Method for preparing a library of peptides or peptides

Abstract

The present invention relates to a method for preparing a library of peptides or isolated peptides, comprising the steps of: (a) releasing one or more linear dithiol peptides, which have a sulfhydryl group at the N-terminal region of the one or more peptides and are immobilized on a solid phase via a disulfide bridge at the C-terminal region of the one or more peptides, from the solid phase with (i) an agent that reduces the disulfide bridge, thereby releasing the one or more linear dithiol peptides from the solid phase, which agent is volatile and removable by evaporation, or (ii) a base that deprotonates the sulfhydryl group at the N-terminal region of the one or more linear dithiol peptides, thereby inducing intramolecular disulfide exchange, thereby releasing the one or more linear dithiol peptides from the solid phase in the form of one or more cyclic peptides.

Description

[Technical field]

[0001] The present invention relates to a method for preparing a library of peptides or isolated peptides, comprising the steps of: (a) releasing one or more linear dithiol peptides, which have a sulfhydryl group at the N-terminal region of the one or more peptides and are immobilized on a solid phase via a disulfide bridge at the C-terminal region of the one or more peptides, from the solid phase with (i) an agent that reduces the disulfide bridge, thereby releasing the one or more linear dithiol peptides from the solid phase, which agent is volatile and removable by evaporation, or (ii) a base that deprotonates the sulfhydryl group at the N-terminal region of the one or more linear dithiol peptides, thereby inducing intramolecular disulfide exchange, thereby releasing the one or more linear dithiol peptides from the solid phase in the form of one or more cyclic peptides. [Background technology]

[0002] Numerous documents, including patent applications and manufacturer's manuals, are cited in this specification. The disclosures of these documents are not considered relevant to the patentability of this invention, but are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] Peptide libraries, especially libraries of cyclic peptides (a type of macrocycle), are of great interest to the pharmaceutical industry because they allow screening of peptides with the ability to bind to challenging targets that have previously been difficult or impossible to achieve through the generation of classical small molecule-based ligands.

[0004] Currently, over 40 cyclic peptides have been approved as drugs, and over 100 cyclic peptides are being evaluated in various stages of clinical trials (A. Zorzi et al., Curr. Opin. Chem. Biol., 2017, 38, 24-29). Innovative strategies to design cyclic peptide ligands based on protein epitopes or to isolate target-specific cyclic peptides from large combinatorial libraries of gene-encoded peptides have further spurred 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).

[0005] Of particular interest are macrocycles that have a sufficiently small size, ideally well below 1 kilodalton (kDA), and a small polar surface area to place intracellular targets within reach. Macrocycles of interest include small cyclic peptides, non-peptide-based macrocycles, or macrocycles that contain peptide and non-peptide components. However, the development of macrocyclic ligands that bind to targets of interest and are membrane permeable to reach intracellular targets is difficult due to the relatively small number of macrocycles in commercially available collections or the lack of methods to efficiently synthesize new macrocyclic libraries.

[0006] The challenging step in the synthesis of macrocycles is the conversion of linear molecules to cyclic molecules. Most reactions have macrocyclization yields well below 90% and show a large variability in yields for different precursors (e.g., different linear peptide sequences), which poses a problem in the synthesis of libraries. Macrocycle libraries offered by major providers such as Asinex, ChemBridge, and Polyphor are all based on molecules that are individually purified after the macrocyclization step, because the product without purification is not pure enough for most compounds. The need for purification limits the number of macrocycles that can be produced in parallel, which in turn limits the size of the library, with commercially available libraries being less than 30,000 molecules.

[0007] Macrocyclization reactions that generally show high cyclization yields, typically above 90%, over a broad substrate range involve the use of bis-electrophiles, e.g., bis-(bromomethyl)benzene (S N 2 reactions), bis-(bromoacetamido)benzene (S N 2 reactions), haloacetones (S N 2 reaction), vinyl sulfoxide (Michael addition) or hexafluorobenzene (S NThe cyclization of peptides is achieved by cyclization of peptides via two thiol groups located at the distal ends of the peptide, such as by the Ar reaction (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; S.S. Kale et al., Nat. Chem., 2018, 10, 715-723). Such reactions have been used to develop (bi)cyclic peptides that mimic linear or non-linear epitopes (P. Timmerman et al., ChemBioChem, 2005, 6, 821-824), to deploy (bi)cyclic peptides by phage display (C. Heinis et al., Nat. Chem. Biol., 2009, 5, 502-507) or other display methods, to stabilize α-helical peptides in helical structures (H. Jo et al., J. Am. Chem. Soc., 2012, 134, 17704-17713), or to cyclize peptides for other purposes.

[0008] Recently, a large library of cyclic peptides with molecular weights below 1 kilodalton was synthesized by combinatorial cyclization of a large number of linear dithiol peptides with about 20 different bis-electrophiles (unpublished results from the Laboratory of Therapeutic Proteins and Peptides, EPFL, Group Heinis). Different combinations of dithiol peptides and cyclization reagents were mixed in separate wells of a 384-microwell plate. The high cyclization yields allowed us to screen cyclic peptides for protein target binding without prior purification. Eliminating the throughput-limiting purification step enabled the screening of large libraries.

[0009] Combinatorial peptide cyclization strategies using bis-electrophiles require multiple dithiol peptides with different sequences. Regardless of the method used to identify lead peptides, peptide library and peptide drug development typically involves multiple iterative cycles of synthesizing tens to hundreds of peptide variants to improve key properties such as binding affinity, specificity, stability, and pharmacokinetic properties, thereby preparing a large number of peptides.

[0010] A further major obstacle in the development of cyclic peptide therapeutics is the chromatographic purification of peptides after synthesis, which, even if automated and optimized, is costly due to the sequential handling of each peptide and the high reagent consumption (solvent). The main reason for the need for purification is the macrocyclization reaction, which is typically the most challenging step in cyclic peptide synthesis (CJ White et al., Nat. Chem., 2011, 3, 509-524). Most macrocyclization reactions exhibit yields less than 90%, and their efficiency often varies wildly depending on the peptide sequence and length. Chromatographic purification is also necessary to remove reagents and scavengers added to liberate the peptide, as well as side-chain protecting group by-products generated during global deprotection. Summary of the Invention

[0011] It is apparent from the above that there is an immediate need for new methods, particularly methods that do not require complex preparative steps, to generate large peptide libraries, or one or more peptides that can be added to a peptide library, which can then be screened for peptide therapeutics. This need is addressed by the present invention.

[0012] In a first aspect, the present invention relates to a method for preparing a library of peptides or isolated peptides, comprising the step of: (a) releasing one or more linear dithiol peptides, which have a sulfhydryl group at the N-terminal region of the one or more peptides and are immobilized on a solid phase via a disulfide bridge at the C-terminal region of the one or more peptides, from the solid phase with (i) an agent that reduces the disulfide bridge, thereby releasing the one or more linear dithiol peptides from the solid phase, the agent being volatile and removable by evaporation, or (ii) a base that deprotonates the sulfhydryl group at the N-terminal region of the one or more linear dithiol peptides, thereby inducing intramolecular disulfide exchange, thereby releasing the one or more linear dithiol peptides from the solid phase in the form of one or more cyclic peptides.

[0013] The term "comprise" is generally used in the sense of include / including, i.e. permitting the presence of one or more features or components. The terms "comprise" and "comprising" also include the more restrictive terms "consist of" and "consisting of".

[0014] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The term "peptide" as used herein refers to a polymer that comprises at least one amino acid and at least one peptide bond. A peptide preferably comprises a plurality of amino acids, for example, two or more, three or more, four or more, or five or more. A peptide may also comprise non-amino acid building blocks and non-peptide bonds. Many of the peptides described in the accompanying examples contain building blocks at their termini, such as mercaptopropanoic acid (MPA) and cysteamine (MEA), which are not amino acids because they do not contain an amino group (in the case of MPA) or a carboxylic acid (in the case of MEA).

[0015] Further examples of non-amino acid building blocks containing thiol groups and suitable for incorporation into peptides (as MEAs), especially at the C-terminus, are 3-aminopropane-1-thiol, 3-(methylamino)propane-1-thiol, (Z)-4-aminobut-2-ene-1-thiol, piperidine-4-thiol, 4-(mercaptomethyl)piperidine, and 3-(mercaptomethyl)azetidine. See the table and formulas below. Of these non-amino acid building blocks, those with secondary amines are particularly preferred in the development of membrane-permeable macrocycles, since coupling of the secondary amine with a carboxylic acid (of an adjacent amino acid) results in an amide bond without a hydrogen bond donor group. [Table A]

[0016] [ka]

[0017] Further examples of non-amino acid building blocks that contain a thiol group and are particularly suitable for incorporation into peptides at the N-terminus (as MPAs) are 2-mercaptoacetic acid, (R)-2-mercaptopropanoic acid, 5-(mercaptomethyl)furan-2-carboxylic acid, and 3-(mercaptomethyl)benzoic acid. See the table and formulas below. [Table B]

[0018] [ka]

[0019] A variety of other non-amino acid building blocks are suitable for incorporation into dithiol peptides, especially at internal positions. For example, building block A-COOH can be attached to the amino group of the previous residue on the solid phase, and then building block B-NFh can be added, in which case the functional groups of A and B react with each other to form a covalent bond. This latter reaction may result in a linkage that is not a peptide bond. An example of such a reaction is the well-established so-called "submonomer" strategy, in which a haloacetic acid, typically bromoacetic acid activated (e.g., by diisopropylcarbodiimide), is first attached to the amino group of the peptide growing on the solid phase (the haloacetic acid represents the building block A-COOH). In a second step and chemical reaction, an amine (B-NH 2 (representing 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)pyrazine-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, as preferred examples. See the table and formulas below. [Table C]

[0020] [ka]

[0021] The term "peptide" preferably refers to a short chain of amino acids linked by peptide bonds. Also, the short chain of amino acids linked by peptide bonds may contain non-amino acid building blocks at its ends, such as MPA and cysteamine MEA. Peptides are distinguished from proteins or polypeptides based on size, and preferably include less than 50 amino acids, preferably less than 40 amino acids, less than 30 amino acids, less than 20 amino acids, less than 10 amino acids, and less than 5 amino acids.

[0022] The term "isolated peptide" as used herein indicates that the peptide is not bound to a solid phase and is released from the solid phase.The isolated peptide is generally in the form of a free peptide, for example, in solution.There may be one or more copies of the isolated peptide in the solution.Therefore, there may also be one or more copies of the peptide to be isolated on the solid phase before the peptide is isolated.

[0023] The term "amino acid" as used herein generally refers to an amine (-NH 2 It refers to an organic compound that consists of a carboxylic acid (-COOH) functional group and a carboxylic acid (-NH) functional group. The simplest amino acid, glycine, has no side chain (the formula is H 2 NCH 2In amino acids with a carbon chain attached to the α-carbon (such as lysine), the carbons are labeled in the order α, β, γ, δ, etc. In some amino acids, the amine group may be attached, for example, to the α-carbon, β-carbon, or γ-carbon, and thus these are called α-amino acids, β-amino acids, or γ-amino acids, respectively. The term "amino acid" preferably refers to a group having the general formula H 2 It refers to α-amino acids (also called 2-amino acids or α-amino acids), which generally have the formula NCHRCOOH, where R is an organic substituent called a "side chain", as well as β-amino acids, γ-amino acids, and δ-amino acids, which contain multiple carbon atoms between the amine and the carboxylic acid. The simplest α-amino acid is alanine (formula H 2 NCHCH 3 In the case of aryloxycarbonyl (COOH), the side chain is a methyl group. Amino acids according to the invention are L-amino acids or D-amino acids.

[0024] Amino acids include the so-called standard or canonical amino acids. These 21 α-amino acids are directly encoded by codons of the universal genetic code. They are the proteinogenic α-amino acids identified in eukaryotes. These amino acids are referred to herein by the so-called one-letter code: [Table D]

[0025] As mentioned above, the side chain of an amino acid is an organic substituent, which in the case of an α-amino acid is linked to the α-carbon atom. Thus, the side chain is a branch from the parent structure of the amino acid. Amino acids are usually classified by the properties of their side chain. For example, the side chain can make an amino acid weakly acidic (e.g., amino acid D and amino acid E) or weakly basic (e.g., amino acid K and amino acid R), hydrophilic if the side chain is polar (e.g., amino acid L and amino acid I), or hydrophobic if the side chain is non-polar (e.g., amino acid S and amino acid C). Aliphatic amino acids have a side chain that is an aliphatic group. The aliphatic group makes the amino acid non-polar and hydrophobic. The aliphatic group is preferably an unsubstituted branched or straight chain alkyl. Non-limiting examples of aliphatic amino acids are A, V, L, and I. In cyclic amino acids, one or more series of atoms in the side chain are linked 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 kept distinct from the ring formed in the case of cyclic peptides, as further detailed herein below. The former ring of cyclic amino acids is part of the side chain of a single amino acid, while the latter ring is formed between two thiol groups of dithiol peptides. Aromatic amino acids are the preferred form of cyclic amino acids. In aromatic amino acids, the ring is aromatic. In terms of the electronic properties of the molecule, aromaticity indicates a directionality in which the conjugated ring of unsaturated bonds, the single pair of electrons, or the empty molecular orbital exhibits stronger stabilization than would be expected by the stabilization of conjugation alone. Aromaticity can be considered a manifestation of cyclic delocalization and resonance. Hydrophobic amino acids have non-polar side chains that make the amino acid hydrophobic. Non-limiting examples of hydrophobic amino acids are M, P, F, W, G, A, V, L, and I. Polar uncharged amino acids have non-polar side chains that do not have charged residues. Non-limiting examples of polar uncharged amino acids are S, T, N, Q, C, U, and Y. A polar charged amino acid has a non-polar side chain with at least one charged residue. Non-limiting examples of polar charged amino acids are D, E, H, K, and R.

[0026] A "dithiol peptide" is a peptide that contains two or more thiol groups, preferably only two thiol groups. The thiol group is part of either an amino acid building block or a non-amino acid building block of the peptide. The amino acid building block or non-amino acid building block bearing the thiol group can be any of the following: Cysteine ​​or cysteine ​​analogues, such as homocysteine, penicillamine, D-cysteine, etc. Building blocks containing thiol groups and carboxylic acids, such as mercaptopropanoic acid (MPA), · Building blocks containing thiol groups and amines, such as cysteamine (MEA).

[0027] When a linear dithiol peptide is immobilized on a solid phase, one thiol group is "liberated" and becomes first a protected sulfhydryl group (RS-PG; PG = protecting group) and then a sulfhydryl group (R-SH) after removal of the thiol protecting group, while the other thiol group forms a disulfide bond (R 1 -SSR 2 ). A disulfide bridge is formed when a sulfur atom of one thiol-containing building block (linked to the solid phase) forms a single covalent bond with a sulfur atom of a thiol-containing building block at the C-terminal region of the peptide. Means and methods for synthesizing peptides via disulfide bonds to the solid phase are known in the art and are described in further detail herein below and illustrated by the accompanying examples.

[0028] The term "linear peptide" refers to a linear short chain of amino acids linked by peptide bonds, which may also include non-amino acids and non-peptide bonds as described herein above in connection with a "peptide" according to the present invention. In a linear peptide, the sequence of atoms in the peptide is not linked to form a macrocyclic ring or ring.

[0029] The term "cyclic peptide" means that a series of 12 or more atoms in a peptide are linked to form a macrocyclic ring or ring. The cyclic peptide is preferably a monocyclic peptide, which means that only two sites in the peptide are linked to form a ring or ring. The ring or ring is formed according to the present invention by the involvement of two thiol groups of a dithiol peptide. When the two thiol groups are directly bonded, the ring or ring is formed by a disulfide bridge. The two thiol groups of a dithiol peptide can also be linked by a bis-electrophile, as described in more detail herein below.

[0030] A library of peptides refers to a composition or article of manufacture that includes a plurality of different peptides. When the library is a composition, the composition comprises a mixture of different peptides. The composition is preferably a solution, more preferably DMSO or an aqueous solution. The solution can be dried, for example, by lyophilization or centrifugal vacuum evaporation (for example, using a SpeedVac system). In this case, the composition can be in the form of a dry powder that can be dissolved by a desired solvent. For such a composition, the number of different peptides in the 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 more different peptides are synthesized.

[0031] The article of manufacture comprises different wells, 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 in parallel (typically on a solid support) in different wells, so that one peptide can be identified (in multiple copies) per well. The multiple wells of the article of manufacture together form a library of peptides. The library in the format of the article of manufacture is preferred because when such a library is screened for binders or inhibitors against a specific target molecule (described later in this specification), it is immediately known in which well the desired library member can be identified, and no further complex isolation or identification of the desired library member is required.

[0032] Although one peptide per well is preferred, it is also possible to synthesize more than one peptide per well, e.g., 2, 3, 4 or 5 different peptides in parallel in each well (typically on a solid support), thereby obtaining wells with 2, 3, 4 or 5 different peptides per well. The number of different peptides per well can be adjusted as required, e.g., by the number of different peptides immobilized on the solid support and / or by mixing solid supports on which one or more different peptides have been synthesized.

[0033] The library of different peptides preferably comprises 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 100, at least 500, at least 1000, at least 1536, at least 3456, at least 10000, and at least 100000 different peptides.

[0034] The N-terminal region preferably refers to one of the up to three N-terminal amino acids or building blocks of one or more linear dithiol peptides, more preferably one of the up to two N-terminal amino acids or building blocks, and most preferably up to one N-terminal amino acid or building block. In the most preferred case, the sulfhydryl group is part of the amino acid or building block at the N-terminal position of one or more linear dithiol peptides.

[0035] Similarly, the C-terminal region preferably means one of up to three C-terminal amino acids or building blocks of one or more linear dithiol peptides, more preferably one of up to two C-terminal amino acids or building blocks, and most preferably one C-terminal amino acid or building block. In the most preferred case, the peptide or peptides are immobilized to the solid phase via a C-terminal disulfide bridge.

[0036] The term "solid phase" refers to any solid material or support on which peptides can be synthesized via disulfide bonds, for example via solid phase peptide synthesis (SPPS).

[0037] The term "agent" as used herein means any molecule capable of reducing disulfide bridges and thereby liberating one or more linear dithiol peptides from the solid phase. The agent may therefore be referred to as a reducing agent or liberating agent. Various reducing agents are known in the prior art. In biochemistry, thiols such as β-mercaptoethanol (β-ME) or dithiothreitol (DTT) function as reducing agents. According to the present invention, the agent is volatile and can be removed by evaporation, preferably by evaporation under vacuum, most preferably by centrifugal vacuum evaporation. Volatile substances are easily converted to gas by evaporation. Evaporation is a type of evaporation that occurs at the surface of a liquid as it changes to a gas phase. The ability of molecules of a liquid to evaporate is primarily based on the amount of kinetic energy that each particle possesses. The rate of evaporation increases with increasing temperature, but even at low temperatures, evaporation is possible if each molecule of the liquid has more than the minimum amount of kinetic energy required for evaporation. The use of an agent that is volatile and can be removed by evaporation is technically advantageous since it can be removed from a composition comprising a library of peptides or isolated peptides produced by the method of the present invention. The removal of one or more peptides from the solid support by an agent, also referred to herein as "reductive release," is illustrated in Figure lb. As is evident from Figure lb, the one or more peptides are released by the agent in the form of one or more linear peptides, in which the two thiol groups are "free" sulfhydryl groups.

[0038] A base is a substance that can accept a proton (such as a Brønsted base), donate an electron (such as a Lewis base), or form hydroxide ions (OH - The base used in accordance with the present invention is any chemical compound that produces one or more linear dithiol peptides (RS -The sulfhydryl group at the N-terminal region of the dithiol peptide can be deprotonated. The deprotonated sulfhydryl group induces intramolecular disulfide exchange, thereby releasing one or more linear dithiol peptides from the solid phase in the form of one or more cyclic peptides. The deprotonated sulfhydryl group contains a reactive S that acts as a nucleophile to induce a thiol-disulfide exchange reaction at the disulfide bond. The removal of one or more peptides from the solid support by base is also referred to herein as "cyclizing release" and is illustrated by FIG. 1a. As is evident from FIG. 1a, one or more peptides are released by base in the form of cyclic peptides, and the two thiol groups of the dithiol peptides are linked by a disulfide bond.

[0039] As discussed above, the method of claim 1, item (i) is a "reductive release" approach that results in linear dithiol peptides, whereas the method of claim 1, item (ii) is a "cyclizing release" approach that results in cyclic dithiol peptides. As will become apparent below, both options represent significant advances over prior art methods for preparing libraries of peptides or isolated peptides.

[0040] The "reductive release" strategy, in which dithiol peptides immobilized via disulfide bridges on a solid support are released from the solid phase by disulfide reduction from the resin, is shown diagrammatically in Figure 1b. In contrast to cyclizing release, the efficiency of reductive release does not depend on the length and amino acid composition of the peptide. In particular, this approach also works efficiently for short peptides that cannot be efficiently cyclized via disulfide formation (but can be cyclized via bis-electrophilic linkers, since this cyclization is less sterically demanding since an additional atom is added to the macrocyclic backbone by the linker). A challenge in reductive peptide release was that a reducing agent had to be added to the peptide, and this reagent prevented subsequent cyclizing reactions with bis-electrophiles (i.e., reactions with electrophilic groups). This drawback is overcome by using volatile reducing agents that are removable from the peptides in a relatively simple step, for example by evaporation under vacuum. For example, volatile reducing agents can be removed by centrifugal vacuum evaporation of the peptides in a 96-well plate.

