Aromatic inhibitors of dehydroquinate synthase: Synthesis, evaluation and implications for gallic acid biosynthesis

Article abstract of DOI:10.1016/S0960-894X(01)00065-8

The role of the active site metal in determining binding to 3-dehydroquinate synthase has been examined. Protocatechuic acid, catechol, and derivatives of these aromatics were synthesized that shared the common element of an ortho dihydroxylated benzene ring. Inhibition constants were determined for each aromatic as well as the variation of this inhibition as a function of whether Co⁺² or Zn⁺² was the active site metal ion.

Full text of DOI:10.1016/S0960-894X(01)00065-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1493±1496
Aromatic Inhibitors of Dehydroquinate Synthase: Synthesis,
Evaluation and Implications for Gallic Acid Biosynthesis
Sunil S. Chandrana and J. W. Frosta,b,
*
aDepartment of Chemistry, Michigan State University, East Lansing, MI 48823-1322, USA
bDepartment of Chemical Engineering, Michigan State University, East Lansing, MI 48823-1322, USA
Received 28 November 2000; accepted 16 January 2001
AbstractÐThe role of the active site metal in determining binding to 3-dehydroquinate synthase has been examined. Protocatechuic
acid, catechol, and derivatives of these aromatics were synthesized that shared the common element of an ortho dihydroxylated
benzene ring. Inhibition constants were determined for each aromatic as well as the variation of this inhibition as a function of
whether Co+2 or Zn+2 was the active site metal ion. # 2001Elsevier Science Ltd. All rights reserved.
3-Dehydroquinate synthase is an enzyme in the com-
mon pathway of aromatic amino acid biosynthesis that
catalyzes the conversion of 3-deoxy-d-arabino-heptulo-
sonic acid 7-phosphate (DAHP, Scheme 1) into 3-dehy-
droquinic acid (DHQ, Scheme 1).1,2 This reaction
assembles the carbocyclic ring that ultimately becomes
the benzene ring of l-phenylalanine, l-tyrosine, l-tryp-
tophan, coenzyme Q, folic acid, and a spectrum of aro-
matic secondary metabolites. Protocatechuic acid,
catechol, and derivatives of these aromatics have been
synthesized and their inhibition examined of both the
Co+2-form and the Zn+2-form of 3-dehydroquinate
synthase. In addition to illustrating the importance of
the metal ion as a determinant of binding to the active
site of 3-dehydroquinate synthase, the ortho dihy-
droxylated benzene ring of these inhibitors represents a
fundamental departure from all previously reported
inhibitors of this enzyme.2b,c,3
3-Dehydroquinate synthase is mechanistically dis-
tinguished by its catalytic use of cofactor NAD+.
Reduction of NAD+ to NADH during the ®rst step of
the enzyme-catalyzed reaction is followed by reoxida-
tion of NADH to NAD+ at a later enzyme-catalyzed
step prior to release of 3-dehydroquinic acid from the
active site (Scheme 1). Increasing attention has recently
been paid to the catalytic role of the metal at the active
site.2a,4 3-Dehydroquinic acid is a metalloenzyme, which
utilizes either Co+2 or Zn+2 as its metal cofactor. A
crystal structure of the Zn+2-form of 3-dehydroquinate
synthase isolated from Aspergillus nidulans has been
solved with a carbaphosphonate analogue of DAHP
bound at the active site.5 The active site Zn+2 is posi-
tioned in close proximity to the backbone vicinal
hydroxyl groups with metal to C-4 and C-5 oxygen
bond distances of 2.3 A and 2.2 A, respectively. There
has been disagreement as to whether the native form of
3-dehydroquinate found in nature binds Co+2 or Zn+2
at its active site. The Co+2-form of the enzyme has been
reported to be more stable and has a higher speci®c activity
relative to the Zn+2-form.4 However, the bioavailability of
Scheme 1.
*Corresponding author. Fax: +1-517-432-3873; e-mail: frostjw@
cem.msu.edu
Zn+2 in nature is much greater than Co+2 6
.
