CA2489186C - Chemical synthesis of reagents for peptide coupling - Google Patents
Chemical synthesis of reagents for peptide coupling Download PDFInfo
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- CA2489186C CA2489186C CA2489186A CA2489186A CA2489186C CA 2489186 C CA2489186 C CA 2489186C CA 2489186 A CA2489186 A CA 2489186A CA 2489186 A CA2489186 A CA 2489186A CA 2489186 C CA2489186 C CA 2489186C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/50—Organo-phosphines
- C07F9/5004—Acyclic saturated phosphines
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/50—Organo-phosphines
- C07F9/5045—Complexes or chelates of phosphines with metallic compounds or metals
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- 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
- C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
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- 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
Abstract
The present invention provides improved methods for synthesis of phosphinothiol reagents, as well as novel protected reagents, for use in formation of amide bonds, and particularly for peptide ligation. The invention provides phosphineborane complexes useful as reagents in the formation of amide bonds, particularly for the formation of an amide bond between any two of an amino acid, a peptide, or a protein.
Description
CHEMICAL SYNTHESIS OF REAGENTS FOR PEPTIDE COUPLING
BACKGROUND
The chemoselective ligation of peptides can be used to effect the total chemical synthesis of proteins.' The most common ligation method, native chemical ligation, relies on the presence of a cysteine residue at the N-terminus of each ligation junction .2,3 Recently, we have reported4 a peptide ligation method, a "Staudinger Ligation," that is universal, i.e., independent of the presence of any particular side chain This method is based on the Staudinger reaction, wherein a phosphine reduces an azide via a stable iminophosphorane intermediate.5 Acylation of this iminophosphorane yields an amide.b'' Scheme I (Figure 1) illustrates our Staudinger Ligation which is further described in PCT application PCT/US01/15440, filed May 11, 2000. A peptide fragment having a C-terminal phosphinothioester (2) reacts with another peptide fragment having an N-terminal azide (3). The resulting iminophosphorane (4) leads, after an S- to N-acyl shift, to an amidophosphonium salt (5). The P-N bond of the amidophosphonium salt is hydrolyzed readily to produce the amide product (6) and a phosphine oxide (7).
Importantly, no residual atoms remain in the amide product.4'6b, so the ligation is traceless. The phosphinothioester (2) is prepared by reaction of a of phosphinothiol reagent (1), such as Ph2CH2-SH, where Ph is a phenyl group. The Staudinger Ligation can generally be employed to form peptide bonds and as such can be employed to ligate two amino acids, a peptide or a protein with an amino acid or peptide or two proteins. More generally, the Staudinger Ligation can be employed to form amide bonds. The amide bond is formed between a thioester and an azide.
In general, the reaction functions for any thioester and any azide. The thioester is converted into a phosphinothioester which then reacts with the azide. For example, the thioester group may be formed, at the carboxy group of an amino acid or at the carboxy terminus of a peptide or protein or at an acid side group of an amino acid or one or more amino acids in a peptide or protein. The azido group may be formed, for example, at the amino group of an amino acid or at the amino terminus of a peptide or protein or at a basic side group of an amino acid or one or more amino acids in a peptide or protein. The Staudinger Ligation may also be employed to ligate an amino acid, peptide or protein group to a carbohydrate group, which may be a mono-, di-, tri- or polysaccharide, or to a nucleoside. The Staudinger Ligation may also be employed to ligate an amino acid, a peptide or protein group to a reporter group, tag or label (e.g., a group whose presence can be detected by optical or mass spectrometry or other instrumental method), including a fluorescent or phosphorescent group, an isotopic label or a radiolabel.
All natural a-amino acids except glycine have a stereogenic center at their a-carbon.8 To be an effective tool for the total chemical synthesis of proteins, a peptide ligation reaction must proceed without epimerization. The coupling of thioesters in native chemical ligation, which like the Staudinger Ligation (Scheme 1, Figure 1) involves transthioesterification followed by an S- to N-acyl shift," 3 is known to proceed without detectable racemization.9 We have demonstrated that the Staudinger Ligation (Scheme 1, Figure 1) proceeds in near quantitative yield without detectable epimerization.
The Staudinger Ligation of Scheme 1(Figure 1) employs a phosphinothiol reagent (1).
Previously reported methods of synthesis of such reagents4a' 4b generally proceed in low yield.
Synthesis of the phosphinothiol of formula 1 where R and R' are phenyl groups requires four synthetic steps, two of which are problematic, with an overall yield of about 39%.
Difficulties can also be encountered in the synthesis of reagents of formula 1 where R and R' are small alkyl groups, such as ethyl groups, due to instability of the reagent itself. Use of the Staudinger Ligation for the formation of amide bonds between a variety of species would be facilitated by the development of improved methods for the synthesis of phosphinothiol reagents and the development of such reagents with increased stability. This invention provides improvements for carrying out the Staudinger Ligation.
SUMMARY OF THE INVENTION
The present invention provides improved methods for synthesis of phosphinothiol reagents, as well as novel protected reagents, for use in formation of amide bonds, and particularly for peptide ligation as exemplified in Scheme I and Scheme 2 (Figures 1 and 2).
In a specific embodiment, the invention provides improved methods for synthesis of phosphinothiols, e.g., I (Scheme 1, Figure 1), which were the most effective known phosphinothiols for effecting the Staudinger Ligation of peptides. In one aspect, the invention provides a synthesis of phosphinothiols themselves. In another aspect, the invention provides a protected phosphinothiol reagent (10, Scheme 2, Figure 2) which are phosphine-borane complexes which can be employed in the Staudinger Ligation to prepare phosphinothioesters.
A phosphinothiol reagent of this invention (1) is synthesized as illustrated in generalized Scheme 3 (Figure 3) by reacting a protected alkylating agent of formula (20) whereRp is a protecting group, particularly an acyl group -COR' (defined below) and X is a leaving group (LG) with a phosphine-borane complex of formula (25, where R, R' and R" are defined as in formula 10, below) on deprotonation of the phosphine-borane complex to generate the protected phosphine-borane addition complex (10). The phosphinothiol reagent (1) is generated by disruption of the phosphine-borane complex (10) and removal of the protecting group (Rp).
In specific embodiments, the protecting group Rp is a -CO-R' where R5 is H, an alkyl group, an aryl group or a substituted alkyl or a substituted aryl group. In specific embodiments, R and R' are aryl groups, particularly phenyl groups. In specific embodiments, R2 and R3 are H or alkyl groups. In specific embodiments, R" are all hydrogen or are all small alkyl groups. In specific embodiments, X is a "good leaving group" as that term is understood in the art and specifically X can be a halogen, or a OTs, Otf, or OMs group.
Alternatively, the phosphine-borane complex of formula 10 can be used The invention also provides phosphine-borane complexes of formula:
R'RP+ XS-Rp B(R")3 10 where:
BACKGROUND
The chemoselective ligation of peptides can be used to effect the total chemical synthesis of proteins.' The most common ligation method, native chemical ligation, relies on the presence of a cysteine residue at the N-terminus of each ligation junction .2,3 Recently, we have reported4 a peptide ligation method, a "Staudinger Ligation," that is universal, i.e., independent of the presence of any particular side chain This method is based on the Staudinger reaction, wherein a phosphine reduces an azide via a stable iminophosphorane intermediate.5 Acylation of this iminophosphorane yields an amide.b'' Scheme I (Figure 1) illustrates our Staudinger Ligation which is further described in PCT application PCT/US01/15440, filed May 11, 2000. A peptide fragment having a C-terminal phosphinothioester (2) reacts with another peptide fragment having an N-terminal azide (3). The resulting iminophosphorane (4) leads, after an S- to N-acyl shift, to an amidophosphonium salt (5). The P-N bond of the amidophosphonium salt is hydrolyzed readily to produce the amide product (6) and a phosphine oxide (7).
Importantly, no residual atoms remain in the amide product.4'6b, so the ligation is traceless. The phosphinothioester (2) is prepared by reaction of a of phosphinothiol reagent (1), such as Ph2CH2-SH, where Ph is a phenyl group. The Staudinger Ligation can generally be employed to form peptide bonds and as such can be employed to ligate two amino acids, a peptide or a protein with an amino acid or peptide or two proteins. More generally, the Staudinger Ligation can be employed to form amide bonds. The amide bond is formed between a thioester and an azide.
In general, the reaction functions for any thioester and any azide. The thioester is converted into a phosphinothioester which then reacts with the azide. For example, the thioester group may be formed, at the carboxy group of an amino acid or at the carboxy terminus of a peptide or protein or at an acid side group of an amino acid or one or more amino acids in a peptide or protein. The azido group may be formed, for example, at the amino group of an amino acid or at the amino terminus of a peptide or protein or at a basic side group of an amino acid or one or more amino acids in a peptide or protein. The Staudinger Ligation may also be employed to ligate an amino acid, peptide or protein group to a carbohydrate group, which may be a mono-, di-, tri- or polysaccharide, or to a nucleoside. The Staudinger Ligation may also be employed to ligate an amino acid, a peptide or protein group to a reporter group, tag or label (e.g., a group whose presence can be detected by optical or mass spectrometry or other instrumental method), including a fluorescent or phosphorescent group, an isotopic label or a radiolabel.
