Method for obtaining a library of peptides or peptide

Abstract

FIELD: medicine.SUBSTANCE: new method for producing a peptide library or an isolated peptide, which comprises: (a) cleaving one or more linear dithiol peptides containing a sulfhydryl group at the N-terminal region of said one or more peptides and immobilized via a disulfide bridge at the C-terminal region of said one or more peptides on a solid phase, from said solid phase by means of (i) an agent that reduces said disulfide bridge, thereby cleaving said one or more linear dithiol peptides from said solid phase, wherein said agent is selected from 1,3-propanedithiol, 1,4-butanedithiol, 2,4-pentanedithiol, ethane-1-thiol, propane-1-thiol, butane-1-thiol, propane-2-thiol, 2-methyl-1-propanethiol, butane-2-thiol, 2-methylpropane-2-thiol, 2-hydroxy-1-ethanethiol, 1,2-ethanedithiol, 2-propene-1-thiol, 3-methyl-1-butanethiol, thiophenol, benzylthiol, 2-butene-1-thiol, 3-butene-1-thiol, 2-methyl-2-propene-1-thiol and 3-methyl-2-butene-1-thiol and is removed by evaporation, or (ii) a base that deprotonates said sulfhydryl group in the N-terminal region of said one or more linear dithiol peptides, which is a trialkylamine, thereby inducing an intramolecular disulfide exchange with cleavage and cyclization thereof of said one or more linear dithiol peptides from said solid phase in the form of one or more cyclic peptides.EFFECT: less complex preparation steps to produce large libraries of peptides or one or more peptides that can be added to peptide libraries, where said peptide libraries can then be screened for peptide therapeutics.14 cl, 30 dwg, 2 tbl, 4 ex

Description

[0001] The present invention relates to a method for producing a peptide library or an isolated peptide, comprising (a) cleaving one or more linear dithiol peptides containing a sulfhydryl group at the N-terminal region of said one or more peptides and immobilized via a disulfide bridge at the C-terminal region of said one or more peptides on a solid phase, from said solid phase by means of (i) an agent that reduces said disulfide bridge thereby cleaving said one or more linear dithiol peptides from said solid phase, wherein said agent is volatile and amenable to removal by evaporation, or (ii) a base that deprotonates said sulfhydryl group at the N-terminal region of said one or more linear dithiol peptides,thereby inducing an intramolecular disulfide exchange with the consequent cleavage of said one or more linear dithiol peptides from said solid phase in the form of one or more cyclic peptides,

[0002] This description cites a number of documents, including patent applications and manufacturer's instructions. The contents of these documents, although not considered relevant to the patentability of the present invention, are hereby incorporated by reference in their entirety. More specifically, all cited documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0003] Peptide libraries, and in particular libraries of cyclic peptides (a class of macrocyclic molecules), have attracted great interest from the pharmaceutical industry because they can be screened to identify peptides that have the ability to bind to complex targets for which it has been difficult or even impossible to design ligands based on classical small molecules.

[0004] Currently, more than 40 cyclic peptides have been approved for use as drugs, and more than 100 are being evaluated in various stages of clinical trials (A. Zorzi et al., Curr. Opin. Chem. Biol., 2017, 38, 24-29). Innovative strategies for designing cyclic peptide ligands based on protein epitopes or for isolating target-specific cyclic peptides from large combinatorial libraries of genetically encoded peptides have given further impetus to the development of this field (A. Luther et al., Curr. Opin. Chem. Biol., 2017, 38, 45-51; C. Sohrabi et al, Nat. Rev. Chem., 2020, 4, 90-101). Of particular interest are macrocyclic molecules that are small enough, ideally significantly less than one kilodalton (kDa), and have a small polar surface area, placing intracellular targets within easy reach. Macrocyclic molecules of high interest include small cyclic peptides, non-peptide-based macrocyclic structures, or macrocyclic structures containing peptide and non-peptide components. However, the development of macrocyclic ligands that bind to targets of interest and have membrane penetration capabilities to reach intracellular targets is hampered by the relatively small number of macrocyclic compounds in commercially available collections or the lack of efficient methods for synthesizing new macrocyclic libraries.

[0006] A challenging step in the synthesis of macrocyclic compounds is the conversion of linear molecules into cyclic ones. Most reactions yield macrocyclizations well below 90% and exhibit wide variation in yields for different precursors (e.g., different linear peptide sequences), which poses a challenge for library synthesis. All macrocyclic libraries offered by leading suppliers such as Asinex, ChemBridge, and Polyphor are based on molecules that have been individually purified after the macrocyclization step, as the products would not be sufficiently pure for most compounds without purification. The need for purification limits the number of macrocycles that can be prepared in parallel and, consequently, library sizes, which for commercially available libraries are less than 30,000 molecules.

[0007] A macrocyclization reaction that generally exhibits high cyclization yields for a wide range of substrates, typically above 90%, is the cyclization of peptides via two thiol groups located at distant ends of said peptides using bis-electrophilic reagents such as bis-(bromomethyl)benzenes (reaction S N 2 (bimolecular nucleophilic substitution)), bis-(bromoacetamide)benzenes (reaction S N 2), halogenated acetones (reaction S N 2), vinyl sulfoxides (Michael addition reaction) or hexafluorobenzene (S reaction NAt (aromatic nucleophilic substitution)) (H. Jo et al., J. Am. Chem. Soc, 2012, 134, 17704–17713; P. Timmerman et al., ChemBioChem, 2005, 6, 821–824; N. Assem et al., Angew. Chemie - Int. Ed., 2015, 54, 8665–8668; SS Kale et al., Nat. Chem., 2018, 10, 715–723). Such reactions have been used to design (bi)cyclic peptides that mimic linear or nonlinear epitopes (P. Timmerman et al., ChemBioChem, 2005, 6, 821–824), to design (bi)cyclic peptides via phage display (C. Heinis et al., Nat. Chem. Biol, 2009, 5, 502–507) or other display methods, to stabilize α-helical peptides in helical conformations (H. Jo et al., J. Am. Chem. Soc, 2012, 134, 17704–17713), or to cyclize peptides for other purposes.

[0008] Large libraries of cyclic peptides with molecular weights of less than one kilodalton were recently synthesized by combinatorial cyclization of a large number of linear dithiol peptides using approximately 20 different bis-electrophilic reagents (unpublished results, Therapeutic Protein and Peptide Laboratory, EPFL, Heinis group). Different combinations of dithiol peptides and cyclization reagents were mixed in separate wells of 384-well microplates. High cyclization yields allowed the present inventors to screen these cyclic peptides for interactions with target proteins without prior purification. Eliminating a throughput-limiting purification step allowed the screening of large libraries.

[0009] A combinatorial cyclization strategy for peptides using bis-electrophilic reagents requires a large number of dithiol peptides with different sequences.

[0010] Regardless of the method used to identify the leader peptide, the development of peptide libraries and peptide drugs typically involves several iterative rounds of synthesizing tens to hundreds of peptide variants to improve key properties such as binding affinity, specificity, stability, pharmacokinetic properties, and others, and thus obtaining a large number of peptides.

[0011] Another major bottleneck in the development of cyclic peptide therapeutics is the chromatographic purification of peptides after synthesis, which is expensive due to the sequential processing of each peptide and the high consumption of reagents (solvents), even with automation and optimization. The main reason for the need for purification is the macrocyclization reaction, which is typically the most challenging step in the synthesis of cyclic peptides (CJ White et al., Nat. Chem., 2011, 3, 509–524). Most macrocyclization reactions exhibit yields below 90%, and their efficiencies often vary greatly depending on the sequence and length of the peptide. Chromatographic purification is also required to remove reagents and scavengers added for peptide cleavage, as well as side-chain protecting group-based byproducts generated during extensive deprotection.

[0012] From the above, it is evident that there is a pressing need for new methods, particularly methods that require less complex preparation steps for producing large libraries of peptides or one or more peptides that can be added to peptide libraries, where said peptide libraries can then be screened for peptide therapeutics. The present invention satisfies this need.

[0013] In a first aspect, the present invention relates to a method for producing a peptide library or an isolated peptide, comprising (a) cleaving one or more linear dithiol peptides containing a sulfhydryl group at the N-terminal region of said one or more peptides and immobilized via a disulfide bridge at the C-terminal region of said one or more peptides on a solid phase, from said solid phase by means of (i) an agent that reduces said disulfide bridge thereby cleaving said one or more linear dithiol peptides from said solid phase, wherein said agent is volatile and can be removed by evaporation, or (ii) a base that deprotonates said sulfhydryl group at the N-terminal region of said one or more linear dithiol peptides,thereby inducing an intramolecular disulfide exchange thereby cleaving said one or more linear dithiol peptides from said solid phase in the form of one or more cyclic peptides.,

[0014] The term "comprise / comprising" is generally used to mean "including / includes," in other words, allowing for the presence of one or more features or components. The terms "comprise" and "comprising" also encompass the narrower terms "consist of" and "consisting of."

[0015] As used in the present description and claims, the singular and definite forms of terms include plural references unless the context clearly dictates otherwise.

[0016] As used herein, the term "peptide" refers to a polymer comprising at least one amino acid and at least one peptide bond. Said peptide preferably comprises several—for example, two or more, three or more, four or more, or five or more—amino acids. Said peptide may also comprise non-amino acid structural units and non-peptide bonds. Many of the peptides described in the attached examples contain structural units such as mercaptopropionic acids (MPA) and cysteamine (MEA) at their ends, which are not amino acids because they do not contain an amino group (in the case of MPA) or are not a carboxylic acid (in the case of MEA).

[0017] Other examples of non-amino acid structural units that contain a thiol group and are suitable for incorporation into peptides, particularly at the C-terminus (like MEA), are 3-aminopropane-1-thiol, 3-(methylamino)propane-1-thiol, (2)-4-aminobut-2-ene-1-thiol, piperidine-4-thiol, 4-(mercaptomethyl)piperidine, and 3-(mercaptomethyl)azetidine; see the table and formulas below. Among these non-amino acid structural units, those containing secondary amines are particularly preferable for the development of membrane-permeable macrocyclic compounds, since the combination of a secondary amine and a carboxylic acid (from an adjacent amino acid) results in the formation of an amide bond without a hydrogen bond donor group.

[0018]

[0019]

[0020] Other examples of non-amino acid structural units that contain a thiol group and are suitable for incorporation into peptides, particularly at the N-terminus (as MPA), are 2-mercaptoacetic acid, (R)-2-mercaptopropionic acid, 5-(mercaptomethyl)furan-2-carboxylic acid, and 3-(mercaptomethyl)benzoic acid; see table and formulas below.

[0021]

[0022]

[0023] Various other non-amino acid structural units are suitable for incorporation into dithiol peptides, particularly at internal positions. For example, the structural unit A-COOH can be coupled to the amino group of a preceding residue on the solid phase, and then the structural unit B-NH2 can be attached, where the functional groups in A and B react with each other to form a covalent bond. This latter reaction can result in the formation of a bond that is not a peptide bond. An example of such a reaction is the well-established so-called "submonomeric" strategy, in which a haloacetic acid, typically bromoacetic acid, which has undergone activation (e.g., with diisopropylcarbodiimide), is first coupled to the amino group of the growing peptide on the solid phase (this haloacetic acid corresponds to the structural unit A-COOH).In the second step and in the second chemical reaction, the amine (corresponding to B-NH2) displaces the halide to form an N-substituted glycine residue (according to the classic SN2 reaction). The submonomer approach allows the use of any commercially available or synthetically achievable amine, which is a major advantage, as multiple amines can be used in parallel, and a wide variety of peptides can be obtained. Many other reagents can be used in place of the haloacetic acids, including, as preferred examples, 4-(bromomethyl)benzoic acid, 3-(bromomethyl)benzoic acid, 2-(chloromethyl)oxazole-4-carboxylic acid, 2-(chloromethyl)thiazole-4-carboxylic acid, 5-(bromomethyl)isoxazole-3-carboxylic acid, 5-(bromomethyl)dimethylazine-2-carboxylic acid, 2-(bromomethyl)furan-3-carboxylic acid, (R,E)-5-chloro-2,4-dimethylpent-3-enoic acid, and (S,E)-5-chloro-2,4-dimethylpent-3-enoic acid; see the table and formulas below.

[0024]

[0025]

[0026] The term "peptide" preferably refers to short chains of amino acids linked by peptide bonds. Furthermore, short chains of amino acids linked by peptide bonds may contain non-amino acid structural units at their ends, such as MPA and cysteamine MEA. Peptides differ from proteins or polypeptides in size and contain, with increasing preference, fewer than 50 amino acids, and with increasing preference, fewer than 40 amino acids, fewer than 30 amino acids, fewer than 20 amino acids, fewer than 10 amino acids, and fewer than 5 amino acids.

[0027] The term "isolated peptide" as used herein is intended to indicate that said peptide is not bound to a solid phase, but has been cleaved from said solid phase. Said isolated peptide is generally in the form of a free peptide, for example, in solution. One or more copies of the isolated peptide may be present in the solution. Accordingly, one or more copies of the peptide to be isolated may also be present on the solid phase prior to isolation of said peptide.

[0028] The term "amino acid" as used herein refers to an organic compound consisting of amine (-NH2 or -NH) and carboxylic acid (-COOH) functional groups, generally together with a side chain that is different for each amino acid. The simplest amino acid, glycine, does not contain a side chain (it has the formula H2NCH2COOH). In amino acids that contain a carbon chain attached to the α-carbon (such as lysine), the carbon atoms are designated in the order α, β, γ, δ, and so on. In some amino acids, the amino group may be attached, for example, to the α-, β-, or γ-carbon, and are therefore called α-, β-, or γ-amino acids, respectively.The term "amino acid" preferably describes α-amino acids (also called 2- or α-amino acids), which generally have the general formula H2NCHRCOOH, where R is an organic substituent called a "side chain," as well as beta-, gamma-, and delta-amino acids, which contain multiple carbon atoms between the amine and the carboxylic acid. In the simplest α-amino acid, alanine (formula: H2NCHCH3COOH), the side group is a methyl group. The amino acids of the present invention are L-amino acids or D-amino acids.

[0029] Amino acids include the so-called standard or canonical amino acids. These 21 α-amino acids are encoded directly by codons of the universal genetic code. They represent the proteinogenic α-amino acids found in eukaryotes. These amino acids are designated in this document by the so-called single-letter code:

[0030]

[0031]

[0032] As mentioned, the side chain of an amino acid is an organic substituent, which in the case of α-amino acids is bonded to the α-carbon atom. Therefore, the side chain is a branch from the parent structure of the amino acid. Amino acids are generally classified by the properties of their side chain. For example, a side chain can make an amino acid a weak acid (e.g., amino acids D and E) or a weak base (e.g., amino acids K and R), and also a hydrophilic substance if said side chain is polar (e.g., amino acids L and I), or a hydrophobic substance if it is non-polar (e.g., amino acids S and C). An aliphatic amino acid has a side chain that is an aliphatic group. Aliphatic groups make the amino acid non-polar and hydrophobic. Said aliphatic group is preferably an unsubstituted branched or linear alkyl.Non-limiting examples of aliphatic amino acids are A, V, L, and I. In a cyclic amino acid, one or more rows of atoms in the side chain are / are joined to form a ring. Non-limiting examples of cyclic amino acids are P, F, W, Y, and H. It should be understood that said ring must be distinguished from the ring that is formed in the case of a cyclic peptide, as will be described in more detail below herein. While the first ring of a cyclic amino acid is part of the side chain of one amino acid, the last ring is formed between two thiol groups of a dithiol peptide. An aromatic amino acid is a preferred form of cyclic amino acid. In an aromatic amino acid, said ring is an aromatic ring.In terms of the electronic nature of a molecule, aromaticity describes how a conjugated ring of unsaturated bonds, lone pairs of electrons, or empty molecular orbitals exhibits stronger stabilization than would be expected from the stabilization of conjugation alone. Aromaticity can be viewed as a manifestation of cyclic delocalization and resonance. A hydrophobic amino acid contains a nonpolar side chain, which makes the amino acid hydrophobic. Non-limiting examples of hydrophobic amino acids are M, P, F, W, G, A, V, L, and I. A polar uncharged amino acid contains a nonpolar side chain and no charged residues. Non-limiting examples of polar uncharged amino acids are S, T, N, Q, C, U, and Y. A polar charged amino acid contains a nonpolar side chain with at least one charged residue. Non-limiting examples of polar charged amino acids are D, E, H, K, and R.

[0033] A "dithiol peptide" is a peptide that contains two or more, and preferably only two, thiol groups. These thiol groups are either part of amino acids or non-amino acid structural units of the peptide. The amino acid or non-amino acid structural units containing the thiol group may be one of the following:

[0034] - Cysteine ​​or a cysteine ​​analog such as homocysteine, penicillamine, or D-cysteine

[0035] - A structural unit containing a thiol group and a carboxylic acid, such as mercaptopropionic acid (MPA)

[0036] - A structural unit containing a thiol group and an amine, such as cysteamine (MEA)

[0037] When the said linear dithiol peptide is immobilized on the solid phase, one thiol group is "free", initially it is a protected sulfhydryl group (RS-PG; PG=protecting group), and after removing the protecting group for the thiol group, it is a sulfhydryl group (R-SH), while the other thiol group is involved in a disulfide bond (R 1 -SSR 2 ), connecting said peptide to said solid phase. A disulfide bridge is formed when a sulfur atom from one thiol-containing structural unit (linked to said solid phase) forms a single covalent bond with a sulfur atom from a thiol-containing structural unit in the C-terminal region of said peptide. Means and methods for synthesizing peptides via disulfide bonds to a solid phase are known in the art and are described in more detail herein below and illustrated by the accompanying examples.

[0038] The term "linear peptide" means a linear short chain of amino acids linked by peptide bonds, which may also contain non-amino acid structural units and non-peptide bonds as described above in connection with the "peptide" of the present invention. In said linear peptides, none of the rows of atoms in the peptide are linked to form a macrocyclic ring or cycle.

[0039] The term "cyclic peptide" means that a series of 12 or more atoms in said peptide are linked to form a macrocyclic ring or cycle. Said cyclic peptide is preferably a monocyclic peptide, meaning that only two sites in said peptide are linked to form a ring or cycle. Said ring or cycle is formed according to the present invention by involving two thiol groups of said dithiol peptides. In the case where said two thiol groups are directly linked, said ring or cycle is formed via a disulfide bridge. Said two thiol groups of said dithiol peptides can also be linked using a bis-electrophilic reagent, as will be explained in more detail below.

[0040] A peptide library refers to a composition or article containing a plurality of different peptides.

[0041] If said library is a composition, said composition comprises a mixture of different peptides. Said composition is preferably a solution, and more preferably a solution in dimethyl sulfoxide (DMSO) or an aqueous solution. Said solution can be dried, for example, by lyophilization or centrifugal vacuum evaporation (for example, using a SpeedVac system). In this case, said composition can be in the form of a dried powder that can be dissolved using the desired solvent. In the case of such a composition, the number of different peptides in said library can be determined by the number of different peptides immobilized on a solid support and / or by mixing solid supports on which one or two or more different peptides have been synthesized.

[0042] The said article comprises various wells and is preferably a microtiter plate, more preferably a 96-well plate, a 384-well plate, a 1536-well plate, or a 3456-well plate. Different peptides are preferably synthesized (generally on a solid support) in parallel in different wells such that one kind of peptide (in multiple copies) can be present in each well. The plurality of wells of the said article collectively form the said peptide library. A library in the article format is preferable because if such a library is screened for a substance that binds to a specific target molecule or an inhibitor of a specific target molecule (described below in this document), it is immediately known in which well the desired library member can be found, and no additional complex isolation or identification of the desired library member is required.

[0043] While one type of peptide per well is preferred, it is also possible to synthesize more than one peptide per well, for example, two, three, four, or five different peptides per well (generally on a solid support), in parallel in each well, thereby obtaining wells with two, three, four, or five different peptides per well. The number of different peptides per well can be adjusted as needed, for example, by adjusting the number of different peptides immobilized on the solid support and / or by mixing solid supports on which one, two, or more different peptides have been synthesized.

[0044] The said library of different peptides contains, with increasing preference, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 96, at least 100, at least 384, at least 500, at least 1000, at least 1536, at least 3456, at least 10000 and at least 100000 different peptides.

[0045] Said N-terminal region preferably means one or one of the three most N-terminal amino acids or structural units, more preferably one or one of the two most N-terminal amino acids or structural units, and most preferably the most N-terminal amino acid or structural unit of said one or more linear dithiol peptides. In the most preferred case, said sulfhydryl group is part of an amino acid or structural unit at the N-terminal position of said one or more linear dithiol peptides.

[0046] Similarly, said C-terminal region preferably means one or one of the three most C-terminal amino acids or structural units, more preferably one or one of the two most C-terminal amino acids or structural units, and most preferably the most C-terminal amino acid or structural unit of said one or more linear dithiol peptides. In the most preferred case, said one or more peptides are immobilized via a disulfide bridge at the C-terminus on a solid phase.

[0047] The term "solid phase" means any solid substance or support on which peptides can be synthesized via a disulfide bond, such as by solid-phase peptide synthesis (SPPS).

[0048] The term "agent" as used herein means any molecule capable of reducing a disulfide bridge, thereby cleaving said one or more linear dithiol peptides from said solid phase. Therefore, said agent may also be referred to as a reducing agent or a cleaving agent. Various reducing agents are known in the art. In biochemistry, thiols such as β-mercaptoethanol (β-ME) or dithiothreitol (DTT) serve as reducing agents. According to the present invention, said agent is volatile and amenable to removal by evaporation, preferably by evaporation under vacuum and most preferably by centrifugal vacuum evaporation. The volatile substance will readily convert to gas as a result of evaporation. Evaporation is a type of vaporization that occurs on the surface of a liquid when it passes into the gas phase.The ability of a liquid molecule to evaporate is largely based on the amount of kinetic energy possessed by an individual particle. Although the rate of evaporation increases at higher temperatures, even at lower temperatures, individual liquid molecules can evaporate if they possess an amount of kinetic energy greater than the minimum amount required for vaporization. The use of an agent that is volatile and amenable to removal by evaporation is technically advantageous, since said agent can be removed from a composition containing a peptide library or an isolated peptide to be obtained by the method of the present invention. Removal of said one or more peptides from said solid support by means of an agent is also referred to herein as "reductive cleavage" and is illustrated in Figure 1b.As can be understood from Figure lb, said one or more peptides are cleaved by said agent in the form of one or more linear peptides, wherein said two thiol groups are "free" sulfhydryl groups.

[0049] A base is a substance that can accept protons (such as Bronsted bases), donate electrons (such as Lewis bases), or any chemical compound that results in the formation of hydroxide ions (OH-) in aqueous solution. The base used in the present invention is capable of deprotonating the sulfhydryl group at the N-terminal region of the said one or more linear dithiol peptides (RS -). The deprotonated sulfhydryl group induces an intramolecular disulfide exchange, resulting in the cleavage of said one or more linear dithiol peptides from said solid phase in the form of one or more cyclic peptides. Said deprotonated sulfhydryl group contains a reactive sulfur atom S, which acts as a nucleophilic agent, causing a thiol-disulfide exchange reaction at the disulfide bond. The removal of said one or more peptides from said solid support by means of a base is also referred to herein as "cleavage with cyclization" and is illustrated in Figure 1a. As can be understood from Figure 1a, said one or more peptides are cleaved by means of said base in the form of a cyclic peptide, wherein two thiol groups of said dithiol peptide are connected by a disulfide bond.