[0041] Only a few studies have described the release of peptides from the solid phase by reductive cleavage of disulfide bridges, all of which liberate peptides bearing a single thiol group with other applications in mind, such as the generation 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 generation of head-to-tail cyclized peptides containing a thiol handle (W. Tegge et al., J. Pept. Sci., 2007, 13, 693-699), or the cyclization of peptides via a single thiol group (A. A. Virgilio et al., Tetrahedron Lett., 1996, 37, 6961-6964), identification of released peptides by 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 rely on the use of the reducing agents tris-(2-carboxyethyl)phosphine (TCEP) (J. Mery et al., Int. J. Pept. Protein Res., 1993, 42, 44-52; AAVirgilio 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) (DS Kemp et al., J. Org. Chem., 1986, 51, 1821-1829), which therefore cannot be removed by evaporation and requires a purification step. In only one of these studies was the volatile reducing agent β-mercaptoethanol (β-Me) used to release disulfide-linked peptides from the solid support (O. Lack et al., Helv. Chim. Acta, 2002, 85, 495-501). However, in this application, peptides were released from single beads, but only in small amounts from a polar (PEGA), poorly loaded (0.2 mmol / gram) resin with a high swelling volume, which is not suitable for the presently envisioned application. No studies have reported the synthesis and reductive release of dithiol peptides, nor have any studies applied this approach to the generation of cyclic peptide libraries.

[0042] To the best of our knowledge, the strategy of cyclic disulfide release is entirely new and has never been used to generate cyclic peptides. The closest strategy to cyclative release is the oxidative release of thioether-immobilized peptides (BH 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 generation due to low yields, dimeric by-products, and the presence of oxidizing agents in the eluted products that require removal by purification. Here, cyclative release is caused by a base that deprotonates the N-terminal sulfhydryl group. If a volatile base is used, it can be removed by evaporation, so that the only product in the reaction tube or well of a microtiter plate is one or more pure cyclic peptides. If a non-volatile base is used, it can also be neutralized by acid. An important requirement for the cyclization strategy is that peptides can be synthesized on the solid phase immobilized via disulfide bridges, which must be sufficiently stable during peptide synthesis, especially during Fmoc deprotection with piperidine. Several studies have reported the solid-phase synthesis of disulfide-bridged immobilized peptides for applications ranging from the generation of peptide-protein conjugates to the reductive release of peptides 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 Immobilization, 2007, 13, 693-699). Lett., 1996, 37, 6961-6964; O. Lack et al., Helv. Chim. Acta, 2002, 85, 495-501; D.S. Kemp et al., J. Org. Chem., 1986, 51, 1821-1829). Mery, J. and coworkers have suggested that the stability of the disulfide bridge depends on the substituent on the carbon atom adjacent to the sulfur and on the linker NH with a bulky methyl group. 2 -CH2 -CH 2 -SSC(CH 3 ) 2 -COOH, which was sufficiently stable for Fmoc peptide synthesis (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). 2 -CH 2 -CH 2 -SS-CH 2 -C(NH 2 ) Disulfide-immobilized peptides via H-COOH have been synthesized, but for short peptides, despite the linker being rather unhindered, it was found that no significant amount of peptide was present, possibly due to the limited number of times the beads were exposed to piperidine (Y. Wu et al., Chem. Commun., 2020, 56, pp. 2917-2920).

[0043] According to a preferred embodiment of the first aspect of the invention, the method comprises, after step (a), a step of removing the agent by evaporation. As mentioned above, the agents used in the claims are volatile and removable by evaporation. According to this preferred embodiment, a step of removing the agent by evaporation forms part of the method. The evaporation is preferably performed under vacuum (e.g. using a SpeedVac system). The agent is most preferably removed by centrifugal vacuum evaporation.

[0044] Also, the base according to the present invention is preferably volatile and removable by evaporation, in which case the process of the first aspect of the invention preferably comprises, after step (a), a step of removing the base by evaporation.

[0045] Instead of removing the drug by evaporation, the drug may also be removed by freeze-drying. According to another preferred embodiment of the first aspect of the invention, the method further comprises the step (b) of cyclizing one or more linear dithiol peptides liberated by the agent.

[0046] As mentioned above, in the case of "reductive release" by an agent, one or more peptides are released by the agent in the form of a linear peptide, in which the two thiol groups are "free" sulfhydryl groups. The two sulfhydryl groups can be used to cyclize the peptide.

[0047] According to a more preferred embodiment of the first aspect of the present invention, the one or more dithiol peptides are cyclized by at least one bis-electrophile or by disulfide oxidation.

[0048] The two sulfhydryl groups can be linked directly to form a disulfide bond (disulfide oxidation) or can be linked via a linking molecule, in particular via a bis-electrophile. A variety of bis-electrophiles are commercially available or can be readily synthesized by routine chemical reactions. Different bis-electrophiles can be used in parallel reactions to generate many different cyclic peptides from one dithiol peptide.

[0049] Electrophiles act as electron pair acceptors in the formation of new bonds. In the case of nucleophilic substitution, the leaving group leaves as a negatively charged species. Bis-electrophiles are compounds that contain at least two functional groups that can react with two sulfhydryl groups, whereby the sulfhydryl groups are linked via the bis-electrophile.

[0050] Disulfide oxidation links two sulfhydryl groups of a dithiol peptide with a direct disulfide bond. According to another preferred embodiment of the first aspect of the present invention, the disulfide bonds of the one or more cyclic peptides of item (ii) are reduced and the peptides are recyclized with a bis-electrophile.

[0051] As described herein above, according to item (ii) of the first aspect of the present invention, one or more peptides are released by a base in the form of a cyclic peptide, the two thiol groups of the dithiol peptide being linked by a disulfide bond.

[0052] These disulfide bonds are preferably reduced by agents as described in connection with item (i) of the first aspect of the invention, resulting in linear peptides with two "free" sulfhydryl groups. These linear peptides can then be recyclized by bis-electrophiles as described herein above.

[0053] According to a further preferred embodiment of the first aspect of the invention, the agent is selected from the group consisting of 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, preferably 1,3-propanedithiol, 1,4-butanedithiol or 2,4-pentanedithiol, most preferably 1,4-butanedithiol (BDT); and / or the 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, preferably a tertiary, secondary or primary amine, more preferably a tertiary amine, even more preferably a tri-alkylamine, most preferably N,N-diisopropylethylamine (DIPEA).

[0054] Preferred examples of reducing agents that are removable by evaporation are shown in Table 1.

[0055] [Table 1]

[0056] [ka]

[0057] 1,4-butanedithiol (BDT) is used as the agent in the accompanying examples and is therefore the most preferred agent. Preferred examples of bases for deprotonating sulfhydryl groups to induce disulfide exchange and release of cyclized peptides are shown in Table 2.

[0058] [Table 2-1]

[0059] [Table 2-2]

[0060] The list in Table 2 is not exhaustive, but gives examples of useful reagents of each type / subtype. Preferred are trialkyl tertiary amines, with particular examples being given. N,N-diisopropylethylamine (DIPEA) is used as the base in the accompanying examples, and is therefore the most preferred base.

[0061] According to a further preferred embodiment of the first aspect of the invention, the method comprises, prior to step (a), a step (a') of synthesising a linear dithiol peptide on a solid phase. Solid-phase peptide synthesis (SPPS) is a common technique for peptide synthesis. Normally, peptides are synthesized from the carbonyl group (C-terminus) to the amino group (N-terminus) of the amino acid chain in the SPPS method, but in cells, peptides are biologically synthesized in the reverse direction. In peptide synthesis, an amino-protected amino acid is coupled to a solid-phase material, forming a covalent bond between the carbonyl group and the solid-phase material. The amino group is then deprotected and reacted with the carbonyl group of the next N-protected amino acid, which is now loaded with a dipeptide. The cycle is repeated to form the desired peptide chain.

[0062] According to another preferred embodiment of the first aspect of the present invention, the side chains of the amino acids of the linear dithiol peptide are protected by protecting groups, and the method further comprises the step of removing the protecting groups prior to step (a) and, if present, after step (a') while the linear dithiol peptide is immobilized on the solid phase.

[0063] Because amino acids have multiple reactive groups, peptide synthesis must be performed carefully to reduce the length of the peptide chain and to avoid side reactions that may lead to branching. To minimize side reactions and facilitate peptide formation, chemical groups have been developed that are attached to amino acid reactive groups and block or protect the functional groups from nonspecific reactions.

[0064] Typically, the purified individual amino acids used in the synthesis of the peptide are reacted with these protecting groups prior to synthesis, and then the specific protecting group is removed from the newly added amino acid immediately after coupling (a step called deprotection) to allow the next amino acid to be attached to the growing peptide chain in the proper orientation. Once peptide synthesis is complete, all remaining protecting groups are removed from the nascent peptide.

[0065] Three types of protecting groups are commonly used in the art depending on the method of peptide synthesis and are described below. The N-terminus of an amino acid is protected by what are called "temporary" protecting groups because they are relatively easily removed to allow the formation of a peptide bond. Two common N-terminal protecting groups are tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), with each group having distinct characteristics that determine their uses. Boc requires a moderately strong acid such as trifluoroacetic acid (TFA) to remove it from the newly added amino acid, while Fmoc is a base-labile protecting group and is removed with a weak base such as piperidine. Boc chemistry requires acidic conditions for deprotection, while Fmoc, which was not reported for another 20 years, is cleaved under mildly basic conditions. Due to the milder deprotection conditions, Fmoc chemistry is more commonly used in commercial situations due to its higher quality and yield, but Boc is preferred for complex peptide synthesis or when base-sensitive unnatural peptides or analogs are required.

[0066] The use of a C-terminal protecting group depends on the type of peptide synthesis used: in solution phase peptide synthesis, protection of the C-terminus of the first amino acid (the C-terminal amino acid) is necessary, whereas in solid phase peptide synthesis, the solid support (e.g., a resin) does not, since it serves as a protecting group for the only C-terminal amino acid that requires protection.

[0067] Amino acid side chains represent a wide range of functional groups and are therefore sites of significant side chain reactivity during peptide synthesis. For this reason, many different protecting groups are required, which are usually based on benzyl (Bzl) or tert-butyl (tBu) groups. The specific protecting groups that can be used during the synthesis of a given peptide depend 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 multiple cycles of chemical treatment during the synthetic steps and are only removed during treatment with strong acids after the synthesis is complete.

[0068] According to a further preferred embodiment of the first aspect of the invention, at least a portion of the linear dithiol peptides comprise a primary or secondary amine, and the method further comprises the step of modifying the primary or secondary amine with a carboxylic acid, and the cyclic peptide comprising a primary or secondary amine and a carboxylic acid is preferably transferred by acoustic dispensing.

[0069] By modifying primary or secondary amines with carboxylic acids, the complexity of peptide libraries can be further increased. That is, the number of different peptides in the library can be greatly increased. This increases the chance of identifying ideal inhibitors or binding molecules when the peptide library is then screened for binders or inhibitors against a particular target molecule. This will be described in more detail in the present specification below in relation to the second aspect of the present invention.

[0070] Acoustic dispensing is a liquid transfer technique that allows reactions to be performed in very small volumes, as little as 80 nanoliters as shown in Example 3. Acoustic dispensing allows for non-contact, high precision, and high speed liquid handling by using acoustic ultrasonic energy to transfer liquids.

[0071] Modification of primary or secondary amines with carboxylic acids, where the cyclic peptide comprises a primary or secondary amine and a carboxylic acid, is also the subject of the second aspect of the invention described herein below. The preferred embodiments and definitions of the second aspect of the invention apply mutatis mutandis to the above further preferred embodiments of the first aspect of the invention.

[0072] According to a further preferred embodiment of the first aspect of the invention, the method further comprises the steps of (c) contacting the peptide library with the target molecule, preferably without prior purification of the peptide library, and (d) screening the peptide library for peptides which bind to and preferably inhibit the target molecule.

[0073] The means and methods for screening peptide libraries for peptides that bind to, modulate, and preferably inhibit target molecules are known in the art.In a preferred embodiment, the target is an amino acid-based target, such as a protein.Generally, the target can also be a non-amino acid-based compound, such as an oligonucleotide or polynucleotide.

[0074] In this context, the term "inhibits a target molecule" means that the biological activity is inhibited, preferably completely eliminated. Biological activity is the ability of a particular molecular entity to achieve a defined biological effect on a target. It is measured in terms of the potency or concentration of the molecular entity required to obtain the effect. Biological activity is measured by biological assays.

[0075] According to a more preferred embodiment of the first aspect of the invention, step (c) and step (d) are carried out in the same well in which the primary or secondary amines are modified with a carboxylic acid.

[0076] This approach saves materials and time. The wells can be in a multi-well plate format, such as, for example, a 96-well plate, a 384-well plate, or a 1536-well plate.

[0077] According to an even more preferred embodiment of the first aspect of the present invention, the target molecule is a protein or a nucleic acid molecule, preferably a protein. Of these options, the target is preferably an amino acid-based target, such as a protein.

[0078] According to another preferred embodiment of the first aspect of the present invention, the solid phase comprises a resin, preferably a non-polar resin, more preferably a polystyrene resin. Several resins for peptide synthesis are known and commercially available. Non-limiting examples are PEGA resins consisting of 2-acrylamidoprop-1-yl-(2-aminoprop-1-yl) polyethylene glycol 800, poly-ε-lysine resins crosslinked with sebacic acid, crosslinked hydroxyethyl polystyrene and polyethylene glycol resins, and polyethylene glycol-based resins. Any of the above resin matrices can be functionalized with reactive groups including, but not limited to, aminomethyl chloride, thiomethyl chloride, thioethyl chloride, chloroalkyl chloride, trityl chloride, HMBA, and Rink amide. Polystyrene resins are preferred as they are used in the examples of this application.

[0079] According to another preferred embodiment of the first aspect of the present invention, the linear dithiol peptide comprises a linear dithiol peptide having a molecular weight of less than 1000 Da, preferably less than 600 Da, and / or a linear dithiol peptide comprising three or four amino acids or building blocks, preferably three amino acids or building blocks.

[0080] The dithiol peptides of this preferred embodiment are particularly suitable for "reductive release" according to item (i) of the claimed method, because the two thiol groups in the dithiol peptide are in close proximity to each other and cannot be effectively released by cyclizing release due to structural constraints.

[0081] In the cyclizing release strategy shown in Figure 1a, peptides are synthesized on the solid phase via a disulfide linker and released via a cyclizing disulfide exchange reaction. The main limitation of the cyclizing release is that especially short dithiol peptides cannot be efficiently generated due to the conformationally constrained disulfide cyclized peptide-mediated route. As shown in Figure 1a, peptides composed of one amino acid and a thiol-containing building block on each side (three building blocks / amino acids in total) are not efficiently released. Even some peptides containing two amino acids between the thiol-containing building blocks (four building blocks / amino acids in total) were not efficiently released when the conformational flexibility of the peptide was limited (e.g., by amino acids with constraints on Phi and Psi angles). The limitation of the cyclizing release approach to deliver only dithiol peptides longer than three or four building blocks prevents the generation of macrocycles with molecular weights less than about 600 Da, thus preventing the generation of macrocycles that are of particular interest in the development of oral or cell-permeable drugs.

[0082] Thus, the dithiol peptides used in the "cyclizing liberation" according to item (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 contain more amino acids.

[0083] In a second aspect, the present invention relates to a method for diversifying a macrocycle library, preferably a cyclic peptide library, wherein at least some of the macrocycles, preferably cyclic peptides, comprise a primary or secondary amine, and the method comprises the step of modifying the primary or secondary amine with one or more carboxylic acids.

[0084] The definitions and preferred embodiments of the first aspect of the invention, insofar as amendable, apply mutatis mutandis to the second aspect of the invention. The term "macrocycle" refers to a molecule in which a series of 12 or more atoms are linked to form a macrocyclic ring or ring. The macrocycle is preferably a cyclic peptide as defined in relation to the first aspect of the invention, however, the macrocycle is not necessarily peptide-based and may also have a structure containing 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.

[0085] The nature of the carboxylic acid used is not particularly limited, and preferred examples are shown in Figures 8c and 10a. The complexity of the library can be adjusted by the number of different carboxylic acids used.

[0086] The cyclic peptide or macrocycle to be modified is preferably contacted with a 2- to 20-fold, preferably a 3- to 15-fold, and most preferably a 4- to 12-fold excess of carboxylic acid, since this excess increases the efficiency of the reaction.

[0087] The carboxylic acid is preferably an activated carboxylic acid, which 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.

[0088] To activate the carboxylic acid, an acid activating agent can be used. It follows that the process of the second aspect of the invention preferably comprises an acid activating agent capable of activating one or more carboxylic acids.

[0089] The acid activator is preferably HBTU ((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; hexafluorophosphate benzotriazole tetramethyluranium), which is used in the examples. Another suitable acid activator that can be used is 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 BF 4 (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate).

[0090] The process of the second aspect of the invention preferably includes a base, which facilitates the activation and coupling of the carboxylic acid, preferred examples of which are described herein below. Cyclic peptides or macrocycles preferably contain primary or secondary amines as peripheral groups (as depicted in FIG. 8) or within the backbone. In the case of cyclic peptides, the amino groups can be introduced through the side chains of amino acids or through the N-terminal amino acid.

[0091] The generation of macrocycle-based ligands is currently hampered by the lack of large libraries of macrocycles, and it has been noted that the greater the number of different potential binding partners in a library, the greater the chance of isolating macrocycle-based ligands from the library that bind with high affinity to a selected target. The limitations in obtaining large libraries of macrocycles are overcome by the method of the second aspect of the present invention. The approach of this method is based on tethering chemically distinct fragments to the peripheral groups of a structurally distinct macrocycle scaffold in a combinatorial manner. As a proof of concept, the generation of a 19,968 macrocycle library by conjugating more than 104 carboxylic acid fragments to 192 macrocycle scaffolds is shown in Example 3. The high reaction efficiency of the acylation reaction and the low by-products allowed high-throughput screening (HTS) of the library without prior purification. Example 3 also shows the generation of low nanomolar thrombin inhibitors (K i = 44 nM) and high nanomolar MDM2 / p53 protein-protein interaction inhibitors (K i = 390 nM) was successfully isolated from the library.

[0092] The approach of the second aspect of the invention offers a dramatic increase in the rate at which macrocycles can be synthesized and screened, and is generally applicable to any target.

[0093] According to a preferred embodiment of the second aspect of the invention, the library is in the article of manufacture format described in relation to the first aspect of the invention. The preferred embodiment of the article of manufacture described in relation to the first aspect applies mutatis mutandis to the second aspect. According to this preferred embodiment of the second aspect of the invention, a cyclic peptide or macrocycle containing a primary or secondary amine is reacted with a carboxylic acid in the well of the article of manufacture.

[0094] According to a more preferred embodiment of the second aspect of the invention, the one or more cyclic peptides, the one or more carboxylic acids, the acid activator, and / or the base are transferred to the article of manufacture by acoustic dispensing.

[0095] Further details regarding acoustic dispensing are described herein above in relation to the first aspect. The use of acoustic dispensing is illustrated in Example 3.2 herein below.

[0096] Acoustic dispensing uses sound waves, preferably acoustic droplet ejection technology, which has the great advantage of being able to transfer reagents contact-free, especially nanomolar amounts, which eliminates the need for pipetting tips, increases dispensing speed, and reduces waste and costs.

[0097] According to a further preferred embodiment of the second aspect of the invention, the macrocycles, preferably cyclic peptides, are screened after diversification without prior purification. The screening preferably comprises the steps of: (a) contacting a macrocyclic compound, preferably a cyclic peptide library, with a target molecule; and (b) screening the macrocyclic compound, preferably a cyclic peptide library, for compounds, preferably peptides, which bind to and preferably inhibit the target molecule as described herein above in relation to the first aspect of the invention.

[0098] In relation to the second aspect of the invention, particularly the preferred embodiment above where no pre-purification is performed, it is preferred that the cyclic peptide library has been generated by cyclizing release of the first aspect of the invention, or that the method of the second aspect comprises the step of generating a cyclic peptide library according to the first aspect of the invention prior to diversifying the cyclic peptide library.

[0099] Also, the cyclic peptide library generated by the cyclizing release of the first aspect of the invention can be screened without prior purification. Thus, if the cyclic peptide library is generated by the cyclizing release of the first aspect of the invention, or if the method of the second aspect includes a step of generating a cyclic peptide library according to the first aspect of the invention before diversifying the cyclic peptide library, the cyclic peptide library can be generated and diversified without the need for a purification step before screening.

[0100] According to a more preferred embodiment of the second aspect of the invention, macrocyclic compounds, preferably cyclic peptides, are therefore modified with one or more carboxylic acids and screened within the same well of the article of manufacture in which they were synthesized.

[0101] This can be achieved by dispensing the target molecule (e.g. a protein) and optionally other required screening assay reagents into the wells and measuring binding, for example by a plate reader. Cyclization in this context is again preferably carried out in accordance with the cyclative release of the first aspect of the invention.

[0102] According to a further preferred embodiment of the second aspect of the invention, the base is DABCO, the sodium salt of HEPES, or NMM, most preferably DABCO. Volatile bases such as DIPEA can also be used, but they may evaporate, especially when sonicated into 2.5 nl droplets. Therefore, the use of non-volatile bases such as DABCO (1,4-diazabicyclo[2.2.2]octane) or the sodium salt of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is preferred herein. Among volatile bases, NMM (N-methylmorpholine) was found to be suitable when applied in greater excess. The best results were obtained by using DABCO. See Example 3.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present patent specification, including definitions, shall control.

