Photoregulation of α-Chymotrypsin Activity by Spiropyran-Based Inhibitors in Solution and Attached to an Optical Fiber

Article abstract of DOI:10.1002/chem.201501488

Here the synthesis and characterization of a new class of spiropyran-based protease inhibitor is reported that can be reversibly photoswitched between an active spiropyran (SP) isomer and a less active merocyanine (MC) isomer upon irradiation with UV and

Full text of DOI:10.1002/chem.201501488

DOI: 10.1002/chem.201501488
Full Paper
&
Chemical Biology
Photoregulation of a-Chymotrypsin Activity by Spiropyran-Based
Inhibitors in Solution and Attached to an Optical Fiber
Xiaozhou Zhang, Sabrina Heng, and Andrew. D. Abell*[a]
Abstract: Here the synthesis and characterization of a new
class of spiropyran-based protease inhibitor is reported that
can be reversibly photoswitched between an active spiropyr-
an (SP) isomer and a less active merocyanine (MC) isomer
upon irradiation with UV and visible light, respectively, both
in solution and on a surface of a microstructured optical
fiber (MOF). The most potent inhibitor in the series (SP-3b)
has a C-terminal phenylalanyl-based a-ketoester group and
inhibits a-chymotrypsin with a Ki of 115 nm. An analogue
containing a C-terminal Weinreb amide (SP-2d) demonstrat-
ed excellent stability and photoswitching in solution and
was attached to the surface of a MOF. The SP isomer of
Weinreb amide 2d is a competitive reversible inhibitor in so-
lution and also on fiber, while the corresponding MC isomer
was significantly less active in both media. The ability of this
new class of spiropyran-based protease inhibitor to modu-
late enzyme activity on a MOF paves the way for sensing ap-
plications.
Introduction
controls the geometry of the azobenzene, and the associated
inhibitory activity.[4,5]
An ability to detect the on/off binding of a bioligand to a com-
plementary surface-bound receptor provides a basis of real-
time sensors of wide applicability.[1] The associated “on” and
“off” states are typically modulated photochemically, by means
of a component azobenzene, or other photochromic substruc-
ture.[2] One problem with switchable sensors of this type is
that they generally lack an ability to be tailored to a range of
ligands, with each specific application requiring a different
base system.[1b,e,2,3] For example, an azobenzene-based molecu-
lar glue has been reported to hybridize DNA strands with mis-
match mutations on a gold surface.[3a] While this permits rever-
sible control of DNA hybridization with an external light stimu-
lus, the molecule only binds to a specific nucleotide sequence
rather than a range of such structures.
The use of azobenzenes to regulate enzyme activity in this
way is, however, somewhat limited since the two photostation-
ary states often exhibit only a small difference in activity.[6] In
addition, the fluorescence intensities of both switchable states
are low,[7] such that detecting and measuring the extent of iso-
merization can be problematic. Finally, the cis-azobenzene usu-
ally undergoes spontaneous thermal isomerization back to the
low-energy trans-isomer.[8] Given these limitations, there is
a need to develop new photoswitchable systems.
We now present a new class of serine protease inhibitor
based on a photoswitchable spiropyran core that mimics a pep-
tide backbone as found in protease inhibitors. This structure is
amenable to functionalization to target a particular protease,
and also for attachment to a microstructured optical fiber
(MOF) core surface for biosensor development (Figure 1). The
fiber has a duel function; it facilitates irradiation of the spiro-
pyran with light of a specific wavelength (532 nm) to bring
about photoswitching and also the detection of changes in
fluorescence produced on binding to the protease. There are
reports on the nonspecific covalent attachment of a spiropyran
to a-chymotrypsin in order to modulate its activity.[9] However,
our system is unique in that we designed this structural unit to
mimic the extended backbone of a competitive inhibitor. Pro-
teases are known to almost universally recognize the backbone
of substrates and inhibitors in a b-strand.[10] Binding in this ge-
ometry is dictated by the juxtaposition of active site binding
pockets that accommodate the side chains of the peptide-
based ligands.[10a]
More general and modular photoswitchable systems capable
of targeting families of biomolecules, in a controlled and pre-
dictable manner, are needed to advance this area. With this in
mind we recently reported a switchable surface containing an
azobenzene core to which was attached a peptidomimetic tri-
fluoromethyl ketone, the backbone structure of which can be
tailored to inhibit a particular class or example of protease.[4]
This system was immobilized onto gold surface by Huisgen
1,3-dipolar cycloaddition to provide an on/off switch and basis
of a biosensor. Irradiation with UV (or visible light) reversibly
[a] X. Zhang, Dr. S. Heng, Prof. A. D. Abell
ARC Centre of Excellence for Nanoscale BioPhotonics
Institute for Photonics and Advanced Sensing and
Department of Chemistry, The University of Adelaide
South Australia, 5005 (Australia)
Irradiation of the spiropyran core of these new inhibitors re-
sults in reversible ring opening (Figure 1), with an associated
change in polarity and geometry[11] to affect inhibitor binding
and hence potency. The change in dipole moment would be
E-mail: andrew.abell@adelaide.edu.au
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501488.
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study as a model protease, since much is known about its
structure and inhibition.[16] The overall system also contains
a spacer to link the photoswitchable inhibitor to the sensing
platform (in grey, Figure 1). A relatively short spacer was
chosen in this study for ease of synthesis and since the active
site of a-chymotrypsin is located close to the enzyme sur-
face.[17]
A MOF (as depicted in Figure S1 in the Supporting Informa-
tion) has additional advantages. Fluorescence of spiropyran is
known to be greatly enhanced when coupled to a MOF,[18] to
provide significantly improved sensitivity compared to the
equivalent solution-based experiments. This is particularly im-
portant when using the small sample volumes required for
subcellular-scale biological samples.[18a] A MOF also provides
a potential platform for performing in vivo assays in confined
and well-defined locations such as would be expected in po-
tential in vivo sensing applications. Furthermore, the air holes
within the MOF can be used to guide light to interact with
both the attached molecules and the sample solution within.
These voids simultaneously act as microsample chambers.
Figure 1. Schematic representation of spiropyran-based a-chymotrypsin in-
hibitors. The photoswitch is highlighted in blue, the sensing platform and
linker grey, amino acid residue red and the C-terminal electrophile green.
expected to be greater than that observed with an azobenze-
ne.[1b,12] A spiropyran has the added advantage that it is resist-
ant to reductive environments and thermal relaxations in aque-
ous environment, while providing excellent fluorescence inten-
sity to allow easy detection of inhibitor binding.[13] In addition,
isomerization of a spiropyran is more efficient and the prod-
ucts are easier to detect compared to other photoswitchable
systems such as an azobenzene.[13]
Synthesis of the inhibitors
The photoswitchable inhibitors prepared for this study are de-
picted in Figure 2. Compounds 2a–d and 3a–c contain a phe-
nylalanine (Phe) analogue at the C terminus for binding to a-
chymotrypsin. Derivatives 3a/3c and 3b also contain a C-ter-
minal electrophilic warhead (an aldehyde and a-ketoester, re-
spectively) that is capable of interacting with the protease
active site once bound. A number of compounds were pre-
pared with functionality for possible surface attachment, for
example, a carboxylic acid (1a–c, 4) for amide coupling, an
azide (1b, 2c, 3c) for Huisgen cycloaddition, and an alcohol
(1c, 2d) for esterification. Compound 2d provides a combina-
tion of the C-terminal Phe mimetic and a suitable C8’ tether
for surface attachment. This compound also contains a chemi-
cally stable Weinreb amide at the C terminus, which is capable
of hydrogen bonding with the enzyme’s active site.
