Compounds and methods for high purity chemiluminescent substrates

Abstract

The present invention relates to substrate molecules suitable for monitoring enzymatic activity via chemiluminescence. The substrates are provided via solid phase peptide synthesis using an aminoluciferin precursor. After solid phase synthesis the precursor is converted to an aminoluciferin moiety. The substrates are of high purity and stability.

Description

[0001] Compounds and methods for high purity chemiluminescent substrates

[0002] Field of the invention

[0003] The present invention relates to substrate molecules suitable for monitoring enzymatic activity via chemiluminescence. The substrates are provided via solid phase peptide synthesis using an aminoluciferin precursor. After solid phase synthesis the precursor is converted to an aminoluciferin moiety. The substrates are of high purity and stability.

[0004] Background art

[0005] In the recent decades luminescent imaging has emerged as a powerful tool to monitor enzymatic activity. Luminescent imaging generally occurs via the oxidation of small molecules, where no external light source is needed, a high signal-to-noise ratio can be obtained, and the background signal is usually low as compared to fluorescence. Numerous methods have been reported for luminescent imaging, as for instance for sensing reactive oxygen and nitrogen species, imaging of cancer cells, and the in vivo quantification of glucose uptake.

[0006] White and co-workers were among the firsts who reported on the use of the d-luciferin derivative aminoluciferin (aLuc), which can be used in proteolytic assays since the amine can be coupled to the C-terminus of a peptide (E. H. White et al., J. Am. Chem. Soc. 1966, 88, 2015-2019.) The free 6'-amino group of aLuc appeared to be crucial to preserve its luminescent properties (as similar for the 6'-hydroxy in d-luciferin), modification of this moiety usually results in quenching of the luminescence. These quenched luminescent probes have also been referred to as caged luciferins and can be used in luminescent enzymatic assays, as upon enzymatic activity the free aLuc is released which can be quantified. aLuc is oxidized into the excited oxyluciferin by the catalytic action of luciferase and subsequent relaxation back into the ground state releases photons. Numerous luminescent probes have been developed according to this uncaging principle and are widely used with a variety of enzymes among which hydrolases and proteases, see for instance W02020 / 079155.

[0007] To extent the applications of luminescent imaging, new caged luciferins are needed which upon enzymatic activity cleave of the luciferin moiety result in a proportional light signal. Current methods for the preparation of luminescent caged peptides mostly rely on the use of solution phase synthesis. Generally, a protected peptide is C-terminally activated with reagents such as isobutyl chloroformate in the presence of a base like as 4-methyl-morpholine (NMM), prior to the addition of the aLuc precursor 6-aminobenzo[c(]thiazole-2-carbonitrile (6-ABTC, Fig. 1A, see D. Sondag et al., ChemBioChem 2022, 23.) After this coupling, the sidechain protecting groups of the peptide are cleaved off and a final condensation reaction with d-cysteine yields the caged luciferin peptides. The use of solid phase peptide synthesis (SPPS) for the synthesis of caged luciferins would drastically lowerthe synthesis steps in solution phase, eliminate multiple purification steps and allow for parallel synthesis. Kovacs et al. (Int. J. Pept. Res. Ther. 2019, 25, 1209-1215) revealed that loading of the carboxylic acid of aLuc on to a solid support and the subsequent elongation of the peptide chain was unsuccessful due to the inherent instability of the thiazoline moiety of aLuc (Fig. 1 B). The thiazoline ring of luciferins is prone to oxidation, which can result in the formation of the dehydrogenated derivative, a well-known luciferase inhibitor. Kovacs et al. therefore recommend to use liquid phase coupling of peptides to aLuc.

[0008] There is a need for improved caged luciferins. There is a need for improved methods for the provision of caged luciferins. There is a need for more pure caged luciferins.

[0009] Summary of the invention

[0010] The inventors developed a solid phase peptide synthesis (SPPS) method for the synthesis of caged luminescent peptides starting with the sidechain anchoring of an amine-bearing side chain such as eithera lysine or an ornithine residue (Fig. 1 C). The amine moieties could be guanidinylated on resin to obtain arginine residues or analogues. The introduction of the aminoluciferin (aLuc) moiety at a late-stage renders this approach compatible with SPPS, because the more stable 6- ABTC moiety at the C-terminus of the first residue is introduced prior to the solid phase reactions. The invention thus provides a method for producing a peptide conjugate, the method comprising the steps of: i) providing a building block of general formula (I): wherein n is 1 , 2, 3, 4, or 5; p' is a protecting group; ii) immobilizing the building block on a peptide synthesis resin; iii) conjugating one or more additional amino acid residues using conventional solid phase peptide synthesis to obtain an immobilized peptide conjugate.

[0011] Preferably, the method further comprising the step of: iv) cleaving the immobilized peptide conjugate off the resin to obtain a free peptide conjugate, and optionally purifying it. Preferably the method further comprising the step of: v) contacting the free peptide conjugate with cysteine to obtain a peptide-aminoluciferin conjugate. In preferred embodiments step ii) comprises reductive amination of the free amine of the building block onto an aldehyde of the peptide synthesis resin to form an immobilizing secondary amine, wherein the immobilizing secondary amine is optionally subsequently protected. Here, in some embodiments the immobilizing secondary amine is guanidinylated, after which the resulting guanidinium moiety is optionally protected.

[0012] Also provided is a compound of general formula (V) or a salt thereof: wherein hi is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; h2 is H, p', C1-4 alkyl, or a peptide synthesis resin; n is 1 , 2, 3, 4, or 5; R is H, p', X, or a polypeptide moiety comprising 1 -10 amino acids; p' is in each instance independently a protecting group; and X is H or -C1-20 alkyl, - SO2-CI-20 alkyl, -C(=O)-O-C1-20 alkyl, or-C1-20 acyl, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy. Preferably R is a polypeptide moiety of general formula (pep): wherein sc1is an amino acid side chain preferably selected from H or a linear, branched, or cyclic C1-12 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, or selected from -(CH2)o-4-[C5-1O (hetero)aryl] that is optionally substituted with halogen; sc2is an amino acid side chain preferably selected from H or a linear, branched, or cyclic C1-12 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, or selected from - (CH2)O-4-[C5-1 O (hetero)aryl] that is optionally substituted with halogen, wherein each instance of sc2is 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, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy. In preferred embodiments m is 1. In preferred embodiments X is -C(=O)-CH2-O-CH3. In some embodiments m is 1 or 2, preferably 1 ; sc2is H for the amino acid moiety adjacent to X; b is -CH2- for the amino acid moiety adjacent to X. In some embodiments h2 is a peptide synthesis resin, preferably an aldehyde functionalized resin, preferably a highly acidic labile resin such as (3- formylindolyljacetamido methyl polystyrene. Preferably hi is H, and preferably n is 2 or 3, more preferably 3. Preferably hi is -C(=N-p')-NH-p' or -C(=NH)-NH2, and preferably n is 2 or 3, more preferably 2.

[0013] Also provided is a kit of parts comprising i) a compound as defined above; and ii) cysteine, preferably D-cysteine. Also provided is a method for determining protease activity, the method comprising the steps of: i) providing one or more compounds as defined above; ii) contacting the provided compounds with cysteine to provide aminoluciferin-peptide conjugates; iii) contacting the aminoluciferin-peptide conjugates with the protease to provide free aminoluciferin; iv) contacting the free aminoluciferin with luciferase to generate a luminescent signal.

[0014] Description of embodiments

[0015] In the art, the synthesis of caged luminescent peptide substrates remains challenging, especially when multiple substrates are required. Most currently available methods rely on solution phase approaches. We herein present a solid phase peptide synthesis (SPPS) method for the synthesis of C-terminally caged aminoluciferin peptides via the sidechain anchoring of the residue that borders the aminoluciferin. The resulting peptides can for instance be as shown in Example 1 .3.1 .1 and in Example 1 .3.2.1 . Accordingly the invention provides a method for producing a peptide conjugate, the method comprising the steps of: i) providing a building block of general formula (I): wherein n is 1 , 2, 3, 4, or 5; p' is a protecting group; ii) immobilizing the building block on a peptide synthesis resin; iii) conjugating one or more additional amino acid residues using conventional solid phase peptide synthesis to obtain an immobilized peptide conjugate.

[0016] Such a method is referred to herein as a method according to the invention. Preferably steps are carried out in ascending numerical order. The method avoids the formation of dehydrogenated aminoluciferin (aLux), which is a known downside of known SPPS approaches towards aLuc peptide conjugates.

[0017] In step i) of the method a building block is provided. The building block has a free primary amine, as part of the side chain of an amino acid residue. This amino acid residue is linked to aminobenzo[cf]thiazole-2-carbonitrile (ABTC) at its C-terminus. This amino acid residue bears a protecting group at its N-terminus, to allow its convenient use in SPPS. This amino acid residue is directly adjacent to the eventual aLuc and can be referred to as Pi. In analogy, the amino acid residue that is directly N-terminal to Pi can be referred to as P2, and the next one as P3, etc. n determines the length of the side chain of the Pi amino acid. In preferred embodiments n is 1 , 2, 3, or 4. It is more preferred that n is 2, 3, or 4. Most preferably, n is 2 or 3, so as to form ornithine or lysine. p' is a protecting group. Preferably p' is a protecting group that is suitable for SPPS strategies. A skilled person can select suitable protecting groups for p'. The protecting group is preferably base-labile. As used herein, a protecting group has its customary meaning of a group that is linked to the heteroatom in such a way as to reduce its reactivity, or to protect it from certain conditions. After having served their function, protecting groups can generally be removed again using routine chemistry. A skilled person is well aware of protecting groups and can select useful protecting groups for particular groups or atoms to be protected, reaction conditions, storage conditions, purification techniques, or available deprotection techniques. A helpful reference in this regard is Greene's Protective Groups in Organic Synthesis, Wuts & Greene, DOI:10.1002 / 0470053488.

[0018] In preferred embodiments p' is 9-fluorenylmethyl carbamate (Fmoc), f-Butyl carbamate (Boc), benzyl carbamate (Cbz), acetamide (Ac), trifluoroacetamide, phthalimide, benzylamine (Bn), triphenylmethylamine (Tr, or Trt), or tosylamide (Ts). Preferred base-labile embodiments for p' are Fmoc, Cbz, Ac, trifluoroacetamide, and phthalimide, more preferably Fmoc or Cbz, most preferably Fmoc. For its reliability in SPPS, it is most preferred that p' be Fmoc.

[0019] The building block of general formula (I) can be synthesized by the conjugation of the appropriate amino acid with ABTC, preferably with 6-ABTC. It is convenient when p' is already present during this coupling, and the free amine in general formula (I) is preferably also protected during this coupling. For this protection, use of a protecting group that is orthogonal to p' is preferred, such as Boc. Detailed embodiments are provided in Example 2.1 .

[0020] In some embodiments a salt of building block (I) is provided. A skilled person understands that for immobilization on a resin (step ii), the primary amine of Pi should be a free amine. In such cases the amine should be freed before immobilization. Preferably, the building block of general formula (I) is of general formula (II), more preferably of general formula (III), most preferably of general formula (IV):

[0021] Step / ' / ' - immobilizing the building block on a peptide synthesis resin

[0022] In step ii) the building block is immobilized on a peptide synthesis resin. A skilled person can select a suitable resin. This immobilization can conveniently be performed via the free primary amine of the building block, forming a secondary amine that connects the building block to the resin. This amine is referred to herein as the immobilizing secondary amine. To obtain free amines after SPPS, it is preferred that the resin is a resin that can release an amine after cleavage. Such a resin can also release a guanidinium moiety when appropriate. Examples of suitable resins are aldehyde- functionalized resins and 2-chlorotrityl chloride resins. Aldehyde-functionalized resins are preferred, particularly highly acid labile aldehyde resins such as (3-formylindolyl)acetamido methyl polystyrene resin. As used herein, a highly acid labile aldehyde resin is a resin where cleavage can be performed using at most 10 vol.-% trifluoroacetic acid (TFA) in water; preferably at most 7 vol.- % is use, more preferably about 1-6 vol.-% is used, most preferably about 2-5 vol.-%. Using a highly acid labile aldehyde resin is attractive because it reduces the risk of acidic hydrolysis of the nitrile moiety present in the building block.