All natural a-amino acids except glycine have a stereogenic center at their a-carbon.8 To be an effective tool for the total chemical synthesis of proteins, a peptide ligation reaction must proceed without epimerization. The coupling of thioesters in native chemical ligation, which like the Staudinger Ligation (Scheme 1, Figure 1) involves transthioesterification followed by an S- to N-acyl shift," 3 is known to proceed without detectable racemization.9 We have demonstrated that the Staudinger Ligation (Scheme 1, Figure 1) proceeds in near quantitative yield without detectable epimerization.
The Staudinger Ligation of Scheme 1(Figure 1) employs a phosphinothiol reagent (1).
Previously reported methods of synthesis of such reagents4a' 4b generally proceed in low yield.
Synthesis of the phosphinothiol of formula 1 where R and R' are phenyl groups requires four synthetic steps, two of which are problematic, with an overall yield of about 39%.
Difficulties can also be encountered in the synthesis of reagents of formula 1 where R and R' are small alkyl groups, such as ethyl groups, due to instability of the reagent itself. Use of the Staudinger Ligation for the formation of amide bonds between a variety of species would be facilitated by the development of improved methods for the synthesis of phosphinothiol reagents and the development of such reagents with increased stability. This invention provides improvements for carrying out the Staudinger Ligation.
SUMMARY OF THE INVENTION
The present invention provides improved methods for synthesis of phosphinothiol reagents, as well as novel protected reagents, for use in formation of amide bonds, and particularly for peptide ligation as exemplified in Scheme I and Scheme 2 (Figures 1 and 2).
In a specific embodiment, the invention provides improved methods for synthesis of phosphinothiols, e.g., I (Scheme 1, Figure 1), which were the most effective known phosphinothiols for effecting the Staudinger Ligation of peptides. In one aspect, the invention provides a synthesis of phosphinothiols themselves. In another aspect, the invention provides a protected phosphinothiol reagent (10, Scheme 2, Figure 2) which are phosphine-borane complexes which can be employed in the Staudinger Ligation to prepare phosphinothioesters.
A phosphinothiol reagent of this invention (1) is synthesized as illustrated in generalized Scheme 3 (Figure 3) by reacting a protected alkylating agent of formula (20) whereRp is a protecting group, particularly an acyl group -COR' (defined below) and X is a leaving group (LG) with a phosphine-borane complex of formula (25, where R, R' and R" are defined as in formula 10, below) on deprotonation of the phosphine-borane complex to generate the protected phosphine-borane addition complex (10). The phosphinothiol reagent (1) is generated by disruption of the phosphine-borane complex (10) and removal of the protecting group (Rp).
In specific embodiments, the protecting group Rp is a -CO-R' where R5 is H, an alkyl group, an aryl group or a substituted alkyl or a substituted aryl group. In specific embodiments, R and R' are aryl groups, particularly phenyl groups. In specific embodiments, R2 and R3 are H or alkyl groups. In specific embodiments, R" are all hydrogen or are all small alkyl groups. In specific embodiments, X is a "good leaving group" as that term is understood in the art and specifically X can be a halogen, or a OTs, Otf, or OMs group.
Alternatively, the phosphine-borane complex of formula 10 can be used The invention also provides phosphine-borane complexes of formula:
R'RP+ XS-Rp B(R")3 10 where:
Rp is a suitable protecting group, which can include, among others, -CO-R5 groups where R5 can be selected from H, alkyl or aryl groups or substituted alkyl or aryl groups, where the substituents do not affect the function of Rp as a protecting group for reactions illustrated herein;
R and R', independently of one another, are alkyl or aryl groups or substituted alkyl or aryl groups where the substituents do not significantly detrimentally affect the reactions as illustrated herein, R and R' may be the same or different groups, R and R' may be covalently linked to each other;
R", independently of other R" in the compound, can be H, an alkyl or aryl group or a substituted alkyl or aryl group where the substituents do not significantly negatively affect the formation of the phosphine-borane complex or significantly negatively affect the properties of B(R")3 as a protective groups for the phosphine; all three of R"
may be the same or each may be different, any two or three of R" may be covalently linked to each other; and R2 and R3, independently of one another, can be selected from H, an alkyl group, an aryl group or a substituted alkyl group or a substituted aryl group, where the substituents do not significantly negatively affect the function of the phosphine-borane complex in the Staudinger Ligation, particularly as illustrated in Scheme 2 (Figure 2); R2 and R3 may be covalently linked to each other.
In specific embodiments, the invention provides phosphine-borane complexes for use as peptide ligation reagents in which R and R' are alkyl groups, particularly ethyl groups, propyl groups or butyl groups, or aryl groups, particularly phenyl groups or substituted phenyl groups; R" are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R2 and R3 are H or small alkyl groups (e.g., methyl, ethyl, propyl, butyl groups) and Rp is a -CO-R5 group where R5 is H, and alkyl group or an ary l group.
In more specific embodiments, the invention provides phosphine-borane complexes of formula 10 in which R and R' are alkyl groups, particularly ethyl groups, propyl groups or butyl groups; R" are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R2 and R3 are H and Rp is a -CO-R5 group where R5 is H, an alkyl group or an aryl group.
In other specific embodiments, the invention provides phosphine-borane complexes of formula 10 in which R and R' are phenyl groups; R" are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R2 and R3 are H and Rp is a -CO-R5 groups where R5 is H, an alkyl group or an aryl group.
In another aspect, the invention provides phosphine-borane complexes of formula 12:
R and R', independently of one another, are alkyl or aryl groups or substituted alkyl or aryl groups where the substituents do not significantly detrimentally affect the reactions as illustrated herein, R and R' may be the same or different groups, R and R' may be covalently linked to each other;
R", independently of other R" in the compound, can be H, an alkyl or aryl group or a substituted alkyl or aryl group where the substituents do not significantly negatively affect the formation of the phosphine-borane complex or significantly negatively affect the properties of B(R")3 as a protective groups for the phosphine; all three of R"
may be the same or each may be different, any two or three of R" may be covalently linked to each other; and R2 and R3, independently of one another, can be selected from H, an alkyl group, an aryl group or a substituted alkyl group or a substituted aryl group, where the substituents do not significantly negatively affect the function of the phosphine-borane complex in the Staudinger Ligation, particularly as illustrated in Scheme 2 (Figure 2); R2 and R3 may be covalently linked to each other.
In specific embodiments, the invention provides phosphine-borane complexes for use as peptide ligation reagents in which R and R' are alkyl groups, particularly ethyl groups, propyl groups or butyl groups, or aryl groups, particularly phenyl groups or substituted phenyl groups; R" are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R2 and R3 are H or small alkyl groups (e.g., methyl, ethyl, propyl, butyl groups) and Rp is a -CO-R5 group where R5 is H, and alkyl group or an ary l group.
In more specific embodiments, the invention provides phosphine-borane complexes of formula 10 in which R and R' are alkyl groups, particularly ethyl groups, propyl groups or butyl groups; R" are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R2 and R3 are H and Rp is a -CO-R5 group where R5 is H, an alkyl group or an aryl group.
In other specific embodiments, the invention provides phosphine-borane complexes of formula 10 in which R and R' are phenyl groups; R" are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R2 and R3 are H and Rp is a -CO-R5 groups where R5 is H, an alkyl group or an aryl group.
In another aspect, the invention provides phosphine-borane complexes of formula 12:
p R2 R3 O
YO/BR'"3 AA S / P\
R R' where R, R', R", R2 and R3 are as defined above and AA is an amino acid, peptide or protein or a fully or partially protected derivative thereof. The AA group can be linked to the thio group of the complex of formula 12 by formation of a thioester at the COOH
group of the amino acid (i.e. RpNHC(RA)COOH --* RpNHC(RA)CO-S-C(RZR3)-O+PRR'(DBR"3, where Rp is an amine protecting group and RA is an amino acid side-group) , at the carboxyl terminus of a peptide or protein (i.e., RpNH-peptide-COOH --- RpNH-peptide-CO-S-C(R2R3)-OPRR'-eBR"3) or at a carboxyl group of an amino acid side group RA. Dependent upon where the thioester linkage is formed the AA group can be protected, if needed, with appropriate Rp groups at its COOH terminus or at a COOH group on an amino acid side-group.
The complex of formula 12 indicates formation of one thioester linkage, however, in cases in which AA is an amino acid with a carboxylate on RA or a peptide or protein containing one or more RA containing one or more carboxylates, multiple phosphine-borane complexes in which two or more carboxyl groups (most generally n) of the amino acid or peptide are ligated to the phosphine borane complex of formula 12 can be formed, as illustrated in formula 12d:
p R2 R3 AA-1~ X(D/ BR"3 S R/ P\R in 12d where n is the number of thioesters linkages in the complex, n can be 1, 2, 3, or more. AA
can be any naturally-occurring or synthetically prepared amino acid or any naturally-occurring or synthetically-prepared peptide, protein or protein fragment.
