Talarodiolide, a New 12-Membered Macrodiolide, and GC/MS Investigation of Culture Filtrate and Mycelial Extracts of Talaromyces pinophilus

Talarodiolide, a new 12-membered macrodiolide, was isolated and characterized from the culture filtrate of strain LT6 of Talaromyces pinophilus. The structure of (Z)-4,10-dimethyl-1,7-dioxa-cyclododeca-3,9-diene-2,8-dione was assigned essentially based on NMR and MS data. Furthermore, several known compounds were isolated and identified in the crude extract of the culture filtrate and mycelium of this strain. EI mass spectrum at 70 eV of all isolated metabolites was acquired and compiled in a custom GC/MS library to be employed to detect metabolites in the crude extracts.


Introduction
With a widespread occurrence in very diverse environmental contexts, from the soil to the sea [1-3], the species Talaromyces pinophilus (=Penicillium pinophilum) (Eurotiales: Trichocomaceae) has received increasing attention in mycological research for its ability to act as a fungal antagonist and plant-growth promoter [1, 4,5], and for possible biotechnological applications based on the production of enzymes [6,7] and bioactive metabolites [8][9][10].
Two strains (LT4 and LT6), possibly deriving from the same wild clone since they were both recovered from the rhizosphere of a tobacco plant cropped near Lecce (Apulia, Southern Italy), have been particularly studied in our laboratories after they were shown to produce a novel fungitoxic and cytostatic compound named 3-O-methylfunicone (OMF) [1,11]. OMF is part of a homogeneous family comprising about 20 structurally related secondary metabolites which have been mainly characterized from cultures of Talaromyces strains [12]. It has notable antitumor properties based on several biomolecular mechanisms of action resulting from a series of preclinical assays [13][14][15][16][17]. Although it represents the main extrolite produced by our strains, other funicone variants have been occasionally extracted [18,19], indicating that some factors act during the culturing cycle which may lead to the accumulation of intermediate or side products. Within our recent activity aiming at the standardization of OMF production, additional compounds were detected from cultures of strain LT6. Among them, a new product with an unusual structure for a natural compound, namely talarodiolide, was purified from its culture filtrates. Furthermore, the present paper reports findings from the talarodiolide, was purified from its culture filtrates. Furthermore, the present paper reports findings from the first GC/MS-based investigation on secondary metabolites in culture filtrate and mycelial extracts of T. pinophilus.
Compound 1, isolated as amorphous solid, has a molecular weight of 224 m/z accounting for a molecular formula of C12H16O4 and the index of hydrogen deficiency is five as deduced from ESI-TOF MS. The 1 H-NMR spectrum (Table 1 and Figure S1) revealed one broad singlet methyl, one broad triplet and one triplet in aliphatic region, and a broad singlet of olefinic signals. In the 13 C-NMR spectrum (Table 1 and Figure S5), only six carbon signals were present indicating a highly symmetric molecule. The 1 H and 13 C resonances of 1 were assigned by combination of COSY and HSQC experiments. The COSY experiment showed homocorrelations among the olefinic proton at δ 5.84 with the methyl at δ 2.03 and methylene at δ 2.40, the latter of which was also correlated with methylene at δ 4.40. The HSQC ( Figure S3) spectrum showed correlations of methyl at δ 2.03 with carbon at δ 22.4, two methylenes at δ 2.40 and 4.40 with carbons at δ 29.2 and 65.8, respectively, and one methine at δ 5.84 with carbon 116.8. The carbons at δ 164.6 and 157.7 were assigned to a carboxyl Compound 1, isolated as amorphous solid, has a molecular weight of 224 m/z accounting for a molecular formula of C 12 H 16 O 4 and the index of hydrogen deficiency is five as deduced from ESI-TOF MS. The 1 H-NMR spectrum (Table 1 and Figure S1) revealed one broad singlet methyl, one broad triplet and one triplet in aliphatic region, and a broad singlet of olefinic signals. In the 13 C-NMR spectrum (Table 1 and Figure S5), only six carbon signals were present indicating a highly symmetric molecule. The 1 H and 13 C resonances of 1 were assigned by combination of COSY and HSQC experiments. The COSY experiment showed homocorrelations among the olefinic proton at δ 5.84 with the methyl at δ 2.03 and methylene at δ 2.40, the latter of which was also correlated with methylene at δ 4.40. The HSQC ( Figure S3) spectrum showed correlations of methyl at δ 2.03 with carbon at δ 22.4, two methylenes at δ 2.40 and 4.40 with carbons at δ 29.2 and 65.8, respectively, and one methine at δ 5.84 with carbon 116.8. The carbons at δ 164.6 and 157.7 were assigned to a carboxyl group and substituted sp 2 carbon, respectively. According to the structure in the HMBC ( Figure S4) spectrum, the H 2 -6/H 2 -12 protons were correlated to the C-8/C-2 at 164.4, C-4/C-10 at 157.7 and C-5/C-11 at 29.2. Furthermore, the H 3 -13/H 3 -14 protons were correlated to C-3/C-9, C-4/C-10 and C-5/C-11 carbons. The analysis of NOESY ( Figure S6) spectrum evidenced NOE of the methyl at δ 2.03 and olefinic H-3 proton indicating a Z configuration at double bond.  Symmetric macrodiolides have been reported from many natural sources, and displayed some interesting effects, such as antibacterial, antifungal and cytotoxic activities ( [22] and literature therein). However, in the light of the current knowledge, no 12-membered macrodiolide has been isolated from natural sources so far.

