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404
BIOCHEMICAL SOCIETY TRANSACTIONS
lished work). The reaction occurs without the addition of
cofactor, in contrast to the hydantoins. Although we
have no further evidence, this observation alone suggests
that a nucleophilic mechanism may be
in the
inhibition of dihydro-orotate dehydrogenase.
Dutler, H. & Branden, C. -I. (1980) Bioorg. Chem. 10, 1-13
Eklund, H., Nordstrom, B., Zeppzauer, E., Soderlund, G., Ohlsson, I.,
Boiwe, T., Sodmberg, B. O., Tapia, O., Branden, C. -I. & Akeson, A.
(1976) J. Mol. Biol. 102, 27-59
Guengerich, F. P. & Macdonald, T. L. (1984) Acc. Chem. Res. 17,
9-16
Hanzlik, R. P. & Tullman, R. H. (1982) J. Am. Chem. SOC. 104,
2048-2050
Laurie, D., Lucas, E., Nonhebel, D. C. & Suckling, C. J. (1985)
Tetrahedron in the press.
Macdonald, T. L., Zirvi, K., Burka, L. T., Peyman, P. & Guengerich,
F.P. (1982) J. Am. Chem. SOC.104, 205C2052
MacInnes, I., Schorstein, D. E., Suckling, C. J. & Wrigglesworth, R.
(1981) J . Chem. SOC.Perkin Trans. 1 1103-1108
MacInnes, I., Nonhebel, D. C., Orszulik, S . T. & Suckling, C. J.
(XVIII)
(XW
(XX)
(1982) J. Chem. SOC.,Chem. Commun. 121-122.
The interplay between mechanism and inhibition is MacInnes, I., Nonhebel, D. C., Orszulik, S. T., Suckling, C. J. &
Wrigglesworth, R. (1983~)J. Chem. SOC.Perkin Trans. 1,2771-2776
one of the fascinations of this field for the organic
chemist, each aspect in turn supporting the other. We MacInnes, I., Nonhebel, D. C., Orszulik, S. T. & Suckling, C. J.
(19836) J. Chem. SOC.,Perkin Trans 1, 2777-2779
have so far only found cyclopropane derivatives that
Parkes,
C. & Abeles, R. H. (1984) Biochemistry 23, 63554363
inhibit enzymes apparently by nucleophilic mechanisms;
B. & Abeles, R. H. (1985) Biochemistry 24, 25962605
such radical reactions as have come along have treated Sherry,
Silverman, R. B. (1983) J. Biol. Chem. 258, 1476614769
our cyclopropanes as substrates. Currently, we are devot- Silverman, R. B. & Yamaski, R. B. (1984) Biochemistry 23, 1322-1332
ing most attention to clarifying the details of alcohol Silverman, R. B. & Zieske, P. A. (1985) Biochemistry 24, 2128-2138
dehydrogenase inhibition and to applying our knowledge Silverman, R. B., Hoffman, S. J. & Catus, W. B., 111 (1980) J. Am.
to the design of inhibitors of chemotherapeutically sigChem. SOC.102, 71267128
nificant enzymes.
Sligar, S. G., Kennedy, K. A. & Peerson, D. C. (1980) Proc. Natl.
Acad. Sci. U.S.A. 77, 124e1244
Bantia, S., Bevins, C. L. & Pollack, R. M. (1985) Biochemistry, 24, Suckling, C . J., Nonhebel, D . C., Brown, L., Sucklng, K . E., Seilman,
262G2609
S . & Wolf, C. R. (1985) Biochem. J. 232, 199-203
Buntain, I. G., Suckling, C. J. &Wood, H. C. S. (1985) J . Chem. SOC. Suckling, K. E., Smellie, C. G., Ibrahim, I. E. S . , Nonhebel, D. C. &
Chem. Commun. 242-244
Suckling, C. J. (1982) FEBS Lett. 145, 179-181
Dijkstra, M., Frank, J., Jongejan, J. A,, & Duine, J. A. (1984) Eur. J. Wiseman, J. S . , Tayrein, G. & Abeles, R. H. (1980) Biochemistry 19,
Biochem. 140, 369-313
4222423 1
Dowd, P., Kaufman, C. & Abeles, R. H., (1984) J. Am. Chem. SOC. Wiseman, J. S., Nichols, J. S. & Kolpakin (1982) J. Biol. chem. 257,
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63284332
A new perspective on pyridoxal chemistry and its relevance to enzyme inhibition
P. ARMSTRONG,* D. T. ELMORE,? R. GRIGG*
and C . H. WILLIAMS?
Departments of *Chemistry and ?Biochemistry
Queen’s University of Belfast, Belfast BT9 SAG,
N . Ireland, U.K.
