Recent Advances in Biocatalytic Dearomative Spirocyclization Reactions

Abstract

Spirocyclic architectures, which feature two rings sharing a single atom, are common in natural products and exhibit beneficial biological and material properties. Due to the significance of these architectures, biocatalytic dearomative spirocyclization has recently emerged as a powerful approach for constructing three-dimensional spirocyclic frameworks under mild, sustainable conditions and with exquisite stereocontrol. This review surveys the latest advances in biocatalyzed spirocyclization of all-carbon arenes (phenols and benzenes), aza-aromatics (indoles and pyrroles), and oxa-aromatics (furans). We highlight cytochrome P450s, flavin-dependent monooxygenases, multicopper oxidases, and novel metalloenzyme platforms that effect regio- and stereoselective oxidative coupling, epoxidation/semi-pinacol rearrangement, and radical-mediated cyclization to produce diverse spirocycles. Mechanistic insights gleaned from structural, computational, and isotope-labeling studies are discussed where necessary to help the readers further understand the reported reactions. Collectively, these examples demonstrate the transformative potential of biocatalysis to streamline access to spirocyclic scaffolds that are challenging to prepare through traditional methods, underscoring biocatalysis as a transformative tool for synthesizing pharmaceutically relevant spiroscaffolds while adhering to green chemistry paradigms to ultimately contribute to a cleaner and more sustainable future.

1. Introduction

Spirocyclic compounds, characterized by their unique three-dimensional frameworks where two rings share a single atom, are widely found in natural products (Figure 1a) [1,2] and have beneficial applications as catalysts and ligands [3,4,5,6,7,8], as well as other materials (Figure 1b) [9,10,11]. They have long captivated chemists and pharmacologists due to their structural complexity and biological relevance for spirocyclic bioactive compounds, in which their rigid conformations enable precise interactions with biological targets, enhancing binding affinity and selectivity [12,13,14,15,16,17]. Notable examples include griseofulvin, a spirocyclic antifungal agent, and yatakemycin, a DNA-alkylating antibiotic with a spirocyclopropane core (Figure 1c). Despite their significance, the synthesis of spirocycles remains a formidable challenge [18,19,20], particularly when stereochemical control and sustainable methodologies are needed [21,22,23,24,25,26,27,28,29].

The dearomatization reaction of planar aromatic substrates [30,31] was considered one of the most promising methods for constructing these three-dimensionally complex chiral skeletons due to the large number of readily available aromatic compounds for this reaction. Transition-metal-catalyzed dearomatization reactions in asymmetric dearomatization strategies for spirocycle construction have been well developed, particularly through asymmetric allylic dearomatization reactions catalyzed by Pd, Ir, and Ru [32], and Pd-catalyzed enantioselective Heck reactions [33]. Additionally, organo-catalyzed dearomatization reactions as a more economical and environmentally friendly approach, such as hypervalent iodine catalysis, have gained widespread recognition over the past two decades [34,35]. Furthermore, a combination of organocatalysis and transition metal catalysis has the advantages of both catalytic methodologies while suppressing their weaknesses, and has also been employed in the field of asymmetric synthetic chemistry for dearomatization reactions [36]. Despite the elegance of these pioneering enantioselective dearomative spirocyclization methods, they frequently rely on harsh conditions (such as high-temperature, strictly anhydrous, and oxygen-free environments) and expensive chiral ligands to achieve enantioselective control for the reactions with residual heavy metals; require complicated reaction mixtures with side-products that require isolation; and have narrow functional group tolerance, especially active hydroxyl, amino, and carboxyl groups.

Compared with transition-metal- and organo-catalyzed dearomatization reactions, biocatalysis has emerged as a transformative paradigm offering precise stereocontrol, mild reaction conditions, and inherent compatibility with green chemistry principles [37,38,39,40,41,42,43,44,45,46,47,48,49,50,51]. However, the topic of biocatalytic dearomative spirocyclization has received less attention in the literature compared to other forms of biocatalysis. Although there is an excellent review on biocatalytic dearomatization [52], it primarily focused on the dearomatization of monocyclic aromatic substrates without the formation of spirocycles, and did not systematically address the mechanistic diversity in biocatalytic dearomative systems. To address this gap, we conducted a comprehensive literature survey using Web of Science and citation tracking, employing keywords including biocatalysis, enzymatic catalysis, dearomatization, spirocyclization, and related terms to access relevant studies published before May 2025. Representative examples found by this strategy focusing on non-enzymatic methods or dearomatization without spirocycle formation were excluded. This review critically evaluated, for the first time, the rapidly evolving field of biocatalytic dearomative spirocyclization. We highlight the breakthroughs in three key aromatic scaffolds, all-carbon arenes (phenols and benzenes), aza-aromatics (indoles and pyrroles), and oxa-aromatics (furans), as well as the mechanistic insights that underpin the reaction selectivity and efficiency. By unifying the scattered advances into a coherent framework, this review provides a timely resource to accelerate the sustainable synthesis of spirocycles, showcasing how biocatalysis can overcome the longstanding limitations of traditional methods. This publication is pivotal as the field reaches maturity, offering chemists strategic insights to harness enzymatic platforms for accessing privileged spiroarchitectures.

