Acetamidoquinone and acetamidohydroxy derivatives as inhibitors for both dihydroxyacetamido epoxidase and dehydrogenase

Article abstract of DOI:10.1016/S0968-0896(01)00403-5

A series of monohydroxy and dihydroxyacetanilides, acetamidoquinones and bromoacetamidoquinones have been synthesised and tested as substrates and/or inhibitors of highly purified dihydroxyacetamido epoxidase (DHAE) and dihydroxy acetamido dehydrogenase (DHADH) from Streptomyces LL-C10337. None was found to act as substrates but many selectively inhibit the enzymes. Kinetic analysis has shown that all the compounds act as reversible competitive inhibitors with respect to the substrates 2,5-dihydroxyacetanilide and 2,3-epoxy-1,4-benzoquinone-5-acetanilide. Monohydroxy acetanilides showed weak inhibition to these enzymes compared to the dihydroxy derivatives while the more powerful inhibitors were the benzoquinoneacetanilide and its 5-bromo equivalent.

Full text of DOI:10.1016/S0968-0896(01)00403-5

Bioorganic & Medicinal Chemistry 10 (2002) 1221–1227
Acetamidoquinone and Acetamidohydroxy Derivatives as
Inhibitors for Both Dihydroxyacetamido Epoxidase
and Dehydrogenase
Chris G. Whiteley*
Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa
Received 2 July 2001; accepted 11 October 2001
Abstract—A series of monohydroxy and dihydroxyacetanilides, acetamidoquinones and bromoacetamidoquinones have been syn-
thesised and tested as substrates and/or inhibitors of highly purified dihydroxyacetamido epoxidase (DHAE) and dihydroxy acet-
amido dehydrogenase (DHADH) from Streptomyces LL-C10337. None was found to act as substrates but many selectively inhibit
the enzymes. Kinetic analysis has shown that all the compounds act as reversible competitive inhibitors with respect to the sub-
strates 2,5-dihydroxyacetanilide and 2,3-epoxy-1,4-benzoquinone-5-acetanilide. Monohydroxy acetanilides showed weak inhibition
to these enzymes compared to the dihydroxy derivatives while the more powerful inhibitors were the benzoquinoneacetanilide and
its 5-bromo equivalent. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
irreversible. Reversible inhibition is characterised by an
equilibrium formed between the enzyme and the inhi-
bitor that depends on the concentration of the inhibitor,
value of the inhibitor constant (Ki), and is time inde-
pendent. The reversible inhibitor binds the enzyme non-
covalently to modulate the enzymatic activity and this
enzymic activity can be restored when the inhibitor is
removed by a suitable procedure such as gel filtrationor
dialysis. Onthe contrary, irreversible inhibitionis charac-
terised by a progressive decrease of enzymatic activity
with time and becomes complete when all the enzyme is
combined with the inhibitor. After irreversible inhibi-
tion the enzyme activity cannot be restored by gel fil-
trationor dialysis.
A large number of epoxyquinone antibiotics exist in
nature as agents against murine leukaemia and asso-
ciated diseases. Antitumour antibiotic (1), isolated and
purified from cultures of Streptomyces LL-C100371 was
found to have activity against murine leukaemia P388
with a 29% increase in the life span of treated mice. The
biosynthetic origin of these molecules, originate from
anthranilic acid with dihydroxyacetamide (2) and the
corresponding epoxyquinone (3) the major precursors
(Scheme 1).2ꢀ4
Two enzymes [dihydroxyacetamido epoxidase (DHAE)
and dihydroxyacetamido dehydrogenase (DHADH)]
were shown to play a major significant role during this
biosynthetic pathway.5
The rational design of inhibitors based on the inhibition
of the dihydroxyacetanilide epoxidase and dehy-
drogenase enzymes has attracted the attention of
Despite the strategic locationof these enzymes relative
to the epoxyquinone antibiotics, it is surprising that
very little is known about them as far as their kinetic
characteristics and inhibition. A useful tool for enzy-
mologists has been to find and use specific inhibitors so
as to follow the effects of these inhibitors on the enzyme
action and probe structure–function relationships. Inhi-
bitors can be divided into two groups: reversible and
Scheme 1. Biosynthesis of the semiquinone antitumour antibiotic (1)
and epoxyquinone (3) from dihydroxyacetanilide (2) with dihydroxy-
acetanilide epoxidase (DHAE) and dihydroxyacetanilide dehy-
drogenase (DHADH).
