Extradiol oxidative cleavage of catechols by ferrous and ferric complexes of 1,4,7-triazacyclononane: Insight into the mechanism of the extradiol catechol dioxygenases

Article abstract of DOI:10.1021/ja004280u

The major oxygenation product of catechol by dioxygen in the presence of FeCl₂ or FeCl₃, 1,4,7-triazacyclononane (TACN), and pyridine in methanol is the extradiol cleavage product 2-hydroxymuconic semi-aldehyde methyl ester (Lin, G.; Reid, G.; Bugg, T. D. H. J. Chem. Soc. Chem. Commun. 2000, 1119-1120). Under these conditions, extradiol cleavage of a range of 3- and 4-substituted catechols with electron-donating substituents is observed. The reaction shows a preference in selectivity and rate for iron(II) rather than iron(III) for the extradiol cleavage, which parallels the selectivity of the extradiol dioxygenase family. The reaction also shows a high selectivity for the macrocyclic ligand, TACN, over a range of other nitrogen- and oxygen-containing macrocycles. Reaction of anaerobically prepared iron-TACN complexes with dioxygen gave the same product as monitored by UV/vis spectroscopy. KO₂ is able to oxidize catechols with both electron-donating and electron-withdrawing substituents, implying a different mechanism for extradiol cleavage. Saturation kinetics were observed for catechols, which fit the Michaelis-Menten equation to give k_cat^app = 4.8 × 10^-3 s^-1 for 3-(2′,3′-dihydroxyphenyl)propionic acid. The reaction was also found to proceed using monosodium catecholate in the absence of pyridine, but with different product ratios, giving insight into the acid/base chemistry of extradiol cleavage. In particular, extradiol cleavage in the presence of iron(II) shows a requirement for a proton donor, implying a role for an acidic group in the extradiol dioxygenase active site.

Full text of DOI:10.1021/ja004280u

5030
J. Am. Chem. Soc. 2001, 123, 5030-5039
Extradiol Oxidative Cleavage of Catechols by Ferrous and Ferric
Complexes of 1,4,7-Triazacyclononane: Insight into the Mechanism
of the Extradiol Catechol Dioxygenases
Gang Lin,† Gillian Reid,‡ and Timothy D. H. Bugg*,†
Contribution from the Department of Chemistry, UniVersity of Warwick, CoVentry CV4 7AL, U.K., and
Department of Chemistry, UniVersity of Southampton, Highfield, Southampton SO17 1BJ, U.K.
ReceiVed December 18, 2000
Abstract: The major oxygenation product of catechol by dioxygen in the presence of FeCl2 or FeCl3, 1,4,7-
triazacyclononane (TACN), and pyridine in methanol is the extradiol cleavage product 2-hydroxymuconic
semi-aldehyde methyl ester (Lin, G.; Reid, G.; Bugg, T. D. H. J. Chem. Soc. Chem. Commun. 2000, 1119-
1120). Under these conditions, extradiol cleavage of a range of 3- and 4-substituted catechols with electron-
donating substituents is observed. The reaction shows a preference in selectivity and rate for iron(II) rather
than iron(III) for the extradiol cleavage, which parallels the selectivity of the extradiol dioxygenase family.
The reaction also shows a high selectivity for the macrocyclic ligand, TACN, over a range of other nitrogen-
and oxygen-containing macrocycles. Reaction of anaerobically prepared iron-TACN complexes with dioxygen
gave the same product as monitored by UV/vis spectroscopy. KO2 is able to oxidize catechols with both
electron-donating and electron-withdrawing substituents, implying a different mechanism for extradiol cleavage.
Saturation kinetics were observed for catechols, which fit the Michaelis-Menten equation to give kcatapp ) 4.8
× 10-3 s-1 for 3-(2′,3′-dihydroxyphenyl)propionic acid. The reaction was also found to proceed using
monosodium catecholate in the absence of pyridine, but with different product ratios, giving insight into the
acid/base chemistry of extradiol cleavage. In particular, extradiol cleavage in the presence of iron(II) shows a
requirement for a proton donor, implying a role for an acidic group in the extradiol dioxygenase active site.
Introduction
The oxidative cleavage of catechol and other dihydroxy
aromatics is a key step in the biodegradation by soil bacteria of
naturally occurring aromatic molecules and many aromatic
environmental pollutants.1 The catechol dioxygenases are a class
of non-heme iron enzymes that catalyze the oxidative cleavage
of catechols. These enzymes can be divided into two subclasses
(Figure 1): the intradiol dioxygenases, which utilize a non-
heme iron(III) cofactor, that catalyze the cleavage of the
carbon-carbon bond between the two catechol hydroxyl oxy-
gens; and the extradiol dioxygenases, which utilize a non-heme
iron(II) cofactor, that catalyze the cleavage of the carbon-
carbon bond adjacent to the catechol oxygens.2
The three-dimensional crystal structures of three extradiol
catechol dioxygenases, 2,3-dihydroxybiphenyl 1,2-dioxygenase
(BphC) from Pseudomonas LB400,3 catechol 2,3-dioxygenase
(XylE) from Pseudomonas putida mt-2,4 and protocatechuate
4,5-dioxygenase (LigAB) from Sphingomonas paucimobilis5
Figure 1. Enzyme-catalyzed reactions of the extradiol and intradiol
dioxygenases.
have been determined, and in each case the mononuclear iron(II)
center is ligated by two histidine ligands and one glutamic acid
ligand. In contrast the mononuclear iron(III) center of the
intradiol-cleaving protocatechuate 3,4-dioxygenase from Pseudo-
monas aeruginosa and catechol 1,2-dioxygenase from Acine-
tobacter calcoaceticus is ligated by two histidine ligands and
two tyrosine ligands.6
Many attempts to model the catechol dioxygenases have been
reported, most of which give products consistent with intradiol
cleavage.7 In all of these cases, the ligands used to prepare the
iron(III) complexes are tetradentate, thus resembling the coor-
dination environment of iron(III) center in the active site of
* Correspondence should be addressed to: Prof. T. D. H. Bugg,
Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.
Telephone: +44 02476-573018. Fax: +44 02476-524112. E-mail:
mssgv@csv.warwick.ac.uk.
† University of Warwick.
‡ University of Southampton.
(5) Sugimoto, K.; Senda, T.; Aoshima, H.; Masai, E.; Fukuda, M.; Mitsui,
Y. Structure 1999, 7, 953-965.
(1) Dagley, S. Essays Biochem. 1975, 11, 81-135.
(2) For reviews of the catechol dioxygenases, see: Que, L., Jr.; Ho, R.
Y. N. Chem. ReV. 1996, 96, 2607-2624; Bugg, T. D. H.; Winfield, C. J.
Nat. Prod. Rep. 1998, 15, 513-530.
(3) Han, S.; Eltis, L. P.; Timmis, K. N.; Muchmore, S. W.; Bolin, J. T.
Science 1995, 270, 976-980.
(4) Kita, A.; Kita, S.; Fujisawa, I.; Inaka, K.; Ishida, T.; Horiike, K.;
Nozaki, M.; Miki, K. Structure 1998, 6, 25-34.
(6) Ohlendorf, D. H.; Lipscomb, J. D.; Weber, P. C. Nature 1988, 336,
403-404; Vetting, M. W.; Ohlendorf, D. H. Structure 2000, 8, 429-440.
(7) Que, L., Jr.; Kolanczyk, R. C.; White, L. S. J. Am. Chem. Soc. 1987,
109, 5373-5380; Jang, H. G.; Cox, D. D.; Que, L., Jr. J. Am. Chem. Soc.