Results and Discussion
The system reported here and depicted in Figure 1, consists of
four components: a photoswitchable spiropyran backbone
core (blue), an amino acid-based group (red) capable of bind-
ing to the active site of a given protease for example, a-chy-
motrypsin (orange), an attached C-terminal electrophile (X,
green) to bring about inhibition once bound, and a microstruc-
tured optical fiber (MOF)-based sensing platform to guide light
and detect changes in fluorescence (grey). The key photo-
switchable spiropyran core was designed to reversibly switch
the inhibitor geometry and polarity to modulate active site
binding. This group is known to provide an excellent fluores-
cence emission profile for sensing purposes,[13] where the ring-
closed spiropyran (SP; Figure 1, left) generally exhibits weak
fluorescence and the switched merocyanine isomer (MC; pro-
duced on UV irradiation, Figure 1, right) has an enhanced fluo-
rescence emission[14] that can be detected through the MOF.
The isomerization from MC to SP is promoted by visible light
typically provided by a halogen lamp. We can then detect
changes in fluorescence through the fiber upon selective
active site binding of one isomer, anticipated to be the SP
isomer given that a-chymotrypsin prefers nonpolar substrates.
The design allows for incorporation of suitable amino acid
and sensing components to target a particular protease and/or
sensing application. Here we use a phenylalanine-based amino
acid mimic since such a group is known to bind in the primary
S1 pocket[15] (and also other neighboring pockets) of our target
protease, a-chymotrypsin. Both an aldehyde and a a-ketoester
were investigated as C-terminal electrophilic groups capable of
interacting with the protease. a-Chymotrypsin was chosen for
Compounds 1a–c were prepared in three steps starting
from 4-hydrazinobenzoic acid (Scheme 1). In particular, reac-
tion with 2-methylbutan-2-one, under acidic conditions, gave
indole 6. This was subsequently N-alkylated on treatment with
methyl iodide in toluene and acetonitrile to give 7 in 87%
yield over two steps. Separate condensation of 7 with the hy-
droxyl nitro-benzaldehydes 8a–c, under reflux, gave the de-
sired spiropyran analogues 1a–c in 45–60% yield.
HATU-mediated coupling of 1a and 1b with either phenyl-
alaninol (9a) or methyl-3-amino-2-hydroxy-4-phenylbutanoate
(9b)[19] then gave 2a–c (Scheme 2). The phenylalaninol groups
of 2a and 2c were oxidized with Dess–Martin periodinane to
give the corresponding aldehydes 3a and 3c, respectively. The
a-ketoester 3b was similarly obtained by Dess–Martin oxida-
tion of the a-hydroxymethylester group of 2b.
The spiropyran 2d was prepared as shown in Scheme 3.
BOP-mediated coupling of commercially available N,O-dime-
thylhydroxylamine hydrochloride with N-Boc-l-phenylalanine,
followed by removal of the Boc group on treatment with TFA,
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Scheme 2. Preparation of C-terminal substituted spiropyrans.
Figure 2. Structures of target spiropyrans.
Scheme 3. Synthesis of 2d.
Table 1. In vitro inhibition of a-chymotrypsin by compounds 1a,b, 2a–d,
3a–c and 4.
Scheme 1. Synthesis of the core spiropyran unit.
gave the key intermediate 11. This was coupled with 1c, in the
presence of HATU, to give the spiropyran 2d.
R
X
Ki [mm]
1a
1b
2a
2b
2c
2d
3a
3b
3c
4
(CH2OCH2)3CH3
CH2N3
(CH2OCH2)3CH3
(CH2OCH2)3CH3
CH2N3
CH2O(CH2)2OH
(CH2OCH2)3CH3
(CH2OCH2)3CH3
CH2N3
–
–
305Æ45
In vitro inhibition assay against a-chymotrypsin
170Æ27
48Æ6
CH2OH
CHOHCO2CH3
CH2OH
CONCH3CH3
CHO
COCO2CH3
CHO
–
The spiropyrans 1–4 were assayed against bovine a-chymo-
trypsin as described in the Supporting Information and the re-
sults are shown in Table 1. Ki values for all the inhibitors were
determined graphically according to the Dixon methodolo-
gy.[20]
75Æ9
23Æ4
86Æ11
1.7Æ0.26
0.12Æ0.016
1.8Æ0.56
>1000
Compounds 1a and 1b, which lack a hydrophobic phenyl-
alanine mimic at their C termini, were weak inhibitors of a-chy-
OCH3
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motrypsin, with similar Ki values of 305 and 170 mm, respective-
ly. Even weak inhibitory activity here is particularly interesting
given that these compounds lack any significant peptide char-
acter normally associated[21] with protease inhibitors. The
nature of the R group at the C8 of the benzopyran ring ap-
pears to have little influence on potency (c.f. 1a and 1b), an
important observation given that this is a potential site for at-
tachment to the MOF as discussed later. On the other hand,
compound 4, with a pentanoic acid linker on the indole nitro-
gen, was inactive against a-chymotrypsin at a concentration of
1000 mm. Thus, this is not a suitable site for surface attach-
ment.
The incorporation of phenylalanine mimetic at C5 of the
indole (2a–c) results in a significant improvement in potency,
with Ki values of 48, 75 and 23 mm, respectively. This suggests
that the substituted spiropyran-core can act as a surrogate of
the backbone of a protease inhibitor or substrate. The intro-
duction of a reactive C-terminal group as in the aldehyde of
3a and 3c further improves potency, with these compounds
having Ki values of 1.7 and 1.8 mm, respectively. Compound 3b,
with a C-terminal a-ketoester as found in other serine protease
inhibitors,[22] proved to be particularly potent with a Ki of
115 nm. This compound is >1000-fold more potent than 1a
that contains the spiropyran base unit only. Gratifyingly, the
spiropyran 2d, with an ethylene glycol substituent at C8 suita-
ble for attachment to a MOF, was a reasonable inhibitor of a-
chymotrypsin in solution with a Ki =86 mm. This compound has
the advantage that it lacks a reactive C-terminal warhead and
as such was chosen for initial surface studies and the associat-
ed characterization. Importantly, compound 2d was shown to
be a competitive reversible inhibitor of a-chymotrypsin by
Dixon plot and Cornish–Bowden plot analyses[20,23] (Figure S2
in the Supporting Information). It thus competes for substrate,
an important observation if the spiropyran is to bind in the
active site as a peptide backbone mimetic.
Scheme 4. Photoisomerization of 2d, where SP depicts the ring-closed non-
fluorescent isomer and MC the ring-opened fluorescent isomer.
(red). The lack of absorption at 550 nm indicates that the
sample consists predominantly of the SP isomer, where the MC
isomer typically has strong absorption in this region.[24] The so-
lution of 2d in methanol was then irradiated with UV light
(365 nm) for 15 min to induce isomerization to the MC isomer
as evidenced by a significant and characteristic increase in ab-
sorption at 550 nm (Figure 3a, blue). The sample was further ir-
radiated with visible light to give a spectrum essentially the
same as the initial one (black and red, Figure 3a). Switching
between the two isomers was apparent after multiple cycles of
alternate irradiation with UV and visible light, without any evi-
dence of photobleaching, as shown in Figure 3b and Figure S5
in the Supporting Information. A significant increase in fluores-
cence of 2d was also observed upon UV irradiation and
switching to the MC isomer, while a decrease in fluorescence
was apparent upon visible light irradiation to switch the com-
pound back to the SP isomer (Figure S3 in the Supporting In-
formation). Consistent with the UV/Vis absorption experiments,
the fluorescence experiments also demonstrate the ability of
compound 2d to reversibly photoisomerize between two iso-
mers.
