• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A process for producing a peptide comprises reacting a substrate selected from amino acid esters, peptide esters, depsipeptides, optionally N-substituted amino acid amides and peptide amides, and optionally N-terminal protected peptides, with an amine component selected from amino acids, optionally N-substituted aminoacid amides and amino acid esters in the presence of a carboxypeptidase enzyme in an aqueous solution or dispersion having a pH from 5 to 10.5.
    公开号:SU1378785A3
    申请号:SU803217951
    申请日:1980-12-05
    公开日:1988-02-28
    发明作者:Таанинг Йохансен Джек；Видлер Фред
    申请人:Карлсберг Биотехнолоджи Лтд А/С (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to methods for the enzymatic production of peptides that can be used in the synthesis of biologically active compounds.
    The purpose of the invention is to simplify the process of enzymatic peptide synthesis.
    Starting materials
    Carbopepsidau-Y, obtained from baker's yeast, was isolated by chromatography using the means proposed by Johansen, and produced as a freeze-dried powder (10% enzyme in citric acid). Before use, the enzyme was desalted with Sephadex 025 fine (1, 5-25 cm), equilibrated and washed with distilled water. Enzyme concentration was determined spectrophotometrically provided
     2von% A stock solution was prepared with a concentration of 7 mg / ml (110 µmol), 5 stored at -21 ° C, in aliquots of 250-500 ml. Benzoyl alanine methyl ether (Bz-Ala-OMe), manufactured by Bachem, Lishtap, Switzerland, was used. Boron trifluoride ester complex (for synthesis), solvents and reagents (all analytical grade) were purchased from Merck, Darmstadt, FRG. All amino acids, amino acid amides and their derivatives were purchased from Sigma Chemical Company, San Luis, C1CA. Carbobenzyl hydroxyphenylalanine methyl ester (Z-Phe OMe) was prepared according to the procedure of Yamada et al. And used as a syrup. Phenylalanine hydrazide, alanine hydrazide and alanine hydrazide hydrochloride were made from ethers in accordance with the description of Los et al. Uncorrected t, pl. were 86-88 and 184-185 s (according to known data 82-83 and 184-185 ° C, respectively). Asparaginamide dihydrochloride was prepared from aspartic acid and diethyl ether according to Fisher using classical aminolysis (t.pl. 210-214 С, according to well-known data 214-215 s). Other raw materials were purchased from specified companies or produced in the same way.
    Definition of output products. Purity was quantified by TLC on silica gel 60.
    0
    Fg, (Merck). The solvent system CllClg (CK (CH ,,), OH (CHjCOOH) (11: 5: 2: 1) was used, and Pt was visually checked for fluorescence quenching to assess the composition of the reagents.
    The reagent composition was quantified on a HPLC phase inverter using a RP-8.10 µm column (Merck) -Hewlett Packard chromatograph 10845 fitted with a variable-wavelength recorder (model 79875, A). Separation was ensured by using appropriate specific
    gradients in elution systems ranging from 10% CHjCN to 10 mmol of NaAc (pH 4) to 100% or from 10 mmol of NaAc (pH 4) to 100%.
    Q The latter system was used for compounds such as Bz Ala-Gly-OH, Bz-Ala-Ala-OH, Bz-Ala-Ser-OH, and the corresponding amides. Flow rate 3 ml / min, column temperature
    5, the working wavelength is 260 nm. The yields were determined from the molar ratio of reagents, which was found over the integrated areas under the washout peaks.
    Identification of products. The spots were identified by cochromatography of thin layers with the corresponding standard compounds. Some products have been identified using a combination method.
    HPLC and amino acid analysis. After 10 min, 1 ml aliquot samples were taken from the reaction mixture and the reaction was stopped by adding 250 µl of 6 M HC1. After this, the pH was adjusted to 4 with NaOH and the mixture was separated by HPLC using Waters equipment including two pumps Solvent Software
     model .660j model b bK injector, model 450 variable wavelength recorder combined with a recorder (radiometer 61), or f Hewlett-Packard integrator,
    0 model 3380A, Washout control was performed by scanning at a suitable wavelength of 255 to 280. Chromatography was carried out using the reverse phase method with the help
    5 pillar waters s-18m-bondapak in
    20 vol% elution system (TEAP (tri-methyl ammonium phosphate buffer) in methanol with appropriate gradients and a flow rate of 1.5-2.0 ml / min.
