WO2011145639A1 - Cristal d'alanylcystéine oxydée et procédé pour produire celui-ci - Google Patents

Cristal d'alanylcystéine oxydée et procédé pour produire celui-ci Download PDF

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Publication number
WO2011145639A1
WO2011145639A1 PCT/JP2011/061361 JP2011061361W WO2011145639A1 WO 2011145639 A1 WO2011145639 A1 WO 2011145639A1 JP 2011061361 W JP2011061361 W JP 2011061361W WO 2011145639 A1 WO2011145639 A1 WO 2011145639A1
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WIPO (PCT)
Prior art keywords
oxidized
alanylcysteine
crystal
aqueous solution
water
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PCT/JP2011/061361
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English (en)
Japanese (ja)
Inventor
哲夫 西村
光隆 城野
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協和発酵バイオ株式会社
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Priority to JP2012515901A priority Critical patent/JP5863647B2/ja
Publication of WO2011145639A1 publication Critical patent/WO2011145639A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala

Definitions

  • the present invention relates to a crystal of oxidized alanylcysteine which is useful as a product, raw material or intermediate for health foods, pharmaceuticals, cosmetics and the like, and a method for producing the crystal.
  • Oxidized alanylcysteine has been reported to be a useful substance that can improve the stability of cysteine and the solubility of cystine (Non-patent Documents 1 and 2). However, there are no crystals of oxidized alanylcysteine on the market including reagents. Therefore, there is no report of the manufacturing method of the crystal.
  • the object of the present invention is to provide crystals of oxidized alanylcysteine which are useful as products, raw materials or intermediates such as health foods, pharmaceuticals and cosmetics, and oxidized alanylcysteine suitable for mass synthesis or industrialization. It is in providing the manufacturing method of crystal
  • the present invention relates to the following (1) to (13).
  • the diffraction angle (2 ⁇ °) is 6.800, 11.520, 13.640, 17.700, 18.700, 19.300, 20.700, 21.940, 22.240, 22.740, 23.180, 24.320, 25.080, 25.420, 26.140, 26.680, 27.560 , 30.480, 31.280, 31.980, 32.240, 34.580, 35.120, 35.880, 37.160, 42.640, 45.480. Oxidized alanylcysteine monohydrate crystals.
  • a method for producing a crystal of oxidized alanylcysteine comprising a step of crystallizing oxidized alanylcysteine from a mixed solution of an oxidized alanylcysteine-containing aqueous solution and a water-miscible organic solvent.
  • a method for producing a crystal of oxidized alanylcysteine comprising a step of adding or dropping a water-miscible organic solvent to an aqueous solution containing oxidized alanylcysteine.
  • a method for producing a crystal of oxidized alanylcysteine comprising a step of adding or dropping an aqueous solution containing oxidized alanylcysteine to a water-miscible organic solvent.
  • the crystal of oxidized alanylcysteine is a crystal of oxidized alanylcysteine n hydrate (wherein n represents an integer of 0 or more and less than 8). Any one of the manufacturing methods.
  • the present invention provides crystals of oxidized alanylcysteine that are useful as products, raw materials, intermediates, and the like such as health foods, pharmaceuticals, and cosmetics, and methods for producing the same.
  • FIG. 1 shows the stability of oxidized alanylcysteine monohydrate crystals ( ⁇ ), cystine ( ⁇ ), cysteine free ( ⁇ ), and cysteine hydrochloride ( ⁇ ) in water at 60 ° C. It is the figure evaluated using the residual rate of each compound as a parameter
  • the oxidized alanylcysteine crystal may be either an anhydride or a hydrate, but the oxidized alanylcysteine n hydrate (wherein n is an integer or fraction of 0 or more and less than 8).
  • the oxidized alanylcysteine-containing aqueous solution may be any aqueous solution containing oxidized alanylcysteine, but is preferably an aqueous solution in which the purity of oxidized alanylcysteine in the dissolved component is 50% or more, 70% The above aqueous solution is more preferable.
  • the aqueous solution may contain other organic solvents such as alcohols such as methanol, ethanol, isopropyl alcohol and n-propanol, and ketones such as acetone and methyl ethyl ketone.
  • the water content in the aqueous solution is preferably 20% or more.
  • an aqueous solution containing oxidized alanylcysteine an aqueous solution in which oxidized alanylcysteine is dissolved is subjected to membrane treatment, gel filtration treatment, activated carbon treatment, ion exchange resin treatment, synthetic adsorption resin treatment, chelate resin treatment, or solvent precipitation.