[0023] For immobilizing a primary amine on an aldehyde resin, it can be convenient to use reductive amination. In preferred embodiments, step ii) comprises reductive amination of the free amine of the building block onto an aldehyde of the peptide synthesis resin to form an immobilizing secondary amine, wherein the immobilizing secondary amine is optionally subsequently protected. Reductive amination is well known in the art. In general, the primary amine of the building block forms an imine with the aldehyde, which is then reduced to form a secondary amine. Reductive amination can be performed with for instance NaBH(OAc)3, NaCNBFta, or NaBH4-Ti(OiPr)4, more particularly with NaBH(OAc)3 in DCE (1 ,2-dichloroethene) and / or TMOF (trimethyl orthoformate), with NaBHsCN in THF (tetrahydrofuran) and / or TMOF and / or AcOH, or with NaBH4-Ti(OiPr)4 in THF. Reductive amination is preferably performed for about 0.5-48 hours, preferably about 1 -36 hours, more preferably about 2-30 hours, more preferably about 2.5-24 hours, more preferably about 3-18 hours, more preferably about 3.5-16 hours, more preferably about 4-14 hours. In some embodiments it is performer for about 2.5-5.5 hours, preferably about 3-5 hours. Reductive amination is preferably performed at a temperature of about 10-40 °C, more preferably about 15- 30 °C, more preferably about 17-25 °C, most preferably about 19-23 °C. It is convenient to perform the reductive amination at room temperature. Good results were obtained when reductive amination was carried out with NaCNBHs in a 1 :1 mixture of tetrahydrofuran and trimethyl orthoformate for 4 hours, at room temperature. When Pi is intended to eventually be a residue such as lysine or ornithine, the immobilizing amine can be subsequently protected to make it compatible with any following SPPS steps. It is convenient to select an acid-labile protecting group for this, such as Boc or triphenylmethylamine. A skilled person knows how to introduce such a protecting group. Boc can for instance be conveniently introduced using di-tert-butyl dicarbonate. When introducing such a protecting group, the presence of a base can be preferred, for instance DIPEA such as 1 equiv. Such a reaction can be allowed to proceed for 2-24 hours, preferably about 8-14 hours, such as overnight.

[0024] When Pi is intended to eventually be a guanidinylated residue such as arginine, the immobilizing amine can be guanidinylated prior to any following SPPS steps. In preferred embodiments, the immobilizing secondary amine is guanidinylated, after which the resulting guanidinium moiety is optionally protected. In preferred embodiments the guanidinium moiety is protected. Guanidinylating agents can be used such as A / , / V'-di-Boc-thiourea or the 5,6-chloro- benzotriazole agent shown below (A / ,A / ’-di-ferf-butoxycarbonyl-5-chloro-1 / 7-benzotriazole-1- carboxamidine & A / ,A / ’-di-ferf-butoxycarbonyl-6-chloro-1 / 7-benzotriazole-1-carboxamidine). The invention provides use of this guanidinylating agent in the guanidinylation of a secondary amine, preferably of a secondary amine that is immobilized on a resin, most preferably of an immobilizing amine. Such agents are preferably in the presence of a base such as DIPEA, preferably about 1 equiv. relative to the guanidinylating agent. Additionally a carbodiimide is preferably present, such as diisopropylmethanediimine (DIPCDI) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), preferably DIPCDI, such as 1 equiv. relative to the guanidinylating agent. Such a reaction is preferably allowed to proceed about 4-20 hours, more preferably about 8-14 hours such as overnight. A skilled person will appreciate that these agents introduce a protected guanidinium moiety, obviating the need for subsequent protection.

[0025] Because the building block is immobilized on a resin, the reaction can be conveniently washed after it has completed, such washings can be performed using routine SPPS techniques, for instance using alternating swelling and contracting washes. Unreacted sites can be capped using acetic anhydride. In preferred embodiments, after immobilization, the resin is washed with DCM (preferably 3x) and DMF (preferably 3x) and unreacted sites are preferably capped by agitating with AC2O (138 pL, 1.46 mmol) and a base such as pyridine (1 18 pL, 1.46 mmol) in a solvent such as DMF, preferably for 10 min. Afterwards, the resin is preferably washed with a solvent such as DMF (preferably 3x).

[0026] Step Hi - solid phase peptide synthesis (SPPS)

[0027] After the building block has been immobilized on the resin, the remainder of the intended peptide can be constructed using otherwise conventional SPPS. For this, generally, p' is deprotected, after which a new amino acid residue is coupled. This new residue is duly protected while it is coupled, and can in turn be deprotected to make a primary amine available. In between steps, the resin can be conveniently washed to remove excess reagents or side products. In preferred embodiments the SPPS follows the Fmoc-strategy. Fmoc can be deprotected using a base, preferably an organic base, preferably a non-nucleophilic base. For instance, it can be deprotected using 20 vol.-% piperidine in DMF, or using 3 vol.-% DBU in DMF, the latter of which was found to be preferred.

[0028] Preferably, at least one additional amino acid residue is added. Preferably, no more than about 25 additional amino acid residues are added, more preferably no more than 20, more preferably no more than 15, most preferably about 1 -10 additional amino acid residues are added.

[0029] In preferred embodiments the amine of the N-terminal residue is capped. Capping agents are known in the art, and are generally carboxylic acids without further reactive groups. Good results were obtained using methoxyacetic acid to obtain a methoxyacetamide cap.

[0030] Step iv - cleaving the peptide conjugate off the resin

[0031] It can be useful to keep the ABTC-peptide conjugates immobilized on their resin, for instance when long term storage is envisaged or when possible further modification is foreseen. When the ABTC-peptide conjugate is to be used, it can be cleaved off of the resin, using conventional SPPS procedures. Thus, in some embodiments, the method further comprises the step of: iv) cleaving the immobilized peptide conjugate off the resin to obtain a free peptide conjugate, and optionally purifying it.

[0032] The free peptide conjugate is as described later in the section on compounds. Cleavage is a routine procedure in SPPS and is preferably performed using acid treatment, more preferably using TFA treatment. The acid treatment is preferably diluted to avoid side product formation. Dilution can be in any inert aprotic solvent, such as solvents known to swell resins, for instance using dichloromethane (CH2CI2, DCM). It was found that good purity was achieved when at most 75 vol.-% TFA was used during cleavage. Cleavage can be for about 0.5-16 hours, preferably about 1-12 hours, more preferably about 1.5-8 hours, or about 2-6 hours. Cleavage is preferably performed at a temperature of about 10-40 °C, more preferably about 15-30 °C, more preferably about 17-25 °C, most preferably about 19-23 °C. It is convenient to perform cleavage at room temperature. Preferably the immobilized peptide conjugate is cleaved from the resin using 50 vol.- % TFA in CH2CI2 for 2 h.

[0033] When the immobilized peptide conjugate comprises certain protecting groups known to produce undesirable side products such as reactive carbocations, scavengers can be used during cleavage. The use of scavengers is well-established and a skilled person can design a composition for use in cleavage. For instance, thiols such as ethanedithiol (EDT) or silanes such as triisopropylsilane (TIS) can be added. Good results were obtained when 2.5 vol.-% TIS and 2.5 vol.- % EDT were used for cleaving peptides comprising Trt protecting groups.

[0034] In some embodiments the free peptide conjugate is purified after cleavage. Purification can be performed in any conventional way, for instance by precipitating the cleavage cocktail in diethyl ether, preferably about three times. The precipitated product can be concentrated by centrifugation, after which it can optionally be washed and / or concentrated in vacuo. In preferred embodiment the free peptide conjugate is lyophilized after cleavage, more preferably after purification following cleavage. Further purification can be achieved via chromatography techniques such as HPLC, preferably preparative or semi-preparative HPLC. Suitable HPLC is RP-HPLC.

[0035] Step v - reaction with cysteine

[0036] The free peptide conjugates, or the immobilized peptide conjugates, comprise an ABTC moiety that has a nitrile group. This group readily reacts with cysteine to form aLuc. In preferred embodiments, the method further comprises the step of: v) contacting the free peptide conjugate with cysteine to obtain a peptideaminoluciferin conjugate.

[0037] Here, the reaction with cysteine is performed after the peptide conjugate has been cleaved off of the resin. This allows the convenient storage of peptide conjugates without risk of aLuc dehydrogenation, because aLuc has not been formed yet. The reaction with cysteine can be performed close to the moment of intended use of the caged aLuc conjugates, which can even be in situ directly prior to an assay. In preferred embodiments, the free peptide conjugate is reacted with cysteine in situ prior to addition of a protease.

[0038] In other embodiments, the reaction with cysteine is performed while the peptide conjugate is still immobilized. This associates the advantages of SPPS with this final reaction step. In such cases, the method comprises the step of: v) contacting the immobilized peptide conjugate with cysteine to obtain a peptideaminoluciferin conjugate. This step is preferably performed in between steps iii) and iv). In such cases the steps are preferably performed in the order i), ii), iii), v), iv).

[0039] When the free peptide conjugate is contacted with cysteine, this is preferably performed in an aqueous buffer such as 25 mM HEPES, 125 mM NaCI, and 0.5 wt.-% BSA, at pH 7.4. Cysteine is preferably present in excess, such as 10-50 equiv., or 20-40 equiv., or more. Contacting can be performed at room temperature or at slightly elevated temperature, such as at 30-50 °C, preferably at 35-40 °C such as at about 37 °C. Contacting is preferably performed for about 1 -600 minutes, preferably about 5-300 minutes, more preferably about 10-180 minutes, more preferably about 15- 120 minutes, more preferably about 20-60 minutes, most preferably about 25-45 minutes such as about 30 minutes.

[0040] When the immobilized peptide conjugate is contacted with cysteine, this is preferably performed in a solvent suitable for SPPS such as DMF or NMP. Other features are preferably as described above.

[0041] For this contacting, a preferred cysteine is D-cysteine. For this contacting, cysteine is preferably free cysteine, without protecting groups. Compounds

[0042] The method according to the invention allows for the convenient production of peptide-aLuc conjugates, and of peptide-ABTC conjugates. These latter are convenient intermediates towards peptide aLuc-conjugates and are not susceptible to the dehydrogenation that can occur in aLuc conjugates. Because aLuc can be formed in situ, the peptide-ABTC conjugates are almost as ready- to-use as the aLuc conjugate. The invention thus provides a compound of general formula (V) or a salt thereof: wherein hi is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; h2 is H, p', C1-4 alkyl, or a peptide synthesis resin; n is 1 , 2, 3, 4, or 5;

[0043] R is H, p', X, or a polypeptide moiety comprising 1-10 amino acids; p' is in each instance independently a protecting group; and

[0044] X is H or -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy. hi is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; preferably, hi is H, -C(=N-p')- NH-p', or -C(=NH)-NH2; most preferably hi is H. In some embodiments hi is -C(=N-p')-NH-p' or - C(=NH)-NH2. In some embodiments hi is H, p', or C1-4 alkyl, preferably H or p'. h2 is H, p', C1-4 alkyl, or a peptide synthesis resin; preferably h2 is H or a peptide synthesis resin. In preferred embodiments, both hi and h2 are H. This allows use ofthe compound according to the invention as building blocks for a method according to the invention. In such cases, R is preferably p'. When p' is comprised in hi or h2 it is preferably acid-labile, more preferably it is Boc.