Pyridoxal phosphate-dependent enzymes occur widely
and are responsible for the synthesis, racemization,
degradation and interconversion of a-amino acids in
living systems. (Bloch, 1972; Davis & Metzler, 1972;
Walsh, 1977). Transamination of the holoenzyme by an
a-amino acid produces the corresponding imine of the
a-amino acid (I). Concurrently, the aza-allylic bonds are
activated to cleavage by the facility with which the
protonated pyridyl ring of the pyridoxal moiety can
delocalize a negative charge. The rich chemistry of the
enzyme-substrate complex derives from this activation
moderated by the steric environment of the enzyme.
Many workers have studied aspects of pyridoxal chemistry (Walsh, 1979; Vederas & Floss, 1980) and strong
interest continues in both systems in vivo (Liu et al., 1984)
and model systems (Weiner et al., 1985). The initial
reactive intermediates (2) or (3) are generated by cleavage
of either bond (a) or (b) in protonation of the pyridine
nitrogen atom. Stereoelectronic control requires that the
breaking bond [bond (a) or bond (b) in (l)] is aligned
with the pyridyl azomethine n-system (Dunathan, 1966;
Fischer & Abbot, 1979).
Abbreviation used: GABA, y-aminobutyric acid.
R-
C -CO,H
R-I-c--co;
I t
I (b’
R-T
I
I/
I
R-CH
II
N
II
I
There are a large number of naturally occurring toxins
which function as suicide substrates for pyridoxal
enzymes (Rando, 1975) of which cycloserine (4) (Roze
& Strominger, 1966) and gabaculine ( 5 ) (Rando &
Bangerter, 1976, 1977; Rando, 1977) are probably the
best known. The former appears to generate an active
acylating agent which blocks the pyridoxal enzyme,
1986
61 5th MEETING, BELFAST
405
whilst the latter becomes aromatized, and hence inert to
cleavage, whilst bound to the pyridoxal.
(4)
(5)
The potential for mechanism-based pyridoxal enzyme
inhibition as a source of novel drugs has focused attention on this area in recent years. For example, inhibition
of alanine racemase could provide novel anti-bacterial
agents since the D-amino acids are key constituents of the
peptidoglycan layer of bacterial cell walls but are not
common metabolites in mammalian cells. Thus bfluoroalanines (Kollonitsch & Barash, 1976; Kollonitsch
et al., 1978; Wang & Walsh, 1981) and 0-acetyl serine
(Wang & Walsh, 1978) have proved effective in this
respect. Until recently, these inhibitors were considered
to function by undergoing conversion into Michael
acceptors (e.g. 6 + 7), that are trapped by nucleophilic
groups at the active site of the enzyme. However, recent
elegant work by Metzler’s group (Likos et al., 1982;
Ueno et al., 1982) using L-serine 0-sulphate has shown
that inactivation occurs by carbon-carbon bond formation between a presumed aminoacrylate intermediate,
and the carbon atom of the imine of pyridoxal with a
lysine side chain of the protein, leading to (8). Subsequently (8) has been shown to arise from b-haloalanines
and pyridoxal enzymes (Badet et al., 1984; Roise et al.,
1984). Thus the long-accepted mechanisms of action of
suicide inactivators of pyridoxal phosphate-dependent
enzymes need re-evaluation.
to generate Michael acceptors in situ. These early interpretations of the mechanism of inhibition must however
now be revised in the light of the work of Metzler’s group
referred to above.
A recent entirely different approach has been used
(Johnston et al., 1980) to inhibit irreversibly both
cystathione y-synthetase, which catalyses a y-replacement reaction in bacterial methionine biosynthesis, and
methionine y-lyase, which catalyses a y-elimination reaction in bacterial methionine breakdown. It is proposed
that (10) effects the inactivation via conversion into the
enzyme-bound ally1 sulphoxide (1 l), which undergoes a
2,3-sigmatropic rearrangement to the electrophilic sulphenate ester (12). The latter is then captured by an
enzymic nucleophile (12, arrows) resulting in irreversible
inhibition of the enzyme.
0
ENZ-NU:
\
Our own work which bears on the mechanism of
action of pyridoxal enzymes has been concerned with the
thermal generation of 1,3-dipoles from X=Y-ZH
systems (13
14) (Grigg, 1984; Grigg et al., 1984a). We
have shown that a novel, formal 1,2-prototropic shift
generating a 1,3-dipole occurs in imines (X = Z = C,
Y = N) (Grigg, 1984; Grigg et al., 1984a), hydrazones
(X = C, Y = Z = N) (Grigg et al., 1978) and oximes
(X = C, Y = N, Z = 0) (Grigg & Thianpatanagul,
1984a).
