2
58
N. B. Pestov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 255–258
2
5
1
4
.
.
.
.
Kirschmann, D. A.; Seftor, E. A.; Fong, S. F.; Nieva, D. R.; Sullivan, C. M.; Edwards,
Lys ꢀ HCl (0.7 g; 40%). ½
1.54 (m, 2H, CH CH CH
CH CD ), 12.98–3.03 (m, 0.04H, CH
CHNH
(CH CHNH
2
a
ꢁ
= +8.8 (c 2.9; H
2
O). H NMR (500 MHz, D
), 1.69–1.72 (m, 2H, CH CHNH ), 1.84–1.93 (m, 2H,
NH CHDNH ), 3.75 (t, J = 6.1 Hz, 1H,
O): d 22.6 (CH CH CH ), 27.5 (CH CD ), 31.1
), 175.8 (CO H).
2
O): d 1.38–
D
E. M.; Sommer, P.; Csiszar, K.; Hendrix, M. J. Cancer Res. 2002, 62, 4478.
Fong, S. F.; Dietzsch, E.; Fong, K. S.; Hollosi, P.; Asuncion, L.; He, Q.; Parker, M. I.;
Csiszar, K. Genes Chromosomes Cancer 2007, 46, 644.
Erler, J. T.; Bennewith, K. L.; Nicolau, M.; Dornhofer, N.; Kong, C.; Le, Q. T.; Chi, J.
T. A.; Jeffrey, S. S.; Giaccia, A. J. Nature 2006, 66, 10238.
Li, W.; Nellaiappan, K.; Strassmaier, T.; Graham, L.; Thomas, K. M.; Kagan, H. M.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12817.
Tang, C. L.; Klinman, J. P. J. Biol. Chem. 2001, 276, 30575.
2
2
2
2
2
5
6
7
2
2
2
2
,
2
3
2
). C NMR (125 MHz, D
), 55.7 (CHNH
2
2
2
2
2
2
2
2
2
25. Yamazaki, S.; Yamazaki, Y. Bull. Chem. Soc. Jpn. 1990, 63, 301.
26. Secrist, J. A.; Logue, M. W. J. Org. Chem. 1972, 37, 335.
27. Wang, C.; Valtavaara, M.; Myllyla, R. DNA Cell Biol. 2004, 19, 71.
28. Eyre, D. R.; Weis, M. A.; Wu, J. J. J. Biol. Chem. 2010, 285, 16675.
29. Nagan, N.; Kagan, H. M. J. Biol. Chem. 1994, 269, 22366.
8
9
.
.
Rodriguez, H. M.; Vaysberg, M.; Mikels, A.; McCauley, S.; Velayo, A. C.; Garcia,
C.; Smith, V. J. Biol. Chem. 2010, 285, 20964.
30. Fmoc-D -Lys(Boc) (5) was prepared according 1to the standard protocol
2
1
1
0. Anderson, C.; Bartlett, S. J.; Gansner, J. Mol. Biosys. 2007, 3, 51.
1. Granchi, C.; Funaioli, T.; Erler, J. T.; Giaccia, A. J.; Macchia, M.; Minutolo, F.
ChemMedChem 2009, 4, 1590.
