1. Introduction
1
1.1
INTRODUCTION
Nitric oxide (NO) has been the subject of thousands of publications in recent years
since it was identified in 1987 as one of the body’s key chemical messengers.1,2 That so
simple a molecule had such an important physiological role came as a great surprise.
Thus, although NO had previously been of interest to inorganic, organometallic and
environmental chemists, the vast majority of NO-related publications now arise from
the biomedical research community. NO is an oxide of nitrogen amongst a family of
seven, some of which are of commercial importance: dinitrogen oxide (N2O) is an
anaesthetic, otherwise known as “laughing gas”, dinitrogen tetroxide (N2O4) is an
oxidising agent in rocket fuels, and NO itself is an intermediate in the synthesis of nitric
acid from ammonia. One of the prominent associations of nitrogen oxides such as
nitrogen dioxide (NO2) and NO, however, has been their contribution as toxic
components of polluted air.
In order to preface a discussion of
NO’s
physiological
role
and
NO
its
z*
function in biochemical pathways, it is
important to understand its chemistry.
x* y* O
N
2p
NO is an odourless and colourless gas
at
ambient
temperature
and
z
atmospheric pressure (b.p.1atm -151.7
x y
ºC). It is a stable radical, Figure 1, that
does not share the tendency of other
2s
radicals to dimerise. This absence of
s*
s
dimerisation has been explained by the
1s
s
s
fact that there would be no net gain in
bond order if two molecules of NO
Figure 1
were to associate to form dinitrogen
dioxide.3 Indeed, the NO molecule has a bond order of 2.5 and N2O2 has a bond order
of 5.
The molecular orbital diagram for NO also shows that the HOMO is an
antibonding orbital, making NO a comparatively easily oxidised molecule. This latter
property is important for some of NO’s biological activities.
As a radical, NO can react with other radical species, the most important one
biologically being dioxygen (O2). The reaction between NO and O2 gives another oxide
of nitrogen, nitrogen dioxide (NO2), and it is this reaction that produces NO2 in polluted
2
air (Equation 1). Nitrogen dioxide dimerises to N2O4, which in turn hydrolyses in
aqueous media to form nitrite (NO2-) and nitrate (NO3-) (Equation 2).
The NO2
produced by oxidation of NO can also react with another NO molecule to form
dinitrogen trioxide N2O3 (Equation 3) which hydrolyses to form nitrite (NO2-)
(Equation 4).
2 NO (g)
O2 (g)
2 NO2 (g)
N2O4 (g)
deep brown gas
colorless
N2O4 (g)
3H2O (l)
NO 3- (aq)
NO
NO 2
N2O3
N2O3
H2O
NO 2-
NO 2- (aq)
Equation 1
Equation 2
Equation 3
H3O+
Equation 4
These reactions of NO (Equations 1-4) show that NO chemistry in aqueous aerobic
solutions is complex, and this complexity explains partly why NO’s biological activity
was identified relatively late.
Thus, the existence of a chemical messenger
“endothelium derived relaxing factor” (EDRF), which is produced by the vascular
endothelium to elicit vasodilation, had been postulated by Furchgott et al4 for more than
a decade before it was finally identified1,2 in 1987 as NO. Further investigations
showed that NO also serves as a neurotransmitter and, more than that, as a cytotoxic
effector molecule of the immune system.5 The elucidation of NO’s physiological and
pathophysiological roles will undoubtedly contribute substantially to major advances in
medicine in the future.
NO is produced in vivo by oxidation of the amino acid L-arginine (Arg), in a process
which is catalysed by the nitric oxide synthase (NOS) enzyme family. In mammals,
three different isoforms of the enzyme exist; these are expression products of distinct
genes but share similar structure and catalytic mechanism. Two of the isoforms are
constitutively present at low levels as part of the normal make up of the cells that
express them. These latter isoforms are the endothelial (eNOS)6 and neuronal (nNOS)7
nitric oxide synthases. Their names indicate the histological location in which they
were first found to be located. The endothelial isoform is present in the cardiovascular
endothelium while the neuronal enzyme is present in nerve cells. The third member of
3
the enzyme family, inducible nitric oxide synthase (iNOS) is expressed by cells of the
immune system. 8 This latter isoform is called inducible because it is synthesised upon
activation of cells of the immune system by cytokines and bacterial products. Once
formed, iNOS produces comparatively high levels of NO which then acts as a cytotoxic
molecule for the destruction of pathogenic organisms and cancer cells.9
NO is produced by the different NOS isoforms for distinct physiological purposes and
is carefully regulated.
Inappropriate over or under production will result in
pathophysiological effects, and the potential involvement of NO dysfunction in a host of
disorders is currently under investigation; these include: cardiovascular disorders
(stroke, ischemia–reperfusion injury), neurodegenerative conditions (Alzheimer's,
Parkinson’s and Huntington’s diseases), inflammatory disease (arthritis), muscular
pathophysiologies (muscular dystrophy), respiratory illness (asthma), and reproductive–
urogenital disease (impotence, pre-eclampsia).10,11
NO may also play a role in
autoimmune disorders and diabetes as well as susceptibility to infection and (separately)
septic shock.12 However, in many cases it is not known whether abnormal levels of NO
production are the cause or effect of these disorders.
Understanding how NO is produced and how this production is regulated should have
an immense significance in the pharmaceutical and biochemical fields. Indeed, useful
clinical applications exist for both NOS inhibitors and NO donor drugs, with some of
the latter having been in use in the clinic for many years.11 NOS inhibitors and NO
donor compounds are also widely used in biochemical studies.
This thesis describes exploratory work in a programme directed towards the
construction of synthetic models that mimic the function of the NOS enzymes. Such
models should throw light on the mechanism of NOS catalysis and could ultimately lead
to technology for clean, highly-regulated and localised generation of NO in a
physiological setting, thus providing a valuable tool for investigating the biological role
of this remarkable molecule.
1.2
THE BIOLOGICAL ROLE OF NO
1.2.1 NO in the cardiovascular system
Physiological role
4
In the cardiovascular system, NO biosynthesis is mediated by eNOS. Its physiological
role involves regulation of tissue conductance, blood flow and blood pressure by
modulating the state of the smooth muscle layer in blood vessels. In simplified terms,
as the muscle relaxes the vessels dilate and the blood pressure falls. Relaxation of
vascular smooth muscle (VSM) is not just the absence of contraction, but is a positive
process mediated by NO which acts as a potent chemical messenger. VSM relaxation is
triggered by a number of mechanisms including neurotransmitters such as bradykinin
and acetylcholine; however, these do not act on the VSM directly, but on the vascular
endothelium, a single layer of cells lining the lumen of the blood vessel (Figure 2).4
vascular smooth muscle
lumen
endothelium
Ca 2+
blood vessel cross-section
ion channel
acetylcholine
receptor
endothelial
cell
Ca 2+
eNOS
L-Arg
NO
NO
GC
VSM cell
PDE
GTP
cGMP
GMP
[cGMP]
[cGMP] 
 relaxation  contraction
O
N
N
O
O P O
O
O
O P O
OH
HO
O
O P O
O
GTP
O
N
NH
N
NH
N
O
O
O P
O
O
OH
cGMP
O
N
NH
N
NH 2
N
O
O
O P
O
NH
N
NH 2
O
HO
OH
GMP
Figure 2
Binding of such messengers to receptors in the endothelial layer triggers opening of
calcium ion channels in the cellular membrane, leading to an increase in intracellular
5
Ca2+ levels within the endothelial cells. This in turn stimulates the biosynthesis of NO
which diffuses into the underlying smooth muscle layer.
There, NO activates an
enzyme, guanylyl cyclase (GC), which catalyses the transformation of guanosine
triphosphate (GTP) into cyclic-3’,5’-guanosine monophosphate (cGMP). This latter
molecule acts as a second messenger which initiates a cascade of biochemical events
leading to the relaxation of the muscle cells. Levels of cGMP are not only regulated by
the rate of its synthesis, but also by its destruction, which is facilitated by its hydrolysis
to guanosine 5’-monophosphate (GMP). This latter transformation is catalysed by
phosphodiesterase (PDE) enzymes. The contractile state of VSM is thus regulated by
the balanced activity of both GC and PDE enzymes, and maintenance of normal blood
pressure requires constant synthesis of low levels of NO by vascular endothelial cells.
In addition to its role in the regulation of blood pressure, NO also contributes to the
control of blood clotting which involves the aggregation and adhesion of blood platelets
to the endothelium.
13
In particular, NO decreases the affinity of blood platelets for
adhesion to the vascular endothelial surface and to one another, thereby functioning as
an endogenous antithrombotic agent.
Pathophysiological role
The pathophysiological effects of NO dysfunction in the cardiovascular system are
immensely complicated and still poorly understood, but probably include some forms of
hypertension.14 When the low levels of NO required to maintain normal blood pressure
are not produced, the VSM does not relax to the appropriate degree. The resulting
vasoconstriction then increases blood pressure. For decades, nitrovasodilatators such as
glyceryl trinitrate and isosorbide dinitrate (Figure 3) have been used to treat
hypertension, even before they were known to serve as a source of plasma NO as a
result of their decomposition.
This type of NO donor drug is now also used
therapeutically for treatment of pre-eclampsia and angina pectoris.15 More recently,
direct inhalation of NO has been used to treat pulmonary hypertension in neonates and
primary hypertension in pregnancy.16,17
O2NO
ONO 2
ONO 2
ONO 2
O
O
ONO 2
Glyceryl trinitrate
Isosorbide dinitrate
Figure 3
6
1.2.2 NOS in the nervous system
Physiological Role
The bioactivity of NO was first identified in the cardiovascular system, but it was soon
found that NO also acts as a messenger in the nervous system.18 Indeed, NO acts as a
neuromodulator and neurotransmitter in the central nervous system (CNS) and the
peripheral nervous system (PNS).
However, NO is far from being a typical
neurotransmitter. Thus, a classical neurotransmitter is synthesised by enzymes and
stored in synaptic vesicles. The process of exocytosis then allows the neurotransmitter
to be released from the synaptic vesicles into a synapse or neuroeffector junction when
required. Once released it binds to a receptor and triggers a response in the postsynaptic cell. Re-uptake or degradation then terminates the activity of this type of
neurotransmitter.
In contrast, NO is synthesised on demand because its
physical
properties — as a short-lived, small, diffusible and membrane-permeable species —
prevent its storage in the manner of a conventional neurotransmitter.
In the nervous system, NO is produced in neurones by nNOS. As with eNOS, nNOS is
constitutively expressed by cells, meaning that the activation of these enzymes to
produce NO does not require new enzyme synthesis. The way nNOS is activated to
produce NO is similar to that for eNOS, but the neurotransmitter that initiates the
process in the CNS is generally glutamate rather than bradykinin or acetylcholine.
Thus, glutamate binding to NMDA receptors opens calcium ion channels in the
postsynaptic cell. The influx of calcium ions activates nNOS and the NO then produced
diffuses into the surrounding cells, Figure 4.11
presynaptic neurone
NMDA receptor
GC
synapse
Ca 2+
ion channel
glutamate
postsynaptic neurone
Ca 2+
nNOS
L-Arg
NO
feedback regulation
Figure 4
7
The particular biological response to NO depends on the nature of the target cells. In
the PNS, for example, nitric oxide may be released from nerve endings that innervate
smooth muscle tissue and subsequently cause relaxation of that tissue (mediated by
activation of GC in the smooth muscle cells). In the CNS, NO may act as a retrograde
messenger. That is, NO is released by the postsynaptic neurone and diffuses back to the
presynaptic neurone. In the presynaptic neurone, GC is activated and levels of cGMP
increase. The accumulation of cGMP then controls the release of neurotransmitter from
the presynaptic neurone.
In this way NO serves as a neuromodulator through a
feedback process as shown in Figure 4. Levels of NO produced for neurotransmission
and neuromodulation are thought to be in the nanomolar concentration range.
In the CNS it has been suggested that the NO-mediated feedback communication
pathway may be implicated in memory formation.19 In the PNS NO has multiple
functions.
For example, in the gastrointestinal tract, NO causes relaxation of the
smooth muscle associated with peristalsis.20 NO is also the neurotransmitter of nerves
that elicit penile erection. 21 Cerebral blood flow might also be regulated by NO.22
Pathophysiological Role
NO dysfunction in the nervous system is implicated in neurotoxicity. 23 It has been
known for some years that excess glutamate acting via NMDA receptors is involved in
neurodegenerative conditions such as Alzheimer’s disease and Huntington’s chorea24
and in ischemic damage25 (infarction associated with stroke). One hypothesis is that
abnormal production of glutamate and consequent NMDA receptor activation leads to
an increase in the intracellular calcium ion concentration. The resulting activation of
NOS in turn produces abnormally high levels of NO. Interestingly, it has been shown
that NO produced by eNOS during ischemia has a neuroprotective effect attributed to its
vasodilator action which helps alleviate impaired cerebral blood flow.11
NO cytotoxicity is thought to occur, at least in part, through its interaction with
another radical species superoxide (O2–) present in low concentration in normal tissue.
The tissue concentration of this radical may increase during pathological states. NO and
O2– react with each other to produce the highly reactive and toxic species peroxynitrite
(ONO2–), which may lead to cell death. 29 However the function of NO in the nervous
system, both normal and pathological, is highly complex and still remains to be fully
elucidated.
8
1.2.3 Functions of NO from iNOS
Physiological Role
Living creatures need to defend themselves against invading organisms, in particular
viruses and bacteria. The primary function of the immune system is to destroy any
foreign matter that penetrates the body. Cells known as macrophages are responsible
for the destruction of these microorganisms and cancer cells, and can do so through a
variety of mechanisms. The predominant mode of action of macrophages involves a
process known as phagocytosis in which foreign matter is engulfed. Subsequent killing
may occur by the release of cytotoxic substances into the foreign organism. NO
produced from iNOS is one of the cytotoxic effectors used to destroy foreign organisms
and cancer cells, Figure 5.26
cytokines/endotoxins
nucleus
macrophage
iNOS mRNA
iNOS
phagocytosis
foreign organism
NO
L-Arg
Figure 5
It might seem strange that the same molecule can act both as an “inoffensive”
messenger (in the cardiovascular and nervous systems) and as a cytotoxic species. The
key to this apparent paradox lies in the differences between the isoforms that produce
NO and in the levels of NO produced. Thus NO in its “non-toxic” messenger role is
produced at low levels by the constitutive endothelial and neuronal NOS isoforms. In
contrast, NO produced as a cytotoxic effector is formed at much higher levels. The
isoform (iNOS) that mediates NO production in macrophages is not normally present in
the cells at detectable levels, but is synthesised in response to stimulation by proteins
called cytokines that act as a local chemical mediator.11 Once iNOS is induced, it
produces large amounts of NO for sustained periods of time.
Another difference
between the NOS isoforms is seen in their dependence on calcium ions. The activity of
the two constitutive forms of NOS (eNOS and nNOS) is stimulated by raised calcium
levels whereas iNOS activity is independent of calcium ions. In this way iNOS exists in
9
a permanently and fully activated state for high output NO production for as long as the
protein is present in the cell.27
Pathophysiological Role
Uncontrolled production of NO from iNOS is seen in both acute infections and chronic
inflammation. An acute condition that is frequently lethal is septic shock. This is a
pathological condition associated with systemic infection where both roles of NO, as a
cytotoxic molecule and as a messenger, come into conflict. At first, as a response to a
massive infection, iNOS is expressed and the NO produced has a protective effect. It
destroys the microbes responsible for the infection and maintains organ perfusion
through its action on the vasculature. NO also prevents microvascular thrombosis by
inhibiting platelet aggregation.
However, prolonged high-level production of NO
induces systemic vascular smooth muscle relaxation leading to massive hypotension,
organ failure and eventually death. 28
High levels of NO derived from iNOS have also been implicated in chronic
inflammatory diseases such as arthritis29 and chronic inflammatory bowel disease30, and
may also play a role10 in the onset of some forms of diabetes.
As explained previously, NO toxicity may occur via its interaction with superoxide
anion to form peroxynitrite, a powerful oxidant that can give rise to reactive NO2 and
OH radicals. However this interaction is not the only one accounting for NO toxicity.
Thus, NO forms complexes with heme-containing proteins and may also interact with
other protein-bound metal centres, altering their activity.31 Cytotoxicity caused by high
local levels of NO may also be due to NO-mediated DNA damage, probably though
nitrosative deamination.32
The pathophysiological conditions associated with iNOS activity are not linked
exclusively to the proteins expression in macrophages. Indeed, iNOS may also be
induced in various other cell types. Induction of iNOS in endothelial cells, for example,
may result in endothelial damage. Within macrophages themselves, NO may inhibit
cellular respiration in mitochondria and cause apoptosis*.11
*
a genetically determined destruction of cells from within - called also programmed cell death.
10
1.3
NO BIOSYNTHETIC PATHWAY
In order to understand the design of our nitric oxide synthase model, it is necessary to
understand the biological pathway for NO synthesis. NO is generated in the body by
oxidation of L-arginine (Arg) to L-citrulline (Cit).33 This process involves two separate
monooxygenations, Scheme 1, and proceeds via a guanidoxime intermediate, Nhydroxy-L-arginine (NHA).34 Both steps are catalysed by NOS and involve reductive
activation of O2 at a cysteine thiolate-liganded heme centre in the enzyme.35 The
electrons required for O2 activation are supplied by nicotinamide adenosine dinucleotide
phosphate (NADPH) as a co-substrate.
H2N
H2N
NH 2
HN
NADPH
NADP +
OH
N
HN
NO
0.5 H+
0.5 NADPH
H2N
0.5 NADP +
2eH3N+
CO 2–
O2
H2O
Arg
O
HN
1eH3N+
CO 2–
NHA
O2
H2O
H3N+
CO 2–
Cit
Scheme 1
The 3:2 NADPH/Arg stoichiometry shown in Scheme 1 is now generally accepted and
the overall process constitutes a five-electron oxidation of the terminal guanidinium
nitrogen atom of Arg.
Arg to Cit turnover is accompanied by consumption of 2
equivalents of O2 and formation of two molecules of water and one molecule of NO.36
In each oxidation step the O2 molecule is exhaustively reduced with the gain of four
electrons. In the first monooxygenation two of the four electrons required by O2 are
supplied by NADPH, the other two being derived by formal oxidation of the substrate.
Here, NOS resembles the cytochrome P450–dependent monooxygenase enzyme family.
These enzymes are heme proteins which catalyse monooxygenation reactions in which
the substrate undergoes a two-electron oxidation and in which NADPH supplies the
remaining two electrons for O2 reduction. The second monooxygenation catalysed by
NOS, however, is unprecedented as only one of the four electrons required for O2
reduction is derived from the co-substrate NADPH; the other three derive from the
oxidation of NHA to Cit and NO.
11
1.3.1 NOS structure
Although the three NOS isoforms derive from different genes, they share a common
structural organisation and catalytic mechanism. The enzymes are catalytically active in
a homodimeric state, each subunit comprising an oxygenase and a reductase domain
(Figure 6).
S S
Zn
S S
Cit, NO
Arg, O2
H4B
NOS oxy
Fe
CaM
e–
NOS red
FMN
e–
FAD
–
NADPH
e
Figure 6
Substrate oxidation occurs at the oxygenase domain, where Arg binds at a catalytic
centre containing the heme unit and a reduced pterin cofactor, tetrahydrobiopterin
(H4B). During catalytic turnover, reductive activation of O2 occurs at the heme iron
centre. The electrons used in this process are supplied by NADPH and delivered via
flavin co-factors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)
in the reductase domain.36 Binding of a small calcium–binding regulatory protein,
calmodulin (CaM) at the interface of the NOS oxygenase and reductase domains
facilitates interdomain electron transfer.37 As shown in Figure 6, the reductase domain
of one subunit probably serves the oxygenase domain of the other. A key feature in
subunit dimerisation is a zinc ion terahedrally coordinated by two cysteine residues in
each subunit.38 The zinc ion is required to maintain the dimeric structure of the enzyme
but does not play a catalytic role.
Possible roles of the tetrahydrobiopterin factor H4B
All NOS enzymes require H4B for catalytic activity.11 This pteridine (Scheme 2) is
thought to have a dual role as an allosteric effector and as a redox-active co-factor.
Studies have shown that both Arg and H4B induce a change in the conformation of
NOS, creating enhanced affinities of the two binding sites for their respective ligands.39
12
This mutual binding enhancement has also been shown to occur with an oxidised form
of the pterin (H2B, Scheme 2).
H2N
N
N
H
N
N
O
OH
+ 2H+ + 2 e- H2N
N
HN
H
OH
- 2H+ - 2 e-
dihydrobiopterin H2B
O
H
N
N
H H
OH
+ e-
H2N
N
HN
OH
tetrahydrobiopterin H4B
- e-
O
H
N
 N
H H
OH
OH
H4B+
Scheme 2
Nevertheless, NO synthesis requires the presence of the fully reduced pterin (H4B),
suggesting a probable redox-active role for the cofactor. No evidence has been found to
support the redox cycling of the pterin between its reduced tetrahydro and oxidised
dihydro states in NOS. However, latest reports have suggested that redox cycling may
involve a one-electron oxidised pterin radical cation (H4B+, Scheme 2).40,41 The pterin
may therefore be involved in the electron transfer pathway that conducts electrons from
NADPH to the heme centre. Indeed, further recent work has been published supporting
the idea that the pterin contributes an electron during the reductive activation of O2 by
NOS.42 It is suggested that the second NOS monooxygenation (NHA  Cit + NO)
initially involves a two-electron reduction of O2 with one electron supplied by the
pterin. The two-electron reduction of O2 in this step would result in the formation of
nitroxyl anion (NO-) rather than NO. However, at a late stage in the mechanism the
one-electron oxidised pterin recovers an electron from the substrate so as to result in the
release of NO rather than NO-.
Calmodulin protein
Calmodulin is a small regulatory protein that binds up to four calcium ions. Calcium
binding to CaM triggers a conformational change in the protein that allows it to bind to
NOS at the junction between its reductase and oxygenase domains. Work on nNOS has
shown that binding of the Ca2+-calmodulin complex (Ca2+-CaM) facilitates electron
transfer at two points: between the reductase and oxygenase domain, and also within the
reductase domain itself between the flavins.37
For eNOS and nNOS, CaM binding is reversible and, as a consequence, the activity of
these constitutive isoforms is sensitive to intracellular levels of Ca2+. In contrast, CaM
remains tightly bound to the inducible iNOS even under nominally Ca2+–free
13
conditions, thus explaining the insensitivity of this isoform to intracellular levels of
Ca2+. This difference in Ca2+-sensitivity reflects the different physiological roles played
by the isoforms. The constitutive enzymes, eNOS and nNOS, are required to produce
NO at low levels, and their activity is regulated by modulation of the Ca2+ concentration
within the cell. These isoforms are finely responsive to the demand for NO and produce
carefully regulated levels.
In contrast iNOS has to produce comparatively large
quantities of NO for sustained periods from macrophages, an output that needs to be
maintained independent of fluctuations in Ca2+ levels which may regulate other
pathways in macrophages. NO production by iNOS is regulated by controlled synthesis
of the enzyme itself rather than by modulation of the enzyme’s activity. In fact CaM
binds to the nascent iNOS polypeptide chain immediately and irreversibly upon its
synthesis. Indeed, complexation with CaM is essential for correct folding of the iNOS
protein as it is formed, and the resulting enzyme is produced in a fully activated state.
NADPH, FAD and FMN.
NADPH, through the flavins, provides the electrons required for the reductive
activation of O2 at the heme centre in the NOS enzymes. These flavins, FAD and FMN,
can store and transfer electrons by reversible redox changes, Scheme 3.
NADPH is an obligate two-electron reductant:
O
+
+H +2e
NH 2
- H+ - 2 e-
N
R
N
N
NH 2
R=
O P O
O
N
N
O P O
O
N
R
NADP+
O
NH2
O
-
O
HO
OH
O
HO
NADPH
O
O P O
O
The Flavins FAD and FMN are two-electron oxidants, and can also accept
or donate (from their reduced forms) one at a time
O
N
+
+H +e
NH
-
+
N
N
O
R
Oxidised form
FAD
FMN
Flavin Adenine
Dinucleotide
H
N
-
N
N
N
O
N
O
CH2
(OH)3
CH2
O
H
N
+ H+ + e-H
+
-
- e
Flavin Mononucleotide R =
O P O P O
O
HO
NH
N
N
O
R
Flavin Semiquinone Form
FADH
FMNH
-H -e
NH2
R=
O
CH2
(OH)3
CH2
O
O
OH
Scheme 3
14
NH
N
N
O
H
R
Reduced form
FADH2
FMNH2
O P O
O
O
Each fully reduced flavin, like NADPH, has the capacity to give up two electrons.
However, unlike NADPH which is an obligate two-electron donor, the flavins can
undergo redox changes in single electron increments (Scheme 3).
Role of NOS heme
As we have seen earlier, NO is produced by the oxidation of the arginine guanidine
function. In order to develop a functional NOS model, it is necessary to understand
how the enzyme catalyses this oxidation. In the laboratory, we have at our disposal a
large variety of oxidising agents. In contrast, Nature frequently harnesses dioxygen
itself as an oxygen donor. However, because of its electronic structure and consequent
reactivity, the use of O2 as an oxidising agent has required the evolution of specialised
biochemical machinery.
Complete oxidation of organic molecules to carbon dioxide and water is an exothermic
process and therefore thermodynamically highly favourable. However, O2 exists in a
triplet ground state and, therefore, its reaction with singlet organic molecules to give
singlet products is a spin forbidden process with a very low rate (Equation 5).
Oxidation of organic molecules by O2 is thus kinetically unfavourable.
1
RH +
3
O2
Thermodynamically favourable
1
CO2
+
1
+
2
H2O
Equation 5
Kinetically unfavourable
1
RH
+
3
O2
Thermodynamically unfavourable
2
Kinetically favourable
R
HO 2
Equation 6
One way of circumventing the kinetic barrier is through the intermediacy of free
radicals. Reaction of a singlet state organic molecule with triplet state dioxygen to give
two doublet state products (i.e. free radicals) is a spin allowed process (Equation 6).
However, the latter is an endothermic process and only works for highly reactive
substrates that form resonance stabilised radicals e.g. flavins.
Another way that nature uses to overcome the spin conservation barrier is to combine
3
O2 with a paramagnetic transition metal ion. The co-ordinated oxygen species that are
created in this way (Scheme 4) are capable of selectively oxidising organic substrates
such as hydrocarbons. In the case of NOS and many other oxygenase enzymes, Nature
15
exploits a metalloporphyrin as the protein’s prosthetic group that is responsible for this
activation of O2.
O
n
n
M + O2
M
O
Mn+1
e
-
H+
O
Mn+1
Mn+3 OH
O
O
O
OH
superoxo
hydroperoxo
oxo
Scheme 4
The NOS mode of action is thought to resemble in some respects that of the
cytochrome P450-dependent monooxygenases, a comparatively well-studied group of
heme-containing monooxygenases.43,44 Cytochrome P450 enzymes incorporate one
oxygen atom from dioxygen into the substrate, the other oxygen atom being reduced to
water. These enzymes catalyse a variety of site selective and sometimes stereoselective
oxidations, including olefin epoxidation and alkane and arene hydroxylation.
The P450 catalytic cycle (Scheme 5) starts with an initial one-electron reduction of the
resting iron(III) porphyrin cytochrome. Subsequent binding of O2 forms a ferrous iron
oxygen complex, which may also be formulated as a ferric superoxide species (A). A
second one-electron reduction is postulated to give a (hydro)peroxoiron(III) species (B),
which heterolytically cleaves to afford an oxoiron(IV) porphyrin radical cation (C) that
is the active oxidant in the P450-mediated oxygenation reactions.45
In NOS the oxoiron species (C, Scheme 5) is thought to mediate the first
monooxygenation step (Arg  NHA), but it is postulated that the second
monooxygenation (NHA  Cit + NO) is effected by the ferric heme superoxide
complex (A).46 Thus NOS appears to be an unusually complicated enzyme that has to
balance peroxoiron and oxoiron chemistry.
The presence of a strongly electron
donating thiolate ligand on the iron centre and an appropriate proton transport chain in
the protein are essential for formation of the transient oxoiron species (C) from the
ferric heme hydroperoxide complex (B). In vivo the electrons required for the catalytic
cycle are provided by NADPH in its role as a co-substrate, albeit through the
intermediacy of the reductase domain flavins and possibly the pterin as discussed
16
earlier.41 The mechanistic details for the two NOS monooxygenations are not discussed
here, but have been extensively studied and are comprehensively reviewed elsewhere.46
substrate-O
e-, H+
FeIII
S-protein
substrate
XO
PEROXIDE
SHUNT
O
FeIV
FeII
S-protein
C S-protein
X
O2
-H 2O
O
OH
O
O
FeIII
FeIII
S-protein
S-protein
B
A
e-, H+
Scheme 5
1.4
PORPHYRINS AND THEIR SYNTHETIC ASPECTS
In the mid seventies, various groups demonstrated that microsomal cytochrome P450
can catalyse the hydroxylation of hydrocarbon substrates by a variety of single-oxygen
donors, such as hydrogen peroxide, hydroperoxides, periodate and iodosylbenzene.
These species serve as surrogates for the combination of O2 and NADPH. This pathway,
which later became known as the “peroxide shunt” pathway, provided a means for
circumventing the need for a co-reductant in such systems (Scheme 5). In 1979, Groves
and co-workers47 were the first to apply the peroxide shunt pathway to a model
porphyrin system, (meso–tetraphenylporpyrinato)iron(III) chloride. This porphyrin was
shown to effect the epoxidation of olefins and hydroxylation of alkanes with the
lipophilic oxygen donor, iodosylbenzene.
17
Following this initial study, extensive investigations were then carried out with
metalloporphyrin-catalysed oxidation of olefins and alkanes using a variety of single
oxygen donors. These studies disclosed some fundamental problems with these systems
arising from the tendency of the metalloporphyrin catalysts to undergo degradation in
oxidising media. Poor catalyst recovery, and therefore reuse, was a serious drawback
given the expense of these catalysts. One solution to this latter problem was found in
anchoring the metalloporphyrin to a solid surface, thus combining the practicality of a
heterogeneous catalyst with the advantages of a homogeneous catalyst.
The poor
durability of porphyrins as oxygenation catalysts was found to be ameliorated to some
extent by halogenation of the porphyrin periphery and by sterically shielding the
porphyrin core against attack by reactive oxygen species, particularly at the vulnerable
meso carbon centres.
Synthetic porphyrins, then, have been widely used as biomimetic models of
cytochrome P450-dependent monooxygenases.45 As will be seen in the following
section, a synthetic porphyrin also constitutes the key feature in our own models
intended to mimic NOS. It is therefore important to review briefly at this point the
chemistry of porphyrins.
Porphyrins (Scheme 6) are macrocyclic tetrapyrrole ligands that play a central role in
life processes.
In living beings, iron porphyrin complexes are used for dioxygen
complexation and redox reactions in a wide variety of fundamental biological processes:

haemoglobin is the protein responsible for the transport of dioxygen from the lungs
to all parts of the organism;

myoglobin is a protein responsible for the storage of O2 in muscle tissue;

cytochromes b and c mediate electron transfer;

cytochrome c oxidase mediates the production of cellular energy by mitochondrial
reduction of dioxygen to water;

and, as we have seen above, cytochrome P450-dependent monooxygenases mediate
key oxidation reactions in the biosynthesis of steroids and detoxification of foreign
substances.
It is therefore unsurprising that considerable interest has been shown in the study of
metalloporphyrins. This interest is stimulated both by the need to understand important
18
natural processes, and by the potential to develop new useful biomimetic oxygenation
catalysts.
The cyclic tetrapyrrolic porphyrin nucleus consists of a 20-carbon skeleton, with the 4
pyrrole rings being linked by single carbon atom bridges. The macrocycle contains 22
pi-electrons of which 18 are in direct conjugation, thus conforming to Hückel’s
aromaticity rule. Two of the pyrrole rings in the porphyrin structure are in an oxidised
state, that is their nitrogen atoms are of the pyridine type with unshared electron pairs
oriented towards the centre of the macrocycle. Ionisation of the remaining two pyrrole
rings results in a highly delocalised, symmetrical dianion, which possesses four
unshared electron pairs in the central cavity.
Porphyrins are therefore capable of
forming tetradentate complexes with metal ions (Scheme 6). A large number of metals
can be co-ordinated to the porphyrin to form a metalloporphyrin.
meso position
-pyrrole
position
N
NH
HN
N
-2 H+
N
N
N
N
M2+
N
N
M
N
N
porphine
Scheme 6
As porphyrins are aromatic in character, electrophilic aromatic substitution features
prominently in their chemistry. Porphine (the parent unsubstituted porphyrin) has two
distinct reactive sites: namely, the eight equivalent -pyrrole positions and the four
meso-bridging carbons. The -substituted porphyrins (Figure 7) resemble the naturally
occurring macrocycles. The meso-substituted porphyrins are not found naturally but are
widely used45 as biomimetic models, and indeed feature in the design of our NOS model
(vide infra, section 1.5). Thus, it is their synthesis that is of particular relevance to this
project.
In general, the meso-substituted porphyrins are more readily synthesised than the –
substituted series and are amenable to synthetic elaboration. These porphyrins can be
prepared conveniently in the one–pot reaction of an aldehyde with pyrrole, where the
aldehyde is the key to synthetic diversity. The ready availability of a wide range of
aldehyde precursors allows groups to be incorporated in the porphyrin which will serve
19
as exploitable sites for further synthetic elaboration. This enables the synthesis of a
wide range of porphyrins without extensive multistep synthetic routes. The synthesis of
less symmetrical porphyrins, and compounds carrying more complex patterns of meso–
and -substituents, does demand more elaborate stepwise approaches to construction of
the macrocycle, and indeed a large body of literature describing such syntheses has been
published.
This section surveys key methods for synthesizing meso-substituted
porphyrins that are of particular relevance to the current programme.
R
R
R
R
N
R
HN
R
N
HN
R
NH
N
NH
N
R
R
R
R
meso-tetrasubstituted porphyrin
R
-octasubstituted
porphyrin
Figure 7
Rothemund method for porphyrin synthesis
Rothemund was the first chemist to synthesise a meso-substituted porphyrin (in
1935).48
He studied the synthesis of meso-tetramethylporphyrin by reaction of
acetaldehyde and pyrrole in methanol at various temperatures in a sealed vessel. Other
aldehydes bearing various alkyl and aryl chains were subsequently used; Scheme 7
illustrates
the
synthesis
CHO
of
meso–tetraphenylporphyrin,
methanol
NH

H2(TPP).
HN
N
NH
N
7.5-9%
1
2
Scheme 7
3
The Rothemund method consists essentially of reacting the aldehyde and pyrrole at high
concentrations (4 M each) and at high temperature in the absence of an added oxidant.
It was later found that adding zinc acetate to the mixture doubled the yield of porphyrin
from 4-5% of the free-base to 10–11% of the zinc chelate.49 This method has fallen into
20
disuse as it produces very low yields of porphyrins and can only be applied to
porphyrins bearing four identical meso substituents.
Adler method for porphyrin synthesis
In the mid 1960’s, Adler, Longo and co-workers developed a method that afforded
higher yields of porphyrins. They initially carried out the condensation of benzaldehyde
and pyrrole at lower concentrations (0.02 M each), in a variety of acidic solvents at
reflux in glassware open to the atmosphere. Acidic solvents originally included acetic
acid (bp 118 ºC) and benzene containing chloroacetic acid or trifluoroacetic acid.50
Further study of these reaction conditions led to the use of propionic acid (bp 141 ºC),
higher concentrations of pyrrole and benzaldehyde (0.27 M each) refluxed for 30
minutes in an open reaction vessel.51,52 This improved procedure is now known as the
Adler or Adler-Longo method, and affords H2(TPP) in yields of around 20%. Although
primarily studied for porphyrins having four identical substituents, the Adler method
can be extended to the synthesis of porphyrins bearing up to four different mesosubstituents using mixed-aldehyde condensations, Scheme 8.
A CHO
Pyrrole
However, even the
B CHO
A
HN
N
B
A
N
NH
A
HN
N
A
NH
A
A4
N
B
cis-A2B2
HN
N
A
B
A
A
NH
NH
N
B
B
A3B
HN
N
A
A
NH
N
B
trans-A2B2
Scheme 8
21
N
HN
N
B
B
A
A
HN
N
B
B
NH
B
B
AB3
B4
N
reaction between pyrrole and a mixture of just two aldehydes can, in principle, generate
a set of six porphyrins (Scheme 8). Therefore, the apparent simplicity of the synthesis
gives way to extremely tedious separations when more complex porphyrins are
required.
Lindsey method for porphyrin synthesis
The two methods described so far for the synthesis of meso-substituted porphyrins
involve relatively harsh conditions such as a large excess of acid and high temperatures.
However, aryl aldehydes can react with certain nucleophiles under mild conditions;
acetal formation, for example, can be carried out at room temperature with a catalytic
amount of acid. Moreover, pyrrole is an aromatic, electron-rich species that exhibits
extremely high reactivity towards electrophilic aromatic substitution.
These
considerations led Lindsey and co-workers to believe that the condensation of pyrrole
and benzaldehyde, for instance, should occur under mild conditions.53,54 Their approach
to porphyrin synthesis was further influenced by the fact that biosynthesis of porphyrins
proceeds via a porphyrinogen intermediate (Scheme 9) which is then oxidised to the
porphyrin in a second step.55 The method developed by Lindsey consists of a two-step,
one-flask procedure, where condensation of pyrrole and benzaldehyde is carried out first
under anaerobic conditions and affords an intermediate porphyrinogen which is
subsequently oxidised to the porphyrin upon addition of an oxidant. Typically, a dry
dichloromethane solution of pyrrole and an aldehyde (10 mM each) at room temperature
is treated with a catalytic quantity of trifluoroacetic acid or borontrifluoride-etherate for
30-60 minutes under an inert atmosphere. A stoichiometric amount of an oxidant is
then added, causing conversion, again at room temperature, of the porphyrinogen to the
porphyrin (Scheme 9).
Ph
Ph
CHO
NH HN
i. cat TFA
NH
Ph
RT
1
ii. [O]
Ph
NH HN
HN
N
Ph
Ph
NH
N
2
Ph
Ph
3
porphyrinogen intermediate
Scheme 9
22
H2(TPP)
This method can also be applied to mixed-aldehyde condensations, but again the
formation of complex congeneric product mixtures means that lengthy separations are
required. Various synthetic strategies have been developed in order to control the
number and type of meso–substituents introduced during porphyrin construction; these
have been reviewed extensively elsewhere56 and will not be discussed in detail here.
The design of our prototypical NOS model though incorporating symmetry to simplify
its construction, does however require the synthesis of porphyrins bearing two different
meso–substituents (of the trans–A2B2 pattern, Scheme 8).
Synthesis of trans–A2B2 meso–tetrasubstituted porphyrins
Clearly application of mixed–aldehyde Alder–Longo or Lindsey methods for synthesis
of trans-A2B2 meso-tetrasubstituted porphyrins would lead to the formation of a
complex product mixture from which separation of the target porphyrin would be
daunting. However, adaptation of the pyrrole–aldehyde condensation chemistry should,
in principle, allow clean synthesis of such porphyrins through the reaction of a mesosubstituted dipyrromethane with an aldehyde (Scheme 10).
B
B
H+ cat.
O
NH
excess pyrrole
BCHO
NH
N
H+
A
HN
A
H
[O]
dipyrromethane
A
NH
N
B
porphyrin
Scheme 10
The synthesis of a porphyrin bearing only meso-substituents by this approach, the
“MacDonald [2+2] synthesis”,57 requires as the starting material a meso-substituted
dipyrromethane.
The preparation of such compounds is usually carried out by
condensation of pyrrole with an aldehyde in the presence of a catalytic amount of acid
(Scheme 10). Many procedures have been reported for the direct condensation leading
to dipyrromethanes and these are reviewed elsewhere.56
However, the facile
protonation of pyrrole can be the source of a significant side reaction, the formation of
“pyrrole-red”, a material so named because of its distinctive colour but which remains
structurally undefined.58 Moreover, condensations, particularly with pyrroles lacking substituents, can proceed to give higher oligomers.
23
To efficiently generate the
dipyrromethane, the optimal strategy is to react the aldehyde with a large excess of
pyrrole in the presence of a catalytic amount of trifluoroacetic acid or boron trifluoride.
Indeed, the pyrrole itself may be used as the solvent and this is probably the most
effective method, giving alkyl- and aryl-dipyrromethanes in moderate to good yields.
Porphyrin synthesis is then carried out by condensation of the meso-substituted
dipyrromethane and an aldehyde under the conditions used in either the Adler or
Lindsey methods. In principle, the MacDonald approach to porphyrin synthesis should
deliver exclusively a trans-A2B2 meso–tetrasubstituted porphyrin product. However,
acid–promoted fragmentation and fragment recombination of the dipyrromethane and
intermediates en route to the porphyrinogen do give scope for the scrambling of
substituents and formation of congeneric porphyrin product mixtures.56
1.5
NOS MODEL DESIGN
A schematic representation outlining the design and principal features of our NOS
models is shown in Figure 8. The model comprises:

a metalloporphyrin that is intended to catalyse the target guanidine oxidation;

acidic guanidine molecular recognition superstructure to bind the substrate close to
the porphyrin’s catalytic centre;

rigid spacer units designed to create a substrate-binding cleft, by supporting the
molecular recognition superstructure while preventing direct contact of the latter
with the catalytic centre (which would lead to its oxidative degradation);

a surface-supported thiol (thiolate) ligand to the metal centre, where the solid
support might be inert (for peroxide shunt studies) or an electrode (for
electrocatalytic operation of the model).
24
R
molecular recognition
superstructure
X
rigid
spacer
NH 2
HN
A
A
NH 2
X
Fe
S
synthetic porphyrin
S
thioether
OR
Si
O O
–
e
e–
OR
Si
O O
–
e
surface-modified
electrode
–
e
Figure 8
Studies carried out by others in our group have suggested that a combination of
phenanthrene spacer units and simple oxyacetic acid chains (X−A = OCH2CO2H)
provides an appropriate cleft size for binding N-alkylguanidinium guest species. The
first NOS model prototype to be synthesised by our group, then, was porphyrin 4,
Figure 9.59 This symmetrical model possesses two identical phenanthrenyloxyacetic
acid units at the 5- and 15-positions of the porphyrin core. The remaining mesopositions are occupied by simple phenyl groups.
The primary remit of the current project was to develop a strategy for covalent fixation
of the porphyrin to a mercaptoalkyl-modified solid surface.
This goal was to be
achieved by replacing the meso-phenyl substituents in 4 with units that could be
connected to surface-bound thiol sites. Thus, the intention was to develop the dual
coordinative and covalent surface attachment regime illustrated in Figure 8, where the
mercaptoalkyl-modified surface simultaneously provides the sites for metal ion
coordination and covalent fixation of the porphyrin. This strategy is intented to permit
robust attachment of the NOS model and prevent surface leaching of the porphyrin, for
example, in thiol-containing biological fluids.
A second goal for the project was, time permitting, to investigate the synthesis of
alternative spacer-superstructure units for construction of new variants of the NOS
model prototype 4. For instance, the oxyacetic acid chains could be replaced by an
amino acid unit (4a) or oxymethylphosphonic acid (4b), Figure 9.
25
variation in superstructure
e.g replacement of
oxyacetic acid chains by:
CO 2H
Cl
O
3
HO2C
Ph 20
2
Cl
HO 2C
R = Bn,
HN
N
5
R
O
N
H
O
(HO) 2P
4a
4b
N
NH
10
Ph
4
modification to allow covalent
attachment to solid surfaces
Figure 9
When work began by this group on NOS models, no porphyrin-based models for NOS
had been reported in the literature. This situation changed in 1999 when a group led by
Groves were the first to describe the use of a metalloporphyrin as a model for NOS.47
The Groves’ group carried out oxidation of fluorenone oxime as a guanidoxime model
for NHA model using hydroxoiron(III) porphyrins bearing four identical meso
substituents such as mesityl and pentafluorophenyl groups.60
More recently a
Hungarian team published a study of the oxidation of NHA using chloro[5,10,15,20tetrakis(perfluorophenyl)porphyrinato]iron(III) as model for NOS and hydrogen
peroxide as an oxygen donor.61 However, neither of these porphyrin models possessed
an elaborate molecular recognition superstructure for guanidinium guest species such as
Arg.
Moreover, they lacked the strongly electron donating thiolate ligand to the
porphyrin iron centre which is present in the NOS enzymes and intended to feature in
the models synthesised by our own group. Therefore, we anticipate that the more
sophisticated thiolate-liganded porphyrins, generated through our programme, will
allow a more effective modelling of the enzyme.
1.6
SURFACE ATTACHMENT OF ORGANOMOLECULES
1.6.1 Introduction
A significant difference between porphyrin-containing enzymes and the models
designed to mimic them is the absence of the protein matrix in the model system. The
protein binds and isolates the catalytic site, preventing inactivation of the enzyme
through aggregation or bimolecular self-oxidation of the porphyrin. The protein matrix
also controls access of the substrate to the active oxidant, hence controlling the
selectivity of the oxidation. The matrix may also provide a hydrophobic environment
26
that can be important for substrate binding, but may also subtly control the redox
potential of the metalloporphyrin prosthetic group.
The lack of these features in simple porphyrin model systems has been partly
overcome through structural modification of the porphyrin rings. Bulky and electronwithdrawing substituents help to prevent inactivation of the catalyst and selfoxidation.62 Picket-fenced, strapped and capped porphyrins have been developed to
control the access of the substrate to the porphyrin’s catalytic centre and thereby to
introduce selectivity into the oxidation.63,64 Finally, the use of chiral superstructures on
the porphyrins can lead to enantioselective oxidation.65
Because of the expense incurred in the synthesis of metalloporphyrins as catalytic
oxidants, two important difficulties will need to be overcome in relation to catalyst
recovery and reuse. One way to do this is to anchor the metalloporphyrins to a solid
surface, which will then allow the easy separation of the catalyst from reactants and
products.
However, this method also presents potential disadvantages: if the
metalloporphyrins are able to move on the surface, the support can, by concentrating the
metalloporphyrins on the surface, lead to increased aggregation and decreased catalyst
efficiency. Consequently, it is necessary that any support should be rigid. Moreover,
interference from the support could also be a problem e.g. the backbone of an organic
polymer support might act as a competing substrate in oxidation.
It is therefore
important for the support to be inert. Another disadvantage of surface attaching a
catalyst is the difficulty in characterising the molecules on the surface of the support.
Indeed, methods available for determining detailed information about the structure of
surface-bound metalloporphyrins are not as well developed for solid samples as they are
for homogeneous systems.
For the same reason determining the structure of
intermediates formed in the catalytic process is very difficult.
Nevertheless,
considerable attention has recently been focused on the immobilisation of
metalloporphyrins on both organic and inorganic supports, the objective being to obtain
a well defined complex that can be highly dispersed throughout the reaction medium
while remaining in a separate phase.46
Organic polymers can serve as solid supports for porphyrins, but their flexibility may
allow bidentate ligation of the metal centre, leading to inactivation of the catalyst
(Figure 10).66
Inert inorganic materials such as silica, alumina and molecular sieves
have also been used as solid supports and, of these, silica has been used most
extensively.
27
N
Fe
N
Schematic diagram of an iron porphyrin supported on
N-imidazylmethylated polystyrene or poly(4-vinylpyridine); a flexible
support can lead to bisligation from both faces of the porphyrin.
Figure 10
1.6.2 Silica as a solid support
Because it is relatively cheap and readily available, silica has been widely studied as a
solid support for catalysts.67,68,69 Even though the nature of silica itself has been
investigated,
70
discrepancies still exist in the literature in regards to current
understanding of surface silanol sites. This can largely be attributed to variation in the
physical properties of the silica studied — i.e. particle size, shape, pore diameter, etc. —
but also to the differences in treatment of the surface prior to analysis i.e. purification,
rehydration. Despite these discrepancies, it is generally agreed that silanol sites can
exist in one of two basic configurations: single isolated and geminal sites71 (Figure 11).
H
O
HO
Si
OH
Si
Single site
Geminal site
Figure 11
However, discrepancies exist in the literature in the number, reactivity and distribution
of each type of silanol.
More recent studies, including
29
Si NMR and FTIR
experiments, have helped to throw light on the nature of silica and modified silica
surfaces.72 These studies have demonstrated that the relative population of geminal
hydroxyl groups to lone hydroxyl silanol sites depends on the type of silica. However,
geminal sites seem to react faster with electrophilic reagents used in surface
modification than the single silanol sites.67
28
Several routes exist for the covalent attachment of organic functionality to a silica
surface. Grafting of organosilanes is, however, the most popular method, mostly due to
its simplicity in terms of experimental procedure.67
The silica is reacted with an
organofunctional silane of the type X4-nSiRn, where R is the organic group and X an
alkoxy or halide function (Scheme 11).
silica surface
SiR3
O
H
HX
O
SiR3
X
Scheme 11
Monofunctional silanes having two methyl side groups (XSiR(CH3)2) yield a substrate
with the most homogeneous silane loading. However, these modified silicas can be
susceptible to undesirable hydrolysis of the silane even under mild conditions.
Polyfunctional silanes (X2SiR2 or X3SiR) tend to cross-link and can be anchored to the
surface at more than one site (Scheme 12).73 Such substrates are consequently less
susceptible to hydrolysis. Trialkoxysilanes are known to readily polymerise in aqueous
solution, and the presence of water in the reaction conditions, therefore, yields thick and
uneven coatings.74 Anhydrous conditions have been shown to yield relatively thin and
even surface coatings of good stability. 75
X
O Si
X
toluene
OH
OH
+
1 siloxane bond
X
O Si
O
RSiX3
OH
Silica
R
R
2 siloxane bonds
O
O Si
O
R
3 siloxane bonds
Scheme 12
29
1.7
OUR STUDIES TOWARDS SURFACE ATTACHMENT OF NOS MODELS
For our studies directed towards surface attachment of NOS models silica gel was
chosen as a support to investigate surface loading of the model. Ultimately, a silica
supported NOS model will be used for peroxide shunt catalysis studies. In the studies
described in this thesis, we investigated the attachment of simple porphyrins to silica
modified with (3-mercaptopropyl)trimethoxysilane. These simple porphyrins contained
two meso–alkenyloxyphenyl groups, the alkenyloxy side chain varying in length. The
strategy adopted for covalent surface fixation of the porphyrins centred on the radical
addition of surface-bound thiol groups to the double bond of the porphyrin’s meso–
alkenylphenyl substituents. In principle, the non–metallated porphyrins could be bound
to the modified silica surface in two different ways. The plane of the porphyrin could
be fixed parallel to the surface if both of the meso–alkenylphenyl groups react with
thiols on the surface.
Alternatively, the plane of the porphyrin could be roughly
perpendicular to the surface if only one of the porphyrin’s meso substituents reacts with
the modified surface (Scheme 13).
n
O
perpendicular attachment mode
parallel attachment mode
n
O
O
n
O
n
n
O
HS
radical initiator
S
O
n
OR
S
S
Scheme 13
In the case of the metalloporphyrin we envisaged that exposure to the thiol-modified
surface could also bring about surface fixation through dative bonding between the
porphyrin core metal atom and a thiol sulfur centre, Scheme 14. Subsequent addition of
a radical initiator might then trigger the radical addition reaction between the remaining
30
surface-tethered thiol sites and the porphyrin’s meso-alkenylphenyl groups. Thus, the
key objective of this project was to develop a dual coordinative-covalent attachment
regime that would simultaneously ensure robust surface fixation and eventually allow
the development of functionally active NOS models, that is, models possessing the
necessary thiolate ligand to the porphyrin metal centre. Although the primary solid
support selected for the surface attachment work was silica gel, we also envisaged that
adaptation of the procedure from silica gel to quartz slides could furnish suitably
modified surfaces for spectroscopic analysis of the bound material.
Ar1
N
NH
n
M2+
HN
N
O
Ar1
O
O
n
n
N
M
N
Ar1
Ar1
Ar1
n
O
S
N
N
M
N
N
S 1
Ar
O
n
N
N
HS
n
O
Si
O O O
S
Coordination
Si
O O O
Si
O O O
Si
O O O
Radical Addition
Scheme 14
At the outset of the project the mode of attachment proposed in Scheme 14 was
unprecedented.
Previous publications report porphyrin surface attachment through
either covalent bonding between a porphyrin side chain and the modified silica or
dative bonding of the porphyrin metal atom to the modified silica but not both.
Moreover, although alkenyloxy side chains have been used previously76 to covalently
attach an organic macrocycle to a mercaptopropyl-modified silica surface, this approach
has never been reported for surface attachment of porphyrins. In fact, in the past decade
the main strategy for covalently binding porphyrins to modified silica surfaces takes
advantage of easy substitution reactions between surface-tethered nucleophiles and
meso(pentafluorophenyl) substituents on the porphyrin substrate.77
Thus, new
supported oxidation catalysts have been prepared by covalently binding mesotetra(pentafluorophenyl)porphyrin derivatives onto various supports bearing an amino
group as a nucleophile (Scheme 15). 69,78,79
31
F
O Si
NH 2
F
F
M
F
F
F
Ar
Ar
O Si
Ar
F
Ar
H
N
M
F
F
Ar
Ar
Scheme 15
Although covalently bound catalysts may achieve higher turnovers than their
homogenous analogues, they are not necessarily as efficient at alkane or alkene
oxidation. Thus, metalloporphyrins tethered by coordination of an axial donor ligand to
the metal centre may perform better.80 Several groups have tried to circumvent this
problem by using pyridine or imidazole as co-catalysts.78 Here we propose a new dual
coordinative-covalent attachment strategy which may not only be more efficient at
preventing catalyst leaching from the surface, but may also provide a better
representation of thiolate-liganded heme in nature.
In addition to providing a robust surface fixation protocol for the porphyrin, then, this
mode of attachment was intended to introduce the thiolate ligation of the metal centre in
our NOS model. The electron donating character of the thiolate ligand is likely to be
important in the formation of the oxoiron species from the ferric heme hydroperoxide
complex in the NOS catalytic cycle (cf. complex B  C, Scheme 5). Supported
porphyrins with sulphur ligation to the metal have not been reported yet; ligation is
usually carried out using substituted imidazole rings.66,81 Use of a thiol or thiolate for
ligation could pose a problem due to the potential instability of the ligand under
oxidising conditions.
We anticipate that replacement of the simple surface-bound
mercaptopropyl chain used in this project with a more robust thiol may ultimately be
required.
Two groups reported the construction of cytochrome P450 porphyrin-
containing models where the axial thiolate ligand is part of the “basket handle”
protecting the porphyrin (Figure 12).82,83 In these cases, the thiolate itself is sterically
shielded and this strategy could be adapted to our studies. However, the synthesis of
such models is extremely complicated and, as a result, this approach would only be
adopted for our studies if the thiolate axial ligand bound to the silica surface proves to
be too sensitive under oxidising conditions. The preferred option for generating more
robust models would be to replace the simple unbranched mercaptopropyl chain used in
this project with the more elaborate, sterically encumbered thiol tethers.
N
N
M
N
N
S
Figure 12
32
In general, porphyrin ring syntheses are intrinsically low yielding and product
purification can be difficult. Therefore, surface attachment studies in this project were
carried out using simple alkenyloxy substituted aromatic compounds, in the first
instance, and then on simplified model porphyrins. This approach was intended to
avoid the risk of losing synthetically expensive metalloporphyrins on a hitherto untested
attachment procedure. At the end of the project the attachment procedure was extended
for surface fixation of the fully developed prototypical NOS model to a silica surface
and also to a thiol-modified gold electrode surface.
1.8
SYNTHESIS OF THE PROTOTYPICAL NITRIC OXIDE SYNTHASE MODEL
The synthetic route that has been developed by our group to the prototypical NOS
model (7, X = R3 = H, R2 = Ph; Scheme 16) requires synthesis of
phenanthrenecarboxaldehydes (5) for condensation with phenyldipyrromethane.56 It
will be apparent that in order to create a guest-binding cleft, it is necessary that the two
spacer units are fixed on the same face of the porphyrin. To prevent rotation of the
spacers and maintain the guest–binding cleft will require introduction of a bulky group
(X) on the phenanthrene units, ortho to the porphyrin linkage. Synthetic strategies to
accomplish this are not the aim of the work described in this project, but are currently
being studied by our group.
O
NH
Cl
O
O
R1
propionic acid
NH
1
CHO
R =H
R1 = O(CH2)nCH=CH2
X
5
6
3
CO 2R
Cl
O
3
R O2C
O
R2
Cl
HN
N
N
NH
R2
7
R3 = t-Bu
R2 = Ph, C 6H4O(CH 2)nCH=CH 2
Scheme 16
The route to the parent phenanthrene spacer unit comprises a six step synthesis starting
from 4-chloro-2-methyl-phenol (8, Scheme 17) and featuring a key stilbene
33
photocyclisation reaction (12  13). Firstly, the phenolic function is protected by
methylation. In the next step the substrate is transformed into a benzyl bromide (10).
Different approaches were tried for this transformation, and the most successful
approach was found to be a photochemical radical bromination. A Wittig reaction of
terephthalaldehyde with the phosphonium salt (11) derived from bromide 10 then leads
to a mixture of the cis and trans stilbenes (12). As the two geometrical isomers
interconvert by photoisomerisation under the cyclisation conditions, the stilbene
mixture itself can be used directly for the photocyclisation step without prior separation
of the two isomers. Photochemical electrocyclic ring closure of the cis–stilbene under
oxidising conditions (in the presence of iodine) leads, via a dihydrophenanthrene (not
shown) to the target phenanthrene product (13). Finally, a nucleophilic demethylation
of 13 using ethanethiolate followed by alkylation of the resulting phenolate ion with
tert–butyl bromoacetate affords the porphyrin precursor 14, which now carries the
oxyacetate side chain destined to form the NOS model’s substrate recognition
superstructure.
Me 2SO4
NaHSO 3, KBrO3
NaOH (aq)
H2O/EtOAc
uv
0 to 70°C
Cl
OH
Cl
OMe
Cl
step 1
OMe
step 2
8
9
Br
10
PPh 3
toluene
step 3
reflux
LDA, THF
I2,
O
uv
Cl
Cl
OMe
CHO
step 6
OMe
step 5
13
Cl
CHO
12
EtSH, NaH, DMF, 100°C
O
then
Br
O
O
Cl
O
O
CHO
14
CHO
OHC
Scheme 17
34
step 4
OMe
PPh 3
11
Br
In order to adapt the prototypical NOS model (7, Scheme 16) for surface attachment, it
was necessary in the current project to replace the phenyldipyrromethane, used in the
original route, with a suitable alkenyloxy substituted analogue (6, R2 =
O(CH2)nCH=CH2).
As noted earlier, however, in this project we also planned to
investigate the synthesis of model variants in which the oxyacetate superstructure chain
is replaced by either an amino acid or oxymethylphosphonic acid. Construction of such
models would require the synthesis of suitably modified phenanthrenecarboxaldehyde
precursors (19 and 20, Scheme 18). During the project, then, we also planned to
investigate the synthesis of these phenanthrenes by alkylation of phenanthrenol 15 as
shown in Scheme 18. The one-step deprotection/alkylation that was previously used
(13  14, Scheme 18) was, therefore, abandoned in favour of a two-step process. The
methoxy group of phenanthrene 13 was to be removed with sodium ethanethiolate as
before, but the resulting phenolate solution was then to be acidified to produce the
phenanthrenol (15). The triflate derivative (16) of this compound was then to be
synthesised in a standard fashion using triflic anhydride.
We then planned to
investigate a palladium-catalysed amination procedure84 on the triflate using a chiral
amino ester (e.g. L-phenylalanine ethyl ester, 18).
Finally, synthesis of the
phosphonate-containing phenanthrene was to be achieved by alkylation of
phenanthrenol 15 with a suitable triflate reagent (17). The latter triflate is known in the
literature and is prepared in two steps from diethyl phosphite.85
Cl
OMe
Cl
i. EtSH, NaH
CHO
OH
ii. H+
Tf2O, DIPEA
CHO
13
DCM
CHO
15
O
EtO P
EtO
17
O
O S CF 3
O
Cl
16
Pd cat.
BINAP
Cs2CO3
toluene
Ph
O
O S CF 3
O
K2CO3
DMF
H2N
CO 2Et
18
O
Cl
P OEt
OEt
O
Ph
Cl
N
H
CHO
19
CHO
20
Scheme 18
35
CO 2Et
2. Results and discussion
36
2.1
SURFACE ATTACHMENT STUDIES
2.1.1 Introduction
The bulk of the work carried out in this project was designed to test the feasibility of the
attachment strategy proposed for surface fixation of our NOS models.
As noted
(section 1.7), in order to facilitate surface attachment of the model, it was planned to
take advantage of the meso(p-alkenyloxyphenyl) groups on the porphyrin for radical
addition to a mercaptoalkyl-modified surface, thus bringing about covalent attachment
of the porphyrin, Scheme 19. However, we also envisaged that the thiol-modified
surface might simultaneously be used to donate an axial ligand to a metal centre in the
porphyrin, allowing the development of functionally active NOS models.
NOS model spacer units and molecular recognition
superstrucutre
n
O
n
M
O
n
O
HS
n
M
S
O
S
S
Scheme 19
To test the radical-mediated attachment strategy it was decided firstly to investigate the
surface attachment of simple alkenyloxybenzene molecules. At the outset of the project
the length of the porphyrin’s alkenyl substituents required to achieve the dual
attachment mode shown in Scheme 19 was unknown.
Thus model studies were
conducted with two different alkene chain lengths using allyl phenyl ether and 4-(pent4-enyloxy)benzaldehyde, corresponding respectively to porphyrin alkenyloxyphenyl
substituents with n = 1 and n = 3 in Scheme 19. Having developed a viable radical
addition strategy, it would then be possible to progress to model porphyrins possessing
the
meso(p-alkenyloxyphenyl)
chain
but
lacking
the
molecular
recognition
superstructure of our prototype NOS model. Introduction of a labelled carbon atom at
the terminal olefinic centre of the side chains in these porphyrin models would allow the
37
mode of surface attachment to be probed by solid-state NMR spectroscopy. Finally, to
carry out preliminary electrochemical studies, a NOS prototype model comprising the
elaborate superstructure would need to be synthesised and attached to a suitable
conducting solid support such as gold wire.
2.1.2 Surface attachment via meso-(p-allyloxyphenyl) groups on the porphyrin
The synthesis of porphyrins carrying complex substituents is typically low yielding
and time consuming. To avoid losing valuable material on a non-viable route for surface
attachment, test studies were undertaken using simple alkenyloxybenzene substrates.
Our first studies were made with allyl phenyl ether (22) for attachment to a silica
surface modified with a mercaptopropyl tether.
Mercaptopropyl-modified silica is
readily prepared by heating silica gel in a dry solvent containing commercially available
(3-mercaptopropyl)trimethoxysilane (21).68
Exposure of the modified silica to a
solution of the alkene substrate (22) in the presence of a radical initiator would then be
expected to bring about the radical addition reaction, Scheme 20. However, with this
simple substrate the surface attachment procedure might also be undertaken by
reversing the order of the surface attachment and radical addition steps. It was decided
to compare the efficacy of both routes for the surface attachment of allyl phenyl ether.
OPh
22
Si(OMe) 3
HS
Ph
O
radical initiator
21
Si(OMe) 3
S
23
silica, toluene