[0045] C1-4 alkyl is preferably C1-3 alkyl, more preferably C1 -2 alkyl. Examples of C1-4 alkyl are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, tert-butyl, and cyclobutyl.

[0046] Preferred compounds according to the invention are of general formula (I), more preferably of general formula (II), still more preferably of general formula (III), most preferably of general formula (IV). n and p' are as defined for compounds of general formula (I). In further preferred embodiments, compounds are of general formula (VI). Compounds and building blocks such as those of general formula (I) preferably match the stereochemistry as shown in general formula (VI). In other words, the Pi residue is preferably an L-residue.

[0047] In particular embodiments h2 is a peptide synthesis resin. In such cases, such a compound is often referred to as a peptide synthesis resin loaded with the compound of general formula (V). Such a resin generally comprises multiple compounds of general formula (V), as resins generally have a plurality of loading sites. A peptide synthesis resin as can be represented by h2 is preferably as defined earlier herein. In preferred embodiments h2 is a peptide synthesis resin, preferably an aldehyde functionalized resin, preferably a highly acidic labile resin such as (3- formylindolyl)acetamido methyl polystyrene.

[0048] It can be preferred that Pi be lysine or ornithine. In such cases, hi is H, and preferably n is 2 or 3, more preferably 3. It can be preferred that Pi be arginine. In such cases, hi is -C(=N-p')- NH-p' or -C(=NH)-NH2, and preferably n is 2 or 3, more preferably 2.

[0049] R is H, p', X, or a polypeptide moiety comprising 1 -10 amino acids. In preferred embodiments, R is H, p', or X, more preferably H or p'. Such compounds can advantageously be used as building blocks, or as precursors thereof.

[0050] When R is a polypeptide moiety, the compound can be a substrate for a protease, or a resin loaded with such a substrate. Generally, when R is a polypeptide moiety, one or more rounds of SPPS have been performed on an initial building block. In such embodiments, R is a polypeptide moiety of general formula (pep): wherein sc1is an amino acid side chain preferably selected from H or a linear, branched, or cyclic 01-12 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, or selected from -(CH2)o-4-[C5-1 O (hetero)aryl] that is optionally substituted with halogen; sc2is an amino acid side chain preferably selected from H or a linear, branched, or cyclic C1-12 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, or selected from -(CH2)o-4-[C5-1 O (hetero)aryl] that is optionally substituted with halogen, wherein each instance of sc2is 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, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy. sc1and sc2are amino acid side chains, and are preferably not optionally substituted; and are preferably H, C2-5(halo)alkyl-N(h1)2, C1-4(halo)alkyl, 5-9-membered (hetero)aryl, C1 - 2(halo)alkyl-[5-9-membered (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-membered (hetero)cycloalkyl]; wherein C2-6(halo)alkyl-N(h1)2is preferably -CH2-CH2-CH2-NH2 or -CH2-CH2-CH2-CH2- NH2; wherein 5-10-membered (hetero)aryl is preferably phenyl; wherein 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; wherein 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, more preferably -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; wherein C2-6(halo)alkyl-N(h1)C(N(h1)2)(=Nh1) is preferably -CH2-CH2-CH2-N-C(=NH)-NH2; wherein C1-6(halo)alkyl-C(0)-N(h1)2is preferably -CH2-CH2-C(O)NH2or -CH2-C(O)NH2. b is -CH2- or absent; when b is not in the moiety adjacent to X, it is preferably absent. In preferred embodiments, b is -CH2- when it is in the moiety directly adjacent to X. This forms a beta amino acid as the N-terminal residue. 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, most preferably it is 1 . This forms a tripeptide conjugated to ABTC.

[0051] X is H or -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy. When X is optionally substituted, it is preferably with methoxy. C1 -20 in alkyl or acyl as defined for X is preferably C1-12, more preferably C1-8, even more preferably C1 -6. For C1-6 acyl, it 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. Examples of acyl moieties are as the alkyl moieties, bearing an oxo-substituent on the carbon atom directly adjacent to the nitrogen to which X is linked. It is highly preferred that X is -C(=O)- CH2-O-CH3.

[0052] In preferred embodiments, m is 1 or 2, preferably 1 ; sc2is H for the amino acid moiety adjacent to X; and b is -CH2- for the amino acid moiety adjacent to X.

[0053] The invention provides a kit of parts comprising a compound as defined above, and cysteine. Preferably, the cysteine is D-cysteine. Such a kit has is useful for performing luciferase assays. The kit can further comprise a luciferase enzyme. The kit can further comprise ATP, or MgCh, or instructions for use. Method for determining protease activity

[0054] The compounds can suitably be used in assays for determining protease activity. Proteases of interest can for instance be blood clotting factors. The method for determining protease activity preferably comprising the steps of: i) providing one or more compounds according to the invention; ii) contacting the provided compounds with cysteine to provide aminoluciferin-peptide conjugates; iii) contacting the aminoluciferin-peptide conjugates with the protease to provide free aminoluciferin; iv) contacting the free aminoluciferin with luciferase to generate a luminescent signal.

[0055] Features and definitions are preferably as defined above. For instance, 6-ABTC conjugated peptides can be incubated with D-cysteine for 30 min at 37 °C in buffer to generate the respective aLuc derivatives prior to the addition of protease, ATP, MgCk and luciferase. Then, luminescence can be recorded at 37 °C for 30 minutes and the area under the curve (AUC) can be calculated. AUC can for instance be calculated via: where t is the time in minutes and y the luminescent signal in RLU / s.

[0056] Luciferase is a generic term for the class of oxidative enzymes that produce (bio)luminescence using luciferin as a substrate. Luciferases do not require an external light source, but do require luciferin and O2, and often also ATP. Mg2+is known to increase luminescent yield of some luciferases. Luciferases and their assays are known in the art, as are suitable conditions for their activity. 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), Nano Luciferase (NLuc), Renilla Luciferase (Renilla), Metridia Luciferase (MetLuc), Lucia Luciferase (Lucia) Gaussia Luciferase (GLuc), or Green Renilla Luciferase (GrRenilla), most preferably it has the activity of NLuc. Many luciferases can be commercially obtained from manufacturers such as Promega, Sigma, and the like.

[0057] General definitions

[0058] In preferred embodiments, compounds and compositions according to the invention are for use in methods according to the invention, or are for use according to the invention. Each embodiment as identified herein may be combined together unless otherwise indicated.

[0059] Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components. It is understood that charge is often associated with equilibrium. A moiety that is said to carry or bear a charge is a moiety that will be found in a state where it bears or carries such a charge more often than that it does not bear or carry such a charge. As such, an atom that is indicated in this disclosure to be charged could be non-charged under specific conditions, and a neutral moiety could be charged under specific conditions, as is understood by a person skilled in the art.

[0060] In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of 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 this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

[0061] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. “Hemostasis” and “Haemostasis” can be used interchangeably herein. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value more or less 1 % of the value. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention.

[0062] When a structural formula or chemical name is understood by the skilled person to have chiral centers, yet no chirality is indicated, for each chiral center individual reference is made to all three of either the racemic mixture (having any enantiomeric excess), the pure R enantiomer, and the pure S enantiomer. Whenever a fragment of a molecule, often referred to as a moiety, is represented, a dotted or wavy line indicates which bond links it to the entirety of the molecule; alternately, an asterisk (*) indicates where the represented moiety is linked to the rest of the molecule. This asterisk does not imply an atom, and neither does a bond that is crossed by a dotted or wavy line convey information about which atom is at the non-moiety side of the bond. All this is known in the art, and is routine practice.

[0063] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0064] Description of Drawings

[0065] Fig. 1A - Conventional solution phase method for the synthesis of aminoluciferin peptides via C- terminal activation of a protected peptide and the subsequent sidechain deprotection and cysteine condensation in solution.

[0066] Fig. 1 B - Known method using SPPS (Kovacs et al., Int. J. Pept. Res. Ther. 2019, 25, 1209-1215). Loading of the aminoluciferin on a solid support furnished the dehydrogenated product (thiazole, indicated by curved block arrow), which is not suitably active in assays. Fig. 1 C - The invention provides the sidechain anchoring of the first amino acid on a resin, and the subsequent regular SPPS chemistry prior to the condensation reaction with cysteine. This yields aminoluciferin caged peptides that have good activity in assays while avoiding formation of dehydrogenated product.

[0067] Fig. 2 - Assay strategy using precursors of aLuc - i. pA-X-R / pA-X-K substrate (1 .0 equiv.) D-cysteine (1 .5 equiv), buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.4), 37 °C, 30 min. ii. Protease (Thrombin I FXa final cone. 10 nM), ATP (1.5 equiv), MgCh (10 equiv), Quantilum® Recombinant firefly Luciferase (final cone. 10 pM, Promega).

[0068] Fig. 3 - Total luminescence of substrate candidates (pA-X-R / pA-X-K) using thrombin and Factor Xa. Library screening of peptide library with the substrates (677 pM) methoxyacetamide-pA-X-R- 6ABTC and methoxyacetamide-pA-X-K-6ABTC after the in situ condensation with cysteine. The proteolytic activity of the substrate towards either FXa or Thrombin was determined and its ratio was plotted (ratio 1 :1 : y=x) compared to the total luminescence for either thrombin or Factor Xa.

[0069] Examples

[0070] Example 1 - material and methods

[0071] 1. 1 General

[0072] NMR spectra were recorded on a Bruker Avance III 400 MHz or a Bruker 500 MHz spectrometer and the compounds were assigned using1H NMR,13C NMR, COSY, HSQCED and HMBC spectra. Chemical shifts were reported in parts per million (ppm.) relative to reference (CDCh:1H: 7.26 ppm. and13C 77.16 ppm.; CD3OD:1H: 3.31 ppm. and13C 49.00 ppm.; D2O:1H: 4.79 ppm.; (CD3)2SO:1H: 2.50 ppm. and13C 39.52 ppm.). NMR data are presented in the following way: chemical shift, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dtd = doublet of triplet of doublets h = heptet, m = multiplet and / or multiple resonances) and coupling constants J in Hz. Peptides were synthesized using Fmoc solid-phase peptide (SPPS) chemistry and on resin coupling and deprotection steps were monitored using Kaiser tests. Mass spectra were recorded on a JEOL AccuTOF CS JMS- T100CS (ESI) mass spectrometer. Automatic flash column chromatography was executed on a Biotage Isolera Spektra One using SNAP or Silicycle cartridges (Biotage, 30-100 pm, 60A) 4 - 50 g. Preparative HPLC was performed on a Phenomenex® Gemini-NX 3u C18 110A reversed-phase column (150 x 21.2 mm) using gradient elution with a constant flow of 10 mL / min at 30 °C. MiliQ (0.1 % TFA) and CH3CN (0.1 % TFA) were used as the solvents. The pure fractions containing product were combined and lyophilized overnight to yield the target compounds. Reactions under protective atmosphere were performed under positive AL / N2 flow using flame-dried glassware. Adenosine 5’-triphosphate (ATP), bovine serum albumin (BSA), 4-(2-hydroxyethyl)-1 -piperazine- ethane-sulfonic acid (HEPES), magnesium chloride hexahydrate (MgCk), sodium chloride (NaCI), 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). Half-area white Greiner Bio-One 96 well plates (# 675075) were obtained from VWR (Amsterdam, the Netherlands).

