WO2003099852A2 - Processus de derivatisation de peptides - Google Patents

Processus de derivatisation de peptides Download PDF

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Publication number
WO2003099852A2
WO2003099852A2 PCT/US2003/016644 US0316644W WO03099852A2 WO 2003099852 A2 WO2003099852 A2 WO 2003099852A2 US 0316644 W US0316644 W US 0316644W WO 03099852 A2 WO03099852 A2 WO 03099852A2
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Prior art keywords
peptide
acid
disulfide
reaction mixture
group
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PCT/US2003/016644
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English (en)
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WO2003099852A3 (fr
Inventor
Kirk E. Cryer
Daniel Strydom
Jin Seog Seo
Barton Holmquist
Fred W. Wagner
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Restoragen Inc.
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Priority to AU2003240796A priority Critical patent/AU2003240796A1/en
Publication of WO2003099852A2 publication Critical patent/WO2003099852A2/fr
Publication of WO2003099852A3 publication Critical patent/WO2003099852A3/fr

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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
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C07K1/126—Aminolysis

Definitions

  • the invention provides a process wherein a peptide having a Cys-His group at its C terminus undergoes nucleophilic addition, such as amidation, to the carbonyl carbon of the a ino acid residue immediately preceding the Cys- His, by reacting the peptide at a pH of 10 or greater with a disulfide agent and a cyanylating agent in the presence of a nucleophile.
  • the peptide must be separated from the leader sequence, purified and recovered in an active form. Separation from the leader sequence may be accomplished by placing a sequence of amino acids at the junction of the leader and the peptide which are specifically recognized and cleaved under appropriate conditions, e.g. acid cleavage or enzymatic cleavage.
  • acid cleavage or enzymatic cleavage e.g. acid cleavage or enzymatic cleavage.
  • natural amino acid modifications such as C-terminal amide group substitution, which occur routinely in vivo, are difficult to affect in vitro.
  • Post-translational amidation usually converts the peptide to a biologically active form most suitable for pharmaceutical use. For many peptides, C-terminal amidation is important for biological activity.
  • Carboxypeptidase enzymes are known to catalyze transpeptidation reactions, yielding C-terminally amidated peptides.
  • wild-type carboxypeptidase enzymes are not useful for C-terminal amidation of many peptides because the inherent substrate specificity of wild-type carboxypeptidase restricts the variety of peptides that may be modified using this enzyme.
  • carboxypeptidase Y displays a strong preference for peptides with a penultimate apolar residue; however, the amino acid sequences of many pharmaceutically important peptides, including growth hormone releasing factor (GRF) or glucagon like peptide- 1 (GLP-1), have a penultimate or ultimate amino acid with a positively charged side chain, thereby making transamidation with carboxypeptidase Y commercially impractical.
  • GRF growth hormone releasing factor
  • GLP-1 glucagon like peptide- 1
  • United States Patent No. 6,251,635 describes the treatment of a chimeric protein, including multiple copies of a target sequence, in a precursor peptide which includes hCA-(MetValAspAs ⁇ AspAspAsn-ECF2) n -Xxx (SEQ ID NO: 17), where hCA is human carbonic anhydrase, ECF2 is apolypeptide fragment having the formula: Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly -Ala-Gly-Thr-Pro (SEQ ID NO: 18); and Xxx is typically a C-terminal carboxylic acid ("—OH"), a C-terminal carboxamide (" — NH 2 "), or group capable of being converted into a C-terminal carboxamide, such as an amino acid residue or a polypeptide group (typically
  • Such a precursor peptide may be treated with CNBr to form Val- Asp- Asp- Asp- Asp- Asn-ECF2-Hse (SEQ ID NO: 19) peptide fragments (where Hse is a homoserine residue produced by the reaction of CNBr with a Met residue).
  • the peptide fragments may then be reacted with a nucleophile such as o- nitrophenylglycine amide (“ONPGA”) in the presence of a peptidase such as carboxypeptidase Y resulting in the replacement of the Hse residue by ONPGA.
  • ONPGA o- nitrophenylglycine amide
  • the transpeptidation product is converted to a C-terminal carboxamide.
  • the ONGPA process requires a three step chemical reaction in which column chromatography is required between each step to separate unreacted compound for use in the subsequent steps.
  • Another method of forming a C-terminal amide on a recombmantly produced polypeptide uses the enzyme peptidyl alpha-amidating enzyme which is present in eukaryotic systems.
