Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases

Article abstract of DOI:10.1016/j.bmc.2011.03.014

Activity-based probes (ABPs) have found increasing use in functional proteomics studies. Recently, ABPs that can be employed in combination with click chemistry gained particular attention due to their flexible application in vitro and in vivo. Moreover,

Full text of DOI:10.1016/j.bmc.2011.03.014

Bioorganic & Medicinal Chemistry 20 (2012) 633–640
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alkyne derivatives of isocoumarins as clickable activity-based probes
for serine proteases
⇑
Ute Haedke, Markus Götz, Philipp Baer, Steven H. L. Verhelst
Center for Integrated Protein Science Munich (CIPSM), Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Activity-based probes (ABPs) have found increasing use in functional proteomics studies. Recently, ABPs
that can be employed in combination with click chemistry gained particular attention due to their flex-
ible application in vitro and in vivo. Moreover, there is a continuous need for new ABPs that target small
subsets of enzymes. We here report novel clickable ABPs based on the 4-chloro-isocoumarin (IC) electro-
phile, a mechanism-based inhibitor scaffold that covalently binds serine proteases. We describe the syn-
thesis of a small library of IC ABPs containing an alkyne function and a set of diverse selectivity elements.
The different substituents on the IC structure determine which proteases are bound, showing good cor-
relation with the preferred substrate preferences. The IC ABPs can detect their target proteases in a pro-
teome background in a sensitive manner (down to 0.007% of total protein). Furthermore, we show
activity-dependent and selective labeling of endogenous proteases in a tissue proteome. These ICs there-
fore represent a valuable extension to already existing ABPs for serine proteases and may be instrumental
in future elucidation of serine protease functions.
Received 14 January 2011
Revised 2 March 2011
Accepted 7 March 2011
Available online 12 March 2011
Keywords:
Activity-based probes
Click chemistry
Serine proteases
Isocoumarins
Functional proteomics
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
types of warheads, because of the lower reactivity and the in-
creased hardness of the serine active site nucleophile. Up to date,
In the post-genomic era, with genome sequences of numerous
organisms available, most proteomics studies are aimed at quanti-
fication of the level of expressed proteins. However, the mere
abundance of a protein does not address its functional state. The
difference between abundance and activity is especially crucial
for enzymes, whose activity is under tight post-translational con-
trol. In order to determine the active partition of expressed en-
zymes, small molecule activity-based probes (ABPs) have become
a valuable tool in functional proteomics. Most ABPs consist of a
certain detection tag conjugated to a mechanism-based inhibitor
(often termed warhead) that forms a covalent bond with the target
enzymes.1
In the last decade, ABPs have proven particularly useful for the
functional analysis of proteases.2–4 The proteasome, for example,
has been studied using ABPs based on vinyl sulfones,5,6 vinyl
amides7 and epoxyketones.8 For cysteine proteases, a wide variety
of warheads have been reported based on mild electrophiles that
selectively react with the active site cysteine. Examples include
acyloxymethyl ketones,9 O-acyl hydroxamates,10 unsaturated ke-
tones11,12 and sulfones,13,14 and several derivatives of epoxysucci-
nates.15–17 For serine proteases, the activity-based proteomics
toolbox is less well equipped. Serine proteases require different
the main serine protease ABPs comprise fluorophosphonates18
and peptidyl diphenyl phosphonates.19–21 We aimed to expand
the ABP toolbox for serine proteases with non-peptidic reagents
based on the 4-chloro-isocoumarin scaffold.
4-Chloro-isocoumarins (ICs) have been investigated as inhibi-
tors for soluble serine proteases,22,23 rhomboids24 (intramembrane
serine proteases) and cholesterol esterase.25 The mechanism of
inactivation involves an attack of the nucleophilic serine residue
in the active site of the protease on the carbonyl group of the iso-
coumarin. Depending on the substituent at the 7-position, the
chloride is eliminated, giving rise to a quinone imine methide,
which can in turn trap a second nucleophile, such as the active site
histidine or a solvent molecule (Fig. 1).26
ICs do not closely mimic a protease substrate. Hence, it is hard
to predict which substituents on the isocoumarin ring are being
recognized by the S1 pocket, which is the primary specificity pock-
et in serine proteases. Interestingly, crystal structures of proteases
in covalent complex with 3,7-disubstituted ICs have shown bind-
ing in different conformations, depending on the inhibitor struc-
ture and their mode of inhibition (i.e., only bound to the active
site serine or doubly bound: to the active site serine and histidine).
A substituent on the 3-position can, for example, dock into the S1
or the S3 pocket of porcine pancreatic elastase,27,28 whereas a re-
cent crystal structure of the Escherichia coli rhomboid GlpG shows
that a substituent at the 7-position can dock into the S1 pocket.24
Previous studies of ICs with positively charged binding elements at
⇑
Corresponding author. Tel.: +49 8161 713505.
E-mail address: verhelst@wzw.tum.de (S.H.L. Verhelst).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.014

634
U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
formate could be used to successfully introduce pentynoic acid as a
HO
OR
O
O
Enzyme
click-tag, yielding the desired target probes 5–7. At the 3-position,
these probes have either a small or a hydrophobic selectivity ele-
ment. The formation of a positively charged group at the 3-position
was accomplished by nucleophilic substitution of the bromide in
compound 7 by thiourea, yielding an isothioureido group, which
mimics the arginine side chain.
H
N
H
N
R'
O
R'
O
Enzyme
CO2R
Cl
O
Cl
O
Ideally, compound 7 could serve as a starting point for broader
diversification at the 3-position through substitution of the bro-
mide by different nucleophiles. Unfortunately, when we tested this
out on a model compound, it turned out that the use of nucleo-
philes other than thiourea led to degradation of the isocoumarin
scaffold, presumably by attack on the electrophilic carbonyl.34
At this point, we turned our attention to the synthesis of the
3-alkyne series (Scheme 2). Selective esterification of 4-nitrohom-
ophthalic acid with 3-butyn-1-ol was accomplished using TsOH as
a catalyst,35 after which reaction with PCl5 yielded the IC 12. The
same reaction sequence for homophthalic acid gave probe 13.
