2H),3.55-3.49(m,2 H),3.37(s,3H),3.27-3.22(m,2H),2.50(t,J=6.7Hz,2H),2.48-2.41(m,2H),2.14-2 .06(m,1H),2.06-2.00(m,2H),1.85(ddt,J=13.9,9.2,4.7Hz,1H),1.78-1.67(m,2H). 13 C NMR(126MHz,CD3OD)δ 176.4,174.4,174.2,172.6,172.3,158.6,150.0,140.4,138.0,136.9,126.0,122.3,114 .0,112.9,72.6,59.5,56.6,54.8,42.0,36.5,36.4,31.1,30.1,27.8,26.3.HRMS(m / z):C 25 H 33 Calculation of N9O7S cases[M+H] + :604.2301, measured value 604.2310.

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[0082] [ka] Methoxyacetamide-βAIR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.72(d,J=2.1Hz,1H),8.16(dd,J=9.0,0.6Hz,1H),7.78(dd,J=9.0,2.1Hz,1H),4.60(dd,J=9.2 ,5.0Hz,1H),4.18(d,J=7.6Hz,1H),3.88-3.86(m,2H),3.59-3.49(m,2H),3.39(s,3H),3.27(q, J=6.6Hz,2H),2.55-2.51(m,2H),2.08-1.98(m,1H),1.92-1.83(m,2H),1.80-1.68(m,2H),1.60 (ddd,J=13.6,7.5,3.6Hz,1H),1.29-1.23(m,1H),0.98(d,J=6.9Hz,3H),0.94(t,J=7.5Hz,3H). 13 C NMR(126MHz,CD3OD)δ 174.4,174.2,172.7,172.5,158.6,149.2,139.2,138.1,138.1,126.0,122.2,114.0,112.8 ,72.6,68.1,60.0,59.5,42.0,37.6,36.5,36.5,30.1,26.4,26.2,15.9,11.3.HRMS(m / z):C 26 H 37 [M+H] calculated for N9O5S +: 588.2716, measured value 588.2714.

[0083] [ka] Methoxyacetamide-βAAR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.71(d,J=2.0Hz,1H),8.14(d,J=9.0Hz,1H),7.81(dd,J=9.0,2.1Hz,1H),4.58(dd,J=9.3,4.8Hz,1H),4.28(q,J=7.2Hz,1H),3.85(d,J=1.9Hz,2 H),3.56-3.50(m,2H),3.36(s,3H),3.28-3.22(m,2H),2.50(td,J=6.7, 2.1Hz,2H),1.91-1.82(m,2H),1.79-1.67(m,2H),1.39(d,J=7.2Hz,3H). 13 C NMR(126MHz,CD3OD)δ 175.7,175.2,172.6,172.4,158.6,150.0,140.4,138.0,136.9,126.0,122.3,114 .0,112.9,72.6,59.5,54.7,51.3,42.0,36.5,36.4,30.1,26.3,17.5.HRMS(m / z):C 23 H 31 [M+H] calculated for N9O5S + : 546.2247, measured value 546.2246.

[0084] [ka] Methoxyacetamide-βAPR-6ABTC: 1H NMR (500MHz, CD3OD)δ 8.72(d,J=2.2Hz,1H),8.14(d,J=8.9Hz,1H),7.84(dd,J=9.0,2.1Hz,1H),4.6 0(dd,J=9.6,4.6Hz,1H),4.44(dd,J=8.7,4.6Hz,1H),3.84(s,2H),3.69-3.62 (m,2H),3.57-3.52(m,2H),3.36(s,3H),3.29-3.23(m,2H),2.71-2.62(m,2H) ,2.32-2.24(m,1H),2.08-2.03(m,3H),1.92-1.82(m,2H),1.80-1.71(m,2H). 13 C NMR(126MHz,CD3OD)δ 175.0,173.0,172.5,172.3,158.6,150.0,140.4,138.0,136.9,126.0,122.3,114.0,11 2.8,72.6,61.8,59.5,54.7,48.4,41.9,35.8,35.3,30.9,30.0,26.4,25.8.HRMS(m / z):C 25 H 33 Calculation of N9O5S cases[M+H] + :572.2403, measured value 572.2397.

