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DOI: 10.1039/D5NP00044K (Highlight) Nat. Prod. Rep., 2025, Advance Article

para-Quinone methides in natural product biosynthesis

Jie Gao a, Qibin Chen a and Qi Zhang *ab
aNational Engineering Research Center for Carbohydrate Synthesis, College ofChemistry and Materials, Jiangxi Normal University, Jiangxi 330022, China. E-mail: qizhang@sioc.ac.cn
bDepartment of Chemistry, Fudan University, Shanghai, 20043, China

Received 19th June 2025

First published on 1st August 2025

Abstract

Covering: up to 2025

para-Quinone methides (p-QMs) are highly reactive Michael acceptors with broad applications in organic synthesis, drug development, and materials science. Nature ingeniously harnesses these intermediates for diverse biochemical processes, ranging from melanization to the biosynthesis of bioactive natural products. While some natural products incorporate stable p-QM moieties, most p-QMs are transient, serving as pivotal intermediates in various metabolic pathways. This highlight examines p-QM-mediated enzymatic transformations in natural product biosynthesis, emphasizing catalytic mechanisms, substrate flexibility, and engineering potential. Understanding these biosynthetic strategies would advance enzyme discovery, inspire biomimetic synthesis, and guide rational enzyme design efforts.

image file: d5np00044k-p1.tif

Jie Gao

Jie Gao received her B.S. in Pharmacy from Central South University in 2011 and completed her PhD in Organic Chemistry at Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences in 2016 under the guidance of Prof. Haji Akber Aisa. She then conducted postdoctoral studies in the laboratory of Prof. Youcai Hu at Institute of Materia Medica, Chinese Academy of Medical Sciences. Currently, she is an assistant professor in Prof. Qi Zhang's team at Jiangxi Normal University. Her research interests focus on the discovery, mining, and biosynthesis of functional natural products.

image file: d5np00044k-p2.tif

Qibin Chen

Qibin Chen obtained his B.S. in Pharmacy from Sichuan University, and earned his M.S. and PhD in Organic Chemistry at Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. He completed postdoctoral training in Prof. Youcai Hu's group at Institute of Materia Medica, Chinese Academy of Medical Sciences in 2019. Currently, he is an associate professor at College of Chemistry and Materials, Jiangxi Normal University. His research focuses on biosynthesis and biocatalysis, with particular emphasis on (1) discovery and biosynthesis of novel antibiotics from microorganisms, and (2) engineering and pharmaceutical application of specialized functional enzymes.

image file: d5np00044k-p3.tif

Qi Zhang

Qi Zhang obtained his B.S. in Chemistry from Fudan University, and earned his PhD in Organic Chemistry from the Shanghai Institute of Organic Chemistry in 2010, working under Professor Wen Liu. Following postdoctoral research with Professor Wilfred van der Donk at the University of Illinois at Urbana-Champaign, he established his independent research group at Fudan University in 2014, and was promoted as a full professor in 2020. He joined Jiangxi Normal University in 2024, where his team focuses on biosynthesis, mechanistic enzymology, antibiotic chemical biology and flow chemistry.

1. Introduction

Quinone methides (QMs), structurally characterized by a cyclohexadienone core with a carbonyl group attached at the para (p-QMs) or ortho (o-QMs) position of the exocyclic C–C double bond, are highly reactive intermediates in chemical and biological systems (Fig. 1).1 Due to their unique zwitterionic aromatic structures, the exocyclic methylene exhibits significant electron deficiency, making QMs excellent Michael acceptors.1
image file: d5np00044k-f1.tif
Fig. 1 Non-aromatic neutral and aromatic zwitterionic resonance structures of para-quinone methide (p-QM) and ortho-quinone methide (o-QM).

In organic synthesis, QMs serve as versatile building blocks. While o-QMs have been extensively utilized in the synthesis of ortho-disubstituted arenes and heterocycles through Michael addition, Diels–Alder reaction, and 6π-electrocyclization,2–4 p-QMs only gained attraction in asymmetric synthesis in the past decade. The broader synthetic application came with the design of stabilized 2,6-disubstituted p-QMs bearing electron-donating groups, which enabled their use in 1,6-addition and annulation reactions across diverse catalytic systems. These transformations facilitate the formation of new C–C and C–heteroatom bonds, providing access to complex and architecturally refined structures.5–8

In biological systems, the electrophilic nature of QMs renders them susceptible to attack by intracellular nucleophiles (e.g., amines, alcohols, and thiols derived from amino acids, nucleobases, and other biomolecules), resulting in covalent adducts with proteins, DNA, and other biomolecules such as glutathione (GSH).9 Notably, QM-mediated DNA alkylation and cross-linking, generated either directly or via metabolic biotransformation, have been exploited in anticancer drug development, as exemplified by selective estrogen receptor modulators (SERMs) like raloxifene and tamoxifen.10,11 More recently, rational design of p-QM-based prodrugs has focused on improving anticancer specificity and inducing irreversible GSH alkylation. This strategy not only overcomes the ROS-scavenging effect of the reversible GSH system but also enables synergistic anticancer activity.12–14

