Cell-Penetrating Cyclodextrins
FULL PAPER
Lyophilization afforded a pale-yellow solid (0.421 g, 79%). 1H NMR
(500 MHz, D2O, 25 8C): d=5.21 ꢀ5.11 (brs, 8H; H1), 3.90 (brt, 16H; H3,
H5), 3.58 (brd, 8H; H2), 3.52 (brt, 8H; H4), 3.01 (brt, 16H; -CH2NH2),
2.95 (br, 16H; H6), 2.76 (br, 16H; -NHCH2-), 1.87 ppm (brt, 16H;
-NHCH2CH2-); 13C NMR (63 MHz, D2O, 25 8C): d=101.0–99.6 (C1),
81.4–80.1 (C4), 72.5 (C3), 71.9 (C2), 69.8 (C5), 49.1 (C6), 46.6 (-NHCH2),
37.9 (-CH2NH2), 26.2 ppm (-NHCH2CH2-); ESI-MS: m/z (%): 219.3 (28)
[M+8H]8+; MALDI-TOF MS: m/z (%): 1768.0 (50) [M+Na]+, 1746.0
(100) [M+H]+, 1671.9 (77) [([M+H]+ꢀH2NCH2CH2CH2NH2 =74)ꢁ
M1], 1597.9 (24) [(M1+H+ꢀ74)ꢁM2], 1523.8 (5) [M2+H+ꢀ74].
(-NHCH2CH2CH2CH2CH2CH2NH-C=NH); ESI-MS: m/z (%): 303.5 (50)
[M+7H]7+
; +
MALDI-TOF MS: m/z (%): 2159.5 (43) ([M+H]+
C(NH)NH2 =43)+, 2117.5 (29) [M+H]+, 1959.4 (61) ([M+H]+ꢀH2N-
C(NH)HN
43].
A
Octakis[6-(2-guanidinoethylamino)-6-deoxy]-gCD (geg): Octakis[6-(2-
aminoethylamino)-6-deoxy]-gCD (ge) (0.163 g, 0.1 mmol) was dispersed
in dry dimethylformamide (5 mL) and to the mixture 1H-pyrazolecarbox-
amidine hydrochloride (0.352 g, 2.4 mmol) and DIPEA (547 mL,
3.2 mmol) were added in three equal portions over a period of 72 h. The
reaction mixture was then reacted and treated as above. Lyophilization
afforded a pale-yellow solid (0.120 g, 61%). 1H NMR (500 MHz, D2O,
258C): d=5.15 (br, 8H; H1), 3.88 (br, 16H; H3, H5), 3.58 (br, 16H; H2,
H4), 3.29 (br, 16H; -CH2NH-C=NH), 3.06 (br, 8H; H6), 2.88 ppm (br,
24H; -NHCH2-, H6’); 13C NMR (126 MHz, D2O, 25 8C): d=157.1 (NH=
C-NH2), 99.6 (C1), 79.9 (C4), 71.7 (C3), 71.3 (C2), 69.6 (C5), 47.7 (C6),
46.9 (-NHCH2-), 39.9 ppm (-CH2NH-C=NH); ESI-MS: m/z (%): 247.2
(44) [M+8H]8+; MALDI-TOF MS: m/z (%): 1992.2 (42) [M+Na]+,
Heptakis[6-(2-guanidinoethylamino)-6-deoxy]-bCD
(beg):
Heptakis-
[6-(2-aminoethylamino)-6-deoxy]-bCD (be) (0.208 g, 0.12 mmol) was dis-
persed in dry dimethylformamide (10 mL) and to the mixture 1H-pyrazo-
lecarboxamidine hydrochloride (1.23 g, 8.4 mmol) and N,N-diisopropyl-
ethylamine (DIPEA) (1.65 mL, 9.7 mmol) were added in three equal por-
tions over a period of 72 h. During this period the mixture was stirred
continuously at 708C under a N2 atmosphere. After cooling, diethyl ether
(150 mL) was added drop-wise and the suspension formed was stirred for
2 h at RT. The solvent was decanted and the collected sticky solid was
dissolved in water (2 mL). Addition of ethanol (200 mL) resulted in the
precipitation of a white substance that was filtered off, washed with etha-
nol (50 mL) and dried under vacuum. This precipitate was dissolved in
doubly distilled water (3 mL), the pH was adjusted to 7 with hydrochloric
acid (1n) and the solution was treated with Dowex Type I resin (Clꢀ ex-
changer) for 1 h. The resin was removed by filtration and the solution
was kept inside dialysis tubing for 72 h to remove the low-molecular-
weight impurities. Subsequent lyophilization afforded a pale-yellow solid
(0.070 g, 27%). 1H NMR (500 MHz, D2O, 25 8C): d=5.04 (brs, 7H; H1),
3.89 (brs, 14H; H3, H5), 3.58–3.41 (brm, 21H; H2, H4, H6), 3.27 (brs,
14H; -CH2NH-C=NH), 2.81 ppm (brt, 21H; H6’, -NHCH2-); 13C NMR
(126 MHz, D2O, 25 8C): d=159.9 (NH=C-NH2), 104.3 (C1), 85.0 (C4),
75.5 (C3), 74.7 (C2), 73.3 (C5), 51.5 (C6), 50.7 (-NHCH2-), 43.6 ppm
+
+
+
C
1970.2 (100) [M+H] , 1954.2 (79) ([M+H ꢀNH2 ), 1869.1 (73) [M+H
C
ꢀ
[H2N-C(NH)HN
(CH2)2NH2 ]=102ꢁM1], 1852 (44) [M1ꢀ16].