Solution-based photoisomerism of compound 2d
Studies on the photoswitching of spiropyran 2d in solution
were undertaken in order to compare the relative potencies of
the two photoswitchable states against a-chymotrypsin. These
two switchable isomers are depicted in Scheme 4 as ring-
closed spiropyran (SP) and ring-opened merocyanine (MC).
A solution of 2d in the assay buffer was irradiated with UV
light at 365 nm, for 5 min, and the resulting sample was incu-
bated with a-chymotrypsin in the dark to minimize any iso-
merization back to the SP isomer. This sample proved to be
a very weak inhibitor of a-chymotrypsin, with an IC50 greater
than 500 mm. Thus, the SP-based photostationary state is sig-
nificantly more potent (>fivefold) than the equivalent MC-
based state. This observation presumably reflects significant
differences in polarity and conformation of the two isomers,
and hence their abilities to bind to the protease.
In silico docking
The SP and MC isomers of 2d were separately docked into
a structure of a-chymotrypsin (Bos Taurus, PDB ID. 1GGD)[25]
using AutoDock4.2[26] in order to gain some insight into the
potential mode of active site binding and associated differen-
ces in potency. Docking studies were carried out by perform-
ing 25 docking runs on each isomer using the Lamarckian Ge-
netic Algorithm[27] and a maximum of 500000 energy evalua-
tions. The results of these studies are showed in Figure 4. The
SP isomer of 2d docked with a binding energy of À7.04 kcal
molÀ1 and docking energy of À13.57 kcalmolÀ1 (Figure 4a)
while the apparently less active MC isomer docked less well
The ability of 2d to photoswitch between its two isomers
was then studied. Specifically, a sample of 2d was irradiated
with visible light (>400 nm) and the resulting UV–visible (UV/
Vis) absorption spectrum was measured as shown in Figure 3
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Figure 3. a) The UV/Vis absorption spectra of 2d in MeOH under visible light
(red), after irradiation with UV light (365 nm) for 15 min (blue). Compound
2d was then isomerized by visible light irradiation again to produce spec-
trum in black. b) Photoswitching of 2d induced by UV (365 nm) and visible
light (>400 nm) irradiations. Cycle 0 represents the state before 2d was irra-
diated with UV light. Cycles 1, 3, 5, 7 and 9 represent the state of 2d after ir-
radiated with UV light for 15 min. Cycles 2, 4, 6, 8, and 10 represent the
state of 2d after irradiated with visible light for 15 min.
with a binding energy of À0.61 and a docking energy of
À8.93 kcalmolÀ1, respectively (Figure 4b). Figure 4a reveals
that the SP isomer sits within the active site, with the amide
carbonyl near the key active site serine as would be expected
for a competitive reversible inhibitor. In comparison, the MC
isomer docks poorly at the periphery of the active site (Fig-
ure 4b).
Figure 4c depicts interactions of SP isomer of 2d with key
binding pockets within the enzyme’s active site as observed in
the docked structures. Interestingly, the benzyl group of Phe
mimic resides in the S2’ binding pocket (highlighted in green).
This pocket is known to bind favorably with aromatic amino
acids,[16c] however this interaction has been little explored in in-
hibitor design. This then positions the spiropyran amide car-
bonyl of 2d in close proximity to the active site serine, with
the indole ring sitting on the outer edge of the S1 binding
pocket. The N-methyl group then projects into this pocket.
The ethylene glycol linker on the benzopyran is positioned on
the surface of the enzyme. The overall result is a somewhat un-
usual mode of binding to a-chymotrypsin, where the spiropyr-
an provides a suitable scaffold for other key interactions to
occur with the active site. It is noteworthy that our original
Figure 4. AutoDock4 results of 2d in the active site of a-chymotrypsin (PDB
ID: 1GGD): a) ring-closed SP 2d; b) ring-opened MC 2d, and c) key binding
interactions of SP 2d with S2’, S1, and S2 binding pockets highlighted in
green, pink and yellow, respectively.
design had the Phe mimetic of 2d interacting with the S1
pocket rather than S2’ as is apparent from these studies. This
difference is of little consequence to the current study, but it
does present some intriguing future inhibitor design possibili-
ties. It is also important to note that the ethylene glycol-based
linker of 2d is positioned toward the enzyme surface of the
protease and is thus suitably located for attachment to the
MOF.
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For comparison, the SP isomers of 3a–c and 4 were also
docked into a-chymotrypsin (see the Supporting Information
for detail). All these structures bind to the active site of the
enzyme with similar binding energies and docking energies
compared to the SP isomer of 2d. Compounds 3b and 3c
bind in essentially the same orientation as 2d, while 3a differs
only in the orientation of the benzyl group (Supporting Infor-
mation, Figures S6–S8).
the solution experiments. Subsequent excitation of this isomer
with 532 nm laser (as before) resulted in an enhanced (sixfold)
average fluorescence relative to the SP isomer. This is consis-
tent with the fluorescence characteristics of the MC isomer,
where this isomer is known to have enhanced fluorescence
due to p-conjugation as reported in literature[13,24,30] (Figure 6,
black, lem ~630 nm). Subsequent exposure of the Fiber-2d to
visible light for 5 min decreased the fluorescence intensity, as
expected for a photostationary state enriched in SP isomer.
Thus, 2d is able to be reversibly photoswitched on fiber as per
in solution. This is an important observation for sensor devel-
opment.
Microstructured optical fiber-based experiments
Compound 2d was next attached to a silica-based suspended-
core MOF (Supporting Information, Figure S1) in order to
probe its surface-based interactions with a-chymotrypsin as
a step toward a functional sensor. All surface functionalization
procedures were carried out under anhydrous conditions in
a sealed metal chamber pressurized with N2.[18a] The fiber was
first treated with pirahna solution (3:1 sulfuric acid/ 30% H2O2
v/v) and the thus liberated free hydroxyl groups were reacted
with 2.5% carboxyethylsilanetriol.[28] Steglich esterification[29] of
the resulting carboxyl groups, with 2d in the presence of DIC,
HOBt, and DMAP, gave the functionalized fiber (Fiber-2d).
The ability of 2d to photoswitch when attached to the MOF
was studied using the setup depicted in Figure 5.
In particular, Fiber-2d (SP isomer) was filled with Mili-Q
water by capillary action and irradiated for 10 ms using
a 532 nm laser and the resulting fluorescence was measured as
shown in Figure 6.[18] The functionalized fiber was next irradiat-
ed with UV light (l=365 nm) for an extended period of 5 min
in order to switch the surface attached SP-2d to MC-2d as per
Figure 6. Fluorescence spectra of Fiber-2d (SP) in water (blue), fiber-bound
2d in water irradiated with UV light (l=365 nm; black) and Fiber-2d subse-
quently irradiated with visible light (red). Fluorescence was induced by ex-
posure of Fiber-2d to 10 cycles of the 532 nm laser irradiation 10 ms per ex-
posure. The spectra are averages of 10 fluorescence measurements of each
sample.
