Biotransformation of Huperzine A by a Fungal Endophyte of Huperzia

Article pubs.acs.org/jnp

Biotransformation of Huperzine A by a Fungal Endophyte of Huperzia serrata Furnished Sesquiterpenoid−Alkaloid Hybrids You-Min Ying, Wei-Guang Shan,* and Zha-Jun Zhan* College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China S Supporting Information *

ABSTRACT: Biotransformation of huperzine A (hupA) by a fungal endophyte, Ceriporia lacerate HS-ZJUT-C13A, afforded compounds 1−5 and three tremulane sesquiterpenoids, 6−8. Huptremules A−D (1−4) feature unusual sesquiterpenoid− alkaloid hybrid structures that integrate the characteristics of fungal metabolites (tremulane sesquiterpenoids) and the exogenous substrate (hupA). These results support the use of fungal endophytes as biocatalysts for the biotransformation of natural products, particularly those originating from the host plant.

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characterization of the AChE inhibitory activity of the transformed products, as well as a discussion of their ecological and pharmacological significance.

ndophytes are microorganisms that spend all or part of their life cycle inter- and/or intracellularly colonizing inside healthy tissues of the host plants, typically causing no apparent symptoms of disease.1 These microorganisms are thought to participate in a complex web of interactions with the host plant and other endophytic microorganisms during colonization. Over the past several decades, the secondary metabolites of endophytes have been investigated rigorously, resulting in the discovery of many structurally diverse and biologically active compounds.2−8 More recently, endophytes and the enzymes responsible for the synthesis of their potent, diverse arsenal of secondary metabolites have gained attention as unexplored or underexplored sources of biocatalysts for the biotransformation of natural products,9,10 such as alkaloids,11,12 flavans,13 lignans,14 and triterpenoids.15 Huperzine A (hupA), a lycopodium alkaloid from the club moss Huperzia serrata, is a potent, highly specific, reversible acetylcholinesterase (AChE) inhibitor that shows promise for the palliative treatment of Alzheimer’s disease.16 The chemical synthesis, structural modification, and biological effects of hupA have been studied extensively since its AChE inhibitory activity was reported in 1986.17 Owing to its rigid molecular configuration, the structural modification of hupA by traditional chemical methods is challenging.18 Microbial transformation can be used as an alternative to traditional chemical synthesis and has proven powerful for expanding the chemical diversity of natural products.19,20 In a previous report, hupA was transformed by the bacterium Streptomyces griseus CACC 200300 to produce three new hupA derivatives that are difficult to obtain by traditional chemical methods.18 To further expand the structural diversity of hupA and investigate the potential of employing endophytes as biocatalysts, it was subjected to biotransformation by the fungal endophyte Ceriporia lacerate HS-ZJUT-C13A. This paper describes the isolation, structural elucidation, and © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Forty-nine fungal endophytes of H. serrata were screened for their ability to transform hupA in liquid potato-dextrose medium, liquid Czapek-Dox medium, and liquid Sabouraud’s medium. The fungus C. lacerate HS-ZJUT-C13A cultivated in the liquid potato-dextrose medium was found to be the most effective and was selected for the preparative-scale biotransformation of hupA. Five transformed products (1−5) and three tremulane sesquiterpenoids (6−8) were isolated and identified (Chart 1). The 1H and 13C NMR spectroscopic data for the new transformed products huptremules A−D (1−4) are shown in Tables 1 and 2. Huptremule A (1) was obtained as a white powder. The molecular formula was determined as C31H40N 2O5 by HRESIMS from the [M + H]+ peak at m/z 521.3017 (calcd for C31H41N2O5, 521.3017) and the 13C NMR data. The IR spectrum indicated the presence of hydroxy (3390 cm−1) and carbonyl (1658 and 1613 cm−1) groups. The 1H and 13C NMR spectra of 1 retained the typical signals of hupA but were far more complicated. Approximately half of the NMR signals of 1 were nearly superimposable with those of hupA. Inspection of the NMR data indicated a hupA residue whose signals were closely similar to those of hupA, with the exception of deviations of +6.7, −9.6, and +2.7 ppm at C-12, C-13, and C14, respectively (Table 1), suggesting that the hupA residue in 1 was substituted at the primary amino group. The remaining signals in 1 shared remarkable similarity with those of Received: May 18, 2014 Published: September 15, 2014 2054

dx.doi.org/10.1021/np500412f | J. Nat. Prod. 2014, 77, 2054−2059

Journal of Natural Products

Article

Chart 1. Structures of Huperzine A and Compounds 1−8

Table 1. 1H NMR Spectroscopic Data of Compounds 1−4 1a

position 1 2 3 4 5 6a 6b 7 8 9c 10 11 12 13 14a 14b 15 16 1′ 2′ 3′ 4′a 4′b 5′a 5′b 6′ 7′ 8′a 8′b 9′ 10′a 10′b 11′a 11′b 12′ 13′ 14′ 15′ 16′

