Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/80—Cytochromes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06086—Dipeptides with the first amino acid being basic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
  • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

• the present invention relates to methods and compositions for extending the technique of native chemical ligation to permit the ligation of a wide range of peptides, polypeptides, other polymers and other molecules via an amide bond.
• Chemical ligation involves the formation of a selective covalent linkage between a first chemical component and a second chemical component.
• Unique, mutually reactive, functional groups present on the first and second components can be used to render the ligation reaction chemoselective.
• the chemical ligation of peptides and polypeptides involves the chemoselective reaction of peptide or polypeptide segments bearing ' compatible unique, mutually- reactive, C-terminal and N-terminal amino acid residues.
• the original native chemical ligation methodology (Dawson et al., supra; and WO 96/34878) has proven a robust methodology for generating a native amide bond at the ligation site.
• Native chemical ligation involves a chemoselective reaction between a first peptide or polypeptide segment having a C-terminal ⁇ - carboxythioester moiety and a second peptide or polypeptide having an N- terminal cysteine residue.
• a thiol exchange reaction yields an initial thioester- linked intermediate, which spontaneously rearranges to give a native amide bond at the ligation site while regenerating the cysteine side chain thiol.
• the primary drawback of the original native chemical ligation approach is that it requires an N- terminal cysteine, i.e., it only permits the joining of peptides and polypeptide segments possessing a cysteine at the ligation site.
• a mercaptoethoxy auxiliary group can successfully lead to amide bond formation only at a glycine residue. This produces a ligation product that upon cleavage generates a glycine residue at the position of the N-substituted amino acid of the second peptide or polypeptide segment.
• this embodiment of the method is only suitable if one desires the ligation product of the reaction to contain a glycine residue at this position, and in any event can be problematic with respect to ligation yields, stability of precursors, and the ability to remove the O-linked auxiliary group.
• other auxiliary groups may be used, for example the HSCH 2 CH 2 NH-[peptide], without limiting the reaction to ligation at a glycine residue, such auxiliary groups cannot be removed from the ligated product.
• the invention is directed to methods and compositions related to extended native chemical ligation.
• the extended native chemical ligation method of the invention comprises: generating an N-substituted amide-linked initial ligation product of the formula:
• J1 is a peptide or polypeptide having one or more optionally protected amino acid side chains, or a moiety of such peptide or polypeptide, a polymer, a dye, a suitably functionalized surface, a linker or detectable marker, or any other chemical moiety compatible with chemical peptide synthesis or extended native chemical ligation
• R1 , R2 and R3 are independently H or an electron donating group conjugated to C1 ; with the proviso that at least one of R1 , R2 and R3 comprises an electron donating group conjugated to C1
• J2 is a peptide or polypeptide having one or more optionally protected amino acid side chains, or a moiety of such peptide or polypeptide, a polymer, a dye, a suitably functionalized surface, a linker or detectable marker; or any other chemical moiety compatible with chemical peptide synthesis or extended native chemical ligation.
• the ligation product is produced by the process of ligating a first component comprising a carboxyl thioester of the formula J1-C(0)SR to a second component comprising an acid stable N-substituted 2 or 3 carbon chain amino alkyl or aryl thiol of the formula:
• such cleavage is facilitated by forming a resonance stabilized cation at C1 under peptide compatible cleavage conditions.
• the removal of the alkyl or aryl thiol chain from the N generates a final ligation product of the formula:
• compositions for effecting such extended native chemical ligation and to cartridges and kits that comprise them.
• the compositions comprise a fully protected, partially protected or fully unprotected acid stable N-substituted, and preferably N ⁇ -substituted, 2 or 3 carbon chain amino alkyl or aryl thiol of the formula:
• X1 is H or an amino protecting group
• X2 is H or a thiol protecting group
• J2, R1 , R2 and R3 are as defined above
• Z2 is any chemical moiety (including, without limitation, an amino acid side chain) compatible with chemical peptide synthesis or extended native chemical ligation.
• the invention also is directed to chiral forms of such compounds of the invention that are substantially free of racemates or diasterioisomers.
• the invention is further directed to solution phase and solid phase methods of producing such fully protected, partially protected or fully unprotected N-substituted 2 or 3 carbon chain amino alkyl or aryl thiols.
• the methods for producing these compounds include halogen-mediated amino alkylation, reductive amination, and preparation of N ⁇ -protected, N-alkylated, S-protected, amino alkyl- or aryl- thiol amino acid precursors compatible with solid phase peptide synthesis methods.
• the J1 moiety of the carboxythioester component can comprise any chemical moiety compatible with the carboxythioester and reaction conditions for extended native chemical ligation, and the N-substituted component of the invention can be provided alone or joined to a wide range of chemical moieties, including 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 near neutral pH and at a range of temperature conditions.
• N-substituted components of the invention also are robust, providing a wide range of synthetic routes to these novel compounds in surprisingly high and pure yields.
• N ⁇ -protected, N-alkylated, S-protected, amino alkyl- or aryl- thiol amino acid precursors of the invention are particularly useful for rapid automated synthesis using conventional peptide synthesis and other organic synthesis strategies.
• the protected N-substituted components of the invention expand the utility of chemical ligation to multi-component ligation schemes, such as when producing a polypeptide involving multiple ligation strategies, such as a three or more segment ligation scheme or convergent ligation synthesis schemes.
• the methods and compositions of the present invention permit one to use a first pair of carboxythioester and N- substituted components 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 suitable deprotection and/or modification) to form the desired molecule.
• compositions of the invention greatly expand the scope of native chemical ligation, and the starting, intermediate and final products of the invention find a wide range of uses.
• Figure 1 illustrates the present invention by showing its ability to mediate the extended native chemical ligation of peptides; the same schemes could be employed to effect the ligation of any suitable molecule.
• a first component containing an ⁇ -carboxyl thioester of the formula J1-HN-CH(Z1)- ⁇ CO-SR, and a second component containing an N-terminal acid stable N ⁇ - substituted 2 carbon chain alkyl or aryl thiol of the formula HS-C2-C1(R1)-NH ⁇ - CH(Z2)-C(0)-J2.
• the 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.
• Z1 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 N ⁇ -substituted amino acid, such as a protected or unprotected side chain of an amino acid.
• R1 is a benzyl moiety (benzyl when referred to in the context of C1 , otherwise referred to as phenyl) substituted with an electron- donating group preferably in the ortho or para position relative to C1 ; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to C1).
• Thiol exchange occurs between the ⁇ COSR thioester component and the amino N- ⁇ alkyl thiol ⁇ component.
• the exchange generates a thioester-linked intermediate ligation product that after spontaneous rearrangement through a 5- membered ring intermediate generates a first ligation product of the formula J1- HN-CH(Z1)-C(0)-N ⁇ (C1(R1)-C2-SH)-CH(Z2)-C(0)-J2 having a removable N ⁇ - substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] at the ligation site.