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[0088] [ka] Methoxyacetamide-βAYR-6ABTC: 1 H NMR(500MHz,CD3OD)δ 8.69(d,J=2.0Hz,1H),8.15(d,J=9.0Hz,1H),7.82(dd,J=9.0,2.1Hz,1H),7.04(d,J= 8.5Hz,2H),6.69(d,J=8.5Hz,2H),4.39-4.31(m,2H),3.81-3.80(m,2H),3.49-3.42(m ,1H),3.35(s,3H),3.24-3.15(m,1H),3.08-3.02(m,1H),3.00-2.94(m,1H),2.86-2.7 8(m,1H),2.42-2.34(m,2H),2.07-2.01(m,2H),1.72-1.65(m,1H),1.66-1.57(m,2H). 13 C NMR(126MHz,CD3OD)δ 174.5,173.9,172.4,170.8,158.9,157.4,150.0,140.3,138.1,137.0,131.1,128.8,126.1,122 .3,116.2,114.0,113.0,72.5,59.5,57.5,56.4,38.8,37.8,36.4,36.3,30.8,26.4.HRMS(m / z):C 29 H 35 [M+H] calculated for N9O6S + : 638.2509, measured value 638.2514.

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[0092] [1.3.2 General Procedure B] A typical 100 mg resin (3-formylindolyl)acetamidomethyl resin (100 mg, 0.073 mmol depending on the amount used) was suspended in dry THF:TMOF (1:1, 2 mL) in a flame-dried flask. Fmoc-Lys-6ABTC (7, 84 mg, 0.146 mmol) was added, and the suspension was stirred at room temperature for 4 hours. NaBH3CN (9 mg, 0.146 mmol) and AcOH (15 μL, 0.256 mmol) in THF (2 mL) were added, and the suspension was stirred for 2 hours. The resin was washed with THF (3 times), DCM (3 times), MeOH (3 times), and Et2O (3 times), and dried under high vacuum for 1 hour. The resin was swollen in DMF for 20 minutes. Di-tert-butyl dicarbonate (48 mg, 0.219 mmol) and DIPEA (38 μL, 0.219 mmol) were added, and the reaction mixture was stirred overnight. The resin was washed with DCM (3 times) and DMF (3 times), and the unreacted sites were capped by stirring for 10 minutes with Ac2O (138 μL, 1.46 mmol) and pyridine (118 μL, 1.46 mmol) in DMF. The resin was washed with DMF (3 times). Subsequently, this resin was used in a standard SPPS using standard Fmoc chemistry. Fmoc amino acid coupling was performed using diisopropylmethandiamine (3.30 equivalents) and hydroxybenzotriazole (3.6 equivalents) in DMF. After each coupling, the resin was washed with DMF (3 times), and the Fmoc groups were removed using 3% DBU (v / v) in DMF. Prior to the final cleavage of the peptide, the resin was washed with DMF (3 times), DCM (3 times), MeOH (3 times), and Et2O (3 times). The peptide was cleaved from the resin using a 1:1 TFA:DCM solution for 2 hours (2.5% TIS and EDT were added for Trt-containing sequences), concentrated under reduced pressure, freeze-dried overnight, and purified by RP-HPLC on a half-portion to obtain the target peptide.

[0093] A typical 10 g of resin (10.00 g, or 7.50 mmol depending on usage) was suspended in dry THF:TMOF (1:1, 150 mL) in a flame-dried flask. Fmoc-Lys-6ABTC (7, 3.55 g, 6.75 mmol) was added, and the suspension was stirred on a rotary evaporator at room temperature for 4 hours. NaCNBH3 (848 mg, 13.5 mmol) and AcOH (1.5 mL, 26.2 mmol) in THF (5 mL) were added, and the suspension was stirred for 2 hours. After washing the resin with DMF (3 times) and DCM (3 times), Boc2O (4.91 g, 22.5 mmol) and DIPEA (3.92 mL, 22.5 mmol) in DMF (100 mL) were added, and the mixture was stirred for 18 hours. Subsequently, the resin was washed with DMF (3 times), DCM (3 times), and Et2O (3 times). The resin was dried under high vacuum and used in a standard SPPS. Multiple batches were prepared with final usage amounts between 0.16 and 0.25 mmol / g (Fmoc quantification at 301 nm).

[0094] [1.3.2.1 Peptide conjugates containing lysine as P1] [ka] Methoxyacetamide-βAVK-6ABTC: 1 H NMR(500MHz,D2O)δ 8.37(d,J=2.1Hz,1H),8.11(d,J=8.9Hz,1H),7.68-7.63(m,1H),4.51(dd,J=8.8, 5.8Hz,1H),4.13(d,J=0.9Hz,1H),3.96-3.95(s,2H),3.54(t,J=6.6Hz,2H),3.40( s,3H),3.04(t,J=7.6Hz,2H),2.66-2.52(m,2H),2.13-2.07(m,1H),2.04-1.97(m ,1H),1.95-1.89(m,1H),1.80-1.73(m,2H),1.64-1.50(m,2H),1.00-0.97(m,6H). 13C NMR(126MHz,D2O)δ 174.2,173.8,172.4,172.4,148.2,137.3,136.8,136.4,124.5,122.2,113.5,112.9,70 .8,59.7,58.9,54.3,39.1,35.3,34.9,30.2,29.9,26.2,22.1,18.3,17.7.HRMS(m / z):C 25 H 35 [M+H] calculated for N7O5S + : 546.2498, measured value 546.2516.