[0104] With regard to the embodiments characterized in this specification, and in particular in the claims, each embodiment mentioned in a dependent claim is intended to be combined with each embodiment of each claim (independent or dependent) from which said dependent claim depends. For example, in the case of independent claim 1 reciting three options A, B and C, dependent claim 2 reciting three options D, E and F, and claim 3 dependent on claims 1 and 2 and reciting three options G, H and I, it will be understood that the specification, unless otherwise indicated, expressly discloses embodiments corresponding to combinations of 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.

[0105] Similarly, it should be understood that if a dependent claim refers to more than one preceding claim, any combination of the subject matter covered thereby is considered to be explicitly disclosed, even if the independent and / or dependent claim does not recite an alternative. For example, in the case of independent claim 1, dependent claim 2 referring to claim 1, and dependent claim 3 referring to both claims 2 and 1, 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. If there is a further dependent claim 4 referring to any one of claims 1 to 3, the combination of the subject matter of claims 4 and 1, the subject matter of claims 4, 2 and 1, the subject matter of claims 4, 3 and 1, and the subject matter of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0106] The above considerations apply mutatis mutandis to all appended claims. The figure is shown. [Brief description of the drawings]

[0107] [Figure 1] Figure 1. Strategy for reductive release of peptides synthesized on solid phase via disulfide bridges. (a) Recently developed "cyclative disulfide release" reaction. Short peptides, e.g., peptides containing only one amino acid between two adjacent thiol-containing structures (three building blocks), cannot be efficiently released due to conformational constraints (shown by dashed lines). (b) Release of short dithiol peptides by reduction of disulfide bridges. [Diagram 2] Figure 1. Reductive release of peptides linked via disulfide bridges to a solid phase. (a) Chemical structures of the four peptides used to test the reductive release of peptides from the solid phase. (b) HPLC chromatograms (left) of the four peptides released by reduction of the disulfide bridges using BDT (100 mM) in DMF containing the base TEA (100 mM TEA). When the same immobilized peptides were exposed to TEA (100 mM) in DMSO, conditions previously used to cleave dithiol peptides via cyclizing release, only minor amounts of disulfide-dimerized peptides were eluted (right). The peak of the desired peptide is highlighted in red. The only by-product observed is the peptide dimerized via the disulfide bridge (dimer). [Diagram 3] Figure 1: Reductive release of dithiol peptides bound to the solid phase via disulfide bridges. (a) Structure of the dithiol peptides on the solid phase. (b) HPLC chromatogram of eight dithiol peptides released by reduction of the disulfide bridges using BDT (100 mM) in DMF containing the base TEA (100 mM). The desired peptide is highlighted in red. BDT and by-products are indicated. [Figure 4-1]Cyclization of dithiol peptides with bis-electrophiles. (a) Cyclization reaction (1) showing the use of Mpa-Trp-Mea and the reagent 2,6-bis(bromomethyl)pyridine. (b) Bis-electrophiles 2–10. The desired cyclic product is highlighted in red. By-products s1–s7 are shown in Supplementary Fig. 7. L = bis-electrophilic cyclization reagent. L* is hydrolyzed L. [Figure 4-2] Continued from Figure 4. (c) Chemical structures of macrocycles and HPLC chromatographic analysis of the cyclization reaction. [Figure 4-3] Continued from Figure 4c. [Diagram 5] Figure 1. Strategy for cyclative disulfide release. (a) Schematic of the strategy. Short peptides are synthesized on the solid phase via disulfide bridges. Removal of the protecting groups (red) on the solid phase allows for efficient removal. Treatment with base deprotonates the N-terminal thiol and induces intramolecular disulfide exchange to give the cyclic product. (b) Chemical structure of test peptide Mpa-Gly-Gln-Trp-Mea disulfide-linked to solid support and the commercial resin used. (c) Recovery of disulfide-cyclized peptide Mpa-Gly-Gln-Trp-Mea synthesized on resin 4 and resin 5 and released with 150 mM DIPEA in DMSO. Concentration was determined by measuring absorbance at 280 nm. Reactions were performed in triplicate. (d) Purity of the disulfide-cyclized peptide in panel d. Purity was determined by LCMS measuring the AUC of all species at UV absorbance at 220 nm. [Figure 6-1] Cyclic release of peptides with variable sequences. (a) Chemical structures of the four desired cyclic peptides. The peptides are based on the linear sequence Mpa-Gly-Ala-Xaa-Mea, where Xaa is an amino acid with variable structural flexibility in the backbone. (b) Analytical HPLC chromatograms of the crude peptides after cyclic release. The chromatogram on the right shows the peptides after disulfide bond reduction with TCEP. For impurities that were not identified, masses are indicated (assuming the species is singly charged). [Figure 6-2]Continued from Figure 6. (c) Examples of chemical structures consistent with the molecular weights of identified impurities. [Figure 7] Library design, peptide recovery quantification by absorbance, and thrombin inhibition of cyclic peptide libraries. (a) Design of a library containing 96 different peptides and structures of the unnatural amino acids used in the library. (b) Scatter plot of cyclic peptide concentration (mM in DMSO) for 96 synthetic cyclic peptides quantified by absorbance at 280 nm. The average recovery and 1.5x and 0.67x of this value are shown on the graph. (c) Thrombin inhibition measured at an average cyclic peptide concentration of 11 μM. Peptides are ordered according to their thrombin inhibitory activity in the first screen (black dots; highest to lowest activity). Green dots show thrombin inhibition of the same cyclic peptides measured in a second screen using the same conditions. The chemical structure of the most active inhibitor is shown (Ki=13±1 μM). [Figure 8] Diversification of macrocycles by combinatorial addition of fragments to peripheral groups. a, General principle of the approach. b, Model macrocycles containing peripheral primary amines are modified by acylation. c, Reaction of model macrocycles with the indicated acids. The top numbers show the transformations in a 4 μL volume by pipetting, the bottom numbers show the transformations in 80 nl and acoustic liquid transfer, the first number is DIPEA and the second number is DABCO. To show the difference in scale, images of two droplets in a 96-well plate are shown. The droplets contain fluorescein and are exposed to UV light for visualization. [Figure 9-1] 1. Preparation of macrocycles containing peripheral amino groups. a, Cyclic disulfide release of side-chain deprotected peptides. b, Scaffold format of Library 1. [Figure 9-2] Continued from Figure 9. c, Amino acids used in the scaffold library synthesis. d, Yields of 45 tryptophan-containing scaffolds determined by absorbance measurements. [Figure 10-1]Screening of macrocycle libraries against thrombin and identification of hits. a, Carboxylic acids 9–16 were used along with acids 1–3 to diversify scaffold library 1a–f (shown in Figure 2). b, Schematic procedure for macrocycle library synthesis by acoustic liquid transfer. Reaction conditions are shown. c, Heat map showing thrombin inhibition of each macrocycle. Amino acid compositions of all macrocycles are shown in Supplementary Table 1. [Figure 10-2] Continuation of Figure 10. d, Replicate reaction and screening of all compounds containing acid 14. e, Chemical structures and activities of the top three hits M1 to M3. Means and SDs of three independent determinations are shown. f, Analysis of fractions of chromatographically separated acylation reactions yielding M1 and thrombin inhibitor species. [Figure 11-1] FIG. 1. Screening against thrombin. 384 macrocycles were synthesized according to previously described procedures (the utilized Fmoc amino acids and their corresponding one-letter codes are shown in Supplementary Fig. 1b). Each macrocycle was reacted with 12 carboxylic acids. After reaction and quenching, thrombin and a fluorogenic substrate were added to the wells and the increase in fluorescence was measured over a period of 30 min. The final macrocycle concentration was 10 μM. Residual thrombin activity was determined by dividing the slope of the fluorescence intensity over time for each well by the slope of the control wells without macrocycle. [Figure 11-2] Continued from Figure 11. [Figure 11-3] Continued from Figure 11. [Figure 12-1] Figure 2: MDM2 binders. a, Analysis of chromatographically separated acylation reactions resulting in M6 through M8 and fractionation of thrombin-inhibited species. b, Binding of purified macrocycles to MDM2 measured by examining displacement of a fluorescent MDM2 probe (linear peptide) using fluorescence polarization. Means and SD of three independent measurements are shown. [Figure 12-2] Continued from Figure 12. c, Chemical structure of fluorescein (F)-labeled macrocycles and binding to MDM2 measured by fluorescence polarization. The mean and SD of three independent measurements are shown. [Figure 13]Diversification of macrocycles by combinatorial addition of fragments to peripheral groups. a, Chemical structures of macrocycles that all contain amino groups. b, Carboxylic acids used for diversification of the macrocycles. c, Yield of the reaction. [Figure 14] Strategy for synthesizing macrocyclic libraries at the edge of Ro5. (a) Previous macrocycle strategy and the improved strategy reported here. The circles represent the three groups of building blocks: amino acids (grey), cysteamine and analogs (red), and bis-electrophilic linkers (white). (b) Schematic of the strategy for cyclizing linear peptides by reacting the terminal thiol group with a bis-electrophilic reagent. The chemical structure of the newly developed cysteamine analog used as the C-terminal building block of the linear precursor is shown. (c) Strategy for synthesizing a polystyrene resin carrying cysteamine analogs linked via disulfide bridges. The cysteamine analogs are loaded onto the resin as activated thiosulfonate building blocks. [Figure 15] Synthesis of phenylsulfone-activated cysteamine building blocks and purification without a chromatography step. (a) Schematic diagram of synthesizing dithiol resins. X=Br or Cl. (b) Simplified diagram of synthesis of dithiol peptides without purification. (c) Stacked HPLC chromatograms (UV220) of crude peptide quality of a model peptide (MPA-Trp-Ala) synthesized with seven different resin building blocks. [Figure 16-1] Design of macrocyclic libraries tailored to generate inhibitors of trypsin-like serine proteases. (a) Format of the designed cyclic peptide library. Predicted physicochemical properties of the macrocyclic library calculated using DataWarrior (vers. 5.5.0). (b) Amino acids and linkers used in library synthesis. (c) Histogram of selected properties of the generated macrocyclic library. Marked in green are regions that are ro5 compliant and / or are within the range predicted to be possible for cell-permeable macrocycles. [Figure 16-2] Continued from Figure 16. [Figure 16-3] Continued from Figure 16. [Figure 17] Figure 1: Experimental steps of library generation. Workflow for synthesizing macrocyclic libraries in microtiter plates. [Figure 18-1] Figure 3. Affinity optimization of MDM2:p53 inhibitors. a, Library 3 scaffold is based on M8 with diversification at the amino acids shown in blue. Amino acid building blocks are shown in three frames. [Figure 18-2] Continued from Figure 18. b, Heat map of MDM2 binding to 63 scaffolds combinatorially acylated with 15 carboxylic acids at the 30 pmol scale. Binding to MDM2 was measured by a displacement assay of a fluorescent peptide probe by the macrocycle at a concentration of 750 nM. [Figure 18-3] Continued from Figure 18. c, Screening of library 4 based on the best 9 scaffolds from the previous screen and 15 additional carboxylic acids. [Figure 18-4] Continued from Figure 18. d, Binding of fluorescein-labeled, HPLC-purified macrocycle M10 (F-M10) to MDM2 measured by FP. Means and SD of three independent measurements are shown. e, Binding of unlabeled macrocycles M8 and M10 to MDM2 measured by SPR. [Figure 19-1] Comparison of acylation reactions by pipetting (4 μl volume) and acoustic transfer (80 nl volume) using DIPEA as base. Reactions were diluted 100-fold with water and 5 μl samples were analyzed by LC-MS using a RP column and a 0-60% MeCN / H20 gradient over 5 min. For the 80 nl reaction volume, two samples were pooled. [Figure 19-2] Continued from Figure 19. [Figure 19-3] Continued from Figure 19. [Figure 19-4] Continued from Figure 19. [Figure 19-5] Continued from Figure 19. [Figure 20-1] Figure 2 shows acylation reactions in 80 nl volume using acoustic dispensing and different bases. Two non-volatile bases (DABCO and HEPES sodium salt) at 80 mM concentration and a volatile base (NMM) at 500 mM concentration were tested as alternatives to 80 mM DIPEA for the acylation of a model scaffold with three carboxylic acids. Reactions were diluted 100-fold with water and 5 μl samples were analyzed by LC-MS using a RP column and a 0-60% MeCN / H20 gradient over 5 min (two pooled reactions). The reaction with DIPEA did not go to completion, but all other bases were quantitatively converted to products. [Figure 20-2] Continued from Figure 20. [Figure 20-3] Continued from Figure 20. [Figure 21] Figure 1. Comparison of acylation reactions in 80 nL volume using acoustic dispensing and DABCO as base. Model scaffold 1 was reacted with 80 nl volume of carboxylic acids 1-8 using DABCO (80 mM) as base. Reactions were diluted 100-fold with water and 5 μl samples analyzed (2 pooled reactions) were analyzed by LC-MS using a RP column and a 0-60% MeCN / H2O gradient run for 5 min. [Figure 22-1] Acylation and acoustic dispensing of model scaffolds 2-5 in 80 nl volumes. a, LC-MS analysis of model scaffolds. Reactions were diluted 100x with water and 5 μl samples were analyzed. Solvent B gradient: 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. Reactions were diluted 100x with water and 5 μl samples were analyzed. Solvent B gradient: 0-60% MeCN, 5 min. TMU = tetramethylurea. [Figure 22-2] Continued from Figure 22. [Figure 22-3]Continued from Figure 22. c-f, UHPLC analysis of the acylation reactions of model scaffolds 2(c), 3(d), 5(e) and 5(f). Reactions were diluted 100-fold with water and 5 μL samples were analyzed. Solvent B gradient: for model scaffold 2, 0–60% MeCN, 5 min. for model scaffolds 3–5, 10–100% MeCN, 5 min. [Figure 22-4] Continued from Figure 22. [Figure 22-5] Continued from Figure 22. [Figure 22-6] Continued from Figure 22. [Diagram 23] Figure 1: Preparation of cyclic peptide scaffolds containing an N-terminal amino group. a, Six different scaffold formats. b, Amino acids used for scaffold synthesis. [Figure 24] Figure 2: Physicochemical properties of library 1 (thrombin screening) and library 2 (MDM2 screening). Properties were calculated using DataWarrior software. Regions conforming to Kielberg's permeability rules (P. Matsson et al., Adv. Drug Deliv. Rev. 2016, 101, pp. 42-61; B. Doak et al., Chem. Biol. 2014, 21, pp. 1115-1142) are colored green. The majority of both libraries lie in spaces predicted to be cell permeable. MW = molecular weight, cLogP = calculated n-octanol / water partition coefficient, HBD = hydrogen bond donor, HBA = hydrogen bond acceptor, PSA = polar surface area, NRotB = number of rotatable bonds. a, library 1 (for thrombin screening). b, library 2 (for MDM2 screening). [Figure 25-1] FIG. 1 shows thrombin inhibitors M1-M5. a, Chemical structures and analytical HPLC chromatograms obtained using a 0-100% MeCN / H2O gradient over 15 min. [Figure 25-2]Continued from Figure 25. b, For all macrocycles, 18-point two-fold serial dilutions were performed in 50 μl volumes. Thrombin was added (50 μl, 2 nM final concentration) followed 10 min later by the addition of the fluorogenic substrate Z-Gly-Gly-Arg-AMC (50 μl, 50 μM final concentration). The increase in fluorescence was measured over a 30 min period. Residual thrombin activity was determined by dividing the slope of the fluorescence intensity over time for each well by the slope of the control wells without macrocycle. Means and SD of three independent measurements are shown. [Figure 26-1] Figure 1 shows the structure of M1 bound to thrombin: a, X-ray structure of M1 bound to thrombin, where M1 is expanded to show the H-bond interactions. [Figure 26-2] Continued from Figure 26. b, Chemical structure of M1 and the H-bond interactions formed with thrombin. [Figure 27] Figure 1: Scaffolds synthesized for library 2 (MDM2 screening). a, Library 2 scaffold format. b, Amino acids used in the synthesis of the scaffold library. All combinations of 4 diamino acids, 4 backbone amino acids, 2 side chain amino acids, and 6 sub-library formats were synthesized. c, Yield of tryptophan-containing scaffolds after cyclizing release. The average concentration of scaffolds was 12.9 mM as measured by Nanodrop absorbance. The average purity was about 90% as measured by LC-MS. [Figure 28-1] FIG. 1 shows an outline of the carboxylic acids used to acylate peripheral amines of the cyclic peptide scaffold. [Figure 28-2] Continued from Figure 28. [Figure 28-3] Continued from Figure 28. [Figure 28-4] Continued from Figure 28. [Figure 28-5] Continued from Figure 28. [Figure 28-6] Continued from Figure 28. [Figure 29]Figure 1: Study of the binding site of M10 by competitive binding experiments: a, Displacement of the FP53 linear peptide probe by M10 and nutlin-3a measured by fluorescence polarization, b, Displacement of the F-M10 macrocyclic peptide probe by M10 and nutlin-3a. [Figure 30-1] Figure 1: Binding of macrocycles to MDM2 measured by SPR. a, Single cycle SPR sensorgrams of fluorescein-labeled macrocycle, positive control nutlin-3a (no fluorescein labeling), and negative control (thrombin inhibitor; no fluorescein labeling). RU, response units. [Figure 30-2] Continued from Figure 30. [Figure 30-3] Continued from Figure 30. b, Single-cycle SPR sensorgrams of macrocycles M6, M7, M8, and M10 (without fluorescein labeling), run in triplicate. [Figure 30-4] Continued from Figure 30. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] The present invention will now be explained by way of examples. EXAMPLES

[0109] Example 1 - Reductive Release 1.1. Reductive release of disulfide-anchored peptides from the solid phase We first evaluated whether peptides immobilized via disulfide bridges on solid supports could be quantitatively released by volatile reducing agents. For this purpose, we synthesized four peptides, Ala-Trp-Mea, Trp-Ala-Mea, Ala-Tyr-Mea, and Tyr-Ala-Mea (Mea = 2-mercapto-ethylamine, also called cysteamine), shown in Figure 2a. The peptides contain tryptophan or tyrosine residues, allowing accurate measurement of the amount of peptide released by absorbance measurement at 220 nm or 280 nm. We omitted the N-terminal thiol group found in dithiol peptides to quantify peptides released by reduction of disulfide bridges, without any other mechanism such as cyclative disulfide exchange. The peptides were synthesized on polystyrene (PS) resin, which has a high loading capacity (approximately 1 mmol / gram) and is commonly used for peptide synthesis. First, disulfide bridges were established by incubating PS thiol resin with excess pyridyldithioethylamine, and peptides were synthesized by standard Fmoc chemistry. All peptides were synthesized in wells of a 96-well plate at a 5 μmol scale to test conditions that are destined for later high-throughput synthesis of dithiol peptide libraries.

[0110] Release of disulfide-immobilized peptides from the PS resin was tested by incubating the beads overnight in 200 μL of DMF containing 20 equivalents of β-Me (0.5 M) and 20 equivalents of trimethylamine (TEA; 0.5 M). LC-MS analysis of the peptides showed efficient release, but also showed that approximately 40% of the product occurred as a disulfide adduct with β-Me. It was reasoned that the extent of the adduct could potentially be reduced by using a larger molar excess of β-Me and / or by repeated reduction, but this was not the route of most interest as it would require additional work-up steps. To release the peptide from the resin and remove the disulfide adduct in a single step, it was proposed to use a reducing agent such as dithiothreitol (DTT), which does not form disulfide adducts as it removes itself by forming cyclic DTT. Incubation of resin bearing either peptide Ala-Trp-Mea or Tyr-Ala-Mea with 4 equivalents of DTT resulted in efficient release of the peptide without formation of a peptide-reducing agent adduct, but DTT could not be removed by vacuum evaporation in a standard SpeedVac. A related 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 / 1_4-Butanedithiol), accessed 20.04.21). 5 μmol of resin-bound peptide was incubated with 200 μl of DMF containing 4 equivalents of BDT (100 mM) and 4 equivalents of TEA (100 mM). As a control, a parallel reaction was performed in which the four resin-bound peptides were treated with the conditions for cyclizing release, 250 mM TEA in DMSO. The peptides were analyzed by LC-MS and were efficiently released by BDT. For all four peptides, a major peak corresponding to the desired product was observed (Figure 2b). The only by-product identified for two peptides was a peptide dimer, which occurred in small amounts less than 3%.The yields of the desired product were 3.4 μmol, 1.3 μmol, 4.2 μmol and 4.3 μmol, corresponding to yields of 68%, 25%, 84% and 86%, respectively (assuming an amount of peptide synthesized on the beads of 5 μmol).

[0111] 1.2. Reductive release of short dithiol peptides from the solid phase Next, a panel of eight short dithiol peptides of the format Mpa-Xaa-Mea (Mpa = mercaptopropanoic acid), where the Xaa amino acids were Trp, Tyr, Ser, His, Phe, Arg, Asp, and Ala, were synthesized. For these peptides, it was expected that they would be released by reduction of the disulfide bridge, but not efficiently via the disulfide cyclization mechanism. As a control, we also synthesized the longer peptide Mpa-Lys-Trp-Gly-βAla-Mea, which is expected to be efficiently released via disulfide cyclization. All peptides were efficiently released when the resin was incubated with the reducing agent BDT (Figure 3). The major product was the desired dithiol peptide. Only small amounts of by-products were identified, which were dithiol peptides with trityl and tert-butyl protecting groups (Figure 3). Incubation of the resin with TEA in DMSO for cyclization release afforded approximately 10-100 times less of the shorter peptide and produced many by-products. As expected, the longer peptide Mpa-Lys-Trp-Gly-βAla-Mea was efficiently released via the cyclization release mechanism, presumably due to its less structural constraint. The yields of peptides Mpa-Trp-Mea and Mpa-Trp-Mea, which could be quantified by absorbance measurements at 280 nm, were 3.6 μmol and 4.3 μmol, respectively, which correspond to 71% and 85% yields, respectively, assuming that the peptides were present on the resin in amounts of 5 μmol.

