Penicillides from Penicillium and Talaromyces: Chemical Structures, Occurrence and Bioactivities

Penicillide is the founder product of a class of natural products of fungal origin. Although this compound and its analogues have been identified from taxonomically heterogeneous fungi, they are most frequently and typically reported from the species of Talaromyces and Penicillium. The producing strains have been isolated in various ecological contexts, with a notable proportion of endophytes. The occurrence of penicillides in these plant associates may be indicative of a possible role in defensive mutualism based on their bioactive properties, which are also reviewed in this paper. The interesting finding of penicillides in fruits and seeds of Phyllanthus emblica is introductory to a new ground of investigation in view of assessing whether they are produced by the plant directly or as a result of the biosynthetic capacities of some endophytic associates.


Introduction
Fungi in the genus Penicillium (Eurotiomycetes, Aspergillaceae) represent one of the most exploited sources of chemodiversity, with a multitude of structural models and compound classes having been reported since the discovery of mycophenolic acid by Gosio [1].Many species of these fungi have been described from all ecological contexts and geographical areas in the world, which are able to produce blockbuster drugs such as penicillin and compactin [2].However, the recent affirmation of the principle 'one fungus, one name' in taxonomy [3] and the widespread employment of biomolecular markers for a more accurate identification have brought the separation of Penicillium species having symmetrical biverticillate conidiophores into the genus Talaromyces (Eurotiomycetes, Trichocomaceae) based on phylogenetic reconstructions [4].
Fifty years ago, a Japanese researcher reported on the finding of a new secondary metabolite from an isolate of Penicillium sp., which was named penicillide (11-hydroxy-3-[(1S)-1-hydroxy-3-methylbutyl]-4-methoxy-9-methyl-5H,7H-dibenzo[b,g] [1,5]dioxocin-5one) (Figure 1) [5].This product is the founder of a class of bioactive products characterized by 2,4-dihydroxybenzilic alcohol and 2-hydroxy-4-methoxy-benzoic acid moieties linked together by ether and ester bonds, forming a typical 8-membered heterocycle in the place of the 7-membered one usually found in depsidones [6].Rather than being one of a kind, more analogues of penicillide were later found by other independent research groups in the early 1990s; possibly due to difficulties in accessing the pertinent literature at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].
at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].
This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.at that time, some of these compounds were given the unrelated names of purpactins [6] and vermixocins [7].This paper offers an overview on the research activity which has been developed in the last five decades on penicillides, with reference to their occurrence in nature as secondary metabolites of Penicillium and Talaromyces species, as well as to their biological properties and possible biotechnological applications.

Chemical Structures
With reference to the general molecular scaffold outlined above, the A ring is invariable in all the known penicillides with the exception of hydroxypenicillide, while the C ring presents a side chain of five carbons at C-3. Substitutions in this chain vary among the ten analogues which have been identified from these fungi so far (Table 1).Besides searching databases, such as PubMed, Google Scholar and Web of Science, an accurate analysis of the available data based on the chemical structure was carried out using Scifinder.With reference to the structures in Table 1, it must be noted that isopenicillide ( 9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5′-hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13].With reference to the structures in Table 1, it must be noted that isopenicillide ( 9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5′-hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13].With reference to the structures in Table 1, it must be noted that isopenicillide ( 9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5′-hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13].With reference to the structures in Table 1, it must be noted that isopenicillide ( 9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5′-hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13].With reference to the structures in Table 1, it must be noted that isopenicillide (9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5′-hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13].With reference to the structures in Table 1, it must be noted that isopenicillide (9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5′-hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13].With reference to the structures in Table 1, it must be noted that isopenicillide (9) is not an isomeric form of penicillide (1) since it has an additional hydroxyl group on the side chain in ring C; rather, it is an isomer of 5 ′ -hydroxypenicillide (7).Moreover, the denomination adopted for compounds 7 and 8 can be misleading, and it is incoherent because compound 8 has an additional hydroxyl group on the A ring.

