Protease responsive nanoprobes with tethered fluorogenic peptidyl 3-arylcoumarin substrates

Article abstract of DOI:10.1039/b816637d

Protease responsive nanosensors were obtained by the attachment of unique green fluorescent bifunctional 3-arylcoumarin-derived fluorogenic substrates to poly(acrylamide-co-N-(3-aminopropyl)methacrylamide) nanoparticles, in which proteolysis results in su

Full text of DOI:10.1039/b816637d

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Protease responsive nanoprobes with tethered fluorogenic peptidyl
3-arylcoumarin substratesw
Katharina Welser,ab Jakob Grilj,c Eric Vauthey,c Jonathan W. Aylott*b and Weng C. Chan*a
Received (in Cambridge, UK) 23rd September 2008, Accepted 13th November 2008
First published as an Advance Article on the web 8th December 2008
DOI: 10.1039/b816637d
Protease responsive nanosensors were obtained by the attachment
of unique green fluorescent bifunctional 3-arylcoumarin-derived
fluorogenic substrates to poly(acrylamide-co-N-(3-aminopropyl)-
methacrylamide) nanoparticles, in which proteolysis results in
substantial signal amplification.
Fluorescence bio-imaging has become an indispensable tool
for monitoring molecular changes in the intracellular environ-
ment. A plethora of ‘smart’ optical devices including fluoro-
genic substrates,1 quantum dots,2,3 near-infrared fluorescence
optical probes4 and optical nanosensors5 have been developed
to provide non-invasive interrogation of enzymatic activity
and concentration changes of analytes such as H+, Ca2+ and
Fig. 1 Model of a protease responsive polymer nanoprobe.
O2. One of the major challenges faced by researchers under-
taking cellular studies is the accurate and rapid detection of
acid (ACA) (5) as the fluorophore, which has previously been
used to prepare protease substrates.8 Although 5 has a high
quantum yield and large Stokes shift, it shows emission
proteolytic processes in vivo.6 This is important since protease
activities are stringently controlled, and their disregulation
maximum in the blue region making it less ideal for general
applications; especially as intracellular probes where the result-
commonly results in many disease conditions.7
By combining the advantages of fluorogenic substrates and
ing autofluorescence of cells and proteins could be problematic.
those of optical nanosensors, we have designed a modular
With the view to shifting both the absorption and emission
tool capable of rapidly and accurately monitoring protease
maximum of 5 to longer wavelengths, we installed (hetero)-
activity. Here we report the synthesis and in vitro validation of
aryl-substituents at the 3-position which extended the p system
of the parent fluorophore. Our synthetic strategy to 5 is based
our so-called ‘protease responsive nanoprobe’ (PRN), which
consists of a bifunctional fluorophore attached to both a
on the Suzuki C–C coupling reaction between a coumarinyl
nanoparticle and
a protease specific peptide substrate
bromide and an aryl boronic acid/ester.9 Thus, the desired
intermediate, ethyl 3-bromo-7-N-(carbethoxy)amino-coumarin-
4-acetate (3) (Scheme 1), was obtained in two steps from 1. We
became aware that protection of the 4-carboxymethyl group as
an ethyl ester was necessary as significant decarboxylation
occurred during the bromination and Suzuki reaction steps.
Optimized Pd-catalyzed cross-coupling of 3 with a judicious
(Fig. 1). Notably, the fluorogenic moiety is of low molecular
weight, displays optimized fluorescence properties and is
covalently tethered to the sensor matrix. Compared to non-
activatable imaging probes, this approach of proteolytic acti-
vation of the imaging probe has the advantage that multiple
fluorophores can be switched on by a single enzyme and that
protease specificity is achieved by the use of different peptide
substrates.4
In the design of our fluorescent PRN, we rationalized that the
fluorogenic moiety should be a bifunctional and relatively non-
toxic reagent with a large Stokes shift and a high quantum yield.
The nanoparticle itself should be mono-functionalized, which
will enable the covalent attachment of the fluorophore, and
should consist of a biocompatible polymer matrix. Thus, we
considered the hetero-bifunctional 7-aminocoumarin-4-acetic
a School of Pharmacy, Centre for Biomolecular Sciences, University of
Nottingham, University Park, Nottingham, UK NG7 2RD.
E-mail: weng.chan@nottingham.ac.uk
b School of Pharmacy, Boots Science Building, University of
Nottingham, University Park, Nottingham, UK NG7 2RD.
E-mail: jon.aylott@nottingham.ac.uk
c
´
Departement de Chimie-physique, Universite de Geneve, 30 Quai
Ernest Ansermet, Geneve 4, Switzerland CH-1211
´
`
`
w Electronic supplementary information (ESI) available: Experimental
protocols and products characterization. See DOI: 10.1039/b816637d/
Scheme 1 Synthesis of new derivatives of 5.
