Matthew T. Salinger: Bioalkylation Strategies to Synthesize Allylated Tetrahydroisoquinolines by Using Norcoclaurine Synthase and O-Methyltransferases

In this work, we have used O-MTs for the allylation of various catechol and tetrahydroisoquinoline substrates via a biocatalytic cascade involving additionally methionine adenosyltransferases (MATs) and a methylthioadenosine nucleosidase (MTAN). Furthermore, we have integrated norcoclaurine synthase into this cascade to stereoselectively generate (S)-THIQs in situ as both intermediates and products in the allylation cascade. Notably, a variation in the order in which NCS and MAT-MT-MTAN are added can significantly affect the regioselective outcome, enabling exquisite control of both stereochemistry and regiochemistry in the products. We also identified Ureaplasma urealyticum MAT as an effective enzyme for the formation of the S-adenosyl-S-allyl-L-homocysteine required as the cofactor for the O-MTs and established that the racemate rather than the single isomer of S-allyl-homocysteine can effectively be used in the cascades with MATs.

1 Introduction

Stereoselective alkylations, such as allylations, are important for the tailored modification of structural motifs to alter compound bioactivities or introduce click handles for use in chemical biology applications.[1] Enzymatic alkylations typically use methyltransferase (MT) enzymes, which in native reactions catalyze the transfer of methyl groups to target molecules and are rapidly gaining favor as a method for green and regioselective chemical synthesis.[2-4] Most MTs require the methyl group donor cofactor S-adenosyl-L-methionine (SAM) as the electrophile, with the reaction operating via an SN2 mechanism. Using MTs in alkylation reactions presents several advantages over traditional chemical methods, such as enhanced regioselectivities, mild reaction conditions, and the avoidance of toxic alkylating reagents such as methyl iodide and dimethyl sulfate. However, the key requirement for stoichiometric quantities of SAM in biomethylations can be a problem in synthetic applications, as it is expensive and relatively unstable.[5] To overcome this issue, SAM can be generated in situ from ATP and L-methionine using methionine adenosyltransferase (MAT).[6] Methylthioadenosine nucleosidase (MTAN) can also be added to hydrolyze the S-adenosyl-L-homocysteine (SAH) by-product and prevent MT feedback inhibition.[6] This three-enzyme MAT-MT-MTAN methylation cascade has been used with several substrates,[6] including tetrahydroisoquinolines (THIQs).[7] Other studies have produced cyclic regeneration systems using SAH hydrolase instead of MTAN, with polyphosphate and kinases to convert the adenosine produced into ATP, and L-homocysteine S-methyltransferase to transform the L-homocysteine into L-methionine.[8, 9] Also, halide methyltransferases (HMTs) have been described to regenerate SAM from SAH, using methyl iodide or less hazardous methyl tosylate as methyl donors.[10, 11] HMTs have additionally been combined into MAT-MT-HMT cascades to avoid priming the methylations with SAM.[12]

There has been a recent expansion in the range of MT-catalyzed alkylations. For instance, ATP and L-ethionine or S-allyl-L-homocysteine (S)-1 were used with a human MAT mutant I322 V hMAT2A to give S-adenosyl-L-ethionine (SAE) and S-adenosyl-S-allyl-L-homocysteine (SAA), respectively, then used in N-MT reactions with THIQs, although conversions were not indicated.[13] Similarly, SAE and SAA were prepared from L-ethionine and S-allyl-L-homocysteine using Thermococcus kodakarensis (Tk) MAT and used to O- or N-alkylate amino nitrophenols, with up to 40% conversions noted.[14] In addition, engineered enzymes were used for O-carboxymethylations of catechols and N-carboxylmethylations of THIQs.[15] HMTs have also been mutated for the in situ generation of SAE and SAA from SAH and then applied in the ethylation of luteolin and allylation of 3,4-dihydroxybenzaldehyde in up to 48% conversions.[16]

THIQs are important pharmacophores due to the various antibiotic, hypotensive, and antitumor properties associated with this scaffold.[17-20] The synthesis of single-isomer THIQs by traditional chemical methods is challenging due to the high density of functional groups present.[21] However, biocatalytic approaches have recently proven to be particularly effective in generating a range of THIQs in high enantiomeric purities using the Pictet–Spenglerase enzyme norcoclaurine synthase (NCS), a key enzyme in the biosynthetic pathway to such alkaloids.[22] NCS catalyzes the stereoselective Pictet-Spengler condensation between dopamine and analogues and a range of aldehydes to form (S)-THIQs.[22, 23] Indeed, mutagenesis of Thalictrum flavum NCS (TfNCS) has led to a wider range of aromatic and aliphatic aldehydes, as well as ketones, being accepted as substrates.[24-26] Recently, studies have successfully combined NCS into in vitro enzyme cascades or in vivo biosynthetically inspired pathways to produce a range of alkaloids.[27-30] Moreover, MTs have been used to perform a range of biocatalytic N- and O-methylations with (S)-THIQs, achieving impressive regioselectivities,[13] some together with NCS in cascades.[7, 28]

While MTs show much promise for the selective methylation of molecular scaffolds, allylations have been less explored. In this work, we report biocatalytic cascades for the O-allylation of (S)-THIQs by Rattus norvegicus catechol O-MT (RnCOMT) and Myxococcus xanthus SafC-O-MT (MxSafC) from the saframycin biosynthetic pathway, demonstrating successful allylations on these complex motifs. A key consideration was at what stage in the biocatalytic pathway the allylation should be performed, either before the NCS step to produce (S)-THIQs (Scheme 1: route A) or after (Scheme 1: route B), and the impact of these two routes on the cascade's stereoselectivity is presented. Additionally, we established that S-allyl-DL-homocysteine (rac-1), which is readily synthesized in one step, can be used as an allyl donor in a MAT-MT-MTAN allylation cascade. Furthermore, we identified that Ureaplasma urealyticum MAT (UuMAT) is highly effective in forming the allyl analogue of SAM,[31] negating the need for higher temperatures, which has been reported with other MATs.[32, 33]

Read full paper here