silica, toluene

Ph
SH
SH
MeO Si OMe
Si OMe
O
O
O O
SH
OPh
Ph
O
O
S
S
S
radical initiator
Si
O
Ph
O
MeO Si OMe
Si OMe
O
O
O O
O
Scheme 20
38
Si
O
O
The solution phase radical addition of (3-mercaptopropyl)trimethoxysilane (21) to allyl
phenyl ether (22) was carried out using AIBN as a radical initiator. The simplicity and
efficiency of similar reactions have been related in recent literature. Thus, Lecamp et
al.
have
studied
AIBN-promoted
radical
additions
of
(3-
mercaptopropyl)trimethoxysilane (21) to allyl and vinyl substituted heterocycles.86 The
molar ratios of thiol and AIBN to olefinic substrate used in this study were respectively
1 and 0.05, and the reactions were carried out over 6 hours in acetonitrile at 80 °C under
an argon atmosphere. At the end of the reaction the solvent and unreacted volatile
compounds were removed in vacuo. All adducts were obtained in yields above 85% and
spectroscopic data showed that only the mono-addition products had been formed.
Thus,
applying
this
method
to
our
studies,
a
1:1
mixture
of
(3-
mercaptopropyl)trimethoxysilane (21) and allyl phenyl ether (22) was treated with
AIBN in boiling acetonitrile under nitrogen for 6 hours. Subsequent evaporation of the
solvent afforded the crude product in 98% yield and greater than 95% purity according
to its 1H NMR spectrum. As expected, the spectrum exhibited no signal for allylic or
thiol groups. Instead the presence of signals consistent with a phenoxypropyl chain was
observed: a pentet and two triplets respectively at H 2.1, 2.7 and 4.1 ppm. The ,  and
 methylenes in the silylpropyl chain came to resonance respectively at H 0.8, 1.7 and
2.6 and the methoxy signal was observed as a singlet at H 3.6. Integration of the
trimethoxysilylpropyl and phenoxypropyl resonances demonstrated unambiguously that
only the monoaddition product was obtained. The clean reaction outcome was fully
consistent with the work published by Lecamp and the crude product (23) was used for
surface attachment without further purification.
The concentration of organosilane residues grafted onto the surface of silica can be
calculated from microanalytical data and the BET surface area* for the bare silica.87 In
principle, either the %C or %H figure might be used to calculate the surface density of
attached residues for silica grafted with alkyltrimethoxysilanes such as 23. However,
use of the %H figure is avoided in practice because calculations are complicated by the
loss of residual silanol groups from the surface under the destructive conditions of the
combustion analysis (T ~1600 °C), Scheme 21.
*
The BET method, which is widely used to determine the surface area of solids, uses an isotherm
derived by Brunauer, Emmett and Teller to treat multilayer adsorption to surfaces. In essence, the
volume of N2 gas necessary to cover a single layer of a given mass of solid is determined. The area
occupied by this volume provides the surface area of the solid.
39
H2 O
OH
O Si
O O
OH
Si O
O O
O Si
O O
O
Si O
O O
Scheme 21
The surface attachment of alkyltrimethoxysilane 23 was carried out with silica gel in
hot anhydrous toluene. A series of reactions was undertaken with variation in the
silica/silane mass ratio used to gauge the effect on surface loading of the quantity of
silane for a given amount of silica.
It was necessary to make some preliminary
estimation of the quantity of silane 23 likely to be required for full surface loading on a
given mass of silica gel. This was possible by referring to previous studies by Kirkland
et al. who modified silica gel with triisopropylchlorosilane in order to investigate the
properties of the resulting material as a stationary phase for HPLC. 88 These workers
found a maximum silane loading of 2.1 mol/m2 using silica gel with a particle and
pore size of 5 m and 8 nm respectively and having a surface area of 180 m2/g. The
Kirkland value for maximum loading of the triisopropylsilyl moiety was used as an
approximate estimate for the quantities of silane 23 required to modify silica gel in our
studies.
However, it was realised that this figure may represent a very loose
approximation given the different steric demand and mode of attachment of this silane
compared to triisopropylchlorosilane. Moreover, the grade of silica gel studied in the
Kirkland work differed from that to be used in our own studies, where TLC grade silica
gel was used (Aldrich catalogue number 28,850–0; particle size 2-25 m, pore diameter
60 Å, pore volume 0.75 cm3/g, surface area 500 m2/g). The uncertainty introduced by
use of a different silica, however, was considered less problematic since previous
studies have shown an approximately linear relationship between silane loading and
surface area across different silicas.68
In order to optimise the surface loading of silane 23, a series of four experiments was
undertaken using firstly 2.0 mol of the silane per m2 of silica and then 5, 10 and 20
times this quantity. Other than the variation in the amount of silane 23 used in these
reactions, the experiments were carried out under identical conditions. To exclude
moisture the silica substrate was first dried at 110 ºC under vacuum (0.1 mmHg) for 24
hours prior to use. Previous studies89 have shown that such conditions are suitable for
drying silica to constant mass, with the loss of all physisorbed water. In the present
study a 5% mass loss was observed for the commercial silica gel after drying for 24 h;
40
no further mass loss was observed when dried for 48 h under the same conditions.
Microanalytical data for the parent unmodified silica gel before and after the drying
periods are shown in Table 1.
Sample
Drying conditions
% mass
loss wrt
sample A
Batch 1
Batch 2
Batch 3
%C
%H
%C
%H
%C
%H
A
none
__
0
0.40
0
0.40
0
0.40
B
24 h, 100 ºC, 0.1 mmHg
4.9
0
0.35
0
0.35
0
0.34
C
48 h, 100 ºC, 0.1 mmHg
4.8
0
0.36
0
0.35
0
0.36
Table 1: Microanalytical data for the unmodified silica gel used in the surface attachment of silane 23.
A: undried commercial silica gel; B: silica gel dried under the conditions used for preparation of the
surface-modified samples; C: silica gel dried for an extended period.
The surface attachment reactions with silane 23 were carried out in anhydrous toluene
at 110 ºC in a flame dried sealable flask over 16 hours. After this period the solvent and
unreacted silane were removed by filtration under argon (to prevent ingress of
moisture), washing with toluene followed by dichloromethane. Identical volumes of
solvent were used in each of the four experiments. The silane-modified silica was then
dried for 24 hours (110 ºC, 0.1 mmHg) and subjected to microanalysis in order to
estimate the silane loading.
Depending on the number of siloxane linkages formed to the surface, there are three
possible attachment modes for individual surface bound-residues, Modes A-C
illustrated in Figure 13. The process of calculating surface loading is illustrated in
Figure 13 (boxed) for the first in the series of surface attachment experiments
undertaken with silane 21 (2.0 mol per square metre of silica gel), which afforded
modified silica with 7.59% and 1.32% carbon and hydrogen contents respectively.
41
Formulae and formula weights of attached organic residues:
Ph
O
C14H23O3SSi
MW = 299.482
S
MeO Si OMe
O
Type A
attachment mode
Ph
Ph
C13H20O2SSi
MW = 268.448
O
S
Si
C12H17OSSi
MW = 237.414
O
S
OMe
Si
O O
O
Type B
attachment mode
O
O
Type C
attachment mode
EXPERIMENT 1: example calculation of surface loading
Mass of silica gel used
–––––––––––––
200 mg
Silica gel surface area
–––––––––––––
500 m2/g
 Surface area of silica gel used
–––––––––––––
100 m2
Quantity of silane 23 used
–––––––––––––
69 mg, 0.21 mmol
Microanlalytical data for modified
silica gel
–––––––––––––
 Quantity of carbon in 1 g of
modified silica gel
–––––––––––––
C, 7.59%; H, 1.32%
0.0759 g of C atoms
 6.32 mmol of C atoms
12.011 g/mol
Assuming that surface-bound residues are attached exclusively in the mode A pattern, each attached residue has
14 C atoms:
Quantity of surface–bound
residues in 1 g of silica gel
–––––––––––––
6.32 mmol of C atoms
 0.451
mmol of silane residues
14 C atoms / residue
FW of surface-bound residues
–––––––––––––
298.482
 Mass of surface-bound
residues in 1 g of modified silica
gel
–––––––––––––
0.451 mmol  299.482 g/mol = 135 mg of silane residues
Mass of actual silica in 1 g of
modified silica gel
–––––––––––––
1000 mg – 135 mg = 865 mg of silica
Silica surface area in 1 g of
modified silica gel
–––––––––––––
2
0.865
 500
/g = residues
432 m2
0.451gmmol
ofm
silane
 Loading of surface-bound
residues
432 m 2
 1.04 mol/m2
–––––––––––––
Figure 13
The surface area for the parent silica substrate (500 m2/g) quoted by the supplier has
been assumed in all calculations of surface densities for grafted residues in this thesis.
Using this value, then, and assuming attachment of surface-bound residues solely
through a single siloxane bond to the surface (Mode A), the calculated surface density
of residues based on the %C figure is 1.04 µmol/m2. The results of calculations made
assuming surface attachment solely by Mode B and solely by Mode C are shown in
Table 2.
42
Residue attachment mode
assumed in the calculation
Calculated surface loading
(see Figure 13)
Based on %C (7.59%)
100% by Mode A
1.04 µmol/m2
100% by Mode B
1.12 µmol/m2
100% by Mode C
1.20 µmol/m2
Table 2:Calculated surface loadings for silane 23 attached to silica gel in an experiment where the
silane to silica gel ratio used was 2.0 µmol / square metre of silica surface. Calculations are based on
microanalytical data for the resulting modified silica gel (%C, 7.59; %H, 1.32) assuming the three
different attachment modes illustrated in Figure 13.
The surface loading of silane 23 is clearly subject to uncertainty because of the lack of
information about the exact proportion of surface-bound residues distributed between
the different attachment modes (type A, B or C) on the modified silica. As shown in
Table 2, however, the estimated silane loading varies from 1.04 to 1.20 µmol/m2 for the
two extremes, full Mode A attachment and full Mode C attachment.
Therefore,
variation in the mode of attachment of individual silane units to the silica surface is
relatively unimportant as far as the uncertainty in the surface loading level is concerned,
provided that the formula weight of attached residues is reasonably large.
Later in the course of the project (vide infra, section 2.1.6) solid-state NMR studies
were carried out on silica gel modified with (3-mercaptopropyl)trimethoxysilane. The
29
Si and 13C NMR spectra of this silane-modified silica showed that surface attachment
had occurred principally through modes involving single and double siloxane linkages.
However, the situation was complicated by the loss of methoxy groups from the
attached organosilane, assumed to occur through exposure to moisture during
subsequent handling of the grafted silica. Partial hydrolysis of the modified silica
substantially complicates the analysis, with several attachment modes now possible for
individual organosilane residues, Figure 14.
Calculations made assuming 100%
attachment by the various modes shown in Figure 14 show that even for the extremes
the surface loading still lies in a narrow range (1.04 – 1.23 µmol/m2).
43
Formulae, formula weights and calculated surface density for organosilane residues attached
2
exclusively by the various modes indicated on silica gel of surface area 500 m /g and
analysing for 7.59% carbon content.
attachment with single silaxane linkage
attachment with double siloxane linkage
Formula
Surface density
(mol/m2)
–––––––––––––––––––––––––––––––––––––––––––
C 14H23O3SSi
298.482
1.04
C13H21O3SSi
285.455
1.13
C 12H19O3SSi
271.429
1.23
C 13H20O2SSi
268.448
1.12
C 12H18O2SSi
254.422
1.22
attachment with triple siloxane linkage
Mode A
Mode D
Ph
MeO
Mode E
Ph
FW
Mode B
Mode F
Ph
Ph
Ph
O
O
O
O
O
S
S
S
S
S
Si OMe
HO
O
Si OMe
HO
O
Si
Si
OH
Si OH
OMe
O O
O
O
O
C 12H17O SSi
C 12H17O1.5SSi
C 12H18O2.5SSi
C 13H20O2.5SSi
237.414
245.416
262.424
276.450
Ph
Ph
1.20
1.21
1.22
1.12
Mode C
Ph
Ph
Ph
Ph
O
O
O
O
O
O
S
S
S
S
S
S
Si
O O O
O
Si O Si OH
O
O O
HO
Si
O
O
Si
O
O
Si OMe
O
Figure 14
The solid-state
29
Si and
13
C NMR spectra acquired for silica grafted with (3-
mercaptopropyl)trimethoxysilane (section 2.1.6) indicate a 1.5 / 1 ratio of attachments
involving the formation of single and double siloxane linkages, with hydrolysis of 60%
of the residual methoxy groups from attached silane units. Assuming a similar picture
for the silica grafted with silane 23, and fitting the various attachment modes shown in
Figure 14 to match these NMR data, gives a silane loading very close to 1.16 µmol/m2
for the surface-modified silica prepared in the first experiment.
44
The microanalytical data and estimated surface loadings for the modified silicas
produced in the series of four experiments undertaken with increasing silane 23/silica
mass ratios are summarised in Table 3. The results show that an increase in the quantity
of silane 23 used does result in higher levels of silane loading, but the relationship is not
linear. Indeed, a twenty-fold increase in the number of equivalents of silane 23 reacted
increased the surface loading only by a factor of 1.7. These results suggested that
acceptable surface loadings should be possible using 2.1 mol of a trimethoxysilane per
square metre of silica to be modified.
Expt
Silane 23 qty reacted
per m2 silica surface*
%C†
%H†
Surface density
(mol/m2)
(mol/m2)
range estimate
fitted to NMR data§
1
2.00
7.59
1.32
1.04 – 1.23
1.16
2
10.0
7.88
1.34
1.09 –1.28
1.21
3
20.0
10.03
1.60
1.45 – 1.71
1.62
4
40.0
11.59
1.85
1.74 – 2.06
1.94
* Calculated for a surface area of 500 m2/g quoted by supplier of the silica substrate.
†
Determined by elemental microanalysis of the surface-modified silicas

Limiting values taken from calculations assuming 100% attachment by modes A and E of Figure 14.
Fitted to solid-state NMR data for silica grafted with (3-mercaptopropyl)trimethoxysilane (section 2.1.6) that indicate a 1.5/1
ratio of attachments involving the formation of single and double siloxane linkages, with hydrolysis of 60% of the residual methoxy
groups from attached silane units.
§
Table 3: Calculated surface loadings for silane 23 attached to silica gel from 4 different experiments in
which the silane to silica ratio used was progressively increased .
Thus, the calculated surface loading for silane 23 fitted to NMR data for silica grafted
with (3-mercaptopropyl)trimethoxysilane (section 2.1.6) varied from 1.16 to 1.94
mol/m2 as the amount of silane 23 used in the reaction was increased from 2 to 40
µmol per square metre of silica substrate surface. How does this compare to the surface
density of silanol sites on the silica substrate?
As shown in Table 1, under the standard conditions used to dry the silica prior to
preparation of the surface-modified materials (0.1 mmHg, 110 °C, 24 hours) a 5% mass
loss was observed and the residual hydrogen content was found to be 0.35% (Sample
B). Silica gel submitted to drying for an extended period of 48 h at 110 °C (Sample C)
showed no significant difference in the mass loss and hydrogen content from that dried
over 24 h. If physisorbed water is fully removed from silica samples B and C prior to
the microanalysis, then the residual hydrogen content found in the analyses ought to be
due to loss of silanol sites, Scheme 21.
45
A recent thermogravimetric and 1H MAS NMR study by Ek et al examined the loss of
physisorbed water and silanol sites from Kieselgel 60, a silica which possesses similar
particle size, pore diameter and surface area characteristics to the silica used in our
study.90 Samples of kieselgel 60 were heated from room temperature under a stream of
argon at a rate of 5 °C/min. Complete loss of physisorbed water was estimated at 130
°C (with a 4.0% mass loss), and at this temperature the surface silanol number of the
silica was estimated to be 5.5 silanol sites per nm2 of surface area. Loss of silanol sites
from the silica was approximately complete at 1000 °C. Thus, assuming complete loss
of silanol sites from our silica samples B and C under the microanalysis conditions (T ~
1600 °C), the surface silanol density in the dried silica (calculated from the
microanalytical hydrogen content) is 6.9 µmol/m2 or 4.2 silanol sites per nm2 of surface
area. This figure is reasonably consistent both with the value obtained by Ek et al for
kieselgel 60 and the values obtained in a wider survey of over 100 fully hydroxylated
silicas undertaken by Zhuravlev, 91 where the silanol number ranged between 4.2 and
5.7 OH/nm2 irrespective of the origin and structural characteristics of the silica.
The experiments summarised in Table 3 showed only a modest increase in the levels
of silane loading as the quantity of trimethoxysilane 23 reacted was increased from 2 to
40 µmol per m2 of the substrate silica surface. One aspect that was not investigated in
this series of experiments was how reproducible the loading level was at any given
silane/silica mass ratio used in the attachment reactions. In order to probe this issue a
further set of four identical experiments was carried out using a reactant ratio of 2.0
µmol of silane 23 per m2 of the substrate silica surface. The results of these experiments
are summarised in Table 4.
Elemental analysis of the four samples of modified silica gave an average %C figure of
6.4 ± 0.3 %. The maximum and minimum values for the surface loading range quoted
in Table 4 for each of the four samples are based on calculations assuming 100%
attachment respectively by Modes A and E shown in Figure 14. Calculations that fit
the surface loading for silane 23 to NMR data for silica grafted with (3mercaptopropyl)trimethoxysilane (section 2.1.6) suggest a surface loading varying
between 0.91 and 1.01 µmol/m2, with an average loading of 0.96 + 0.05 µmol/m2. Thus
the surface density of silane coverage in these experiments shows coherence and the
attachment is reproducible.
46
Silane 23 qty reacted per
m2 of silica surface*
(µmol/m2)
%C†
5
2.00
6.14
0.82 – 0.96
0.91
6
2.00
6.30
0.84 – 0.99
0.94
7
2.00
6.71
0.91 – 1.07
1.01
8
2.00
6.62
0.89 – 1.05
0.99
Expt
Surface density
(µmol/m2)
range estimate** fitted to NMR data§
* Calculated for a surface area of 500 m2/g quoted by the supplier of the silica substrate.
†
Carbon content determined by microanalysis of the surface-modified silica.
** Limiting values taken from calculations assuming 100% attachment by modes A and E of Figure 14.
§
Fitted to solid-state NMR data for silica grafted with (3-mercaptopropyl)trimethoxysilane (section 2.1.6) that indicate a 1.5 / 1
ratio of attachments involving the formation of single and double siloxane linkages, with hydrolysis of 60% of the residual
methoxy groups from attached silane units.
Table 4:Calculated surface loadings for silane 23 attached to silica gel from four identical experiments
in which the silane to silica gel ratio used was fixed at 2.10 µmol per m 2 of silica surface.
However, the silane loading was somewhat lower than that obtained originally under
Experiment 1 of Table 3, where the calculated surface density was 1.16 µmol/m2. The
reason for this discrepancy is unclear and may indicate that surface attachment is quite
sensitive to the exact drying conditions applied in the preparation of the silica gel
substrate. Indeed, the silica used for Experiments 5-8 was taken from a common batch
that was dried separately from that used for Experiments 1-4. Nevertheless the surface
coverage calculated for silica grafted with silane 23 is in the same micromolar range as
the results published previously in the Kirkland study with triisopropylchlorosilane.
These results suggest that oligomerisation of the silane has not occurred during the
grafting process through loss of the methoxy groups. Had polymerisation occurred, the
quantity of silane loaded on the surface may not have been controlled and the results
would not have been reproducible.92 However, as discussed later in section 2.1.6, it is
likely that the residual methoxy groups of the attached organosilane units are replaced
by silanols on handling of the silica after the grafting reaction.
Having successfully demonstrated the solution phase radical addition of (3mercaptopropyl)trimethoxysilane to allyl phenyl ether and surface attachment of the
resulting silane (23), we next examined the radical addition to allyl phenyl ether with
the mercaptopropyl chain already bound to the silica surface, Scheme 22
47
HS
Ph O
22
Si(OMe) 3
, AIBN
O
acetonitrile, 

21
S
23
Si(OMe) 3
Experiments 1-8,
Tables 3-4
PhMe

PhMe

silica
silica
Ph
SH
SH
MeO Si
OH OH
Si
OO
O OO
silane-modified silica-I
Ph
O
O
S
S
MeO Si
, AIBN
Ph O
22
acetonitrile, 
OH
OH
Si
OO
O OO
silane-modified silica-II
Scheme 22
The surface attachment strategy intended for our NOS model metalloporphyrins
involves simultaneous coordinative and covalent fixation of the porphyrin. Indeed the
presence of a thiolate ligand attached to the metal centre is a vital design feature of the
models. It was proposed at the outset of this project to accomplish this dual attachment
strategy (Scheme 23) by first exposing a mercaptopropyl-modified surface to a solution
of the metalloporphyrin, in order to initiate attachment by coordination, and then to
trigger the covalent fixation by addition of a radical initiator. Thus, the reaction of allyl
phenyl ether with the mercaptopropyl-modified silica was intended to model the
covalent attachment of porphyrins carrying allyloxyphenyl substituents.
n
O
HS
metalloporphyrin
MeCN
SH
n
M
O
S
HS
coordinative attchment
AIBN
MeCN
n
O
S
Scheme 23
48
M
S
n
O
S
According to Scheme 22, then, dried silica was treated with the (3mercaptopropyl)trimethoxysilane (21; 2.1 mol per m2 of substrate silica) in hot
toluene under conditions similar to those employed in Experiments 1–8 summarised in
Tables 3 and 4. This procedure afforded a sample of mercaptopropyl-modified silica,
Silica-I, which analysed for 2.64 %C and 0.83 %H. Silica-I was then treated with allyl
phenyl ether (2.1 mol per m2 of substrate unmodified-silica) and a catalytic amount of
AIBN in boiling acetonitrile for 6 hours. The final modified silica, Silica-II, was
isolated by filtration, scrupulously washed with toluene and dichloromethane and then
thoroughly dried (0.1 mmHg, 110 ºC, 24 hours) before being subjected to
microanalysis. Silica-II analysed for 5.78% and 1.15% carbon and hydrogen contents
respectively. The carbon content of modified Silica-II prepared by this route is clearly
somewhat lower than that prepared by the direct surface modification with silane 23.
The latter typically afforded a surface loading of 0.9-1.1 mol/m2. Seemingly, this
could suggest a lower overall surface loading of silane units. However, it is more
likely that a proportion of surface bound mercaptoalkyl groups failed to undergo
addition to the allyl ether — perhaps because of their inaccessibility or perhaps because
of loss of some thiols to disulfide formation or the formation of 2/1 thiol-alkene
adducts. This latter possibility might arise through the chain-terminating combination
of adjacent surface bound thiyl radicals during the radical chain reaction, Scheme 24.
PhO
PhO
S
S
S
S
S
S
chain
termination
Si OMe
O O
Si
OOO
Si OMe
O O
Si
OOO
Si OMe Si
O O
OOO
chain termination
S
S
MeO Si
Si
O O OO O
Scheme 24
Calculations based on the carbon content of Silica-I, the substrate for preparation of
Silica-II, suggested that the surface loading of mercaptopropyl silane units lies within
the range 0.95–1.63 mol/m2, where these two limits are calculated, respectively,
assuming uniform attachment of silane residues solely by modes A and E of Figure 15.
49
The calculated surface loading range for silane units on Silica-I is wider than for the
loading of silane 23 (Table 3). This reflects the lower formula weight of attached
mercaptopropyl species and, therefore, the greater percentage difference in formula
weight of possible attached units.
Formulae, formula weights and calculated surface density for mercaptopropyl residues attached
to Silica-I exclusively by the various modes indicated (surface area of parent unmodified silica =
2
500 m /g; microanalytical carbon content for Silica-I = 2.64%).
attachment with single silaxane linkage
attachment with double siloxane linkage
Formula
Surface density
(mol/m2)
–––––––––––––––––––––––––––––––––––––––––––
C 5H13O2SSi
165.307
0.95
C 4H11O2SSi
151.280
1.20
C 3H9 O2 SSi
137.254
1.63
C 4H10O SSi
134.273
1.19
C 3H8 OSSi
120.246
1.61
attachment with triple siloxane linkage
Mode A
Mode D
SH
MeO Si OMe
Mode E
SH
HO
O
Mode B
HO
Si
O
Mode F
SH
SH
Si OMe
FW
Si OH
OMe
Si
OH
SH
O O
O
O
C 3H7SSi
C 3H7O0.5SSi
C 3H8O1.5SSi
C 4H10O1.5SSi
O
103.239
111.239
128.246
142.273
1.59
1.59
1.62
1.29
Mode C
SH
SH
Si
O O O
O
SH
SH
HO
Si O Si OH
O
O O
Si
SH
SH
O
O
Si
O
O
Si OMe
O
Figure 15
Solid-state
NMR
data
for
the
silica
grafted
in
this
way
with
(3-
mercaptopropyl)trimethoxysilane (section 2.1.6) indicate a 1.5/1 ratio of attachments by
single and double siloxane linkages, with hydrolysis of 60% of the residual methoxy
groups from attached silane units. Fitting combinations of attachment modes A-F from
Figure 15 to these data narrows the surface loading range for mercaptopropyl silane
units in Silica-I to 1.36-1.41 µmol/m2. This calculated surface density is slightly higher
than that produced by direct modification of silica with silane 23 under comparable
conditions,
suggesting
that
the
longer
50
and
more
flexible
3-(3-
phenoxypropylsulfanyl)propyl chain of 23 with its bulky head results in less efficient
surface packing of the silane units than the shorter mercaptopropyl chain of silane 21.
On the other hand, radical addition of the surface bound mercaptopropyl group to allyl
phenyl ether must be significantly less efficient than the corresponding solution phase
reaction between mercaptopropylsilane 21 and the allyl ether.
SILICA–II: surface loading calculation for mercaptopropyl silyl residues
Silica gel surface area
–––––––––––––
Microanalytical data for
modified silica gel
–––––––––––––
500 m2/g
C, 5.78%; H, 1.15%
2
Let X = surface loading of long chains (mol/m ) of averaged formula C12.6H19.9O2.6SSi and MW 273.145. Assume a
surface density of 1.39 mol/m2 for the mercaptopropylsilanyl units of the parent Silica–I with the averaged formula
C3.6H10.1O1.6SSi and MW 138.867.
Calculation based on carbon content (5.78%) determined by elemental microanalysis:
C content 
(mass of C in ureacted mercaptopropylsilylresidues)  (mass of C in long chain residues)
total mass of sample
Mass of C atoms in residual mercaptopr opy lsily lresidues
Mass of C atoms in long chain residues attached
(3.6 C f or each av eraged silane residue attached)
(each av eraged silane residue has 12.6 C atoms)