Selective reduction of the nitro-group in 12 in the presence of
the alkyne function was achieved by reaction with iron powder
in acetic acid. The resulting free amino group now allowed the
introduction of different substituents at the 7-position, either
small (15), hydrophobic (16) or basic (17 and 18).
H
N
N
R'
O
R'
O
Enzyme
Enzyme
CO2R
CO2R
Nu
Figure 1. The mechanism of covalent modification of proteases by 4-chloro-
isocoumarins.
either the 3- or the 7-position have shown inhibition of proteases
with tryptic activity.29 These results suggest that elements in both
positions can potentially dock into the S1 pocket.
Despite their application as protease inhibitors, ICs have only
been used as ABPs to a very limited extent. The few IC ABPs re-
ported to date carry a biotin tag at the 7-position and a low variety
of functional groups at the 3-position.30,31 Recently, one of these
biotinylated ICs has been used to identify a protease involved in
host cell rupture by the malaria parasite Plasmodium falciparum,32
illustrating that IC ABPs are promising tools for the functional char-
acterization of serine protease activities.
In order to exploit ICs to their full potential, we decided to syn-
thesize a set of IC ABPs with a variety of recognition elements and a
versatile alkyne handle amenable for the introduction of detection
tags by click chemistry (Fig. 2). Two versions of IC ABPs were de-
signed – either with a selectivity element on the 3-position and
an alkyne handle on the 7-position (A), or the other way around
(B). We here report the synthesis of these probes and show that
they react with trypsin-like, elastase-like and chymotrypsin-like
serine proteases depending on the substituents on the IC scaffold.
Furthermore, these reagents selectively label proteases in complex
proteomes, demonstrating the potential of alkyne IC ABPs in future
functional proteomics studies.
2.2. Labeling specificity
With the desired molecules at hand, we first tested out their
labeling efficiency using members from different subclasses of ser-
ine proteases: bovine chymotrypsin, human cathepsin G and por-
cine pancreatic elastase, known for their specificity for different
types of hydrophobic residues, and trypsin and urokinase-type
plasminogen activator (uPA) as examples of trypsin-like proteases.
ICs were used at a concentration of 2 lM (see Supplementary
Fig. 1) and visualized using Cu(I)-mediated click chemistry with a
tetramethylrhodamine (TAMRA) derivative carrying an azide
function.
Probe 13 (Fig. 3a) reacted with all tested proteases. We attribute
this to the lack of a 7-substituent that would induce selectivity.
Bovine chymotrypsin is labeled by all probes with approxi-
mately the same band intensity, including by the ICs with posi-
tively charged substituents ( Fig. 3a). The reaction occurs in an
activity-dependent manner, since pretreatment with an active
site-directed covalent inhibitor abrogates labeling. We hypothe-
sized that the alkyne function, which was intended to serve solely
as a tagging handle, docks into the hydrophobic S1 pocket of chy-
motrypsin, which may explain its promiscuous behavior. To test
this hypothesis, we modified the labeling experiment by perform-
ing the azide–alkyne click reaction before adding the enzyme. We
assumed that once modified to a triazole ring holding a bulky fluo-
rophore, the former alkyne group would no longer fit into the S1
pocket. As shown in Figure 3b, this holds true for compound 8:
in contrast to the alkyne, its ‘clicked’ version does not label chymo-
trypsin (lanes 3 and 4). IC 16 binds both before and after modifica-
tion, probably because the 3-alkyne as well as the 7-benzoyl group
can serve as recognition site for chymotrypsin (lanes 1 and 2). The
bands resulting from compound 18, which carries a positively
charged 7-substituent, show that the hypothesized binding mode
may not be true for all tested probes (lanes 5 and 6).
2. Results and discussion
2.1. Synthesis
Scheme 1 outlines the synthesis of the 7-alkyne series. The reac-
tion sequence starts with an acid-catalyzed mono-esterification of
4-nitrohomophthalic acid using slight modifications from known
procedures.26,33 The mono-esters 2a–c were subsequently con-
verted into the ICs 3a–c upon reaction with PCl5.26 In order to al-
low further modification, the 7-nitro substituent was reduced to
an aniline by catalytic hydrogenation. Due to the low reactivity
of the aniline function, amide-bond formation with carboxylic
acids proved to be cumbersome using standard peptide coupling
reagents such as DIC/HOBt or HATU/DIEA. However, the mixed
anhydride method with the condensation reagent isobutyl chloro-
O
O
7
R
Human cathepsin G reacted with fewer probes than chymotryp-
sin (Fig. 3a). 3-Alkynes with small and hydrophobic substituents at
the 7-position (14 and 16) labeled as well as 7-alkynes with small
to medium-sized hydrophobic substituents at the 3-position (5
and 7). This finding is consistent with the chymotrypsin-like activ-
ity of cathepsin G. No reactivity towards probes with positively
charged substituents was observed, although human cathepsin G
also displays tryptic activity.36
O
3
O
OR
O
4
Cl
7-alkyne group
R = selectivity element
Cl
A
B
3-alkyne group
Figure 2. General structure of the target clickable isocoumarin ABPs.

