SUPPLEMENTARY MATERIAL
A New Glutarimide Derivative from Marine Sponge-Derived
Streptomyces anulatus S71
Dandan Sun1, Wei Sun1, Yinxian Yu2, Zhiyong Li1, Zixin Deng1, and
Shuangjun Lin1*
1
State Key Laboratory of Microbial Metabolism, School of Life Sciences
and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan
Road, Shanghai 200240, China
2
Department of Orthopedic Surgery, Shanghai Frist People's Hospital,
School of Medicine, Shanghai Jiao Tong University, 100 Haining Road,
Shanghai 200080, China
*Corresponding author: Prof. Shuangjun Lin
E-mail: linsj@sjtu.edu.cn
A New Glutarimide Derivative from Marine Sponge-Derived
Streptomyces anulatus S71
Four glutarimide-derived compounds including one new 3-[2-[2-hydroxy-3methylphenyl-5-(hydroxymethyl)]-2-oxoethyl] glutarimide (1) and three
known 3-[2-(2-hyroxy-3,5- dimethylphenyl)-2-oxoethyl] glutarimide (2,
Actiphenol), 3-hydroxy-3-[2-(2-hydroxy-3,5-dimethylphenyl)-2-oxoethyl]
glutarimide (3) and 3-[2-[2-hydroxy-3-(hydroxymethyl)-5-methylphenyl]-2oxoethyl] glutarimide (4), along with a known indole alkaloid 3-(2Hydroxyacetyl) indole (5) were isolated from ethyl acetate extract of the
fermentation broth of the marine sponge-derived Streptomyces anulatus S71.
Their structures were deduced by extensive studies of NMR and mass spectra.
Keywords: marine sponge; actinomycete; glutarimide; Streptomyces anulatus
S71; polyketide synthase
1. Experimental
1.1. General
The strain S71 was grown at 28 °C in M1 agar plate, and GYM4 medium for
production of secondary metabolites containing 10 g glucose, 4 g yeast extract, 4 g
malt extract, 1 liter water, and adjusted to pH7.2 before autoclave.
Structures of the isolated compounds were elucidated by NMR spectroscopy
and mass spectrometry. 1H (500 MHz) and 13C NMR (125 MHz) spectra were
recorded on Bruker AV 500 instruments. LC-QTOF-MS analysis was performed on
an Agilent 1200 series LC/MSD trap system in tandem with a 6530 Accurate-Mass QTOF MASS Spectrometer with an ESI source (600-1500 m/z mass range, positive
mode). Chromatography was carried out on silica gel (200-300 mesh, Qingdao
Haiyang Chemical Factory), Sephadex LH-20 (GE Healthcare), reversed-phase silica
gel (YMC*GEL C18) and reversed-phase HPLC (Agilent 1260, ZORBAX SB-C18, 5
μm, 4.6x150 mm), respectively.
1.2. Isolation of actinomycetes
Five culture media were used for the isolation of sponge-associated actinomycetes.
Compositions of these media are given in Table S5. All media were supplemented
with K2Cr2O7 (50 µg ml-1) to inhibit fungi and nalidixic acid (15 µg ml-1) to inhibit
Gram-negative bacteria. Sponge samples were rinsed with sterile artificial seawater
(26.52 g NaCl, 5.228 g MgCl2 6H2O, 3.305 g MgSO4, 1.141 g CaCl2, 0.725 g KCl,
0.202 g NaHCO3, 0.083 g NaBr, 1L distilled water) to remove the bacteria loosely
attached on the surface. Subsequently, a few tissue cubes were excised from different
sections (cortex and endosome) of the sponge samples. They were cut into pieces and
aseptically ground using sterilized pestles and mortars. Actinomycetes were isolated
by means of serial dilution and plating techniques. The inoculated plates were
incubated at 28 °C for 3–6 weeks. The colonies bearing distinct morphological
characteristics were picked up and transferred onto freshly prepared media until pure
cultures were obtained. Strain S71 was picked up from M1 medium.
1.3. Sequence comparison and phylogenetic analysis
The 16S rRNA gene sequence of S71 was proofread using Chromas, version 1.62
(Technelysium), and compared with the sequences available in NCBI
(http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool
(BLAST). Multiple sequence alignment was performed using CLUSTALX.
Phylogenetic tree was constructed using Mega 4.1. The consistency of the tree was
verified by bootstrapping (1,000 replicates) for parsimony (Figure S6).