[0112] 1.3. Evaporation of Solvent and Reducing Agent under Vacuum Next, it was tested whether the reducing agent BDT could be removed by centrifugation under vacuum. Peptides synthesized on a 5 μmol scale and released in 200 mL of DMF containing 100 mM BDT and 100 mM TEA were centrifuged in wells of a 96-well plate at 0.1 mbar, 30 °C and 400 × g. The solvent was efficiently removed in 1 h when all wells of the microwell plate were filled. LC-MS analysis of the peptides revealed that all BDT had been removed. However, it was also confirmed that for some peptides, a portion of the product, up to 10%, was reverse-oxidized. Since it was speculated that oxidation could occur due to high pH, ​​2 equivalents of TFA relative to TEA (200 mM TFA) were added to each well before centrifugal vacuum evaporation. This procedure allowed efficient suppression of fractionation of oxidized peptides.

[0113] 1.4. Cyclization of dithiol peptides with biselectrophiles As shown in Figure 4a, the short dithiol peptides Mpa-Trp-Mea and Mpa-Tyr-Mea were tested for their ability to be cyclized by bis-electrophiles. Cyclization of peptides through two or three cysteines with electrophilic linker reagents is very efficient and clean when the peptide is performed at a dilute concentration of about 1 mM (or less) and the cyclization reagent is applied in small excess (P. Timmerman et al., ChemBioChem, 2005, 6, 821-824; SS Kale et al., Nat. Chem., 2018, 10, 715-723). The peptide was dissolved in 1 ml of 1:1 acetonitrile:water and cyclized with 3 ml of 90% NH 4 HCO 3Buffer (100 mM, pH 8.0), 10% acetonitrile was added, and 1 ml bis-electrophile in acetonitrile (10 mM) was immediately added. A total of 10 bis-electrophiles shown in Figure 4b were tested. The final concentrations of peptide and cyclization reagent were approximately 1 mM and 2 mM, respectively. HPLC chromatograms of cyclization reactions with peptide Mpa-Trp-Mea are shown in Figure 4c. In most reactions, the dithiol peptides were cyclized almost quantitatively, with yields exceeding 90%. The small amount of by-products were mainly peptides that reacted with only one thiol group, since one of the thiol groups was protected with trityl. To quench the excess bis-electrophile, 6 equivalents (relative to peptide) of β-Me were added, which reacts with these reagents but does not affect the macrocycle.

[0114] 1.5. Discussion Herein, a solid-phase peptide synthesis and elution strategy is established that provides dithiol peptides in high purity and concentration, which can be readily cyclized with bis-electrophiles to synthesize macrocycles and libraries. The key elements in this strategy are i) synthesis of peptides via disulfide linkers, ii) deprotection of side chains on the solid phase, iii) release of peptides by disulfide reduction, and iv) use of a volatile reducing agent that can be removed by evaporation, thereby enabling cyclization with bis-electrophiles. The omission of purification steps after synthesis of dithiol peptides and cyclization with bis-electrophiles allows access to a large number of macrocycles and their application to high-throughput screening.

[0115] 1.6. Materials and Methods Synthesis of 2-(2-pyridyldithio)ethylamine hydrochloride To a stirred solution of 2,2'-dipyridyl disulfide (4.41 g, 20 mmol) in MeOH (16 ml containing 2% [v / v] AcOH), cysteamine hydrochloride (1.14 g, 10 mmol) dissolved in MeOH (10 ml containing 2% (v / v) AcOH) was added dropwise and this was dissolved in 10 ml MeOH containing 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 greasy oil. The residue was dissolved in MeOH (16 ml), dispensed into eight 50 ml Falcon tubes and precipitated by adding ice-cold diethyl ether (48 ml per tube). The tubes were incubated at -20°C for 30 min and centrifuged at 3400xg (4000 rpm in a Thermo Scientific Heraeus Multifuge 3L-R centrifuge with a Sorvall 75006445 rotor, radius = 19.2 cm, explosion-proof) for 30 min at 4°C. After precipitation was repeated eight times, the product was obtained as a colorless product (yield = 90%).

[0116] Preparation of cysteamine-polystyrene resin The following procedure describes the preparation of polystyrene resin with approximately 2 mmol of cysteamine immobilized via a disulfide linker, sufficient for the synthesis of 4 × 96 peptides on a 5 μmol scale. 563 mg of resin (Rapp Polymere Polystyrene A SH resin, 200-400 mesh, 0.95 mmol / gram loading) was added to each of four 20 ml plastic syringes. The resin was washed with DCM (15 ml) and then swollen in MeOH / DCM (7:3; 15 ml) for 20 min. Pyridyl-cysteamine 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. A 19 ml volume of this solution was injected into each syringe, which was then shaken at RT for 3 h. The pyridyl-cysteamine solution was discarded and the resin was washed with MeOH / DCM (7:3; 2×20 ml) and then with DMF (2×20 ml). The resin was combined as a suspension in DMF into a single syringe and washed with a 1.2 M solution of DIPEA in DMF (11.8 ml) for 5 minutes to ensure all amines were neutralized. This solution was discarded and the resin was washed with DMF (2×20 ml) and then with DCM (4×20 ml) and kept under vacuum overnight to give a free-flowing powder.

[0117] Fmoc peptide synthesis in 96-well plates Peptides were synthesized in a 96-well peptide synthesis filter plate (Orochem, cat.# OF1100) at 5 μmol scale 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). At this and all subsequent washing steps, the resin was incubated for 1 min. The following reagents were transferred to tubes in the order indicated, 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 eq.), 5 μl N-methylpyrrolidone (NMP), 12.5 μl N-methylmorpholine (NMM in DMF, 4 M, 10 eq.) and 53 μl amino acid (500 mM in DMF, 5.3 eq.). The final volume of the coupling reaction was 120.5 μl and the final concentrations of the reagents were 208 mM HATU, 415 mM NMM and 220 mM amino acid. Couplings were performed in duplicate. The resin was washed with DMF (1×225 μl). Unreacted amino groups were capped by incubation with 5% acetic anhydride and 6% 2,6-lutidine in DMF (100 μl) for 5 min without shaking. The resin was washed with DMF (8×225 μl). The Fmoc group was removed by incubation twice with DMF (120 μL) containing 20% ​​(v / v) piperidine for 5 min each time without shaking. For the synthesis of longer peptide sequences, the incubation time was reduced from 5 to 2 min to reduce exposure to bases. The resin was washed with DMF (8 × 225 μL). At the end of peptide synthesis, the resin was washed with DCM (2 × 200 μL).

[0118] Peptide side-chain deprotection in 96-well plates To remove the protecting groups from the amino acid side chains as well as from Mea, the bottom of a 96-well synthesis plate was sealed by pressing the plate against a 6 mm thick soft ethylene-vinyl acetate pad and the resin in each well was incubated with a solution of TFA:TIPS:H20 (95:2.5:2.5 [v / v / v], approximately 300 μl). The plate was covered with adhesive sealing film (iST Scientific, QuickSeal Micro, cat.# IST-125-080-LS) and weighted down with a weight (1 kg) on ​​top to prevent leakage. After 1 h of incubation, the synthesis plate was placed on a 2 ml deep-well plate and the TFA mixture was drained off. The synthesis plate was resealed and the deprotection procedure was repeated. The wells were washed with DCM (3 × 500 μl; added by syringe) which was allowed to flow into the wells by gravity flow. The resin was dried by placing the synthesis plate on a vacuum manifold for 5 min.

[0119] Cyclic peptide release with β-Me, DTT, or BDT To release the peptides from the resin, the bottom of the 96-well synthesis plate was sealed by pressing the plate against a 6 mm thick soft ethylene-vinyl acetate pad, and the resin in each well was incubated for 4 hours at RT with 200 ml of DMF solution containing 500 mM β-Me, or 100 mM DTT, or 100 mM BDT, and 100 mM TEA, after which the samples were collected in the 96 deep-well plate by centrifugation at 250×g (1100 rpm in a Thermo Scientific Heraeus Multifuge 3L-R centrifuge with a Sorvall 75006445 rotor, radius=19.2 cm rotor) for 2 hours at RT.

[0120] Cyclic release of peptides The 96-well synthesis plate was sealed as above and the peptides were released from the resin by incubating with 200 μl of DMSO solution containing 250 mM TEA (10 equiv.) overnight at RT, after which the samples were collected in the 96 deep-well plate by centrifugation at 250×g (1100 rpm in a Thermo Scientific Heraeus Multifuge 3L-R centrifuge with a Sorvall 75006445 rotor, radius=19.2 cm rotor) for 2 h at RT.

[0121] LC-MS analysis of peptides after solid-phase release or cyclization For peptides released from the solid phase (concentration up to 25 mM in DMF or DMSO), 1 μl of peptide was diluted with 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 Å, 50 × 2.1 mm) and a linear gradient of solvent B (MeCN, 0.05% formic acid) to solvent A (H2O, 0.05% formic acid) at a flow rate of 1 ml / min from 0 to 60% in 5 min. Absorbance was recorded at 220 nm and mass was analyzed in positive mode.

[0122] Vacuum centrifugal evaporation of reducing agents and solvents The following example describes a peptide that had a concentration of 20 mM after reductive release. Of the 200 μl peptide (in DMF containing 100 mM BDT and 100 mM TEA) released from the solid phase by reduction, 5 μl (0.1 μmol) were transferred to a well of a V-bottom 96-well plate (Ratiolab, 6018321, PP, non-sterile). A volume of 7 μl of 1% TFA in water (v / v) was added to each well, resulting in 2 equivalents of TFA relative to TEA. The sample was subjected to vacuum centrifugal evaporation using a Christ RVC 2-33 CDplus IR instrument to remove the solvent (DMF) and reducing agent (BDT). The sample was centrifuged at 0.1 mbar, 30 °C, 400 × g (1750 rpm, radius = 10.5 cm in a Christ 124700 rotor with 124708 plate holder insert). The peptide was no longer visible after this step.

[0123] Peptide cyclization The reduced and dried peptide (0.1 μmol) was dissolved in 20 μl of 50% acetonitrile, 50% HO to a concentration of 5 mM. To this solution, 60 μl of reaction buffer (100 mM ammonium bicarbonate, pH 8.0 containing 10% acetonitrile [v / v]) was added, followed by 20 μl (2 equivalents) of 10 mM cyclized linker in acetonitrile. The final concentrations of the reaction were 1 mM peptide, 2 mM cyclized linker, 60 mM ammonium bicarbonate buffer, and 35% acetonitrile. The plate was covered with a foil seal and the reaction was incubated at RT for 2 h.

[0124] Quenching of linker reagents in cyclization reactions After completion of the cyclization reaction, 4 μl of 150 mM β-Me in acetonitrile (0.6 μmol, 6 equivalents relative to peptide) was added to the reaction mixture and incubated for 1 h at RT. Solvent (MeCN), buffer (bicarbonate) and excess β-Me were removed by vacuum centrifugal evaporation using a Christ RVC 2-33 CDplus IR instrument. Samples were centrifuged at 0.1 mbar, 30 °C, 400 × g (1750 rpm, radius = 10.5 cm in a Christ 124700 rotor with 124708 plate holder insert). The peptide was no longer visible after this step. EXAMPLES

[0125] Example 2 - Cyclic Release 2.1. Results and Discussion In a first experiment, various resins were tested for the synthesis of short peptides tethered to the solid phase via disulfide bridges. The peptide Mpa-Gly-Gln-Trp-Mea was synthesized on five different resins, where Mpa is mercaptopropanoic acid (a cysteine ​​without an amino group) and Mea is 2-mercapto-ethylamine (cysteamine; a cysteine ​​without a carboxylic acid) (Figure 5b). Two polyethylene glycol (PEG) resins (1, 2), which are polar, one PEG-modified polystyrene (PS) resin, which is also polar, and two PS resins (4 and 5), which are non-polar, were used. Resin 5 already contained a thiol group, and for resins 1–4, the thiol group was introduced by adding a trityl-protected Mpa via amidation. Disulfide-linked Mea was introduced by incubating the resins with an excess of 2-(2-pyridinyldithio)-ethanamine. Disulfide exchange reactions were tested in methanol (MeOH) / dichloromethane (DCM) mixtures and in DMF, in the presence of base or acid, and conditions of 30% MeOH, 70% DCM, and 1 equivalent of N,N-diisopropylethylamine (DIPEA; for 2-(2-pyridinyldithio)-ethanamine) were found to be optimal. The three amino acids Trp, Gin, Gly, and Mpa were added using standard Fmoc chemistry, and the side chain protecting groups were removed by incubating the resin with 95% TFA, 2.5% TIS, and 2.5% water for 1 h.

[0126] Cyclic disulfide release was then tested by deprotonating the sulfhydryl groups at the N-terminus of the peptides using DIPEA as a base. Incubation of the resins in DMSO containing 150 mM DIPEA led to very efficient release for the non-polar resins 4 and 5 (Figure 5c). The concentrations of peptide in the eluate were 5.1 mM (resin 4) and 11.6 mM (resin 5), respectively. The amount of peptide released was 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 likely not quantitative, the percentage of peptide recovered by the cyclicative release mechanism would likely have been higher than 44%, even for the resin already bearing a thiol group (resin 5). LC-MS analysis of the products showed high purity of 95 ± 4% (resin 4) and 93 ± 1% (resin 5) for the disulfide-cyclized peptides (Figure 5d). The only by-product was a cyclic peptide dimer, which was identified in small amounts, averaging 6%. This dimeric cyclic product was most likely formed by the transfer of one peptide to an adjacent peptide on the resin via disulfide exchange, followed by cyclative release of the cyclic dimer.

[0127] To evaluate the substrate scope of the cyclizing disulfide release strategy, four peptides of the format Mpa-Gly-Ala-Xaa-Mea were synthesized on a 25 μmol scale (Figure 6a). In three of the four peptides, an amino acid building block was inserted at the "Xaa" position, which imparts rigidity to the peptide backbone. HPLC analysis of the products obtained by base-induced cyclizing release showed major peaks for each of the four peptides, and MS analysis confirmed that these major products were the desired cyclic peptides. Absorbance measurements revealed that all four peptides were obtained in double-digit millimolar concentrations. Gravimetric quantification of the products after extensive lyophilization revealed yields ranging from 13.5 to 26 μmol, which corresponds to 54% to 100% of the expected yield based on the resin loading. Only a limited number of by-products were identified for all four peptides, and they were in small amounts (Figure 6b and Figure 6c). The main by-product was a cyclic dimer, this time eluting as two closely spaced peaks, likely corresponding to two possible dimers, one linked head-to-head / tail-to-tail and the other linked head-to-tail. Linearization of the products with TCEP and HPLC analysis showed linear peptide products with purities of 96%, 96%, 100% and 92% for peptides 1–4, respectively (Fig. 6b, right chromatograms). The TCEP linearization experiments suggested that the dimeric cyclic peptides could be removed by reduction followed by oxidative recyclization at concentrations favoring intramolecular cyclization. To test whether even shorter peptides could be generated following the cyclative release strategy, the experiment was repeated with four peptides in the format Mpa-Ala-Xaa-Mea, and thus one amino acid shorter. The four peptides were also efficiently released, and the main peaks in the HPLC profile were the desired cyclic peptides. The percentage of cyclic dimers was slightly higher overall, most likely due to less efficient cyclization hindered by the short backbone and the resulting conformational constraints.

[0128] To evaluate whether the cyclative disulfide release strategy could be applied to library synthesis and screening, cyclic peptides were designed and synthesized at a 5 μmol scale in 96-well plates. Ninety-six random disulfide-cyclized peptides in three formats were prepared as shown in FIG. 7a. The peptides contain three random amino acids Xaa flanked by Mpa and Mea. Two of the random amino acids in each peptide were selected from four structurally diverse amino acids resulting in highly diverse cyclic peptide backbones. One of the random amino acids was Trp or Tyr, allowing quantification of peptide yields by absorbance measurements at 280 nm. Cyclative release of peptides by addition of 150 mM DIPEA in 200 μl DMSO, followed by absorbance measurements, showed a high average peptide concentration of 13.3 mM and a narrow concentration distribution between 8.9 mM (1.5-fold lower than the average) and 20 mM (1.5-fold higher than the average) for the 90 peptides. Three of the peptides were not synthesized or released at all. Analysis of 12 randomly selected cyclic peptides by LC-MS showed a high average purity of 84%.

[0129] Despite the relatively small number of cyclic peptides, only 96, and therefore the low probability of finding an active compound, we performed a screen against thrombin, an important target for anticoagulant therapeutics development, using a compound concentration of approximately 10 μM. The most active peptide reduced thrombin activity by 52%, which was remarkable considering that the peptide does not contain the amino acids Arg and Lys that bind to the thrombin specificity pocket S1 (Figure 7c). Repeated screening identified the same cyclic peptide as the most active hit (green dot in Figure 7c). The HPLC-purified disulfide-cyclized peptide Mpa-Tyr-II-Pro-Mea had a K of 13 ± 1 μM. iThe cyclic peptides inhibited thrombin at 1000 ng / mL. Small scale screening revealed that most cyclic peptides did not affect thrombin activity, suggesting that there were no components co-eluted with the peptides that would interfere with the biological assay, including DMSO and DIPEA present in the peptide stock after cyclizing release. Biological screening is typically performed at compound concentrations of approximately 10 μM, meaning that 100% DMSO and 150 mM DIPEA in a peptide stock of approximately 10 mM diluted 1000-fold to reach concentrations of 0.1% and 150 μM, respectively, is unlikely to affect most biological assays.

[0130] 2.2. Conclusion In summary, a cyclizing peptide release strategy based on disulfide exchange reaction has been developed herein, which gives high purity disulfide-cyclized peptides directly from solid support. To the best of our knowledge, this is the first approach in which cyclic peptide libraries are released with high purity and with a cleavage reagent that can be removed by evaporation, so that the peptides can be easily screened using bioassays without prior purification. Importantly, the yields of peptides with different sequences showed a narrow distribution, allowing the screening of cyclic peptides without the need to determine or adjust the concentration. This approach shows that it is applicable to the generation of libraries containing hundreds of peptides. EXAMPLES

[0131] Example 3 - Large-scale expansion of the size of a cyclic peptide library 3.1. Acylation of cyclic peptide scaffolds via peripheral amines For the combinatorially diversifying macrocyclic library shown in Figure 8a, we chose to modify the fragments with amines acting as peripheral groups and carboxylic acids. The N-acetylation reaction is efficient and selective and has been widely applied in the synthesis of DNA-encoded chemical libraries (P.R.Fitzgerald et al., Chem.Rev., 2020). N-acetylation was also used to diversify lead structures by a panel of carboxylic acids in solution followed by activity screening of crude reactions (A.Brik et al., Chem.Biol., 2002, 9, 891-896) or by X-ray crystallographic analysis (M.R.Bentley et al., J.Med.Chem., 2020, 63, 6863-6875). The reaction was tested with the model scaffold cyclo(Mea-Lys-Xaa-Mpa) with primary amines as peripheral groups (Figure 8b) and eight structurally diverse carboxylic acids (Figure 8c). To efficiently convert the scaffold to the desired product, we chose to react the scaffold with a 4-fold molar excess of carboxylic acid. Near-quantitative conversion of the scaffold to product was desired because if the unmodified scaffold was more abundant than the acylated scaffold, binding would be weaker and could interfere with screening. The excess of carboxylic acid would lead to unreactive carboxylic acids that may be present during screening, but most carboxylic acids would not bind to the target by themselves due to their small size. The only expected by-products of the acylation reaction would be excess carboxylic acid, HOBt, and tetramethylurea, none of which should be compatible with biochemical assays. The cyclic peptide scaffold (final concentration 10 mM) was incubated with a 4-fold molar excess of eight acids in a volume of 4 μl for 3 h using HBTU as the activator and DIPEA as the base, confirming complete conversion of the scaffold with almost all acids (upper numbers in Figure 8c).

[0132] 3.2. Macrocyclic Library Synthesis in Nanoliter Volumes Combinatorial diversification of the same cyclic peptide scaffold was subsequently tested in a volume of 80 nl, thus 50 times smaller. This step was essential since we planned to generate libraries in small nanomolar scale volumes, but as a result, micromolar amounts of scaffolds, which can be easily synthesized in a well of a 96-well plate (5 μmol scale), were sufficient to synthesize more than 100 macrocycles from one scaffold. Furthermore, we aimed to apply an acoustic dispensing technique to transfer the reagents, which is suitable for transferring nanoliter volumes but not microliter volumes. Acoustic dispensing has the great advantage of being able to transfer reagents contact-free, without the need for pipetting tips, allowing a high speed of dispensing, reducing waste and costs. Application of the same reaction conditions resulted in a significant decrease in yield (first of the two lower figures in Figure 8c), calling for optimization of the acylation reaction. We hypothesized that the low yields were related to the use of DIPEA, since the base is relatively volatile and is partially lost when dispensed as 2.5 nl droplets by sonication. Tests were performed with the sodium salts of the non-volatile bases DABCO and HEPES, and with the volatile NMM, which has a high solubility in the solvent system used and can be applied in higher concentrations. With all three bases, the macrocycle was quantitatively acylated with the three acids tested, and application of DABCO to further acids showed that the conditions were suitable for efficient modification of peripheral amines of cyclic peptides (second of two lower figures in Figure 8c).

