A BODIPY-Tagged Phosphono Peptide as Activity-Based Probe for Human Leukocyte Elastase

Article abstract of DOI:10.1021/acsmedchemlett.7b00533

Human leukocyte elastase plays a crucial role in a variety of inflammatory disorders and represents an important subject of biomedical studies. The chemotype of peptidic phosphonates was employed for the design of a new activity-based probe for human leukocyte elastase. Its structure combines the phosphonate warhead with an adequate peptide portion and a BODIPY fluorophore with a clickable ethinylphenyl moiety at meso position. The probe 6 was assembled by copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition. It was characterized as an active site-directed elastase inhibitor exhibiting a second-order rate constant of inactivation of 88400 M^-1s^-1. The suitability of 6 as a fluorescent probe for human leukocyte elastase was demonstrated by in-gel fluorescence analysis. Labeling experiments and inhibition data toward a panel of related proteases underlined the selectivity of the probe for the targeted leukocyte elastase.

Full text of DOI:10.1021/acsmedchemlett.7b00533

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
A BODIPY-Tagged Phosphono Peptide as Activity-Based Probe for
Human Leukocyte Elastase
Anna-Christina Schulz-Fincke,† Michael Blaut,‡ Annett Braune,*,‡ and Michael Gutschow*,†
̈
†Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
‡Department of Gastrointestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee
114-116, 14558 Nuthetal, Germany
S
* Supporting Information
ABSTRACT: Human leukocyte elastase plays a crucial role in a variety of
inflammatory disorders and represents an important subject of biomedical
studies. The chemotype of peptidic phosphonates was employed for the
design of a new activity-based probe for human leukocyte elastase. Its
structure combines the phosphonate warhead with an adequate peptide
portion and a BODIPY fluorophore with a clickable ethinylphenyl moiety at
meso position. The probe 6 was assembled by copper-catalyzed alkyne−azide
1,3-dipolar cycloaddition. It was characterized as an active site-directed
elastase inhibitor exhibiting a second-order rate constant of inactivation of
88400 M−1s−1. The suitability of 6 as a fluorescent probe for human
leukocyte elastase was demonstrated by in-gel fluorescence analysis. Labeling
experiments and inhibition data toward a panel of related proteases
underlined the selectivity of the probe for the targeted leukocyte elastase.
KEYWORDS: BOPIPY derivatives, copper-catalyzed azide−alkyne cycloaddition, human leukocyte elastase, phosphonates,
serine proteases
uman leukocyte elastase (HLE) is a serine protease
of the active-site serine at the carbonyl carbon of the scissile
Hstored as an active enzyme within the azurophilic bond and the subsequent hydrolysis of the resulting acyl
enzyme.1,2,6 The only synthetic HLE inhibitor that has reached
granules of polymorphonuclear neutrophils. After the fusion
the market, sivelestat, interacts similarly in the course of an acyl
transfer. Its ester bond is enzymatically cleaved by HLE, and a
pivaloylated enzyme is generated.9
of azurophilic granules with vacuoles carrying phagocytosed
bacteria, HLE functions in intracellular host defense by
degrading bacterial membrane proteins. Beside this primary
role, HLE also acts extracellularly. Upon neutrophil activation
by chemokines, chemoattractants, or bacterial lipopoly-
saccharides, HLE is released extracellularly. As a membrane
bound or free protease, it can then break down matrix proteins,
such as elastin, fibronectin, laminin, and collagen. Moreover,
HLE activates other proteases or deactivates their endogenous
inhibitors, releases cytokines from their precursors, or liberates
growth factors.1−5
The catalytic activity of HLE is controlled by endogenous
serine protease inhibitors, such as α1-proteinase inhibitor, α2-
macroglobulin, or secretory leukocyte peptidase inhibitor.