[0050] As noted above, the method of claim 1, subparagraph (i), of the claims represents an approach called "cleavage with reduction" that results in the production of linear dithiol peptides, while the method of claim 1, subparagraph (ii), of the claims represents an approach called "cleavage with cyclization" that results in the production of cyclic dithiol peptides. As will become apparent from the following, both embodiments significantly improve upon the prior art methods for producing a peptide library or an isolated peptide.

[0051] The "reductive cleavage" strategy, in which the specified dithiol peptides immobilized via a disulfide bridge on a solid support are cleaved from said solid phase via disulfide reduction from the resin, is schematically shown in Figure 1b. Unlike cyclization-assisted cleavage, the efficiency of reductive cleavage is independent of the length and amino acid composition of the peptides. In particular, this approach also works effectively with short peptides that cannot be efficiently cyclized via disulfide formation (but can be cyclized using bis-electrophilic linkers, since said cyclization is sterically less demanding due to the additional atoms added to the macrocycle backbone by said linker).The problem with peptide cleavage with reductive cleavage was that a reducing agent must be added to the peptide, and this reagent interferes with the subsequent cyclization reaction using bis-electrophilic reagents (i.e., it reacts with electrophilic groups). This drawback is overcome by using a volatile reducing agent that can be removed from the peptides by evaporation, for example, under vacuum, in a relatively simple step. For example, the volatile reducing agent can be removed by centrifugal vacuum evaporation of the peptides in 96-well plates.

[0052] Several studies have described the cleavage of peptides from the solid phase via reductive disulfide bridge cleavage, all of which cleave peptides containing a single thiol group and imply other applications such as the preparation of peptide-protein conjugates (J. Mery et al., Int. J. Pept. Protein Res., 1993, 42, 44-52), the synthesis of peptide heterodimers (A. Taguchi et al., Org. Biomol. Chem., 2015, 13, 3186-3189), the preparation of head-to-tail cyclized peptides containing a thiol handle (W. Tegge et al., J. Pept. Sci., 2007, 13, 693-699), peptide cyclization via a single thiol group (AA Virgilio et al., Tetrahedron Lett., 1996, 37, 6961-6964), identification of cleaved peptides using mass spectrometry (O. Lack et al., Helv. Chim. Acta, 2002, 85, 495-501) and temporary immobilization during peptide synthesis (DS Kemp et al., J. Org. Chem., 1986, 51, 1821-1829).Most of these approaches used the reducing agents tris-(2-carboxyethyl)phosphine (TCEP) (J. Mery et al., Int. J. Pept. Protein Res., 1993, 42, 44–52; A. A. Virgilio et al., Tetrahedron Lett., 1996, 37, 6961–6964), dithiothreitol (DTT) (J. Mery et al., Int. J. Pept. Protein Res., 1993, 42, 44–52; W. Tegge et al., J. Pept. Sci., 2007, 13, 693–699) and tri-n-butylphosphine (P(n-Bu)3) (D. S. Kemp et al., J. Org. Chem., 1986, 51, 1821–1829), i.e., reagents that cannot be removed by evaporation and require a purification step. Only one of the cited studies used a volatile reducing agent, β-mercaptoethanol (β-Me), to cleave disulfide-linked peptides from a solid support (O. Lack et al., Helv. Chim. Acta, 2002, 85, 495–501).However, in the case of the stated application, the peptides were cleaved from individual beads and in small quantities from a resin that was polar (polyacrylamide-polyethyleneglycol copolymer (PEGA)), had a low loading (0.2 mmol / g), and a high swelling volume, which was not suitable for the intended application of the present invention. No studies have reported the synthesis and reductive cleavage of dithiol peptides, and no studies have used this approach to generate cyclic peptide libraries.

[0053] To the best of the present inventors' knowledge, the disulfide cleavage with cyclization strategy is entirely new and has not been applied to the preparation of cyclic peptides. The closest strategies to this cleavage with cyclization are the oxidative cleavage of thioether-immobilized peptides (B.H. Rietman et al., Int. J. Pept. Protein Res., 1994, 44, 199-206; T. Zoller et al., Tetrahedron Lett., 2000, 41, 9989-9992). However, these approaches are not suitable for library preparation due to low yields, dimeric by-products, and the presence of oxidizing agents in the eluted product, which would need to be removed by purification. In the present invention, the cyclization cleavage is initiated by a base that deprotonates the N-terminal sulfhydryl group.If a volatile base were used, it could be removed by evaporation so that the only product in the reaction tube or microtiter plate well would be one or more pure cyclic peptides. If a non-volatile base were used, it could also be neutralized with acid. An important requirement for this cyclization strategy was that the peptides could be synthesized on a solid phase immobilized via a disulfide bridge, where the disulfide bridge had to be sufficiently stable during peptide synthesis, and in particular during removal of the Fmoc protecting group with piperidine. Several studies have reported solid-phase synthesis of disulfide-linked peptides for applications ranging from the preparation of peptide-protein conjugates to reductive peptide cleavage for mass spectrometric identification (J. Mery et al., Int. J. Pept. Protein Res., 1993, 42, 44-52; A. Taguchi et al., Org. Biomol. Chem., 2015, 13, 3186-3189; W. Tegge et al., J. Pept. Sci., 2007, 13, 693-699; A. A. Virgilio et al., Tetrahedron Lett, 1996, 37, 6961-6964; O. Lacket al., Helv. Chim. Acta, 2002, 85, 495-501; D. S. Kemp et al., J. Org. Chem., 1986, 51, 1821-1829). Mery, J. and colleagues reported that the stability of the disulfide bridge depends on the substituents at the carbon atoms next to the sulfur atoms, and the NH2-CH2-CH2-SS-C(CH3)2-COOH linker with bulky methyl groups was stable enough for the synthesis of peptides with Fmoc (J. Mery et al., Int. J. Pept. Protein Res., 1993, 42, 44-52; W. Tegge et al., J. Pept. Sci., 2007, 13, 693-699; J. Mery et al., Pept. Res., 1992, 5, 233-240).Peptides disulfide-immobilized via the disulfide linker NH2-CH2-CH2-SS-CH2-C(NH2)H-COOH were previously synthesized, and no significant amount of peptide was found in the case of short peptides despite a rather sterically unhindered linker, possibly due to the limited number of times the beads were exposed to piperidine (Y. Wu et al., Chem. Commun., 2020, 56, 2917-2920).

[0054] According to a preferred embodiment of the first aspect of the present invention, said method comprises, after step (a), a step of removing said agent by evaporation.

[0055] As noted above, the agent to be used in the claimed invention is volatile and amenable to removal by evaporation. According to said preferred embodiment, the step of removing said agent by evaporation forms part of said method. Said evaporation is preferably carried out under vacuum (e.g., using a SpeedVac system). Said agent is most preferably removed by centrifugal vacuum evaporation.

[0056] Furthermore, the base according to the present invention is preferably volatile and amenable to removal by evaporation. In this case, the method according to the first aspect of the present invention preferably comprises, after step (a), the step of removing said base by evaporation.

[0057] As an alternative to removing said agent by evaporation, said agent may also be removed by lyophilization.

[0058] According to another preferred embodiment of the first aspect of the present invention, said method further comprises the step (b) of cyclizing said one or more linear dithiol peptides cleaved by said agent.

[0059] As noted above, in the case of "cleavage with reduction" using the said agent, the said one or more peptides are cleaved using the said agent in the form of linear peptides, where the said two thiol groups represent "free" sulfhydryl groups. The said two sulfhydryl groups can be used for cyclization of the said peptides.

[0060] According to a more preferred embodiment of the first aspect of the present invention, said one or more dithiol peptides are cyclized using at least one bis-electrophilic reagent or by disulfide oxidation.

[0061] The two sulfhydryl groups can be linked either directly by forming a disulfide bond (disulfide oxidation) or through a linker, specifically a bis-electrophile reagent. A variety of bis-electrophiles are commercially available or can be easily synthesized using routine chemical reactions. Different bis-electrophiles can be used in parallel reactions to produce multiple different cyclic peptides from a single dithiol peptide.

[0062] Electrophilic reagents act as electron pair acceptors during bond formation. In nucleophilic substitutions, the leaving group is released as a negatively charged species. A bis-electrophilic reagent is a chemical compound containing at least two functional groups that can react with two sulfhydryl groups, resulting in the sulfhydryl groups joining through the bis-electrophilic reagent.

[0063] Disulfide oxidation results in the formation of a direct disulfide bond linking the two sulfhydryl groups of the dithiol peptide.

[0064] According to another preferred embodiment of the first aspect of the present invention, the disulfide bonds of one or more cyclic peptides according to subparagraph (ii) are reduced and said peptides are recyclized using a bis-electrophilic reagent.

[0065] As noted herein above, according to subparagraph (ii) of the first aspect of the present invention, said one or more peptides are cleaved using said base in the form of a cyclic peptide, wherein two thiol groups of said dithiol peptide are linked by disulfide bonds.

[0066] These disulfide bonds can be reduced preferably using the agent described in subparagraph (i) of the first aspect of the present invention, so that linear peptides with two "free" sulfhydryl groups are obtained. These linear peptides can then be recyclized using a bis-electrophilic reagent, as explained herein above.

[0067] According to another preferred embodiment of the first aspect of the present invention, said agent is selected from 1,3-propanedithiol, 1,4-butanedithiol, 2,4-pentanedithiol, ethane-1-thiol, propane-1-thiol, butane-1-thiol, propane-2-thiol, 2-methyl-1-propanethiol, butane-2-thiol, 2-methylpropane-2-thiol, 2-hydroxy-1-ethanethiol, 1,2-ethanedithiol, 2-propene-1-thiol, 3-methyl-1-butanethiol, thiophenol, benzylthiol, 2-butene-1-thiol, 3-butene-1-thiol, 2-methyl-2-propene-1-thiol and 3-methyl-2-butene-1-thiol, and is preferably 1,3-propanedithiol, 1,4-butanedithiol or 2,4-pentanedithiol, and most preferably is 1,4-butanedithiol (BDT), and / or said base is selected from a tertiary, secondary or primary amine, boron, aluminum or silicon hydride and a base with an oxygen, nitrogen or carbon anion and preferably is a tertiary, secondary or primary amine, more preferably a tertiary amine, even more preferably a trialkylamine and most preferably N,N-diisopropylethylamine (DIPEA),

[0068] Preferred examples of reducing agents that can be removed by evaporation are shown in Table 1.

[0069]

[0070]

[0071] 1,4-Butanedithiol (BDT) is used as the said agent in the accompanying examples, and therefore it is the most preferred agent.

[0072] Preferred examples of bases for deprotonation of sulfhydryl groups to induce disulfide exchange and peptide cleavage with cyclization are shown in Table 2.

[0073]

[0074]

[0075]

[0076] The list in Table 2 is not exhaustive, but provides examples of suitable reagents of each type / subtype. Preferred are trialkyl tertiary amines, and in particular specific examples thereof. N,N-Diisopropylethylamine (DIPEA) is used as the reference base in the accompanying examples and is therefore the most preferred base.

[0077] According to another preferred embodiment of the first aspect of the present invention, said method comprises, before step (a), step (a') of synthesizing said linear dithiol peptides on said solid phase.

[0078] Solid-phase peptide synthesis (SPPS) is a common method for synthesizing peptides. In SPPS, peptides are typically synthesized from the carbonyl side (C-terminus) to the amino side (N-terminus) of the amino acid chain, although peptides are biologically synthesized in cells in the opposite direction. In peptide synthesis, an amino-protected amino acid binds to a solid-phase substance, forming a covalent bond between the carbonyl group and the solid-phase substance. The amino group is then deprotected, and the amino group reacts with the carbonyl group of the next N-protected amino acid. The solid phase now contains a dipeptide. This cycle is repeated to produce the target peptide chain.

[0079] According to another preferred embodiment of the first aspect of the present invention, the amino acid side chains of said linear dithiol peptides are protected with protecting groups, and said method further comprises, before step (a) and, if present, after step (a'), removing said protecting groups, while the linear dithiol peptides are immobilized on said solid phase.

[0080] Because amino acids contain several reactive groups, peptide synthesis must be carried out carefully to avoid side reactions that can shorten and branch the peptide chain. To facilitate the formation of peptides with minimal side reactions, chemical groups have been developed that bind to reactive groups of amino acids and block or protect the functional group from nonspecific reactions.

[0081] Generally, purified individual amino acids used for peptide synthesis are reacted with designated protecting groups prior to synthesis. Specific protecting groups are then removed from the newly added amino acid (a step called deprotection) immediately after coupling, allowing the next incoming amino acid to bind to the growing peptide chain in the correct orientation. After peptide synthesis is complete, any remaining protecting groups are removed from the resulting peptides.

[0082] In the present art, three types of protecting groups are generally used depending on the method of synthesizing peptides, and they are described below.

[0083] The N-termini of amino acids are protected with groups called "temporary" protecting groups because they are relatively easy to remove to allow the formation of peptide bonds. Two common N-terminal protecting groups are tert-butoxycarbonyl (B0e) and

[0084] 9-fluorenylmethoxycarbonyl (Fmoc) groups, and each group has different characteristics that determine its application. Removing Boe from a newly added amino acid requires a moderately strong acid, such as trifluoroacetic acid (TFA), while Fmoc is a base-labile protecting group that is removed with a weak base, such as piperidine. Boc chemistry requires acidic conditions for protecting group removal, while Fmoc, which was not reported for another twenty years, is cleaved under mild alkaline conditions. Due to the mild conditions for protecting group removal, Fmoc chemistry is more commonly used commercially due to its higher quality and yield, while Boe is preferred for the synthesis of complex peptides or when unnatural peptides or analogs that are sensitive to bases are required.

[0085] The use of a C-terminal protecting group depends on the type of peptide synthesis used; while liquid-phase peptide synthesis requires protection of the C-terminus of the first amino acid (C-terminal amino acid), solid-phase peptide synthesis does not require this because the solid support (e.g., resin) acts as a protecting group for the single C-terminal amino acid that requires protection.

[0086] Amino acid side chains represent a wide range of functional groups and, therefore, are the site of significant side chain reactivity during peptide synthesis. Consequently, a variety of protecting groups are required, although they are typically based on the benzyl (Bzl) or tert-butyl (tBu) group. The specific protecting groups that can be used during the synthesis of a particular peptide vary depending on the peptide sequence and the type of N-terminal protection used. Side chain protecting groups are known as permanent protecting groups because they can withstand several cycles of chemical treatment during the synthesis phase and are removed only by treatment with strong acids after completion of the synthesis.

[0087] According to a further preferred embodiment of the first aspect of the present invention, at least some of said linear dithiol peptides contain a primary or secondary amine, and said method further comprises modifying said primary or secondary amine using a carboxylic acid, wherein the cyclic peptides containing a primary or secondary amine and carboxylic acids are preferably transferred by acoustic dosing.

[0088] By modifying said primary or secondary amine using a carboxylic acid, the complexity of the peptide library can be further increased; that is, the number of different peptides in said library can be significantly increased. This, in turn, increases the chances of identifying an ideal inhibitor or binding molecule when said peptide library is screened for a substance that binds to a specific target molecule or an inhibitor of a specific target molecule. This will be explained in more detail in connection with the second aspect of the present invention below.

[0089] Acoustic dosing is a liquid transfer method that enables reactions to be carried out in very small volumes, as illustrated in Example 3, in volumes as small as 80 nanoliters. Acoustic dosing enables contactless, high-precision, high-speed liquid handling by transferring liquids using acoustic ultrasound energy.

[0090] Modification of said primary or secondary amine using a carboxylic acid, wherein the cyclic peptides comprise a primary or secondary amine and carboxylic acids, is also the subject of the second aspect of the present invention described herein below. The preferred embodiments and definitions of the second aspect of the present invention apply mutatis mutandis to the above additional preferred embodiment of the first aspect of the present invention.

[0091] According to another preferred embodiment of the first aspect of the present invention, said method further comprises (c) bringing said peptide library, preferably without prior purification thereof, into contact with a target molecule and (d) screening said peptide library to identify a peptide that binds to said target molecule and preferably inhibits it.

[0092] Means and methods for screening a peptide library for a peptide that binds to and modulates a specified target molecule, and preferably inhibits said target molecule, are known in the art. In a preferred embodiment, said target is an amino acid-based target, such as a protein. In general, said target may also be a non-amino acid-based compound, such as an oligo- or polynucleotide.

[0093] In this regard, the term "inhibition of a target molecule" means that biological activity is inhibited and preferably completely eliminated. Biological activity is the ability of a specific molecular structure to achieve a specific biological effect on a target. It is measured in terms of the potency or concentration of a given molecular structure required to achieve the specified effect. Biological activity is determined using a biological assay.

[0094] According to a more preferred embodiment of the first aspect of the present invention, steps (c) and (d) are carried out in the same wells in which said primary or secondary amine has been modified using a carboxylic acid.

[0095] This approach saves both materials and time. These wells can be in a multi-well plate format, such as a 96-well plate, a 384-well plate, or a 1536-well plate.

[0096] According to another more preferred embodiment of the first aspect of the present invention, said target molecule is a protein or a nucleic acid molecule, and is preferably a protein.

[0097] Among these embodiments, said target is preferably an amino acid-based target such as a protein.

[0098] According to another preferred embodiment of the first aspect of the present invention, said solid phase comprises a resin, preferably a non-polar resin and more preferably a polystyrene resin.

[0099] Some resins for peptide synthesis are known and commercially available. Non-limiting examples are PEGA resins, which are composed of 2-acrylamidoprop-1-yl-(2-aminoprop-1-yl)polyethyleneglycol 800, a resin of poly-ε-lysine cross-linked with sebacic acid, a resin of cross-linked hydroxyethylpolystyrene and polyethyleneglycol, a resin based on polyethyleneglycol. Any of the above-mentioned resin matrices can be functionalized with reactive groups, including, but not limited to, aminomethyl, thiomethyl, thioethyl, chloroalkyl, trityl chloride, 4-(hydroxymethyl)benzoic acid (HMBA), and Rink amide. Polystyrene resin is preferred since it is used in the examples according to the present application.

[0100] According to another preferred embodiment of the first aspect of the present invention, said linear dithiol peptides include linear dithiol peptides having a molecular weight of less than 1000 Da, preferably less than 600 Da, and / or linear dithiol peptides containing 3 or 4 amino acids or structural units, and preferably 3 amino acids or structural units.

[0101] The dithiol peptides according to the said preferred embodiment are particularly suitable for the "cleavage with reduction" according to subparagraph (i) of the claimed method. This is due to the fact that in the case of such peptides, the two thiol groups in the dithiol peptides are located close to each other, so that they cannot be effectively cleaved by cleavage with cyclization due to conformational restrictions.

[0102] In the cyclization-assisted cleavage strategy shown in Figure 1a, peptides are synthesized on solid phase via a disulfide linker and cleaved via a cyclization-assisted disulfide exchange reaction. The main limitation of cyclization-assisted cleavage is that particularly short dithiol peptides cannot be efficiently prepared due to the formation of conformationally constrained disulfide-cyclized peptides. Peptides consisting of a single amino acid and a thiol-containing structural unit on each side (a total of three structural units / amino acids), as shown in Figure 1a, are not efficiently cleaved. Even some of the peptides containing two amino acids between thiol-containing structural units (4 structural units / amino acids in total) were not cleaved efficiently if these peptides were limited in their conformational flexibility (e.g., by amino acids having constraints on the Phi and Psi angles).The limitation of the cyclocleavage approach to delivering only dithiol peptides longer than three or four structural units precludes the preparation of macrocyclic compounds with molecular weights less than approximately 600 Da, and thus macrocyclic compounds that would be particularly attractive for the development of oral or cell-penetrating drugs.

[0103] Therefore, the dithiol peptides to be used for the "cleavage with cyclization" according to subparagraph (ii) of the claimed method preferably have a molecular weight of more than 600 Da and / or contain more than 3 amino acids, more preferably more than amino acids.

[0104] In a second aspect, the present invention relates to a method for diversifying a library of macrocyclic compounds, preferably a library of cyclic peptides, wherein at least some of the macrocyclic compounds, preferably cyclic peptides, contain a primary or secondary amine, wherein said method comprises modifying said primary or secondary amine using one or more carboxylic acids.

[0105] The definitions and preferred embodiments of the first aspect of the present invention, to the extent that they are adjustable in the second aspect of the present invention, apply mutatis mutandis to the second aspect of the present invention.

[0106] The term "macrocyclic molecule" refers to a molecule wherein a series of 12 or more atoms are linked to form a macrocyclic ring or cycle. Said macrocyclic molecule is preferably a cyclic peptide as defined in connection with the first aspect of the present invention; however, said macrocyclic molecule is not necessarily based on peptides, and the macrocyclic molecule may also have a structure comprising a peptide and non-peptide components. Examples of non-peptide components include hydrocarbons, ethers, esters, amides, aryls, sugars, ketones, epoxides, and amines. Examples of non-peptide macrocycles include rapamycin, macrolide antibiotics, lorlatinib, and simeprevir.

[0107] The nature of the carboxylic acids to be used is not particularly limited, and preferred examples are shown in Figures 8c and 10a. The complexity of said library can be adjusted by using a variety of different carboxylic acids to be used.

[0108] Cyclic peptides or macrocyclic compounds to be modified are preferably brought into contact with 2 to 20 times, preferably

[0109] 3-15-fold and most preferably 4-12-fold excess of carboxylic acids, since the said excess increases the efficiency of the reaction.

[0110] The specified carboxylic acids are preferably activated carboxylic acids. An activated carboxylic acid is a derivative of a carboxyl group that is more susceptible to nucleophilic attack than the free carboxyl group, such as acid anhydrides, acyl chlorides, thioesters, and esters.

[0111] An acid activating agent can be used to activate the carboxylic acids. It follows that the method according to the second aspect of the present invention preferably includes an acid activating agent capable of activating the said one or more carboxylic acids.

[0112] The acid activating agent is preferably HBTU ((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; benzotriazoletetramethyluronium hexafluorophosphate) used in the examples of the present invention. Other suitable acid activating agents that can be used are HATU ((1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), HSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate), TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate), TPTU (O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) and DMTMM BF4 (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate).

[0113] The method according to the second aspect of the present invention preferably includes a base. Said base promotes the activation and binding of said carboxylic acid. Preferred examples of said base will be described herein below.

[0114] The cyclic peptides or macrocyclic compounds preferably contain a primary or secondary amine as a peripheral group (as illustrated in Figure 8) or a secondary amine as part of the backbone. In the case of cyclic peptides, the amino groups can be incorporated via amino acid side chains or via N-terminal amino acids.

[0115] The production of macrocycle-based ligands is currently hampered by the lack of large macrocycle libraries, although the chances of isolating a macrocycle-based ligand that binds with high affinity to a selected target from a library increase with the number of different potential binding partners in said library. The limitation of producing large macrocycle libraries is overcome by the method according to the second aspect of the present invention. The approach of said method is based on the attachment of chemically diverse moieties to the peripheral groups of structurally diverse macrocyclic backbones in a combinatorial manner. As a proof of concept, Example 3 illustrates the production of a library of 19,968 macrocycles by conjugating more than 104 carboxylic acid moieties to 192 macrocyclic backbones.The high reaction efficiency and low number of byproducts from the acylation reactions allowed high-throughput screening (HTS) of this library to be performed without prior purification. Example 3 also illustrates the successful isolation of a low-nanomolar thrombin inhibitor (K). i =44 nM) and a high-nanomolar inhibitor of the protein-protein interaction MDM2 / p53 (K i =390 nM) from the specified library.

[0116] The approach according to the second aspect of the present invention provides a dramatic increase in the speed at which macrocycles can be synthesized and screened, and is generally applicable to any target.

[0117] According to a preferred embodiment of the second aspect of the present invention, said library is in the format of the article described in connection with the first aspect of the present invention.