In a specific embodiment, AA is a naturally-occurring (D-, L-, achiral or racemic)amino acid and specifically can be selected from the group consisting of any one or more of (D-, L-, achiral or racemic) glycine, alanine, valine, leucine, isoleucine, phenylalanine, serine, methionine, proline, tyrosine, tryptophan, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, cysteine, methionine, hydroxyproline, y-carboxyglutamate, O-phosphoserine, ornithine, homoarginine and various protected derivatives thereof. Amino acid protecting groups can be selected from any of those known in the art including, but not limited to, Mtr, Pmc, Tos, Mts, Mbh, Tmob, Trt, Xan, tBu, Bzl, OcHEX, Acm, S-tBu, MeBzl, Mob, Bum, Drip, Bom, Z, CIZ, Boc, CHO or BrZ where conventional abbreviations have been employed to name protecting groups. Those of ordinary skill can select from among the known amino acid protecting groups including those specifically listed, a protecting groups appropriate for a given amino acid and a given moiety within a given amino acid and for a group that is chemically compatible for use in the reactions of this invention.
In additional specific embodiments, the invention provides phosphine-borane complexes of formula 12d where R and R' are alkyl groups or aryl groups which may optionally be substituted and particularly those in which R and Ware both ethyl groups. In other specific embodiments, the invention provides phosphine-borane complexes of formula 12d where R2, R3 are H. In other specific embodiments, the invention provides phosphine-borane complexes of formula 12d where R" are all H or all small alkyl, e.g., methyl or ethyl.
In other specific embodiments, the invention provides phosphine-borane complexes of formula 10 in which R and R' are alkyl groups or phenyl groups; R" are all H;
R2 and R3 are H and-Rp is a -CO-R5 groups where R5 is H, an alkyl group or an aryl group.
The invention further provides kits for the ligation of amino acids, peptides or proteins which comprise one or more phosphine-borane complexes of formula 10, 12 in combination with instructions for carrying out a Staudinger Ligation as illustrated in Scheme 2 or more generally for formation of an amide bond between a thioester and an azide. The phosphine-borane reagents of formula 10, 12 can be provided in one or more suitable containers or receptacles in the kit and may be pre-weighed to provide sufficient reagent for conducting a ligation reaction on a selection scale for a selected amount of starting amino acids, peptide or proteins to be ligated. The kits may additional contain one or more protected amino acid starting materials or other starting materials for ligation. The kit may further contain one or more solvents for conducting the reaction, a deprotecting agent for deprotecting the phosphine-borane complex or other useful reagents or materials useful in the purification of starting materials for ligation or end-products of ligation.
YO/BR'"3 AA S / P\
R R' where R, R', R", R2 and R3 are as defined above and AA is an amino acid, peptide or protein or a fully or partially protected derivative thereof. The AA group can be linked to the thio group of the complex of formula 12 by formation of a thioester at the COOH
group of the amino acid (i.e. RpNHC(RA)COOH --* RpNHC(RA)CO-S-C(RZR3)-O+PRR'(DBR"3, where Rp is an amine protecting group and RA is an amino acid side-group) , at the carboxyl terminus of a peptide or protein (i.e., RpNH-peptide-COOH --- RpNH-peptide-CO-S-C(R2R3)-OPRR'-eBR"3) or at a carboxyl group of an amino acid side group RA. Dependent upon where the thioester linkage is formed the AA group can be protected, if needed, with appropriate Rp groups at its COOH terminus or at a COOH group on an amino acid side-group.
The complex of formula 12 indicates formation of one thioester linkage, however, in cases in which AA is an amino acid with a carboxylate on RA or a peptide or protein containing one or more RA containing one or more carboxylates, multiple phosphine-borane complexes in which two or more carboxyl groups (most generally n) of the amino acid or peptide are ligated to the phosphine borane complex of formula 12 can be formed, as illustrated in formula 12d:
p R2 R3 AA-1~ X(D/ BR"3 S R/ P\R in 12d where n is the number of thioesters linkages in the complex, n can be 1, 2, 3, or more. AA
can be any naturally-occurring or synthetically prepared amino acid or any naturally-occurring or synthetically-prepared peptide, protein or protein fragment.
In a specific embodiment, AA is a naturally-occurring (D-, L-, achiral or racemic)amino acid and specifically can be selected from the group consisting of any one or more of (D-, L-, achiral or racemic) glycine, alanine, valine, leucine, isoleucine, phenylalanine, serine, methionine, proline, tyrosine, tryptophan, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, cysteine, methionine, hydroxyproline, y-carboxyglutamate, O-phosphoserine, ornithine, homoarginine and various protected derivatives thereof. Amino acid protecting groups can be selected from any of those known in the art including, but not limited to, Mtr, Pmc, Tos, Mts, Mbh, Tmob, Trt, Xan, tBu, Bzl, OcHEX, Acm, S-tBu, MeBzl, Mob, Bum, Drip, Bom, Z, CIZ, Boc, CHO or BrZ where conventional abbreviations have been employed to name protecting groups. Those of ordinary skill can select from among the known amino acid protecting groups including those specifically listed, a protecting groups appropriate for a given amino acid and a given moiety within a given amino acid and for a group that is chemically compatible for use in the reactions of this invention.
In additional specific embodiments, the invention provides phosphine-borane complexes of formula 12d where R and R' are alkyl groups or aryl groups which may optionally be substituted and particularly those in which R and Ware both ethyl groups. In other specific embodiments, the invention provides phosphine-borane complexes of formula 12d where R2, R3 are H. In other specific embodiments, the invention provides phosphine-borane complexes of formula 12d where R" are all H or all small alkyl, e.g., methyl or ethyl.
In other specific embodiments, the invention provides phosphine-borane complexes of formula 10 in which R and R' are alkyl groups or phenyl groups; R" are all H;
R2 and R3 are H and-Rp is a -CO-R5 groups where R5 is H, an alkyl group or an aryl group.
The invention further provides kits for the ligation of amino acids, peptides or proteins which comprise one or more phosphine-borane complexes of formula 10, 12 in combination with instructions for carrying out a Staudinger Ligation as illustrated in Scheme 2 or more generally for formation of an amide bond between a thioester and an azide. The phosphine-borane reagents of formula 10, 12 can be provided in one or more suitable containers or receptacles in the kit and may be pre-weighed to provide sufficient reagent for conducting a ligation reaction on a selection scale for a selected amount of starting amino acids, peptide or proteins to be ligated. The kits may additional contain one or more protected amino acid starting materials or other starting materials for ligation. The kit may further contain one or more solvents for conducting the reaction, a deprotecting agent for deprotecting the phosphine-borane complex or other useful reagents or materials useful in the purification of starting materials for ligation or end-products of ligation.
The Staudinger Ligation can be employed generally to form amide bonds. The amide bond is formed between a thioester and an azide and the reaction most generally functions, for any thioester and any azide. In the reaction, the thioester group may be formed, at the carboxy group of an amino acid or at the carboxy terminus of a peptide or protein or at an acid side group of an amino acid or one or more amino acids in a peptide or protein. The azido group may be formed, for example, at the amino group of an amino acid or at the amino terminus of a peptide or protein or at a basic side group of an amino acid or one or more amino acids in a peptide or protein. The reagents of this invention may also be employed to ligate an amino acid, peptide or protein group to a carbohydrate group, which may be a mono-, di-, tri- or polysaccharide, or to a nucleoside. The reagents of this invention may also be employed to ligate an amino acid, a peptide or protein group to a reporter group, tag or label (e.g., a group whose presence can be detected by optical or mass spectrometry or other instrumental method), including a fluorescent or phosphorescent group, an isotopic label or a radiolabel. The invention provides kits comprising one or more phosphine-borane complexes of formula 10, 12 for formation of an amide bond and more specifically for ligation of an amino acid, peptide or protein to a carbohydrate, a nucleoside or to a reporter group, tag or label.
This invention also provides an improved method for forming an amide bond by Staudinger Ligation which employees a phosphine-borane reagent of formula 10, 12.
DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates Scheme 1 the Staudinger ligation described in PCT
application PCT/USOI/15440, ftled May 11, 2000.
Figure 2 illustrates Schemes 2 and 2A. Scheme 2 shows the use of complexes of formula 10 to form amide bonds on reaction with azides. Scheme 2A shows disruption of the phosphine-borane complex 12.
Figure 3 illustrates Schemes 3A and 313. Scheme 3A shows a generalized method for synthesis of a phosphinothiol reagent 1 of this invention. Scheme 3B shows the synthesis of a specific phosphinothiol 1b.
Figure 4 illustrates Scheme 4A. Scheme 4A shows two related generalized methods for synthesis of protected reagents 10.
Figure 5 illustrates Scheme 4B. Scheme 4B shows synthesis of a specific phosphine-borane complex 10c.
This invention also provides an improved method for forming an amide bond by Staudinger Ligation which employees a phosphine-borane reagent of formula 10, 12.
DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates Scheme 1 the Staudinger ligation described in PCT
application PCT/USOI/15440, ftled May 11, 2000.
Figure 2 illustrates Schemes 2 and 2A. Scheme 2 shows the use of complexes of formula 10 to form amide bonds on reaction with azides. Scheme 2A shows disruption of the phosphine-borane complex 12.
Figure 3 illustrates Schemes 3A and 313. Scheme 3A shows a generalized method for synthesis of a phosphinothiol reagent 1 of this invention. Scheme 3B shows the synthesis of a specific phosphinothiol 1b.
