7
16
Kojima and Irie
salt) d2.01 and 2.41 (br, H , and H for Pro), 3.40 (br, H for solution (210 mL) was added, and the crude compounds were
b
c
d
Pro), 4.05 (m, H for Gly), 4.45 (br, H for Pro), 5.23 (s, Bzl), extracted with ethyl acetate. The organic phase was washed
a
a
13
7.46 (s, phenyl). C NMR (400 MHz, D O containing 3-(tri- with 10% citric acid aqueous solution, water, 4% sodium
2
methylsilyl)propionic-2,2,3,3-d acid, sodium salt) d26.1 (C
hydrogen carbonate, and then water. The compound was puri-
4
c
for Pro), 32.5 (C for Pro), 44.5 (C for Gly), 49.5 (C for fied with a silica gel column (elute: ethyl acetate/hexane 5 1/
b
a
d
1
Pro), 62.6 (C for Pro), 70.8 (Bzl), 131.5, 131.7, 131.8, and 2). Yield 83%. H NMR (400 MHz, DMSO) d 0.82 and 0.85
a
138.1 (phenyl), 173.2 and 173.9 (C 5 O).
(m, H for Val), 1.38 (s, Boc), 1.92 (br, H for Val), 3.83, 3.87,
c b
3
.94, and 3.98 (m, H for Val and H for Gly), 5.12 (s, Bzl),
a a
13
Synthesis of Boc-Val-Pro-Gly-OBzl. TFAÁH-Pro-Gly-OBzl 7.36 (s, C H ). C NMR (400 MHz, DMSO) d 18.0 and 19.1
6
5
(
6.2 mmol) was dissolved in distilled acetonitrile (50 mL) and (C for Val), 28.1 ((CH ) C for Boc), 30.4 (C for Val), 40.6
c 3 3 b
TEA (15 mmol). Boc-Val (6.2 mmol), followed by HBTU (6.2 (C for Gly), 59.4 (C for Val), 65.8 (Bzl), 77.9 ((CH ) C for
3 3
a
a
mmol), were added and stirred for 4 days at room tempera- Boc), 127.9, 128.0, 128.3, and 135.8 (phenyl), 155.3, 169.6, and
ture. Saturated NaCl solution (150 mL) was added, and the 171.9 (C 5 O).
crude compounds were extracted with ethyl acetate. The or-
ganic phase was washed with 10% citric acid aqueous solution,
Synthesis of TFAÁH-Val-Gly-OBzl. Boc-Val-Gly-OBzl (7.4
water, 4% sodium hydrogen carbonate, and then water. The
ꢀ
mmol) was dissolved in TFA (6 mL) and incubated at 4 C for
compound was purified with a silica gel column (elute: ethyl
1
acetate/hexane 5 1/1). Yield 72%. H NMR (400 MHz,
4
h. The reaction mixture was evaporated. After the addition
of water, evaporation was performed four separate times. The
DMSO) d0.83 and 0.89 (m, H for Val), 1.34 (s, Boc), 1.80 and
c
crude compound was recrystallized from ethyl acetate/hexane.
1
Yield 96%. H NMR (400 MHz, D O containing 3-(trimethyl-
2.02 (br, H , and H for Pro), 1.90 (br, H for Val), 3.57 and
b c b
2
3
.67 (br, H for Pro), 3.78, 3.83, 3.94, and 3.98 (m, H for Gly
d
a
silyl)propionic-2,2,3,3-d acid, sodium salt) d 1.00 (br, H for
4
c
and H for Val), 4.36 (br, H for Pro), 5.11 (s, Bzl), 7.37 (s,
a
a
Val), 2.19 (br, H for Val), 3.85 (br, H for Val), 4.03, 4.08,
b
13
a
phenyl). C NMR (400 MHz, DMSO) d18.3 and 19.1 (C for
c
4
.19, and 4.23 (m, H for Gly), 5.23 (s, Bzl), and 7.45 (s, phe-
a
Val), 24.3 (C for Pro), 28.1 ((CH ) C for Boc), 29.1 and 29.7
c 3 3
13
nyl). C NMR (400 MHz, D O containing 3-(trimethylsilyl)-
2
(C for Pro and Val), 40.6 (C for Gly), 46.9 (C for Pro), 57.2
b a d
propionic-2,2,3,3-d acid, sodium salt) d 19.7 and 20.2 (C for
4
c
and 59.0 (C for Pro and Val), 65.8 (Bzl), 77.9 ((CH ) C for
a 3 3
Val), 32.8 (C for Val), 44.2 (C for Gly), 61.4 (C for Val),
b
a
a
Boc), 127.9, 128.0, 128.4 and 135.8 (phenyl), 155.5, 169.6,
70.3 and 172.1 (C 5 O).
