HK1012012A - Hirudine derivatives and processes for their preparation - Google Patents
Hirudine derivatives and processes for their preparation Download PDFInfo
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- HK1012012A HK1012012A HK98113072.7A HK98113072A HK1012012A HK 1012012 A HK1012012 A HK 1012012A HK 98113072 A HK98113072 A HK 98113072A HK 1012012 A HK1012012 A HK 1012012A
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Description
The invention relates to hirudin derivatives, the production of which involves the selective enzymatic exchange of a molecule part by an amin derivative and the use of the hirudin derivatives as medicinal products.
Hyrudo medicinalis is a protein consisting of 65 amino acid residues that can be isolated from the salivary glands of the leech (Hirudo medicinalis). As a selective thrombin inhibitor, it acts as an anticoagulant (Pharmacy 36, 633-660 (1981)). The single-chain polypeptide has three disulfide bridges that stabilize the domain between the amino acids at positions 1 to 49 as a highly folded, compact protein interstitial structure. The C-terminal amino acid sequence 55 to 65 is linearised as an anchor sequence to bind to thrombin. Native hirudin is a sulfate at the phenol group of the tyrosine at position 63, which is enhanced by the cluster of acids without disulfide causing brain dysregulation.
Chemically and enzymatically produced hirudine derivatives are found in EP 0 493 588 (nitro group, halogen substitution) and DE 4 224 213 (C-terminals, enzymatic amidation).
For use as long-acting medicinal products, conventional hirudin polyethylene glycol derivatives are produced by chemically coupling a functional group of an amino acid in hirudin with a polyethylene glycol residue (PEG) (see EP 0 345 616), without creating defined compounds, as the residue can usually be bound at several functional sites on the hirudin molecule.
Hirudin contains the amino acids lysine at three positions, tyrosine at two positions, and phenylalanine at one position in the molecule. Such positions in a protein are usually cleaved by serine proteases such as trypsin or chymotrypsin. Hitherto known enzymatic semisynthesis processes (The Peptides, S. Udenfriend and J. Meienhofer (ed.), Vol. 9, Acad. Press, NY. 1987, 103-165) therefore did not appear to be transferable to hirudin.
It has been found that under certain reaction conditions, selective transpeptidization occurs in enzymatic semisynthesis reactions of hirudin with chymotrypsin, trypsin or a comparable enzyme (e.g. lysylendopeptidase) leading to new compounds.
The invention relates to compounds of formula or II:
The Commission has concluded that the use of the active substance in the Union is not likely to lead to a significant increase in the level of adverse events.
where Y is an amino derivative,A1 and A2 are independently an amino acid residue, and AO is an amino acid residue or hydrogen atom.
The nomenclature of formulae I and II refers to the publication of D. Tripier in Folia Haematol. (Leipzig) 115, 30-35 (1988).
The comma in formula I means that the amino acid sequence of the underlying hirudine is interrupted between positions 36 and 37 and the compound contains two hirudine fragments connected by disulfide bridges.
Formula II shows a compound containing an amine derivative Y linked by an amide bond to the amino acid at position 63 of the original sequence of the underlying hirudin.
Aminderivatives Y are preferably compounds of formula Illa or Illb:
H2 N-R-X (Illa),A-R1-X (Illb), wherein:
A
(b) a peptide containing 2 to 10 amino acid residues;R
(C1-C10) alkyl, branched or straight-chain, (C1-C10) alkyl, branched or straight-chain, independently substituted one or more times by:
1) Phenyl, 2) Indolyl, 3) Imidazolyl or4) Phenyl, one or more times substituted by hydroxyl, c) Phenyl or) Naphthyl, R
(a) Hydrogen atom (b) Covalent bond (c) Sugar such as glucose, fructose, mannose, galactose, ribose, ribulose or xylose (d) Polysaccharide containing 2 to 10 sugars, other than:
(a) Hydrogen atom, (b) -OR2, (c) -SR2, (d) -NHR2, (e) -COOR2 or (f) A, where: R21) Hydrogen atom, (2) (C1-C10) alkyl, branched or straight chain, (3) (C1-C10) alkyl, branched or straight chain, replaced by one or more of the following:
3.1 Phenyl,3.2 Indolyl,3.3 Imidazolyl or3.4 Phenyl, replaced by hydroxyl one or more times,4) Phenyl or5) Naphthyl.
The preferred amine derivatives Y of formula Illa or Illb are:
A a peptide with 2 to 5 amino acid residues, R ethyl, replaced by
It consists of: 1) phenyl, 2) indoyl, 3) imidazolyl or 4) 4-hydroxyphenyl, R-[O-CH2-m]n-, with:
m the number 2 andn an integer from 20 to 50,X
(a) hydrogen atom, (b) -OR2, (c) -NHR2 or) -COOR2, wherein:R21) is hydrogen atom, (2) (C1-C5) alkyl or (3) phenyl.