• the N ⁇ -substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-j at the ligation site is amenable to being removed, under peptide-compatible conditions, to generate a final ligation product of the formula J1-HN-CH(Z1)-CO-NH-CH(Z2)- CO-J2 having a native amide bond at the ligation site.
• Figure 2 illustrates the present invention by showing its ability to mediate the extended native chemical ligation of peptides; the same schemes could be employed to effect the ligation of any suitable molecule.
• a first component containing ⁇ -carboxyl thioester of the formula J1-HN-CH(Z1)- ⁇ CO-SR and a second component containing an acid stable N ⁇ -substituted 3 carbon chain alkyl or aryl thiol of the formula HS-C3(R3)-C2(R2)-C1(R1)-NH ⁇ - CH(Z2)-C(0)-J2.
• the 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.
• Z1 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 N ⁇ -substituted amino acid, such as a protected or unprotected side chain of an amino acid.
• R1 is other than hydrogen
• R2 and R3 are hydrogen
• R1 is a phenyl moiety, unsubstituted or substituted with an electron-donating group in the ortho or para position relative to C1 ; a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to 01); a methanethiol; or a sulfoxymethyl.
• R1 is hydrogen
• R3 and R2 form a benzyl group that is substituted with an electron-donating group in the ortho or para position relative to 01 ; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to 01).
• Thiol exchange occurs between the COSR thioester component and the amino alkyl thiol component.
• the exchange generates a thioester-linked intermediate ligation product that after spontaneous rearrangement through a 6- membered ring intermediate generates a first ligation product of the formula J1- HN-CH(Z1)-C(0)-N ⁇ (C1-C2(R2)-C3(R3)-SH)-CH(Z2)-J2 having a removable N ⁇ - substituted 3 carbon chain alkyl or aryl thiol [HS-C3(R3)-C2(R2)-C1(R1)-] at the ligation site.
• N ⁇ -substituted 3 carbon chain aryl thiol [HS-C3(R3)-C2(R2)- 01 (R1)-] at the ligation site is amenable to being removed, under peptide- compatible conditions, to generate a final ligation product of the formula J1-HN- CH(Z1)-CO-NH-CH(Z2)-CO-J2 having a native amide bond at the ligation site.
• Figure 3 illustrates a multi-component extended native chemical ligation scheme.
• a polypeptide ⁇ -carboxyl thioester with an N ⁇ -protected N-terminal polypeptide N ⁇ -substituted 2 carbon chain alkyl or aryl thiol of the formula HS-C2- C1(R1)-N ⁇ (PG1)-CH(Z2)-C(0)-J2 as embodied in Figure 1 is reacted with a peptide that contains an N-terminal Cys residue.
• R1 is a phenyl, unsubstituted, or substituted with an electron-donating group, preferably in the ortho or para position relative to 01 ; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to 01).
• the protecting group (PG1) may be any suitable protecting group, such as an alkylcarbonyl protecting group (e.g., benzyloxycarbonyl (Z), Boc, Bpoc, Fmoc, etc.), a triphenylmethyl protecting group (Trt), a 2-nitrophenylsulfenyl protecting group (Nps), etc. The protecting group is removed after the first ligation reaction.
• a first native chemical ligation reaction is carried out between the polypeptide ⁇ -carboxyl thioester with an N ⁇ -protected N-terminal polypeptide N ⁇ - substituted 2 carbon chain alkyl or aryl thiol of the formula HS-C2-C1(R1)- N ⁇ (PG1)-CH(Z2)-C(0)-J2 as embodied in Figure 1 and the N-termi ⁇ al Cys- peptide to give a first ligation product of formula: HS-C2-C1(R1)-N ⁇ (PG1)-CH(Z2)- C(0)-Peptide2-Peptide3 .
• the protecting group PG1 is then removed to give the ligation product of formula HS-C2-C1(R1)-N ⁇ (H)-CH(Z2)-C(0)-Peptide2-Peptide3. This species is then reacted with a third, thioester-containing component. Thiol exchange occurs between the COSR thioester component and the amino NJalkyl thiol ⁇ component.
• the exchange generates a thioester-linked intermediate ligation product that after spontaneous rearrangement through a 5-membered ring intermediate generates a second ligation product of the formula Peptidel -C(O)- N ⁇ (C1(R1)-C2-SH)-CH(Z2)-C(0)Peptide2-Cys-Peptide3, having a removable N ⁇ - substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1 (R1)-] at the second ligation site.
• N ⁇ -substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1 (R1)-] at the second ligation site is amenable to being removed, under peptide- compatible conditions, to generate a final ligation product of the formula peptidel - C(0)-N ⁇ H-CH(Z2)-C(0)Peptide2-Cys-Peptide3, having a native amide bond at the first and second ligation sites.
• Figure 4 illustrates a general ligation strategy employing two different 1- phenyl-2-mercaptoethyl auxiliaries of the invention.
• Figures 5A and 5B shows analytical High Performance Liquid Chromatography (HPLC) results of a ligation reaction for cytochrome b562 as described in Example 21 using an N ⁇ -1-(4-methoxyphenyl)-2-mercaptoethyl auxiliary.
• Figure 5B shows the status of the ligation after the reaction is allowed to proceed overnight.
• two ligation products are observed that result from the achiral center at C1 of the N ⁇ -1-(4-methoxyphenol)-2- mercaptoethyl auxiliary.
• Figure 6A and 6B shows reconstructed electrospray mass spectra (MS) of the ligation product Cytochrome b562 residues 1-106 formed by using extended native chemical ligation with an N ⁇ - ⁇ 1-(4-methoxyphenyl) 2-mercaptoethano ⁇ - modified N-terminal segment.
• Cytochrome b562 residues 1-63 bearing a C- terminal ⁇ thioester was ligated with Cytochrome b562 residues 64-1.06 bearing an N-terminal N ⁇ - ⁇ 1-(4-methoxyphenyl) 2-mercaptoethano ⁇ glycine.
• Figure 6A shows MS reconstruct of the initial ligation product that includes a removable N ⁇ - ⁇ 1-(4-methoxyphenyl) 2-mercaptoethano ⁇ group at the ligation site.
• Figure 6B shows a MS reconstruct of ligation product following hydrogen fluoride (HF) treatment to remove the N ⁇ - ⁇ 1-(4-methoxyphenyl) 2-mercaptoethano ⁇ group to generate a native amide bond at the ligation site.
• the observed masses were 11948 ⁇ 1 Da (before HF treatment) and 11781 ⁇ 1 Da (after HF treatment), i.e.
