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WO2013029075A1 - Agent et procédé d'alkylation - Google Patents

Agent et procédé d'alkylation Download PDF

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Publication number
WO2013029075A1
WO2013029075A1 PCT/AT2012/000228 AT2012000228W WO2013029075A1 WO 2013029075 A1 WO2013029075 A1 WO 2013029075A1 AT 2012000228 W AT2012000228 W AT 2012000228W WO 2013029075 A1 WO2013029075 A1 WO 2013029075A1
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WO
WIPO (PCT)
Prior art keywords
methyltransferase
group
sam
alkyl
alkenyl
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.)
Ceased
Application number
PCT/AT2012/000228
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German (de)
English (en)
Inventor
Mandana GRUBER
Harald STECHER
Martin TENGG
Peter Remler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ACIB GmbH
Technische Universitaet Graz
Original Assignee
ACIB GmbH
Technische Universitaet Graz
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by ACIB GmbH, Technische Universitaet Graz filed Critical ACIB GmbH
Publication of WO2013029075A1 publication Critical patent/WO2013029075A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the present invention relates to means and processes for alkylation.
  • Alkylating agents for carrying out the above-described reactions are alkyl halides and sulfonates which are highly toxic. Very often, aggressive conditions are required for the implementation and the implementation is unselective. The result is a product mixture which reduces the yield of the desired product. Isolation and cleaning of the product requires expensive
  • Methyltransferases are enzymes that perform alkylation reactions. These enzymes need cofactors.
  • SAM S-adenosyl-L-methionine
  • the present invention relates to a process for the transfer of an alkyl, alkenyl or alkynyl group to a small molecule having a nucleophilic center comprising the step of contacting the compound with an S-adenosyl-L-methionine ( SAM) -dependent methyltransferase in the presence of an alkylated
  • X is an organic or inorganic anion and R, R and R are each independently a substituted or unsubstituted alkyl, alkenyl and alkynyl group containing from 1 to 10 carbon atoms.
  • Alkyl sulfoxonium salts are capable of acting as cofactors for enzymatic reactions involving methyltransferases. This allows the establishment of enzymatic alkylation methods that do not require SAM as a cofactor.
  • the method according to the present invention be carried out at a temperature at which the used
  • Methyltransferase shows the highest reaction rate, preferably between 25 and 45 ° C, more preferably between 30 and 35 ° C.
  • small molecule refers to a low molecular weight, low molecular weight organic compound (molecular weight less than or equal to 1 kDa, preferably less than or equal to 0, 8 kDa), which is by definition not a polymer
  • small molecule also refers to a molecule bound to a biopolymer, such as a protein, nucleic acid, polysaccharide or a resin, and additionally the activity or function of the "Small Molecules” can have a variety of biological functions, such as signaling molecules, as tools in molecular biology, as medicines, as pesticides, and much more. These compounds can be more natural (such as secondary metabolites) or artificial (such as antivirals).
  • the target atoms in the substrate are oxygen, nitrogen, sulfur and carbon.
  • the nucleophilic centers in the substrate are -OH, -NH 2 , -NHR, -CONH 2 , -CONHR, -SH, -SR, and electron-rich carbon atoms, preferably an sp 2 -hybridized carbon as part of an aliphatic or aromatic compound.
  • Preferred methyltransferases are O-methyltransferases, N-methyltransferases, S-methyltransferases and C-methyltransferases.
  • O-methyltransferases catalyze the methylation of a hydroxyl (-OH) or carboxyl (-COOH) functionality.
  • S-methyltransferases catalyze the methylation of thiols (-SH) and thioethers (-SR).
  • C-methyltransferases transfer the methyl group on an electron-rich carbon atom.
  • the substituted or unsubstituted alkyl, alkenyl and alkynyl group comprises 1 to 8 carbon atoms, preferably 1 to 7 carbon atoms, more preferably 1 to 3 carbon atoms, especially 1 carbon atom.
  • Ci to Ci 0 preferably Ci to C 8 , more preferably i to C 7 , even more preferably Ci to C 3 , in particular Ci, alkyl, alkenyl or alkynyl used.
  • one or more alkyl, alkenyl and alkynyl groups may be substituted with at least one carboxyl, carbonyl and / or amino group.
  • An alkyl group of the sulfonium salts used according to the invention can not be an adenosyl group.
