Synthesis and activity of pyrrolidinyl- and thiazolidinyl-dipeptide derivatives as inhibitors of the Tc80 prolyl oligopeptidase from Trypanosoma cruzi

Article abstract of DOI:10.1016/S0223-5234(00)00118-5

Pyrrolidinyl- and thiazolidinyl- dipeptide derivatives, featuring either a vinyl sulfone-, a 2-ketobenzothiazole-, a nitrile-, or a benzimidazole group at the C-terminus, were designed and synthesized as potential inhibitors of the prolyl-specific Tc80 proteinase from Trypanosoma cruzi, the agent of Chagas' disease. These compounds were evaluated in vitro towards the target enzyme which was classified as a serine protease belonging to the prolyl oligopeptidase family (EC 3.4.21.26). A peptidyl nitrile and two peptidyl α-ketobenzothiazoles were shown to be potent reversible and competitive inhibitors of Tc 80 proteinase, with K(i) values in the range 38- 219 nM, and compared advantageously with some known mammalian prolyl oligopeptidase inhibitors. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00118-5

Eur. J. Med. Chem. 35 (2000) 257−266
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001185/FLA
257
Preliminary communication
Synthesis and activity of pyrrolidinyl- and thiazolidinyl-dipeptide derivatives as
inhibitors of the Tc80 prolyl oligopeptidase from Trypanosoma cruzi
Roger Joyeaua*, Chanfi Maoulidaa, Cindy Guilleta, François Frappiera, Antonio R.L. Teixeirac,
Joseph Schrévelb, Jaime Santanac, Philippe Grellierb
aLaboratoire de chimie des substances naturelles, MNHN, ESA CNRS 8041, 63 rue Buffon, 75005 Paris, France
bLaboratoire de biologie parasitaire, MNHN, EP CNRS 1790, 61 rue Buffon, 75005 Paris, France
cLaboratorio Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasilia, 70919-970 Brasilia -DF, Brazil
Received 29 May 1999; revised 22 July 1999; accepted 5 August 1999
Abstract – Pyrrolidinyl- and thiazolidinyl- dipeptide derivatives, featuring either a vinyl sulfone-, a 2-ketobenzothiazole-, a nitrile-, or a
benzimidazole group at the C-terminus, were designed and synthesized as potential inhibitors of the prolyl-specific Tc80 proteinase from
Trypanosoma cruzi, the agent of Chagas’ disease. These compounds were evaluated in vitro towards the target enzyme which was classified
as a serine protease belonging to the prolyl oligopeptidase family (EC 3.4.21.26). A peptidyl nitrile and two peptidyl α-ketobenzothiazoles
were shown to be potent reversible and competitive inhibitors of Tc 80 proteinase, with Ki values in the range 38–219 nM, and compared
advantageously with some known mammalian prolyl oligopeptidase inhibitors. © 2000 Éditions scientifiques et médicales Elsevier SAS
prolyl oligopeptidase / Trypanosoma cruzi / Chagas’ disease / inhibitor / peptidyl nitrile / peptidyl α-ketobenzothiazole
1. Introduction
parasite attachment site, an unusual process that results in
the formation of a parasitophorous vacuole with lysoso-
mal properties [1]. T. cruzi trypomastigotes may also
enter macrophages by interfering with the host immune
response. Whatever the type of cell being infected, a
limited set of T. cruzi proteins and target cell components
probably play an important role in the parasite host cell
relationship. Several proteolytic activities have recently
been identified in T. cruzi beside the major cysteine
proteinase, cruzipain. A 120 kDa serine endopeptidase [2]
has been shown to be required for the generation of Ca2+
signalling in mammalian cells [3] and an acidic 30 kDa
cysteine proteinase was characterized, which exhibited an
ability to hydrolyse human type I collagen and bovine
serum albumin [4].
The haemoflagellate Trypanosoma cruzi is the caus-
ative agent of Chagas’ disease in Latin America. Despite
vector-control programmes in many of the affected coun-
tries, this illness represents a major public health concern
notably because of its clinical variability, lack of efficient
non-toxic drug and its transmission by infected donor
blood.
Entry of T. cruzi into non-phagocytic mammalian cells
occurs by recruitment and fusion of host lysosomes at the
*Correspondence and reprints: joyeau@mnhn.fr
Abbreviations: AMC, 7-amino-4-methylcoumarin; DCC, dicyclo-
hexylcarbodiimide; 3,4-DCI, 3,4-dichloroisocoumarin; DCU, dicy-
clohexylurea; DFP, diisopropyl fluorophosphate; DIPEA, diisopro-
pylethylamine; DMF, dimethylformamide; DMSO, dimethyl
sulfoxide; HOBT, 1-hydroxybenzotriazole; pCMB, p-chloro-
mercuribenzoate; PMSF, phenylmethanesulfonyl fluoride; PyBOP,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophos-
phate; pyr, pyrrolidinyl; N-Suc-Gly-Pro-Leu-Gly-Pro-AMC,
N-succinyl-glycyl-prolyl-leucyl-glycyl-prolyl-7-amido-4-methylcou-
marin; TFAA, trifluoroacetic anhydride; THF, tetrahydrofuran; thia,
A third specific proteinase activity was identified and
characterized in cell-free extracts of amastigote, trypo-
mastigote and epimastigote forms of T. cruzi using the
collagenase-like peptidase fluorogenic substrate N-Suc-
Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin (-AMC)
[5]. This T. cruzi 80 kDa endopeptidase (Tc80 proteinase)
is secreted by the infective trypomastigotes. It mediates
thiazolidinyl;
glycine.
Z-Leu-Gly-OH,
N-benzyloxycarbonyl-leucyl-

258
specific degradation of purified human type I and IV
collagens, and native type I collagen hydrolysis in rat
mesentery at physiological pH. Taken together, the data
on this enzyme provide evidence that T. cruzi contains a
specific proteinase capable of degrading collagens of the
extracellular matrix of the cell being infected and suggest
that Tc80 proteinase inhibition would represent a thera-
peutic approach to the treatment of Chagas’ disease. At
the same time, the development of specific inhibitors
would serve as key tools to further investigate the role of
the proteinase in the host cell invasion process by T.
cruzi.