2a

3b

4b

hupAa

6.35, d (9.5) 7.66, d (9.5)

6.32, d (9.5) 7.65, d (9.5)

6.36, d (9.5) 7.74, d (9.5)

6.38, d (9.0) 7.77, d (9.0)

6.40, d (9.5) 7.89, d (9.5)

2.89, 2.60, 3.65, 5.47,

2.91, 2.60, 3.65, 5.47,

2.83, 2.57, 3.58, 5.34,

2.91, 2.61, 3.60, 5.38,

2.89, 2.75, 3.59, 5.40,

brd (18.0) brd (16.5) m d (5.0)

dd (18.0, 4.5) brd (16.5) m d (5.0)

dd (16.0, 4.5) brd (16.5) m d (5.0)

dd (16.5, 5.0) brd (16.5) m d (4.0)

dd (16.5, 5.0) dd (17.0, 1.0) m d (5.0)

1.61, d (7.0) 4.63, q (7.0)

1.62, d (6.5) 4.77, q (6.5)

1.67, d (6.5) 5.88, q (7.0)

1.67, d (6.5) 5.85, q (6.5)

1.66, d (7.0) 5.47, q (7.0)

2.96, d (16.5) 2.36, d (16.5)

2.86, d (16.5) 2.61, d (16.5)

2.63, d (16.0) 1.49, d (16.0)

2.65, d (16.0) 2.01, d (16.0)

2.14, d (16.5) 2.10, d (16.5)

1.60, s

1.60, s

1.54, s

1.55, s

1.53, s

2.90, dd (18.0, 6.0) 2.61, dd (18.0, 5.0) 3.76, ddd (10.0, 5.5, 5.5)

2.84, dd (18.0, 5.5) 2.67, dd (18.0, 5.5) 3.73, ddd (9.5, 5.0, 5.0)

2.40, dd (14.5, 4.5) 1.85, dd (15.0, 2.0) 4.11, m

2.07, 2.39, 1.72, 1.42,

2.04, 2.36, 1.69, 1.42,

2.08, m 3.73, m 1.54, m

1.95, 1.82, 2.23, 1.90, 2.25, 2.21, 3.57,

m m m m m m d (9.5)

1.96, 1.84, 4.34, 3.87, 4.43, 0.90, 0.86, 1.08,

d (16.0) brd (16.0) d (13.5) d (13.5) s d (7.0) s s

m m dd (12.5, 7.0) dd (13.5, 13.0)

1.92, d (14.0) 1.78, d (14.0)

5.54, 1.14, 1.13, 0.99, 3.49,

s d (7.0) s s s

m m m dd (13.0, 13.0)

1.97, d (14.0) 1.78, d (14.0)

5.52, 1.14, 1.13, 0.99,

1.94, m 4.36, 3.92, 4.39, 0.84, 0.94, 1.12,

s d (7.0) s s

d (12.5) d (12.5) s d (7.0) s s

a

Data were measured in CDCl3. bData were measured in methanol-d4. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on 1H−1H COSY, HMQC, and HMBC experiments. cThe numbering of hupA does not include C-9.

