WO2010111737A1

WO2010111737A1 - Peptide and phosphopeptide synthesis via phosphate assisted ligation

Info

Publication number: WO2010111737A1
Application number: PCT/AU2010/000366
Authority: WO
Inventors: Richard Payne; Gemma Thomas
Original assignee: The University Of Sydney
Priority date: 2009-03-31
Filing date: 2010-03-31
Publication date: 2010-10-07

Abstract

Methods of preparing polypeptides and proteins. In particular, the invention has been developed as a method of preparing peptides and phosphopeptides through formation of a peptide bond via phosphate assisted peptide ligation

Description

PEPTIDE AND PHOSPHOPEPTIDE SYNTHESIS VIA PHOSPHATE ASSISTED LIGATION

FIELD OF THE INVENTION

The present invention relates to a method of preparing polypeptides and proteins. In particular, the invention has been developed as a method of preparing peptides and phosphopeptides through formation of a peptide bond via phosphate assisted peptide ligation and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

Phosphorylation is a ubiquitous post-translational modification reported to occur on 30- 50% of human proteins. This reversible process is known to be intimately involved in signal transduction and the control of a host of biological processes that are critical for normal cellular function. Unlike protein synthesis, phosphorylation events are not under template control, but rather are dictated by the combined activities of a range of kinase (phosphorylation) and phosphatase (dephosphorylation) enzymes. To date, the study of phospho proteins has been significantly hampered by the fact that they exist as heterogeneous mixtures of phosphoforms. It is currently accepted that chemical synthesis may provide a viable avenue for the construction of post-translationally modified peptides and proteins in a homogeneous fashion and thus has been the focus of intense research efforts over the past decade. To date, the most efficient strategy for the generation of these biomolecules has relied on chemoselective ligation methods. Native chemical ligation (NCL) currently represents a useful strategy and involves the condensation of a C-terminal peptide thioester and a peptide bearing an TV-terminal cysteine residue to afford a native peptide bond in a rapid and chemoselective manner. The major limitation of this method, however, is the requirement for the scarce amino acid cysteine (1.9% occurrence) at the iV-terminus of a peptide fragment. A number of alternative approaches have since been developed to overcome this prerequisite cysteine, with a view to expanding the number of ligation sites that can be accessed by peptide ligation chemistry. Recent examples include the traceless Staudinger ligation, NCL at phenylalanine and valine residues, sugar-assisted ligation and thiol free "direct aminolysis" methods.

However, there remains a need for the development of new ligation strategies that can be implemented in the synthesis of native and post-translationally modified peptides and proteins for biological study.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

It is an object of the invention in its preferred form to provide a method of preparing peptides and phosphopeptides via phosphate-assisted ligation.

SUMMARY OF THE INVENTION

In a broadest aspect, the invention provides a method of forming a peptide bond between a C terminal of a first amino acid, peptide or analogue thereof and an N terminal of a second amino acid, peptide or analogue thereof, the second amino acid, peptide or analogue thereof comprising a pendant phosphate bearing linker attached to a carbon α to the N terminal. The N and C terminals or the phosphate may be independently substituted or unsubstitued with protecting or leaving groups or similar.

According to a first aspect of the invention there is provided a method of preparing a compound of Formula (I):

(I) including a stereoisomer, tautomer, solvate, hydrate, protonated form, or a salt thereof; wherein,

R^a and R^b are each independently at each occurrence H or alkyl; R^c is H or optionally substituted alkyl;

R^d is H or optionally substituted alkyl; X is a covalent bond or aryl;

R¹ together with carbonyl to which it is attached is an optionally substituted amino acid or peptide; and R² is an optionally substituted amino acid, peptide; the method comprising: contacting a compound of Formula (II):

(II) and a compound of Formula (III)

(III) wherein LG is a leaving group selected from the group consisting of -OR, -SR and -NR₂, wherein R is independently at each occurrence optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, or each R together with the N to which it is attached, forms an optionally substituted heterocyclalkyl or heteroaryl; to provide a compound of Formula (I).

It will be appreciated that R¹ together with the carbonyl to which it is attached is an optionally substituted amino acid or peptide. That is, the carbonyl (C=O) forms part of the carboxylic acid (COOH) group of the amino acid. The carboxylic acid -OH of the - A - amino acid or the C-terminal end of the peptide is substituted by LG or is absent by way of formation of an amide bond. Conversely, the optionally substituted amino acid or peptide R² is connected by the N-terminus of the amino acid or peptide. That is, the carbonyl to which it is attached does not form part of the carboxylic acid group of the N- terminal amino acid.

The compound of formula (II) is preferably selected from the group consisting of

More preferably, the compound of formula (II) is selected from the group consisting of

Preferably R^c is H. R^d is preferably H. In one embodiment preferably LG is -SR. -SR is preferably -S(CH₂)2CO₂Et. In a further embodiment preferably LG is -NR₂. -NR₂ is

preferably ^■ < oλ ^NH . In yet another embodiment preferably LG is -OR. -OR is

preferably A

The compound of formula (III) is preferably an amino acid, peptide or polypeptide or a protected amino acid, peptide or polypeptide. Preferably R¹ together with the carbonyl to which it is attached is an amino acid, or an optionally substituted peptide commencing at the C- terminus with an amino acid, selected from the group consisting of Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and VaI.

Preferably R¹ together with the carbonyl to which it is attached is an amino acid, or an optionally substituted peptide commencing at the C-terminus with an amino acid, selected from the group consisting of GIy, Ala, Met, Phe, Tyr, Ser and VaI.

R¹ is preferably N-Acetyl-Leu-Tyr-Arg-Ala-Y-Z, wherein Y is Asn or a covalent bond, and Z is selected from the group consisting of GIy, Ala, Met, Phe, Tyr, Ser and VaL

Preferably R² is an amino acid, or an optionally substituted peptide commencing at the N-terminus with an amino acid, selected from the group consisting of Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and VaI.

R² is preferably Ser-Pro-Gly-Tyr-Ser-NH₂.

Preferably contacting comprises contacting in an aqueous solution. The aqueous solution preferably has a pH in the range between about 6 and about 14,

Preferably the aqueous solution further comprises a water miscible aprotic solvent. The aprotic solvent is preferably selected from the group consisting of NMP, DMF, HMPA and DMSO, or mixtures thereof.

Preferably the aqueous solution further comprises 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) and guanidine.HCl. More preferably the aqueous solution comprises JV-methylpyrrolidinone (NMP), 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) and guanidine.HCl.

Preferably the aqueous solution further comprises a thiol. The preferred thiol is thiophenol. Preferably contacting is carried out at a temperature range of between about -80 ⁰C to about 150 ⁰C. More preferably the temperature is in the range between about 35 ⁰C to about 40 ⁰C.

Preferably the compound of formula (II) and the compound of formula (III) are employed in a molar ratio of about 0.8 to about 1.2.

The method preferably further comprises purifying the compound of formula (I) by HPLC, reverse phase column chromatography, anion exchange chromatography, size exclusion chromatography or by crystallisation.

