Cyclised peptides

Abstract

The invention relates to a method for preparing a cyclised peptide from a modified peptide comprising an acyl azide group; the use of the method to prepare a library of two or more cyclised peptides; and to a library of cyclised peptides prepared using the method.

Description

Cyclised Peptides

[0001] This invention relates to a method for preparing a cyclised peptide from a modified peptide comprising an acyl azide group. This invention also relates to a method of generating a library of cyclised peptides.BACKGROUND

[0002] Natural product cyclic peptides are a vital source of therapeutics, including clinically used antibiotics and anticancer agents (RSC Chem. Biol., 2022, 3, 18, 2022). Cyclic peptides have restricted conformations, giving them a defined surface to interact with a target and are more resistant to proteases. Thus, substantial research efforts have been made towards their synthesis using chemical and biochemical methodologies.

[0003] Naturally occurring cyclic peptides are biosynthesized via two different routes: ribosomal and non-ribosomal. Ribosomal peptides are genetically encoded and post translationally modified, and their cyclization is catalyzed by pathway specific cyclases (J. Am. Chem. Soc. 141, 4167-4181, 2019). On the other hand, non-ribosomal peptide biosynthesis employs modular mega enzymes known as non-ribosomal peptide synthetases (NRPS), which act as an assembly line in which each module is responsible for sequentially adding a new amino acid to the covalently bound linear peptide. (Chem. Rev. 106, 3468- 3496, 2006; Science 303, 1805-1810, 2004).

[0004] While the chemical synthesis of linear peptides is often straight forward, regio- and chemoselective peptide macrocyclization remains a significant synthetic challenge. Several thioesterase (TE) domains and ribosomal peptide cyclases have been characterized in vitro and have potential as peptide cyclization biocatalysts, including for the production of natural product peptide derivatives (Nature, 407, 215-218, 2000; Nat. Catal. 3, 507-515, 2020; Nat. Chem. Biol. 10, 732-738, 2014; Angew. Chem. Int. Ed. 53, 14171-14174, 2014; J. Am.Chem. Soc. 137, 3494-3497, 2015). However, these enzymes are generally selective for one natural product or allow only limited structural diversity.

[0005] On the other hand, several synthetic methods have been developed to cyclize peptides via amide bond formation. These include the use of standard coupling reagents which have been widely applied to the total synthesis of several natural products, e.g. rufomycin (Org. Lett. 20, 6166-6169, 2018; Mar. Drugs 20, 632, 2022), cyclosporine (J. Am. Chem. Soc. 132, 4098-4100, 2010), cyclomarine (Org. Lett. 18, 204-207, 2016) and surugamide B (Angew. Chem. Int. Ed. 57, 9447-9451 , 2018). However, these methods require extensive protecting group chemistry, long reaction times, and often result in lowoverall yield. This is also the case with other cyclisation methods, including silver assisted cyclization (in which the N terminus and a thioester C-terminus are chelated to promote reaction) and metal templating of cyclization involving chelation of backbone residues.

[0006] Besides direct aminolysis, alternative chemo-selective ligations have been developed - most notably native chemical ligation (NCL), which also employs a C terminal thioester. Cyclization occurs via trans-thioesterification and S- to N- acyl shift (ChemBioChem 13, 542-546, 2012), however it requires an N-terminal cysteine in the sequence. While a few traceless approaches have been reported, they have yet to find general use for natural product peptide synthesis.

[0007] The solution phase methods of cyclised peptide synthesis generally do not easily lend themselves to production of derivative libraries, while synthetic handles for semisynthesis are limited. These are problems hindering the development of many non- ribosomal peptides as therapeutics.

[0008] It is an aim of the present invention to provide a method of synthesising cyclised peptides that is rapid, operationally simple and high yielding.

[0009] It is another aim of the present invention to provide a method of synthesising cyclised peptides that can be applied to a diverse range of cyclised peptides.

[0010] It is a further aim of the invention to provide a method of synthesising cyclised peptides that can be used to produce a library of cyclised peptides.BRIEF SUMMARY OF THE DISCLOSURE

[0011] In a first aspect of the present invention, there is provided a method of preparing a cyclised peptide from a modified peptide comprising an acyl azide group, the method comprising increasing the pH of a reaction medium comprising an acidic aqueous solution of the modified peptide to generate the cyclised peptide.

[0012] In an embodiment of the first aspect, there is provided a method of preparing a cyclised peptide from a modified peptide comprising an acyl azide group, the method comprising increasing the pH of a reaction medium comprising an acidic aqueous solution of the modified peptide to generate the cyclised peptide via the formation of an amide bond or an ester bond between the carbonyl of the acyl azide group of the modified peptide and another functional group on the modified peptide, wherein the reaction medium further comprises a first solvent.

[0013] The inventors have also developed a method that is suitable for forming a cyclic peptide via an ester bond. In a second aspect of the present invention, there is provided a method of preparing a cyclised peptide from a modified peptide comprising an acyl azidegroup, the method comprising increasing the pH of a reaction medium comprising an acidic solution of the modified peptide to generate the cyclised peptide, wherein the acyl azide group forms an ester bond with a hydroxyl group on the modified peptide.

[0014] The inventors have found that increasing the pH of a reaction medium comprising a peptide modified with an acyl azide group results in cyclisation of the modified peptide via reaction of the acyl azide group with a functional group (e.g. an amine group or a hydroxyl group) elsewhere on the modified peptide, e.g. at the N-terminus or on a side chain of the peptide backbone.

[0015] Where the functional group with which the acyl azide group reacts is an amine, an amide linkage is formed between the carbonyl of the acyl azide group and the nitrogen of the amine group.

[0016] Where the functional group with which the acyl azide group reacts is a hydroxyl, an ester linkage is formed between the carbonyl of the acyl azide group and the oxygen of the hydroxyl group.

[0017] The methods of the invention are high yielding, with high conversion of modified peptide starting material and high selectivity towards cyclisation (i.e. over dimerization or peptide hydrolysis). Thus, the methods of the invention lead to reduced production of undesirable by-products and therefore require minimal purification. The methods also provide a rapid, operationally simple route to cyclised peptides compared to prior art methods. Further, the methods can be broadly applied to the synthesis of a broad range of cyclised peptides without the need for protecting groups or chelation on backbone residues or the installation of specific linkers between the resin and peptide C-terminus.

[0018] In a third aspect of the present invention, there is provided a method of generating a library of cyclised peptides, the method comprising performing the method of the first aspect or second aspect on at least two different modified peptides comprising acyl azide groups.

[0019] In a fourth aspect of the present invention, there is provided a peptide library obtained by the method of the third aspect.

[0020] Certain cyclised peptides produced by methods of the invention are novel compounds and these compounds form a further aspect of the invention.

[0021] Further aspects and features of the invention are set out in the detailed description below.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:Figure 1 shows examples of bioactive non-ribosomal cyclic peptides, as well as typical head to tail macrocyclization strategies known in the art. (A) Representative bioactive head-to-tail cyclic peptide cyclized via amide bond. (B) Biosynthetic macrocyclization strategies of ribosomal peptides (RiPPs) which require a C or N terminal sequence for recognition by a dedicated cyclase enzyme, and non-ribosomal peptides (NRPs) which are cyclized by a thioesterase where the peptide is covalently bound as a thioester to a carrier protein via phosphopantetheine (pPant). A / -acetyl cysteamine thioesters can act as pPant mimics in in vitro assays. (C) Macrocyclisation via direct aminolysis using: a. coupling reagents, b. metal assisted templating, and c. case dependent C-terminal linker or on resin. (D) Peptide cyclization via native chemical ligation which requires an N-terminal cysteine in the sequence.Figure 2 shows a general overview of the peptide synthesis methods of the invention (i.e. acyl azide peptide to cyclic peptide, or acyl-hydrazide peptide to cyclic peptide via acyl azide peptide), as well as a comparative route to cyclic peptide via SNAC peptide. Cyclic peptide may be further modified via enzymatic functionalisation to product postmodified peptides.Figure 3 shows a sequence of synthetic acyl hydrazide peptides. Acyl hydrazide peptide 8a is the natural sequence of rufomycin. Acyl hydrazide peptides 9a, 10a, 10b, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a and 36a include modified side chains compared to native rufomycin. Peptide 25a is the acyl hydrazide peptide precursor to tyrocidine.Figure 4 shows a comparison of silver assisted peptide macrocyclization and the bioinspired cyclisation method of the invention. A. Silver assisted cyclization of SNAC- peptide, 9b (200 pM). B. Bioinspired one-pot chemical macrocyclization. Acyl-hydrazide peptide 9a (2.2 mM), organic solvent is MeCN, buffer (50 mM NaH2PC>4, 6 M GdmCI, 1.5 mM EDTA). C. HPLC analysis (355 nm) of silver assisted cyclization, (i) the starting material SNAC peptide 9b, and (ii) the crude mixture after 1 h. 9d is the hydrolyzed linear peptide. D. HPLC analysis (355 nm) of bioinspired one pot cyclization, (i) the starting material hydrazide peptide 9a. (ii) the crude mixture 16 min after NaNO2mediated oxidation. Azide intermediate S9 was fully converted to 9c within 16 min.Figure 5 shows the pH and solvent dependence of bioinspired macrocyclization methods of the invention.Figure 6 shows peptide backbone controlled chemical cyclization. A. Reaction scheme of 14a cyclization to 14c. B. HPLC analysis of the reaction of 14a to 14c.Chromatogram SM is the purified acyl-hydrazide peptide 14a before oxidation. C. Reaction scheme of 15a cyclization to 15c. D. HPLC analysis of the reaction of 15a to 15c.Chromatogram SM is the purified acyl-hydrazide peptide 15a before oxidation. E. Reaction scheme of the attempted cyclisation for 16a to 16c, which was unsuccessful due to precipitation of the peptide.Figure 7 shows a biphasic macrocyclization reaction of the invention and illustrates the role of pH and organic solvent in facilitating cyclization.Figure 8 shows the chemical cyclization of rufomycin B 8c via a method of the invention. A. One-pot style fast macrocyclization of rufomycin B including oxidation of C- terminal acyl hydrazide peptide 8a to acyl azide followed by in-situ head to tail cyclisation. B. HPLC comparison of rufomycin B 8c from enzymatic cyclization (I), rufomycin 8c purified from extracts of Streptomyces atratus (II), chemical cyclization to form rufomycin 8c using methods of the invention (III), and (IV) co-injection of above three. C. HPLC analysis of chemical cyclization of 8a. (II) = acyl hydrazide peptide crude starting material, (I) = reaction mixture after extraction. All UV-vis traces were recorded at 355 nm.Figure 9 shows the side chain and steric effects on macrocyclization using peptides derived from rufomycin. A. Head to tail peptide cyclization. B&C. Peptide sequences based on rufomycin (including positions of backbone modifications) used to probe the relationship of sequence to cyclization efficiency. D. Relative cyclization rates for peptides 9c & 19c-22c. Data was fitted using a non-linear regression. (Time points 1 min, 21 min, 41 min and 61 min). % conversion was determined by HPLC analysis of reactions. E. Conversion after 21 min of linear peptide to cyclized or hydrolysed products was calculated using HPLC at 214 nm or 355 nm for peptide containing 3-nitro tyrosine. (areaction time 41 min,breaction time 81min)Figure 10 shows the application of bioinspired macrocyclisation to the synthesis of cyclic peptides with varying composition, ring size & cyclisation mode to produce peptides , 25c, 26c, 27c, 28c, 29c, 31c, 32c and 33c. Below each structure the reaction time and isolated yield is provided.Figure 11 shows A. the total synthesis of colistin analogue 30c via regioselective head to side chain macrocyclization.3Organic solvent is MeCN; B. HPLC analysis (214 nm) of chemical cyclization, i. crude starting linear peptide 30a. ii. Crude cyclisation reaction 1 min after neutralization. Hi. commercial standard colistin sold as a mixture of polymyxin Ei 30g, polymyxin E2indicated with *. iv. co-injection of cyclisation reaction mixture and commercial standard (1:1 mix); C. Comparison of MSEspectra demonstrates that 30c and 30g have identical fragmentation patterns.Figure 12 shows HPLC analysis (214 nm) of peptide cyclization of peptides 19a, 20a, 21a and 22a conducted on analytical scale (reaction volume 1 mL). S19-22 indicates peptide azide formation, 19-22c indicates cyclic peptide. 20c appears as two peaks, likely resulting from the presence of cis - trans proline isomers.Figure 13 shows HPLC analysis (214 nm) of peptide cyclization of 25a and 26a over time conducted on analytical scale. 26d is hydrolysed linear peptide. S26 indicates intermediate acyl azide formationFigure 14 shows HPLC analysis (214 nm) of peptide cyclization of 27a and 28a conducted on analytical scale. S27 indicates acyl azide formation. 27d is hydrolysed peptide.Figure 15 shows HPLC analysis (214 nm) of an analytical scale (1 mL reaction buffer) peptide cyclization study of 31a and 29a.Figure 16 shows HPLC analysis (214 nm) of peptide cyclization (1 mL reaction buffer) of 30a.Figure 17 shows the comparison of MSEspectra of colistin analogues resulting from the cyclisation reaction of 30a. The results demonstrate that 30c and 30g have identical mass fragmentation patterns. 30g is commercial colistin.Figure 18 shows the effect of solvent conditions on the cyclization reaction of 32a conducted on analytical scale (1mL). The results highlight the role of co-solvent in both product selectivity and conversion during the cyclization. The peak label * is an artefact of HPLC analysisFigure 19 shows HPLC analysis (214 nm) of peptide cyclization of 33a.Figure 20 shows the cyclisation of valhidepsin A analogue 35c in an analytical scale reaction (2mM 35a in 1mL reaction buffer) and analysis by LC-HRMS.Figure 21 shows HPLC analysis (214nm) of the cyclisation of macolacin analogue acyl hydrazide precursor 37a in an analytical scale reaction (2mM 37a in 1mL reaction buffer) to cyclic peptide 37c.Figure 22 shows HPLC analysis (214nm) of bremelanotide acyl hydrazide precursor 38a in an analytical scale reaction (2mM 38a in 1mL reaction buffer) to give bremelanotide 38c.Figure 23 shows the analysis of HPLC (280 nm) analysis of cyclisation of microcin J2541a in an analytical scale reaction (2mM 41a in 1ml_ reaction buffer) precursor to microcin J2541c.Figure 24 shows the HPLC analysis (214nm) of the cyclisation of a backbone- modified polyalanine peptide 44a in an analytical scale reaction (2mM 44a in 1mL reaction buffer) to 44cDETAILED DESCRIPTIONDefinitions

[0023] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0024] The term “amino acid” refers to an organic compound composed of amine and carboxylic acid functional groups, along and optionally a side-chain specific to each amino acid. The amino acids may be proteinogenic or non-proteinogenic amino acids. The term amino acid typically refers to a-amino acids, i.e. having the generic formula H2NCHRC(O)OH. The amino acids may also refer to beta-, gamma-, and delta-amino acids, i.e. that contain multiple carbon atoms between the amine and the carboxylic acid.

[0025] The term “amino acid residue” refers to an individual amino acid that has been attached to one or more other amino acids by a peptide bond. Each amino acid residue may be unmodified or modified (as described below).

[0026] The term “peptide” refers to polymer a chain of amino acids linked by peptide bonds. The peptide may also comprise one or more non-amino acid building blocks and / or nonpeptide linkages. Peptides are distinguished from proteins or polypeptides on the basis of size, and comprise no more than 50 amino acid residues. In the context of the present invention, the peptide may comprise a primary or secondary amine at the N-terminus or a primary or secondary amine within the peptide backbone.

[0027] The “N-terminus” refers to the free amine group located at the start of the peptide chain which does not form part of a peptide bond.

[0028] The “C-terminus” refers to the carboxylic acid group located at the end of the peptide chain which does not form part of a peptide bond.

[0029] The term “modified peptide” refers to a peptide in which one or more amino acid residues in the peptide has been modified. For the avoidance of doubt, the modifying of an amino acid residue may refer to the chemical transformation of one or more functional groups on the amino acid residue. For example, in the methods of the invention, themodified peptide comprises a peptide modified with an acyl azide group. The acyl azide group may be introduced via modification of the carboxylic acid group at the C-terminus of the peptide. Alternatively, the acyl azide group may be introduced via modification of a side chain of an amino acid residue in the peptide chain. The modified peptide may also comprise one or more additional modified moieties elsewhere in the peptide chain, thereby comprising multiple acyl azide groups.

[0030] The term “modified linear peptide” refers to a modified peptide comprising a straight chain portion of at least 2 amino acid residues. For example, the modified linear peptide may comprise a straight chain portion of at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 amino acid residues. In addition to the straight chain portion, the modified linear peptide may further include one or more cyclic or branched portions.

[0031] The term “cyclised peptide” refers to a peptide comprising at least one cyclic portion formed from cyclisation of the modified peptide via the reaction between the acyl azide group on the modified peptide and another functional group on the modified peptide (e.g. at the N-terminus to form an amide bond, or via an amino group on a side chain to form an amide bond, or via a hydroxyl group on a side chain to form an ester bond) to form an amide bond (i.e. -C(O)NR-) or an ester bond (i.e. -C(O)O-).

[0032] The term “peptide library” refers to a composition or an article of manufacture comprising a plurality of different peptides. For example, the peptide library may be an article comprising different wells, such as a microtiter plate (e.g. a 96-well plate, a 384-well plate, a 1536-well plate, or a 3456-well plate), wherein each well comprises one peptide species. Alternatively, each well may comprise more than one peptide species. The number of different peptide species per well may be adjusted as needed. The plurality of wells together forms the peptide library.

[0033] The term “solid phase” designates any solid material or support to which peptides can be synthesized, e.g. via solid-phase peptide synthesis (SPPS).

[0034] The term “base” refers to a substance which can accept protons (i.e. Brensted bases), donate electrons (i.e. Lewis bases), or yield hydroxide ions (OH-) in aqueous solution. In the context of the present invention, a base may be employed to facilitate the formation of the cyclised peptide. The base in the present invention may be capable of neutralising a protonated functional group (e.g. at the N-terminus or on a side chain of the modified peptide) to facilitate the reaction between the acyl azide group and said functional group to generate the cyclised peptide. The base may be a non-nucleophilic base. The base may be a water-compatible base.

[0035] The term “water-compatible base” refers to a species that, when added to the reaction medium, is capable of increasing the pH of the reaction medium, and is fully or partially soluble in water, or is partially / wholly insoluble in water but still has basicity to enable the desired reaction to take place.

[0036] The term “peptide compatible” as used herein refers to a species which does not covalently react with a peptide and / or cause degradation of a peptide or prevent a peptide from reacting with another component.

[0037] The term “immiscible” refers to two substances being incapable of combining with one another to form a homogeneous mixture. Unless otherwise stated, when used in the context of the invention, two substances are deemed to be immiscible if they do not form a homogenous mixture under standard temperature and pressure. In the context of the present invention, a substance may be said to be “immiscible” with an aqueous solution if the substance has a solubility of less than about 10 g per 100 mL of aqueous solution under standard temperature and pressure, and is present in a sufficient amount such that a two- phase mixture with an aqueous solution is obtained.

[0038] The term “water miscible” refers to a substance being capable of combining with water to form a homogeneous mixture.

[0039] The term “protecting group” as used herein is given its ordinary meaning which is readily understandable to those of skill in the art. It is used herein to refer to a group suitable for protecting a side chain on the modified peptide. Exemplary protecting groups which may be employed include: / V-Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)isovaleryl (ivDde), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), phthalimide, sulfonamide, 2,4,6-Triisopropylsulfonamide (Trs), tert-butoxycarbonyl (Boc), 2-(4-biphenylyl)-2-propyloxycarbonyl (Bpoc), monomethoxytrityl (MMT), dimethoxytrityl (DMT), 2,4-Dimethoxybenzyl (DMB), A / - Diphenylmethyleneamine, 2-(Trimethylsilyl)-ethoxycarbonyl (Teoc), 2- Trimethylsilylethoxymethyl (SEM), 6-nitroveratryloxycarbonyl (NVOC), 2,2’-bis(2- nitrophenyl)ethoxycarbonyl (diNPEOC), benzoyl, benzyl, tetrahydropyran (THP), dimethoxybenzyl, monomethoxymethyl (MOM), acetyl, Levulinate (Lev), 2,2,2- Trichloroethoxycarbonyl (Troc), tert-Butyldimethylsilyl (TBDMS), tert-Butyldiphenylsilyl (TBDPS), Trimethylsilyl (TMS), Triisopropylsilyl (TIPS), Pivaloyl (Pv), allyl, and 4-Azido-3- chlorobenzyl (CIAzb).

[0040] The term “solubiliser” refers to any substance that increases the solubility of another species in a solution. In the context of the present invention, a solubiliser may be added tothe reaction medium to increase the solubility of the modified peptide and / or modified peptide precursor.