In the field of chemical biology, QMs have been employed to modify proteins via covalent bond formation with nucleophilic amino acid residues in living cells.15 This approach has proven effective for developing both enzyme inhibitors (including mechanism-based inactivators and suicide inhibitors) and self-immobilizing fluorogenic probes, the latter enabling single-cell-resolution fluorescence imaging.16,17

In materials chemistry, QMs function as versatile linkers that covalently conjugate stimuli-responsive functional molecules to construct self-immolative polymers. These polymers have found broad applications in drug delivery, signal amplification, degradable materials, biomedicine, and related fields.16,18 Once the elimination of QMs is triggered, the polymers are disassembled from head to tail via a domino-like fragmentation, resulting in reported luminescence, drug release, or material degradation, depending on the designed functional molecules.18

Through evolutionary refinement, nature has evolved to harness QMs as key intermediates in diverse biochemical processes, including melanization, cuticle sclerotization, lignin formation, and the biosynthesis of numerous secondary metabolites.19 Some natural products (predominantly diterpenes, triterpenes and pigments) possess a p-QM moiety in their final structures,20 as exemplified by celastrol,21 pristimerin,22 taxodione,23 kendomycin,24 and neocandenatone25 (Fig. 2). These compounds exhibit remarkable biological activities, including anti-cancer, anti-inflammatory, anti-obesity, and anti-bacterial properties.


image file: d5np00044k-f2.tif
Fig. 2 Examples of natural products with p-QM moiety in their final existing structures.

A prominent example is the triterpenoid celastrol, derived from the medicinal plant Tripterygium wilfordii Hook F, which modulates multiple signaling pathways (e.g., NF-κB, miRNA, and PI3K/Akt) and demonstrates broad pharmacological effects against cancer, inflammation, neurodegeneration, obesity, diabetes, and cardiovascular diseases.21 Similarly, the polyketide kendomycin, produced by various Streptomyces strains, exhibits diverse bioactivities, such as endothelin receptor antagonism, anti-osteoporotic, antibacterial, and cytotoxic effects.26 In most cases, however, QMs exist as transient intermediates that are difficult to be isolated as pure compounds due to their inherent reactivity toward adjacent nucleophiles, which complicates biosynthetic pathway elucidation.

While o-QM-generating enzymes were extensively studied and comprehensively reviewed in 2022,27 recent advances have revealed numerous enzymes that exploit p-QM chemistry to drive specialized reactions in natural product biosynthesis. Notable examples include DynA-mediated C–N coupling between tyrosine and histidine residues in dynobactin A biosynthesis, PcpXY-catalyzed tyramine excision that introduces a distinctive α-keto-β-amide moiety into spliceotides, and LdpA-driven dehydration and deformylation in lignostilbene biosynthesis. These p-QM-mediated reactions exhibit high efficiency and regio-/stereo-selectivity, which are usually inaccessible in traditional chemical synthesis approaches. Understanding these biosynthetic logic and enzymatic mechanisms not only facilitates discovery of functionally analogous enzymes and related metabolites, but also inspires biomimetic synthetic strategies and provides a framework for rational enzyme design and engineering.

In this highlight, we summarize diverse p-QM-dependent enzymatic transformations in natural product biosynthesis, with emphasis on their underlying catalytic mechanisms, substrate specificity and catalytic versatility, and enzyme engineering. Based on p-QM intermediate formation, these reactions are grouped into three major categories, oxidative generation, C–O bond cleavage, and isomerization of p-hydroxystyrene derivatives.

2. Oxidative formation of p-QM intermediates

In natural product biosynthesis, p-QM formation usually occurs through free radical-mediated reactions or cofactor-dependent redox reactions. These oxidative transformations serve as key gateways for p-QM generation in biochemistry.

2.1 Radical S-adenosylmethionine (rSAM) enzymes

The radical S-adenosylmethionine (rSAM) enzyme superfamily is one of the largest protein families, with over 700[thin space (1/6-em)]000 members found in all three kingdoms of life.28,29 These enzymes catalyze a wide range of reactions, ranging from nucleic acid and protein modifications to the biosynthesis of various small molecule metabolites. Despite this diversity, rSAM enzymes share a common mechanism by using a [4Fe–4S] cluster to bind and reductively cleave SAM, producing L-Met and a highly reactive 5′-deoxyadenosyl radical (dAdo˙), the latter then initiates various radical-based reactions, typically by abstracting a H-atom from the substrate.28

Apparently, one of the effective strategies to obtain p-QM intermediates is to abstract the Hα or Hβ of a tyrosine to generate corresponding image file: d5np00044k-t1.tif or image file: d5np00044k-t2.tif radical, which further undergo a protein-coupled electron transfer (PCET) process to form a p-QM intermediate. Recent studies have identified several rSAM enzymes that utilize p-QM intermediates generated through this approach to form critical structural scaffolds in natural product biosynthesis.