    The buffer solution TEAR produced by the method of Riviera. In many cases, satisfactory results were provided in the system with 0.1 M HAC, pH 3, 20 vol%, and 0.1 M HAC, pH 3 in methanol.
    The effluent containing L-acyldipeptides was collected manually and adjusted to dryness by lyophilization or using a Buchi Rotowap apparatus at 35-45 ° C. Small residue samples were hydrolyzed in 6 M HC1 under vacuum for 36 hours. Evaporated hydrolysates were analyzed on the Darrum L-500 type amino acid analyzer.
    Synthesis with free amino acids as amine component
    Example 1.2 ml 0.6 M solution of valine 0.1 M KCl, 1 m EDTA at pH 9.8 is mixed with 100 μl (0.1 mmol) of 1 M solution of Bz-Ala-OMe (dissolved in 96% ethanol) . The reaction is carried out in a pH-stat at 35 ° C and a pH of 9.8, the pH being maintained at all times by the automatic addition of 0.5 M IOH. The reaction is initiated by the addition of 0.7 mg carboxypeptidase-U (150i / mg, manufactured by De ForeNedé Briegerie). After the reaction has proceeded for 30 minutes, the reaction is stopped by adjusting the pH to 1 with 6 M HC1. The reaction product is purified and separated by chromatography under high pressure. The yield of Bz-Ala-Val-OH is 40%. A quantitative analysis of the amino acid after hydrolysis of the product in 6 M HC1 for 24 hours gives, respectively, 1.0 mol of alanine and 1.0 mol of valine. .
    The effect of pH, temperature, and concentration of the substrate component, the amine component, and CPD-Y on the yield of Bz-Ala-Val-OH + CHjOH was studied using five sets of experiments carried out similarly to the procedure described. In each of the experiments, one of the indicated parameters was changed when the other four were constant: 0.6 M valine; 55 mmol Bz-Ala-OM 4.5 μmol CPD-Y; pH 9.7; 35 ° C. The results showed that the range of optimal pH values is rather narrow and is only 0.5 units. The pH, the increase in temperature, the reaction leads to an almost linear increase in synthesis due to the relatively weak hydrolysis. At temperature
    15
    25


    20 30 35 40
    45 Q
    five
    A pax above 45 decrease in enzyme activity and nonenzymatic hydrolysis of esters becomes unacceptable. At lower temperatures at pH up to 10.0, the hydrolysis of the esters was negligible during the 10 min reaction. However, it became significant with a decrease in the rate of the enzymatic reaction, when the reaction duration increased to 2-5 hours.
    The yield of Bz-Ala-Val-OH increased with increasing concentration of the amine component. An inverse relationship was observed between the yield and the concentration of the substrate component for Bz-Ala-OMe. This circumstance, taking into account the dependence of the yield on a high concentration of the enzyme, makes it possible to find the optimal ratio of the substrate to the concentration of the enzyme.
    In the presence of 0.5 M valine, the substrate Bz-Ala-OMe was quickly transformed (within 20 minutes) into 38% Br- / i.a-Val-OK and 62% Bz-Ala-OH. These numbers also indicate that the dipeptide is not. hydrolyzed at pH 9.7 in the presence of excess valine. If p11 was adjusted to 5-8, then the whole Bz-Ala-Val-OH hydrolyzed in a few seconds. Such a selective behavior of CPD-Y at high pH is a property that is important in the synthesis of peptides.
    Example2. 2 ml of a solution of 3 M lysine, 0.1 M KCl, 1 mM EDTA at pFi 9.8 are shifted from 400 μl of 100% methanol and 100 μl {0.1 mmol) of 1 M Z-Phe-OMe (dissolved in 100 % methanol). The reaction is carried out analogously to example 1, ionizing with the addition of 0.7 mg carboxypeptidase-U. After 30 minutes, the pH was adjusted to 1 with 6 M HC1. The reaction product is purified and obtained by high pressure chromatography. The yield of Phc-Lys-OH reaches 60%. A quantitative analysis of the amino acids produced as in Example 1 showed that the product contained 1 mole of lysine and 1 mole of phenylalanine.
    The peptides shown in Table 1 were made from the indicated starting materials, similarly to Examples 1 and 2. Experiments were performed in a radiometric pH-stat, and the yields were determined on an HPLC unit. The values maintained were: 4.5 µM CPD-Y; RP 9.7; .
    Synthesis with amides of amino acids as an amine component.
