A. Domzalski et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116254
rRNA amplicon sequencing using a primer pair targeting bacterial V4-V5
(515F: GTGYCAGCMGCCGCGGTAA, 926R: CCGYCAATTYMTT-
TRAGTTT).39,40 The sequencing was conducted with Illumina MiSeq at
IMR. The resulting paired-end Illumina reads were analyzed with
Quantitative Insights into Microbial Ecology 2 (Qiime2) to characterize
the microbial communities in MMCs.41 Briefly, sequence quality control
and feature table construction were carried out with DADA2.42 Taxo-
nomic analysis was conducted using a classifier that was trained on the
Greengenes 13_8 99% OTUs, where sequences had been trimmed to the
16S rRNA region amplified by the 515F/926R primer pair.
5.8. (S)-(+)-O-acetylmandelic acid derivatives of 12-HSA methyl ester
(4)
To a solution of 12-HSA methyl ester (4 mg, 12.7 µmol) dry
dichloromethane (1 mL) was added (S)-(+)-O-acetylmandelic acid
(14.6 mg, 75 µmol), EDC (14.4 mg, 78 µmol), DMAP (1.5 mg, 12.3 µmol)
The reaction mixture was stirred at room temperature for 24 h. The
mixture was concentrated in vacuo and the residue was purified by silica
gel chromatography to obtain the (S)-(+)-O-acetylmandelic acid deriv-
ative (3 mg, 5.8 µmol, 46%). The racemic mixture (4): 13C NMR (125
MHz, CDCl3) δ 174.60, 174.59, 170.52, 168.95, 134.34, 129.31, 128.86,
127.82, 76.23, 74.96, 51.69, 34.35, 34.33, 34.08, 31.89, 31.77, 29.69,
29.66, 29.63, 29.61, 29.59, 29.56, 29.45, 29.37, 29.33, 29.23, 29.10,
25.37, 25.32, 25.17, 24.92, 24.85, 22.78, 22.64, 20.99, 14.31, 14.27.
The (R)-isomer: 1H NMR (500 MHz, CDCl3) δ 7.45 (2H, m), 7.35 (3H,
m), 5.85 (1H, s), 4.85 (1H, quint, J = 6.2 Hz), 3.65 (3H, s), 2.28 (2H, t, J
= 7.6 Hz), 2.18 (3H, s), 1.61 (2H, m), 1.50 (2H, m), 1.35 (2H, m),
1.30–1.20 (16H, m), 1.12 (2H, m), 1.01 (4H, m), 0.86 (2H, m), 0.80 (3H,
t, J = 7.4 Hz). 13C NMR (125 MHz, CDCl3) δ 174.61, 170.52, 168.95,
134.33, 129.31, 128.86, 127.82, 76.23, 74.97, 51.69, 34.36, 34.34,
34.08, 31.77, 29.70, 29.66, 29.63, 29.46, 29.36, 29.11, 25.37, 25.18,
24.85, 22.64, 20.99, 14.27. HRMS (ESI; m/z) Calcd for C29H47O6
[M+H]+ 491.3367; found 491.3371.
5.5. LC/Ms
The culture broths of Control and Wheatgrass MMCs were extracted
with ethyl acetate. Solvent was removed in vacuo. Crude extracts were
weighed and dissolved in 100 µL methanol: 500 µL acetonitrile. Average
sample concentrations were 0.5 mg/mL. 10 µL of sample was diluted
into 40 µL acetonitrile/water (1:1). Blank controls were prepared from
the methanol/acetonitrile (1:5) mixture and diluted with acetonitrile/
water (1:1) as described above. 5 µL sample was injected into Agilent
iFunnel 6550 Q-ToF coupled to Agilent 1290 Infinity LC system equip-
ped with Agilent Eclipse C18 column, 1.8 µm, 2.1 × 50 mm. Method:
Solvent A, water with 0.1% formic acid, solvent B, acetonitrile with
0.1% formic acid; gradient of 2–98% acetonitrile in 18 min; flow rate of
5.9. Assays for planktonic growth and biofilm formation
◦
0.4 mL/min; column temperature at 30 C. MS/MS settings: MS scan
range 100–1200 m/z, where two most abundant precursors selected for
MS/MS (range 50–800); collision energy of 50 eV (positive mode) and
35 eV (negative mode). Source settings: gas temperature at 250 ◦C,
drying gas at 17 L/min, nebulizer at 30 psi, sheath gas temperature at
250 ◦C, and sheath gas flow at 12 L/min.