2. Biocatalytic Dearomative Spirocyclization Reactions of All-Carbon Aromatic Rings

2.1. Dearomatization of Phenols

Phenols and their derivatives, which are ubiquitously distributed in natural products, bioactive molecules, and lignin, represent a vital class of chemically versatile and readily accessible feedstocks. Their intrinsic keto-enol tautomeric equilibrium, wherein the enol form dominates due to aromatic stabilization, endows phenols with unique reactivity profiles. Dearomatization reactions of phenols thus emerged as a transformative strategy for converting planar aromatic precursors into three-dimensionally complex cyclohexadienones [53,54,55,56,57,58,59], which are frequently embedded in bioactive natural products and pharmaceuticals. Consequently, phenol dearomatization bridges the gap between simple aromatic building blocks and architecturally intricate molecules, offering a streamlined route to access medicinally relevant scaffolds while preserving the inherent sustainability of phenol-derived feedstocks.

2.1.1. Dearomatization of Phenols via P450 Enzymes

Griseofulvin (1c) with an intriguing spirocyclic core shows versatile antifungal, anticancer, and antiviral activities [60,61]. Its biosynthetic genes from Penicillium aethiopicum were reported by Tang’s group in 2010 [62], and the complete biosynthetic pathway was elucidated in 2013 [63]. The cytochrome P450 GsfF was found to perform intramolecular oxidative coupling between the orcinol and the phloroglucinol rings of griseophenone B (1a) as the precursor (Scheme 1). Later in 2016, Houk and Tang elucidated the mechanism of this P450-catalyzed dearomatization reaction using density functional theory (DFT) calculations [64]. First, the high-valent iron(IV)-oxo heme intermediate compound I (cpd I) abstracts a hydrogen atom from the hydroxyl group to form the reduced intermediate compound II (cpdII) and an oxygen radical, which directly attacks the neighboring arene to generate the spirocyclic radical intermediate. Subsequent phenolic O-H abstraction by cpdII delivers the final product desmethyl-dehydrogriseofulvin A (1b). These studies not only clarify the intricate biosynthetic pathway of griseofulvin, but also demonstrate the catalytic mechanism of cytochrome P450 GsfF in oxidative dearomatization and spirocyclization, providing a blueprint for engineering spirocyclic natural product analogs via biocatalysis.

Recently, in 2024, Yang and Liu’s group developed P450 enzymes as a metalloenzyme platform for the dearomatization of a broad spectrum of aromatic substrates, mainly including phenol 2a (Scheme 2) [65]. The engineered variant P450_rad4 (P450arc1-L266H/G438T/L78C/L75F/G268P/L436A/F437P) converted racemic phenol derivative 2a to the enantioenriched dearomatized spirocyclic product cyclohexadienone 2b in moderate to excellent yields (45–98%) with high enantioselectivities (56–90% ee). When unsymmetric phenol was employed, the dearomatization platform produced a corresponding product bearing an additional spiro quaternary carbon stereocenter, achieving 81:19 dr value and 68% ee value. The catalytic cycle initiates when the ferrous heme catalyst engages the α-bromocarbonyl substrate (2a). This interaction proceeds via either halogen atom abstraction or electron transfer, generating an electrophilic radical species (2-I) while oxidizing the heme iron to the ferric state simultaneously. The highly reactive radical 2-I then undergoes intramolecular cyclization with the proximal electron-rich aromatic moiety. This key step forms a dearomatized radical intermediate (2-II), which delivers the dearomatized product 2b through oxidative radical–polar crossover with concomitant proton transfer. Finally, the active ferrous heme catalyst is regenerated to the next catalytic cycle. This work highlights the power of unnatural metalloredox biocatalysts that can be precisely tuned for different situations to solve long-standing problems in asymmetric catalysis.

2.1.2. Dearomatization of Phenols via Copper-Dependent Oxidases

Geodin (3c), a fungal natural product featuring a conserved grisan backbone with griseofulvin (reviewed in Section 2.1.1), was discovered in 1936 [66], and exhibits diverse pharmacological properties, including antimicrobial activity, modulation of glucose uptake in rat adipocytes, cytotoxicity against tumor cell lines, fibrinolytic enhancement, and antiviral effects [67,68,69]. Despite its early discovery and broad bioactivity profile, the biosynthetic machinery of geodin remained enigmatic until Mortensen and colleagues achieved a pivotal breakthrough in 2013 by heterologously reconstituting its entire gene cluster in Aspergillus nidulans (Scheme 3) [70].