    And pimer 3, 2 ml of a solution of 0.6 M methioninamide (Met-NH), 0.1 M KCl, 1 mM EDTA at pH 9.8 are mixed with 100 μl (0.1 mmol) of a solution of 1 M Bz - Ala-OMe in 96% methanol. The reaction is carried out as in Example 1, initiated by the addition of 0.7 mg carboxypeptidase-Y. 30 minutes after the start of the reaction, the pH was adjusted to 1 with 6 M HC1. The reaction product is purified and isolated by high pressure chromatography. The output of Bz-Ala-Met-NH 95%. A quantitative amino acid analysis, as in Example 1, showed that the product contained 1.0 mol Ala, 1.0 mol Met, and 1.0 mol NH.
    Within 20 minutes, the substrate was almost completely converted to a dipeptide (95%), and 5% was hydrolyzed. to Bz-Al OH,
    11 example4. 2 ml of a solution of 0.4 valinamide (Val-NH), 0.1 M KCl, 1 Ml l EDTA at pH 9.5 are mixed with 100 μl (0.1 mmol) of a solution of 1 M Bz-A1a-OMe in 96% methanol. The reaction is carried out as in Example 1, The product of the reaction Bz-Ala-Val-NHg is precipitated during the reaction. 20 minutes after the start of the reaction, the pH was adjusted to 1 and the precipitate was separated by centrifugation. The product was dissolved in 1 ml of methanol and 5 purified and separated by high pressure chromatography. The yield of Bz-Ala-Val-NH is 95%, while in example I, when using the free amino acid as the amine component, it was equal to only 40%. A quantitative analysis of the amino acid, which is carried out as in example 1 , showed that the product contained 1.0 mol Ala, 1.0 mol Val, and 1.0 mol NH.
    Dependence of the reaction on the pH value and on the concentration of valinamide.
    Reactions) were carried out similarly to the described synthesis, and the constant parameters were: 0.6 M valines and; 55 mM Bz-Ala-OMe; 4.5 μM CPD-Y pH 9.7; 35 ° C.
    The results showed that the yield is almost independent of the concentration of valinamide.
    The effective pH value of the vapinos does not exceed 9, the pH value is 9.8
    0
    five
    0
    5 30 5
    0
    five
    50
    five
    is the upper limit, after which the output immediately decreases.
    Analogously to examples 3 and 4, the peptides are given in table. 2, is prepared from the specified source materials. Experiments are performed under the following conditions: 4 µM CPD-Y; pH 9.6; 35 C. The concentration of the substrate is given in table. 2
    Etc. and measure 5. Synthesis with amino acid hydrazides as the amine component.
    Analogously to examples 3 and 4, the peptides listed in Table 3 were prepared. Wali from the specified starting materials. Experiments were performed at 35 ° C, pH 9.6 in the presence of 4.5 μM CPD-Y. ..
    PRI me R 6. Synthesis with amino acid esters as the amine component.
    Experiments with amino acid esters are carried out in the same manner as in Examples 1-4: in a radiometric pH-stat at pH from 9.0 to 9.7 and 23-35 C. The products and the yield are determined by HPLC. The CPD-Y concentration ranged from 4.5 to 11 μM.
    In tab. 4 shows peptides derived from the indicated starting materials. It can be seen that in some cases some oligomerization took place. Since yields are usually very high, amino acid esters can be considered suitable for further limiting oligomerization.
    Example 7b-and D-stereoisomers as an amine component.
    The peptides listed in table. 5. were obtained from the original products in the same way as in examples 1-4 and 6.
    It can be seen that only the L-isomers are included. As a result, the process becomes economical, since the need to purify the starting amino acids to obtain a pure L isomer is eliminated.
    Example 8: Changes in the substrate ester group.
    The peptides listed in table. 6 were prepared from the indicated starting materials in the same manner as in Examples 1-4.
    The results prove the flexibility of the proposed method with respect to the choice of substrates.
    PRI me R 9. Depsipeptides as substrates.
    As in examples 3 and 4, the peptides listed in table. 7 were obtained from the indicated starting materials. The process was carried out in the presence of 4.5 μM CFD-Y at pH 7.6 25 ° C.
    From tab. 7 shows that very high yields are provided.
    Example 10. Peptides as a substrate component.
    The peptides listed in .tabl. 8 were obtained in the same manner as in Examples 1-4. The experiments were performed in a radiometric pH-stat; yields were determined by HPLC. The following parameters were constantly supported: 4-25 μM CPD-Y, pH 7.6 at.