2.1.1 DynA. Dynobactin A (1), belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) family,30,31 exhibits potent activity against various Gram-negative bacteria through targeting of the β-barrel assembly machinery (Bam) complex on their outer membranes.32 The β-strand-like conformation of dynobactin A, essential for its antibacterial activity, is formed by the N–C crosslink between His6 and Tyr8, and the C–C crosslink between Trp1 and Asn4 (Fig. 3a). The N–C crosslink at the Nτ position of a His residue in dynobactin A is highly unique among known RiPP family members. Recent studies have shown that both N–C and C–C crosslinks of dynobactin A are formed by the rSAM enzyme DynA, which proceeds in a stepwise fashion with N–C coupling occurring prior to C–C coupling.33,34
image file: d5np00044k-f3.tif
Fig. 3 Biosynthesis of dynobactin A. (a) DynA catalyzed N–C coupling between His6 and Tyr8. (b) Proposed mechanism of the N–C coupling through p-QM intermediate.33,34 PCET: protein-coupled electron transfer.

The dAdo˙ radical generated by reductive cleavage of SAM abstracts the Hβ atom of the Tyr8 residue (2) to form the image file: d5np00044k-t3.tif radical (3), and a following PCET step afford the key p-QM intermediate (Fig. 3b). Since Hα in Tyr8 does not undergo solvent exchange in DynA assays performed in 2H2O or with [α-2H]Y8-DynB, it appears that the N–C crosslinking is unlikely via an α,β-desaturated dehydrotyrosine (dhTyr) intermediate.33,34 Mutagenesis studies of DynB, such as the Tyr-to-Phe mutant (Y8F), provide further evidence for the involvement of a p-QM intermediate. These results demonstrated that DynA catalyzes the oxidation of the Tyr8 to generate a key p-QM intermediate, which is then nucleophilically attacked by His-Nτ to form the N–C crosslink.33

The involvement of the p-QM intermediate is further validated by the biochemical assays with the H6A-DynB mutant.34 The results revealed that small-molecule nucleophiles like imidazole could covalently modify Tyr8, indicating that Tyr8 is converted to p-QM. It appears that DynA prefers to use charge-neutral nucleophiles (e.g., imidazole derivatives, thiols, and phosphine), and the reactivity does not correlate with the nucleophilicity of the heterocycles. DynA might adopt a conformation to stabilize the specific tautomer (i.e. protonation of Nπ) of His6 for the C–N crosslinking.

As for the C–C crosslinking between Trp1 and Asn4, a series of evidence have shown that the Trp1–Asn4 coupling only occurs after the formation of His6–Tyr8 crosslinking or modification of Tyr8-Cβ with imidazole or its derivatives.34 DynA is hypothesized to selectively recognize the Tyr8-Cβ-imidazole group to initiate C–C bond formation.33,34 However, the detailed mechanisms governing coupling reaction order and radical initiation warrant further structural and functional investigation.

2.1.2 PcpXY. PcpXY, a tyrosine splicease belonging to the rSAM superfamily, catalyzes the post-translational installation of α-keto-β-amide residues into the precursor peptide PcpA.35 This type of enzymes act on the XYG motifs (where X represents variable residues), mediating a splicing reaction that excises a tyrosine-derived tyramine unit. Due to this activity, these enzymes are classified as spliceases, and their ketoamide-containing peptide products are termed spliceotides.36 Recent studies have revealed that spliceotide biosynthesis proceeds via a p-QM intermediate (Fig. 4a).37
image file: d5np00044k-f4.tif
Fig. 4 PcpXY-catalyzed excision of tyramine unit to introduce α-keto-β-amide residues into peptide backbones. (a) The splicease PcpXY recognizes MYG motif in the PcpA precursor as the splicing target. (b) Proposed mechanism of for the splicease reaction.37

PcpXY from Pleurocapsa sp. PCC7327 specifically recognizes the MYG motif at Tyr15 in the PcpA precursor as its splicing target.37 Through a multidisciplinary approach, researchers have proposed that PcpXY initially abstracts the Cα hydrogen of tyrosine (6) to form the peptide radical intermediate (7), which undergoes further oxidation to generate the p-QM intermediate 9 (Fig. 4b). The formation of 7 was verified by deuterium labeling experiments and detection of the isomeric shunt product 8 (containing D-Tyr).

