Demystifying racemic natural products in the homochiral world

Abstract

Natural products possess structural complexity, diversity and chirality with attractive functions and biological activities that have significantly impacted drug discovery initiatives. Chiral natural products are abundant in nature but rarely occur as racemates. The occurrence of natural products as racemates is very intriguing from a biosynthetic point of view; as enzymes are chiral molecules, enzymatic reactions generating natural products should be stereospecific and lead to single-enantiomer products. Despite several reports in the literature describing racemic mixtures of stereoisomers isolated from natural sources, there has not been a comprehensive review of these intriguing racemic natural products. The discovery of many more natural racemates and their potential enzymatic sources in recent years allows us to describe the distribution and chemical diversity of this 'class of natural products' to enrich discussions on biosynthesis. In this Review, we describe the chemical classes, occurrence and distribution of pairs of enantiomers in nature and provide insights about recent advances in analytical methods used for their characterization. Special emphasis is on the biosynthesis, including plausible enzymatic and non-enzymatic formation of natural racemates, and their pharmacological significance.

Keywords: Biosynthesis; Natural products; Pharmacology; Structure elucidation.

Fig. 1. Distribution and diversity of racemates in nature according to up-to-date published data.

Labels of chemical structures are coloured to highlight the sources in nature: green for plants, purple for microorganisms and orange for animals. a | Distribution of naturally occurring racemates that have been discovered across different kingdoms: 66% in plants, 25% in microorganisms and 9% in animals. b | Distribution of racemic compounds according to their main chemical classes or biosynthetic origins and across different source kingdoms. Alkaloids = nitrogen-containing compounds including amides; polyketides = keto-methylene chains (cyclic or not) including phloroglucinols; terpenes = mainly linear (or monocyclic) sesquiterpenes but also monoterpenes, diterpenes and sesterterpenes; miscellaneous = compounds belonging to other classes not listed in the row. Supplementary Fig. S1 shows the structures of 778 compounds and 248 reference articles.

Fig. 2. Main groups and examples of natural racemates.

Labels of chemical structures are coloured to highlight the sources in nature: green for plants, purple for microorganisms and orange for animals. a | Racemic alkaloids peganumaline A (1), tengerensine (2), oxazinin A (3), nigegladine A (4), bialternacin A (5), eudistidine C (6), lihouidine (7), xylaridine A (8), macathiohydantoin H (9) and purealin (10). b | Racemic polyketides kingianin E (11), sancti C (12) and tsavoenone C (13). c | Racemic meroterpenes drychampone A (14) and hypulatone A (15). d | Phenylpropanoid-condensed racemates (E)-mesocyperusphenol A (16) and aspongdopamine B (17). e | Racemic rotamers parvistemin A (18), selaginellin A (19) and (S)-dioncophyllacine A (20). f | Other phenolics and miscellaneous paulownione C (21), cratosumatranone D (22), regiolone (23), (4R*,5S*,6S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5-methyl-2-cyclohexen-1-one (24), (4S*,5S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one (25), penicilliode A (26), penicilliode B (27) and cnidimonin A (28).

Fig. 3. Analysis approaches for natural racemates.

Labels of chemical structures are coloured to highlight the sources in nature: green for plants and purple for microorganisms. A | Structures of analysed racemates peplidiforone B (29), stritidas A–C (30–32), xanthiifructin B (33), xanthiifructin C (34) and penicilliode C (35). B | Analysis approaches for natural racemates, from extraction and separation to structure elucidation with absolute configuration determination. Detailed reviews on these techniques are published elsewhere^,,,. Ba | Extraction/preparation: during dryness, sun light/energy and high temperature (>24 °C) could induce transformations including racemization. Uses of liquid nitrogen should avoid such transformations leading to artefacts, allowing extraction of genuine natural racemates. Bb | Separation process: basic chromatographic methods such as column chromatography and thin layer chromatography can afford unnatural racemic mixtures (artefacts) from complex extracts. High performance liquid chromatography (HPLC), online liquid chromatography and liquid chromatography–mass spectrometry (LC-MS) improve the isolation avoiding possible racemization due to silica–sample or inter-sample interactions. Bc | Structure determination: the gross structure of a racemate could be achieved by means of nuclear magnetic resonance (NMR), mass spectrometry and LC-MS/tandem, HPLC coupled with ultraviolet photodiode array (HPLC-UV), infrared, nuclear Overhauser effect spectroscopy/rotating frame Overhauser effect spectroscopy and X-ray diffraction (XRD) providing required data to define the relative configuration. Bd | Enantiomeric ratio determination/resolution: the resolution of racemates could be achieved by various techniques, including chiral HPLC (Table 1). Be | Absolute configuration determination: several methods were commonly used including electronic circular dichroism (ECD) and vibrational circular dichroism (VCD), experimental and calculated spectra using density functional theory (DFT), Mosher’s method (chemical reaction), NMR (¹H, ¹³C), quantum mechanics (for example, DP4+ method), X-ray diffraction, microcrystal electron diffraction (MicroED) and crystalline sponge X-ray diffraction (CS-XRD).