[Houben-Weyl: Vol. E 22 Synthesis of Peptides] H NMR: 7.78 (d, J = 7.2 Hz,
2H, Fmoc), 7.58 (m, 2H, Fmoc), 7.38 (t, 7.2 Hz, 2H, Fmoc), 7.28 (t, 7.2 Hz, 2H),
6.29–6.45 (br, 1H, NH), 5.78–5.90 (br, 1H, NH), 4.37–4.44 (m, 3H, CHNH, CH
CH), 4.20 (m, 1H, CHNH, CH , CH), 4.2 (m, 1H), 1.26–1.87 (m, 15H, CH , CH
Boc). 13C NMR: 22.2 (CH
), 28.4 (3C, CH ), 29.3 (CH ), 31.7 (CH ), 47.1 (CH-
Fmoc), 53.7 (CHNH), 67.0 (CH O), 79.5 (Me CO), 119.9 (Fmoc), 125.1 (Fmoc),
2
,
1
1
2. Gordon, M.; Zykorie, S.; Gardner, B.; Patti, J.; Gray, H. Cancer 1972, 29, 509.
3. Wang, S. X.; Mure, M.; Medzihradszky, K. F.; Burlingame, A. L.; Brown, D. E.;
Dooley, D. M.; Smith, A. J.; Kagan, H. M.; Klinman, J. P. Science 1996, 273, 1078.
4. Lucero, H. A.; Kagan, H. M. Cell. Mol. Life Sci. 2006, 63, 2304.
5. Williamson, P. R.; Kagan, H. M. J. Biol. Chem. 1986, 261, 9477.
6. Williamson, P. R.; Kagan, H. M. J. Biol. Chem. 1987, 262, 8196.
7. Elison, C.; Rapoport, H.; Laursen, R.; Elliott, H. W. Science 1961, 134, 1078.
8. Belleau, B.; Moran, J. Ann. N.Y. Acad. Sci. 1963, 107, 822.
2
2
3
-
2
3
2
2
2
3
1
1
1
1
1
1
127.0 (Fmoc), 127.6 (Fmoc), 141.2 (Fmoc), 143.7 (Fmoc), 143.8 (CO), 156.3
(CO), 175.4 (COOH).
31. The peptide substrates for LOX were custom made, HPLC-purified and MS-
analysed by JPT Peptide Technologies GmbH (Germany) using synthon 5: Ac-
Arg-(Gly)
(Gly) -Gly-CONH
32. Lysyl oxidase. LOX was isolated from lamb aorta,
4
-Glu-(D
2
-Lys)-(Gly)
4
-Gly-CONH
2
;
Ac-Arg-(Gly)
4 2
-Glu-(H -Lys)-
9. Dourish, C. T.; Dewar, K. M.; Dyck, L. E.; Boulton, A. A. Psychopharmacology
4
2
.
33,34
1
983, 81, 122.
0. Yu, P. H.; Barclay, S.; Davis, B. A.; Boulton, A. A. Biochem. Pharmacol. 1981, 30,
089.
1. Yu, P. H.; Kazakoff, C.; Davis, B. A.; Boulton, A. A. Biochem. Pharmacol. 1982, 31,
697.
by extraction with a urea
2
2
solution followed by IE chromatography, ammonium sulphate precipitation,
and gel filtration. To prepare recombinant LOX, cDNA encoding the catalytic
domain (Asp163-Tyr411 of the full-length preproenzyme) of mouse LOX was
amplified by RT-PCR with Mu-MLV reverse transcriptase and Phusion High
Fidelity DNA polymerase (Finnzymes Oy, Finland) from mouse lung RNA and
cloned into BamH I/Sph I sites of pQE30 bacterial expression vector (Qiagen,
USA). The expression of N-terminally hexahistidine-tagged LOX and
purification by immobilized nickel chelate affinity chromatography under
denaturing conditions was performed as previously described for unrelated
3
3
2
2
2. Williamson, P. R.; Kagan, H. M. J. Biol. Chem. 1987, 262, 14520.
3. Shah, M. A.; Scaman, C. H.; Palcic, M. M.; Kagan, H. M. J. Biol. Chem. 1993, 268,
1
1573.