Figure 4 illustrates Scheme 4A. Scheme 4A shows two related generalized methods for synthesis of protected reagents 10.
Figure 5 illustrates Scheme 4B. Scheme 4B shows synthesis of a specific phosphine-borane complex 10c.
Figure 6 illustrates Scheme 5. Scheme 5 shows the preparation of several non-glycyl a-azido acids used to examine epimerization during Staudinger ligation.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates generally to improved methods for forming amide bonds and more specifically to improved synthetic methods for forming reagents useful in forming amide bonds and improved reagents for forming amide bonds.
The following terms are defined for use herein:
Alkyl groups refer to saturated hydrocarbon groups which may be linear, branched or cyclic. Small alkyl groups are those having from one to six carbon atoms.
Alkyl groups may be substituted so long as the substituents do not significantly detrimentally affect the function of the compound or portion of the compound in which it is found.
Aryl groups refer to groups which contain at least one aromatic ring which can be a five-member or a six-member ring. The one or more rings of an aryl group can include fused rings. Aryl groups may be substituted with one or more alkyl groups which may be linear, branched or cyclic. Aryl groups may also be substituted at ring positions with substituents that do not significantly detrimentally affect the function of the compound or portion of the compound in which it is found. Substituted aryl groups also include those having heterocyclic aromatic rings in which one or more heteroatoms (e.g., N, 0 or S, optionally with hydrogens or substituents for proper valence) replace one or more carbons in the ring.
Scheme 3A (Figure 3) provides a generalized method for synthesis of a phosphinothiol reagent (1) of this invention. The synthesis is based on the reaction of an alkylating agent 20 and a borane-organophosphine complex 25. The phosphine of the phosphine-borane complex 25 is deprotonated with base followed by alkylation with 20 to give phosphine-borane complex 10. Phosphine-borane complexes 10 are stable to air and moisture and can be stored at room temperature for months without any sign of oxidation or decomposition.
The borane complex 10 is disrupted by mild heating, in the presence of an amine, preferably with DABCO in toluene for 4 hr to generate a protected phosphinothiol 11 (Rp in this case should be resistant to deprotection under the conditions of complex disruption Ilia).
A preferred protecting group is an -CO-RS group, particularly an acyl group, which can be removed as previously described4b to give the phosphinothiol 1. The use of and methods for removal of other suitable protecting groups is known in the art.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates generally to improved methods for forming amide bonds and more specifically to improved synthetic methods for forming reagents useful in forming amide bonds and improved reagents for forming amide bonds.
The following terms are defined for use herein:
Alkyl groups refer to saturated hydrocarbon groups which may be linear, branched or cyclic. Small alkyl groups are those having from one to six carbon atoms.
Alkyl groups may be substituted so long as the substituents do not significantly detrimentally affect the function of the compound or portion of the compound in which it is found.
Aryl groups refer to groups which contain at least one aromatic ring which can be a five-member or a six-member ring. The one or more rings of an aryl group can include fused rings. Aryl groups may be substituted with one or more alkyl groups which may be linear, branched or cyclic. Aryl groups may also be substituted at ring positions with substituents that do not significantly detrimentally affect the function of the compound or portion of the compound in which it is found. Substituted aryl groups also include those having heterocyclic aromatic rings in which one or more heteroatoms (e.g., N, 0 or S, optionally with hydrogens or substituents for proper valence) replace one or more carbons in the ring.
Scheme 3A (Figure 3) provides a generalized method for synthesis of a phosphinothiol reagent (1) of this invention. The synthesis is based on the reaction of an alkylating agent 20 and a borane-organophosphine complex 25. The phosphine of the phosphine-borane complex 25 is deprotonated with base followed by alkylation with 20 to give phosphine-borane complex 10. Phosphine-borane complexes 10 are stable to air and moisture and can be stored at room temperature for months without any sign of oxidation or decomposition.
The borane complex 10 is disrupted by mild heating, in the presence of an amine, preferably with DABCO in toluene for 4 hr to generate a protected phosphinothiol 11 (Rp in this case should be resistant to deprotection under the conditions of complex disruption Ilia).
A preferred protecting group is an -CO-RS group, particularly an acyl group, which can be removed as previously described4b to give the phosphinothiol 1. The use of and methods for removal of other suitable protecting groups is known in the art.
Useful bases for deprotonation of the phosphine-borane complex 25 in the first step of the synthesis of Scheme 3A (Figure 3) are NaH, LiH, KH, KOtBu, NaOMe, NaOEt and the use of NaH is preferred. Amine bases are not preferred, as they can remove the protecting group. While DMF is a preferred solvent for this first step, other useful solvents include THF, toluene, DMA and more generally any solvent that will dissolve the various components and not significantly detrimentally affect the desired reaction.
Solvents including thiols, thioethers, and amines should not be used.
The preferred reagent for disrupting the borane complex 10 is DABCO (1,4-diazabicyclo[2.2.2]octane), but primary, secondary, tertiary or aromatic amines, thiols or thioethers can also be used in this step. Amines such as pyridine, N,N,N , N-tetra-methylethylenediamine, diethylamine, triethylenediamine, or dimethylsulfide can specifically be used. The preferred solvent for this step is toluene, but benzene, THF, or any solvent that will dissolve the reagents can be used.
The protecting group of the acyl phosphinothiol can be removed by reaction with base in alcohol. Preferred deprotection agent is I eq. NaOH in MeOH. Other reagents include excess NaOH, (I equivalent or excess) LiOH, KOH, NH3, NHZOH NaHCO3 in H20/THF, LiAIH4 in ether, AgNO3 in MeOH or a lipase enzyme. Methanol is the preferred solvent, but other alcohols (including EtOH, iPrOH) or H2O can be employed. Oxygen gas should be removed from the solvent to prevent oxidation of the phosphene to a phosphene oxide.
DMSO should be avoided as a solvent in this step.
The protecting group of the acyl phosphinothiol can be removed by reaction with base in alcohol. Preferred deprotection agent is I eq. NaOH in MeOH. Other reagents include excess NaOH, (1 equivalent or excess) LiOH, KOH, NH3 or NH2OH. Methanol is the preferred solvent, but other alcohols (including EtOH, iPrOH) or H2O can be employed. 02 (g) should be removed from the solvent to prevent oxidation of phosphene to a phosphene oxide. DMSO should be avoided as a solvent in this step.
Scheme 3B illustrates the synthesis of a specific phosphinothiol lb where R
and R' are both phenyl groups and R2 and R3 are both hydrogens. The illustrated synthesis of Scheme 3B gave an overall yield of about 74%. See the Examples for experimental details.
With respect to starting reagents in Scheme 3A, R and R' can generally be any organic moiety (including alkyl and aryl) that does not contain an amine, thiol or thioether. The Rand R groups can be linked to P via a C-P or O-P bond. More specifically, Rand R are optionally substituted alkyl group alkoxide group, aryl group or aryloxy group. Alkyl groups include straight-chain, branched or cyclic alkyl groups. Aryl groups may contain one or more (preferably one or two) aromatic rings which may be carbocyclic or heterocylic rings.
Preferred alkyl groups are optionally substituted ethyl groups. Preferred alkoxy groups are ethoxy groups. Preferred aryl groups are optionally substituted phenyl groups including phenyl groups and halogen (particularly fluorine)-substituted or carboxy-substituted phenyl groups.
Various known protecting groups (Rp) can be employed in the starting reagents of Scheme 3A (Figure3). One or ordinary skill in the art in view of the teachings herein and what is well-known in the art can selected appropriate protecting groups from those available in the art. Preferred protecting groups are -CO-R5 groups where R5 is hydrogen, alkyl, aryl or substituted alkyl or substituted aryl groups (R5 can specifically be hydrogen, methyl, ethyl or other small alkyl group, a -CH2-Ph group (Ph = phenyl), a -CH2-Ph(Y)n group where Y is a substituent and n is the number of substituents (Y can, for example, be a halogen, including fluorine, or -OR7 where R7 is an optionally substituted alkyl or aryl group.) In all cases, optional substituents include halogens and alkoxy groups and for appropriate groups can be alkyl, and or aryl substituents. Optional substituents do not include amines, thiols or thioester groups.
In Scheme 3A (Figure 3) in the thioester reagent is a "good leaving group", as that term is generally known and accepted in the art, that does not include an amine, a thiol or a thioether group. Preferred X are halogens (Br, Cl or 1), OTs (tosyl, CH3C6H4SO2-), OTf (triflate, CF3SO2-), or OMs (mesyl, CH3SO2-). The most preferred X is Br.
The leaving group X is preferably separated from S by a -CR2R3- group, e.g., -where R2 and R3are both hydrogens, as illustrated in Schemes 3A and 3B (Figure 3).
However, the linker to S can also be -CH2-CH2- or an o-substituted Phe group as in R2 R3 X S-Rp YX S'_1 or X Rp Ar where R2 and R3 groups, independent of other R2 and R3 groups in the same molecule, are as defined above and where Ar is an optionally substituted aryl group which may contain one or more aromatic rings. Rp is preferably an acyl group, e.g., a -COR5 group as defined above.