• Figure 7A and 7B illustrate a representative analytical HPLC of linear cytochrome b562 material (Figure 7A) depicted in Figure 6B, and an ion exchange chromatogram ( Figure 7B) of the material following folding.
• the invention is directed to methods and compositions related to extended native chemical ligation.
• the method involves ligating a first component comprising a carboxyl thioester, and more preferably, an ⁇ -carboxyl thioester with a second component comprising an acid stable N-substituted, and preferably, N ⁇ -substituted, 2 or 3 carbon chain amino alkyl or aryl thiol.
• Chemoselective reaction between the carboxythioester of the first component and the thiol of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol of the second component proceeds through a thioester-linked intermediate, and resolves 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 ligation product that after spontaneous rearrangement through a 5-membered or 6-membered ring intermediate generates an amide-linked first ligation product of the formula:
• J1 , J2, R1 , R2 and R3 are as defined above.
• N-substituted 2 or 3 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] or [HS-(C3(R3)-C2(R2)-C1(R1)-] at the ligation site is amenable to being removed, under peptide-compatible conditions, without damage to the product, to generate a final ligation product of the formula:
• J1-C(0)-HN-J2 V where J1 , J2, R1 , R2, and R3 are as defined above.
• the final ligation product has a native amide bond at the ligation site
• the extended native chemical ligation method of the invention comprises chemical ligation of: (i) a first component comprising an ⁇ - carboxyl thioester of the formula J1-C(0)SR and (ii) a second component comprising an acid stable N-substituted 2 or 3 carbon chain amino alkyl or aryl thiol of the formula:
• J1 , J2, R1 , R2, and R3 are as defined above.
• the R1 , R2 and R3 groups are selected to facilitate cleavage of the N-C1 bond under peptide compatible cleavage conditions.
• electron donating groups particularly if conjugated to C1 , can be used to form a resonance stabilized cation at C1 that facilitates cleavage.
• the chemical ligation reaction preferably includes as an excipient a thiol catalyst, and is carried out around neutral pH conditions in aqueous or mixed organic-aqueous conditions. Chemical ligation of the first and second components may proceed through a five or six member ring that undergoes spontaneous rearrangement to yield an N-substituted amide linked ligation product. Where the first and second components are peptides or polypeptides, the N-substituted amide linked ligation product has the formula:
• J1 , J2 and R1 , R2, R3 and Z2 are as defined above
• the conjugated electron donating groups R1 , R2 or R3 of the N- substituted amide bonded ligation product facilitate cleavage of the N-C1 bond and removal of the 2 or 3 carbon chain alkyl or aryl thiol from the N-substituted amide-linked ligation product. Removal of the alkyl or aryl thiol chain of the N under peptide-compatible cleavage conditions generates a ligation product having a native amide bond at the ligation site. If the first and second components were peptides or polypeptides, the ligation product will have the formula:
• the present invention provides multiple advantages over previous chemical ligation approaches. Several such advantages relate to the finely tuned nature of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol component of the present invention.
• the unligated N-substituted component is stable to acidic conditions, which permits its robust synthesis and storage.
• the regenerated alkyl or aryl thiol moiety at the N ⁇ position of the ligation site of the initial ligation product can be selectively removed under conditions fully compatible with unprotected, partially protected or fully protected peptides, polypeptides or other moieties, i.e., the alkyl or aryl thiol moiety can be removed without damaging the desired ligation product.
• the selective cleavage reaction can be readily performed under standard peptide-compatible cleavage conditions such as acidic, photolytic, or reductive conditions, depending on the particular N-substituted alkyl or aryl thiol moiety chosen for ligation.
• another advantage of the invention is that one or more groups on remaining portions of the ligation components, if present, can be unprotected, partially protected or fully protected depending on the intended end use. Moreover, given the chemoselective nature and solubility properties of the carboxyl thioester and N-substituted 2 or 3 carbon chain alkyl or aryl thiol, the ligation reaction can be carried out rapidly and cleanly to give high product yields at around pH 7 under aqueous conditions at around room temperature. This makes the invention particularly flexible for ligating partially or fully unprotected peptides, polypeptides or other polymers under mild conditions.
• this compound has the formula:
• 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- donating group in the ortho or para position relative to C1 ; or a picolyl group (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to C1).
• Positioning of the phenyl and picolyl electron-donating substituents R1', R3' and R5' in the ortho or para positions is necessary to maintain electronic conjugation to the C1 carbon to enhance cleavage of the N-C1 bond following ligation.
• Preferred electron-donating groups for R1', R3' and R5' include strong electron-donating groups such as methoxy (-OCH3), thiol (-SH), hydroxyl (-OH), ethylthio (-SCH3), and moderate electron-donators such as methyl (-CH3), ethyl (-CH2- CH3), propyl (-CH2-CH2-CH3), isopropyl (-CH2(CH3)3).
• R1 ⁇ R3' and R5' maybe H.
• the strong electron-donating groups enhance the sensitivity of the 2-carbon chain alkyl or aryl thiol to cleavage following ligation.
• a single electron-donating group is present as a R1', R3' or R5' substituent, the ligation reaction may proceed at a faster rate, whereas cleavage is slower or requires more stringent cleavage conditions.
• two or more electron-donating groups are present as a R1 ⁇ R3' or R5' substituent, the ligation reaction may be slower, whereas cleavage is faster or requires less stringent cleavage conditions.
• _Thus a particular electron-donating group can be selected accordingly.
• N-substituted 2 carbon chain compounds which include a thiol as a substituent of R1 in the R1' and R5' positions.
• a thiol as a substituent of R1 in the R1' and R5' positions.
• introduction of a thiol at one or both of these locations enables the compounds to ligate through a 6-member ring mediated through the R1 group (as well as through a 5-member ring by the N ⁇ -2 carbon chain alkyl thiol). It also increases the local concentration of available thiols for reacting with the ⁇ -carboxy thioester, and provides for additional conformations in terms of structural constraints that can improve ligation.
• this compound has the formula HS-C3(R3)-C2(R2)- C1 (R1)-NH ⁇ -CH(Z2)-C(0)-J2, which is depicted below in Table II.
• J2 can be any chemical moiety compatible with the chemical peptide synthesis or extended native chemical ligation
• Z2 is any side chain group compatible with an N-substituted amino acid, such as a side chain of an amino acid.
• R1 is other than hydrogen
• R2 and R3 are hydrogen
• R1 is a phenyl moiety, unsubstituted, or more preferably, substituted with an electron- donating group in the ortho or para position relative to C1 ; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to C1).
• R1 is hydrogen
• R3 and R2 form a benzyl group that is substituted with an electron-donating group in the ortho or para position relative to C1 ; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the ortho or para position relative to C1).
• R1', R3' and R5' are phenyl and picolyl electron-donating substituents in the ortho or para positions.
