Method for synthesizing s-adenosyl-l-methionine analogues

Abstract

A method is disclosed for chemo-enzymatic production of the functionally active stereoisomer of S-adenosyl-L-methionine analogs with extended transferable moieties of formula (I) and (II) from corresponding alkylbromides and S-adenosyl-L-homocysteine.

Description

[0001] METHOD FOR SYNTHESIZING S-ADENOSYL-L -METHIONINE ANALOGUES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to chemoenzymatic method for production of S-adenosyl-L- methionine (AdoMet) analogues (cofactors of AdoMet-dependant methyltransferases) containing extended moieties useful for biotechnological applications, namely relates to halide methyltransferase-directed synthesis of stereomerically pure AdoMet cofactor analogues using corresponding alkylhalides and S-adenosyl-L-homocysteine.

[0004] BACKGROUND TO THE INVENTION

[0005] Methylation of DNA cytosines, histone amino acids, and RNA adenines or riboses are fundamental epigenetic mechanisms involved in many crucial biological processes including embryonic development, transcription, chromatin structure, disease pathogenesis, etc. The enzymatic reaction is catalysed by a large class of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that transfer a methyl group from the co-substrate (cofactor) AdoMet to N-, C-, O- or S-nucleophiles (Moore et al. 2013). The MTases with alkyltransferase activity engineered in recent years enable the transfer of larger moieties with functional or reporter groups from AdoMet analogues onto specific molecules or targets and has tremendous potential in areas of chemical and synthetic biology (Struck et al. 2012; Tomkuviene et al. 2019). These enzymes serve as tools for deciphering epigenetic / epitranscriptomic / biological processes and their regulation, and even for the synthesis of small chemical compounds such as pharmaceuticals, foods, and other natural products (Mickute et al. 2021 ; Stankevicius et al. 2022; Mo et al. 2017; Kunjapur et al. 2016).

[0006] Diverse functional AdoMet analogues were designed and utilized for labelling of DNA, RNA, proteins, or small molecules for isolation, visualization, and analysis of these tagged biomolecules by the next-generation sequencing and other technologies (Lukinavicius et al., 2012 and 2013; Stankevicius et al. 2022; Mickute et al. 2021 ; Stasevskij et al. 2017; Osipenko et al. 2017). The majority of current approaches use AdoMet analogues bearing a long transferable moiety with a triple bond next to the reactive carbon for effective DNA MTases’ reaction (Lukinavicius et al., 2013; Tomkuviene et al. 2019).

[0007] Regrettably, chemical synthesis of artificial AdoMet analogues is expensive, often very challenging, and suffers from poor stereoselectivity and low yields, while most biological applications require milligram quantities. Due to difficulties encountered during chemical synthesis, the chemoenzymatic production of cofactors would be considered advantageous, offering: improved site-, stereo-, chemoselectivity; higher stability due to ambient temperatures, mild and aqueous conditions which are crucial for enzymes; more sustainable and bioorthogonal; reduced costs (Bennett et al. 2017; Mordhorst et al. 2017; Zhang et al. 2021 ; Liao and Seebeck 2020; Doyon and Narayan 2020).

[0008] Recently, two distinct enzymes were used in chemoenzymatic synthesis. Methionine adenosyltransferases employing ATP and L-methionine (L-Met) analogues can produce a wide range of cofactors (Huber et al. 2020; Shu et al. 2020; Hartstock et al. 2018). Halogenases - adenosyl-chloride synthase and adenosyl-fluoride synthase, catalyse a reverse reaction to produce cofactors using L-Met or its derivatives and 5'-chloro- or 5'-fluoro-5'-deoxyadenosine (Davis et al. 2018; Bennett et al. 2017; Thomsen et al. 2013). Nonetheless, these chemoenzymatic approaches encountered substantial obstacles, including cumbersome chemical synthesis of L-Met analogues; competition of cellular L-Met with the L-Met analogues in biological milieus, which demands cultivation of cells in unnatural methionine-deficient media (Tang et al. 2021b).

[0009] Utilization of halide methyltransferases (HMTs) allows the use of S-adenosyl-L-homocysteine (AdoHcy) and haloalkanes, the synthesis of which is less complicated (Liao and Seebeck 2019). (Bengel et al. 2021 ; Tang et al. 2021a; Hoffmann et al. 2023). Previously reported production of a series of AdoMet analogues using Aspergillus clavatus HMT (Schulke et al. 2022) demonstrated conversion yields below 10% for alkyl halides with moieties larger than 5 carbon atoms. In the closest prior art (Hoffmann et al. 2023), the alkylation yield with benzyl iodide reached 29% (using 2.5 equivalents of the haloalkane, 0.1 equivalent of enzyme to AdoHcy and 2 h incubation at 37°C), but the compound also conferred a high (10%) non-specific background reactivity (chemical non-stereo-selective alkylation) in control reactions. Altogether, other prior arts reported stereoselective production of cofactors with sulfonium-bound moieties larger than 5 carbon atoms carrying functional groups at yields below 50%. The solution to this technical problem is achieved by providing examples shown in Figures 1-5 which demonstrate production of the functionally active stereomers of AdoMet analogues with extended moieties larger than 5 carbon atoms at conversion yields exceeding 60 — 95%.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides a chemoenzymatic method for producing of AdoMet analogues bearing moieties suitable for effective modification reactions using AdoMet-dependent methyltransferases. The method comprising synthesis of AdoMet analogues bearing moieties with a reporter group or functional group, which can be applied for targeted DNA methyltransferase-directed labeling of specific targets on proteins, RNA or DNA;