  • one or more of the following compounds is used in the process according to the invention:
  • the method according to the present invention involves the use of SAM-dependent methyltransferases.
  • This group of enzymes includes methyltransferases that are responsible for their
  • SAM-dependent methyltransferases of the present invention accept methionine methyl sulfonium as a cosubstrate or cofactor to a significantly lesser extent than SAM, or even are unable to transfer the methyl group from the methionine methyl sulfonium to the substrate.
  • SAM-dependent methyltransferases of the present invention show a reduced or no affinity for methionine methylsulfonium.
  • a methyltransferase such as homocysteine S-methyltransferase EC 2.1.1.10, which shows a higher affinity to cosubstrates other than SAM, is not to be used as a preferred methyltransferase in the method according to the present invention.
  • the Km value of SAM in the SAM-dependent methyltransferases used in the method according to the present invention is preferably less than 10 -3 M, more preferably less than 10 -4 M and even more preferably less than 10 -5 M.
  • Preferred methyltransferases are: EC 2.1.1.1 nicotinamide N-methyltransferase, EC 2.1.1.2
  • Methyltransferase EC 2.1.1.164 Demethylrebeccamycin D-Glucose O-Methyltransferase, EC 2.1.1.165 Methyl Halide Transferase, EC 2.1.1.169 Tricetin S ' ⁇ ' ⁇ 'O-Trimethyltransferase, EC 2.1.1.175 Tricine Synthase, EC 2.1.1.195 Cobalt Precorrin 5B (Cl) -Methyltransferase, EC 2.1.1.196 Cobalt Precorrin-7 (C15) -Methyltransferase, EC 2.1.1.197 Malonyl CoA O-Methyltransferase, and EC 2.1.1.201 2-Methoxy-6-Polyprenyl- 1,4-benzoquinol methylase.
  • the substrates ("small molecules") and cosubstrates of all of the methyltransferases mentioned herein are known, however, the method of the present invention can be used to alkylate, alkenylate, and alkynylate other substrates (other "small molecules"). Methods for the identification of such substrates are known in the art and include the investigation of whether a potential substrate according to the present invention is obtained by incubation of
  • Methyltransferase and corresponding Cosubstrat was modified.
  • the organic or inorganic anion is selected from the group of halides and sulfonates.
  • the substrate when the substrate is in a concentration of 0.5 to 2 mM, preferably 1 mM, the cosubstrate according to the general formulas (I) and (II) according to the present invention is in a concentration of 12 to 200mM, preferably 25 to 100mM.
  • the methyltransferases used in the method according to the present invention have been produced recombinantly.
  • Culture medium comprising cells capable of SAM-dependent methyltransferases produce, can be used. Alternatively, it is also possible to use the supernatant of the disrupted cells which produce SAM-dependent methyltransferases.
  • kits for transferring an alkyl, alkenyl or alkynyl group to a substrate comprising an S-adenosyl-L-methionine (SAM) -dependent methyltransferase and an alkylated sulfonium salt having the general formula (I)
  • R 1 , R 2 and R 3 are each independently a substituted or unsubstituted alkyl, alkenyl and alkynyl group comprising 1 to 10 carbon atoms.
  • FIG 1 shows S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH) and SAM analogues. It has also been shown that DNA methyltransferases also accept non-natural cofactors (SAM analogs) and sequence-specific DNA alkylation was observed using S-adenosyl-L-homocysteine derivatives. In all known and published results, the adenosyl part of the cofactor remains untouched and can interact with the enzyme.
  • SAM S-adenosyl-L-methionine
  • SAH S-adenosyl-L-homocysteine
  • Figure 2 shows sulfur containing salts tested as possible methyl donors for CouO and NovO.
  • Trimethylsulfonium 1 and trimethylsulfoxonium 2 were used as iodides
  • Methionine methyl sulfonium 3 as chloride.
  • Figure 3 shows the synthesis of the substrates for CouO and NovO: a) Ac 2 O (acetic anhydride), pyridine, DMAP (4-dimethylaminopyridine), room temperature (rt), 24h, 93%. b) SOCl 2, CH 2 Cl 2, reflux, 5h. c) KOH, MeOH, rt, 3h, 73%. d) Et 3 N, MgCl 2 , THF, 2h at 0 ° C, 16h at rt, 73%. e) aq. NaOH, MeOH, rt, 16h, 92%. f) IN HCl, MeOH, rt, 24h, 67%.
  • Figure 4 shows the methylation of coumarin benzamide 4a catalyzed by CouO at different concentrations of the sulfonium and sulfoxonium salts.
  • Me 3 S trimethylsulfonium iodide;
  • Me 3 S 0 trimethylsulfoxonium iodide;
