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Biomimetic Extradiol Cleavage of Catechols Insights into the Enzyme Mechanism.

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mate ratio of 1 :6. If the electric dipole moment p for H,N . . . F,
is assumed to be about 1.6 D (that is slightly in cxcess of that of
the free NH,), the implied electric dipole moment of
[(CH,),NF]+ . . . F - is roughly 10 D.
Finally, the observed moments of inertia of the three isotopomers of the complex (Table 1) can be fitted to give the distances r ( N . . . FJ and r(Fi... F,) (i = inner; o = outer) under the
assumption that the geometry of the (CH,),N subunit is unchanged when F, approaches along the C3 axis to produce
[(CH,),NF]+...F- . The results of the least squares fit are
r(N . . . F,) = 1.29(4) A and r(Fi. . . F,) = 2.32(4) A. Although the
errors are large because these distances are highly correlated,
it is significant that r(N . . . Fi) and r(Fi. . . F,) are, respectively,
similaro to and much larger than the quantjties r(N-F) =
1.365 A in NF,[16] and r(F-F) =1.41744(6) A in F,.I'21 The
bond lengths r(N . . Fi) and r(Fi. . . F,) are evidently also consistent with the conclusion from the other evidence adduced here:
that is that F, is heterolyzed as it approaches the nitrogen atom
along the C , axis of the (CH,),N unit to form a N-F bond and
the ion pair [(CH,),NF]+ . . F-.
A possible mechanism for formation of the ion pair is shown
in Scheme 1, in which the weak, outer complex (as observed for
S. A. Cooke. G Cotti. C M. Evans, J. H. Holloway, A. C. Legon, Chwn. Pl7j.s.
L r / / . 1996. 260. 388-394
G Cotti. C. M. Evans. J H. Holloway. A. C. Legon. Clic~iii.P l i j x Let/ 1997,
264, 513-521.
S. A. Cooke. G. Cotti, J H. Holloway, A C. Legon, h g w . C h i . 1997. fO9,
81 -83; A ~ ~ IC/rw?7.
I . . In!. Ed Engl. 1997, 36,129 130.
A. C. Legon in Aroiirir. rml ,Moleculur Bmrn M ~ l h ~ d Ks ),/ , 2 (Ed.: G Scoles)
Oxford University Press. Oxford, 1992, Chapter 9, p. 289
A. C . Legon, C. A. Rego, J Clieiw Soc. Furridq, R i m 1990, 86, 1915-1921.
See. for example, L W. Buxtoii, E.J. Campbell, M. R. Keenan, T. J. Balle,
W. H. Flygare, Clien7. P l i ~ s 1981,
54, 173 -181.
D. J. Millen. Chn..I Clrerii 1985, 63, 1477-1479.
J. E Wollrab and V W Laurie, J CIiom. Ph,~..s.1969, 5 f , 1580.
H. G. M. Edwards, E. A. M. Good, D. A. Long. J Clin71.Soc. F u r u d q l7rni.s.
2 1976, 72. 984 - 987
C. A. Rego, R. A. Batten, A. C. Legon, J. Cl~eni P h w . 1988, 89, 696- 702.
A C. Legon. C. A. Rego, J. Mol. S/ruc./, 1988, 189, 137-152.
A. C. Legon. Cheii?. Sor. Reri1ws. 1993, 22, 153 163.
M. Otake, C. Matsumura. Y. Morino, J Mol. Spc( 1968, 28, 316.
E.J Campbell, L W Buxton, T. J. Balle. W H Flygare, J Cheitn. P/?y.s 1981.
Biomimetic Extradiol Cleavage of Catechols:
Insights into the Enzyme Mechanism**
Masami Ito and Lawrence Que, Jr.*
H,N . . . F212])is formed first but nucleophilic attack on F, then
occurs, accompanied by synchronous charge transfer. The
present investigation is noncommittal about the existence of the
outer complex at a minimum in the potential energy surface.
The transfcr of F + from F, to (CH,),N implies that the F f
affinity (the analogue of proton affinity) of trimethylamine is
large. We note that the mechanism in Scheme 1 is identical to
that which would be written for formation of the isoelectronic
molecule (CH,),CH,. . . F- from trimethylamine and methyl
The catechol dioxygenases are a class of nonheme iron enzymes that catalyze the oxidative cleavage of catechols as part of
nature's biodegradation pathway for aromatic rnolecules.l']
These enzymes can be divided into two subclasses : intradiolcleaving enzymes, which break the catechol C1 -C2 bond, and
extrddiol-cleaving enzymes, which break the C2-C3 bond
(Scheme I).[',
E.xperirnen tal Sect ion
Rotational spectra of the complexes [(CH,),N, F,] were observed by using a pulsednozzle, Fourier-transform microwave spectrometer [7] fitted with a fast-mixing
nozzle [8].A mixture of approximately 2 % of fluorine (99.8% pure, Distillers
M. G.) in argon was pulsed down the outer of the two concentric tubes that constitute the fast-mixing nozzle at a rate of 2 Hz from 21 stagnation pressure of 3 bar.
Trimethylamine vapor (Aldrich) was flowed continuously from the inner (glass.
0 3 mni internal diameter) tube to yield a nominal pressure of 8 x lo-' mbar in the
evacuated Fabry-Perot cavity of the spectrometer Gas pulses produced in this way
were rotationally polarized and the subsequent free induction decay at ro[ational
transition frequencies collected and processed as described elsewhere [8]. Samples of
['5N]ti-imethylamine(99 atom%) and [D,]trimcthylamine (99.9 atom%) were supplied by CK Gas Products Ltd.
Received. January 20, 1997 [Z10011 IE]
German version: A n p w Clienz. 1997, fO9, 1399 1401
Keywords: fluorine gas-phase chemistry
pairs rotational spectroscopy
. ion
[I] The dissociation energy for the process F2 = F' + F - is given by Do + IP
~ E Awhere
is the zero-point dissociation energy for Fz = 2F, and IP and
EA are the first ionization energy and electron affinity of F, respectively. The
values D,,=154.4(4)kJmol-', IP =1513.5(6)kJinol-' and EA = 297(10) kJ
mol I are given in: Gini4in Nundl~~iclz
dei- Anorgunischm Clzeinie, 8th ed..
Fluorine, Supplcn7ai?i fi~lunie2. Springer. Berlin. 1980, pp. 51-69
[2] H. 1 Bloemink. K . Hinds. J H Holloway, A C. Legon, Clrem. Pl7.w Lrrr
1995, 245. 598-604.
[ 3 ] S. A Cooke. G CotIi. C. M Evans. J. H. Holloway, A. C. Legon. Clzein Pkys.
L e / / . 1996. 262. 308-314.
Scheme 1 The two modes of oxidative cleavage of catechols in nature.
The intradiol-cleaving enzymes require Fe"' and are the inorc
extensively characterized. Thc persistencc of the Fe"' oxidation
state in the reaction cycle has led to a proposed mechanism in
which the substrate cdtechol is activated by coordination to the
Fe"' center for direct electrophilic attack by 0, .I4, 51 In contrast,
the extradiol-cleaving enzymes, which typically require Fe",
are proposed to utilize an oxygen activation mechanism,I2.61
though this mechanism is not as well understood. Interestingly,
extradiol-cleavage can also be elicited by catechol 1,2-dioxygenase with some substrate analogues,[7,81 which demonstrates
that even an Fe"' enzyme can carry out this chemistry.
[*] Prof. Dr. L Que, Jr., Dr. M. Ito
Department of Chemistry and Center for Metals in Biocatalysis
University of Minnesota
207 Pleasant St. SE, Minneapolis, MN 55455 (USA)
Fax: Int. code +(612)624-7029
e-mail- que@chem.unin edu
[**I This work was supported by a grant froin the National Institutes of Health
(GM-33162, L. Q.) and a fellowship from the Japan Society for the Promotion
of Science (M. I.).
Biomimetic [Fe"'(L)dbc] complexes (L = tetradentate tripodal ligand ; dbc = 3,5-di-tert-butylcatecholate dianion) react
with 0, to afford excellent yields of intradiol cleavage
product.[5.91 These complexes have played an important role in
supporting and refining the novel substrate activation mechanism for intradiol cleavage. We have thus embarked on an
analogous effort to elicit extradiol cleavage from a metal catecholate. Thus far, there are only two reported examples of extradiol cleavage by synthetic iron complexes. Funabiki et al. found
that FeCl, in THF/H,O cleaves dbcH, to extradiol cleavage
products in 40% yield,"'] but the relevant reactive species was
not identified. On the other hand, Dei et al. found that the
complex [Fe"'(tacn)Cl(dbc)] (1, tacn = 1,4,7-triazacyclononane)
afforded extradiol cleavage product upon exposure to 0, in
35% yield.["] Neither of these studies featured a systematic
investigation that shed light on the extradiol cleavage mechanism. We have thus followed up on the study by Dei et al. and
report in this paper conditions under which extradiol cleavage
of the coordinated catecholate in 1 can be achieved quantitatively. Our observations provide the basis for a proposed mechanism for extradiol cleavage and the principles underlying the
regioselectivity of catechol cleavage.
[Fe"'(tacn)Cl(dbc)] (1, Scheme 2) is a six-coordinate complex
with a structure akin to that recently reported for [Fe"'(tacn)-
0 2
Scheme 2. The reaction of 1 with 0, gives 2-4.
N,(Cl,cat)] (Cl,catH,, tetrachlorocatechol) .[12] When exposed
to 0, in CH,CN, 1 afforded three products: 3,5-di-tertbutylquinone (2) in 50% yield, and the two isomeric di-tertbutyl-2-pyrones (3 and 4) in 29 YOcombined yield (Table 1). The
pyrones 3 and 4 derive from the extradiol cleavage of dbc with
insertion of one oxygen atom into either the CI-C6 or the
C2-C3 bond followed by loss of CO.l'o. 1 3 , 1 4 1 In pyridine solvent the complex afforded a comparable yield of extradiol cleavage products, though the isomer ratio differs. In both cases
3,5-di-t~rt-butylmuconicacid anhydride, the intradiol cleavage
product, was observed in only trace amounts, confirming the
proclivity of this complex towards extradiol cleavage.
Complex 1 differs from the [Fe(L)dbc] complexes that afford
intradiol cleavage products in having a tridentate tacn ligand
(instead of a tetradentate L) plus an additional chloride. Dei et
al. have suggested that the presence of the labile chloride in 1
may be the essential factor for achieving extradiol cleavage." '1
We have thus investigated the reactivity properties of 1 in the
presence of silver salts to remove the chloride (Table 1). No
extradiol cleavage products were formed upon addition of an
equivalent of AgOAc; instead about a 40% yield of quinone 2
was obtained in both CH,CN and pyridine. However the use of
AgBF, significantly favored the oxidative cleavage reaction;
indeed, the conversion was 90 YOin pyridine. The contrasting
effects of AgOAc and AgBF, on the oxidation of the coordinatAngeM-. Chwm 1111 E d Enyl. 1997. 36. N o . I2
Table 1 Product distributions for the reaction of complex 1 with 0, [a]
1 equiv AgOAc
1 equiv AgBF,
1 equiv AgOAc
1 equiv AgBF4
1 equiv AgOAc
1 equiv AgBF4
1 equiv AgBF,
20 equiv pyridine
1 equiv AgBF,
20 equiv 2-methylpyridine
1 equiv AgBF,
20 equiv 4-methylpyridine
1 equiv AgBF,
20 equiv N-methylimidazole
1 equiv AgBF,
20 equiv 2.6-dimethylpyridine
[a] Reaction time f = 12 h. ca. 25 ' C . In all cases, only trace amounts ( < 1 % yield)
of the intradiol cleavage product, 3,5-di-terf-butylmuconic anhydride, could be
detected. Numbers reported are the average of three runs. For the redctions wlth low
product yields, the balance is unchanged starting complex.
ed catecholate suggest that the availability of a coordination site
on the metal center plays a key role in the extradiol cleavage
mechanism. Extradiol cleavage is inhibited in the presence of a
coordinating anion such as acetate but favored in the presence
of a noncoordinating anion such as BF,.
The reaction of 1 with 0, was also investigated in CH,Cl, to
determine whether the coordinating solvents used thus far may
play a role in extradiol cleavage. The major product observed in
the presence of AgBF, was the quinone 2 (82 % yield, Table 1).
In the presence of AgBF, and 20equiv pyridine, only trace
amounts of quinone 2 were found, and the major products were
the two isomeric pyrones 3 and 4 (78% yield). Under these
conditions, " 0 labeling studies showed that the extradiol cleavage products formed had incorporated one atom of " 0 . The
prominent ions at m/z 208 [M'], 193 [ ( M - CH,)+], and 180
[ ( M - CO)'] in the mass spectra of the two pyrone isomers
shifted by two mass units in the corresponding '80-labeled
products. Comparison of their intensities with the residual l6O
peaks indicated that the pyrone ring oxygen atoms of the products were approximately 90% labeled.
Interestingly, the use of stronger Lewis bases such as 2methylpyridine, 4-methylpyridine, or N-methylimidazole afforded even higher conversions; indeed the latter two gave nearly quantitative (98 'YO) extradiol cleavage of the bound
catecholate, which suggests that coordination of this base to the
metal center is an important feature of the mechanism. In accord with this observation, use of a sterically hindered pyridine
such as 2,6-dimethylpyridine afforded a low yield of extradiol
cleavage products. Thus, extradiol cleavage is elicited in the
absence of chloride and in the presence of an additional coordinating aromatic nitrogen ligand.
The nearly quantitative extradiol cleavage elicited from the
dbc complex 1 with tridentate tacn stands in striking contrast to
the quantitative intradiol cleavage observed for the complexes
with tetradentate ligand~.~'.
