New synthetic models of cytochrome P450: How different are they from the natural species?

Article abstract of DOI:10.1055/s-2005-863724

Soluble and matrix-bound P450 enzyme models have been synthesized carrying a SO₃^- ligand coordinating to iron. These complexes display features very similar to cofactors of enzymes such as P450_cam with respect to electrochemistry and UV/Vis spectroscopy. Further they catalyze epoxidation reactions with turnover numbers up to 1800. DFT calculations revealed that the coordination of SO₃^- to Fe(III) produces an active species that displays allylic hydroxylation and epoxidation reactivity patterns that are nearly indistinguishable from those calculated for the natural active species of the enzyme cytochrome P450.

Full text of DOI:10.1055/s-2005-863724

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675
New Synthetic Models of Cytochrome P450: How Different Are They from the
Natural Species?
P
450 Enzyme
e
M
odels bastian Kozuch,a Tycho Leifels,b Dominik Meyer,b Laura Sbaragli,b Sason Shaik,*a Wolf-D. Woggon*b
a
Department of Organic Chemistry and The Lise Meitner-Minerva Center for Computational Quantum Chemistry, Givat Ram Campus,
Jerusalem 91904, Israel
b
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland
Fax +41(61)2671102; E-mail: wolf-d.woggon@unibas.ch
Received 3 December 2004
man liver P450s exhibit a rather broad substrate tolerance
hydroxylating drugs and other xenobiotics and rendering
them into easily excretable compounds.
Abstract: Soluble and matrix-bound P450 enzyme models have
been synthesized carrying a SO3– ligand coordinating to iron. These
complexes display features very similar to cofactors of enzymes
such as P450cam with respect to electrochemistry and UV/Vis spec-
troscopy. Further they catalyze epoxidation reactions with turnover
numbers up to 1800. DFT calculations revealed that the coordina-
tion of SO3– to Fe(III) produces an active species that displays allyl-
ic hydroxylation and epoxidation reactivity patterns that are nearly
indistinguishable from those calculated for the natural active spe-
cies of the enzyme cytochrome P450.
Ever since these enzymes were first identified by the shift
of the Soret band from ca. 410 nm [Fe(III)·S– resting state]
to ca. 450 nm [OC·Fe(II)·S–]2 the significance of the
unique thiolate ligand has been debated. In this context the
investigation of synthetic active site analogues provided
interesting clues.3 The bridged Fe(III)·S– porphyrins 1–3
(Figure 1)4 are resting state models not only with respect
to spectroscopy but also concerning reactivity, since for 1
it was possible indeed to demonstrate the intramolecular
hydroxylation of a non-activated position in the alkane
bridge spanning the distal side of the porphyrin.5
Key words: catalysis, enzyme models, DFT calculations, heme-
thiolate proteins, iron porphyrins
Cytochromes P450 are naturally abundant heme-thiolate
proteins that catalyze a broad spectrum of stereoselective
oxidation reactions through reductive cleavage of molec-
ular oxygen bound to iron and ‘insertion’ of a single oxy-
gen atom into activated or non-activated positions of the
substrate.1 Accordingly, these enzymes are the prototype
of a monooxygenase system. The significance of these
proteins derives from their participation in the metabolism
of endogenous compounds and xenobiotics, e.g. in human
glands highly substrate specific P450s catalyze the forma-
tion of steroid hormones from cholesterol, whereas in hu-
Interestingly, however, the redoxpotentials of 1–36 were
shown to be far more negative than the resting state of
P450cam (Eo = –290 mV)7 a cytosolic crystallizable pro-
tein from Pseudomonas putida. From these results it was
concluded that the charge at the thiolate ligand of P450cam
is somewhat ‘shielded’. In fact this was shown to be true
8
using a refined X-ray structure of P450cam revealing H-
bonding of several amino acids of the protein backbone to
the thiolate (Figure 2).
H3COOC
O
O
O
N
H
COOCH3
H
O
O
O
N
N
N
N
3+
3+
O
O
3+
N
N
N
Fe
Fe
Fe
N
N
N
O
O
N
O
O
N
N
–
–
S
S
S–
O
O
O
Eo = - 704 mV
Eo = - 714 mV
3
Eo = - 607 mV
1
2
Figure 1 Enzyme models of the resting state of cytochrome P450.
SYNLETT 2005, No. 4, pp 0675–0684
0
4.
0
3.
2
0
0
5
Advanced online publication: 22.02.2005
DOI: 10.1055/s-2005-863724; Art ID: Y04604ST
© Georg Thieme Verlag Stuttgart · New York

676
S. Kozuch et al.
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Subsequently, model compounds 4 and 5 were reported ready for iron insertion. Treatment of 13 with FeBr2
mimicking this situation, confirming the idea that indeed yielded 14, which on exposure to oxygen furnished 6 car-
–
the redoxpotential of P450cam is largely dependent on the rying a SO3 ligand coordinating to iron. According to
charge density at the thiolate coordinating to iron. Due to EPR spectroscopy enzyme models 6 and 14 are both high-
the non-covalent attachment of the fifth ligand complexes spin complexes with g-values of 5.76 and 7.45, 4.50, 3.40
4 and 5 are very elegant models for analytical purposes respectively. They display distinct UV/Vis spectra with
but are not suitable for catalytic reactions.9
lmax = 422 nm (14) and lmax = 414 nm (6).
Cyclic voltammetry of complexes 14 and 6 revealed re-
versible redox behavior attributed to Fe(III)/Fe(II), see
Figure 3, from which characteristic Eo were calculated. In
comparison to complexes 1–3 it is obvious that the addi-
tion of electron-withdrawing groups in the meso-phenyl
rings causes an anodic shift of Eo = 140–250 mV. The
increment resulting from an exchange of thiolate coordi-
–
nation (14) for the SO3 ligand (6) is +280 mV.