Given the observed tyrosine-derived coproduct 4-hydroxybenzaldehyde 18 and the putative involvement of the cyanide intermediate 16, density functional theory (DFT) calculations suggested two possible mechanisms via the p-QM radical intermediate 9, involving 1,3-acyl radical migration (pathway A) or an azetine four-membered-ring transition state (pathway B) (Fig. 4b). For both pathways, fragmentation of the intermediate 11 would give the α-keto-β-amide product 15 and the cyano quinone methide 16, and the latter is further transformed to hydroxybenzaldehyde 18 via a putative 4-hydroxymandelonitrile intermediate 17 (Fig. 4b).37

As a member of the rSAM-SPASM protein family, PcpXY contains an elongated C-terminal SPASM domain (PF13186, named for the enzymes involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively) harboring two auxiliary [4Fe–4S] clusters.37 It is proposed that the catalytic process involves an auxiliary I [4Fe–4S] cluster with an open Fe-coordination site, which is speculated to assist in the formation of the C–C bond and the extrusion of the product 16. However, the specific function of the auxiliary [4Fe–4S] clusters and whether there are other non-cysteine iron-ligation partners or external redox cofactors need further investigation.

2.1.3 MftC. Mycofactocin, a putative redox cofactor belonging to the RiPPs family, is primarily produced by Mycobacteria.38 The biosynthesis gene cluster of mycofactocin (mftABCDEF) includes three conserved genes: mftA, mftB, and mftC.39 MftA, a peptide with 30–60 amino acids, possess a strictly conserved C-terminal region with the sequence of IDGXCGVY.39 MftB, identified as the RiPP recognition element (RRE),40 specifically binds the precursor peptide MftA and is essential for catalysis by the mycofactocin maturase MftC.41 MftC, a rSAM enzyme harboring a SPASM domain, catalyzes the first modifying step in the biosynthesis of mycofactocin.38,39,42

Current evidence suggests that MftC utilizes two equivalents of SAM to catalyze two distinct reactions on MftA (Fig. 5).39,42 In the first step, the dAdo˙ radical produced via reductive cleavage of SAM by MftC abstracts the Hβ atom of the C-terminal Tyr30 (19), generating the image file: d5np00044k-t4.tif radical (20). This intermediate undergoes oxidative decarboxylation via the p-QM intermediate 21 to form MftA** (22). Mutational analysis confirmed the essential role of the Tyr para-phenol group, as the MftA Y30F mutant was not modified by MftC.


image file: d5np00044k-f5.tif
Fig. 5 MftC catalyzes the oxidative decarboxylation of MftA followed by a C–C bond formation between Val29 and Tyr30.42

In the second step, MftC consumes another equivalent of SAM to generate a second dAdo˙ radical, which abstracts the Hβ atom from the penultimate Val29 to form the alkyl radical (23) (Fig. 5). This radical attacks the α,β-unsaturated bond of Tyr30 at Cα, forming a C–C crosslink between Val29 and Tyr30.39 The resulting radical intermediate (24) undergoes a PCET process to yield MftA* (25), which contains a 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP) moiety.38 It is noteworthy that MftC represents the first reported SPASM member of rSAM enzymes capable of catalyzing both oxidative decarboxylation and redox-neutral C–C bond formation.42,43

2.2 Other oxidases

2.2.1 Vanillyl-alcohol oxidase. Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum is the first characterized and most extensively studied member of the VAO/PCMH (para-cresol methylhydroxylase) flavoprotein family, serving as a model enzyme for this class.44 VAO contains a covalently bound FAD cofactor, which uses molecular oxygen as an electron acceptor to oxidize para-substituted phenols, producing hydrogen peroxide and a p-QM intermediate; the latter is then transformed into diverse products (Fig. 6a).45,46 While its putative physiological role involves the oxidative demethylation of 4-(methoxymethyl)phenol to generate 4-hydroxybenzaldehyde and methanol, VAO displays remarkable substrate versatility, tolerating side chains of up to 7 carbons and ortho substituents (e.g. H, OH, or OCH3).45 The para-phenolic hydroxyl group is strictly essential, consistent with its p-QM-dependent reaction mechanism. Beyond oxidative ether cleavage, VAO also mediates oxidative deamination, hydroxylation, and dehydrogenation of 4-allylphenols.46
image file: d5np00044k-f6.tif
Fig. 6 Vanillyl alcohol oxidase (VAO) and eugenol oxidases (EUGOs) catalyzed reactions. (a) Proposed mechanisms for VAO-catalyzed dehydrogenation and hydration reactions through p-QM intermediates.52 (b) Reactions catalyzed by EUGO and its engineered variants.