 


0.0578

[500m 2 (1.39  X )10
6
mol/m 2  3.6  12.011 g/mol]  [500m 2  ( X  10  6 mol/m 2 )  12.6  12.011 g/mol
1  [ 500m 2  (1.39  X )10
 6
 138.867 g/mol]  [500m  ( X  10
)  273.145g/mol

mol/m


 mol/m



6
2
2
Mass of residual mercaptopr opy lsily lresides attached
(av eraged silane residue has f ormula C
Mass of core silica
H
3.6 10.1
Mass of long chain residues attached
SSi )
O
2
(av eraged residue has f ormula C
16
H
12.6 19.9
O
SSi)
2.6
From elemental microanalysis
This results in: Long chain surface loading (X) = 0.67 mol/m2
Figure 16
Given the surface loading of mercaptopropyl silane residues in Silica-I and
microanalytical data for Silica-II, it is possible, in principle, to calculate the proportion
of unreacted thiol sites present in the latter.
The calculation is complicated by
uncertainty in the distribution of attachment modes for surface-bound silane units, but
an approximate figure is calculated in Figure 16 using averaged molecular formulae for
surface-bound residues that fit the solid-state NMR data previously referred to. The
surface coverage of long chain residues in Silica-II issuing from this calculation is 0.67
µmol/m2, commencing from a surface density of 1.36-1.41 µmol/m2 for the
mercaptopropylsilyl residues in the parent Silica-I. Thus, it is estimated that 50% of the
surface-bound mercaptopropyl chains remain unreacted in Silica-II.
51
In summary, then, model studies undertaken with allyl phenyl ether demonstrated that
the proposed mode for covalent attachment of our NOS models was a feasible option.
The radical addition of a thiol group to the allyl ether proceeds both in solution and with
the surface-bound mercaptopropyl chain, though in the latter case the addition is
somewhat less efficient. Surface loading of silane units can be estimated from the
carbon content of the modified silicas as determined by elemental microanalysis. Silane
loadings were found to be in the range 0.9-1.9 µmol/m2 of surface, consistent with the
formation of a surface-bound layer without oligomerisation of the trimethoxysilane
reagent.
2.1.3 Surface attachment studies with porphyrins carrying allyloxy groups
Synthesis of a suitable porphyrin substrate
Having successfully tested the covalent fixation strategy with allyl phenyl ether the
next phase of the project extended the surface attachment to porphyrin systems. This
study required the construction of a suitably substituted porphyrin (26) carrying olefinic
side chains, which we planned to prepare by reaction of an aryldipyrromethane (24)
with benzaldehyde in a Macdonald 2+2 porphyrin synthesis, Scheme 25. Although, in
principle, this synthetic approach should generate solely the target 5,15-diaryl-10,20diphenylporphyrin (26), scrambling of the meso substituents is typically encountered in
such porphyrin syntheses, leading to formation of minor porphyrin congeners along
with the desired product.56 Scrambling occurs through acid-catalysed fragmentation
and subsequent recombination of the aryldipyrromethane and other intermediates en
route to the porphyrin, Scheme 25.
In the hope of suppressing this problem it was decided to work with an alkoxycarbonyl
side chain on the aryldipyrromethane in the first instance (i.e. where the connecting
function X in Scheme 25 is CO2). It was hoped that the electron-withdrawing ester on
the phenyl ring would disfavour formation of some of the carbocationic intermediates
assumed to participate in the scrambling.
52
X
X
X
NH HN
PhCHO
NH HN
NH HN
H+
NH HN
NH HN
X
X
Ph
Ph
Ph
24
25
H+
X
oxidation
Ar
X
N
NH
Ph
Ph
N
HN
HN
NH HN
Ph
Ar
HN
26
Ar =
Ar
Ph
N
NH
Ph
NH
Ph
N
X
HN
Ar
N
Ph
N
HN
N
NH
Ar
Ph
Ph
N
HN
Ph
Ar
Ph
27
28
29
Scheme 25
Our first target porphyrin was, therefore, 5,15-bis(4-allyloxycarbonylphenyl)-10,20diphenylporphyrin (34), Scheme 26. For simplicity, the synthesis of this porphyrin was
planned by a route involving transesterification of a precursor porphyrin (32) carrying
methyl rather than allyl esters. The methyl 4-formylbenzoate starting material (30)
required for preparation of the dipyrromethane (31) en route to this latter porphyrin was
commercially available. Porphyrin 32 might also be prepared by an alternative route,
through the reaction of methyl 4-formylbenzoate with phenyldipyrromethane.
However, we chose the synthetic strategy that built the ester function into the
dipyrromethane because the same dipyrromethane might then be used with our more
53
elaborate phenanthrenecarboxaldehydes (in place of benzaldehyde) for construction of a
NOS model (33) suitable for surface attachment.
CO 2Me
excess pyrrole HN
OHC
PhCHO
CO 2Me
TFA
propionic
acid
HN
30
N
NH
Ph
CO 2Me
Ph
N
HN
31
Cl
OMe
CO 2Me
32
CHO
transesterification
14
O
O
OBut
O
NH
N
ButO
O
Cl
O
O
O
O
N
Cl
HN
Ph
NH
O
N
O
33
Ph
N
HN
O
O
34
surface attachment
Scheme 26
Dipyrromethane 31 was prepared by acid-catalysed condensation of methyl 4formylbenzoate with pyrrole, the latter present in excess (typically 5 equivalents) to
suppress formation of higher order condensed products such as 35 and 36, Scheme 27.
After removal of the solvent the black tarry residue was subjected to flash column
chromatography to afford the target dipyrromethane as a crystalline solid in 77% yield.
The formation of higher order side products is a common problem with this type of
aryldipyrromethane preparation, and indeed the minor products 35 and 36 were
identifiable within the residual fractions from the column.
54
pyrrole (5 eq.)
OHC
CO 2Me
TFA cat.
30
CO 2Me
N
H
N
H
31
N
H
CO 2Me
CO 2Me
N
H
N
H
35
N
H
CO 2Me
CO 2Me
CO 2Me
N
H
N
H
N
H
36
Figure 27
The condensation between dipyrromethane 31 and benzaldehyde was carried out in
boiling propionic acid exposed to the air so that the intermediate porphyrinogen (37),
Scheme 28, underwent direct aerial oxidation to afford porphyrin 32. This reaction
proceeded with rapid discolouration and afforded a black tarry residue after evaporation
of the propionic acid.
The target porphyrin (32) was isolated by flash column
chromatography in 11% yield as a purple solid along with minor porphyrin congeners
27, 38 and 39 in 1.5%, 2.2% and 8.2% yields respectively. These compounds were not
fully characterised but were identified from their 1H NMR spectra by comparative
integration of the porphyrin -hydrogen/aryl signals and the methoxy resonances. The
formation of congeneric porphyrins therefore remains problematic notwithstanding the
electron withdrawing ester present in the aryldipyrromethane building block.
Transesterification of the methyl ester to the allyl ester was then attempted with
porphyrin 32. Transesterifications are commonly carried out with an alcohol under
acidic conditions. The ultimate goal of the project, however, was to adapt the route
shown in Scheme 26 to our more elaborate NOS models comprising the phenanthrene
spacer units. These spacer units possess acid-labile tert-butyl ester groups of uncertain
stability to the transesterification conditions.
Therefore, to avoid unwanted side
reactions we chose to examine the use of a mild Lewis acid catalyst. Transesterification
reactions have been reported for esters attached to porphyrin meso aryl substituents
using titanium isopropoxide as a catalyst.93 Thus, porphyrin 32 was treated with 10
equivalents of allyl alcohol and 0.1 equivalent of titanium isopropoxide in boiling
dichloromethane, but returned the starting material unreacted after 1.5 hours.
55
Ph
PhCHO
HN
NH HN
MeO 2C
CO 2Me
propionic
acid
HN
CO 2Me
NH HN
31
Ph
37
Ph
N
Ph
HN
Ph
N
Ph
NH
N
CO 2Me
NH
Ph
N
HN
MeO 2C
N
Ph
27 1.5%
32 11%
CO 2Me
CO 2Me
N
HN
Ph
NH
HN
MeO 2C
Ph
N
CO 2Me
NH
N
Ph
Ph
38 2.2%
39 8.2%
Scheme 28
Reactions in toluene (110 °C, sealed tube, 1.5 h) and eventually in neat allyl alcohol (70
°C, 3 h) also returned the porphyrin unreacted with high material recovery. Attempts to
effect the transesterification with para-toluenesulfonic acid (0.2 equivalents) and allyl
alcohol (10 equivalents) in boiling dichloromethane (5.5 h) similarly returned the
starting material. A closer inspection of the literature revealed that other groups have
found titanate-mediated transesterifications with allyl alcohol to be problematic. Thus,
the instability of allyl alcohol frustrated attempts by Seebach et al to transesterify an
ethyl ester using titanium isopropoxide.94
Rather than pursue the transesterification approach attention was turned to the
introduction of the allyl ester prior to the dipyrromethane-forming step of Scheme 26.
We envisaged that the dipyrromethane precursor 31 might be prepared by alkylation of
4-formylbenzoic acid with allyl bromide, Scheme 29.
commercially available but expensive.
4-Formyl benzoic acid is
We chose to prepare this compound by
oxidation of p-toluic acid using a procedure previously applied in our laboratories for
56
the preparation of 3-formylbenzoic acid from m-toluic acid. Using this procedure,
which had been adapted from the literature,95,96 p-toluic acid (44) was oxidised with
cold chromic acid in the presence of acetic anhydride to afford diacetate 43 in 37%
yield.
Hydrolysis of the latter was achieved in boiling ethanol containing dilute
sulphuric acid and afforded aldehyde 41 91% yield.
O
excess pyrrole
HN
O
O
TFA
HN
O
OHC
40
31
allyl bromide
K2CO 3
acetone
HO
OHC
O
CO 2H
O
O
41
91%
AcO
AcO
43
O
O
NH
N
42
H2SO4 (aq)
EtOH

H2SO4, AcOH
Ac2O, CrO3,
5 °C
CO 2H
Me
37%
CO 2H
Ph
Ph
N
HN
44
O
O
34
Scheme 29
Preparation of the allyl ester 40 was attempted by alkylation of 4-formylbenzoic acid
with allyl bromide in boiling acetone using potassium carbonate as a base over 5.5
hours. After removal of solvent and salts the crude product was subjected to flash
chromatography leading to isolation of an oil which was shown from its NMR spectra
not to be the target material (40), but rather the product (42). The latter compound,
isolated in 60% yield, arose from the aldol reaction of the target material with acetone.
The 1H NMR spectrum of 42 exhibited signals consistent with the formation of the allyl
ester but lacked a formyl signal. Instead signals arising from the hydroxybutanone
chain were observed at  2.16 (3 H, s),  2.81 (2 H, d) and  3.63 (l H, br m).
The preparation of aldehyde 40 may perhaps have been successfully accomplished by
switching solvents, for example, from acetone to DMF. However, it was decided
57
instead to focus efforts on a porphyrin possessing simple allyloxyphenyl meso
substituents and abandon the allylic ester.
Attention was thus turned to the synthesis of 5,15-bis(4-allyloxyphenyl)-10,20diphenylporphyrin (47), Scheme 30. Synthesis of this porphyrin required construction
of a dipyrromethane (46) for reaction with benzaldehyde. If studies with the model
porphyrin showed promise, then the dipyrromethane might also be reacted with the
phenanthrenecarboxaldehyde (14) for construction of a NOS model (48) suitable for
surface attachment.
O
excess pyrrole
OHC
PhCHO
HN
O
TFA
propionic
acid
HN
45
NH
N
Ph
O
Ph
N
HN
46
t
CO 2Bu
Cl
O
O
CHO
47
14
O
OBu
t
ButO
O
Cl
O
O
O
N
Cl
HN
surface attachment
NH
N
O
48
Scheme 30
Dipyrromethane 46 was prepared by acid-catalysed condensation of para–
allyloxybenzaldehyde (45) with excess pyrrole in boiling toluene containing a trace
amount of TFA. After removal of the solvent the black tarry residue was subjected to
flash column chromatography to afford the target dipyrromethane (46) as a crystalline
solid in 60% yield.
58
Synthesis of the bis(4-allyloxyphenyl) substituted porphyrin (47) was accomplished
under conditions identical to those used for construction of porphyrin 32.
Thus,
reaction of benzaldehyde with dipyrromethane 46 in boiling propionic acid afforded a
black tarry material from which the target porphyrin was isolated by flash column
chromatography in 3.5% yield as a purple solid. Once again three minor porphyrin
congeners 27, 49 and 50 were also isolated, Figure 17, in 1.3%, 1.8% and 1.3% yields
respectively. These compounds were identified from their 1H NMR spectra but were
not fully characterised.
O
O
Ph
N
NH
Ph
N
N
NH
Ph
HN
Ph
27 1.3%
Ph
N
N
NH
Ph
HN
O
O
N
Ph
HN
Ph
49 1.8%
50
1.3%
Figure 17
The porphyrin preparation was repeated several times in order to obtain enough
material to carry out surface attachment studies. Yields were consistently low but
ranged from 3.5 to 7.0% for the target porphyrin (47). The minor congeners 27, 49 and
50 were also consistently formed in the reaction, although the proportions of the
isolated porphyrins varied despite following a single procedure with identical quantities
of starting materials each time. This reaction illustrates the difficulties inherent to
porphyrin synthesis — low yields with the formation of congeners that are tedious to
separate. Nonetheless, the desired porphyrin 47 was obtained in sufficient quantities for
surface attachment studies to be undertaken.
Surface attachment of porphyrin 47
In principle both routes investigated for surface fixation of the model allyl phenyl ether
might be applied for covalent surface attachment of porphyrin 47. Thus, the porphyrin
could be reacted first with (3-mercaptopropyl)trimethoxysilane in solution and the
resulting silylated porphyrin (51) then exposed to silica (Route 1, Scheme 31).
59
Ph
HN
NH
AIBN
MeCN
21
N
O
SH
S
S
Ph
51
Si(OMe) 3
Si(OMe) 3
Ph
N
O
O
N
(MeO)3Si
HN
NH
O
N
Route 1
OH OH OH OH OH OH
Ph
47
OR
SH
Route 2
SH
R=
N
HO
Si OMe
Si OMe
O OH O O OH
Ph
HN
NH
AIBN
MeCN
Route 2
Ph
N
or
Si(OMeO) 3
"perpendicular" attachment mode
"parallel" attachment mode
O
Ph
N
O
HN
NH
O
N
S
HO Si
O
OH
OHOH
HO Si
O
S
Ph
HS
S Route 1
OMe
OH
HS
S
HO Si MeO Si MeO Si OMe
OH OH OH O O
O
O O
Scheme 31
Alternatively, the silica surface might first be modified with the mercaptopropylsilanyl
group and subsequently subjected to the radical reaction with the porphyrin (Route 2,
Scheme 31). We envisaged that this latter route would be adopted for the generation of
functional NOS models with more elaborate metallated porphyrins.
Thus, the
mercaptopropyl-modified surface would be used to deliver a ligand to the metal centre,
bringing about coordinative attachment before initiating the radical reaction for covalent
fixation as shown in Scheme 19. In the case of the simple unmetallated porphyrin (47),
however, it was interesting to compare the efficacy of the two strategies, Route 1 and
Route 2, for the surface attachment.
Attempts to achieve the solution phase addition of (3-mercaptopropyl)trimethoxysilane (21) to bis(allyloxyphenyl)porphyrin 47 were made under the same conditions
used for reaction of the mercaptan with allyl phenyl ether. Thus, the porphyrin was
60
treated with 2 mole equivalents of the thiol (one for each allyl group present) and
approximately 0.1 equivalent of AIBN in boiling acetonitrile under a nitrogen
atmosphere. However, to our surprise the 1H NMR spectrum of the residue following
evaporation of the solvent indicated that the porphyrin’s allyl groups remained
completely unreacted. Repetition of the reaction with 10 equivalents of the mercaptan
and with 0.3 equivalents of AIBN added in batches also failed to give any radical
addition to the porphyrin’s allyl groups. The failure of these reactions was surprising
given the highly efficient addition of mercaptan 21 to allyl phenyl ether (22). This
result suggests that in some way the porphyrin inhibits the radical chain mechanism.
For example, if the porphyrin effectively catalysed the trapping of thiyl radicals as
disulfide then the addition reaction would not take place.
In
contrast
to
the
solution
phase
reaction,
radical
addition
of
bis(allyloxyphenyl)porphyrin 47 to a mercaptopropyl-modified silica gel surface (Route
2, Scheme 31) was highly successful. The silica surface was first silylated with (3mercaptopropyl)trimethoxysilane (21) using the procedure described previously and,
after drying in vacuo for 24 hours (0.1 mmHg, 110 °C), was then boiled in acetonitrile
with the porphyrin and trace AIBN for 6 hours. To estimate a reasonable quantity of
porphyrin to use in this experiment, molecular modelling studies were carried out to
estimate the approximate surface area required for occupancy of a single porphyrin.
This study, Figure 18, suggested length by breadth dimensions of 24.5 x 17.6 Å which
equates to an area of 431 Å2 and a maximum surface coverage of 0.3 µmol/m2 (0.1
mmol/g of silica), assuming a “parallel” mode of attachment with the allyloxy chains
fully extended (Scheme 31). To provide a reasonable margin, the reaction was carried
out with nearly ten times this quantity of porphyrin and with 0.1 equivalent of AIBN
based on the amount of porphyrin.
Thus, in a typical experiment, 200 mg of
mercaptopropyl-modified silica was reacted with 145 mg of the porphyrin. After the
reaction physisorbed material was removed by the usual thorough washing procedure
with toluene followed by dichloromethane until the filtrate was colourless.
Ph
H
NH
4.3 Å
H
O
H
N
H
N
HN
O
H
H
Ph
24.5 Å
Figure 18
61
17.6 Å
Elemental analysis was carried out on the mercaptopropyl-modified parent silica
substrate and on the porphyrin-modified material (Table 5). The former analysed for
2.64 %C which gives a silane coverage in the range of 0.95-1.63 µmol/m2 but probably
nearer to 1.4 µmol/m2 as discussed above (section 2.1.2). To our delight the porphyrinmodified material, which was a dark purple colour, analysed for 28.9 %C with a clear
N content (2.35%). The high carbon and nitrogen content of this material suggested a
surprisingly high surface density, which was rationalised by assuming that radical
addition had taken place at just one of the porphyrin’s two allyloxyphenyl substituents.
This would therefore provide a material, illustrated in Figure 19, with the porphyrin
units organised such that they stack with the plane of the porphyrin approximately
perpendicular to the surface.
Approximate formula and formula weight for pophyrin residues assuming perpendicular attachment by a
single side chain, a 1.5/1 ratio of single and double siloxane connections and loss of 60% of the residual
moethoxy groups from the silicon anchor.
C53.6H48.2N4O3.6SSi
FW = 866.130
Ph
O
Si
O
OMe
S
N
O
NH
OH
HN
N
O
Ph
Ph
OH
O
Si
S
N
O
NH
OH OMe
HN
N
O
Ph
Figure 19
Surface modified silica
%C†
%H†
%N†
Surface density (mol/m2)
Mercaptopropyl–modified silica
2.64
0.85
0
Based on
%C
1.36–1.41§
Porphyrin–modified silica
28.89
2.43
2.35
1.47*
†
§
*
Based on
%N
–
1.32*
Determined by elemental microanalysis of the surface–modified silicas.
See discussion on page 50.
Calculated assuming the attachment shown in Figure 19. Uncertainty in the mode of connection for
the silicon anchor group has very little impact on the calculated surface density in this case because of
the high formula weight of the attached residues.
Table 5: Calculated surface loading for porphyrin 47 attached to mercaptopropyl-modified Silica-I
The estimated porphyrin coverage, ignoring any unreacted surface-bound thiol chain
(which has a much lower formula weight), is 1.47 mol/m2 based on the carbon figure.
This calculated surface loading could be somewhat higher than the surface density of
62
mercaptopropyl chains on the parent silica gel which clearly should not be possible for
the attachment mode proposed in Figure 19. However, calculations based on the %N
figure give a surface density of 1.32 µmol/m2.
This latter figure may be more
representative of the porphyrin coverage. Either way, the picture that emerges from
these data is that radical addition of the porphyrin has taken place to a very high
proportion of the surface-bound thiol chains attached to the parent silica.
Although at the time we had no direct evidence to support the proposed mode of
surface attachment (Figure 19), two control reactions were carried out. These test
reactions strongly suggested that the porphyrin was covalently bound to the surface
rather than simply physisorbed to the silica. Firstly, unmodified silica gel was treated
with porphyrin 47 under the same conditions (including AIBN) used to react the
mercaptopropyl-modified silica. Elemental analysis, after washing the silica until the
filtrate was colourless, indicated no attachment of the porphyrin.
Secondly,
mercaptopropyl-modified silica was reacted with the porphyrin, but in the absence of
the radical initiator AIBN. Once again microanalysis of the resulting silica showed the
absence of attached porphyrin (0% N content). Therefore, the mercaptopropyl-modified
surface and the presence of AIBN are both necessary for attachment of the porphyrin,
thus corroborating circumstantially the proposed covalent fixation.
There are two singular features about the fixation of porphyrin 47 to the
mercaptopropyl-modified silica that require discussion.
Firstly, why does the
attachment occur at all given that the porphyrin fails to react with (3mercaptopropyl)trimethoxysilane in solution? Secondly, why is the surface density of
attached porphyrin so high? One possible solution to the first question could be that the
organisation of mercaptopropyl chains on the surface of the silica is such that the
propagation steps of the radical chain reaction, once initiated, are particularly efficient.
For example, if the carbon radical resulting from thiyl attack on the porphyrin’s allyl
chain is close to an adjacent mercapto group, then hydrogen abstraction to create an
adjacent thiyl radical might be particularly efficient as shown in Scheme 32.
63
AIBN
porphyrin
porphyrin
N2
H3 C
CN
O
H3C
H3 C
CN
O
H
H3 C
H
HS
HS
Si
OH
O O
chain
propagation
S
HS
Si
OCH 3
Si
O O
OH
O O
Si
OCH 3
Si
O O
O O
porphyrin
porphyrin
porphyrin
porphyrin
O
O
O
O
H
H
H
S
Si
O O
Si
OH
Si
OCH 3
O O
H
H
S
OH
S
S
S
S
OCH 3
Si
O O
O O
OH
Si
OCH 3
O O
Scheme 32
The second surprising feature about the porphyrin fixation is the high surface coverage
achieved.
This results contrasts with fixation of allyl phenyl ether to the
mercaptopropyl-modified silica where it was estimated that 50% of the surface-bound
mercaptan underwent addition to the allyl ether. It may be that stacking interactions
between adjacent porphyrin units together with an efficient radical chain propagation
mechanism (see Scheme 32 above) result in formation of a surface dense monolayer.
Molecular modelling studies, Figure 18, suggest a rectangular cross sectional area of
4.3 Å  17.6 Å for the porphyrin 47 if linked perpendicular to the silica surface by a
single side chain. If we allow a Van der Waals contact radius of between 1.1 and 1.5 Å
around this cross section, then the predicted area occupied by one porphyrin unit lies in
the range 130-150 Å2. This corresponds to a maximum surface coverage of 1.1-1.3
mol/m2.
Though a gross approximation, it is interesting that porphyrin coverage
estimated from the observed nitrogen percentage falls within this range.
64
One possibility that the studies described above have not excluded is that a degree of
radical olefin polymerisation, see Scheme 33, may have occurred so that the porphyrin
is not actually attached in a strict monolayer.
porphyrin
porphyrin
porphyrin
porphyrin
porphyrin
porphyrin
H atom source O
H
O
O
O
O
O
n
n
S
S
Si
OH
O O
Si
OH
O O
Scheme 33
We needed to corroborate our hypothesis that attachment had proceeded with formation
of a single layer of vertically aligned porphyrin units on the silylated silica surface. To
do so attempts were made to etch the silica with 48% hydrofluoric acid. Unfortunately,
only a very small amount of organic material was recovered by chloroform extraction
and the material obtained exhibited a featureless 1H NMR spectrum. It may be that the
hydrofluoric acid did not simply cleave the surface siloxane bonds, but released
porphyrin units with variable amounts of polysiloxane clusters still attached. Therefore,
to probe the mode of attachment for the non-metallated porphyrins it was planned to
carry out solid-state NMR studies with
13
C labelled porphyrins and these are described
in section 2.1.6.
Surface attachment studies with the zinc(II) complex of porphyrin 47
The strategy planned for surface attachment of our NOS models involves simultaneous
covalent and coordinative fixation. Thus, exposure of the mercaptopropyl-modified
silica to a solution of the metalloporphyrin is intended to bring about coordination of the
porphyrin in the horizontal mode and then allow subsequent radical-mediated covalent
attachment. It was decided to test this strategy using the zinc(II) derivative (52) of
bis(allyloxyphenyl)porphyrin 47.
65
Metallation of bis(allyloxyphenyl)porphyrin 47 was accomplished with zinc acetate in
boiling dichloromethane and proceeded quantitatively in ten minutes.
The zinc
porphyrin 52 was then reacted with mercaptopropyl-modified silica (Scheme 34).
However, the zinc porphyrin was only available in small quantities and it was necessary
to carry out the attachment reaction with a lower porphyrin to silica ratio than had been
used for the non-metallated parent porphyrin.
Ph
N
O
Approximate formula and formula weight for porphyrin
residues coordinatively attached with two covalently linked
tethers:
HN
NH
O
N
C60.9H64.6N4O6.8S3Si3Zn 1207.23
Ph
The formula is calculated assuming that the silicon anchoring
group is connected by modes involving single and double
siloxane linkages in 1.5/1 ratio with replacement of 60% of
residual methoxy groups by hydroxyls.
47
Ph
Zn(OAc)2
DCM, 
N
O
N
Zn
N
O
N
Ph
52
i. mercaptopropyl-modified silca
ii. AIBN, MeCN, 
Ph
Ph
N
O
N
S
HO Si
O
Zn
N
HO Si
OH OH
O
N
S
S Ph
OH
N
O
O
N
OMe
OH
OH
HO Si
O
Zn
N
S Ph
HO Si
OH
OH OH
O
O
N
S
OMe
HO Si
OH OH OH
O O
Scheme 34
In the study with the non-metallated porphyrin, 200 mg of the mercaptopropylmodified silica had been treated with 145 mg of the porphyrin. This afforded a silica
with a calculated surface coverage of 1.3 mol of porphyrin per m2, corresponding to
the attachment of 94 mg of porphyrin. The mercaptopropyl-modified silica substrate for
reaction of the zinc porphyrin was prepared by treating silica with (3mercaptopropyl)trimethoxysilane (2.1 mol per m2 of silica surface) in hot toluene
under the conditions used previously, washing and drying in the normal manner. The
porphyrin-attachment reaction was carried out by treating 200 mg of the resulting
modified silica with 58 mg of the zinc-porphyrin (52) and catalytic AIBN in boiling
acetonitrile. The silica was recovered, washed and dried in the normal way and then
66
submitted for elemental analysis together with a sample of the mercaptopropyl-modified
silica substrate, Table 6.
%C†
Surface modified silica
%H†
%N†
Surface density (mol/m2)
Based on
%C
Based on
%N
Mercaptopropyl–modified silica
3.00
0.93
0
1.56–1.62§
–
Porphyrin–modified silica
5.99
1.10
0.17
0.18*
0.063*
†
§
*
Determined by elemental microanalysis of the surface-modified silicas.
The maximum and minimum surface density limits for mercaptopropyl chains on this silica are 1.09
and 1.88 µmol/m2 (calculated assuming attachment exclusively by the type A and type E modes
shown in Figure 15). Solid-state NMR data discussed in section 2.1.6 suggest that attachment is likely
by a mixture of modes involving one and two siloxane bonds in 1.5/1 ratio with replacement of 60%
of residual methoxy groups by hydroxyls. The range quoted in the table assumes this latter situation.
Calculated assuming the triply-tethered attachment shown in Scheme 34. Uncertainty in the mode of
connection for the silicon anchor group has very little impact on the calculated surface density in this
case because of the high formula weight of the attached residues. The values quoted are for
calculations that do not take into account the presence of residual unreacted mercaptopropyl chains .
Table 6: Calculated surface loading for zinc(II) porphyrin 52 attached to mercaptopropyl-modified
silica.
The porphyrin-modified silica analysed for 5.99 %C and 0.17% N, indicating that the
surface loading with the metallated porphyrin was substantially lower that with the nonmetallated porphyrin (47). This outcome is consistent with a horizontal rather than
perpendicular attachment mode for the porphyrin. Evaluation of the porphyrin loading
based on the %N content of the sample, and ignoring any unreacted mercaptopropyl
units suggested a surface density of 0.063 mol/m2. The latter value was obtained
assuming that the porphyrin units are attached exclusively by the triply-tethered mode
shown in Scheme 34.
Ignoring residual unreacted mercaptopropyl silane units
introduces only small errors into the calculation because of their low formula weight
compared to the porphyrin-containing units. For example, the presence of these chains
at the level of 1.60 µmol/m2 on the silica alters the calculated surface density of
porphyrin units from 0.063 µmol/m2 to 0.070 µmol/m2. The small increase in porphyrin
coverage seen here is caused by a reduction in the estimated silica surface area to which
the porphyrin is attached. Thus, increasing the amount of mercaptopropylsilanyl units
in the surface-modified material reduces the mass proportion (and associated surface
area) of the core silica itself in the material.
Evaluation of the porphyrin loading using the %C figure from the microanalysis
without taking into account residual unreacted mercaptopropyl silane units gives a value
of 0.18 µmol/m2 (Table 6) for the surface density of the metalloporphyrin units, again
67
assuming that these are attached exclusively by coordinative linkage with two covalent
tethers. A more complicated calculation to take into account residual mercaptopropyl
silane units that also fits surface loading levels to both the %N and %C figures was
made by setting up a Microsoft Excel worksheet. As previously noted, the precise
attachment of the silanyl units to the surface is subject to some uncertainty in the
number of siloxane bonds formed and the degree to which residual methoxy groups
have been lost. This uncertainty has very little impact on calculated surface loadings for
the high formula weight porphyrin residues but is problematic for less massive residues
such as the mercaptopropylsilanyl units.
Figure 15 illustrates that the limiting
maximum and minimum values for the surface loading can be calculated by assuming
exclusive attachment through modes A and E respectively. Table 7 presents calculated
surface loading levels of mercaptopropylsilanyl and porphyrin units with these limiting
attachment regimes that are required to give the %C and %N figures observed in the
microanalytical data for the silica modified with metalloporphyrin 52. The various
attachment modes and associated formulae that form the basis for these calculations are
illustrated in Figure 20. In addition to the two limiting attachment modes (modes I and
II) for the silicon anchor group a third, intermediate attachment regime (mode III) has
been included. This latter regime reflects solid-state NMR data (discussed in section
2.1.6) for one sample of mercaptopropyl-modified silica that suggested attachment is
likely by a mixture of modes involving one and two siloxane bonds in 1.5/1 ratio with
replacement of 60% of residual methoxy groups by hydroxyls. The surface loadings
shown in Table 7 are calculated where the porphyrin units are assumed to be attached
exclusively with a coordinative linkage and two covalent tethers (modes I-III) and by
coordinative linkage with a single covalent tether (modes IV-VI).
68
Ph
N
O
Zn
N
S
Si
O
Ph
HS
N
O
HS
S
RO Si
RO
O
RO Si
RO
O
S Ph
OR
RO
OR RO Si
O
Si
OR
OR
O
N
O
N
N
SH
S
Zn
N
O
N
HS
S Ph
OR
Si OR RO Si
RO
O
O
OR
Si OR
Si
OR
O OR O
SH
Si OR
OR
O
Attachment Mode I (R = Me)
formula / FW for porphyrin unit:
C65H71N4O8S3Si3Zn
1282.13
Attachment Mode IV (R = Me)
formula / FW for porphyrin unit:
C60H59N4O6S2Si2Zn
1117.83
formula / FW for residual mercaptopropylsilanyl unit:
C5H12O2SSi
164.299
Attachment Mode II (R = H)
formula / FW for porphyrin unit:
C59H59N4O8S3Si3Zn
1197.97
formula / FW for residual mercaptopropylsilanyl unit:
C5H12O2SSi
164.299
Attachment Mode V (R = H)
formula / FW for porphyrin unit:
C59H51N4O6S2Si2Zn
1061.72
formula / FW for residual mercaptopropylsilanyl unit:
C3H8O2SSi
136.246
formula / FW for residual mercaptopropylsilanyl unit:
C3H8O2SSi
136.246
Ph
N
O
N
S
HS
OR
Si
OR
O O
Zn
Ph
N
O
N
N
HS
S Ph
RO
OR
RO Si
OR O O
O O
Si
N
O
S
S
RO
RO
Si
Si OR
OR
OO
O O
HS
Zn
N
O
N
S Ph
HS
HS
RO
RO
RO
Si
RO Si
Si
Si OR Si
OR
OR
O O OR O O OR
OO
O O OR O O
Attachment Mode III (R = H / Me)
formula / FW for porphyrin unit:
C60.9H60.3N4O6.8S3Si3Zn
1202.90
Attachment Mode VI (R = H / Me)
formula / FW for porphyrin unit:
C57.3H50.9N4O5.2S2Si2Zn
1064.43
formula / FW for residual mercaptopropylsilanyl unit:
C3.6H8.4O1.6SSi
137.726
formula / FW for residual mercaptopropylsilanyl unit:
C3.6H8.4O1.6SSi
137.726
Figure 20
Surface loadings of porphyrin-containing and residual mercaptopropylsilanyl
units required to fit observed microanalytical data (5.99 %C, 0.17 %N) for the
surface attachment of porphyrin 52 to mercaptopropyl-modified silica
Entry
Attachment mode
assumed in calculation†
Porphyrin units
(µmol/m2)
Residual mercaptan units
(µmol/m2)
1
Mode I
0.070
1.40
2
Mode II
0.074
2.60
3
Mode III
0.072
2.06
4
Mode IV
0.070
1.47
5
Mode V
0.074
2.68
6
Mode VI
0.072
2.12
† Calculated assuming the attachment modes (I-VI) shown in Figure 20. Solid-state NMR data
discussed in section 2.1.6 suggest that attachment is likely by a mixture of modes involving one and
two siloxane bonds in 1.5/1 ratio with replacement of 60% of residual methoxy groups by hydroxyls;
attachment modes III and VI assume this composite attachment regime.
Table 7:Calculated surface loading for zinc(II) porphyrin 52 attached to mercaptopropyl-modified
silica taking into account residual unreacted mercaptopropyl residues.
69
The surface loadings shown in Table 7 illustrate that differences in attachment mode
for the silicon anchoring groups and the formation of two covalent tethers or just a
single tether have little impact on the calculated surface coverage for the porphyrin —
porphyrin surface density lies in the range 70-74 nmol/m2. On the other hand, levels of
residual mercaptopropyl units are heavily dependent on the assumed attachment mode,
varying from 1.40 to 2.68 µmol/m2.
Calculated minimum and maximum surface
loading of these residues in the parent mercaptopropyl-modified silica used as the
substrate for attachment porphyrin 52 were 1.09 and 1.88 µmol/m2, Table 6.
Unfortunately the microanalytical studies do not permit conclusive definition of the
mode of porphyrin attachment and at this point in the project, therefore, it was unclear
to what extent the double covalent and simultaneous coordinative linkages had formed.
Indeed, it was not even certain that the surface density of mercaptopropyl groups on the
silica substrate was sufficient to permit this mode of attachment. It was of interest,
therefore, to examine the surface attachment of a second porphyrin model possessing
longer alkenyl substituents.
Therefore, we chose to study 5,15-bis[4-(pent-4-
enyloxy)phenyl]-10,20-diphenylporphyrin 56, with a plan to probe the attachment mode
by solid-state NMR studies on 13C labelled material.
2.1.4 Model surface attachment studies with 4-(pent-4-enyloxy)benzaldehyde
To preface the planned work with 5,15-bis[4-(pent-4-enyloxy)phenyl]-10,20diphenylporphyrin (56), and to avoid losing synthetically expensive material on nonviable chemistry, the covalent attachment strategy was again probed using a simplified
compound containing a chain of the appropriate length.
This time 4-(pent-4-
enyloxy)benzaldehyde (53) was selected for the studies because the same compound
could potentially be used as a building block en route to the model porphyrin (56). As
before, two routes for surface attachment could be studied: firstly, by carrying out the
radical addition in solution and subsequently reacting the resulting silane (52) with
silica
gel
(Route
1,
Scheme
35),
and
secondly,
by
first
reacting
(3-
mercaptopropyl)trimethoxysilane (21) with silica and then carrying out the radical
addition at the surface of the mercaptopropyl-modified silica (Route 2, Scheme 35). As
in previous studies with allyl phenyl ether Route 1 was studied in the first instance,
requiring preparation of silane 54.
70
O
CHO
53
Si(OMe) 3
HS
O
(MeO)3Si
AIBN, MeCN
84%
21
CHO
S
54
Route 1
silica, toluene