*Fax:+27-46-622-3984; e-mail: c.whiteley@ru.ac.za
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00403-5

1222
C. G. Whiteley / Bioorg. Med. Chem. 10 (2002) 1221–1227
medicinal chemists in the search for new antitumour
and antiviral agents. Furthermore these studies can also
lead to views on their enzymatic mechanisms. As a
continuation of research in these laboratories on
enzymes associated with cell proliferation,6ꢀ8 we deci-
ded to investigate the isolation, purification, kinetic
properties and inhibition of both the dihydroxy-
acetanilide epoxidase and the dehydrogenase enzymes
from Streptomyces LL-C10037 species.
activity of the sample eluted from the Sephacryl S-400
was 3460.3 nM gꢀ1 with anoverall yield of 12% and a
8.8-fold purification(Table 1).
Dihydroxyacetanilide dehydrogenase. The steps for the
purificationof this dehydrogenase are summarised in
Table 2. The specific activity of the crude homogenate
was 438.3 nM gꢀ1 gradually increasing by ammonium
sulphate precipitationdan DEAE–Sephadex ion
exchange chromatography to give a specific activity of
Results
Enzyme isolation and purification
Dihydroxyacetanilide epoxidase. The steps for the puri-
ficationof this epoxidase are summarised inTable 1. The
specific activity of the crude homogenate was 391.4 nM
gꢀ1 gradually increasing by ammonium sulphate pre-
cipitationand Sephadex ionexchange chromatography
to give a specific activity of 895.7 nM gꢀ1, a 22% yield
and a 2.3-fold purification. The results of the chroma-
tographic separations on DEAE–Sephadex are shown
inFigure 1. Elutionof the active enzyme ina sharp
peak is readily accomplished by a gradient application
in ionic strength to 1 M NaC1. Removal of extraneous
proteins is achieved at low ionic strength. Other ion-
exchange resins were found to be totally inadequate in
purifying the enzyme giving a broad elution peak over a
wide range of ionic strength indicating a poor resolu-
tion. The pooled purified samples from the DEAE–
Sephadex columnwere subjected to chromatography
and molecular weight determination on Sephacryl S-400
equilibrated inphosphate buffer (50 mM, pH 7) con-
taining glycerol (20%) and EDTA (0.2 mM) (Fig. 1,
inset). Two peaks (one major) eluted. The first at Ve of
70 mls representing a Mr=12,600 and a second smaller
peak at 85 mls corresponding to a Mr=43,000. The
column was standardised with proteins of known mole-
cular weights–Blue Dextran( Mr=2ꢁ106, lactate dehy-
drogenase (Mr=140,000), Dnase (Mr=63,000) and
carboxypeptidase A (Mr=34,300). The final specific
Figure 1. Ionexchagne chromatography of purified dihydroxy-
acetanilide epoxidase on DEAE–Sephadex. The absorbance is mon-
. . . .
itored at 280 nm and the NaCl gradient is indicated ( - - - ). The inset
represents chromatography of the epoxidase on Sephacryl S-400. The
absorbance is monitored at 280 nm. Standard proteins eluted through
the same column under the same conditions are superimposed: (1)
Blue dextran( Mr=2ꢁ106). (2) lactate dehydrogenase (Mr=140,000);
(3) deoxyribonuclease (Mr=63,000); (4) carboxypeptidase
(Mr=34,300).
A
Table 1. Purification of dihydroxyacetanilide epoxidase by DEAE–Sephadex and Sephacryl S-400
Purificationstep
Protein
Enzyme
Vol. (mL)
Concn (mg mLꢀ1
)
Total (mg)
Activity (nM minꢀ1
)
Sp. Act (nM gꢀ1
)
Fold purific
Yield (%)
Crude
(NH4)SO4
DEAE–Sephadex
Sephacryl-S400
500
200
160
38
50
25,000
7880
2432
9804
391.4
776.5
895.7
1
2
2.3
8.8
100
62.4
22.2
12
39.4
15.2
8.9
6118.7
2176.5
1176.5
338.2
3460.3
Table 2. Purification of dihydroxyacetanilide dehydrogenase by DEAE–Sephadex and Sephacryl S-400
Purificationstep
Protein
Enzyme
Vol. (mL)
Concn (mg mLꢀ1)
Total (mg)
Activity (nM minꢀ1
)
Sp. Act (nM gꢀ1
)
Fold purific
Yield %
Crude
(NH4)SO4
DEAE–Sephadex
Sephacryl-S400
325
90
60
16
30
28
23.5
8.3
9750
2520
1410
130
4273
1329
1176
515.4
438.3
527.4
834
1
100
1.2
1.9
9.2
31.1
27.5
11.6
4026.6

C. G. Whiteley / Bioorg. Med. Chem. 10 (2002) 1221–1227
1223
834 nM gꢀ1, 27.5% overall yield and a 1.9-fold puri-
fication. The results of the chromatographic separations
onDEAE–Sephadex are showninFigure 2. Againthe
removal of extraneous proteins was affected by low
ionic strength and pure active dehydrogenase enzyme
eluted ina sharp peak by means of a gradient applica-
tionof 1 M NaC1. Molecular weight determinationwas
determined on Sephacryl S-400 equilibrated in phos-
phate buffer (50 mM, pH 7) (Fig. 2, inset). A single peak
eluted at Ve=61 mls accounting for a Mr=134,000.
