Phosphonic analogues of glutamic acid as irreversible inhibitors of Staphylococcus aureus endoproteinase GluC: An efficient synthesis and inhibition of the human IgG degradation

Article abstract of DOI:10.1016/j.bmcl.2012.12.074

Endoproteinase GluC (V8 protease) is one of many virulence factors released by the Staphylococcus aureus species in vivo. The V8 protease is able to hydrolyze some serpins and all classes of mammalian immunoglobulins. The application of specific and potent inhibitors of V8 protease may lead to the development of new antibacterial agents. Herein, we present the synthesis and the inhibitory properties of novel peptidyl derivatives of a phosphonic glutamic acid analogue. One of the compounds Boc-Phe-Leu-Glu^P(OC ₆H₄)₂ displayed an apparent second-order inhibition rate value of 8540 M^-1 s^-1. The Boc-Phe-Leu-Glu^P(OC₆H₄)₂ compound with the highest inhibitory potency showed the ability to prevent V8-mediated human IgG proteolysis in vitro.

Full text of DOI:10.1016/j.bmcl.2012.12.074

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1412–1415
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phosphonic analogues of glutamic acid as irreversible inhibitors
of Staphylococcus aureus endoproteinase GluC: An efficient synthesis
and inhibition of the human IgG degradation
⇑
´
´
Ewa Burchacka, Marcin Skorenski, Marcin Sienczyk, Józef Oleksyszyn
_
Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspian´skiego 27, 50-370 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Endoproteinase GluC (V8 protease) is one of many virulence factors released by the Staphylococcus aureus
species in vivo. The V8 protease is able to hydrolyze some serpins and all classes of mammalian immu-
noglobulins. The application of specific and potent inhibitors of V8 protease may lead to the development
of new antibacterial agents. Herein, we present the synthesis and the inhibitory properties of novel pep-
tidyl derivatives of a phosphonic glutamic acid analogue. One of the compounds Boc-Phe-Leu-Glu-
P(OC6H4)2 displayed an apparent second-order inhibition rate value of 8540 Mꢀ1 sꢀ1. The Boc-Phe-Leu-
GluP(OC6H4)2 compound with the highest inhibitory potency showed the ability to prevent V8-mediated
human IgG proteolysis in vitro.
Received 17 October 2012
Revised 17 December 2012
Accepted 21 December 2012
Available online 5 January 2013
Keywords:
V8 protease
Staphylococcus aureus
Ó 2013 Elsevier Ltd. All rights reserved.
a-Aminoalkylphosphonates
Serine protease inhibitors
Extracellular bacterial proteases belong to the group of secreted
virulence factors which are important for effective pathogenesis.
Proteolysis, for which bacterial proteases are responsible, targets
different elements of extracellular structures in infected organisms
where fibronectin, collagen, elastin are just a few examples.1–3 The
presence of the active proteases allows circumvention of host de-
fense mechanisms and modulation of the immune system during
infection and inflammation by bacteria.4,5 Moreover, most of the
proteases released by bacteria are not only susceptible to inactiva-
tion by endogenous inhibitors present in plasma, but also can rel-
inactivation of 1-PI
α
converting kininogen to kinin
digestion of IgA, E, D, M, G
V8 protease
degradation of desmoglein-1
S. aureus
Scheme 1. The role of V8 protease during infections.
atively quickly inactivate serpins, including
a1-PI, antithrombin III,
and
a
1-microglobulin.2,6 Several bacterial species produce prote-
Staphylococcus aureus contributes to globally important infec-
tions such as pneumonia, meningitis, osteomyelitis, endocarditis,
toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis.11
It has been shown that the activity of GluV8 protease increases
the growth and survival of S. aureus in animal models.12 Addition-
ases (V8, 56K protease) capable of immunoglobulin degradation
which in turn facilitates the progression of the infection and en-
hances the pathogenesis of infectious diseases.2,7,8
Endoproteinase GluC (also called V8 protease) from Staphylo-
coccus aureus is one of the proteases that play an important role
in evading host immune defense mechanisms (Scheme 1). V8 pro-
tease (EC 3.4.21.19) is a member of the glutamyl endopeptidase I
family of the Staphylococcus aureus V8 strain (GluV8).9,10 It specif-
ically cleaves the peptide and ester bonds on the carboxylic side of
Glu and Asp residues where the rate of Glu residue hydrolysis is
approximately 100–1000 times more effective than hydrolysis of
Asp residue.