[0073] 1.2 Building block synthesis

[0074] 1.2.1 synthesis of 6-aminobenzo[dlthiazole-2-carbonitrile (6-ABTC)

[0075] Scheme S1.2.1 - Synthesis of 6-ABTC from 2-chlorobenzo[d]thiazole

[0076] 2-chloro-6-nitrobenzo[cf]thiazole (8): 2-Chlorobenzo[cf]thiazole (15.00 g, 88.43 mmol) was added portion wise to concentrated H2SO4 (90 mL) on an ice bath. KNO3 (9.834 g, 97.27 mmol) was added portion wise and the reaction was stirred at 0 °C for 30 min. The reaction mixture was allowed to warm up to rt and stirred for 18 h. at this temperature. The mixture was poured on ice water (300 mL) and the formed precipitate was collected by filtration. The crude product was rinsed with ice cold water and aqueous NaHCCh until acid free. The product was dried under reduced pressure overnight and recrystallized from EtOH (650 mL) to afford 8 (16.81 g, 89%) as an off-white solid. TLC (EtOAc / n- heptane, 1 :4 v / v): Rf= 0.60.1H NMR (500 MHz, CDCh) 6 8.75 (d, J = 2.3 Hz, 1 H), 8.38 (dd, J = 9.0, 2.3 Hz, 1 H), 8.07 (d, J = 9.0 Hz, 1 H).13C NMR (126 MHz, CDCh) 6 158.9, 154.9, 136.6, 123.4, 122.3, 1 17.8.

[0077] 6-nitrobenzo[d]thiazole-2 -carbonitrile (9): 2-Chloro-6- nitrobenzo[c(]-thiazole (8, 10.78 g, 50.23 mmol) was dissolved in ACN (1000 mL) and triethylenediamine (845.2 mg, 7.53 mmol) was added.

[0078] NaCN (2.78 g, 56.83 mmol) was dissolved in water (100 mL) and added dropwise to the stirred reaction mixture. After 24 h. the reaction mixture was quenched with aqueous iron(iii) chloride hexahydrate (0.3M, 50 mL) and diluted with water (350 mL). The reaction mixture was extracted with EtOAc (3 x 400 mL) and the combined organic layers were washed with brine (100 mL), dried with MgSC and concentrated in vacuo. The crude product was loaded on a silica plug and flushed with CHCh (2000 mL), concentrated and dried under high vacuum to afford 9 (8.30 g, 81 %) as a yellow solid. TLC (CHCh): Rf = 0.33.1H NMR (500 MHz, CDCh) 6 8.95 (d, J = 2.2 Hz, 1 H), 8.52 (dd, J = 9.1 , 2.2 Hz, 1 H), 8.38 (d, J = 9.1 Hz, 1 H).13C NMR (126 MHz, CDCh) 6 155.4, 147.4, 141 .9, 135.7, 126.2, 123.2, 118.6, 112.1. 6-aminobenzo[<y]thiazole-2 -carbonitrile (1): 6-Nitrobenzo[c(]- 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 was stirred for 24 h. The reaction was diluted with water (1400 mL) and filtered over Celite. The aqueous solution was extracted with EtOAc (3 x 700 mL) and washed with brine (400 mL). The crude product was loaded on a silica plug and flushed with CHCh (2000 mL), concentrated and dried under high vacuum to afford 1 (3.45 g, 53%) as a yellow solid. TLC (DCM, 1 :1 v / v): Rf = 0.26.1H NMR (500 MHz, CDCI3) 6 7.95 (d, J = 8.9 Hz, 1 H), 7.08 (d, J = 2.2 Hz, 1 H), 6.95 (dd, J = 8.9, 2.3 Hz, 1 H).13C NMR (126 MHz, CDCI3) 6 147.6, 145.5, 138.0, 131.0, 125.9, 117.6, 103.8, 77.2. HRMS (m / z): [M + H]+calcd. for CsHsNsS: 176.0282, found 176.0294.

[0079] 1 .71 mmol) was added in dry pyridine (3 mL) and the reaction mixture was stirred for 3 h. at 40 °C. The reaction mixture was allowed to cool down to rt, diluted with EtOAc (150 mL) and washed with 10% aqueous citric acid (150 mL). The aqueous phase was re-extracted with EtOAc (75 mL). The combined organic layers were washed with 10% aqueous citric acid (100 mL), saturated aqueous NH4CI (2 x 100 mL) and brine (100 mL). The combined organic layers were dried with MgSO4, concentrated in vacuo and purified through silica gel column chromatography (20 80 % EtOAc / n- heptane) to afford Fmoc-Orn(Boc)-6ABTC (4, 1.03 g, 98%) as a yellow solid. TLC (EtOAc / n- heptane, 1 :1 v / v): Rf= 0.69.1H NMR (500 MHz, CDCI3) 6 9.26 (s, 1 H), 8.66 (d, J = 2.1 Hz, 1 H), 8.09 (d, J = 8.9 Hz, 1 H), 7.76 (d, J = 7.6 Hz, 2H), 7.60 (t, J = 7.2 Hz, 2H), 7.57 - 7.53 (m, 1 H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 5.5 Hz, 2H), 5.76 - 5.71 (m, 1 H), 4.87 - 4.83 (m, 1 H), 4.68 - 4.64 (m, 1 H), 4.42 (d, J = 7.1 Hz, 2H), 4.22 (t, J = 7.0 Hz, 1 H), 3.63 - 3.50 (m, 1 H), 3.16 - 3.06 (m, 1 H), 2.04 (s, 2H), 1.67 (s, 2H), 1.45 (s, 9H).13C NMR (126 MHz, CDCI3) 6 171.3, 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. as a yellow solid.1H NMR (400 MHz, CD3OD) 6 8.64 (d, J = 2.1 Hz, 1 H), 8.12 (d, J = 9.0 Hz, 1 H),

[0080] 7.78 (d, J = 7.6 Hz, 2H), 7.70 (dd, J = 9.0, 2.1 Hz, 1 H), 7.66 (t, J = 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.33 - 7.24 (m, 2H), 4.42 (qd, J = 10.6, 6.7 Hz, 2H), 4.35 - 4.31 (m, 1 H), 4.21 (t, J = 6.7 Hz, 1 H), 2.97 (t, J = 7.3 Hz, 2H), 1 .88 - 1 .73 (m, 3H).13C NMR (101 MHz, CD3OD) 6 177.3, 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,

[0081] 112.9, 67.9, 56.5, 39.7, 30.1 , 24.2, 22.1. HRMS (m / z): [M + H]+calcd. for C28H25N5O3S: 512.1756, found 512.1752. . reaction mixture was stirred for 3 h. at 40 °C. The reaction mixture was allowed to cool down to rt, diluted with EtOAc (150 mL) and washed with 10% aqueous citric acid (150 mL). The aqueous phase was re-extracted with EtOAc (75 mL). The combined organic layers were washed with 10% aqueous citric acid (100 mL), saturated aqueous NH4CI (2 x 100 mL) and brine (100 mL). The combined organic layers were dried with MgSO4, concentrated in vacuo and purified through silica gel column chromatography (0 70 % EtOAc / n -heptane) to afford Fmoc-Lys(Boc)-6ABTC (5, 1 .38 g, 77%) as a yellow solid. TLC (EtOAc / n-heptane, 9:1 v / v): Rf= 0.83.1H NMR (500 MHz, CD3OD) 6 8.65 - 8.60 (m, 1 H), 8.08 (dd, J = 9.2, 4.3 Hz, 1 H), 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, 1 H), 4.22 - 4.18 (m, 1 H), 3.07 - 3.00 (m, 2H), 1 .89 - 1 .82 (m, 1 H), 1 .80 - 1 .71 (m, 1 H), 1 .55 - 1 .47 (m, 4H), 1 .39 (s, 9H).13C NMR (126 MHz, CDCI3) 6 173.7, 158.6, 149.8, 145.2, 145.1 , 142.5, 140.5, 138.0, 136.7, 128.7, 128.1 , 126.2, 125.9, 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.

[0082] Hz, 1 H), 8.07 (d, J = 9.0 Hz, 1 H), 8.28 (s, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.70 - 7.60 (m, 3H), 7.35 (t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 4.40 (dd, J = 6.7, 3.4 Hz, 2H), 4.28 (dd, J = 8.9, 5.3 Hz, 1 H), 4.19 (t, J = 6.7 Hz, 1 H), 2.93 - 2.88 (m, 2H), 1.94 - 1.83 (m, 1 H), 1 .83 - 1.62 (m, 3H), 1.58 - 1.40 (m, 2H).13C NMR (101 MHz, CD3OD) 6 172.0, 157.2, 148.5, 143.7, 141.2, 139.1 , 136.6, 135.4, 127.4, 126.7, 124.8, 124.6, 120.9, 1 19.6, 112.7, 11 1.6, 66.5, 55.6, 47.0, 39.1 , 31 .3, 26.8, 22.5.

[0083] HRMS (m / z): [M + H]+calcd. C29H27N5O3S: 526.1912, found 526.1909.

[0084] 1.3 Solid Phase Peptide Synthesis

[0085] 1.3. 1 General Procedure A

[0086] Typical scale 100mg of resin: (3-Formylindolyl)acetamidomethyl resin (100 mg, 0.073 mmol according to loading) was suspended in dry THF:TMOF (1 :1 , 2mL) in a flame-dried flask. Fmoc- Orn-6ABTC (6, 82 mg, 0.146 mmol) was added and the suspension was stirred for 4 h. at rt. NaBHsCN (9 mg, 0.146 mmol) in THF (2 mL) and AcOH (15 pL, 0.256 mmol) were added and the suspension was stirred for 2 h. The resin was washed with THF (3x), DCM (3x), MeOH (3x) and Et2O (3x) and dried under high vacuum for 1 h. The resin was swollen in dry DCM for 20 min. / V, / V'- di-Boc-thiourea (40 mg, 0.146 mmol), DIPEA (25 pL, 0.146 mmol) and diisopropylmethanediimine (23 pL, 0.146 mmol) were added and the reaction was agitated overnight. The resin was washed with DCM (3x) and DMF (3x) and unreacted sites were capped by agitating with AC2O (138 pL, 1 .46 mmol) and pyridine (118 pL, 1.46 mmol) in DMF for 10 min. The resin was washed with DMF (3x). Subsequently, the resin was used for regular SPPS using standard Fmoc chemistry. Couplings of the Fmoc amino acid were done using diisopropylmethanediimine (3.30 equiv) and Hydroxybenzotriazole (3.6 equiv) in DMF After each coupling the resin was washed with DMF (3x) and the Fmoc group was removed using 3% DBU in DMF (v / v). Before the final cleavage of the peptide the resin was washed with DMF (3x), DCM (3x), MeOH (3x) and Et2O (3x). The peptides were cleaved from the resin using 1 :1 TFA:DCM for 2 h. (addition of 2.5% TIS and EDT for Trt containing sequences), concentrated in vacuo, lyophilized overnight and purified via semipreparative RP-HPLC to obtain the target peptides.

[0087] Typical scale 10 g of resin: (3-Formylindolyl)acetamidomethyl resin (10.00 g, 7.50 mmol according to loading) 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 agitated for 4 h. at rt. on a rotary evaporator. NaBHsCN (848 mg, 13.5 mmol) in THF (5 mL) and AcOH (1.5 mL, 26.2 mmol) were added and the suspension was agitated for 2 h. The resin was washed with DMF (3x) and DCM (3x) before the addition of 10 (2.82 g, 6.75 mmol) and DIPEA (3.26 mL, 18.7 mmol) in DMF (100 mL) and agitated for 18 h. Hereafter, the resin was washed with DMF (3x), DCM (3x) and Et2O (3x). The resin was dried under high vacuum and used for regular SPPS. Multiple batches were prepared with a final loading between 0.20 - 0.22 mmol / g (Fmoc quantification at 301 nm).