70.7 (Bzl), 131.4, 131.7, and 137.9 (phenyl), 172.7 and 173.7
1
(C 5 O).
Synthesis of Boc-Val-Pro-Gly-OH. Boc-Val-Pro-Gly-OBzl
(
4.5 mmol) was dissolved in ethanol (74 mL). Pd-C (1.5 Synthesis of Boc-Val-Pro-Gly-Val-Gly-OBzl. TFAÁH-Val-Gly-
mmol) and 1,4-cyclohexadiene (45 mmol) were added and OBzl (4.2 mmol) was dissolved in distilled acetonitrile (36 mL)
reduced with H gas. The reaction mixture was filtered in the and TEA (11 mmol). Boc-Val-Pro-Gly-OH (4.2 mmol), fol-
2
presence of celite (545RVS) and evaporated. Coevaporation lowed by HBTU (4.2 mmol), were added and stirred for 4 days
with ethanol was performed four times, and the residual was at room temperature. Saturated NaCl solution (120 mL) was
1
dried under vacuum. Yield 95%. H NMR (400 MHz, DMSO) added, and the crude compounds were extracted with ethyl ac-
d 0.84 and 0.90 (m, H for Val), 1.37 (s, Boc), 1.85 and 2.03 etate. The organic phase was washed with 10% citric acid
c
(br, H , and H for Pro), 1.91 (br, H for Val), 3.57 and 3.67 aqueous solution, water, 4% sodium hydrogen carbonate, and
b c b
(
br, H for Pro), 3.62, 3.66, 3.80, 3.84, and 3.97 (H for Gly then water. The compound was purified with a silica gel col-
d
a
13
and H for Val), 4.37 (br, H for Pro). C NMR (400 MHz, umn (elute: ethyl acetate/methanol 5 95/5) and a Sephadex
a
a
DMSO) d 18.3 and 19.1 (C for Val), 24.4 (C for Pro), 28.2 LH-20 column (GE Healthcare Life Sciences) (elute: metha-
c
c
1
(
(CH ) C for Boc), 29.1 and 29.7 (C for Val and Pro), 40.6 nol). Yield 28%. H NMR (400 MHz, DMSO) d 0.78 (m, H
b c
3
3
(
C for Gly), 46.9 (C for Pro), 57.2 and 59.0 (C for Val and for Val), 1.35 (s, Boc), 1.82 and 2.05 (br, H , and H for Pro),
a
d
a
b
c
Pro), 77.9 ((CH ) C for Boc), 155.5, 169.6, 170.3, and 172.1 (C 1.96 (br, H for Val), 3.57 and 3.80 (br, H for Pro), 3.73, 3.88,
3
3
b
d
5
O).
3.91, 3.93, 3.97, and 4.18 (m, H for Val and H for Gly), 4.29
a a
13
(m, H for Pro), 5.11 (s, Bzl), and 7.34 (s, phenyl). C NMR
a
Synthesis of Boc-Val-Gly-OBzl. Boc-Val (9.0 mmol), Gly- (400 MHz, DMSO) d 18.1, 18.5, and 19.1 (C for Val), 24.6
c
OBzl tosylate (9.0 mmol), and TEA (22 mmol) were dissolved (C for Pro), 28.2 ((CH ) C for Boc), 29.1, 29.7, and 30.4 (C
b
c
3 3
in distilled acetonitrile (72 mL). HBTU (9.0 mmol) was added for Pro and Val), 40.7 and 42.1 (C for Gly), 47.1 (C for Pro),
a
d
and stirred for 30 h at room temperature. Saturated NaCl 57.6, 57.7, and 59.6 (C for Val and Pro), 65.8 (Bzl), 78.0
a
Biopolymers (Peptide Science)