Facile Synthesis of Aminomethyl Phosphinate Esters as Serine Protease Inhibitors with Primed Site Interaction

Article abstract of DOI:10.1021/acsmedchemlett.0c00284

Serine proteases comprise about one-third of all proteases, and defective regulation of serine proteases is involved in numerous diseases. Therefore, serine protease inhibitors are promising drug candidates. Aminomethyl diphenyl phosphonates have been regularly used as scaffolds for covalent serine protease inhibition and the design of activity-based probes. However, they cannot make use of a protease's primed site. Therefore, we developed a facile two-step synthesis toward a set of phenyl phosphinates, which is a related scaffold but can interact with the primed site. We tested their inhibitory activity on five different serine proteases and found that a phenyl group directly attached to the phosphorus atom leads to superior activity compared with phosphonates.

Full text of DOI:10.1021/acsmedchemlett.0c00284

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Letter
Facile Synthesis of Aminomethyl Phosphinate Esters as Serine
Protease Inhibitors with Primed Site Interaction
Jan Pascal Kahler, Stijn Lenders, Merel A. T. van de Plassche, and Steven H. L. Verhelst*
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ABSTRACT: Serine proteases comprise about one-third of all
proteases, and defective regulation of serine proteases is involved in
numerous diseases. Therefore, serine protease inhibitors are
promising drug candidates. Aminomethyl diphenyl phosphonates
have been regularly used as scaffolds for covalent serine protease
inhibition and the design of activity-based probes. However, they
cannot make use of a protease’s primed site. Therefore, we
developed a facile two-step synthesis toward a set of phenyl
phosphinates, which is a related scaffold but can interact with the
primed site. We tested their inhibitory activity on five different
serine proteases and found that a phenyl group directly attached to
the phosphorus atom leads to superior activity compared with
phosphonates.
KEYWORDS: Covalent inhibitors, phosphinates, protease inhibitor, serine proteases
erine proteases comprise about one-third of all proteases.
A multitude of them are implicated in human diseases.
active species.18,19 Because of their close resemblance to amino
acid residues, peptidyl diphenyl phosphonates allow for a
rational inhibitor design, using knowledge of the protease
substrate specificity, which is determined by subsites
neighboring the active site. The subsites interact with side
chains of individual amino acid residues around the cleaved
peptide bond (scissile bond).20 For S1 family proteases, the
biggest family of serine proteases, the S1 pocket is the primary
recognition site, but other subsites also contribute to
selectivity. Therefore, extending phosphonate inhibitors
beyond the P1 position can improve selectivity. For example,
inclusion of non-natural amino acids in the P2−P4 position
has led to highly active and selective inhibitors for the serine
protease human neutrophil elastase (HNE).21,22
S
Neutrophil elastase, for instance, plays a role in inflammatory
diseases such as inflammatory bowel disease,1 chronic
obstructive pulmonary disease,2 and cystic fibrosis.3 Further-
more, it is a component of neutrophil extracellular traps
(NETs), a network of chromatin and granular proteins that is
secreted by neutrophils to trap and kill pathogens.4 Chymase is
linked to atherosclerosis and gastric cancer, and numerous
other serine proteases are involved in carcinogenesis, tumor
progression, and neurodegeneration.5 Hence, serine proteases
are attractive drug targets and protease inhibitors represent
promising drug candidates. For example, camostat is an
approved drug for treatment of chronic pancreatitis.
The development of covalent inhibitors is a re-emerging
topic in drug development.6,7 Various recently approved drugs,
such as ibrutinib for the treatment of leukemia, have a covalent
inhibitory mechanism.7 In the past, several covalent inhibitor
scaffolds for serine proteases have been developed, such as
isocoumarins,8 benzoxazinones,9 N-(sulfonyloxy) and N-
(acyloxy) phtalimides,10 sulfonyl11 and phosphonyl fluorides,12
1,2,5-thiadiazolidin-3-one 1,1-dioxides,13 diphenyl phospho-
nates,14 β-lactams, and more recently oxolactams.15,16 Interest-
ingly, these can not only be used as inhibitors but also as
warheads for activity-based probes (ABPs).