[0088] 1.3. 1.1 peptide conjugates with arginine as Pi . , , . , . , , .

[0089] (t, J = 6.8 Hz, 2H), 2.12 - 1 .96 (m, 3H), 1 .90 - 1 .83 (m, 1 H), 1.81 - 1 .68 (m, 3H), 1 .05 - 0.95 (m, 6H).13C NMR (126 MHz, CD3OD) 6 174.4, 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

[0090] (m / z): [M + H]+calcd. for C25H35N9O5S: 574.2560, found 574.2557.

[0091] 3.24 (td, J = 7.0, 3.7 Hz, 2H), 2.52 (t, J = 6.6 Hz, 2H), 1 .89 - 1 .80 (m, 2H), 1 .76 - 1 .66 (m, 2H).13C

[0092] NMR (126 MHz, CD3OD) 6 174.9, 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): [M + H]+calcd. for C22H29N9O5S: 532.2090, found 532.2113.

[0093] J = 11 .0, 6.7 Hz, 2H), 3.37 (s, 3H), 3.27 - 3.22 (m, 2H), 2.93 (dd, J = 13.9, 5.6 Hz, 1 H), 2.86 (dd, J

[0094] = 13.9, 7.0 Hz, 1 H), 2.53 (t, J = 6.7 Hz, 2H), 2.07 - 2.01 (m, 1 H), 1 .86 (dp, J = 14.0, 4.7 Hz, 1 H), 1.81 - 1.68 (m, 2H).13C NMR (126 MHz, CD3OD) 6 174.5, 172.8, 172.6, 172.2, 158.6, 150.0, 140.3, 138.0, 136.9, 126.0, 122.3, 114.0, 1 12.9, 72.6, 59.5, 57.8, 55.0, 41 .9, 36.6, 36.4, 30.0, 26.4, 26.4.

[0095] HRMS (m / z): [M + H]+calcd. for C23H31 N9O5S2: 578.1967, found 578.1975. 2H), 2.37 (t, J = 7.4 Hz, 2H), 2.12 - 1 .94 (m, 3H) , 1 .90 - 1 .81 (m, 1 H), 1 .79 - 1 .67 (m, 2H).13C

[0096] NMR (126 MHz, CD3OD) 6 177.7, 174.3, 174.3, 172.6, 172.4, 158.7, 150.0, 140.4, 138.0, 136.9, 126.0, 122.3, 114.0, 1 12.9, 72.6, 59., 54.9, 54.8, 42.0, 36.6, 36.4, 32.4, 30.1 , 28.4, 26.3. HRMS

[0097] (m / z): [M + H]+calcd. for C25H34N10O6S2: 603.2461 , found 603.2458.

[0098] = 15.7, 8.1 Hz, 1 H), 2.74 (dd, J = 15.7, 5.6 Hz, 1 H), 2.49 (t, J = 6.7 Hz, 2H), 1.87 - 1.79 (m, 2H),

[0099] 1 .78 - 1 .68 (m, 2H).13C NMR (126 MHz, CD3OD) 6 174.6, 174.0, 173.9, 172.5, 172.5, 158.6, 150.0, 140.5, 137.9, 136.9, 125.8, 122.7, 114.0, 113.1 , 72.6, 59.5, 54.8, 52.0, 41.9, 37.4, 36.5, 36.4, 29.6, 26.3. HRMS (m / z): [M + H]+calcd. for C24H32N10O6S: 589.2305, found 589.2324.

[0100] (m, 2H), 3.37 (s, 3H), 3.25 (q, J = 6.7 Hz, 2H), 2.58 - 2.54 (m, 2H), 2.10 - 2.02 (m, 1 H), 1 .89 - 1 .82

[0101] (m, 1 H), 1 .79 - 1 .70 (m, 2H), 1 .24 (d, J = 6.4 Hz, 3H).13C NMR (126 MHz, CD3OD) 6 174.5, 172.9,

[0102] 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,

[0103] 54.8, 41.9, 36.5, 36.5, 30.0, 26.3, 20.0. HRMS (m / z): [M + H]+calcd. for C24H33N9O6S: 576.2352, found 576.2357. Hz, 1 H), 3.86 - 3.85 (m, 2H), 3.81 (dd, J= 10.7,

[0104] 6.3 Hz, 1 H), 3.56 - 3.51 (m, 2H), 3.37 (s, 3H), 3.25 (td, J = 6.9, 3.8 Hz, 2H), 2.53 (dt, J = 6.6, 3.2 Hz, 2H), 2.16 - 2.09 (m, 1 H), 1 .88 - 1 .82 (m, 1 H), 1 .80 - 1 .70 (m, 2H).13C NMR (126 MHz, CD3OD) 6 174.5, 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,

[0105] 62.8, 59.5, 57.4, 54.8, 41.9, 36.6, 36.4, 29.7, 26.4. HRMS (m / z): [M + H]+calcd. for C23H31N9O6S: 562.2196, found 562.2206.

[0106] Hz, 2H), 2.48 - 2.41 (m, 2H), 2.14 - 2.06 (m, 1 H), 2.06 - 2.00 (m, 2H), 1.85 (ddt, J = 13.9, 9.2, 4.7 Hz, 1 H), 1.78 - 1.67 (m, 2H).13C NMR (126 MHz, CD3OD) 6 176.4, 174.4, 174.2, 172.6, 172.3,

[0107] 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): [M + H]+calcd. for C25H33N9O7S: 604.2301 , found 604.2310.

[0108] 7.5 Hz, 1 H), 2.82 - 2.78 (m, 1 H), 2.49 (t, J = 6.8 Hz, 2H), 2.15 - 2.09 (m, 1 H), 1 .87 - 1 .81 (m, 1 H),

[0109] 1.79 - 1.68 (m, 2H).13C NMR (126 MHz, CD3OD) 6 175.3, 174.3, 174.1 , 173.7, 172.3, 156.4, 150.0,

[0110] 140.3, 137.9, 136.9, 125.9, 122.5, 114.0, 113.1 , 72.6, 59.5, 54.8, 51.8, 41.9, 36.6, 36.4, 36.3, 29.7,

[0111] 26.3. HRMS (m / z): [M + H]+calcd. for C24H31N9O7S: 590.2145, found 590.2162.

[0112] - 3.22 (m, 2H), 2.50 (td, J = 6.7, 2.1 Hz, 2H), 1.91 - 1 .82 (m, 2H), 1 .79 - 1 .67 (m, 2H), 1 .39 (d, J =

[0113] 7.2 Hz, 3H).13C NMR (126 MHz, CD3OD) 6 175.3, 174.3, 172.5, 172.3, 158.6, 150.0, 140.4, 138.8,

[0114] 137.0, 126.0, 122.2, 114.0, 112.8, 72.6, 59.5, 54.7, 53.9, 42.0, 41.5, 36.4, 36.4, 26.4, 25.9, 23.3,

[0115] 21.9. HRMS (m / z): [M + H]+calcd. for C26H37N9O5S: 588.2716, found 588.2706. . (m, ), . (s, ), . (q, . z,

[0116] 2H), 2.55 - 2.51 (m, 2H), 2.08 - 1 .98 (m, 1 H), 1 .92 - 1 .83 (m, 2H), 1 .80 - 1 .68 (m, 2H), 1 .60 (ddd, J = 13.6, 7.5, 3.6 Hz, 1 H), 1 .29 - 1 .23 (m, 1 H), 0.98 (d, J = 6.9 Hz, 3H), 0.94 (t, J = 7.5 Hz, 3H).13C NMR (126 MHz, CD3OD) 6 174.4, 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.

[0117] HRMS (m / z): [M + H]+calcd. for C26H37N9O5S: 588.2716, found 588.2714. Hz, 2H), 3.56 - 3.50 (m, 2H), 3.36 (s, 3H),

[0118] 3.28 - 3.22 (m, 2H), 2.50 (td, J = 6.7, 2.1 Hz, 2H), 1.91 - 1 .82 (m, 2H), 1 .79 - 1 .67 (m, 2H), 1 .39 (d, J = 7.2 Hz, 3H).13C NMR (126 MHz, CD3OD) 6 175.7, 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): [M + H]+calcd. for C23H31N9O5S: 546.2247, found 546.2246.

[0119] (m, 2H), 3.36 (s, 3H), 3.29 - 3.23 (m, 2H), 2.71 - 2.62 (m, 2H), 2.32 - 2.24 (m, 1 H), 2.08 - 2.03 (m,

[0120] 3H), 1.92 - 1.82 (m, 2H), 1.80 - 1.71 (m, 2H).13C NMR (126 MHz, CD3OD) 6 175.0, 173.0, 172.5,

[0121] 172.3, 158.6, 150.0, 140.4, 138.0, 136.9, 126.0, 122.3, 114.0, 112.8, 72.6, 61.8, 59.5, 54.7, 48.4,

[0122] 41.9, 35.8, 35.3, 30.9, 30.0, 26.4, 25.8. HRMS (m / z): [M + H]+calcd. for C25H33N9O5S: 572.2403, found 572.2397. 1 H), 3.78 (s, 2H), 3.50 - 3.42 (m, 2H), 3.32 (s, 3H),

[0123] 3.30 - 3.28 (m, 2H), 3.27 - 3.19 (m, 1 H), 3.11 (dd, J = 14.5, 7.9 Hz, 1 H), 2.46 - 2.33 (m, 2H), 2.06 - 1.98 (m, 1 H), 1.78 - 1.69 (m, 1 H), 1.63 - 1.53 (m, 2H).13C NMR (126 MHz, CD3OD) 6 175.0, 174.0, 172.5, 170.9, 158.9, 150.0, 140.3, 138.1 , 138.0, 137.0, 128.6, 126.1 , 124.5, 122.5, 122.3, 119.8, 119.2, 113.9, 113.0, 112.3, 110.8, 72.5, 59.5, 56.8, 56.4, 38.8, 36.5, 36.2, 30.7, 28.7, 26.3. HRMS (m / z): [M + H]+calcd. for C31H36N10O5S: 661 .2669, found 661 .2650. 3H), 3.27 - 3.22 (m, 3H), 3.18 - 3.10 (m, (t, J = 6.8 Hz, 2H), 1 .90 - 1 .67 (m, 4H).13C

[0124] NMR (126 MHz, CD3OD) 6 174.0, 172.5, 172.5, 172.3, 159.2, 150.0, 141.2, 140.4, 138.1 , 137.0, 126.1 , 122.1 , 122.1 , 118.6, 114.0, 112.8, 72.6, 59.5, 55.2, 53.8, 42.0, 36.6, 36.4, 30.3, 27.9, 26.4.