  • Etc. preferably prepared by pretreatment such as activated carbon treatment, ion exchange resin treatment, synthetic adsorption resin treatment, or solvent precipitation, more preferably ion exchange resin treatment or synthetic adsorption resin treatment.
  • the aqueous solution containing oxidized alanylcysteine is preferably an aqueous solution having a concentration of oxidized alanylcysteine contained of 100 to 900 g / L. Of these, aqueous solutions of 400 to 600 g / L are preferable, and these are prepared by appropriately concentrating.
  • the water-miscible organic solvent may be any organic solvent having a property of being miscible with water.
  • alcohols such as methanol, ethanol, isopropyl alcohol and n-propanol
  • ketones such as acetone and methyl ethyl ketone are used.
  • Etc. preferably methanol, ethanol, and acetone.
  • Examples of the synthetic adsorption resin include non-polar, porous adsorption resins, and the like.
  • Diaion HP series for example, HP10, HP20, HP21, HP30, HP40, HP50, etc .; manufactured by Mitsubishi Chemical
  • diamond Ion SP800 series for example, SP800, SP825, SP850, SP875, etc .
  • Diaion SP200 series for example, SP205, SP206, SP207, SP207SS, etc .; manufactured by Mitsubishi Chemical
  • Amberlite XAD series for example, XAD4, XAD7HP, XAD16, XAD1600, etc .; manufactured by Rohm and Haas).
  • the ion exchange resin examples include a cation exchange resin and an anion exchange resin.
  • cation exchange resins strong acid cation exchange resins such as 124Na and 252Na such as Amberlite IR series (manufactured by Organo), Marathon C, Dowex such as XUS-40232.01 (manufactured by Dow Chemical), etc. are weak.
  • acidic cation exchange resins include Amberlite IRC series (Rohm and Haas) such as IRC50 and IRC70, WK series such as WK40 (Mitsubishi Chemical).
  • anion exchange resins strong basic anion exchange resins such as PA306, PA312 and PA412 and other diamond ion PA series (manufactured by Mitsubishi Chemical Corporation), etc., and weak basic anion exchange resins such as WA10, WA20 and WA30 No. Diaion WA series (Mitsubishi Chemical Co., Ltd.).
  • strong basic anion exchange resins such as PA306, PA312 and PA412 and other diamond ion PA series (manufactured by Mitsubishi Chemical Corporation), etc.
  • weak basic anion exchange resins such as WA10, WA20 and WA30 No. Diaion WA series (Mitsubishi Chemical Co., Ltd.).
  • Type alanylcysteine is produced and oxidized with a known method, for example, an enzyme that oxidizes oxygen, hydrogen peroxide, ascorbic acid, or the like to obtain an aqueous solution in which oxidized alanylcysteine is dissolved.
  • This aqueous solution can be subjected to pretreatment as necessary so that the purity of oxidized alanylcysteine in the dissolved component is 50% or more, preferably 70% or more.
  • Oxidized alanylcysteine is concentrated by concentrating the pretreated solution or the untreated solution so that the concentration of oxidized alanylcysteine is, for example, 100 to 900 g / L, preferably 400 to 600 g / L.
  • a containing aqueous solution is obtained.
  • an oxidized alanylcysteine-containing aqueous solution can be obtained by converting a pretreated solution or an untreated solution into a powder by freeze-drying and dissolving the obtained powder in water so as to have a similar concentration.
  • Examples of pretreatment methods for obtaining an oxidized alanylcysteine-containing aqueous solution include membrane treatment, gel filtration treatment, activated carbon treatment, ion exchange resin treatment, synthetic adsorption resin treatment, chelate resin treatment (chelate resin used for chelate resin treatment) As, for example, Duolite C467; manufactured by Sumitomo Chemical Co., Ltd.), solvent precipitation, etc., preferably activated carbon treatment, ion exchange resin treatment, synthetic adsorption resin treatment, chelate resin treatment, solvent precipitation, etc.
  • Preferred examples include synthetic adsorption resin treatment or ion exchange resin treatment, and these treatment methods may be appropriately combined.
  • an aqueous solution in which oxidized alanylcysteine is dissolved is passed through a synthetic adsorption resin, preferably SP207, and water or a water-miscible organic solvent (the water-miscible organic solvent is as defined above) alone.
  • a synthetic adsorption resin preferably SP207
  • water or a water-miscible organic solvent the water-miscible organic solvent is as defined above
  • oxidized alanylcysteine is dissolved as a strongly acidic cation exchange resin (H + type), preferably After passing through Marathon C (manufactured by Dow Chemical Co., Ltd.), high purity oxidized alanylcysteine is contained by separating and purifying oxidized alanylcysteine by elution with basic solvents such as aqueous ammonia and sodium chloride.
  • An aqueous solution can be prepared.