[0012] A chemoenzymatic method of producing S-adenosyl-L-methionine (AdoMet) analogues bearing moieties with a functional group or reporter group, according to formula I and II the method comprising contacting an AdoMet precursor molecule of formula III with an alkylhalide R-Hal and halide methyltransferase (HMT) under conditions suitable for converting AdoHcy or SeAdoHcy to AdoMet analogues, wherein n is 0,1 , 2, 3, or 4

[0013] Z is S, or Se

[0014] X is functional or reporter group: N3, C=CH, NH2, or 4-Y-2,3,5,6-Tetrazin-1-yl, 4-(4’Y- 2,3,5,6-tetrazin-1 ’-yl)-W-1 -yl wherein

[0015] W is para-Arylene whose scaffold consists of any number and combination of: N, CH, C- OAlkyl (C1-C12).

[0016] Y is H, CH3, OCH3INH2INHCH3IN(CH3)2ICH2CH3ICH(CH3)2ICH2CH2CH3IPh, 4- CH3OPh, 3,4-diCH3OPh, or 2,6-diCH3OPh.

[0017] R is CH2-(CH2)n-X or CH2-C=C-(CH2)n-X and it is connected to Z (formula I, II, III) The inventors have found that the halide methyltransferase from the fungus Aspergillus clavatus can direct the transfer of an extended and bulky halide-bound moiety from certain haloalkanes (R-Hal) onto the S-atom of S-adenosyl-L-homocysteine. In one embodiment, halogenide R-Hal1 with a bromine-bound 6-azidohex-2-ynyl moiety carries functional azide groups that can be applied for analysis of the ssRNA, dsRNA and DNA targets of DNA or RNA methyltransferases using “Click” conjugation reactions (refs). In another embodiment, halogenide R-Hal2 carries a bromine-bound extended moiety with a functional 1 ,2,4,5-tetrazine group. 1 ,2,4,5-tetrazines have gained wide utility for bioorthogonal conjugation reactions (Oliveira et al. 2017).

[0018] DESCRIPTION OF DRAWINGS

[0019] The drawings are provided as a reference to possible embodiments and are not intended to limit the scope of the invention. Neither of the drawings nor the graphs presented herein should be construed as limiting the scope of the invention, but merely as an example of a possible embodiment.

[0020] Fig. 1. General schematic for stereoselective S-alkylation of S-adenosyl-L-homocysteine (AdoHcy) or Se-alkylation of Se-adenosyl-L-homoselenocysteine (SeAdoHcy) with alkylhalogenides (R-Hal) carrying extended moieties catalysed by Aspergillus clavatus halidemethyltransferase (HMT).

[0021] Fig. 2. shows that HMT can catalyse the transfer of an extended moiety from the halogen atom in azido-1-bromohex-2-yne (R-Hal1 ) to the sulphur atom of AdoHcy to produce AdoMet cofactor analogue Ado-6-azide. A. HPLC analysis of specified time-points of the HMT-directed synthesis of Ado-6-azide from AdoHcy. B. MS analysis of chromatographic peaks in A corresponding to precursor AdoHcy, C14H21N6O5S (theoretical 385.1294, observed 385.1291 ) and product Ado-6- azide, C2oH28N905S+(theoretical 506.1929, observed 506.1929). C. Time course of HMT-directed conversion of AdoHcy to Ado-6-azide cofactor containing an extended 6-azidohex-2-ynyl moiety. All reactions were performed using 1 mM AdoHcy, 10 mM R-Hal1 and 100 pM enzyme at 25 °C. Reactions were quench by 0.5 M phosphoric acid and centrifugation at 20000 g for 30 min at 4 °C. Prepared samples were loaded on an integrated HPLC / ESI-MS / MS system (Agilent 1290 Infinity / 6410 Triple Quad LC / MS) equipped with a Supelco DiscoveryOHS C18 column (7.5 cm x 2.1 mm, 3 pm) and resolved by elution with a linear gradient of solvents (A) 0.02% formic acid in water and (B) 0.02% formic acid in acetonitrile following 0-2 min, 0 % B; 2-8 min, 0-15 % B; 8- 9 min, 15-95% B; 9-14min 100 % B; 14-15 min, 100-0 % B; 15-20 min, 0 % B. Mass spectrometer was operating in the positive ion mode and MRM counts of the following ion transitions were recorded: AdoHcy 385.1 136.1 , Ado-6-azide 506.2 250.1 or 506.2 136.1.