α-Aminomethyl diphenyl phosphonates are of particular
interest due to their ease of synthesis by a one-step three-
component reaction.17 Although usually synthesized as an
enantiomeric mixture, the R-configuration, which corresponds
to the natural L-configuration of amino acids, is usually the
Aminomethyl diphenyl phosphonates make no efficient use
of the primed site of the protease. Although adding
substituents to the phenyl group has been shown to influence
the activity,23,24 this may be a result of the leaving group
capacity, possible primed site interaction, or a combination
thereof. However, the leaving groups are not observed in
crystal structures of the compounds due to “aging” (Figure 1,
upper part). Knowledge of the interaction with the primed site,
Received: May 26, 2020
Accepted: August 10, 2020
Published: August 10, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00284
© XXXX American Chemical Society
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starts with a three-component Mannich-type reaction between
commercially available aldehydes 6, dichlorophosphines 7, and
benzyl carbamate (Scheme 1). From these materials, the
aldehyde R1 substituent forms the eventual amino acid side
chain mimic, whereas the R2 substituent on the phosphine
constitutes the potential primed site binding element. The
formed phosphinyl chloride intermediate 8 is then reacted with
phenol and triethylamine to yield the final phosphinate ester
10. Although successful for benzaldehyde as a starting material,
the one-pot procedure did not succeed with aliphatic
aldehydes. As an alternative, we directed our efforts toward a
two-step synthesis, using the same starting materials (Scheme
2). Here, we isolated intermediate phosphinic acids 9, which
were then reacted with phenol in a separate, Steglich-type
esterification using DMAP and DIC (Scheme 2). Satisfyingly,
this yielded the desired compounds 10−13, as a mixture of
diastereomers. Since no purification of the intermediate is
necessary, this two-step synthesis is a facile way to access α-
amino phosphinate esters with aliphatic as well as aromatic
amino acid side chains.
Figure 1. Phosphonate esters (1) and phosphinate esters (2) bind
with their side chain to the protease S1 subsite. Phosphinates can
potentially make use of the primed sites with their R2 group. Diphenyl
phosphonates (upper) react with the nucleophilic active site serine of
the protease, thereby expelling a phenoxy leaving group. The second
phenoxy group is later lost in an “aging” process, and the negatively
charged oxygen occupies the oxyanion hole. Phosphinates react in the
same manner but do not undergo the aging process.
Since one of our aims was to identify the influence of R2, we
used different dichlorophosphines to vary this substituent.
Unfortunately, our choice was limited by the small amount of
commercially available dichlorophosphines. Eventually, we
selected three different ones, each with a distinct carbon
substituent: 7a with a phenyl substituent would most closely
resemble the diphenyl phosphonate counterparts and was the
only commercially available dichlorophosphine with an
aromatic substituent (1, see Figure 1B). Besides this, 7b with
n-propyl as a sterically small and 7c with t-butyl as a bulky,
sterically demanding substituent were chosen. The aldehydes
were selected according to the proteases to be targeted. We
decided for phenylacetaldehyde, benzaldehyde, isovaleralde-
hyde, and isobutyraldehyde leading to analogues of Phe, Phg,
Leu, and Val, respectively.
The free acid intermediates 9 were generally obtained as
pure compounds after a simple aqueous workup. Unfortu-
nately, despite several efforts, the reaction with 7b or 7c and
phenylacetaldehyde did not yield the desired product. Overall,
the obtained yields are generally higher for reactions with n-
propyldichlorophosphine and lower for the sterically more
demanding t-butyldichlorophosphine, whereas the ones for
phenyldichlorophosphine are found in between.
Having the small series of phosphinates in hand, we tested
their inhibitory activity on different serine proteases from the
S1 family of serine proteases. We decided for neutrophil
elastase (NE), porcine pancreas elastase (PPE), and proteinase
3 (PR3) which possess a preference for small hydrophobic
amino acids in their S1 site as well as for chymotrypsin (ChT)
and cathepsin G (CatG) which in turn prefer cleavage after
large hydrophobic amino acids. In order to select active
compounds, we first performed a competitive activity-based
protein profiling experiment (competitive ABPP) at high
however, could lead to a potential gain in selectivity.
Phosphinates are structurally similar to phosphonates but
have one of the ester bonds replaced by a carbon substituent.