-02cYo
--&-
H2N7
R
Co2H
(9a) R = CH,F
(9b) R = CHF,
( 9 ~ )R = CH=CH,
(9d) R = C-CH
The occurrence of pyridoxal enzymes in the central
nervous system and their role in controlling y-aminobutyric acid (GABA) brain levels provides another area
for the design of novel drugs. For example, elevation of
GABA brain levels by inhibition of GABA-transaminase
could provide treatments for epilepsy and Huntington’s
disease (Jung, 1980; Allan & Johnston, 1983). Here again
substrates such as (9a-d) (Metcalf, 1979; Loscher, 1980;
Bey et al., 1981; Silverman & Levy, 1981) were designed
Vol. 14
This observation of the formation of a dipole from a
neutral precursor is both mechanistically interesting and
synthetically rewarding since the dipoles can be trapped
in cycloaddition reactions (14 + 15) to give a range of
interesting heterocycles, usually in excellent yield.
The ease of 1,3-dipoleformation will clearly depend on
both the basicity of Y, the central nitrogen atom, and the
pK, of the ZH proton. These properties will, in turn, be
influenced by the nature of the substituents on X and Z.
To study the effect of structure on rate of dipole formation, it is necessary to make the dipole-forming step
rate-determining. This can be done for imines of a-amino
406
BIOCHEMICAL SOCIETY TRANSACTIONS
acid esters by varying the dipolarophile. Thus, when
N-phenylmaleimide, a very reactive dipolarophile, is
used (Scheme 1) k, < k,,whilst with less active dipolarophiles such as dimethyl fumarate, k, > k,.
/
kl . \
Dipolarophile
0
Rate-determining
step
kl < k2
DIPOLE
FORMATION
Ph
Meo2C
Table 1 . Efect of structure on rate of cycloaddition when dipole
.formation is rate-determining
-
CYCLOADDITION
k2 < kl
R
NMe,
Me0
H
CN
NO2
Rate constant (s-')
44.6
7.8
3.55
0.72
0.80
x
x
x
x
x
Isotope effect
2.21
2.14
2.70
2.75
2.17
10-5
10-5
10-~
10-~
Scheme 1
In reactions in which dipole formation is rate-determining, the effect of structure on reactivity does follow
the expected order and some rate data are shown in Table
1. The variation in rate between (R = NMe,) and
(R = NO,) is not large, but is in the direction expected for
a decrease in imine basicity. The deuterium isotope effect
is comparatively small and the precise reason for this is
uncertain at present (Grigg et al., 1984~).The formation
of a 1,3-dipole from neutral imines is catalysed by both
Bronsted and Lewis acids (Grigg & Gunaratne, 1982)
and the catalysed reactions display the same regio- and
stereo-chemistry as the uncatalysed processes. It is
apparent from our studies that dipole formation is stereospecific and that an endo transition state is favoured for
the cycloaddition. Thus some property inherent in the
imine system imparts a kinetic bias to one dipole. The
simplest explanation of this observation is shown in
H
Ar4
R
i cy
CO, Me
zgh
H N T \
H
N
H..._____ 0
CO, Me
The 1,3-dipoles undergo cycloaddition with a wide
range of dipolarophiles, invariably via endo transition
states, providing many novel heterocyclic compounds
(e.g. 16 -, 17).
The formation of 1,3-dipoles from imines of both uamino acids and a-amino acid esters has relevance, we
believe, to the biochemistry of pyridoxal. The pyridoxal
imines of a-amino acids function, in the case of racemases and transaminases, by removal of the proton u to
the carboxyl group and protonation of the pyridine
nitrogen atom (1 + 2). This sequence of events, coupled
with intramolecular hydrogen bonding between the
phenolic proton and the imine nitrogen atom, suggested
to us that (2) might be more properly regarded as a
1,3-dipole (18) analogous to those previously discussed.
H _______.
0
Scheme 2
Scheme 2 and involves an intermediate hydrogen-bonded
enol (uncatalysed route) or a hydrogen-bonded protonated imine (acid-catalysed route). We have shown, by
appropriate trapping experiments, that the same dipole is
generated in the racemization of u-amino acids in the
presence of aldehydes (Grigg & Gunaratne, 1983b).
1986
407
61 5th MEETING. BELFAST
0
R-CHC0,Me
II
I
Ph
'Vo
*OH
HO
v
110"-140"C
N'
(20)
R = H, Me, CMe,,
CH,OH, CH,Ph, etc.
