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University

5

I m ' fix‘ 4 “'5
i

This is to certify that the

dissertation entitled

DISTAL STERIC AND HYDROGEN BONDING EFFECTS
IN HEME-DIOXYGEN INTERACTIONS

presented by

Michail Kondylis

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in ChemiStry

 

 

Cid/c.

Major professor

Date IQ/ICQ/gb’

 

)MSU is an Affirmative Action/Equal Opportuniry Instilun'on 0-12771

 

 

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RETURNING MATERIALS:
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DISTAL STERIC AND HYDROGEN BONDING EFFECTS
IN HEHE-DIOXYGEN INTERACTIONS

BY

Hichail Kondylis

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1985

ABSTRACT

DISTAL STERIC AND HYDROGEN BONDING EFFECTS
IN HEHE-DIOXYGEN INTERACTIONS

BY

Michail Kondylis

The synthetic analog approach has been used very often

to elucidate the structure-function relationships in many

proteins. The same approach was also used in this study.
The proposed distal steric effect of hemoglobin and
myoglobin was probed using specifically designed and

synthesized porphyrins with an off-the-center strap on one
of the porphyrin faces, and an intramolecularly appended
proximal imidazole base on the other. The kinetic and
thermodynamic parameters of O2 and CO binding to these
synthetic heme models were determined using the standard
flash photolysis and spectrophotometric titration methods.
A small distal steric effect was observed reducing the CO
association rate by a factor of not more than 3 compared to
02. This small effect cannot account for the large
discrimination against CO binding exhibited by the proteins.
On the other hand, the polarity near the binding site was

proven to be more important, reducing the affinity ratio (M)

by an order of magnitude in the models studied.

In order to quantify hydrogen-bonding effects on
dioxygen binding to hemes and thus to understand its
importance to proteins, the synthesis of Co(II) porphyrins
with functional groups of varying hydrogen-bonding ability
near the coordination site was undertaken. The realization
of this goal was made possible with the synthesis of meso l-
naphthyl porphyrins substituted at the 8-naphthyl position.
The study of meso l-anthryl porphyrins substituted at the 8-
anthryl position was also included. The affinity constants

for this series of Co(II) porphyrins were determined at

different low temperatures and their thermodynamic
parameters (AH, AS) were calculated. There is a large
enhancement in 02 affinity which correlates well with the

hydrogen bond strength of each model. The 3 orders of
magnitude variation of the binding constants, gave us a
clear picture of the importance of the hydrogen bonding in

dioxygen binding.

To my mother Irini

and the memory of my father Panayiotis

ii

ACKNOHLEDGHENTS

First of all I would like to thank Professor C. K. Chang
for his constant encouragement and guidance during the
course of this work.

I would also like to thank Mr. M. S. K00 and all the
present and past members of our group for many helpful
discussions and friendship. They are all bery-bery good
students, although they could be better.

My deep appreciation is due to my parents and my family
who put my education as a very high priority in their lives.

My thanks should also be given to Dr. Nick Leventis and
the rest of the Greek students who together with the Greek-
American community of Lansing made my stay here a little
more enjoyable.

I'm also grateful to Eleni for definitely making my new

world adventure worthwhile.

iii

LIST OF TABLES

LIST OF FIGURES

CHAPTER 1:

CHAPTER 2:

TABLE OF CONTENTS

Kinetics of CO and 02 Binding to Imidazole

Appended Strapped Hemes

Introduction ...............................

Results and Discussion .....................

Conclusion .................................

Intramolecular Hydrogen Bonding Affecting

Dioxygen Binding to Cobalt (II) Porphyrins

Introduction ...............................

Results and Discussion

A. Oxygen Binding .........................

B. Decomposition of Co(II) Naphthoic Acid

Porphyrin ..............................
Experimental ...............................
A. Dioxygen Binding to CO(II) Porphyrins...

B. Decomposition of the CO(II) Naphthoic Acid

Porphyrin [N(C02H)P] ....................

C. Fe Insertion to Naphthoic Acid

Porphyrin ......... . .....................

iv

..33
..38

..38

41

,,45

CHAPTER 3:

REFERENCES

Synthesis of Novel Porphyrins

A.

Synthesis of Models of Myoglobin

Introduction ............................. 46
Synthesis ................................ 48
Experimental ............................. 51

Synthesis of Meso Substituted Porphyrins

Introduction ............................. 62
Synthesis ................................ 63
Experimental ............................. 67

Synthesis of Novel Highly Functionalized

Porphyrins

Introduction ............................. 76
Synthesis ................................ 80
Experimental ............................. 83
....................... ........................123

Table

Table

Table

Table

Table

1‘3

LIST OF TABLES

Kinetic and equilibrium constants for CO and

O2 binding to unsymmetrically strapped and

unstrapped five coordinate hemes ................ 11

CO and O2 binding constants of diphenyl five

coordinate hemes ................................ 12

Experimental data for kinetic and thermodynamic
constants of CO and O2 binding to five-

coordinate hemes ................... . ............. 23

Thermodynamic properties for 0 binding to

cobalt porphyrins .... ........................... 29

Experimental data for the thermodynamic

properties for 0 binding to cobalt

2
porphyrins.. ..................................... 43

vi

Figure 1-1

Figure 1-2

Figure 1-3

Figure l-A

Figure 1-5

Figure 2-1

Figure 2-2

Figure 2-3

LIST OF FIGURES

Tertiary structure of myoglobin ............ 3
Graphic representation of C0 distortion in
symmetrically and side strapped hemes ...... 7
Structures of Fe(CH2)2Im, FeBzIm, FeSSP-l2
and FeSSP-l3 hemes ......................... 8
The change in dipole orientation upon
introduction of a tight strap across the

heme face .................................. 15
Spectrophotometric titration of Fe(CH2)2Im

in toluene, with CO at R.T.; [C0] x 108M =

0.0, l.A0, 4.18, 7.90, 12.5“ and 1920 in

1-6 respectively. Inset: plot of A-Ao/Am-A

vs. [CO] at 413 nm ......................... 22
The structures of NP, N(C02H)P, N(CH20H)P,
N(CONH2)P, N(CONHNH2)P, and A(C02H)P ....... 26

Graphic representation of the proximity of
the acidic proton to the bound 02 in
N(C02H)P and A(C02H)P ...................... 32

Decomposition of N(002H)P .................. 37

vii

Figure 2-A

Figure 2-5

Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure

Figure

3-10
3-11
3-12
3‘13

14

LA)
I

Spectrophotometric titration of A(CH 0H)P

2
in DMF with 02 at -30°C; P02 (Torr) =

0.0, 0.79, 2.09, 6.28, 13.61, 24.1, ”5.0,

and >760 in 1-8 respectively. Inset:

P02 at u19 nm ............. 40

plot
of A-Ao /Am-A Vs .
Van't Hoff plots for the oxygen binding of

cobalt porphyrins:

A. O: NP in DMF;A.: A(CH2OH)P in DMF.
B. O: N(CONH2)P in DMF; A N(CHZOH)P in

DMF
C. O: N(C02H)P in DMF; A N(C02H)P in

Ph3CIm/CH2C12 .......................... 42
'H-NMR spectrum of porphyrin 2 ............. 93
'H-NMR spectrum of porphyrin 3 ....... . ..... 94
'H-NMR spectrum of porphyrin A ............. 95
'H-NMR spectrum of porphyrin 8 ............. 96
'H-NMR spectrum of porphyrin ll ............ 97
'H-NMR spectrum of porphyrin l6 ............ 98
'H~NMR spectrum of porphyrin l7 ............ 99
'H-NMR spectrum of porphyrin 18 ........... .100
'H-NMR spectrum of porphyrin 19 ............ 101
'H-NMR spectrum of Dipyrromethane 21 ....... 102
'H-NMR spectrum of porphyrin 23 .......... ..103
'H~NMR spectrum of porphyrin 2A .......... ..104
'H-NMR spectrum of porphyrin 25a ........... 105
'H-NMR spectrum of porphyrin 26 ............ 106

viii

Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure

Figure

'H-NMR

'H-NMR

'H-NMR

'H-NMR

'H-NMR

'H-NMR

'H-NMR

'H-NMR

'H-NMR

'HrNMR

'HrNMR

'H-NMR

'H-NMR

'H~NMR

'H—NMR

'H-NMR

spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum
spectrum

spectrum

ix

of

of

of

of

of

of

of

of

of

of

of

of

of

of

of

Of

porphyrin 27 ............ 107
dipyrromethane 28a ...... 108
dipyrromethane 28c ...... 109
porphyrin 29 ............ 110
porphyrin 29a ........... 111
pyrrole 31 .............. 112
pyrrole 32 .............. 113
pyrrole 33 .............. 114
tripyrrin 37 ............ 115
biladiene 38 ............ 116
porphyrin 39 ............ 117
tripyrrin A0 ............ 118
biladiene A1 ............ 119
porphyrin A2 ............ 120
dipyrromethane A3b ...... 121
Porphyrin AA ............ 122

H.._._-_‘

CHAPTER 1

KINETICS OF CO AND O BINDING TO

2
IMIDAZOLE APPENDED STRAPPED HEMES

Introduction

 

The ability of the single compound protoheme to either
reversibly bind oxygen, catalyze oxidation of organic
compounds, decompose hydrogen peroxide or transport
electrons, depending upon its environment, is remarkable.1

Even among the first class of compounds, the oxygen

carriers, there are large variations in the kinetics and

equilibria of oxygenation, which have been the subject of
extensive investigations. Hemoglobin is one of the few
extensively studied proteins. It was the first protein
crystallized (1849); the first protein with a recognized
physiological purpose (02 transport 1864; 002 transport
1904); one of the first proteins whose molecular weight and

primary sequence were established (0. 1930); and one of the
first proteins whose tertiary and quartenary structures were
determined by x-ray crystallography (1960).1d Hemoglobin
(Hb) is a tetrameric protein consisting of 2a- and 28-
subunits (141 and 146 amino acids, respectively) with one
protoheme IX (iron (ii) protoporphyrin IX complex) per
subunit. Myoglobin (Mb) consists of a polypeptide chain of
153 residues and one protoheme, which closely resembles one
tsubunit of a hemoglobin, tetramer. In myoglobin the iron
atom is coordinated by four porphyrin nitrogen atoms, and

1

the nitrogen atom of the proximal imidazole (from a

histidine).2’3 (Fig. l-l).

To examine the relationships between function and
structure of dioxygen binding heme proteins, the proteins
themselves offer rather limited possibilities. One

productive and thus very useful approach, used very often,
is the synthetic analog approach.2 That is, the design,
synthesis, characterization and study of "small" models
((2000 amu) of metalloproteins which are structurally and
functionally similar to the active site of metalloproteins.
Due to its flexibility, the synthetic analog approach can
lead to a better understanding of the active site than can
be obtained from the protein itself. It allows us to change
the structure of the model and by comparison of its
spectroscopic and reactivity properties, to recognize
previously unrevealed functions of the protein. The study

of metalloproteins many times follows the sequence:2b

1. Isolation and purification of the metalloprotein.
2. Measurement of physical properties of the active site.
3. Synthesis and isolation of analog complexes.
4. Characterization of structural, spectroscopic and

chemical reactivity properties of the synthetic analogs.
5. Comparison between the protein and the analogs and among
the analogs to reveal new structure-function

relationships.

 

 

M YOGLoazN

 

Tertiary structure of myoglobin.

Figure 1-1

 

4

One may argue for this sequence that molecular modeling
occurs only after a great deal about the proteins is already
known. That's because it is difficult to design and
synthesize active site model compounds unless one already
has enough information about the active site. Synthetic
models can provide a detailed understanding of the structure
and functioning of the active site which cannot be obtained
(or at least not without extreme difficulty) by studying the
hemoprotein itself. The modification of the metal
reactivity by the protein can also be revealed by the study
of simple models where the metal is not influenced by the
protein. Finally, the synthetic analogs can give us a
better understanding of the function and mechanism of
biological systems.

The so-called distal steric effect in Hb and Mb is one
of the problems currently being probed by means of synthetic
models. The idea that non-bonding interactions with protein
residues, like the distal imidazole, can after the binding
of ligands to hemoglobin and myoglobin, began with the
demonstration by St. George and Pauling“ of reduced binding
of isocyanides (RNC) with increasing steric bulk of the R
group. X-ray crystallography showed that the structures of
carbon monoxide liganded hemoglobin and myoglobin exhibit a
bent or tilted FeCO linkage with respect to the porphyrin
ring,5 whereas in heme model compounds the FeCO bond is
linear and perpendicular to the heme plane.6 The origin of

the distorted configuration in the proteins is attributed

primarily to nonbonding steric interactions of the axial
ligand with residues at the distal side. An assumption is

made that ligands such as O which preferentially form bent

2 1
complexes, should encounter less steric hindrance when bound

in the heme pocket.7

It has been proposed that in Hb and Mb,
the distal side steric effect would decrease the affinity
ratio of C0 vs. 02, and is responsible for the
detoxification of CO poisoning in respiratory systems.8 A
comparison of ligand binding constants of proteins and model
compounds often shows that many heme models have a larger CO
vs. 02 affinity ratio (M value) than the proteins. However,
such a comparison does not necessarily constitute a
correlation between the distal steric effect and affinity,
as the ligand binding constants of heme models can be
drastically altered by medium effects.9 Indeed, Traylor and
coworkers have shown that a five-coordinate protoheme-
imidazole model binds both 02 and CO in aqueous suspensions
with equilibrium and kinetic parameters almost identical to

9’10 which led them to

R—state isolated hemoglobin chains,
the conclusion that R-state Hb simply maintains the
protoheme five-coordinated, soluble in water and protected
against oxidation, without any steric hindrance. In other
cases, for example, T-state Hb and notably myoglobins have
very small M values which cannot be duplicated by simple
heme compounds and so they must be subjected to some distal

side steric hindrance.

It is, therefore, of importance to examine the steric

 

6
effects on ligand affinity using synthetic models equipped
with varying degrees of steric hindrance at the distal side.
Several porphyrin models of this kind have been preparedll
but failed to show the degree of discrimination against C0,
that is present in myoglobin. All the existing models have
a covalently bound alkyl or aromatic group residing exactly

above the center of the porphyrin ring, protecting the

distal side of the heme and at the same time providing

steric hindrance for the incoming gaseous ligands. And
indeed reduced association rate constants for CO and 02 have
been observed for these models, but without being able to
reproduce the differentiation, Mb shows, in favor of 02. The

fact that for those models both 02 and CO association rates
are reduced to values that in some cases are even lower than
that for myoglobin and yet, the proper differenziation
between them is not observed led us to believe that the
differentiation may not be proportional to the steric
hindrance but that it rather reaches a maximum and then
decreases, as the steric effect becomes too great. In order
to test this hypothesis new model compounds with more
"delicate" steric hindrance should be synthesized. It is
reasonable to assume that strapped porphyrins with the strap
off-the-center of the ring would impose the desired gentle
steric effect (Fig. 1-2). It was with these thoughts that

the strapped porphyrins shown in Fig. 1-3 were synthesized

and studied.

