GB2220938A - Use of PAM enzyme in solid phase peptide synthesis - Google Patents
Use of PAM enzyme in solid phase peptide synthesis Download PDFInfo
- Publication number
- GB2220938A GB2220938A GB8817265A GB8817265A GB2220938A GB 2220938 A GB2220938 A GB 2220938A GB 8817265 A GB8817265 A GB 8817265A GB 8817265 A GB8817265 A GB 8817265A GB 2220938 A GB2220938 A GB 2220938A
- Authority
- GB
- United Kingdom
- Prior art keywords
- peptide
- gly
- enzyme
- synthesis
- trp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000004190 Enzymes Human genes 0.000 title claims description 15
- 108090000790 Enzymes Proteins 0.000 title claims description 15
- 238000010647 peptide synthesis reaction Methods 0.000 title description 2
- 239000007790 solid phase Substances 0.000 title description 2
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 title 1
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 54
- 238000000034 method Methods 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 150000001408 amides Chemical class 0.000 claims description 7
- 210000004899 c-terminal region Anatomy 0.000 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 6
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- 208000037196 Medullary thyroid carcinoma Diseases 0.000 claims description 4
- 208000023356 medullary thyroid gland carcinoma Diseases 0.000 claims description 4
- 208000013818 thyroid gland medullary carcinoma Diseases 0.000 claims description 4
- 102000008109 Mixed Function Oxygenases Human genes 0.000 claims description 3
- 108010074633 Mixed Function Oxygenases Proteins 0.000 claims description 3
- 229920001192 peptidylglycine Polymers 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims 2
- 239000011347 resin Substances 0.000 description 18
- 229920005989 resin Polymers 0.000 description 18
- 102000004196 processed proteins & peptides Human genes 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 9
- 230000007017 scission Effects 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- UHPQFNXOFFPHJW-UHFFFAOYSA-N (4-methylphenyl)-phenylmethanamine Chemical compound C1=CC(C)=CC=C1C(N)C1=CC=CC=C1 UHPQFNXOFFPHJW-UHFFFAOYSA-N 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 238000002523 gelfiltration Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 102000016938 Catalase Human genes 0.000 description 3
- 108010053835 Catalase Proteins 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940072107 ascorbate Drugs 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- MGHPNCMVUAKAIE-UHFFFAOYSA-N diphenylmethanamine Chemical compound C=1C=CC=CC=1C(N)C1=CC=CC=C1 MGHPNCMVUAKAIE-UHFFFAOYSA-N 0.000 description 3
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000002953 preparative HPLC Methods 0.000 description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000000637 Melanocyte-Stimulating Hormone Substances 0.000 description 2
- 108010007013 Melanocyte-Stimulating Hormones Proteins 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 238000005915 ammonolysis reaction Methods 0.000 description 2
- 150000008064 anhydrides Chemical class 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- -1 phenylacetamidomethyl Chemical group 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- ZQEBQGAAWMOMAI-ZETCQYMHSA-N (2s)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)(C)OC(=O)N1CCC[C@H]1C(O)=O ZQEBQGAAWMOMAI-ZETCQYMHSA-N 0.000 description 1
- SZXBQTSZISFIAO-ZETCQYMHSA-N (2s)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)OC(C)(C)C SZXBQTSZISFIAO-ZETCQYMHSA-N 0.000 description 1
- VRPJIFMKZZEXLR-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(O)=O VRPJIFMKZZEXLR-UHFFFAOYSA-N 0.000 description 1
- HBAHZZVIEFRTEY-UHFFFAOYSA-N 2-heptylcyclohex-2-en-1-one Chemical compound CCCCCCCC1=CCCCC1=O HBAHZZVIEFRTEY-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N Aspartic acid Chemical compound OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 229910021583 Cobalt(III) fluoride Inorganic materials 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 229920001367 Merrifield resin Polymers 0.000 description 1
- NFVNYBJCJGKVQK-ZDUSSCGKSA-N N-[(Tert-butoxy)carbonyl]-L-tryptophan Chemical compound C1=CC=C2C(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)=CNC2=C1 NFVNYBJCJGKVQK-ZDUSSCGKSA-N 0.000 description 1
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 1
- 239000007994 TES buffer Substances 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- WZJQNLGQTOCWDS-UHFFFAOYSA-K cobalt(iii) fluoride Chemical compound F[Co](F)F WZJQNLGQTOCWDS-UHFFFAOYSA-K 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- RJSZPKZQGIKVAU-UXBJKDEOSA-N peptide f Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C(C)C)C(C)C)C1=CC=CC=C1 RJSZPKZQGIKVAU-UXBJKDEOSA-N 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Description
USE OF PAM ENZYME IN SOLID PHASE PEPTIDE SYNTHESIS no,938 222- The present
invention relates to the preparation of amidated peptides.