In 1987, Sankawa and Ebizuka’s group found that the dihydrogeodin oxidase (DHGO) GedJ catalyzed the regio- and stereospecific formation of (+)-geodin (3c) [71] from dihydrogeodin (3b) [72]. During the investigation of the biosynthetic pathway of (+)-geodin, an additional grisandiene analog, bisdechlorogeodin (3d), was discovered, which was proposed to also be formed by GedJ from sulochrin directly without the double chlorination catalyzed by the sulochrin halogenase GedL [73]. Meanwhile, a highly homologous gene cluster of A. fumigatus and A. fischerianus was found to produce trypacidin (3f) via monomethylsulochrin (3e). It could be formed through the methylation of sulochrin by O-methyltransferase TpcH and ring closure by multicopper oxidase (MCO) TpcJ. Notably, MCO from different hosts showed opposite stereospecificity for bisdechlorogeodin [74], and other laccases or peroxidases could also produce geodin, but without enantioselectivity [75], highlighting the crucial role of MCOs in determining stereospecificity. These results indicate that sulochrin as the branch point intermediate and MCO as key enzymes are both crucial to the formation of grisan structures in the desired configuration.

2.1.3. Dearomatization of Phenols via Vanadium-Dependent Chloroperoxidase

In 2014, Moore’s group discovered a vanadium-dependent chloroperoxidase Mcl24 from the biosynthetic pathway of the merochlorin meroterpenoids (merochlorin A and B) (Scheme 4) [76]. These compounds with attracting antimicrobial activities were isolated from Streptomyces sp. CNH-189 in 2012 [77,78]. Mcl24, as a multifunctional chloroperoxidase, unexpectedly catalyzes site-selective naphthol chlorination and an oxidative dearomatization/terpene cyclization cascade reaction to produce merochlorin A (4b) and B (4c). However, this enzyme exhibited strict substrate specificity, showing no activity toward structural analogs. To shed light on the mechanism of Mcl24 with such a complicated process, they developed a synthetic chlorination protocol to mimic Mcl24 reactivity.

The proposed mechanism involves several key steps. First, a chlorinated amino acid residue is formed like the rebeccamyin flavin-dependent chlorinase RebH [79], which then chlorinates the substrate at the 2 position to form the intermediate 4-I. The chlorine atom transfer from chlorinated amino acid to the substrate is proposed to be responsible for the high regioselectivity of Mcl24. Subsequently, a second chlorination step occurs to form the aromatic hypochlorite species 4-II, and it transforms to the cation intermediate 4-III following chloride ion departure. This intermediate undergoes cation-induced terpene cyclization to deliver the spiro intermediate 4-IV. Finally, two different pathways of the nucleophilic attack from the C atom of enolate or directly from the O atom furnishes merochlorin A or B, respectively. Notably, in sharp contrast to the Mcl24-catalyzed process with high site-selectivity for halogenation, high substrate specificity, and controllable timing of the chlorination reaction, the developed chemical reaction with the NCS/iPr₂NH system predominantly produced other derivatives including deschloro-merochlorin A and B, isochloro-merochlorin B, and dichloro-merochlorin B. This comparison highlights this powerful enzymatic synthesis strategy, especially in terms of the regioselectivity and substrate specificity.

2.1.4. Dearomatization of Phenols via Methyltransferases

Yatakemycin (YTM), CC-1065, and duocarmycins belong to the spirocyclopropane family of antibiotics, which function as naturally derived DNA-alkylating agents [80,81,82]. Their excellent bioactivities, which attract continuous attention for chemical, biological, and pharmaceutical studies, primarily stem from their cyclopropane moiety. The biosynthetic origin of their structural features was studied until 2012 by Tang’s group (Scheme 5) [83]. They proposed the sadenosylmethionine (SAM)-dependent enzyme YtkT-catalyzed methylation/cyclopropanation cascade reaction of YTM-T (5a) towards YTM (5b), according to the accumulation of the precursor YTM-T after the inactivation of ytkT. Although this work characterized the radical SAM-dependent C-methyltransferase YtkT as being responsible for the methylation/cyclopropanation cascade reaction, the detailed reaction mechanism is still unknown.

Later in 2018, Tang’s group reported their further investigation on the mechanism of cyclopropane formation process based on their continuing interest in spirocyclopropane-containing antibiotics (Scheme 6) [84]. The two-component cyclopropanase system including a HemN-like radical SAM enzyme C10P and a methyltransferase C10Q was identified to catalyze cyclopropanation in the biosynthesis of the antitumor antibiotic CC-1065 (6b), which is quite challenging for chemical reactions. Then, the mechanism was proposed by the authors. The first SAM1 molecule is reductively cleaved by the HemN-like radical SAM enzyme C10P to yield the Ado-CH₂ radical, abstracting the hydrogen atom from the second SAM₂ molecule to form the SAM methylene radical (6-I). Then, it reacts with the C11 position of the substrate to form the adduct radical 6-II, which is protonated by the solvent to give the intermediate 6-III, and then undergoes intramolecular S_N2 cyclopropanation by C10Q to produce the final product CC-1065 with S-adenosyl homocysteine (SAH) as the coproduct.

