AT511856A1 - MEDIUM AND METHOD OF ALKYLATION - Google Patents

Abstract

The present invention relates to a method for transferring 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 sulfonium salt having the general formula (I) or an alkylated sulfoxonium salt having the general formula (II) where 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 1 to 10 carbon atoms.

Description

The present invention relates to means and processes for alkylation.

The formation of C-O bonds for the formation of esters and ethers, the formation of CN bonds for the formation of secondary and tertiary amines and amides, the formation of CS bonds for the formation of thioethers and sulfonium compounds and the formation of C-C bonds for the chain extension of aliphatic Substances and alkylation of aromatic compounds (Friedel-Crafts reaction) are widely used in the synthesis of organic compounds. Common alkylating agents for carrying out the reactions described above 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 separation steps and increases the amount of waste produced.

Therefore, alternative methods of alkylation under environmentally friendly processes are desirable. Enzymatic methods usually meet these requirements. Methyltransferases are enzymes that perform alkylation reactions. These enzymes need cofactors. S-adenosyl-L-methionine (SAM) is one of the most commonly used cofactors in nature and highly conserved among SAM-dependent methyltransferases. More than 150 reactions are known to be catalyzed by SAM-dependent methyltransferases. The enzymes are classified according to their substrates. These are low molecular weight compounds ("small molecules"), proteins, nucleic acids and polysaccharides. The target atoms in these molecules are carbon, oxygen, nitrogen, sulfur or halogen. Recently, it has been reported that two enzymes besides SAM also accept analogous compounds in which the methyl group of SAM has been replaced by alkenyl, alkynyl and benzyl residues (Stecher H. et al., Angew Chem. Int. Ed, 48 (2009) : 9546 to 9548). Nevertheless, both SAM and its analog connections are very expensive. Therefore, such cofactors can not be used to establish economic processes, to alkylate large amounts of substrate.

It is an object of the present invention to provide methods and means for alkylating low molecular weight compounds ("small molecules") containing nucleophilic centers. These methods and means are intended to overcome the disadvantages of the known methods. Therefore, the present invention relates to a method of transferring 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 sulfonium salt having the general formula (I) Ί © R1 [

Or an alkylated sulfoxonium salt having the general formula (II) R 1 i r 2 -s = .gamma. (X) ## STR5 ## O

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.

It has surprisingly been found that simple alkylsulfonium and alkylsulfoxonium salts, as indicated above, 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 efficiency of enzymatic reactions depends not only on the enzymes, substrates and cofactors used, but also on the reaction conditions under which the reactions were carried out. Therefore, it is preferred that the method according to the present invention be carried out at a temperature at which the methyltransferase used shows the highest reaction rate, preferably between 25 and 45 ° C, more preferably between 30 and 35 ° C.

The term "low molecular weight compound" ("Small molecule") used herein refers to a low molecular weight, low molecular weight organic compound (molecular weight less than or equal to 1 kDa, preferably molecular weight less than or equal to 0.8 kDa), which is produced by Definition is not a polymer. The term "small molecule" also refers to a molecule which is bound to a biopolymer, such as a protein, nucleic acid, polysaccharide or a resin, and in addition may alter the activity or function of the polymer. "Small molecules " can have a variety of biological functions, such as signaling molecules, as tools in molecular biology, as medicines, as pesticides, and more. These compounds may be more natural (such as secondary metabolites) or artificial (such as antiviral drugs). They may have positive effects against diseases (e.g., drugs) or deleterious effects (e.g., teratogens and carcinogens). The target atoms in the substrate are oxygen, nitrogen, sulfur and carbon. The nucleophilic centers in the substrate are -OH, -NH2, -NHR, -CONH2, -CONHR, -SH, -SR, and electron-rich carbon atoms, preferably an sp2-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, N-methyltransferases catalyze the methylation of primary and secondary amines (-ΝΗ2, -NHR), imines (= NH) and amides (- CONH2, -CONHR), S-methyltransferases catalyze the methylation of thiols (-SH) and thioethers (-SR). C-methyltransferases transfer the methyl group on an electron-rich carbon atom.

According to a preferred embodiment of the present invention, 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. Particular preference according to the invention thus substituted or unsubstituted C3 to C10, preferably C2 to Cg, more preferably Ci to C7, even more preferably Ci to C3, in particular C1 (alkyl, alkenyl or alkynyl used.

According to another preferred embodiment of the present invention, 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.