0960-894X/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00065-8

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S. S. Chandran, J. W. Frost / Bioorg. Med. Chem. Lett. 11 (2001) 1493±1496
To explore the role of the divalent ion as a determinant
of active site binding, several derivatives of proto-
catechuic acid were prepared to exploit the potential
complexation of the ortho hydroxyl groups of these
putative inhibitors with the active site metal ion. Both
the Co+2-form and the Zn+2-form of 3-dehydroquinate
synthase were also prepared. These two avenues facili-
tated exploration of how active site binding as measured
by inhibition constant (Ki) varied as a function of inhibitor
structure and enzyme-bound metal cofactor.
Inhibitors 3±5 facilitated a stepwise dissection of the
role of complexation with the active site metal as a
determinant of binding to 3-dehydroquinate synthase.
Phosphonoethyl catechol 4, which was synthesized
(Scheme 4) from commercially available 2,3-dimethoxy
benzaldehyde, lacks a carboxylic acid group. Active site
binding for 4 must then be derived from active site
interactions of the phosphonoethyl group and the metal
binding of the ortho hydroxyl groups. Protocatechuic
acid 3 is missing a substituent mimicking the phosphate
monoester of DAHP. Its binding to 3-dehydroquinate
synthase must therefore be derived from active site
interactions with its carboxylate and metal complexa-
tion to the ortho hydroxyl groups. Di€erences in the
inhibition of 3-dehydroquinate synthase for phospho-
noethyl catechol 4 and protocatechuic acid 3 can be
used to infer the importance of the carboxylate relative
to the phosphate monoester analogue to active site
binding. Catechol 5 constitutes the minimized set of
active site binding capabilities. The only apparent basis
for binding to 3-dehydroquinate synthase is complexa-
tion between the ortho hydroxyl groups of 5 and the
active site metal.
A trianionic ionization state in substrate analogues has
been demonstrated to be important for binding to the
active site of 3-dehydroquinate synthase.3c This was the
ionization state for both phosphonomethyl proto-
catechuate 1 and phosphonoethyl protocatechuate 2.
The phosphono-ethyl ring substituent of 2 is isosteric
while the phosphonomethyl substituent of 1 is non-
isosteric to the phosphate monoester of substrate
DAHP. More potent inhibition of 3-dehydroquinate
synthase is often, but not always, observed for non-
isosteric phosphate monoester analogues.3 For this rea-
son, it is generally advisable to synthesize both
phosphonomethyl and phosphonoethyl substrate ana-
logues when a new class of 3-dehydroquinate synthase
inhibitor is examined. A precursor synthesized (Scheme 2)
from commercially available 3,4-dimethoxybenzoic acid
was used for the synthesis (Scheme 3) of both phos-
phonomethyl protocatechuate 1 and phosphonoethyl
protocatechuate 2.
3-Dehydroquinate synthase puri®ed from Escherichia
coli was employed for all inhibition studies.7 Puri®ca-
tion of the Co+2-form of 3-dehydroquinate synthase,
generation of apoenzyme and ®nal reconstitution with
Zn+2 resulted in an unstable enzyme preparation. A
stable Zn+2 form of 3-dehydroquinate synthase was
obtained when the 250 mM CoCl2 normally present in
all bu€ers beginning with cell lysis and extending
through each step of the enzyme puri®cation process
was replaced with 75 mM ZnSO4. Inhibition constants
(Ki) were obtained from Dixon plots of the reciprocal
initial velocities plotted against varying inhibitor con-
centrations in the presence of a ®xed concentration of
substrate. For phosphonomethyl protocatechuate 1,
phosphonoethyl protocatechuate 2, phosphonoethyl
catechol 4, and protocatechuic acid 3, replots of the
Dixon data gave a line passing through the origin indi-
cating competitive inhibition. Lineweaver±Burke and
Hanes±Woolf plots also gave results consistent with
competitive inhibition. Inhibition by catechol 5 was
estimated from an I50 determination where reciprocal
initial velocities were plotted against inhibitor con-
centrations using a concentration of substrate equal to
the Km (18 mM) for the Co+2-form or the Km (16 mM)
for the Zn+2-form of 3-dehydroquinate synthase. One
half of the value of the x-intercept was taken as an
estimate of Ki.
Scheme 2. (a) MeOH, H2SO4, 98%; (b) Br2, AcONa, AcOH, 70 ꢀC,
97%; (c) Bu3Sn(CHˆCH2), Pd(PPh3)4, HMPA, 65 ꢀC, 63%; (d) O3,
CH2Cl2, À78 ꢀC, 91%.