U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
635
O
O
O
O
O2N
O2N
a)
b)
O2N
c)
H2N
OH
OH
OH
OR
O
O
OR
OR
Cl
Cl
O
O
1
2a
2b
3a
3b
4a
4b
R = Me
R = CH2CH2Ph
R = Me
R = CH2CH2Ph
R = Me
R = CH2CH2Ph
2c R = CH2CH2Br
3c R = CH2CH2Br
4c R = CH2CH2Br
d)
O
O
O
H
N
H
N
e)
O
O
S
NH2
NH
O
O
OR
Cl
Cl
8
5
6
R = Me
R = CH2CH2Ph
7 R = CH2CH2Br
Scheme 1. Synthesis of the 7-alkyne IC ABPs. Reagents and conditions: (a) R-OH, H2SO4 (cat), toluene, 70 °C; (b) PCl5, toluene, 70 °C; (c) H2, Pd–C, THF/CHCl3; (d) 4-pentynoic
acid, isobutyl chloroformate, N-methyl morpholine, THF, ꢀ20 °C ? rt; (e) thiourea, THF, 65 °C.
O
O
O
O
R
R
a)
b)
R
c)
H2N
OH
OH
OH
O
O
O
O
O
Cl
Cl
O
O
1
9
10
11
12
13
14
R = NO2
R = H
R = NO2
R = H
R = NO2
R = H
d)
NH
O
O
H
N
H
N
R
e)
H2N
N
H
O
O
O
O
O
O
Cl
Cl
18
15
16
R = Me
R = Ph
17 R = CH2NH2
Scheme 2. Synthesis of the 3-alkyne IC ABPs. Reagents and conditions: (a) 3-butyn-1-ol, p-TsOH, toluene, 70 °C; (b) PCl5, toluene, 70 °C; (c) Fe(s), AcOH, 70 °C; (d) Ac2O/N-
methyl morpholine/DMAP (cat), or BzCl/pyridine/THF, or Boc-Gly-OH, isobutylchloroformate, N-methyl morpholine, THF, ꢀ20 °C ? rt, then 25% TFA in DCM; (e) (1) di-Boc-
guanidine-triflate, N-methyl morpholine, (2) 25% TFA in DCM.
Elastase was strongly labeled by the 7-alkynes with small and
medium-sized hydrophobic 3-substituents (5 and 7), in accordance
with the substrate preference of this protease for small hydropho-
bic amino acids. Compound 18, with a positively charged 7-substi-
tuent, also reacted with elastase. All these labeling events could be
blocked by inhibitor pretreatment, demonstrating the reaction’s
activity-dependency. In contrast, probes 14 and especially 12 label
regardless of the presence of an inhibitor. Apparently, binding to a
residue distant from the active site takes place, likely due to the
high reactivity caused by the electron withdrawing nitro-
substituent.
and trypsin was achieved by probes 12–14, although only for
uPA the reaction occured in an activity-dependent manner.
Overall, the majority of alkyne IC ABPs display activity-depen-
dent labeling of their target proteases. The reaction reflects in most
cases the P1 selectivity of the labeled protease. However, there are
also some exceptions, making it difficult to rationally design and
optimize IC ABPs for specific proteases without a crystal structure
available.
2.3. Labeling in proteomes
As expected, uPA, a serine protease with tryptic activity, is
strongly labeled by probes 8, 17 and 18, which all have positively
charged 3- or 7-substituents. The same pattern is seen for trypsin.
This observation suggests that both the 3- and the 7-position can
display a positively charged selectivity element to the S1 pocket
of tryptic proteases, in line with previous data.29 Labeling of uPA
At this stage, we aimed to find out what the labeling capacity of
the IC APBs is in the context of a complex proteome. Therefore, we
titrated decreasing amounts of selected proteases into a lysate of
mammalian cells (EL4 mouse lymphoma) and added the ICs that
displayed strong activity-based labeling in the previous
experiments.

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U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
Figure 3. Labeling of representative members of serine protease subclasses using isocoumarin (IC) activity-based probes (ABPs). (a) 100 ng of protease per lane were
incubated for 30 min with (+) or without (ꢀ) an active site-directed inhibitor (100 M Ala-Ala-Phe-diphenylphosphonate for chymotrypsin, 1 mM PMSF for cathepsin G,
100 M DFP for uPA and 1 mM dichloroisocoumarin for trypsin and elastase) and subsequently labeled with 2 M of the indicated IC. The enzyme-probe complexes were
l
l
l
detected by Cu(I)-catalyzed click chemistry and fluorescence scanning. (b) The Cu(I)-catalyzed click reaction of selected ICs with a fluorophore azide was performed before
(pre) and after (post) reaction with chymotrypsin. Note that labeling by IC 8 is strongly diminished when functionalized with a fluorophore prior to labeling, indicating that
the alkyne function is involved in recognition by chymotrypsin. (c) Overview of the used IC ABPs.
The protease uPA can be visualized by IC 18 at a concentration
of 0.03% of total protein content (Fig. 4, right panel). IC 18 yielded
some additional weak bands, which could not be blocked by DFP
pretreatment indicating that these are off-target proteins that do
not represent serine protease activities. IC 5 detected elastase
without any background labeling at an amount as low as 1 ng
(40 fmol) or 0.007% of total protein. Labeling of cathepsin G with
IC 14 did also not lead to any off-target reactions, but was slightly
less efficient, with a detection limit of 0.03% of total protein
content.
To verify that the IC ABPs can be utilized to detect endogenously
expressed, active proteases, a selection of IC ABPs with different
substituents at the 7-position was reacted with a rat liver prote-
ome, followed by click chemistry-mediated fluorescent detection.
Compound 16 shows an exquisitely selective labeling of a band
of approximately 25 kDa (Fig. 5, left panel, arrowhead). Pretreat-
ment of the lysate with N-tosyl-phenylalanine-chloromethyl ke-
tone (TPCK),
a
known mechanism-based inhibitor of
chymotrypsin-like proteases, completely prevents detection of this
band confirming the activity-dependent nature of the labeling.