These studies elucidate a novel two-component cyclopropanase system, comprising a HemN-like radical SAM enzyme C10P and a methyltransferase C10Q, which collaboratively catalyze the chemically challenging cyclopropanation in the biosynthesis of YTM and CC-1065. The system operates through radical-mediated methylene transfer followed by an unprecedented intramolecular S_N2 cyclization mechanism. These findings thereby expand the enzymatic repertoire of SAM-dependent biochemistry, and offer critical insights into the biosynthesis of pharmacologically significant spirocyclopropane in drug development.

2.2. Dearomatization of Benzenes

Although benzene rings as prototypical non-activated arenes are a structural skeleton that is more widely distributed than phenol, they exhibit inherently low reactivity due to their high aromatic stabilization energy for the robust aromatic π-conjugation system. This renders these planar hydrocarbons chemically inert under conventional reaction conditions. This electronic and structural resilience poses significant challenges in dearomatization processes, which requires substantial energy input or sophisticated catalytic strategies to disrupt the delocalized π-system, further compounded by the need to overcome kinetic barriers while controlling regio- and stereochemical outcomes in the transformation from planar aromatic precursors to three-dimensional molecular architectures [35,85]. The fundamental characteristics render benzene derivatives particularly challenging substrates in dearomative chemistry, necessitating innovative approaches to achieve selective π-system perturbation without compromising molecular integrity.

In 2023, Lewis’ group repurposed an engineered variant of B₁₂-dependent transcription factor CarH (termed CarH*) to realize non-native radical cyclization reactions, including dearomatization of pendant arenes 7a, and redox-neutral or reductive ring closure reactions of α-chlorodifluoroacetamides bearing phenyl, alkenyl, and alkynyl groups (Scheme 7) [86]. Interestingly, different from the existing methods for 1,4-dienes, this biocatalytic reaction produces bicyclic 1,3-diene product 7b in 8–98% yields. The mechanism initiates with oxidative addition of substrate 7a to Co(I)-H to form the Co(III) intermediate 7-I. Then, they recombine to Co(II) with an alkyl radical generating the complex 7-II, which cyclizes to the spirocyclic radical complex 7-III. Then, they dissociate to the Co(II) with spirocyclic radical 7-IV, which abstracts a hydrogen atom from the Co(III)-H complex through hydrogen atom transfer (HAT).

This study demonstrates that the engineered B₁₂-dependent enzyme CarH* is a versatile biocatalyst enabling unprecedented non-native radical cyclization, including the dearomatization of inert arenes and redox-neutral or reductive annulation of α-chlorodifluoroacetamides, which uniquely furnishes spirobicyclic 1,3-dienes through a Co(I)-initiated radical cascade involving oxidative addition, cyclization, and hydrogen atom transfer. This work expands the synthetic utility of B₁₂ enzymes and provides a biocatalytic platform for accessing structurally complex, three-dimensional architectures from planar aromatic precursors with controlled selectivity.

3. Biocatalytic Dearomative Spirocyclization Reactions of Aza-Aromatic Rings

3.1. Dearomatization of Indoles

Indoles represent a cornerstone of heterocyclic chemistry, whose aromatic bicyclic framework serves as the structural basis for natural products, pharmaceuticals, and agrochemicals [87,88,89,90]. Dearomatization of indoles into indolines or 3H-indoles provides access to structurally complex scaffolds, notably spirocyclic indoline motifs that are pervasive in biologically active alkaloids with activities ranging from cytotoxicity and antimalarial efficacy to opioid receptor modulation [91,92,93,94]. However, the inherent stability of the indole π-system makes controlled dearomatization exceptionally challenging, demanding finely tuned catalysts, precise substrate design, and rigorously optimized reaction conditions. Consequently, the development of robust dearomative methodologies is both a formidable synthetic endeavor and a strategic imperative to achieve atom-economical access to spirocyclic indole derivatives that would otherwise require lengthy multistep sequences.