However, an out-of-balance activity might produce deleterious
effects and contributes to the onset and progression of
inflammatory lung diseases. Thus, the upregulated HLE activity
is involved in the pathophysiology of chronic obstructive
pulmonary disease, acute lung injury, respiratory distress
syndrome, and inflammatory bowel disease.1,2,6 HLE represents
an enzyme of diagnostic potential,7 e.g., as a fecal marker of
intestinal inflammation.8
The crucial role of HLE in multiple forms of inflammatory
disorders has provided the impetus for the development of
fluorescent reporters,10,11 as well as synthetic inhibitors. The
different chemotypes of HLE inhibitors include peptidic
chloromethyl and trifluoromethyl ketones,12 4-oxo-ß-lactams,13
kojic acid derivatives,14 saccharines,15 and isothiazolones.16
The α-aminoalkylphosphonates represent phosphonic ana-
logues of naturally occurring amino acids. Combined with a
tailored peptide portion, sufficiently stable and potentially
selective serine protease inhibitors can be achieved.17 Due to
the tetrahedral configuration of the phosphorus atom, α-
aminophosphonates mimic the transition state of the enzymatic
reaction. Two aryloxy groups on the phosphorus atom were
introduced to maintain reactivity toward the active-site serine.
The nucleophilic attack of the catalytic serine residue and the
release of a phenoxy group forms a tetravalent derivative. This
serine phosphono diester undergoes a slow “aging” process
upon the loss of the second phenoxy group to form a stable
HLE has a primary substrate specificity to cleave peptides
with small aliphatic residues such as alanine or valine in the P1
position.1,2 The cleavage is initiated by the nucleophilic attack
Received: December 22, 2017
Accepted: March 4, 2018
Published: March 4, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.7b00533
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of the Activity-Based Probe 6
a
Reagents and conditions: (a) 50% TFA/CH2Cl2 (v/v), rt; (b) HBTU, DIPEA, MeCN, rt; (c) CuSO4·5 H2O, sodium ascorbate, H2O, DMSO, rt.
Table 1. Inhibition of Proteases by Phosphono Peptides 1A, 1B, and 6
b
kinac/Ki (M−1s−1
)
bovine
chymotrypsin
bovine factor
human
thrombin
bovine
trypsin
human
cathepsin B
human
cathepsin L
a
compd
HLE
PPE
Xa
c
1A (S,S,R) 399000 55000
7990 1180
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
1B (S,S,S)
6 (S,S,R)
1000 150
NI
88400 10400
1440 70
565 52
a
The stereochemistry is given in parentheses. The first entry refers to the configuration at Val, the second at Pro, and the third at the
b
aminophosphonate. Progress curves were analyzed using the equation [P] = vi (1 − exp(−kobst))/kobs + d, where [P] is the product concentration,
vi the initial rate, kobs the observed first-order rate constant, and d the offset. The kobs values (means from duplicate measurements) were plotted
against the inhibitor concentrations, [I]. Second-order rate constants of inactivation, kinac/Ki, were determined according to kinac/Ki = kobs/[I](1 +
[S]/Km) + d, where [S] is the substrate concentration, Km the Michaelis−Menten constant, and d the offset. Standard errors ( SEM) refer to this
c
linear regression. NI: No inhibition relates to more than 90% product formation after 60 min at an inhibitor concentration of 1 μM in duplicate
measurements.
serine phosphono monoester, leading to irreversible inhi-
bition.18
addition (click reaction). Click chemistry has already been
successfully utilized for the development of activity-based
probes for serine proteases.13,22 It was intended to equip our
probe with a boron-dipyrromethene (BODIPY) label, which
should be introduced through the aforementioned click
reaction. BODIPYs have excellent features for biological
applications, such as chemical robustness, photochemical
stability, high quantum yields, and appropriate excitation and
emission properties.31,32 The linear synthesis of our activity-
based probe started with an Oleksyszyn reaction employing the
three components tris(4-(methylthio)phenyl)phosphite, avail-
able from phosphorus trichloride and 4-(methylthio)phenol,
isobutyraldehyde, and benzyl carbamate. The resulting rac
aminoalkylphosphonate was deprotected with HBr in acetic
acid and coupled in turn with Boc-D-Pro-OH and, after
deprotection, with Boc-D-Val-OH. This led to a diastereomeric
mixture, which was separated by column chromatography to
obtain the epimers 1A (Scheme 1) and 1B, respectively.30 The
code A or B refers to the differences in 31P NMR spectra with A
exhibiting the stronger upfield shifted signal (1A, 17.87 ppm;
1B, 18.25 ppm).