Although compound 13 reacted with all tested purified proteases
(see Fig. 3) it displays a remarkable selectivity in the context
of the rat liver proteome, where it labels one major band at
26 kDa (Fig. 5, middle panel, open arrowhead). Interestingly, this
band can be competed by pretreatment with TPCK, but is distinct
from the band tagged by 16 (see Supplementary Fig. 2), suggesting
that the two IC probes can distinguish between different chymo-
tryptic activities. Treatment of the rat liver proteome with IC 14
resulted in labeling of an intense band around 50 kDa, which could
not be competed with TPCK, but disappeared by pretreatment with
Figure 4. Labeling of serine proteases in a proteome background. Selected IC ABPs were used to determine the detection limits of three representative serine proteases.
Decreasing amounts of pure proteases (0.7%, 0.2%, 0.03% and 0.007% of total protein content for human cathepsin G and uPA, and 0.2%, 0.03%, 0.007% and 0.001% for porcine
pancreatic elastase) were spiked into a mammalian cell lysate (EL4 mouse lymphoma, 1 mg/mL). After labeling with the indicated IC ABPs, detection took place by
Cu(I)-catalyzed click chemistry using a fluorophore-azide. + and ꢀ indicate the presence or absence of an active site-directed inhibitor. Note that elastase was detected down
to a concentration of 0.007%, cathepsin G and uPA down to 0.03% of total protein content.

U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
637
electrospray TOF. NMR data were recorded on a Bruker 500 MHz
Avance III.
4.2. Synthesis
4.2.1. Esterification of homophthalic acid and 4-
nitrohomophthalic acid
A suspension of the homophthalic acid and the appropriate
alcohol (3 equiv) in toluene (2 mL/mmol) was heated to 70 °C.
Several drops of 98% sulfuric acid were added, except for the alco-
hol 3-butyn-1-ol, for which 0.1 equiv pTsOH was used. The mixture
was stirred for 4–6 h when TLC indicated complete consumption of
the starting acid. After cooling to room temperature, the reaction
mixture was quenched with diluted sodium bicarbonate and
extracted twice with ethyl acetate. Next, the aqueous layer was
acidified using concentrated HCl and extracted twice with ethyl
acetate. The resulting organic layer was dried (MgSO4), filtered
and concentrated under reduced pressure. The material was used
in the next step without further purification.
4.2.2. Formation of the 3-alkoxy-4-chloro-isocoumarin
scaffolds
Figure 5. In vitro labeling of a rat liver proteome at pH 7.4 with ICs 13, 14 and 16.
Target proteins were detected by click chemistry and fluorescent scanning. All
proteome samples were pretreated with DMSO (ꢀ) or the indicated active-site
directed serine protease inhibitor (+).
PCl5 (2.5 equiv) was added to a solution of the mono-esterified
homophthalic acid derivatives in toluene (10 mL/mmol) and the
resulting solution was stirred overnight at 70 °C. The reaction mix-
ture was cooled down, diluted with ethyl acetate and extracted
with sodium bicarbonate. Water layers were backextracted with
ethyl acetate and the combined organic layers were washed with
sodium bicarbonate and brine, dried (MgSO4), concentrated under
reduced pressure and subjected to silica column chromatography
(50–0% petroleum ether in toluene).
the more broad spectrum serine protease inhibitor PMSF (Fig. 5,
right panel, star). Overall, these experiments show that the IC ABPs
can label endogenous proteins in a selective and competable man-
ner, dependent on the substituent at the 7-position.
3. Conclusion
In this study, we describe the synthesis and evaluation of alkyne
derivatives of ICs as ABPs for serine proteases. We demonstrate that
these ABPs can react with the different subclassesof serine proteases
dependent on the substituents displayed on the IC scaffold. How-
ever, rational design of IC ABPs for specific proteases remains a chal-
lenge due to the lack of a clear structure–activity relationship.
Our ICs can detect target proteases in a whole proteome back-
ground down to 40 fmol or 0.007% of total protein content. These
sensitivities are in the same order of magnitude as previously re-
ported click ABPs.37,38 Furthermore, experiments in a rat tissue
proteome show clean, selective, activity-dependent labeling.
The alkyne handle on the described IC ABPs offers advantages
over previously reported biotinylated ICs: the versatile choice of
tags creates flexibility in the use of detection methods. Moreover,
biotinylated molecules generally have poor cell permeability. Al-
kyne versions may circumvent this unfavorable property. Overall,
we think that the clickable IC ABPs represent valuable compounds
for the use in future functional proteomics studies of serine
proteases.
4.2.3. Coupling of pentynoic acid to the aniline function
Pentynoic acid (dried by coevaporation of water with dioxane)
was pre-activated in THF (2 mL/mmol) at ꢀ20 °C for 30 min with
isobutyl chloroformate (1.1 equiv) and N-methylmorpholine
(1.2 equiv with respect to pentynoic acid). Next, 2 equiv of acti-
vated pentynoic acid were added to a stirred solution of the aniline
in THF (final concentration 0.2–0.25 M) and the reaction was al-
lowed to warm up to room temperature. After TLC analysis re-
vealed dissapearance of the starting material, the reaction
mixture was diluted with EtOAc and subsequently washed with
1 M KHSO4, saturated NaHCO3 and brine. The organic layer was
dried (MgSO4), filtered and concentrated under reduced pressure.
The crude material was purified by HPLC.
4.2.4. 4-Chloro-3-methoxy-7-nitro-isocoumarin (3a)
The title compound was isolated as a yellow solid in 51% yield
from its mono-esterified nitro-homophthalic acid precursor. 1H
NMR (500 MHz, CDCl3) d 9.05 (d, 1H, J = 2.4 Hz), 8.53 (dd, 1H,
J = 2.4 Hz, J = 9.0 Hz), 7.84 (d, 1H, J = 8.9 Hz), 4.18 (s, 3H). 13C DEPT-
NMR (126 MHz, CDCl3): d 129.8, 126.4, 123.4, 57.3. ESI-HRMS:
[M+H]+ m/z 255.9904 (found), C10H7ClNO5 requires 256.0007.