[0022] Fig. 3. shows that HMT can catalyze the transfer of an extended moiety from the halogen atom in 3-(2-bromoethyl)-6-methyl-1 ,2,4,5-tetrazine (R-Hal2) to the sulphur atom of AdoHcy to produce the AdoMet cofactor analogue Ado-2-Tet. A. HPLC analysis of the HMT-directed synthesis of Ado-2-Tet from AdoHcy. Presented chromatogram shows AdoHcy conversion to Ado-2-Tet after 60 min. B. MS analysis of chromatographic peaks in A corresponding to precursor AdoHcy, C14H21N6O5S (theoretical 385.1294, observed 385.1291 ) and product Ado-2-Tet, Ci9H27Ni0O5S+(theoretical 507.1881 , observed 507.1891 ). C. Time course of HMT-directed conversion of AdoHcy to Ado-2-Tet cofactor containing an extended methyl-tetrazine-ethyl moiety. All reactions were performed using 1 mM AdoHcy, 10 mM R-Hal2 and 100 pM enzyme at 25°C. Reactions were quenched by 0.5 M phosphoric acid and centrifugation at 20000 g for 30 min at 4°C. Prepared samples were loaded on an integrated HPLC / ESI-MS / MS system (Agilent 1290 Infinity / 6410 Triple Quad LC / MS) equipped with a Supelco DiscoveryOHS C18 column (7.5 cm x 2.1 mm, 3 pm) and resolved by elution with a linear gradient of solvents (A) 0.02% formic acid in water and (B) 0.02% formic acid in acetonitrile following 0-2 min, 0 % B; 2-8 min, 0-15 % B; 8-9 min, 15-95% B; 9-14min 100 % B; 14-15 min, 100-0 % B; 15-20 min, 0 % B. Mass spectrometer was operating in the positive ion mode and MRM counts of the following ion transitions were recorded: AdoHcy 385.1 136.1 , Ado-2-Tet 507.2 250.1 or 507.2 136.1.

[0023] Fig. 4 shows that HMT-directed synthesis of Ado-6-azide is stereo-specific leading to production of the functionally active S-epimer (the assignment of sulfonium epimers is based on that of AdoMet). Chemo-enzymatic reaction was prepared containing 1 mM AdoHcy, 10 mM 6-Azido-1- bromohex-2-yne (R-Hal1 ), 100 pM AcIHMT at 25°C for 30 min (line 2). Chemically synthesized Ado-6-azide cofactor containing a mixture of sulfonium S- and R- epimers (Lukinavicius et al., 2013) was used as a control (line 1 ). Prepared samples were loaded on an integrated HPLC / ESI- MS / MS system (Agilent 1290 Infinity / 6410 Triple Quad LC / MS) equipped with a Supelco DiscoveryOHS C18 column (7.5 cm x 2.1 mm, 3 pm) and resolved by elution with a linear gradient of solvents (A) 0.0075% formic acid in water and (B) 0.0075% formic acid in acetonitrile following 0-5 min, 0% B; 5-15 min, 0-10% B; 15-20 min, 10-100% B; 20-24% min, 100% B; 24-24.5 min, 100-0% B; 24.5-30 min, 0% B. Mass spectrometer was operating in the positive ion mode and the counts of the Ado-6-azide 506.2 —> 250.1 ion transition were recorded. Fig. 5 shows that the Ado-6-azide cofactor produced in the HMT-directed reaction is in situ utilized by the eM.Hhal DNA cytosine-5 methyltransferase, thereby converting 2’-deoxycytidine to 5-(6- azidohexynyl)-2’-deoxycytidine (N3-m5dC) in its GCGC target sites in DNA. One-pot reactions containing 50 pM AcIHmt, 100 pM AdoHcy, 10 mM 6-azido-1-bromohex-2-yne (R-Hal1 ), 500 ng pUC19 plasmid DNA, 1 pM eM.Hhal (the Q82A / Y254S / N304A mutant of the Hhal DNA cytosine- 5 methyltransferase as described in Lukinavicius et aL, 2012) were incubated for 1 h at 37°C and quenched by heating for 10 min at 80°C (line 2). Negative control contained no AcIHMT protein (line 3); positive control contained 500 ng plasmid DNA, 1 pM eM.Hhal and 100 pM chemically synthesized Ado-6-azide (line 1) as described in Lukinavicius et al. 2013. After purification, 500 ng of modified DNA was hydrolyzed to nucleosides with 0.01 U / pL of P1 nuclease for 4 h at 50°C followed by an overnight incubation with 0.01 U / pL thermosensitive alkaline phosphatase at 37°C. Reactions were quenched by heating for 10 min at 80°C and centrifugation at 20000 g for 30 min at 4°C. Prepared samples were loaded on an integrated HPLC / ESI-MS / MS system (Agilent 1290 Infinity / 6410 Triple Quad LC / MS) installed with a Supelco DiscoveryOHS C18 column (7.5 cm x

[0024] 2.1 mm, 3 pm) and resolved by elution with a linear gradient of solvents (A) 0.0075% formic acid in water and (B) 0.0075% formic acid in acetonitrile following 0-5 min, 0% B; 5-15 min, 0-10% B; 15-20 min, 10-100% B; 20-24% min, 100% B; 24-24.5 min, 100-0% B; 24.5-30 min, 0% B. Mass spectrometer was operating in the positive ion mode and the counts of the N3-m5dC 349.2 —>

[0025] 233.1 ion transition was recorded.