In particular, preference is given to the amine derivatives Y of formula Illa or Illb, where:
A a peptide containing 2 to 5 amino acid residues from the group Thr or Arg,R
(a) Hydrogen atom, (b) Covalent bond, (c) Glucose or other) Polyethylene glycol with a molecular weight of 100 to 3000 g/mol,
(a) hydrogen atom or (b) -OR2, where R2 is tertiary butyl.
In particular, -[0-CH2]m]n residues with a molecular weight of 1500 g/mol are preferred.
Amino acid means a natural, genetically coded, non-natural, non-coded L or D amino acid.
Examples of an L or D amino acid are alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cysteine, methionine, ornithine, citrulline, arginine, lysine, asparagine, aspartic acid, glutamic acid, glutamine, phenylalanine, tyrosine, thyroxine, proline, hydroxyproline, tryptophan, histidine. Glycine and gamma-aminobutyric acid are other examples of an amino acid.
The invention also relates to a process for the preparation of compounds of formula I and 11, characterized by the transformation of aminer derivatives and hirudine in the presence of proteases, preferably serine proteases, preferably chemotrypsin, trypsin, trypsin-like enzymes or lysylendopeptidase.
Hirudines, known from:
The sequence protocol ID No: 1 is an example of the structure of a hirudin. The sequence protocol ID No: 1 shows the structure of a hirudin.
For example, the compounds described in the literature cited above, in particular those described in EP 171 024, EP 158 986, EP 209 061 and DE 38 05 540.6, are considered to be hirudines, e.g.:
The Commission shall, by means of implementing acts, lay down the rules for the application of this Regulation and the procedures for implementing it, in particular the rules on the application of the rules on the application of the rules on competition to undertakings, the rules on the application of the rules on competition to undertakings, the rules on the application of the rules on competition to undertakings, the rules on the application of the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the rules on competition to undertakings, the application of the rules on competition to undertakings, and the rules on competition to undertakings.
These hirudines may be produced by generally known methods of peptide chemistry or by equivalent known methods, or alternatively, the hirudines may be produced by genetically engineered methods known to the professional.
Err1:Expecting ',' delimiter: line 1 column 780 (char 779)
Compounds of formula II are formed from a hirudin and a suitable amine derivative of formula Illa or Illb in excess with the aid of chemotrypsin or a similar enzyme. Suitable amine derivatives are those that do not carry a free tyrosyl side chain. A transpeptidization takes place at the C-terminus of the tyrosine residue at position 63 of the amino acid sequence of the hirudin used. The dipeptide, consisting of the amino acids at positions 64 and 65 of the amino acid sequence of the underlying hirudin, is replaced by an amine derivative.
The amine derivative is used in excess. It is preferable to use a 50- to 150-fold molar excess of the amine derivative to the amount of hirudin. The amine derivative is preferably used in the highest possible concentration. The solvent is water or a mixture of water and a non-protonating organic solvent such as dimethylformamide. The mixture preferably contains less than 25% vol. (v/v) water.
The enzymes used may be of different biological origin, e.g. trypsin may be derived from pigs or bovine animals.
The enzyme catalyzed transformation of hirudin results in the corresponding esters of the hirudin derivative when amino acid or oligopeptide esters are used as the amine derivative. If desired, the ester groups can be separated by conventional methods.
The enzyme-catalyzed semisynthesis of hirudin derivatives according to the invention is a process that complements genetic engineering methods to modify the structure of hirudin. In particular, the semisynthesis can be used to target the introduction of non-coding amino acids such as D-amino acids or any amino derivatives.
The method of the invention allows, for example, the production of a defined hirudin polyethylene glycol derivative of formula 1 or 11, which can be used as a medical deposit; a polyethylene glycol esterified with an amino acid or an oligopeptide can be converted into an amine derivative with a hirudin very selectively, so that the polyethylene glycol residue is bound only once at a defined position.
Other hirudin derivatives manufactured according to the invention may be of therapeutic use for transdermal application, e.g. lontophoresis.
The invention also relates to medicinal products containing an effective amount of at least one compound of formula I and/or formula II.
The invention also relates to the use of the compounds of formula I and formula II as anticoagulants.