3.1.1. Dearomatization of Indoles via p450 Enzymes

In 2013, Watanabe’s group elucidated two distinct enzymatic mechanisms for spiro-carbon formation in the biosynthesis of spirotryprostatins (Scheme 8) [95], a class of pharmaceutically important indole alkaloids from Aspergillus fumigatus [96,97]. The first pathway involves cytochrome P450 FtmG from the fumitremorgin pathway, which catalyzes the formation of spirotryprostatin B (8b-1) through a radical-mediated mechanism involving sequential hydroxylation and dehydration of compound 8a. FtmG first abstracts the hydrogen of 8a to produce radical 8-I, followed by hydroxylation to form intermediate 8-II. Subsequent hydrogen abstraction of 8-II delivers radical 8-III, which isomerizes to radical 8-IV. Second hydroxylation followed by semi-pinacol rearrangement finally generates 8b. This pathway also produces spirotryprostatin G (8b-2), a novel intermediate identified for the first time, demonstrating the versatility of FtmG in substrate recognition. The other pathway involving flavin adenine dinucleotide (FAD)-dependent monooxygenase (FqzB) will be discussed later in Section 3.1.2.

In 2015, Sattely’s group discovered that cytochrome P450 CYP71CR1 from Brassica rapa (Chinese cabbage), as a member of a novel CYP71 subfamily, could transform the brassinin 9a to (S)-(−)-spirobrassinin 9d through unprecedented S-heterocyclization (Scheme 9) [98]. Through the isotope labeling study, the authors proposed that spirobrassinin is produced via an epoxide intermediate 9b, which is attacked by a S atom at the C3 position through S-heterocyclization to afford spirobrassinol 9c, followed by dehydration driven by indole aromaticity restoration.

Later, in 2021, Nakamura’s group pioneered a whole-organism biocatalytic approach using turnip as both a reaction medium and enzymatic source, producing (S)-(−)-spirobrassinin with a 91% ee value and (S)-(−)-5-methylspirobrassinin with a 24% ee value from L-tryptophan and 5-methyl-DL-tryptophan, respectively, through coordinated biocatalytic steps using endogenous enzymes (Scheme 9) [99]. More importantly, more unnatural derivatives of spirobrassinin were obtained with different alkyl groups substituted on the S atom. The authors not only achieved the biocatalytic production of (S)-(−)-spirobrassinin and its derivatives, but also developed a novel method using fresh turnip in line with green and sustainable chemistry principles. These advances provide crucial genetic and biochemical insights that enable targeted metabolic engineering of health-promoting indole-derived phytoalexins in edible crops.

In 2024, Yang’s group extended their previous work on phenol dearomatization (Section 2.1.1) by developing the biocatalytic radical dearomatization of indoles (10a) catalyzed by P450_rad1 (P411-CIS-A78C/V87L/L181V/P248T/I263G/F437A) (Scheme 10) [65]. Various spirocyclic dearomatized 3H-indoles (10b) bearing vicinal quaternary–quaternary carbon centers were successfully obtained in 39–90% yields with 64–94% ee via this powerful artificial metalloenzyme platform. This work overcomes longstanding challenges in stereocontrol in spirocyclic 3H-indoles featuring vicinal quaternary–quaternary stereocenters through radical-mediated dearomatization, and greatly expands the toolbox for three-dimensional heterocyclic scaffold construction in drug discovery and bioactive molecule development.

3.1.2. Dearomatization of Indoles via Flavin-Dependent Oxygenases

In 2020, Sherman and Williams’ group characterized NotI from Aspergillus protuberus and NotI’ from Aspergillus amoenus as flavin-dependent monooxygenases [100], which could catalyze the dearomatization of indoles for the formation of the spiro-oxindole moiety in the biosynthesis of notoamides, a class of fungal indole alkaloids with anticancer properties (Scheme 11) [101,102,103]. The K_m and v_max values of NotI were determined to be 37.4 μM and 1.19 μM/min, respectively. Moreover, these enzymes exhibited broad substrate tolerance, converting stephacidin A derivatives (11a, 11c, and 11e) into enantiomeric notoamides, such as (−)- and (+)-notoamide B (11b and 11d), as well as (+)-versicolamide B (11f). Mechanistic insight revealed a two-step process mediated by NotI/NotI’, involving the stereoselective epoxidation of 11a and subsequent semi-pinacol rearrangement of 11-I, which generates cation 11-II with the characteristic spirocyclic center. In the spirocyclization process, the conserved catalytic arginine (Arg195) was proposed to be critical for directing stereoselectivity via structural analysis.

In the same year, Sherman and coworkers elucidated the enzymatic mechanism of spiro-oxindole formation in paraherquamides (Scheme 12) [104], a class of antifungal and anthelmintic fungal indole alkaloids. The flavin-dependent monooxygenase PhqK is responsible for the stereoselective epoxidation of 12b and 12c, derived from preparahequamide (12a). Meanwhile, PhqK also catalyzes the following semipinacol rearrangement of 12d and 12e to generate the spirocyclic core of paraherquamide M (12f) and paraherquamide N (12g), which could be further functionalized to 12h-12k. The K_m and k_cat/K_m of PhqK for 12c were determined to be 19.4 μM and 0.05 μM⁻¹ min⁻¹, respectively. Apart from these primary substrates, several halogenated malbrancheamides were also compatible for generating spirocyclized products. Moreover, DFT calculations modeled by a guanidinium moiety for Arg192 revealed that the enzyme favored the 1,2-shift of the C3 hydroxyl intermediate. Using a combination of biochemical assays, structural biology, and computational modeling, the authors demonstrated that PhqK operates via a dual epoxidase–pinacolase mechanism.

