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EtOH); 1H NMR (CDCl3) d 1.45 (9H, s, Boc), 2.07–2.49
(2H, m, Pro C3H2), 3.52–3.65 (2H, m, Pro C5H2), 3.77
(3H, s, COOCH3), 4.27–4.40 (2H, m, Pro aCH and
Pro C4H). Anal. Calcd for C11H19NO5: C, 53.87; H,
7.81; N, 5.71. Found: C, 53.90; H, 7.80; N, 5.74.
7.2.11. HClÆHLeu-Phe-OMe. Thionyl chloride
(0.078 mL, 1.06 mmol) was added to a solution of
Boc-Leu-Phe-OMe (0.4 g, 1.02 mmol) in dry methanol
(2.5 mL) and cooled at ꢁ15 ꢁC. After stirring at
ꢁ15 ꢁC for 40 min and at 45 ꢁC for 3 h, the solution
was evaporated under reduced pressure to give the title
hydrochloride as a foam. This was used in the next step
without further purification.
7.2.8. N-Boc-cis-4-mesyloxy-L-proline methyl ester (15).
To a solution of N-Boc-cis-4-hydroxy-L-proline methyl
ester (14) (0.070 g, 0.286 mmol) and TEA (0.048 mL)
in dry CH2Cl2 (5 mL) cooled in ice bath was added
MsCl (0.027 mL, 0.343 mmol) and the resulting mixture
was stirred for 20 min. The solution was diluted with
CH2Cl2 and washed with 1 N HCl, Na2CO3 ss and
brine. The organic layer was dried (Na2SO4) and con-
centrated to yield 0.090 g (97%) of pure 15 as a pale-yel-
low oil that solidified in the freezer: 1H NMR (CDCl3) d
1.44 (9H, s, Boc), 2.48–2.60 (2H, m, Pro C3H2), 3.03
(3H, s, CH3SO2), 3.75 and 4.45 (2H, m, Pro C5H2),
3.77 (3H, s, COOCH3), 4.44 and 5.29 (2H, m, Pro
aCH and Pro C4H). Anal. Calcd for C12H21NO7S: C,
44.57; H, 6.55; N, 4.33; S, 9.92. Found: C, 44.55; H,
6.52; N, 4.31; S, 9.91.
7.2.12. N-Boc-cis-4-methylthio-Pro-Leu-Phe-OMe (1).
i-BuOCOCl (0.065 mL, 0.5 mmol) was added at
ꢁ15 ꢁC to a stirred solution of 10 (0.130 g, 0.5 mmol)
and NMM (0.042 mL, 0.6 mmol) in dry CH2Cl2
(4 mL). The temperature was maintained at ꢁ15 ꢁC
for 10 min, and HClÆHLeu-Phe-OMe (0.5 mmol),
NMM (0.042 mL, 0.6 mmol) and dry CH2Cl2 (4 mL)
were added. The mixture was stirred at ꢁ15 ꢁC for
15 min and then allowed to warm to rt. Dry DMF (10
drops) was added and the solution was stirred overnight.
EtOAc was added and the organic layer was washed
with 1 N HCl, brine, saturated aqueous NaHCO3 and
brine. The organic phase was dried (Na2SO4) and evap-
orated under reduced pressure to give crude solid tripep-
tide 1, which was purified by silica gel chromatography
(CH2Cl2/EtOAc 4:1 and 1:1 as eluants) to give pure tri-
peptide 1 as an amorphous white solid (0.210 g, 78%),
mp 153–154.5 ꢁC; [a]D ꢁ37 (c 1.0, CHCl3); IR (KBr)
7.2.9. N-Boc-trans-4-(acetylthio)-L-proline methyl ester
(16). A solution of 15 (0.578 g, 1.79 mmol) and potassi-
um thioacetate (0.263 g, 2.3 mmol) in DMF (7.8 mL)
was stirred at 68 ꢁC for 4 h. 1 N HCl was added to ad-
just the aqueous layer to pH 4. EtOAc (100 mL) was
added and the solution was washed with ice-cooled
brine (100 mL). The organic layer was separated and
successively washed with another portion of saturated
brine, dried over Na2SO4 and evaporated under reduced
pressure. The residue was purified by silica gel chroma-
tography (n-hexane/EtOAc 4:1, as eluant) to give pure
16 as an orange-coloured oil (0.450 g, 83%):
1
3290, 1705, 1660, 1549, 1402 cmꢁ1, H NMR (CDCl3)
d
0.91 [6H, m, CH(CH3)2], 1.44–1.74 [3H, m,
CH2CH(CH3)2], 1.48 [9H, s, C(CH3)3], 2.14 (3H, s,
SCH3), 2.45 (2H, br, Pro C3H2), 3.04–3.35 (4H, m,
Pro C4H, 1H of Pro C5H2 and Phe bCH2), 3.71 (3H,
s, COOCH3), 3.85 (1H, br, 1H of Pro C5H2), 4.29
(1H, m, Pro aCH), 4.40 (1H, m, Leu aCH), 4.82 (1H,
m, Phe aCH), 6.78–7.35 (7H, m aromatic, Leu and
Phe NH). Anal. Calcd for C27H41N3O6S: C, 60.54; H,
7.71; N, 7.84; S, 5.99. Found: C, 60.59; H, 7.72; N,
7.80; S, 6.01.