The catalytic mechanism of VAO have been elucidated through comprehensive kinetic studies, mutational and structural analysis.45–49 The reaction is initiated with the deprotonation of the phenolic hydroxyl group, mediated by two tyrosine residue (i.e. Tyr108 and Tyr503), to form a phenolate–enzyme complex (26).47 Subsequent hydride transfer from the substrate to FAD generates the p-QM intermediates (27, 28). The highly reactive p-QM intermediates then undergo divergent reactions depending on their structures (Fig. 6a).46,48 When the X group is a poor leaving group such as an alkyl chain, the p-QM intermediate (27) can be dehydrogenated to form the corresponding alkene (29), or hydrated by a water molecule (activated by a protein residue such as Asp170) to yield an alcohol (30).48,49 When the X group is an ether or amine, hydration produces a hemiacetal (31), which rapidly cleaves to form the corresponding aldehyde (32).46

Owing to their remarkable catalytic promiscuity, VAO and its homologous enzymes have been broadly applied in chemoenzymatic synthesis50 and bio-engineering studies (Fig. 6b).51–54 For example, eugenol oxidase (EUGO) from the bacterial Rhodococcus sp., sharing 45% sequence identity with the fungal VAO, can also catalyze the dehydrogenation and hydration of 4-allylphenols.55 Recently, several engineered EUGOs have been reported as biocatalysts for different purposes, including PROGO (for dehydrogenation of 4-propylguaiacol (35) to yield isoeugenol (36)),51 EUGO D4 (for oxidative cleavage of para-hydroxy benzyl ethers (37)),52 EUGO10X (for oxidation of dihydrosinapyl alcohol (39) to sinapyl alcohol (40)),53 and EUGO-DTT (mediating glycine amination of 4-propylguaiacol (35)).54

2.2.2 Laccase & dirigent protein. p-QMs are also involved in the oxidative coupling of phenolic compounds derived from the phenylpropanoid biosynthesis pathway to construct lignans. During the biosynthetic process, oxidases such as laccases and peroxidases catalyze the single electron oxidation of phenylpropanoid units like coniferyl, sinapyl, and p-coumaryl alcohols to form the corresponding phenoxy radicals, where the lone electron can delocalize over the entire conjugated aromatic systems, rendering several positions reactive.56 By coupling radicals carrying p-QM groups, a lignan or neolignane skeleton can be formed. The electrophilicity of the p-QM moiety thus enables diverse structures to be constructed through intramolecular Michael addition reactions.

In the biosynthesis of the furofuran lignan pinoresinol, the bisquinonmethide generated by C8–C8′ coupling of phenoxy radicals derived from coniferyl alcohol (34) undergoes a double intramolecular oxa-Michael addition, leading to furan ring cyclization and rearomatization to form the diarylfurofuran moiety (Fig. 7).57,58 Interestingly, the oxidative coupling reactions in the plants imparted highly regio- and stereo-selectivity, such as 8,8′-linked (+)-pinoresinol (46) from Forsythia intermedia57 or (−)-pinoresinol (47) from Arabidopsis thaliana,59 whereas the in vitro dimerization experiments with laccases or peroxidases led to racemic (±)-8,8′-, (±)-8,5′-, and (±)-8-O-4′- linked mixtures (Fig. 7).57 These differences are attributed to dirigent protein (DIR or DP), which bind oxidase-generated radicals and enforce regio- and stereo-selectivity during coupling.60 (+)-Pinoresinol-forming DIRs (such as FiDIR1,57 PsDRR206,61 and PhDIR58) promote si–si face coupling of coniferyl alcohol radicals, while (−)-pinoresinol-forming DIR (such as AtDIR6[thin space (1/6-em)]59,62) favor re–re face coupling, resulting in (R,R)- or (S,S)-bisquinonmethides and further generation of (+) or (−)-pinoresinol, respectively (Fig. 7). DIRs may also promote the formation of the diarylfurofuran moiety, and this process is likely activated through protonation or hydrogen bonding.62


image file: d5np00044k-f7.tif
Fig. 7 Laccase-catalyzed oxidation of E-coniferyl alcohol and bimolecular radical coupling products with/without dirigent proteins (DIRs).60,61

Recently, a DIR from Podophyllum hexandrum (PhDIR) has been utilized in combination with a laccase to promote the regio- and stereoselective heterocoupling of coniferyl alcohol (34) and its synthetic analogues, resulting in a series of analogues of (+)-pinoresinol (46).58 Since 46 is an intermediate in the biosynthetic pathway of etoposide aglycone, which is a semi-synthetic precursor of the chemotherapeutic drug etoposide, replacing the substituents on the phenyl ring of 34 (while retaining the para-hydroxyl group, as it is crucial for p-QM formation) can yield diverse etoposide analogues.58 Therefore, by expanding the substrate versatility of DIRs, optimizing them through protein engineering, and combining with conventional synthetic chemistry, more unnatural lignan analogues can be obtained in the future.

3. Formation of p-QM intermediates by C–O bond fission

For substrates bearing a para-phenolic hydroxyl group with a hydroxyl or ester substitute at the Cα position of the phenyl ring, several enzymes can convert them into p-QM intermediates via concerted deprotonation of the phenolic hydroxyl group and elimination of the oxygen-containing substituent. This process can be classified as either a dehydration or deesterification reaction. The resulting p-QM intermediates then enables diverse transformations such as Michael additions, cofactor-dependent reductions, deformylations, and Diels–Alder reactions.