Preferably the method further comprises dephosphorylating the compound of formula (I). Preferably dephosphorylating the compound of formula (I) comprises contacting the compound of formula (I) with alkaline phosphatase.

According to a second aspect of the invention there is provided an intermediate compound of formula (IV)

(IV)

including a stereoisomer, tautomer, solvate, hydrate, protonated form, or a salt thereof; wherein,

R^d is H or optionally substituted alkyl;

X is a covalent bond or aryl; R¹ together with carbonyl to which it is attached is an optionally substituted amino acid or peptide; and

R is an optionally substituted amino acid or peptide.

According to a third aspect of the invention there is provided a compound of formula (I) when prepared by the method of the first aspect of the invention.

The methods of the present invention can include a further step of dephosphorylating the compound of formula I.

(I)

DEFINITIONS

The term "amino acid," includes but is not limited to the ribosomal amino acids (e.g. Ala, Arg, Asn, Asp, Cys, GIu₅ Gin, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI), with the proviso that each of these may be in the D or the L form, as well as naturally occurring but non-ribosomal amino acids (e.g. phospho serine, phospho threonine, phosphotyrosine, hydro xypro line, hydroxylysine, gammacarboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l₅2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α- methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Further, the term encompasses α-amino acids that have not been found in nature, such as for example fluoro-substituted amino acids, as well as amino acids wherein the amino group is not in an α relationship with the carboxyl group, such as β-, γ-, and δ-amino acids.

It will also be appreciated by those skilled in the art that the term amino acid or peptide, especially as used with reference to R¹ and R², includes within its scope amino acid or peptide analogues. The term "amino acid" also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (Ci-C₆)alkyl, phenyl or benzyl ester or amide; or as an α-methylbenzyl amide), as well as amino acids with sidechains such as carboxyl, carboxamide, amino, guanido, thio, hydroxyl, and other groups bearing protecting groups. Additional suitable protecting groups of all these types are known to those skilled in the art (See for example, Greene, T. W.; Wutz, P.G.M. Protecting Groups In Organic Synthesis, 2nd edition, John Wiley & Sons, Inc., New York (1991)).

An amino acid sidechain refers to a monovalent radical bonded to a carbon atom of an amino acid that is other than the carboxy lie acid (COOH) group of the amino acid. The amino acid sidechain can refer to the group R, as exemplified in the following structure of a ribosomal α-amino acid:

O

H₂N _^ J_x CH ^OH

R

wherein R is methyl (Ala); R is 3-guanidopropyl (Arg); R is carboxamidomethyl (Asn); R is carboxymethyl (Asp); R is thiolmethyl (Cys); R is carboxyethyl (GIu); R is carboxamidoethyl (GIn); R is hydrogen (GIy); R is (4-imidazolyl)methyl (His); R is isobutyl (He); R is sec-butyl (Leu); R is 4-aminobutyl (Lys); R is 2-(methylthio)ethyl (Met); R is benzyl (Phe); R is hydroxymethyl (Ser); R is 1 -hydroxyethyl (Thr); R is 3- indolylmethyl (Trp); R is p-hydroxybenzyl (Tyr); and R is isopropyl (VaI). It is understood that additional carbon atoms may exist between the carboxyl group and the amino group of an amino acid, and the R group may reside on any of those carbon atoms. In an α-amino acid, the structure is as shown above, but the "amino acid sidechain" may be disposed on a carbon atom other than the carboxyl carbon of a β-, γ-, or δ-amino acid.

The term "peptide" describes a sequence of two or more amino acids (e.g. as defined hereinabove) wherein the amino acids are sequentially joined together by amide (peptide) bonds. The sequence may be linear or cyclic. When the sequence is cyclic, the peptide may further comprise other bond types connecting the amino acids, such as an ester bond (a depsipeptide) or a disulfide bond. For example, a cyclic peptide can be prepared or may result from the formation of a disulfide bridge between two cysteine residues in a sequence. Peptide sequences specifically recited herein are written with the amino or N-terminus on the left and the carboxy or C-terminus on the right.

A "peptide residue" refers to a sequence of amino acids, that is, amino acids connected by amide bonds, wherein the N-terminus and the C-terminus are not necessarily in free form but may be further linked to additional amino acids or to other radicals. Thus a single peptide may include a large set of possible peptide residues as defined herein.

Optionally substituted amino acids and peptides include, although are not limited to phosphoamino acids, phosphopeptides, methylated amino acids, methylated peptides, glycoamino acids, glycopeptides, acylated amino acids, acylated peptides, isoprenylated amino acids, isoprenylated peptides, alkylated amino acids, alkylated peptides, sulfated amino acids, sulfated peptides, glycophosphatidylinositol (GPI anchor) amino acids, glycophosphatidylinositol peptides, ubiquitinated amino acids and ubiquitinated peptides.

"Alkyl" refers to a Ci to about a Ci g hydrocarbon containing primary, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH₃), ethyl (Et, -CH₂CH₃), 1-propyl («-Pr, w-propyl, -CH₂CH₂CH₃), 2-propyl (/-Pr, /-propyl, -CH(CH₃)₂), 1-butyl (n-Bu, »-butyl, -CH₂CH₂CH₂CH₃), 2-methyl- 1-propyl (/-Bu, /-butyl, -CH₂CH(CH₃)₂), 2-butyI (j-Bu, j-butyl, -CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (/-Bu, /-butyl, -C(CH₃)₃), and so forth. The alkyl can be a monovalent radical, capable of bonding to a single radical as described and exemplified above, or it can be a divalent radical capable of bonding to two distinct monovalent radicals or to a single divalent radical. The alkyl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, halo alkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, hetero cycle, cyclo alkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, car boxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzene sulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, phosphate, phosphonate, sulfamoyl, sulfinamoyl, sulfino, sulfa, sulfoamino, thiosulfo, NR^jR^k and/or COOR^j, wherein each R^J and R^k are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. The alkyl can optionally be interrupted with one or more non-peroxide oxy (-O-), thio (-S-), imino (-N(H)-), methylene dioxy (-OCH₂O-), carbonyl (-C(=O)-), carboxy (-C(=O)O-), carbonyldioxy (- OC(=O)O-), carboxylato (-OC(=O)-), imine (C=NH), sulfinyl (SO) or sulfonyl (SO₂). Additionally, the alkyl can optionally be at least partially unsaturated, thereby providing an alkenyl.

The term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to about 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Preferred aryls include phenyl, naphthyl and the like.

The aryl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoro methyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, phosphate, phosphonate, sulfamoyl, sulfinamoyl, sulfino, sulfa, sulfoamino, thiosulfo, NR'R¹⁴ and/or COOR*, wherein each R^J and R^k are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. The term "cycloalkyl" refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings; e.g. bicyclo, tricyclo, and higher polycyclic entities. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclo butyl, cyclopentyL cyclooctyl, and the like, or multiple ring structures such as adamantanyl, bicyclo structures such as pinanes, and the like. The cycloalkyl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthϊo, alkylsulfinyl, alkyl sulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarboiiyl, benzylthio, carbamoyl, carbamate, isocyannato, phosphate, phosphonate, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^jR^k and/or COOR^J, wherein each R^j and R^k are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.

