MXPA03001450A - Extended native chemical ligation. - Google Patents

Abstract

The invention is directed to methods and compositions for chemical ligation of components comprising a first component having a carboxythioester, and preferable an agr;-carboxythioester, moiety and a second component having an N-substituted, and preferably an Nagr;-substituted, 2 or 3 carbon chain alkyl or aryl thiol to give a ligation product having an N-substituted amide bond at the ligation site. The reactants of the invention are chemoselective, and the alkyl or aryl thiol moiety is removable from the ligation product. Removal of the alkyl or aryl thiol gives a native amide bond at the ligation site. The methods and compositions of the invention are particularly useful for ligation of peptides and polypeptides. The ligation system of the invention is applicable to a wide variety of molecules, and thus can be exploited to generate peptides, polypeptides and other amino acid containing polymers having a native amide bond at the ligation site.

Description

EXTENDED NATIVE CHEMICAL LIGAMENT FIELD OF THE INVENTION The present invention relates to methods and compositions for extending the native chemical ligation technique to allow the ligation of a broad range of peptides, polypeptides, other polymers and other molecules by means of an amide bond. BACKGROUND OF THE INVENTION Chemical linkage involves the formation of a selective covalent bond between a first chemical component and a second chemical component. Single, mutually reactive, functional groups present in the first and second components can be used to arrive at the chemoselective ligation reaction. For example, chemical linkage of peptides and polypeptides involves the chemoselective reaction of peptide and polypeptide segments carrying unique and mutually reactive N-terminal and C-terminal amino acid residues. Several different chemistries have been used for this purpose, examples of which include native chemical linkage (Dawson, et al., Science (1994) 266: 776-779; ent, et al., WO 96/34878; Kent, et al. ., WO 98/28434), chemical linkage forming oxime (Rose, et al., J. Amer. Chem. Soc. (1994) 116: 30-34), ligation forming the thioester (Schnolzer, et al., Science (1992) 256: 221- REF: 144205 225), linkage that forms thioether (Englebretsen, et al., Tet. Letts. (1995) 36 (48): 8871-8874), linkage that forms hydrazone (Gaertner, et al. al., Bioconj. Chem. (1994) 5 (4): 333-338), and thiazolidine-forming linkage and oxazolidine-forming linkage (Zhang, et al., Proc. Nati. Acad. Sci. (1998) 95 ( 16): 9184-9189; Tam, et al., WO 95/00846; U.S. Patent No. 5,589,356). Of these methods, only the native chemical ligation procedure produces a ligation product which has a native amide bond (ie, peptide) at the ligation site. The original native chemical linkage methodology (Dawson et al., Supra; and WO 96/34878) has proven to be a robust methodology for generating a native amide bond at the linkage site. Native chemical linkage involves a chemoselective reaction between the first segment of peptide or polypeptide having a C-terminal oc-carboxythioster portion and a second peptide or polypeptide having an N-terminal cis-residue. A thiol exchange reaction produces an intermediate bound to the initial thioester, which rearranges spontaneously to give a native amide bond at the ligation site while the thiol in the side chain of the tank is regenerated. The primary disadvantage of the original native chemical linkage method is that it requires an N-terminal cysteine, i.e. it only allows the joining of segments of peptides and polypeptides that possess a cysteine at the linkage site. Despite this disadvantage, native chemical linkage has been reported for peptides with N-terminal amino acids other than cysteine (W098 / 28434). In this method, ligation is performed using a first segment of peptide or polypeptide which has a C-terminal ot-carboxythioster and a second segment of peptide or polypeptide which has an N- group. { thiol - substituted with auxiliary} N-terminal represented by the formula HS-CH2-CH2-0-NH- [peptide]. After ligating, the group N- is removed. { thiol substituted with auxiliary} by cleaving the auxiliary group HS ~ CH2-CH2-0- to generate a native amide bond at the ligation site. One limitation of this method is that the use of a mercaptoethoxy-auxiliary group can successfully lead to the formation of the amide bond only in a glycine residue. This produces a ligation product which, when excised, generates a glycine residue at the position of the N-substituted amino acid of the second segment of peptide or polypeptide. As such, this mode of the method is only suitable if one wishes the binding product of the reaction to contain a glycine residue in this position, and in any. case may be problematic with respect to linkage yields, stability of the precursors, and the ability to remove the 0-linked auxiliary group. Although other auxiliary groups can be used, for example HSCH2CH2NH- [peptide], without limiting the reaction for ligation in a glycine residue, such auxiliary groups can not be removed from the bound product. Therefore, what is needed is a widely applicable and robust chemical linkage system that extends the native chemical linkage to a wide variety of amino acid residues, peptides, polypeptides, polymers and other molecules by means of an effective thiol-containing auxiliary group. , easily removable, and that binds such molecules together with a native amide bond at the linkage site. The present invention handles these and other needs. SUMMARY OF THE INVENTION The invention is directed to methods and compositions related to. extended native chemical linkage. The extended native chemical linkage method of the invention comprises: generating an initial linkage product linked by N-substituted amide of the formula: J1-C (0) -N (C1 (R1) -C2-SH) -J2 or J1 -C (0) -N (C1 (R1) -C2 (R2) -C3 (R3) -SH) -J2 II wherein J1 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such a peptide or polypeptide, a polymer, a dye, a suitably functionalized surface, a detectable linker or label, or any other chemical portion compatible with chemical synthesis of peptide or native chemical linkage -extended; R1, R2 and R3 are independently H or a group that donates electrons conjugated to Cl; with the proviso that at least one of R1, R2 and R3 comprises an electron donor group conjugated to Cl; and J2 is a peptide or polypeptide which has one or more side chains of optionally protected amino acids, or a potion of such peptide or polypeptide, a polymer, a dye, a suitably functionalized surface, a detectable linker or label, or any other portion chemistry compatible with chemical synthesis of peptide. or extended native chemical linkage. The ligation product is produced by the process of ligating a first component which comprises a carboxyl thioester of the formula J1-C (0) SR to a second component which comprises an amino alkyl or arylthiol of chain of 2 or 3 carbons N - Stably substituted with the acid of the formula: HS-C2-C1 (R1) -HN-J2 III or HS-C3 (R3) -C2 (R2) -C1 (R1) -HN-J2 IV wherein J2, R1, R2 and R3, are as defined above, and then optionally remove the 2 or 3 carbon chain alkyl or aryl thiol from the linkage product bonded by N-substituted amide. In a preferred embodiment, such cleavage is facilitated by forming a cation stabilized at resonance in Cl under cleavable conditions compatible with peptide. Removal of the alkyl or aryl thiol chain from N generates a final linkage product of the formula: J1-C (0) -HN-J2. V wherein J1, J2, R1, R2 and R3 are as defined above. The invention is also directed to compositions for effecting such extended native chemical linkage, and to cartridges and equipment comprising them. The compositions comprise an amino alkyl or aryl thiol of the N-substituted and preferably Na-substituted chain of 2 or 3 carbons fully protected or partially protected, or totally unprotected stable to the acid, and preferably one of the formula: SX2-C2- C1 (R1) -X1 N-CH (Z2) -C < 0) -J2 VI or SX2-C3 (R3) -C2 (R2) -C1 (R1) -X1 N-CH (Z2) -C (0) -J2 VII wherein XI is H or an amino protecting group; X2 is H or a thiol protecting group; J2, R1, R2 and R3 are as defined above; and Z2 is any chemical moiety (which includes, without limitation, an amino acid side chain) compatible with chemical synthesis of peptide or extended native chemical linkage. The invention is also directed to chiral forms of such compounds of the invention that are substantially free of racemic or diastereoisomeric mixtures. The invention is further directed to solution phase and solid phase methods to produce such N-substituted 2-or 3-carbon chain alkyl or arylthioles fully protected, partially protected or totally unprotected. Methods for producing these compounds include halogen-mediated amino alkylation, reductive amination, and preparation of amino-alkyl or aryl-thiol-protected, N-alkylated, S-protected amino acid precursors compatible with solid-phase peptide synthesis methods . Portion Jl of the carboxytioster component can comprise any carboxythioester compatible chemical moiety and the reaction conditions for extended native chemical ligation, and the M-substituted component of the invention can be provided alone or linked to a broad range of chemical moieties, which they include amino acids, peptides, polypeptides, nucleic acids or other chemical moieties, such as dyes, haptens, carbohydrates, lipids, solid support, biocompatible polymers or other polymers and the like. The extended native chemical ligation method of the invention is robust and can be performed in an aqueous system of almost neutral pH and in a range of temperature conditions. The methods for producing the N-substituted components of the invention are also robust, they provide a wide range of synthetic routes for these novel compounds in surprisingly high and pure yields. The amino acid, or A-protected, N-alkylated, S-protected amino acid arylthiol precursors of the invention are particularly useful for rapid automated synthesis using conventional peptide synthesis and other organic synthesis strategies. On the other hand, the protected N-substituted components of the invention expand the chemical ligation utility to multi-component ligation schemes, such as when a polypeptide is produced which involves multiple ligation strategies, such as a three linkage scheme. or more segments or convergent link synthesis schemes. For example, the methods and compositions of the present invention allow a first pair of carboxythioester and N-substituted components to be used to synthesize a first portion of a desired molecule, and to use additional pairs of carboxythioester and N-substituted components to synthesize additional portions. of the molecule. The ligation products of each such synthesis can then be ligated together (after appropriate deprotection and / or modification) to form the desired molecule. Accordingly, the methods and compositions of the invention greatly expand the scope of the native chemical linkage, and the starting, intermediate and final products of the invention find a wide range of uses. BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the present invention for showing its ability to mediate the extended native chemical linkage of the peptides; the same schemes can be employed to effect the ligation of any suitable molecule. As the picture shows, a first component which contains a ct-carboxyl thioester of the formula J1-HN-CH (Z1) -aCO-Sr, and a second component which contains a 2-carbon alkyl or arylthiol of Na-substituted chain stable to acid N-terminal of the formula HS-C2-C1 (RI) -NHot-CH (Z2) -C (o) -J2. Components J1 and J2 can be any chemical moiety compatible with the chemoselective ligation reaction, such as a protected or unprotected amino acid, peptide, polypeptide, other polymer, dye, linker, and the like. Zl is any side chain group compatible with the CO-SR thioester, such as a protected or unprotected side chain of an amino acid. Z2 is any side chain group compatible with an? -substituted amino acid, such as a protected or unprotected side chain of an amino acid. R1 is a benzyl portion (benzyl when referring to the context of Cl, otherwise referred to as phenyl) substituted with an electron donor group preferably in the ortho or para position relative to Cl; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to Cl). The thiol exchange occurs between the COSR thioester component and the N- amino component. { alkylthiol} . The exchange generates a thioester-linked intermediate linkage product that after spontaneous rearrangement through a 5-membered ring intermediate generates a first linkage product of the formula J1-HN-CH (Z1) -C (O) - Na (Cl (RI) -C2-SH) -CH (Z2) -C (O) -J2 which has an alkyl or aryl thiol of 2-carbon chain Na-substituted removable [HS-C2-C1 (RI) -] in the linkage site. The 2-carbon Na-substituted alkyl or aryl thiolide chain [HS-C2-C1 (Rl) -] at the ligation site is capable of being removed, under peptide-compatible conditions, to generate a final linkage product of the formula J1-HN-CH (Z1) -CO-NH-CH (Z2) -C0-J2 which has a bond by native amide at the linkage site. Figure 2 illustrates the present invention for showing its ability to mediate the extended native chemical linkage of the peptides; the same schemes can be used to effect the ligation of any suitable molecule. As shown in the Figure, a first component which contains a -carboxythioester of the formula J1-HN-CH (Z1) -aCO-SR, and a second component which contains an alkyl or aryl thiol of 3 carbon chain Na -substituted stable to the acid of the formula HS-C3 (R3) -C2 (R2) -Cl (RI) -NHa-CH (Z2) -C (O) -J2. Components Jl and J2 can be any chemical moiety compatible with the chemoselective ligation reaction, such as a protected or unprotected amino acid, peptide, polypeptide, other polymer, dye, linker, and the like. Zl is any side chain group compatible with the aCO-SR thioester, such as a protected or unprotected side chain of an amino acid. Z2 is any group, side chain compatible with a Na-substituted amino acid, such as a protected or unprotected side chain of an amino acid. When R1 is different from hydrogen, R2 and R3 are hydrogen, and R1 is a phenyl portion, unsubstituted or substituted with an electron donor group in the ortho or para position relative to Cl; a picolyl (unsubstituted or substituted with hydroxyl) 0 thiol in the ortho or para position in relation to a Cl); a methanethiol; or a sulfoxymethyl. When R2 and R3 are different from hydrogen; R1 is hydrogen and R3 and R2 form a benzyl group which is substituted with an electron donor group in the ortho or para position relative to Cl; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to Cl). The thiol exchange occurs between the COSR thioester component and the amino alkyl thiol component. The exchange generates a thioester-linked intermediate linkage product that after spontaneous re-rotation through a 6-membered ring intermediate generates a first linkage product of the formula J1-HN-CH (Z1) -C (0) -N (C1-C2 (R2) -C3 (R3) -SH) -CH (Z2) -J2 which has a removable Na-substituted 3-chain alkyl or aryl thiol [HS-C3 (R3) -C2 (R2) -Cl (Rl) -] at the linkage site. The Na-substituted 3-carbon chain aryl thiol [HS-C3 (R3) -C2 (R2) -Cl (RI) -1 is capable of being removed at the ligation site, under conditions compatible with the peptide to generate a final linkage product of the formula J1-HN-CH (Z1) -CO-NH-CH (Z2) -C0-J2 which has a native amide bond at the linkage site. Figure 3 illustrates an extended native chemical linkage scheme of multiple components. A cc-carboxyl thioester polypeptide is reacted with an alkyl or aryl thiol of Na-substituted 2-chain Na-substituted polypeptide Na-protected polypeptide of the formula HS-C2-C1 (R1) -Na (PGI) -CH (Z2 ) -C (0) -J2 as exemplified in Figure 1 is reacted with a peptide containing an N-terminal Cys residue. R 1 is a phenyl, unsubstituted, or substituted with an electron donor group, preferably in the ortho or para position for Cl, or a picolyl (unsubstituted or substituted with hydroxyl or tiql in the ortho or para position with respect to to Cl) The protecting group (PG1) can be any suitable protecting group, such as an alkylcarbonyl protecting group (eg, benzyloxycarbonyl (Z), Boc, Bpoc, Fmoc, etc.), a triphenylmethyl protecting group (Trt) , a 2-nitrophenylsulfenyl protecting group (Nps), etc. The protective group is removed after the first ligation reaction.A first native chemical ligation reaction is performed between the α-carboxyl thioester polypeptide with an alkyl or aryl thiol chain 2 α -substituted α-substituted polypeptide carbons of the formula HS-C2-Cl (R1) -Na (PG1) -CH (Z2) -C (0) -J2 as exemplified in Figure 1 and the N-terminal peptide-Cys to give a first linkage product of Formula: HS-C2-C1 (R1) -Na (PG1) -CH (Z2) -C (0) -peptide2-peptide3. The protecting group PG1 is then removed to give the linking product of the formula HS-C2-Cl (RI) -N (H) -CH (Z2) -C (0) -Peptido2 -Peptido3. This species is then reacted. with a third component which contains thioester. Thiol exchange occurs between the COSR thioester component and the aminoN- component. { alkylthio} . The exchange generates a thioester-linked intermediate ligation product which after spontaneous rearrangement through a 5-membered ring intermediate generates a second ligation product of the formula Peptidol -C (O) -Nct (Cl (Rl) -C2 -SH) -CH (Z2) -C (O) peptide '2 -Cys-Peptido3-, which has an a-substituted 2-carbon chain alkyl or aryl thio [HS-C2-C1 (Rl) -] in the second linkage site. The 2-carbon Na-substituted alkyl or aryl thio chain [HS-C2-Cl (RI) -] at the second ligation site is capable of being removed, under conditions compatible with the peptide, to generate a final ligation product of the formula peptidol-C (O) -α to β-CH (Z 2) -C (O) Peptido 2 -Cys-Peptido 3, which has a native amide bond in the first and second linkage sites. Figure 4 illustrates a general linkage strategy which employs two different auxiliaries of 1-phenyl-1-mercaptoethyl of the invention Figures 5A and 5B show results of analytical high-resolution liquid chromatography (HPLC) of a binding reaction for cytochrome b562 as described in Example 21 using an auxiliary Nct -1- (4-methoxyphenyl) -2-mercaptoethyl. Figure 5A shows the state of the linkage reaction at time = 0. Figure 5B shows the linkage state after the reaction is allowed to proceed overnight. As shown also in 'Figure 5B, there are two ligating products that result from the achiral center in Cl of the auxiliary Nct-1- (4-methoxyphenol) -2-mercaptoethyl. Figure 6A and 6B show reconstructed electroaspersion mass (MS) spectra of the cytochrome b562 residue ligation product formed by using extended native chemical linkage with an N- terminal segment modified with Na-. { 1- (4-methoxyphenyl) -2-mercaptoethane} . Residues 1-63 of cytochrome b562 carrying a C-terminal α-thioester are linked to residues 64-106 of cytochrome b562 bearing an Nct-. { 1- (4-methoxyphenyl) 2-mercaptoethane} N-terminal glycine. Figure 6A shows MS reconstruction of the product, of initial linkage that includes a Na- group. { 1- (-methoxyphenyl) 2-mercaptoethane} removable in the linkage site. Figure 6B shows a MS reconstruction of the ligation product following treatment with hydrogen fluoride (HF) to remove the Na- group. { 1- (4-methoxyphenyl) 2-mercaptoethane} to generate a native amide link in the linkage site. The observed masses are 11948 + 1 Da (before treatment with HF) and 11781 + 1 Da (after treatment with HF), ie a loss of 167 + 2 Da, in good agreement with the loss of 166 Da expected for elimination of the auxiliary group 1- (4-methoxyphenyl) 2-mercaptoethane. Figure 7A? 7B illustrates an analyte HPLC representative of the linear cytochrome b562 material (Figure 7A) depicted in Figure 6B, and an ion exchange chromatogram (Figure 7B) of the folloWing material. DESCRIPTION OF SPECIFIC MODALITIES The invention is directed to methods and compositions related to extended native chemical linkage. In general, the method involves ligating a first component which comprises a carboxyl thioester, and more preferably, an a-carboxyl thioester with a second component which comprises an N-substituted amino or aryl thiol of 2 or 3 carbon chain, and preferably, α-substituted acid stable. The chemoselective reaction between the carboxythioester of the first component and the thiol of the N-substituted 2 or 3 carbon alkyl or aryl thiol of the second component proceeds through the thioester-linked intermediate, and develops into an initial ligation product. More specifically, the thiol exchange occurring between the COSR thioester component and the amino alkyl thiol component generates a thioester-linked intermediate linkage product that after spontaneous rearrangement through a 5-member or 6-member ring intermediate generates a first product of linkage linked by amide of the formula: J1-C (0) -N (C1 (R1) -C2-SH) -J2. I C J1 -C (0) -N (C1 (R1) -C2 (R2) -C3 (R3) -SH) -J2 II where J1, J2, I, R2 and R3 are as defined above. The alkyl or aryl thiol chain of 2 or 3 carbons N-substituted [HS-C2-C1 (RI) -] or [HS- (C3 (R3) -C2 (R2) -Cl (RI) -] at the ligation sis capable of being removed, under conditions compatible with peptide, without damage to the product, to generate a final linkage product of the formula: J1-C (0) -HN-J2 where J1, J2, R1, R2 and R3 are as defined above. has a native amide bond at the linkage sMore particularly, the extended native chemical linkage method of the invention comprises chemical linkage of: (i) a first component which comprises an o-carboxyl thioester of the formula J1-C (0) SR and (ii) a second component which comprises a N-substituted 2 or 3 carbon chain alkyl or aryl thiol stable to the acid of the formula: J1-C (0) -N (C1 (R1) -C2-SH) -J2 | 0 J1-C (0) -N (C1 (R1) -C2 (R2) -C3 (R3) -SH) -J2 | where · Jl, J2, Rl, R2 and R3 are as defined above. The groups R1, R2, and R3 are selected to facilitate cleavage of the N-Cl bond under cleavable conditions compatible with peptide. For example, electron donating groups, particularly if conjugated to Cl, can be used to form a cation stabilized at resonance at, C1 which facilitates cleavage. The chemical ligation reaction preferably includes as an excipient a thiol catalyst, and is carried out around neutral pH conditions under mixed aqueous or aqueous organic conditions. The chemical linkage of the first and second components can proceed with a five or six member ring that undergoes spontaneous rearrangement to produce a linkage product bonded by N-substituted amide. Where the first and second components are peptides or polypeptides, "the N-substituted amide bonded linkage product has the formula: J1-C (0) -Na (C1 (R1) -C2-SH) -CH (Z2) -C (0) -J2 VIII or J1-C (0) -Na (Cl (R1) -C2 (R2) -C3 (R3) -SH) -CH (Z2) -C (0) -J2 IX where J1, J2 and R1, R2, R3 and Z2 are as defined above. The conjugated electron donor groups Rl (2 or R3 of the linkage product bonded by N-substituted amide facilitate cleavage of the N-Cl linkage and removal of the alkyl or aryl thiol chain of 2 or 3 cations from the bonded linkage product by N-substituted amide Removal of the alkyl or aryl thiol chain of N under cleavage conditions compatible with peptide generates a ligation product which has a native amide bond at the ligation s are peptides or polypeptides, the ligation product will have the formula: J1-CONaH-CH (Z2) -C (0) -J2 X The present invention provides multiple advantages over previous chemical ligation procedures. to the finely harmonic nature of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol component of the present invention. First, the unbound N-substituted component is stable to the acidic conditions, which allows its robust synthesis and storage. Second, it reacts selectively with the carboxytioster component to generate an initial ligation product which has an N-substituted amide bond at the ligation s Third, the alkyl or aryl thiol moiety regenerated at the Na position of the ligation sof the initial ligation product can be selectively removed under conditions wholly compatible with -peptides, polypeptides or other unprotected, partially protected or fully protected portions, i.e. . The alkyl or aryl thiol portion can be removed without damaging the desired ligation product. The selective cleavage reaction can be easily performed under conditions of cleavage compatible with standard peptide such as acidic, photolytic, or reductive conditions, depending on the particular N-substituted alkyl or aryl thiol portion chosen for ligation. ThusAnother advantage of the invention is that one or more groups in remaining portions of the linkage components, if present, may be unprotected, partially protected or fully protected depending on the proposed end use. On the other hand, given the chemoselective nature and solubility properties of carboxyl thioester and N-substituted 2 or 3 carbon chain or alkyl or aryl thiol, "the ligation reaction can be carried out quickly and cleanly to give high product yields in about of pH 7 under aqueous conditions at about room temperature This makes the invention particularly flexible for binding peptides, polypeptides or other partially or totally unprotected polymers under subtle conditions For a peptide component comprising the alkyl or aryl component N-substituted 2-carbon chain thiol of the invention, this compound has the formula: HS-C2-C1 (R1) -NHa-CH (Z2) -C (0) -J2 XI as shown below in Table 1 J2 and R2 are as described above; Z2 is any side chain group compatible with an N-substituted amino acid, such as a side chain of an amino acid. R1 is preferably a phenyl group substituted with an electron donor group in the ortho or para position relative to Cl; or a picolyl group (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to Cl).

