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A R T I C L E S
Qiao et al.
Figure 2. Inhibition of BPAO by 2,3-butadienal (3) at 30 °C, 100 mM,
pH 7.2, phosphate buffer.
Figure 3. Hydrolysis of 2,3-butadienal (3) in 100 mM, pH 7.2, phosphate
buffer at 30 °C showing isosbestic conversion to 5. Time interval: 10 min.
Inset: ln[(A∞ - A0)/(A∞ - At)] vs time plot for the increase of absorbance
at 276 nm due to 5.
mechanisms for the inactivating effect of 1, it was desirable to
evaluate the effect of incubating the enzyme with the theoretical
turnover product of 1, 3-butynal (2). The acetal precursor, 4,4-
diethoxy-1-butyne was successfully prepared by the reaction
of propargyl bromide with triethyl orthoformate in the presence
of aluminum amalgam as catalyst.10 We knew that to avoid
tautomerization of 2 to its allenic form, 2,3-butadienal (3), the
subsequent acidic hydrolysis would have to be carried out under
mild conditions. However, all efforts to generate 2 in good yield
failed. Although many different acids were tried, either no
hydrolysis occurred or, once there was sufficient acid strength
to begin to convert the acetal, a substantial amount of 2
underwent tautomerization to 3. Another route to aldehydes is
the cleavage of dithioacetals by mercury(II), ferric(III), or
cerium(III) salts under neutral conditions. 4,4-Diethoxy-1-butyne
was successfully converted to 4,4-bis(ethylsulfanyl)-1-butyne,11
but even mild oxidative cleavage conditions failed to afford the
desired aldehyde 2. A summary of these experiments confirms
the high instability of 3-butynal with respect to its allenyl
tautomer and is consistent with the fact that 2 has been reported
in the literature only as a mixture with 3.12
At lower concentrations of 3 (e1.3 mM), the loss of activity
with time reached a plateau after 1.5 h. Assuming 3 inactivates
BPAO by a simple bimolecular alkylation reaction (or via an
E‚I complex characterized by a high Ki), the cessation of further
inactivation after some time suggests that little 3 remains at
this point, even though its initial concentration was far in excess
over enzyme. In fact, aldehyde 3 was expected to hydrolyze in
aqueous solution to 3-oxobutanal (5).
The hydrolysis of 3 was monitored over time spectrophoto-
metrically under the conditions used for the enzyme incubations
(pH 7.2, 100 mM potassium phosphate buffer, 30 °C). As judged
by the isosbestic behavior shown in Figure 3, consumption of
3 (λmax 222 nm) occurs concomitant with the formation of 5
1
(λmax 276 nm), which was shown by H NMR to exist at least
partly as the enol.14 Under these conditions, 3 has a half-life of
38 min. The authentic compound 5 was prepared15 and shown
not to inhibit BPAO at a concentration up to 2 mM. Thus, the
plateau behavior in Figure 2 at lower concentrations of 3 can
be understood in terms of the predominance of the unimolecular
hydrolysis reaction (converting inactivator 3 to noninactivator
5) in competition with the bimolecular enzyme inactivation
reaction. In contrast, at higher concentrations of 3, the bi-
molecular inactivation reaction proceeds to near completion
within the lifetime of 3. The sensitivity of this competition to
relatively small changes in concentration of 3 is remarkable.
Irreversible Inactivation of BPAO by 1-Amino-2,3-buta-
diene (4). The strong inactivation of BPAO by 1 coupled with
the observation of enzyme inactivation by 3 prompted us to
explore the activity of 1-amino-2,3-butadiene (4), since this is
the amine that would theoretically give 3 as its normal turnover
product. The known amine 4 was prepared16 and was found to
The facile tautomerization reaction of 2 to 3 suggests that if
2 were truly the initial direct product of metabolic turnover of
1, it would soon be converted to 3. Also, it is possible that the
tautomerization could occur at the product Schiff base stage
prior to hydrolytic release of aldehyde. In either case, enzyme
inactivation could then be arising from alkylation of an active-
site nucleophile by the highly reactive electrophile 2,3-butadienal
(3). Thus, it seemed prudent to evaluate the interaction of BPAO
with 3, which was already in hand from attempted synthesis of
2, but was prepared in excellent yield independently by a known
procedure.13 Data shown in Figure 2 demonstrate that 3 exerts
time- and concentration-dependent inactivation of BPAO,
though only at relatiVely high concentrations.
(10) Vereshchagin, L. I.; Gavrilov, L. D.; Titova, E. I.; Vologdina, L. P. Zh.
Org. Khim. 1973, 9, 247-252.
(11) Rothstein, E.; Whiteley, R. J. Chem. Soc., Abstr. 1953, 4012-4017.
(12) Blankespoor, R. L.; Smart, R. P.; Batts, E. D.; Kiste, A. A.; Lew, R. E.;
Van der Vliet, M. E. J. Org. Chem. 1995, 60, 6852-6859.
(13) (a) Shostakovskii, M. F.; Khomenko, A. K. Bull. Acad. Sci. USSR, DiV.
Chem. Sci. 1960, 1022-1026. (b) Burgers, P. C.; Holmes J. L.; Lossing,
F. P.; Mommers, A. A.; Povel, F. R.; Terlouw, J. K. Can. J. Chem. 1982,
60, 2246-2255.
(14) George, W. O.; Mansell, V. G. J. Chem. Soc., B 1968, 132-134.
(15) Vavilova, A. N.; Trofimov, B. A.; Volkov, A. N.; Keiko, V. V. J. Org.
Chem. USSR 1981, 17, 809-812.
(16) Casara, P.; Jund, K.; Bey, P. Tetrahedron Lett. 1984, 25, 1891-1894.
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8040 J. AM. CHEM. SOC. VOL. 126, NO. 25, 2004