[0041] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0042] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0043] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.Methods of Preparing a Cyclised Peptide

[0044] The first aspect of the invention provides a method of preparing a cyclised peptide from a modified peptide comprising an acyl azide group, the method comprising increasing the pH of a reaction medium comprising an acidic aqueous solution of the modified peptide to generate the cyclised peptide. For the avoidance of doubt, the cyclised peptide is generated via the formation of a bond between the carbonyl of the acyl azide group of the modified peptide and another functional group (e.g. an amine group or a hydroxyl group) on the modified peptide to form either an amide bond or an ester bond. In preferred embodiments, the cyclised peptide is generated via the formation of an amide bond (- C(O)NR-) between the carbonyl of the acyl azide group and another functional group (e.g. an amine group). In such embodiments, the functional group may be an amine group at theN-terminus or an amine group on a side chain of the modified peptide backbone. In other embodiments, the cyclised peptide is generated via the formation of an ester bond (-C(O)O-) between the carbonyl of the acyl azide group and another functional group (e.g. a hydroxyl group).

[0045] For the avoidance of doubt, in embodiments wherein the cyclised peptide is generated via the formation of an amide bond or ester bond between the carbonyl of the acyl azide of the modified peptide and another functional group on the modified peptide, the cyclised peptide may be formed directly from the modified peptide in a single reaction step. Thus, in these embodiments, the cyclised peptide is not formed from the acyl azide of the modified peptide via a multi-step process that proceeds via an intermediate species.

[0046] The second aspect of the invention provides a method of preparing a cyclised peptide from a modified peptide comprising an acyl azide group, the method comprising increasing the pH of a reaction medium comprising an acidic solution of the modified peptide to generate the cyclised peptide, wherein the acyl azide group forms an ester bond with a hydroxyl group on the modified peptide. For the avoidance of doubt, where the term “increasing the pH of a reaction medium” is used in relation to a reaction medium which does not include an aqueous component, it is meant that an amount of base is added to the reaction medium.

[0047] In embodiments of the second aspect, the acidic solution may be an acidic aqueous solution.

[0048] The following embodiments may refer to the method of either the first aspect or the second aspect.

[0049] The acidic aqueous solution may comprise an acidic buffer solution.

[0050] The acidic aqueous solution may be selected from the group comprising: acidified water, phosphate buffer, HEPES buffer, tris buffer, carbonate buffer, or acetate buffer. The acidic aqueous solution may be selected from the group comprising: phosphate buffer, HEPES buffer, tris buffer, carbonate buffer, or acetate buffer. The acidic aqueous solution may be a phosphate buffer.

[0051] Where the acidic aqueous solution is an acidic buffer solution, the acidic buffer solution may have a concentration of from about 1 to about 100 mM.

[0052] The acidic buffer solution may have a concentration of from about 10 mM to about100 mM. The acidic buffer solution may have a concentration of from about 20 mM to about100 mM. The acidic buffer solution may have a concentration of from about 30 mM to about100 mM. The acidic buffer solution may have a concentration of from about 40 mM to about100 mM. The acidic buffer solution may have a concentration of from about 50 mM to about100 mM.

[0053] The acidic buffer solution may have a concentration of from about 1 mM to about 90 mM. The acidic buffer solution may have a concentration of from about 1 mM to about 80 mM. The acidic buffer solution may have a concentration of from about 1 mM to about 70 mM. The acidic buffer solution may have a concentration of from about 1 mM to about 60 mM. The acidic buffer solution may have a concentration of from about 1 mM to about 50 mM.

[0054] The acidic buffer solution may have a concentration of from about 10 mM to about 90 mM. The acidic buffer solution may have a concentration of from about 20 mM to about 80 mM. The acidic buffer solution may have a concentration of from about 30 mM to about 70 mM. The acidic buffer solution may have a concentration of from about 40 mM to about 60 mM. The acidic buffer solution may have a concentration of about 50 mM.

[0055] The acidic aqueous solution may have a pH of no more than about 4. The acidic aqueous solution may have a pH of no more than about 3.5.

[0056] The acidic aqueous solution may have a pH of from about 1.0 to about 3.5. The acidic aqueous solution may have a pH of from about 2.0 to about 3.5. The acidic aqueous solution may have a pH of from about 2.5 to about 3.5. The acidic aqueous solution may have a pH of about 3.0.

[0057] The reaction medium may further comprise a first solvent. For the avoidance of doubt, the first solvent is a different species to the acidic aqueous solution present in the reaction medium. For example, it may be that the first solvent is an organic solvent.

[0058] The first solvent may be a polar, non-chlorinated organic solvent. For example, the first solvent may be selected from the group comprising: acetonitrile, ethyl acetate, tetrahydrofuran, methyl ethyl ketone, butanol, dimethylformamide, tetrahydropyran, 1,4- dioxane, 1 ,3-dioxolane, 2,2,2-trifluoroethanol, 2-methyltetrahydrofuran, dimethylsulfoxide, and mixtures thereof.

[0059] Without wishing to be bound by theory, it is thought that the presence of an organic solvent shifts the equilibrium in the reaction. In particular, when the functional group with which the acyl azide reacts is an amine group, said amine group will be protonated under the acidic conditions of the reaction. Upon increasing the pH of the reaction medium, it is thought that a fraction of the modified peptide becomes neutral via deprotonation of said protonated amine group. It is thought that this uncharged modified peptide moves into the organic phase where cyclisation may occur, and that said cyclisation shifts the equilibrium ofthe amine protonation / deprotonation, resulting in the movement of more modified peptide into the organic phase (Fig. 7). Without wishing to be bound by theory, it is thought that the uncharged modified peptide adopts a favourable conformation in the organic phase such that the acyl azide group and the other functional group with which the acyl azide group reacts are in close proximity, thereby facilitating rapid cyclisation. Additionally, without wishing to be bound by theory, it is thought that this process reduces the possibility of dimer formation by diluting the peptide.

[0060] The first solvent may be immiscible with the acidic solution or acidic aqueous solution. For example, the first solvent may be selected from: ethyl acetate, methyl ethyl ketone, butanol, toluene, and mixtures thereof. It may be that the first solvent is ethyl acetate, methyl ethyl ketone, butanol, and mixtures thereof. It may be that the first solvent is ethyl acetate. Thus, in these embodiments, the reaction medium is biphasic.

[0061] Without wishing to be bound by theory, it is thought that a biphasic reaction medium further facilitates preorganisation of the modified peptide in the reaction medium, thereby increasing the proximity between the acyl azide group and the other functional group with which the acyl azide group reacts (e.g. an amine group or hydroxyl group) and facilitating rapid cyclisation.

[0062] The first solvent may be miscible with the acidic solution or acidic aqueous solution. For example, the first solvent may be selected from the group comprising: acetonitrile, tetrahydrofuran, dimethylformamide, tetrahydropyran, 1 ,4-dioxane, 1 ,3-dioxolane, 2,2,2- trifluoroethanol, 2-methyltetrahydrofuran, dimethylsulfoxide, N-methylmorpholine, and mixtures thereof. In these embodiments, the reaction medium is a monophasic system.

[0063] The reaction medium may comprise water in an amount of at least about 5% by volume. The reaction medium may comprise water in an amount of at least about 10% by volume. The reaction medium may comprise water in an amount of at least about 25% by volume. The reaction medium may comprise water in an amount of at least about 50% by volume.

[0064] The pH of the reaction medium prior to increasing the pH may be no more than about 4. The pH of the reaction medium prior to increasing the pH may be from about 2.5 to about 3.5, more preferably about 3.0.

[0065] The pH of the reaction medium may be increased to a pH of from about 6 to about 8. The pH of the reaction medium may be increased to a pH of from about 7 to about 7.5.

[0066] The pH of the reaction medium may be increased using a base. The base may be a non-nucleophilic, water-compatible base. For example, the base may be selected from:sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, an inorganic phosphate, potassium carbonate, triethylamine, and N,N- diisopropylethylamine. It may be that the base is sodium bicarbonate.

[0067] The base may be added to the reaction medium in a concentration of from about 100 mM to about 140 mM of the reaction mixture.

[0068] The acyl azide may be present at the C-terminus of the modified peptide. The acyl azide may be present on a side chain of the modified peptide.

[0069] The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be at least 1 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be at least 1.5 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be at least 2 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be at least 5 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be at least 10 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be at least 15 mM.

[0070] The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 30 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 25 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 20 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 15 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 10 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 9 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 8 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 7 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 6 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 5 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be no more than about 4 mM.

[0071] The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be from about 1 mM to about 30 mM. The concentration of themodified peptide comprising the acyl azide group in the reaction medium may be from about 1 mM to about 20 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be from about 1 mM to about 10 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be from about 1 mM to about 8 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be from about 2 mM to about 6 mM. The concentration of the modified peptide comprising the acyl azide group in the reaction medium may be from about 1.5 mM to about 4 mM.

[0072] The method of preparing the cyclised peptide from the modified peptide may be performed at a temperature of from about -20 °C to about 25 °C. The method of preparing the cyclised peptide from the modified peptide may be performed at a temperature of from about -10 °C to about 25 °C. The method of preparing the cyclised peptide from the modified peptide may be performed at a temperature of from about 0 °C to about 25 °C.

[0073] The method of preparing the cyclised peptide from the modified peptide may be performed for a time period of from about 1 minute to about 120 minutes.

[0074] The method of preparing the cyclised peptide from the modified peptide may be performed at atmospheric pressure.

[0075] The method of preparing the cyclised peptide from the modified peptide may be performed under an inert atmosphere. Alternatively, method of preparing the cyclised peptide from the modified peptide may be performed in air.

[0076] The modified peptide may be a modified linear peptide.

[0077] The modified peptide may comprise at least 2 amino acid residues. The modified peptide may comprise at least 3 amino acid residues. The modified peptide may comprise at least 4 amino acid residues. The modified peptide may comprise at least 5 amino acid residues. The modified peptide may comprise at least 6 amino acid residues. The modified peptide may comprise at least 7 amino acid residues.

[0078] The modified peptide may comprise no more than 40 amino acid residues. The modified peptide may comprise no more than 35 amino acid residues. The modified peptide may comprise no more than 30 amino acid residues. The modified peptide may comprise no more than 25 amino acid residues. The modified peptide may comprise no more than 24 amino acid residues. The modified peptide may comprise no more than 23 amino acid residues. The modified peptide may comprise no more than 22 amino acid residues. The modified peptide may comprise no more than 21 amino acid residues. The modified peptide may comprise no more than 20 amino acid residues.

[0079] The modified peptide may comprise from 2 to 40 amino acid residues. The modified peptide may comprise from 4 to 30 amino acid residues. The modified peptide may comprise from 5 to 25 amino acid residues. The modified peptide may comprise from 5 to 21 amino acid residues. The modified peptide may comprise from 5 to 15 amino acid residues. The modified peptide may comprise from 5 to 11 amino acid residues.

[0080] The modified peptide may comprise a peptide chain length of least 2 amino acid residues. The modified peptide may comprise a peptide chain length of least 3 amino acid residues. The modified peptide may comprise a peptide chain length of least 4 amino acid residues. The modified peptide may comprise a peptide chain length of least 5 amino acid residues. The modified peptide may comprise a peptide chain length of least 6 amino acid residues. The modified peptide may comprise a peptide chain length of least 7 amino acid residues.

[0081] The modified peptide may comprise a peptide chain length of no more than 40 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 30 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 20 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 15 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 14 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 13 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 12 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 11 amino acid residues. The modified peptide may comprise a peptide chain length of no more than 10 amino acid residues.

[0082] The modified peptide may comprise a peptide chain length of from 2 to 40 amino acid residues. The modified peptide may comprise a peptide chain length of from 4 to 30 amino acid residues. The modified peptide may comprise a peptide chain length of from 4 to 20 amino acid residues. The modified peptide may comprise a peptide chain length of from 4 to 15 amino acid residues. The modified peptide may comprise a peptide chain length of from 4 to 11 amino acid residues.

[0083] The modified peptide may have a logP value in the range of from about -3 to about 2. The modified peptide may have a logP value in the range of from about -3 to about 1.8.

[0084] The modified peptide may have a positive logP value. Thus, the modified peptide may be hydrophobic. Thus, the modified peptide may be hydrophobic. For example, the modified peptide may have a logP value of at least about 0.5, optionally at least about 1.

[0085] The modified peptide may comprise at least 20% hydrophobic amino acid residues. The modified peptide may comprise at least 40% hydrophobic amino acid residues. The modified peptide may comprise at least 60% hydrophobic amino acid residues. The modified peptide may comprise from about 20% to about 100% hydrophobic amino acid residues.

[0086] The modified peptide may comprise one or more moieties that force the modified peptide into a curved conformation. The one or more moieties may be a bulky moiety that exerts a steric effect on the modified peptide chain. Without wishing to be bound by theory, it is thought that the curved conformation increases the spatial proximity of the acyl azide group and the other functional group with which the acyl azide group reacts (e.g. the amine or hydroxyl group), thereby facilitating cyclisation of the peptide.

[0087] Each of the one or more moieties may be substituted onto a nitrogen of an amino acid residue in the modified peptide (e.g. N-modified or N-alkylated), substituted onto a side chain of an amino acid residue in the modified peptide, or may form part of the modified peptide backbone (e.g. an L-proline residue or other turn-inducing residue).

[0088] The modified peptide may comprise at least one N-alkylated amino acid residue. For example, the modified peptide may comprise at least one, at least two, at least three, at least four, at least five or at least six N-alkylated amino acid residues. It may be that each instance of N-alkylated amino residue is a N-methylated amino acid residue. Without wishing to be bound by theory, it is thought that N-alkylation to form cis-amide bonds in the modified peptide forces the modified peptide into a curved conformation, which may aid cyclisation.

[0089] The modified peptide may comprise at least one amino acid residue that is N- substituted with a cleavable group. In this context, the term “cleavable group” refers to a chemical moiety which may be added to the modified peptide prior to cyclisation and subsequently removed after cyclisation is complete. Without wishing to be bound by theory, it is thought that functionalising the modified peptide in this manner facilitates cyclisation by forcing the modified peptide into a curved conformation. Exemplary cleavable groups include: tert-butyl, 2-hydroxy-4-methoxybenzyl, 2,4-dimethoxybenzyl and pseudo-proline derivatives.

[0090] The modified peptide may comprise at least one L-proline residue or other turn inducing residue, such as a D-amino acid (e.g. D-proline), a 2-aminobenzoic acid derivative, a heterocycle (e.g. oxazole, thiazole), a cyclic gamma amino acid or a gamma-carbonyl containing amino acid. Without wishing to be bound by theory, it is thought that the inclusion of a turn inducing residue (such as L-proline) in the modified peptide forces the modified peptide into a curved conformation, which may aid cyclisation.

[0091] Additionally, without wishing to be bound by theory, it is thought that interactions between functional groups on the side-chains of different amino acid residues in the modified peptide may lead to the modified peptide adopting a curved conformation that favours cyclisation.

[0092] The modified peptide may comprise one or more protecting groups on at least one amino acid residue. Alternatively, the modified peptide may not include any protecting groups.

[0093] The modified peptide may have a molecular weight of from about 200 Da to about 4000 Da.

[0094] The distance between the acyl azide group of the modified peptide and the functional group on the modified peptide with which the acyl azide group reacts (e.g. an amine group or a hydroxyl group) may be at least about 3 A.

[0095] The distance between the acyl azide group of the modified peptide and the functional group on the modified peptide with which the acyl azide group reacts (e.g. an amine group or a hydroxyl group) may be no more than about 6 A.

[0096] The modified peptide may comprise any proteinogenic amino acid and / or derivative thereof, any naturally occurring non-proteinogenic amino-acid and / or derivative thereof, and / or any non-naturally occurring amino acid and / or derivative thereof.

[0097] The modified peptide may be derived from a naturally occurring cyclised peptide. For example, the modified peptide may be derived from a naturally occurring cyclised peptide selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, ambactin, macolacin, lassomycin, valhidepsin, microcin, colistin, gramicidin S, planktocyclin, bremelanotide or derivatives thereof. In preferred embodiments, the modified peptide may be derived from a naturally occurring cyclised peptide selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, colistin, gramicidin S, planktocyclin, bremelanotide or derivatives thereof. In further preferred embodiments, the modified peptide may be derived from a naturally occurring cyclised peptide selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, colistin, or derivatives thereof.

[0098] The modified peptide may be selected from the group comprising:

[0099] The modified peptide may be selected from the group comprising:

[0100] It may be that the modified peptide is not:

[0101] It may be that the modified peptide is not:

[0102] The cyclised peptide may be selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, ambactin, macolacin, lassomycin, microcin, valhidepsin, colistin, gramicidin S, a C5aR antagonist, planktocyclin, bremelanotide,selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, colistin, gramicidin S, a C5aR antagonist, planktocyclin, bremelanotide,eof. The cyclised peptide may be selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, colistin,reof.

[0103] The cyclised peptide may be selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, ambactin, macolacin, lassomycin, microcin, valhidepsin, colistin, gramicidin S, a C5aR antagonist, planktocyclin, bremelanotide, or derivatives thereof. The cyclised peptide may be selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, colistin, gramicidin S, a C5aRantagonist, planktocyclin, bremelanotide, or derivatives thereof. The cyclised peptide may be selected from: rufomycin, tyrocidine, pseudostellarin, cyclosporin, cyclomarine, cyclopurpuracin, colistin, or derivatives thereof.

[0104] The modified peptide comprising the acyl azide group may be prepared by treating a precursor reaction medium comprising a modified peptide precursor with an oxidising agent, wherein the modified peptide precursor comprises a precursor functional group. For example, the modified peptide precursor may comprise a precursor functional group selected from the group comprising: aldehyde, carboxylic acid, ester, or hydrazide. It may be that the modified peptide precursor comprises a hydrazide.

[0105] For the avoidance of doubt, the modified peptide precursor differs from the modified peptide in that the modified peptide precursor includes the precursor functional group referred to above in the position corresponding to the position of the acyl azide group of the modified peptide. Thus, the step of preparing the modified peptide from the modified peptide precursor involves converting the precursor functional group to an acyl azide group. Other than the precursor functional group in place of the acyl azide group, the structure of the modified peptide precursor may be identical to the structure of the modified peptide. Thus, other than the precursor functional group in place of the acyl azide group, the structure of the modified peptide precursor may be as defined in any of the embodiments above relating to the modified peptide.

[0106] For the avoidance of doubt, the precursor reaction medium differs from the reaction medium in that the precursor reaction medium comprises the modified peptide precursor in place of the modified peptide, and further comprises the oxidising agent. The remaining components of the precursor reaction medium may be the same as those of the reaction medium.

[0107] Accordingly, in embodiments of the first aspect, the precursor reaction medium may comprise an acidic aqueous solution of the modified peptide precursor and oxidising agent. Alternatively, in embodiments of the second aspect, the precursor reaction medium may comprise an acidic solution of the modified peptide precursor and oxidising agent. The acidic aqueous solution or acidic solution in the precursor reaction medium thus may be as defined above in relation to the reaction medium.

[0108] The precursor reaction medium may not comprise the first solvent. For the avoidance of doubt, in these embodiments, the first solvent may be added to the reaction medium after formation of the acyl azide. Without wishing to be bound by theory, it is thought that adding the first solvent to the reaction medium after the acyl azide has formed can result in improved yield of the cyclised azide and can reduce the number of undesirablebyproducts, thereby resulting in improved purity. In these embodiments, the first solvent is preferably immiscible with the acidic solution or acidic aqueous solution. For example, the first solvent may be ethyl acetate.

[0109] The oxidising agent may be selected from sodium nitrite, nitrous acid, N- bromosuccinimide, and A / -chlorosuccinimide. The oxidising agent may be sodium nitrite.

[0110] The precursor reaction medium and / or reaction medium may further comprise a solubiliser which maintains the modified peptide precursor and / or the modified peptide comprising the acyl azide group in solution. The solubiliser may be present when the modified peptide precursor and / or the modified peptide are insoluble or sparingly soluble in the precursor reaction medium and / or reaction medium.

[0111] The solubiliser may be selected from: guanidine or a salt thereof, urea or a salt thereof, arginine or a salt thereof, or a second solvent. The solubiliser may be guanidine or a salt thereof, e.g. guanidinium chloride.

[0112] The modified peptide precursor may further comprise a removable tag. The removable tag may be a protein tag. For example, the removable tag may be selected from a polyhistidine tag (e.g. His6tag), a lysine tag, a polylysine tag, or an arginine tag.Alternatively, the removable tag may be a polyethylene glycol tag or a Fmoc-Ddae-OH tag. Without wishing to be bound by theory, it is thought that the presence of such a removable tag leads to improved solubility of the modified peptide precursor in aqueous media.

[0113] The second solvent may be a water-miscible organic solvent. For example, the second solvent may be selected from: acetonitrile, ethyl acetate, tetrahydrofuran, methyl ethyl ketone, butanol, dimethylformamide, tetrahydropyran, 1 ,4-dioxane, 1,3-dioxolane, 2,2,2-trifluoroethanol, 2-methyltetrahydrofuran, dimethylsulfoxide, sodium dodecyl sulfate, polysorbate 20 (Tween-20), polysorbate 80 (Tween-80), octylphenoxypolyethoxyethanol (IGEPAL), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X- 100), 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or similar peptide compatible detergents / surfactants (e.g. dodecylphosphocholine, octylglycoside).

[0114] The precursor reaction medium may comprise an acidic aqueous solution having a pH of no more than about 4.0. The precursor reaction medium may comprise an acidic aqueous solution having a pH of from about 2.5 to about 3.5. The precursor reaction medium may comprise an acidic aqueous solution having a pH of about 3.0.