    The dependence of peptide synthesis on pH.
    B. In those cases where the synthesized peptides are unsuitable dihydrates for CPD-Y, synthesis can be performed at pH values of preferably 9-10.5.
    Example 11. In the same way as in examples 1 and 3, the peptides are given in table. 9, were obtained in a pH-stat at the indicated pH values.
    The experiments were carried out at 25 ° C in the presence of 15 μmol CPD-Y.
    Other amine components.
    Example 12. The peptides are given in table. 10 were obtained in the same manner as in Examples 3 and 4. Experiments were performed in pH-stat, the yields were determined using KPLC. The experiments were performed with a pH of 4.5 μM CPD-Y, pH 9.5,.
    Example 13. Synthesis of peptides with barley carboxypeptidases.
    Germinating barley, for example, in the form of malt, contains two different carboxypeptidases, denoted by CP-1-1 and CP-2-1.
    CP-1-1 and CP-2-1 isolated by the method of Ren; the peptides listed in table. 11 were prepared in the same manner as in Examples 1-4, from the indicated starting materials. The following conditions were maintained: 6 μM CP-1-1 or
    CP-2-1, pH 8.0. 25 ° C.
    Example 14. Diamidation of peptide amides with carboxypeptidase catalysis.
    To 2 ml of a solution of 15 mM Bz-Ala-Leu-NHg in O, M KCl, 1 mM EDTA (pH 9.7, in 10% dimethylformamide, 2 mg of CPD-Y was added. The results were determined by HPLC as in at
    ten
    five
    20
    five
    five
    0
    0
    five
    0
    five
    Measure 1. It can be seen that Bz-Ala-Leu-NH after 20 minutes was completely transformed into 68% Bz-Ala-Leu-OH and 32% Bz-Ala-OH. Amino acid analysis of the reaction medium has shown that Bz-Ala-OH is mainly formed by the elimination of Leu-NH from Bz-Ala-Leu-1JH 2.
    Example 15. Comparison of peptide-ether substrates with and without terminal protective groups.
    In the same way as in examples 3 and 4, the peptides are given in table. 12 were prepared under the following conditions: 50 mM substrate, 5 µM CPD-Y, pH 9.5, 25 ° C.
    Example 16. Synthesis in the presence of organic solvents.
    Just as in examples 1-4 and 6, the peptides are given in table. 13-15. were obtained from the indicated starting materials at the indicated concentrations of organic solvents.
    The following conditions were maintained: 50 M of substrate, 5 µM CPD-Y, pH 9.6,
    . .
    PRI me R 17. Insoluble (immobile) carboxypepsidase-Y.
    CPD-Y was added to Konkavalin-A-Sepharose 4B reagent, manufactured by Pharmacna Fain Chemicals, after which they formed cross-links between CPD-Y and Concalin A with glutaraldehyde, as described by Hsias and Royer. The concentration of CPD-Y was 3 mg per pre-packaged Sepharose.
    The peptides listed in table. 16, were obtained in the same manner as in Examples 1 and 3 under the following conditions: 50 mM substrate, 0.25 ml CPD-Y gel {5 µm CPD-Y); pH 9.7; . After removal of the enzyme by filtration, products and yields were determined by HPLC.
    Example 1c. Amides of peptides as substrates.
    A solution of 5 ml of Bz-Ala-Leu-NH in 0.10 M KCL, I ml E) TA in the presence of 10% dimethylformamide at pH 10, and 25 C mixed with 0.25 M vapin-amide.
    The reaction was initiated by the addition of 40 mM CPD-Y and was carried out similarly to examples 3 and 4. Bz-Ala-Leu-Yal-NH was prepared by HPLC with a yield of about 65%.
    The reaction was repeated using Bz-Ala-Flifr-NHj as the substrate component. Bz-Ala-Phe-Yal-NH, was isolated by HPLC with a yield of about 44%.
    PRI me R 19. Synthesis of met-enkephalin.
    Stepwise synthesis of met-enkephalin when using as starting material 5 g of Bz-Ai-g-Oet and in the presence of CPD-Y as a catalyst was carried out according to the following scheme: synthesis of met-enkephalin using CPU-Y as a catalyst; the output for each stage is shown in brackets.