According to a particularly preferred embodiment of the present invention, one or more of the following compounds is used in the process according to the invention:

1 2 3

The method according to the present invention involves the use of SAM-dependent methyltransferases. This group of enzymes includes methyltransferases that use and / or require SAM as cosubstrate (cofactor) for their enzymatic activity. The 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. Thus, the SAM-dependent methyltransferases of the present invention show a reduced or no affinity for methionine methylsulfonium. Therefore, a methyltransferase such as homocysteine S-methyltransferase EC 2.1.1.10, which shows a higher affinity to co-substrates other than SAM, is not to be used as a preferred methyltransferase in the method according to the present invention. 4 * «f * * * * * *» • i «« · · · # · · «· t · * · ··

The Km value of SAM in the SAM-dependent methyltransferases used in the method according to the present invention is preferably less than 103 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 guanidinoacetate N-methyltransferase, EC 2.1.1.4 acetylserotonin O-methyltransferase, EC 2.1.1.6 catechol O-methyltransferase, EC 2.1.1.7 nicotinate N-methyltransferase, EC 2.1.1.8 histamine N-methyltransferase, EC 2.1.1.9 thiol S-methyltransferase, EC 2.1.1.12 methionine S-methyltransferase, EC 2.1.1.15 fatty acid O-methyltransferase, EC 2.1.1.16 methylene fatty acid acyl phospholipid synthase, EC 2.1.1.17 phosphatidylethanolamine N-methyltransferase, EC EC 2.1.1.20 glycine N-methyltransferase, EC 2.1.1.22 carnosine N-methyltransferase, EC 2.1.1.25 phenol O-methyltransferase, EC 2.1.1.26 iodophenol O-methyltransferase, EC 2.1.1.27 Tyramine N-Methyltransferase, EC 2.1.1.28 Phenylethanolamine N-Methyltransferase, EC 2.1.1.38 0-Demethylpuromycin O-Methyltransferase, EC 2.1.1.39 Inositol 3-Methyltransferase, EC 2.1.1.40 Inositol 1-Methyltransferase, EC 2.1.1.41 Sterol 24- C-Methyltransfera se, EC 2.1.1.42 luteolin 0-methyltransferase, EC 2.1.1.44 dimethylhistidine N-methyltransferase, EC 2.1.1.46 isoflavone 4'-0-methyltransferase, EC 2.1.1.47 indolepyruvate C-methyltransferase, EC 2.1.1.49 amine N-methyltransferase, EC 2.1.1.50 Loganate O-methyltransferase, EC 2.1.1.53 Putrescine N-methyltransferase, EC 2.1.1.59 [cytochrome c] lysine N-methyltransferase, EC 2.1.1.64 3-demethylubiquinone-9 3-O-methyltransferase, EC 2.1.1.65 Licodione 2'-O-Methyltransferase, EC 2.1.1.67 Thiopurine S-Methyltransferase, EC 2.1.1.68 Caffeate O-Methyltransferase, EC 2.1.1.69 5-Hydroxyfurancoumarin 5-0-Methyltransferase, EC 2.1.1.70 8- Hydroxyfurancoumarin 8-0-methyltransferase, EC 2.1.1.71 phosphatidyl-N-methylethanolamine N-methyltransferase, EC 2.1.1.75 apigenin 4'-0-methyltransferase, EC 2.1.1.76 quercetin 3-O-methyltransferase, EC 2.1.1.78 isoorientin 3 ' -0-methyltransferase, EC 2.1.1.79 cyclopropane-fatty acid acyl-phospholipid synthase, EC 2.1.1.82 3-methylquercetin 7-0-methyltransferase, EC 2.1.1.83 3,7 -Dimethylquercetin 4'-O-methyltransferase, EC 2.1.1.84 methylquercetagetine 6-0-methyltransferase, EC 2.1.1.87 pyridine N-methyltransferase, EC 2.1.1.88 8-hydroxyquercetin 8-0-methyltransferase, EC 2.1.1.89 tetrahydrocolumbamine 2 -0-methyltransferase, EC 2.1.1.91 isobutyraldoxime O-methyltransferase, EC 2.1.1.94 tabersonine 16-0-methyltransferase, EC 2.1.1.95 tocopherol O-methyltransferase, EC 2.1.1.96 thioether S-methyltransferase, EC 2.1.1.97 3 -Hydroxyanthranilate 4-C-methyltransferase, EC 2.1.1.98 diphthin synthase, EC 2.1.1.99 3-hydroxy-16-methoxy-2,3-dihydrotabersonine N-methyltransferase, EC 2.1.1.101 macrocin O-methyltransferase, EC 2.1.1.102 demethylmacrocin 0-methyltransferase, EC 2.1.1.103 phosphoethanolamine N-methyltransferase, EC 2.1.1.104 caffeoyl-CoA O-methyltransferase, EC 2.1.1.105 N-benzoyl-4-hydroxyanthranilate 4-O-methyltransferase, EC 2.1.1.106 tryptophan 2-C- Methyltransferase, EC 2.1.1.107 Uroporphyrinogen III C-methyltransferase, EC 2.1.1.108 6-Hydroxymellein O-methyltran sferase, EC 2.1.1.109 Demethylsterigmatocystin 6-O-methyltransferase, EC 2.1.1.110 Sterigmatocystin 8-0-methyltransferase, EC 2.1.1.111 Anthranilate N-methyltransferase, EC 2.1.1.112 Glucuronoxylan 4-O-methyltransferase, EC 2.1.1.114 Polyprenyldihydroxybenzoate Methyltransferase , EC 2.1.1.115 (RS) -l-benzyl-l, 2,3,4'-tetrahydroisoquinoline N-methyltransferase, EC 2.1.1.116 3'-hydroxy-N-methyl- (S) - «· · · ·« · * * · · • φ »φ φ · φ # ··« · • φ · * «· φ φ · * · J ♦ φ φ φ # φ φ φ * φ