Because of their irreversible mode of action, peptidic
phosphonates provide an excellent structural basis for the
design of activity-based probes for different serine pro-
teases.19−25 Such probes contain a phosphonic analogue of an
amino acid (AAP) in P1 position. Phosphonate-based probes
for elastase and the related neutrophil enzyme proteinase-3
have been reported with rac-ValP carrying either biotin or
cyanine 5 as label.26,27 An extended P3−P1 portion, Val−Pro−
rac-LeuP, was employed for a biotinylated probe targeting the
neutrophil serine proteases cathepsin G and elastase.28 Another
biotin-labeled probe for elastase contains a tetrapeptide unit
composed of unnatural amino acids with the phosphonate
analogue of 4-aminobutyric acid in P1 position.29
In this study, we have designed a new activity-based probe
for neutrophil elastase with a defined configuration at the α-
carbon of the aminophosphonate substructure. The tripeptidic
P3−P1 sequence, Val−Pro−ValP, was chosen on the basis of
highly active elastase inactivators bearing two 4-methylthio-
phenoxy groups at the phosphonate warhead.30 The structure
of our probe was further assembled to contain a P4 glycine
moiety whose nitrogen is incorporated into a triazole ring
accessible via copper-catalyzed alkyne−azide 1,3-dipolar cyclo-
In order to elucidate the effect of the configuration on the
biological activity, the tripeptidic phosphonates 1A and 1B
were evaluated for their inhibitory potency against HLE and for
B
DOI: 10.1021/acsmedchemlett.7b00533
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 1. Detection of HLE after labeling with the fluorescent probe 6 and SDS-PAGE. (A, C, E) In-gel fluorescence detection. (B, D, F) Coomassie
staining. (A, B) Increasing amounts of HLE were incubated with 2.5 μM 6. (C, D) HEK cell lysate (9 μg, first lane) or HLE (600 ng, second lane)
was incubated with 2.5 μM 6. HEK cell lysate (9 μg) was added after (third lane) or before (fourth lane) incubation of HLE (600 ng) with 2.5 μM 6.
The horizontal line indicates the incubation period; the HEK cell lysate was added either before or after the incubation time. (E, F) HLE (600 ng)
was incubated with or without 5 μM sivelestat (Siv) followed by incubation with 2.5 μM 6. M, molecular mass marker.
their selectivity versus five serine proteases, i.e., porcine
pancreatic elastase (PPE), chymotrypsin, factor Xa, thrombin,
and trypsin, as well as two cysteine proteases, i.e., cathepsin B
and L (Table 1). The protease activities were assayed
photometrically or fluorometrically and the reactions were
followed over 60 min.16,23,33
Epimer 1A showed a strong inhibition of HLE with a second-
order rate constant of 399000 M−1s−1, a moderate effect against
PPE, and inactivity against the other proteases (Table 1).
Compound 1B exhibited no inhibitory activity against the
investigated proteases except HLE, which was inhibited with a
weak second-order rate constant of 1000 M−1s−1 (Table 1).
The obtained progress curves with HLE showed time-
dependent inhibition (Figures S1 and S2, Supporting
Information), which is in accordance with the expected
irreversible mode of action. Analysis of the progress curves
gave the aforementioned second-order rate constants, kinac/Ki,
as the main characteristic of irreversible inhibitors.
achieved by molecular docking,23 (iii) literature data on
aminophosphonates as serine protease inhibitors in which, in
four of the five cases, the active, (R)-configured epimer had 31P
NMR resonances at higher field than the (S)-epimer,30 (iv) an
asymmetric synthesis of aminophosphonates with the (R)-
enantiomers as superior inactivators of human cathepsin G,
chymotrypsin, and HLE.34 The assignment of the respective
configuration at the CHPO chiral center for 1A and 1B can
moreover be expected because the (R)-configured amino-
phosphonate substructures have the same molecular geometry
as natural (S)-configured amino acids.
