Organic & Biomolecular Chemistry
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1H), 4.59 (s, 2H), 4.46 (s, 1H), 4.22 (s, 4H), 4.13 (dd, J = 16.3, 2-yl)vinyl)-4H-pyran-2-yl)vinyl)-7-(diphenylamino)-9-(2-(2-ethoxy-
8.9 Hz, 1H), 3.32–3.19 (m, 6H), 3.15–3.01 (m, 5H), 2.87–2.80 ethoxy)ethyl)-9H-fluoren-9-yl)propanoic acid (K, 50 mg,
(m, 2H), 2.73 (dtd, J = 24.7, 9.5, 5.9 Hz, 2H), 2.47 (d, J = 0.8 Hz, 0.025 mmol) was dissolved in DMF (3 mL) under N2; then,
3H), 2.23 (s, 4H), 2.12 (tt, J = 13.5, 6.6 Hz, 2H), 1.64 (s, 3H), N-hydroxysuccinimide (5 mg, 0.043 mmol) was added to the
1.49–1.33 (m, 2H), 1.22 (d, J = 7.4 Hz, 2H), 0.99 (t, J = 7.0 Hz, mixture at room temperature. Next N,N′-dicyclohexyl-
3H). 13C NMR (126 MHz, DMSO) δ (ppm): 172.42, 171.80, carbodiimide (8 mg, 0.043 mmol) was added to the mixture
171.77, 171.80, 171.77, 171.27, 170.88, 164.57, 160.53, 157.15, and stirred at room temperature for 36 h. The mixture was
157.12, 151.47, 150.02, 148.05, 147.65, 142.73, 138.49, 134.92, then filtered and redissolved in a minimum volume of DMF,
133.73, 129.97, 129.39, 128.43, 126.45, 124.21, 123.54, 122.84, and then filtered again. The product was dried under vacuum
122.11, 120.29, 119.02, 118.43, 115.95, 107.19, 106.27, 69.78, to afford red oil (45 mg, 85% yield). 1H NMR (400 MHz, CDCl3)
69.58, 67.13, 65.90, 56.03, 55.27, 52.64, 49.06, 43.37, 41.05, δ (ppm): 7.60 (d, J = 8.4 Hz, 2H), 7.56–7.43 (m, 8H), 7.34–7.18
39.96, 39.88, 39.79, 39.62, 39.51, 39.46, 38.85, 38.71, 30.22, (m, 9H), 7.17–6.943 (m, 16H), 6.82–6.69 (m, 2H), 6.58 (d, J =
25.68, 24.95, 19.86, 15.50. HRMS (MALDI-DTL, [M + H]+) calcd 2.0 Hz, 1H), 3.69 (d, J = 4.7 Hz, 76H), 3.51 (q, J = 7.1 Hz, 2H),
for C72H80N12O11+ 1289.6149, found 1289.6109.
3.24 (hept, J = 5.5 Hz, 6H), 2.87 (m, 8H), 2.39–2.09 (m, 4H),
1.81–1.45 (m, 6H), 1.10 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ (ppm): 173.20, 169.88, 169.47, 161.96, 159.35, 156.35,
150.42, 149.00, 148.63, 147.66, 143.32, 138.38, 133.95, 132.72,
129.41, 127.82, 124.74, 123.32, 122.96, 122.48, 121.22, 119.71,
117.68, 117.05, 115.18, 115.09, 106.94, 106.39, 70.59,
70.54, 70.47, 70.39, 70.09, 69.63, 69.01, 67.31, 66.54, 63.43,
Synthesis of 3-(2-((E)-2-(4-(dicyanomethylene)-6-((E)-
2-(7-(diphenylamino)-9,9-di(3,6,9,12,15,18,21,24,27,30-
decaoxadotriacontyl)-9H-fluoren-2-yl)vinyl)-4H-pyran-
2-yl)vinyl)-7-(diphenylamino)-9-(2-(2-ethoxyethoxy)ethyl)-
9H-fluoren-9-yl)propanoic acid (K)
A
mixture of (E)-3-(2-(2-(4-(dicyanomethylene)-6-methyl-4H- 59.01, 52.28, 50.24, 39.05, 34.65, 29.60, 25.55, 15.07.
pyran-2-yl)vinyl)-7-(diphenylamino)-9-(2-(2-ethoxyethoxy)ethyl)- HRMS-MALDI-DTL: high resolution MS shows a series of ions
9H-fluoren-9-yl)propanoic acid (H, 0.12 g, 0.175 mmol), and centered around m/z 2052 [C79H69N5O7(C2H4O)20]+ which are
7-(diphenylamino)-9,9-di(3,6,9,12,15,18,21,24,27,30 decaoxado- fragments related to the various ethoxylate oligomers that
triacontyl)-9H-fluorene-2-carbaldehyde (J, 0.15 g, 0.115 mmol) differ by Δm/z 44, an ethoxy unit.