As a result, phenyl phosphinate esters only possess one leaving
group (Figure 1, lower part) but have an additional
stereocenter at the phosphorus atom. With their carbon
substituent, phosphinates may have the ability to interact with
the primed site, thereby potentially increasing selectivity.
A small number of studies have reported phosphinate serine
protease inhibitors,25−27 but these studies only incorporated
one carbon substituent, preventing a thorough analysis of their
influence on activity. Additionally, the synthesis of most of
these compounds requires three to five synthetic steps from
commercially available starting materials.26
Here we report a facile one-pot (for benzaldehydes) and
two-pot (for other aldehydes) two-step synthesis of α-amino
phenyl phosphinate esters as covalent, irreversible serine
protease inhibitors. We show that they display better inhibition
of various S1 family serine proteases compared with diphenyl
phosphonates and we present a possible binding mode of the
different carbon substituents in the active site of several serine
proteases.
Inspired by work on the synthesis of phosphinodepsipep-
tides by Meng and Xu,28 we explored a two-step one-pot
synthesis of the phosphinate scaffold in a pseudo-four-
component condensation reaction (Scheme 1). The procedure
a
Scheme 1. Two-Step, One-Pot Synthesis of Phosphinate Esters
a
b
Reagents and conditions: (a) CbzNH2, acetonitrile, reflux; (b) TEA, phenol. Diastereomeric ratio as determined after silica chromatography.
c
Mimic of amino acid: Phg = phenylglycine.
B
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a
Scheme 2. Two-Step, Two-Pot Synthesis of Phosphinate Esters
a
b
Reagents and conditions: (a) CbzNH2, acetonitrile, reflux, aqueous workup; (b) phenol, DIC, DMAP, toluene, 80 °C, 4 h. Diastereomeric ratio
c
as determined after silica chromatography. Mimic of amino acid: Phg = phenylglycine.
Figure 2. Initial testing of compounds by competitive activity-based protein profiling. Proteases were treated with the indicated compound at 100
μM or DMSO control (−), after which residually active enzyme was fluorescently labeled with the activity-based probe FP-Rh. Hence, a
disappearance of a band corresponds to an active compound. NE = human neutrophil elastase, PR3 = human proteinase 3, ChT = bovine
chymotrypsin, PPE = porcine pancreatic elastase, CG = human cathepsin G.
Table 1. Kinetic Values of the Synthesized Inhibitors
a
b
entry
enzyme
NE
compound
AA
kinact (10−3 s−1
)
KI (μM)
k
inact/KI (M−1 s−1
)
DR
1
2
3
4
5
6
7
8
12a
Val
Val
Val
Val
Val
Val
Val
Val
Phg
Phg
Leu
Leu
Phe
Phe
13.0 3.0
0.59 0.04
11.0 1.6
1.75 0.20
51.0 15.4
2.96 0.85
26.5 6.2
83.8 18.3
255 96
200 59
415 115
21
n.i.
59 10
n.i.
409 50
(3.4 0.6) × 103
404 122
978 176
188 48
(16.4 3.6) × 103
(8.1 0.9) × 103
11:89
1a (ValP)
12a
PR3
11:89
54:46
12b
5
c
c
d
1a (ValP)
12a
n.d.
n.d.
PPE
ChT
7.80 0.79
132 19
11:89
c
c
d
1a (ValP)
12a
n.d.
n.d.
3.04 0.15
1.57 0.07
5.84 0.9
21.1 2.0
2.44 0.31
23.0 1.7
5.63 0.24
7.42 0.83
0.46 0.076
14.5 3.8
21.6 3.3
13.0 2.9
1.40 0.28
0.69 0.07
11:89
20:80
55:45
44:56
53:47
39:61
9
10a
10b
11a
11b
13a
1b (PheP)
10
11
12
13
14
a
b
The tested compound mimics the indicated amino acid. Compounds were tested as indicated mixtures of diastereomers (either as obtained in
the reaction or as a mixture with a DR of approximately 50:50). n.d.: not determined; since no inhibition was seen, these values were not
determined. n.i.: no inhibition; no good inhibition was seen in a dilution series between 200 and 26.7 μM.