[0118] The preferred embodiments of the article described in connection with said first aspect apply mutatis mutandis to said second aspect. According to said preferred embodiment of the second aspect of the present invention, said cyclic peptides or macrocyclic compounds containing a primary or secondary amine are reacted with said carboxylic acids in the wells of said article.

[0119] According to a more preferred embodiment of the second aspect of the present invention, said one or more cyclic peptides, said one or more carboxylic acids, said acid activating agent and / or said base are transferred into said article by means of acoustic dosing.

[0120] More detailed information on acoustic dosing was provided above in this document in connection with the first aspect mentioned. The application of acoustic dosing is illustrated in Example 3.2, given later in this document.

[0121] Acoustic dispensing uses acoustic waves and is preferably a sonic droplet ejection technology. Acoustic dispensing offers the significant advantage of being able to transfer reagents, particularly those in nanomolar volumes, contactlessly, eliminating the need for pipetting tips, increasing transfer speed and reducing waste and costs.

[0122] According to another preferred embodiment of the second aspect of the present invention, said macrocyclic compounds, preferably cyclic peptides, are screened after diversification without prior purification.

[0123] Said screening preferably comprises (a) bringing said library of macrocyclic compounds, preferably a library of cyclic peptides, into contact with a target molecule and (b) screening said library of macrocyclic compounds, preferably a library of cyclic peptides, for a compound, preferably a peptide, that binds to said target molecule and preferably inhibits said target molecule, as described herein above in connection with the first aspect of the present invention.

[0124] In connection with the second aspect of the present invention, and in particular the above-mentioned preferred embodiment without preliminary purification, it is preferable that said library of cyclic peptides is obtained by cleavage with cyclization according to the first aspect of the present invention, or that the method according to the second aspect includes, before diversifying the library of cyclic peptides, the steps of obtaining a library of cyclic peptides according to the first aspect of the present invention.

[0125] Furthermore, the cyclic peptide library obtained by the cleavage with cyclization according to the first aspect of the present invention can be screened without prior purification. Therefore, if said cyclic peptide library was obtained by the cleavage with cyclization according to the first aspect of the present invention, or the method according to the second aspect includes the steps of obtaining a cyclic peptide library according to the first aspect of the present invention before diversifying the cyclic peptide library, said cyclic peptide library can be obtained and diversified without the need for a purification step before screening.

[0126] Therefore, according to a more preferred embodiment of the second aspect of the present invention, said macrocyclic compounds, preferably said cyclic peptides, are modified using said one or more carboxylic acids and screened in the same wells of said article in which they were synthesized.

[0127] This can be achieved by transferring said target molecule (e.g., a protein) and, optionally, other necessary reagents for the screening assay and binding measurement, for example, using a plate reader, into the wells. In this context, cyclization is preferably carried out in accordance with the cyclization-cleavage method according to the first aspect of the present invention.

[0128] According to another preferred embodiment of the second aspect of the present invention, said base is 1,4-diazabicyclo[2.2.2]octane (DABCO), sodium salt of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or N-methylmorpholine (NMM), and most preferably DABCO.

[0129] Although volatile bases such as DIPEA can also be used, volatile bases may evaporate during transfer, particularly in the form of 2.5 nL droplets under the action of acoustic waves. Therefore, the use of non-volatile bases such as DABCO (1,4-diazabicyclo[2.2.2]octane) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) sodium salt is preferred in the present invention. Among the volatile bases, NMM (N-methylmorpholine) was found to be suitable when used in greater excess. The best results were obtained using DABCO; see Example 3.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present description, including definitions, will prevail.

[0131] With regard to the embodiments characterized in the present application, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) on which said dependent claim depends. For example, in the case of independent claim 1, which sets forth 3 alternatives A, B and C, dependent claim 2, which sets forth 3 alternatives D, E and F, and claim 3, dependent on claims 1 and 2, and which sets forth 3 alternatives G, H and I, it should be understood that the said description unambiguously discloses embodiments corresponding to the combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless otherwise specifically stated.

[0132] Similarly, and in cases where independent and / or dependent claims do not set out alternatives, it is understood that if dependent claims refer to a plurality of preceding claims, any combination of the subject matter covered by them is considered to be clearly disclosed. For example, in the case of independent claim 1, dependent claim 2 referring to claim 1, and dependent claim 3 referring to claims 2 and 1, it follows that the combination of the subject matter of claims 3 and 1 is clearly and unambiguously disclosed, as is the combination of the subject matter of claims 3, 2 and 1. In the case where there is another dependent claim 4 that refers to any of claims 1-3, it follows that the combination of the subject matter of claims 4 and 1, claims 4, 2 and 1, claims 4, 3 and 1, as well as claims 4, 3, 2 and 1 are clearly and unambiguously revealed.

[0133] The above considerations apply mutatis mutandis to all appended claims. Brief description of the drawings.

[0134] Figure 1. Disulfide bridge-mediated cleavage-reduction strategy for solid-phase synthesized peptides. (a) The newly developed “disulfide cleavage-cyclization” reaction. Short peptides, such as those containing only one amino acid between two flanking thiol-containing structures (three structural units), are unable to undergo efficient cleavage due to conformational constraints (indicated by dashed lines). (b) Cleavage of short dithiol peptides by disulfide bridge reduction.

[0135] Figure 2. Reductive cleavage of disulfide-linked peptides attached to the solid phase. (a) Chemical structures of four peptides used to test reductive cleavage of peptides from the solid phase. (b) HPLC chromatograms of the indicated four peptides cleaved by disulfide bridge reduction using BDT (100 mM) in DMF containing TEA base (100 mM TEA) (left). Exposure of the same immobilized peptides to TEA (100 mM) in DMSO, the conditions previously used to separate dithiol peptides via cyclization cleavage, eluted only small amounts of disulfide-dimerized peptide (right). Target peptide peaks are highlighted in red. The only observed by-product is a peptide dimerized through a disulfide bridge (dimer).

[0136] Figure 3. Reducing cleavage of dithiol peptides attached to the solid phase via a disulfide bridge. (a) Structure of dithiol peptides on the solid phase. (b) HPLC chromatograms of eight dithiol peptides cleaved by disulfide bridge reduction using BDT (100 mM) in DMF containing TEA base (100 mM). Target peptides are highlighted in red. BDT and byproducts are indicated.

[0137] Figure 4. Cyclization of dithiol peptides using bis-electrophilic reagents, (a) Cyclization reaction using Mpa-Trp-Mea and 1,3-bis(bromomethyl)benzene reagent (1) as an example. (b) Bis-electrophilic reagents 2-10. (c) Chemical structures of the macrocycles and HPLC analysis of the reaction mixtures during cyclization. The target cyclic products are highlighted in red. The si-s7 byproducts are shown in Supplementary Figure 7. L = bis-electrophilic cyclization reagent. L* represents hydrolyzed L.

[0138] Figure 5. Disulfide elimination strategy with cyclization, (a) Schematic representation of the strategy. Short peptides are synthesized via a disulfide bridge on the solid phase. Removal of the protecting groups (highlighted in red) on the solid phase allows for efficient elimination. Base treatment deprotonates the N-terminal thiol, which induces intramolecular disulfide exchange to yield the cyclic product. (b) Chemical structure of the studied peptide Mpa-Gly-Gln-Trp-Mea linked to the solid support via a disulfide, and the commercial resins used, (c) Yield of the disulfide-cyclized peptide Mpa-Gly-Gln-Trp-Mea synthesized on resins 4 and 5 and cleaved using 150 mM DIPEA in DMSO. Concentrations were determined by measuring optical density at 280 nm. Reactions were performed in triplicate.(d) The purity of the disulfide-cyclized peptide is shown in panel d. Purity was assessed by LCMS by determining the AUC of all compounds for UV absorbance at 220 nm.

[0139] Figure 6. Cleavage with cyclization of peptides with variable sequences. (a) Chemical structures of four target cyclic peptides. The peptides have a linear sequence Mpa-Gly-Ala-Xaa-Mea, where Xaa is an amino acid with variable conformational flexibility in the backbone.

[0140] (b) Analytical HPLC chromatograms of the crude peptide after cyclization cleavage. The chromatograms on the right show the peptides after disulfide bond reduction with TCEP. For impurities that were not identified, the mass is indicated (assuming the species is singly charged).

[0141] (c) Examples of chemical structures corresponding to the molecular weights of the identified impurities.

[0142] Figure 7. Library design, peptide yield quantification by optical density, and thrombin inhibition by a cyclic peptide library, (a) Design of the library containing 96 different peptides and the structures of the unnatural amino acids used in the library. (b) Scatterplot of cyclic peptide concentrations (mM in DMSO) for the 96 synthesized cyclic peptides quantified by optical density at 280 nm. The plot shows the average yield, as well as 1.5-fold and 0.67-fold values, (c) Thrombin inhibition determined at an average cyclic peptide concentration of 11 μM. Peptides are ordered according to their thrombin inhibition activity in the first screening (black dots; from highest to lowest activity). Green dots indicate thrombin inhibition for the same cyclic peptides determined in a second screening using the same conditions.The chemical structure of the most active inhibitor (K) is shown. i =13±1 μM).

[0143] Figure 8. Diversification of macrocycles by combinatorial attachment of moieties to peripheral groups. a, General principle of the method. b, A model macrocycle containing a peripheral primary amine is modified by acylation. c, Reaction of the model macrocycle with the indicated acids. The top number shows the conversion in a 4 μL volume by pipetting, and the bottom number in an 80 nL volume by acoustic liquid dispensing, the first number using DIPEA and the second number using DABCO. Images of two droplets in a 96-well plate are shown to demonstrate the difference in scale. The droplets contain fluorescein and are exposed to UV light for visualization.

[0144] Figure 9. Preparation of macrocycles containing a peripheral amino group, a, Disulfide elimination with cyclization for peptides with an unprotected side chain. b, Format of the backbones in library 1. c, Amino acids used for the synthesis of the backbone library, d, Yields of 45 tryptophan-containing backbones according to optical density measurements.

[0145] Figure 10. Screening of a macrocyclic compound library against thrombin and identification of hit compounds, a, Carboxylic acids 9-16 used along with acids 1-3 to diversify the scaffold library 1a-f (shown in Figure 2). b, Schematic representation of the macrocycle library synthesis procedure using acoustic liquid dosing. Reaction conditions are indicated, c, Heat map showing thrombin inhibition for each macrocycle. The amino acid composition of all macrocycles is listed in Supplementary Table 1. d, Replica reaction and screening of all compounds containing acid 14. e, Chemical structures and activities of the three top hit compounds M1-M3. Mean values ​​and SD of three independent determinations are shown, f, Chromatographic separation of the reaction mixture upon acylation to give M1 and analysis of fractions to identify thrombin inhibitory substances.

[0146] Figure 11. Thrombin screening. A total of 384 macrocycles were synthesized according to our previously described method (the Fmoc amino acids used and the corresponding single-letter codes are shown in Supplementary Fig. b). Each macrocycle was reacted with 12 carboxylic acids. After the reaction was completed and stopped, thrombin and a fluorogenic substrate were added to the wells, and the increase in fluorescence was determined over 30 min. The final macrocycle concentration was 10 μM. Residual thrombin activity was determined by dividing the slope of the fluorescence intensity versus time plot for each well by the slope for the control wells without the macrocycle.

[0147] Figure 12. MDM2-binding compounds, a, Chromatographic separation of the acylation reaction mixture to yield M6 to M8 and analysis of fractions to identify thrombin inhibitory compounds. b, Binding of purified macrocycles to MDM2 determined by fluorescence polarization displacement analysis of the MDM2 fluorescent probe (linear peptide). The mean values ​​and SD of three independent determinations are shown. c, Chemical structures of fluorescein (F)-labeled macrocycles and binding to MDM2 determined by fluorescence polarization. The mean values ​​and SD of three independent determinations are shown.

[0148] Figure 13. Macrocycle diversification by combinatorial addition of fragments to peripheral groups, a, Chemical structures of macrocyclic compounds, all containing an amino group. b, Carboxylic acids used for macrocycle diversification, c, Reaction yields.

[0149] Figure 14. Synthetic strategy for the Ro5 edge macrocycle library. (a) The previous macrocycle synthesis strategy and the improved strategy presented here. Circles represent three groups of building blocks: amino acids (gray), cysteamine and analogs (red), and bis-electrophilic linkers (white). (b) Schematic representation of the cyclization strategy for linear peptides by reacting terminal thiol groups with bis-electrophilic reagents. The chemical structures of the newly developed cysteamine analogs used as C-terminal building blocks in the linear precursors are shown. (c) Synthetic strategy for polystyrene resin bearing cysteamine analogs linked via a disulfide bridge. The cysteamine analogs are added to the resin as activated thiosulfonate building blocks.

[0150] Figure 15. Synthesis of phenylsulfone-activated cysteamine building blocks and purification without a chromatography step, (a) Schematic representation of the synthesis of dithiol resins. X=Br or Cl (b) Simplified representation of the synthesis of dithiol peptides without a purification step, (c) Pooled HPLC (UV220) chromatograms characterizing the quality of the crude peptide for a model peptide (MPA-Trp-Ala) synthesized using 7 different resin building blocks.

[0151] Figure 16. Design of a macrocycle library tailored for the generation of trypsin-like serine protease inhibitors. (a) Format of the designed cyclic peptide library. (b) Amino acids and linkers used for library synthesis, (c) Histograms of selected properties of the resulting macrocycle library. Predicted physicochemical properties of the macrocycle library calculated using DataWarrior (ver. 5.5.0). The region in green corresponds to go5 and / or lies in the range predicted to correspond to the ability of the macrocycles to enter the cell.

[0152] Figure 17. Experimental stages during library preparation. Process of synthesizing macrocycle libraries in microtiter plates.

[0153] Figure 18. Affinity optimization of the MDM2:p53 inhibitor. a, Scaffolds of M8-based library 3, where amino acids highlighted in blue are diversified. The amino acid structural units are shown in three boxes. b, Heat maps of MDM2 binding to 63 scaffolds that were combinatorially acylated with 15 carboxylic acids at a 30 pmol scale. Binding to MDM2 was determined by a fluorescent peptide probe displacement assay with macrocycles at a concentration of 750 nM. c, Screen of library 4 based on the nine best scaffolds from previous screens and 15 additional carboxylic acids, d, Binding of fluorescein-labeled and HPLC-purified macrocycle M10 (F-M10) to MDM2 determined by FP. The mean values ​​and SD of three independent determinations are shown. e, Binding of unlabeled macrocycles M8 and M10 to MDM2 determined by SPR.

[0154] Figure 19. Comparison of acylation reactions by pipetting (4 μL volume) and acoustic dosing (80 nL volume) using DIPEA as a base. The reaction mixtures were diluted 100-fold with water, and 5 μL samples were analyzed by LC-MS using an RP column and a 0-60% MeCN / H2O gradient over 5 minutes. For a reaction volume of 80 nL, the two samples were pooled.

[0155] Figure 20. Acylation reactions in 80 nL volumes using acoustic dosing and different bases. Two non-volatile bases (DABCO and sodium HEPES) at 80 mM and a volatile base (NMM) at 500 mM were tested as alternatives to 80 mM DIPEA in the acylation of a model backbone with three carboxylic acids. Reaction mixtures were diluted 100-fold with water, and 5 μL samples (the two combined reaction mixtures) were analyzed by LC-MS using an RP column and a 0-60% MeCN / H2O gradient over 5 min. While reactions with DIPEA did not go to completion, all other bases provided quantitative conversion to product.

[0156] Figure 21. Comparison of acylation reactions in 80 nL volumes using acoustic dosing and DABCO as a base. Model scaffold 1 was reacted with carboxylic acids 1–8 in 80 nL volumes using DABCO as a base (80 mM). The reaction mixtures were diluted with water 100-fold, and 5 μL of the analyzed samples (the two combined reaction mixtures) were analyzed by LC-MS using an RP column and a 0-60% MeCN / H2O gradient over 5 min.

[0157] Figure 22. Acylation of model scaffolds 2–5 in 80 nL volumes using acoustic dosing. a, LC-MS analysis of model scaffolds. Reaction mixtures were diluted 100-fold with water, and 5 μL samples were analyzed. Solvent gradient B: 0–60% MeCN, 5 min for model scaffold 2; 10–100% MeCN, 5 min for model scaffolds 3–5. b, LC-MS analysis of acids. Reaction mixtures were diluted 100-fold with water, and 5 μL samples were analyzed. Solvent gradient B: 0–60% MeCN, 5 min. TMU = tetramethylurea. cf, UHPLC analysis of reaction mixtures during the acylation of model scaffolds 2(c), 3(d), 5(e), and 5(f). Reaction mixtures were diluted 100-fold with water, and 5 μL samples were analyzed. Solvent gradient B: 0-60% MeCN, 5 min for model scaffold 2; 10-100% MeCN, 5 min for model scaffolds 3-5.

[0158] Figure 23. Preparation of cyclic peptide backbones containing an N-terminal amino group, a, Six different backbone formats. b, Amino acids used for backbone synthesis.

[0159] Figure 24. Physicochemical properties of Library 1 (screened against thrombin) and Library 2 (screened against MDM2). The properties were calculated using DataWarrior software. Regions meeting the Kihlberg rules for permeability (P. Matsson et al., Adv. Drug Deliv. Rev. 2016, 101, 42–61; B. Doak et al., Chem. Biol. 2014, 21, 1115–1142) are colored green. The majority of both libraries are within the range predicted to be cell permeable. MW=molecular weight, cLogP=estimated n-octanol / water partition coefficient, HBD=hydrogen bond donors, HBA=hydrogen bond acceptors, PSA=polarized surface area, NRotB=number of rotatable bonds, a, Library 1 (screened against thrombin). b, Library 2 (screened against MDM2).

[0160] Figure 25. Thrombin inhibitors M1-M5. a. Chemical structures and analytical HPLC chromatograms obtained using a 0 100% MeCN / H2O gradient over 15 min. b. For all macrocycles, a two-fold serial dilution was prepared to yield 18 spots in 50 μL volumes. Thrombin (50 μL, final conc. 2 nM) was added, and after 10 min, the fluorogenic substrate Z-Gly-Gly-Arg-AMC (50 μL, final conc. 50 μM) was added. The increase in fluorescence was determined over 30 min. Residual thrombin activity was determined by dividing the slope of the fluorescence intensity versus time plot for each well by the slope for the control wells without the macrocycle. The mean values ​​and SD of three independent determinations are shown.

[0161] Figure 26. Structure of M1 bound to thrombin, a, X-ray crystallographic structure of M1 bound to thrombin. M1 is enlarged and H-bond interactions are shown. b, Chemical structure of M1 and H-bond interactions formed with thrombin.

[0162] Figure 27. Scaffolds synthesized for library 2 (screening against MDM2). a, Scaffold format in library 2. b, Amino acids used to synthesize the scaffold library. All combinations of four diamino acids, four backbone amino acids, two side chain amino acids, and six sublibrary formats were synthesized. c, Yields of tryptophan-containing scaffolds after cyclization cleavage. The average scaffold concentration was 12.9 mM, as determined by optical density in the nanodroplet. The average purity determined by LC-MS was approximately 90%.

[0163] Figure 28. Overview of carboxylic acids used to acylate peripheral amines in cyclic peptide backbones.

[0164] Figure 29. Probing the M10 binding site using competition binding experiments, a, Displacement of the linear peptide probe FP53 by M10 and nutlin-3a as determined by fluorescence polarization. b, Displacement of the F-M10 macrocycle probe by M10 and nutlin-3a.

[0165] Figure 30. Macrocycle binding to MDM2 determined by SPR. a, Single-cycle SPR sensograms for fluorescein-labeled macrocycles, the positive control nutlin-3a (not fluorescein-labeled), and negative controls (thrombin inhibitors; not fluorescein-labeled). RU, response unit. b, Single-cycle SPR sensograms for macrocycles M6, M7, M8, and M10 (not fluorescein-labeled), performed in triplicate.

[0166] The present invention is illustrated by examples.

[0167] Example 1 - Elimination with Reduction

[0168] 1.1. Cleavage with reduction of disulfide-immobilized peptides from the solid phase.

[0169] We first assessed whether peptides immobilized on solid supports via a disulfide bridge could be quantitatively cleaved using a volatile reducing agent. For this purpose, four peptides were synthesized: Ala-Trp-Mea, Trp-Ala-Mea, Ala-Tyr-Mea, and Tyr-Ala-Mea (Mea = 2-mercaptoethylamine, also known as cysteamine), shown in Figure 2a. These peptides contain a tryptophan or tyrosine residue, allowing for precise determination of the amount of cleaved peptide by measuring the absorbance at 220 or 280 nm. The present inventors did not consider the N-terminal thiol group found in dithiol peptides to quantify peptides that were cleaved by disulfide bridge reduction rather than by some other mechanism such as disulfide exchange with cyclization.Peptides were synthesized on polystyrene (PS) resin, which has a high loading capacity (approximately 1 mmol / gram) and is commonly used for peptide synthesis. A disulfide bridge was first introduced by incubating the thiol PS resin with excess pyridyldithioethylamine, and the peptides were synthesized using a standard Fmoc chemistry strategy. All peptides were synthesized in 96-well plates at a 5 μmol scale to validate the conditions under which high-throughput dithiol peptide libraries were later synthesized.

[0170] Cleavage of a disulfide-immobilized peptide from a PS resin was tested by incubating the beads overnight with 200 µL of DMF containing 20 equiv. β-Me (0.5 M) and 20 equiv. trimethylamine (TEA; 0.5 M). LC-MS analysis of the peptides demonstrated efficient cleavage, but also that approximately 40% of the product was present as a disulfide adduct with β-Me. It was hypothesized that the adduct content could potentially be reduced by using a larger molar excess of β-Me and / or repeating the reduction, but this would require additional process steps and was therefore not the most desirable route. To cleave peptides from resins and eliminate disulfide adducts in a single step, the use of a reducing agent such as dithiothreitol (DTT), which does not form disulfide adducts because it is eliminated by the formation of cyclic DTT, was proposed. Incubation of the resin carrying the Ala-Trp-Mea or Tyr-Ala-Mea peptide with 4 equiv.DTT provided efficient peptide cleavage without the formation of peptide-reducing agent adducts, but DTT could not be removed by vacuum evaporation in a standard Speedvac concentrator. A similar reducing agent that evaporates at 195°C and therefore has a lower boiling point is 1,4-butanedithiol (BDT) (PubChem, https: / / pubchem.ncbi.nlm.nih.gov / compound / l 4-Butanedithiol, accessed 20.04.21). 5 μmol of resin-bound peptides were incubated with 200 μL of DMF containing 4 equiv. BDT (100 mM) and 4 equiv. TEA (100 mM). As a control, parallel reactions were performed in which four resin-bound peptides were treated under cyclization cleavage conditions, namely, 250 mM TEA in DMSO. LC-MS analysis showed that the peptides were efficiently cleaved using BDT. A major peak corresponding to the target product was observed for all four peptides (Figure 2b).The only by-product detected for the two peptides was a peptide dimer, which was present in small amounts, less than 3%. The yields of the target products were 3.4, 1.3, 4.2, and 4.3 μmol, respectively, corresponding to yields of 68, 25, 84, and 86% (assuming that the amount of peptide synthesized on the beads was 5 μmol).

