To explore
Rhodotorula species with bioactive and diverse metabolomes, we focused our isolation efforts on an underexplored deep-sea region of the Mid-Atlantic Ridge. An initial bioactivity screening against infectious human pathogens, plant pathogens and cancer cell lines of a small library of the six deep-sea
Rhodotorula strains cultured on solid PDA (high carbon medium) and solid WSP-30 (medium with high salt content), highlighted one strain,
R. mucilaginosa 50-3-19/20B, to exhibit interesting bioactivity and complex chemical UPLC-QToF-MS profiles (
Table S1,
Figure S1). Crude extracts of this strain exhibited anticancer activity only when cultured on PDA medium, while only in WSP-30 extracts antimicrobial activity was observed (
Table S1). No inhibition of the phytopathogens, yeasts, and dermatophytes could be detected (
Table S1); therefore, in this study, we focused only on positive bioactivity results, specifically the anticancer activity.
2.1. Culture Medium-Dependent Bioactivity Profiles and Metabolomics
We set out to examine the media-dependent metabolomic output of
R. mucilaginosa 50-3-19/20B when grown on PDA and WSP-30 media to identify metabolites that may contribute to the differential bioactivity. Therefore, we up-scaled our cultivation efforts and grew the strain on 200 PDA and 200 WSP-30 agar plates. The extraction with EtOAc gave notably different yields; 3.5 g of extract was obtained from the PDA culture and only 0.7 g of extract was recovered from the WSP-30 culture. The two extracts were subjected to a modified Kupchan liquid–liquid partition scheme to yield
n-hexane (K-hexane), dicholoromethane (K-DCM), and aqueous MeOH (K-MeOH) subextracts. The crude and the Kupchan subextracts were tested for their anticancer activity against two very common and deadly cancer types, using lung carcinoma cell line A549 and human breast cancer line MDA-MB-231). The antimicrobial potential was assessed against methicillin-resistant
Staphylococcus aureus (MRSA) and
Enterococcus faecium, whereas the general toxicity was assessed against a well-established non-cancerous human keratinocyte line HaCaT. The anticancer activity of
R. mucilaginosa 50-3-19/20B could be tracked to the PDA-K-DCM subextract (
Table 1), which exhibited 73% inhibition of the breast cancer line MDA-MB-231 at a test concentration of 100 µg/mL and 99% inhibitory activity against the lung carcinoma cell line A549. No toxicity against the non-cancerous human keratinocyte cell line HaCaT was detected in any of the extracts (
Table 1). After fractionation, both K-hexane subextracts showed antimicrobial activity against both pathogens. The WSP-30-K-hexane extract showed specifically high inhibition values (90% and 98%) against MRSA and
E. faecium, respectively, while the PDA-K-hexane extract inhibited MRSA at 74% (
Table 1) at the test concentration of 100 µg/mL.
Next, the Kupchan subextracts were profiled via UPLC-QToF-MS/MS and the data were used to construct a molecular network through the Global Natural Product Social (GNPS) online platform [
13]. This open-access tool does not only facilitate rapid and automated dereplication of chemical profiles against a number of MS/MS fragment databases, but also serves as a useful comparative tool to investigate the chemical diversity of different samples. Therefore, we utilized molecular networking to compare the metabolomes of the differentially active subextracts to identify the compound classes that are responsible for their anticancer and/or antimicrobial activities. The molecular network (MN,
Figure 1) consisted of three molecular families (clusters of more than two nodes). Dereplication workflows on GNPS and UNPD
in silico database matching did not provide meaningful hits. Only one singleton with
m/
z 295.2271 [M + H]
+ was putatively identified as an unsaturated fatty acid, 9-oxo-octadeca-10,12-dienoic acid (9-OxoODE,
Figure 1 and
Figure S3). Thus, we largely relied on manual dereplication against the Dictionary of Natural Products (
http://dnp.chemnetbase.com), DEREP_NP [
14], and SciFinder (
https://scifinder.cas.org/) in order to annotate the MN. The largest molecular family in the MN could be identified as glycolipids via manual dereplication against relevant literature data [
15,
16,
17]. These glycolipids belong to a class of polyol esters of fatty acids (PEFAs) with differential degrees of acetylation and have been proposed as biosurfactants [
17]. The cluster consisted of 100 ions (
Figure 1), which were predominantly expressed in PDA subextracts (K-DCM and K-hexane). 99 of the PEFA ions were detected in the PDA extracts (K-DCM and K-hexane) of which 68 ions were unique to PDA extracts (K-DCM, K-hexane, and K-MeOH) and 31 were shared between WSP-30 and PDA extracts (predominantly K-hexane subextracts); only one node (
m/
z 697.5872 [M + Na]
+) in the glycolipid cluster originated solely from the WSP-30-K-hexane subextract. Only two low molecular weight ions of this cluster were detected in the K-MeOH subextracts (
m/
z 373.1109 [M + Na]
+ and
m/
z 373.1111 [M + Na]
+). In total, 36 nodes were unique to the PDA-K-DCM subextract and were thus annotated to potentially cause anticancer activity and are presented as larger nodes in
Figure 1.