 

 

® ® ,0
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Figure 1-2 Graphic representation of C0 distortion in

symmetrically and side strapped hemes.

 

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Results and Discussion

 

Here is presented the equilibria and kinetic rates of C0

and 0 binding of a system which possesses both an off-the-

2
center strap and a covalentely bound imidazole. Two
unprotected hemes were also studied for reasons of
comparison with the protected system. The equilibria

constants for C0 binding were determined by means of direct

Spectrophotometric titrations_ of a degassed and reduced16

solution of the hemes in toluene. The C0 association rate

CO) were also determined directly by flash

*CO

constants (k

photolysis. The CO dissociation rates (k ) were

calculated from the C0 equilibrium constants and the CO

association rates. 02 affinities were determined by C0

competition measurements and calculated according to the

Gibson equation:la

l/R = 1/k~02 + K02/kCOEC0]

The 0 association rate constants (k02) were determined

2
directly by means of flash photolysis, while the 02
dissociation rate constants (k-O2) were calculated from the
O

02 affinities and the k 2. A detailed description of how
the kinetic and equilibria values were obtained is described
in the "Materials and Methods" section of this chapter. All
the rates and equilibrium constants for the model compounds

studied and relevant heme proteins, are tabulated in Table

1-1.

10
The first somewhat unexpected result one can observe is
the higher than usual, although not entirely

llf’lub rate constants for the two

unprecendented,
unprotected hemes. (Compare for example Tables 1-1 and 1-2).
If these values are compared with the very similar series of
diaryl substituted hemes studied earlier12 (Table l-2), it
is apparent that all the rate constants are 2-5 fold larger
in the present system. The two heme systems are very
similar (same imidazole tail, same solvent and temperature,
same electronic effect from the porphyrin periphery), the
only difference being the second aryl group. The electronic
nature of the porphyrin ring cannot account for this
difference simply because there is hardly any difference
between the two system, and even more, in cases where
appreciable changes in the electronic nature of the heme
periphery have been introduced only the 02 dissociation rate
is affected.luC For the same reasons one cannot expect
differences at either polarity of the ligand binding site or
solvation effects to account for this unexpected kinetic
behavior of our new models. Another effect which can
influence the kinetic behavior of the hemes is the heme
deformation effect which is due to a movement of the iron
atom either toward (proximal push) or away from (proximal
pull), the incoming ligand.”b the proximal push, where the
iron atom is "pushed" by the proximal base (imidazole)

towards the plane of the porphyrin, thus making it more

accessible to the incoming sixth ligand, would be expected

 

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13

to enhance both C0 and 02 affinities and provide an
attractive explanation for the observed rates for FeBzIm.
However, this cannot be true. The fact that the two
unhindered hemes, FeBzIm and Fe(CH2)21m, with the different
imidazole tails but very similar kinetic parameters leaves
no doubt that the proximal base's position and orientation
cannot be responsible for the kinetic behavior of these
hemes. So, after all, it seems reasonable to assume that
the different kinetic behavior of FeBzIm and Fe(CH2)2Im can
be attributed to the unsymmetrical substitution of the
porphyrin ring which in turn can cause distortion of the
porphyrin ring, thus altering its reactivity.lua Structural
studies have shown that the binding of a ligand to a five-
coordinate iron (II) porphyrin is accompanied by movement of
the iron into the porphyrin macrocycle. So an "unusual"
conformation of the heme ring is reflected in the rate
constants. At present there is not sufficient structural
data available to unequivocally substantiate this, prc:-sa1,
but it seems the only plausible explanation fitting all the
experimental observations.

Comparing now, the unhindered hemes to FeSSP-l3, one can
clearly see that the CO affinity is decreased while the 02
affinity increased. And as a result M is reduced from 4300
and 6500 for FeBzIm and Fe(CH2)2Im respectively, to 860 for
FeSSP-l3. This is a significant reduction, but it does not
completely reflect the magnitude of the steric effect the

strap ‘imposes on the binding site of the heme, as a closer

 

 

 

l4
look at the kinetic parameters reveals. Previous work from
our laboratory as well as from others has demonstratedlz’lu
that any distal side steric effect will be reflected in the

association rates of CO and 0 leaving the dissociation

2.
rates unaffected. In the present case though, even the CO
dissociation rate is reduced going from the unprotected
systems to the strapped ones. Having in mind that the CO
dissociation rate is practically insensitive to any distal
steric effect, one has to conclude that the tight strap
which is introduced over the porphyrin ring forces it in a
different conformation, thus cancelling the effect of its
unsymmetrical substitution. So the reduction of the CO
dissociation rate can be attributed mainly to a
conformational change of the porphyrin ring. If one now
considers the change of the 02 dissociation rate, it is
evident that it is reduced too, but reduced 2-3 times more

than the CO dissociation rate. There must be a different

reason for this greater reduction of the O dissociation

2
rate, in addition to the conformational change of the
11,12,13

porphyrin. As it has been shown before where amide

linkages are holding straps or caps over the porphyrin ring,
there is a constructive dipolar interaction between the

amide dipole and the Fe-O dipole which stabilizes the bound

2

02 and, of course, reduces its dissociation rate (Fig. 1-4).

So it is not surprising that the 0 dissociation rate is

2

reduced much more than the CO on going from the unhindered

heme to the strapped one. So far it has been clear that by

15

 

 

Figure 1-4 The change in dipole orientation upon
introduction of a tight strap across the

heme face.

 

16

comparing only the affinity ratios of different hemes it is
impossible to determine the magnitude of the distal steric
effect accurately, since many other effects also play
important roles in determining M. It would be better to
compare the ratio of the association rates of 02 vs. CO, as
Traylor suggested,lu because it is the association rates
that are primarily affected by distal side steric effect.
Indeed when the ratios of the association rates for the two
models are compared, it is evident that there is a 2-3 fold
differentiation against CO (Table l-l). So for FeSSP-l3
there certainly is a small steric differentiation in favor

of 0 and although its magnitude cannot account for the

2;
much greater difference in affinity ratios between
myoglobins and unhindered heme models it is nevertheless
significant. Now if the tighter strapped heme FeSSP-l2
which might be expected to show a greater steric hindrance
is compared to FeSSP-13, certain important differences can
be observed. First of all, M is reduced by almost an order
of magnitude to come very close to that of myoglobin. But
again a closer look at the individual rate constants reveals
that this improvement is not due to an enhanced steric
differentiation but rather due to a large stabilization of

the bound 0 which is evident by the more than 10-fold

2 9

reduction of the O2 dissociation rate. In fact, although

the strap of FeSSP-l2 is tighter than FeSSP-l3 even the

small but significant distal steric effect of FeSSP-l3 is

completely lost (kO2/kCO for FeSSP-lZ is practically the

17
same with the one of the unprotected hemes). As the study
of the CPK models for these two compounds revealed, the
reason for this loss of steric effect is that because the
tighter strap in FeSSP-lz is forced to assume a conformation
with the bulk of it lying away from the center of the heme.
The steric interaction between the strap and the incoming
gaseous ligands is thus diminished. It is also the fact
that the strap of FeSSP-12 consists of an even number of
atoms without any carbon at the center of the strap (as
opposed to FeSSP-13), combined with the different
conformation of the strap, that brings about the complete
loss of the distal side steric effect. An alternative
explanation for the loss of steric hindrance can be put
forward by using Traylor's proposa1.lu This proposal states
that the distal side steric effect is not directly related
to repulsion in the bound state but is governed by the
limited access to the heme face. In other words, the
transition state and bound state are equally affected by
steric encumbrance, the mechanism comprising a rapid
conformational equilibrium among almost equal energy states,
some of which deny access to the heme and thus slow down the
association rates. In the present case of FeSSP-12 one may
argue that the strap is very short for any of its low-energy
conformation to reach far enough and protect the heme
center. Consequently, no distal steric effect is observed.
The validity of this explanation, though, could not be

decisively confirmed by this study.

18
The decrease, now, in the O2 dissociation rate can be
explained in terms of better and stronger dipole-dipole
interactions between the bound 02 and the amide groups of
the strap. The shorter strap, on the one hand, changes the

orientation of the amide to better align itself with the

bound 0 dipole, and on the other hand, brings the dipoles

2
closer, for a stronger interaction12 (Fig. 1-4). The result
is a greater stabilization of the bound 02 which is
reflected in the reduced 0 dissociation rate. And it is

2

this reduction of k-OZ that overcomes the loss of steric

differentiation and which gives rise to a net decrease of

the affinity ratio.

Conclusion

 

At first it is noteworthy that with the exception of the

O2 dissociation rate, FeSSP-13 has CO and O2 kinetic rate

constants very similar to Mb. Secondly, it is also evident
from the kinetic parameters of FeSSP-12, that 02 binding is
greatly affected by the constructive, head-to-tail, dipole-
dipole, interaction with the polar amide linkages. Hydrogen
bonding to the bound oxygen has also been shown to be even
more effective in reducing the O2 dissociation rate.12 30 it
is not surprising that FeSSP-l3, which does not have the
capability to form hydrogen bond(s) to the bound 02 and in
which the dipole-dipole interaction is not very strong,

exhibits a much larger k‘02 value than does oxomyoglobin,

where a hydrogen bond between the bound O2 and the distal

19

3a

imidazole NH has been shown to exist. So from this work,

as well as from earlier studies,12 it is evident that the

influence of different factors like the medium, local
polarity, hydrogen bonding, porphyrin deformation etc., is
greater than the steric effect. So in general our results

reaffirm the notion“l that distal side steric effects cannot

account for more than a 3-fold differentiation of CO and O

2
and that the proper differentiation cannot effectively be
brought about by distal side steric effect alone.lu The

present study also indicates that it would be a unique
synthetic challenge to prepare heme models that match Mb's
kinetic behavior. Such a model should possess some distal
steric hindrance like the one of FeSSP-13 and at the same
time a functional group capable of forming a hydrogen bond

(like -OH or -CONH2) with the bound 0 at the appropriate

2 3

distance from the center of the heme.

Materials and Methods

 

The synthesis of the models discussed here is outlined

v

in tne synthetic part of this work (Chapter 3). Iron

insertions were accomplished by the ferrous bromide
method.15 Toluene was purified by stirring at R.T. with
several changes of cone. H280“ followed by drying over

anhydrous sodium carbonate and distillation from lithium
aluminum hydride just prior to solution preparation. Sample
solutions for kinetics and CO titrations were prepared by

dissolving the ferric compounds in approximately 4 mL of

20

u

5 M) containing 10- M of benzophenone. The

toluene ( 10-

solutions were degassed in a 120 mL tonometer by freeze-

pump-thaw cycles at 10..5 Torr. The hemin chlorides were

reduced by photolysis according to the previously described
16

method. Kinetic rates were measured in toluene at R.T. by

flash photolysis17 according to:

 

 

 

Flash photolysis was carried out with either a xenon
photographic flash gun (Braun 2000) or a flash lamp pumped
dye laser (Phase-RDL2100) with rhodamine 6G dye. Decay
constants were calculated from transmittance vs. time
measurements at 407 nm (oxyheme appearance). CO association
was monitored at 413 nm and the output of the
photomultiplier was recorded on a Bascom-Turner recorder
through a log amplifier in absorbance units then directly

computed as pseudo-first order rate constants. CO and O

5

2
concentrations ranged from 1 x 10- M to 2 x 10-” M and 3 x

-5 -u

10 to 8 x 10 M, respectively. CO association rates

(kCO

) were calculated from plots of the observed pseudo-
first order rate constants vs. CO concentration which
typically had correlation coefficients of 0.996 to 1.000 and
varied between experiments by less than 10% O2 association

rates (kO2) were calculated from similar plots with

21

correlation coefficients of not less than 0.92. Oxygen
affinities were determined by CO competition measurements

and calculated according to the Gibson equation:la

l/R = l/k-OZ + KO2/kCOECO]

where kCOECO] was the observed pseudo-first order rate
constant determined before the introduction of 02. Oxygen
dissociation was calculated from the observed oxygen
association rate and equilibrium constant. Carbon monoxide
affinities were determined by direct titration of the heme
with a gas mixture containing 0.73% CO in argon, at R.T.,
using a standard Spectrophotometric procedure used by
Halpern and coworkers18 (Fig. 1-5). The experimental data
for kinetic and thermodynamic constants are tabulated in
Table 1-3. CO dissociation rates (k_CO) were calculated

-CO kCO/KCO.

from k = Optical spectra were recorded on a

Cary 219 spectrometer.

22

 

 

 

 

 

 

 

 

 

 

 

'04" J ./
IA} .
fl '24
I04
LZ-i f . ,
.334 ,/
2.1
J
'00"I 31' .
l /
2
O . .
_T 2 a 6 a 0 z
“(2)1 .8" 0 (cozxuosu
e
O
a)
Q .6-
.4- X4
43-
Q-
I If T I
400 500 600
A nm
Figure 1-5 Spectrophotometric titration of Fe(CH2)ZIm

in toluene, with CO at R.T.; [CO] x 108M =

0.0, 1.40, 4.18, 7.90, 12.54 and 1920 in
1~6 respectively. Inset: plot of A-Ao/Am-A

vs. [CO] at 413 nm.

23

Table l-3. Experimental Data For Kinetic and Thermodynamic Constants

 

 

of CO and 02 Binding to Five-Coordinate Hemes
Compound R%?O _lkCO -l RQOZ _lk02 -1
Torr M sec Torr M sec
6 8
FeBzIm 0.0069 3.97 x 10 26.0 1.8 x 10
0.0072 4.23 x 106 33.2 2.2 x 108
4.63 x 106 30.2
Fe(CH2)2Im 0.0074 3.81 x 106 49.6 1 62 x 108
0.0084 4.19 x 106 54.1 1 97 x 108
FeSSP-l3 0.0135 2.56 x 105 11.5 3.73 x 107
0.0200 2.99 x 105 11.8 3 00 x 107
0.0094 2.09 x 105
3.40 x 105
FeSSP-12 0.0050 10.30 .787 4 42 x 107
0.0067 6.36 .677 4 71 x 107
0.0076 8.02

 

CHAPTER 2
INTRAHOLECULAR HYDROGEN BONDING AFFECTING DIOXYGEN

BINDING TO COBALT (II) PORPHYRINS

Introduction

 

The binding of molecular oxygen to cobalt complexes has
been the subject of considerable interest in the last 10-15
25,26 . . . .
years. During that time the thermodynamic properties of
many Co(II) complexes have been investigated. It was well

established by the early studies in this field, that the

pentacoordinate Co(II) porphyrin complexes (LCoP), where the

fifth ligand is an external ligand (pyridine, imidazole,
DMF, piperidine, etc.), can reversibly bind 02 at low
temperature in a variety of solvents. Dioxygen has been

shown to form a 1:1 adduct with LCoP according to the

equation:

LCoP + 02 -——£——-rLCoP(02)

The cobalt-oxygen bond was also probed and it was found to

PR25a,d

be very polar. In fact E studies clearly suggest

III 7
02,
where cobalt is oxydized and O bears a negative charge,

2
II-O . It is not surprising that the 0 adduct of

2 2
27

that it is actually more accurately represented by 00

than Co

II
0

LC P is stabilized in polar solvents. One also expects

to see a much greater effect on the O2 binding to Co(II)

complexes by hydrogen bonding. In addition, the hydrogen

24

25

bonding ability of the distal imidazole to CoMb (Myoglobin

reconstituted with CoP), may enhance its 02 binding ability

by several orders of magnitude in comparison with CoP.
While direct evidence such as neutron diffraction

3a 30 has left no

studies of oxymyoglobin and oxyhemoglobin
doubt that heme-bound dioxygen has a tendency to form
hydrogen-bond with proton donors, quantitative estimation of
how much this interaction contributes to the overall
stability of the metal-dioxygen complex has been scarce, due
to the inavailability of suitable models. Using a cobalt

33 observed a 400 fold

salicylidenimine complex, Drago et a1.
increase in oxygen affinity when trifluoroethanol was added
to the CH2012 solution, suggesting intermolecular hydrogen-
bonding. The abnormally high oxygen affinity observed for

Co(II) "picket-fence" porphyrin26e was also attributed to a

H-bonding effect of the o-anilido groups,27 although the
rather long 4 A distance separating N-H ""02 seems to
render this interpretation less tenable. In an effort to

create an ideal environment for such a hydrogen-bond to
occur, we have designed porphyrin models in which an
intramolecular hydrogen donor is juxtaposed to the terminal
oxygen atom of the coordinated dioxygen (Fig 2-1). These
[models should provide a means of quantitatively determining
the influence of H-bond on the formation of dioxygen
adducts. Barring electronic effects, the strength of
hydrogen-bond is largely dependent on steric constraints.

