bath was removed and stirring was continued for 18 h at room
temperature. The resulting solution was washed with 1 M HCl
(2 × 100 mL), dried over Na2SO4 and then evaporated. The oily
yellow residue was crystallized from diethyl ether–petroleum
ether to produce the ester as a white solid (7.4 g, 85%). Mp
124–125 ЊC (Found: C, 61.49; H, 6.82; N, 4.79%. C15H19NO5
requires C, 61.40; H, 6.53; N, 4.78%); [α]2D0 = Ϫ41 (c 1
in CHCl3); νmax(KBr)/cmϪ1 3300 (br NH), 3200–2500 (OH),
1738 (CO2Me), 1700 (CO2H), 1636 (CONH); δH(200 MHz,
CDCl3) 8.70 (1H, br s, CO2H, exchange with D2O), 7.99–
7.49 (4H, m, ArH), 6.73 (1H, d,J 8.4 Hz, NH, exchange with
D2O), 4.87 (1H, m, NCH), 3.76 (3H, s, CO2CH3), 1.77–1.68
(3H, m, CHCH2), 1.00 (3H, d, J 6.46 Hz, CH3), 0.97 (3H, d,
J 6.55 Hz, CH ); δ (50.3 MHz, CDCl ) 173.50, 169.91(C᎐O),
᎐
3
C
3
169.81, 137.19, 132.46, 131.23, 130.05, 128.87 and 127.91 (Ar),
52.47 (CHNH), 51.28 (OCH3), 41.38 (CH2), 24.84 (CH), 22.73
and 21.92 ((CH3)2); m/z calculated 293.17, found 294.15
(M ϩ 1)ϩ
.
ؒ
N-(o-Carboxybenzoyl)-L-leucine (NCBL)18
A solution of NCBL-ME (3.48 mmol) in 1 M aqueous NaOH
(11 mL) was stirred at room temperature for 2.5 h. The reaction
mixture was acidified with 1 M hydrochloric acid up to pH 1
and extracted with ethyl acetate (3 × 50 mL). The organic layers
were combined, washed with water (2 × 50 mL) and brine
(2 × 50 mL), dried over Na2SO4 and evaporated. The resulting
solid residue was suspended in CHCl3 (50 mL), stirred for 1 h,
and the diacid 4 as a white amorphous solid was collected by
filtration and dried under vacuum to give the title compound
(0.66 g, 68%). Mp 127–128 ЊC (Found: C, 60.34; H, 6.00; N,
5.09%. C14H17NO5 requires C, 60.21; H, 6.13; N 5.02%);
[α]D20 = Ϫ42 (c 1 in EtOH); νmax(KBr)/cmϪ1 3300 (br NH), 3200–
2500 (OH), 1750 and 1690 (CO2H), 1616 (CONH); δH(200
MHz, DMSO-d6) 12.80 (1H, br s, CO2H, exchange with D2O),
8.63 (1H, d, J 8.00 Hz, NH, exchange with D2O), 7.78–7.39
(4H, m, ArH), 4.40–4.30 (1H, m, NCH), 1.70 (3H, m,
CHCH2), 0.98 (6H, d, J 6.37 Hz, (CH3)2); δC(50.3 MHz,
Fig. 1 Ultraviolet spectra in 5 M HCl, at 50 ЊC: Fig. 1A. Spectral
variation obtained during the reaction of NCBL (1.0 × 10Ϫ3 M). The
time interval between each scan (a1–a6) is 3 min. Fig. 1B shows the
spectra of (b1) N-phthaloylleucine (1.0 × 10Ϫ3 M); (b2), solid line, refers
to the observed spectra of the products of the NCBL reaction; the
symbol (᭹) corresponds to the absorbance calculated for a solu-
tion composed of 6.67 × 10Ϫ4 M phthalic acid and 3.33 × 10Ϫ4
M
DMSO-d ) 174.07, 168.31, 167.89 (C᎐O), 138.05, 131.04,
᎐
6
N-phthaloylleucine; and (b3) corresponds to the spectra of phthalic
acid (1.0 × 10Ϫ3 M). Fig. 1C shows repetitive scans obtained during the
reaction of N-phthaloylleucine, 1.0 × 10Ϫ3 M. The time interval
between each scan (c1–c9) is 24 hours.
130.80, 129.18, 129.06 and 127.76 (Ar), 50.51 (CHNH), 39.79
(CH2), 24.16 (CH), 23.01 and 21.24 ((CH3)2); m/z calculated
279.28, found 261.15 (M Ϫ 18)ϩ
.
ؒ
L-Phthaloylleucine (NPL)
using the HP 8452 kinetics software, which showed, for at least
4–5 half-lives, an excellent first-order kinetics behavior.
This was prepared by a direct reaction between -leucine and
phthalic anhydride in toluene, according to a method given in
the literature20 (85%, mp 115–116 ЊC, lit.21 116 ЊC). νmax and
δH data were consistent with the structure.
Results and discussion
The kinetic behavior of N-(o-carboxybenzoyl)--leucine,
NCBL, in aqueous solution was studied as a function of hydro-
gen ion concentration from H0 = Ϫ3.59 to pH 5. Fig. 1A shows
the spectral variation observed in the 250–350 nm region, upon
addition of NCBL to a 5 M HCl solution. Spectrum (a1)
corresponds to NCBL at the starting time: the intermediate
spectra were collected at 3 minute intervals and the final
spectrum (a6) upon completion of the reaction. As can be
seen, there is a smooth absorbance variation from NCBL to
products, with an isosbestic point at 278 nm, permitting the use
of changes in absorbance at 310 nm to follow the kinetics of the
reaction.
Fig. 1B shows the spectrum obtained after 10 half lives for
the reaction of NCBL in the presence of 5.0 M HCl. The final
spectrum b2 seems to correspond to a mixture between that
of the imide N-phthaloylleucine (NPL), spectrum b1, and that
of phthalic acid, spectrum b3, which are included in Fig. 1B for
comparison purposes. The dots marked in spectrum b2 corre-
spond to the calculated absorbances, assuming that the final
mixture corresponds to 3.33 × 10Ϫ4 M N-phthaloylleucine
(NPL) and 6.67 × 10Ϫ4 M phthalic acid, and that they agree
(within 3%) with the final kinetic spectrum. The intensity of the
Kinetic measurements
All kinetic work was carried out using a Hewlett Packard
diode-array spectrophotometer fitted with a thermostated
water-jacketed cell holder (Microquímica MQBTZ99-20). All
solutions were prepared using distilled water which was boiled
and cooled under nitrogen to remove dissolved CO2. The
kinetic solutions were prepared from reagent-grade HCl,
chloroacetic acid and acetic acid (Merck). Measurements of pH
were made using a Beckman Model Φ 71 pH meter and were
checked before use. In the pH region 1 to 5, the stock buffers
were made to a total ionic strength of 0.1 M by addition of
KCl. A typical kinetic run, 2.7 mL of the appropriate solution,
was kept in equilibrium at 50 ЊC for ten minutes. The reaction
was then initiated by adding 0.3 mL of a stock solution of
NCBL (1.0 × 10Ϫ2 M) prepared in acetonitrile, and placed
in the thermostated cell compartment (50 0.1 ЊC). This pro-
cedure thus added 10% v/v acetonitrile into the reaction
mixture. The reaction was monitored following changes in
absorbance at 310 nm as a function of time. The absorbance
values were stored directly on a microcomputer and analyzed
1864
J. Chem. Soc., Perkin Trans. 2, 2001, 1863–1868