[0095] [ka] Methoxyacetamide-βAGK-6ABTC: 1 H NMR(500MHz,D2O)δ 8.35(d,J=2.0Hz,1H),8.08(d,J=9.0Hz,1H),7.64(dd,J=9.0,2.0Hz,1H),4.52(dd,J=9.0,5.5Hz,1H),4.00(s,2H),3.93(s,2H),3.58-3.52( m,2H),3.35(s,3H),3.03(t,J=7.7Hz,2H),2.59(t,J=6.6Hz,2H),2.08 -1.96(m,1H),1.93-1.87(m,1H),1.79-1.70(m,2H),1.61-1.48(m,2H). 13 C NMR(126MHz,D2O)δ 174.7,172.6,172.4,171.6,148.2,137.2,136.7,136.3,124.4,122.3,113.6, 112.9,70.7,58.9,54.3,42.5,39.1,35.3,34.9,30.4,26.2,22.1.HRMS(m / z):C 22 H 29 [M+H] calculated for N7O5S + : 504.2029, measured value 504.2035.

[0096] [ka] メトキシアセトアミド-βACK-6ABTC: 1 H NMR(500MHz,D2O)δ 7.65-7.63(m,1H),7.54(d,J=9.0Hz,1H),7.32(dd,J=8.9,2.2Hz,1H),4.71-4.67(m,1H),4.57-4.50(m,1H),4.03(s, 2H),3.62-3.55(m,2H),3.46(s,3H),3.01(t,J=7.8Hz,4H),2.66-2.58(m,2H),1.80-1.70(m,4H),1.56-1.46(m,2H). 13 C NMR(126MHz,D2O)δ 173.5,172.5,170.8,170.6,147.8,136.4,136.2,135.7,124.0,120.4,115.1,111 .7,70.8,59.0,54.1,53.1,39.1,35.3,35.0,30.9,26.2,26.2,21.9.HRMS(m / z):C 23 H 31 Calculation of N7O5S2 cases[M+H] + :550.1906, measured value 550.1902.

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[0098] [ka] Methoxyacetamide-βANK-6ABTC: 1 1H NMR (500 MHz, D2O) δ 8.42-8.40(m,1H),8.16(d,J=9.0Hz,1H),7.70(dd,J=9.1,2.1Hz,1H),4.50 (dd,J=9.2,5.3Hz,1H),4.08(d,J=6.0Hz,1H),3.97(s,2H),3.57-3.50(m,2H ),3.40(s,3H),3.04(t,J=7.5Hz,2H),2.91-2.83(m,1H),2.82-2.74(m,1H), 2.61-2.54(m,2H),1.95-1.85(m,2H),1.79-1.72(m,2H),1.62-1.47(m,2H). 13 C NMR(126MHz,D2O)δ 174.2,173.9,172.9,172.8,172.4,148.4,137.2,136.9,136.4,124.5,122.5,113.8, 112.9,70.8,58.9,54.4,50.6,39.1,36.2,35.2,34.9,30.2,26.2,22.1.HRMS(m / z):C 24 H 32 [M+H] calculated for N8O6S + : 561.2243, measured value 561.2246.

[0099] [ka] メトキシアセトアミド-βATK-6ABTC: 1 H NMR(500MHz,D2O)δ 8.38(d,J=2.0Hz,1H),8.11(dd,J=9.0,1.2Hz,1H),7.68-7.64(m,1H),4.54(dd,J=8.9, 5.7Hz,1H),4.35(dd,J=5.4,1.1Hz,1H),4.20(ddd,J=6.5,5.2,1.1Hz,1H),3.96(s,2H), 3.55(t,J=6.6Hz,2H),3.39(s,3H),3.04(t,J=7.5Hz,2H),2.67-2.59(m,2H),2.06-1.98 (m,1H),1.96-1.87(m,1H),1.79-1.72(m,2H),1.63-1.47(m,2H),1.25(d,J=1.1Hz,3H). 13 C NMR(126MHz,D2O)δ 174.4,172.4,172.3,172.1,148.3,137.2,136.8,136.4,124.5,122.3,113.5,112.9 ,70.8,66.9,59.4,58.9,54.3,39.1,35.3,34.9,30.3,26.2,22.1,18.8.HRMS(m / z):C 24 H 33 Calculation of N7O6S cases[M+H] + :548.2291, measured value 548.2290.

