RSC Advances
Paper
+
+
HRMS m/z [M + Na] calcd for C31
6
H
38
N
8
NaO
7
657.2756; found 124.8, 125.0, 129.6, 130.3, 134.2, 139.8, 141.7, 144.8, 149.0,
ꢂ1
+
57.2762. IR (nmax, cm ): 3307 w, 2929 w, 2873 w, 3057 w, 1711 150.6, 163.9, 166.2, 168.2, 168.3, 173.6. HRMS m/z [M + Na]
w, 1658 m, 1577 w, 1535 w, 1428 w, 1410 w, 1366 w, 1310 w, 1266 calcd for C H N NaO S 882.3691; found 882.3713. IR
w, 1210 w, 1196 w, 1120 m, 1103 m, 1062 m, 1036 w, 983 w, 934 (nmax, cm ): 3300 (m), 2925 (m), 2870 (m), 2110 (w), 2099 (w),
+
4
1
53 11
8
ꢂ1
1
13
w, 918 w, 862 w, 832 w, 732 s, 702 m, 665 w. H-NMR, C-NMR 1940 (w), 1887 (w), 1801 (w), 1656 (s), 1544 (m), 1440 (m), 1323
spectra and signal assignments (ESI S-23†). (m), 1307 (m), 1265 (m), 1088 (m), 1037 (m), 976 (w), 920 (w), 867
Preparation of ligand DIBO-PEG -FAPi. To a solution of (w), 834 (w), 760 (w). Detailed synthetic procedure, H-NMR,
compound 7 (1 equiv., 0.016 mmol, 10 mg) in anhydrous DCM
0.5 mL), were added 4-nitrophenyl chloroformate (2 equiv., 13†).
.032 mmol, 6.5 mg) and anhydrous Py (2 equiv., 0.08 mmol, 6.5
1
3
1
3
C-NMR spectra and signal assignments, UPLC trace (ESI S-
(
0
mL). The solution was stirred for 16 h at rt. The solvent was Functionalization of BFO-PEG NPs
evaporated in vacuo and the crude product was puried by FCC
To a suspension of BFO-PEG NPs (4 mg) in EtOH (4 mL) was
(
(
DCM/MeOH 10 : 1) to afford intermediate (S,E)-2-(2-(2-(2-(4-((3-
4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-
added distilled water (4 mL). A solution of DIBO-PEG -FAPi
3
(
17.4 mmol, 17 mg) in DMF (200 mL) was added and the
yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)
ethoxy)ethyl (4-nitrophenyl) carbonate as white solid
0.011 mmol, 8.7 mg, 68%). To a solution of this intermediate (1
ꢁ
suspension was ultra-sonicated for 16 h at 40 C. The mixture
was then divided into eppendorfs and centrifuged (10 min,
a
(
13 000 rpm). The supernatant was discarded and the NPs were
equiv., 0.063 mmol, 50 mg) in anhydrous DMF (2 mL), were
added NEt (3 equiv., 0.19 mmol, 26 mL) and DIBO derivative 8
resuspended in EtOAc (1 mL) and centrifuged (10 min, 13 000
3
rpm). The procedure was repeated 3 times. BFO-PEG-FAPi NPs
(
1.1 equiv., 0.069 mmol, 21 mg). The solution was stirred for 5 h
at rt. The solvent was evaporated in vacuo and the crude product
was puried by FCC (DCM/MeOH 15 : 1) to afford DIBO-PEG
ꢂ1
were stored in EtOH at a concentration of 1 mg mL . A sample
(10 mL) was diluted with distilled water (1 mL) and ultra-
3
-
sonicated for 30 min, before being analyzed with a Malvern
NanoZ instrument to determine mean hydrodynamic diameter
by dynamic light scattering and zeta potential.