C-Terminal amidated peptides account for about 50% of secretory peptides of biological interest. The OC-amide group confers a degree of stability against "in vivo" proteolytic degradation and is, often, essential for the biological activity. The-substitution of the amidated amino acid residue with a non-amidated one results in most cases in drastic reduction of the biological activity.
The solid-phase synthesis of peptide amides is accomplished either by ammonolysis of peptides anchored on a Merrifield resin or, more often, by HF cleavage of peptides linked to benzhydrylamine-type resins. Neither method is without drawbacks.
- The ammonolysis procedure is unsatisfactory if the C-terminal residue is sterically hindered (as in the case of Ile or Val). The procedure cannot be applied to peptides containing aspartic or glutamic acid with the side-chain carboxy functions protected by esterification, because they would be converted to the corresponding asparagine and glutamine residues. These problems have led to the development of amine resins suitable for the direct synthesis of C-terminal amidated peptides. The benzyhydrylamine (BHA) or the p-methylbenzhydrylamine (MBHA) i resins, which have been widely used as support for the synthesis of peptide amides,-give the desiredOL-carboxyamide by RF cleavage. However, the cleavage of the peptide-resin bond depends markedly upon the nature of the C-terminal residue and the length of the sequence.
Usually, the greater is the length of the peptide chain and the steric hindrance of the amino acid residue attached to the solid support, the lower are the cleavage yields. Peptides ending with glycine (the least bulky amino acid residue) are generally cleaved from the resin more easily than analogous peptides terminating with a different amino acid residue, resulting in higher yields and better quality products.
The present invention provides a two-steps process for the synthesis of an amidated peptide, which process comprises preparing the corresponding C-terminal Glyextended peptide by solid-phase synthesis and converting enzymatically the said Gly-extended peptide to the corresponding desglycine peptide amide.
The enzyme used in the present invention is the enzyme responsible in nature for the post-translational amidation of secretory peptides. The enzyme, "peptidylglycine,CC-amidating monooxygenase" (PAM), occurs in many -tissues of different animal species. Originally discovered in the pig pituitary (Bradbury et al., Nature, 1 Vol, 298, Pages 686-688, 1982), it catalyzes the transformation of a C- terminal glycine to glyoxylic acid, leaving the amino nitrogen of the Gly- residue as the amide function of the preceding amino acid.
The affinity of the enzyme for peptide substrates is largely dependent upon the penultimate amino acid residue. Bulky lipophilic substituents, difficult to cleave from a polymeric support, are thosse with a higher affinity for the enzyme. The enzyme may be used in solution or in immobilised form. Typically, ascorbate, copper 2+ ions and catalase are present when the enzyme is used in solution.
Peptides which end at their C-terminus by the group -CONE 2 rather than by a carboxyl group can thus be readily prepared. The present invention not only permits better quality products to be obtained in higher yields but also makes it possible to use a single type of resin for the preparation of both C-terminal amidated and C-terminal free carboxyl peptides.
As an example, the pentapeptide H-Trp-Gly-Lys-ProVal-NH 2 [bL-MSH-(9-l3)1 has been prepared by automatic solidphase synthesis both directly and via enzymatic conversion of the corresponding Gly-extended precursor. In the first case the peptide was synthesized on a MBHA resin, followed by EF cleavage. In the second one, the Gly-extended precursor was obtained by HF cleavage of-the peptide f assembled on a phenylacetamidomethyl (PAM) resin and converted to the corresponding des-glycine peptide amide by means of an OC-amidating enzyme solution containing ascorbate, copper and catalase. The enzymatic reaction was performed at 37 0 C and monitored by HPLC. In both cases the product was recovered using standard procedures such as gel filtration, ion-exchange chromatography and preparative HPLC.
