Novel flow synthesis method using native chemical ligation

Abstract

To provide methods that allow rapid and facile preparation of native peptides and proteins and furthermore are clean, robust and scalable.SOLUTION: The present invention provides a method relating to the flow synthesis of an amide-containing compound, in particular, the flow synthesis of polypeptides via native chemical ligation, which is a method of preparing an amide-containing compound in flow and comprises the step of reacting (i) an ester with (ii) molecules comprising a terminal amino acid selected from the group consisting of cysteine, cystine, a thiol-derivatized amino acid, a disulfide-derivatized amino acid, selenocysteine, selenocystine, a selenol-derivatized amino acid, and a diselenide-derivatized amino acid; where the ester is a thioester or selenoester. The present invention also provides a method for selective desulfurization or deselenization of amide-containing compounds each comprising a thiol, disulfide, selenol or diselenide functional group, particularly polypeptides.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to the flow synthesis of amide-containing compounds. In particular, the present disclosure relates to the flow synthesis of polypeptides by native chemical ligation. The present disclosure also relates to the selective desulfurization or deselenization of amide-containing compounds, particularly polypeptides, each containing a thiol, disulfide, selenol or diselenide functional group.

Background Art

[0002] Peptides and proteins are ubiquitous molecules in biological systems and generally exhibit exquisite selectivity for their targets, a property that has led to renewed interest in polypeptides as therapeutic agents. These so-called "biologics" have been reported to have an approval rate twice that of small molecule therapeutics (Hay 2014) and currently account for 10% of approved drugs (Fosgerau 2015; Usmani 2017). As a result of this resurgence in polypeptide-based therapies, the development of methods for efficient access to these biomolecules has received attention.

[0003] Solid-phase peptide synthesis (SPPS) represents the most efficient means for generating peptides via chemical synthesis (Merrifield 1963; Kent 1988). However, there are significant limitations to the size of the target that can be produced in one batch (typically 40-50 residues). Most of these limitations of SPPS have been addressed by the development of native chemical ligation (NCL), a transformation technique that enables the convergent and chemoselective ligation of unprotected peptide fragments (Dawson 1994; Kent 2009). NCL is carried out between a peptide containing an N-terminal cysteine (Cys) residue and a peptide functionalized as a C-terminal thioester, and typically involves a rate-limiting trans-thioesterification step with a reactive thioester (SR 2requires the use of appropriate thiol additives for its generation (Johnson 2006; Thompson 2014). Mechanistically, the reaction proceeds through an initial trans-thioesterification step, followed by a rapid S→N acyl shift to give the native peptide bond. NCL has dramatically increased the size of target polypeptides that can be assembled by total chemical synthesis.

[0004] Important advances with this powerful methodology have been the development of metal-based desulfurization (Yan 2001), and subsequently, milder radical-based protocols (Wan 2007; Jin 2017) that facilitate the conversion of the minimal amount of proteinogenic amino acid Cys to alanine (Ala) residues at the ligation junction. The subsequent development of thiolated amino acids (as Cys surrogates) has helped to expand the number of targets accessible via the NCL technique (Dawson 2011; Kulkarni 2018; Malins 2015; Malins 2014; Malins 2015; Bondalapati 2016). A further innovation has been the development of one-pot ligation-desulfurization chemistries using thiol additives (e.g., trifluoroethanethiol (TFET)) (Thompson 2014; Huang 2016). These additives help to enhance the rate of the NCL reaction, but unlike traditionally used aryl thiols, do not interfere with subsequent radical desulfurization chemistries and can thus be carried out without intermediate purification.

[0005] Despite the impact that NCL and related methodological advances have had on the field of peptide and protein science, this technology has not yet been used in the manufacture of any approved peptide or protein pharmaceuticals. There is still a need to develop methods that allow for the rapid and facile preparation of native peptides and proteins, and that are clean, robust, and scalable.

[0006] References to prior art in this specification are not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would reasonably be expected to be understood, regarded as relevant, and / or combined by a person skilled in the art with other prior art.

Summary of the Invention

[0007] The present disclosure relates to (i) reacting an ester (ii) a molecule comprising a terminal amino acid selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids; The method comprising reacting, wherein the ester is a thioester or a selenoester, and relates to a method for producing an amide-containing compound in a flow.

[0008] The amide-containing compound is preferably a polypeptide. Preferably, the polypeptide has the formula (I):

[0009]

Chemical formula

[0010]

Chemical formula

[0011]

Chemical formula

[0012]

Chemical formula

[0013]

Chemical formula

[0014] The ester is preferably a "reactive ester". A reactive ester can react with the terminal amino acid in the molecule in the absence of a thiol additive or a selenol additive, and promotes the rate-determining transesterification step. Preferably, the reactive ester is selected from the group consisting of trifluoroethyl thioester, 4-mercaptophenylacetic acid thioester, mercaptoethyl sulfonate thioester, methylthioglycolic acid thioester, thiophenyl thioester, benzyl mercaptan thioester, phenyl selenoester, and 4-selenophenylacetic acid selenoester. Preferably, when the ester is a reactive ester, the reaction is carried out in the absence of a thiol additive or a selenol additive.

[0015] In another embodiment, the ester may be a less reactive ester. By reacting the ester with a thiol additive or a selenol additive, the ester can be converted into a reactive ester. Examples of less reactive esters include ethyl 3-mercaptopropionate thioester, reduced L-glutathione (GSH) thioester, dithiothreitol (DTT) thioester, mercaptopropionic acid-leucine thioester, tert-butylthiol thioester, mercaptopropanoyl glycine thioester, selenoacetamide selenoester, selenopropionic acid-isoleucine selenoester, and (9-fluorenylmethyl) selenoester. In this embodiment, the reaction is carried out in the presence of a thiol additive or a selenol additive. In one embodiment, the ester is a thioester and the reaction is carried out in the presence of a thiol additive or a selenol additive. Preferably, the thiol additive is selected from the group consisting of trifluoroethanethiol, 4-mercaptophenylacetic acid, mercaptoethyl sulfonate, methyl thioglycolate, thiophenol, and benzyl mercaptan. Preferably, the selenol additive is aryl selenol, more preferably phenyl selenol or 4-selenophenylacetic acid.

[0016] In another embodiment, the ester is a selenoester and the reaction is carried out in the presence of a selenol additive. Preferably, the selenol additive is an arylselenol, more preferably phenylselenol or 4-selenophenylacetic acid.

[0017] Preferably, the reaction is carried out in an aqueous solution.

[0018] In one embodiment, the reaction is carried out in the presence of a first nucleophile. The first nucleophile can accelerate the rate of the ligation reaction. Preferably, the first nucleophile comprises imidazole. More preferably, the first nucleophile is selected from the group consisting of 2-methylimidazole, imidazole, and combinations thereof.

[0019] In one embodiment, a second nucleophile can be used to thiolytically cleave, aminolytically cleave, hydrolyze, or hydrazinolyze the product ester formed between the amide-containing compound and the ester. In a preferred embodiment, the second nucleophile is selected from the group consisting of reduced glutathione (GSH), dithiothreitol (DTT), cysteine, imidazole, amines, hydroxide ions, hydrazine, and combinations thereof. Preferably, the second nucleophile is GSH. Preferably, the second nucleophile is added to the ligation reaction mixture after completion of the ligation reaction.

[0020] The reaction can be carried out using a concentration of the molecule containing the terminal amino acid of about 5 mM to about 20 mM. In another embodiment, the reaction can be carried out at high dilution using a concentration of the molecule containing the terminal amino acid of less than about 1 mM, preferably less than about 500 μM, more preferably less than about 100 μM, and even more preferably less than about 50 μM. Preferably, the reaction contains at least about 1.2 molar equivalents, more preferably about 2 molar equivalents of the ester.

[0021] In one embodiment, the method further comprises a step of desulfurizing or deselenizing the amide-containing compound in a flow. In a preferred embodiment,

[0022]

Chem.

[0023]

Chem.

[0024] In another aspect of the present invention, a method for desulfurizing an amide-containing compound containing a thiol group or a disulfide group in a flow is provided. Preferably, the amide-containing compound is a reaction product of a native chemical ligation reaction. Preferably, the desulfurization includes exposing the compound to ultraviolet irradiation in the presence of a phosphine source.

[0025] In yet another aspect of the present invention, a method for deselenizing an amide-containing compound containing a selenol group or a diselenide group in a flow is provided. Preferably, the amide-containing compound is a reaction product of a native chemical ligation reaction. Preferably, the deselenization includes exposing the compound to ultraviolet irradiation in the presence of a phosphine source.

[0026] In another aspect of the present invention, there is provided a method for desulfurizing an amide-containing compound containing a thiol group or a disulfide group, the method comprising exposing the compound to ultraviolet irradiation in the presence of a phosphine source. This method can be carried out batchwise or in a flow.

[0027] In yet another aspect of the present invention, there is provided a method for deselenizing an amide-containing compound containing a selenol group or a diselenide group, the method comprising exposing the compound to ultraviolet irradiation in the presence of a phosphine source. This method can be carried out batchwise or in a flow.

[0028] The phosphine is preferably water-soluble. Preferably, the phosphine source is TCEP.

[0029] In another preferred embodiment, the desulfurization or deselenization further comprises a hydrogen atom source. Preferably, the hydrogen atom source is selected from the group consisting of reduced L-glutathione (GSH), dithiothreitol (DTT), tert-butylthiol, cysteine, and combinations thereof. More preferably, the hydrogen atom source is GSH.

[0030] In another preferred embodiment, the desulfurization or deselenization is carried out in the absence of a chemical radical initiator.

[0031] The present invention also relates to an amide-containing compound prepared by the method described herein. The present invention also relates to a desulfurized or deselenized amide-containing compound prepared by the method described herein.

[0032] In yet another aspect, the present invention (i) an ester with (ii) a molecule containing a terminal amino acid selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids A step of reacting in the presence of a reducing agent, optionally including a radical scavenger, The ester is a thioester or a selenoester, The concentration of the molecule is less than about 1 mM, It relates to a method for producing an amide-containing compound in batch.

[0033] In a preferred form of this embodiment, the concentration of the molecule containing the terminal amino acid is less than about 500 μM, more preferably less than about 100 μM, and even more preferably less than about 50 μM. Preferably, the reaction contains at least about 1.2 molar equivalents, more preferably about 2 molar equivalents of the ester.

[0034] In a preferred form of this embodiment, the reducing agent may be selected from the group including a thiol group-containing reducing agent, a selenol group-containing reducing agent, a phosphine group-containing reducing agent, or a combination thereof. Examples of thiol group-containing reducing agents include, but are not limited to, MPAA (4-mercaptophenylacetic acid), thiophenol, TFET (2,2,2-trifluoroethanethiol), methylthioglycolic acid methyl, benzyl mercaptan, and MESNa (sodium 2-mercaptoethanesulfonate). Examples of selenol group-containing reducing agents include, but are not limited to, phenylselenol, 4-selenophenylacetic acid, and methylselenol. Examples of phosphine group-containing reducing agents include, but are not limited to, TCEP (tris(2-carboxyethyl)phosphine), tributylphosphine, and THPP (tris(3-hydroxypropyl)phosphine). In another embodiment, the reducing agent may be selected from DTT (dithiothreitol), glutathione, NaBH 4 , NaHBH 3 CN, and ascorbic acid. In another embodiment, the reducing agent may be a non-chemical reducing agent such as an electrochemical reducing agent.

[0035] In one embodiment, the radical scavenger may be selected from the group consisting of aryl selenols and diselenides, more specifically phenyl selenol, diphenyl diselenide, and 4-selenophenylacetic acid; aryl thiols, more specifically MPAA and thiophenol; more specifically ascorbic acid, 2,2,6,6-tetramethylpiperidine, tetrahydroxy-1,4-benzoquinone, phenyl N-t-butyl nitrone, 2,2-diphenyl-1-picrylhydrazyl, and naringenin.

[0036] As used herein, unless the context otherwise requires, the terms "comprise", "comprising", and "include" and variations thereof such as "includes" and "including" are not intended to exclude further additives, components, integers or steps.

[0037] Further aspects of the invention, and further embodiments of the aspects described in the previous paragraph, will become apparent from the following specification, given by way of example, and with reference to the accompanying drawings.

Brief Description of the Drawings

[0038]

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Mode for Carrying Out the Invention

[0039] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings generally understood by those skilled in the art. Also, unless particularly required by the context, singular terms shall include plural ones, and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a protein" includes multiple proteins, and reference to "a cell" includes a population of multiple cells.

[0040] As used herein, the term "additive" refers to any means for promoting a reaction and / or preventing side reactions that is added separately to the reaction mixture from an external source. The reaction mixture contains reagents. The reaction mixture can include a solvent. One of ordinary skill in the art will understand that the solvent can include an aqueous solution. The aqueous solution can include buffer salts and denaturants. By way of example, an additive can refer to an exogenous molecule added to the reaction mixture. An additive can also refer to the addition of electrons as required in an electrochemical reduction. Some non-limiting examples of additives are nucleophiles and reducing agents. For illustration, some thiol group-containing reducing agents that have been traditionally used in NCL are MPAA, thiophenol, trifluoroethanethiol (TFET), methyl thioglycolate, benzyl mercaptan, and MESNa. Reducing agents that can be used in the ligation reactions disclosed herein include TCEP (tris(2-carboxyethyl)phosphine), THPP (tris(3-hydroxypropyl)phosphine), DTT (dithiothreitol), NaBH 4 4, NaHBH 3 4CN, and ascorbic acid, but are not limited thereto. Alternatively, thiol-based or selenol-based nucleophiles or reducing agents, and / or imidazole-based nucleophiles can be used. The methods disclosed herein can be carried out in the absence of additives unless otherwise specified.

[0041] In the context of this specification, "thiol additive" refers to a thiol-based additive or a disulfide-based additive, and "selenol additive" refers to a selenol-based additive or a diselenide-based additive. The "thiol additive" and the "selenol additive" can accelerate the rate-determining transesterification step in the ligation reaction. The "thiol additive" or the "selenol additive" is used to convert a less reactive ester into a reactive ester. Specifically, the "thiol additive" is used to convert a less reactive thioester into a reactive thioester, and the "selenol additive" is used to convert a less reactive thioester into a reactive selenoester or a less reactive selenoester into a reactive selenoester. Examples of thiol additives include, but are not limited to, trifluoroethanethiol (TFET), 4-mercaptophenylacetic acid (MPAA), mercaptoethyl sulfonate, methyl thioglycolate, thiophenol, and benzyl mercaptan. Examples of selenol additives include aryl selenols, more specifically phenyl selenol or 4-selenophenylacetic acid.

[0042] As used herein, "amino acid" refers to a molecule containing both an amino group and a carboxy group. For example, in an α-amino acid, there is an "α-amino group" directly bonded to the carbon atom having both an amino group and a carboxyl group, and an "α-carboxyl group" directly bonded to the carbon atom having both an amino group and a carboxyl group. The term "carboxyl" refers to either a COOH group or a -COO- group. An α-amino acid has the general formula H 2 N-CHR-COOH (wherein R is a side chain or H). The side chain is generally an alkyl chain, which may be substituted, and the substitution is usually at the distal end but not necessarily so. The N-terminus of an amino acid (or peptide) is the end at which an amine functional group (optionally ionized or substituted / protected) is located, and the C-terminus is the end at which a carboxyl functional group (optionally ionized or substituted / protected) is located.

[0043] As used herein, the term "peptide" refers to a chain containing (or consisting of) at least two amino acid residues linked by amide bonds. They can be dipeptides, oligopeptides, polypeptides, proteins, glycopeptides, glycoproteins, etc. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and include molecular chains of two or more amino acids covalently linked via peptide bonds. These terms do not refer to a specific length of the product. This term includes post-translational modifications of peptides, such as glycosylation, acetylation, biotinylation, 4-pentynoylation, PEGylation, phosphorylation, sulfation, etc. Furthermore, protein fragments, analogs, mutant or variant proteins, fusion proteins, etc. are included in the meaning of polypeptide. This term also includes molecules containing one or more amino acid analogs or non-canonical or non-natural amino acids. It will be understood that any natural or synthetic protein falls within the scope of the term "peptide". In the context of the present invention, natural proteins and peptides include recombinant expressed proteins and peptides, such as enzymes, hormones, structural proteins, transport proteins, signal proteins, antigens, and antibodies. Preferably, "peptide" includes synthetic and expressed peptides and proteins, such as selenoproteins, cysteinyl proteins, protein hydrazides, protein selenoesters, and protein thioesters.Furthermore, for example, by well-known organic chemistry techniques described in Smith, M. B. March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Seventh Edition; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; Fmoc Solid Phase Peptide Synthesis, A Practical Approach; Chan, W.C., White, P. D., Eds.; Oxford University Press, 2000 (chapter 6, pages 137-178; chapter 9, pages 215-227, chapter 11, pages 243-262); and Peptide Synthesis and Applications, Second Edition (Methods in Molecular Biology); Jensen, K. J., Shelton, P. T., Pedersen, S. L., Eds.; Humana Press, 2013 (chapter 8, pages 119-130), peptides can be derivatized as described herein.

[0044] The term "aryl" alone or in combination means a carbocyclic aromatic moiety containing one, two or even three rings, such rings may be joined together in a fused form. Thus, the term "aryl" includes aromatic groups such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, anthracenyl, and indanyl. The said "aryl" group may have one or more substituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, and lower alkylamino. -O-CH 2 Phenyl substituted with -O- forms an arylbenzodioxolyl substituent. As used herein, aryl means a completely unsaturated ring.

[0045] A "replaceable group" that can be replaced generally refers to a group that can be replaced from a molecule during the course of a reaction.