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Table 2. 13C NMR Spectroscopic Data of Compounds 1−4 position 1 2 3 4 5 6 7 8 9c 10 11 12 13 14 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′

1a

2a

3b

4b

hupAa

166.0, 118.2, 141.9, 123.5, 143.6, 35.6, 34.7, 125.3,

qC CH CH qC qC CH2 CH CH

166.1, 118.2, 142.2, 123.6, 143.8, 35.2, 34.7, 125.6,


164.6, 117.2, 141.6, 123.8, 142.1, 35.2, 33.3, 124.0,


165.0, 116.7, 141.9, 124.0, 142.5, 35.2, 33.2, 124.1,


165.4, 116.9, 140.2, 122.8, 142.5, 35.2, 32.8, 124.3,


12.7, 113.6, 136.5, 63.6, 46.4, 134.6, 23.2, 148.1, 139.2, 83.1, 36.5, 71.9, 38.9, 56.0, 41.5, 37.0, 56.8, 173.8, 90.9, 20.7, 30.1, 31.4, 52.1,

CH3 CH qC qC CH2 qC CH3 qC qC qC CH2 CH CH CH CH2 qC CH2 qC CH CH3 CH3 CH3 CH3

12.7, 113.6, 136.3, 63.1, 47.1, 134.7, 23.2, 149.6, 136.5, 83.0, 36.6, 71.6, 39.2, 56.1, 41.5, 37.1, 57.1, 172.7, 85.2, 20.4, 30.1, 31.4,

CH3 CH qC qC CH2 qC CH3 qC qC qC CH2 CH CH CH CH2 qC CH2 qC CH CH3 CH3 CH3

12.7, 113.7, 140.4, 57.5, 44.0, 133.6, 22.9, 141.0, 133.7, 80.2, 32.9, 74.7, 38.2, 38.6, 44.8, 38.0, 48.0, 65.7, 93.3, 11.7, 27.2, 28.7,

CH3 CH qC qC CH2 qC CH3 qC qC qC CH2 CH CH CH CH2 qC CH2 CH2 CH CH3 CH3 CH3

12.5, CH3 113.6, CH 140.3, qC 57.5, qC 44.4, CH2 133.5, qC 22.9, CH3 135.6,d qC 135.1,d qC 78.4, qC 27.7, CH2 30.7, CH2 29.2, CH 53.3, CH 81.7, CH 39.9, qC 44.4, CH2 65.1, CH2 93.6, CH 11.9, CH3 19.9, CH3 26.2, CH3

12.3, 111.2, 143.2, 54.3, 49.1, 134.0, 22.6,

CH3 CH qC qC CH2 qC CH3

a

Data were measured in CDCl3. bData were measured in methanol-d4. The assignments were based on 1H−1H COSY, HMQC, and HMBC experiments. cThe numbering of hupA does not include C-9. dData may interchange due to the overlap of signals.

Figure 1. Key 1H−1H COSY (bold bonds) and HMBC (blue arrows) correlations of 1−4.

Figure 2. Key NOE correlations (green dashed arrows) in the sesquiterpenoid moieties of 1−4.