4. Synthesis of D
sodium (9.24 g; 402 mmol) in 350 ml anhydrous EtOH,
00 mmol) was added with stirring. In 1 h, the precipitate of NaCl was filtered
off, and reaction mixture was cooled to 5 °C, then ethyltrifluoroacetate
39.76 g; 280 mmol) was added dropwise over 30 min with stirring. The
mixture was stirred for 1.5 h at 5 °C and 1 h at rt, then AcOH (12.12 g,
02 mmol) was added and the mixture was stirred for 10 min. The precipitate
e
2
2
-Lys (Fig. 1). N -trifluoroacetyl-
L-lysine (1): To a solution of
L-lysine ꢀ HCl (36.5 g;
2
proteins.35 For refolding, the purified LOX (0.65
lg/ll) in TBS (50 mM Tris–HCl,
pH 7.6, 140 mM NaCl) with 8 M urea was incubated with 2.5 mM tris-(2-
carboxyethyl) phosphine for 1 h at 25 °C. After that, dry sodium lauryl
sarcosinate was added to 2% followed by dialysis against TBS with 2 M urea
solutions: for 3 h at 25 °C against pure TBS with 2 M urea, with 10 mM 2,2 -
2
dipyridine overnight at 25 °C, with 0.5 mM CuCl for 2 days at 4 °C, with 1 mM
(
0
2
e
was collected, washed and dried in vacuo to give N -trifluoroacetyl-
L-lysine
2
5
(
a
27.3 g; 56%). Tm 252–254 °C (decomp), ½
aꢁ
= +16.8 (n = 1.0; 1iꢂHCl).
e
EDTA for 3 h at 25 °C and against TBS-2 M urea overnight at 4 °C. The proteins
isolated from aorta and from E. coli were probed by immunoblotting with a
D
e
-N-Boc-N -trifluoroacetyl-
L-lysine (2): N -trifluoroacetyl-L-lysine (3.63 g;
1
5 mmol) was suspended in DMF (40 ml), and di-tert-butyl dicarbonate
mouse monoclonal antibody (Fig. 3). LOX activity was measured as H
release coupled to oxidation of 10-acetyl-3,7-dihydrophenoxazine by
horseradish peroxidase;36 the assays were run at 37 °C on
Fusion
microplate analyzer (Perkin Elmer) using 100 l volume, with kex = 535 and
em = 620 nm. Enzyme-independent oxidation rate of 10-acetyl-3,7-
dihydrophenoxazine by atmospheric oxygen was taken into account and the
exposure to light was minimized. LOX activity was fully sensitive to 100
2 2
O
(
3.8 ml; 16.5 mmol) was added. The mixture was stirred at rt for 2 h, diluted
100 ml H
O), and extracted with EtOAc (2 ꢀ 50 ml). The organic phase was
(2 ꢀ 100 ml), NaClsatd (50 ml), dried (Na SO and
evaporated. The oil solidified into a white solid (4.7 g; 92%).
S)-2-BOC-Amino-(5-cyano)pentanoic acid (3): acid (2) (2.349 g; 7 mmol) and
NaOH (0.56 g; 14 mmol) were dissolved in H O (40 ml). To this, a solution of
NiSO O (0.2 g; 0.7 mmol) in H O (1 ml) was added, followed by Na
ꢂ7H
3.332 g; 14 mmol). Additional NaOH (1.12 g in 5 ml H O) was added in five
portions over 3 h. The mixture was stirred overnight. A little Na SO was added
(
2
a
washed with
H
2
O
2
4
)
l
k
(
2
lM
4
2
2
2
2
S O
8
BAPN. Curves were fitted with Prism 4 software package (GraphPad, USA).
(
2
33. Kagan, H. M.; Cai, P. Methods Enzymol. 1995, 258, 122.
34. Ohkawa, K.; Fujii, K.; Nishida, A.; Yamauchi, T.; Ishibashi, H.; Yamamoto, H.
Biomacromolecules 2001, 2, 773.
35. Pestov, N. B.; Gusakova, T. V.; Kostina, M. V.; Shakhparonov, M. I. Russ. J. Bioorg.
Chem. 1996, 22, 567.
36. Palamakumbura, A. H.; Trackman, P. C. Anal. Biochem. 2002, 300, 245.
37. Olivieri, A.; O’Sullivan, J.; Fortuny, L. R.; Vives, I. L.; Tipton, K. F. Biochim.
Biophys. Acta 2010, 1804, 941.
