hrs while the temperature was maintained at less than 10°C.
The reaction mixture was diluted with ethyl acetate and
washed with 0.1 M HCl, saturated sodium bicarbonate solu-
tion and brine successively. The organic layer was dried over
sodium sulfate and purified by silica gel column chromatog-
raphy using hexanes and EtOAc in a 95:5 ratio, yielding a
white solid (Rf = 0.44, Hexanes/EtOAc (80:20), 46.3% yield).
The characterization was found to be consistent with the pre-
viously reported NMR spectra.108
Lys(Dnp)-D-Arg-NH2 (BML-P145), Z-Val-Val-Arg-AMC
(BML-P199).
(BAPNA)
N-alpha-Benzoyl-DL-arginine-p-nitroanilide
(B4875), trypsin (T1426), para-
nitrophenylphosphate were purchased from Sigma–Aldrich
(St. Louis, MO, USA). All the biological grade buffers were
used and were purchased from Sigma–Aldrich (St. Louis, MO,
USA). All enzyme kinetics experiments (unless otherwise
stated) were carried out at 30°C in appropriate buffer condi-
tion with 5% DMSO concentration. For measuring the initial
rates of enzyme catalyzed reaction, a temperature-controlled
steady-state arc lamp fluorometer equipped with Felix 32
software (Photon Technology Instrument, Birmingham, NJ,
USA) and a UV–Vis spectrophotometer (Modelk25; Perkin
Elmer Inc., Waltham, MA, USA) was used.
(iii) (((4-ethynylbenzyl)oxy)carbonyl)-L-phenylalanine (8):
Subsequent deprotection to the carboxylic was carried out by
following the literature reported protocol and its characteriza-
tion was found to be consistent with the previously reported
4.2.2 Determination of Second Order Inactivation
Rate Constant of KDP-1 Reactivity with Human Cathepsin
L and its Selectivity Profile: A previously reported cathepsin
L assay procedure was adopted with suitable modifications.86
Briefly, recombinant human liver cathepsin L (Enzo Life Sciences,
USA) was first activated by incubation in sodium acetate buffer
(50mM, pH 5.5) for 30 minutes at 30 °C. The inactivation reaction
at pH 5.5 was then continuously monitored for 3 minutes under
pseudo first order condition ([I]>>[E]; Net [E] 160 pM; [I] 5
nM – 100 nM) in a fluorescence (excitation:365 nm; emission:
440 nm) quartz cuvette containing Z-FR-AMC substrate (net 5
μM) maintained at 30°C. The progress curves thus obtained at
various KDP-1 concentration were fitted using a non-linear
regression method to the following equation:110
(iv) Ethyl (E)-3-amino-5-methylhex-1-ene-1-sulfonate: This
compound was synthesized by following a literature reported
(v)
Ethyl
(S,E)-3-((S)-2-((((4-
ethynylbenzyl)oxy)carbonyl)amino)-3-phenylpropanamido)-5-
methylhex-1-ene-1-sulfonate (9): This compound was synthe-
sized by following an analogous
literature reported
protocol.109 1H NMR (400 MHz CDCl3) δ ppm: 0.87 (m, 6H),
1.31 (m, 2H), 1.38 (t, J=7.0 Hz, 3H), 1.50 (m, 1H), 3.02 (dd,
J= 13.5 and 8.1 Hz, 1H), 3.12 (dd, J= 13.5 and 8.1 Hz, 1H),
3.09 (s, 1H), 4.14 (m, 2H), 4.36 (m, 1H), 4.62 (m, 1H), 5.08
(m, 2H), 5.33 (b, 1H), 5.75 (b, 1H), 5.94 (d, J=15.2 Hz, 1H),
6.60 (dd, J= 15.2 and 5.1 Hz, 1H), 7.18 (d, J=7.0 Hz, 2H),
7.30 (m, 5H), 7.48 (d, J=8.2 Hz, 2H). 13C NMR (100 MHz
CDCl3) δ ppm: 14.9, 21.8, 22.6, 24.6, 38.5, 42.7, 48.1, 56.6,
66.7, 66.9, 77.8, 83.2, 122.2, 124.6, 127.6, 127.9, 129.1,
129.2, 132.4, 135.9, 136.6, 147.6, 155.9, 170.4.