One isolate of B. mediterranea (SDH6) and three strains of A. oceani
(SD3, SD8 and SD9) were recovered as described previously.26 Indole
(Alfa Aesar A14427, Haverhill, MA) and 12-HSA (Alfa Aesar 44810,
Haverhill, MA) were purchased to have sufficient quantities for our as-
says. Since compounds were dissolved in dimethyl sulfoxide (DMSO),
DMSO was used as the vehicle control. Planktonic growth was quantified
by OD600, whereas biofilm formation was characterized with crystal
violet staining, as described previously.43 Assays were performed at two
different temperatures (19 ◦C and 27 ◦C). In the assays at 19 ◦C, bacteria
were incubated with the compounds for 96 h, whereas the assays at
27 ◦C used a shorter incubation period (48 h). In the planktonic growth
assay of 12-HSA, the addition of the compound turned the culture broth
turbid. As such, OD600 readings of cultures treated with 12-HSA were
corrected by OD600 readings of 12-HSA in media without culture. For
each treatment condition, 12 data points were obtained.
5.6. 12-HSA methyl ester
To a solution of commercial (R)-12-HSA (1) (50 mg, 0.17 mmol) in
toluene/methanol (4:1) (1 mL) was added 200 µL of 2 M TMS diazo-
methane in hexane (Sigma-Aldrich). The mixture was concentrated in
vacuo and the residue was purified by silica gel chromatography to
1
obtain the purified product (40 mg, 0.13 mmol, 75%). H NMR (500
MHz, CDCl3) δ 3.64 (3H, s), 3.55 (1H, m), 2.27 (2H, t, J = 7.6 Hz), 1.59
(2H, m), 1.50–1.20 ppm (27H, m), 0.86 (3H, t, J = 7.0 Hz). 13C NMR
(125 MHz, CDCl3) δ 174.61, 72.19, 51.69, 37.69, 37.67, 34.33, 32.07,
29.89, 29.79, 29.71, 29.62, 29.59, 29.45, 29.35, 25.86, 25.84, 25.16,
22.85, 14.33. HRMS (ESI; m/z) Calcd for C19H38O3Na [M+Na]+
337.2719; found 337.2715.
5.10. Data availability
Sequence files are available from the NCBI Sequence Read Archive
(SRA) with accession number PRJNA672656. Mass spectrometric data
sets are available from GNPS under accession number MSV000086394.
5.7. Racemic 12-HSA methyl ester
Declaration of Competing Interest
To a solution of commercial (R)-12-HSA methyl ester (31 mg, 0.1
mmol) in dry dichloromethane (1 mL) was added Dess-Martin period-
inane (42 mg, 0.1 mmol). The reaction mixture was stirred at room
temperature for 3 h. The mixture was concentrated in vacuo and the
residue was purified by silica gel chromatography to obtain the keto
product 2 (25 mg, 0.08 mmol, 80%). 1H NMR (500 MHz, CDCl3) δ 3.59
(3H, s), 2.31 (4H, m), 2.23 (2H, t, J = 7.5 Hz), 1.55 (2H, m), 1.48 (4H,
m), 1.15–1.28 ppm (18H, m), 0.80 (3H, t, J = 7.0 Hz). 13C NMR (125
MHz, CDCl3) δ 211.82, 174.37, 51.48, 42.85, 42.81, 34.11, 31.63, 29.40,
29.25, 29.23, 29.14, 28.95, 24.95, 23.86, 23.85, 22.52, 14.06. HRMS
(ESI; m/z) Calcd for C19H37O3 [M+H]+ 313.2737; found 313.2737. 2
(18 mg, 0.06 mmol) was then dissolved in dry methanol (1 mL). NaBH4
(3 mg, 0.081 mmol) was added to the solution, and the mixture was
stirred at RT for 1 h. The mixture was concentrated in vacuo and the
residue was purified by silica gel chromatography to obtain 3 (16 mg,
0.05 mmol, 85%).
The authors declare that they have no known competing financial
interest or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
AK thanks PSC CUNY Grant TRADA-50-661 for the funding of this
study. Purchase of the NEO-500 NMR spectrometer used to obtain re-
sults included in this publication was supported by the National Science
Foundation under the award CHE MRI 1900509. VV and AV thank
Hunter College MARC and RISE Programs, which are supposed by NIH/
NIGMS awards T34GM007823 and R25GM060665, respectively. ACD
thanks Early Research Initiative (ERI) Provost’s Pre-Dissertation
Research Fellowship at The CUNY Graduate Center and the Japan So-
ciety for the Promotion of Science (JSPS) Short-Term Summer Fellow-
ship. AK and ACD thank Ray Domzalski Jr. for his assistance in
7