[0046] A "leaving group" generally refers to a group that can be replaced by a nucleophile. Such leaving groups are known in the art. Examples of leaving groups include halides (e.g., I, Br, F, Cl), sulfonates (e.g., mesylate, tosylate), sulfides (e.g., SCH 3 ), thiolates, selenoates, N-hydroxysuccinimide, N-hydroxybenzotriazole, and the like, but are not limited thereto.

[0047] A "nucleophile" is a species that can attack a molecule at the attachment point of a leaving group and cause replacement of the leaving group. Nucleophiles are known in the art. Examples of nucleophilic groups include amines, thiols, alcohols, selenols, Grignard reagents, anionic species (e.g., alkoxides, amides, carbanions), and the like, but are not limited thereto.

[0048] In the context of this specification, a "first nucleophile" refers to a nucleophile that accelerates the rate of a ligation reaction. Examples of suitable first nucleophiles include imidazole. In a preferred embodiment, the first nucleophile is selected from the group consisting of 2-methylimidazole, imidazole, and combinations thereof. In the context of this specification, the first nucleophile is distinguished from a thiol additive or a selenol additive. The first nucleophile can contain a thiol or selenol moiety.

[0049] In the context of the specification, the "second nucleophile" means a nucleophile that can be used to thiolytically cleave, aminolytically cleave, hydrolyze or hydrazinolyze the product ester formed between the amide-containing compound and the ester. Examples of suitable second nucleophiles include, but are not limited to, reduced glutathione (GSH), dithiothreitol (DTT), cysteine, imidazole, amines, hydroxide ions, hydrazine, and combinations thereof. The second nucleophile may be added before, during, or after the ligation reaction, or in combinations thereof. Thus, the ligation reaction may be carried out in the presence of the second nucleophile. Alternatively, the thiolytic cleavage, aminolytic cleavage, hydrolysis or hydrazinolysis may be an additional step carried out after the completion of the ligation reaction. In a preferred embodiment, the second nucleophile is added to the ligation reaction mixture after the completion of the ligation reaction.

[0050] The thiol additive or selenol additive, the first nucleophile and the second nucleophile may be used independently. In one embodiment, when the ester is a reactive ester, the reaction is carried out in the absence of the thiol additive or selenol additive, but in the presence of the first nucleophile. In this form of the embodiment, the first nucleophile may contain a thiol or selenol moiety. In another embodiment, when the ester is a less reactive ester, the reaction is carried out in the presence of the thiol additive or selenol additive and in the presence of the first nucleophile.

[0051] The first nucleophile and the second nucleophile may be used independently. In one embodiment, the flow ligation method comprises the first nucleophile rather than the second nucleophile. In another embodiment, the flow ligation method comprises the second nucleophile rather than the first nucleophile. In yet another embodiment, the flow ligation method comprises both the first nucleophile and the second nucleophile. When further describing the peptides described herein, one-letter abbreviations are often applied to indicate the identity of the 20 "canonical" or proteinogenic amino acid residues and the 21st amino acid selenocysteine, which are commonly incorporated into naturally occurring peptides and proteins (Table 1). Such one-letter abbreviations are completely interchangeable in meaning with three-letter abbreviations or non-abbreviated amino acid names.

[0052] Non-canonical or non-proteinogenic amino acid residues can be incorporated into peptides by using the techniques disclosed herein. The term "non-canonical amino acid residue" refers to an amino acid residue of the D or L type that is not among the 20 canonical amino acids commonly incorporated into naturally occurring proteins, such as, for example, β-amino acids, homoamino acids, cyclic amino acids, selenoamino acids, thioamino acids, and amino acids with derivatized side chains (such as those described in US2015 / 0023988).

[0053]

Table 1

[0054] The nomenclature and symbols for amino acids and peptides by the UPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are published in the following references: Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following page 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pp. 39-69, all of which are hereby incorporated by reference in their entirety.

[0055] As described above herein, in accordance with the present disclosure, the described peptides can also be chemically derivatized with one or more amino acid residues by known organic chemical techniques. "Derivative" or "derivatization" refers to a target peptide having one or more residues that are chemically derivatized by reaction of functional side chain groups. Such derivatized molecules include, for example, molecules in which the free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. The free carboxyl group may be derivatized to form a salt, methyl ester and ethyl ester, or other types of esters or hydrazides. The free hydroxyl group may be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine can be derivatized to form N-benzylhistidine. Also included as chemical derivatives are peptides containing one or more naturally occurring amino acid derivatives of the 20 canonical amino acids, regardless of whether they are in the L- or D-form. For example, 4-hydroxyproline may be substituted for proline, 5-hydroxylysine may be substituted for lysine, 3-methylhistidine may be substituted for histidine, homoserine may be substituted for serine, and ornithine may be substituted for lysine.

[0056] Useful derivatizations include the modification of the N-terminal free amino group to attach a contrast agent, such as a fluorescent dye, an MRI contrast agent, a PET contrast agent, or a therapeutic agent that adds activity to the potential therapeutic activity of the peptide. The N-terminus can be modified to an acylated or substituted amine, or to an aromatic moiety (e.g., indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn 2) or derivatized with another functional group such as benzyloxycarbonyl (Cbz or Z), N,N-dimethylglycine or creatine. For example, in some embodiments, but not limited to, an acyl moiety such as formyl, acetyl (Ac), propanoyl, butanoyl, pentanoyl, heptanoyl, hexanoyl, octanoyl, or nonanoyl can be covalently attached to the N-terminus of the peptide. Other exemplary N-terminal derivative groups include -NRR 1 (-NH 2 other than), -NRC(O)R 1 , -NRC(O)OR1, -NRS(O) 2 R 1 , -NHC(O)NHR 1 , succinimide, or benzyloxycarbonyl-NH-(Cbz-NH-), where R and R 1 are each independently hydrogen or lower alkyl or phenyl, and the phenyl ring is C 1 ~C 4 alkyl, C 1 ~C 4 alkoxy, chloro, and bromo and may be substituted with 1-5 substituents selected therefrom.

[0057] In some embodiments, one or more peptidyl [-C(O)NR-] bonds (linkages) between amino acid residues may be replaced by non-peptidyl linkages. Exemplary non-peptidyl linkages are -CH 2 -carbamate [-CH 2 -OC(O)NR-], phosphonate, -CH 2 -sulfonamide [-CH 2 -S(O) 2 NR-], thiourea [-NHC(S)NH-], urea [-NHC(O)NH-], -CH 2 -secondary amine, and alkylated peptide [-C(O)NR 6 (wherein R 6 is lower alkyl)].

[0058] The above examples of derivatization are not intended to be an exhaustive treatment, but are merely illustrative. One of ordinary skill in the art will be able to derivatize one or more individual amino acids, for example, by well-known organic chemistry techniques such as those described in Smith, M. B. March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Seventh Edition; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; and that various derivatizing agents are known to react with selective side chains and terminal residues as described, for example, in Fmoc Solid Phase Peptide Synthesis, A Practical Approach; Chan, W.C., White, P. D., Eds.; Oxford University Press, 2000; Peptide Synthesis and Applications, Second Edition (Methods in Molecular Biology); Jensen, K.

[0059] It will be understood that the invention, as disclosed and defined herein, extends to all those features of the text or drawings which are mentioned or apparent and to all the alternative combinations of two or more of those distinct features. All of these different combinations constitute various alternative aspects of the invention.

[0060] The present invention (i) reacting an ester (ii) a molecule comprising a terminal amino acid selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids; comprising the step of reacting, wherein said ester is a thioester or a selenoester, relates to a method for producing an amide-containing compound in a flow.

[0061] The ester may be a molecule containing an ester functional group. The ester can be selected from polymers (including peptides), small molecules (including therapeutic agents or diagnostic agents such as fluorescent dyes, radioisotopes, MRI contrast agents, or PET contrast agents), or antibodies. Preferably, the ester is a peptide.

[0062] The ester is preferably a reactive ester. The reactive ester can react with the terminal amino acid in the molecule in the absence of a thiol additive or a selenol additive, accelerating the rate-determining transesterification step. Preferably, the reactive ester is selected from the group consisting of trifluoroethyl thioester, 4-mercaptophenylacetic acid thioester, mercaptoethyl sulfonate thioester, methylthioglycolic acid thioester, thiophenyl thioester, benzyl mercaptan thioester, phenyl selenoester, and 4-selenophenylacetic acid selenoester. Preferably, when the ester is a reactive ester, the reaction is carried out in the absence of a thiol additive or a selenol additive.

[0063] In another embodiment, the ester may be a low-reactivity ester. The ester can be converted into a reactive ester by reacting the ester with a thiol additive or a selenol additive. Examples of low-reactivity esters include thioesters of ethyl 3-mercaptopropionate, reduced L-glutathione (GSH) thioester, dithiothreitol (DTT) thioester, leucine thioester of mercaptopropionic acid, tert-butyl-butylthiol thioester, mercaptopropanoyl glycine thioester, selenoacetamide selenoester, isoleucine selenoester of selenopropionic acid, and (9-fluorenylmethyl) selenoester. In this embodiment, the reaction is carried out in the presence of a thiol additive or a selenol additive. In one embodiment, the ester is a thioester and the reaction is carried out in the presence of a thiol additive or a selenol additive. Preferably, the thiol additive is selected from the group consisting of trifluoroethanethiol, 4-mercaptophenylacetic acid, mercaptoethyl sulfonate, methyl thioglycolate, thiophenol, and benzyl mercaptan. In another embodiment, the ester is a selenoester and the reaction is carried out in the presence of a selenol additive. Preferably, the selenol additive is aryl selenol, more preferably phenyl selenol or 4-selenophenylacetic acid.

[0064] Molecules containing a terminal amino acid selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids can be selected from polymers (including peptides), small molecules (including therapeutic agents, or diagnostic agents such as fluorescent dyes, radioisotopes, MRI contrast agents, or PET contrast agents), or antibodies. Preferably, the molecule is a peptide.

[0065] It is understood that an amino acid containing a thiol functional group can be either in the reduced form (cysteine or thiol-derivatized amino acid) or in the oxidized form (cystine or disulfide-derivatized amino acid). In the oxidized form, the thiol functional group may form a disulfide bond with a second amino acid containing an oxidized thiol functional group, forming a dimer. For example, the molecule may form a dimer of formula (IV):

[0066] [Chemical formula] wherein AA lig is selected from cystine and disulfide-derivatized amino acids.

[0067] It is understood that an amino acid containing a selenol functional group can be either in the reduced form (selenocysteine or selenol-derivatized amino acid) or in the oxidized form (selenocystine or diselenide-derivatized amino acid). In the oxidized form, the selenol functional group may form a diselenide bond with a second amino acid containing an oxidized selenol functional group, forming a dimer. For example, the molecule may form a dimer of formula (IV):

[0068] [Chemical formula] wherein AA lig is selected from selenocystine and diselenide-derivatized amino acids.

[0069] The amide-containing compound is the product of a ligation reaction between an ester and a molecule. Preferably, the amide-containing compound is a polypeptide. Preferably, the ester and the molecule are independently selected from synthetic and natural peptides. The present disclosure contemplates the synthesis of natural and non-natural peptides. Natural peptides contain naturally occurring amino acids having L-stereochemistry. Non-natural peptides include peptides containing one or more non-natural amino acids having D stereochemistry, or derivatized amino acids incorporating a diagnostic agent (e.g., a fluorescent dye, a radioisotope, an MRI contrast agent, or a PET contrast agent) or a therapeutic agent (e.g., a drug, or an antibody). Non-natural peptides also include peptides in which one or more of the peptidyl bonds between amino acid residues are replaced by non-peptidyl bonds. The amino acids used in the present invention may be L, D, or racemic. The chemistry currently described can preserve the stereochemistry of the amino acids.

[0070] In a particularly preferred embodiment, the polypeptide is defined by formula (I):

Chemical formula

[0071]

Chemical formula

[0072]

Chemical formula

[0073]

Chemical formula

[0074] Wherein, N term is the N-terminus of the polypeptide; C term is the C-terminus of the polypeptide; AA is an amino acid; N is an integer; (AA) n represents a polypeptide containing n number of amino acid monomers; DG is a replaceable group selected from thiolate and selenoate;

[0075] [Chemical formula]

[0076] is a thiol, disulfide, selenol or diselenide functionalized amino acid residue of a ligation site selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids.

[0077] It is understood that the polypeptide of formula (I) can be either in the reduced or oxidized form. In the reduced form, the polypeptide of formula (I) is a monomer. In the oxidized form, AAlig of the polypeptide of formula (I) may form a bond with AAlig of the second polypeptide of formula (I) to form a dimer.

[0078] Preferably, the reaction is carried out using a concentration of molecules containing terminal amino acids of about 5 mM to about 20 mM. In another embodiment, the reaction can be carried out at high dilution using a concentration of molecules containing terminal amino acids of less than about 1 mM, preferably less than about 500 μM, more preferably less than about 100 μM, and even more preferably less than about 50 μM. Preferably, the reaction contains at least about 1.2 molar equivalents, more preferably about 2 molar equivalents of ester.

[0079] Preferably, the reaction is carried out in an aqueous solution. The aqueous solution is preferably a buffer selected from HEPES, Na 2 HPO 4 , MOPS, and Tris, more preferably HEPES.

[0080] Preferably, the aqueous solution has a pH in the range of about 4 to 9, preferably about 6 to 8.

[0081] The aqueous solution can optionally contain a denaturant. Preferably, the denaturant is 6M guanidine hydrochloride or urea, more preferably 6M guanidine hydrochloride.

[0082] The aqueous solution may optionally contain a reducing agent. Preferably, the reducing agent is selected from the group consisting of tris-(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and L-glutathione (GSH), more preferably TCEP.

[0083] The reaction can be carried out at a moderately elevated temperature, or at room temperature or below. Generally, the ligation reactions described herein are carried out at room temperature. Nevertheless, one of ordinary skill in the art will understand that the reaction can be carried out at a lower temperature to minimize side reactions, or, for example, at an elevated temperature to further accelerate the reaction rate. Suitable lower temperatures are below room temperature, below 0 °C to about -100 °C, for example -10, -20, -50, -70 °C, or about -100 to about 0 °C, or about -100 to -50, -100 to -70, -50 to 0, -20 to 0, or -80 to -60 °C, for example about -100, 90, -80, -78, -70, -60, -50, -40, -30, -20, -10, or 0 °C. Suitable elevated temperatures are above room temperature, above about 30, 40, 50, 60, 70, up to about 80 °C. About 10 to about 80 °C, or about 20 to 80, 50 to 80, 70 to 80, 10 to 30, 10 to 50, 30 to 60, 30 to 40, 40 to 70 or 50 to 70 °C, for example about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 °C.

[0084] The ligation reaction is preferably initiated by combining a solution of an ester with a solution of a molecule containing a terminal amino acid selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenol-derivatized amino acids in a flow, and then allowed to proceed to completion while being measured by an analytical technique. Preferably, the ligation reaction may be allowed to proceed to about 100% completion. The ligation reaction can be allowed to proceed to at least 90% completion, at least 85% completion, at least 80% completion, at least 75% completion, at least 70% completion, at least 65% completion, at least 60% completion, at least 55% completion, at least 50% completion, at least 45% completion, at least 40% completion, at least 35% completion, at least 30% completion, at least 25% completion. It is understood that preferably, the ligation reaction can be allowed to proceed to at least 90% completion, more preferably to about 100% completion.

[0085] Except for the reaction where AAlig is selected from Ile or Val and the reaction is completed within about 40 minutes while being measured by HPLC-MS, the reaction is preferably completed within about 10 minutes while being measured with attention to the formation of the product by HPLC-MS.

[0086] In one embodiment, the reaction is carried out in the presence of a first nucleophile. The first nucleophile accelerates the rate of the ligation reaction. Preferably, the first nucleophile contains imidazole. More preferably, the first nucleophile is selected from the group consisting of 2-methylimidazole, imidazole, and combinations thereof.

[0087] The ligation reaction between the ester and the molecule produces an amide-containing compound. The amide-containing compound can react with the excess ester in the ligation reaction mixture to form a product ester.

[0088] In one embodiment, a second nucleophile can be used to thiolytically cleave, aminolytically cleave, hydrolyze, or hydrazinolyze the product ester formed between the amide-containing compound and the ester. In a preferred embodiment, the second nucleophile is selected from the group consisting of reduced glutathione (GSH), dithiothreitol (DTT), cysteine, imidazole, amines, hydroxide ions, hydrazine, and combinations thereof. Preferably, the second nucleophile is GSH. Preferably, the second nucleophile is added to the ligation reaction mixture after completion of the ligation reaction.

[0089] The ligation reaction carried out in flow proceeds rapidly at room temperature in an aqueous buffer having a wide range of pH values using unprotected peptide fragments.

[0090] Following the ligation reaction, the crude reaction mixture can be subjected to appropriate desulfurization or deselenization conditions and reagents without intermediate purification (however, optionally with at least partial removal of at least one reagent or catalyst used in the ligation reaction). The resulting ligated and selectively desulfurized or deselenized product peptide can be obtained from the resulting reaction mixture following a time appropriate for the reaction.

[0091] In one embodiment, the method further comprises the step of desulfurizing or deselenizing the amide-containing compound in flow. In a preferred embodiment, when AAlig is a thiol or disulfide-functionalized amino acid residue at a ligation site selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, and disulfide-derivatized amino acids, the method further comprises the step of desulfurizing the amide-containing compound in flow.