• R2 and R3 form a benzyl group with C2 and C3
• at least one of R1' and R3' comprises a strong electron donating group, where R1' or R3' is selected from methoxy (- OCH3), thiol (-SH), hydroxyl (-OH), and thiomethyl (-SCH3).
• R1 comprises a phenyl or picolyl group in which R1 ⁇ R3' and R5' include either strong or moderate electron-donating groups, or a combination thereof.
• the strong electron-donating groups enhance the sensitivity of the 3 carbon chain alkyl or aryl thiol to cleavage following ligation.
• a particular electron-donating group or combination thereof can be selected accordingly.
• the N-substituted 3 carbon chain compounds of the present invention may include a thiol as a substituent of R1 in the R1' and R5' positions when available for substitution in a construct of interest.
• the electron-donating thiol group is conjugated to C1 and its introduction at these locations enables the compounds to have two routes for the 6-member ring forming ligation event. It also increases the local concentration of available thiols for reacting with the ⁇ -carboxy thioester, and provides for additional conformations in terms of structural constraints that can improve ligation.
• the preferred approach employs N alpha protected N alkylated S-protected amino alkyl- or aryl- thiol amino acid precursors.
• the reagents utilized for synthesis can be obtained from any 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 later use, provided in kits and the like.
• protecting group strategies are employed.
• the preferred protecting groups (PG) utilized in the various synthesis strategies in general are compatible with Solid Phase Peptide Synthesis ("SPPS"). In some instances, it also is necessary to utilize orthogonal protecting groups that are removable under different conditions.
• suitable protecting groups include, but are not limited to, benzyl, 4-methylbenzyl, 4- methoxybenzyl, trityl, Acm, TACAM, xanthyl, disulfide derivatives, picolyl, and phenacyl.
• N ⁇ -substituted 2 or 3 carbon chain alkyl or aryl thiols can be prepared in accordance with Scheme I (Solid-Phase preparation of the N ⁇ -substituted precursor), Scheme II (Solution-Phase preparation of the N ⁇ - substituted precursor).
• Scheme I N ⁇ -substituted 2 or 3 carbon chain alkyl or aryl thiols are assembled directly on the solid phase using standard methods of polymer-supported organic synthesis, while the N ⁇ -protected, N-alkylated, S- protected, aminoalkyl or arylthiol amino acid precursor of Scheme II are coupled to the resin using standard coupling protocols.
• X is a halogen
• R1 and R2 are as described above and can be preformed as protected or unprotected moieties or elaborated on-resin
• J2 is preferably attached to halogen as X-CH(R)-J2-Resin, where R is hydrogen or other side chain.
• J2 can be a variety of groups, for example where 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.
• glyoxalic moiety HC(O)-C(O)- J2-Resin
• resulting side chain R is hydrogen.
• X is a halogen
• R1 and R2 are as described above and can be preformed as protected or unprotected moieties or elaborated in solution or on-resin, and where R is hydrogen or other side chain.
• R is hydrogen or other side chain.
• glyoxalic acid moiety HC(0)-C(0)-OH
• the resulting side chain R is hydrogen.
• Schemes I and II can be applied in the synthesis of the 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 use in extended native chemical ligation.
• this component has the formula J1-CO-SR.
• the more preferred carboxy thioester component comprises an ⁇ -carboxyl thioester amino acid of the formula J1-NH-C(Z1)-CO-SR.
• the group J1 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.
• Z1 is any side chain group compatible with the ⁇ CO-SR thioester, such as a side chain of an amino acid.
• R is any group compatible with the thioester group, including, but not limited to, aryl, benzyl, and alkyl groups.
• examples of R include 3-carboxy-4-nitrophenyl thioesters, benzyl thioesters, and mercaptopropionic acid leucine thioesters (See, e.g., Dawson et al., Science (1994) 266:776-779; Canne et al. Tetrahedron Lett.
• ⁇ -carboxythioesters can be generated by chemical or biological methods following standard techniques known in the art, such as those described herein, including the Examples.
• ⁇ -carboxythioester peptides can be synthesized in solution or from thioester-generating resins, which techniques are well known (See, e.g., Dawson et al., supra; Canne et al., supra;hackeng et al., supra, Hojo H, Aimoto, S. (1991) Bull Chem Soc Jpn 64:111-117).
• thioester peptides can be made from the corresponding peptide ⁇ -thioacids, which in turn, can be synthesized on a thioester-resin or in solution, although the resin approach is preferred.
• the peptide- ⁇ -thioacids 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 of 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 alkyl thioesters.
• a trityl- associated mercaptoproprionic acid leucine thioester-generating resin can be utilized for constructing C-terminal thioesters (Hackeng et al., supra).
• C-terminal thioester synthesis also can be accomplished using a 3- carboxypropanesulfonamide safety-catch linker by activation with diazomethane or iodoacetonitrile followed by displacement with a suitable thiol (Ingenito et al., supra; Shin et al., (1999) J. Am. Chem. Soc, 121, 11684-11689).
• Peptide or polypeptide C-terminal ⁇ -carboxythioesters also can be made using biological processes.
• intein expression systems with or without labels such as affinity tags can be utilized to exploit the inducible self- cleavage activity of an "intein" protein-splicing element in the presence of a suitable thiol to generate a C-terminal thioester peptide or polypeptide segment.
• the intein undergoes specific self-cleavage in the presence of thiols such as DTT, ⁇ -mercaptoethanol, ⁇ -mercaptoethanesulfonic acid, or cysteine, which generates a peptide segment bearing a C-terminal thioester.
• Ligation of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol components of the invention with the first carboxythioester component generates a ligation product having an N-substituted amide bond at the ligation site, as depicted in Figures 1, 2 and 3.
• the ligation 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 moiety.
• the ligation reaction is carried out in a buffer solution having pH 6-8, with the preferred pH range being 6.5-7.5.
• the buffer solution may be aqueous, organic or a mixture thereof.
• the ligation reaction also may include one or more catalysts and/or one or more reducing agents, lipids, detergents, other denaturants or solubiiizing reagents and the like.
• catalysts are thiol and phosphine containing moieties, such as thiophenol, benzylmercaptan, TCEP and alkyl phosphines.
• denaturing and/or solubiiizing agents examples include guanidinium, urea in water or organic solvents such as TFE, HFIP, DMF, NMP, acetonitrile admixed with water, or with guanidinium and urea in water.
• the temperature also may be utilized 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.
• the ligation reactions proceed well in a reaction system having 2% thiophenol in 6M guanidinium at a pH between 6.8 and 7.8.
• the ligation event results from a thiol exchange that occurs between the COSR thioester component and the amino alkyl thiol component.