Scheme 3. (a) [(CH3)2HSi]2O, TMSCl, NaI, AcCN, re¯ux; (b)
P(OiPr)3, toluene, re¯ux, 23%; (c) (i) BBr3, CH2Cl2, À78 ꢀC to rt; (ii)
concd HCl, 62%; (d) n-BuLi, [(iPrO)2P(O)]2CH2, THF, À78 ꢀC to rt,
34%; (e) H2, 10% Pd/C, MeOH, 76%; (f) (i) BBr3, CH2Cl2, À78 ꢀC to
rt; (ii) concd HCl, 88%.
Scheme 4. (a) n-BuLi, [(iPrO)2P(O)]2CH2, THF, À78 ꢀC to rt, 95%;
(b) H2, 10% Pd/C, MeOH, 75%; (c) (i) BBr3, CH2Cl2, À78 ꢀC to rt;
(ii) concd HCl, 97%.

S. S. Chandran, J. W. Frost / Bioorg. Med. Chem. Lett. 11 (2001) 1493±1496
1495
Table 1. Inhibition constants (mM) for binding to the Co+2 and
tocatechuic acid 3 in the microbe-catalyzed conversion
of glucose to gallic acid. Apparently, the protocatechuic
acid 3 is inhibiting 3-dehydroquinate synthase. DAHP
accumulating in the cytoplasm due to inhibition of 3-
dehydroquinate synthase is dephosphorylated, which
results in export and accumulation of 3-deoxy-d-ara-
bino-heptulosonic acid as a major contaminant in fer-
mentation broths. Given that protocatechuic acid 3 is
an intermediate in numerous biocatalytic syntheses,
inhibition of 3-dehydroquinate synthase by this ortho
dihydroxylated aromatic constitutes a signi®cant impe-
diment to the elaboration of high-yielding microbial
syntheses of cis,cis-muconic acid, catechol, vanillic acid,
and gallic acid.8
Zn+2 forms of 3-dehydroquinate synthasea
Metalloform
Inhibitor
1
2
3
4
5
Co+2
Zn+2
215
0.35
44
1.7
200
65
880
90
630
aConditions: DHQ synthase activity was assayed in a 1.0 mL solution
of 3-(N-morpholino)propanesulfonate (MOPS) bu€er (50 mM, pH
7.5) containing either 50 mM CoCl2 or 75 mM ZnSO4 depending on the
form of enzyme being studied, NAD+ (10 mM), dehydroquinase (1
unit), and varying concentrations of DAHP and inhibitor. After
equilibration at rt, puri®ed DHQ synthase (0.024 units) was added,
and the increase in absorbance at 234 nM was monitored over time.
Initial rates were determined by linear square ®ts of the progress
curves and were used to determine inhibition constants.
Acknowledgements
A pronounced di€erence in the pattern of inhibition of
the Co+2-form relative to the Zn+2-form of 3-dehy-
droquinate synthase was observed (Table 1). Proto-
catechuate 2, whose phosphonoethyl substituent is
isosteric to a phosphate monoester, was the most potent
inhibitor of the Co+2-form (Table 1) of the enzyme. By
Research was funded by a grant from the National
Science Foundation.
contrast,
nonisosteric
phosphonomethyl
proto-
-
References and Notes
catechuate 1 was the most potent inhibitor of the Zn+2
form (Table 1) of 3-dehydroquinate synthase. The
Zn+2-form of 3-dehydroquinate synthase bound phos-
phonomethyl protocatechuate 1 more tightly than the
Co+2-form of the enzyme by a factor of 60-fold (Table
1). Binding of the carboxylate and the phosphonoethyl
substituents by the Zn+2-form of the enzyme was
approximately the same based on the similar extent to
which enzyme inhibition was reduced for phospho-
noethyl catechol 4 and protocatechuic acid 3 relative to
phosphonoethyl protocatechuate 2 (Table 1). The
Co+2-form of 3-dehydroquinate, by comparison, dis-
played a more pronounced di€erence (Table 1) in bind-
ing of carboxylate and phosphonoethyl substituents
based on the di€erent extent to which enzyme inhibition
was reduced for phosphonoethyl catechol 4 and proto-
catechuic acid 3 relative to phosphonoethyl proto-
catechuate 2. Although both the Co+2-form and the
Zn+2-form of 3-dehydroquinate synthase were inhibited
(Table 1) only weakly by catechol 5, the fact that this
structurally simple aromatic was an inhibitor at all
attests to the important role that the active site metal
ion plays in substrate binding.