The efficient inactivation of HLE by the phosphono peptide
1A provided the impetus for the design and synthesis of an
activity-based probe of this chemotype. For this purpose, an N-
terminal elongation of valine and the incorporation of a
fluorescence tag through click chemistry was performed. The
1,2,3-triazole-containing linker structure may participate
actively in hydrogen bond formation and dipole−dipole
interactions within the active site cleft.13,35 The synthesis of
the probe is outlined in Scheme 1 and started with the
deprotection of compound 1A. The azido component 3 was
prepared from bromoacetic acid, and a HBTU-mediated
coupling reaction with the ammonium salt 2 yielded compound
4. For the synthesis of ethynyl-BODIPY 5, 4-ethynylbenzalde-
hyde was reacted with 4-dimethylpyrrole, and the resulting
The (S,S,R)- and (S,S,S)-configuration was assigned to 1A
and 1B, respectively, for the following reasons: (i) the full
agreement of our data (Table 1) with those of a previous report
by Winiarski et al.30 with respect to the much stronger anti-
elastase activity of the (S,S,R)-configured 1A compared to the
(S,S,S)-configured epimer 1B, (ii) the assignment of the active
phosphonate epimer as matriptase-2 and trypsin inhibitors
C
DOI: 10.1021/acsmedchemlett.7b00533
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
intermediate was further treated with boron trifluoride diethyl
etherate in the presence of p-chloranil in a base-catalyzed
complexation.36 Compound 5 has been investigated in several
physicochemical and synthetic studies and utilized for the
generation of one DNA probe.37,38 To combine the fluorescent
tag with the inhibitor substructure, a click-chemistry approach
was performed through the reaction of the azido peptide 4 and
ethynyl-BODIPY 5, using a CuII salt and sodium ascorbate as
reducing agent.39 This yielded the desired activity-based probe
6 with a phosphonate warhead for an irreversible modification
of HLE, a peptidic linker structure capable of interacting with
the enzyme’s binding pockets, a BODIPY as fluorescence
reporter group, and the preferred (R)-configuration at the
crucial amino phosphonate moiety. The NMR data of 6 (e.g.,
31P NMR, δ = 17.85 ppm) resembled that of the
aforementioned precursor 1A.
The results of the kinetic evaluation of probe 6 are given in
Table 1. Again, no inhibition was observed for factor Xa,
thrombin, trypsin, and the two cathepsins. Chymotrypsin was
inhibited to some extent. The inactivation of HLE and PPE
confirmed the desired preference of the probe for elastases.
The progress curves of the substrate consumption of both
elastase enzymes in the presence of different concentrations of
6 revealed that 6 maintained the irreversible mode of action, a
prerequisite for an activity-based probe (see Figure S3 for
HLE). Probe 6 was approximately 5-fold less potent than its
precursor 1A (Table 1). However, with the kinac/Ki value of
88400 M−1s−1, our probe showed a still extraordinary potency
against the target HLE. The strong selectivity of 1A for HLE
versus PPE was retained in the case of 6 (88400 M−1s−1 versus
1440 M−1s−1).
bands have been demonstrated to represent distinct, catalyti-
cally active HLE isoforms, which differ in their carbohydrate
content and have been resolved by SDS-PAGE.41−43 Moreover,
the self-cleavage of elastase from murine and human
neutrophils generates variants whose catalytic activity was
shown by means of a biotinylated probe.44 The occurrence of
the pattern of three elastase bands (Figure 1) has been
observed in several previous studies.27−29,41−44 Thus, it can be
attributed to either differences in the carbohydrate contents or
to an autoprocessing of elastase. Compared to the Coomassie
Brilliant Blue staining, probe 6 produced a much stronger
labeling of HLE (Figure 1A versus B).
The selectivity of activity-based probe 6 was investigated by
adding HEK cell lysate after or prior to the incubation time of
20 min to two mixtures containing HLE and 6. SDS-PAGE and
subsequent fluorescence analysis revealed a selective elastase
labeling as only HLE was imaged (Figure 1C). The following
Coomassie staining (Figure 1D) indicated that the ratio
between HLE and the HEK lysate proteins was appropriate
to demonstrate the selective labeling by 6. In addition, the
binding mode of 6 was explored in a competition experiment
(Figure 1E,F). HLE was preincubated with the active site-
directed, covalent inhibitor sivelestat in order to protect the
enzyme from a reaction with 6. As a control, 6 was incubated
with HLE in the absence of sivelestat. A clearly reduced
fluorescence signal was observed in the presence of sivelestat
due to the competition of our probe with sivelestat for the
active site of the target HLE.
