3.2.1. Synthesis of PAMAM G0 Dendrimer Substituted with 8 Glycidol Molecules, G0gl
, 0.9 mL, 0.967 g, 13.0 mmol) was added dropwise to a solution of PAMAM G0 (0.884 g, 1.63 mmol) in 20 mL of methanol with magnetic stirring. The mixture was left at room temperature for 2 days. Then methanol and excess gl
were removed by vacuum rotary evaporation. Yellow syrup was obtained, which was identified using 1
H NMR as G0gl
; for atom numbering see Scheme 1
): chemical shift [ppm] (intensity, multiplicity, assignment): 7.83 ([4H], bs, H-6’); 4.47 ([16H], bs, OH
); 3.51 ppm ([8H], q, Hb’
); 3.22 ppm ([16H], m, Ha’
); 3.09 ppm ([8H], H-7’); 2.63 ppm ([8H], t, H-2’); 2.51 ppm ([8H], m, H-8’); 2.42 ppm ([4H], s, H-1’); 2.33–2.40 ppm ([16H], m, Hc’
); 2.18 ppm ([8H], t, H-4’).
3.2.2. Synthesis of PAMAM G3 Dendrimer Substituted with 64 Glycidol Molecules (G3gl): The Megamer Macromolecular Core
Dendrimer PAMAM G3 in methanol (525 mg, 0.076 mmol in 5 mL; 15.2 mM solution) was added dropwise into a 25 mL round bottom flask containing glycidol (0.35 mL, 374 mg, 5.10 mmol) in 2 mL of methanol. The mixture was left at room temperature for 2 days. After two days, the solution was transferred into a nitrocellulose dialytic bag (MWcutoff = 3 kDa) and dialyzed against water for three days. Water was removed under reduced pressure by rotary evaporation and the resulting solid residue was dried under 0.2 mm Hg pressure overnight. A 90% yield (810 mg) of G3gl was obtained as a brown syrup and was characterized using 1H NMR spectroscopy. The isolated G3gl macromolecular core was found to be very soluble in water and in dimethyl sulfoxide.
; for atom numbering see Scheme 2
): chemical shift [ppm] (intensity, multiplicity, assignment): 7.80 ([60H], bs NH
); 4.47 ([128H], bs, OH
); 3.51 ([64H], bs, Hb’
); 3.33 ([128H] Ha’
+ [120H] PAMAM); 3.10 ([120H], bs, PAMAM); 2.65 ([120H], bs, PAMAM); 2.30–2.45 ([128H], m, Hc’
+ [4H], s, PAMAM); 2.20 ([120H], bs, PAMAM).
3.2.3. Synthesis of N-(4-Nitrophenoxycarbonyl) Nimesulid: N-(4-Nitrophenoxycarbonyl), N-(4-Nitro-2-Phenoxyphenyl) Methanesulfonamide, 1
Nimesulide (220 mg, 0.713 mmole) was dissolved in chloroform (5 mL) and triethylamine (TEA, 0.4 mL). Then 4-nitrophenylchloroformate was added (288 mg, 1.43 mmoles) in portions with continuous stirring and the mixture was left under nitrogen at room temperature for 24 hrs. The chloroform layer was washed three times with water (25 mL) and the solvent was removed under reduced pressure. The mixture was chromatographed with chloroform: ethyl acetate (7:1) on silica gel. 1 was eluted as first fraction as identified using NMR and MS. Crystals of 1 were grown in an NMR tube upon layering the solution (0.05 M) with hexane. The percent yield of 1 was 52.9% (250 mg).
Analytical data (for atom numbering see Scheme 3
1H NMR (CDCl3): 8.25 (d, J16–17 = 9.2 Hz, [2H], H-17,19); 8.04 (dd, J6–7 = 8.7 Hz, J6–4 = 2.5 Hz, [1H], H-6); 7.70 (d, [1H], H-7); 7.69 (d, 1H], H-4); 7.46 (t, [1H], H-10,12); 7.32 (t, J11-10 = 7.3 Hz, [1H], H-11); 7.27 (d, [2H], H-16,20); 7.10 (d, J9-10 = 7.5 Hz, [2H], H-9,13); 3.54 (s, [3H], H-1).
13C NMR (CDCl3): 154.7 (C-5); 154.2 (C-18 and C-8); 149.7 (C-14); 149.4 (C-3); 146.0 (C-15); 132.9 (C-6); 131.0 (C-10,12); 130.4 (C-2); 126.4 (C-11); 125.4 (C-17,19); 122.1 (C-16,20); 119.8 (C-9,13); 118.2 (C-7); 112.6 (C-4); 41.9 (C-1).
AuNPET LDI MS: 396.07 (90%, Au+), 237.50 (30%, [1 + 2H]2+); 393.93 (100%, Au2+); 496.04 [25%, 1 + Na]+; 590.90 (60%, Au3+). Theoretical molecular mass for C20N3O9SH15-473.05.
IR (in KBr): 1: ν(CO) = 1756 cm−1; νas(NO2) = 1526 cm−1; νas(SO2) = 1301 cm−1, for comparison N: νas(NO2) = 1522 cm−1; νas(SO2) = 1293 cm−1.
3.2.4. Synthesis of PAMAM G0-Bis-Carbonylnimesulide (G02N) and Single Fluorescein-Labeled PAMAM G0 (G0F)
150 mg (0.32 mmol) of 1 in chloroform (1 mL) was added to PAMAM G0 (83 mg, 0.16 mmol in 4 mL methanol) at room temperature with vigorous stirring for 24 h. Then the solution was heated to reflux for one hour and solvents were evaporated in vacuo. G02N was rinsed with chloroform and isolated as pure compound in 35% yield.