[0092] In another preferred embodiment, when AAlig is a selenol or diselenide functionalized amino acid residue at a ligation site selected from the group consisting of selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids, the method further comprises the step of de-selenating the amide-containing compound in a flow.

[0093] In one embodiment, the desulfurization or de-selenation step comprises adding a chemical radical initiator to the amide-containing compound in a flow. Non-limiting examples of water-soluble radical initiators are 2,2'-azobis(2-(2-imidazolin-2-yl)propane) dihydrochloride (VA-044).

[0094] In a preferred embodiment, the desulfurization or de-selenation step comprises exposing the compound to ultraviolet irradiation in the presence of a phosphine source.

[0095] Preferably, the photo-desulfurization or photo-de-selenation step is carried out in the absence of a chemical radical initiator.

[0096] In another aspect of the present invention, a method for desulfurizing an amide-containing compound containing a thiol group or a disulfide group in a flow is provided. Preferably, the amide-containing compound is a reaction product of a native chemical ligation reaction. In one embodiment, the desulfurization comprises adding a chemical radical initiator to the amide-containing compound in a flow. Preferably, the desulfurization comprises exposing the compound to ultraviolet irradiation in the presence of a phosphine source.

[0097] In yet another aspect of the present invention, a method for de-selenating an amide-containing compound containing a selenol group or a diselenide group in a flow is provided. Preferably, the amide-containing compound is a reaction product of a native chemical ligation reaction. In one embodiment, the de-selenation comprises adding a chemical radical initiator to the amide-containing compound in a flow. Preferably, the de-selenation comprises exposing the compound to ultraviolet irradiation in the presence of a phosphine source.

[0098] In another aspect of the present invention, there is provided a method for desulfurizing an amide-containing compound containing a thiol group or a disulfide group, the method comprising exposing the compound to ultraviolet irradiation in the presence of a phosphine source. This method can be carried out in batch or flow.

[0099] In yet another aspect of the present invention, there is provided a method for deselenizing an amide-containing compound containing a selenol group or a diselenide group, the method comprising exposing the compound to ultraviolet irradiation in the presence of a phosphine source. This method can be carried out in batch or flow.

[0100] The desulfurization or deselenization reaction preferably starts in flow by exposing a solution containing an amide-containing compound containing a thiol group, a disulfide group, a selenol group or a diselenide group, preferably in the presence of a phosphine source, to ultraviolet irradiation, and then proceeds to completion while being measured by an analytical technique. Preferably, the desulfurization or deselenization may be allowed to proceed to about 100%. It should be understood that the desulfurization or deselenization reaction can proceed to at least 90% completion, at least 85% completion, at least 80% completion, at least 75% completion, at least 70% completion, at least 65% completion, at least 60% completion, at least 55% completion, at least 50% completion, at least 45% completion, at least 40% completion, at least 35% completion, at least 30% completion, at least 25% completion. Preferably, the desulfurization or deselenization may be allowed to proceed to at least 90% completion, more preferably to about 100% completion.

[0101] The phosphine is preferably water-soluble. Examples of suitable water-soluble phosphines include dipotassium bis(p-sulfonatophenyl)phenylphosphine dihydrate, bis(4,6-dimethyl-3-sulfonatophenyl)(2,4-dimethylphenyl)phosphine disodium salt hydrate, 1,2-bis(di-4-sulfonatophenylphosphino)benzene tetrasodium salt, bis(3-sulfonatophenyl)(3,5-di-trifluoromethylphenyl)phosphine disodium salt monohydrate, dipotassium bis(p-sulfonatophenyl)phenylphosphine dihydrate, bis(3-sulfonatophenyl)(2-trifluoromethylphenyl)phosphine disodium dihydrate, bis(3-sulfonatophenyl)(4-trifluoromethylphenyl)phosphine disodium dihydrate, di-t-butyl(3-sulfonatopropyl)phosphine, 2'-dicyclohexylphosphino-2,6-dimethoxy-3-sulfonato-1,1'-biphenyl hydrate sodium salt, 2'-dicyclohexylphosphino-2,6-di-i-propyl-4-sulfonato-1,1'-biphenyl hydrate sodium salt, diphenyl(m-sulfonatophenyl)phosphine dihydrate sodium salt, 2-(dicyclohexylphosphino)ethyl]trimethylammonium chloride, diphenyl(p-sulfonatophenyl)phosphine monohydrate dimethyl sulfoxide adduct potassium salt, dicyclohexyl-{9-[3-(4-sulfonylphenyl)propyl]-2-sulfonylfluoren-9-yl}phosphonium hydrogen sulfate, tetrabutylphosphonium chloride, 1,3,5-triaza-7-phosphaadamantane, tris(2-carboxyethyl)phosphine (TCEP), tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate, tris(hydroxymethyl)phosphine, tris(3-hydroxypropyl)phosphine, and tris(3-sulfonatophenyl)phosphine hydrate sodium salt, but are not limited thereto. Preferably, the phosphine source is TCEP.

[0102] In another preferred embodiment, the desulfurization or deselenization further comprises a hydrogen atom source. Preferably, the hydrogen atom source is selected from the group consisting of reduced L-glutathione (GSH), dithiothreitol (DTT), tert-butylthiol, cysteine, and combinations thereof. Preferably, the hydrogen atom source is GSH.

[0103] In another preferred embodiment, the photodesulfurization or photodeselenization is carried out in the absence of a chemical radical initiator.

[0104] The present invention also relates to an amide-containing compound prepared by reacting an ester with a molecule containing a terminal amino acid selected from the group consisting of cysteine, cystine, thiol-derivatized amino acids, disulfide-derivatized amino acids, selenocysteine, selenocystine, selenol-derivatized amino acids, and diselenide-derivatized amino acids in a flow.

[0105] The present invention also relates to a desulfurized or deselenized amide-containing compound prepared by the method described herein.

Examples

[0106] Experimental items The following examples relate to the use of synthetic peptides. Those skilled in the art will understand that the present disclosure is not limited to synthetic peptides and includes expressed peptides and proteins.

[0107] General synthetic procedure Nuclear magnetic resonance (NMR) spectra were recorded at 300 K using either a Bruker Avance DPX 400, 500, or 800 spectrometer as specified in the figure legends for each individual spectrum in D 2 O or d 6 -DMSO calibrated with the residual solvent peak as an internal standard. 3:7 MeCN:H containing 0.1% TFA (TA30) 2High-resolution mass spectra were acquired in the reflectron mode on a Bruker (MA, USA) Autoflex™ Speed MALDI-TOF using either a matrix of saturated α-cyano-4-hydroxycinnamic acid in O or 2’,4’,6’-trihydroxyacetophenone (THAP) / diammonium hydrogen citrate (18 mg / 7 mg in 500 μL of TA30). Equal amounts of the sample in TA30 and the matrix were thoroughly mixed, spotted onto a polished steel MALDI plate, and then dried. Data were acquired using a Protein 1 calibrant (Bruker) and then analyzed using Flexanalysis (Bruker) software.

[0108] Analytical UPLC was performed on a Waters Acquity UPLC system equipped with a Sample Manager FTN, a Quaternary Solvent Manager (H-Class), and a PDA eλ detector (λ = 210–400 nm). Analyses were carried out using a Waters Acquity UPLC-18BEH (130 Å, 1.7 μm) 2.1 mm × 50 mm column operating at 0.60 mL / min. Preparative reverse-phase HPLC (RP-HPLC) was carried out on a Waters 2535 quaternary gradient system coupled with a Waters 2489 UV / Vis detector module operating at 230 nm and 280 nm and a Waters fraction collector III. Unless otherwise specified, a Waters Sunfire C-18 OBD (100 Å, 5 μm), 30 × 150 mm column was used at a flow rate of 38.0 mL / min. Both apparatuses were operated using a mobile phase consisting of 0.1 vol% TFA in Milli-Q water (A / solvent A) and 0.1 vol% TFA in HPLC-grade acetonitrile (B / solvent B) with a linear gradient. Chromatogram analysis was carried out using Empower3 Pro software (2010).

[0109] UPLC-MS was performed using a Shimadzu LC-30AD liquid chromatography pump module with a DGU-20A5R degassing unit and an SPD-M30A diode array detector. A CTO-20A column oven was used with a Waters C-18 BEH (130 Å, 1.7 μm) 2.1×50 mm column operating at 0.60 mL / min. These components were coupled with a CBM-20A communication bus module, along with a Nexera X2 SIL-30AC autosampler and a Shimadzu LCMS-2020 mass spectrometer operating in positive mode. Peptides were analyzed using a linear gradient with a mobile phase of milliQ water and 0.1 volume% formic acid and 0.1 volume% formic acid in HPLC grade acetonitrile. Analysis was performed using Shimadzu LabSolutions software. The ESI-MS spectra of the purified proteins were obtained by averaging the total ion counts over the entire gradient and wash cycle.

[0110] Flow experiments were carried out using a Vapourtec RS-200 automated control system, together with an R4 convection heating module equipped with a stainless steel R2 series pumping module and a Vapourtec standard coil tube reactor. These modules were combined with an R-2S pumping module having two V-3 peristaltic pumps and pre-installed Flow Commander software. Polytetrafluoroethylene (PTFE) tubing (0.50 mm id×1.60 mm od) or polyfluoroalkoxy (PFA) tubing (0.50 mm id×1.60 mm od) was used together with PTFE injection loops of 100 μL, 500 μL, 2.0 mL (0.50 mm id×1.60 mm od) and 5.0 mL (0.75 mm id×1.60 mm od) volumes as specified. A Rayonet RPR-100 photoreactor was used at λ = 254 nm, 302 nm, and 365 nm (35 W).

[0111] Absorbance of tyrosine residues in the model peptides used (εTyr = 1280 M -1 cm-1 ) Based on the integration of the peaks in the UPLC chromatogram at λ = 280 nm corresponding to , the ligation progress of the model system was calculated. The reported reaction yield was calculated from the mass of the isolated peptide relative to the theoretical reaction yield. The yield was adjusted to account for any aliquots removed for reaction monitoring.

[0112] Materials Commercially available materials were used as received unless otherwise specified. Amino acids, coupling reagents, and resins were obtained from Novabiochem or GL Biochem. Reagents not commercially available were synthesized according to literature procedures as indicated in the experimental section. N,N-Dimethylformamide (DMF) was obtained in peptide synthesis grade from Merck or Labscan

[0113] Compound S1 was synthesized as reported in Sayers2015.

Chemical formula

[0114]

Chemical formula

[0115] Fmoc SPPS procedure All reagent equivalents are reported relative to the number of moles of amino acid loaded onto the resin.

[0116] Resin loading Link amide resin Link amide resin (copoly(styrene-1%DVB) 100 - 200 mesh) (200 μmol) was swollen in CH 2 Cl 2 for 30 minutes and then washed with DMF (5 × 5 mL). The resin was washed with DMF (5 × 5 mL), CH 2 Cl 2Before washing with DMF (5 × 5 mL) and DMF (5 × 5 mL), it was Fmoc-deprotected with 20 vol.% piperidine in DMF (2 × 5 mL, 3 min). PyBOP (4 eq) and NMM (8 eq) were added to Fmoc-Xaa-OH (4 eq) (0.125 M Fmoc-Xaa-OH) in DMF and added to the above resin at room temperature with stirring for 2 h. The loading solution was removed and the resin was washed with DMF (5 × 5 mL), CH 2 Cl 2 (5 × 5 mL) and DMF (5 × 5 mL), then capped with Ac 2 O (0.125 M) and iPr 2 EtN (0.125 M) in DMF (5 mL). The resin was washed with DMF (5 × 5 mL), CH 2 Cl 2 (5 × 5 mL) and DMF (5 × 5 mL).

[0117] 2-Chlorotrityl chloride resin 2-Chlorotrityl chloride resin (copoly(styrene-1% DVB) 100 - 200 mesh) (200 μmol) was swollen in CH 2 Cl 2 for 30 min, and a solution of Fmoc-Xaa-OH (2 eq) and iPr 2 EtN (4 eq) in CH 2 Cl 2 (0.125 M of Fmoc-Xaa-OH) was added to the resin and stirred at room temperature for 16 h. The loading solution was removed and the resin was washed with DMF (5 × 5 mL), CH 2 Cl 2 (5 × 5 mL), and DMF (5 × 5 mL). The resin was treated with a solution of CH 2 Cl 2 / CH 3 OH / iPr 2 EtN (17:2:1 v / v / v, 5 mL) for 1 h and washed with DMF (5 × 5 mL), CH 2 Cl 2 (5 × 5 mL), and DMF (5 × 5 mL).

[0118] Derivatization of 2-chlorotrityl chloride resin with hydrazine This procedure was adopted from Zheng et al. 2013. 2-Chlorotrityl chloride resin (copoly(styrene-1%DVB) 100-200 mesh) (200 μmol) was swollen in CH 2 Cl 2 for 30 minutes. A 5 vol.% N 2 H 4 ·H 2 O DMF solution was added to the resin and gently stirred for 30 minutes. The resin was washed with DMF (5×5 mL), CH 2 Cl 2 (5×5 mL), and DMF (5×5 mL), followed by repeated hydrazine treatment and washing. A 5 vol% CH 3 OH capping solution in DMF was added to the resin and stirred for 10 minutes. The resin was then washed with DMF (5×5 mL), CH 2 Cl 2 (5×5 mL), and DMF (5×5 mL) to obtain a pale yellow-green resin. Then, the standard peptide coupling procedure was immediately carried out.

[0119] Quantification of resin loading amount The resin (10 mg) was treated with a 2 vol% 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) DMF solution (2 ml), stirred for 30 minutes, and then 8 ml of acetonitrile was added. A 1 mL aliquot of the resulting solution was taken and diluted to 12.5 mL with acetonitrile. The absorbance (λ = 304, ε = 9254 M -1 cm -1 ) of the DBU-fullerene adduct was measured to estimate the loading.

[0120] Iterative peptide assembly (Fmoc SPPS) Deprotection The resin (initial loading of 200 μmol) was treated with 20 volume% piperidine in DMF (2×5 mL, 3 minutes) and washed with DMF (5×5 mL), CH 2 Cl 2 (5×5 mL), and DMF (5×5 mL).

[0121] General amino acid coupling A DMF solution of Fmoc-Xaa-OH (4 equivalents), PyBOP (4 equivalents) and NMM (8 equivalents) (0.125 M Fmoc-Xaa-OH) was added to the resin (200 μmol initial loading), and the mixture was stirred at room temperature for 1 hour. Then, the resin was washed with DMF (5 × 5 mL), CH 2 Cl 2 (5 × 5 mL), and DMF (5 × 5 mL).

[0122] Capping Ac 2 A DMF solution of O (0.125 M) and iPr 2 EtN (0.125 M) (5 mL) was added to the resin. After 3 minutes, the resin (200 μmol initial loading) was washed with DMF (5 × 5 mL), CH 2 Cl 2 (5 × 5 mL), and DMF (5 × 5 mL).

[0123] Cleavage and workup from resin without side-chain deprotection The resin (200 μmol initial loading) was treated with HFIP / CH 2 Cl 2 (3:7, v / v, 5 mL) and stirred at room temperature for 40 minutes. The resin was filtered and washed with CH 2 Cl 2 (5 × 5 mL). The combined filtrate was concentrated under a nitrogen stream.

[0124] Cleavage and workup from resin with complete side-chain deprotection A mixture of TFA / iPr 3 SiH / H 2 O (90:5:5 v / v / / v) was added to the resin (200 μmol initial loading). After 2 hours, the resin was filtered and washed with TFA (3 × 5 mL). The combined filtrate was concentrated to less than 5 mL under a nitrogen stream. Diethyl ether (40 mL) was added, and the solution was cooled to -20 °C for 15 minutes. The precipitate was pelleted by centrifugation at 4000 rcf for 8 minutes, and the supernatant was decanted.

[0125] Automated SPPS Automated Fmoc-SPPS was performed on a Gyros Protein Technologies Symphony Automated Synthesizer. Fmoc-deprotection using 20 vol% piperidine in DMF, and Ac 2 O (0.3 M) and iPr 2 A general synthetic protocol for capping using EtN (0.3 M) was performed as described above. The coupling solution was prepared with Fmoc-Xaa-OH (0.3 M) in DMF, OxymaPure® (0.3 M) in DMF, and DIC (0.3 M) in DMF.

[0126] Microwave-assisted peptide synthesis Microwave-assisted peptide synthesis was performed on a CEM Liberty Blue automated microwave peptide synthesizer (USA, NC) at either 90 °C or 50 °C. High-temperature coupling used a 4-minute coupling method: [2-minute coupling (90 °C), 1-minute deprotection (90 °C), 1-minute related washing and liquid handling] On the other hand, synthesis on 2-chlorotrityl chloride resin (N-terminal segment) was performed at 50 °C using a 30-minute coupling method: [20-minute coupling (50 °C), 2 × 3-minute deprotection (room temperature), 4-minute related washing and liquid handling]. Both methods used a 4-fold excess of Fmoc-Xaa-OH, Oxyma, and DIC. Capping of unreacted N-terminal amines was achieved with a 5-fold molar excess using N-acetylglycine, Oxyma, and DIC with 1-minute 90 °C coupling or 3-minute 50 °C coupling.

[0127] Solution-phase thioesterification The crude side-chain protected peptide was dissolved in dry DMF (20 mM) and cooled to -30 °C. To this, ethyl 3-mercaptopropionate (30 equivalents) was added, followed by PyBOP (5 equivalents) and iPr 2EtN (5 equivalents) was added with stirring for 3 hours under an argon atmosphere. The solution was warmed to room temperature and concentrated under a nitrogen stream. Deprotection and workup were achieved using the acidic deprotection conditions described for the fully protected peptide on resin. No epimerization was observed using this procedure.