c
d
inhibitor concentration. To this end, each of these enzymes
was incubated with 100 μM of the compounds, and residual
activity was measured by labeling with the general serine
hydrolase ABP FP-rhodamine.29 As can be seen in Figure 2,
NE, PR3, and PPE are best inhibited by the valine derivative
12a, in agreement with their P1 preference. The substituent on
the phosphorus, however, seems crucial. Whereas the phenyl
substituent results in an active compound (12a), the n-propyl
substituent renders the compound inactive (NE and PPE) or
dramatically reduces the inhibitory capacity (PR3) and a t-
butyl inactivates it completely. For CatG, the Phe derivative
13a is the most active compound. All compounds with the
exception of the t-butyl-substituted ones and 12b show some
degree of inhibition of ChT, in agreement with the general
preference of the digestive protease ChT for large hydrophobic
residues in the P1 position. Overall, the compounds with the
phenyl substituent on the phosphorus seem most active,
followed by the n-propyl ones. The t-butyl leads to inactivity
for all tested proteases. In addition to the general screening, we
tested 12a as the best compound with a small aliphatic residue
in different concentrations on NE and, for comparison, on
ChT to get an insight into the compound’s activity and
selectivity. Furthermore, we performed a titration of 13a, the
best compound with a large hydrophobic residue on ChT as
well as NE. To our delight, both of these experiments reveal
high selectivity of the individual compound for their respective
target protease over the off-target protease (see Figure S1).
Past reports about phosphinate esters as serine protease
inhibitors in comparison with phosphonate esters have been
contradictory in their results. Boduszek et al. reported di- and
tripeptidyl phosphinate esters to be nearly unreactive with NE
and PPE, whereas similar phosphonate esters were found to be
C
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active.25 In contrast, Walker et al. found that phosphinate
esters with a single amino acid in the P1 position displayed
consistently higher activity than their phosphonate counter-
parts.26
To shed more light on the difference in activity of
phosphinate and phosphonate esters, we made a comparable
assessment of the potency of the here described compounds as
well as their diphenyl phosphonate ester counterparts 1a and
1b. To this end, we performed inhibition kinetics experiments.
Because of the covalent, irreversible nature of the inhibitors, we
used a two-step binding model (Figure S2) consisting of a first,
reversible, non-covalent interaction and a second, irreversible
reaction between enzyme and inhibitor. Applying this binding
model, we determined kinact and KI. kinact is the rate constant of
the second, irreversible step in this process at infinite inhibitor
concentration and as such an indicator of compound reactivity.
The inhibitor concentration that results in an observed rate
constant of 1/2kinact is called KI. It reflects the reversible binding
process between inhibitor and enzyme before reaction. The
overall inhibitory potency is reflected in the ratio of these two
values with better inhibitors possessing higher kinact/KI values.
Valine derivatives 12a and 1a were tested against three
enzymes with elastase-like activity (NE, PR3, and PPE).
Additionally, 12b was tested against PR3. Whereas 12a showed
activity with the highest kinact/KI against PR3, the diphenyl
phosphonate was mostly inactive, in line with previous
reports,23 except against NE (Table 1, entries 1−7).
Compound 12b, which only showed activity against PR3 in
the gel-based experiments (Figure 2), displayed much lower
activity than 12a.
Figure 3. Proposed binding models by in silico covalent docking.
Shown are close-ups of the active site with the protein in cartoon
format (α-helices in cyan, β-sheets in magenta, loops in pink),
molecular surface depicted as gray transparent, and inhibitors as stick
models. All phosphinates are colored yellow. Water molecules and
inhibitor molecules originally present in the structure were removed
prior to docking. (a) Compound 12a covalently bound to S195 of
human NE (PDB code: 4WVP). (b) Compound 12a covalently
bound to S195 of human PR3 (PDB code: 1FUJ). (c) Compound
13a and diphenyl phosphonate 1b (in magenta) bound to S195 of
bovine α-chymotrypsin (PDB code: 4Q2K). (d) Chirality at the
phosphorus atom with priorities of the substituents (the PO bond
is treated as a normal single-bonded substituent according to IUPAC
rules). Here, the S-enantiomer is depicted. Note that the chirality is
inverted upon reaction with the enzyme in an SN2P-type mechanism.