  • the enzyme has been used to form an amide on the C-terminal amino acid of recombinantly produced peptides, like human growth hormone releasing hormone in vitro, as described by J. Engels, Protein Engineering, 1:195-199 (1987). While effective, the enzymatic method is time consuming, expensive, gives unpredictable yields, and requires significant post- reaction purification.
  • the enzymatic method is also limited to modifying the recombinantly produced peptide by C-terminal amidation only.
  • United States Patent No. 5,861,284 ('"284 patent”) describes the use at low pH of S-cyanylating agents such as 2-nitro-5-thiocyanobenzoic acid (NTCB) or a l-cyano-4-(dimethylamino)pyridinium salt (CDMAP) and subsequent amidation by aminolysis with ammonia.
  • S-cyanylating agents such as 2-nitro-5-thiocyanobenzoic acid (NTCB) or a l-cyano-4-(dimethylamino)pyridinium salt (CDMAP) and subsequent amidation by aminolysis with ammonia.
  • NTCB 2-nitro-5-thiocyanobenzoic acid
  • CDMAP l-cyano-4-(dimethylamino)pyridinium salt
  • Catsimpoolas and Wood disclose S-cyanylation at mildly basic pH using cyanide ion as the cyanylating agent.
  • the cyanylation method of Catsimpoolas and Wood is very sensitive to factors such as time, temperature, cyanide concentration, and particularly pH. High temperatures (57 °C) and high concentrations of cyanide (100-fold excess) are necessary to induce maximal reaction, typically in 18 hours or more. The major limitation, however, was that the pH needed to be in the 6 - 8 range or iminothiazolidine ring formation was greatly diminished in favor of thiocyanate ion and dehydroalanine formation. Furthermore, Catsimpoolas and Wood disclose that unless the cyanide ion is present in greater than 10-fold excess, oxidation of any sulfhydryl group becomes difficult without producing CNO ⁇ and other derivatives.
  • the instant invention provides a novel peptide C-terminal derivatization process that can be done one- pot and that is essentially not sequence specific.
  • the invention enables the economic production of a wide variety of biologically active peptides.
  • a peptide having a Cys-His group with free thiol or a metal-thiol complex, or incompletely oxidized disulfide, at its C terminus undergoes nucleophilic addition (such as amidation) by reacting the peptide in a reaction mixture comprising a nucleophile (such as ammonia or an amine-containing base), a disulfide reagent, and a cyanylating agent, wherein the pH of the reaction mixture is 10 or greater and the molar concentration of the disulfide reagent is approximately two or more times greater than that of the peptide.
  • a nucleophile such as ammonia or an amine-containing base
  • a disulfide reagent such as ammonia or an amine-containing base
  • a cyanylating agent wherein the pH of the reaction mixture is 10 or greater and the molar concentration of the disulfide reagent is approximately two or more times greater than that of the peptide.
  • Nucleophiles useful in the instant invention include but are not limited to any primary amine, ammonia, hydroxides, alkoxides, hydrazides, hydroxamates, hydrazines or hydroxylamines.
  • Disulfide reagents useful in the instant invention include but are not limited to cystine, homocystine, dithiodinicotinic acid, dithiodipropionic acid, dithiodiglycolic acid, dithiodibutyric acid, dithiodibenzoic acid, cystamine, oxidized dithiothreitol, oxidized dithioerythritol, 2,2'-dipyridyldisulfide, 4,4'-dipyridyldisulfide, 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), penicillamine disulfide, oxidized leucine thiol, guanidino ethyldisulfide,
  • Cyanylating agents useful in the instant invention include but are not limited to cyanide anion. cyanogen halides or cyanogen.
  • Reaction media of the instant invention can be completely aqueous and include but are not limited to a denaturing solvent such as urea or guanidine, or mixtures of urea and organic solvents such as alcohols or acetonitrile. The process may be used to derivatize naturally occurring peptides, synthetically prepared peptides, a single-copy recombinant polypeptide, a multicopy recombinant polypeptide or a single or multi-copy recombinant cliimeric protein construct.
  • a polypeptide When a polypeptide is prepared by recombinant techniques, one can add a Cys-His group to the C- terminus of the amino acid sequence defining the peptide product by incorporating or mutating the appropriate nucleotides into the encoding nucleic acid by any of various methods including, for example, site-directed mutagenesis. Recombinant methods can also be used to generate a nucleic acid encoding a protein with a repeating polypeptide sequence, with each sequence separated by a cleavage site and having a Cys-His group at the carboxy terminus of each sequence to facilitate derivatization.
