L. Zhai, K.-W. Yang / Dyes and Pigments 120 (2015) 228e238
231
product was taken up by pyridine (80 mL) in a 250 mL round
diisopropylethylamine (DIEA) (175
m
L, 1 mmol). The solution was
ꢀ
bottomed flask and refluxed with stirring for an hour. The solution
left to stand for 30 min at 0 C with stirring, brought to room
temperature and stirred for 20 h. The reaction was quenched by
adding 20 mL of acetone. A deep brown solid precipitated, was
filtered out, and was washed by 5 mL acetone. The crude product
was dried in vacuo at room temperature to give 175 mg brown
solid, which was not soluble in common organic solvent. The solid
ꢀ
was allowed to cool and stand for 24 h at 0 C. The mixture was
filtered, and the collected solid was washed with acetone until the
rinsings were colourless. The dark purple product was dried in
vacuo at room temperature to give 1.285 g H (p-NO )TPP (1) with a
2 2
total yield around 12.9%. The product was used for the next reaction
directly without further purification.
2
was suspended in 1 mL H O, and the pH was adjusted using 0.5%
Elemental analysis: C44
4.10% found: C, 64.39; H, 2.68; N, 12.61%.
H
26
N
8
O
8
, calcd. C, 66.50; H, 3.30; N,
NaOH until the mixture became a clear yellow solution (pH
approximately 8.0). After centrifugation (8000 rpm, 15 min), the
1
supernatant was loaded onto
a
Sephadex G-25 column
2
.2.2. Synthesis of 5,10,15,20-tetrakis(para-aminophenyl)
(250 ꢁ 3.0 mm), and a 0.5% NaOH solution was employed as the
mobile phase at a flow rate of 1.0 mL/min. Fractions were collected
every 2 mL per tube and detected by UVevisible spectrometer
simultaneously to gather the targeted eluent. Identified fractions
were neutralized with 1 M HCl. Precipitates were collected by
centrifugation (11,000 rpm, 15 min) and dried in vacuo at room
porphyrin [26]
2 2
An amount of 700 mg H (p-NO )TPP (0.88 mmol) was dissolved
in concentrated HCl (80 mL) in a 250 mL three-neck round
bottomed flask. A solution of SnCl $2H O (5.96 g, 26.4 mmol) in
concentrated HCl (20 mL) was added to the reaction system. The
2
2
ꢀ
solution was brought to 70 C in a water bath and kept stirring for
h. The hot-water bath was removed and replaced by a cold-water
temperature to obtain 16 mg H
yellowish-green solid with a yield of 22.0%.
MALDI-TOF-MS (molecular ion, m/z): obsd. 2904.8615 [M] ,
calcd. 2904.0304 (M ¼ C154
UVevis (acetone, max, nm): 280, 428, 525, 567, 660.
Elemental analysis: C154 28 calcd. C, 63.62; H, 5.27; N,
2 2
(p-NH )TPP-Van conjugate (4) as a
7
þ
bath and then an ice-water bath. The reaction mixture was
neutralized with concentrated NaOH to a pH approximately 7.0.
During this course, the resulting solution gradually changed from a
deep green solution to a drab suspension. The resultant basic so-
lution was filtered, and the greenish solid was washed twice with
water, dried in vacuo at room temperature and then Soxhlet
extracted with acetone. The solvent was removed under reduced
3 25
H152Cl N O28).
l
3 25
H152Cl N O
12.04% found: C, 62.53; H, 4.92; N, 11.85%.
2.3. Minimum inhibitory concentration determination
pressure to give 520 mg isolated H
crystal with a yield of 87.6%.
2 2
(p-NH )TPP (2) as a purple
The in vitro antibacterial activity of 4 was investigated by
determining the MIC values against S. aureus, MRSA-1, MRSA-2,
E. faecalis, VRE and B. subtilis strains in the dark. The MIC value
determination was conducted by the Clinical and Laboratory
Standards Institute (CLSI) macrodilution (tube) broth method [27].