Chem. Commun., 2009, 671–673 | 671
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Fig. 2 Fluorescent characteristics of 5 and its derivatives 5a–5f.
Table 1 Photophysical properties of 5 and its derivatives 5a–5f
a
a
lem
Reagent
lmax/ex
log eb
Ffac
tad/ns
5
345
355
350
355
360
370
380
450
460
460
457
484
490
496
4.11
4.33
4.18
4.41
4.47
4.21
4.32
0.99
0.83
0.68
0.75
0.11
0.26
0.30
5.0
3.7
3.5
3.6
5a
5b
5c
5d
5e
5f
0.6
2.9 (0.7)/0.4 (0.3)
3.2 (0.6)/0.4 (0.4)
a
b
Measurements carried out in PBS–10% DMSO. Molar extinction
coefficient at lmax (L molÀ1 cmÀ1; EtOH–5% DMSO). Quantum
yields. Fluorescence lifetime; the numbers in parentheses represent
relative amplitudes.
c
d
Fig. 3 Chemical structures of fluorogenic probes and nanoprobe.
is a known fluorogenic substrate for subtilisin10 and was used as
a standard. In our model substrates, the ACA 4-carboxylic acid
functionality is extended by an amide bond to a b-alanine unit,
which is exploited both as a spacer and to endow chemical
stability. Because of the poor nucleophilicity of the anilino group
in 5, 5a and 5e, it was possible to regioselectively react the
fluorophores under mild coupling conditions to b-Ala-preloaded
Wang resin without previous Fmoc-protection.11,12 This was
followed by coupling of the C-terminal amino acid residue using
HATU/2,4,6-collidine.8 Assembly of the peptides using a
standard Fmoc/tBu solid-phase peptide synthesis method13 and
their subsequent acidolytic release then proceeded smoothly. As
anticipated, the prepared fluorogenic substrates displayed blue
shifted lmax and lem when compared to the parent fluorophores
(see Fig. 3 and 4).
selection of (hetero)aryl boronic acids, followed by saponifica-
tion afforded the unique 3-(hetero)aryl-substituted coumarin
derivatives 5a–5f.
Screening of the derivatives 5a–5f for their spectroscopic
properties (Fig. 2 and Table 1) showed that the modification
of 5 in the 3-position with heteroaryls (5d–5f) resulted in
a significant red shift of both the absorption/excitation
(lmax/ex) and emission (lem) maxima. Compared to 5, the shift
in lmax/ex and lem was found to be 15–35 nm (1200–2670 cmÀ1
)
and 34–46 nm (1560–2060 cmÀ1), respectively. In contrast,
only a minor shift of lmax/ex and lem was observed for the
phenyl, tolyl and naphthyl derivatives. Interestingly, changing
the substituent at the 3-position resulted in a general decrease
in quantum yields and lifetimes, especially when heteroaryls
were incorporated into the ACA scaffold. Similar results have
Optimized conditions were then established for the spectro-
fluorimetric monitoring of the enzymatic cleavage reaction.
The specific lex and lem were chosen such that the intact
substrate showed a minimal absorbance and emission, thus
ensuring optimal amplification of the fluorescent signal
upon proteolysis. The determined optimized conditions were:
6 (lex,opt = 380 nm, lem,opt = 460 nm), 6a (lex,opt = 390 nm,
been reported by Bauerle and co-workers with coumarin.9
¨
The presence of a new deactivation pathway shows up as a
shortening of the fluorescence lifetime for 5d–5f. The
bi-exponential decay for 5e and 5f suggests the existence of
two different structural forms: one where this new deactivation
pathway is operative (hence the shorter t) and the other where
it is inactive (long t).
From the newly synthesized ACA derivatives, the green
fluorophores 5d–5f were identified as ‘hits’ due to their highly
favourable lmax/ex and lem. In fact, compared to the fluores-
cein dyes (lmax/ex = 490 nm, lem = 514 nm), their Stokes
shifts (116–124 nm; 6150–7120 cmÀ1 versus 24 nm; 950 cmÀ1
for fluorescein) are particularly noteworthy. Further com-
parative investigations were then carried out with 5e because
of its ease of synthesis and higher quantum yield when
compared to 5d.
lem,opt = 470 nm), 6e (lex,opt = 430 nm, lem,opt = 510 nm). At
these stringently optimized conditions, we observed an
approximately 10-fold decrease in the relative fluorescence
(Frel) for the ACA(furyl)-based compounds compared to the
ACA and ACA(phenyl) derivatives (see Fig. S2 and S3, and
Employing subtilisin as the model protease, an efficient
enzyme widely used for studying enzyme–substrate interactions,
we proceeded to synthesize the fluorogenic substrates 6, 6a and
6e (Fig. 3). The commercially available Z-Gly-Gly-Leu-AMC (7)
Fig. 4 Normalized excitation (ex., dashed line) and emission spectra
(em., solid line) of (a) 6 and 8 and (b) 6e and 8e in Tris–HCl buffer.