enzyme activity. As shown (Fig. 3) the inactivation of
the enzyme by 4-acetamidophenol showed a 31% inhi-
bitionafter 30 minwhile 2-acetamido-phenol had little
or no effect. The 2,3-dihydroxyacetanilide and 3,4-
dihydroxyacetanilide produced inhibition at 37 and
41%, respectively, while more powerful inhibitors were
1,4-benzoquinoneacetanilide (57%) and its bromoana-
logue (72%).
Dihydroxyacetanilide dehydrogenase. Incubation of the
dehydrogenase with inhibitor also resulted in pro-
gressive loss of enzyme activity (results not shown). The
inactivation of the enzyme by 4-acetamidophenol
The final specific activity of the dehydrogenase enzyme
ꢀ1
eluted from the S-400 columnwas 4026.6 nM g
with
anoverall yield of 11.6% and a fold purificationof 9.2
(Table 2).
showed
a 25% inhibition after 30 min, 2-acet-
amidophenol (10%), 2,3-dihydroxyacetanilide (31%),
and 3,4-dihydroxyacetanilide (41%) while the quinone
inhibitors were 1,4-benzoquinone acetanilide (50%) and
its bromoanalogue (65%).
Irreversible inhibition studies
Dihydroxyacetanilide epoxidase. Incubation of the
epoxidase with inhibitor resulted in progressive loss of
Reversible inhibition studies
Dihydroxyacetanilide epoxidase. The evidence of a
reversible inhibitory mechanism was realised after
characteristic Lineweaver–Burk plots (Fig. 4) were pro-
duced, indicating that all of the inhibitors studied were
competitive with respect to the natural substrate 2,5-
dihydroxyacetanilide. A replot of the slopes of this plot
versus inhibitor concentration is linear. (Fig. 4, inset)
and Ki, the inhibitor constant, can be calculated by
extrapolationof the graph slope versus hinibitor
concentration (Fig. 4, inset). All the relevant kinetic
Figure 2. Ionexchagne chromatography of purified dihydroxy-
acetanilide dehydrogenase on DEAE–Sephadex. The absorbance is
monitored at 280 nm and the arrow indicates elution with 1 M NaCl.
The inset represents chromatography of the dehydrogenase on Sepha-
cryl S-400. The absorbance is monitored at 280 nm. Standard proteins
eluted through the same column under the same conditions are super-
imposed: (1) Blue dextran( Mr=2ꢁ106); (2) lactate dehydrogenase
(Mr=140,000).
Figure 3. Inactivation of dihydroxyacetanilide epoxidase by 2-acet-
amidophenol (*); 4-acetamidophenol (*); 2,3-dihydroxacetanilide
(~); 3,4-dihydroxyacetanilide (~); 1,4-benzoquinoneacetanilide (&);
5-bromo-1,4-benzoquinone (&).