[0171] 1.2. Cleavage with reduction of short dithiol peptides from the solid phase

[0172] A panel of eight short dithiol peptides of the Mpa-Xaa-Mea format (Mpa = mercaptopropanoic acid) was then synthesized, where the Xaa amino acids were Trp, Tyr, Ser, His, Phe, Arg, Asp, and Ala. These peptides were expected to be cleaved by disulfide bridge reduction, but cleavage via disulfide cyclization was ineffective. As a control, a longer peptide, Mpa-Lys-Trp-Gly-pAla-Mea, was also synthesized, which was expected to be efficiently cleaved via disulfide cyclization. Incubation of the resins with the reducing agent BDT resulted in efficient cleavage of all peptides (Figure 3). The target dithiol peptides were the main products. By-products were detected only in small amounts and were dithiol peptides containing trityl and tert-butyl protecting groups (Figure 3).Incubation of the resin with TEA in DMSO for cyclization cleavage yielded short peptides in approximately 10- to 100-fold lower quantities and resulted in more byproducts. As expected, the longer peptide Mpa-Lys-Trp-Gly-pAla-Mea was efficiently cleaved via the cyclization cleavage mechanism, most likely due to less conformational constraint. The yields of Mpa-Trp-Mea and Mpa-Trp-Mea peptides, which could be quantified by determining the absorbance at 280 nm, were 3.6 and 4.3 μmol, respectively, corresponding to yields of 71% and 85%, respectively, assuming the peptides were present on the resin at 5 μmol.

[0173] 1.3. Evaporation of solvent and reducing agent in vacuum

[0174] Next, it was tested whether the reducing agent BDT could be removed by vacuum centrifugation. Peptides synthesized at a 5 μmol scale and cleaved in 200 μL of DMF containing 100 mM BDT and 100 mM TEA were centrifuged at 0.1 mbar, 30°C, and 400 × g in the wells of 96-well plates. The solvent was effectively removed within 1 hour if all wells of the microplate were filled. According to LC-MS analysis of the peptides, all BDT was removed. However, for some peptides, it was also found that up to 10% of the product was back-oxidized. It was hypothesized that the high pH allowed this oxidation, and therefore 2 equiv of TEA were added to each well before vacuum centrifugation. TFA relative to TEA (200 mM TFA). Using this procedure, the proportion of oxidized peptide could be effectively reduced.

[0175] 1.4. Cyclization of dithiol peptides using bis-electrophilic reagents

[0176] We examined whether cyclization of the short dithiol peptides Mpa-Trp-Mea and Mpa-Tyr-Mea could be achieved using bis-electrophilic reagents, as shown in Figure 4a. Cyclization of peptides through two or three cysteine ​​residues using electrophilic linker reagents occurs highly efficiently and cleanly when performed at low concentrations, with peptide concentrations of approximately 1 mM (or lower) and using a slight excess of the cyclization reagents (P. Timmerman et al., ChemBioChem, 2005, 6, 821–824; SS Kale et al., Nat. Chem., 2018, 10, 715–723). Peptides were dissolved in 1 mL of 1:1 acetonitrile:water, 3 mL of 90% NH4HCO3 buffer (100 mM, pH 8.0), 10% acetonitrile were added, and 1 mL of bis-electrophilic reagents in acetonitrile (10 mM) was immediately added. A total of ten bis-electrophilic reagents were tested, shown in Figure 4b. The final concentrations of the peptide and cyclization reagent were approximately 1 mM and 2 mM, respectively.HPLC chromatograms of the reaction mixtures during the cyclization of the Mpa-Trp-Mea peptide are shown in Figure 4c. In most of the reactions, cyclization of the dithiol peptides occurred almost quantitatively with yields exceeding 90%. Small amounts of byproducts were mainly peptides that reacted with only one thiol group, since one of them was protected by trityl. To block excess bis-electrophilic reagents, 6 equivalents (relative to the peptide) of β-Me were added; it interacted with these reagents but did not affect the macrocycles.

[0177] 1.5. Discussion

[0178] This example defines a solid-phase peptide synthesis and elution strategy that yields high-purity dithiol peptides at high concentrations, making them readily cyclizable with bis-electrophilic reagents for the synthesis of macrocyclic compounds and libraries. The key elements of the strategy are: i) peptide synthesis using a disulfide linker, ii) solid-phase deprotection of side chains, iii) peptide cleavage by disulfide reduction, and iv) the use of a volatile reducing reagent that can be removed by evaporation, enabling cyclization with bis-electrophilic reagents. The elimination of purification steps after dithiol peptide synthesis and cyclization with bis-electrophilic reagents provides access to a large number of macrocyclic compounds and their application in high-throughput screening.

[0179] 1.6. Materials and Methods

[0180] Synthesis of 2-(2-pyridyldithio)ethylamine hydrochloride

[0181] To a stirred solution of 2,2'-dipyridyl disulfide (4.41 g, 20 mmol) in MeOH (16 mL supplemented with 2% [v / v] AcOH) was added dropwise cysteamine hydrochloride (1.14 g, 10 mmol) dissolved in MeOH (10 mL supplemented with 2% [v / v] AcOH) over 15 min. The reaction mixture was stirred overnight at room temperature (rt) and concentrated under reduced pressure to give a yellow fatty oil. The residue was dissolved in MeOH (16 mL), distributed into eight 50 mL Falcon tubes, and precipitated by adding ice-cold diethyl ether (48 mL to each tube). The tubes were incubated at -20°C for 30 min and centrifuged at 3400 × g (4000 rpm in a Thermo Scientific Heraeus Multifuge 3L-R centrifuge with a Sorvall 75006445 rotor, radius = 19.2 cm, explosion-proof) at 4°C for 30 min. After repeating the sedimentation eight times, a colorless product was obtained (yield = 90%).

[0182] Preparation of cysteamine polystyrene resin

[0183] The following procedure describes the preparation of a polystyrene resin containing approximately 2 mmol of cysteamine immobilized via a disulfide linker, which is sufficient for the synthesis of 4 × 96 peptides at a 5 μmol scale. 563 mg of resin (Rapp Polymere Polystyrene A SH resin, 200-400 mesh, loading 0.95 mmol / gram) was added to each of four 20 mL plastic syringes. The resin was washed with DCM (15 mL) and then swelled in MeOH / DCM (7:3; 15 mL) for 20 min. Pyridylcysteamine disulfide (2.10 grams, 9.42 mmol, 4.4 equiv) was dissolved in MeOH (23 mL) and then in DCM (53 mL). N,N-Diisopropylethylamine (DIPEA; 410 μL) was then added. 19 mL of this solution was drawn into each syringe, and the syringes were then shaken at RT for 3 h. The pyridylcysteine ​​solution was discarded, and the resin was washed with MeOH / DCM (7:3; 2 × 20 mL), then DMF (2 × 20 mL).The resin was combined into a single syringe as a suspension in DMF and washed with a solution of 1.2 M DIPEA in DMF (11.8 mL) for 5 min to ensure neutralization of all amines. This solution was discarded, and the resin was washed with DMF (2 × 20 mL), then DCM (4 × 20 mL), then stored under vacuum overnight to obtain a free-flowing powder.

[0184] Fmoc-Mediated Peptide Synthesis in 96-Well Plates Peptides were synthesized at 5 μmol scale in 96-well peptide synthesis filter plates (Orochem, catalog #OF1100) using an automated peptide synthesizer (Intavis MultiPep RSi). Cysteamine-PS resin (approximately 5 mg, 0.95 mmol / g, 5 μmol scale) was dispensed as a powder into each well of the plate. The resin was washed with DMF (3 × 225 μL). In this and all subsequent wash steps, the resin was incubated for 1 min. The following reagents were transferred to the tubes in the listed order, mixed, incubated for 1 min, transferred to the resin in the microwell plate, and incubated for 45 min without shaking. Reagents: 50 µl HATU (500 mM in DMF, 5 equiv), 5 µl N-methylpyrrolidone (NMP), 12.5 µl N-methylmorpholine (NMM in DMF, 4 M, 10 equiv), and 53 µl amino acid (500 mM in DMF, 5.3 equiv).The final volume of the coupling reaction mixture was 120.5 μL, and the final concentrations of the reagents were 208 mM HATU, 415 mM NMM, and 220 mM amino acid. The coupling was performed twice. The resin was washed with DMF (1 × 225 μL). Unreacted amino groups were blocked by incubation with 5% acetic anhydride and 6% 2,6-lutidine in DMF (100 μL) without shaking for 5 min. The resin was washed with DMF (8 × 225 μL). Fmoc groups were removed by incubation twice for 5 min with DMF (120 μL) containing 20% ​​(v / v) piperidine without shaking. For the synthesis of longer peptide sequences, the incubation time was reduced from 5 min to 2 min to reduce base exposure. The resin was washed with DMF (8 × 225 μl). After completion of peptide synthesis, the resin was washed with DCM (2 × 200 μl).

[0185] Deprotection of Peptide Side Chains in 96-Well Plates To deprotect the amino acid side chains as well as Mea, the bottom of the 96-well synthesis plate was sealed by pressing the plate onto a 6 mm thick soft ethylene vinyl acetate backing, and the resin in each well was incubated with a TFA:TIPS:H2O solution (95:2.5:2.5 [v / v / v], approximately 300 μL). The plates were covered with an adhesive sealing film (iST scientific, QuickSeal Micro, catalog number IST-125-080-LS) and then pressed down with a weight (1 kg) placed on top to prevent leakage. After incubation for 1 h, the synthesis plate was placed on a 2 mL deepwell plate, and the TFA mixture was allowed to drain off. The synthesis plate was resealed, and the deprotection procedure was repeated. The wells were washed with DCM (3 x 500 µL; added via syringe), which was gravity-fed. The resin was dried by placing the synthesis plate in a vacuum manifold for 5 min.

[0186] Cleavage of peptides with reduction using β-Me, DTT or BDT

[0187] To cleave the peptides from the resin, the bottom of the 96-well synthesis plate was sealed by pressing the plate onto a 6 mm thick soft ethylene vinyl acetate backing, and the resin in each well was incubated with 200 μL DMF solution containing 500 mM β-Me, or 100 mM DTT, or 100 mM BDT, and 100 mM TEA for 4 h at RT. After this time, samples were collected in a 96-well deep-well plate by centrifugation at 250 × g (1100 rpm in a Thermo Scientific Heraeus Multifuge 3L-R centrifuge with a Sorvall 75006445 rotor, rotor radius = 19.2 cm) for 2 min at RT.

[0188] Cleavage of peptides with cyclization

[0189] A 96-well synthesis plate was sealed as described above, and peptides were cleaved from the resin by incubation with 200 μL DMSO containing 250 mM TEA (10 equiv) overnight at RT. After this time, samples were collected in a 96-well deep-well plate by centrifugation at 250 × g (1100 rpm in a Thermo Scientific Heraeus Multifuge 3L-R centrifuge with a Sorvall rotor 75006445, rotor radius = 19.2 cm) for 2 min at RT.

[0190] LC-MS analysis of peptides after cleavage from the solid phase or cyclization

[0191] For peptides cleaved from the solid phase (concentration up to 25 mM in DMF or DMSO), 1 μL of peptide was diluted in 60 μL of milliQ H2O containing 0.05% formic acid. For peptides from cyclization reactions (concentration approximately 1 mM), 10 μL of the reaction mixture was mixed with 10 μL of milliQ H2O containing 0.05% formic acid. Samples (10 µL injection) were analyzed on a Shimadzu 2020 single quadrupole LC-MS system using a reversed-phase C18 column (Phenomenex Kinetex®, 2.6 µm, 100 Ǻ, 50 × 2.1 mm) and a linear gradient of solvent B (MeCN, 0.05% formic acid) to solvent A (H2O, 0.05% formic acid) from 0 to 60% over 5 min at a flow rate of 1 mL / min. Optical density was recorded at 220 nm, and masses were analyzed in positive ion mode.

[0192] Vacuum Centrifugal Evaporation of Reducing Agent and Solvent The following example describes a peptide whose concentration after reductive cleavage was 20 mM. 5 μL (0.1 μmol) of 200 μL of the peptide cleaved from the solid phase by reduction (in DMF containing 100 mM BDT and 100 mM TEA) were transferred to a well of a 96-well V-bottom plate (Ratiolab, 6018321, PP, non-sterile). 7 μL of 1% TFA in water (v / v) was added to each well to provide 2 equiv of TFA to TEA. This sample was subjected to vacuum centrifugal evaporation using a Christ RVC 2-33 CDplus IR instrument to remove solvent (DMF) and reducing agent (BDT). Samples were centrifuged at 0.1 mbar, 30°C, and 400 × g (1750 rpm in a Christ 124700 rotor with 124708 plate holder inserts, radius = 10.5 cm). After this step, peptides were no longer visible.

[0193] Peptide Cyclization

[0194] The recovered and dried peptide (0.1 μmol) was dissolved in 20 μL of 50% acetonitrile, 50% H2O to reach a concentration of 5 mM. To this solution, 60 μL of running buffer (100 mM ammonium bicarbonate, pH 8.0, containing 10% acetonitrile [v / v]) was added, followed by 20 μL of 10 mM cyclization linker in acetonitrile (2 equiv). The final concentrations in the reaction mixture were 1 mM peptide, 2 mM cyclization linker, 60 mM ammonium bicarbonate buffer, and 35% acetonitrile. The plate was covered with foil, and the reaction mixture was incubated for 2 h at RT.

[0195] Quenching of Linker Reagents in Cyclization Reaction Mixtures After completion of the cyclization reaction, 4 μL of 150 mM β-Me in acetonitrile (0.6 μmol, 6 equiv relative to the peptide) were added to the reaction mixture and incubated for 1 h at rt. Solvent (MeCN), buffer (bicarbonate), and excess β-Me were removed by vacuum centrifugation using a Christ RVC 2-33 CDplus IR instrument. Samples were centrifuged at 0.1 mbar, 30°C, and 400 × g (1750 rpm in a Christ rotor 124700 with plate holder inserts 124708, radius = 10.5 cm). After this step, the peptides were not visible.

[0196] Example 2 - Cleavage with Cyclization 2.1 Results and Discussion In the first experiment, different resins were tested for the synthesis of short peptides that were attached to the solid phase via a disulfide bridge. The peptide Mpa-Gly-Gln-Trp-Mea, where Mpa is mercaptopropanoic acid (cysteine ​​without an amino group) and Mea is 2-mercaptoethylamine (cysteamine; cysteine ​​without a carboxylic acid group), was synthesized on five different resins (Figure 5b). Two polyethylene glycol (PEG) resins, which are polar (1, 2), one PEG-modified polystyrene (PS) resin, which is also polar, and two PS resins, which are non-polar (4 and 5), were used. Resin 5 already contained a thiol group, and in the case of resins 1-4, the thiol group was introduced by amidation of trityl-protected Mpa. Disulfide-linked Mea was introduced by incubating the resins with excess 2-(2-pyridinyldithio)-ethanamine.The disulfide exchange reaction was tested in methanol (MeOH) / dichloromethane (DCM) and in DMF, in the presence of either base or acid, and conditions comprising 30% MeOH, 70% DCM, and one equivalent of N,N-diisopropylethylamine (DIPEA; compared to 2-(2-pyridinyldithio)ethanamine) were found to perform best. Three amino acids, Trp, Gin, and Gly, as well as Mpa, were coupled using standard Fmoc chemistry, and side-chain protecting groups were removed by incubating the resins with 95% TFA, 2.5% TIS, and 2.5% water for one hour.

[0197] Next, disulfide cleavage with cyclization by deprotonation of the sulfhydryl group at the N-terminus of the peptide using DIPEA as a base was tested. Incubation of the resins in DMSO with 150 mM DIPEA resulted in highly efficient cleavage for non-polar resins 4 and 5 (Figure 5c). The peptide concentrations in the eluate were 5.1 mM (resin 4) and 11.6 mM (resin 5), respectively. The amounts of cleaved peptide were 21 ± 5% (resin 4) and 44 ± 4% (resin 5) of the amount expected to be synthesized based on the resin loading. Given that the introduction of the disulfide linker was most likely not quantitative even for the resin already bearing thiol groups (resin 5), the percentage of peptide recovered via the cyclization-cleavage mechanism likely exceeded 44%. LC-MS analysis of the products showed high purities of 95±4% (resin 4) and 93±1% (resin 5) for the disulfide-cyclized peptide (Figure 5d).The only byproduct was a cyclic peptide dimer, which was detected in only small amounts, averaging 6%. The dimeric cyclic product most likely formed through disulfide-mediated transfer of one peptide to an adjacent one on the resin and subsequent cleavage with cyclization of the cyclic dimer.

[0198] To evaluate the substrate range of the disulfide cleavage-cyclization strategy, four peptides of the Mpa-Gly-Ala-Xaa-Mea format were synthesized at a 25 μmol scale (Figure 6a). Structural amino acid units were introduced at the Xaa position of three of the four peptides, imparting rigidity to the peptide backbones. HPLC analysis of the products obtained by base-induced cyclization-induced cleavage revealed a dominant peak for each of the four peptides, and MS analysis confirmed that these major products represent the target cyclic peptides. Optical density determination revealed that all four peptides were obtained at double-digit millimolar concentrations. Quantification of the product by weighing after over-lyophilization showed yields ranging from 13.5 to 26 µmol, corresponding to yields of 54% to 100% of those expected based on resin loading.For all four peptides, only a limited number of byproducts were observed, and these were detected in small amounts (Figure 6b and 6c). The major byproducts were cyclic dimers, in this case eluting as two closely spaced peaks that most likely corresponded to two possible dimers, one with units linked head-to-head / tail-to-tail and the other with units linked head-to-tail. TCEP linearization of the products and HPLC analysis revealed linear peptide products with purities of 96, 96, 100, and 92% for peptides 1–4, respectively (Figure 6b, chromatograms on the right). The TCEP linearization experiment indicates that the dimeric cyclic peptide can be removed by reduction and subsequent oxidative recircularization at concentrations that promote intramolecular cyclization.To test whether even shorter peptides could be obtained using the cleavage-cyclization strategy, the experiment was repeated with four Mpa-Ala-Xaa-Mea peptides, each shorter by one amino acid. These four peptides were also efficiently cleaved, and the main peak in the HPLC profile corresponded to the target cyclic peptide. The overall proportion of cyclic dimer was slightly higher, most likely due to less efficient circularization, which was hindered by the short scaffold and the resulting conformational constraints.

[0199] To assess whether the disulfide cleavage-cyclization strategy could be applied to library synthesis and screening, cyclic peptides were designed and synthesized in a 96-well plate at a 5 μmol scale. Ninety-six random disulfide-cyclized peptides were obtained in three formats, as shown in Figure 7a. These peptides contained three random amino acids, Xaa, flanked by Mpa and Mea. Two of the random amino acids in each peptide were chosen from four structurally distinct amino acids, providing highly diverse cyclic peptide scaffolds. One of the random amino acids was Trp or Tyr, allowing peptide yields to be quantified by determining absorbance at 280 nm.Cleavage of cyclized peptides by adding 150 mM DIPEA to 200 µL of DMSO and subsequent optical density determination revealed a high average peptide concentration of 13.3 mM and a narrow concentration distribution for 90 peptides, ranging from 8.9 mM (1.5-fold lower than average) to 20 mM (1.5-fold higher than average). Three peptides were not synthesized or cleaved to any extent. LC-MS analysis of 12 randomly selected cyclic peptides revealed a high purity, averaging 84%.

[0200] Despite the relatively small number of 96 cyclic peptides and therefore the low probability of detecting active compounds, we screened for thrombin, an important target for the development of anticoagulant therapeutics, using a compound concentration of approximately 10 μM. The most active peptide reduced thrombin activity by 52%, which was remarkable given that the peptides did not contain the amino acids Arg and Lys, which bind to the thrombin S1 specificity pocket (Figure 7c). Repeating the screening identified the same cyclic peptide, which was the most active hit compound (green dots in Figure 7c). HPLC-purified disulfide-cyclized peptide Mpa-Tyr-II-Pro-Mea inhibited thrombin with a K i13±1 μM. Small-scale screening showed that most cyclic peptides did not affect thrombin activity, indicating that no components co-elute with the peptides that would interfere with the bioassay, including DMSO and DIPEA, which are present in peptide stock solutions after cyclization cleavage. Biological screenings are typically performed at compound concentrations of approximately 10 μM, meaning that 100% DMSO and 150 mM DIPEA in approximately 10 mM peptide stock solutions are diluted 1000-fold to achieve concentrations of 0.1% and 150 μM, respectively, which are unlikely to interfere with most bioassays.

[0201] 2.2. Conclusion

[0202] Thus, the present invention develops a strategy for cyclizing peptides based on a disulfide exchange reaction, which enables the production of highly pure disulfide-cyclized peptides directly from a solid support. To our knowledge, this is the first approach in which cyclic peptide libraries are cleaved with high purity using a cleaving reagent that can be removed by evaporation, thereby enabling easy screening of peptides using biological assays without prior purification. Importantly, the yields of peptides with different sequences showed a narrow distribution, enabling screening of cyclic peptides even without concentration determination or adjustment. The proposed approach has been demonstrated to be applicable to the production of libraries containing hundreds of peptides.

[0203] Example 3. Scaling up the size of a cyclic peptide library

[0204] 3.1. Acylation of cyclic peptide backbones via peripheral amines

[0205] For combinatorial diversification of macrocycle libraries, as shown in Figure 8a, it was decided to modify amines, which serve as peripheral groups, and moieties, which are carboxylic acids. jV-acetylation reactions are efficient and selective and are widely used in the synthesis of DNA-encoded compound libraries (P.R. Fitzgerald et al., Chem. Rev., 2020). N-acetylation has also been used to diversify leader structures using a panel of carboxylic acids in solution, followed by activity screening of crude reaction mixtures (A. Brik et al., Chem. Biol., 2002, 9, 891-896) or even X-ray crystallographic analysis (M.R. Bentley et al., J. Med. Chem., 2020, 63, 6863-6875). The reaction was tested on the model cyclo(Mea-Lys-Xaa-Mpa) scaffold containing a primary amine as a peripheral group (Figure 8b) and eight structurally different carboxylic acids (Figure 8c).To efficiently convert the scaffolds to the target products, it was decided to react them with a 4-fold molar excess of carboxylic acid. Near-quantitative conversion of the scaffold to product was preferred, as the unmodified scaffold could potentially bind weakly and interfere with screening if present in greater amounts than the acylated scaffold. The excess carboxylic acid ensures the presence of unreacted carboxylic acid, which will be present during screening; however, it was believed that most carboxylic acids as such do not bind to the target due to their small size. The only expected byproducts of the acylation reaction were excess carboxylic acid, HOBt, and tetramethylurea, none of which should be incompatible with biochemical analysis methods. Cyclic peptide backbone (final conc.10 mM) were incubated with a 4-fold molar excess of the eight acids in 4 μL volumes using HBTU as the activating agent and DIPEA as the base for three hours, and complete backbone conversion was observed with almost all acids (upper numbers in Figure 8c).

[0206] 3.2. Synthesis of a Macrocycle Library in Nanoliter Volumes Next, we explored the combinatorial diversification of the same cyclic peptide backbone in 80 nL, i.e., in a 50-fold smaller volume. This step was crucial, as the library was planned to be produced at the nanomolar scale in small volumes so that micromolar amounts of the backbone, which could be easily synthesized in the wells of 96-well plates (5 μmol scale), would be sufficient for the synthesis of more than 100 macrocycles based on a single backbone. In addition, the goal was to use acoustic pipetting technology for reagent transfer, which is suitable for transferring nanoliter, but not microliter, volumes. Acoustic pipetting has the significant advantage of allowing reagents to be transferred contactlessly, eliminating the need for pipette tips, increasing transfer speed and reducing waste and costs.Using the same reaction conditions resulted in significantly lower yields (the first of the two lower numbers in Figure 8c) and required optimization of the acylation reaction. It was hypothesized that the low yields were due to the use of DIPEA, as this base is relatively volatile and is partially lost during acoustic wave dosing as 2.5 nL droplets. Tests were performed with the non-volatile bases DABCO and sodium HEPES, as well as with the volatile NMM, which has high solubility in the solvent system used and can be used at higher concentrations. Using all three bases, the macrocycles were quantitatively acylated with the three acids tested, and the use of DABCO with additional acids showed that the conditions were suitable for efficient modification of peripheral amines in cyclic peptides (the second of the two lower numbers in Figure 8c).