Table 8
OHC
silica, toluene

Route 2
O
O
SH
CHO
S
53
(OH)
MeO Si
O O
AIBN, MeCN
MeO Si OMe
(OH)
O O (OH)
OMe
(OH)
Scheme 35
Preparation of silane 54 was achieved following the procedure previously described
for the synthesis of [3-(3-phenoxypropylsulfanyl)propyl]trimethoxysilane (23).
A
solution of 4-(pent-4-enyloxy)benzaldehyde, (3-mercaptopropyl)trimethoxysilane and
AIBN (trace) in acetonitrile under nitrogen was heated at 85 °C for 6 hours. The
solvent and unreacted volatile materials were evaporated affording the target silane (54)
in 84% yield and greater than 95% purity according to its 1H NMR spectrum. The latter
exhibited resolved signals for the methylene  to the Si atom and for the methoxy
groups at H 0.62 (t, J 8.2) and H 3.43 (s) respectively. Signals for the methylenes  to
the Si and ,  and  to the ether O were unresolved as an overlapping multiplet (8H, H
1.4-1.6). The two methylenes adjacent to the sulfanyl centre and  to the ether O were
observed at H 2.4 (4H, 2  t overlapping, J 7.2) and H 3.90 (2H, t, J 6.3) respectively.
Having synthesised silane 54 successfully, surface attachment studies could be
instigated. Following the same procedure as the one described for attachment of [3-(3phenoxypropylsulfanyl)propyl]trimethoxysilane (23), silane 54 and silica were heated in
toluene overnight (2.1 mol/m2). After washing and drying (0.1 mmHg, 110 ºC, 24
hours) the resulting modified silica was submitted for elemental analysis. The calculated
surface density of silane 54 per m2 of silica was found to be comparable to the surface
density of silane 23 in Experiments 1-8, Tables 3 and 4. As before, the calculated
surface loading is subject to uncertainty in regards to the attachment mode for the
silicon anchor group.
71
Several modes are possible, Figure 21, and calculations made assuming grafting
exclusively by type A and type E attachment modes provides the limiting values of 0.90
and 1.03 µmol/m2 for surface coverage. Uncertainty in regards to the attachment mode,
then, has little impact on the calculated surface loading because of the reasonably high
formula weight of the attached residues. When the calculation is fitted to solid-state
NMR data for silica grafted with (3-mercaptopropyl)trimethoxysilane (discussed in
section 2.1.6) the calculated surface density is 0.98 µmol/m2.
Formulae, formula weights and calculated surface density for organosilane residues attached
2
exclusively by the various modes indicated on silica gel of surface area 500 m /g and analysing
for 7.92% carbon content.
attachment with single silaxane linkage
attachment with double siloxane linkage
Formula
Surface density
(mol/m2)
–––––––––––––––––––––––––––––––––––––––––––
C 17H27O4SSi
355.545
0.90
C16H25O4SSi
341.519
0.96
C 15H23O4SSi
327.492
1.03
C 16H24O3SSi
324.511
0.95
C15H22O3SSi
310.485
1.02
attachment with triple siloxane linkage
Ar =
CHO
FW
Mode A
OAr
Mode D
OAr
Mode E
OAr
Mode B
OAr
Mode F
OAr
S
S
S
S
S
MeO Si OMe
O
HO
Si OMe
HO
O
Si
Si OH
OMe
Si
OH
O O
O
O
C 15H21O2SSi
C15H21O2.5SSi
C 15H22O3.5SSi
C 16H24O3.5SSi
O
293.478
301.482
318.490
332.417
Mode C
OAr
OAr
OAr
OAr
OAr
OAr
S
S
S
S
S
S
Si
O O O
O
Si O Si OH
O
O O
HO
Figure 21
72
Si
O
O
Si
O
O
Si OMe
O
1.01
1.01
1.02
0.96
Silica
Microanalytical data
Surface density
(mol/m2)
Modified with silane 54 (2.1 mol/m2)
%C
%H
based on %C
7.92
1.28
0.98*
* The maximum and minimum surface density limits for organic residues on this silica are 0.90 and 1.03
µmol/m2 (calculated assuming attachment exclusively by the type A and type E modes shown in
Figure 21). Uncertainty in the mode of connection for the silicon anchor group has little impact on the
calculated surface density in this case because of the reasonably high formula weight of the attached
residues. Solid-state NMR data discussed in section 2.1.6 suggest that attachment is likely by a
mixture of modes involving one and two siloxane bonds in 1.5/1 ratio with replacement of 60% of
residual methoxy groups by hydroxyls. The figure quoted in the table assumes this latter situation.
Table 8: Calculated surface loading for silane 54 attached to silica
The second route (Route 2, Scheme 35) was next explored. Thus, silica gel was first
modified with (3-mercaptopropyl)trimethoxysilane (21) in boiling toluene using 2.1
mol of 21 per m2 of substrate silica surface. The resulting modified silica was washed
and
dried
under
standard
conditions.
Surface
attachment
of
4-(pent-4-
enyloxy)benzaldehyde (53) — 2.1 mol/m2 of core silica — was undertaken in
acetonitrile in the presence of trace AIBN, washing and drying as previously. Samples
of each mercaptan 21- and silane 53-modified silica were submitted for elemental
analysis, and the results are summarised in Table 9.
Surface-modified silica
Microanalytical data
Surface density
(mol/m2)
%C
%H
based on %C
Mercaptopropyl-modified silica
2.88
0.93
1.49-1.55*
Alkene 53-modified silica
7.53
1.27
0.78†
* The maximum and minimum surface density limits for mercaptopropyl chains on this silica are 1.04
and 1.79 µmol/m2 (calculated assuming attachment exclusively by the type A and type E modes
shown in Figure 21). Solid-state NMR data discussed in section 2.1.6 suggest that attachment is likely
by a mixture of modes involving one and two siloxane bonds in 1.5/1 ratio with replacement of 60%
of residual methoxy groups by hydroxyls. The range quoted in the table assumes this latter situation.
†
Calculated assuming attachment by a mixture of modes involving one and two siloxane bonds in 1.5/1
ratio with replacement of 60% of residual methoxy groups by hydroxyls and starting from a surface
density of 1.51 µmol/m2 for the mercaptan residues on the parent mercaptopropyl-modified silica.
Table 9: Calculated surface loading for silane 53 attached to mercaptopropyl-modified silica
Surface loading calculations for the parent mercaptopropyl-modified silica prior to the
radical addition step with alkene 53 give a limiting range of 1.04-1.79 µmol/m2 for the
grafted mercaptopropylsilanyl units. When the calculation is fitted to solid-state NMR
data (discussed in section 2.1.6) the calculated surface density lies in the range 1.4973
1.55 µmol/m2. Following radical addition of the alkene, the calculated surface loading
of residues containing the benzaldehyde unit is 0.78 µmol/m2. This calculation is based
on the assumption that the surface density of mercaptopropyl units (taken as 1.51
µmol/m2) for the parent silica is valid.
These figures suggest that some 53% of
mercaptan chains in the parent mercaptopropyl-modified silica have undergone the
radical addition process, a result which is very similar to the outcome for the reaction of
allyl phenyl ether with the mercaptopropyl-modified silica. These studies suggested,
therefore, that a simple model porphyrin possessing p-(pent-4-enyloxy)phenyl groups in
the 5- and 15-positions and phenyl rings in the 10- and 20-positions ought to participate
effectively in the radical-mediated surface attachment process.
2.1.5 Surface attachment of the pentenyloxyphenyl-substituted porphyrin (56)
The synthesis of porphyrin 56, Scheme 36, was accomplished following the procedures
previously described for bis(allyloxyphenyl)porphyrin 47 and in comparable yield.
Thus,
[4-(pent-4-enyloxy)phenyl]dipyrromethane
(55)
was
condensed
benzaldehyde in hot propionic acid to afford the porphyrin in 2.3% yield.
with
The
dipyrromethane was obtained in 60% yield by condensation of 4-(pent-4enyloxy)benzaldehyde (53) with excess pyrrole in the presence of catalytic
trifluoroacetic acid.
OHC
excess pyrrole
HN
TFA
HN
O
53
O
55
PhCHO
CH3CH2CO2H

Ph
N
O
HN
NH
N
O
Ph
56
Zn(OAc)2
DCM, 
Ph
N
O
N
Zn
N
N
Ph
Scheme 36
74
57
O
For consistency, one attempt was made to carry out the radical addition between the
olefinic side chains of porphyrin 56 and mercaptan 21 in solution. Not surprisingly,
given the outcome with the allyloxyphenyl substituted porphyrin (47), this reaction was
unsuccessful, returning the porphyrin unreacted. Attention was then turned to the
radical addition using mercaptopropyl-modified silica.
For comparative purposes,
surface attachment studies were undertaken both with the free porphyrin and its zinc(II)
derivative (57). These studies were carried out under identical conditions to those
established for the bis(allyloxyphenyl)porphyrin (47) with the exception that the
mercaptopropyl-modified silica substrate was prepared using a higher mass ratio of (3mercaptopropyl)trimethoxysilane to silica gel (5.1 µmol per m2 of substrate silica
surface).
It was thought that increasing the silane-to-silica ratio, and thereby the
mercaptopropyl loading, would allow a greater porphyrin surface density to be achieved
in the radical addition step. To check for homogeneity four separate samples were
withdrawn from the batch of mercaptopropyl-modified silica and subjected to
microanalysis, Table 10.
Mercaptopropyl-modified silica
Entry
Sample No
Microanalytical data
%C
Surface density (mol/m2)
Limiting range*
NMR fitted†
1
A
4.05
1.51-2.66
2.19-2.28
2
B
4.41
1.67-2.94
2.41-2.52
3
C
4.59
1.75-3.08
2.53-2.64
4
D
4.82
1.85-3.27
2.68-2.79
5
mean
4.47
1.70-2.99
2.45-2.56
* The maximum and minimum surface density limits for mercaptopropyl chains on this silica are
calculated by assuming, respectively, attachment exclusively through the type A and type E modes
shown in Figure 21.
† Solid-state NMR data discussed in section 2.1.6 for a sample of silica gel grafted with
mercaptopropylsilanyl units suggest that attachment is likely by a mixture of modes involving one and
two siloxane bonds in 1.5/1 ratio with replacement of 60% of residual methoxy groups by hydroxyls.
The range quoted in the last column of this table assumes this latter situation
Table 10: Calculated surface loading for the mercaptopropyl-modified silica used in the attachment
studies with porphyrin 56.
The microanalysis results obtained showed significant variation in the percentage
carbon value (4.05-4.82 %C) and gave a calculated mean surface coverage of silane
units within the limiting range 1.70-2.99 µmol/m2 (entry 5). When fitted to solid-state
NMR data for a sample of silica gel grafted with mercaptopropylsilanyl units (section
75
2.1.6) the calculated silane loading lies in the range 2.45-2.56 µmol/m2. The surface
density of mercaptopropyl groups, at about 2.49 µmol/m2, was approximately double
that in the silica substrate used for attachment of bis(allyloxyphenyl)porphyrin 47.
Fixation of the non-metallated bis(pentenyloxyphenyl)porphyrin (56; 0.13 mmol)† was
undertaken with 200 mg of the mercaptopropyl-modified silica. After the standard
reaction conditions, clean up and drying procedure the resulting porphyrin-modified
silica was subjected to microanalysis. Again four samples from the same batch of silica
were withdrawn to check for homogeneity.
Nitrogen and carbon contents for the
porphyrin-modifed silica were found respectively in the ranges 1.43-1.62% and 21.0221.47%, Table 11, with mean values of 1.55 %N and 21.24 %C. The carbon content
was
noticeably
lower
than
found
for
the
silica
modified
with
bis(allyloxyphenyl)porphyrin 47, Table 5, suggesting that a significant proportion of the
mercaptan residues in the parent mercaptopropyl-modified silica did not undergo the
radical addition. It was unclear to what extent the porphyrin units might be attached
through the radical addition having occurred at both of the porphyrin’s alkenyl side
chains.
With the corresponding reaction of bis(allyloxyphenyl)porphyrin 47, the
doubly-tethered parallel attachment mode (Scheme 31) was assumed to make a
negligible contribution because of the very high surface density of porphyrin units in the
resulting silica, requiring a singly-tethered perpendicular attachment mode, Figure 19.
Given the lower loading of the pentenyloxyphenyl-substituted porphyrin, surface
density calculations were made for several possible attachment regimes, modes I-VI of
Figure 22, assuming radical addition to one or both of the pentenyl chains of the
porphyrin substrate with alternative connection modes for the anchoring silicon atom of
the mercaptopropyl chain.
†
The corresponding reaction with the bis(allyloxyphenyl)porphyrin 47 had been carried out with 0.20
mmol on 200 mg of mercaptopropyl–modified silica.
76
O
N
Ph
HN
NH
Ph
N
Ph
O
N
O
HN
NH
S
HS
S
RO
O
Si RO Si
O O
O
RO Si
OR
OH
Attachment Mode I (R = Me)
S
Ph
HS
RO Si OR
O
N
O
OR
OH
OH
RO
Si
O O
Attachment Mode IV (R = Me)
formula / FW for porphyrin unit:
C59H58N4O4SSi
947.265
formula / FW for residual mercaptopropylsilanyl unit:
C5H12O2SSi
164.299
formula / FW for porphyrin unit:
C64H70N4O6S2Si2
1111.567
formula / FW for residual mercaptopropylsilanyl unit:
C5H12O2SSi
164.299
Attachment Mode II (R = H)
Attachment Mode V (R = H)
formula / FW for porphyrin unit:
C57H54N4O4SSi
919.212
formula / FW for porphyrin unit:
C60H62N4O6S2Si2
1055.461
formula / FW for residual mercaptopropylsilanyl unit:
C3H8O2SSi
136.246
formula / FW for residual mercaptopropylsilanyl unit:
C3H8O2SSi
136.246
Attachment Mode VI
Attachment Mode III
Perpendicular singly-tethered mode as above but
with anchoring silicon atom attached by a mixture of
single and double siloxane linkages and with 60%
of residual methoxy groups replaced by hydroxyls
formula / FW for porphyrin unit:
C57.6H54.4N4O5.6SSiZn
Parallel doubly-tethered mode as above but with
anchoring silicon atom attached by a mixture of
single and double siloxane linkages and with 60%
of residual methoxy groups replaced by hydroxyls
formula / FW for porphyrin unit:
C61.2H62.8N4O5.2S2Si2
1057.878
920.422
formula / FW for residual mercaptopropylsilanyl unit:
C3.6H8.4O1.6SSi
137.726
formula / FW for residual mercaptopropylsilanyl unit:
C3.6H8.4O1.6SSi
137.726
Figure 22
The silane attachment modes chosen for the basis of these calculations were selected,
as with earlier calculations, because they provide the maximum and minimum limits for
possible surface loading. Thus, modes I and IV give the lower limit for attachment with
singly- and doubly-tethered porphyrin; modes II and V give the upper limit for singlyand doubly-tethered porphyrin. Modes III and VI assume a mixture of attachment
modes for the anchoring silicon atom with a 1.5/1 ratio of attachments by single and
77
double siloxane linkages and hydrolysis of 60% of residual methoxy groups.
Calculations were performed to fit surface loadings of porphyrin units and residual
mercaptopropyl chains to %C and %N figures for each of the four porphyrin 56modified silica samples (A-D) that were separately analysed as well as to the mean of
%C and %N values. In each case the calculation was repeated for the six hypothetical
attachment models, modes I-VI of Figure 22. The results are summarised in Table 11.
Porphyrin units (µmol/m2)
Residual mercaptopropyl
chains (µmol/m2)
Porphyrin units (µmol/m2)
Residual mercaptopropyl
chains (µmol/m2)
Porphyrin units (µmol/m2)
Residual mercaptopropyl
chains (µmol/m2)
Mean†
Porphyrin units (µmol/m2)
Sample D
Residual mercaptopropyl
chains (µmol/m2)
Sample C
Calculation*
Porphyrin units (µmol/m2)
Sample B
ATTACHMENT MODE ASSUMED IN THE
Sample A
I
0.75
1.44
0.80
0.64
0.81
0.68
0.83
0.47
0.80
0.79
II
0.81
3.12
0.83
1.67
0.85
1.75
0.86
1.34
0.84
1.94
III
0.78
2.35
0.81
1.21
0.83
1.27
0.84
0.98
0.82
1.42
IV
0.75
0.69
0.81
0
0.82
0
0.85
0
0.80
0
V
0.81
2.31
0.83
0.84
0.85
0.89
0.86
0.53
0.84
1.10
VI
0.78
1.58
0.81
0.40
0.83
0.44
0.84
0.14
0.82
0.61
1.43
21.07
1.56
21.39
1.58
21.47
1.62
21.24
1.55
%C
%N
%C
%N
%C
%N
%C
%N
%C
%N
Residual mercaptopropyl
chains (µmol/m2)
21.02
* The attachment modes used as the basis for the calculations in this table are illustrated in Figure 22.
† Calculations based on the mean %C and %N values averaged across samples A-D.
Table 11:Calculated surface loading for porphyrin 56-modified silica for four separate samples
withdrawn from a common batch.
The figures in Table 11 show that the calculated porphyrin coverage is little affected
by the assumption of a singly-tethered or doubly-tethered attachment regime. The
assumption of different attachment modes for the anchoring silicon atom also has little
impact on the calculated porphyrin loading but does, of course, significantly affect the
estimated loading of the low formula weight residual mercaptopropyl chains.
Calculated surface loadings made using the mean microanalysis figures indicate that the
surface density of porphyrin units is close to 0.82 µmol/m2 and, assuming an
exclusively singly-tethered perpendicular attachment (Mode III), gives a surface
coverage of 1.42 µmol/m2 for unreacted mercaptopropylsilanyl residues. The total
78
loading of surface-attached residues (both porphyrin and residual mercaptopropyl
chains) is 2.24 µmol/m2 in this instance, which correlates reasonably well with the
calculated surface density of chains in the parent mercaptopropyl-modified silica at
about 2.49 µmol/m2, Table 10.
The coverage obtained with the bis(pentenyloxyphenyl)porphyrin (56) was, therefore,
rather lower than that obtained with the bis(allyloxyphenyl)porphyrin (47), Table 5,
regardless of the greater quantity of thiol sites present at the surface of the parent
mercaptopropyl-modified silica used for the attachment reaction. The lower loading of
porphyrin 56 may be due to the greater chain length conferring additional flexibility and
perhaps lower packing efficiency for the porphyrin units. However, the attachment
reaction was also undertaken with a somewhat lower porphyrin to silica ratio than used
for the allyloxyphenyl-substituted porphyrin.
Metallation of porphyrin 56 was accomplished as before, using zinc acetate in hot
dichloromethane. The silica substrate for fixation of the resulting zinc porphyrin 57
was
prepared
by
the
usual
treatment
of
dried
silica
gel
with
(mercaptopropyl)trimethoxysilane (2.1 mol/m2 of substrate silica surface) in hot
toluene.
After the standard clean up and drying procedure the resulting
mercaptopropyl-modified silica analysed for 2.80% carbon content, corresponding to a
surface coverage within the limiting range of 1.01-1.74* µmol/m2 but probably close to
1.47† µmol/m2. In order to demonstrate definitively that the presence of a metal center
in the porphyrin alters the attachment mode, this silica was divided into two batches of
200 mg and then, under identical reaction conditions, one batch was reacted with 0.12
mmol of zinc porphyrin 57 whilst the other was treated with 0.12 mmol of the nonmetallated parent porphyrin 56. The two batches of porphyrin-modified silicas resulting
from this parallel experiment analysed respectively for 5.49 %C / 0.00 %N and 21.07
%C / 1.43 %N. The latter result is clearly very similar to the microanalytical data
summarised in Table 11 and demonstrates the reproducibility for attachment of the nonmetallated porphyrin.
The calculated porphyrin surface coverage, using the same
procedure adopted for the data in Table 11 and assuming a type III attachment mode is
0.78 µmol/m2. Interestingly this loading is very close to that issuing from the earlier
experiment even though the loading of mercaptan units (~2.49 µmol/m2) in the parent
*
†
Calculated using the limiting attachment modes A and E illustrated in Figure 15.
Calculated assuming that the anchoring silicon atom is attached by a range of modes with a 1.5/1 ratio
of single to double siloxane linked forms and loss of 60% of residual methoxy groups.
79
mercaptopropyl-modified silica used for that experiment was substantially higher than
here (~1.47 µmol/m2).
The silica modified with the metallated porphyrin (57) analysed for a much lower
carbon
content,
consistent
with
the
result
obtained
with
the
zinc
bis(allyloxyphenyl)porphyrin (52), Table 6, and consistent with a switch in the
porphyrin’s attachment from a substantially perpendicular mode to a parallel mode.
Thus, porphyrin attached parallel to the silica surface through initial coordination
followed by covalent fixation, Scheme 37, occupies a much greater area on the surface.
Ph
O
N
HN
NH
N
O
Approximate formula and formula weight for porphyrin residues
coordinatively attached with two covalently linked tethers:
Ph
C64.9H68.3N4O6.8S3Si3Zn
56
Zn(OAc)2
DCM, 
The formula is calculated assuming that the silicon anchoring group
is connected by modes involving single and double siloxane
linkages in 1.5/1 ratio with replacement of 60% of residual methoxy
groups by hydroxyl
Ph
O
N
N
Zn
N
N
1258.51
O
Ph
Ph
N
O
HN
NH
57
O
N
S
S
Ph
HS
1. mercaptopropyl-modified silica
2. AIBN, MeCN, 
HO Si
O
OH
MeO Si
OH
O
OMe
OH
OH
HO
Si
O O
Scheme 37
The failure to detect any nitrogen in the zinc porphyrin 57-modified silica was not
unduly disturbing; the silica sample had remained purple after thorough washing and
the %N figure expected was very low (of the order of 0.1 or 0.2%). Moreover, the silica
showed a clear increase in carbon content from 2.80% for the parent mercaptopropylmodified silica to 5.49%. Evaluation of the porphyrin loading based purely on the
carbon content, assuming an initial mercaptopropyl coverage of 1.47 µmol/m2 in the
silica substrate and the attachment mode shown in Scheme 37, suggests a porphyrin
loading of 0.085 µmol/m2. This value is a little lower than the surface loading of zinc
bis(allyloxyphenyl)porphyrin 52, at 0.10 µmol/m2, calculated in the same way from the
%C figures for the porphyrin-modified silica (5.99 %C) and its parent mercaptopropyl-
80
modified silica (3.00 %C). This result may reflect the larger area occupied by
porphyrin 57.
These studies, though informative, did not conclusively show that the dual
coordinative and the doubly-tethered attachment strategy had been achieved. The
loadings of metalloporphyrins 52 and 57 were substantially lower than that of the nonmetallated parent porphyrins, consistent with the coordinative bonding between the
metal and surface bound thiols, but this was not tangible proof that the radical addition
between the double bond of the two porphyrin side chains and the surface-bound thiols
had occurred concomitantly. To definitively probe the mode of porphyrin attachment on
mercaptopropyl-modified silica, solid-state NMR studies were undertaken with a
porphyrin labelled with 13C on the terminal olefinic centre.
2.1.6 Solid-state NMR studies
Standard pulsed Fourier Transform NMR techniques commonly used for soluble
samples can not be used for solid samples because of the low mobility of the latter. In
solution, the rapid motion of the molecules averages the local magnetic field created by
the applied magnetic field and chemical shift anisotropy to 0. To increase homogeneity
the sample is rotated at approximately 15 Hz and, as a result, sharp signals can be
observed.97 In contrast, the absence of movement in the solid phase creates a great deal
of line broadening due to magnetic dipole-dipole interactions and chemical-shift
anisotropy. Nevertheless, research has allowed these difficulties to be partly overcome
and techniques are now available for reducing the linewidths of solid samples. To
average the dipolar and chemical shift anisotropies to zero, the solid sample is rotated at
an angle to the applied magnetic field. This angle, called the “magic angle” is equal to
54.74º and the technique is known as magic angle spinning (MAS) spectroscopy.98 The
difficulty with MAS spectroscopy is that the required spinning frequency of the solid
sample depends on the width of the resonance, which is of the order of the kHz.
However, gas-driven sample spinners that can be rotated at 4 to 5 kHz are now routinely
available and can often produce narrower resonances.