Next, the fluorescence of Fiber-2d (SP isomer) in the pres-
ence of a-chymotrypsin was measured to investigate enzyme
binding. A new section of Fiber-2d was filled with a solution
of a-chymotrypsin in water (1 mm) over 5 min and the fluores-
cence was measured at 0, 10, 25, and 85 min by excitation
with a 532 nm laser (10”10 ms pulses) in each case. After
10 min the fluorescence was observed to be sixfold lower (c.f.
Figure 7a, red and brown lines), which we suggest is the result
of active site binding of the SP isomer of 2d that would
quench the inhibitor’s fluorescence. A similar decrease in fluo-
rescence was observed in the solution-based experiments with
compound 2d and a-chymotrypsin (see Figure S8 in the Sup-
porting Information for detail). The fluorescence intensity in-
creased gradually over the ensuring measurements (25,
85 min, see Figure 7a, blue and black curves, respectively).
This is consistent with slow but incomplete isomerism to the
MC isomer in the dark,[31] with this isomer having an inherent
Figure 5. Optical set-up for measuring the fluorescence of a functionalized
MOF. The area of illumination is shown by the dotted rectangle. (a) A SEM
cross section of the silica suspended-core fiber.
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Figure 8. Fluorescence emissions of compound 2d incubated in Mili-Q
water for 0 (blue) and 85 min (black).
peated using water in place of a-chymotrypsin as a negative
control. No significant change in fluorescence was apparent
after 85 min (Figure 8), which confirms that the earlier changes
in fluorescence on fiber were due to the enzyme binding to
2d.
The binding of fiber-bound MC isomer with a-chymotrypsin
was similarly investigated. The solution-filled section of the
fiber was cleaved and discarded and the remaining section
was infused with a fresh sample of a-chymotrypsin by capillary
action over 5 min with concomitant UV (365 nm) irradiation in
order to give a photostationary state enriched in the MC
isomer. Fluorescence was measured at 0, 10, 25, and 85 min
with laser irradiations (10”10 ms, 532 nm) as before, without
any apparent significant change in fluorescence over the time
course (Figure 7b). This is consistent with the earlier solution-
based observation that the MC isomer has significantly re-
duced activity and does not effectively bind to the protease.
Small variations in fluorescence are likely a result of incomplete
isomerization from the SP isomer to the MC isomer. This dem-
onstrates that attaching 2d to fiber does not affect the relative
binding affinities of the SP and MC isomers.
Figure 7. Fluorescent emissions of: a) SP of fiber-bound 2d with a-chymo-
trypsin left at room temperature for 0 min (red), 10 min (brown), 25 min
(blue), and 85 min (black). The fiber-bound compound was excited by 10
pulses of 532 nm laser irradiation before each measurement; and b) MC of
fiber-bound 2d (isomerized under UV irradiation; l=365 nm) with a-chymo-
trypsin left at room temperature for 0 (red), 10 (brown), 25 (blue), and
85 min (black).
Conclusions
Here we report the development of the first examples of spiro-
pyran-based protease inhibitors, which are able to undergo re-
versible photoisomerization between an active nonpolar SP
isomer and a significantly less active polar MC isomer upon
UV–visible light irradiations. The inhibitors reported here were
specifically designed to target a-chymotrypsin as a first step
toward a more modular approach to a range of proteases.
The spiropyrans 1a–c, 2a–d and 3a–c were synthesized and
assayed against a-chymotrypsin. The spiropyran cores alone
(1a–c) were active against a-chymotrypsin. The incorporation
of phenylalanyl mimetic at the C terminus (2a–d, 3a–c) signifi-
enhanced fluorescence while also dissociating from the
enzyme due to its weaker binding affinity as demonstrated in
the solution-based experiments. In support, it is known that a-
chymotrypsin has a greater affinity for nonpolar inhibitors
(e.g., the SP isomer) compared to polar inhibitors (e.g., MC
isomer).[32] Repeated photoswitching between the SP and MC
isomer produced similar changes in fluorescence intensities
(see Figure S9 in the Supporting Information for detail), which
demonstrates that MOF is a suitable platform for monitoring
chymotrypsin activity reversibly. Finally, the experiment was re-
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cantly improved the binding affinity. Compound 3b with an
additional C-terminal a-ketoester, was found to be the most
active inhibitor in this series, with a Ki of 115 nm.
Synthesis and characterization
8-(2-(2-Ethoxyethoxy)ethoxy)-1’,3’,3’-trimethyl-6-nitrospiro[chro-
mene-2,2’-indoline]-5’-carboxylic acid (1a): To an ice-cooled solu-
tion of NaH (371 mg, 8.90 mmol) in anhydrous THF (15 mL) was
added diethylene glycol monoethyl ether (747 mg, 5.57 mmol)
dropwise under N2. The mixture was stirred for 15 min with ice
cooling and then a solution of 3-(chloromethyl)-2-hydroxy-5-nitro-
benzaldehyde (1 g, 4.64 mmol) in anhydrous THF (5 mL) was
added dropwise. The mixture was allowed to warm to room tem-
perature with stirring for 18 h under N2. The mixture was cooled in
ice before H2O (160 mL) was added slowly to quench the reaction.
The mixture was washed with CH2Cl2 (2”90 mL). The aqueous
layer was acidified to pH 1 with 1m aqueous HCl and extracted
with ethyl acetate (3”90 mL). The organic phase was combined,
dried over Na2SO4 and concentrated in vacuo to give 8a (1.12 g,
77%), which was used in the subsequent step without further pu-
The Weinreb amide 2d was chosen as a stable analogue
suitable for fiber functionalization while exhibiting reasonable
activity against a-chymotrypsin (Ki =86 mm). This compound
was shown to bind as a competitive inhibitor by the Dixon
plot and the in silico docking experiments. The UV irradiation
of 2d in solution gave a photostationary state enriched in the
MC isomer that was sixfold less active against the enzyme than
the SP isomer. This is consistent with the general observations
that a-chymotrypsin has a greater affinity for nonpolar inhibi-
tors compared to polar inhibitors. A sample of 2d attached to
a MOF (Fiber-2d) could be reversibly photoisomerized to the
MC isomer on fiber as per in solution.
1
rification. H NMR (300 MHz, CDCl3): d=10.02 (s, 1H), 8.62 (s, 1H),
Quenching of fluorescence (sixfold) was observed for the
fiber-bound SP isomer of 2d after 10 min incubation with
a-chymotrypsin, which is consistent with active site binding.
No significant change in fluorescence was apparent over
85 min incubation of the MC fiber-bound isomer with a-chy-
motrypsin. This is consistent with the earlier solution-based ob-
servation that the MC isomer has significantly reduced binding
affinity compared to the SP isomer.
8.49 (s, 1H), 4.70 (s, 2H), 3.90–3.54 (m, 10H), 1.26–1.19 ppm (t, J=
4.5 Hz, 3H); and MS: [M]+calcd =313.12, [M]+found =313.12.
To
a solution of 8a (181 mg, 0.58 mmol) and 7 (125 mg,
0.58 mmol) in anhydrous ethanol (17 mm) was added Et3N (59 mg,
0.58 mmol). The mixture was refluxed for 3 h under N2 and concen-
trated in vacuo. The crude mixture was purified using C-18 reverse
phase silica gel (35% MeCN in H2O) to give 1a (80 mg, 27%).