The two other main clusters could not be annotated and might constitute of new metabolites. Furthermore, based on the MN analysis, it did not become clearly apparent which compounds contributed to the antimicrobial activity. None of the main molecular families and only several singletons originated exclusively from the WSP-30-K-hexane subextract. It is likely that the antimicrobial activity is caused by synergy of multiple lipids. Hence, we decided to focus on the K-DCM subextract of the yeast cultured on PDA medium and its anticancer activity as the MN did not reveal any explicit antimicrobial target compounds in the K-hexane subextract.
2.2. Full Genome Sequencing and Analysis of Biosynthetic Gene Clusters
The
de novo genome of marine
R. mucilaginosa 50-3-19/20B was assembled. The genome assembly has a size of 20.02 Mb and an N50 of 498.8 kb (
Figure S2). It possessed 2.14% of repetitive element contents (
Figure S2). This genome size is comparable to other known
Rhodotorula genomes (
Table 2) as reported in previous studies [
18,
19]. Based on AntiSMASH 4.0 analysis, we discovered 19 biosynthetic gene clusters (BGCs) in total. Out of these, 15 BGCs are unknown, while two terpene BGCs and one NRPS, as well as one fatty acid based BGC were identified (
Table 2). Numbers and types of BGCs in the different
Rhodotorula genomes showed high inter-species similarities. All other
Rhodotorula strains exhibited also one NRPS cluster; two terpene BGCs were specifically associated with
R. mucilaginosa strains and
R. taiwanensis MD1149 had three such BGCs. All
Rhodotorula genomes, except for
Rhodotorula sp. FNED7-22, contained a fatty acid BGC. We propose that this BGC is most likely involved in the synthesis of PEFAs that we detected through the metabolomics analysis and we thus further investigated this gene cluster.
We identified BGC3 (
Figure 2) as the putative fatty acid producing cluster. BGC3 represents the only fatty acid producing gene cluster in the genome. Its core enzyme is a malonyl CoA-acyl carrier protein transacylase, which is a type of fatty acid synthase [
20]. This synthase is a potential producer of fatty acids as it is characterized by the presence of a huge multifunctional enzyme complex with several protein domains (
Figure 2A), like (i) the fatty acid synthase meander beta sheet domain (Pfam ID—PF17951.1); (ii) N-terminal half of MaoC dehydratase (PF13452.6); (iii) MaoC like domain (PF01575.19); (iv) acyl transferase domain (PF00698.21); (v) two fatty acid synthase subunit alpha acyl carrier domains (PF18325.1); (vi) fatty acid synthase type I helical domain (PF18314.1); (vii) beta-ketoacyl synthase, N-terminal domain (PF00109.26); (viii) beta-ketoacyl synthase, C-terminal domain (PF02801.22); and (ix) 4′-phosphopantetheinyl transferase superfamily domain (PF01648.20). These domains have previously been reported from several fatty acid producing core enzymes [
21].
Table 2 depicts the presence of a single BGC (Cf_fatty_acid) in several
Rhodotorula strains. Phylogenetic analysis further suggests that a single core enzyme is conserved across several
Rhodotorula spp. (
Figure 2B).
This is the only bona fide identified fatty acid producing biosynthetic gene cluster in different
Rhodotorula strains (
Table 2) suggesting that at least the fatty acid moiety of PEFA production is dependent on this BGC. However, the exact role of this BGC in the production of PEFA remains unclear.
