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M. Sedlak et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2554–2557
2557
6. Janoff, A. S.; Perkins, W. R.; Saleton, S. L.; Swenson, C.
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apparatus, respectively; The IR spectra were measured
using Nicolet Impact 400 spectrometer. The samples
(0.2 mg) were measured in KBr pellets (200 mg).
23. Harada, A.; Cammas, S.; Kataoka, K. Macromolecules
1996, 29, 6183.
24. Poly(ethylene glycol)-b-poly(N ꢁ (4-formylphenyl-carbox-
amino)-L-lysine) (PEG-b-PFLL) (Mw = 15,500),: yield
92%; 1H NMR (400 MHz, CDCl3): d = 1.40 (m; 30H;
(CH2)3), 2.76 (br s; 2H, e-CH2, (a-helix)), 3.09 (br s; 8H, e-
CH2, (random coil)), 3.70 (m, 114H, (CH2)2O), 5.39 (br s,
5H, CH), 6.92 (d, J = 7.7 Hz, 14H, arom.), 7.83 (d; J = 7.7
Hz, 14H, arom.), 10.11 (m, 6H, CHO), 10.20 (br s, 5H,
CONH); IR (KBr) m: 3341, 3116, 2942, 2866, 2337, 1852,
1814, 1701,1783, 1657, 1630, 1537, 1455, 1258, 1139, 923,
748, 696,963, 531 cmꢁ1; for C612H1096N22O274 (%): C,
55.92; H, 8.35; N, 2.35; found: C, 55.81; H, 8.13; N, 2.56;
Mw/Mn = 1.18.
7. Moribe, K.; Mararuyama, K.; Iwatsuru, M. Int. J. Pharm.
2000, 201, 37.
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8. Sedlak, M.; Buchta, V.; Kubicova, L.; Simunek, P.;
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Holcapek, M.; Kasparova, P. Bioorg. Med. Chem. Lett.
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2001, 11, 2833.
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9. (a) Conover, C. D.; Zhao, H.; Clifford, B.; Longley, C. B.;
Shum, K. L.; Greenwald, R. B. Bioconjugate Chem. 2003,
14, 661; (b) Greenwald, R. B.; Choe, Y. H.; McGurie, J.;
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Cancer Chemother. Pharmacol. 2006, 57, 120.
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13. Sedlak, M.; Staud, F.; Pravda, M.; Kubicova, L.; Tycova,
K.; Ventura, K.; Bioorg. Med. Chem. submitted for
publication.
25. Shou, W. G.; Yang, Y. Y.; Wang, Y. G. Synthesis 2005,
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14. (a) Harris, J. M. In Poly(ethylene glycol) Chemistry:
Biotechnical and Biomedical Application; Harris, J. M.,
26. Poly(ethylene glycol)-[b-poly((L-lysine)5]2-(AMB)12 (PEG-
b-PFLL- AMB) (Mw = 26 700), yield: 86%; IR (KBr) m:
3341, 3116, 2945, 2886, 2741,2337, 1852, 1814, 1719,1693,
1637, 1630, 1537, 1467, 1256, 1201,1139, 923, 748,696, 963,
531 cmꢁ1; UV–vis (kmax: 346, 367, 386, 409 nm); for
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Ed.; Plenum Press: New York, 1992; pp 1–13; (b) Sedlak,
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1998, 199, 247; (c) Sedlak, M. Collect. Czech. Chem.
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15. (a) Thunemann, A. F.; Beyermann, J.; Kukula, H.
¨
C1176H1948N34O466 (%) calculated: C, 58.82; H, 8.13; N,
1.98; found: C, 58.99; H, 8.36; N, 2.20; Mw/Mn = 1.11.
27. A micro-test-tube was charged (from a pipette) with 500 ll
sample of plasma or serum (from healthy donors of
plasma at transfusion station) and 500 ll aqueous solution
of conjugate PEG-b-PFLL-AMB to obtain the final
concentration of the conjugate equal to 1 · 10ꢁ1 M. The
samples were incubated at 37 ꢀC. After the incubation, at
definite intervals: 0 (immediately), 1, 2 –10 h and after 24 h
they were stabilised with 100 ll of 4% ZnSO4Æ7H2O. The
mixture was shaken for 1 min and then centrifuged for
3 min at 14000 rpm. The supernatant (20 ll) was intro-
duced to a column LiChroCARTꢂ 125 · 4 mm packed
with LiChrospherꢂ 100 RP-18e 5 lm (MERCK); mobile
phase: acetonitrile, 2 · 10ꢁ2 M chelaton II.
Macromolecules 2000, 33, 5906; (b) Harada, A.; Togawa,
H.; Kataoka, K. Eur. J. Pharm. Sci. 2001, 13, 35; (c)
Thunemann, A. F.; Kubowitz, S.; Burger, C.; Watson, M.