1991, 113, 9200-9204; Fuji, S.; Duda, M.; Pascaly, M.; Krebs, B. J. Chem.
Soc. Chem. Commun. 1997, 835-836; Viswanathan, R.; Palaniandavar, M.;
Balasubramarian, T.; Muthiah, T. D. Inorg. Chem. 1998, 37, 2943-2951;
10.1021/ja004280u CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/03/2001

Mechanism of the Extradiol Catechol Dioxygenases
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5031
the intradiol dioxygenases. Que and co-workers have found a
correlation between the Lewis acidity of the iron center in a
series of iron(III) complexes of tripodal ligands, with the product
yield and reactivity,7 and proposed a substrate activation rather
than oxygen activation mechanism. In this mechanism an iron-
(III)-catecholate species undergoes ligand to metal charge
transfer to give an iron(II)-semiquinone intermediate. The attack
of O2 on the activated substrate yields a transient alkyl-peroxy
radical which combines with the equally short-lived iron(II)
center to generate an alkyl-peroxy iron(III) species. The peroxy
adduct decomposes by a Criegee-type rearrangement to muconic
anhydride.
However, there are a few reported examples of extradiol-
type cleavage by synthetic iron complexes to give pyrone
products. Funabiki et al. found that FeCl2/FeCl3 complexes with
bipyridine/pyridine prepared in situ cleave 3,5-di-tert-butyl
catechol to 2-pyrones, which are believed to derive from
decarbonylation of the extradiol-cleavage intermediate R-keto
lactone.8 Dei et al. found that the complex [FeIII(TACN)Cl-
(dbc)] yielded 2-pyrones upon exposure to O2 in 35% yield.9
Que et al. used the same complex to give an almost quantitative
yield of 2-pyrone using a modified procedure.10 We have
reported that the oxygenation of catechol by O2 in the presence
of FeCl2/FeCl3, TACN (1,4,9-triazacyclononane), and pyridine
in methanol gave authentic 2-hydroxymuconic semi-aldehyde
methyl ester in about 50% yield.11 In these model reactions,
ligands TACN and bipyridine/pyridine are tridentate, resembling
the coordination environment of the iron(II) center in the active
site of extradiol dioxygenases. However, the mechanism of this
model reaction remains unclear. Extradiol catechol cleavage has
also been reported using KO2.14
Previous studies in our laboratory have focused on the
reaction mechanism of the extradiol enzyme 2,3-dihydroxyphen-
yl propionate 1,2-dioxygenase (MhpB) from Escherichia coli.
Mechanistic studies using substrates containing cyclopropyl
radical traps have established that, following the ligation of the
catechol and dioxygen by the iron(II) center, the catalytic
mechanism proceeds via single electron transfers to give a
semiquinone-iron(II)-superoxide intermediate.12 18O-labeling
studies have established the existence of a seven-membered ring
lactone intermediate, formed by Criegee rearrangement of the
hydroperoxide.13 Synthesis and evaluation of carba-analogues
of putative proximal and distal hydroperoxide intermediates have
provided evidence in support of a proximal hydroperoxide
intermediate for MhpB. The reaction is completed by attack of
iron(II)-hydroxide upon the lactone to give the extradiol ring
fission product.13
Scheme 1. Macrocyclic Ligands, Synthetic Route for
N-(3′-Pyridyl)-TACN (4)a
a Reagents and conditions: i) Boc-ON,CHCl3, 92%; ii) K2CO3,
CH3CN, 3-BrCH2-pyridine, 45%; iii) CF3CO2H, CH2Cl2, quant.
substrate specificity, kinetics, and regioselectivity. These studies
give new insights into the questions of why iron(II) and iron(III)
are employed respectively by the extradiol and intradiol
dioxygenases, and why they exhibit different active-site coor-
dination chemistry.
Results
Synthesis of Macrocyclic Ligands. TACN was synthesized
according to a literature procedure,16 and commercially available
12ane-N3 (1,5,9-triazacyclododecane) was tested for activity.
Since the extradiol dioxygenase active site contains N, N, O
coordination, 9ane-N2O and 12ane-N3O were synthesized ac-
cording to literature methods.17,18 Since the active site of BphC
also contains additional acid/base residues3, monosubstituted
N-(3′-pyridylmethyl)-TACN 4, was designed and synthesized
as shown in Scheme 1. N,N′-DiBoc-TACN 2 was obtained by
reacting TACN 1 with 2 equiv of Boc-ON reagent in 92%
yield.19 Treatment of 2 with 3-bromomethyl pyridine gave 3 in
45% yield. Removal of Boc groups was achieved by stirring
with 2% trifluoroacetic acid in dichloromethane to give 4 in
quantitative yield.
Assay of Monosubstituted TACN and N2O, 12ane-N3 in
Model Reaction. We have developed a simple method to scan
for the extradiol cleaving activity of specific ligands on the basis
of the observation that extradiol cleavage of catechol in buffer
pH 8.0, yields a bright yellow color with UV/vis absorption at
375 nm. When catechol was used in the model reaction
catalyzed by TACN/FeCl2, the initial extradiol cleavage product
shows an absorbance at 315 nm in methanol and then shifts to
405 nm by adding pH 8.0 Tris buffer; with addition of NaOH
solution, the absorbance completely shifts to 378 nm after 2 h
(Figure 2).
All assays with other macrocyclic ligands showed no absor-
bance above 300 nm, which indicates that (1) three nitrogen
atoms here are essential for the occurrence of the reaction; (2)
substitution of H on one nitrogen atom of TACN disrupts the
extradiol-cleaving activity; (3) the increase of one carbon in
each bridge between two nitrogen atoms also leads to the loss
of activity. The extradiol cleavage reaction is therefore highly
selective for the macrocyclic ligand.
We report here, the syntheses of tridentate ligands and studies
of the TACN/FeCl2 model reaction, including evidence of
formation of an intradiol cleavage product and an extradiol
cleavage product; the preference of iron(II) over iron(III),
Funabiki, T.; Yamazaki, T.; Fukui, A.; Tanaka, T.; Yashida, S. Angew.
Chem., Int. Ed. 1998, 37, 513-515; Koch, W. O.; Kru¨ger, H-J. Angew.
Chem., Int. Ed. Engl. 1995, 34, 2671-2674; Mialane, P.; Tchertanov, L.;
Banse, F.; Sainton, J.; Girerd, J-J. Inorg. Chem. 2000, 39, 2440-2444.
(8) Funabiki, T.; Mizoguchi, A.; Sugimoto, T.; Tada, S.; Tsugi, M.;
Sakamoto, H.; Yoshida, S. J. Am. Chem. Soc. 1986, 108, 2921-2932.
(9) Dei, A.; Gatteschi, D.; Pardi, L. Inorg. Chem. 1993, 32, 1389-1395.
(10) Ito, M.; Que, L., Jr. Angew. Chem., Int. Ed. Engl. 1997, 36, 1342-
1344.
Metal Dependence. The replacement of FeCl2/FeCl3 by
MnCl2, CoCl2, CuCl showed no reaction at all. The replacement
(15) Wieghardt, K.; Pohl, K.; Gebert, W. Angew. Chem., Int. Ed. Engl.
1983, 22, 727.
(11) Lin, G.; Reid, G.; Bugg, T. D. H. J. Chem. Soc. Chem. Commun.
2000, 1119-1120.
(16) Erhardt, J. M.; Grover, E. R.; Wuest, J. D. J. Am. Chem. Soc. 1980,
102, 6365-6369.