[0115] The concentration of the modified peptide precursor in the precursor reaction medium may be at least about 1 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be at least about 1.5 mM. The concentration of themodified peptide precursor in the precursor reaction medium may be at least about 2 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be at least about 5 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be at least about 10 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be at least about 15 mM.

[0116] The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 30 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 25 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 20 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 15 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 10 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 9 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 8 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 7 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 6 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 5 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be no more than about 4 mM.

[0117] The concentration of the modified peptide precursor in the precursor reaction medium may be from about 1 mM to about 30 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be from about 1 mM to about 20 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be from about 1 mM to about 10 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be from about 1 mM to about 8 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be from about 2 mM to about 6 mM. The concentration of the modified peptide precursor in the precursor reaction medium may be from about 1.5 mM to about 4 mM.

[0118] It may be that the modified peptide comprising the acyl azide group is not isolated from the precursor reaction medium prior to increasing the pH of the reaction medium to generate the cyclised peptide.

[0119] The modified peptide precursor may be prepared by cleavage of a modified peptide starting material from a solid support. For example, where the precursor functional group ofthe modified peptide precursor is a hydrazide, the modified peptide starting material may be covalently bonded to the solid support via a hydrazide linker.

[0120] The modified peptide starting material may be covalently bonded to the solid support at the C-terminus of the modified peptide starting material. The modified peptide starting material may be covalently bonded to the solid support at a side chain of the modified peptide starting material.

[0121] The solid support may be a resin support. For example, the solid support may be a resin support selected from: chlorotrityl resin and cross-linked polystyrene resin (e.g. divinyl benzene cross-linked polystyrene resin).

[0122] The method of preparing the modified peptide from the modified peptide precursor may be performed at a temperature of from about -15 °C to about 0 °C.

[0123] The method of preparing the modified peptide from the modified peptide precursor may be performed for a time period of from about 1 minute to about 60 minutes, optionally from about 1 minute to about 20 minutes.

[0124] The method of preparing the modified peptide from the modified peptide precursor may be performed at atmospheric pressure.

[0125] The method of preparing the modified peptide from the modified peptide precursor may be performed under an inert atmosphere. Alternatively, method of preparing the modified peptide from the modified peptide precursor may be performed in air.Method of Generating a Library

[0126] In a third aspect of the invention, there is provided a method of generating a library of cyclised peptides, the method comprising performing the method of the first aspect on at least two different modified peptides comprising acyl azide groups.

[0127] The library of cyclised peptides may comprise at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 96, at least 100, at least 384, at least 500, at least 1000, at least 1536, at least 3456, at least 10000, and at least 100000 different cyclised peptides.

[0128] A precursor modified peptide library may be synthesised using an automated solid phase peptide synthesiser for parallel synthesis of peptides resulting in a library of precursor modified peptides in well plate or similar format. The precursor modified peptides can then undergo cyclisation using a parallel reaction vessel apparatus i.e. in well plates or multiple reaction vessels, well plates or columns.EXAMPLESGeneral Methods

[0129] All the reagents and solvents were obtained from commercial suppliers and were used without further purification. Dry reactions were conducted in oven-dried glassware under a nitrogen atmosphere. Starting materials were purchased from commercial sources or synthesised according to the methos described herein or using literature processes.

[0130] Anhydrous THF, MeOH and DCM were obtained from the Inert Solvent Purification System, all other solvents were supplied as Sureseal® bottles by Sigma Aldrich, DMF (sequencing grade) was purchased from Cambridge Reagent Ltd used for Solid Phase Peptide Synthesis (SPPS).AbbreviationsDCM dichloromethaneTHF tetrahydrofuranTE thioesteraseGdmCI guanidinium chlorideNRPS non-ribosomal peptide synthetaseHATU hexafluorophosphate azabenzotriazole tetramethyl uraniumDIPEA N,N-DiisopropylethylamineIPTG isopropyl-p-D-thiogalactopyranosideAnalytical MethodsNMR Spectroscopy

[0131] Nuclear Magnetic Resonance (NMR) spectra were recorded using a Bruker UltraShieldTM 400 MHz or 700 MHz and at default temperature (18 °C). The chemical shifts (5) are reported in parts per million (ppm) using the abbreviations: s, singlet; d, doublet; dd, double of doublet; t, triplet; q, quartet. Resonances that could not be easily interpreted were designated multiplets (m). Chemical shifts (5) are referenced to the residual solvent signal. Spin-spin coupling constants J are quoted in Hz.Column Chromatography

[0132] Flash column chromatography was performed using 60 A (40-64 micron) silica and solvent mixtures of hexane and ethyl acetate or DCM and MeOH. Analytical thin layer chromatography was performed on TLC Silica gel 60 F254 (Aluminium sheets). Visualization was assisted with 254 nm UV-lamp, potassium permanganate (KMnC ) stain and ninhydrin stain.High-Resolution Mass Spectrometry

[0133] High-resolution mass spectra were recorded on a Waters Acquity UPLC-Class I equipped with an ACQUITY UPLC column. The detector was a Waters Xevo-G2-XS Qtof with electrospray ionization source. The instrument was operated in positive mode full-scan with detection window set from 50 to 2000 Da. For peptide fragmentation, a collision energy ramp from 15 V to 40 V was employed. Optical rotation readings were recorded using Anton Parr MCP100 Polarimeter. Specific rotations ([a]25D) were recorded at the sodium D line (589 nm) in methanol or chloroform and are quoted in: deg cm2g-1. Solution concentration (c) are given in 0.1. Temperatures are in degrees Celsius (°C). The prefixes (+) and (-) indicate the sign of the optical rotation.Reverse-Phase Chromatography

[0134] Analytical high-performance liquid chromatography (HPLC) was performed on an Agilent 1260 Infinity II instrument equipped with a DAD detector with an Agilent Eclipse XDB-C18 5 pm 4.6 x 150 mm column or Eclipse XDB-C8 5 pm 4.6 x 150 mm column at a constant flow rate of 1 mL / min at 40 °C. The UV-vis absorbance of the eluent was monitored at 220 nm, 280 nm and 355 nm.

[0135] Semi-preparative HPLC was performed on an Agilent Eclipse XDB-C8 5 pm 9.4 x 250 mm column or Agilent ZORBAX 300SB-C18 5 pm 9.4 x 250 mm column was used at a constant flow rate of 3.5 mL / min at 60 °C.

[0136] HPLC purification of the peptides was performed on an Agilent 1260 Preparative HPLC system using a reversed phase Agilent Zorbax 300SB-C18 PrepHT 21.2 x 150 mm 7 pm column was used at a constant flow rate of 20 mL / min at room temperature.

[0137] All hydrazide peptides were analyzed via ultra-performance liquid chromatography (UPLC) on a Waters ACQUITY UPLC BEH C8, 1.7 pm, 2.1 x 50 mm column, the rest of peptides on a Waters ACQUITY UPLC BEH C18, 1.7 pm, 2.1 x 50 mm column, the proteins were analyzed on a Waters ACQUITY UPLC BEH C4, 300 A, 1.7 pm, 2.1 x 50 mm column with constant flow of 0.4 mL / min 40 °C.

[0138] Typical gradients using mixture of two solvents are described below (with the solvents abbreviated as Solvents A-H).Solvent A: H2O containing 0.1% TFASolvent B: MeCN containing 0.1 % TFASolvent C: 20% MeCN in H2O containing 0.1% TFASolvent D: 70% MeCN in H2O containing 0.1% TFASolvent E: 30% MeCN in H2O containing 0.1% TFASolvent F: 80% MeCN in H2O containing 0.1% TFASolvent G: H2O containing 0.1% FASolvent H: MeCN containing 0.1% FAAnalytical HPLCGradient 1: linear gradient from 5-100% B over 40 min, wash 5 min with 100% B, then back to 5% B over 3 min. (C184.6 x 150 mm)Gradient 2: 2 min with 5% B, linear gradient from 5-100% B over 40 min, wash 5 min with 100% B, then back to 5% over 2 min. (C84.6 x 150 mm)Gradient s: 2 min with 5% B, linear gradient from 5-40% B over 1 min, linear gradient from 40- 95% B over 9 min, wash 1 min with 95% B, then back to 5% B over 0.5 min, calibrate with 5% B over 1.5 min. (C84.6 x 150 mm)Gradient 4: 2 min with 5% B, linear gradient from 5-50% B over 13 min, linear gradient from 50-95% B over 1 min, wash 2 min with 95% B, then back to 5% B over 1 min, calibrate with 5% B over 1 min. (C84.6 x 150 mm)Gradient 5: linear gradient from 0-20% B over 15 min, linear gradient from 20-95% B over 1 min, wash 1 min with 95% B, then back to 0% B over 0.5 min, calibrate with 0% B over 2.5 min. (C8 4.6 x 150 mm)Gradient 6: 1 min with 5% B, linear gradient from 5-90% B over 29 min, wash 2 min with 90% B, back to 5% B over 3 min. (C8 4.6 x 150 mm)Gradient 7: linear gradient 5-95% B over 15 min, wash 2 min with 95% B, back to 5% B over 1 min, calibrate with 5% B over 2 min. (C84.6 x 150mm)Gradient 8: linear gradient 5-95% B over 20 min, wash 1 min with 95% B, back to 5% B over 1 min, calibrate with 5% B over 1 min. (C84.6 x 150mm)Gradient 9: 2 min with 5% B, linear gradient from 5-50% B over 28 min, linear gradient from 50-95% B over 1 min, wash 2 min with 95% B, then back to 5% B over 1 min, calibrate with 5% B over 1 min. (C84.6 x 150 mm)Semi-preparative HPLCIn semi-preparative mode, an Agilent Eclipse XDB-C8 5 pm 9.4 x 250 mm column or Agilent ZORBAX 300SB-C18 5 pm 9.4 x 250 mm column or Agilent ZORBAX 300-C35 pm 9.4 x 250 mm column was used at a constant flow rate of 3.5 mL / min at 60 °C.Gradient 10: linear gradient 25%~100% D in C over 37 min, then wash 2 min with 100% D, then back to 25% D in C over 2 min. (C8 9.4 x 250 mm)Gradient 11: 2 min with 35% B, linear gradient 35%~65% B over 25 min, then 65%~95% B over 1 min, wash 5 min with 95% B, then back to 35% B over 2 min. (C8 9.4 x 250 mm) Gradient 12: 2 min with 30% B, linear gradient from 30%~50% B over 35 min, then 50%~95% B over 1 min, wash 5 min with 95% B, then back to 30% B over 2 min. (C8 9.4 x 250 mm) Gradient 13: 2 min with 20% B, linear gradient from 20%~95% B over 40 min, then wash 2 min with 95% B, then back to 20% B over 2 min. (C18 9.4 x 250 mm)Gradient 19: 2 min with 5% B, linear gradient from 5~45% B over 1 min, then linear gradient from 45%~85% B over 25 min, then linear gradient from 85~95% over 0.5 min, then wash 2 min with 95% B, then back to 5% over 2 min. (C3 9.4 x250 mm)Gradient 22: 3 min with 2% B, linear gradient from 2~15% B over 1 min, then linear gradient from 15%~40% B over 40 min, then linear gradient from 40~95% over 1 min, then wash 2 min with 95% B, then back to 2% over 2 min. (C8 9.4 x250 mm)Gradient 23: linear gradient 30% C in D to 100% C over 43 min, then wash 1 min with 100% C, then back to 30% D in C over 2 min. (C18 9.4 x 250 mm)Gradient 27: 1 min of 20 % B in A, then a linear gradient of 20 % - 50 % of B in A over 25 min, then a linear gradient of 50 % - 95 % B in A over 1 min, then wash with 95 % B in A over 2 min, then back to 20 % B in A over 3 min. (C8 9.4 x 250 mm, flow rate = 5 mL / min)Preparative HPLCHPLC purification of the peptides was performed on an Agilent 1260 Preparative HPLC system using a reversed phase Agilent Zorbax 300SB-C18 PrepHT 21.2 x 150 mm 7 pm column was used at a constant flow rate of 20 mL / min at room temperature.Gradient 14: linear gradient from 0%~100% F in E over 40 min, then wash 5 min with 100% F, then back to 100% E over 2 min.Gradient 15: linear gradient from 20%~70% B over 30 min, then 70%~95% B over 1 min, wash 2 min with 95% B, then back to 20% B over 2 min.Gradient 16: linear gradient from 30%~70% B over 40 min, then 70%~95% B over 1 min, wash 2 min with 95% B, then back to 30% B over 2 min.Gradient 17: 2 min with10% B, linear gradient from 10~50% B over 17 min, then linear gradient from 50~95% B over 1 min, wash 2 min with 95% B, then back to 10% B over 2 min.Gradient 20: 2 min with 2% B, linear gradient from 2~30% B over 30 min, then linear gradient from 30~95% B over 0.5 min, wash 2 min with 95% B, then back to 2% B over 2 min.Gradient 21: 2 min with 2% B, linear gradient from 2~15% B over 2 min, then linear gradient from 15~40% B over 30 min, then linear gradient from 40-95% B over 0.5 min, wash 2 min with 95% B, then back to 2% B over 2 min.Gradient 24: 5 min of 5% B in A, then a linear gradient of 5 % - 40 % of B in A over 20 min, then a linear gradient of 40 % - 95 % B in A over 2 min, then wash with 95 % B in A over 3 min, then back to 5% B in A over 2 min. (C1821.2 x 150 mm, flow rate = 20 mL / min)Gradient 25: 5 min of 5% B in A, then a linear gradient of 5 % - 50 % of B in A over 20 min, then a linear gradient of 50 % - 95 % B in A over 2 min, then wash with 95 % B in A over 3 min, then back to 5% B in A over 2 min. (C1821.2 x 150 mm, flow rate = 20 mL / min)Gradient 26: 5 min of 15% B in A, then a linear gradient of 15 % - 45 % of B in A over 20 min, then a linear gradient of 45 % - 95 % B in A over 2 min, then wash with 95 % B in A over 3 min, then back to 15% B in A over 2 min. (C18 21.2 x 150 mm, flow rate = 20 mL / min)UPLC-HRMSAll hydrazide peptides were analyzed on a Waters ACQUITY UPLC BEH C8, 1.7 pm, 2.1 x 50 mm column, the rest of peptides on a Waters ACQUITY UPLC BEH C18, 1.7 pm, 2.1 x 50 mm column, the proteins were analyzed on a Waters ACQUITY UPLC BEH C4, 300 A, 1.7 pm, 2.1 x 50 mm column with constant flow of 0.4 mL / min 40 °C. High-resolution mass spectra were recorded on a Waters Acquity UPLC-Class I equipped with an ACQUITY UPLC column. The detector was a Waters Xevo-G2-Xs QTof with electrospray ionization source. The instrument was operated in positive mode full-scan with detection window set from 50 to 2000 Da. For peptide fragmentation, a collision energy ramp from 15 V to 40 V was employed. Typical gradients are described below.Gradient 18: 0.5 min with 5% solvent H in solvent G, linear gradient from 5-95% solvent H over 4 min, wash 1 min with 95% solvent H, then back to 5% over 0.5 min and then 0.5 min with 5% solvent H.Example 1 : Synthesis of Non-Proteinogenic Amino Acids7. 1. Synthesis of tert-butyl 2-((diphenylmethylene)amino)acetate 2

[0139] To a stirred suspension of glycine tert-butylester hydrochloride 1 (1.8 g, 10.73 mmol, 1 equiv.) in dichloromethane (43 mL), benzophenone imine (1.946 g, 10.73 mmol, 1 equiv.) was added and the mixture left to stir at room temperature for 24 h. The organic layer was then washed with water (2 x 20 mL), dried over magnesium sulfate and concentrated in vacuo to give the crude title compound 2 as a white solid in quantitative yield. The crude product was directly used for the next step without any purification.1H NMR (CDCI3, 400 MHz): 5 7.68 (d, J = 7 Hz, 2H, ortho-H), 7.47 - 7.40 (m, 3H, ortho-H & para-H), 7.39 - 7.34 (m, 1 H, para-H) 7.31 (dd, J = 8.3, 6.5Hz, 2H, meta-H), 7.20 - 7.15 (m, 2H, meta-H), 4.14 (s, 2H, NCH2CO2tBu), 1.47 (s, 9H, HtBu).13C NMR (CDC , 101 MHz): 5 171.33, 169.68, 139.26, 136.04, 130.25, 128.64, 128.63, 128.51 , 127.91 , 127.57, 80.83, 77.48, 77.16, 76.84, 56.20, 27.99.HRMS (ESI+) calc, for C19H21NO2 calc, for [M+H]+: 296.1650 m / z, found: 296.1654 m / z. (21 = 1.35 ppm).The spectroscopic data agree with those reported in the literature (Fanelli, R. et al.. Stereoselective synthesis of unsaturated a-amino acids. Amino Acids 47, 1107-1115 (2015)).1.2. Synthesis of tert-butyl (S,E)-2-((diphenylmethylene)amino)hex-4-enoate 3

[0140] To a stirred suspension of intermediate 2 (200 mg, 0.68 mmol, 1 equiv.) and Corey-Lygo catalyst O-allyl- / V-(9-anthracenylmethyl) cinchonidinium bromide (41 mg, 0.068 mmol, 10 mol%.) in 3.4 mL of toluene / dichloromethane (v / v, 7:3), a solution of 9M potassium hydroxide (1.96 mL, 17.64 mmol, 26 equiv.) was added. The mixture was cooled to 0 °C and crotyl bromide (85% purity, mixture of cis & trans) (110 mg, 0.816 mmol, 1.2 equiv.) was slowly added. The reaction mixture was vigorously stirred for 24 h at 0 °C. After concentration, the residue was diluted in water (30 mL) and diethyl ether (30 mL). The aqueous layer was then extracted with diethyl ether (3 x 45 mL), and the organic layer was washed with water (2 x 45 mL), dried over magnesium sulfate and concentrated to give the title compound 3 as a pale-yellow oil in quantitative yield, the crude was directly used for the next step without any purification.1H NMR (CDCI3, 400 MHz): 5 7.66 (d, J = 8.0 Hz, 2H), 7.48 - 7.41 (m, 3H), 7.40 - 7.35 (m, 1 H), 7.36 - 7.29 (m, 2H), 7.22 - 7.13 (m, 2H), 5.57 - 5.43 (m, 1 H, CH-C4), 5.34 (m, 1H, CH-C3), 4.05 - 3.80 (m, 1 H, CH-C1), 2.58 (m, 2H, CH2-C2), 1.62 (d, J = 6.32 Hz, 3H, CH3- C5), 1.45 (s, 9H, HtBu).13C NMR (CDCh, 101 MHz): 5 171.20, 169.90, 139.91, 136.89, 130.21 , 129.13, 128.90, 128.55, 128.44, 128.33, 128.07, 128.02, 127.90, 127.19, 80.92, 66.49, 37.10, 28.19, 18.09.HRMS (ESI+) calc, for C23H27NO2 calc, for [M+H]+: 350.2120 m / z, found: 350.2122 m / z. (21 = 0.58 ppm)[a]D25= - 59.5 (c 1.0, CHC )

[0141] To a solution of intermediate 3 (267 mg, 0.7 mmol, 1 equiv.) in tetrahydrofuran (1.4 mL) was added a solution of 15% citric acid (4.2 ml_). The mixture was stirred at room temperature for 7 h. Then the THF was evaporated, and the residue was washed with diethyl ether (3 x 10 mL). The pH of aqueous solution was increased to 9 with 1M sodium carbonate before extracted with ethyl acetate (4 x 15 mL). The combined organic phases were concentrated at room temperature in vacuo. The residue and Fmoc- OSu (306 mg, 0.91 mmol, 1.3 equiv.) were dissolved in ethyl acetate (3.5 mL) then triethylamine (213 mg, 2.1 mmol, 3 equiv.) was added. The reaction mixture was stirred at room temperature overnight. The mixture was then washed with saturated sodium bicarbonate (3 x 15 mL). The organic phase was washed with brine (20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification of the residue by flash column chromatography on silica gel (hexane : ethyl acetate, 6:1) afforded the title compound S1 (181 mg, 64%, E : Z = 4 : 1 , 97% e.e.) as a colourless oil. (Supplementary Fig. 28)1H NMR (CDCI3, 400 MHz): 5 7.77 (d, J = 7.5 Hz, 2H), 7.62 (dd, J = 7.5, 2.9 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 5.74 - 5.50 (m, 1H, CH-C4), 5.42 (d, J = 8.1 Hz, 1 H, NW), 5.49 - 5.30 (m, 1 H, CH-C3), 4.51 - 4.30 (m, 3H), 4.25 (t, J = 7.2 Hz, 1 H), 2.73 - 2.42 (m, 2H, CH2-C2), 1.66 (d, J = 6.7 Hz, 3H, CH3-C5), 1 .48 (s, 9H, Heu).13C NMR (CDCI3, 101 MHz): 6 171.08, 155.72, 144.02, 143.93, 141.35, 129.87, 127.74, 127.10, 125.22, 125.20, 124.64, 120.03, 82.09, 66.98, 54.02, 47.24, 35.86, 28.11, 18.08.HRMS (ESI+) calc, for C25H29NO4 calc, for [M+H]+: 408.2175 m / z, found: 408.2173 m / z (A = -0.5 ppm)HPLC (AD-H chiral column, hexane (5%)-isopropyl isocratic): 27.889 min.1.4. Synthesis of tert-butyl (E -2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hex-4-enoate S3(Synthesis of racemic mixture for chiral HPLC comparison)