The cycloalkyl can optionally be at least partially unsaturated, thereby providing a cycloalkenyl.

The term "heterocycloalkyl" refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced by a heteroatom, such as oxygen, nitrogen, phosphorus, or sulfur. The term encompasses structures wherein two or more carbon atoms are replaced, each by a different type of heteroatom.

The term "heteroaryl" is defined herein as a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted. Examples of heteroaryl groups include, but are not limited to, 2//~pyrrolyl, 3//-indolyl, 4//-quinolizinyl, 4n#^"-carbazolyl, acridinyl, benzo[ό]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnaolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, naptho[2,3-&], oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl. In one embodiment the term "heteroaryl" denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from the group non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl. In another embodiment heteroaryl denotes an ortho-fhsed bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, or tetramethylene diradical thereto.

The heteroaryl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkyl sulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, phosphate, phosphonate, sulfamoyl, sulfinamoyl, sulfuio, sulfo, sulfoamino, thiosulfo, NR^jR^k and/or COOR", wherein each R^j and R^k are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.

The term "amino" refers to -NH₂, and the term "alkylamino" refers to -NR₂, wherein at least one R is alkyl and the second R is alkyl or hydrogen. The term "acylamino" refers to RC(=O)N, wherein R is alkyl or aryl.

The terms "carboxy" or "carboxyl" refer to a monovalent radical -CO₂H or -CO₂M, wherein M is a cation and the -CO₂ portion bears a negative charge; both groups having a single additional valance to be filled by formation of a single bond to a substituent. A C(=O) radical is termed a "carbonyl" and is a divalent radical, having two valances that are filled by single bonding to two separate substituents or double bonding to a single substituent. The term "thiol" or "mercapto" refers to the monovalent radical -SH. With a single unfilled valance, it may be bonded to a single substituent.

The terms "hydroxyl" or "hydroxy" as used herein refer to the monovalent OH radical, typically bonded to a carbon atom, although the term encompasses an OH group bonded to a heteroatom. A "hydroxyl protecting group" refers to any of the groups that may replace the hydrogen atom of the -OH group such as to render the group less reactive or unreactive under certain conditions. See, for example, Greene, T. W.; Wutz, P.G.M. Protecting Groups In Organic Synthesis, 2nd edition, John Wiley & Sons, Inc., New York (1991)). A" hydroxyl protecting group" encompasses esters such as acetates and benzoates, carbonates such as methyl carbonates, urethanes such as dimethylaminocarbamates, acetals such as tetrahydropyranyl ethers and methoxymethyl ethers, sulfonate esters such as p-toluenesulfonyl esters, and silylethers such as tert- butyldiphenylsilyl ethers.

The term "oxo" or "an oxo group" as used herein refers to a divalent radical of the formula =0, wherein a single oxygen atom is bonded via a covalent double bond to another atom. When an oxo group is bonded to a carbon atom, that group is a carbonyl radical.

Abbreviations

DCM d ichloro methane

DIC diisopropylcarbodiimide

DIEA di-isopropylethylamine

DMF N, N-d imethylformamide

DMSO dimethyl sulfoxide

DVB divinylbenzene

ESI electrospray ionisation

Fmoc 9-fluo reny lmetho xycarbony 1

HATU O-(7-azabenzotriazo 1- 1 -yl)-Λζ N, N ',N '-tetramethyluronium hexafluor opho sphate

HEPES 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid

HMPA hexamethylphosphoramide HOBt N-hydroxybenzotriazole

HPLC high performance liquid chromatography

LC-MS liquid chromatography - mass spectrometry

NMM 7V-methylmorpholine PyBOP benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate

TFA trifluoro acetic acid

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows a synthetic scheme for preparation of a phosphopeptide according to the present invention; and

Figure 2 shows the kinetics of the phosphate-assisted peptide ligation between peptide thioester 5 and peptides 1-4; ■ = 1, ♦ = 2, A = 3, # = 4. f glycine thioester 5 was completely hydrolysed after 72h.

PREFERRED EMBODIMENT OF THE INVENTION

The present invention provides a method for the ligatory synthesis of phosphopeptides and peptides, which utilizes the inherent reactivity of a peptide bearing a side chain phosphorylated hydroxy amino acid such as serine or threonine as the iV-terminal amino acid to facilitate amide bond formation with a C-terminal peptide thioester (-C(O)SR). The present invention also anticipates the suitability of this method with reactive C- terminal peptide esters -C(O)OR and amide groups -C(O)NR₂ wherein R is independently at each occurrence optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. The present invention also anticipates the suitability of this method with other phosphorylated hydroxyl amino acids such as a side chain phosphorylated tyrosine as the JV-terminal amino acid. In a preferred embodiment the C-terminal peptide thioester is -C(O)S(CH₂)₂CO₂Et. In

another embodiment the preferred C-terminal peptide ester is

. In yet another embodiment of the present invention, the preferred C-

terminal peptide amide is

The cysteine-free ligatory synthesis of peptides and phosphopeptides of the present invention, namely phosphate-assisted ligation, harnesses the nucleophilicity of a phosphate moiety on a side chain phosphorylated amino acid at the iV-terminal amino acid of a peptide. Without wishing to be bound by theory, the applicant postulates that, when reacted with a peptide thioester under suitable conditions, the phosphate generates an acyl phosphate intermediate. A subsequent O→N acyl shift via a 7- membered ring transition state forms the desired peptide bond, as shown in Scheme 1.

Scheme 1. Proposed mechanism of the phosphate-assisted ligation. The reaction of the present invention as exemplified by the reaction between sidechain phosphorylated serine or threonine as the ΪV-terminal amino acid of a peptide displayed impressive scope with a broad range of thioesters to provide the corresponding phosphopeptide products in good yield. Serine and threonine as linkers, with abundances of 7% and 6%, make up 13% of the amino acids of all proteins. The method of the present invention therefore provides an enhanced ability to find suitable linking sites in a large number of proteins. Enzymatic dephosphorylation of the phosphopeptide ligation products using alkaline phosphatase consequently generated the corresponding native (unphosphorylated) peptides.

The present invention also offers an increased number of ligation junctions over native chemical ligation (NCL) as serine and threonine are more common in peptides than cysteine. (NCL is a ligation that can be carried out between a peptide thioester and a peptide with the iV-terminal amino acid being cysteine, wherein a thioester bond is first formed with the cysteine, which is followed by intramolecular S to N acyl migration).

