Organic & Biomolecular Chemistry
Page 10 of 12
Org. Biomol. Chem.
ARTICLE
2.27 (dd, J = 8.8, 6.4 Hz, 2H, CH2), 2.57 (m, 3H, CH2, CH2), Binding assay (
2.83 (m, 1H, Phe-Hβ), 3.03 (m, 1H, Phe-Hβ), 3.15 (ddd, J = 8.0,
3 8)
7.2, 5.5 Hz, 2H, CH2), 4.29 (m, 1H, Phe-Hα), 6.57 (2 AB-d, , The synthesis of the fluorescent probe wDasOIc: a10rr.1i0e3d9o/Cu4tOoBn01s9o2l6iAd
2H, HC=CH), 7.05 (m, 12H, ArH), 7.44 (dm, J = 7.6 Hz, 1H, phase using Fmoc/t-Bu protection scheme. The fluorescent
ArH), 7.83 (br s, 1H, Phe-NH), 7.92 (t, J = 5.5 Hz, 1H, Trigger- probe high throughput screening (FT-HTS) of
3
8
was
NH).
performed with a 2102 EnVision multilabel plate reader
(PerkinElmer). Corning Costar 384-well black plates were used
for the assay. To each well was added 200 nM R1, 50 nM
MS (ESI, 30 eV) m/z (%): 485.5 (100, [M+H]+).
1
E
-Ac-Trigger-Asp-Phe-OH (7) (4,1 mg, 6.83 µmol, 7%). H dansylated heptapeptide, inhibitors (in the concentration range
NMR: (500 MHz, DMSO-d6) δ =1.80 (s, 3H, CH3), 2.42 (m, from 10 to 100 µM) and the reaction buffer (0.05 % Triton x-
3H, Hβ-Asp, CH2), 2.64 (dd, J = 16.3, 5.9 Hz, 1H, Hβ-Asp), 100, 10 mM NDSB195 and 5 mM Hepes pH 7) to a final
2.72 (t, J = 7.5 Hz, 2H, CH2), 2.80 (t, J = 7.5 Hz, 2H, CH2), reaction volume of 50 µl. The mixture was incubated for 2 min
2.91 (dd, J = 13.7, 7.6 Hz, 1H, Hβ-Phe), 3.02 (dd, J = 13.7, 5.3 at room temperature. Exitation was done with polarized light
Hz, 1H, Hβ-Phe), 3.30 (dt, J = 7.5, 5.6 Hz, 2H, CH2), 4.40 (ddd, with a wavelength of 333 nm at room temperature, and
J = 8.3, 7.6, 5.3 Hz, 1H, Hα-Phe), 7.11 (d, J = 7.7 Hz, 2H, ArH), detection of the emission was done at 518 nm. Fluorescence
7.21 (m, 7H, ArH), 7.22 (2 AB-d, 2H, HC=CH), 7.40 (m, 4H, polarization was calculated according to the function FP = (I= -
ArH), 7.63 (ddd, J = 8.3, 7.4, 5.9 Hz, 1H, Hα-Asp), 7.92 (d, J = I┴)/(I= + I┴) where I= and I┴ is the intensity of the emitted
8.3 Hz, 1H, NH-Phe), 7.94 (t, J = 5.6 Hz, 1H, NH-Trigger), parallel and vertical beam.
8.16 (d, J = 8.3 Hz, 1H, NH-Asp).
Acknowledgements
Z
-isomer: 2.5 mg of E-isomer was irradiated for 240 min to Financial support by the Swedish Foundation for Strategic
afford a 72:28 mixture, Ф= 0.20; δ = 1.76 (s, 3H, CH3), Research (SSF) and by the Swedish Research Council (VR) are
Z:E
2.31 (m, 3H, Hβ-Asp, CH2), 2.59 (t, J = 7.4 Hz, 1H, CH2), 2.68 gratefully acknowledged. We also thank Per I. Arvidsson (UU)
(m, 3H, Hβ-Asp, CH2), 2.92 (m, 1H, Hβ-Phe), 2.99 (m, 1H, Hβ- and Gert Kruger (UKZN) for supporting an exchange visit at
Phe), 3.19 (dt, J = 13.0, 6.4 Hz, 2H, CH2), 4.19 (m, 1H, Hα- the University of KwaZulu-Natal through their bi-lateral
Phe), 4.56 (ddd, J = 7.4, 7.4, 7.4 Hz, 1H, Hα-Asp), 6.58 (2 AB- Sweden-South Africa Research Link program. Gert Kruger is
d, 2H, HC=CH), 7.17 (m, 12H, ArH), 7.45 (m, 1H, ArH), 7.69 also acknowledged for his hospitality during the stay at UKZN.