[0142] To a stirred suspension of intermediate 2 (100 mg, 0.34 mmol, 1 equiv.) in dry tetra hydrofuran (2 ml_) at -78 °C, lithium diisopropylamide (2 M in THF) (0.25 mL, 0.51 mmol, 1.5 equiv.) was slowly dropwise added, then the mixture was incubate for 30 min, crotyl bromide (85% mixture of cis & trans) (55 mg, 0.41 mmol, 1.2 equiv.) was added. The reaction mixture was stirred for 12 h at -78 °C. The reaction was quenched with 2 mL of water, then tetrahydrofuran was evaporated, the residue was diluted in diethyl ether (5 mL). The aqueous layer was then extracted with diethyl ether (3 x 5 mL), combined organic layer dried over magnesium sulfate and concentrated. Then the resulting crude mixture S2 was directly used to prepare S3 with the same procedure as synthesis of S1.1H NMR (CDCh, 400 MHz): 5 7.77 (d, J = 7.5 Hz, 2H), 7.61 (dd, J = 7.5, 2.9 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 5.60 - 5.51 (m, 1 H, CH-C4), 5.45 - 5.24 (m, 2H, CH-C3 & NH), 4.52 - 4.28 (m, 3H), 4.24 (t, J = 7.2 Hz, 1H), 2.50 (m, 2H, CH2-C2), 1.67 (d, J = 6.6 Hz, 3H, CH3-C5), 1.48 (s, 9H, HtBu).13C NMR (CDCh, 101 MHz): 5 171.12, 155.75, 144.07, 143.98, 141.40, 129.96, 127.79, 127.15, 125.27, 125.24, 124.64, 120.08, 82.16, 67.03, 54.04, 47.29, 35.91, 28.19, 28.16, 18.13.HRMS (ESI+) calc, for C25H29NO4 calc, for [M+H]+: 408.2175 m / z, found: 408.2173 m / z (A = -0.5 ppm)1.5. Synthesis of (S.E)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hex-4-enoic acid 4FmocHN

[0143] To a solution of intermediate S1 (363 mg, 0.89 mmol, 1 equiv.) in dichloromethane (5 mL) at 0 °C, trifluoroacetic acid (3.4 mL) was slowly added. The resulting mixture was stirred at 0 °C and monitored by thin layer chromatography (hexane :ethyl acetate, 6:1). Once all the starting material consumed, the mixture was concentrated in vacuo to afford the title product 4 as a white solid in quantitative yield. The crude was directly used for the solid phase peptide synthesis without any purification.1H NMR (CD3OD, 400 MHz): 5 7.76 (d, J = 7.8 Hz, 2H), 7.64 (dd, J = 7.4, 4.7 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.29 (td, J = 7.4, 1.3 Hz, 2H), 5.62 - 5.52 (m, 1 H, CH-C4), 5.47 - 5.28 (m, 1H, CH-C3), 4.40 - 4.26 (m, 2H), 4.23 - 4.12 (m, 2H), 2.65 - 2.44 (m, 1 H, CH-C2), 2.42 - 2.26 (m, 1 H, CH-C2), 1.66 - 1.58 (d, J = 6.4 Hz, 3H, CH3-C5).13C NMR (CD3OD, 101 MHz): 6 175.57, 158.43, 145.29, 145.19, 142.54, 129.86, 128.74, 128.12, 127.00, 126.22, 120.88, 67.97, 55.62, 48.36, 35.97, 18.13.HRMS (ESI+) calc, for C2IH2I NO4calc, for [M+H]+: 352.1549 m / z, found: 352.1543 m / z (4 = -1.7 ppm)[a]o25= + 4.08 (c 1.0, CHC )1.6. Synthesis of methyl Na-(tert-butoxYcarbonyl)-1-(2-methylbut-3-en-2-yl)-L- tryptophanate 66

[0144] To a stirred suspension of methyl (fert-butoxycarbonyl)- / _-tryptophanate 5 (200 mg, 0.63 mmol, 1 equiv.), copper (II) acetate (229 mg, 1.26 mmol, 2 equiv.), silver trifluoromethanesulfonate (324 mg, 1.26 mmol, 2 equiv.) and palladium (II) acetate (14 mg, 0.063 mmol, 10 mol%.) in dry acetonitrile (30 ml_), 2-methyl-2-butene (133 mg, 1.89 mmol, 3 equiv.) was added at 36 °C. Then followed by three sequential additions of palladium (II) acetate (10 mol% after each hour). The resulting mixture was stirred at 36 °C for 24 h. The mixture then concentrated in vacuo and directly purified by flash column chromatography on silica gel (hexane : ethyl acetate, 6:1) afforded the title compound 6 (136 mg, 59%) as a yellowish oil.1H NMR (CDCI3, 400 MHz): 6 7.52 (ddd, J = 18.3, 7.3, 2.0 Hz, 2H), 7.15 - 7.05 (m, 3H), 6.14 (dd, J = 17.5, 10.7 Hz, 1 H, CH-C4), 5.26 - 5.10 (m, 2H, CH2-C5), 5.07 (d, J = 8.4 Hz, 1 H, NH), 4.66 (dt, J = 8.8, 5.8 Hz, 1 H, CH-C1), 3.68 (s, 3H), 3.38 - 3.16 (m, 2H, CH2-C2), 1.73 (s, 6H, CH3-C6&C7), 1.44 (s, 9H, HBoc).13C NMR (CDCh, 101 MHz): 5 172.97, 155.33, 144.21 , 135.65, 129.77, 123.92, 120.98, 119.06, 118.93, 113.89, 113.61 , 108.26, 79.85, 59.10, 54.47, 52.24, 28.47, 28.29, 28.05.HRMS (ESI+) calc, for C22H30N2O4 calc, for [M+H]+: 387.2284 m / z, found: 387.2281 m / z. (21 = - 0.77 ppm)1.7. Synthesis of Na-(((9H-fluoren-9-yl)methoxy)carbonyl)- 1-(2-methylbut-3-en-2-yl)-L- tryptophan 7

[0145] To a solution of intermediate 6 (211 mg, 0.546 mmol, 1 equiv.) in dry methanol (4 mL), thionyl chloride (0.06 mL, 0.819 mmol, 1.5 equiv.) was slowly added at 0 °C. The solution was gently heated up to 50 °C, then left for 2 h. The mixture was concentrated in vacuo then diluted with dichloromethane and water (10 mL each) and adjust to pH 12 with 1M sodium hydroxide. The organic layer was separated, washed with water and brine (15 mL each), dried over magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was then dissolved in tetrahydrofuran (26 mL), 1M lithium hydroxide (8.2 mL, 8.2 mmol, 15 equiv.) was added over 3 min at 0 °C. The mixture was left for 2 h, then tetrahydrofuran was evaporated. The residues were suspended in dichloromethane (19 mL) and 1 M sodium carbonate (19 mL), followed by addition of Fmoc-CI (fluorenylmethyloxycarbonyl chloride) (155 mg, 0.6 mmol, 1.1 equiv.). The resulting mixture was stirred vigorously for 15 min, then was acidified to pH 5 using sulfuric acid. Then dichloromethane was evaporated, the residues were extracted with ethyl acetate (3 x 30 mL), the combined organic phase washed with water (100 mL) and brine (100 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo to afford the title compound 7 as a brown solid. The crude was directly used for the solid phase peptide synthesis without any purification.1H NMR (CD3OD, 400 MHz) 5 7.75 (d, J = 7.6 Hz, 2H), 7.61 - 7.57 (m, 1 H), 7.55 (d, J = 7.6 Hz, 2H), 7.47 - 7.42 (m, 1 H), 7.34 (t, J = 7.1 Hz, 2H), 7.26 (s, 1 H), 7.25 - 7.17 (m, 2H), 7.08 - 6.94 (m, 2H), 6.08 (dd, J = 17.5, 10.7 Hz, 1 H, CH-C4), 5.16 - 5.01 (m, 2H, CH2-C5), 4.51 (dd, J = 8.8, 4.7 Hz, 1 H, CH-C1), 4.30 - 4.15 (m, 2H), 4.11 (t, J = 7.0 Hz, 1 H), 3.36(dd, J = 14.8, 5.0 Hz, 1 H, CH-C2), 3.13 (dd, J = 14.7, 9.0 Hz, 1 H, CH-C2), 1.66 (s, 6H) ppm.13C NMR (CD3OD, 101 MHz) 6 175.66, 158.43, 145.52, 145.19, 145.16, 142.48, 136.98, 130.80, 128.73, 128.15, 126.28, 126.21, 125.24, 121.62, 120.87, 119.82, 119.58, 114.96, 113.82, 110.13, 68.04, 60.02, 56.25, 48.28, 28.68, 28.33, 28.30.HRMS (ESI+) calc, for C31H30N2O4 calc, for [M+H]+: 495.2284 m / z, found: 495.2272 m / z. (21 = -2.4 ppm)Example 2: General Procedure for Solid Phase Synthesis of Linear Rufomycin Peptides and Derivatives

[0146] Solid phase peptide synthesis was performed either manually in a Bio-Rad Poly-Prep® polypropylene column with nitrogen swelling or on a Biotage I nitiator+ Alstra peptide synthesizer at room temperature, using standard Fluorenylmethoxycarbonyl (Fmoc) strategy.2. 1. General procedure of hydrazine functionalizing 2-chlorotrityl resin

[0147] 2-chlorotrityl chloride resin (substitution: 1.06 mmol / g) (200 mg, 0.2 mmol, 1 equiv.) were gently stirred in dimethylformamide (DMF) (1 mL) at 0 °C for 10 min. A mixture of triethylamine (84 pL, 0.6 mmol, 3 equiv.) and hydrazine hydrate (22 pL, 0.4 mmol, 2 equiv.) in DMF (1 mL) was added dropwise at 0 °C, and then the resulting mixture was gently stirred for 1 h at room temperature. Methanol (2 mL) was added and stirred for 15 min to quench the excess of reactive chloride sites on the resin. The resin was filtered by vacuum, washed with DMF (2 x 5 mL), water (2 x 5 mL), DMF (2 x 5 mL), methanol (2 x 5 mL) and DMF (2 x 5 mL).4Then, the C-terminal Fmoc amino acid was loaded manually, a mixture of Fmoc amino acid (0.4 mmol, 2 equiv.), K-Oxyma (72 mg, 0.4 mmol, 2 equiv.) and / V, / V'-Diisopropylcarbodiimide (DIG) (63 pL, 0.4 mmol, 2 equiv.) in DMF (1 mL) was added to the resin then swelling the mixture with nitrogen flow for 45 min. The resin was filtered and double coupled using the same procedure. After that, the resin was filtered and washed with DMF (5 x 2 mL) (Batch-Flow-Flow-Batch with DMF, batch washes for 1 min, flow washes for 30 sec).2.2. General manual peptide coupling method and Fmoc deprotection (50 mg resin scale)

[0148] Typically, to a solution of the Fmoc-amino acid (4 equiv.) and hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (3.9 equiv.) in DMF, / V. / V-diisopropylethylamine (DI PEA) (8 equiv.) was added and mixed by shaking for 2 min.The resulting pre-activated amino acid solution was added to the resin mixture followed by swelling under nitrogen for 45 min at room temperature. A small portion of resin (~ 2 mg) was taken into a test tube to check the reaction completion by Kaiser Test (Kaiser et al., Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598, 1970) or Chloranil Test (Vojkovsky, T., Detection of secondary amines on solid phase. Pept. Res. 8, 236-237, 1995) for secondary amine. Recoupling was carried out for incomplete coupling result. For Fmoc deprotection, the resin was swelled in 20% piperidine in DMF (volume used is 3 times resin volume), swelling twice at room temperature (the first deprotection for 3 min followed by an additional deprotection for 12 min). The resin was filtered and washed with DMF (5 x 2 mL) (Batch-Flow-Flow-Batch with DMF, one batch wash for 1 min, one flow wash for 30 sec).2.3. General automatic peptide coupling method and Fmoc deprotection (200~3Q0 ma resin scale)

[0149] Automatic peptide synthesis was carried out on a Biotage I nitiator+ Alstra, typically, each Fmoc-amino acid (4 equiv.) was coupled at room temperature for 1 h with DIC (4 equiv.) and oxyma (4 equiv.). Fmoc deprotection was accomplished with 20% piperidine in DMF twice (one deprotection for 1 min followed by an additional deprotection for 15 min).2.4. General peptide cleavage from resin

[0150] Peptidyl resins (300 mg) were treated with 6 mL of cleavage cocktail. Cocktail A (1% TFA, 2% TIS and 97% DCM) for peptides containing / V-prenylated tryptophan and Cocktail B (95% TFA, 2.5% TIS and 2.5% water) for other peptides. The resulting solution was shaken at room temperature for 1 h. The resin was filtered and washed with DCM (2 x 6 mL) and the filtrates were evaporated under a nitrogen stream. Cold diethyl ether (3 x 10 mL) was added to precipitate hydrazide peptide as a yellowish powder (color from the presence of 3-nitro tyrosine). The resulting peptides were directly used for further synthesis without any purification. Except for hydrazide peptide 9a, 14a, 15a and 16a, which were used for later chemical cyclization study. The purification of a small portion was carried out on semi-prep C8 column use HPLC using different gradient.Gradient 4 for hydrazide peptide 9aGradient 5 for hydrazide peptide 14a and 15aGradient 6 for hydrazide peptide 16a2.5 General procedure for SNAC-peptide synthesis (comparative example)

[0151] All the buffer solutions were freshly prepared and degassed before using. It should be noted 5 M sodium nitrite stock was prepared as using degassed water to dissolve sodium nitrite. Typically, to a suspension of crude hydrazide linear peptide (1 equiv., 1.5 - 4 mM) in phosphate buffer pH 3 (6 M GdmHCI, 50 mM NaHzPC , 1.5 mM EDTA) was added sodium nitrite stock (20 equiv.) at -15 °C. Then the solution was left to stir for 20 min before / V-acetylcysteamine (50 equiv.) was added. The resulting mixture was stirred for 15 min at 0 °C, then neutralized with saturated sodium bicarbonate and extracted with ethyl acetate (3 x 10 mL). Additonal water was added to facilitate better separation. Combined organic layers were concentrated in vacuo at room temperature and redissolved in 80% acetonitrile in H2O, then purified through HPLC using different gradient.Gradient 7 for SNAC peptide 8bGradient 10 for SNAC peptide 10b (Both D-1Me-Trp and Z--1Me-Trp)Gradient 11 for SNAC peptide 12bGradient 12 for SNAC peptide 9b, 14b and 15bGradient 13 for SNAC peptide 11 b and 13bRepresentative Example 3: General Procedure for RufT-TE catalysed peptide cyclisationSNAC peptide b Cyclic peptide c Hyrfrofyz&d peptide 4

[0152] Analytical enzymatic cyclization was performed at a total volume of 200 pL Typically, to SNAC peptide b (50 pM final cone.) in Tris Buffer (25 mM, 300 mM NaCI, pH = 8) containing 5 % DMSO, RufT-TE (12.5 pM final cone.) was added to initialize the reaction. The resulting mixture was incubated for 3 h at 30 °C with shaking 800 rpm. The reaction was extracted with ethyl acetate (3 x 400 pL). The combined organic layers were dried and redissolved in 40 pL 80% acetonitrile in H2O. The sample was analyzed viaHPLC using Gradient 1. All bioassays were carried out using the same batch of the purified enzymes. The negative control used the same conditions as above but RufT-TE was heat inactivated by heating the protein at 100 °C for 15 min.Representative Example 4: Silver assisted peptide cyclisation.

[0153] The procedure used was developed by Tam and co-workers J. Am. Chem. Soc. 121, 3311-3320, 1999), as depicted in Figure 4A. SNAC peptide 9b (final cone. 0.2 mM) was dissolved in 1 mL of a 1 :1 mixture of DMSO and sodium acetate buffer (0.5 M, pH 7.5). Large amount of DMSO essential for SNAC peptide solubility. Silver trifluoroacetate (3 equiv.) was added. The resulting mixture was shaken at room temperature for 1h. The crude mixture was directly analyzed using Gradient 1. HPLC analysis after 1 hr at room temperature showed that the reaction was incomplete and produced a mixture of products resulting from cyclization (15% 9c), hydrolysis (13% 9d) and dimerization (11% dimer). This is demonstrated in Figure 4C.Example 5: General Procedure for Bioinspired Peptide Cyclisation (Analytical scale)Acyt-hydrazide peptide a Acyl-az fe peptide S Cyclic peptide c

[0154] All buffers and solutions were freshly prepared and degassed before using. It should be noted 5 M sodium nitrite stock was prepared using degassed water to dissolve sodium nitrite. To a solution of purified hydrazide linear peptide (2.2 pmol, 1 equiv.) in 1 mL of pH 3 phosphate buffer (50 mM NaH2PC>4, 1.5 mM EDTA) was added sodium nitrite stock (44 pmol, 20 equiv.) at -15 °C under nitrogen, optionally in the presence of 6 M GdmHCl. Then the solution was stirred for 15 min at -15 °C. The mixture was gently neutralized with saturated sodium bicarbonate to pH 7~7.5. 500 pL was removed and diluted with 1 mL of 50% solvent B in A in a HPLC sample vial. Then the reaction was analysed by HPLC using Gradient 2 or Gradient 3.

[0155] Figure 4B depicts this general procedure being applied to acyl-hydrazide peptide 9a to produce cyclised peptide 9c via the reactive acyl-azide intermediate S9. Theacyl-hydrazide peptide 9a (2.2 mM) was oxidized using sodium nitrite in guanidinium containing phosphate buffer at pH 3, to generate intermediate S9. In the same pot, the pH was raised to 7~7.5 using saturated sodium bicarbonate, initiating cyclization by partially neutralizing the terminal amine. The acyl-azide peptide was consumed shortly after oxidation, resulting in cyclization (63% 9c) and hydrolysis (37% 9d) and no dimerization was detected. Without wishing to be bound by theory, it is thought that the low yield observed was due to issues with regards to the solubilities of both the hydrazide- and azide-peptides 9a and S9. Many peptides are rich in hydrophobic amino acids and so have poor aqueous solubility. Where this is the case, a solubiliser such as guanidinium may be added to the reaction medium to maintain the solubility of both hydrazide- and azide-peptides. Therefore, the reaction mixture was diluted with 50% acetonitrile in water and 6 M GdmCI. At around pH 7, acyl-azide peptide S9 was converted to cyclic peptide 9c (90%) after 16 mins with low levels of hydrolyzed peptide 9d (10%), as demonstrated in Figure 4D and Figure 5.Example 6: Peptide Conformation Studies

[0156] To demonstrate the effect of N-methylation on cyclisation, macrocyclization reactions of two peptides 14a and 15a were monitored (Figures 6A and 6C). Removing one methyl group from the peptide backbone was sufficient to slow down the reaction, allowing the detection of the acyl azide intermediate (S14 and S15) after 16 min (Figures 6B and 6D). This data confirms that backbone methylation and thus peptide conformation can affect macrocyclization.Example 7: General Procedure for Bioinspired Peptide Cyclization (Semi-prep Scale)

[0157] To produce mg of cyclic peptide and facilitate full characterization and enzymatic derivatization, the macrocyclization reaction was scaled up. A biphasic process was employed due to the improved conversion and separation of the cyclic peptide into the organic phase that was previously observed.

[0158] To test this on a small scale, a HPLC purified hydrazide peptide 9a was neutralized to pH 7 and ethyl acetate (1 vol.) was added to the reaction flask, creating two phases. The reaction was maintained at moderate temperature (-15 °C to 10 °C) to minimize side reactions e.g. Curtius rearrangement and hydrolysis. After 16 min, the ethyl acetate was separated and evaporated. HPLC analysis of the crude reaction indicated complete consumption of the acyl azide. Without wishing to be bound by theory, it is though that the selectivity of the cyclization of the reaction (cyclization over dimerization) ispartly explained by the preorganization of the peptide in solution. Additionally, it is thought that the biphasic conditions further improve the reaction profile. Without wishing to be bound by theory, it is thought that preorganization increases the proximity between the N and C termini of peptide S9 and aids rapid cyclisation.

[0159] For peptides S14 and S15, where the termini are more distant, the acyl azide was still detectable after 1h under room temperature. It is thought that the presence of organic solvent shifts the equilibrium in the reaction in a process analogous to Schotten Baumann conditions (Schotten, C. Ber. Dtsch. Chem. Ges. 1884, 17, 2544-2547; Baumann, E. Ber. Dtsch. Chem. Ges. 1886, 19, 3218-3222). Thus, on neutralizing the reaction to pH 7, it is thought that a fraction of the linear peptide becomes neutral via deprotonation of the terminal amine (pKaof free L-Trp amine is 9.39 but predicted pKa of / V terminal amine of peptide 9a is 7.3 (Data for Biochemical Research, Oxford, Clarendon Press, 1959) (Figure 7). This uncharged peptide moves into the organic phase where, isolated from guanidinium, it cyclizes quickly by adopting a preferred conformation. This process also further dilutes the peptide, reducing the possibility of dimer formation. Irreversible cyclization in turn shifts the equilibrium of the amine protonation, pulling more peptide into the organic phase.