Table 3. Inhibitor constants (Ki) of various inhibitors with respect to dihydroxyacetanilide epoxidase and dihydroxyacetanilide dehydrogenas
Dihydroxyacetanilide epoxidase Dihydroxyacetanilide dehydrogenase
Inhibitor
Inhibitor
Ki (nM)
Ki (nM)
2-Acetamidophenol
4-Acetamidophenol
2,3-Dihydroxyacetanilide
3,4-Dihydroxyacetanilide
1,4-Benzoquinoneacetanilide
5-Bromo-1,4-benzoquinone
nd
20
27
18
12
4
2-Acetamidophenol
4-Acetamidophenol
2,3-Dihydroxyacetanilide
3,4-Dihydroxyacetanilide
1,4-Benzoquinoneacetanilide
5-Bromo-1,4-benzoquinone
nd
24
32
26
19
9

1224
C. G. Whiteley / Bioorg. Med. Chem. 10 (2002) 1221–1227
highly specific recognition and purification parameters
of biological specificity and thus the degree of purifica-
tion. In some protocols that follow ion-exchange chro-
matography and/or sucrose gradient centrifugation
there have been complaints of rather low yields and
decreased resolutionwith modificationof the properties
of the purified protein. Purification and fractionation of
proteins is facilitated by their different isoelectric points
and the possibility of producing a pH gradient using an
ion-exhange column has provided techniques for
separationwith resolutionand recovery comparable to
those of electrophoretic systems. With ampholyte dis-
placement column chromatography a pH gradient is
produced either by mixing buffers of different pH or by
employing the buffering action of an ion-exchanger and
a running buffer initially adjusted to one pH through a
column adjusted to another pH. The advantages of this
system are that ordinary chromatographic equipment
may be used; there is no restriction on the size of the
column, mixtures of ordinary buffers may be used and
proteins are not subjected to more extreme pH values
Figure 4. Competitive inhibition of dihydroxyacetanilide epoxidase by
5-bromo-1,4-benzoquinone: 0 nM (*), 3.3 nM (&), 6.7 nM (~), and
10 nM (*).The secondary plot represents the slopes of the primary
plot versus concentration of 5-bromo-1,4-benzoquinone.
parameters for the inhibitors are represented (Table 3).
With variable substrate concentrations values for Km
and Vmax for the epoxidase were determined at 110 nM
and 0.8 nM minꢀ1, respectively. The single substituted
hydroxy-acetanilide showed relatively weak inhibition
whencompared to the dihydroxy-acetainlides. The
bromo-1,4-quinone was the most potent inhibitor
(Ki=4 nM).
13
thantheir pI. As part of a study inthese laboratories
we required large amounts of purified protein and in
view of the foregoing discussion an improved method
for purificationwas sought. Earlier attempts to purify
involved ‘classical’ biochemical methods such as salt
formation, gel filtration and polymer partitioning. None
of these techniques nor any combination of them would
achieve substantial purification of the enzymes with a
reasonable yield. Our attempts to purify the epoxidase
enzyme by DEAE–Cellulose afforded the enzyme in a
yield of 6.2%, a fold purificationof 6.8 and a specific
activity of 2383.3 nM gꢀ1. Furthermore, attempted
purificationonhydroxyapatite affinity chromatographic
resinlead to poor yields (4.1%), a fold purificationof 3
Dihydroxyacetanilide dehydrogenase. A reversible com-
petitive inhibition was also seen with the dehydrogenase
enzyme (results not shown) and the relevant kinetic
parameters for these inhibitors are represented (Table 3).
Discussion
and a specific activity of 1152.7 nM gꢀ1
.
Current techniques for the preparative isolation of pro-
teins use the chromatrographic recognition of a variety
of physical parameters. The ideal separationmethod
By using a combination of DEAE–Sephadex followed
by Sephacryl S-400 we managed to purify the epoxidase
enzyme with a specific activity of 3460 nM gꢀ1, 8.8-fold
purificationand a 12% overall yield. Incomparisonthe
dihydroxyacetanilide-dehydrogenase was purified to
specific activity of 4026.6 nM gꢀ1, 9.2-fold purification
and 11.6% yield.
employs an easily identifiable physical parameter unique
10
to the proteinof interest.
Gel filtration, salt fraction-
ation, affinity chromatography with biospecific or group
specific adsorbent, ion exchange chromatography and
preparative electrophoresis have provento be versatile
separation techniques. Seldom does any of these pro-
cedures provide sufficient resolution or characterisation
to obtaina homogeneous proteinfrom a complex bio-
logical material. For this reasonpurificationprocedures
usually combine some or all of these techniques to
increase purity in a series of chromatographic or elec-
trophoretic steps. Each of these steps, however, suffer
from several disadvantages. The resolving power of gel
filtrationis limited by chromatographic factors adn
cannot yield fractions of discrete molecular size. Ion-
exchange chromatography, a technique based on the
This article has shownthat all the inhibitors studied
inhibit the epoxidase and the dehydrogenase reversibly
and competitively with respect to the respective sub-
strates. A kinetic analysis of the inhibition of the
enzyme by the bromobenzoquinone acetanilide is pre-
sented (Fig. 4) using standard graphical techniques of
Lineweaver–Burk.14 The lines are drawn by computer
calculation for linear competitive inhibition [eq (2)] and
least square analysis:15
nature and degree of available ionisable groups on the
11
À
À
ÁÁ
vꢀ1 ¼ V ꢀ1max Km 1 þ Kiꢀ1:I Sꢀ1 þ V
ð2Þ
ꢀ1
proteinmolecule
is not sufficiently characteristic and
max
many proteins elute under similar conditions. The
interaction between proteins and biospecific affinity
adsorbents may be too strong, causing problems in de-
sorption or too weak making an inefficient adsorbent.
