Fungal secondary metabolites are rich sources of unique compounds and a lot of useful compounds have already been discovered. However, it has been proposed that there is an immeasurable number of microbial metabolites not yet discovered [1]. Therefore, our group has continued to investigate fungal metabolites, which has already resulted in the discovery of novel compounds such as the virgaricins A and B, cinatrins D and E, and cladomarine [2,3,4]. Recent research led us to discover a new dipeptide, tolyprolinol (1), containing a l-phenylalanine and a l-prolinol, from a culture broth of Tolypocladium sp. FKI-7981. Prolinol is a rare moiety among natural products and this is the first report of a natural product containing a prolinol moiety isolated from Tolypocladium species. We detail here the taxonomy of producing strain, as well as the fermentation, isolation, structure elucidation, and some biological properties of 1.

The fungal strain FKI-7981 had 95.2% similarity with the internal transcribed spacer sequence of CBS 869.73 (ex-type of Tolypocladium album). From this information, combined with morphological characteristics, FKI-7981 was identified to be a member of the genus Tolypocladium family [5, 6].

The EtOAc extract of a 14-day static cultured broth was subjected to column choromatographies and high-performance liquid chromatography purification to afford to 1 (22.3 mg). The detailed fermentation and isolation procedure for 1 is summarized in Schemes S1 and S2 in the Supplementary Information.

The physico-chemical properties of 1 are summarized in Table 1. It is soluble in methanol with ultraviolet (UV) absorption at 206 nm, as well as dimethyl sulfoxide (DMSO), chloroform, and water, but insoluble in n-hexane. The characteristic infrared absorptions at 3440, 3267, 1624, and 1454 cm−1 suggested the presence of an amidocarbonyl group moiety.

Table 1 Physico-chemical properties of tolyprolinol (1)
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The molecular formula of 1 was elucidated by HR-ESI-MS to be C16H22N2O3 with a molecular ion peak [M + Na]+ at m/z 313.1521 (calcd. m/z 313.1528). Two conformers were observed with the ratio of 3:2 in 1H and 13C nuclear magnetic resonance spectroscopy (NMR) spectral data of 1 in DMSO-d6 at room temperature (Figs. S2-1 and S2-2). The spectra did not change dramatically, even at 80 °C. Amide signals and α-proton signals in 1H and 13C NMR spectral data of 1 suggested 1 is a peptide compound. When 1 was measured in CDCl3, two conformers were observed with the ratio of 5:2 at room temperature (Figs. S2-6 and S2-7). The structure of major and minor conformers of 1 in CDCl3 was elucidated by 1D and 2D NMR, as shown in Table 2.

Table 2 1H and 13C NMR data of the major and minor conformers of tolyprolinol (1) in CDCl3
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Combined analysis of the 1H-1H correlation spectroscopy (COSY) and heteronuclear multiple-bond correlation (HMBC) spectra identified phenylalanine (Phe) and prolinol residues in 1 (Fig. 1b), which were finally confirmed by their 1-fluoro-2,4-dinitrophenyl-5-d-leucineamide (FDLA) derivatives described below. In the major conformer of 1, HMBC correlations observed from CH3 (δH 1.98) and Phe NH (δH 6.92) to C-8′ (δC 169.7) indicated that the amino group in Phe in 1 was acetylated. The planar structure of 1 was established by HMBC correlations from prolinol H2-5 (δH 2.67 and 3.65) to Phe C-1′ (δC 172.1) as N-acetylphenylalanylprolinol. Compound 1 has not been previously reported and we designated it as tolyprolinol. The minor conformer of 1 was also elucidated in the same manner (Table 2). This minor conformer is suggested as being derived from the regioisomeric amide bond of prolinol residue in 1.

Fig. 1
figure 1

a Structure of tolyprolinol (1). b Key correlations of 1H-1H COSY and HMBC in 1. Bold lines show proton spin networks; arrows show 1H-13C long-range correlations

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The absolute configuration of amino acids in 1 was elucidated by Advanced Marfey’s method after acid hydrolysis [7]. Compound 1 was hydrolyzed and derivatized with FDLA and analyzed by an ultraperformance liquid chromatography coupled with ESI-MS. As the result of the comparison of retention time with FDLA derivatives of standard Phe and prolinol, both Phe and prolinol were elucidated to be the l configuration (Table S3 and Fig. S3).

Compound 1 was tested for antimalarial activity against both a chloroquine-resistant K1 strain and chloroquine-sensitive FCR3 strain of P. falciparum, as well as cytotoxicity against nine human cell lines. Compound 1 showed half-maximal inhibitory concentration values of 163 and 285 µM against the K1 strain and the FCR3 strain of P. falciparum, respectively. However, 1 did not display any cytotoxicity against the human MRC-5 cell at 345 µM and other eight human cancer cell lines, HL-60, Jarkat, THP-1, HeLa S3, A549, Panc1, HT29, and H1299 at 100 µM.

The antimicrobial activity of 1 was assessed against six microorganisms, Bacillus subtilis KB 211 (ATCC 6633), Kocuria rhizophilia KB 212 (ATCC 9341), Escherichia coli KB 213 (NIHJ), Xanthomonas oryzae pv. oryzae KB 88, Candida albicans KF 1 (ATCC 64548), and Mucor racemosus KF 223 (IFO 4581) using a disk diffusion assay with 8-mm paper disks, as previously described [8]. Compound 1 was inactive against all microorganisms tested, even at 50 µg per disk.

In summary, we have discovered a new dipeptide, named tolyprolinol, consisting of l-Phe and l-prolinol, from secondary metabolites of Tolypocladium sp. FKI-7981. We found 1 showed moderate antimalarial activity, but did not show cytotoxicity or other antimicrobial activity against the microbes tested. There have been a few reports of compounds containing prolinol, such as actinonin, viriditin, scalusamides, asperelines, and barmumycin [9,10,11,12,13,14]. Most of the reported compounds produced by genus Tolypocladium were cyclosporin-like polypeptides [15]. Therefore, the isolation of the new dipeptide 1 suggests that Tolypocladium may be a potential source of unique compounds that could prove to be interesting lead compounds for future drug discovery.