Although in this particular example the enzymatic conversion was performed using a protease-free preparation of enzyme extracted from a rat medullary thyroid carcinoma, c_-amidating enzymes from different sources or obtained by r-DNA techniques, both in solution and in immobilized form, can be used for the same purpose.
Yields were calculated taking into account either the net peptide content and purity (theoretical yields) or the net peptide content only (actual overall yield)r and are referred to the commercially available starting materials: 0.5 mmol of MBHA resin and 0.5 mmol of Boc-Gly-PAM resin, respectively. Net peptide content, determined by amino acid analysis, represents -the exact quantity of peptide to the exclusion of the associated counter-ions (such as trifluoroacetate or hydrofluoride),, and humidity or traces of non-peptidic material resulting from the preparation process.
i Peptide purity was determined by HPLC analysis, using a Hewlett-Packard 1084 B apparatus equipped with a uv detector operating at 210 nm. Separation was carried out on a 3.9 x 250 mm Bondapak C 18 column by the pair of solvent A (0. 01M VaH 2 PO 4 adjusted to pH 7.0 with 1N NaOH) and solvent B (CH 3 CN). The elution was programmed with a linear gradient from 10% B to 70% B over a period of 10 min and then isocratically for 15 min, with a flow rate of 1.5 ml/min. 'The peptides were characterized by their retention time.
Symbols and abbreviations are those commonly used in peptide chemistry (see Eur. J. Biochem. (1984) 138, 9-37). Consequently, the three-letter amino acid symbols denote the L configuration of chiral amino acids. Other symbols and abbreviations are: AA, amino acid; BHA, benzhydrylamine; BOC, tert-butyloxycarbonyl; DCC, N,N'-dicyclohexylcarbodiimide; DIPEAr N, N-,diisopropylethylamine; DMF, dimethy1formamide; FAB-MS, fast atomic bombardment-mass spectrometry; HPLC, high performance liquid chromatography; MBHA, 4-methylbenzhydrylamine; MSH, melanocyte stimulating hormone; PAM (resin), phenylacetamidomethyl; PAM (enzyme), peptidylglycine 6C-amidating monooxygenase; TES, Ntris[hydroxymethyllmethyl-2-aminoethanesulfonic acid; TFA, trifluoroacetic acid; n.d., not determined; Z, benzyloxycarbonyl.
The direct synthesis of Ot-MSH-(9-13) is described in the Reference Example below. The Example below describes the synthesis of vC-MSH-(9-13) according to the invention.
REFERENCE EXAMPLE One-step synthesis of H-TrpGly-Lys-Pro-Val-NH 2 The peptide was prepared starting from 0.5 mmol of MBHA resin (1.04 g7 0. 48 mmol/g) by step-wise addition of Boc-Val-OH, Boc-Pro-OH, Boc-Lys(Cl-Z)OHI Boc-Gly-OH and Boc-Trp-OH, according to the following protocols. The synthesis was carried out in an Applied Biosystems 430A automatic peptide synthesizer.
f 1 - 6 Protocol for step-wise synthesis Step Reagents and Operations Mix times Min.
1 CH.Cl. wash, 6 ml (3 times) 2 60% TFA in CHzC1n, 7 mI 1 3 60% TFA in CH2C12, 7 ml 4 CH.Cl,, wash, 6 ml (3 times) 10% DIPEA in DMF, 10 ml 6 10% DIPEA in DMF, 10 ml 2 7 DMF wash, 10 ml (4 times) 1 8 Pre-formed Boc-AA symmetrical anhydride (1 mmol) in 10-15 ml 20 to 30 of DMF - 7 Protocol for-symmetrical anhydride preparation Step Reagents and operations Mix times Min.