[0160] To test the applicability of the method to the synthesis of other non- ribosomal peptides, linear tyrocidine corresponding to the final covalently bound peptide on the NRPS (TycC) was synthesised. It was found that tyrocidine was similarly cyclized within 16 minutes, with even higher conversion to tyrocidine A 18c (Figures 7B and 7C). Although, tyrocidine contains an internal nucleophile which could compete with the macrocyclization reaction, no branched cyclisation products were detected. Without wishing to be bound by theory, it is thought that the macrocyclization of 18a to 18c is conformationally driven by intra molecule hydrogen bonds within the linear peptide and also the macrolactamization between D-amino acid and L-amino acid (D-Phe to L-Leu) facilitate cyclization. This method compares very favorably with previous reports of tyrocidine chemical synthesis, which have reaction times of 2 h, 6 h and overnight (Org. Lett. 4, 2893-2895, 2002; J. Comb. Chem. 5, 353-355, 2003; and J. Comb. Chem. 11 , 1066-1072, 2009).Example 8: Impact of side chains and sterics

[0161] To investigate the impact of side chains, a series of linear rufomycin derivatives were synthesized and were subjected to chemical cyclization in their crude form (Fig. 9A-C). The conversion and rate of macrocyclization were compared (Fig. 9D, 5E).

[0162] Peptides 19 and 21 were synthesized by changing replacing residues on peptide 9 with either leucine (L), alanine (A) or glycine (G). Both peptides were cyclized with comparable conversion but with different reaction times (Fig. 9B & 9D), indicating that A / -methylation influences cyclisation in rufomycins, but also that side-chain interactions play a role.

[0163] In peptide 20, the methylated amides were replaced with the turn-inducing residue L-proline (P). The cyclization of peptide 20 was accomplished, but at a slower rate (requiring 40 min to complete) than compounds 19 and 21. Without wishing to be bound by theory, it is thought that this difference in reaction time is the result of interconversion between proline cisltrans isomers, negatively impacting the ability of the peptide to achieve a pro-cyclisation conformation.

[0164] Peptide 22 introduces L-glycine at the C-terminus, thereby removing the chiral centre to reduce steric hindrance and increase flexibility. An increased rate of reaction of the cyclisation of peptide 22 was observed relative to peptide 19, but the ratio of cyclic to hydrolyzed products was unaffected.Example 9: Varying ring size and modes of cyclisation

[0165] The applicability of this methodology to targets of varying ring size and modes of cyclisation was investigated. Without wishing to be bound by theory, high N- methylation of the modified peptide backbone is thought to conformationally drive cyclization.

[0166] Two head to tail cyclized peptides of varying ring size were synthesized: tyrocidine A 18c and gramicidin S 29c each contain internal alternative nucleophiles in the form of L-lysine or L-ornithine side chains. The linear peptides were synthesized corresponding to the final covalently non-ribosomal peptide synthetase bound peptide (; Mootz et al., J Bacteriol 1997, 179 (21), 6843-6850, Kratzschmar J et al J Bacteriol. 1989, 171 , 5422-9 and subjected to macrocyclization. Both produced >80% conversion in 20 min. No branched cyclization products were detected. Without wishing to be bound by theory, this suggests that conformation and controlled pH conditions enable selective regiocontrol; due to well-understood differences in pK3H between the / V-terminal and side chain amines, only the / V-terminus will be deprotonated to a sufficient degree at pH 7. It is thought that this regiocontrol may be difficult to achieve in pure organic solvent where the N terminus and amine sidechains would be similarly deprotonated. While tyrocidine contains no backbone / V-methylation, the linear peptide is highly organized via intra-molecular hydrogen bonds and the presence of an L-proline residue (Bu et al., Org. Let. 2002, 4 (17), 2893-2895).

[0167] Head-to-side chain cyclisation was investigated using the linear fully deprotected acyl-azide peptide 30a, synthesized according to biosynthetic logic (Tambadou et al., Arch Microbiol 2015, 197 (4), 521-532). A commercially available lipid was used (a regio isomer of the natural lipid) (Fig. 11 A). The peptide was subjected to cyclisation, and HPLC analysis at 1 min revealed complete conversion of the acyl-azide and formation of 3 major products (Fig. 11B (ii)). The most intense peak correlated to the cyclized product 30c (56%) identified by MSEfragmentation and by comparison to a commercial standard, the native cyclized product 30g (Fig. 11 B & 11C). Without wishing to be bound by theory, it is thought that the two byproducts 30e and 30f formed likely represent a larger macrocyclization (17 %) and a smaller macrolactam likely formed by the adjacent Dab residue as a competing nucleophile (21%), respectively. It is thought that this regioselectivity and reaction rate is achieved due to conformational bias.

[0168] The cyclization method was expanded other compounds for which the linear biosynthetic peptide precursors, and thus the point of cyclisation, was unknown. In particular, the plant ribosomal natural product pseudostellarin A 24c, the reversed natural product cyclopurpuracin 26c and the cyanobacterial natural product planktocyclin 27c were all produced in good yield. Reversed cyclopurpuracin 26c contains two proline residues; the slow rate of reaction for 26c (i.e. complete conversion of the linear acyl azide was only achieved after 100 minutes) agrees with the slower cyclisation rate of the rufomycin analogue 20, which also contained two proline residues.Example 10: Synthesis of cyclised peptides 8c, 9c, 10c, 11c, 12c, 13c, 17c and 18cPeptide 8c