Coclaurine 4'-O-methyltransferase, EC 2.1.1.117 {S) -coulerin 9-O-methyltransferase, EC 2.1.1.118 columbamine O-methyltransferase, EC 2.1.1.119 10-hydroxydihydrosanguinarine 10-0-methyltransferase, EC 2.1.1.120 12 -Hydroxydihydrochelirubin 12-0-methyltransferase, EC 2.1.1.121 6-0-Methylnorlaudanosolin 5'-O-methyltransferase, EC 2.1.1.122 (S) -tetrahydroprotoberberin N-methyltransferase, EC 2.1.1.123 [cytochrome c] -methionine S-methyltransferase , EC 2.1.1.124 [cytochrome c] arginine N-methyltransferase, EC 2.1.1.128 (RS) norcodaurin 6-O-methyltransferase, EC 2.1.1.129 inositol 4-methyltransferase, EC 2.1.1.130 precorrin-2 C20 methyltransferase, EC 2.1.1.131 precorrin-3B C17 methyltransferase, EC 2.1.1.132 precorrin-6Y C5,15-methyltransferase (decarboxylating), EC 2.1.1.133 precorrin-4 Cll methyltransferase, EC 2.1.1.136 chlorophenol 0-methyltransferase, EC 2.1. 1.137 arsenite methyltransferase, EC 2.1.1.139 3'-demethylstaurosporine O-methyltransferase, EC 2.1.1.140 (S) -coramaur-N-methyltransferase , EC 2.1.1.141 Jasmonate O-Methyltransferase, EC 2.1.1.142 Cydoartenol 24-C-Methyltransferase, EC 2.1.1.143 24-Methylene Esterol C-Methyltransferase, EC 2.1.1.144 Trans-Aconity 2-Methyltransferase, EC 2.1.1.145 Trans-Aconitat 3-methyltransferase, EC 2.1.1.146 (iso) eugenol O-methyltransferase, EC 2.1.1.147 corydaline synthase, EC 2.1.1.149 myricetin O-methyltransferase, EC 2.1.1.150 isoflavone 7-O-methyltransferase, EC 2.1.1.151 cobalt Factor II C20 Methyltransferase, EC 2.1.1.152 Precorrin-6A Synthase (Deacetylation), EC 2.1.1.153 Vitexin 2 " -0-Rhamnoside 7-O-Methyltransferase, EC 2.1.1.154 Isoliquiritigenin 2'-O-methyltransferase, EC 2.1 .1.155 Kaempferol 4'-O-methyltransferase, EC 2.1.1.156 glycine / sarcosine N-methyltransferase, EC 2.1.1.157 sarcosine / dimethylglycine N-methyltransferase, EC 2.1.1.158 7-methylxanthosine synthase, EC 2.1.1.159 theobromine synthase, EC 2.1 .1.160 caffeine synthase, EC 2.1.1.161 dimethylglycine N-methyltransferase, EC 2.1.1.162 glycine / sarcosine / dimethylglycine N-met hyltransferase, EC 2.1.1.163 demethylmenaquinone 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 3 ', 4', 5'-0-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 the methyltransferases mentioned here are known. However, the method of the present invention can be used to alkylate, alkenylate and alkynylate other substrates (other "small molecules") as well. 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 has been modified by incubation of methyltransferase and corresponding cosubstrate.

According to a preferred embodiment of the present invention, the organic or inorganic anion is selected from the group of halides and sulfonates.

In a particularly preferred embodiment of the present invention, the alkylated sulfonium salt having the general formula (I) and the alkylated sulfoxonium salt having the general formula (II) in a co-substrate to substrate ratio of 10: 1 to 200: 1, preferably 15: 1 to 150: 1, more preferably from 20: 1 to 125: 1, especially from 25: 1 to 100: 1, has been added to the reaction mixture. For example, in the reaction mixture, when the substrate is in a concentration of 0.5 to 2 mM, preferably 1 mM, the cosubstrate according to general formulas (I) and (II) according to the present invention is in a concentration of 12 to 200mM, preferably 25 to 100mM.