The following examples using genetically engineered desulphateTyr63-hirudin (sequence protocol ID No: 1) are intended to illustrate the invention, but are not limited to them.
Err1:Expecting ',' delimiter: line 1 column 410 (char 409)
3 g of genetically produced desulphate tyr63-hirudin (sequence protocol SEQ ID NO: 1) together with 20 g of Thr ((But) OBut acetate (threonine with tert. butyl protective groups; see EP 0056951 for manufacture) are dissolved in 6 ml of water and mixed with 190 mg of chemotrypsin (Merck, Darmstadt) and the pH of the solution is set to 4.5 and the reaction mixture is stored at 4°C for 72 hours.
The resulting precipitate is centrifuged, the residue is washed again with a methanol/diethyl ether mixture (1:2 v/v), centrifuged with ether and dried at reduced pressure.
The raw product consists of unrealized hirudin, the desired hirudin derivative and by-products obtained by enzymatic cleavage.
The raw product is chromatographically separated on a preparative HPLC column (5 x 25 cm, stationary phase is a reverse phase C,8-material) by gradient elution with acetonitrile/water/trifluoroacetic acid. (Hirudin 1-63)-Thr (But) OBut elutes due to lipophilic tert. butyl protecting groups with significantly delayed retention time. The fraction containing the pure intermediate is collected, acetonitrile is evaporated under reduced pressure and the aqueous solution is dried.
The resulting hirudin derivative is dissolved in 20 ml of 95% TFAC and kept at room temperature for 45 minutes, then mixed with 100 ml of dimethyl ether, which is frozen.
The following information is provided for the purpose of the analysis:
The amino acid analysis of the product corresponds to the composition expected from the amino acid sequence.
3 g of genetically derived desulphate-Tyr63-hirudin (sequence protocol ID No 1) is converted as in example 2 to 41 g of Thr ((But) arg-arg (synthesised by standard methods) and 170 mg of chemotrypsin (Merck, Darmstadt).
After the reaction is completed, the mixture is mixed with 300 ml of methanol and 150 ml of dimethyl ether. The decentrifying precipitate is washed three times with methanol/ether and then with ether and dried in a vacuum. Output: 4.8 g of raw product. The HPLC (stationary phase C↑ε silica gel, solvent acetonitrile/H20/trifluoroacetic acid) gives the following composition: 65.2% (hirudin 1-63)-thr ((But) (64)-argg ((65)-argg ((66)-OH); 3.9% (hirudin-65)); 18.7% cleavage products. Chromatographic cleaning and isolation of the product is carried out as in example 2.
The following shall be added to the list of substances which are to be used for the purposes of the determination of the concentration of the active substance:
Purity: 90.3%
The separation of the tertiary butyl protective group from the threonine side chain is known as in Example 2. yield: 1,2 g (hirudinl-63) -Thr(64) -Arg(65) -Arg(66) -OH (see sequence protocol SEQ ID NO:3).
The amino acid analysis of the product corresponds to the composition expected from the amino acid sequence.
In the determination of anticoagulant effects after intravenous administration in rhesus monkeys (1 mg/kg), this hirudin derivative shows almost the same efficacy as the starting hirudin despite changes in the isoelectric point of the hirudin derivative (see test conditions: J. Jürgens, German Arch. Klin. Med. 200, 67 (1952).
3 g of genetically produced desulphate-Tyr63-hirudin (sequence protocol SEQ ID NO: 1) is converted as in example 2 with 20 g of Thr ((But) OBut acetate and 200 mg of trypsin (Merck, Darmstadt).
After 72 hours, the reaction is interrupted and processed as in example 2. yield: 4.2 g of raw product with the following composition: 55 % (hirudin 1-36) -Thr (But) OBut, (hirudin 37-65); 31 % unrealized hirudin; 15 % cleavage products.
The separation of the tert-butyl protective groups from the threonine residue is as in example 2, yield: 1,3 g (hirudin 1-36) -Thr, (hirudin 37-65). The structure of the first chain of the hirudin derivative, (hirudin 1-36) -Thr, is represented by SEQ ID NO:4, the structure of the second chain of the hirudin derivative, (hirudin 37-65), by sequence protocol SED ID NO:5.
The amino acid analysis of the product corresponds to the composition expected from the amino acid sequence. Edman sequence analysis yields 2 N-terminal amino acid residues Leu (1) and Asn (37). In determining the anticoagulant effect after intravenous administration in rhesus monkeys, this hirudin derivative surprisingly shows almost the same potency as the parent shirudin despite significant structural changes.