Unfortunately, the kinetic experiments with cathepsin G
gave no good progression curves and thus no good fitting was
possible in that case. Hence, phosphinates with large
hydrophobic P1 residues (Leu, Phe, Phg) were tested against
chymotrypsin, as this protease was inhibited by the largest
number of compounds (Figure 2) and may be able to give
more insight into the primed site effect. Phe in the P1 position
is clearly preferred with its kinact/KI value being 5−20-fold
higher than the values of the corresponding Phg and Leu
derivatives with the same primed site element (Table 1, entries
9−13). Additionally, it is twice as active as the corresponding
diphenyl phosphonate inhibitor 1b, which is especially due to a
higher kinact. Unfortunately, the Phe phosphinates could only
be synthesized with one R2 group. Therefore, comparison of
the primed site binding elements can only be made with the
Leu and Phg derivatives. In both cases, the inhibitors with a
phenyl R2 substituent displayed higher potency. Interestingly,
for the Leu phosphinate esters, the difference was mainly
caused by a higher kinact for the R2=Ph, whereas, for the Phg
phosphinate esters, the underlying potency difference had its
cause in a better KI for the R2=Ph compound.
For further insight into the different binding modes, we
performed covalent docking with AutoDock 4.230 of some
selected compounds in crystal structures of NE, PR3, and ChT
(Figure 3). In these experiments, only the R-configurations at
the α-carbon to the phosphorus were assessed, because these
reflect the natural L-configuration of amino acids and
correspond to the active species, as previously shown in
structural and kinetics studies.27,31 However, we included both
configurations at the phosphorus atom: the bound RR- and RS-
diastereomers. Note that the R-configuration at the phospho-
rus bound to the enzyme originates from the S-configuration at
the phosphorus in the inhibitor, because the stereochemistry
inverts upon reaction with the active site serine in an SN2P-
type mechanism with trigonal bipyramidal transition state
(Figure 3D) Strikingly, we found the RS-diastereomer to be
consistently superior to the RR-diastereomer in binding energy,
with the only exception being 11a (Table S1). Additionally, a
visual inspection of the top scoring poses showed that the RR-
derivatives generally displayed unlikely binding modes (Figure
S3). Therefore, we propose that the RS-diastereomers are
probably the (most) active species. Future efforts in synthesis
toward enantiomerically pure phosphinate esters may address
this further and result in compounds with higher potency.
For NE and PR3 we took a closer look at the interactions of
12a. In both proteases the valine side chain docks well into the
S1 pocket and the Cbz group occupies the S2 pocket, whereas
the phenyl substituent at the phosphorus atom reaches toward
the primed site (Figure 4A,B). The better KI value seen for
PR3, as measured in the kinetics experiments, may be
explained by the somewhat different orientation of the Cbz
and Ph groups as well as the preference of PR3 for slightly
larger P1 and P2 residues, as has been shown before.21,22,31
For ChT, we compared the most active compound 13a with
the reference diphenyl phosphonate 1b. For both compounds,
the Phe side chain reaches deep into the S1 pocket and the
carboxybenzyl substituent interacts with the S2 pocket. The
phenoxy as well as the phenyl group are both oriented toward
the S1′ pocket. The overall very similar binding mode may
explain their comparable KI values.
In conclusion, we report a straightforward synthetic
procedure to synthesize phenyl phosphinates in one to two
steps from commercially available aldehydes and dichloro-
phosphines. These compounds inhibit S1 family serine
D
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00284.
Supplemental figures, supplemental tables, experimental
methods, and copies of NMR spectra and LC-MS
chromatograms (PDF)
Figure 4. Proposed explanation for the higher reactivity of
phosphinate esters with a phenyl ring attached to the phosphorus.
In the SN2P reaction mechanism, the orbitals on the phosphorus that
form bonds to the incoming nucleophile and outgoing leaving group
have overlap with the π-orbitals of the phenyl ring (left), thereby
stabilizing this structure and lowering the transition state energy. This
stabilization is not present with aliphatic substituents on the
phosphorus (right).