Octakis[6-(3-guanidinopropylamino)-6-deoxy]-gCD (gpg): Octakis[6-(3-
aminopropylamino)-6-deoxy]-bCD (gp) (0.106 g, 0.05 mmol) was dis-
persed in dry dimethylformamide (5 mL) and to the mixture 1H-pyrazo-
lecarboxamidine hydrochloride (0.236 g, 1.6 mmol) and DIPEA (370 mL,
2.1 mmol) were added in three equal portions over a period of 72 h. The
reaction mixture was then reacted and treated as above. Lyophilization
afforded a pale-yellow solid (0.0125 g, 13%). 1H NMR (500 MHz, D2O,
258C): d=5.24–5.14 (br, 8H; H1), 3.99 (br, 8H; H5), 3.90 (br, 8H; H3),
3.59 (br, 8H; H2), 3.55 (br, 8H; H4), 3.23 (br, 16H; -CH2NH-C=NH),
3.03 (br, 16H; H6), 2.87 (br, 16H; -NHCH2-), 1.93–1.88 ppm (br, 16H;
-NHCH2CH2-); 13C NMR (126 MHz, D2O, 25 8C): d=156.8 (NH=C-
NH2), 98.5 (C1), 79.3 (C4), 71.1 (C3), 70.7 (C2), 68.0 (C5), 48.1 (C6), 45.3
(-NHCH2-), 37.9 (-CH2NH-C=NH), 25.0 ppm (-NHCH2CH2-); ESI-MS:
m/z (%): 261.3 (5) [M+8H]8+; MALDI-TOF MS: m/z (%): 2082.4 (23)
(-CH2NH-C=NH); ESI-MS: m/z (%): 247.3 (37) [M+7H]7+; MALDI-
+
TOF MS m/z (%): 1781 (78) ([M+H]+ +2[CNH2 =28]) , 1752.1 (100)
C
[M+H]+, 1966.0 (36) [M+H+ꢀH2N-C(NH)HN
Fluorescein labeling
A
+
[([M+H]+ +CNH2 =28)ꢁM1], 1736.0 (52) (M1ꢀNH2 ) , 1724.1 (34)
C
C
+
[M+H] , 1665 (33) ([MꢀH2NCH(NH)NH) ]+ =58).
C
Heptakis[6-(3-guanidinopropylamino)-6-deoxy]-bCD (bpg): Heptakis[6-
(3-aminopropylamino)-6-deoxy]-bCD (bp) (0.200 g, 0.11 mmol) was dis-
persed in dry dimethylformamide (5 mL) and to the mixture 1H-pyrazo-
lecarboxamidine hydrochloride (0.678 g, 4.6 mmol) and DIPEA
(0.93 mL, 5.5 mmol) were added in three equal portions over a period of
72 h. The reaction mixture was then reacted and treated as above. Lyo-
Mono(6-amino-6-deoxy)-bCD·Fl (bNH2·Fl): Mono(6-amino-6-deoxy)-
bCD[18] (0.057 g, 50 mmol) was dispersed in dry DMF (5 mL) mixed with
DIPEA (9 mL, 50 mmol), to which a solution of fluorescein isothiocyanate
(isomer I) (0.001 g, 2.5 mmol) in DMF (5 mL) was added drop-wise over
1 h. The mixture was stirred and heated at 708C under N2 for 3 h. Cool-
ing to RT and solvent removal in vacuo gave a solid that was dissolved in
doubly distilled water (5 mL) and the pH was adjusted to 11 using 1n
ammonium hydroxide. The solution was extracted with chloroform (3
50 mL) to remove DIPEA and the pH was adjusted to 7 with 1n HCl, so
that unreacted fluorescein precipitated and could be removed carefully
after centrifugation. The final solution was concentrated to dryness, dis-
solved in doubly distilled water (5 mL) and dialysed. After water removal
philization afforded
a
pale-yellow solid (0.027 g, 13%). 1H NMR
(500 MHz, D2O, 25 8C): d=5.34–4.90 (br, 7H; H1), 3.84 (br, 14H; H3,
H5), 3.50 (br, 7H; H2), 3.36 (br, 7H; H4), 3.16 (br, 14H; -CH2NH-C=
NH), 2.94–2.73 (br, 14H; H6), 2.61 (br, 14H; -NHCH2-), 1.72 ppm (br,
14H; -NHCH2CH2); 13C NMR (126 MHz, D2O, 25 8C): d=157.9 (NH=C-
NH2), 101.6 (C1), 83.6 (C4), 73.1 (C3), 72.7 (C5), 72.3 (C2), 49.2 (C6),
45.7 (-NHCH2-), 38.7 (-CH2NH-C=NH), 27.8 ppm (-NHCH2CH2-); ESI-
MS: m/z (%): 261.3 (97) [M+7H]7+, 304.3 (22) [M+6H]6+; MALDI-TOF
MS m/z (%): 1851.0 (70) [M+CNH2 =28]+, 1823.1 (100) [M+H]+, 1806.1
a
yellow solid was obtained (0.055 g) (fluorescein content ~20%).