H•Thr-O-PEG1500 is produced by conversion of polyethylene glycol monomethylether and tert-butoxycarbonyl-threonine, e.g. in a single-shot reaction with hydroxybenzotriazole and dicyclohexylcarbodiimide by known peptide chemical methods.
3 g of genetically modified desulphate tyr63-hirudin (sequence protocol SEQ ID NO: 1) are reacted with 80 g H•Thr-O-PEG1500 as in example 2 with 250 mg of chemotrypsin.
After completion of the reaction, dilute the reaction mixture with 200 ml of methanol and remove unrefined hirudin and PEG-hirudin by addition of dimethyl ether.
The decentrifying raw product is washed three times with a mixture of methanol/ether (2:1, V/V) and dried at reduced pressure. The separation of PEG-hirudin and hirudin can be done as in example 2 by the preparative HPLC or better by chromatographic cleaning at a cation exchange column (Fractogel-EMD-S03; Merck, Darmstadt).
The amino acid analysis of the product corresponds to the composition expected from the amino acid sequence.
The presence of PEG conjugation is demonstrated by chymotryptic digestion in dilute aqueous solution and subsequent thin-film chromatographic comparison with Thr-O-PEG and free threonine, where no free threonine can be detected within the analytical limits.
3 g of genetically modified desulphate tyr63-hirudin (sequence protocol SEQ ID NO:1) together with 25 g of D-glu ((OBut) 2-acetate (Sigma, Deisenhofen) are dissolved in 6 ml of water and added to 190 mg of trypsin.
For the structure of the product consisting of two peptide chains, see sequence protocols for the sub-sequences SEQ ID NO:7 and SEQ ID NO: 5.
3 g of genetically engineered desulphate tyr63-hirudin (sequence protocol SEQ ID NO:1) together with 30 g of D-glu ((OBut) 2-acetate (Sigma, Deisenhofen) are dissolved in 6 ml of water and mixed with 190 mg of chemotrypsin.
The further processing and processing is carried out in the same way as in example 2.
The structure of the product consists of a chain (sequence protocol SEQ ID NO: 8.
(1) GENERAL INFORMATION: The following information is provided for the purpose of the examination:
(i) The applicant:
(A) NAME: Hoechst Aktiengesellschaft (B) STREET: (C) LOCATION: Frankfurt am Main (E) COUNTRY: Federal Republic of Germany (F) POST CALL: 65926 (G) TELEPHONE: 069-305-6047 (H) TELEFACS: 069-35-7175 (I) TELEX: 041234-700 (ii) RECORDING TITLE: Hirudine derivatives and the method of their manufacture (iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER-LESBARE FORM:
(a) Data carrier: floppy disk (b) computer: IBM PC compatible (c) operating system: PC-DOS/MS-DOS (d) software: patent in release #1.0, version #1.25 (EPA) (e) information about the SEQ
(i) Sequence characteristics:
(a) LENGTH: 65 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (vi) Origin:
(A) Organic: Desulphate of tyr63-hirudin (ix) Characteristics:
(A) NAME/KEY: Protein (B) LOCATION: 1..65 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 The following is a list of the active substances that are to be used in the preparation of the product:
The Commission shall, by means of implementing acts, lay down the rules for the application of this Regulation and the procedures for the application thereof.
(i) Sequence characteristics:
(a) LENGTH: 64 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (vi) Origin:
(A) Organism: Hirudin derivative (ix) Characteristics: The substance is a mixture of the following:
(A) NAME/KEY: Protein (B) Location: 1..64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 is replaced by the following:
The Commission shall, by means of implementing acts, lay down the rules for the application of this Regulation and the procedures for implementing it.
(i) Sequence characteristics:
(a) LENGTH: 66 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (vi) Origin:
(A) Organism: Hirudin derivative (ix) Characteristics: The substance is a mixture of the following:
(A) NAME/KEY: Protein (B) Location: 1..66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 is replaced by the following:
The Commission shall, by means of implementing acts, lay down the rules for the application of this Regulation and the procedures for the application thereof.
(i) Sequence characteristics:
(a) LENGTH: 37 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (ix) Characteristic:
(A) NAME/KEY: Protein (B) Location: 1..37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 . The following is a list of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the seeds of the se the seeds of the se se se se se se of the se se se of the se se .
The following information is provided for the purpose of the application:
(i) Sequence characteristics:
(a) LENGTH: 29 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (ix) Characteristic:
(A) NAME/KEY: Protein (B) LOCATION: 1..29 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 The following is a list of the active substances that are to be used in the preparation of the product:
The following information is provided for the purposes of this Regulation:
(i) Sequence characteristics:
(a) LENGTH: 64 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (vi) Origin:
(A) Organism: Hirudin derivative (ix) Characteristics: The substance is a mixture of the following:
(A) NAME/KEY: Protein (B) Location: 1..64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 is replaced by the following:
The Commission shall, by means of implementing acts, lay down the rules for the application of this Regulation and the procedures for implementing it.