Collectively, these studies elucidate the enzymatic mechanisms underpinning indole dearomatizative spirocyclization through epoxidation and semi-pinacol rearrangement catalyzed by flavin-dependent monooxygenases. It also expands the substrate scope to diverse heteroaromatic systems and provides a robust biocatalytic platform for constructing complex spirocyclic architectures with contiguous quaternary stereocenters, which advances synthetic strategies for bioactive alkaloids and underscores the versatility of enzyme engineering in addressing challenges in asymmetric catalysis.

In 2021, Gao and Zhu’s group elucidated the enzymatic mechanism of the flavin-dependent monooxygenase CtdE, which stereoselectively transformed indole derivatives 13a toward the 3S-spirooxindole (13b) in the biosynthesis of 21R-citrinadin A (13c) (Scheme 13) [105], an anticancer fungal indole alkaloid first isolated from a marine-derived Penicillium citrinum strain [106]. The k_cat/K_m of CtdE for 13a was determined to be 27.9 min⁻¹ mM⁻¹. Like PhqK, CtdE first catalyzes the epoxidation of the indole via the hydroxylation of 13a and the intramolecular addition of intermediate 13-I, followed by the regioselective collapse of the epoxide from the C3 side by protonation to give cation 13-III. Finally, the semipinacol rearrangement of 13-III generates the spirocyclic product. In contrast to NotI and PhqK as α-face epoxidases to form the 3R-spirooxindole, CtdE is identified as the β-face epoxidase to form a product with a different configuration (3S spirocyclization). During the catalysis, FAD adopts an “in” conformation on the β-face of the substrate, which was observed in the high-resolution crystal structures. By integrating structural biology, enzymology, and computational modeling, the multidisciplinary study advances the understanding of stereochemical control in complex natural product assembly, offering a platform for engineering biocatalysts to synthesize enantiopure spirooxindoles for drug discovery.

Beyond the P450 FtmG-mediated pathway to spirotryprostatin B (Section 3.1.1), the second pathway involves FAD-dependent monooxygenase FqzB in the biosynthesis of spirotryprostatins, in which FqzB catalyzes the epoxidation of fumitremomgin C (14a) to form 14b (Scheme 14) [95]. This triggers a semipinacol-type rearrangement, forming the spiro-carbon in spirotryprostatin A (14c) through a non-enzymatic dehydration step. The reaction mimics halogen-assisted synthetic strategies, but achieves stereochemical control via enzyme-mediated epoxide geometry.

Both FqzB (epoxidation) and FtmG (radical chemistry) exemplify how oxygenating enzymes diversify spirocyclic natural product scaffolds through distinct dearomatization strategies of aromatic indole rings. These studies underscore the role of enzyme promiscuity and pathway crosstalk in natural product diversification. By leveraging oxygenases from unrelated pathways, A. fumigatus expands its chemical repertoire, offering insights for the engineered biosynthesis of spirocyclic pharmaceuticals.

3.1.3. Dearomatization of Indoles via Uncharacterized Enzymes

In 2018, Lin’s group achieved the biosynthesis of FR900452 [107] and two new analogs in Streptomyces sp. B9173, as well as maremycin G (15) featuring the distinctive spirocyclic skeleton (Scheme 15) [108]. Although FR900452 and its analogs lack spirocyclic structures, they might share the same node intermediate as maremycin G due to the similar building blocks. Thus, the authors proposed the biosynthetic pathways of these products based on the gene annotations and homolog comparisons. Notably, within the mar gene cluster, all the products originate from common precursors: a MarM-tethered linear Trp-Cys dipeptide (15-I) and a MarL-tethered enolate (15-II). The key innovation lies in the discovery that MarP selectively generates an enolate at C-5′, directing a nucleophilic attack on the dipeptide to form 15-III. In contrast, maremycin G arises via spontaneous enolate formation at C-2′, leading to spirocyclization through dearomatization of the indole ring, a similar transformation to the above-reviewed examples. This work not only deciphers the enzymatic logic behind divergent indole alkaloid biosynthesis, but also showcases the role of SnoaL-like proteins in controlling dearomatization-driven skeletal rearrangements, providing a blueprint for engineering spirocyclic natural products and pharmaceutically relevant scaffolds.