Starting materials and reagents for the reactions of Scheme 3A (Figure 3) are readily available either from commercial sources, by use of known synthetic methods or by routine adaptation of known synthetic methods.
Scheme 4A (Figure 4) provides two related generalized methods for synthesis of protected reagents 10 of this invention. The synthesis is based on the addition of an aldehyde or ketone (31) to a phosphine-borane complex (not specifically shown) to form the phosphine-borane complex alcohol 35. The phosphine of the initial phosphine-borane complex formed is deprotonated with base followed by addition of the aldehyde or ketone to give the derivatized alcohol phosphine-borane complex 35. The alcohol -containing phosphine-borane complex (35) is activated by introduction of an activating group A by reaction with AX
(37). The alcohol is activated for reaction with a protected thiol (39), such as an acyl thiol, particularly thioacetic acid to form the phosphine-borane complex 10. Complexes 10 are stable to air and moisture and can be stored at room temperature for months without any sign of oxidation or decomposition.
Alternatively, reaction IVB can be used to directly prepare complex 10 by replacing aldhyde or ketone 31 with the sulfur analogs 32.
The borane complex 10 can be disrupted by mild heating, in the presence of an amine, preferably with DABCO in toluene to generate a protected phosphinothiol 11 (Rp in this case should be resistant to deprotection under the conditions of complex disruption). A preferred protecting group is an acyl group, e.g., a -CO-R5 group, particularly an acetyl group, which can be removed as described in references 4a and b to give a phosphinothiol of formula 1 which can be employed as illustrated in Scheme 3A in the Staudinger Ligation to form a thioester. The use of and methods for removal of other suitable protecting groups is known in the art.
However, rather than generating the phosphinothiol 1, complex 10 can be used to generate derivatized amino acid, peptide or protein reagents of formula 12, which can be employed in the Staudinger Ligation as illustrated in Scheme 2 (Figure 2). The complex of formula 10 is coupled with an amino acid, peptide or protein that is activated by incorporation of a good leaving group LG. The amino acid, peptide or protein of the complex of formula 12 is provided with appropriate protecting groups to allow the reaction to proceed as indicated in the first reaction of Scheme 2 (Figure 2). It has been found that derivatized amino acid, peptide or protein complexes of formula 12, on disruption of the phosphine-borane complex, react with azides as indicated in Scheme 2 (Figure 2). Scheme 2 (Figure 2) illustrates the Staudinger Ligation to form an amide bond between two peptides. Complexes of formula 12d can also be employed to form amide bonds between any two of an amino acid, peptide or protein. Complexes of formula 12d can further be employed to form an amide bond between an amino acid, a peptide or a protein and a carbohydrate, a nucleoside or a suitable reporter, tag or label.
The method of Scheme 2 (Figure 2) is preferred for use with phosphinothiols that are unstable, for example those in which R and R' are ethyl groups.
Amine bases are not preferred, for use in the reactions of Schemes 2 (Figure 2) and 4A (Figure 4), unless otherwise stated, as they can remove the BH3 protecting group. In additional to the preferred base KOH, for formation of complexes 35 and 36, other bases including NaH, LiH, KH, KOtBu, NaOMe, and NaOEt can be used. While THF is a preferred solvent for this reaction, other useful solvents include THF, toluene, DMA and more generally any solvent that will dissolve the various components and not significantly detrimentally affect the desired reaction. Solvents including thiols, thioethers, and amines should not be used in the reactions of Schemes 2 (Figure 2) and 4A (Figure 4), unless otherwise indicated.
The preferred reagent for disrupting the phosphine-borane complexes is DABCO
(1,4-diazabicyclo[2.2.2]octane), but primary, secondary, tertiary or aromatic amines, thiols or thioethers can also be used in this step. Amines such as pyridine, N,N,N , N -tetra-methylethylenediamine, diethylamine, triethylenediamine, or dimethylsulfide can specifically be used. The preferred solvent for this step is toluene, but benzene, THF, or any solvent that will dissolve the reagents can be used.
The protecting group of the protected complex 10 can be removed, if desired, for example by reaction with base in alcohol. Other reagents include excess NaOH, (1 equivalent or excess) LiOH, KOH, NH3, NH20H NaHCO3 in H20/THF, LiAIH4 in ether, AgNO3 in McOH or a lipase enzyme. Oxygen gas should be removed from the solvent to prevent oxidation of the phosphene to a phosphene oxide.
Scheme 4B (Figure 5) illustrates the synthesis of a specific phosphine-borane complex 10c where R and R' are both ethyl groups and R2 and R3 are both hydrogens.
As noted above complexes of formula 12 and 12d can be employed as illustrated, for example, in Scheme 2 (Figure 2) to form amide bonds between amino acids, peptides or proteins or between an amino acid and another species, such as a carbohydrate (e.g., a saccharide) a nucleoside or simply to an appropriate reporter group, tag or label. If desired, the complex 12 or 12d can be disrupted as illustrated in Scheme 2 (page 2) employing DABCO or other amine.
Starting materials and reagents for the reactions of Scheme 4A are readily available either from commercial sources, by known synthetic methods or routine adaptation of known synthetic methods.
Several non-glycyl a-azido acids were prepared to examine epimerization during the Staudinger ligation. The azido benzamides of both the D and L enantiomers of phenylalanine, serine, and aspartic acid were prepared (Scheme 5, Figure 6).
The azido group was prepared by diazo transfer;10 the benazmide was prepared by DCC/HOBt coupling with benzyl amine. Phenylalanine, aspartic acid, and serine were chosen as being representative of three distinct side chains and moderate (phenylalanine) to high (aspartate and serine) propensity to epimerize during standard peptide couplings.16 Each of these azido acids was coupled with phosphinothioester 51 (which is AcGIySCH2PPh2;Table 1). The couplings were carried out in THF/H20 (3:1) for 12 hat room temperature with a 1:1 stoichiometry of starting materials. The resulting peptides were purified by flash chromatography to give a nearly quantitative yield of each product (Table 1). The high yield of this equimolar reaction of phosphinothiol 1 with non-glycyl azides is consistent with those observed previously4b. The preparation of phosphinothioester 51 was modified from that described in references 4a and b in which coupling using DCC alone led to lower yields and several undesired side products. Pretreatment of N-acetyl glycine with HOBt and DCC followed by addition of the phosphinothiol 1 improved the yield dramatically. See the Examples.
The chirality of the Staudinger Ligation products from the reaction of the D
and L a-azido acids was analyzed by HPLC using a D-phenylglycine chiral column. The chromatographic conditions enabled the baseline resolution of the two possible enantiomeric products (Figure 1). Materials to be analyzed were injected onto a D-phenylglycine analytical HPLC column and eluted with 30% (v/v) isopropanol in hexanes (isocraticO for 20 min followed by a shallow gradient to 50% (v/v) isopropanol for 40 min. After reaction of the D epimer, there was no evidence of product containing the L epimer, and vice versa.
Thus, the Staudinger Ligation proceeds without detectable epimerization of the a-carbon of the azido acid. The detection limit of the HPLC chromatographic analysis used is estimated to be _<0.5%, so that the Staudinger Ligation proceeds with >_99.5% retention of chirality.
Those or ordinary skill in the art will appreciate that starting materials, reagents, solvents, temperature and other reaction conditions other than those specifically disclosed, can be employed in the practice of this invention without resort to undue experimentation.
All such art-recognized equivalents are included to be encompassed by this invention. In particular, published PCT application WO 01 /87920 is cited herein to provide details of the Stauding Ligation and method for amide bond formation using the phosphinothiol reagents (1) and the phosphine-borane complexes 10, 12 and 12d.
Solvents including thiols, thioethers, and amines should not be used.
The preferred reagent for disrupting the borane complex 10 is DABCO (1,4-diazabicyclo[2.2.2]octane), but primary, secondary, tertiary or aromatic amines, thiols or thioethers can also be used in this step. Amines such as pyridine, N,N,N , N-tetra-methylethylenediamine, diethylamine, triethylenediamine, or dimethylsulfide can specifically be used. The preferred solvent for this step is toluene, but benzene, THF, or any solvent that will dissolve the reagents can be used.
The protecting group of the acyl phosphinothiol can be removed by reaction with base in alcohol. Preferred deprotection agent is I eq. NaOH in MeOH. Other reagents include excess NaOH, (I equivalent or excess) LiOH, KOH, NH3, NHZOH NaHCO3 in H20/THF, LiAIH4 in ether, AgNO3 in MeOH or a lipase enzyme. Methanol is the preferred solvent, but other alcohols (including EtOH, iPrOH) or H2O can be employed. Oxygen gas should be removed from the solvent to prevent oxidation of the phosphene to a phosphene oxide.
DMSO should be avoided as a solvent in this step.
The protecting group of the acyl phosphinothiol can be removed by reaction with base in alcohol. Preferred deprotection agent is I eq. NaOH in MeOH. Other reagents include excess NaOH, (1 equivalent or excess) LiOH, KOH, NH3 or NH2OH. Methanol is the preferred solvent, but other alcohols (including EtOH, iPrOH) or H2O can be employed. 02 (g) should be removed from the solvent to prevent oxidation of phosphene to a phosphene oxide. DMSO should be avoided as a solvent in this step.
