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M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
4.11. N-Acetyl-
D
-allo- and
L
-isoleucine benzyl ester 1j
(NHCOH)COOCH3]; 1.25 ppm [m, 1H, CH3CH2CH-
(CH3)CH(NHCOH)COOCH3]; 2.0 ppm, [m, 1H, CH3-
CH2CH(CH3)CH(NHCOH)COOCH3]; 3.9 ppm, [s, 3H,
RCOOCH3]; 4.95 ppm, [dd, J1=9.4 Hz, J2=3.5 Hz,
1H, CH3CH2CH(CH3)CH(NHCOH)COOCH3]; 6.2
ppm, [d broad, J=9.4 ppm, 1H, CH3CH2CH(CH3)-
CH(NHCOH)COOCH3]; 8.15 ppm, [s,1H, CH3-
CH2CH(CH3)CH (NHCOH) COOCH3]. Anal. calcd
for C8H15NO3: C, 55.47%; H, 8.73%; N, 8.09%. Found:
C, 55.40%; H, 8.71%; N, 8.11%
To 5.0 g (23 mmol) of 1d, dissolved in acetic anhydride
(25 ml) in an ice-bath, dimethylaminopyridine (1 mg)
and triethylamine (3.5 ml) were added and the solution
left at rt for 2.5 h. After evaporation of the solvent, the
residue was taken up in cold brine (50 ml) and the
product extracted with ethyl acetate (3×50 ml). The
combined organic layers were washed successively with
0.2 M aqueous hydrochloric acid (50 ml), aqueous 5%
sodium hydrogen carbonate (50 ml) and with sodium
chloride (50 ml). Drying over sodium sulphate and
evaporation of the solvent gave 5.74 g (22 mmol, 96%)
of a white solid. HPLC (Method 2c) tR: 13.7 min 3j
(54%) and 15.1 min 2j (46%).
4.15. N-Formyl-D-alloisoleucine chloroethyl ester 3g
[Substrate]: 0.18 M [Enzyme]: 0.043 (g/ml) The reaction
was stopped after 2.5 h (59% of sodium hydroxide
consumption). After recovery by extraction the unre-
acted substrate was purified by chromatography (hex-
ane/ethyl acetate 1:1). Yield: 40% (pale yellow oil)
d.e.(HPLC, method 2b): 96% [h]D=−13.3 (c 1 in
CHCl3); NMR (CDCl3) l: 0.9 ppm, [d, J=7.1 Hz, 3H,
CH3CH2CH(CH3)CH(NHCOH)COOR]; 0.95 ppm, [t,
J=7.5 Hz, 3H, CH3CH2CH(CH3)CH(NHCOH)-
COOR]; 1.1–1.3 ppm, [dq, J1=7.1 Hz, J2=not deter-
mined, CH3CH2CH(CH3)CH(NHCOH)COOR]; 1.35–
1.55 ppm, [dq, J1=7.1 Hz, J2=not determined,
CH3CH2CH(CH3)CH(NHCOH)COOR]; 2.0 ppm, [m,
1H, CH3CH2CH(CH3)CH (NHCOH)COOR]; 3.70
ppm, [t, J=5.6 Hz, RCOOCH2CH2Cl]; 4.40 ppm, [m,
2H, RCOOCH2 CH2Cl]; 4.85 ppm, [dd, J1=9.4 Hz,
J2=4.1 Hz, 1H, CH3CH2CH(CH3)CH(NHCOH)-
COOR]; 6.35 ppm, [d (broad), J=9.0 Hz, 1H,
CH3CH2CH(CH3)CH(NHCOH)COOBn]; 8.25 ppm, [s,
1H, CH3 CH2CH(CH3)CH(NHCOH)COOCH3]. Anal.
calcd for C9H16ClNO3: C, 48.76%; H, 7.27%, Cl
15.99%; N, 6.32%. Found: C, 48.65%; H, 7.22%, Cl
15.76%; N, 6.33%.