(12) Spence, E. L.; Langley, G. J.; Bugg, T. D. H. J. Am. Chem. Soc.
1996, 118, 8336-8343.
(17) Hancock, R. D.; Thoem, V. J. J. Am. Chem. Soc. 1982, 104, 291-
292.
(13) Sanvoisin, J.; Langley, G. J.; Bugg, T. D. H. J. Am. Chem. Soc.
1995, 117, 7836-7837.
(14) Muller, R.; Lingens, F. Z. Naturforsch. 1989, 44c, 207-211.
(18) Rasshofer, W.; Voegtle, F. Justus Liebigs Ann. Chem. GE. 1977,
1340-1343
(19) Kovacs, Z.; Sherry, A. D. Tetrahedron Lett. 1995, 36, 9269-9272.

5032 J. Am. Chem. Soc., Vol. 123, No. 21, 2001
Lin et al.
Figure 2. Intermediates and respective λmax values for FeCl2/TACN model reaction.
Table 1. Metal Dependence of Model Reactiona
ring R-keto lactone, is becoming more rate-determining upon
changing solvent from methanol to ethanol then to 2-propanol,
due to increased steric hindrance. No reaction was observed
with the reaction carried out in tert-butyl alcohol. Also, no
reaction was observed when solvent was changed to H2O, THF,
or Et2O.
Substrate Specificity. The TACN/FeCl2 model reaction was
found to operate on a range of substituted catechols. Highest
rates were observed with electron-donating substituents, whereas
catechols with electron-withdrawing substituents were not
processed. This indicates that an electronic effect plays an
important role in the TACN/FeCl2 model reaction. A similar
pattern of results was found for the reaction catalyzed by E.
coli 2,3-dihydroxyphenyl propionate 1,2-dioxygenase (MhpB):
catechols with electron-withdrawing substituents were not turned
over. The results are listed in Table 3. The similar substrate
dependence is consistent with a similar mechanism for enzy-
matic and model reactions.
In contrast, the KO2/DMSO reaction reacts with catechols
containing either electron-donating substrates or electron-
withdrawing substrates at similar rates (Table 3). Furthermore,
all of the catechols with electron-donating alkyl substituents give
products that have an absorbance maximum at 377 nm. This
means that KO2 reacts with 3-substituted catechols via 1,6-
cleavage to give substituted 2-hydroxymuconic semi-aldehydes
rather than ketone derivatives. It suggests a different mechanism
for the KO2/DMSO reaction, probably via a dioxetane inter-
mediate.
When 3,5-di-tert-butyl catechol was used as substrate in the
TACN/FeCl2 reaction, mixtures of 2-pyrones were obtained
(Scheme 2), as observed by Dei et al.9 No 2-hydroxymuconic
semi-aldehyde derivatives were found, suggesting that the
mechanism for 2-pyrone formation diverges from the extradiol
mechanism prior to the formation of the R-keto lactone
intermediate.
Assay of FeIII(TACN)Cl3 in the Model Reaction. FeIII-
(TACN)Cl3 was prepared using the method of Wieghardt et al.,15
and was used in place of FeCl3 and TACN in the model reaction.
The complex was not soluble in methanol; however, when
pyridine was added to the suspension of FeIII(TACN)Cl3 and
catechol in methanol, the complex gradually dissolved with time,
and finally gave the characteristic absorbance of the extradiol
cleavage product at λmax 315 nm (Scheme 3).
Characterization of Reaction Products and Regioselec-
tivity. The identity of the reaction products was confirmed by
carrying out the reaction of 11 mg of catechol with 1 equiv of
TACN, 1 equiv of FeCl2‚4H2O, and 3 equiv of pyridine in 500
mL of methanol by bubbling O2 under the surface of the solution
for 3 h. The 1H NMR spectrum of the reaction extraction product
displayed signals corresponding to 2-hydroxymuconic semi-
aldehyde methyl ester 5 as the major product by comparison to
the extradiol cleavage product of enzymatic reaction of 2,3-
dihydroxyphenylpropionic acid and 2,3-dihydroxyphenylpropi-
onate 1,2-dioxygenase. Muconic acid monomethyl ester 6 was
identified as the minor product confirmed by comparison to the
NMR spectrum of an authentic sample (Scheme 4). The ratio
of extradiol cleavage product to intradiol cleavage product
metal salta
basea
substratea solvent rate (∆A/min)
FeCl2
pyridine
pyridine
pyridine
pyridine
DBU
catechol CH3OH
DHP CH3OH
catechol CH3OH
DHP CH3OH
catechol CH3OH
0.033
0.098
0.053
0.133
FeCl2
FeBr2
FeBr2
FeCl2
FeCl2
FeCl2
MnCl2
CoCl2
CuCl
0.026
2,6-lutidine catechol CH3OH
DMAP
pyridine
pyridine
pyridine
pyridine
no reaction
no reaction
no reaction
no reaction
no reaction
no reaction
no reaction
catechol CH3OH
catechol CH3OH
catechol CH3OH
catechol CH3OH
catechol CH3OH
catechol CH3OH
FeSO4
(NH4)2Fe(SO4)2 pyridine
a Reactions were carried out with 0.1 mM of metal salt, 1 mM of
TACN, 0.3 mM of base, and 0.1 mM of substrate in methanol (DHP
) 3-(2′,3′-dihydroxyphenyl)propionic acid) at 23 °C.
Table 2. Effect of Changing Reaction Conditions on Ratea
concentration (mM)
solvent FeCl3 TACN pyridine catechol ∆A/min nmoles/min
MeOH 0.1 0.1 0.3 0.1 0.017 0.94
solvent FeCl2 TACN pyridine catechol ∆A/min nmoles/min
ratea
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
0.1
0.1
0.1
0.1
0.1
0.1
0.05 0.05
0.05 0.1
0.1
0.1
0.1
0.1
0.1
0.05
0.05
0.3
0.15
0.1
0.1
0.3
0.15
0.3
0.3
0.3
0.3
0.3
0.1
0.1
0.1
0.05
0.1
0.1
0.1
0.1
0.1
0.1
0.1b
0.1b
0.033
0.036
0.024
0.018
0.021
0.021
0.013
0.036
0.026
0.016
0.047
0.046
1.83
2.00
1.33
1.00
1.17
1.17
0.72
2.00
1.44
0.89
2.61
2.56
0.1
0.1
0.1
0.1
2-Propanol 0.1
MeOH
MeOH
0.1
0.1
-
a Methanol, FeCl2 or FeCl3, TACN, pyridine, catechol were added
in this order into a cuvette, and extradiol product formation was
measured at 315 nm over a total of 6 min at 23 °C. b Catechol was
replaced by monosodium catecholate.
of FeCl2 by FeSO4 or Fe2(NH4)2(SO4)2 also showed no reaction.
FeBr2 gave the characteristic UV absorbance of 315 nm under
the standard reaction conditions, and kinetic studies showed that
reaction with FeBr2 is faster than that with FeCl2 (Table 1).
Since FeBr2 is a softer Lewis acid than FeCl2, this may indicate
a preference for a soft Lewis acid for extradiol cleavage.
Optimization of the TACN/FeCl2 Model Reactions. Opti-
mization of the reaction was carried out by changing the ratio
between all the four reagents, and by changing the base. The
results are listed in Table 2. No extradiol ring cleavage was
observed when either TACN, pyridine, or FeCl2/FeCl3 was
omitted. The ratio of reagents found to give optimum activity
was a 1:1:1.5:1 ratio of catechol:TACN:pyridine:FeCl2. The
reaction was found to proceed using DBU in place of pyridine
but at a lower rate. The absorption maximum at 315 nm was
still observed using ethanol or 2-propanol instead of methanol.
