Chinese Journal of Oceanology and Limnology   2016, Vol. 34 issue(2): 367-371     PDF
Institute of Oceanology, Chinese Academy of Sciences

Article Information

YANG Haibin(杨海滨), LI Fang(李昉), JI Naiyun(季乃云)
Alkaloids from an algicolous strain of Talaromyces sp.
Journal of Oceanology and Limnology, 34(2): 367-371

Article History

Received Dec. 25, 2014
accepted in principle Apr. 2, 2015
Alkaloids from an algicolous strain of Talaromyces sp.
YANG Haibin(杨海滨)1,2, LI Fang(李昉)1, JI Naiyun(季乃云)2        
1 College of Chemistry and Molecular Engineering, Qingdao University of Science & Technology, Qingdao 266042, China;
2 Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
ABSTRACT: Compounds isolated and identified in a culture of the alga-endophytic fungus Talaromyces sp. cf-16 included two naturally occurring alkaloids, 2-[(S)-hydroxy(phenyl)methyl]-3-methylquinazolin-4(3H)-one(1a) and 2-[(R)-hydroxy(phenyl)methyl]-3-methylquinazolin-4(3H)-one(1b), that were identified for the first time. In addition, seven known compounds(2-8) were obtained from the culture. Following chiral column chromatography, compounds 1a and 1b were identified as enantiomers by spectroscopic analyses and quantum chemical calculations. Bioassay results showed that 5 was more toxic to brine shrimp than the other compounds, and that 3-6 could inhibit Staphylococcus aureus.
Key words: Talaromyces sp.     alkaloid     chiral separation     quantum chemical calculation     bioactivity    

Stereogenic centers occur widely in the secondary metabolites of plants,animals,and microbes,where they add to the molecular diversity of these natural products and result in different bioactivities (Batishta Jr et al.,2011; Ji et al.,2014; Rufino et al.,2014). Several chiroptical techniques,such as electronic circular dichroism (ECD)(Stephens et al.,2008b; Batishta Jr et al.,2011; Miao et al.,2012; Mazzeo et al.,2013; Santoro et al.,2013; Wu et al.,2014),vibrational circular dichroism (Stephens et al.,2008b; Batishta Jr et al.,2011; Mazzeo et al.,2013),optical rotatory dispersion (Mazzeo et al.,2013; Santoro et al.,2013),and specific optical rotation ([α] D)(Stephens et al.,2008ab; Miao et al.,2012),have often been used to establish the absolute configurations of chiral compounds. Quantum chemical calculations greatly expand the application of these techniques. Using these techniques and computational calculations,a series of structurally interesting compounds have been discovered from marine algae and algicolous fungi (Li et al.,2013; Ji et al.,2013; Miao et al.,2014). The aim of the present study was to investigate the alga-endophytic fungus Talaromyces sp. cf-16. The isolation,structural elucidation,and bioactivity assays of the identified compounds are described. These compounds included two naturally occurring alkaloids,2-[(S)-hydroxy (phenyl) methyl]- 3-methylquinazolin-4(3H)-one (1 a) and 2-[(R)- hydroxy (phenyl) methyl]-3-methylquinazolin-4(3H)- one (1b)(Tennant,1966),that were identified for the first time,and seven known compounds,including roquefortine C (2)(Scott et al.,1979; Musuku et al.,1994),Z -roquefortine C (3)(Scott et al.,1979),viridicatol (4)(Fremlin et al.,2009),penitrem A (5)(Rundberget and Wilkins,2002),penijanthine A (6)(Itabashi et al.,2009),paspaline (7)(Nozawa et al.,1988),and 3-deoxo-4b-deoxypaxilline (8)(Fan et al.,2013)(Fig. 1).

Figure 1 Chemical structures of the isolated alkaloids 1a/b–8
2 EXPERIMENTAL 2.1 General

Ultraviolet-visible and ECD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd.,Leatherhead,UK). Infrared spectra were obtained on a JASCO FT/IR-4100 spectrometer (JASCO,Tokyo,Japan). NMR spectra were recorded at 500 and 125 MHz for 1 H and 13 C,respectively,on a Bruker Avance III 500 NMR spectrometer (Bruker Corp.,Billerica MA) using tetramethylsilane as an internal standard. Low and high resolution electron ionization mass spectra were obtained using an Autospec Premier P776 mass spectrometer (Waters Corp.,Milford MA). High performance liquid chromatography (HPLC) was carried out on an Agilent 1260 HPLC system (Agilent Technologies Inc.,Santa Clara,CA) using an Eclipse SB-C18 column (9.4×250 mm,5 μm) or a Pirkle Whelk-O1 column (4.6×250 mm). Column chromatography (CC) was performed with silica gel (200–300 mesh,Qingdao Haiyang Chemical Co.,Qingdao,China) and Sephadex LH-20(GE Healthcare,Little Chalfont,UK). Thin layer chromatography was carried out with precoated silica gel plates (GF-254,Qingdao Haiyang Chemical Co.). All solvents were of analytical grade,except for MeOH for HPLC,which was of spectral grade. Quantum chemical calculations were performed using Gaussian 09 software (IA32W-G09RevC.01; Gaussian Inc.,Wallingford,CT)(Frisch et al.,2010).

