Reversible photoregulation of binding of α-chymotrypsin to a gold surface

Article abstract of DOI:10.1021/ja0766674

An ability to optically modulate the interactions of surfaces with functional biomolecules provides an important basis for generating new technologies including reversible biosensors, advanced medical implants, and biomolecular computers. Here we report t

Full text of DOI:10.1021/ja0766674

Published on Web 11/10/2007
Reversible Photoregulation of Binding of r-Chymotrypsin to a Gold Surface
David Pearson, Alison J. Downard, Andrew Muscroft-Taylor, and Andrew D. Abell*,‡
Department of Chemistry, UniVersity of Canterbury, Christchurch, New Zealand
Received September 12, 2007; E-mail: andrew.abell@adelaide.edu.au
An ability to optically modulate interactions of surfaces with
functional biomolecules provides an opportunity to develop new
reversible biosensors, advanced medical implants, biomolecular
computers, and the like. However, existing photoswitch-based
systems1-3 are quite specific and lack generality. For example, the
reported photoregulated binding of an RNA aptamer to a surface-
attached peptide3 is limited to the aptamer specifically developed
to bind to it. A real advance would come from being able to target
a given RNA sequence with a general class of photoisomerizable
peptide; however, this is a difficult prospect.
Approaches are required that allow photoswitchable binding of
families of biomolecules to a surface in predictable and controllable
ways. In this paper, we report the reversible photoregulation of
binding of a protease (a class of enzyme that accepts a range of
related peptide-based substrates) to a functional surface as the first
step toward this goal. Our modular approach, using an inhibitor
containing a tether for surface attachment and an azobenzene core
Figure 1. Photoswitch of R-chymotrypsin. (a) Inhibitor (E)-1. (b) Schematic
of surface photoswitching showing surface-attached (E)-1 and R-chymot-
to which can be attached a range of peptide groups, provides a
means to target a given protease with such a family of related
inhibitors. Photochemical irradiation with UV (or visible) light
reversibly controls the geometry of the azobenzene, which alters
the affinity of the inhibitor and hence controls protease binding to
the surface.
rypsin-bound (Z)-1.
Scheme 1. Synthesis of Photoswitch Inhibitor 1
Previous work in our laboratories and elsewhere has demon-
strated the generality of this approach in the solution phase by
targeting both serine proteases (R-chymotrypsin4,5 and subtilisin5)
and cysteine proteases (papain5 and calpain6) with inclusion of
appropriate enzyme binding groups in the inhibitor. Reversible
surface photoregulation is now demonstrated for R-chymotrypsin
using the phenylalanine-based trifluoromethylketone inhibitor 1
(Figure 1) containing an azobenzene switch (core), an ethylene
glycol tether (to extend the inhibitor into solution), and a terminal
alkyne for attachment to a surface-bound azide using Huisgen 1,3-
dipolar cycloaddition (“click chemistry”).7,8 An internal alkyne was
included adjacent to the azobenzene since we had already shown
that R-chymotrypsin inhibitors of this general structure exhibit
excellent enzyme photoswitching in solution.9
The synthesis of 1 was carried out as shown in Scheme 1. One
terminus of dialkyne 210 was protected by reaction with TMSCl to
give 3, which was reacted with iodoazobenzene 411 in a Sonogashira
coupling to give 5. Treatment with base then gave the carboxylic
acid 6, which was coupled to amine 712 in the presence of EDCI to
give 8. Finally, the alcohol was oxidized with Dess-Martin
periodinane to give 1.
irradiated with visible light (>360 nm) to return to an E-enriched
solution (E:Z ratio ) 92:8). An aliquot was removed for each
photostationary state, and a spectrophotometric assay against
R-chymotrypsin was carried out. The initial sample (94 E:6 Z) gave
an inhibition constant (IC50) of >51 µM (limited by solubility).
The UV-irradiated sample (15 E:85 Z) was significantly more
potent, with an IC50 of 14 µM.13 Subsequent irradiation with visible
light gave a sample with a returned IC50 >51 µM. This represents
a greater than 3.6-fold reversible change in enzyme activity on
photoisomerization.
A two-step procedure was used to attach 1 to commercially
available surface plasmon resonance (SPR) surfaces containing
carboxylic acid groups in a dextran polymer matrix (Scheme 2). A
solution of EDCI/NHS was injected over the surface, followed by
solutions of amine 9, then ethanolamine to block unreacted sites.
Attachment of 9 to the surface was detected by an increase in SPR
response (corresponding to a mass increase at the surface) following
injection. The resulting azide-modified surface was removed from
the instrument, and 1 was attached by Huisgen 1,3-dipolar cy-
Photoisomerization and inhibition assays were initially carried
out in solution to assess the ability of 1 to photoswitch and inhibit
the enzyme. A solution of 1 in acetonitrile-d3 (E:Z isomeric ratio
) 94:6, determined by 1H NMR analysis) was irradiated with UV
light from a Hg arc lamp (320-380 nm) to give a solution enriched
in the Z isomer (E:Z ratio ) 15:85). This solution was then
‡ Current address: School of Chemistry and Physics, University of Adelaide,
SA 5005, Australia.
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14862
J. AM. CHEM. SOC. 2007, 129, 14862-14863