þ
4. Experimental
4.1. General
4.2.5. 4-Chloro-3-(2-phenyl-ethoxy)-7-nitro-isocoumarin (3b)
The title compound was isolated as a yellow solid in 38% yield
from its mono-esterified nitro-homophthalic acid precursor. 1H
NMR (500 MHz, DMSO-d6) d 8.71 (d, 1H, J = 2.4 Hz), 8.59 (dd, 1H,
J = 2.5 Hz, J = 8.9 Hz), 7.81 (d, 1H, J = 8.9 Hz), 7.37–7.19 (m, 5H),
4.67 (t, 2H, J = 6.6 Hz), 3.11 (t, 2H, J = 6.6 Hz). 13C DEPT-NMR
(126 MHz, DMSO-d6): d 129.6, 128.8, 128.1, 126.3, 124.7, 122.8,
71.0, 34.4.
4-Nitrohomophthalic acid was synthesized as described in Ref.
33. All other reagents were obtained from commercial suppliers
and used without further purification, unless noted otherwise. All
solvents were of analytical grade, except for solvents used in silica
column chromatography. Reactions were monitored by TLC (silica-
gel 60) using UV light and/or treatment with cerium ammonium
molybdate followed by heating. Preparative HPLC purification
was performed on a Waters 515 HPLC system using an X-bridge
C18 column. High resolution mass spectrometry analysis was
performed on an Agilent 6210 LC–MS equipped with an
4.2.6. 4-Chloro-3-(2-bromo-ethoxy)-7-nitro-isocoumarin (3c)
The title compound was isolated as a yellow solid in 52% yield
from its mono-esterified nitro-homophthalic acid precursor. 1H

638
U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
NMR (500 MHz, CDCl3) d 9.07 (d, 1H, J = 2.3 Hz), 8.57 (dd, 1H,
J = 2.3 Hz, J = 8.9 Hz), 7.89 (d, 1H, J = 8.9 Hz), 4.77 (t, 2H,
J = 6.1 Hz), 3.71 (t, 2H, J = 6.1 Hz). 13C DEPT-NMR (126 MHz,
CDCl3): d 129.8, 126.3, 123.9, 69.6, 27.7. ESI-HRMS: [M+H]+ m/z
4.2.12. 3-(2-Bromoethoxy)-4-chloro-7-(4-pentynoylamino)-
isocoumarin (7)
The title compound was isolated as a fluffy, slightly yellow solid
in 30% yield after HPLC purification. 1H NMR (500 MHz, DMSO-d6)
d 10.39 (s, 1H), 8.51 (d, 1H, J = 2.2 Hz), 8.01 (dd, 1H, J = 2.3 Hz,
J = 8.7 Hz), 7.68 (d, 1H, J = 8.7 Hz), 4.64 (t, 2H, J = 5.5 Hz), 3.85 (t,
2H, J = 5.5 Hz), 2.81 (t, 1H, J = 2.6 Hz), 2.57 (t, 2H, J = 6.9 Hz),
þ
347.9303 (found), C11H8BrClNO5 requires 347.9269.
4.2.7. 7-Amino-4-chloro-3-methoxy-isocoumarin (4a)
2.53–2.46 (m, 2H).). 13C DEPT-NMR (126 MHz, DMSO-d6):
d
A mixture of 3a in THF/CHCl3 4:1 and palladium on carbon was
stirred under an atmosphere of hydrogen until TLC revealed com-
plete disappearance of the starting material. The reaction mixture
was filtered and concentrated in vacuo. The title compound was iso-
lated quantitatively as a yellow powder. 1H NMR (500 MHz,
126.8, 122.6, 117.9, 83.2, 71.3, 69.9, 34.9, 30.4, 13.7. ESI-HRMS:
[M+H]+ m/z 397.9796 (found), C16H14BrClNO4 requires 397.9795.
þ
4.2.13. 4-Chloro-7-(4-pentynoylamino)-3-(2-
isothioureidoethoxy)-isocoumarin (8)
A solution of compound 7 (6.62 mg) and thiourea (2.6 mg,
2 equiv) in THF/DMF (1:1, 350 lL) was stirred at 65 °C for 2 d.
CDCl3 + methanol-d4)
d 7.56 (d, 1H, J = 8.6 Hz), 7.48 (d, 1H,
J = 2.5 Hz), 7.22 (dd, 1H, J = 2.5 Hz, J = 8.6 Hz), 4.05 (s, 3H). 13C
DEPT-NMR (126 MHz, CDCl3 + methanol-d4): d 124.8, 124.0, 113.9,
57.6. ESI-HRMS: [M+H]+ m/z 226.0173 (found), C10H9ClNO3
The reaction mixture was purified by HPLC and the title compound
was isolated as a fluffy, slightly yellow solid in 30% yield. 1H NMR
(500 MHz, DMSO-d6 + methanol-d4) d 8.54 (d, 1H, J = 2.0 Hz), 8.04–
7.97 (m, 1H), 7.69 (d, 1H, J = 8.7 Hz), 4.57 (t, 2H, J = 5.8 Hz), 3.63 (t,
2H, J = 5.8 Hz), 2.80 (t, 1H, J = 2.6 Hz), 2.60–2.56 (m, 2H), 2.53–2.48
(m, 2H). 13C DEPT-NMR (126 MHz, DMSO-d6 + methanol-d4): d
126.8, 122.6, 117.9, 83.2, 71.3, 68.1, 34.9, 29.5, 13.7. ESI-HRMS:
þ
requires 226.0265
4.2.8. 7-Amino-4-chloro-3-(2-phenyl-ethoxy)-isocoumarin (4b)
A mixture of 3b in THF/CHCl3 4:1 and palladium on carbon was
stirred under an atmosphere of hydrogen until TLC revealed com-
plete disappearance of the starting material. The reaction mixture
was filtered and concentrated in vacuo. The title compound was
isolated as a yellow powder in 87% yield. 1H NMR (500 MHz,
DMSO-d6) d 7.40 (d, 1H, J = 8.6 Hz), 7.34–7.19 (m, 6H), 7.15 (dd
1H, J = 2.5 Hz, J = 8.6), 4.47 (t, 2H, J = 6.8 Hz), 3.05 (t, 2H,
J = 6.8 Hz). 13C DEPT-NMR (126 MHz, DMSO-d6): d 128.7, 128.0,
126.2, 122.9, 122.7, 110.7, 71.1, 34.8. ESI-HRMS: [M+H]+ m/z
[M+H]+ m/z 394.0725 (found), C16H14BrClNO4 requires 394.0623.