Occurrence
Conforming to the above-introduced separation of symmetrically biverticillate species in Talaromyces, most of the strains producing penicillides are found to belong to this genus (Table 2).Similar to Penicillium, the latter taxon has been characterized as an outstanding source of chemodiversity [8][9][10][11], exhibiting some peculiar biosynthetic models which have been only or predominantly found in these fungi, such as the funicones [12,13]. 1 This strain should be more correctly ascribed to Talaromyces based on an updated GenBank blast of its deposited ITS sequence. 2This strain was originally identified as Penicillium asperosporum.
In light of the above, it is quite possible that the isolates provisionally identified as Penicillium sp.actually belong to Talaromyces.This can be directly verified for strain MA-37 through a blast in GenBank of the reported rDNA-ITS sequence [17], while the doubt remains in the other cases.However, apart from these incompletely identified strains, the fact that penicillides have been reported in three unrelated species, namely P. chrysogenum P. montanense and P. simplicissimum, represents an indication that these compounds may be common secondary metabolites in Penicillium, too.Yet, the connotation of penicillides as products characterizing the biosynthetic abilities of Talaromyces is well supported by the remark that as many as 18 species of this genus are listed in Table 1 as documented sources.
The assumption that penicillides are typical fungal products has been impaired by an intriguing report on the extraction in equable amounts of penicillide, purpactin A and the novel analogue 1 ′ S-11-dehydroxypenicillide from fruits of the Indian gooseberry (Phyllanthus emblica) (Malpighiales, Phyllanthaceae) [66].This finding has been followed by the detection of penicillide in seeds of the same plant by an independent research group [67].Indeed, recent progresses in natural product research have disclosed the ability of taxonomically diverse endophytic fungi to synthesize compounds originally characterized from plants [68], which, in many cases, has postulated the transfer of gene clusters encoding for their synthesis [69,70].By extension of this concept, horizontal gene transfer (HGT) could also have operated in the case of P. emblica, but in which direction?Did the plant acquire the genetic base from any endophytic fungus, or rather, did it occur that several endophytes borrowed this gene cluster from the plant and later spread to the various ecological contexts from which the penicillide producers have been reported?Alternatively, the extraction from P. emblica could be consequential to its production and accumulation in the plant tissues as resulting from the biosynthetic capacities of one or more endophytic associates, as has been demonstrated in the case of defensive mutualists of ryegrass and other plants [71].Indeed, both Talaromyces and Penicillium species are at the forefront among endophytic fungi, having been reported from many and diverse plants in all environments [11,72], and the hypothesis that a systematic association established with P. emblica could lead to the accumulation of penicillides in its fruits and seeds deserves to be verified.The very recent report of the species Talaromyces atroroseus and Penicillium choerospondiatis from gooseberries [73] opens a new ground of investigation in this respect.
Finally, while this manuscript was in preparation, another paper was published reporting on the detection of purpactin A in the aqueous extract obtained from the Chinese vine Sargentodoxa cuneata (Ranunculales, Lardizabalaceae) [74]; undoubtedly, this last finding reinforces the need to more thoroughly examine plants as possible sources of penicillides.