For effective hydrogen-bonding to take place, it is

26

HOOC

"P wccoome

 

moowwpe

   

N<C0NH~H,>P
A<CH,0H)P

Figure 2-1

  

The structures of NP, N(C02H)P, N(CH20H)P,

N(CONH2)P. N(CONHNH2)P, and A(C02H)p. A(COOH)P

27

essential to minimize the distance and the freedom of motion
between the proton source and the acceptor. The realization
of this goal was possible with the synthesis and study of

intramolecularly hydrogen-bonded cobalt-O complexes.

2

Results and Discussion

 

A. Oxygen Binding.
Here is presented the equilibrium constants and

thermodynamic parameters of 0 binding to a series of Co(II)

2
porphyrins with functional groups capable of forming
hydrogen-bonds of different strength, at very close
proximity to the metal center. For comparison, a similar
porphyrin with the same substitution but without any
functionality near the metal center was also synthesized
and studied. The equilibrium constants were determined at 3
different low temperatures (-42°, -30° and 0°C) in DMF, and
in some cases CHZClz/imidazole. The dioxygen adduct formed
in DMF, without extraneous nitrogen base, has a DMF molecule
weakly bound trans-axially. In the presence of imidazole
base, the O2 affinity increased significantly as a result of
the stronger axial base, but this affinity remained constant
throughout the range of base concentration tested (0.1 -

2 mM). The reluctance of CO(II) porphyrins to form a

25f

6-coordinate bis-imidazole complex thus allows us to

relate the 02 binding of models with that of proteins. The
equilibrium constants and thermodynamic parameters of these

model compounds were obtained as described in the "Materials

28

and Methods" section of this chapter. All the equilibrium
constants, Pl/Z and AH and AS values are tabulated in Table
2-1.

Our results clearly demonstrate that the presence of a
protic group near the dioxygen binding site, greatly
increases the Co-O2 formation constant, producing a free
energy gain between about 3 kcal/mole for N(002H)P to 1.1
kcal/mol for N(CH20H)P, with reference to NP in DMF at -
42°C. The enthalpy change (obtained from Van't Hoff plots)
for formation of these intramolecular hydrogen-bonded
dioxygen complexes also increases significantly. More
importantly, both the affinity increase and the enthalpic
gain correlate well with the hydrogen-bond strength.
Considering the literature thermodynamic parameters of
hydrogen-bonding for the following pairs: benzoic-acid and
DMSO: AH = -8.8 kcal/mole, AS = -20.7 eu; benzoic acid and
tripropylamine: AH = ‘12.9, AS = -26.2; benzyl alcohol and
amines: AH = ~10; the large gain in enthalpy for our model
compounds may be entirely attributable to the intramolecular
hydrogen bond. The large negative entropy is also
consistent with the loss of rotational degree of freedom of
the metal-bound 0 .

2

It is also evident from the results on Table 2-1 that

the difference between the naphthalene and anthracene
substituted porphyrins is negligible. The anthracene acid
and naphthoic acid, as well as the anthracene alcohol and

naphthalene alcohol porphyrins have practically indentical

29

 

 

m + H0 mug 0130.0 .... d: 0
swmm- Hum? mm.0ns.= TS x 2.0H:.mv 0m-
0.0 ..1 0.4 TB 0. 8.0 H #3 ms- 020 izomzovzou
500.0 M 000.0 H .+. 2 m7 020 010120208200
CNHS ~13 x Am0.0ua.: 0
5M0? SAME- m.0...0.: 8.0....000 0m;
00.0 M 0m.0 0.0 M m.m ms. .120 imzzoovzoo
m00.0 H 000.0 A + om ms. 020 immoozoo
5H0: ~13 x Am.0H0.m0 0
00.0 H 3.0 70 H :4 0T
m H 00- m; H mm- 000.0 .+. 000.0 0 u. 0m 0:. 0:0 .190000200
0 H 0» 0-04 x $0. M 9.: 0m-
0 H mm- 3.0 ... 2.. .10 H a 0-04 x 30. ..1 0:; N7 020 0200
A000 Aaoe\amoxv v
mmc mz< ALLOHVAE Aaucgoevx Aoove uco>aom ncsoaeoo
m

mCHL>2QLom namnoo 00 mcfiocwm

o no» wmfiueoqoem ofiemczuosnmce

.HIN manmk

 

 

3O

 

. Fm @OCQLQQQL SOL.”— .U .Om QOCQL®.HOL SOLE .0
.00 000 :0 0000 000000000000 .0 00000 a .00000 00000000 .0
0.0 1 m. H1 m. m 00 00:00
o o z :m1
. 0 .m . 00:00
0 0 1 0 H1 m =0 00 :01
0:1 m.-1 0m mm ofimmLocv 0200
001 0.001 00 mm 0:0:003 000000 0:00
0A0: mav
00.0.” 0.0 0100 x :0.0.H 0.00 0
0 H.001 H H.001 000.0 H.0m0.0 0 H.00 0m1
. 1. . . 1. . m 0 m m
010: x :00 0 + m :0 010: 0:0 m + 0 000 001 00 :0\eH0 00 0:: 000200
. I. . . I. . m m m
m 0 + 0 0 m 0 + 0 a 001 H0 :0\eH0 00 0200
m “.00 010: x A00.0 H.0.00 0
N H.001 0.0.H 001 0.0.” 0.0 :10: x 00.0 H.0.m0 001
0.0 H.N.N 00 x Am.0 H.0.00 001 020 00:00:00000

H11

31

binding constants. This fact, first, is surprising because
one would expect to see some differentiation due to the
longer distance between the metal center and the functional
group is located in the anthracene case. Nevertheless, a
closer look at the structure of these compounds reveals that
the distance difference is not critical and the anthracene
substituted porphyrins have the same capability to form
hydrogen-bond with the bound oxygen as their naphthalene
counterparts (Fig. 2-2).

The 0 binding affinity of N(C02H)P and NP was also

2

studied in CHZCI2 using a substituted imidazole as the fifth

ligand for the Co complex. This system resembles the
proteins because an imidazole is the proximal base in the

protein case, too. The difference between the O2 binding

affinity at the two solvent systems, is about an order of
magnitude and it is attributed to the fact that imidazole is
a much stronger ligand than DMF. So the strong hydrogen-
bonding ability of 23 provided the necessary stabilization

for the bound O2 and for the first time the equilibrium

constant of CoMb can be reproduced by a simple model.
Nevertheless, an important difference between the model and

protein cannot escape attention. While the high 0 affinity

2
exhibited by CoMb and Cpo using a single hydrogen-bond in

model porphyrins has been duplicated, the thermodynamic data

suggest that the enhanced O affinity of the protein has

2

more subtle cause than the mere formation of a hydrogen-bond

36

with the distal histidine. It has been pointed out that

32

.01000000 000 01000002

:0 No 0:200 0:0 00 000000 000000 0:0

00 >0~e~xona 0:0 00 coaumucmnccacn 0020000

OAQIIC

NIN mLzmqm

 

33

the enhancement of O2 affinity caused by the apoprotein can
be the result of a delicate balance between enthalpic and
entropic components which differ from protein to protein.
This study makes it very clear that a moderate hydrogen—bond
involving the histidine N-H can easily supply the 3
kcal/mole AH(negative) needed for stabilizing the 02 adduct.
However, the accompanying loss in AS observed in models is
not present in the protein, possibly because of other
compensatory factors. Some of the positive entropy may come
from conformational changes distributed throughout the
protein. Also unlike models, a pre-formed protein pocket
has the advantage of not showing the solvent reorganization
entropic loss upon oxygenation. Indeed, these seemingly
small contributions are often the major obstacles, as well

as challenges, in our quest for mimicking enzymes with

synthetic models.

B. Decomposition of the Co(II) Naphthoic Acid Porphyrin.
From the numerous studies of O2 binding to CO(II)
porphyrin complexes it is well established that for a Co(II)
porphyrin to bind oxygen it must be 5-coordinate and even
then low temperatures are needed to achieve appreciable
binding. The tetracoordinate Co(II) - porphyrins are very
stable at ambient temperatures when dissolved in non-

26,27 It

coordinating solvents such as methylene chloride.
is, thus, a unique property of the Co(II) naphthoic acid

porphyrin (N(COZH)P) not only to become oxydized in CH2Cl2

 

 

34

at R.T., but to decompose as well. The activation of
dioxygen by a Co(II) porphyrin under such mild conditions
and the complete destruction of the porphyrin ring, are
completely unprecedented. This intriguing phenomenon was
studied and efforts to elucidate its mechanism were made.

In this effort to understand the mechanism, the
necessity of the dioxygen had to be established. And
indeed, when the CO(II) complex of N(COZH)P was kept
dissolved in CH2C12 under argon, no decomposition took
place, even after prolonged periods of time. As soon as
this solution was exposed to air, the decomposition (evident
from the color change) would start immediately. So it is
beyond any doubt that oxygen is needed for the
decomposition. The second piece of experimental evidence
that indicates that the carboxylic acid group is essential
is that if ammonia is passed through the methylene chloride
solution before it is exposed to air, N(COZH)P is perfectly
stable. That means that the acidic proton of the porphyrin
sitting close to the metal is necessary. That should not be
surprising though, since the difference between this
unstable Co(II) porphyrin and all the other ones is exactly
this carboxylic group, so that is only natural that it does
participate in the reaction. The solvent is also important
for the decomposition. If DMF is used instead of methylene
chloride, then, the porphyrin ligand does not decompose

III
0

although the cobaltous ion oxidized to C immediately.

This solvent effect can be attributed to the mild basicity

35

of DMF and its polarity which effectively "diffuse" the
strength of the acidic proton rendering it unable to promote
the decomposition.

The effect of external acid on the system is also very
profound. The decomposition takes place significantly
faster when a strong acid is present. In the presence of

HCl gas in CH Cl

2 2, the decomposition takes place with a

tW of about 1 min. while in the case of the untreated
solution tw‘ is about 10 min.

Another important finding is that radical traps, such as
duroquinone and galvinoxyl,, do not inhibit the reaction, so
that any radical chain mechanism can be ruled out.

The most important result, though, is the isolation and
characterization of one of the major products of this
decomposition, namely the Etiobiliverdin IV (EBV IV, Fig.
2-3) which suggests that one of the major pathways of the
reaction involves an attack of the meso position of the
porphyrin where the napthyl group is attached by the
activated 0 . This is contradictory to the previous

2
examples of metallo-porphyrins' decomposition by H202 where
the phenyl-substituted meso positions are the most stable
towards oxygen attack (because of steric reasons).38 In a
separate experiment, when the Co(II) complex of meso-
monophenyl porphyrin was decomposed in methylene chloride
with H202 and the H1 NMR of the mixture of the colored

products was taken, the phenyl group could be seen attached

to the tetrapyrroles. That means that 02 attacks preferably

 

 

36

at the unsubstituted meso positions as opposed to the
N(C02H)P case, where no napthyl protons were detected. The
importance of the position of the carboxyl group is also
demonstrated by the inability of A(C02H)P to initiate the
same sequence of reactions, although it, too, binds to
dioxygen with the same affinity as N(C02H)P (Table 2-1).
This observation clearly shows that the carboxyl group being
so close to the ring cleavage position is not coincidental
but that it should play a dominant role in the sequence of
reactions. A plausible mechanism for the decomposition of
N(002H)P consistent with the experimental facts is thus
proposed, and is outlined in Fig. 2-3. The first step is
the O2 binding to the Co center and the intramolecularly
protonation of the bound oxygen by the carboxylic proton.
We suggest that in the presence of an acid catalyst, the
carboxylic group would react with the metal-bound ~02H
species much the same way as it would reaction with hydrogen
peroxide to yield a peracid.

The peracid. being closer to the substituted meso
position of the porphyrin will, of course, preferentially
hydroxylate this methine position rather than any of the
other three, to cause the collapse of the porphyrin ring.
The actual mechanism of the ring cleavage cannot be
determined at this time but may involve a reaction sequence
very similar to the biodegradation of heme molecules that
39

lead to the formation of bile pigments.

In conclusion, it is evident that the strength of a

 

 

 

37

 

NH ,

MN I ‘17

 

 

 

 

 

Ei

 

 

Figure 2-3 Decomposition of N(COZH)P.

____. _

38

hydrogen donor, as well as its position relative to the

binding site of 02, have a very large effect on the

stability of the 0 adduct and the activation of dioxygen.

2

Experimental

 

IA. Dioxygen Binding to Co(II) Porphyrins.

The synthesis of the porphyrin-nathoic acid (N(COOH)P)
and its derivatives is outlined in the synthetic part of
this work (Chapter 3). The synthesis of the porphyrin

anthracene acid and its derivatives as well as the

23

naphthalene porphyrin is described elsewhere. Cobalt ion

was incorporated into the prophyrins as follows: 5 mg of

porphyrin free base were dissolved in CH2C12 (10 mL) and the

solution was degassed by passing argon through it for 5 min.

Then a solution of excess CoCl2 and anhydrous sodium acetate

in CH OH (5 mL) was added. The mixture was heated on a

3

steam bath under argon, until all the solvent was

evaporated. CH2012 was added (x.5 mL) and the CoP was

transferred to a syringe containing a degassed solution of

sodium dithionite in H20. The reduced Co(II) porphyrins

were then dissolved anaerobically in freshly distilled DMF

“5

(approximately 4 mL, 1 x 10 M). The solutions were

degassed in a 60 mL tonometer by freeze-pump-thaw cycles at

10“5 Torr. Oxygenation was monitored spectrophotometrically

at several temperatures. Binding constants in 1 mM solution

of l-tritylimidazole in CH2012 were similarly determined.