(br s, 1H, NH-Phe), 7.97 (t, J = 6.4 Hz, 1H, NH-Trigger), 8.01 Torsten Unge (UU) is acknowledged for providing access to the
(br s, 1H, NH-Asp).
MS (ESI, 30 eV) m/z (%): 600.5 (100, [M+H]+).
binding assay.
Notes and references
Electronic Supplementary Information (ESI) available:
[experimental details for syntheses and computations, NMR
E
-Ac-Asp-Trigger-Asp-Phe-OH (8) (4.3 mg, 6.02 µmol 7%).
1H NMR: (500 MHz, DMSO-d6) δ = 1.82 (s, 3H, CH3), 2.41
(m, 4H, Hβ-Asp1, Hβ-Asp2, CH2), 2.59 (dd, J = 16.0, 5.7 Hz,
1H, Hβ-Asp1), 2.63 (dd, J = 16.0, 5.1 Hz, 1H, Hβ-Asp2), 2.71
(t, J = 7.2 Hz, 2H, CH2), 2.79 (t, J = 7.9 Hz, 2H, CH2), 2.91 (dd,
J = 14.0, 8.0 Hz, 1H, Hβ-Phe), 3.02 (dd, J = 14.0, 5.1 Hz, 1H,
Hβ-Phe), 3.28 (dt, J = 7.2, 5.6 Hz, 2H, CH2), 4.36 (ddd, J = 8.0,
7.4, 5.5 Hz, 1H, Hα-Phe), 4.51 (ddd, J = 8.0, 7.4, 5.7 Hz, 1H,
Hα-Asp1), 4.60 (ddd, J = 8.0, 7.4, 5.1 Hz, 1H, Hα-Asp2), 7.10
(d, J = 7.3 Hz, 2H, ArH), 7.20 (m, 7H, ArH), 7.21 (2 AB-d, 2H,
HC=CH), , 7.42 (m, 4H, ArH), 7.87 (d, J = 7.4 Hz, 1H, NH-
Phe), 7.92 (t, J = 5.6 Hz, 1H, NH-Trigger), 8.11 (d, J = 8.0 Hz,
1H, NH-Asp1), 8.17 (d, J = 8.0 Hz, 1H, NH-Asp2).
spectra of compounds
3 8]. See DOI: 10.1039/b000000x/
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Z
-isomer: 2.3 mg of E-isomer was irradiated for 330 min to
afford a 81:28 mixture, Ф=0.18; δ = 1.81 (s, 3H, CH3), 2.43
Z:E
(m, 4H, Hβ-Asp1, Hβ-Asp2, CH2), 2.54 (m, 4H, Hβ-Asp1, Hβ-
Asp2, CH2), 2.63 (m, 2H, CH2), 2.92 (m, 1H, Hβ-Phe), 3.01 (m,
1H, Hβ-Phe), 3.12 (m, 2H, CH2), 4.21 (m, 1H, Hα-Phe), 4.52
(m, 2H, Hα-Asp1, Hα-Asp2), 6.58 (2 AB-d, 2H, HC=CH), 7.15
(m, 12H, ArH), 7.42 (m, 1H, ArH), 7.67 (br s, 1H, NH-Phe),
7.96 (br s, 1H, NH-Trigger), 8.13 (m, 2H, NH-Asp1, NH-
Asp2).
MS (ESI, 30 eV) m/z (%): 715.4 (100, [M+H]+).
10 | Org. Biomol. Chem., 2014, 00, 1‐3
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