20
D
1
½aꢂ = ꢁ33 (c 0.9, CHCl3); H NMR (CDCl3) d 1.41
(9H, s, Boc), 2.10 and 2.47 (2H, m, Pro C3H2), 2.34 (s,
3H, CH3CO); 3.31–4.10 (3H, m, Pro C5H2 and Pro
C4H), 3.75 (3H, s, COOCH3), 4.40 (1H, m, Pro aCH).
Anal. Calcd for C13H21NO5S: C, 51.47; H, 6.98; N,
4.62; O, 26.37; S, 10.57. Found: C, 51.39; H, 6.90; N,
4.60; O, 26.40; S, 10.55.
7.2.13. N-For-4-cis-methylthio-Pro-Leu-Phe-OMe (2).
The N-Boc-tripeptide (1) (0.093 g, 0.174 mmol) was dis-
solved in formic acid (1 mL) and the mixture was stirred
at room temperature for 24 h. After removal of the ex-
cess of formic acid under reduced pressure, the residue
was dissolved in 1 mL of dry chloroform. EEDQ
(0.052 g, 0.208 mmol) was added and the solution was
stirred at room temperature for 24 h. Evaporation under
reduced pressure afforded a crude residue, which was
purified by PLC (chloroform/EtOAc 3:1, as eluant) to
give 0.080 mg (99%) of pure N-For-cis-4-methylthio-
Pro-Leu-Phe-OMe (2) as a foam; [a]D ꢁ55.5 (c 0.83,
7.2.10. N-Boc-trans-4-(methylthio)-L-proline (17).
A
solution of 16 (0.440 g, 1.45 mmol) in methanol
(7.6 mL) was treated successively with 1 N NaOH
(1.6 mL) and dimethyl sulfate (0.150 mL, 1.6 mmol) at
rt. After stirring for 30 min, an additional 1 N NaOH
(3.15 mL) portion was added and the reaction mixture
was stirred for 3.5 h at rt. Volatiles were removed under
reduced pressure, and the residue was partitioned be-
tween 10% KHSO4 and EtOAc. The combined organic
layers were washed with brine, dried (Na2SO4) and con-
centrated under reduced pressure to give 0.360 g of
crude product 7 as an orange oil. Purification of the
crude residue by flash chromatography (2% acetic acid
CHCl3), IR (KBr) 3297, 1746, 1654, 1542, 1383 cmꢁ1
;
1H NMR (CDCl3) d 0.88–0.91 [6H, two d, J = 6.3 Hz,
CH(CH3)2], 1.42–1.75 [3H, m, CH2 (CH3)2], 2.18 (3H,
s, SCH3), 2.46 (2H, m, Pro C3H2), 3.05–3.25 (3H, m,
Phe bCH2 and Pro C4H), 3.37 and 3.97 (2H, two m,
Pro C5H2), 3.73 (3H, s, COOCH3), 4.36 (1H, m, Leu
aCH), 4.46 (1H, t, J = 8.1 Hz, Pro aCH), 4.84 (1H, m,
Phe aCH), 6.68 (1H, d, J = 7.7 Hz Phe NH), 7.06 (1H,
d, J = 7.7 Leu NH), 7.13 (5H, m, aromatic), 8.28 (1H,
s, HCO). Anal. Calcd for C23H33N3O5S: C, 59.59; H,
7.17; N, 9.06; S, 6.92. Found: C, 59.40; H, 7.18; N,
9.03; S, 6.90.
in EtOAc/n-hexane 1:4) gave 0.330 g of pure 17 (87%)
20
D
as a pale-yellow oil; ½aꢂ = 37.2 (c 0.46, DMF); 1H
NMR (CDCl3) d 1.42 (9H, s, Boc), 2.15 and 2.55 (5H,
m, CH3SO2 and Pro C3H2), 3.23–4.47 (4H, m, Pro
C5H2, Pro C4H, and Pro aCH). Anal. Calcd for
C11H19NO4S: C, 50.56; H, 7.33; N, 5.36; S, 12.27.
Found: C, 50.40; H, 7.35; N, 5.32; S, 12.28.