As a conclusion, the activity-based probe 6 was designed on
the basis of a highly potent elastase inhibitor. The BODIPY
fluorophore was introduced through click chemistry yielding a
compound with a second-order rate constant of 88400 M−1s−1,
making it one of the most potent fluorescently labeled
inactivators of human leukocyte elastase. We have demon-
strated that the phosphono peptide 6 reacts with the active site
of HLE and allows for a selective in-gel fluorescence labeling of
low amounts, down to 160 ng, of this protease. The new probe
is expected to serve as a valuable tool to further investigate the
pathophysiological role of leukocyte elastase in several
inflammatory disorders.
In contrast to compounds 1A, 1B, and 6, sivelestat showed a
kinetic “slow binding” behavior, with concentration-dependent
steady-state rates for the HLE-catalyzed reaction (Figure S4).
This is in agreement with the covalent-reversible mode of
action of sivelestat.9 We obtained a Ki value of 37 nM (kon
=
30300 3600 M−1s−1, koff = 0.00113 0.00021 s−1). Next, we
investigated the reactivation of HLE inhibited by sivelestat, 1A,
and 6, respectively. Unlike in the case of sivelestat, the
enzymatic activity was not regained when a large excess of
substrate was added after incubation of HLE with the
phosphonates 1A and 6 (Figure S5). This finding confirmed
the irreversible character of the interaction of probe 6 with
HLE.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00533.
The spectroscopic properties of 6 were determined in
methanol, dichloromethane, and water as solvents (Figure S6).
The fluorescence spectra showed excitation maxima at 495−
509 nm and emission maxima at 503−513 nm, typical for
BODIPY derivatives. Structure 6 features four methyl groups at
pyrrolic positions and a biaryl moiety as meso substituent in an
expected orthogonal configuration relative to the BODIPY
core, giving rise to a bathochromic shift and a more efficient
fluorescence.31,40
The characteristics of our compound 6 encouraged us to
evaluate its suitability as an activity-based probe for HLE. To
assess the direct in-gel fluorescence visualization, varying
amounts of HLE were incubated with 2.5 μM probe 6 and
subjected to SDS-PAGE. Fluorescent bands at approximately
29 kDa were detected, and their intensities correlated with the
amount of HLE (Figure 1A). Even the lowest protease amount
employed in this experiment, i.e., 160 ng, could be effectively
visualized. Moreover, in each lane, three fluorescent bands,
representing three labeled forms of HLE, were observed. Such
General methods and materials. Synthetic procedures
and characterization of all compounds. General enzy-
matic methods. Enzyme inhibition assays. Inhibition of
HLE with 1A, 1B, probe 6, and sivelestat. Reactivation
experiments. Normalized absorption and emission
spectra of probe 6. Detection of HLE with probe 6.
NMR spectra and LC/MS data of synthesized com-
pounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: guetschow@uni-bonn.de. Phone: +49 228 732317
(M.G.).
■
*E-mail: braune@dife.de. Phone: +49 33200 882402 (A.B.).
ORCID
Michael Gutschow: 0000-0002-9376-7897
̈
D
DOI: 10.1021/acsmedchemlett.7b00533
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Optimization of O3-acyl kojic acid derivatives as potent and selective
human neutrophil elastase inhibitors. J. Med. Chem. 2013, 56, 9802−
9806.
Author Contributions
The manuscript was written through contributions of all
authors, who have given approval to the final version of the
manuscript.
(15) Martyn, D. C.; Moore, M. J.; Abell, A. D. Succinimide and
saccharin-based enzyme-activated inhibitors of serine proteases. Curr.
Pharm. Des. 1999, 5, 405−415.
Notes
The authors declare no competing financial interest.
(16) Gutschow, M.; Pietsch, M.; Themann, A.; Fahrig, J.; Schulze, B.
̈
2,4,5-Triphenylisothiazol-3(2H)-one 1,1-dioxides as inhibitors of
human leukocyte elastase. J. Enzyme Inhib. Med. Chem. 2005, 20,
341−347.
ACKNOWLEDGMENTS
The authors thank Martin Mangold for providing cell lysates
■
and Anke Guhler for technical assistance.
́
(17) Sienczyk, M.; Oleksyszyn, J. Irreversible inhibition of serine
̈
proteases - design and in vivo activity of diaryl alpha-amino-
phosphonate derivatives. Curr. Med. Chem. 2009, 16, 1673−1687.