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tremulenolide A,21 a tremulane sesquiterpenoid produced by this fungus in our previous study.22 Compared with the NMR data for tremulenolide A, the carbon signals for C-3′, C-5′, and C-12′ in 1 were deshielded. On the basis of previous knowledge of tremulane sesquiterpenoids,22,23 the remaining signals in 1 were elucidated as representing a 5-hydroxy-3-methoxytremulenolide A residue, which was verified by 1H−1H COSY correlations between H3-13′/H-6′, H-6′/H-5′, and H-5′/H2-4′ and HMBC correlations from H3-16′ (δH 3.49, s) to C-12′ (δC 90.9) and from H-12′ (δH 5.54, s) to C-2′ and C-11′ (Figure 1). The linkage between the two moieties was established on the basis of the resonances assignable to the hemiaminal group at C-12′ (δH 5.54, s; δC 90.9) and verified by the HMBC correlation from H-12′ to C-13 and the NOE correlations of H12′/H-11 and H-12′/H2-14 (Supporting Information). The relative configuration of 1 was determined from the NOESY spectrum. The configuration of the hupA residue in 1 was assumed to be the same as those of hupA, which was confirmed by the observed NOESY correlations. In the sesquiterpenoid moiety, H3-14′, H-7′, H-6′, H-5′, H3-16′, and H-12′ were deduced to be cofacial by the NOE correlations of H3-14′/H7′, H-7′/H-6′, H-6′/H-5′, H-6′/H3-16′, and H3-16′/H-12′ (Figure 2). Thus, the structure of compound 1 was elucidated as N-[5α-hydroxy-3β-methoxy-1-tremulen-11(12)-olide-12α-]huperzine A, and 1 was named huptremule A. The molecular formula of huptremule B (2) was established as C30H38N2O5 based on the HRESIMS quasi-molecular ion [M + H]+ at m/z 507.2866 (calcd for C30H39N2O5, 507.2859) and the 13C NMR data. The NMR spectra of 2 showed similar signals to those of 1, with the exception of the disappearance of the methoxy signals and the shielded C-12′ in 2 compared with 1. Hence, the structure of huptremule B (2) was proposed to be N-[3β,5α-dihydroxy-1-tremulen-11(12)-olide-12α-]huperzine A and was confirmed by 2D NMR (Figure 1). Huptremule C (3), a white powder, was assigned the molecular formula C30H40N2O4 based on the HRESIMS quasimolecular ion [M + H]+ at m/z 493.3079 (calcd for C30H41N2O4, 493.3066) and the 13C NMR data. The NMR spectra of 3 displayed signals characteristic of both 1 and 2, suggesting that 3 was also a sesquiterpenoid−alkaloid hybrid. After excluding the signals belonging to the hupA residue, typical NMR signals of a tremulane sesquiterpenoid moiety were evident. The structure of the sesquiterpenoid moiety was identified as a 3-hydroxy-12-demethoxyceriponol L residue by comparing the NMR data with those for ceriponol L23 and was verified by interpreting the 1H−1H COSY and HBMC correlations (Figure 1). 1H−1H COSY plots of H-7′/H2-8′ and H3-13′/H-6′/H-5′/H2-4′ suggested the presence of two partial fragments, CH(7′)−CH2(8′) and CH3(13′)−CH(6′)− CHOH(5′)−CH2(4′), respectively. On the basis of the molecular formula, the HMBC correlations from H2-4′ and H-12′ (δH 4.39) to the oxygenated tertiary carbon (δC 80.2) implied the presence of a hydroxy group at C-3′. The two moieties of 3 were linked similarly as in 1 and 2 based on the characteristic hemiaminal resonances (δH 4.39, s; δC 93.3) in the sesquiterpenoid moiety, the chemical shifts of C-12, C-13, and C-14 in the hupA residue, and the NOE correlations of H12′/H-11 and H-12′/H2-14 (Supporting Information). The NOESY spectrum of 3 (Figure 2) displayed correlations of H314′/H-7′ and H-7′/H-6′, indicating a β-orientation of H-6′ and H-7′ and, consequently, an α-orientation of H3-13′ and H-5′ based on the NOE correlation of H-5′/H 3 -13′. The configurations at C-3′ and C-12′ could not be determined by

NOESY and remained undefined. Hence, compound 3 was deduced to possess the structure N-(3,5β-dihydroxy-11,12epoxy-1-tremulene-12-)huperzine A and was named huptremule C. Huptremule D (4) was obtained as an isomer of huptremule C (3) with the same molecular formula, C30H40N2O4, based on HRESIMS data (m/z 493.3079 [M + H]+, calcd for C30H41N2O4, 493.3066) and the 13C NMR data. Comparing the NMR data of 3 and 4 indicated that the structure of huptremule D (4) was closely related to that of huptremule C (3). The key differences in the NMR data for 3 and 4 involved the resonances attributed to an oxygenated methine (δH 3.57, d, J = 9.5 Hz; δC 81.7 for 4 and δH 4.11, m; δC 74.7 for 3) of the sesquiterpenoid moiety, suggesting a different location of the oxygenated methine in 4. The 1H−1H COSY correlation between H-7′ and the oxygenated methine proton (δH 3.57, d (9.5)) and the HMBC correlations from H3-14′ and H3-15′ to the oxygenated methine carbon (δC 81.7), along with the molecular formula, indicated a hydroxy group at C-8′ (Figure 1). The NOE correlations of H3-14′/H-7′ and H-7′/H-6′ suggested a β-orientation of these protons. Thus, H-8′ was in an α-orientation based on the correlation of H-8′/H3-15′. As in the case of huptremule C (3), the configurations at C-3′ and C12′ could not be determined. Thus, the structure of compound 3 was established as N-(3,8β-dihydroxy-11,12-epoxy-1-tremulene-12-)huperzine A, and 3 was named huptremule D. The known compounds were identified as 8α,15αepoxyhuperzine A (5),18 tremulenediol A (6),21 conocenol B (7),24 and 11,12-dihydroxy-1-tremulen-5-one (8)25 by comparing their spectroscopic data with reported data. In our previous work, the fungus C. lacerate HS-ZJUT-C13A was isolated as a fungal endophyte from the medicinal plant H. serrata and was found to produce a series of tremulane sesquiterpenoids and lanostane triterpenoids.22,23,26 In the present work, hupA was subjected to biotransformation by this fungus. Interestingly, the transformed products, huptremules A−D (1−4), featured unusual sesquiterpenoid−alkaloid hybrid structures that integrated the characteristics of fungal metabolites (tremulane sesquiterpenoids) and the exogenous substrate (hupA). 8α,15α-Epoxyhuperzine A (5), the known hupA derivative reported to protect PC12 cells against SNPinduced apoptosis,27 was obtained with a yield (>27%) higher than reported.18 The AChE inhibitory activities of 1−5 were significantly lower than that of hupA (Table 3). However, huptremules A−D (1−4) share similar structural features with ZT-1, a sustained-release prodrug of hupA in a clinical trial for the treatment of Alzheimer’s disease.28 Further studies would reveal whether they possess the same pharmacological and pharmacokinetic properties as ZT-1. Table 3. Acetylcholinesterase Inhibitory Activity of Compounds 1−5a compound 1 2 3 4 5