Referring to Figure 1, a synthetic scheme of an embodiment according to the present invention is shown. The peptide referred to as "Peptide chain 1 " that will form the N- terminal segment of the product is prepared with a C-terminal carboxy thioester, which serves to activate the segment for amide (peptide) bond formation. Peptide chain 1 may optionally be protected at its N-terminus, for example with an N-acetyl group, or may have a free amino group. Sidechains comprising free amino groups as in lysine, free hydroxy 1 groups as in tyrosine, serine and threonine, free thio groups as in cysteine, free guanido groups as in arginine, carboxyl groups as in aspartate or glutamate, and free carboxamido groups as in asparagine and glutamine may be present during the coupling reaction in unprotected form. Cysteine residues, however, can be blocked during the coupling reaction, for instance as acetamido methyl or tert-butyl derivatives. To prepare the Peptide chain 1 thioester for use as a reactant in the method of the present invention, the C-terminal carboxyl group may be obtained in thioester form using any suitable method. Alternatively, an activated carboxylic acid (-C(O)OR) or amide (-C(O)NR₂), wherein R is not H, may be employed. Preferably, the C-terminal thioester group is provided using known solid phase peptide synthesis procedures, wherein the anchoring polymer is derivatised with a group that will yield the desired thioester upon final removal of the assembled peptide from the polymer. The peptide segment shown as "Peptide chain 2" in Figure 1 will form the C-terminal portion of the coupled (phospho)peptide. Peptide chain 2 comprises an amino acid bearing a side chain to which a phosphate moiety is covalently bonded. Peptide chain 2 comprises an α-amino group which will be coupled with the carboxyl thioester of Peptide 1 by the method of the invention.

The peptide 1, bearing an /V-terminal phospho serine residue and peptide thioester 5 possessing a C-terminal glycine residue, were added to a mixed solvent buffer comprising 4: 1 v/v N-methylpyrrolidinone: 1.25M Gn.HCl, 0.2 M HEPES, pH 7.5 in the presence of thiophenol at 37 ⁰C. After 48 h the reaction was complete and the desired phosphopeptide product was isolated in 91% yield (entry I₅ Table 1). Peptide 2, bearing an /V-terminal phosphothreonine residue, also reacted with thioester 5 in a facile manner to afford the desired phosphopeptide in 95% yield (entry 2, Table 1). The importance of the phosphate moiety for the efficiency of this ligation reaction was verified by conducting control ligations with peptides 3 and 4, bearing TV-terminal serine and threonine residues respectively. These were reacted with 5 under the same mixed solvent system to afford ligated peptide products in only 45 and 36% yields respectively after an increased reaction time of 72 h (entries 3 and 4, Table 1). Given the increased steric bulk of the phosphate group, it was surprisingly found the phosphate moiety was crucial to the efficiency of the ligation reactions. Kinetic studies quantified this increase in reaction rate (Figure 2). After 1O h, reactions with 1 and 2 provided a 50% yield of the desired ligation product and reactions were complete within 48 h. In contrast, unphosphorylated peptides 3 and 4 underwent sluggish ligation reactions, corroborating that the phosphate moiety in 1 and 2 was assisting the formation of the peptide bond and that these reactions were not proceeding via a direct amino lysis reaction alone. Thus, the Applicant has found that N-terminal phosphorylamino acids enhance the rate of ligation reactions.

Peptide thioesters were synthesized bearing a range of amino acids at the C-terminus (Ficht, S.; Payne, R. J.; Guy, R. T.; Wong, C. H. Chem. Eur. J. 2008, 14, 3620-3629). GIy, Ala, Met, Phe, Tyr, Ser and VaI were selected as a representative range of the 20 proteinogenic amino acids to incorporate into peptide thioesters (5-11). These were reacted with phosphopeptides 1 and 2 under the previously described conditions (Table 1). The phosphate-assisted ligation reactions with 1 were high yielding in almost all cases (entries 5-10, Table 1). Indeed, phosphate-assisted ligations with thioesters 5-10 bearing C-terminal GIy, Ala, Met, Phe, Tyr and Ser residues gave the desired phosphopeptides in yields ranging from 62-91%. In line with reactivity patterns observed in NCL, the ligation with C-terminal valine thioester 11 resulted in a much slower reaction, requiring 14 days to reach completion. The valine thioester was significantly more stable than 5-10 under the ligation conditions. This led to slower hydrolysis and allowed for a satisfactory ligation yield (47%, entry 10, Table 1). Ligations with phosphopeptide 2 containing an //-terminal phospho threonine residue provided lower yields. Without wishing to be bound by theory the Applicant believes this to be due to the increased steric bulk of the threonine residue. Isolated reaction yields were moderate to high in all cases and represent synthetically useful reactions (entries 11-16, Table 1).

Ac-LYRAX-S(CH₂)₂CO₂Et

1 : R = PO₃H, R² = H 5: X = AsnGly; 6: X = AsπAla; 7: X = AsnMet; 8: X = AsnPhe; 2: R = PO₃H, R² = CH₃ 9: X = Tyr; 10: X = Ser; 11: X = VaI 3: R = H, R² = H 4: R = H, R² = CH₃

Table 1. Scope of the phosphate-assisted ligation reaction. Reaction times: ^a 48 h; ^b 72 h; C 14 d.

Dephosphorylation reactions on the abovementioned ligation products afforded unmodified peptides. Phosphopeptide ligation products were treated with alkaline phosphatase in 50 mM Tris buffer at pH 8.6 to provide dephosphorylated peptide products in high yields in all cases (74-98% isolated yields).

The dephosphorylation reaction significantly expands the utility of the phosphate- assisted ligation, whereby the phosphate moiety can be introduced as a traceless ligation auxiliary with the view to incorporating native serine and threonine residues into target peptides or proteins. The generation of unmodified peptides by post-ligatory enzymatic dephosphorylations therefore provides a direct disconnection at serine and threonine residues.

The method of the present invention displays impressive scope for a range of amino acids at the ligation junction, and, as such, should serve as a useful tool for the construction of biologically relevant peptides, phosphopeptides, proteins, phosphoproteins and their modified equivalents e.g. for example by phosphorylation, methylation, glycosylation, acylation or isoprenylation. EXAMPLES

Embodiments of the invention are described below with reference to the following non- limiting examples.

General materials and methods

Analytical reverse-phase HPLC was performed on a Waters System 2695™ separations module with an Alliance™ series column heater at 30 °C and 2996 photodiode array detector. A Waters Sunfire™ 5 μm, 2.1 x 150 mm column was used at a flow rate of 0.2 niL min^"1 and results analysed with Waters Empower™ software. Preparative reverse- phase HPLC was performed using a Waters 600 Multisolvent Delivery System™ and Waters 500™ pump with 2996 photodiode array detector or Waters 490E™ Programmable wavelength detector operating at 230 and 280 nm. A Waters Sunfire™ 5 μm, 19 x 150 mm column was used at a flow rate of 7 niL min^"1 using a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B). Semi- preparative HPLC was performed on a Waters 600 Multisolvent Delivery System™ and Waters 500™ pump with 2996 photodiode array detector or Waters 490E™ Programmable wavelength detector operating at 230 and 280 nm. A Grace Vydac™ "Protein and Peptide C 18", 250 x 10 mm, 10-15 μM particle size column was used at a flow rate of 4 mL min^'1 using a mobile phase of 0.1% TFA in water (Solvent A) and 0.1 % TFA in acetonitrile (Solvent B).