[0133] In the next step, the acylation reaction was tested using other macrocycles. In particular, macrocycles with less exposed amino groups than the one in the model scaffold cyclo(Mpa-Lys-Xaa-Mea) were selected. For this purpose, four macrocycles provided by Enamine were ordered (13a). All these compounds are non-peptide macrocycles, containing building blocks other than amino acids. The acylation reaction (Figure 13b), using the same reaction conditions and acids, showed an efficient conversion of the macrocycle scaffold to a carboxylic acid-bearing macrocycle (Figure 13c).

[0134] 3.3. Synthesis of cyclic peptide scaffolds bearing amine groups We then synthesized cyclic peptide scaffolds with random structures and one peripheral amino group using a recently developed approach for the efficient generation of large numbers of small cyclic peptides in 96-well plates. Briefly, short peptides are synthesized on the solid phase and liberated by a disulfide cyclization reaction to obtain essentially pure scaffolds with no need for further purification (Figure 9a). We generated a first library of cyclic peptide scaffolds containing three altered amino acids: one with a primary amine in the side chain (selected from 7 aa), one α-amino acid with a random side chain (selected from 15 aa), and one with a random backbone structure (selected from 6 aa) (Figure 9b and Figure 9c; sub-libraries 1a-f). We also synthesized a second library in which the primary amino group was introduced via a cysteine ​​residue. Of the 3,240 (library 1) and 540 (library 2) different scaffolds that could theoretically be assembled in a combinatorial manner using the indicated amino acids, 384 were randomly selected and synthesized in four 96-well plates. Quantification of 45 cyclic peptides containing Trp residues by absorption showed that most molecules were obtained in sufficient amounts (average concentration = 8.1 mM; Figure 9d). Given the relatively narrow distribution of yields, concentrations were not normalized for further use.

[0135] 3.4. Synthesis of Combinatorial Macrocyclic Libraries and Thrombin Screening The 384 scaffolds were combinatorially reacted with 12 carboxylic acids (Figure 10a), resulting in 4,608 different macrocycles, and this library was screened against the coagulation proteases and therapeutic target thrombin. Thrombin inhibitors are already used in the clinic as antithrombotic drugs, but suffer from poor oral availability. As carboxylic acids, structurally diverse molecules were selected, including several fragments that could bind to the S1 (H-bond) and S2 (hydrophobic interaction) specificity pockets of thrombin (Figure 10a). Although groups containing positive charges, such as guanidine, are known to bind particularly well to the S1 subsite, they were omitted because we were interested in developing uncharged macrocycles with limited polar surface that could be applied orally. Indeed, the active forms of approved thrombin inhibitors contain such positively charged groups and need to be applied as prodrugs, which could be the basis for limiting their oral availability. Using acoustic dispensing, 20 nl of scaffold (average 8.1 mM) and 20 nl of pre-activated acid (80 mM) were combined to reach final concentrations of approximately 4 mM scaffold and 40 mM carboxylic acid (Figure 10b). We chose to apply a 10-fold molar excess of carboxylic acid (versus 4-fold as before) because some of the amino groups in the scaffold are less accessible than the α-amino groups of the model peptides above. After 5 h, the overnight reaction was quenched by adding 5 μl of 100 mM Tris buffer, a 5 μl volume of thrombin (final concentration 2 nM) was added, and the remaining thrombin activity was measured using a fluorogenic substrate (5 μl). The concentration of macrocycles in the screen was approximately 10 μM. A fraction of 0.2% of the reactions (9 / 4277) inhibited thrombin by more than 50%, all of which were macrocycles containing chlorothiopheneic acid (14) (Figure 10c). Repeated runs of all macrocyclic synthesis reactions involving chlorothiophenic acids (384 scaffolds × 14) and thrombin in independent experiments identified essentially the same top hits, thus confirming high reproducibility in both the diversification reactions and activity screening (Figure 10d).

[0136] The chlorothiophene acid (14), which gave most of the hits, was previously reported to function as the S1 subsite binding group of the trypsin-like serine protease FXa (P.M. Fischer, J. Med. Chem., 2018, 61, 3799-3822), and therefore the hits were expected to be macrocycles directing the chlorothiophene group to the S1 pocket. Not all macrocycles modified with acid 14 inhibited thrombin, suggesting that the macrocycle contributes substantially to binding. Given the 10-fold excess of carboxylic acid used in the acylation reaction, unreacted 14 was present at a concentration of approximately 100 μM in all wells of the thrombin screen. At this concentration, 14 did not inhibit thrombin, as evidenced by the control reactions in which 14 but not the macrocyclic scaffold was added, and by the majority of the 384 wells that contained 14 and the scaffold but showed no thrombin inhibition (Figure 10c). Since 14 is generated as a carboxamide after reacting with the amino group, the inhibition of thrombin was tested by chlorothiophene amide. i = 380 μM. The weak inhibition confirmed that the macrocyclic scaffold contributed substantially to the activity of the identified macrocycles. The best three hits, M1, M2, and M3, were highly related in structure and were all based on the formatcyclo (Mpa-D3-B5-Xaa-Mea) scaffold, where the amino acid Xaa was all an α-amino acid with a hydrophobic side chain (D-Val, L-Phe, L-Val; Figure 10e).

[0137] 3.5. Acylated scaffolds are active species We next assessed whether the activity observed in the screen was derived from the expected macrocycle or from by-products, such as, for example, carboxylic acid or macrocyclic dimer adducts. To this end, the reaction of the scaffold with carboxylic acid 14 was repeated on a 250-fold larger scale (same concentration but larger volume) for the two top hits M1 and M2, the reactions were run on a RP-HPLC column, 20 fractions each combining products eluted within 1 min were isolated, the fractions were lyophilized, and thrombin inhibitory activity was measured (Figure 10f). In both reactions, the fractions containing the desired macrocycle product showed by far the highest activity, indicating that the hits were identified based on the activity of the macrocycles M1 and M2.

[0138] The purified macrocycles M1, M2, and M3 had K of 44 nM, 165 nM, and 125 nM, respectively. i In some therapeutic applications, the reducible disulfide bond present in all scaffolds screened herein is undesirable, so we tested whether it could be replaced with a more stable bond. M4 and M5 were synthesized containing dithioacetal or thioether linkers. M4 and M5 have K values ​​of 83 ± 8 nM and 135 ± 16 nM. i inhibited thrombin at 0.05 and therefore the affinity was only two- and three-fold weaker.

[0139] 3.6. Development of protein-protein interaction inhibitors Macrocycles have attracted much interest as inhibitors of protein-protein interactions (PPIs), and many attempts have been made to develop inhibitors for the prototype PPI disease target, MDM2:p53 (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 suppressor p53, and MDM2 binders that block the MDM2-p53 interaction have been of interest in the development of new anticancer therapies (P. Chene, Nat. Rev. Cancer, 2003, 3, 102-109). To test new approaches with this challenging PPI target, 192 structurally diverse cyclic peptide scaffolds were synthesized, all based on three random amino acids, one of which contained a lateral diversified amino group. To increase the chances of identifying binders, all scaffolds contained either tryptophan or phenylalanine, the two amino acids that form the key interactions of the stapled peptides that bind to MDM2 and inhibit the MDM2:p53 interaction. Cyclic peptide scaffolds were synthesized in 96-well plates as described for library 1 above, and obtained at an average concentration of 12.9 mM and an average purity of 90%. As before, this time 104 carboxylic acids were used, and the scaffolds were modified by acylation with fragments in a combinatorial manner. Thus, the size of the scaffold library was expanded from 192 structures to 19,968 macrocycles, thus expanding by more than 100-fold.

[0140] The library was screened by dispensing the target protein MDM2 and a reporter peptide that measures binding of the macrocycle into reactions in 384-microwell plates. The fluorescent reporter peptide binds to the PPI interface on MDM2 (K d= 0.5 μM), whose displacement by the macrocycle can be followed by measuring fluorescence polarization. The screening results were again displayed in an array of scaffold (vertical) and fragment (horizontal) combinations, with the color indicating the degree of reporter peptide displacement from MDM2 (Figure 11). Two hit groups were most interesting, one identified in the horizontal orientation and therefore macrocycles sharing the same scaffold, cyclo(Mpa-Trp-D3-B4-Mea), and the other identified on a vertical line and therefore macrocycles sharing the same carboxylic acid (9). The acylation reaction and screening were repeated using all 192 scaffolds and the four carboxylic acids that gave the best hits (9, 14, 25, 91), confirming the results of the first screen. Analysis of the active species in the three reactions for each of the two hit groups revealed that in the first group (hits M6, M7, and M8), the active species was the expected macrocyclic structure, but in the second group this was not the case (Figure 12a).

[0141] 3.7. Identification of Nanomolar MDM2 Binders Purified macrocycles M6, M7 and M8 efficiently displaced the fluorescent peptide probe from MDM2, with similar IC of approximately 1 μM. 50 Although we demonstrated binding constants of 650 ± 50 nM (F-M6), 790 ± 80 nM (F-M7), and 340 ± 40 nM (F-M8), the competition assay was not suitable for determining binding constants in the low micromolar or nanomolar range due to the high MDM2 concentration required for the assay (1.2 μM). Therefore, three macrocycles were synthesized as conjugates with fluorescein linked to the N-terminal region of the peptide scaffold, and binding affinities were measured in a direct fluorescence polarization assay (Figure 12c). The conjugates showed K values ​​of 650 ± 50 nM (F-M6), 790 ± 80 nM (F-M7), and 340 ± 40 nM (F-M8). d The values ​​were shown.

[0142] 3.8 Picomole-scale iterative synthesis and screening The facile synthesis of macrocycles allows for the iterative synthesis of sub-libraries based on hit compounds that can improve binding affinity. To enhance the potency of macrocycle M8, which we identified in the initial screen, we synthesized 63 scaffolds (Figure 18a) using amino acids similar to those of M8, including three analogs of nipecotic acid (Nip), three analogs of diaminopropionic acid (Dap), and seven analogs of tryptophan (Trp) (3 x 3 x 7 = 63). We then diversified the 63 scaffolds with 14 carboxylic acids, including repeats from the initial library hits (carboxylic acids 14, 25, 91), as well as related structures that are analogs of cinnamic acid 91, the carboxylic acid of macrocyclic hit M8 (105-115; Figure 28). To identify binders with nanomolar affinity, we screened 882 macrocycles (63 × 14) at a 13-fold lower concentration (750 nM) than in the first screen, which corresponds to a 30 pmol scale (Figure 18b). Although the most active macrocycle was based on the original scaffold, we identified in this screen a more potent macrocycle, namely a carboxylic acid resulting in 109 (macrocycle M9) that displaced the reporter peptide by 68% at 750 nM, compared to 21% for M8 (acid 91; Figure 18b). We did not improve on this scaffold, but obtained meaningful structure-activity relationship data for the macrocycle from a screen using 63 scaffolds, showing that all building blocks were essential.

[0143] We then performed a third cycle of library synthesis and screening, acylating the nine most promising scaffolds from the previous screen with 15 additional carboxylic acids, most of which were cinnamic acid derivatives with larger substituents. We then identified M10, a macrocycle based on the original scaffold and acylated with acid 120, which displaced the fluorescent probe used, F-M8, by 84% from MDM2 at 750 nM, more efficiently than the parent compounds M8 and M9 (Figure 18c). We conjugated the macrocycle M10 to fluorescein and measured binding to MDM2 using FP with a Kd of 43 ± 18 nM (Figure 18d). Using the fluorescein conjugate, we also performed competition experiments with Nutlin-3a, which binds to a defined hydrophobic pocket of MDM2 (B. Anil et al., Acta Crystallogr. Sect. D Biol. Crystallogr. 2013, 69, 1358-1366) and is the precursor of a nutlin-based clinical candidate. The two ligands did not compete, suggesting that the macrocycles bind to different sites and may have novel inhibitory mechanisms (Figure 29). Repeated binding measurements of the macrocycles by surface plasmon resonance (SPR) as an orthogonal method showed Kd of 600 ± 300 nM (M6), 550 ± 190 nM (M7), 169 ± 93 nM (M8), and 29 ± 14 nM (M10), confirming the affinity ranges identified by the FP assay (Figure 18e and Figure 30).

[0144] 3.9 Conclusion Herein, we describe a method for diversifying macrocyclic scaffolds on the picomole scale using acoustic dispensing 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 directly screened against protein targets to successfully identify inhibitors. The acylation reactions in nanoliter volumes were robust, resulting in high conversion to product for many different carboxylic acids and macrocycles. No unexpected by-products were observed. Our automated platform allows this final diversification step to be performed within an hour. This is in contrast to traditional synthesis of macrocyclic libraries, which require laborious purification. Acoustic dispensing has been used in synthesis in the past, but it has been limited to elaboration of synthetic reaction scope rather than direct synthesis of screening compounds. Our small-scale reactions, due to their relatively high purity, allowed for direct screening of macrocycles against protein targets in the same microtiter plate. It was demonstrated that inhibitors could be identified from these screens and that the observed activity was due to the desired product and not impurities. Screening of crude mixtures remains limited in the literature. We are not aware of any existing efforts at the same scale or specifically related to macrocycles. Given the facile nature of our method, it should be applicable to screening many protein targets. Furthermore, many different chemical reactions can be utilized for ADE-based diversification. Amide bond formation was chosen as the first example due to its simplicity and previous use as a crude screening reaction. The largest library synthesized by us was 20,000 macrocycles, but could be relatively easily expanded to hundreds of thousands. To generate more lead-like compounds in the future, this method could be applied to scaffold structures cyclized via non-reducing bonds. To further enhance the drug-like properties of the library, the building block set could be expanded beyond amino acids.This method can be used to screen for more therapeutically relevant targets for which it is hoped to develop clinical candidates.

[0145] 3.10 Supplementary results Overall structure of human α-thrombin complexed with M1 Human α-thrombin consists of two polypeptide chains of 36 amino acid residues (light chain) and 259 amino acid residues (heavy chain) covalently linked through a disulfide bridge (Cys122 in the heavy chain and Cys1 in the light chain). X-ray structural analysis of crystals formed by α-thrombin (light and heavy chains) and macrocycle M1 showed that there are four nearly identical copies of the heavy and light chains of human α-thrombin in the asymmetric unit. The four light / heavy chains of α-thrombin are designated A / B, C / D, E / F, and H / L. The structure of H / L was used for all calculations and construction of the structure diagram. The light chain of human α-thrombin can be clearly traced from Glu1C to Ile14K. The amino-terminal residues (Thr1H to Gly1D) and the carboxyl-terminal residues Asp14L (except for light chains C and E), Gly14M, and Arg15 are not clear and are 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 flexible autolysis loop at the surface (Trp148 to Val149C). The carboxyl-terminal residue Glu247 lacks adequate electron density. The only minor differences occur at the level of the flexible, less well-defined loops or in the orientation of the exposed peripheral side chains. The overall structure of human α-thrombin bound to the macrocycle, whether in the apo form or in complex with inhibitors, does not show any significant rearrangement of the backbone when compared to other human α-thrombin structures.

[0146] Overall structure of macrocyclic molecule M1 The electron density of the macrocycle M1 is well defined and allows unambiguous assignment of group orientations for all four protein complexes present in the asymmetric unit. The atom numbering of M1 is shown in Figure 26b. Classical secondary structure elements and non-covalent intramolecular interactions are not identified in the macrocycle. The molecule appears to adopt a chair-like conformation that fits well with the shape of the catalytic pocket.

[0147] Interaction of human α-thrombin with M1 The M1 macrocycle is located 400.5 Å from the active site. 2 It fits well into the gap formed by its surrounding substrate pockets covering the protein surface of the macrocycle (N. Voss et al., Nucleic Acids Res. 2010, 38, pp. W555-W562). The conformation and interactions of the macrocycle are equivalent in the four active sites of the four thrombin molecules present in the asymmetric unit. The majority of the interaction of M1 with human α-thrombin is mediated by a 5-chlorothiophene-2-carboxamide functional group that fits into the primary specificity S1 pocket. This group is mediated by a H group that bridges the main chain of Gly219 (M1 N7 to Gly219 O) and the oxygen 09 of M1 to the main chain nitrogen of Gly193 N and the main chain nitrogen of Ser195. 2It is trapped in the pocket by hydrogen bonding with the O molecule. The 5-chlorothiophene-2-carboxamide is further involved in a network of polar contacts with the main chains near Cys191 (M1 09 with Cys191 O), Glu192 (M1 09 with Glu192 N), Gly216 (M1 N7 with Gly216 O and M1 S15 with Gly216 N), Trp215 (M1 S15 with Trp215 N), and the side chain of Cys220 (M1 N7 with Cys220 S). The chlorine atom of the 5-chlorothiophene-2-carboxamide functional group points towards the bottom of the S1 pocket, where it appears to form a halogen-aromatic π interaction (4.0 Å) with the aromatic ring of Tyr228. The main chain nitrogen N4 and oxygen O17 of M1 form hydrogen bonds with the main chain oxygen of Gly216 (Gly216 O) and the nitrogen of Gly216 (Gly216 N), respectively. In addition, the main chain nitrogen N4 and oxygen O17 of M1 can form polar contacts with the main chain nitrogen of Gly219 (Gly219 N) and the oxygen of Gly216 (Gly216 O), respectively. Similarly, the main chain nitrogen N18 of M1 can form two polar contacts with the side chain carboxyl group of Glu192 (Glu192 OE1 and OE2). Finally, the H 2 One molecule of O bridges the main chain nitrogen N27 of M1 with the main chain oxygen of Glu97A (Glu97A O). Importantly, binding of M1 to human α-thrombin is mediated by multiple hydrophobic contacts by the main chain and side chains of adjacent enzyme residues. The macrocyclic backbone (C20-C24) containing the disulfide bridge S21-S22 lies towards the hydrophobic cage formed by the side chains of residues His57, Tyr60A, Trp60D (proximal S2 pocket) and Leu99 (distal S3 pocket). The valine side chain (C28-C31) bends the other side of the ring towards the hydrophobic pocket formed by Ile174 and Trp215. Finally, the phenyl ring of C35-C40 extends onto the top of the thrombin loop (Gly216-Cys220).

[0148] 3.11 Supplementary Materials and Methods General Considerations Unless otherwise stated, all reagents were purchased from commercial sources and used without further purification. Solvents were not anhydrous or dried prior to 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-O-(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).

[0149] Synthesis of model scaffold 1 Cyclic peptide model scaffold 1 was synthesized using a cyclic disulfide release strategy (CDR) previously described (S. Habeshian et al., ACS Chem. Biol. 2022, 17, pp. 181-186). Linear peptide precursors were prepared using Rapp Polymere HA40004.0 polystyrene A SH resin (200-400 mesh), 0.95 mmol / gram loaded resin and Habeshian, S. et al., 2022 2 The peptide was synthesized on a 25 μmol scale in a 5 ml polypropylene synthesis column (MultiSyntech GmbH, V051PE076) using the following procedure described in. The peptide was released as follows: To deprotect the side chains, the resin was diluted with 2 ml of 38:1:1 TFA / TIS / ddH 2 The resin was incubated with 1 ml of DMSO containing 150 mM DIPEA (6 eq.) overnight to release the cyclic peptide. The resin was removed by filtration. The crude mixture was purified using a Waters HPLC system (2489 UV detector, 2535 pump, fraction collector III) with a 19 mm x 250 mm Waters XTerra MS C18 OBD Prep Column C18 column (125 Å pore, 10 μm particles), solvent system A (H 2The model scaffolds were purified by RP-HPLC using solvent system A (MeCN, 0.1% v / v TFA) and solvent system B (MeCN, 0.1% v / v TFA), and a gradient of 0–25% solvent B over 30 min. Fractions containing the model scaffolds were lyophilized and dissolved in DMSO to reach a concentration of 40 mM.

[0150] Acylation of model scaffold 1 by pipetting of reagents Model scaffolds were acylated in 4 μl volumes at a 40 nmol scale as follows: Scaffold (20 μl of 40 mM stock in DMSO) was supplemented with base (20 μl of 160 mM DIPEA dissolved in DMSO) and 2 μl of the mixture was transferred to a well of a PCR plate. Carboxylic acids were prepared as 160 mM stocks in DMSO containing 160 mM DIPEA. An equal amount of HBTU (160 mM in DMSO) was added to each acid stock and 2 μl of the resulting active ester (80 mM) was added to the same PCR plate. Reactions were allowed to proceed for 3 hours at room temperature. After this, 1 μl of the reaction was transferred to 99 ml of 100 mM aqueous Tris-HCl pH 7.5 and incubated for 6 hours, the activated acid was quenched with Tris and the reaction was analyzed by LC-MS.

[0151] Acylation of model scaffold 1 by acoustic reagent transfer Model scaffolds were acylated on an 800 pmol scale in a volume of 80 nl as follows: Scaffold 1 (20 μL of 40 mM stock in DMSO) was supplemented with base (20 μL of 160 mM DIPEA, 20 μL of 160 mM DABCO, 20 μL of 160 mM HEPES sodium salt, or 20 μL of 1 M NMM dissolved in DMSO) and 10 μl of the mixture was transferred to an ECHO source plate (Labcyte Echo Qualified 384-well low dead volume microplate). The concentrations in the source plate were 20 mM model scaffold and 80 mM DIPE (4 equiv.), or 80 mM DABCO (4 equiv.), or 800 mM NMM (40 equiv.). Carboxylic acids were prepared as 160 mM stocks 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, and the active ester (80 mM) was added to the same source plate. The source plate was centrifuged at 950 g (2,000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 3 min to remove potential air bubbles. Using a Labcyte Echo 650 acoustic dispenser, 40 nl of model scaffold 1 (800 pmol) was transferred to a Nunc 384-well low volume polystyrene plate, followed by 40 nl of the active ester (3.2 nmol, 4 equiv). Transfers were performed in duplicate to obtain sufficient material for LC-MS analysis. The plate was sealed and the reaction was allowed to proceed at room temperature for 6 h. After this, 8 μl of 100 mM aqueous Tris-HCl (pH 7.5) was added to each of the duplicate reactions, the duplicate reactions were pooled and incubated for 3 h, the activated acid was quenched with Tris, and the reactions were analyzed by LC-MS.