ally, this enzyme inactivates a1-Proteinase Inhibitor (a1-PI) which
protects tissues from inflammatory mediators and controls the
activity of host proteases such as neutrophil elastase, trypsin and
plasminogen activator.6 Moreover, V8 protease converts kininogen
to kinin which leads to blood vessel dilation and increases vascular
permeability.13 In vitro, staphylococcal glutamyl endopeptidase de-
grades human immunoglobulin A, IgG, IgM, IgE and IgD which re-
sults in immune system dysfunction.7,8 GluV8 contributes to skin
blistering disease via degradation of the desmoglein 1 protein.14
Development of potent and specific V8 protease inhibitors may
lead to potential therapeutics targeting Staphylococcus aureus
infections.
⇑
Corresponding author. Tel.: +48 71 320 4027; fax: +48 71 320 2427.
E-mail address: jozef.oleksyszyn@pwr.wroc.pl (J. Oleksyszyn).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.074

E. Burchacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1412–1415
1413
In this Letter we present the synthesis and biological evaluation
of phosphonic di- and tripeptides towards V8 Staphylococcus aur-
eus protease. The a-aminoalkylphosphonate diaryl esters and their
peptidyl derivatives are known selective, potent and specific inhib-
itors which bind to the active site of serine proteases in an irrevers-
ible manner.15–17
The first Letter showing the application of aminophosphonates
as V8 protease inhibitors was presented by Hamilton et al.18
Simple acetylated phosphonic analogues of glutamic and aspartic
acid displayed second-order inhibition rate values of 5.3 ꢁ 103
and 5.0 ꢁ 102 Mꢀ1 sꢀ1 for Ac-GluP(OC6H5)2 and Ac-AspP(OC6H5)2,
respectively. However, the inhibitory activity of peptidyl deriva-
tives of phosphonic glutamic and aspartic acid against V8 protease
has not yet been presented. The preferred substrate sequence of
Staphylococcus aureus V8 protease is Phe/Leu/Ala-Leu-Asp/Glu
(P3–P2–P1, Schechter nomenclature).19,20 Based on the substrate
recognition pattern we synthesized a series of di- and tripeptidyl
For the synthesis of compound 4, which was further used for the
peptide derivative synthesis, we applied the procedure described
by Hamilton et al. with modification in the synthesis of intermedi-
ate 2 (Scheme 1).18 Briefly, an
a-amidoalkylation reaction of triaryl
phosphite with paraformaldehyde and benzyl carbamate led to
Cbz-GlyP(OC6H5)2 (1) in which the protective group was removed
using 33% hydrobromic acid solution in acetic acid. The resulting
hydrobromide salt of H-GlyP(OC6H5)2 was condensed with benzo-
phenone imine to obtain compound 2.
Alkylation of compound 2 with benzyl-3-bromopropanoate and
KHMDS gave compound 3 which, after treatment with 1 M HCl in
diethyl ether solution, gave hydrochloride salt of H-Glu(OBzl)-
P(OC6H5)2 4, a starting compound used for peptide derivative syn-
thesis. The synthesis of Cbz-GluP(OPh)2 (6), which was used as the
reference inhibitor in our studies, started with the deprotection of
a benzyl group by hydrogenolysis over 10% palladium on carbon
leading to intermediate H-GluP(OC6H5)2. To avoid phenyl ester
group hydrolysis under basic conditions associated with the appli-
cation of benzyl chloroformate (Cbz-Cl) as observed previously,18
the introduction of a Cbz-protective group was achieved using N-
(benzyloxycarbonyloxy)succinimide (CbzOSu) in the presence of
N,N0-diisopropylethylamine (DIPEA) in acetonitrile which led to
Cbz-GluP(OC6H5)2 (6). In addition, we synthesized Ac-GluP(OC6H5)2
(5) which was used as the control inhibitor (under the conditions
of our assay the measured k2/Ki value of Ac-GluP(OC6H5)2 was iden-
tical to the value published by Hamilton group18).