We reasoned that, in the absence of currently known
structural requirements concerning the Tc80 proteinase
active site, except substrate specificity, a preliminary
work aimed at inhibiting the proteinase could be achieved
with peptidyl derivatives based on the substrate recogni-
tion sequence, the C-terminus of which would have been
modified with functional groups likely to interact with the
protease active site. In this paper, we report the synthesis
and inhibitory properties (IC50 and Ki) of a series of
derivatives 1–5 against Tc80 proteinase, featuring either a
vinyl sulfone (1), a 2-ketobenzothiazole (2 and 3), a
nitrile (4) or a benzimidazole group (5) as surrogate of the
P1–P'1 scissile bond in the substrate (figure 1). As part of
this work, the Tc80 proteinase was classified as a serine
protease belonging to the prolyl oligopeptidase family
(EC 3.4.21.26) using a standard serine inhibitor, and a
limited series of mammalian prolyl oligopeptidase inhibi-
tors was studied and compared to derivatives 1–5.
2. Chemistry
Making use of the well-known properties of Weinreb
amides [6, 7], both the peptidyl vinyl sulfone 1 and
peptidyl α-ketobenzothiazole 2 could be synthesized
from the N-methyl-O-methyl hydroxamate derivative
6 [8] (figure 2). Vinyl sulfone pyrrolidine 9 was obtained
through the Wittig-Horner reaction [9] with excellent
yield from diethyl phenylsulphonyl methylphosphonate
and prolinal 8, readily avalaible by reduction of 6 with
LiAlH4 [10]. Coupling of the N-deprotected pyrrolidine 9
with Z-Leu-Gly-OH in the presence of PyBOP/DIPEA
reagents provided the desired peptidyl vinyl sulfone 1.
Compounds 2 and 3 were prepared via nucleophilic
addition of the benzothiazole anion to Weinreb amides 6
and 7 [11]. (S)-N-methoxy-N-methylcarbamoyl-thiazo-
lidine 7 was obtained from the corresponding free car-
boxylic acid and N,O-dimethylhydroxylamine using
DCC in 78% yield. Amides 6 or 7 were added to the
benzothiazole anion (2 equivalents) in THF at –78 °C.
Quenching the reaction mixture with the required amount
Figure 1. Tc80 proteinase inhibitors, based on the Leu-Gly-Pro
recognition sequence, the C-terminus of which has been repla-
ced by a functional group likely to interact reversibly or
irreversibly with the proteinase active site.
of saturated aqueous NH4Cl for neutralization and sub-
sequent work-up provided α-amino ketones 10 and 11 in
74 and 71% yield, respectively, after silica gel chroma-
tography. Amino alcohols 12 and 13 were obtained
through reduction of 10 and 11 by NaBH4 in methanol at
–20 °C [12] in 83 and 73% yield with 92 and 95%
diastereoselectivity, respectively, and used as a diastereo-
meric mixture in the following step. After removal of the
t-butyl group with 5.6 N HCl in AcOEt, the resulting
N-deprotected amino alcohols were coupled with Z-Leu-

259
Figure 2. Synthetic scheme for the preparation of peptidyl vinyl sulfone 1 and peptidyl α-ketobenzothiazoles 2 and 3: (a) LiAlH4,
ether; (b) NaH, diethyl phenylsulphonyl methylphosphonate, THF; (c) Z-Leu-Gly-OH, DIPEA, PyBOP, CH3CN; (d) benzothiazole,
BuLi, THF, –78 °C; (e) NaBH4, MeOH, –20 °C; (f) (COCl)2, DMSO, CH2Cl2, –60 °C.
Gly-OH in the presence of PyBOP/DIPEA reagents
giving the peptidyl alcohols 14 and 15 in 66 and 32%
yields, respectively. Finally, Swern oxidation of the
hydroxy function provided the expected peptidyl
α-ketobenzothiazoles 2 and 3 in 70 and 56% yields,
respectively. The 13C-NMR spectrum of compound 2
showed only chemical shifts relevant to the presence of
rotamers, indicating that essentially no epimerization
occured at the chiral carbon of the five-membered ring
during the Weinreb amide alkylation step.
The tripeptidylamide 16 was prepared as a precursor of
peptidyl nitrile 4 (figure 3), since there are several dehy-
drating methods to convert amide into nitrile under
relatively mild conditions. However, attempts to dehy-
drate amide 16 according to the Swern oxidation protocol
[13] or with Ac2O- [14] or TFAA-pyridine methods [15,
16] mostly led to recovered starting material. Fortunately,
the peptidyl nitrile 4 could be easily obtained by treat-
ment of the same peptidyl amide with SOCl2/DMF in the
presence of N-methylmorpholine [17] (figure 3).
Synthesis of the peptidyl benzimidazole 5 required
preparation of pyrrolidinyl benzimidazole 17 which was
readily available by condensation of Z-Pro-OH and
1,2-phenylenediamine with DCC/HOBT reagents in
THF/CH2Cl2 and subsequent cyclization in AcOH at
65 °C [18] (figure 3). Hydrogenolysis of compound 17
under H2 (1 atm.) with 10% Pd/C in MeOH followed by
coupling of the resulting N-deprotected pyrrolidine and
Z-Leu-Gly-OH using DCC/HOBT reagents in THF/
CH2Cl2 provided the desired peptidyl heterocycle 5 in
82% yield after silica gel chromatography.
3. Biological evaluation
Tc80 proteinase was purified from T. cruzi epimasti-
gote forms as previously described [5]. Proteolytic activ-
ity was determined using the fluorogenic substrate N-Suc-
Gly-Pro-Leu-Gly-Pro-AMC. The specific activity of the
purified Tc80 proteinase was 0.26 nmol of AMC released/

260
Figure 3. Synthetic scheme for the preparation of peptidyl nitrile 4 and peptidyl benzimidazole 5: (a) Z-Leu-Gly-OH, HOBT, DCC,
THF; (b) N-methylmorpholine, SOCl2, DMF; (c) (i) HOBT, 1,2-diaminobenzene, DCC, CH2Cl2; (ii) AcOH, 65 °C.
min per µg of protein. The IC50 value was defined as the
inhibitor concentration which causes a 50% decrease of
the activity. Data presented are the mean ± standard
deviation of at least 3 independent experiments.
oligopeptidase is representative of a new serine peptidase
family (EC 3.4.21.26). The enzymes of this family have
been reported to have an unusual inhibitor profile char-
acterized by their inactivation by DFP [22, 23] but not by
several reagents for the recognition of a serine peptidase
(3,4-DCI, PMSF) [24]. Surprisingly, they are also very
susceptible to pCMB, a cysteine-protease inhibitor and
partially inhibited by the smaller thiol reagent, iodoaceta-
mide. These data suggest the presence of a cysteine
residue at or near the active site so that a large reagent
attached to this thiol excludes the substrate from the
catalytic centre [25–28]. A previous study did not provide
conclusive evidence with regard to the Tc80 proteinase
class, whether serine- or cysteine-protease. Thus, the
enzyme was inhibited by some cysteine reagents (Z-Phe-
Ala-CHN2, pCMB) but remained insensitive to the serine
inactivator, PMSF [5]. To clarify the situation with re-
spect to Tc80 proteinase, we examined the effect of DFP
on its activity. Indeed, a strong inactivation was observed
in the presence of this reagent (IC50 value of about 60
nM). On the basis of its overall inhibition profile, the
protozoan proteinase can now be unambiguously classi-
fied as a serine enzyme belonging to the prolyl oligopep-
tidase family.