LC-MS was performed on a Thermo Separation Products: Spectra System™ consisting of P400 Pump and a UV6000LP Photodiode array detector on a Phenomenex Jupiter™ 5 μm, 2.1 x 150 mm column at a flow rate of 0.2 mL min^"1 coupled to a Thermoquest Finnigan LCQ Deca™ mass spectrometer (ESI) operating in positive mode. Separations involved a mobile phase of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B).

ESI mass spectrometry was performed on a Thermoquest Finnigan LCQ Deca™ mass spectrometer operating in positive mode.

Commercial materials were used as received unless otherwise noted. Amino acids, coupling reagents and resins were obtained from Novabiochem. DCM and methanol were distilled from calcium hydride. DMF was obtained as peptide synthesis grade from Auspep or Labscan.

Synthesis of peptides and phosphopeptides (1-4) via the Fmoc strategy Solid-phase peptide synthesis was carried out in syringes, equipped with teflon filters, purchased from Torviq.

Preloading Rink Amide resin

Rink amide resin was initially washed with DCM (x 5) and DMF (x 5), followed by removal of the Fmoc group by treatment with 10% piperidine/DMF (2 x 5 min). The resin was washed DMF (x 5), DCM (x 5), DMF (x 5). PyBOP (4 eq.) and NMM (8 eq.) was added to a solution of Fmoc-Ser(0tBu)-OH (4 eq.) in DMF (final concentration 0.1 M). After 5 min of pre-activation, the mixture was added to the resin. After 2 h the resin was washed DMF (x 5), DCM (x 5), DMF (x 5), capped with acetic anhydride/pyridine ( 1 :9 v/v) (2 x 5 min) and washed DMF (x 5), DCM (x 5) and DMF (x 5).

Iterative peptide assembly: Deprotection: The resin was treated with 10% piperidine/DMF (2 x 5 min) and washed with DMF (x 5), DCM (x 5) and DMF (x 5). Amino acid coupling: A preactivated solution of protected amino acid (4 eq.), PyBOP (4 eq.) and NMM (8 eq.) in DMF (final concentration 0.1 M) was added to the resin. After 45 min, the resin was washed with DMF (x 5), DCM (x 5) and DMF (x 5). Capping: Acetic anhydride/pyridine (1 :9 v/v) was added to the resin. After 5 min the resin was washed with DMF (x 5), DCM (x 5) and DMF (x 5). Cleavage: A mixture of TFA, thioanisole, triisopropylsilane and water (17:1:1 :1 v/v/ v/v) was added to the resin. After 2 h, the resin was washed with TFA (3 x 2 mL) Work-up: The combined solutions were concentrated in vacuo. The residue was dissolved in water containing 0.1% TFA and purified by preparative HPLC (gradient 0 to 30% B over 40 min) and analyzed by LC- MS (0 to 30% B over 30 min) and ESI mass spectrometry. Analytical Data for peptides and phosph op ep tides 1-4

₍A_Ubso_ran H-pSSPGYS-NH₂ (l)

50 μM scale, Yield - 32 mg, 95%; ESI (m/z): Calculated Mass [M+H]⁺: 676.2, Mass Found 676.1; HPLC: t_R: 14.2 min (Gradient 0 to 50% B over 40 min.).

H-pTSPGYS-NH₂ (2)

50 μM scale, Yield = 30 mg, 87%; ESI (m/z): Calculated Mass [M+H]⁺: 690.3, Mass Found 690.0; HPLC: t_R: 14.3 min (Gradient 0 to 50% B over 40 min.).

H-SSPGYS-NH₂ (3)

10 50 μM scale, Yield = 29 mg, 97%; ESI (m/z): Calculated Mass [M+H]⁺: 596.3, Mass Found 596.1; HPLC: t_R: 13.3 min (Gradient 0 to 50% B over 40 min.).

15

H-TSPGYS-NH₂ (4)

0

50 μM scale, Yield = 28 mg, 92%; Calculated Mass [M+H]⁺: 610.3, Mass Found 610.2; HPLC: t_R: 14.7 min (Gradient 0 to 50% B over 40 min.).

Synthesis of peptide thioesters (5-11) via the side chain anchoring strategy

Side Chain Anchoring of amino acids onto Rink Amide Resin:

Rink Amide resin co. (100-200 mesh; 1% DVB) (290 mg, loading = 0.69 mmol/g, 200 μmol) was initially washed with DMF (x 5), DCM (x 5) and DMF (x 5), treated with DMF/piperidine (9:1) (2x 5 min) and washed with DMF (x 5), DCM (x 5) and DMF (x 5). A solution of Fmoc-Asp-OAllyl (800 μmol, 4 eq.) in dry DMF (4 ml) containing PyBOP (416 mg, 800 μmol, 4 eq.) and JV-methylmorpholine (176 μl, 1600 μmol, 8 eq.) was preactivated for 4 min and then added to the resin. After two hours of shaking, the resin was washed with DMF (x 5), DCM (x 5) and DMF (x 5), treated with Ac₂O/Py (1 :9 v/v) for 10 min and then washed with DMF (x 5), DCM (x 5) and DMF (x 5). Treatment of the resin with 10% piperidine/DMF (2 x 5 min) and measurement of the resulting fulvene-piperidine adduct at λ = 302 nm showed that the yield of the side chain anchoring was quantitative. Side Chain Anchoring onto Bromo-(4-methoxyphenyl)methylpoϊystyrene:

Bromo-(4-methoxyphenyl)methylpolystyrene resin (174 mg, loading = 2.3 mmol/g, 400 μmol) was initially washed with DMF (x 5) and DCM (x 10) and the resin swelled with DCM (5 mL) for 1 h with the exclusion of light. A solution of Fmoc-Ser-Oallyl (Ficht, S.; Payne R. J.; Guy R. T.; Wong C. H. Chem. Eur. J. 2008, 14, 3620-3629) or Fmoc- Tyr-Oallyl (Ficht, S.; Payne R. J.; Guy R. T.; Wong C. H, Chem. Eur. J. 2008, 14, 3620- 3629) (1200 μmol, 3 eq.), DIEA (2400 μmol, 397 μL, 6 eq.) in DCM (4 mL) was added to the resin, which was shaken for 18-46 h at rt with the exclusion of light. The resin was washed with DCM (x 5) and DMF (x 10). Treatment of the resin with 10% piperidine/DMF (2x 5 min) and measurement of the resulting fulvene-piperidine adduct at λ = 302 nm was used to determine the loading. NB: The commercially available resin is sold with a very high loading, therefore preloadings of the resins were conducted for a length of time that allowed for the resin to be loaded no more than 1.4 mmol/g to prevent resin crowding and product aggregation. Final loadings: Fmoc-Ser-OAllyl (18 h): loading = 1.28 mmol/g; Fmoc-Tyr-OAllyl (46 h): loading = 1.02 mmol/g;

Solid Phase Peptide Assembly

Peptide assembly was conducted as described for the iterative peptide synthesis above.