• the exchange generates a thioester-linked intermediate ligation product that after spontaneous rearrangement through a 5- membered ring intermediate generates a first ligation product of the formula J1- HN-CH(Z1)-C(0)-N ⁇ (C1 (R1)-C2-SH)-CH(Z2)-J2 having a removable N- substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] at the ligation site, where the substituents are as defined above.
• the N-substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-j at the ligation site is amenable to being removed, under peptide-compatible conditions, to generate a final ligation product of the formula J1-HN-CH(Z1)-C0-NH-CH(Z2)-C0-J2 having a native amide bond at the ligation site.
• the thiol exchange between the COSR thioester component and the amino alkyl thiol component generates a thioester-linked intermediate ligation product that after spontaneous rearrangement through a 6-membered ring intermediate generates a first ligation product of the formula J1-HN-CH(Z1)-C(O)-N ⁇ (01-C2(R2)-C3(R3)-SH)-CH(Z2)-J2 having a removable N-substituted 3 carbon chain alkyl or aryl thiol [HS-C3(R3)- C2(R2)-C1(R1)-j at the ligation site.
• the N-substituted 3 carbon chain aryl thiol [HS-C3(R3)-C2(R2)-C1(R1)-j at the ligation site is amenable to being removed, under peptide-compatible conditions, to generate a final ligation product of the formula J1-HN-CH(Z1)-C0-NH-CH(Z2)-C0-J2 having a native amide bond at the ligation site.
• Removal of the N-substituted alkyl or aryl thiol group is preferably performed in acidic conditions to facilitate cleavage of the N-C1 bond, yielding a stabilized, unsubstituted amide bond at the ligation site.
• peptide-compatible cleavage conditions is intended physical-chemical conditions compatible with peptides and suitable for cleavage of the N-linked alkyl or aryl thiol moiety from the ligation product.
• Peptide-compatible cleavage conditions in general are selected depending on the N ⁇ -alkyI or aryl thiol moiety employed, which can be readily deduced through routine and well known approaches (See, e.g., "Protecting Groups in Organic Synthesis", 3rd Edition, T.W. Greene and P.G.M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic 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.W.
• the more universal method for removal involves acidic cleavage conditions typical for peptide synthesis chemistries. This includes cleavage of the N-C1 bond under strong acidic conditions or water-acidic conditions, with or without reducing reagents and/or scavenger systems (e.g., acid such as anhydrous hydrogen fluoride (HF), triflouroacetic acid (TFA), or trifluoromethanesulfonic acid (TFMSA) and the like).
• acid such as anhydrous hydrogen fluoride (HF), triflouroacetic acid (TFA), or trifluoromethanesulfonic acid (TFMSA) and the like.
• More specific acidic cleavage systems can be chosen to optimize cleavage of the N ⁇ -C1 bond to remove the aryl or alkyl thiol moiety for a given construct. Such conditions are well known and compatible with maintaining the integrity of peptides.
• Another method for cleavage involves the inclusion of a thiol scavenger where tryptophans are present in a peptide or polypeptide sequence to avoid reaction of the tryptophan side chain with the liberated aryl or alkyl thiol moiety.
• thiol scavengers include ethanediol, cysteine, beta-mercaptoethanol and thiocresol.
• another embodiment of the invention is the addition of a thiol scavenger when cleaving the N-C1 bond to remove the aryl or alkyl thiol moiety.
• Other specialized cleavage conditions include light or reductive-cleavage conditions when the picolyl group is the substituent.
• photolysis e.g., ultraviolet light
• zinc/acetic acid or electrolytic reduction may be used for cleavage following standard protocols.
• R1 of the N-substituted 2 carbon chain thiol comprises a thiomethane at R1
• mercury(ll) or HF cleavages can be used.
• the cleavage system also can be used for simultaneous cleavage from a solid support and/or as a deprotection reagent when the first or second ligation components comprise other protecting groups.
• N-picolyl groups can be removed by dissolving the polypeptide in a 10% acetic acid/water solution, with activated zinc ( ⁇ 0.5g/ml).
• Thiomethane groups such as 2-mercapto, 1-methylsulfinylethane groups (HS-C2-C1 (S(0)-CH3)-N ⁇ ), can be removed after ligation by reduction and mercuric, mercaptan-mediated cleavage.
• the methylsulfinylethane group can be removed by dissolving the polypeptide in an aqueous 3% acetic acid solution containing N-methylmercaptoacetamide (MMA) (e.g., 1 mg polypeptide in 0.5ml of acetic acid/water and 0.05 ml of MMA), for reduction to the thiomethane form, followed by freezing and lyophilization of the mixture after overnight reaction.
• MMA N-methylmercaptoacetamide
• the reduced auxiliary can then be removed in an aqueous solution of 3% acetic acid containing mercury acetate (Hg(OAC) 2 ) (e.g., 0.5ml of acetic acid in water and 10 mg of Hg(OAC) for about 1 hour), followed by addition of beta-mercaptoethanol (e.g., 0.2ml beta-mercaptoethanol).
• Hg(OAC) 2 acetic acid containing mercury acetate
• beta-mercaptoethanol e.g., 0.2ml beta-mercaptoethanol
• one or more catalysts and/or excipients may also be utilized in the cleavage system, such as one or more scavengers, detergents, solvents, metals and the like.
• selection of specific scavengers depends upon the amino acids present.
• the presence of scavengers can be used to suppress the damaging effect that the carbonium ions, produced during cleavage, can have on certain amino acids (e.g., Met, Cys, Trp, and Tyr).
• Other additives like detergents, polymers, salts, organic solvents and the like also may be employed to improve cleavage by modulating solubility. Catalysts or other chemicals that modulate the redox system also can be advantageous. It also will be readily apparent that a variety of other physical-chemical conditions such as buffer systems, pH and temperature can be routinely adjusted to optimize a given cleavage system.
• the present invention also provides protected forms of the N ⁇ -substituted 2 or 3 carbon chain alkyl or aryl thiols of the invention. These compounds are especially useful for automated peptide synthesis and orthogonal and convergent ligation strategies.
• compositions comprise a fully protected, partially protected or fully unprotected acid stable N ⁇ -substituted 2 or 3 carbon chain amino alkyl or aryl thiol of the formula (PG2)S-C2-C1(R1)-N ⁇ (PG1 )-CH(Z2)-C(0)- J2 or (PG2)S-C3(R3)-C2(R2)-C1(R1)-N ⁇ (PG1)-CH(Z2)-C(0)-J2, which are depicted below in Table III and Table IV.
• one or more of R1 , R2 and R3 comprises an electron donating group conjugated to C1 that, following conversion of the N ⁇ -substituted amino alkyl or aryl thiol to an N ⁇ -substituted amide alkyl or aryl thiol, is capable of forming a resonance stabilized cation at C1 that facilitates cleavage of the N ⁇ -C1 bond under peptide compatible cleavage conditions.