[0169] To a suspension of crude hydrazide linear peptide 8a (1 equiv.) in 10 mL of phosphate buffer (6 M GdmHCI, 50 mM NaFkPC , 1.5 mM EDTA, pH 3) was added sodium nitrite (20 equiv.) at -15 °C under nitrogen. The resulting solution was stirred for 20 min at -15 °C before gently raising up the pH to 7~7.5 with saturated sodium bicarbonate. After 5 min, the solution was extracted with ethyl acetate (3 x 15 mL). The combined organic layers were concentrated in vacuo at room temperature and redissolved in 80% acetonitrile in water, then purified through preparative HPLC using Gradient 13. After lyophilization, 11 mg of rufomycin B 8c was harvested as a yellowish powder. (HPLC yield: 84%, isolated yield: 54% upon assuming crude hydrazide is 70% pure).1H NMR(CD3OD, 700 MHz) 57.79 (d, J =2.2 Hz, 1H, CH-C42), 7.54-7.50 (m, 2H, CH- 062 & CH-C65), 7.40 (dd, J= 8.7, 2.3 Hz, 1H, CH-C46), 7.13 (s, 1H, CH-C61), 7.09 - 7.00 (m, 3H, CH-C45, CH-063 & CH-C64), 6.16- 6.10 (m, 1H, CH-C67), 5.52-5.44 (m, 1H, CH-C52), 5.23 - 5.13 (m, 3H, CH-051 & CH2-C68), 4.94 - 4.91 (m, 1H, CH-C28), 4.89 - 4.87 (m, 1H, CH-C2, overlap with water), 4.89-4.84 (m, 1H, CH-C12, overlap with water), 4.72-4.69 (m, 1H, CH-C22), 4.63-4.50 (m, 3H, CH-C31, CH-C17, CH-C36), 3.42 (dd, J = 13.3, 11.2 Hz, 1H, CH-C39), 3.16 (dd, J= 13.3, 4.4 Hz, 1H, CH-C39), 3.05 (dd, J= 13.3, 9.6 Hz, 1H, CH-C40), 2.75 - 2.72 (m, 5H, CH-C38, CH-C40, CH3-CIO), 2.67 (s, 3H, CH3- C26), 2.47-2.40 (m, 1H, CH-C38), 1.98-1.95 (ddd, J= 13.7, 7.5, 5.9 Hz, 1H, CH-C1), 1.81 -1.69 (m, 9H, CH2-C37, CH-C54, CH3-C69 & CH3-C70), 1.64 - 1.61 (m, 1 H, CH- 023), 1.58-1.55 (m, 3H, CH3-C53), 1.54-1.51 (m, 1H, CH-C1), 1.51 - 1.45 (m, 1H, CH- 05), 1.23 (d, J= 6.8 Hz, 3H, CH3-C15), 1.10-1.04 (m, 1H, CH-048), 1.00-0.91 (m, 12H, CH3-C55, CH3-C56, CH3-C7 & CH3-C8), 0.38 (d, J = 6.8 Hz, 3H, CH3-C50 or CH3-C49), 0.18 (d, =6.7 Hz, 3H, CH3-C49 orCH3-C50), -0.60- -0.66 (m, 1H, CH-023).13C NMR (CD3OD, 176 MHz) 5175.08 (011), 174.00 (C34), 173.95 (025), 173.48 (C30), 172.31(016), 171.55 (C3), 169.80 (C20), 154.65 (044), 145.41 (C67), 139.27 (C46), 136.94 (059), 135.53 (C43), 131.34 (052), 130.42 (058), 129.53 (C41), 126.80 (042), 125.47 (C61), 125.30 (C51), 122.15 (064), 121.14 (C45), 120.36 (C63), 119.46 (062), 115.32 (C65), 114.12 (C68), 108.94 (057), 60.21 (C66), 59.99 (C2), 59.79 (022), 55.55 (017), 55.00 (C36), 53.45 (C31), 51.90 (C28), 46.76 (C12), 44.38 (037), 38.72 (C1), 38.49 (023), 38.20 (C40), 36.22 (C38), 29.88 (C10), 29.82 (C26), 28.53 (069 or C70), 28.42 (069 or C70), 28.28 (039), 25.98 (C54), 25.79 (C5), 25.47 (048), 23.49 (C50 or C49), 23.39 (C7 or C8), 23.25 (055 or C56), 23.06 (07 or 08), 21.96 (055 or C56), 21.02 (049 or 050), 18.64 (C53), 17.01 (015).As previously reported for natural rufomycins, this cyclic peptide appears as more than one conformer. The major species ~ 80% by NMR is assigned.HRMS (ESI) calc, for C54H77N9O10 calc, for [M+H]+: 1012.5872 m / z, found: 1012.5898 m / z.(Z1 = 2.56 ppm)Purification: Purified through preparative HPLC using Gradient 14.Peptide 9cSynthetic method:: Peptide 9a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 9a was present in solution at 3 mM concentration (10 mL reaction buffer).9c was isolated after HPLC purification as a yellowish powder. (HPLC conversion (90%), isolated yield (56%)).1H NMR (CD3OD, 700 MHz) 57.79 (d, J = 2.2 Hz, 1 H, CH-C42), 7.54 - 7.50 (m, 2H, CH- 061 &CH-C64), 7.42 (dd, J= 8.6, 2.3 Hz, 1H, CH-C46), 7.12 (s, 1H, CH-C60), 7.08-7.01 (m, 3H, CH-C45, CH-C62 & CH-C63), 6.16-6.11 (m, 1H, CH-C66), 5.65-5.57 (m, 1H, CH-C51), 5.22 - 5.14 (m, 2H, CH2-C67), 5.13 - 5.06 (m, 2H, CH2-C52), 4.93 (ddd, J = 11.2, 4.5, 2.1 Hz, 1H, CH-C28), 4.89 (dd, J= 8.2, 5.9 Hz, 1H, CH-C2), 4.86 (m, 1H, CH- 012, overlap with water), 4.72 - 4.65 (m, 2H, CH-C22 & CH-C31), 4.65 - 4.56 (m, 2H, CH- 017 &CH-C36), 3.43 (dd, J= 13.3, 11.1 Hz, 1H, CH-C39), 3.16 (dd, J= 13.3, 4.3 Hz, 1H, CH-C39), 3.07 (dd, J = 13.4, 9.2 Hz, 1 H, CH-C40), 2.83 - 2.77 (m, 1 H, CH-C38), 2.76 - 2.71 (m, 4H, CH-C40 & CH3-CIO), 2.60 (s, 3H, CH3-C26), 2.56- 2.49 (m, 1H, CH-C38), 1.96 (ddd, J= 13.6, 7.4, 5.7 Hz, 1H, CH-C1), 1.79- 1.68 (m, 9H, CH2-C37, CH-C53, CH3- C68 & CH3-C69), 1.64-1.58 (m, 1H, CH-C23), 1.57-1.52 (m, 1H, CH-C1), 1.52-1.45 (m, 1H, CH-C5), 1.24 (d, J= 6.9 Hz, 3H, CH3-C15), 1.10-1.05 (m, 1H, CH-C48), 1.00- 0.93 (m, 12H, CH3-C54, CH3-C55, CH3-C7 & CH3-C8), 0.38 (d, J= 6.7 Hz, 3H, CH3-C50 or CH3-C49), 0.18 (d, J= 6.6 Hz, 3H, CH3-C49 orCH3-C50), -0.59- -0.67 (m, 1H, CH-C23).13C NMR (CD3OD, 176 MHz) 5175.14 (C11), 174.05 (C34), 173.79 (C25), 173.36 (C30), 172.47 (C16), 171.56 (C3), 169.69 (C20), 154.63 (C44), 145.44 (C66), 139.26 (C46),136.97 (C58), 135.52 (C43), 133.15 (C51), 130.43 (C57), 129.76 (C41), 126.78 (C42), 125.48 (C60), 122.15 (C63), 121.10 (C45), 120.44 (C52), 120.35 (C62), 119.46 (C61), 115.32 (C64), 114.10 (C67), 109.00 (C56), 60.21 (C65), 60.03 (C2), 59.81 (C22), 55.60 (C17), 54.89 (C36), 53.26 (C31), 51.77 (C28), 46.80 (C12), 44.37 (C37), 38.70 (C1), 38.63 (C23), 37.90 (C40), 37.52 (C38), 29.89 (C10), 29.73 (C26), 28.52 (C68 or C69), 28.42 (C68 or C69), 28.21 (C39), 26.03 (C53), 25.83 (C5), 25.49 (C48), 23.49 (C50 or C49), 23.41 (C7 or C8), 23.25 (C54 or C55), 23.05 (C7 or C8), 21.95 (C54 or C55), 21.01 (C49 or C50), 17.05 (C15).As for natural rufomycins, this cyclic peptide appears as more than one conformer. The major species ~ 80% by NMR is assigned HRMS (ESI) calc, for C53H75N9O10 calc, for [M+H]+: 998.5715 m / z, found: 998.5717 m / z. (ZJ = 0.20 ppm)Purification: Purified through preparative HPLC using Gradient 14.Peptide 10cSynthetic method: Peptide 10a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 10a was present in solution at 3 mM concentration (10 mL reaction buffer). 10c was isolated after HPLC purification as a yellowish powder. (HPLC conversion (93%), isolated yield (58%)1H NMR (CD3OD, 600 MHz) 5 7.79 (d, J = 2.3 Hz, 1 H, CH-C42), 7.51 (d, J = 7.9 Hz, 1H, CH-C61), 7.42 (dd, J = 8.5, 2.2 Hz, 1 H, CH-C46), 7.35 (d, J = 8.2 Hz, 1H, CH-C64), 7.17 (t, J = 7.7 Hz, 1 H, CH-C62), 7.09 - 7.01 (m, 2H, CH-C63 & CH-C45), 6.95 (s, 1H, CH- C60), 5.64 - 5.57 (m, 1 H, CH-C51), 5.15 - 5.06 (m, 2H, CH2-C52), 4.96 - 4.92 (m, 1 H, CH-C28), 4.89 - 4.86 (m, 1 H, CH-C2, overlap with water), 4.83 - 4.78 (m, 1H, CH-C12), 4.68 - 4.64 (m, 1 H, CH-C31), 4.64 - 4.55 (m, 3H, CH-C17, CH-C22 & CH-C36), 3.74 (s, 3H, CH3-C65), 3.45 (dd, J = 13.5, 10.8 Hz, 1 H, CH-C39), 3.14 (dd, J = 13.4, 4.1 Hz, 1 H,CH-C39), 3.07 (dd, J = 13.2, 9.4 Hz, 1 H, CH-C40), 2.86 - 2.77 (m, 1 H, CH-C38), 2.77 - 2.70 (m, 4H, CH-C40 & CH3-C10), 2.56 - 2.54 (m, 4H, CH-C38 & CH3-C26), 1.98 - 1.92 (m, 1H, CH-C1), 1.82 - 1.67 (m, 3H, CH2-C37 & CH-C53), 1.60 - 1.51 (m, 2H, CH-C23, CH-C1), 1.50 - 1.43 (m, 1 H, CH-C5), 1.22 (d, J = 6.8 Hz, 3H, CH3-C15), 1.06 - 0.99 (m, 1 H, C48), 0.99 - 0.91 (m, 12H, CH3-C54, CH3-C55, CH3-C7 & CH3-C8) 0.44 (d, J = 6.6 Hz, 3H, CH3-C50 or CH3-C49), 0.21 (d, J = 6.5 Hz, 3H, CH3-C49 or CH3-C50), -0.56 (ddd, J = 13.1, 9.4, 3.5 Hz, 1H, CH-C23).13C NMR (CD3OD, 151 MHz) 0 175.14 (C11), 174.18 (C34), 173.83 (C25), 173.22 (C30), 172.43 (C16), 171.61 (C3), 169.83 (C20), 154.61 (C44), 139.23 (C46), 138.52 (C58), 135.51(043), 133.14 (C51), 129.83 (C41), 129.06 (C60), 129.05 (C57), 126.74 (C42), 122.96 (C63), 121.08 (C45), 120.43 (C52), 120.30 (C62), 119.61 (C61), 110.67 (064), 109.87 (C56), 60.13 (02), 59.92 (C22), 55.61 (C17), 55.01 (036), 53.31 (C31), 52.14 (028), 46.78 (C12), 44.56 (037), 38.63 (C1), 38.25 (C23), 37.86 (040), 37.46 (C38), 32.81 (065), 29.89 (C10), 29.80 (026), 28.37 (C39), 26.02 (C53), 25.82 (05), 25.42 (C48), 23.40 (050 or C49), 23.43; 23.22; 23.03; 21.96 (07, C8, C54 & C55), 21.19 (049 or C50), 17.04 (015).As for natural rufomycins, this cyclic peptide appears as more than one conformer. The major species ~ 80% by NMR is assigned.HRMS (ESI) calc, for C49H59N9OIO calc, for [M+H]+: 944.5245 m / z, found: 944.5281 m / z. (21 = 3.81 ppm)Purification: Purified through preparative HPLC using Gradient 15.Peptide 11cSynthetic method: Peptide 11a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 9a was present in solution at 3.5 mMconcentration (10 mL reaction buffer). 11c was isolated after HPLC purification as a yellowish powder (HPLC conversion (87%), isolated yield (52%))1H NMR (CD3OD, 700 MHz) 5 7.78 (d, J = 2.2 Hz, 1 H, CH-C42), 7.50 (d, J = 8.19 Hz, 1 H, CH-C59), 7.41 (dd, J = 8.6, 2.2 Hz, 1 H, CH-C46), 7.35 (d, J = 8.19 Hz, 1 H, CH-C62), 7.12 - 7.08 (m, 1H, CH-C61), 7.07 - 7.04 (m, 1 H, CH-C45), 7.03 - 6.98 (m, 2H, CH-C60 & CH- 058), 4.91 - 4.87 (m, 2H, CH-C28 & CH-C2, overlap with water), 4.83 - 4.79 (m, 1 H, CH- 012, overlap with water), 4.65 - 4.60 (m, 1 H, CH-C17), 4.57 - 4.53 (m, 3H, CH-C31 , CH- 022 & CH-C36), 3.41 (dd, J = 13.5, 10.9 Hz, 1H, CH-C39), 3.18 - 3.14 (m, 1 H, CH-C39), 3.03 (dd, J = 13.5, 8.6 Hz, 1 H, CH-C40), 2.80 - 2.75 (m, 1H, CH-C40), 2.74 (s, 3H, CH3- C10), 2.47 (s, 3H, CH3-C26), 1.95 (ddd, J = 13.7, 7.6, 5.7 Hz, 1H, CH-C1), 1.81 - 1.66 (m, 3H, CH2-C37 & CH-C51), 1.57 - 1.44 (m, 3H, CH-C1 , CH-C23, CH-C5), 1.41 (d, = 6.9 Hz, 3H, CH3-C38), 1.25 (d, J = 6.9 Hz, 3H, CH3-C15), 1.03 - 0.99 (dd, J = 9.3, 6.4 Hz, 1H, CH-C48), 0.99 - 0.91 (m, 12H, CH3-C53, CH3-C52, CH3-C7 & CH3-C8), 0.43 (d, J = 6.6 Hz, 3H, CH3-C50 or CH3-C49), 0.27 (d, J = 6.6 Hz, 3H, CH3-C49 or CH3-C50), -0.51 (ddd, J = 13.1, 9.3, 3.6 Hz, 1H, CH-C23).13C NMR (CD3OD, 176 MHz) 5 175.31 (C11), 174.66 (C30), 174.19 (C25), 173.95 (C34), 172.33 (C16), 171.69 (C3), 170.00 (C20), 154.43 (C44), 139.33 (C46), 137.91 (C56), 135.52 (C43), 129.72 (C41), 128.50 (C55), 126.85 (C42), 124.67 (C58), 122.76 (C61), 120.99 (C45), 120.18 (C60), 119.20 (C59), 112.68(062), 110.20 (C54), 60.07 (C2), 59.91 (C22), 54.99 (C17), 54.55 (C36), 52.33 (C28), 49.72 (C31), 46.81 (C12), 43.39 (C37), 38.68 (C1), 37.95 (C40), 37.82 (C23), 29.98 (C10), 29.76 (C26), 28.71 (C39), 26.00 (C51), 25.78 (C5), 25.48 (C48), 23.37 (C52 or C53), 23.27 (C50 or C49), 23.26 (C7 or C8), 23.01 (C7 or C8), 21.97 (C52 or C53), 21.28 (C49 or C50), 19.53 (C38), 17.03 (C15).As for natural rufomycins, this cyclic peptide appears as more than one conformer. The major species ~ 80% by NMR is assigned.HRMS (ESI) calc, for C46H55N9Oio calc, for [M+H]+: 904.4933 m / z, found: 904.4977 m / z. ( 1 = 4.86 ppm)Purification: Purified through preparative HPLC using Gradient 15.Peptide 12cSynthetic method: Peptide 12a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 12a was present in solution at 2 mM concentration (10 mL reaction buffer). 12c was isolated after HPLC purification as a yellowish powder. (HPLC conversion (63%), isolated yield (22%)1H NMR (CD3OD, 700 MHz) 58.76 (d, J = 8.6 Hz, 1 H, NH-N19), 7.65 (d, J = 2.2 Hz, 1 H, CH-C42), 7.56-7.51 (m, 2H, CH-C60 & C63), 7.29-7.26 (m, 1H, CH-C46), 7.19-7.15 (m, 3H, CH-C70, CH-C71 & CH-C72), 7.14 - 7.11 (m, 2H, C73 & C69), 7.09 - 7.03 (m, 3H, CH-C61 , CH-C62 & CH-C59), 7.03 - 7.00 (dd, J = 8.6, 2.5 Hz, 1 H, CH-C45), 6.18 - 6.11 (m, 1H, CH-C65), 5.24-5.15 (m, 2H, CH2-C66), 4.97-4.93 (m, 1H, CH-C28), 4.90 -4.86 (m, 1H, CH-C2), 4.84-4.81 (m, 1H, CH-C31, overlap with water), 4.71 (q, =6.9 Hz, 1 H, CH-C12), 4.67 - 4.60 (m, 2H, CH-C36 & CH-C22), 4.44 - 4.38 (m, 1 H, CH-C17), 3.52 (dd, J= 13.2, 11.2 Hz, 1H, CH-C39), 3.50-3.45 (m, 1H, CH-C38), 3.25-3.19 (m, 2H, CH-C39 & CH-C38), 2.75 (s, 3H, CH3-C26), 2.68 (s, 3H, CH3-CIO), 2.29 - 2.24 (m, 1H, CH-C40), 2.17-2.11 (m, 1H, CH-C40), 1.97-1.91 (m, 1H, CH-C1), 1.76-1.70 (m, 6H, CH3-C67 & CH3-C68), 1.70 - 1.63 (m, 2H, CH-C37 & CH-C52), 1.63 - 1.56 (m, 2H, CH-C37 & CH-C23), 1.53- 1.46 (m, 2H, CH-C1 &CH-C5), 1.17 (dd, J = 6.8, 4.1 Hz, 3H, CH3-C15), 1.11-1.04 (m, 1H, CH-C48), 0.99-0.91 (m, 12H, CH3-C53, CH3-C54, CH3-C7 & CH3-C8), 0.37 (d, J= 6.8 Hz, 3H, CH3-C50 or CH3-C49), 0.13 (d, J = 6.7 Hz, 3H, CH3- C49 or CH3-C50), -0.63 - -0.67 (m, 1 H, CH-C23).13C NMR (CD3OD, 176 MHz) 5175.04 (C11), 174.08 (C34), 173.44 (C25), 172.94 (C30), 172.37 (C16), 171.55 (C3), 169.52 (C20), 154.51 (C44), 145.46 (C65), 139.19 (C46), 136.99 (C51), 136.97 (C57), 135.45 (C43), 131.62 (C69 & C73), 130.47 (C56), 129.94 (C41), 129.33 (C70 & C72), 128.24 (C59), 126.63 (C42), 125.57 (C71), 122.19 (C62), 120.95 (C45), 120.40 (C61), 119.47 (C60), 115.35 (C63), 114.12 (C66), 108.91 (C55), 60.24 (C64), 60.04 (C2), 59.97 (C22), 55.31 (C17), 54.94 (C36), 54.61 (C31), 51.95 (C28),46.64 (C12), 44.53 (C37), 38.68 (C1), 38.66 (C23), 37.92 (C38), 37.86 (C40), 29.91 (C26 or C10), 29.90 (C10 or C26), 28.55 (C67 or C68), 28.44 (C67 or C68), 28.31 (C39), 25.96 (C52), 25.77 (C5), 25.51 (C48), 23.46 (C50 or C49), 23.42 (C7 or C8 and C53 or C54), 23.03 (C7 or C8), 21.67 (C53 or C54), 20.94 (C49 or C50), 17.04 (C15).As for natural rufomycins, this cyclic peptide appears as more than one conformer. The major species ~ 80% by NMR is assigned.HRMS (ESI) calc, for C57H77N9O10 calc, for [M+H]+: 1048.5872 m / z, found: 1048.5884 m / z. (71 = 1.14 ppm)Purification: Purified through semi-preparative HPLC using Gradient 13.Peptide 13cSynthetic method: Peptide 13a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 13a was present in solution at 3 mM concentration (10 mL reaction buffer). 13c was isolated after HPLC purification as a yellowish powder. (HPLC conversion (88 %), isolated yield (40%))1H NMR (CD3OD, 700 MHz) 6 7.73 (d, J = 2.2 Hz, 1 H, CH-C42), 7.33 (dd, J = 8.6, 2.2 Hz, 1 H, CH-C46), 7.21 - 7.19 (m, 2H, CH-C56 & CH-C58), 7.16 - 7.13 (m, 2H, CH-C55 & CH- 059), 7.13 - 7.09 (m, 1 H, CH-C57), 7.05 (dd, J = 8.5, 1.2 Hz, 1H, CH-C45), 4.91 - 4.86 (m, 2H, CH-C22, CH-C28), 4.82 - 4.81 (m, 1 H, CH-C2), 4.69 - 4.59 (m, 4H, CH-C31, CH- C17, CH-C12 & CH-C36), 3.44 (dd, J = 14.1, 5.8 Hz, 1 H, CH-C38), 3.18 - 3.12 (m, 1H, CH-C38), 2.70 (s, 3H, CH3-C26), 2.68 (s, 3H, CH3-CIO), 2.50 - 2.44 (m, 1H, CH-C40), 2.40 (dd, J = 13.8, 6.2 Hz, 1 H, CH-C40), 1.99 - 1.93 (m, 1H, CH-C1), 1.88 - 1.83 (m, 1 H, CH-C23), 1.64 - 1.54 (m, 4H, CH-C23, CH2-C37 & CH-C52), 1 .55 - 1.44 (m, 3H, CH-C1 , CH-C5 & CH-C48), 1.42 (d, J = 6.8 Hz, 3H, CH3-C39), 1.24 (d, J = 6.9 Hz, 3H, CH3-C15),1.00 - 0.95 (m, 6H, CH3-C49 & CH3-C50), 0.95 - 0.93 (m, 6H, CH3-C7 & CH3-C8), 0.93 - 0.88 (m, 6H, CH3-C53 & CH3-C54).13C NMR (CD3OD, 176 MHz) 5 175.17 (C11), 174.85 (C25), 174.01 (C34), 172.63 (C30), 172.35 (C16), 171.37 (C3), 170.70 (C20), 154.43 (C44), 139.26 (C46), 137.00 (C51), 135.48 (C43), 131.63 (C55 & C59), 130.25 (C41), 129.19 (C56 & C58), 128.09 (C57), 126.60 (C42), 120.92 (C45), 60.43 (C22), 60.10 (C2), 54.80 (C36), 54.75 (C17), 54.61 (C31), 46.63 (C12), 46.30 (C28), 43.93 (C37), 38.56 (C23 or C1), 38.55 (C1 or C23), 37.95 (C38), 37.50 (C40), 29.96 (C26), 29.92 (C10), 25.92 (C52), 25.76 (C5), 25.58 (C48), 23.58 (C49 or C50), 23.44 & 23.42 (C53 or C54 and C7 or C8), 22.99 (C7 or C8), 22.82 (C49 or C50), 21.65 (C53 or C54), 17.22 (C39), 17.11 (C15).As for natural rufomycins this cyclic peptide appears as more than one conformer, only the major species is assigned.HRMS (ESI) calc, for C44H64N8OIO calc, for [M+H]+: 865.4824 m / z, found: 865.4812 m / z. (A = -1.38 ppm)Purification: Purified through preparative HPLC using Gradient 15.Peptide 17cSynthetic method: Peptide 17a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 17a was present in solution at 3 mM concentration (10 mL reaction buffer). 17c was isolated after HPLC purification as a yellowish powder (HPLC conversion (84%), isolated yield (42%)1H NMR (400 MHz, (CD3)2SO) 5 10.88 (d, J = 2.4 Hz, 1 H, NH-N57), 10.78 - 10.60 (brs, 1 H, OH-O65), 9.22 (d, J = 7.0 Hz, 1H, NH-N29), 9.00 (d, J = 4.8 Hz, 1H, NH-N14), 8.51 (d, = 8.7 Hz, 1H, NH-N4), 8.24 (d, J = 7.7 Hz, 1 H, NH-N19), 7.62 - 7.56 (m, 2H, NH-N33 & CH-C42), 7.48 (d, J = 7.9 Hz, 1 H, CH-C59), 7.32 (d, J = 7.8 Hz, 1 H, CH-C62), 7.18 (dd, J= 8.6, 2.2 Hz, 1 H, CH-046), 7.07 - 7.03 (m, 2H, CH-C58 & CH-C61), 6.98 - 6.92 (m, 2H, CH-C60 & CH-C45), 5.59-5.43 (m, 1H, CH-C63), 5.02-4.90 (m, 2H, CH2-C64), 4.87- 4.71 (m, 3H, CH-C28, CH-C12 & CH-C2), 4.68 (dd, J= 10.2, 4.3 Hz, 1H, CH-C22), 4.60 (td, J= 7.4, 5.8 Hz, 1H, CH-C17), 4.49-4.39 (m, 1H, CH-C31), 4.15-4.05 (m, 1H, CH- 036), 3.61 -3.49 (m, 2H, CH2-C15), 3.28 (dd, J= 13.6, 9.1 Hz, 1H, CH-C39), 3.09-3.01 (m, 1H, CH-C39), 2.95 (dd, J= 13.8, 7.6 Hz, 1H, CH-C40), 2.71 -2.65 (m, 1H, CH-C40), 2.66 (s, 3H, CH3-CIO), 2.45 - 2.39 (m, 1 H, CH-C38), 2.39 (s, 3H, CH3-C26), 2.30 - 2.23 (m, 1H, CH-C38), 1.97-1.76 (m, 1H, CH-C1), 1.66-1.53 (m, 2H, CH2-C37), 1.52-1.42 (m, 3H, CH-C5, CH-C23& CH-051), 1.40-1.30 (m, 1H, CH-C1), 1.03- 0.94 (m, 1H, CH- 048), 0.93 - 0.86 (m, 6H, CH3-C7 & CH3-C8), 0.86 - 0.74 (m, 6H, CH3-C52 & CH3-C53), 0.44 (d, J = 6.6 Hz, 3H, CH3-C49 or CH3-C50), 0.33 (d, J = 6.6 Hz, 3H, CH3-C49 or CH3- C50), -0.16- -0.25 (m, 1H, CH-C23).13CNMR(101 MHz, (CD3)2SO) 5171.43 (C30), 171.34 (C25), 170.66 (C16), 170.51 (C34), 170.05 (011), 169.07 (C3), 167.50 (020), 151.05 (C44), 136.49 (C46), 136.02 (054), 135.93(055), 133.03(063), 127.73(041), 126.97 (aromatic C-Trp), 125.74(042), 123.80 (aromatic C-Trp), 121.03 (aromatic C-Trp), 118.91 (aromatic C-Trp), 118.41 (aromatic C- Trp), 118.19 (aromatic C-Trp), 118.08 (064), 111.41 (aromatic C-Trp), 109.23 (aromatic C- Trp), 60.87 (015), 57.49 (C2), 57.37 (C22), 53.01 (C17 &C36), 52.03 (C12), 51.24 (C31), 49.90 (C28), 40.89 (C37), 38.07 (C1), 36.92 (C23), 36.76 (C40), 35.75 (038), 28.67 (C10), 28.40 (C26), 27.23 (C39), 24.40 (C5), 24.27(051), 23.75 (048), 23.21, 22.90, 22.78, 22.47, 21.44 & 20.95 (07, C8, C52, 053, C49 & C50).As for natural rufomycins this cyclic peptide appears as more than one conformer only the major species is assigned.HRMS (ESI) calc, for C48H57N90II calc, for [M+H]+: 946.5038 m / z, found: 946.5082 m / z. (4 = 4.64 ppm)Purification: Purified through preparative HPLC using Gradient 15.Peptide 18cSynthetic method: Peptide 18a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 18a was present in solution at 8 mM concentration (40 mL reaction buffer). 18c was isolated after HPLC purification as a yellowish powder. (HPLC conversion (78%), isolated yield (35%)).1H NMR (400 MHz, (CD3)2SO) 5 10.88 (d, J = 2.4 Hz, NH-N57), 9.22 (d, J = 6.8 Hz, 1H, NH-N29), 8.88 (d, J = 5.1 Hz, 1H, NH-N14), 8.52 (d, J = 8.7 Hz, 1 H, NH-N4), 8.26 (d, J = 8.1 Hz, 1 H, NH-N19),7.66 - 7.61 (m, 2H, NH-N33 & CH-C42), 7.47 (d, J = 7.9 Hz, 1 H, CH-C59), 7.32 (d, J = 8.1 Hz, 1 H, CH-62), 7.20 (dd, J = 8.6, 2.2 Hz, 1H, CH-C46), 7.07 - 7.02 (m, 2H, CH-C58 & CH-C61), 6.97 - 6.91 (m, 2H, CH-C60 & CH-C45), 5.56 - 5.47 (m, 1 H, CH-C63), 5.01 - 4.92 (m, 2H, CH2-C64), 4.83 - 4.73 (m, 3H, CH-C28, CH-C2 & CH-C17), 4.72 - 4.62 (m, 1 H, CH-C22), 4.54 (t, J = 6.0 Hz, 1 H, CH-C12), 4.46 - 4.39 (m, 1 H, CH-C31), 4.12 (q, J =7.6 Hz, 1H, CH-C36), 3.84 - 3.76 (m, 1 H, CH-C15), 3.28 (dd, J = 13.6, 9.3 Hz, 1 H, CH- C39), 3.03 (dd, J = 13.5, 5.1 Hz, 1 H, CH-C39), 2.94 (dd, J = 13.6, 6.5 Hz, 1 H, CH-C40),2.66 (m, 4H, CH-C40 & CH3-C10), 2.45 - 2.39 (m, 1H, CH-C38), 2.32 - 2.28 (m, 1 H, CH- CSS), 2.32 (s, 3H, CH3-C26), 2.14 - 2.04 (m, 1H, CH-C1), 1.63 - 1.42 (m, 5H, CH2-C37, CH-C51 , CH-C23 & CH-C5), 1.19 - 1.10 (m, 1 H, CH-C1), 1.06 (d, J = 6.3 Hz, 3H, CH3- C66), 1.00 - 0.95 (m, 1 H, CH-C48), 0.94 - 0.86 (m, 6H, CH3-C7 & CH3-C8), 0.85 - 0.78 (m, 6H, CH3-C52 & CH3-C53), 0.43 (d, = 6.6 Hz, 3H, CH3-C49 or CH3-C50), 0.32 (d, J =6.6 Hz, 3H, CH3-C49 or CH3-C50), -0.19 - -0.34 (m, 1 H, CH-C23).13C NMR (101 MHz, (CD3)2SO) 5 171.42 (C25), 171.35 (C30), 171.02 (C16), 170.42 (C34), 169.78 (C11), 168.61 (C3), 167.49 (C20), 151.08 (C44), 136.57 (C46), 136.04 (C54), 135.91 (C55), 133.20 (C63), 127.96 (C41), 127.00 (aromatic C-Trp), 125.74 (C42), 123.83 (aromatic C- Trp), 121.05 (aromatic C-Trp), 118.93 (aromatic C-Trp), 118.43 (aromatic C-Trp), 118.21 (aromatic C-Trp), 118.05 (C64), 111.43 (aromatic C-Trp), 109.25 (aromatic C-Trp), 66.43 (C15), 57.46 (C2), 57.38 (C22), 55.77 (C12), 53.09 (C36), 52.77 (C17), 51.38 (C31), 50.04 (C28), 40.96 (C37), 38.55 (C1), 37.17 (C40), 36.91 (C23), 35.61 (C38), 28.84 (C10), 28.29(C26), 27.31 (C39), 24.83 (C5), 24.31 (C51), 23.82 (C48), 23.57, 22.91, 22.70, 22.11, 21.64 & 20.96 (C7, C8, C52, C53, C49 & C50), 20.23 (C66).As for natural rufomycins this cyclic peptide appears as more than one conformer. Only the major species is assigned.HRMS (ESI) calc, for C49H69N90II calc, for [M+H]+: 960.5195 m / z, found: 960.5178 m / z. (21 = -1.76 ppm)Purification: Purified through preparative HPLC using Gradient 15.Example 11 : Synthesis of cyclised peptides 25c, 26c, 27c, 28c, 29c, 31c, 32c, 33c, 36c and 41cSynthetic method:: To the linear peptide 25a (0.02 mmol, 1 equiv., 4 mM) was dissolved in 5 mL of reaction buffer (6 M GdmCI, 50 mM Nab^PC , 1 .5 mM EDTA) was added sodium nitrite stock (0.4 mmol, 20 equiv.) at -15 °C under nitrogen. Then the solution was stirred for 20 min at -15 °C. The mixture was gently neutralized with saturated sodium bicarbonate. Then the solution was diluted with 10 mL of 50% solvent B in A. To a final pH 6.8 ~ 7.2. After stirring at room temperature for 15 min, the reaction was flited and directly purified by prep HPLC using Gradient 14. 