  • FIGURE 1 illustrates a possible mechanism for amidation in accordance with the process of the instant invention.
  • FIGURE 2 illustrates the DNA (SEQ ID NO:25) and peptide (SEQ ID NO: 26) sequence of the GRF chimeric protein.
  • the signal sequence, Vg sequence, linker sequence, and GRF(1-44)CH sequence are indicated by bracketed lines.
  • the stop codon is indicated by stars.
  • FIGURE 3 illustrates ammonolysis of a Cys-Gly containing peptide.
  • Chimeric proteins employed in the instant invention may be expressed in a microbial host cell using known techniques of recombinant DNA production. Any suitable host cell known to be useful for the expression of proteins by recombinant DNA methods may be employed, including prokaryotic and eukaryotic host cells and cell lines. E. coli is a preferred host cell.
  • the host cell contains an expression vector that encodes the chimeric protein under the control of a regulatory sequence which is capable of directing its expression in the host, as well as an origin of replication that is functional in the host cell.
  • the vector may contain other DNA sequences conventionally employed in recombinant DNA technology such as sequences encoding selectable markers.
  • Example 1 provides a detailed description of the preparation of a T7tag-based expression system useful for high-level expression of mammalian proteins in E. coli.
  • the host cell containing the expression vector is grown and the chimeric protein expressed under appropriate conditions.
  • the conditions for growth of the host cell and expression of the chimeric protein will vary depending on factors such as the host cell employed, the promoter and the particular chimeric protein being expressed. Those skilled in the art are capable of determining the appropriate conditions for the particular host/vector system employed. Methods for expressing a foreign gene in a host organism also are well known in the art (see, e.g., Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd ed., 1989).
  • the gene encoding a particular polypeptide can be constructed by chemically synthesizing the entire nucleotide sequence, by amplification, such as by the polymerase chain reaction (PCR), or by cloning the gene of interest. The gene is then subcloned into an appropriate expression vector.
  • PCR polymerase chain reaction
  • Cloning vectors, expression vectors, plasmids, and viral vectors are well known in the art (see, e.g., Maniatis et al., supra, and Goedell, Methods in Enzymology, Vol. 185 (Academic Press 1990)).
  • Example 1 provides a detailed description of the preparation of a T7-based expression system useful for high-level expression of mammalian proteins in E. coli.
  • a polypeptide When a polypeptide is prepared by recombinant techniques, one can add a Cys-His group to the C-terminus of the amino acid sequence defining the peptide product by incorporating or mutating the appropriate nucleotides into the encoding nucleic acid by any of various methods including, for example, site- directed mutagenesis.
  • Recombinant methods can also be used to generate a nucleic acid encoding a protein with a repeating polypeptide sequence, with each sequence separated by a cleavage site and having a Cys-His group at the C- terminus of each sequence to facilitate derivatization.
  • cleavage by an appropriate technique can occur at multiple cleavage sites as defined above in the polypeptide, releasing multiple copies of the desired peptide ending in Cys- His.
  • the polypeptide comprises Y-cysteine- histidine-X n , wherein Y comprises the polypeptide of interest, and X n comprises an optional amino acid or sequence of amino acids.
  • the polypeptide should have at least one Cys-His sequence at its C terminus and have a molecular weight of between about 400 to about 100,000 daltons or greater (preferably between 1,000 and 50,000 daltons).
  • "Protein,” “polypeptide,” and “peptide” are used interchangeably herein and are intended to refer to any sequence of two or more amino acids, regardless of length.
  • Polypeptides suitable for cleavage can comprise any of the natural amino acids, such as Ala (A), Arg (R), Asp (D), Asn (N), Glu (E), Gin (Q), Gly (G), His (H), Leu (L). lie (I), Lys (K), Met (M), Cys (C), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V) (single letter amino acid code in parentheses), or may comprise any side chain-modified amino acid derivative commonly used in peptide chemistry.
  • the latter amino acid derivatives include, for example, nipecotic acid, 1- or 2-napthylalanines and p-benzoylamino-L-phenylalanine, among others.
  • the process of the instant invention is applicable to natural polypeptides, synthetic polypeptides, or polypeptides produced using recombinant techniques.
  • Methods for preparing synthetic polypeptides are well known in the art and include, for example, Merrifield solid phase peptide synthesis.
  • Methods for expressing a foreign gene in a host organism also are well known in the art (see, e.g., Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd ed., 1989).