Group specific affinity adsorbents have eliminated this
problem12 but indoign so they have decreased the
The data demonstrates reversible competitive inhibition
for the system, implying that the inhibitors bind at the
substrate binding site of the enzymes.

C. G. Whiteley / Bioorg. Med. Chem. 10 (2002) 1221–1227
1225
The potency of a particular inhibitor in the interaction
with the enzymes is determined by the dissociation con-
stant for the enzyme–inhibitor complex. The fit into the
active site of the enzyme, reflected by the values of Ki is
largely determined by the size, structure and configura-
tion of the inhibiting molecule. The capability of the
inhibitor to bind non-covalently at, or close to, the
active site could also influence these values.
The CFE was brought to 0.01% with protamine sul-
phate (to remove nucleic acids) by a dropwise addition
of a 2% aqueous solution. The resulting solution was
stirred (30 min) and centrifuged (38,400g, 4 ꢂC, 20 min).
The supernatant was brought to 52% saturation by the
additionof solid ammonium sulphate, stirred (60 min),
the precipitate brought to 72% saturationwith a further
addition of ammonium sulphate and after stirring for a
further 60 minthe active pellet was collected by cen-
trifugation(38,400 g, 4 ꢂC, 20 min). These active frac-
tions were applied to a DEAE–Sephadex column
equilibrated with the same phosphate–glycerol buffer.
After the A280 had returned to baseline the column was
eluted (1 mL/min) with a gradient containing the buffer
and 0–1 M NaC1 in this buffer. Active fractions were
collected, dialysed, freeze dried and the residue dis-
solved inphosphate buffer (50 mM, pH 7, 11.5 mL)
containing 20% glycerol and 0.2 mM EDTA to remove
metal ions and reduce disulphide formation. This was
thenapplied to a Sephacryl S-400 columnthat had been
equilibrated in the same buffer and fractions collected
(7.0 mL) at a flow rate of 13 mL hꢀ1. Active fractions
were pooled dialysed against distilled water, freeze dried
and the residue redissolved in buffer.
Detectionof spectroscopic changes ina proteinupon
binding with ligands or inhibitors is one of the simplest
methods to study induced conformational changes. In
the present investigation the changes in optical proper-
ties of the enzyme upon binding with the inhibitors may
be due to the presence of non polar regions around the
substrate binding site of the enzyme. The inhibitors
themselves cannot, however, increase the basicity of the
micro-environment of the binding locus. It is assumed
that the binding of inhibitors in the active site leads to
exposure of new groups and a transmission of con-
formational changes from one subunit to another. The
dihydroxyacetanilide, quinone and bromoquinone bind
strongly to a hydrophobic domain on the enzyme sur-
face increasing the rigidity of the molecule and, at the
same time, influencing the solvating properties of the
catalytic site and hence accelerating the binding process.
Assay
The strong inhibitory power of the benzoquinones
especially the bromobenzoquinone are reflected in the
electrophilic centres and Michael acceptors, creating
hydrophobic binding between the inhibitor and the
lipophilic regionof the enzymes. The possibility that the
epoxidase and dehydrogenase enzymes were metallo-
enzymes was ruled out from data obtained with the
potential chelating 2,3- and 3,4-dihydroxyacetanilide
inhibitors giving only limited inhibitions.
This enzyme was assayed by following the consumption
of the dihydroxyacetamide (2) and the production of the
epoxyquinone (3) simultaneously by HPLC on a Beck-
manGold and a C
reverse-phase columnwith H O/
2
18
MeCN (85/15) as the mobile phase. The assay (400 mL)
contained aliquots of substrate, phosphate buffer and
enzyme and was incubated at 30 ꢂC for 5 min. The
enzymatic reaction was terminated by the addition of
trifluoroacetic acid/acetonitrile/water/(1:1:8, 100 mL)
(TFA/MeCN/H2O).
Conclusion
Kinetic study
Insummary, the ihnibitionof both the dihydroxy-
acetamido epoxidase and the dihydroxyacetamido
dehydrogenase, which have been isolated and purified
from Streptomyces species, has been investigated and
evaluated with respect to a series of monohydroxy and
dihydroxyacetanilides, and acetamidoquinones and its
bromo analogue. All inhibitors were reversibly compe-
titive and the most powerful was the halogenated acet-
amidoquinone reflected in a Ki value of 4 nM.