a
-aminophosphonate diaryl ester derivatives of phosphonic glu-
tamic acid with various P2 and P3 residues in order to determine
their influence on the inhibitory activity towards V8 and selectivity
over different proteases produced by S. aureus, for example, the
SplB protease. The SplB protease sequence reveals a high homology
to the S. aureus V8 protease and epidermolytic toxins.8–10 Introduc-
ing the particular amino acid residue into the structure of phos-
phonic peptide derivatives allowed
a comparison of their
inhibitory activity towards both serine proteases produced by
Staphylococcus aureus. Studies have shown that V8 protease has a
narrow substrate specificity (S1 binding pocket accommodated
only/exclusively by Asp and Glu) whereas SplB protease mainly
prefers Asp, Asn and Gln residues at P1.10 Interestingly, investiga-
tions of SplB protease substrate mapping revealed that the glu-
tamic acid side chain was not accepted at the P1 position. In this
Letter we present the selectivity of novel peptidyl phosphonate
inhibitors—analogues of glutamic acid—towards staphylococcal
SplB and V8 protease.
The general synthetic procedure for generation of di- and trip-
eptidyl derivatives of
a-aminoalkylphosphonate diphenyl esters,
analogues of glutamic acid, is outlined in Scheme 1. Briefly, com-
pound 4 was first coupled with N-Boc-protected amino acid using
HBTU as the coupling agent in the presence of triethylamine, and
the benzyl group in the resulting dipeptidyl derivatives was re-
moved under standard conditions (H2, 10% Pd/C) leading to the
derivatives 7a–c. Elongation of the peptidyl chain was achieved
in a similar fashion—after removal of the N-Boc-protective group
O
O
O
O
O
a
b,c
O
N
H
P
N
P
P(OPh)3
O
O
O
O
1
2
d
O
OH
O
O
O
O
O
O
O
O
O
f,g
e
N
H
P
HCl*H2N
P
N
P
O
O
O
5
4
3
h,g
i,g
i,j,k,g
O
OH
O
O
OH
O
OH
O
O
O
Boc-AA1
O
O
Boc-AA2-AA1
O
O
O
N
P
N
H
P
N
P
H
H
O
O
8a-j
6
7a-c
Scheme 2. General synthesis of phosphonic glutamic acid derivatives. Reagents and conditions: (a) benzyl carbamate, acetic anhydride, paraformaldehyde, AcOH, 60–70 °C,
3 h; (b) 33% HBr/AcOH, rt, 2 h; (c) benzophenone imine, CH2Cl2, rt, 24 h; (d) KHMDS, benzyl-3-bromopropanoate, THF, ꢀ78 °C, 3.5 h; (e) 1 M HCl in Et2O, rt, 24 h; (f) AcOH,
HBTU, Et3N, MeCN, rt, 12 h; (g) 10% Pd/C, H2, MeOH, rt, 6 h; (h) CbzOSu, MeCN, DIPEA, rt, 48 h; (i) N-Boc-L-AA1-OH, HBTU, Et3N, MeCN, rt, 12 h; (j) 50% TFA/DCM (v/v), rt, 2 h;
(k) N-Boc-L-AA2-OH, HBTU, Et3N, MeCN, rt, 12 h.

1414
E. Burchacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1412–1415
Table 1
The inhibition constant values of
a
-aminoalkylphosphonate diphenyl ester derivatives towards V8 and SplB proteases
V8 protease
No
Compound
SplB protease
k2/Ki (Mꢀ1 sꢀ1
)
Ki (l
M)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (lM)
5
6
Ac-GluP(OC6H5)2
11.07 1.2
8.1 0.95
7.8 1.2
23.3 5.5
39%a
5300
1384
792
102
nd
8540
Nd
314
1708
nd
238
5012
nd
1002
—
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
—
—
—
—
—
—
—
—
—
—
—
—
Cbz-GluP(OC6H5)2
7a
7b
7c
8a
8b
8c
8d
8e
8f
8g
8h
8i
Boc-Leu-GluP(OC6H5)2
Boc-Val-GluP(OC6H5)2
Boc-Ala-GluP(OC6H5)2
Boc-Phe-Leu-GluP(OC6H5)2
Boc-Asp-Leu-GluP(OC6H5)2
Boc-Glu-Leu-GluP(OC6H5)2
Boc-Phe-Ala-GluP(OC6H5)2
Boc-Asp-Ala-GluP(OC6H5)2
Boc-Glu-Ala-GluP(OC6H5)2
Boc-Phe-Val-GluP(OC6H5)2
Boc-Asp-Val-GluP(OC6H5)2
Boc-Glu-Val-GluP(OC6H5)2
6.2 0.98
41%a
9.5 1.1
9.4 0.99
26%a
12.9 0.19
8.15
50%a
—
9.95 0.82
NI
—
Boc-Glu-Leu-GlnP(OC6H5)2
4.2 0.4b
500b
a
9
Bold values represent the most active compound in the presented studies.