Scheme 3B illustrates the synthesis of a specific phosphinothiol lb where R
and R' are both phenyl groups and R2 and R3 are both hydrogens. The illustrated synthesis of Scheme 3B gave an overall yield of about 74%. See the Examples for experimental details.
With respect to starting reagents in Scheme 3A, R and R' can generally be any organic moiety (including alkyl and aryl) that does not contain an amine, thiol or thioether. The Rand R groups can be linked to P via a C-P or O-P bond. More specifically, Rand R are optionally substituted alkyl group alkoxide group, aryl group or aryloxy group. Alkyl groups include straight-chain, branched or cyclic alkyl groups. Aryl groups may contain one or more (preferably one or two) aromatic rings which may be carbocyclic or heterocylic rings.
Preferred alkyl groups are optionally substituted ethyl groups. Preferred alkoxy groups are ethoxy groups. Preferred aryl groups are optionally substituted phenyl groups including phenyl groups and halogen (particularly fluorine)-substituted or carboxy-substituted phenyl groups.
Various known protecting groups (Rp) can be employed in the starting reagents of Scheme 3A (Figure3). One or ordinary skill in the art in view of the teachings herein and what is well-known in the art can selected appropriate protecting groups from those available in the art. Preferred protecting groups are -CO-R5 groups where R5 is hydrogen, alkyl, aryl or substituted alkyl or substituted aryl groups (R5 can specifically be hydrogen, methyl, ethyl or other small alkyl group, a -CH2-Ph group (Ph = phenyl), a -CH2-Ph(Y)n group where Y is a substituent and n is the number of substituents (Y can, for example, be a halogen, including fluorine, or -OR7 where R7 is an optionally substituted alkyl or aryl group.) In all cases, optional substituents include halogens and alkoxy groups and for appropriate groups can be alkyl, and or aryl substituents. Optional substituents do not include amines, thiols or thioester groups.
In Scheme 3A (Figure 3) in the thioester reagent is a "good leaving group", as that term is generally known and accepted in the art, that does not include an amine, a thiol or a thioether group. Preferred X are halogens (Br, Cl or 1), OTs (tosyl, CH3C6H4SO2-), OTf (triflate, CF3SO2-), or OMs (mesyl, CH3SO2-). The most preferred X is Br.
The leaving group X is preferably separated from S by a -CR2R3- group, e.g., -where R2 and R3are both hydrogens, as illustrated in Schemes 3A and 3B (Figure 3).
However, the linker to S can also be -CH2-CH2- or an o-substituted Phe group as in R2 R3 X S-Rp YX S'_1 or X Rp Ar where R2 and R3 groups, independent of other R2 and R3 groups in the same molecule, are as defined above and where Ar is an optionally substituted aryl group which may contain one or more aromatic rings. Rp is preferably an acyl group, e.g., a -COR5 group as defined above.
Starting materials and reagents for the reactions of Scheme 3A (Figure 3) are readily available either from commercial sources, by use of known synthetic methods or by routine adaptation of known synthetic methods.
Scheme 4A (Figure 4) provides two related generalized methods for synthesis of protected reagents 10 of this invention. The synthesis is based on the addition of an aldehyde or ketone (31) to a phosphine-borane complex (not specifically shown) to form the phosphine-borane complex alcohol 35. The phosphine of the initial phosphine-borane complex formed is deprotonated with base followed by addition of the aldehyde or ketone to give the derivatized alcohol phosphine-borane complex 35. The alcohol -containing phosphine-borane complex (35) is activated by introduction of an activating group A by reaction with AX
(37). The alcohol is activated for reaction with a protected thiol (39), such as an acyl thiol, particularly thioacetic acid to form the phosphine-borane complex 10. Complexes 10 are stable to air and moisture and can be stored at room temperature for months without any sign of oxidation or decomposition.
Alternatively, reaction IVB can be used to directly prepare complex 10 by replacing aldhyde or ketone 31 with the sulfur analogs 32.
The borane complex 10 can be disrupted by mild heating, in the presence of an amine, preferably with DABCO in toluene to generate a protected phosphinothiol 11 (Rp in this case should be resistant to deprotection under the conditions of complex disruption). A preferred protecting group is an acyl group, e.g., a -CO-R5 group, particularly an acetyl group, which can be removed as described in references 4a and b to give a phosphinothiol of formula 1 which can be employed as illustrated in Scheme 3A in the Staudinger Ligation to form a thioester. The use of and methods for removal of other suitable protecting groups is known in the art.
However, rather than generating the phosphinothiol 1, complex 10 can be used to generate derivatized amino acid, peptide or protein reagents of formula 12, which can be employed in the Staudinger Ligation as illustrated in Scheme 2 (Figure 2). The complex of formula 10 is coupled with an amino acid, peptide or protein that is activated by incorporation of a good leaving group LG. The amino acid, peptide or protein of the complex of formula 12 is provided with appropriate protecting groups to allow the reaction to proceed as indicated in the first reaction of Scheme 2 (Figure 2). It has been found that derivatized amino acid, peptide or protein complexes of formula 12, on disruption of the phosphine-borane complex, react with azides as indicated in Scheme 2 (Figure 2). Scheme 2 (Figure 2) illustrates the Staudinger Ligation to form an amide bond between two peptides. Complexes of formula 12d can also be employed to form amide bonds between any two of an amino acid, peptide or protein. Complexes of formula 12d can further be employed to form an amide bond between an amino acid, a peptide or a protein and a carbohydrate, a nucleoside or a suitable reporter, tag or label.
The method of Scheme 2 (Figure 2) is preferred for use with phosphinothiols that are unstable, for example those in which R and R' are ethyl groups.
Amine bases are not preferred, for use in the reactions of Schemes 2 (Figure 2) and 4A (Figure 4), unless otherwise stated, as they can remove the BH3 protecting group. In additional to the preferred base KOH, for formation of complexes 35 and 36, other bases including NaH, LiH, KH, KOtBu, NaOMe, and NaOEt can be used. While THF is a preferred solvent for this reaction, other useful solvents include THF, toluene, DMA and more generally any solvent that will dissolve the various components and not significantly detrimentally affect the desired reaction. Solvents including thiols, thioethers, and amines should not be used in the reactions of Schemes 2 (Figure 2) and 4A (Figure 4), unless otherwise indicated.
The preferred reagent for disrupting the phosphine-borane complexes is DABCO
(1,4-diazabicyclo[2.2.2]octane), but primary, secondary, tertiary or aromatic amines, thiols or thioethers can also be used in this step. Amines such as pyridine, N,N,N , N -tetra-methylethylenediamine, diethylamine, triethylenediamine, or dimethylsulfide can specifically be used. The preferred solvent for this step is toluene, but benzene, THF, or any solvent that will dissolve the reagents can be used.
The protecting group of the protected complex 10 can be removed, if desired, for example by reaction with base in alcohol. Other reagents include excess NaOH, (1 equivalent or excess) LiOH, KOH, NH3, NH20H NaHCO3 in H20/THF, LiAIH4 in ether, AgNO3 in McOH or a lipase enzyme. Oxygen gas should be removed from the solvent to prevent oxidation of the phosphene to a phosphene oxide.
Scheme 4B (Figure 5) illustrates the synthesis of a specific phosphine-borane complex 10c where R and R' are both ethyl groups and R2 and R3 are both hydrogens.
As noted above complexes of formula 12 and 12d can be employed as illustrated, for example, in Scheme 2 (Figure 2) to form amide bonds between amino acids, peptides or proteins or between an amino acid and another species, such as a carbohydrate (e.g., a saccharide) a nucleoside or simply to an appropriate reporter group, tag or label. If desired, the complex 12 or 12d can be disrupted as illustrated in Scheme 2 (page 2) employing DABCO or other amine.
Starting materials and reagents for the reactions of Scheme 4A are readily available either from commercial sources, by known synthetic methods or routine adaptation of known synthetic methods.
Several non-glycyl a-azido acids were prepared to examine epimerization during the Staudinger ligation. The azido benzamides of both the D and L enantiomers of phenylalanine, serine, and aspartic acid were prepared (Scheme 5, Figure 6).
The azido group was prepared by diazo transfer;10 the benazmide was prepared by DCC/HOBt coupling with benzyl amine. Phenylalanine, aspartic acid, and serine were chosen as being representative of three distinct side chains and moderate (phenylalanine) to high (aspartate and serine) propensity to epimerize during standard peptide couplings.16 Each of these azido acids was coupled with phosphinothioester 51 (which is AcGIySCH2PPh2;Table 1). The couplings were carried out in THF/H20 (3:1) for 12 hat room temperature with a 1:1 stoichiometry of starting materials. The resulting peptides were purified by flash chromatography to give a nearly quantitative yield of each product (Table 1). The high yield of this equimolar reaction of phosphinothiol 1 with non-glycyl azides is consistent with those observed previously4b. The preparation of phosphinothioester 51 was modified from that described in references 4a and b in which coupling using DCC alone led to lower yields and several undesired side products. Pretreatment of N-acetyl glycine with HOBt and DCC followed by addition of the phosphinothiol 1 improved the yield dramatically. See the Examples.