3.1 Dehydration

3.1.1 γ-Formaldehyde lyase LdpA. During lignin degradation in microbial-mediated plant decay, bacterial and fungal enzymes facilitate the cleavage of C–C bonds in recalcitrant aromatic dimers, converting them into monomers. For instance, the γ-formaldehyde lyases NaLdpA (from Novosphingobium aromaticivorans) and SpLdpA (from Sphingobium sp. SYK-6) cleave the diarylpropane lignin derivative erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (50), releasing formaldehyde and lignostilbene (52); the latter can then be further degraded by lignostilbene-α,β-dioxygenase (LsdA) to yield two equivalents of vanillin.63,64 The p-QM intermediate (51) plays a key role in the LdpA-catalyzed reaction by acting as an electron sink to facilitate the deformylation reaction (Fig. 8).64
image file: d5np00044k-f8.tif
Fig. 8 γ-Formaldehyde lyase LdpA-catalyzed dehydroxylation and subsequent deformylation to form the C–C double bond in lignostilbene.64

LdpA adopts a characteristic twisted α + β barrel fold and belongs to the nuclear transport factor 2 (NTF-2)-like structural superfamily. Similar to other dehydratases and decarboxylases in this family, LdpA employs a His–Asp dyad for acid-base catalysis and two tyrosine residues to stabilize the oxyanion intermediate. Furthermore, a conserved Glu–Tyr–Gln triad facilitates the dehydration of 50 to form the p-QM intermediate 51.64 Subsequently, His97 acts as a catalytic base, abstracting the Cγ hydroxyl proton to initiate Cβ–Cγ bond cleavage of 51, resulting in formaldehyde elimination and lignostilbene (52) production.64

3.1.2 Dihydroflavonol-4-reductases. DcDFR-1 and DcDFR-2, identified in Dracaena cambodiana, are dihydroflavonol-4-reductases (DFRs) involved in flavonoid biosynthesis. These enzymes catalyze the reduction of the 4-keto carbonyl group in dihydrochalcone-M272 (53, loureirin C), a bioactive dihydrochalcone from the traditional medicine called dragon's blood, which is the red resin produced by injured Dracaena trees.65 The resulting chalcone alcohol 54 is unstable and undergoes spontaneous dehydration in aqueous solution to form the p-QM intermediate 55. Incubation of 53 with resveratrol (56) led to the production of the dimer cochinchinenene D (58), likely proceeding via a Michael addition process (Fig. 9). Incubation of 53 with other nucleophiles (including flavonoids, oxyresveratrol, phloroglucinol, phloroglucide, benzoic acids, coumarins, naphthalenes, quinolines, and indoles) yielded a series of C–C or C–N coupled chalcone-containing dimers. These results suggest that many of the approximately 50 reported oligomers in dragon's blood may form through analogous coupling pathways.65
image file: d5np00044k-f9.tif
Fig. 9 Dihydroflavonol-4-reductases (DFRs) DcDFR-1 and DcDFR-2 catalyze the reduction of the dihydrochalcone loureirin C and the subsequent spontaneous dimer formation.65
3.1.3 Leucoanthocyanidin reductase LAR1. Leucoanthocyanidin reductase 1 from Vitis vinifera (VvLAR1), belonging to the superfamily of short-chain dehydrogenases/reductases (SDRs), catalyzes the NADPH-dependent deoxygenation of leucocyanidin (59) to produce (+)-catechin (61).66 As a member of the PIP subfamily of enzymes (named after three first discovered enzymes, pinoresinol–lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase), VvLAR1 catalyzes the reductive cleavage of C–O bonds via a p-QM intermediate, a characteristic reaction of this enzyme class.66,67 Based on the VvLAR1 crystal structure, a two-step mechanism was proposed (Fig. 10):66 first, Lys140 deprotonates the C7–OH of 59 via a water bridge, while His122 then orchestrates substrate orientation and acid-catalyzed water elimination (assisted by the C5–OH) to form the p-QM intermediate 60. Second, pro-R hydride transfer from NADPH to the C4 of 60 affords (+)-catechin 61.
image file: d5np00044k-f10.tif
Fig. 10 Leucoanthocyanidin reductase 1 (LAR1) catalyzed NADPH-dependent reductive deoxygenation of leucocyanidin to (+)-catechin.66
3.1.4 A dehydratase domain in ElaP. Elansolid A3 (65), featuring a p-QM moiety, is a polyketide-derived antibiotic isolated from the metabolites of gliding bacterium Chitinophaga sancti (formerly Flexibacter spec.).68 The isolation process requires strictly anhydrous aprotic solvents, as the electrophilic p-QM readily undergoes nucleophilic addition with protic solvents (e.g., water or methanol), yielding artifact products elansolids B1 and B2.68 Elansolid A3 (65) could further cyclize by a Michael-type attack from its carboxylate onto the p-QM to obtain the 19-membered macrolactones, elansolids A1 and A2, which are stable and separable atropisomers.68,69