(18) Oleksyszyn, J.; Boduszek, B.; Kam, C. M.; Powers, J. C. Novel
amidine-containing peptidyl phosphonates as irreversible inhibitors for
blood coagulation and related serine proteases. J. Med. Chem. 1994, 37,
226−231.
ABBREVIATIONS
■
AA, amino acid; BODIPY, boron-dipyrromethene; DIPEA,
N,N-diisopropylethylamine; HBTU, 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; HEK, human
embryonic kidney; HLE, human leukocyte elastase; PPE,
porcine pancreatic elastase
́ ́
(19) Sabido, E.; Tarrago, T.; Giralt, E. Towards the identification of
unknown neuropeptide precursor-processing enzymes: Design and
synthesis of a new family of dipeptidyl phosphonate activity probes for
substrate-based protease identification. Bioorg. Med. Chem. 2010, 18,
8350−8355.
(20) Zou, F.; Schmon, M.; Sienczyk, M.; Grzywa, R.; Palesch, D.;
Boehm, B. O.; Sun, Z. L.; Watts, C.; Schirmbeck, R.; Burster, T.
Application of a novel highly sensitive activity-based probe for
detection of cathepsin G. Anal. Biochem. 2012, 421, 667−672.
(21) Guarino, C.; Legowska, M.; Epinette, C.; Kellenberger, C.;
REFERENCES
■
(1) Korkmaz, B.; Moreau, T.; Gauthier, F. Neutrophil elastase,
proteinase 3 and cathepsin G: Physicochemical properties, activity and
physiopathological functions. Biochimie 2008, 90, 227−242.
(2) Korkmaz, B.; Horwitz, M. S.; Jenne, D. E.; Gauthier, F.
Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic
targets in human diseases. Pharmacol. Rev. 2010, 62, 726−759.
(3) Pipoly, D. J.; Crouch, E. C. Degradation of native type IV
procollagen by human neutrophil elastase. Implications for leukocyte-
mediated degradation of basement membranes. Biochemistry 1987, 26,
5748−5754.
(4) Owen, C. A.; Campbell, E. J. Neutrophil proteinases and matrix
degradation. The cell biology of pericellular proteolysis. Semin. Cell
Biol. 1995, 6, 367−376.
(5) Ungurs, M. J.; Sinden, N. J.; Stockley, R. A. Progranulin is a
substrate for neutrophil-elastase and proteinase-3 in the airway and its
concentration correlates with mediators of airway inflammation in
COPD. Am. J. Physiol. Lung Cell. Mol. Physiol. 2014, 306, L80−L87.
(6) Lucas, S. D.; Costa, E.; Guedes, R. C.; Moreira, R. Targeting
COPD: Advances on low-molecular-weight inhibitors of human
neutrophil elastase. Med. Res. Rev. 2013, 33, E73−101.
́
Dallet-Choisy, S.; Sienczyk, M.; Gabant, G.; Cadene, M.; Zoidakis, J.;
Vlahou, A.; Wysocka, M.; Marchand-Adam, S.; Jenne, D. E.; Lesner,
A.; Gauthier, F.; Korkmaz, B. New selective peptidyl di(chlorophenyl)
phosphonate esters for visualizing and blocking neutrophil proteinase
3 in human diseases. J. Biol. Chem. 2014, 289, 31777−31791.
(22) Serim, S.; Baer, P.; Verhelst, S. H. Mixed alkyl aryl phosphonate
esters as quenched fluorescent activity-based probes for serine
proteases. Org. Biomol. Chem. 2015, 13, 2293−2299.
(23) Haußler, D.; Mangold, M.; Furtmann, N.; Braune, A.; Blaut, M.;
̈
Bajorath, J.; Stirnberg, M.; Gutschow, M. Phosphono bisbenzguani-
̈
dines as irreversible dipeptidomimetic inhibitors and activity-based
probes of matriptase-2. Chem. - Eur. J. 2016, 22, 8525−8535.
(24) Penczek, A.; Sienczyk, M.; Wirtz, C. R.; Burster, T. Cell surface
cathepsin G activity differs between human natural killer cell subsets.
Immunol. Lett. 2016, 179, 80−84.
(7) Henriksen, P. A. The potential of neutrophil elastase inhibitors as
anti-inflammatory therapies. Curr. Opin. Hematol. 2014, 21, 23−28.