IC50 (μM) 0.99 2.17 0.11 0.06 12.11

± ± ± ± ±

0.03 0.05 0.01 0.00 0.34

HupA was used as a positive control with an IC50 value of 0.54 ± 0.02 nM. a

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activity was measured spectrophotometrically using a Bio-RAD iMark microplate reader. All reagents used for biological evaluation were provided by Sigma-Aldrich. All solvents used were of analytical grade and obtained from commercially available sources. Substrate. The substrate (hupA) was isolated and purified from H. serrata by Runyu Biotechnology Co., Ltd. in Shanxi Province, China. Its purity was determined to be 99% by HPLC analysis. Fungal Strain and Culture Media. The fungus was isolated from the medicinal plant H. serrata collected in Pan-An County, Zhejiang Province, China, in July 2010.22,23,26 It was identified as C. lacerate based on DNA sequence analysis conducted by Sangon Biotech (Shanghai) Co. Ltd. The original culture (voucher number HS-ZJUTC13A) was deposited at the China Center for Type Culture Collection with the deposit number CCTCC M 2012433. All cultures were maintained on potato dextrose agar (PDA) slants and stored in a refrigerator at 4 °C until use. Prior to biotransformation, the fungi were precultivated on PDA in Petri dishes for 6 days at 28 °C. Fermentation and Biotransformation. The preparative-scale fermentation and biotransformation were performed in liquid potatodextrose medium (200 g of potato extracts, 20 g of glucose, 1 L of distilled H2O). A four-day-old seed culture was used to inoculate 350 Erlenmeyer flasks (250 mL containing 100 mL of medium), which were cultivated at 28 °C on rotary shakers (185 rpm) for 6 days. Each flask was fed with 3 mg of hupA (dissolved in EtOH) and incubated for another 20 days without agitation. Thus, the total culture volume was 35 L, and a total of 1.05 g of hupA was fed as substrate. Extraction and Isolation. At the end of the fermentation and biotransformation, the cultures were filtered through cheesecloth and centrifuged to separate the mycelium. The supernatant was condensed at 40 °C to approximately 3 L. The final pH of the condensed supernatant was adjusted with ammonia to approximately 10 and extracted with CHCl3 (2 L × 5). The CHCl3-soluble fraction (8.3 g) was subjected to CC on MCI gel (4 × 40 cm) to yield seven main fractions (Fr. A−G) after elution with a MeOH−H2O gradient (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, v/v, 1 L of each gradient solution). Fr. A was purified on Fractogel HW-40C gel (2 × 75 cm) eluted with MeOH to furnish 5 (310 mg). Fr. D was first separated on silica gel CC (3.5 × 30 cm) eluted with CHCl3−MeOH (15:1, v/v), followed by successive CC on Fractogel HW-40C (2 × 75 cm) eluted with MeOH and ODS C-18 (2.5 × 40 cm) employing a MeOH−H2O gradient (50:50 and 55:45, v/v, 400 mL of each gradient solution) as the eluent to give 4 (3.6 mg), 6 (5.1 mg), and 7 (7.9 mg). Fr. E was also purified by silica gel CC (3.5 × 25 cm) eluted with CHCl3−MeOH (15:1, v/v), followed by successive CC on Fractogel HW-40C eluted with MeOH and ODS C-18 (2.5 × 40 cm) eluted with a MeOH−H2O gradient (65:35, 70:30, 75:25, and 80:20, v/v, 400 mL of each gradient solution) to afford 1 (4.0 mg), 2 (2.4 mg), 3 (2.4 mg), and 8 (14.7 mg). All products (1−8) were identified on the basis of spectroscopic data. The 1H and 13C NMR data are presented in Tables 1 and 2. Huptremule A (1): white powder; [α]20 D +55 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 211 (3.40), 236 (3.40), 313 (3.59) nm; IR νmax 3390, 2929, 2862, 1658, 1378, 1350, 1303, 1184, 1057, 804 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS [M + H]+ m/z 521.3017 (calcd for C31H41N2O5, 521.3017). Huptremule B (2): white powder; [α]20 D +83 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 211 (3.58), 232 (3.58), 310 (3.52) nm; IR νmax 3374, 2926, 2857, 1657, 1612, 1401, 1032, 1049, 837 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; HRESIMS [M + H]+ m/z 507.2866 (calcd for C30H39N2O5, 507.2859). Huptremule C (3): white powder; [α]20 D −50 (c 0.02, CHCl3); UV (MeOH) λmax (log ε) 244 (3.62), 310 (3.71) nm; IR νmax 3389, 2924, 2858, 1653, 1610, 1453, 1298, 1250, 1109, 1064, 1014, 924, 840 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS [M + H]+ m/z 493.3079 (calcd for C30H41N2O4, 493.3066). Huptremule D (4): white powder; [α]20 D +7 (c 0.03, CHCl3); UV (MeOH) λmax (log ε) 221 (3.36), 233 (3.36), 310 (3.55) nm; IR νmax 3363, 2921, 2854, 1653, 1609, 1453, 1298, 1250, 1109, 1064, 1014, 924, 840 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS [M + H]+ m/z 493.3079 (calcd for C30H41N2O4, 493.3066).