  • the gene encoding a particular polypeptide can be constructed by chemically synthesizing the entire nucleotide sequence, by amplification, such as by the polymerase chain reaction (PCR), or by cloning the gene of interest.
  • PCR polymerase chain reaction
  • Example 1 provides a detailed description of the preparation of a T7 -based expression system useful for high-level expression of mammalian proteins in E. coli.
  • the process of the instant invention provides for the production of biologically active (derivatized) peptides which include but are not limited to glucagon-like peptide-2 (GLP-2), glucagon-like peptide- 1 (GLP-1), growth hormone-releasing factor (GRF), parathyroid hormone (PTH) (including, but not limited to PTH(l-34) or PTH(l-84)), parathyroid hormone related peptide, adrenocorticotropic hormone (ACTH), enkephalins, endorphins, exendins, amylins, various opioid peptides, frog skin antibiotic peptides, such as magainin, gaegurins 5 and 6, brevinin 1, the ranatuerins 1 through 9, and the esculetins, glucose-dependent insulinotropic polypeptide (GIP), glucagon, motilin, thymopoietins, thymosins, ubiquitin, serum thymic factor,
  • Precursor non-amidated or reduced forms of the following peptides can also be expressed as a fusion construct with a Cys-His derivatization site and subjected to cleavage and derivatization in accordance with the process of the instant invention: gastrin, glucagon-like peptide-2 (GLP-2), glucagon-like peptide- 1 (GLP-1), growth hormone-releasing factor (GRF), calcitonin, luteinizing-hormone-releasing hormone, pancreatic polypeptide, endothelin, corticotropin releasing factor, neuropeptide Y, atrial natriuretic peptide, amylin, galanin, somatostatins, vasoactive intestinal peptide, insulin, and fragments and derivatives of these peptides.
  • GLP-2 glucagon-like peptide-2
  • GLP-1 glucagon-like peptide- 1
  • GRF growth hormone-releasing factor
  • calcitonin lute
  • leader sequences which can be employed with chimeric proteins include a signal sequence such as that used to direct secretion of a protein from a cell, the N-terminal portion of a mature protein sequence, such as from a structural gene, a linker sequence, or combinations thereof.
  • a leader sequence can be obtained from the genes encoding glutathione-S-transferase or carbonic anhydrase.
  • Vestigial and polyhedrin leaders which can be used include the following, in which sequences 1-15 are vestigial leader sequences and sequence 16 is a polyhedrin leader sequence:
  • Linkers that are particularly susceptible to cleavage may be used with chimeric proteins employed in peptide production in accordance with the process of the instant invention.
  • the chimeric protein has a molecular weight of between about 400 to about 100,000 daltons or greater (preferably between 1,000 and 50,000 daltons) and can comprise any of the natural amino acids, such as Ala (A), Arg (R), Asp (D), Asn (N), Glu (E), Gin (Q), Gly (G), His (H), Leu (L), He (I), Lys (K), Met (M), Cys (C), Phe (F), Pro (P), Ser (S), Thr (T), Tip (W), Tyr (Y), Val (V) (single letter amino acid code in parentheses), or may comprise any side chain-modified amino acid derivative commonly used in peptide chemistry.
  • the latter amino acid derivatives include, for example, 1- or 2-napthylalanines andp-benzoylamino-L-phenylalanine, among others.
  • the chimeric protein After the chimeric protein has been expressed, it can be recovered (in the form of inclusion bodies) from the host cells by known methods such as, for example, lysing the cells chemically or mechanically and separating the inclusion bodies (or soluble chimeric protein) by centrifugation.
  • Recovered inclusion bodies or soluble precursor peptides are thereafter subjected to appropriate cleavage techniques which cleave the peptide from the leader sequence and any associated linker.
  • the cleaved peptide produced in accordance with the process of the instant invention can be recovered by ultrafiltration, precipitation, or more preferably, by reverse phase chromatography. Any commercially-available reverse phase column suitable for the peptide being isolated may be employed. In many cases, the peptide recovered from the reverse phase column will refold into its native confo ⁇ nation, however, additional steps (e.g., oxidation) may be required to restore the peptide to a biologically active form, particularly when the peptide requires the formation of internal disulfide bonds for activity.
  • nucleophilic addition such as amidation
  • Figure 1 details a possible mechanism for amidation in accordance with the process of the instant invention.
