Fumarprotocetraric Acid Lactones Identified and Characterized by UHPLC ‐ PDA / ESI / ORBITRAP / MS / MS from the Antarctic Lichen Cladonia metacorallifera

Lichens are symbiotic organisms between algae and fungi, which are makers of secondary compounds named as lichen substances. Hyphenated techniques have significantly helped natural product chemistry, especially UHPLC/ESI/MS/MS  in  the  identification, separation, and  tentative characterization of secondary metabolites from natural sources. Twenty‐five compounds were de‐ tected from the Antarctic lichen Cladonia metacorallifera for the first time using UHPLC‐PDA/ESI/Or‐ bitrap/MS/MS. Compounds 5 and 7 are reported as new compounds, based on their MS/MS frag‐ mentation routes, and considered as fumarprotocetraric acid derivatives. Besides, ten known phe‐ nolic identified as orsellinic acid, ethyl 4‐carboxyorsellinate, psoromic acid isomer, succinprotoce‐ traric acid, siphulellic acid, connorstictic acid, cryptostictic acid, lecanoric acid, lobaric acid and gy‐ rophoric acid are noticed for the first time in the Cladonia genus.

Analyses of crude extracts were performed using chromatographic methods such as high-performance liquid chromatography (HPLC) in combination with various detection spectroscopic methods [8][9][10][11][12]. However, HPLC coupled to UV spectroscopy provide limited structural information when compounds are unknown [13]. Mass spectrometry (MS) provides rapid identification of unknown compounds while electro-spray ionization (ESI) is the most successful interface used in LC-MS coupled to time of flight (ToF), quadrupole or orbitrap [14]. Currently, tandem mass spectrometers operate within 1-5 ppm mass accuracy with high resolution and the quality of MS/MS spectra depend on the parameters such as precursor ion isolation width, intensity threshold, collision energy, total acquisition speed, accumulation time on MS/MS spectrum, and others [14]. Therefore, LC-MS is a dominant analytical technique to identify secondary metabolites, and unknown constituents, in plant extracts giving information on elemental composition and structural fragmentation patterns, but are unable to distinguish positional isomers [14]. The hyphenated Q-exactive instrument with a high-resolution collision cell has significantly contributed in the area of lichens chemistry for the identification of unknown compounds based on structural characterization by MS/MS [8,12,15].
Herein, we describe the chromatographic fingerprinting of the Antarctic lichen Cladonia metacorallifera by UHPLC/PDA/ESI/MS/MS, which revealed the presence of two new metabolites.

Collection and Identification of Lichen
A 5 g aliquot of C. metacorallifera was collected in "Peninsula Fildes", King George Island, Antarctica during March, 2020. Vouchers specimens (reference numbers: CM-010420) were deposited at the Extreme natural product laboratory, Universidad de Chile.

Sample Preparation
A 1.0 g aliquot of C. metacorallifera was extracted with methanol (3 times, 10 mL each time, using a sonicator 30 min). The organic solutions were evaporated to obtain 18.0 mg of dark green gummy extract.

Instrument
The Thermo Scientific Dionex Ultimate 3000 UHPLC system, hyphenated with a Thermo Q exactive focus, was already reported [8,12]. For the analysis, 2 mg of each lichen extracts were first dissolved in 2 mL of methanol, then filtered (PTFE filter) and, finally, 10 μL were injected in the instrument, with all specifications set as previously reported [8].

MS Parameters
The HESI parameters were setup as follows: gas flow rate 75 units; capillary temperature at 400 °C; auxiliary gas unit flow rate at 20 unit (N2); auxiliary gas heater temperature 500 °C; spray voltage 2500 V (for ESI − ); and S lens RF level 30. For the compounds of interest, a scan range of m/z 100-1000 was chosen; the automatic gain control (AGC) was set at 3 x 10 6 and the injection time was set to 200 ms. Collision energy was setup at 30 kV. Detection was performed based on calculated exact mass and on retention time. The mass tolerance window was set to 5 ppm for the two analysis modes.