Reversibility of the inhibition was evaluated after
complete enzyme inhibition by dialysis at 4 °C for 24 h
and determination of the recovered enzyme activity. The
inhibition constants were determined by the progress
curve method in which the inhibitor is added to a
steady-state enzyme–substrate reaction [19, 20]. The in-
hibitor was at least in a 20-fold molar excess over the
enzyme. The reactions were carried out under experimen-
tal conditions where less than 5% of substrate was
hydrolysed. Under these conditions, v0/vi = 1 + [I]/Ki (app)
where v0 is the enzyme activity without inhibitor and vi
the enzyme activity in the presence of inhibitor. Ki (app)
was obtained for several concentrations of inhibitor. The
Ki values were calculated from Ki (app) by the relationship
Ki (app) = Ki (1 + [S]/Km). The Km value of N-Suc-Gly-
Pro-Leu-Gly-Pro-AMC (18.5 µM) and the type of inhi-
bition were determined graphically according to the plot
[S]/V versus [S] [21].
4. Results and discussion
Although the specific activity of the Tc80 proteinase on
the pentapeptidyl substrate N-Suc-Gly-Pro-Leu-Gly-Pro-
AMC was seven-fold higher than that on the dipeptide
N-Suc-Gly-Pro-AMC [5], we thought that the Leu-Gly-
Pro sequence would provide a sufficient potential binding
By its property to cleave peptide bonds at the carboxyl
side of proline residues, Tc80 proteinase could be classi-
fied as a prolyl oligopeptidase, previously called prolyl
endopeptidase or postproline cleaving enzyme. Prolyl

261
Table I. Inhibition of Tc80 proteinase activity by compounds 1–5,
14 and three mammalian prolyl oligopeptidase inhibitors: IC50 and
Ki values.
for the purpose of our study. Inhibitors were designed by
replacing the C terminus of the P3–P1 sequence by a
functional group, Y, likely to interact irreversibly (1, Y:
vinyl sulfone) or reversibly (2, 3, Y: 2-ketobenzothiazole;
4, Y: nitrile; 5, Y: benzimidazole) at the active site of the
enzyme (figure 1). Vinyl sulfone as a Michael acceptor
specific for cysteine proteinase was first described by Liu
and Hanzlik [29] and since developed towards several
thiol peptidases. The presumed presence of a cysteine
residue near the active site of the Tc80 proteinase and the
reported covalent modifications of the reactive cysteine
thiol- and serine hydroxy groups of the mammalian prolyl
oligopeptidase by an O-acylhydroxamate derivative and
Ac-Ala2-Pro-CHN2, respectively, both thiol protease spe-
cific inhibitors, [30, 31], prompted us to examine the
effect of a peptidyl vinyl sulfone on the Tc80 peptidase
activity. We retained nitrile and 2-ketobenzothiazole
groups for similar reasons. The peptidyl α-keto-
heterocycle concept was introduced by Edwards and
co-workers towards the serine protease elastase [32], and
subsequently applied to cysteine- and other serine-
proteases including prolyl endopeptidase [33]. Nitrile
derivatives have also been reported to exert potent
inhibitory effects towards thiol proteases and prolyl
oligopeptidase, possibly by mimicking the initial cova-
lent enzyme adduct through a thioimidate and imidate
function, respectively [34]. Lastly, we planned to inves-
tigate the effect of a benzimidazole moiety which may be
regarded as a hydrolytically stable peptide bond isostere
with both a potential hydrogen acceptor and a donor [35].
The functionalized peptides 1–5 were easily prepared
by liquid phase synthesis. Compounds 1–3 were obtained
using the Weinreb amide methodology, with alkylation of
the appropriate N-methyl-N-methoxy amide by lithiated
benzothiazole as a key step in the case of 2 and 3 [11].
The nitrile group in 4 and the benzimidazole moiety in 5
were introduced by standard methods.
Compound
IC50 (nM)
Ki (nM)
1
2
3
4
5
14
> 10 000
200 ± 27
251 ± 22
52 ± 17
> 10 000
> 10 000
6 700
35 000
219 ± 28
139 ± 64
38 ± 14
n.d.a
n.d.
Z-Pro-L-prolinal dimethyl acetal
Boc-Asn-Phe-Pro-aldehyde
Z-Pro-Pro-OH
1 120 ± 120
280 ± 110
n.d.
4 300
> 10 000
Data are the mean ± standard deviation of at least 3 independent
experiments except for the IC50 values of Z-Pro-L-prolinal di-
methyl acetal and Boc-Asn-Phe-Pro-aldehyde which are the mean
a
of 2 independent experiments. n.d.: not determined.
shown). Ki values of compounds 2, 3 and 4 were
219 ± 28, 139 ± 64 and 38 ± 14 nM, respectively
(table I). According to our data, the binding mode was
fast since no difference in potency was found with or
without preincubation of the enzyme with the inhibitors.
Nitrile derivative 4 was the most potent inhibitor of the
Tc80 proteinase activity.
