Vol. 26, No. 19 (2014)
Heating Reactions of N-t-Butyloxycarbonyl-Asparagine and Related Compounds 6543
MW=17,800; clupeine: MW = 4110; insulin A-chain: MW =
2340) and glycine hexamer (Gly6: MW = 360) for the
calibration of the molecular weight of the peptides were
purchased from SIGMA-Aldrich (St. Louis, USA). The
pentamer of prolyl-prolyl-glycine ((Pro-Pro-Gly)5: MW =
1274) was purchased from the Peptide Institute, Inc. (Osaka,
Japan).
(amide I), 1538 (amide II). Elementary analysis: Calcd. for
C7H13NO4: C, 47.99; H, 7.48; N, 8 %. Found: C, 48.01; H,
7.40; N, 7.99 %.
N-t-Butyloxycarbonyl-L-alanine (4c):Yield: 28.8 g (76 %).
1
m.p. 87-88 °C (lit.22) 83-84 °C). H NMR (DMSO-d6): δ =
1.38 ppm (9H, s, CH3), 2.09 (1H, s, NH), 1.18-1.26 (3H, d,
CH3), 3.77-4.07 (1H, q, CH), 7.03-7.11 (1H, d, NH). IR (KBr,
ν
max, cm-1): 3386 (NH), 1742 (COOH), 1690 (amide I), 1520
A Hitachi model 260-50 infrared spectrophotometer
(Hitachi, Tokyo, Japan) was used to record the infrared spectra.
A Hitachi Model 200-10 Spectrophotometer was used to record
the ultraviolet-visible spectra. Optical rotation of the synthetic
compounds from L-aspartic acid was determined with a Jasco
DIP-181 digital polarimeter.A Hitachi 835 amino acid analyzer
(Hitachi, Tokyo, Japan) was used for amino acid analysis.
Molecular weight determination was performed using a high-
performance liquid chromatography (HPLC) system composed
of an ultraviolet detector, Jasco Uvdec-100-V UV spectro-
photometer and a flow pump, Jasco TRI Rotor-V, equipped
with a TSK GEL G3000PW (300 mm × 7.5 mm I.D.). The
flow rate was 0.7 mL/min (0.1 M sodium phosphate, pH 6.9)
and the absorbance of the samples was recorded at 230 nm.
The peaks on the chromatograms were integrated with an SIC
Chromatocorder II. Enantiomer separation of amino acids was
performed with a Hitachi 163 gas chromatograph equipped
with a chiral glass capillary column (Chirasil-Val16-19), 25 m ×
0.3 mm I.D.). Nitrogen was used as the carrier gas at a flow
rate of 30 mL/min. The temperature was programmed from
80 to 170 °C at a rate of 4 °C/min. The detection was carried
out with a flame thermionic detector. A Shimadzu DT-40
(Shimadzu, Kyoto, Japan) was used for thermal analysis. A
Shimadzu GCMS-QP 1000A mass spectrometer was used for
measuring mass spectra.A JEOL EX-270 NMR system (JEOL,
(amide II). Elemental analysis: Calcd. for C8H15NO4: C, 50.78;
H, 7.99; N, 7.40 %. Found: C, 50.78; H, 8.10; N, 7.27 %.
[α]D15-24.2° (c = 1.30, acetic acid) (lit.21) [α]D20-22.4 (c = 2.1,
acetic acid).
N-t-Butyloxycarbonyl-L-valine (4d): Yield: 23.4 g (93
%). m.p. 78-79 °C (lit.22) 77-79 °C). 1H NMR (DMSO-d6): δ =
0.83-0.91 ppm (6H, q, CH3), 1.39 (9H, s, CH3), 1.84-2.11 (1H,
m, CH), 3.72-3.87 (1H, q, CH), 6.85-6.94 (1H, d, NH). IR
(KBr, νmax, cm-1): 3312 (NH), 1740 (COOH), 1649 (amide I),
1500 (amide II). Elemental analysis: Calcd. for C10H19NO4: C,
55.28; H, 8.81; N, 6.45 %. Found: C, 55.42; H, 8.86; N, 6.45
%. [α]D15-5.1° (c = 0.97, acetic acid) (lit.21) [α]D20-5.8 (c = 1.2,
acetic acid).