  • aqueous solution containing oxidized alanylcysteine obtained in step 1 is adjusted with hydrochloric acid or sulfuric acid, or a sodium hydroxide aqueous solution, if necessary, so that the pH value becomes, for example, 5.5 to 7.5, and then mixed with water.
  • a solvent is added for 1 minute to 10 hours, preferably 1 to 6 hours, and crystals are precipitated by stirring and crystallization.
  • the precipitated crystals are separated using, for example, centrifugal filtration, decantation, etc., washed with water or a water-miscible organic solvent, and then dried under reduced pressure or ventilation to dry the oxidized alanyl. Cysteine crystals can be obtained.
  • the aqueous solution containing oxidized alanylcysteine obtained in step 1 is adjusted with hydrochloric acid or sulfuric acid, or an aqueous sodium hydroxide solution so that the pH value becomes 5.5 to 7.5, for example.
  • Oxidized alanylcysteine crystals can also be obtained by crystallization while adding to a miscible organic solvent.
  • the aqueous solution containing oxidized alanylcysteine obtained in step 1 is adjusted with hydrochloric acid or sulfuric acid, or an aqueous sodium hydroxide solution to a pH value of, for example, 5.5 to 7.5, and allowed to stand for 24 hours or longer.
  • the crystals of oxidized alanylcysteine can also be obtained by depositing the crystals. When performing the crystallization described above, a seed crystal may be added as necessary.
  • Oxidized alanylcysteine crystals obtained by the above production method may be obtained as adducts with various water-miscible organic solvents, but these adducts with various water-miscible organic solvents are also included in the crystals of the present application.
  • the oxidized alanylcysteine crystals obtained by the above production method may have different crystal forms or different particle sizes, and these may be obtained alone or as a mixture. Single or mixtures of different crystal forms or of different particle sizes are also included in the crystals of the present application.
  • the saturated solubility and storage stability of the oxidized alanylcysteine crystals of the present invention will be specifically described with reference to test examples.
  • Test example 1 Comparison of Saturated Solubility of Oxidized Alanylcysteine Monohydrate and Cystine About Oxidized Alanylcysteine Monohydrate Crystal Obtained in Example 2 and Commercially Available Cystine (manufactured by Wako Pure Chemical Industries, Ltd.) The saturation solubility in water at 20 ° C. was measured. As shown in Table 1, the oxidized alanylcysteine monohydrate crystals showed 120 times or more solubility compared to cystine.
  • Test example 2 Comparison of Storage Stability of Oxidized Alanylcysteine Monohydrate with Cystine, Cysteine Free Form, and Cysteine Hydrochloride Crystals of Oxidized Alanylcysteine Monohydrate Obtained in Example 2
  • the stability of commercially available cystine manufactured by Wako Pure Chemical Industries, Ltd.
  • cysteine-free body manufactured by Wako Pure Chemical Industries, Ltd.
  • cysteine hydrochloride manufactured by Wako Pure Chemical Industries, Ltd.
  • alanylcysteine was eluted using a 1.0 mol / l aqueous sodium hydroxide solution, and fractions containing reduced alanylcysteine were collected. 2 equivalents of hydrogen peroxide was added to the fraction and stirred at room temperature for 1 hour to convert reduced alanylcysteine to oxidized alanylcysteine. Thereafter, the pH was adjusted to 7.0 and concentrated under reduced pressure to 500 g / l to obtain an oxidized alanylcysteine-containing aqueous solution. Crystals of oxidized alanylcysteine were precipitated by adding 6 L of ethanol to the aqueous solution at room temperature over 6 hours.
  • the crystals were dried to obtain oxidized alanylcysteine crystals.
  • the Karl Fischer moisture content and differential scanning calorimetry (DSC) of the oxidized alanylcysteine crystals were measured to confirm that the crystals were oxidized alanylcysteine monohydrate. The results are shown below together with the powder X-ray diffraction and melting point analysis results.
  • oxidized alanylcysteine crystals useful as products, raw materials or intermediates such as health foods, pharmaceuticals and cosmetics, and oxidized alanylcysteine crystals suitable for mass synthesis or industrialization.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention concerne un cristal d'alanylcystéine oxydée. Un cristal d'alanylcystéine oxydée, en particulier un cristal d'alanylcystéine oxydée monohydratée est produit par un procédé comprenant une étape de cristallisation d'alanylcystéine oxydée dans une solution mixte d'une solution aqueuse contenant de l'alanylcystéine oxydée et un solvant organique miscible avec l'eau.
PCT/JP2011/061361 2010-05-18 2011-05-18 Cristal d'alanylcystéine oxydée et procédé pour produire celui-ci WO2011145639A1 (fr)

