Microbial transformation for Region-selective Hydroxylation of Z-Butylidenephthalide by Aspergillus niger CGMCC 3.739

Jin Zou,Yongheng Wng,Zhengqun Zhou,Rongrong He,c＊,Xinsheng Yo,Ho Go＊

a Institute of Traditional Chinese Medicine &Natural Products,College of Pharmacy/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research,Jinan University,Guangzhou 510632,China;

b Integrated Chinese and Western Medicine Postdoctoral Research Station,Jinan University,Guangzhou 510632,China;

c School of Traditional Chinese Medicine,Jinan University,Guangzhou 510632,China

Abstract Microbial transformation is a useful tool for structure modification of active natural products.Z-Butylidenephthalide is the main active constituent of Angelica sinensis.In the present work,a pair of new oxidized metabolites,namely (–) (11R)-(Z)-11-hydroxybutylidenephthalide (1a) and (+) (11S)-(Z)-11-hydroxy-butylidenephthalide (1b),were obtained from microbial transformation for region-selective 11-hydroxylation of Z-butylidenephthalide by Aspergillus niger CGMCC 3.739.Their structures,including absolute configurations,were elucidated by extensive analysis of HR-ESI-MS,NMR spectra,and the modified Mosher’s method.This is the first study on the microbial transformation of Z-butylidenephthalide,and these findings offer a tool for region-selective 11-hydroxylation of Z-butylidenephthalide.

Keywords:microbial transformation;Z-butylidenephthalide;Aspergillus niger CGMCC 3.739;region-selective hydroxylation

1 Introduction

Microbial transformation is a remarkable tool for the generation of pharmaceutical lead compounds from natural products in recent years [1].A lot of natural products,including flavonoids,terpenoids,alkaloids,and steroids,can be transformed by multi-enzyme systems in the microorganisms [2-5].Biochemical reactions,including hydroxylation,hydrogenation,and methylation,are typically stereoand regio-selective,which are difficult to achieve by chemical methods [4].Microbial transformation can improve the chemical properties of active compounds and provide candidates for novel drug development,especially for hydroxylation.For example,microbial transformation of artemisinin by

Cunninghamella echinulata

and

Aspergillus niger

efficiently yielded specific hydroxylation metabolites [6].

Angelica sinensis

(Dang-gui in Chinese),belonging to the family Apiaceae,

has been used as a Traditional Chinese Medicine (TCM) for the treatment of gynecological disorders for thousands of years in China [7].

It has also been marketed as a dietary supplement in many countries and regions,including China,Japan,Korea,Malaysia,Indonesia,Europe,and United States [8,9].

Z

-Butylidenephthalide is the main active constituent of

Angelica sinensis

and has been reported to exhibit a series of interesting bioactivities,including anticancer,anti-inflammatory,anti-Alzheimer’s disease(AD),and anti-fungal [10-14].In our preliminary research on Dang-gui,abundant phthalide monomers(such as

Z

-ligustilide and

Z

-butylidenephthalide)and six pairs of active phthalide polymers with new skeleton were isolated [15,16].In order to find more diversified phthalide derivatives,the microbial transformation of

Z

-butylidenephthalide was studied.Twenty-four strains were screened for their capacities to transform

Z

-butylidenephthalide.Screening results showed that a strain of

Aspergillus niger

CGMCC 3.739 specifically converted

Z

-butylidenephthalide into one metabolite (Fig.1).Besides,one pair of regio-selective 11-hydroxylation transformation products were obtained,namely (–)(11

R

)-(

Z

)-11-hydroxy-butylidenephthalide (1a) and(+) (11

S

)-(

Z

)-11-hydroxy-butylidenephthalide (1b).The absolute configuration of compound 1b was determined by the modified Mosher’s method.

Fig.1 HPLC profile of the biotransformed product (1) from Z-butylidenephthalide (tR=28.5 min) by Aspergillus niger CGMCC 3.739

2 Experimental

2.1 General experimental procedures

Optical rotations were measured on a JASCO P1020 digital polarimeter (Jasco International Co.Ltd,Tokyo,Japan),and UV data were recorded on a JASCO V-550 UV/VIS spectrometer (Jasco International Co.,Ltd,Tokyo,Japan).IR data were recorded using a JASCO FT/IR-480 plus spectrometer (Jasco International Co.,Ltd,Tokyo,Japan).ESI-IT-MS spectra were obtained on a Finnigan LCQ Advantage MAX mass spectrometer(Thermo Fisher Scientific,Inc.,Waltham,MA,USA),and HR-ESI-MS spectra were obtained on Waters Synapt G2 mass spectrometer (Waters Corporation,Milford,MA,USA).H andC-NMR spectra were acquired with Bruker AV 600 (Bruker BioSpin Group,Faellanden,Switzerland) using solvent signal (CDCl:

δ

7.26/

δ

77.0) as the internal standard.The analytical HPLC was performed on a Shimadzu HPLC (Shimadzu Co.,Ltd.,Kyoto,Japan) system equipped with an LC-20AB pump,an SPD-M20A diode array detector,a SIL-20A autosampler using a Cosmosil-Ccolumn (4.6 mm ×250 mm,5