In 2019, Lopes’ group realized the biosynthesis of unnatural spirocyclic oxindole alkaloids in Uncaria guianensis through precursor-directed strategies feeding fluorinated and methylated tryptamine analogs to plantlets (Scheme 16a) [109]. The biosynthesis involves a stereospecific oxidation of ajmalicine (16a), followed by a dehydration process to deliver 16-II. Then, pinacol–pinacolone rearrangement as a key dearomatization process transforms the aromatic indole moiety into the spirocyclic framework of 16b and 16c. Although the complete biosynthetic pathway remains uncharacterized, ¹³C-labeled feeding experiments support a proposed pinacol–pinacolone rearrangement to form the spiro-oxindole scaffold, which is frequently occurring in limonoid biosynthesis [94]. Notably, fluorinated and methylated precursors exhibit superior incorporation efficiency, underscoring the flexibility of U. guianensis enzymes in transforming unnatural substrates to unnatural oxindole alkaloids (Scheme 16b). This work highlights a pivotal dearomatization step central to the formation of the spiro-oxindole scaffold with the rational design of “new-to-nature” oxindole alkaloids via metabolic engineering, advancing both natural product chemistry and drug discovery paradigms.

In 2024, Qi’s group discovered six novel prenylated indole diketopiperazine alkaloids, talaromyines A-F, from the marine-derived fungus Talaromyces purpureogenus SCSIO 41,517 (Scheme 17) [110]. Among the newly isolated natural products, talaromyines A-E feature unprecedented spirocyclic indole diketopiperazines formed via a key dearomatization-driven pathway. They are biosynthesized through the condensation of L-tryptophan and L-alanine, diverging from the canonical L-tryptophan-L-proline scaffold observed in known spirotryptostatins [111]. Similarly to the flavin-dependent oxygenases discussed in Section 3.1.2, the formation of their spirocyclic cores involves a stereospecific oxidative dearomatization of talaromyines F (17a) via epoxidation to form the intermediate 17b, followed by a semipinacol rearrangement, a critical step that facilitates the transition from planar aromaticity to a complex spiro system of talaromyines A (17c-1). Then, the common intermediate 17c-1 diversifies into talaromyines B-E (17c-2 to 17c-5) sharing the same spirocyclic skeleton. Notably, talaromyines B, D, and E exhibit selective inhibition against protein tyrosine phosphatases (TCPTP and MEG2), while talaromyines D and E demonstrate mild cytotoxicity against H1975 and HepG-2 cancer cell lines. This work not only reveals a novel biocatalytic strategy for dearomatization in fungal secondary metabolism, but also provides structurally unique scaffolds for cytotoxic activities, bridging chemical innovation with bioactivity-driven drug discovery.

3.2. Dearomatization of Pyrroles

Pyrroles represent privileged scaffolds in heterocyclic chemistry, with their five-membered, π-rich framework serving as the structural foundation for numerous natural products, pharmaceuticals, and materials [112,113]. The aromatic stabilization of the pyrrole nucleus, however, renders its selective dearomatization exceptionally challenging. Yet, access to partially saturated pyrroline and pyrrolidine scaffolds is of paramount importance, as these motifs confer enhanced three-dimensionality, new stereocenters, and valuable functional handles for medicinal chemistry [114,115,116].

3.2.1. Dearomatization of Pyrroles via P450

In 2024, Yang’s group further extended their metalloenzyme platform to additional N-heterocycles, such as the pyrrole motif in 18a (Scheme 18) [65]. More importantly, the enantiodivergent dearomatization of pyrrole could be achieved through the careful regulation of the enzymes. P450_rad2 (P411-CIS-L75A/L181A/A82V) furnished (R)-18b in 94% yield with 2320 TTN and an 82% ee value, while P450_rad3 (P411_Diane2-W263I/G268A/P327T/V328A/E267L) delivered (S)-18b in 76% yield with 3230 TTN and an 84% ee value.

3.2.2. Dearomatization of Pyrroles via Uncharacterized Enzymes

Kosinostatin (KST), as one of the aromatic polyketide natural products, was originally isolated from Streptomyces aureofaciens in the 1950s [117], and subsequently reisolated from marine Micromonospora sp. TP-A0468 in Japan and S. violaceusniger HAL64 in Egypt. KST contains a unique spiro pyrrolopyrrole moiety, which exhibits potent inhibition against Gram-positive bacteria, along with moderate activity against Gram-negative bacteria and yeasts. Although its biosynthetic pathway attracts the interest of scientists, and several elegant studies have been reported to elucidate its biosynthetic pathway, the complete pathway still remains elusive. In 2013, Tang’s group [118] revealed that the biosynthesis of KST (19d) involves uncharacterized enzyme-catalyzed coupling and dearomative spirocyclization from 19a and PCP-tethered building block 19b, as well as late-stage functionalization of key quinocycline spiro intermediate 19c (Scheme 19) [119]. This study elucidates a novel biosynthetic strategy for constructing the spirocyclic core of KST, where an unprecedented dearomative spirocyclization process converges independently biosynthesized polyketide and pyrrolopyrrole building blocks, circumventing conventional oxidative C-C bond cleavage mechanisms. The discovery of this unique dearomative pathway, facilitated by a hybrid PKS/NRPS system utilizing unconventional precursors and tryptophan-like enzymes for pyrrolopyrrole dearomatization, significantly expands the enzymatic logic of forming complex spirocyclic natural products.