The introduction of a sulfur atom in the ring of several
pyrrolidinyl derivative inhibitors of mammalian prolyl
The IC50 values for Tc80 inhibition by the compounds
synthesized are listed in table I. Peptidyl α-ketobenzo-
thiazoles 2, 3 and nitrile 4 were revealed as reversible
inhibitors since after a complete enzyme inhibition by
10 µM of each derivative, an extensive dialysis by
ultrafiltration allowed the recovery of more than half of
the initial enzymatic activity. These three derivatives
displayed competitive inhibition characteristics as deter-
mined by the plots [S]/V versus [S] [21]. Kinetics of
inhibition of Tc80 proteinase by compound 4 are depicted
in figure 4. The intercept on the [S]/V axis (Km/V)
increased with increasing concentrations of inhibitor, and
equivalent slopes (1/V) were determined, indicating a
competitive inhibition. Similar data were obtained with
the 2-ketobenzothiazole derivatives 2 and 3 (data not
Figure 4. Cornish-Bowden plots [21]: determination of the
inhibition type of the Tc80 proteinase by compound 4 at 333.3
nM ([), 111 nM (▲), 33.3 nM (■), and in the absence of 4 (●).

262
oligopeptidase has been reported to afford compounds
with remarkably increased inhibition potency but, unfor-
tunately, we did not notice a significant increase when
evaluating thiazolidinyl compound 3 compared to 2.
Compound 1 exhibited only a weak inhibition of the Tc80
peptidase, even after a lengthy preincubation (Ki =
35 000 nM). The vinyl sulfone group could be misposi-
tioned for reacting with the putative cysteine near the
active centre. While our work was in progress, an X-ray
study of the porcine prolyl oligopeptidase complexed
with a peptidyl aldehyde was reported in which this
cysteine residue was identified as Cys 255 and situated
closer to the S3- than the S1-subsite [36]. Not surpris-
ingly, hydroxy compound 14 did not inhibit Tc80 pro-
teinase activity (IC50 values > 10 µM) [37]. The absence
of inhibition activity observed wih 5 (IC50 value > 10
µM) suggests that the imidazole moiety poorly mimics
the scissile bond: the oxyanion hole of the enzyme might
not accommodate the imine nitrogen instead of C=O due
to electronic effects and steric hindrance. Inhibitory
activity of 2 and 3 compared to 5 strongly suggests that
the presence of a carbonyl α to the heterocycle is
essential for an interaction to take place in the active site.
Several mammalian prolyl oligopeptidase inhibitors are
now available, three of them, Z-Pro-L-prolinal dimethyl
acetal [38], Boc-Asn-Phe-Pro-H (unpublished data), and
Z-Pro-Pro-OH [37] were also tested against Tc80 pepti-
dase (table I). Boc-Asn-Phe-Pro-H (Ki = 280 ± 110 nM)
is the most potent inhibitor of this limited series, however
its Ki value remains 5–6-fold higher than that of 4 and
slightly higher than that of 2 and 3.
Analyses indicated by the symbols of the elements were
within ± 0.4% of theoretical values. Mass spectra were
recorded on a Jeol MS 700 apparatus. TLC analyses were
performed on thin layer analytical plates 60F264 (Merck).
Flash column chromatography was performed on silica
gel 60 (Merck).
5.1.1. 3-tert-Butyloxycarbonyl-4(S)-N-methyl-
N-methoxycarbamoyl-1,3-thiazolidine 7
To a suspension of N,O-dimethylhydroxylamine hydro-
chloride (1.844 g, 18.9 mmol) in dry CH2Cl2 (30 mL) at
5 °C under argon, was added successively, neat DIPEA
(2.44 g, 18.9 mmol), N-Boc-L-thiazolidine-4-carboxylic
acid (2.517 g, 18.9 mmol) and DCC (4.094 g, 19.8 mmol)
as solids. The mixture was stirred overnight at room
temperature, DCU was filtered off, the filtrate was con-
centrated and the residue was taken up in ether (40 mL).
The organic phase was washed successively by 1 N HCl
(2 × 10 mL), H2O (10 mL), saturated NaHCO3 and brine
before flash chromatography on silica gel (pentane;
ether/pentane, 1:3; 1:2, 4:5, 1:1): colourless oil 2.159 g
(78.2%), [α]20D –103 (c = 4.7 AcOEt); 1H-NMR (CDCl3)
δ 1.43 (s, 9H, tert-C4H9); 2.97–3.08 (m, 1H, 5-CH(thia));
3.18 (s, 3H, NCH3); 3.30–3.43 (m, 1H, 5-CH(thia)); 3.73
(bs, 1H, OCH3); 4.47–4.50 (m, 1H, 2-CHH(thia));
4.63–4.76 (m, 1H, 2-CHH(thia)), 5.07 (m, 1H,
4-CH(thia)); 13C-NMR (CDCl3) δ 26.51, 32.13, 33.09,
49.11, 59.06, 61.06, 80.47, 152.90, 170.93. Anal.
C11H20N2O4S (C, H, N).
5.1.2. 1-tert-Butyloxycarbonyl-2(S)-[phenylsulphonyl-
(E)-ethenyl]pyrrolidine 9
The three newly synthesized compounds, 2–4, likely to
act by transition state analogue adduct formation, exhibit
potent reversible and competitive inhibition of the Tc80
proteinase. Collectively, the findings presented herein are
consistent with classifying this T. cruzi peptidase as a
member of the prolyl oligopeptidase family. Our results,
together with those disclosed recently by a combinatorial
approach towards Tc80 proteinase [39, 40], may serve to
design new potent inhibitors of this enzyme. The study of
the novel synthetic inhibitors 2–4 towards the T. cruzi-
cell invasion process is under way.
To NaH (92 mg, 60% dispersion in oil, 2.3 mmol)
suspended in dry THF (3 mL), was slowly added drop-
wise a solution of diethyl phenylsulphonylmethylphos-
phonate (598 mg, 2.3 mmol) in THF (3 mL) under argon
at room temperature. When hydrogen had evolved, the
mixture was stirred for an additional 10 min and a
solution of freshly prepared Boc-L-prolinal [8] (458 mg,
2.3 mmol) in THF (3 mL) was added. After stirring for
1 h, ice cold water was added (20 mL), the mixture was
extracted with AcOEt (5 × 10 mL) and the combined
organic layers were dried (MgSO4). The crude product
was flash chromatographed on silica gel (ether/pentane,
5. Experimental protocols
1:1): 590 mg (76%) white solid, m.p. 82–83 °C; [α]20
D
–39.4 (c = 2 AcOEt); IR (KBr): 1 683, 1 478, 1 404,
5.1. Chemistry
1
1 367, 1 309 cm–1; H-NMR δ 1.15 (s, 9H, tert-C4H9),
Melting points are uncorrected. IR spectra were re-
1.74–1.90 (m, 3H, 3-CH2(pyr), 4-CH(pyr)), 2.00–2.18
(m, 1H, 3-CH(pyr)), 3.28–3.46 (m, 2H, 5-CH2(pyr)),
4.28–4.41 (m, 1H, 2-CH(pyr)), 6.27 (d, J = 14.0 Hz,
C=CHSO2), 6.80 (dd, J = 5.4, 15.0 Hz, CH=CSO2),
7.44–7.65 (m, 3Harom), 7.86 (d, J = 7.2 Hz; 2Harom); MS
1
corded in the FT-mode. H- and 13C-NMR spectra were
recorded at 300 and 75 MHz, respectively, and only
chemical (δ) shifts of the major rotamers are reported
(ppm relative to solvent signal as the internal reference).

