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or three orders of magnitude smaller than those for other (5a, in Table 2.
(tert-Butoxycarbonyl)amino Acid p-(Amidinomethyl)phenyl Esters p-
Toluenesulfonic Acid Salt (5a—c) General Procedure: A solution of
(tert-butoxycarbonyl)amino acid p-{[(acetoxy)imino]-2-aninoethyl}phenyl
ester (4) (1.0 mmol) and p-toluenesulfonic acid monohydrate (1 mmol) in
EtOH (10 ml) was hydrogenated over 10% Pd–C (10 mg) at room tempera-
ture for overnight with vigorous stirring. The catalyst was filtered away, and
the filtrate was concentrated in vacuo. The residue was washed with absolute
ether, and the solid was recrystallized to give 5 as a colorless solid. Physical
properties and spectral data are given in Table 3.
3-Hydroxybenzyl Cyanide (6) This compound was prepared from (3-
methoxyphenyl)acetonitrile according to the reported procedure.15)
meta-Series Compounds These compounds were synthesized by a pro-
cedure similar to that employed for the corresponding p-compounds (5a—
c). Physical properties and spectral data are given in Tables 1—3.
Kinetic Measurements Enzyme concentration was determined by ac-
tive site titration using p-nitrophenyl pꢄ-guanidinobenzoate.16) Analysis of
kinetic parameters was carried out by potentiometrically using a pH-stat
under steady-state conditions following the reported procedure.1,9) Determi-
nation of kcat and Km were carried out in 0.1 M KCl, pH 8.0, containing
0.02 M CaCl2 at 25 °C. In these experiments the enzyme concentration
was 1.95ꢃ10ꢀ8—1.50ꢃ10ꢀ6 M, and the substrate concentration was
1.76ꢃ10ꢀ5—1.07ꢃ10ꢀ4 M.
5b, 10a).
The kinetic parameters for N-Boc-Aib-OAM (5c, 10c)
were also compared with those for N-Boc-L-Ala-OpAm (15).
The Ks (Km) value for 5c and 10c was two orders of magni-
tude larger than those for 15, respectively, as shown in Table
4. The k3 (kcat.) value of 5c and 10c was two orders to four or-
ders of magnitude smaller than that of 15, respectively. This
result indicated that the bulky a,a-dialkyl substituent im-
pedes the placement of 5c and 10c in juxtaposition with the
active site of trypsin. The kcat./Km value for 10c showed a
much less favorable interaction. However, we previously
reported that even a,a-dialkyl amino acid m-(guanidi-
nomethyl)phenyl ester could be used in a Streptomyces
griseus trypsin-catalyzed coupling reaction.14) We conclude
that all new synthetic inverse substrates examined in this
study could be expected as acyl donor components in
trypsin-catalyzed peptide synthesis.
Experimental
General The melting points were measured on a Yanaco micro melting
point apparatus. IR spectra were taken on a JASCO VALOR-III FT-IR spec-
trometer. 1H-NMR spectra were recorded on a JEOL JNM-FX-400 FT NMR
spectrometer. Chemical shifts are quoted in parts per million (ppm) with
tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are
given in Hz. The following abbreviations are used: s, singlet; d, doublet; t,
triplet; q, quartet; br s, broad singlet; dd, doublet of doublets; m, multiplet.
The optical rotations were measured with a JASCO DIP-360 digital po-
larimeter in a 5 cm cell. Kinetic parameters were determined with a Ra-
diometer TTT-80 pH-stat. Flash column chromatography was performed
using Silica Gel 60N (Kanto Chemical Co., Inc.) as a solid support in the
immobile phase. Kieselgel 60 F-254 plates (Merck) were used for thin-layer
chromatography (TLC). Bovine pancreas trypsin (EC 3.4.21.4) was pur-
chased from Worthington Biochemical Corp. (twice crystallized, lot TRL).
p-{[(Hydroxy)imino]-2-aminoethyl}phenol (2) To a solution of 4-hy-
droxybenzyl cyanide (1) (01.65 g, 80 mmol) in EtOH (80 ml) was added to a
solution of hydroxylamine hydrochloride (19.46 g, 0.28 mol) and potassium
carbonate (19.34 g, 0.14 mol) in H2O (120 ml), and the mixture was refluxed
for 4 h. The mixture was concentrated to half volume and then cooled to
room temperature. The formed precipitate was collected by filtration to give
2 (13.17 g, 99%) as brownish needles. Physical properties and spectral data
are given in Table 1.