1.4. Fermentation, Extraction and Isolation
To analyse the products of the strain S71, a two-stage fermentation process was
adopted. Briefly, a spore collection (50 μL) of the strain S71 was inoculated into 50
mL of GYM4 medium in a 250 mL flask and incubated at 28 °C and 220 rpm for 1
day, and 10 mL GYM4 cultured broth was transferred into 500 mL of GYM4
production medium in a 2 L which was flask incubated at the same cultured
conditions for 7 days before extraction.
The culture broth (66 L in total) was centrifuged at 6000 rpm for 20 min. The
culture supernatant was extracted with ethyl acetate for three times. The mycelial cake
was extracted with acetone. After removal of acetone, the aqueous solution was
extracted three times with ethyl acetate. The organic extracts from culture supernatant
and mycelia were combined and concentrated in vacuo to dryness.
The crude extract (12.5 g) dissolved in methanol was subjected to silica gel
flash column. The separation was accomplished by a step gradient from Petroleum
ether-CHCl3 (1:1), CHCl3 to CHCl3-CH3OH (2:1) to give 12 fractions. Fraction 5-7
(1.1 g) was purified by silica gel column which was eluted with Petroleum etherEtOAc (2:1-1:5) and Sephadex LH-20 column chromatography (CH3OH) to yield
compound 2 (45 mg). Fraction 8 (6.3 g) was purified by silica gel column eluted with
Petroleum ether-EtOAc (2:3-1:5), Sephadex LH-20 column chromatography
(CH3OH) to yield compound 4 (30 mg) , and HPLC (ACN-H2O）to obtain
compounds 3 (5 mg) and 5 (3 mg). Fraction 9 (1.9 g) was purified by reversed-phase
silica gel column eluted with CH3OH-H2O, Sephadex LH-20 column chromatography
(CH3OH) and HPLC (ACN-H2O）to yield compound 1 (3mg).
Compound 5: HR-ESI-MS m/z [M+H]+ 176.0706 ; 1H NMR (DMSO-d6,
400MHz) δ 12.0 (brs), 8.35(s, 1H), 8.17(dd, J=8.0, 2.4Hz, 1H), 7.45(dd, J=8.0, 2.4Hz,
1H), 7.22(m, 2H), 4.58(d, J=6.0Hz, 2H).
References
Abdelmohsen UR, Pimentel-Elardo SM, Hanora A, Radwan M, Abou-El-Ela SH,
Ahmed S, Hentschel U. 2010. Isolation, phylogenetic analysis and antiinfective activity screening of marine sponge-associated actinomycetes. Mar
Drugs. 8:399-412.
Mincer TJ, Jensen PR, Kauffman CA, Fenical W. 2002. Widespread and Persistent
Populations of a Major New Marine Actinomycete Taxon in Ocean
Sediments. Appl Environ Microbiol. 68:5005-5011.
Zhang H, Lee YK, Zhang W, Lee HK. 2006. Culturable actinobacteria from the
marine sponge Hymeniacidon perleve: isolation and phylogenetic diversity by
16S rRNA gene-RFLP analysis. Antonie van Leeuwenhoek. 90:159-169.
Table S1. NMR data for compound 1 in DMSO-d6 (δ ppm, 1H NMR 400MHz, 13C NMR
100MHz)
Position
δH(multi, Hz)
1
2
δC
172.8 s
2.59 (dd, J=16.0, 4.0Hz,
2H)
2.40 (dd, J=16.0,
10.0Hz, 2H)
37.0 t
3
2.66 (m, 1H)
25.9 d
4
3.20 (d, J=6.5Hz, 2H)
42.2 t
5
205.3 s
6
118.0 s
7
158.7 s
8
125.9 s
9
7.42 (s, 1H)
10
136.1 d
126.0 s
11
7.71 (s, 1H)
132.5 d
12
2.18 (s, 3H)
15.2 q
13
4.42 (d, J=3.5Hz, 2H)
62.2 t
7-OH
12.33 (s, 1H)
NH