The lower rate of the reaction in ethanol and 2-propanol
indicates that the final step, alcoholysis of the seven-membered

Mechanism of the Extradiol Catechol Dioxygenases
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5033
Table 3. Substrate Selectivity for FeCl2/TACN and KO2 Reactions and for MhpB-Catalyzed Enzymatic Reactionc
a See Spence E. L.; Kawamukai M.; Sanvoisin J.; Braven H.; Bugg T. D. H.; J. Bacteriol. 1996, 178, 5249. b See refs 12 and 13. c NT, not
tested; RFP, ring fission product.
Scheme 2. Reaction of 3,5-Di-tert-butyl Catechol with
TACN/FeCl2/Pyridine
Scheme 4. Product Ratios for Cleavage of Catechol and
Substituted Catechols
Scheme 3. Reaction of Catechol with Fe(TACN)Cl3
Complex
1
determined by integration of H NMR signals was 7:1. When
FeCl2 was replaced by FeCl3, the major product was still the
extradiol cleavage product, but the ratio between extradiol and
intradiol products was reduced to 2:1. When the solvent was
changed from methanol to ethanol, the reaction slowed, and
the 1H NMR spectrum of the reaction product displayed peaks
consistent with the ethyl ester of 2-hydroxymuconic semi-
aldehyde.
The regioselectivity of the TACN/FeCl2 reaction was inves-
tigated using 3-methylcatechol and 4-methylcatechol. In the case
of 3-methylcatechol, only extradiol cleavage products were
observed: the ketone product of 2,3-cleavage and the aldehyde
product of 1,6-cleavage were obtained in a 35:1 ratio, indicating
a highly regioselective reaction. In the case of 4-methyl catechol,
the NMR spectrum of the crude product showed only one
extradiol product, arising from 2,3-cleavage, and the intradiol
products. Again, the extradiol cleavage is highly regioselective,
but in this case intradiol cleavage is a competing reaction. When
3-(2′,3′-dihydroxyphenyl)propionic acid was used as substrate,
the aldehyde product was not detectable in the NMR spectrum,
only the ketone product was obtained. These data indicate that
the reaction shows high regioselectivity for extradiol cleavage.
Reaction Kinetics. The addition of the four reagents and
solvent was always carried out in the order: methanol, FeCl2/
FeCl3, TACN, catechol, pyridine. The rate of product formation
at 315 nm is linear over time up to 6 min when the final
concentration of FeCl2/FeCl3, TACN, catechol are each 0.1 mM.
The effect of changing the relative concentration of the four
reagents led to varied behavior. There was no change of initial
rate when the concentration of pyridine was reduced from 3 to
1.5 equiv, but in the presence of 1.0 equiv, a reduced rate was
observed. Halving the concentration of TACN gave a reduced
rate; halving the FeCl2 concentration gave no change in rate at
0.1 mM TACN, but a reduction in rate at 0.05 mM TACN was
observed.
The initial rates of FeCl2-containing reaction were slower than
that of FeBr2-containing reaction with catechol, DHP, respec-
tively. All of the results are shown in Table 2.
The variation of rate with changing catechol concentration
was measured at 23 °C, keeping the final concentrations of
FeCl2‚4H2O (0.1 mM), TACN (0.1 mM), pyridine (0.3 mM)

5034 J. Am. Chem. Soc., Vol. 123, No. 21, 2001
Lin et al.
Figure 3. Kinetic behavior of model reaction with substrate 2,3-dihydroxyphenylpropionic acid. Assays carried out at constant final concentrations
of FeCl2‚4H2O (0.1 mM), TACN (0.1 mM), pyridine (0.3 mM), varying the concentration of DHP, recording the initial rates at each concentration
up to 6 min at 23 °C.
Table 4. Steady-State Kinetic Parameters for (A) FeCl2‚4H2O (0.1 mM) + TACN (0.1 mM) + Pyridine (0.3 mM) + DHP in MeOH
(apparent Km, kcat Values; See Figure 3); (B) MhpB-Catalyzed Reaction (Ref 24)
ꢀRFP
(M-1 cm-1
Km
(µM)
kcat
kcat/Km
∆∆G
(kJ/mol)
substrate
)
(s-1
)
(M-1 s-1
)
3-(2′,3′-dihydroxyphenyl)propionate
A
B
8100 (317 nm)
15600 (394 nm)
140
26
4.8 ×10-3
34.3
26.0
29
1.1 × 106
constant, but changing the concentration of DHP. Saturation
kinetics were observed by plotting initial rates versus concentra-
tion of catechol (Figure 3). Apparent Km (0.14 mM) and kcat
(4.8 × 10-3 s-1) values were obtained by applying Lineweaver-
Burk or Eadie-Hofstee plots shown in Table 4. This strongly
suggests that the complex of iron-TACN, the “active site” of
the model reaction, is formed before combining with the
substrate.
Investigation of Intermediate Catechol-Iron-TACN Com-
plexes. Dei et al. have previously described the preparation of
FeIII(TACN)(3,5-di-tert-butylcatechol)Cl from FeIII(TACN)Cl3
under anaerobic conditions; using the same procedure we
prepared anaerobically FeIII(TACN)(catechol)Cl and FeII-
(TACN)(catechol)Cl from FeCl3 and FeCl2 respectively, using
triethylamine as base.
The FeIII(TACN)(catechol)Cl complex was found to exhibit
a UV/vis spectrum containing peaks at 451 and 720 nm (see
Figure 4A), similar to the UV/vis data (λmax 480, 520, 782 nm)
reported by Funabiki et al. for a complex of 3,5-di-tert-butyl-
catechol with FeIII(bipyridine)(pyridine), due to a ligand-to-metal
charge-transfer interaction.8 When the FeIII(TACN)(catechol)-
Cl complex was exposed to O2, the peaks at 451 and 720 nm
decayed, and a new peak at 315 nm appeared, corresponding
to the extradiol cleavage product.
When the TACN/FeCl3 reaction was carried out at 0.4 mM
TACN, catechol, FeCl3‚6H2O concentration, as shown in Figure
4B, the same absorbance peaks were observed immediately and
decreased with time, as the extradiol product was formed. These
observations demonstrate the formation of the same FeIII-
(TACN)(catechol) complex in the FeCl3 model reaction.
pyridine as base. In this case a different FeII(TACN)(catechol)
complex was obtained, which showed no λmax in the range 300-
800 nm, but was converted rapidly to extradiol product on
exposure to O2. Attempts to prepare single crystals of this
complex using a range of substituted catechols were unsuc-
cessful; however, the FeII(TACN)(catechol) complex was
1
characterized by H NMR spectroscopy. The NMR spectrum
showed signals in the range 0-10 ppm, characteristic of a low-
spin iron(II) complex. Signals were observed for TACN (2.9-
3.6 ppm), catechol (6.68, 6.76 ppm), and pyridine (7.6, 7.95,
8.6 ppm) ligands, implying a mononuclear FeII(TACN)(cat-
echol)(pyridine) complex.