The chirality of the Staudinger Ligation products from the reaction of the D
and L a-azido acids was analyzed by HPLC using a D-phenylglycine chiral column. The chromatographic conditions enabled the baseline resolution of the two possible enantiomeric products (Figure 1). Materials to be analyzed were injected onto a D-phenylglycine analytical HPLC column and eluted with 30% (v/v) isopropanol in hexanes (isocraticO for 20 min followed by a shallow gradient to 50% (v/v) isopropanol for 40 min. After reaction of the D epimer, there was no evidence of product containing the L epimer, and vice versa.
Thus, the Staudinger Ligation proceeds without detectable epimerization of the a-carbon of the azido acid. The detection limit of the HPLC chromatographic analysis used is estimated to be _<0.5%, so that the Staudinger Ligation proceeds with >_99.5% retention of chirality.
Those or ordinary skill in the art will appreciate that starting materials, reagents, solvents, temperature and other reaction conditions other than those specifically disclosed, can be employed in the practice of this invention without resort to undue experimentation.
All such art-recognized equivalents are included to be encompassed by this invention. In particular, published PCT application WO 01 /87920 is cited herein to provide details of the Stauding Ligation and method for amide bond formation using the phosphinothiol reagents (1) and the phosphine-borane complexes 10, 12 and 12d.
Claims (59)
1. A phosphine-borane complex of formula:
where:
Rp is an acyl group, R and R', independently, are optionally substituted alkyl, aryl or alkoxy groups, where R and R' are the same or different groups or R and R' are optionally covalently linked to each other;
each R", independently, is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group, where R" are the same or different groups, or any two or three of R" are optionally covalently linked to each other; and R2 and R3, independently, are hydrogen, optionally substituted alkyl or optionally substituted aryl groups, where R2 and R3 are optionally covalently linked to each other.
where:
Rp is an acyl group, R and R', independently, are optionally substituted alkyl, aryl or alkoxy groups, where R and R' are the same or different groups or R and R' are optionally covalently linked to each other;
each R", independently, is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group, where R" are the same or different groups, or any two or three of R" are optionally covalently linked to each other; and R2 and R3, independently, are hydrogen, optionally substituted alkyl or optionally substituted aryl groups, where R2 and R3 are optionally covalently linked to each other.
2. The complex of claim 1 wherein R and R' are the same group or are covalently linked to each other.
3. The complex of claim 2 wherein R and R' are alkyl groups.
4. The complex of claim 1 wherein Rp is a-CO-R5 group, where R5 is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group.
5. The complex of claim 1 wherein Rp is an acetyl group.
6. The complex of claim 1 wherein R and R' are optionally substituted alkyl groups.
7. The complex of claim 1 wherein R and R' are optionally substituted aryl groups.
8. The complex of claim 1 wherein each R" is the same group and is hydrogen, a methyl, an ethyl, a propyl, or a butyl group.
9. The complex of claim 1 wherein R2 and R3 are hydrogen, a methyl group, an ethyl group, a propyl group, or a butyl group.
10. The complex of claim 1 wherein R and R' are ethyl groups.
11. A phosphine-borane complex of formula:
where R and R', independently, are optionally substituted alkyl, aryl or alkoxy groups, where R and R' are the same or different groups or R and R' are optionally covalently linked to each other;
each R", independently, is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group, where R" are the same or different groups, or any two or three of R" are optionally covalently linked to each other;
R2 and R3, independently, are hydrogen, optionally substituted alkyl or optionally substituted aryl groups, where R2 and R3 are optionally covalently linked to each other;
n is a integer ranging from 1 to the number of carboxyl groups in AA; and AA is an amino acid, peptide or protein or a protected derivative thereof.
where R and R', independently, are optionally substituted alkyl, aryl or alkoxy groups, where R and R' are the same or different groups or R and R' are optionally covalently linked to each other;
each R", independently, is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group, where R" are the same or different groups, or any two or three of R" are optionally covalently linked to each other;
R2 and R3, independently, are hydrogen, optionally substituted alkyl or optionally substituted aryl groups, where R2 and R3 are optionally covalently linked to each other;
n is a integer ranging from 1 to the number of carboxyl groups in AA; and AA is an amino acid, peptide or protein or a protected derivative thereof.
12. The complex of claim 11 wherein the AA group is an amino acid or a protected derivative thereof.
13. The complex of claim 11 wherein the AA group is an amino acid, peptide or protein.
14. The complex of claim 11 wherein the AA group is glycine, alanine, valine, leucine, isoleucine, phenylalanine, serine, methionine, proline, tyrosine, tryptophan, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, cysteine, methionine, hydroxyproline, .gamma.-carboxyglutamate, O-phosphoserine, ornithine, or homoarginine.
15. The complex of claim 11 wherein the AA group is a peptide or a protected derivative thereof.
16. The complex of claim 11 wherein n is 1, 2 or 3.
17. The complex of claim 11 wherein n is 1 and AA is a protected amino acid.
18. The complex of claim 17 wherein R and R' are phenyl groups.
19. The complex of claim 17 wherein R and R' are alkyl groups.
20. The complex of claim 17 wherein R2 and R3 are hydrogen.
21. The complex of claim 20 wherein R and R' are phenyl groups.
22. The complex of claim 21 wherein all R" are hydrogen.
23. The complex of claim 11 wherein AA is a side-group protected amino acid.
24. The complex of claim 23 wherein AA is a protected aspartate, a protected glutamate, a protected asparagine, or a protected glutamine.
25. The complex of claim 11 wherein AA is an amino acid having a side-group carrying a carboxyl group and wherein the thioester is formed at the carboxyl group of the amino acid side group.
26. The complex of claim 25 wherein AA is aspartate or glutamate.
27. The complex of claim 11 which is:
where Boc is t-butyloxycarbonyl, Bn is benzyl and Et is ethyl.
where Boc is t-butyloxycarbonyl, Bn is benzyl and Et is ethyl.
28. The complex of claim 11 wherein AA is glycine, alanine, valine, leucine, isoleucine, phenylalanine, serine, methionine, proline, tyrosine, tryptophan, lysine, arginine, histidine, asparate, glutamate, asparagine, glutamine, cysteine, methionine, hydroxyproline, .gamma.-carboxyglutamate, O-phosphoserine, ornithine, homoarginine or protected derivatives thereof.
29. The complex of claim 11 wherein AA is a naturally-occurring amino acid, a synthetically prepared amino acid, or a protected derivative thereof.
30. The complex of claim 11 wherein R and R' are optionally substituted alkyl, alkoxy or aryl groups.
31. The complex of claim 11 wherein R and R' are ethyl groups.
32. The complex of claim 11 wherein R2 and R3 are hydrogen.
33. The complex of claim 11 wherein all R" are hydrogen.
34 The complex of claim 11 wherein R" are methyl, ethyl, propyl or butyl groups.
35. The complex of claim 11 wherein R and R' are the same group or are covalently linked to each other.
36. The complex of any one of claims 11, 12, 15-24 or 28-35 wherein the protected amino acid, peptide or protein is protected with one or more protecting groups selected from Mtr (2,3,6-trimethyl-4-methoxybenzenesulfonyl), Pmc (2,2,5,7,8-pentamethychroman-6-sulfonyl), Tos (p-toluenesulfonyl), Mts (2,4,6-trimethylbenenesulfonyl), Mbh (4,4'-dimethyoxybenzhydryl), Tmob (trimethyloxybenzyl), Trt (trityl), Xan (9-xanthenyl), tBu (t-butyl), Bn (benzyl), OcHEX (-O-cyclohexyl), Acm (acetamidomethyl), S-tBu (t-butylmercapto), MeBzl (methylbenzyl), Mob (p-methoxybenzyl), Bum (t-butoxymethyl), Dnp (2,4-dinitophenyl), Bom (benzyloxymethyl), Z (benzyloxycarbonyl), ClZ (2-chlorobenzyloxycarbonyl), Boc (t-butyloxycarbonyl), CHO (formyl), BrZ (2-bromobenzyloxycarbonyl) or Ac (acetyl).
37. A method for synthesis of a phosphinothiol reagent which comprises the steps of:
(a) reacting a phosphine-borane complex of formula:
with an alkylating agent of formula:
to form a phosphine-borane complex of formula:
(b) disrupting the phosphine-borane product of step a to form:
(c) deprotecting the product of step b to form a phosphinothiol reagent wherein:
Rp is an acyl group;
X is a leaving group;
R and R', independently, are optionally substituted alkyl, aryl or alkoxy groups, where R and R' are the same or different groups or R and R' are optionally covalently linked to each other;
each R", independently, is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group, where R" are the same or different groups, or any two or three of R" are optionally covalently linked to each other; and R2 and R3, independently, are hydrogen, optionally substituted alkyl groups or optionally substituted aryl groups, where R2 and R3 are optionally covalently linked to each other.
(a) reacting a phosphine-borane complex of formula:
with an alkylating agent of formula:
to form a phosphine-borane complex of formula:
(b) disrupting the phosphine-borane product of step a to form:
(c) deprotecting the product of step b to form a phosphinothiol reagent wherein:
Rp is an acyl group;
X is a leaving group;
R and R', independently, are optionally substituted alkyl, aryl or alkoxy groups, where R and R' are the same or different groups or R and R' are optionally covalently linked to each other;
each R", independently, is hydrogen, an optionally substituted alkyl or an optionally substituted aryl group, where R" are the same or different groups, or any two or three of R" are optionally covalently linked to each other; and R2 and R3, independently, are hydrogen, optionally substituted alkyl groups or optionally substituted aryl groups, where R2 and R3 are optionally covalently linked to each other.