In recent years, fungi of the genus Ceriporia have shown promise in the field of environmental bioremediation,29−32 because they can produce a panel of extracellular enzymes such as laccase, lignin peroxidase, and manganese peroxidase.33 Laccases are ligninolytic enzymes with biological functions ranging from depolymerization of lignin, coal, and humic acids to polymerization reactions and pigment formation in microbial cells or spores.34 Laccases have also gained attention for their versatile capabilities in catalyzing C−O, C−S, or C−N coupling reactions between two different molecules to create new hybrid structures.34 Laccases can accept a wide range of compounds as substrates.35 Hence, it was presumed that the formation of huptremules A−D (1−4) may be catalyzed by laccases or laccase-like enzymes, although neither tremulane sesquiterpenoids nor hupA possesses the classical structural features of a laccase substrate. Given the potential of such coupling reactions in expanding the chemical diversity of natural products, indepth studies are warranted to clarify the underlying enzymes responsible for the generation of huptremules A−D (1−4). These enzymes may serve as important biocatalysts for combinatorial chemical synthesis of hybrid structures. The fungal endophyte C. lacerate HS-ZJUT-C13A seems to have a broad ability to transform hupA, one of the main constituents of its host plant H. serrata, to produce products with unique structures. This ability is likely a product of the endophytic nature of this fungus. Two characteristics of endophytes suggest their promise as new sources of biocatalysts for the biotransformation of natural products, particularly those derived from the host plant. First, endophytes can produce secondary metabolites with novel and diverse structures, indicating that the biosynthetic pathways of those compounds contain enzymes with versatile functions. The in vitro application of these enzymes in the biotransformation of natural products may also lead to diverse transformed products. Second, endophytes likely suffer environmental stress from the defense constituents of the host plants. As a consequence, they may produce a series of specific enzymes for detoxification, e.g., transforming the toxic plant constituents to nontoxic substances. Such an endophyte self-defense mechanism may also contribute to their capability in transforming natural products, particularly those originating from the host plant. Similarly, microorganisms with petroleum oil-degrading abilities have been detected in depleted oil wells or oil-polluted soil where environmental stress was imposed by related chemicals. Hence, screening endophytes for microorganisms that can potently transform the host-derived natural product represents a type of intellectual selection.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained with a Rudolph Research Autopol III automatic polarimeter. UV spectra were measured on a Shimadzu UV-2450 spectrometer. IR spectra were recorded on a Thermo Nicolet 6700 FT-IR microscope instrument (FT-IR microscope transmission). 1D and 2D NMR spectra were obtained at 500 MHz for 1H and 125 MHz for 13C on a Bruker Avance 500 spectrometer, in methanol-d4 or CDCl3, with solvent peaks used as references. ESIMS and HRESIMS data were measured on an Agilent Technologies 6210 LC/TOF instrument. Column chromatography was performed with silica gel (200−300 mesh, Qingdao Marine Chemical Inc. Qingdao, China), Toyopearl HW-40C gel (Tosoh Corporation, Japan), and YMC ODS C-18 gel (50 μm, YMC Co. Ltd., Kyoto, Japan). TLC was performed on precoated silica gel GF254 plates. Spots were visualized by spraying with 10% H2SO4 in 95% EtOH followed by heating. AChE inhibitory 2058