Briefly, a single colony of the above bacteria on solid LB-agar
plates was transferred to 10 mL of MH culture medium. The cul-
NMR: 1H NMR (400 MHz, CDCl
, 298 K)
.98 (d, J ¼ 7.9 Hz, 8H); 7.06 (d, J ¼ 7.4 Hz, 8H); 4.01 (s, 8H); -2.74 (s,
H).
MALDI-TOF-MS (molecular ion, m/z): obsd. 674.8339 M , calcd.
).
max, nm): Soret band: 346e445, Q band: 495,
d (ppm) 8.88 (s, 8H);
3
7
2
6
5
þ
74.2906 (M ¼ C44
34 8
H N
UVevis (acetone,
l
ꢀ
8
25, 567, 660.
tures were grown at 35 C for 3e5 h to obtain 10 colony-forming
units (CFU)/mL bacterium cultures according to Maxwell turbi-
Elemental analysis: C44
34
H N
8
, calcd. C, 78.32; H, 5.08; N, 16.61%
found: C, 78.29; H, 4.96; N, 16.75%.
dimetry. Compounds 2, 3 and 4 were dissolved in ddH
2
O to prepare
1280 mg/L stock solutions (to obtain a clear solution, the pH of 4
was adjusted to 7.80 by NaOH, and 2 was adjusted to 6.72 by HCl).
The stock solutions were diluted to 1 mL by MH medium to offer
concentration series with 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and
2
.2.3. Synthesis and purification of 5,10,15,20-tetrakis(para-
aminophenyl) porphyrin vancomycin conjugate
2
In a 50 mL round bottomed flask, H (p-NH )TPP (33.74 mg,
2
0
.25 mg/L, respectively. The 1 mL portion of bacterial culture
0
0
.05 mmol) and vancomycin hydrochloride (3) (315.16 mg,
6
(~10 CFU/mL) in MH medium was added sequentially into the
.2 mmol) were dissolved in 5 mL freshly distilled dimethyl sulf-
ꢀ
ꢀ
prepared solutions. Mixtures were incubated at 37 C for 24 h and
kept away from light.
oxide (DMSO). The mixture was cooled to 0 C with stirring, and o-
0
(
7-azabenzotriazol-1-yl)-N,N,N ,N’-tetramethyluronium
hexa-
fluorophosphate (HATU) (57.03 mg, 0.15 mmol) dissolved in 2 mL
dry N,N-dimethylformamide (DMF) was added, followed by N,N-
2.4. Photodynamic inactivation evaluation
Photodynamic inactivation was investigated by a traditional
surface plating method [20,28,29]. In addition to E. coli, the same
bacteria used in the MIC determination were employed.
A single colony of bacteria on LB-agar plates was transferred to
ꢀ
1
0 mL of LB culture medium and was grown at 37 C overnight.
ꢀ
Cells were collected by centrifugation (4 C, 4000 rpm, 10 min),
washed 3e6 times with phosphate-buffered saline (PBS) buffer (pH
7
.4), and diluted with the same buffer to an O.D.600 of 0.5. Con-
centration series of 2, 3, and 4 dissolved samples with 2, 4, 6, 8, 10,
and 12 M were prepared. A 0.5 mL portion of the samples was
m
added into 0.5 mL diluted bacterium fluid, and co-cultures were
ꢀ
incubated in the dark at 37 C for 30 min with shaking. All samples
2
were illuminated for 5 min with light (400e800 nm, 350 mW/cm ),
which was produced by a xenon lamp. For each compound, paral-
leled experiments were conducted. Furthermore, the light dose-
dependent photoinactivation of each compound with 10
mM was
Fig. 3. UVevis absorption spectra in monitoring the purification of conjugate 4.
tested. The illumination time ranged from 0 to 10 min, respectively.