1. (a) Rotenberg, S.; Sprinson, D. B. J. Biol. Chem. 1978, 253,
2210. (b) Maitra, U. S.; Sprinson, D. B. J. Biol. Chem. 1978,
253, 5426.
2. (a) Bender, S. L.; Mehdi, S.; Knowles, J. R. Biochemistry
1989, 28, 7555. (b) Bender, S. L.; Widlanski, T.; Knowles, J. R.
Biochemistry 1989, 28, 7560. (c) Widlanski, T.; Bender; S. L.;
Knowles, J. R. Biochemistry 1989, 28, 7572.
3. (a) Le Marechal, P.; Azerad, R. Biochimie 1976, 58, 1123.
(b) Montchamp, J.-L.; Frost, J. W. J. Am. Chem. Soc. 1997,
119, 7645. (c) Tian, F.; Montchamp, J.-L.; Frost, J. W. J. Org.
Chem. 1996, 61, 7373. (d) Montchamp, J.-L.; Frost, J. W. J.
Org. Chem. 1994, 59, 7596. (e) Montchamp, J.-L.; Peng, J.;
Frost, J. W. J. Org. Chem. 1994, 59, 6999. (f) Montchamp, J.-
L.; Piehler, L. T.; Tolbert, T. J.; Frost, J. W. Bioorg. Med.
Chem. Lett. 1993, 3, 1403.
4. More, J. D.; Skinner, M. A.; Swatman, D. R.; Hawkins,
A. R.; Brown, K. A. J. Am. Chem. Soc. 1998, 120, 7105.
5. Carpenter, E. P.; Hawkins, A. R.; Frost, J. W.; Brown,
K. R. Nature 1998, 394, 299.
6. Lambert, J. M.; Boocock, M. R.; Coggins, J. R. Biochem.
J. 1985, 226, 817.
7. Frost, J. W.; Bender, J. L.; Kadonaga, J. T.; Knowles, J. R.
Biochemistry 1984, 23, 4470.
8. (a) Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 1994,
116, 399. (b) Draths, K. M.; Frost, J. W. J. Am. Chem. Soc.
1995, 117, 2395. (c) Li, K.; Frost, J. W. J. Am. Chem. Soc.
1998, 120, 10545. (d) Kambourakis, S.; Draths, K. M.; Frost,
J. W. J. Am. Chem. Soc. 2000, 122, 9042.
A valuable dividend of the inhibition of 3-dehydro-
quinate synthase by ortho dihydroxylated aromatics is
the ability to now explain the accumulation of 3-deoxy-
d-arabino-heptulosonic acid as a major contaminant in
the biocatalytic synthesis of gallic acid.8d Because it is
known to be an impediment to the ¯ow of carbon
through the common pathway of aromatic amino acid
biosynthesis, expression of aroB-encoded 3-dehy-
droquinate synthase is routinely ampli®ed in hydroaro-
matic- or aromatic-synthesizing Escherichia coli
biocatalysts. However, even with ampli®ed expression
of aroB-encoded 3-dehydroquinate synthase in gallate-
synthesizing recombinant E. coli, accumulation of 3-
deoxy-d-arabino-heptulosonic acid has been observed.
This can now be explained by the intermediacy of pro-
1H NMR spectra were recorded on a 300 MHz spectrometer.
Chemical shifts for 1H NMR spectra are reported (in parts per
million) relative to internal tetramethyl silane (Me4Si,
d=0.0 ppm) with CDCl3 as the solvent, and to sodium 3-(tri-
methylsilyl) propionate-2,2,3,3-d4 (TSP, d=0.0 ppm) when
D2O was the solvent. 13C NMR were recorded at 75 MHz.
