New Insights into Chemical and Biological Properties of Funicone-like Compounds

Funicone-like compounds are a homogeneous group of polyketides that, so far, have only been reported as fungal secondary metabolites. In particular, species in the genus Talaromyces seem to be the most typical producers of this group of secondary metabolites. The molecular structure of funicone, the archetype of these products, is characterized by a γ-pyrone ring linked through a ketone group to a α-resorcylic acid nucleus. This review provides an update on the current knowledge on the chemistry of funicone-like compounds, with special emphasis on their classification, occurrence, and diverse biological activities. In addition, their potential relevance as mycotoxins is discussed.


Introduction
Research on fungal secondary metabolites is mainly driven by remarks concerning their bioactive properties, which can either be inherent to their role in biocenotic interrelations or their effects on human health, the latter depending on either their possible accumulation in foodstuffs as mycotoxins, or eventual pharmaceutical relevance.
Funicones and structurally related compounds represent a homogeneous group of fungal polyketides that were initially characterized as determinants of the antagonistic abilities by the producers against other microorganisms, but were later found to possess remarkable biological properties that have promoted their consideration as drug prospects. Considering that these properties are partly based on observations concerning cytostatic and antiproliferative effects on human cells, these products should be also evaluated with reference to toxicological aspects related to possible contamination of foodstuffs by the producing fungi.
In light of the novel knowledge developed in over a decade since the publication of a previous review [1], this paper offers an update on the state of the art concerning occurrence, bioactivities, structural, synthetic, and biosynthetic aspects of funicone-like compounds.

Structures and Chemical Properties
Funicone-like compounds include natural products characterized by a molecular structure that is built on a γ-pyrone ring linked through a ketone group to a α-resorcylic acid nucleus. A total of 34 chemically defined compounds, which are referable to this basic structural model, have been identified and characterized so far. Among them, 13 can be considered true funicones because the typical moieties are present without alterations. The other compounds, showing modifications on the α-resorcylic acid nucleus, on the γ-pyrone ring, or on both moieties, are grouped in three subclasses, namely phthalide, furopyrone, and pyridone types, depending on peculiar substructural variations (Table 1).

Phthalide Type
The molecular structure of compounds in this subclass includes dimethoxyphthalide moiety ( Figure 4). Vermistatin (15) is the reference compoun group, deriving its name from a strain of Talaromyces flavus identified in anamorph Penicillium vermiculatum [47]. This metabolite was later isolated as a pro Pseudocercospora (=Mycosphaerella) fijiensis and wrongly reported as a new compou the name fijiensin [30]. This is not surprising because the attribution of different n the same chemical structure represents a recurring nomenclatural issue in natural research [54].
Neosarphenol (24) is an isomer of hydroxyvermistatin, which was named basis of the producing fungus, Neosartorya glabra (currently reclassified as As neoglaber), rather than with reference to its chemical structure [40].

Phthalide Type
The molecular structure of compounds in this subclass includes a dimethoxyphthalide moiety ( Figure 4). Vermistatin (15) is the reference compound of group, deriving its name from a strain of Talaromyces flavus identified in anamorphic-s Penicillium vermiculatum [47]. This metabolite was later isolated as a produc Pseudocercospora (=Mycosphaerella) fijiensis and wrongly reported as a new compound the name fijiensin [30]. This is not surprising because the attribution of different nam the same chemical structure represents a recurring nomenclatural issue in natural pro research [54].
Neosarphenol (24) is an isomer of hydroxyvermistatin, which was named on basis of the producing fungus, Neosartorya glabra (currently reclassified as Asperg neoglaber), rather than with reference to its chemical structure [40].

Phthalide Type
The molecular structure of compounds in this subclass includes a 4,6-dimethoxyphthalide moiety ( Figure 4). Vermistatin (15) is the reference compound of this group, deriving its name from a strain of Talaromyces flavus identified in anamorphic-stage Penicillium vermiculatum [47]. This metabolite was later isolated as a product of Pseudocercospora (=Mycosphaerella) fijiensis and wrongly reported as a new compound with the name fijiensin [30]. This is not surprising because the attribution of different names to the same chemical structure represents a recurring nomenclatural issue in natural product research [54].
Neosarphenol (24) is an isomer of hydroxyvermistatin, which was named on the basis of the producing fungus, Neosartorya glabra (currently reclassified as Aspergillus neoglaber), rather than with reference to its chemical structure [40].