1H NMR (600 MHz, CD3OD): d=8.14 (d, J=2.6 Hz, 1H), 8.06 (d, J=
2.7 Hz, 1H), 7.91 (d, J=8.2, 1H), 7.74 (s, 1H), 7.12 (d, J=10.4 Hz,
1H), 6.63 (d, J=8.2 Hz, 1H), 6.00 (d, J=10.4 Hz, 1H), 4.37–4.25 (m,
2H), 3.59–3.38 (m, 10H), 2.79 (s, 3H), 1.32 (s, 3H), 1.20 (s, 3H),
1.14 ppm (t, J=7.0 Hz, 3H); 13C NMR (151 MHz, CD3OD): d=156.2,
151.5, 140.8, 136.1, 131.0, 128.5, 128.4, 125.8, 124.3, 122.9, 121.6,
120.4, 118.6, 106.5, 105.9, 70.2, 70.0, 69.4, 66.2, 51.4, 39.0, 27.6,
24.8, 18.7, 14.0 ppm; HRMS (ESI) found [M+H]+ 513.2230,
In this work we report a new class of spiropyran-based pro-
tease inhibitor, the inhibitory activity of which can be con-
trolled by irradiation with light of a specific wavelength, both
in solution and on attachment to a MOF. This provides an im-
portant basis for probing and modulating enzyme activity in
a confined and well-defined environment, and paves the way
for potential real-time biosensing applications for in vivo dis-
ease diagnosis.
+
C27H33N2O8 requires 513.2237.
8-(Azidomethyl)-1’,3’,3’-trimethyl-6-nitrospiro[chromene-2,2’-in-
doline]-5’-carboxylic acid (1b): To a solution of sodium azide
(166 mg, 2.60 mmol) in anhydrous DMSO (4.6 mL) was added 3-
(chloromethyl)-2-hydroxy-5-nitrobenzaldehyde
(500 mg,
2.30 mmol) in one portion. The mixture was stirred at room tem-
perature under N2 for 18 h. H2O (5 mL) was added dropwise and
the mixture was extracted with ethyl acetate (2”20 mL). The com-
bined organic phase was washed with H2O (20 mL), dried over
Na2SO4 and concentrated in vacuo to yield 8b as a pale yellow
solid (389 mg, 76%), which was subsequently used without further
purification. 1H NMR (500 MHz, CDCl3): d=12.01 (s, 1H), 10.02 (s,
1H), 8.55 (s, 1H), 8.48 (s, 1H), 5.30 (s, 1H), 4.56 (s, 2H), 1.55 ppm (s,
1H); and MS: [M]+calcd =222.04, [M]+found =222.04.
Experimental Section
General information
Unless otherwise indicated, all starting materials, enzymes, chemi-
cals and anhydrous solvents were purchased from Sigma–Aldrich
(NSW, Australia) and were used without further purification. UNI-
BOND C-18 reverse phase silica gel was purchased from Analtech
Inc. (USA). 3-(Chloromethyl)-2-hydroxy-5-nitrobenzaldehyde and
compound 4 were prepared as previously described.[18a] 1H and
13C NMR spectra were recorded on a Varian 500 MHz and a Varian
Inova 600 MHz instruments in the indicated solvents. Chemical
shifts are reported in ppm (d). Signals are reported as s (singlet),
brs (broad singlet), d (doublet), dd (doublet of doublets), t (triplet)
or m (multiplet). High-resolution mass spectra were collected using
an LTQ Orbitrap XL ETD with flow injection, with a flow rate of
5 mLminÀ1. Where indicated compounds were analyzed and puri-
fied by reverse phase HPLC, using an HP 1100 LC system equipped
with a Phenomenex C-18 column (250”4.6 mm) for analytical
traces and a Gilson GX-Prep HPLC system equipped with a Phenom-
enex C18 column (250”21.2 mm). H2O/TFA (100:0.1 by v/v) and
MeCN/TFA (100:0.08 by v/v) solutions were used as aqueous and
organic buffers. All graphs were generated using GraphPad Prism 6
To
a solution of 8b (611 mg, 2.75 mmol) and 7 (593 mg,
2.75 mmol) in anhydrous ethanol (17 mm) was added Et3N
(278 mg, 2.75 mmol). The mixture was refluxed for 3 h under N2
and then concentrated in vacuo. The mixture was purified using C-
18 reverse phase silica gel (25% MeCN in H2O) to give 1b (150 mg,
1
13%). H NMR (500 MHz, [D6]DMSO): d=8.26 (s, 1H), 8.17 (s, 1H),
7.80 (d, J=8.3 Hz, 1H), 7.68 (s, 1H), 7.28 (d, J=10.5 Hz, 1H), 6.68
(d, J=8.1 Hz, 1H), 6.06 (d, J=10.5 Hz, 1H), 4.27 (d, J=13.6 Hz, 1H),
4.23 (d, J=13.6 Hz, 1H), 2.75 (s, 3H), 1.27 (s, 3H), 1.15 ppm (s, 3H);
13C NMR (151 MHz, [D6]DMSO): d=167.7, 157.0, 151.0, 140.2, 135.8,
130.9, 128.5, 125.9, 123.1, 122.9, 121.1, 119.0, 106.7, 106.5, 51.6,
48.1, 28.5, 25.8, 19.6 ppm. HRMS (ESI) found [M]+ 421.1381,
+
C21H19N5O5 requires 421.1386.
software.
A
mercury lamp (365 nm) and
a
halogen lamp
8-((2-Hydroxyethoxy)methyl)-1’,3’,3’-trimethyl-6-nitrospiro[chro-
mene-2,2’-indoline]-5’-carboxylic acid (1c): To an ice-cooled solu-
tion of NaH (63 mg, 1.59 mmol) in anhydrous THF (2.8 mL) was
(>450 nm) were used as the UV and visible light sources in photo-
switching experiments.
Chem. Eur. J. 2015, 21, 10703 – 10713
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
added diethylene glycol monoethyl ether (99 mg, 1.59 mmol) drop-
wise at under N2. The mixture was stirred for 15 min with ice cool-
ing and then a solution of 3-(chloromethyl)-2-hydroxy-5-nitroben-
zaldehyde (200 mg, 0.80 mmol) in anhydrous THF (0.7 mL) was
added dropwise. The mixture was allowed to warmed to room
temperature with stirring for 18 h under N2. The mixture was
cooled in ice before H2O (35 mLmmolÀ1 aldehyde) was added
slowly to quench the reaction. The mixture was washed with
CH2Cl2 (2”20 mL). The aqueous layer was acidified to pH 1 with
1m aqueous HCl and extracted with ethyl acetate (3”20 mL). The
organic phase was combined, dried over Na2SO4 and concentrated
in vacuo to give 8c (115 mg, 60%), which was used in the subse-
quent step without purification. 1H NMR (500 MHz, CDCl3): d=
11.92 (s, 1H), 10.01 (s, 1H), 8.57 (s, 2H), 8.50 (s, 1H), 4.85 (s, 2H),
2.25–2.11 (m, 2H), 1.58 (s, 1H), 1.25 ppm (s, 1H); and MS:
[M]+calcd =241.06, [M]+found =241.06.
mene-2,2’-indoline]-5’-carboxamide (2b): Compound 2b (54 mg,
47%) was prepared by coupling of compound 1a with (2R,3S)-
methyl 3-amino-2-hydroxy-4-phenylbutanoate (9b)[19] according to
general procedure A. 1H NMR (599 MHz, CD3OD): d=8.17 (s, 1H),
8.08 (s, 1H), 7.90 (s, 1H), 7.72–7.62 (m, 1H), 7.53 (s, 1H), 7.40–7.27
(m, 4H), 7.26–7.18 (m, 1H), 7.15 (d, J=10.4 Hz, 1H), 6.65 (d, J=
8.2 Hz, 1H), 6.01 (d, J=10.4 Hz, 1H), 4.71 (m, 1H), 4.58 (brs, 1H),
4.32 (s, 2H), 4.22 (s, 1H), 3.68 (s, 3H), 3.58–3.39 (m, 10H), 3.15–2.94
(m, 2H), 2.78 (s, 3H), 1.34 (s, 3H), 1.22 (s, 3H), 1.15 ppm (t, J=
7.0 Hz, 3H); 13C NMR (151 MHz, CD3OD): d=175.2, 170.2, 157.7,
152.3, 142.4, 139.7, 137.9, 130.6, 130.1, 129.7, 127.8, 127.4, 126.7,
125.8, 123.1, 122.3, 122.0, 120.2, 108.1, 107.6, 72.1, 71.7, 71.6, 71.0,
67.7, 55.6, 53.1, 52.8, 38.5, 29.2, 26.3, 20.2, 15.6 ppm; HRMS (ESI)
+
found [M]+ 703.3104, C38H45N3O10 requires 703.3105.