25c was isolated after HPLC purification as a white powder (HPLC conversion (95%), isolated yield (68%))1H NMR (CD3OD, 700 MHz) 5 9.42 (brs, 1 H, NH-N48), 9.19 (brs, 1 H, NH-N83), 8.77 (d, J = 9.5 Hz, 1 H, NH-N65), 8.59 (brs, 1 H, NH-N8), 7.89 (d, J = 9.0 Hz, 1 H, NH-N71), 7.52 (d, J = 9.1 Hz, 1 H, NH-N1), 7.35 (d, J = 7.6 Hz, 2H, HAr), 7.31 - 7.25 (m, 5H, HAr), 7.23 (m, 2H, HAr), 7.19 - 7.12 (m, 6H, HAr), 6.82 (d, J = 8.0 Hz, 2H, CH-C73 & CH-C77), 6.45 (d, J = 8.0 Hz, 2H, CH-C74 & CH-C76), 5.88 (t, J = 10.8 Hz, 1 H, CH-C39), 5.51 (td, = 9.1, 5.5 Hz,1 H, CH-C88), 5.01 -4.93 (m, 1H, CH-C7), 4.89-4.86 (m, 1H, CH-C79, overlap with residual water peak), 4.68 (t, J= 3.9 Hz, 1H, CH-C50), 4.64-4.59 (m, 1H, CH-C67), 4.59 -4.55 (m, 1H, CH-C2), 4.49 (dd, J= 11.1, 5.1 Hz, 1H, CH-C28), 4.17-4.13 (m, 1H, CH- 015), 4.06 (t, J= 5.7 Hz, 1H, CH-C58), 3.37 (t, J= 9.3 Hz, 1H, CH-C18), 3.34-3.30 (m, 2H, CH-C40 & CH-C52), 3.28 - 3.20 (m, 2H, CH2-C29), 3.20 (dd, J = 16.8, 4.7 Hz, 1 H CH- 052), 3.13-3.06 (m, 2H, CH2-C68), 3.03-2.95 (m, 1H, CH-C91), 2.91 (t, J= 13.1 Hz, 1H, CH-C40), 2.84-2.79 (m, 1H, CH-C91), 2.45-2.38 (m, 1H, CH-C3), 2.32-2.24 (m, 1H, CH-C3), 2.24-2.14 (m, 3H, CH-018, CH-C80 & CH-C89), 2.02- 1.94 (m, 1H, CH- 061), 1.92-1.85 (m, 1H, CH-C89), 1.84-1.78 (m, 5H, CH2-C6O, CH-C61 &CH2-C90), 1.74-1.65 (m, 2H, CH-C9 & CH-C10), 1.55-1.49 (m, 1H, CH-C9), 1.46 (ddd, J= 8.8, 6.7, 4.3 Hz, 1H, CH-016), 1.34-1.28 (m, 1H, CH-C16), 1.15 (d, J = 6.7 Hz, 3H, CH3-C84 orCH3-C85), 1.13 (d, J = 6.8 Hz, 3H, CH3-C84 orCH3-C85), 1.10 (d, J = 6.4 Hz, 3H, CH3- C11 orCH3-C12), 1.07 (d, J = 6.4 Hz, 3H, CH3-C11 orCH3-C12), 1.05- 1.02 (m, 1H, CH- 17), 0.49-0.37 (m, 1H, CH-17).13C NMR (CD3OD, 176 MHz) 6178.32 (C62), 175.18 (C53), 174.12 (C59), 174.00 (C69), 173.91 (C4), 173.67 (C41), 173.48 (C5), 173.34 (C30), 173.06 (C86), 172.63 (C25), 172.26 (C81), 172.09 (C51), 157.22 (C75), 139.10 (C19), 138.91 (C42), 136.96 (C28), 131.02 (C73 &C77), 130.69 (CAr), 130.19 (CAr), 129.71 (CAr), 129.41 (CAr), 129.37 (CAr), 129.23 (C72), 128.59 (CAr), 127.84 (CAr), 127.77 (CAr), 116.29 (C74 & C76), 61.57 (C15), 59.66 (C79), 58.57 (C67), 56.77 (C58), 56.16 (C28), 55.40 (02), 54.86 (C39), 52.74 (C88), 52.42 (C7), 51.06 (C50), 47.73 (018), 43.17 (C9), 41.52 (C40), 40.49 (C91), 38.91 (C3), 38.39 (C68), 38.11 (C29), 36.60 (C52), 33.39 (C80), 32.87 (089), 31.68 (C61), 29.91 (016), 26.90 (C60), 26.41 (C10), 24.45 (C90), 23.97 (C11 orC12), 23.43 (C17), 23.01 (011 orC12), 19.71 (084 orC85), 19.26 (C84 orC85).The spectroscopic data agree with those reported in the literature.HRMS (ESI) calc, for C66H87NI3OI3calc, for [M+H]+: 1270.6625 m / z, found: 1270.6639 m / z. (71 = 1.10 ppm)Purification: Purified through preparative HPLC using Gradient 14.Peptide 26cSynthetic method: Peptide 26a was synthesised using the same cyclisation procedure as used for linear peptide 25a. The linear peptide 26a was present in solution at 3.88 mM concentration (15 mL reaction buffer). Following HPLC purification and lyophilisation, 26c was isolated as a white powder (HPLC conversion (58%); isolated yield (42%)).Purification: Purified through preparative HPLC using Gradient 21.1H NMR (400 MHz, (CD3)2SO) 5 8.21 - 8.11 (m, 1 H, amide H), 7.98 - 7.77 (m, 3H, amide H), 7.63 - 7.53 (m, 2H, amide H), 7.32 - 7.13 (m, 5H, aromatic H, CH-C37, CH-C38, CH- C39, CH-C40 & CH-C41), 4.59 (qd, J = 9.3, 5.2 Hz, 1 H), 4.49 - 4.38 (m, 2H), 4.25 - 4.16 (m, 1H), 4.07 - 3.99 (m, 2H), 3.91 - 3.80 (m, 3H), 3.79 - 3.71 (m, 4H), 3.69 - 3.28 (m, 28H), 3.10 (dt, J = 13.7, 5.7 Hz, 1 H, CH-C9), 2.88 (td, J = 14.0, 9.5 Hz, 1 H, CH-C9), 2.54 (s, 1 H), 2.17 (tdd, J = 12.6, 8.0, 5.5 Hz, 1 H), 2.11 - 2.04 (m, 1 H), 1.98 (ddt, J = 14.3, 9.5, 4.8 Hz, 2H), 1.91 - 1.74 (m, 5H), 1.69 - 1.57 (m, 1 H), 1.24 (dddd, J = 16.2, 12.8, 8.2, 4.6 Hz, 1 H), 0.97 (tq, J = 6.6, 3.5 Hz, 1 H), 0.89 (td, J = 6.5, 4.0 Hz, 3H), 0.82 (d, J = 6.7 Hz, 2H), 0.75 (t, J = 6.8 Hz, 4H), 0.56 (dd, J = 17.6, 6.7 Hz, 3H).13C NMR (101 MHz, (CD3)2SO) 5 171.77, 170.69, 170.59, 170.51, 170.34, 169.81 , 168.63, 168.42, 137.31 , 129.20, 128.13, 126.41, 60.94, 59.91 (C42), 59.68, 58.00, 56.41 , 53.78, 47.40, 45.66, 41.82, 41.54, 40.43, 37.88, 36.40, 30.33, 28.57, 26.45, 24.87, 24.47, 24.28, 19.20, 18.91, 17.55, 15.25, 10.98.This cyclic peptide 26c appears as conformers only the major species is assigned.HRMS (ESI) calc, for C37H54N8O9 calc, for [M+H]+: 755.4092 m / z, found: 755.4119 m / z. (A - 3.57 ppm)Peptide 27cSynthetic method: Peptide 27a was synthesised using the same cyclisation procedure as used for linear peptide 25a. The linear peptide 27a was present in solution at 4.2 mM concentration (10 mL reaction buffer). Following HPLC purification and lyophilisation, 27c was isolated as a white powder (HPLC conversion (38%); isolated yield (12%)).Purification: Purified through preparative HPLC using Gradient 12.1H NMR (400 MHz, (CD3)2SO) 5 8.73 (dd, J = 7.5, 4.8 Hz, 1 H, NH-N20), 8.35 - 8.26 (m, 1 H, NH-N9), 8.20 - 8.05 (m, 3H, NH-N39, NH-N4 & NH-N36), 7.64 (d, J = 8.8 Hz, 1 H, NH- N13), 7.30 - 7.14 (m, 5H, aromatic, CH-C43, CH-C44, CH-C45, CH-C46 & CH-C47), 7.09 (d, J = 9.0 Hz, 1 H, NH-N28), 4.64 - 4.50 (m, 2H, CH-C12 & CH-C27), 4.28 (m, 1 H, CH- CSS), 4.14 - 4.05 (m, 2H, CH23 & CH-C2), 3.96 (dd, J = 16.8, 7.5 Hz, 1 H, CH-C18), 3.86 - 3.69 (m, 3H, CH-C34, CH-C7 & CH-C54), 3.67 - 3.42 (m, 2H, CH-C34, CH-C54), 3.38 (dd, J = 16.8, 4.7 Hz, 1 H, CH-C18), 3.25 (dd, J = 14.0, 4.2 Hz, 1 H, CH-C41), 2.90 (dd, J = 14.0, 10.9 Hz, 1H, CH-C41), 2.39 - 2.22 (m, 2H, CH2-C21), 2.17 - 1.98 (m, 4H, CH-C10, CH-C29, CH-C5 & CH-C55), 1.96 (s, 3H, CH3-C53), 1.85 (m, 1 H, CH-C55), 1.79 - 1.64 (m, 3H, CH-C5 & CH2-C56), 1 .59 - 1.43 (m, 3H, CH2-C15 & CH-C16), 0.98 - 0.75 (m, 18H, CH3-C30, CH3-C31, CH3-C50, CH3-C51 , CH3-C48 & CH3-C49).13C NMR (101 MHz, (CD3)2SO) 5 173.41 (C11), 172.89 (C6), 172.41 (C22), 171.55 (C1), 171.30 (C37), 169.95 (C26), 168.56 (C17), 168.39 (C33), 138.02 (C42), 128.82 (C43 & C47), 128.09 (C44 & C46), 126.41 (C45), 61.20 (C7), 60.97 (C23), 54.75 (C27), 54.66 (C38), 53.40 (C2), 50.33 (C12), 47.48 (C54), 43.17 (C34), 42.83 (C18), 40.55 (C15), 36.12 (C41), 30.31 (C29), 29.45 (C21), 28.81 (C56), 28.49 (C10), 25.01 (C55), 24.00 (C16), 23.12 (C48 or C49), 22.01 (C48 or C49), 19.47, 19.23, 18.93, 17.65 (Vai C50, C51 , C30, C31), 14.36 (C53).The spectroscopic data agree with those reported in the literature.HRMS (ESI) calc, for CsgHsoNsOs calc, for [M+H]+: 801.4333 m / z, found: 801.4362 m / z. (21 = 3.62 ppm)Peptide 28cSynthetic method: Peptide 28a was synthesised using the same cyclisation procedure as used for linear peptide 25a. The linear peptide 26a was present in solution at 4.9 mM concentration (10 mL reaction buffer). Following HPLC purification and lyophilisation, 28c was isolated as a white powder (HPLC conversion (77%); isolated yield (51%)).Purification: Purified through preparative HPLC using Gradient 20.1H NMR (400 MHz, (CD3)2SO) 5 9.18 (brs, 1 H, OH-O31), 8.51 (d, J = 7.8 Hz, 1H, NH- N35), 8.01 (d, J = 7.6 Hz, 1 H, NH-N4), 7.83 (t, J = 4.8 Hz, 1 H, NH-N30), 7.28 (d, J = 9.3 Hz, 1 H, NH-N12), 6.97 - 6.91 (m, 2H, CH-C18 & CH-C22), 6.65 - 6.60 (m, 2H, CH-C19 & CH-C21), 4.47 - 4.35 (m, 1 H, CH-C10), 4.14 - 4.05 (m, 1 H, CH-C33), 4.05 - 3.90 (m, 3H, CH-C14, CH-C29 & CH-C2), 3.85 - 3.76 (m, 1 H, CH-C24), 3.56 - 3.46 (m, 2H, CH-C29 & CH-C24), 2.82 (dd, J = 13.6, 6.9 Hz, 1 H, CH-C16), 2.72 (dd, J = 13.6, 9.1 Hz, 1 H, CH- C16), 2.07 - 1.94 (m, 1 H, CH-C26), 1.83 (p, J = 6.8 Hz, 2H, CH2-C25), 1.66 - 1.56 (m, 1 H, CH-C5), 1.54 - 1.38 (m, 2H, CH-C26, CH-C5), 1.37 - 1.27 (m, 1 H, CH-C6), 1 .24 (d, J = 7.1 Hz, 3H, CH3-C36), 0.93 - 0.75 (m, 6H, CH3-C7 & CH3-C8).13C NMR (101 MHz, (CD3)2SO) 6 172.56 (C32), 171.42 (C1), 170.40 (C9 & C13), 167.68 (C27), 155.82 (C20), 129.88 (C18 & C22), 127.30 (C17), 114.81 (C19 & C21), 61.18 (C14), 55.31 (C10), 53.81 (C2), 48.71 (C33), 46.57 (C24), 41.90 (C29), 40.09 (C5, overlap with solvent peaks), 36.88 (C16), 29.10 (C26), 24.43 (C6), 24.22 (C25), 22.49 (C7 or C8), 22.08 (C7 or C8), 16.66 (C36).HRMS (ESI) calc, for C25H35N5O6 calc, for [M+H]+: 502.2666 m / z, found: 502.2666 m / z. (21 = 0 ppm)Peptide 29cSynthetic method: Peptide 29a was synthesised using the same cyclisation procedure as used for linear peptide 25a. The linear peptide 29a was present in solution at 5 mM concentration (10 mL reaction buffer). Following HPLC purification and lyophilisation, 29c was isolated as a white powder (HPLC conversion (92%); isolated yield (63%)).Purification: Purified through preparative HPLC using Gradient 14.1H NMR (400 MHz, (CD3)2SO) 5 9.09 (d, J = 3.6 Hz, 2H, NH-N8 & NH-N50), 8.72 (d, J = 9.2 Hz, 2H, NH-N75 & NH-N32), 8.35 (d, J = 9.1 Hz, 2H, NH-N41 & NH-N7), 7.79 (br, 6H, NH-N40 & NH-N82), 7.32 - 7.20 (m, 12H, NH-N26 & NH-N68, aromatic CH-C14-18 & CH- C55-59), 4.78 (td, J = 9.4, 5.2 Hz, 2H, CH-C34 & CH-C76), 4.64 - 4.54 (m, 2H, CH-C2 & CH-C43), 4.43 - 4.33 (m, 4H, overlap, CH-C28, CH-C69, CH-C10 & CH-C51), 4.33 - 4.26 (m, 2H, CH-C21 & CH-C62), 3.59 (overlap, 2H, CH-C24 & CH-C65), 2.98 (dd, J = 12.8, 5.4 Hz, 2H, CH-C53, CH-C11), 2.92 - 2.80 (m, 4H, CH-C53, CH-C11, CH-C39 & CH-C81), 2.80 - 2.72 (m, 2H, CH-C39 & CH-C81), 2.48 - 2.39 (m, 2H, CH-C24 & CH-C65), 2.08 (dt, J = 13.8, 6.9 Hz, 2H, CH-C29 & CH-C71), 1.99 - 1.89 (m, 2H, CH-C22 & CH-C63), 1.81 - 1.69 (m, 2H, CH-C37 & CH-C79), 1.68 - 1.57 (m, 4H, CH-C23, CH-C64, CH-C80 & CH- CSS), 1.57 - 1.44 (m, 8H, CH-C23, CH-C64, CH-C22, CH-C63, CH-C80, CH-C38, CH-C37 & CH-C79), 1.45 - 1.35 (m, 2H, CH-C3 & CH-C46), 1.35 - 1.22 (m, 4H, CH2-C1 & CH2- C45), 0.91 - 0.63 (m, 24H, CH3-C4, CH3-C5, CH3-C31, CH3-C35, CH3-C47, CH3-C48, CH3-C72 & CH3-C73).13C NMR (101 MHz, (CD3)2SO) 6 171.69 (C44 & C6), 171.21 (C12 & C52), 170.92 (C30 & C70), 170.17 (C36 & C77), 169.74 (C25 & C66), 136.18 (C13 & C54), 129.34 & 128.29 (aromatic C14, C15, C17, C18, C55, C56, C58 & C59), 126.98 (C16 & C57), 59.98 (C21 & C62), 56.92 (C28 & C69), 53.96 (C10 & C51), 50.82 (C34 & C76), 49.48 (C2 & C43), 46.11 (C24 & C65), 40.92 (C1 & C45), 38.65 (C39 & C81), 35.68 (C11 & C53), 31.03 (C29 & C71), 29.71 (C79 & C37), 29.09 (C22 & C63), 23.97 (C3 & C46), 23.11 & 23.05 (C64,C23, C38 & C80), 22.83 (Methyl Vai or Leu), 22.52 (Methyl Vai or Leu), 18.97 (Methyl Vai or Leu), 18.10 (Methyl Vai or Leu).HRMS (ESI) calc, for C60H92NI2OI0calc. for [M+H]+: 1141.7137 m / z, found: 1141.7142 m / z. (Z1 = 0.44 ppm)The spectroscopic data agree with those reported in the literature.Peptide 31cSynthetic method: Peptide 31a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 31a was present in solution at 4 mM concentration (20 mL reaction buffer). Following HPLC purification and lyophilisation, 31c was isolated as a white powder (HPLC conversion (61%); isolated yield (22%)).Purification: Purified through preparative HPLC using Gradient 19.1H NMR (400 MHz, (CD3)2SO) 5 8.32 - 8.22 (m, 2H), 7.93 (d, J = 8.8 Hz, 1 H), 7.47 (d, J = 6.0 Hz, 1H), 5.40 (dd, J = 11.2, 3.7 Hz, 1 H), 5.26 (dd, J = 8.9, 5.7 Hz, 1 H), 5.20 - 5.11 (m, 3H), 5.10 - 4.96 (m, 3H), 4.78 - 4.65 (m, 2H), 4.42 - 4.30 (m, 1 H), 4.18 - 4.09 (m, 1 H), 2.90 (s, 3H), 2.86 - 2.77 (m, 15H), 2.68 (s, 3H), 2.28 - 2.14 (m, 2H), 2.01 - 1.86 (m, 3H), 1.81 - 1.09 (m, 20H), 1.00 - 0.63 (m, 45H).13C NMR (101 MHz, (CD3)2SO) 5 172.80, 172.65, 171.96, 171.23, 170.53, 170.18, 170.02, 169.57, 168.77, 57.33, 54.35, 53.94, 53.48, 53.22, 51.97, 50.88, 49.98, 49.51 , 48.30, 43.69, 37.65, 37.53, 37.15, 36.85, 33.45, 31.20, 30.26, 29.57, 29.52, 29.36, 24.67, 24.53,24.48, 24.24, 24.00, 23.61, 23.48, 23.37, 23.34, 23.22, 23.17, 23.08, 22.23, 22.14, 21.20,20.92, 20.85, 20.50, 19.31, 18.99, 18.79, 18.13, 17.94, 17.02, 10.32.HRMS (ESI) calc, for C59H107N11O11 calc. for [M+H]+: 1146.8230 m / z, found: 1146.8220 m / z. (Z1 = -0.87 ppm)Peptide 32cSynthetic method: Peptide 32a was synthesised using the same cyclisation procedure as used for linear peptide 8a. The linear peptide 32a was present in solution at 2 mM concentration (20 mL reaction buffer). Following HPLC purification and lyophilisation, 32c was isolated as a white powder (HPLC conversion (95%); isolated yield (50%)).Purification: Purified through preparative HPLC using Gradient 14.1H NMR (400 MHz, (CD3)2SO) 5 9.13 (d, J = 7.5 Hz, 1 H, NH-N12), 9.06 (d, J = 4.6 Hz, 1 H, NH-N43), 8.37 (d, J = 8.1 Hz, 1 H, NH-N30), 8.03 (d, J = 7.8 Hz, 1 H, NH-N4), 7.65 (d, J = 8.1 Hz, 1H, NH-N19), 7.46 (d, J = 8.0 Hz, 1 H, CH-C54), 7.37 (d, J = 8.0 Hz, 1 H, CH-C51), 7.21 - 6.93 (m, aromatic, 8H, CH-C59 CH-C63 CH-C47 CH-C60 CH-C62 CH-C61 , CH- C52 & CH-C53), 5.58 (ddt, J = 17.2, 10.1 , 7.1 Hz, 1H, CH-C21), 5.17 - 5.07 (m, 1 H, CH- C7), 4.94 - 4.80 (m, 3H, CH2-C22 & CH-C41), 4.65 - 4.54 (m, 2H, CH-C14 & CH-C1), 4.33 (dd, = 10.1 , 7.7 Hz, 1 H, CH-C11), 4.25 (dd, J = 10.5, 4.3 Hz, 1H, CH-C33), 3.98 - 3.90 (m, 1 H, CH-C28), 3.72 (s, 3H, CH3-C55), 3.19 - 2.99 (m, 2H, CH2-C45), 2.99 - 2.91 (m, 2H, CH2-C20), 2.68 (s, 3H, CH3-C57), 2.62 (s, 3H, CH3-C44), 2.26 - 2.05 (m, 5H, CH- C17, CH2-C5, CH2-CI6), 1.52 - 1.37 (m, 2H, CH-C36 & CH-C23), 1.08 (d, = 7.0 Hz, 3H, CH3-C31), 1.00 - 0.78 (m, 13H, CH3-C26, CH3-C56, CH-C37, CH3-C24, CH3-C25), 0.48 (d, J = 6.6 Hz, 3H, CH3-C38 or CH3-C39), 0.33 (d, J = 6.6 Hz, 3H, CH3-C38 or CH3-C39), - 0.34 (ddd, J = 12.9, 8.9, 4.1 Hz, 1H, CH-C36).13C NMR (101 MHz, (CD3)2SO) 5 172.35 (C13), 171.83 (C40), 171.10 (C27), 171.06 (C9), 170.51 (C2), 168.60 (C32), 167.53 (C6), 137.35 (C58), 136.49 (C49), 133.52 (C21), 129.54 (C59 & C63), 128.43 (aromatic C), 127.91 (C60 & C62), 127.66 (aromatic C), 126.34 (aromatic C), 121.34 (C47), 118.77 (C54), 118.60 (aromatic C), 117.76 (C22), 109.59 (C51), 108.25 (aromatic C), 57.28 (C7), 57.12 (C33), 55.33 (C11), 53.35 (C14), 51.38 (C1), 50.55 (C41), 49.85 (C28), 38.71 (C16), 37.65 (C5), 37.25 (C36), 37.14 (C20),32.37 (C55), 29.32 (C17), 28.83 (C44), 28.60 (C57), 27.47 (C45), 24.66 (C23), 23.99 (C37), 23.44 (C24 or C25), 23.18 (C38 or C39), 22.04 (C24 or C25), 20.76 (C38 or C39), 19.73 (C56 or C26), 19.21 (C56 or C26), 16.78 (C31).HRMS (ESI) calc, for C48H58N8O7 calc, for [M+H]+: 869.5289 m / z, found: 869.5289 m / z. (21 = 0 ppm)Peptide 33cSynthetic method: Peptide 33a was synthesised using the same cyclisation procedure as used for linear peptide 25a. The linear peptide 33a was present in solution at 3 mM concentration (10 mL reaction buffer). Following HPLC purification and lyophilisation, 33c was isolated as a white powder (HPLC conversion (95%); isolated yield (62%)).Purification: Purified through preparative HPLC using Gradient 17.1H NMR (400 MHz, (CD3)2SO) 510.86 (d, J = 2.4 Hz, 1 H, NH-N36), 8.39 (d, J = 6.5 Hz, 1H, NH-N23), 8.11 -8.06 (m, 2H, NH-N6 & NH-N3), 7.89 (d, =6.5 Hz, 1H, NH-N18), 7.82 (d, J= 8.4 Hz, 1H, NH-N43), 7.70 (t, J= 5.8 Hz, 1H, NH-N49), 7.54 (d, J= 7.8 Hz, 1H, CH-C42), 7.35 (d, J= 8.1 Hz, 1H, CH-C39), 7.27-7.14 (m, 6H, CH-C59, CH-C60, CH-C61, CH-C62, CH-C63 & CH-C35), 7.13-7.07 (m, 1H, CH-C40), 7.07-6.99 (m, 2H, CH-C4 & NH-N54), 6.40 (d, J = 8.5 Hz, 2H, CH-C29 & CH-C25), 6.29 (d, J = 8.5 Hz, 2H, CH-C26 & CH-C28), 4.58 (dd, J= 7.9, 2.5 Hz, 1H, CH-C12), 4.51-4.47 (m, 2H, CH-C7 & CH-C2), 4.41 (q, J=5.9Hz, 1H, CH-C19), 4.14-4.07 (m, 2H, CH-C31 &CH-C45), 3.58 (m, 1H, CH-C15), 3.42 (m, 1H, CH-C15), 3.35 - 3.23 (m, 1H, CH-C53), 3.20 - 3.12 (m, 1H, CH-C33), 3.11 -3.04 (m, 2H, CH2-C50), 3.05-2.98 (m, 1H, CH-C33), 2.94 (dd, J = 13.8, 4.4 Hz, 1H, CH-C1), 2.71-2.66 (m, 2H, CH-C1 & CH-C53), 2.64-2.52 (m, 2H, CH2-C21), 2.08-1.95 (m, 1H, CH-C13), 1.83 (m, 3H, CH2-C14, CH-C47 & CH-C8), 1.77- 1.66 (m, 5H, CH-C13), 1.65 - 1.52 (m, 2H, CH-C47 & CH-C8), 1.52 - 1.23 (m, 5H, CH2- C51, CH2-C52).13C NMR (101 MHz, (CD3)2SO) 0 171.78 (C32), 171.47 (C20), 170.90 (C4), 170.69 (C16), 170.64 (C55), 170.51 (C9), 169.23 (C64), 156.85 (C46), 155.76 (C27), 137.94 (C58), 136.35 (C37), 130.12 (C29 & C25), 129.19 (C59 & C63), 128.04 (C60 & C62), 127.01 (C35), 126.53 (C24), 126.28 (C61), 124.18 (C38), 121.16 (C40), 118.53 (C41), 118.09 (C42), 114.77 (C26 & C28), 111.60 (C39), 109.99 (C34), 58.64 (C12), 55.78 (C31), 54.76 (C19), 53.80 (C2), 52.80 (C45), 49.95 (C7), 46.68 (C15), 40.43 (C50), 38.19 (C53), 37.55 (C1), 36.60 (C21), 28.37 (methylene), 27.87 (methylene), 26.94 (C33), 26.79 (C13), 25.49 (methylene), 24.58 (methylene), 24.37 (C14), 22.48 (C66).HRMS (ESI) calc, for C47H59N11O8 calc, for [M+H]+: 906.4626 m / z, found: 906.4641 m / z. (4 = 1.65 ppm)Peptide 36cSynthetic method: Purified and dried linear peptide 36a (15.07 pmol, 15 mg, 1 equiv.) was dissolved in 10 mL cyclisation buffer and sodium nitrite stock solution (300 uL) was added -15 °C under nitrogen. The solution was stirred for 20 minutes at -15 °C. The mixture was gently neutralised using saturated sodium bicarbonate ( 1mL) to pH 7.5. 15 mL of ethyl acetate was added, and the mixture stirred vigorously for 45 minutes at room temperature. The mixture was further extracted with ethyl acetate (4 x 10 mL) and the combined organic layers were concentrated in vacuo, before being dissolved in 80% acetonitrile in water and purified by semi-preparative HPLC. Purified peptide was lyophilised and isolated as a yellow powder. (Isolated yield (56%)).Purification: Purified through semi-preparative HPLC using Gradient 23.1H NMR (CD3OD, 600 MHz) 6 8.84 (d, J = 8.4 Hz, 1H, NH-N19), 7.77 (d, J = 2.2 Hz, 1H, CH-C42), 7.56 - 7.50 (m, 2H, CH-C60 & CH-C63), 7.44 (dd, J = 8.6, 2.2 Hz, 1 H, CH-C46),7.13 (s, 1 H, CH-C59), 7.09 - 7.00 (m, 3H, CH-C45, CH-C61 & CH-C62), 6.16 - 6.10 (m, 1H, CH-C65), 5.23 - 5.15 (m, 2H, CH2-C66), 4.98 - 4.89 (m, 2H, CH-C28 & CH-C2), 4.89 - 4.86 (m, 1 H, CH-C12), 4.72 (dd, J = 11.8, 3.3 Hz, 1H, CH-C22), 4.65 - 4.56 (m, 3H, CH-C31 CH- 036 & CH-C17), 3.45 (dd, J = 13.3, 11.2 Hz, 1H, CH-C39), 3.18 - 3.13 (m, 1 H, CH-039),3.13 - 3.06 (m, 2H, CH-040 & CH-C38), 2.90 - 2.82 (m, 1H, CH-C38), 2.74 (dd, J = 13.3,4.7 Hz, 1 H, CH-040), 2.70 (s, 3H, CH3-CIO), 2.64 (s, 3H, CH3-C26), 2.35 (t, J = 2.5, 1 H, CH-C69), 1.95 (ddd, J = 13.7, 7.6, 5.7 Hz, 1 H, CH-C1), 1.87 - 1.78 (m, 2H, CH-C37), 1.77 - 1.67 (m, 7H, CH-C52, CH3-C67 & CH3-C68), 1.65 - 1.59 (m, 1H, CH-C23), 1.58 - 1.51 (m, 1 H, CH-C1), 1.51 - 1.45 (m, 1H, CH-C5), 1.22 (d, J= 6.9 Hz, 3H, CH3-C15), 1.14 - 1.05 (m, 1H, CH-C48), 1.02 - 0.88 (m, 12H, CH3-C53, CH3-C54, CH3-C7 & CH3-C8), 0.37 (d, J =6.7 Hz, 3H, CH3-C50 or CH3-C46), 0.16 (d, J = 6.7 Hz, 3H, CH3-C49 or CH3-C50), -0.64 - - 0.7 (m, 1 H, CH-C23).13C NMR (CD3OD, 151 MHz) 6 175.02 (011), 174.47 (034), 173.38 (025), 172.61 (030), 172.18 (016), 171.39 (03), 169.64 (020), 154.62 (044), 145.43 (065), 139.28 (046), 136.94 (057), 135.51 (043), 130.45 (056), 129.89 (041), 126.77 (042), 125.55 (059), 122.15 (062), 121.10 (045), 120.35 (061), 119.48 (060), 115.31 (063), 114.10 (068), 108.94 (055), 79.59 (051), 73.02 (069), 60.23 (066), 60.20 (02), 59.82 (022), 55.78 (017), 55.05 (036), 52.26 (031), 51.83 (028), 46.64 (012), 44.63 (037), 38.76 (023), 38.43 (01), 38.40 (040), 29.79 (010 & 026), 28.53 (067 or 068), 28.43 (067 or 068), 28.09 (039), 25.96 (052), 25.76 (05), 25.48 (048), 23.53 (07 or 08), 23.49 (050 or 049), 23.00 (038), 22.99 (053 or 054), 22.91 (07 or 08), 22.40 (053 or 054), 20.92 (049 or 050), 17.06 (015).This cyclic peptide has rotamers, only major peaks are assigned.HRMS (ESI) calc, for C53H73N9O10 calc, for [M+H]+: 996.5558 m / z, found: 996.5568 m / z. (Z\ = 1.00 ppm)Peptide 41cSynthetic method: Peptide 41a was synthesised using the same cyclisation procedure as used for linear peptide 25a. The linear peptide 41a was present in solution at 3 mM concentration (10 mL reaction buffer). Following HPLC purification and lyophilisation, 41c was isolated.HRMS: (ESI) calc for C101H139N23O27 [M+2H]2+= 1054.0183 m / z, found = 1054.0182 m / z (Z1 = -0.09 ppm).Example 12: Synthesis of cyclised peptides 45c, 46c, 47c and 48cPeptide 45cSynthetic method: Linear peptide hydrazide 45a (64 mg, 1 eq) was dissolved in cyclisation buffer (10 mL) (6 M GdmHCI, 50 mM NaH2PO4, 1.5 mM EDTA, pH 3)) and cooled to - 15 °C under nitrogen. A 5 M sodium nitrite stock was prepared using degassed water and was added (20 eq) to the linear peptide hydrazide solution at - 15 °C under nitrogen. The solution was stirred for 15 min then the pH was raised to pH 7 - 8 through the addition of a 1 M sodium bicarbonate solution (monitored with pH indicator paper). MeCN was added (5 mL) and the reaction was stirred for 60 min. The reaction mixture was then purified using preparative HPLC with Gradient 24, yielding the cyclic peptide 45c as a colourless solid (8.0 mg) after lyophilisation (HPLC conversion = 57 %, isolated yield = 13 % (loss due to instrument).Purification: Purified through preparative HPLC using Gradient 24.1H NMR (800 MHz, (CD3)2SO) 5 12.36 (bs, 3H, 052 - H, 057 - H, 069 - H), 9.17 (s, 1 H, 063 - H), 8.37 - 8.31 (m, 2H, N11 - H, N27 - H), 8.30 (d, J = 7.2 Hz, 1 H, N35 - H), 8.25 (d, J = 7.9 Hz, 1 H, N15- H / N19 - H), 8.12 (d, J = 7.6 Hz, 1 H, N15 - H / N19 - H), 8.10 - 8.00 (m, 3H, N7 - H, N31 - H, N39 - H), 7.76 (d, J = 7.9 Hz, 1H, N23 - H), 7.51 (bs, 1 H, N31 - H), 7.00 (d, J = 8.5 Hz, 2H, C60 - 1 H, C65 - 1H), 6.62 (d, J = 8.5 Hz, 2H, C61 - H, C64 - H), 5.25 (qd, J = 6.6, 3.8 Hz, 1 H, C81 - H), 4.59 - 4.53 (m, 2H, C16 - H / C20 - H, C28 - H), 4.50 - 4.45 (m, 1H, C16 - H / C20 - H) 4.36 - 4.31 (m, 1H, C24 - H), 4.25 -4.21 (m, 2H, C12 - H, C36 - H), 4.20 - 4.14 (m, 3H, C4 - H, C32 - H, 040 - H), 3.72 (d, J = 5.7 Hz, 1H, C8-H), 3.47-3.41 (m, 1H, C41 - H), 3.34-3.28 (m, 1H, C41 - H), 3.12- 3.01 (m, 2H, C76 - 2H), 2.89 - 2.82 (m, 1 H, C58 - H), 2.75 - 2.64 (m, 3H, C58 - H, C66 - 2H), 2.60-2.37 (m, 4H, C50-2H, C54-2H), 2.19-2.12 (m, 1H, C43 - H), 2.05-1.99 (m, 1H, C44- H), 1.99-1.92 (m, 1H, C47- H), 1.91 - 1.83 (m, 2H, C42-H, C43- H), 1.82- 1.76 (m, 1H, C42-H), 1.73 - 1.64 (m, 2H, C74 - H, C70 - H), 1.62- 1.55 (m, 1H, C74- H), 1.53-1.43 (m, 2H, 075 -2H), 1.43-1.36 (m, 1H, 072 - H), 1.30 (d, J= 6.5 Hz, 3H, C82-3H), 1.10-1.01 (m, 1H, C72 - H), 0.91 (dd, J = 9.0, 6.9 Hz, 6H, C45 - 3H, C46 - 3H), 0.86 (dd, J = 17.0, 6.8 Hz, 6H, C48 - 3H, C49 - 3H), 0.82 (d, J = 6.8 Hz, 3H, C71 - 3H), 0.79 (t, J = 7.4 Hz, 3H, C73 - 3H).13CNMR13C N MR (201 MHz, (CD3)2SO) 5171.81 (C51 / C55), 171.72 (C51 / C55), 171.09 (017 / C33), 170.83 (C33), 170.58 (017 / C33), 170.38 (021 / C37 / C29), 170.35(021 / C37 I C29), 170.29 (021 / C37 / C29), 169.58 (05), 167.72 (C9), 163.23 (01), 156.77 (C78), 155.82 (C62), 130.09 (060, C65), 127.58 (059), 114.91 (C61, C64), 68.74 (C81), 58.37 (04), 57.76 (040), 57.60 (C12), 57.10 (08), 56.61 (32), 54.38 (C24), 51.71 (036), 49.63 (016 / 020), 49.34 (016 / 020), 44.80 (041), 40.36 (076), 36.89 (070), 36.50 (058), 36.04 (050 / 054), 35.95 (050 / 054), 30.80 (047), 30.03 (044) 27.94 (043, 074), 24.70 (075), 24.12 (072), 22.08 (042), 19.20 (0451046 / 048 / 049), 18.36 (045 / 046 I 048 / 049), 18.16 (045 / 046 / 048 I 049), 17.64 (045 / 046 / 048 / 049), 16.56 (082), 15.20 (071), 11.12 (073).HRMS: (ESI) calc for C52H78N13O18 [M+2H]2+= 587.7911 m / z, found = 587.7933 m / z (71 = - 3.7 ppm)Synthetic method: Linear peptide hydrazide 46a (63 mg, 1 eq) was dissolved in cyclisation buffer (5 mL) (6 M GdmHCI, 50 mM NaH2PO4, 1.5 mM EDTA, pH 3)) and cooled to -15 °C under nitrogen. A 5 M sodium nitrite stock was prepared using degassedwater and was added (20 eq) to the linear peptide hydrazide solution at - 15 °C under nitrogen. The solution was stirred for 15 min then the pH was raised to pH 7 - 8 through the addition of a 1 M sodium bicarbonate solution (monitored with pH indicator paper). MeCN was added (2.5 mL) and the reaction was stirred for 60 min. The reaction mixture was then purified using preparative HPLC using Gradient 25, yielding the cyclic peptide 46c as a colourless solid (14 mg) after lyophilisation (HPLC conversion = 81 %, isolated yield = 23 % (loss due to instrument).Purification: Purified through preparative HPLC using Gradient 25.HRMS (ESI) calc, for C^HsiNgOsS for [M+H]+: 840.4442 m / z, found: 840.4456 m / z (71 = - 1.7 ppm)Peptide 47cSynthetic method: Peptide 47a (65 mg) was cyclised using the same procedure as used for linear peptide 46a. HPLC purification was performed using Gradient 26 then Gradient 27, yielding peptide 47c as a colourless solid (8.2 mg) after lyophilisation (HPLC conversion = 30 %, isolated yield = 13 %).Purification: Purified through -preparative HPLC using Gradient 26.HRMS (ESI) calc, for C43H51N11O7 for [M+H]+= 834.4051 m / z, found: 834.4048 m / z (71 = 0.36 ppm); [M+2H]2+= 417.7065 m / z, found: 417.7042 (71 = 5.5 ppm).Synthetic method: Peptide 48a (102 mg) was cyclised using the same procedure as used for linear peptide 46a. HPLC purification was performed using Gradient 26 then Gradient 27, yielding peptide 47c as a colourless solid (5.6 mg) after lyophilisation (HPLC conversion = 28 %, isolated yield = 6 %) Purification: Purified through preparative HPLC using Gradient 26 then semi preparative HPLC using gradient 27.HRMS (ESI) calc, for CssHs+NsOg for [M+H]+= 767.4092 m / z, found: 767.4093 m / z (4 = - 0.13 ppm).