91 This difference suggests that the
key to the extradiol cleavage mechanism lies in the availability
of a coordination site. While intradiol cleavage must involve the
direct attack of an electrophilic 0, molecule on the enediolate
moiety of the coordinated c a t e c h ~ l a t e ,51~in
~ ,extradiol cleavage
VCH Ver/
mhH. D-6945i Weinberm. 1997
R i7 S O +
the initial C - 0 bond-forming step must occur at the carbon
adjacent to the enediolate moiety. This distinct regiochemistry
can only be achieved by a different mode of attack, which we
propose to be that of a nucleophilic superoxide on an electrophilic site on the aromatic ring (Scheme 3). The superoxide
would be generated from the binding of 0, to the five-coordinate iron center.
tallography,['6- 1 8 ] show that the enzyme-substrate complex
has a five-coordinate iron active site with a bidentate catecholate. This result supports the conclusions from our
biomimetic studies.
Experimental Section
1 was synthesized as previously described [ll]. The oxygenation reactions were
carried out with 50 mg (0.l mmol) of 1 in 10 mL solvent under 0, over 12 h. The
products were then extracted in ether and subjected to GC analysis (Hewlett Packard 5890 Series I1 gas chromatograph fitted with a J & W Scientific DB-1 column;
injection temperature 250'C; initial column temperature 200 "C for 1 min, then
increasing at a rate of 2'Cmin-' to 260"C, which was maintained for 10min);
retention times: 7.5 min for 3, 8.4 min for 4, 9.7 min for 3,5-di-tert-butylmuconic
acid anhydride, and 10.8 min for 2. The 3.5-isomer 4 was identified by independent
synthesis [13]; the 4.6-isomer 3 exhibited similar NMR and mass spectra but could
be distinguished from the 3,5-isomer by comparison of their GC retention times.
Mass spectral analysis was carried out on a Hewlett-Packard Model 5989B mass
Received: January 21, 1997 [Z10015IE]
German version: Angew. Chem. 1997, 109, 1401-1403
Keywords: bioinorganic chemistry . biomimetic synthesis
dioxygenases . extradiol cleavage . iron
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Dekker, New York, 1984.
J. D. Lipscomb, A. M Orville, Meral Ions Biol S y t . 1992, 28, 243-298.
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York, 1989, pp. 467- 524.
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1977.485. 60-74.
a) H. G. Jang, D. D. Cox. L. Que, Jr., J. Am. Chem. Soc. 1991,113,9200-9204;
b) D . D. Cox. L. Que, Jr., ;hid. 1988, 110, 8085-8092.
L. Shu, Y-M. Chiou, A. M. Orville, M. A. Miller, J. D. Lipscomb, L. Que, Jr.,
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M. Fujiwara, L. A. Golovleva, Y. Saeki, M. Nozaki, 0. Hayaishi, J Biol.
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M. Matsumoto, K. Kuroda, J. Am. Chem. SOC.1982, 104, 1433-1434.
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Scheme 3. Proposed mechanism for the reaction of 1 with 0,. The first step, the
freeing of a coordination site on cleavage of the chloride ligand from 1, is not shown.
Such a mechanistic scheme raises an important question: how
does 0, bind to an Fe"' center? We propose that the Fell'-catecholate complex is in resonance with its Fell-semiquinone form,
as proposed for the intradiol cleavage mechanism.151The loss of
the halide ligand from 1 enhances the covalency of the Fe"'catecholate interaction, which increases the Fe" character of the
metal center. The availability of an iron coordination site allows
0, to bind to the iron center, thereby forming an Fell'-superoxide complex. In the absence of the added pyridine, electron
transfer occurs between the semiquinone and the superoxide
through the iron center to produce quinone, as observed in
CH,CI,. When present, the pyridine binds to the metal-0,
adduct by displacing the more weakly bound carbonyl oxygen
of the semiquinone. The conversion of the semiquinone into a
monodentate ligand brings the bound superoxide in close proximity to a ring carbon adjacent to the enediol unit. The nucleophilic superoxide can then attack the ring at this carbon to
effect a Michael addition on the dienone function; subsequent
decomposition of the resultant peroxide affords the observed
pyrone products. The regiospecificity of oxidative cleavage is
thus determined by the nature of the dioxygen species that attacks the bound catechol: nucleophilic attack by superoxide for
extradiol cleavage versus electrophilic attack by 0, for intradiol
The emerging detaiis of the a c t i v e site of extradioi-cleaving
dioxygenases derived from spectroscopy
' 5 1 and
X-ray crys-
Veriugsgeseiischuff mhH. 0-69451 Wiinheim. 1997
0570-0833/Y7/3612-1344 X 17.5Oi SO10
Angen. Chem. Int. Ed. Engl. 1997, 36, No. 12
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catechol, cleavage, enzymes, mechanism, insights, extradiol, biomimetic
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