• PG1 and PG2 are protecting groups that are present individually or in combination or are absent and can be the same or different, where Z2 is any chemical moiety compatible with chemical peptide synthesis or extended native chemical ligation, and where J2 is any chemical moiety compatible with chemical peptide synthesis or extended native chemical ligation.
• PG1 (or X1) is a group for protecting the amine.
• PG2 (or X2) is a group for protecting the thiol.
• Many such protecting groups are known and suitable for this purpose (See, e.g., "Protecting Groups in Organic Synthesis", 3rd Edition, T.W. Greene and P.G.M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic 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.W.
• Examples of preferred protecting groups for PG1 and X1 include, but are not limited to [Boc(t-Butylcarbamate), Troc(2,2,2,-Trichloroethylcarbamate), Fmoc(9-Fluorenylmethylcarbamate), Br-Z or CI-Z(Br- or Cl-Benzylcarbamate), Dde(4,4,-dimethyl-2,6-dioxocycloex1-ylidene), MsZ(4-
• Methylsulfinylbenzylcarbamate Msc(2-Methylsulfoethylcarbamate) Nsc(4- nitrophenylethylsulfonyl-ethyloxycarbonyl].
• Preferred PG1 and X1 protecting groups are selected from "Protective Groups in Organic Synthesis,” Green and Wuts, Third Edition.Wiley-lnterscience, (1999) with the most preferred being Fmoc and Nsc.
• Examples of preferred protecting groups for PG2 include, but are not limited to [Acm(acetamidomethyl), MeOBzl or Mob(p-Methoxybenzyl), MeBzl(p- Methylbenzyl), Trt(Trityl),Xan(Xanthenyl),tButhio(s-t-butyl),Mmt(p-Methoxytrityl),2 or 4 Picolyl(2 or 4 pyridyl)),Fm(9-Fluorenylmethyl), tBu(t- Butyl)Jacam(Trimethylacetamidomethyl)]
• Preferred protecting groups PG2 and X2 are selected from "Protective Groups in Organic Synthesis," Green and Wuts, Third Edition,Wiley-lnterscience, (1999), with the most preferred being Acm, Mob, MeBzl, Picolyl .
• Orthogonal protection schemes involves two or more classes or groups that are removed by differing chemical mechanisms, and therefore can be removed in any order and in the presence of the other classes. Orthogonal schemes offer the possibility of substantially milder overall conditions, because selectivity can be attained on the basis of differences in chemistry rather than reaction rates.
• the protected forms of the N ⁇ -substituted 2 or 3 chain alkyl or aryl thiols of the invention can be prepared as in Schemes I and II above.
• the compounds of the present invention may be produced by any of a variety of means, including halogen-mediated amino alkylation, reductive amination, and by the preparation of N ⁇ -protected, N-alkylated, S-protected, amino alkyl- or aryl- thiol amino acid precursors compatible with solid phase or solution amino acid or peptide synthesis methods. When desirable, resolution of the racemates or diastereisomers produced to give compounds of acceptable chiral purity can be carried out by standard methods.
• N ⁇ -substituted 2 or 3 carbon chain alkyl or aryl thiols of acceptable chiral purity are preferred in some instances.
• use of the N ⁇ -1-(4-methoxyphenyl)-2-mercaptoethyl auxiliary in the preparation of cytochrome b562 yielded two ligation products (diastereoisomers) with overlapping purification profiles. Although removal of the N ⁇ -auxiliary yields a single major product, a small percentage of deletion and side-reactant products will be present in the final product, which may be undesirable.
• the reductive amination synthetic route as described in Examples 4 through 6 employed for synthesis the N ⁇ -1-(4-methoxyphenyl)-2- mercaptoethyl auxiliary employed in the cyt b562 synthesis inherently results in the production of both epimers at the chiral center C1.
• resolution of the racemates or diastereisomers produced to give compounds of acceptable chiral purity can be carried out by standard methods.
• Standard approaches for obtaining N ⁇ -auxiliaries of the invention of acceptable chiral purity are: (1) chiral chromatography; (2) chiral synthesis; (3) use of a covalent diasteriomeric conjugate; and (4) crystallization or other traditional separation methods to give enantiomerically pure chiral auxiliary.
• chiral chromatography e.g., Ahuja, Satinder. 'Chiral separations.
• a racemic mixture produced by the reductive amination route for the total synthesis of chiral N ⁇ -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 amino acid side chain):
• Either enantiomer may be obtained in chirally pure form, or both may be obtained in chirally pure form.
• Either enantiomer may be used to form chirally-pure auxiliary modified components, such as peptide segments (i.e. two chirally pure epimers), which can be rigorously purified without interference from the presence of the other epimer and its impurities. Note that unless some provision is made for using both enantiomers, 50% of the total mass of the auxiliary will be wasted. For example, the two chirally pure auxiliary-modified peptide segments can then used in separate ENCL reactions, to give chirally pure auxiliary-modified ligation product mixtures.
• auxiliary group is removed from the epimer ligation products (either separately or after being combined) to give the same native structure, ligated product, which is then subjected to purification.
• a preferred method employs an enantiomerically pure, chiral starting material, as illustrated below for a para-methoxyphenyl substituted
• N ⁇ -2 carbon auxiliary N ⁇ -2 carbon auxiliary
• the resulting chirally pure precursor compound can then be used to make either a protected (N-substituted) amino acid, viz.:
• auxiliary-modified peptide on a polymer support.
• deprotection/cleavage gives the auxiliary-modified peptide segment in chirally- pure form, viz.:
• N ⁇ -auxiliaries of the invention can be made from the readily available para-substituted phenylgylcine(s) of known chirality, thus predetermining the chirality and chiral purity of the resulting auxiliary.
• Another preferred embodiment employs enantioselective synthesis employing asymmetric reduction to yield the auxiliary, for example as illustrated below:
• R -H, or -CH 3 , or -CH 2 COOH
• Asymmetric reduction can also be used for enantioselective synthesis to yield an N ⁇ -auxiliary-modified amino acid, such as for glycine illustrated below, viz.:
• Another preferred standard technique is resolution by use of a covalent diasteriomeric conjugate.
• this approach 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, such as illustrated below.
• the racemic auxiliary 1 can be converted to a mixture of diastereomers by covalent incorporation of a second chiral center:
• the protected N ⁇ -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 ligation to multi-component ligation schemes, such as when producing a polypeptide involving orthogonal ligation strategies, such as a three or more segment ligation scheme or convergent ligation synthesis schemes.
• the extended native chemical ligation method and compositions of the invention can be employed in conjunction with nucleophile stable thioester generating methods and thioester safety-catch approaches, such as the orthothioloester and carboxyester thiols described in co-pending application PCT application Serial No.
