Supplementary Information
Figure 1S Isolation and purification of JS-2 under guidance of DD-peptidase 64-575 II
inhibition reaction.
Figure 2S 1H NMR spectrum of a natural product of JS-2.
Figure 3S COSY spectrum of a natural product of JS-2.
Figure 4aS Part of the 1H {13C}HSQC spectrum of a natural product of JS-2.
Figure 4bS Part of the 1H {13C}HSQC spectrum of a natural product of JS-2.
Figure 5S IR spectrum of compound JS-2.
Figure 6S UV-Vis spectrum of compound JS-2.
Figure 7S Time- and concentration- dependent effect of JS-2 on growth of bacteria S. aureus
ATCC 29213 (susceptible to methicillin).
We report the isolation of a second bioactive metabolite, named JS-2, from the supernatant of
a Streptomyces sp. 8812 culture. JS-2 exhibited slight inhibitory properties on the DDpeptidases 64-575 II and R39 from Actinomadura R39.i It showed antimicrobial activity
against B. bronchiseptica, S. epidermidis and S. aureus (methicillin, vancomycin resistant).
Experimental
Purification of JS-2
Preliminary purification steps were done as described earlierii, with modification of second
step of purification (anion exchange resin IRA-400 (AcO-) of the active compound, which
included elution with 2 M aqueous acetic acid. Lyophilization yielded a partially purified
material (240 mg).
Further purification of JS-2 was carried out using Backerbond SPE on PolarPlus C18 column
2g, (J.T. Baker, USA). The material (30 mg) was dissolved in 0.1 % TFA, applied to the
column and eluted stepwise with 0.0, 1.0, 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 % CH3CN
containing aqueous TFA (0.1 %) (3 x 2 ml of each solvent). The most active fractions
obtained at 7.5 % of CH3CN, exhibiting inhibition of DD-peptidase 64-575 II activity, were
collected, pooled and lyophilized. This procedure was repeated 8 times and resulted in
obtaining 10 mg of powder sample.
The sample was then subjected to HPLC (Atlantis, dC18, 4.6x250 mm, 5 μm, Waters, USA);
detection by UV at 214 nm; flow rate of 1 ml/minute; mobile phase, phase B [B: CH3CN-0.1
% TFA, 20:80 (v/v)], phase A, 0.05% TFA, linear gradient from 0 to 100 % of phase B
during 100 min. Under these conditions, JS-2 was eluted at a retention time of 50 minutes.
Fractions eluted in 20 repeated runs were pooled and lyophilized to give 3 mg of white-cream
powder.
NMR procedure
The NMR spectra were recorded at 298K on Varian INOVA 500 MHz spectrometer operated
at 499.8, 125.7 and 50.5 MHz for 1H, 13C, and 15N, respectively. The compound was dissolved
in 0.7 mL of H2O/D2O (9:1) and transferred to 5 mm NMR tube. The 1H and 13C chemical
shifts, , given in ppm, were referenced against internal reference TSPA-d4 (deuterated
sodium trimethylsilylpropionate). For the 15N chemical shifts, CH3NO2 was used as an
external reference. The spectrometer was equipped with an TRIPLE-500 TR-PFG 5 mm
probe with an actively shielded z-gradient coil. The 2D experiments: GCOSY and GHSQC
were run by using the standard Varian software.
1H
NMR A standard single-pulse experiment was used to acquire the 1H spectrum using a
6000 Hz spectral window, 90o pulse width, an acquisition time of 2.7 s, 16K complex data
points with presaturation of water signal (2 s). The FIDs were processed without zero-filling
or appodisation functions application.
The GCOSY iii,
iv
(low power presaturation 1.0 s prior to acquisition was used to suppress the residual
water signal) spectra were acquired using 6000 Hz spectral width in both dimensions, an acquisition
time of 0.17 s, 16 transients per 512 increments and 1K complex data points in F2 domain. Prior to
Fourier transformation the data was processed using sine bell multiplication in both dimension.
13C
NMR The echo-antiecho phase sensitive, gradient-selected, HSQC iv,v,vi,vii
(Heteronuclear Single Quantum Coherence, adiabatic version) correlation spectrum was
obtained with an acquisition time of 0.17 s, a sweep width of 6000 Hz, 1K complex data
points in the 1H dimension and 20 000 Hz, 400 x 2 increments in the 13C dimension; 64
transients per t1 increment with a relaxation delay of 1.2 s and 1J(C, H)=135 Hz. The proton
and carbon /2 pulse lengths were 7.2 and 14.8 s, respectively. The data were zero filled to 4
K in F1 before Fourier transformation.
The HMBC (Heteronuclear Multiple Bond Correlation) technique with pulse-field-gradient
(PFG) coherence selection using two PFG pulses of relative amplitudes of +(I + S) : -(I S) and -(I - S) : +(I + S), for heteronuclear echo and antiecho selection, respectively was
used. The S-spin 90 deg. pulse low-pass filter was used to suppress one-bond correlation Such
implementation enabled us to obtain a nearly pure absorption lineshapes along F1, combined
with absolute value mode in F2 dimension.viii, ix The 1H {13C} GHMBC spectrum was
recorded with the following experimental conditions: an acquisition time of 0.20 s, spectral
windows of 5000 (F2) and 25000 (F1) Hz, 1K complex data points in the 1H dimension, 256
x2 increments in the 13C dimension, nJ(C,H) = 8 Hz, a 1.3 s relaxation delay and 512
transients per increment. The data were zero filled to 4096 points in F1 and processed using
sine bell multiplication in t2 and cosine window function in t1 dimensions prior to Fourier
transformation. The proton and carbon /2 pulse lengths were 7.2 and 14.8 s, respectively.
