M. E. Fait et al.
0.25 % v/v water. Papain adsorbed onto polyamide (1 g) was
added as biocatalyst. Individual reactions were performed
under nitrogen atmosphere in Erlenmeyer flasks (100 ml)
placed on an orbital shaker (250 rpm) at 37 °C for 72 h.
After incubation, the biocatalyst was removed by filtration
and washed with acetonitrile (3 × 10 ml) and diethyl ether
(3 × 10 ml), to eliminate the excess of fatty amine. Finally,
reaction products were extracted from the biocatalyst sur-
face using MeOH/H2O 4:1 (3 × 10 ml) and filtered through
a nylon membrane filter (0.22 μm, Osmonics). Purification
of each surfactant was achieved in an Äkta Purifier 10 (GE
Healthcare) by cation exchange chromatography as follows:
aliquots of the filtrate (1 ml) were loaded onto a SP Sepha-
rose Fast Flow column (10 ml bed volume, GE Healthcare)
preequilibrated with boric acid-sodium borate buffer 0.01 M
pH 8.5/ethanol 1:1 (solvent A). Unbound material was
eluted after washing the column with 2 bed volumes (BV)
of the same solvent. Elution of each condensation product
was accomplished by a step of 31 % of solvent B, consisting
in boric acid-sodium borate 0.01 M pH 8.5 buffer/ethanol
(1:1) with 1 M NaCl (2 BV). The column was washed with
100 % of solvent B (2 BV) and reequilibrated to the start-
ing conditions. Flow rate was kept at 1 ml/min throughout
the whole process. Eluted peaks were detected at 215 and
254 nm. Collected fractions containing the purified products
were pooled and the solvent was evaporated. The resulting
solid was desalted by precipitation with absolute ethanol
and centrifugation. Supernatant was evaporated under vac-
uum. The purity of the products obtained was analysed by
HPLC under the above conditions. Identities of both com-
Bz-Arg-NHC10 1H NMR (400 MHz, CD3OH): δ 7.80–7.76
(m, 2H, Ph, H-2′, H-6′), 7.48–7.43 (m, 1H, Ph, H-4′), 7.40–
7.34 (m, 2H, Ph, H-3′, H-5′), 4.74 (s, 6H, NH, COCHNH),
4.46 (dd, J = 8.9, 5.5 Hz, 1H, (NH)2CHNH), 3.18–3.12
(m, 2H, CH3(CH2)8CH2NHCO), 3.10 (t, J = 7.0 Hz, 2 H),
2.84–2.74 (m, 1H), 1.91–1.80 (m, 1H), 1.79–1.69 (m, 1H),
1.68–1.48 (m, 2H), 1.48–1.34 (m, 2 H), 1.33–1.11 (m, 18
H), 1.10–1.03 (m, 1H), 0.79 (m, 4H, CH3, NH). 13C NMR
(101 MHz, CD3OD) δ 172.45 (NHCHCONH), 168.79
(PhCO), 157.17 (Ph, C-1), 133.63 (NHC(NH)(NH2), 131.55
(Ph, C-4), 128.14 (Ph, C-3, C-5), 127.11 (Ph, C-2, C-6),
53.47, 40.56, 39.06, 31.60, 29.26, 29.24, 29.01, 28.98, 28.93,
28.89, 26.52, 25.13, 22.28, 12.98.
Antimicrobial assays
Determination of minimum inhibitory and bactericidal
concentrations
Antimicrobial activity of Bz-Arg-NHC10 and Bz-Arg-
NHC12 against several bacterial strains was determined by
the microdilution assay in 96-well flat-bottom plastic tis-
sue culture plates (TCP-96T-SI, Axygen, USA). In each
case, 125 µl of sterile nutritive broth (Biokar Diagnostics,
Beauvais, France) was placed into second column’s wells
of the microplate. Subsequently, 125 µl of the correspond-
ing surfactant solution in sterile nutritive broth (800 µg/
ml for Bz-Arg-NHC10 and 200 μg/ml for Bz-Arg-NHC12)
was added to the first and second columns’ wells of the
microplate. From the second well, 125 µl was transferred
serially to the subsequent wells, discarding 125 µl of the
mixture in the tenth column, so that the final volume con-
tained in each well was 125 µl. This process resulted in
twofold serial dilutions of the surfactant solution within the
first ten columns. Columns 11 and 12 did not contain sur-
factant and served as negative and positive growth controls,
respectively. All wells (except for the 11th column) were
inoculated with 2.5 µl of a bacterial suspension adjusted to
0.5 McFarland (1.5 × 108 CFU/ml) in physiological solu-
tion. Microplates were covered and incubated for 24 h at
37 °C. Optical density at 600 nm (OD600) was determined
with a spectrophotometer (ELISA Plate Reader SLT Lab
instruments Rainbow Reader, Vienna, Austria). The mini-
mum inhibitory concentration (MIC) was determined for
each strain as the lowest concentration of surfactant that
completely inhibits measurable growth (OD600 = 0 and
GI % >95 %, where GI % means growth inhibition per-
centage). Samples of those wells that did not show visible
growth were transferred to nutrient agar plates. Presence
of colonies was analysed for after incubation at 37 °C for
24 h. Minimum bactericidal concentration (MBC) was
defined as the lowest concentration of surfactant that com-
pletely inhibits bacterial growth (absence of colonies).
1
pounds were confirmed by H-NMR and 13C-NMR, using
bidimensional techniques (HSQC and COSY, see Supple-
mentary Material), as well as ESI MS. Theoretical exact
masses were obtained using the ChemCalc online service
(Patiny and Borel 2013) and compared with the experimen-
tal values obtained by ESI MS. Bz-Arg-NHC10, ESI (+)-
TOF–MS [M + 1] calculated for C23H40N5O2: 418.31820;
found: 418.31889. Bz-Arg-NHC12, ESI (+)-TOF–MS
[M + 1] calculated for C25H44N5O2: 446.34950; found:
446.34924.
Bz-Arg-NHC12 1H NMR (400 MHz, CH3OD) δ 7.88–
7.85 (m, 2 H, Ph, H-2′, H-6′), 7.58–7.50 (m, 1 H, Ph, H-4′),
7.46 (ddt, J = 8.2, 6.7, 1.3 Hz, 2 H, Ph, H-3′, H-5′), 4.83
(s, 6 H, NH, COCHNH), 4.54 (dd, J = 8.9, 5.7 Hz, 1 H,
(NH)2CHNH), 3.28–3.10 (m, 2 H), 2.89 (t, J = 7.0 Hz,
1H), 2.02–1.56 (m, 6 H), 1.49 (dd, J = 13.8, 6.9 Hz, 2
H), 1.40–1.22 (m, 20 H), 1.16 (t, J = 7.1 Hz, 1 H), 0.87
(t, J = 6.8 Hz, 3 H, CH3). 13C NMR (101 MHz, CH3OD)
δ 172.47 (NHCHCONH), 168.79 (PhCO), 157.17(Ph,
C-1), 133.62 (Ph, C-4), 128.13 (Ph, C-3, C-5), 127.12 (Ph,
C-2, C-6), 53.50, 40.56, 39.36, 39.06, 31.62, 29.34, 29.29,
29.27, 29.26, 29.03, 28.92, 27.20, 26.52, 26.02, 25.14,
22.28, 12.99.
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