[0026] Fig. 6 shows that HMT-directed synthesis of Ado-6-azide cofactor is nearly 3-fold as efficient at pH 6.5 as compared to pH 7.4. The chemo-enzymatic synthesis was carried out in a reaction containing 1 mM SAH, 10 mM R-Hal1 , and 100 pM AcIHMT at 25°C for 3 hours at pH 6.5 or 7.4. Equal reaction aliquotes were subsequently analyzed using HPLC-MS as described in Fig. 4 Trace 1 depicts the amount of Ado-6-N3 produced at pH 6.5, trace 2 shows the amount of Ado- 6-N3 produced in the reaction at pH 7.4.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] As indicated above the present invention provides, in a first aspect, a halide methyltransferase- controlled chemoenzymatic method for preparing biology active epimers of AdoMet analogues that are well suited for AdoMet-dependent enzymatic reactions and following analysis of modification of biomolecules in vitro and in vivo, wherein the part of the moiety transferred comprises a reporter group or a functional group. Biocatalytic production of AdoMet analogues of formula I and II from AdoHcy and a corresponding alkylhalide R-Hal1 or R-Hal2 is carried out according to the general scheme shown in Fig.1. Production of AdoMet analogues of formula I in epimerically pure form is exemplified by synthesis of Ado-6-azide from AdoHcy and 6-azido-1-bromohex-2-yne as shown in Figures 2 and 4. The utility of such produced analogue Ado-6-azide for MTase-directed derivatization of DNA is shown in Figure 5.

[0029] Biocatalytic production of AdoMet analogues of formula II is exemplified by synthesis of Ado-2- Tet from AdoHcy and 3-(2-bromoethyl)-6-methyl-1 ,2,4,5-tetrazine as shown in Fig. 3.

[0030] The examples demonstrate that using appropriate precursor bromides and the reaction conditions described in Fig. 2 and Fig. 3 (10 equivalents of alkylbromide, 0.1 equivalents of enzyme to AdoHcy and 0.5-1 h incubation time at 25°C), biocatalytic production of the Ado-6-azide and Ado- 2-tet cofactors can be achieved with conversion yields of >90% and >60%, respectively. These reactions take into consideration the following 3 factors:

[0031] 1) AdoMet and its analogs are known to readily undergo temperature-dependent decomposition upon prologed incubations. Shorter incubation times (0.5-1 hour), lower reaction temperatures (25°C) and lower pH values of the reaction buffer (pH=6.5) as described in the presented examples render minimal chemical decomposition and thus improved yields of the produced cofactor;

[0032] 2) alkyl iodides used in prior art examples (Bengel et al. 2021 ; Tang et al. 2021a; Hoffmann et al. 2023; Schulke et al. 2022) are in general more reactive both in the direct and reverse reactions, which may lead to the generation of non-enzymatic alkylation products (nonstereospecific product (Hoffmann et al. 2023)) and lower yields due to reaction reversal by the accumulated coproduct iodide anion. We chose to use alkyl bromide as the reaction substrates to avoid the above complications.

[0033] 3) our analysis of available crystal structures suggested that AdoMet analogs with certain longer transferable moieties may be favourably accommodated in the catalytic centre of the AcIHMT enzyme. We propose that a high transfer efficiency of the longer moieties can be achieved if combined with proper electronic activation of the SN2 reaction (due to presence of a nearby triple bond (cofactor of formula I.) or a tetrazine ring (cofactor of formula II.) in the sulfonium-bound moiety).

[0034] Enzyme The present inventors have found that halide methyltransferase enzymes are able to transfer long alkynes bearing a reporter group or a functional group from a co-substrate alkylhalide to AdoHcy. The halide-methyltransferase enzyme to be used in the method described herein may be obtained from plants, fungus, bacteria and is preferably AcIHMT encoded by a gene from Aspergillus clavatus. The sequences of the wild type thiol methyltransferase AcIHMT can be found in GenBank, Accession No XP_001272206 (thiol methyltransferase, putative).