(i) Sequence characteristics:
(a) LENGTH: 37 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (ix) Characteristic:
(A) NAME/KEY: Protein (B) Location: 1..37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 is replaced by the following:
The following information is provided for the purpose of the application:
(i) Sequence characteristics:
(a) LENGTH: 64 amino acids (b) TYPE: amino acid (d) Topology: linear (ii) TYPE of molecule: protein (vi) Origin:
(A) Organism: Hirudin derivative (ix) Characteristics: The substance is a mixture of the following:
(A) NAME/KEY: Protein (B) Location: 1
Claims (9)
1. compound of formula I or II,
Other
The Commission has received comments from the Member States on the use of the active substance in the Union.
The following substances are to be classified as 'chemical agents' in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council:
A1 is an amino acid residue,
A2 is an amino acid residue,
AO is an amino acid residue or hydrogen atom,
Y is an amine derivative of the formula Illa or Illb:
H2N-R-X (Illa),
The following shall be used for the calculation of the maximum mass of the vehicle:
The following shall be included:
Other
A
Other
(a) an amino acid residue,
(b) a peptide containing 2 to 10 amino acid residues,
R
a. Alkyl (C1-C10) branched or straight-chain,
(C1-C10) alkyl, branched or straight-chain, with one or more independent substituents:
Other
1) Phenyl,
2) Indolyl,
3) Imidazolyl or
4) Phenyl, one or more times substituted by hydroxyl,
(c) Phenyl or
(d) Naphthyl,
R
Other
(a) hydrogen atom,
(b) covalent bonding,
(c) sugars such as glucose, fructose, mannose, galactose, ribose, ribulose or xylose;
(d) Polysaccharide containing 2 to 10 sugars, or
(e) -[O-CH2) n-, where m is an integer of 2, 3, 4 or 5 and n is an integer from 1 to 100, and
X
Other
(a) hydrogen atom,
(b) -OR2,
(c) -SR2,
(d) -NHR2,
(e) -COOR2 or
(f) A, which includes:
Other
R2
1) hydrogen atom,
2) (C1-C10) alkyl, branched or straight-chain,
3) (C1-C10) alkyl, branched or straight-chain, with one or more substituents:
Other
3.1 Phenyl
The following is a list of the active substances:
3.3 Imidazole or
3.4 Phenyl, single or multiple substitution of hydroxyl,
4) Phenyl or
(5) Naphthyl.
2. compound of formula I or II as described in claim 1, having:
Other
A a peptide with 2 to 5 amino acid residues,
Rethyl, replaced by
Other
1) Phenyl,
2) Indolyl,
3) Imidazolyl or
4) 4-hydroxyphenyl,
-[O]] (CH2) m]n-, wherein:
Other
m the number 2 and
n is an integer from 20 to 50,
X
Other
(a) hydrogen atom,
(b) -OR2,
(c) -NHR2 or
(d) COOR2 is:
Other
R2
1) hydrogen atom,
2) (C1-C5) alkyl or
3) Phenyl.
3. compound of formula I or II as described in claim 1 or 2, where:
Other
A a peptide containing 2 to 5 amino acid residues from the group Thr or Arg, R1
(a) hydrogen atom,
(b) covalent bonding,
(c) Glucose or
(d) Polyethylene glycol with a molecular weight of 100 to 3000 g/mol,
X
Other
(a) hydrogen atom or
(b) -OR2, where R2 is tertiary butyl.
4. compound of formula I or II according to one or more of claims 1 to 3, where R is a polyethylene residue with a molecular weight of 1500 g/mol.
5. a process for the preparation of the compounds described in claims 1 to 4, characterised by the reaction of an amine derivative of formula Illa or Illb with hirudin in the presence of proteases.
6. a process as described in claim 5, characterised by the use of a protease from the group of chemotrypsins, trypsins or trypsin-like enzymes such as lysyl endopeptidase.
7. medicinal products containing a compound as defined in claims 1 to 4.
Use of the compound of formula I or II according to claims 1 to 4 to produce a medicinal product with anticoagulant properties.
Use of the product as claimed for transdermal application, in particular by lontophoresis.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4404168 | 1994-02-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HK1012012A true HK1012012A (en) | 1999-07-23 |
Family
ID=
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