General Procedure for the C-terminal introduction of amino acid thioesters (Ficht, S.; Payne R. J.; Guy R. T.; Wong C. H. Chem. Eur. J. 2008, 14, 3620-3629):

The resin (25 μmol) was swollen in dry DCM for 30 min, followed by the addition of a solution of Pd(PPh₃ )₄ (25 mg, 22 μmol) and PhSiH₃ (123 μl, 108 mg, 1 mmol, 40 eq.) in dry DCM (2 mL). The resin was shaken for 1 h and the procedure was repeated once. Afterwards, the resin was washed with DCM (x 10), DMF (x 5) and DCM (x 5). The amino acid thioester (GIy-SR, AIa-SR, Met-SR, Phe-SR, Ser-SR, VaI-SR) (Ficht, S.; Payne R. J.; Guy R. T.; Wong C. H. Chem. Eur. J. 2008, 14, 3620-3629) (250 μmol, 10 eq.) and DIEA (86 μl, 65 mg, 500 μmol, 20 eq.) were dissolved in dry DCM (1 ml) and added to the resin. HATU (95 mg, 250 μmol, 10 eq.) was added as a solid and the resin was shaken for 1 h. The procedure was repeated once and the resin was washed with DCM (x 5), DMF (x 5) and DCM (x 10). Cleavage: A mixture of TFA, thioanisole, triisopropylsilane and water (17:1 :1 :1 v/v/v/v) was added to the resin. After 2 h, the resin was washed with TFA (4x 4 mL) Work-up: The combined solutions were concentrated in vacuo. The residue was dissolved in water containing 0.1% TFA and purified by preparative HPLC (0 to 50% B over 40 min.) to afford the desired peptide thioesters as white fluffy solids which were analyzed by HPLC (0 to 50% B over 40 min) and ESI mass spectrometry.

Analytical Data for peptide thioesters 5-8 and 11:

Ac-LYRANG-S(CH₂) ₂CO₂Et (5)

25 μmol of fully assembled Rink amide resin was treated with H-Gly-S(CH₂)₂CO₂Et according to the general procedure described above. Yield: 21.3 mg, quant.; Calculated Mass [M+H]⁺: 851.4, Mass Found 851.5; HPLC: t_κ: 28.5 min (Gradient 0 to 50% B over 40 min.).

Ac-LYRANA-S(CH₂) ₂CO₂Et (6)

25 μmol of fully assembled Rink amide resin was treated with H-Ala-S(CH₂)₂CO₂Et according to the general procedure described above. Yield: 21.7 mg, quant.; Calculated Mass [M-HH]⁺: 865.4, Mass Found 865.4; HPLC: t_R: 29.5 min (Gradient 0 to 50% B over 40 min.).

Ac-LYRANM-S(CH₂) ^CO₂Et (7)

25 μmol of fully assembled Rink amide resin was treated with H-Met-S(CH₂)₂CO₂Et according to the general procedure described above. Yield: 20.6 mg, 90%; Calculated Mass [M+H]⁺: 925.4, Mass Found 925.4; HPLC: fe: 32.6 min (Gradient 0 to 50% B over

K_Ivee 40 min.).

Ac-LYRANF-S(CH₂) ₂CO₂Et (8)

25 μmol of fully assembled Rink amide resin was treated with H-Phe- S (CH₂^C O₂Et according to the general procedure described above. Yield: 22.6 mg, 96%; Calculated Mass [M+H]⁺: 941.5, Mass Found 941.5; HPLC: t_R: 34.9 min (Gradient 0 to 50% B over 40 min.).

Ac-LYRANV-S(CH₂) ₂CO₂Et (11)

K^Itaeⁱ

25 μmol of fully assembled Rink amide resin was treated with H-VaI-S(CH₂^CO₂Et according to the general procedure described above. Yield: 18.5 mg, 83%; Calculated Mass [M+H]⁺: 893.5, Mass Found 893.6; HPLC: t_κ: 32.5 min (Gradient 0 to 50% B over 40 min.).

General procedure for the direct thioesterification on solid support:

Bromo-(4-methoxyphenyl)methylpolystyrene resin (25 μmol) fully assembled with the desired peptide sequence was swollen in dry DCM for 30 min, followed by the addition of a solution of Pd(PPh₃)₄ (25 mg, 22 μmol) and Ph₃SiH (123 μl, 108 mg, 1 mmol) in dry DCM (2 mL). The resin was shaken for 1 h and the procedure was repeated once. Afterwards, the resin was washed DCM (x 10), DMF (x 5), DCM (x 5) and a solution of ethyl 3-mercaptopropionate (77 μl, 600 μmol, 24 equiv.), anhydrous HOBt (101 mg, 750 μmol, 30 eq.), DIEA (161 μl, 121 mg, 938 μmol, 37.5 eq.) and DIC (116 μl, 750 μmol, 30 eq.) in DCM/DMF (4:1 v/v, 1.5 ml) was added and the resin shaken for 1 h. This thioesterification step was repeated once before washing the resin with DCM (x 5), DMF (x 5) and DCM (x 10). Cleavage: A mixture of TFA, thioanisole, triisopropylsilane and water (17:1 : 1:1 v/v/v/v) was added to the resin. After 2 h, the resin was washed with TFA (4x 4 mL) Work-up: The combined solutions were concentrated in vacuo. The residue was dissolved in water containing 0.1% TFA and purified by preparative HPLC (0 to 50% B over 40 min.) to afford the desired peptide thioesters as white fluffy solids which were analysed by HPLC (0 to 50% B over 40 min.) and ESI mass spectrometry. Analytical Data for peptide thioesters 9 and 10:

Ac-LYRAY-S(CH₂) ₂CO₂Et (9)

25 μmol of fully assembled Rink amide resin was treated with ethyl 3- mercaptopropionate according to the general procedure described above. Yield: 15.6 mg, 74%; Calculated Mass [M+H]⁺: 843.4, Mass Found 843.5; HPLC: t_κ: 34.2 min (Gradient 0 to 50% B over 40 min.).

Ac-LYRAS-S(CH₂) ₂CO₂Et (10)

25 μmol of fully assembled Rink amide resin was treated with ethyl 3- mercaptopropionate according to the general procedure described above. Yield: 13.3 mg, 70%; Calculated Mass [M+H]⁺: 767.4, Mass Found 767.4; HPLC: fe: 29.4 min (Gradient o_ra 0 to 50% B over 40 min.).

General Procedure for the Phosphate-Assisted Ligation

Phosphopeptides 1 and 2 (1.5 mg, approx. 2.18-2.22 μmol) were dissolved in 150μL of ligation buffer [4:1 v/v iV-methyl-2-pyrrolidinone (NMP): 6 M guanidine hydrochloride, IM HEPES, pH = 8.5] ^[a]. This solution was transferred to an eppendorf tube containing the peptide thioester (5 - 11) (1.2 eq., 2.62-2.66 μmol). Thiophenol (2% by volume, 3 μL) was added and the reaction mixed gently. The ligation mixture was incubated at 37 °C with gentle mixing every 12 h until the reaction was confirmed to be complete by LC-MS. The ligation reactions were quenched by the addition of 0.1% TFA in water (0.6 mL). The products were purified by semi-preparative HPLC (0 to 50% B over 40 min.).