263
m/z = 281 (MH+ –C4H9), 205 (M+ –C4H9O), 205, 170,
149, 114, 95, 71, 58, 42. Anal. C17H23NO4S (C, H, N).
tained for 40 min during which the temperature was
allowed to warm to –10 °C and the mixture was quenched
with water and quickly extracted with ether (8 × 4 mL).
After washing with brine (2 × 4 mL) and drying
(MgSO4), the title compound was obtained as an amor-
phous solid, 140 mg (83%), m.p. 132–133 °C, and used
5.1.3. 2-[Benzo[d][1,3]thiazol-2-yl-carbonyl]-
1-tert-butyloxycarbonyl-(2S)pyrrolidine 10
To benzothiazole (812 mg, 6 mmol) in dry THF (7 mL)
at –78 °C under argon, was added dropwise BuLi (1.6 M,
6 mmol). The mixture was stirred for 10 min after the end
of addition and a solution of N-methoxy-N-methyl-Nα-
Boc-L-prolinamide [8] (520 mg, 2 mmol) in THF (2 mL)
was added dropwise, maintaining the temperature at
–78 °C. After stirring for an additional 30 min at the same
temperature, the reaction mixture was quenched with
saturated aqueous NH4Cl (20 mL) and allowed to warm
to 0 °C before partitioning with AcOEt (20 mL). The
organic layer was quickly washed with chilled 1 N HCl
(3 × 6 mL), water (3 mL), saturated NaHCO3 (3 × 3 mL)
and dried (MgSO4). Purification by flash chromatography
on silica gel (cyclohexane/AcOEt, 98:2; 96:4) yielded the
expected compound, 557 mg (84%), as a yellowish solid,
1
for the next reaction without further purification. H-
NMR (CDCl3) δ 1.50 (s, 9H, tert-C4H9), 1.54–2.24 (m,
4H, 3-CH2(pyr) and 4-CH2(pyr)), 3.40–3.53 (m, 2H,
5-CH2(pyr)), 4.19 (m, 1H, 2-CH(pyr)), 4.97 (m, 1H,
CHOH), 6.60 (bs, 1H, CHOH), 7.36–7.45 (m, 2Harom),
7.87–7.98 (m, 2Harom).
5.1.6. 4-[Benzo[d][1,3]thiazol-2-yl-hydroxymethyl]-
3-tert-butyloxycarbonyl-1,3-(4S)-thiazolidine 13
Compound 11 (350 mg, 1 mmol) was submitted to the
conditions described in the preparation of 12. Flash
chromatography (cyclohexane; AcOEt/cyclohexane,
10:90; 1:6): 256 mg (73%) as a yellowish oil; [α]20
D
–25.6 (c = 5.6 AcOEt); 1H-NMR (CDCl3) δ 1.46 (s, 9H,
tert-C4H9), 3.04–3.15 (m, 1H, 5-CHH(thia)), 3.32–3.46
(m, 1H, 5-CHH(thia)), 4.31 (d, J = 9.2 Hz, 1H, CHOH),
4.58–4.69 (m, 2H, 2-CH2(thia)), 5.20 (m, 1H,
4-CH(thia)), 6.32 (bs, 1H, CHOH), 7.33–7.47 (m,
2Harom), 7.88 (m, 1Harom), 7.96 (m, 1Harom); 13C-NMR
(CDCl3) δ 26.42, 27.68, 49.03, 64.01, 72.92, 80.71,
121.27, 122.42, 124.54, 125.49, 134.45, 152.40, 152.59,
173.91; MS (DCI) m/z = 353 (MH+ ). DCI-HRMS calcd.
for C16H21N2O3S2, 353.0994; found, 353.0996.
m.p. 81–84 °C; [α]20 –24.5 (c = 2.1 MeOH); IR (KBr)
D
1
1 705, 1 558, 1 485 cm–1; H-NMR (CDCl3) δ 1.17 (s,
9H, tert-C4H9) 1.89 (m, 2H, 4-CH2(pyr)), 1.99–2.40 (m,
2H, 3-CH2(pyr)), 3.53 (m, 2H, 5-CH2(pyr)), 5.54 (dd, J =
4.9, 7.9 Hz, 1H, 2-CH(pyr)), 7.48 (m, 2Harom), 7.90 (m,
1Harom), 8.10 (m, 1Harom); MS m/z = 332 (M+ ), 259,
232, 170, 166, 152, 135, 114. HRMS calcd. for
C17H20N2O3S, 332.11946; found, 332.11945.