[0207] In the next step, acylation reactions were investigated using other macrocyclic compounds. Specifically, macrocyclic backbones were selected in which the amino groups were less exposed on the surface than in the model cyclo(Mpa-Lys-Xaa-Mea) backbone. For this purpose, four macrocyclic compounds offered by Enamine were ordered (Figure 13a). All of these compounds were non-peptide macrocyclic compounds containing structural units other than amino acids. Acylation reactions using the same reaction conditions and acids (Figure 13b) showed efficient conversion of the macrocyclic backbones into macrocycles bearing carboxylic acids (Figure 13c).

[0208] 3.3. Synthesis of cyclic peptide backbones bearing amino groups

[0209] Cyclic peptide backbones having random structures and a single peripheral amino group were then synthesized using a recently developed approach for the efficient production of large quantities of small cyclic peptides in 96-well plates. Briefly, short peptides were synthesized on solid phase and cleaved via a disulfide cyclization reaction to yield substantially pure backbones that did not require further purification (Figure 9a). The first cyclic peptide backbone library was obtained, containing three amino acids that were different: one was an amino acid with a primary amine in the side chain (selected from seven amino acids), another was an α-amino acid with a random side chain (selected from 15 amino acids), and the third had a random backbone structure (selected from six amino acids) (Figures 9b and 9c; sublibraries 1a-f).A second library was also synthesized in which primary amino groups were introduced via cysteine ​​residues. Of the 3240 (Library 1) and 540 (Library 2) different scaffolds that could theoretically be assembled combinatorially using the indicated amino acids, 384 were randomly selected and synthesized in four 96-well plates. Quantification of 45 cyclic peptides containing the Trp residue by optical density showed that most molecules were obtained in sufficient quantity (average conc. = 8.1 mM; Figure 9d). Given the relatively narrow distribution of yields, concentration normalization was not performed for further use.

[0210] 3.4. Combinatorial Synthesis of a Macrocycle Library and Screening for Thrombin

[0211] 384 scaffolds were combinatorially reacted with 12 carboxylic acids (Figure 10a) to yield 4608 different macrocycles, and this library was screened against the blood coagulation protease and therapeutic target thrombin. The thrombin inhibitor is already used in clinical practice as an antithrombotic drug, but its oral availability is limited. Structurally diverse molecules were chosen as carboxylic acids, including several moieties that could potentially bind to the thrombin specificity pockets S1 (via H-bonds) and S2 (hydrophobic interactions) (Figure 10a). Positively charged groups such as guanidines are known to bind particularly well to the S1 subsite, but they were excluded because the interest was in developing macrocycles with limited polar surface area that are uncharged and could potentially be administered orally.Essentially, the active form of the approved thrombin inhibitor contains such a positively charged group and must be formulated as a prodrug, which may result in limited oral availability. Using an acoustic pipette, 20 nL of the backbone (average 8.1 mM) and 20 nL of preactivated acid (80 mM) were combined to achieve final concentrations of approximately 4 mM backbone and 40 mM carboxylic acid (Figure 10b). The decision to use a 10-fold molar excess of carboxylic acid (as opposed to the 4-fold used previously) was made because some of the amino groups in the backbones are less accessible than the α-amino group in the model peptide described above. After five hours, the reaction was stopped overnight by adding 5 μl of 100 mM Tris buffer, 5 μl of thrombin (final concentration 2 nM) was added, and residual thrombin activity was determined using a fluorogenic substrate (5 μl).The concentration of macrocyclic compounds in the screening was approximately 10 μM. A proportion of the reaction mixtures, 0.2% (9 / 4277), inhibited thrombin by >50%, all of which were macrocycles containing chlorothiophene acid (14) (Figure 10c). Repeating all macrocycle synthesis reactions involving chlorothiophene acid (384 scaffolds × 14) and reactions with thrombin in an independent experiment revealed essentially the same top hit compounds and thus demonstrated high reproducibility for both the diversification reactions and activity screening (Figure 10d).

[0212] Chlorothiophene acid (14), which produced the majority of the hit compounds, was previously reported to serve as a binding group for the S1 subsite in the trypsin-like serine protease FXa (P. M. Fischer, J. Med. Chem., 2018, 61, 3799–3822), and therefore the hit compounds were expected to be macrocycles that direct the chlorothiophene group to the S1 pocket. Not all macrocycles modified with acid 14 inhibited thrombin, indicating that the macrocycle significantly contributes to the binding. Given the 10-fold excess of carboxylic acid used in the acylation reactions, unreacted acid 14 was present in all wells at a concentration of approximately 100 μM during thrombin screening.At this concentration, it did not inhibit thrombin, as demonstrated by a control reaction mixture containing 14 but no macrocyclic backbone, as well as numerous 384-well reaction mixtures containing 14 and the backbone, which showed no thrombin inhibition (Figure 10c). Since 14 is present as a carboxamide after reaction with amino groups, thrombin inhibition by chlorothiophenamide was tested and inhibition with K was observed. i =380 μM. Weak inhibition confirmed that the macrocyclic backbones contributed significantly to the activity of the identified macrocyclic compounds. The top three hit compounds, M1, M2, and M3, were very similar in structure; all were synthesized based on cyclo(Mpa-D3-B5-Xaa-Mea) scaffolds, where all Xaa amino acids were α-amino acids with hydrophobic side chains (D-Val, L-Phe, L-Val; Figure 10e).

[0213] 3.5. Acylated scaffolds are active compounds It was then assessed whether the activity observed in the screens was due to the putative macrocyclic compounds or byproducts, such as carboxylic acid adducts or macrocycle dimers. For this purpose, the reactions of the scaffold and carboxylic acid 14 were repeated for the two best hit compounds M1 and M2 at a 250-fold larger scale (identical concentrations but larger volume), and the reactions were performed on an RP-HPLC column with 20 fractions separated, each of which was pooled for one minute of elution, the fractions were lyophilized, and the thrombin inhibition activity was determined (Figure 10f). For both reactions, fractions containing the target macrocyclic products showed the highest activity, indicating that the hit compounds were identified based on the activities of macrocycles M1 and M2.

[0214] Purified macrocycles M1, M2 and M3 inhibited thrombin with K i44, 165, and 125 nM, respectively (Figure 10e). Depending on the therapeutic application, the reducible disulfide bonds present in all scaffolds screened may be undesirable, and therefore it was tested whether they could be replaced with more stable bonds. M4 and M5 were synthesized, containing dithioacetal or thioether linkers. M4 and M5 inhibited thrombin with K i 83±8 nM and 135±16 nM, therefore, had only 2- and 3-fold weaker affinity. 3.6. Development of inhibitors of protein-protein interactions

[0215] Macrocycles have attracted great interest for their inhibition of protein-protein interactions (PPIs), with MDM2:p53 being a prototypical PPI target in disease and the subject of numerous inhibitor development efforts (M. Konopleva et al., Leukemia, 2020, 34, 2858-2874; L. Skalniak et al., Expert Opin. Ther. Pat., 2019, 29, 151-170). Overexpression of MDM2 inhibits the activity of the tumor repressor p53, and MDM2-binding compounds that block MDM2-P53 interactions are of interest for the development of novel anticancer therapeutics (P. Chene, Nat. Rev. Cancer, 2003, 3, 102-109). To test the new approach against this challenging PPI target, 192 structurally diverse cyclic peptide backbones were synthesized, all based on three random amino acids, one of which contained an amino group for side chain diversification.To increase the likelihood of identifying binding compounds, all scaffolds included tryptophan or phenylalanine, two amino acids that form key interactions in fusion peptides that bind MDM2 and inhibit the MDM2:p53 interaction. These cyclic peptide scaffolds were synthesized in 96-well plates as described above for Library 1 and were obtained at an average concentration of 12.9 mM and an average purity of 90%. Similarly, the scaffolds were modified by acylation with moieties in a combinatorial manner, in this case using 104 carboxylic acids. Thus, the size of the scaffold library was increased from 192 structures to 19,968 macrocyclic compounds, a more than 100-fold increase.

[0216] Library screening was performed by adding the target protein MDM2 and a reporter peptide to reaction mixtures in 384-well microplates to determine macrocycle binding. The fluorescent reporter peptide binds to the PPI interface on MDM2 (K d= 0.5 μM), and its displacement by the macrocycles can be monitored by fluorescence polarization. The screening result was again displayed as an array of backbone (vertical) and fragment (horizontal) combinations, with color indicating the degree of displacement of the reporter peptide from MDM2 (Figure 11). Two sets of hit compounds were of greatest interest: one horizontally aligned and hence the macrocycles share the same backbone, cyclo(Mpa-Trp-D3-B4-Mea), and the other vertically aligned and hence the macrocycles contain the same carboxylic acid (9). Repeating the acylation reaction and screening using all 192 backbones and the four carboxylic acids that provided the best hit compounds (9, 14, 25, 91) confirmed the results of the initial screening.Analysis of the active substances in the three reaction mixtures for each of the two groups of hit compounds showed that in the case of the first group (hit compounds M6, M7 and M8) the active substances were the expected macrocyclic structures, but not in the case of the second group (Figure 12a).

[0217] 3.7. Identification of compounds that bind MDM2 with nanomolar affinity

[0218] Purified macrocycles Mb, M7, and M8 efficiently displaced the fluorescent peptide probe from MDM2 and had similar IC50 values ​​of approximately 1 μM (Figure 12b), but the competition assay was not suitable for determining binding constants in the low micromolar or nanomolar range, since the assay required high concentrations of MDM2 (1.2 μM). Therefore, these three macrocycles were synthesized as conjugates with fluorescein, which was linked to the N-terminal region of the peptide backbone, and the binding affinity was determined using direct fluorescence polarization assay (Figure 12c). The conjugates exhibited Kd values ​​of 650±50 nM (F-M6), 790±80 nM (F-M7), and 340±40 nM (F-M8).

[0219] 3.8 Iterative synthesis at the picomolar scale and screening

[0220] Facile synthesis of macrocyclic compounds enables iterative synthesis of sublibraries based on hit compounds, which can improve binding affinity. To improve the efficiency of macrocycle M8, which was identified in the initial screening, we synthesized 63 scaffolds (Figure 18a) using amino acids similar to those in M8, which were three nipecotic acid (Nip) analogs, three diaminopropionic acid (Dap) analogs, and seven tryptophan (Trp) analogs (3×3×7=63). The present inventors then diversified these 63 scaffolds using 14 carboxylic acids that included repeats from the original library hit compounds (carboxylic acids 14, 25, 91) as well as related structures that were analogs of cinnamic acid 91, the carboxylic acid of the hit macrocycle M8 (105, 115; Figure 28).To identify binders with nanomolar affinity, we screened 882 macrocycles (63×14) at a concentration 13-fold lower (750 nM) than in the first screen, corresponding to a 30 pmol scale (Figure 18b). Although the most active macrocycles were generated from the original backbone, in this screen we identified carboxylic acids that yielded more potent macrocycles, namely acid 109, which yielded a 68% reporter peptide bias at a concentration of 750 nM (macrocycle M9) compared to 21% for M8 (acid 91; Figure 18b). Although the present inventors did not refine the scaffold, they obtained significant structure-activity relationship data for the macrocyclic ring from screens with 63 scaffolds and showed that all structural units are essential.

[0221] We then performed a third round of library synthesis and screening, acylating the nine most promising scaffolds from the previous screens with 15 additional carboxylic acids, most of which were cinnamic acid derivatives with larger substituents. We subsequently identified M10, a macrocycle based on the parent scaffold acylated with acid 120, which provided 84% displacement of the used fluorescent probe F-M8 from MDM2 at a concentration of 750 nM, more efficiently than the parent compounds M8 and M9 (Figure 18c). We conjugated the macrocycle M10 to fluorescein and determined its binding to MDM2 using FP with a Kd of 43±18 nM (Figure 18d).For the fluorescein conjugate, we also conducted a competition experiment with nutlin-3a, which binds to a specific hydrophobic pocket of MDM2 (B. Anil et al., Acta Crystallogr. Sect. D Biol. Crystallogr. 2013, 69, 1358-1366) and is a precursor to nutlin-based clinical candidates. The two ligands did not compete, indicating that the macrocycle binds to a different site and potentially has a novel mechanism of inhibition (Figure 29). Repeating the macrocycle binding determination using surface plasmon resonance (SPR) as an independent method demonstrated Kd of 600±300 nM (Mb), 550±190 nM (M7), 169±93 nM (M8), and 29±14 nM (M10), confirming the affinity range found in the FP assay (Figure 18e and Figure 30).

[0222] 3.9 Conclusion

[0223] This paper describes a method for diversifying macrocyclic scaffolds at the picomolar scale using acoustic dosing of activated carboxylic acids. Starting with SPPS, thousands of diverse macrocycles were obtained as crude reaction mixtures in just four days. These mixtures were then subjected to direct screening against protein targets, and inhibitors were successfully identified. The nanoliter-scale acylation reaction was stable and yielded high product conversions for a variety of different carboxylic acids and macrocycles. No unexpected byproducts were detected. Our automated platform enabled this final diversification step to be completed in less than an hour, in contrast to traditional macrocycle library synthesis methods that require labor-intensive purification.Although acoustic dosing has been used for synthesis previously, its application has been limited to the development of synthetic reactions rather than the direct synthesis of compounds for screening. The small scale of the reactions proposed in this invention, along with their relative purity, enabled direct screening of macrocycles against protein targets in the same microtiter plate. It was demonstrated that these screens can identify inhibitors and that the observed activity is due to the target products and not impurities. Information on screening crude mixtures in the literature remains limited. The present inventors are unaware of any existing work performed at a similar scale or related specifically to macrocycles. Given the simplicity of the method proposed in this invention, it should be applicable to screening against many protein targets.Furthermore, a variety of different chemistries can be used for ADE diversification; amide bond formation was chosen as an initial example due to its simplicity and its previous use as a crude product screening reaction. Although the largest library synthesized by the present inventors contained 20,000 macrocycles, it could be relatively easily expanded to hundreds of thousands. To obtain more lead-like compounds in the future, the method proposed herein can be applied to scaffolds cyclized through non-reducible bonds. To further enhance the drug-like properties of the library, the sets of structural units can be expanded beyond amino acids.The method proposed in the present invention can be applied to screen for a larger number of therapeutically significant targets for which it is desirable to develop clinical candidate compounds.

[0224] 3.10 Additional Results

[0225] General structure of human α-thrombin in complex with M1

[0226] Human α-thrombin consists of two polypeptide chains of 36 (light chain) and 259 amino acid residues (heavy chain), covalently linked via a disulfide bridge (Cys122 of the H chain to Cys1 of the L chain). X-ray crystallographic analysis of crystals formed by α-thrombin (light and heavy chain) and the M1 macrocycle revealed four virtually identical copies of the heavy and light chains of human α-thrombin in the asymmetric region of the unit cell. The four light / heavy chains of α-thrombin are named A / B, C / D, E / F, and H / L. The H / L structure was used for all calculations and for preparing the structural images. The light chain of human α-thrombin can be unambiguously traced from Glu1C to Ile14K. The amino-terminal residues (Thr1H to Gly1D) and carboxy-terminal residues Asp14L (except light chain C and E), GlyHM, and Arg15 are undefined and not visible in the Fourier map.The electron density of the heavy chain is clearly visible for all residues except for a few amino acids that are part of the surface flexible autolysis loop (Trp148 to Vall49C). The carboxy-terminal residue Glu247 lacks corresponding electron density. Minor differences arise at the level of the flexible and less pronounced loops or in the orientation of the surface-exposed peripheral side chains. The overall structure of human α-thrombin bound to the macrocycle does not exhibit any significant rearrangements of the backbone compared to other human α-thrombin structures, either in the apo form or in complex with inhibitors.

[0227] General structure of the M1 macrocycle

[0228] The electron density of the M1 macrocycle is clearly defined, allowing for unambiguous determination of the orientation of the groups for all four protein complexes present in the asymmetric region of the cell. The atomic numbering in M1 is shown in Figure 26b. No classical secondary structure elements or non-covalent intramolecular interactions were detected in this macrocycle. The molecule appears to adopt a chair conformation, which closely matches the shape of the catalytic pocket.

[0229] Interaction between human α-thrombin and M1

[0230] The M1 macrocycle fits well into the cleft formed by the active site and surrounding substrate pockets, occupying a protein surface area of ​​400.5 A 2(N. Voss et al., Nucleic Acids Res. 2010, 38, W555-W562). The conformations and interactions of the macrocycles are equivalent at the four active sites of the four thrombin molecules present in the asymmetric compartment. Most of the interactions of M1 with human α-thrombin are mediated by the 5-chlorothiophene-2-carboxamide functional group, which is located in the primary specificity pocket of S1. This group is held in the pocket by a hydrogen bond with the backbone of Gly219 (N7 of M1 with the O of Gly219) and an H2O molecule that forms a bridge between the O9 oxygen atom of M1 and the backbone nitrogen atom of N Gly193, as well as the backbone nitrogen atom of Ser195. 5-chlorothiophene-2-carboxamide is also involved in a network of polar contacts with the backbone of nearby Cys191 (09 M1 with O Cys191), Glul92 (09 M1 with N Glu192), Gly216 (N7 M1 with O Gly216 and S15 M1 with N Gly216), Trp215 (S15 M1 with N Trp215) and the side chain of Cys220 (N7 M1 with S Cys220).The chlorine atom functional group of 5-chlorothiophene-2-carboxamide is directed toward the bottom of the S1 pocket, where it likely forms a halogen-aromatic π interaction (4.0 Å) with the aromatic ring of Tyr228. The nitrogen atom N4 and the oxygen atom O17 of the main chain of M1 form hydrogen bonds with the main chain oxygen atom of Gly216 (O of Gly216) and the nitrogen atom of Gly216 (N of Gly216), respectively. In addition, the nitrogen atom N4 and the oxygen atom O17 of the main chain of M1 can form polar contacts with the main chain nitrogen atom of Gly219 (N of Gly219) and the oxygen atom of Gly216 (O of Gly216), respectively. Similarly, the N18 nitrogen atom of the M1 backbone can form two polar contacts with the carboxyl group of the Glul92 side chain (OE1 and OE2 of Glul92). Finally, an H2O molecule forms a bridge between the N27 nitrogen atom of the M1 backbone and the oxygen atom of the Glu97A backbone (O of Glu97A).Importantly, M1 binding to human α-thrombin is mediated by multiple hydrophobic contacts between the backbone and side chains of adjacent residues of the enzyme. The macrocycle framework (C20-C24), including the S21-S22 disulfide bridge, is directed toward the hydrophobic cage formed by the side chains of residues His57, Tyr60A, Trp60D (proximal pocket S2), and Leu99 (distal pocket S3). The valine side chain (C28-C31) bends the other side of the ring toward the hydrophobic pocket formed by Ile174 and Trp215. Finally, the phenyl ring C35-C40 is located on top of the thrombin loop (Gly216-Cys220).

[0231] 3.11 Additional Materials and Methods

[0232] General Provisions

[0233] Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents were not anhydrous and were not dried before use. The following abbreviations are used: DIPEA (N,N-diisopropylethylamine), DABCO (1,4-diazabicyclo[2.2.2]octane), NMM (4-methylmorpholine), HBTU (N,N,N',N'-tetramethyl-(9-(1H-benzotriazol-1-yl)-uronium hexafluorophosphate), HATU (N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)-uronium hexafluorophosphate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid)

[0234] Synthesis of the model skeleton

[0235] The model backbone of cyclic peptide 1 was synthesized using the cyclization-disulfide elimination (CDR) strategy described previously (S. Habeshian et al., ACS Chem. Biol. 2022, 17, 181-186). The linear peptide precursor was synthesized at 25 μmol scale in a 5 mL polypropylene synthesis column (MultiSyntech GmbH, V051PE076) using Rapp Polymere HA40004.0 Polystyrene A SH resin (200-400 mesh), resin loading 0.95 mmol / g, according to the method described in Habeshian, S. et al. 2022 2The peptide was cleaved as follows. To remove side chain protecting groups, the resin was incubated with 2 mL TFA / TIS / ddH2O 38:1:1 v / v / v for 1 h and then washed with 5 × 4 mL DCM. To cleave the peptide with cyclization, the resin was treated with 1 mL DMSO containing 150 mM DIPEA (6 equiv) overnight. The resin was removed by filtration. The crude mixture was purified by RP-HPLC using a Waters HPLC system (2489 UV detector, 2535 pump, fraction collector III), a Waters XTerra MS C18 OBD Prep Column, 19 mm × 250 mm (125 Ǻ pore size, 10 μm particle size), solvent system A (H2O, 0.1% TFA v / v) and B (MeCN, 0.1% TFA v / v), and a gradient of 0-25% solvent B over 30 min. The fraction containing the model backbone was lyophilized and dissolved in DMSO to provide a concentration of 40 mM.

[0236] Acylation of model backbone 1 using pipetting of reagents

[0237] The model backbone was acylated at the 40 nmol scale in 4 µl volumes as follows. The backbone (20 µl of 40 mM stock solution in DMSO) was supplemented with the base (20 µl of 160 mM DIPEA dissolved in DMSO), and 2 µl of the resulting mixture was transferred to the wells of a PCR plate. The carboxylic acids were prepared as 160 mM stock solutions in DMSO containing 160 mM DIPEA. Equal volumes of HBTU (160 mM in DMSO) were added to each acid stock solution, and 2 µl of the resulting active esters (80 mM) were added to the same PCR plate. The reaction was allowed to proceed for 3 hours at room temperature. After this time, 1 μl of the reaction mixture was transferred to 99 μl of 100 mM Tris-HCl in water, pH 7.5, incubated for 6 hours to block activated acids under the action of Tris, and the reaction mixtures were analyzed by LC-MS.

[0238] Acylation of model scaffold 1 using acoustic reagent dosing

[0239] The model backbone was acylated at 800 pmol scale in 80 nL volumes as follows. Backbone 1 (20 μL of 40 mM stock solution in DMSO) was mixed with base (20 μL of 160 mM DIPEA, 20 μL of 160 mM DABCO, 20 μL of 160 mM sodium HEPES, or 20 μL of 1 M NMM dissolved in DMSO), and 10 μL of the resulting mixtures were transferred to an ECHO master plate (384-well low dead volume microplate meeting Echo criteria, Labcyte). The concentrations in the master plate were 20 mM model backbone and 80 mM DIPEA (4 equiv), or 80 mM DABCO (4 equiv), or 800 mM NMM (40 equiv). The carboxylic acids were prepared as 160 mM stock solutions in DMSO containing either 160 mM DIPEA, 160 mM DABCO, or 1 M NMM. An equal volume of HBTU (160 mM in DMSO) was added to each acid stock solution, and the active esters (80 mM) were added to the same master plate. The master plate was centrifuged at 950 g (2000 rpm). / min in a Thermo Heraeus Multifuge 3L-R centrifuge for 3 min to remove any bubbles. Using a Labcyte Echo 650 acoustic pipette, 40 nL of model scaffold 1 (800 pmol) was transferred to a Nunc low-volume 384-well polystyrene plate, followed by 40 nL of active esters (3.2 nmol, 4 equiv). Transfers were performed in duplicate to ensure sufficient material for LC-MS analysis. The plates were sealed and allowed to react for 6 h at room temperature. After this time, 8 μl of 100 mM Tris-HCl in water, pH 7.5, were added to each of the duplicate reactions, the duplicates were combined, incubated for 3 hours to block activated acids under the action of Tris, and the reaction mixtures were analyzed by LC-MS.