2.3. Identification and Characterization of an Exo-Inulinase Enzyme
The knowledge on the PEFA biosynthetic pathway and its components is limited. A recent study suggested that an exo-inulinase enzyme holds key roles in the PEFA production via a mannitol biosynthesis pathway using the polysaccharide inulin as the carbon source [
23]. Hence, we used the recently identified protein sequence of exo-inulinase enzyme from
R. paludigena P4R5 [
23] to detect enzymes in the marine
R. mucilaginosa 50-3-19/20B genome aided by BLAST suite [
22]. Herein, we report the existence of an exo-inulinase gene (as g1629.t1) and its genomic locus including its flanking genes deduced from the marine
R. mucilaginosa 50-3-19/20B genome (
Table S2).
The corresponding peptide (g1629.t1) is 679 amino acids long and it has a molecular weight of 71.24 kDa. It harbors a disordered region of 159 amino acid in the N-terminus, plus two glycoside hydrolase family 32 (GH32) domains as Glyco_hydro_32N (Pfam ID—PF00251.20) and Glyco_hydro_32C (PF08244.12) mapped from 178–485 and 506–673 amino acids, respectively (
Figure 3 and
Table S2). Hence, this protein is a member of the glycoside hydrolase family 32 (GH32). We performed homology detection in NCBI and found several fungi to possess homologs of this putative exo-inulinase enzyme (
Table S3). This result demonstrates that the exo-inulinase enzyme (g1629.t1) has orthologs in several fungi with annotation as either GH32 protein or
β-fructofuranosidase (
Table S3). This putative exo-inulinase harbors known conserved motifs of exo-inulinases [
23,
24,
25] as
189FMNDPNGC
189,
209Q,
250FS
251,
315RDP
317, and
366ECP
368 (
Figure 3, marked by stars, numbering according to amino acid numbering of exo-inulinase enzyme of marine
R. mucilaginosa). Out of these residues, three amino acids
192D-
316D-
366E form the conserved catalytic triad (marked by red stars in
Figure 3) with
192D serving as the nucleophile,
316D as a stabiliser of the transient state and
366E as the acid-base catalyst.
We further constructed a three-dimensional structural model of the enzyme using the crystal structure of fructofuranosidase from
Schwanniomyces occidentalis [
26] as template (PDB ID-3u75.1, chain A). This template shows 37.89% sequence identity with the identified exo-inulinase protein. It features the typical
β-propeller with the conserved catalytic triad as
192D-
316D-
366E, which is known in several members of the GH32 protein family [
23,
26,
27].
2.4. In-Depth Metabolome Analysis and Anticancer Activity
In order to analyze the chemical constituents of the PDA-derived DCM extract of
R. mucilaginosa 50-3-19/20B, we further fractionated the DCM subextract by C18-MPLC to obtain 31 fractions. Fractionation allows detection of minor ions that are often masked in crude and subextracts by UPLC-QToF-MS/MS. Therefore, we also performed anticancer screening (
Table S4) and bioactive molecular networking (BMN) of the fractions (
Figure 4). In BMN, features (in this case peak area) in the UPLC-QToF-MS/MS chromatograms are correlated with bioassay results to determine a bioactivity score. This score can be visualized through different node sizes in the MN (the larger the node, the higher its bioactivity score) and thus allows prediction of molecules or compound classes that contribute to the bioactivity.
The BMN revealed 39 different molecular clusters (
Figure 4). The GNPS dereplication workflow revealed a diketopiperazine cluster, which included cyclo-(Leu-Phe) (
m/
z 261.1304 [M + H]
+) that could be matched with high confidence against the GNPS library (
Figure S4). This compound class, which is likely linked to the identified NRPS gene cluster (
Section 2.2.), was not detected in our initial comparative MN analysis (
Figure 1) and thus highlights the effectiveness of fractionation to enhance the detection of minor ions in extracts. A second cluster was found to comprise indole-containing compounds, such as
DL-indole-3-lactic acid (
m/
z 206.0829 [M + H]
+) (
Figure 4 and
Figure S5) and methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (
m/
z 220.0983 [M + H]
+). These compounds clustered with other larger ions with molecular weights above 400 Da, these ions are potentially representing additional small peptidic compounds synthesized via the NRPS pathway.
A small cluster for steroids, which are truncated terpenes could also be putatively annotated (
Figure 4 and
Figure S6). Also associated with terpene biosynthesis are carotenoids that represented the largest cluster in the BMN. Molecular weights of several nodes were in agreement with those reported for carotenoids derived from microorganisms including
Rhodotorula sp. [
28]. These were tetrahydroxydihydrolycopene (
m/
z 603.5312 [M + H]
+), dihydroxylycopene (
m/
z 571.6362 [M + H]
+), and cryptoxanthin (
m/
z 553.5593 [M + H]
+), however, the observed fragmentation patterns did not provide definite confirmation of their presence in the extract. Further GNPS annotations included C17-sphinganine, (
m/
z 288.2906 [M + H]
+) belonging to the aminolipid cluster, (
Figure 3 and
Figure S7) and the fatty acid derivative 9,10-epoxy-12-octadecenoic acid (EpOMe,
m/
z 279.2330 [M-H
2O + H]
+) (
Figure 4 and
Figure S8).