[0035] The nucleotide sequence of the methyltransferase gene is optimized based on E. coli preferred codons without changing the encoded amino acids:

[0036] CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTCTACTCCC

[0037] TCACTGATTCCGAGCGGTGTGCACGAGGTTCTTGCAAAGTATAAGGATGGGAATTACGTG GACGGCTGGGCGGAATTGTGGGATAAATCTAAGGGTGATCGCCTGCCGTGGGACCGTGG CTTTCCGAATCCGGCACTTGAGGATACACTTATTCAAAAGCGTGCCATTATTGGCGGGCCG TTAGGTCAGGATGCCCAAGGTAAAACTTATCGTAAGAAAGCCCTCGTTCCTGGGTGTGGG CGTGGCGTCGATGTACTTTTACTGGCATCGTTTGGTTATGACGCGTACGGGCTGGAATATT CAGCAACCGCCGTTGACGTTTGCCAAGAAGAACAAGCCAAGAATGGCGACCAGTATCCAG TCCGCGACGCAGAAATTGGTCAAGGTAAAATTACGTTCGTTCAAGGGGACTTCTTCGAAGA CACGTGGTTAGAGAAATTGAACCTTACCCGTAATTGTTTTGATGTTATCTATGACTATACCTT CTTTTGTGCTCTGAATCCGAGCATGCGTCCTCAGTGGGCGCTGCGCCATACCCAACTTTTA GCGGACAGCCCGCGTGGCCATCTGATTTGTCTGGAGTTCCCGCGCCATAAAGACCCGAGT GTGCAGGGCCCGCCCTGGGGATCGGCAAGTGAAGCCTATCGTGCGCACCTCAGTCATCC TGGTGAAGAGATTCCGTATGACGCGTCTCGCCAGTGTCAATTCGATTCTTCCAAAGCACCG TCCGCGCAAGGGCTGGAACGCGTGGCCTACTGGCAGCCAGAACGCACTCACGAGGTCGG CAAGAACGAGAAAGGCGAAGTGCAGGACCGCGTGTCAATCTGGCAACGTCCCCCGCAGT CTAGCCTGTAA

[0038] Amino acid sequence of AcIHMT has N-terminal HisTag (in bold) and are as follows: HHHHHHSSGLVPRGSH^STPSLIPSGVHEVLAKYKDGNYVDGWAELWDKSKGDRLPWDRGF PNPALEDTLIQKRAIIGGPLGQDAQGKTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAV DVCQEEQAKNGDQYPVRDAEIGQGKITFVQGDFFEDTWLEKLNLTRNCFDVIYDYTFFCALNP SMRPQWALRHTQLLADSPRGHLICLEFPRHKDPSVQGPPWGSASEAYRAHLSHPGEEIPYDA SRQCQFDSSKAPSAQGLERVAYWQPERTHEVGKNEKGEVQDRVSIWQRPPQSSL The alkylbromides for use in the methods described herein comprise a functional group or a reporter group in an extended moiety, which can be transferred onto the AdoHcy or Se-AdoHcy by the enzyme described above.

[0039] Synthesis of 6-Azido-1-bromohex-2-yne (R-Hal1 )

[0040] 6-Chlorohex-2-yn-1-ol. To the stirred solution of dry THF (120 ml) 5-chloro-1 -pentyne (10 ml, 94.4 mmol) was added under argon and cooled to -78 °C. Then butyllithium 2.5 M in hexanes (37,8 ml, 93 mmol) was added and stirred for 30 min. After that, paraform (3.36 g, 104 mmol) was added to the reaction mixture and stirred for another 30 min. Then the cooling bath was removed and the reaction vessel was left to warm to room temperature. The reaction was stopped when 100 ml of cool (0 °C) water was added. The mixture was poured to separating funnel and organic phase was separated. The aqueous layer was extracted with diethyl ether (2 x 40 ml). The organic layers were combined, dried over sodium sulphate and evaporated. The product was purified by distillation under vacuum. Colourless oil is obtained (9.51 g; 76%).1H NMR (400 MHz, CDCh): 1.98 (m, 3H, CH2CH2CH2 + OH); 2.44 (tt, 2H,3J = 6.3 Hz,5J = 2.1 Hz, C CCH2); 3.67 (t, 2H,3J = 6.3 Hz, CH2CI); 4.27 (t, 2H,5J = 2.1 Hz, CH2OH).

[0041] 6-Azidohex-2-yn-1-ol. To a stirred solution of 6-chlorohex-2-yn-1-ol (2.266 g, 2 ml, 17.1 mmol, 1 equiv) in DMF (15 ml) sodium azide (3.33 g, 51.3 mmol; 3 equiv) and tetrabutylammonium bromide (0.551 g, 1 .71 mmol) were added. The reaction mixture was left to stir for 24 h at 80 °C (sand bath). Then DMF was removed under reduced pressure and 20 ml of water was added. The product was extracted with diethyl ether (3 x 30 ml), organic layers were combined, dried over sodium sulphate and evaporated. Product is purified by dry column vacuum chromatography (DCVC), firstly eluting with CH2CI2 and after then with CH2Cl2:EtOAc(20:1). Colourless oil is obtained (2.13 mg; 90%).1H NMR (400 MHz, CDCh): 5 1.78 (m, 3H, CH2CH2CH2+ OH); 2.34 (tt, 2H,3J = 6.9 Hz,5J = 2.4 Hz, C CCH2); 3.41 (t,3J = 6.9 Hz, 2H, CH2N3); 4.24 (t, 2H,5J = 2.4 Hz, CH2OH).13C NMR (100 MHz, CDCh): 5 16.04; 27.69; 50.16; 51.12; 79.49; 84.36.