1
FAPi as a white oil (0.063 mmol, 61 mg, quant.). H NMR (400
MHz, acetonitrile-d
) d 2.05–2.24 (4H, m), 2.75 (1H, dd, J ¼ 4.0,
5.0 Hz), 3.07–3.18 (5H, m), 3.39–3.53 (11H, m), 3.62–3.69 (1H,
m), 3.79 (2H, t, J ¼ 5.1 Hz), 4.03 (2H, t, J ¼ 4.7 Hz), 4.20 (2H, dd, J
2.1, 5.7 Hz), 4.44 (2H, t, J ¼ 5.0 Hz), 4.51 (2H, d, J ¼ 5.8 Hz),
.68–4.75 (1H, m), 5.28 (1H, s), 5.92 (1H, s), 6.26 (1H, s), 6.77
1H, d, J ¼ 15.7 Hz), 7.19 (1H, t, J ¼ 5.8 Hz), 7.24–7.39 (7H, m),
.44–7.55 (3H, m), 7.63 (1H, d, J ¼ 15.7 Hz), 7.76 (1H, s), 7.86
3
1
Inhibition of hrFAP, hrDPP IV and hrPREP
¼
The human recombinant enzymes were purchased from
commercial sources: hrDPP IV and hrPREP (Enzo Life Sciences,
Lausen, Switzerland), hrFAP (R&D systems, Abingdon, UK). The
enzymatic activities were measured in at bottom 96-well plates
(Costar) containing in each well 0.01 mg of the enzymes and 50
mM of the appropriate substrates: Z-Gly-Pro-AMC for hrFAPa
and hrPOP; H-Gly-Pro-AMC for hrDPP IV (both substrates from
Bachem, Vionnaz, Switzerland) diluted in their respective assay
4
(
7
(
1
1H, dd, J ¼ 1.9, 8.9 Hz), 8.00 (1H, d, J ¼ 8.8 Hz), 8.54 (1H, d, J ¼
1
3
.9 Hz), 8.86 (1H, d, J ¼ 4.3 Hz). C NMR (101 MHz, acetonitrile-
d
3
) d 25.9, 30.6, 35.8, 41.6, 41.8, 43.0, 46.7, 47.0, 47.7, 50.9, 64.8,
7
1
1
1
0.0, 70.1, 71.0, 71.1, 71.2, 77.1, 110.7, 113.5, 120.1, 120.6, 121.8,
24.15, 124.20, 124.3, 125.0, 125.5, 126.78, 126.84, 127.1, 128.1,
28.2, 129.27, 129.34, 131.1, 131.2, 135.0, 140.1, 143.2, 145.8,
ꢂ1
buffers (50 mM Tris, 1 M NaCl, 1 mg mL BSA, pH 7.5, for
ꢂ1
hrFAP; 50 mM Tris, 1 mg mL BSA, pH 7.5, for hrPREP; 25 mM
q
49.8, 151.8, 152.4, 153.3, 156.8, 166.3, 168.2, 168.5. Two C are
ꢂ1
+
+
Tris and 1 mg mL BSA, pH 8.0 for hrDPP IV). The enzyme
not resolved. HRMS m/z [M + Na] calcd for C51
9
w, 2878 w, 2159 w, 1716 m, 1657 m, 1521 m, 1443 w, 1431 w,
1
8
H
54
N
10NaO10
ꢁ
ꢂ1
solutions were incubated for 30 min at 37 C with increasing
89.3917; found 989.3920. IR (nmax, cm ): 3319 w, 3061 w, 2922
concentrations (5, 10, 20, 50 and 100 nM) of compounds 5 or
Biotin-PEG
3
-FAPi. The residual enzymatic activity was deter-
288 w, 1260 m, 1121 m, 1105 m, 1053 w, 1037 w, 981 w, 916 w,
ꢁ
1
13
mined by measuring uorescence increase for 60 min at 37 C
60 w, 832 w, 764 m, 733 s, 699 m, 1970 w, 1840 w. H-NMR, C-
in a uorescence multi-well plate reader (l /l ¼ 360/460 nm,
ex em
NMR spectra and signal assignments (ESI S-26†).
Synergy HT). Experiments were conducted in triplicate wells
and repeated twice. The half maximal inhibitory concentrations
Characterization of ligand Biotin-PEG -FAPi. Analytical
3
1
UPLC: R
1
t
¼ 1.32 min. H NMR (400 MHz, chloroform-d) d 1.42–
(
IC50) were graphically determined and the inhibition constants
.67 (6H, m), 1.93–2.07 (2H, m), 2.19–2.37 (4H, m), 2.49 (1H, d, J
49
i
(K ) were calculated.
¼
12.8 Hz), 2.69 (1H, dd, J ¼ 4.9, 13.0 Hz), 2.86–2.93 (1H, m),
3
.30–3.51 (2H, m), 3.50–3.67 (11H, m), 3.74–3.82 (1H, m), 3.81–
.92 (3H, m), 4.14 (1H, t, J ¼ 6.4 Hz), 4.32 (2H, dd, J ¼ 2.3, 5.5
Evaluation of cytocompatibility on MucilAir™-HF tissue samples
3
Hz), 4.47–4.54 (2H, m), 4.58 (2H, t, J ¼ 6.7 Hz), 4.78–4.85 (1H, Airway cells were obtained from patients undergoing surgical
m), 5.16 (1H, s), 6.63 (1H, s), 6.75 (1H, d, J ¼ 15.7 Hz), 7.22 (1H, t, polypectomy. All experimental procedures were explained in full,
J ¼ 5.5 Hz), 7.47 (1H, d, J ¼ 4.4 Hz), 7.67 (1H, d, J ¼ 15.6 Hz), 7.78 and all subjects provided informed consent. The study was con-
(
(
1H, dd, J ¼ 1.8, 8.9 Hz), 7.91 (1H, s), 8.01–7.97 (1H, m), 8.03 ducted according to the declaration of Helsinki on biomedical
1H, d, J ¼ 8.7 Hz), 8.44 (1H, s), 8.56 (1H, t, J ¼ 5.5 Hz), 8.87 (1H, research (Hong Kong amendment, 1989), and received approval
1
3
d, J ¼ 4.3 Hz). C NMR (101 MHz, chloroform-d) d 25.4, 25.6, from local ethics commission (Commission cantonale d' ´e thique
2
6
8.2, 28.4, 29.8, 34.9, 35.8, 39.5, 40.6, 42.4, 46.1, 47.1, 50.4, 55.6, de la recherche scientique de Gen `e ve [CCER]). Airway epithelia
0.0, 61.9, 69.4, 70.0, 70.3, 70.6, 70.7, 118.3, 119.5, 123.1, 124.0, co-cultured with broblasts were isolated from a mixture of
31666 | RSC Adv., 2019, 9, 31659–31669
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