AUTHOR INFORMATION
■
Corresponding Author
Steven H. L. Verhelst − Laboratory of Chemical Biology,
Department of Cellular and Molecular Medicine, KU Leuven −
University of Leuven, 3000 Leuven, Belgium; AG Chemical
Proteomics, Leibniz Institute for Analytical Sciences − ISAS,
44227 Dortmund, Germany; orcid.org/0000-0002-7400-
1319; Email: Steven.Verhelst@kuleuven.be
proteases depending on their P1 and primed site binding
elements. As expected, for the P1 position, the compounds
follow the known substrate specificity preferences of the
targeted enzymes: phosphinates with a P1 valine inhibit
elastase-like proteases, whereas those with large, hydrophobic
P1 elements are active against proteases with chymotryptic
activity.
Authors
Jan Pascal Kahler − Laboratory of Chemical Biology,
Department of Cellular and Molecular Medicine, KU Leuven −
University of Leuven, 3000 Leuven, Belgium
Stijn Lenders − Laboratory of Chemical Biology, Department of
Cellular and Molecular Medicine, KU Leuven − University of
Leuven, 3000 Leuven, Belgium; orcid.org/0000-0002-
8833-9353
As a primed site binding element (R2), a phenyl substituent
was generally preferred. More particular, these compounds
were superior to phosphonate esters, in line with a previous
study by Walker et al.26 In all compounds, the phenyl was also
preferred over the n-propyl substituent, and all compounds
with t-Bu substituents on the phosphorus atom were inactive.
The last observation may be explained by a steric clash: the
bulky t-Bu group could either prevent attack of the active site
serine or substantially disturb the interaction with the enzyme
(i.e., raising KI). However, steric hindrance is not a logical
explanation for the lower activity of the compounds with n-Pr
substituents. Interestingly, the reason for the activity difference
is mainly rooted in a better kinact of the phosphinates with the
Ph substituent. Hence, it is not the initial binding to the
enzyme’s active site but rather the reaction rate with the active
site serine residue that underlies the difference in activity.
Remarkably, the kinact was also higher than the one for diphenyl
phosphonates. This may be counterintuitive, because the
electrophilicity of the phosphorus in phosphinate esters is
lower than that for phosphonate esters, indicated by a lower
partial Gasteiger charge as calculated during ligand preparation
for in silico docking. As a possible explanation for the increased
reactivity, we here propose that the R2 phenyl ring stabilizes
the transition state in the reaction with the enzyme (Figure 4).
We also found in compounds 12 (the Val series of
phosphinates) that the n-Pr substituent only showed activity,
albeit with low potency, against PR3 but not against NE. This
result may offer future possibilities to obtain inhibitors that are
selective for PR3 over the closely related NE. It also illustrates
the capacity of the primed site to influence the binding to the
target protease. Overall, we expect that phosphinate inhibitors
with extended primed site elements as well as residues that
bind the S2−S4 sites will lead to more specific inhibitors and
activity-based probes. Work along these lines is currently being
investigated and will be reported in due course.
Merel A. T. van de Plassche − Laboratory of Chemical Biology,
Department of Cellular and Molecular Medicine, KU Leuven −
University of Leuven, 3000 Leuven, Belgium
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00284
Author Contributions
S.H.L.V. and J.P.K. developed the project and designed
compounds, S.L. synthesized compounds, J.P.K. performed
biochemical analysis, M.A.T.v.d.P. performed covalent dock-
ing, J.P.K. wrote the manuscript, and S.H.L.V. revised the
manuscript. All authors have given approval to the final version
of the manuscript.
Funding
We acknowledge funding by the FWO (project grant
G0D8617N and PhD fellowship 1S18520N to J.P.K.), the
Ministerium fu
Nordrhein-Westfalen, the Regierende Bu
Berlin−inkl. Wissenschaft and Forschung, and the Bundesmi-
nisterium fur Bildung and Forschung.
Notes
̈
r Kultur and Wissenschaft des Landes
̈
rgermeister von
̈
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Luc Baudemprez for recording NMR spectra.
ABBREVIATIONS
■
ABPP, activity-based protein profiling; CatG, cathepsin G;
Cbz, carboxybenzyl; ChT, chymotrypsin; DIC, N,N′-diisopro-
pylcarbodiimide; DMAP, 4-dimethylaminopyridine; DR, dia-
stereomeric ratio; HNE, human neutrophil elastase; NET,
neutrophil extracellular trap; Phg, phenyl glycine; PPE, porcine
pancreas elastase; PR3, proteinase 3
E
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Uneven Location of Serine Proteases in Neutrophils. J. Am. Chem. Soc.
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