^[a] An aqueous buffer containing 6 M Gn.HCl and 1 M HEPES was prepared and adjusted to pH 8.5 using 25% aqueous sodium hydroxide solution. The resulting solution (1 mL) was diluted with NMP (4 mL) to produce the final buffer for use in the phosphate assisted ligation reactions. Final concentrations are 1.25 M Gn. HCl and 0.2 M HEPES.

Although NMP was employed as the organic co-solvent in the ligation buffer, any suitable water miscible aprotic co-solvent may be employed. Suitable aprotic co- solvents include, although are not limited to NMP, DMF, HMPA and DMSO, and mixtures thereof. Kinetics of the Phosphate- Assisted Ligations

Phosphopeptides 1 and 2 and peptides 3 and 4 were reacted with peptide thioester 5 (Ac- LYRANG-S(CIHb)₂CO₂NH₂) under the ligation conditions described above. Aliquots of 10 μL were removed from the reaction mixture and quenched with water containing 0.1% TFA (90 μL). These aliquots were taken every 2 h for 12 h then every 12 h until peptide thioester 2 had been completely consumed (by peptide bond formation or hydrolysis). These samples were analyzed by analytical HPLC (0 to 50% B over 40 min.) and the percentage yield calculated by integrating the peaks (at λ = 280 nm) corresponding to starting peptide and the ligated peptide/phosphopeptide product.

Analytical Data for phosphate-assisted ligations

12 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide thioester 5)

Analytical HPLC trace and ESI mass spectrometry data for 12.

Yield: 2.8 mg, 91%; ESI (m/z): Calculated Mass [M+H]⁺: 1392.4, Mass Found 1392.5; HPLC: /_R: 23.3 min (Gradient 0 to 50% B over 40 min.). 13 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide thioester 6)

Analytical HPLC trace and ESI mass spectrometry data for 13.

Yield: 2.8 mg, 90%; ESI (m/z): Calculated Mass [M+H]⁺: 1406.4, Mass Found 1406.5; HPLC: t_R: 23.5 min (Gradient 0 to 50% B over 40 min.).

14 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide thioester 7)

Analytical HPLC trace and ESI mass spectrometry data for 14.

Yield: 2.3 mg, 71%; ESI (ra/z): Calculated Mass [M+H]⁺: 1466.5, Mass Found 1466.5; HPLC: /_R: 25.1 min (Gradient 0 to 50% B over 40 mia).

15 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide thioester 8)

Analytical HPLC trace and ESI mass spectrometry data for 15.

Yield: 2.72 mg, 82%; ESI (m/z): Calculated Mass [M+H]⁺: 1482.5, Mass Found 1482.5; HPLC: /_R: 26.7 min (Gradient 0 to 50% B over 40 min.). 16 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide

Yield: 1.8 mg, 62%; ESI (m/z): Calculated Mass [M+H]⁺: 1308.3, Mass Found 1308.5; HPLC: fa: 23.6 min (Gradient 0 to 50% B over 40 min.).

17 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide thio ester 9)

Analytical HPLC trace and ESI mass spectrometry data for 17.

Yield: 2.7 mg, 82%; ESI (m/z): Calculated Mass [M+H]⁺: 1384.4, Mass Found 1384.5; HPLC: t_R: 25.5 min (Gradient 0 to 50% B over 40 min.). l R^lt _tea_vveea

18 (Product of the phosphate-assisted ligation between phosphopeptide 1 and peptide thioester 11)

Analytical HPLC trace and ESI mass spectrometry data for 18.

Yield: 1.5 mg, 47%; ESI (m/z): Calculated Mass [M+H]⁺: 1434.5, Mass Found 1434.6; HPLC: /_R: 23.6 min (Gradient 0 to 50% B over 40 min.). 19 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thioester 6)

Analytical HPLC trace and ESI mass spectrometry data for 19.

Yield: 1.9 mg, 61%; ESI (m/z): Calculated Mass [M+H]⁺: 1420.4, Mass Found 1420.5; HPLC: t_R: 20.8 min (Gradient 0 to 50% B over 40 rain.)-

20 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thioester 5)

Analytical HPLC trace and ESI mass spectrometry data for 20.

Yield: 2.9 mg, 95%; ESI (m/z): Calculated Mass [M+H]⁺: 1406.4, Mass Found 1406.5; HPLC: /_R: 23.4 min (Gradient 0 to 50% B over 40 min.).

21 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thio ester 7)

Analytical HPLC trace and ESI mass spectrometry data for 21.

Yield: 1.6 mg, 50%; ESI (m/z): Calculated Mass [M+H]⁺: 1480.5, Mass Found 1480.6; HPLC: fa: 25.1 min (Gradient 0 to 50% B over 40 min.). _{b (}A_lso_rcean

22 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thio ester 9)

Analytical HPLC trace and ESI mass spectrom _t A_bvanceeunetry data for 22.

Yield: 1.7 mg, 52%; ESI (m/z): Calculated Mass [M+H]⁺: 1496.5, Mass Found 1496.7; HPLC: t_κ: 27.3 min (Gradient 0 to 50% B over 40 min.).

23 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thioester lO)

Analytical HPLC trace and ESI mass spectrometry data for 23.

Yield: 1.8 mg, 62%; ESI (m/z): Calculated Mass [M+H]⁺: 1322.3, Mass Found 1322.7; HPLC: /_R: 23.4 min (Gradient 0 to 50% B over 40 min.).

24 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thioester 9)

Analytical HPLC trace and ESI mass spectrometry data for 24.

Yield: 2.1 mg, 90%; ESI (m/z): Calculated Mass [M+H]⁺: 1398.4, Mass Found 1398.6; HPLC: /_R: 25.7 min (Gradient 0 to 50% B over 40 min.). 25 (Product of the phosphate-assisted ligation between phosphopeptide 2 and peptide thioester 11)

Analytical HPLC trace and ESI mass spectrometry data for 25.

Yield: 1.5 mg, 47%; ESI (m/z): Calculated Mass [M+H]⁺: 1448.5, Mass Found 1448.5; HPLC: t_R: 24.5 min (Gradient 0 to 50% B over 40 min.).

Genera] Procedure for Dephosphorylation of Phosphopeptide Ligation Products

Phosphopeptide ligation products (1.0 mg) were dissolved 50 mM Tris buffer at pH 8.6 (50 μL). Alkaline phosphatase (Sigma™: Alkaline phosphatase from bovine intestinal mucosa, P5521) (7.5 units) was added and the reactions incubated at 37 ⁰C. The dephosphorylation reactions were monitored by LC-MS and took between 2 and 24 h to reach completion.