According to a preferred embodiment of the present invention, the methyltransferases used in the method according to the present invention have been prepared recombinantly. In addition, it is particularly preferred to use non-purified or substantially non-purified SAM-dependent methyltransferases in the process according to the invention. That is, in the method of the present invention, a crude lysate of a culture medium comprising cells capable of producing SAM-dependent methyltransferases can be used. Alternatively, it is also possible to use the supernatant of the disrupted cells which produce SAM-dependent methyltransferases.

In the most preferred embodiment, frozen and thawed or disrupted recombinant cells overexperimating SAM-dependent methyltransferases have been used for methylation of substrates.

Another aspect of the present invention relates to a kit 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) 1 © R1 _ Θ. R R J x (I) or sulfoxomium salt having the general formula (II) R 1 i r 2 -s = o

ΘXί wherein R1, R2 and R3 are each independently a substituted or unsubstituted alkyl, alkenyl and alkynyl group comprising 1 to 10 carbon atoms. The present invention is further illustrated with the following figures and examples, but without being limited thereto.

Figure 1 shows S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH) and SAM analogues. DNA methyltransferases have also been shown to accept non-natural cofactors (SAM analogues) and sequence-specific DNA alkylation has been demonstrated using S-adenosyl L- + * 4 * ····· ♦ * ♦ 9 I «·» . In all known and published results, the adenosyl part of the cofactor remains untouched and can interact with the enzyme.

Figure 2 shows sulfur containing salts tested as possible methyl donors for CouO and NovO. Trimethylsulfonium 1 and Trimethylsulfoxoniurn 2 were used as iodides, Methioninmethylsulfonium 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) Et3N, MgCl2, THF, 2h at 0 ° C, 16h at rt, 73%. e) aq. NaOH, MeOH, rt, 16h, 92%. f) IN HCl, MeOH, rt, 24h, 67%. g) Pyrrole-2-carboxylic acid, PyBOP ((benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate), NMM (N-methylmorpholine), DMF, rt, Ar atmosphere, 48h, 78%. h) PhCOCl, pyridine, DMF, rt, 24h. ί) 3N NaOH / MeOH 1: 1, rt, 72h. (h + i> 99%).

Figure 4 shows the methylation of coumarin benzamide 4a catalyzed by CouO at different concentrations of the sulfonium and sulfoxonium salts. Me3S trimethylsulfonium iodide; Me3S = 0 trimethylsulfoxonium iodide; MeMet DL-methionine methylsulfonium chloride

Figure 5 shows the methylation of coumarin benzamide 4a catalyzed by NovO at different concentrations of the sulfonium and sulfoxonium salts. Me3S trimethylsulfonium iodide; Me3S = 0 trimethylsulfoxonium iodide; MeMet DL-methionine methylsulfonium chloride

Figure 6 shows the influence of the addition of SAH to the reaction mixtures. Concentrations of Me3S + f and Me3SO * V are each 25 mM, concentration of methionine methyl sulfonium chloride is 100 mM. Me3S trimethylsulfonium iodide; Me3S = 0 trimethylsulfoxonium iodide; MeMet DL-methionine methylsulfonium chloride

Examples

Two S-adenosyl-L-methionine (SAM) -dependent C-methyltransferases from Streptomyces rishiriensis (CouO) and Streptomyces spheroides (NovO) were cloned and overexpressed in E. coli. The methyltransferases CouO and NovO are involved in the biosynthesis of the aminocoumarin antibiotics coumermycin Al in Streptomyces rishiriensis and novobiocin in 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. 8 ·· ·· »ι * ···

SAM-dependent methyltransferase, involved in the biosynthesis of the antibiotic novobiocin, was discovered in 1960. The entire gene cluster for the biosynthesis of coumermycin Al in Streptomyces rishiriensis and the gene CouO, which codes for the methyltransferase, have already been extensively studied, likewise 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. Here, methylsulfonium and methylsulfoxonium salts (FIG. 2) were shown for the first time, which proved to be advantageous in a methylation catalyzed by SAM-dependent methyltransferase. L Experimental Part 1.1. Materials 1.1.1. Cloning and Expression For the cloning and heterologous expression of the methyltransferase-encoding genes NovO and CouO, the genomic DNA of the strains Streptomyces spheroides (DSMZ 40292) and Streptomyces rishiriensis (DSMZ 40489) was determined according to the protocol of Kieser et al. (Practical Streptomyces Genetics (Eds .: T. Kieser, et al.), The John Innes Foundation, Norwich, 2000).