1 Boc-AA (2 mmol) and DCC (1 mmol) about 7 in CH..Cl, (8-10 ml) 2 Transfer to the concentrator vessel and evaporation 3 Transfer to the reaction vessel after DMF solubilization At the end of the synthesis 1.4 g of dried peptide-resin was obtained.
1.0 g of this peptide resin were treated with anisole (2 rnl), thioanisole (3 ml) and HF distilled over CoF3 (20 ml) for 1 hour at OOC. After EF elimination under vacuum, the residue was washed with diethyl ether (100 ml). The peptide was recovered from the resin by extraction with 10% acetic acid (50 ml) and lyophilization (203 mg; net peptide content, 67.9%; purity, 66.6%; theoretical yield, 44%).
1 8 - After desalting on a 2.5 x 80 cm Sephadex G-15 column, crude product (104 mg) were obtained by lyophilization. The material was further purified by preparative HPLC (20-25 mgIbatch) on a 2.5 x 25 cm Lichrosorb RP-18 71L column using the following eluents: A, 0.05% TFA; B, 0.05%TFAICH3W 3:7 by vol. Elution was accomplished by a linear gradient f rom 10% to 90% B in 15 min. Fractions of comparable purity were pooled and lyophilized. 24.2 mg of H-Trp-Gly-Lys-Pro-ValNH.. as di-trifluoroacetate were obtained (net peptide content, 64.1%; actual overall yield, 7.4% for a peptide of 95% purity).
Amino acid ratios after 6 N HCl hydrolysis: Pro, 1.14 (1); Gly, 0.82 (1); Val 1; Lys, 0.97 (1); Trp, n.d.; FAB-MS, 585 (MH'); RT 8.9.
EXAMPLE Two-steps synthesis of H-Trp-Gly-Lys-Pro-Val-NH, i) Synthesis of H-Trp-Gly-Lys-Pro-Val-Gly-OH The peptide was prepared starting from 0.5 mmol of Boc-Gly-PAM resin (0. 62 g; 0.68 mmollg), operating as described in example I.
At the end of the synthesis, 0.89 g of dried peptide resin were obtained. A 0.5 g sample of this peptide resin, treated as described in example I, gave 220 mg of crude H-Trp-Gly-Lys-Pro-Val-Gly-OH as di-hydrofluori- - 9 de (netpeptide content, 83.9%; purity, 76.3%; theoretical yield in Gly-extended peptide referred to the starting Boc-Gly-PAM resin, 78.0%). By gel-filtration on a Sephadex G 15 column, 127.6 mg of E-Trp-Gly-LysPro-Val-Gly-OH were obtained (net peptide content, 60.4%; purity, 97.0%; theoretical yield, 41.4%).
ii) Enzymatic conversion to H-Trp-Gly-Lys-Pro-Val-KH2 -_ -W -10,.mg of gel-filtered H-Trp-Gly-Lys-Pro-Val-Gly-OH were..". 9. -, _A%' I Z iieated with a semi-purified protease-free preparation of PAM enzyme from rat medullary thyroid carcinoma (MTC) containing 39.4 mU of enzyme.
The conversion was performed in 0.05 M TES buffer at pH 7.0 containing 0. 01% Tween, 50 mM KI, 2liM CuSO,, 3 mM ascorbate, 1% EtOH and 800;ig catalase. After 5 hours the reaction mixture was diluted 1 to 10 with water, filtered and the pH adjusted to 6.7. The solution was loaded on a 1 x 7 cm CM-Sephadex C-25 column previously equilibrated with 0.02 M ammonium acetate at pH 6.7, and eluted with a linear gradient from 0.02 to 0.5 M ammonium acetate. Fractions containing the target peptide were pooled and lyophilized: 5.2 mg of H-Trp-Gly-Lys-Pro-Val-NH, were obtained (net peptide content, 70.1%; actual overall yield, 28.9% for a peptide of 98% purity).
Amino acid ratios: Pro 0.97 (1); Gly 1.07 (1); Val 1; Lys 0.98 (1); Trp n. d.; FAB-MS, 585 (MH'); RT 8.9.