þ
4.2.14. 3-(3-Butynoxy)-4-chloro-7-nitro-isocoumarin (12)
The title compound was isolated as a yellow solid in 59% yield.
1H NMR (500 MHz, CDCl3) d 9.03 (d, 1H, J = 2.4 Hz), 8.54 (dd, 1H,
J = 2.4 Hz, J = 8.9 Hz), 7.86 (d, 1H, J = 8.9 Hz), 4.59 (t, 2H,
J = 6.8 Hz), 2.78 (dt, 2H, J = 2.7 Hz, J = 6.8 Hz), 2.08 (t, 1H,
þ
316.0674 (found), C17H15ClNO3 requires 316.0735.
J = 2.7 Hz). 13C DEPT-NMR (126 MHz, CDCl3):
d 129.8, 126.3,
123.7, 78.6, 71.1, 68.3, 19.7. ESI-HRMS: [M+H]+ m/z 294.0155
4.2.9. 7-Amino-3-(2-bromoethoxy)-4-chloro-isocoumarin (4c)
A mixture of 3c in THF/AcOH 2:1 and palladium on carbon was
stirred under an atmosphere of hydrogen until TLC revealed com-
plete disappearance of the starting material. The reaction mixture
was filtered, concentrated under reduced pressure and purified by
flash column chromatography (10–50% EtOAc in toluene), yielding
the title compound as a yellow powder in 56% yield. 1H NMR
þ
(found) C13H9ClNO5 requires 294.0164.
4.2.15. 3-(3-Butynoxy)-4-chloro-isocoumarin (13)
The title compound was isolated as an off-white solid in 42%
yield. 1H NMR (500 MHz, DMSO-d6) d 8.12 (dd, 1H, J = 0.9 Hz,
J = 7.9 Hz), 7.91 (ddd, 1H, J = 1,3 Hz, J = 7.3 Hz, J = 8.5 Hz), 7.74–
7.64 (m, 1H), 7.52 (ddd, 1H, J = 1.1 Hz, J = 7.4 Hz, J = 8.2 Hz), 4.43
(t, 2H, J = 6.4 Hz), 2.92 (t, 1H, J = 2.7 Hz), 2.70 (dt, 2H, J = 2.7 Hz,
J = 6.4 Hz). 13C DEPT-NMR (126 MHz, DMSO-d6): 135.8, 129.4,
126.4, 121.6, 80.1, 72.6, 68.0, 18.8. ESI-HRMS: [M+H]+ m/z
(500 MHz, DMSO-d6)
d 7.44 (d 1H, J = 8.6 Hz), 7.29 (d, 1H,
J = 2.4 Hz), 7.17 (dd, 1H, J = 2.5 Hz, J = 8.6 Hz), 4.56 (t, 2H,
J = 5.5 Hz), 3.80 (t, 2H, J = 5.5 Hz). 13C DEPT-NMR (126 MHz,
DMSO-d6): d 123.1, 122.7, 110.8, 70.3, 30.5. ESI-HRMS: [M+H]+
þ
þ
m/z 317.9637 (found), C11H10BrClNO3 requires 317.9527.
294.0155 (found) C13H9ClNO5 requires 294.0164.
4.2.16. 7-Amino-3-(3-butynoxy)-4-chloro-isocoumarin (14)
Powdered iron (9 equiv) was added to solution of compound 12
(130 mg) in acetic acid (2.5 mL) and stirred at 70 °C until TLC re-
vealed complete conversion of the starting material to a lower run-
ning spot (30 min). The reaction mixture was filtered over a layer of
sand, which was subsequently washed with methanol. The filtrate
was concentrated under reduced pressure and the residue was puri-
fied by flash column chromatography (0–50% EtOAc in toluene). The
title compound was isolated as a yellow solid (82 mg, 71% yield). 1H
NMR (500 MHz, CDCl3 + methanol-d6): d 7.54 (d, 1H, J = 8.6 Hz), 7.43
(d, 1H, J = 2.5 Hz), 7.10 (dd, 1H J = 2.5 Hz, J = 8.6 Hz), 4.39 (t, 2H,
J = 7.0 Hz), 2.69 (dt, 2H, J = 2.7 Hz, J = 6.9 Hz), 2.02 (t, 1H J = 2.7 Hz).
13C DEPT-NMR (126 MHz, CDCl3 + methanol-d6): d 124.3, 124.2,
4.2.10. 4-Chloro-3-methoxy-7-(4-pentynoylamino)-
isocoumarin (5)
The title compound was isolated as a fluffy, slightly yellow solid
in 35% yield after HPLC purification. 1H NMR NMR (500 MHz,
DMSO-d6) d 10.37 (s, 1H), 8.50 (d, 1H, J = 2.2 Hz), 7.99 (dd, 1H,
J = 2.3 Hz, J = 8.8 Hz), 7.64 (d, 1H, J = 8.7 Hz), 4.02 (s, 3H), 2.81 (t,
J = 2.6 Hz, 1H), 2.59–2.55 (m, 2H), 2.52–2.48 (m, 2H). 13C DEPT-
NMR (126 MHz, DMSO-d6): d 126.8, 122.2, 117.8, 83.2, 71.3, 57.0,
39.7, 39.5, 39.3, 34.9, 13.7.