Acknowledgments This work was supported in part by a Grant-in-Aid
for High Technology Research Program from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan, and by a grant from the
Japan Private School Promotion Foundation.
References and Notes
1) Tanizawa K., Kasaba Y., Kanaoka Y., J. Am. Chem. Soc., 99, 4485—
4488 (1977).
2) Itoh K., Sekizaki H., Toyota E., Fujiwara N., Tanizawa K., Bioorg.
Chem., 24, 59—68 (1996).
3) Sekizaki H., Itoh K., Toyota E., Tanizawa K., Chem. Pharm. Bull., 44,
1577—1579 (1996).
4) Sekizaki H., Itoh K., Toyota E., Tanizawa K., Chem. Pharm. Bull., 44,
1585—1587 (1996).
5) Sekizaki H., Itoh K., Toyota E., Tanizawa K., Chem. Pharm. Bull., 46,
846—849 (1998).
6) Sekizaki H., Itoh K., Toyota E., Tanizawa K., Amino Acids, 17, 285—
291 (1999).
7) Sekizaki H., Itoh K., Toyota E., Tanizawa K., Chem. Pharm. Bull., 47,
104—110 (1999).
8) Brian D. J., David G. A., Tracey A. C., Synth. Commun., 26, 4351—
4367 (1996).
p-{[(Acetoxy)imino]-2-aminoethyl}phenol (3) p-{[(Hydroxy)imino]-2-
aminoethyl}phenol (2) (1.66 g, 10 mmol) was soluble in hot pyridine
(20 ml), and to a solution was added dropwise acetic anhydride (946 ml) at
0 °C, and the mixture was stirred at room temperature for 1 h. To the mixture
was added heptane, and azeotropic mixtures were concentrated in vacuo.
The resulting residue was recrystallized from hexane/EtOH to give 3 (1.79 g,
86%) as colorless needles. Physical properties and spectral data are given in
Table 1.
9) Nozawa M., Tanizawa K., Kanaoka Y., J. Pharmacobio-Dyn., 3, 213—
219 (1980).
10) Nakano M., Tanizawa K., Nozawa M., Kanaoka Y., Chem. Pharm.
Bull., 28, 2212—2216 (1980).
11) Sekizaki H., Itoh K., Murakami M., Toyota E., Tanizawa K., Comp.
Biochem. Physiol., 127B, 337—346 (2000).
12) The kcat./Km value is equivalent to k2/Ks where kcat. and Km denote the
overall catalytic rate constant and the Michaelis constant, respectively,
as reported; Ryan J. T., Fenton W. J., II, Chang T.-I., Feinman D. R.,
Biochemistry, 15, 1337—1441 (1976).
(tert-Butoxycarbonyl)amino Acid p-{[(Acetoxy)imino]-2-aminoethyl}-
phenyl Esters (4a—c) General Procedure: To
a solution of p-
{[(acetoxy)imino]-2-aminoethyl}phenol (3) (312 mg, 1.5 mmol), N-Boc-
amino acid (1.7 mmol), and DMAP (24 mg, 0.2 mmol) in DMF (5 ml) and
EtOAc (5 ml) was added DCC (351 mg, 1.7 mmol) at 0 °C, and the mixture
was stirred at the same temperature for 1 h. Then the mixture was stirred at
room temperature for overnight and concentrated. The resulting residue was
chromatographed on a silica gel column (benzene/EtOAcꢁ1 : 3). The ben-
zene/EtOAcꢁ1 : 3 elute was evaporated to dryness in vacuo, and the solid
was recrystallized to give 4. Physical properties and spectral data are given
13) Brot E. F., Bender M. L., J. Am. Chem. Soc., 91, 7187—7191 (1969).
14) Sekizaki H., Itoh K., Toyota E., Tanizawa K., Tetrahedron Lett., 38,
1777—1780 (1997).
15) Kametani T., Kigasawa K., Hiiragi M., Ishimaru H., Saito S., Yaku-
gaku Zasshi, 89, 1482—1487 (1969).
16) Chase T., Jr., Shaw E., Biochem. Biophys. Res. Commun., 29, 508—
514 (1967).
Sekizaki, Haruo