[0240] Acylation of model scaffolds 2-5 using acoustic reagent dosing

[0241] Model backbones 2–5, purchased from Enamine, were obtained as powders weighing 1.1–1.2 mg. These backbones were dissolved in 62–88 µL of DMSO to obtain 40 mM stock solutions. The backbones were acylated using DABCO as the base as described above for model backbone 1, with the following differences: The reaction time was 6 hours. Before LC-MS analysis, 720 nL of DMSO was added to each well, followed by the addition of 7.2 µL of 100 mM Tris-HCl in water, pH 7.5. Blocking was performed overnight.

[0242] Scaffold and amino acid sequence design

[0243] The cyclic peptide backbones used for Library 1 were prepared by randomly selecting amino acid sequences. The number of different sequences that could theoretically be obtained based on the chosen backbone formats and amino acid structural units was much greater than the number of backbones synthesized for Library 1 (384), as described below:

[0244] Theoretical number of backbones for library 1:

[0245] 1. Scaffolds containing diamino acids: 3240

[0246] number of backbone formats (6) × number of diamino acids (6) × number of backbone amino acids (6) × number of side chain amino acids (15)

[0247] 2. Cysteine-containing scaffolds: 540

[0248] number of backbone formats (6) × number of backbone amino acids (6) × number of side chain amino acids (15)

[0249] To randomly select 384 amino acid sequences, all structural units were assigned an alphanumeric identifier, and all possible combinations were numbered manually. Peptides were assigned numbers from 1 to 3780. A random sequence generator (https: / / www.random.org / sequences / ) was then used to reorder the numbers, and the first 384 were selected for synthesis.

[0250] Preparation of polystyrene-E-E-cysteamine resin for library synthesis To prepare polystyrene-8-8-cysteamine resin for the synthesis of 4 × 96 peptides at a 5 μmol scale in four 96-well plates, which was necessary for the synthesis of the backbones for library 1 (thrombin screening), the following procedure was used. 589 mg of resin (Rapp Polymere HA40004.0 Polystyrene A SH resin, 200-400 mesh) was added to each of four 20 mL plastic syringes (CEM, 99.278) at a loading rate of 0.85 mmol / g, which corresponds to a 0.5 mmol scale. The resin was washed with 15 mL of DCM and then swelled in 15 mL of MeOH / DCM 3:7 v / v for 20 min. 2-(2-pyridinyldithio)-ethanamine hydrochloride (1.96 g, 8.8 mmol, 4.4 equiv) was dissolved in 21.12 mL of MeOH, then 49.28 mL of DCM and 1.53 mL of DIPEA were added. 17.7 mL of this solution was drawn into each syringe and then shaken at room temperature for 3 h.After this time, the 2-(2-pyridinyldithio)-ethanamine solutions were discarded, and the resins were washed with 2 × 20 mL MeOH / DCM 3:7 v / v, then 2 × 20 mL DMF. The resins were combined into one syringe as a suspension in DMF, then washed with 11.8 mL of a 1.2 M DIPEA solution in DMF for 5 min to ensure neutralization of all amines. This solution was discarded, and the resin was washed with 2 × 20 mL DMF, 4 × 20 mL DCM, and then kept under vacuum overnight to obtain a free-flowing powder. For library backbone synthesis (MDM2 screening), the resin loading was 0.95 mmol / gram, and therefore 526 mg of resin was added to each of the two syringes.

[0251] Synthesis of a peptide library in 96-well plates

[0252] Automated solid-phase peptide synthesis was performed on an Intavis Multipep RSi synthesizer. For the thrombin-targeted library, 565 mg of polystyrene-SS-cysteamine resin (0.48 mmol cysteamine, assuming that the thiol groups were quantitatively modified with cysteamine) and 20 mL of DMF were added to a 50 mL tube. For the MDM2-targeted library, 505 mg of the functionalized resin were added instead. The tube was shaken to ensure uniform resin suspension, and 200 μL (5.88 mg resin, 5 μM) was transferred to each well of a 96-well solid-phase synthesis plate (Orochem OF 1100). The resin was washed 6 × with 150 μL of DMF. The coupling was performed with 53 μl of amino acids (500 mM, 5.3 equiv), 50 μl of HATU (500 mM, 5 equiv), 12.5 μl of N-methylmorpholine (4 M, 10 equiv), and 5 μl of N-methylpyrrolidone. All components were premixed for 1 min and then added to the resin (reaction for 1 hour, without shaking).The final volume of the coupling reaction mixture was 120.5 μL, and the final reagent concentrations were 220 mM amino acid, 208 mM HATU, and 415 mM N-methylmorpholine. The coupling was performed twice, then the resin was washed with 6 × 225 μL DMF. Fmoc deprotection was performed using 120 μL piperidine / DMF 1:5 v / v for 5 min and was performed twice. The resin was washed with 8 × 225 μL DMF. After completion of peptide synthesis, the resin was washed with 2 × 200 μL DCM.

[0253] Removing protecting groups from library side chains

[0254] To remove the side chain protecting groups, the bottom of the 96-well synthesis plate was sealed by pressing the plate onto a 6 mm thick soft ethylene vinyl acetate foam backing (Rayher Hobby GmbH, 78 263 01), and the resin in each well was incubated with approximately 500 μL of TFA / TIS / ddH2O 38:1:1 v / v / v for 1 hour. The plates were covered with adhesive polypropylene film and then pressed down with a weight (1 kg) on ​​top to ensure no leakage. After 1.5 hours, the synthesis plates were placed on 2 mL deepwell plates (Thermo Scientific, 278752) and the TFA mixture was allowed to drain off. Wells were washed with 3 x 500 µl DCM (added via syringe) and then allowed to air dry for 3 h.

[0255] Cyclization of library peptides in 96-well plates

[0256] The plates were pressed into foam pads as described above to seal the holes, and 200 µL of 150 mM DABCO in DMSO (6 equiv) was added to each well. The plates were sealed with adhesive foil, pressed (1 kg), and left overnight. The next day, the synthesis plates were placed on 2 mL deep-well plates (Thermo Scientific, 278752) and centrifuged at approximately 200 g (1000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 1 min to collect the cleaved macrocycles in DMSO.

[0257] Quantification of library peptides by optical density

[0258] Optical density was determined using a Nanodrop 8000 spectrophotometer (Thermo Scientific) at a wavelength of 280 nm and an optical path length of 10 mm. Cleaved peptides containing Trp and D-Trp were diluted 250-fold with water for the thrombin-targeted library and 125-fold with water for the MDM2-targeted library. The Beer-Lambert law was used to calculate peptide concentrations. The extinction coefficient of Trp, S280 = 5500 M, was used. -1 cm -1 .

[0259] LC-MS analysis

[0260] The peptides were analyzed by LC-MS using a UHPLC and single quadrupole MS system (Shimadzu LCMS-2020) using a reversed-phase C18 column (Phenomenex Kinetex column, 2.1×50 mm, C18, pore size 100 Ǻ, particle size 2.6 μm) and a linear gradient of solvent B (acetonitrile, 0.05% formic acid) to solvent A (H2O, 0.05% formic acid) at a flow rate of 1 mL / min. Mass analysis was performed in positive ion mode.

[0261] For LC-MS analysis, samples from different experiments were prepared as follows. For the analysis of proof-of-concept acylation reactions, 160 nL of reaction mixtures were diluted in 16 µL of Tris-HCl buffer, pH 7.5, to yield a peptide concentration of 100 µM. For the analysis of the scaffolds synthesized for library 1, 1 µL of DMSO / DABCO eluates was diluted in 80 µL of water to yield a cyclic peptide concentration of approximately 120 µM. For the analysis of the scaffolds synthesized for library 2, 1 µL of DMSO / DABCO eluates was diluted in 128 µL of water to yield a cyclic peptide concentration of approximately 120 µM. For all analyses, 5 µL of samples were injected, typically using a solvent B gradient from 0 to 60% over 5 min.

[0262] Calculation of physicochemical properties of macrocycles

[0263] Physicochemical properties: molecular weight, calculated water / n-octanol partition coefficient (cLogP), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), polarized surface area (PSA), and number of rotatable bonds (NRotB) were calculated using DataWarrior software (www.openmolecules.org). The structures of the backbones and carboxylic acids were constructed in ChemDraw and saved as SMILES strings in SD files, one for the backbones and one for the acids. Both SD files were opened in DataWarrior. The “enumerate combinatorial library” functionality was used to determine the target reaction of amide bond formation between the macrocyclic backbones and carboxylic acids. The following definitions were made: amide was defined as an “excluded group”. The nitrogen atom was defined as not being part of an aromatic ring and having a hydrogen atom count greater than 0.The carbon atom adjacent to the amine was identified as non-aromatic and lacking pi electrons. The starting material and product atoms were then mapped. Following combinatorial enumeration, target properties were calculated based on the structures.

[0264] Acylation of backbones to obtain library 1

[0265] Cyclic peptide backbones in the solvent used for peptide cleavage from the resin (DMSO containing 150 mM DABCO) were transferred to a Labcyte Echo-compliant 384-well low-dead-volume microplate (10 μL per well). The cyclic peptide backbone concentrations averaged approximately 8.1 mM. Carboxylic acids were dissolved to a concentration of 160 mM in DMSO containing 160 mM DABCO. An equal volume of HBTU (160 mM in DMSO) was added to each acid stock solution. Active esters (80 mM) were added to another low-dead-volume stock plate. The original plates were centrifuged at 850 g (2000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 3 min to remove any bubbles. Using a Labcyte Echo 650 acoustic pipette, 20 nL of the backbones (160 pmol) were transferred to a 384-well low-volume polystyrene plate (Nunc, 264705), followed by 20 nL of the active esters (1.6 nmol, 10 equiv).The plates were sealed and allowed to react for 6 hours at room temperature. After this time, 5 µl of Tris buffer (100 mM Tris-Cl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.1% w / v BSA, 0.01% v / v Triton-X100) were added to each well using a BioTek MultiFlo microplate dispenser. Reactions were stopped overnight at room temperature.

[0266] Thrombin inhibition screening

[0267] Thrombin inhibition by library 1 macrocycles was assessed by determining the residual thrombin activity in the presence of cyclic peptides at an average final concentration of 11 μM. The studies were carried out in 384-well plates using Tris buffer at pH 7.4 (100 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.1% w / v BSA, 0.01% v / v Triton-X100, and 0.6% v / v DMSO), thrombin at a final concentration of 2 nM, and the fluorogenic substrate Z-Gly-Gly-Arg-AMS at a final concentration of 50 μM. Thrombin (5 µl, 6 nM) in the Tris-Cl buffer described above was added to each peptide using a BioTek MultiFlo microplate dispenser and incubated for 10 min at room temperature.Fluorogenic substrate (5 μl, 150 μM) was added in the same buffer using a BioTek MultiFlo microplate dispenser, and fluorescence intensity was determined using a Tecan Infinite M200 Pro plate reader (excitation at 360 nm, emission at 465 nm) at 25°C for 30 min, recording every 3 min. The slope of each activity curve was calculated using Excel. The average slope was calculated for negative controls (20 wells containing DMSO but no macrocycle). Percent thrombin inhibition was calculated by dividing the slopes and multiplying the results by 100. Acylation of backbones to produce library 2.

[0268] Cyclic peptide backbones in the solvent used for peptide cleavage from the resin (DMSO containing 150 mM DABCO) were transferred to a Labcyte 384-well Echo-compliant polypropylene microplate (40 µL per well). The cyclic peptide backbone concentrations averaged approximately 12.9 mM. Carboxylic acids were dissolved to a concentration of 184 mM in 184 mM DABCO in DMSO. An equal volume of HBTU (184 mM in DMSO) was added to each acid stock solution. Active esters (92 mM) were added to the same polypropylene stock plate. The original plates were centrifuged at 950 g (2000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 3 min to remove any bubbles. Using a Labcyte Echo 650 acoustic pipette, 12.5 nL of macrocycles (161 pmol) were transferred to a 384-well low-volume polystyrene plate (Nunc, 264705), followed by 17.5 nL of active esters (1.61 nmol, 10 equiv).The plates were sealed and left to react for 6 hours at room temperature. After this time, 5 µl of Tris buffer was added to each well using a Gyger Certus Flex liquid dispenser, and the reaction was stopped overnight at room temperature.

[0269] MDM2 Binding Screening

[0270] MDM2 binding to cyclic peptides was assessed by determining the displacement of the p53 fluorescent peptide probe in the presence of cyclic peptides at an average final concentration of 11 μM. The assays were performed in 384-well plates using PBS buffer, pH 7.4 (100 mM Na2HPO4, 18 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.01% v / v Tween-20, and 3% v / v DMSO), MDM2 at a final concentration of 1.2 μM, and the p53 fluorescent peptide probe (FP53, sequence = 5(6)-FAM-GSGSSQETFSDLWKLLPEN) at a final concentration of 25 nM. Premixed MDM2 and FP53 (10 μl, 1.8 mM MDM2, 37.5 nM FP53) in the PBS buffer described above were added to each peptide using a Gyger Certus Flex liquid dispenser and incubated for 30 minutes in the dark at room temperature. A single fluorescence anisotropy value was obtained using a Tesal Infinite F200 Pro plate fluorometer (excitation at 485 nm, emission at 535 nm) at 25°C.The percentage of probe displacement was calculated using the following formula.

[0271]

[0272] where N is the average anisotropy of the negative control (without inhibition), X is the value obtained for each well, and P is the average anisotropy of the probe separately. Identification of active substances in reaction mixtures of hit compounds Macrocycles identified as hit compounds in the thrombin screening were resynthesized at 40 nmol scale by reacting 5 μL of 8 mM cyclic peptide backbones in DMSO containing 150 mM DABCO with 5 μL of 80 mM carboxylic acid, 80 mM HBTU, and 80 mM DABCO for 5 hours at room temperature. The remaining activated ester was blocked by adding 1.25 ml Tris buffer (100 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2) and incubating overnight.The next day, 240 µL of MeCN and 1 mL of water were added, and the reaction mixtures were passed through a C18 column (Waters NovaPak C-18 column, 7.8 mm × 300 mm, 60 Å pore size, 6 µm particle size) on a Thermo Dionex HPLC system using solvent A (H2O, 0.1% v / v TFA) and a 10-80% gradient of solvent B (MeCN, 0.1% v / v TFA) over 20 min, with fractions collected every minute. The fractions were lyophilized and dissolved in 120 µL of 2% DMSO in water. The activities of the products in the fractions were determined using the same assays described above, but in 96-well plates. Fifty microliters of each fraction were transferred to a 96-well assay plate (Greiner, 655101), followed by 50 microliters of thrombin (6 nM in buffer). After 10 minutes of incubation, 50 microliters of fluorogenic thrombin substrate (Z-Gly-Gly-Arg-AMC, 150 μM in buffer, 1% DMSO) were added. The signal in the plates was read, and the data were processed as described above. Compounds in the active fractions were identified by mass spectrometry.

[0273] For the hit compounds from the MDM2 screening, reactions were performed in the same manner but at 50 nmol scale and purified in the same manner but with a 10-80% solvent B gradient for 22 min. Fractions were lyophilized and dissolved in 40 μL DMSO, 160 μL water was added, and 5 μL of each fraction was transferred to a 384-well plate (Nunc, 264705) and 15 μL premixed MOM2 / FP53 peptide (final concentrations: 1.2 μM MDM2, 50 nM FP53, 5% DMSO) were added.

[0274] Crystallization of thrombin with M1

[0275] Human α-thrombin was purchased from Hematologic Technologies (catalog number: HCT-0020). The protein stabilizing agent was removed using a PD-10 desalting column (GE Healthcare) equilibrated with 20 mM Tris-HCl, 200 mM NaCl, pH 8.0, and the same buffer as the solvent. After buffer exchange, human α-thrombin was incubated with macrocycle M1 at a molar ratio of 1:3 and then concentrated to 7.5 mg / mL using a Vivaspin ultrafiltration device with an MWCO of 3000 (Sartorius-Stedim Biotech GmbH). During concentration, an additional amount of macrocycle M1 was added to ensure a 3-fold molar excess. Crystallization studies of the complex were performed at 293 K in a 96-well 2-drop MRC plate (Hampton Research, California, USA) using the sessile drop vapor diffusion method and the Morpheus and LMB crystallization screening kits (Molecular Dimensions Ltd, Suffolk, UK).600 nL droplets (with a protein:precipitant ratio of 1:1) were prepared using an Oryx 8 crystallization robot (Douglas Instruments Ltd, Berkshire, UK) and equilibrated with 80 µL of reservoir solution. The best crystals were obtained by adding microseeds to fresh droplets that had been left to equilibrate for 2–3 days, using the following mixture as the precipitating agent: 20 mM sodium formate, 20 mM ammonium acetate, 20 mM sodium citrate tribasic dihydrate, 20 mM potassium sodium tartrate tetrahydrate, 20 mM sodium oxamate, 100 mM MOPS / sodium HEPES, pH 7.5, 12.5% ​​w / v PEG 1000, 12.5% ​​w / v PEG 1000. PEG 3350, 12.5% ​​v / v MPD.

[0276] Crystallization, data collection and structure determination

[0277] For X-ray diffraction data collection, crystals were mounted on LithoLoops (Molecular Dimensions Ltd, Suffolk, UK) and flash-cooled with liquid nitrogen. X-ray diffraction data of human α-thrombin in complex with M1 were collected at beamline i04 of Diamond Light Source Ltd (DLS, Oxfordshire, UK). The best crystals diffracted to a maximum resolution of 2.27 Ǻ. The crystals belong to space group P21 with the unit cell parameters a = 56.25 Ǻ, b = 100.57 Ǻ, c = 108.90 Ǻ and a = 90°, β = 90.11°, γ = 90°. The asymmetric part of the cell contains four molecules, which corresponds to a Matthews coefficient of 2.78 Ǻ. 3 / Yes and a solvent content of approximately 48% of the crystal volume. Frames were indexed and integrated using XIA2 software, merged and scaled using AIMLESS (CCP4i2 crystallographic package) (M. Winn et al., Acta Crystallogr. D 2011, 67, 235-242). The structure was solved by molecular replacement using PHASER software (A. McCoy et al., J. Appl. Crystallogr. 2007, 40, 658-674), using the 6GWE model (S. Kale et al., Sci. Adv. 2019, 5, eaaw2851) as a matrix. Refinement was performed using REFMAC (A. Vagin et al. Acta Crystallogr. D 2004, 60, 2184–2195) and PHENLX (P. Adams et al., Acta Crystallogr. D 2010, 66, 213–221). From the first refinement runs, a broad electron density corresponding to the bound ligand was clearly visible in the electron density map. Macrocycle construction was performed using Molview, and the constraint file was generated and optimized using Phenix eLBOW.The macrocycle was manually fitted using the COOT graphical software (P. Emsley et al., Acta Crystallogr. D 2010, 66, 486-501). The final model contains 9098 protein atoms, 160 macrocycle atoms, and 4 Na atoms. + and 399 water molecules. The resulting crystallographic R-factor was 0.189 (Rfree 0.243). The geometric parameters of the model are as expected or better for this resolution. The excluded solvent volume and the corresponding buried surface were calculated using the PISA software and a spherical probe of radius 1.4 Ǻ. Intramolecular and intermolecular interactions due to hydrogen bonds were analyzed using PROFUNC (R. Laskowski et al., Nucleic Acids Res. 2005, 33, W89-W93), LIGPLOT+ (R. Laskowski et al., J. Chem. Inf. Model. 2011, 51, 2778-2786), and PYMOL software.

[0278] Acylation of backbones to obtain libraries 3 and 4

[0279] The cyclic peptide backbones required for libraries 3 and 4 were synthesized as described for the backbones used in library 2. Due to the presence of multiple N-methylated amino acids, which are more difficult to couple, 200 mM HOAt was used along with HATU. The backbones were diluted to 2 mM in DMSO and transferred with 15 nL by acoustic pipetting, followed by 15 nL of DMSO containing carboxylic acids (40 mM, 20 equiv), HBTU (40 mM), and DABCO (40 mM). After reacting for 6 h at room temperature, 370 nL of DMSO was added to each well, followed by 5 μL of 100 mM Tris-Cl, pH 7.4, containing 0.01% v / v. Tween 20 for overnight blocking. To screen for MDM2 binding, F-M8 was used as a fluorescent probe due to its higher affinity for MDM2, allowing for the use of a lower concentration of the target protein (720 nM, resulting in approximately 55% probe binding).To each well, 35 μl of PBS buffer, pH 7.4 (100 mM Na2HPO4, 18 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.01% v / v Tween-20) containing 28.6 nM F-M8 and 823 nM MDM2 were added, and the reporter probe displacement was determined as described above. Macrocycle concentrations were 750 nM. Macrocycle synthesis on a milligram scale.

[0280] Automated solid-phase peptide synthesis was performed on an Intavis Multipep RSi synthesizer. 25 μmol polystyrene-SS-cysteamine resin was added to a 5 mL syringe (MultiSyntech GmbH, V051PE076). The resin was washed with 6 × 150 μL DMF. Coupling was performed with 210 μL amino acids (500 mM, 4.2 equiv), 200 μL HATU (500 mM, 4 equiv), 50 μL N-methylmorpholine (4 M, 8 equiv), and 5 μL N-methylpyrrolidone. All components were premixed for 1 min and then added to the resin (reaction for 1 hour, without shaking). The final volume of the coupling reaction mixture was 465 μL, and the final reagent concentrations were 226 mM amino acid, 215 mM HATU, and 430 mM methylmorpholine. The coupling was performed twice, and the resin was then washed with 2 × 600 μL of DMF. Fmoc deprotection was performed using 450 μL of piperidine / DMF (1:5 v / v) for 5 min and was repeated twice. The resin was washed with 7 × 600 μL of DMF.After completion of peptide synthesis, the resin was washed with 2 × 600 µl DCM.

[0281] After SPPS, the resin was incubated with 2 mL of TFA / TLS / ddH2O 38:1:1 (v / v / v) for 1 h. The TFA solution was discarded, and the resin was washed with 5 × 4 mL of DCM. After air drying for 3 h, 1 mL of 150 mM DIPEA in DMSO was added, and the syringes were shaken overnight at room temperature. The next day, these DMSO solutions were transferred to 50 mL conical tubes.

[0282] Carboxylic acid coupling was typically performed by adding 500 µL of premixed acids (100 mM, 2 equiv), HBTU (100 mM), and DABCO (100 mM) to DMSO. After 3 hours at room temperature, 8 mL of water was added, and the tubes were frozen and then lyophilized for 2 days to remove DMSO. The tube contents were dissolved in 3 mL of MeCN, followed by the addition of 7 mL of water.

[0283] The crude mixtures were purified by RP-HPLC using a Waters HPLC system (2489 UV detector, 2535 pump, fraction collector III), a Waters XTerra MS C18 OBD preparative column, 19 mm*250 mm (pore size 125 Ǻ, particle size 10 μm), a solvent system of Ǻ (H2O, 0.1% TFA v / v) and B (MeCN, 0.1% TFA v / v), and typically a gradient of 30-70% solvent B over 30 minutes.