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[0102] [ka] Methoxyacetamide-βADK-6ABTC: 1 1H NMR (500 MHz, D2O) δ 8.30-8.26(m,1H),8.01(d,J=9.0Hz,1H),7.58(dd,J=9.0,2.1Hz,1H),4.43( dt,J=9.3,5.8Hz,2H),3.87(s,2H),3.49-3.42(m,2H),3.31(s,3H),2.95(t,J =7.5Hz,2H),2.90-2.85(m,1H),2.82(d,J=7.4Hz,1H),2.49(t,J=6.6Hz,2H) ,1.98-1.91(m,1H),1.87-1.78(m,2H),1.69-1.60(m,1H),1.52-1.38(m,2H). 13 C NMR(126MHz,D2O)δ 174.0,173.9,172.7,172.5,172.3,148.3,137.2,136.8,136.4,124.5,122.3,113.5, 112.9,70.8,58.9,54.3,50.2,39.1,35.3,35.0,30.2,26.2,22.1,21.7.HRMS(m / z):C 24 H 31 [M+H] calculated for the N7O7S case. + : 562.2083, measured value 562.2086.

[0103] [ka] メトキシアセトアミド-βALK-6ABTC: 1 H NMR(500MHz,D2O)δ 8.38(d,J=2.1Hz,1H),8.13(d,J=9.0Hz,1H),7.66(ddd,J=8.9,2.1,0.8Hz,1H),4.52(dd,J= 8.9,5.6Hz,1H),4.36(dd,J=9.2,5.4Hz,1H),3.95(s,2H),3.53(t,J=6.6Hz,2H),3.39(s,3H ),3.04(t,J=7.6Hz,2H),2.63-2.50(m,2H),2.07-1.96(m,1H),1.95-1.86(m,1H),1.78-1.7 2(m,2H),1.69-1.60(m,3H),1.57-1.48(m,2H),0.96(d,J=5.8Hz,3H),0.91(d,J=5.9Hz,3H). 13 C NMR(126MHz,D2O)δ 175.0,174.1,172.4,172.3,148.3,137.3,136.8,136.4,124.5,122.2,113.5,112.9,70.8 ,58.9,54.2,52.6,39.7,39.1,35.3,34.9,30.2,26.2,24.3,22.1,22.0,20.7.HRMS(m / z):C 26 H 37 Calculation of N7O5S cases[M+H] + :560.2655, measured value 560.2651.

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[0107]

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[0108] [ka] Methoxyacetamide-βAHK-6ABTC: 1 H NMR(500MHz,D2O)δ 8.58(s,1H),8.34(s,1H),8.04(dd,J=9.0,1.2Hz,1H),7.61(dd,J=9.0,1.0Hz,1H),7.30(s ,1H),4.74-4.69(m,1H),4.52-4.46(m,1H),3.95(s,2H),3.49(q,J=6.4Hz,2H),3.39(s,3H) ,3.27(dd,J=15.3,7.0Hz,1H),3.17(dd,J=15.4,8.0Hz,1H),3.02(t,J=7.6Hz,2H),2.53(t ,J=6.7Hz,2H),2.01-1.92(m,1H),1.91-1.83(m,1H),1.78-1.70(m,2H),1.60-1.43(m,2H). 13 C NMR(126MHz,D2O)δ 173.8,172.4,172.1,171.7,148.1,137.4,136.6,136.4,133.8,128.2,124.5,121.9,117.2, 113.0,112.9,70.8,58.9,54.4,52.5,39.1,35.2,34.9,30.4,26.3,26.3,22.1.HRMS(m / z):C 26 H 33 [M+H] calculated for N9O5S + : 584.2403, measured value 584.2397.

[0109] [ka] Methoxyacetamide-βAKK-6ABTC: 1H NMR(500MHz,D2O)δ 8.39(d,J=2.1Hz,1H),8.14(d,J=8.9Hz,1H),7.68(dd,J=9.0,2.1Hz,1H),4 .51(dd,J=8.8,5.8Hz,1H),4.32(dd,J=8.3,6.1Hz,1H),3.97(s,2H),3.59- 3.50(m,2H),3.41(s,3H),3.04(t,J=7.6Hz,2H),2.98(t,J=7.7Hz,2H),2.5 7(q,J=6.3Hz,2H),1.95-1.82(m,2H),1.82-1.65(m,5H),1.65-1.42(m,5H). 13 C NMR(126MHz,D2O)δ 174.1,174.0,172.5,172.4,148.3,137.3,136.8,136.4,124.6,122.2,113.5,112.9,70.8 ,58.9,54.3,53.7,39.1,39.1,35.3,34.9,30.4,30.4,27.2,26.3,22.1,22.0.HRMS(m / z):C 26 H 38 Calculation of N8O5S cases[M+H] + :575.2764, measured value 575.2751.