[0042] 6-Azidohex-2-yn-1-yl 4-methylbenzenesulfonate. In an argon filled round-bottom flask 6- azidohex-2-yn-1-ol (800 mg; 5,75 mmol; 1 eq.) is dissolved in 15 ml of THF and the mixture is cooled to 0 °C in an ice bath. Potassium tert-butoxide (632 mg; 5,63 mmol; 0,98 eq.) is added, then after 5 min., 4-methylbenzenesulfonyl chloride (1 ,10 g; 5,75 mmol; 1 eq.) is added and stirred. After 1 hour solvent is evaporated with rotary evaporator. To the remainder 40 ml of dielthyl ether is added the organic layer is washed with 20 ml of sat. NaHCO3, then with 20 ml of saturated sodium chloride solution. Organic phase is dried with anhydrous Na2SO4, filtered and evaporated with rotary evaporator. The remainder is purified by column chromatography using toluene as eluent.1H NMR (400 MHz, CDCb): 5 2,46 (s, 3H, CH3); 3,48 (t, J = 5,1 Hz, 2H, CH2N3); 5,16 (t, J = 5,1 Hz, 2H, CH2O); 7,37 (d, J = 8,2 Hz, 2H, Ar3-H); 7,81 (d, J = 8,2 Hz, 2H, Ar2-H) NMR (100 MHz, CDCb): 5 21 ,8; 49,7; 68,2; 128,1 ; 130,1 ; 132,7; 145,4.

[0043] 6-Azido-1 -bromohex-2 -yne. In a round-bottom flask 6-azidohex-2-yn-1-yl 4- methylbenzenesulfonate. (1 ,40 g; 4,71 mmol; 1 eq.) and lithium bromide (2,07 g; 23,8 mmol; 5 ekv.) is dissolved in 50 ml of acetone. The reaction mixture is refluxed for 18 hours. After the reaction, acetone is evaporated under reduced pressure not bellow 100 mbar with a rotary evaporator. To the residue 30 ml of water is added and washed with diethyl ether (3 x 20 ml), organic layers combined, dried with anhydrous MgSCb, filtered and evaporated under reduced pressure. Product is purified by dry column vacuum chromatography (DCVC) using petrol ether, then the product eluted with PE:DCM (3:1 ). Fractions containing the product are evaporated under reduced pressure using a rotary evaporator and the product dried in 50 mbar pressure. A colourless oil is obtained (810 mg; 84%).1H NMR (400 MHz, CDCb): 5 1 ,79 (q, J = 6,8 Hz, 2H, CH2CH2CH2); 2,32-2,41 (m, 2H, C CCH2CH2); 3,41 (t, J = 6,8 Hz, 2H, CH2N3); 3,88-3,95 (m, 2H, CH2Br).13C BMR (100 MHz, CDCb): 6 15,2; 16,3; 27,6; 50,1 ; 76,5; 86,1.

[0044] 2-(6-methyl-1,2,4,5-tetrazin-3-yl)ethan-1-ol. In a round-bottom flask 3-hydroxypropanenitrile (2,00 g; 1 ,87 ml; 28,1 mmol; 1 eq.), acetonitrile (9,24 g; 11 ,8 ml; 0,255 mol; 8 eq.), 3- mercaptopropanoic acid (2,99 g; 2,45 ml; 28,1 mmol; 1 eq.) and hydrazine hydrate (22,54 g; 21 ,84 ml; 0,450 mol; 16 eq.) are mixed and the solution is stirred for 18 hours. After that, the mixture is transferred to a 500 ml round bottom flask, cooled in an ice bath to 0 °C and sodium nitrite (29,12 g; 0,422 mol; 15 eq.) solution in 80 ml of water is added. While strongly stirring, 3M HCI is slowly added with a dropping funnel (approx. 250 ml in 2 hours) until gas evolution ceases and reaction pH drops below <3. The aqueous solution is evaporated by rotary evaporator at 55 °C. The remaining pink crystals are washed with DCM (4 x 50 ml). Organic layer is dried with anhydrous Na2SO4, filtered and evaporated off with rotary evaporator. The remainder is purified by column chromatography as eluent using DCM:MeOH (40:1 ). A purple oil is obtained (1 ,48 g; 37%).1H NMR (400 MHz, CDCh): 5 2,37 (pl. s, 1 H, OH); 3,05 (s, 3H, CH3); 3,55 (t, J = 5,8 Hz, 2H, CH2CH2OH); 4,24 (t, J = 5,8 Hz, 2H, CH2CH2OH).13C BMR (100 MHz, CDCh): 521 ,1 ; 37,4; 60,0; 167,8; 168,3.