5.1.4. 4-[Benzo[d][1,3]thiazol-2-yl-carbonyl]-
3-tert-butyloxycarbonyl-1,3-(4S)-thiazolidine 11
5.1.7. 2-[Benzo[d][1,3]thiazol-2-yl-hydroxymethyl]-1-(N-
benzyloxycarbonyl-leucyl-glycyl)-(2S)-pyrrolidine 14
Compound 12 (150 mg, 0.45 mmol) was treated by
HCl/AcOEt (5.6 N, 2 mL) for 10 min at 4 °C. The
hydrochloride obtained after evaporation of the solvent
under vacuum was taken up in CH3CN (3.5 mL) to which
were added successively, Z-Leu-Gly-OH (153 mg,
0.47 mmol) as a solid, neat DIPEA (241 µL, 1.41 mmol)
and PyBOP (234 mg, 0.45 mmol) as a solid at 4 °C under
argon. After stirring for 1 h, the mixture was concentrated
under reduced pressure, taken up by AcOEt (10 mL),
washed with 1 N HCl (3 × 2 mL), water (2 mL), saturated
NaHCO3 (2 mL) and dried (MgSO4). Flash chromatog-
raphy on silica gel (CH2Cl2; CH2Cl2/MeOH, 99.5:0.5;
This compound was obtained as described for the
preparation of 10 except starting from N-methyl-N-
methoxy amide 7 (1.38 g, 5 mmol) instead of N-methoxy-
N-methyl-Nα-Boc-L-prolinamide. Flash chromatography
on silica gel (cyclohexane; AcOEt/cyclohexane 0.5:99.5;
1:99; 1.25:98.75; 1.5:98.5; 2:98): yellowish oil, 1.755 g,
(71.6%); [α]20 –35.5 (c = 5.8 AcOEt); IR (KBr) 3 407,
1 702, 1 663 cDm–1; 1H-NMR (CDCl3) δ 1.26 (s, 9H,
tert-C4H9), 3.33 (dd, J = 4.8, 12.1 Hz, 1H, 5-CHH(thia)),
3.61–3.69 (m, 1H, 5-CHH(thia)), 4.59–4.78 (m, 2H,
2-CH2(thia)), 5.83 (m, 1H, 4-CH(thia)), 7.5–7.62 (m,
2Harom), 7.96 (m, 1Harom), 8.14 (m, 1Harom); 13C-NMR
(CDCl3) δ 27.72, 34.71, 49.61, 64.08, 80.73, 122.11,
125.21, 126.91, 127.78, 136.73, 152.60, 153.05, 163.48,
190.40; MS (DCI) m/z = 351 (MH+ ). DCI-HRMS calcd.
for C16H19N2O3S2, 351.0837; found, 351.0837.
99:1; 98.5:1.5): 160 mg (66%) as an amorphous solid,
1
m.p. 79–81 °C; [α]20 –50.8 (c = 2 AcOEt); H-NMR
D
(CDCl3) δ 0.89 (d, J = 5.7 Hz, 6H, C(CH3)2), 1.44–2.18
(m, 7H, â-CH2(Leu), γ-CH(Leu), 3-CH2(pyr),
4-CH2(pyr)), 3.06–3.32 (m, 2H, 5-CH2(pyr)), 3.95–4.17
(m, 2H, CH2(Gly)), 4.47 (m, 2H, α-CH(Leu),
2-CH(pyr)), 4.99–5.10 (m, 2H, CH2Ar), 5.28 (m, 1H,
CHOH), 5.76 (bs, 1H, CHOH), 5.97 (d, J = 8.4 Hz, 1H,
5.1.5. 2-[Benzo[d][1,3]thiazol-2-yl-hydroxymethyl]-
1-tert-butyloxycarbonyl-(2S)-pyrrolidine 12
A solution of compound 10 (166 mg, 0.5 mmol) in dry
MeOH (4 mL) cooled to –20 °C under argon was treated
with NaBH4 (20 mg, 0.55 mmol). Stirring was main-

264
NH), 6.15 (d, J = 3.8 Hz, 1H, NH), 7.21–7.40 (m,
7Harom), 7.80–7.92 (m, 2Harom).
N-(2-aminophenyl)-Nα-Z-prolinamide which was treated
by AcOH at 65 °C for 90 min. After evaporation of the
solvent under reduced pressure, the residue was taken up
by AcOEt and washed with saturated NaHCO3. Flash
chromatography on silica gel (CH2Cl2; CH2Cl2/MeOH,
99.5:0.5) yielded the title compound as a white solid,
5.1.8. 4-[Benzo[d][1,3]thiazol-2-yl-hydroxymethyl]-
3-(N-benzyloxycarbonyl-leucyl-glycyl)-1,3-
(4S)-thiazolidine 15
397 mg (61%), m.p. 93–94 °C; [α]20 –109.2 (c = 2
Compound 13 (146 mg, 0.42 mmol) was submitted to
the conditions described in the preparation of 14. Flash
chromatography on silica gel (CH2Cl2; CH2Cl2/MeOH,
99.6:0.4; 99.4:0.6; 99.2:0.8): 76 mg (32%) as an amor-
D
AcOEt); 1H-NMR (CDCl3) δ 1.54–2.29 (m, 4H,
3-CH2(pyr), 4-CH2(pyr)), 3.04 (m, 1H, 2-CH(pyr)),
3.47–3.60 (m, 2H, 5-CH2(pyr)), 5.12–5.25 (m, 2H,
CH2Ar), 7.14–7.33 (m, 8Harom), 7.70 (bs, 1H, NH). Anal.
C19H19N3O2.0.5H2O (C: calcd 69.07 found 69.67, H, N).
phous solid, m.p. 81–83 °C; [α]20 –33.7 (c = 1.1
D
1
AcOEt); H-NMR (CDCl3) δ 0.90 (d, J = 5.2 Hz, 6H,
C(CH3)2), 1.48–1.69 (m, 3H, â-CH2(Leu), γ-CH(Leu)),
2.99–3.49 (m, 2H, 5-CH2(thia)), 4.02–4.86 (m, 7H,
CH2(Gly), α-CH(Leu), 2-CH2(thia) and 4-CH(thia)),
5.01-5.10 (m, 2H, CH2Ar), 5.31 (m, 1H, CHOH), 5.64
(bs, 1H, CHOH), 5.68 (bs, 1H, NH), 5.95 (bs, 1H, NH),
7.26–7.48 (m, 7Harom), 7.84–7.97 (m, 2Harom); MS (DCI)
m/z = 557 (MH+ ). DCI-HRMS calcd. for C27H33N4O5S2,
557.1892; found, 557.1893.
5.1.11. 1-(N-Benzyloxycarbonyl-leucyl-glycyl)-2(S)-
[phenylsulphonyl-(E)-ethenyl]pyrrolidine 1
Obtained according to the procedure described for the
preparation of 14 except starting from compound 9
(168 mg, 0.5 mmol) instead of 12. Flash chromatography
on silica gel (AcOEt/cyclohexane, 97:3) yielded the title
compound 1 as an amorphous solid, 100 mg (40%), m.p.
