M. Schmittel, M. Lal, R. Lal, A. Haeuseler
Universität Siegen, Organische Chemie I,
Adolf-Reichwein-Str. 2, D-57068 Siegen,
Tel: +49 271 740 4212
e-mail: m.lal@chemie.uni-siegen.de
Universität Siegen
Design and Synthesis of Substrates to Probe Radical and Radical
Ionic Intermediates in Coenzyme B12 Dependent Enzymes Such as
Diol and Glycerol Dehydratase.
Does the ET concept work? How can we test it in solution?11
Introduction:
Folkers1,
Since the isolation of Vitamin B12 by
it has been found to serve as a
cofactor in many enzymatic reactions that include carbon skeleton
rearrangements, heteroatom eliminations and intramolecular amino group
migrations.2 Diol dehydratase is one such enzyme converting vicinal diols into
aldehydes.3,4
! The concept was tested by intermolecular trapping of enol radical cations with a
life time in the millisecond regime12 using ferrocene as the donor. The whole process
was monitored by UV-Vis spectroscopy.
OH
FC
FC
FC
-H+
OH
Fc+•
O
+H+
FC
Abs
R
OH
D ioldehydratase
R
R
R
! Does the concept work for enol radical cations
having short life times (10-3-10-6 s)?
O
OH
550
750
nm
!
In order to generate enol radical cations having life times ranging between 10-310-6 s Barton ester 1 was photolysed in presence of trifluoracetic acid (and ferrocene).
Our Approach:
! Translating short-lived radical and radical ionic intermediates into relati-
O
vely long-lived ones either via fast β-cleavage or electron transfer
process5 (ET process) directly at the active site of the enzyme.
O
I [rel]
Fc+•
O
O
O
O
N
S
1
H
enzyme
hv
Z
D/A/N
X
OH
Substrate for enzyme
X
D/A/N
X
OH
H
-HX
H
β−Cleavage
Z
OH
3440
X
photocleavable or
thermally cleavable
substrate for homogeneous probes
X
ET
OH
decarboxylation
of
Barton ester 1 during
photolysis as observed by time resolved
EPR
X
500
Wavelength [nm]
700
! LFP experiments performed on Barton ester 1 confirmed the formation of enol/enol
N = Persistent radical source
(piperidinoxyl group, thiophenyl)
ether radical cation13 with λmax around 580 nm, which was quenched by addition of
methanol. Thus even short-lived enol/enol ether radical cations can be trapped by ET
process.
OH
Advantages: Easy, efficient and rapid detection of radical and radical
How fast are the β-cleavage processess?
ionic intermediates
! Theoretical calculation were performed on model compounds to get an overview of βcleaving ability of various groups.
What factors can potentially influence the ET process at the active site of
the enzyme?
!
Hydrogen bonding at the active site of enzyme is commonly observed
and utilised by the enzymes to anchor and preorient substrates, transition
states and products during enzymatic action.
! Using cyclic voltammetry we have been able to demonstrate that hydrogen
bonding can indeed modulate reduction or oxidation potentials of precursors of
ketyl radical anion6,7,8 (~300 mV) and enol radical cations9,10 respectively(~500 mV).
H
mes
mes
OH
mes
H
N
mes
OH
δ−
O H δ+
N
mes
O H
mes
N
Ñ
mes
O H
sem
Ï
Ï
OH
N
mes
Ñ
O H
N
0,8
Epa [V]
R
HO
R
H
H
H
H
Substrate
Radical
#
Transition state (TS)
R group
Activation Energy
Exo/Endothermicity of
[Kcal/mol]
reaction [Kcal/mol]
-ONMe2
11.0
-6.8
-NO
1.2
-20.9
-SPh
0.5
-1.5
-SePh
2.8
-0.2
4.3
-4.1
-SO2Me
-SO2Ph
3.4
-5.0
Gaussian 98, UB3LYP/6-31G(d, p)
HO
+
R
Persistent
Radical
Vibrational freequency of
TS [cm-1]
505
624
191
261
283
275
mes
Acknowledgments:
! We are greatly indebted to Dr. Götz Bucher for LFP experiments, Dr. R.
Ï
mes
N
mes
H O
N
0,0
mes
HO
mes
mes
N H
I
! Easy monitoring of
trapping process using
UV-Vis spectroscopy
by photolysis of Barton
ester 1 in presence of
ferrocene and trifluoroacetic acid.
300
D
O
3540
Δ
ET
A = Acceptors (benzoquinone,
benzophenone)
A
3520
0.008
OH
D = Donors (ferrocene,
N,N-dimethylaminophenyl)
3500
!Radical formed after
D
O
3480
H [G]
A
N
3460
Δ OD
D/A/N
1,6
0,0
sem
Ñ
Ï
0,8
Epa [V]
1,6
0,0
0,8
Epa [V]
1,6
Jaquet for calculations and finally the Deutsche Forschungsgemeinschaft for
financial support in the priority programme „Radicals in enzymatic catalysis“
and Fonds der Chemischen Industrie for continued assistance.
References:
1) K. Folkers, Vitamin B12, B. Zagalak, W. Friedrich (eds), Walter de Gruyter, Berlin, 1979, 7-18. 2) J. Stubbe, W. A. van der Donk, Chem. Rev. 1998, 98, 705-762. 3)
T. Toraya, J. Mol. Catal. B 2000, 10, 87-106. 4) D. M . Smith, B. T. Golding, L. Radom, J. Am. Chem. Soc. 2001, 123, 1664-1675. 5) M. Schmittel, M. K. Ghorai,
Electron Transfer in Chemistry, V. Balzani (ed), Wiley VCH, 2001, 5-54. 6) M. Oelgemöller, A. Haeuseler, M. Schmittel, A. G. Griesbeck, J. Lex, Y. Inoue, J. Chem.
Soc. Perkin Trans. 2 2002, 676-686. 7) A. G. Griesbeck, M. Oelgemöller, J. Lex, A. Haeuseler, M. Schmittel, Eur. J. Org. Chem. 2001, 1831-1843. 8) M. Oelgemöller,
A. G. Griesbeck, J. Lex, A. Haeuseler, M. Schmittel, M. Niki, D. Hesek, Y. Inoue, Org. Lett. 2001, 3, 1593-1596. 9) M. Lal, A. Langels, H.-J. Deiseroth, J. Schlirf, M.
Schmittel, J. Phys. Org. Chem. 2003, 16, 373-379. 10) M. Schmittel, M. Lal, W. A. Schenk, M. Hagel, N. Burzlaff, A. Langels, Z. Naturforsch. B 2003, in press. 11) M.
Schmittel, M. Lal, G. Bucher, manuscript in prepartaion. 12) M. Schmittel, A. Burghart, Angew. Chem. Int. Ed. 1997, 36, 2550-2589. 13) E. Taxil, L. Bagnol, J. H.
Horner, M. Newcomb, Org. Lett. 2003, 5, 827-830.