(8) Langhorst, J.; Elsenbruch, S.; Koelzer, J.; Rueffer, A.; Michalsen,
A.; Dobos, G. J. Noninvasive markers in the assessment of intestinal
inflammation in inflammatory bowel diseases: performance of fecal
lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices.
Am. J. Gastroenterol. 2008, 103, 162−169.
(25) Haußler, D.; Schulz-Fincke, A. C.; Beckmann, A. M.; Keils, A.;
̈
Gilberg, E.; Mangold, M.; Bajorath, J.; Stirnberg, M.; Steinmetzer, T.;
Gutschow, M. A fluorescent-labeled phosphono bisbenzguanidine as
̈
an activity-based probe for matriptase. Chem. - Eur. J. 2017, 23, 5205−
5209.
(9) Nakayama, Y.; Odagaki, Y.; Fujita, S.; Matsuoka, S.; Hamanaka,
N.; Nakai, H.; Toda, M. Clarification of mechanism of human sputum
elastase inhibition by a new inhibitor, ONO-5046, using electrospray
ionization mass spectrometry. Bioorg. Med. Chem. Lett. 2002, 12,
2349−2353.
(10) Gehrig, S.; Mall, M. A.; Schultz, C. Spatially resolved monitoring
of neutrophil elastase activity with ratiometric fluorescent reporters.
Angew. Chem., Int. Ed. 2012, 51, 6258−6261.
(11) Avlonitis, N.; Debunne, M.; Aslam, T.; McDonald, N.; Haslett,
C.; Dhaliwal, K.; Bradley, M. Highly specific, multi-branched
fluorescent reporters for analysis of human neutrophil elastase. Org.
Biomol. Chem. 2013, 11, 4414−4418.
(26) Gilmore, B. F.; Quinn, D. J.; Duff, T.; Cathcart, G. R.; Scott, C.
J.; Walker, B. Expedited solid-phase synthesis of fluorescently labeled
and biotinylated aminoalkane diphenyl phosphonate affinity probes for
chymotrypsin- and elastase-like serine proteases. Bioconjugate Chem.
2009, 20, 2098−2105.
(27) Edgington-Mitchell, L. E.; Barlow, N.; Aurelio, L.; Samha, A.;
Szabo, M.; Graham, B.; Bunnett, N. Fluorescent diphenylphospho-
nate-based probes for detection of serine protease activity during
inflammation. Bioorg. Med. Chem. Lett. 2017, 27, 254−260.
(28) Grzywa, R.; Burchacka, E.; Łęcka, M.; Winiarski, Ł.; Walczak,
M.; Łupicka-Słowik, A.; Wysocka, M.; Burster, T.; Bobrek, K.;
́
Csencsits-Smith, K.; Lesner, A.; Sienczyk, M. Synthesis of novel
(12) Wei, A. Z.; Mayr, I.; Bode, W. The refined 2.3 Å crystal structure
of human leukocyte elastase in a complex with a valine chloromethyl
ketone inhibitor. FEBS Lett. 1988, 234, 367−373.
phosphonic-type activity-based probes for neutrophil serine proteases
and their application in spleen lysates of different organisms.
ChemBioChem 2014, 15, 2605−2612.
(13) Ruivo, E. F.; Gonca̧ lves, L. M.; Carvalho, L. A.; Guedes, R. C.;
(29) Kasperkiewicz, P.; Poreba, M.; Snipas, S. J.; Parker, H.;
Winterbourn, C. C.; Salvesen, G. S.; Drag, M. Design of ultrasensitive
probes for human neutrophil elastase through hybrid combinatorial
substrate library profiling. Proc. Natl. Acad. Sci. U. S. A. 2014, 111,
2518−2523.
Hofbauer, S.; Brito, J. A.; Archer, M.; Moreira, R.; Lucas, S. D.
Clickable 4-oxo-β-lactam-based selective probing for human neutro-
phil elastase related proteomes. ChemMedChem 2016, 11, 2037−2042.
(14) Lucas, S. D.; Gonca̧ lves, L. M.; Carvalho, L. A.; Correia, H. F.;
Da Costa, E. M.; Guedes, R. A.; Moreira, R.; Guedes, R. C.