[0110]

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[0111]

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[0112]

Chem.

[0113]

Chem.

[0114] [1.3 N,N'-di-tert-butoxycarbonyl-5-chloro-1H-benzotriazole-1-carboxamidine and N,N'-di-tert-butoxycarbonyl-6-chloro-1H-benzotriazole-1-carboxamidine (10)] [ka] These compounds were prepared in multiple batches. Typical procedure: N,N'-di-Boc-thiourea (7.7 g, 27.9 mmol) and 5-chlorobenzotriazole (4.3 g, 27.9 mmol) were dissolved in anhydrous ACN (250 mL), and DIPEA (14.6 mL, 83.6 mmol) was added. The reaction mixture was cooled to 0°C, EDCl (10.7 g, 55.7 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. This mixture was diluted with toluene (600 mL) and washed with 10% aqueous citrate (300 mL) and brine (300 mL). The organic layer was dried over MgSO4, concentrated under reduced pressure, and purified by silica gel column chromatography (0 → 15% toluene / n-heptane) to obtain a mixture of 5'- and 6' isomers (4, 3.49 g, 32%) as a white solid. TLC (toluene / n-heptane, 1:4 v / v):R f = 0.32 (6' isomer), 0.27 (5' isomer). 1 H NMR(500MHz,CDCl3)δ 8.99(s,2H),8.39(s,1H),8.32(d,J=8.8Hz,1H),8.09(d,J=1.9Hz,1H),8.03(d ,J=8.8Hz,1H),7.63-7.58(m,1H),7.51-7.45(m,1H),1.52(d,J=17.5Hz,27H). 13 C NMR(126MHz,CDCl3)δ 131.1,127.4,121.1,119.8,116.3,115.2,28.1.HRMS(m / z):C 17 H 22 [M+H] calculated for the case of ClN5O4Na + : 418.1258, measured value 418.1244.

[0115] [1.4 Protease assay conditions] Luminescent enzyme activity assay: D-cysteine ​​(final concentration 1 mM) and assay buffer (25 mM HEPES, 125 mM NaCl, 0.5% BSA, pH=7.4) were added to wells containing methoxyacetamide-βA-XR-6ABTC or methoxyacetamide-βA-XK-6ABTC (final concentration 666.7 μM), for a total volume of 30 μL. After incubation at 37°C for 30 minutes, 30 μL of detection mixture containing luciferase (final concentration 10 μM), MgCl2 (final concentration 6.7 mM), ATP (final concentration 1 mM), protease enzyme (factor Xa or thrombin) (final concentration 10 nM), and assay buffer (25 mM HEPES, 125 mM NaCl, 0.5% BSA, pH=7.4) was added. Using a SpectraMax M3 plate reader (Molecular Devices, San Jose, California, USA), luminescence was recorded in units of relative luminescence (RLU) every minute for 30 minutes at 37°C with an integration time of 1000 ms. The area under the curve (AUC; RLU) was:

number

[0116] [Example 2 - Provision of a library of caged luminescent probes] [2.1 Provision of constituent units] A peptide library of 40 caged luminescent probes, each containing either an arginine or lysine residue on P1, was synthesized and subsequently screened using two different trypsin-like serine proteases as a test of the synthesis method. By using a more stable aminoluciferin precursor and subsequently in-situ condensation with cysteine ​​in the final step before enzyme assay, our method is highly suitable for SPPS.

[0117] To develop an improved synthetic method for preparing luminescent peptides using SPPS, a more stable 6-ABTC precursor from aLuc was used during synthesis. The inventors aimed to address the inherent unreactive nature of the amino group of 6-ABTC(1) by first coupling it with a P1 residue in solution (Scheme 2) to realize the constituent units. Initially, 1 was synthesized in small quantities of a few grams starting from 2-chlorobenzo[d]thiazole using a procedure described in the well-known literature by Bon et al. (Beilstein J. Org. Chem. 2016, 12, 2019-2025). All initial coupling reactions of 1 with either Fmoc-Orn(Boc)-OH(2) or Fmoc-Lys(Boc)-OH(3) under standard peptide coupling conditions (HOBt, DIC / HATU, DIPEA), or C-terminal activation using isobutyl chloroformate and NMM, were in low yield and accompanied by considerable amounts of byproducts. High yields were obtained by switching to pre-activation of 1 with phosphorus trichloride in pyridine before adding 2 or 3. Coupling reactions of 1 with 2 or 3 yielded protected structural units 4 and 5, respectively (Scheme 2). Subsequently, the Boc side-chain protecting group was removed with formic acid, and the products were freeze-dried to obtain ornithine and lysine structural units 6 and 7, respectively.