• nucleophile-stable thioester generating compounds comprise an orthothioloester or a carboxyester thiol; these compounds have wide applicability in organic synthesis, including the generation of peptide-, polypeptide- and other polymer-thioesters.
• the nucleophile-stable thioester generating compounds are particularly useful for generating activated thioesters from precursors that are made under conditions in which strong nucleophiles are employed, such as peptides or polypeptides made using Fmoc SPPS, as well as multi-step ligation or conjugation schemes that require (or benefit from the use of) compatible selective-protection approaches for directing a specific ligation or conjugation reaction of interest.
• the nucleophile- stable orthothioloesters have the formula X-C(OR') 2 -S-R, where X is a target molecule of interest optionally comprising one or more nucleophile cleavable protecting groups, R' is a nucleophile-stable protecting group that is removable under non-nucleophilic cleavage conditions, and R is any group compatible with the orthothioloester -C(OR')-S-.
• Nucleophile-stable orthothioloester thioester- generating resins also are provided, and have the formula X-C(OR') 2 -S-R-linker- resin or X-C((OR 1 '-linker-resin )(OR 2 '))-SR, where X, R' and R are as above, and where the linker and resin are any nucleophile-stable linker and resin suitable for use in solid phase organic synthesis, including safety-catch linkers that can be subsequently converted to nucleophile-labile linkers for cleavage.
• the nucleophile-stable orthothioloesters can be converted to the active thioester by a variety of non-nucleophilic conditions, such as acid hydrolysis conditions.
• Nucleophile-stable carboxyester thiol-based thioester-generating resins also are provided, and have the formula X- 0(O)-O-CH(R")-CH2n-S-linker-resin or X-C(0)-0-CH(R"-linker-resin)-CH2n-S-R'", where X, R", n and R'" are as above, and where the linker and resin are any nucleophile-stable linker and resin suitable for use in solid phase organic synthesis.
• the nucleophile-stable carboxyester thiols can be converted to the active thioester by addition of a thiol catalyst, such as thiophenol.
• the extended native chemical ligation methods and compositions of the present invention can be employed in multi-segment convergent ligation techniques, where a one end of a target compound can bear a protected or unprotected N ⁇ -2 or 3 carbon chain alkyl or aryl thiol of the present invention, and the other end a orthothioloester or carboxyester thiol moiety for subsequent conversion to the active thioester and ligation.
• N ⁇ -2 or 3 carbon chain alkyl or aryl thiol of the present invention can be employed in combination with other ligation methods, for example, such as native chemical ligation (Dawson, ef al., Science (1994) 266:776-779; Kent, ef al., WO 96/34878), extended general chemical ligation (Kent, et al., WO 98/28434), oxime-forming chemical ligation (Rose, ef al., J. Amer. Chem. Soc.
• Also contemplated by the present invention is the substitution of selenium in place of the thiol sulfur in the N ⁇ -2 or 3 carbon chain 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 ligating peptides, polypeptides and other polymers.
• the ability to carry out native chemical ligation at practically any amino acid, including the naturally occurring as well as unnatural amino acids and derivatives expands the scope of native chemical ligation to targets that are missing suitable cysteine ligation sites.
• the invention also can be used to ligate polymers in addition to peptide or polypeptide segments when it is desirable to join such moieties through a linker having an N ⁇ - substituted or totally native amide bond at the ligation site.
• the invention also finds use in the production of a wide range of peptide labels for expressed-protein ligation (EPL) applications.
• EPL-generated thioester polypeptides can be ligated to a wide range of peptides via an N ⁇ -substituted alkyl or aryl thiol amide bond or a completely native amide bond, depending on the intended end use.
• the invention also can be exploited to produce a variety of cyclic peptides and polypeptides, having a native amide bond at the point of cyclization even for peptides and polypeptides that do not contain cysteine. For instance, this is significant as 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 cyclizing (i.e., head-to-tail) ligation site.
• HATU ( ⁇ /-[(dimethylamino)-1 H-1 , 2, 3-triazol [4, 5-b] pyridiylmethylene]-/V- methylmethanaminium hexafluorophosphate ⁇ /-oxide).
• HMP resin 4-hydroxymethylphenoxy resin; palkoxybenzyl alcohol resin; or
• PEG-PS polyethylene glycol-polystyrene
• Tacam Trimethylacetamidomethyl fBoc fert-butyloxycarbonyl
• Peptides were synthesized in stepwise fashion on a modified ABI 430A peptide synthesizer by SPPS using in situ neutralization/HBTU activation protocols for Boc-chemistry on PAM resin or thioester-generating resin following standard protocols (Hackeng et al., supra; Schnolzer et al., (1992) Int.J.Pept.Prot.Res., 40:180-193; and Kent, S.B.H. (1988) Ann. Rev. Biochem. 57, 957-984). After chain assembly the peptides were deprotected and simultaneously cleaved by treatment with anhydrous hydrogen fluoride (HF) with 5% p-cresol and lyophilized and purified by preparative HPLC.
• HF hydrous hydrogen fluoride
• Boc protected amino acids were obtained from Peptides International and Midwest Biotech. Trifluoroacetic acid (TFA) was obtained by Halocarbon. Other chemicals were from Fluka or Aldrich.
• Analytical and preparative HPLC were performed on a Rainin HPLC system with 214 nm UV detection using Vydac C4 analytical or preparative.
• Peptide and protein mass spectrometry was performed on a Sciex API-I electrospray mass spectrometer.
• BocGlycineOSuccinimide was coupled to the resin. After the coupling was completed Boc group was removed and the resin was neutalized with 2 washes with 10% Diisopropylethylamine in DMFJhe resin was then washed with DMF and DMSO. Then 9mg of 2(4'methoxybenzylthio)benzylbromide in 0.2 ml of DMSO and 0.01ml of Diisopropylethylamine were added. The mixture reacted for 12 hrs at room temperature. The peptide was cleaved and deprotected in HF conditions using standard protocols. The peak with correct mass of 2,079 Da was about 12% (measured by HPLC) of all peptidic material. The correct peptide was purified using standard semi-preparative HPLC.
• Example 5 Preparation of 1 amino,1(4-methoxyphenyl),2(4- methylbenzylthio) ethane 4'-Methoxy 2(4'methylbenzylthio) acetophenone 1.44 mmol, 411 mg and aminoxyacetic acid 4.3 mmol, 941 mg were dissolved in 20ml of TMOF and 0.047ml of methanesulfonic acid was added as catalyst at room temperature. After 48 hours, the solvent was evaporated and the residue taken up in ethylacetate, washed with 1M monohydrogenpotassium sulfate and dried over sodium sulfate.
• the crude product was purified with silica gel chromatography, and 200mg of oxime complex obtained.