38. The method of claim 37 wherein Rp is an acetyl group.
39. A method for forming an amide bond which comprises the step of reacting an azide with the phosphine-borane complex of claim 11 followed by hydrolysis of the combined reactants to form an amide bond.
40. The method of claim 39 wherein R and R' are optionally substituted alkyl groups.
41. The method of claim 40 wherein R and R' are ethyl groups.
42. The method of claim 39 wherein n is 1 and AA is an amino acid.
43. The method of claim 39 wherein AA is a peptide or a protein.
44. The method of claim 39 wherein n is 1, 2 or 3.
45. The method of claim 39 wherein n is 1.
46. The method of claim 39 wherein the azide is an azide of an amino acid, a peptide or a protein.
47. The method of claim 39 wherein the azide is an azide of a carbohydrate.
48. The method of claim 39 wherein the azide is an azide of a monosaccharide, disaccharide or trisaccharide.
49. The method of claim 39 wherein all R" are hydrogen.
50. The method of claim 39 wherein all R" are methyl, ethyl, propyl or butyl groups.
51. The method of claim 39 wherein R2 and R3 are both hydrogen.
52. A kit for forming an amide bond which comprises one or more phosphine-borane complexes of claim 1 and a deprotecting agent for deprotecting the one or more phosphine-borane complexes to form a phosphinothiol reagent for forming the amide bond.
53. A kit for forming an amide bond which comprises one or more phosphine-borane complexes of claim 1 and an azide for reaction with the one or more phosphine-borane complexes to form an amide bond.
54. The kit of claim 53 wherein the azide is an azide of an amino acid, a peptide, a protein or a carbohydrate.
55. The kit of claim 53 wherein the azide is an azide of a monosaccharide, a disaccharide, a trisaccharide, a nucleoside or a reporter.
56. A kit for forming an amide bond which comprises one or more phosphine-borane complexes of claim 11 and an azide for reaction with the one or more phosphine-borane complexes to form an amide bond.
57. The kit of claim 56 wherein the azide is an azide of an amino acid, a peptide, a protein, or a carbohydrate.
58. The kit of claim 56 wherein the azide is an azide of a monosaccharide, a disaccharide, or a trisaccharide.
59. The kit of claim 56 wherein the azide is an azide of a nucleoside or a reporter.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US38717102P | 2002-06-07 | 2002-06-07 | |
| US60/387,171 | 2002-06-07 | ||
| PCT/US2003/017885 WO2003104243A1 (en) | 2002-06-07 | 2003-06-06 | Chemical synthesis of reagents for peptide coupling |
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| Publication Number | Publication Date |
|---|---|
| CA2489186A1 CA2489186A1 (en) | 2003-12-18 |
| CA2489186C true CA2489186C (en) | 2013-01-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| CA2489186A Expired - Lifetime CA2489186C (en) | 2002-06-07 | 2003-06-06 | Chemical synthesis of reagents for peptide coupling |
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| Country | Link |
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| US (2) | US6974884B2 (en) |
| EP (1) | EP1534718A4 (en) |
| JP (1) | JP2005529166A (en) |
| AU (1) | AU2003248633A1 (en) |
| CA (1) | CA2489186C (en) |
| WO (1) | WO2003104243A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050214209A1 (en) * | 2001-05-25 | 2005-09-29 | Human Genome Sciences, Inc. | Antibodies that immunospecifically bind to TRAIL receptors |
| EP1534718A4 (en) * | 2002-06-07 | 2006-06-07 | Wisconsin Alumni Res Found | Chemical synthesis of reagents for peptide coupling |
| US8242058B2 (en) * | 2006-07-21 | 2012-08-14 | Wisconsin Alumni Research Foundation | Reagents and methods for appending functional groups to proteins |
| US7943728B2 (en) | 2006-12-26 | 2011-05-17 | National Cheng Kung University | Disintegrin variants and their use in treating osteoporosis-induced bone loss and angiogenesis-related diseases |
| US8183201B2 (en) * | 2006-12-26 | 2012-05-22 | National Cheng Kung University | Methods of treating αvβ3 integrin-associated diseases by administering polypeptides selective for αvβ3 integrin |
| US8415453B2 (en) * | 2007-02-13 | 2013-04-09 | Academia Sinica | Lung cancer-targeted peptides and applications thereof |
| US8088887B2 (en) * | 2007-02-13 | 2012-01-03 | Academia Sinica | Peptide-conjugates that bind to VEGF-stimulated or tumor vasculature and methods of treatment |
| US8541543B2 (en) * | 2007-11-20 | 2013-09-24 | Academia Sinica | Peptides specific for hepatocellular carcinoma cells and applications thereof |
| US8410247B2 (en) * | 2008-08-22 | 2013-04-02 | Wisconsin Alumni Research Foundation | Water-soluble phosphinothiol reagents |
| WO2010057220A1 (en) | 2008-11-17 | 2010-05-20 | Wisconsin Alumni Research Foundation | Preparation of diazo and diazonium compounds |
| GB0908711D0 (en) * | 2009-05-20 | 2009-07-01 | Isis Innovation | Preparation of labelled compounds |
| CA2768360A1 (en) * | 2009-07-20 | 2011-01-27 | National Cheng Kung University | Polypeptides selective for .alpha.v.beta.3 integrin conjugated with a variant of human serum albumin (hsa) and pharmaceutical uses thereof |
| AR079706A1 (en) | 2009-12-23 | 2012-02-15 | Univ Nat Cheng Kung | COMPOSITIONS, METHODS FOR THE TREATMENT OF EYE DISEASES RELATED TO ANGIOGENESIS AND USE |
| WO2013110005A1 (en) | 2012-01-18 | 2013-07-25 | Wisconsin Alumni Research Foundation | Boronate-mediated delivery of molecules into cells |
| US9732101B2 (en) | 2012-01-18 | 2017-08-15 | Wisconsin Alumni Research Foundation | Bioreversible boronates for delivery of molecules into cells |
| EP2814472A4 (en) | 2012-02-15 | 2015-11-04 | Wisconsin Alumni Res Found | Dithioamine reducing agents |
| US9206271B2 (en) | 2012-03-25 | 2015-12-08 | Wisconsin Alumni Research Foundation | Fully backbone degradable and functionalizable polymers derived from the ring-opening metathesis polymerization (ROMP) |
| US9371521B2 (en) | 2014-06-30 | 2016-06-21 | Wisconsin Alumni Research Foundation | Substituted pyrazinedithiol reducing agents |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4066684A (en) * | 1974-04-15 | 1978-01-03 | The Dow Chemical Company | Preparation of peptides |
| EP0403524B1 (en) * | 1988-03-09 | 1993-02-10 | Cis Bio International | Preparation of nitruro compounds usable as radio-pharmaceutical products |
| US5543389A (en) * | 1990-11-01 | 1996-08-06 | State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University, A Non Profit Organization | Covalent polar lipid-peptide conjugates for use in salves |
| US5541289A (en) * | 1994-03-30 | 1996-07-30 | Washington University | Phosphine containing amino acids and peptides and methods of preparing and using same |
| EP1263771B1 (en) | 2000-03-16 | 2006-06-14 | The Regents Of The University Of California | Chemoselective ligation by use of a phosphine |
| JP4694085B2 (en) | 2000-05-12 | 2011-06-01 | ウィスコンシン アルムニ リサーチ ファンデイション | Ligation methods and reagents for forming amide bonds |
| KR20030046389A (en) | 2000-09-01 | 2003-06-12 | 그리폰 테라퓨틱스, 인코포레이티드 | Nucleophile-stable thioester generating compounds, methods of production and use |
| EP1534718A4 (en) | 2002-06-07 | 2006-06-07 | Wisconsin Alumni Res Found | Chemical synthesis of reagents for peptide coupling |
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2003
- 2003-06-06 EP EP03757381A patent/EP1534718A4/en not_active Withdrawn
- 2003-06-06 JP JP2004511312A patent/JP2005529166A/en not_active Withdrawn
- 2003-06-06 WO PCT/US2003/017885 patent/WO2003104243A1/en active Application Filing
- 2003-06-06 AU AU2003248633A patent/AU2003248633A1/en not_active Abandoned
- 2003-06-06 CA CA2489186A patent/CA2489186C/en not_active Expired - Lifetime
- 2003-06-06 US US10/456,988 patent/US6974884B2/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| US6974884B2 (en) | 2005-12-13 |
| EP1534718A1 (en) | 2005-06-01 |
| EP1534718A4 (en) | 2006-06-07 |
| CA2489186A1 (en) | 2003-12-18 |
| AU2003248633A1 (en) | 2003-12-22 |
| WO2003104243A1 (en) | 2003-12-18 |
| US20040030105A1 (en) | 2004-02-12 |
| US20060025578A1 (en) | 2006-02-02 |
| US7317129B2 (en) | 2008-01-08 |
| JP2005529166A (en) | 2005-09-29 |
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