μ

m,Nacalai Tesque Inc.,Kyoto,Japan) and a Phenomenex Lux™ Amylose-2 column (4.6 mm ×250 mm,5

μ

m,Phenomenex Inc.,Los Angeles,CA,USA).Semi-preparative HPLC was carried out on a Shimadzu LC-6AD (Shimadzu Co.,Ltd.,Kyoto,Japan) system equipped with a UV detector using a YMC-Ccolumn (10.0 mm × 250 mm,5

μ

m,YMC Co.,Ltd.,Kyoto,Japan) and a Phenomenex Lux™ Amylose-2 column (4.6 mm ×250 mm,5

μ

m,Phenomenex Inc.,Los Angeles,CA,USA).TLC was performed on precoated silica gel plates (SGF254,0.2 mm,Yantai Chemical Industry Research Institute,Yantai,China).

2.2 Substrate

The substrate

Z

-butylidenephthalide (with purity >98%) was isolated from the root of

Angelica sinensis

.Its structure was characterized by comparing theH-NMR andC-NMR data with the data reported in the literature [17].

2.3 Microorganism and culture medium

All 24 microorganism strains were obtained from Prof.Feng Qiu in Tianjin University of Traditional Chinese Medicine,Tianjin,China.These strains were screened for their abilities to transform

Z

-butylidenephthalide.They are

Absidia coerulea

CGMCC 3.3389,

Alternaria alternata

CGMCC 3.4578,

Alternaria longipes

CGMCC 3.2875,

Aspergillus cabonarius

CICC 2087,

Aspergillus flavus

CGMCC 3.3554,

Aspergillus fumigatus

CICC 2168,

Aspergillus niger

CGMCC 3.1858,

Aspergillus niger

CGMCC 3.739,

Aspergillus niger

CGMCC 3.795,

Cunninghamella blakesleana

CGMCC 3.970,

Cunninghamella elegans

CGMCC 3.1207,

Fusarium avenaceum

CGMCC 3.4594,

Mucor polymorphosporus

CGMCC 3.3443,

Mucor spinosus

CICC 40243,

Mucor subtilissimus

CGMCC 3.2454,

Paecilomyces variotii

CICC 4024,

Penicillium adametzii

CGMCC 3.4470,

Penicillium citrinum

CICC 4013,

Penicillium decumbens

CGMCC 3.5255,

Penicillium janthinellum

CGMCC 3.514,

Penicillium melinii

CGMCC 3.4474,

Penicillium urticae

CICC 4015,

Sporotrichum

sp.CGMCC 3.2882,and

Syncephalastrum racemosum

CGMCC 3.264.In an Eyela LTI-700 conventional incubator(Tokyo Rikakikai Co.Ltd,Tokyo,Japan),stock cultures were maintained at 4 °C on agar slants containing PDA (Potato Dextrose Agar).All initial screening experiments were performed in conical flasks (500 mL) containing 100 mL of PDB (Potato Dextrose Broth),which was sterilized by autoclaving at 121 °C for 30 min using an MLS3750 autoclave(Sanyo Electric Co.Ltd,Moriguchi,Japan).

2.4 Biotransformation of Z-butylidenephthalide and isolation of metabolites

The preliminary screening experiment of

Aspergillus niger

CGMCC 3.739 was carried out on potato dextrose medium.Fermentations were carried out according to two-stage fermentation.Stage I:the seed cultures were grown in 500 mL Erlenmeyer flasks containing 100 mL of medium and incubated at 28 °C and 180 rpm in the shaker (HDL Apparatus Co.Ltd,Beijing,China) for 3 d.Stage Ⅱ:10 mg of the substrate (dissolved in methanol) was added into the flask for another 3 d and harvested.The fermented broths of flasks were filtered,and then the filtrate was extracted with ethyl acetate (3 times).Mycelia were ultrasonically extracted with the same solvent for 40 min (3 times).The two extracts were combined and evaporated under reduced pressure on a rotary evaporator (38 °C) and then detected by analytical HPLC.Substrate controls with substrate but without fungi and culture controls with fungi but without substrate were incubated under the same conditions as described above.The preparative scale biotransformation of

Z

-butylidenephthalide by

Aspergillus niger

CGMCC 3.739 was carried out in two (3000 mL) flasks,each containing 500 mL of medium.After incubation for 3 d,a total of 100 mg of the substrate (dissolved in methanol) was added to the cultures,followed by incubation for another 3 d.The incubation conditions and extraction process were the same as those in the preliminary screening experiment.500 mg of the crude extract was separated on preparative HPLC (CHOH/HO,45 :55,3.0 mL/min,254 nm)to produce 1 (

t

:10.5 min,8.0 mg).Compound 1 was further separated on a Phenomenex Lux™Amylose-2 column (4.6 mm × 250 mm,5

μ

m)(CHCN-HO 30 :70,1 mL/min) to obtain 1a (

t

:10.1 min,1.0 mg) and 1b (

t

:11.1 min,1.5 mg).