Table I It is necessary to position the phenyl and picolyl electron donor substituents Rl ', R3' and R5 'in the ortho positions or to maintain the electronic conjugation to the Cl carbon to increase the cleavage of the N-Cl bond following the ligation. Preferred electron donor groups for R1 ', R3' and R5 'include strong electron donating groups such as methoxy (-OCH3), thiol (-SH), hydroxyl (-OH), methylthio (-SCH3), and moderate electrons such as methyl (-CH3), ethyl (-CH2-CH3), propyl (-CH2-CH2-CE3), isopropyl (-CH2 (CH3) 3). With the proviso that any or all of R1 ', R3' and R5 'can be H. A general observation is that strong electron donor groups increase the sensitivity of the 2-carbon chain alkyl or arylthiol to cleavage after ligation. When a simple electron donor group is present as a substituent R1", R3" or R5", the linkage reaction can proceed at a faster rate, while the cleavage is slower or requires more stringent cleavage conditions. or more electron donating groups are present as a substituent Rl ', R3' or R5", the ligation reaction may be slower, while the cleavage is faster or requires less stringent cleavage conditions. In this way, a particular electron donor group can be selected accordingly. Another embodiment of the invention relates to 2-carbon N-substituted chain compounds, which include a thiol as a substituent of Rl at the Rl "and R5" positions. In addition to being an electron donor group conjugated to Cl, the introduction of a thiol into one or both of these locations allows the compounds to bind through a 6-membered ring mediated through the Rl group (as well as through of a 5-membered ring by the 2-carbon chain alkyl thiol Na). It also increases the local concentration of available thiols to react with the α-carboxy thioester, and provides additional ions in terms of structural constrictions that can improve linkage. With reference to the Na-substituted 3-carbon alkyl chain or aryl thiol component of the invention, this compound has the formula HS-C3 (R3) -C2 (R2) -Cl (RI) -NHa-CH (Z2) -C (O) -J2, which is represented later in Table II. Table II As described above, J2 can be any chemical portion compatible with the chemical synthesis of peptide or extended native chemical linkage, Z2 is any side chain group compatible with an N-substituted amino acid, such as an amino acid side chain. When l is different from hydrogen, R 2 and R 3 are hydrogen, and R 1 is a phenyl portion, unsubstituted, or more preferably, substituted with an electron donor group in the ortho or para position relative to Cl; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to Cl). When R2 and R3 are different from hydrogen, R1 is hydrogen, and R3 and R2 form a benzyl group which is substituted with an electron donor group in the ortho or para position relative to Cl; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to Cl). As with the 2-carbon N-substituted chain compounds, it is necessary to place the electron donating substituents phenyl and picolyl Rl ', R3' and R5 'in the ortho or para positions to maintain the electronic conjugation to the carbon Cl for robust cleavage of the? a-Cl link following linkage. However, when R2 and R3 form a benzyl group with C2 and C3, at least one of R1 'and R3' comprises a strong electron donor group wherein R1 'or R3' is selected from methoxy (-OCH3), thiol ( -SH), hydroxyl (-0H) and thiomethyl (-SCH3). For the 3-carbon N-substituted chain thiols in which R2 and R3 are hydrogens, R1 comprises a phenyl or picolyl group in which R1", R3" and R5 'include electron donor groups either strong or moderate, or a combination thereof. As with the 2-carbon N-substituted chain alkyl or aryl thiols, the strong electron donating groups increase the sensitivity of the 3-carbon alkyl or aryl thiol chain to cleavage after ligation. In this way a particular electron donor group or combination thereof can be selected accordingly. Similar to the 2-carbon N-substituted chain compounds, the 3-carbon N-substituted chain compounds of the present invention may include a thiol as a substituent of Rl at the Rl "and R5" positions when they are available for substitution at a construction of interest. Here again the electron donating thiol group is conjugated to Cl and its introduction in these locations allows the compounds to have two routes for the linkage event that forms the 6-membered ring. It also increases the local concentration of available thiols to react with the -carboxythioester, and provides additional conformations in terms of structural constructs that can improve ligation. The synthesis of the alkyl or aryl thiol N-terminal N-substituted 2 or 3 carbon chain amino acids of the invention can be carried out as described herein, for example, in Reaction Scheme I and Reaction Scheme II, Examples, and in accordance with standard organic chemistry techniques known in the art. See, for example, "Advanced Organic Chemistry, Reactions, Mechanisms, and Structures," 4th Edition, J. March (Ed.) John iley &; Sons, New York, NY. 1992; "Comprehensive Organic Transformations, A Guide to Functinoal Group Preparations", R. Larock (Ed.), VCH Publishers, New York, NY, 1989. They can be synthesized in solution, by synthesis supported on polymer, or a combination thereof. The preferred process employs protected N-alkylated N-alpha amino or arylthiol amino acid S-protected amino acid precursors. The reagents used for the synthesis can be obtained from a number of commercial sources. Also, it will be well understood that the starting components and various intermediates, such as the individual amino acid derivatives can be stored for late use, provided in equipment and the like. When preparing the alkyl or aryl thiol N-terminal α-substituted 2 or 3 carbon chain amino acids of the invention, protective group strategies are employed The preferred protective groups (PG) used in the various synthetic strategies in general They are compatible with Solid Phase Peptide Synthesis ("SPPS") .In some cases, it is also necessary to use orthogonal protective groups that are removable under different conditions.Many of such protective groups are known and suitable for this purpose (See, for example, "Protecting Groups in Organic Synthesis", 3rd Edition, TW Greene and PGM Wuts, Eds., John Wiley &Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic Peptides, A User's Guide," GA, Grant, Ed. , WH Freeman &Company, New York, NY, 1992; "Advanced Chemtech Handbook of Combinatorial &Solid Phase Organic Chemistry", WD Bennet, J. Christensen, LK Hamaker, ML Peterson, M.Rhodes, and HH Saneii , E ds., Advanced Chemtech, 1998; "Principies of Peptide Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting Groups" P.J. Ocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994). Examples include benzyloxycarbonyl (Z), Boc, Bpoc, Trt, Nps, FmocCl-Z, Br-Z; NSC; MSC, Dde, etc. For the sulfur moieties, examples of suitable protecting groups include, but are not limited to, benzyl, 4-methylbenzyl, 4-methoxybenzyl, trityl, Acm, TACAM, xanthyl, disulfide, picolyl and phenacyl derivatives.