The genes were amplified according to standard PCR conditions using the forward primer 5'-AATCTGCATATGAAGATTGAACCGATTAC-3 'and the reverse primer 5'-TAGGTAAAGC lTTCAGGCAGCGGCCCGACGGG-3'. These primers each introduced the restriction sites Ndel and Hindill (see underlined part of the primer) for subsequent cloning into the expression vector pET26b (+). The PCR products were gel purified, digested with Ndel and HindIII, and ligated into the linearized pET26b (+) vector. The use of these primers for both genes resulted in two amino acid changes in the case of NovO (A5P and S223C).

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 pg / ml kanamycin to an OD600 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. 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 disrupted by sonication for 6 minutes.) The insoluble cell debris were removed by centrifugation (60 minutes at • 4 * ·· * * · · * * VI · «* *« * * * * «« «*« * * * 1 * * 0 * * * * * · * * * * 50000 xg) are separated and removed The supernatant was analyzed by SDS-PAGE and stored for enzymatic reactions at 4 ° C. The insoluble cell cake was resuspended in buffer A and also analyzed on SDS-PAGE

While stirring and protected from light, to a solution of 5.0 g of 2,4-dihydroxybenzoic acid (32.4 mmol, 1 eq.) In 30 mL of pyridine, 15.3 mL of acetic anhydride (16.6 g, 162 mmol, 5 eq. ) and 0.40 g of DMAP (3.24 mmol, 0.1 eq.). After 24 hours, ice was added to the reaction solution. The solution was acidified with aqueous 3N HCl. The resulting suspension was extracted 3x with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and then dried with anhydrous Na 2 SO 4. The solution was concentrated to ca. 15 mL and the product crystallized on standing. The filtered product was dried over CaCl 2. A second batch of product precipitated from the mother liquor. In total, 7.2 g (93%) of cream-white crystals were collected. * H NMR (300MHz, DMSO-d6) δ ppm: 13.12 (br, 1H, -COOH), 7.98 (d, J = 8.6Hz, 1H, -CHar-}, 7.18 (dd, J1 = 8.6, J2 = 2.3Hz, 1H, - CHar-), 7.09 (d, J = 2.2Hz, 1H, -CHar-), 2.29 (s, 3H, -CH3), 2.25 (s, 3H, -CH3). 13C (75MHz) δ ppm: 169.0, 1668.6, 165.0, 154.0, 151.1, 132.5, 121.6, 119.7, 117.6, 20.8 , 20, 8, 2.4-diacetoxybenzoyl chloride 2.0 g 2,4-diacetoxybenzoic acid (8.4 mmol, 1 eq) was dissolved in 20 mL CH 2 Cl 2, 12 mL SOCl 2 (20 g, 165 mmol, 20 eq) became The mixture was refluxed for 5 h then CH2CIZ and SOCl2 were distilled off and the resulting brown solid was used for the next step without further characterization 2- (tert-butoxycarbonylamino) -3-ethoxy-3-oxopropanoic acid

To a suspension of 4.49 g KOH (80 mmol, 1 eq.) In 100 mL EtOH was added 20.4 mL diethyl 2- (7-tert-butyloxycarbonylamino) propanedioic acid ester (22.0 g, 80 mmol, 1 eq.). The mixture was stirred for 3 h and then removed about 90% of the solvent. 150 mL of an aqueous NaHCO 3 solution was added and washed 2 × with ethyl acetate to remove the unreacted starting material. The solution was cooled to 0 ° C, acidified with KHSO4 and extracted 3x with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over Na 2 SO 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%). ! H NMR (500MHz, CDCl3) δ ppm: 7.73 (d, J = 4.4Hz, 0.6H, -NH-), 5.65 (d, J = 6.8Hz, 0.4H, -NH -), 4.98 (d, J = 7.3Hz, 0.4H, -CH-), 4.77 (d, J = 4.9Hz, 0.6H, -CH-), 4.23-4 , 31 (m, 2H, -CH2-), 1.42 (s, 9H, 3 -CH3), 1.31 (t, J = 7.3Hz, 3H, -CH3). 13C (125MHz) δ ppm: 168.5, 166.9, 156.4, 83.0, 63.0, 58.9, 28.3, 14.3. 4- (2- (tert-butoxycarbonylamino) -3-ethoxy-3-oxopropanoyl) -1,3-phenylenediacetate * * · #. «♦ ·ο · .. * * .. * * .. *. * * * .. *.:.