10.78 (s, 1H)
Table S2. NMR data for compound 2 in DMSO-d6 (δ ppm, 1H NMR 500MHz, 13C NMR
125MHz)
Position
δH(multi, Hz)
1
2
δC
172.9 s
2.50 (dd, J=16.5, 4.0Hz,
2H)
2.38 (dd, J=16.5,
10.5Hz, 2H)
37.0 t
3
2.64 (m, 1H)
26.0 d
4
3.18 (d, J=6.5Hz, 2H)
42.2 t
5
205.3 s
6
118.3 s
7
157.2s
8
138.3 s
9
7.27 (s, 1H)
10
138.2 d
127.2 s
11
7.58 (s, 1H)
127.8 d
12
2.14 (s, 3H)
15.1 q
13
2.24 (s, 3H)
19.9 q
7-OH
12.24 (s, 1H)
NH
10.78 (s, 1H)
Table S3. NMR data for compound 3 in DMSO-d6 (δ ppm, 1H NMR 400MHz, 13C NMR
100MHz)
Position
δH(multi, Hz)
δC
1
171.8 s
1’
174.4 s
2
4.05 (dd, J=8.0, 5.5Hz,
1H)
70.3 d
5.79 (d, J=5.5Hz, OH)
2’
2.62 (m,2H)
36.0 t
3
2.60 (m, 1H)
33.6 d
4
3.47 (dd, J=17.0, 3.0Hz,
1H)
3.03 (dd, J=17.0, 8.0Hz,
1H)
40.4 t
5
205.7 s
6
118.4 s
7
157.7 s
8
126.0 s
9
7.29 (s, 1H)
10
138.2 d
127.1 s
11
7.58 (s, 1H)
127.8 d
12
2.15 (s, 3H)
15.1 q
13
2.25 (s, 3H)
20.0 q
7-OH
12.26 (s, 1H)
NH
10.83 (s, 1H)
Table S4. NMR data for compound 4 in DMSO-d6 (δ ppm, 1H NMR 500MHz, 13C NMR
125MHz)
Position
δH(multi, Hz)
1
2
δC
172.8 s
2.59 (dd, J=16.0, 4.0Hz,
2H)
2.38 (dd, J=16.0,
10.0Hz, 2H)
37.0 t
3
2.65 (m, 1H)
26.0 d
4
3.19 (d, J=6.5Hz, 2H)
42.3 t
5
205.4 s
6
118.2 s
7
156.4 s
8
127.2 s
9
7.48 (s, 1H)
10
134.9 d
128.4 s
11
7.64 (s, 1H)
130.7 d
12
4.5 (s, 2H)
57.2 t
13
2.29 (s, 3H)
20.2 q
7-OH
12.16 (s, 1H)
NH
10.78 (s, 1H)
Table S5. Composition of 5 media for the isolation of actinomycetes from the
South China Sea sponges
Medium formula
Reference
M1 10 g soluble starch, 4 g yeast extract, 2 g peptone,
(Mincer et al. 2002)
18 g agar, and 1 l of artificial seawater
M2 2 ml glycerol, 8 g yeast extract, 4 g malt extract,
This study
10 g manitol, 10 g glucose, 0.2 mg ZnSO4, 0.2 mg
MnSO4, 0.2 mg CuSO4, 2 mg FeSO4, 18 g agar, and 1
l of artificial seawater
M3 0.1 g L-asparagine, 0.5 g K2HPO4, 0.001 g
(Zhang et al. 2006)
FeSO4, 0.1 g MgSO4, 2 g peptone, 4 g sodium
propionate, 18 g agar, and 1 l of artificial seawater
M4 0.5 g yeast extract, 0.25 g tryptone, 0.75 g
(Abdelmohsen et al. 2010)
peptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g
K2HPO4, 0.024 g MgSO4, 0.3 g sodium propionate, 18
g agar, and 1 l of artificial seawater
M5 6 ml glycerol, 1 g L-arginine, 1 g K2HPO4, 0.5 g
MgSO4, 18 g agar, and 1 l of artificial seawater
(Mincer, et al. 2002)
Figure S1. IR spectrum of compound 1
Figure S2. 1H NMR spectrum of compound 1 (DMSO-d6, 400MHz)
Figure S3. 13C NMR spectrum of compound 1 (DMSO-d6, 100MHz)
Figure S4. 1H NMR spectrum of compound 1 (DMSO-d6 + D2O, 400MHz)
Figure S5. HMBC spectrum of compound 1 (DMSO-d6 + D2O, 400MHz)
Figure S6. Phylogenetic tree based on 16S rRNA gene sequences (ca.1,400 bp) of
strain S71 and its Streptomyces relatives. The number is the percentage indicating the
level of boot strap support, based on a neighbor-joining analysis of 1,000 resampled
data sets. The scale bar represents 0.01 substitutions per nucleotide position.
Micromonospora aurantiaca (NR_074415) is used as the outgroup.