[0128] Diazotization and thioesterification of peptide hydrazide The conversion of the peptide hydrazide to the peptide thioester was carried out using a procedure slightly modified from that reported by Zheng2013. The fully deprotected peptide with a C-terminal acyl hydrazide was solvated to a concentration of 40 mM in aqueous ligation buffer (6 M Gdn·HCl, 0.1 M HEPES). This solution was adjusted to pH 3.0 and cooled to -15 °C. To this, an aqueous solution of NaNO 2 (12 M, 10 equivalents) was added. After 10 minutes, distilled TFET (10 equivalents for Ala thioester, 20 equivalents for Val thioester) was added (Warning: TFET is irritating and acutely toxic and should be used in a fume cupboard). The solution was adjusted to pH 6.8 - 7.0 and warmed to room temperature. After 10 minutes, the resulting peptide trifluoroethyl thioester was purified by HPLC.

[0129] Native chemical ligation in flow Native chemical ligation is carried out between a peptide containing an N-terminal cysteine (Cys) residue and a peptide functionalized as a C-terminal thioester (Figure 1). Mechanistically, it proceeds through an initial trans-thioesterification step, followed by a rapid S→N acyl shift to give the native peptide bond. Typically, a suitable thiol additive is required to generate a reactive thioester (SR 2 ) that accelerates the rate-determining trans-thioesterification step.

[0130] Model peptide H-CSPGYS-NH 2 (1, SEQ ID NO: 1) and model peptide thioester Ac-LYRANX-S(CH 2 )2 CO 2 The model NCL reaction was carried out in the flow between CO and Et(2: X = A (SEQ ID NO: 2), 3: X = V (SEQ ID NO: 32)) (Figs. 2 to 4).

[0131] Any additives used were solvated in a degassed ligation buffer (500 mM MPAA, 500 mM TFET, 5.0 M 2-MIM) (pH 7.4 to 7.5) (Note: TFET is irritating and acutely toxic and should be used in a fume hood) containing 6 M Gdn·HCl, 0.1 M HEPES and 50 mM TCEP. This solution was added to model peptide 1 (10 mM, 1.0 equivalent). A second solution of model peptide thioester 2 or 3 (1.2 equivalents or 2.0 equivalents) in a degassed ligation buffer (10 mM) (pH 7.1 to 7.2) containing 6 M Gdn·HCl, 0.1 M HEPES and 50 mM TCEP was prepared. These solutions were inserted into an injection loop and combined at a T-piece on a 150 μL scale using petroleum ether as the system solvent (Fig. 3A). The resulting reaction stream was fed into a PTFE coil reactor I (0.5 mm × 1.6 mm) at 37 °C (6 bar BPR). An equal volume of 7 vol.% N 2 H 4 ·H 2 O in MilliQ water was used to directly quench the reaction solution upon recovery. The ligation time course was obtained by varying the flow rate to adjust the volume of the reactor I and the residence time. A 10 μL aliquot of the quenched solution was taken, diluted with 40 μL of 0.1% TFA in MilliQ water, and then analyzed by UPLC. The estimated conversion was based on the relative peak areas of starting material 1 and the desired ligation product 6 or 7 at λ = 280 nm [ε280(1) = 1280 M -1 cm -1 −1, ε280(6,7) = 2560 M -1 cm -1 −1].

[0132] Native chemical ligation in batch Model peptide H-CSPGYS-NH 2(1, SEQ ID NO: 1) and the model peptide thioester Ac-LYRANX-S(CH 2 ) 2 CO 2 Et (2: X = A (SEQ ID NO: 2), 3: X = V (SEQ ID NO: 3)) were subjected to a comparative NCL reaction in batches.

[0133] Any additives used were solvated in a degassed ligation buffer (500 mM MPAA, 500 mM TFET, 5.0 M 2-MIM) (pH 7.4 - 7.5) (Note: TFET is irritating and acutely toxic and should be used in a fume cupboard) containing 6 M Gdn·HCl, 0.1 M HEPES and 50 mM TCEP. This solution was added to model peptide 1 (10 mM, 1.0 equivalent). A second solution of model peptide thioester 2 or 3 (1.2 equivalents or 2.0 equivalents) was prepared in a degassed ligation buffer (pH 7.1 - 7.2) containing 6 M Gdn·HCl, 0.1 M HEPES and 50 mM TCEP. These solutions were combined (1:1 vol / vol), gently stirred and then incubated at 37 °C (Scheme S1). The progress of the ligation was plotted by taking 5 μL aliquots at various time points and quenching with 5 μL of 7 vol% N 2 H 4 ·H 2 O in MilliQ water. This solution was diluted with 40 μL of 0.1% TFA in MilliQ water and then analyzed by UPLC. The estimated conversion values were based on the relative peak areas of starting material 1 and the desired ligation products 6 or 7 at λ = 280 nm [ε280(1) = 1280 M -1 cm -1 , ε280(6,7) = 2560 M -1 cm- 1 .

[0134] Figure 5 shows the kinetics of the ligation reaction between model peptide 1 and model peptide thioester 2 in batch and flow, either in the absence or presence of a thiol additive selected from trifluoroethanethiol (TFET), 4-mercaptophenylacetic acid (MPAA) and MESNa.

[0135] Native chemical ligation in flow using preformed reactive thioesters Preformed trifluoroethyl thioester Ac-LYRANX-SCH 2 CF 3 (4: X = A (SEQ ID NO: 4), 5: X = V (SEQ ID NO: 5)) was ligated to 1 using 2-methylimidazole (2-MIM) as an optional additive to develop an NCL flow strategy (Figure 4B). Using the same flow settings as above, these conditions resulted in complete ligation between peptide 1 and thioester 4 in 2 minutes and between thioester 5 in 11 minutes (see Figures 4B and 4C, Figure 6).

[0136] Native chemical ligation in batch using preformed reactive thioesters Preformed trifluoroethyl thioester -LYRANX-SCH conjugated to 1 using 2-methylimidazole (2-MIM) as an optional additive 2 CF 3 Comparative NCL reactions were performed in batches of (4: X = A (SEQ ID NO: 4), 5: X = V (SEQ ID NO: 5)).

[0137] The reaction rates of batch and flow ligation reactions between 1 and thioester 3 using TFET as an additive and between 1 and pre-activated trifluoroethyl thioesters 4 or 5 were compared (Figures 6 and 7). General batch and flow ligations were performed according to the above procedure with either 1.2 equivalents or 2.0 equivalents of thioesters 4 and 5, with or without the addition of 2.5 M 2-MIM. Rate acceleration was observed in experiments performed in flow vs. batch with the addition of 2-MIM as an additive for 2.0 equivalents or 1.2 equivalents. 2 CF 3 Rate acceleration was observed in experiments performed in flow vs. batch with the addition of 2-MIM as an additive for 2.0 equivalents or 1.2 equivalents.

[0138] The optimized flow procedure using the pre-activated thioester was demonstrated to be substantially faster (4 - 50 fold) than the corresponding batch reaction, and the rate difference was more pronounced at a larger scale (20 μmol) despite vigorous mixing (Figure 7A). The reaction endpoint of flow ligation did not change upon scale-up (see Figure 7B for the crude UPLC traces of ligation between 1 - 5 after quenching with 3 vol% hydrazine), and excellent isolation yields were obtained upon direct HPLC purification of the flow eluent (18 mg, 67% for 6 and 18 mg, 65% for 7).

[0139] Native chemical ligation in flow upon dilution Two 0.5 mL solutions were prepared: one was a solution containing H-CSPGYS-NH 2 1 (1 mM or 0.1 mM) and TCEP (50 mM) in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 6.0), and the other was a solution containing 2 equivalents of Ac-LYRANA-SCH 2 CF 3 4 (SEQ ID NO: 4) (2 mM or 0.2 mM) and TCEP (50 mM) in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 6.0). These solutions were inserted into the injection loop and combined at a T-piece using petroleum ether as the system solvent. The resulting reaction stream was introduced into a PTFE coil reactor I (0.5 mm × 1.6 mm) at 37 °C (6 bar BPR). The reaction solution was collected in a quenching solution of aqueous hydrazine monohydrate (7 vol.%) and analyzed by UPLC and UPLC-MS. The estimated conversion was based on the relative peak areas of the starting material and the desulfurized product at λ = 280 nm [ε280(1) = 1280 M -1 cm -1 , ε280(6) = 2560 M -1 cm -1 .

[0140] The kinetics of batch and flow ligation reactions without thiol or selenol additives between dilute solutions of model peptide 1 and model peptide thioester 2 are shown in Figure 8.

[0141] Standard desulfurization in flow To establish an optimized protocol for NCL in flow, the inventors investigated whether desulfurization could be carried out under a similar manifold. The general scheme for desulfurization of Cys-containing peptides 1, 6, or 7 is shown in Figure 9.

[0142] Cys-containing peptides 1, 6, or 7 were solvated in a degassed ligation buffer (5 mM) (pH 7.3 - 7.4) containing 6 M Gdn·HCl, 0.1 M HEPES, 50 mM TCEP, and 2.5 M 2-MIM. A second solution containing 40 mM VA-044 and 80 mM GSH was prepared in a degassed ligation buffer (pH 7.4 - 7.5) containing 6 M Gdn·HCl, 0.1 M HEPES, and 350 mM TCEP. These two solutions were inserted into an injection loop and combined at a T-piece on a 150 μL scale using petroleum ether as the system solvent (Figure 10). Next, the resulting reaction stream was placed into a PTFE coil reactor I heated to 37 °C. The desulfurization time course was obtained by varying the reactor volume and flow rate to adjust the residence time. The reaction solution exited the system through a 6 bar BPR and was quenched with an equal volume of a neutral 15 mM MPAA solution in milliQ water. A 5 μL aliquot of this solution was diluted with 40 μL of 0.1% TFA in milliQ water and analyzed by UPLC. The estimated conversion values were based on the relative peak areas of the starting material and desulfurization product at λ = 280 nm [ε280(1,8) = 1280 M -1 cm -1 、ε280(6,7,9,10)=2560M -1 cm -1 and the desulfurization product.

[0143] Standard desulfurization in batch Comparative desulfurization reactions of Cys-containing peptides 1, 6, or 7 were carried out in batch.

[0144] Cys-containing peptide 1, 6, or 7 was solvated in a degassed ligation buffer (5 mM) (pH 7.3 - 7.4) containing 6 M Gdn·HCl, 0.1 M HEPES, and 50 mM TCEP. A second solution containing 40 mM VA-044 and 80 mM reduced L-glutathione (GSH) in 6 M Gdn·HCl, 0.1 M HEPES, and 350 mM TCEP was prepared (pH 7.4 - 7.5). These solutions were combined in a polypropylene microcentrifuge tube at a 150 μL scale (1:1 vol / vol), gently stirred, and then incubated at 37 °C (Figure 9). The desulfurization time course was obtained by quenching 5 μL aliquots at various time points with 3 μL of neutral 25 mM MPAA MilliQ aqueous solution. This solution was diluted with 40 μL of 0.1% TFA in MilliQ water and analyzed by UPLC. The estimated conversion values were based on the relative peak areas of the starting material and desulfurized products at λ = 280 nm [ε280(1,8) = 1280 M -1 cm -1 , ε280(6,7,9,10) = 2560 M -1 cm -1 .

[0145] The results of desulfurization performed in batch and flow using the chemical radical initiator VA-044 (20 mM) and 40 mM GSH are shown in Figure 11.

[0146] Photochemical desulfurization in flow A protocol for desulfurization in the presence or absence of a chemical radical inhibitor was developed.

[0147] Cys-containing peptide 1, 6, or 7 was solvated in a degassed ligation buffer (5 mM) (pH 7.3 - 7.4) containing 6 M Gdn·HCl, 0.1 M HEPES, 50 mM TCEP, and 2.5 M 2-MIM. A second solution containing 80 mM GSH with or without 40 mM VA-044 was prepared in a degassed ligation buffer containing 6 M Gdn·HCl, 0.1 M HEPES, and 350 mM TCEP. These two solutions were inserted into an injection loop and combined at a T-piece on a 150 μL scale using petroleum ether as the system solvent (Figs. 12 - 17). The resulting reaction stream entered a PFA coil reactor in a Rayonet RPR-100 UV photoreactor at λ = 254 nm, 302 nm, or 365 nm (35 W, room temperature). The desulfurization time course was obtained by varying the reactor volume and flow rate to adjust the residence time. The reaction solution exited the system through a 6 bar BPR and was quenched with an equal volume of neutral 25 mM MPAA solution in Milli-Q water. A 5 μL aliquot of this solution was diluted with 40 μL of 0.1% TFA in Milli-Q water and analyzed by UPLC. The estimated conversion values were based on the relative peak areas of the starting material and the desulfurized product at λ = 280 nm [ε280(1,8) = 1280 M -1 cm -1 , ε280(6,7,9,10) = 2560 M -1 cm -1 .

[0148] Photochemical desulfurization in batch Comparative photodesulfurization reactions of Cys-containing peptide 1, 6, or 7 in the batch were performed.

[0149] Peptide 1, 6, or 7 containing Cys was solvated in degassed ligation buffer (5 mM) (pH 7.3 - 7.4) containing 6 M Gdn·HCl, 0.1 M HEPES, 50 mM TCEP, and 2.5 M 2-MIM. A second solution containing 80 mM GSH was prepared in degassed ligation buffer (pH 7.4 - 7.5) containing 6 M Gdn·HCl, 0.1 M HEPES, and 350 mM TCEP, with (Figure 12) or without (Figure 13) 40 mM VA-044. These solutions were combined in 150 μL scale (1:1 vol / vol) in polypropylene microcentrifuge tubes, gently stirred, and irradiated with light at λ = 254 nm, 302 nm, or 365 nm (35 W, room temperature) using a Rayonet RPR-100 UV photoreactor. The desulfurization time course was obtained at various time points by quenching 5 μL aliquots with 3 μL of a neutral 25 mM MPAA solution in MilliQ water. This solution was diluted with 40 μL of 0.1% TFA in MilliQ water and analyzed by UPLC. The estimated conversion values were based on the relative peak areas of the starting material and desulfurized products at λ = 280 nm [ε280(1,8) = 1280 M -1 cm -1 , ε280(6,7,9,10) = 2560 M -1 cm -1 and the desulfurized product.

[0150] In the absence of the chemical radical initiator VA-044, clean conversion of 1 to desulfurized product 8 was observed only upon irradiation with light at λ = 254 nm (35 W) for 15 s at room temperature (Figures 17A and B). Products 6 and 7 (having internal Cys residues) from the initial flow NCL reaction were also subjected to these photodesulfurization conditions and cleanly produced 9 and 10 in 60 - 90 s (Figure 17C). The reaction was also chemoselective in the presence of potentially reactive side chains (e.g., methionine and side-chain protected Cys residues) (Figures 18 - 20). Finally, peptides having penicillamine and β-thiol-aspartic acid residues were cleanly photodesulfurized in <1 min to give natural valine and aspartic acid residues, respectively (Figures 21 and 22). Irradiation under flow minimized product decomposition compared to batch reactions.

[0151] The inventors have developed a novel method for desulfurizing amide-containing compounds containing a thiol group. This method involves exposing the compound to ultraviolet irradiation in the presence of a phosphine source and optionally further containing a hydrogen atom source. This method is suitable for batch and flow. Surprisingly, a chemical radical initiator is not required.

[0152] Ligation-photochemical desulfurization in flow After optimizing the ligation and photodesulfurization reactions in flow, these were combined to perform an “in-line” ligation-photodesulfurization 20 μmol flow experiment.

[0153] A 20 μmol flow experiment was performed, whereby a solution of 1 (in ligation buffer containing 10 mM, 5 M 2-MIM and 50 mM TCEP) was mixed with thioester 4 or 5 (in ligation buffer containing 20 mM, 50 mM TCEP, Figure 23) at a T-piece. After passing a PTFE coil reactor at 37 °C (4:3 minutes, 5:11 minutes), the product was mixed at a second T-piece with a stream of 250 mM GSH and 350 mM TCEP in ligation buffer and directed towards a second PTFE reactor at 37 °C (4:1 minutes, 5:6 minutes), where the product thioester generated by trans-thioesterification between the internal Cys of the ligation product 6 or 7 and the excess thioester 4 or 5 was thiolysed (Figures 24 - 28). Finally, the reaction mixture was irradiated with light for 1 minute (λ = 254 nm) to promote photodesulfurization prior to recovery (see Figures 23B and 23C and Figures 29 - 30). After HPLC purification, the desired products were isolated in excellent yields over two steps (9: 17 mg, 65%, 10: 17 mg, 63%). Peptides were also generated on a rapid time scale; the total reaction time for 9 was only 5 minutes and for 10 was only 18 minutes, the latter representing one of the most difficult ligation bonds (Val thioester).

[0154] One-pot ligation-photochemical desulfurization in batch A one-pot ligation-photodesulfurization procedure was performed in batch on a 20 μmol scale.

[0155] Two 2.0 mL solutions were prepared, one containing 10 mM H-CSPGYS-NH in degassed ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5), 2 50 mM TCEP and 5.0 M 2-MIM, and the other containing 20 mM Ac-LYRANX-SCH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.1 - 7.2) 2 CF 3 (4: X = A (SEQ ID NO: 4), 5: X = V (SEQ ID NO: 5)) and 50 mM TCEP. A desulfurization solution containing GSH (250 mM) and TCEP (350 mM) was prepared in 5.0 ml of ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5). During degassing, the two ligation solutions were combined and mixed vigorously at 37 °C. At complete ligation as determined by UPLC (X = A: 6 min, X = V: 40 min), the reaction solution was transferred to a 15 mL Corning (TM) Falcon (TM) polypropylene conical centrifuge tube and combined with 4.0 mL of the desulfurization solution. This mixture was irradiated with light at λ = 254 nm (35 W, room temperature) (Figure 31). Upon completion as determined by UPLC (X = A: 2.5 h; X = V: 2.5 h), the product was immediately isolated using RP-HPLC (X = A: 9, total processing time 2.6 h, isolation yield 66%; X = V: 10, total processing time 3.2 h, isolation yield 66%. The processing time refers to the total time taken for complete injection and reaction).