[0045] 2-(6-Methyl-1,2,4,5-tetrazin-3-yl)ethyl methanesulfonate. In a round-bottom flask 2-(6-methyl- 1 ,2,4,5-tetrazin-3-yl)ethan-1-ol (782 mg; 5,58 mmol; 1 eq.) and methanesulfonyl chloride (1 ,28 g; 864 pl; 11 ,2 mmol; 2 eq.) are dissolved in 30 ml of DCM. The prepared solution is cooled to 0 °C in an ice bath, triethylamine (1 ,13 g; 1 ,56 ml; 11 ,2 mmol; 2 eq.) is added and stirred in the ice bath for 10 minutes. After that, the ice bath is removed and the mixture is additionally stirred for 20 min. at room temperature. The reaction mixture is washed with 20 ml of sat. NaHCO3and 20 ml of sat. sodium chloride solution. Organic phase is dried with anhydrous MgSO4, filtered, transferred into a round-bottom evaporating flask and 10 ml of silica is added. The mixture is evaporated onto silica with a rotary evaporator. The product is purified by dry column vacuum chromatography (DCVC), firstly eluting the side-product with DCM, then eluting the product with DCM:MeOH (50:1). A purple oil is obtained (1 ,08 g; 89%).1H NMR (400 MHz, CDCh): 5 3,02 (s, 3H, SCH3); 3,07 (s, 3H, CCH3); 3,76 (t, J = 6,2 Hz, 2H; CCH2); 4,87 (t, J = 6,2 Hz, 2H, OCH2).13C NMR (100 MHz, CDCh): 5 21 ,3; 34,7; 37,7; 66,1 ; 166,2; 168,3.

[0046] 3-(2-Bromoethyl)-6-methyl-1,2,4,5-tetrazine. In a round-bottom flask 2-(6-methyl-1 , 2,4,5- tetrazin-3-yl)ethyl methanesulfonate (208 mg; 0,953 mmol; 1 eq.), lithium bromide (414 mg; 4,77 mmol; 5 eq.) and 10 ml of acetone is added. The mixture is refluxed for 3 hours, after that, acetone is removed with a rotary evaporator. To the residue 20 ml of diethyl ether is added, the organic layer is washed with 10 ml of sat. NaHCO3, 10 ml sat. sodium chloride solution. The organic phase is dried with anhydrous Na2SO4, filtered and evaporated with a rotary evaporator. The product is purified by column chromatography using DCM as an eluent. A purple oil is obtained (181 mg; 95%).1H NMR (400 MHz, CDCI3): 5 3,07 (s, 3H, CH3); 3,89 (t, J = 6,2 Hz, 2H, CCH2); 3,97 (t, J = 6,2 Hz, 2H, CH2Br).13C NMR (100 MHz, CDCI3): 5 21 ,3; 28,2; 37,8; 167,4; 168,2.

[0047] Cofactors synthesized by the chemoenzymatic method

[0048] The cofactors produced in the methods described herein is based on the molecule S-adenosyl- L-methionine (AdoMet) and is an S-adenosyl-L-methionine or Se-adenosyl-L-selenomethionine analogue which comprises a functional group in an extended moiety, which can be transferred onto the target.

[0049] In particular, the AdoMet analogues may have the following formula: n = 0-4

[0050] Z= S, Se

[0051] X= N3, C CH, NH2I4-Y-2,3,5,6-Tetrazin-1-yl, 4-(4’Y-2,3,5,6-tetrazin-1’-yl)-W-1-yl where in

[0052] W= para-Arylene whose scaffold consists of any number and combination of: N, CH, C-OAIkyl (C1-C12). Y= H, CH3, OCH3INH2, NHCH3IN(CH3)2, CH2CH3ICH(CH3)2ICH2CH2CH3IPh, 4-CH3OPh, 3,4- diCH3OPh, 2,6-diCH3OPh

[0053] In one preferred embodiment is Compound of formula I, wherein n=2, Z= S and X= N3 (Ado-6- azide as described in Fig. 2).

[0054] In other preferred embodiments is Compound of formula I, wherein n=2, Z= S and X= C=CH or NH2.

[0055] In another preferred embodiment is Compound of formula I, wherein n=0, Z= S and X= 4-(4’Y- 2,3,5,6-tetrazin-1’-yl)-W-1-yl, W= para-phenyl and Y= CH3.

[0056] In one preferred embodiment is Compound of formula II, wherein n= 1 , Z= S, X= 4-Y-2, 3,5,6- tetrazin-1 -yl and Y= CH3(Ado-2-Tet as described in Fig. 3).

[0057] In another preferred embodiment is Compound of formula II, wherein n= 1 , Z= S, X= 4-(4’Y-

[0058] 2,3,5,6-tetrazin-1’-yl)-W-1-yl, W= para-phenyl and Y= CH3.