    Bz-Arg-Oet pH 9.6 Earl
    I H-Tyr-NH2 BZ-Arg-Tyr-NH (85 ° / o) pH 9.6 SRGN

    BZ-Arg-Tyr-OH (90%) EtOH / HCl
    H-Tyg-Gly-Gl / -Phe-Mel-OH (95%) pH8.0 f Trypsin
    Bz-Arg-Tyt -Gly-Gl: y-Pbe-Met-OH (75o / oV. PH9.5 1CPD-Y
    IH-Met-OH
    Bz-Arg-Tyr-Gly-Gl-Pet-Oel (80Vo) IrtOH / HCl
    Bz-Arg-Tyi: -Oei (80% Bz-Arg-T / r-Qiy-Gly-Phe-OH (95%)
    pH9,0CPD-ypH9,5 ICPD-y
    IH-Gly-OeiB7. -Arg-Tyr-Gly-Gly-Oet (60%) Bz-Atg-Tyr-Gly-Gly-Phe-iqH2 (65%) CPD-y, pH-8.0
    The stages of CPD-Y catalyzed condensation and deamidation were carried out in the pure aqueous phase, and Bz-Arg was selected as a solubilizing protective 1 group that can be removed at the last stage using trypsin. After expansion of the peptide under the influence of the amide of the amino acid, followed by deamidation, the next peptide-ester substrate was synthesized with ab- sated ethanol and anhydrous HC1. After each step, reagents were isolated by preparative HPLC. In this way, an excess of the ash component and by-products of the hydrolysis can also be regenerated.
    The proposed method allows the process of enzymatic production of peptides to be enhanced by using an exopeptide instead of previously used enzymes, each of which showed a dominant or, at least, significant endopeptidase activity, which allows to expand the set of amino acids and substrate components, to use enzymatic
    the process for the synthesis of multilink biologically active peptides, for example enkephalin, will significantly reduce the use of protective groups, carry out stenospecific synthesis, reduce the consumption of the enzyme to a concentration of 2-25 micromoles, carry out the process in a wider pH range (7.6-10.5 to obtain a stable high yield of the target product.
    权利要求:
    Claims (3)
    [1]
    1. A method for enzymatically producing peptides of the general formula AB, where A: N is the terminal protected Ij-amino acid residue or peptide residue of 2-6 L-amino acids, containing L-amino acid at its C-terminal end and optionally a protecting group at N-terminal end, B: T. 1 - amino acid residue, which may be C-terminally protected, by reacting the amino acid and substrate components in solution in the presence of an enzyme, characterized in that.
    In order to simplify the process, a component selected from the group containing amino acid esters, peptide esters and depipeptides of the formula A-OR, where the values of A are as described above, is used as a substrate component, R is C-C "alkyl benzyl or alpha - a desamino fragment of Gly, Ala or Phe-, amino acid amides or peptides of the formula A-NH, where the values of A are as indicated above, peptides of the formula A, -X, where L-amino acid or dipantapeptide, X is L-amino acid, and As an amine component, a component selected from the group consisting of total amino acids is used. of formula H-B-OH, where the values of B are indicated above, the amides of the optionally N-substituted amino acid of the formula HE-NHR, where the values of B are given you - Dje, Rj is hydrogen, hydroxy, amino, C -C-alkyl, complex esters of amino acids of the formula H-B-OH., where the values of B are as indicated above.
      P-4-C, -C-alkyl, and the process is carried out in the presence of L-specific serine or thioalkyl Tabl and c a 1
    Synthesis of peptides catalyzed by carboxypeptidase-Y using free amino acids as the amine component
    Leucine (0.17 M) Phenylalanine (0.16 M)
    Serine (3.2 M) Treonil (0.7 M) Methionine (0.6 M) Lysine (1.5 M) Arginine (i, 8 M)
    Aspartic acid (1.0 M)
    0
    five
    0
    five
    carboxypeptidase enzyme, which is introduced into the reaction mass in a concentration of from 2 to 25 µmol, the process is carried out in an aqueous solution or dispersion at pH 7.6-10.5, 25-45 0 with the initial concentration of the substrate component 0.01-0.15 mole, amine component, 0.05-3,000 mol, followed by isolation of an optionally protected peptide, after which, if necessary, R 2 or K groups are cleaved with a serine carboxypeptidase enzyme.
    [2]
    2. A method according to claim I, characterized in that yeast carboxypeptidase-U, carboxypeptidase or CP-2-1 from barley seedlings are used as the carboxypeptidase enzyme.