To an ice-cooled solution of 2- (tert-butyloxycarbonylamino) -3-ethoxy-3-oxo-propanoic acid (2.91 g, 11.8 mmol, 1.4 eq.) In dry THE (30 mL) was added 7.4 mL of triethylamine ( 5.36 g, 52.8 mmol, 6.3 eq.) And 2.69 g MgCl 2 (28.3 mmol, 3.4 eq.) Were added and the resulting suspension stirred vigorously for 2 h. The crude substance 2,4-diacetoxybenzoyl chloride (theoretical amount 8.4 mmol, 1 eq.) In 50 mL dry THE was added to the solution and the resulting suspension was stirred overnight at room temperature. The reaction was quenched with saturated aqueous NHdCl solution until a clear solution formed. The solution was extracted 3x with ethyl acetate. The combined organic phases were dried over Na 2 SO 4. After removal of the solvent remained 4.31 g (121%) of a dark brown oil, which was used without further purification for the next stage.

Tert-butyl 4/7 -dihydroxy-2-oxo-2H-chromen-3-ytcarbamate 4

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 SOd and the solvent removed under vacuum.

The crude product was purified by chromatography. The yield was 1.24 g (50% over three steps) of an orange solid. ] H NMR (500MHz, DMSO-d6) δ ppm: 7.78 (s, 1H, -NH-), 7.64 (d, J = 8.8Hz, 1H, -CHar-), 6.77 (dd , J1 = 8.8, J2 = 2.0Hz, 1H, -CHar-), 6.66 (d, i = 2Hz, 1H, -CHar-), 1.40 (s, 9H, -CH3). 13C NMR (125MHz) δ ppm: 166.4, 161.9, 161.6, 155.4, 154.1, 125.7, 113.5, 108, .7, 102.5, 100.8, 79 , 3, 28.9. 3-amino-4,7-dihydroxy-2H-chromen-2-one hydrochloride

To a cooled solution of 25 mL Et20 and 5 mL MeOH was carefully added 4.3 mL acetyl chloride. The acidic solution was added to a cooled and stirred solution of 1.24 g of tert-butyl 4,7-dihydroxy-2-oxo-2H-chromen-3-ylcarbamate in 25 mL Et 2 O and 5 mL MeOH (IN HCl in the reaction solution). cast. The solution was stirred for a further 24 h at room temperature. The precipitate was collected and dried. The yield was 0.52 g (53%) of a light brown solid. * H NMR (500MHz, DMSO-d6) O ppm: 7.96 (d, J = 8.8Hz, 1H, -CHar), 6.84 (dd, J1 = 8.8Hz, J2 = 2.0Hz, 1H , -CHar-), 6.75 (d, J = 2.0Hz, 1H, -CHar-). 13C NMR (125MHz) δ ppm: 162.7, 161.5, 160.4, 154.2, 126.4, 113.9, 108.7, 103.0, 95.5. N- (4,7-Dibydroxy-2-oxo-2H-chromen-3-yl) benzamide 4a

To a stirred solution of 200 mg of 3-amino-4,7-ihydroxy-2H-chromen-2-one hydrochloride (0.87 mmol, 1 eq.) In 10 mL of ethyl acetate was added 0.51 mL of benzoyl chloride (0.61 g , 4.4 mmol, 5 eq.) And 1.22 mL triethylamine (0.88 g, 8.7 mmol, 10 eq.). The suspension was stirred at room temperature overnight. The precipitate was filtered off and the solvent evaporated. The remaining solid was dissolved in 10 mL MeOH and 10 mL IN NaOH added.

The reaction solution was stirred for 24 h, then acidified to pH 3 with IN HCl and extracted 3x with ethyl acetate. The combined organic phases were washed with saturated NaCl solution, dried over Na2504, celite added and the solvent removed under vacuum. The fine brown powder was purified by chromatography. The yield was 243 mg (94%) of an orange solid. * H NMR (500MHz, DSM0-d6) δ ppm: 11.82 (s, 1H, -OH), 10.56 (s, 1H, -OH), 9.42 (s, 1H, -NH-), 8.00 (d, J = 7.3Hz, 2H, -CHar-), 7.72 (d, J = 8.8Hz, 1H, -CHar-), 7.58 (t, J = 7.3Hz, 1H, -CHar-), 7.51 (t, J = 7.6Hz, 2H, -CHar-), 6.83 (dd, J1 - 8.8Hz, J2 = 2.0Hz, 1H, -CHar-) , 6.74 (d, J = 2.4Hz, 1H, -CHar-). 13C (125MHz) δ ppm: 166.6, 161.4, 160.8, 160.3, 153.5, 133.9, 131.6, 128.2, 128.0, 125.0, 113.0 , 107.9, 101.9, 100.1. N- (4,7-D // 7cycloxy-2Oxo-2H-cf-rom-3-y / i) -1H-pyrrole / -2-corf) oxom / d 4b