[0125] HRMS (m / z): [M + H]+calcd. for C26H33N11O5S: 612.2465, found 612.2474. = 6.8 Hz, 2H), 1.90 - 1.81 (m, 3H), 1.77 - 1.66 (m,

[0126] 5H), 1.56-1.44 (m, 2H).13C NMR (126 MHz, CD3OD) 6174.5, 174.2, 172.6, 172.5, 158.6, 150.0,

[0127] 140.4, 138.0, 136.9, 126.1, 122.2, 114.0, 112.8, 72.6, 59.5, 54.9, 54.9, 42.0, 40.4, 36.6, 36.4, 32.1,

[0128] 30.3, 28.1 , 26.4, 23.7. HRMS (m / z): [M + H]+calcd. for C26H38N10O5S: 603.2825, found 603.2830. 3.24 - 3.15 (m, 1H), 3.08 -3.02 (m, 1H), 3.00 -2.94 (m,

[0129] 1 H), 2.86 - 2.78 (m, 1 H), 2.42 - 2.34 (m, 2H), 2.07 - 2.01 (m, 2H), 1.72 - 1.65 (m, 1 H), 1.66 - 1.57 (m, 2H).13C NMR (126 MHz, CD3OD) 6174.5, 173.9, 172.4, 170.8, 158.9, 157.4, 150.0, 140.3,

[0130] 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,

[0131] 36.4, 36.3, 30.8, 26.4. HRMS (m / z): [M + H]+calcd. for C29H35N9O6S: 638.2509, found 638.2514. 2H), 3.35 (s, 3H), 3.10-3.01 (m, 3H), 2.92 (dd, J= 13.7,

[0132] 8.4 Hz, 1 H), 2.37 (td, J = 6.8, 4.8 Hz, 2H), 2.07 - 1.97 (m, 1 H), 1.80 - 1.72 (m, 1 H), 1.60 (d, J = 6.2 Hz, 2H).13C NMR (126 MHz, CD3OD) 6174.3, 173.9, 172.4, 170.8, 158.9, 150.0, 140.3, 138.2, 138.1, 137.0, 130.2, 129.5, 127.8, 126.1, 122.3, 114.0, 113.0, 72.5, 59.5, 57.1, 56.4, 38.8, 38.6,

[0133] 36.4, 36.2, 30.8, 26.4. HRMS (m / z): [M + H]+calcd. for C29H35N9O5S: 622.2560, found 622.2572. . . , , . . , , . ,

[0134] = 6.7 Hz, 2H), 2.08 (s, 3H), 2.05 - 1.95 (m, 3H), 1.90 - 1.82 (m, 2H), 1.78 - 1.69 (m, 2H).13C NMR (126 MHz, CD3OD) 6 174.4, 174.3, 172.6, 172.3, 158.6, 150.0, 140.4, 138.1 , 136.9, 126.0, 122.2, 114.0, 112.8, 72.6, 59.5, 54.9, 54.6, 42.0, 36.5, 36.4, 32.2, 31 .1 , 30.1 , 26.4, 15.2. HRMS (m / z): [M + H]+calcd. for C25H35N9O5S2: 606.2280, found 606.2281 . 1.92 - 1.84 (m, 2H), 1.81 - 1.66 (m, 6H). C NMR

[0135] (126 MHz, CD3OD) 6 174.2, 174.2, 172.6, 172.5, 158.6, 158.5, 150.0, 140.4, 137.8, 136.9, 126.1 , 122.2, 114.0, 112.8, 72.6, 59.5, 55.0, 54.6, 42.0, 42.0, 36.6, 36.4, 30.3, 29.9, 26.4, 26.2. HRMS (m / z): [M + H]+calcd. for C26H38N12O5S: 631 .2887, found 631 .2866.

[0136] 1.3.2 General Procedure B

[0137] Typical scale 100mg of resin: (3-Formylindolyl)acetamidomethyl resin (100 mg, 0.073 mmol according to loading) was suspended in dry THF:TMOF (1 :1 , 2mL) in a flame-dried flask. Fmoc- Lys-6ABTC (7, 84 mg, 0.146 mmol) was added and the suspension was stirred for 4 h. at rt. NaBHsCN (9 mg, 0.146 mmol) in THF (2 mL) and AcOH (15 pL, 0.256 mmol) were added and the suspension was stirred for 2 h. The resin was washed with THF (3x), DCM (3x), MeOH (3x) and Et2O (3x) and dried under high vacuum for 1 h. The resin was swollen in DMF for 20 min. Di-tert- butyl dicarbonate (48 mg, 0.219 mmol) and DIPEA (38 pL, 0.219 mmol) were added and the reaction was agitated overnight. The resin was washed with DCM (3x) and DMF (3x) and unreacted sites were capped by agitating with AC2O (138 pL, 1 .46 mmol) and pyridine (118 pL, 1 .46 mmol) in DMF for 10 min. The resin was washed with DMF (3x). Subsequently, the resin was used for regular SPPS using standard Fmoc chemistry. Couplings of the Fmoc amino acid were done using diisopropylmethanediimine (3.30 equiv) and Hydroxybenzotriazole (3.6 equiv) in DMF After each coupling the resin was washed with DMF (3x) and the Fmoc group was removed using 3% DBU in DMF (v / v). Before the final cleavage of the peptide the resin was washed with DMF (3x), DCM (3x), MeOH (3x) and Et2O (3x). The peptides were cleaved from the resin using 1 :1 TFA:DCM for 2 h. (addition of 2.5% TIS and EDT for Trt containing sequences), concentrated in vacuo, lyophilized overnight and purified via semi-preparative RP-HPLC to obtain the target peptides.

[0138] Typical scale 10 g of resin: (3-Formylindolyl)acetamidomethyl resin (10.00 g, 7.50 mmol according to loading) 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 agitated for 4 h. at rt. on a rotary evaporator. NaCNBHs (848 mg, 13.5 mmol) in THF (5 mL) and AcOH (1.5 mL, 26.2 mmol) were added and the suspension was agitated for 2 h. The resin was washed with DMF (3x) and DCM (3x) before the addition of B0C2O (4.91 g, 22.5 mmol) and DIPEA (3.92 mL, 22.5 mmol) in DMF (100 mL) and agitated for 18 h. Hereafter, the resin was washed with DMF (3x), DCM (3x) and Et2O (3x). The resin was dried under high vacuum and used for regular SPPS. Multiple batches were prepared with a final loading between 0.16 - 0.25 mmol / g (Fmoc quantification at 301 nm).

[0139] = 6.6 Hz, 2H), 3.40 (s, 3H), 3.04 (t, J = 7.6 Hz, 2H), 2.66 - 2.52 (m, 2H), 2.13 - 2.07 (m, 1 H), 2.04

[0140] - 1 .97 (m, 1 H), 1 .95 - 1 .89 (m, 1 H), 1 .80 - 1 .73 (m, 2H), 1 .64 - 1 .50 (m, 2H), 1 .00 - 0.97 (m, 6H).13C NMR (126 MHz, D2O) 6 174.2, 173.8, 172.4, 172.4, 148.2, 137.3, 136.8, 136.4, 124.5, 122.2, 113.5, 1 12.9, 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): [M + H]+calcd. for C25H35N7O5S: 546.2498, found 546.2516.

[0141] 3H), 3.03 (t, J = 11 Hz, 2H), 2.59 (t, J = 6.6 Hz, 2H), 2.08 - 1 .96 (m, 1 H), 1 .93 - 1 .87 (m, 1 H), 1 .79 - 1.70 (m, 2H), 1.61 - 1.48 (m, 2H).13C NMR (126 MHz, D2O) 6 174.7, 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): [M + H]+calcd. for C22H29N7O5S: 504.2029, found 504.2035.

[0142] Hz, 4H), 2.66 - 2.58 (m, 2H), 1 .80 - 1 .70 (m, 4H), 1 .56 - 1 .46 (m, 2H).13C NMR (126 MHz, D2O) 6 173.5, 172.5, 170.8, 170.6, 147.8, 136.4, 136.2, 135.7, 124.0, 120.4, 115.1 , 11 1.7, 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): [M + H]+calcd. for C23H31N7O5S2: 550.1906, found 550.1902. (t, J = 7.6 Hz, 2H), 2.61 - 2.54 (m, 2H), 2.41 (t, J = 7.6 Hz, 2H), 2.17 - 2.09 (m, 1 H), 2.06 - 1 .98 (m, 2H), 1.96 - 1.86 (m, 1 H), 1.76 (p, J = 7.8 Hz, 2H), 1.65 - 1 .49 (m, 2H).13C NMR (126 MHz, D2O) 6 177.7, 174.1 , 173.6, 172.4, 148.2, 137.3, 136.7, 136.4, 124.5, 122.2, 113.4, 112.9, 70.8, 58.9, 54.4, 53.2, 39.1 , 35.3, 34.9, 31.0, 30.3, 26.8, 26.2, 22.1. HRMS (m / z): [M + H]+calcd. for C25H34N8O6S2: 575.2400, found 575.2387.

[0143] 7.5 Hz, 2H), 2.91 - 2.83 (m, 1 H), 2.82 - 2.74 (m, 1 H),

[0144] 2.61 - 2.54 (m, 2H), 1 .95 - 1 .85 (m, 2H), 1 .79 - 1 .72 (m, 2H), 1 .62 - 1 .47 (m, 2H).13C NMR (126

[0145] MHz, D2O) 6 174.2, 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): [M + H]+calcd. for C24H32N8O6S: 561 .2243, found 561 .2246.

[0146] 1 H), 3.96 (s, 2H), 3.55 (t, J = 6.6 Hz, 2H), 3.39 (s, 3H), 3.04 (t, J = 7.5 Hz, 2H), 2.67 - 2.59 (m, 2H),

[0147] 2.06 - 1 .98 (m, 1 H), 1 .96 - 1 .87 (m, 1 H), 1 .79 - 1 .72 (m, 2H), 1 .63 - 1 .47 (m, 2H), 1 .25 (d, J = 1 .1 Hz, 3H).13C NMR (126 MHz, D2O) 6 174.4, 172.4, 172.3, 172.1 , 148.3, 137.2, 136.8, 136.4, 124.5,

[0148] 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

[0149] (m / z): [M + H]+calcd. for C24H33N7O6S: 548.2291 , found 548.2290. Hz, 2H), 3.55 (t, J = 6.9 Hz, 2H), 3.39 (s,

[0150] 3H), 3.04 (t, J = 7.3 Hz, 2H), 2.61 (t, J = 6.6 Hz, 2H), 2.06 - 2.02 (m, 1 H), 1 .96 - 1 .87 (m, 1 H), 1 .80 - 1.72 (m, 2H), 1.63 - 1.49 (m, 2H).13C NMR (126 MHz, D2O) 6 174.4, 172.4, 172.4, 172.2, 148.4,

[0151] 137.2, 136.9, 136.4, 124.6, 122.4, 113.7, 112.9, 70.8, 60.9, 58.9, 55.7, 54.3, 39.1 , 35.3, 34.9, 30.2,

[0152] 26.2, 22.1 . HRMS (m / z): [M + H]+calcd. for C23H31N7O6S: 534.2134, found 534.2136. 3.04 (t, J = 7.5 Hz, 2H), 2.59 - 2.55 (m, 2H), 2.52 (t,

[0153] J = 7.5 Hz, 2H), 2.18 - 2.13 (m, 2H), 2.04 - 2.02 (m, 1 H), 1 .94 - 1 .89 (m, 1 H), 1 .78 - 1 .72 (m, 2H), 1.62 - 1.51 (m, 2H).13C NMR (126 MHz, D2O) 6 176.8, 174.1 , 173.6, 172.4, 172.4, 148.2, 137.3, 136.7, 136.4, 124.5, 122.2, 113.4, 112.9, 70.8, 58.9, 54.3, 53.1 , 39.1 , 35.3, 34.9, 30.3, 29.8, 26.2,

[0154] 26.1 , 22.1 . HRMS (m / z): [M + H]+calcd. for C25H33N7O7S: 576.2240, found 576.2230. 2.85 (m, 1 H), 2.82 (d, J = 7.4 Hz, 1 H), 2.49 (t, J =

[0155] 6.6 Hz, 2H), 1 .98 - 1 .91 (m, 1 H), 1 .87 - 1 .78 (m, 2H), 1 .69 - 1 .60 (m, 1 H), 1 .52 - 1 .38 (m, 2H).13C

[0156] NMR (126 MHz, D2O) 6 174.0, 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): [M + H]+calcd. for C24H31N7O7S: 562.2083, found 562.2086. . z, ), . (s, ), . ( , .

