Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1187
under reduced pressure. The residue was recrystallized from (1H, m), 1.82–1.88 (1H, m), 2.04–2.12 (2H, m), 2.45 (2H, t,
ethyl acetate/n-hexane to obtain Boc-Val-Gly-OBzl (13.2g, J=7.40Hz), 3.79 (1H, t, J=6.36Hz), 4.31–4.45 (1H, m), 4.57
36.2mmol) as white crystals.
Boc-Val-Gly-OBzl (5.47g, 15.0mmol) was added to a 4N
(2H, s). ESI-MS m/z: 291.10 (M+H)+.
Large-Scale Synthesis of γ-L-Glutamyl-L-valylglycine
HCl/dioxane solution (40mL) and the mixture was stirred (35)
at room temperature for 50min. Dioxane was removed by
concentration under reduced pressure, n-hexane (30mL) was
[Step 1]
N-Hydroxysuccinimide (NHS; 60.1kg, 522mol, 1.05 equiv.)
added to the residue, and the mixture was concentrated under and N,N′-dicyclohexylcabodiimide (DCC (107.8kg, 522mol,
reduced pressure. This procedure was repeated thrice to quan- 1.05 equiv) in ethyl acetate (147L) were added to Cbz-Val
titatively obtain H-Val-Gly-OBzl·HCl.
(102, 125kg, 498mol) in ethyl acetate (675L) and the mixture
H-Val-Gly-OBzl·HCl (15.0mmol) and N-α-carbobenzoxy- was stirred under ambient conditions. After completion of the
L-glutamic acid α-benzyl ester (Cbz-Glu-OBzl, 5.57g, reaction, insoluble solids (dicyclohexylurea) were removed by
15.0mmol) were dissolved in methylene chloride (50mL) filtration to afford Cbz-Val N-hydroxysuccinimide ester (103)
and the solution was kept at 0°C. Triethylamine (2.30mL, in ethyl acetate. The resultant solution was added to glycine
16.5mmol), HOBt (2.53g, 16.5mmol), and WSC·HCl (3.16g, (39.2kg, 522mol, 1.05 equiv) in water (225L). The mixture
16.5mmol) were added to the solution and the mixture was was stirred at 10°C while maintaining slightly basic conditions
stirred at room temperature for 2d. The reaction mixture was by adding an aqueous sodium hydroxide solution (25%). The
concentrated under reduced pressure and the resulting residue water layer was then separated to afford Cbz-Val-Gly (104) in
was dissolved in heated ethyl acetate (1500mL). The solution water. Ethyl acetate (375L) was added to the 104 solution and
was washed with water (200mL), 5% aqueous citric acid solu- the Cbz group was deprotected via hydrogenation in the pres-
tion (200mL×twice), saturated brine (150mL), 5% aqueous ence of a palladium catalyst (10% Pd/C, 1.3kg). After remov-
sodium bicarbonate solution (200mL×twice), and saturated ing the palladium catalyst by filtration, the water phase was
brine (150mL). The organic layer was dried over anhydrous separated to afford a solution of Val-Glu (105).
magnesium sulfate, filtered, and the filtrate was concentrated
[Step 2]
under reduced pressure. The deposited crystals were collected
DCC (112.9kg, 547mol, 1.10 equiv.) in ethyl acetate (153L)
by filtration and dried under reduced pressure to obtain Cbz- was added to Cbz-Glu (106, 146.9kg, 522mol, 1.05 equiv.)
Glu(Val-Gly-OBzl)-OBzl (6.51g, 10.5mmol) as white crystals. in ethyl acetate (514L) at 10°C. After stirring the solution
Cbz-Glu(Val-Gly-OBzl)-OBzl (6.20g, 10.03mmol) was under ambient conditions overnight, the resultant solids (di-
suspended in ethanol (200mL), to which 10% palladium/ cyclohexylurea) were removed by filtration to afford Cbz-Glu
carbon (1.50g) was added. The reduction reaction was per- anhydride (107) in ethyl acetate. The resultant solution was
formed at 55°C for 5h under a hydrogen atmosphere. During added to the 105 solution obtained in step 1 in the presence
the reaction, a total of 100mL of water was gradually added. of sodium bicarbonate (43.9kg, 522mol, 1.05 equiv.). After
The catalyst was removed by filtration using a Kiriyama fun- completion of the reaction, tetrahydrofuran (625L) was added
nel and the filtrate was concentrated under reduced pressure and the pH of the water layer was acidified with concentrated
to half volume. The reaction mixture was further filtered hydrochloric acid. Peptide 108 was then extracted into the
through a membrane filter and the filtrate was concentrated organic layer (ethyl acetate and tetrahydrofuran) and washed
under reduced pressure. The resulting residue was dissolved with 10% aqueous sodium chloride solution (625L) and 3%
in a small volume of water and ethanol was added to deposit aqueous sodium chloride solution (625L×twice). Water (537L)
the crystals, which were then collected by filtration and dried was then added to the solution, and the Cbz group was depro-
under reduced pressure to obtain 35 (2.85g, 9.40mmol) as a tected via hydrogenation in the presence of a palladium cata-
white powder.