[0156] In particular, when pre-activated thioesters (4: 2.6 h, 5: 3.2 h, Figures 31 and 32) were used, a single-digit increase in reaction time was observed in batch compared to flow. Furthermore, the flow NCL-photodesulfurization process was two orders of magnitude faster than the conventional ligation-desulfurization reaction in batch (using thioesters 2 or 3 and TFET as additives) but gave comparable isolation yields (Figures 33 - 35).

[0157] Synthesis of enfuvirtide Enfuvirtide is a clinically approved 36-residue peptide HIV entry inhibitor. It is commercially manufactured by condensation of three protected fragments in an organic solvent. Enfuvirtide was prepared by NCL-photodesulfurization in flow by reacting peptide 11 (enfuvirtide 1-18, SEQ ID NO: 11) having a C-terminal lysine derivatized as a trifluoroethyl thioester with peptide 12 (enfuvirtide 19-36, SEQ ID NO: 12) having an N-terminal β-thiol asparagine residue (Figure 36). Both peptides were made using Fmoc-SPPS.

[0158] [Chemical formula]

[0159] Hydrazine-derived 2-chlorotrityl chloride resin (copoly(styrene-1% DVB) 100-200 mesh) was loaded with Fmoc-Lys(Boc)-OH (125 μmol, 1.6 mmol / g), and the peptide was elongated using automated Fmoc-SPPS as described in the general procedure. The fully assembled resin-bound peptide was cleaved from the resin via treatment with TFA / iPr 3 SiH / H 2 O (90:5:5 v / v / v) and simultaneously all side-chain protecting groups were deprotected. The reaction was stirred at room temperature for 2 h and concentrated in vacuo. The fully deprotected peptide acyl hydrazide was solvated to 10 mM in aqueous ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 3.0) and cooled to -15 °C. To this, NaNO 2An aqueous solution (1 M, 10 equivalents, 1.25 mL) was added. After 10 minutes, TFET (20 equivalents, 221 μL) was added (Warning: TFET is irritating and acutely toxic and should be used in a fume cupboard). The solution was warmed to room temperature and stirred for 45 minutes. Complete conversion to the thioester was confirmed by UPLC-MS and the crude material was purified by HPLC (20 - 60% B, 0.1% TFA over 40 minutes) to give peptide 11 as a white solid after lyophilization (70 mg, 22% yield based on the initial resin load). UPLC: Rt 4.52 minutes (λ = 230 nm, 0 - 60% B, 0.1% TFA over 5 minutes); calculated mass values [M+2H]2+: 1144.5, [2M+3H]3+: 1525.7, [3M+4H]4+: 1716.0, [4M+5H]5+: 1830.6. Observed mass values (ESI+): 1144.7 [M+2H]2+, 1526.0 [2M+3H]3+, 1716.7 [3M+4H]4+, 1831.0 [4M+5H]5+ (ESI-MS data was collected over the entire gradient and wash cycle of the UPLC-MS). 19F{1H}-NMR (376 MHz, d6-DMSO) δ -65.1 ppm. The NMR spectra of enfuvirtide (1 - 18) 11 are shown in Figures 59 - 61.

[0160]

Chemical formula

[0161] Fmoc-Phe-OH was loaded onto Rink amide resin (copoly(styrene - 1% DVB) 100 - 200 mesh) (100 μmol) and the peptide was elongated using automated Fmoc-SPPS as outlined in the general procedure. A pre-activated solution of β-thiolasparagine S11 (1.20 equivalents), PyAOP (1.20 equivalents), and NMM (2.40 equivalents) in DMF (final concentration 0.1 M) was added to the resin. After gentle stirring at room temperature for 2 hours, the resin was washed with DMF (5 × 3 mL), CH 2 Cl 2 (5 × 3 mL) and DMF (5 × 3 mL). The fully protected resin-bound peptide was cleaved from the solid support with TFA / iPr 3SiH / H 2 Deprotection was carried out using a solution of O / EDT (89:5:5:1 v / v / v). The reaction mixture was stirred at room temperature for 2 h and concentrated in vacuo. The crude peptide 12 was precipitated from cold Et 2 O and the crude product was purified by preparative reverse phase (20 - 80% B over 40 min, 0.1% TFA) to give peptide 12 as a white solid after lyophilization (51.6 mg, 20% yield based on the initial resin load). UPLC: Rt 4.43 min (λ = 214 nm, 0 - 60% B, 0.1% TFA over 5 min); calculated mass values [M+2H]2+: 1177.1, [2M+3H]3+: 1569.1. Observed mass (ESI+): 1177.2 [M+2H]2+, 1569.3 [2M+3H]3+ (ESI - MS data were collected over the entire gradient and wash cycle of the UPLC - MS). The NMR spectrum of enfuvirtide (19 - 36) 12 is shown in Figure 62.

[0162]

Chemical Structure

[0163] A solution of enfuvirtide (19 - 36) 12 (5 μmol, 12.9 mg, 10 mM) was prepared in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES) containing 50 mM TCEP and 5.0 M 2 - MIM (pH 7.0). A second solution of enfuvirtide (1 - 18) 11 (10 μmol, 25.1 mg, 20 mM) was prepared in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES) containing 50 mM TCEP (pH 6.2). A desulfurization solution was prepared in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES) (pH 7.5) containing 800 mM TCEP and 250 mM GSH. The ligation solution was inserted into a 0.50 mL injection loop and the desulfurization solution was inserted into a 2.0 mL injection loop (Figure 36). The two peptide solutions were first combined at a 5 μmol scale with a T - piece using petroleum ether as the system solvent (0.20 mL / min). The resulting reaction stream was placed into a PTFE coil reactor (0.5 mm × 1.6 mm, 12.0 mL) at 37 °C. The reaction solution was then combined with the desulfurization solution (0.40 mL / min) with a second T - piece and placed into a second PTFE coil reactor (0.5 mm × 1.6 mm, 1.0 mL) at 37 °C, followed by placement into a final PFA coil reactor (0.5 mm × 1.6 mm, 2.5 mL) within a Rayonet RPR - 100 photoreactor (λ = 254 nm, 35 W, room temperature). The reaction mixture was recovered through a 6 bar BPR (total processing time 38 min) and immediately purified using RP - HPLC to obtain a fluffy white solid. (9.7 mg, 40%, 0.8 μmol / min). UPLC: Rt 5.75 min (λ = 214 nm, 0 - 70% B over 5 min, 0.1% TFA); calculated mass values [M + 3H]3+: 1498.1, [M + 4H]4+: 1123.8; measured mass values (ESI+); 1498.1 [M + 3H]3+, 1124.0 [M + 4H]4+. (ESI - MS data were collected over the entire gradient and wash cycle of the UPLC - MS.) (Figure 37). The NMR spectra of enfuvirtide 13 are shown in Figures 63 and 64.

[0164] This result clearly demonstrates the utility of the flow methodology in complex target assembly, as well as the compatibility with thiolated amino acids in addition to Cys.

[0165] Synthesis of somatorelin Somatorelin is a 44-residue peptide used as a diagnostic agent for determining growth hormone deficiency. Somatorelin was prepared by flow NCL photodesulfurization (Figure 38) by reacting a peptide 14 having a C-terminal thioester (Somatorelin 1-18, SEQ ID NO: 14) with a peptide 15 containing an N-terminal Cys residue (Somatorelin 19-44, SEQ ID NO: 15). Both peptides were prepared using Fmoc-SPPS.

[0166]

Chemical formula

[0167] Fmoc-Ser(OtBu)-OH (250 μmol, 1.1 mmol / g) was loaded onto hydrazine-derived 2-chlorotrityl chloride resin (copoly(styrene-1%DVB) 100-200 mesh), and the peptide was extended using microwave-assisted automated Fmoc-SPPS as described in the general procedure. Double coupling was performed at positions 1-8, 11-13, and 15-16 of the N-terminal segment, and N-acetylglycine capping was performed starting from position 13 (valine). After synthesis, the resin was washed with DMF (5 × 10 mL) and CH 2 Cl 2 (5 × 10 mL) and then dried in vacuo. The full-length resin-bound peptide was then cleaved from the resin by treatment with TFA / iPr 3 SiH / H 2 O (90:5:5 v / v / v) for 2 h, which resulted in the simultaneous deprotection of all side-chain protecting groups. The cleavage cocktail was concentrated in vacuo, and the crude peptide was then precipitated from cold Et 2 O and dried in vacuo. The fully deprotected peptide acyl hydrazide (225 mg) was solvated to 10 mM in aqueous ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 3.0) and cooled to -15 °C. To this, NaNO 2An aqueous solution (1 M, 10 eq, 1.125 mL) was added. After 10 minutes, TFET (20 eq, 186 μL) was added, the solution was warmed to room temperature and stirred for 45 minutes (Warning: TFET is irritating and acutely toxic and should be used in a fume cupboard). Complete conversion to the thioester was confirmed by UPLC-MS and the product was purified by preparative reverse-phase HPLC (20 - 35% B over 40 minutes, 0.1% TFA) to give peptide 14 as a fluffy white solid after lyophilization. (3 × 250 μmol synthesis; 272.0 mg, 14.6% yield based on initial resin load). UPLC: Rt 4.25 minutes (λ = 214 nm, 0 - 50% B over 5 minutes, 0.1% TFA); calculated mass values [M+3H]3+: 715.7, [M+2H]2+: 1073.0, [2M+3H]3+: 1430.4. Observed mass values (ESI+): 715.8 [M+3H]3+, 1073.3 [M+2H]2+, 1430.7 [2M+3H]3+ (ESI-MS data was collected over the entire gradient and wash cycle of the UPLC-MS). 19F{1H} NMR (471 MHz, D2O) δ - 66.4 ppm. The NMR spectra of somatrelin (1 - 18) 14 are shown in Figures 65 and 66.

[0168]

Chem.

[0169] Fmoc-Leu-OH was loaded onto rink amide resin (copoly(styrene - 1%DVB) 100 - 200 mesh) (250 μmol) and the peptide was elongated using microwave-assisted automated Fmoc-SPPS as outlined in the general procedure. All residues in the sequence were double-coupled except for the 32-position Fmoc-(DMB)Gly-OH installed by single coupling. After incorporation of this residue, capping was performed using N-acetylglycine. After synthesis, the resin was washed thoroughly with DMF (5 × 10 mL) and then CH 2 Cl 2 (5 × 10 mL) and then dried in vacuo, the fully protected resin-bound peptide was cleaved from the solid support with TFA / iPr 3SiH / H 2 Deprotection was carried out using a solution of O / thioanisole / 2,2'-(ethylenedioxy)diethanol (90:2.5:2.5:2.5:2.5:2.5:2.5 / v / v / v / v / v / v / v). The reaction mixture was stirred at room temperature for 2 h and concentrated in vacuo. The crude peptide 15 was precipitated from cold Et 2 O and the product was purified by preparative reverse-phase HPLC (5 - 30% B over 40 min, 0.1% TFA) to give peptide 15 as a white solid after lyophilization (234.0 mg, 24% yield based on the initial resin loading). UPLC: Rt 3.35 min (λ = 214 nm, 0 - 50% B over 5 min, 0.1% TFA); calculated mass values [M+6H]6+: 508.1, [M+5H]5+: 609.5, [M+4H]4+: 761.7, [M+3H]3+: 1015.2, [M+2H]2+: 1522.3. Observed mass values (ESI+): 508.2 [M+6H]6+, 609.7 [M+5H]5+, 761.9 [M+4H]4+, 1015.5 [M+3H]3+, 1522.6 [M+2H]2+. (ESI-MS data were collected over the entire gradient and wash cycle of the UPLC-MS.). The NMR spectra of somatrelin (19 - 44) 15 are shown in Figures 67 and 68.

[0170]

Chemical Structure

[0171] A solution of somatrelin(19 - 44)15(30 μmol, 115.2 mg, 10 mM) was prepared in a ligation buffer(6 M Gdn·HCl, 0.1 M HEPES)(pH 7.0) containing 50 mM TCEP and 5.0 M 2 - MIM. A second solution of somatrelin(1 - 18) trifluoroethyl thioester 14(60 μmol, 149.1 mg, 20 mM) was prepared in a ligation buffer(6 M Gdn·HCl, 0.1 M HEPES)(pH 6.2) containing 50 mM TCEP. A desulfurization solution was prepared in a ligation buffer(6 M Gdn·HCl, 0.1 M HEPES)(pH 7.5) containing 800 mM TCEP and 250 mM GSH. The peptide solution was inserted into a 3.0 mL injection loop, and the desulfurization solution was inserted into a 7.0 mL injection loop(Scheme S24). The two peptide solutions were first combined at a 30 μmol scale with a T - piece using petroleum ether as the system solvent(0.25 mL / min). The resulting reaction stream was placed in a PTFE coil reactor(0.5 mm×1.6 mm, 5.0 mL) at 37 °C. The reaction solution was then combined with the desulfurization solution(0.10 mL / min) at a second T - piece and placed in a second PTFE coil reactor(0.5 mm×1.6 mm, 1.0 mL) at 37 °C, and then placed in a final PFA coil reactor(0.5 mm×1.6 mm, 2.5 mL) inside a Rayonet RPR - 100 photoreactor(λ = 254 nm, 35 W, room temperature). The reaction mixture was recovered through a 6 bar BPR(total processing time 27 min) and immediately purified using RP - HPLC to obtain a fluffy white solid.(102.9 mg, 57%, 1.4 μmol / min). UPLC: Rt 4.20 min(λ = 214 nm, 0 - 70%B over 5 min, 0.1% TFA); calculated mass values[M + 7H]7+: 720.8, [M + 6H]6+: 840.8, [M + 5H]5+: 1008.7, [M + 4H]4+: 1260.7, [M + 3H]3+: 1680.6. Measured mass value(ESI+): 720.9[M + 7H]7+, 840.9[M + 6H]6+, 1008.8[M + 5H]5+, 1260.9[M + 4H]4+, 1680.9[M + 3H]3+.(ESI - MS data were collected over the entire gradient and wash cycle of the UPLC - MS.)(Figure 39) The NMR spectra of somatrelin 16 are shown in Figures 69 and 70.

[0172] Model peptides and peptide thioesters

Chem.

[0173] Peptide 1 was synthesized (730 μmol) using the general procedure of Fmoc-strategy SPPS on rink amide resin. The peptide was cleaved from the resin and deprotected under acidic conditions according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 20% B, 0.1% TFA over 40 min), followed by lyophilization, gave Peptide 1 as a white solid (257 mg, 57% yield). UPLC: Rt 3.99 min (0 - 15% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H] + : 612.2. Measured mass value (ESI + ): 612.5 [M + H] + . (ESI-MS data was collected over the entire gradient and wash cycle of the UPLC-MS.) H-CSPGYS-NH 2 (1, SEQ ID NO: 1) of 1 1H-NMR spectrum (D 2 2O, 500 MHz) is shown in Figure 45.

[0174]

Chem.

[0175] Fully side-chain protected Peptide 2 was synthesized (730 μmol) using the general procedure of Fmoc-strategy SPPS on 2-CTC resin. The protected peptide was cleaved from the resin, thioesterified, and deprotected according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave Peptide 2 as a white solid (288 mg, 46% yield). UPLC: Rt 3.63 min (0 - 30% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H] + : 865.4, [M + 2H]2+: 433.2. Measured mass value (ESI +): 865.8 [M+H]+, 433.4 [M+2H] 2+ 。(ESI-MS data was collected over the entire gradient and wash cycle of UPLC-MS.) Ac-LYRANA-S(CH 2 ) 2 CO 2 Et (2, SEQ ID NO: 2)'s 1 H-NMR spectrum (D 2 O, 500 MHz) is shown in Figure 46.

[0176]

Chemical Structure

[0177] The fully side-chain protected peptide 3 was synthesized using the general procedure of Fmoc-strategy SPPS on 2-CTC resin (350 μmol). The protected peptide was cleaved from the resin, thioesterified, and deprotected according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 minutes), followed by lyophilization, gave peptide 3 as a white solid (128 mg, 41% yield). UPLC: Rt 3.89 minutes (0 - 30% B, 0.1% TFA, λ = 214 nm over 5 minutes); calculated mass value [M+H] + : 893.5, [M+2H] 2+ : 447.2. Measured mass value (ESI + ): 893.9 [M+H] + , 447.4 [M+2H] 2+ 。(ESI-MS data was collected over the entire gradient and wash cycle of UPLC-MS.) Ac-LYRANV-S(CH 2 ) 2 CO 2 Et (3, SEQ ID NO: 3)'s 1 H-NMR spectrum (D 2 O, 500 MHz) is shown in Figure 47.