- 10 In another experiment, starting from -5 mg of H-Trp-GlyLys-Pro-Val-Gly-OH directly before gel-filtration, 3.3 mg of H-Trp-Gly-Lys-Pro-Val-NH2 were obtained (net peptide content, 65.0%; purity, 100%; actual overall yield, 57.4% for a peptide of 100% purity).
1. Comparlson of purity wid yields In the preparation of a-WR-(9-13) by ffifferent synthetic procedures.
product Peptide content HPLC purity theoreticalactual Overall % % yields % yield % one, M_process NF cleavage H-'ftp-(;IT-LIS-PrO-Val-)IH2 67.9 66.6 44.0 gel filtration, U n.d. n.d. U.d.
preparative HPLC to 64.1 95.0 7.0 7.4 two-stem Vrocess w cleavage H-Trp--Gly-LTS-PrG-V&1--GlT-OH 83.9 76.3 78.0 gel filtration of If 60.4 97.0 41.4 - enzymatic conversion 8D4 H-Trp-Gly-LTS-Pm-Val-NH, 70.1 98.0 28.3 28.9 ion-exchange dummatograpby HP cleavage H-Trp-(;Jy-Lys-Pro-GIT-M 83.9 76.3 78 enzygntic cmvmian and H-Trp-Gly-Lys-PW-Val-NH2 65.0 100 SM 57.4 ion-embange t The results reported in Table 1, relevant to the pentapeptide amide cL- MSH-(9-13), are suggestive of better yields and higher purity for the two- steps process based on the enzymatic conversion of the glycine-extended precursor. The advantage of this process over the direct solid-phase synthesis of cL-amidated peptides is expected to be greater and greater with the increase of the peptide, chain length.
t
Claims (4)
1. A process for preparing an amidated peptide, which process comprises preparing the corresponding C-terminal Gly-extended peptide by solidphase synthesis and converting enzymatically the said Gly-extended peptide to the corresponding des-glycine peptide amide.
2. A process according to claim 1 in which the enzymatic conversion is carried out with the peptidylglycine OC-amidating monooxygenase.
3. A process according to claim 2 in which the enzyme has been extracted from rat medullary thyroid carcinoma.
4. A process for preparing an amidated peptide, said process being substantially as hereinbefore described in the Example.
Published 1990atTl?e Patent Office. State House. 6671 High Holborn, LondonWCIR4TP. Further copies maybe obtainedfrom The Patent Office.
Sales Branch, St Mary Cray. Orpington, Kent BR5 3AD. Printed by Multiplex techniques ltd. St Mary Cray, Kent. Con. V87
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8817265A GB2220938B (en) | 1988-07-20 | 1988-07-20 | Use of pam enzyme in solid phase peptide synthesis |
| DE3923583A DE3923583A1 (en) | 1988-07-20 | 1989-07-17 | USE OF PAM ENZYME IN SOLID PHASE PEPTIDE SYNTHESIS |
| IT8921210A IT1231149B (en) | 1988-07-20 | 1989-07-17 | USE OF PAM ENZYME IN THE SOLID PHASE SYNTHESIS OF PEPTIDES. |
| JP1187225A JPH0297398A (en) | 1988-07-20 | 1989-07-19 | Use of pam enzyme in solid phase peptide synthesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8817265A GB2220938B (en) | 1988-07-20 | 1988-07-20 | Use of pam enzyme in solid phase peptide synthesis |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB8817265D0 GB8817265D0 (en) | 1988-08-24 |
| GB2220938A true GB2220938A (en) | 1990-01-24 |
| GB2220938B GB2220938B (en) | 1992-01-29 |
Family
ID=10640781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8817265A Expired - Fee Related GB2220938B (en) | 1988-07-20 | 1988-07-20 | Use of pam enzyme in solid phase peptide synthesis |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JPH0297398A (en) |
| DE (1) | DE3923583A1 (en) |
| GB (1) | GB2220938B (en) |
| IT (1) | IT1231149B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2233978A (en) * | 1989-07-17 | 1991-01-23 | Erba Carlo Spa | Cofactors for enzymatic amidation |
| GB2246133A (en) * | 1990-07-12 | 1992-01-22 | Erba Carlo Spa | Amidated human pro-urokinase enzymes and their precursors |