Biological Properties
Biological properties have been essentially evaluated for the two most common products, penicillide and purpactin A; the available data are shown in Table 3.
It is generally agreed that the bioactivities of microbial secondary metabolites are related to their competitive interactions in the biocenosis.In this respect, both penicillide and purpactin A exhibit moderate antibacterial properties, which anyway could have an ecological impact considering the possible synergism with other antimicrobial compounds produced by Penicillium and Talaromyces species.Although various strain panels have been used in the antibacterial assays, the same MICs for both products generally resulted when they were concomitantly tested (e.g., against Klebsiella pneumoniae, Pseudomonas aeruginosa and Vibrio parahaemolyticus) [12].The highest activities were detected for penicillide at 0.78 µg mL −1 against the methicillin and oxacillin resistant strain ATCC 43300 of Staphylococcus aureus [27], while purpactin A was active at a 5-fold higher concentration (4 µg mL −1 ) against E. coli [26].In the latter study, a positive correlation was observed between the acetylation of the side chain and antibacterial efficacy [26].
Antifungal activity was evaluated for penicillide against the opportunistic pathogenic Basidiomycete Cryptococcus neoformans, but effectiveness only resulted at a quite high concentration [20].High active concentration also resulted in assays of this compound against brine shrimps [4], while it displayed antifouling properties against Balanus amphitrite at a lower concentration than purpactin A [34].Conversely, antiplasmodial effects were observed at lower concentrations for purpactin A [20].
Assays for cytotoxic activity yielded quite variable results.In fact, on Hep G2 (hepatocellular carcinoma) cells, cytotoxicity was higher for penicillide, as assessed in two different laboratories [6,15], while incongruent values were obtained in two other laboratories for purpactin A on MCF-7 (breast cancer) cells [17,20].In assays carried out at the same laboratory, it was slightly higher for penicillide against KB (epidermoid carcinoma) cells and slightly higher for purpactin A against Vero (kidney epithelium of African green monkey) cells [20].Finally, the same values were measured for the two compounds in terms of incorporation of uridine, thymidine and valine in P388 (murine leukemia) cells [25].
Table 3. Biological activities of penicillide and purpactin A.

Biological Activity Concentration
Results and Further Details Ref.

Biosynthesis of Penicillide and Purpactin A
Penicillides are structurally related compounds biosynthetically derived from the polyketide pathway.It has been proposed that the skeleton of these natural products, characterized by 2,4-dihydroxybenzilic alcohol acid and 2,4-dihydroxybenzoic acid moieties linked by ether and ester bonds, derives from chrysophanol anthrone after the decarboxylation of a single octaketide chain.The subsequent oxidative cleavage of the B ring of chrysophanol anthrone is assumed to generate a benzophenone intermediate which is then oxidized to produce the tricyclic skeleton (spirobenzofuran-l,2 ′ -cyclohexa-3 ′ ,5 ′ -diene-2 ′ ,3dione).Further structural modifications (i.e., prenylation, acetylation and methylation) of this latter compound generate purpactin B which is synthesized as an intermediate; then, the oxidation of its hydroxymethyl group leads to purpactin A [78,79].

Related Products
Several products of Penicillium and Talaromyces are structurally related to penicillides, but they cannot be strictly considered members of this class of natural products because of the lack of the typical structural features (Figure 3).This is the case of purpactins B and C which, despite having been named as purpactin A homologues, are characterized by

Related Products
Several products of Penicillium and Talaromyces are structurally related to penicillides, but they cannot be strictly considered members of this class of natural products because of the lack of the typical structural features (Figure 3).This is the case of purpactins B and C which, despite having been named as purpactin A homologues, are characterized by spirobenzofuran-l,2 ′ -cyclohexa-3 ′ ,5 ′ -diene-2 ′ ,3-dione instead of dibenzo[b,g][1,5]dioxocin-5(7H)-one.Considering the biosynthetic pathway in Figure 2, purpactin B is an intermediate in the biosynthesis of purpactin A, which is formed by the acetylation and rearrangement of the former compound.The common biosynthetic origin of purpactins could explain why these compounds were frequently reported from the same fungal sources [6, 37,52].
Molecules 2024, 29, x FOR PEER REVIEW 10 of 14 intermediate, followed by esterification [46].Secopenicillides A and B were isolated for the first time from P. simplicissimum [23], while secopenicillide C was detected as a new compound from T. pinophilus, and its production was found to be enhanced in co-culture with Trichoderma harzianum [45].It must be noted that all these metabolites were detected along with other known penicillides.