[0152] Acylation of model scaffolds 2-5 by acoustic reagent transfer Model scaffolds 2-5 were purchased from Enamine and obtained as 1.1-1.2 mg powder. Scaffolds were dissolved in 62-88 μl DMSO to obtain 40 mM stocks. Scaffolds were acylated using DABCO as the base as described for model scaffold 1 above, with the following differences: reaction time was 6 h. Prior to LC-MS analysis, 720 nl DMSO was dispensed into each well, followed by 7.2 μl of 100 mM aqueous Tris-HCl (pH 7.5). Quenching was performed overnight.

[0153] Scaffold and amino acid sequence design The cyclic peptide scaffolds used in Library 1 were prepared by randomly selecting amino acid sequences. The number of different sequences that could theoretically be generated based on the selected scaffold format and amino acid building blocks was much greater than the number of scaffolds (384) synthesized in Library 1, as described below.

[0154] Number of theoretical scaffolds in Library 1: Diamino acid-containing scaffolds: 3,240 Number of scaffold formats (6) × number of diamino acids (6) × number of backbone amino acids (6) × number of side chain amino acids (15) Cysteine-containing scaffolds: 540 Number of scaffold formats (6) × number of backbone amino acids (6) × number of side chain amino acids (15).

[0155] To randomly select 384 amino acid sequences, we assigned alphanumeric identifiers to all building blocks and manually enumerated all possible permutations. Peptides were assigned numbers from 1 to 3,780. We then used a random sequence generator (https: / / www.random.org / sequences / ) to permute the numbers and select the first 384 for synthesis.

[0156] Preparation of polystyrene-SS-cysteamine resin for library synthesis The following procedure was applied to prepare polystyrene-SS-cysteamine resin for the synthesis of 4x96 peptides at 5 μmol scale in four 96-well plates required for the synthesis of scaffolds for Library 1 (thrombin screening). To each of four 20 ml plastic syringes (CEM, 99.278) was added 589 mg of resin (Rapp Polymere HA40004.0 Polystyrene A SH resin, 200-400 mesh) at a loading of 0.85 mmol / gram, corresponding to a 0.5 mmol scale. The resin was washed with 15 mL of DCM and then swollen in 15 ml of 3:7 MeOH / DCM v / v for 20 min. 2-(2-pyridinyldithio)-ethanamine hydrochloride (1.96 grams, 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 placed into each syringe, which was then shaken for 3 hours at room temperature. After this, the 2-(2-pyridinyldithio)-ethanamine solution was discarded and the resin was washed with 2×20 ml of 3:7 MeOH / DCM v / v, then 2×20 ml of DMF. The resin was combined into a single syringe as a suspension in DMF, then washed with 11.8 ml of a 1.2 M solution of DIPEA in DMF for 5 minutes to ensure that all amines were neutral. This solution was discarded and the resin was washed with 2×20 ml of DMF, 4×20 ml of DCM, then kept under vacuum overnight to obtain a free-flowing powder. In the synthesis of the library scaffold (MDM2 screening), the resin loading was 0.95 mmol / gram, so 526 mg of resin was added to each of the two syringes.

[0157] Peptide library synthesis in 96-well plates Automated solid-phase peptide synthesis was performed on an Intavis Multipep RSi synthesizer. For the thrombin library, 565 mg of polystyrene-SS-cysteamine resin (0.48 mmol of 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 library, 505 mg of functionalized resin was added instead. The tube was shaken to ensure that the resin was evenly suspended, and 200 μl (5.88 mg resin, 5 μmol) was transferred to each well of a 96-well solid-phase synthesis plate (Orochem OF 1100). The resin was washed with 6 × 150 μl of DMF. Coupling was performed with 53 μl amino acid (500 mM, 5.3 equiv.), 50 μl HATU (500 mM, 5 equiv.), 12.5 μl N-methylmorpholine (4 M, 10 equiv.), and 5 μl N-methylpyrrolidone. All components were premixed for 1 min and then added to the resin (1 h reaction, no shaking). The final volume of the coupling reaction was 120.5 μl, with final concentrations of reagents of 220 mM amino acid, 208 mM HATU, and 415 N-methylmorpholine. Coupling was performed twice, then the resin was washed with 6×225 μl DMF. Fmoc deprotection was performed using 120 μl 1:5 piperidine / DMF v / v for 5 min and performed twice. The resin was washed with 8×225 μl DMF. At the end of peptide synthesis, the resin was washed with 2×200 μl DCM.

[0158] Removal of library side chain protecting groups To remove side-chain protecting groups, the bottom of a 96-well synthesis plate was sealed by pressing the plate onto a soft 6 mm thick ethylene-vinyl acetate foam pad (Rayher Hobby GmbH, 78 263 01) and the resin in each well was filled with approximately 500 μl of 38:1:1 TFA / TIS / ddH 2The plates were then covered with a polypropylene adhesive seal and then weighted by placing a weight (1 kg) on ​​top to ensure no leakage occurred. After 1.5 hours, the synthesis plate was placed on a 2 ml deep well plate (Thermo Scientific, 278752) and the TFA mixture was drained off. The wells were washed with 3×500 μl DCM (added via syringe) and then air dried for 3 hours.

[0159] Library cyclization release of library peptides in 96-well plates The plate was pressed against a foam pad as above to seal the openings, and 200 μl of 150 mM DABCO (6 equiv.) in DMSO was added to each well. The plate was sealed with adhesive foil, weighted (1 kg), and left overnight. The next day, the synthesis plate was placed onto a 2 ml deep-well plate (Thermo Scientific, 278752) and centrifuged at approximately 200 g (1,000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 1 min to recover the cleaved macrocycles in DMSO.

[0160] Quantification of library peptides by absorbance Absorbance measurements were performed using a Nanodrop 8000 spectrophotometer (Thermo Scientific) at a wavelength of 280 nm using a path length of 10 mm. Truncated peptides containing Trp and D-Trp were diluted 250-fold in water for the thrombin library and 125-fold in water for the MDM2 library. The concentration of peptides was calculated using the Beer-Lambert law. The extinction coefficient Trp ε 280 =5,500M -1 cm -1 was used.

[0161] LC-MS analysis Peptides were purified using a C18 reversed-phase column (Phenomenex Kinetex 2.1 mm × 50 mm C18 column, 100 Å pore, 2.6 μm particles) and solvent A (H 2The fractions were analyzed by UHPLC using a linear gradient of solvent B (acetonitrile, 0.05% formic acid) to solvent C (O, 0.05% formic acid) at a flow rate of 1 ml / min and LC-MS analysis on a single quadrupole MS system (Shimazu LCMS-2020). Mass spectrometry was performed in positive ion mode.

[0162] For LC-MS analysis, samples of the different experiments were prepared as follows: To analyze the proof-of-concept reaction of acylation, 160 nl of the reaction mixture was diluted with 16 μl of Tris-HCl buffer pH 7.5 to obtain a peptide concentration of 100 μM. To analyze the scaffolds synthesized for library 1, 1 μl of the DMSO / DABCO eluate was diluted with 80 μl of water to obtain a cyclic peptide concentration of approximately 120 μM. To analyze the scaffolds synthesized for library 2, 1 μl of the DMSO / DABCO eluate was diluted with 128 μl of water to obtain a cyclic peptide concentration of approximately 120 μM. In all analyses, 5 μl of sample was injected, typically using a 0-60% gradient of solvent B over 5 min.

[0163] Calculation of physicochemical properties of macrocyclic molecules The physicochemical properties molecular weight, calculated water / n-octanol partition coefficient (cLogP), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), polar surface area (PSA), and number of rotatable bonds (NRotB) were calculated using DataWarrior software (www.openmolecules.org). The scaffold and carboxylic acid structures were drawn in ChemDraw and saved as SMILES strings in SD files, one for the scaffold and one for the acid. Both SD files were opened in DataWarrior. The “Enumerate combinatorial library” function was used to define the desired amide bond formation reaction between the macrocyclic scaffold and the carboxylic acid. The following definitions were made: amide was defined as an “excluded group”. Nitrogen atoms were defined as not being part of an aromatic ring and having a hydrogen atom number greater than 0. The carbon atom next to the amine was defined as not aromatic and not containing pi electrons. The atoms of the starting material and the product were then mapped. The desired properties were calculated from the structures after combinatorial enumeration.

[0164] Acylation of scaffolds to generate library 1 Cyclic peptide scaffolds in the solvent used to release the peptides from the resin (DMSO containing 150 mM DABCO) were transferred to a Labcyte Echo Qualified 384-well low dead volume microplate (10 μl per well). The concentration of cyclic peptide scaffolds was approximately 8.1 mM on average. Carboxylic acids were dissolved to 160 mM in DMSO containing 160 mM DABCO. An equal volume of HBTU (160 mM in DMSO) was added to each acid stock. Active esters (80 mM) were added to a separate low dead volume source plate. This source plate was centrifuged at 850 g (2,000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 3 min to remove potential air bubbles. Using a Labcyte Echo 650 acoustic dispenser, 20 nl of scaffold (160 pmol) was transferred to a 384-well low volume polystyrene plate (Nunc, 264705), followed by 20 nl of active ester (1.6 nmol, 10 equiv). The plate was sealed and the reaction was allowed to proceed for 6 h at room temperature. This was followed by 5 μl of Tris buffer (100 mM Tris-Cl, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 1 mM CaCl 2 , 0.1% w / v BSA, 0.01% v / v Triton-X100) was dispensed into each well using a BioTek MultiFlo microplate dispenser. The reaction was quenched overnight at room temperature.

[0165] Thrombin Inhibition Screening Thrombin inhibition by Library 1 macrocycles was assessed by measuring the residual activity of thrombin in the presence of cyclic peptides at an average final concentration of 11 μM. Assays were performed in 384-well plates in Tris buffer, pH 7.4 (100 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl 2 , 1 mM CaCl 2, 0.1% w / v BSA, 0.01% v / v Triton-X100, and 0.6% v / v DMSO) with thrombin at a final concentration of 2 nM and the fluorogenic substrate Z-Gly-Gly-Arg-AMC at a final concentration of 50 μM. Thrombin (5 μl, 6 nM) in the above Tris-Cl buffer was added to each peptide using a BioTek MultiFlo microplate dispenser and incubated for 10 min at room temperature. Fluorescent substrate (5 μl, 150 μM) in the same buffer was added using a BioTek MultiFlo microplate dispenser and fluorescence intensity was measured using a Tecan Infinite M200 Pro fluorescent plate reader (excitation at 360 nm, emission at 465 nm) by reading every 3 min for 30 min at 25°C. The slope of each activity measurement curve was calculated in Excel. For the negative controls (20 wells containing DMSO but no macrocycle), the average slope was calculated. The percent of thrombin inhibition was calculated by dividing the slope and multiplying by 100.

[0166] Acylation of scaffolds to generate library 2 Cyclic peptide scaffolds in the solvent used to release the peptides from the resin (DMSO containing 150 mM DABCO) were transferred to a Labcyte Echo Certified 384-well polypropylene microplate (40 μL per well). The concentration of cyclic peptide scaffolds was approximately 12.9 mM on average. Carboxylic acids were dissolved to 184 mM in a 184 mM DMSO solution of DABCO. An equal volume of HBTU (184 mM in DMSO) was added to each acid stock. Active esters (92 mM) were added to the same polypropylene source plate. The source plate was centrifuged at 950 g (2,000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 3 min to remove potential air bubbles. Using a Labcyte Echo 650 acoustic dispenser, 12.5 nl of macrocycle (161 pmol) was transferred to a 384-well low volume polystyrene plate (Nunc, 264705), followed by 17.5 nl of active ester (1.61 nmol, 10 equiv). The plate was sealed and the reaction was allowed to proceed for 6 hours at room temperature. After this, 5 μml of Tris buffer was dispensed into each well using a Gyger Certus Flex liquid dispenser and the reaction was quenched overnight at room temperature.

[0167] MDM2 binding screening MDM2 binding by cyclic peptides was assessed by measuring the displacement of a fluorescent p53 peptide probe in the presence of cyclic peptides at an average final concentration of 11 μM. The assay was performed in 384-well plates with PBS buffer (100 mM NaCl), pH 7.4. 2 HPO 4 , 18 mM KH 2 PO 4, 137 mM NaCl, 2.7 mM KCl, 0.01% v / v Tween-20, and 3% v / v DMSO), a final concentration of 1.2 μM MDM2, and a final concentration of 25 nM fluorescent p53 peptide probe (FP53, sequence=5(6)-FAM-GSGSSQETFSDLWKLLPEN). MDM2 and FP53 (10 μl, 1.8 mM MDM2, 37.5 nM FP53), premixed in the PBS buffer described above, were added to each peptide using a Gyger Certus Flex liquid bulk dispenser and incubated for 30 min in the dark at room temperature. A single reading of fluorescence anisotropy was taken at 25° C. using a Tecan Infinite F200 Pro fluorescent plate reader (excitation at 485 nm, emission at 535 nm). The percentage of probe displacement was calculated using the following formula:

[0168]

number

[0169] where N is the mean anisotropy of the negative controls (no inhibition), X is the value obtained for each well, and P is the mean anisotropy of the probe alone. Identification of active species in hit reactions Macrocycles identified as hits in the thrombin screen were resynthesized on a 40 nmol scale by reacting 5 μl of 8 mM cyclic peptide scaffold in DMSO containing 150 mM DABCO with 5 μl of 80 mM carboxylic acid, 80 mM HBTU and 80 mM DABCO for 5 h at room temperature. The remaining activated esters were cleaved by filtration in 1.25 ml of Tris buffer (100 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl). 2 , 1 mM CaCl 2 The reaction was quenched by adding 240 μl MeCN and 1 ml water and incubated overnight. The next day, 240 μl MeCN and 1 ml water were added and the reaction was quenched by adding solvent A (H 2The run was performed on a C18 column (7.8 mm × 300 mm Waters NovaPak C-18 column, 60 Å pore, 6 μm particles) on a Thermo Dionex HPLC using a 10-80% gradient of solvent B (MeCN, 0.1% v / v TFA) and solvent C (MeCN, 0.1% v / v TFA) over 20 min, with fractions collected every minute. Fractions were lyophilized and dissolved in 120 μl of 2% aqueous DMSO. Activity of products in fractions was measured using the same assay as above, except in 96-well plates. 50 μl of each fraction was transferred to a 96-well assay plate (Greiner, 655101), followed by 50 μl of thrombin (6 nM in buffer). After 10 min incubation, 50 μl of fluorogenic thrombin substrate (Z-Gly-Gly-Arg-AMC, 150 μM in 1% DMSO buffer) was added, the plate was read, and the data was processed as above. Compounds in the active fractions were identified by mass spectrometry.

[0170] For hits from the MDM2 screen, reactions were performed in the same way except on a 50 nmol scale and purified in the same way except for a 10-80% gradient of solvent B and over 22 min. Fractions were lyophilized, dissolved in 40 μl DMSO, 160 μl water was added, 5 μl of each fraction was transferred to a 384-well plate (Nunc, 264705) and 15 μl of premixed MDM2 / FP53 peptide was added (final concentrations: 1.2 μM MDM2, 50 nM FP53, 5% DMSO).

[0171] Crystallization of thrombin by M1 Human α-thrombin was purchased from Haematologic Technologies (catalog number: HCT-0020). Protein stabilizers were 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. Buffer-exchanged human α-thrombin was incubated with macrocycle M1 in a 1:3 molar ratio and subsequently concentrated to 7.5 mg / ml using a 3,000 MWCO Vivaspin ultrafiltration device (Sartorius-Stedim Biotech GmbH). Further M1 macrocycle was added during the concentration to ensure that a 3-fold molar excess was retained. Crystallization studies of the complex were performed at 293 K in 96-well 2-drop MRC plates (Hampton Research, CA, USA) using the sitting-drop vapor diffusion method and Morpheus and LMB crystallization screens (Molecular Dimensions Ltd, Suffolk, UK). Droplets of 600 nl volume (with a 1:1 protein:precipitant ratio) were set up using an Oryx 8 crystallization robot (Douglas Instruments Ltd, Berkshire, UK) and equilibrated against 80 μl of reservoir solution. The best crystals were obtained by applying microseeding to fresh droplets equilibrated for 2-3 days using the following mixture as precipitant: 20 mM sodium formate, 20 mM ammonium acetate, 20 mM citrate tribasic dihydrate, 20 mM sodium potassium 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 3350, 12.5% ​​v / v MPD.

[0172] Crystallization, data collection and structure determination For X-ray data collection, crystals were mounted in LithoLoops (Molecular Dimensions Ltd, Suffolk, UK) and flash cooled in liquid nitrogen. X-ray diffraction data for human α-thrombin complexed with M1 were collected on the i04 beamline at Diamond Light Source Ltd (DLS, Oxfordshire, UK). The best crystals diffracted to a maximum resolution of 2.27 Å. Crystals were P2 1 It belongs to the space group 114, with the unit cell dimensions a = 56.25 Å, b = 100.57 Å, c = 108.90 Å, α = 90°, β = 90.11°, and γ = 90°. The asymmetric unit contains four molecules and has a length of 2.78 Å. 3 This corresponds to a Matthews coefficient of 100 / Da and a solvent content of about 48% of the crystal volume. Frames were indexed and integrated with the software XIA2, and merged and scaled with AIMLESS (CCP4i2 crystallography package) (M. Winn et al., Acta Crystallogr.D 2011, 67, pp. 235-242). The structure was solved by molecular replacement with the software PHASER (A. McCoy et al., J. Appl. Crystallogr. 2007, 40, pp. 658-674), using the model 6GWE (S. Kale et al., Sci. Adv. 2019, 5, eaaw2851) as template. Refinement was performed using REFMAC (A. Vagin et al. Acta Crystallogr.D 2004, 60, 2184-2195) and PHENIX (P. Adams et al., Acta Crystallogr.D 2010, 66, 213-221). From the first cycle of refinement, a broad area of ​​electron density corresponding to the bound ligand was clearly recognized in the electron density map. Building of the macrocycle was performed by Molview and the restraint files were generated and optimized by Phenix eLBOW. The macrocycle was fitted manually by the graphics software COOT (P. Emsley et al., Acta Crystallogr.D 2010, 66, 486-501). The final model contained 9,098 protein atoms, 160 macrocycle atoms, 4 Na +The final crystallographic R-factor is 0.189 (R free The resolution reached 0.243). The geometric parameters of the model are as expected or better at this resolution. The solvent excluded volume and the corresponding buried surface were calculated using PISA software and a spherical probe of radius 1.4 Å. The intra- and intermolecular hydrogen bonding interactions were analyzed by PROFUNC (R. Laskowski et al., Nucleic Acids Res. 2005, 33, pp. W89-W93), LIGPLOT+ (R. Laskowski et al., J. Chem. Inf. Model. 2011, 5, pp. 2778-2786), and PYMOL software.

[0173] Acylation of scaffolds to generate libraries 3 and 4 The cyclic peptide scaffolds required for libraries 3 and 4 were synthesized as described for those used in library 2. Due to the presence of many N-methylated amino acids that are more difficult to couple, 200 mM HOAt was applied together with HATU. The scaffolds were diluted to 2 mM in DMSO and 15 nL was transferred using acoustic dispensing, followed by 15 nl of DMSO containing carboxylic acid (40 mM, 20 equiv.), HBTU (40 mM) and DABCO (40 mM). After 6 h of reaction at room temperature, 370 nl of DMSO was added to each well, followed by quenching overnight by the addition of 5 μl of 100 mM Tris-Cl pH 7.4 containing 0.01% v / v Tween 20. For the MDM2 binding screen, F-M8 was used as a fluorescent probe due to its high affinity for MDM2, allowing the target protein to be used at low concentrations (720 nM, approximately 55% bound probe). 35 μl volume of pH 7.4 PBS buffer (100 mM Na 2 HPO 4 , 18 mM KH 2 PO 4, 137 mM NaCl, 2.7 mM KCl, 28.6 nM F-M8, and 823 nM MDM2 in 0.01% v / v Tween-20 was added to each well, and displacement of the reporter probe was determined as described above. The macrocycle concentration was 750 nM.

[0174] Synthesis of macrocycles on the mg scale 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, V051 PE076). The resin was washed with 6 × 150 μl DMF. Coupling was performed with 210 μL amino acid (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 (1 h reaction, no shaking). The final volume of the coupling reaction was 465 μl, with final concentrations of reagents of 226 mM amino acid, 215 mM HATU, and 430 mM N-methylmorpholine. Coupling was performed twice and then the resin was washed with 2×600 μl DMF. Fmoc deprotection was performed using 450 μl 1:5 piperidine / DMF (v / v) for 5 min, which was performed twice. The resin was washed with 7×600 μl DMF. At the end of peptide synthesis, the resin was washed with 2×600 μl DCM.

[0175] After SPPS, the resin was diluted with 2 ml of 38:1:1 TFA / TIS / ddH 2 The resin was incubated with 1000 ml of 10000 ml of DMSO v / v / v for 1 hour. The TFA solution was discarded and the resin was washed with 5×4 ml of DCM. After air drying for 3 hours, 1 ml of 150 mM DIPEA in DMSO was aspirated and the syringe was shaken overnight at room temperature. The next day, the DMSO solution was pushed into a 50 mL conical tube.