As already observed in the comparative metabolomics studies of the Kupchan subextracts of the PDA and WSP-30 cultures, the metabolome of
R. mucilaginosa 50-3-19/20B was dominated by acetylated polyol (sugar alcohol) esters of fatty acids (PEFAs), hence we established a rapid identification approach for this compound class. Because several PEFAs have the same molecular weight, MS/MS fragmentation data is highly valuable in differentiating the PEFAs. We first used a list of theoretical PEFA molecular weights that allowed immediate determination whether the PEFA contained a mannitol or arabitol polyol head group (
Table S5). Second, the number of acetyloxy groups in the compound was established based on the number of MS/MS fragments that indicated the loss of 60 Da (one acetyloxy group). In PEFAs, acetylation can occur on the polyol unit as well as C-3 position of the fatty acid, thus the fragment produced by the natural loss of the fatty acid becomes highly useful in the identification of the PEFA. A table with the observed fragment ions for the residual polyols with different degrees of acetylation can be found in the supplementary information (
Table S6). It should be considered that there are several isomers of moderately acetylated PEFAs and the position of the acetylation could not be established through this approach.
In the molecular network of the fractionated K-DCM subextract (
Figure 4), the GNPS algorithm clustered the PEFAs depending on their degree of acetylation. We identified 49 different PEFA derivatives in
R. mucilaginosa 50-3-19/20B containing either mannitol or arabitol as sugar alcohols (
Table S7). Nine clusters had a mannitol head group while five contained arabitol (
Figure 4).
Interestingly, the MN and manual MS/MS fragment analyses revealed that the PEFAs in
R. mucilaginosa 50-3-19/20 had fatty acid chain lengths of C10, C12, C14, C16, C18, and C20 with either 3-hydroxy or 3-acetoxy substitutions, while prior reports of PEFAs derived from
Rhodotorula spp. only reported chain lengths of C12, C14, C16, C18, and C20 [
16,
17,
23,
29]. Therefore, this is the first report of a PEFA containing decanoic acid (C10). Arabitol-tetraacetate-3-methoxy-tetradecanoate, a PEFA derivative bearing a 3-methoxy fatty acid was also detected.
The anticancer activity of the K-DCM subextract was tracked to the non-polar MPLC fractions (F21–F24,
Table S4) containing complex mixtures of PEFAs and 22 nodes showed strong correlations (
r > 0.5) with bioactivity (
Figure 4). Therefore, we decided to purify PEFAs from the active
R. mucilaginosa 50-3-19/20B MPLC fractions in order to determine their chemical structures and anticancer activities.
2.5. Compound Isolation and Bioactivity Testing
The purification of PEFA glycolipids from
Rhodotorula-derived extracts is highly challenging, as the yeast generally synthesizes a diverse range PEFAs (including different isomers) with high chemical resemblance and similar retention times. In the DCM subextract of
R. mucilaginosa 50-3-19/20B, 99 nodes were attributed with the PEFA glycolipid cluster and all PEFAs eluted within 2.5 min in the UPLC-QToF-MS/MS chromatogram. Even after further MPLC fractionation, the most bioactive fractions, F22 and F24, were both highly complex in PEFAs. Based on visual inspection of the chromatographic data, F22 appeared to be better resolved, thus judged more promising for isolation of pure compounds. PEFAs lack a strong UV chromophore; therefore, fractionation was performed by time to afford four pure PEFA compounds,
1–
4 (
Figure 5) as clear oils in minor amounts. Their structure elucidations were based on extensive HR-MS/MS and NMR analyses.