    [3]
    3. A method according to claim 1, characterized in that an aqueous solution is used containing 0-20% organic solvent selected from the group consisting of lower alkanol, dimethylformamide or polyethylene glycol.

    24 27
    50 24 46 56 26
    0
    131378785.4
    Continued table. one
    Glutamic acid
    (1.2M) Bz-ALa-Glu-OHO
    Methyl ether glutamine-. New acid (1, OM) Bz-Ala-Glu (OMe) -OH 30
    Bz-Ala-OMe Isoleucine (0.15M) Bz-Ala-He-OH40
    (50 #M)
    Glutamine (0.5 M) Bz-AJ.a-Cln-OH 20
    0 tert-butyl ether
    aspartic acid
    (0.3 M) Bz-Ala-Asp / 0 U / -OH 30
    Tryptophan (0.05 M) Bz-Ala-Trp-OH I O Histidine (1, OM) Bz Ala-His-OH15
    Z.-Phe-OMe Valine (M) Z-Phe-Val-OH6
    (55 mM)
    Lysine (3.0 M). Z-Phe-Lys-OH60
    (example 2)
    Bz-Phe-Gly-OMe (30 mM) Valine (0.6 M) Bz-Phe-Gly-rVal-OH 40
    Z-Ala-OMe Glycine (2.0 M) Z-Ala-Gly-OH30
    (10 mM)
    Alanya (0.7 M). Z-Ala-Ala-OH60
    Leucine (0.15 M) Z-Ala-Leu-OH2
    Lysine (1.5 M) Z-Ala-Lys-OH 60
    Bz-Gly-OMe Glycine (2.0 M) Bz-Gly-Gly-OH63
    (20 mM)
    Ac-Phe-OFt. .
    (50 mM) Alanya, (0.8 M) Bz-Phe-Ala-OH85
    Z-Ala-Ala- Glycine (2.0 M) Z-Ala-Ala-Gly-OH 40 -OMe (10 mM)
    Leucine (0.15 M) Z-Ala-Ala-Leu-OH O
    Phenylalanine (0.15 M) Z-Ala-Ala-Pfte-OH O
    Z-Ala-Phe- Glycine (2.0 M) Z-Ala-Phe-Gly-OH 35 -OMe (10 mM)
    Leucine (0.15 M) Z-Ala-Phe-Leu-OH O
    Z-Ala-Ser-.
    -OMe (Yu mM) Leucine (0.15 M). Z-Ala-Ser-Leu-OH 40
    15



    Leucine (0.15 M)
    Lysine (1.5 M) Glycine (2.0 M)
    Leucine {o, 15 Mj Glycine (2.0 M) Leucine (0.15 M) Glycine (2.0 M) Leucine (0.15 M) Phenylalanine (0.15 Mj Lysine (1.5 M) Leucine 0, 15 M Lysine (l, 5 M / Alanin (1.7 M j
    table 2
    Synthesis of peptides using amino acid amides as the amine component; catalyst - carboxypeptidase-Y
    (example 4)
    Leucinamide (0.3 M) Bz-Ala-Leu-NH
    Methioninamide (0.6 M)
    (example 3), Bz-Ala-Met-NH
    Phenylalaninamide (0.3 M) Bz-ALa-Phe-NH
    Tyrosine (0.6 M) Bz-Ala-Tyr-NH Asparaginamide (0.3 M) Bz-Ala-Asn-NH.
    1378785
    sixteen
    Continued table. I
    3
    Z-Ala-Val-Cu-OH
    Z Ala-Val-Lys-OH
    Z-Ala-NLe i-Gly-OH
    Z-ALa-NLeu-I eu-OH
    Z-ALa-Met-Gly-OH
    Z-ALa-Met-Le i-OH
    Z-Ala-Trp-Gly-OH
    Z Ala-Trp-Leu-OH
    Z-Ala-Trp-Phe-OH
    Z-Ala-Trp-Lys-OH
    Z Ala-Ala-Tj r-Leu-OH
    Z-Ala-Ala-Tyr-Lys-OH
    Z-Ala-Ala-Tyr-Ala-OH
    85
    95 90 90. 80
    -OME (1 O mM)
    Z-Thr-Pro-OOM (25 mM)
    Z-Ala-Ala- -Tug-OMe (10 mM)
    Z-Tyr-Gly- -Gly-OMe (20 mM)
    Valaminamide (0.2.M) Glycinamide (O, 15 M) Leuciumamide (0.15 M)
    Z-Thr-Pro-Val-NHj 95 Z-Ala-Ala-Tyr-Gly-NH .J 100 Z-Ala-Ala-Tyr-Leu-NHj 100
    Phenyl al aninamide (0.2 M) Z-l -r-Gly-Gly-Phe-NH 40
     The product precipitated.