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Acetylcholinesterase Inhibition Assay. The acetylcholinesterase inhibitory activities of compounds 1−5 were assayed according to Ellmann’s method36 with slight modifications, employing hupA as a positive control. Briefly, 100 μL of 0.1 M Na3PO4 buffer (pH = 8.0), 20 μL of enzyme solution (50 mU/mL in buffer), and 20 μL of sample diluted in 0.2% DMSO−buffer solution were added to a microplate and incubated at 37 °C for 20 min. Then, 40 μL of 0.6 mM DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] was added. The reaction was initiated by adding 20 μL of 1.2 mM acetylthiocholine. After incubating the reaction solution at 37 °C for 30 min, the optical densities were immediately measured in a 96-well plate reader at 405 nm. The percentage inhibition was evaluated using the equation I (%) = [1 − (Asample − Abackground)/Ablank] × 100%, where Asample is the absorbance of each test compound, Ablank is the absorbance of the blank without test compound, and Abackground is the absorbance of the background without enzyme. The concentrations of test samples that inhibited the hydrolysis of acetylthiocholine by 50% (IC50) were determined by monitoring the effect of increasing concentrations of these samples in assays on the inhibition values and are presented as means ± SD of three individual determinations, each performed in triplicate.

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(12) Shibuya, H.; Kitamura, C.; Maehara, S.; Nagahata, M.; Winarno, H.; Simanjuntak, P.; Kim, H. S.; Wataya, Y.; Ohashi, K. Chem. Pharm. Bull. 2003, 51, 71−74. (13) Agusta, A.; Maehara, S.; Ohashi, K.; Simanjuntak, P.; Shibuya, H. Chem. Pharm. Bull. 2005, 53, 1565−1569. (14) Verza, M.; Arakawa, N. S.; Lopes, N. P.; Kato, M. J.; Pupo, M. T.; Said, S.; Carvalho, I. J. Braz. Chem. Soc. 2009, 20, 195−200. (15) Fu, S. B.; Yang, J. S.; Cui, J. L.; Feng, X. Chem. Pharm. Bull. 2011, 59, 1180−1182. (16) Kozikowski, A. P.; Tückmantel, W. Acc. Chem. Res. 1999, 32, 641−650. (17) Ma, X. Q.; Tan, C. H.; Zhu, D. Y.; Gang, D. V.; Xiao, P. G. J. Ethnopharm. 2007, 113, 15−34. (18) Zhang, X. Y.; Zou, J. H.; Dai, J. G. Tetrahedron. Lett. 2010, 51, 3840−3842. (19) Donova, M. V.; Egorova, O. V. Appl. Micribiol. Biotechnol. 2012, 94, 1423−1447. (20) Parshikov, I. A.; Netrusov, A. I.; Sutherland, J. B. Appl. Micribiol. Biotechnol. 2012, 95, 871−889. (21) Ayer, W. A.; Cruz, E. R. J. Org. Chem. 1993, 58, 7529−7534. (22) Ying, Y. M.; Shan, W. G.; Zhang, L. W.; Zhan, Z. J. Phytochemistry 2013, 95, 360−367. (23) Shan, W. G.; Liang, D. E.; Ying, Y. M.; Zhan, Z. J. J. Chem. Res. 2012, 36, 15−16. (24) Liu, Z. D.; Wang, F.; Liu, J. K. J. Nat. Prod. 2007, 70, 1503− 1506. (25) Zhou, Z. Y.; Tang, J. G.; Wang, F.; Dong, Z. J.; Liu, J. K. J. Nat. Prod. 2008, 71, 