F. P. J. T. Rutjes et al.
General procedure for the enzymatic reactions: Enzymatic acyl-
transfer reactions were performed at 258C in a total volume of
375 mL containing HEPES buffer (0.2m, pH 8.0), NaCl (0.2m), CaCl2
(20 mm), 10% DMF and p-toluenesulfonic acid (pTSA; 2 mm) as an
internal standard. Stock solutions of Z-XAA-OGp esters (50 mm) in
DMF and nucleophiles (30 mm) in buffer were prepared. The final
concentrations of acyl donor and acyl acceptor were 2 and 15 mm,
respectively. The latter was calculated as the free, Na-unprotonated
nucleophile concentration [HN]0 according to the Henderson–Has-
selbalch Equation:
Experimental Section
Synthesis: see the Supporting Information for a detailed descrip-
tion of the synthetic procedures and product characterization.
p-[N’,N’’-Di(Boc)guanidino]phenol (7): N,N’-Di(Boc)-S-methyliso-
thiourea (2.90 g, 10.0 mmol, 1 equiv) and p-aminophenol (1.64 g,
15.0 mmol, 1.5 equiv) were dissolved in dry THF (60 mL), and this
mixture was cooled to 08C before HgCl2 (2.99 g, 11.0 mmol,
1.1 equiv) was added. After the mixture had been stirred for
20 min under argon, its temperature was raised to 258C, and it
was stirred for 20 h. The white precipitate that was formed during
the reaction was filtered off, and the filtrate was concentrated
under reduced pressure. Recrystallization from methanol yielded
1.53 g (43%) of the pure product. The mother liquor was then
evaporated to dryness, and the remaining solid was purified by
column chromatography (EtOAc/heptane, 1:9!1:2) to afford 7
(753 mg, 65% yield) as an off-white solid. Rf =0.37 (EtOAc/heptane
1:2); m.p. decomp. >2408C; 1H NMR (CDCl3, 300 MHz): d=11.61
(s, NH), 10.04 (s, NH), 7.14 (d, J=8.6 Hz, 2H), 6.68 (d, J=8.3 Hz,
2H), 6.22 (brs, OH), 1.54 (s, 9H), 1.46 (s, 9H); 13C NMR (CDCl3,
75 MHz): d=163.2, 155.9, 155.3, 153.2, 127.0, 126.3, 116.1, 83.7,
79.8, 28.1; IR (film): n˜ =3264, 2979, 2737, 1720, 1647, 1517, 1409,
1227, 1152, 1112, 1059 cmꢀ1; HRMS (ESI) m/z calcd for C17H26N3O5:
352.1873 [M+H]+, found: 352.1875.
½HNꢁ0 ¼ ½Nꢁ0=ð1 þ 10pKꢀpH
Þ
Papain (4 mg) was activated before use by adding dithiothreitol
(0.6 mg) and phosphate buffer (1 mL, 0.1m, pH 6.5) containing
EDTA (2.5 mm) and shaking the mixture for 10 min at 258C. This so-
lution was stored on ice and was freshly prepared daily. Following
thermal equilibration of assay mixtures, the enzymatic reactions
were started by the addition of papain at a final concentration of
3.5 mm. Blank reactions with Milli-Q instead of papain were run in
parallel. From this control experiment, the spontaneous ester hy-
drolysis could be determined, as well as nonenzymatic aminolysis
of the acyl donor esters; the latter could be ruled out. At regular
intervals, aliquots (20 mL) were withdrawn and quenched with
glacial acetic acid (20 mL). The reactions were monitored for 3 h by
HPLC and checked once more for changes in reaction mixture
composition after 24 h. The values reported are the average of at
least two separate experiments. The identity of the peptide prod-
ucts was established by chemical synthesis of reference com-
pounds and LC-MS.
General procedure for the DCC coupling of Cbz-protected
amino acids with phenol 7 as illustrated by the synthesis of Na-
Cbz-glycine p-[N’,N’’-di(Boc)guanidino]phenyl ester (8G): DCC
(867 mg, 4.21 mmol, 1.4 equiv) was added slowly to a cooled (08C)
solution of Z-Gly-OH (879 mg, 4.20 mmol, 1.4 equiv), p-[N’,N’’-di-
(Boc)guanidino]phenol (7, 1.05 g, 3.00 mmol, 1 equiv), and p-(dime-
thylamino)pyridine (73 mg, 0.61 mmol, 0.2 equiv) in EtOAc (10 mL).