62–63 °C; [α]20 –85.1 (c = 1 MeOH); 1H-NMR
D
5.1.9. Nα-Benzyloxycarbonyl-leucyl-glycyl-prolinamide 16
To a suspension of L-prolinamide (228 mg, 2 mmol) in
dry THF (4 mL) at 4 °C under argon, were successively
added, neat Z-Leu-Gly-OH (644 mg, 2 mmol), neat
HOBT (540 mg, 4 mmol) and a solution of DCC
(434 mg, 2.1 mmol) in THF (2 mL). After being stirred
for 1 h at 4 °C, the whole was concentrated under
vacuum, the residue was taken up with CH2Cl2 (15 mL)
and DCU was filtered off. The filtrate was washed
successively with 1 N HCl (3 × 4 mL), water (4 mL),
saturated NaHCO3 (3 × 4 mL) and brine (4 mL), dried
(MgSO4) and flash chromatographed on silica gel
(CH2Cl2; CH2Cl2/MeOH, 99:1; 98:2; 97:3): 614 mg
(73%) as a white solid, m.p. 87–89 °C; [α]20D –68.7 (c =
2, CH2Cl2); 1H-NMR (CDCl3) δ 0.87 (d, J = 5.9 Hz, 6H,
C(CH3)2), 1.49–2.14 (m, 7H, â-CH2(Leu), γ-CH(Leu),
â-CH2(Pro), γ-CH2(Pro)), 3.34–3.58 (m, 2H,
δ-CH2(Pro)), 4.00 (d, J = 3.7 Hz, 2H, CH2(Gly)),
4.31–4.42, (m, 2H, α-CH(Leu), α-CH(Pro)), 4.99–5.10
(m, 2H, CH2Ar), 5.78 (d, J = 8.0 Hz, 1H, NH(Leu)), 6.18
(bs, 1H, NH), 6.84 (bs, 1H, NH), 7.28 (s, 5Harom), 7.64
(bs, 1H, NH). Anal. C21H30N4O5.0.5H2O (C, H, N).
(DMSO-d6) δ 0.84 (d, J = 5.7 Hz, 6H, C(CH3)2),
1.41–1.47 (m, 1H, γ-CH(Leu)), 1.56–2.01 (m, 6H,
â-CH2(Leu), 3-CH2(pyr), 4-CH2(pyr)), 3.31–3.59 (m,
2H, 5-CH2(pyr)), 3.77–3.98 (m, 2H, CH2(Gly)),
4.03–4.25 (m, 1H, α-CH(Leu)), 4.63–4.68 (m, 1H,
2-CH(pyr), 5.00 (s, 2H, CH2Ar), 6.73 (d, J = 15 Hz,
C=CHSO2), 6.85 (dd, J = 4.5, 15 Hz, 1H, CH=CSO2),
6.90 (bs, 1H, NH), 7.42–7.94 (m, 6H, 5Harom, NH).
FAB-HRMS calcd. for C28H36O6N3S, 542.2325; found,
542.2310.
5.1.12. 2-[Benzo[d][1,3]thiazol-2-yl-carbonyl]-1-
(benzyloxycarbonyl-leucyl-glycyl)-(2S)pyrrolidine 2
To a solution of oxalyl chloride (10 µL, 0.12 mmol) in
CH2Cl2 (300 µL) at –60 °C under argon, was added a
solution of dry DMSO (18 µL, 0.24 mmol) in CH2Cl2
(36 µL). Stirring was maintained for 10 min, then a
solution of compound 14 (22 mg, 0.04 mmol) in CH2Cl2
(100 µL), was added and the mixture was stirred for a
further 30 min before treatment with triethylamine
(46 µL, 0.33 mmol). The resulting slurry was quickly
warmed to 0 °C, stirred for 4 h at that temperature before
being taken up in AcOEt (10 mL), washed with brine and
dried. Purification on silica-gel thin layer chromatogra-
phy (CH2Cl2/MeOH, 99:1) yielded the title compound as
5.1.10. 2-Benzimidazol-2-yl-1-benzyloxycarbonyl-
(2S)-pyrrolidine 17
To Z-Pro-OH (498 mg, 2 mmol) in CH2Cl2 (6 mL)
were added successively, a solution of HOBT (604 mg,
4 mmol) in THF (6 mL), neat 1,2-phenylenediamine
(216 mg, 2 mmol) and a solution of DCC (452 mg,
2.2 mmol) in CH2Cl2 (5 mL). After being stirred for 4h,
the whole was filtered and the crude product was purified
through a silica pad (CH2Cl2/MeOH, 99.5:0.5), providing
an amorphous solid, 15 mg (70%), m.p. 73–74 °C; [α]20
–55.7 (c = 1.5 MeOH); H-NMR (CDCl3) δ 0.87 (d J =
D
1
5.8 Hz, 6H, C(CH3)2), 1.51–1.76 (m, 3H, â-CH2(Leu),
γ-CH(Leu)), 2.06–2.51 (m, 4H, 3-CH2(pyr), 4-CH2(pyr)),
3.54–3.71 (m, 2H, 5-CH2(pyr)), 4.00–4.22 (m, 4H,
CH2(Gly), α-CH(Leu), NH), 5.00–5.10 (m, 2H, CH2Ar),

265
5.22 (d J = 8.2 Hz, 1H, NH), 5.82 (d, J = 3.9, 8.6 Hz, 1H,
2-CH(Pyr)), 6.89 (bs, 1H, NH), 7.25–7.30 (m, 5Harom),
7.49–7.60 (m, 2Harom), 7.95–8.00 (m, 1Harom), 8.15–8.20
(m, 1Harom); 13C-NMR (CDCl3) δ 21.71, 22.90, 24.66,
29.11, 41.90, 41.96, 46.14, 53.54, 61.82, 66.96, 122.40,
125.64, 127.06, 127.91, 128.01, 128.04, 136.21, 137.29,
153.53, 156.01, 164.11, 166.39, 172.20, 191.78. FAB-
HRMS calcd. for C28H33N4O5S, 537.2172; found,
537.2181.
5.1.15. 2-Benzimidazol-2-yl-1-(N-benzyloxycarbonyl-
leucyl-glycyl)-(2S)- pyrrolidine 5
Compound 17 (76 mg, 0.24 mmol) was hydrogeno-
lysed for 2 h under H2 (1 atm.) using 10% Pd/C in
MeOH. After filtration and evaporation to dryness, the
free amine was treated for coupling with Z-Leu-Gly-OH
using the procedure described for the preparation of 9.
Flash chromatography on silica gel (CH2Cl2; CH2Cl2/
MeOH, 99:1; 98:2; 97:3; 96:4) provided the title com-
pound 5 as an amorphous solid, 96 mg (82%); m.p.