    Table3
    Carbroxypeptidase-Y catalyzed peptide synthesis with amino acid hydraides as the amine component
    Br-A1a-OMe Alaninghydrazide (0.6 M) Bz-Ala-Ala-NH-NHg
    PhenylalaningiDrazid ..
    (0.3 M). Bz-Ala-Phe-NH-NH,
    Z-Thr-Pro-Val-NHj 95 Z-Ala-Ala-Tyr-Gly-NH .J 100 Z-Ala-Ala-Tyr-Leu-NHj 100
    37
    80
    Table 4
    Carboxypeptidase-Y catalyzed peptide synthesis with amino acid esters as the amine component
    Glycinpropyl ether (0.5 M)
    Glycinisopropyl ether (0.5 M)
    Glycinbutyl ether (0.5 M)
    Leucine methyl ester
    (0.5 M)
    Leucine ethyl ester (0.25 M).
    Leucine 1 butyl ether (0.25 M)
    Phenyl alanine methyl ether (0.25 M)
    Phenylalanine ethyl ether (0.15 M)
    Methyl ester (t-Bu) glutamic acid (0.5 M)
    (if-t-Bu) t-butyl ester of glutamic acid (0.25 M)
    Methionine methyl ester. (0.2 M)
    Methionine ethyl ester (0.2 M)
    Bz-Ala-Gly-OH
    Bz, -Ala-Gly-OH
    Bz-Ala Gly-OH
    Bz-Ala - Leu-OH Bz Ala Leu-Leu-OH Bz-Ala-Leu-Leu-Leu-OH Bz-Ala-Leu-Leu-Leu-Leu- -OH
    Bz-Ala-Leu-OH Bz-Ala-Leu-Iieu-OH 50
    Bz-AJ-a-Leu-OHO
    Bz-Ala-Phe-OH
    Bz-Ala-Phe-Phe-OH 80 Bz-Ala-Phe-Phe-Phe-OH
    Bz-Ala-Phe-OH Bz-Ala Phe-Phe-OH
    70
    Bz-Ala-Glu (-t-Bu) -OH 35
    Bz-Ala-Glu (-t-Bu) -OH O
    Bz-Ala-Met-OH Bz-Aba-Met-Met-OH Bz-Ala Met-Met-Met-OH Bz-Ala-Met-Met-Met-Met OH
    Bz-Ala-Met-OH40
    c-Phe-OEt 50mM)
    z-Gly-OMe 50 mM)
    Me thioninis opyl ether (0.2 M)
    Valine methyl ether (0.7 M)
    Serine methyl ether (0.5 M)
    Tyrosine methyl ether (0.5 M)
    Arginine-type eLir (I, 5 M)
    Arginine (NOj) methyl ester (0.5 K)
    Histidine methyl ester (0.5 M)
    Threonine methyl ester
    (0.5 M)
    Alanine methyl esAir (0.5 M)
    Bz-Ala-Met-OH
    Bz-Ala-Val-OH Bz-Ala-Val-Va
    Bz-Ala-Ser-OH
    Bz-Ala-Tyr-OH
    Bz-Ala-Arg-OH
    Br-Ala-Arg (NO.
    Bz-Ala-His-OH
    Bz-Ala-Thr-OH
    Ac-Fne-Ala-OH Ac-Phe-Ala-Ala
    Histidine methyl ester Bz-Gly-His-OH
    Table 5
    Carboxypyptindase-Y catalyzed peptide synthesis with L- and D-isomers as amine component
    Bz-Ala-Met-OH
    Bz-Ala-Val-OH Bz-Ala-Val-ValBz-Ala-Ser-OH
    Bz-Ala-Tyr-OH
    Bz-Ala-Arg-OH
    Br-Ala-Arg (NO.) -OH
    Bz-Ala-His-OH
    Bz-Ala-Thr-OH
    Ac-Fne-Ala-OH Ac-Phe-Ala-Ala-OH
     Bz-Gly-His-OH
    Table 6
    Output (%) in the synthesis of peptides using various ether groups as substrate components in the catalysis of carboxypeptidase-Y
    Table 7
    Catalyzed carboxypeptidazole-Y synthesis with depsipeptides as substrate components.