4.12. N-Carbobenzyloxy-D-allo- and L-isoleucine benzyl
ester 1m
To 100 ml of a 1 M NaHCO3, 5.0 g (23 mmol) of 1d,
benzyl chloroformate (4.2 ml, 30 mmol) were added
dropwise and the mixture stirred at 5–10°C. After 45
min, the reaction was quenched with 2 M aqueous
hydrochloric acid (21 ml) and, after separation of the
phases, the aqueous one was extracted with ethyl ace-
tate (2×100 ml). The combined organic layers were
washed with 0.2 M aqueous hydrochloric acid (50 ml)
and with brine (2×50 ml). Drying over sodium sulphate
and evaporation of the solvent gave a residue, which
yielded 7.65 g (22 mmol, 96%) of a pale yellow oil after
chromatography. NMR (CDCl3) l: 0.75–1.0 ppm, 6H,
m; 1.0–1.25 ppm, 1H, m; 1.25–1.5 ppm, 1H, m; 1.95
ppm, 1H, m; 4.40 ppm, 0.5H, dd; 4.95 ppm, 0.5H, dd;
5.0–5.4 ppm, 5H, m; 7.35 ppm, 10H, m. MS (EI) m/z
356 (M+1), 220 (M−PhCH2OCO), 91 (PhCH2). HPLC
(Method 2c) tR: 9.4 min 2m (47%) and 24.0 min 3m
(53%).
4.13. Enzymatic hydrolysis of compounds 1f–m
4.16. N-Formyl-D-alloisoleucine benzyl ester 3h
A suspension of the substrate in distilled water (0.18 or
0.36 M) was heated at 39°C and adjusted to pH 7.8 by
the addition of NaOH 0.35 M. Alcalase 2.5L was then
added (0.21 or 0.43 g/ml) and the reaction mixture was
kept at pH 7.8 by automatic addition of NaOH 0.35 M.
When the consumption of NaOH reached about 50%
(or more) the reaction was stopped and the unreacted
substrate was recovered by extraction with tert-butyl-
methylether. The acidic product could be recovered by
extraction or precipitation after acidification of the
aqueous phase with citric acid or dilute hydrochloric
acid.
[Substrate]: 0.18 M [Enzyme]: 0.021 (g/ml) The reaction
was stopped after 22.5 h (47% of sodium hydroxide
consumption). After recovery by extraction the unre-
acted substrate was purified by chromatography (hex-
ane/ethyl acetate 6:4). Yield: 41% (pale yellow oil) d.e.
(HPLC, method 2c): 98% [h]D=−7.7 (c 1 in CHCl3);
NMR (CDCl3) l: 0.8 ppm, [d, J=7.2 Hz, 3H,
CH3CH2CH(CH3)CH(NHCOH)COOBn]; 0.9 ppm, [t,
J=7.2 Hz, 3H, CH3CH2CH(CH3)CH(NHCOH)-
COOBn]; 1.1–1.3 ppm, [dq, J1=7.2 Hz, J2=6.4 Hz,
CH3CH2CH(CH3)CH(NHCOH)COOBn]; 1.3–1.5 ppm,
[dq, J1=7.2 Hz, J2=6,4 Hz, CH3CH2CH (CH3)CH-
(NHCOH)COOBn]; 1.95 ppm, [m, 1H, CH3CH2CH-
(CH3)CH(NHCOH) COOBn]; 4.85 ppm, [dd, J1=8.8
Hz, J2=4.0 Hz, 1H, CH3CH2CH(CH3)CH(NHCOH)
COOBn]; 5.15 ppm, [AB system, JAB=16.0 Hz, 2H,
RCOCH2Ph]; 6.30 ppm, [d (broad), J=8,0 Hz, 1H,
CH3CH2CH (CH3)CH(NHCOH)COOBn]; 7.35 ppm,
[m, 5H, RCOCH2Ph]; 8.20 ppm, [s, 1H,
CH3CH2CH(CH3) CH(NHCOH)COOCH3]. Anal.
calcd for C14H19NO3: C, 67.45%; H, 7.68%; N, 5.62%.
Found: C, 67.35%; H, 7.72%; N, 5.60%.
4.14. N-Formyl-D-alloisoleucine methyl ester 3f
[Substrate]: 0.18 M; [Enzyme]: 0.043 (g/ml) The reac-
tion was stopped after 23 h (64% of sodium hydroxide
consumption). Yield: 30% (pale yellow oil) d.e.(HPLC,
Method 2a): 98% [h]D=−32.5 (c 1 in CHCl3) NMR
(CDCl3) l: 0.85 ppm [d, J=7.0 Hz, 3H,
CH3CH2CH(CH3)CH(NHCOH)COOCH3]; 0.95 ppm,
[t, J=7.0 Hz , 3H, CH3CH2CH(CH3)CH(NHCOH)-
COOCH3]; 1.1 ppm, [m, 1H,CH3CH2CH(CH3) CH