F. R. Pinsetta et al. / Carbohydrate Research 373 (2013) 97–102
101
Bruker Daltonics UltrOTOF-Q-ESI-TOF mass spectrometer. Optical
rotation measurements were performed in a JASCO P-2000 digital
polarimeter.
(C-3, C-1), 31.2 (C-2). ESI HRMS: m/z 399.12406 [M+Na+]. Calcd
for C12H20N6O8 399.1241
3.1.4. 4-O-(b-
D-Galactopyranosyl)-1,3-diazido-1,2,3-trideoxy-
3.1. 4-O-(b-D-Glucopyranosyl)- and (1) and 4-O-(b-D-
myo-inositol (2)
galactopyranosyl)-1,3-diazido-1,2,3-trideoxy-myo-inositol (2)
½
a 2D5 +10.2 (c 1.0 MeOH); 1H NMR (300 MHz/D2O): d 4.62 (1H, d,
ꢁ
J1 ,2 7.8 Hz, H-10), 3.91 (1H, d, J3 ,4 3.4 Hz, H-40), 3.78 (1H, dd, J5 ,6 a
0
0
0
0
0
0
General procedure: Compounds 5 (48 mg, 0.16 mmol) and 9 (or
10) (150 mg, 0.30 mmol) were dissolved in anhydrous dichloro-
methane under N2 at 0 °C, treated with a trimethylsilyl triflate
8.1 Hz; J6 a,6 b 11.5 Hz, H-60a), 3.71 (1H, dd, J5 ,6 b 3.7 Hz; J6 a,6 b
11.5 Hz, H-60b), 3.69–3.35 (8H, m, H-1, H-30, H-50, H-3, H-4, H-5, H-
6, H-20), 2.36 (1H, dt, J1,2eq = J2eq,3 4.3 Hz; J2eq,2ax 12.8 Hz, H-2 eq),
1.48 (1H, q, J 12.8 Hz, H-2ax). 13C NMR (75 MHz/D2O): d 102.3 (C-
10), 81.5 (C-4), 74.5, 74.4, 72.6, 71.8, 70.2 (C-30, C-6, C-5, C-50, C-20),
68.0 (C-40), 60.5 (C-60), 59.6, 59.4 (C-1, C-3), 30.6 (C-2). ESI HRMS:
m/z 399.1235 [M+Na+]. Calcd for C12H20N6O8 399.1241.
0
0
0
0
0
0
solution (1.78 mg, 1.5 lmol) in dichloromethane (1 mL) and the
mixture stirred for 40 min. After neutralization with Et3N, the solu-
tion was concentrated and purified by flash chromatography (ethyl
acetate–hexane 2:8 v/v) to give hexa-acetylated pseudodisaccha-
rides 11 or 12 in 89 and 86% yields, respectively (approx. 87 mg,
0.14 mmol). This compound was dissolved in methanol and com-
pletely deprotected (30 min, rt) by the addition of sodium methox-
ide methanolic solution (1 mol Lꢀ1, pH 9–10). The solution was
neutralized using DowexÒ 50WX8 (H+), filtered and concentrated
to quantitatively yield the desired pseudodisaccharides 1 and 2,
which was used in the otoxicity assays without further chromato-
graphic purification.
3.2. Ototoxicity assays
Albino guinea pigs were handled according to the guidelines for
the care and use of laboratory animals of the Institute of Laboratory
Animal Resources, Commission on Life Sciences, of the US National
Research Council. The animals were selected from the central ani-
mal house of the University of São Paulo (USP), Campus at Ribeirão
Preto, on the basis of the determination of the Preyer reflex.36 After
a hearing rest for 24 h, they were reevaluated using manual otos-
copy. Albino guinea pigs showing signs of otitis external or acute
otitis media, wax of difficult removal, inflammatory changes of
the external auditory meatus, or a meatus too narrow to ade-
quately accommodate the probe of the otoacoustic emission equip-
ment were discarded.
The selected animals were then submitted to auditory screening
using distortion product otoacoustic emissions (DPOAEs) and
brainstem evoked auditory potential (BEAP) in an acoustically iso-
lated cabin under anesthesia with 65 mg/kg ketamine hydrochlo-
ride and 6.5 mg/kg xylazine. Seven albino guinea pigs that
showed the presence of DPOAEs in both ears and thresholds values
to induce BEAP responses of 5 dB were then chosen for the trans-
tympanic application of the pseudodisaccharides 1 or 2 in the right
ear, while the left ear was used as control (distilled water).
A 0.222 mol Lꢀ1 aqueous solution of the pseudodisaccharides
3.1.1. 4-O-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-5,6-di-O-
acetyl-1,3-diazido-1,2,3-trideoxy-myo-inositol (11)
1H NMR (500 MHz/CDCl3): 5.22 (1H, tapp, J 9.4 Hz, H-30), 5.08
(1H, tapp, J 9.7 Hz; H-40), 4.91–4.99 (2H, m, H-5, H-6), 4.94 (1H,
dd, J´ 9.3 Hz, J1 ,2 8.0 Hz, H-20), 4.80 (1H, d, J1 ,2 8.0 Hz, H-10),
0
0
0
´
´
2,3
´
4.40 (1H, dd, J5 ,6 a 4.3 Hz; J6 a.6 b 12.5 Hz, H-60a), 4.05 (1H, dd,
0
0
0
0
J5 ,6 b 1.8 Hz; J6 a.6 b 12.5 Hz, H-60b), 3.70 (1H, ddd, J5 ,6 a 4.3 Hz;
0
0
0
0
0
0
J5 ,6 b 1.8 Hz; J4´,5´ 9.6 Hz H-50), 3.59–3.46 (3H, m, H-1, H-3, H-4),
2.37 (1H, dt, J1,2eq 4.4 Hz, J2eq,3 4.1 Hz; J2eq,2ax 12.9 Hz, H-2 eq),
2.10–2.01 (18H, 6s, 6 COCH3), 1.55 (1H, q, J 12.9 Hz, H-2ax). 13C
NMR (125 MHz/CDCl3) d 169.9, 169.7 (CO), 101.0 (C-10), 80.4 (C-
4), 73.9, 73.5 (C-20, C-50), 72.2, 71.9, 71.5 (C-30, C-5, C-6) 68.3 (C-
40), 62.0 (C-60), 61.2, 58.2 (C-1, C-3), 32.6 (C-2), 21.0, 20.9 (COCH3).
0
0
3.1.2. 4-O-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-5,6-di-
O-acetyl-1,3-diazido-1,2,3-trideoxy-myo-inositol (12)
were prepared, filtered through a 0.22 lm membrane filter and
1H NMR (500 MHz/CDCl3): 5.36 (1H, d, J3 ,4 3.3 Hz, H-40), 5.10
an aliquot of 0.1 mL administered transtympanically in the right
ears of the seven albino guinea pigs immediately after otoscopic
and audiological examinations. The animals were kept in the su-
pine position with the head tilted 45° to the healthy side for 3 h
in order to prevent the leaking of the inoculated substance from
the ear cleft to the Eustachian tube. Otoacoustic emissions and
BEAP were determined before treatment and after 11 days, when
animal were sacrificed for the electron microscopy study.
The function of outer cochlear hair cells was determined by
DPOAEs using the Intelligent Hearing Systems (Miami, FL, USA).
For the test, the animals were anesthetized with ketamine hydro-
chloride and xylazine. Before the recording of evoked otoacoustic
emissions (EOAEs), the animals were submitted to manual otos-
copy for the evaluation of the auditory meatus and the tympanic
membrane. None of the seven albino guinea pigs showed signs of
otitis or wax of difficult removal, which could cause them to be ex-
cluded from the test.
The DPOAE test was performed before treatment and immedi-
ately before sacrifice, following the relation of the 2F1–F2 fre-
quency, with an F1:F2 ratio = 1.22, with a resolution of two
points per octave. We considered otoacoustic emissions starting
from 1.5 kHz, as the dimensions of the external auditory meatus
of the guinea pig make it difficult to detect otoacoustic emissions
below this frequency, with the occurrence of responses that coin-
cide with noise responses, which were maintained at 10 dB for
0
0
(1H, dd, J1 ,2 7.9 Hz; J2 3 10.4 Hz, H-20), 5.02 (1H, dd, J3 ,4 3.3 Hz;
0
0
0
0
0
0
J2 ,3 10.4 Hz, H-30), 5.00–4.94 (2H, m, H-5, H-6), 4.74 (1H, d, J1 ,2
0
0
0
0
7.9 Hz, H-10), 4.16 (1H, dd, J5 ,6 a 6.6 Hz; J6 a.6 b 11.4 Hz, H-60a),
0
0
0
0
4.10 (1H, dd, J5 ,6 b 7.1 Hz, J6 a.6 b 11.4 Hz, H-60b), 3.90 (1H, dt,
0
0
0
0
J5 ,6 a 6.6 Hz; J5 ,6 b 7.1 Hz, H-50), 3.59–3.47 (3H, m, H-1, H-3, H-4),
2.34 (1H, dt, J1,2eq = J2eq,3 4.3 Hz; J2eq,2ax 12.7 Hz, H-2 eq), 2.14–
1.97 (18H, 6s, 6 COCH3), 1.51 (1H, q, J 12.7 Hz, H-2ax). 13C NMR
(125 MHz/CDCl3): dC 169.5 (CO), 101.4 (C-10), 80.0 (C-4), 73.9,
71.7, 71.4, 71.0 (C-6, C-5, C-30, C-50), 69.5 (C-20), 67.2 (C-40), 61.3
(C-60), 61.0 (C-3), 58.3 (C-1), 32.7 (C-2), 21.1 (COCH3), 21.0
(COCH3), 21.0 (COCH3), 20.9 (2 COCH3), 20.9 (COCH3).
0
0
0
0
3.1.3. 4-O-(b-D-glucopyranosyl)-1,3-diazido-1,2,3-trideoxy-myo-
inositol (1)
½
a 2D5
ꢁ
ꢀ4.9 (c 1.0 MeOH); 1H NMR (300 MHz/D2O): d 4.58 (1H, d,
0
J1 ,2 7.8 Hz, H-1 ), 3.79 (1H, dd, J5 ,6 a 1.5 Hz, J6 a,6 b 12.5 Hz; H-60a),
0
0
0
0
0
0
3.61 (1H, dd, J5 ,6 b 5.3 Hz, J6 a,6 b 12.5 Hz, H-60b), 3.54 (1H, J1,2eq or
J2eq,3 4.4 Hz, J1,2ax or J2ax,3 12.1 Hz, H-1 or H-3), 3.57–3.25 (7H, m,
0
0
0
0
H-40, H-30, H-5, H-4, H-50, H-6, H-1 or H-3), 3.20 (1H, dd, J1 ,2
0
0
7.8 Hz, J2 ,3 9.3 Hz, H-20), 2.27 (1H, dt, J1,2eq = J2eq,3 4.4 Hz, J2eq,2ax
12.8 Hz, H-2 eq), 1.48 (1H, qapp, J 12.4 Hz; H-2ax). 13C NMR
(75 MHz/D2O) d 102.6 (C-10), 82.3 (C-4), 75.8, 75.4, 75.1, 73.1,
73.0, 69.3 (C-40, C-5, C-6, C-20, C-30, C-50), 60.5 (C-60), 60.1, 60.0
0
0