Results and Discussion
The identification of unknown secondary metabolites in metabolomics is the main bottleneck on the structural interpretation based on MS/MS spectra, making the identification an arduous time-consuming task. As a strategy, many researches are limited to library matching, software, databases, algoritms and matching learning. Among the most known, both publicly and commercially, MS/MS software tools and databases including Mass Frontier, SmileMS, Mass ++ , XCMS 2 , NIST, METLIN, MassBank, MoNA, Wiley MSforID, CFM-ID, and MS-DIAL. For the generation of molecular structures of unknown compounds, it is important to follow the MS fragmentation ruler analysing the main small fragments. The biosynthetic pathway of secondary metabolite should also be considered if it applies [13,14].
During our study, twenty-five compounds were characterized for the first time in the methanolic extract of C. metacorallifera (Table 1) using UHPLC/PDA/ESI/MS/MS in negative ion mode. Among the identified compounds, two fumarprotocetraric acid lactones (peaks 5 and 7) are reported here for the first time. Their structures ( Figure 1) are proposed based on biosynthetic pathways, UV data and MS/MS fragmentations. identified by UHPLC/ESI/MS/MS from C. metacorallifera. It proposes interconversion mediated by natural oxidation from peak 13 (fumarprotocetraric acid) to peak 7 and after to peak 5, as supported by mass spectrometry and UV spectroscopy.
Peak 1 was identified as orsellinic acid (C8H8O4). The electrospray TOF mass fragmentation of this metabolite has been previously reported [10,15]. In the present study, orsellinic acid is identified, for the first time, in Cladonia genus. Orsellinic acid showed antioxidant activity in the β-carotene-linoleate model, and in the nitric oxide radical scavenging assay [6]. Peak 2 with an ion at m/z 239.0546 showed UV absorbance at max 249, 308 nm, which is a similar to the one showed by ethyl orsellinate (max 219, 265, 302 nm). Their fragmentation yielded a diagnostic MS ion at m/z 195.0649 and 149.0229 (see Supplementary Material). Considering that peak 2 displayed diagnostic losses of CO2 and C2H6O, we identified peak 2 as ethyl 4-carboxyorsellinate. From Picea schrenkiana has isolated this compound, and it is reported for the first time from lichens [16].
Peak 3 was characterized as squamatic acid (molecular anion at m/z 389.0860), whose fragmentation indicated diagnostic MS ions at m/z 209.0442 and 181.0490 [9,15]. This depside has been identified in Cladonia uncialis, C. crispata, C. bellidiflora, C. squamosa, and C. cenotea from Poland samples [17,18]. So far, squamatic acid has shown to be inactive against Staphylococcus aureus, Escherichia coli and Candida albican [19]. . This fragmentation indicated that peak 5 is a new compound related to fumarprotocetraric acid (peak 13). The key for this proposal implied the presence of the residue butendioic acid (C4H3O4 -; 115.0023) along with the residue C17H9O8 -(341.0303). Considering that peak 5 showed UV absorbance at max 215, 248 and 315 nm, which is similar to the one of peak 13 (max 212, 238, 314 nm), and according to biosynthetic considerations, we tentatively identified peak 5 as hydroxy fumarprotocetraric acid lactone ( Figure 2). Finally, the presence of fumarprotocetraric acid in Cladonia species supported this idea to be a derivative [17,18]. Peak 6 was identified as the depsidone connorstictic acid (m/z 373.0547) and its fragmentation produced ions at 329.0666 and 181.0555 [9,10,15]. To the best of our knowledge, there is no paper of connorstictic acid being present Cladonia genus. To support this fact, the presence of a related compound norstictic acid has been reported in the Cladonia genus [17,18]. No biological activity has been reported so far.
Peak 7 showed an [M-H] − ion at m/z 485.0336 (C22H13O13). According to their fragmentation pathway indicated that peak 7 is a new depsidone related to fumarprotocetraric acid (peak 13). The proposal implied the presence of three daughter fragments at m/z 369.0252 (C18H9O9 − ), m/z 115.0023 (butendioic acid; C4H3O4 − ) and the residue C17H9O7 − (325.0354). Besides, peak 7 showed UV absorbance at max 248 and 311 nm, which is similar to peak 13. Considering this evidence and based on the biosynthesis of lichen metabolites, we tentatively identified peak 7 as a fumarprotocetraric acid lactone ( Figure 3). It is well known that depsides as fumarprotocetraric acid are synthesized by the acetate-polymalonate pathway, which are formed by the bonding of two β-orcinoltype phenolic unit with ether, ester and C-C link [20]. In this context, both peak 5 and peak 7 derived from fumarprotocetraric acid are probably formed by lactonisation for peak 7 and oxidation and lactonisation for peak 5. Peak 8 with an ion at m/z 401.0514 was identified as siphulellic acid, which showed diagnostic daughter ions at m/z 123.0444, 149.0238, and 253.0505. To the best of our knowledge, this is the first information of its presence in the Cladonia genus [17,18]. No biological activity has been reported according to Scopus so far. Peak 9 was detected as lecanoric acid, which showed an [M-H] − ion at m/z 317.0668 according to published data [9][10][11]. Therefore, this is the first report of the presence of lecanoric acid in the Cladonia genus [17,18]. Lecanoric acid has displayed antioxidant, antibacterial and anticancer activities [5][6][7]. Peak 10 showed an [M-H] − ion at m/z 473.0701 (max 250 and 311 nm). Their fragmentation produced daughter ions at m/z 355.0441, 311.0545 and 117.0179 indicating that peak 10 is not confumarprotocetraric acid [17]. Considering these fragments, we identified as succinprotocetraric acid [21]  respectively. They were tentatively characterized as the fatty acids pentahydroxytetracosanoic acid (C24H48O7), tetrahydroxydocosanoic acid (C22H44O6), tetrahydroxytricosanoic acid (C23H46O6), dihydroxyoxodocosanoic acid (C22H42O5), and dihydroxyheptadecatrienoic acid (C17H28O4), respectively. Compounds related to these peaks have also been reported [17].
Some reports from the genus Cladonia including C. metacorallifera var. reagens KoLRI002260 as a rare lichen from Korean. They have been studied as mycobiont producing red naphthoquinonic pigments as cristazarin, and 6-methylcristazarin in culture media with 1% fructose and/or light [25]. Besides, these two compounds are produced by Cladonia cristatella mycobiont, but not in lichen thalli [26]. From Cladonia cariosa fumarprotocetraric acid was isolated, which showed 50 μM decrease on the β sheet content, demonstrated through Thioflavin T assays. In addition, the oligomers formed in that study with fumarprotocetraric acid were not toxic in N2a neuroblastoma cells [27]. A study based on UHPLC/QTOF/HRMS/MS of Algerian lichen Cladonia rangiformis detected the presence of the following thirteen metabolites highlighting ethyl orsellinate, squamatic acid, atranorin, evernic acid, usnic acid, roccellic acid, jackinic acid, norrangiformic acid, isorangiformic acid, and rangiformic acid [28]. In this context, our study detected twenty five metabolites using UHPLC/Q/Orbitrap/MS/MS. According to our search on SciFinder, the compounds identified with peaks 5 and 7 are reported here for the first time. As mentioned above, fumarprotocetraric acid display considerable pharmacological activity. Therefore, these two compounds could be isolated to evaluate their potential as pharmacological agent.