8-(Azidomethyl)-N-((R)-1-hydroxy-3-phenylpropan-2-yl)-1’,3’,3’-
trimethyl-6-nitrospiro[chromene-2,2’-indoline]-5’-carboxamide
(2c): Compound 2c (120 mg, 87%) was prepared by coupling of
compound 1b with l-phenylalaninol 9a according to general pro-
To
a solution of 8c (115 mg, 0.47 mmol) and 7 (101 mg,
0.47 mmol) in anhydrous ethanol (17 mm) was added Et3N (47 mg,
0.47 mmol). The mixture was refluxed for 3 h under N2 and concen-
trated in vacuo. The crude mixture was purified using C-18 reverse
phase silica gel (30% MeCN in H2O) to give 1c (93 mg, 45%).
1H NMR (500 MHz, CD3OD): d=8.19 (s, 1H), 8.07 (s, 1H), 7.92 (d, J=
8.2 Hz, 1H), 7.75 (s, 1H), 7.14 (d, J=10.4 Hz, 1H), 6.64 (d, J=8.2 Hz,
1H), 6.01 (d, J=10.4 Hz, 1H), 4.57 (brs, 1H), 4.32 (s, 2H), 3.62–3.52
(m, 2H), 3.45–3.35 (m, 2H), 2.80 (s, 3H), 1.33 (s, 3H), 1.22 ppm (s,
3H); 13C NMR (151 MHz, CD3OD): d=157.7, 152.8, 142.5, 137.6,
132.4, 130.1, 127.5, 125.7, 124.4, 123.1, 122.1, 120.2, 108.1, 107.4,
73.7, 67.7, 62.4, 40.6, 29.2, 26.3, 20.3 ppm; HRMS (ESI) found [M]+
1
cedure A. H NMR (500 MHz, CD3OD): d=8.12 (s, 1H), 8.09 (s, 1H),
7.67 (d, J=8.2 Hz, 1H), 7.56 (d, J=6.0 Hz, 1H), 7.27 (m, 4H), 7.16
(m, 2H), 6.62 (d, J=8.2 Hz, 1H), 6.02 (d, J=10.4 Hz, 1H), 4.58 (s,
1H), 4.38–4.29 (m, 1H), 4.26 (d, J=13.8 Hz, 1H), 4.18 (d, J=13.8 Hz,
1H), 3.65 (d, J=5.3 Hz, 2H), 3.10–2.85 (m, 2H), 2.81 (s, 3H), 1.34 (s,
3H), 1.21 ppm (s, 3H); 13C NMR (126 MHz, CD3OD): d=168.9, 156.8,
150.5, 140.7, 138.7, 136.01, 128.9, 128.4, 128.1, 127.9, 125.9, 125.6,
125.2, 123.0, 122.4, 120.7, 120.7, 119.2, 107.1, 106.0, 62.9, 53.4, 51.7,
36.6, 27.6, 24.9, 24.8, 18.7 ppm; HRMS (ESI) found [M]+ 554.2278,
+
C30H30N6O5 requires 554.2278.
+
440.1581, C23H24N2O7 requires 440.1584.
8-((2-Hydroxyethoxy)methyl)-N-((R)-1-(methoxy(methyl)amino)-
1-oxo-3-phenylpropan-2-yl)-1’,3’,3’-trimethyl-6-nitrospiro[chro-
mene-2,2’-indoline]-5’-carboxamide (2d): Compound 2d (74 mg,
57%) was prepared by coupling of compound 1c with compound
11 according to general procedure A. 1H NMR (500 MHz, CD3OD):
d=8.19 (s, 1H), 8.07 (s, 1H), 7.69 (d, J=7.5 Hz, 1H), 7.58 (s, 1H),
7.29 (d, J=3.4 Hz, 4H), 7.21 (brs, 1H), 7.13 (d, J=10.4 Hz, 1H), 6.63
(d, J=8.4 Hz, 1H), 6.00 (d, J=10.5 Hz, 1H), 5.35 (brs, 1H), 4.30 (s,
1H), 3.80 (s, 3H), 3.56 (t, J=4.6 Hz, 2H), 3.35 (d, J=4.5 Hz, 2H),
3.20 (s, 3H), 3.18–3.02 (m, 2H), 2.78 (s, 3H), 1.34 (s, 3H), 1.21 ppm
(s, 3H); 13C NMR (151 MHz, CD3OD): d=156.1, 150.9, 140.9, 136.3,
128.9, 128.6, 128.3, 128.0, 126.4, 125.8, 124.1, 121.5, 120.8, 120.4,
118.6, 106.5, 106.0, 72.1, 66.1, 60.8, 56.07, 51.5, 41.0, 29.4, 27.6,
General procedure A for HATU-mediated peptide coupling
To a solution of carboxylic acid (1 equiv) in anhydrous DMF
(40 mm) was sequentially added HATU (1 equiv) and the amine
(1.2 equiv). The mixture was stirred under N2 for 15 min, cooled in
an ice bath, and DIPEA (4 equiv) was added dropwise. The mixture
was allowed to warm to room temperature while stirring under N2
for 18 h. Aqueous HCl (1m; 10 mLmmolÀ1 of carboxylic acid) was
added and the mixture was extracted with ethyl acetate (3”
50 mLmmolÀ1 of carboxylic acid). The combined organic phase
was washed with 1m aqueous HCl (10 mLmmolÀ1 of carboxylic
acid), saturated NaHCO3 (10 mLmmolÀ1 of carboxylic acid) and
brine (20 mLmmolÀ1 of carboxylic acid). The organic phase was
dried over Na2SO4 and concentrated in vacuo. The crude product
was purified using C-18 reverse phase silica gel (30–50% MeCN in
H2O).
+
23.0, 18.6, 16.0 ppm; HRMS (ESI) found [M]+ 630.2694, C34H38N4O8
requires 630.2690.