10.1021/ja0766674 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Attachment of 1 to a Gold Surface
the SPR instrument and irradiated with UV light (carried out as
above for solution-phase irradiations) to photoisomerize surface-
attached (E)-1 to (Z)-1. The surface was returned to the instrument,
and injection of enzyme (2.0 µM) was repeated to assess the extent
of enzyme binding after this photoisomerization (see Figure 2b,
curve 2). A significantly higher response was observed compared
to that before irradiation (curve 1). Therefore, significantly more
enzyme binds to the surface after UV irradiation. After removal of
attached enzyme as above, the surface was irradiated with visible
light to “switch back” to the less active E-enriched state [(E)-1],
and injection of enzyme was repeated. The response (curve 3) was
essentially identical to the initial response before UV irradiation,
corresponding to a similar amount of enzyme binding. These results
show that the surface can be reversibly photoswitched between two
states, one of which binds a significantly larger amount of enzyme.
Thus, photochemical switching can modulate binding of R-chy-
motrypsin to a surface.
cloaddition. In particular, a solution of compound 1, CuBr, sodium
ascorbate, 2,2′-bipyridine, and 2,6-lutidine in 1:1 DMF/H2O was
placed onto the gold surface, and after 1 h, the surface was rinsed
with 1:1 DMF/H2O, 0.1 M EDTA, and H2O to ensure complete
removal of unbound reactants. Successful attachment of 1 was
inferred since this modified surface gave photoregulated enzyme
binding consistent with the solution-phase photoswitching of 1 (see
below).
The binding of R-chymotrypsin to the surface with attached 1
was then monitored by SPR. Solutions of enzyme (0, 2.0, 6.0, and
18 µM) were separately injected over the surface for periods of
300 s to monitor enzyme binding to the surface, and after each
experiment, buffer was run over the surface for 300 s to monitor
detachment of enzyme (Figure 2a, enzyme injection at t ) 70 s).
Remaining enzyme was washed from the surface by injections of
guanidine (6 M, 5 µL), followed by acetic acid (1 M, 10 µL), to
rapidly regenerate the surface after each measurement. For each
nonzero enzyme concentration, the SPR response increased over
the period enzyme was injected relative to the surface control
lacking attached 1. Furthermore, the response was enhanced for
each increase in enzyme concentration (0 f 18 µM in Figure 2a),
which is consistent with attachment of enzyme to the surface. This
represents binding of enzyme to approximately 10% of the
theoretical maximum number of surface-attached inhibitor mol-
ecules (see Supporting Information S5), at the highest enzyme
concentration. Next, the enzyme-free surface was removed from
Reversible photoswitching of R-chymotrypsin binding to a
surface-attached inhibitor has been demonstrated. The approach is
simple, efficient, and the inhibitor design modular. Ongoing studies
are focused on further improving photoswitching, extension of the
system to a range of other proteases, and attachment to other solid
supports.
Acknowledgment. The authors thank Nathan Alexander for the
synthesis of compound 7, and Fiona Clow for help with SPR.
Financial support from the Royal Society of NZ Marsden Fund
and the ARC (DP0771901) are also gratefully acknowledged.
Supporting Information Available: Experimental procedures,
calculation of the maximum possible SPR response on binding of
R-chymotrypsin to the modified surface, synthesis of 1, 3, 5, 6, and 8,
1H and 13C NMR spectra for 1, 3, 5, 6, and 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Auernheimer, J.; Dahmen, C.; Hersel, U.; Bausch, A.; Kessler, H. J. Am.
Chem. Soc. 2005, 127, 16107.
(2) Blonder, R.; Levi, S.; Tao, G.; Ben-Dov, I.; Willner, I. J. Am. Chem.
Soc. 1997, 119, 10467.
(3) Hayashi, G.; Hagihara, M.; Dohno, C.; Nakatani, K. J. Am. Chem. Soc.
2007, 129, 8678.
(4) Pearson, D.; Abell, A. D. Org. Biomol. Chem. 2006, 4, 3618.
(5) Westmark, P. R.; Kelly, J. P.; Smith, B. D. J. Am. Chem. Soc. 1993, 115,
3416.
(6) Abell, A. D.; Jones, M. A.; Neffe, A. T.; Aitken, S. G.; Cain, T. P.; Payne,
R. J.; McNabb, S. B.; Coxon, J. M.; Stuart, B. G.; Pearson, D.; Lee, H.
Y.-Y.; Morton, J. D. J. Med. Chem. 2007, 50, 2916.
(7) The use of amine chemistry for surface attachment was deemed inap-
propriate since it is incompatible with the constituent trifluoromethylketone
group.
(8) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004.
Figure 2. SPR sensorgrams for binding of R-chymotrypsin to surfaces
modified with 1. SPR data simultaneously recorded for a control surface
were subtracted from these plots. (a) Binding of a range of R-chymotrypsin
concentrations (0, 2.0, 6.0, 18 µM) to the surface. (b) Photoswitching of
R-chymotrypsin (2.0 µM) binding to the surface. Key: (1) surface before
irradiation; (2) surface after UV irradiation; (3) surface after UV then visible
irradiation.
(9) Pearson, D. Ph.D. Thesis, University of Canterbury, 2007.
(10) McPhee, M. M.; Kerwin, S. M. Bioorg. Med. Chem. 2001, 9, 2809.
(11) Coleman, G. H.; McCloskey, C. M. J. Am. Chem. Soc. 1943, 65, 1588.
(12) Peet, N. P.; Burkhart, J. P.; Angelastro, M. R.; Giroux, E. L.; Mehdi, S.;
Bey, P.; Kolb, M.; Neises, B.; Schirlin, D. J. Med. Chem. 1990, 33, 394.
(13) See ref 4 for a discussion on relative binding of E and Z isomers.
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