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chlorhexidine for preservation, Mw ꢁ972 kDamolÀ1) were kindly
washed with saturated brine (50 mL). After drying with anhydrous
Na2SO4, the solvent was evaporated under reduced pressure, and
the product was allowed to dry under high vacuum overnight. The
intermediate was used without further purification. Yield: 3.20 g
provided by Lohmann & Rauscher (Neuwied, Germany).
1
Synthesis
(8.9 mmol, 40%) of a dark red solid. H NMR (400.13 MHz, CDCl3):
3
d=9.62 (s, br, 1H; CarÀOH), 8.23 (d, J(H,H)=7 Hz, 1H; CarH), 7.71
(3-Azidopropyl)triethoxysilane (1)
3
3
(t, J(H,H)=7 Hz, 1H; CarH), 7.65 (t, J(H,H)=7 Hz, 1H; CarH), 7.28 (d,
3J(H,H)=7 Hz, 1H; CarH), 6.96 (d, J(H,H)=7 Hz, 2H; CarH), 6.87 (s,
3
A
suspension of TBAB (3.20 g, 9.9 mmol) and NaN3 (10.0 g,
3
3
2H; CarH), 6.79 (d, J(H,H)=7 Hz, 2H; CarH), 3.97 (q, J(H,H)=7 Hz,
2H; CH2), 0.89 ppm (t, 3J(H,H)=7.0 Hz, 3H; CH3); 13C NMR
(100.61 MHz, CDCl3): d=175.6 (C=O), 165.2 (C=O), 157.9 (CarÀO),
156.2 (Cq), 134.1 (Cq), 132.5 (CarH), 131.2 (CarH), 130.6 (CarH), 130.2
(CarH), 129.8 (CarH), 122.1 (CarH), 114.9 (Cq), 103.7 (CarH), 61.4 (CH2),
13.6 ppm (CH3); FTIR: n˜ =2980 (CÀH), 1715 (C=O), 1639, 1567,
1452, 1381, 1247, 1202, 1103, 1076, 912, 845 cmÀ1; UV/Vis (MeOH):
lmax =502 nm; TLC (acetone): Rf =0.91; m.p. 129–1308C (CH2Cl2).
153.8 mmol) in anhydrous acetonitrile was prepared and stirred
under N2. (3-Chloropropyl)triethoxysilane (10 mL, 41.5 mmol) was
added with constant flushing with N2, and the reaction mixture
was allowed to stir for 18 h under reflux conditions. The reaction
mixture was filtered to remove the bulk of the salt. After evapora-
tion of the solvent, the product was dissolved in dichloromethane
(50 mL), filtered through Celiteꢃ, and washed with water and brine
(2ꢄ20 mL each). The organic phase was dried carefully over
Na2SO4 and evaporated. The remaining colorless oil (which con-
tained a small portion of oily yellowish drops) was allowed to
stand overnight at RT for complete separation of the phases. The
yellowish phase was discarded and the product was dried under
high vacuum at RT for 5 h. Yield: 6.15 g (24.9 mmol, 60%) of color-
Ethyl 2-[3-oxo-6-(prop-2-yn-1-yloxy)-3H-xanthen-9-yl]benzoate
(4)
DMF was added to a suspension of 3 (3.20 g, 8.9 mmol) in acetone
(50 mL) until complete dissolution of the reagent. Finely ground
K2CO3 (1.85 g, 13.4 mmol) was added followed by the addition of
propargyl bromide solution (80 wt% in toluene, 1.50 mL,
13.4 mmol) at RT. The suspension was allowed to stir at 608C for
18 h. The reaction mixture was poured into cold distilled water
(300 mL), and the voluminous orange precipitate was collected by
filtration. The raw product was washed with water until the initially
brownish filtrate became colorless, followed by careful washing
with cold acetone (20 mL). After a final washing step with water
(20 mL), the product was recrystallized from acetone to remove
traces of remaining reagent impurities. The obtained crystals were
washed with cold acetone and dried under high vacuum at RT
overnight. The crystallization procedure was repeated successively
with the mother liquor. Yield: 3.05 g (7.6 mmol, 86% combined
1
3
less oil. H NMR (400.13 MHz, CDCl3): d=3.84 (q, J(H,H)=7 Hz, 6H;
3
CH2), 3.28 (t, J(H,H)=7 Hz, 2H; g-CH2), 1.73 (m, 2H; b-CH2), 1.24 (t,
3J(H,H)=7 Hz, 9H; CH3), 0.69 ppm (m, 2H; a-CH2); 13C NMR
(100.61 MHz, CDCl3): d=58.4 (CH2), 53.8 (g-CH2), 22.7 (b-CH2), 18.3
(CH3), 7.6 ppm (a-CH2); 29Si NMR (79.49 MHz, CDCl3): d=
À46.2 ppm; FTIR: n˜ =2973 (CÀH), 2929 (CÀH), 2879 (CÀH), 2094
(N3), 1444, 1390, 1242, 1166, 1101, 1074, 954, 775 cmÀ1
.
Preparation of preactivated BC-N3 (2)
A mixture of 1 (4.0 mL, 16.2 mmol) in acetone (32 mL) and H2O
(4 mL) was prepared in a wide Petri dish. A wet BC sheet (Ø=
13 cm, ꢁ505 mg of dry cellulose, ꢁ3.1 mmol anhydroglucose unit
(AGU)) was put into the mixture, and it was allowed to shake at RT
for 18 h. Oily drops were formed during solvent exchange. The BC
sheet was washed with acetone to remove nonadsorbed reagent
on the surface and then predried at 408C for 3 h. The predried
sheet was subsequently cured at 1058C for 2 h. To remove the
noncondensed reagent, the silane-grafted sheet was subjected to
Soxhlet extraction with EtOH for 18 h followed by drying at 1058C
for 1 h. Yield: 699 mg of a white BC sheet. 13C NMR (100.68 MHz,
CP/TOSS, swollen in H2O): d=108.3–105.0 (AGU-C1), 92.6–88.9
(AGU-C4), 79.5–71.3 (AGU-C2+C3+C5), 68.2–65.5 (AGU-C6), 56.8–
54.2 (g-CH2), 25.7–23.0 (b-CH2), 14.2–9.8 ppm (a-CH2); 29Si NMR
(79.53 MHz CP/TOSS, swollen in H2O): d=À54.0 to À59.3, À61.1 to
À71.5 ppm; FTIR: n˜ =3346 (OH), 2895 (CÀH), 2098 (N3), 1428, 1314,
1160, 1107, 1054, 1031, 1003, 664 cmÀ1; elemental analysis (%)
found C 42.54, H 6.32, N 3.17, S <0.05, O 41.71; grafting amount of
1
yield) of an orange solid. H NMR (400.13 MHz, CDCl3): d=8.23 (d,
3J(H,H)=7 Hz, 1H; CarH), 7.71 (t, 3J(H,H)=7 Hz, 1H; CarH), 7.65 (t,
3
3J(H,H)=7 Hz, 1H; CarH), 7.28 (d, J(H,H)=7 Hz, 1H; CarH), 7.05 (d,
3
4J(H,H)=2 Hz, 1H; CarH), 6.91 (d, J(H,H)=9 Hz, 1H; CarH), 6.85 (d,
3J(H,H)=10 Hz, 1H; =CH), 6.79 (dd, 3J(H,H)=9 Hz, 4J(H,H)=2 Hz,
3
1H; CarH), 6.54 (d, J(H,H)=10 Hz, 1H; =CH), 6.48 (s, 1H; =CH), 4.78
(d, 4J(H,H)=2 Hz, 2H; CH2), 4.01 (m, 2H; CH2), 2.59 (t, 4J(H,H)=
2 Hz, 1H; ꢀCH), 0.95 ppm (t, 3J(H,H)=7 Hz, 3H; CH3); 13C NMR
(100.61 MHz, CDCl3): d=185.5 (C=O), 165.3 (C=O), 161.8 (CarÀO),
158.8 (CqÀO), 154.0 (CarÀO), 150.2 (Cq), 134.1 (Cq), 132.5 (CarH), 131.2
(CarH), 130.8 (Cq), 130.4 (CarH), 130.3 (=CH), 129.9 (=CH), 129.7
(CarH), 129.0 (CarH), 118.1 (Cq), 115.6 (Cq), 113.7 (CarH), 105.8 (=CH),
101.6 (CarH), 77.1 (ꢀCÀ), 76.8 (ꢀCH), 61.4 (CH2), 56.4 (CH2), 13.6 ppm
(CH3); FTIR: n˜ =3198 (ꢀCH), 2121 (CꢀC), 1718 (C=O), 1591, 1509,
1480, 1441, 1247, 1210, 1107, 856 cmÀ1; UV/Vis (MeOH): lmax
456 nm; TLC (acetone): Rf =0.73; m.p. 222–2248C (acetone).
=
ÀN3 calculated from the N content: 755 mmolgÀ1
.
Ethyl 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate (3)
Click reaction of BC-N3 and 4 to yield fluorescein-modified BC
(5)
Fluorescein (free acid, 7.46 g, 22.5 mmol) was suspended in EtOH
(50 mL) and concentrated H2SO4 (2 mL) as the catalyst was added
slowly with stirring at RT. The suspension was heated to reflux for
6 h. An additional funnel packed with 3 ꢂ molecular sieves (60 mL)
was located between the round-bottomed flask and the condenser
to dry the condensed ethanol continuously. After cooling, the reac-
tion mixture was concentrated on a rotary evaporator, water
(50 mL) was added, and the suspension was extracted with CH2Cl2
(3ꢄ50 mL). The organic phases were combined, washed carefully
with saturated NaHCO3 solution and water until neutral, and
Azide-modified BC 2 (107 mg) was cut into pieces of approximately
1.5ꢄ1.5 cm and suspended in ACN (10 mL) in a resealable glass
bottle. Alkyne-modified fluorescein derivative
4
(10.3 mg,
25.9 mmol) and N,N-diisopropylethylamine (100 mL, 1.02 mmol)
were added. The reaction mixture was degassed by flushing with
Ar for 5 min, CuI (7 mg, 37 mmol) was added as a catalyst and the
bottle was capped and sealed tightly with Parafilm. After the glass
bottle was covered with aluminum foil to protect against light, the
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