General procedure B for Dess–Martin oxidation of alcohol
8-(2-(2-Ethoxyethoxy)ethoxy)-N-((R)-1-hydroxy-3-phenylpropan-
2-yl)-1’,3’,3’-trimethyl-6-nitrospiro[chromene-2,2’-indoline]-5’-
carboxamide (2a): Compound 2a (96 mg, 62%) was prepared by
coupling 1a with l-phenylalaninol 9a according to general proce-
dure A. 1H NMR (600 MHz, CD3OD): d=8.19 (s, 1H), 8.08 (s, 1H),
7.79–7.61 (m, 1H), 7.51 (s, 1H), 7.33–7.21 (m, 2H), 7.19–7.15 (m,
1H), 7.13 (d, J=10.4 Hz, 1H), 6.62 (d, J=8.2 Hz, 1H), 6.00 (d, J=
10.4 Hz, 1H), 4.59 (brs, 1H), 4.41–4.23 (m, 3H), 3.75–3.59 (m, 2H),
3.55–3.37 (m, 10H), 3.13–2.95 (m, 1H), 2.95–2.83 (m, 1H), 2.78 (s,
3H), 2.65 (s, 2H), 1.37 (s, 3H), 1.22 (s, 3H), 1.14 ppm (t, J=7.0 Hz,
3H); 13C NMR (151 MHz, CD3OD): d=168.8, 156.2, 150.6, 140.8,
138.7, 136.3, 128.9, 128.5, 128.0, 127.9, 125.9, 125.8, 125.5, 124.2,
121.6, 120.7, 120.5, 118.6, 106.5, 106.0, 70.1, 70.0, 69.4, 66.2, 63.0,
53.4, 51.5, 39.0, 36.6, 27.60, 24.70, 18.7, 14.0 ppm; HRMS (ESI)
To a solution of alcohol (1 equiv) in anhydrous CH2Cl2 (7 mm)
under N2 was added Dess–Martin periodinane (3 equiv) in one por-
tion. The mixture was stirred at room temperature under N2 for
1.5 h. The mixture was diluted with CH2Cl2 (100 mLmmolÀ1 of alco-
hol) and then Na2S2O3 in saturated NaHCO3 (10% w/v,
10 mLmmolÀ1 of alcohol) was added. The mixture was stirred for
15 min and the aqueous phase was separated and extracted with
CH2Cl2 (50 mLmmolÀ1 of alcohol). The combined organic phase
was dried over Na2SO4 and concentrated in vacuo. The crude prod-
uct was purified using RP-HPLC.
8-((2-(2-Ethoxyethoxy)ethoxy)methyl)-1’,3’,3’-trimethyl-6-nitro-N-
((R)-1-oxo-3-phenylpropan-2-yl)spiro[chromene-2,2’-indoline]-5’-
carboxamide (3a): Compound 2a was oxidized according to gen-
eral procedure B to give compound 3a (3 mg, 8%). 1H NMR
(500 MHz, CDCl3): d=9.74 (s, 1H), 8.20 (s, 1H), 7.95 (d, J=2.6 Hz,
1H), 7.54 (m, 2H), 7.40–7.18 (m, 5H), 6.96 (d, J=10.4 Hz, 1H), 6.52
(d, J=8.0 Hz, 1H), 5.84 (d, J=10.2 Hz, 1H), 4.89 (s, 1H), 4.29 (s,
+
found [M]+ 645.3055, C36H43N3O8 requires 645.3050.
8-(2-(2-Ethoxyethoxy)ethoxy)-N-((2R,3S)-1-carbomethoxy-1-hy-
droxy-3-phenylpropan-2-yl)-1’,3’,3’-trimethyl-6-nitrospiro[chro-
Chem. Eur. J. 2015, 21, 10703 – 10713
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10711
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
2H), 3.67–3.44 (m, 10H), 3.29 (m, 2H), 2.75 (s, 3H), 1.29 (s, 3H),
1.23–1.12 ppm (m, 6H); 13C NMR (151 MHz, CD3OD): d=168.9,
156.2, 150.6, 140.8, 139.2, 129.0, 128.9, 128.5, 128.1, 127.9, 127.3,
125.8, 125.8, 124.2, 121.6, 120.8, 118.6, 106.5, 105.9, 70.1, 70.0, 69.8,
66.1, 51.5, 39.0, 34.7, 34.6, 27.6, 24.7, 24.7, 18.7, 14.0 ppm; HRMS
d=8.36 (s, 1H), 8.17 (d, J=8.0 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 3.97
(s, 3H), 2.79 (s, 3H), 1.55 ppm (s, 6H).
(S)-2-Amino-N-methoxy-N-methyl-3-phenylpropanamide (11): To
a
solution of N-(tert-butoxycarbonyl)-l-phenylalanine (500 mg,
1.88 mmol) in anhydrous CH2Cl2 (8 mL) was added BOP (833 mg,
1.88 mmol) and Et3N (188 mg, 1.87 mmol). solution of
+
(ESI) found [M]+ 643.2894, C36H41N3O8 requires 643.2898.
A
CH3ONHCH3.HCl (220 mg, 2.3 mmol) and Et3N (155 mg, 1.5 mmol)
in anhydrous DCM (4.3 mL) was added to the mixture. The mixture
was stirred under N2 at room temperature for 21 h before
quenched by 1m aqueous HCl (20 mL). The mixture was diluted
with DCM (50 mL) and the aqueous layer was separated and ex-
tracted with CH2Cl2 (2”50 mL). The combined organic phase was
washed with 1m aqueous HCl (50 mL), sat. NaHCO3 (50 mL) and
brine (50 mL), dried with Na2SO4 and concentrated in vacuo to
give the crude product (630 mg), which was then dissolved in
CH2Cl2 (24 mL). The solution was cooled on ice and then TFA
(3 mL) was added. The mixture was stirred with ice cooling for 1 h.
The volatiles were removed in vacuo to give the desired analogue
(3R)-Methyl 3-(8-((2-(2-ethoxyethoxy)ethoxy)methyl)-1’,3’,3’-tri-
methyl-6-nitrospiro[chromene-2,2’-indoline]-5’-ylcarboxamido)-
2-oxo-4-phenylbutanoate (3b): To a solution of 2b (33 mg,
0.047 mm) in anhydrous CH2Cl2 (2.4 mL) under N2 was added Dess–
Martin periodinane (20 mg, 0.047 mm) in one portion. The mixture
was stirred at room temperature under N2 for 3 h. The mixture was
diluted with CH2Cl2 (4.7 mL) and Na2S2O3 in saturated NaHCO3
(10% w/v, 0.5 mL) was added. The mixture was stirred for 15 min
and the aqueous phase was separated and extracted with CH2Cl2
(2.4 mL). The combined organic phase was dried over Na2SO4 and
concentrated in vacuo to give the crude product as a dark red oil,
which was purified by RP-HPLC to give 3b as a purple solid (2 mg,
6%). 1H NMR (500 MHz, [D6]DMSO): d=8.15 (s, 1H), 8.06 (s, 1H),
7.68 (d, J=6.7 Hz, 1H), 7.56 (s, 1H), 7.28–7.20 (m, 5H), 7.12 (d, J=
10.4 Hz, 1H), 6.63 (d, J=8.2 Hz, 1H), 5.99 (d, J=10.3 Hz, 1H), 5.08
(s, 1H), 4.30 (s, 1H), 3.72 (s, 3H), 3.54–3.36 (m, 10H), 3.28–3.06 (m,
2H), 2.78 (s, 3H), 1.32 (s, 3H), 1.20 (s, 3H), 1.14 ppm (t, J=7.0 Hz,
3H); 13C NMR (151 MHz, CD3OD): d=173.0, 168.8, 156.2, 149.4,
140.8, 138.5, 136.2, 129.0, 129.0, 128.5, 128.1, 127.8, 127.8, 126.4,
125.8, 125.8, 124.9, 124.2, 121.6, 120.7, 120.4, 118.6, 106.0, 105.9,
70.2, 70.1, 70.0, 70.0, 69.5, 66.2, 56.1, 51.5, 33.5, 27.6, 24.7, 18.6,
1
11 as a white wax (188 mg, quant.). H NMR (500 MHz, CDCl3): d=
7.35–7.11 (m, 5H), 5.15 (s, 1H), 4.95 (s, 1H), 3.65 (s, 3H), 3.16 (s,
3H), 3.10–2.81 ppm (m, 2H).