The assay mixture consisted of phosphate buffer (1 M,
pH 6.5, 50 mL), MeOH (40 mL), 2,5-dihydroxy-
acetanilide (DHA) (60 mM, 25 mL), enzyme (40 mL) and
distilled water (245 mL). The enzyme reaction was
quenched after 5 min incubation with trifluoroacetic
acid/MeCN/H2O (1:1:8) (100 mL) and the activity of the
enzyme assayed, by the production of the epoxyquinone
(EQ) (3) at 225 nm using HPLC with a C18 reverse-
phase columnand H 2O/MeCN (85:15) mobile phase.
Enzyme activity was determined from a standard curve
prepared from 2.0, 5.0, 10.0 and 20.0 mL of a 1 mM
solution of epoxyquinone (3) onanHPLC C
phase columnwith H O/MeCN (85:15) mobile phase.
2
reverse-
18
Experimental
Dihydroxyacetamido epoxidase
The slope of the curve represented an integration area
of 1 nmol mLꢀ1. The rate of the enzymatic reaction (v)
(nM minꢀ1) was determined from equation 1.
Isolation and purification. Cells from S. LL-C100379
were harvested at 120 h by centrifugation (13,800g, 30
min, 4 ꢂC). They were washed with KC1 (1 M) followed
by NaC1 (0.8 M) to remove surface proteases, sus-
pended in potassium phosphate buffer (pH 7, 0.01 M)
and sonicated (2 min) with cooling. Centrifugation
(26,700g, 20 min) yielded a cell-free extract (CFE) (10%
v/v from the original broth).
v ¼ ðEQꢁt VtÞ=ðEQꢁs ViꢁtÞ
ð1Þ
where EQt is the integration area of the epoxyquinone
(3) produced, EQs the integration area of the epoxy-

1226
C. G. Whiteley / Bioorg. Med. Chem. 10 (2002) 1221–1227
Kinetic studies
The assay mixture contained NADPH (2.0 mM, 100 mL),
epoxyquinone (16.26 mM, 100 mL), Tris buffer (100 mM,
pH 8.5, 1.7 mL), enzyme (100 mL, 132 mg/mL). The
enzyme activity was monitored by the decrease in A340
.
Inhibition studies
The assay mixture contained NADPH (2.0 mM, 100
mL), epoxyquinone (0–20 mM, 100 mL), Tris buffer (200
mM, pH 8.5, 1.7 mL), enzyme (100 mL, 132 mg mLꢀ1),
and inhibitor (0–10 nM, 10 mL). The enzyme activity
Scheme 2. Synthesis of 2,3-dihydroxyacetanilide (4).
was monitored by the decrease in A340
.
quinone standard (nmol mLꢀ1), Vt is the total volume of
the assay (500 mL), Vi is the injection volume (10 mL)
and t is the assay time (5 min).
Synthesis of inhibitors
2,3-Dihydroxyacetanilide (4) (Scheme 2). 2,3-Dimethoxy-
benzoic acid (Aldrich) (1.82 g, 10 mM) was dissolved in
dry benzene (10.0 mL). To this was added thionyl chlo-
ride (1.45 g, 1.0 mL 12 mM) and the whole was heated
under reflux (2 h). The solvents and lower boiling
liquids were removed under reduced pressure and the
residue dissolved indry acetone (10 mL). This solution
was thenadded to a cold (5 ꢂC) stirred solutionof
sodium azide (780 mg, 12 mM) and ꢂwater (10 mL)
maintaining the temperature below 15 C. Stirring was
continued (60 min) and then the whole was extracted
into benzene. The benzene solution was heated (60 ꢂC, 2
h) (gas evolution), evaporated and then treated with
concentrated hydrochloric acid (15 mL) and heated at
80 ꢂC, 18 h. The solutionwas cooled, enutralised
(NaHCO3) and thoroughly extracted into ethylacetate,
dried (Na2SO4) and concentrated to afford crude 2,3-di-
methoxy-aniline (1.303 g, 85%). All attempts to purify this
by distillation under reduced pressure lead to considerable
loss inproduct. The crude dimethoxyaniline was dissolved
in dichloromethane (50.0 mL) containing triethylamine
(1.4 mL), cooled to 5 ꢂC thentreated dropwise with a 33%
(v/v) dichloromethane solution of acetylchloride (4 mL).