We therefore prepared a range of pyridoxal imines of
a-amino acid esters and examined their suitability as
1,3-dipole precursors. We were rewarded with a series of
smooth, stereospecific cycloadditions (1 9 -+ 20) which,
except for one or two cases, occur in excellent yield
(Grigg & Kemp, 1978; R. Grigg & J. Kemp, unpublished
work). Two further observations with arylidene imines of
a-amino acid esters bear on the possibility of inhibiting
pyridoxal enzymes. 1,3-Dipoles generated by prototropy
can be trapped intramolecularly to produce fused ring
systems (e.g. 21 -+ 22) (Armstrong et af., 1985)and imines
of vinyl amino acid esters undergo cyclization via 1,5dipole formation (23 -+ 24) (Grigg & Gunaratne,
1983~).
a
'
H
=Ax N AC,H
Ar+
The pyridoxal-dependent decarboxylases mediate
important biological processes leading to formation of
the so-called biogenic amines. This process is often called
decarboxylative transamination Model studies have led
to the proposal that the non-enzymic process occurs by
a concerted mechanism (28 -+ 29) (Buehler & Pearson,
1977) analogous to that established for the decarboxylation of b,y-unsaturated acids. We have recently shown
this to be incorrect and provided evidence for the intervention of a 1,3-dipole (Scheme 3). (Grigg & Thianpatanagul,
1984b; Grigg et al., 1984b). This new proposal rationalizes the literature data on model reactions and explains
how mixtures of imines can result since the final prototropy depends on the electron density at (a) and (b)
(Scheme 3).
4
CO, Me
CO, Me
(22a) a. 73%
b. 92%
(21a) Ar = C,H,, X = CH,
(21b) Ar = 2-naphthy1, X = S
Q
Scheme 3
RCH
I
I
CO, H
(25a) X
(25b) X
=
=
NLH
CH,
S
Thus pentenyl glycine (25a) and S-ally1 cysteine (25b)
are promising candidates for inhibition of pyridoxal
enzymes and a-vinyl a-amino acids such as vinyl glycine
and 4-vinyl-GABA may function as inhibitors via initial
cyclization to pyrrolines (26 -+ 27).
n
Vol. 14
=
pyridoxyl
I
4
I
-CHR
It follows from Scheme 3 that pyridoxal decarboxylases might also be regarded as producing 1,3dipoles. Thus (3) might be more properly regarded as the
1,3-dipole (30). Similar trapping experiments to those
described above were therefore carried out. However, in
this case it is not necessary or usually desirable to form
the imine in a separate step. Merely allowing the amino
acid and carbonyl compound to react in the presence of
a dipolarophile is sufficient. Reaction temperatures range
from room temperature to 120°C.
n
(26) py
A
\
Ar
(27)
80
408
BIOCHEMICAL SOCIETY TRANSACTIONS
HoeoH
H
Ph
v
-;I+
Ph
,CHCO,H
I
~~
%
YNH
70%
Scheme 4
Thus pyridoxal reacts in hot methanol or aqueous
acetonitrile to give a single cycloadduct from phenylglycine and N-phenylmaleimide (Scheme 4). Adduct
stereochemistry is assigned on the basis of nuclear Overhauser enhancement difference spectroscopy and signal
enhancement values are indicated in the scheme. Analogous processes can be carried out intramolecularly (e.g.
31 + 32).
In summary, pyridoxal imines of a-amino acids and
esters are shown to participate in cycloaddition reactions
and this suggests a new approach to the inhibition of
pyridoxal enzymes by trapping the intermediate dipole.
Possible suicide substrates therefore include pentenylglycine (25a), S-allylcysteine (25b) and related amino
acids. However, since the proposed mechanism of inactivation results in the suicide substrate being bound to the
coenzyme rather than the enzyme protein it may be more
successful with enzymes that bind pyridoxal phosphate
tightly.
We thank the M.R.C., S.E.R.C. and Queen's University for support.
Allan, R. D. &Johnston, G. A. R. (1983) Med. Res. Rev. 3, 91-118
Armstrong, P., Grigg, R., Jordan, M. W. & Malone, J. F. (1985)
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Bey, P., Jung, M. J., Gehat, F., Sehirlin, D., Van Dorsselaer, V. &
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Anti-convulsant effects of manipulation of brain 4-aminobutyrate concentrations by selective
enzyme inhibition
ROBERT A. JOHN* and LESLIE J. FOWLER?
?Department of Pharmacology, School of Pharmacy,
Cardif CFI IXL, Wales U.K., and
f Department of Pharmacology, School of Pharmacy,
Brunswick Square, London WCl, U.K.
After the original isolation of 4-amino butyrate from
brain (Awapara et al., 1950; Roberts & Frankel, 1950)
and its identification as an important neurotransmitter
(Bazemore et al., 1956), it became clear that experi-
mentally induced convulsions could be suppressed by
direct introduction of the compound into the brain either
by topical application to the cerebral cortex or by injection into the cerebral ventricles (Hayashi, 1959; Purpura
et al., 1959). These observations pointed the way to a
possible means of controlling epileptic seizures in human
subjects. However, although orally administered 4-aminobutyrate appears to be effective in a small number of
epileptic patients (Tower, 1960), brain 4-aminobutyrate
cannot readily be increased by systemic administration of
1986