Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam - An Investigation Using Active Site Analogues

Article abstract of DOI:10.1002/adsc.200303011

The crystal structure of the resting state of cytochrome P450 _cam (CYP101), a heme thiolate protein, shows a cluster of six water molecules in the substrate binding pocket, one of which is coordinating to iron(III) as sixth ligand. The resting

Full text of DOI:10.1002/adsc.200303011

FULL PAPERS
Remote Effects Modulating the Spin Equilibrium of the Resting
State of Cytochrome P450cam
Site Analogues
An Investigation Using Active
Martin Lochner, Linjing Mu, Wolf-D. Woggon*
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland
Fax: ( ‡ 41)-61-267-1102, e-mail: wolf-d.woggon@unibas.ch
Received: January 14, 2003; Accepted: March 20, 2003
Abstract: The crystal structure of the resting state of sites mimicking proposed remote effects have been
cytochrome P450cam (CYP101), a heme thiolate prepared and studied by cw-EPR. The results indicate
protein, shows a cluster of six water molecules in that the low-spin of the resting state of P450cam is due
the substrate binding pocket, one of which is coordi- to the fact that the water molecule coordinating to
nating to iron(III) as sixth ligand. The resting state is iron has an OHÀ-like character because of hydrogen
low-spin and changes to high-spin when substrate bonding and polarisation of the water cluster, re-
camphor binds and H2O is removed. In contrast to the spectively.
protein, previously synthesised enzyme models such
III
À
À
as H2O Fe (porph)(ArS ) were shown to be purely Keywords: cytochrome P450; enzyme models; EPR
high-spin. Iron(SÀ)porphyrins with different distal spectroscopy; porphyrinoids; spin states
Introduction
A common feature of the P450 molecular structure is
an iron(III) protoporphyrin IX (heme b) cofactor in the
After more than 30 years of research P450-catalysed active site (Figure 1). The fifth proximal cysteinate
reactions have been recognised as one of nature×s most ligand is provided by the polypeptide chain of the
common and sophisticated methods to oxidise endog- globular protein and is responsible for the unique
enous compounds in prokaryotic and eukaryotic cells.[1] feature of these enzymes to activate molecular dioxygen
Accordingly, P450 s can be isolated from bacteria,[2] towards insertions into C-H bonds.
plants[3] and different tissues of mammals,[4] where
Among these enzymes, the P450 s responsible for
numerous isozymes participate highly specifically in epoxidation of alkenes and the hydroxylation of non-
the metabolism of compounds as diverse as steroids, activated C-H bonds (RH), utilising either NADH or
fatty acids and alkaloids. The same reactivity is also NADPH as electron sources (Equation 1), have been
common to those P450 s acting on xenobiotics like toxic studied extensively.[1,2] Experiments with labelled 18O2
compounds and drugs and they can be isolated, e.g., have shown that one of the oxygen atoms is incorporated
from liver microsomes. The membrane-bound hepatic into the substrates and the other is reduced to water
P450 s generally accept a broad spectrum of substrates, (monooxygenase reaction).
which is understood as a method of detoxification in
order to render lipophilic compounds water soluble and
excretable.
ꢀ1†
The cytochrome P450cam (CYP101) isolated from the
soil bacterium Pseudomonas putida is a soluble cytosolic
enzyme and by using camphor as the only carbon source
this organism employs P450cam in order to catalyse the
stereospecific hydroxylation at the 5-exo position
(Scheme 1) as a first step in a cascade of energy
supplying reactions. Its feasible isolation, solubility
and the possibility to overexpress this enzyme in other
Figure 1. Molecular structure of the P450 active site.
bacteria has made P450cam one of the best studied P450
Adv. Synth. Catal. 2003, 345, 743 765
DOI: 10.1002/adsc.200303011
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Scheme 1. Hydroxylation of (‡)-camphor by P450cam from
Pseudomonas putida.
systems and for a number of years the high-resolution
crystal structure of P450cam (Figure 2) solved by Poulos
and co-workers[5a] in 1986 was the only available three-
dimensional structure of a P450 enzyme.
The information gained from the X-ray structures of
the substrate-free resting state (Figure 2),[5a] the E ¥ S
complex with camphor[5b] and substrate analogues,[5c]
the structure of the CO adduct,[5d] and the E ¥ P complex
with 5-exo-hydroxycamphor[5e] of P450cam had a great
impact on P450 research. The comparison of these
™derivatives∫ with respect to changes of the spatial
arrangements at the active site not only confirmed
former indirect spectroscopic evidence but provided
Figure 3. cw-EPR spectra of the resting state (bottom trace)
and camphor-bound P450cam (top trace). Spectra were
measured in 50 mM potassium phosphate buffer, pH 7.1,
3.5 mM camphor. EPR conditions: n ˆ 9.2 GHz, P ˆ 0.5 mW,
Tˆ 12 K, modulation frequency 100 kHz, modulation
amplitude 12.5 G.
important details of substrate binding and clues con- hydrogen bond formed between Tyr96 and the carbonyl
cerning the mechanism of P450 action. Very recently, in group of camphor.
a landmark publication, Schlichting, Sligar and their co-
Before the three-dimensional structure of P450cam was
workers have made ™snap-shots∫ of the catalytic cycle of elucidated the substrate binding process was studied
[6]
P450cam by using rapid data-collection techniques, extensively by spectroscopic methods such as UV-
cryogenic temperatures and long-wavelength X-rays. visible spectroscopy,[7] cw-EPR,[8] Mˆssbauer spectro-
This work revealed crystal structures of some inter- scopy[9] and electrochemical methods[10] which revealed
mediates of the catalytic cycle which filled important significant changes upon camphor binding to the resting
white spots on the map of the P450cam mode of action.
state of P450cam. Substrate binding, for instance, causes a
A closer look at the active site of the crystal structure 26 nm blue shift of the Soret band in the UV/Vis
of the resting state of P450cam[5a] (Figure 2) revealed that spectrum of the P450cam resting state.
the substrate binding pocket is occupied by a cluster of
six hydrogen-bonded water molecules, one of which is inantly low-spin (S ˆ 1/2) with EPR g values at 2.45,
coordinating to iron as sixth ligand. 2.26, and 1.91, but addition of substrate is accompanied
At 12 K the native cytochrome P450cam is predom-
Upon binding of the substrate the water cluster, by 60% conversion to a rhombically distorted high-spin
including the sixth distal water ligand, is completely (S ˆ 5/2) form with g ˆ 7.85, 3.97, and 1.78 (Figure 3).[8]
removed and the camphor molecule is bound by a Mˆssbauer studies by Sharrock and co-workers con-
firmed that the binding of camphor to P450cam causes a
partial conversion from low- to high-spin.[9] Their data
also showed that this spin equilibrium is temperature
dependent and the high-spin fraction of camphor-bound
P450cam measured at 215 K is approximately 1.6 times
that at 4 K which would result in>96% high-spin at
room temperature.
The fact that the resting state 1 (Scheme 2) of P450cam
is low-spin was very surprising once its crystal structure
was solved (Figure 2). It seems unlikely that the thiolate
and one of the water molecules, both known to be weak
ligands, could establish a low-spin iron complex.
Also, the binding of the camphor alters dramatically
the redox potential of the heme iron of P450cam from
À 300 mV for the resting state to À 170 mV with
substrate-bound[10] (see 2, Scheme 2) rendering the
Figure 2. Structure of the active site of the P450cam resting
state (PDB entry 1PHC).
latter to accept an electron from NADH via the redox
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
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Scheme 2. The consensus mechanism of P450 hydrocarbon hydroxylations.
proteins putidaredoxin reductase (flavoprotein) and of the resting state 1 is crucial for the function and
putidaredoxin ([Fe-S] protein) which gives rise toa high- efficiency of the P450 enzyme since this control mech-
spin pentacoordinated FeII complex 3 (Scheme 2).
anism prevents unnecessary flow of electrons into the
The ferrous species 3 is now able to bind molecular catalytic cycle if no substrate is bound and hence
dioxygen which results in the diamagnetic low-spin oxy suppresses uncoupling (formation of H2O2). Numerous
intermediate 4. A second electron transfer from puti- theoretical studies of the Shaik group suggest that
daredoxin to 4 forms the ferric peroxo complex 5. In the compound I (7) is a chameleon species, that may tune its
rebound mechanism proposed by Groves et al.[11] the reactivity and selectivity patterns in response to the
distal oxygen atom of the peroxo complex 5 is proto- substrate and the protein environment in which it is
nated twice, first to give a hydroperoxide complex 6 and accommodated.[13] According to their calculations, com-
À
then to induce a heterolytic O O bond cleavage, pound I of P450 is a two-state reagent having closely
releasing water and generating the oxo-ferryl (FeIV) lying quartet (4A2u, high-spin) and doublet states (2A2u,
porphyrin radical cation intermediate 7 which is equiv- low-spin). Moreover, C-H hydroxylation is following
alent to the high-valent iron-oxo species of peroxidase the two-state reactivity (TSR) scenario[14] and reactivity
enzymes, historically described as compound I.[12] The patterns and product distribution are determined by the
highly reactive species 7 abstracts a hydrogen atom from interplay of the two states which itself depends on the
the enzyme bound substrate yielding a carbon-centred spin-crossover from high-spin to low-spin, on the
radical with a finite lifetime and hydroxo ferryl complex environment (the shape of the substrate binding pocket
8. In the following oxygen rebound step the oxygen atom of the enzyme used, its acidity, polarity, etc.[13a, d]) and
is transferred to the enzyme-bound substrate radical finally on the substrate. Hence, spin state changes also
which results in the formation of the low-spin ferric trigger the mode/reactivity of oxygen insertion by the
enzyme-product complex 9. Finally, dissociation of the compound I species.
product closes the catalytic cycle.
Contradictory arguments have been published to
It is evident that the spin equilibrium triggers the entry explain the origin of the low-spin resting state of
into the catalytic cycle. Furthermore, the low-spin state P450cam. On one hand, Poulos et al.[15] suggested that
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Results and Discussion
Synthesis and Study of the Bis(tetrahydrofuranyl)
Iron(III) Porphyrin a First Model for Mimicking the
Polarisation Effect in the Water Cluster
The bis-THF model 11 was considered being suitable to
polarise a coordinated water molecule via hydrogen
bonding whereby the ether oxygens act as H-bond
acceptors (Scheme 3). Therefore, 11 should be an active
site analogue to mimic the polarisation effect in the
water cluster of the native enzyme. Retrosynthetic
analysis leads to bis(aminophenyl)porphyrin 12 and
tetrahydrofurancarbonyl chloride 13[19] which, in turn,
would be synthesised from the corresponding acid 14.
Figure 4. Previous high-spin active site analogue 10-H2O for
the resting state of P450cam
.
polarisation of the coordinated water molecule through The bis(aminophenyl)porphyrin can be disconnected to
the hydrogen bonding network in the water cluster could the following three fragments: dipyrromethane 15[18a]
,
result in a distinctive hydroxide character (strong protected aldehyde 16 and activated and protected
ligand) and thus lead to a low-spin complex. On the thiophenyl bridge 17.[18]
other hand by comparing simulated and experimental
The synthesis started from commercially available m-
17O ESEEM and pulsed ENDOR/four-pulse ESEEM anisidine (18) the amino function of which was first
spectra of the resting state of P450cam, Goldfarb et al.[16] protected as the tert-butoxycarbamate (Scheme 4). For-
concluded that the distal ligand is a water molecule mylation of 19[20] was achieved following a protocol by
rather than a hydroxide ligand. Semiempirical calcula- Stanetty et al.[21] whereby the methoxy and the carba-
tions by Loew and Harris[17a] suggested a significant mate group direct the formylation to the 2-position.
contribution of the electrostatic field of the surrounding Saponification of 20[22] and subsequent decarboxylation
protein stabilising the low-spin resting state. It was of the resulting diacid 21 yielded the air- and light-
proposed by the same authors that a point charge above sensitive dipyrromethane 15 which was immediately
the porphyrin plane would simulate the electrostatic used in the next step. Classical porphyrin condensa-
field of the protein and push the spin equilibrium tion[23] of dipyrromethane 15 and aldehyde 16 under acid
towards low-spin.[17b]
catalysis with p-toluenesulphonic acid gave after sub-
Using various EPR techniques it was previously sequent oxidation of the formed porphyrinogens with
shown in our group that the active site analogue 10-
H2O (Figure 4)[18a] is definitely high-spin and only
changes to the low-spin state if the coordinating water
is replaced by 1,2-Me2Im.[18b] These results clearly
indicated that the coordination of water to Fe(III) of
the heme thiolate cofactor is insufficient to stabilise the
low-spin state of the resting state of P450cam
.
In order to unravel the origin of the low-spin resting
state of P450cam our aim was to synthesise and character-
ise active site analogues of this enzyme that mimic (i) the
proposed polarisation effect[15] in the water cluster and
(ii) the influence of the electrostatic field from the
surrounding protein as suggested by calculations.[17a]
From the design point of view we thought that the
former could be achieved by placing hydrogen bond
acceptors near the distal site of the porphyrin which can
form hydrogen bonds with the water molecule coordi-
nating to the iron. The latter effect was thought to be
mimicked by attaching a point charge above the
porphyrin plane which would push the spin equilibrium
towards low-spin.
Scheme 3. Retrosynthetic analysis of tetrahydrofuranyl iron
porphyrin model 11.
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
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Scheme 5. Chemical derivatisation of bisphenol porphyrin
aa-25 for atropisomer assignment. Reagents and conditions:
(a) AlCl3 (38 equivs.), EtSH/CH2Cl2 (3:2), rt, 43 h, 73%. (b)
Cs2CO3 (30 equivs.), DMF, 608C, 30 min; then Br(CH2)11Br
(1.5 equivs.), DMF, 608C, high-dilution, 3 h, 63%.
this stage it was possible to separate the two atropisom-
ers by flash chromatography on silica gel which yielded
pure aa-24 in 49% as the more polar fraction and ab-24
in 24% as the less polar compound. This results in a ratio
of aa/ab ~ 67:33 which was virtually the same ratio as
deduced from RP-HPLC of the atropisomeric mixture
aa/ab-24.
If one is looking at the overall yield of aa-24 (20%
from aldehyde 16) it seems very low at first glance but it
is in the typical range of porphyrin condensations (10
30%) compared to examples in the literature. The
synthesis was now continued with the aa-atropisomer
of 24 and cleavage of the methyl ethers was carried out
with anhydrous aluminium chloride in a solvent mixture
of CH2Cl2 and EtSH. This gave bisphenol porphyrin aa-
25 in good yield (Scheme 5).
It is worth noting that, in contrast to isomers such as
aa-26[18a], the atropisomers of this series of porphyrins,
due to steric congestion between the substituents at the
b-pyrrole positions and the substituents at the 2'- and 6'-
positions of the phenyl rings, are configurationally
stable during manipulations or purification (Figure 5).
To distinguish between atropisomers such as aa-24 and
ab-24 1H NMR analysis is insufficient due to very small
differences in chemical shift pattern. Also polarity
arguments taken from TLC behaviour of similar com-
pounds[24] proved to be ambiguous.
Scheme 4. Synthesis of tetrahydrofuranyl model 11
por-
phyrin condensation. Reagents and conditions: (a) Boc2O (1.6
equivs.), THF, rt, overnight, quant. (b) t-BuLi (2.2 equivs.),
Et2O, À 108C, 3 h; then DMF (2 equivs.), Et2O, À 788C ! rt,
overnight, 82%. (c) NaOH (5 equivs.), EtOH/H2O, reflux,
4 h, 92%. (d) H2N(CH2)2OH, 1208C, 45 min, 57%. (e) cat. p-
TsOH, MeCN, rt, overnight; then DDQ (2 equivs.) in THF, rt,
1.5 h. (f) TFA (82 equivs.), CH2Cl2, rt, 12.5 h, 40% (2 steps).
(g) AcCl (2.5 equivs.), pyridine (3.3 equivs.), cat. DMAP,
CH2Cl2, rt, 18 h; then flash chromatography, 49% aa-24, 24%
ab-24.
Therefore it was decided to pursue a macrocyclisation
which could be easily done with aa-25 but would be
impossible to perform with ab-25. In the event, the
DDQ a mixture of two atropisomers, aa-22 and ab-22, porphyrin, tentatively assigned as aa-25 was bridged
respectively. This mixture was treated with trifluoro- with 1,11-dibromoundecane in the presence of Cs2CO3
acetic acid to remove the protecting groups and the in hot DMF under high-dilution conditions (Scheme 5).
liberated amino functions of aa-23 and ab-23 were It is known that eleven carbon atoms are sufficient to
reprotected with the acid-stable acetylamino group. At span the porphyrin plane[25] and furthermore the ab-
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reaction of diallylthiophenol carbamate 28.[25] The distal
acetylamino groups were hydrolysed by refluxing 30 in a
mixture of 6 M HCl and MeOH to give bis(aminophen-
yl) porphyrin 12 in high yield.
The bis(aminophenyl) porphyrin 12 (9% overall yield
for longest linear sequence starting from m-anisidine 18)
is a very useful building block because the free amino
groups can be functionalised in many different ways and
lead to thiophenolate-bridged iron(III) porphyrins with
various functionalities in the distal site. By this strategy
it is possible to have access to model compounds
Figure 5. Comparison of configurational stability of porphyr-
ins with different substitution at the meso-phenyl rings.
isomer of 25 cannot form a macrocyclic structure. As mimicking different active sites of enzymes having a
1
observed in H NMR successful bridging of aa-25 was heme cysteinate cofactor like cytochrome P450 s, chloro-
confirmed by the high-field shifted signals for the peroxidase (CPO) and nitric oxide synthase
methylene groups of the undecanylene strap of 27 due (NOS).[18,25,26]
to the ring current of the porphyrin (a-CH2 3.82 ppm, b-
CH2 0.70 ppm, g-CH2 À 0.34 ppm, d-CH2 À 0.67 ppm, amino groups were acylated with tetrahydrofurancar-
bonyl chloride 13 which was obtained from the corre-
In the case of the bis(tetrahydrofuranyl) model 11 the
e-CH2 and z-CH2 À 1.61 ppm, respectively).
Accordingly, the correct atropisomer was identified sponding acid 14 by treatment with oxalyl chloride
and the synthesis continued with the bridging of aa-25 (Scheme 7). Cleavage of the thiocarbamate protecting
with activated and protected thiophenylene 17 under group of 31 liberated the thiophenol function and
high-dilution conditions in hot DMF in the presence of subsequent iron insertion using FeBr2 in the presence
Cs2CO3 (Scheme 6). The latter reagent acts on one hand of 2,6-lutidine as base in refluxing toluene completed
as a base to preform the bisphenolate and on the other the synthesis of bis(tetrahydrofuranyl) model 11.
hand has a templating effect for macrocyclisation.[25]
Upon iron insertion the Soret band in the UV/Vis
The dimesylate 17 was synthesised from the correspond- blue-shifts from 412 nm for 32 to 406 nm for iron(III)
ing diol 29 which was obtained via hydroboration compound 11. Furthermore, the correct mass of the
Scheme 6. Synthetic route to bridged bis(aminophenyl) por-
phyrin 12. Reagents and conditions: (a) Cs2CO3 (30 equivs.),
DMF, 608C, 30 min; then 17 (1.5 equivs.), DMF, 608C, 4.5 h,
high-dilution, 85%. (b) BH3 ¥ Me2S (3.6 equivs.), THF, rt, 3 h;
then 3 M NaOH, 10% aqueous H2O2, reflux, 1 h, 77%. (c)
MsCl (4.7 equivs.), Et3N (8 equivs.), CH2Cl2, 08C ! rt, 3 h,
96%. (d) 6 M HCl/MeOH, reflux, 2 h, 91%.
Scheme 7. Completion of the synthesis of tetrahydrofuranyl
model 11. Reagents and conditions: (a) 13 (4 equivs.), Et3N
(4.8 equivs.), cat. DMAP, CH2Cl2, rt, 13 h, 92%. (b) oxalyl
chloride, rt, 22 h, 94%. (c) KOMe in MeOH (2.2 M, 59
equivs.), dioxane, reflux, 15 min, 73%. (d) FeBr2 (9 equivs.),
2,6-lutidine (17 equivs.), toluene, reflux, 1 h, 92%.
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
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Figure 7. cw-EPR spectra of imidazole complex 11-Im in
DMF. Conditions: n ˆ 9.39 GHz, P ˆ 20 mW, Tˆ 104 K,
modulation frequency 100 kHz, modulation amplitude 5.2 G.
Figure 6. cw-EPR spectra of tetrahydrofuranyl model 11 in
dry toluene (top trace) and in water-saturated toluene
(bottom trace). Conditions: n ˆ 9.46 GHz, P ˆ 20 mW, Tˆ
100 K, modulation frequency 100 kHz, modulation amplitude
5.2 G.
bond acceptors, polarising the water molecule which
coordinates to the central metal and hence leading to a
hydroxide character.
A purely rhombic low-spin cw-EPR spectrum, al-
though with slightly different g values (2.42, 2.21, 1.92),
isolated iron(III) porphyrin model compound 11 was can be obtained if iron(III) porphyrin 11 is treated with
confirmed by ESI-MS and MALDI-TOF-MS. an excess of imidazole which is known to be a strong
The cw-EPR spectrum of 11 in dry toluene at low ligand with a high affinity towards iron(III) porphyrins
temperature showed g values (8.33, 5.32, 3.08, 2.08 and (Scheme 8 and Figure 7). This result confirms a general
2.00) characteristic of pure high-spin iron(III) porphyr- trend obvious from an earlier experiment using 10-H2O,
ins with a strong rhombic distortion (Figure 6, top see Figure 4, which on treatment with 1,2 dimethylimi-
trace). If this compound was stirred in water-saturated dazole also changes completely to a low-spin system.[18b]
toluene at room temperature for 2.5 days (Scheme 8)
It is essential to carry out the deprotection reaction of
cw-EPR analysis of the resulting solution revealed very 31 and the iron insertion under strict exclusion of
weak rhombic low-spin signals with g values of 2.22, 2.15 dioxygen because it is well known from previous work in
and 1.97 (Figure 6, bottom trace). Even though the our group that the sulphur of the thiophenolateÀis prone
amount of low-spin signals was very small this result to oxidation to give the FeIII(porph)(ArSO3 ) com-
clearly showed that ether oxygens, when placed in a pound[27]. In practice, the deprotection reactions, the
proper distance in the distal site, can act as hydrogen work-up and the purification of 31 were done under
argon with degassed solvents and reagents. The iron
insertions and all further reactions with the iron(III)
porphyrins were performed in a glove box with inert
atmosphere and degassed solvents and reagents.
Of course, 31 consists of a mixture of four stereo-
isomers. Separation of diastereoisomers was not done at
this stage since the subsequent deprotection reaction
under strongly basic conditions (59 equivalents of
KOMe in refluxing dioxane) leads to epimerisation at
the stereogenic centres. The resulting RR,SS-32 and
RS,SR-32 were separated by flash chromatography
displaying distinct 1H NMR spectra due to their differ-
ent symmetries. Pure RR,SS-32 was used for iron
insertion to yield RR,SS-11; note that 2,6-lutidine is
too weak a base to cause epimerisation at chirality
centres of the tetrahydrofuran subunits. The spectro-
scopic characteristics and the cw-EPR behaviour in the
presence of water were the same as for the diastereoiso-
meric mixture of 11, see above.
Scheme 8. Complexes of active site model 11. Reagents and
conditions: (a) water-saturated toluene, rt, 62 h. (b) ImH (100
equivs.), DMF, rt, 20 h.
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Crown-Capped Iron Porphyrins Models for the
Electrostatic Field Effect and the Polarisation Effect in
the Water Cluster
On one hand the results with the tetrahydrofuranyl
model 11 pointed out that ether oxygens near the distal
site of an iron(III) porphyrin can act as hydrogen bond
acceptors for a coordinated water molecule. On the
other hand we were looking for a model which mimics
the electrostatic field effect. Crown-capped iron(III)
porphyrins 33 (n ˆ 1) and 34 (n ˆ 2) combine these two
required criteria in one molecule (Scheme 9).
The 1,10-diaza-18-crown-6 is known to bind divalent
cations such as Ba2‡[28] which would place a point charge
above the porphyrin plane and allow the investigation of
the electrostatic field effect on the iron(III) spin state. It
was considered to choose a divalent cation in order to
have a larger amount of charge since upon binding of
cations to crown ethers the charge is normally slightly
shielded. In addition, the element providing the cation
ˆ
should not have isotopes with nuclear spin I 1/2 or 3/2
which would complicate cw-EPR analysis or provoke
spin-spin interactions with the paramagnetic iron(III).
Moreover, the oxygens of the crown ether can act as
hydrogen bond acceptors[29] to water coordinated to the
iron(III) if placed in a proper distance. Because this
critical distance is difficult to estimate by molecular
ˆ
modelling, a crown-capped model 33 (n 1) with a C1-
linker and active site model 34 (nˆ2) with a C2-linker
were designed and synthesised. Retrosynthetic discon-
nections are shown in Scheme 9 and simplified crown-
capped models 33 and 34 to 1,10-diaza-18-crown-6 (35), 2-
chloroacetyl chloride for 33, acryloyl chloride for 34 and
the already synthesised bis(aminophenyl) porphyrin 12.
For the capping of the distal face of the porphyrin we
took advantage of published procedures of Collman
et al.[30] and Guilard et al.[29] In the event, bis(amino-
phenyl)porphyrin 12 was acylated with either 2-chloro-
acetyl chloride or acryloyl chloride which provided the
bisamide porphyrins 36 (R ˆ CH2Cl) and 37 (R ˆ
Scheme 10. Synthesis of crown-capped active site analogues
33 and 34. Reagents and conditions: (a) for n ˆ 1:2-chloro-
acetyl chloride (3.8 equivs.), Et3N (4.8 equivs.), cat. DMAP,
CH2Cl2, rt, 15 min, 61% 36; for n ˆ 2: acryloyl chloride (2.4
equivs.), Et3N (27 equivs.), CH2Cl2, rt, 4.5 h, 72% 37. (b) for
R ˆ CH2Cl: 35 (5 equivs.), EtOH, reflux, 21 h, 68% 38; for
ˆ
R ˆ CH CH2: 35 (5 equivs.), MeOH/CH2Cl2 25:1, reflux,
44 h, 66% 39. (c) KOMe in MeOH (10 equivs.), dioxane,
reflux, 10 min, 48% 40, 81% 41. (d) FeBr2 (11 equivs.), 2,6-
lutidine (20 equivs.), toluene, reflux, 30 min, 92% 33, 58% 34.
(e) solid KOMe (60 equivs.), dioxane, reflux, 10 min, 54%.
ˆ
CH CH2) as depicted in Scheme 10. Both compounds,
especially bis(acrylamido)porphyrin 37 are quite sensi-
tive and should be used as soon as possible in the next
step. The capping with diazacrown ether 35 proceeded
via a congruent doublesubstitution reactionto give 38 or
via a congruent double Michael addition which furnish-
ed 39. In both cases the capped products 38 and 39 were
obtained in surprisingly good yields (68% and 66%,
respectively).
Regarding the yield for reactions on a larger scale it
proved to be advantageous in the case of 38 to use the
crude bis(amidophenyl)porphyrin 36 in the subsequent
reaction and to purify after the capping step (see
Experimental Section for details).
Scheme 9. Retrosynthetic analysis of crown-capped porphyr-
ins 33 and 34.
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
Table 1. Proton chemical shifts[a] of crown ether methylene
groups of 40 and 41.
which leads to the conclusion that the oxidation happens
only at sulphur. In contrast, oxidation of the porphyrin
ring (e.g., at the meso-positions) should result in a
spectrum with reduced symmetry and different UV/Vis
and mass spectra.
Compound
dH a-NCH2
dH b-OCH2
dH c-OCH2
40
41
0.45, À 0.17
1.44, 1.59
1.04
1.76
1.98
0.10
After having assured the structures of 40 and 41 iron
insertion was carried out under standard conditions
affording crown-capped iron(III) porphyrins 33 and 34
(Scheme 10). The yield was much lower if using crown-
capped porphyrin 41 with the C2-linker and dropped
drastically if the reaction was carried out on a larger
scale ( > 10 mg). Judging from TLC decomposition is
taking place during the iron insertion reaction with 41.
One reason could be a retro-Michael reaction which is
initiated by the base present (20 equivalents of 2,6-
lutidine) giving the bis(acrylamidophenyl)porphyrin 37
which as such is unstable as already experienced during
the synthesis of the crown-capped models (vide supra).
a All chemical shifts in ppm relative to residual solvent peak.
Spectra were recorded at 298 K in CDCl3.
Figure 8. Atom numbering of the diazacrown cap.
As in the case of tetrahydrofuranylporphyrin 31 the The observation that iron insertion of crown-capped
thiocarbamate protecting groups of 38 and 39 were porphyrin 40 with the C1-linker gives very good yields
cleaved off by treatment with KOMe in refluxing and is not accompanied by decomposition supports this
dioxane. Again, it is absolutely essential that this suggestion since such asidereaction is not possible in the
reaction is carried out under strict exclusion of dioxygen case of 40. Both iron(III) porphyrins 33 and 34 showed
and using a freshly prepared KOMe solution (in MeOH) characteristic physical data (Table 2).
instead of solid KOMe. For instance, a substantial
amount of or even only the S-oxidised product 42 was
isolated if the cleavage reaction of 38 was not done cw-EPR Study of Crown-Capped Iron(III) Porphyrins
carefully enough. Furthermore, the oxidised product 42 and of Their Water Complexes Mimicking the
was very difficult to separate from 40.
Polarisation Effect of the Water Cluster
Due to the bulky thiocarbamate protecting group the
conformational freedom of the sulphur bridge tethered In dry toluene both 33 and 34 showed cw-EPR spectra
to the porphyrin macrocycle is largely reduced. This characteristic of pure high-spin iron(III) porphyrins
results in a reduced symmetry and thus to very with a strong rhombic contribution from the ligand field
1
complicated H NMR spectra, especially in the cases (Figure 9a and 9b, top traces).
of 31, 38 and 39. Once the sulphur protecting group is
The cw-EPR spectrum of 34 in water-saturated
cleaved off the symmetry increases and allows inter- toluene was identical with the spectrum in dry toluene
1
pretation of the H NMR spectra. In addition, homo- (Figure 9b, bottom trace). In contrast, when 33 was
and heterocorrelated 2D NMR techniques were very measured in water-saturated toluene low-spin signals in
helpful to assure the full assignment of all signals and the cw-EPR spectrum appeared (Figure 9a, bottom
together with supportive data from MALDI-TOF-MS trace). These g values are very similar to those obtained
and ESI-MS to confirm the structures. Due to the ring for the low-spin resting state of cytochrome P450cam (g ˆ
current of the porphyrin the methylene groups of the 2.45, 2.26, 1.91, Figure 3)[8] and to those obtained for the
crown ether are high-field shifted (Table 1 and Figure 8) imidazole complex 11-Im (g ˆ 2.42, 2.21, 1.92, Figure 7).
which is another indication of a successfully capped A sample of 33 in water-saturated toluene was periodi-
structure.
cally measured over atime period of24 h tosee if there is
1
The H NMR of the S-oxidised crown-capped por- an increase in intensity of the low-spin signals but this
phyrin 42 also shows a spectrum with high symmetry was not the case. It was calculated that in a typical 5 mM
Table 2. Physical data of iron(III) porphyrins 33 and 34.
Compound
UV/Vis[a] [nm]
MALDI-TOF-MS [m/z]
ESI-MS[b] [m/z]
‡
‡
‡
33
34
404
406
1447 [M ‡ 2]
1469 [M ‡ Na]
‡
1475 [M ‡ 1]
1497 [M ‡ Na]
[a]
In toluene at 298 K.
In MeOH.
[b]
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Martin Lochner et al.
FULL PAPERS
Figure 10. Different water binding motives of crown-capped
models 33 and 34.
toluene. In contrast, due to the different length of the
linker of crown-capped iron(III) porphyrin 34 hydrogen
bonding is not possible and therefore only high-spin
signals are observed in the cw-EPR spectrum.
As for to the bis(tetrahydrofuranyl)iron(III) porphyr-
in 11 we tried to generate low-spin imidazole complexes
of the crown-capped iron(III) porphyrin models. The
model compound 34 was chosen due to the longer C2-
linkers and hence better accessibility of the imidazole to
the central metal and treated with a large excess of the
ligand (Scheme 11). The analysis by cw-EPR showed
low-spin signals with g values of 2.45, 2.21 and 1.91 which
were very similar to g values found for the imidazole
complex 11-Im, however, displaying much lower inten-
sity (Figure 11).
For yet unknown reasons attempts to prepare the
hydroxide complex (HOÀ Fe(III) SÀ) from either 33-
H2O or 34-H2O failed. In contrast we have been able to
prepare the required adduct from 10-H2O, see Figure 4,
by deprotonation with ethyldiisopropylamine. As ex-
pected the cw-EPR of 10-OHÀ showed a complete
conversion to a low-spin system displaying g values ˆ
2.20, 2.02, 1.95. The coordination features of the ™open-
face∫ iron thiolate porphyrins 10 and 11 versus the
Figure 9. cw-EPR spectra of crown-capped models 33 (a) and
34 (b) in dry toluene (top traces) and in water-saturated
toluene (bottom traces). Conditions: n ˆ 9.46 GHz, P ˆ 20
mW, Tˆ 100 K, modulation frequency 100 kHz, modulation
amplitude 5.2 G.
cw-EPR iron(III) porphyrin sample in water-saturated ™crown-capped∫ complexes 33 and 34 suggest that the
toluene only ~ 3 4 equivalents of water are present, latter are sterically much more congested such that the
thus it is not surprising that the system does not convert sixth coordination site of iron(III) is much less acces-
completely to low spin. In water-saturated 2-MTHF cw- sible than molecular modelling predicts.
EPR spectra of 33 (not shown) displayed the same low-
spin signals albeit with lower intensity.
Guilard and co-workers have prepared a crown-
capped zinc porphyrin[29] which has the same distal
architecture as crown-capped model 33 and they were
able to solve the crystal structure of the corresponding
water complex. This analysis revealed that the water
molecule was bound to the central Zn(II) ion and was
hydrogen bonded to two crown ether oxygens with
hydrogen bond distances of 1.95 ä and 1.83 ä. It is
reasonable to assume that water binds in the same
fashion to crown-capped iron(III) porphyrin 33 (Fig-
ure 10) and these hydrogen bonds presumably polarise
the coordinated water molecule leading to a distinctive
hydroxide character which gives rise to low-spin signals
Scheme 11. Synthesis of crown-capped imidazole complex 34-
Im. Reagents and conditions: (a) ImH (100 equivs.), DMF, rt,
in the cw-EPR spectrum of 33 in water-saturated 39 h.
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
Figure 11. cw-EPR spectra of reaction mixture in DMF after
treatment of 34 with excess of imidazole. Conditions: n ˆ 9.39
GHz, P ˆ 20 mW, Tˆ 104 K, modulation frequency 100 kHz,
modulation amplitude 5.2 G.
cw-EPR and ESI-MS Studies of Barium Complexes of
Crown-Capped Iron(III) Porphyrins Mimicking the
Electrostatic Field Effect
If solutions of crown-capped iron(III) porphyrins 33 and
34 in acetone were treated with 5 equivalents of
Ba(ClO4)2 and the resulting solutions were ultrasoni-
cated the ESI-MS analysis of the mixtures revealed mass
peaks for the corresponding complexes next to the [M ‡
‡
‡
H] and [M ‡ Na] peaks (Figure 12, insets). However,
binding of barium(II) ions did not change the cw-EPR
spectra of the compounds. In fact, model compound 34-
H2O/Ba2‡ with C2-linkers remains high-spin in water-
saturated toluene as well as in acetone (Figure 12b) and
33-H2O/Ba2‡ shows the same content of low-spin in
water-saturated toluene as 33-H2O (Figure 12a).
Figure 12. cw-EPR spectra of 33 in water-saturated toluene
(a) and of 34 in acetone (b) after addition of 5 equivalents of
Ba(ClO4)2. Insets: corresponding ESI-MS in acetone. EPR
conditions: n ˆ 9.47 GHz, P ˆ 20 mW, Tˆ 100 K, modulation
frequency 100 kHz, modulation amplitude 5.2 G.
It is difficult to estimate to what extent the charge of
the bound barium(II) ion is shielded by the diazacrown
ether and in organic solvents like toluene the counterion
perchlorate is probably in close proximity to the
barium(II) which may further decrease the point charge.
‡
Such ion paired [M ‡ Ba ‡ ClO4] species are even
detectable in ESI-MS (Figure 12, insets). Therefore, in
order to increase the dipole-distance between the
barium(II) and the counterion the perchlorates were
exchanged by the large, lipophilic TRISPHAT anions
developed by Lacour et al.[31] Barium(II) complex 33-
Ba2‡ was treated with (Bu3NH)[TRISPHAT][32] in
CH2Cl2 and the resulting solution was used for the
analysis. Gratifyingly, the desired mass peaks could be
detected by ESI-MS (Figure 13) but the cw-EPR
spectrum of this solution in water-saturated toluene
showed a high content of rhombic high-spin signals and
Figure 13. Positive ion mode ESI-MS in acetone after anion
exchange of 33-Ba2‡ with
2 equivalents of (Bu3NH)
very little to no content of low-spin signals (not shown). [TRISPHAT]; inset: same sample at negative ion mode.
Taken together, the experiments point to the fact that
a positively charged barium(II) ion attached above the
In order to study an alternative model for the electro-
porphyrin ring does not have a significant influence on static field effect our attention was drawn to the idea
the spin state of the iron(III).
with a fixed charge above the porphyrin plane. Inspired
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Scheme 12. Synthesis of N-alkylated crown-capped iron(III)
porphyrin 43 and generation of S-alkylated side product 44.
Reagents and conditions: (a) MeI (291 equivs.) in portions
over a time period of 20 h, toluene, rt, 24% 43 and 76% 44.
by the idea of Collman et al.[33] who intended to
synthesise water-soluble polyazacrown-capped por-
phyrins as oxygen carriers by alkylating the nitrogens
of the polyazacrown caps, iron(III) porphyrin model
compound 33 was reacted with an excess of iodo-
methane (Scheme 12). This yielded two products, both
having the same mass according to ESI-MS and
MALDI-TOF-MS but different colour, UV/Vis spectra
and Rf values on TLC (Table 3).
Figure 14. cw-EPR of N-methylated 43 (a) and S-methylated
44 (b) in dry toluene (top traces) and water-saturated toluene
(bottom traces). Conditions: n ˆ 9.47 GHz, P ˆ 20 mW, Tˆ
100 K, modulation frequency 100 kHz, modulation amplitude
5.2 G.
Because of its brown colour, the very similar UV/Vis
and cw-EPR spectra (vide infra) compared to the
starting material 33 the less polar spot on TLC was Soret band in the region of 415 nm.[35] Accordingly, the
assigned to the N-methylated compound 43. Presum- assignment of the red compound presented here may be
ably, the more polar red compound which had a rather inappropriate and 44 should be synthesised independ-
unusual UV/Vis and cw-EPR spectrum is S-methylated ently for comparison. However, it is also difficult to
44. An S-methyl thiophenyliron(III) porphyrin was think of an alternative structure. Formation of a carbene
prepared in our group by Matile as a model compound species as observed with iron(II) porphyrins and CDCl3
for cytochrome c[34] and it displayed a Soret band at by St‰ubli[27] seems rather unlikely.
424 nm. On the other hand, an S-methyl cytochrome c
As already mentioned the cw-EPR spectrum of brown
model compound synthesised by Nagano et al. showed a product 43 in dry toluene was very similar to the
Table 3. Physical data of N- and S-methylated iron(III) porphyrins 43 and 44.
Compound
Colour[a]
Rf[b]
UV/Vis[c] [nm (%)]
ESI-MS[d] [m/z (%)]
‡
‡
43
44
brown
red
0.58
0.41
404 (100); 513 (11)
365 (sh, 58); 405 (100); 573 (18)
1461 (100, M )
1461 (100, M )
[a]
Toluene solution.
[b]
Silica gel, toluene/THF, 2: 1.
In toluene at 298 K.
In acetone.
[c]
[d]
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
spectrum of starting material 33 (Figure 14a, top trace)
showing characteristic peaks for a rhombically distorted
high-spin iron(III) species. However, when 43 was
measured in water-saturated toluene the already known
low-spin signals (Figure 14a, bottom trace) were ob-
served to more or less the same extent as in the water
complex 33-H2O and the Ba2‡/water complex 33-H2O/
Ba2‡ (vide supra).
The cw-EPR spectrum of the red compound 44 in dry
toluene also shows signals for a pure rhombic high-spin
iron(III) but with a completely different pattern (Figure
14b, top trace). Measuring the sample in water-saturated
toluene results in an identical spectrum (Figure 14b,
bottom trace). An interesting fact is that the spectra of
44 show a very high similarity with the high-spin portion
of the cw-EPR spectrum of camphor-bound cytochrome
P450cam (Figure 3).[7,8]
Figure 16. Model compounds for studying both effects simul-
taneously.
The results withtheN-methylated crown-capped model
43 again point to the fact that a charge above the equilibrium of P450cam. In contrast, it was qualitatively
porphyrin plane does not influence the iron(III) spin state. but clearly demonstrated that the spin equilibrium of the
heme-thiolate cofactor shifts towards low-spin if the
water coordinating to iron(III) adopts a distinctive
hydroxide character due to hydrogen bonding interac-
tions. This conclusion is also supported by calcula-
Conclusion
The water adducts of the crown-capped iron(III) tions.[36]
thiolate porphyrins 33 and 34 proved to be active site
In summary, experiments support that polarisation in
analogues of the resting state of P450cam. 33-H2O and 34- the water cluster plays an important role for the
H2O, their corresponding Ba2‡ complexes and 43-H2O observed low-spin resting state of P450cam. Accordingly,
are versatile model compounds to distinguish between the water coordinating to iron has a functional role[37]
two effects that could possibly modulate the spin state of not only for adjusting Eo to À 300 mV but also fine-
heme-thiolate proteins (Figures 15 and 16).
tuning the spin state of the cofactor.
Taking together the results presented in this work
suggest that a distant charge above the porphyrin has no
or very little effect on the spin state of the iron(III).
Accordingly, the results do not support the predictions
by Harris and Loew[17a] that the electrostatic field of the
protein and the coordination of water modulate the spin
Experimental Section
General Remarks
Reagents were used as received from Fluka AG (Buchs,
Switzerland), Merck AG (Darmstadt, Germany) and Aldrich
(Buchs, Switzerland) unless otherwise stated. Chemicals of the
quality purum, purum p. a. or>98% were used without further
purification. 2,6-Lutidine for iron insertions was purchased
from Aldrich, dried over solid KOH, distilled from BaO and
degassed in high-vacuum by four freeze-pump-thaw cycles.
Dry dichloromethane (CH2Cl2) was distilled from CaH2, Et2O
and THF from Na/benzophenone, MeOH from Mg, DMF was
dried over solid KOH and distilled from BaO, dioxane and
toluene were distilled from Na. All freshly dried solvents were
stored over activated molecular sieves. Further solvents used
for reactions corresponded to the quality puriss p. a., abs., over
Molecular Sieves from Fluka AG. Degassed solvents for
reactions under oxygen-free condition (e.g., in the glove box)
were obtained by at least four freeze-pump-thaw cycles. For all
non-aqueous reactions glassware was flame dried and the
atmosphere was exchanged by argon. Iron insertions and all
other reactions with iron porphyrins were carried out in a
Labmaster 130 glove box (MBRAUN). The levels of dioxygen
(<2 ppm) and water (<0.1 ppm) were measured with a
Figure 15. Separation of the remote charge and the polar-
isation effect by the model compounds 33-H2O and 34-H2O/
Ba2‡.
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Martin Lochner et al.
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to-bulb (1808C/0.1 mbar) which gave the title compound as
light yellow oil which solidified on standing at 48C; yield: 3.7 g
(82%); Rf ˆ 0.41 (hexane/ethyl acetate, 5:1). IR (KBr): n ˆ
3218 m, 3001 m, 2979 m, 2939 m, 2894 m, 1731 s, 1647 s, 1612 s,
1588 s, 1534 s, 1521 s, 1477 s, 1454 s, 1419 s, 1401 s, 1369 s, 1293 s,
1270 s,1229 s, 1199 s, 1157 s, 1049 s, 1027 s, 760 m, 718 m cmÀ1;
1H NMR (300 MHz, CDCl3): d ˆ 10.96 (br, s, 1H, NH), 10.48 (s,
1H, CHO), 8.00 (d, J ˆ 10.1 Hz, 1H, aryl H-5), 7.44 (t, J ˆ
9.8 Hz, 1H, aryl H-4), 6.53 (d, J ˆ 9.8, 1H, aryl H-3), 3.90 (s,
3H, OCH3), 1.47 (s, 9H, t-Bu); EI-MS (70 eV): m/z (%) ˆ 251
Figure 17. Atom numbering for NMR assignment.
‡
(14, M ); 195 (25); 178 (25); 167 (16); 152 (5); 151 (44); 150 (7);
133 (6); 123 (15); 122 (43); 106 (6); 94 (9); 93 (16); 92 (5); 65 (7);
58 (6); 57 (100); 41 (27); 39 (9).
combined H2O/O2-analyser (MBRAUN). All solvents and
reagents used in the glove box were dried and degassed in high-
vacuum. For flash chromatography silica gel 60 from Merck
(0.043 0.06 mm, 230 400 mesh) or aluminium oxide 90 from
Merck (standardised (activity II III), 0.063 0.2 mm, 70 230
mesh) were used. Analytical RP-HPLC was performed on
aa/ab-10,20-Bis[6-(tert-butoxycarbonyl)amino-2-
methoxyphenyl]-3,7,13,17-tetrabutyl-2,8,12,18-
tetramethylporphyrin (aa/ab-22)
¾
LiChrospher 100 RP-18 silica gel from Merck (5 mm particle
size, 4 Â 250 mm column) with nanopure water and HPLC-
grade MeOH or acetonitrile. Ultra violet-visible absorption
spectra were recorded on a Hewlett-Packard 8452A Diode
Array spectrophotometer and an Agilent 8453 Diode Array
spectrophotometer. Infrared spectra were measured on a
Perkin-Elmer 1600 series FTIR spectrometer in KBr (1% w/
w). NMR was performed using either a Bruker av250
(250 MHz), Varian Gemini 300 (300 MHz), Bruker DPX-
NMR (400 MHz), Bruker DRX-500 (500 MHz) or Bruker
DRX-600 (600 MHz) spectrometer. CDCl3 was filtered
through basic alumina prior to use. dH and dC in ppm relative
to residual solvent peaks. Porphyrin models atom numbering
for NMR assignment see Figure 17.
EI-MS was measured on a Varian double focusing VG-70-
250 spectrometer and ESI-MS were recorded on a Finnigan
Mat LCQ-700. For MALDI-TOF-MS a Perseptive Biosystems
Vestec Mass Spectrometry Products VoyagerTM Elite Biospec-
trometryTM Research Station was used. cw-EPR was carried
out using a Bruker ESP-300 X-band (nmicrowave ˆ 9.485 GHz)
spectrometer equipped with a T102 cell and an ER4111VT
liquid nitrogen cryostat to assure low temperature during
measurement. Sample concentration was typically 5 mM and
spectra were measured with a microwave power of 20 mW,
modulation frequency of 100 kHz, and a modulation amplitude
of 5.2 G.
A solution of tert-butyl N-(2-formyl-3-methoxyphenyl)carba-
mate (16) (4.59 g, 18.3 mmol) in MeCN (400 mL) was degassed
by bubbling with argon for 15 min. Then 3,3'-dibutyl-4,4'-
dimethyl-2,2'-dipyrrylmethane (15) (5.24 g, 18.3 mmol) was
added with a spatula and the reaction mixture was bubbled
with argon for another 15 min. After that p-toluenesulphonic
acid (346 mg, 1.7 mmol) was added and the solution turned
immediately dark-brown. Stirring was continued in the dark
overnight at room temperature. For the subsequent oxidation
of the formed porphyrinogens DDQ (8.29 g, 36.6 mmol) was
dissolved in THF (80 mL) and added to the reaction mixture.
The black mixture was stirred under air at room temperature
and completion of the oxidation was followed with UV/Vis
(CH2Cl2 ‡ 1% Et3N) by the disappearance of the porphyrino-
gen absorption at 483 nm and the appearance of the porphyrin
Soret absorption at 406 nm. After 1.5 h all the volatiles were
removed under vacuum which gave 20 g of crude product that
was dissolved in CH2Cl2 and filtered through aluminium oxide.
Fractions were controlled by TLC (silica gel, hexane/ethyl
acetate, 3:1 ‡ 1% Et3N) and MALDI-TOF-MS and those
‡
‡
displaying mass peaks at m/z ˆ 1033 (M ), 933 ([M Boc] )
‡
and 833 ([M 2 Boc] ) were combined. After removing the
solvent under reduced pressure this gave 5.26 g of porphyrin
mixture as purple solid which was used without further
purification for the next step.
tert-Butyl N-(2-Formyl-3-methoxyphenyl)carbamate
(16)
aa/ab-10,20-Bis(6-amino-2-methoxyphenyl)-3,7,13,17-
tetrabutyl-2,8,12,18-tetramethylporphyrin (aa/ab-23)
tert-Butyl N-(3-methoxyphenyl)-carbamate (19) (4 g,
17.9 mmol) was dissolved in dry Et2O (20 mL) and cooled to
À 308C. To this solution was added dropwise a 1.5 M solution
of t-BuLi (in hexane, 26.72 mL, 39.4 mmol) and the temper-
ature was kept below À 158C. After having finished the
addition the solution was stirred for 3 h at À 108C. Then the
reaction mixture was cooled to À 788C and DMF (2.86 mL,
37.1 mmol) in dry Et2O (5 mL) was added dropwise. After-
wards it was warmed to room temperature and stirring was
continued overnight. The reaction was quenched with brine
and extracted three times with Et2O. The organic layers were
separated, washed with water, dried over Na2SO4 and concen-
trated under vacuum. The viscous yellow oil was distilled bulb-
The porphyrin mixture (3.28 g, 3.17 mmol) from the previous
step was dissolved in CH2Cl2 (300 mL) and a solution of
trifluoroacetic acid (20 mL, 261 mmol) in CH2Cl2 (50 mL) was
added within 20 min via a dropping funnel at room temper-
ature. The greenish-purple solution was stirred for 12.5 h at
room temperature. The reaction mixture was cooled in an ice
bath and quenched with saturated aqueous NaHCO3
(300 mL). After phase separation, the organic layer was
washed again with saturated aqueous NaHCO3 (200 mL).
The combined aqueous phases were extracted with CH2Cl2
(2 Â 100 mL) until colourless. The combined organic phases
were finally washed with water (300 mL) and brine (200 mL),
dried over Na2SO4 and concentrated under vacuum. The crude
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
product was purified by flash chromatography (silica gel,
hexane/ethyl acetate, 6:1 ‡ 1% Et3N) which yielded 1.91 g
(72%) of a 6:4-mixture of aa-23 and ab-23, respectively
(estimated from 1H NMR) as a purple solid. Rf ˆ 0.34 (hexane/
ethyl acetate, 3:1 ‡ 1% Et3N); UV/Vis (CH2Cl2 ‡ 1% Et3N):
lmax (%) ˆ 410 (100, Soret), 508 (12), 542 (8), 574 (9), 586 nm
(7). IR (KBr): n ˆ 3462 m, 3445 m, 3378 m, 2954 s, 2928 s, 2868 s,
2858 s, 1609 s, 1596 s, 1584 s, 1576 s, 1569 s, 1559 m, 1471 s, 1437 s,
1256 s, 1131 s, 1087 s, 762 m cmÀ1; 1H NMR (400 MHz, CDCl3):
d ˆ 10.16 (s, 2H, meso-H), 7.55 (t, J ˆ 8.2 Hz, 2H, aryl H-4'),
6.76 (m, 4H, aryl H-3', aryl H-5'), 4.00 (m, 8H, PyCH2), 3.63 (s,
6H, aa-OCH3), 3.55 (s, 6H, ab-OCH3), 3.36 (br, s, 2H, ab-
NH2), 3.23 (br, s, 2H, aa-NH2), 2.74 (s, 12H, PyCH3), 2.17 (m,
8H, PyCH2CH2), 1.75 (m, 8H, Py(CH2)2CH2), 1.09 (t, J ˆ
7.3 Hz, 12H, Py(CH2)3CH3), À 2.32 (br, s, 2H, NH pyrrole);
2H, aryl H-4'), 7.13 (d, J ˆ 8.3 Hz, aryl H-3'), 6.49 (br, s,
NHAc), 4.01 (m, 8H, PyCH2), 3.57 (s, 6H, OCH3), 2.60 (s, 12H,
PyCH3), 2.18 (m, 8H, PyCH2CH2), 1.73 (m, 8H, Py(CH2)2CH2),
1.09 (s, 6H, COCH3), 1.09 (t, J ˆ 7.4 Hz, 12H, Py(CH2)3CH3),
À 2.30 (br, s, 2H, NH pyrrole); MALDI-TOF-MS: m/z (%) ˆ
‡
‡
917 (100, [M ‡ 1] ), 939 (9, [M ‡ Na] ).
aa-10,20-Bis(6-acetylamino-2-hydroxyphenyl)-
3,7,13,17-tetrabutyl-2,8,12,18-tetra-methylporphyrin
(aa-25)
Porphyrin aa-24 (1.03 g, 1.12 mmol) was dissolved in dry
CH2Cl2 (75 mL) and to this solution was added ethanethiol
(110 mL) followed by AlCl3 (5.7 g, 42.7 mmol) in portions. The
solution turned grass-green and it was stirred at room temper-
ature for 43 h. The course of the reaction was followed by
taking aliquots from the reaction mixture and injecting them
into the RP-HPLC after a mini-work up [RP-HPLC: 90 100%
MeCN/H2O in 10 min, then 100% MeCN for 10 min, flow
1.5 mL/min, ldet ˆ 407 nm, Tˆ 408C; Rt (product) ˆ 4.4 min;
Rt (monomethoxy side product) ˆ 6.1 min; Rt (starting mate-
rial) ˆ 9.4 min]. The reaction was stopped by cooling the
mixture in an ice bath and slow addition of saturated aqueous
NaHCO3 (200 mL). After separating the two phases the
organic phase was washed with of saturated aqueous NaHCO3
(2 Â 140 mL). The aqueous phases were extracted with CH2Cl2
(2 Â 200 mL) to minimise loss of material. The combined
organic phases were washed with water (400 mL) and brine
(300 mL), dried over Na2SO4 followed by removal of volatiles
under vacuum. The crude amorphous purple solid was purified
by flash chromatography (silica gel, hexane/ethyl acetate/
CH2Cl2, 4:2:1 ‡ 1% Et3N) which yielded 726 mg (73%) of the
title compound as purple solid; Rf ˆ 0.34 (hexane/ethyl
acetate, 1:1 ‡ 1% Et3N); RP-HPLC (90 100% MeCN/H2O
in 10 min, then 100% MeCN for 10 min, flow 1.5 mL/min,
ldet ˆ 407 nm, Tˆ 408C): Rt ˆ 4.4 min; UV/Vis (CH2Cl2 ‡ 1%
Et3N): lmax (%) ˆ 408 (100, Soret), 508 (8), 542 (4), 578 (4),
626 nm (2); IR (KBr): n ˆ 3530 s, 3403 s, 2955 s, 2928 s, 2859 s,
1676 s, 1588 s, 1527 s, 1466 s, 1370 s, 1339 s, 1273 s, 1178 m, 1156 s,
1138 m, 1100 m, 1038 m, 983 m, 950 m, 894 w, 846 w, 758 m, 735
m, 618 m, 576 w cmÀ1; 1H NMR (400 MHz, CDCl3): d ˆ 10.34
(s, 2H, meso-H), 8.38 (d, J ˆ 8.1 Hz, 2H, aryl H-5'), 7.75 (t, J ˆ
8.3 Hz, 2H, aryl H-4'), 7.19 (dd, J ˆ 8.3 Hz, J ˆ 0.8 Hz, 2H, aryl
H-3'), 6.44 (s, 2H, NHAc), 4.03 (m, 8H, PyCH2), 2.69 (s, 12H,
PyCH3), 2.19 (m, 8H, PyCH2CH2), 1.77 (m, 8H, Py(CH2)2CH2),
1.14 (s, 6H, CH3CO), 1.11 (t, J ˆ 7.5 Hz, 12H, Py(CH2)3CH3),
À 2.31 (br, s, 2H, NH pyrrole); MALDI-TOF-MS: m/z (%) ˆ
‡
MALDI-TOF-MS: m/z (%) ˆ 834 (100, [M ‡ 1] ).
aa-10,20-Bis(6-acetylamino-2-methoxyphenyl)-
3,7,13,17-tetrabutyl-2,8,12,18-tetramethyl-porphyrin
(aa-24) and ab-10,20-Bis(2-acetylamino-6-
methoxyphenyl)-3,7,13,17-tetrabutyl-2,8,12,18-
tetramethylporphyrin (ab-24)
A solution of the atropisomeric mixture of porphyrin aa/ab-23
(1.91 g, 2.29 mmol) in dry CH2Cl2 (150 mL) was prepared and
dry pyridine (0.4 mL, 7.64 mmol) and DMAP (60 mg,
0.49 mmol) added. Then a solution of acetyl chloride
(0.4 mL, 5.63 mmol) in dry CH2Cl2 (75 mL) was added drop-
wise within 25 min at room temperature. The reaction mixture
was stirred at room temperature for 18 h and quenched with
saturated aqueous NaHCO3 (150 mL) while cooling in an ice
bath. The layers were separated and the aqueous phase was
extracted with CH2Cl2 (2 Â 75 mL). The combined organic
layers were washed with water (250 mL) and brine (150 mL),
dried over Na2SO4 and the solvents were evaporated under
vacuum. Purification of the crude product and separation of
the two atropisomers was achieved by repetitive flash chro-
matography (silica gel, hexane/ethyl acetate, 3:1 ‡ 1% Et3N,
and hexane/ethyl acetate/CH2Cl2, 6:2:1 ‡ 1% Et3N). This
yielded 1.02 g (49%) of aa-24 and 512 mg (24%) of ab-24 as
purple solids (combined yield: 73%).
Physical data of aa-24: Rf ˆ 0.18 (hexane/ethyl acetate/
CH2Cl2, 6:2:1 ‡ 1% Et3N); RP-HPLC (90 100% MeOH/
H2O in 25 min, then 100% MeOH for 5 min, flow 1.5 mL/min,
ldet ˆ 410 nm, Tˆ 408C): Rt ˆ 12.2 min; UV/Vis (CH2Cl2 ‡ 1%
Et3N): lmax (%) ˆ 410 (100, Soret), 508 (7), 542 (2), 574 (2),
628 nm (4); 1H NMR (400 MHz, CDCl3): d ˆ 10.26 (s, 2H,
meso-H), 8.45 (d, J ˆ 8.3 Hz, 2H, aryl H-5'), 7.81 (t, J ˆ 8.5 Hz,
2H, aryl H-4'); 7.14 (d, J ˆ 8.3 Hz, aryl H-3'), 6.51 (br, s,
NHAc), 4.01 (m, 8H, PyCH2), 3.60 (s, 6H, OCH3), 2.61 (s, 12H,
PyCH3), 2.19 (m, 8H, PyCH2CH2), 1.75 (m, 8H, Py(CH2)2CH2),
1.11 (t, J ˆ 7.3 Hz, 12H, Py(CH2)3CH3), 1.10 (s, 6H, COCH3),
À 2.36 (br, s, 2H, NH pyrrole); MALDI-TOF-MS: m/z (%) ˆ
‡
‡
889 (100, [M ‡ 1] ); 910 (12, [M ‡ Na] ).
Strapped porphyrin 27
To a solution of bisphenol porphyrin aa-25 (6.9 mg, 7.8 mmol)
in dry DMF (4 mL) was added Cs2CO3 (77 mg, 236 mmol,
previously dried overnight at 1008C in high vacuum). This
suspension was heated to 608C and stirred for 30 min. Then a
solution of 1,11-dibromoundecane (2.7 mL, 11 mmol) in dry
DMF (1 mL) was added via a syringe pump within 3 h at 608C.
Stirring was continued at 608C for 1 h after which the reaction
mixture was poured into a ice-cooled mixture of 1 M HCl
(2 mL) and CH2Cl2 (5 mL). The layers were separated and the
‡
‡
917 (100, [M ‡ 1] ), 939 (8, [M ‡ Na] ).
Physical data of ab-24: Rf ˆ 0.26 (hexane/ethyl acetate/
CH2Cl2, 6:2:1 ‡ 1% Et3N); RP-HPLC (90 100% MeOH/
H2O in 25 min, then 100% MeOH for 5 min, flow 1.5 mL/min,
ldet ˆ 410 nm, Tˆ 408C): Rt ˆ 10.6 min; UV/Vis (CH2Cl2 ‡ 1%
Et3N): lmax (%) ˆ 410 (100, Soret), 508 (10), 542 (5), 574 (5),
588 nm (4); 1H NMR (400 MHz, CDCl3): d ˆ 10.25 (s, 2H,
meso-H), 8.44 (d, J ˆ 8.6 Hz, 2H, aryl H-5'), 7.81 (t, J ˆ 8.5 Hz,
Adv. Synth. Catal. 2003, 345, 743 765
asc.wiley-vch.de
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
757

Martin Lochner et al.
FULL PAPERS
aqueous phase was extracted with CH2Cl2 (2 Â 5 mL). The
combined organic phases were washed with saturated aqueous
NaHCO3 (2 Â 20 mL), water (10 mL) and brine (10 mL), dried
over Na2SO4 and all volatiles were removed under vacuum.
Purification by flash chromatography (silica gel, hexane/ethyl
acetate, 2:1 ‡ 1% Et3N) yielded 5.1 mg (63%) of the title
compound as purple solid; Rf ˆ 0.56 (hexane/ethyl acetate,
1:1 ‡ 1% Et3N); UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 410
C-1, C-9), 135.5 (s, C-11, C-19), 130.8 (d, aryl CH-4'), 123.3 (s,
aryl C-2'', aryl C-6''), 123.0 (d, aryl CH-3'', aryl CH-5''), 120.6 (s,
aryl C-6'), 113.3 (d, aryl CH-5'), 109.3 (d, aryl CH-3'), 107.4 (s,
C-10, C-20), 97.0 (d, meso-CH), 96.0 (d, meso-CH), 69.9 (t, a-
CH2), 35.6 (t, PyCH2CH2), 35.1 (t, PyCH2CH2), 34.0 (s,
C(CH3)3), 32.5 (q, NCH3), 31.0 (q, C(CH3)3), 30.6 (t, g-CH2),
28.6 (t, b-CH2), 26.4 (t, PyCH2), 26.1 (t, PyCH2), 24.7 (q,
COCH3), 23.3 (t, Py(CH2)2CH2), 23.2 (t, Py(CH2)2CH2), 14.2
(q, Py(CH2)3CH3), 14.1 (q, Py(CH2)3CH3), 13.8 (q, PyCH3),
13.4 (q, PyCH3); MALDI-TOF-MS: m/z (%) ˆ 1135 (11, [M
1
(100, Soret), 508 (5), 542 (1), 580 (2), 628 nm (1); H NMR
(400 MHz, CDCl3): d ˆ 10.23 (s, 2H, meso-H), 8.46 (d, J ˆ
8.3 Hz, 2H, aryl H-5'), 7.77 (t, J ˆ 8.3 Hz, 2H, aryl H-4'), 7.08
(dd, J ˆ 8.3 Hz, J ˆ 0.8 Hz, 2H, aryl H-3'), 6.86 (s, 2H, NHAc),
4.00 (m, 8H, PyCH2), 3.82 (m, 4H, a-CH2), 2.61 (s, 12H,
PyCH3), 2.20 (m, 8H, PyCH2CH2), 1.77 (m, 8H, Py(CH2)2CH2),
1.21 (s, 6H, CH3CO), 1.11 (t, J ˆ 7.3 Hz, 12H, Py(CH2)3CH3),
0.70 (m, 4H, b-CH2), À 0.34 (m, 4H, g-CH2), À 0.67 (m, 4H, d-
CH2), À 1.61 (br, s, 6H, e-CH2, z-CH2), À 2.32 (br, s, 2H, NH
‡
‡
CONMe2] ); 1207 (100, [M ‡ 1] ).
Thiocarbamoylbisaminoporphyrin 12
‡
pyrrole); MALDI-TOF-MS: m/z (%) ˆ 1042 (100, [M ‡ 1] ).
Bis(acetylamido)porphyrin 30 (222 mg, 184 mmol) was dis-
solved in MeOH (57 mL) and to this solution was added 6 M
HCl (87 mL). The resulting grass-green solution was heated to
reflux and stirred for 2 h after which the reaction mixture was
cooled in an ice bath and the acid was neutralised dropwise
with aqueous 50% NaOH (32 mL). The basic aqueous phase
was extracted with CH2Cl2 (3 Â 140 mL). The combined
organic phases were washed with water (2 Â 150 mL) and
brine (140 mL), dried over Na2SO4 and all volatiles were
removed under vacuum. After purification by flash chroma-
tography (silica gel, hexane/ethyl acetate, 3:1 ‡ 1% Et3N)
188 mg (91%) of pure bisamino porphyrin 12 was obtained as
purple solid; Rf ˆ 0.66 (hexane/ethyl acetate, 1:1 ‡ 1% Et3N);
RP-HPLC (90 100% MeOH/H2O in 10 min, then 100%
MeOH for 10 min, flow 1.5 mL/min, ldet ˆ 414 nm, Tˆ 408C):
Rt ˆ 11.1 min (broad); UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax
(%) ˆ 414 (100, Soret), 510 (9), 544 (3), 578 (4), 631 nm (2);
IR (KBr): n ˆ 3468 w, 3362 m, 2956 s, 2870 m, 1707 w, 1654 s,
1596 s, 1459 s, 1364 m, 1258 s, 1094 m cmÀ1; 1H NMR (400 MHz,
CDCl3): dH ˆ 10.07 (s, 1H, meso-H), 9.65 (s, 1H, meso-H), 7.53
(t, J ˆ 8.2 Hz, 2H, aryl H-4'), 6.87 (d, J ˆ 8.3 Hz, 2H, aryl H-5'),
6.61 (d, J ˆ 8.3 Hz, 2H, aryl H-3'), 6.32 (s, 2H, aryl H-3'', aryl H-
5''), 4.29 (br, s, 4H, NH2), 4.02 (m, 4H, PyCH2), 3.88 (m, 2H,
PyCH2 and a-CH2), 3.79 (m, 2H, PyCH2 and a-CH2), 3.57 (m,
4H, PyCH2 and a-CH2), 2.92 (s, 6H, PyCH3), 2.69 (s, 6H,
PyCH3), 2.26 (m, 4H, PyCH2CH2), 1.95 (m, 2H, PyCH2CH2),
1.85 (m, 6H, PyCH2CH2 and Py(CH2)2CH2), 1.79 (br, s, NCH3),
1.71 (m, 4H, Py(CH2)2CH2), 1.16 (t, J ˆ 7.3 Hz, 6H,
Py(CH2)3CH3), 1.15 (t, J ˆ 7.3 Hz, 6H, Py(CH2)3CH3), 1.06 (s,
9H, t-Bu), 0.60 (m, 4H, b-CH2), 0.48 (m, 2H, g-CH2), À 0.04 (m,
2H, g-CH2), À 1.16 (br, s, 3H, NCH3), À 2.07 (br, s, 2H, NH
pyrrole); 13C NMR (150 MHz, CDCl3): d ˆ 167.1 (s, CONMe2),
160.9 (s, aryl C-2'), 150.2 (s, aryl C-4''), 147.1 (s, aryl C-6'), 145.7
(s, C-12, C-18), 145.5 (s, C-2, C-8); 144.5 (s, arylC-1''); 142.9 (s,
C-4, C-6, C-14, C-16); 141.3 (s, C-3, C-7); 140.9 (s, C-13, C-17),
136.1 (s, C-1, C-9), 135.6 (s, C-11, C-19), 130.3 (d, aryl CH-4'),
123.7 (s, aryl C-2'', aryl C-6''), 123.0 (d, aryl CH-3'', aryl CH-5∫),
117.5 (s, aryl C-1'), 109.5 (s, C-10, C-20), 108.7 (d, aryl CH-5'),
103.9 (d, aryl CH-3'), 96.4 (d, meso-CH), 95.5 (d, meso-CH),
69.9 (t, a-CH2), 35.8 (t, PyCH2CH2), 35.1 (t, PyCH2CH2), 34.0
(s, C(CH3)3), 32.2 (q, NCH3), 31.1 (q, C(CH3)3), 31.08 (t, g-
CH2), 28.8 (t, b-CH2), 25.6 (t, PyCH2), 26.2 (t, PyCH2), 23.4 (t,
Py(CH2)2CH2), 14.3 (q, Py(CH2)3CH3), 14.1 (q, Py(CH2)3CH3),
13.4 (q, PyCH3), 13.1 (q, PyCH3); MALDI-TOF-MS: m/z
Thiocarbamoylbis(acetylamido)porphyrin 30
To a solution of bisphenol porphyrin aa-25 (200 mg, 225 mmol)
in dry DMF (90 mL) was added Cs2CO3 (2.21 g, 6.78 mmol)
which was previously dried at 1008C in high-vacuum overnight.
The resulting suspension was heated to 608C and stirred for
30 min. Then a solution of dimesylate 17 (172 mg, 337 mmol) in
dry DMF (28 mL) was added via a syringe pump within 4 h at
608C. Stirring was continued for 30 min at 608C after which the
reaction mixture was cooled with an ice bath and the reaction
was quenched with 1 M HCl (55 mL). For better phase
separation CH2Cl2 (110 mL) was added. After phase separa-
tion the aqueous phase was extracted with CH2Cl2 (2 Â 85 mL).
The combined organic layers were washed with saturated
aqueous NaHCO3 (2 Â 200 mL), water (170 mL) and brine
(100 mL), dried over Na2SO4 and concentrated under reduced
pressure. The crude product was purified by flash chromatog-
raphy (silica gel, hexane/ethyl acetate, 2:1 ‡ 1% Et3N) which
gave 230 mg (85%) of the bridged porphyrin as purple solid;
Rf ˆ 0.49 (hexane/ethyl acetate, 2:1 ‡ 1% Et3N); RP-HPLC
(90 100% MeOH/H2O in 10 min, then 100% MeOH for
10 min, flow 1.5 mL/min, ldet ˆ 414 nm, Tˆ 408C): Rt ˆ
10.3 min; UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 414 (100,
Soret), 511 (9), 543 (2), 578 (4), 632 nm (2); IR (KBr): n ˆ 3401
m, 2956 s, 2870 s, 1704 s, 1660 s, 1602 s, 1588 s, 1520 s, 1462 s. 1365
m, 1256 s, 1087 cmÀ1 s; 1H NMR (400 MHz, CDCl3): d ˆ 10.15
(s, 1H, meso-H), 9.72 (s, 1H, meso-H), 8.54 (d, J ˆ 8.3 Hz, 2H,
aryl H-5'), 7.79 (s, 2H, NHAc), 7.78 (t, J ˆ 8.3 Hz, 2H, aryl H-
4'), 6.97 (d, J ˆ 8.3 Hz, 2H, aryl H-3'), 6.32 (s, 2H, aryl H-3'', aryl
H-5∫), 4.06 (m, 2H, PyCH2), 3.99 (m, 2H, PyCH2), 3.84 (m, 4H,
PyCH2 and a-CH2), 3.61 (m, 4H, PyCH2 and a-CH2), 2.72 (s,
6H, PyCH3), 2.54 (s, 6H, PyCH3), 2.24 (m, 4H, PyCH2CH2),
1.94 (m, 2H, PyCH2CH2), 1.87 (m, 2H, PyCH2CH2), 1.79 (m,
4H, Py(CH2)2CH2), 1.76 (br, s, NCH3), 1.71 (m, 4H,
Py(CH2)2CH2), 1.54 (s, 6H, COCH3), 1.15 (t, J ˆ 7.3 Hz, 6H,
Py(CH2)3CH3), 1.13 (t, J ˆ 7.3 Hz, 6H, Py(CH2)3CH3), 1.06 (s,
9H, t-Bu), 0.59 (m, 6H, b-CH2 and g-CH2), À 0.04 (m, 2H, g-
CH2), À 1.17 (br, s, 3H, NCH3), À 2.13 (br, s, 2H, NH pyrrole);
13C NMR (150 MHz, CDCl3): d ˆ 168.8 (s, CO), 159.9 (s, aryl C-
2'), 150.4 (s, aryl C-4''), 145.4 (s, C-12, C-18), 145.1 (s, C-2, C-8),
144.1 (s, aryl C-1''), 143.7 (s, C-3, C-7), 143.6 (s, C-13, C-17),
141.7 (s, C-14, C-16), 141.1 (s, C-4, C-6), 138.9 (s, C-1'), 135.9 (s,
‡
‡
(%) ˆ 1052 (9, [M ‡ 1 CONMe2] ); 1123 (100, [M ‡ 1] ).
758
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2003, 345, 743 765

Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
amide), 8.63 (d, J ˆ 8.6 Hz, 2H, aryl H-5'), 7.78 (t, J ˆ 8.3 Hz,
2H, aryl H-4'), 6.96 (d, J ˆ 8.3 Hz, 2H, aryl H-3'), 6.39 (s, 2H,
aryl H-3'', aryl H-5''), 4.12 (dd, J ˆ 8.6 Hz, J ˆ 4.0 Hz, 2H, a-
CH), 3.97 (m, 4H, PyCH2), 3.91 (m, 2H, PyCH2), 3.87 (m, 2H,
a-CH2), 3.59 (m, 4H, PyCH2 and a-CH2), 2.70 (s, 6H, PyCH3),
2.54 (s, 6H, PyCH3), 2.33 (q, J ˆ 7.5 Hz, 2H, d-CH2), 2.21 (m,
4H, PyCH2CH2), 2.15 (m, 2H, b-CH2), 1.95 (m, 2H, b-CH2),
1.85 (m, 8H, PyCH2CH2 and Py(CH2)2CH2), 1.71 (m, 7H,
Py(CH2)2CH2 and NCH3), 1.59 (m, 2H, d-CH2), 1.42 (m, 4H, c-
CH2), 1.19 (t, J ˆ 7.3, 6H, Py(CH2)3CH3), 1.14 (t, J ˆ 7.3, 6H,
Py(CH2)3CH3), 1.08 (s, 9H, t-Bu), 0.78 (dt, J ˆ 3.5, J ˆ 11.4, 2H,
g-CH2), 0.66 (m, 2H, b-CH2), 0.55 (m, 2H, b-CH2), 0.13 (dt, J ˆ
4.3, J ˆ 13.1, 2H, g-CH2), À 1.44 (br, s, 3H, NCH3), À 1.92 (br,
s, 2H, NH pyrrole); 13C NMR (125 MHz, CDCl3): d ˆ 172.3 (s,
CO amide), 160.0 (s, arylC-2'), 146.2 (s, C-12, C-18), 145.3 (s, C-
2, C-8), 144.4 (s, arylC-4''), 143.8 (s, C-13, C-17), 143.6 (s, C-3, C-
7), 143.4 (s, aryl C-1''), 140.9 (s, C-14, C-16), 140.4 (s, C-4, C-6),
138.4 (s, aryl C-1'), 135.6 (s, C-11, C-19), 135.1 (s, C-1, C-9),
131.0 (d, aryl CH-4'), 123.3 (d, aryl CH-3'', aryl CH-5''), 122.3 (s,
aryl C-2'', aryl C-6∫), 120.7 (s, aryl C-6'), 112.9 (d, aryl CH-5'),
109.2 (d, aryl CH-3'), 107.4 (s, C-10, C-20), 97.1, 95.8 (d, meso-
CH), 78.3 (d, a-CH), 69.7 (t, a-CH2), 68.4 (t, d-CH2), 35.7, 35.1
(t, PyCH2CH2), 34.1 (s, C(CH3)3), 31.6 (q, NCH3), 31.1 (q,
C(CH3)3), 31.2 (t, g-CH2), 30.3 (t, b-CH2), 28.9 (t, b-CH2), 26.5,
26.1 (t, PyCH2), 24.7 (t, c-CH2), 23.6, 23.4 (t, Py(CH2)2CH2),
14.2, 14.1 (q, Py(CH2)3CH3), 13.7, 13.4 (q, PyCH3); MALDI-
Thiocarbamoylbis(THF)porphyrins RR,SS-31 and
RS,SR-31
To a solution of bisaminoporphyrin 12 (117 mg, 104 mmol) in
dry CH2Cl2 (22 mL) were added Et3N (70 mL, 503 mmol) and
DMAP (1.5 mg, 12 mmol). Then a solution of carbonyl chloride
13 (58 mg, 431 mmol) in dry CH2Cl2 (3 mL) was added
dropwise at room temperature and the reaction mixture
started to get cloudy (Et3N ¥ HCl). Stirring was continued for
13 h after which the reaction mixture was cooled in an ice bath
and the reaction was quenched with some pieces of ice and
saturated aqueous NaHCO3 (15 mL). After phase separation
the organic layer was washed with saturated aqueous NaHCO3
(2 Â 20 mL) and water (20 mL). In order to reduce loss of
material the aqueous phases were extracted with CH2Cl2 (2 Â
10 mL). The combined organic phases were dried over Na2SO4
and concentrated under vacuum. The obtained residue was
purified by flash chromatography (silica gel, hexane/ethyl
acetate, 2:1 ‡ 1% Et3N) which yielded 97 mg (71%) of RR,SS-
31, 17 mg (12%) of RS,SR-31 and 12 mg (9%) of RR,SS/RS,SR-
31, (92% combined yield) as purple solids. Actually, it is not
necessary to separate the diastereoisomers since in the next
step an epimerisation at a-CH of the tetrahydrofuranyl ring
takes place.
Physical data of RR,SS-31: Rf ˆ 0.37 (Et2O); UV/Vis
(CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 413 (100, Soret), 510 (9), 544
(4), 581 (4), 633 nm (2); 1H NMR (400 MHz, CDCl3): d ˆ 10.07
(s, 1H, meso-H), 9.66 (s, 1H, meso-H), 9.27 (br, s, 2H, NH
amide), 8.55 (d, J ˆ 8.6 Hz, 2H, aryl H-5'), 7.78 (t, J ˆ 8.8 Hz,
2H, aryl H-4'), 6.98 (d, J ˆ 7.3 Hz, 2H, aryl H-3'), 6.33 (s, 1H,
aryl H-5''), 6.30 (s, 1H, aryl H-3''), 4.09 (m, 2H, a-CH), 4.00 (m,
4H, PyCH2), 3.92 (m, 1H, PyCH2), 3.85 (m, 2H, a-CH2), 3.74
(m, 1H, PyCH2), 3.58 (m, 4H, PyCH2 and a-CH2), 2.72 (s, 3H,
PyCH3), 2.67 (s, 3H, PyCH3), 2.55 (s, 3H, PyCH3), 2.49 (s, 3H,
PyCH3), 2.28 (m, 2H, d-CH2), 2.23 (m, 4H, PyCH2CH2), 2.13
(m, 2H, b-CH2), 1.96 (m, 1H, b-CH2), 1.92 (m, 4H, PyCH2CH2),
1.82 (m, 4H, Py(CH2)2CH2), 1.71 (m, 4H, Py(CH2)2CH2), 1.68
(m, 1H, b-CH2), 1.60 (m, 2H, d-CH2), 1.17 (m, 12H,
Py(CH2)3CH3), 1.07 (s, 9H, t-Bu), 0.89 (m, 4H, c-CH2), 0.60
(m, 6H, b-CH2 and g-CH2), À 0.05 (m, 2H, g-CH2), À 1.07 (br,
s, 3H, NCH3), À 1.98 (br, s, 2H, NH pyrrole); 13C NMR
(125 MHz, CDCl3): d ˆ 172.2 (s, CO amide), 166.8 (s, CO
thiocarbamate), 160.4, 160.0 (s, aryl C-2'), 150.4 (s, aryl C-4''¥),
147.2 (s, C-12, C-18), 147.0 (s, C-2, C-8), 144.3 (s, C-13), 144.2 (s,
C-17), 144.1 (s, C-3), 144.0 (s, C-7), 143.4 (s, C-14), 143.3 (s, C-
16), 142.9 (s, C-4), 142.8 (s, C-6), 138.4 (s, arylC-6'), 136.0 (s, C-
9), 135.6 (s, C-19), 135.2 (s, C-1), 134.9 (s, C-11), 130.8 (d, aryl
CH-4'), 123.5 (s, aryl C-1''), 123.0 (d, aryl CH-3'', aryl CH-5''),
122.8 (s, aryl C-2'', aryl C-6''), 121.9, 121.2 (s, aryl C-1'); 113.2,
112.8 (d, aryl CH-5'); 110.3, 110.1 (d, aryl CH-3'), 107.5, 107.4
(s, C-10, C-20), 96.9, 96.1 (d, meso-CH), 78.4, 78.3 (d, a-CH),
69.7 (t, a-CH2), 68.5, 68.3 (t, d-CH2), 35.8, 35.2, 35.1 (t,
PyCH2CH2), 34.1 (s, C(CH3)3), 31.6 (q, NCH3), 31.1 (q,
C(CH3)3), 30.8 (t, g-CH2), 30.3, 30.1 (t, b-CH2), 28.8, 28.7 (t,
b-CH2), 26.6, 26.4, 26.1, 26.0 (t, PyCH2), 24.2 (t, c-CH2), 23.6,
23.5, 23.4, 23.3 (t, Py(CH2)2CH2), 14.3, 14.1 (q, Py(CH2)3CH3),
13.75, 13.63, 13.3, 13.2 (q, PyCH3); MALDI-TOF-MS: m/z
‡
TOF-MS: m/z (%) ˆ 1249 (5, [M ‡ 2 CONMe2] ); 1320 (100,
‡
[M ‡ 2] ).
Bis(THF)thiophenolporphyrins RR,SS-32 and RS,SR-
32
In a oxygen-free atmosphere thiocarbamoylporphyrin 31
(51 mg, 39 mmol) was dissolved in dry and degassed dioxane
(10 mL) and the resulting solution was heated to 908C. Then a
freshly prepared and degassed 2.18 M KOMe solution (in
MeOH, 1 mL, 2.2 mmol) was added dropwise and the colour of
the reaction mixture turned slightly green. The reaction
mixture was heated to reflux and by blowing argon into the
solution MeOH was evaporated until the solution was grass-
green. Stirring at reflux was continued for 15 min after which
the reaction was cooled in an ice bath and quenched with
saturated aqueous NH4Cl (6 mL). All of the used solvents and
solutions for the aqueous work up were quickly degassed by
bubbling with argon for 5 10 min prior to use. The obtained
suspension was diluted with water (12 mL) and CH2Cl2
(20 mL). The layers were separated and the aqueous phase
was extracted with CH2Cl2 (2 Â 20 mL). The combined organic
phases were washed with water (2 Â 10 mL), dried over
Na2SO4 and evaporated under reduced pressure. The crude
product was purified by flash chromatography (silica gel,
hexane/ethyl acetate, 3:1 ‡ 1% Et3N, under argon, solvents
were quickly degassed by bubbling with argon for 15 min prior
to use) and preparative TLC (silica gel, hexane/ethyl acetate,
3:1 ‡ 1% Et3N, under argon, solvents were quickly degassed
by bubbling with argon for 15 min prior to use) which yielded
25.2 mg (52%) of RR,SS-32 and 7.7 mg (16%) of RS,SR-32
(combined yield: 68%) as purple solids.
‡
‡
(%) ˆ 1248 (5, [M ‡ 1 CONMe2] ); 1320 (100, [M ‡ 2] ).
Physical data of RS,SR-31: Rf ˆ 0.31 (Et2O); UV/Vis
(CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 414 (100, Soret), 510 (10),
544 (5), 581 (5), 633 nm (1); 1H NMR (400 MHz, CDCl3): d ˆ
10.03 (s, 1H, meso-H), 9.71 (s, 1H, meso-H), 9.38 (s, 2H, NH
Important note: the addition of 1% of Et3N to the solvent
system for chromatography is essential to achieve separationof
the diastereoisomers.
Adv. Synth. Catal. 2003, 345, 743 765
asc.wiley-vch.de
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
759

Martin Lochner et al.
FULL PAPERS
Physical data for RR,SS-32: Rf ˆ 0.29 (hexane/ethyl acetate,
2:1 ‡ 1% Et3N); UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 412
chloride (1.1 mL, 13.8 mmol) in dry CH2Cl2 (0.2 mL) was added
dropwise via a syringe at room temperature. The reaction
mixture was stirred at room temperature for 15 min after which
it was cooled in an ice bath and the reaction was quenched with
saturated aqueous NaHCO3 (1 mL). The biphasic mixture was
diluted with CH2Cl2 (2 mL), the phases were separated and the
organic phase was washed with saturated aqueous NaHCO3
(2 Â 1 mL) and water (1 mL). In order to reduce loss of
material the aqueous wash phases were extracted with CH2Cl2
(2 Â 2 mL). The combined organic layers were dried over
Na2SO4 and the solvent was evaporated under reduced
pressure. The crude porphyrin was purified by preparative
TLC (silica gel, hexane/ethyl acetate, 2:1 ‡ 1% Et3N) which
yielded 2.8 mg (61%) of pure title compound as purple solid.
However, for the synthesis of larger amounts a reported
procedure[30] was used: 12 (10.5 mg, 9.4 mmol) was dissolved in
dry CHCl3 (4 mL, filtered through basic aluminium oxide prior
to use) and to this solution was added freshly distilled acetyl
chloride (3 mL, 37.7 mmol) in dry CHCl3 (0.4 mL). The result-
ing green solution was stirred at room temperature for 2.5 h
after which the reaction was quenched with 0.1 M KOH (2 mL)
in an ice bath. Furthermore, saturated aqueous NaHCO3
(1 mL) and CH2Cl2 (2 mL) were added and the phases were
separated. The organic layers were washed with water (5 mL)
and brine (1 mL), dried over Na2SO4 and concentrated under
vacuum. The obtained crude product (13 mg) was used without
further purification for the next step. Rf ˆ 0.31 (hexane/ethyl
acetate, 2:1 ‡ 1% Et3N); RP-HPLC (90 100% MeOH/H2O
in 10 min, then 100% MeOH for 10 min, flow 1.5 mL/min,
ldet ˆ 414 nm, Tˆ 408C): Rt ˆ 11.3 min.; UV/Vis (CH2Cl2 ‡
1% Et3N): lmax (%) ˆ 413 (100, Soret), 510 (9), 544 (4), 579
(4), 633 nm (3); 1H NMR (250 MHz, CDCl3): d ˆ 10.15 (s, 1H,
meso-H), 9.74 (s, 1H, meso-H), 9.09 (s, 2H, NH amide), 8.58 (d,
J ˆ 8.0 Hz, 2H, aryl H-5'), 7.83 (t, J ˆ 8.3 Hz, 2H, aryl H-4'),
7.04 (d, J ˆ 7.5 Hz, 2H, aryl H-3'), 6.38 (s, 2H, aryl H-3'', aryl H-
5''), 4.09 (m, 2H, PyCH2), 3.97 (m, 2H, PyCH2), 3.88 (m, 4H,
PyCH2 and a-CH2), 3.78 (d, J ˆ 14.9 Hz, 2H, COCH2Cl), 3.68
(d, J ˆ 14.9 Hz, 2H, COCH2Cl), 3.62 (m, 4H, PyCH2 and a-
CH2), 2.74 (s, 6H, PyCH3), 2.55 (s, 6H, PyCH3), 2.26 (m, 4H,
PyCH2CH2), 1.79 (m, 15H, PyCH2CH2, Py(CH2)2CH2 and
NCH3), 1.17 (t, J ˆ 7.3, 6H, Py(CH2)3CH3), 1.16 (t, J ˆ 7.2 Hz,
6H, Py(CH2)3CH3), 1.10 (s, 9H, t-Bu), 0.68 (m, 6H, b-CH2 and
g-CH2), 0.06 (m, 2H, g-CH2), À 1.17 (br, s, 3H, NCH3), À 1.97
(br, s, 2H, NH pyrrole); MALDI-TOF-MS: m/z (%) ˆ 1206 (29,
1
(100, Soret), 510 (8), 545 (3), 577 (4), 632 nm (2); H NMR
(400 MHz, CDCl3): d ˆ 10.13 (s, 2H, meso-H), 9.38 (s, 2H, NH
amide), 8.65 (d, J ˆ 7.9 Hz, 2H, aryl H-5'), 7.82 (t, J ˆ 8.3, 2H,
aryl H-4'), 7.01 (d, J ˆ 7.7 Hz, 2H, aryl H-3'), 6.18 (s, 2H, aryl H-
3'', aryl H-5''), 4.15 (dd, J ˆ 8.5 Hz, J ˆ 3.9 Hz, 2H, a-CH), 4.00
(m, 4H, PyCH2), 3.91 (m, 4H, PyCH2), 3.66 (m, 4H, a-CH2),
2.66 (s, 6H, PyCH3), 2.62 (s, 6H, PyCH3), 2.26 (m, 2H, d-CH2),
2.13 (m, 8H, PyCH2CH2), 2.10 (m, 2H, b-CH2), 1.94 (m, 2H, b-
CH2), 1.77 (m, 8H, Py(CH2)2CH2), 1.53 (m, 2H, d-CH2), 1.23
(m, 4H, c-CH2), 1.12 (t, J ˆ 7.4, 12H, Py(CH2)3CH3), 0.88 (s, 9H,
t-Bu), 0.57 (m, 8H, b-CH2 and g-CH2), À 2.13 (br, s, 2H, NH
pyrrole); À 3.12 (s, 1H, SH); 13C NMR (150 MHz, CDCl3): d ˆ
172.3 (s, CO amide), 159.5 (s, aryl C-2'), 146.6 (s, C-2, C-8, C-12,
C-18), 146.2 (s, aryl C-4''), 144.1 (s, C-14, C-16), 143.8 (s, C-4, C-
6), 143.2 (s, C-13, C-17), 143.0 (s, C-3, C-7), 140.2 (s, C-10, C-
20), 138.6 (s, aryl C-6'), 137.9 (s, aryl C-2'', aryl C-6''), 135.5 (s,
C-11, C-19), 135.1 (s, C-1, C-9), 130.7 (d, aryl CH-4'), 126.5 (s,
aryl C-1''), 122.9 (d, aryl C-3'', aryl C-5''), 121.5 (s, aryl C-1'),
113.1 (d, aryl CH-5'), 109.0 (d, aryl C-3'), 97.0 (d, meso-CH),
78.3 (d, a-CH), 68.5 (t, a-CH2), 68.3 (t, d-CH2), 35.6, 35.5 (t,
PyCH2CH2), 33.6 (s, C(CH3)3), 30.9 (q and t, C(CH3)3 and g-
CH2), 30.3 (t, b-CH2), 28.9 (t, b-CH2), 26.4, 26.5 (t, PyCH2), 24.5
(t, c-CH2), 23.4 (t, Py(CH2)2CH2), 14.4 (q, Py(CH2)2CH3), 13.5,
13.4 (q, PyCH3); MALDI-TOF-MS: m/z (%) ˆ 1248 (100,
‡
[M ‡ 1] ).
Physical data for RS,SR-32: Rf ˆ 0.24 (hexane/ethyl acetate,
2:1 ‡ 1% Et3N); UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 413
1
(100, Soret), 510 (9), 544 (4), 577 (4), 631 nm (2); H NMR
(400 MHz, CDCl3): d ˆ 10.12 (s, 1H, meso-H), 10.10 (s, 1H,
meso-H), 9.41 (s, 2H, NH amide), 8.65 (d, J ˆ 7.9 Hz, 2H, aryl
H-5'), 7.81 (t, J ˆ 8.3 Hz, 2H, aryl H-4'), 6.99 (d, J ˆ 7.5 Hz, 2H,
aryl H-3'), 6.21 (s, 2H, aryl H-3'', aryl H-5''), 4.16 (dd, J ˆ
8.5 Hz, J ˆ 4.2 Hz, 2H, a-CH), 3.95 (m, 8H, PyCH2), 3.65 (m,
4H, a-CH2), 2.64 (s, 12H, PyCH3), 2.34 (q, J ˆ 7.6 Hz, 2H, d-
CH2), 2.12 (m, 10H, PyCH2CH2 and b-CH2), 1.96 (m, 2H, b-
CH2), 1.73 (m, 10H, Py(CH2)2CH2 and d-CH2), 1.26 (m, 4H, c-
CH2), 1.15 (t, J ˆ 7.4 Hz, 6H, Py(CH2)3CH3), 1.04 (t, J ˆ 7.4, 6H,
Py(CH2)3CH3), 0.89 (s, 9H, t-Bu), 0.76 (m, 2H, g-CH2), 0.54 (m,
6H, b-CH2 and g-CH2), À 2.08 (br, s, 2H, NH pyrrole), À 3.07
(s, 1H, SH); 13C NMR (150 MHz, CDCl3): d ˆ 172.3 (s, CO
amide), 159.4 (s, aryl C-2'), 146.2 (s, aryl C-4''), 145.5 (s, C-12, C-
18), 145.0 (s, C-2, C-8), 143.7 (s, C-14, C-16), 143.5 (s, C-4, C-6),
141.8 (s, C-10, C-20), 143.4 (s, C-3, C-7, C-13, C-17), 138.6 (s,
aryl C-6'), 138.0 (s, aryl C-2'', aryl C-6''), 135.5 (s, C-11, C-19),
135.1 (s, C-1, C-9), 130.7 (d, aryl CH-4'), 126.4 (s, aryl C-1''),
123.1 (d, aryl C-3'', aryl C-5''), 121.2 (s, aryl C-1'), 113.0 (d, aryl
CH-5'), 108.6 (d, aryl C-3'), 97.3, 96.6 (d, meso-CH), 78.3 (d, a-
CH), 68.4 (t, d-CH2), 67.1 (t, a-CH2), 35.6, 35.5 (t, PyCH2CH2),
33.6 (s, C(CH3)3), 31.2 (t, g-CH2), 30.9 (q, C(CH3)3), 30.2 (t, b-
CH2), 29.0 (t, b-CH2), 26.4 (t, PyCH2), 24.5 (t, c-CH2), 23.4, 23.3
(t, Py(CH2)2CH2), 14.5 (q, Py(CH2)2CH3), 13.5 (q, PyCH3);
‡
‡
‡
[M ‡ 2 2 Cl] ), 1242 (61, [M ‡ 2 Cl] ), 1276 (100, [M ‡ 2] );
‡
ESI-MS (MeOH): m/z (%) ˆ 1275 (20, [M ‡ H] ), 1297 (100,
‡
[M ‡ Na] ).
Crown-Capped Thiocarbamoylporphyrin 38
To a solution of bis(chloroacetylamido)porphyrin 36 (3 mg,
2.4 mmol) in dry EtOH (2.5 mL) was added 1,10-diaza-18-
crown-6 (35) (3.1 mg, 11.8 mmol) in portions. The resulting red
solution was heated to reflux and stirred for 21 h after which
the reaction mixture was concentrated to dryness under
reduced pressure. The crude residue was taken up in CH2Cl2
(4 mL) and washed with saturated aqueous NaHCO3 (2 mL)
and water (2 mL). In order to reduce loss of material the
aqueous wash phases were extracted with CH2Cl2 (2 mL). The
combined organic phases were dried over Na2SO4 and volatiles
evaporated under vacuum. Purification by preparative TLC
‡
MALDI-TOF-MS: m/z (%) ˆ 1248 (100, [M ‡ 1] ).
Thiocarbamoylbis(chloroacetylamido)porphyrin 36
In a experiment on a small scale bisaminoporphyrin 12 (4 mg,
3.6 mmol) was dissolved in dry CH2Cl2 (0.8 mL) and to this
solution were added Et3N (2.4 mL, 17.2 mmol) and one crystal
of DMAP. Then a solution of freshly distilled chloroacetyl
760
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2003, 345, 743 765

Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
(silica gel, CH2Cl2/MeOH, 95:5 ‡ 1% Et3N) yielded 2.4 mg
(68%) of crown-capped porphyrin 38 as a purple varnish.
Alternatively, crude bis(chloroacetylamido)porphyrin 36
(11.8 mg, 9.25 mmol) (vide supra) was dissolved in dry EtOH
(9.4 mL). To this solution was added 1,10-diaza-18-crown-6
(35) (12.3 mg, 46.9 mmol) in portions. The resulting reaction
mixture was refluxed for 24 h and after analogous work-up and
purification by flash chromatography (silica gel, CH2Cl2/
MeOH 98:2) 7.6 mg (55% from 12, 2 steps) of pure crown-
capped porphyrin 38 were obtained. Rf ˆ 0.51 (CH2Cl2/
MeOH, 95:5 ‡ 1% Et3N); RP-HPLC (90 100% MeOH/
H2O in 10 min, then 100% MeOH for 10 min, flow 1.5 mL/
min, ldet ˆ 414 nm, Tˆ 408C): Rt ˆ 11.5 min; UV/Vis
(CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 414 (100, Soret), 510 (9), 542
(3), 578 (3), 632 nm (1); 1H NMR (400 MHz, CDCl3): d ˆ 10.19
(s, 1H, meso-H), 9.98 (s, 2H, NH amide), 9.64 (s, 1H, meso-H),
8.70 (dd, J ˆ 7.9 Hz, J ˆ 0.8 Hz, 2H, aryl H-5'), 7.79 (t, J ˆ
8.3 Hz, 2H, aryl H-4'), 6.99 (dd, J ˆ 8.0 Hz, J ˆ 0.8 Hz, 2H,
aryl H-3'), 6.29 (s, 2H, aryl H-3'', aryl H-5''), 3.98 (m, 4H,
PyCH2), 3.88 (m, 2H, PyCH2 and a-CH2), 3.79 (m, 2H, PyCH2
and a-CH2), 3.66 (m, 2H, PyCH2 and a-CH2), 3.59 (m, 2H,
PyCH2 and a-CH2), 2.84 (d, J ˆ 17.4, COCH2N), 2.74 (d, J ˆ
17.4, COCH2N), 2.72 (s, 6H, PyCH3), 2.53 (s, 6H, PyCH3), 2.29
(m, 4H, PyCH2CH2), 2.03 (m, 4H, PyCH2CH2), 1.92 (m, 8H,
Py(CH2)2CH2 and c-CH2), 1.75 (m, 11H, Py(CH2)2CH2, c-CH2
and NCH3), 1.62 (m, 2H, b-CH2), 1.33 (m, 2H, b-CH2), 1.23 (t,
J ˆ 7.3, 6H, Py(CH2)3CH3), 1.23 (m, 2H, b-CH2), 1.19 (t, J ˆ 7.5,
6H, Py(CH2)3CH3), 1.14 (m, 2H, b-CH2), 1.10 (s, 9H, t-Bu), 0.99
(m, 2H, a-CH2), 0.62 (m, 4H, b-CH2), 0.40 (m, 2H, g-CH2), 0.07
(m, 2H, a-CH2), À 0.14 (m, 2H, g-CH2), À 0.28 (m, 2H, a-CH2),
À 0.53 (m, 2H, a-CH2), À 0.68 (br, s, 3H, NCH3), À 2.12 (s, 2H,
NH pyrrole); MALDI-TOF-MS: m/z (%) ˆ 1395 (12, [M ‡ 2
In the worst case arylsulphonic acid-porphyrin (SO3H) 42 is the
main product.
Physical data of thiophenol porphyrin 40: Rf ˆ 0.41
(CH2Cl2/MeOH 95:5 ‡ 1% Et3N); UV/Vis (CH2Cl2 ‡ 1%
Et3N): lmax (%) ˆ 413 (100, Soret), 510 (9), 543 (4), 576 (4),
630 (2), 658 nm (1); 1H NMR (250 MHz, CDCl3): d ˆ 10.20 (s,
2H, meso-H), 10.05 (s, 2H, NH amide), 8.76 (d, J ˆ 7.7 Hz, 2H,
aryl H-5'), 7.82 (t, J ˆ 8.4 Hz, 2H, aryl H-4'), 7.03 (d, J ˆ 7.5 Hz,
2H, aryl H-3'), 6.13 (s, 2H, aryl H-3'', aryl H-5''), 3.98 (m, 8H,
PyCH2), 3.66 (m, 4H, a-CH2), 2.84 (s, 4H, COCH2N), 2.65 (s,
12H, PyCH3), 2.22 (m, 8H, PyCH2CH2), 1.81 (m, 8H,
Py(CH2)2CH2), 1.76 (m, 8H, c-CH2), 1.59 (m, 4H, b-CH2),
1.44 (m, 4H, b-CH2), 1.16 (t, J ˆ 7.3, 12H, Py(CH2)3CH3), 0.84
(s, 9H, t-Bu), 0.45 (m, 12H, b-CH2, g-CH2 and a-CH2), À 0.17
(m, 4H, a-CH2), À 2.25 (s, 2H, NH pyrrole), À 3.26 (s, 1H, SH);
‡
MALDI-TOF-MS: m/z (%) ˆ 1395 (100, [M ‡ 2] ).
Physical data of S-oxidised (SO3H) porphyrin 42: Rf ˆ 0.39
(CH2Cl2/MeOH, 95:5 ‡ 1% Et3N); UV/Vis (CH2Cl2 ‡ 1%
Et3N): lmax (%) ˆ 412 (100, Soret), 508 (8), 542 (3), 577 (3),
632 nm (2); 1H NMR (250 MHz, CDCl3): d ˆ 10.50 (s, 2H,
meso-H), 9.75 (s, 2H, NH amide), 8.73 (d, J ˆ 8.2 Hz, 2H, aryl
H-5'), 7.87 (t, J ˆ 8.4 Hz, 2H, aryl H-4'), 7.11 (d, J ˆ 7.9 Hz, 2H,
aryl H-3'), 6.08 (s, 2H, aryl H-3'', aryl H-5''), 4.07 (m, 4H,
PyCH2), 3.98 (m, 4H, PyCH2), 3.62 (m, 4H, a-CH2), 2.81 (s, 4H,
COCH2N), 2.71 (s, 12H, PyCH3), 2.24 (m, 8H, PyCH2CH2),
1.88 (m, 8H, Py(CH2)2CH2), 1.74 1.57 (m, 16H, crown-CH2),
1.22 (t, J ˆ 7.4 Hz, 12H, Py(CH2)3CH3), 0.88 (s, 9H, t-Bu), 0.57
(m, 4H, b-CH2), 0.37 (m, 4H, g-CH2), 0.09 (m, 4H, crown-CH2),
À 2.48 (s, 2H, NH pyrrole); MALDI-TOF-MS: m/z (%) ˆ 1443
‡
‡
(100, [M ‡ 2] ), 1465 (15, [M ‡ 1 ‡ Na] ), 1481 (6, [M ‡ 1 ‡
‡
K] ); ESI-MS (MeOH): positive ion mode: m/z (%) ˆ 1442 (15,
‡
‡
‡
[M ‡ H] ), 1464 (100, [M ‡ Na] ), 1480 (17, [M ‡ K] ), 1486
‡
‡
‡
CONMe2] ), 1466 (100, [M ‡ 2] ); ESI-MS (MeOH): positive
(62, [M H ‡ 2 Na] ); negative ion mode: m/z (%) ˆ 1440 (100,
‡
ion mode: m/z (%) ˆ 1487 (100, [M ‡ Na] ), 1503 (9, [M ‡
MÀ).
‡
K] ); negative ion mode: m/z (%) ˆ 1462 (100, [M H]À).
Thiocarbamoylbis(acrylamido)porphyrin 37
Crown-Capped Thiophenolporphyrin 40
Bisaminoporphyrin 12 (15 mg, 13.4 mmol) was dissolved in dry
CH2Cl2 (2 mL) and to this solution was added Et3N (50 mL,
359 mmol). Then a solution of freshly distilled acryloyl chloride
(2.6 mL, 32 mmol) in dry CH2Cl2 (0.5 mL) was added dropwise
via a syringe within 4 min at room temperature and stirring was
continued for 4.5 h in the dark. The reaction mixture was
cooled in an ice bath and saturated aqueous NaHCO3 (2 mL)
was added to quench the reaction. After phase separation the
organic phase was washed with saturated aqueous NaHCO3
(3 Â 1 mL) and water (2 mL). To minimise loss of material the
aqueous phases were extracted with CH2Cl2 (2 Â 5 mL). The
combined organic phases were dried over Na2SO4 and
evaporated under reduced pressure. The obtained residue
was purified by flash chromatography (silica gel, hexane/ethyl
acetate, 2:1 ‡ 1% Et3N) which yielded 11.9 mg (72%) of the
title compound as purple solid. It should be noted that this
compound seems to be unstable, especially in solution, and
therefore should be used as soon as possible for the next step.
Rf ˆ 0.28 (hexane/ethyl acetate, 2:1 ‡ 1% Et3N); RP-HPLC
(90 100% MeOH/H2O in 10 min, then 100% MeOH for
10 min, flow 1.5 mL/min, ldet ˆ 414 nm, Tˆ 408C): Rt ˆ
10.3 min; UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 414 (100,
Soret), 510 (6), 546 (2), 578 (1), 630 nm (1); 1H NMR
(400 MHz, CDCl3): d ˆ 10.14 (s, 1H, meso-H), 9.72 (s, 1H,
In an oxygen-free atmosphere a solution of crown-capped
porphyrin 38 (6.3 mg, 4.3 mmol) in dry and degassed dioxane
(1.1 mL) was heated to 1008C. To this hot solution was added a
freshly prepared and degassed 2.18 M KOMe solution (in
MeOH, 20 mL, 44 mmol) and the resulting grass-green solution
was heated at reflux for 10 min. The reaction mixture was
cooled in an ice bath and the reaction was quenched with
saturated aqueous NH4Cl (1 mL). All of the used solvents and
solutions for the aqueous work up were quickly degassed by
bubbling with argon for 5 10 min prior to use. For better phase
separation the biphasic mixture was diluted with water (1 mL)
and CH2Cl2 (2 mL). The aqueous layer was extracted with
CH2Cl2 (2 Â 2 mL), the combined organic layers were washed
with water (3 mL), dried over Na2SO4 and concentrated under
vacuum. The obtained crude residue was purified by prepara-
tive TLC (silica gel, CH2Cl2/MeOH, 95:5 ‡ 1% Et3N, under
argon, solvents were quickly degassed by bubbling with argon
for 5 10 min prior to use) which yielded 2.9 mg (48%) of pure
thiophenolporphyrin 40. It should be noted that it is essential to
work under oxygen-free conditions and freshly prepared
KOMe solution (instead of solid KOMe) should be used
otherwise substantial amounts of S-oxidised products are
generated and these are difficult to separate from the product.
Adv. Synth. Catal. 2003, 345, 743 765
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Martin Lochner et al.
FULL PAPERS
meso-H), 8.68 (d, J ˆ 8.6 Hz, 2H, aryl H-5'), 8.02 (s, 2H, NH
amide), 7.80 (t, J ˆ 8.5 Hz, 2H, aryl H-4'), 6.99 (dd, J ˆ 8.2 Hz,
J ˆ 0.6 Hz, 2H, aryl H-3'), 6.33 (s, 2H, aryl H-3'', aryl H-5''), 5.84
PyCH2), 24.5 (t, Py(CH2)2CH2), 14.1 (q, Py(CH2)3CH3), 13.8,
13.4 (q, PyCH3); MALDI-TOF-MS: m/z (%) ˆ 1494 (100,
‡
‡
[M ‡ 1] ), 1531 (12, [M ‡ K] ).
(dd, Jtrans ˆ 16.8 Hz, J ˆ 0.9 Hz, olefin H-2''cis); 5.37 (dd, Jtrans
ˆ
16.9 Hz, Jcis ˆ 10.4 Hz, olefin H-1''), 5.13 (dd, Jcis ˆ 11.0 Hz, J ˆ
0.8 Hz, olefin H-2''trans), 4.05 (m, 2H, PyCH2), 3.98 (m, 2H,
PyCH2), 3.86 (m, 4H, PyCH2 and a-CH2), 3.61 (m, 4H, PyCH2
and a-CH2), 2.72 (s, 6H, PyCH3), 2.55 (s, 6H, PyCH3), 2.22 (m,
4H, PyCH2CH2), 1.94 (m, 2H, PyCH2CH2), 1.86 (m, 2H,
PyCH2CH2), 1.77 (m, 4H, Py(CH2)2CH2), 1.70 (m, 4H,
Py(CH2)2CH2), 1.14 (t, J ˆ 7.3 Hz, 6H, Py(CH2)3CH3), 1.11 (t,
J ˆ 7.3 Hz, 6H, Py(CH2)3CH3), 1.07 (s, 9H, t-Bu), 0.59 (m, 6H,
b-CH2 and g-CH2), À 0.02 (m, 2H, g-CH2), À 1.02 (br, s, 3H,
NCH3), À 2.14 (br, s, 2H, NH pyrrole); MALDI-TOF-MS: m/z
Crown-Capped Thiophenolporphyrin 41
In an oxygen-free atmosphere thiocarbamoylporphyrin 39
(126 mg, 85 mmol) was dissolved in dry and degassed dioxane
(15 mL). The resulting solution was heated at 1008C and then a
freshly prepared and degassed 1.8 M KOMe solution (in
MeOH, 0.48 mL, 860 mmol) was added via a syringe. The
colour of the reaction mixture turned immediately grass-green
and it was heated at reflux for 10 min. The reaction mixture was
cooled with an ice bath and the reaction was quenched with
saturated aqueous NH4Cl (6 mL). All of the used solvents and
solutions for the aqueous work-up were quickly degassed by
bubbling with argon for 5 10 min prior to use. The layers were
separated and the aqueous phase was extracted with CH2Cl2
(2 Â 50 mL). The combined organic phases were washed with
water (2 Â 50 mL), dried over Na2SO4 and concentrated under
vacuum. Purification of the residue by flash chromatography
(silica gel, CH2Cl2/MeOH, 97:3, under argon, solvents were
quickly degassed by bubbling with argon for 15 min prior to
use) yielded 98 mg (81%) of crown-capped thiophenolpor-
phyrin 41 as purple solid. Rf ˆ 0.54 (silica gel, CH2Cl2/MeOH,
95:5); UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 415 (100,
Soret), 510 (7), 544 (2), 576 (3), 630 nm (1); 1H NMR
(400 MHz, CDCl3): d ˆ 10.89 (s, 2H, NH amide), 10.12 (s, 2H,
meso-H), 8.74 (dd, J ˆ 8.2 Hz, J ˆ 0.8 Hz, 2H, aryl H-5'), 7.75 (t,
J ˆ 8.5 Hz, 2H, aryl H-4'), 6.93 (dd, J ˆ 8.1 Hz, J ˆ 0.8 Hz, 2H,
aryl H-3'), 6.10 (s, 2H, aryl H-3'', aryl H-5''), 3.98 (m, 4H,
PyCH2), 3.90 (m, 4H, PyCH2), 3.80 (t, J ˆ 4.7 Hz, 4H, a-CH2),
2.65 (s, 12H, PyCH3), 2.21 (q, J ˆ 5.5 Hz, 4H, 2'''-CH2), 2.17 (m,
8H, PyCH2CH2), 2.05 (t, J ˆ 5.6 Hz, 4H, 1'''-CH2), 1.98 (m, 8H,
c-CH2), 1.82 (sext, J ˆ 7.0 Hz, 8H, Py(CH2)2CH2), 1.12 (t, J ˆ
7.3 Hz, 6H, Py(CH2)3CH3), 1.04 (m, 8H, b-CH2), 0.82 (s, 9H, t-
Bu), 0.41 (m, 4H, g-CH2), 0.34 (m, 4H, b-CH2), 0.10 (t, J ˆ
5.4 Hz, 8H, a-CH2), À 2.27 (br, s, 2H, NH pyrrole), À 3.33 (s,
1H, SH); 13C NMR (125 MHz, CDCl3): d ˆ 172.1 (s, CO
amide), 159.7 (s, aryl C-2'), 146.2 (s, aryl C-4''), 145.9 (s, C-1,
C-9, C-11, C-19), 143.2 (s, C-3, C-7, C-13, C-17), 141.6 (s, C-4, C-
6, C-14, C-16), 140.3 (s, aryl C-6'), 137.8 (s, aryl C-2'', aryl C-6''),
136.0 (s, C-2, C-8, C-12, C-18), 130.5 (d, aryl CH-4'), 126.5 (s,
aryl C-1''), 123.0 (d, aryl CH-3'', aryl CH-5''), 121.2 (s, aryl C-1'),
114.2 (d, aryl CH-5'), 109.2 (s, C-10, C-20), 108.3 (d, arylCH-3'),
96.9 (d, meso-CH), 68.5 (t, a-CH2), 68.3 (t, b-CH2), 65.3 (t, c-
CH2), 49.3 (t, a-CH2), 47.5 (t, 2'''-CH2), 36.0 (t, PyCH2CH2), 33.9
(t, 1'''-CH2), 33.6 (s, C(CH3)3), 31.0 (t, g-CH2), 30.1 (q,
C(CH3)3), 29.0 (t, b-CH2), 26.5 (t, PyCH2), 23.5 (t,
Py(CH2)2CH2), 14.3 (q, Py(CH2)3CH3), 13.6 (q, PyCH3);
‡
‡
(%) ˆ 1160 (10, [M ‡ 1 CONMe2] ); 1231 (100, [M ‡ 1] ).
Crown-Capped Thiocarbamoylporphyrin 39
To a solution of bis(acrylamido)porphyrin 37 (167 mg,
136 mmol) in MeOH/CH2Cl2 25:1 (25 mL) was added 1,10-
diaza-18-crown-6 (35) (180 mg, 686 mmol) in portions at room
temperature. The resulting solution was heated at reflux for
44 h. The completion of the reaction was controlled by RP-
HPLC (90 100% MeOH/H2O in 10 min, then 100% MeOH
for 10 min, flow 1.5 mL/min, ldet ˆ 414 nm, Tˆ 408C; Rt
(starting material) ˆ 10.3 min, Rt (product) ˆ 12.4 min). The
reaction mixture was concentrated to dryness under vacuum
and the crude product was purified by flash chromatography
(aluminium oxide, CH2Cl2/MeOH, 100:1) which gave 133 mg
(66%) of pure crown-capped porphyrin 39 as purple solid. Rf ˆ
0.49 (aluminium oxide, CH2Cl2/MeOH, 100:1); RP-HPLC
(90 100% MeOH/H2O in 10 min, then 100% MeOH for
10 min, flow 1.5 mL/min, ldet ˆ 414 nm, Tˆ 408C): Rt ˆ
12.4 min; UV/Vis (CH2Cl2 ‡ 1% Et3N): lmax (%) ˆ 416 (100,
Soret), 512 (6), 546 (1), 580 nm (1); 1H NMR (400 MHz,
CDCl3): d ˆ 10.80 (s, 2H, NH amide), 10.15 (s, 1H, meso-H),
9.59 (s, 1H, meso-H), 8.70 (d, J ˆ 8.2 Hz, 2H, aryl H-5'), 7.75 (t,
J ˆ 8.3 Hz, 2H, aryl H-4'), 6.94 (d, J ˆ 7.8 Hz, 2H, aryl H-3'),
6.29 (s, 2H, aryl H-3'', aryl H-5''), 4.08 (m, 2H, PyCH2), 3.90 (m,
2H, PyCH2), 3.80 (m, 2H, a-CH2), 3.74 (m, 2H, PyCH2), 3.59
(m, 4H, PyCH2 and a-CH2), 2.75 (s, 6H, PyCH3), 2.58 (s, 6H,
PyCH3), 2.28 (m, 4H, PyCH2CH2), 2.20 (m, 4H, 2'''-CH2), 2.14
(m, 4H, c-CH2), 2.03 (m, 4H, 1'''-CH2), 1.95 (m, 8H, b-CH2),
1.97 (m, 4H, PyCH2CH2), 1.91 (sext, J ˆ 7.2 Hz, 4H,
Py(CH2)2CH2), 1.85 (br, s, 3H, NCH3), 1.78 (sext, J ˆ 7.3 Hz,
4H, Py(CH2)2CH2), 1.21 (t, J ˆ 7.5 Hz, 6H, Py(CH2)3CH3), 1.19
(t, J ˆ 7.6, 6H, Py(CH2)3CH3), 1.14 (m, 4H, c-CH2), 1.11 (s, 9H,
t-Bu), 0.56 (m, 4H, b-CH2), 0.39 (dt, J ˆ 4.4 Hz, J ˆ 13.0 Hz, 2H,
g-CH2), 0.17 (m, 4H, a-CH2), À 0.08 (m, 4H, a-CH2), À 0.22
(dt, J ˆ 4.6 Hz, J ˆ 13.2 Hz, 2H, g-CH2), À 0.69 (br, s, 3H,
NCH3), À 2.09 (br, s, 2H, NH pyrrole); 13C NMR (125 MHz,
CDCl3): dC ˆ 172.1 (s, CO amide), 166.6 (s, CO thiocarbamate),
160.5 (s, aryl C-2'), 150.4 (s, aryl C-4''), 146.1, 145.9 (s, C-1, C-9,
C-11, C-19), 144.1 (s, aryl C-2''), 143.1, 141.7, 141.1 (s, C-4, C-6,
C-14, C-16), 140.2 (s, aryl C-6'), 136.5, 135.9 (s, C-2, C-8, C-12,
C-18), 130.9 (d, aryl CH-4'); 123.4 (s, arylC-1''), 122.5 (d, aryl
CH-3'', aryl CH-5''), 121.2 (s, aryl C-1'), 114.2 (d, aryl CH-5'),
109.7 (d, aryl CH-3'), 109.2 (s, C-10, C-20), 96.7, 96.3 (d, meso-
CH), 70.2, 70.0 (t, 1'''-CH2), 68.2 (t, b-CH2), 65.2 (t, c-CH2), 49.3
(t, a-CH2), 47.5 (t, 2'''-CH2), 36.2, 35.5 (t, PyCH2CH2), 34.1 (s,
C(CH3)3), 33.9 (t, a-CH2), 31.2 (q, C(CH3)3), 26.6, 26.1 (t,
‡
MALDI-TOF-MS: m/z (%) ˆ 1422 (100, [M ‡ 1] ); ESI-MS
(MeOH/H2O, 10 : 1 ‡ 0.1% NH3): m/z (%) ˆ 1422 (100, [M ‡
‡
H] ).
Bis(THF)iron Porphyrins RR,SS/RS,SR-11 and
RR,SS-11
To
a solution of thiophenolporphyrin RR,SS/RS,SR-32
(11.8 mg, 9.5 mmol) in dry and degassed toluene (3.5 mL) was
added dry and degassed 2,6-lutidine (19 mL, 164 mmol) and the
762
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Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam
FULL PAPERS
resulting red solution was heated to reflux. Then a suspension
of FeBr2 (17.9 mg, 83 mmol) in dry and degassed toluene
(0.5 mL) was added and the dark brown reaction mixture was
stirred at reflux for 1 h. After cooling to room temperature the
reaction mixture was filtered through a small pad of celite and
the residue was washed with small toluene portions until the
filtrate was colourless. The combined brown filtrates were
concentrated under vacuum and the obtained residue was
purified by column chromatography (silica gel, hexane/ethyl
acetate, 2:1) which gave 11.4 mg (92%) of iron porphyrin
RR,SS/RS,SR-11 as a brown solid.
Generation of Iron(III) Porphyrin Water Complexes;
General Procedure
A biphasic mixture of degassed toluene and degassed water
(2:1 v/v) was stirred vigorously for 30 min at room temper-
ature. Stirring was stopped and phase separation awaited. The
upper organic phase was used for dissolving the iron porphyr-
ins, such that 5 mM solutions in wet toluene were obtained.
These solutions were used directly for cw-EPR measurements.
cw-EPR (water-saturated toluene, Tˆ 100 K): Data for 33-
H2O: g ˆ 8.20, 6.00, 4.82, 3.38, 2.04, 2.01 (rhombic, high-spin);
2.42, 2.22, 1.93 (rhombic, low-spin). Data for 34-H2O: g ˆ 8.20,
6.00, 4.82, 3.38, 2.04, 2.01 (rhombic, high-spin). Data for RR,SS-
11-H2O: g ˆ 8.26, 5.17, 3.08, 2.03, 2.00 (rhombic, high-spin);
2.22, 2.15, 1.97 (rhombic, low-spin).
The same experiment was carried out with the pure
diastereoisomer RR,SS-32 (7 mg, 5.6 mmol). Treatment with
dry and degassed 2,6-lutidine (13 mL, 112 mmol) and FeBr2
(12 mg, 55.6 mmol) yielded 5.9 mg (79%) of iron porphyrin
RR,SS-11 as a brown solid after work-up and purification. Rf ˆ
0.16 (hexane/ethyl acetate, 2:1); UV/Vis (toluene): lmax (%) ˆ
404 (100, Soret), 517 nm (12); MALDI-TOF-MS: m/z (%) ˆ
Bis(THF)iron Porphyrin Imidazole Complex RR,SS/
‡
‡
1301 (100, [M ‡ 1] ), 1324 (11, [M ‡ 1 ‡ Na] ); ESI-MS RS,SR-11-Im
‡
(MeOH): positive ion mode: m/z (%) ˆ 1301 (6, [M ‡ H] ),
Iron porphyrin RR,SS/RS,SR-11 (3 mg, 2.3 mmol) was dis-
‡
1323 (100, [M ‡ Na] ); negative ion mode: m/z (%) ˆ 1300
solved in a dry and degassed 0.2 M imidazole solution (in DMF,
1 mL, 200 mmol) and the resulting brown solution was stirred at
room temperature for 20 h. The reaction mixture was used
directly for analysis. Note: judging from cw-EPR the imidazole
complex RR,SS/RS,SR-11-Im is not stable during column
chromatography. UV/Vis (toluene): lmax (%) ˆ 404 (100,
Soret);, 520 nm (11); MALDI-TOF-MS: m/z (%) ˆ 1301 (100,
(100, MÀ); cw-EPR (toluene, Tˆ 100 K): g ˆ 8.33, 5.32, 3.08,
2.08, 2.00 (rhombic, high-spin).
‡
Crown-Capped Iron Porphyrin 33
[M ‡ 1 ImH] ); ESI-MS (MeOH): m/z (%) ˆ 1301 (36, [M ‡
‡
‡
H
ImH] ), 1323 (100, [M ‡ Na ImH] ); cw-EPR (DMF, Tˆ
As for 11 thiophenolporphyrin 40 (2.9 mg, 2.1 mmol) was
treated with dry and degassed 2,6-lutidine (5 mL, 43 mmol) and
FeBr2 (5.4 mg, 25 mmol) in dry and degassed toluene at reflux
for 30 min. Work-up and purification by column chromatog-
raphy (silica gel, conditioned with toluene, eluted with toluene/
THF, 3:1) yielded 2.8 mg (92%) of iron porphyrin 33 as abrown
solid. Rf ˆ 0.39 (toluene/THF, 3:1); UV/Vis (toluene): lmax
(%) ˆ 404 (100, Soret), 517 (12), 644 nm (5); MALDI-TOF-
104 K): g ˆ 2.42, 2.21, 1.92 (rhombic, low-spin).
Crown-Capped Iron Porphyrin Imidazole Complex 34-
Im
Similarly, crown-capped iron porphyrin 34 (3 mg, 2 mmol) in a
dry and degassed 0.2 M imidazole solution (in DMF, 1 mL,
200 mmol) was stirred at room temperature for 39 h. The
resulting reaction mixture was use for analysis directly. Note:
the imidazole complex is not stable during column chroma-
tography. UV/Vis (toluene): lmax (%) ˆ 406 (100, Soret),
517 nm (8); MALDI-TOF-MS: m/z (%) ˆ 1475 (82, [M ‡ 1
‡
‡
MS: m/z (%) ˆ 1447 (100, [M ‡ 2] ), 1469 (29, [M ‡ Na] ); ESI-
‡
MS (MeOH): m/z (%) ˆ 1469 (100, [M ‡ Na] ); cw-EPR
(toluene, Tˆ 100 K): g ˆ 8.20, 6.00, 4.82, 3.38, 2.04, 2.01
(rhombic, high-spin).
‡
‡
‡
‡
‡
ImH] ), 1497 (100, [M ‡ Na ImH] ), 1514 (16, [M ‡ 1 ‡ K
ImH] ); ESI-MS (MeOH): m/z (%) ˆ 1475 (10, [M ‡ H
‡
ImH] ), 1497 (100, [M ‡ Na ImH] ), 1513 (7, [M ‡ K
ImH] ); cw-EPR (DMF, Tˆ 104 K): g ˆ 8.25, 5.20, 3.20, 2.08,
Crown-Capped Iron Porphyrin 34
2.00 (rhombic, high-spin); 2.45, 2.21, 1.91 (rhombic, low-spin).
Analogously, thiophenolporphyrin 41 (10.9 mg, 7.7 mmol) was
reacted with dry and degassed 2,6-lutidine (17 mL, 71 mmol)
and FeBr2 (15.2 mg, 71 mmol) in dry and degassed toluene at
reflux for 30 min. Work-up and purification by column
chromatography (silica gel, conditioned with toluene, eluted
with toluene/THF 1:1) furnished 5 mg (44%) of iron porphyrin
34 as a brown solid. Important note: if this reaction is carried
out on a larger scale ( > 10 mg of 41) or if applying prolonged
reaction periods the yield drops drastically (decomposition).
Rf ˆ 0.48 (toluene/THF, 1:1); UV/Vis (toluene): lmax (%) ˆ
406 (100, Soret), 516 nm (11); MALDI-TOF-MS: m/z (%) ˆ
Barium Crown Ether Complexes; General Procedure
To 1 equivalent of crown-capped iron porphyrin were added 5
equivalents of Ba(ClO4)2 as a dry and degassed 4 mM solution
(in acetone). The resulting mixture was treated with ultrasound
for 5 min at room temperature after which it was used directly
for the analysis. In order to study the influence of the
counteranion the mixture of crown-capped iron porphyrin 33
and Ba(ClO4)2 in acetone was evaporated to dryness under
reduced pressure. The obtained residue was dissolved in a dry
and degassed 7 mM n-Bu3N(TRISPHAT) solution (in CH2Cl2,
2 equivalents) and for reasons of solubility dry and degassed
acetone was added also. The resulting mixture was stirred at
‡
‡
1475 (100, [M ‡ 1] ), 1498 (19, [M ‡ 1 ‡ Na] ); ESI-MS
‡
(MeOH): m/z (%) ˆ 1497 (100, [M ‡ Na] ); 1513 (5, [M ‡
‡
K] ); cw-EPR (toluene, Tˆ 100 K): g ˆ 8.39, 7.50, 5.47, 4.91,
3.15, 2.47, 2.04, 2.00 (rhombic, high-spin).
Adv. Synth. Catal. 2003, 345, 743 765
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¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
763

Martin Lochner et al.
FULL PAPERS
room temperature for 2 h after which it was used directly for
the analysis.
References and Notes
Data for 33-H2O/Ba2‡: ESI-MS (acetone): m/z (%) ˆ 792
[1] W.-D. Woggon, Top. Curr. Chem. 1996, 184, 40.
[2] P. R. Ortiz de Montellano, in Cytochrome P450: Struc-
ture, Mechanism, and Biochemistry, (Ed.: P. R. Ortiz de
Montellano), 2nd edn., Plenum Press, New York, 1995.
[3] F. Durst, in Frontiers in Biotransformation, (Eds.: K.
Ruckpaul, H. Rein), Akademie Verlag, Berlin, 1991,
p. 191.
‡
‡
(83, [M ‡ Ba]2‡), 1447 (100, [M ‡ H] ), 1469 (19, [M ‡ Na] ),
‡
1683 (11, [M ‡ Ba ‡ ClO4] ); cw-EPR (water-saturated tol-
uene, Tˆ 100 K): g ˆ 8.20, 6.00, 4.78, 3.39, 2.04, 2.00 (rhombic,
high-spin); 2.42, 2.21, 1.92 (rhombic, low-spin).
Data for (33-H2O/Ba2‡)[TRISPHAT]: ESI-MS (acetone):
‡
positive ion mode: m/z (%) ˆ 186 (100, [Bu3NH] ), 724 (25,
[M ‡ 2 H]2‡), 792 (34, [M ‡ Ba]2‡), 1447 (30, [M ‡ H] ), 1683
‡
‡
[4] F. P. Guengerich, in Mammalian cytochrome P450, Vol.
(16, [M ‡ Ba ‡ ClO4] ); negative ion mode: m/z (%) ˆ 769
(100, [TRISPHAT]À); cw-EPR (water-saturated toluene, Tˆ
100 K): g ˆ 8.08, 5.80, 4.69, 3.45, 2.03, 2.00 (rhombic, high-
spin); 2.21 (rhombic, low-spin).
1
2, CRC, Boca Raton, Florida, 1987.
[5] a) T. L. Poulos, B. C. Finzel, A. J. Howard, Biochemistry
1986, 25, 5314; b) T. L. Poulos, B. C. Finzel, A. J. Howard,
J. Mol. Biol. 1987, 195, 687; c) R. Raag, T. L. Poulos,
Biochemistry 1991, 30, 2674; d) R. Raag, T. L. Poulos,
Biochemistry 1989, 28, 7586; e) H. Li, S. Narasimhulu,
L. M. Havran, J. D. Winkler, T. L. Poulos, J. Am. Chem.
Soc. 1995, 117, 6297.
Data for 34-Ba2‡: ESI-MS (acetone): m/z (%) ˆ 738 (9,
[M ‡ 2 H]2‡), 806 (37, [M ‡ Ba]2‡), 1475 (50, [M ‡ H] ), 1497
‡
‡
‡
(100, [M ‡ Na] ), 1711 (90, [M ‡ Ba ‡ ClO4] ); cw-EPR (ace-
tone, Tˆ 100 K): g ˆ 8.26, 5.47, 3.25, 2.75, 2.03, 2.00 (rhombic,
high-spin).
[6] I. Schlichting, J. Berendzen, K. Chu, A. M. Stock, S. A.
Maves, D. E. Benson, R. M. Sweet, D. Ringe, G. A.
Petsko, S. G. Sligar, Science 2000, 287, 1615.
N-Methylated Crown-Capped Iron Porphyrin (43)
[7] S. G. Sligar, Biochemistry 1976, 15, 5399.
Crown-capped iron porphyrin 33 (1.2 mg, 0.83 mmol) was
dissolved in dry and degassed toluene (200 mL) and to this
solution was added MeI (15 mL, 240 mmol) in portions over a
time period of 20 h while stirring at room temperature. The
course of the reaction was followed ESI-MS. The reaction
mixture was concentrated to dryness under vacuum and the
brown residue was purified by column chromatography (silica
gel, conditioned with toluene, eluted with toluene/THF, 5:1).
This yielded 0.4 mg (24%) of N-methylated crown-capped iron
porphyrin 43 as a brown microcrystalline powder and 1.3 mg of
a red compound which is presumably the S-methylated iron
porphyrin 44.
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Angew. Chem. Int. Ed. 2002, 41, 1947; b) F. Ogliaro, S.
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Physical data of N-methylated crown-capped 43: Rf ˆ 0.58
(toluene/THF, 2:1); UV/Vis (toluene): lmax (%) ˆ 404 (100,
Soret), 513 nm (11); ESI-MS (acetone): m/z (%) ˆ 1446 (37, [M
‡
‡
CH3] ), 1461 (100, M ); MALDI-TOF-MS: m/z (%) ˆ 1447
‡
‡
(100, [M ‡ 1 CH3] ), 1461 (86, M ), 1470 (27, [M ‡ 1 ‡ Na
‡
‡
CH3] ), 1485 (13, [M ‡ K CH3] ); cw-EPR (toluene, Tˆ
100 K): g ˆ 7.55, 6.07, 4.65, 3.16, 2.04, 2.00 (rhombic, high-
spin); cw-EPR (water-saturated toluene, Tˆ 100 K): g ˆ 8.20,
6.07, 4.71, 3.34, 2.04, 2.00 (rhombic, high-spin); 2.42, 2.22, 1.93
(rhombic, low-spin).
Physical data of red compound 44: Rf ˆ 0.41 (toluene/THF,
2:1); UV/Vis (toluene): lmax (%) ˆ 365 (sh, 58), 405 (100,
[15] R. Raag, T. L. Poulos, in Frontiers in Biotransformation,
(Eds.: K. Ruckpaul, H. Rein), Vol. 7, Akademie Verlag,
Berlin, 1992, p. 1.
‡
Soret), 573 nm (18); ESI-MS (acetone): m/z ˆ 1461 (100, M ),
‡
1501 (64); MALDI-TOF-MS: m/z ˆ 1461 (100, M ); cw-EPR
(toluene, Tˆ 100 K): g ˆ 7.60, 5.87, 4.18, 2.02, 1.81 (rhombic,
high-spin); cw-EPR (water-saturated toluene, Tˆ 100 K): g ˆ
7.66, 5.84, 4.18, 2.03, 1.81 (rhombic, high-spin).
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Acknowledgements
Financial support from the Swiss National Science Foundation
is gratefully acknowledged.
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764
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