Conclusions
As can be concluded from the available information on their occurrence and structures examined in this review, species of Penicillium and Talaromyces from various ecological contexts represent the main natural source of penicillides.However, the reported extraction of these depsidones from two unrelated plant species introduces the opportunity for further investigations in the aim to assess whether, or not, this biosynthetic aptitude results from any endophytic associates.In the latter alternative, further studies are also advisable to investigate if the inter-organism spread derives from the transfer of a pertinent gene cluster through HGT, which may eventually confer an ecological advantage depending on the bioactive properties of these compounds.
The broad-range laboratory investigations carried out so far have pointed out the multifaceted bioactivities of penicillides.Particularly, the best antibiotic effects have been documented against methicillin and oxacillin resistant S. aureus [27], deserving further assessments in the perspective to integrate the available panel of these highly demanded drugs.In this respect, a relevant contribution is expected by the pharmaceutical industry through the realization of more potent semi-synthetic derivatives, as a follow-up of the pioneering research activity carried out in some laboratories [80,81].Considering the increasing impact of pulmonary diseases, a notable contribution in the achievement of new drugs is also expected from further investigations on the above-outlined effects on mucus hypersecretion [42].Finally, following the preliminary evidence obtained for depsidones [82,83], more valuable opportunities could result from the exploration of the antiviral effects of these compounds.It has been proposed that secopenicillides A, B and C could share the same biosynthetic route of penicillide and purpactin A [46].However, these compounds, and the related 7-Oacetylsecopenicillide C [17], are characterized by the presence of a diphenylether moiety which does not conform to the genuine structural model of penicillides.In particular, Wu and coworkers proposed that secopenicillide A originates from purpactin C which is converted to purpactin A via reduction of the aldehydic group to form an alcohol intermediate, followed by esterification [46].Secopenicillides A and B were isolated for the first time from P. simplicissimum [23], while secopenicillide C was detected as a new compound from T. pinophilus, and its production was found to be enhanced in co-culture with Trichoderma harzianum [45].It must be noted that all these metabolites were detected along with other known penicillides.

Conclusions
As can be concluded from the available information on their occurrence and structures examined in this review, species of Penicillium and Talaromyces from various ecological contexts represent the main natural source of penicillides.However, the reported extraction of these depsidones from two unrelated plant species introduces the opportunity for further investigations in the aim to assess whether, or not, this biosynthetic aptitude results from any endophytic associates.In the latter alternative, further studies are also advisable to investigate if the inter-organism spread derives from the transfer of a pertinent gene cluster through HGT, which may eventually confer an ecological advantage depending on the bioactive properties of these compounds.
The broad-range laboratory investigations carried out so far have pointed out the multifaceted bioactivities of penicillides.Particularly, the best antibiotic effects have been documented against methicillin and oxacillin resistant S. aureus [27], deserving further assessments in the perspective to integrate the available panel of these highly demanded drugs.In this respect, a relevant contribution is expected by the pharmaceutical industry through the realization of more potent semi-synthetic derivatives, as a follow-up of the pioneering research activity carried out in some laboratories [80,81].Considering the increasing impact of pulmonary diseases, a notable contribution in the achievement of new drugs is also expected from further investigations on the above-outlined effects on mucus hypersecretion [42].Finally, following the preliminary evidence obtained for depsidones [82,83], more valuable opportunities could result from the exploration of the antiviral effects of these compounds.

14 Figure 2 .
Figure 2. The proposed biosynthetic schemes of penicillide and purpactin A.

Figure 2 .
Figure 2. The proposed biosynthetic schemes of penicillide and purpactin A.

Figure 3 .
Figure 3. Compounds structurally and/or biosynthetically related to penicillides reported from Penicillium and Talaromyces.

Figure 3 .
Figure 3. Compounds structurally and/or biosynthetically related to penicillides reported from Penicillium and Talaromyces.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.

Table 2 .
Penicillium and Talaromyces species/strains reported as producers of penicillides.