[0178]

Chemical Structure

[0179] Peptide acyl hydrazide 4 was synthesized (480 μmol) using the general procedure of Fmoc-strategy SPPS on 2-CTC resin functionalized with hydrazine. Cleavage from the resin and overall deprotection were carried out under acidic conditions according to the general procedure, followed by diazotization and thioesterification of the crude product. Purification by preparative reverse-phase HPLC (0 - 40% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide 4 as a white solid (351 mg, 86% yield). UPLC: Rt 3.73 min (0 - 30% B over 5 min, 0.1% TFA, λ = 214 nm); calculated mass values [M + H] + : 847.4, [M + 2H] 2+ : 424.2. Measured mass (ESI + ): 847.7 [M + H]+, 424.4 [M + 2H] 2+ (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.) 19 F{ 1 H}NMR (471 MHz, D 2 O) δ -66.5 ppm. The NMR spectra of Ac-LYRANA-SCH 2 CF 3 (4, SEQ ID NO: 4) are shown in FIGS. 48 - 50.

[0180]

Chemical Structure

[0181] Peptide acyl hydrazide 5 was synthesized (640 μmol) using the general procedure of Fmoc-strategy SPPS on 2-CTC resin functionalized with hydrazine. Cleavage from the resin and overall deprotection were carried out under acidic conditions according to the general procedure, followed by diazotization and thioesterification of the crude product. Purification by preparative reverse-phase HPLC (0 - 40% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide 5 as a white solid (431 mg, 77% yield). UPLC: Rt 4.05 min (0 - 30% B over 5 min, 0.1% TFA, λ = 214 nm); calculated mass values [M + H] + : 875.4, [M + 2H] 2+: 438.2. Measured mass value (ESI + ): 875.8 [M+H] + , 438.4 [M+2H] 2+ (ESI-MS data was collected over the entire gradient and wash cycle of UPLC-MS.). 19 F{ 1 H}NMR (471 MHz, D 2 O) δ -66.5 ppm. Ac-LYRANV-SCH 2 CF 3 (5, SEQ ID NO: 5) The NMR spectra are shown in FIGS. 51-53.

[0182]

Chemical Structure

[0183] Batch synthesis of 6: H-CSPGYS-NH 2 1 (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANA-SCH 2 CF 3 4 (33.9 mg, 40 μmol, 10 mM) was ligated in batch (2.5 M 2-MIM, 37 °C, t = 6.0 min) following a general procedure while vigorously stirring the reaction solution. Purification by preparative reverse-phase HPLC (0-40% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide 6 as a white solid (18.3 mg, 68% yield). UPLC: Rt 5.23 min (0-28% B over 5 min, 0.1% TFA, λ = 214 nm); calculated mass value [M+H] + : 1342.6, [M+2H] 2+ : 671.8. Measured mass value (ESI + ): 1343.6 [M+H] + , 672.4 [M+2H] 2+ . (ESI-MS data was collected over the entire gradient and wash cycle of UPLC-MS.).

[0184] Flow synthesis of 6: H-CSPGYS-NH 2 1(12.2 mg, 20 μmol, 5 mM) and Ac-LYRANA-SCH 2 CF 3 4 (33.9 mg, 40 μmol, 10 mM) and the ligation was carried out in a flow manner with 2.5 M 2-MIM according to a general procedure (pumps A, B: 0.50 mL / min, reactor I: 3.0 mL, T = 37 °C, τI (retention time) = 3.3 min). Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide 6 as a white solid (18.0 mg, yield 67%, 3.4 μmol / min). The analytical data was the same as that of 6 prepared above. Ac-LYRANACSPGYS-NH 2 (6, SEQ ID NO: 6) of 1 The 1H-NMR spectrum (D 2 2O, 500 MHz) is shown in Figure 54.

[0185]

Chemical formula

[0186] Batch synthesis of 7: H-CSPGYS-NH 2 1 (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANV-SCH 2 CF 3 5 (35.0 mg, 40 μmol, 10 mM) and the peptide ligation in batch (37 °C, 2.5 M 2-MIM, t = 40.0 min) was carried out according to a general procedure while vigorously stirring the reaction solution. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide 7 as a white solid (18.6 mg, yield 67%). UPLC: Rt 5.80 min (0 - 28% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H] + : 1370.7, [M + 2H] 2+ : 685.8. Measured mass value (ESI + ): 1372.1 [M + H] + , 686.3 [M + 2H] 2+(ESI-MS data was collected over the entire gradient and wash cycles of UPLC-MS.)

[0187] Flow synthesis of 7: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANV-SCH 2 CF 3 5 (SEQ ID NO: 5) (35.0 mg, 40 μmol, 10 mM) was ligated with it by flow in 2.5 M 2-MIM according to a general procedure (pumps A, B: 0.50 mL / min, reactor I: 10.0 mL, T = 37 °C, τI = 10.8 min). Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide 7 as a white solid (18.0 mg, yield 65%, 3.3 μmol / min). The analytical data was the same as that of 7 prepared above. Ac-LYRANVCSPGYS-NH 2 (7, SEQ ID NO: 7) 1 1H-NMR spectrum (D 2 2O, 500 MHz) is shown in Figure 55.

[0188]

Chemical Structure

[0189] H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (3.2 mg, 5.2 μmol, 2.5 mM) was subjected to photodesulfurization in batch according to the general procedure already outlined. (λ = 254 nm, 125 mM GSH, t = 1.0 min). Purification by preparative reverse-phase HPLC (0 - 20% B, 0.1% TFA over 30 min), followed by lyophilization, gave peptide 8 as a white solid (2.5 mg, yield 82%). UPLC: Rt 3.72 min (0 - 15% B, 0.1% TFA, λ = 214 nm over 5 min); calculated mass value [M + H] + : 580.3. Measured mass value (ESI + ): 580.4 [M + H] +. (ESI-MS data was collected over the entire gradient and wash cycles of UPLC-MS.) H-ASPGYS-NH 2 (8, SEQ ID NO: 8) of 1 The H-NMR spectrum (D 2 2O, 400 MHz) is shown in Fig. 56.

[0190] [Chemical formula]

[0191] Batch synthesis of 9, i: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANA-S(CH 2 ) 2 CO 2 Et 2 (SEQ ID NO: 2) (34.6 mg, 40 μmol, 10 mM) in a batch ligation (250 mM TFET, 25 min) (Warning: TFET is irritating and acutely toxic and should be used in a fume hood), followed by in situ desulfurization (20 mM VA-044, 40 mM GSH, 37 °C, t = 16 h) was carried out according to a general procedure with the reaction solution being vigorously stirred. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide 9 as a white solid (18.1 mg, 69% yield). UPLC: Rt 5.15 min (0 - 28% B, 0.1% TFA, λ = 214 nm over 5 min); calculated mass value [M + H] + : 1310.6, [M + 2H] 2+ : 655.8. Measured mass value (ESI): 1311.4 [M + H] + , 656.4 [M + 2H] 2+ . (ESI-MS data was collected over the entire gradient and wash cycles of UPLC-MS.).

[0192] Batch synthesis of 9, ii: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANA-SCH2 CF 3 4 (SEQ ID NO: 4) (33.9 mg, 40 μmol, 10 mM) was ligated in a batch with stirring (2.5 M 2-MIM, 37 °C, 6.0 min), followed by on-site photodesulfurization (125 mM GSH, λ = 254 nm, room temperature, t = 2.5 h) in a 15 mL Corning® Falcon® polypropylene conical centrifuge tube according to a general procedure. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min) was followed by lyophilization to obtain peptide 9 as a white solid (17.3 mg, 66% yield). The analytical data was the same as that of 9 prepared above.

[0193] Flow synthesis of 9: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANA-SCH 2 CF 3 4 (SEQ ID NO: 4) (33.9 mg, 40 μmol, 10 mM) was ligated in a flow (2.5 M 2-MIM, pumps A, B: 0.25 mL / min, reactor I: 1.5 mL, 37 °C), followed by a novel on-site photodesulfurization (125 mM GSH, pump C: 0.50 mL / min, reactor II: 1.0 mL, reactor III: 1.0 mL, λ = 254 nm, room temperature) according to a general procedure (τtotal = 5.0 min). Purification by preparative reverse-phase HPLC was performed (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization to obtain peptide 9 as a white solid (17.0 mg, 65% yield, 1.6 μmol / min). The analytical data was the same as that of 9 prepared above. Ac-LYRANAASPGYS-NH 2 (of 9, SEQ ID NO: 9) 1 1H-NMR spectrum (D 2 2O, 500 MHz) is shown in Figure 57.

[0194]

Chemical Structure

[0195] Batch synthesis of 10, i: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANV-S(CH 2 ) 2 CO 2 Et 3 (SEQ ID NO: 3) (35.7 mg, 40 μmol, 10 mM) in a batch ligation (250 mM TFET, t = 9 h) (Warning: TFET is irritating and acutely toxic and should be used in a fume hood), followed by in situ desulfurization (20 mM VA-044, 40 mM GSH, 37°C, t = 16 h) was carried out according to a general procedure with the reaction solution being vigorously stirred. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization gave peptide 10 as a white solid (16.4 mg, 61% yield). UPLC: Rt 5.47 min (0 - 28% B, 0.1% TFA, λ = 214 nm over 5 min); calculated mass values [M + H] + : 1338.7, [M + 2H] 2+ : 669.8. Measured mass value (ESI + ): 1339.6 [M + H] + , 670.3 [M + 2H] 2+ . (ESI-MS data were collected over the entire gradient and wash cycle of the UPLC-MS.)

[0196] Batch synthesis of 10, ii: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANV-SCH 2 CF 3 5(SEQ ID NO: 5) (35.0 mg, 40 μmol, 10 mM) was ligated in a batch with vigorous stirring (2.5 M 2-MIM, 37 °C, t = 40.0 min), followed by in situ photocleavage (125 mM GSH, λ = 254 nm, room temperature, t = 2.5 h) in a 15 mL Corning® Falcon® polypropylene conical centrifuge tube according to a general procedure. Purification by preparative reverse-phase HPLC (0 - 40% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide 10 as a white solid (17.7 mg, 66% yield). The analytical data was the same as that of 10 prepared above.

[0197] Flow synthesis of 10: H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (12.2 mg, 20 μmol, 5 mM) and Ac-LYRANV-SCH 2 CF 3 5 (SEQ ID NO: 5) (35.0 mg, 40 μmol, 10 mM) was ligated in a flow (2.5 M 2-MIM, pump A, B: 0.20 mL / min, reactor I: 4.0 mL, T = 37 °C), followed by a novel in situ photocleavage (125 mM GSH, pump C: 0.40 mL / min, reactor II: 5.0 mL, reactor III: 1.0 mL, λ = 254 nm, room temperature) according to a general procedure (τtotal = 17.5 min). Purification by preparative reverse-phase HPLC (0 - 40% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide 10 as a white solid (16.9 mg, 63% yield, 1.3 μmol / min). The analytical data was the same as that of 10 prepared above. Ac-LYRANVASPGYS-NH 2 (of 10, SEQ ID NO: 10) 1 1H-NMR spectrum (D 2 2O, 500 MHz) is shown in Figure 58.

[0198] Additional property evaluation

Chemical Structure

[0199] H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (4.1 mg, 6.70 μmol, 5 mM) 1 and Ac-LYRANA-SCH 2 CF 3 4 Peptide ligation in a batch with (SEQ ID NO: 4) (11.3 mg, 13.4 μmol, 10 mM) was carried out without hydrazinolysis of the product thioester as detailed in the general procedure (37 °C, t = 12.0 min). Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave the peptide S3 as a white solid (8.6 mg, 62% yield). UPLC: Rt 3.54 min (0 - 30% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass values [M + H] + : 2073.0, [M + 2H] 2+ : 1037.0, [M + 3H]3 + : 691.7. Measured mass value (ESI + ): 1037.9 [M + 2H] 2+ , 692.2 [M + 3H] 3+ . (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.)

[0200]

Chemical Structure

[0201] H-CSPGYS-NH 2 1 (SEQ ID NO: 1) (4.4 mg, 7.2 μmol, 5 mM) and Ac-LYRANV-SCH 2 CF 3 5Peptide ligation in a batch with (sequence number 5) (12.6 mg, 14.4 μmol, 10 mM) was carried out without hydrazinolysis of the product thioester as detailed in the general procedure (37 °C, t = 60.0 min). Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S4 as a white solid (8.9 mg, 58% yield). UPLC: Rt 3.62 min (0 - 30% B, 0.1% TFA, λ = 214 nm over 5 min); calculated mass values [M+2H] 2+ : 1065.0, [M+3H] 3+ : 710.4. Measured mass value (ESI + ): 1066.2 [M+2H] 2+ , 711.0 [M+3H] 3+ . (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.)

[0202]

Chemical formula

[0203] 1.5 equivalents of Boc-anhydride (439 mg, 2.01 mmol) was added to L-penicillamine (200 mg, 1.34 mmol) in 2 mL of THF and 2 mL of saturated sodium bicarbonate solution, and the reaction was carried out for 18 h (room temperature). The resulting solution was cooled to 0 °C and 0.5 equivalent of hydrogen peroxide (30 wt%, 68 μl, 0.67 mmol) was added dropwise with mixing, and the reaction was carried out for 5 min. Next, the solution was acidified with 1 M aqueous HCl, and the compound was extracted 3 times with an equal volume of ethyl acetate. The organic layer was dried over MgSO 4 and concentrated, and then purified by silica gel flash chromatography (0 - 10% methanol in CH 2 Cl 2 containing 1% acetic acid) to give compound S5 as an amorphous white solid (185 mg, 55%). [α] D +127.9 (c 1.0, CH 2 Cl 2 ); IR ν max2977, 2931, 1712, 1655, 1498, 1456, 1393, 1367, 1327, 1247, 1158, 1116, 1050, 1027; 1H-NMR (400 MHz, MeOD) δ 4.23 (s, 2H), 1.45 (s, 18H), 1.41 (s, 6H), 1.38 (s, 6H) ppm; 13 C NMR (101 MHz, MeOD) δ 173.5, 157.6, 80.9, 62.4, 52.0, 28.7, 26.8, 24.9 ppm; HRMS (ESI+) C 20 H 36 N 2 O 8 S 2 Calculated mass value of Na [M + Na] + : 519.1805; Measured mass value [M + Na] + : 519.1813.

[0204]

Chem.

[0205] Peptide S6 was synthesized using the general procedure of Fmoc-strategy SPPS on a rink amide resin (100 μmol). The peptide was cleaved from the resin and deprotected under acidic conditions according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 20% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S6 as a white solid (17.4 mg, 22% yield). UPLC: Rt 4.26 min (0 - 15% B, 0.1% TFA over 5 min, λ = 214 nm); Calculated mass value [M + H] + : 686.8, [2M + H] + : 1371.5. Measured mass value (ESI + ): 686.4 [M + H] + , 1372.0 [2M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.) H-CSPMYS-NH 2 (S6, SEQ ID NO: 18) The NMR spectra are shown in FIGS. 71 and 72.

[0206]

Chem.

[0207] H-CSPMYS-NH 2 S6 (SEQ ID NO: 18) (14.77, 18.4 μmol, 2.5 mM) was subjected to photodesulfurization in flow according to the general procedure already outlined (λ = 254 nm, 125 mM GSH, t = 1.0 min). Purification by preparative reverse-phase HPLC (2 - 20% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide S7 as a white solid (10.5 mg, 74% yield). UPLC: Rt 5.10 min (0 - 15% B over 5 min, 0.1% TFA, λ = 214 nm); calculated mass values [M + H] + : 654.3, [M + Na] + : 677.3, [2M + H] + : 1307.6. Observed mass (ESI + ): 654.4 [M + H] + , 676.4 [M + Na] + , 1308.3 [2M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of the UPLC-MS.) H-ASPMYS-NH 2 (S7, SEQ ID NO: 19) The NMR spectra are shown in FIGS. 73 and 74.

[0208]

Chemical Structure

[0209] Peptide S8 was synthesized using the general procedure of Fmoc-strategy SPPS on rink amide resin (100 μmol). The peptide was cleaved from the resin and deprotected under acidic conditions according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 20% B over 40 min, 0.1% TFA), followed by lyophilization, gave the peptide S8 as a white solid (39.8 mg, 47% yield). UPLC: Rt 4.89 min (0 - 15% B over 5 min, 0.1% TFA, λ = 214 nm); calculated mass values [M + H] +: 729.8, [2M + H] + : 1457.5. Measured mass value (ESI + ): 729.5 [M + H] + , 1457.9 [2M + H] + . (ESI - MS data was collected over the entire gradient and wash cycle of UPLC - MS.) H - CSPC(Acm)YS - NH 2 (S8, SEQ ID NO: 20)'s NMR spectra are shown in FIGS. 75 and 76.

[0210]

Chemical Structure

[0211] H - CSPC(Acm)YS - NH 2 S8 (SEQ ID NO: 20) (38.9 mg, 46.2 μmol, 2.5 mM) was subjected to photodesulfurization in flow according to the general procedure already outlined (λ = 254 nm, 125 mM GSH, t = 1.0 min). Purification by preparative reverse - phase HPLC (2 - 20% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S9 as a white solid (30.6 mg, 82% yield). UPLC: Rt 4.41 min (0 - 15% B, 0.1% TFA, λ = 214 nm over 5 min); calculated mass value [M - Acm + H] + : 626.3, [M + H] + : 697.3, [M + Na] + : 719.3, [2M + H] + : 1393.6. Measured mass value (ESI + ): 626.4 [M - Acm + H] + , 697.5 [M + H] + , 719.4 [M + Na] + , 1393.5 [2M + H] + . (ESI - MS data was collected over the entire gradient and wash cycle of UPLC - MS.) H - ASPC(Acm)YS - NH 2 (S9, SEQ ID NO: 21)'s NMR spectra are shown in FIGS. 77 and 78.

