J. Wrzesien, D. Graham / Tetrahedron 68 (2012) 1230e1240
1237
recorded on a Bruker DPX 400 MHz spectrometer with the appro-
priate solvent peak as a reference. J values are quoted in hertz. Mass
spectrometry was carried out as a service by the EPSRC National
Mass Spectrometry Service Centre, Swansea.
4.2.3. 2-{9-[5-Amino-2-(ethoxycarbonyl)phenyl]-3-oxo-3H-
xanthen-6-yloxy}acetic acid (3) (2-{9-[2-(ethoxycarbonyl)phenyl]-3-
oxo-3H-xanthen-6-yloxy}acetic acid (9)). TFA (3 ml) was added to
the solution of 2 (0.79 g, 1.61 mmol) or 8 (0.66 g, 1.40 mmol) in
DCM (3 ml). After stirring for 3 h at room temperature DCM and
TFA were evaporated. The residue was solidified by addition of
diethyl ether, then it was filtered and washed with the same sol-
vent giving after drying 0.36 g of the title compound as an orange
solid (52%) (3) or 0.33 g of the title compound as a dark yellow solid
(57%) (9).
4.2. Synthesis of the 6-aminofluorescein linker
4.2.1. Ethyl 4-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate
(1)
(ethyl
2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate
(7)). H2SO4 (A: 0.77 ml, 14.4 mmol; B: 0.80 ml, 15.00 mmol) was
added dropwise to the suspension of 6-aminofluorescein (A)
(1.00 g, 2.88 mmol) or fluorescein (B) (1.000 g, 3.0 mmol) in EtOH.
After stirring at reflux for 24 h the EtOH was removed under re-
duced pressure. The residue was diluted in CHCl3 with a few drops
of MeOH. Solid NaHCO3 was added until gas evolution ceased. The
resulting mixture was filtered, then the organic phase was dried
over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was subjected to silica gel chromatography
eluting with EtOAc, then MeOH/DCM (1:9) to afford the title
compound as a dark orange solid (1) (1.082 g, 99%) or brown solid
(7) (0.814 g, 75%).
Compound 3: 1H NMR (400 MHz, DMSO)
d 0.85 (3H, t, 5.7, CH3),
3.82e3.89 (2H, m, CH2), 4.96 (2H, s, CH2), 6.46 (1H, s, CH), 6.55 (1H,
s, CH), 6.64 (1H, d, 7.7, CH), 6.81 (1H, d, 7.0, CH), 7.05 (1H, d, 7.2, CH),
7.13e7.16 (2H, m, 2ꢁCH), 7.33 (1H, s, CH), 7.91 (1H, d, 8.7, CH). 13C
NMR (100.62 MHz, DMSO)
d 13.54, 59.74, 65.23, 101.16, 103.78,
113.77, 113.97, 115.07, 115.34, 116.42, 131.44, 132.84, 135.55, 152.89,
154.51, 158.69, 164.54, 169.21. LRMS 434.20 [(MþH)þ] (C24H20O7N
requires 434.70).
Compound 9: 1H NMR (400 MHz, DMSO)
d 0.86 (3H, t, 7.1, CH3),
3.93e4.01 (2H, m, CH2), 4.95 (2H, s, CH2), 6.49 (1H, s, CH), 6.57 (1H,
d, 9.6, CH), 6.95e7.04 (3H, m, 3ꢁCH), 7.32 (1H, s, CH), 7.52 (1H, d,
7.6, CH), 7.79 (1H, t, 7.7, CH), 7.87 (1H, t, 7.5, CH), 8.21 (1H, d, 8.2,
Compound 1: 1H NMR (400 MHz, DMSO)
d
0.86 (3H, t, 7.1, CH3),
CH). 13C NMR (100.62 MHz, DMSO)
d 13.32, 60.96, 65.16, 101.28,
3.71 (2H, q, 7.1, CH2), 6.43 (1H, s, CH), 6.66 (4H, d, 7.4, 4ꢁCH), 6.78
104.22, 114.62, 129.42, 129.83, 130.19, 130.49, 130.71, 130.87, 169.26.
(1H, d, 8.7, CH), 7.01 (2H, d, 9.7, 2ꢁCH), 7.89 (1H, d, 8.7, CH). 13C NMR
HRMS 419.40 [(MþH)þ] (C24H19O7 requires 419.20).
(100.62 MHz, DMSO)
d 13.50, 15.09, 59.62, 61.07, 102.92, 113.57,
113.97, 114.60, 115.15, 130.50, 132.81, 135.81, 152.83, 164.58. LRMS
4.2.4. tert-Butyl 1-{[9-(5-amino-2-ethoxycarbonyl)phenyl]-3-oxo-
3H-xanthen-6-yloxy}-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oate
(4). Compound 3 (0.36 g, 0.83 mmol) was dissolved in small
amount of DMF. HATU (0.378 g, 0.995 mmol) and DIPEA
(0.32 g, 2.500 mmol) were added. After stirring for 15 min at
room temperature tert-butyl-12-amino-4,7,10-trioxadodecanoate
(0.23 g, 0.83 mmol) was added. After stirring for 3 h at room
temperature another portions of HATU (0.378 g, 0.995 mmol) and
DIPEA (0.32 g, 2.50 mmol) were added. After stirring for another
20 h at room temperature the reaction mixture was diluted with
water, then extracted with Et2O (3ꢁ20 ml). The organic phase
was dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The residue was subjected to silica gel
chromatography eluting with MeOH/DCM 1e20% giving 0.142 g
of the title compound (25%) as an orange solid. 1H NMR
376.20 [(MþH)þ] (C22H18O5N requires 376.62).
Compound 7: 1H NMR (400 MHz, DMSO)
d 0.83 (3H, t, 8.0,
CH3), 3.91 (2H, q, 8.0, CH2), 5.75 (1H, s, OH), 7.07 (2H, d, 10.0,
2ꢁCH), 7.22 (2H, s, 2ꢁCH), 7.29 (2H, d, 12.0, 2ꢁCH), 7.54 (1H, d,
6.0, CH), 7.86 (1H, t, 8.0, CH), 7.93 (1H, t, 8.0, CH), 8.28 (1H, d, 8.0,
CH). 13C NMR (100.62 MHz, DMSO)
d 13.29, 15.10, 61.11, 61.24,
102.35, 116.13, 120.56, 129.43, 130.15, 130.88, 130.97, 132.62,
133.18, 158.32, 164.50, 171.35. HRMS 361.11 [(MþH)þ] (C22H17O5
requires 361.17).
4.2.2. Ethyl
4-amino-2-[(6-tert-butoxy-2-oxoethoxy)-3-oxo-3H-
xanthen-9-yl]benzoate (2) (ethyl 2-[6-(2-tert-butoxy-2-oxoethoxy)-
3-oxo-3H-xanthen-9-yl]benzoate (8)). tert-Butyl bromoacetate (2:
0.47 ml, 3.17 mmol; 8: 0.37 ml, 2.48 mmol) was added to the mix-
ture of compound 1 (1.082 g, 2.88 mmol) or 7 (0.814 g, 2.26 mmol)
and K2CO3 (1:0.44 g, 3.17 mmol; 7: 0.34 g, 2.48 mmol) in the min-
imum amount of DMF. After stirring for 3 h at room temperature
the reaction mixture was diluted with H2O and extracted with
EtOAc (3ꢁ20 ml). The organic phase was washed with 1 M NaHCO3
and satd NaCl, then dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The residue was subjected to
silica gel chromatography eluting with EtOAc, then MeOH/DCM
(1:9) giving 0.79 g (2) or 0.66 g (8) of title compound as orange
solids (2: 56%; 8: 62%).
(400 MHz, DMSO)
d 0.86 (3H, t, 7.1, CH3), 1.42 (9H, s, C(CH3)3),
2.40 (2H, t, 6.2, CH2), 2.85 (2H, t, 5.4, CH2), 3.48e3.52 (10H, m,
5ꢁCH2), 3.56 (2H, t, 6.2, CH2), 3.81e3.91 (2H, m, CH2), 4.32 (2H, s,
CH2), 6.20 (1H, s, CH), 6.30 (2H, s, NH2), 6.36 (1H, d, 9.7, CH), 6.42
(1H, s, CH), 6.76e6.82 (2H, m, 2ꢁCH),6.89e6.93 (3H, m, 3ꢁCH),
7.88 (1H, d, 8.7, CH). 13C NMR (100.62 MHz, DMSO)
d 13.63, 27.76,
35.81, 66.22, 69.65. HRMS 693.3018 [(MþH)þ] (C37H44O11N2 re-
quires 693.3020).
4.2.5. tert-Butyl
[(ethoxycarbonyl)phenyl]-3-oxo-3H-xanthen-6-yloxy}-2-oxo-6,9,12-
trioxa-3-azapentadecan-15-oate (5). Thioctic acid (0.107 g,
1-{9-[5-(1,2-dithiolan-3-yl)pentanamido]-2-
Compound 2: 1H NMR (400 MHz, DMSO)
d 0.84(3H, t, 7.1, CH3),
1.43 (9H, s, C(CH3)3), 3.81e3.89 (2H, m, CH2), 4.86 (2H, s, CH2), 6.21
(1H, s, CH), 6.39e6.45 (2H, m, 2ꢁCH), 6.77 (1H, d, 7.6, CH), 6.90 (3H,
m, 3ꢁCH), 7.16 (1H, s, CH), 7.89 (1H, d, 8.7, CH). 13C NMR
0.210 mmol) and DIC (0.132 g, 0.520 mmol) were added to the so-
lution of 4 (0.142 g, 0.210 mmol) in DCM. After stirring for 48 h at
room temperature the reaction mixture was concentrated under
reduced pressure. The residue was subjected to silica gel chroma-
tography eluting with MeOH/DCM (0.1:9.9), then (0.5:9.5) giving
0.18 g of title compound as a yellow solid (97%). 1H NMR (400 MHz,
(100.62 MHz, DMSO)
d 13.54, 27.68, 59.62, 81.80, 104.44, 113.42,
129.01. LRMS 490.76 [(MþH)þ] (C28H28O7N requires 490.20).
Compound 8: 1H NMR (400 MHz, DMSO)
d 0.85 (3H, t, 8.0, CH3),
1.42 (9H, s, C(CH3)3), 3.92e4.05 (2H, m, CH2), 4.87 (2H, s, CH2), 6.44
(1H, s, CH), 6.38 (1H, d, 8.0, CH), 6.80e6.92 (3H, m, 3ꢁCH), 7.20 (1H,
s, CH), 7.50 (1H, d, 8.0, CH), 7.76 (1H, t, 4.0, CH), 7.79 (1H, t, 8.0, CH),
acetone) d 0.95 (3H, t, 7.2, CH3), 1.49 (9H, s, C(CH3)3), 1.69e1.75 (6H,
m, 3ꢁCH2), 1.90e1.95 (2H, m, CH2), 2.35 (2H, t, 6.5, CH2), 2.45 (2H, t,
6.5, CH2), 3.11e3.20 (2H, m, CH2), 3.50e3.64 (12H, m, 6ꢁCH2), 3.55
(2H, t, 5.2, CH2), 3.95e4.01 (2H, m, CH2), 4.60 (2H, s, CH2), 6.44 (1H,
s, CH), 6.48 (1H, s, CH), 6.55 (1H, d, 9.7, CH), 6.79 (1H, d, 8.9, CH),
6.83 (1H, d, 8.6, CH), 6.97e7.07 (3H, m, 3ꢁCH), 8.08 (1H, d, 8.6, CH).
8.18 (1H, d, 8.0, CH). 13C NMR (100.62 MHz, CDCl3)
d 13.86, 13.92,
28.40, 53.73, 61.74, 104.14, 114.01, 115.33, 122.43, 129.60, 130.08,
130.60, 130.79, 130.91, 131.02, 131.52, 131.61, 132.76, 132.91, 134.38,
135.52, 155.47, 158.01, 165.70, 175.75. HRMS 475.1751 [(MþH)þ]
(C28H27O7 requires 475.1751).
13C NMR (100.62 MHz, DMSO)
d 25.64, 26.28, 26.74, 28.34, 29.41,