GC/MS Analysis
In this study, an EI mass spectrum at 70 eV of all isolated metabolites was acquired and compiled in a custom MS target library to be employed to detect metabolites separated in the crude extracts. GC/MS measurements served several purposes within our strategy. First, when the mass spectrum of the metabolite could be retrieved from a MS database, the acquired mass spectrum provided a definitive proof of its identity, as in the case of cyclo-(S-Pro-R-Leu).
When no mass spectrum satisfactorily matches the acquired mass spectrum could be inferred from a database, the unknown metabolite had to be otherwise identified (e.g., via ESI-TOF MS and 1 H/ 13 C-NMR mono-and bi-dimensional), but interpretation of the acquired mass spectrum served as a guide in the identification process by setting restrictions on possible structures.
In all cases, the acquired mass spectrum was incorporated into the custom MS library to be used for interpreting GC/MS measurements to be performed directly on samples of mycelium and culture filtrates extracts obtained. Table 2 shows data collected via GC/MS of the identified metabolites.    Apart from the isolated metabolites, Figure 2b shows the presence of some fatty acids and their methyl esters in the mycelial extract. In fact, due to the high sensitivity of this technique, GC/MS was able to detect them, combining the retention indices and the reference mass spectra gathered in NIST 14 Mass Spectral library (2014) [27]. Within the framework of the overall strategy, a very important outcome of the procedures arises from the fact that crude extracts were analyzed by GC/MS to check the presence of the isolated metabolites. Notwithstanding some metabolites were not isolated from the culture filtrate, AMDIS attributes peaks in the TIC, as in the case of penicillide, vermistatin and penisimplicissin. Hence, GC/MS analysis is very useful in assessing the possible diversity in the pattern of metabolites extracted from the different sources. With exception of talarodiolide, 1-glycerol-linoleate and the diketopiperazines, all metabolites were detected in both crude extracts, while fatty acids and their esters (10)(11)(12)(13) are present in the mycelial extract only. This is in line with the reported occurrence of the latter compounds in the cell membrane of fungi [28].

General Experimental Procedures
Optical rotations were measured in CHCl3, CH3OH, and C2H5OH on a Jasco P-1010 digital polarimeter; 1 H and 13 C-NMR spectra were recorded at 400/100 MHz in CDCl3 or in CD3OD on Bruker (Bremen, Germany) spectrometers. The same solvent was used as internal standard. 2D NMR experiments were performed using Bruker microprograms. ESI-TOF mass spectra have been measured on an Agilent Technologies QTOF 6230 in the positive ion mode (Milan, Italy).
GC/MS measurements were performed with an Agilent 6850 GC equipped with an HP-5MS capillary column (5% phenyl methyl polysiloxane stationary phase) and the Agilent 5973 Inert MS detector (used in the scan mode). Helium was employed as the carrier gas, at a flow rate of 1 mL/min. The injector temperature was 250 °C and during the run a temperature ramp raised the column temperature from 70 °C to 280 °C: 70 °C for 1 min; 10 °C min −1 until reaching 170 °C ; and 30 °C min −1 until reaching 280 °C . Then it was held at 280 °C for 5 min. The electron impact (EI) ion source was operated at 70 eV and at 200 °C. The quadrupole mass filter was kept at 250 °C and was programmed to scan the range 45-550 m/z at a frequency of 3.9 Hz. Apart from the isolated metabolites, Figure 2b shows the presence of some fatty acids and their methyl esters in the mycelial extract. In fact, due to the high sensitivity of this technique, GC/MS was able to detect them, combining the retention indices and the reference mass spectra gathered in NIST 14 Mass Spectral library (2014) [27].
Within the framework of the overall strategy, a very important outcome of the procedures arises from the fact that crude extracts were analyzed by GC/MS to check the presence of the isolated metabolites. Notwithstanding some metabolites were not isolated from the culture filtrate, AMDIS attributes peaks in the TIC, as in the case of penicillide, vermistatin and penisimplicissin. Hence, GC/MS analysis is very useful in assessing the possible diversity in the pattern of metabolites extracted from the different sources. With exception of talarodiolide, 1-glycerol-linoleate and the diketopiperazines, all metabolites were detected in both crude extracts, while fatty acids and their esters (10)(11)(12)(13) are present in the mycelial extract only. This is in line with the reported occurrence of the latter compounds in the cell membrane of fungi [28].