The reaction mixture was stirred at 08C for 1 h and for an addition-
al 2 h at room temperature. The solid N,N’-dicyclohexylurea was fil-
tered off, and the solvent was evaporated in vacuo. The product
was obtained as a white solid after purification by column chroma-
tography (1!4% MeOH in CH2Cl2). Yield: 1.33 g (82%); Rf =0.67
HPLC analyses: Samples were analyzed on a Shimadzu LC 2010
analytical HPLC system equipped with a RP C18 column (Varian, In-
ertsil ODS-3, 5 mm, 150ꢁ4.6 mm) and eluted with various mixtures
of acetonitrile/water containing 0.1% trifluoroacetic acid under iso-
cratic and gradient conditions at a flow rate of 1.0 mLminꢀ1. The
wavelength of detection was 254 nm. Product yields were calculat-
ed from peak areas of the substrate esters and the hydrolysis and
aminolysis products.
1
(4% MeOH in CH2Cl2); m.p. 1118C; H NMR (CDCl3, 300 MHz): d=
11.62 (s, NH), 10.35 (s, NH), 7.62 (d, J=8.5 Hz, 2H), 7.41–7.28 (m,
5H), 7.07 (d, J=8.3 Hz, 2H), 5.34 (m, NH), 5.15 (s, 2H), 4.22 (d, J=
5.3 Hz, 2H), 1.53 (s, 9H), 1.50 (s, 9H); 13C NMR (CDCl3, 75 MHz): d=
168.6, 163.4, 156.2, 153.5, 153.3, 146.8, 136.1, 134.8, 128.6, 128.2,
128.1, 123.2, 121.6, 83.9, 79.8, 67.3, 42.9, 28.2, 28.1; IR (film): n˜ =
2978, 2928, 1779, 1720, 1640, 1508, 1412, 1240, 1154, 1114,
1057 cmꢀ1; HRMS (ESI) m/z calcd for C27H34N4NaO8: 565.2274
[M+Na]+, found: 565.2274.
Molecular modeling of the papain–peptide complex: The molec-
ular model of papain bound to the hexapeptide LLRLSL was con-
structed on the basis of the crystal structure of a papain–leupeptin
complex (PDB ID: 1POP) solved at 2.1 ꢂ resolution.[17] This structure
contains an LLR peptide bound only to the S subsites. To gain
more insight into peptide binding to the S’ subsites, a hybrid
model was built by using an LSL peptide fragment bound to the S’
subsites of another papain crystal structure (PDB ID: 2CIO) solved
at 1.5 ꢂ resolution.[22] First, the two crystal structures were aligned
by using the MOTIF algorithm,[23] after which the coordinates of
the LSL peptide were transferred to the papain–leupeptin complex.
Subsequently, a peptide bond between the LLR and LSL peptide
fragment was added manually by using the Yasara program[24]
and finally the resulting complex was energy minimized by using
the Yasara2 force field.[25]
General procedure for the acidic Boc deprotection of guanidino-
phenyl esters (8) with TFA as illustrated by the synthesis of Na-
Cbz-Glycine p-guanidinophenyl ester (1G): Boc-protected com-
pound 8 (100 mg) was dissolved in CH2Cl2 (2 mL), and TFA (0.5 mL)
was added. The reaction mixture was stirred at room temperature
overnight. The solvents were removed under reduced pressure and
co-evaporated with tBuOH (3ꢁ10 mL). The obtained oil was lyophi-
lized from H2O/dioxane (10 mL) in the presence of HCl (2m,
0.5 mL) to give the product as a sticky oil (quant.). Rf =0.50 (CHCl3/
Molecular docking of papain substrates: All molecular-docking
studies in papain were performed by using the flexible docking
program Fleksy.[16] The crystal structure of a papain–leupeptin com-
plex (PDB ID: 1POP) solved at 2.1 ꢂ resolution[17] was used as the
receptor structure. The structure was prepared for docking by re-
moving leupeptin and all water molecules from the complex. Sub-
sequently, hydrogen atoms were added to the structure, and their
positions were optimized by using the Yasara program.[24] In the
applied docking protocol only those docking poses in which the
1
MeOH/NH4OH, 65:45:20); H NMR ([D6]DMSO, 300 MHz): d=10.03
(s, NH), 7.89 (t, J=5.9 Hz, NH), 7.56 (brs, 4NH), 7.40–7.25 (m, 7H),
7.21–7.15 (m, 2H), 5.08 (s, 2H), 4.07 (d, J=6.1 Hz, 2H); 13C NMR
([D6]DMSO, 75 MHz): d=169.0, 156.5, 156.0, 148.2, 136.8, 132.9,
128.3, 127.8, 127.7, 125.7, 122.7, 65.6, 42.4; IR (film): n˜ =3309, 3166,
2950, 1770, 1706, 1671, 1629, 1588, 1507, 1455, 1280, 1201, 1166,
1053 cmꢀ1; HRMS (ESI) m/z calcd for C17H18N4NaO4: 365.1226
[M+Na]+, found: 365.1231.
2206
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 2201 – 2207