2.2 Fungal material and fermentation

The fungal strain Talaromyces sp. cf-16 was isolated from fresh tissue of surface-sterilized marine brown alga Sargassum sp. collected from the coast near Yantai (China) in July,2009. The fungus was identified by analysis of the internal transcribed spacer regions of its rDNA. Sequence data was deposited at GenBank with the accession number KM014498. The strain was preserved at the Yantai Institute of Coastal Zone Research,Chinese Academy of Sciences. The initial cultures were maintained on potato dextrose agar plates. Pieces of mycelia were cut into small segments and aseptically inoculated into 50 Erlenmeyer flasks (1 L),each containing 300 mL of Jerusalem artichoke (Helianthus tuberosus)-dextrose broth culture medium. The medium was prepared as follows: Jerusalem artichoke broth,500 mL/L; dextrose,10 g/L; NaNO3,2 g/L; natural sea water from the coast of Yantai,500 mL/L. Static fermentations were performed at room temperature for 30 d.

2.3 Extraction and isolation

Whole cultures (300 mL×50 flasks,30 d) were filtered through cheesecloth to separate the mycelia from the broth. The dried mycelia were homogenized and extracted with a mixture of CHCl3 and MeOH (1:1,v/v). The extract was evaporated to dryness,then partitioned between EtOAc and H2O to yield an EtOAc-soluble extract (5.4 g). The broth was directly extracted with EtOAc to yield 3.2 g. Based on their identical thin layer chromatography profiles,these two extracts were combined then subjected to silica gel CC. Separation was achieved with a solvent gradient of 0–100% petroleum ether (PE)-EtOAc and then 100% MeOH to afford 10 fractions (Frs. 1–10). Fraction 7 eluted with PE/EtOAc (2:1,v/v) and was further purified by CC on silica gel (PE/EtOAc,6:1 to 0:1,v/v) and Sephadex LH-20(CHCl3 /MeOH,1:1,v/v) and semipreparative HPLC (MeOH/H2O,9:1,v/v) to yield compounds 7(8.4 mg) and 8(0.9 mg). Fraction 8 eluted with PE/EtOAc (1:1,v/v) and was further purified by CC on silica gel (PE/EtOAc,4:1 to 0:1,v/v) and Sephadex LH-20(CHCl3 /MeOH,1:1,v/v) and semipreparative HPLC (MeOH/H2O,3:1,v/v) to afford 5(6.1 mg),6(8.7 mg),and a mixture of 1a and 1b,which was further purified by semipreparative HPLC with a chiral Pirkle Whelk-O1(4.6×250 mm) column (hexane/ethanol: 19:1,v/v) to give 1a (3.2 mg) and 1b (3.4 mg) based on their weak ECD and [α] D signals. Fraction 9 eluted with EtOAc and was further purified by CC on silica gel (PE/ EtOAc,2:1 to 0:1,v/v) and Sephadex LH-20(CHCl3 / MeOH,1:1,v/v) to afford two subfractions (Frs. 9-1 and 9-2). Fraction 9-1 was purified by semipreparative HPLC (MeOH/H2O,13:7,v/v) to yield 3(21.9 mg),and Fr. 9-2 was also purified by semipreparative HPLC (acetonitrile/H2O,2:3) to yield 4(5.3 mg). Fraction 10 eluted with MeOH and was further purified by CC on silica gel (EtOAc/MeOH,5:1 to 0:1,v/v) and Sephadex LH-20(CHCl3 /MeOH,1:1,v/v) and semipreparative HPLC (MeOH/H2O,13:7,v/v) to give 2(6.7 mg).

2.4 Computational details

Conformational searches for 1a and 1b were performed using the Dreiding force field in MarvinSketch (2014)regardless of rotations of methyl and hydroxy groups,and the energyminimized conformers were further optimized at the gas-phase B3LYP/6-31G (d) level using Gaussian 09 software (Frisch et al.,2010). The optimized conformers were subjected to calculations of ECD spectra using time-dependent density functional theory at the gas-phase B3LYP/6-31G (d) level using SpecDic software with sigma equal to 0.2(Bruhn et al.,2013).

2.5 Bioactivity assay

Compounds 1a / b – 8 were assayed for their toxicities to brine shrimp (Artemia salina) and inhibitory activities against bacteria (Escherichia coli and Staphylococcus aureus)(Miao et al.,2012).