More particularly, the a-substituted 2 or 3 carbon alkyl or aryl chains can be prepared according to Reaction Scheme I (Solid preparation Phase of the α-substituted precursor). Reaction Scheme II (preparation of the solution phase of the Na-substituted precursor). In Reaction Scheme I, Na-substituted 2 or 3 carbon alkyl or aryl thiols are directly assembled into the solid phase using standard methods of polymer-supported organic synthesis, while the aminoalkyl or arylthiol amino acid precursor? Protected, N-alkylated, S-protected from Reaction Scheme II is coupled to the resin using standard coupling protocols. In Reaction Scheme I, X is halogen, R1, and R2 are as described above and can be preformed as protected or unprotected portions or made in resin, and J2 is preferably bonded to halogen as X-CH (R) - J2-Resin, where R is hydrogen or another side chain. It will be appreciated that J2 can be a variety of groups, for example where the halogen X and J2 -Resin are separated by more than one carbon, such as in synthesis of beta or gamma amino acids or similar molecules. Where the glyoxal portion (HC (O) -C (0) -J2-Resin) is employed, the resulting side chain R is hydrogen. In Reaction Scheme II is a halogen, R1 and R2 are as described above and can be preformed as protected or unprotected portions or made in solution or resin, and wherein R is hydrogen or another side chain. Where the glyoxalic acid portion (HC (O) -C (O) -OH) is employed, the resulting side chain R is hydrogen. As indicated above, it will be appreciated that Reaction Schemes I and II can be applied in the synthesis of 3-carbon chain alkyl or aryl thiols. Where racemic or diastereomeric products are produced, it may be necessary to separate these by standard methods before using in the extended native chemical linkage. Reaction scheme I Resin Reaction scheme II First component used for the extended native chemical ligation method of the invention, this component has the formula J1-C0-SR. The most preferred carboxy thioester component comprises an α-carboxyl thioester amino acid of the formula Jl-H-C (Zl) -CO-SR. Group J1 can be any chemical moiety compatible with the chemoselective linkage reaction, such as an amino acid, peptide, polypeptide, other protected or unprotected polymer, dye, linker and the like. Zl is any side chain group compatible with the ocCO-SR thioester, such as an amino acid side chain. R is any group compatible with the thioester group, which includes, but is not limited to, aryl, benzyl and alkyl groups. Examples of R include 3-carboxy-nitrophenyl thioesters, benzyl thioesters and mercaptopropionic acid leucine thioesters (See, for example, Dawson et al., Science (1994) 266: 776-779; Canne et al., Tetrahedron Lett. (1995) 36: 1217-1220; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434; Ingénito et al., JACS (1999) 121 (49): 11369-11374, - and Hackeng et al. al., Proc. Nati, Acad. Sci. USA (1999) 96: 10068-10073). Other examples include dithiothreitol, or alkyl or aryl thioesters, which can be produced by inteina-mediated biological techniques, which are also well known (See, for example, Chong et al., Gene (1997) 192: 277-281; Chong et al., Nucí Acids Res (1998) 26: 5109-5115; Evans et al., Protein Science (1998) 7: 2256-2264; and Cotton et al., Chemistry &Biology (1999) 6 (9 ): 247-256). The CC-carboxythioesters can be generated by chemical or biological methods following standard techniques known in the field, such as those described herein, including the Examples. They can be synthesized by chemical synthesis, cc-carboxythioester peptides in solution or from thioester-generating resins, techniques that are well known (See, for example, Dawson et al., Supra).; Canne et al., Supra; Hackeng et al., Supra, Hojo H, Aimoto, S. (1991) Bull Chem Soc Jpn 64: 111-117). For example, chemically synthesized thioester peptides can be made from the corresponding α-thioacid peptides, which in turn can be synthesized in a thioester resin or in solution, although a resin process is preferred. The α-thioacid peptides can be converted to the corresponding 3-carboxy-4-nitrophenyl thioesters, to the corresponding benzyl ester, or, to any of a variety of alkyl thioesters. All these thioesters provide satisfactory leaving groups for the ligation reactions, with the 3-carboxy-4-nitrophenyl thioesters demonstrating a somewhat faster reaction rate than the corresponding benzyl thioesters, which in turn may be more reactive than the corresponding ones. alkyl thioesters. As another example, a resin that generates mercaptopropionic acid leucine thioester associated with trityl can be used to construct C-terminal thioesters (Hackeng et al., Supra). The C-terminal thioester synthesis can also be performed using a carboxypropane sulfonamide safety capture linker by activation with diazomethane or iodoacetonitrile followed by displacement with a suitable thiol (Ingénito et al., Supra; Shin et al., (1999) J. Am. .