The biosynthesis of elansolids starts from chorismate-derived p-hydroxybenzoic acid, as confirmed by isotopically labeled feeding experiments in Chitinophaga sancti.69 The PKS module 10 has a hybrid structure, consisting of the C-terminal portion of ElaP (containing KS, DH, and KR domains) and the N-terminal region of ElaQ (containing MT and ACP domains).69 The dehydratase (DH) domain within ElaP is proposed to catalyze the dehydration of the OH group at C23, generating the p-QM intermediate 63. This intermediate then undergoes intramolecular Diels–Alder cyclization to construct the characteristic bicyclo[4.3.0]nonane scaffold (64) (Fig. 11).69 Later synthetic studies using model compounds supported this proposed mechanism.70 However, the exact timing of the intramolecular Diels–Alder cyclization (whether it occurs on PKS or after chain release) remains to be investigated. This p-QM-mediated intramolecular Diels–Alder cycloaddition has attracted considerable interest in natural product chemistry, serving as a key strategic element in biomimetic asymmetric total synthesis of elansolids.71,72


image file: d5np00044k-f11.tif
Fig. 11 p-QM initiated intramolecular Diels–Alder reaction in the biosynthesis pathway of the polyketide elansolid A3.69

3.2 Deesterification reaction

3.2.1 Hinokiresinol synthase. (Z)- and (E)-hinokiresinols (66, 67) represent the structurally simplest norlignans, featuring a diphenylpentane skeleton derived from the coupling of two phenylpropane units (Fig. 12).73 In Asparagus officinalis, hinokiresinol synthase (HRS) catalyzes the decarboxylative rearrangement of 4-coumarate (68) to produce (Z)-hinokiresinol (66). In contrast, the homologous enzyme from Cryptomeria japonica generates (E)-hinokiresinol (67).73,74
image file: d5np00044k-f12.tif
Fig. 12 Hinokiresinol synthase (HRS)-catalyzed decarboxylative rearrangement to obtain hinokiresinols.76

Hinokiresinol synthase (HRS) from Asparagus officinalis possessing two subunits, HRSα and HRSβ. While individually expressed the HRSα or HRSβ subunits show low enantioselectivity in converting 68 to (E)-hinokiresinol (67), their equimolar combination achieves near-perfect stereoselectivity and enantioselectivity, yielding exclusively (Z)-hinokiresinol (66).73,75

The mechanism of the HRS-catalyzed decarboxylative rearrangement converting 68 to 66 in A. officinalis was elucidated through X-ray crystallography, computational analysis, and site-directed mutagenesis.76 The reaction initiates with C4′-phenolic oxygen deprotonation, triggering cleavage of 68 to generate coumarate 69 and p-QM 70, which subsequently undergo C8–C7′ coupling to form 71. Mutation of Asp165 to Ala in HRSα alters product selectivity, yielding both 67 and the decarboxylated product 75 (derived from intermediate 74 formed through C8–C9′ coupling). These results establish the essential role of α-Asp165 in controlling both the regioselectivity and stereoselectivity of the reaction. DFT calculations suggested that intermediate 71 may transiently dissociate from the heterodimeric HRS active cavity, undergo conformational rearrangement, and subsequently rebind to undergo decarboxylation, ultimately yielding 66 with the desired (Z)-configuration.76

3.2.2 Eugenol synthase. Eugenol synthase (EGS), a short-chain dehydrogenase/reductase (SDR) from Ocimum basilicum, catalyzes the reductive cleavage of the C–O bond in the side chain of coniferyl acetate (76) through a p-QM intermediate 77, resulting in the allyl-phenylpropene product of eugenol (33, Fig. 13).67,77
image file: d5np00044k-f13.tif
Fig. 13 Reductive elimination of acetate from coniferyl acetate to form eugenol and isoeugenol by eugenol synthase (EGS) and isoeugenol synthase (IGS).67,77

The p-QM intermediate 77 is generated through deprotonation of the para-phenolic hydroxyl group, a process mediated by a conserved proton-relay network. In this system, Lys132 in EGS positions a bridging water molecule that facilitates both substrate hydroxyl coordination and proton abstraction, thereby enabling acetate elimination and p-QM formation. Notably, similar water-mediated proton-relay network is also found in VvLAR1 (Fig. 10), an SDR enzyme sharing 36% sequence identity with EGS that also belongs to the PIP protein subfamily.66 Mutational analysis confirms the essential catalytic role of Lys132, as both Lys132Ala and Lys132Gln mutants completely lost enzyme activity.