2-[(S)-hydroxy (phenyl) methyl]-3-methylquinazolin- 4(3H)-one and 2-[(R)-hydroxy (phenyl) methyl]-3- methylquinazolin-4(3H)-one (1a and 1b):

White amorphous powder; UV (MeOH)λmax (log ε)204(4.65),225(4.52),267(3.94),276(3.91),303(3.58),314(3.47) nm; IR (KBr) vmax 3 398,3 313,2924,1 685,1 604,1 049,775,702/cm; 1 H NMR (500 MHz,CDCl3H 8.30(1H,brd,J =8.1,H-6),7.53(1H,ddd,J=8.1,6.2,2.2,H-7),7.81(1H,m,H-8),7.81(1H,dd,J=6.2,1.2,H-9),5.68(1H,s,H-11),7.33(2H,m,H-13 and 17),7.36(2H,m,H-14 and 16),7.36(1H,m,H-15),3.34(1H,s,H-18); 13 C NMR (125 MHz,CDCl3C 156.6(C,C-2),162.1(C,C-4),120.4(C,C-5),127.1(CH,C-6),127.4(CH,C-7),134.7(CH,C-8),126.7(CH,C-9),145.3(C,C-10),71.8(CH,C-11),138.9(C,C-12),127.7(CH,C-13/17),129.3(CH,C-14/16),129.1(CH,C-15),29.9(CH3,C-18); EI-MS m/z(%)266(100),189(55),175(43),160(39),77(51); HREI-MS m/z 266.106 2 [M] +,calcd for C 16 H 14 N 2 O 2,266.105 5.

4 RESULT AND DISCUSSION 4.1 Structure elucidation of compound 1

Compound 1 was originally isolated as a white amorphous powder by achiral CC and HPLC. Its molecular formula was calculated as C 16 H 14 N 2 O 2 from high resolution electron ionization mass spectrometry (m/z 266.106 2 [M] +,calcd for C 16 H 14 N 2 O 2,266.105 5),requiring eleven degrees of unsaturation. The 1 H NMR spectrum showed one methyl singlet,one singlet ascribable to an oxygenated methine,and signals for nine aromatic protons. The 13 C and DEPT NMR spectra along with heteronuclear single quantum coherence data demonstrated the presence of sixteen carbon atoms,which could be classified as one methyl,10 methines,and five non-protonated carbons. The NMR data were similar to those reported for 3,4-dihydro-2-(α-hydroxybenzyl)-4-oxoquinazoline,except for the presence of signals for a methyl group and the lack of a broad singlet for NH (Catir et al. 2009). Thus,compound 1 was deduced to be a methylated derivative of 3,4-dihydro-2-(α- hydroxybenzyl)-4-oxoquinazoline at N-3,which was confirmed by the heteronuclear multiple bond correlations from H-18 to C-2 and C-4. Further heteronuclear multiple bond correlations from H-11 to C-2,C-12,C-13,and C-17,from H-6 to C-4,C-8,and C-10,and from H-7 to C-5 and C-10 further corroborated the structure of 1 to be 3,4-dihydro-2-(α-hydroxybenzyl)-3-methyl-4-oxoquinazoline. This compound has been identified previously as a synthetic intermediate (Tennant,1966).

To establish the absolute configuration of compound 1 with only one stereogenic carbon atom (C-11),its ECD spectrum and [α] D value were determined. Unfortunately,no obvious Cotton effects were observed,and its [α] D value was also close to zero. Hence,1 was proposed to be an enantiomeric mixture. This was confirmed,and the mixture was further separated by chiral HPLC to yield 1a and 1b (Fig. 2). The ECD spectra of 1a and 1b were determined,and they matched well with the computed ones (Figs. 3 and 4). Thus,the absolute configurations at C-11 were assigned to be 11 S for 1 a and 11 R for 1b . However,the optical rotations of 1a and 1b were too low to be recorded,and even their signs could not be low to be recorded,and even their signs could not be confirmed. confirmed.

Figure 2 HPLC profile for the chiral separation of 1a and 1b
Figure 3 Experimental and calculated ECD spectra of 1a with an 11 S configuration
Figure 4 Experimental and calculated ECD spectra of 1b with an 11 R configuration
4.2 Other chemical constituents of Talaromyces sp.

The remaining seven known alkaloids,including roquefortine C (2)(Scott et al.,1979; Musuku et al.,1994),Z -roquefortine C (3)(Scott et al.,1979),viridicatol (4)(Fremlin et al.,2009),penitrem A (5)(Rundberget and Wilkins,2002),penijanthine A (6)(Itabashi et al.,2009),paspaline (7)(Nozawa et al.,1988),and 3-deoxo-4b-deoxypaxilline (8)(Fan et al.,2013),were identified by comparison of spectral data with literature values.

4.3 Bioactivity

Investigation of the bioactivities (Table 1) showed that 5(LC 50 =3.1 μg/mL) was the most toxic to A . salina among the isolates. The enantiomers 1a and 1b exhibited similar toxicities to A . salina,but roquefortine C (2) was more toxic than its Z isomer (3). Additionally,only 3 – 6 inhibited S . aureus,and none of the compounds inhibited E . coli .

Table 1 The bioassay results for compounds 1a/b–8

Chemical investigation of the alga-endophytic fungus Talaromyces sp. cf-16 resulted in the isolation and identification of nine alkaloids (1a / b8),including two naturally occurring enantiomers that were identified for the first time (1a and 1b). The enantiomers were successfully separated and identified by the chiral chromatographic and spectral techniques. These results provide a valuable reference for studies on enantiomeric mixtures with low or no signals in chiroptical determinations.

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