Fungal Sources
The data summarized in Table 2 show that the fungi reported as funicone producer have been recovered from various substrates, often in association with plants or othe organisms, and in diverse environments, both terrestrial and marine. They are also quit heterogeneous in taxonomic terms, as they belong to two Ascomycetes classes: th Dothideomycetes and Eurotiomycetes. Members in the first class are sparse, bein ascribed to five orders, with each of them represented by a single strain. Even considerin

Fungal Sources
The data summarized in Table 2 show that the fungi reported as funicone producers have been recovered from various substrates, often in association with plants or other organisms, and in diverse environments, both terrestrial and marine. They are also quite heterogeneous in taxonomic terms, as they belong to two Ascomycetes classes: the Doth-ideomycetes and Eurotiomycetes. Members in the first class are sparse, being ascribed to five orders, with each of them represented by a single strain. Even considering the approximate taxonomic identification of three strains, which were only identified at the genus level, it is clear that funicone biosynthetic aptitudes occur among Dothideomycetes, and might be more widespread than currently known. Conversely, the Eurotiomycetes look to be much more abiding producers and taxonomically homogeneous, with about 31 strains belonging to three genera in two families. Again, some uncertainty in identification is to be noted, deriving from the absence of adequate support by sequencing of valid DNA markers, and by the provisional ascription to Penicillium sp. of some strains prior to the formal separation of the biverticillate Penicillium species and their assignment to the genus Talaromyces [55]. In this respect, the identification of strain IFM53375 as Penicillium simplicissimum was considered unreliable by leading taxonomists of these fungi based on a secondary metabolite profile more respondent to Talaromyces [55]. In another case, the producing strain (AF1-2) was not identified at all [26]; however, the image provided by the authors showing its bright yellow mycelium and the overlying green sporulation in culture on agar medium unequivocally allows its ascription to Talaromyces. In any case, species in the genus Talaromyces are the most typical producers of funicone-like compounds; with reference to the recent affirmation of the horizontal gene transfer concept [56,57], it cannot be excluded that the other fungal species may have occasionally acquired their funicone-biosynthetic abilities through this intriguing biological mechanism.   Talaromyces verruculosus CMI294548 Unknown Pakistan 15 [31] Recently, some independent studies have reported that production of funicone-like compounds may occur in co-cultures of various microbial strains (Table 3). Again, the Eurotiomycetes are more represented in these few studies, and can be thought to provide the genetic base for biosynthesis, which is eventually stimulated by the co-cultured strain in the course of an antibiotic struggle, as clearly demonstrated in the case of the pairing between Talaromyces siamensis and Phomopsis sp. (Sordariomycetes, Diaporthaceae) [43]. In two cases, the partner microbe was represented by Streptomyces strains (Actinomycetota), which are well-known for their capacity to modulate the metabolic potential of fungi [60].

Biosynthesis
The potential biosynthetic pathways of funicone-like compounds have been investigated by two independent research groups [21,28]. Figure 6 shows a possible scheme for each type of compound proposed in the previous section. Funicone-like compounds are epta and octaketides, originating from units of acetate-mevalonate. The main structural differences can be caused by the folding of the eptaketidic and octaketidic chains, which produce structures with a methyl or a propenyl group, respectively, on the γ-pyrone ring. The presence of an amino group in compounds belonging to the pyridone type suggests a possible transamination process during the biosynthesis of γ-pyridone. The origin of the phthalide type can be attributed to the lactonization of the carboxylic group in the α-resorcylic ring, with the hydroxyl group produced through the reduction of the exocyclic ketone group. Subsequent functional modifications (e.g., reduction, epoxydation, hydroxylation, methylation, and acetylation) are responsible for the ample structural variability observed in the group of funicone-like compounds.

Bioactivities
As previously introduced, the biological activity of funicones was initially evaluated with reference to antibiotic properties, generally evidencing poor effects against bacteria and yeasts, and more relevant activities against filamentous fungi. Subsequent investigations on antiproliferative properties against human cells line have become prevalent, underlining the potential of these compounds as antitumor drugs. Additional data have been gathered on the antiviral and the insecticidal properties, and the inhibitory effects toward several enzymes; moreover, some minor bioactivities have been described. The outcomes of this wide-ranging investigational work, as assessed in quantitative terms, are summarized in Table 4.