A mixture of 200 mg of 3-amino-4,7-dihydroxy-2H-chromen-2-one hydrochloride (0.87 mmol, 1 eq.), 97 mg of ltf-pyrrole-2-carboxy acid (0.87 mmol, 1 Eq.), 454 mg (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.87 mg, 1 eq.) And 770 μL N-methylmorpholine (707 mg, 7.0 mol, 8 eq.) In 12 mL DMF was stirred for 27 h at room temperature under argon atmosphere. An additional 227 mg of PyBOP (0.44 mmol, 0.5 eq.) Was added to the solution and stirred for a further 66 h. The reaction solution was acidified with IN HCl solution and extracted 3x with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over Na 2 SO 4. The solvent was removed in vacuo. The resulting brown oil was taken up in ethyl acetate. It formed a suspension. The filtered solid formed the desired product. From the mother liquor, a product was further recovered and recrystallized in cyclohexane. The dried product gave a yield of 180 mg (72%) of a brown powder. ] H NMR (500MHz, DMSO-d6) δ ppm: 12.01 (br, 1H, -OH), 11.65 (s, 1H, -NH-) 10.57 (br, 1H, -OH), 9 , 30 (s, 1H, -NH-), 7.69 (d, J = 8.3Hz, 1H, -CHar-) 7.01 (s, 1H, CHpyrr), 6.94 (s, 1H, - CHpyrr-), 6.81 (dd, J1 = 8.8, J2 = 2.0Hz, 1H, -CHar-), 6.71 (d, J = 2Hz, 1H, -CHar-), 6.15 ( s, 1H, -CHpyrr-). 13C NMR (125Hz) δ ppm: 162.0, 161.6, 161.5, 160.3, 154.0,

1.1.3. Activity assay of methyltransferase

The enzymatic conversion was carried out according to the scheme shown (enzymatic synthesis of alkyl coumarin derivatives):

OH

NovO or CouO 4

Buffer / DMSO 9: 1 30 ° C, 24h

OH

For the activity study, model substrates with high similarity to the natural substrates of these enzymes were selected and synthesized (FIG. 3). The possible methyl sources (Figure 2) were tested for activity with both CouO and NovO enzymes. At the beginning * * * * 12 * of the studies on alternative cofactors concentrations of sulfonium salts of 1 M were chosen to work in a similar concentration range reported by the working group Klimasauskas (see S. Klimasauskas et al., Nat. Chem Biol. 5, 2009, 400-402). This group was able to show a hydroxyalkylation catalyzed by DNA methyltransferase using formaldehyde and acetaldehyde as a SAM substituent at 0.8 M concentration. For the enzymatic conversion harvested cells of the recombinant E. coli were used, which were frozen in liquid nitrogen lx and then thawed. 0.1 g of disrupted cells per mL buffer (100 mM phosphate buffer pH 6.5 for NovO and pH 7.0 for CouO) were used for the investigations. Various negative controls were performed to exclude chemical product formation. No product formation was detected when no enzyme was added or prior to addition the enzyme was thermally inactivated (inactivation before use for 30 minutes at 85eC). At a concentration of 1M, trimethylsulfoxonium iodide is not soluble while trimethylsulfonium iodide and methionine methylsulfonium chloride are completely dissolved at that concentration. The conversion was 5-20% (highest conversion with compound 2). The influence of the concentration of alternative cofactors on the turnover was investigated in further experiments. The concentration range was examined in two independent experiments from 2 mM to 1 M. The results are summarized in FIGS. 4 and 5.

The reactions were carried out in 0.2 mL scale in a thermomixer at 30eC and 1000 rpm for 24 h. The solutions contained 1 mM (mmolar) substrate of a 10 mM stock solution in DM50 (dimethyl sulfoxide), 2 mM SAM (A7007 from Sigma) from stock solution in 50 mM phosphate buffer (positive control) or the sulfonium salts at various concentrations, 1 mg / mL BSA (Bovine serum albumin A3294 from Sigma) and disrupted cells containing the recombinant proteins CouO and NovO, respectively. The reaction was stopped by heating the reaction vessels to 85 ° C for 15 minutes. Subsequently, the reaction vessels were placed in the refrigerator for 15 minutes. Thereafter, the mixtures were centrifuged at 14,000 rpm for 15 minutes. 3 μl of the aqueous solution were injected into the HPLC without further dilution. This screening was repeated 3 times. The analysis was performed on a 1200 Agilent HPLC system equipped with autosampler, Multiwave UV and SingleQuad MS detector. The M $ 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. Rf values are: 4a: 2.3 minutes; 8-methyl-4a: 4.9 minutes

To investigate whether the methyl donors recycle via the methylation of SAH, SAM, experiments with two different SAH concentrations were performed. The results are shown in FIG. 2. Results and conclusions

Two SAM-dependent methyltransferases CouO and NovO have been discovered that surprisingly accept alternative alkyl donors as cofactors for methylation. A single product was formed, although a very large amount of the methylating reagent was used, which is cheap, commercially available and non-toxic. The alternative cofactors are trimethylsulfonium halide, trimethylsulfoxonium halide and Dl-methionine methylsulfonium halide.

