N. Wimmer et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2635–2637
2637
Acknowledgements
(1H benzotriazole-1-yl)-1,3,3-tetramethyluronium hexafluoro
phosphate, 0.5 M in DMF, 3 equiv) and DIEA (0.442 mL, 4
equiv) was shaken with the resin for 12 min. Negative ninhy-
drin reaction (5 min) showed nearly quantitative coupling
(ꢂ99.98%) and the Boc protecting group was subsequently
removed using 100% TFA. Between all manipulations the
resin was washed intensively with DMF. Coupling and
deblocking of Boc protecting groups were done in an analo-
gous manner for peptides 4–7. Upon completion of the synth-
esis and removal of the terminal Boc groups, the resin was
washed with DMF, methanol and DCM. The resin was dried
to constant weight over KOH in vacuum. The peptides were
cleaved from the resin using a high HF method and p-cresol
as scavenger. The cleaved peptide was precipitated using die-
thyl ether, redissolved in 2.5% aqueous acetic acid and lyo-
philised to afford an amorphous powder.
23. Analytical RP-HPLC was performed on a Shimadzu
instrument (LC-10ATliquid chromatograph, SC l-10A system
controller, SPD-6A UV detector, a SIL-6B auto injector with
a SCL-6B system controller, and columns C4, C18; 25 cm
Vydac C4, C18 column with 5 nm pore size and 4.6 mm
internal diameter) in order to optimize the appropriate
gradient for the preparative HPLC and to identify the synthe-
sised peptide. The amount of 100 mg of each crude peptide
(4–7) was preparative separated on a Waters HPLC system
(Model 600 controller, 490E UV detector, F pump, and TSK-
GEL C4/C18 columns with 10 nm pore size and 2.5 cm
internal diameter) using a acetonitrile/water gradient and
characterised by electrospray-MS (Perkin Elmer API 3000
instrument). The resulting peptides were used as diaster-
eomeric mixtures.
This project was supported by NH & MRC Australia
and Wellcome Trust UK.
References and Notes
1. Gibbons, W. A.; Hughes, R. A.; Chralambous, M.; Szeto,
A.; Aulabaugh, A. E.; Mascagni, P.; Toth, I. Liebigs Ann.
Chem. 1990, 1175.
2. Toth, I.; Flinn, N.; Hillery, A. M.; Gibbons, W. A.;
Artursson, P. Int. J. Pharm. 1994, 105, 241.
3. Florence, A. T.; Wilderspin, A. F.; Toth, I.; Sakthivel, T.;
Bayele, H. K. PCT Int. Appl. 2000, 48 pp. WO 0016807.
4. Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos,
G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polymer J. 1985,
17, 117.
5. Brown, M. D.; Schatzlein, A. G.; Uchegbu, I. F. Int. J.
Pharm. 2001, 229, 1.
6. Segura, T.; Shea, L. D. Annu. Rev. Mater. Rev. 2001, 31, 25.
7. Godbey, W. T.; Mikos, A. G. J. Controlled Release 2001,
72, 115.
8. Pouton, C. W., Seymour, L. W. Adv. Drug Del. Rev. 2001,
46, 187, and references cited therein.
9. Toth, I.; Sakthivel, T.; Wilderspin, A. F.; Bayele, H.;
O’Donnel, M.; Perry, D. J.; Pasi, K. J.; Lee, C. A.; Florence,
A. T. S. T. P. Pharma. Sci. 1999, 9, 93.
10. Garrett, K. L.; Shen, W. Y.; Rakoczy, P. E. J. Gene Med.
2001, 3, 373, and references cited therein.
11. Shen, W. Y. Unpublished results, 2002.
12. Persidis, A. Nat. Biotechnol. 1998, 17, 403.
24. Complex formation: to manufacture complexes with the
required molar charge ratio, oligodeoxynucleotide ODN1 (0.5
mg/mL) was added to each dendrimer (1.5 mg/mL) mixed for
15 min, diluted with 250 mL sterile water and lyophilised. The
optimal molar charge ratio (+/ꢀ) for the complex was adjus-
ted at 5:1 (dendrimer/ODN1).16 ITC experiments were per-
13. Akhtar, S.; Hughes, M.; Khan, A.; Bibby, M.; Hussain,
M.; Nawaz, Q.; Double, J.; Sayyed, P. Adv. Drug Del. Rev.
2000, 44, 3.
14. Rojanasakul, Y. Adv. Drug Del. Rev. 1996, 3, 3.
15. Garcia Chaumont, C.; Seksek, O.; Grzybowski, E.;
Bolard, J. Pharmac. Ther. 2000, 87, 255.
formed in
a MicroCal VP-ITC microcalorimeter. The
16. Cohen, J. S. Pharmac. Ther. 1991, 52, 211.
oligonucleotide solution (2 mM) was placed in the sample cell
and the dendrimer (243 mM) was placed in the syringe. The cell
temperature was maintained at 30 ꢃC and the dendrimer was
added to the solution of oligonucleotide using 25 ꢄ 4 mL
injections, each injection was 4 min apart.
17. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
18. Shah, D. S.; Sakthivel, T.; Toth, I.; Florence, A. T.;
Wilderspin, A. F. Int. J. Pharm. 2000, 208, 41.
19. Murphy, E. A.; Waring, A. J.; Murphy, J. C.; Willson,
R. C.; Longmuir, K. J. Nucleic Acids Res. 2001, 29, 3694.
20. Lobo, B. A.; Rogers, S. A.; Wiethoff, C. M.; Choosa-
koonkriang, S.; Bogdanowich-Knipp, S.; Middaugh, C. R.
Methods Mol. Med. 2001, 65, 319.
25. In vitro assay Cells of human RPE origin, D407 were
grown to 80% confluency in a 24-well plate and transfected
with 1 mM (final concentration) of ODN1 in quadruplet sets
TM
using either cytofectin GSV (as per manufactures instruc-
21. Tame, J. R. H.; O’Brien, R.; Ladbury, J. E. In Biocalori-
metry, Applications of Calorimetry in the Biological Sciences;
Ladbury, J. I., Chowdhry B. Z., Eds.; John Wiley and Sons,
Chichester, 1998, pp 27–38.
22. General experimental procedure for the synthesis of com-
pounds 4–7 in detail: MBHA resin (4-methyl benz-
hydrylamine, substitution ratio, 0.62 mmol/g, 1000 mg) was
swelled in dimethylformamide in a sintered glass peptide
synthesis vessel for 90 min. An activation mixture consisting
of Boc-amino acid (3 equiv per mol amino-group), HBTU (2-
tions) or the dendrimer/ODN1–complexes. The cells were
grown under hypoxic conditions (5% CO2/2% O2) for 24 h
after which the media was removed for analysis. 500 mL of
media from each of the sample was placed in a microcon-30TM
concentrator and centrifuged to a volume of 10 mL. 500 mL of
phosphate buffer (pH 7.2) was then applied to wash the sam-
ple which was centrifuged to a final volume of 100 mL. The
samples were then used in an sandwich ELISA assay as per the
manufactures instructions (CytelisaTM Human VEGF kit,
CYTIMMUNE Sciences Inc., Maryland, USA).