Chem. Soc, 121, 11684-11689). C-terminal α-carboxythioester peptides or polypeptides can also be made using biological processes. For example, intein expression systems, with or without labels such as affinity tags, can be used to exploit the induced self-cleaving activity of an "internal" protein splicing element in the presence of a suitable thiol to generate a segment of C-terminal thioester peptide or polypeptide. In particular, the internal one suffers from specific self-cleavage in the presence of thiols such as DTT, β-mercaptoethanol, β-mercaptoethanesulfonic acid, or cistern, which generates a peptide segment which carries a C-terminal thioester. See, for example, Chong et al., (1997) supra; Chong et al., (1998) supra; Evans et al., Supra; and Cotton et al., supra. The ligation of the N-substituted 2 or 3 carbon alkyl or aryl thiol chain components of the invention with the first carboxithioester component generates a ligation product which has an N-substituted amide bond at the ligation site, as shown in Figures 1, 2 and 3. The linkage conditions of the reaction are chosen to maintain the selective reactivity of the thioester with the N-substituted 2 or 3 carbon chain alkyl or aryl thiol portion. In a preferred embodiment, the ligation reaction is performed in a buffer solution which has a pH of 6-8, with the preferred pH range being 6.5-7.5. The buffer solution can be aqueous, organic or a mixture thereof. The ligation reaction may also include one or more catalysts and / or one or more reducing agents, lipids, detergents, other denaturants or solubilizing reagents and the like. Examples of preferred catalysts are thiol and phosphine containing portions, such as thiophenol, benzyl mercaptan, TCEP and alkyl phosphines. Examples of denaturing agents and / or solubilizers include guanidinib, urea in water or organic solvents such as TFE, FFIP, DMF, NMP, acetonitrile in admixture with water, or with guanidinium and urea in water. Temperature can also be used to regulate the rate of the ligation reaction, which is usually between 5 ° C and 55 ° C, with the preferred temperature being between 15 ° C and 40 ° C. As an example, the ligation reactions proceed well in a reaction system which has 2% thiophenol in 6M guanidinium at a pH between 6.8 and 7.8. For the N-substituted 2-carbon alkyl or aryl thiols, the linkage event results from a thiol exchange occurring between the COSR thioester component and the amino alkyl thiol component. The exchange generates a thioester-linked intermediate linkage product which after spontaneous rearrangement through a 5-membered ring intermediate generates a first linkage product of the formula Jl-HN-CH (Zl) -C (O) -Na (Cl (Rl) -C2-SH) -CH (Z2) -J2 which has an N-substituted 2-carbon alkyl or aryl thiol [HS-C2-C1 (Rl) -] removable at the linkage site , wherein the substituents are as defined above. The N-substituted 2-carbon alkyl chain or aryl thiol [HS-C2-C1 (RI) -] at the ligation site is capable of being removed, under peptide-compatible conditions, to generate a final linkage product of the Jl-HN-CH (Zl) -CO-NH-CH (Z2) -CO-J2 formula which has a native amide bond at the linkage site. For the aryl or N-substituted 3-chain alkyl thiols, the thiol exchange between the COSR thioester component and the amino-alkyl thiol compound generates an intermediate linkage product linked by thioester that after spontaneous rearrangement through an intermediary of 6-membered ring generates a first linkage product of the formula Jl-HN-CH (Zl) -C (O) -N (Cl-C2 (R2) -C3 (R3) -SE) -CH (Z2) - J2 which has a removable N-substituted 3-chain alkyl or aryl thiol [HS-C3 (R3) -C2 (R2) -Cl (RI) -] at the linkage site. The N-substituted 3-carbon chain aryl thiol [HS-C3 (R3) -C2 (R2) -C1 (R1) -] in the ligation site is capable of being removed, under conditions compatible with peptide, to generate a final linkage product of the formula J1-HN-CH (Z1) -C0-H-CH (Z2) -C0-J2 which has a native amide bond at the linkage site . Removal of the N-substituted alkyl or aryl thiol group is preferably performed under acidic conditions to facilitate cleavage of the N-Cl bond, which produces an unsubstituted amide bond, stabilized at the ligation site. By "peptide-compatible cleavage conditions", physico-guymic conditions compatible with peptides and suitable for cleavage of the N-linked alkyl or aryl thiol portion from the ligation product are proposed. Peptide-compatible cleavage conditions are generally selected depending on the α-alkyl or aryl thiol moiety used, which can be easily deduced through routine and well-known procedures (See, for example, "Protecting Groups in Organic Synthesis ", 3rd edition, TW Greene and PGM Wuts, Eds., John Wiley & amp;; Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synt etic Peptides, a User's Guide" G.A. Grant, Ed., W.H. Freeman & Company, New York, NY, 1992; "Advanced Chemtech Handbook of Combinatorial &Solid Phase Organic Chemistry", W.D. Bennet, J. Christensen, L.K. Hamaker, M.L. Peterson, M.R.Rhodes, and H.H. Saneii, Eds., Advanced Chemtech, 1998; "Principles of Peptide Synthesis 2nd edition, M. Bodanszky, Ed. Springer-Verlag, 1993", The Practice of Peptide Synthesis, 2nd edition ", Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; and" Protecting Groups ", PJ ocienski, Ed., Georg Thieme Veriag, Stuttgart, Germany, 1994. For example, where the substituents R 1", R 2 'or R 3' comprise a methoxy, hydroxyl, thiol or thiomethyl, methyl and the like, the more universal for elimination involves typical acid cleavage conditions for peptide synthesis chemistries. This includes cleavage of the N-Cl bond under strong acidic conditions or water and acid conditions, with or without reduction reagents and / or purifying systems (eg, acid such as anhydrous hydrogen fluoride (HF), trifluoroacetic acid (TFA)). , or trifluoromethanesulfonic acid (TFMSA) and the like). More specific acidic cleavage systems can be chosen to optimize cleavage of the Nct-Cl bond to remove the aryl or alkylthiol portion for a given construct. Such conditions are well known and compatible to maintain the integrity of the peptides. Another method of cleavage involves the inclusion of a thiol scrubber where the trophthaphants are present in a peptide or polypeptide sequence to avoid the reaction of the side chain of threophophan with the "aryl or alkyl thiol moiety released." Examples of thiol scrubbers include ethanediol, cysteine, beta-mercaptoethanol and thiocresol Therefore, another embodiment of the invention is the addition of a thiol scavenger when the N-Cl bond is cleaved to remove the aryl or alkyl thiol moiety Other specialized excision conditions include light cleavage conditions or reductive when the substituent is the picolyl group As an example, when the substituents R 1, or R 2 and R 3 comprise a picolyl portion, photolysis (e.g., ultraviolet light), zinc / acetic acid or electrolytic reduction can be used for cleavage following standard protocols, where Rl of the 2-carbon N-substituted chain thiol comprises a thiomethane in R l, then excisions with mercury (II) or HF can be used. The cleavage system can also be used for simultaneous cleavage from a solid support and / or as a deprotection reagent when the first or second linkage component comprises other protecting groups. For example, N-picolyl groups can be removed by dissolving the polypeptide in a solution of 10% acetic acid / water, with activated zinc (~ 0.5 g / ml). The thiomethane groups, such as the 2-mercapto, 1-methylsulfinylethane groups (HS-C2-C1 (S (O) -CH3) -Na), can be removed after ligation by reduction and cleavage mediated with mercuric mercaptan. As an example, the methylsulfinylethane group can be removed by dissolving the polypeptide in an aqueous solution of 3% acetic acid which contains N-methylmercaptoacetamide (???) (eg, lmg of polypeptide in 0.5 ml of acetic acid / water). and 0.05 ml of MMA), by reduction to the thiomethane form, followed by freezing and lyophilization of the mixture after the reaction overnight. The reduced auxiliary can then be removed in an aqueous solution of 3% acetic acid which contains mercury acetate (Hg (0AC) 2) (for example, 0.5 ml of acetic acid in water and 10 mg of Hg (0AC) 2). for about 1 hour), followed by the addition of beta-mercaptoethanol (for example, 0.2 ml of beta-mercaptoethanol). The products can then be purified by standard methods, such as reverse phase HPLC (RPHPLC). As can be seen, one or more catalysts and / or excipients may also be used in the cleavage system, such as one or more scrubbers, detergents, solvents, metals and the like. In general, the selection of specific debuggers depends on the amino acids present. For example, the presence of scavengers may be used to suppress the effect of damage that the carbonium ions produced during cleavage may have on certain amino acids (e.g., Met, Cys, Trp and Tyr). Other additives such as detergents, polymers, salts, organic solvents, and the like can also be employed to improve cleavage by modulating the solubility. Catalysts or other chemicals that modulate the redox system can also be advantageous. It will also be readily apparent that a variety of other physicochemical conditions such as buffer systems, pH and temperature can be adjusted routinely to optimize a given cleavage system. The present invention also provides protected forms of Na-substituted 2 or 3 carbon chain alkyl or aryl thiols of the invention. These compounds are especially useful for automated synthesis of peptides and convergent and orthogonal linkage strategies. These compositions comprise a stable or partially deprotected or fully deprotected amino or aryl thiol chain of 2 or 3 carbons a-substituted acid stable of the formula (PG2) S-C2-C1 (RI) -Noc (PGI) - CH (Z2) -C (O) -J2 or (PG2) S-C3 (R3) -C2 (R2) -Cl (RI) -Na (PG1) -CH (Z2) -C (O) -J2, which are subsequently represented in Table III and Table IV. In particular, one or more of R 1, R 2 and R 3 comprises an electron donor group conjugated to Cl which, after the conversion of the amino alkyl or aryl thiol α-substituted to alkyl or aryl thiol α-substituted amide, is capable of forming a cation stabilized at resonance in Cl that facilitates the cleavage of the Ncc-Cl bond under cleavage conditions compatible with peptide. PG1 and PG2 are protecting groups that are present individually or in combination or are absent and may be the same or different, where Z2 is any chemical moiety compatible with chemical synthesis of peptides or extended native chemical linkage, and where J2 is any compatible chemical moiety with chemical synthesis of peptides or extended native chemical linkage. PG1 (or XI) is a group to protect the amine. PG2 (or X2) is a group to protect the thiol. Many such protective groups are known and suitable for this purpose (See, for example, "Protecting Groups in Organic synthesis", 3rd edition, T.W. Greene and P.G.M. Wuts, Eds., John iley & amp;; Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic Peptides, A üser's Guide2, G.A., Grant, Ed., H. Freeman &Company, New York, NY, 1992;" Advanced Chemtec Handbook of Combinatorial & Solid Phase Organic Chemistry, "WD Bennet, JW Christensen, LK Hamaker, ML Peterson, MR Rhodes, and HH Saneii, Eds., Advanced Chemtech, 1998;" Principies of Peptide Synthesis, 2nd ed., "M. Bodanszky, Ed. , Springer-Verlag, 1993; "The Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994, and "Protecting Groups" PJ ocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994). Table III Examples of preferred protecting groups for PG1 and XI include, but are not limited to [Boc (t-Butylcarbamate), Troc (2, 2, 2, -tricloroetilcarbamato), Fmoc (9- fluorenylmethylcarbamate), Br-Z or Cl-Z (Br- or Cl- Bencilcarbaraato), Dde (4, 4-dimethyl-2, 6-dioxocicloexl- ylidene) MsZ (4-metilsulfinilbencilcarbamato), Msc (2- metilsulfoetilcarbamato), Nsc (4-nitrofeniletilsulfonil- ethyloxycarbonyl]. Groups Preferred PG1 and XI protectants are selected from "Protective Groups in Organic Synthesis", Green and Wuts, Third Edition, Wiley-Interscience (1999) with the most preferred being Fmoc and Nsc Examples of preferred protecting groups for PG2 include, but not limited to [Acm (acetamidomethyl), MeOBzl or Mob (p-methoxybenzyl), MeBzl (p-methylbenzyl), Trt (Trityl), Xan (xanthenyl), tButio (st-butyl), Mmt (p-methoxytrityl), 2 or 4 picolyl (2 or 4 pyridyl)), Fm (9-fluorenylmethyl), tBu (t-butyl), Tacam (Trimethylacetamidomethyl)} . The preferred protecting groups PG2 and X2 are selected from "Protective Groups in Organic Synthesis", Green and Wuts, Third Edition, Wiley-interscience, (1999), with the most preferred being Acm, Mob, MeBzl, Picolyl. Orthogonal protection schemes involve two or more classes or groups that are removed by different chemical mechanisms, and therefore can be removed in any order and in the presence of other classes. Orthogonal schemes offer the possibility of substantially smoothing the total conditions, because the selectivity on the basis of differences in chemistry can be obtained more than the reaction rates.

The protected forms of the Na-substituted 2 or 3 carbon chain alkyl or aryl thiols of the invention can be prepared as in the above Reaction Schemes I and II. The compounds of the present invention can be produced by any of a variety of means, which include halogen-mediated amino alkylation, reductive amination, and by the preparation of amino-aryl thiol amino acid precursors α-protected, N-alkylated , S-protected compatible with the methods of synthesis of amino acids or peptides of solid phase or solution. When desired, the resolution of the racemic or diastereomeric mixtures produced to give compounds of acceptable chiral purity can be accomplished by standard methods. As indicated above, the 2 or 3 carbon Na-substituted alkyl or aryl thiols of acceptable chiral purity are preferred in some cases. As it is shown in Example 21, and in Figure 5B, the use of auxiliary Na-1- (4-methoxyphenyl) -2 -mercaptoetilo in preparing cytochrome B562 produces two ligation products (diastereoisomers) with profiles purification overlap Although the elimination of a Na auxiliary produces only one main product, a small percentage of reaction products will be present lateraly and elimination in the final product, which may be undesirable. For example, the reductive synthetic amination route as described in Examples .4 to 6 used for the synthesis of the auxiliary Na-1- (4-methoxyphenyl) -2-mercaptoethyl used in the synthesis cyt b562 is inherently in the production of both epimers in the chiral center Cl. As indicated above, when desired, resolution of the racemic mixtures or diastereomers produced to give the compounds of acceptable chiral purity by standard methods can be performed. The standard procedures for obtaining Na auxiliaries of the invention of acceptable chiral purity are: (1) chiral chromatography; (2) chiral synthesis; (3) use of a covalent diastereomeric conjugate; and (4) crystallization or other traditional separation methods to give the enantiomerically pure chiral auxiliary. (See, for example, Ahuja, Satinder. "Chiral separations." An Overview "ACS Symp. Ser. (1991), 471 (Chiral Sep. Liq. Chromatogr.), 1-26; Collet, Andre." Separation and Purification of Enantiomers by Crystallization Methods ", In: Enantiomer (1999) 4: 157-172; Lopata et al., J. Chem. Res. Minipprint (1984) 10: 2930-2962; Lopata et al., J. Chem. Res. (1984) 10: 2953-2973; Ahuja, Satinder. 'Quiral separations and technology: an overview'.

Chiral Sep. (1997), 1-7; Chiral Separations: Applications and Technology. Ahuja, Satinder; Editor. USES. (1597), 349 pag. Publisher: (ACS, Washington, D.C.)). All these procedures of standard methods can be used for the resolution of racemic mixtures or diastereomers to give compounds of acceptable chiral purity. For example, crystallization can be used for optical resolution of enantiomers. For chiral chromatography, it is well known that racemic mixtures can be separated into chirally pure enantiomers by means of preparative chromatography using chiral means. In this way, a racemic mixture produced by the reductive amination pathway for the total synthesis of chiral Na auxiliaries can be used to prepare each enantiomer in chirally pure form, for example, as illustrated below for an amino acid auxiliary (e.g. where R is an amino acid side chain): molar priority Any enantiomer can be obtained in chirally pure form, or both can be obtained in chirally pure form. Any enantiomer can be used to form components modified with chirally pure auxiliaries, such as peptide segments (ie two chirally pure epimers), which can be "purified rigorously without interference from the presence of the other epimer and its impurities. that unless a condition is made to use both enantiomers, 50% of the total mass of the auxiliary will be discarded For example, the two peptide segments modified with chirally pure auxiliaries can then be used in separate ENCL reactions, to give mixtures of Ligation products modified with chirally pure auxiliaries After separate purifications, the auxiliary group is removed from the epimer bonding products (either separately or after being combined) to give the bound product of the same native structure, which is then subjected to purification.For chiral synthesis, a preferred method or employs a chiral, enantiomerically pure starting material, as illustrated below for a 2-carbon Na-para-substituted-methoxyphenyl auxiliary.

[PGx = Boc or Fmoc; PG2 = (4Me) benzyl or (4MeO) benzyl The resulting pure chirally precursor compound can then be used to make both an (N-substituted) protected amino acid, ie: directly in the path of 1 peptoid submonomer, ie:? G ?? 2 ???? + activating agent (PEPTIDE,) - RESIN · - BrCH2CO- N. (PEPTID02) -RESINA to form the peptide modified with auxiliary on a polymer support. Subsequent deprotection / cleavage gives the modified peptide segment with helper in chirally pure form, ie: Deprotection (PEPTIDE,) - RESrr. HN-CHzCO-NH-PEPTIDE;, 1PG > "Cleavage CH 2 / S% In this way the chirally pure Na-uxiliares of the invention can be made from readily available substituted phenylglycine derivatives of known chirality, thereby determining the chirality and chiral purity of the resulting auxiliary. preferred embodiment employs enantioselective synthesis which employs asymmetric reduction to produce the auxiliary, for example as illustrated below: R = -H, or-CH3, or -CH2COOH (or, opposite enantiomer) The asymmetric reduction can also be used for enantioselective synthesis to produce an α-auxiliary modified amino acid, such as for glycine illustrated below, ie: (u, another enantiomer) While an asymmetric synthesis performed suitably will give a considerable excess of one enantiomer over the other, however it is expected that there will be present quantities of the other enantiomer. This can be handled using a chiral purification step in order to obtain the majority of the enantiomer in pure form. The main benefits of an enantioselective synthetic route are that chiral chromatographic separation is easier, and that large amounts of material (waste) are not discharged. Another preferred standard technique is the resolution by use of a covalent diastereomeric conjugate. In general, this method employs a chiral amino acid (e.g., Ala) to modify a racemic auxiliary mixture, and separation of the resulting diastereomers by standard (non-chiral) chromatography methods, as illustrated below. For example, the racemic auxiliary 1 can be converted to a mixture of diastereomers by covalent incorporation of a second chiral center: ENANTIO EROS DIASTEREÓMEROS (racemic mixture) In this case, the racemic mixture is reacted, by means of a nucleophilic reaction SN2 (with inversion), with (2) Br-propionic acid, to produce the pair of diastereomers shown. Indeed, L-Ala has been generated here with an auxiliary containing the N-linked chiral thiol. As diastereomers, these two compounds will typically exhibit different chromatographic behavior under achiral chromatography conditions, and thus be separable under practical preparative conditions to give the pure, different epimers. After adequate protection of the Na portion, each compound can be used to generate a segment of peptide modified with pure auxiliary chirally unprotected or partially protected with an N-terminal Ala residue. The protected Na-substituted components of the invention are particularly useful for rapid automated synthesis using conventional peptide synthesis and other organic synthesis strategies. They also expand the utility of chemical linkage to multiple component linkage schemes, such as when a polypeptide is produced which involves orthogonal linkage strategies, such as three or more segment linkage schemes or convergent linkage synthesis schemes.

For example, the extended native chemical ligation method and compositions of the invention can be employed in conjunction with nucleophilic stable thioester-generating methods and thioester safety capture methods, such as orthotioloester and carboxy ester thiols described in the co-pending application of US Pat. PCT application no. Series [not yet assigned] filed on August 31, 2001, and United States Provisional Patent Application Serial No. 60 / 229,295 filed on September 1, 2000, which are incorporated herein by reference. Briefly, the nucleophilically stable thioester-generating compounds comprise an orthotioloester or a thiol carboxester; These compounds have broad applicability in organic synthesis, which includes the generation of peptide, polypeptide and other polymer thioesters. Compounds that generate thioester stable to nucleophiles are particularly useful for generating activated thioesters of precursors that are made under conditions in which strong nucleophiles are employed, such as peptides or polypeptides made using Fmoc, SPPS, as well as linkage or conjugation schemes. multiple stages that require (or benefit from the use of) compatible selective protection procedures to direct a specific binding or conjugation reaction of interest. The nucleophilic stable ortho-yolo-esters have the formula XC (OR ') 2-SR, where X is a target molecule of interest optionally comprising one or more nucleophilic cleavable protecting groups, R' is a stable nucleophilic protecting group that is removable under non-nucleophilic cleavage conditions, and R is any group compatible with the orthotioloester -C (OR ') -S-. Resins which generate nucleophilic stable thioester orthotioloester are also provided, and have the formula XC (OR ') 2-SR-linker-resin or XC ((ORi' -linker-resin) (0R2 ')) -SR, where X, R 'and R are as above, and wherein the linker and the resin are any stable nucleophilic linker and resin suitable for use in solid phase organic synthesis, which includes safety pick-up linkers that can be subsequently converted to labile linkers. nucleophiles for cleavage. Stable nucleophilic orthotioloes can be converted to the active thioester by a variety of non-nucleophilic conditions, such as acid hydrolysis conditions. The nucleophilic stable carboxy ester thiols have the formula X-C (0) -0-CH (R ") - (CH2) n-S-R, where X is a target molecule of interest comprising one or more nucleophilic labile protecting groups, R "'is a group not stable to nucleophile, n is 1 or 2, with n = 1 preferred, and R" "is hydrogen, a protecting group or a capture linker-safety or labile to reduction or acid bound to a resin or protecting group that is removable under non-nucleophilic conditions.The resins are also provided which generate thioester based on carboxyl ester, stable to nucleophilic, and have the formula XC (0) -0-CH (R ") -CH2n-S -linker-resin or XC (0) -0-CH (R "-linker-resin) -CH2n-SR, where X, R", n and R, are as above, and where the linker and resin are any linker and resin stable to nucleophile suitable for use in solid phase organic synthesis. The nucleophilic stable carboxy ester thiols can be converted to the active thioester by the addition of a thiol catalyst, such as thiophenol. Thus, the extended native chemical linkage methods and compositions of the present invention can be employed in convergent multisigment linkage techniques, where one end of a target compound can carry a 2 or 3 carbon alkyl or aryl thiol chain? -protected or unprotected of the present invention, and the other end an orthotioloester or carboxy ester thiol portion for subsequent conversion to the active thioester and ligation. It will also be appreciated that the 2 or 3 Na alkyl or aryl thiol of the present invention can be used in combination with other ligation methods, for example, such as native chemical ligation (Dawson, et al., Science (1994)). 266: 776-779; Kent, et al., WO 96/34878), extended general chemical linkage (Kent, et al., WO 98/28434), chemical linkage forming oxime (Rose, et al., J. Amer. Chem. Soc. 1994) 116: 30-33), thioester-forming linkage (Schnolzer, et al., Science (1992) 256: 221-225), linkage that forms thioether (Englebretsen, et al., Tet. Letts. (1995) 36 (48): 8871-8874), hydrazone-forming linkage (Gaertner, et al., Bioconj. Chem. (1994) 5 (4): 333-338) and thiazolidine-forming linkage and oxazolidine-forming linkage (Zhang, et al., Proc. Na. Acad. Sci. (1998) 95 (16): 9184-9189; Tam, et al, WO95 / 00846) or by other methods (Yan, LZ and Dawson, PE, "Synthesis of Peptides and Proteins without Cysteine Residues by Native Chemical Ligation Combined with Desulfurization, "J. Am. Chem. Soc. 2001, 123, 526-533, incorporated herein by reference, Gieselnan et al., Org. Lett., 2001 3 ( 9): 1331-1334; Saxon, E. et al., "Traceless" Staudinger Ligation for the Chemoselective Synthe sis of Amide Bonds, Org. Lett. 2000, 2, 2141-2143). Also contemplated by the present invention is the substitution of selenium instead of the sulfur of the thiol in the 2 or 3 carbon Na alkyl or aryl thiol of the invention. The methods and compositions of the invention have many uses. The methods and compositions of the invention are particularly useful for binding peptides, polypeptides and other polymers. The ability to perform the native chemical linkage in virtually any amino acid, w includes the one found naturally as well as also non-natural amino acids and derivatives expands the. extent of the native chemical linkage for targets that are lost cysteine linkage sites. The invention can also be used to bind polymers in addition to peptide or polypeptide segments when it is desirable to link such portions · through a linker w has a Na-substituted or fully-native amide bond at the linkage site. The invention also finds use in the production of a wide range of peptide tags for expressed protein ligation (EPL) applications. For example, thioester polypeptides generated by EPL can be ligated to a wide range of peptides by means of an amide bond and alkyl or aryl thiol-substituted or a completely native amide bond, depending on the proposed end use. The invention can also be exploited to produce a variety of cyclic peptides and polypeptides, w have a native amide bond at the point of cyclization even for peptides and polypeptides that do not contain cysteine. For example, this is significant since most cyclic peptides, such as antibiotics and other drugs generated by industry standards do not contain a cysteine residue that can be used to form a native amide bond at the cyclization linkage site (i.e. , head to tail). All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated for reference. EXAMPLES The following preparations and examples are given to enable those skilled in the art to understand and practice the present invention more clearly. They should not be considered to limit the scope of the invention, but simply as being illustrative and representative of it. Abbreviations Acm Ac.tamidomethyl Aloe Alioxycarbonyl BOP Hexafluorophosphate benzotriazole-1-yloxytris (dimethylamino) phosphonium Br, Cl, Z Benzylcarbamate Br, Cl DCM Dichloromethane DDE, 4-dimeitl-2,6-dioxocycloex-l-ylidene DIPCDI N, N -diisopropylcarbodiimide DIPEA N, -diisopropylethylamine D AP 4-dimethylaminopyridine- DMF N, -dimethylformamide DMSO Dimethylsulfoxide EtOH Ethanol Fmoc 9-Fluorenylmethoxycarbonyl FM 9-Fluorenylmethyl HATU (N- [(dimethylamino) -1H-1, 2-hexafluorophosphate N-oxide , 3-triazole [4,5-b] -pyridylmethylene] -N-methylmethanaminium). HBTU N - [(lH-benzotriazol-1-yl) (dimethylamine) methylene] -N-methylmethanaminium N-oxide hexafluorophosphate previously named O- (benzotriazol-1-yl) - 1, 3, 3- hexafluorophosphate tetramethyluronium HF Hydrofluoric acid Resin HMP 4-hydroxymethylphenoxy resin; p-alkoxybenzyl alcohol resin; or resin Wang HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole Mbh dimethoxybenzhydril Resin MBHA resin 4-methylbenzhydrylamine eb p-methylbenzyl MMA N-methylmercaptoacetamide Mmt p-methoxytrityl or p-methoxybenzyl Msc 2-methylsulfoethylcarbamate Msz 4-methylsulfinylbenzylcarbamate tr 4- Methoxy-2, 3-6-trimethylbenzenesulfonyl NMM N-methylmorpholine NMP N-methylpyrrolidine, N-methyl-2-pyrrolidone Nsc 4 - nitrofeniletilsulfonil-ethyloxycarbonyl OPfp ester pentafluorophenyl ester OtBu tert-butyl PAC acid linker peptide PAL linker peptide amide Pbf 2,2,4,6, 7-pentamethyldihydrobenzofuran-5- sulfonyl PEG-PS polyethyleneglycol -polystyrene picolyl Pmc 2,2,4,6-pyridyl methyl, 8-pentamethylchroman-6-sulfonyl PyAOP hexafluorophosphate 7-azabenzotroazol-l liloxtris (pyrrolidino) phosphonium S-tBu tert-butyl-thio Tacam trimetilacetamidometilo tBoc tertbutyloxycarbonyl TBTU O - (benzotriazol-l-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate tBu tert-butyl TFA trifluoroacetic acid Tis Trisisopropilsilano Tmob 2,4, 6-trimehoxibencilo T OF trimethylorthoformate Troc 2, 2, 2-trichloroethylcarbamate Trt yl trifenilme Example 1: General Materials and Methods The peptides were synthesized stepwise on a peptide synthesizer ABI 430A modified by SPPS using in situ activation protocols BUT / neutralization in Boc-chemistry or resin- PAM thioester generating resin following protocols standard (Hackeng et al., supra; Schnolzer et al., (1992) Int. J. Pept. Prot. Res., 40: 180-193; and Kent, SBH (1988) Ann. Rev. Biochem. 57, 957 -984). After assembly of the chain the peptides are deprotected and simultaneously cleaved by treatment with anhydrous hydrogen fluoride (HF) with 5% p-cresol and lyophilized and purified by preparative HPLC. Protected amino acids are obtained with Boc from Peptides International and Mid est Biotech. Trifluoroacetic acid (THA) is obtained by Halocarbon. Other chemicals from Fluka or Aldrich. Analytical and preparative HPLC is performed in a Rainin KPLC system with 214 nm UV detection using analytical or preparative Vydac C4. Peptide and protein mass spectrometry is performed in a Sciex API-I electroaspersion mass spectrometer. Example 2: Preparation of 2 ( "methoxybenzylthio) benzylbromide is reacted 2 -hidroximetiltiofenol, 10 mmol, 1.4 g in 10 ml of DMF with 10 mmols of 4-methoxybenzylchloride and 1.75 mi of D1EA at room temperature the reaction is completed in 10. minutes, 50 ml of water are added at pH 3. The product is extracted with ethyl acetate and dried over sodium sulfate, then the crude oil obtained is purified with 11 mmol of carbon tetrabromide (3.64 g) and 11 mmol. of triphenylphosphine (2.88 g) in 20 mL of THF After the overnight reaction the THF is evaporated, the product is purified with silica gel chromatography using hexanes / eitlo acetate 6/1 as the mobile phase. g Example 3: Preparation of Na (2-mercaptobenzyl) glycine-peptide A pure TFA is added to a Boc-protected N-terminal Ala resin-bound peptide (78 mg) to remove the Boc group. Using standard chemistry protocols, BocGlicineO-Succinimide is coupled to the resin. After the coupling is complete, the Boc group is removed and the resin is neutralized with 2 washes with 10% diisopropylethylamine in DMF.

The resin is then washed with DMF and DMSO. 9 mg of 2 ('methoxybenzylthio) benzylbromide are added in 0.2 ml of DMSO and 0.01 ml of diisopropylethylamine. The mixture is reacted for 12 hours at room temperature. The peptide is cleaved and deprotected under conditions with HF using standard protocols. The peak with the correct mass of 2.079 Da is approximately 12% (measured with HPLC) of all the peptide material. The correct peptide is purified using standard semipreparative HPLC. Example 4: Preparation of 4'-methoxy-2 (4'methylbenzylthio) acetophenone are dissolved 4-metilbencilmercaptano, 4 mmol, 0.542 mi and 4'-bromoacetophenone 4 mmol methoxy-2, 916.3 mg in 4 ml of DMF. Then add diisopropylethylamine, 4 mmole 0.7 ml. The mixture is stirred at room temperature for one hour. The mixture is poured into dilute HCl and extracted with ethyl acetate and dried over sodium sulfate. The oil is dissolved in ethyl acetate and precipitated by the addition of petroleum ether., with. recovery of 450 mg of a white solid. Example 5: Preparation of 1-amino, 1 (4-methoxyphenyl), 2 (4-methylbenzylthio) ethane 4"-methoxy-2- (4'-methylbenzylthio) acetophenone 1.44 mmoles, 411 mg and aminoxyacetic acid 4.3 mmoles, 941 mg are dissolved in 20 mg. of TMOF and 0.047 ml of methanesulfonic acid are added as catalyst at room temperature.After 48 hours, the solvent is evaporated and the residue is taken up in ethyl acetate, washed with 1 M monohydrogenpotassium sulfate and dried in Sodium sulfate Purify the product without purification with silica gel chromatography, and obtain 200 mg of oxime complex, 200 mg of this oxime complex, 0.556 mmoles in 2 ml of THF, are dissolved, followed by the addition of 1.67 ml of the BH3 / THF 1M complex After 27 hours there is no starting material, add 3 ml of water and add 1.5 ml of 10 N sodium hydroxide.The mixture is refluxed for 1 hour. then mix with ethyl acetate (4x) and dry in sodium sulfate. the final product (40 mg) using silica gel chromatography. Example 6: Preparation of Nal - (4-methoxyphenyl) 2-mercaptophan ethano glycine-peptide A model peptide bound to a resin with a N-terminal Ala protected with Boc (78 mg), pure TFA is added to remove the Boc group. . Using standard chemistry protocols bromoacetic acid is coupled to the resin. Then, lamino, 1 (4-methoxyphenyl), 2 (4-methylbenzylthio) ethane 17 mg is added in 0.3 ml of DMSO with 0.010 ml of diisopropylethylamine to the resin. After the reaction overnight the resin is washed and the peptide is cleaved and deprotected using standard HF protocol. The desired product is then purified using semipreparative HPLC.

Preparation of another Nal- (4-methoxyphenyl) 2-mercaptoethaneglycine-peptide It is coupled to a resin with peptide model sequence S-Y-R-F-L-polymer 0.1 mmol, bromoacetic acid using standard coupling protocol. After the coupling the resin is washed with DMSO. Lamino, 1- (4-methoxyphenyl), 2- (4-methylbenzylthio) ethane 32.5 mg, 0.12 mmol in 0.3 ml of DMSO and 0.025 ml of diisopropylethylamine are then added. The mixture is kept in reaction overnight. The peptide is cleaved and deprotected using standard HF method. HPLC of the cleavage without purification shows the desired product (M, 2.122) in 60% of the total product. It is found that the n-alkylethanethio group is 97% stable in HF. Example 7: Preparation of 2", 4" -dimethoxy-2- (4'-methylbenzylthio) acetophenone. 4-Methylbenzylmercaptan, 3.94 mmol, 0.534 mmol and 2", 4'-dimethoxy-2-bromoacetophenone 3.86 mmol, 1 g in 4 ml of DMF are dissolved. The diisopropylethylamine, 3.94 mmoles 0.688 ml is then added, the mixture is stirred at room temperature for 24 hours, and the mixture is poured into a 1 M solution of potassium monohydrogensulfate and extracted with ethyl acetate and dried over sodium sulfate. After evaporation, the residual oil is dissolved in ethyl acetate and precipitated by addition of petroleum ether, which yields 616 mg of a white solid Example 8: Preparation of 1-amino, 1 (2,4-dimethoxyphenyl), 2 (4-methylbenzylthio) ethane The 2 ', 4'-dimethoxy2 (4'-methylbenzylthio) acetophenone is dissolved in 0.526 mmol, 166 mg and aminoxyacetic acid 1.59 mmol, 345 mg in 6 ml of TMOF and 0.034 ml of methanesulfonic acid as catalyst in ambient temperature After 31 'hours, the The solvent is taken up in ethyl acetate, washed with 1M monohydrogenpotassium sulfate and dried over sodium sulfate. The product is then purified without purification with silica gel chromatography, which yields 126 mg (61% yield) of the oxime complex. 126 mg of the oxime complex is dissolved, 0.324 mmoles in 1.5 ml of THF. Then 0.973 ml of the BH3 / THF 1M complex is added. After 54 hours there is still starting material and 0.5 ml of the BH3 / THF 1M complex is added. After 3 more days, in total 6 days of reaction, add 3 ml of water and add 1 ml of 10 N sodium hydroxide. The mixture is refluxed for 1 hour. The mixture is then extracted with ethyl acetate (4x) and dried over sodium sulfate. The final product (43 mg) is then purified using silica gel chromatography.

Example 9: Preparation of Nal- (2, 4-dimethoxyphenyl) 2-mercaptoethane glycine-peptide It is coupled to a resin-bound peptide of the S-Y-R-F-L-polymer sequence, 0.1 mraoles, of bromoacetic acid using a standard coupling protocol. After the coupling the resin is washed with DMSO. Lamino, 1 (2 ', 4'-dimethoxyphenyl), 2 (4-methylbenzylthio) ethane 36 mg, 0.12 mmol in 0.3 ml of DMSO and 0.025 ml of diisopropylethylamine are then added. The mixture is kept in reaction overnight. The peptide is cleaved and deprotected using standard HF method. HPLC of the cleavage without purification shows the desired product (M 938) in 42% of the total product. It is found that the n-allylethanediol group is 92% stable in HF. Example 10: Chemical linkage Ala-Gly of SDFl-alanine-thioester C-terminal and N (2-mercaptobenzyl) glycine N-terminal peptide For a 6-member rearrangement ligation, 1 mg of C-terminal thioester Ala fragment is dissolved (MW 4429) of SDF1-a and 0.6 mg of the N-terminal Na (2-mercaptobenzyl) glycine fragment (MW 2079) of SDF1-a, in 100 μ? 6M guanidinium buffer pH 7.0 and 1 μ? of thiophenol. After 2 days at room temperature (-25 ° C), the formation of the desired ligation product (MW 5472) is confirmed by ES- S. The reaction mixture is then incubated at 40 ° C for an additional 24 hours, and determines the yield of the desired ligation product based on the ratio between the product and the unreacted C-terminal fragment as measured by HPLC integration. The observed performance is approximately 40%. Example 11: Ala-Gly chemical linkage of C-terminal SDFl-alanine-thioester and 1- (4-methoxyphenyl) 2-mercaptoethane glycine-N-terminal peptide For 5-member rearrangement ligation, 1 mg of the C-terminal alanine thioester fragment (MW 4429) of SDF1 and 1 mg of N-terminal Peptide Model (MW 2122) which has an N-terminal glycine which comprises a Nal- (4) group is dissolved. -methoxyphenyl) 2 -mercaptoethane, in 100 μ? of guanidinium buffer 6M pH 7.0 [and 1 μ? of thiophenol] The reaction mixture is incubated at room temperature (-25 ° C) and the ligation reaction is monitored. After 8 hours, the formation of the desired ligation product (MW 5515) is confirmed by ES-MS. After 3 days, the yield of the desired ligation product is about 45% based on the ratio between the product and the unreacted N-terminal fragment. After 3 days at room temperature, followed by additional incubation at 40 ° C for an additional 24 hours, the yield is 65% ·. After 3 days at room temperature, followed by further incubation at 40 ° C for an additional 48 hours, the yield increases to about 70%. For 5-member rearrangement linkage, 0.5 mg of the C-terminal Ala thioester fragment (MW 4429) of SDFl-cc is dissolved, and 0.5 mg of the N-terminal fragment (MW 2122) which has an N-terminal glycine which comprises a Nal- (4-methoxyphenyl) 2-mercaptoethane group, in 100 μ? of buffer of guanidinium 6 M pH 8.2 and 1 μ? of thiophenol. The reaction mixture is then incubated at room temperature (~ 25 ° C), followed by the addition of another 1 μ? of thiophenol after 5 hours. After 24 hours, the yield of the desired product is approximately 60%. Example 12: C-terminal Glycine-thioester Gly-Gly chemical linkage and N- terminal Na- (2-mercaptobenzyl) glycine-peptide For a 6-member rearrangement ligation, 3.5 mg of the C-terminal Gly thioester fragment is dissolved ( MW 1357) of a decamer model peptide and 2 mg of the N-terminal Na (2-mercaptobenzyl) glycine fragment (MW 2079) of a model peptide with 3HisDnp, in 200 μ? of 6 M guanidinium buffer at pH 7.9 and '2 μ? of thiophenol. The mixture is incubated at 33 ° C for 60 hours. The formation of the desired ligation product (MW 2631) is confirmed by ES-MS, with an observed yield of about 40% based on the ratio between the product and the unreacted N-terminal fragment. Chemical linkage of C-terminal glycine-thioester peptide and Nal- (4-methoxyphenyl) 2-mercaptoethanol glycine-peptide For 5-member rearrangement ligation, 2 mg of the C-terminal Gly thioester fragment (MW 1357) and 2.5 mg are dissolved. of the N-terminal fragment (MW 2122) of a model peptide with 3 HisDnp which comprises a Na 1- (4-methoxyphenyl) -2-mercaptoethane group in 100 μ? of 6 M guanidinium buffer pH 7.0 with 1 μ? of thiophenol. The reaction mixture is incubated at room temperature (~ 25 ° C), and the ligation reaction is monitored. The formation of the desired linkage product (MW 2675.9) is confirmed by ES-MS after 3 and 8 hours of incubation. After 24 hours, the yield of the desired ligation product is about 40% based on the ratio between the product and the unreacted N-terminal fragment. The pH is then increased to 8.2 by the addition of solid sodium bicarbonate, and the reaction mixture is incubated for an additional 24 hours, which results in a yield of 88%. Example 13: Chemical linkage Ala-Gly of Lar-alanine-thioester with a 1- (4-methoxyphenyl) 2 -mercaptoethane glycine-peptide A thioester of C-terminal peptide Larc 1-31 Ala of mouse 3mg (MW 3609 ) and peptide model to 1-, (4-methoxyphenyl) 2-mercaptoethane glycine-SYRFL 1 mg (MW 908) in 0.15 ml of 6 molar guanidinium buffer pH 8.2 and 0.03 ml of thiophenol. After stirring overnight the linkage is 81% complete and after 40 hours 92% complete based on the consumption of the peptide thioester. Expected bound product 4312 Da, found 4312 Da. Example 14: Chemical linkage Ala-Gly of Larc 1-31-alanine-thioester with a 1- (2, -dimethoxyphenyl) 2-mercaptoethane glycine-peptide The thioster of C-terminal peptide Larc 1-31 Ala of mouse 3 is dissolved mg (MW 3609) and peptide model Na l- (2, 4-dimethoxyphenyl) 2-mercaptoethane glycine-S-Y-R-F-L 1 mg (MW 938) · in 0.15 ml of 6 molar guanidinium buffer pH 8.2 and 0.03 ml of thiophenol. After stirring overnight the ligation is 73% complete, and after 40 hours 85% complete based on the peptide thioester consumption. The calculated and experimental masses of the linkage product are both 4342 Da. Example 15: Chemical linkage Gly-Gly of tripeptide glycine C-terminal thioester and Na-1- (2,4-dimethoxyphenyl) 2-mercaptoethane glycine-N-terminal peptide FGG-thioester peptide fragment 0.8 mg and peptide are dissolved model to 1- (2,4-dimethoxyphenyl) 2-mercaptoethane glycine-S. Y-R-F-L 1 mg (MW 938) in 0.1 ml of 6 M guanidinium buffer pH 8.2 and 0.02 ml of thiophenol. After stirring overnight the reaction is completed quantitatively. The calculated and experimental masses of the linkage product are 1199.4 Da and 1195.5 Da, respectively. Example 16: Removal of 1- (2,4-dimethoxyphenyl) 2-mercaptoethane from the ligation product 1 mg of the purified ligation product is dissolved from Example 15 in 0.95 ml of TFA and 0.025 ml of water and 0.025 ml. ml of TIS. After one hour the solvent is evaporated and a 50% solution of water / acetonitrile is added and the mixture is lyophilized. Cleavage is greater than 95% complete by HPLC. The experimental and calculated mass is 1003 Da. Example 17: Chemical linkage Gly-Gly of tripeptide glycine C-terminal thioester and Na 1-, (4-methoxyphenyl) 2-mercaptoethane glycine-N-terminal peptide A fragment of tripeptide peptide thioester, FGG-thioester 1.6 mg and peptide model Na 1- (4-methoxyphenyl) 2-mercaptoethane glycine-SYRFL 2 mg (MW 908) is dissolved in 0.2 ml of 6M guanidium buffer pH 8.2 and 0.04 ml of thiophenol is added. After stirring overnight the reaction is completed quantitatively. MW expected for the bound product 1169.4 Da, found 1169.5 Da. Example 18: Removal of the 1- (4-methoxyphenyl) 2-mercaptoethane group after ligation The purified ligation product of Example 17 is treated with 5% c-cresol in HF at -2 ° C for one hour. After evaporation, the product is precipitated with ether. The crude peptide is taken in 50% water / acetonitrile 0.1% TFA and injected on HPLC. The main peak >; 80% shows the expected molecular weight for the cleaved peptide (expected mass 1003 Da, found 1003 Da). Example 19: His-Gly chemical linkage of histidine-thioester C-terminal and 1- (2,4-dimethoxyphenyl) 2-mercaptoethane glycine N-terminal peptide Dissolves peptide fragment TBP-A 1-67 CaHis thioester 4 mg and peptide model Na 1- (2, -dimethoxyphenyl) 2-mercaptoethane Glycine-SYRFL 1 mg (MW 938) in 0.1 ml of guanidinium buffer 6M pH 8.2 and 0.02 ml of thiophenol. After stirring overnight the reaction is 87% complete based on the consumption of the peptide thioester. The expected molecular weight for the ligated product 9220 Da, and found 9220 Da. Example 20: Removal of the 1- (2,4-dimethoxyphenyl) 2-mercaptoethane group after ligation The purified peptide fragment is dissolved after ligation of HG 2 mg in 0.95 ml of TFA and 0.025 ml of water and 0.025 ml of TIS . After 1 hour, the solvent is evaporated and a 50% water / acetonitrile solution is added to the residue and the mixture is lyophilized. The excision is > 90% complete by HPLC. The expected MW 9023 Da, found 9024 Da. Example 21: Synthesis of cytochrome b562 by extended native chemical ligation. 3 mg of C-terminal thioester of cytochrome 1-63 MW 7349 0.4 μt ??? β? and 1.5 mg of residues 64-106 of a 1- (4-methoxyphenyl) 2-mercaptoethane glycine cytochrome b562 N-terminal MW 4970 0.3 μp ??? eß in 0.1 ml of guanidinium linkage buffer 6M pH 7 with 0.002 ml of thiophenol as a catalyst. See Figure 5A. After 24 hours, 0.025 ml of 2-mercaptoethanol is added to the mixture and the reaction is maintained for 45 minutes, then 15 mg of TCEP are added and after an additional 30 minutes of. The mixture is loaded onto a semipreparative HPLC. After penetration is eluted and then desalted the mixture all the components of the ligation mixture are eluted by ramming the gradient to 65% of B and collecting in a vial alone. Analytical HPLC of the desalted material shows that ligation is greater than 90% complete based on the consumption of the C-terminal peptide. HPLC shows two main peaks (diastereoisomers) with calculated and expected mass of 11.946 Da. See Figures 5B and 6A. The amino acid sequence for cytochrome b562 (1-106) ADLEDNMETL NDNLKVIEKA DNAAQVKDAL TKMRAAALDA is shown below QKATPPKLED KSPDSPEM D FRHGFDILVG QIDDAL LAN EG V EAQAA AEQLKTTRNA YHQ YR (SEQ ID NO: l) Calculated mass (average isotope composition) 11780. 3 Da N-terminal group: hydrogen C-terminal group: free acid MH + monoisotopic mass = 11774.0088 amu HPLC index = 249.80 MH + average mass = 11781.2781 amu Valor Bull & Bréese = 1.5360 Elemental composition: C508 H830 N147 0168 S3 User-defined amino acid residues: B- His-DNP Example 22: Removal of the 1- (4-methoxyphenyl) 2-mercaptoethane group from cytochrome b562 residues ligated 1-106 and native protein generation The desalted solution is then lyophilized before elimination of the 1- (4-methoxyphenyl) -2-mercaptoethane group. The lyophilized material is treated with 95% HF and 5% anisole and 1 mmol of cistern (121 mg) for 1 hour at -2 ° C. The HF is evaporated using standard protocols and then 100 ml of 50% B buffer is added and the mixture is lyophilized. The mixture is then purified using semipreparative HPLC to give 2 mg of purified single peak of native cytochrome 1-106 (56% yield after purification), with calculated and experimental mass of 11.780 Da. See Figures 6B and 7A. A wild-type cytochrome b562 analog in the same form is also synthesized, and is designated Slm7 cyt b562. The mutant Slm7 cyt b562 differs from the wild type by replacing the methionine at position 7 with a selenomethionine (the sulfur of methionine is replaced by its lower selenium cognere). The circular dichroism indicating high content of a-élice in both b562 wild-type apo and b562 Slm apo'y del (data not shown) is performed. ESMS also shows that both b562 of the wild type apo and b562 Slm7 apo have the expected molecular masses (data not shown). Apo proteins are reconstituted with heme (heme pH 7 NaPi overnight, room temperature), and the resulting purified proteins. with FPLC with ion exchange (FPLC Resource Q purification, Tris HCL pH 8, NaCl gradient). For example, see Figure 7B. It was found that the UV-visible (optical) spectra of the reconstituted heme proteins are consistent with coordination of sulfur or selenium to Fe (data not shown).