P = vz [1- exp(-kobs* t)] / kobs
where [P] is the product, vz is the initial velocity (at time zero),
and kobs is the pseudo-first order rate constant.
(vi)
4-bromophenyl
(S,E)-3-((S)-2-((((4-
The pseudo-first order rate constants kobs thus obtained from
the fit above were plotted against corresponding inhibitor con-
centrations [I]. The 2nd order enzyme inactivation rate con-
stant, kinact, was calculated (Figure S1, Supporting Infor-
mation) from the slope of the plot according to following
ethynylbenzyl)oxy)carbonyl)amino)-3-phenylpropanamido)-5-
methylhex-1-ene-1-sulfonate (KDP-1): This compound was
synthesized by following an analogous literature reported
protocol.109 1H NMR (400 MHz CDCl3) δ ppm: 0.82 (m, 6H),
1.22 (m, 2H), 1.41 (m, 1H), 2.99 (dd, J= 13.5 and 8.4 Hz, 1H),
3.10 (dd, J= 13.5 and 8.4 Hz, 1H), 3.10 (s, 1H), 4.37 (m, 1H),
4.53 (m, 1H), 5.06 (m, 2H), 5.44 (d, J=7.1 Hz, 1H), 5.89 (d,
J=5.2 Hz, 1H), 6.04 (d, J=15.1 Hz, 1H), 6.47 (dd, J= 15.1 and
5.3 Hz, 1H), 7.08 (m, 2H), 7.13 (m, 2H), 7.28 (m, 5H), 7.49
(m, 4H). 13C NMR (100 MHz CDCl3) δ ppm: 21.8, 22.5, 24.6,
38.4, 42.3, 48.2, 56.6, 66.7, 77.8, 83.2, 120.8, 122.2, 123.8,
124.4, 127.6, 127.9, 129.1, 129.1, 132.3, 132.9, 135.8, 136.5,
148.3, 149.9, 155.9, 170.5. HRMS (m/z): [M+H]+ for molecu-
lar formula C32H33BrN2O6S: calculated 653.1315; found
653.1318.
kobs = kinact [I] / [1+ ([S]/KM)]
where kobs is the pseudo-first order rate constant, [I] is the cor-
responding inhibitor concentration, [S] is the substrate concen-
tration with a given KM. KaleidaGraph (version 3.52) was used
for plotting and analyzing the data. All assays involving se-
lectivity profile of various enzymes were performed fol-
4.3 Tandem Mass-spectrometric Analysis of Hu-
man Cathepsin L Labelled with KDP-1:
4.2 Steady State Inactivation Kinetics:
4.2.1 General: The following enzymes and their
substrates were purchased from Enzo Life Sciences (Farming-
dale, NY, USA): cathepsin L (BML-SE201), cathepsin K
(BML-SE553), cathepsin B (BML-SE198), cathepsin D
A 10 µg KDP1-reacted and 5 µg unreacted human
Cathepsin L samples were run on a 4-12% % SDS-PAGE gel
and stained with colloidal coommassie blue (Thermo Scien-
tific). Bands were excised manually, and digested by sequenc-
ing grade chymotrypsin (Promega) at 27 °C for 6h. Chymo-
trypsin was added at the beginning of the digestion to a final
substrate-to-enzyme ratio of ~30:1 in a buffer containing 100
mM Tris, 10 mM CaCl2, pH 8.0 and supplemented at a sub-
strate-to-enzyme ration of ~60:1 halfway through the reaction.
After digestion, samples were prepared as previously de-
(BML-SE199), cathepsin
S (BML-SE453), cathepsin H
(BML-SE200), cathepsin G (BML-SE283), human PTP1B
(BML-SE332), Z-Arg-Arg-pNA (BML-P138), Z-Gly-Pro-
Arg-AMC (BML-P142), Z-Phe-Arg-AMC (BML-P139), H-
Arg-AMC.2HCl (BML-P135), Suc-Ala-Ala-Pro-Phe-pNA
(BML-P141),
Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-