15N
NMR The 1H {15N} GHMQC spectra were recorded with the following parameters: an
acquisition time of 0.20 s, spectral windows of 5000 Hz (F2) and 7600 (F1) Hz; 1K complex
data points in the 1H dimension, 2 x 64 increments in the 15N dimension, nJ(C,H) = 4 Hz, 256
transients per increment, a relaxation delay of 1.8 s. Low pass filter (see above) was not used.
The proton and nitrogen /2 pulse lengths were 7.2 and 34 s, respectively.
NMR data
In particular, we used 1H {13C} HMBC spectrum to confirm the position of the –C(3)H2C(2)H- moiety connection to the aromatic ring. In the 1H-{13C} HMBC spectrum we observed
strong two and three-bond correlations from H3a and H3b protons at 2.80 and 3.06 to
quaternary carbon atom and to two CH carbon atoms at 133.2, 124.2 and 119.5 ppm,
respectively. Proton H2 at 4.38 ppm had strong three-bond correlation to quaternary carbon
atom at 133.2 ppm. These correlations confirmed the substitution position of the –C(3)H2C(2)H- moiety to the aromatic ring and also allowed for the unambiguous assignment of C
133.2, 124.2 and 119.5 ppm to their positions in the structure, respectively, C1’, C6’ and C2’.
Protons H2’at 6.79 ppm and H5’ at 6.85 ppm gave strong correlations to quaternary carbon
atoms at 145.2 ppm and 146.3 ppm. Proton H6’at 6.70 ppm gave strong correlation to
quaternary carbon atom at 145.2 ppm. These correlations allowed for the unequivocal
assignment of C 145.2 and 146.3 ppm to their positions, C4’ and C3’, respectively.
In the 1H {13C} HMBC spectrum we also observed strong cross peaks for H3a and H3b
protons with CH carbon atom at 59.0 ppm (C2) and between H2 protons and CH2 carbon atom
at 39.6 ppm (C3). Additionally, protons H3a and H3b gave weak cross peaks with carboxyl
carbon atom at 180.9 ppm (C1). These observation confirmed the existence of the –C(3)H2C(2)H-C(1)OOH moiety in the studied compound.
The existence of the –NH-C(=O)CH3 moiety was confirmed by observation in the HMBC
spectrum the correlation of protons at 1.93 ppm (-CH3 group) to the carbon atom at 175.9
ppm.
Bioassay
DD-peptidase inhibition assay
The amount of 100 L of the reaction mixture contained 7 L of DD-peptidase 64-575 II or 20
L DD-peptidase R39 (Penzym, Neogen, USA/Canada), 5L of 10 mM substrate solution
(thioester: benzoyl-D-alanyl-thioglycollate (synthesized at Siedlce University)), 5-10 L of
fresh inhibitor’s solution at various concentration and phosphate buffer, pH 8.0 or destilled
H2O.
JS-2 (N-Ac-L-Dopa), L-Dopa (Sigma), D-Dopa (Sigma), N-Ac-D-Dopa (synthesized at
Siedlce University) were used as inhibitors. D-Dopa and L-Dopa solutions were prepared at
acidic environment.
Hydrolysis of thioester was monitored spectrophotometrically (Jasco V-630, Japan) at 250 nm
in 37 oC during 3000s.
Bacterial strains
A panel of Gram-positive and Gram-negative bacterial strains was used: S. aureus ATCC
29213 (susceptible to methicillin), S. aureus ATCC 43300 (resistant to methicillin), S. aureus
ATCC 700787 (resistant to vancomycin), S. epidermidis ATCC 12228, Enterococcus faecalis
ATCC 29219, B. bronchiseptica ATCC 4617, Acinetobacter baumanii ATCC 19606,
Klebsiella pneumoniae ATCC 13882, Burkholderia cepacia ATCC 25416, Pseudomonas
aeruginosa ATCC 27853, Stenotrophomonas maltophilia ATCC 13637, Proteus vulgaris
ATCC 6896, Proteus mirabilis ATCC 12453, Escherichia coli ATCC 25922, Escherichia coli
ATCC BAA-198 and Helicobacter pylori ATCC 43504.
i
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ii
Solecka, J., Rajnisz, A. & Laudy, A. E. J. Antibiot. 62, 575-580 (2009).
iii
Aue, W.P., Karhan, J. &. Ernst, R.R . J. Chem. Phys. 64, 4226-4227 (1976).
iv
Hurd, R. E. J. Magn. Reson. 87, 422-428 (1990).
v
Bodenhausen, G. & Ruben, D. J. Chem. Phys. Lett. 69, 185–188 (1980).
vi
Kupce, E., & Freeman, R.. J. Magn. Reson. Ser. A , 117, 246-256 (1995).
vii
Willker, W., Leibfritz, D., Kerrsebaum, R. & Bermel, W. Magn. Reson. Chem. 31, 287–292
(1993).
viii
ix
Meissner, A. & Sørensen, O.W. Magn. Reson. Chem. 38, 981-984 (2000).
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