Chemical shifts for 13C NMR spectra are reported (in parts
per million) relative to CDCl3 (d=77.0 ppm) or internal ace-
tonitrile (CH3CN, d=3.69 ppm) in D2O. FAB and CI mass
spectra were obtained on a double-focusing mass spectro-
meter. Elemental analysis were performed by Atlantic Micro-
lab Inc. (Norcross, GA).

1496
S. S. Chandran, J. W. Frost / Bioorg. Med. Chem. Lett. 11 (2001) 1493±1496
1
5-Bromo-3,4-dimethoxy methybenzoate 7. H NMR (CDCl3) d
3.90 (s, 3H), 3.92 (s, 6H), 7.10 (s, 1H), 7.42 (s, 1H); 13C NMR
(CDCl3) d 165.8, 151.9, 147.7, 122.7, 116.8, 114.1, 113.8, 56.2,
56.1, 52.2. Anal. calcd for C10H11BrO4: C, 43.84; H, 4.05.
Found: C, 43.94; H, 4.07.
149.3, 146.0 (d, J=8 Hz), 131.0 (d, J=25 Hz), 122.0, 117.2 (d,
J=191 Hz), 112.8, 109.3, 70.6 (d, J=6 Hz), 70.5, 56.0, 52.1,
24.1, 24.0, 23.9, 23.8. Anal. calcd for C18H27O7P: C, 55.95; H,
7.05. Found: C, 55.40; H, 6.83.
Homophosphonate intermediate 14. 1H NMR (CDCl3) d 1.34
(s, 12H), 2.06 (m, 2H), 3.20 (m, 2H), 3.89 (s, 3H), 3.91 (s, 3H),
3.93 (s, 3H), 4.72 (m, 2H), 6.78 (s, 1H), 7.48 (s, 1H); 13C NMR
(CDCl3) d 166.9, 151.9, 146.8, 137.7 (d, J=18 Hz), 120.4,
113.5, 113.4, 69.9 (d, J=6 Hz), 28.8 (d, J=123 Hz), 27.8 (d,
J=11 Hz), 23.9. Anal. calcd for C18H29O7P: C, 55.66; H, 7.53.
Found: C, 55.03; H, 7.50.
1
5-Vinyl-3,4-dimethoxy methyl benzoate 8. H NMR (CDCl3) d
3.90 (s, 3H), 3.92 (s, 3H), 3.96 (s, 3H), 5.30 (dd, J=1, 11 Hz,
1H), 5.57 (dd, J=1, 17 Hz, 1H), 7.03 (s, 1H), 7.43 (s, 1H), 7.54
(dd, J=11, 17 Hz, 1H); 13C NMR (CDCl3) d 167.1, 151.8, 147.8,
135.8, 134.2, 120.2, 114.9, 112.6, 109.2, 55.8, 55.7, 51.8. Anal.
calcd for C12H14O4: C, 64.86; H, 6.35. Found: C, 64.79; H, 6.33.
1
3-Formyl-4,5-dimethoxy methyl benzoate 9. H NMR (CDCl3)
Protocatechuic homophosphonate 2. 1H NMR (D2O) d 2.00 (m,
2H), 3.02 (m, 2H), 6.76 (s, 1H), 7.37 (s, 1H); 13C NMR (D2O)
d 172.9, 151.3, 144.7, 140.3 (d, J=19 Hz), 122.3, 121.7, 120.6,
31.1 (d, J=131 Hz), 29.9; HRMS (FAB) calcd for C9H10PO7
261.0164, found 261.0158.
d 3.97 (s, 3H), 3.99 (s, 3H), 4.01(s, 3H), 7.47 (s, 1H), 7.51(s,
1H), 10.65 (s, 1H); 13C NMR (CDCl3) d 191.2, 166.2, 152.3,
151.9, 131.2, 125.9, 112.5, 109.5, 56.3, 56.2, 52.6. Anal. calcd
for C11H12O5: C, 58.96; H, 5.40. Found: C, 58.57; H, 5.49.
Phosphonate intermediate 10. 1H NMR (CDCl3) d 3.92 (s, 3H),
3.93 (s, 3H), 3.94 (s, 3H), 4.96 (s, 2H), 6.86 (s, 1H), 7.48 (s,
1H); 13C NMR (CDCl3) d 166.4, 152.0, 148.0, 135.4, 120.0,
113.7, 113.3, 56.02, 52.03, 5.2.