  • Nucleophiles useful in the instant invention include but are not limited to any primary amine, ammonia, hydroxides, alkoxides, hydrazides, hydroxamates, hydrazines or hydroxylamines.
  • Disulfide reagents useful in the instant invention include but are not limited to cystine; homocystine; dithiodinicotinic acid; dithiodipropionic acid; dithiodiglycolic acid; dithiodibutyric acid; dithiodibenzoic acid; cystamine, oxidized dithiothreitol; oxidized dithioerythritol; 2,2'-dipyridyldisulfide; 4,4'-dipyridyldisulfide; 5,5'- dithiobis(2-nitrobe ⁇ zoic acid) (DTNB); penicillamine disulfide; oxidized leucine thiol; guanidinoethyl disulfide; oxidized glutathione (GSSG); 2,2'- dithiodiethanol; 6-hydroxy-2-naphthyl disulfide; and 1, l'-c amino- 2,2'dinaphthyldisulfide.
  • Cyanylating agents useful in the instant invention include but are not limited to cyanide anion, cyanogen halides or cyanogen.
  • cystine dithiodipropionic acid (dtdp), cystamine, and dithiodinicotinic acid (dtdn) as disulfide reagents
  • cyanide anion as cyanylating reagent and ammonia as nucleophile
  • use of cystamine resulted in the highest amidation yield and dtdp resulted in the lowest amidation yield.
  • the construct T7tag-Ng- D 4 KCH-GRF(1-44)CH (SEQ ID ⁇ O:20) was expressed recombinantly inE. coli and was recovered in the form of an inclusion body. This inclusion body was then cleaved through palladium-promoted cleavage in malonic acid to yield the non-amidated peptide GRF(1-44)CH, which was desalted by reversed phase chromatography. GRF(1-44)CH was thereafter solubilized in urea (4 - 8 M) to form a reaction mixture in which the concentration of GRF(1-44)CH was approximately 1 mg/mL (0.2 niM) or greater. A range of 0.01 to 2.0 mM has been shown to be effective.
  • the concentration of disulfide is in five to ten-fold molar excess over peptide.
  • T7tag-Vg-D 4 KCH-GRF(l-44)-CH (SEQ ID NO:20; Figure 2 (SEQ ID NO:25 and SEQ ID NO:26) was recombinantly expressed inE. coli as follows.
  • E. coli bacteria containing expression plasmids encoding the leader -CH- GRF(1 -44)CH (S ⁇ Q ID N 0:21 ) polypeptide were grown in 500 mL shake flasks containing tryptone, yeast, glucose, batch salts (sodium and potassium mono- and diphosphate salts and ammonium sulfate), and antibiotic. Inoculated shake flasks were subject to orbital shaking (200 rpm, 37 °C). Incubation was completed when the culture reached an optical density (OD) of 0.8-1.8 at 540 nm.
  • OD optical density
  • a fermentor of 5 L production capacity was seeded using a shake flask culture.
  • the media included batch salts, glucose, and chelated metals solution (potassium citrate, sodium citrate, magnesium sulfate, phosphoric acid ferric chloride, zinc chloride, cobalt chloride, sodium molybdate, manganese chloride, calcium chloride, and copper sulfate).
  • the pH of the medium was adjusted to 6.9 prior to inoculation and the pH was maintained at 6.9 during culture.
  • Dissolved oxygen was maintained at approximately 40 %, via agitation and supplemental oxygen.
  • Either silicone-based or polypropylene glycol-based "antifoam” was added aseptically on an "as needed" basis to reduce foaming in the fermentation culture.
  • the whole cells containing inclusion bodies of precursor peptide from 5 L fermentation were collected by centrifugation, suspended in Tris-EDTA buffer (pH 8.0, 10 mM and 1 mM, respectively) and then broken in a homogenizer.
  • the isolated precursor peptide inclusion bodies were further washed with deionized water until the conductivity of the wash became less than 0.1 mS/cm.
  • These crude precursor peptides were further purified from the inclusion bodies by solubilization in 1.5 M citric acid followed by precipitation by titration of the acid with NaOH.
  • the precipitate obtained from pH 4.0 was washed with deionized water until the conductivity of the solution became less than 0.1 mS/cm.
  • Precursor peptide comprising T7tag-Vg-D 4 KCH-GRF(l-44)-CH was cleaved in 5 M malonic acid as follows.
  • the precursor peptide (101.6 g, wet weight) was solubilized in 5 M malonic acid at a concentration of approximately 5 mg/mL (0.45 mM).