General Experimental Procedures
Optical rotations were measured in CHCl 3 , CH 3 OH, and C 2 H 5 OH on a Jasco P-1010 digital polarimeter; 1 H and 13 C-NMR spectra were recorded at 400/100 MHz in CDCl 3 or in CD 3 OD on Bruker (Bremen, Germany) spectrometers. The same solvent was used as internal standard. 2D NMR experiments were performed using Bruker microprograms. ESI-TOF mass spectra have been measured on an Agilent Technologies QTOF 6230 in the positive ion mode (Milan, Italy).
GC/MS measurements were performed with an Agilent 6850 GC equipped with an HP-5MS capillary column (5% phenyl methyl polysiloxane stationary phase) and the Agilent 5973 Inert MS detector (used in the scan mode). Helium was employed as the carrier gas, at a flow rate of 1 mL/min. The injector temperature was 250 • C and during the run a temperature ramp raised the column temperature from 70 • C to 280 • C: 70 • C for 1 min; 10 • C min −1 until reaching 170 • C; and 30 • C min −1 until reaching 280 • C. Then it was held at 280 • C for 5 min. The electron impact (EI) ion source was operated at 70 eV and at 200 • C. The quadrupole mass filter was kept at 250 • C and was programmed to scan the range 45-550 m/z at a frequency of 3.9 Hz.

Culture Filtrate Preparation
Liquid cultures were prepared by inoculating mycelial plugs from actively growing cultures of strain LT6 in 1 L-Erlenmayer flasks containing 500 mL potato-dextrose broth (PDB, Himedia) which were kept in darkness on stationary phase at 25 • C. After 21 days, cultures were filtered at 0.45 µm, and the culture filtrates were concentrated in a lyophilizer until reduction to 1/10 of the starting volume. The mycelial cake floating on the broth was collected separately and stored at −20 • C.

Extraction and Isolation of Metabolites from Liquid Cultures
The freeze-dried culture filtrates (6 L) were dissolved in 600 mL of pure water (pH 4) and extracted with same volume of CHCl 3 for three times. The organic extracts were combined, dried on Na 2 SO 4 , and evaporated under reduced pressure to give a yellowish oil residue (75.3 mg).

Extraction and Isolation of Metabolites from Mycelium
Fresh mycelium was homogenized in a mixer with 440 mL of MeOH-H 2 O (NaCl 1%) mixture (55:45, v/v). The suspension was stirred in the dark at room temperature for 4 h. After this period, the suspension was centrifuged (40 min at 7000 rpm, 10 • C) and separated from the supernatant. The residue was overnight extracted with 250 mL of the mixture reported above. The suspension was centrifuged, and both supernatants were combined for the subsequent extraction with CHCl 3 . The organic extracts were combined, dried on anhydrous Na 2 SO 4 , and evaporated under reduced pressure yielding crude extract as a red oil (230.2 mg). The extract was fractionated by CC on silica gel (1.5 × 40 cm i. d.), eluting with CHCl 3 /iso-PrOH (97:3, v/v). The last fraction was eluted with MeOH. Seven homogeneous fraction groups were collected (A 16.0 mg, B 16.4 mg, C 12.2 mg, D 14.2 mg, E 9.8 mg, F 29.1 mg, G 66.2 mg). The residue of fraction B was identified as OMF (2). Fraction C was purified by TLC on silica gel eluted with n-hexane/acetone (6:4, v/v) to afford a further amount of OMF (5.6 mg), a crystalline compound identified as vermistatin (6, 1.5 mg, R f 0.37 on TLC on silica gel eluent n-hexane-acetone (6:4, v/v)), and an amorphous solid identified as penisimplicissin (7, 0.5, mg, R f 0.29 on TLC on silica gel eluting with n-hexane-acetone (6:4, v/v)). Fraction D was purified using the same condition described for C giving penicillide (8, 6.9, mg, R f 0.29 on TLC on silica gel eluent n-hexane-acetone (6:4, v/v)) as amorphous solid. Finally, the residue of fraction F was further purified on TLC on silica gel eluting with CHCl 3 /iso-PrOH (9:1, v/v) giving 1-glycerol-linoleate (9, 1.5 mg, R f 0.40 on TLC on silica gel eluent CHCl 3 /iso-PrOH (9:1, v/v)) as soft solid.