+
+
was reduced to a residue as described above. The hydrolysate residue
The flow rate was 1 mL/min. The mobile phase consisted of a mixture
of 50 mM dihydrogen potassium phosphate buffer (pH 3) and
acetonitrile. The volume ratio of the eluting solvent and the selected
wavelength varied with the application.
HP LC-MS-MSsSolution aliquots of synthetic standards and
isolated hydrolysates were injected into a Finnigan TSQ-70 mass
spectrometer with a Waters 590 HPLC pump at a flow rate of 0.8
mL/min without a column. The mobile phase consisted of 50 mM
ammonium acetate buffer (pH 5) and methanol (75:25 v/v). The
thermospray vaporizer temperature was 80 °C, the jet temperature
was 220 °C, and the block temperature reading was 175 °C.
was subjected to flash chromatographic separations as previously
described. Hydrolysates PAGA and PAA were isolated. The chro-
matographic and spectroscopic data of the isolates matched the data
of corresponding standards.
Syn th esissPAG, PAIG, and PAGA were synthesized as shown in
Scheme 3 by coupling of phenylacetyl chloride with the appropriate
glutamic acid derivative. As an example, the synthesis of PAIG is
given: Into 50 mL of water were added isoglutamine (1 g, 6.8 mmol)
and sodium bicarbonate (2.8 g) with vigorous stirring until a clear
solution developed. Phenylacetyl chloride (1.24 g, 8 mmol) was added
slowly with stirring. The solution was stirred for 2 h. The pH of the
solution was adjusted to 2.5 with 12 M HCl. The solution was
extracted twice with 25 mL portions of dichloromethane. The aqueous
layer was concentrated under reduced pressure to a white precipitate,
which was dissolved in 10 mL of eluting solvent and purified by two
passes through a flash chromatography column (described above). The
eluting solvent consisted of aqueous acetic acid (pH 3) and methanol
(85:15 v/v). The product PAIG resulted as a white solid (600 mg, 33%
yield). The spectroscopic data of the synthetic standards are reported
in Figures 2 and 3.
References and Notes
1. Green, S. J . Am. Med. Assoc. 1992, 267, 2924-2928.
2. Burzynski, S.; Houston, R.; Waneb, H.; Green, S. J . Am. Med.
Assoc. 1993, 269, 475-476.
3. Burzynski, S.; Mohabbat, M.; Burzynski, B. Drugs Exp. Clin.
Res. 1984, 10, 611.
4. Burzynski, S.; Mohabbat, M.; Burzynski, B. Drugs Exp. Clin.
Res. 1984, 10, 891.
NMRsSolid isolates of PAG (60 mg) and PAIG (8 mg) were
dissolved in water in separate NMR tubes, and the pH was adjusted
to 6.4 by addition of potassium hydroxide solution. D2O was added
(approximately 10% v/v) to the solutions for a field frequency lock.
5. Burzynski, S.; Hendry, L.; Mohabbat, M.; Liau, M.; Khalid, M.;
Burzynski B. Proc. Int. Congr. Chemother., 13th 1983, 17, 11-
14.
6. Burzynski, S.; Hai, T. Drugs of the Future 1985, 10, 103-105.
7. Burzynski, S. Recent Adv. Chemother., Proc. Int. Congr. Chemo-
ther., 14th 1985, 586-587.
1
HMQC, HMBC, H, and 13C experiments were carried out at 2 °C on
a Unity-Plus NMR spectrometer (Varian) operating at a proton
resonance frequency of 500 MHz. Collection conditions were as
follows: proton pulse length ) 10.5 µs, sweep width ) 4287 Hz,
acquisition time ) 1.9 s, number of transients ) 8; carbon pulse length
) 12 µs, sweep width ) 21 231 Hz, acquisition time ) 1.35 s, number
of transients ) 300; HMQC sweep width in f2 ) 4287 Hz, sweep width
in f1 ) 21 231, acquisition time in t2 ) 0.2455 s, acquisition time in
t1 ) 0.006 55 s, number of increments in t1 ) 256, number of
transients ) 8, number of complex data points in t2 ) 2048, Gaussian
apodization employed in both dimensions prior to Fourier transforma-
tion; HMBC conditions were identical to those for HMQC except 64
(PAG) and 128 (PAIG) transients were collected at each t1 increment.
Additionally, for HMBC, sine-bell apodization was employed in the
t2 dimension using absolute-value processing and Gaussian filtering
was used in t1 in the phase-sensitive mode. Suppression of the intense
H2O signal by transmitter preirradiation was carried out for all except
the 13C NMR experiments.
8. Burzynski, S. Drugs Exp. Clin. Res. 1986, 12, 1-9.
9. Michalska, D. Spectrochim. Acta, Part A 1993, 49, 303-314.
10. Lehner, A.; Burzynski, S.; Hendry, L. Drugs Exp. Clin. Res.
1986, 12, 57-72.
11. Hendry, L.; Muldoon, T.; Burzynski, S.; Copland, J .; Lehner, A.
Drugs Exp. Clin. Res. 1987, 13, 77-81.
12. Burzynski, S.; Mohabbat, M.; Lee, S. Drugs Exp. Clin. Res. 1986,
12, 11-16.
13. Ashraf, A.; Liau, M.; Mohabbat, M.; Burzynski, S. Drugs Exp.
Clin. Res. 1986, 12, 37-45.
14. Lee, J .; Lee, Y; Shin, S.; Choi, B. Arch. Pharmacal Res. 1995,
18, 75-78.
Acknowledgments
We thank Roger T. Wilson of the U.S. Department of Agriculture,
FSIS Midwestern Laboratory, St. Louis, MO, for mass spectral
support. We thank J ohn C. Reepmeyer of the Division of Drug
Analysis, St. Louis, MO, for reading the manuscript and offering
perceptive suggestions.
HP LC-UVsSolution aliquots of synthetic standards, commercial
standards, and hydrolysates were injected manually onto a 3 µm,
Ultracarb, C-18 column (150 × 4.6 mm) attached to a Hewlett-
Packard 1090M HPLC system equipped with a diode array detector.
J S960120Y
1052 / Journal of Pharmaceutical Sciences
Vol. 85, No. 10, October 1996