(CH2C12 was distilled over LAH just prior to use.) The low

39

temperatures were achieved by immersing the tonometer in a
dewar filled with liquid propane (b.p.: -42°C) liquid freon-
12 (b.p.: -30°C) or water/crushed ice (0°C). The dioxygen
adducts were very stable at -42°C but underwent autoxidation
rapidly at room temperature (half lifes at 0°C in most cases
were about 4 min.). The O affinities were determined by

2

direct titration of GOP with either pure 02 or air, using a

standard spectrophotometric procedure used by Halpern and

coworkers18 (Fig. 2-4). The O2 binding constant of NP at
0°C was impossible to determine because even at higher than
atmospheric O2 pressures the complex is not fully
oxygenated. This observation, though, is in agreement with

25,26

previous studies of simple Co(II) porphyrins. The

solubility of O in DMF at low temperatures is not known and

2
all the equilibrium constants are calculated at the standard
state of 1 Torr. The titration curves typically had
correlation coefficients of .990 to .999 and varied between
experiments by less than 18% (given the instability of the
cobalt dioxygen adducts at 0°C the error margin is somewhat
larger than usual). The equilibrium constants given on
Table 2-1 are the average value of 2-4 runs(Table 2-2).
Since the UV-vis spectrum of the oxygenated complexes is
very similar with the one of the oxydized Co(III) complex,
in order to prove that no oxidation had taken place it was
necessary to pump the solution again after the titration to

see the spectrum returning to its original form.

The values of the thermodynamic parameters (AH, AS) were

4O

 

1H

'01
I.6 - l
H

 

 

 

 

 

 

 

 

U
S
1<
O
8
(D
9
1 1 110
400 500 600
A.nhn
Figure 2-4 Spectrophotometric titration of A(CH20H)P

in DMF with O at -30°C; P02 (Torr) -

2
0.0, 0.79. 2.09. 6.28, 13.61. 24.1, 45.0,

and >760 in 1-8 respectively. Inset: plot

of A-Ao/Au-A Vs. P02 at 419 nm.

l

 

41

obtained by the Van't Hoff plots of l/T vs In R (Fig 2-5).
In the case of NP, although the Van't Hoff plot consists of
only two points, the AH and AS values agree very nicely with
those of previously studied simple Co(II) porphyrins.25’26

Optical spectra were recorded on a Cary 219

spectrometer.

B. Decomposition of the Co(II) Naphthoic Acid Porphyrin

[N(COZH)P].

CoN(002H)P (10 mg., 0.015 mmol) was dissolved in methylene
chloride (5mL) under Ar, and this solution was then added to
CH2C12 (5 mL) containing one drop of benzoyl chloride. The
mixture was left for 30 min., open to air, during which time
a very characteristic color change from pinkish-red to dark

green, took place. The green solution was washed with 25%

NaOH (3 x 10 mL), conc. HCl (2 x 10 mL) and saturated aq.

NaHCO3. The organic layer was then separated, dried over
anhydrous Na2003 and evaporated to dryness under reduced
pressure. TLC revealed many colored bands; the major band

was separated on a preparative silica gel plate and proved
to be Etiobiliverdin IV by means of NMR, MS, and UV-vis
spectroscopies (15%). The isolated product was identical in
all respects to an authentic sample; MS m/e 498 (13, M+);
NMR 6ppm 1.17 (6H, t, Et), 1.22 (6H, t, Et), 1.83 (6H, S,
Me), 2.08 (6H, S, Me), 2.53 (4H, q, St), 2.60 (4H, q, Et),

5.94 (2H, 8, meso), 6.66 (2H, S, meso), 8.3 (hr, NH).

 

 

m

0000:0\000 00

5 02000002 0 e “020 5 0300002 "0 .0

mzo

5 00:00:00: 0 0 00:0 5 0:05.000: 0 .0

.020 S 0900:0000 "a £20 5 02 0 .0

uncucazqcoa uamnoo

 

42

 

 

 

00 0:00:00 ccmaxo 000 000 00000 000: 0.:0> mum 003000
00.0.»)
.HV _ awn _ fin no . x: . n
. . ma” 0.
mu. 0 ..¢ _ _ _ p
TWI
r
inuw
4
F
< 1.1
00.xpx.
.0. 1 m. _ 1..
TVI
IN...“ 0
T X1
1o
m '-
.r~

 

 

43

Table 2-2. Experimental Data For the Thermodynamic Properties

For 02 Binding to Cobalt Porphyrins

 

 

Compound Solvent T(°C) Py(Torr)
2
CONP DMF -u2 21.5
20.9
-30 68.7
71.1
CON(C02H) DMF ~42 0.0280
0.0349
0.0225
0.0271
‘30 0.770
0.691
0 ”3.0
47.0
57.3
COA(002H)P DMF “U2 0.0179
0.0229
CON(CONH2)P DMF “”2 0.26
0.34
H.13
O 92.8
89.3
CON(CONHNH2)P DMF -U2 0.0758
' 0.0851
CON(CH20H)P DMF ~42 1.75
2.07
‘30 3.93
H.28

44

COA(CH20H)P DMF -H2 2.08
2.30
"30 “.83
5.00
O 8903
92.8
CONP Ph3Im/CH2012 ~N2 0.893
1.1Nl
CON(C02H)P Ph3Im/CH2C12 -NZ 0.00128
0.0013”
‘30 0.030
0.0U2

.81

 

45

C. Fe Insertion To Naphthoic Acid Prophyrin.
Porphyrin (20 mg) was dissolved in 1:1 THF/benzene (20

mL), containing collidine (2 drops) and FeBr (#0 mg). The

2
solution was heated under argon for ca. 30 min. and the
solvent was removed in vacuo. The residue was redissolved
in CH2C12, extracted twice with 10% HCl washed with H20 and
eluted on silica gel column. To obtain the ferric chloride
form, the solution was washed with saturated NaCl in 0.1 N

HCl. UV-vis Amax 6&5, 541, 505, 388.

CHAPTER 3

SYNTHESIS OF NOVEL PORPHYRINS

A. SYNTHETIC MODELS OF MYOGLOBIN

Introduction

 

The construction of synthetic metalloporphyrin
complexes which mimic heme containing proteins has been one
of the most powerful methods in studying reaction mechanisms
and structure-function relationships, of hemoproteins and
especially myoglobin. Most model systems trying to mimic
the distal steric hindrance that myoglobin exhibits are
based on two families of porphyrins; the B-substituted
porphyrins (e.g. protoporphyrin) and the meso-substituted
tetraphenyl (TPP) and diphenyl (DPE) derivatives. These two
types of porphyrins have been manipulated extensively and in
the last 10-15 years a large number of interesting model

systems with colorful names, have been created.l2’19

The B-
substituted compounds resemble more closely the naturally
occurring hemes. However, the excessive floppiness of the
side chains used in functionalization is often undesirable.
The tetraphenyl systems, particularly those functionalized
with o-anilido groups (e.g. "picket fence" heme) are
structurally more rigid. Nevertheless, they suffer from the
fact that synthetically it is very difficult to derivatize

one particular phenyl group (out of four in TPP) on the

porphyrin ring in order to attach special appendaces. The

46

47

diphenyl systems, although a little easier to derivatize,
offer little control over systematic change of the steric
hindrance at the distal side of the porphyrin. For that
reason, meso-monophenyl and at the same time B-substituted
systems were designed and synthesized in an attempt to
prepare better models, which on one hand would be easier to
selectively derivatize at the proper position and on the
other hand would give us a greater flexibility in the design
of the kind and magnitude of the steric hindrance.

The strapped porphyrins 2, 3 and u (Scheme I) were at
first synthesized from the easily obtained B-substituted
porphyrin (l),20 but proved to be unsuitable for kinetic and
equilibria studies of CO and O2 binding, since even the
bulky external bases used to form the penta-coordinate heme
necessary for these studies formed hexa-coordinate complexes
instead. So the more lengthy synthesis of the elaborate
porphyrins 5 and 6 (Scheme III) was undertaken. The new
system should have a rather rigid imidazole linkage from the
opposite side of the strap in order to form a stable five-
coordinate heme and to thus allow a detailed study of 02 and
CO kinetic and equilibria parameters. At the same time two
more unprotected porphyrins with no strap were prepared for
reason of comparison to the stericly hindered ones (Scheme
II). These synthetic hemes were indeed applied in modeling

studies of myoglobin and the results are discussed elsewhere

in this work.

48

Synthesis

 

The parent compound 1 for the simple strapped porphyrins
was synthesized according to the literature.20 The porphyrin
was converted to its diacid-chloride and then, without
isolation, coupled with equivalent amount of the proper
diamine (Scheme I). The two reactants were dissolved
separately in CH2C12 and mixed slowly under high-dilution
conditions in order to reduce di- and oligomerization. The
purification of the final product was carried out on silica
gel plates.

For the synthesis of the meso-(o-aminophenyl) porphyrin
9 the 5,5'-unsubstituted dipyrromethane 7 had to be prepared
first (Scheme 11). This was accomplished by the acid
catalysed reaction of a-free pyrrole 5 with o~acetamido~
benzaldehyde. The 5,5'-unsubstituted dipyrromethane was
obtained from an one pot hydrolysis and decarboxylation of 7
So by condensing 7 and 5,5'-diformyldipyrromethane 15 in
CH2C12 with a catalytic amount of p-toluenesulfonic acid in
the presence of zinc acetate, Zn-porphyrin (9) was produced
which was demetalated after purification (it is easier to
purify the Zn-porphyrin as it travels on silica gel faster
than its free base). The two different imidazole tails were
finally attached to the porphyrin according to a high yield
literature procedure.15

A different dipyrromethane had to be designed for the

synthesis of porphyrins with imidazole tail, as well as,

strap. Pyrrole 12 equipped with an ethyl propionate group

fin—_4.._ _

49

 

_ wimxow

szxqzovzf a

 

A

NTooov 2

IO

:0

50'

H+
/ \ + -—>
H N 0005* NHCOCH,

 

. H HO
(5)
(SW -COOEt x-cocu3
<7>R - H x-H
+
OHC / \ / \ CHO

12
12

(IS)

H+
0 fi «— 0
HN H,N
“ (8)
(9)

Br
notoc©/

croc\/\N/\\N + A
\:-_/ 2) N NNa

SCHEME 2

51

served this purpose. It was condensed with o-acemido
benzaldehyde in a similar fashion with the previous case
(Scheme III). The hydrolysis and decarboxylation of the
diester dipyrromethane 13 was once again done in one step.
The condensation of the two dipyrromethanes was in this case
done in methanol because 14 was insoluble in CH2C12. To the
mono-anilido porphyrin 16, the tail was attached first so
that its bulkyness would force the strap to "close" from the
opposite face of the porphyrin. Finally the ester groups
were acid hydrolyzed (the amide bond was unaffected), the so
produced di~acid porphyrin was converted to the di-acid
chloride in situ, and subsequently coupled to the diamine,

followed by the attachment of imidazole without isolation of

the intermediate.

Experimental

 

'H NMR spectra were recorded on a Bruker WM-250 MHz

instrument in CDCl . Absorption spectra were measured in

3

CH2C12 using a Cary 219 spectrometer. Mass spectra were

measured with a Finnigan 4021 GC-MS (direct insertion probe,
70 eV, ZOO-300°C), or a Varian MAT CH5 equipped with ionteck

FAB gun, operated at 8 KV. Methylene chloride was distilled

from Cal-I2 and THF from LiAlHu before use.

(3,7-Diethyl-2,8,13,l7-tetramethylporphyrin-12,18)-butane-

l,4-[4(1-aza-2~oxo),4(1-aza-2-oxo)-Cyclophane] (2).

20

Diacid-porphyrin l (190 mg, 0.34 mmol) was suspended

 

 

52

 

 

 

COOEt
H4-
\ + —+
u COOEt NHCOCH,
HO
(I2) (13) R‘.COOEt 82-05! x-COCH3
(14) n,- H Ra— 0” x'”
+ / \ / \
OHC N N CHO
H H
(l5)
NH 01:13 I PCH’
0c (I6)
(I?)
DHCOOH/HCL
2)(COCL),
3) H2N(CH,),, NH 2
V 4)Nalm

 

 

(I8) X'4
(l9) X-5

SCHEME 3

53

in 30 mL dry methylene chloride, excess oxalyl chloride (1
mL) was added and the mixture was refluxed gently on an oil
bath for 2 h. protected from the moisture. The homogenized
solution was then evaporated to dryness under vacuum, and
the diacyl-chloride porphyrin was redissolved in dry
methylene chloride (50 mL) and used without isolation for
the coupling reaction. This solution was placed under argon
in one syringe of a two—syringe drive. In the other one was
placed under argon a solution of 1,4-diamino-butane (35.5
mg, 0.40 mmol) and 0.5 mL of triethylamine, in methelene
chloride (50 mL). These two solutions were injected at a
rate of 1 mL/min per syringe at R. T. into 400 mL of dry
methylene chloride with magnetic stirring. The stirring
continued overnight in the dark and then the solvent was
evaporated under reduced pressure. The residue was taken in
CH Cl (100 mL) washed with 103 HCl (2 x 50 mL). H 0 (50 mL)

2 2 2
and saturated NaHCO (2 x 50 mL). Finally the organic layer

3
was chromatographed on silica gel plate (3% CH3OH~CH2012).
The crude product was recrystallized from CH3OH/CHZCL2; (4O

mgr, 19.3%); MS, m/e 618 (10, M+); NMR dppm - 3.51 (2H, s,

pyr. NH), -1.0, -0.7 (each 2H, br, -NHCH2CH2-), 1.6 (2H, br,

NHCHZ“), 1.88 (6H, t, Et), 2.3 (2H, m, ~CH2CO-), 2.42 (2H,

br, 'CONH-), 2.6 (2H, br,-NHCH ~), 2.9 (2H, d,-CH CH C0“),

2 2 2
3.57 (6H, S, Me), 3.62 (6H, S, Me), 4.10 (4H, q, Et), 4.11

(4H, q, Et), 4.3 (2H, m, -CH2CH2CO~), 4.6 (2H, d, ~CH2CH2C0-).

9.93 (2H, S, meso), 10.02 (1H, S, meso), 10.09 (1H, 8,

meso); UV-vis Amax (EM) 620 nm (5600), 565 (7200), 533

54

(11100), 497 (13200), 399 (165400).

(3,7-Diethy1-2,8,13,l7-tetramethy1porphyrin)~pentane-l.5-

 

[5(1-aza-2-oxo). 5(1-aza-2-oxo)-cyclophane]. (3)

 

This strapped porphyrin was prepared in a manner
analogous to 2 using 1.5-diamino-pentane; (yield 18.9%). MS

m/e 632(10,M+); NMR dppm ~ 3.7 (2H, br, pyr NH), ~2.8, ~1.6

(each: 2H, br, -NHCHZCH2-), ~1.l, -O.6 (each: 1H, br, -
NHCHZCHZCH2~), 1.2 (2H, br, ~NHCH2~), 1.90 (6H, t, Et), 2.5
(2H, br, ~NHCH -), 3.1 (4H, m,-CH CO"), 3.5 (2H, br, -CONH-

2 2
), 3.61 (6H, S, Me), 3.65 (6H, S, Me), 4.10 (4H, q, Et), 4.4
(2H, m,-CH20H200-), 4.6 (2H, m, ~CH2CH2C0-), 10.01 (2H, S,
meso), 10.06 (1H, S, meso). 10.12 (1H, S, meso); UV-vis Amax
(EM) 620 nm (6700). 563 (8600), 531 (12800), 496 (15900),

398 (152600).