After stirring (60 min) the reaction mixture was quenched
with water and extracted into ethyl acetate. The solution
was dried (Na2SO4) and the solvent removed to afford
2,3-dimethoxy acetanilide. 13C NMR (CDC13,
100 MHz) d 112.81–156.98, aromatic C; 170.7 amide C;
56–57.2 (2ꢁOCH3); 24.24 (C–CH3).
Inorder to obtainvalues for Km and Vmax, a series of
kinetic experiments were performed with the variation
in the concentration of the substrate (DHA).
Inhibition studies
Assay mixtures were set up containing phosphate buffer
(pH 6.5, 100 mM, 50 mL), MeOH (40 mL), enzyme (50
mL), 2,5-dihydroxyacetanilide (2,4 mM Lꢀ1 inphos-
phate buffer, 0–50 mM), inhibitor (10 mM Lꢀ1 inphos-
phate buffer, 0–10 nM) and water to a total volume of
400 mL. After 5 min incubation terminating solution
(TFA/MeCN/H2O, 1:1:8, 100 mL) was added. The rate
of the enzymatic reaction v (nmol minꢀ1) was deter-
mined by HPLC using a C18 reverse-phase columnwith
a mobile phase (MeCN/H2O, 85/15) and using eq (1)
above.
Dihydroxyacetamido dehydrogenase
Isolation and purification. The protamine sulphate of
cell free extract (CFE) (see Dihydroxyacetanilide epoxi-
dase above) was brought to 40% saturationby the
addition of solid ammonium sulphate, the suspension
stirred (60 min) and the precipitate removed by cen-
trifugation(13,800 g, 10 min). The resultant supernatant
was brought to 60% saturationammonium sulphate,
stirred (60 min) and the active pellet collected by cen-
trifugation(38,400 g, 10 min). All subsequent purifica-
tion steps involving column chromatography on
DEAE– Sephadex and Sephacryl S-400 were identical as
for the epoxidase enzyme.
This dimethoxyanilide (380 mg, 2 mM) was dissolved in
dry dichloromethane and cooled to ꢀ78 ꢂC (dry ice/
acetone) under a stream of nitrogen. Boron tribromide
(5.0 mL, 1 M solutionindichloromethane, 5 mM) was
added slowly. After the additionthe pale-yellow/green
solutionwas allowed to reach room temperature. The
reaction mixture was quenched with brine (10%),
extracted into methylene chloride, dried (Na2SO4) an d
the solvents evaporated to produce a light brown oil
(300 mg, 90%) identified as 2,3-dihydroxyacetanilide.
13C NMR (CDC13, 100 MHz) d 113.26–146.84 (aro-
matic C); 170.7 (amide C) 24.24 (C–CH3).
Assay
The dehydrogenase was assayed by the disappearance of
the epoxyquinone (3) using HPLC with a C18 reverse-
phase columnand H 2O/MeCN (85:15) mobile phase,
monitoring the effluent at 225 nm. The assay solution
(400 mL) contained NADPH, substrate and enzyme,
incubated at 30 ꢂC, 5 min, then the enzymatic activity
quenched with 100 mL terminating solution (MeCN/
H2O/TFA). Comparative assays were performed by
monitoring the disappearance of the absorbance of
NADPH at 340 nm.
3,4-Dihydroxyacetanilide (5) (Scheme 3). 3,4-Dimethoxy-
aniline (Aldrich) (1.47 g, 10 mM) was dissolved in di-
chloromethane (5.0 mL) containing triethylamine (1.4

C. G. Whiteley / Bioorg. Med. Chem. 10 (2002) 1221–1227
1227
100 MHz) d 109.07–151.52 (aromatic C); 108.44 (C–Br)
56.4 (2ꢁOCH3); 170.7 (amide c); 24.24 (C–CH3).
This bromoacetanilide (68.5 mg, 250 mM) was dissolved
inacetonitrile (10.0 mL) thentreated with excess ceric
ammonium nitrate (CAN) (1.1 g) in water (10.0 mL).
After stirring (30 min) a yellow crystalline solid pre-
cipitated. This was extracted into ethyl acetate, washed
with sodium chloride, dried (Na2SO4) and evaporated
to afford a yellow crystalline material (52 mg, 85%), mp
138 ꢂC. 13C NMR (CDC13, 100 MHz) d 181.43 (Br–C–
CO); 177.55 (Br–C=C–CO); 171.76 (amide C); 136.32
(C¼C–NH); 134.12 (Br–C¼C–CO), 132.72 (Br–C¼);
24.1 (C–CH3).