[0176] Carboxylic acids were typically coupled by adding 500 μl of premixed acid (100 mM, 2 eq.), HBTU (100 mM) and DABCO (100 mM) in DMSO. After 3 hours at room temperature, 8 ml of water was added, the tube was frozen and then lyophilized for 2 days to remove DMSO. The contents of the tube were dissolved in 3 ml of MeCN followed by the addition of 7 ml of water.

[0177] The crude mixture was purified using a Waters HPLC system (2489 UV detector, 2535 pump, Fraction Collector III), a 19 mm × 250 mm Waters XTerra MS C18 OBD Prep column (125A pore, 10 μm particles), and solvent system A (H 2 The extracts were purified by RP-HPLC using B (MeCN, 0.1% v / v TFA) and B (MeCN, 0.1% v / v TFA), typically a gradient of 30–70% solvent B over 30 min.

[0178] K of thrombin inhibitors i Decision Purified thrombin inhibitor (10 mM in DMSO) was dissolved in 125 μl of Tris buffer (100 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl) containing 0.1% w / v BSA, 0.01% v / v Triton-X100 and 0.2% DMSO. 2 , 1 mM CaCl 2 ) to 80 mM. Macrocycles were diluted 2-fold in Tris buffer containing 0.1% w / v BSA, 0.01% v / v Triton-X100 and 1% DMSO in the buffer. Thrombin activity was measured in 96-well plates (Greiner, 655101) and residual activity was calculated as described above for the assay used to measure activity of HPLC separated fractions of screening hits. Residual activity was plotted against the Log of the corresponding macrocycle concentration and fitted to a sigmoidal curve in GraphPad Prism 6 using the following four-parameter equation:

[0179]

number

[0180] K i Values ​​were calculated using the Cheng-Prusoff equation 50 The K m = 168 μM):

[0181]

number

[0182] IC of MDM2-binding macrocycles 50 Decision The concentration at which the MDM2 macrocycle displaces the reporter peptide for 50% of the protein (IC 50 ) was determined by the fluorescence polarization competition assay described above. 5 μL volumes of purified macrocycles (20 mM in DMSO) were serially diluted 2-fold in 100% DMSO in a low dead volume ECHO source plate. 150 nl of each dilution was transferred to a 384-well low volume polystyrene plate (Nunc, 264705) using acoustic droplet transfer. PBS buffer pH 7.4 (100 mM Na) containing 1% v / v DMSO was used to transfer the dilutions. 2 HPO 4 , 18 mM KH 2 PO 4 A 15 μL volume of MDM2 / FP53 probe premix (1.2 μM MDM2, 25 nM FP53 probe) in 10 mM NaCl, 137 mM NaCl, 2.7 mM KCl, 0.01% v / v Tween-20 was added to each well and incubated for 30 min in the dark. Fluorescence anisotropy was measured as described above. The percentage of bound inhibitor was calculated using the following formula:

[0183]

number

[0184] where N is the average anisotropy of the DMSO control, X is the anisotropy value obtained for each well, and P is the average anisotropy of the unbound probe. 50 was determined by plotting the percent inhibitor bound against the logarithm of the corresponding macrocycle concentration, and curves were fitted in GraphPad Prism 6 as described above.

[0185] Synthesis of fluorescein-labeled macrocycles Fluorescein-labeled macrocycles were synthesized essentially as described in the mg-scale macrocycle synthesis procedure. For 5(6)-FAM, manual coupling was performed using 4 equivalents of acid (180 mM, 556 μl), 4 equivalents of HATU (500 mM, 200 μl), 10 equivalents of NMM (4 M, 62.5 μl), all in DMF. Coupling was performed for 1×2 h and then washed as previously described.

[0186] K of fluorescein-labeled MDM2 binders by FP d Decision Fluorescein-labeled macrocycle stocks (20 mM in DMSO) were diluted to a concentration of 10 μM by adding 0.5 μl to 999.5 ml 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 was transferred to wells of a 384-well low-volume polystyrene plate (Nunc, 264705). A 7.5 μL volume of a two-fold dilution of MDM2 in PBS was pipetted into the wells. The final concentration of fluorescent macrocycle was 50 nM. After incubating the plate in the dark at room temperature for 30 min, the fluorescence anisotropy was measured at 25 °C on a Tecan Infinite F200 Pro fluorescence plate reader (excitation at 485 nm, emission at 535 nm). The anisotropy was plotted against the logarithm of the corresponding MDM2 concentration and a sigmoidal curve was fitted as described above.

[0187] Synthesis of thrombin inhibitors containing thioether bonds. A linear peptide containing three amino acids and a C-terminal cysteamine was synthesized by automated SPPS on a 50 μmol scale as described above for the mg-scale synthesis of cyclic peptides, except that cysteamine 4-methoxytrityl resin (Novabiochem 856087, 200-400 mesh, 1% DVB, 0.92 mmol / gram) was used. To this peptide, still on the resin, 4-bromobutyric acid (500 μL, 500 mM, 10 equiv.) was manually coupled 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 (1 h reaction, with shaking). The final volume of the coupling reaction was 1 ml, and the final concentrations of the reagents were 250 mM amino acid, 250 mM DIC. Coupling was performed twice and then the resin was washed with 4×4 ml DMF and then 2×4 ml DCM.

[0188] Removal of side chain protecting groups and cleavage were performed using 2 ml of 38:1:1 TFA / TIS / ddH 2 This was performed by incubating the resin with 0.1% O v / v / v for 1 h with shaking. After this, 50 ml of cold diethyl ether was added to the solution to precipitate the peptide. The mixture was stored at -20°C for 30 min and then centrifuged at 3,800g (4,000 rpm in a Thermo Heraeus Multifuge 3L-R centrifuge) for 30 min at 4°C. The ether was decanted and the peptide pellet was air-dried for 15 min.

[0189] The peptide was dissolved in 50 ml of freshly degassed 1:4 water / acetonitrile and 200 μl (1.15 mmol, 23 equiv.) of neat DIPEA was added. The cyclization reaction was allowed to proceed for 90 min at room temperature, then frozen and lyophilized.

[0190] Carboxylic acid 14 was attached as follows: the macrocycle was redissolved in 1 ml DMSO containing 100 mM DABCO. Carboxylic acids were typically coupled by adding 500 μL of premixed acid (100 mM, 2 eq.), HBTU (100 mM) and DABCO (100 mM) in DMSO. After 3 h at room temperature, 8 ml water was added, the tube was frozen and lyophilized for 2 days to remove DMSO. The contents of the tube were dissolved in 3 ml MeCN followed by the addition of 7 ml water. The crude mixture was purified by RP-HPLC as above.

[0191] K of MDM2-binding agents by SPR d Decision Experiments were performed using a Biacore 8K instrument from GE Healthcare. MDM2 (10 μg / mL) was dissolved in 10 mM MES buffer (pH 6.0) and immobilized onto three channels of a CM5 Series S chip (Cytiva, 29104988) using EDC / NHS amine coupling conditions in running buffer (10 mM PBS pH 7.4, 150 mM NaCl, 3 mM KCl, and 0.005% v / v Tween-20). Typically, immobilization levels were between 6,000 and 7,000 resonance units (RU). A reference cell was treated in the same way without MDM2. For measurements of binding kinetics and dissociation constants, five serial dilutions (3-fold) of the macrocycles and a DMSO blank were prepared in running buffer (10 mM PBS pH 7.4, 150 mM NaCl, 3 mM KCl, and 0.005% v / v Tween-20, and 0.5% v / v DMSO) and analyzed in single-cycle kinetics mode with contact and dissociation times of 120 and 60 s, respectively. EXAMPLES

[0192] Example 4 – Synthesis and screening of a large macrocycle library at the edge of the rule of five 4.1. Results In Example 1, 1 (Figure 14b; also called cysteamine) was coupled onto a thiol-functionalized resin by a dithiol exchange reaction with an excess of pyridyldithioethylamine. Here, activated thiosulfonates were used instead, since they can be synthesized without a chromatographic purification step and can be synthesized more easily in large quantities. To this end, we sought out commercial sources of building blocks from which N-Boc alkyl halogens could be obtained, and a substitution reaction with sodium benzenethionosulfonate was performed to obtain the Boc-protected precursor in excellent yield on the gram scale (87% quantitative, see Materials and Methods section for synthesis). Despite the limited commercial availability of amino halogens, six new diversified building blocks were synthesized without the need for silica column purification. These could be taken further and directly loaded onto highly loaded SH PS resin to give 2-7 immobilized on the resin by dithiol crosslinking (Figure 15a).

[0193] To test whether the building blocks could be adapted for 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). After synthesis and removal of protecting groups by TFA treatment, the resin-bound peptide was incubated with 2x200 μl of reductive release cleavage cocktail (1,4-butanedithiol (BDT) and NEt3, both at 100 mM in DMF). The peptide was released from the resin and analyzed by HPLC-MS after removal of DMF and volatile BDT by rotary vacuum concentration (RVC). The peptides were efficiently released and analyzed by LC-MS. For all peptides, the desired products were confirmed in excellent quality (Figure 15c).

[0194] These fragments were utilized in the synthesis of a macrocyclic library, with resin-bound diversification elements under investigation, in pursuit of binders against trypsin-like serine proteases involved in the blood coagulation pathway. Due to the shear load of trypsin-like serine proteases and their role in various diseases, these proteases serve as important targets for the development of potent and specific inhibitors against this class of proteases. The complexity of this task arises from the structural identity shared by many members of this group, due to their ability to recognize and cleave positively charged residues, and a conserved aspartate buried deep in the S1 pocket. It was therefore anticipated that this model system could be used to develop potent and selective macrocyclic inhibitors, as the results could be applied in future to other parts of cyclic peptide research.

[0195] Thus, 384 dithiol peptides were prepared at a 5 μmol scale by automated SPPS in a 96-well filter plate. The peptides were synthesized on three different scaffolds (1a-c, Figure S16a) containing one of seven different diversification elements (1–7), random amino acids (from 27 different α, β, γ, and N-methylated amino acids), and randomly selected sequences of S1 pocket binding motifs known from the literature.

[0196] Peptides were synthesized on solid support and the associated protecting groups were removed by acidic treatment with TFA. The peptides were then released from the resin by reductive release to give the linear dithiol peptides. After acidification and removal of the volatile reducing agent by RVC, the concentration of the peptides was measured using Ellman's reagent (average concentration = 24.9 mM, 20% average from the resin loading to evenly distribute the peptides by all 7 different derivatives).

[0197] Our latest approach in the lab focused on preparing libraries at the picomole scale for direct use in assay-ready microtiter plates. Since compound library preparation was envisioned to be used to screen multiple targets at once, an approach was needed that would allow for large-scale compound preparation without the need for cumbersome and laborious pipetting, while still allowing for large-scale compound diversification with multiple linkers. Therefore, we focused on the use of Acoustic Droplet Ejection (ADE) technology for dispensing. Although this technology is primarily utilized for the transfer of nl volumes, it has already been shown that even larger μl volumes can be transferred using this system, thus eliminating the need for pipetting. Thus, 40 nmol of linear peptide was dispensed into multiple Echo® ADE-enabled 384 polypropylene (PP) plates (Figure 17). Since reduced dithiol peptides slowly undergo oxidation when dissolved in DMSO, the peptides were quickly re-reduced by adding a DMF:BDT solution to the wells, and after acidification and RVC, they were immediately cyclized in MeCN / NH4HCO3 buffer with seven diverse linkers (see Figure 16B for a selection). This is in contrast to the traditionally used method of first solubilizing the linear species, followed by the addition of the linker solution. The new methodology minimizes the amount of reoxidation that occurs, resulting in a purer reaction mixture. Finally, excess linker was quenched with β-mercaptoethanol (β-ME).

[0198] Materials and Methods All reagents and solvents were of analytical grade and were used without further purification as obtained from commercial suppliers. Reactions were performed on silica gel-coated plates (analytical grade SiO 2The elution was monitored by thin layer chromatography (TLC) using a 100-mL HPLC column (F-60, F-254) and / or by HPLC-MS analysis. The TLC plates were visualized under UV light or immersed in potassium permanganate solution (10 g / L) followed by visualization with a heat gun. Rotary evaporation of the solvent was performed under reduced pressure at temperatures below 40 °C. HPLC-MS analysis was performed on a UHPLC using a C18 reversed-phase column (Phenomenex Kinetex 2.1 × 50 mm C18 column, 100 Å pore, 2.6 μm particles) and a single quadrupole MS system (Shimadzu LCMS-2020). A linear gradient of solvent B (0.05% HCOOH in MeCN) against solvent A (0.05% HCOOH in water) increasing linearly from 0% to 60% between t = 1.00 and 6.00 min was applied at a flow rate of 1.00 ml / min. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III equipped with a cryogenically cooled probe ( 1 H NMR and 13 C NMR was recorded at 400 MHz and 101 MHz, respectively. All spectra were recorded at 298 K. Chemical shifts are reported in ppm relative to the deuterated solvent as internal standard (δ H DMSO-d 6 2.50 ppm; δ C DMSO 39.52 ppm; δ H CDCl 3 7.26 ppm; δ C CDCl 3 77.16 ppm).

[0199] High-resolution mass spectrometry (HRMS) measurements were recorded on a maXis G3 quadrupole time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) source.

[0200] Chemical synthesis of building blocks and amino acids S-(3-((tert-butoxycarbonyl)amino)propyl)benzenesulfonothioate (S1, ALN-1-31)

[0201] [ka]

[0202] 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 benzylthiosulfonate (7.48 g, 38.0 mmol, 1.5 equiv; tech. 85%) and the solution was stirred at 80° C. overnight. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 ml) and extracted with EtOAc:hexane (2×150 ml, 10:1, v / v). The combined organic layers were washed with water (3×150 ml) and brine (150 ml) and diluted with anhydrous Na 2 SO 4 Drying at 40° C. and concentration under reduced pressure gave crude S1 (8.21 g, 24.8 mmol, 98%) as a yellowish oil. TLC (25% EtOAc in hexanes): R f =0.25(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3 ) 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 = 7.2 Hz, 2H), 1.83 (p, J = 6.8 Hz, 2H), 1.43 (s, 9H). tert-Butyl (3-chloropropyl) (methyl)carbamate (S2, VC-1-1)

[0203] [ka]

[0204] CH 2 Cl 2A 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 NEt (150 ml) was cooled to 0° C. under an argon atmosphere. 3 (4.16 ml, 30.0 mmol, 1.0 equiv) was added dropwise over 5 min and the solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure and resuspended in EtOAc (120 ml). The solution was diluted with aqueous HCl (1M, 2×120 ml), saturated NaHCO 3 (120 ml) and brine (120 ml), and then washed with anhydrous Na 2 SO 4 After drying at 40° C. and concentrating under reduced pressure, crude S2 (6.22 g, 30.0 mmol, quant.) was obtained as a colorless crystalline solid. TLC (25% EtOAc in hexanes): R f =0.45(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3 ) 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.

[0205] S-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)benzenesulfonothioate (S3, VC-1-3)

[0206] [ka]

[0207] To a stirred solution of VC-1-1 (3.51 g, 16.9 mmol) in DMF (25 ml) was added sodium benzylthiosulfonate (5.50 g, 27.9 mmol, 1.65 equiv; tech. 85%) and the solution was stirred at 80° C. overnight. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 ml) and extracted with EtOAc:hexane (2×75 ml, 10:1, v / v). The combined organic layers were washed with water (3×75 ml) and brine (75 ml) and diluted with anhydrous Na 2 SO 4 After drying at 40° C. and concentrating under reduced pressure, crude S3 (5.07 g, 16.2 mmol, 95%) was obtained as a yellowish oil. TLC (12.5% ​​EtOAc in hexanes): R f =0.30(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3 ) 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, 3H), 1.85 (p, J = 6.8 Hz, 2H), 1.42 (s, 9H). tert-Butyl (Z)-(4-chlorobut-2-en-1-yl)carbamate (S4, P1_N2_E29 / E46)

[0208] [ka]

[0209] CH 2 Cl 2 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 NEt (75 mL) was cooled to 0° C. under an argon atmosphere. 3 (2.45 ml, 17.6 mmol, 1.0 equiv) was added dropwise over 5 min and the solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure and CH 2 Cl2 (100 ml). The solution was diluted with aqueous HCl (1M, 2×100 ml), saturated NaHCO 3 (100 ml) and brine (100 ml), and then washed with anhydrous Na 2 SO 4 After drying at 40° C. and concentration under reduced pressure, crude S4 (3.55 g, 17.3 mmol, 98%) was obtained as a light brown solid. TLC (25% EtOAc in hexanes): R f =0.30(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3 ) 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, 2H), 1.44 (s, 9H).CAS RN:123642-28-4. NMR spectrum consistent with literature.

[0210] (Z)-S-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)benzenesulfonothioate (S5, P1_N2_E30)

[0211] [ka]

[0212] To a stirred solution of S4 (3.55 g, 17.3 mmol, 1.0 equiv) in DMF (70 ml) was added sodium benzylthiosulfonate (6.81 g, 34.5 mmol, 2.0 equiv; tech. 85%) and the solution was stirred at 80° C. overnight. 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) and diluted with anhydrous Na 2 SO 4 and concentrated under reduced pressure to give crude S5 (5.03 g, 14.6 mmol, 85% * ) was obtained as a brown oil. TLC (25% EtOAc in hexanes): Rf =0.20(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3 ) 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). * Although the purity of the crude compound is lower than other thiosulfonate building blocks, it provides superior resin quality for the subsequent resin loading step.

[0213] tert-Butyl 3-(((phenylsulfonyl)thio)methyl)azetidine-1-carboxylate (S6, ALN-1-57)

[0214] [ka]

[0215] 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 benzylthiosulfonate (2.43 g, 12.3 mmol, 1.6 equiv; tech. 85%) and the solution was stirred at 80° C. overnight. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 ml) and extracted with EtOAc:hexane (2×75 ml; 10:1, v / v). The combined organic layers were washed with water (2×75 ml), saturated NaHCO 3 (75 ml) and brine (75 ml), and then washed with anhydrous Na 2 SO 4 After drying at 40° C. and concentrating under reduced pressure, crude S6 (2.65 g, 7.71 mmol, quant.) was obtained as a yellowish oil. TLC (33% EtOAc in hexanes): R f =0.30(KMnO 4 staining). 1H NMR (400 MHz, CDCl 3 ) 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). tert-Butyl 4-((phenylsulfonyl)thio)piperidine-1-carboxylate (S7, ALN-1-41)

[0216] [ka]

[0217] 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 benzylthiosulfonate (2.77 g, 14.0 mmol, 1.65 equiv; tech. 85%) and the solution was stirred at 80° C. overnight. As the reaction was incomplete overnight (monitored by TLC), additional sodium benzylthiosulfonate (1.38 g, 7.00 mmol; tech. 85%) was added and the reaction was stirred at 80° C. for another 24 h. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 mL) and extracted with EtOAc:hexane (2×75 mL; 10:1, v / v). The combined organic layers were washed with water (2×75 ml), saturated NaHCO 3 (75 ml) and brine (75 ml), and then washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C. and concentrated under reduced pressure to give crude S7 (2.65 g, 7.43 mmol, 87%). * ) was obtained as a yellowish oil. TLC (33% EtOAc in hexanes): R f =0.44(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3) 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 proceeds significantly slower than in the preparation of other thiosulfonate building blocks. Furthermore, the crude purity is low, but excellent resin quality is obtained in the subsequent resin loading step.

[0218] tert-Butyl 4-(((phenylsulfonyl)thio)methyl)piperidine-1-carboxylate (S8, ALN-1-38)

[0219] [ka]

[0220] 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 benzylthiosulfonate (2.52 g, 12.8 mmol, 1.65 equiv; tech. 85%) and the solution was stirred at 80° C. overnight. After cooling, the reaction mixture was concentrated under reduced pressure, resuspended in water (100 ml) and extracted with EtOAc:hexane (2×75 ml; 10:1, v / v). The combined organic layers were washed with water (2×75 ml), saturated NaHCO 3 (75 ml) and brine (75 ml), and then washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C. 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): f =0.30(KMnO 4 staining). 1 H NMR (400 MHz, CDCl 3) 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). (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-chlorofuran-2-carboxamido)propanoic acid (Thio, ALN-1-77).

[0221] [ka]

[0222] This synthesis was adapted from the previous procedure of the analogous amino acid: 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 atmosphere. The solution was cooled to 0° C., after which a solution of DCC (2.89 g, 14.0 mmol, 1.4 equiv.) dissolved in THF (30 ml) was slowly added to the reaction mixture. The solution gradually became turbid and was stirred overnight towards room temperature, after which the solution was filtered. Meanwhile, Fmoc-Dap(Boc)-OH (4.26 g, 10.0 mmol, 1.0 equiv.) was dissolved in CH 2 Cl 2 (20 ml; cloudy solution) and TFA (20 ml) was added slowly to the solution. The solution immediately turned yellowish clear and foaming occurred. After the foaming ceased, the solution was stirred at room temperature for a further 30 min, after which the solvent was removed under a stream of nitrogen. Excess TFA was removed with CH 2 Cl 2 The residue was resuspended in THF (40 ml) and then i-Pr 2NEt (5.75 ml, 60.0 mmol, 6.0 equiv) was added. The solution was then poured into the mother liquor containing the NHS-activated thiophene and stirred at room temperature overnight. Upon completion, the solution was concentrated under reduced pressure, redissolved in EtOAc:Hexanes (300 ml, 10:1, v / v), and washed twice with water (100 ml) and brine (100 ml). The organic layer was washed with anhydrous Na 2 SO 4 The crude product was purified by silica column chromatography to give the Fmoc-protected amino acid building block (4.06 g, 8.62 mmol, 86%) as an off-white solid. * TLC(CH 2 Cl 2 in 5% MeOH and 0.5% AcOH): R f =0.2(UV). 1 H NMR (400 MHz, DMSO-d 6 ) 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). 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 (overlapped with solvent peak).C 23 H 2 OClN 2 0 5 S + [M+H] +HRMS m / z calculated, 471.0776; found, 471.0786.