1423−1426. (26) Ying, Y. M.; Shan, W. G.; Zhang, L. W.; Chen, Y.; Zhan, Z. J. Helv. Chim. Acta 2013, 96, 2092−2097. (27) Ning, N.; Hu, J. F.; Yuan, Y. H.; Zhang, X. Y.; Dai, J. G.; Chen, N. H. Acta. Pharmacol. Sin. 2012, 33, 34−40. (28) Wei, G. L.; Xiao, S. H.; Lu, R.; Liu, C. X. J. Chromatogr. B 2006, 830, 120−125. (29) Lin, Y. H.; He, X. B.; Han, G. M.; Tian, Q. J.; Hu, W. Y. J. Environ. Sci-China 2011, 23, 2055−2062. (30) Suharar, H.; Daikoku, C.; Takata, H.; Suzuki, S.; Matsufuji, Y.; Sakai, K.; Kondo, R. Appl. Microbiol. Biotechnol. 2003, 62, 601−607. (31) Tang, L.; Wei, W.; Wang, Y. W.; Deng, W.; Li, Z. G. Indian J. Biochem. Biophys. 2010, 47, 348−352. (32) Xu, L.; Zhu, Y.; He, X. B.; Han, G. M.; Tian, X. J. World J. Microbiol. Biotechnol. 2008, 24, 3097−3104. (33) Hong, C. Y.; Kim, H. Y.; Lee, S. Y.; Kim, S. H.; Lee, S. M.; Choi, I. G. J. Environ. Sci. Health A 2013, 48, 1280−1291. (34) Mikolasch, A.; Schauer, F. Appl. Microbiol. Biotechnol. 2009, 82, 605−624. (35) Jeon, J. R.; Chang, Y. S. Trends Biotechnol. 2013, 31, 335−341. (36) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88−90.

ASSOCIATED CONTENT

* Supporting Information S

1D and 2D NMR, HRESIMS, IR, and UV spectra of 1−4. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(W.-G. Shan) Tel: +86-0571-88320554. Fax: +86-057188320913. E-mail: [email protected]. *(Z.-J. Zhan) Tel: +86-0571-88871030. Fax: +86-057188871075. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was financially supported by the National Natural Science Foundation of China (No. 21402174), the Foundation of Zhejiang Educational Committee (No. Y201329702), and the Scientific Foundation of Zhejiang University of Technology (No. 1301116075408).

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REFERENCES

(1) Petrini, O. In Microbial Ecology of Leaves; Andrews, J.; Hirano, S., Eds.; Springer-Verlag: New York, 1991; pp 179−497. (2) Aly, A. H.; Debbab, A.; Proksch, P. Appl. Micribiol. Biotechnol. 2011, 90, 1829−1845. (3) Aly, A. H.; Debbab, A.; Proksch, P. Pharmazie 2013, 68, 499− 505. (4) Kharwar, R. N.; Mishra, A.; Gond, S. K.; Stierle, A.; Stierle, D. Nat. Prod. Rep. 2011, 28, 1208−1228. (5) Prado, S.; Li, Y. Y.; Nay, B. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier: The Netherlands, 2012; pp 249−296. (6) Strobel, G.; Daisy, B.; Castillo, U.; Harper, J. J. Nat. Prod. 2004, 67, 257−268. (7) Tan, R. X.; Zou, W. X. Nat. Prod. Rep. 2001, 18, 448−459. (8) Zhang, H. W.; Song, Y. C.; Tan, R. X. Nat. Prod. Rep. 2006, 23, 753−771. (9) Suryanarayanan, T. S.; Thirunavukkarasu, N.; Govindarajulu, M. B.; Gopalan, V. Fungal Diversity 2012, 54, 19−30. (10) Warley de, S. B.; Keyller, B. B.; Pierina, S. B.; Suraia, S.; Mônica, T. P. Curr. Org. Chem. 2009, 13, 1137−1163. (11) Kumar, A.; Ahmad, A. Biocatal. Biotransfor. 2013, 31, 89−93. 2059


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