(3.7-Diethyl-2,8,13,l7-tetramethylporphyrin)-hexane-1,6-

 

[6(1-aza-2-oxo), 6(1-aza-2-oxo)-cyclophane] (4)

 

This strapped porphyrin was prepared in a manner
analogous to 2 using 1,6-diamino-hexane; (yield 16%); MS m/e
646 (16, M+); NMR Gppm —3.7 (2H, br, pyr NH), -o.9 (2H, br,

~NHCH CH CH2“), “0.6 (4H, br ~NHCH CH CH ~), 0.2 (2H, br,

2 2 2 2 2
-NHCH2CH2-), 1.8 (2H, br,-NHCH2-), 1.89 (6H, t, Et). 3.0-3.3
(6H, m, -CH2C0NHCH2), 3.57 (6H, S, Me), 3.64 (6H, S, Me).

3.8 (2H, br, ~CONH"), 4.12 (2H, 0, Et), 4.13 (2H, q, Et),

4.4 (2H, m, ~CH20H2CO-), 4.6 (2H, d, ~CH2CH2CO-), 10.02 (3H,

S, meso), 10.12 (1H, S, meso); UV-vis Amax (EM) 620 nm

_.

55

(5900). 565 (7600), 530 (11300), 496 (14400). 398 (157100).

Zinc 5-[6-amino~l~pheny1)-3.7.12,18-tetramethyl-2,8,l3,17-

tetraethylporphyrin (8).

 

Ethyl 3-ethyl-4-methyl-2-pyrrolecarboxy1ate21 (5.0 g,

27.6 mmol) and o-acetamido benzaldehyde (2.25 g, 13.8 mmol)
were dissolved in ethanol (100%, 100 mL); catalytic amount
of concentrated H280,4 was added and the solution was
refluxed for 5 h. on a steam bath. The pressumably formed
dipyrromethane diethyl ester 6 was hydrolyzed and
decarboxylated in the same pot, as follows: To the above
reaction mixture a 30% aq. KOH solution was added (100 mL)
and refluxing continued for 3 h. on an electric heating
mantle. The condenser was then removed and the volume of
the solution was reduced to 1/2 by evaporation. Vigorous
refluxing continued overnight during which a dark brown
viscous oil separated from the solution. The oil solidified
upon cooling to R. T. The bulk of the solution was dicanted
carefully and what remained in the flask was partioned
between saturated NaCl and CH2012. The organic layer was
evaporated to dryness and the crude product was used in the
next reaction without purification.

Diformyl-dipyrromethane 1522 (180 mg, 0.63 mmol) and the
above crude 5,5'-unsubstituted dipyrromethane 7 (230 mg)

were dissolved in CH2C1 (100 mL) and a solution of p-

2
toluolo-sulfonic acid (0.5 g) in CH30H (5 mL) was added.

After stirring for 6 h. in the dark at R. T., 5 mL of

 

56

saturated methanolic zinc acetate was added to the reaction

mixture and stirring continued overnight. The reaction
mixture was then washed with water (2 x 30 mL), evaporated
to dryness and chromatographed on silica gel. The product

was finally recrystallized from CHZClz-CH3OH; (40 mg, 10%
based on 15); MS m/e 631/633/635 (31/19/17, M+); NMR 8ppm
1.75 (6H, t, Et), 1.77 (6H, t, Et), 2.56 (6H, S, Me), 3.25
(2H, br, NH ), 3.48 (6H, S, Me), 3.84 (4H, q, Et), 3.95~4.06
(4H, m, Et), 6.79 (1H, d, Ar). 7.15 (1H, t, Ar), 7.52 (1H,
t, Ar), 7.61 (1H, 0, Ar). 9.66 (1H, S, meso). 9.97 (2H, 8,
meso), UV-vis Amax (EM) 569 nm (19300). 532 (19200), 405

(364500).

5-(6-Amino-l-phenyl)-3,7,12,18-tetramethyl-2,8,13,17-
tetraethylprophyrin (9).

Zinc porphyrin 8 was dissolved in CH2012 and washed with
15% HCl twice, H20 and saturated NaHC03. The organic layer
was dried over anhydrous NaZSOLl and evaporated to dryness
under reduced pressure. The residue was recrystalized from
CH2C12/CH3OH to afford a practically quantitative yield of
9; MS m/e 569 (17, M+), 285 (20, M2+); NMR 6ppm —3.27 (2H,
br, pyr NH), 1.77 (6H, t, Et), 1.88 (6H, t, Et), 2.71 (6H,
3, Me), 3.63 (6H, 3, Me), 4.02 (4H, q, Et), 4.10 (4H, q,
Et), 7.06-7.68 (4H, m, Ar), 9.98 (1H, S, meso), 10.17 (2H,

S, meso).

5-{0-[3-(N-Imidazolyl)-propylamido]phenyl}~2,8,13,l7-

 

57

tetraethyl-3,7,12,18-tetramethy1prophyrin (10).

 

l5

Excess 3-(N-Imidazolyl)propionic acylchloride was
dissolved in CHBCN (20 mL) and added dropwise to a refluxing
solution of porphyrin 9 (40 mg, 0.07 mmol) in CH Cl (50

2 2
mL), containing 2 drops of triethylamine. After 3 h. the

reaction mixture was poured into ice, the organic layer

separated and washed successively with 5% HCl (30 mL), H20

(30 mL), saturated NaHCO (30 mL) and H 0 (30 mL). After

3

drying over anhydrous Na2SO4

2

the organic layer was
evaporated to dryness and the crude product purified on a
thick layer silica gel plate with 3% MeOH-CH2C12. The major
band was porphyrin (10); MS m/e 691 (M+); NMR 6ppm ~3.25
(2H, br, pyr NH), 1.76 (6H, t, Et). 1.90 (6H, t, Et), 2.48
(6H, S, Me). 3.67 (6H, S, Me), 3.79 (2H, t, CH2), 4.07 (4H,
q, Et), 4.09 (4H, q, Et), 6.27, 6.67, 6.84, 7.03 (each: 1H,
S, 3 Im~H and -CONH-). 7.54 (1H, t, Ar). 7.80-7.92 (2H, m,
Ar), 8.72 (1H, 0, Ar), 10.01 (1H, S, meso), 10.20 (2H, S,

meso); UV-vis Amax (EM) 622 nm (2900). 568 (12300), 536

(9900). 503 (11500). 403 (139100).

5-{o~[m-[a-N-Imidazolyl)-toluamido]phenyl}-2,8,l3,l7-

 

tetraethyl-3,7,12,l8-tetramethylporphyrin (ll).

 

A solution of the o~amino-phenyl pr0phyrin 9 (40 mg,

0.070 mmol) in CH2C12 (20 mL) was added to an excess of a-
15

bromo-m-toluic acyl chloride (0.4 mmol) and heated to
reflux for 2 h. An even greater excess of sodium imidazolate

(135 mg, 1.5 mmol) in CHBCN (20 mL) was then added all at

58

once. The mixture refluxed for 3 h. after which it was

diluted with water, the organic layer separated and
successively extracted with 5% HCl (100 mL), H20 (100 mL),
saturated NaHCO3 (100 mL) and H20 (100 mL). After drying
over anhydrous Na2SO4 the organic layer was evaporated to

dryness and the crude product purified on a thick layer
silica gel plate with 3% CHBOH-CHZClZ. The major band was
porphyrin 11; MS m/e 753 (M+); NMR 6ppm -3.15 (2H, br, pyr
NH), 1.72 (6H, t, Et), 1.87 (6H, t, Et), 2.56 (6H, 8, Me),
2.98 (2H, S, ArCHZ), 3.61 (6H, 3, Me), 4.03 (4H, q, Et),
4.09 (4H, q, Et). 5.24 (1H, s, Im-H), 5.45 (1H, s, Im-H).
6.30 (1H, S, Im-H), 6.2-6.6 (4H, m, ArNH). 7.5-8.2 (4H, m,

Ar), 8.95 (1H, 0, Ar). 9.99 (1H, S, meso), 10.16 (2H, 8,

Methyl 5-(6-amino~1-phenyl)-l3,17-diethyl-3,7,12,18~

 

tetramethyl-Z,8,-dipropionateporphyrin (16).

 

Ethyl 4~methyl~3-(2-methoxycarbonyl ethyl)~2-pyrrole
carboxylate 12 (6.0 g, 33.1 mmol) and o-acetamido
benzaldehyde (2.70 g., 16.6 mmol) were dissolved in 100%
ethanol (100 mL), catalytic amount of conc. H230” was added
and the solution was refluxed for 5 h. on a steam bath. The
pressumably formed dipyrromethane diethyl ester 13 was
hydrolyzed and decarboxylated in the same pot without
isolation, as follows: To the above reaction mixture a 30%

KOH solution was added (100 mL) and refluxing continued for

3 h. on an electric heating mantle. The condenser was then

bah—..i

59

removed and the solution's volume was reduced to 1/2 by
evaporation. Vigorous refluxing continued overnight. The
dark red solution was then cooled down in an ice~bath and
carefully neutralized with acetic acid. The precipitate
that formed was filtered, washed with water and air dried.
An attempt to positively identify this crude product was
unsuccessful, and so it was used without purification for
the next reaction.

Diformyl-dipyrromethane 15 (500 mg, 1.75 mmol) and the
above crude product (700 mg) were dissolved in methanol (300
mL) and p-toluolo-sulfonic acid (1.0 g) was added. After
the reaction mixture was stirred for 6 h. in the dark at
R.T., 5 mL of saturated methanolic zinc acetate were added
and stirring continued overnight. The reaction mixture was
then evaporated to dryness under reduced pressure. The
residue was dissolved in methylene chloride (150 mL) and

washed successively with H 0 (50 mL), 15% HCl (50 mL), H 0

2 2
(50 mL and saturated NaHCO3 (50 mL). The organic layer was
then chromatographed on a silica gel column (3% CH30H-

CH2C12). The desired product, 16 was the only porphyrin in
the reaction mixture (41 mg, 3.5% based on 15); NMR éppm ~
3.32 (1H, br, pyr NH), -3.18 (1H, br, pyr NH), 1.87 (6H, t,
Et), 2.69 (6H, S, Me), 3.15 (4H, t,-CHZCO2Me), 3.65 (6H, 8,
Me), 3.68 (6H, 8, Me), 4.07 (4H, q, Et), 4.37 (4H, t,

“CHZCHZCOZMe), 6.35-8.35 (4H, m, Ar), 9.96 (1H, S, meso),

10.16 (2H, 8, meso).

60

Methyl 5-{o-[m-(a-Bromo)-tu1uamido]pheny1]-l3,l7-diethyl-

 

3,7,12,18-tetramethyl-2,8-dipropionateporphyrin (l7)

 

Porphyrin 16 (50 mg, 0.073 mmol) in CH C1 (50 mL) was

2 2
15 (

added to an excess of a-Bromo-m-tuluic acyl chloride .4

mmol), and the mixture was heated to reflux for 2 h. It was

then washed with saturated NaHCO3 (30 mL) and

chromotographed on a thick silica gel plate (3% CH30H~

CH2012), the desired product being the only major band; NMR

Gppm; 1.89 (6H, t, Et), 2.58 (6H, S, Me), 3.06 (2H, S, -

CH2Br), 3.12 (2H, t, ~CH2002M8), 3.65 (12H, S, Me), 4.08

(4H, q, Et), 4.35 (4H, t, ~CH2CH2C02Me), 6.2-9.0 (9H, m, Ar

and ~CONH-), 10.00 (1H, S, meso), 10.21 (2H, S, meso).

(5-{o-[m~(a-N-Imidazolyl)-toluamido]phenyl}-l3,17-diethyl-

 

3,7,12,l8-tetramethy1porphyrin)~butane-l,4[4(1-aza-2—oxo),

 

4(1-aza-2~oxo)-cyclophane) (18)

 

Porphyrin 17 (20 mg, 0.023 mmol) was dissolved in 88%
formic acid (50 mL) and 2 mL of conc. HCl was added. The
mixture was heated on a steam bath for 3 h. and evaporated
to dryness under reduced pressure. The residue was
suspended into dry methylene chloride (30 mL) and 0.5 mL of
oxalyl chloride was added. The mixture was refluxed gently
on an oil bath for 1-1/2 h. during which it was homogenized.
It was then once again evaporated to dryness and the crude
diacyl chloride porphyrin was used without purification for
the coupling reaction with 1,4-diamino-butane using the high

dilution method described earlier (see synthesis of 2).

Il‘

61

After the coupling was completed the crude product was

dissolved in CH2C12 (20 mL) and an excess of sodium

imidazolate (10 mg, 0.1 mmol) in CH3CN (20 mL) was added all

at once. The mixture was heated to refluxing for 3 h. after

which the solution was diluted with H20. The organic layer

was separated and successively extracted with 5% HCl (100

mL), H20 (100 mL), saturated NaHCO3 (10 mL), H20 (100 mL)

and evaporated to dryness. The product was separated on a
thick silica gel plate (5% CH30H~CH2C12); MS m/e 892 (M+);
NMR dppm ~2.5 (2H, br, pyr NH), -1.0, -0.8 (each: 2H, br, -

NHCH CH2), 1.4 (2H, br, -NHCH2-), 1.88 (6H, t, Et), 2.5-2.85

2
(8H, m, *CHZCONHCH2—), 2.75 (6H, S, Me), 3.60 (6H, 8, Me),
4.06 (4H, q, Et), 4.12 (2H, S, ArCH2-), 4.25, 4.6 (each: 2H,
br, -CH2CHZCO-), 5.05 (1H, S, Im-H), 5.9-8.8 (10H, m, Ar and

2 Im-H), 9.94 (1H, 8, meso), 10.06 (2H, S, meso); UV-vis
lmax (EM) 624 nm (2500), 571 (5600), 539 (6900), 506

(10100), 405 (179900).

(5-{o-[m-(a-N~Imidazolyl)-toluamido]phenyl}—13,17-diethyl-

 

3.7,12,18-tetramethylprophyrin)~pentane—l.5-[5(l-aza-2-

 

oxo).5-(l-aza-2-oxo)-cyclophane] (19)

This strapped porphyrin was prepared in a manner
analogous to the previous one 18, using 1,5-diamino-pentane.
The final product in this case, was the only major product
of this sequence of reactions; MS m/e 907 (M+); NMR 6ppm -
2.9 (2H, br, pyr NH), -2.2, -2.0 (each: 2H, br, ~NHCH CH ~).