  • Figure 5 shows the methylation of coumarin benzamide 4a catalyzed by NovO at different concentrations of the sulfonium and sulfoxonium salts.
  • Figure 6 shows the influence of the addition of SAH to the reaction mixtures.
  • SAM S-adenosyl-L-methionine
  • CouO Streptomyces rishiriensis
  • NovO Streptomyces spheroides
  • Aminocoumarins form part of the antibiotic molecules that are produced in the previously mentioned strains.
  • the aminocoumarin part is the substrate for methyl transfer from the natural cofactor SAM.
  • Streptomyces rishiriensis and the gene CouO coding for methyltransferase have been extensively studied, as has the gene cluster of novobiocin synthesis in Streptomyces spheroides. These methyltransferases catalyze the transfer of the methyl residue from SAM selectively to position 8 of the coumarin ring. This was the first time methylsulfonium and
  • the expression constructs pET26b (+) - CouO and pET26b (+) - NovO were each transformed into electrocompetent E. coli BL21Gold (DE3) cells for protein expression.
  • Transformants with the desired constructs were cultured at 30 ° C in LB medium supplemented with 40 g / ml kanamycin to an OD 6 oo of 0.8. Subsequently, the temperature was lowered to 25 ° C and induced with 100 mM final concentration of isopropyl D-thiogalactopyranoside (IPTG). Growth continued for 16h at 25 ° C.
  • the cells were harvested by centrifugation (10 minutes at 4000 xg), resuspended in Buffer A (50 mM Na phosphate buffer pH 7 (6.5 for NovO), and passed through
  • the solution was cooled to 0 ° C, acidified with KHSO 4 and extracted 3x with ethyl acetate.
  • the combined organic phases were washed once with saturated NaCl solution and dried over Na 2 S0 4 .
  • the solvent was removed under reduced pressure (bath temperature 20 ° C). The remaining oil was dried under vacuum.
  • the product is a white powder and was 14.45 g (73%).
  • the crude substance 4- (2- (tert-butyloxycarbonylamino) -3-ethoxy-3-oxopropanoyl) -1,3-phenylene diacetate (4.31 g) was dissolved in 24 mL MeOH and 30 mL 1.5 N NaOH added. The solution was stirred for 3 h at room temperature. The solution was then acidified to pH 3 with 1 N HCl and extracted 3x with ethyl acetate. The combined organic phases were washed with saturated NaCl solution, dried over Na 2 S0 4 and the solvent removed under vacuum.
  • Trimethylsulfoniumiodid and Methioninmethylsulfoniumchlorid are completely dissolved at this concentration.
  • the conversion was 5-20% (highest conversion with compound 2).
  • the reactions were performed in 0.2 mL scale in a thermomixer at 30 ° C and 1000 rpm for 24 h.
  • the solutions contained 1 mM (mmolar) substrate of a 10 mM stock solution in DMSO (dimethylsulfoxide), 2 mM SAM (A7007 from Sigma) from a stock solution in 50 mM
  • the MS signals were collected in scan and SIM ESI positive mode.
  • a 100 x 2 mm Chromolith ® Performance RP-18e column from Merck was used.
  • the eluent consisted of 10 mM ammonium acetate buffer pH 5.5 and acetonitrile 95: 5 and 96: 4, respectively.
  • the flow was 0.7 mL / min at 40 ° C column temperature.
  • R r values are:
  • Cofactors are trimethylsulfonium halide, trimethylsulfoxonium halide and DL-methionine methylsulfonium halide.
  • DL-methionine-methylsulfonium halide behaves differently than the other two alternative cofactors.
  • Compound 3 has a concentration optimum of about 100 mM for both enzymes and is higher than the other two compounds ( Figures 4 and 5). Similarly, compound 3 shows the highest conversion of both enzymes compared to the other two compounds. For salts 1 and 2, a concentration of 25 mM shows the best results. Very high concentrations of sulfonium or sulfoxonium salts seem to inhibit the enzymes.
  • SAM S-adenosyl-L-methionine
  • Saccharomyces cerevisiae mutants have been generated that produce large quantities of SAM up to 13.5 g / L. Nevertheless, methyltransferases, as enzymes for C-C bond formation, have not yet found access to industrial biotechnology. The main reason for this is the high price. The use of inexpensive and commercially available alkyl donors could pave the way for the use of SAM-dependent methyltransferases for selective alkylation.
  • SAM S-adenosyl-L-methionine
  • Can transfer methyl group from SAM to homocysteine can be used, e.g. L-homocysteine S-methyltransferase described by Shapiro and Stanley (Methods Enzymol 17 Pt.B, Sulfur Amino acids, pp. 400-405 (1971)) and Shapiro (Biochim Biophys Acta 29: 405-9 ( 1958)) and even more preferred are the SAM-dependent homocysteine S-methyltransferases from E. coli (Thanbichler et al., J.