  • a palladium cleavage promotor, Na 2 PdCl 4 was added in 5-fold molar excess over Cys-His, i.e., to reach a palladium promotor concentration in the cleavage solution of around 4 mM.
  • the cleavage reaction then proceeded at 61 °C for 90 min to yield approximately 1.9 g cleaved GRF(l-44)-CH.
  • the solution was diluted 4 fold with water and filtered.
  • HPLC method A Microsorb-MV d 8 300 A, 5 ⁇ m, 4.6 x 250 mm column was used.
  • the gradient used was as follows: 30 - 40 %B (14 min.), 40 -100 %B (0.5 min.), 100 - 30 %B (0.1 min.), 30 %B (5 min) at 1 mL/min at ambient temperature. Absorbance was monitored from 210 - 320 nm.
  • EXAMPLE 3 Amidation of rGRF (1-44VCH rGRF(l-44)CH solution, prepared as described in Example 1, was adjusted to 6 M urea, to form a reaction mixture in which the concentration of rGRF(l-44)CH was approximately 1 mg/mL (0.2 mM). Strong ammonium hydroxide solution (27 %, 14 M) was added to this reaction mixture in an amount sufficient to raise the final concentration of arnmom ' um hydroxide to 3 ⁇ 0.5 M, thereby raising the pH of the reaction mixture to about 12 ⁇ 0.3.
  • a disulfide reagent selected from the group consisting of cystine, dithiodipropionic acid (dtdp), cystamine, or dithiodinicotinic acid (dtdn) was then introduced to the reaction mixture (without additional added oxidant) and, after a short period to allow disulfide exchange to reach equilibrium, KCN was added.
  • the disulfide concentration was varied between 0.075 - 4 mM and the KCN concentration was maintained at about 5 - 8.5 mM.
  • Approximately 26 - 60 % of the GRF(1-44)CH was converted to GRF(l-44)amide in 2 hours, depending on which disulfide reagent was used, as analyzed by the HPLC Method of Example 2, and as summarized in Table 1. Tablel. Yields of GRF(l-44)amide formed by action of various disulfides in cyanylation/amidation of GRF(1-44)CH in 2 hrs.
  • the peptide Bz-Lys-Gly-Arg-Cys-Gly-Lys-Tyr (SEQ ID NO:22) was cyanylated to yield Bz-Lys-Gly-Arg-Cys(CN)-Gly-Lys-Tyr (SEQ ID NO:23). This cyanylated peptide was reacted with 3 M ammonium hydroxide at pH 9.0 - 11.5 at 0 °C and the resultant reactants were identified and quantified by C 18 HPLC.
  • the primary product yielded by the reaction was the dehydroalanine(Dha) derivative Bz-Lys-Gly-Arg-Dha-Gly-Lys-Tyr (SEQ ID NO:24).
  • the amide Bz-Lys-Gly-Arg-NH 2 was produced in substantially smaller amounts.
  • Figure 3 shows the peak area of identified peptide species as a function of pH from 9.5 to 11.5.

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Abstract

L'invention concerne un procédé de dérivatisation d'un peptide ayant un groupe Cys-His à sa terminaison carboxyle, selon lequel il est prévu de faire réagir ledit peptide à un pH supérieur à 10 ou davantage, avec un réactif bisulfure et un agent de cyanylation en présence d'un nucléophile.
PCT/US2003/016644 2002-05-24 2003-05-23 Processus de derivatisation de peptides WO2003099852A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021137870A1 (fr) * 2020-01-03 2021-07-08 The Texas A&M University System Technique de ligature de polypeptide dirigée par la cystéine activée

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985627A (en) * 1997-02-28 1999-11-16 Carlsberg Laboratory Modified carboxypeptidase
US6051399A (en) * 1994-12-07 2000-04-18 Bionebraska, Inc. Production of C-terminal amidated peptides from recombinant protein constructs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6051399A (en) * 1994-12-07 2000-04-18 Bionebraska, Inc. Production of C-terminal amidated peptides from recombinant protein constructs
US5985627A (en) * 1997-02-28 1999-11-16 Carlsberg Laboratory Modified carboxypeptidase

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021137870A1 (fr) * 2020-01-03 2021-07-08 The Texas A&M University System Technique de ligature de polypeptide dirigée par la cystéine activée
EP4085139A4 (fr) * 2020-01-03 2023-11-29 The Texas A&M University System Technique de ligature de polypeptide dirigée par la cystéine activée

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