[0118] Scheme 2 - Synthesis of constituent units 6 and 7. i. 6-ABTC (1, 1.0 equivalents), PCl3 (0.51 equivalents), Pyr, 3.5 hours, 40°C, 77-98%. ii. HCOOH, freeze-dried, quantification. [ka]

[0119] [2.2 Immobilization of constituent units on peptide synthesis resin] Since constituent units 6 and 7 were obtained, the inventors attached these constituent units to an aldehyde-functionalized resin by reductive amination. Because a tendency for partial acid hydrolysis of nitriles may occur, the inventors aimed to use an acid-unstable (3-formylindolyl)acetamidomethyl polystyrene resin (A. Hamze et al., J. Org. Chem 2004, 69, 8394-8402). Reductive amination of either 6 or 7 onto the aldehyde-functionalized resin was carried out at room temperature for 4 hours using NaBH3CN in a 1:1 mixture of tetrahydrofuran and trimethyl orthoformate as a water scavenger (Scheme 3). Extending the reaction time or heating did not increase the inventors' yield, because it often resulted in more byproducts, some of which involved hydrolysis of nitriles to amides.

[0120] Scheme 3 - Solid-phase peptide synthesis method for luminescent peptides by side-chain anchoring of compound 6 or 7 by reductive amination on (3-formylindolyl)acetamidomethyl resin. i. 6 or 7 (2 equivalents), THF:TMOF (1:1), 4 hours, room temperature. ii. NaBH3CN (2 equivalents), AcOH (3.5 equivalents), 2 hours, room temperature. iii. For n=3: N,N'-di-Boc-thiourea (2 equivalents), DIC (2 equivalents), DCM, room temperature, 16 hours. For n=4: Boc2O (3 equivalents), DIPEA (3 equivalents), DMF, room temperature, 16 hours. iv.a. 3% DBU in DMF, DMF (3 times). b. HOBt (3.6 equivalents), DIC (3.3 equivalents), Fmoc-AA-COOH / Cap-COOH (3.0 equivalents) (3 times), DMF. v. TFA:DCM (1:1, v / v), 2 hours. In this scheme, AA2 and AA3 represent further amino acid residues. R1 is -Boc or -C(=NBoc)NHBoc; R2 is H or -C(=NH)NH2. [ka]

[0121] After 6 and 7 were successfully mounted on a solid support, the second binding amine was further functionalized before the N-terminal peptide extension progressed. In the case of lysine derivatives (n=4), the inventors temporarily protected the secondary amine with di-tert-butyl dicarbonate and DIPEA in DMF, stirring overnight. To obtain arginine derivatives from the inventors' ornithine-binding resin, since highly toxic HgCl2 is unsuitable for SPPS due to the formation of insoluble HgS, the inventors guanidylated the secondary amine overnight with 2 equivalents of N,N'-di-Boc-thiourea in DCM along with N,N'-diisopropylcarbodiimide (DIC) as a desulfurization reagent. This yielded an arginine-functionalized resin, which could be used for subsequent N-terminal extension.

[0122] Following the initial synthesis of an arginine library by the inventors, they found, as confirmed by mass spectrometry, that this guanidylation reaction, following standard literature methods, yielded a considerable amount of thiourea byproduct (see Figure 2). Early attempts to shorten the reaction or add new reagents failed to overcome the formation of this byproduct. The use of guanidylation promoted by different activators, such as EDC, Cu(I)Cl or Cu(II)Cl or TCT, also did not reduce the formation of the byproduct. Evaluation of different guanidylation reagents, such as N,N'-di-Boc-1H-pyrazole-1-carboxamidine or Goodman's reagent, also resulted in a decrease in conversion rate, mainly due to the formation of the starting ornithine product.

[0123] In our initial library, we were still able to purify the resulting final product using the N,N'-di-Boc-thiourea DIC method, but at the same time, we sought a convenient solution to overcome the formation of this byproduct. Ultimately, we evaluated the use of a benzotriazole-based reagent (H.-J. Musiol, L. Moroder, Org. Lett. 2001, 3,3859-3861). After optimization, we were able to find that this reagent could be readily synthesized in small quantities (as a mixture of 5' and 6'-isomers) without using toxic HgCl2. This remarkable guanidylation reagent is now being used and evaluated in our laboratory for the synthesis of similar peptides by SPPS, and initial results show that a purer conversion with fewer byproducts is obtained compared to the use of N,N'-di-Boc-thiourea. [ka]

