282 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Juli et al.
(S)-1-(Benzylsulfonyl)piperidine-2-carboxylic Acid (S-3b).18
S-3b was obtained as a yellow oil (0.57 g, 90%). 1H NMR
(CDCl3, δ [ppm], J [Hz]): 7.49-7.33 (m, 5H), 4.60-4.55 (m, 1H),
4.27 (s, 2H), 3.49-3.41 (m, 1H), 3.15 (dt, 1H, 3J = 12.4, 3J =
3.1); 2.20-2.12 (m, 1H), 1.73-1.52 (m, 3H), 1.51-1.18 (m, 2H).
IR [cm-1]: 1060, 1118, 1317, 1455, 1728, 2854, 2952, 3192.
General Procedure for the Synthesis of Compounds 4a-f and
S-4c. The syntheses of compounds 4a-f and S-4c were per-
formed using a procedure described by Holt et al.9 for analogous
substances. Compounds 3 (1 equiv), the corresponding alcohol
(1.1 equiv), DCC (1.5 equiv), DMAP (0.3 equiv), and p-toluene
sulfonic acid (0.3 equiv) were dissolved in 50 mL of anhydrous
CH2Cl2. This mixture was stirred for 72 h at RT. After the
reaction had ceased (TLC control), the precipitate was filtered
off and the solvent was removed in vacuo. The residue was
suspended in ethyl acetate, and the resulting precipitate was filtered
off again. This procedure was repeated as long as a precipitate was
formed. Afterward, the filtrate was dried over Na2SO4 and subse-
quently the solvent removed in vacuo. If it was necessary, a
subsequent purification by silica gel column chromatography
was performed to obtain the compounds 4a-f and S-4c.
(S)-3-(3,4,5-Trimethoxyphenyl)propyl 1-(benzylsulfonyl)-
piperidine-2-carboxylate (S-4c). After purification by column chro-
matography (aluminum oxide; chloroform: ethyl acetate = 1:1)
S-4c was obtained as a yellow waxy substance (0.12 g, 21%). 1H
NMR (CDCl3, δ [ppm], J [Hz]): 7.49-7.33 (m, 5H), 6.41 (s, 2H),
4.55-4.50 (m, 1H), 4.27 (s, 2H), 4.24-4.15 (m, 2H), 3.84 (s, 6H),
3.82 (s, 3H), 3.46-3.40 (m, 1H), 3.17 (dt, 1H, 3J = 12.4, 3J = 3.1),
2.68-2.62 (m, 2H), 2.20-2.12 (m, 1H), 2.03-1.95 (m, 2H),
1.75-1.50 (m, 3H), 1.51-1.18 (m, 2H). IR [cm-1]: 1124, 1335,
1455, 1589, 1733, 2857, 2938. Anal. Calcd for C25H33NO7S (MW:
491.60 g/mol): C, 61.08; H, 6.77; N, 2.85; S, 6.52. Found: C, 60.86;
H, 6.87; N, 3.06; S, 6.13.
Normalized chemical changes were expressed as weighted
geometric average of chemical shift changes (1H and 15N) by
normshift = (δ(1H)2 þ (0.1δ(15N))2)1/2. Normalized chemical
shift changes larger than 0.03 ppm were considered as signifi-
cant.
Infection Assay of U937 Cells. The human macrophage-like
cell line U937 was infected as described previously.20 In brief,
cells were infected with a multiplicity of infection (MOI) of 10
with or without 50 μM of substance B (S-4c) or rapamycin. The
used bacterial strains were L. pneumophila Philadelphia-1 JR32
(wild type, WT), the MIP-negative mutant L. pneumophila
Philadelphia-1 JR32-23, and the E. coli strain HB101. Two
hours after infection, extracellular bacteria were killed by gen-
tamicin and infection samples were plated on BCYE agar to
determine CFU counts immediately and after 24 and 48 h.
During incubation of the respective samples, 50 μM of rapamy-
cin or substance B (S-4c) were always present. Control experi-
ments showed that rapamycin and substance B (S-4c) do not
alter replication rates of bacterial cultures.
Acknowledgment. Financial support by the Deutsche
Forschungsgemeinschaft (SFB 630) is gratefully acknowledged.
Supporting Information Available: Experimental details of the
synthesis, analytical and spectral data of all synthesized com-
pounds. This material is available free of charge via Internet at
References
(1) Steinert, M.; Hentschel, U.; Hacker, J. Legionella pneumophila: an
aquatic microbe goes astray. FEMS Microbiol. Rev. 2002, 26 (2),
149–162.
(2) Cianciotto, N. P.; Stamos, J. V.; Kamp, D. W. Infectivity of
Legionella pneumophila MIP mutant for alveolar epithelial cells.
Curr. Microbiol. 1995, 30 (4), 247–250.
(3) Wintermeyer, E.; Ludwig, B.; Steinert, M.; Schmidt, B.; Fischer,
G.; Hacker, J. Influence of site specifically altered MIP proteins on
intracellular survival of Legionella pneumophila in eucaryotic cells.
Infect. Immun. 1995, 63 (12), 4576–4583.
(4) Wagner, C.; Khan, S. A.; Kamphausen, T.; Schmausser, B.; Unal,
C.; Lorenz, U.; Fischer, G.; Hacker, J.; Steinert, M. Collagen
binding protein MIP enables Legionella pneumophila to transmi-
grate through a barrier of NCI-H292 lung epithelial cells and
extracellular matrix. Cell. Microbiol. 2007, 9, 450–462.
General Procedure for the Synthesis of Compounds 5a-b. The
reduction of 4e and 4f was performed according to Bellamy
et al.19 Compound 4e and 4f (1 equiv), respectively, was sus-
pended in 50 mL of anhydrous ethanol and SnCl2 (5 equiv)
added in portions. This reaction was refluxed for 2 h. The
reaction was monitored by TLC (silica gel; triethylamine:ethyl-
acetate = 5:95). Thirty mL of water and NaHCO3 were added to
neutralize the mixture. The aqueous phase was washed with 4 ꢀ
30 mL ethyl acetate. The combined organic layers were dried
over anhydrous Na2SO4, and the solvent was removed in vacuo.
If necessary, a subsequent purification by silica gel column
chromatography was performed to obtain compounds 5a-b.
Protease-Coupled PPIase Assay. The PPIase activity of MIP
(and FKBP12, respectively) was determined by using the pep-
tide substrate Succinyl-Ala-Phe-Pro-Phe-4-nitroanilide in the
protease-coupled PPIase assay as described by Fischer et al.6 To
study the effect of compounds 2a-d, 4a-f, S-4c, and 5a-b on
PPIase activity, 40 nM MIP (20 nM FKBP12), and 40 μM
peptide substrate were preincubated in the presence or absence
of various inhibitor concentrations for 4 min at 10 °C in 35 mM
HEPES/NaOH buffer (pH 7.8). Then reaction was started by
addition of 100 μg/mL of the isomer specific protease chymo-
trypsin, and the release of 4-nitroaniline was monitored by
measuring the absorbance at 390 nm.
€
(5) Gelse, K.; Poschl, E.; Aigner, T. Collagens structure, function
;
and biosynthesis. Adv. Drug Delivery Rev. 2003, 12, 1531–1546.
(6) Fischer, G.; Bang, H.; Ludwig, B.; Mann, K.; Hacker, J. MIP
protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans
isomerase (PPIase) activity. Mol. Microbiol. 1992, 6 (10), 1375–
1383.
(7) Ceymann, A.; Horstmann, M.; Ehses, P.; Schweimer, K.; Paschke,
A.; Steinert, M.; Faber, C. Solution structure of the Legionella
pneumophila MIP-rapamycin complex. BMC Struct. Biol. 2008,
8, 17.
€
(8) Riboldi-Tunnicliffe, A.; Konig, B.; Jessen, S.; Weiss, M. S.;
Rahfeld, J.; Hacker, J.; Fischer, G.; Hilgenfeld, R. Crystal struc-
ture of MIP, a prolylisomerase from Legionella pneumophila.
Nature Struct. Biol. 2001, 8, 779–783.
(9) Holt, D. A.; Luengo, J. I.; Yamashita, D. S.; Oh, H.; Konialian,
A. L.; Yen, H.; Rozamus, L. W.; Brandt, M.; Bossard, M. J.; Levy,
M. A.; Eggleston, D. S.; Liang, J.; Schultz, L. W.; Stout, T. J.;
Clardy, J. Design, Synthesis, and Kinetic Evaluation of High-
Affinity FKBP Ligands and the X-ray Crystal Structures of Their
Complexes with FKBP12. J. Am. Chem. Soc. 1993, 115, 9925–9938.
(10) Holt, D. A.; Konialian-Beck, A. L.; Oh, H.; Yen, H.; Rozamus,
L. W.; Krog, A. J.; Erhard, K. F.; Ortiz, E.; Levy, M. A.; Brandt,
M.; Bossard, M. J.; Luengo, J. Structure-activity studies of
synthetic FKBP Ligands as peptidyl-prolyl isomerase inhibitors.
Bioorg. Med. Chem. Lett. 1994, 4, 315–320.
NMR Spectroscopy: HSQC Experiments. 15N labeled MIP77-213
was expressed and purified as described previously.7 NMR
samples contained 0.1 mM 15N labeled MIP77-213 in 20 mM
potassium phosphate buffer, pH 6.5, and 10% D2O for field/
1
frequency lock. Standard H, 15N FHSQC experiments were
recorded at 298 K with a Bruker Avance 800 MHz NMR
spectrometer equipped with a cryogenically cooled probe.
Ligands were dissolved in deuterated acetonitrile to a concen-
tration of 23 mM. For each ligand, HSQC experiments were
recorded with protein:ligand ratios of 1:5, 1:10, and 1:20. A set
of reference experiments was recorded by adding the same
amount of deuterated acetonitrile without any ligand to the
protein solution.
€
(11) Fischer, G.; Mech, C.; Bang, H. Nachweis einer Enzymkatalyse fur
die cis/trans Isomerisierung der Peptidbindung in prolinhaltigen
Peptiden. Biomed. Biochim. Acta. 1984, 43, 1101–1111.
€
€
€
€
(12) Kohler, R.; Fanghanel, J.; Konig, B.; Luneberg, E.; Frosch, M.;
Rahfeld, J. U.; Hilgenfeld, R.; Fischer, G.; Hacker, J.; Steinert, M.
Biochemical and functional analyses of the MIP protein: influence
of the N-terminal half and of peptidylprolyl isomerase activity on