38. Lyles, G. A.; Singh, I. J. Pharm. Pharmacol. 1985, 37, 637.
39. McGrath, A. P.; Hilmer, K. M.; Collyer, C. A.; Shepard, E. M.; Elmore, B. O.;
Brown, D. E.; Dooley, D. M.; Guss, J. M. Biochemistry 2009, 48, 9810.
40. Palamakumbura, A. H.; Jeay, S.; Guo, Y.; Pischon, N.; Sommer, P.; Sonenshein, G.
E.; Trackman, P. C. J. Biol. Chem. 2004, 279, 40593.
2
3
to decolorize the reaction mixture, followed by acidification by HCl to pH 2–3.
The mixture was extracted (EtOAc, 80 ml), washed with NaClsatd (50 ml), dried
over MgSO
CHCl
) to give the title compound (1.42 g; 84%). ½
NMR (DMSO): d 1.40 (s, 9H, Me C), 1.58–1.70 (m, 3H, CH
CH ), 2.49–2.55 (m, 2H, CH CN), 3.92 (dt, J = 7.9, 4.6 Hz, 1H, CHNHBoc), 7.13 (d,
.9 Hz, 1H, NH). C NMR (DMSO) d 15.8 (CH ), 21.6 (CH ),
), 28.11 (3 ꢀ C, CH
9.8 (CH ), 52.7 (CHNHBoc), 78.0 ((CH C), 120.2 (CN), 155.5 (CO NH), 173.6
H).
,6-Dideuterolysine (4): nitrile (3) (2 g; 8.13 mmol) was dissolved in a mixture
O (20 ml) and stirred for 5 min. The solvent was
evaporated and residue dissolved in a mixture of MeOD (15 ml) and D
25 ml), to which PtO (100 mg) was added. The mixture was kept under D gas
4
and evaporated. The residue was purified by CC (0–8% MeOH in
25
1
3
a
ꢁ
D
= +12.0 (c 4.1; CHCl
3
).
H
3
2
), 1.73–1.82 (m, 1H,
2
2
13
7
2
2
2
3
2
3
)
3
2
(
CO
2
6
of MeOD (10 ml) and D
2
2
O
4 5 4 5
41. N-Ac-(Gly) -Glu-Lys-(Gly) is oxidised faster than N-Ac-(Gly) -Lys-Glu-(Gly) .
(
2
2
Replacement of the Gly residue with Asp further reduces the efficiency of LOX.
42. Kagan, H. M.; Williams, M. A.; Williamson, P. R.; Anderson, J. M. J. Biol. Chem.
1984, 259, 11203.
43. Thomas, M. R.; Irving, C. S.; Reeds, P. J.; Malphus, E. W.; Wong, W. W.; Boutton,
T. W.; Klein, P. D. Eur. J. Clin. Nutr. 1991, 45, 227.
overnight. The catalyst was filtered off, and reaction mixture evaporated. The
residue was dissolved in a mixture of dioxane (11 ml) and 20% HCl (3 ml), kept
overnight, and then evaporated. The residue was dissolved in EtOH (8 ml), and
2
the title compound precipitated as dihydrochloride by addition of Et O (10 ml).
The precipitate was filtered, dried in vacuo, dissolved in EtOH (20 ml), brought
to a boil, and then pyridine (0.45 ml) was added. The mixture was left to
recrystallize overnight at 2 °C. The crystals were collected and dried to give D -
2
44. Kushner, D. J.; Baker, A.; Dunstall, T. G. Can. J. Physiol. Pharmacol. 1999, 77, 79.
45. Irving, C. S.; Thomas, M. R.; Malphus, E. W.; Marks, L.; Wong, W. W.; Boutton, T.
W.; Klein, P. D. J. Clin. Invest. 1986, 77, 1321.