When the FeCl2-containing reaction at 0.4 mM TACN/FeCl2/
catechol was monitored by UV/vis spectroscopy (Figure 4D),
immediate extradiol product formation was observed at 315 nm,
and the only other observed species was the gradual formation
of the FeIII(TACN)(catechol) complex at 449/720 nm. These
data can be rationalized by the formation of an FeII(TACN)-
(catechol) complex (no λmax), followed by its gradual oxidation
to an FeIII(TACN)(catechol) complex (λmax 449, 720 nm) under
aerobic conditions. These observations give some insight into
the oxidation state of the iron center during the reaction. Since
oxidation of iron(II) to iron(III) occurs on a slower time scale
than extradiol product formation, these observations imply that
extradiol cleavage is mediated by an iron(II) complex. The
appearance of the same FeIII(TACN)(catechol) complex in the
FeCl2/TACN reaction also suggests that the active iron(II) and
iron(III) complexes are structurally related.
Reaction of Monocatecholate with TACN/Iron. To explore
the role of pyridine in this model reaction, monosodium
catecholate and disodium catecholate were prepared by reacting
catechol with 1 equiv of NaOH and 2 equiv of NaOH in
methanol, respectively. No reaction was observed with disodium
catecholate upon treatment with TACN/FeCl2. When sodium
monocatecholate was reacted with TACN/FeCl2 or TACN/FeCl3
The FeII(TACN)(catechol)Cl complex, prepared anaerobically
using triethylamine as base, was found to exhibit a UV/vis
spectrum containing one peak at 550 nm (see Figure 4C);
however, upon exposure to O2 only very slow extradiol product
formation was observed. The complex was then prepared using

Mechanism of the Extradiol Catechol Dioxygenases
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5035
Figure 4. UV/vis spectra of TACN/iron/catechol complexes (A) FeIII(TACN)(catechol) complex generated anaerobically in methanol, using
triethylamine as base. (B) UV/vis scanning of TACN/FeCl3 reaction at 0.4 mM concentration in methanol. Scans recorded at 1-min intervals over
0-10 min. Arrows indicate increase in A315 (extradiol product formation), and decrease in A451 and A719 (complex). (C) FeII(TACN)(catechol)
complex generated anaerobically in methanol using triethylamine as base. (D) UV/vis scanning of TACN/FeCl2 reaction at 0.4 mM concentration
in methanol. Scans recorded at 1-min intervals over 0-11 min. Arrows indicate increase in A315 (extradiol product formation), and increase in A451
and A719 (complex).
Scheme 5. Product Ratios for the Reaction of Monosodium
Catecholate with TACN/FeCl2 and TACN/FeCl3
Figure 5. Similar regioselectivity of model reaction to that of extradiol
catechol dioxygenases.
Discussion
These results show that the TACN/FeCl2 and TACN/FeCl3
reactions show considerable scope, reactivity, and selectivity
for the extradiol oxidative cleavage of catechol substrates. The
studies in this paper give new insight into the mechanism of
in methanol, in the absence of pyridine, UV/vis spectra showed
in both reactions the formation of extradiol ring fission product
(RFP) with absorbance at 315 nm. kobs showed the reaction
running at the same rate, with or without pyridine (Table 2).
No extradiol cleavage was observed in the absence of TACN.
extradiol cleavage by these complexes, and by inference, the
catalytic mechanism of the extradiol catechol dioxygenases.
This model reaction is highly selective for the TACN
macrocyclic ligand, indicating that the coordination chemistry
and precise alignment is important for the extradiol reaction.
The tridentate coordination of TACN matches the His, His, Glu
tridentate coordination of Fe(II) at the active site of the extradiol
dioxygenases.3 The fact that the N,N,N-TACN ligand works in
this reaction suggests that coordination geometry is more
important than ligand type. The observation that the mono-N-
substituted derivative gives no sign of formation of extradiol
cleavage product indicates that all three NH’s are required for
activity.
To determine the ratios of products, large-scale reactions were
carried out using monosodium catecholate in the absence of
pyridine (Scheme 5). With TACN/FeCl3, the NMR spectrum
of the crude product indicated that extradiol RFP and intradiol
RFP were both formed, in a ratio of 3.2:1. With TACN/FeCl2,
the NMR spectrum of the crude product showed that although
both extradiol RFP and intradiol RFP were formed, the intradiol
RFP was by far the major product. The ratio of intradiol RFP
and extradiol RFP was 15:1. When 1 equiv of pyridine
hydrochloride was added to the reaction of TACN/FeCl2 and
monosodium catecholate, the extradiol RFP was obtained as
the major product, and the ratio of extradiol RFP and intradiol
RFP was 4:1 (Figure 6). Thus, the presence of 1 equiv of
pyridine hydrochloride leads to a 60-fold increase in ratio of
extradiol/intradiol products in the TACN/FeCl2 reaction.
Since the extradiol catechol dioxygenases are selective for
Fe(II) while the intradiol dioxygenase are selective for Fe(III),
it is of considerable interest that both TACN/FeCl2 and TACN/
FeCl3 are capable of extradiol cleavage. However, the TACN/
FeCl2 reaction shows higher rates and higher selectivity for

5036 J. Am. Chem. Soc., Vol. 123, No. 21, 2001
Lin et al.
1
Figure 6. 300 MHz H NMR spectra of reaction products: (A) FeCl2 + TACN + Catechol + Pyridine; (B) FeCl2 + TACN + Monosodium
catecholate; (C) FeCl2 + TACN + Monosodium catecholate + pyridine hydrochloride (1 equiv).
extradiol cleavage, suggesting that Fe(II) has an inherently
higher chemical reactivity for extradiol cleavage than Fe(III).
Also, highly regioselective cleavage is observed for 3-methyl-
catechol, and 3-(2′,3′-dihydroxyphenyl)propionic acid in the
TACN/FeCl2 reaction, the same selectivity as observed in
extradiol catechol dioxygenases which process 3-substituted
catechols (Figure 5). It is interesting to note that there are
reported examples of catechol 1,2-dioxygenases which yield
some extradiol cleavage product with certain substrates; thus,
there are biological examples of Fe (III)-dependent extradiol
cleavage.25,26
This reaction has wide substrate selectivity for catechols with
electron-donating substituents, but not electron-withdrawing
substituents, as found for MhpB. The very different pattern of
results observed for the KO2 reaction, suggests that this reaction
proceeds via a different mechanism. Since the KO2 reaction is
performed under basic conditions, it probably proceeds via a
dioxetane intermediate. Similarities between the TACN/FeCl2
reaction and enzymatic reaction suggests that they probably
follow the same reaction mechanism. Both enzymatic and model
reactions are sensitive to electron-withdrawing substituents,
which could be rationalized by the inhibitory effect of an
electron-withdrawing substituent on the Criegee rearrangement
step. Saturation kinetics are also consistent with equilibrium
binding of catechol to the Fe(II)-TACN complex, similar to
the enzyme-catalyzed reaction.
the catechol binding to the iron center as the monoanion. This
observation explains why the model reaction does not proceed
using stronger bases such as (dimethylamino)pyridine and 2,6-
lutidine. One consequence of catechol monoanion binding is
that the semiquinone intermediate will be uncharged, perhaps
a neutral semiquinone is required for C-O bond formation with
superoxide.