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D. J. Am. Chem. Soc. 2003, 125, 352; (d) Harada, A.;
Kataoka, K.; Furusho, M.; Tanaka, F.; Wada, H.;
Ikenaka, K. J. Gene Med. 2004, 6, 471; (e) Katakura,
H.; Harada, A.; Auguste, D. T.; Armes, S. P.; Brzezinska,
K. R.; Deming, T. J.; Kohn, J.; Prud’homme, R. K.
Biomaterials 2006, 27, 2599; (f) Thunemann, A. F.;
¨
Schuett, D.; Sachse, R.; Schlaad, H.; Mo¨hwald, H.
Langmuir 2006, 22, 2323.
16. (a) Co¨lfen, H. Macromol. Rapid Commun. 2001, 22, 219;
(b) Co¨lfen, H.; Mann, S. Angew. Chem., Int. Ed. 2003, 42,
2350; (c) Kasparova, P.; Antonietti, M.; Co¨lfen, H.
Colloids Surf. A Physicochem. Eng. Asp. 2004, 250, 153.
17. Paquet, V.; Zumbuehl, A.; Carreira, E. M. Bioconjugate
Chem. 2006, 17, 1460.
18. (a) Mellman, I.; Fuchs, R.; Helenius, A. Annu. Rev.
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Biochem. 1986, 55, 773; (b) Ulbrich, K.; Subr, V. Adv.
Drug Delivery Rev. 2004, 56, 1023.
19. Nasti, T. H.; Khan, M. A.; Owais, M. J. Antimicrob.
Chemother. 2006, 57, 349.
20. Dimitrov, I.; Schlaad, H. Chem. Commun. 2003, 2944.
21. Poly(ethylene glycol)-b-poly(L-lysine) (PEG-b-PLL) (Mw =
13 200): yield 98%; 1H NMR (400 MHz, CDCl3): d = 1.38
(m; 30 H; (CH2)3), 2.74 (br s; 4H, e-CH2, (a-helix)), 3.01
(br s; 6H, e-CH2, (random coil)), 3.69 (m, 114H, (CH2)2O),
5.38 (br s, 5H, CH); IR (KBr) m: 3339, 3114, 2942, 2866,
2337, 1852,1814, 1783, 1657, 1630, 1537, 1455, 1258, 1139,
923, 748, 696,963, 531 cmꢁ1; for C516H1036N22O238 (%): C,
54.58; H, 9.13; N, 2.71; found: C, 54.81; H, 9.32; N, 2.98;
Mw/Mn = 1.16.
28. The kinetic measurements were performed on an HP VU/
VIS 8453 Diode Array apparatus using 1 cm closable
quartz cells in thermostat block at 37 ꢀC. The cell was
charged with 2 ml phosphate buffer (pH 7.4, 7 · 10ꢁ2 M or
pH 5.5, 7 · 10ꢁ2 M), and after attaining the mentioned
temperature, 15 ll methanolic solution of conjugate PEG-
b-PFLL-AMB was injected into the cell to obtain the final
concentration of substrate equal to approximately
5 · 10ꢁ5 M. The measured absorbance-time (A–t) depen-
dences were used to calculate the observed rate constants
of pseudo-first order (kobs (sꢁ1)) from the equation:
kobst = ln DA + const., where DA = (A1 ꢁ At) or
(At ꢁ A ).
1
29. (a) Cordes, E. H.; Jencks, W. P. J. Am. Chem. Soc. 1963,
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85, 2843; (b) Hoffmann, J.; Sterba, V. Collect. Czech.
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Chem. Commun. 1972, 38, 2043; (c) Hoffmann, J.; Sterba,
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30. Gaboriau, F.; Cheron, M.; Leroy, L.; Bolard, J. Biophys.
Chem. 1997, 66, 1.
22. Gel permeation chromatography (GPC) was used for
estimation Mw of polymers. The measurements were
performed with HEMA-BIO columns (hydrophilic mod-
ified HEMA-Gel, particle size 10 lm porosity 40/100/300/
1000) at room temperature using RI detector and UV/Vis
detector. Redistilled water (pH 7.1) was used as the eluent.
The columns were calibrated with a series of standard
PEGs with varying molecular weights (PSS, Polymer
Standard Service GmbH, Mainz, Germany); the 1H NMR
spectra were measured in deuteriochloroform (CDCl3), on
a Bruker AMX 360 apparatus and a Bruker 500 Avance
31. Acute toxicity was tested for amphotericin B (Bristol-
Myers Squibb) as a control and for PEG-b-PFLL-AMB.
For both the compounds tested, the mice were divided into
six groups of 10 males, and the compound was adminis-
tered intravenously via the tail vein at doses carefully
determined after a series of preliminary trials. Toxic signs
and mortality were monitored for 14 days after adminis-
tration. LD50 values and the corresponding confidence
limits were determined by non-linear regression using
GraphPad Prism (version 4; GraphPad software Inc., San
Diego, CA, USA).