[0157] Hz, 2H), 2.63 - 2.50 (m, 2H), 2.07 - 1 .96 (m, 1 H), 1 .95 - 1 .86 (m, 1 H), 1 .78 - 1 .72 (m, 2H), 1 .69 - 1 .60 (m, 3H), 1 .57 - 1 .48 (m, 2H), 0.96 (d, J = 5.8 Hz, 3H), 0.91 (d, J = 5.9 Hz, 3H).13C NMR (126 MHz, D2O) 6 175.0, 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): [M + H]+calcd. for C26H37N7O5S: 560.2655, found 560.2651 . 2 H), 3.40 (s, 3H), 3.04 (t, J = 7.7 Hz, 2H), 2.65 - 2.51 (m, 2H), 1 .95 - 1 .85 (m, 2H), 1 .76 (ddd, J = 10.8, 8.9, 6.8 Hz, 2H), 1 .65 - 1 .46 (m, 4H), 1 .27

[0158] - 1 .18 (m, 1 H), 0.95 (d, J = 6.8 Hz, 3H), 0.88 (t, J = 6.6 Hz, 3H).13C NMR (126 MHz, D2O) 6 174.1 , 173.9, 172.4, 172.3, 148.3, 137.3, 136.8, 136.4, 124.6, 122.3, 113.5, 112.9, 70.8, 58.9, 58.5, 54.3, 39.1 , 35.9, 35.4, 34.9, 30.2, 26.2, 24.6, 22.1 , 14.7, 10.0. HRMS (m / z): [M + H]+calcd. for C26H37N7O5S: 560.2655, found 560.2644. , , , , , ,

[0159] 2H), 3.38 (s, 3H), 3.04 (t, J = 7.6 Hz, 2H), 2.55 (td, J = 6.6, 2.9 Hz, 2H), 2.06 - 1 .97 (m, 1 H), 1 .95 - 1.86 (m, 1 H), 1.80 - 1.72 (m, 2H), 1.63 - 1.49 (m, 2H), 1.42 (d, J = 7.2 Hz, 3H).13C NMR (126 MHz, D2O) 6 175.4, 174.0, 172.5, 172.4, 148.2, 137.3, 136.7, 136.4, 124.5, 122.2, 113.4, 112.9, 70.7, 58.9, 54.2, 49.9, 39.2, 35.3, 34.8, 30.3, 26.6, 22.1 , 15.5. HRMS (m / z): [M + H]+calcd. for C23H31N7O5S: 518.2185, found 518.2190. , . . , , . , , . ,

[0160] 7.5 Hz, 2H), 2.68 - 2.61 (m, 2H), 2.27 (dd, J = 12.6, 7.2 Hz, 1 H), 2.00 - 1.79 (m, 5H), 1.73 - 1.63 (m, 2H), 1.57 - 1.37 (m, 2H).13C NMR (126 MHz, D2O) 6 174.7, 172.6, 172.6, 172.4, 148.4, 137.3, 136.9, 136.4, 124.6, 122.3, 113.6, 112.9, 70.7, 60.3, 58.9, 54.2, 48.0, 39.2, 34.6, 33.4, 30.2, 29.7,

[0161] 26.2, 24.4, 22.2. HRMS (m / z): [M + H]+calcd. for C25H33N7O5S: 544.2342, found 544.2341.

[0162] Hz, 2H), 3.36 (d, J = 0.9 Hz, 3H), 3.20 (d, J = 7.7 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 2.54 (q, J = 6.5 Hz, 2H), 1 .83 - 1 .73 (m, 1 H), 1 .70 - 1 .58 (m, 3H), 1 .40 - 1 .25 (m, 2H).13C NMR (126 MHz, D2O) 6 173.7, 173.4, 172.3, 171.2, 147.9, 137.3, 136.3, 136.2, 135.8, 126.7, 124.2, 124.1 , 121.9, 121.6, 119.1 , 117.9, 112.9, 112.9, 111.5, 108.5, 70.7, 58.9, 55.1 , 53.8, 39.1 , 35.3, 34.9, 30.8, 26.9, 26.2,

[0163] 21.8. HRMS (m / z): [M + H]+calcd. for C31H36N8O5S: 633.2607, found 633.2597. 3H), 3.27 (dd, J = 15.3, 7.0 Hz, 1H), 3.17 (dd, J =

[0164] 15.4, 8.0 Hz, 1 H), 3.02 (t, J = 7.6 Hz, 2H), 2.53 (t, J = 6.7 Hz, 2H), 2.01 - 1.92 (m, 1 H), 1.91 - 1.83 (m, 1 H), 1.78 - 1.70 (m, 2H), 1.60 - 1.43 (m, 2H).13C NMR (126 MHz, D2O) 6173.8, 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,

[0165] 52.5, 39.1, 35.2, 34.9, 30.4, 26.3, 26.3, 22.1. HRMS (m / z): [M + H]+calcd. for C26H33N9O5S: 584.2403, found 584.2397.

[0166] 2 H), 2.98 (t, J = 7.7 Hz, 2H), 2.57 (q, J = 6.3 Hz, 2H),

[0167] 1.95- 1.82 (m, 2H), 1.82- 1.65 (m, 5H), 1.65- 1.42 (m, 5H).13C NMR (126 MHz, D2O) 6174.1, 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): [M + H]+calcd. for C26H38N8O5S: 575.2764, found 575.2751.

[0168] 3.50 (t, J = 6.6 Hz, 2H), 3.40 (s, 3H), 3.04 - 2.97 (m, 3H), 2.91 (dd, J = 13.6, 9.1 Hz, 1 H), 2.53 (q, J = 7.0 Hz, 2H), 1.92-1.82 (m, 1H), 1.80-1.66 (m, 3H), 1.50-1.35 (m, 2H).13C NMR (126 MHz, D2O) 6173.7, 173.1, 172.4, 171.4, 154.4, 148.3, 137.2, 136.8, 136.5, 130.3, 127.3, 124.5, 122.2, 115.2, 113.6, 113.0, 70.7, 58.9, 55.6, 53.8, 46.6, 39.1, 36.1, 35.3, 34.8, 30.4, 26.2, 21.9. HRMS (m / z): [M + H]+calcd. for C29H35N7O6S: 610.2447, found 610.2426. , , , , , , ,

[0169] 6.6 Hz, 2H), 3.39 (s, 3H), 3.06 (d, J = 8.0 Hz, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.52 (td, J = 6.5, 3.3 Hz, 2H), 1 .94 - 1 .85 (m, 1 H), 1 .82 - 1 .76 (m, 1 H), 1 .71 (p, J = 8.3 Hz, 2H), 1 .51 - 1 .36 (m, 2H).13C NMR (126 MHz, D2O) 6 173.7, 173.0, 172.4, 171.5, 148.2, 137.3, 136.7, 136.4, 135.8, 129.0, 128.6, 127.1 , 124.5, 122.2, 113.4, 112.9, 70.7, 58.94, 55.37, 54.04, 39.16, 37.04, 35.29, 34.86, 30.41 , 26.27, 21 .96. HRMS (m / z): [M + H]+calcd. for C29H35N2O5S: 594.2498, found 594.2490. dissolved in anhydrous ACN (250 mL) and DIPEA (14.6 mL, 83.6 mmol) was added. The reaction mixture was cooled to 0 °C and EDCI (10.7 g, 55.7 mmol) was added and the reaction was stirred for 18 h. at rt. The mixture was diluted with EtOAc (600 mL) and washed with 10% aqueous citric acid (300 mL) and brine (300 mL). The organic layer was dried with MgSC , concentrated in vacuo and purified through silica gel column chromatography (0 -^ 15 % EtOAc / n -heptane) to afford the product as a mixture of the 5’- and 6’ isomers (4, 3.49 g, 32%) as a white solid. TLC (EtOAc / n- heptane, 1 :4 v / v): Rf= 0.32 (6’ isomer), 0.27 (5’ isomer).1H NMR (500 MHz, CDCh) 6 8.99 (s, 2H), 8.39 (s, 1 H), 8.32 (d, J = 8.8 Hz, 1 H), 8.09 (d, J = 1 .9 Hz, 1 H), 8.03 (d, J = 8.8 Hz, 1 H), 7.63 - 7.58 (m, 1 H), 7.51 - 7.45 (m, 1 H), 1 .52 (d, J = 17.5 Hz, 27H).13C NMR (126 MHz, CDCh) 6 131 .1 , 127.4,

[0170] 121 .1 , 1 19.8, 116.3, 115.2, 28.1 . HRMS (m / z): [M + H]+calcd. for Ci7H22CIN5O4Na: 418.1258, found 418.1244.

[0171] 1.4 Protease assay conditions

[0172] Luminescent enzymatic activity assay: D-Cysteine (final cone. 1 mM) and assay buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.4) were added to a well containing Methoxyacetamide- PA-X-R-6ABTC or Methoxyacetamide-pA-X-K-6ABTC (final cone. 666.7 pM) with a total volume of 30 pL. After incubation for 30 minutes at 37 °C, 30 pL of the detection mix was added containing luciferase (final cone. 10 pM), MgCh (final cone. 6.7 mM), ATP (final cone. 1 mM), protease enzyme (Factor Xa or thrombin) (final cone. 10 nM), and assay buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.4). The luminescence was recorded in relative light units (RLU) every minute for 30 minutes at 37 °C with an integration time of 1000 ms using a SpectraMax M3 plate reader (Molecular Devices, San Jose, CA, USA). The Area Under the Curve (AUC; RLU) was calculated via: where t is the time in minutes and y the luminescent signal in RLU / s. All measurements were performed in duplicate, of which the mean AUC was calculated and plotted using GraphPad Prism (version 9.0).

[0173] Example 2 - provision of a library of caged luminescent probes

[0174] 2.1 Provision of building blocks

[0175] A peptide library of 40 caged luminescent probes was synthesized containing either an arginine or lysine residue on P1 and subsequently screened with two different trypsin-like serine proteases as proof of the synthetic methodology. The use of a more stable aminoluciferin precursor followed by the in situ condensation with cysteine in a final stage prior to an enzymatic assay renders our approach highly suitable for SPPS.

[0176] To develop an improved synthesis method for the preparation of luminescent peptides via SPPS the more stable 6-ABTC precursor of aLuc was used during synthesis. We aimed to address the inherent unreactive character of the amino group of 6-ABTC (1), by first coupling it to the Pi residue in solution (Scheme 2) to achieve a building block. First, 1 was synthesized on multigram scale starting from 2-chlorobenzo[c(]thiazole according to a known literature procedure by Bon et al. (Beilstein J. Org. Chem. 2016, 12, 2019-2025). Initial coupling reactions of either Fmoc- Orn(Boc)-OH (2) or Fmoc-Lys(Boc)-OH (3) with 1 under standard peptide coupling conditions (HOBt, DIC / HATU, DIPEA) or C-terminal activation with isobutyl chloroformate and NMM all appeared to be low yielding with significant amounts of byproducts. When switching to preactivation of 1 with phosphorus trichloride in pyridine, prior to the addition 2 or 3, high yields were obtained. The coupling reaction of 1 with 2 or 3 was executed to yield protected building blocks 4 and 5 respectively (Scheme 2). The Boc sidechain protecting groups were subsequently removed with formic acid and the products were lyophilized to obtain the ornithine and lysine building blocks 6 and 7 respectively.

[0177] 2.2 immobilizing building blocks on a peptide synthesis resin

[0178] With building blocks 6 and 7 in hand, we attached the building blocks on an aldehyde functionalized resin via reductive amination. Since the nitrile moiety can be prone to acidic hydrolysis, we aimed to use the highly acidic labile (3-formylindolyl)acetamido methyl 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 with NaBHsCN in a 1 :1 mixture of tetrahydrofuran and trimethyl orthoformate as water scavenger (Scheme 3) for 4 h. at room temperature. Prolongation of the reaction time or heating did not increase our yields, as often in our hands more byproducts were obtained among which hydrolysis of the nitrile into the amide.