[0212] [Chemistry]

[0213] Peptide S10 was synthesized (100 μmol) using the general procedure of Fmoc-strategy SPPS on rink amide resin. The final coupling was carried out at room temperature for 18 h using 1.1 equivalents of N,N'-bis(tert-butyloxycarbonyl)-L-penicillamine dimer S5 (54.6 mg, 110 μmol) with 1.1 equivalents of HOAt (15.0 mg, 110 μmol) and 1.1 equivalents of DIC (17.0 μL, 110 μmol). The peptide was cleaved from the resin and deprotected under acidic conditions according to the general procedure. The crude product (6 mM) was dissolved in aqueous TCEP (300 mM, 8 mL), adjusted to pH 12, and subsequently incubated at room temperature. Purification by preparative reverse-phase HPLC (0 - 20% B over 40 min, 0.1% TFA), followed by lyophilization, gave peptide S10 as a white solid (18.1 mg, 28% yield). UPLC: Rt 4.40 min (0 - 15% B over 5 min, 0.1% TFA, λ = 214 nm); calculated mass values [M + H] + : 640.7, [2M + H] + : 1279.5. Measured mass value (ESI + ): 640.3 [M + H] + , 1279.6 [2M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of the UPLC-MS.) The NMR spectra of H-PenSPGYS-NH 2 (S10, SEQ ID NO: 22) are shown in FIGS. 79 and 80.

[0214] [Chemistry]

[0215] H-PenSPGYS-NH 2 S10Photodesulfurization of (SEQ ID NO: 22) (16.9 mg, 22.4 μmol, 2.5 mM) was carried out in flow following the general procedure already outlined (λ = 254 nm, 125 mM GSH, t = 1.0 min). Purification by preparative reverse-phase HPLC (2 - 20% B, 0.1% TFA over 40 min, followed by lyophilization) gave peptide S11 as a white solid (13.5 mg, 83% yield). UPLC: Rt 4.24 min (0 - 15% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass values [M + H] + : 608.3, [M + Na] + : 630.3, [2M + H] + : 1215.6. Observed mass values (ESI + ): 608.3 [M + H] + , 630.4 [M + Na] + , 1216.2 [2M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of the UPLC-MS.) H-VSPGYS-NH 2 (S11, SEQ ID NO: 23) NMR spectra are shown in Figures 81 and 82.

[0216]

Chem.

[0217] Peptide S12 was synthesized (50 μmol) using the general procedure of Fmoc-strategy SPPS on rink amide resin. For the final coupling, 1.1 equivalents of N-(tert-butoxycarbonyl)-3-(2,4,6-trimethoxybenzylthiol)-L-aspartic acid (27.6 mg, 55 μmol) was coupled with 1.1 equivalents of HOAt (7.5 mg, 55 μmol) and 1.1 equivalents of DIC (8.6 μL, 55 μmol) at room temperature for 18 h. The peptide was cleaved from the resin and deprotected under acidic conditions according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 20% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S12 as a white solid (11.8 mg, 36% yield). UPLC: Rt 3.59 min (0 - 15% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H] + : 656.2, [2M + H] + : 1311.5. Measured mass value (ESI + ): 656.4 [M + H] + , 1311.4 [2M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.) H-(β-SH)DSPGYS-NH 2 (S12, SEQ ID NO: 24) The NMR spectra are shown in FIGS. 83 and 84.

[0218]

Chemical Structure

[0219] H-(β-SH)DSPGYS-NH 2 S12 (SEQ ID NO: 24) (11.1 mg, 14.4 μmol, 2.5 mM) Photodesulfurization was carried out in flow according to the general procedure already outlined (λ = 254 nm, 125 mM GSH, t = 0.5 min). Purification by preparative reverse-phase HPLC (2 - 20% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S13 as a white solid (5.7 mg, 50% yield). UPLC: Rt 3.65 min (0 - 15% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H]+ : 624.3, [M+Na] + : 646.2, [2M+H] + : 1247.5. Measured mass value (ESI+): 625.3 [M+H] + , 647.0 [M+Na] + , 1248.6 [2M+H] + . (ESI-MS data was collected over the entire gradient and wash cycle of UPLC-MS.) H-DSPGYS-NH 2 (S13, SEQ ID NO: 25) NMR spectra are shown in FIGS. 85 and 86.

[0220] Thiolysis of product thioesters It was observed that product thioester species were formed during ligation.

[0221] Kinetic Studies of Thiolysis and Hydrazine Decomposition in Batch A solution of product thioester S3 or S4 was prepared in a degassed ligation buffer (5 mM) (pH 7.3 - 7.4) containing 6 M Gdn·HCl, 0.1 M HEPES, 50 mM TCEP, and 2.5 M 2-MIM. A second solution containing a thiolysis reagent (250 mM GSH or 250 mM DTT) or 7 vol.% N2H4·H2O was prepared in a ligation buffer (pH 7.4 - 7.5) containing 6 M Gdn·HCl, 0.1 M HEPES, and 350 mM TCEP. Equal volumes of the two solutions were combined and incubated at 37°C. 5 μL aliquots were taken at multiple time points, diluted with 45 μL of 0.1% TFA in MilliQ water, and then immediately analyzed by UPLC. The conversion estimates were based on the relative peak areas of the starting material thioester and the resulting ligation peptide at λ = 280 nm [ε280(S2,S3) = 3840 M -1 cm -1 , λ 280 (6,7) = 2560 M -1 cm- 1 .

[0222] Kinetic Studies of Thiolysis in Flow An aqueous stream of model peptide thioester 4 or 5 (20 mM) in ligation buffer (pH 7.1 - 7.2) containing 6 M Gdn·HCl, 0.1 M HEPES and 50 mM TCEP was mixed at the T-piece at a 150 μL scale with an aqueous stream of model peptide 1 (10 mM) in ligation buffer (pH 7.4 - 7.5) containing 6 M Gdn·HCl, 0.1 M HEPES, 50 mM TCEP and 5.0 M 2-MIM using petroleum ether as the system solvent (individual pump rates; X = A: 0.25 mL / min, X = V: 0.20 mL / min) (see Figure 27 for the flow system used). This reaction stream was introduced into a PTFE coil reactor I (0.5 mm × 1.6 mm, X = A: 1.5 mL, X = : 4.0 mL) operated at 37 °C. A third solution of TCEP (350 mM) and GSH (250 mM) in ligation buffer (pH 7.4 - 7.5) containing 6 M Gdn·HCl and 0.1 M HEPES was combined with the ligation mixture at the second T-piece (individual pump rates; X = A: 0.50 mL / min, X = V: 0.40 mL / min). This reaction stream was introduced into a PTFE coil reactor II (0.5 mm × 1.6 mm, 37 °C) and then into a PFA reactor III (0.5 mm × 1.6 mm, 1.0 mL) and irradiated in a Rayonet RPR-100 photoreactor at λ = 254 nm (35 W, room temperature). The reaction solution was recovered through a 6 bar BPR into an equal volume of neutral MPAA (15 mM) in MilliQ water. 5 μL aliquots were taken at multiple time points and diluted with 45 μL of 0.1% TFA in MilliQ water. UPLC analysis was performed immediately. The thiolysis time course was obtained by varying the volume of reactor II. The estimated conversion was based on the relative peak areas of the starting material thioester (S2,S3) and the resulting ligation product (6,7) for the desulfurized peptide (9,10) at λ = 280 nm [ε280(S2,S3) = 3840 M -1 cm -1 、λ 280 (6,7) = 2560 M -1 cm- 1 and was based on the relative peak areas of the starting material thioester (S2,S3) and the resulting ligation product (6,7) for the desulfurized peptide (9,10).

[0223] Preparative-Scale Reactions in Model Systems The novel native chemical ligation procedure was carried out in batch and flow at a 20 μmol scale.

[0224] Native Chemical Ligation in Batch Two 2.0 mL solutions were prepared, one containing 10 mM H-CSPGYS-NH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5), 2 1 (SEQ ID NO:1), 50 mM TCEP and 5.0 M 2-MIM, and the other containing 20 mM Ac-LYRANX-SCH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.1 - 7.2) 2 CF 3 (4: X = A (SEQ ID NO:4), 5: X = V (SEQ ID NO:5)) and 50 mM TCEP. These reaction solutions in equivalent amounts were vigorously mixed in batch, and upon completion, quenched with hydrazine monohydrate (7 vol.%) in milliQ water and immediately purified using RP-HPLC (X = A: 6.6 min, isolated yield 68%; X = V: 7.40 min, isolated yield 67%).

[0225] Native Chemical Ligation in Flow Two 2.0 mL solutions were prepared, one containing 0 mM H-CSPGYS-NH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5), 2 1 (SEQ ID NO:1), 50 mM TCEP and 5.0 M 2-MIM, and the other containing 20 mM Ac-LYRANX-SCH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.1 - 7.2) 2 CF 3(4: X = A (array number 4), 5: X = V (array number 5)) and 50 mM TCEP are included. These solutions were inserted into an injection loop and joined with a T-piece at a 20 μL scale using petroleum ether as the system solvent (0.50 mL / min). The resulting reaction stream entered a PTFE coil reactor I (0.5 mm × 1.6 mm, X = A: 3.0 mL, X = V: 10.0 mL) at 37 °C (6 bar BPR). These reaction solutions were collected in a quenching solution of aqueous hydrazine monohydrate (7 vol.%) and immediately purified using RP-HPLC (X = A: 6 , total processing time 6 minutes, isolation yield 67%, productivity 3.4 μmol / min; X = V: 7 , total processing time 15 minutes, isolation yield 65%, productivity 3.3 μmol / min. The processing time refers to the total time taken for complete injection and reaction.)

[0226] Standard One-Pot Ligation-Photodesulfurization in Batch The standard one-pot ligation-photodesulfurization batch procedure developed by Thompson et al. was investigated here to provide a comparison with the flow method reported here. This standard procedure was carried out in batch at a 20 μmol scale at the junction of both alanine and valine. Two ligation solutions were prepared, one containing H-CSPGYS-NH 2 1 (array number 1) (10 mM) and TCEP (50 mM) in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5), and the other containing Ac-LYRANX-S(CH 2 ) 2 CO 2 Et (20 mM, X = A: 2 (array number 2), X = V: 3VA-044 (40 mM), GSH (80 mM), and TCEP (50 mM) containing desulfurization solution were also prepared in 4.0 mL of ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.5). All solutions were degassed for 10 minutes, and then the two ligation buffers were combined and TFET (250 mM) was added immediately (Warning: TFET is irritating and acutely toxic and should be used in a fume hood). This ligation mixture was vigorously mixed at 37 °C and the progress of the reaction was monitored by UPLC. At completion (X = A: 25 minutes, X = V: 9 hours), the desulfurization solution was added and the reaction mixture was vigorously mixed at 37 °C. At completion as determined by UPLC analysis (X = A: 16 hours, X = V: 16 hours), the pure desulfurized peptide was isolated using RP-HPLC. (X = A: 9 , total processing time 16.5 hours, isolation yield 69%; X = V: 10 , total processing time 25.0 hours, isolation yield 61%. The processing time refers to the total time taken for complete injection and reaction).

[0227] One-Pot Ligation-Photodesulfurization in Batch The one-pot ligation-photodesulfurization procedure was performed in batch and flow at a 20 μmol scale.

[0228] Two 2.0 mL solutions were prepared: one containing 10 mM H-CSPGYS-NH in degassed ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5) 2 1 (SEQ ID NO: 1), 50 mM TCEP, and 5.0 M 2-MIM, and the other containing 20 mM Ac-LYRANX-SCH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.1 - 7.2) 2 CF 3(4: X = A (Array No. 4), 5: X = V (Array No. 5)) and 50 mM TCEP are included. A desulfurization solution containing GSH (250 mM) and TCEP (350 mM) was prepared in 5.0 ml of ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5). During degassing, the two ligation solutions were combined and mixed vigorously at 37 °C. At the time of complete ligation determined by UPLC (X = A: 6 minutes, X = V: 40 minutes), the reaction solution was transferred to a 15 mL Corning (trademark) Falcon (trademark) polypropylene conical centrifuge tube and combined with 4.0 mL of the desulfurization solution. This mixture was irradiated with light at λ = 254 nm (35 W, room temperature). Upon completion as determined by UPLC (X = A: 2.5 hours; X = V: 2.5 hours), the product was immediately isolated using RP-HPLC (X = A: 9, total processing time 2.6 hours, isolation yield 66%; X = V: 10, total processing time 3.2 hours, isolation yield 66%. The processing time refers to the total time taken for complete injection and reaction).

[0229] Ligation-Photodesulfurization in Flow Two 2.0 mL solutions were prepared: one containing 10 mM H-CSPGYS-NH in degassed ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5) 2 1 (Array No. 1), 50 mM TCEP and 5.0 M 2-MIM, and the other containing 20 mM Ac-LYRANX-SCH in ligation buffer (6 M Gdn·HCl, 0.1 M HEPES pH 7.1 - 7.2) 2 CF 3(4:X = A (SEQ ID NO: 4), 5:X = V (SEQ ID NO: 5)) and contains 50 mM TCEP. A desulfurization solution containing GSH (250 mM) and TCEP (350 mM) was prepared in 5.0 mL of degassed ligation buffer (6 M Gdn·HCl, 0.1 M HEPES, pH 7.4 - 7.5). These solutions were loaded into the injection loop, and the two peptide solutions were combined at the T-piece on a 20 μL scale using petroleum ether as the system solvent (X = A: 0.25 mL / min, X = V: 0.20 mL / min). The resulting reaction stream entered a PTFE coil reactor I (0.5 mm × 1.6 mm, X = A: 1.5 mL, X = V: 4.0 mL) at 37°C. Subsequently, the reaction solution was combined with the desulfurization solution at the second T-piece (X = A: 0.5 mL / min; X = V: 0.40 mL / min), placed in a second PTFE coil reactor II (0.5 mm × 1.6 mm, X = A: 1.0 mL, X = V: 5.0 mL) at 37°C, and then placed in a PFA reactor III (0.5 mm × 1.6 mm, 1.0 mL) in a Rayonet RPR-100 photoreactor (λ = 254 nm, 35 W, room temperature). The reaction mixture was recovered through a 6 bar BPR and immediately purified using RP-HPLC (X = A: 9, total processing time 0.26 h, isolation yield 65%, productivity 1.6 μmol / min; X = V: 10, total processing time 0.46 h, isolation yield 63%, productivity 1.3 μmol / min. The processing time refers to the total time taken for complete injection and reaction).

[0230] Native Chemical Ligation in Flow Using Selenoesters Model peptide H-CSPGYS-NH 2 1 (SEQ ID NO: 1) and the model peptide Ac-LYRANV-SePh S15 A model NCL reaction in the flow between (SEQ ID NO: 27) was carried out (Figure 40A).

[0231] Model peptide H-CSPGYS-NH 2 1 (SEQ ID NO: 1) was solvated in a ligation buffer (10 mM, 1.0 equivalent) containing 6 M Gdn·HCl, 0.1 M HEPES, and 50 mM TCEP (pH 6.2 or 7.0). The model peptide Ac-LYRANV-SePhS15 (SEQ ID NO: 27)'s second solution was prepared in a ligation buffer (20 mM, 2.0 equivalents; or 15 mM, 1.5 equivalents) (pH 6.2 or 7.0) containing 6 M Gdn·HCl, 1 M HEPES, and 50 mM TCEP. Model peptide Ac-LYRANV-SePh S15 (SEQ ID NO: 27) was prepared according to the method described in WO2016 / 138563. These solutions were inserted into an injection loop and combined at a T-piece on a 150 μL scale using petroleum ether as the system solvent. The resulting reaction stream was introduced into a PTFE coil reactor (0.5 mm × 1.6 mm) at 37 °C (6 bar BPR). N in an equal volume of Milli-Q water 2 H 4 ·H 2 O at 7 Vol% was used to quench the reaction solution upon recovery. The ligation time course was obtained by varying the reactor volume and flow rate to adjust the residence time. A 10 μL aliquot of the quenched solution was taken, diluted with 40 μL of 0.1% TFA in Milli-Q water, and then analyzed by UPLC. The estimated conversion value was at λ = 280 nm [ε 280 (H-CSPGYS-NH 2 ) = 1280 M -1 cm - −1, ε 280 (Ac-LYRANVCSPGYS-NH 2 ) = 2560 M -1 cm - −1]. Based on the relative peak areas of the starting material H-CSPGYS-NH 2 1 (SEQ ID NO: 1) and the desired ligation product Ac-LYRANVCSPGYS-NH 2 (SEQ ID NO: 7).

[0232] Native Chemical Ligation in Batch Using Selenoesters The model peptide H-CSPGYS-NH 2 1 (SEQ ID NO: 1) was solvated in a ligation buffer (10 mM, 1.0 equivalent) containing 6 M Gdn·HCl, 0.1 M HEPES, and 50 mM TCEP (pH 6.2 or 7.0). Model peptide Ac-LYRANV-SePhS15 (SEQ ID NO: 27) The second solution was prepared in ligation buffer (pH 6.2 or 7.0) containing 6M Gdn·HCl, 0.1M HEPES and 50mM TCEP (20mM, 2.0 equivalents; or 15mM, 1.5 equivalents; or 12.5mM, 1.25 equivalents). The model peptide Ac-LYRANV-SePh (SEQ ID NO: 27) was prepared according to the method described in WO2016 / 138563. These solutions were combined and incubated at 37°C. The progress of the ligation time was determined by quenching 5 μL aliquots in MilliQ water containing an equal volume of 7 vol% N 2 H 4 ·H 2 O. This solution was diluted with 40 μL of 0.1% TFA in MilliQ water and then analyzed by UPLC. The estimated conversion value was λ = 280 nm [ε 280 (H-CSPGYS-NH 2 ) = 1280 M -1 cm - -1, ε 280 (Ac-LYRANVCSPGYS-NH 2 ) = 2560 M -1 cm - -1] for the starting material H-CSPGYS-NH 2 1 (SEQ ID NO: 1) and was based on the relative peak areas of the desired ligation product Ac-LYRANVCSPGYS-NH 2 (SEQ ID NO: 7).