4. Biocatalytic Dearomative Spirocyclization Reactions of Oxa-Aromatic Rings

Dearomatization of Furans

Furans are versatile five-membered heterocycles distinguished by their high electron density and pronounced aromatic stabilization, with widespread occurrence in biomass-derived feedstocks and bioactive natural products [102,120,121]. Oxa-spirolactones containing spirocyclic ketal and γ-butenolide motifs, which could be formed through the dearomatization of furans, are widely found in biologically active natural products and pharmaceuticals [122,123]. Owing to the relatively weak aromatic stabilization energy, furans behave more like an alkene, enol ether, or conjugated diene, rendering them highly susceptible to dearomatization.

However, knowledge of the biocatalytic dearomatization strategy of furans is quite scarce. In 2023, Deska and Liu’s group designed a one-pot multienzymatic pathway comprising chloroperoxidase (CPO), a glucose oxidase (GOx), and an alcohol dehydrogenase (ADH) to construct the chiral spirolactone building blocks (Scheme 20) [124]. CPO with oxygenase activities on many kinds of aromatic compounds (20a) was employed to catalyze the enzymatically driven Achmatowicz oxidation to produce the intermediate 20b, and GOx was used for the in situ generation of hydrogen peroxide from glucose and air to provide hydrogen peroxide as the cosubstrate for CPO. Then, ADH oxidized the intermediate 20b to the final δ-lactone 20c, simultaneously regenerating NAD(P)H through the reduction of the sacrificial electron acceptors, such as acetone. This exclusive biocatalytic dearomatization of furans towards spirolactones, including natural spirocyclic lactones lanceolactone A and crassalactone D in 30–81% yields, 1–5.7:1 dr values, and 17–99% ee values, shows an excellent demonstration of the application value of biocatalytic method development.

5. Conclusions and Perspective

Biocatalytic dearomative spirocyclization emerges as a powerful paradigm for constructing stereochemically intricate spirocycles with sustainable precision. Enzymatic systems, including cytochrome P450s, multicopper oxidases, and engineered metalloenzymes, are reviewed to demonstrate the unparalleled stereocontrol and functional group tolerance, enabling the transformation of inert aromatic scaffolds (phenols, indoles, furans, etc.) into enantioenriched spiroarchitectures under mild conditions.

Through iterative rounds of structure-guided mutagenesis and directed evolution, the advantages of biocatalysis could be reflected in expanding substrate scopes well beyond native metabolites, enabling the dearomative spirocyclization of diverse non-natural phenols, indoles, pyrroles, and furans with impressive turnover numbers, especially for well-investigated P450 enzymes. In many cases, altering just a handful of active-site residues not only enhances enantioselectivity but can also invert the stereochemical outcome entirely, while judicious remodeling of binding pockets has endowed enzymes with reactivities, such as enantiodivergent radical cyclizations and radical–polar crossovers. Nevertheless, outcomes for sterically shielded substrates remain largely empirical, constrained by our limited capacity to predict how distal mutations cooperatively modulate active-site topology. Engineered metalloenzymes unlock radical-mediated spirocyclizations unattainable in nature, yet controlling the trajectory of fleeting radical intermediates demands deeper understanding of confinement effects within synthetic active sites. Moreover, enzyme engineering might address the scalability bottlenecks, such as enzyme stability under industrial conditions and cost-effective cofactor regeneration, which will be critical for transitioning these methodologies from lab-scale to industrial applications.

Mechanistic breakthroughs, such as radical relay cascades and two-component cyclopropanase systems, illuminate pathways for uncommon spiropropane formation. Nevertheless, mechanistic elucidation of enzymatic processes and confinement effects using advanced in situ characterization coupled with multiscale quantum mechanics/molecular mechanics (QM/MM) simulations is still highly desirable. It could help scientists to develop more novel biocatalytic reactions, such as hybrid systems merging enzymatic precision with chemocatalytic or photoredox catalysis to expand reaction horizons.

By synergizing biocatalytic precision with synthetic versatility, synthetic biology holds immense potential to redefine spirocyclic drug discovery and sustainable chemical manufacturing. Bridging enzymatic logic with cutting-edge technologies, such as AI-driven enzyme design and biohybrid materials, will unlock new reactivity frontiers, ultimately advancing green chemistry and precision synthesis in the era of carbon neutrality.