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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • General Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne un procédé pour transférer un groupe alkyle, alkényle ou alkinyle à un composé à faible poids moléculaire ("petite molécule") présentant un centre nucléophile, ledit procédé comprenant les étapes suivantes: mettre en contact le composé avec une méthyltransférase dépendant de S-adenosyl-L-méthionine (SAM) en présence d'un sel de sulfonium alkylé de formule générale (I) ou en présence d'un sel de sulfoxonium de formule générale (II). A cet effet, XΘ désigne un anion organique ou inorganique et R1, R2 et R3 désignent chacun indépendamment l'un de l'autre un groupe alkyle, alkényle et alkinyle substitué ou non substitué comprenant entre 1 et 10 atomes de carbone.
PCT/AT2012/000228 2011-09-01 2012-09-03 Agent et procédé d'alkylation Ceased WO2013029075A1 (fr)

Applications Claiming Priority (2)

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ATA1254/2011 2011-09-01
ATA1254/2011A AT511856A1 (de) 2011-09-01 2011-09-01 Mittel und verfahren zur alkylierung

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148430A (zh) * 2015-04-02 2016-11-23 中国科学院大连化学物理研究所 一种生物催化儿茶酚类化合物烃基化的方法
CN106866804A (zh) * 2015-12-11 2017-06-20 中国科学院植物研究所 一种与植物光合作用相关的cam01蛋白及其编码基因和应用
WO2020053196A1 (fr) 2018-09-10 2020-03-19 Universität Basel Procédé pour l'alkylation biocatalytique d'un substrat

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WO2010115846A1 (fr) * 2009-04-02 2010-10-14 Biotechnologijos Institutas Dérivation de biomolécules par couplage covalent de composés non-cofacteurs à l'aide de méthyltransférases

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AU2008334087A1 (en) * 2007-11-30 2009-06-11 The Regents Of The University Of California Biological systems for production of commercially valuable compounds

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EP1712557A1 (fr) * 2005-04-14 2006-10-18 RWTH Aachen Analogues de s-adénosyl-L-méthionine avec des groupes activés, allongés pour tranfert avec méthyltranférases
WO2010115846A1 (fr) * 2009-04-02 2010-10-14 Biotechnologijos Institutas Dérivation de biomolécules par couplage covalent de composés non-cofacteurs à l'aide de méthyltransférases

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Title
HARALD STECHER ET AL: "Biocatalytic Friedel-Crafts Alkylation Using Non-natural Cofactors", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 48, no. 50, 7 December 2009 (2009-12-07), pages 9546 - 9548, XP055048957, ISSN: 1433-7851, DOI: 10.1002/anie.200905095 *
KIESER ET AL.: "Practical Streptomyces Genetics", 2000, THE JOHN INNES FOUNDATION
MARTIN TENGG ET AL: "Molecular characterization of the C-methyltransferase NovO of Streptomyces spheroides, a valuable enzyme for performing Friedel-Crafts alkylation", JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC, vol. 84, 1 December 2012 (2012-12-01), pages 2 - 8, XP055049092, ISSN: 1381-1177, DOI: 10.1016/j.molcatb.2012.03.016 *
S. KLIMASAUSKAS ET AL., NAT. CHEM. BIOL., vol. 5, 2009, pages 400 - 402
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SHAPIRO; STANLEY, METHODS ENZYMOL. 17 PT.B, SULFUR AMINO ACIDS, 1971, pages 400 - 405
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148430A (zh) * 2015-04-02 2016-11-23 中国科学院大连化学物理研究所 一种生物催化儿茶酚类化合物烃基化的方法
CN106148430B (zh) * 2015-04-02 2019-09-06 中国科学院大连化学物理研究所 一种生物催化儿茶酚类化合物烃基化的方法
CN106866804A (zh) * 2015-12-11 2017-06-20 中国科学院植物研究所 一种与植物光合作用相关的cam01蛋白及其编码基因和应用
CN106866804B (zh) * 2015-12-11 2021-03-05 中国科学院植物研究所 一种与植物光合作用相关的cam01蛋白及其编码基因和应用
WO2020053196A1 (fr) 2018-09-10 2020-03-19 Universität Basel Procédé pour l'alkylation biocatalytique d'un substrat

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