| US10196601B2 (en) | 2013-08-20 | 2019-02-05 | Lek Pharmaceuticals D.D. | Cell culture medium and process for controlling α-amidation and/or C-terminal amino acid cleavage of polypeptides |
-
1988
- 1988-07-20 GB GB8817265A patent/GB2220938B/en not_active Expired - Fee Related
-
1989
- 1989-07-17 DE DE3923583A patent/DE3923583A1/en not_active Withdrawn
- 1989-07-17 IT IT8921210A patent/IT1231149B/en active
- 1989-07-19 JP JP1187225A patent/JPH0297398A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2233978A (en) * | 1989-07-17 | 1991-01-23 | Erba Carlo Spa | Cofactors for enzymatic amidation |
| GB2233978B (en) * | 1989-07-17 | 1993-03-03 | Erba Carlo Spa | Cofactors for enzymatic amidation |
| GB2246133A (en) * | 1990-07-12 | 1992-01-22 | Erba Carlo Spa | Amidated human pro-urokinase enzymes and their precursors |
| US10196601B2 (en) | 2013-08-20 | 2019-02-05 | Lek Pharmaceuticals D.D. | Cell culture medium and process for controlling α-amidation and/or C-terminal amino acid cleavage of polypeptides |
Also Published As
| Publication number | Publication date |
|---|---|
| IT8921210A0 (en) | 1989-07-17 |
| JPH0297398A (en) | 1990-04-09 |
| DE3923583A1 (en) | 1990-01-25 |
| IT1231149B (en) | 1991-11-19 |
| GB8817265D0 (en) | 1988-08-24 |
| GB2220938B (en) | 1992-01-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| SU1651787A3 (en) | Method for preparation of polypeptide with properties of factor release of growth hormone | |
| USRE29842E (en) | Untriakontapeptide with opiate activity | |
| Andreu et al. | Solid‐phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa‐(4–24)] from skin secretion of Xenopus laevis | |
| Atherton et al. | Peptide synthesis. Part 7. Solid-phase synthesis of conotoxin G1 | |
| EP0477885A2 (en) | Parathyroid hormone derivatives | |
| OA11501A (en) | Process for the preparation of resin-bound cyclic peptides. | |
| CA1175372A (en) | Process for enzymatic replacement of the b-30 amino acid in insulins | |
| WO1993003056A1 (en) | Lanthionine bridged peptides | |
| Breddam et al. | Amidation of growth hormone releasing factor (1–29) by serine carboxypeptidase catalysed transpeptidation | |
| US4473555A (en) | Nona- and dodecapeptides for augmenting natural killer cell activity | |
| CN109306366B (en) | Method for synthesizing PT141 | |
| GB2220938A (en) | Use of PAM enzyme in solid phase peptide synthesis | |
| US6448031B1 (en) | Process for producing LH-RH derivatives | |
| Quibell et al. | Identification of coupling conditions proceeding with low C-terminal epimerization during the assembly of fully protected backbone-substituted peptide segments | |
| US5252705A (en) | Peptide derivatives | |
| JP2777193B2 (en) | Method for producing peptide | |
| IE53459B1 (en) | Antigenic linear peptide compounds | |
| CN111018963B (en) | Preparation method of glucagon | |
| TENO et al. | Amino Acids and Peptides. XIV. Synthesis and Biological Activity of Three S-Peptide Analogues of Bovine Pancreatic Ribonuclease A (RNase A) | |
| AU647796B2 (en) | An enzymatic process for the preparation of derivatives of growth hormone releasing factor and peptides useful as intermediates in the process | |
| Haskell-Luevano et al. | Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions | |
| DANHO et al. | Human Proinsulin, IV. Synthesis of a Protected Peptide Fragment Corresponding to the Sequence 1-23 of the Prohormone | |
| WO1992015695A1 (en) | Process for the c-terminal modification of peptides having a c_terminal penultimate proline residue | |
| AU3708697A (en) | Process for producing lh-rh derivatives | |
| GB2118190A (en) | Peptides with sauvagine-like activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19940720 |