Figure 2 H-bonding of the thiolate ligand of P450cam and corre-
sponding model compounds 4 and 5, which mimic the proximal coor-
dination site.
To find more suitable models, we sought for an alternative
species that would be synthetically convenient and theo-
retically appropriate, i.e. to replace the thiolate ligand for
–
SO3 . In this case the charge donating character of the
ligand should be reduced to an extent similar to -H·S–·H-
and one could predict a considerable anodic shift of Eo and
further an increase in stability of the model complex in the
presence of strong co-oxidants. In this communication we
wish to report the preparation of suitable compounds com-
paring characteristic features of these new complexes
with earlier Fe(III)·S– enzyme models and present DFT
calculations that at least in part support the synthetic con-
cept.
Figure 3 Cyclic voltammograms of 6 (trace above) and 14 (trace
below); glassy carbon/Pt vs. SCE, 0.1 M LiClO4 in LiBr sat. DMF.
Accordingly, complex 6 exhibits a redoxpotential identi-
–
cal to Eo of the E.S complex of P450cam. Further the SO3
ligand (6) has a considerable practical advantage over
thiolate coordination (14) concerning catalytic reactions.
In this context it is important to note that despite consid-
erable steric congestion at the thiolate ligand in complexes
1–3 and 14 oxidants used for the ‘shunt pathway’ such as
PhIO, PhF5IO, and H2O2 and in particular O2 oxidize the
Synthesis of Iron Porphyrins
It is known that electron-withdrawing groups in the pe-
riphery of the porphyrin shift the redox potential of the
central metal towards more positive values. Thus, we de-
cided to attach Cl to phenyl groups linked to the meso-po-
sition of the macrocycle and prepared target compound 6
according to Scheme 1. The starting material dipyrro-
methane 7 and the protected aldehyde 8 were prepared
following standard procedures.4 The resulting porphyrin 9
was deprotected and the free phenol 10 obtained as a mix-
ture of atropisomers a,a-10 and a,b-10 that are intercon-
vertible at room temperature. The mixture of atropisomers
10 was then condensed under diluted conditions with the
dimesylate 11, which has been prepared according to our
own protocol.4 The product 12 was treated with strong
base to give the mono-bridged free-base porphyrin 13
–
–
thiolate slowly via -SO–, -SO2 to SO3 creating an un-
desired mixture of oxidants during reactions. For 6 having
a stable coordination site it is even possible to work out-
side the glove box.
To compare homogenous reactions of catalyst 6 with
heterogeneous conditions we prepared the polymer bound
porphyrin 15 according to Scheme 2. The aldehyde 16
was condensed with the dipyrromethane 7 to yield the
prophyrin 17 as mixture of a,a-and a,b-atropisomers.
BBr3 treatment liberated the phenolic OH groups ready
for macrocyclization with the dimesylate 11. The mono-
bridged porphyrin 19 was deprotected at sulfur and at the
peripheric esters to furnish the free base porphyrin diacid
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P450 Enzyme Models
677
Cl
Cl
Cl
RO
Cl
OHC
N
H
N
H
N
Cl
H N
O
N
H
Cl
CHO
TFA
H
N
Cl
N
N
H
N
NH
N
+
Cl
RO
NH
O
Cl
7
Cl
OR
8
OR
Cl
Cl
α,β-9, R: CH2Ph
α,β-10, R: H
BBr3
α,α-9, R: CH2Ph
α,α-10, R: H
BBr3
O
α,α-/αβ-10
S
N
Cs2CO3
MsO
OMs
Cl
Cl
11
Cl
Cl
N
N
3+
H
N
N
Fe
N
N
FeBr2
O
O
N
NH
Cl
Cl
S-
SR
O
O
Cl
Cl
Cl
14
12, R: CO-N(CH3)2
13, R: H
KOMe
Cl
O2
N
3+
N
Fe
N
O
N
Cl
-
SO3
O
Cl
6
Scheme 1 Synthesis of the P450 enzyme mimic 6 carrying a SO3– ligand coordinating to iron.
20. Iron insertion gave 21 and subsequent oxidation achieved with 6 and 15 exceed those obtained with model
–
yielded the desired iron(III) complex carrying a SO3
compounds 1–3, 14 by a factor of ca. 50. Hence, these
ligand. Activation of the acid and coupling to the amino experiments demonstrate that iron porphyrins carrying a
–
groups of RAPP Tentagel finally gave the immobilized SO3 ligand are valuable P450 mimics with respect to
catalyst 15. The epitopic density of 15 was determined by electrochemistry and reactivity.
UV (420 nm) 20 mmol porphyrin/g Tentagel which was
confirmed by a Ninhydrin test after acetylation of the
residual amino groups at the polymer and subsequent
Results of Theoretical Modeling
derivatization of the free COOH groups at 15.
The iron-oxo porphyrin species 6Ph and 6Me, which serve
as models for 6, are shown in Figure 4 in two different co-
ordination modes, one with an Fe-O bond between the
heme and the RSO3– ligand [6Ph (A) and 6Me(A)], and the
other with an Fe-S bond [6Ph(B) and 6Me(B)]. It is apparent
Catalytic reactions were pursued under standardized con-
ditions with substrates such as 22–25 using PhIO as the
‘O’-source. Some results are shown in Scheme 3 demon-
strating the capability of 6 and 15 to catalyze epoxidations
even in a regioselective fashion. In general turnovers
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678
S. Kozuch et al.
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Cl
O
CHO
O
O
Cl
Cl
TFA
O
N
H
O
N
7
+
O
O
N
O
NH
Cl
O
Cl
BBr3
N
H
N
O
O
N
HO
NH
16
Cl
O
Cl
α,α- and α,β-17
O
OH
Cl
O
α,α- and α,β-18
S
N
Cl
Cl
O
MsO
OMs
COOH
Cl
O
Cl
N
N
H
N
N
H
N
N
O
11
NH
S
Cl
O
O
NH
Cl
KOMe
Cl
O
19
HOOC
SH
O
O
N
O
Cl
Cl
19
COOH
Cl
N
3+
20
N
Fe
N
O
N
Cl
FeBr2
S-
HOOC
O
Cl
21
RAPP
Tentagel
Cl
RAPP
Tentagel
2.
1. O2
O
Cl
N
DCC
N
H
N
Fe
N
H2N
N
O
Cl
-
HOOC
SO3
O
Cl
15
Scheme 2 Synthesis of the polymer-bound P450 mimic.
that the second option (mode B) is highly unfavorable, the have two low lying states, a high-spin (HS) quartet and a
Fe-S bond being too long and the species laying 19–31 low-spin (LS) doublet. The energy differences are rather
kcal/mol higher than the one with the Fe-O bond (mode small, 0.16–0.31 kcal/mol, and the closely lying HS-LS
A). In addition, frequency calculations show that 6Ph(B) pair of states is much like the situation in the analogous
and 6Me(B) are not really stable minima but are saddle species in the P450 enzyme,10 known also as Compound I
points for the exchange of the three different Fe-O bonds. (Cpd I). Thus, the synthetic model resembles in this sense
Thus the synthetic model involves an iron coordinated to the natural species, and moreover, 6Me(A) is a faithful
an oxygen, compared with the natural enzyme, which model of 6Ph(A), and hence can replace the latter in the
possesses an Fe-S bond. The species 6Ph(A) and 6Me(A) subsequent reactivity calculations.
Synlett 2005, No. 4, 675–684 © Thieme Stuttgart · New York

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P450 Enzyme Models
679
Reaction
O
6
Catalyst
15
Figure 5 shows the spin-density distribution of the HS
states of 6Ph(A) and 6Me(A). It is apparent that the species
are both tri-radicaloids, with three unpaired electrons
residing in p*-orbitals and in a porphyrin centered orbital
called a2u (the a2u orbital, as shown in the orbital diagram,
depicted in the figure alongside the spin density diagrams.
The LS states show analogous distribution, with the
exception that the spin state of the a2u electron is now cou-
pled antiferromagnetically to the triplet pair in the p*xz-
and p*yz-orbitals. In terms of electronic structure there is
difference between the model species and the species of
P450, where the a2u electron is highly delocalized over the
sulfur in a manner that is very sensitive to the polarity and
hydrogen bonding capability of the environment.11
0.06 mol%,
PhIO
turnover: 1810
0.01 mol%,
PhIO
turnover: 574
22
O
0.06 mol%,
PhIO
turnover: 345
0.1 mol%;
PhIO
turnover: 930
23
24
0.1 mol%,
PhIO
turnover: 134
O
COOCH3
COOCH3
0.1 mol%,
PhIO
turnover: 189
O
25
Scheme 3 Catalytic epoxidations with P450 mimics 6 and 15.
Figure 5 Spin density distribution in the HS states of the stable
complexes 6Ph(A) and 6Me(A) and in the corresponding P450 model
6. Alongside the structures we show a schematic orbital diagram of
the key orbitals.
In summary, we may safely conclude that the synthetic
reagent contains in fact an Fe-O bond, unlike the natural
system that contains an Fe-S bond. Furthermore, the mod-
el system has a pure porphyrin radical-cationic situation,
as opposed to the natural system, which is a mixed sulfur-
porphyrin radical species. On the other hand, much as the
iron-oxo species of the natural system, here too, the active
species of the synthetic model is a two-state oxidant and
will be expected to exhibit two-state reactivity (TSR).12
With the above differences and similarities between the
two iron-oxo species of the synthetic model, it would be
Figure 4 Structures of the iron-oxo porphyrin complexes [optimi-
zed with LACVP+*(Fe)6-31+G*(C,H,N,O)], 6Ph and 6Me, of heme
with RSO3– (R = Ph, Me) in the coordination modes A and B. Energy
data underneath the structures show the relative energies of the low-
spin state complexes in kcal/mol. Shown is the corresponding P450 interesting to compare the reactivity of the synthetic
species, too.10 Note that each complex possesses high-spin (HS) and
model 6Me(A) with that of the P450 Cpd I model.
varieties. Data obtained in LACVP*+ basis set.
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Reactivity Patterns of the Active Species
The reaction of Cpd I with propene has been studied
extensively in the past,10 and this will allow us to compare
the reactivity patterns of a P450 species with those of
6Me(A). The reaction profile for allylic hydroxylation of
propene by 6Me(A) depicted in Figure 6 (a) shows the
commonly encountered bi-phasic pathway, with a bond
activation phase followed by radical rebound.13 The dou-
bling of the profile corresponds to TSR that arises from
the HS and LS states of the iron-oxo active species.13 As
before, here too, the HS mechanism is truly stepwise with
a significant barrier for rebound, while the LS profile is
effectively concerted with a negligible barrier (0.1 kcal/
mol) for rebound. Figure 6 (b) shows the corresponding
TSR profiles for epoxidation, and here both HS and LS
pathways are effectively concerted; no intermediates
could be detected. The experimental results, indeed, show
only stereospecific epoxidations, see Scheme 3.
To project the similarities and differences between the re-
activity of the synthetic model and the natural P450 spe-
cies, Figure 7 shows a superimposition of the two profiles,
using color-coding. There are some minor differences be-
tween the two profiles; the most notable difference is the
rebound step, which exhibits higher rebound barriers for
the native P450; this feature has been addressed in a recent
review article, where a valence bond model for the re-
bound was used to rationalize trends in the rebound barri-
ers.13 This means in turn that the synthetic model should
be a slightly more stereoselective reagent compared to the
natural species. In addition, the synthetic model shows a
small preference for C-H hydroxylation over C=C epoxi-
dation, while the natural species has an opposite slight
preference. Other than these differences, the reaction pro-
files show almost a perfect superimposition that discloses
the fundamental similarity between the two reagents.
Figure 6 High-spin (HS) and low-spin (LS) energy profiles for the
reaction of 6Me(A) with propene, (a) C-H hydroxylation, and (b) C=C
epoxidation. All energies are given in kcal/mol relative to the LS
reactants. The energy data in parentheses include zero point energy
correction. All calculations were done with the LACVP basis set.
Figure 7 Color-coded and superimposed high-spin (HS) and low-spin (LS) energy profiles for the reaction of 6Me(A) (red) and of Cpd I
(black) with propene. The reactants are placed in the mid-diagram. The energies (in kcal/mol) are given relative to the separated reactants.
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P450 Enzyme Models
681
13C NMR (75 MHz, CDCl3): d = 137.1 (2 C), 135.9, 129.4 (2 C),
In summary, comparison of experimental data to theoret-
ical calculations shows that the action of P450 enzymes
can be mimicked by synthetic iron porphyrins designed to
control the redox potential and reactivity by electron with-
drawing subunits at the porphyrin’s periphery and through
128.7 (2 C), 117.0 (2 C), 108.7 (3 C), 107.4 (2 C), 40.1.
5,15-Bis-(2,6-dichlorophenyl)-10,20-bis(2-benzyloxy)porphy-
rin (9)
To a solution of dipyrromethane 7 (5.306 g, 18.22 mmol) and ben-
zaldehyde 8 (2.579 g, 12.15 mmol) in 150 mL CH2Cl2, 0.1 mL TFA
were added and the solution was stirred at r.t. for 16 h. Subsequent-
ly, 3,4,5,6-tetrachloro-1,2-benzoquinone (4 g) were added and the
mixture was heated under reflux for 1 h. The solvent was evaporated
and the residue chromatographed on silica (CH2Cl2) to obtain 1.340
g (1.388 mmol, 22.8%) 9 as a red microcrystalline powder; mp
>250 °C.
–
changing the thiolate coordination for a SO3 ligand to
iron.
Theoretical Methods
The calculations were performed with the hybrid functional,
B3LYP[Ref], using the basis set combination LACVP*+(Fe)6-
31G*+(H,C,N,O) and LACVP(Fe)6-31G(H,C,N,O), both of them
giving virtually the same geometric and electronic characteristics,
as reported in previous calculations of the Jerusalem group.13 Ini-
tially, we calculated the iron-oxo porphyrin species 6Ph including
the phenyl group. Subsequently, we tested a smaller model system,
with methyl replacing phenyl, and labeled as 6Me. A few different
states were calculated to ascertain the nature of the ground state for
both species. The paper describes only the ground states, while the
rest of the data are relegated to the supplementary material. Since
6Ph and 6Me were found to be very similar, the reaction profiles were
studied with 6Me, using propene as a substrate that can undergo both
C-H hydroxylation and C=C epoxidation.13 The results with the
iron-oxo porphyrin species of P450 were taken from a previous
computational work.10 All critical species (intermediates, transition
states) were ascertained by frequency calculation. The programs
JAGUAR 4.214 and GAUSSIAN 9815 were used throughout; the
former is faster in geometry optimization, the latter is more efficient
and accurate in frequency calculations.
Rf = 0.23 (silica, CH2Cl2–hexane, 1:1).
1H NMR (300 MHz, CDCl3): d = 8.84 (d, J = 4.8 Hz, 4 H, pyrrole),
8.65 (d, J = 4.8 Hz, 4 H, pyrrole), 8.09–8.05 (m, 2 H, H-4¢), 7.83–
7.79 (m, 4 H, H-3¢,5¢), 7.78–7.34 (m, 12 H, H-3¢,5¢,3¢¢,4¢¢,5¢¢,6¢¢),
6.88–6.60 (m, 10 H, H-2¢¢¢,3¢¢¢,4¢¢¢,5¢¢¢,6¢¢¢), 5.00 (s, 4 H, -O-CH2-).
MS (LDI-TOF): m/z = 964.6.
UV/Vis (CH2Cl2): lmax (%) = 418 (100), 514 (8), 586 (5) nm.
The purification of 9 can be improved by preparing the correspond-
ing Zn complex Zn-9 followed by purification and final Zn remov-
al. For this purpose porphyrin 9 (1.340 g, 1.388 mmol) was
dissolved in 100 mL CH2Cl2 and after addition of 3 g
Zn(OAc)2·6H2O in 50 mL MeOH the mixture was heated under re-
flux for 2 h. The organic phase was washed with H2O, dried over
Na2SO4 and evaporated yielding a residue which was chromato-
graphed on silica (CH2Cl2–hexane, 1:1); 973.4 mg (946.0 mmol,
68%) Zn-9 were obtained as a red solid; mp >250 °C.
Rf = 0.30 (silica, CH2Cl2–hexane, 1:1).
1H NMR (300 MHz, CDCl3): d = 9.0 (d, J = 4.8 Hz, 4 H, pyrrole),
8.8 (d, J = 4.8 Hz, 4 H, Pyrrole), 8.2 (m, 2 H, H-4¢), 7.9–6.9 (m, 12
H, H-3¢,5¢,3¢¢,4¢¢,5¢¢,6¢¢), 6.8–6.4 (m, 10 H, H-2¢¢¢,3¢¢¢,4¢¢¢,5¢¢¢,6¢¢¢),
5.3 (s, 4 H, -O-CH2-).
2-Benzyloxy-benzaldehyde (8)
Salicylaldehyde (11.00 g, 90.0 mmol), (18.80 g, 103 mmol) potas-
sium carbonate and (17.04 g, 99.6 mmol) benzyl bromide were dis-
solved in 400 mL MeOH and heated under reflux for 4 h. The
residue was filtered off, the filtrate taken up in CH2Cl2 and washed
with 1 N HCl. The water phase was extracted three times with
CH2Cl2, the combined organic phases were partially reduced at the
rotavapor and washed with brine, dried over Na2SO4 and evaporat-
ed. The residue was chromatographed on silica (CH2Cl2–hexane,
1:1) and recrystallized in MeOH–hexane to obtain 9.93 g (46.8
mmol, 52%) of 8 as colorless needles; mp 46–47 °C. Rf = 0.22
(silica, CH2Cl2–hexane, 1:1).
1H NMR (300 MHz, CDCl3): d = 10.55 (s, 1 H, CHO), 7.86–6.99
(m, 9 H, aryl), 5.17 (s, 2 H, benzyl).
13C NMR (75 MHz, CDCl3): d = 189.8, 160.5, 136.2, 136.0, 128.5-
127.4 (6 C), 125.3, 121.1, 113.1, 70.5.
UV/Vis (CH2Cl2): lmax (%) = 422 (100), 400 (14), 548 (8) nm.
For Zn removal 973.4 mg (946 µmol) Zn-9 were dissolved in 100
mL CH2Cl2 and after addition of 100 mL concd HCl the mixture
was stirred for 30 min. The organic phase was neutralized with sat.
NaHCO3 solution, dried over Na2SO4, evaporated and the residue
chromatographed on silica (CH2Cl2–hexane, 1:1); 780.6 mg (809
µmol, 86%) of the pure free base porphyrin 9 were isolated as a red
solid.
5,15-Bis(2,6-dichlorophenyl)-10,20-bis(2-hydroxyphenyl)-
porphyrin (aa and ab 10)
A solution of 780.6 mg (809.1 mmol) porphyrin 9 in 100 mL CHCl3
was cooled to –78 °C, and then 1.5 mL BBr3 were added and the so-
lution stirred for 30 min. The reaction was quenched with sat.
NaHCO3 solution and extracted twice with CH2Cl2. the organic
phase was dried over Na2SO4, evaporated and the residue chromato-
graphed on silica (CH2Cl2) to obtain 268.0 mg (aa) and 338.2 mg
(ab) of 10; mp >250 °C.
UV/Vis (CH2Cl2): lmax (%) = 252 (100), 318 (57) nm.
(2,6-Dichlorophenyl)-bis(2-pyrryl)methane (7)
2,6-Dichlorobenzaldehyde (1.600 g, 9.141 mmol) was dissolved in
50 mL freshly distilled pyrrole and 50 mL TFA were added. The
mixture was stirred for 6 h at r.t. and then quenched with 200 mL of
CH2Cl2–sat. NaHCO3 solution (1:1). The organic phase was evapo-
rated and the residue chromatographed on silica (CH2Cl2–hexane,
1:1) under exclusion of light and oxygen to obtain 1.916 g (6.581
mmol, 72%) of 7 as a viscous oil, which crystallized after 24 h at
4 °C.
Atropisomerization occurs at 40 °C well below the temperature of
the subsequent macrocyclization. Therefore the two isomers were
combined for further use.
Rf = 0.79 (aa, silica, CH2Cl2).
Rf = 0.25 (ab, silica, CH2Cl2).
1H NMR (300 MHz, CDCl3): d = 8.92 (m, 4 H, pyrrole), 8.73 (m, 4
H, pyrrole), 8.10–7.20 (m, 14 H, aryl), 5.07 (aa) or 5.02 (ab) (s, 2
H, OH), –2.55 (s, 2 H, NH).
Rf = 0.60 (silica, CH2Cl2–hexane, 1:1).
1H NMR (300 MHz, CDCl3): d = 8.27 (br s, 2 H, HN), 7.34–7.31
(m, 2 H, H-3¢,5¢), 7.15–7.08 (m, 1 H, H-4¢), 6.72–6.67 (m, 2 H, H-
3,9), 6.48 (br s, 1 H, H-6), 6.20–6.16 (m, 2 H, H-5,7), 6.08–6.04 (m,
2 H, H-4,8).
MS (LDI-TOF): m/z = 785.3.
UV/Vis (CH2Cl2): lmax (%) = 418 (100), 512 (6) nm.
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S. Kozuch et al.
CLUSTER
5,15-({[5-(t-Butyl)-2-(N,N-dimethylcarbamoyl)thio-1,3-phe-
nylene]bis(trimethyleneoxy)}-di-2,1-phenylene)-10,20-bis(2,6-
dichlorophenyl)porphyrin (12)
[5,15-({[5-(t-Butyl)-2-sulfonato-1,3-phenylene]bis(trimethyl-
eneoxy)}-di-2,1-phenylene)-10,20-bis(2,6-dichlorophenyl)por-
phyrinato]iron(III) (6)
To a solution of porphyrin 10 (242.4 mg, 308 mmol) in 150 mL dry
DMF 5.5 g Cs2CO3 and 40 mg DMAP were added and the mixture
stirred for 30 min at 80 °C. Then, dimesylate 11 (157.4 mg, 308
mmol) in 10 mL DMF was added via automatic syringe during 3 h
at 80 °C. Heating was continued for further 1.5 h. The solvent was
evaporated, the residue redissolved in CH2Cl2, washed with H2O,
dried over Na2SO4 and finally chromatographed on silica (CH2Cl2)
to obtain 270.3 mg (245 mmol, 79%) of 12 as a purple solid; mp
>250 °C.
Bubbling air through a solution of the porphyrin 14 (42.9 mg, 39.6
mmol) in 10 mL CH2Cl2 at r.t. during 15 min gave according to con-
trol by UV spectroscopy and MS spectrometry quantitative conver-
sion to 6.
Compound 6 can also be obtained by iron insertion into the sul-
fonate corresponding to 13.
Rf = 0.30 (silica, CH2Cl2–MeOH, 95:5).
HPLC (RP18, hexane–i-PrOH, 90:10, flow rate 1 mL/min, l = 410
nm): t = 12.3 min.
Rf = 0.16 (silica, CH2Cl2).
1H NMR (300 MHz, CDCl3): d = 9.18–8.46 (m, 8 H, pyrrole), 8.76–
7.18 (m, 14 H, H-3¢,4¢,5¢,6¢,3¢¢,4¢¢,5¢¢), 6.64 (s, 2 H, H-3¢¢¢,5¢¢¢), 3.93–
3.81 (m, 4 H, H-a¢), 2.00–1.50 (br s, 3 H, N-CH3), 1.48–1.30 (m, 4
H, H-b¢), 1.21 (s, 9 H, H-2¢¢¢¢¢), 1.12–0.93 (m, 4 H, H-g¢), –1.10 to
–1.50 (br s, 3 H, N-CH3), –1.95 (s, 2 H, NH).
EPR (CDCl3; T = 121 K): g = 5.76.
MS (LDI TOF): m/z = 1135.
UV/Vis (CH2Cl2): lmax (%) = 414 (100), 512 (9.8) nm.
UV/Vis (THF): lmax = 416 (100), 510 (17) nm.
13C NMR (75MHz, CDCl3): d = 116.3, 159.9, 150.8, 145.2, 139.9,
139.5, 138.8, 138.2, 137.2, 134.0, 130.4, 130.3, 130.0, 129.9, 128.9,
128.1, 128.0, 127.8, 127.5, 127.3, 123.6, 119.5, 116.0, 112.2, 68.7,
34.1, 31.6, 31.0, 30.3.
5,15-Bis(2-benzyloxy-4-carboxyethylphenylene)-10,20-bis(2,6-
dichlorophenyl)porphyrin (17)
To a solution of 2,6-dichlorophenyl dipyrromethane 7 (1.9168 g,
6.583 mmol) and 3-benzyloxy-4-formyl-ethylbenzoate (16, 1.5424
g, 6.583 mmol) in 50 mL CH2Cl2 0.5 mL TFA were added and the
solution was stirred at r.t. for 2 h. Subsequently, 3 g of 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone were added and the mixture stirred
for 1 h at r.t. The solvent was largely evaporated at 15 Torr and the
residue filtered over silica. The resulting solution was evaporated
and subjected to column chromatography on silica (CH2Cl2–
hexane, 7:3). The porphyrin 17 (722.6 mg, 651.7 mmol, 20%) was
obtained as a red microcrystalline powder; mp >250 °C.
MS (ESI, MeOH): m/z = 1102.
MS (LDI-TOF): m/z = 1108.0 [M+], 1035.5.
UV/Vis (CH2Cl2): lmax (%) = 422 (100), 400 (14), 548 (8) nm.
5,15-({[5-(t-Butyl)-2-mercapto-1,3-phenylene]bis(trimethyle-
neoxy)}-di-2,1-phenylene)-10,20-bis(2,6-dichlorophenyl)por-
phyrin (13)
To a solution of porphyrin 12 (121.9 mg, 110.6 mmol) in 15 mL dry
oxygen-free dioxane 3.3 mL (11.06 mmol) 25% MeOK solution
were added and the solution heated for 2 h at 70 °C. After quench-
ing with 10% HCl solution the mixture was extracted three times
with CH2Cl2, the organic layer washed with sat. NH4Cl solution,
dried over Na2SO4 and evaporated. The residue was chromato-
graphed on silica (CH2Cl2–hexane, 1:1) to obtain 53.1 mg (51.51
mmol, 46%) 13 as a red solid; mp >250 °C.
1H NMR (300 MHz, CDCl3): d = 8.88–6.67 (m, 20 H, aryl and pyr-
rol), 6.90–6.60 (m, 10 H, benzyl), 5.10 (s, 4 H, benzyl-CH2), 4.58
(q, 4 H, COOCH2CH3), 1.55 (t, 6 H, COOCH2CH3).
MS (LDI-TOF): m/z = 1109.6.
UV/Vis (CH2Cl2): lmax (%) = 420 (100), 486 (5), 514 (8), 548 (3),
592 (4).
UV/Vis (CH2Cl2, HCl): lmax (%) = 366 (25), 400 (23), 422 (23), 448
(100), 486 (18), 656 (19) nm.
Rf = 0.57 (silica, CH2Cl2–hexane, 1:1).
1H NMR (300 MHz, CDCl3): d = 8.90–8.63 (m, 8 H, pyrrole), 7.83–
7.18 (m, 14 H, H-3¢,5¢,6¢), 6.33 (s, 2 H, H-3¢¢¢,5¢¢¢), 3.74 (m, 4 H,
CH2-a¢), 1.02 [s, 9 H, C(CH3)3¢¢¢¢¢), 0.95 (m, 4 H, CH2-b¢), 0.67 (m,
4 H, CH2-g¢), –2.29 (s, 2 H, NH), –2.59 (s, 1 H, SH).
The purification of 17 can be improved by preparing the corre-
sponding Zn complex Zn-17 followed by purification and final Zn
removal. For this purpose porphyrin 17 (400 mg, 360.7 mmol) was
dissolved in 50 mL CH2Cl2 and after addition of
2 g
MS (ESI, CH2Cl2–MeOH, 1:1): m/z = 1031.4.