A b562 cyt mutant with non-coordinating isotero norleucine is also prepared in the same way. In this way, synthetic cryochromes are made using extended native chemical linkage, reconstituted with their active heme sites and fully characterized by biophysical methods. Accordingly, this example further demonstrates that peptides and proteins lacking cysteines suitable for the native native chemical ligation procedure can be made by extended native chemical ligation, and standard amino acids are not incorporated herein. For example, the folding and reactivity of many b562 cyt mutants have been studied, but in this way the non-natural axial ligands have remained unexplored. Along with the extended native chemical linkage, the vast array of non-natural amino acids available must allow the systematic harmony of the properties of these and other proteins. Example 23: Chemical Lys-Gly Linkage of MCP 1-35-Lysine-thioester with Na 1-, (4-methoxyphenyl) 2-mercaptoethane glycine-peptide C-terminal peptide thioester 1-35 is dissolved Lys MCP 3 mg and peptide model Na 1- (4-methoxyphenyl) 2-mercaptoethane glycine-SYRFL 1 mg (MW 908) in 0.15 ml of 6-molar guanidinium buffer pH 7 and adjusted to pH 7.2 by addition of triethylamine and 0.03 ml of thiophenol. After stirring overnight the linkage is 69% complete and after 40 hours 76% complete based on the thioester peptide consumption. Expected bound product 4893 Da, found 4893 Da. Example 24: Removal of the 1- (4-methoxyphenyl) 2-mercaptoethane group after ligation) The freeze-dried, freeze-dried product of Example 23 (Lys-Gly linkage) is dissolved in 1 mg of TFA, 25 μ? of ethane di thiol, 50 μ? of TIS. Then 150 μ? of bromotrimethylsilane. The reaction is allowed to proceed for 2 hours at room temperature ("rt"). The volatile components of the mixture are evaporated in vacuo, and the resulting oil is taken in guanidinium buffer .6 M pH 7.5. The organic matter is extracted with CHC13. The HPLC does not show more starting material, therefore the auxiliary group is removed successfully. The mass expected for the native sequence is 4726 Da, and a mass of 4725 Da is found. Example 25: Comparison of Linkage Studies with GSYRFL Peptides The comparison of 5-member rearrangement linkage studies with GSYRFL a peptides is summarized. from Examples 13-20 and 24 later in Table V.