    Table 8
    Carboxypeptidase-Y catalyzed peptide synthesis using peptides as substrates
    Glycinamide f1,3 M)
    Table 10
    Carboxypeptidase-catalyzed peptide synthesis at various amino acid derivatives as an amine component
     -Alaninamide (0.2 M)
    Glycinhydroxamic acid (0.2 M)
    D, B-alanine hydroxamic acid (0.2 M)
    Glycine ti -methylamide (0.50 M)
    Table
    Synthesis of peptides using carboxypeptidases from barley seedlings
    CP-1- or CP-2-1
    NK5, 0 9.5 9.0
    5 77 95
    8.0 100 7.0 97 6.0 44
    Bz-Ala-Ala-NHj 80
    Bz-Ala-Gly-NH-OH 75
    Bz-Ala-Ala-NH-OH 50
    Bz-Ala-Gly-NH-CH; | 20
    31137878532
    Table 12
    Comparison of carboxypeptidose-catalyzed-U peptide synthesis using peptide esters as substrates with and without N-terminal protecting groups
    Amine component (concentration)
    Leucinamide (0.15 M) Leucinamide (0.15 M)
    Table 13
    Carboxypeptidase-Y catalyzed peptide synthesis using 50 mM Bz-Ala-OMe as a substrate and free amino acids as an amine component in the presence and absence of 20% methanol (MeOH) in 0.1 M KCl, 1 mM EDTA
    Product
    Output,
    %
    Ac-A1a-A1a-A1a-Leu-NHj90
    Ala-Ala-Ala-Leu- -NH,
    75
    Table 14
    Carboxypeptidae-Y catalyzed Bz-Alk-Tlir-OH synthesis from B7. (50 mM) and threonine at various concentrations of polyethylene glycol-300 (PEG-300). Conditions: CPD-Y 4 μM; pH 9.6; 25 seconds
    ABOUT
    10 20 30 40
    Table 15
    Carboxypeptidase-Y catalyzed peptide synthesis from 50 mM Bz-A.a-OMe and the indicated amino acid esters as the amine component in 3% polyethylene glycol. O, 1 M KS1, 1 mM E1DTA Other conditions: CPD-Y 8 μM; pH9.6; 25 ° С
    Glutamic acid Bz-Ala-Gly-OH (-OBut) -OMe (0.5 M) (jf-OBut) -OH
    Phenylalanine-OMe (0.5 M)
    L-Methionine-OMe (0.5 M)
    D-Methionine-OMe (0.5 M)
    36 34 31 29 28
    38
    Bz-Ala-Phe-OH Br-Ala-Phe-Phe-OH
    Bz-Ala-Met-OH Bz-Ala-Met-blet-OH
    Bz-Ala-Met-Met-Met
    Bz-Ala-D-Met-OH
    Table 16,
    Catalyzed by insoluble carboxypeptidase-Y synthesis
    peptides.
    Bz-Ala-OMe Phenylalanine (0.15 M) Bz-Ala-Phe 21 (50 mM). ,
    . Leucinamide (0.2 M) Bz-Ala-Leu-NH. 91
    类似技术:
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    同族专利:
    公开号 | 公开日
    RO80806A|1983-02-01|
    EP0017485B1|1984-09-26|
    US4806473A|1989-02-21|
    EP0017485A1|1980-10-15|
    DE3069259D1|1984-10-31|
    IE50256B1|1986-03-19|
    AU545416B2|1985-07-11|
    NZ193375A|1983-06-14|
    WO1980002157A1|1980-10-16|
    FI70597C|1986-09-24|
    FI801035A|1980-10-07|
    ZA801929B|1981-11-25|
    JPS56500519A|1981-04-23|
    BR8008024A|1981-03-31|
    IL59776D0|1980-06-30|
    IL59776A|1985-01-31|
    CA1160973A|1984-01-24|
    CS254954B2|1988-02-15|
    JPS6339237B2|1988-08-04|
    AT9595T|1984-10-15|
    HU186734B|1985-09-30|
    CS239980A2|1987-07-16|
    IE800694L|1980-10-06|
    FI70597B|1986-06-06|
    US4339534A|1982-07-13|
    AU5981580A|1980-10-22|
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    法律状态:
    优先权:
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    DK144379|1979-04-06|
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