Bioactivities
As previously introduced, the biological activity of funicones was initially evaluated with reference to antibiotic properties, generally evidencing poor effects against bacteria and yeasts, and more relevant activities against filamentous fungi. Subsequent investigations on antiproliferative properties against human cells line have become prevalent, underlining the potential of these compounds as antitumor drugs. Additional data have been gathered on the antiviral and the insecticidal properties, and the inhibitory effects toward several enzymes; moreover, some minor bioactivities have been described. The outcomes of this wide-ranging investigational work, as assessed in quantitative terms, are summarized in Table 4.

Potential Role of Funicone-like Compounds as Mycotoxins
The applicative aspects of studies concerning fungal bioactive secondary metabolites involve their accumulation in food products and ensuing possible impact on consumers' health. Within the multitude of such compounds described so far, a very small number have been considered mycotoxins, based on the results of toxicological studies that noted their noxious effects on humans and animals [69]. This implies that a high number of compounds yet to be examined for these aspects may represent a potentially underestimated concern [70,71].
Funicones are one of the classes of fungal secondary metabolites for which very limited assessments have been carried out in this regard so far. Most of the producing species are not established pathogens of crops, with the exception of Pseudocercospora (=Mycosphaerella) fijiensis, a vermistatin producer that is known as the agent of black sigatoka disease of banana [72]. However, this is a leaf pathogen that is not known to spread to fruit, implying that it is unlikely that bananas can be contaminated with vermistatin. Nevertheless, a search for this compound in some fruit products carried out in Nigeria evidenced its presence at low levels (0.30 µg kg −1 ) in pineapple and mixed juices [73]. This is not at all surprising, as several Talaromyces spp. are commonly found in association with both healthy and diseased pineapples, including T. purpureogenus, T. funiculosus, and T. flavus, which may even survive pasteurization [74][75][76][77]. Conversely, a preliminary search carried out in Italy on marketed pineapple juices yielded negative results with reference to the eventual presence of 3-O-methylfunicone [78]. Recently, vermistatin was also detected in the analysis of grains used as cattle and poultry feed in Kenya [79], indicating that it may also occur as a cereal contaminant. Moreover, the finding of vermistatin as a product in co-cultures of strains of Alternaria alternata and Streptomyces exfoliatus [37] deserves to be further investigated, particularly in view of verifying the biosynthetic capacities by the first species. It is known as a pathogen of many crops and a saprophyte able to proliferate in several kinds of foodstuffs, with very important implications as a mycotoxin producer [80].
Considering the widespread endophytic occurrence of Talaromyces spp. [23,81], which are the dominant producers of funicones, the possible release of these compounds in plant products may arise during the postharvest phase, where the biosynthetic aptitudes can be boosted along with the saprophytic development. Recent reports of these fungi as postharvest pathogens concern T. albobiverticillius on pomegranate [82], T. rugulosus on grapes [83], T. minioluteus on onion bulbs and quince, orange, and tomato fruit [84], and both of the latter two species on pears [85]. Although none of these species are known to produce funicones, it is quite possible that other Talaromyces spp. producers of these compounds may affect fruit and other crop products, likewise documented for pineapple. This conclusion is supported by the finding of T. funiculosus as an agent of fruit core rot of peach [86].
Among the other funicone sources, Ramichloridium apiculatum, generally recorded as a soil saprophyte and only known as a producer of rapicone [27], was reported as an agent of sooty blotch and flyspeck of apples and pears in China [87], which may represent an indication for possible contamination of these fruits and their derived transformation products.

Conclusions
The present review provides an update on the recent developments concerning the distribution, chemical diversity, bioactivity and implications of occurrence of funicone-like compounds. The structures and properties of 34 funicone-like compounds extracted from different fungal species were reviewed. In particular, species in the genus Talaromyces seem to be the most typical producers of this group of secondary metabolites, soliciting consideration in view of possible chemotaxonomic implications.
In addition to outlining the general anti-inflammatory, antifungal, antiviral, and cytotoxic activities of these compounds, the available data indicate vermistatin as the most credited candidate to be added to the list of mycotoxins currently considered as food contaminants, with reference to its more common occurrence amongst the known funicone producers. The majority of these taxonomically heterogeneous fungi can perform its biosynthesis, implying that its presence in crop products may be more than just occasional. Whether this represents a threat or, conversely, can eventually be beneficial to consumers' health based on the described bioactivities, deserves thorough further assessments.