4.2.11. 4-Chloro-7-(4-pentynoylamino)-3-(2-phenylethoxy)-
isocoumarin (6)
The title compound was isolated as a fluffy, slightly yellow solid
in 59% yield after HPLC purification. 1H NMR (500 MHz, DMSO-d6)
d 10.37 (s, 1H), 8.48 (d, 1H, J = 2.2 Hz), 7.99 (dd, 1H, J = 2.3 Hz,
J = 8.8 Hz), 7.63 (d, 1H, J = 8.7 Hz), 7.36–7.26 (m, 4H), 7.25–7.18
(m, 1H), 4.55 (t, 2H, J = 6.7 Hz), 3.07 (t, 2H, J = 6.7 Hz), 2.80 (t, 1H,
J = 2.6 Hz), 2.56 (d, 2H, J = 6.6 Hz), 2.53–2.46 (m, 2H). 13C DEPT-
NMR (126 MHz, DMSO-d6): d 128.7, 128.0, 126.8, 126.2, 122.4,
117.83 83.2, 71.3, 70.8, 34.9, 34.7, 13.7. ESI-HRMS: [M+H]+ m/z
113.0, 70.7, 69.0, 19.9. ESI-HRMS: [M+H]+ m/z 264.0409 (found)
þ
C
13H9ClNO5 requires 264.0427.
4.2.17. 7-Acetylamino-3-(3-butynoxy)-4-chloro-isocoumarin (15)
To a solution of compound 14 (10.1 mg) in THF (250 L) were
added acetic anhydride (9.6 L, 2.5 equiv), N-methylmorpholine
(4.3 L, 1.2 equiv) and a catalytic amount of dimethylamino–pyri-
dine. After TLC revealed complete disappearance of the starting
l
l
l
þ
396.0912 (found), C22H19ClNO4 requires 396.0997.

U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
639
material, the reaction mixture was diluted with acetonitrile/water/
4.3. Click chemistry
TFA 50:50:0.1 and purified by HPLC. Peak fractions were pooled
and lyophilized, yielding the title compound as a fluffy, slightly
yellow solid in 53%. 1H NMR (500 MHz, CDCl3 + methanol-d4): d
8.28 (d, 1H, J = 8.8 Hz), 8.09 (d, 1H, J = 1.9 Hz), 7.69 (d, 1H,
J = 8.8 Hz), 4.46 (t, 2H, J = 6.9 Hz), 2.77–2.71 (m, 2H), 2.19 (s, 3H),
2.07 (t, 1H, J = 2.6 Hz). 13C DEPT-NMR (126 MHz, CDCl3 + metha-
nol-d4): d 128.1, 123.6, 119.3, 79.0, 70.7, 68.5, 24.1, 19.8. ESI-
We used Cu(I)-catalyzed click chemistry to visualize the com-
plexes of IC ABPs bound to their target enzymes. After the labeling
reactions, the following reagents were added: 25 lM TAMRA-PEG-
N3, 50 lM tris(3-hydroxypropyltriazolyl-methyl)amine (THPTA) li-
gand39 (both fluorophore and ligand from 5 mM DMSO stocks),
1 mM CuSO4 (from a freshly prepared 50 mM stock in H2O) and
HRMS: [M+H]+ m/z 306.0509 (found) C15H13ClNO4 requires
500 l
M Na+ ascorbate (from a freshly prepared 25 mM stock in
þ
306.0528.
H2O). The reaction was allowed to proceed for 30 min at room tem-
perature (for pure proteases) or on ice (for lysates) and was
stopped by addition of 4ꢁ sample buffer and heating for 3 min at
95 °C. Protein samples were separated on 12% or 15% SDS poly-
acrylamide gels. Fluorescent bands were detected by scanning with
a Typhoon TRIO + fluorescent scanner (excitation at 532 nm, emis-
sion at 580 nm).
4.2.18. 7-Benzoylamino-3-(3-butynoxy)-4-chloro-isocoumarin
(16)
Benzoyl chloride (9
were added to a solution of compound 14 (10 mg, dried by coeva-
poration of water with dioxane) in THF (100 L). After 30 min, the
lL, 2 equiv) and pyridine (9.2 lL, 3 equiv)
l
reaction mixture was diluted with acetonitrile and water, and puri-
fied by HPLC. Peak fractions were pooled and lyophilized, yielding
the title as a fluffy, slightly yellow solid in 46%. 1H NMR (500 MHz,
DMSO-d6): d 10.63 (s, 1H), 8.69 (d, 1H, J = 2.2 Hz), 8.30 (dd, 1H,
J = 8.8 Hz, J = 2.3 Hz), 8.00 (dd, 2H, J = 5.3, J = 3.3 Hz), 7.72 (d, 1H,
J = 8.7 Hz), 7.65–7.53 (m, 3H), 4.43 (t, 2H, J = 6.4 Hz), 2.93 (t, 1H,
J = 2.6 Hz), 2.71 (dt, 2H, J = 6.4 Hz, J = 2.7 Hz). 13C DEPT-NMR
(126 MHz, DMSO-d6): d 132.6, 131.6, 129.0, 128.3, 128.2, 127.9,
127.4, 122.3, 119.2, 80.1, 72.6, 68.2, 18.8.
