Regioselective Enzymatic Carboxylation of Bioactive (Poly)phenols

Abstract In order to extend the applicability of the regioselective enzymatic carboxylation of phenols, the substrate scope of o‐benzoic acid (de)carboxylases has been investigated towards complex molecules with an emphasis on flavouring agents and polyphenols possessing antioxidant properties. o‐Hydroxycarboxylic acid products were obtained with perfect regioselectivity, in moderate to excellent yields. The applicability of this method was proven by the regioselective bio‐carboxylation of resveratrol on a preparative scale with 95% yield.

Thec arboxylation of (hetero)aromatic and phenolic compounds is ac onvenient method to obtain aromatic carboxylic acids useda sp harmaceuticals [1,2] (e.g., salicylic and m-aminosalicylic acid) as well as building blocks for organic synthesis.T he traditional chemical (Kolbe-Schmitt) carboxylation process performed on an industrial scale requires high pressure and temperature (~90 bar, 120-300 8 8C) and often suffers from incomplete regioselectivities resulting in product mixtures. [3] In ordert oc ircumventt hese limitations,v arious chemical CO 2 fixationc onceptsu sing heterogeneous,( transition) metal and organic catalysts have been established. [4] An ovela ttractive biocatalytic alternativei st he use of (de)carboxylases,w hich act at ambient reactionc onditions and show perfect regioselectivities. [5,6,7] To date,abiocatalytic toolbox for the regioselective ortho-, [1,2,8] para- [9] and b-carboxylation [10] of phenols and hydroxystyrenes,r espectively,h as been established,w hich employs decarboxylases acting in the (reverse)c arboxylation direction using bicarbonatea s CO 2 source.I na ddition,e lectron-rich heteroaromatics,s uch as pyrrole and indole were successfully carboxylated. [11] Enzymatic carboxylation of phenolic substances is ac ommon detoxification pathway in an anaerobic environment [,12] andh ence the corresponding enzymes are expected to possess ar elaxed substrate tolerance,w hich is indeed true for the o-carboxylation of phenols [5,8f] andt he b-carboxylation of hydroxystyrenes. [10] However, the substratesr eported so far are predominantly small or medium-sized phenold erivatives,w hich were converteda tl ow substrate loading, except for ar ecently reported study on hydroxystilbenes and the naturally occurringp olyphenol resveratrol. [8g] Based on our previous studies on the enzymatic ortho-carboxylation of phenols,w ea imedt oe xpand the scope of this method towards more complex (poly)phenolic substrates (Scheme 1, Figure 1). The latter compounds are well known for theirb iological activitiess uch as antioxidant, anti-inflammatory and antimicrobial properties,w hich promote them as promising targetsf or the pharmaceutical industry as well as for the cosmetic and food fields. [13,14,15] Enzymatic carboxylation of these compounds would provide an efficienta ccess to more polard erivatives with enhanced water solubility thereby facilitating their formulation and modulating their bioavailability.I n addition,t he soft electron-withdrawing effect of the newly introduced carboxylate group should render these products more stable towards autoxidation in analogy to the beneficial effect of the e À -withdrawing carbonylg roup of green tea polyphenols. [16] Furthermore,p henolic acids are expected to impede light-induced degradationa sd emonstrated in the case of photolabile substances in food or feed, such as vitamins [17a] or nucleic acids. [17b] Thes ubstrate scope of this studyr anges from the flavouring agent vanillin (1a)t os econdary plant me-tabolites, such as p-hydroxybenzaldehyde (2a), esters of p-hydroxycinnamic( p-coumaric) (3a)a nd m-hydroxyphenylacetic acid (4a), as well as representatives of flavonoids such as dihydrochalcones( 5a, 6a)a nd hydroxystilbenes,s uch as resveratrol (7a)a nd resveratrol-likep olyphenols (8a, 9a). Phloretin (6a)w hich is abundantly present in apples is describedt op ossess antioxidant properties and to act as ap eroxynitrite scavenger and an inhibitor of lipid peroxidation. [13b,18] Them ost famous example among this group of substrates is the natural product resveratrol (7a)w hich has fostered research andd evelopment towards cosmetic, food, nutraceutical andpharmaceutical applications,d ue to its uniquea bility to modulate physiological as well as pathologicalp athways. [13,19] In particular, resveratrol (7a)e xhibitsa ntimicrobial [13c] anda ntioxidant [13d] properties that are expected to be retained in the more soluble carboxylated form 7b.F urthermore, the g-resorcylic acid moiety found in carboxylation products 7b-9b would bring about new biological activities that are not displayed by the corresponding parentc ompounds 7a-9a.F or instance, g-resorcylic acid derivatives bearing al ipophilics ubstituenta re known as thrombolytic [20] anda sa nti-inflammatory agents throughu ncoupling of phosphorylation. [21] In particular, g-resorcylic acids with an alkylaryl substituenti nt he 4-position were shown to reduce inflammation in vivo in amice model. [22] In order to examinet he synthetic potential of the method,t he most promising substrate candidates were subjected to preparative-scale carboxylation.
In order to convert substratesc arrying af ree carboxylic acid group,w here decarboxylation wouldb e favoured, the corresponding methyle stersw ere applied as masking groups.T his strategy proved to be successful, as cinnamic and phenylacetic acid esters 3a and 4a were carboxylated by 2,3-DHBD_Ao and SAD_Tm with up to 66% conversion (entries 3a nd 4).
Surprisingly,a ll enzyme candidates showed significantly enhancedc onversion by further expanding the complexity of the substrates containing twoa romatic moieties (entries5-9).
Products 1b-9b and 9c were characterized by 1-D ( 1 Ha nd 13 C) and 2-DN MR (COSY,H SQC and HMBC,s ee the Supporting Information) and HR-MS.

Analytics
HPLC analysis: HPLC/UV experiments were performed on an HPLC Agilent 1260 Infinitys ystem with ad iodea rray detector and ar eversed phase Phenomenex Luna column C18 (100 ,2 50 4.6 mm, 5 mm, column temperature 24 8 8C). Conversions were determined by comparison with calibration curves for products and substrates prepared with authenticr eference material. All compounds were spectrophotometrically detected at 254, 280 and 310 nm, respectively.
Method A was runo ver 22 min with H 2 O/TFA( 0.1%) as the mobile phase (flow rate 1mLmin À1 )a nd aM eCN/TFA