In terms of turnover, DL-methionine-methylsulfonium halide behaves differently than the other two alternative cofactors. Compound 3 has an optimum concentration of about 100 mM for both enzymes and is higher than the other two compounds (FIGS. 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.

A strong background reaction was found. When broken cells were used, product formation could be detected even without added methyl donor. Nevertheless, the addition of one of the three methyl donors increases the conversion compared to the experiments without the addition of alternative methyl donors. At the moment, the reaction mechanism of these "alternative cofactors" is not clarified. Whether the methyl group is transported directly from the alternative cofactor to the substrate or whether a transmethylation takes place before substrate methylation is the subject of further investigations.

The alkyltransferase activity of CouO and NovO shows the first case of an enzyme-catalyzed Friedel-Crafts reaction. These S-adenosyl-L-methionine (SAM) -dependent methyltransferases are able to be "non-natural". To accept cofactors (alkylated SAH derivatives) and to transfer the alkyl moiety to the aromatic system in a regioselective manner and with a high yield.The substrate scope of these enzymes is broader than previously expected.The implementation of completely different substrates than natural substrates makes these enzymes attractive Catalysts for Organic Synthesis For SAM, the natural compound, approaches to increasing the production of metabolic pathways have already been made: Pichia pastoris and Saccharomyces cerevisiae mutants have been generated, with large SAM levels up to 13.5 g / L Nevertheless, methyltransferases, as enzymes for CC bond formation, have not yet found access to industrial biotechnology, mainly because of the high price tag.

The use of inexpensive and commercially available alkyl donors could pave the way for the use of SAM-dependent methyltransferases for selective alkylation. To replace this "high energy" compound, simple alkylsulfonium salts were used successfully for the first time in this study. The specific activity of these methyltransferases for unnatural cofactors is not as high as for the natural cofactor SAM. At the same time, replacing SAM with simple methyl donors drastically increases the atom economy. The selectivity of the 14- 14- ψ · * * ♦ * »·

Enzymes leads to the avoidance of by-products. The use of enzymes as catalysts brings old general advantages of enzymatic reactions, such as mild reaction conditions and waste reduction, with it. The alkylsulfonium salts are inexpensive, commercially available and non-toxic alkylating reagents.

Any S-adenosyl-L-methionine (SAM) -dependent homocysteine S-methyltransferase which can transfer a 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. Bacteriol., 181 (2): 662-5 (1999)) or from Saccharomyces cerevisiae Chem., 239 (5): 1551-6 (1964) and Thomas et al., J. Biol. Chem., 275 (52): 40718-24 (2000)).

Claims (10)

Claims 1. A method of transferring 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 Presence of an alkylated sulfonium salt having the general formula (I) Ί © L R1 i Θ X (i) or an alkylated sulfoxonium salt having the general formula (II) R 1, i R 2-S = O R 3 Θ Θ X (ID wherein X @ is an organic or inorganic anion and R 1, R 2 and R 3 each independently represent a substituted or unsubstituted alkyl, alkenyl and alkynyl group comprising 1 to 10 carbon atoms. 2. The method according to claim 1, characterized in that the substituted or unsubstituted alkyl, alkenyl and alkynyl group 1 to 8 carbon atoms, preferably 1 to 7 carbon atoms, more preferably 1 to 3 carbon atoms, in particular 1 carbon atom. 3. The method according to claim 1 or 2, characterized in that the alkyl, alkenyl and alkynyl group is substituted by at least one carboxyl, carbonyl and / or amino group. 4. The method according to any one of claims 1 to 3, characterized in that the alkyl group is selected from the group consisting of methyl and 2-aminobutyric acid. 5. The method according to any one of claims 1 to 4, characterized in that the low molecular weight compound is an aliphatic or aromatic substrate having a molecular mass less than 1 kDa (1000 g / mol). 6. The method according to claim 5, characterized in that the aromatic compound comprises at least one homocyclic or heterocyclic aromatic ring. 7. The method according to any one of claims 1 to 6, characterized in that the SAM-dependent methyltransferase is a methyltransferase which methylates low molecular weight compounds. 8. The method according to any one of claims 1 to 7, characterized in that the organic or inorganic anion is selected from the group of halides or sulfonates. • ft ··· * * ♦ · • φ φ «· - *» t · ft φ * · · * i6 * ·· ** ·· *** · * A kit 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) Ί © R1 i, -SV " R3 R2 Θ X (I) or sulfoxomium salt having the general formula (II) R1 r2 -s = o R3 Ί © (II) f characterized in that R1, R2 and R3 each independently represent a substituted or unsubstituted alkyl, alkenyl and alkynyl group comprising 1 to 10 carbon atoms.