a
Percentage of inhibition observed after 30 min of measurement at a 200 lM concentration of the tested compound.
b
Compound previously reported;24 nd—not determined; NI—0% of inhibition observed after 30 min of measurement at a 200
lM concentration of the tested compound.
in dipeptidyl derivatives using 50% trifluoroacetic acid in dichloro-
methane, N-Boc-protected amino acid was coupled using the
HBTU/Et3N system. Finally, tripeptides 8a–j were obtained by the
hydrogenolysis of their benzyl-protected parent compounds over
Pd/C in methanol (see Scheme 2).
displayed a k2/Ki value of 792 Mꢀ1 sꢀ1. Replacing Leu residue with
Val at P2 position decreased the inhibitory activity by about 8-
times (7b, k2/Ki = 102 Mꢀ1 sꢀ1). For dipeptide Boc-Ala-Glu-
P(OC6H5)2 (7c) only 39% of V8 inhibition was observed at 200
lM.
Among tripeptidyl derivatives of phosphonic glutamic acid the
All of the synthesized derivatives (5, 6, 7a–c, 8a–j) were evalu-
ated for their inhibitory activity against V8 and SplB proteases.
The obtained inhibition kinetic data (Ki and k2/Ki values) towards
V8 and SplB proteases are summarized in Table 1. The observed rate
of inhibition constants was determined by the progress curve
method.21 All measurements were performed using a Spectra Max
Gemini XPS spectrofluorometer (Molecular Devices, USA) at 37 °C.
The kinetics of V8 and SplB protease inhibition was determined
by addition of the enzyme into the solution of substrate and tested
inhibitor ([E]0 ꢂ [I]0) according to the following mechanism:
highest potency of action was observed for Boc-Phe-Leu-Glu-
P(OC6H5)2 (8a) which displayed a k2/Ki value of 8540 Mꢀ1 sꢀ1
.
Replacing Phe residue at P3 position with glutamic acid (8c) de-
creased the inhibitory activity by approximately 27-times (k2/
Ki = 315 Mꢀ1 sꢀ1). Interestingly, for derivative 8b, which contains
an aspartic acid residue at position P3, only 41% of V8 protease
inhibition was observed. Additionally, replacing Leu residue at P2
position with Ala (8d) decreased the inhibitory activity by 5-times
(k2/Ki = 1708 Mꢀ1 sꢀ1). The obtained results indicate the impor-
tance of P3 and P1 residues in the inhibitor sequence. The highest
inhibitory activity was observed for compounds containing Phe-X-
GluP motif—hydrophobic residue occupying the S3 binding pocket
and acidic side chain of glutamate interacting with the protease S1
pocket. Decreasing the length of the P2 residue aliphatic side chain
decreases the inhibitor ability for V8 protease inactivation (Ala <
Val < Leu). Importantly, all of the synthesized analogues of
glutamic acids presented in this study were obtained as diastereo-
isomeric mixtures. Since only one diastereoisomer reacts with
the protease active site, the true inhibitory potency should be at
least twofold greater than the potency of the diastereoisomer
mixture.23
Ki
k2
E þ I ¢ EI ! Ei
ð1Þ
where Ki is the reversible enzyme–inhibitor complex (EI) dissocia-
tion constant, and k2 is the rate of an irreversible complex (Ei)
formation.
The rates of V8 protease inhibition (75 nM, Sigma–Aldrich) and
SplB protease (100 nM, expressed and purified as previously de-
scribed 22) were measured in 0.1 M Tris–HCl, 0.05% Tween 20
(pH 7.8) at 37 °C and 0.1 M Tris–HCl, 0.01% Triton X-100 buffer
(pH 7.6) at 37 °C, respectively. Both proteases were assayed using
fluorogenic substrates: Ac-Phe-Leu-Glu-ACC (15
Ex. 355 nm, Em. 460 nm), and Ac-Trp-Glu-Leu-Gln-ACC (10
SplB protease, Ex. 355 nm, Em. 460 nm). The calculated Michaelis
constant (KM) values for the substrate of V8 and SplB protease were
l
M, V8 protease,
The reason for introducing a particular amino acid residue into
the structure of phosphonic peptide inhibitors was the generation
of selective inhibitors which do not react with Staphylococcus aur-
eus SplB protease, which specifically recognizes Gln at P1 position.