Note that the calculated surface loading for porphyrin 52 based on the %N figure lies in the range 0.0700.074 µmol/m2, Table 7. This value is likely to be more reliable because the calculation based purely on
the %C figures for the porphyrin-modified silica and its parent mercaptopropyl-modified silica is subject
to a high degree of uncertainty in the mercaptopropylsilanyl surface density due to the poorly defined
attachment mode of these units. The absence of a usable %N figure for the silica modified with zinc
porphyrin 57, however, leaves no option but to use the %C-based calculation.
81
Solid-state NMR techniques have been applied to the studies of silica gel surfaces and
their silylated derivatives.89 Thus, silica and surface-modified silica systems have been
characterised by
29
Si and
13
C solid-state NMR. It was therefore decided to subject the
parent silica gel, the mercaptopropyl-modified silica and silica modified with a
labelled porphyrin (58) and its metallated derivative (74), Scheme 43, to
29
Si and
13
C-
13
C
solid-state NMR investigation.
Solid-state NMR studies of silica gel and its mercaptopropyl derivative.
As mentioned in the introduction of this thesis, silica gel has been studied by various
techniques and it is now widely accepted that the surface of silica is composed of two
types of silanol sites: single hydroxyl sites and geminal sites.70 It is also agreed that the
bulk of silica is formed by silicon atoms surrounded by 4 siloxane bonds, and these sites
are known as lattice sites, Figure 23. These three types of sites (single and geminal
silanol sites, lattice sites) have different environments and therefore give different
29
Si
resonances in solid-state NMR studies. Maciel and co-workers carried out extensive
investigations of silica gel using MAS solid-state 29Si NMR and have published data for
silicas where resonances at -110, -100 and -91 ppm were attributed to lattice sites (Q4)§,
single silanol sites (Q3) and geminal sites (Q2) respectively.99 The 29Si NMR spectrum
of the silica gel substrate used in our attachment studies is shown in Figure 23 and
clearly exhibits the three resonances (at -92, -101 and -111 ppm) described by Maciel et
al. Quantitative analysis of the spectrum by deconvolution of the resonances showed
that single silanol sites constituted 92% of the total hydroxyl site population.
§
The Qn notation refers to the number of siloxane bonds connected to a core silicon atom: Q n =
Si(OSi)n(OH)4-n.
82
single hydroxyl sites OH
geminal hydroxyl sites
OH
OH (-92 ppm)
(-101 ppm)
Si
Si
O
O
O
Schematic representation of sites present
O
in silica
Si
Si
Si
O
O
O
lattice sites (-111 ppm)
Si
29
Si solid–state NMR spectrum of the unmodified silica used for surface attachment in our
studies
Signal deconvolution for quantitative analysis of the proportion of lattice, single silanol and
geminal silanol sites.
Figure 23
A mercaptopropyl-modified silica was prepared according to the method described
previously. Thus, dried silica was treated with (3-mercaptopropyl)trimethoxysilane (2.1
µmol/m2) in hot toluene. The resulting mercaptopropyl-modified silica was washed and
dried in the normal way and analysed for 2.89% and 0.88% carbon and hydrogen
contents respectively, corresponding to a surface coverage within the limiting range of
1.05-1.80* µmol/m2 but probably close to 1.52† µmol/m2. The
29
Si solid-state NMR
spectrum of this material, shown in Figure 24, exhibited signals at Si -111, -102, -92,
*
Calculated using the limiting attachment modes A and E illustrated in Figure 15.
†
Calculated assuming that the anchoring silicon atom is attached by a range of modes with a 1.5/1 ratio
of single to double siloxane linked forms and loss of 60% of residual methoxy groups.
83
-57, -47 and -37. The resonances at  -111, -102 and -92 correspond respectively to
silicon atoms in lattice sites (Q4), single silanol sites (Q3) and geminal silanol sites (Q2)
as seen for the parent unmodified silica gel, Figure 24.
The ratio of silanol silicon atoms (from both single and geminal sites) to lattice silicon
29
Si solid-state NMR spectrum of the mercaptopropyl-modified silica
SH
SH
(SiO)4Si (-111 ppm)
HO OH OH
OH
Si
Si
Si
Si
O
O
O
O
O
O
Si
Si O Si
Si
Si
O
O
O
Si
Si(OH) (-100 ppm)
Si(OH) (-90 ppm)
Signal deconvolution for quantitative analysis.
OCH3
O Si
SH
-47 ppm
SH
-57 ppm
OCH3
OCH3
O Si
O
Figure 24
atoms changes from 1:2.5 in the unmodified silica (Figure 22) to 1:3.5 in the
mercaptopropyl-modified silica (Figure 24), corresponding to a 26% loss in silanol
hydroxyl groups from the core silica on grafting with the mercaptopropylsilanyl chains.
Given a silanol density‡ of 6.9 µmol/m2 for the unmodified silica gel, and assuming a
surface mercaptopropylsilanyl density† of 1.52 µmol/m2 the expected loss in hydroxyl
groups is 31%. Thus, the degree of hydroxyl loss from the core silica observed by NMR
‡
Estimated from microanalytical data (page 47)
†
Calculated assuming that the anchoring silicon atom is attached by a range of modes with a 1.5/1 ratio
of single to double siloxane linked forms and loss of 60% of residual methoxy groups.
84
(26%) correlates reasonably well with the estimated loss (31%) based on
microanalytical data.
One of the objectives of the solid-state NMR studies with the mercaptopropylmodified silica was to probe the attachment mode for the silicon anchoring atom of the
grafted residues — several attachment modes are possible, Figure 15. In their solidstate 29Si NMR studies of silica gel modified with alkyltrimethoxysilanes, Maciel et al
attributed signals at Si -49, -57 and -65 to T1, T2 and T3 attachment modes which
involve respectively one, two and three anchoring siloxane linkages.¶ These resonances
are highly diagnostic and are now routinely used for analysis of silicas grafted with
alkyltrimethoxysilanes.100,101
Low intensity resonances at  -47 and -57 in the
29
Si NMR spectrum of our
mercaptopropyl-modified silica (Figure 24) can be attributed, therefore, to T1 and T2
type grafted silicon atoms respectively, and these are present in approximately 1.5/1
ratio. T3 type grafted silicon centres are not detectable from the noise in the spectrum,
and a third very low intensity signal (marked at  -37) is probably not real. Silicon
atoms that would give rise to T1, T2 and T3 resonances in the NMR spectrum of
mercaptopropyl-modified silica are highlighted, respectively, with blue, red and green
boxes in Figure 15. Note that, although the chemical shift of the T1, T2 and T3
resonances is sensitive to successive replacement of MeO groups by SiO siloxane
linkages, it is insensitive to the replacement of a methoxy group by a hydroxy group.101
Thus, the replacement of carbon by electropositive silicon (ie Si-OCH3 to Si-OSi)
causes an upfield movement in the position of the observed Si resonance as the
attachment mode changes from T1 through T2 to T3. However, the silicon centres in
grafted residues described as modes A, D and E in Figure 15 would all contribute to the
same T1 resonance observed at Si -47. The predominance of T1 and T2 type attachment
modes in our mercaptopropyl-modified silica is typical for the grafting of an
alkyltrimethoxysilane. Thus, silica gel treated with (3-chloropropyl)trimethoxysilane in
boiling toluene for 24 h gives mainly T1 and T2 type attachments with very little T3
linked residues observed; grafting under basic conditions, however, reportedly shifts the
distribution in favour of T2 and T3 attachments with little or no T1 linked species.123
The quantitative solid-state 13C NMR spectrum of our mercaptopropyl-modified silica
is shown in Figure 25. Signals were observed at  48.8, 27.1 and 10.2, which can be
85
attributed respectively to the residual methoxy methyl groups, the  and  chain
methylenes (together), and the  chain methylene adjacent to the silicon atom.
Interestingly, the ratio of  methylenes to residual methoxy groups in this
mercaptopropyl-modifed silica is 1.6/1. Given the 1.5/1 ratio for T1 and T2 grafted
silane residues observed in the
29
Si spectrum, the expected  methylene-to-methoxy
ratio in the 13C spectrum is 1/1.6. The ratio actually observed, however, is the reverse,
1.6/1.
Taken together these data suggest that approximately 60% of the residual
methoxy groups have been replaced by hydroxyls in this sample of mercaptopropylmodifed silica. Clearly, the hydrolysis of grafted methoxysilane centres should not be
possible under strictly anhydrous conditions, and the loss of methoxy groups observed
here must have arisen during post-preparation handling of the material exposed to the
atmosphere. It is unclear how representative this sample is of the various batches of
mercaptopropyl-modified silica that were prepared for surface attachment studies during
this project. Certainly some modest variation was seen in the microanalytical data for
different batches of the silica prepared under identical conditions with the same reacting
ratio of silica to (3-mercaptopropyl)trimethoxysilane. As an approximate guideline,
however, the attachment regime indicated from these solid-state NMR studies has been
used (where indicated) as the basis for the surface density calculations discussed earlier
in this thesis.
11 ppm
O
Si
O
OCH3
SH
27 ppm
50 ppm
Figure 25
The main objective of the solid-state NMR studies in this project was to probe the
attachment of model porphyrins to a mercaptopropyl-modified silica surface.
Preliminary solid-state NMR studies were carried out on the bis(pentenyloxyphenyl)porphyrin (56)-modified silica discussed previously in section 2.1.5. It was hoped that
¶
The Tn notation refers to the number of siloxane bonds connected to the grafted alkylsilanyl silicon
atom: Tn = RSi(OSi)n(OMe)3-n.
86
any residual olefinic signals from 56 would be detectable in the solid-state
13
C and 1H
NMR spectra of the silica modified with this porphyrin. Such signals would confirm
the singly-tethered perpendicular attachment mode proposed for the non metallatedporphyrin (ie covalently bound to the surface through radical addition to just one of the
pentenyloxy side chains). Unfortunately, due to the breadth of resonances in both
spectra and low intensity of signals in the 13C spectrum, it was impossible to distinguish
olefinic resonances from those arising from other centres in the surface-bound residue.
This problem led us to introduce a 13C-labelled carbon at the terminal olefinic centre of
the pentenyl chains in porphyrin 58, Scheme 38. Thus, any radical addition of a surfacebound mercaptan to the olefinic centre should be evident in the replacement of the
labelled olefinic resonance in the 13C spectrum with an upfield thioether signal. In this
way we hoped to probe the degree of covalent tethering with porphyrin 58 and its zinc
derivative (74).
Syntheses of 13C-labelled porphyrin 58 and its zinc derivative (74)
As discussed in the introduction of this thesis, porphyrin syntheses are typically
accompanied by the formation of congeneric compounds. If the labelled carbon atom
were to be introduced before the synthesis of the porphyrin, then unwanted labelled
porphyrin congeners would be formed. Therefore, to avoid losing expensive labelled
material, it was planned to synthesise bis(4-hydroxyphenyl)porphyrin 59 and
subsequently alkylate the meso phenol substituents with 5-bromo-pent-1-ene (60)
labelled at the terminal olefinic centre, Scheme 38.
introduction
of
the
13
C
label
involved
the
The first route selected for
Wittig
reaction
between
4-
bromobutyraldehyde (62) and the triphenylphosphonium salt (61) prepared from
commercially available
13
C labelled iodomethane. The aldehyde is not commercially
available but we thought might be conveniently prepared through ozonolysis of
commercial, unlabelled 5-bromopent-1-ene (63).
87
Ph
N
Ph
N
HN
O
NH
HN
HO
O
OH
NH
N
Ph
*
N
Ph
*
58
59
Br
*
60
O
Br
Br
* H 3C
H
63
62
P(Ph) 3
I
61
Scheme 38
Synthesis of porphyrin 59 was achieved by reaction of phenyldipyrromethane (65) and
4-hydroxybenzaldehyde (64) in neat propionic acid, Scheme 39. Unfortunately this
porphyrin 59 could not be separated from its congener (66) by flash chromatography.
Separation might perhaps have been accomplished by HPLC, but >100 mg quantities of
this material were required in order to support surface attachment studies. As we knew
from previous experiments that the congeneric pentenyloxyphenyl-substituted
porphyrins could be separated comparatively easily, it was decided to carry out the
alkylation step on the porphyrin mixture.
CHO
NH
propionic acid
N
HN
HO
NH
OH
NH
N
OH
64
65
R
59, R = H
66, R = OH
Scheme 39
Ozonolysis of 5-bromopent-1-ene (63) was carried out by bubbling ozone through a
dichloromethane solution of the substrate at -78 ºC. Decomposition of the resulting
ozonide was achieved by adding triphenylphosphine to the solution at -78 ºC. The
88
target aldehyde was isolated by flash chromatography as a colourless oil in 77% yield
(Scheme 40). This aldehyde has also been prepared by reduction of 4-bromobutanoyl
chloride with HCr(CO)5 (95%) and by ring opening of THF with boron tribromide
followed by oxidation of the resulting borate ester with pyridinium chlorochromate
(70%).102, 103 The 1H NMR spectrum of 62 exhibited an aldehydic resonance at  9.81
and the loss of the starting material’s olefinic signals. The aldehyde was found to be air
sensitive and gradually oxidised to the corresponding acid on exposure to air; storage
was thus necessary under an inert atmosphere.
13
Initial route:
CH 3I
67
PPh 3
I Ph 3P
i. O3, DCM, -78 °C
ii. PPh3
Br
Br
H
63
CH 3
61
O
Br
13
62
*
60
LDA
Scheme 40
The
Wittig
reaction
between
aldehyde
62
and
13
C-labelled
methyltriphenylphosphonium iodide 61 was then attempted, Scheme 40. The latter was
prepared by heating
13
C-labelled iodomethane and triphenylphosphine in toluene in a
sealed tube overnight. The resulting white precipitate was collected by filtration and
dried in vacuo over P2O5. A THF suspension of phosphonium salt 61 was deprotonated
under standard conditions using n-butyllithium as a base at -78 ºC. The resulting dark
red ylide solution was then transferred to a flask containing a solution of aldehyde 62 in
dry THF at –78 ºC and stirred overnight at room temperature. The 1H NMR spectrum
of the organic residue obtained following a standard aqueous work-up exhibited no
signals in the olefinic region and indicated that a complex mixture of components had
been formed. Chromatographic fractionation of this mixture did not allow the isolation
and identification of any definable compound. The reason for the failure of the Wittig
reaction on aldehyde 62 was unclear, though the bromide is a possible site for
substitution or elimination (with basic reagents). A publication by a Swiss group
reported the failure of a Wittig reaction with similar bromo-aldehyde, 4-bromo-2hydroxybutanal, though in this case the compound possessed an acidic hydroxyl  to
the aldehyde which had not been protected.104
89
The disappointing failure of the Wittig reaction between aldehyde 62 and
phosphonium salt 61 led us to abandon this route for construction of the labelled
pentenyl chain.
Literature procedures for the preparation 5-bromopent-1-ene have
involved zinc-mediated debromination105,106,107 of 1,2,5-tribromopentane (71%),
dehydrobromination108 of 1,2-dibromopentane in hexamethylphosphoramide (57%) and
bromination109 of pent-4-en-1-ol with phosphorus tribromide (65%).
Of these
procedures the latter seemed most amenable to adaptation with the 13C label. However,
the modest yield of bromide from the PBr3 treatment prompted us to consider
transforming the pentenol into a triflate derivative rather than the bromide, since the
triflate would also serve as a good leaving group for alkylation of porphyrin 59 and
might conceivably be prepared in better yield. Thus, a synthetic route was required for
preparation of [5-13C ]-pent-4-en-1-ol (72). It was decided to start from the symmetrical
1,4-butandiol (68), protect one of the two alcohol functions as the t-butyldimethylsilyl
ether, oxidise the remaining hydroxyl to the aldehyde, and then undertake the Wittig
reaction with the labelled phosphonium salt (61), Scheme 41.
Successful route
i. Oxalyl chloride
DMSO, -78 °C
i. NaH, THF, RT
OH
HO
ii. TBSCl, RT
68
O
OTBS
HO
ii. Et 3N, -78 °C
69
99%
OTBS
H
70
71%
67%
Ph 3P
CH 3 I
13
61
n-BuLi, RT
OTf
H213C
73
(TfO)2, Et 3N
OH
H213C
60%
72
TBAF, THF
OTBS
H213C
70%
71
Scheme 41
Monoprotection of symmetrical diols can be carried under statistical control with tertbutyldimethylsilyl chloride using imidazole as a base.
Better yields are obtained,
however, when a solution of the diol in THF is treated with one equivalent of sodium
hydride followed by the silyl chloride.110 The mono-sodium salt of the parent diol is
formed under these conditions, facilitating selective protection of just one of the alcohol
groups. Accordingly, a solution of 1,4-butandiol in THF was treated with sodium
hydride, TBSCl and after an aqueous work-up and purification by flash chromatography
silyl ether 69 was isolated in nearly quantitative yield.
90
Partial oxidation of the monoprotected diol (69) under standard Swern conditions
afforded aldehyde 70 in yields around 70%. As with the bromobutanal (62), this
aldehyde was also air sensitive and storage under argon was again necessary.
In
contrast to the bromobutanal, Wittig reaction between aldehyde 70 and the ylide derived
from the
13
C labelled methylphosphonium salt (61) was successful. The
13
C-labelled
alkene (71) was obtained as a colourless oil in yields of nearly 70%. As expected, the
1
H NMR spectrum of olefin 71 exhibited a large coupling between the terminal olefinic
protons and the
13
C atom (spin quantum number = 1/2). Thus, the characteristic dq
pattern for both terminal hydrogens was doubled with an additional doublet splitting
(1JCH 155 Hz). The 13C spectrum was, unsurprisingly, dominated by the resonance for
the labelled centre (C 114.5) with very low intensity signals for the other carbons of the
molecule.
With the successful introduction of the labelled olefinic centre, the route progressed to
deprotection of the silyl ether. This was achieved by treatment with a hot solution of
tetrabutylammonium fluoride (TBAF) in THF. At the end of the reaction, solvent was
removed by conventional distillation rather than rotary evaporation as a precaution
against loss of the target alcohol — the boiling point of the corresponding unlabelled
pent-1-en-5-ol is 131 °C. The triflate (73) was then obtained in 60% yield by reacting
freshly distilled trifluoromethanesulfonic anhydride with alcohol 72 and triethylamine
in ice-cooled dichloromethane.
Synthesis of the target labelled bis(pentenyloxyphenyl)porphyrin (58), Scheme 42, was
successfully achieved by reaction of the porphyrin 59/66 mixture, prepared according to
Scheme 39, with the triflate and triethylamine in dichloromethane. As previously noted,
the two phenolic porphyrin congeners were inseparable by flash chomatography.
Moreover, their proportions in the starting mixture were unclear from the 1H NMR
spectrum, and consequently it is not possible to give a defined yield for this reaction.
The target labelled porphyrin (58; 153 mg, 0.19 mmol) was isolated by flash
chromatography following treatment of the starting mixture (470 mg, 0.71-0.73 mmol)
with 0.71 mmol of the triflate.
91
OTf
N
*
HN
HO
73
N
NH
N
HN
O
OH
O
NH
DIPEA, DCM

N
*
*
R
58
59, R = H
66, R = OH
Scheme 42
The zinc derivative (74) of porphyrin 58 was synthesised by boiling a solution of the
porphyrin with zinc acetate in dichloromethane (Scheme 43). The metallated porphyrin
was obtained as a lustrous purple powder in 95% yield. Its 1H NMR spectrum was
identical to that of the non-metallated parent porphyrin with the exception that the
characteristic core NH signal at  -2.68 was absent. In the UV-visible spectrum a red
shift was observed in the Soret band of the zinc porphyrin compared to the nonmetallated parent. With sufficient quantities of the labelled porphyrin to hand, it was
possible to progress to the surface attachment studies.
*
*
O
O
ZnOAc, DCM

N
NH
Ph
N
HN
N
N
Ph
Ph
Zn
N
95%
O
O
*
*
74
58
Scheme 43
92
Ph
N
Solid-state NMR studies of silica modified with porphyrin 58 and the zinc(II)
derivative 74.
A sample of mercaptopropyl-modified silica (110 mg) from the batch prepared
previously for solid-state NMR study (pages 83-84) was reacted with the non-metallated
porphyrin (58; 0.33 mmol) in hot acetonitrile containing a trace of AIBN. The silica
recovered, following the usual washing and drying procedure, analysed for 9.10, 1.44
and 0.23% carbon, hydrogen and nitrogen contents respectively.
This result was
surprising, given that the mercaptopropyl-silicas previously modified with the nonmetallated porphyrins (47 and 56) gave carbon contents above 20%. Unfortunately it
was not possible to repeat the attachment reaction with the labelled porphyrin because
of its limited availability. Thus, the solid-state
13
C NMR spectrum, Figure 26, was
acquired on the material functionalised with the labelled non-metallated porphyrin from
this one experiment.
13
Signal deconvolution for quantitative analysis of C solid–state NMR spectrum for the porphyrin
58-modified silica
Solid-state NMR spectrum of porphyrin 58-modified silica
Figure 26
In addition to signals arising from the more abundant mercaptopropylsilanyl residues
attached to the silica, the NMR spectrum shown in Figure 26 exhibited three prominent
93
resonances that could be assigned to
13
C labelled centres at  113, 51 and 32. Clearly
the signal at  113 originates from the residual labelled olefinic centre that must be
associated with the singly-tethered porphyrin as shown in Figure 27.
*
O
NH
Ph
N
N
Ph
HN
Ph
O
3
3
*
*
S
Si OMe
O O
NH HN
NH HN
O
S
Si OMe
O O
SH
Si OMe
O O
Ph
O
3
*
SH
S
Si OMe MeO Si
O O
O O
Figure 27
The resonance at  32 is likely to be derived from the labelled olefinic centres which
have undergone the radical addition to form a thioether linkage (-13CH2SCH2-). Signals
from the  and  methylenes in the abundant unreacted mercaptopropylsilanyl chains
are also expected close to this resonance, and indeed a shoulder at  27-28 may arise
from these carbons.
Of considerable surprise, however, was the strong resonance
observed at  51. This signal is clearly derived from the labelled olefinic centres of the
porphyrin substrate, although the methoxy groups of the parent mercaptopropylmodified silica give a resonance at  49, Figure 25. The intensity of the  51 resonance,
however, when compared to the mercaptopropyl  methylene at  11, is far too strong
simply to arise from the residual methoxy groups. Indeed close inspection reveals a
shoulder on the upfield side of the  51 resonance which might arise from the residual
methoxy groups present in the material. The origin of the  51 resonance is obscure but
might potentially be due to aerial oxidation of the thioether linkage, giving rise to a
sulfoxide species [-13CH2S(O)CH2].
Predicted chemical shifts for the methylene
adjacent to the sulfoxide are consistent with this view, Figure 28.
94
Predicted shifts
72.3
25.5 33.1 36.3 9.7
Si(OMe) 3
S
Ar O
29.9 32.3
72.3
42.1
25.8 52.3 55.5 9.8
Ar O
Si(OMe) 3
S
O
30.8 27.9
16.8
42.1
12.4
Figure 28
Using the upfield SiCH2 resonance at  11 as a reference it is possible to estimate the
proportion of total aliphatic signals in the spectrum that are derived from the porphyrin.
The ratio of signals in the
13
C spectrum of the mercaptopropyl-modified silica (Figure
25) was 0.6 ( 49) / 2.0 ( 27) / 1.0 ( 11). Subtracting the mercaptopropyl component
from the spectrum in Figure 26, then, gives a ratio of 1/3.3 for the olefinic  113
resonance to porphyrin-derived aliphatic signals. Ignoring the small contribution from
the unlabelled aliphatic centers in the porphyrin, this olefin to aliphatic signal ratio
suggests that the porphyrin is attached by a 1/1.1 mixture of singly- and doubly-tethered
modes, see Figure 27. The comparatively high proportion of units with the doublytethered parallel arrangement is consistent with the lower porphyrin surface density
observed with this modified silica. An important question, then, is why in earlier
preparations of the unlabelled porphyrin 56-modified silica, Table 11, the loading level
was so much higher? Conceivably the concentration of porphyrin used in the surface
attachment reaction might hold the key to this discrepancy. Indeed, with the labelled
porphyrin the attachment reaction was carried out at half the concentration used for
fixation of the unlabelled porphyrin (56).
The 13C spectrum of the labelled porphyrin 58-modified silica (Figure 26) shows some
further complications in addition to the  51 resonance. Thus, the presence of signals
centred at approximately at  22 and  40 requires explanation. One possibility is that
the field induced by the porphyrin’s ring current perturbs the chemical shift of the /
methylenes of some mercaptopropyl chains and perhaps the labelled center in some of
the porphyrin tethers. Upfield or downfield displacements might be induced.
Indeed, strong support for the proposed porphyrin-induced chemical shift perturbation
in the aliphatic region is seen in the solid-state
13
C spectrum of mercaptopropyl-
modified silica treated with the zinc porphyrin (74) in the absence of a radical initiator.
A sample of mercaptopropyl-modified silica (110 mg) from the batch prepared
previously for solid-state NMR study (pages 83-84) was treated with 0.059 mmol of
95
zinc porphyrin 72 in acetonitrile. The material was washed and dried in the usual way,
and on submission for microanalysis returned carbon, hydrogen and nitrogen contents
of 5.52, 1.23 and 0.01% respectively. Not surprisingly, with the porphyrin attached
necessarily in the parallel mode (Figure 29), the surface loading in this material was
comparatively low.
Ph
O
*
N
N
SH
N
N
O
HS
S
Ph