Scheme 3. Synthesis of 3,4-dihydroxyacetanilide (5).
2-Acetamido-1,4-benzoquinone (7) (Scheme 4). 2,5
Dimethoxyacetanilide (250 mM) was dissolved inaceto-
nitrile (10.0 mL) then treated with excess ceric ammo-
nium nitrate (CAN) (1.1 g) in water (10.0 mL). After
stirring (30 min), the crystalline solid was collected,
extracted into ethyl acetate, dried (Na2SO4) and evapo-
rated to afford a solid product (194 mg, 81%) mp
126 ꢂC. 13C NMR (CDC13, 100 MHz) d 136.32–132.72
(aromatic C); 181.43–177.55 (aromatic CO); 171.76
(amide C); 24.1 (C–CH3).
Scheme 4. Synthesis of 2-acetamido-1,4-benzoquinone (7) an d 5-
bromo-2-acetamido-1,4-benzoquinone (6).
mL). The reactionmixture was cooled to 5 ꢂC, treated
dropwise with a 33% (v/v) dichloromethane solution of
acetyl chloride (4 mL). After stirring (60 min) the reac-
tionmixture was quenched with water, extracted with
ethyl acetate, dried (Na2SO4) and the solvents removed
under reduced pressure to afford crude 3,4-dimethoxy-
acetanilide (1.42 g, 82%). 13C NMR (CDC13, 100 MHz)
d 101.91–151.18 (aromatic C), 170.45 (amide C); 55.85,
55.95 (2ꢁOCH3, 23.95 (C–CH3).
References and Notes
1. Lee, M. D.; Fantini, A. A.; Morton, O.; James, J. C.; Bor-
der, D. B.; Testa, R. T. J. Antibiot. 1984, 37, 1149.
2. Gould, S. J.; Shen, B. J. Am. Chem. Soc. 1991, 113, 684.
3. Gould, S. J.; Shen, B.; Whittle, Y. G. J. Am. Chem. Soc.
1989, 111, 7932.
4. Shen, B.; Whittle, Y. G.; Gould, S. J.; Keseler, D. A. J.
Org. Chem. 1990, 55, 4422.
5. Gould, S. J.; Kirchmeier, M. J.; La Fever, R. E. J. Am.
Chem. Soc. 1996, 118, 7663.
6. Whiteley, C. G.; Mmutle, T. B. J. Enz. Inhibit. 1994, 8, 1.
7. Whiteley, C. G.; Daya, S. Bioorg. Med. Chem. Lett. 1996,
6, 2801.
8. Whiteley, C. G.; Ngwenya, D. S. Biochem. Mol. Biol.
Intern. 1995, 36, 1107.
The crude anilide (380 mg, 2 mM) was dissolved in dry
dichloromethane and cooled to ꢀ78 ꢂC (dry ice/acetone)
under a stream of nitrogen. Boron tribromide (5.0 mL,
1 M solutionindichloromethane, 5 mM) was added
slowly. After the additionthe pale-yellow/greensolution
was allowed to reach room temperature. The reaction
mixture was quenched with brine (10%) and extracted
into dichloromethane. This was dried (Na2SO4) and the
solvents evaporated to produce an oil (275 mg, 85%).
13C NMR (CDC13, 100 MHz) d 111.46–146.74 (aro-
matic C); 170.45 (amide c); 23.95 (C–CH3).
9. A gift from the Department of Chemistry, Oregon State
University, Corvallis, OR, USA.
2-Acetamido-5-bromo-1,4-benzoquinone (6) (Scheme 4).
2,5-Dimethoxy acetanilide (195 mg, 1 mM) was dis-
solved in dichloromethane (10.0 mL) and glacial acetic
acid (1.0 mL) added. The whole was treated with slight
excess bromine (176 mg), 55 mL, 1.1 mM) and the pale-
orange solution stirred for a further 30 min. The organic
layer was washed with sodium dithionite then sodium
bicarbonate dried (Na2SO4) and evaporated to yield the
final product (120 mg, 66%). 13C NMR (CDC13,
10. Richey, J.; Beedling, L. Int. Lab. 1982, 12, 56.
11. Kirkegaard, L. H. Anal. Biochem. 1972, 50, 122.
12. Page, M. Can. J. Biochem. 1988, 51, 1213.
13. Sluyteman, L. A.; Elgersma, O. J. Chromatogr. 1978, 150,
17.
14. Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56,
658.
15. Any suitable computer graphics package may be used.
The author used Quattro Pro 6 (Borland International).