NB: peptides containing phosphorylated threonine residues were slower to dephosphorylate with alkaline phosphatase

Analytical Data for Dephosphorylation Reactions

Synthesis of 26

Analytical HPLC trace and ESI mass spectrometry data for 26.

Phosphopeptide 16 was dephosphorylated using the conditions described above to afford 26 as a white fluffy solid. Yield: 98%; ESI (m/z): Calculated Mass [M+H]⁺: 1228.3, Mass Found 1228.6; HPLC: t_R: 23.7 min (Gradient 0 to 50% B over 40 min.).

Synthesis of 27

Analytical HPLC trace and ESI mass spectrometry data for 27.

Phosphopeptide 15 was dephosphorylated using the conditions described above to afford 27 as a white fluffy solid. Yield: 74%; ESI (m/z): Calculated Mass [M+H]⁺: 1402.5, Mass Found 1402.7; HPLC: fo: 27.0 min (Gradient 0 to 50% B over 40 min.).

Synthesis of 28

Analytical HPLC trace and ESI mass spectrometry data for 28.

Phosphopeptide 13 was dephosphorylated using the conditions described above to afford 28 as a white fluffy solid. Yield: 85%; ESI (m/z): Calculated Mass [M+H]⁺: 1242.3, Mass Found 1242.6; HPLC: f_R: 27.5 min (Gradient 0 to 50% B over 40 min.).

Synthesis of 29

Analytical HPLC trace and ESI mass spectrometry data for 29.

Phosphopeptide 22 was dephosphorylated using the conditions described above to afford 29 as a white fluffy solid. Yield: 89%; ESI (m/z): Calculated Mass [M+H]⁺: 1416.5, Mass Found 1416.5; HPLC: /_R: 23.9 min (Gradient 0 to 50% B over 40 min.).

Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of forming a peptide bond between a C terminal of a first amino acid or peptide and an N terminal of a second amino acid or peptide, the second amino acid, or peptide comprising a pendant phosphate bearing linker attached to a carbon α to the N terminal.

2. A method accordng to claim 1 wherein the N terminal, C terminal or phosphate is independently substituted or unsubstitued.

3. A method of preparing a compound of Formula (I):

R^a and R^b are each independently at each occurrence H or alkyl; R^c is H or optionally substituted alkyl; R^d is H or optionally substituted alkyl; X is a covalent bond or aryl;

R¹ together with carbonyl to which it is attached is an optionally substituted amino acid or peptide; and

R² is an optionally substituted amino acid or peptide; the method comprising: contacting a compound of Formula (II) :

and a compound of Formula (III)

(III) wherein LG is a leaving group selected from the group consisting of -OR, -SR and -NR₂, wherein R is independently at each occurrence optionally substituted alkyl, cycloalkyl, hetero eye Io alkyl, aryl or heteroaryl, or each R together with the N to which it is attached, forms an optionally substituted hetero cyclalkyl or heteroaryl; to provide a compound of Formula (I).

4. The method according to claim 2 wherein said compound of formula (II) is selected from the group consisting of

5. The method according to claim 4 wherein said compound of formula (II) is selected from the group consisting of

6. The method according to any one of claims 3 to 5 wherein R^c is H.

7. The method according to any one of claims 3 to 6 wherein R^d is H.

8. The method according to any one of claims 3 to 7 wherein said LG is -SR.

9. The method according to claim 8 wherein -SR is -S(CH₂)₂CO₂Et.

10. The method according to any one of claims 3 to 7 wherein said LG is -NR₂.

11. The method according to claim 10 wherein -NR₂ is

cr ^Nri

12. The method according to any one of claims 3 to 7 wherein LG is -OR.

13. The method according to claim 12 wherein -OR i .s

14. The method according to any one of claims 3 to 13 wherein R together with the carbonyl to which it is attached is an amino acid, or an optionally substituted peptide commencing at the C-terminus with an amino acid, selected from the group consisting of Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and VaL

15. The method according to claim 14 wherein R¹ together with the carbonyl to which it is attached is an amino acid, or an optionally substituted peptide commencing at the C-terminus with an amino acid, selected from the group consisting of GIy, Ala, Met, Phe, Tyr, Ser and VaI.

16 The method according to any one of claims 3 to 15 wherein R¹ is N-Acetyl-Leu- Tyr-Arg-Ala-Y-Z, wherein Y is Asn or a covalent bond, and Z is selected from the group consisting of GIy, AIa, Met, Phe, Tyr, Ser and VaL

17. The method according to any one of claims 3 to 16 wherein R² is an amino acid, or an optionally substituted peptide commencing at the N-terminus with an amino acid, selected from the group consisting of Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and VaI.

18. The method according to any one of claims 3 to 17 wherein R² is Ser-Pro-Gly- Tyr-Ser-NH₂.

19. The method according to any one of claims 3 to 18 wherein said contacting comprises contacting in an aqueous solution.

20. The method according to claim 19 wherein said aqueous solution has a pH in the range between about 6 and about 14.

21. The method according to claim 19 or claim 20 wherein the aqueous solution further comprises a water miscible aprotic solvent.

22. The method according to claim 21 wherein said aprotic solvent is selected from the group consisting of NMP, DMF, HMPA and DMSO, or mixtures thereof.

23. The method according to any one of claims 19 to 22 wherein said aqueous solution further comprises 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and guanidine.HCl.

24. The method according to any one of claims 19 to 23 wherein said aqueous solution comprises 7V-methylpyrrolidinone (NMP), 4-(2-hydroxyethy I)-I- piperazineethanesulfonic acid (HEPES) and guanidine.HCI.

25. The method according to any one of claims 19 to 24 wherein said aqueous solution further comprises a thiol.

26. The method according to claim 25 wherein said thiol is thiophenol.

27. The method according to any one of claims 3 to 26 wherein said contacting is carried out at a temperature range of between about -80 ⁰C to about 150⁰C.

28. The method according to claim 27 wherein said temperature is in the range between about 35 ⁰C to about 40 ⁰C.

29. The method of any one of claims 3 to 28 wherein the compound of formula (II) and the compound of formula (III) are employed in a molar ratio of about 0.8 to about

1.2.

30. The method of any one of claims 3 to 29 further comprising purifying the compound of formula (I) by HPLC, reverse phase column chromatography, anion exchange chromatography, size exclusion chromatography or by crystallisation.

31. The method according to any one of claims 3 to 30 further comprising dephosphorylating said compound of formula (I).

32. The method according to claim 31 wherein dephosphorylating said compound of formula (I) comprises contacting said compound of formula (I) with alkaline phosphatase.

33. An intermediate compound of formula (IV)

(IV)

R^a and R^b are each independently at each occurrence H or alkyl; R^c is H or optionally substituted alkyl; R^d is H or optionally substituted alkyl; X is a covalent bond or aryl; R¹ together with carbonyl to which it is attached is an optionally substituted amino acid or peptide; and

R² is an optionally substituted amino acid or peptide.

34. A compound of formula (I) when prepared by the method of any one of claims 3 to 32.