MS (LDI TOF): m/z = 1030.3.
Zn(OAc)2·6H2O in 5 mL MeOH the mixture was heated under re-
flux for 1.5 h. The organic phase was washed with H2O, dried over
Na2SO4 and evaporated at 15 Torr yielding a residue, which was
chromatographed on silica (CH2Cl2). After chromatography Zn-17
(410.1 mg, 350.0 mmol, 97%) was obtained as a mixture of atrop-
isomers. Rf = 0.52 (aa) und 0.60 (ab) (silica, CH2Cl2–hexane, 80:20).
UV/Vis (CH2Cl2): lmax (%) = 422 (100), 518 (7) nm.
[5,15-({[5-(t-Butyl)-mercapto-1,3-phenylene]bis(trimethylene-
oxy)}-di-2,1-phenylene)-10,20-bis(2,6-dichlorophenyl)por-
phyrinato]iron(III) (14)
Mp >250 °C.
In a glove box porphyrin 13 (51 mg, 49.47 mmol) and approx. 200
mg iron(II)bromide were dissolved in toluene and heated under re-
flux for 1 h after addition of 20 mL 2,6-lutidine. The solvent was
evaporated and the residue chromatographed on silica (CH2Cl2–
MeOH, 95:5) to obtain 42.9 mg (39.6 mmol, 80%) 14 as a brown sol-
id; mp >250 °C.
UV/Vis (CH2Cl2): lmax (%) = 422 (100), 496 (14), 552 (11) nm.
For Zn removal Zn-17 (410.1 mg, 350.0 mmol) was dissolved in 20
mL CH2Cl2 and after addition of 5 mL concd HCl the mixture was
stirred for 1 h. The green solution was diluted with 50 mL H2O, and
washed with 100 mL sat. NaHCO3 solution NaHCO3. The resulting
red solution was evaporated and the residue chromatographed on
silica (CH2Cl2–hexane, 7:3); 376.3 mg (339.3 mmol, 97%) of the
pure free base porphyrin 17 were isolated.
Rf = 0.30 (silica, CH2Cl2–MeOH, 95:5).
HPLC (RP18, hexane–i-PrOH, 90:10; flow rate 1.0 mL/min,
l = 410 nm): t = 12.4 min.
5,15-Bis(4-carboxyethyl-2-hydroxyphenyl)-10,20-bis(2,6-di-
chlorophenyl)porphyrin (18)
A solution of porphyrin 17 (187.2 mg, 169.2 mmol) in 30 mL abs.
CH2Cl2 was cooled to –78 °C (acetone/CO2), BBr3 (0.3 mL) was
added and the green solution stirred for 1 h. The reaction was
EPR (CDCl3; T = 121 K) bromide complex: g = 7.45, 4.50, 3.40.
MS (LDI TOF): m/z = 1083.
UV/Vis (CH2Cl2): lmax (%) = 422 (100), 518 (7).
Synlett 2005, No. 4, 675–684 © Thieme Stuttgart · New York

CLUSTER
P450 Enzyme Models
MS (ESI, MeCN): m/z = 1119.5.
683
quenched with 100 mL H2O, the organic layer washed with sat.
NaHCO3 and dried over Na2SO4. Finally, the product was purified
on silica (CH2Cl2–MeOH, 97:3) yielding 134.3 mg (145 mmol,
86%) red, microcrystalline 18; mp >250 °C.
UV/Vis (CH2Cl2): lmax (%) = 302 (9), 422 (100), 516 (9), 548 (3),
592 (3) nm.
Rf = 0.4 (ab) and 0.6 (aa) (silica, CH2Cl2–MeOH, 97:3).
[5,15-Bis(4-carboxyl-{[5-(tert-butyl)-2-mercapto-1,3-phe-
nylene]bis(trimethyleneoxy)}di-2,1-phenylen)-10,20-bis(2,6-
dichlorophenyl)porphyrinato]iron(III) (21)
In a glove box (<20 ppm O2) porphyrin 92 (111.9 mg, 100 mmol)
were dissolved in 20 mL DMF and after addition of 50 mL 2,6-luti-
dine and 300 mg iron(II)bromide the mixture was heated to 160 °C
for 3 h. DMF was evaporated and the residue purified on silica
(CH2Cl2–MeOH–HOAc, 96:3:1). The iron(III) complex 21 was iso-
lated as a brown solid (71.3 mg, 60.8 mmol) in 61% yield.
1H NMR (300 MHz, CDCl3): d = 8.79–7.22 (m, 20 H, aryl and pyr-
rol), 4.46 (q, 4 H, COOCH2CH3), 1.47 (t, 6 H, COOCH2CH3), –1.20
(2 s, 2 H, NH).
MS (LDI-TOF): m/z = 932.6.
UV/Vis (CH2Cl2): lmax (%) = 418 (100), 512 (6) nm.
5,15-Bis(4-carboxyethyl-{[5-(tert-butyl)-2-N,N-dimethyl-car-
bamoyl]-thio-1,3-phenylene}-bis-[(trimethyleneoxy)di-2,1-phe-
nylene]-10,20-bis-(2,6-dichlorophenyl)porphyrin (19)
Rf = 0.3 (silica, CH2Cl2–MeOH–HOAc, 96:3:1).
Mp >250 °C.
To a solution of porphyrin 18 (134.3 mg, 145 mmol) in 70 mL dry
DMF (degassed) 2.0 g Cs2CO3 and 20 mg DMAP were added and
the mixture heated at 80 °C for 30 min. The dimesylate 11 (81.3 mg,
159 mmol) dissolved in 5 mL DMF was subsequently added via au-
tomatic syringe during 3 h at 80 °C. Heating was continued for fur-
ther 1.5 h. After evaporation of DMF the residue was dissolved in
CH2Cl2, washed with H2O, dried over Na2SO4 and finally chro-
matographed on silica (CH2Cl2–MeOH, 99:1). The mono-bridged
porphyrin 19 (152.29 mg, 122 mmol) was obtained as a red micro-
crystalline substance in 84% yield; mp >250 °C.
MS (ESI): m/z = 1168.1.
UV/Vis (CH2Cl2): lmax (%) = 338 (20), 422 (100), 514 (8) nm.
UV/Vis (MeOH): lmax (%) = 422 (50.8), 518 (4.6), 548 (1.9), 592
(1.9) nm.
Tentagel-linked Fe––OSO2 porphyrin (15)
A solution of iron porphyrin 21 (9.0 mg, 7.680 mmol) in 5 mL DMF
was aired for 15 min and the complete oxidation at sulfur controlled
by UV/Vis (lmax = 415 nm) and by ESI-MS spectrometry (m/z =
1220.2). Subsequently, 201.1 mg RAPP Tentagel-S-NH2, 0.3 mL
DCC and 2 mL CH2Cl2 were added. The mixture was stirred for 12
h at r.t. and finally the polymer collected by filtration and washed
with CH2Cl2, MeOH, DMF and HOAc (5 mL each). The volume of
the combined filtrates was adjusted to 200 mL, and from UV absor-
bance an epitopic density on the gel was calculated 18.1 mmol/g
RAPP Tentagel. This value was confirmed through re-isolation of
the uncoupled 21 from the filtrate.
Rf = 0.5 (silica, CH2Cl2–MeOH, 99:1).
IR (KBr): 3500 (br), 2928.9 (m), 1717.3 (s), 1427.7 (m), 1285.6 (s),
1102.8 (m), 801.4 (m) cm–1.
1H NMR (300 MHz, CDCl3): d = 9.11–7.55 [m, 20 H, H-b(pyrrol)
and H-3¢, H-4¢, H-5¢, H-6¢, H-3¢¢,H-4¢¢, H-5¢¢), 6.64 (s, 2 H, H-3¢¢¢,
H-5¢¢¢), 4.63 (q, 4 H, COOCH2CH3), 4.01, 3.90 [m, 6 H, N(CH3)2],
1.60 (t, 6 H, COOCH2CH3), 1.37 (br, 4 H, H-a¢), 1.20 [s, 9 H,
(CH3)3], 1.00 (br, 4 H, H-b¢), 0.79 (br, 4 H, H-g¢), –1.34 (br, 2 H,
NH).
13C NMR (75 MHz, CDCl3): d = 166.8, 166.4, 160.0, 151.2, 145.3,
140.0, 139.4, 139.3, 138.9, 138.3, 137.3, 135.3, 134.0, 132.4, 130.6,
130.2, 128.2, 128.1, 127.9, 127.8, 127.5, 123.9, 123.8, 121.1, 115.2,
113.0, 69.1, 61.5, 34.3, 31.7, 31.3, 31.2, 30.9, 30.2, 14.7, 14.6.
Then, 30 mg of 15 was suspended in CH2Cl2 and the unmodified
amino groups acetylated with an excess of HOAc and DCC; the re-
sulting sample was negative in a ninhydrin test. The polyacetylated
gel was then reacted with an excess of 1,2 diamino ethane and DCC
in order to derivatize the free COOH groups at the porphyrin.
Quantitative analysis of the Ninhydrin test revealed an epitopic
density of 22 mmol/g.
MS (ESI, MeCN): m/z = 1246.5.
MS (LDI-TOF): m/z = 1247.6.
The good agreement of the two values of the epitopic density indi-
cates the absence of cross-linking of 21 with the RAPP Tentagel.
UV/Vis (CH2Cl2): lmax (%) = 422 (100), 516 (6), 548 (3), 592 (2)
nm.
General Procedure for Epoxidations
In a 20 mL Schlenk-tube the substrate, PhIO, porphyrin 6 (RAPP-
porphyrin 15), and decane (internal standard) were dissolved in 5
mL CH2Cl2. Concentrations: catalysts: <0.1 mol%, olefin: 0.5
mmol, PhIO: 1 mmol; reaction time 24 h at r.t. The reaction was
controlled by GC-FID and the turnover was quantified in reference
to the internal standard.
5,15-Bis(4-carboxyl-{[5-(tert-butyl)-2-mercapto-1,3-phe-
nylene]-bis(trimethyleneoxy)}di-2,1-phenylene)-10,20-bis(2,6-
dichlorophenyl)porphyrin (20)
The porphyrin 19 (143.0 mg, 114.7 mmol) was dissolved in 20 mL
dry oxygen-free dioxane. After addition of 3.5 mL 25% MeOK–
MeOH the mixture was heated for 2.5 h at 65 °C. The reaction was
quenched with 50 mL 10% HCl and extracted three times with 50
mL CH2Cl2. The organic layer was washed with sat. aq NH4Cl and
dried over Na2SO4. Chromatography on silica (CH2Cl2–MeOH–
HOAc, 96:3:1) furnished the diacid 20 (111.9 mg, 100 mmol) in
87% yield; mp >250 °C.
Acknowledgment
WDW gratefully acknowledges financial support by the Swiss
National Foundation.
Rf = 0.4 (silica, CH2Cl2–MeOH–HOAc, 96:3:1).
IR (KBr): 3440.2 (br), 2923.9 (m), 1683.8 (s), 1591.1 (m), 1364.4
(m), 800.8 (w), 764.1 (m), 698.7 (w) cm–1.
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Synlett 2005, No. 4, 675–684 © Thieme Stuttgart · New York

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Synlett 2005, No. 4, 675–684 © Thieme Stuttgart · New York