H NMR (DMSO-d6
): 8.14 (bs, [2H], NH
(G0); 7.80 (dd, [2H], H-6); 7.47 (d, J6–4
= 2.8 Hz, [2H], H-4); 7.31 (d, J6–7
= 9.4 Hz, [2H], H-7); 7.28 (t, [4H], H-10,12); 6.99 (t, J10–11
= 7.3 Hz, [2H], H-11); 6.84 (d, J9–10
= 7.9 Hz, [4H], H-9,13); 6.02 and 5.89 (both bs, [1H], NH
); 3.13–2.98 (bm, [12H], G0); 2.62 (bm, [12H], G0); 2.58 (s, [6H], CH3
-1); 2.41 (s, [4H], G0); 2.19 (t, [8H], G0, CH2
-CO-). IR (Figure A1
): ν(CO) = 1644 cm−1
Fluorescein labeled G0 was synthesized on a 0.10 mmolar scale. To the solution of 51.7 mg (100 µmoles) PAMAM G0 in methanol (2 mL), fluorescein isothiocyanate (FITC, 38.9 mg, 100 µmoles in 2 mL methanol) was added stepwise with vigorous stirring. The red precipitate of G0F was collected using filtration, washed with methanol and dried in vacuo.
H NMR (DMSO-d6
): 8.50–7.94 (bm, [5H], NH
); 7.17–6.25 (m, [9H], ar(F)); 3.53 (bs, [2H], G0); 3.23 (bs, [2H], G0); 3.09 (bs, [6H], G0); 2.70–2.50 (overlapped m, [14H], G0); 2.34 (bs, [4H], G0); 2.24–2.00 (overlapped m, [8H], G0). IR (Figure A1
): ν(CO) = 1634 cm−1
3.2.5. Synthesis of megamers G3gl-12G02N and G3gl-4G0F
: 100 mg G3gl
(8.5 µmoles) was dissolved in 2 mL of DMSO and 0.5 mL of TEA. To this solution, solid NPCF (28.2 mg, 102 µmoles) was added with vigorous stirring. The obtained mixture was immediately used to react with 119 mg (100 µmoles) of G02N
in 2 mL DMSO at room temperature overnight. Then the mixture was dialyzed in nitrocellulose tubing (MWcutoff
= 3 kDa) against water for 4 days. Water was removed under reduced pressure and the 1
H NMR spectrum was recorded. Based upon integral intensity of N
versus PAMAM resonances the stoichiometry of conjugate was determined to be G3gl–12G02N
(see Figure 2
1H NMR (DMSO-d6): N resonances: 7.98 (dd, J6–7 = 8.8 Hz, J6–4 = 2.5 Hz, [24H], H-6); 7.63 (d, [24H], H-7); 7.52 (d, [24H], H-4); 7.44 (t, [48H], H-10,12); 7.21 (t, J11–10 = 6.4 Hz, [24H], H-11); 7.09 (d, J9–10 = 7.1 Hz, [48H], H-9,13); 3.05 (s, [72H], H-1), PAMAM G3gl + G0 resonances: 3.53 (bs, [64H], Hb’); 3.33, 3.12, 2.97, 2.85, 2.67, 2.57, 2.45, 2.21 (unresolved broad resonances, ca [1200H], theor. [1172H] with contribution from G3 [484H], Ha’ and Hc’ of 64 gl [256H], and 12 G0 [432H]).
The weight-averaged molecular weight MW by GPC was ca 22.4 kDa with 1.63 Mw/Mn dispersity (and 37 kDa from MZ) vs theoretical average molecular weight 26,249 kDa.
The G3G02N conjugate was tested for toxicity against cancer cells. Additionally, ca 1 µmol of G3G02N (26 mg) was labeled with one equivalent of FITC and used for confocal microscopic monitoring in cell lines (G3G02N*, vide infra).
G3G0F: 31 mg G3gl (2.7 µmoles) was dissolved in 1.5 mL of DMSO and 0.3 mL of TEA. To this solution, solid NPCF (2.3 mg, 11.4 µmoles) was added with vigorous stirring. Then, 0.115 mL of 0.10 M G0F solution in DMSO (11.5 µmoles) was added and the mixture left for 24 h at room temperature. Then the mixture was dialyzed against water for 3 days in the dark. The solvents were removed under reduced pressure, yielding 35 mg of G3gl. G3gl was conjugated with 4 equivalents of G0F based upon integral intensity of F aromatic resonances versus total intensity of G3 and G0 resonances. The estimated yield was 84%. The average molecular weight determined using GPC was ca 15.5 kDa vs theoretical molecular weight 15,383 for G3–4G0F stoichiometry. The aqueous solution of G3G0F was used to deposit the monomolecular layer in mica to estimate molecular size using AFM (vide infra).
1H NMR (DMSO-d6): 7.91 + 7.82 (bs, bs, ca [70H], NH); 6.71–6.50 (unresolved multiplets, [36H], aromatic CH from fluorescein); 4.47 (bs, ca [110H], OH); PAMAM G3gl + G0 resonances: 3.57–2.13 (total integrity ca [950H] versus theoretical [938H] with contribution from G3gl [824H] and 4 G0 [144H].