Photoswitch Ligands
FULL PAPER
1
were determined by H NMR analysis by use of the integrals of well-sep-
Enzyme binding to this modified surface was monitored by running a
sensorgram in Tris buffer at 30 mLminꢀ1. A range of a-chymotrypsin solu-
tion concentrations were injected over the surface and monitored as fol-
lows:
arated peaks. The major isomer was assigned the thermodynamically
more stable E configuration on the basis of literature precedence.[37,38]
The minor Z isomer characteristically gave rise to upfield signals for the
aryl protons, which were enhanced on irradiation. 2D NMR was used to
confirm the presence of minor isomers in some representative cases. All
experiments involving photoisomerization were carried out in dim light-
ing conditions (that is, glassware wrapped in foil and with the lights
turned off).
An a-chymotrypsin solution (0, 2.0, 6.0, or 18 mm in Tris buffer, 150 mL)
was injected over a period of 5 min, flow rate 30 mLminꢀ1. After the in-
jection was complete, buffer was run for 5 min to monitor dissociation of
enzyme from the surface, and then the surface was regenerated. Regener-
ation was carried out at 5 mLminꢀ1 in Tris buffer by the injection of gua-
nidine hydrochloride (6m, 5 mL) followed by acetic acid (1m, 10 mL), and
buffer was then run for 5 min.
Enzyme assays: Buffer solution (Tris): tris(hydroxymethyl)aminomethane
(1.21 g), CaCl2·6H2O (0.44 g) and Triton X-100 (0.05 g) were dissolved in
Milli-Q deionized water (75 mL), adjusted to pH 7.8 with HCl solution
(1m), and made up to 100 mL with Milli-Q water.
For photoswitching experiments, the above enzyme binding assay was
carried out, and the chip was then regenerated, removed from the instru-
ment, rinsed with water, and irradiated with UV light (as detailed above
for solution-phase photoisomerization) for 10 min. The chip was then re-
placed in the SPR instrument, and the enzyme binding assay was repeat-
ed. The photoisomerization process was repeated with visible light irradi-
ation and this assay was repeated.
Substrate solution: N-Succinyl-(Ala)2-Pro-Phe-4-nitroanilide (21 mg) was
dissolved in Tris buffer solution (10 mL) with the aid of ultrasonication.
The solution was stored at ꢀ188C for up to two weeks. The concentration
of the solution was determined at the start of each day from its UV spec-
trum (e315 =14000 Lmolꢀ1 cmꢀ1).
Enzyme solution: A stock solution was prepared from a-chymotrypsin
(15 mg) in HCl solution (10 mL, pH 3, made up by dilution of conc. HCl
with Milli-Q water). The stock solution was stored at ꢀ188C for up to 1
month. Each day an enzyme solution was prepared. Stock solution
(200 mL) and Triton X-100 (25 mg) were made up to 50 mL with Milli-Q
water.
Details of synthesis and NMR spectra are given in the Supporting Infor-
mation.
Acknowledgements
Inhibition of a-chymotrypsin was determined with Suc-Ala-Ala-Pro-Phe-
4-nitroanilide as the substrate by an assay procedure developed from the
technique described by Geiger,[39] except that the order of addition of
enzyme and substrate was inverted, and only one substrate concentration
was used, in order to obtain inhibition constants as IC50 values rather
than Ki values. Briefly, inhibitors were dissolved in CD3CN at a series of
dilutions ranging from 0.5–500 mm as appropriate for each compound. For
each rate measurement, inhibitor solution (or acetonitrile blank, 50 mL),
substrate solution (60 mL), and buffer solution (910 mL) were mixed in a
cuvette and incubated for 5 min at 258C. Enzyme solution (30 mL) was
added, and the absorbance at 405 nm was monitored for 5 min for a-keto
esters or 10 min for trifluoromethyl ketones. Absorbance vs. time plots
were obtained, and the slopes were used to find the initial rates for a-
keto esters or the final rates for trifluoromethyl ketones. From the differ-
ences between these rates and the rates for the acetonitrile blanks, the
percent inhibitions were calculated. Each experiment was repeated over
as many inhibitor concentrations as required to obtain a good straight-
line plot of percent inhibition vs. log (inhibitor concentration), from
which the IC50 value was interpolated.
The authors thank Andrew Muscroft-Taylor for synthesis of compound
26, Nathan Alexander for synthesis of compound 10, and Fiona Clow for
help with SPR. Financial support from the Royal Society of NZ, the
Marsden Fund and the ARC (DP0771901) is also gratefully acknowl-
edged. D.P. was funded by a TEC Bright Future Top Achiever Doctoral
Scholarship.
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Surface plasmon resonance experiments: SPR was performed with a Bia-
core 2000 instrument. All solutions were filtered through a 0.22 mmfilter
and centrifuged (1300 rpm, 10 s) before use.
Buffers used: HEPES [0.01m, pH 7.4 containing NaCl (0.15m) and
EDTA (3 mm)], Tris [0.1m, pH 7.8, containing CaCl2 (0.02m) and P20 sur-
factant (12.5 mL)].
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Compound 1 was immobilized to an SPR chip as follows. A Biacore CM5
chip was docked in the SPR instrument and prepared by priming and
running a sensorgram in HEPES buffer at 100 mLminꢀ1 flow rate, with
injection of NaOH (50 mm, 20 mL ꢁ 2), HCl (10 mm, 20 mL ꢁ 2), and SDS
(0.1%, 20 mL ꢁ 2). Surface modification was then carried out at
5 mLminꢀ1 flow rate by injection of EDCI/N-hydroxysuccinimide
(35 mL), followed by 11-azido-3,6,9-trioxaundecan-1-amine (32,
11 mgmLꢀ1 in HEPES buffer, 3 ꢁ 5 mL) and then ethanolamine (1m,
pH 8.5, 35 mL). During attachment of 32, the SPR resonance increased
by 147 RU. The chip was then undocked and removed from the instru-
ment and rinsed with water. A solution of 1 (0.3 mg, 0.44 mmol), CuBr
(0.063 mg, 0.44 mmol), 2,6-lutidine (0.094 mg, 0.88 mmol), 2,2’-bipyridine
(0.14 mg, 0.88 mmol), and sodium ascorbate (0.17 mg, 0.88 mmol) in DMF/
H2O (1:1, 150 mL) was pipetted onto the surface and left for 1 h at room
temperature. The surface was then rinsed with DMF/H2O (1:1), H2O,
EDTA (0.1m), DMF/H2O (1:1), and H2O and was then redocked in the
SPR instrument. A sensorgram was run in Tris buffer for several hours in
order to obtain a stable baseline. A reference cell was prepared by the
same method except with omission of the injection of 32.
Chem. Eur. J. 2010, 16, 6983 – 6992
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6991