The reactions using monosodium catecholate as substrate
showed significant differences between the iron(II) and iron(III)
reactions. In the iron(III) reaction, the major product is extradiol
RFP in both cases, the ratio of extradiol RFP and intradiol RFP
being 2:1 with pyridine and 3.2:1 without pyridine. In the
reaction of sodium monocatecholate with TACN/FeCl2, intradiol
RFP was found as major product, and the ratio of extradiol RFP
and intradiol RFP is 1:15, while in the TACN/FeCl2/pyridine
model reaction, extradiol RFP was found as major product, in
a 7:1 ratio. The only difference between these two reactions is
that there was pyridine in the former reaction and no pyridine
in the latter reaction. This indicates that pyridine has two roles
in the reaction: one equivalent is required as a base to abstract
a proton from catechol, and the resulting pyridinium salt is
subsequently required as the proton donor for the Criegee
rearrangement. This suggestion is confirmed by the addition of
pyridine hydrochloride to the reaction of monosodium catecho-
late with TACN/FeCl2, which resulted in the formation of the
extradiol RFP as the major product in a ratio of 4:1 (Figure 6).
This result clearly demonstrates the requirement for a proton
donor in the extradiol cleavage reaction catalyzed by iron(II),
presumably to assist cleavage of the O-O bond in the Criegee
rearrangement. It is interesting to note that in each of the
extradiol dioxygenase X-ray crystal structures there are ad-
ditional acid-base residues in the vicinity of the iron(II)
center.3-5 For example, in Pseudomonas LB400 BphC, His-
195 is located 4.0 Å from the iron(II) center, while on the other
side of the active site, Tyr-250 is situated 3.8 Å from the iron(II)
The reaction of monosodium catecholate with TACN/FeCl2
yielded oxidative cleavage products, whereas no extradiol
cleavage was observed with the catecholate dianion, which
indicates that catechol binds to the iron center as the monoanion.
These observations are consistent with EXAFS studies of the
2,3-catechol dioxygenase: catechol complex,20 which show that
the coordination of catechol to the iron(II) center is asymmetric,
(20) Shu, L.; Chiou, Y-M.; Orville, A. M.; Miller, M. A.; Lipscomb, J.
D.; Que, L., Jr. Biochemistry 1995, 34, 6649-6659.

Mechanism of the Extradiol Catechol Dioxygenases
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5037
Figure 9. Possible π-participation mechanism for alkenyl migration,
which rationalizes the regioselectivity for R ) CH3, and the electronic
effect for electron-withdrawing R groups.
ate. Recombination of superoxide forms a proximal peroxy
intermediate. By Criegee rearrangement assisted by protonation
from a pyridium salt formed earlier, a seven-membered R-keto
lactone is obtained. After exchange of solvent methanol with
Fe(II)-hydroxide, it is attacked by MeO- to yield the ring-
opened methyl ester.
The regioselectivity of the TACN/FeCl2 can be rationalized
by this mechanism. It is known that 1,2-rearrangements such
as the Baeyer-Villiger oxidation are assisted by adjacent alkyl
substituents,22 so the methyl substituent of 3-methylcatechol will
favor 2,3-cleavage rather than 1,6-cleavage. In the case of
4-methyl catechol, 2,3-cleavage will result in a transient
carbocation intermediate adjacent to the 4-methyl group, which
would therefore reduce the activation energy for this pathway
(Figure 9).
Figure 7. RASMOL representation of BhpC active-site residues, in
vicinity of iron(II) cofactor. Darkly shaded residues are the iron(II)
ligands His146, His210, and Glu260. Distance in Å: His195-Fe 4.0;
Tyr250-Fe 3.8; His241-Fe 4.8; His241-Tyr250 2.7; His209-Fe 6.8;
His209-His210 2.8.
The mechanism for extradiol cleavage in the iron(III)/TACN
reaction must be slightly different from that of iron(II) in view
of the different product ratios obtained. The model reaction of
TACN/FeCl3 and the reaction of monosodium catecholate with
TACN/FeCl3 showed a similar extradiol RFP selectivity and
similar ratios between extradiol RFP and intradiol RFP. This
indicates that pyridine acts only as a base in the iron(III) reaction
and that no proton donor is required in this case. As the
iron(III)-TACN complex forms, the catechol monoanion binds
with the complex to form iron(III)-TACN-catechol monoanion
complex. The attack of O2 on the activated substrate yields a
transient alkyl-peroxy radical (Figure 10). There are two ways
the reaction may proceed from this intermediate. One is to form
the extradiol RFP by Criegee rearrangement of the hydroper-
oxide which has ligated to the iron center. The stronger Lewis
acidity of Fe(III) will facilitate cleavage of the O-O bond; it
then does not require a pyridium salt to act as a proton donor.
Proton transfer in this case may either be from the catechol OH
ligand or from solvent methanol. The other route is to form
intradiol RFP by decomposition of this peroxy adduct via acyl-
migration. The increased yield of extradiol RFP in the absence
of pyridine may be due to the partial displacement of one ligand
in the tridentate hydroperoxide complex by pyridine.
Figure 8. Proposed mechanism for TACN/FeCl2 model reaction (X
) pyridine).
center, with a nearby His-241 residue (Figure 7).3 From this
work it seems likely that one of these groups acts as a base to
deprotonate the catechol substrate, while the other functions as
proton donor for the Criegee rearrangement.
We propose that the mechanism for the iron(II) model
reaction is similar to the mechanism of extradiol catechol
dioxygenases, since (1) a similar substrate dependence is shown
by model and enzymatic reactions and (2) cleavage in the
presence of a series of alcohols gives the respective alkyl esters,
consistent with a common R-keto lactone intermediate, arising
from Criegee rearrangement. We have recently demonstrated
the inhibition data of E. coli MhpB by carba-analogues of the
proximal hydroperoxide, supporting the existence of a proximal
hydroperoxide intermediate in the extradiol catechol dioxygenase
mechanism.21 Following the formation of iron(II)-TACN
complex, the catechol monoanion combines with the iron center
of the complex as shown in Figure 8. A 1-electron transfer from
iron(II) to dioxygen forms a transient iron(III) species, and then
1-electron transfer from catechol monoanion to the iron center
forms a iron(II)-TACN-semiquinone-superoxide intermedi-
In conclusion, we have shown that both iron(II) and iron(III)
are capable of supporting extradiol catechol cleavage but that
highest selectivity is observed in the iron(II) reaction in the
presence of a tridentate ligand and a proton donor. These
observations provide a convincing model for the enzymatic
extradiol reaction and suggest roles for other active-site residues
in dioxygenase catalysis.
Experimental Section
Chemicals. Deuterated compounds D2O, CDCl3 were obtained from
Isotope Laboratories. Starting materials and reagents FeCl2‚4H2O,
FeCl3‚6H2O, p-toluenesulfonyl chloride (TsCl), NaH in mineral oil,
3-methylbenzoic acid, 1,5,9-triazacyclododecane (12ane-N3), 1,4,7,10-
tetraazacyclododecane (12ane-N4), 3-methylcatechol, 4-methylcatechol,
3-methoxy catechol, 2,3-dihydroxybenzaldehyde, 2,3-dihydroxybenzoic
(21) Winfield, C. J.; Al-Mahrizy, Z.; Gravestock, M.; Bugg, T. D. H. J.
Chem. Soc., Perkin Trans. 1 2000, 3277-3289.
(22) March, J. AdVanced Organic Chemistry, 3rd ed.; John Wiley &
Sons: New York, 1985; p 991.

5038 J. Am. Chem. Soc., Vol. 123, No. 21, 2001
Lin et al.