2.4.1 (Z)-11-hydroxy-butylidenephthalide

CHO,colorless oil;UV (MeOH)

λ

(log

ε

):205 (5.80),235 (5.64),260 (5.59),311 (5.16) nm;IR (KBr)

v

:3416,2923,1721,1567,1383,1287 cm;ESI-MS (positive):

205 [M+H];HR-ESI-MS(positive):

m/z

205.0858 [M+H](calcd.for CHO,

m/z

205.0865);H (600 MHz,CDCl) andC (150 MHz,CDCl) NMR data are presented in Table 1.

(+) (11

S

)-(

Z

)-11-hydroxy-butylidenephthalide(1b):[

a

]=+10.4 (

c

0.5,MeOH);UV,NMR,and HR-ESI-MS are the same as those of 1.

2.5 Preparation of (S)-and (R)-MTPA esters of 1b

The solution of 1b (0.5 mg) in pyridine-

d

(0.5 mL) was treated with (

S

)-MTPA chloride(10

μ

L) under the atmosphere of nitrogen in an NMR tube.The mixture was stirred at room temperature for 4 h to obtain (

R

)-MTPA ester (1b2).The same procedure was used to prepare (

S

)-MTPA ester (1b1)with (

R

)-MTPA chloride.The Δ

δ

values (Δ

δ

H-12:-0.06012;Δ

δ

Ha-10:+0.07147;Δ

δ

Hb-10:+0.08049) of (

S

)-and (

R

)-MTPA esters of 1 (1b1 and 1b2) indicated the

S

configuration for C-11 in 1b.

3 Results and discussion

Compound 1 was isolated as colorless oil.Its molecular formula CHO(seven degrees of unsaturation) was determined by HR-ESI-MS at

m/z

205.0858 [M+H](calcd.for CHO,205.0865).TheC-NMR spectrum showed 12 carbon signals,which was consistent with the HRESI-MS deduction.Combined with the DEPT-135 and HSQC experiments (Table 1),these carbons were categorized into one carbonyl carbon (

δ

167.0),eight olefinic/aromatic carbons (

δ

147.0,139.3,134.3,129.7,125.3,124.5,119.9,and 105.0),one oxygenated spmethine carbon (

δ

67.6),one spmethylene carbon (

δ

35.4),and one methyl carbon (

δ

23.4).TheH-NMR spectrum revealed that 1 possessed five olefinic or aromatic protons[

δ

7.90 (

br

d

,

J

=7.7 Hz),7.69 (overlapped),7.68(overlapped),7.53 (

ddd

,

J

=7.7,6.0,1.7 Hz),and 5.76 (

t

,

J

=7.8 Hz)],one oxygenated methine proton[

δ

4.05 (m)],one methylene group Ha-10/Hb-10[

δ

2.69 (

ddd

,

J

=14.8,7.8,4.8 Hz)/2.62 (

dt

,

J

=14.8,7.8 Hz)],and one methyl group [

δ

1.30 (

d

,

J

=6.2 Hz)].Analysis of theH,H-COSY spectrum led to the identification of two subunits (C-3–C-4–C-5–C-6 and C-9–C-10–C-11–C-12) (Fig.3).Combined with the analysis ofH,H-COSY and unsaturation requirement,the key HMBC signals (Fig.3) from H-3 to C-1/C-7,from H-4 to C-2,from H-5 to C-7,from H-6 to C-2/C-8,from H-9 to C-7,and from Ha-10/Hb-10 to C-8 established its planar structure as shown in Fig.2.The geometry of the double bond of Δin 1 was assigned as

Z

configuration by the ROESY correlation between H-9 and H-6 (Fig.3).Thus,the structure of compound 1 was identified as (

Z

)-11-hydroxy-butylidenephthalide.The assignments of all proton and carbon resonances are presented in Table 1.

Fig.2 Chemical structures of 1

Fig.3 Key HMBC,1H-1H COSY,and ROESY correlations of compound 1

Table 1 1D and 2D NMR data (600 MHz for 1H,150 MHz for 13C) for compound 1 in CDCl3 (δ in ppm,J in Hz)

Fig.4 Chiral HPLC chromatogram of compound 1

Fig.5 Δδ(δS – δR) values (in ppm) obtained for (S)-and(R)-MTPA esters (1b1 and 1b2)

4 Conclusion

In conclusion,

Z

-butylidenephthalide was transformed by

Aspergillus niger

CGMCC 3.739 to specifically afford one pair of new hydroxylation metabolites,(–) (11

R

)-(

Z

)-11-hydroxy-butylidenephthalide (1a) and (+) (11

S

)-(

Z

)-11-hydroxy-butylidenephthalide (1b).This is the first report on the microbial transformation of

Z

-butylidenephthalide,and our discovery provided a new tool for the regio-selective 11-hydroxylation of

Z

-butylidenephthalide.

Acknowledgments

This work was financially supported by grants from the National Key Research and Development Program of China (2018 YFA 0903200/2018YFA0903201),the National Natural Science Foundation of China (81925037/ 31900284),Chang Jiang Scholars Program (Young Scholar) from the Ministry of Education of China (Hao Gao,2017),National High-level Personnel of Special Support Program (2017RA2259),Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y036),and K.C.Wong Education Foundation (Hao Gao,2016).