L-Aspartic acid β-benzyl ester (5): L-Aspartic acid (1b)
(50 g, 0.38 mol), benzyl alcohol (125 g, 1.16 mol), a mixture
of concentrated sulfuric acid (40.5 g) and water (10 g) were
mixed in a water bath at 70 °C for 0.5 h to give a clear solution,
which was stirred for s further 2 h. The resulting solution was
evaporated in vacuo to an oily product, which was added to a
cold solution containing sodium hydrogen carbonate (70 g)
and water (250 mL) at 0 °C. The obtained cooled solution was
mixed with ether (150 mL) to give a precipitate, which was
filtered and washed with cold water to give another precipitate
(88 g). This was recrystallized twice with water to give 34 g
(41 %). m.p. 218-219 °C (lit.23) 222 °C).
1
Tokyo, Japan) was used for the measurement of H NMR
spectra.
N-t-Butyloxycarbonyl-L-aspartic acid β-benzyl ester
(6): L-Aspartic acid β-benzyl ester (5) (3 g, 13.4 mmol) was
dissolved in 1 M sodium hydroxide (13 mL) and tetrahydro-
furan-acetonitrile (1:1 (v/v), 26 mL). To the cooled solution
was added dropwise an acetonitrile solution containing di-t-
butyloxy-dicarbonate (3.22 g, 14.7 mmol) over 0.5 h. The
solution was adjusted to pH 8 by adding 1 M sodium hydroxide
(5 mL). The reaction solution was stirred for 24 h and evapo-
rated in vacuo to give an oily product. The cooled oil was
acidified with 10 % potassium hydrogen sulfate (30 mL) to
pH 2 and the resulting solution was extracted with ethyl acetate
(30 mL × 3). The extracts were combined and dried with
anhydrous magnesium sulfate and the evaporation of the filtrate
gave a precipitate. Twice recrystallization with ethyl acetate-
petroleum ether gave 2.5 g (56 %). m.p. 98 °C (lit.24) 99 °C).
N-t-Butyloxycarbonyl-L-aspartic acid β-benzyl ester
α-amide23(7): N-t-butyloxycarbonyl-L-aspartic acid β-benzyl
ester (11.3 g, 35 mmol), ammonium hydrogen carbonate (7.99
g,105 mmol), N-ethoxycarbonyl-2-ethoxy-1 and 2-dihydro-
quinolone (9.52 g, 38.5 mmol) were suspended in chloroform
(65 mL). The reaction mixture was stirred overnight at room
temperature. The reaction mixture was extracted with water
and the resulting chloroform layer was evaporated in vacuo to
give a precipitate, which was recrystallized with ethyl acetate-
hexane to give 6.48 g (58 %). m.p. 153-155 °C. (lit. 157-160).
[α]D25-2.1 (c = 1, ethanol). (lit.25)[α]D25 -2.6 (c = 1, ethanol)).
Preparation of substrates
N-t-Butyloxycarbonyl-L-asparagine (4a): To a solution
containing L-asparagine monohydrate (1a) (15 g, 0.10 mol)
in a mixture of 1 M sodium hydroxide (100 mL) and water
(180 mL) was added a solution containing di-t-butyloxy-
dicarbonate (24 g, 0.11 mol) in 1,4-dioxane (170 mL) dropwise
with stirring at 10 °C. After overnight stirring at room tempe-
rature, the reaction mixture was evaporated in vacuo to an
oily product, which was cooled and acidified to pH 2 with
10 % potassium hydrogen sulfate to give a precipitate. The
precipitate was filtered, washed with water and recrystallized
with 500 mL ethanol to give 14.8 g of crystals (64 %). m.p.
177-179 °C (lit.20) 181-182). 1H NMR (DMSO-d6): δ = 1.38
ppm (9H, s, CH3), 2.09 (1H, s, NH), 2.48 (2H, d, CH2), 4.22
(1H, q, CH), 6.88 (2H, d, NH2). IR (KBr, νmax, cm-1): 1720
(COOH), 1690 (amide I), 1663 (amide II (side chain)), 1591
(amide II (side chain)), 1535 (amide II). Elemental analysis:
Calcd. for C9H16N2O5: C, 46.54; H, 6.94; N, 12.07 %. Found:
C, 46.70; H, 7.05; N, 11.99 %. [α]D15-7.8° (c = 1.22, DMF)
(lit.21) [αD]20 -7.8 (c = 1, DMF)).
N-t-Butyloxycarbonyl-glycine (4b): Yield: 12.6 g
(72 %), m.p. 89-91 °C (lit.21) 88.5-89 °C). 1H NMR (DMSO-
d6): δ = 1.38 ppm (9H, s, CH3), 3.58 (2H, d, CH2), 7.04 (1H, t,
NH). IR (KBr, νmax, cm-1): 1750 (COOH), 1690 (amide I), 1671