2 2

“1.0 (2H, br, -NHCH2CH2CH2-), 1.55 (4H, br, *NHCH2-), 1.87

h... -__._____..__

62

(6H, t, Et), 2.59 (6H, S, Me), 2.92 (4H, t, -CH2C0-), 3.59
(6H, S, Me), 3.65 (2H, S, ArCH2—), 4.04 (4H, q, Et), 4.20,
4.61 (each: 2H, m, ‘CHZCH200“). 5.05 (1H, 8, Im-H), 6.0”8.9

(10H, m, Ar and 21m-H). 9.95 (1H, S, meso), 10.13 (2H, S,
meso); UV-vis Amax (EM) 624 nm (2000). 570 (4300), 539
(5100), 505 (8900), 405 (110300).

B. SYNTHESIS OF MESO-NAPHTHYL SUBSTITUTED PORPHYRINS

Introduction

 

Hydrogen bonding has been thought to play a very

important role in the ability of cobalt (II) macrocyclic

complexes to reversibly bind dioxygen.25 So far, although

many Co (11) porphyrin complexes have been prepared over the

last 15 years,25b_f’26 and their 02 binding abilities

studied, the effect of hydrogen bonding was not fully

investigated because of the lack of appropriate models, and
. . 250-e . . .

it was left open to speculations as to how Significant
it can be. Only recently Jameson and Drago27 have studied

the effect of weak H-bonding present in the "picket fence"
porphyrin. In order for one to systematically examine the
effect of hydrogen bonding, a series of porphyrin models
should be available with substituents which, on the one
hand, would have functional groups capable of forming
hydrogen bonds of different strength to be bound 0 and on

2
the other hand, would be in the right position, close to the

63

center of the porphyrin for maximum effect. For those
reasons, meso substituted porphyrins with a naphthyl group
possessing a functional group at its 8th position, seemed to
be ideal. As CPK models suggest, the functional group of
this system would be held over the porphyrin ring and in
very close proximity to its center. The synthesis of such a

series of porphyrins was undertaken and is described here.

Synthesis

 

The recently developed method for the synthesis of mono-

23 had to be modified in order to

aryl substituted porphyrins
prepare the naphthyl porphyrin 23 because the 8-formyl-1-
naphthoic acid214 first used was unreactive. The loss of its
reactivity was attributed to its tautomeric form where the
formyl group is lost. So to overcome this problem
acenaphthaquinone was first condensed with the a-free
pyrrole 5 (Scheme IV), to give the meso substituted
dipyrromethane 21 which is a very stable X'talline solid.
For the cleavage of the C-C bond to form the 8~
dipyrromethane~1-naphthoic acid, the method of basic
hydrolysis by Fuson and Munn,2L4 for the cleavage of
acenaphthaquinone was used. This method turned out to be
very convenient because at the same time the C-C bond
cleaves, the carboxylic esters of the dipyrromethane are
hydrolyzed and decarboxylated, too. So the dipyrromethane

22 produced from this reaction was ready to use for the one

step synthesis of the porphyrin. The coupling reaction of

64

9 ° H + ’°
“6 + ZH/A\°°°E’ 1" O (M
GK»

(5) 1‘ H

 

OHC N CHO

H 4
HOOC

(23)

 

(coco2

 

   
 

(25a) x: NH,
(256) X: NHNHz

SCHEME 4

65

the two dipyrromethanes was carried out smoothly, under mild
conditions and the desired porphyrin produced in good yield.
The polar carboxylic group inevitably renders this porphyrin
slow moving on silica gel and its purification tedious.
Nevertheless, it can be converted to its acyl chloride
without purification and separated after it is derivatized
to a less polar, easier to purify amides or esters.

The esterification of the carboxylic group of the parent
compound 23 was surprisingly difficult when the CH OH/conc.

3

H 80 method was used. At the same time the hydrolysis of

2 4
the methyl carboxylate 24 was also very slow and incomplete.
The reason for this peculiarity is not clearly understood
but it seems possible that the carboxylic group, being
enclosed in the hydrophobic pocket of the aromatic porphyrin
ring and the naphthyl group, is not easily accessible to
other polar groups. So 23, had to be first converted to its
acyl chloride 23a which then could be quenched by CH30H to
give the methyl ester 24 in good yield. From this compound
one could also prepare the alcohol 26 and aldehyde 27, using
standard procedures. Finally quenching of the acyl chloride
with ammonia produced a small amount of the corresponding
amide, while most the porphyrin decomposed.

An even more functionalized system was also synthesized
by condensing dipyrromethane 22 with another meso~aryl
substituted one 280 (Scheme V). The new system's behavior

and handling was identical to the previous one. The cis-

trans isomers of this comound were impossible to separated

66

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IOOOHm ANNV

 

010 a z 3va

z - £88

5000 .. a 88

I m

 

“m0

0:

1 "meme
£0 u a as

A9

$000

21:

 

I

67

even in small scale. However if this porphyrin is
derivitized, such a separation should be possible, as our

experience with similar systems dictate.15

Experimental

 

l,1-Bis(5~ethoxycarbony1~4~ethy1-3-methyl-2-pyrryl)-2“oxo-

 

acenaphthene (21)

 

To a suspension of acenaphthenequinone 20 (10.0 g, 55.0
mmol) in 100% ethanol (700 mL) was added ethyl 3-ethy1-4-
methyl-Z-pyrrole carboxylate21 5 (20.0 g, 110.0 mmol) and
concentrated H280“ (1 mL). The mixture was refluxed
overnight on a steambath and 20 was completely dissolved.
The solution was then reduced to 250-300 mL under reduced
pressure and cooled in an ice~bath. The yellow crystilline
solid which precipitated was collected by filtration and
washed with 30% H20 in ethanol (3 x 30 mL) (15.0 g). The
filtrate was concentrated to one half of its volume and
cooled again to give a second darker crop (2.5 g), (total
yield 17.5 g, 61%); m.p. 108—110°C; MS m/e 526 (100, M+).
480 (53), 453 (40), 437 (61), 346 (88), 240 (78), 217 (94);
NMR 6 ppm 1.06 (6H, t, Et), 1.28 (6H, t, Et), 1.62 (6H, S,
Me), 2.68 (4H, q, Et), 4.24 (4H, q, OEt), naphthyl: 7.49
(1H, d), 7.69 (1H, t), 7.80 (1H, t). 7.95 (1H, d), 8.03 (1H,

d), 8.19 (1H, d), 8.44 (2H, br, NH).

8-[(4,4'~diethyl-3,3'-dimethy1-2,2'-dipyrryl)methle-l-

 

naphthoic acid (22)

 

68

Dipyrromethane 21 (10.0 g, 19.1 mmol) was suspended in
30% KOH (100 mL) and refluxed overnight under Argon. The
resulted dark red, homogeneous solution was poured into acid
and was neutralized by the careful addition of acetic acid.
Dipyrromethane 22 precipitated as pink amorphous solid,
collected by suction filtration and washed with water and
several times (6.5 g, 85%). This solid was used in the next
step without further purification; NMR 6 ppm 1.1 (6H, t,
Et), 1.6 (6H, S, Me), 2.4 (4H, q, Et), 6.4 (1H, S, methane
CH), 7.2 (2H, d, 5,5'-pyrrole) 7.4-8.4 (8H, m, naphthyl and

2NH).

5-(8-carboxy1-1-naphthy1)‘2,8,13,17-tetraethy1-3,7,12,l8-

 

tetramethylporphyrin (23)

 

To a solution of the decarboxylated dipyrrylmethane 22
(2.1 g, 5.2 mmol) and the diformyldipyrromethane 1522 (1.5
g, 5.2 mmol) in dry methylene chloride (900 mL) was added a
solution of p-toluenesulfonic acid monohydrate (5 g) in

methanol (40 mL). The solution was stirred for 6 h. at R.T.

in the dark; after which a saturated solution of zinc
acetate in methanol (40 mL) was added, and stirring in the
dark continued for another 8 h. This solution was then

washed with H20 (2 x 100 mL) and evaporated to dryness,

under reduced pressure. The crude product was
chromatographed from silica gel column twice (starting with
pure CH2C12 and slowly increasing the percentage of methanol
up to 10%). After the purification, a methylene chloride

69

solution of the porphyrin was shaken with 15% HCl (100 mL)
to demetalate it, washed with saturated NaHCO3 dried over

anhydrous Na SO and evaporated to dryness (500 mg, 14.8%);

2 4
NMR 6 ppm -2.3 (1H, br, NH), -1.1 (1H, br, NH), 1.7 (6H, t,
Et), 1.9 (6H, t, Et), 2.1 (6H, S, Me), 3.7 (6H, S, Me), 3.9
(2H, q, Et), 4.0 (2H, q, Et), 4.1 (4H, q, Et), naphthyl: 7.0
(1H, d), 7.5 (1H, t), 7.9 (1H, t), 8.2 (1H, d), 8.2 (1H, d),
8.3 (1H, d), meso: 10.1 (1H, S), 10.3 (2H, S); UV-vis Amax
(EM) 626 nm (2800), 571 (7600), 538 (7600), 504 (11400), 407

(157100).

5-[8-(methoxycarbonyl)-1-naphthy1]-2,8,l3,17-tetraethyl-

 

3.7.12,l8-tetramethylporphyrin (24)

 

Porphyrin 23 (100 mg, 0.15 mmol) was dissolved in dry
methylene chloride (70 mL) and excess oxalyl chloride (1 mL)
was added. The solution was refluxed for 1-1/2 h. and the
solvent together with the excess of oxalyl chloride, was
removed under reduced pressure. The acyl chloride porphyrin
produced was immediately quenched with large excess of
anhydrous methanol (50 mL). After stirring the mixture for
5 min., methanol was removed and the residue dissolved in
CH Cl (100 mL), washed with saturated NaHCO (30 mL) and

2 2 3

purified by recrystallization from CH2C12/CH30H to give

purple crystals in quantitative yield; MS m/e 662 (60, M+),
331 (g, M2+); NMR 6 ppm ~3.14, -3.07 (each: 1H, br, NH),
0.09 (3H, 3. "0Me). 1.70 (6H, t, Et), 1.89 (6H, t, Et), 2.11

(6H, S, Me), 3.64 (6H, S, Me), 3.96 (4H, q, Et), 4.08 (4H,

70

q, Et), naphthyl: 7.30 (1H, d), 7.60 (1H, t), 7.85 (1H, t),
8.03 (1H, d), 8.27 (1H, d) 8.35 (1H, d), meso: 9.97 (1H, S),
10.14 (2H, S); UV-vis Amax (EM) 625 nm (2900). 572 (5900),

537 (6900). 503 (13900), 405 (178000).

5-[8-(Hydroxymethy1)-1-naphthy1]~2,8,13,17-tetraethy1-

 

3,7,12,l8-tetramethylporphyrin (26)

 

The methyl ester porphyrin 24 (100 mg, 0.15 mmol), was
dissolved in dry THE (150 mL) and a 3-fold excess of LiAlHu
was added carefully. The mixture was magnetically stirred
at R.T. for 2 h. (The progress of the reaction can easily be

monitored by TLC, as the product's R value is significantly

f
smaller than that of the starting material). The solvent
was then evaporated almost to dryness and the residue was

carefully partitioned between CH2C1 and 10% HCl. The

2
organic layer was then washed with saturated NaHC03, dried
over anhydrous Na280u and evaporated to dryness.
Recrystallization of the crude product from CHZClZ/CH3OH

gave 26 as purple crystals (67 mg, 70%); MS m/e 634 (16,
M+); NMR 6 ppm —3.17 (1H, br, NH), -2.98 (1H, br, NH), 0.19
(1H, t, 0H), 1.70 (6H, t, EC), 1.87 (6H, t, Et), 2.12 (6H,
S, Me), 3.09 (2H, d, ‘CH20H), 3.64 (6H, S, Me), 3.93 (2H, q,
Et), 3.99 (2H, q, Et), 4.08 (4H, q, Et), naphthyl: 7.60 (1H,
S), 7.62 (1H, d), 7.78 (1H, t), 8.00 (1H, d), 8.16 (1H, t).
8.34 (1H, d), meso: 9.96 (1H, S), 10.15 (2H, S); Uv—Vis Amax
(EM) 625 nm (2100), 572 (6400). 538 (6500). 503 (15000), 405

(190000).

71

5-(8-Formyl-1-naphthyl)‘2,8,13,l7-tetraethy1-3,7,12,18-

 

tetramethylporphyrin (27)

 

Porphyrin 26 (45 mg, 0.08 mmol) was dissolved in dry
pyridine (3 mL) and it was added in one portion to an ice-
cooled solution of CrO3 (27 mg) in pyridine. The mixture
was stirred for 20 min. after which the ice-bath was removed
and stirring continued for 2 more h., at R. T. Then the
solution was partitioned between CH2012 and H20. The
organic layer was first washed with 10% HCl (2 x 20 mL), and
then with saturated NaHC03. Finally it was evaporated to
dryness and the product separated on a thick silica gel
plate (1% CH3OH~CH2C12), to afford 27 in practically
quantitative yield; NMR 6 ppm -3.08 (2H, br, NH), 1.69 (6H,
t, Et), 1.87 (6H, t, Et), 2.10 (6H, S, Me), 3.63 (6H, S,
Me), 3.93 (4H, m, Et), 4.08 (4H, q, Et), 5.82 (1H, S, -CHO).
7.3-8.5 (6H, m, naphthyl), 9.96 (1H, S, meso), 10.15 (2H, S,

meso).

5-(8-Acetamido-1-naphthyl)-2,8,13,17-tetraethyl-3,7,12,18-

 

tetramethylporphyrin (25a)

 

Porphyrin 23 (100 mg, 0.15 mmol) was dissolved in dry
methylene chloride (70 mL), and oxalyl chloride (1 mL) was
added. The solution was refluxed for 1-1/2 h. and the
solvent together with the excess of oxalyl chloride was
removed under reduced pressure. The residue was redissolved

in dry methylene chloride (70 mL) and dry ammonia was

72

briefly passed through the stirred solution. The reaction
mixture was then washed with water (30 mL), dried over
anhydrous Nazsou and evaporated to dryness. The crude
product was chromatographed on a silica gel column (3%
CH30H~CH2C12) and then recrystallized from CH3OH-CH2C12 (10
mg, 10%); MS m/e 647 (12, M+); NMR 6 ppm -3.15 (2H, br, NH),
1.69 (6H, t, Et), 1.87 (6H, t, Et), 2.12 (6H, S, Me), 2.60
(2H, br, -C0NH2), 3.62 (6H, S, Me), 3.90 (4H, m, Et), 4.07
(4H, q, Et), naphthyl: 7.30 (1H, d), 7.56 (1H, t), 7.89 (1H,
t), 8.17 (1H, d), 8.23 (1H, d), 8.35 (1H, d), 9.95 (1H, 3.
meso), 10.12 (2H, S, meso); UV-vis Amax (EM) 627 mm (2800),

573 (7100). 541 (7300). 506 (8800), 409 (106500).