1H NMR (500 MHz, D2O, 25 8C): d=7.85 (brs, Hfl), 7.75 (brm, Hfl), 7.20
(brs, Hfl), 6.83 (brd, Hfl), 6.72 (brd, Hfl), 4.99 (d, J=3.4 Hz, 7H; H1),
3.84 (t, J=9.5 Hz, 7H; H3), 3.80–3.75 (brm, 21H; H5, H6), 3.57 (dd, J=
3.4, 9.5 Hz, 7H; H2), 3.50 (t, J=9.5 Hz, 7H; H4), 3.12 (brs, 14H; H8),
3.04–2.93 ppm (br, 28H; H6, H7).
+
+
+
C
C
(46) [MꢀNH2 ] , 1790 (35) [Mꢀ2NH2 ) , 1764 (23) [M+H ꢀguani-
dine=59], 1734, 1692.
Heptakis[6-(6-guanidinohexylamino)-6-deoxy]-bCD (bhg): Heptakis[6-(6-
aminohexylamino)-6-deoxy]-bCD (bh) (0.091 g, 0.05 mmol) was dis-
persed in dry dimethylformamide (3 mL) and to the mixture 1H-pyrazo-
lecarboxamidine hydrochloride (0.205 g, 1.4 mmol) and DIPEA (300 mL,
1.75 mmol) were added in four equal portions over a period of 96 h. The
reaction mixture was then reacted and treated as above. Lyophilization
afforded a white solid that includes approximately 50% of free diamine
Heptakis[6-(2-aminoethylamino)-6-deoxy]-bCD·Fl (be·Fl): The amine be
(0.084 g, 50 mmol) was dispersed in dry DMF (5 mL) containing DIPEA
(60 mL, 0.35 mmol), to which a solution of fluorescein isothiocyanate
(0.001 g, 2.5 mmol) in DMF (10 mL) was added drop-wise over a period
of 1 h. After identical treatment to that described above an orange-red
product was obtained (0.080 g) (fluorescein content ~5%). 1H NMR
(500 MHz, D2O, 25 8C): d=8.28–8.22 (m, Hfl), 8.14 (brs, Hfl), 7.93 (d, J=
7 Hz, Hfl), 7.60 (brt, Hfl), 7.51 (brt, Hfl), 5.15 (brs, 7H; H1), 3.97 (br,
14H; H3, H5), 3.68 (brd, 14H; H2, H4), 3.12 (brs, 14H; H8), 3.04–
2.93 ppm (br, 28H; H6, H7).
1
(0.075 g, 30%). H NMR (500 MHz, D2O, 25 8C): d = 5.05 (br, 7H; H1),
4.00–3.76 (br, 14H; H3, H5), 3.61 (br, 7H; H2), 3.46 (br, 7H; H4), 3.10
(br, 14H; -CH2NH-C=NH), 2.95 (br, 14H; H6), 2.71 (br, 14H; -NHCH2-
), 1.52 (br, 28H; -NHCH2CH2CH2CH2CH2CH2NH-C=NH), 1.30 ppm (br,
28H; -NHCH2CH2CH2CH2CH2CH2NH-C=NH); 13C NMR (126 MHz,
D2O, 25 8C): d = 156.6 (NH=C-NH2), 100.8 (C1), 83.6–82.4 (C4), 71.9
(C3), 70.7 (C2), 68.4 (C5), 48.5 (-NHCH2-), 48.1 (C6), 40.3 (-CH2NH-C=
Heptakis[6-(3-aminopropylamino)-6-deoxy]-bCD·Fl (bp·Fl): The amine
bp (0.036 g, 20 mmol) was dispersed in dry DMF (6 mL) containing
DIPEA (24 mL, 0.14 mmol) and to it, fluorescein isothiocyanate was
added (0.0004 g, 1 mmol) in DMF (2 mL). After identical treatment to
NH),
27.0
(-NHCH2CH2CH2CH2CH2CH2NH-C=NH),
25.0 ppm
Chem. Eur. J. 2008, 14, 4188 – 4200
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4197