[0284] K Definition i thrombin inhibitors

[0285] Purified thrombin inhibitors (10 mM in DMSO) were diluted to 80 μM in 125 μl Tris buffer (100 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2) containing 0.1% w / v BSA, 0.01% v / v Tnton-X100, and 0.2% DMSO. Macrocycles were diluted two-fold in Tns buffer containing 0.1% w / v BSA, 0.01% v / v Tnton-X100, and 1% DMSO in the buffer. Thrombin activity was determined in 96-well plates (Gremer, 655101), and residual activity was calculated as described above for the assay used to determine the activity of HPLC-separated fractions of screened hit compounds. Residual activity was plotted against the log of the corresponding macrocycle concentrations, and sigmoidal curves were fitted using the following four-parameter equation in GraphP ad Prism 6:

[0286]

[0287] Ki values ​​were determined based on IC values 50 with and using the Cheng-Prusov equation (Km =168 μM for thrombin and the substrate used):

[0288]

[0289] IC Definition 50 MDM2-binding macrocycles

[0290] Concentrations at which MDM2 macrocycles caused reporter peptide displacement for 50% of the protein (IC 50), were determined using the competitive fluorescence polarization assay described above. Serial two-fold dilutions of 5 µl of purified macrocycles (20 mM DMSO) were prepared in 100% DMSO in a low-dead-volume ECHO master plate. 150 nl of each dilution were transferred by acoustic droplet dispensing into a 384-well low-well-volume polystyrene plate (Nunc, 264705). To each well, 15 μl of a pre-prepared MDM2 / FP53 probe mix (1.2 μM MDM2, 25 nM FP53 probe) in PBS buffer, pH 7.4 (100 mM Na2HPO4, 18 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.01% v / v Tween-20) containing 1% v / v DMSO was added and incubated for 30 min in the dark. Fluorescence anisotropy was determined as described above. The percentage of bound inhibitor was calculated using the following equation

[0291]

[0292] where N is the average anisotropy of the DMSO controls, X is the anisotropy value obtained for each well, and P is the average anisotropy of the unbound IC probe 50 was determined by plotting the percentage of bound inhibitor versus the logarithm of the corresponding macrocycle concentration, and the resulting curves were fitted in GraphPad Prism 6 as described above.

[0293] Synthesis of fluorescein-labeled macrocycles

[0294] Fluorescein-labeled macrocycles were synthesized essentially as described in the milligram-scale macrocycle synthesis procedure. For 5(6)-FAM, manual coupling was performed using 4 equiv. of acid (180 mM, 556 µL), 4 equiv. of HATU (500 mM, 200 µL), 10 equiv. of NMM (4 M, 62.5 µL), all in DMF. Coupling was performed for 1×2 h, then washed as described previously.

[0295] K Definition d fluorescein-labeled MDM2-binding compounds by the FP method

[0296] Stock solutions of fluorescein-labeled macrocycles (20 mM in DMSO) were diluted to a concentration of 10 μM by adding 0.5 μL to 999.5 μL of PBS. These dilutions were further diluted to a concentration of 100 nM by transferring 10 μL to 990 μL of PBS, and 7.5 μL were transferred to the wells of a 384-well low-volume polystyrene plate (Nunc, 264705). 7.5 μL of 2-fold dilutions of MDM2 in PBS were pipetted into the wells. The final concentrations of fluorescent macrocycles were 50 nM. After incubating the plate for 30 minutes in the dark at room temperature, fluorescence anisotropy was determined using a Tecan Infinite F200 Pro plate fluorometer (excitation at 485 nm, emission at 535 nm) at 25°C. Anisotropy was plotted as a function of the logarithm of the corresponding MDM2 concentrations, and sigmoidal curves were fitted as described above.Synthesis of thrombin inhibitors containing thioether linkages Linear peptides containing three amino acids and a C-terminal cysteamine were synthesized by automated SPPS as described above for the milligram scale cyclic peptide synthesis, but at 50 μmol scale and using cysteamine-4-methoxytrityl resin (Novabiochem 856087, 200-400 mesh, 1% DVB, 0.92 mmol / gram). 4-Bromobutyric acid (500 μM, 500 mM, 10 equiv) was manually coupled to this peptide on the resin using N,N'-diisopropylcarbodiimide (DIC, 500 μL, 500 mM, 10 equiv) as the activating reagent and DMF as the solvent. The acid and coupling reagent were premixed for 1 min and then added to the resin (reaction for 1 hour with shaking). The final volume of the coupling reaction mixture was 1 mL, and the final concentrations of the reagents were 250 mM amino acid, 250 mM DIC.The coupling was performed twice, then the resin was washed with 4 × 4 mL DMF, then 2 × 4 mL DCM.

[0297] Side chain deprotection and cleavage were accomplished by incubating the resin with 2 mL of TFA / TIS / ddH2O 38:1:1 v / v / v for 1 hour with shaking. After this time, 50 mL of cold diethyl ether was added to the solution to precipitate the peptide. The mixture was kept at -20°C for 30 minutes and then centrifuged for 30 minutes at 3800 g (4000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) at 4°C. The ether was separated by decantation, and the peptide precipitate was allowed to air dry for 15 minutes.

[0298] The peptide was dissolved in 50 mL of freshly degassed water / acetonitrile 1:4 mixture and 200 μL (1.15 mmol, 23 equiv) of neat DIPEA was added. The reaction mixture was allowed to cyclize at room temperature for 90 min, then frozen and lyophilized.

[0299] The coupling of carboxylic acid 14 was carried out as follows. The macrocycle was redissolved in 1 mL of DMSO containing 100 mM DABCO. The coupling of carboxylic acids was typically performed by adding 500 μL of premixed acids (100 mM, 2 equiv), HBTU (100 mM), and DABCO (100 mM) in DMSO. After 3 h at room temperature, 8 mL of water was added, the tubes were frozen, and lyophilized for 2 days to remove DMSO. The tube contents were dissolved in 3 mL of MeCN, followed by the addition of 7 mL of water. The crude mixtures were purified by RP-HPLC as described above.

[0300] K Definition d MDM2-binding compounds using the SPR method

[0301] Experiments were performed using a Biacore 8K instrument, GE Healthcare. MDM2 (10 μg / mL) was dissolved in 10 mM MES buffer (pH 6.0) and immobilized on three channels of the CM5 S-series chip (Cytiva, 29104988) using amine immobilization conditions with EDC / NHS in the running buffer (10 mM PBS, pH 7.4, 150 mM NaCl, 3 mM KCl, and 0.005% v / v Tween-20). The typical immobilization level was between 6000 and 7000 resonance units (RU). The reference cuvette was processed in the same manner without MDM2. To determine the binding kinetics and dissociation constants, five serial (3-fold) dilutions of the macrocycles and a DMSO control solution were prepared in running buffer (10 mM PBS, pH 7.4, 150 mM NaCl, 3 mM KCl, and 0.005% v / v Tween-20, as well as 0.5% v / v DMSO) and analyzed in single-cycle kinetic mode with contact and dissociation times of 120 seconds and 60 seconds, respectively.

[0302] Example 4. Synthesis and analysis of a large library of macrocyclic compounds within the rule of five

[0303] 4.1. Results

[0304] In Example 1, 1 (Figure 14b; also referred to as cysteamine) was conjugated to a thiol-functionalized resin via a dithiol exchange reaction with excess pyridyldithioethylamine. In this case, activated thiosulfonates were used instead, as they can be synthesized without a chromatographic purification step and, therefore, more easily in large quantities. To this end, commercial suppliers searched for possible building blocks for N-Boc-alkyl halides that could undergo a substitution reaction with sodium benzenethionosulfonate to afford Boc-protected precursors in very good yield on a gram scale (87% - quant., see Materials and Methods for synthesis). Despite the limited commercial availability of aminohalogens, six new structural units were synthesized for diversification without the need for silica gel column purification.It was possible to proceed to the next step and deposit them directly onto the SH PS resin with high loading to obtain 2 7 immobilized on the resin via a dithiol bridge (Figure 15a).

[0305] To test whether the reported structural units are compatible with automated Fmoc-based SPPS, a model dithiol peptide, MPA-Trp-Ala-(1-7) (MPA = 3-mercaptopropionic acid), was synthesized in a 96-well plate format (Figure 15b). Following synthesis and deprotection with TFA, the resin-bound peptides were incubated with 2×200 µL of reductive cleavage solution (1,4-butanedithiol (BDT) and NEt3, both 100 mM in DMF). The peptides were released from the resin and analyzed by HPLC-MS after removal of DMF and volatile BDT using rotary vacuum concentration (RVC). The peptides were efficiently cleaved and analyzed by LC-MS. For all peptides, excellent target product quality was observed (Figure 15c).

[0306] Resin-linked elements for diversification were now available. These fragments were used to synthesize a macrocycle library to generate binding agents targeting trypsin-like serine proteases involved in blood coagulation pathways. Due to the large number of trypsin-like serine proteases and their role in various diseases, these proteases are an important target for the development of potent and specific inhibitors targeting this class of proteases. The difficulty of this task stems from the structural identity shared by many members of this group, due to their ability to recognize and cleave positively charged residues, as well as a conserved aspartate residue buried deep in their S1 pocket. Therefore, it was envisaged that this model system would be used to develop potent and selective macrocyclic inhibitors, as the results could later be applied to other areas of cyclic peptide research.

[0307] Therefore, 384 dithiol peptides were prepared at a 5 μmol scale using automated SPPS in 96-well filtration plates. The peptides were synthesized with three different backbones (1a–c, Figure 16a) containing randomly selected sequences consisting of one of seven different diversification elements (1–7), a random amino acid (from 27 different α, β, γ, and N-methylated amino acids), and an S1 pocket-binding motif known from the literature.

[0308] These peptides were synthesized on a solid support, and the corresponding protecting groups were removed by acidic treatment using TFA. The peptides were then released from the resin by reductive cleavage to yield linear dithiol peptides. After acidification and removal of the volatile reducing agent using RVC, the peptide concentration was determined using Ellman's reagent (average conc. = 24.9 mM, an average of 20% of the resin loading with equal peptide distribution for all 7 different derivatives).

[0309] The present inventors' novel laboratory approaches involve the generation of picomolar-scale libraries for direct use in ready-to-assay microtiter plates. Compound library generation was designed to be used for screening multiple targets simultaneously, and therefore, an approach was needed that would still provide a high diversity of compounds with multiple linkers while allowing for the generation of large quantities of compounds without the need for labor-intensive and resource-intensive pipetting. Therefore, attention was drawn to the use of acoustic droplet ejection (ADE) technology. Although this method is primarily used for transferring nanoliter volumes, it has previously been shown that this system can transfer even larger, microliter volumes, thereby eliminating pipetting.Therefore, 40 nmol of the linear peptide was distributed into multiple Echo® ADE-compatible 384-well polypropylene (PP) plates (Figure 17). Because the reduced dithiol peptides slowly underwent oxidation when dissolved in DMSO, the peptides were rapidly re-reduced by adding a DMF:BDT solution to the wells, which, after acidification and RVC, was immediately cyclized in MeCN / NH4HCO3 buffer with 7 different linkers (see Figure 16B for selection). This differs from previously used methods, where the linear compounds were first solubilized and then the linker solution was added. The new methodology minimizes the extent of re-oxidation that occurs, resulting in purer reaction mixtures. Finally, excess linkers were blocked with β-mercaptoethanol (β-ME).

[0310] 4.2. Materials and Methods

[0311] All reagents and solvents were of analytical grade and were used without further purification as received from commercial suppliers. Reaction progress was monitored by thin-layer chromatography (TLC) using silica gel-coated plates (analytical SiO2-60, F-254) and / or HPLC-MS analysis. TLC plates were visualized under UV light or by immersion in potassium permanganate solution (10 g / L) followed by visualization with a heat gun. Rotary evaporation of solvents was carried out under reduced pressure and temperatures below 40°C. HPLC-MS analyses were performed using UHPLC and a single quadrupole MS system (Shimadzu LCMS-2020) using a reversed-phase C18 column (Phenomenex Kinetex column, 2.1x50 mm, C18, pore size 100 Ǻ, particle size 2.6 μm).A linear gradient of solvent B (0.05% HCOOH in MeCN) to solvent A (0.05% HCOOH in water) was used, increasing linearly from 0% to 60% over t = 1.00 6.00 min at a flow rate of 1.00 mL / min. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III spectrometer (. 1 N NMR and 13 NMR spectra were recorded at 400 and 101 MHz, respectively, using a cryogenically cooled probe. All spectra were recorded at 298 K. Chemical shifts are reported in ppm relative to the deuterated solvent as an internal standard (δ H DMSO-d62.50 ppm; δ C DMSO 39.52 ppm; δ H CDCl37.26 ppm; δ C CDCl377.16 ppm).

[0312] High-resolution mass spectrometry (HRMS) determination was performed on a maXis G3 quadrupole time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) source.

[0313] Chemical synthesis of structural units and amino acids

[0314] 8-(3-((tert-butoxycarbonyl)amino)propyl)-benzenesulfonothioate(S1, ALN-1-31)

[0315]

[0316] To a stirred solution of 3-(Boc-amino)-propyl bromide (6.02 g, 25.3 mmol, 1.0 equiv.) in DMF (120 mL) was added sodium benzyl thiosulfonate (7.48 g, 38.0 mmol, 1.5 equiv.; tech. 85%), and the solution was stirred overnight at 80°C. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 mL), and extracted with EtOAc:hexanes (2×150 mL, 10:1, v / v). The combined organic layers were washed with water (3×150 mL) and brine (150 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S1 (8.21 g, 24.8 mmol, 98%) as a yellowish oil. TLC (25% EtOAc in hexanes): Rf = 0.25 (KMnO4 stain). 1H NMR (400 MHz, CDCl3) δ 7.98-7.90 (m, 2H), 7.70-7.61 (m, 1H), 7.60-7.52 (m, 2H), 3.16 (q, J=6.4 Hz, 2H), 3.02 (t, J=6.4 Hz, 2H), 3.02 (t, J=7.23 Hz), (p, J=6.8 Hz, 2H), 1.43 (s, 9H).

[0317] tert-butyl(3-chloropropyl)(methyl)carbamate (S2, VC-1-1)

[0318]

[0319] A stirred solution of 3-chloropropyl-N-methylamine hydrochloride (4.32 g, 30.0 mmol, 1.0 equiv.) and di-tert-butyl dicarbonate (6.58 g, 30.0 mmol, 1.0 equiv.) in CH2Cl2 (150 mL) was cooled to 0 °C under argon atmosphere. NEt3 (4.16 mL, 30.0 mmol, 1.0 equiv.) was added dropwise over 5 min, and the resulting solution was stirred overnight while warming to ambient temperature. The reaction mixture was concentrated under reduced pressure and resuspended in EtOAc (120 mL). The resulting solution was washed with aq. HCl (1 M, 2×120 mL), sat. NaHCO3 (120 mL) and brine (120 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S2 (6.22 g, 30.0 mmol, quant.) as a colorless crystalline solid. TLC (25% EtOAc in hexanes): R f =0.45 (KMnO4 staining). 1 H NMR (400 MHz, CDCl3) δ 3.55 (t, J=6.5 Hz, 2H), 3.36 (t, J=6.8 Hz, 2H), 2.87 (s, 3H), 2.06-1.91 (m, 2H), 1.46 (s, 9H). CAS RN: 114326-14-6.

[0320] S-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-benzenesulfonothioate(83, VC-1-3)

[0321]

[0322] To a stirred solution of VC-1-1 (3.51 g, 16.9 mmol) in DMF (25 mL) was added sodium benzyl thiosulfonate (5.50 g, 27.9 mmol, 1.65 equiv; tech. 85%), and the solution was stirred overnight at 80 °C. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 mL), and extracted with EtOAc:hexanes (2×75 mL, 10:1, v / v). The combined organic layers were washed with water (3×75 mL) and brine (75 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S3 (5.07 g, 16.2 mmol, 95%) as a yellowish oil. TLC (12.5% ​​EtOAc in hexanes): R f =0.30 (KMnO4 staining). 1H NMR (400 MHz, CDCl3) δ 8.01-7.88 (m, 2H), 7.69-7.50 (m, 3H), 3.24 (t, J=6.7 Hz, 2H), 2.96 (t, J=7.3 Hz, 2H), 2.77 (s, 1,85), p J=6.8 Hz, 2H), 1.42 (s, 9H).

[0323] tert-butyl-(Z)-(4-chlorobut-2-en- 1-yl)-carbamate (S4, P 1_N2_E29 / E46)

[0324]

[0325] A stirred solution of cis-4-chloro-2-butenylamine hydrochloride (2.50 g, 17.6 mmol, 1.0 equiv.) and di-tert-butyl dicarbonate (3.84 g, 17.6 mmol, 1.0 equiv.) in CH2Cl2 (75 mL) was cooled to 0 °C under argon. NEt3 (2.45 mL, 17.6 mmol, 1.0 equiv.) was added dropwise over 5 min, and the resulting solution was stirred overnight while warming to ambient temperature. The reaction mixture was concentrated under reduced pressure and resuspended in CH2Cl2 (100 mL). The resulting solution was washed with aq. HCl (1 M, 2×100 mL), sat. NaHCO3 (100 mL) and brine (100 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S4 (3.55 g, 17.3 mmol, 98%) as a light brown solid. TLC (25% EtOAc in hexanes): R f =0.30 (KMnO4 staining). 1H NMR (400 MHz, CDCl3) δ 5.82-5.70 (m, 1H), 5.70-5.56 (m, 1H), 4.59 (br s, 1H), 4.12 (d, J=7.8 Hz, 2H), 3.83 (t, J=6.6 Hz, 1.24 Hz), H 9H). CAS RN: 123642-28-4. The NMR spectrum is consistent with the literature data.

[0326] (Z)-S-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-benzenesulfonothioate (S5, P1_N2_E30)

[0327]

[0328] To a stirred solution of S4 (3.55 g, 17.3 mmol, 1.0 equiv.) in DMF (70 mL) was added sodium benzyl thiosulfonate (6.81 g, 34.5 mmol, 2.0 equiv.; tech. 85%), and the solution was stirred overnight at 80 °C. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (400 mL), and extracted with EtOAc (3×100 mL). The combined organic layers were washed with water (3×200 mL) and brine (200 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S5 (5.03 g, 14.6 mmol, 85%*) as a brown oily substance. TLC (25% EtOAc in hexanes):R f =0.20 (KMnO4 staining). 1H NMR (400 MHz, CDCl3) δ 7.94-7.87 (m, 2H), 7.68-7.60 (m, 1H), 7.60-7.49 (m, 2H), 5.67-5.57 (m, 1H), 5.51-5.40 (m, 1H), 4.43 (br s, 1H), 3.67 (dq, J=7.0, 1.1 Hz, 2H), 3.60 (t, J=6.1 Hz, 2H), 1.43 (s, 9H). *The purity of the crude compound is lower than that of other thiosulfonate building blocks, but provides superior resin quality for subsequent resin loading steps.

[0329] tert-butyl-3-(((phenylsulfonyl)thio)methyl)azetidine-1-carboxylate (S6, ALN-1-57)

[0330]

[0331] To a stirred solution of 1-Boc-3-bromomethylazetidine (1.93 g, 7.71 mmol, 1.0 equiv.) in DMF (25 mL) was added sodium benzyl thiosulfonate (2.43 g, 12.3 mmol, 1.6 equiv.; tech. 85%), and the solution was stirred overnight at 80 °C. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 mL), and extracted with EtOA:hexanes (2×75 mL; 10:1, v / v). The combined organic layers were washed with water (2×75 mL), sat. NaHCO3 (75 mL) and brine (75 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S6 (2.65 g, 7.71 mmol, quant.) as a yellowish oil. TLC (33% EtOAc in hexanes): R f =0.30 (KMnO4 staining). 1 H NMR (400 MHz, CDCl3) δ 7.93 (d, J=7.5 Hz, 2H), 7.66 (t, J=7.4 Hz, 1H), 7.58 (t, J=7.6 Hz, 2H), 3.96 (t, J=8.6 Hz, 2H), 3.52 (dd, J=9.0, 5.3 Hz, 2H), 3.23 (d, J=7.8 Hz, 2H), 2.85 2.70 (m, 1H), 1.41 (s, 9H).

[0332] tert-butyl 4-((phenylsulfonyl)thio)piperidine-1-carboxylate (S7, ALN-1-41)

[0333]

[0334] To a stirred solution of 1-N-Boc-4-bromopiperidine (2.25 g, 8.51 mmol, 1.0 equiv.) in DMF (25 mL) was added sodium benzyl thiosulfonate (2.77 g, 14.0 mmol, 1.65 equiv.; tech. 85%), and the solution was stirred overnight at 80 °C. Since the reaction was not complete overnight (as determined by TLC), additional sodium benzyl thiosulfonate (1.38 g, 7.00 mmol; tech. 85%) was added, and the solution was stirred for another 24 h at 80 °C. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 mL), and extracted with EtOAc:hexanes (2 × 75 mL; 10:1, v / v). The combined organic layers were washed with water (2 × 75 mL), saturated NaHCO3 (75 mL), and brine (75 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford crude S7 (2.65 g, 7.43 mmol, 87%*) as a yellowish oil. TLC (33% EtOAc in hexanes): R f =0.44 (KMnO4 staining). 1H NMR (400 MHz, CDCl3) δ 8.01 7.89 (m, 2H), 7.71-7.61 (m, 1H), 7.60-7.50 (m, 2H), 3.89-3.63 (m, 2H), 3.55-3.39 (m, 1H), 3.11-2.91 (m, 2H), 1.96-1.86 (m, 2H), 1.64-1.51 (m, 2H), 1.42 (s, 9H). *The substitution reaction is significantly slower than that for other thiosulfonate building blocks. In addition, the purity of the crude product is lower, but ensures superior resin quality for subsequent resin loading steps.

[0335] tert-butyl 4-(((phenylsulfonyl)thio)methyl)piperidine-1-carboxylate(88, ALN-1-38)

[0336]

[0337] To a stirred solution of 1-N-Boc-4-(bromomethyl)piperidine (2.16 g, 7.76 mmol, 1.0 equiv.) in DMF (50 mL) was added sodium benzyl thiosulfonate (2.52 g, 12.8 mmol, 1.65 equiv.; tech. 85%), and the solution was stirred overnight at 80 °C. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 mL), and extracted with EtOA:hexanes (2×75 mL; 10:1, v / v). The combined organic layers were washed with water (2×75 mL), sat. NaHCO3 (75 mL) and brine (75 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude S8 (2.76 g, 7.76 mmol, quant.) as a clear oil. TLC (25% EtOAc in hexanes): R f =0.30 (KMnO4 staining). 1 H NMR (400 MHz, CDCl3) δ 8.01-7.87 (m, 2H), 7.70-7.61 (m, 1H), 7.61-7.51 (m, 2H), 4.20-3.94 (m, 2H), 2.91 (d, J=6.5 Hz, 2H), 2.58 (t, J=12.8 Hz, 2H), 1.73-1.57 (m, 3H), 1.43 (s, 9H), 1.14 0.98 (m, 2H).

[0338] (5)-2-((((9 / / -fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-chlorofuran-2-carboxamido)-propanoic acid (Thio, ALN-1-77).