Replacing phosphonic glutamine by phosphonic glutamic acid at
P1 position made these compounds completely inactive against
SplB protease leading to an absolute selectivity of action against
V8 protease.
lM,
116
screened for inhibitory activity against V8 and SplB protease at a
concentration of 200 M. For compounds for which less than 50%
lM and 135 lM, respectively. All the compounds were
l
of inhibition was observed after 30 min of kinetic measurement,
we expressed the percentage of inhibition (Table 1).
The influence of compound 8a on human IgG degradation by V8
protease was evaluated using the following method. First, V8 pro-
The kinetic investigation of the inhibitory potency towards V8
protease showed that the most active structure of phosphonic glu-
tamic acid derivative was peptide 8a (Boc-Phe-Leu-GluP(OC6H5)2,
k2/Ki = 8.5 ꢁ 103 Mꢀ1 sꢀ1) which was almost twice as active against
V8 protease than control inhibitor Ac-GluP(OC6H5)2 (5, k2/
Ki = 5.3 ꢁ 103 Mꢀ1 sꢀ1). Among the obtained phosphonic dipep-
tides the highest activity was observed for compound 7a which
tease (at final 1
lM) was pre-incubated for 1 h at 37 °C with 8a (at
final 100 M) before addition of human IgG (at final 160
l
l
g/ml,
Sigma–Aldrich). The mixture was then incubated for 24 h at
37 °C. The control sample was prepared similarly with the excep-
tion that DMSO was added instead of the inhibitor solution. Con-
trol V8 protease and human IgG solutions were incubated under

E. Burchacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1412–1415
1415
Acknowledgments
This work was supported by Grants N N401 596340 (to J.O.)
from the Polish Ministry of Science and Higher Education. M.S. is
thankful to Wroclaw University of Technology Statute Funds
(S10156/0313).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.074.
References and notes
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Figure 1. Inhibition of V8-mediated hydrolysis of human IgG: molecular weight
marker (MWM, Lane 1); Control V8 and IgG samples (Lanes 2 and 6, respectively);
degradation of IgG by V8 protease (Lane 5); the inhibitory effect of 8a on IgG
degradation (Lane 4).
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the same conditions. All samples were quenched by the addition of
non-reducing gel loading buffer and analyzed using SDS–PAGE (4–
12%). Visualization of the bands was performed via the silver stain-
ing method (Fig. 1).
The results clearly demonstrated that compound 8a prevents
V8-mediated IgG proteolysis. IgG incubated with V8 protease in
the absence of inhibitor undergoes proteolysis leading to formation
of a 30 kDa band which corresponds to the FC domain (monomer)
of IgG (Fig. 1, Lane 5). These observations are in agreement with
previously reported results.8 Addition of 8a efficiently prevents
IgG degradation due to the V8 inhibition (Fig. 1, Lane 4).
15. Oleksyszyn, J.; Powers, J. C. Biochem. Biophys. Res. Commun. 1989, 161, 143.
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In order to examine the antibacterial activity of 8a the growth
inhibition assay was performed. The results indicated that synthe-
sized V8 inhibitor had no influence on Staphylococcus aureus
growth under the conditions of our assay in which gentamicin dis-
played MIC value of 2 mg/L. The obtained data was not surprising
since the activity of V8 protease is localized outside the cell. To
establish the true potency of 8a its activity needs to be examined
in vivo and will be the subject of further investigations.
In summary, the most potent among all synthesized peptidyl
derivatives of phosphonic glutamic acid was Boc-Phe-Leu-Glu-
P(OC6H5)2 (8a, k2/Ki = 8540 Mꢀ1 sꢀ1) being the most active phos-
phonic V8 inhibitor published to date. Moreover, the results
demonstrated that compound 8a prevents V8-mediated IgG degra-
dation in vitro. The presented preliminary data on new and selec-
tive inhibitors of V8 protease and it relationship to SplB protease
activity could help to establish its role in vivo during staphylococcal
infections, especially its influence on immune system dysfunction.