[0233] The kinetics of the ligation reaction between model peptide 1 and model peptide S15 without thiol or selenol additives in batch and flow are shown in FIGS. 40B-E.

[0234] Photodearsenylation in Flow H-USPGYS-NH 2 Dimer S14(SEQ ID NO: 26) was solvated in degassed ligation buffer (2.5 mM) containing 6 M Gn HCl, 0.1 M HEPES (pH 6.2-6.3). Ac-LYRANF-SePh solvated in degassed ligation buffer (pH 6.2-6.3) containing 6 M Gn HCl, 0.1 M HEPES S16 A second solution was prepared containing (SEQ ID NO:28) (6 mM, 1.2 equivalents). These two solutions were mixed and incubated at 37° C. for 15 minutes, at which point UPLC-MS analysis (H 2 Dilute 1 / 10 in HO, gradient: HO over 5 min. 2 Ligation was shown to have proceeded to completion by elution with 0–40% MeCN in HO [0.1% FA]. An equal volume of petroleum ether was added to the ligation mixture, followed by decantation to remove precipitated DPDS. The extracted ligation mixture was then inserted into an injection loop, and a second injection loop was inserted with a solution of tris(2-carboxyethyl)phosphine (TCEP) (50 mM, 10 volumes) in 6 M Gn·HCl, 0.1 M HEPES (pH 7.0–7.2) buffer. These loops were then injected into a flow system at individual flow rates to give the desired residence time, using petroleum ether as the immiscible system solvent. The two solutions were mixed in a T-piece, and the resulting reaction flow was placed into a PFA coil reactor in a Rayonet RPR-100 UV photoreactor at λ = 254 nm (35 W, 37 °C) for a residence time of 2 min. The reaction solution was then vented from the system via a 6 bar back pressure regulator (BPR) and the reaction was determined to be complete by UPLC-MS analysis (H 2 (Diluted 1 / 10 in HO, Gradient: 0–40% MeCN in HO [0.1% FA] over 5 min).

[0235] Diselenide Selenoester Ligation-Photodearsenylation in Flow Two 0.5 mL solutions were prepared: one containing 5 mM dimer (H-USPGYS-NH) in degassed ligation buffer (6 M Gdn HCl, 0.2 M BIS-TRIS, pH 4.7); 2 ) 2 S14(SEQ ID NO: 26), and the other contains 12.5 mM Ac-LYRANV-SePh in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 4.7) S15 (SEQ ID NO: 27). A deselenization solution containing TCEP (10 mM or 100 mM) in 1.0 mL of degassed ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 5.5) was prepared. These solutions were inserted into the injection loop, and the two peptide solutions were combined at a T-piece using petroleum ether as the system solvent. The resulting reaction stream was introduced into a PTFE coil reactor I (0.5 m × 1.6 mm, 5 min) at 37 °C. Subsequently, the reaction solution was combined with the deselenization solution at a second T-piece and introduced into a second PTFE coil reactor II (0.5 mm × 1.6 mm) in a Rayonet RPR-100 photoreactor (λ = 254 nm, 35 W, room temperature) at 37 °C. The reaction mixture was collected through a 6 bar BPR and immediately analyzed using UPLC. The estimated conversion values were based on the relative peak areas of the ligation product (Ac-LYRANVUSPGYS-NH 280 ((Ac-LYRANVUSPGYS-NH 2 )) 2 ) = 5120 M -1 m - 1, ε 280 (Ac-LYRANVASPGYS-NH 2 ) = 2560 M -1 cm - 1] at λ = 280 nm, and the desired deselenization product Ac-LYRANVASPGYS-NH 2 ) 2 (SEQ ID NO: 29) and Ac-LYRANVASPGYS-NH 2 (SEQ ID NO: 10).

[0236] Reductive Diselenide-Selenoester Ligation in Flow Two 0.5 mL solutions were prepared: one containing (H-USPGYS-NH 2 ) 2 S14(SEQ ID NO: 26) (5 mM, 0.5 mM, 0.05 mM; for dimer), containing TCEP (50 mM) and DPDS (10 mM), and the other was 1.25 equivalents in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 6.0). Ac-LYRANV-SePh S15 (SEQ ID NO: 27) (12.5 mM, 1.25 mM, 0.125 mM), containing TCEP (50 mM) and DPDS (10 mM). These solutions were loaded into the injection loop, and the two peptide solutions were combined at the T-piece using petroleum ether as the system solvent. The resulting reaction stream was introduced into a PTFE coil reactor I (0.5 mm × 1.6 mm) at 37 °C. The reaction mixture was recovered through a 6 bar BPR and immediately analyzed using UPLC. The estimated conversion value was at λ = 280 nm [ε 280 (S14) = 2560 M -1 cm - 1, ε 280 ((Ac-LYRANVUSPGYS-NH 2 ) 2 ) = 5120 M -1 cm - 1] for the starting material (H-USPGYS-NH 2 ) 2 S14 and the desired ligation product (Ac-LYRANVUSPGYS-NH 2 ) 2 (SEQ ID NO: 29) based on the relative peak areas.

[0237] Solution-Phase Selenoesterification The crude side-chain protected peptide was dissolved in dry DMF (20 mM) and cooled to 0 °C. To this, DPDS (30 equivalents) was added followed by Bu 3 P (30 equivalents) under an argon atmosphere with stirring for 3 h. The solution was warmed to room temperature and concentrated under a nitrogen stream. Deprotection and workup were achieved using the acidic deprotection conditions described for the fully protected peptide on resin. No epimerization was observed using this procedure.

[0238] Reductive Diselenide-Selenoester Ligation in Batch upon Dilution An optimized protocol for the reduction-type diselenide-selenoester ligation in a high-dilution flow was established, and the inventors investigated whether the reduction-type diselenide-selenoester ligation could be carried out in batch. Surprisingly, the inventors found that the reduction-type diselenide-selenoester ligation in batch at high dilution proceeds in the presence of additives.

[0239] Two 0.5 mL solutions were prepared: one containing (H-USPGYS-NH 2 ) 2 S14 (SEQ ID NO: 26) (5 mM, 0.1 mL; 0.5 mM, 0.1 mL; or 0.05 mM, 1.0 mL; relative to the dimer), TCEP (30 mM), and DPDS (20 mM) in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 5.2), and the other containing Ac-LYRANV-SePh S15 (SEQ ID NO: 27) (20 mM, 0.1 mL; 2 mM, 0.1 mL; or 0.2 mM, 1.0 mL), TCEP (30 mM), and DPDS (20 mM) in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 5.2). These solutions were combined and incubated at 37 °C. The time course of ligation of the reaction mixture containing the final concentration of 5 mM or 0.5 mM (H-USPGYS-NH 2 ) 2 (SEQ ID NO: 26) monomer was obtained by quenching 5 μL aliquots of the reaction with an equal volume of 7 vol% N 2 H 4 ·H 2 O in MilliQ water. After 10 minutes, these solutions were diluted with 20 μL of 1.5% TFA in MilliQ water and analyzed by UPLC. For the reaction mixture containing 0.05 mM (H-USPGYS-NH 2 ) 2 monomer, 100 μL aliquots were taken in 30 vol% N 2 H 4 ·H 2Quenched with 10 μL, then diluted with 11 μL of 30 vol% TFA in Milli-Q water after 10 minutes, and subsequently analyzed by analytical HPLC coupled with an FLR detector. The conversion estimate was based on the relative peak areas of the starting material (H-USPGYS-NH 2 ) 2 S14 (SEQ ID NO: 26) and the desired ligation product (Ac-LYRANVUSPGYS-NH 2 ) 2 (SEQ ID NO: 29) at λ = 280 nm.

[0240]

Chemical formula

[0241] Peptide S14 was synthesized using the general procedure of Fmoc-strategy SPPS on a rink amide resin. The peptide was cleaved from the resin and deprotected under acidic conditions according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 20% B, 0.1% TFA over 40 minutes), followed by lyophilization, gave peptide S14 as a white solid (78 mg, 61% yield). UPLC: Rt 3.79 minutes (0 - 40% B, 0.1% TFA, λ = 214 nm over 5 minutes); calculated mass values [M + H] + : 1317.4, [M + 2H] 2+ : 659.2. Measured mass value (ESI + ): 1317.3 [M + H] + , 659.3 [M + 2H] 2+ . (ESI-MS data was collected over the entire gradient and wash cycle of UPLC-MS.)

[0242]

Chemical formula

[0243] The fully side-chain protected peptide S15 was synthesized using the general procedure of Fmoc-strategy SPPS on 2-CTC resin. The protected peptide was cleaved from the resin and subjected to selenoesterification and deprotection according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S15 as a white solid (428 mg, yield 77%). UPLC: Rt 4.71 min (0 - 60% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H] + : 917.4. Observed mass value (ESI + ): 917.4 [M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.)

[0244]

Chemical Structure

[0245] The fully side-chain protected peptide S16 was synthesized using the general procedure of Fmoc-strategy SPPS on 2-CTC resin. The protected peptide was cleaved from the resin and subjected to selenoesterification and deprotection according to the general procedure. Purification by preparative reverse-phase HPLC (0 - 40% B, 0.1% TFA over 40 min), followed by lyophilization, gave peptide S16 as a white solid (13.2 mg, yield 37%). UPLC: Rt 4.52 min (0 - 60% B, 0.1% TFA over 5 min, λ = 214 nm); calculated mass value [M + H] + : 965.4. Observed mass value (ESI + ): 965.3 [M + H] + . (ESI-MS data were collected over the entire gradient and wash cycle of UPLC-MS.)

[0246] Those skilled in the art will understand that numerous variations and / or modifications can be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Therefore, this embodiment should be considered exemplary in all respects and not restrictive.

[0247] References 1. Hay, M.; Thomas, D. W.; Craighead, J. L.; Economides, C.; Rosenthanl J. Nat. Biotechnol. 2014, 32, 40. 2. Fosgerau, K; Hoffman, T. Drug Discov. Today 2015, 20, 122. 3. Usmani, S. S.; Bedi, G.; Samuel, J. S.; Singh, S.; Kalra, S.; Kumar P.; Ahuja, A. A.; Sharma, M.; Gautam, A.; Raghava, G. P. PloS One 2017, 12, e0181748. 4. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149. 5. Kent, S. B. Ann. Rev. Biochem. 1988, 57, 957. 6. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994 , 266, 776. 7. Kent, S. B. H. Chem Soc. Rev. 2009, 38, 338. 8. Johnson, E. C. B.; Kent, S. B. H. J. Am. Chem. Soc. 2006, 128, 6640. 9. Thompson, R. E.; Liu, X.; Alonso-Garcia, N.; Pereira, P. J. B.; Jolli ffe, K. A.; Payne, R. J. J. Am. Chem. Soc. 2014, 136, 8161. 10.Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526. 11.Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46, 9248. 12. Jin, K.; Li, T.; Chow, H. Y.; Liu, H.; Li, X. Angew Chem. Int. Ed. 2017, 56, 14607. 13.Dawson, P. E. Isr. J. Chem. 2011, 51, 862. 14.Kulkarni, S. S.; Sayers, J.; Premdjee, B.; Payne, R. J. Nat. Rev. Ch em. 2018, 2, 112. 15.Malins, L. R.; Payne, R. J. Aust. J. Chem. 2015, 68, 521. 16.Malins, L. R.; Payne, R. J. Curr. Opin. Chem. Biol. 2014, 22, 70. 17.Malins, L. R.; Payne, R. J. Top. Curr. Chem. 2015, 362, 27. 18.Bondalapiti, S; Jbara, M.; Brik, A. Nat. Chem. 2016, 8, 407. 19. Huang, Y. C.; Chen, C. C.; Gao, S; Wang, Y. H.; Xiao, H.; Wang, F.; Tian, C. L.; Li, Y. M. Chem. Eur. J. 2016, 22, 7623. 20. Sayers, J.; Thompson, R. E.; Perry, K. J.; Malins, L. R.; Payne, R. J. Org. Lett. 2015, 17, 4902. 21. Thompson, R. E.; Chan, B. Radom, L.; Jolliffe, K. A.; Payne, R. J. A ngew. Chem. Int. Ed. 2013, 52, 9723. 22. Zheng, J. S.; Tang, S.; Qi, Y.-K.; Wang, Z.-P.; Liu, L. Nat. Protoc. 2013, 8, 2483 - 2495. 23. Sakamoto, K.; Tsuda, S.; Mochizuki, M.; Nohara, Y.; Nishio, H.; Yosh iya, T. Chem. Eur. J. 2016, 22, 17940. 24. Hoffmann, F. W.; Ess, R. J.; Simmons, T. C.; Hanzel, R. S. J. Am. Ch em. Soc. 1956, 78, 6414.

Claims

**Claim 1** A method for desulfurizing an amide-containing compound containing a thiol group or a disulfide group and / or for de-selenizing an amide-containing compound containing a selenol group or a diselenide group in a flow, comprising: The method, wherein the desulfurization and / or de-selenization comprises irradiating the amide-containing compound with light in the presence of a phosphine source. **Claim 2** The method according to claim 1, wherein the light irradiation comprises exposing the amide-containing compound to ultraviolet irradiation in the presence of the phosphine source. **Claim 3** The method according to claim 1 or 2, wherein the phosphine source is water-soluble. **Claim 4** The phosphine source is selected from the group consisting of bis(p-sulfonatophenyl)phenylphosphine dipotassium salt dihydrate, bis(4,6-dimethyl-3-sulfonatophenyl)(2,4-dimethylphenyl)phosphine disodium salt hydrate, 1,2-bis(di-4-sulfonatophenylphosphino)benzene tetrasodium salt, bis(3-sulfonatophenyl)(3,5-di-trifluoromethylphenyl)phosphine disodium salt monohydrate, bis(p-sulfonatophenyl)phenylphosphine dipotassium salt dihydrate, bis(3-sulfonatophenyl)(2-trifluoromethylphenyl)phosphine disodium dihydrate, bis(3-sulfonatophenyl)(4-trifluoromethylphenyl)phosphine disodium dihydrate, di-t-butyl(3-sulfonatopropyl)phosphine, 2'-dicyclohexylphosphino-2,6-dimethoxy-3-sulfonato-1,1'-biphenyl hydrate sodium salt, 2'-dicyclohexylphosphino-2,6-di-i-propyl-4-sulfonato-1,1'-biphenyl hydrate sodium salt, diphenyl(m-sulfonatophenyl)phosphine dihydrate sodium salt, 2-(dicyclohexylphosphino)ethyl]trimethylammonium chloride, diphenyl(p-sulfonatophenyl)phosphine monohydrate dimethyl sulfoxide adduct, potassium salt, dicyclohexyl-{9-[3-(4-sulfonylphenyl)propyl]-2-sulfonylfluoren-9-yl}phosphonium hydrogen sulfate, tetrabutylphosphonium chloride, 1,3,5-triaza-7-phosphaadamantane, tris(2-carboxyethyl)phosphine (TCEP), tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate, tris(hydroxymethyl)phosphine, tris(3-hydroxypropyl)phosphine, tris(3-sulfonatophenyl)phosphine hydrate sodium salt, and combinations thereof, the method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein the phosphine source is tris(2-carboxyethyl)phosphine (TCEP).

6. The method according to any one of claims 1 to 5, further comprising a hydrogen atom source.

7. The method according to claim 6, wherein the hydrogen atom source is selected from the group consisting of reduced L-glutathione (GSH), dithiothreitol (DTT), tert-butylthiol, cysteine, and combinations thereof.

8. The method according to claim 7, wherein the hydrogen atom source is reduced L-glutathione (GSH).

9. The method according to any one of claims 1 to 8, wherein the method is carried out in the absence of a chemical radical initiator.

10. The method according to any one of claims 1 to 8, comprising contacting the amide-containing compound with a chemical radical initiator in a flow.

11. The method according to any one of claims 1 to 10, wherein the method is carried out in a solvent containing an aqueous solution and / or the method is carried out in an aqueous solution.

12. The method according to any one of claims 1 to 11, wherein the amide-containing compound is a polypeptide.

13. The method according to claim 12, wherein the polypeptide or a fragment thereof is recombinantly expressed.

14. The method according to claim 12 or 13, wherein the polypeptide comprises one or more post-translational modifications and / or the polypeptide comprises one or more amino acids selected from the group consisting of amino acid analogs, non-canonical amino acids, and non-natural amino acids.

15. The method according to any one of claims 1 to 14, wherein the amide-containing compound is a reaction product of a native chemical ligation reaction.

16. The method according to any one of claims 1 to 14, wherein the amide-containing compound is a reaction product of a native chemical ligation reaction in a flow.

17. The method according to claim 15 or 16, wherein the reaction product of the native chemical ligation reaction is subjected to the method without purification after the native chemical ligation reaction.

18. The method according to claim 15 or 16, wherein the reaction product of the native chemical ligation reaction is subjected to the method with only partial purification after the native chemical ligation reaction. The method according to claim 1, wherein the amide-containing compound is selected from cysteine-containing peptides and selenocysteine-containing peptides, the light irradiation is ultraviolet irradiation, the phosphine source is tris(2-carboxyethyl)phosphine (TCEP), and further includes a hydrogen atom source which is reduced L-glutathione (GSH).

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