• T200mg of this oxime complex 0.556mmol was dissolved in 2ml of THF, followed by the addition of 1.67ml of 1 M BH3/THF complex. After 27 hours no starting material was left. 3ml of water were added and 1.5 ml of 10N sodium hydroxide was added. The mixture was refluxed for 1 hour. The mixture was then extracted with ethylacetate (4x) and dried over sodium sulfate. The final product (40 mg) was then purified using silica gel chromatography.
• Example 10 Ala-GIy chemical ligation of C-terminal SDF1-alanine-thioester and N-terminal N ⁇ (2-mercaptobenzyl) glycine-peptide
• Example 11 Ala-GIy chemical ligation of C-terminal SDF1-alanine-thioester and N-terminal N ⁇ 1-(4-methoxyphenyl) 2-mercaptoethane glycine-peptide
• yield of the desired ligation product was about 45% based on the ratio between product and the non-reacted N-terminal fragment. After 3 days at room temperature, followed by further incubation at 40°C for an additional 24 hours, the yield was 65%. After 3 days at room temperature, followed by further incubation at 40°C for an additional 48 hours, the yield increased to about 70%.
• Example 12 Gly-Gly chemical ligation of C-terminal glycine-thioester and N-terminal N ⁇ -(2-mercaptobenzyl) glycine-peptide
• C-terminal Gly thioester fragment (MW 1357) of a decamermodel peptide 3.5 mg
• 2 mg of N-terminal N ⁇ (2- mercaptobenzyl) glycine fragment (MW 2079) of a model peptide with 3 HisDnp were dissolved in 200 ⁇ l of 6 M guanidinium buffer pH 7.9 and 2 ⁇ l of thiophenol was added. The mixture was incubated at 33°C for 60 hours. Formation of the desired ligation product (MW 2631) was confirmed by ES-MS, with an observed yield of about 40% based on the ratio between product and the non-reacted N- terminal fragment.
• a mouse Larc 1-31 Ala C terminal peptide thioester 3mg (MW 3609) and model peptide N ⁇ 1-,(4-methoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 1 mg (MW 908) were dissolved in 0.15 ml 6molar guanidinium buffer pH8.2 and 0.03 ml thiophenol. After overnight stirring the ligation was 81% complete and after 40 hrs 92% complete based on consumption of peptide thioester. Expected ligated product 4312Da, found 4312Da.
• Example 14 Ala-GIy chemical ligation of Larc 1-31-aIanine-thioester with N ⁇ 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane glycine- peptide
• Mouse Larc 1-31 Ala C terminal peptide thioester 3mg (MW 3609) and model peptide N ⁇ 1-(2,4-dimethoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 1 mg (MW 938) were dissolved in 0.15 ml ⁇ molar guanidinium buffer pH8.2 and 0.03 ml thiophenol. After overnight stirring the ligation was 73% complete, and after 40 hrs 85% complete based on consumption of peptide thioester. The calculated and experimental masses of the ligation product were both 4342Da.
• Example 15 Gly-Gly chemical Ligation of C-terminal tripeptide glycine- thioester and N-terminal N ⁇ -1-(2,4-dimethoxyphenyl) 2- mercapto ethane glycine-peptide
• Peptide fragment FGG-thioester O. ⁇ mg and model peptide N ⁇ 1-(2,4- dimethoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 1 mg (MW 938) were dissolved in 0.1 ml of 6M guanidinium buffer pH 8.2 and 0.02 ml of thiophenol. After overnight stirring the reaction was completed quantitatively. The calculated and experimental masses of the ligation product were 1199.4Da and 1195.5Da, respectively.
• Example 17 Gly-Gly chemical Ligation of C-terminal tripeptide glycine- thioester and N-terminal N ⁇ 1-,(4-methoxyphenyl) 2-mercapto ethane glycine-peptide
• Example 19 His-GIy chemical ligation of C-terminal histidine-thioester and N-terminal N ⁇ 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane glycine-peptide
• Example 22 Removal of the 1-,(4-methoxyphenyl) 2-mercapto ethane group from ligated Cytochrome b562 residues 1-106 and generation of the native protein
• the Slm7 cyt b562 mutant differed from the wild type by replacing methionine at position 7 with a selenomethionine (sulfur of methionine replaced with its lower cogener selenium). Circular dichroism was performed that indicated high ⁇ -helical content in both apo wild type b562 and apo Slm7 b562 (data not shown). ESMS also showed that both apo wild type b562 and apo Slm7 b562 had the expected molecular masses (data not shown).
• the apo proteins were reconstituted with heme (heme pH7 NaPi overnight, room temperature), and the resulting proteins purified with ion exchange FPLC (FPLC purification Resource Q, Tris HCL pH 8, NaCl gradient).
• FPLC purification Resource Q Tris HCL pH 8, NaCl gradient.
• UV-visible (optical ) spectra of the heme-reconstituted proteins were found to be consistent with sulfur or selenium coordination to Fe (data not shown).
• a cyt b562 mutant with the non-coordinating isotere norleucine also is prepared in the same manner.
• synthetic cytochromes were made using extended native chemical ligation, reconstituted with their heme active sites and fully characterized by biophysical methods.
• this example further demonstrates that peptides and proteins devoid of suitable cysteines for the original native chemical ligation approach can be made by extended native chemical ligation, and non- standard amino acids incorporated therein.
• extended native chemical ligation the folding and reactivity of many cyt b562 mutants have been studied, but thus far unnatural axial ligands have remained unexplored.
• extended native chemical ligation the vast array of unnatural amino acids available should allow systematic tuning of the properties of these and other proteins.
• Example 23 Lys-Gly chemical ligation of MCP 1-35-Lysine-thioester with N ⁇ 1-,(4-methoxyphenyl) 2-mercapto ethane glycine-peptide
• Lyophilized de-salted crude from Example 23 (Lys-Gly ligation) was dissolved in 1mg of TFA, 25 ⁇ l of Ethane di thiol , 50 ⁇ l of TIS. Then 150 ⁇ l of bromotrimethylsilane was added. The reaction was allowed to proceed for 2 hrs at room temperature ("rt"). The volatile components of the mixture were evaporated in vacuo, and the remaining oil was taken up in 6M Guanidinium buffer pH 7.5. The organic material was extracted with CHCI3. HPLC showed no more starting material, therefore the auxiliary group was successfully removed. The expected mass for the native sequence was 4726Da, and a mass of 4725Da was found.
• N-terminal auxiliary I Ncc-1-(4-methoxyphenyl)-2-mercaptoethane glycine- SYRFL
• N-terminal auxiliary II N ⁇ -1-(2, 4-methoxyphenyl)-2-mercaptoethane glycine-SYRFL
• Example 26 Preparation of BocGlycine N-1(4'-methoxyphenyl),2(4'- methylbenzylthio) ethane

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