Catechol homophosphonate intermediate 15. 1H NMR (CDCl3)
d 1.33 (s, 3H), 1.35 (s, 3H), 1.37 (s, 3H), 1.39 (s, 3H), 3.86 (s,
3H), 3.87 (s, 3H), 4.72 (m, 2H), 6.35 (dd, J=18, 19 Hz, 1H),
6.93 (dd, J=2, 8 Hz, 1H), 7.06 (dd, J=8, 8 Hz, 1H), 7.13 (dd,
J=2, 8 Hz, 1H), 7.78 (dd, J=18, 23 Hz, 1H); 13C NMR
Phosphonate intermediate 11. 1H NMR (CDCl3) d 1.17 (d,
J=6 Hz, 6H), 1.27 (d, J=6 Hz, 6H), 3.77 (d, J=2.3 Hz, 2H),
3.89 (s, 3H), 3.91(s, 3H), 3.93 (s, 3H), 4.61(m, 2H), 6.95 (d,
J=2.4 Hz, 1H), 7.46 (s, 1H); 13C NMR (CDCl3) d 167.2,
151.3, 146.9, 127.8, 121.6, 114.3, 113.2, 70.2 (d, J=7.0 Hz),
55.8, 51.7, 31.4 (d, J=138 Hz), 24.0, 23.8, 23.7, 23.6. Anal.
calcd for C17H27O7P: C, 54.54; H, 7.27. Found: C, 54.40; H,
7.34.
(CDCl3) d 153.0, 148.0, 142.1 (d, J=8 Hz), 129.1 (d,
J=23 Hz)), 124.0, 118.8, 117.1 (d, J=190 Hz), 113.5, 70.3 (d,
J=6 Hz), 61.1, 55.8, 24.1, 24.0, 23.9, 23.8. Anal. calcd for
C16H25O5P: C, 58.53; H, 7.68. Found: C, 57.91; H, 7.70.
Catechol homophosphonate intermediate 16. 1H NMR (CDCl3)
d 1.32 (s, 3H), 1.33 (s, 3H), 1.34 (s, 3H), 1.35 (s, 3H), 2.01 (m,
2H), 2.90 (m, 2H), 3.84 (s, 3H) 3.85 (s, 3H), 4.72 (m, 2H), 6.79
(m, 2H), 6.99 (dd, J=8, 8 Hz, 1H); 13C NMR (CDCl3) d 152.6,
146.9, 135.0 (d, J=18 Hz), 123.9, 121.4, 110.6, 69.9 (d,
J=6 Hz), 60.5, 55.6, 27.9 (d, J=138 Hz), 23.9, 23.6 (d,
J=4 Hz). Anal. calcd for C16H27O5P: C, 58.17; H, 8.24.
Found: C, 57.56; H, 8.24.
Protocatechuic phosphonate 1. 1H NMR (D2O) d 3.5 (d,
J=22 Hz, 2H), 6.85 (s, 1H), 7.38 (s, 1H); 13C NMR (D2O) d
174.4, 150.8, 145.1, 130.9 (d, J=10 Hz), 124.8 (d, J=6 Hz),
121.9, 121.4, 34.9 (d, J=130 Hz); HRMS (FAB) calcd for
C8H8PO7 247.0008, found 246.9998.
1
Catechol homophosphonate 4. H NMR (D2O) d 2.10 (m, 2H),
1
Homophosphonate intermediate 12. H NMR (CDCl3) d 1.38
2.90 (m, 2H), 6.82 (m, 3H); 13C NMR (D2O) d 147.2, 144.6,
132.2 (d, J=17 Hz), 123.8 (d, J=27 Hz), 117.0, 29.5 (d,
J=132 Hz), 25.768; HRMS (FAB) calcd for C8H12PO5
219.0422, found 219.0416.
(s, 12H), 3.91 (s, 3H), 3.95 (s, 3H), 3.96 (s, 3H), 4.76 (m, 2H),
6.12 (dd, J=17, 18 Hz, 1H), 7.03 (s, 1H), 7.46 (s, 1H), 8.23
(dd, J=17, 22 Hz, 1H); 13C NMR (CDCl3) d 166.6, 151.8,