Table V. Results of linkage studies with peptides GSYRFL Reacci Peptide C-Auxilia Time Rendimien Terminal condition r N- of model changes (peptide ends reactio ligamient elimination thioester) l "on (h) o (%) ion assistant 1 Phe-Gly-Gly I 16 > 98% HF 2 Phe-Gly-Gly II 16 > 98% TFA 3 TBP-A 1-67 II 16 87 TFA (His) 4 Larc 1-31 I 16 81 (Ala) of 40 92 HF mouse 5 Larc 1-31 II 16 73 (Ala) of 40 85 TFA mouse 6 MCP1 1- 35 I 16 69 (Lys) 40 76 TFA / T SB r Note: N-terminal auxiliary I = Na-l- (4-methoxyphenyl) -2-mercaptoethane glycine-SYRFL, and auxiliary N-terminal II = Na-1- (2-methoxyphenyl) -2-mercaptoethane glycine-SYRFL Example 26: Preparation of BocGlycin Nl (4'-methoxyphenyl), 2 ('-methylbenzylthio) ethane 4"-methoxy is suspended 2 (4'-methylbenzylthio) acetophenone 2 mmol, 572 mg, and glycine ethyl ester salt of HC1 2 mmol, 139.5 mg in 15 ml of DCM. DIEA 6 mmoles, 1 mg is added slowly and under nitrogen 1 ml of titanium tetrachloride (1M solution) is added. The reaction is maintained for 2 days at room temperature. The sodium cyanoborohydride is then added 6 mmoles, 0.4 g in 2.5 ml of anhydrous methanol. TLC shows about 40% of a new product spot which after the purification is identified by NMR as N-l (4- 'methoxyphenyl), 2- (4-methylbenzylthio) ethane glycine ethyl ester. Then 1 mmol 374 mg of N-l (4-methoxyphenyl), 2 (4-methylbenzylthio) ethane glycine ethyl ester is dissolved in 2 ml of THF and 2 mmoles of hydrated LiOH 83 mg are added to the solution. After stirring overnight the ester has been completely hydrolyzed. The THF is removed in vacuo and the product is taken in 2 ml of DMF., 5 mmoles of dibutyl dicarbonate are added 1.1 g and finally 3 mmoles of DIEA, 0.45 ml. After the reaction overnight, water and dilute HC1 solution is added and the final product (3X) is extracted with ethyl acetate. The invention will now be fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (1)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An N-substituted amide compound characterized in that it has the formula: J1-C (0) -N (C1 (R1) -C2-SH) -J2 IG J1-C (0) -N (C1 (R1) -C2 (R2) -C3 (R3) -SH) -J2 II wherein J1 and J2 are independently a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such peptide or polypeptide, a polymer, a dye, a suitably functionalized surface, a detectable linker or label, or any other chemical portion compatible with chemical peptide synthesis or extended native chemical linkage; and R1, R2 and R3 are independently H or a group that donates electrons conjugated to Cl; with the proviso that at least one of R1, R2 and R3 comprises an electron donor group conjugated to Cl. 2. The N-substituted amide compound according to claim 1, characterized in that the compound has the formula I. 3. The N-substituted amide compound according to claim 2, characterized in that C1 (R1) is selected of the group which consists of A, B and C: A B c wherein R 1 ', R 3' and R 5 'comprise electron donating groups which may be the same or different. 4. The N-substituted amide compound according to claim 1, characterized in that the compound has the formula II. 5. The N-substituted amide compound according to claim 4, characterized in that Cl (RI) -C2 (R2) -C3 (R3) is selected from the group consisting of D, E, F, G, H and I: I wherein one or more of R1 ', R3"and R5' comprise an electron donor group which may be the same or different 6. The N-substituted amide compound according to any of claims 1-5, characterized in that the substituted N is substituted Na amide 7. The N-substituted amide compound according to any of claims 3 or 5, characterized in that at least one of R1 ', R3' and R5 'comprises a strong electron donor group. 8. The N-substituted amide compound according to claim 7, characterized in that the strong electron donor group is selected from the group which consists of methoxy (-OCH3), thiol (-SH), hydroxyl (-OH) ", and thiomethyl (-SCH3). 9. The N-substituted amide compound according to any of claims 3 to 6, characterized in that at least one of R1 ', R3' and R5 'comprises a moderate electron donor group. 10. The N-substituted amide compound according to claim 8, characterized in that the moderate electron donor group comprises methyl (-CH3), ethyl (-CH2-CH3), propyl (-CH2-CH2-CH3) and isopropyl (-CH2 (C¾) 3). 11. The N-substituted amide compound according to any of claims 1, 2 or 4, characterized in that J1 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or is a portion of such a peptide or polypeptide. 12. The N-substituted amide compound according to any of claims 1, 2 or 4, characterized in that J1 is a polymer. 13. The N-substituted amide compound according to any of claims 1, 2 or 4 characterized in that J1 is a dye. 14. The N-substituted amide compound according to any of claims 1, 2, or 4, characterized in that J1 is a functionalized surface. 15. The N-substituted amide compound according to any of claims 1, 2 or 4, characterized in that J1 is a detectable linker or label. 16. The N-substituted amide compound according to any of claims 1, 2 or 4, characterized in that J2 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such a peptide or polypeptide. 17. The N-substituted amide compound according to any of claims 1, 2, or 4, characterized in that J2 is a polymer, a dye, a funcicnalized surface, a detectable linker or label.; or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage. 18. A N-substituted acid or 2-carbon chain alkyl amino or aryl thiol compound stable to the acid of the formula: HS-C2-C1 (R1) -HN-J2 lli or HS-C3 (R3) -C2 ( R2) -C1. { R1) -HN-J2 IV wherein J1 and J2 are independently a peptide or polypeptide which has one or more side chains of the amino acid optionally protected, or a portion of such peptide or polypeptide, a polymer, a dye, a functionalized surface, a detectable linker or label, or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage; and R1, R2 and R3 are independently H or a group that donates electrons conjugated to Cl; with the proviso that at least one of R1, R2 and R3 comprises an electron donor group conjugated to Cl; 19. The acid-stable N-substituted compound according to claim 18, characterized in that the compound has the formula III. 20. The acid-stable N-substituted compound according to claim 19, characterized in that Cl (Rl) is selected from the group which consists of A, B and C: A B C wherein one or more of R1", R3 'and R5" comprises an electron donor group which may be the same or different. 21. The acid-stable N-substituted compound according to claim 18, characterized in that the compound has the formula IV. 22. The acid-stable N-substituted compound according to claim 21, characterized in that C3 (R3) -C2 (R2) -Cl (l) is selected from the group which consists of D, E, F, G, H yl: I wherein one or more of R1", R3" and R5"| comprises an electron donor group which may be the same or different 23. The acid-stable N-substituted compound according to any of claims 18-22, characterized in that the substituted N-compound is Na substituted 24. The acid-stable N-substituted compound according to any of claims 20 or 22, characterized in that at least one of Rl ', R3' and R5 'comprises a donor group Strong electrons 25. The acid-stable N-substituted compound according to claim 24, characterized in that the strong electron donor group is selected from the group which consists of methoxy (-OCH3), thiol (-SH), hydroxyl (-0H), and thiomethyl (-SCH3) 26. The acid-stable N-substituted compound according to any of claims 20 or 22, characterized in that at least one of Rl ', R3"and R5' comprises an electron donor group moderate 27. The acid-stable N-substituted compound according to claim 26, characterized in that the moderate electron donor group comprises methyl (-CH3), ethyl (-CH2-CH3), propyl (-CH2-CH2-CH3) and isopropyl (-CH2 (CH3) 3) - 28. The acid-stable N-substituted compound according to any of claims 18, 19 or 21, characterized in that J2 is a peptide or polypeptide which has one or more side chains of optionally protected amino acid, or is a portion of such a peptide or polypeptide. 29. The acid-stable N-substituted compound according to any of claims 18, 19 or 21, characterized in that J2 is a polymer, a dye, a functionalized surface, a detectable linker or label, or any other compatible chemical moiety. with the chemical synthesis of peptides or extended native chemical linkage. 30. An N-substituted amide compound characterized in that it has the formula J1-C (0) -N (C1 (R1) -C2-SH) -J2 l Q J1-C (0) -N (C1 (R1) - C2 (R2) -C3 (R3) -SH) -J2 II wherein J1 and J2 are independently a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such a peptide or polypeptide, a polymer , a dye, a functionalized surface, a detectable linker or marker, or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage; and R1, R2 and R3 are independently H or a group that donates electrons conjugated to Cl; with the proviso that at least one of R1, R2 and R3 comprises an electron donor group conjugated to Cl; produced by the process of ligation of a first component which comprises an α-carboxyl thioester of the formula J1-C (0) SR to a second component which comprises an amino alkyl or aryl thiol of chain of 2 or 3 carbons N- Stable substituted to the acid of the formula: Hñ-C2-C1 (R1) -HN-J2 III or HS-C3 (R3) -C2 (R2) -C1 (R1) -HN-J2 IV wherein R1, R2 and R3 are independently H or a group that donates electrons conjugated to Cl; with the proviso that at least one of R1, R2 and R3 comprises an electron donor group conjugated to Cl. 31. The N-substituted amide compound according to claim 30, characterized in that the N-substituted compound stable to the acid has the formula III. 32. The N-substituted amide compound according to claim 31, characterized in that C1 (R1) of the acid-stable N-substituted compound is selected from the group consisting of A, B and C: where one or more of Rl ", R3". and R5 'comprises electron donating groups which may be the same or different. 33. The N-substituted amide compound according to claim 30, characterized in that the compound has the formula IV. 34. The N-substituted amide compound according to claim 33, characterized in that Cl (RI) -C2 (R2) -C3 (R3) is selected from the group consisting of D, E, F, G, H and I: Wherein one or more of R 1", R 3 'and R 5' comprises an electron donor group which may be the same or different 35. The N-substituted amide compound according to any of claims 30-34, characterized in that the substituted N is a substituted Na compound. 36. The N-substituted amide compound according to any of claims 32 or 34, characterized in that at least one of Rl ', R3' and R5 'comprise a strong electron donor group. 37. The N-substituted amide compound according to claim 36, characterized in that the strong electron donor group is selected from the group which consists of methoxy (-0C¾), thiol (-SH), hydroxyl (-0H), and thiomethyl (-SCH3.). 38. The N-substituted amide compound according to any of claims 32 or 34, characterized in that at least one of Rl ', R3' and R5 'comprises a moderate electron donor group. 39. The N-substituted amide compound according to any of claims 38, characterized in that the moderate electron donor group comprises methyl (-CH3), ethyl (-CH2-CH3), propyl (-CH2-CH2-C¾) and isopropyl (-CH2 (CH3) 3). 40. The N-substituted amide compound according to any of claims 30, 31 or 33, characterized in that J1 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or is a portion of such a peptide or polypeptide. 41. The N-substituted amide compound according to any of claims 30, 31 or 33, characterized in that J1 is a polymer. 42. The N-substituted amide compound according to any of claims 30, 31 or 33, characterized in that J1 is a dye. 43. The N-substituted amide compound according to any of claims 30, 31 or 33, characterized in that J1 is a functionalized surface. 44. The N-substituted amide compound according to any of claims 30, 31 or 33, characterized in that J1 is a detectable linker or label. 45. The .N-substituted amide compound according to any of claims 30, 31 or 33, characterized in that J2 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such a peptide or polypeptide. 46. The amide compound -N-substituted according to any of claims 30, 31, or 33, characterized in that J2 is a polymer, a dye, a functionalized surface, a detectable linker or label; or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage. 47. A compound characterized in that it has the order: J1-C (0) -HN-J2 V wherein J1 and J2 are independently a peptide or polypeptide which has one or more side chains of the optionally protected amino acid, or a portion of such a peptide or polypeptide, a polymer, a dye, a functionalized surface,. a detectable linker or label, or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage; wherein the compound is produced by the process of: (A) ligating a first component which comprises an α-carboxyl thioester of the formula J1-C (0) SR to a second component which comprises a chain amino alkyl or arylthiol 2 or 3 carbon atoms stable to the acid of the formula: HS-C2-C1 (R1) -HN-J2 III wherein: R1 is an electron donor group conjugated to Cl to thereby form a linkage product linked by N-substituted amide of the formula: J1-C (0) -N (C1 (R1) -C2-SH) -J2 l and (B) removing the 2-carbon chain alkyl or aryl thiol from the linkage product linked by N-substituted amide by cleaving the N-Cl bond. 48. A compound characterized in having the formula: J1-C (0) -HN-J2 V wherein J1 and J2 are independently a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion thereof peptide or polypeptide, a polymer, a dye, a functionalized surface, a detectable linker or label, or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage; wherein the compound is produced by the process of: (A) ligating a first component which comprises a -carboxyl thioester of the formula J1-C (0) SR to a second component which comprises an amino alkyl or aryl thiol or 3 carbon N-substituted stable to the acid of the formula: HS-C3 (R3) -C2 (R2) -C1 (1) -HN-J2 IV where R1, R2 and R3 are independently H or a conjugated electron donor group a Cl, - with the proviso that at least one of R1, R2, and R3 comprises an electron donor group conjugated to Cl; to thereby form a linkage product linked by N-substituted amide of the formula: J1-C (0) -N (C1 (R1) -C2 (R2) -C3 (3) -SH) -J2 II and (B) removing the 3-carbon alkyl or aryl thiol from the linkage product linked by N-substituted amide by cleaving the N-Cl bond. 49. The compound according to claim 47, characterized in that Cl (R1) of the acid-stable N-substituted compound is selected from the group consisting of A, B and C: ABC wherein one or more of R1 ', R3"and R5' comprises electron donating groups which may be the same or different 50. The compound according to claim 48, characterized in that C3 (R3) - C2 is selected ( R2) -Cl (R1) of the acid-stable N-substituted compound of the group which consists of D, E, F, G, H and I: D H I wherein one or more of R 1", R 3 'and R 5' comprise an electron donor group which may be the same or different 51. The acid-stable N-substituted compound according to any of claims 47-50, characterized because the substituted N-compound is a substituted Na-compound 52. The compound according to any of claims 49-6, characterized in that at least one of Rl ", R3" and R5 'comprises a strong electron donor group. 53. The compound according to claim 52, characterized in that the strong electron donor group is selected from the group which consists of methoxy (-OCH3), thiol (-SH), hydroxyl (-0H), and thiomethyl (-SCH3 ). 54. The compound according to any of claims 49 or 50, characterized in that at least one of Rl ',. R3"and R5" comprises a moderate electron donor group. 55. The compound according to any of claims 54, characterized in that the moderate electron donor group comprises methyl (-C¾), ethyl (-CH2-CH3), propyl (-CH2-CH2-CH3) and isopropyl (-C¾). (CH3) 3). 56. The compound according to any of claims 47 or 48, characterized in that J1 is a peptide or polypeptide which has one or more side chains of optionally protected amino acids. 57. The compound of. according to any of claims 47 or 48, characterized in that J1 is a polypeptide which has one or more optionally protected amino acid side chains. 58. The compound according to any of claims 47 or 48, characterized in that J1 is - a polymer. 59. The compound according to any of claims 47 or 48, characterized in that Jl is a dye. 60. The compound according to any of claims 47 or 48, characterized in that J1 is a functionalized surface. 61. The compound according to any of claims 47 or 48, characterized in that J1 is a detectable linker or label. 62. The compound according to any of claims 47 or 48, characterized in that J2 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such a peptide or polypeptide. 53. The compound according to any of claims 47 or 48, characterized in that J2 is a polymer, a dye, a functionalized surface, a detectable linker or label; or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage. 64. A method to produce a compound- of the formula: j1.C (0) -HN-J2 V wherein J1 and J2 are independently a peptide "or polypeptide which has one or more side chains of the optionally protected amino acid, or a portion of such a peptide or polypeptide, a polymer, a dye, a functionalized surface, a detectable linker or marker, or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage, the method characterized in that it comprises the steps of: (A) ligating a first component which comprises a -carboxyl thioester of the formula J1-C (0) SR to a second component which comprises a N-substituted acid or 2-carbon chain alkyl or aryl thiol stable to the acid of the formula: HS-C2-C1 (R1 ) -HN-J2 wherein Rl is an electron donor group conjugated to Cl, to thereby form an N-substituted amide linked linkage product of the formula: J1-C (0) -N (C1 ( R1) -C2-SH) -J2 ' (B) removing the 2-carbon chain alkyl or aryl thiol from the linkage product linked by N-substituted amide by cleaving the N-Cl bond. 65. A method for producing a compound of the formula: J1-C (0) -HN-J2 V wherein J1 and J2 are independently a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such a peptide or polypeptide, a polymer, a dye, a functionalized surface, a detectable linker or label, or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage; wherein the method is characterized in that it comprises the steps of: (A) ligating a first component which comprises an a-carboxyl thioester of the formula J1-C (0) SR to a second component which comprises a N-substituted amino or aryl thiol of 2 or 3 carbon chain stable to the acid of the formula: HS-C3 (R3) -C2 (R2) -C1 (R1) -HN-J2 IV where R1, R2 and R3 are independently H or an electron donor group conjugated to Cl; with the proviso that at least one of R1, R2, and R3 comprises an electron donor group conjugated to Cl; to thereby form a linkage product linked by N-substituted amide of the formula: J1-C (0) -N (C1 (R1) -C2 (R2) -C3 (R3) -SH) -J2 II (B ) removing the 3-carbon chain alkyl or aryl thiol from the linkage product linked by N-substituted amide by cleaving the Na-Cl bond. 66. The method according to claim 64, characterized in that Cl (l) of the acid-stable N-substituted compound is selected from the group consisting of A B C wherein one or more of R 1 ', R 3' and R 5 'comprises electron donating groups which may be the same or different. 67. The method according to claim 65, characterized in that Cl (Rl) -C2 (R2) -C3 (R3) is selected from the acid-stable N-substituted compound of the group which consists of D, E, F, G , H and I: I wherein one or more of R1 ', R3"and R5" comprises an electron donor group which may be the same or different. 68. The method according to any of claims 64-67, characterized in that the substituted N-compound is a substituted Na compound. 69. The method according to any of claims 66 or 67, characterized in that at least one of Rl ', R3' and R5 'comprises a strong electron donor group. 70. The method according to claim 66, characterized in that the strong electron donor group of the N-substituted compound is selected from the group consisting of methoxy (-0CH3), thiol (-SH), hydroxyl (-OH), and thiomethyl (-SCH3). 71. The method according to any of claims 66 or 67, characterized in that at least one of Rl ', R.3' and R5 'of the N-substituted compound comprises a moderate electron donor group. 72. The method of compliance with the claim 71, characterized in that the moderate electron donor group of the N-substituted compound comprises methyl (-C¾), ethyl (-CH2-CH3), propyl (-CH2-CH2-CH3) and isopropyl (-CH2 (C3) 3). 73. The method according to any of claims 66 or 67, characterized in that J1 is a peptide or polypeptide which has one or more side chains of optionally protected amino acids, or a portion of such peptide or polypeptide. 7 The method according to any of claims 66 or 67, characterized in that J1 is a polymer. 75. The method according to any of claims 66 or 67, characterized in that J1 is a dye. 76. The method according to any of claims 66 or 67, characterized in that J1 is a functionalized surface. 77. The method according to any of claims 66 or 67, characterized in that J1 is a detectable linker or label. 78. The method according to any of claims 66 or 67, characterized in that J2 is a peptide or polypeptide which has one or more optionally protected amino acid side chains, or a portion of such peptide or polypeptide. 79. The method according to any of claims 66 or 67, characterized in that J2 is a polymer, a dye, a functionalized surface, a detectable linker or label; or any other chemical portion compatible with chemical synthesis of peptide or extended native chemical linkage. 80. The method according to any of claims 66 or 67, characterized in that the compound is synthesized in solution. 81. The method according to any of claims 66 or 67, characterized in that the compound is synthesized immobilized to a solid support.