4.4. Labeling of purified proteases
Cathepsin G was diluted in HEPES buffer (50 mM pH 7.4 con-
taining 100 mM NaCl), all other proteases in phosphate buffer
(50 mM, pH 7.4), or EL4 cell lysate (1 mg/mL protein in 50 mM
phosphate buffer, pH 7.4). Lysates were prepared by incubating
EL4 mouse lymphoma cells in RIPA buffer on ice for 30 min and
separating insoluble cell debris by centrifugation). Where indi-
cated, proteases were pre-blocked with active site-directed inhib-
itors: 1 mM 3,4-dichloroisocoumarin for elastase and trypsin,
4.2.19. 3-(3-Butynoxy)-4-chloro-7-glycylamino-isocoumarin (17)
Boc-glycine (88 mg, dried by coevaporation of water with tolu-
100
for chymotrypsin and 1 mM PMSF for cathepsin G. Subsequently,
2 lM IC ABP was added and the mixtures were allowed to react
lM DFP for urokinase, 100 l
M of Ala-Ala-Phe-phosphonate40
ene) was activated at ꢀ20 °C with isobutyl chloroformate (72
lL,
1.1 equiv) and N-methylmorpholine (60 L, 1.2 equiv) in THF
l
(0.5 mL) and stirred for 30 min. To a solution of compound 14
(dried by coevaporation of water with toluene) and N-methylmor-
pholine (8.4 lL) in THF (0.1 mL) was added 0.3 mL of the activated
for 15 min at room temperature (for pure proteases) or on ice
(for lysates). Click chemistry was then performed for visualization
as described under 4.3. The amount of 100 ng of purified proteases
glycine mixture (2 equiv with respect to compound 14), and the fi-
nal solution was allowed to warm up to room temperature. After
1 h, the reaction was diluted with EtOAc, washed with 1 M KHSO4,
water, saturated NaHCO3 and brine. The organic layer was dried
(MgSO4), filtered and evaporated under reduced pressure. The
crude material was then treated with 25% TFA in DCM for 1 h
and subjected to HPLC purification. Peak fractions were pooled
and lyophilized, giving the title compound as a fluffy, slightly yel-
low solid in 39% yield over two steps. 1H NMR (500 MHz, DMSO-
d6): d 8.50 (d, 1H, J = 2.2 Hz), 8.24 (s, 1H), 7.99 (dd, 1H, J = 2.3 Hz,
J = 8.7 Hz), 7.73 (d, 1H, J = 8.7 Hz), 4.42 (t, 2H, J = 6.4 Hz), 3.84 (s,
2H), 2.92 (d, 1H, J = 2.2 Hz), 2.7 (dt, 2H, J = 2.7 Hz, J = 6.4 Hz). 13C
DEPT-NMR (126 MHz, DMSO-d6): d 126.8, 122.9, 118.1, 80.1,
or 15 lg of total protein (for proteases in a proteome background)
were loaded per gel lane.
4.5. Labeling of rat liver proteome
Part of a rat liver was passed through a 70
l
M cell strainer,
0.5% nonidet P-40 substitute in PBS was added (ꢂ2 mL/g of tissue)
and and cells were incubated on ice. After 1 h, cell debris was spun
down in a tabletop eppendorf centrifuge at 15,000 rpm for 10 min.
The supernatant was collected, snap-frozen in liquid nitrogen and
stored at ꢀ80 °C until usage.
Rat liver proteome (diluted to 1 mg/mL total protein with
50 mM phosphate buffer) was pretreated with TPCK (100
PMSF (1 mM) or DMSO for 30 min on ice. Then, proteomes were
treated with the IC ABP (1 M for 13 and 16; 0.5 M for 14) for
lM),
72.6, 68.3, 40.7, 18.8. ESI-HRMS: [M+H]+ m/z 321.0658 (found)
þ
C
15H14ClN2O4 requires 321.0637.
l
l
30 min on ice. Click chemistry with N-TAMRA-3-azido-propyl-
amine was performed as described in section 4.3.
4.2.20. 3-(3-Butynoxy)-4-chloro-7-[guanidinoacetyl]amino-
isocoumarin (18)
To a solution of compound 17 (7.95 mg) in DCM/DMF 1:1
Acknowledgments
(0.4 mL) were added N, N0-di-Boc-guanidine-N00-triflate (9.7 mg,
1 equiv) and N-methylmorpholine (2.7
l
L, 1 equiv). After 30 min,
We thank Dr. Oliver Frank for recording NMR spectra and Dr.
Victoria Albrow for discussions about the isocoumarin synthesis.
We acknowledge financial support from an Emmy Noether grant
of the Deutsche Forschungsgemeinschaft(DFG), the Center for Inte-
grated Protein Science Munich (CIPSM) and the Fonds der Chemis-
chen Industrie.
the reaction was diluted with EtOAc and subsequently washed
with 1 M KHSO4, water, dilute NaHCO3 and brine. The organic
phase was dried on MgSO4, filtered and concentrated under re-
duced pressure. The crude material was treated with 25% TFA in
DCM for 1 h and HPLC purified, giving the title compound as a fluf-
fy, slightly yellow solid in 29% yield. 1H NMR (500 MHz, methanol-
d4): d 8.48 (bs, 1H), 7.97 (d, 1H, J = 8.7 Hz), 7.71 (d, 1H, J = 8.7 Hz),
4.42 (t, 2H, J = 6.6 Hz), 4.11 (s, 2H), 2.68 (t, 2H, J = 6.6 Hz), 2.32 (bs,
1H). 13C DEPT-NMR (126 MHz, methanol-d4): d 126.8, 122.9, 118.1,
80.1, 72.6, 68.3, 40.7, 18.8. ESI-HRMS: [M+H]+ m/z 363.0684
Supplementary data
Supplementary data (two extra figures and copies of all 1H NMR
spectra) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2011.03.014.
þ
(found) C16H16ClN4O4 requires 363.0855.

640
U. Haedke et al. / Bioorg. Med. Chem. 20 (2012) 633–640
24. Vinothkumar, K. R.; Strisovsky, K.; Andreeva, A.; Christova, Y.; Verhelst, S.;
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7.58 (t, 1H, J = 7.7 Hz), 7.43, (t, 1H, J = 7.6 Hz), 6.07 (s, 1H), 4.42 (t, 2H,
J = 6.3 Hz), 3.77 (t, 2H, J = 4.42 Hz), 3.45 (t, 2H, J = 6.2 Hz), 3.21 (dt, 2H,
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