Zn
*

Si OH
O O
Si OMe
O O
MeO
Si OH
O
Figure 29
The solid-state 13C spectrum of this modified silica (Figure 30) was much simpler than
the spectrum for the covalently attached porphyrin discussed above (Figure 26). A
strong signal was observed for the labelled olefinic center of the coordinatively attached
porphyrin at  113.
Three resonances seen in the spectrum of the parent
mercaptopropyl-modified silica (cf Figure 25: C 11, 27 and 49) were also prominent in
the porphyrin-modified sample. The intensity of these signals, derived as they are from
unlabelled mercaptopropyl chains, compared to the labelled olefinic resonance is
consistent with their presence in large excess over the porphyrin units. The unlabelled
aliphatic methylenes of the porphyrin side chains are lost in the baseline noise.
Interestingly, two new minor resonances at  22 and  32 are now seen in the spectrum,
and it is possible that these arise from / methylenes in mercaptopropyl chains
perturbed by the proximity of porphyrin units.
Their intensity relative to the
porphyrin’s labelled olefinic resonance clearly indicates that each individual porphyrin
unit must affect several nearby mercaptopropyl chains.
96
13
C solid-state NMR spectrum of porphyrin 74-modified silica where no covalent tethers were
present
residual
*
O
Si
O
S
H3CO
Si
Figure 30
Having examined the covalently and coordinatively attached porphyrin materials, we
next prepared a sample of porphyrin-modified silica using the dual coordinativecovalent tethering strategy for comparison. This material was prepared, as before, using
a sample of mercaptopropyl-modified silica (110 mg) from the batch previously used
for solid-state NMR study (pages 84-85). An acetonitrile solution of the zinc porphyrin
(74; 0.27 mmol) was reacted with this silica in the presence of AIBN. The resulting
porphyrin-modified silica analysed for carbon, hydrogen and nitrogen contents of 7.84,
1.29 and 0.21% respectively. The comparatively low surface loading of this porphyrin
was again consistent with a substantially parallel mode of attachment and, to our
delight, the 13C spectrum of this material (Figure 31) showed only a low proportion of
residual labelled olefinic centers at  113. Prominent labelled aliphatic resonances at 
32 and  51 were again observed, matching the signals seen in the spectrum of the
covalently attached non-metallated porphyrin (58). With the metallated porphyrin,
however, the ratio of olefinic to porphyrin-derived aliphatic resonances was 1/7.3. Thus
approximatively 88% of the porphyrin alkene chains have undergone the radical
addition process.
97
13
C solid-state NMR spectrum of zinc(II) porphyrin 74-modified silica with covalent tethers
13
Signal deconvolution for analysis of the C solid–state NMR spectrum of zinc (II) porphyrin 74modified silica.
Figure 31
Given that the radical-mediated attachment reactions of the non-metallated and
metallated porphyrins (58 and 74) were carried out under the same conditions with very
similar concentrations of porphyrin, these results clearly demonstrate that the presence
of the metal center substantially increases the degree of covalent tethering. This is
consistent with the establishment of a coordinative linkage to the metallated porphyrin
which brings about a parallel attachment mode, thus promoting radical addition of
surface-bound mercaptans to both of the porphyrin’s olefinic side chains. We have
shown that 88% of these side chains in the metallated bis(pentenyloxyphenyl)porphyrin undergo the radical addition process. To the best of our knowledge,
this is the first study to demonstrate a dual coordinative-covalent tethering strategy for a
metalloporphyrin with a thiol ligand to the metal center.
98
2.2
ATTACHMENT OF A PROTOTYPICAL NOS MODEL TO A MERCAPTOPROPYLMODIFIED GOLD SURFACE
2.2.1 Synthesis of the prototypical NOS model
The surface attachment studies above with simple porphyrin analogues of our NOS
model demonstrated the feasibility of anchoring the porphyrin core to a
mercaptopropyl-modified surface through both covalent and coordinative bonding.
However, the ultimate goal of the Heriot-Watt programme is the construction of an
electrocatalytically functional model, and to do this NOS models will have to be
attached to an electrode surface. It was therefore decided to carry out preliminary
studies towards the attachment of a prototype NOS model to a mercaptopropyl-modified
gold surface. Initial assessment of the redox properties of the resulting material might
then be possible.
A gold surface was chosen over other possible surfaces (e.g.
platinum) because introduction of the mercaptopropyl group used throughout the
preliminary studies with silica, is readily achieved by exposure to di-mercaptan 75,
Scheme 45.
O
Cl
O
OBut
Ar =
Au Au Au Au Au
Ar
HS
SH
75
N
O
N
HS
HS
HS
S
prototypical NOS model
S
S
S
Au Au Au Au Au
HS
HS
Fe
N
S Ar
O
N
HS
HS
S
AIBN, MeCN, 
S
S
S
S
S
S
S
Au Au AuAu Au Au Au Au Au Au Au Au Au Au Au Au Au Au
Scheme 45
As described in section 1.5, the prototype NOS model possesses meso
phenanthrenyloxyacetate units on the porphyrin moiety.
Synthesis of the model
comprising these but lacking meso alkenyloxyphenyl substituents has been achieved by
other members of the group.60 To carry out preliminary surface attachment studies then,
it was decided to adopt the model synthesis, (Scheme 46), in order to introduce the
alkenyl side chain required for the thiol addition process.
99
Me 2SO4
NaHSO 3, KBrO3
NaOH (aq)
H2O/EtOAc
uv
0 to 70 °C
Cl
OH
Cl
OMe
Cl
step 1
OMe
step 2
8
9
Br
10
PPh 3
toluene
step 3
reflux
LDA, THF
I2,
O
uv petrol
Cl
CHO
OHC
Cl
OMe
OMe
Cl
OMe
-78 °C
CHO
step 5
13
step 6
step 4
CHO
PPh 3
12
Br
11
EtSH, NaH, DMF, 100°C
O
then
Br
O
O
Cl
O
NH
O
O
propionic acid
NH
CHO
14
55
CO 2R
Cl
O
RO2C
O
R'
R = t-Bu
R'=
HN
N
Cl
O
N
NH
R'
76
Scheme 46
Methylation of 4-chloro-3-methylphenol (8) was achieved under basic conditions
using dimethyl sulfate as an electrophilic methylating agent.
Aqueous sodium
hydroxide was used as a base to generate the phenolate anion as the reactive
nucleophile. The product, 1-chloro-4-methoxy-2-methylbenzene (9), was obtained in
88% yield following extraction of the aqueous reaction mixture. Formation of anisole 9
was confirmed by observation of a singlet methoxy resonance at  3.75 in its 1H NMR
spectrum. The spectrum showed no detectable impurities and the anisole was used for
subsequent reactions without any further purification.
In order to obtain the benzyl bromide (10) required for synthesis of the phosphonium
salt (11) destined for the Wittig reaction leading to stilbenes 12, anisole 9 had to be
100
selectively monobrominated on its 2-methyl group.
Side chain halogenation of
aromatic compounds is possible with a brominating agent in the absence of a Lewis acid
catalyst (the presence of a catalyst favours ring bromination). Common reagents used
for benzylic bromination are molecular bromine and N-bromosuccinimide (NBS) in the
presence of a radical initiator or under photolysis conditions. Studies by others within
our laboratories, to be reported in detail elsewhere,111 showed that bromination of
anisole 9 using one equivalent of NBS and a catalytic amount of benzoyl peroxide in
carbon tetrachloride afforded target benzyl bromide 10 together with gem-dibromide 77
in 2.5/1 ratio, Scheme 47.
NBS (1eq)
Cl
Cl
OMe
9
benzoyl peroxide
CCl 4
 or UV
OMe
Cl
OMe
Br
Br
10
Br
77
Scheme 47
Separation of the mixture of monobromide 10, dibromide 77 and unreacted anisole 9
issuing from the conventional benzylic bromination with NBS was problematic.
Bromination with molecular bromine under photolysis conditions produced similar
results. The formation of dibromide 77 could be suppressed using the anisole in excess,
and the target bromide was rather more readily isolated from the resulting binary
mixture with the excess anisole than from the ternary mixture including 77. The irritant
properties of monobromide 10 and use of a toxic, environmentally hazardous solvent
prompted a search for an improved preparative method however — one that would
eliminate use of carbon tetrachoride and also generate the bromide efficiently enough to
eliminate need for an elaborate separation procedure.
A literature survey led us to a recent publication by a Japanese group relating the use
of a bromate-bisulfite mixture for benzylic bromination of p-tert-butyltoluene in a twophase ethyl acetate-water medium.112 The mechanism tentatively proposed is a radical
process in which the active bromine atom radical is produced by decomposition of
hypobromous acid (HOBr). The latter would be produced by reduction of bromate with
bisulfite in the aqueous phase. It was proposed that dissociation of HOBr could produce
the free bromine atom which could then cross into the organic phase and participate in
the radical bromination process, Scheme 48. In this scheme the hydrogen bromide
produced by benzylic bromination returns to the aqueous phase where its reaction with
the hydroxyl radical again produces a bromine radical that cycles back into the organic
101
phase. The overall stoichiometry associated with this process is illustrated in Scheme
48 and requires consumption of one bromate and two bisulfite ions per mole of
substrate.
Ar CH 2
Ar CH 3
Br
HBr
Ar CH 2Br
Br
Organic Phase
Aqueous phase
BrO3
2 HSO3
Br
HBr
Br
H2 O
2 HSO4
BrO
OH
HOBr
H2 O
OH
BrO3
BrO3
2 HSO3
BrO
H2 O
BrOH
OH
HSO 4
OH
SO42
H2 O
HOBr
ArCH3
ArCH2Br
H2 O
2 HSO3
ArCH3
BrO
ArCH2Br
2 HSO4
HSO 4
SO42
Scheme 48
Extensive studies were carried out by another member of the group to adapt the
published protocol to our studies.111 Optimal conditions were found using a substrate to
bromate to metabisulfite ratio of 1/1.2/0.8, where an aqueous bisulfite solution was
added slowly under photolysis conditions to a vigorously agitated mixture of aqueous
bromate and substrate in ethyl acetate. Under these conditions, now used routinely in
the group, 2-bromomethyl-1-chloro-4-methoxybenzene (10) is obtained in yields
ranging from 50 to 70%. For the studies described in this thesis, the reaction was
carried out on 136 g of substrate (9); pure bromide 10 was obtained in 45% yield by
separation of the organic layer, evaporation and crystallisation. The somewhat lower
yield, probably due to lack of experience with the protocol, was not an issue because of
the scale on which the reaction was carried out, and bromide 10 was obtained in
quantities sufficient to carry out the ensuing steps in the route to phenanthrene 14.
A key step in the route to phenanthrene 14 is the Wittig reaction between
terephthalaldehyde and the ylide derived from triphenylphosphonium salt 11. The latter
was prepared in essentially quantitative yield by reaction of bromide 10 with
102
triphenylphosphine in boiling toluene over 4 hours, Scheme 49.
The salt, which
precipitated from solution, was collected by filtration and dried in vacuo over
phosphorus pentoxide in preparation for the subsequent Wittig reaction.
PPh3, PhCH3

Cl
Cl
OMe
OMe
99%
PPh 3
Br
10
Br
11
Scheme 49
The Wittig reaction of phosphonium salt 11 and terephthalaldehyde was carried out
using the protocol previously employed within our group.60 Thus, the phosphorus ylide
was prepared by addition of LDA to a suspension of phosphonium salt 11 in dry THF.
The resulting solution was then cannulated into a flask containing terephthalaldehyde in
slight excess (1.2 equivalents) to suppress formation of the diadduct 78, Scheme 51.
The 1H NMR spectrum of the crude material obtained after aqueous work-up of the
reaction mixture showed that an impure mixture of E- and Z-stilbenes 12 had been
obtained. Purification by flash chromatography afforded a 1:2 mixture of the E- and Zisomers of 12 in 42% combined yield. Smaller scale reactions carried out within the
group have previously afforded stilbenes 12 in up to 80% yield.
However, with
sufficient quantities of the stilbene to hand the route proceeded to the photocyclisation
step leading to phenanthrene 13.
1. LDA, THF, RT, Ar
2. OHC
Cl
CHO
Cl
OMe
PPh 3
Cl
OMe
OMe
CHO
Br
11
(E/Z 1:2)
MeO
Cl
12
78
42%
Scheme 50
Separation of the stilbene isomers prior to the photocyclisation was unnecessary
because, although the electrocyclic ring closure takes place on the Z-isomer, stilbene
photoisomerisation
also
occurs
under
the
reaction
conditions.
Stilbene
photocyclisations are typically carried out at low concentrations to minimise competing
[2+2] photocycloadditions of the stilbene with itself or across the 9,10-double bond of
103
the phenanthrene.113,114
In principle, two products might be formed from the
photocyclisation of stilbene 12 — phenanthrenes 13 and 81, respectively, through
dihydrophenanthrene intermediates 79 and 80, Scheme 51. Iodine added to the reaction
mixture, however, efficiently oxidises dihydrophenanthrene 79 leading cleanly to the
desired phenanthrene (13). Other phenanthrene products such as 81, which would be
formed by elimination of HCl from dihydrophenanthrene 80, have not been isolated
from the photocyclisation.
Cl
OMe
CHO
12
MeO
MeO
- HCl
Cl
Cl
CHO
81
H
CHO
H
OMe
H
80
79
[O]
Cl
OMe
CHO
CHO
13
Scheme 51
As with other steps in the route, then, the photocyclisation reaction was carried out
using the protocol previously employed within our group.74 Thus, a dilute solution of
the E/Z-stilbene mixture in light petroleum containing one equivalent of iodine and
excess of 1,2-epoxybutane was irradiated at room temperature with a medium pressure
mercury lamp, Scheme 51. The epoxide added to the reaction mixture is used as a nonbasic scavenger for HI generated during the oxidation of dihydrophenanthrene 79. To
accumulate sufficient quantities of phenanthrene 13 several batches of stilbene were
irradiated in a 1 L quartz immersion well reactor using a 400W lamp. The phenanthrene
partially precipitated during the reaction and was collected by concentration of the
reaction mixture and filtration. Overall, the target material was obtained as a colourless
solid in 69% yield from the combined batches. The 1H NMR spectrum of the product
exhibited a distinctive resonance at  10.51 for the deshielded phenanthrene bay
hydrogen (H-4).
104
I2, UV, petroleum ether
Cl
OMe
Cl
O
CHO
OMe
CHO
69%
12
13
Scheme 52
The next step of the route to the phenanthrene spacer unit involved removal of the Omethyl group and its replacement by an acetic acid chain. Oxyacetate chains had been
designed into the prototype NOS model to provide a functional cleft intended to bind a
guanidinium substrate.60 The procedure developed for introduction of these chains
involved nucleophilic demethylation of phenanthrene 13 using ethanethiolate in hot
DMF and direct alkylation of the resulting phenolate (82) using t-butyl bromoacetate,
Scheme 53. The ethanethiolate was generated by reaction of ethanethiol with sodium
hydride.
O
Cl
i. NaH
EtSH
DMF
OMe
CHO
Cl
Na
O
ii. 100 ºC
BrCH 2CO 2But Cl
O
O
CHO
13
CHO
82
14
Scheme 53
An initial attempt to repeat the one-pot demethylation-alkylation procedure of Scheme
54 was unssuccessful. Two products were isolated, both of which retained the methoxy
resonance in their 1H NMR spectra but lacked the aldehyde. Although one of the
compounds did exhibit signals derived from the bromoacetate, the other possessed a
hydroxymethyl group. Full characterisation of the compounds using two-dimensional
NMR experiments identified them as ester 83 and alcohol 84, Scheme 54. These
compounds clearly arise from a Cannizzaro reaction on the phenanthrenecarbaldehyde
starting material, with the carboxylate component being alkylated by the bromoacetate
added at the end of the reaction.
i. NaH, EtSH
inadequately dry DMF
Cl
OMe
ii. BrCH 2CO 2But
Cl
OMe
Cl
OMe
O
CHO
CH 2OH
O
O
13
83
Scheme 54
105
O
84
The above Cannizzaro reaction suggested that the DMF used in the reaction was
inadequately dry and led to formation of sodium hydroxide from the hydride added to
generate the ethanethiolate nucleophile. The synthesis of phenanthrenyloxyacetate 14
via the one-pot demethylation-alkylation procedure of Scheme 52 is now routinely
carried out in our laboratories with yields in excess of 80% using scrupulously dried
solvent.
In the case of this particular project, however, the one-pot reaction was
abandoned in favour of a separate two step process proceeding with the isolation of
phenanthrenol 15, Scheme 55. The isolation of phenanthrenol 15 was planned in order
to allow its use for construction of new variants of the phenanthrene- superstructure (cf
section 1.8) in addition to the building block (14) incorporated into the original
prototype model. Thus, demethylation of 13 was carried out using ethanethiol in the
presence of sodium hydride as before, but the intermediate phenolate (82) was
protonated by addition of dilute hydrochloric acid at the end of the reaction rather than
trapping it with bromoacetate. Phenanthrenol 15 was isolated in 52% yield and the
alkylation reaction leading to 14 was carried out in a separate step using sodium hydride
in THF with t-butyl bromoacetate; compound 14 was obtained in 45% yield. Sufficient
quantities of phenanthrenol 15 were also produced to investigate the construction of
more elaborate phenanthrene spacer units with alternative chains to the oxyacetate (vide
infra, section 2.3).
O
Cl
NaH, THF  
1. EtSH, NaH, DMF Cl

OMe
CHO
OH
2. H+
BrCH 2CO 2But
Cl
O
CHO
52%
13
O
CHO
45%
15
14
contruction of more elaborate phenanthrene
superstructures for the NOS model
Scheme 55
Construction of the prototype NOS model (76) carrying pentenyloxyphenyl chains for
surface attachment to gold was achieved by boiling phenanthrenecarbaldehyde 14 with
dipyrromethane 55 in propionic acid. Porphyrin 76 was obtained in 57% yield as a dull
purple powder, Scheme 56.
The 1H NMR spectrum of this material showed the
presence of the two atropisomers, ,-76 and ,-76, in a 1/1 ratio.
A similar
observation had been made with original NOS model prototype (7, R3 = t-Bu, X = H;
Scheme 16), where interconversion of the two atropisomers was slow enough on the
106
NMR timescale at room temperature to allow the resolution of two sets of signals.60
However, the energy barrier for interconversion is too low to lock the two atropisomers
and permit their isolation.
O
Cl
O
NH
O
O
propionic acid, reflux
NH
55
CHO
14
CO 2R
Cl
O
RO2C
CO 2R
O
R'
Cl
Cl
HN
N
R'
N
N
NH
O
R'
76
,-atropisomer
R = t-Bu
R'=
HN
N
NH
O
R'
O
Cl
RO2C
76
,-atropisomer
Scheme 56
In order to carry out the surface attachment study with gold and investigate the
model’s redox properties it was decided to introduce the iron centre required for
generation of a functional NOS model. The porphyrin might in principle be metallated
to introduce either a ferrous or ferric iron centre, the former providing the active
oxygen-coordinating species that could participate in substrate oxidation.
Iron(II)-
porphyrins, however, are typically unstable and difficult to work with. 115 Metallation of
porphyrin 76 was therefore carried out with ferrous chloride under conditions that are
known to yield the iron(III)-porphyrin i.e. with oxidation occurring under the
metallation reaction conditions.116
Thus, the parent porphyrin (76) was boiled with ferrous chloride tetrahydrate in
acetonitrile, Scheme 57, monitoring the reaction by UV-visible spectroscopy.
The
reaction appeared to be complete after 3 hours and acetonitrile was then removed in
vacuo. The metallated porphyrin (85) was isolated by flash column chromatography
using CHCl3 as an eluent, eluting after residual traces of the non-metallated starting
material (76). To ensure that the porphyrin was homogenously associated with an axial
chlorine ligand following the chromatography, a solution of the porphyrin in CH2Cl2
was treated with Amberlite IRA-400 (Cl–) resin. The metallated NOS model was
obtained as a dull green powder in 47% yield. Although only very small quantities of
107
porphyrin 83 were available, the few milligrams obtained appeared to be sufficient to
carry out surface attachment to gold wire.
O
O
O
O
N
HN
NH
N
O
O
O
O
Cl
O
O
O
O
Cl
Cl
O
O
Cl
N Cl N
FeCl2.4H20
MeCN
reflux
Fe
N
O
N
O
76
85
Scheme 57
2.2.2 Surface attachment of chloroiron(III)porphyrin 85 on a mercaptopropyl-modified
gold surface and subsequent preliminary electrochemical studies.
To the best of our knowledge, only one set of experiments has been published that
describe surface attachment of porphyrin derivatives to a mercaptopropyl-modified gold
surface.
In this work Pilloud et al studied the electrochemistry of self-assembled
monolayers of iron protoporphyrin IX attached to modified gold electrodes through a
coordinated thiol linkage.117 They used propane dithiol (75) to modify gold coated
quartz slides (0.8  1.5 cm) and subsequently reacted a range of iron(III)porphyrins with
the mercaptopropyl–modified slides, Scheme 58.
Of course, in these studies the
metallated porphyrins were bound to the surface solely through coordinative bonding of
the metal to a mercaptan on the surface.
Cyclic voltammetry then allowed the
American team to characterise their material at the electrode surface. They concluded
that a homogeneous single monolayer of porphyrin had been formed at the surface and
that the redox state of the material could be controlled.
108
Au Au Au Au Au
HS
porphyrin
SH
75
HS
HS
HS
FeIII
porphyrin
Fe
S
S
porphyrin
FeIII
HS
S
III
S
S
S
Au Au Au Au Au
S
S
Au Au Au Au Au
Scheme 58
For our preliminary studies, assistance was sought from others in the Department with
experience in the electrochemical area, and gold wire readily available from Aldrich
was used as the supporting electrode. Prior to use, a 20 cm long wire of 0.5 mm
diameter was divided into three segments of 6 cm each which were then coiled to
provide an electrode. These gold wire segments were then sonicated in CH2Cl2 to
remove grease and subsequently dried with a nitrogen gas gun equipped with a filter to
remove any dust. Each of the three gold filaments was then dipped into a 10-2 M
CH2Cl2 solution of dimercaptan 75 under an argon atmosphere for 2 hours at room
temperature. Two of the gold segments were then reacted in parallel experiments with
chloroiron(III)porphyrin 85 in acetonitrile, one experiment carried out with AIBN added
and one without. It was hoped in this way to bring about coordinative attachment of the
porphyrin on one wire while with the other the dual coordination-covalent attachment
strategy developed with mercaptopropyl-modified silica was reproduced. Both the
modified gold wires were then used as electrodes in cyclovoltammetric experiments.
To our great disappointment, no useful cyclic voltammogram could be obtained and this
may be because insufficient material was present on the gold wires. The approach
followed by Pilloud et al, who used gold coated slides with a larger surface area than
wires used in our studies might provide a more fruitful approach in future work.
These disappointing results concluded the surface attachment studies towards the
construction of biomimetic NOS models in this project. However, the results should not
overshadow the encouraging findings with the dual coordination-covalent attachment
strategy envisaged at the outset of the project for fixation of a porphyrin to a thiolmodified surface. Indeed, we have demonstrated the success of this dual attachment
strategy with mercaptopropyl-modified silica and, to the best of our knowledge, this is
the first time that such an approach has been applied for surface fixation of porphyrins.
109
2.3
SYNTHESIS OF NEW SPACER-SUPERSTRUCTURE UNITS FOR THE NOS MODEL.
Although the majority of work in this project was focussed on the surface attachment
studies, some work was also directed towards the synthesis of new variants of the
phenanthrenyloxyacetate units built into the original NOS model prototype. A range of
models variant in the spacer-superstructure unit might usefully allow the dependency of
substrate oxidation on its bound orientation and proximity to the catalytic centre to be
investigated in future studies. Two variants of the oxyacetate chain of the prototype
model were envisaged in the first instance. Firstly, a phosphonic acid replacement for
the carboxylic acid was considered, requiring the synthesis of phosphonate 19 (Scheme
18), to investigate the effect of a stronger acid on substrate binding. Secondly, an
amino acid derivative (20, Scheme 18) was considered in order test the feasibility of
introducing more sophisticated, branched superstructure chains derived from chiral
amino acids into the model.
Synthesis of phosphonate 19
The planned route to phosphonate 19 (Scheme 18) involved reaction of phenanthrenol
15 with diethyl phosphonomethyl triflate (17). Triflate 17 was prepared according to
the method of Phillion et al 86 who reacted it with a variety of nucleophiles to form more
elaborate
phosphonates
in
good
yields
(above
90%).
Thus,
diethyl
hydroxymethylphosphonate (87) was prepared by reaction of neat diethyl phosphite
(86) with paraformaldehyde in the presence of catalytic triethylamine at 50ºC, Scheme
59. Ester 87 was obtained in 97% yield and was used without purification as its 1H
NMR spectrum showed that it had been formed in >95% purity. Evidence for the
formation of 87 was seen in the resonance for the -methylene protons which was split
by the adjacent phosphorus atom ( 3.79, 2H, J 6.2). Triflate 17 was then prepared by
reaction of 87 with trifluoromethanesulfonyl chloride. This reaction was achieved by
pretreatment of 87 with sodium hydride in ether; the resulting alkoxide suspension was
then cannulated into a solution of the sulfonyl chloride in Et2O at -78 ºC. Following an
aqueous work-up triflate 17 was obtained in 21% yield. As expected, the signal for the
methylene protons adjacent to the phosphonate exhibited a downfield shift compared to
the corresponding signal in the precursor phosphonate 87.
110
O
(EtO) 2P
CH 2O, Et 3N, 
H
95 %
O
(EtO) 2P
86
O
(EtO) 2P
i. NaH, Et 2O, RT
OH
ii. CF3SO2Cl, Et2O, -78 C
87
O
O S CF 3
O
17
Scheme 60
The triflate thus obtained was then reacted with phenanthrenol 15 in CH2Cl2 in the
presence of diisopropylethylamine (DIPEA) in a sealed tube, Scheme 60.
After
purification by flash column chromatography, the target phosphonate was obtained as a
solid in 12% yield.
Though disappointingly low-yielding, this first attempt
demonstrated that a phenanthrene bearing phosphonate functionality could indeed be
synthesised as initially planned. Optimisation of the reaction conditions in order to
improve the yield will be required in further studies if phanthrene 19 is to be taken
forward for construction of the porphyrin.
O
(EtO) 2P
Cl
OH
CHO
O
O S CF 3
O
17
Cl
O
DIPEA, DCM