HR-MS/MS data of compound
1 revealed a sodium adduct ion at
m/
z 627.3346 [M + Na]
+ that corresponded to the molecular formula C
30H
52O
12. Analysis of the HR-MS/MS spectrum revealed fragment ions at
m/
z 567.3137,
m/
z 507.2916 and
m/
z 447.2723 (
Figure S9) arising from the natural loss of three acetyloxy groups (−60 Da each). The fragment
m/
z 313.0904 [M + Na]
+ originated from mannitol-triacetate; thus compound
1 was tentatively identified as
D-mannitol-triacetyloxy-3-acetyloxyhexadecanoate. 2D NMR data were used to confirm the planar structure of
1 (
Figures S10–S15). The
1H NMR data (
Table 3,
Figure S10) together with the HSQC spectrum of
1 (
Figure S11) revealed the presence of a terminal methyl group (H
3-16′,
δH 0.90, t,
J = 6.9 Hz/
δC 14.1) and four acetyl methyl groups resonating between
δH 2.02–2.08 and
δC 20.3–20.8, five oxygenated methine resonances H-2 (m,
δH 3.79/
δC 70.3), H-3 (m,
δH 3.69/
δC 72.7), H-4 (m,
δH 3.48/
δC 70.4), H-5 (m,
δH 3.87/
δC 70.0), H-3′ (m,
δH 5.22/
δC 71.8), and four methylene groups H
2-1 (m,
δH 3.63 and 3.80/
δC 64.8), H
2-6 (m,
δH 4.16 and 4.37/
δC 67.7), H
2-2′ (m,
δH 2.61 and 2.65/
δC 39.8), H
2-4′ (m,
δH 1.61/
δC 34.7) and highly overlapped methylene signals around
δH 1.30 (m)/
δC 23.5–32.9 characteristic of an aliphatic fatty acyl chain. Four additional acetyl carbonyl signals (
δC 172.1,
δC 172.3,
δC 172.9, and
δC 173.1) and another carbonyl resonating at
δC 172.3 (C-1′) were extracted from the HMBC spectrum of
1 (
Figure S13). Detailed analysis of the 2D NMR data (
Table S8) revealed an isolated spin system belonging to the mannitol-triacetate. Key COSY correlations were observed between H
2-6 (
δH 4.16, and 4.37)/H-5 (
δH 3.87), H-5/H-4 (
δH 3.48), H-4/H-3 (
δH 3.69) plus H-3 and H-2 (
δH 3.79), which in turn correlated with the H
2-1 methylene protons (
δH 3.63 and 3.80) (
Figure 6). Based on key HMBC cross-peaks (
Figure 6,
Table S8), the site of acetylations on the mannitol unit was assigned to C-2, C-3, and C-6. The remaining signals were attributed to the C16-fatty acyl chain. Position C-3′ was identified as the site of acetylation based on HMBC correlations of H-3′ (
δH 5.22) with C-17′ (
δC 172.3), C-1′ (
δC 172.3), C-2′ (
δC 39.8), C-4′ (
δC 34.7), and C-5′ (
δC 25.9).
The NOESY spectrum did not allow deduction of the stereochemical configurations within
1. NMR chemical shifts in (
S)- and (
R)-3-hydroxy- and acetoxyhexadecanoic acids are identical and the two respective isomers only differ by their optical rotations [
30]. This is the first time that a PEFA is chemically characterized by NMR and optical rotations, therefore, there is no data for comparison in order to unequivocally assign the stereochemistry of
1. However, a previous study on the extracellular glycolipids of
R. babjevae [
15] used extensive derivatization experiments and chiral separation via GC-MS. This assigned an (
R)-configuration at C-3′ (which is the site of
O-acetyl substitution) of the fatty acid unit in PEFAs, while the polyol components were determined to be
d-arabitol and
d-mannitol. Additional studies by Wang et al. [
23] also described that
Rhodotorula exclusively synthesized
d-mannitol. Our genomics analysis found the fatty acid synthase core enzyme is conserved across several
Rhodotorula spp., thus it is biosynthetically reasonable to assume that the PEFAs in
R. mucilaginosa 50-3-19/20B have the same configuration at all stereocenters. On this basis, we propose the structure of the new compound
1 as
D-mannitol-2,3,6-triacetyloxy-(
R)-3′-acetyloxyhexadecanoate.