[0223] Synthesis of dithiol resin Preparation of highly loaded thiol resin

[0224] [ka]

[0225] Pre-cleaning: Fill each 25 ml fritted syringe with approximately 0.8 g (1.11 mmol) of aminomethyl polystyrene resin (AM PS resin; 1.39 mmol / g, 100-200 mesh; Aapptec, cat.#RAZ001) and wash with MeOH (2 × 10 ml), CH 2 Cl 2 (3 x 10 ml), CH 2 Cl 2 1% (v / v) TFA (2 × 10 ml), CH 2 Cl 2 i-Pr 2 NEt (1.2 M; 2 × 10 ml, 5 min), CH 2 Cl 2 (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 reacted with i-Pr 2 The resin was activated with NEt (1.10 ml, 6.66 mmol, 6.0 equiv.), added to a fritted syringe, and stirred at room temperature for 3 h. The resin was filtered and washed with DMF (3×10 ml) and CH 2 Cl 2 (3×10 ml) and the beads were subsequently dried, first under suction and then under reduced pressure (<0.5 mbar) overnight. The loading of the MPA(Trt) resin was approximately 1.20 mmol / g * (weight basis) was determined. Cap addition: 5% Ac 2A solution of 20 and 6% lutidine in DMF (12 ml; v / v / v) was added to the resin and incubated at room temperature for 5 min. The resin was drained and washed with DMF (3 × 10 ml) and CH 2 Cl 2 (3×10 ml). Deprotection: CH with 10% TFA and 1% TIPS. 2 Cl 2 (15 ml, v / v / v) solution was added to the resin and stirred at room temperature for 1 h. The resin was then washed with CH 2 Cl 2 (3×10 ml) and repeating this procedure once gave a highly loaded thiol resin of polystyrene (SH PS resin) which was available for subsequent disulfide exchange and loading of cysteamine derivatives.

[0226] Preparation of resin 1 (res1)

[0227] [ka]

[0228] Dithiol exchange: Fill each 25 mm frit syringe with approximately 0.4 g (0.48 mmol) of SH PS resin and centrifuge in CH 2 Cl 2 (10 ml) and then drained. 2-Pyridylthiocysteamine hydrochloride (0.48 g, 0.96 mmol, 2.0 equiv.) was dissolved in MeOH:CH 2 Cl 2 (19 ml, 3:7, v / v), followed by i-Pr 2 NEt (167 μl, 0.96 mmol, 2.0 equiv) was added. The solution was added to the resin and stirred at room temperature for 3 h. The resin was drained and diluted with MeOH:CH 2 Cl 2 (2 × 10 ml, 3:7, v / v), DMF (2 × 10 ml), i-Pr in DMF 2 NEt (1.2 M; 10 ml for 5 min), DMF (3 × 10 ml) and CH 2 Cl 2(2 x 10 ml) and subsequently drying the beads (first under suction, then under reduced pressure (<0.5 mbar) overnight). Qualitative controls: (1) Kaiser test; complete purple / blue coloration of the beads. (2) Ellman's reagent on the beads; no coloration.

[0229] Preparation of resins 2–7 (res2–7)

[0230] [ka]

[0231] Deprotection: The N-Boc protected thiosulfonate intermediate (S2, S3, S5, S6, S7 or S8; ca. 7.0 mmol) was dissolved in CH 2 Cl 2 (10 ml), followed by vigorous CO 2 TFA was added dropwise until the formation of bubbles was observed (approximately 10 ml). The solution was stirred at room temperature for a further hour, after which the solvent was removed under a stream of nitrogen. Excess TFA was removed under reduced pressure with CH 2 Cl 2 The TFA salts were removed by coevaporation with NEt:MeOH solution (1:1, v / v) to give the thiosulfonate salts for attachment to the resin. Dithiol exchange: Approximately 0.8 g (0.96 mmol) of SH PS resin was loaded into each of 25 ml fritted syringes and swollen with THF (15 ml) and then drained. The desired thiosulfonate TFA salts (2.40-2.88 mmol, 2.5-3.0 equiv.) were dissolved in THF (15 ml) and NEt 3 (803 μl, 5.76 mmol, 6.0 equiv.) was added. This solution was added to the resin and stirred at room temperature overnight. The resin was drained and washed with THF (3×15 ml) and CH 2 Cl 2 (2 x 15 ml) and subsequently drying of the beads (first under suction, then under reduced pressure (<0.5 mbar) overnight). Qualitative controls: (1) Kaiser test; complete purple / blue coloration of the beads. (2) Ellman's reagent on the beads; no coloration.

[0232] How to Use the Plate Polypropylene (PP) 96-well filter plates were equipped with 5 μmol / well of polystyrene dithiol resin (res1–res7; estimated loading ~1.20 mmol / g) and washed with DMF (6 × 225 μl). Couplings were performed with 53 μl of amino acid (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 couplings of Thio, t-acha, 4amPip, and 2am, 75 μl of amino acid (170 mM, 2.55 equiv.), 25 μl of HATU (500 mM, 2.5 equiv.), 7 μl of N-methylmorpholine (4 M, 5.6 equiv.), and 5 μl of N-methylpyrrolidone were used. All components were premixed for 1 min and then added to the resin (2 h reaction, no shaking). Coupling was performed twice and the resin was washed with DMF (6 x 225 μl). Fmoc deprotection was performed using 20% ​​(v / v) piperidine in DMF (120 μl, 2 x 2 min) and the resin was washed with DMF (6 x 225 μl). At the end of peptide synthesis, the resin was washed with CH 2 Cl 2 (2×200 μl) and the resin beads were dried under vacuum.

[0233] How to Use a Syringe Polystyrene dithiol resin (res1-res7; estimated loading approx. 1.20 mmol / g) was added to a 5 ml syringe reactor and the resin was washed with DMF (6 × 150 μl). Couplings were performed with 210 μl of amino acid (500 mM, 4.2 equiv.), 200 μl of HATU (500 mM, 4.0 equiv.), 50 μl of N-methylmorpholine (4.0 M, 8.0 equiv.) and 5 μl of N-methylpyrrolidone. All components were premixed for 1 min and then added to the resin (2 h reaction, with shaking). Couplings were performed twice and then the resin was washed with DMF (2 × 600 μl). Fmoc deprotection was performed using 20% ​​piperidine in DMF (450 μl, 2 × 2 min) and the resin was washed with DMF (7 × 600 μl). At the end of peptide synthesis, the resin was washed with CH 2 Cl 2(2×600 μl) and the resin beads were dried under vacuum.

[0234] Reductive Release Procedure Removal of side-chain protecting groups: After automated SPPS (5 μmol / well scale), the bottom of the 96-well synthesis plate was sealed by pressing it against a 6 mm thick soft ethylene vinyl acetate pad. The resin was then purified by TFA / TIPS / H 2 The resin was incubated with 100 μl of HO (300 μl, 95:2.5:2.5, v / v / v) for 1 h and covered with an adhesive PP plate lid. The TFA solution was discarded and the resin was washed with CH 2 Cl 2 (3 x 300 μl) and this procedure was repeated once. Reductive release: After air drying for at least 1 h, 1,4-butanedithiol (BDT) and NEt 3 in DMF (both 100 mM, 200 μl, 4 equivalents relative to resin loading) were added to the resin and the plate was agitated overnight at room temperature. The next day, the DMF solutions were forced into a 96-well deep-well plate by centrifugation (1000 rpm) and the reductive release procedure was repeated once for 5 h and pooled in the same 96-well deep-well plate. Concentration: TFA in milliQ water (10% (v / v), 62 μl, NEt 3 2 equivalents per unit volume) was added to the wells and the peptides were dried using a Speedvac concentrator (30°C, 1750 rpm, 0.1 mbar). Resolubilization and transfer: The dried peptide pellet was dissolved in DMSO (40 μl) and transferred to an Echo certified 384-well PP source plate. Concentration measurement: An Ellman's assay was performed to determine the concentration of the dithiol peptide stocks. Ellman's reagent 5 (DTNB) in assay buffer (150 mM NHCl in water) 4 HCO 3The dithiol peptides were dissolved in DMSO (90:10, v / v), pH 8) to a concentration of 10 mM. 135 nl of dithiol peptides in DMSO were transferred to a 384-well black microplate with clear bottom by Echo using acoustic droplet ejection (ADE). Assay buffer (24 μl) was dispensed using CERTUS, followed by addition of DTNB solution (6 μl). The plates were centrifuged (400 g, 2 min) and absorbance (412 nm) was measured on a TECAN M200 plate reader. The concentration of dithiol peptides was calculated using the previously recorded calibration curve:

[0235]

number

[0236] How to Use a Syringe Removal of side-chain protecting groups: After automated SPPS (25 μmol scale), the fritted syringe containing the resin was immersed in TFA / TIPS / H 2 The resin was incubated with 100 mL of TFA in 100 mL of CH4OH (4 ml, 95:2.5:2.5, v / v / v) for 2 h at room temperature. The TFA solution was discarded and the resin was washed with CH4OH. 2 Cl 2 (5×4 ml) and DMF (4 ml). Reductive release: After air drying for at least 1 h, the reaction mixture was washed with BDT and NEt 3 in DMF (both 100 mM, 2.0 ml, 4 equivalents relative to resin loading) were added to the syringe and stirred overnight at room temperature. The next day, the DMF solution was pushed into a 50 ml conical Falcon tube. Concentration: TFA in milliQ water (10% (v / v), 312 μl, NEt 3 (2 equivalents relative to the peptide) was added to the peptide solution, which was dried using a Speedvac concentrator (30° C., 1750 rpm, 0.1 mbar) to give the crude linear dithiol peptide, ready for cyclization in the next step.

[0237] Macrocyclization Procedure How to Use the Plate Transfer to microtiter plates: Based on the determined concentration of each dithiol peptide in DMSO, 40 nmol of dithiol peptide in DMSO was transferred to a 384PP plate (one plate per linker) using ADE. Peptide reduction: As dithiol peptides oxidize over time in DMSO, they were reduced with BDT and NEt. 3 Complete reduction of the peptide was confirmed by adding a solution of TFA in milliQ water (10% (v / v), 6 μl, NEt 3 (2 equivalents per unit area) was added to each well and the peptide was dried using a Speedvac concentrator (30 °C, 1750 rpm, 0.1 mbar) to give a fully reduced dithiol peptide pellet. Cyclization: Bis-electrophilic linkers (L1–L7) were catalyzed by the NH 4 HCO 3 MeCN:H 2 The prepared linker solution (40 μL, 4 equivalents relative to dithiol peptide) was added to a 384PP plate using a liquid dispenser, which was sealed with an adhesive PP lid and stirred at room temperature for 2 h. Quenching of the linker: β-mercaptoethanol (β-ME) was dissolved in the prepared cyclization buffer to a final concentration of 32 mM. The prepared solution (20 μL, 4 equivalents relative to linker) was added to each well and incubated at room temperature for 1 h without covering the plate. Concentration and resolubilization: The solvent was removed using a Speedvac concentrator (40° C., 1750 rpm, 0.1 mbar) to give the peptide macrocycles as a pellet, which was dissolved in DMSO (10 μl) and transferred to a 384LDV plate to give a 4 mM macrocyclic peptide library ready for subsequent application in protease screening assays.

[0238] Conical tube method: Cyclization: The dielectrophilic linker is substituted with an NH 4 HCO 3 MeCN:H 2The linker was dissolved in a degassed 60 mM solution in 20H2O (1:1 (v / v), pH 8) to a final concentration of 4 mM. The prepared linker solution (12.5 ml, 2 equivalents relative to dithiol peptide) was added to the conical tube containing the desired dithiol peptide pellet and the solution was stirred at room temperature for 2 h. Quenching of the linker: After reaction completion (determined by LCMS), excess linker was quenched by adding β-ME (14 μl, 200 μmol, 4 equivalents relative to linker), added to the conical flask, stirred for at least 1 h, and subsequently purified. Purification of the macrocycle: Samples were purified on a preparative HPLC equipped with a C18 RP Waters OBD column. A linear gradient of solvent B (0.1% TFA in MeCN) against solvent A (0.1% TFA in water) was applied at a flow rate of 14.0 ml / min, increasing linearly from 15% to 60% from t = 2.00 to 32.00 min. Pure fractions containing the desired product were combined and lyophilized to obtain the product as a colorless fluffy material. DMSO stocks: The purified macrocycles were transferred to Eppendorf tubes and DMSO was added to obtain 5 mM or 20 mM compound stocks.

[0239] Biochemical assays Protease screening of macrocyclic libraries Enzyme inhibition of the compound library was assessed by measuring the residual enzyme activity in the presence of cyclic peptides (average concentration 10 μM for thrombin, 20 μM for FXI, FXII, KLK5 and PKal) at 1% final DMSO concentration. The crude cyclic library (4 mM DMSO stock in 384-well LDV plates) was transferred to 1536-well microtiter OptiPlates via ADE. The applied buffer solutions were prepared by filtering through PTFE syringe filters (0.22 μm) and the assay was initiated by adding the protease (4.41 μm / well) in the appropriate buffer (see list below) supplemented with bovine serum albumin (BSA; 0.1% w / v) and dispensed using a CERTUS automated liquid handler. The plate was incubated at room temperature for 10 min before the addition of fluorogenic substrate (4.5 μl) in the appropriate buffer using a CERTUS automated liquid handler. Plates were centrifuged (800 g, 2 min) and fluorescence intensity was measured over 15 min at 150 s time increments using a PHERAstar plate reader (excitation 384 nm, emission 440 nm). The slope (m) of the fluorescence increase was calculated in Microsoft Excel (vers. 16.56). A negative control was prepared without macrocycle. The mean value of the 12 negative controls was used to calculate the residual activity using the following formula I:

[0240]

number

[0241] [Table 7]

[0242] Identification of active species of crude cyclic products from hits Selected hits from the library screening (compounds 195_L6, 237_L6 and 293L6) were resynthesized and cyclized (40 nmol scale). The dried macrocyclic products were purified by MeCN:H 2The eluates were dissolved in HO (1.5 ml, 1:1, v / v) and fractionated on a Thermo Fisher Dionex UltiMate 3000 system using a C18 NovaPak reversed-phase column (10 × 150 mm, 125 Å pore, 5 μm particles). A linear gradient of solvent B (0.1% TFA in MeCN) against solvent A (0.1% TFA in water) increasing linearly from 0% to 80% (for thrombin hit) or 0% to 95% (for PKal hit) between t = 2.00 and 22.0 min was applied at a flow rate of 4.00 mL / min. Fractions (1 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 source 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 the assay was subsequently carried out in a black 384-well clear-bottom polystyrene plate. The DMSO fraction solution (0.5 μl) was pipetted into the microtiter plate and the appropriate enzyme buffer (49.5 μl; composition similar to that described on the previous page, exactly 2 nM thrombin was used) was added and incubated for 10 min at room temperature. Substrate in buffer (25 μl, composition similar to that described on the previous page) was added, the plate was centrifuged (800 g, 2 min) and the fluorescence intensity was measured using a PHERAstar plate reader (excitation 384 nm, emission 440 nm) at 150 s time increments over 15 min. The slope (m) of the fluorescence increase was calculated in Microsoft Excel (vers. 16.56). Negative controls were prepared without DMSO (0.5 μl) instead of fractionated samples. The average of the six negative controls was used to calculate the residual activity using formula I.

[0243] I C 50 Decision Half-maximal inhibitory concentration (IC 50) values ​​were determined from protease inhibition in an assay similar to that in which library screening was performed. A two-fold dilution series of purified macrocycles was prepared in a 384-well LDV source plate and transferred to a 1536-well OptiPlate using ADE (final volume: 45 nl macrocycle / DMSO). Enzyme solution (4.5 μl) in buffer was added using a Certus and incubated for 10 min. Substrate (4.5 μl) in buffer was then added, the plate was centrifuged (700 g, 2 min) and fluorescence intensity was measured using a PHERAstar plate reader (excitation 384 nm, emission 440 nm) at 150 s time increments over 15 min. The slope (m) of the fluorescence increase was calculated in Microsoft Excel (vers. 16.56). Negative controls were prepared without macrocycles. The mean of the 12 negative controls was used to calculate residual activity using formula I. IC 50 The values ​​were obtained by fitting the data to the concentration-response equation [number of constraints?] using GraphPad Prism (version 6.0.1), k i The value is calculated using the Cheng-Prusoff formula 11 Using IC 50 Calculated based on:

[0244]

number

[0245] where [S] is the initial substrate concentration and K M is the Michaelis-Menten constant for the enzyme and substrate 12 It is.

Claims

1. 1. A method for preparing a library of peptides or isolated peptides, comprising: (a) one or more linear dithiol peptides having a sulfhydryl group at the N-terminal region of the one or more peptides and immobilized on a solid phase via a disulfide bridge at the C-terminal region of the one or more peptides; (i) an agent that reduces disulfide bridges, thereby liberating one or more linear dithiol peptides from the solid phase, which agent is volatile and removable by evaporation; or (ii) a base that deprotonates sulfhydryl groups at the N-terminal region of the linear dithiol peptide(s), thereby inducing intramolecular disulfide exchange, thereby liberating the linear dithiol peptide(s) from the solid phase in the form of one or more cyclic peptides. The method of claim 1, further comprising the step of releasing the solid phase by

2. 10. The method of claim 1, further comprising, after step (a), removing the agent by evaporation.

3. 10. The method of claim 1, further comprising the step (b) of cyclizing one or more linear dithiol peptides liberated by the agent.

4. 4. The method of claim 3, wherein the one or more dithiol peptides are cyclized by at least one bis-electrophile or by disulfide oxidation.

5. 2. The method of claim 1, wherein the disulfide bonds of the one or more cyclic peptides of item (ii) are reduced and the peptides are recyclized with a bis-electrophile.

6. The drug is 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-ethanedithiol, 4-methyl-1-ethanedithiol, 5-methyl-1-ethanedithiol, 6-methyl-1-ethanedithiol, 7-methyl-1-ethanedithiol, 8-methyl-1-ethanedithiol, 9-methyl-1-ethanedithiol, 10-methyl-1-ethanedithiol, 11-methyl-1-ethanedithiol, 12-methyl-1-ethanedithiol, 13-methyl-1-ethanedithiol, 14-methyl-1-ethanedithiol, 15-methyl-1-ethanedithiol, 16-methyl-1-ethanedithiol, 17-methyl-1-ethanedithiol, 18-methyl-1-ethanedithiol, 19-methyl-1-ethanedithiol, 20-methyl-1-ethanedithiol, 21-methyl-1-ethanedithiol, 22-methyl-1-ethanedithiol, 23-methyl-1-ethanedithiol, 24-methyl-1-ethanedithiol, 25-methyl-1-ethanedithiol, 26-methyl-1-ethanedithiol, 27-methyl-1-ethanedithiol, 28-methyl-1-ethanedithiol, 29-methyl-1-ethanedithiol, 30-methyl-1-ethanedithiol, 31-methyl-1-ethanedithiol, 32-methyl-1-ethane thiol, thiophenol, benzylthiol, 2-butene-1-thiol, 3-butene-1-thiol, 2-methyl-2-propene-1-thiol and 3-methyl-2-butene-1-thiol, preferably 1,3-propanedithiol, 1,4-butanedithiol or 2,4-pentanedithiol, most preferably 1,4-butanedithiol (BDT), and / or 6. The process according to any one of claims 1 to 5, wherein the base is selected from tertiary, secondary or primary amines, boron, aluminium or silicon hybrids, and bases with oxygen, nitrogen or carbon anions, preferably tertiary, secondary or primary amines, more preferably tertiary amines, even more preferably tri-alkylamines, and most preferably N,N-diisopropylethylamine (DIPEA).

7. 2. The method of claim 1, comprising, prior to step (a), a step (a') of synthesizing a linear dithiol peptide on a solid phase.

8. 2. The method of claim 1, wherein the side chains of the amino acids of the linear dithiol peptide are protected by protecting groups, the method further comprising removing the protecting groups before step (a) and, if present, after step (a') while the linear dithiol peptide is immobilized on the solid phase.

9. 2. The method of claim 1, wherein at least a portion of the linear dithiol peptides contain primary or secondary amines, the method further comprising the step of modifying the primary or secondary amines with a carboxylic acid, and wherein the cyclic peptides containing primary or secondary amines and carboxylic acids are transferred, preferably by acoustic dispensing.

10. (c) contacting the peptide library with the target molecule, preferably without prior purification of the peptide library; (d) screening the peptide library for peptides that bind to and preferably inhibit the target molecule; The method of claim 1 further comprising:

11. 11. The method of claim 10, wherein steps (c) and (d) are performed in the same well in which the primary or secondary amines are modified with a carboxylic acid.

12. The method of claim 10, wherein the target molecule is a protein, a peptide, a nucleic acid molecule, a carbohydrate, or a fatty acid, preferably a protein or a peptide.

13. The method of claim 1 , wherein the solid phase comprises a resin, preferably a non-polar resin, more preferably a polystyrene resin.

14. A linear dithiol peptide, Linear dithiol peptides with a molecular weight of less than 1000 Da, preferably less than 600 Da, and / or Linear dithiol peptides containing 3 or 4 amino acids, preferably 3 amino acids. The method of claim 1 , comprising:

15. A method for diversifying a macrocycle library, preferably a cyclic peptide library, wherein at least some of the macrocycles, preferably cyclic peptides, contain a primary or secondary amine, and the method comprises the step of modifying the primary or secondary amine with a carboxylic acid.

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