[0339]

[0340] The synthesis was based on a previous method for similar amino acids: 5-chlorothiophene-2-carboxylic acid (2.44 g, 15.0 mmol, 1.5 equiv) and N-hydroxysuccinimide (1.61 g, 14.0 mmol, 1.4 equiv) were dissolved in THF (100 mL) and stirred under argon. The solution was cooled to 0°C, after which a solution of DCC (2.89 g, 14.0 mmol, 1.4 equiv) in THF (30 mL) was slowly added to the reaction mixture. The solution slowly became cloudy and was left to stir overnight, warming to ambient temperature, after which the solution was filtered. Meanwhile, Fmoc-Dap(Boc)-OH (4.26 g, 10.0 mmol, 1.0 equiv) was stirred in CH2Cl2 (20 mL; cloudy solution), and TFA (20 mL) was slowly added to the solution. The solution immediately turned yellowish-transparent, and bubbles formed. After bubbling ceased, the solution was stirred for another 30 min at ambient temperature, after which the solvent was removed under a stream of nitrogen.Excess TFA was removed by coevaporation with CH2Cl2:toluene (50 mL, 1:1, v / v). The residue was resuspended in THF (40 mL) followed by the addition of i-Pr2NEt (5.75 mL, 60.0 mmol, 6.0 equiv). The solution was then poured into the mother liquor containing NHS-activated thiophene and stirred overnight at ambient temperature. After completion of the reaction, the resulting solution was concentrated under reduced pressure, the residue was redissolved in EtOAc:hexane (300 mL, 10:1, v / v) and washed twice with water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to afford the Fmoc-protected amino acid unit (4.06 g, 8.62 mmol, 86%) as an off-white solid*. TLC (5% MeOH and 0.5% AcOH in CH2Cl2): R. f =0.2 (UV). 1H NMR (400 MHz, DMSO-d6) δ 12.74 (br s, 1H), 8.68 (t, J=5.8 Hz, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.70 (d, J=7.4 Hz, 2H), 7.65 (d, J=8.2 Hz, 1H), 7.61 (d, J=4.1 Hz, 1H), 7.41 (t, J=7.4 Hz, 2H), 7.30 (q, J=7.6 Hz, 2H), 7.18 (d, J=4.0 Hz, 1H), 4.43-4.11 (m, 4H), 3.68-3.50 (m, 2H, overlaps with residual water signal). 13 C NMR (101 MHz, DMSO) δ 171.9, 160.5, 156.0, 143.80, 143.77, 140.7, 138.7, 133.1, 128.2, 128.1, 127.6, 127.1, 125.24, 125.21, 120.1, 65.7, 53.5, 46.6, 40.3 (overlaps with solvent peak), m / z HRMS calculated for C 23 H 20 ClN2O5S + [M+N] + , 471.0776; experimental 471.0786.

[0341] Synthesis of dithiol resins

[0342] Preparation of thiol resin with high capacity

[0343]

[0344] Pre-wash: Each 25 mL fritted syringe was charged with ~0.8 g (1.11 mmol) of aminomethyl polystyrene resin (AM PS resin; 1.39 mmol / g, 100 200 mesh; Aapptec, catalog no. RAZ001) and pre-washed with MeOH (2×10 mL), CH2Cl2 (3×10 mL), 1% (v / v) TFA in CH2Cl2 (2×10 mL), i-Pr2NEt in CH2Cl2 (1.2 M; 2×10 mL for 5 min), CH2Cl2 (2×10 mL), and DMF (2×10 mL). Coupling: A solution of 3-(tritylthio)propionic acid (1.16 g, 3.33 mmol, 3.0 equiv) and HBTU (1.27 g, 3.33 mmol, 3.0 equiv) in DMF (10 mL) was activated with i-Pr2NEt (1.10 mL, 6.66 mmol, 6.0 equiv) and added to a fritted syringe and shaken for 3 h at ambient temperature. The resin was filtered and washed with DMF (3 x 10 mL) and CH2Cl2 (3 x 10 mL), and the beads were then dried (first by aspiration and then under reduced pressure overnight (<0.5 mbar)). The resin loading of MPA(Trt) was determined to be ~1.20 mmol / g* (weight basis).Capping: A solution of 5% Ac2O and 6% lutidine in DMF (12 mL, v / v / v) was added to the resin and incubated for 5 min at ambient temperature. The resin was dried and washed with DMF (3 x 10 mL) and CH2Cl2 (3 x 10 mL). Removal of protective groups: A solution of 10% TFA and 1% TIPS in CH2Cl2 (15 mL, v / v / v) was added to the resin and shaken for 1 h at ambient temperature. The resin was washed with CH2Cl2 (3 x 10 mL) and the above procedure was repeated once to obtain a high loading polystyrene thiol resin (SH PS resin), which can be used for subsequent disulfide exchange and loading of cysteamine derivatives.

[0345] Resin Preparation 1 (res1)

[0346]

[0347] Dithiol exchange: 0.4 g (0.48 mmol) of SH PS resin swollen with CH2Cl2 (10 mL) was placed into each 25 mL fritted syringe, then the resin was dried. 2-Pyridylthiocysteamine hydrochloride (0.48 g, 0.96 mmol, 2.0 equiv) was dissolved in MeOH:CH2Cl2 (19 mL, 3:7, v / v), followed by the addition of i-PnNEt (167 µL, 0.96 mmol, 2.0 equiv). The solution was added to the resin and shaken for 3 h at ambient temperature. The resin was dried and washed with MeOH:CH2Cl2 (2 × 10 mL, 3:7, v / v), DMF (2 × 10 mL), i-Pr2NEt in DMF (1.2 M; 10 mL for 5 min), DMF (3 × 10 mL), and CH2Cl2 (2 × 10 mL), and then the beads were dried (first by aspiration and then under reduced pressure overnight (<0.5 mbar)). Quality control: (1) Kaiser test; completely violet / blue coloration of beads. (2) Ellman's reagent on beads; no coloration.

[0348] Resin Preparation 2-7 (res2-7)

[0349]

[0350] Deprotection: N-Boc-protected thiosulfonate intermediate (S2, S3, S5, S6, S7, or S8; ~7.0 mmol) was dissolved in CH2Cl2 (10 mL), followed by dropwise addition of TFA until CO2 effervescence began (~10 mL). The solution was stirred for another 1 h at ambient temperature, after which the solvent was removed under a stream of nitrogen. Excess TFA was removed under reduced pressure by coevaporation with CH2Cl2:MeOH (1:1, v / v) solution to obtain the thiosulfonate salt for resin support. Dithiol exchange: Each 25 mL fritted syringe was charged with 0.8 g (0.96 mmol) of SH PS resin swollen in THF (15 mL), then the resin was dried. The TFA salt of the desired thiosulfonate (2.40–2.88 mmol, 2.5–3.0 equiv) was dissolved in THF (15 mL), and NEt3 (803 μL, 5.76 mmol, 6.0 equiv) was added. The solution was added to the resin and shaken overnight at ambient temperature.The resin was dried, washed with THF (3 x 15 mL) and CH2Cl2 (2 x 15 mL), and the beads were then dried (first by aspiration and then under reduced pressure overnight (<0.5 mbar)). Quality control: (1) Kaiser test; completely violet / blue coloration of beads. (2) Ellman's reagent on beads; no coloration.

[0351] Method using tablets

[0352] Polystyrene dithiol resins (resl-res7; estimated loading -1.20 mmol / g) were added to 96-well polypropylene (PP) filtration plates and washed with DMF (6×225 μL). Coupling was performed with 53 μL of amino acids (500 mM, 5.3 equiv), 50 μL of HATU (500 mM, 5.0 equiv), 13 μL of N-methylmorpholine (4 M, 10 equiv), and 5 μL of N-methylpyrrolidone. For coupling of Thio, t-acha, 4amPip, and 2am, 75 μL of amino acids (170 mM, 2.55 equiv), 25 μL of HATU (500 mM, 2.5 equiv), 7 μL of jV-methylmorpholine (4 M, 5.6 equiv), and 5 μL of N-methylpyrrolidone were used. All components were premixed for one minute and then added to the resin (reaction for two hours, without shaking). The coupling was performed twice, and the resin was washed with DMF (6 × 225 μL). Fmoc deprotection was performed using 20% ​​(v / v) piperidine in DMF (120 μL, 2 × 2 min), and the resin was washed with DMF (6 × 225 μL). After completion of peptide synthesis, the resin was washed with CH2Cl2 (2×200 µl) and the resin beads were dried by aspiration.

[0353] Method using syringes

[0354] Polystyrene dithiol resins (resl-res7; estimated loading -1.20 mmol / g) were added to 5 mL syringe reactors, and the resin was washed with DMF (6 × 150 μL). Coupling was performed with 210 μL amino acids (500 mM, 4.2 equiv), 200 μL HATU (500 mM, 4.0 equiv), 50 μL N-methylmorpholine (4.0 M, 8.0 equiv), and 5 μL N-methylpyrrolidone. All components were premixed for 1 min and then added to the resin (reaction for 2 hours, with shaking). Coupling was performed twice, then the resin was washed with DMF (2 × 600 μL). Removal of the Fmoc protecting group was performed using 20% ​​piperidine in DMF (450 µL, 2×2 min), and the resin was washed with DMF (7×600 µL). After completion of peptide synthesis, the resin was washed with CH2Cl2 (2×600 µL), and the resin beads were dried by aspiration.

[0355] Cleavage-reduction technique

[0356] Side Chain Removal: After automated SPPS (at a scale of 5 μmol / well), the bottom of the 96-well synthesis plate was sealed by pressing the plate onto a 6 mm thick soft ethylene vinyl acetate backing. The resin was incubated with TFA / TIPS / H2O (300 μL, 95:2.5:2.5, v / v / v) for 1 hour and covered with an adhesive PP plate lid. The TFA solution was discarded, and the resin was washed with CH2Cl2 (3×300 μL), and the procedure was repeated once. Reductive Cleavage: After air drying for at least an hour, a solution of 1,4-butanedithiol (BDT) and NEt3 in DMF (both 100 mM, 200 µL, 4 equiv relative to resin loading) was added to the resin, and the plates were shaken overnight at ambient temperature. The following day, the DMF solutions were transferred to a 96-well deep-well plate by centrifugation (1000 rpm). / min) and repeated the cleavage and reduction procedure once for 5 h, combining the resulting products in the same 96-well deep-well plate. Concentration: A solution of TFA in milliQ water (10% (v / v), 62 µl, 2 equiv relative to NEt3) was added to the wells and the peptides were dried with a Speedvac concentrator (30°C, 1750 rpm, 0.1 mbar). Resolubilization and transfer: The dried peptide pellets were dissolved in DMSO (40 µl) and transferred to a 384-well PP master plate meeting the criteria for Echo. Concentration determination: Ellman's reagent assay was used to determine the concentrations of the dithiol peptide stock solutions. Ellman's reagent. 5(DTNB) was dissolved in assay buffer (150 mM NH4HCO3 in water:MeCH (90:10, v / v), pH 8) to a concentration of 10 mM. The dithiol peptide in DMSO (135 nL) was transferred to a 384-well black microplate with a clear bottom using the Echo system via sonic drop ejection (ADE). Assay buffer (24 μL) was applied using CERTUS, after which the DTNB solution (6 μL) was added. The plates were centrifuged (400 g, 2 min), and the optical density (412 nm) was determined using a TECAN M200 plate reader. The concentration of dithiol peptides was calculated using the previously constructed calibration curve:

[0357]

[0358] Method using syringes

[0359] Side Chain Deprotection: Following automated SPPS (25 μmol scale), the fritted syringe containing the resin was incubated with TFA / TIPS / H2O (4 mL, 95:2.5:2.5, v / v / v) for 2 h at ambient temperature. The TFA solution was discarded, and the resin was washed with CH2Cl2 (5 x 4 mL) and DMF (4 mL). Reductive Cleavage: After air drying for at least 1 h, a solution of BDT and NEt3 in DMF (both 100 mM, 2.0 mL, 4 equiv. to resin loading) were added to the syringe, and the syringe was shaken overnight at ambient temperature. The next day, these DMF solutions were transferred to a 50 mL falcon conical tube. Concentration enhancement: A solution of TFA in milliQ-water (10% (v / v), 312 µL, 2 equiv relative to NEt3) was added to the peptide solution, which was dried using a Speedvac concentrator (30°C, 1750 rpm, 0.1 mbar) to yield the crude linear dithiol peptide ready for immediate cyclization in the next step.

[0360] Macrocyclization Method

[0361] Method using tablets

[0362] Transfer to microtiter plates: Given a specific concentration of each individual dithiol peptide in DMSO, 40 nmol of dithiol peptides in DMSO were transferred to 384PP plates (one plate per linker) using ADE. Peptide recovery: Since dithiol peptides oxidize over time in DMSO, complete recovery of the peptides was ensured by adding BDT and NEt3 in DMF (both 100 mM, 20 µL) to each well, followed by incubation for 30 min at ambient temperature. A solution of TFA in milliQ water (10% (v / v), 6 µl, 2 equiv relative to NEt3) was added to each well, and the peptides were dried using a Speedvac concentrator (30°C, 1750 rpm, 0.1 mbar) to obtain precipitates of fully reduced dithiol peptides. Cyclization: Bis-electrophilic linkers (LI L7) were dissolved in degassed 60 mM NH4HCO3 in MeCN:H2O (1:1 (v / v), pH 8) to a final concentration of 4 mM.The prepared linker solutions (40 μl, 4 equiv relative to the dithiol peptide) were added to 384PP plates using a liquid dispenser, which were sealed with adhesive polypropylene lids and shaken for 2 h at ambient temperature. Linker blocking: p-mercaptoethanol (β-ME) was dissolved in the prepared cyclization buffer to a final concentration of 32 mM. The prepared solution (20 μl, 4 equiv relative to the linker) was added to each well and incubated for 1 h at ambient temperature in unlidded plates. Concentration and resolubilization: Solvent was removed using a Speedvac concentrator (40°C, 1750 rpm, 0.1 mbar) to yield peptide macrocycles as pellets, which were dissolved in DMSO (10 µL) and transferred to 384LDV plates to generate 4 mM macrocyclic peptide libraries that could be immediately used in subsequent protease screening assays.Method in conical tubes.

[0363] Cyclicization: The bis-electrophilic linker was dissolved in degassed 60 mM NH4HCO3 in MeCN:H2O (1:1 (v / v), pH 8) to a final concentration of 4 mM. The resulting linker solutions (12.5 mL, 2 equiv relative to the dithiol peptide) were added to a conical tube containing the target dithiol peptide pellet, and the resulting solution was shaken for 2 h at ambient temperature. Linker Capping: After completion of the reaction (as determined by LCMS), excess linker was capped by adding β-ME (14 μL, 200 μmol, 4 equiv relative to the linker), added to the conical flask, and stirred for at least 1 h, followed by further purification. Macrocycle purification: Samples were purified by preparative HPLC on a Waters C18 RP OBD column. A linear gradient of solvent B (0.1% TFA in MeCN) to solvent A (0.1% TFA in water) was used, increasing linearly from 15% to 60% over t = 2.00–32.00 min at a flow rate of 14.0 mL / min.Pure fractions containing the target product were pooled and lyophilized to yield colorless, powdery solids. DMSO Stock Solutions: Purified macrocycles were transferred to Eppendorf tubes, and DMSO was added to obtain stock solutions of the compounds at 5 mM or 20 mM concentrations.

[0364] Biochemical research

[0365] Screening a Macrocycle Library for Proteases

[0366] Enzyme inhibition by compound libraries was assessed by determining residual enzyme activity in the presence of cyclic peptides (mean concentration for thrombin 10 μM, mean concentration for FXI, FXII, KLK5, and PKal 20 μM) at a final DMSO concentration of 1%. Crude macrocycle libraries (stock solutions of 4 mM in DMSO in 384-well LDV plates) were transferred to 1536-well OptiPlates microtiter plates via ADE. The buffer solutions used were prepared by filtration through PTFE syringe filters (0.22 μm), and the assay was started by adding protease (4.41 μl / well) in the appropriate buffer (see list below) containing bovine serum albumin (BSA; 0.1% w / v) and dispensed using a CERTUS automated liquid dispenser.The plates were incubated for 10 min at ambient temperature, after which the fluorogenic substrate in the appropriate buffer (4.5 μl) was added using a CERTUS automated liquid dispenser. The plates were centrifuged (800 g, 2 min), and the fluorescence intensity was determined using a PHERAstar plate reader (excitation 384 nm, emission 440 nm) with a time step of 150 s for 15 min. The slopes of fluorescence increase (τ) were calculated using Microsoft Excel (version 16.56). Negative controls were prepared without the addition of the macrocycle. The average value for 12 negative controls was used to calculate the residual activity using equation I below:

[0367]

[0368]

[0369] The hit compounds (compounds 195 L6, 237 L6, and 293L6) selected from the library screening were resynthesized and cyclized at a 40 nmol scale. The dried macrocyclic product was dissolved in MeCN:H2O (1.5 mL, 1:1, v / v) and fractionated on a Thermo Fisher Dionex UltiMate 3000 system using a NovaPak C18 reverse-phase column (10 × 150 mm, pore size 125 Å, particle size 5 μm). A linear gradient of solvent B (0.1% TFA in MeCN) over solvent A (0.1% TFA in water) was used, increasing linearly from 0% to 80% (for thrombin-targeted hit compounds) or from 0% to 95% (for PKal-targeted hit compounds) over t = 2.00–22.0 min at a flow rate of 4.00 mL / min. Fractions (one fraction / min) were collected in collection tubes, and the solvent was removed using a SpeedVac concentrator (30°C, 1750 rpm, 0.1 mbar).The dried contents were redissolved in DMSO (50 μl), transferred to a 384-well PP master plate, and dried using a SpeedVac concentrator (30°C, 1750 rpm, 0.1 mbar). Fractions were redissolved in DMSO (5 μl for thrombin, 2 μl for PKal), and subsequent assays were performed in black 384-well polystyrene plates with a clear bottom. A solution of the fraction in DMSO (0.5 μl) was pipetted into a microtiter plate, a solution of the enzyme in the appropriate buffer (49.5 μl; composition similar to that described on the previous page, technically using 2 nM thrombin) was added, and incubated for 10 min at ambient temperature. The substrate in buffer (25 µl, composition similar to that described on the previous page) was added, the plates were centrifuged (800 g, 2 min) and the fluorescence intensity was determined using a PHERAstar plate analyzer (excitation 384 nm, emission 440 nm) with a time step of 150 s for 15 min.Fluorescence increase slopes (τ) were calculated using Microsoft Excel (version 16.56). Negative controls were prepared by adding DMSO (0.5 μl) instead of the sample fraction. The average value for the six negative controls was used to calculate residual activity using equation I.

[0370] IC Definition 50

[0371] Half-maximal inhibitory concentration (IC) values 50) was determined by protease inhibition in an assay similar to that used for library screening. Dilution series of purified macrocyclic compounds were prepared in 384-well low dead volume (LDV) master plates and transferred to 1536-well OptiPlates using ADE technology (final volume: 45 nL macrocycle / DMSO). Enzyme solution in buffer (4.5 μL) was added using Certus and incubated for 10 min. Substrate in buffer (4.5 μL) was then added, plates were centrifuged (700 g, 2 min), and fluorescence intensity was determined using a PHERAstar plate reader (excitation 384 nm, emission 440 nm) with a 150 s time step for 15 min. The fluorescence increase slopes (τ) were calculated using Microsoft Excel (version 16.56). Negative controls were prepared without the addition of the macrocycle.The mean value for the 12 negative controls was used to calculate residual activity using equation I. IC50 values ​​were obtained by fitting the obtained data to a concentration-response equation [limitations?] using GraphPad Prism (version 6.0.1), and K values. i calculated based on IC 50 using the Cheng-Prusov equation 11 :

[0372]

[0373] where [S]0 is the initial concentration of the substrate, and K M - Michaelis-Menten constant 12 for a given enzyme and substrate.

[0374] --->

[0375] SEQUENCE LISTING

[0376] <110> ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL)

[0377] <120> Method for obtaining a library of peptides or a peptide

[0378] <130> AE1383 PCT

[0379] <140> PCT / EP2022 / 061138

[0380] <141> 2022-04-27

[0381] <150> EP 21 174 036.0

[0382] <151> 2021-05-17

[0383] <160> 1

[0384] <170> BiSSAP 1.3.6

[0385] <210> 1

[0386] <211> 19

[0387] <212> PRT

[0388] <213> Artificial Sequence

[0389] <220>

[0390] <223> p53 fluorescent peptide probe

[0391] <400> 1

[0392] Gly Ser Gly Ser Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu

[0393] 1 5 10 15

[0394] Pro Glu Asn

[0395] <---

Claims

1. A method for producing a library of peptides or an isolated peptide, comprising (a) cleaving one or more linear dithiol peptides containing a sulfhydryl group at the N-terminal region of said one or more peptides and immobilized via a disulfide bridge at the C-terminal region of said one or more peptides on a solid phase, from said solid phase by (i) an agent that reduces said disulfide bridge, thereby cleaving said one or more linear dithiol peptides from said solid phase, wherein said agent is selected from 1,3-propanedithiol, 1,4-butanedithiol, 2,4-pentanedithiol, ethane-1-thiol, propane-1-thiol, butane-1-thiol, propane-2-thiol, 2-methyl-1-propanethiol, butane-2-thiol, 2-methylpropane-2-thiol, 2-hydroxy-1-ethanethiol, 1,2-ethanedithiol, 2-propene-1-thiol, 3-methyl-1-butanethiol, thiophenol, benzylthiol, 2-butene-1-thiol, 3-butene-1-thiol, 2-methyl-2-propene-1-thiol and 3-methyl-2-butene-1-thiol and is removed by evaporation, or (ii) a base that deprotonates the said sulfhydryl group in the N-terminal region of said one or more linear dithiol peptides, which is a trialkylamine, thereby inducing an intramolecular disulfide exchange with the resulting cleavage and cyclization of said one or more linear dithiol peptides from said solid phase in the form of one or more cyclic peptides.

2. The method of claim 1, further comprising the step (b) of cyclizing said one or more linear dithiol peptides cleaved by said agent.

3. The method according to claim 2, wherein said one or more dithiol peptides are cyclized using at least one bis-electrophilic reagent or by disulfide oxidation.

4. The method according to claim 1, wherein the disulfide bonds of said one or more cyclic peptides according to subparagraph (ii) are reduced and said peptides are recyclized using a bis-electrophilic reagent.

5. The method according to any of paragraphs 1-4, where said agent is selected from 1,3-propanedithiol, 1,4-butanedithiol or 2,4-pentanedithiol, and is preferably 1,4-butanedithiol (BDT) or The base in question is N,N-diisopropylethylamine (DIPEA).

6. The method according to any one of claims 1 to 5, comprising, before step (a), step (a') of synthesizing said linear dithiol peptides on said solid phase.

7. The method according to any one of claims 1 to 6, wherein the amino acid side chains of said linear dithiol peptides are protected with protecting groups, and said method further comprises, before step (a) and, if present, after step (a'), removing said protecting groups while said linear dithiol peptides are immobilized on said solid phase.

8. The method according to any one of claims 1 to 7, wherein at least some of said linear dithiol peptides contain a primary or secondary amine, and said method further comprises modifying said primary or secondary amine using a carboxylic acid, wherein the cyclic peptides containing primary or secondary amine and carboxylic acids are preferably transferred by acoustic dosing.

9. The method according to any one of claims 1 to 8, wherein said solid phase comprises a resin, preferably a non-polar resin and more preferably a polystyrene resin.

10. The method according to any one of claims 1-9, wherein said linear dithiol peptides comprise linear dithiol peptides having a molecular weight of less than 1000 Da, and preferably less than 600 Da, and / or linear dithiol peptides containing 3 or 4 amino acids, and preferably 3 amino acids.

11. A method for screening a peptide library obtained by the method according to any one of claims 1-10, comprising (c) contacting said library of peptides with a target molecule, and (d) screening said peptide library to identify a peptide that binds to said target molecule.

12. The method according to claim 11, wherein step (c) is carried out without prior purification of said peptide library, and / or the peptide inhibits said target molecule in step (d).

13. The method of claim 11 or 12, wherein steps (c) and (d) are carried out in the same wells in which said primary or secondary amine was modified using a carboxylic acid.

14. The method according to any one of claims 11-13, wherein said target molecule is a protein, a peptide, a nucleic acid molecule, a carbohydrate or a fatty acid, and is preferably a protein or a peptide.

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