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JP2010114028 2010-05-18

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59172444A (ja) * 1983-03-18 1984-09-29 Ajinomoto Co Inc ジペプチド結晶及びその製造方法
WO2001088175A1 (fr) * 2000-05-17 2001-11-22 Instituto Superior Técnico Procede de production continue de cristaux dipeptidiques dans une membrane et un reacteur hydrocyclone a peptidase
WO2006104186A1 (fr) * 2005-03-29 2006-10-05 Kyowa Hakko Kogyo Co., Ltd. Cristal de dipeptide et son procede de production

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59172444A (ja) * 1983-03-18 1984-09-29 Ajinomoto Co Inc ジペプチド結晶及びその製造方法
WO2001088175A1 (fr) * 2000-05-17 2001-11-22 Instituto Superior Técnico Procede de production continue de cristaux dipeptidiques dans une membrane et un reacteur hydrocyclone a peptidase
WO2006104186A1 (fr) * 2005-03-29 2006-10-05 Kyowa Hakko Kogyo Co., Ltd. Cristal de dipeptide et son procede de production

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MAKOTO YAGASAKI ET AL.: "Development of Efficient Dipeptide Manufacturing", BIO. IND., vol. 23, no. 9, 2006, pages 26 - 34, XP003021904 *
STEHLE, P. ET AL.: "In vivo utilization of cysteine-containing synthetic short-chain peptides after intravenous bolus injection in the rat.", J. NUTR., vol. 118, no. 12, 1988, pages 1470 - 1474 *
SUKUMAR, N. ET AL.: "Crystal structure and conformation of N-(t-butoxycarbonyl)-L-alanyl- S-benzyl-L-cysteine methyl ester.", BULL. CHEM. SOC. JPN., vol. 67, no. 7, 1994, pages 1976 - 1979 *

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