Figure 10. Proposed mechanism for TACN/FeCl3 model reaction (X ) Cl or pyridine).
acid, 3,4-dihydroxybenzoic acid, 1,2,3-trihydroxybenzene, 48% HBr
(hydrobromic acid) were purchased from Aldrich Chemical Co. and
used as obtained without further purification. Catechol was recrystallized
from toluene. 1,4,7-Triazacyclononane (TACN) was prepared according
to the method of Erhardt et al.16 1-Oxo-4,7-diazacyclononane was
prepared according to the method of Hancock et al.17 N,N′-DiBoc-
TACN was prepared according to the method of Kovacs and Sherry.20
HPLC grade methanol and ethanol were used without further purifica-
tion, DMF was dried over anhydrous MgSO4. Fe(TACN)Cl3 was
prepared according to the method of Wieghardt.15 FeIII(TACN)-
(catechol)Cl was prepared using the method of Dei et al.9
atmosphere. Then catechol (22 mg, 0.2 mmol) was added to the
resulting suspension, followed by pyridine (15.8 mg, 0.2 mmol). The
resulting violet solution was stirred at room temperature for 4 h.
Methanol was removed under vacuum to give a purple solid. 1H NMR
(CD3OD, 300 MHz) 8.6 (2H, pyr-RH), 7.95 (1H, pyr-γH), 7.6 (2H,
pyr-âH), 6.76 (2H, cat-H), 6.68 (2H, cat-H), 2.92-3.62 (6 × 2H,
TACN-H) ppm.
Procedure A: Oxygenation of Catechol Catalyzed by FeCl2‚
4H2O. Catechol (11 mg, 0.1 mmol), FeCl2‚4H2O (20 mg, 0.1 mmol),
pyridine (24 mg, 0.3 mmol), and TACN (12.9 mg, 0.1 mmol) were
dissolved in methanol (500 mL) in a single-necked round-bottom flask.
Oxygen gas was bubbled through the reaction mixture with stirring
for 3 h, the color of the reaction solution changed from deep purple to
yellow-green over 3 h. The solvent was removed in vacuo, 10% HCl
(2 mL) was added to decompose the iron complex. The products were
extracted with diethyl ether. The organic layer was dried (Na2SO4) and
1
Physical Measurements. H and 13C NMR spectra were recorded
on a Bruker 300 spectrometer in deuterated solvents as described below.
Mass spectra were recorded on a Micromass AutoSpec mass spec-
trometer. UV/visible spectra were recorded on a Beckmann DU 7400
UV/visible spectrophotometer equipped with a temperature-control unit.
Preparation of 1,4-DiBoc-7-(3′-pyridylmethyl)-TACN (3). N,N′-
DiBoc-TACN (164.5 mg, 0.5 mmol), anhydrous K2CO3 (276 mg, 2
mmol) were suspended in 25 mL of acetonitrile. The suspension was
heated to gentle reflux (85 °C). The solution of 3-bromomethyl pyridine-
(103.2 mg, 0.6 mmol) in 5 mL of acetonitrile was added dropwise to
it. The reaction mixture was heated at reflux for 2 days. The resulting
solid was removed by filtration. The acetonitrile was removed in vacuo
and the residue dissolved in dichloromethane and purified by flash
column to give 4 as a colorless oil (94.5 mg, 45%). 1H NMR (CDCl3,
300 MHz) δH 8.38 (2H, m, pyr-H), 7.65 (1H, d, J ) 7.7, pyr-H), 7.16
(1H, m, pyr-H), 3.52 (2H, s, -pyr-CH2-N), 3.25-3.5 (4H, m, TACN),
2.9-3.2 (4H, m, TACN), 2.4-2.6 (4H, m, TACN), 1.37, 1.38 (9H, 2
x s, Boc-H), 1.30 (9H, s, Boc-H) ppm δC 194.8, 194.6, 149.9, 148.3,
136.7, 136.1, 123.1, 79.5, 79.3, 57.6, 54.0, 52.6, 52.2, 50.9, 50.3, 49.6,
28.4, 28.3 ppm; MS (FAB, m/z) 420[M]+, 364[M - CH2dCH(CH3)2]+.
Preparation of N-(3′-Pyridylmethyl)-TACN (4). 1,4-DiBoc-7-(3′-
pyridylmethyl)-TACN (50 mg, 0.24 mmol) was treated with trifluo-
roacetic acid (0.1 mL) in dichloromethane (5 mL) and stirred at room-
temperature overnight. The solvent and excess trifluoroacetic acid were
1
evaporated in vacuo. The crude product was analyzed by H NMR,
and two products were identified. 2-Hydroxymuconic semi-aldehyde
methyl ester (5), yield 50% (based on DMF as the internal standard):
1H NMR (300 MHz, CDCl3) 9.55 (d, 1H, J ) 7.8 Hz, 6-CHO), 7.51
(dd, 1H, J ) 15.3, 11.5 Hz, 4-H), 6.31 (d, 1H, J ) 11.5 Hz, 3-H),
6.21 (dd, 1H, J ) 15.3, 7.8 Hz, 5-H), 3.86 (s, 3H, 1-COOCH3) ppm;
13C NMR (75 MHz, CDCl3) 205.1, 194.1, 164.2, 144.6, 139.6, 133.3,
51.2 ppm. Muconic acid monomethyl ester (6), yield 7.5% (based on
DMF as the internal standard): δH (300 MHz, CDCl3) 7.93 (dd, 0.15H,
J ) 10.8, 10.8 Hz, 3-H), 7.80 (dd, 0.15H, J ) 10.8, 10.8 Hz, 4-H), 6.0
(m, 0.3H, 2-H and 5-H), 3.7 (s, 0.45H, 1-COOCH3) ppm. MS(CI, m/z)
188[M + NH4]+, 171[M + H]+, 128[M - 43]+.
Oxygenation of Catechol Catalyzed by FeCl3‚6H2O. As procedure
A, except FeCl2‚4H2O was replaced by FeCl3‚6H2O, and the reaction
time extended to 5 h. NMR spectrum of crude product showed the
formation of extradiol RFP and intradiol RFP, in a ratio of 2:1
Oxygenation of Catechol Catalyzed by TACN/FeCl2 in Ethanol.
As procedure A, except methanol was replaced by ethanol. The product
2-hydroxy-6-oxo-hexa-2,4-dienoic acid ethyl ester was identified by
1
1
removed in vacuo, to give 4. H NMR (D2O, 300 MHz) 7.92 (s, 1H,
NMR analysis as the major product. H NMR (CDCl3, 300 MHz) δH
2′-H), 7.91 (d, 1H, J ) 6.0 Hz, 6′-H), 7.59 (d, 1H, J ) 7.5 Hz, 4′-H),
7.45 (dd, 1H, J ) 7.5, 6.0 Hz, 5′-H), 3.85 (s, 2H,), 3.53 (s, 4H), 3.12
(dd, 4H, J ) 6.0, 5.1 Hz), 2.91 (dd, 4H, J ) 6.0, 5.1 Hz) ppm; MS
(FAB, m/z) 221[M + H]+.
9.56 (d, 1H, J ) 8.1 Hz, -CHO), 7.52 (dd, 1H, J ) 15.6, 11.6 Hz,
4-H), 6.31 (d, 1H, J ) 11.6 Hz, 3-H), 6.2 (dd, 1H, J ) 15.6, 8.1 Hz,
5-H), 4.31 (q, 2H, J ) 7.2 Hz, -OCH2-), 1.32 (t, 3H, J ) 7.2 Hz,
-CH3) ppm. MS (CI, m/z) 188[M + NH4]+, 171[M + H]+, 153[M +
H-CO]+.
Preparation of FeII(TACN)(Catechol) Complex. A modification
of the method of Dei et al. was used.9 A solution of TACN (25.8 mg,
0.2 mmol) in 1 mL of degassed methanol was added to a degassed
solution of FeCl2‚4H2O (39.6 mg, 0.2 mmol) in 3 mL of methanol.