5-(8-Acetohydrizide-l-naphthyl)-2,8,13,17-tetraethyl-

 

3,7.12,18-tetramethylporphyrin (25b)

 

Porphyrin 23 (100 mg, 0.15 mmol) was dissolved in dry
methylene chloride (20 mL) and excess oxalyl chloride (1 mL)
was added. The solution was refluxed for l to 1-1/2 h. and
the solvent together with the excess of oxalyl chloride was
removed under reduced pressure. The residue was redissolved
in dry methylene chloride (50 mL) and hydrazine monohydrate
(0.5 mL) was added. The mixture was stirred for 5 min.

washed with water (30 mL), dried over anhydrous Na SO and

2 4

evaporated to dryness. The crude product was
chromatographed on a silica gel column (3% CH30H-CH2C12).
The desired product eluted first and the unreacted starting

material second. The final product was recrystallized from

 

73

CH30H-CH2012 (45 mg, 44%); MS m/e 662 (M+); NMR Gppm — 1.79,
-l.30 (each: 2H, br, pyr -NH), 1.76, 1.94 (each: 6H, t, Et),
2.01, 3.52 (each: 6H, S, Me). 3.92 (4H, q, Et), 4.09 (4H, q,
Et), 7.5 - 8.5 (6H, m, naphthyl), 10.16 (1H, S, meso), 10.28
(2H, S, meso). UV-vis Amax (EM) 625 (2900), 572 (6900). 541

(7800), 506 (13000), 407 (169200).

6-Ethoxy-l-E(5,5'-dicarboxy1-4,4'-diethy1-3,3'-dimethy1~

 

2,2'-dipyrry1)methy1]benzene (283)

 

Ethyl 3-ethy1-4—methyl~2~pyrrolecarboxylate (5)21 (4.83
g, 26.7 mmol) and o-ethoxybenzaldehyde (2 g, 13.3 mmol) were
4 was added (5

drops) and the solution was refluxed on a steam bath

dissolved in 100% Ethanol (50 mL), conc. H230

overnight. Then, without isolation of the intermediate,
dipyrromethane 28, a 30% solution of potassium hydroxide was
added in the same flask (50 mL) and the mixture was refluxed
on an electric heating mantle for another 5 h. The solution
was then poured into crushed ice, neutralized carefully by
the slow addition of acetic acid and the precipitated pink
amorphous solid was collected by suction filtration washed
with H20 several times and air dried (5 g, 86%). This
product was used in the next step without further
purification. MS m/e 394 (2, M+~C02), 350 (29, M+—2C02), 44.
(100, 002+); NMR 5 ppm 1.13 (6H, t, Et), 1.32 (3H, t, -0Et),
1.88 (6H, S, Me), 2.75 (2H, q, Et), 2.78 (2H, q, Et), 4.00
(2H, q, ~0Et), 5.68 (1H, S, methane CH), 6.89-7.24 (4H, m,

Ar), 8.85 (2H, S, NH).

 

74

6-Ethoxy-l-[(4,4'-diethy1-3,3'-dimethy1—2,2'-dipyrryl)methy1]
benzene (28b)

Dipyrromethane 28a (5 g, 11.4 mmol), was dissolved in
ethanolamine (20 mL) and refluxed for 2 h. The solution was
then poured into crushed ice and the mixture extracted with
CH2C12 (3 x 30 mL). The organic layer was then evaporated
to dryness and the crude product - a brown oil — was
purified by passing it through a short silica gel column
(CH2C12). The purified product was a yellow viscous oil
(2.8 g, 70%); NMR 6 ppm 1.15 (6H, t, Et), 1.23 (3H, t, -
OEt), 1.79 (6H, S, Me), 2.41 (4H, q, Et), 3.90 (2H, q, -

OEt), 5.74 (1H, S, methane CH), 6.33 (2H, d, 5,5'-pyrrole),

6.80-7.20 (4H, m, Ar), 7.55 (2H, br, NH).

6-Ethoxy-l—E(4,4'~diethy1-3,3'-dimethyl-5,5'—diformy1-2,2'-

 

dipyrryl)methyl]benzene (280)

 

Phosphoryl chloride (1.5 mL) was added dropwise over a
period of 15 min. to a stirred, ice—cooled, solution of
dipyrromethane 28b (5 g, 14.3 mmol), in N,N'-
dimethylformamide (10 mL). The solution was then stirred at
R. T. for 1.5 h. and then poured into ice and H20. To the

acidic solution, saturated NaHCO3 was slowly added until it
became basic. Then it was heated on a steam bath for 15
min. and the product precipitated upon standing for 2-3
days, as brown amorphous crystals (75%); MS m/e 406 (100,

M+), 377 (53, M+-CHO); NMR 8 ppm 1.19 (6H, t, Et), 1.25 (3H,

 

75

t, -0Et), 1.85 (6H, 8, Me), 2.69 (4H. 9. Et). 3.98 (2H. 9. -
OEt), 5.76 (1H, 8, methane CH), 6.87-7.30 (4H, m, Ar). 9.15

(2H, br, NH), 9.48 (2H, S, -CHO).

Cis/trans 5-[8-(Methoxycarbony1)~1-naphthyl1-15-[6-ethoxy-

 

l-pheny1]-2,8,12,18-tetraethyl-3,7,13,17-tetramethy1-

 

porphyrin (29)

 

To a solution of diformyl dipyrromethane 28c (2 g, 4.9
mmol) and 5,5'-unsubstituted dipyrromethane 22 (1.97 g, 4.9
mmol) in dry methylene chloride (900 mL) was added a
solution of p-toluene sulfonic acid monohydrate (5 g) in
methanol (40 mL). The solution was magnetically stirred for
6 h. at R. T. in the dark, after which a saturated solution
of zinc acetate in methanol (40 mL) was added and stirring
continued in the dark for another 8 h. at R. T. The
solution was then washed with H20 (2 x 100 mL) and
evaporated to dryness under reduced pressure. The crude
product was passed through a silica gel column (5% CH CH—

3

CH2012) to separate the porphyrin from most but not all the

impurities. This partially purified porphyrin was dissolved
in dry CH2C12 (100 mL), excess oxalyl chloride was added (2
mL) and the mixture refluxed on an oil bath for 2 h. Then
it was evaporated to dryness under reduced pressure and the
acyl chloride was quenched by anhydrous methanol (100 mL).
The solution was once again evaporated to dryness, the
residue redissolved in CH2012 washed with saturated NaHCO3
and chromatographed on a silica gel column (CH2012).

76

Finally the product was recrystallized from CH30H-0H2C12 (50
mg, 13%). The final product was a mixture of the cis and
trans isomers as expected. Attempts to separate the two
isomers even in small scale (preparative silica gel TLC
plate) were unsuccessful. If we assume that for steric
reasons the trans isomer should be the major product then
the cis/trans ratio is l/l.4 as the NMR reveals; MS m/e 391

112+

(22. ); NMR 6 ppm -2.20 (2H, br, NH), 0.25 (S, Co Me

2

013), 0.34 (S, C02Me trans), 0.80 (t, -0Et 018). 1.00 (t, ~
OEt tranS), 1.72 (6H, t, Et), 1.79 (6H, t, Et), 2.15 (6H, S,
Me), 2.57 (6H, 8, Me), 3.96 (2H, q, Et), 4.01 (2H, q, Et),
4.15 (4H, q, Et), 7.26-8.40 (10H, m, Ar), 10.18 (2H, S,

meso).

C. SYNTHESIS OF NOVEL HIGHLY FUNCTIONALIZED PORPHYRINS

Introduction

 

A plethora of synthetic porphyrins have been prepared
over the last 25 years from simple highly symmetric ones to
more complicated unsymmetric ones. Depending on the
complexity of the target molecule syntheses can be
approached from a variety of directions.28 If laborious
separation of mixtures are to be avoided, totally
unsymmetrical porphyrins must usually be synthesized by

29

cyclization of a preformed open-chain tetrapyrrole. In

this procedure (Scheme VII) an unsymmetrically substituted

77

0000+),

 

«71,000 / \
H
82H,
(30) \ 0H
001,000 /N\
H
(3|)
¢CHZOOC /N\
H
(32)
I) H, ,P /0 Br
2) TFA
3) H(OC,H5 3 0H / N\
H
(33)

SCHEME 6

 

78

2 i + \ \ /
2 59000 /N\ H mCOOt-Bufi \ N N /
H OAC H EtOOC H H COOt—Bu

<5) (34,

(.35)

 

 

79

and differentially protected ethyl tert-butyl pyrromethane-
5.5'-dicarboxylate (35) is selectively hydrolysed and
decarboxylated to give pyrromethane 35a which in turn, can

be transformed into a tripyrrin salt by condensation with a

2—formy1pyrrole. Condesnation with a second 2-
formylpyrrole, after hydrolysis and decarboxylation of the
second ester, gives an a,c-biladiene salt. Changing the
sequence of the introduction of the two formylpyrroles, one

can get the a,c-biladiene salt with a different geometry.
Cyclization of the two l',8’~dimethyl-a,c-biladiene salts
gave the two isomeric porphyrins 39 and 42.

These two porphyrins were designed in this way so that
either symmetric or asymmetric straps of different lengths
could be built across the pr0phyrin plane. Such a
flexibility was hoped to give us a better control over the
magnitude of steric hindrance at the binding site of the
heme. By functionalizing one side chain of the pr0phyrin
periphery with the bromide one can later attach an imidazole
tail necessary for myoglobin models. Unfortunately,
although the incorporation of the straps went smoothly
following the same pathway described in part A of this
chapter, the substitution of the bromide as well as the
physical studies of those models did not work quite as
smoothly. The substitution of the bromide by
(CH3)2CHNH(CH2)3Im was carried out in refluxing xylenes with
excess of the substituted imidazole present. The product

was very hard to purify though, because it was nearly

80

impossible to elute on silical gel or alumina columns.
Nevertheless, the NMR of the small quantities that were
purified, although very complex and difficult to assign, did
show the imidazole hydrogen peaks between 5.5 - 7.5 ppm as
expected. That let us believe that the right compounds were
made. But the even more disappointing result was that these
hemes, when an attempt to measure their C0 association was
made, did not show first order kinetics. The reason for
this behavior is believed to be the high flexibility of the
imidazole tail which on one hand cannot discriminate between
the two different heme faces and on the other can also form
an intermolecular complex with a second heme. So the
completion of the synthesis of these models was abandoned
and the emphasis was shifted to the construction of models
with a more rigid imidazole tail which indeed proved to be
successful (see part A).

Finally, the recently developed method for the synthesis
of monoaryl-substituted porphyrins was also used for the

preparation of o-methoxy phenyl porphyrin 44.

Synthesis

 

The stepwise synthesis of porphyrins 39 and 42, requires

the use of four pyrroles (Scheme VII). Three of them 5.21

3430 and 3631 were synthesized according to the literature
while for the fourth one 33, the modification of Benzyl 4-
(2-ethy1 carboxymethyl)~3,5-dimethy1-2-pyrrolecarboxylate

>32

(30 was necessary. In the first step the methyl ester

81

was converted to the corresponding alcohol in quantitative
yield using 82H6' Reaction of the alcohol with phosphorous
tribromide yields the bromopropylpyrrole 32. The catalytic
cleavage of the benzyl ester was finally followed by
decarboxylation in trifluoroacetic acid and formylation with
triethyl orthoformate in one pot, to afford the 2-formy1—4-
(3-bromopropy1)pyrrole (33), in good yield.

For the synthesis of the a,c-biladienes, Smith's
procedure was followed.29 The t-butyl ester of
dipyrromethane 35 (Scheme VII) was firstly cleaved in TFA
and a formyl pyrrole was added, and condensed with it to
afford the corresponding tripyrrolic salt. In a similar
step, the second ester was cleaved (under much more rigorous
conditions), and the fourth pyrrole was added. Finally, the
a,c-biladiene salts were heated briefly in DMF, in the
presence of Cu(II) chloride to produce the porphyrins 39 and
42, each one as the major poryphyrin product.

For the synthesis of the o-methoxy-phenyl porphyrin 44,
dipyrromethane 43b had to be synthesized first. This was
done in ethanol in the presence of an acid catalyst,
followed by basic hydrolysis. The diacid-dipyrromethane 433
was finally decarboxylated in refluxing ethanolamine and was
coupled to diformyldipyrromethane 15 to afford porphyrin 44

in relatively good yield (Scheme VIII).

82

 

H
(43) a - COOEt
(43a) R -COOH
/ \ / \ (436) R - H
OHC N N CHO
H H
(l5)

 

SCHEME 8

83

Experimental

 

Benzyl 4-(3-hydroxypropy1)-3,5-dimethyl-2-pyrrolecarboxylate
(31)

In a three-neck, round bottom, flask, was placed sodium
boron hydride (3.6 g, 95 mmol) and THF (25 mL). Boron
trifluoride etherate (1M, 17.4 mL) diluted in anhydrous
ether (100 mL) was then added dropwise through a dropping
funnel, to the stirred suspension of NaBHu. A slow stream
of Argon transferred the generated diborane into another R.
B. flask, containing a solution of Benzyl 4-(2-methoxy
carbonylethyl)-3,5-dimethyl-2~pyrrolecarboxylate (30)32 in
anhydrous THE (200 mL). The solution of the pyrrole was
stirred throughout the addition of diborane and was
continued for 1 more hour. The reaction was easily
monitored by means of TLC (the product moves slower than the
starting material). The excess of the diborane was
destroyed by cautiously adding methanol. The solvents were
then removed under reduced pressure and the crude product
was recrystallized from CH30H to give white crystals, in
quantitative yield; m.p. 95°~96°C; MS m/e 287 (14, M+), 242

(17, M+ -(CH2)20H), 91 (100, 608 +); NMR 6 ppm 1.70 (2H, m,

2
~CH2CHZCH20H), 1.72 (1H, br, -0H), 2.18 (3H, S, Me), 2.28
(3H, S, Me), 2.44 (2H, t, -CH2-CH20H20H). 3.62 (2H, t, -

CHZOH), 5.28 (2H, 8, "CH20), 7.3-7.5 (5H, m, Ar), 8.90 (1H,

br, NH).

Benzyl 4-(3-bromopropy1)-3.5-dimethyl-2-pyrrolecarboxylate

 

(32)

84

Benzyl 4-(2-methoxy carbonylethy1)-3.5-dimethy1-2-
pyrrolecarboxylate (31). (6 g, 20.9 mmol) was dissolved in
dry methylene chloride (50 mL). Pyridine (2 mL) was added

and the protected from the moisture solution was cooled down

in an ice-salt bath. To the cooled, magnetically stirred
solution, an excess of phosphorous tribromide (11.3 g, 41.7
mmol), was added dropwise over a period of 30 min., and

stirring continued overnight with gradual warming up of the
mixture. It was then washed with 2N HCl (2 x 20 mL), and

saturated NaHCO3 (2 x 20 mL), and the organic layer was

dried over anhydrous NaZSOLI and evaporated to dryness under

reduced pressure to give a green oil. This crude product

was passed through an alumina column (CH C12), and the new ~

2
lighter colored - oil, solidified upon standing for a few
hours. Finally the product was recrystallized from methanol

to give pale-white crystals (1.54 g, 21%); m.p. 96-97°C; MS

m/e 349/351 (48/58, M+), 242 (90, M+ - CH CH Br), 91 (100,

2 2
¢CH2+); NMR 8 ppm 1.98 (2H, quintet, ~CHZCH28r), 2.21 (3H,
8, Me), 2.28 (3H, 3, Me), 2.53 (2H, t, -CH2CH2CHZBr), 3.37
(2H, t, -CH28r), 5.29 (2H, s, ¢CH2~), 7.2 - 7.5 (5H, m, Ar),

8.81 (1H, br, NH).