1
103–105 °C; [α]20 –120.8 (c = 2 AcOEt); H-NMR δ
0.91 (d, J = 5.8 HzD, 6H, C(CH3)2), 1.47–2.40 (m, 7H,
â-CH2(Leu), γ-CH(Leu), 3-CH2(pyr), 4-CH2(pyr)), 2.97
(m, 1H, 2-CH(pyr)), 3.42–3.54 (m, 2H, 5-CH2(pyr)),
3.82–4.04 (m, 2H, CH2(Gly)), 4.30 (m, 1H, α-CH(Leu)),
5.10 (s, 2H, CH2Ar), 5.27 (d, J = 6.8 Hz, 1H, NH), 5.37
(d, J = 8.2 Hz, 1H, NH), 7.18–7.31 (m, 9 Harom), 7.53 bs,
1H, NH). FAB-HRMS calcd. for C27H34N5O4, 492.2611;
found, 492.2586.
5.1.13. 4-[Benzo[d][1,3]thiazol-2-yl-carbonyl]-3-
(benzyloxycarbonyl-leucyl-glycyl)-1,3-(4S)-
thiazolidine 3
Compound 15 (27 mg, 0.048 mmol) was submitted to
the conditions described in the preparation of 2. Purifi-
cation on silica-gel thin layer chromatography (CH2Cl2/
MeOH, 99:1): 9 mg (34%) whitish solid, m.p. 73–75 °C;
1
[α]20 –38.6 (c = 0.9 MeOH); H-NMR (CDCl3) δ 0.89
(d, JD= 5.2 Hz, 6H, C (CH3)2), 1.45–1.70 (m, 3H,
â-CH2(Leu),
γ-CH(Leu)),
3.35–3.43
(m,
1H,
5.2. Biological evaluation
5-CHH(thia)), 3.61–3.76 (m, 1H, 5-CHH(thia)),
4.05–4.32 (m, 1H, α-CH(Leu)), 4.64–4.70 (m,
CH2(Gly)), 4.90–5.18 (m, 4H, CH2Ar, 2-CH2(thia)), 6.18
(m, 1H, 4-CH(thia)), 6.83 (bs, 1H, NH), 7.30 (m, 5Harom),
7.51–7.61 (m, 2Harom), 7.97–8.19 (m, 2Harom); MS (DCI)
m/z = 555 (MH+ ). DCI-HRMS calcd. for C27H31N4O5S2,
555.1736; found, 555.1735.
Z-Pro-L-Prolinal-dimethylacetal, Boc-Asn-Phe-Pro al-
dehyde and Z-Pro-Pro-OH were purchased from Bachem.
The fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-
AMC was from Sigma and DFP from ICN. Fluorescence
was measured in a Hitachi 2000 spectrofluorimeter at
excitation and emission wavelengths of 380 and 440 nm,
respectively.
5.1.14. 1-(N-Benzyloxycarbonyl-leucyl-
glycyl)-2-cyanopyrrolidine 4
5.2.1. Enzyme assay and determination of IC50 values
To tripeptide amide 16 (126 mg, 0.3 mmol) in dry
DMF (0.4 mL) at 4 °C, were added successively,
N-methylmorpholine (92 µL, 2.8 eq.) and SOCl2 (44 µL,
2 eq.). The mixture was stirred for 2h at room tempera-
ture and poured onto a chilled saturated NaHCO3 solution
(10 mL). After extraction with AcOEt (4 × 2 mL), the
combined organic phases were washed with 1 N HCl
(4 × 2 mL), water (2 mL), brine (2 mL) and dried
(MgSO4). Purification by flash chromatography (CH2Cl2;
CH2Cl2/MeOH, 99.5:0.5) yielded the expected peptidyl
nitrile 4 as an amorphous solid, 69 mg, (57%), m.p.
Enzymatic reactions were carried out in 25 mM Tris-
HCl, pH 7.5, 125 mM NaCl, at 24 °C. Substrate and
inhibitor stock solutions were prepared in DMSO, stored
at –20 °C and diluted in the reaction buffer just prior to
assays. IC50 values were determined after pre-incubation
for 10 min with 30 µL of purified enzyme (1–5 nM
solution in the reaction), with 30 µL of inhibitor solution
at a concentration three times the final concentration
desired and then by addition of 30 µL of a 100 µM
substrate solution. The reaction was stopped 15 min later
by addition of ethanol (1 mL) and the fluorescence
measured. The final inhibitor concentrations used ranged
from 1 nM to 100 µM. The percentage of inhibition was
calculated by comparison with the fluorescence measured
in samples containing reaction buffer instead of inhibitor.
The IC50 value was estimated from the inhibition versus
inhibitor concentration curve. Data presented are the
mean ± standard deviation of at least three independent
experiments.
57–59 °C; [α]20 –82.9 (c = 2.6 CH2Cl2); 1H-NMR
D
(CDCl3) δ 0.89 (d, J = 5.9 Hz, 6H, C(CH3)2), 1.48–1.65
(m, 3H, â-CH2(Leu), δ-CH-(Leu)), 2.01–2.24 (m, 4H,
3-CH2(pyr), 4-CH2(pyr)), 3.35–3.56 (m, 2H, 5-CH2(pyr)),
3.90–4.10 (m, 2H, CH2(Gly)), 4.33 (m, 1H, α-CH(Leu)),
4.70 (m, 1H, 2-CH(pyr)), 5.00–5.10 (m, 2H, CH2Ar), 5.68
(d, J = 8.1 Hz, 1H, NH(Leu)), 7.29 (s, 6H, 5Harom, NH).
FAB-HRMS calcd. for C21H29N4O4 401.2189; found
401.2227.

266
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Hydrolyses were standardized using a 0.05–0.5 µM
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300 µL of substrate prepared in the same buffer, before
adding 3 µL of the inhibitor solution or the equivalent
amount of DMSO. The hydrolysis kinetics were recorded
up to 45 min and slopes expressed in nmol of AMC
released per min using standard curves. The final sub-
strate concentrations used ranged from 3.3–166 µM, and
the inhibitor concentrations from 20 nM to 1 µM.
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30 µL of inhibitor (20 µM) were incubated with 30 µL
of purified enzyme for 2 h at 37 °C. The sample was then
dialysed by ultrafiltration through five successive steps of
dilution in 2 mL reaction buffer, and of concentration
using an Amicon unit (cut off 30 kDa). After a last
concentration to 100 µL, the enzymatic activity was
measured and compared to that measured in samples
incubated without inhibitor and processed in a same way.
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Acknowledgements
This work was partially supported by the PFRMMIP
program.
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