The solution was gently warmed to 40 °C for 0.5 h under N2
Oxygenation of Substituted Catechols Using TACN/FeCl2 in
Methanol. As procedure A, except catechol was replaced by a
substituted catechol. In each case, the products and product ratios were
1
determined by H NMR spectroscopy.

Mechanism of the Extradiol Catechol Dioxygenases
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5039
(a) 3-Methylcatechol. The product was identified by NMR as a 35:1
mixture of 2-hydroxy-6-oxo-hepta-2,4-dienoic acid methyl ester (7a)
and 2-hydroxy-3-methyl-6-oxo-2,4-dienoic acid methyl ester (8a). 7a
1H NMR (CDCl3, 300 MHz) 7.52 (dd, 1H, J ) 16.0, 11.5 Hz, 4-H),
6.23 (d, 1H, J ) 11.5 Hz, 3-H), 6.19 (d, 1H, J ) 16.0 Hz, 5-H), 3.84
(s, 3H, -OCH3), 2.26 (s, 3H, 6-CH3) ppm.
(b) 4-Methylcatechol. The product was identified as a mixture of
2-hydroxy-5-methyl-6-oxo-hexa-2,4-dienoic acid methyl ester (9) and
intradiol cleavage products. 1H NMR (CDCl3, 300 MHz) of 9: δH 9.46
(s, 1H, -CHO), 7.28 (d, 1H, J ) 11.7 Hz, 4-H), 6.52 (d, 1H, J ) 11.7
Hz, 3-H), 3.87 (s, 3H, -OCH3) ppm.
(c) 3-(2′,3′-Dihydroxyphenyl)propionic Acid. The product was
identified as 2-hydroxy-6-oxo-nona-2,4-diene 1,9-dioic acid 1-methyl
ester (7a),23 1H NMR (CDCl3, 300 MHz) 7.65-7.56 (dd, 1H, J ) 15.8,
11.4 Hz, 4-H), 6.29 (d,1H, J ) 11.6 Hz, 3-H), 6.27 (d, 1H, J ) 15.8
Hz, 5-H), 3.90 (s, 3H, -OCH3), 2.95 (t, 2H, J ) 6.7 Hz, 7-H), 2.67 (t,
2H, J ) 6.7 Hz, 8-H) ppm.
FeCl3. NMR spectrum of crude product showed the formation of
extradiol RFP and intradiol RFP, in a ratio of 3.2:1.
Oxygenation of Sodium Monocatecholate Catalyzed by TACN/
FeCl2/Pyridine Hydrochloride in Methanol. Monosodium catecholate
(26.4 mg, 0.2 mmol), FeCl2‚4H2O (40 mg, 0.2 mmol), TACN (25.8
mg, 0.2 mmol), pyridine hydrochloride (23.1 mg, 0.2 mmol) were
dissolved in MeOH (400 mL) in a single-necked round-bottom flask.
Dioxygen was bubbled through the reaction mixture with stirring for 4
h. After removing the solvent, 10% HCl (2 mL) was added to
decompose the iron complex, the product was extracted by diethyl ether.
The organic layer was dried (Na2SO4) and evaporated to give crude
products. NMR spectrum displayed the formation of extradiol RFP and
intradiol RFP, in a ratio of 4:1.
UV/Vis Screening of the Oxidation Reaction of Catechols by KO2.
The procedure followed was described by Muller and Lingens.14 To
KO2 (50 mg) was added dimethyl sulfoxide (50 mL) in a mortar. The
KO2 was ground carefully until a fine suspension was obtained. The
solution was filtered through a glass wool plug to obtain a clear filtrate.
In a 1 mL quartz cuvette was placed 1 mL of this solution, with an
addition of 50 µL of substrate solution (DMSO, 2 mg/mL) to begin
the reaction. The results were tabulated in Table 3.
Kinetics. The extinction coefficient of 2-hydroxymuconic semi-
aldehyde methyl ester (5) (λmax315 nm) was calculated to be 18 000
M-1 cm-1 by quantitative conversion to 2-hydroxymuconic semi-
aldehyde (λmax375 nm; ꢀRFP 48 400 M-1 cm-1) by alkaline hydrolysis,
and comparison of UV/vis spectra.24 The extinction coefficient of DHP
extradiol cleavage product 2-hydroxy-6-keto-nona-2,4-diene 1,9-dioic
acid 1-methyl ester was calculated to be 8100 M-1 cm-1 by alkaline
hydrolysis to 2-hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid (ꢀRFP
15 600 M-1 cm-1).24 Kinetic data for alternative substrates, reaction
conditions, and catechol concentrations were measured at 315 nm,
except where noted. All of the components were added to a quartz
cuvette in the order: methanol, iron chloride, TACN, catechol, pyridine
and mixed and data was collected every minute for 6 min at 23 °C.
(d) 3,5-Di-tert-butyl Catechol. The residue was separated by flash
column on silica gel. Three products were determined by comparison
1
with literature H NMR data.8 3, 5-Di-tert-butyl-2-pyrone: 1H NMR
(300 MHz, CDCl3) 7.14 (d, J ) 2.6 Hz, 1H), 7.04 (d, J ) 2.6 Hz, 1H),
1
1.32 (s, 9H), 1.2 (s, 9H) ppm. 4,6-Di-tert-butyl-2-pyrone: H NMR
(300 MHz, CDCl3) 6.01 (s, 2H), 1.26 (s, 9H), 1.19 (s, 9H) ppm.
Preparation of Monosodium Catecholate and Disodium Mono-
catecholate. Monosodium catecholate and disodium monocatecholate
were prepared by stirring catechol with 1 equiv and 2 equiv of NaOH
in MeOH under a N2 atmosphere, respectively. After 0.5 h, methanol
was removed in vacuo, and the residue was dried under high vacuum
for 2 h.
Procedure B: Oxygenation of Monosodium Catecholate Cata-
lyzed by TACN/FeCl2 in Methanol. Monosodium catecholate (26.4
mg, 0.2 mmol), FeCl2‚4H2O (40 mg, 0.2 mmol), and TACN (25.8 mg,
0.2 mmol) were dissolved in MeOH (400 mL) in a single-necked round-
bottom flask. Oxygen gas was bubbled through the reaction mixture
with stirring for 3 h. The solvent was removed in vacuo, 10% HCl (2
mL) was added to decompose the iron complex. The product was
extracted by diethyl ether. The organic layer was dried (Na2SO4) and
Acknowledgment. This work was supported by BBSRC
Grant B10351.
1
evaporated to give crude product, whose H NMR spectrum showed
JA004280U
the formation of intradiol RFP and extradiol RFP, in a ratio of 15:1.
Oxygenation of Monosodium Catecholate Catalyzed by TACN/
FeCl3 in Methanol. As procedure B, except FeCl2 was replaced by
(24) Bugg, T. D. H. Biochim. Biophys. Acta 1993, 1202, 258-264.
(25) Fujiwara, M.; Golovleva, L. A.; Saeki, Y.; Nozaki, M.; Hayaishi,
O. J. Biol. Chem. 1975, 250, 4848-4855.
(26) Murakami, S.; Okuno, T.; Matsumura, E.; Takenaka, S.; Shinke,
R.; Aoki, K. Biosci. Biotechnol. Biochem. 1999, 63, 859-865.
(23) Lam, W. W. Y.; Bugg, T. D. H. J. Chem. Soc., Chem. Commun.
1994, 1163-1164.