Surface attachment of 2d to a MOF (Fiber-2d)
The MOFs used in this work were fabricated in-house. Details of
the fiber and functionalization set-up have been reported pre-
viously.[18a] The fiber was sealed into a metal chamber with a posi-
tive pressure of 50 psi applied to force solutions through the fiber.
The inner surfaces of the fiber was washed with piranha solution
(3:1 sulfuric acid/30% H2O2 v/v, 1.5 mL) and carboxyethylsilanetriol
in water (2.5% v/v, 9 mL) was forced through the fiber to function-
alize the core surface by silanization.[28] A solution of 2d (0.5 mm),
DIC (0.5 mm), HOBt (0.5 mm) and DMAP (0.3 mm) in MeCN (10 mL)
was then applied through the fiber, overnight. The functionalized
fiber (Fiber-2d) was washed thoroughly with MeCN (10 mL) and
water (10 mL) for 6 h to remove unreacted 2d and other reagents.
+
14.0 ppm; HRMS (ESI) found [M]+ 701.2932, C38H43N3O10 requires
701.2948.
8-(Azidomethyl)-1’,3’,3’-trimethyl-6-nitro-N-((R)-1-oxo-3-phenyl-
propan-2-yl)spiro[chromene-2,2’-indoline]-5’-carboxamide (3c):
Compound 2c was oxidized according to general procedure B to
give 3c (7 mg, 10%). 1H NMR (500 MHz, [D6]DMSO): d=9.57 (s,
1H), 8.64 (d, J=6.1 Hz, 1H), 8.25 (s, 1H), 8.16 (s, 1H), 7.70 (d, J=
8.2 Hz, 1H), 7.62 (s, 1H), 7.24 (m, 5H), 6.67 (d, J=8.3 Hz, 1H), 6.04
(d, J=10.4 Hz, 1H), 4.41 (m, 1H), 4.26 (d, J=13.3 Hz, 1H), 4.18 (d,
J=13.3 Hz, 1H), 3.32–3.23 (m, 1H), 2.99–2.84 (m, 1H), 2.72 (s, 3H),
1.25 (s, 3H), 1.13 ppm (s, 3H); 13C NMR (151 MHz, CD3OD): d=
169.1, 156.8, 150.5, 140.7, 138.7, 136.0, 1290.0, 128.4, 128.1, 127.9,
127.2, 125.8, 125.2, 123.0, 122.4, 120.7, 119.1, 107.1, 106.0, 97.8,
97.5, 55.8, 55.6, 51.7, 34.7, 27.6, 24.8, 18.7 ppm; HRMS (ESI) found
Microstructured optical fiber (MOF) experiments
All MOF experiments were conducted in the dark unless stated
otherwise.
+
[M]+ 552.2138, C30H28N6O5 requires 552.2121.
Fiber-2d was first coupled to an optical set-up as previously re-
ported by Heng et al.[18a] The fiber was filled with Mili-Q water for
5 min through capillary action. Fiber-2d was excited by 10 pulses
of laser irradiation (532 nm, 10 ms per pulse) and the resulting
fluorescence emission after each pulse was measured by a spec-
trometer. Fiber-2d was then irradiated with UV light (365 nm) gen-
erated from a mercury lamp for 5 min. Fiber-2d was again subject-
ed to 10 pulses of laser irradiation (532 nm, 10 ms per pulse) with
the resulting fluorescence emissions measured similarly. Fiber-2d
was then irradiated with visible light generated from a halogen
lamp (>450 nm) and was similarly subjected to laser irradiation
with the fluorescence measured. The filled section of the fiber was
cleaved and removed.
3,3-Dimethyl-2-methyleneindoline-5-carboxylic acid (6): To a solu-
tion of 4-hydrazinobenzoic acid (2.00 g, 13.2 mmol) and 3-methyl-
2-butanone (1.26 g, 14.4 mmol) in anhydrous ethanol was added
98% H2SO4 (0.4 mL) slowly. The reaction mixture was refluxed
under N2 for 18 h. The mixture was allowed to cool to room tem-
perature and the resulting precipitate was removed by vacuum fil-
tration. The filtrate was basified with saturated NaHCO3 and
washed with CH2Cl2 (2”15 mL). The pH of the aqueous phase was
adjusted to 4 with 1m aqueous HCl and extracted with CH2Cl2 (2”
30 mL). The combined organic phase was dried over Na2SO4 and
concentrated in vacuo to give 6 as a dark red solid (2.43 g, 91%).
1H NMR (500 MHz, CDCl3): d=8.13 (d, J=8.0 Hz, 1H), 8.04 (s, 1H),
7.63 (d, J=8.5 Hz, 1H), 2.36 (s, 3H), 1.36 ppm (s, 6H).
Fiber-2d (SP isomer) was then filled with a solution of a-chymo-
trypsin in Mili-Q water (1 mm) for 5 min and excited with 10 pulses
of laser light (532 nm) as before for fluorescence measurements.
Fiber-2d (SP isomer) with a-chymotrypsin solution was left at
room temperature for a total of 85 min with fluorescence mea-
sured at 10, 25 and 85 min. Ten pulses of 532 nm laser light were
used to induce fluorescence before each measurement. The solu-
tion-filled section of the fiber was cleaved and discarded and the
remaining section of the fiber was filled with Mili-Q water over
5-Carboxy-1,2,3,3-tetramethyl-3H-indolium iodide (7): To a solu-
tion of 6 (1.12 g, 5.5 mmol) in a mixture of anhydrous toluene and
MeCN (2:1, 30 mL) was added methyl iodide (782 mg, 5.5 mmol)
dropwise. The mixture was refluxed for 18 h. After cooling to room
temperature, the red precipitate was collected by vacuum filtration
and washed with ethanol (5 mL) and hexane (30 mL) to yield 7 as
a light grey solid (1.19 g, quant.). 1H NMR (500 MHz, [D6]DMSO):
Chem. Eur. J. 2015, 21, 10703 – 10713
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10712
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Full Paper
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5 min. Fiber-2d was similarly excited with ten laser pulses and the
resulting fluorescence emission was measured. Fiber-2d was left
at room temperature for 85 min before the fluorescence was mea-
sured again. The solution-filled section of the fiber was again
cleaved and the remaining section was filled with a-chymotrypsin
in Mili-Q water (1 mm) for 5 min. Fiber-2d was concomitantly irradi-
ated with UV light (365 nm) to allow isomerization to the corre-
sponding MC isomer. Fiber-2d (MC isomer) with a-chymotrypsin
solution was left at room temperature for a total of 85 min with
laser-induced fluorescence measured at 0, 10, 25 and 85 min. The
solution filled section of the fiber was cleaved and discarded.
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We would like to thank Mr. Roman Kostecki, Dr. Herbert Foo,
Mr. Daniel Stubing and Dr. Erik Schartner for their assistance
with the fiber functionalization and all the fiber-based experi-
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Keywords: enzymes · peptidomimetics · photoswitching ·
spiropyran · surface chemistry
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Received: April 16, 2015
Published online on June 23, 2015
Chem. Eur. J. 2015, 21, 10703 – 10713
www.chemeurj.org
10713
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim