4
96
S. Naveh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 493–496
Table 2
⁄
⁄
a
Fragmentation of c(HS4-4), 65–79 0401 and 65–79 0404 after degradation by trypsin/chymotrypsin
Name
Deduced sequence of fragment
Observed MH+
969.53
O
HN
CH2)4
C
NH
(CH )
c(HS4-4)
(
2 4
H Trp N CH CO Gln Lys Arg N CH CO Ala NH2
2
2
⁄
6
5–79 0401
K-D-L-L-E-Q-K-R-A-A-V-D-T-Y-C
K-D-L-L-E-Q-K-R-A-A-V-D-T-Y
C
K-D-L-L-E-Q-K-R
A-A-V-D-T-Y
1752.98
1649.93
##
Fragment 1
Fragment 2
Fragment 3
Fragment 4
Fragment 5
1029.64
638.51
872.53
795.43
1778.84
1676.84
##
1057.23
638.59
901.71
795.40
K-D-L-L-E-Q-K
Fragment 6
R-A-A-V-D-T-Y
⁄
65–79 0404
K-D-L-L-E-Q-R-R-A-A-V-D-T-Y-C
K-D-L-L-E-Q-R-R-A-A-V-D-T-Y
C
K-D-L-L-E-Q-R-R
A-A-V-D-T-Y
Fragment 1
Fragment 2
Fragment 3
Fragment 4
Fragment 5
Fragment 6
K-D-L-L-E-Q-R
R-A-A-V-D-T-Y
a
Fragments were identified by MS ## complementary fragment
2
0. Ahn, J. M.; Boyle, N. A.; MacDonald, M. T.; Janda, K. D. Mini. Rev. Med. Chem.
002, 2, 463.
1. Gante, J. Angew. Chem. Int. Ed. Engl. 1994, 33, 1699.
Acknowledgments
2
2
We thank Timothy Samuel Haug from the Department of
Chemical Engineering, University of Michigan for technical assis-
tance. We thank Professor Assaf Friedler and Dr. Deborah E. Shalev
from the Hebrew University for helpful discussions.
22. Naider, F.; Goodman, M. In Synthesis of Peptides and Peptidomimetics; Goodman,
M., Toniolo, C., Moroder, L., Felix, A., Eds.; Thieme: Stuttgart New York, 2002;
Vol. E22a, p 1.
23. Vagner, J.; Qu, H. C.; Hruby, V. J. Curr. Opin. Chem. Biol. 2008, 12, 292.
24. Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G. Biopolymers 1991, 31, 745.
25. Hruby, V. J.; al-Obeidi, F.; Kazmierski, W. Biochem. J. 1990, 268, 249.
26. Demmer, O.; Frank, A. O.; Kessler, H. In Peptide and Protein Design for
Biopharmaceutical Applications; Keee, J., Ed.; John Wiley and Sons Ltd., 2009;
p 133.
References and notes
1
.
The following abbreviations were used throughout the text. The abbreviations
nitrile; AGBU, Alloc glycine building units; Alloc, allyloxycarbonyl; BTC, bis
27. Ovadia, O.; Greenberg, S.; Laufer, B.; Gilon, C.; Hoffman, A.; Kessler, H. Exp.
Opin. Drug Discovery 2010, 5, 655.
28. Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. Nat. Chem. Biol. 2010, 6,
595.
(
trichloromethyl)carbonate; CD, Circular dichroism; DCM, dichloromethane;
DIPEA, diisopropylethylamine; Fmoc, 9-fluorenylmethyloxy carbonyl; HATU,
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-nium hexafluoro-
2
3
3
9. Haridas, V. Eur. J. Org. Chem. 2009, 5112.
0. Garner, J.; Harding, M. M. Org. Biomol. Chem. 2007, 5, 3577.
(
1. All the peptides were synthesized using standard Fmoc SPPS procedures on
Rink amide MBHA resin as the solid support. The urea backbone cyclic
peptides, designated c(HSn-4), were synthesized according to the procedures
described by Hurevich et al., (J. Pept. Sci. 2010, 16, 178) using various AGBU,
where n stands for the number of atoms in the N-alkyl chain on the glycine at
phosphate); HBTU, (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate); HLA, human leukocyte antigen; MBHA, methyl
benzhydrylamine; NMP, 1-methyl-2-pyrrolidinone; NO, nitric oxide; PPIs,
Protein–protein interactions; RA, Rheumatoid arthritis; RP-HPLC, reverse phase
high pressure liquid chromatography; SAR, structure activity relationship; SE,
shared epitope; SPPS, solid phase peptide synthesis; TDW, tri-distilled water;
TFA, trifluoroacetic acid.
position 2. The sequences of the linear 15-mer peptides are as follows: 65–
⁄
7
9 0401: H-Lys-Asp-Leu-Leu-Glu-Gln-Lys-Arg-Ala-Ala-Val-Asp-Thr-Tyr-Cys-
⁄
NH2 65–79 0402: H-Lys-Asp-Ile-Leu-Glu-Asp-Glu-Arg-Ala-Ala-Val-Asp-Thr-
2
3
4
5
.
.
.
.
Benyamini, H.; Friedler, A. Future. Med. Chem. 2010, 2, 989.
Jones, S.; Thornton, J. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13.
Guharoy, M.; Chakrabarti, P. Bioinformatics 2007, 23, 1909.
⁄
Tyr-Cys-NH2 65–79 0404: H-Lys-Asp-Leu-Leu-Glu-Gln-Arg-Arg-Ala-Ala-Val-
2
Asp-Thr-Tyr-Cys-NH .
3
3
2. Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 319.
3. All analytical HPLC were recorded at 220 nm at a flow of 1 ml/min on a RP-18
Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.;
Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004, 305, 1466.
Moellering, R. E.; Cornejo, M.; Davis, T. N.; Bianco, C. D.; Aster, J. C.; Blacklow, S.
C.; Kung, A. L.; Gilliland, D. G.; Verdine, G. L.; Bradner, J. E. Nature 2009, 462, 182.
Gregersen, P. K.; Silver, J.; Winchester, R. J. Arthritis Rheum. 1987, 30, 1205.
Ou, D.; Mitchell, L. A.; Tingle, A. J. Hum. Immunol. 1998, 59, 665.
Gourraud, P. A.; Boyer, J. F.; Barnetche, T.; Abbal, M.; Cambon-Thomsen, A.;
Cantagrel, A.; Constantin, A. Arthritis Rheum. 2006, 54, 593.
column (5
lm 250 Â 4.6 mm, 110 Å), eluents A (0.05% TFA in TDW) and B
6
.
(
0.05% TFA in ACN) were used in a linear gradient (95% A?5% A in 35 min).
3
3
4. Samples of each peptide were prepared by dissolving a lyophilized peptide in
TDW. Far-UV CD spectra were collected over 190–260 nm at room temperature
using a J-810 spectropolarimeter (Jasco) in a 0.1 cm quartz cuvette for far-UV
CD spectroscopy.
7.
8.
9.
5. Human fibroblast M1 cells were plated at a density of 1 Â 105 cells per well in
1
1
0. n Mil, A. H.; Huizinga, T. W.; Schreuder, G. M.; Breedveld, F. C.; de Vries, R. R.;
Toes, R. E. Arthritis Rheum. 2005, 52, 2637.
1. Ruiz-Morales, J. A.; Vargas-Alarcon, G.; Flores-Villanueva, P. O.; Villarreal-
Garza, C.; Hernandez-Pacheco, G.; Yamamoto-Furusho, J. K.; Rodriguez-Perez, J.
M.; Perez-Hernandez, N.; Rull, M.; Cardiel, M. H.; Granados, J. Hum. Immunol.
96-well plates the day prior to the Nitric Oxide assay. To determine the rate of
NO production in fibroblast, cells were first loaded with 20
fluorescent NO probe 4,5-diaminofluorescein diacetate (DAF-2DA), incubated
in the dark at 37 °C for 1 h and washed in 100 L of DMEM/phenol red-free
medium. The fluorescence level was recorded every 5 minutes over a period of
00 min, using Fusion HT system (PerkinElmer Life Sciences) at an
lM of the
l
2
004, 65, 262.
5
a
a
1
1
1
2. Winchester, R. J.; Gregersen, P. K. Springer Semin. Immunopathol. 1988, 10, 119.
3. Kappes, D.; Strominger, J. L. Annu. Rev. Biochem. 1988, 57, 991.
4. Shookster, L.; Matsuyama, T.; Burmester, G.; Winchester, R. Hum. Immunol.
excitation wavelength of 488 nm and emission wavelength of 515 nm. The
NO production rate is expressed as the mean ± SEM fluorescence units per
minute.
1
987, 20, 59.
36. The trypsin stability assay was conducted as previously described by Tal-Gan
15. Holoshitz, J.; Ling, S. Ann. N.Y. Acad. Sci. 2007, 1110, 73.
16. Holoshitz, J.; De Almeida, D. E.; Ling, S. Ann. N.Y. Acad. Sci. 2010, 1209, 91.
17. Ling, S.; Li, Z.; Borschukova, O.; Xiao, L.; Pumpens, P.; Holoshitz, J. Arthritis Res.
Ther. 2007, 9, R5.
et al., (Bioorg. Med. Chem. 2010, 18, 2976). 400
l
L of each peptide (1 mM)
L of
dissolved in 200 mM NH HCO buffer solution (pH 8) were mixed with 1 l
4
3
trypsin and chymotrypsin (porcine pancreas, Biological Industries Israel, Beit
Haemek Ltd) solution (2.5 mg/1 ml). The peptides were incubated at 37 °C,
1
1
8. Ling, S.; Pi, X.; Holoshitz, J. J. Immunol. 2007, 179, 6359.
9. Ling, S.; Lai, A.; Borschukova, O.; Pumpens, P.; Holoshitz, J. Arthritis Rheum.
30
lL samples were taken every 30 min and mixed with 30
lL of 2% TFA and
30% ACN in water. Samples were analyzed by HPLC and by MALDI-TOF MS.
2
006, 54, 3423.