lyst (10% Pd/C, 2.1kg). After removing the palladium catalyst,
the water layer was separated and concentrated. Addition of
methanol (1687L) to the residue afforded 35 as crystals, which
were collected and dried (86kg, 283mol, α/γ <1/99).
1H-NMR (D2O) δ: 0.87 (3H, d, J=6.8Hz), 0.88 (3H, d,
J=6.8Hz), 1.99–2.09 (3H, m), 2.38–2.51 (2H, m), 3.72 (1H, t,
J=6.35Hz), 3.86 (1H, d, J=17.8Hz), 3.90 (1H, d, J=17.8Hz),
4.07 (1H, d, J=6.8Hz). ESI-MS m/z: 304.1 (M+H)+.
1H-NMR (D2O) δ: 0.88 (3H, d, J=6.8Hz), 0.89 (3H, d,
γ-L-Glutamyl-L-α-aminobutyrylglycine (45, γ-Glu-Abu-Gly) J=6.8Hz), 1.99–2.10 (3H, m), 2.40–2.50 (2H, m), 3.72 (1H,
1H-NMR (D2O) δ: 0.89 (3H, t, J=7.48Hz), 1.63–1.69 (1H, m), t, J=6.4Hz), 3.86 (1H, d, J=17.8Hz), 3.91 (1H, d, J=17.8Hz),
1.73–1.79 (1H, m), 2.04–2.10 (2H, m), 2.41–2.47 (2H, m), 4.08 (1H, d, J=6.8Hz). 13C-NMR (D2O) δ: 17.7, 18.7, 26.5,
3.74 (1H, t, J=6.36Hz), 3.87 (1H, d, J=17.8Hz), 3.90 (1H, d, 31.5, 41.7, 54.2, 60.0, 173.9, 174.0, 174.5, 175.3. IR (KBr)
J=17.8Hz), 4.14 (1H, dd, J=5.66 and 8.34Hz). ESI-MS m/z: cm−1: 3321 (N–H), 3282 (N–H), 2940 (C–H), 1712 (C=O),
290.10 (M+H)+.
1654 (C(C=O)–O), 1919 (amide I band), 1541 (amide II band),
γ-L-Glutamyl-L-tert-leucylglycine (49, γ-Glu-Tle-Gly) 1238 (C–C(C=O)–O). ESI-MS m/z: 304.2 (M+H)+. FAB-MS
1H-NMR (D2O) δ: 0.95 (9H, s), 2.04–2.08 (2H, m), 2.45–2.48 m/z: 304.1505 (M+H) (Calcd for C12H22N3O6: 304.1509). mp:
(2H, m), 3.73 (1H, t), 3.87–3.90 (2H, m), 4.07 (1H, s). ESI-MS 225–228°C. [α]D20−29 (c=1.0, H2O).
m/z: 318.10 (M+H)+.
Synthesisof γ-L-Glutamyl-L-norvalylglycine(48) γ-L-Glu-
γ-L-Glutamyl-L-α-aminobutyryl-glycolic Acid (97, γ-Glu- L-Nva-Gly was prepared in the same manner as γ-L-Glu-
Abu-Gly Acid) In the first step of this synthesis, condensa- L-Val-Gly.
tion of Boc-Abu and glycolic acid benzyl ester was carried out
with WSC·HCl (1.1 equiv.) and 4-dimethylaminopyridine (0.3 m), 1.55–1.73 (2H, m), 2.02–2.10 (2H, m), 2.35–2.48 (2H, m)
equiv.). 3.72 (1H, t, J=6.2Hz), 3.84 (1H, d, J=17.6Hz), 3.89 (1H, d,
1H-NMR (D2O) δ: 0.86 (3H, t, J=7.40Hz), 1.60–1.74 J=18.0Hz), 4.20 (1H, dd, J=5.2 and 5.6Hz). 13C-NMR (D2O)
1H-NMR (D2O) δ: 0.81 (3H, t, J=7.4Hz), 1.22–1.38 (2H,