Claims

CLAIMS1. A method of preparing a cyclised peptide from a modified peptide comprising an acyl azide group, the method comprising increasing the pH of a reaction medium comprising an acidic aqueous solution of the modified peptide to generate the cyclised peptide via the formation of an amide bond or an ester bond between the carbonyl of the acyl azide group of the modified peptide and another functional group on the modified peptide, wherein the reaction medium further comprises a first solvent.

2. A method of preparing a cyclised peptide from a modified peptide comprising an acyl azide group, the method comprising increasing the pH of a reaction medium comprising an acidic solution of the modified peptide to generate the cyclised peptide, wherein the acyl azide group forms an ester bond with a hydroxyl group on the modified peptide, optionally wherein the reaction medium further comprises a first solvent.

3. The method of claim 2, wherein the acidic solution is an acidic aqueous solution.

4. The method of any of claims 1 to 3, wherein the modified peptide comprising the acyl azide group is prepared by treating a precursor reaction medium comprising a modified peptide precursor with an oxidising agent, wherein the modified peptide precursor comprises a precursor functional group.

5. The method of any of claims 1 or 3 to 5, wherein the acidic aqueous solution and / or precursor reaction medium comprises an acidic buffer solution, optionally wherein the acidic buffer solution is a phosphate buffer.

6. The method of any preceding claim, wherein the reaction medium comprises water in an amount of at least 10% by volume.

7. The method of any preceding claim, wherein the first solvent is immiscible with the acidic solution or acidic aqueous solution.

8. The method of any preceding claim, wherein the first solvent is a polar, nonchlorinated organic solvent.

9. The method of claim 8, wherein the first solvent is selected from acetonitrile, ethyl acetate, tetrahydrofuran, methyl ethyl ketone, butanol, dimethylformamide, tetrahydropyran, 1 ,4-dioxane, 1,3-dioxolane, 2,2,2-trifluoroethanol, 2- methyltetrahydrofuran, dimethylsulfoxide, and mixtures thereof, preferably wherein the solvent is ethyl acetate.

10. The method of any preceding claim, wherein the pH of the reaction medium prior to increasing the pH is no more than about 4; optionally wherein the pH is from about 2.5 to about 3.5, more preferably about 3.0.

11. The method of any preceding claim, wherein the pH is increased to a pH of from about 6 to about 8, preferably wherein the pH of the reaction medium is increased to a pH of from about 7 to about 7.5.

12. The method of any preceding claim, wherein the pH is increased using a base, optionally wherein the base is sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, an inorganic phosphate, potassium carbonate, triethylamine, A / , / V-diisopropylethylamine, further optionally wherein the base is sodium bicarbonate.

13. The method of any preceding claim, wherein the acyl azide is present at the C- terminus of the modified peptide.

14. The method of any preceding claim, wherein the concentration of the modified peptide comprising the acyl azide group in the reaction medium is at least about 1 mM, preferably at least about 2 mM; optionally wherein the concentration of the modified peptide comprising the acyl azide group in the reaction medium is from about 2 to about 6 mM.

15. The method of any preceding claim, wherein the modified peptide is a modified linear peptide.

16. The method of claim 1 or any of claims 4 to 15 when dependent on claim 1 , wherein the method forms an amide bond.

17. The method of claim 4, wherein the modified peptide precursor comprises a hydrazide group.

18. The method of claim 4 or claim 17, wherein the oxidising agent is selected from sodium nitrite, nitrous acid, / V-bromosuccinimide, / V-chlorosuccinimide, optionally wherein the oxidising agent is sodium nitrite.

19. The method of any of claims 4, 17 or 18, wherein the precursor reaction medium further comprises a solubiliser which maintains the modified peptide precursor and the modified peptide comprising the acyl azide group in solution.

20. The method of claim 19, wherein the solubiliser is selected from: guanidine or a salt thereof, urea or a salt thereof, arginine or a salt thereof, or a second solvent;optionally wherein the second solvent is a water- miscible organic solvent selected from acetonitrile, ethyl acetate, tetrahydrofuran, methyl ethyl ketone, butanol, dimethylformamide, tetrahydropyran, 1 ,4-dioxane, 1 ,3-dioxolane, 2,2,2-trifluoroethanol, 2- methyltetrahydrofuran, dimethylsulfoxide, sodium dodecyl sulfate, polysorbate 20 (Tween- 20), polysorbate 80 (Tween-80), octylphenoxypolyethoxyethanol (IGEPAL), 2-[4-(2,4,4- trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or other peptide compatible detergent / surfactant.

21. The method of claim 4 or any of claims 17 to 20, wherein the precursor reaction medium has a pH of no more than about 4.0; optionally wherein the precursor reaction medium has a pH of from about 2.5 to about 3.5, more preferably about 3.0.

22. The method of claim 4 or any of claims 17 to 21, wherein the concentration of the modified peptide precursor in the precursor reaction medium is at least about 1 mM, preferably at least about 2 mM; optionally wherein the concentration of the modified peptide precursor in the precursor reaction medium is from about 2 to about 6 mM.

23. The method of claim 4 or any of claims 17 to 22, wherein the modified peptide comprising the acyl azide group is not isolated from the precursor reaction medium prior to increasing the pH of the reaction medium to generate the cyclised peptide.

24. The method of any preceding claim, the method further comprising, after generating the cyclised peptide, modifying one or more functional groups of the cyclised peptide.

25. A method of generating a library of cyclised peptides, the method comprising performing the method of any preceding claim on at least two different modified peptides comprising acyl azide groups.

26. A peptide library obtained using the method of claim 25.

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