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Table 2 Kinetic parameters for subtilisin specific fluoroprobes 6, 6a
and 6e. Kinetic constants for the AMC standard 7 were taken from
ref. 10
Fluoroprobes
KM/mM
kcat/sÀ1
kcat/KM/MÀ1 sÀ1
6
0.136 Æ 0.021
0.241 Æ 0.037
0.263 Æ 0.014
0.740
0.087 Æ 0.005
0.086 Æ 0.011
0.189 Æ 0.005
0.65
650 Æ 62
359 Æ 10
720 Æ 19
880
6a
6e
7
Table S1, ESIw). However, this decrease in Frel is accompanied
by significant bathochromic shifts of the excitation and
emission maxima as well as an extended Stokes shift, which
aids utility of the fluorogenic substrate.
To determine if the introduction of 3-(hetero)aryl-substituents
in the ACA structure has an effect on the enzyme–substrate
interaction, we carried out enzyme kinetic measurements
(Table 2). Compared to the AMC-based substrate 7,10 the three
ACA substrates are characterized by lower KM and kcat values.
However, among the ACA substrates, 6e stands out due to a
higher kcat value and a kcat/KM value that is similar to 7. These
findings together with the feature of having red shifted lmax and
lex highlight the significant utility of our green fluorophore
5e and its corresponding fluorogenic substrate 6e for bio-imaging.
To obtain the PRNs, the fluorogenic peptides 6 and 6e were
tethered to amine-functionalized nanoparticles using a stan-
dard DIC/HOAt coupling procedure. The nanoparticles de-
rived from acrylamide and N-(3-aminopropyl)methacrylamide
were prepared via an inverse microemulsion polymerization
process, which is known to produce nm-sized particles with a
narrow size distribution.14 Characterization by dynamic light
scattering (DLS) showed that the nanoparticles were on
average approximately 47 nm in diameter (Fig. 5a).
Fig. 5 (a) DLS of polyacrylamide nanoparticles (NPs); (b) subtilisin
mediated cleavage reaction of NP-bound 6e (lex
= 430 nm);
(c) enzyme kinetic of NP-bound 6e at different subtilisin con-
centrations; (d) initial velocity (v0) versus subtilisin concentration of
NP-bound 6 and 6e.
tunable PRN should find application in diagnostics and high-
throughput screening of protease inhibitors. We are currently
determining the general utility of different configurations of
bio-imaging agents based on our new bifunctional green
fluorescent coumarin dye, especially in a cellular and in vivo
context.
KW would like to acknowledge Engineering & Physical
Sciences Research Council and Royal Society of Chemistry,
UK for the funding of an analytical science studentship.
Notes and references
Proof of principle for the concept of PRN was indeed
confirmed by a significant increase in fluorescence when the
nanoprobe was incubated with subtilisin at 37 1C in Tris–HCl
buffer (pH = 8.20) (Fig. 5b). The enzyme reaction slowed
after 10 minutes, indicating a fast initial protease response
(Fig. 5c). In fact, after 10 minutes, ca. 80-fold rise in the
fluorescence signal was observed when subtilisin was present at
10 mM. A linear relationship between the initial velocity of
nanoparticle-bound 6 and 6e and subtilisin concentration was
observed up to a measured enzyme concentration of 10 mM
(Fig. 5d). With initial rates three times as high, PRN com-
prising 6e are better enzyme substrates than 6-derived PRN.
These findings are in agreement with our initial enzyme kinetic
results outlined in Table 2. Control experiments revealed that
no significant change in the fluorescence was observed in
the absence of subtilisin or in the presence of chymotrypsin.
The concept presented here is modular and hence can easily be
extended to other endopeptidases by choosing the appropriate
substrates (e.g., see Fig. S4, ESIw).
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In conclusion, using rational design principles we have
synthesized a new family of fluorescence-based PRNs for the
efficient detection of protease activity. These devices consist of
a nanoparticle that is covalently attached to fluorogenic pep-
tide substrates comprising spectrofluorimetrically improved
bifunctionalized 3-arylcoumarin (5e). These customized and
Chichester, 2003.
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