[0179] Scheme 3 - Solid phase peptide synthesis approach for luminescent peptides via sidechain anchoring of compounds 6 or 7 via reductive amination onto a (3-formylindolyl)acetamido methyl resin, i. 6 or 7 (2 equiv), THF:TMOF (1:1), 4 h, rt. ii. NaBH3CN (2 equiv), AcOH (3.5 equiv), 2 h, rt. Hi. torn = 3: N,N’-di-Boc-thiourea (2 equiv), DIC (2 equiv), DCM, rt, 16 h. torn = 4: Boc2O (3 equiv), DIPEA (3 equiv), DMF, rt, 16 h. iv. a. 3% DBU in DMF, DMF (3 times), b. HOBt (3.6 equiv), DIC (3.3 equiv), Fmoc-AA-COOH / Cap-COOH (3.0 equiv.) (3 times), DMF. v. TFA:DCM (1:1, v / v), 2 h. In this scheme AA2 and AA3 represent additional amino acid residues. Ri is -Boc or is - C(=NBoc)NHBoc; R2is H oris -C(=NH)NH2.

[0180] After successful attachment of 6 and 7 on a solid support, the secondary bound amines were further functionalized before proceeding with the N-terminal peptide elongation. For the lysine derivatives (n = 4) we temporary protected the secondary amine with di-tert-butyl dicarbonate and DIPEA in DMF while agitating overnight. To obtain the arginine derivatives from our ornithine bound resin, we guanidinylated the secondary amine with two equivalents of A / , / V'-di-Boc-thiourea in DCM overnight with A / ,A / '-diisopropylcarbodiimide (DIC) as desulfurization reagent, since the highly toxic HgCh is not compatible with SPPS due to the formation of insoluble HgS. This yielded the arginine functionalized resin which could be used for subsequent N-terminal prolongation.

[0181] After the synthesis of our initial arginine library we found that this guanidinylation reaction following standard literature methods yielded significant amounts of the thiourea byproduct (see Figure 2) as observed by mass spectrometry. Initial attempts to shorten the reaction time or addition of fresh reagent did not overcome the formation of this byproduct. Neither did the use of different activators such as EDC, Cu(l)CI or Cu(ll)CI or TCT-promoted guanidinylation lower byproduct formation. Different guanidinylation reagents such as A / ,A / '-di-Boc-1 / 7-pyrazole-1-carboxamidine or Goodman's reagents were also evaluated but gave rise to even lower conversion, as primarily the starting ornithine product was found.

[0182] In our initial library we were still able to purify the resulting end products using the / V, / V -di- Boc-thiourea DIC method, but simultaneously we were looking for a convenient solution to overcome this side product formation. Eventually, we evaluated the use of a benzotriazole based reagent (H.-J. Musiol, L. Moroder, Org. Lett. 2001 , 3, 3859-3861). We were glad to find out that this reagent could after optimization easily be synthesized on multi-gram scale (as a mixture of the 5' and 6'-isomers) without the use of the toxic HgCh. This surprising guanidinylation reagent is currently being used and evaluated for the synthesis of similar peptides via SPPS in our laboratory and initial results reveal a cleaner conversion with minimal byproducts as compared to using N,N'- di-Boc-thiourea. mixture of the 5’ and 6’-isomers of N,N’-diBoc-amino-immino-1-(5 / 6-chlorobenzotriazolyl)methane

[0183] 2.3 conjugating one or more additional amino acid residues

[0184] With the lysine and arginine functionalized resins obtained, we aimed to demonstrate our method via the synthesis of a small peptide library. We were inspired by the positional scanning substrate combinatorial library (PS-SCL) approach as shown by Por^ba et al. (M. Por^ba et al., Caspases, Paracaspases, and Metacaspases: Methods and Protocols (Eds.: P. V. Bozhkov, G. Salvesen), Springer New York, 2014, pp. 41 -59) to define enzyme substrate specificity. In our experience the substrate methoxyacetamide-p-Ala-Gly-Arg-aLuc appeared to be an excellent substrate for trypsin-like serine proteases such as thrombin. We used this substrate as a model sequence for testing our methodology by synthesizing a tripeptide library with altering P2 residues (here: the glycine) for all canonical amino acids, to solely study P2 dependence. We elongated the peptide chains of our resin coupled products on the N-terminus via standard Fmoc SPPS chemistry. The Fmoc deprotections were executed with 3% DBU in DMF, as these conditions gave considerably better results than the use of 20% piperidine in DMF. The subsequent couplings of the three equivalents of Fmoc protected amino acids or the N-terminal cap were conducted under standard coupling conditions (3.6 equivalents of HOBt and 3.3 equivalents of DIC in DMF). When the sequence was finished, the crude peptides were cleaved from the resin with trifluoroacetic acid in DCM (1 :1 , v / v, addition of 2.5% TIS and EDT for Trt containing sequences) and lyophilized followed by RP-HPLC purification to obtain the purified 6-ABTC conjugated peptides which were analyzed by ESI-MS and NMR (typical scale of 100 mg unfunctionalized resin per substrate gave a couple mg of pure product). With this method we obtained a library comprising 40 peptides with a methoxyacetamide-p-Ala-R2-Arg / Lys-aLuc sequence (with P2 = one of all of the 20 naturally occurring L-amino acids, and R2 is as defined in scheme 3 to form either lysine or arginine). The synthesized compounds are or formula F2.3 and are shown in table T2.3. Pi refers to the combination of R2 and side chain length to form either Lys or Arg. P2 refers to the amino acid adjacent to that Lys or Arg. Amino acids listed in table T2.3 are L-amino acids where appropriate. Table T2.3 - compounds prepared in this example

[0185] 2.4 compounds are stable and functional

[0186] The synthesized 6-ABTC functionalized peptides were significantly more stable than the respective aLuc peptides. Their use does require a cysteine condensation reaction prior to enzymatic conversion, preferably using D-cysteine. Since the click reaction of D-cysteine and 6- ABTC has extensively been described in the literature and generally solely yields the aLuc functionalized derivative, we hypothesized that we could perform this reaction in situ in the most final stage of our assay prior to the protease addition. As proof, we screened our peptide library against the two trypsin-like serine proteases Factor Xa (FXa) and thrombin (FXIIa). First, we incubated our 6-ABTC conjugated peptides with D-cysteine for 30 min at 37 °C in buffer to generate the respective aLuc derivatives prior to the addition of protease, ATP, MgCk and luciferase. Then, the luminescence was recorded at 37 °C for 30 minutes and the area under the curve (AUC) was calculated. The ratio of substrate hydrolysis towards either thrombin or FXa was determined and depicted in Fig. 2. With altering the P2 position in our library we were able to obtain a significant difference in selectivity of our substrates towards either thrombin or FXa, demonstrating that our synthetic principle and the in-situ cysteine condensation are robust. We for instance observed that the incorporation of a proline at P2 revealed a significant increase in selectivity towards thrombin.

[0187] 2.5 conclusion

[0188] We developed an SPPS method which allows the rapid synthesis of 6-ABTC C-terminally modified peptides via the side chain anchoring of a first residue. These products were stable. The use of 6-ABTC and the in situ condensation reaction with cysteine were important steps to achieve this stability. Ornithine residues could be converted into arginine residues via a special on resin guanidinylation reaction, avoiding side-products. After cleavage of the peptide from the resin and straightforward purification such as RP-HPLC, the subsequent condensation reactions with cysteine (such as D-cysteine) to yield the aLuc caged peptides were done in situ priorto the addition of protease. We synthesized a peptide library of 40 tripeptides which was screened for specificity for either one of the trypsin-like serine proteases Factor Xa or thrombin. The results presented in this work would allow for the (automated) synthesis of larger luminescent peptide libraries with for instance either an arginine or lysine residue on the Pi position, especially interesting for proteases which prefer positively charged amino acids in the Pi.

Claims

Claims1 . Method for producing a peptide conjugate, the method comprising the steps of: i) providing a building block of general formula (I):wherein n is 1 , 2, 3, 4, or 5; p' is a protecting group; ii) immobilizing the building block on a peptide synthesis resin; iii) conjugating one or more additional amino acid residues using conventional solid phase peptide synthesis to obtain an immobilized peptide conjugate.

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

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

4. The method according to any one of claims 1 -3, wherein step ii) comprises reductive amination of the free amine of the building block onto an aldehyde of the peptide synthesis resin to form an immobilizing secondary amine, wherein the immobilizing secondary amine is optionally subsequently protected.

5. The method according to step 4, wherein the immobilizing secondary amine is guanidinylated, after which the resulting guanidinium moiety is optionally protected.

6. Compound of general formula (V) or a salt thereof:wherein hi is H, p', C1-4 alkyl, -C(=N-p')-NH-p', or -C(=NH)-NH2; h2 is H, p', C1-4 alkyl, or a peptide synthesis resin; n is 1 , 2, 3, 4, or 5;R is H, p', X, or a polypeptide moiety comprising 1-10 amino acids; p' is in each instance independently a protecting group; andX is -C1-20 alkyl, -SO2-C1-20 alkyl, -C(=O)-O-C1-20 alkyl, or -C1-20 acyl, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy.

7. The compound according to claim 6, wherein R is a polypeptide moiety of general formula(Pep):wherein sc1is an amino acid side chain preferably selected from H or a linear, branched, or cyclic C1-12 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, or selected from -(CH2)o-4-[C5-1O (hetero)aryl] that is optionally substituted with halogen; sc2is an amino acid side chain preferably selected from H or a linear, branched, or cyclic C1-12 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, or selected from -(CH2)o-4-[C5-1O (hetero)aryl] that is optionally substituted with halogen, wherein each instance of sc2is 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, wherein alkyl and acyl are linear, branched, or cyclic, are optionally unsaturated, and are optionally substituted with halogen or C1-4 alkoxy.

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

9. The compound according to claim 7 or 8, wherein X is -C(=O)-CH2-O-CH3.

10. The compound according to any one of claims 7-9, wherein m is 1 or 2, preferably 1 ; sc2is H for the amino acid moiety adjacent to X; b is -CH2- for the amino acid moiety adjacent to X.11 . The compound according to claim 10, wherein m is 1 .

12. The compound according to any one of claims 7-11 , wherein h2 is a peptide synthesis resin, or wherein X is -C(=O)-CH2-O-CH3, or wherein b is -CH2- for the amino acid moiety adjacent to X, or whereinX is -C(=O)-CH2-O-CH3 and b is -CH2- for the amino acid moiety adjacent to X.

13. The compound according to any one of claims 6-12, wherein h2 is a peptide synthesis resin, preferably an aldehyde functionalized resin, preferably a highly acidic labile resin such as (3-formylindolyl)acetamido methyl polystyrene.

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

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

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

17. The compound according to any one of claims 6-13, wherein hi is -C(=N-p')-NH-p' or -C(=NH)-NH2, and preferably n is 2 or 3, more preferably 2.

18. The compound according to any one of claims 6-13, wherein hi is -C(=N-p')-NH-p' or - C(=NH)-NH2, and n is 2.

19. Kit of parts comprising i) a compound according to any one of claims 6-18; and ii) cysteine, preferably D-cysteine.

20. Method for determining protease activity, the method comprising the steps of: i) providing one or more compounds according to any one of claims 6-18; ii) contacting the provided compounds with cysteine to provide aminoluciferin-peptide conjugates; iii) contacting the aminoluciferin-peptide conjugates with the protease to provide free aminoluciferin; iv) contacting the free aminoluciferin with luciferase to generate a luminescent signal.

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