Fig. 4. Proposed biosynthetic basis to some racemates.

Labels of chemical structures are coloured to highlight the sources in nature: green for plants. a | Hypothetical biosynthesis of (±)-paracaseolide A (36) proposed by Wang and Hoye through biomimetic synthesis. A key step is the non-enzymatic and spontaneous [4 + 2] Diels–Alder dimerization (cycloaddition) of the butanolide precursor at ambient temperature. b | Proposed biosynthesis of cycloneolignans (±)-piperhancins B/A (37/38) by Yang et al.. Alkylphenol precursors undergo a radical coupling to produce neolignan intermediates. The key non-enzymatic event features the intramolecular [2 + 2] photocycloaddition of the cycloenone with a double bond (red) in both intermediates to yield 37 and 38. c | Biosynthetic basis to racemic pyrrolidinone-containing flavonoids (39) as proposed by Wang et al.. This bioprocess involves chalcone synthases (CHS) and epimerases as bioengineers. Epimerases such as chalcone isomerases (CHI) have been characterized in the conversion of chalcone to racemic flavanones/flavones. It could also be hypothesized that considering the fermentation process of Wang et al. to produce pyrrolidinone catechin derivatives, an epimerase likely catalyses the cyclization of 4-oxobutanamide to pyrrolidinone. The condensation of flavanone/flavone and pyrrolidinone intermediates afford 39. d | Hypothetical intervention of Diels-alderases in cycloaddition. Intermolecular Diels-alderases similar to the recently discovered Morus alba Diels-alderase (MaDA) could catalyse the formation of racemic natural products such as 40 through [4 + 2] cycloaddition^,. e | Proposed oxidative coupling to (±)-hypulatone B (41). The terpene and phloroglucinol derivatives undergo an enzymatic oxidative coupling (catalysed by an oxidase) followed by subsequent cyclization and oxidation to give 41. f | Proposed enzymatic biosynthesis of racemic lignans in the presence or absence of dirigent proteins (DIRs). Hypothetical intervention of two DIRs of opposite stereoselectivities could yield racemate (±)-syringaresinol (42) preferentially with regioselectivity and stereoselectivity^,,.

Fig. 5. Chemical structures of racemic natural products with biological activities.

Labels of chemical structures are coloured to highlight the sources in nature: green for plants, purple for microorganisms and orange for animals. a–h | Compounds within the same shape are the most active racemates (evaluated by the values of IC₅₀, EC₅₀, minimum inhibitory concentration (MIC) or MIC₈₀) of the entitled pharmacological activities as reported in the literature for antineoplastic (lihouidine (7), involucratusin E (43), pyrisulfoxin D (44)) (part a), antifungal (selaginisoquinoline A (45), citridone A (46)) (part b), antiviral (integrastatin A (47), tiegusanin N (48)) (part c), antibacterial (purealin (10), cratosumatranone D (22)) (part d), antiprotozal (cordiaquinol J (51), gaudichaudianic acid (52), brachangobinan B (53)) (part e), anti-inflammatory (paulownione C (21), eupatonin A (49)) (part f), antioxidant ((4R*,5S*,6S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5-methyl-2-cyclohexen-1-one (24), (4S*,5S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one (25), 2-acetamido-3-(2,3-dihydroxybenzoylthio)propanoic acid (50)) (part g) and antidiabetic (quinolactacin B (54), oxypenicinoline A (55)) (part h) activities.