were dissolved in acetonitrile (15 mL). After adding piperidine
(0.5 mL) slowly via syringe while stirring, the reaction mixture
Synthesis of fluorene-RGD Conjugate (2a)
was refluxed for 48 h. The solution color gradually changed Fluorene-NHS 2 (20 mg, 0.010 mmol) and c(RGDfK) (5 mg,
from light yellow to deep red. The solvent was removed under 0.008 mmol) were dissolved in DMF (1.5 mL), and the solution
vacuum, and purification was carried out by silica gel column stirred for 24 h under nitrogen at room temperature. Solvent
chromatography using first CH2Cl2/MeOH (95 : 15) as eluent, was removed under vacuum, and crude product was then
then CH2Cl2/MeOH (90 : 10) to obtain red solid (0.12 g, 58% passed through a size exclusion column (12 cm length,
1
yield). H NMR (400 MHz, CDCl3) δ (ppm): 7.61 (d, J = 8.4 Hz, Bio-Rad Econo-Pac 10DG, MWCO) in pure water to allow
2H), 7.57–7.44 (m, 8H), 7.34–7.18 (m, 9H), 7.18–6.94 (m, 16H), collection of pure RGD conjugate fractions, yielding a red oil
6.80–6.66 (m, 2H), 6.54 (d, J = 2.0 Hz, 1H), 3.64 (d, J = 4.7 Hz, (15 mg, 60% yield). HRMS-MALDI-DTL: high resolution
76H), 3.41 (q, J = 7.1 Hz, 2H), 3.24 (hept, J = 5.5 Hz, 6H), 2.87 MS shows
a series of ions centered around m/z 2540
(ddq, J = 24.6, 9.7, 5.6, 4.7 Hz, 4H), 2.39–2.09 (m, 4H), [C102H105N13O11(C2H4O)20]+ which are fragments related to
1.81–1.45 (m, 6H), 1.13 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, the various ethoxylate oligomers that differ by Δm/z 44, an
CDCl3) δ (ppm): 176.99, 173.20, 161.96, 159.35, 156.30, 150.45, ethoxy unit.
149.03, 148.67, 147.49, 143.35, 138.36, 133.96, 132.74, 129.38,
127.78, 124.60, 123.35, 122.93, 122.52, 121.23, 119.72, 117.70,
117.07, 115.14, 115.05, 106.96, 106.41.70.56, 70.50, 70.43,
Synthesis of (E)-2-(2-(2-(7-(diphenylamino)-9,9-dihexyl-9H-
fluoren-2-yl)vinyl)-6-methyl-4H-pyran-4-ylidene)malononitrile (N)
70.31, 70.10, 69.67, 69.01, 67.34, 66.58, 63.46, 59.01, 58.97, 7-(Diphenylamino)-9,9-dihexyl-9H-fluorene-2-carbaldehyde
52.32, 50.24, 39.09, 34.65, 29.68, 15.09. HRMS-MALDI-DTL: (M, 0.2 g, 0.37 mmol) was mixed with the pyran (0.255 g,
high resolution MS shows a series of ions centered around m/z 1.48 mmol) and dissolved in 100 mL distilled CH3CN. Piper-
1955 [C75H66N4O5(C2H4O)20]+ which are fragments related to idine (0.15 mL) was added and the reaction mixture refluxed
the various ethoxylate oligomers that differ by Δm/z 44, an for 72 h to yield reddish solution. The solvent was removed
ethoxy unit.
under reduced pressure and the crude product purified on
silica gel using hexanes/ethyl acetate (7 : 3) to yield brown solid
(0.25 g, 48%). δ H (400 MHz, Chloroform-d) 0.36–0.86 (10 H,
m), 0.84–1.26 (12 H, m), 1.61–1.94 (4 H, tt, J 13.2, 6.8),
2.16–2.66 (3 H, s), 4.98–5.39 (1 H, d, J 0.7), 6.44–6.51 (1 H, d,
J 1.5), 6.61–6.67 (1 H, m), 6.67–6.72 (0 H, s), 6.91–7.00 (3 H, td,
J 8.4, 8.0, 1.7), 7.01–7.09 (4 H, m), 7.13–7.24 (6 H, m),
Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(2-((E)-
2-(4-(dicyanomethylene)-6-((E)-2-(7-(diphenylamino)-
9,9-di(3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-
9H-fluoren-2-yl)vinyl)-4H-pyran-2-yl)vinyl)-7-(diphenylamino)-
9-(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-9-yl)propanoate (2)
(3-(2-((E)-2-(4-(Dicyanomethylene)-6-((E)-2-(7-(diphenylamino)- 7.34–7.39 (1 H, s), 7.39–7.45 (1 H, d, J 8.5), 7.45–7.52 (1 H, m),
9,9-di(3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-9H-fluoren- 7.52–7.59 (1 H, d, J 7.9). 13C NMR (101 MHz, CDCl3) δ 162.31,
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