[0124] [2.3 Attachment of one or more additional amino acid residues] Having obtained lysine and arginine-functionalized resins, the inventors aimed to demonstrate their method by synthesizing a small peptide library. The inventors conceived of the positional scanning substrate combinatorial library (PS-SCL) method, as described by Poreba et al. (M. Poreba et al., Caspases, Paracaspas, and Metacaspas: Methods and Protocols (Eds.: P.V. Bozhkov, G. Salvesen), Springer New York, 2014, pp. 41-59), to define enzyme substrate specificity. In the inventors' experience, the substrate methoxyacetamide-β-Ala-Gly-Arg-aLuc appeared to be an excellent substrate for trypsin-like serine proteases such as thrombin. The inventors used this substrate as a model sequence to test their method by synthesizing a tripeptide library by simply altering the P2 residue (here: glycine) of all standard amino acids in order to study P2 dependence. The inventors extended the peptide chain of the product coupled with their resin at the N-terminus by standard Fmoc SPPS chemistry. Deprotection of Fmoc was carried out using 3% DBU in DMF because these conditions yielded much better results than using 20% ​​piperidine in DMF. Subsequently, coupling of the amino acids or N-terminal cap protected with 3 equivalents of Fmoc was performed under standard coupling conditions (3.6 equivalents of HOBt and 3.3 equivalents of DIC in DMF). Once the sequence was complete, the crude peptide was cleaved from the resin using trifluoroacetic acid in crude DCM (1:1, v / v; 2.5% TIS and EDT were added for Trt-containing sequences), lyophilized, and subsequently purified by RP-HPLC to obtain a pure 6-ABTC-binding peptide, which was analyzed by ESI-MS and NMR (a typical amount of 100 mg of unfunctionalized resin per substrate yielded several milligrams of pure product).Using this method, the inventors obtained a library containing 40 peptides having the sequence methoxyacetamide-β-Ala-P2-Arg / Lys-aLuc (where P2 = one of all 20 native L-amino acids, and R2 forms either lysine or arginine, as defined in Scheme 3). The synthesized compounds are of formula F2.3 and are shown in Table T2.3. P1 represents the combination of R2 and side chain length to form either Lys or Arg. P2 represents the amino acid adjacent to that Lys or Arg. The amino acids listed in Table T2.3 are L-amino acids where appropriate. [ka]

[0125] [Table 1]

[0126] [2.4 The compound is stable and functional.] The synthesized 6-ABTC-functionalized peptides were far more stable than the individual aLuc peptides. Their use does not require a cysteine ​​condensation reaction prior to enzymatic conversion, preferably using D-cysteine. Since the click reaction of D-cysteine ​​and 6-ABTC is documented in the literature and generally yields only aLuc-functionalized derivatives, the inventors hypothesized that this reaction could be performed in situ at the near final stage of their assay, before the addition of proteases. As proof, the inventors screened their peptide library against two trypsin-like serine proteases, factor Xa (FXa) and thrombin (FXIIa). First, the inventors incubated their 6-ABTC-binding peptides with D-cysteine ​​in buffer at 37°C for 30 minutes to obtain the respective aLuc derivatives, after which proteases, ATP, MgCl2, and luciferase were added. Next, the luminescence was recorded at 37°C for 30 minutes, and the area under the curve (AUC) was calculated. The ratio of substrate hydrolysis to either thrombin or FXa was determined and is shown in Figure 2. By changing the position of P2 in our library, we were able to obtain a significant difference in the selectivity of our substrate to either thrombin or FXa, demonstrating the robustness of our synthesis principle and in-situ cysteine ​​condensation. We confirmed, for example, that incorporating proline at P2 significantly increased the selectivity to thrombin.

[0127] [2.5 Conclusion] The inventors developed an SPPS method that enables the rapid synthesis of C-terminally modified peptides of 6-ABTC by side-chain anchoring of the first residue. These products were stable. The use of 6-ABTC and the in-situ condensation reaction with cysteine ​​were key steps in achieving this stability. By a guanidylation reaction on a special resin, the ornithine residue can be converted to an arginine residue, thus avoiding by-products. After cleavage of the peptide from the resin and direct purification such as RP-HPLC, a subsequent condensation reaction with cysteine ​​(such as D-cysteine) to obtain aLuc-caged peptide was carried out in situ before the addition of the protease. The inventors synthesized a peptide library of 40 tripeptides screened for selectivity to either trypsin-like serine protease factor Xa or thrombin. The results presented in this study may enable the (automated) synthesis of larger luminescent peptide libraries, for example, those having either an arginine or lysine residue at the P1 position, and proteases that prefer positively charged amino acids at P1 are of particular interest.

Claims

1. A method for producing peptide conjugates: i) General formula (I): 【Chemistry 1】 (In the formula, n is 1, 2, 3, 4, or 5; p' is a protecting group. A step of providing a constituent unit; ii) Immobilizing the constituent units onto a peptide synthesis resin; iii) A step of obtaining an immobilized peptide conjugate by attaching one or more additional amino acid residues using conventional solid-phase peptide synthesis, Methods that include...