E
DOI: 10.1021/acsmedchemlett.7b00533
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
́
(30) Winiarski, Ł.; Oleksyszyn, J.; Sienczyk, M. Human neutrophil
elastase phosphonic inhibitors with improved potency of action. J.
Med. Chem. 2012, 55, 6541−6553.
(31) Ulrich, G.; Ziessel, R.; Harriman, A. The chemistry of
fluorescent bodipy dyes: Versatility unsurpassed. Angew. Chem., Int.
Ed. 2008, 47, 1184−1201.
(32) de Bruin, G.; Xin, B. T.; Kraus, M.; van der Stelt, M.; van der
Marel, G. A.; Kisselev, A. F.; Driessen, C.; Florea, B. I.; Overkleeft, H.
S. A set of activity-based probes to visualize human (immuno)-
proteasome activities. Angew. Chem., Int. Ed. 2016, 55, 4199−4203.
(33) Frizler, M.; Lohr, F.; Lulsdorff, M.; Gutschow, M. Facing the
̈
̈
gem-dialkyl effect in enzyme inhibitor design: preparation of
homocycloleucine-based azadipeptide nitriles. Chem. - Eur. J. 2011,
17, 11419−11423.
(34) Walker, B.; Wharry, S.; Hamilton, R. J.; Martin, S. L.; Healy, A.;
Walker, B. J. Asymmetric preference of serine proteases toward
phosphonate and phosphinate esters. Biochem. Biophys. Res. Commun.
2000, 276, 1235−1339.
(35) Horne, W. S.; Yadav, M. K.; Stout, C. D.; Ghadiri, M. R.
Heterocyclic peptide backbone modifications in an alpha-helical coiled
coil. J. Am. Chem. Soc. 2004, 126, 15366−15367.
(36) Li, Z.; Bittman, R. Synthesis and spectral properties of
cholesterol- and FTY720-containing boron dipyrromethene dyes. J.
Org. Chem. 2007, 72, 8376−8382.
(37) Kim, K. T.; Kim, B. H. A fluorescent probe for the 3′-overhang
of telomeric DNA based on competition between two interstrand G-
quadruplexes. Chem. Commun. 2013, 49, 1717−1719.
(38) Ast, S.; Fischer, T.; Muller, H.; Mickler, W.; Schwichtenberg,
̈
M.; Rurack, K.; Holdt, H. J. Integration of the 1,2,3-triazole ″click″
motif as a potent signalling element in metal ion responsive fluorescent
probes. Chem. - Eur. J. 2013, 19, 2990−3005.
(39) Pedersen, D. S.; Abell, A. 1,2,3-Triazoles in peptidomimetic
chemistry. Eur. J. Org. Chem. 2011, 2011, 2399−2411.
(40) Chen, Y.; Wang, H.; Wan, L.; Bian, Y.; Jiang, J. 8-
Hydroxyquinoline-substituted boron-dipyrromethene compounds:
synthesis, structure, and OFF-ON-OFF type of pH-sensing properties.
J. Org. Chem. 2011, 76, 3774−3781.
(41) Twumasi, D. Y.; Liener, I. E. Proteases from purulent sputum.
Purification and properties of the elastase and chymotrypsin-like
enzymes. J. Biol. Chem. 1977, 252, 1917−1926.
(42) Green, B. G.; Weston, H.; Ashe, B. M.; Doherty, J.; Finke, P.;
Hagmann, W.; Lark, M.; Mao, J.; Maycock, A.; Moore, V.; Mumford,
R.; Shah, S.; Knight, W. N. PMN elastases: A comparison of the
specificity of human isozymes and the enzyme from other species
toward substrates and inhibitors. Arch. Biochem. Biophys. 1991, 286,
284−292.
(43) Watorek, W.; van Halbeek, H.; Travis, J. The isoforms of human
neutrophil elastase and cathepsin G differ in their carbohydrate side
chain structures. Biol. Chem. Hoppe-Seyler 1993, 374, 385−393.
(44) Dau, T.; Sarker, R. S.; Yildirim, A. O.; Eickelberg, O.; Jenne, D.
E. Autoprocessing of neutrophil elastase near its active site reduces the
efficiency of natural and synthetic elastase inhibitors. Nat. Commun.
2015, 10, 6722.
F
DOI: 10.1021/acsmedchemlett.7b00533
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX