Molecules 2021, 26, 879
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4.10. Aggregation Inhibition Assay
Lysozyme (20
µ
M) was dissolved in Tris-HCl buffer (50 mM, pH 7.5) containing NaCl
◦
(
300 mM) and an additive, and the mixture was incubated at 96 C for 20 min followed by
air cooling to room temperature.
4.11. Synthesis of 1
To a dry EtOH solution (31 mL) of 2-(2-aminoethoxy)ethanol (1.52 g, 14.5 mmol),
◦
di-tert-butyl decarbonate (3.28 g, 15.0 mmol) was added at 0 C under N . After being
2
◦
stirred for 4 h at 25 C, CH Cl (100 mL) was added to the reaction mixture, and the
2
2
mixture was washed with water (100 mL, twice) and brine (50 mL, once). The organic
extract was dried over anhydrous Na SO and filtered off from insoluble substances. The
2
4
◦
filtrate was evaporated to dryness under reduced pressure at 30 C, and the residue was
chromatographed on silica gel (Silica Gel 60) with CH Cl /MeOH (100/0 to 90/10 v/v) to
2
2
allow isolation of 1 (2.81 g, 13.7 mmol) in 94% yield.
1
TLC R (Merck 60 F254, CH Cl /MeOH = 90/10 v/v): 0.56; H-NMR (400 MHz,
f
2
2
◦
CDCl , 25 C):
δ = 5.18 (brs, 1H), 3.73 (brs, 2H), 3.59–3.54 (m, 4H), 3.33 (q, J = 5.0 Hz,
H), 2.84 (brs, 1H), 1.45 (s, 9H) ppm; C-NMR (100 MHz, CDCl , 25 C):
3
13
◦
2
δ
= 156.24,
3
79.41, 72.33, 70.37, 61.70, 40.44, 28.46 ppm; MALDI-TOF MS (gentisic acid, positive mode):
m/z = 228.132 (calculated m/z on the basis of the monoisotopic mass of C H NNaO
4
9
19
+
[
M + Na] = 228.121).
4.12. Synthesis of 2
To a dry THF solution (31 mL) of
1
(2.68 g, 13.1 mmol), 15% NaOH aq. (10 mL) and a
◦
THF solution (10 mL) of TsCl (2.94 g, 15.4 mmol) were added dropwise at 0 C under N .
2
◦
After being stirred for 12 h at 25 C, water (100 mL) was added to the reaction mixture. The
resulting mixture was extracted with CH Cl (50 mL, three times). The collected organic
2
2
extract was washed with brine (50 mL, once), dried over anhydrous Na SO , and filtered off
2
4
from insoluble substances. The filtrate was evaporated to dryness under reduced pressure
◦
at 30 C to allow isolation of 2 (4.06 g, 11.3 mmol) in 86% yield.
1
◦
TLC R (Merck 60 F254, CH Cl ): 0.25; H-NMR (400 MHz, CDCl , 25 C):
δ
= 7.80
f
2
2
3
(d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 4.86 (brs, 1H), 4.18–4.15 (m, 2H), 3.63 (t, J =
4
.6 Hz, 2H), 3.45 (t, J = 5.0 Hz, 2H), 3.24 (q, J = 5.0 Hz, 2H), 2.45 (s, 3H), 1.45 (s, 9H) ppm;
1
3
◦
C-NMR (100 MHz, CDCl , 25 C):
δ
= 156.24, 144.98, 133.09, 129.91, 128.01, 79.36, 70.40,
3
6
=
9.18, 68.41, 40.29, 28.46, 21.68 ppm; MALDI-TOF MS (gentisic acid, positive mode): m/z
382.114 (calculated m/z on the basis of the monoisotopic mass of C H NNaO S [M +
16
25
6
+
Na] = 382.130).
4.13. Synthesis of 3
To a dry DMF solution (35 mL) of
2
(3.98 g, 11.1 mmol), AcSK (4.03 g, 35.3 mmol) was
◦
◦
added at 25 C under N . After being stirred for 13 h at 90 C, the reaction mixture was
2
◦
cooled to 25 C. To the resulting mixture, water (300 mL) was added, and the mixture was
extracted with CH Cl (100 mL, four times). The collected organic extract was washed with
2
2
brine (100 mL, four times), dried over anhydrous Na SO , and filtered off from insoluble
2
4
◦
substances. The filtrate was evaporated to dryness under reduced pressure at 30 C, and
the residue was chromatographed on silica gel (Silica Gel 60) with AcOEt/hexane (20/80
v/v) to allow isolation of 3 (2.69 g, 10.2 mmol) in 92% yield.
1
TLC R (Merck 60 F254, EtOAc/hexane = 50/50 v/v): 0.54; H-NMR (400 MHz, CDCl ,
5 C): δ = 4.87 (brs, 1H), 3.57 (t, J = 6.4 Hz, 2H), 3.51 (t, J = 5.0 Hz, 2H), 3.30 (q, J = 5.0 Hz,
f
3
◦
2
2
2
13
H), 3.08 (t, J = 6.4 Hz, 2H), 2.35 (s, 3H), 1.45 (s, 9H) ppm; C-NMR (100 MHz, CDCl3,
◦
5 C):
δ
= 195.46, 155.99, 79.36, 69.99, 69.53, 40.42, 30.62, 28.95, 28.48 ppm; MALDI-TOF
MS (gentisic acid, positive mode): m/z = 286.113 (calculated m/z on the basis of the
+
monoisotopic mass of C H NNaO S [M + Na] = 286.109).
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