[0059] Table 1. Sequence listing Literature

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[0071] Lukinavicius G, Tomkuviene M, Masevicius V, Klimasauskas S. 2013. Enhanced Chemical Stability of AdoMet Analogues for Improved Methyltransferase-Directed Labeling of DNA. ACS Chem. Biol. 8: 1134-1139.

[0072] Mickute M, Kvederaviciute K, Osipenko A, Mineikaite R, Klimasauskas S, Vilkaitis G. 2021. Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs. BMC Biol 19: 129. Mo X, Gui C, Wang Q. 2017. Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin biosynthetic pathway. Bioorg Med Chem Lett 27: 4431-4435.

[0073] Moore LD, Le T, Fan G. 2013. DNA methylation and its basic function. Neuropsychopharmacol OffPublAm Coll Neuropsychopharmacol 38: 23-38.

[0074] Mordhorst S, Siegrist J, Muller M, Richter M, Andexer JN. 2017. Catalytic Alkylation Using a Cyclic S-Adenosylmethionine Regeneration System. Angew Chem Int Ed Engl 56: 4037^1041 .

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[0077] Schulke KH, Ospina F, Hdrnschemeyer K, Gergel S, Hammer SC. 2022. Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes. Chembiochem EurJ Chem Biol 23: e202100632.

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Claims

CLAIMS1. A chemoenzymatic method of producing S-adenosyl-L-methionine (AdoMet) analogues bearing moieties with a functional group or reporter group, according to formula I and IIthe method comprising contacting an AdoMet precursor molecule of formula IIIwith an alkylhalide R-Hal and halide methyltransferase (HMT) under conditions suitable for converting AdoHcy or SeAdoHcy to AdoMet analogues, wherein n is 0,1 , 2, 3, or 4Z is S, or SeX is functional or reporter groupR is CH2-(CH2)n-X or CH2-C C-(CH2)n-X.

2. The method of claim 1 , wherein X is N3, C=CH, NH2, or 4-Y-2,3,5,6-Tetrazin-1-yl, 4-(4’Y- 2,3,5,6-tetrazin-1’-yl)-W-1 -yl whereinW is para-Arylene whose scaffold consists of any number and combination of: N, CH, C- OAlkyl (Ci-Ci2).Y is H, CH3, OCH3INH2INHCH3IN(CH3)2ICH2CH3ICH(CH3)2ICH2CH2CH3IPh, 4- CH3OPh, 3,4-diCH3OPh, or 2,6-diCH3OPh.

3. The method of claim 1, wherein modification of AdoHcy or SeAdoHcy is catalysed by Aspergillus clavatus halide-methyltransferase (HMT)4. The method of claim 1 , wherein R-Hal is alkylbromide.

5. The method of claim 1 , wherein R-Hal is 6-Azido-1-bromohex-2-yne (R-Hal1) or 3-(2- Bromoethyl)-6-methyl-1 ,2,4,5-tetrazine (R-Hal2).

6. The method of any one of claims 1 to 3, wherein the method comprises incubation of 10 molar equivalents of alkylhalide, 0.1 molar equivalents of HMT and 1 molar equivalent of AdoMet precursor molecule at 25°C and pH 6.5 for the duration of 0.5-1 h.

7. The method of any one of claims 1 to 3, wherein the method comprises incubation of 10 molar equivalents of alkylbromide, 0.1 molar equivalents of HMT and 1 molar equivalent of AdoMet precursor molecule at 25°C and pH 6.5 for the duration of 0.5-1 h.

8. The AdoMet analogue, according to formula IIwherein n is 0, 1 , 2, 3, or 4Z is S or SeX is N3, C CH, NH2Ior 4-Y-2,3,5,6-Tetrazin-1-yl, 4-(4’Y-2,3,5,6-tetrazin-1’-yl)-W-1-yl whereinW is para-Arylene whose scaffold consists of any number and combination of: N, CH, C- OAlkyl (C1-C12).Y is H, CH3, OCH3INH2INHCH3IN(CH3)2ICH2CH3ICH(CH3)2ICH2CH2CH3IPh, 4- CH3OPh, 3,4-diCH3OPh, or 2,6-diCH3OPh.

9. The AdoMet analogue, obtained by the method of claims 1-7, and having formula II, wherein a) n is 1 , Z is S, X is 4-Y-2,3,5,6-tetrazin-1-yl and Y= CH3; or b) n is 1 , Z is S, X is 4-(4’Y-2,3,5,6-tetrazin-1’-yl)-W-1-yl, W= para-phenyl and Y= CH3.

10. The AdoMet analogue I and II, according to claims 1-9 for use as cofactor of AdoMet- dependant methyltransferase.

11. Use of AdoMet analogues II according to claims 1-10 for a selective labeling of specific targets on proteins, RNA or DNA using corresponding AdoMet-dependent methyltransferases.

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