4-(3-Bromopropy1)-3,5-dimethy1-2—formylpyrrole (33)

 

In a solution of Benzyl 4-(3~bromopropyl)-3,5-dimethy1-
2-pyrrolecarboxylate (32), (5.8 g, 16.6 mmol), in THF (150
mL), was suspended 10% Pd on Carbon (4 g) and the mixture

stirred under H2 until the uptake of 1 equivalent of H2 was

85

complete (about 1 h.). The suspension was then gravity
filtered to separate the catalyst and the solvent of the
clear solution was evaporated under reduced pressure. The
resulting yellow oil was used in the next reaction without
any purification. To the above crude product,
trifluoroacetic acid was added (17 mL), and the solution was
magnetically stirred fro 5 min. at 40°C (oil bath), under
Ar. The evolution of a gas was evident (C02).
Triethylorthoformate was then added (5.1 mL) in one portion
and stirring continued at 40°C for another 5 min. After
that 85 mL of water were added to the reaction mixture and
the separated oil which soon, solidified was collected and
dissolved in ethanol (55 mL). Aqueous ammonia (2N, 35 mL),
was added slowly with stirring, followed by water (50 mL),
10 min. later. Finally, the product was collected by
suction filtration as yellow crystals. (Overall: 7.6 g,

64%); m.p. 123—125°c; MS m/e 243/245 (26/25, M+), 136 (100,

+ a
M -CH20H28r); NMR 6 ppm 2.00 (2H, quintet, CHZCHZCHZBr),
2.28 (6H, S, Me), 2.55 (2H, t, -CH2CH20HZBr), 3.39 (2H, t,
“CH Br). 9.46 (1H, S, -CHO), 10.05 (1H, br, NH).

2

Ethyl 3,4'-dimethyl-4-ethyl-3'~(2-methoxycarbony1ethyl)-5'-

 

t-butoxycarbonyl-dipyrromethane-5~carboxylate (35)

 

A suspension of ethyl 3-ethy1-4-methyl-2-
pyrrolecarboxylate (5)21 (225 mg, 1.24 mmol) and t-Butyl 2-
acetoxymethy1-3-(2-methoxycarbonylethyl)-4-methy1-5-

pyrrolecarboxylate (34)30 (421 mg, 1.24 mmol) in methanol (5

86

mL), was added p-toluolosulfonic acid monohydrate (10 mg)
and heated with stirring under argon at 40°C for 5 h. The
homogenized mixture was then partitioned between H20 (20 mL)
and methylene chloride (30 mL) and the organic layer was
dried over anhydrous NaZSOLl and evaporated to dryness. The
product was‘ used in the next reaction without further
purification; MS m/e 460 (16, M+), 194 (74, CllHl6N02+), 57
(100, t-but+); NMR 8 ppm 1.1 (3H, t, Et), 1.3 (3H, t, -
C02Et), 1.6 (9H, S, t-But), 2.0 (3H, S, Me), 2.2 (3H, S,

Me), 2.5 (2H, t, ~CH2-), 2.6 (2H, t, -CH2-), 2.7 (2H, q,

Et), 3.6 (3H, S, -002Me), 3.8 (2H, S, methane CH2), 4.2 (2H,

q, -C02Et), 8.8 (1H, br, NH), 9.1 (1H, br, NH).
Ethyl l',2,3,5-tetramethy1-l,4-di(2-methoxycarbonylethy1)-6-
ethyltripyrrin~a-6'-carboxylate Hydrobromide (37)
Dipyrromethane 35 (4.12 g, 8.95 mmol), was treated with
trifluoro acetic acid (30 mL) under Argon atmosphere at
ambient temperature, for 5 min. A solution of formyl
pyrrole 3631 (1.88 g, 9.0 mmol) in methanol (200 mL) was
then added all at once and the dark red solution was stirred
an additional 90 min., followed by addition of a 30% HBr~
CH3COOH solution (1.5 mL) and ether (250 mL). Continued
stirring for 15 min. resulted in the formation of orange-
crystals which were collected by means of suction filtration
and washed thoroughly with ether (1.74 g, 31.7%). The

mother liquor was evaporated to approximately 100 mL, and

ether (200 mL) was added to give a second crop of the

87

product (1 g, 17.6%) (overall: 49.3%); m.p.: decomposes at
221~223°c; MS m/e 551 (2, M+ -HBr); NMR 5 ppm 1.07, 1.36
(each: 3H, t, Et), 2.04, 2.26, 2.31, 2.72 (each: 3H, 8, Me),

2.48 (2H, t, -CH CO Me), 2.73 (8H, m, -CH2CH200 Me and Et),

2 2 2

3.67 (6H, s, -C02Me), 4.28 (2H, q, -C02Et), 4.37 (2H, s,
meso CH2), 7.09 (1H, S, methine bridge), 10.22 (1H, br, NH),

13.21 (2H, br, NH).

6-Ethy1-l',8',2,3,5,7-hexamethyl-l,4-di(2-methoxycarbonyl-

 

ethyl)-8-(3-bromopropyl)-a,c-biladiene Dihydrobromide (38)

 

Tripyrrin 37 (3.45 8. 5.46 mmol) was stirred in a
mixture of 48% HBr (30 mL), acetic acid (20 mL) and

trifluoroacetic acid (30 mL) under N at 65-70°C for 6 h.

2.
A solution of formylpyrrole 33 (1.41 g, 5.78 mmol) in
methanol (300 mL) was then added all at once and stirring
continued for l h. at R. T. The reaction mixture was
evaporated under reduced pressure and the residue was
partitioned between CH2Cl2 and saturated NaBr. The organic
layer was evaporated to dryness, the residue redissolved in
the minimum amount of methanol possible (about 60 mL) and
ether was added (500 mL). This mixture was stirred for 10
min. during which the product precipitated as brown
crystals, which were collected by suction filtration, washed
with ether and air dried (3.4 g, 72%); NMR 6 ppm 1.14 (3H,
t, Et), 2.05 (2H, m -CH2CHZCHZBr), 2.29, 2.32, 2.33, 2.71,

2.72, 3.45 (each: 3H, S, Me), 2.50 (2H, t, CH2), 2.6-2.85

(10H, m, CH2), 3.42 (2H, t, -CH28r), 3.70 (6H, s, -C02Me),

88

5.22 (2H, S, meso CH2), 7.12, 7.13 (each: 1H, 8, methine
bridge), 13.20, 13.28 (each: 1H, br, NH), 13.40 (2H, br,

NH).

2-Ethyl-3,8,12,18—tetramethy1-7,13-di(2-methoxycarbonylethyl)-

 

17-(3-bromopropyl)porphyrin (39)

 

a,c-Biladiene dihydrobromide 38 (164 mg, 0.19 mmol), was
dissolved in dry DMF (19 mL) containing copper (II) chloride
(1.07 g, 8.0 mmol). The solution was stirred for 4 min. at
145°C (oil bath) under argon. After cooling, the solution
was poured into water and extracted with methylene chloride.
The organic layer was then dried over anhydrous sodium
sulfate and evaporated to dryness, followed by column
chromatography (silica gel ~CH2C12). The red eluants were

evaporated to dryness and the residue was treated with conc.

H280” (30 mL) in order to dimetalate the copper-porphyrin

which is the initial product of the reaction. The mixture
was then partitioned very carefully between CH2012 and
water. The organic layer was washed with water (2 x 20 mL)

and saturated NaHCO3 (2 x 20 mL), dried over anhydrous

Nazsou and evaporated to dryness. This crude product was

further purified by column chromatography (silica gel -

CH2C12) and recrystallized from CH30H-CH2C12 (30 mg, 23%);
MS m/e 686/688 (2/2, M+), 606 (41, M+ ~HBr); NMR 5 ppm -3.8
(2H, br, NH), 1.87 (3H, t, Et), 2.86 (2H, quintet, ~

CHZCHZBP), 3.27 (2H, t, ~CH2C02Me), 3.28 (2H, t, ~CH2002Me),

3.62, 3.64, 3.67, 3.68 (each: 3H, S, Me), 3.74 (2H, t, -

89

CHZBr), 4.10 (2H, q, Et), 4.25 (2H, t, ~CH20H20HZBr), 4.39

(2H, t, ~'CHZCHZCOZMe), 4.43 (2H, t, ‘CHZCH2C02Me), 10.06

(1H, S, meso), 10.08 (2H, S, meso), 10.14 (1H, S, meso).

Ethyl 1',2,3,5-tetramethyl-4-(2-methoxycarbonylethyl)-6-ethy1-

 

1-(3-bromopropyl)-tripyrrin-a-6'-carboxy1ate Hydrobromide (40)

 

Tripyrrin 40 was synthesized in a manner analogous to
the synthesis of tripyrrin 37 using formyl pyrrole 33
(overall yield: 45.7%); NMR 6 ppm 1.07 (3H. t. Et). 1.36

(3H, t, -C02Et), 2.01 (2H, quintet, ~CH2CH20HZBr), 2.05.

2.27 (each: 3H, S, Me), 2.30 (2H, t, ~CH2CH20H2Br), 2.32

(3H, S, Me), 2.59 (2H, t, ‘CHZCH2C02Me), 2.72 (3H, S, Me),

2.62-2.76 (4H, m, Et and “CHZCH2C02M8). 3.40 (2H, t,-CH2

3.67 (3H, S, -C02Me), 4.27 (2H, q, C02Et), 4.37 (2H, S, meso

CH2). 7.10 (1H, s, meso CH), 10.23 (1H, br, NH), 13.20 (2H,

br, NH).

Br),

6-Ethyl-l',8',2,3,5,7-hexamethy1~4,8-di(2-methoxycarbony1-

 

ethyl)-l-(3~bromopropyl)-a,c-biladiene Dihydrobromide (41)

 

a,c-Biladiene salt 41 was prepared in a manner analogous
to the synthesis of a,c-Biladiene salt 38 using
formylpyrrole 37 instead (overall yield: 76.9%); NMR 6 ppm

1.14 (3H, t, Et), 2.06 (2H, quintet, “CH CH CH Br), 2.01,

2 2 2
2.49, 2.82 (each: 2H, t, CH2), 2.67 (4H, t, CH2), 2.87 (2H,
q, Et), 3.42 (2H, t, -CH28r), 2.26, 3.45 (each: 3H, S, Me),

2.34, 2.73 (each: 6H, 8, Me), 3.48, 3.71 (each: 3H, S,

~C02Me), 5.22 (2H, 8, meso CH2), 7.13. 7.16 (each: 1H, 8,

90

methine bridge), 13.35, 13.44 (each: 1H, br, NH), 13.57 (2H,

br, NH).

2-Ethyl-3,8,12,18-tetramethy1-7,17-di(2-methoxycarbonylethyl)-

 

13-(3-bromopropyl)porphyrin (42)

 

Porphyrin 42 was synthesized in a manner analogous to
the syntehsis of porphyrin 39 using a,c-biladiene salt 41
instead (yield: 24.4%); MS m/e 606 (4, M+ -HBr); NMR 6 ppm

-3.79 (2H, br, NH), 1.89 (3H, t, Et); 2.87 (2H, quintet,

-CH2CH2CHZBr), 3.29 (4H, t, “CHZCOZMe), 3.64, 3.65, 3.67
(3H, 3H, 6H, each: S, Me), 3.72 (6H, 8, CO2Me)' 3.88 (2H, t,
*CHZBr), 4.14 (2H, q, Et), 4.30 (2H, t, *CHZCHZCHZBr), 4.41,
4.44 (each: 2H, t, -CHZCH2C02Me), 10.10 (3H, S, meso), 10.16

(1H, S, meso); UV-vis Amax (EM) 619 nm (5500), 565 (8000).

530 (11100), 496 (16100). 399 (174400).

6~Methoxy~l~[(4,4'~diethyl~3,3'-dimethy1~2,2'-dipyrryl)

 

methlebenzene (43b)

 

a-Free pyrrole 521 (5.32 g, 29.4 mmol) and o-

anisaldehyde (2 g, 14.7 mmol) were dissolved in ethanol
(100%, 50 mL), 0.5 mL conc. HCl was added and the solution
was refluxed on a steam bath for 2 h. Then without
isolation of the intermediate dipyrryl methane diethyl ester
43, a 30% NaOH solution (50 mL), was added and refluxing
continued on an electric heating mantle for another 5 h.
The mixture was poured into crushed ice and neutralized

carefully by the slow addition of glacial acetic acid. The

91

solid, which precipitated was collected by suction
filtration washed with H20 several times and air dried.
This product (without purification) was dissolved in

ethanolamine (20 mL) and refluxed for 2 h. The solution was
then poured into crushed ice, extracted with methylene
chloride (2 x 30 mL) and the organic layer was evaporated
under reduced pressure. The product was a brown oil and was
used without purification in the subsequent reaction (3 g,
60%); MS m/e 336 (42, M+), 228 (100); NMR 6 ppm 1.15 (6H, t,
Et), 1.77 (6H, 3, Me), 2.40 (4H, q, Et), 3.66 (3H, s, -0Me),
5.77 (1H, 8, methane CH), 6.26 (2H, d, 5,5'-pyrrole), 6.8-

7.20 (4H, m, AP). 7.40 (2H, br, NH).

5-(6-methoxy-l-pheny1)-2,8,13,17-tetraethyl~3,7,12,l8-tetra-

 

methylporphyrin (44)

 

5,5'~Diformyldipyrromethane 1522 (286 mg, 1 mmol), and
5,5-free-dipyrromethane 43b (336 mg, 1 mmol) were dissolved

in CH2C12 (300 mL). To this solution, methanolic p-

toluolosulfonic acid (1 g in 20 mL CH30H) was added and

after stirring for 6 h. in the dark, it was treated with
saturated methanolic zinc acetate (20 mL), and was set aside
overnight. It was then washed with 15% HCl (2 x 50 mL),

saturated NaHCO3 (2 x 50 mL), and chromatographed on silica

gel column (CH C1

2 2). The product which was the only

porphyrin in the reaction mixture was recrystallized from

CH2C12-CH30H. (205 mg. 30%); MS m/e 585 (100, M+), 292 (12,

M2+); NMR 6 ppm ~3.2 (2H, br, NH), 1.76 (6H, t, Et), 1.88

92

(6H. t. Et). 2.53 (6H. S. Me). 3.63 (6H. 3. Me). 3.73 (3H.
S, “OCH3), 4.0-4.2 (8H, m, Et), 7.3-7.9 (4H, m, Ar). 9.93
(1H, S, meso), 10.13 (2H, S, meso); UV-vis Amax (EM) 622 nm

(3000). 568 (6800), 533 (7700). 498 (15000), 403 (168400).

93

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