In the reductive step, the pro-R hydride from NADPH is precisely positioned opposite the C7 of 77, enabling direct hydride transfer to yield the allylphenylpropene product eugenol (33).77 For biosynthesis of its structural isomer isoeugenol (36), isoeugenol synthase (IGS) is proposed to mediate analogous hydride attack at C9 of the side chain, producing the isophenylpropene derivative isoeugenol 36.67,77

4. Formation of p-QM intermediates by isomerization of para-hydroxystyrene

Phenolic acid decarboxylases (PADs) are cofactor-free enzymes widely distributed in bacteria78–80 and fungi81 that catalyze the decarboxylation of para-hydroxycinnamic acids (including p-coumaric, ferulic, and caffeic acids) to their corresponding hydroxystyrene derivatives (Fig. 14a). This unique activity makes PADs promising biocatalysts for industrial applications, particularly in polymer production and food flavor enhancement.82 Notably, certain PADs from Lactobacillus plantarum (LpPAD) and Bacillus amyloliquefaciens (BaPAD) exhibit remarkable catalytic versatility beyond their native decarboxylase activity. These enzymes can catalyze both the reverse β-carboxylation of para-hydroxystyrenes to produce (E)-cinnamic acid derivatives using bicarbonate (2–40% conversion, Fig. 14b),83 and stereoselective asymmetric hydration of para-hydroxystyrenes in carbonate buffer (up to 82% conversion, Fig. 14c).84 All three catalytic activities (i.e. decarboxylation, carboxylation, and hydration) strictly require the para-phenolic hydroxyl group, reflecting their shared mechanistic dependence on the p-QM intermediate.78,83,84
image file: d5np00044k-f14.tif
Fig. 14 Phenolic acid decarboxylases (PADs)-catalyzed decarboxylation (a), carboxylation (b), and hydration (c) reactions through p-QM intermediates.85,86

In the natural decarboxylation reaction, deprotonation of the phenolic hydroxyl group of 78 produces the p-QM intermediate 79. Subsequent C–C bond cleavage then yields vinyl phenol 80 with concomitant CO2 release.85,86 PAD structures show a conserved β-barrel with flexible loops mediating open/closed transitions.78–81 Early models proposed that the substrate carboxylate interacted with Tyr11/Tyr13 (BsPAD numbering), and the phenolic hydroxyl was deprotonated by Glu64 with Arg41 assistance.78–81 Faber et al. later proposed that it is the conserved tyrosines coordinate the phenolic hydroxyl group, instead of the carboxylate.83 Further DFT calculations established the revised mechanism (Fig. 14a), identifying Glu64 as the general acid, while Tyr19 stabilizes the carboxylate of the p-QM intermediate (79) through hydrogen bonding, thereby lowering the activation barrier for decarboxylation.86

DFT calculations also revealed mechanisms for both carboxylation and hydration (Fig. 14b and c).85 In the carboxylation pathway, bicarbonate first decomposes to form free CO2, which represents the rate-determining step of this transformation. The hydroxystyrene substrate then reacts with this liberated CO2 (rather than bicarbonate), leading to formation of the p-QM intermediate 81. For the hydration reaction, computational analysis ruled out mechanisms involving C–C bond formation between bicarbonate and substrate due to prohibitively high energy barriers. Instead, bicarbonate serves as a proton shuttle, and the reaction is initiated with Glu64-mediated protonation of the Cα–Cβ double bond to generate the p-QM intermediate 83. The rate-limiting step involves nucleophilic attack by water at the Cα position of this p-QM intermediate. The enantioselectivity of this transformation arises from preferential orientation of the newly formed methyl group toward the less sterically encumbered Val70 and Ile85 residues in the active site, ultimately yielding the S-configured alcohol product 84.85

5. Conclusions

p-QMs serve as versatile reactive intermediates in the biosynthesis of diverse natural products, including RiPPs, phenylpropanoids, lignans, neolignans, chalcones, flavan(ol)s, and polyketides, and apparently many other p-QM-mediated natural products remain to be characterized, such as C30-terpene taxodisone A,87 meroterpenoids,88 and bromophenol symphyocladin L.89 Enzymes strategically generate these electrophilic moieties through oxidation, dehydration, deesterification, or isomerization of para-substituted phenolic precursors. The resulting p-QM intermediates then trigger diverse transformations such as Michael additions, carbon chain rearrangement, decarboxylations, deformylations, radical couplings, or Diels–Alder reactions. These enzymatic processes outperform traditional chemical synthesis in efficiency, regio- and stereo-selectivity, and environmental compatibility, often accessing scaffolds unattainable through conventional methods. These unique advantages of p-QM-mediated transformations render them exceptionally valuable for chemoenzymatic synthesis, biomimetic strategies, and biocatalyst engineering. Given their remarkable potential, discovering new p-QM-utilizing enzymes and their associated natural products could significantly expand the toolbox for biosynthetic and pharmaceutical applications.

6. Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

7. Conflicts of interest

There are no conflicts to declare.

8. Acknowledgements

This work is supported by grants from the National Natural Science Foundation of China (22167016, 22477049, U22A20451), and from West Light Foundation of the Chinese Academy of Sciences (xbzg-zdsys-202105).

9. Notes and references

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