2. iv) The method according to claim 1, further comprising the step of cleaving the immobilized peptide conjugate from the resin to obtain a free peptide conjugate, and optionally purifying it.

3. v) The method according to claim 1 or 2, further comprising the step of contacting the free peptide conjugate with cysteine ​​to obtain a peptide-aminoluciferin conjugate.

4. The method according to any one of claims 1 to 3, wherein step ii) is to reductively aminate the free amine of the constituent unit onto the aldehyde of the peptide synthesis resin to form an immobilized secondary amine, the immobilized secondary amine is optionally subsequently protected.

5. The method according to step 4, wherein the immobilization secondary amine is guanidylated, and the resulting guanidinium moiety is optionally protected.

6. Compounds of general formula (V) or salts thereof: 【Chemistry 2】 (In the formula, h1 is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH ２ And; h2 is H, p', C1-4 alkyl, or peptide synthesis resin; n is 1, 2, 3, 4, or 5; R is a polypeptide moiety containing H, p', X, or 1 to 10 amino acids; p' is a protecting group independently in each case; X is -C1-20 alkyl, -SO ２ The alkyl and acyl groups are -C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where the alkyl and acyl groups are linear, branched, or cyclic, possibly unsaturated, and possibly substituted with halogens or C1-4 alkoxy groups.

7. R is given by the general formula (pep): 【Transformation 3】 (In the formula, sc １ is preferably H, or an amino acid side chain selected from linear, branched, or cyclic C1-12 alkyl groups, where the alkyl is optionally unsaturated and optionally substituted with a halogen, or optionally substituted with a halogen-(CH ２ ) ０－４ - Selected from [C5-10 (hetero)aryl]; sc ２ is an amino acid side chain preferably selected from H, or linear, branched, or cyclic C1-12 alkyl, where the alkyl is optionally unsaturated and optionally substituted with halogen, or optionally substituted with halogen - (CH ２ ) ０－４ - [C5-10 (hetero)aryl], and in each case of sc ２ is independently selected; b is -CH ２ - Either it is, or it does not exist; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; X is H, or -C1-20 alkyl, -SO ２ -C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, where alkyl and acyl are linear, branched, or cyclic, possibly unsaturated, and possibly substituted with halogens or C1-4 alkoxys. The compound according to claim 6, which is the polypeptide portion.

8. The compound according to claim 7, wherein m is 1.

9. X is -C (=O) -CH ２ -O-CH ３ The compound according to claim 7 or 8.

10. m is 1 or 2, preferably 1; sc ２ In the case of an amino acid adjacent to X, it is H; b is -CH if it is the amino acid portion adjacent to X. ２ - The compound according to any one of claims 7 to 9.

11. The compound according to claim 10, wherein m is 1.

12. h2 is a peptide synthesis resin, or X is -C (=O)-CH ２ -O-CH ３ is, or b is -CH if it is the amino acid portion adjacent to X. ２ - is, or X is -C (=O)-CH ２ -O-CH ３ And b is -CH in the case of an amino acid portion adjacent to X. ２ - The compound according to any one of claims 7 to 11.

13. The compound according to any one of claims 6 to 12, wherein h2 is a peptide synthesis resin, preferably an aldehyde-functionalized resin, preferably a highly acid-unstable resin, for example, (3-formylindolyl)acetamidomethylpolystyrene.

14. The compound according to any one of claims 6 to 13, wherein h1 is H, and preferably n is 2 or 3, more preferably 3.

15. The compound according to any one of claims 6 to 13, wherein h1 is H and n is 2.

16. The compound according to any one of claims 6 to 13, wherein h1 is H and n is 3.

17. h1 is -C(=N-p')-NH-p' or -C(=NH)-NH ２ The compound according to any one of claims 6 to 13, wherein n is preferably 2 or 3, more preferably 2.

18. h1 is -C(=N-p')-NH-p' or -C(=NH)-NH ２ The compound according to any one of claims 6 to 13, wherein n is 2.

19. i) A compound according to any one of claims 6 to 18; ii) Cysteine, preferably D-cysteine, A kit of parts including [the specified component].

20. A method for determining protease activity: i) Providing one or more compounds according to any one of claims 6 to 18; ii) The step of contacting the provided compound with cysteine ​​to obtain an aminoluciferin-peptide conjugate; iii) The step of contacting the aminoluciferin-peptide conjugate with the protease to obtain free aminoluciferin; iv) A step of contacting the free aminoluciferin with luciferase to generate a light emission signal, Methods that include...

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