Compound
2 was also purified as a colorless oil. The molecular formula C
26H
48O
10 was assigned based on the sodium adduct ion observed in the HR-ESIMS spectrum of
2 at
m/
z 543.3141 [M + Na]
+ (
Figure S16). On the basis of the molecular networking analysis, this compound also belonged to the PEFA glycolipids. The fragment ions at
m/
z 483.2929 [M + Na]
+ and
m/
z 423.27 [M + Na]
+ indicated the presence of two acetyloxy groups and the ion at
m/
z 229.0757 [M + Na]
+ was indicative of a monoacetylated mannitol. The NMR data of
2 was almost identical to that of compound
1 (
Table 3,
Table S9, Figures S17–S21). COSY and HMBC correlations (
Table S9,
Figure 6,
Figures S19 and S21) revealed that
2 also contained an acetyloxy group at position 3′ of the C16-fatty acyl chain. Further, the HMBC correlation from H-4 (
δH 3.47) to a carbonyl resonance at
δC 172.9 (C-7) (
Figure 6,
Figure S21) indicated the final acetyl substitution to be at the C-4 position of the mannitol unit. Hence, the new compound
2 was determined as D-mannitol-4-monoacetyloxy-(
R)-3′-acetyloxyhexadecanoate.
The HR-ESIMS spectrum of
3 (
Figure S22) revealed a sodium adduct ion at
m/
z 697.3789 [M + Na]
+ that corresponded to the molecular formula C
34H
58O
13. HR-MS/MS fragment ions at
m/
z 637.3586,
m/
z 577.3372,
m/
z 517.3150, and
m/
z 457.2921 indicated the presence of four acetyloxy groups while the ions at
m/
z 335.1011 and
m/
z 295.0796 (difference of 60 Da) pointed to an additional acetyloxy group in
3. The presence of tetraacetylated mannitol was identified based on the residual ion at
m/
z 355.1011 [M + Na]
+ originating from the loss of acetyloxyoctadecanoic acid. Due to poorly resolved NMR data for
3 the exact acetylation sites of the polyol could not be assigned. As optical rotations of
3 indicated the same configuration as in compounds
1 and
2, we proposed compound
3 as a D-mannitol-tetraacetyloxy-(
R)-3′-acetyloxyoctadecanoate derivative. The structure of
3 shown in
Figure 5 is only a tentative structure and needs to be confirmed in the future.
The last PEFA glycolipid
4 with the molecular formula C
33H
56O
12 (
m/
z 667.3669 [M + Na]
+) was also isolated from the bioactive MPLC fraction F22. Similar to PEFA
3, the
1H NMR spectrum of
4 was poorly resolved; thus, we solely used HR-MS/MS data for structural analysis. Analysis of the HR-MS/MS spectrum showed fragment ions at
m/
z 607.34763,
m/
z 547.3257, and
m/
z 487.2971 (
Figure S23) arising from the natural loss of three acetyloxy groups. Fragment ions at
m/
z 525.3370 and 465.3260 as well as
m/
z 505.3015 and 445.5472 indicated the presence of two more acetyl functions. The ion at
m/
z 325.0901 [M + Na]
+ originated from tetraacetylated arabitol. Thus, the chemical structure of
4 was determined as D-arabitol-2,3,4,5-tetraacetyloxy-(
R)-3′-acetyloxyoctadecanoate. This compound was previously identified in the crude extract of
R. babjevae by LC-MS as well as GC-MS with various derivatization methods [
15].
The remaining bioactive MPLC fractions contained complex mixtures of the PEFA type glycolipids that could not be separated by different chromatography columns and solvent gradients.
The molecular network indicated that
R. mucilaginosa 50-3-19/20B produced indole alkaloids (
Figure 4), which can be attributed to the detected NRPS cluster by AntiSMASH analysis. These molecules occurred in earlier eluting brown-colored fractions (F1-F8), which were inactive against the cancer cell line MDA-MB-231. To confirm the presence of indole type compounds, we performed rapid isolation of the major compound (
5) in F2. HR-MS/MS data of
5 (
Figure S24) revealed a pseudomolecular ion at
m/
z 220.0983 [M + H]
+ C
12H
14NO
3 (calculated for 220.0974), this molecular formula corresponded to methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (
Figure 5). 1D and 2D NMR data were used to confirm the structure of
5 (
Table S10,
Figures S25–S30), which was previously isolated by Cimmino et al. from
Diaporthella cryptica, a fungus derived from a hazelnut branch [
31].
1H NMR and specific rotation data of
5 (
−1.7,
c 0.15, CHCl
3) were in agreement with those reported in the literature (
−2.6,
c 0.58, CHCl
3) therefore we conclude that
5 is in (
S)-(–)-form.
Compounds 1–5 were tested in vitro for their cytotoxic activity against the breast cancer cell line MDA-MB-231 and the non-cancerous human keratinocyte cell line HaCaT, however, in their pure form compounds 1–5 were devoid of any inhibitory activity at the test concentration of 100 µg/mL.