15
OEt
P O
EtO
CHO
19
12%
Scheme 61
Synthesis of amino acid 20.
Introduction of a chiral amino acid in place of the prototype’s oxyacetate chain could
open substantial opportunities for development of the model’s molecular recognition
superstructure. In principle, this could lead to enhanced binding features for Arg as
substrate or allow us to broaden the diversity of substrates recognised by the model. A
method was therefore sought for introduction of an amino acid chain through
phenanthrenol 15. Previous work carried out in our group on a different project had
successfully applied the palladium-catalysed Buchwald-Hartwig procedure for
amination of quinolyl halides and triflates.118 Therefore it was decided to synthesise the
triflate derivative (16) of phenanthrenol 15 as a substrate for amination with an -amino
ester, Scheme 61.
111
Ph
H2N
Cl
OH
Tf 2O, DIPEA
Cl
CO 2Et
CsCO 3, Pd 2(dba)3
racemic BINAP
OTf
Cl
N
H
toluene, 
DCM, 0 ºC
CHO
15
18
CHO
16
CO 2Et
CHO
20
Scheme 61
Triflate 16 was prepared in 96% yield from phenanthrenol 15 by reaction with freshly
distilled trifluoromethanesulfonic anhydride and DIPEA, Scheme 62. The conditions
previously applied in our group141 for amination of haloquinolones were then used for
synthesis of amino ester 20. Thus, a solution of triflate 16, L-phenylalanine ethyl ester,
Cs2CO3 and racemic BINAP in dry toluene was heated at 140 ºC in a sealed Wheaton
flask in the presence of Pd2(dba)3. The reaction mixture was then directly transferred
onto a silica gel column and chromatographed. The target amino ester (20) was isolated
in 7.5% yield. Unfortunately, the quantity of material obtained from this single reaction
attempt was insufficient to allow characterisation of the compound beyond acquisition
of its 1H NMR spectrum, and the optical integrity of the product is unclear at present.
The product was clearly identified by its 1H NMR spectrum, however, which exhibited
all the distinctive phenanthrene signals (including the aldehydic resonance at H 9.80) as
well as signals corresponding to the L-phenylalanine ethyl ester derivative — H 3.34
(2H, AB system) for the benzylic methylene; H 5.15 (1H, m) for the -methine. The
intensities of both sets of signals, phenanthrene and amino ester resonances, integrated
in proportion consistent with the structure of amino ester 20. As with the synthesis of
phenanthrene-phosphonate (19) the low yield of 20 was disappointing. However, these
preliminary investigations provide a foundation for future optimisation which could
allow construction of NOS models with more advanced superstructure.
112
3. Conclusion
113
The two main approaches for anchoring porphyrins reported in the literature involve
either covalent or coordinative fixation of the macrocycle to a surface. The former
mode of attachment is generally achieved by substitution of a good leaving group on the
porphyrin’s side chains by a suitable nucleophile bound to the surface. This method is
efficient for preventing catalyst leaching from the surface, however functional operation
of the porphyrin for oxidation requires a suitable axial donor ligand, as observed in
Nature. Coordinatively bound models, where the metalloporphyrin’s core metal centre
is linked by a surface-tethered ligand (generally through a nitrogen donor centre), may
provide more efficient catalysts.
In this thesis we have proposed a novel approach for surface fixation of porphyrins, an
approach that may allow the development of porphyrin-based models for the nitric
oxide synthase enzymes.
Our porphyrin models were anchored through a dual
coordinative-covalent strategy, Scheme 62, where a surface-tethered mercaptopropyl
chain simultaneously serves as a donor ligand to the metal centre and provides a site for
covalent attachment of the porphyrin. This approach is intended to minimise catalyst
leaching from the surface and, moreover, may lead to better NOS mimetics in which the
axial donor ligand centre for the metal is a sulfur atom. To achieve the dual attachment
meso alkenyloxyphenyl side chains were exploited in a radical addition to the surfacebound thiol chains.
NOS model spacer units and molecular recognition
superstrucutre
n
O
M
n
O
n
O
HS
S
M
S
n
O
S
Scheme 62
To avoid losing the synthetically expensive prototype NOS model during development
of the attachment strategy, preliminary studies were carried out on simple porphyrins
possessing allyloxyphenyl or pentenyloxyphenyl side chains (respectively, 47 and 56).
114
These unmetallated porphyrins were successfully bound to a mercaptopropyl-modified
silica by radical addition mediated with the initiator AIBN in acetonitrile. In both cases
high surface loadings were obtained — estimated at up to 1.3 µmol/m2. Surface loading
may be sensitive to the concentration of porphyrin substrate used, with lower
concentrations potentially favouring a doubly-tethered, parallel attachment mode. The
high loading levels achieved with 47 and 56, however, are consistent with a
predominantly singly-tethered, perpendicular attachment, Figure 32.
Ph
O
Si
O
OMe
N
O
S
n
HN
NH
OH
O
N
n
Ph
Ph
OH
O
Si
N
O
S
n
OH OMe
NH
HN
N
O
n
Ph
Figure 32
The zinc derivatives of porphyrins 47 and 56, respectively 52 and 57, were
successfully coordinatively attached to mercaptopropyl-modified silica. The porphyrin
surface loadings were substantially lower than those obtained for the parent free-base
porphyrins and were consistent with parallel binding of the metalloporphyrins, Figure
33. However, with these porphyrins it was not possible to evaluate the percentage of
porphyrin alkene side chains that had undergone the radical addition to become attached
to the surface through the dual mode.
Ph
Ph
N
O
N
S
HO Si
O
Zn
N
HO Si
OH OH
O
N
S
S Ph
OH
N
O
O
N
OMe
OH
OH
HO Si
O
Zn
N
S Ph
HO Si
OH
OH OH
O
O
N
S
OMe
HO Si
OH OH OH
O O
Figure 33
These studies were encouraging but provided no tangible proof that the dual
attachment (i.e. coordinative and covalent) had occurred.
To obtain this proof,
porphyrin 58 and its zinc derivative 74, both labelled at their terminal olefinic centres,
were synthesised and reacted with mercaptopropyl-modified silica. The solid-state
115
NMR experiments carried out on the resulting silicas demonstrated clearly that the dual
attachment mode initially foreseen had indeed taken place, with an estimated 88% of
alkene chains having undergone radical addition in the case of zinc porphyrin 74,
Figure 34.
*
O
NH
Ph
N
N
Ph
HN
Ph
O
*
3
3
*
S
Si OMe
O O
O
NH HN
NH HN
O
S
Si OMe
O
SH
Ph
Si OMe
O O
O
3
*
SH
Si
O O
S
OMe MeO Si
O O
Figure 34
Following the success of the surface attachment studies on silica, preliminary
electrochemical studies were attempted using a mercaptopropyl-modified gold wire
reacted with a prototype NOS model (85). Unfortunately, the surface attachment in this
case was inconclusive and these studies will require further development. For instance,
a larger surface than the gold wire used here could be employed in order to maximise
the quantity of metalloporphyrin to be analysed at the surface. The disappointing
outcome from our preliminary electrochemical studies should not eclipse the excellent
results obtained with the studies carried out on silica however. To the best of our
knowledge this is the first report of such a dual coordinative-covalent attachment
strategy and the way now lies open for attachment of the Heriot-Watt NOS model
prototypes for assessment of their catalytic potential.
A secondary aim of the project was the synthesis of phenanthrene spacer units carrying
acidic superstructure chains differing from the those of the first Heriot-Watt NOS model
prototype. Variation of the original oxyacetic acid superstructure might lead to models
with different guanidinium binding characteristics.
116
Such models might allow the
dependency of substrate oxidation on its bound orientation and proximity to the
catalytic centre to be investigated in future studies. Thus, a chiral amino ester, Lphenylalanine ethyl ester (18) was introduced on phenanthrenol 15 by amination of its
triflate derivative (16) using a palladium-catalysed pocedure.
The synthesis of
phenanthrene 19 possessing a phosphonate-containing superstructure chain was also
achieved by nucleophilic substitution of the phenolate of 15 on triflate 17. These two
procedures, Scheme 63, remain to be improved.
Cl
OH
Tf2O, DIPEA
DCM
CHO
CHO
15
O
EtO P
EtO
17
O
O S CF 3
O
Cl
16
Pd cat.
BINAP
Cs2CO3
toluene
Ph
O
O S CF 3
O
K2CO3
DMF
H2 N
CO 2Et
18
O
Cl
O
Ph
P OEt
OEt
Cl
N
H
CHO
CO 2Et
CHO
19
20
Scheme 63
The work described in this thesis represents a significant step forward in the NOS
models programme at Heriot-Watt University. However, synthetic challenges remain to
be overcome. For instance, a bulky group remains to be introduced on the phenanthrene
ortho to the porphyrin (X, Figure 35). This will prevent rotation of the spacer units for
the construction of models with a fixed cleft. Work has already been directed towards
introduction of a locking halogen atom atom at this position, but did not lead to a
practicable synthetic route to the required spacer units. Current studies by others in our
group with an additional fused benzo ring attached to the lower ring (i.e. a pyrene
spacer) are more promising. The drawback with blocking spacer rotation in these
models, however, is of course the generation of atropisomers — the ,-atropisomer is
required for construction of our models. Work is currently being undertaken using strap
methodology for construction of suitable models with locked spacer units. In this work
a temporary strap between the superstructure chains of two spacer units is established
prior to the porphyrin-forming step. Construction of the porphyrin then gives rise to
structures of the type shown in Figure 35. Removal of the strap subsequently unmasks
117
the porphyrin’s guanidinium-binding cleft. These studies are currently in progress for
the synthesis of more advanced models to be used in detailed solution phase host-guest
binding studies with guanidinium substrates.
strap
Cl
O
O
R
Cl
HN
N
N
NH
X
X
R
n
R=
O
Figure 35
As described above, then, the work towards construction of a functional biomimetic
model for NOS is far from completion. However, substantial progress has been made
towards this goal through the studies in this thesis and by others in our group. NOS
plays a key role in physiological and pathophysiological processes. An advanced model
could usefully throw light on the reactions catalysed by this remarkable enzyme and, in
principle, provide technology for clean, highly-regulated and directed generation of NO
from Arg. Such technology would provide a powerful tool for biomedical research in
the NO field.
118
4. Experimental
119
4.1
GENERAL EXPERIMENTAL
Reactions were routinely carried out under an inert atmosphere of N2 or Ar unless
otherwise stated. Chemicals were purchased Aldrich, Avocado, Strem, Sigma and
Lancaster chemical companies and were used as supplied. THF, Et2O and toluene were
dried by distillation from sodium-benzophenone under Ar.
DCM was dried by
distillation from CaH2 or P2O5 under Ar. Other solvents were purified by distillation
prior to use. Meting points were determined using Reichert Micro Hot Stage apparatus
and are uncorrected. Mass spectra were obtained on a Kratos Concept 15 (electron
impact) instrument.
1
H NMR spectra were recorded at 200 and 400 MHz on Bruker
AC200 and DPX400 spectrometers using tetramethylsilane as an internal standard.
NMR spectra were recorded at 50 and 101 MHz on the same instruments.
13
13
C
C and 29Si
solid-state NMR spectra were carried out by the analytical service of the chemistry
department of Durham Universiy. Chemical shift data are reporteded part per million (
in ppm) where s, d, dd, t, q and m designate singlet, doublet, double doublet, triplet,
quartet and multiplet respectrively. J values are given in Hz. IR spectra were recorded
on a Perkin Elmer 1600 FT IR spectrometer. Flash coloumn chromatography was
performed using Fisher Matrex silica gel 60 (35-70 m). TLC was performed on Merck
silica gel 60 F254 precoated sheets (Art. 1.05554) with detection by UV light and/or
alkaline KMNO4 dip. Elemental analyses were carried out by the analytical service of
the chemistry department at Heriot-Watt University. Silica gel used for the surface
attachment studies was purchased from Aldrich: [28,850-0], BET surface area ~500 m2,
particule size 2-25m, pore volume 0.75 cm3/g, pore diameter 60 Å. Gold wire was
purchased from Aldrich: [31,098-0], 0.5 mm diameter, 99.99%.
120
4.2
SURFACE ATTACHMENT VIA MESO-(P-ALLYLOXYPHENYL) GROUPS.
Preparation of allyl phenyl ether (22)
Phenol (47.0 g, 0.50 mol), potassium carbonate (69.1 g, 0.50 mol)
O
and allyl bromide (60.5 g, 0.50 mol) were refluxed in acetone (100
22
C9H10O
Mol. Wt.: 134.1751
ml) for 8 hours. The reaction mixture was poured onto water (500 ml)
and washed with Et2O (3 x 100 ml). The combined organic layers
were further washed with 2M aqueous NaOH solution, and dried over potassium
carbonate.
The ether was removed on the rotary evaporator and the residue was
distilled under reduced pressure to afford allyl phenyl ether 22119 (47.0 g, 0.35 mol;
70%); bp 19 mmHg 85 ˚C; H (200 MHz, CDCl3) 4.54 (2 H, dt, J 5.3 and 1.5, OCH2), 5.29
(1 H, dq, J 10.4 and 1.3, vinylic-CH2), 5.42 (1 H, dq, J 17.4 and 1.6, vinylic-CH2), 6.07
(1 H, ddt, J 17.3, 10.4 and 5.4, vinylic-CH), 6.89-6.99 (3 H, m, Ph-H), 7.24-7.34 (2 H,
m, Ph-H).
Preparation of [3-(3-phenoxypropylsulfanyl)propyl]trimethoxysilane (23)
Allyl phenyl ether (2.28 g, 17.1 mmol), (3mercaptopropyl)trimethoxysilane
(3.35
g,
O
Si(OCH 3)3
23
C15H26O4SSi
Mol. Wt.: 330.5160
mmol) and AIBN (0.11 g, 0.4 mmol) were heated in
acetonitrile (15 ml) at 85 ºC under nitrogen for 6
hours.
S
17.1
At the end of the reaction solvent and unreacted volatile materials were
evaporated
under
reduced
pressure.
[3-(3-
phenoxypropylsulfanyl)propyl]trimethoxysilane 23 was afforded as a pale yellow oil
(5.62 g, 17.0 mmol; 99% in > 95% purity): Rf (CHCl3) 0.17; H (200 MHz, CDCl3)
0.72-0.80 (2 H, m, SCH2CH2CH2Si), 1.64–1.79 (2 H, m, SCH2CH2CH2Si), 2.10 (2 H,
p, J 6.6, OCH2CH2CH2S), 2.55 (2 H, t, J 7.3, SCH2CH2CH2Si), 2.70 (2 H, t, J 7.1,
OCH2CH2CH2S), 3.55 (9 H, s, 3  OCH3), 4.10 (2 H, t, J 6.1, OCH2CH2CH2Si), 6.886.97 (3 H, m, Ph-H), 7.24-7.32 (2 H, m, Ph-H).
4.2.1 Drying silica
A sample of silica gel (sample A) that had undergone no treatment was submitted for
elemental analysis: (Found 0 %C, 0.40% H). Two batches of this silica gel of identical
121
weight (14.680 g) were dried under the conditions used for all surface the attachment
studies described in this thesis: in vacuo (0.1 mmHg) at 110 ºC for different lengths of
time. Sample B was dried under those conditions for 24 h, weighed 130.955 g at the
end of the treatment and was analysed by elemental analysis: (Found 0 %C, 0.35% H).
The second sample, sample C was dried for 48 h and weighed 130.964 g at the end of
the drying treatment: (Found 0 %C; 0.36% H).
4.2.2 General method for surface attachment of organic molecules onto silica
Method A:
(MeO) 3Si
S
O
R
OH
O
OH
O
Si
OMe
S
O
R
The radical addition between (3-mercaptopropyl)trimethoxysilane and an alkenyloxyfunctionalised organic residue was carried out (vide infra) in solution before surface
attachment.
-
The silica was dried in vacuo (0.1 mm Hg) at 110 ºC for 24 hours.
-
The silane and the silica were heated in dry toluene (5 ml of toluene for 200 mg of
silica) at 100 ºC in a sealed tube overnight.
-
The tube was opened under a nitrogen atmosphere and the mixture was then
filtered, washing consecutively with dry toluene (5 ml for 200 mg silica) and dry
CH2Cl2 (5 ml for 200 mg silica).
-
The resulting silica was then dried in vacuo for (110 ºC, 24 hours, 0.1 mmHg)
before being submitted for elemental analysis.
Method B:
The radical addition was this time carried out on the surface between a silica-bound
alkanethiol and an alkenyloxy-functionalised organic residue.
-
The silica was reacted with (3-mercaptopropyl)trimethoxysilane according to
method A.
-
The resulting mercaptopropyl–modified silica was then heated with the
alkenyloxy-functionalised compound in acetonitrile (5 ml for 200 mg silica) in the
presence of catalytic AIBN in a sealed tube at 100 ºC for 6 hours.
122
-
The reaction mixture was filtered under nitrogen and washed subsequently with
dry toluene (5 ml for 200 mg of silica) and dry CH2Cl2 (5 ml for 200 mg of silica)
or until the filtrate ran clear if the reactions were carried out with porphyrins.
-
The silica was then dried in vacuo (0.1 mmHg, 110 ºC, 24 h.) before being
submitted for elemental analysis.
4.2.3 Attachment of allyl phenyl ether
(MeO)3Si
OH
OH
S
O
23
O
Si
O
OMe
S
O
According to general method A:
General method A was followed with different silane/silica ratios to optimise silane
loading. Four experiments were carried out using 2.0 mol, 10.0 mol, 20 mol and 40
mol per m2 of substrate silica (Experiments 1–4, Table A). Silica gel was dried under
standard conditions prior to use (0.1 mmHg, 100 ºC, 24 hours). Silica gel (200 mg, 100
m2) was then reacted with with silane 23 (66.0 mg, 0.20 mmol, experiment 1); (330 mg,
1.0 mmol, experiment 2); (660 mg, 2.0 mmol, experiment 3); (1.32 g, 4.00 mmol,
experiment 4) in boiling toluene (5 ml) overnight. The resulting modified silicas were
collected by filtration, washing with toluene (5 ml) and CH2Cl2 (5ml), and dried under
standard conditions (0.1 mmHg, 100 ºC, 24 hours). The samples were submitted for
elemental analysis and the results are summarised in Table A.
OPTIMISATION STUDIES
Experiment number
Carbon percentage
Hydrogen percentage
(quantity of silane reacted in mg
per 200 mg of silica substrate)
(from elemental analysis)
(from elemental analysis)
1 (66.0)
7.59
1.32
2 (330)
7.88
1.34
3 (660)
10.03
1.60
4 (1320)
11.59
1.85
Table A
123
Reproducibility of the results was also investigated by carrying out 4 experiments
(experiments 5–8, Table B) using 2 mol of silane per m2 of silica for each experiment.
Silica gel (200 mg, 100 m2) was dried (0.1 mmHg, 110 ºC, 24 hours) and was then
treated with silane 23 (66.0 mg, 20.0 mol) in boiling toluene (5 ml) overnight. The
resulting modified silica was filtered, washed with toluene (5 ml) and CH2Cl2 (5 ml)
and dried under standard conditions (0.1 mmHg, 100 ºC, 24 hours). The samples were
submitted for elemental analysis and the results are summarised in Table B.
REPRODUCIBILITY STUDIES
Experiment number
Carbon percentage
Hydrogen percentage
(quantity of silane reacted in mg)
(from elemental analysis)
(from elemental analysis)
5 (66.0)
6.14
1.13
6 (66.0)
6.3
1.14
7 (66.0)
6.71
1.17
8 (66.0)
6.62
1.23
Table B
According to general method B:
OH
OH
(MeO) 3Si
21
Method A
O
22
SH
O
Si
O
OMe
SH
Method B
O
O
Si
OMe
S
O
Attachment of (3-mercaptopropyl)trimethoxysilane on silica was followed by radical
addition of phenyl allyl ether on the surface. Dry silica gel (200 mg) was heated with (3mercaptopropyl)trimethoxysilane (41.0 mg, 0.21 mmol) in dry toluene (5 ml). The
silica was filtered and dried under the conditions described in the general method (0.1
mmHg, 110 ºC, 24 hours) and submitted for elemental analysis: Found C, 2.64; H,
0.83%. The resulting mercaptopropyl-modified silica was heated with allyl phenyl
ether (29.0 mg, 0.21 mmol) in acetonitrile (5 ml) in the presence of AIBN (2.00 mg,
7.30 mol). This silane 22-modified silica was filtered, washed with toluene (5 ml) and
CH2Cl2 (5 ml) and dried (0.1 mm Hg, 110 ºC, 24 hours) and submitted for elemental
analysis: Found C, 5.70; H, 1.15%.
124
4.2.4 Surface attachment studies with porphyrins carrying allyloxy groups.
Preparation of (4-methoxycarbonylphenyl)dipyrromethane (31)
Methyl 4-formylbenzoate (500 mg, 3.05 mmol), pyrrole (2.04
were refluxed in dry toluene (15 ml) under nitrogen for 1.5
3' 2'
O
g, 30.46 mmol) and trifluoroacetic acid (34.0 mg, 0.35 mmol)
4'
H3CO
1'
NH
1
NH
5' 6'
31
C17H16N2O2
Mol. Wt.: 280.3212
hours. The solvent and unreacted pyrrole were then removed by
conventional distillation on a water pump. The crude material
was then submitted to a flash column chromatography on silica gel (Et2O/light
petroleum, 1:9-3:7 gradient).
After recrystallisation from toluene, the target
dipyrromethane 31 was obtained as colourless needles (390.8 mg, 1.39 mmol; 47%):
m.p. 164 ºC; Rf (2:3, EtOAc-light petroleum) 0.61; max (KBr disk)/cm-1 3341, 3121,
2952, 1705, 1606, 1557, 1433, 1417, 1260, 1196, 1180, 1031; H (200 MHz, CDCl3)
3.83 (3 H, s, OCH3), 5.45 (1 H, s, H-1), 5.81-5.83 (2 H, m, pyrrole H), 6.09 (2 H, q, J
2.8, pyrrole H), 6.63-6.66 (2 H, m, pyrrole H), 7.20 (2 H, ~ d, J 8.3, H-2’ and H-6’),
7.91 (2 H, ~ d, J 8.4, H-3’ and H-5'), 7.91 (2 H, bs, 2  NH); C (50 MHz, CDCl3) 43.88
(CH-1), 52.04 (OCH3), 107.42 (pyrrole CH), 108.49 (pyrrole CH), 117.48 (pyrrole CH),
128.34 (CH-2’ and CH–6’), 128.78 (C), 129.84 (CH-3’ and CH–5’), 131.49 (C), 147.23
(C), 168.82 (CO2); m/z (EI) 280 (100%, M+), 249 (17%, [M – OCH3]+), 214 (42%, [M –
C4H4N]+) (Found M – 31: 249.1011. C16H13N2O requires 249.1028); (Found: C, 72.73;
H, 5.73; N, 10.02%. C17H16N2O2 requires C, 72.84; H, 5.75; N, 9.99%).
Preparation of 5,15-bis(4-carboxymethylphenyl)-10,20-diphenylporphyrin (32)
(4-Methoxycarbonylphenyl)dipyrromethane
31
(448.4
mg,
1.60
mmol)
and
benzaldehyde (188.4 mg, 1.76 mmol) were refluxed in propionic acid (15 ml) for 30
minutes. The reaction was carried out in air to ensure
oxidation of the porphyrinogen intermediate. Propionic
2
acid was removed under reduced pressure and the crude
3
4
material was submitted to a flash chromatography column
5
on silica (CH2Cl2/light petroleum, 2:3-4:1 gradient). Four
1
N
NH
20
HN
N
15
10
porphyrin congeners were eluted and identified from their
1
H NMR spectra.
5,10,15,20-Tetraphenylporphyrin 2751 (15.6 mg, 25.3
125
27
C44H30N4
Mol. Wt.: 614.7360
mol, 1.5 %) was eluted first in 1:1 CH2Cl2-petroleum: Rf (CH2Cl2) 0.87; H (200 MHz,
CDCl3) <0 (2 H, bs, 2  NH), 7.69-7.78 (12 H, m, 8  meta Ph-H and 4  para Ph-H),
8.20-8.25 (8 H, m, 8  ortho Ph-H), 8.79 (8 H, s, pyrrolic-H).
5-(4-carboxymethylphenyl)-10,15,20-triphenylporphyrin 38 (23.8 mg, 35.4 mol,
2.2%) eluted second in 3:2 CH2Cl2-petroleum: Rf
(CH2Cl2) 0.83; max (CHCl3)/nm (log ε) 251
2'
(5.27), 320 (4.98), 419 (6.57), 514 (5.08), 534
O
(4.26), 550 (4.62); H (200 MHz, CDCl3) <0 (2 H,
bs, 2  NH), 4.03 (3 H, s, OCH3), 7.62-7.74 (9 H,
2
3'
4'
3
1
N
4
5'
H3CO
1'
20'
HN
NH
N
15'
10'
m, 6  meta Ph-H and 3  para Ph-H), 8.12-8.16
(6 H, m, 6  ortho Ph-H), 8.23 (2 H, d, J 8.2, H-2
38
C46H32N4O2
Mol. Wt.: 672.7720
and H-6), 8.37 (2 H, d, J 8.2, H-3 and H-5), 8.78–
8.81 (8 H, m, pyrrolic-H); C (50 MHz, CDCl3)
52.35 (OCH3), 119.16 (C), 120.29 (C), 126.63 (meta Ph-CH and para Ph-CH), 127.76
(CH-2 and CH-6), 129.45 (C), 131.29 (pyrrolic-CH), 134.46 (CH-3 and CH-5, ortho
Ph-CH), 141.95 (C), 146.97 (C), 167.25 (CO2); m/z (ESI) 673 (100%, MH+).
The target 5,15-bis(4-carboxymethylphenyl)-10,20-diphenylporphyrin 32 (129.2 mg,
0.20 mmol; 11%) was eluted third in 7:3
CH2Cl2-petroleum: Rf (CH2Cl2) 0.28; max
2'
(CHCl3)/nm (log ε) 306 (3.93), 332 (2.78),
419 (5.42), 514 (3.78), 552 (3.08); max
(KBr disk)/cm-1 3318, 2924, 1720, 1604,
O
2
3'
4'
3
1
H3CO
1559, 1473, 1434, 1400, 1349, 1310, 1276,
4
5'
1'
N
NH
20'
HN
N
O
15'
OCH 3
10'
1177, 1110, 1020, 964, 799, 755, 730, 701;
H (200 MHz, CDCl3) <0 (2 H, bs, 2  NH),
4.03 (6 H, s, 2  OCH3), 7.71-7.81 (6 H, m,
32
C48H34N4O4
Mol. Wt.: 730.8081
4  meta Ph-H and 2  para Ph-H), 8.19-8.24 (4 H, m, 4  ortho Ph-H), 8.23 (4 H, d, J
8.2, 2  H-2 and 2  H-6), 8.37 (4 H, d, J 8.2, 2  H-3 and 2  H-5), 8.80 (8 H, d, J 4.8,
pyrrolic-H), 8.88 (8 H, d, J 4.9, pyrrolic-H); C (50 MHz, CDCl3) 52.37 (OCH3), 118.86
(C), 120.51(C), 126.68 (meta Ph-CH and para Ph-CH), 127.84 (CH-2 and CH-6),
129.53 (C), 131.38 (pyrrole CH), 134.46 (CH-3 and CH-5, ortho Ph-CH), 141.81 (C),
146.84 (C), 167.24 (CO2); m/z (ESI) 731 (55%, MH+); (Found C, 78.62; H, 4.99; N,
7.27%. C48H34N4O4 requires C, 78.89; H, 4.69; N, 7.67%).
126
5,10,15-tris(4-carboxymethylphenyl)-20-phenylporphyrin 39 (102.7 mg, 0.13 mmol;
8.2%)
eluted
petroleum:
Rf
fourth
in
(CH2Cl2)
4:1
CH2Cl2-
0.21;
max
(CHCl3)/nm (log ε) 306 (4.51), 323 (4.26),
420 (5.84), 516 (4.45), 535 (3.62), 552
2'
O
2
1
H3CO
3'
4'
3
4
5'
1'
N
NH
(4.05); H (400 MHz, CDCl3) -2.81 (2 H,
20'
HN
O
15'
N
OCH 3
10'
bs, 2  NH), 4.03 (9 H, s, 3  OCH3), 7.76
(3 H, m, 2  meta Ph-H and para Ph-H),
8.20-8.24 (2 H, m, 2  ortho Ph-H), 8.23 (6
H, d, J 8.2, 3  H-2 and 3  H-6), 8.37 (6 H,
O
OCH 3
39
C50H36N4O6
Mol. Wt.: 788.8442
d, J 8.2, 3  H-3 and 3  H-5), 8.81–8.90 (8
H, m, pyrrolic-H); C (100 MHz, CDCl3) 52.43 (CH3, OCH3), 119.16 (C), 121.01 (C),
126.77 (meta Ph-CH and para Ph-CH), 127.95 (CH-2 and CH-6), 129.71 (C), 131.17
(pyrrole CH), 134.52 (CH-3 and CH-5, ortho Ph-CH), 141.79 (C), 146.76 (C), 167.25
(CO2); m/z (ESI) 789 (100%, MH+).
Preparation of 4-(diacetoxymethyl)benzoic acid (43)
In a three-necked round bottom flask equipped with a
AcO
mechanical stirrer and immersed in an ice-salt bath were placed
AcO
glacial acetic acid (96.0 ml, 1.68 mol), acetic anhydride (116 ml,
1.23 mol) and p-toluic acid (10.0 g, 73.5 mmol). To this solution
3
4
2
1
CO 2H
43
C12H12O6
Mol. Wt.: 252.2201
was added slowly with stirring concentrated sulfuric acid (18.0 ml, 0.33 mol). When
the mixture had cooled to 5 ºC, chromium trioxide (20.0 g, 0.20 mol) was added in
small portions at such a rate that the temperature did not rise above 10 ºC. The
chromium trioxide was added over an hour and the reaction was left to stir for three
more hours at 5 ºC. The reaction mixture was then poured onto crushed ice (200 ml)
and the resulting mixture was then extracted with toluene (4  50 ml). The combined
organic layers were dried (MgSO4) and the toluene was evaporated in vacuo. The
residue was recrystallised from hot toluene, affording the target material, 4(diacetoxymethyl)benzoic acid 43 as colourless needles (6.65 g, 26.4 mmol, 37%): Rf
(2:3, Et2O-CHCl3) 0.53; m.p. 86-87 ºC; max (KBr disk)/cm-1 2500-3040, 3039, 1772,
1692, 1619, 1585, 1494, 1426, 1375, 1290, 1245, 1200, 1126, 1064, 1007, 968, 937; H
(200 MHz, C2D6SO) 2.12 (6 H, s, 2  CH3), 7.61 (1 H, s, CH(OAc)2), 7.63 ( 2 H, d, J
127
8.3, H-3 and H-5), 8.00 (1 H, d, J 8.4, H-2 and H-6); C (50 MHz, C2D6SO) 20.96 (2 
CH3), 104.6 (CH(OAc)2), 127.15 (CH-3’ and CH-5’), 130.02 (CH-2’ and CH-6’),
140.06 (C-4), 167.20 (CO2H), 169.11 (C–acetate); m/z (EI) 252 (2%, M+); (Found C,
57.27; H, 4.79%. C12H12O6 requires C, 57.14; H, 4.80%).
Preparation of 4-formylbenzoic acid (41)
3
A solution of diacetate 43 (5.00 g, 19.8 mmol), ethanol (11.1 ml),
water (11.1 ml, 0.62 mmol) and concentrated H2SO4 (1.1 ml, 20.1
mmol) was refluxed for 30 minutes. The reaction mixture was
cooled and the resulting crystals were collected by filtration and
OHC
2
4
1
CO 2H
41
C 8 H 6 O3
Mol. Wt.: 150.1314
washed with water. Recrystallisation from hot methanol afforded 4-formylbenzoic
acid 41 as colourless needles (2.71 g, 18.1 mmol; 91%): Rf (Et2O-CHCl3, 2:3) 0.21;
m.p. 146-147 ºC; max (KBr disk)/cm-1 2500-3200, 1690, 1575, 1504, 1431, 1393,
1290, 1205, 1125, 1110, 1015, 931, 851, 790; H (200 MHz, C2D6SO) 5.50 (1 H, bs,
CO2H), 8.01 (2 H, d, J 8.3, H-3 and H-5), 8.13 (2 H, d, J 8.2, H-2 and H-6), 10.01 (1 H,
s, CHO); C (50 MHz, C2D6SO) 129.99 (CH-3 and CH-5), 130.36 (CH-2 and CH-6),
136.04 (C-1), 139.31 (C-4), 167.01 (CO2H), 193.45 (CHO); m/z (EI) 149 (8%, [M –
H]+).
Formation of 4-(1-hydroxy-3-oxobutyl)benzoic acid allyl ester(42) during the
preparation of 4-formylbenzoic acid allyl ester (40)
A suspension of 4-formylbenzoic acid (41; 1.50 g, 10.0
O
mmol), allyl bromide (1.45 g, 12.0 mmol) and potassium
O
carbonate (1.38 g, 9.99 mmol) in acetone (20 ml) was
refluxed for 48 hours. The reaction mixture was then filtered
2 3
1
4
OH
O
6 5
42
C14H16O4
Mol. Wt.: 248.2744
through celite and the filtrate was evaporated under reduced
pressure. The residue was taken up in CHCl3 and washed with saturated NaHCO3
solution (2  20 ml). The aqueous layers were extracted with CHCl3 (20 ml). The
combined organic layers were dried (MgSO4) and the solvent was evaporated under
reduced pressure, affording a yellow oil which was identified as being 4-(1-hydroxy-3-

commercial reference 147 ºC (Aldrich)
128
oxobutyl)benzoic acid allyl ester 42 (1.50 g, 6.04 mmol; 60%): Rf (CHCl3) 0.33; max
(neat)/cm-1 3483, 3017, 2940, 1712, 1648, 1611, 1577, 1509, 1414, 1362, 1266, 1176,
1076, 1076, 1017, 969, 937, 858, 762, 706; H (200 MHz, CDCl3) 2.16 (3 H, s,
COCH3), 2.79–2.83 (2 H, m, J 4.0, CHOHCH2CO), 3.63 (1 H, bs, OH), 4.78 (2 H, d, J
5.5, CO2CH2), 5.14-5.20 (1 H, m, CHOH), 5.26 (1 H, dd, J 10.4 and 0.8, vinylic-CH2),
5.37 (1 H, dd, J 17.4 and 1.4, vinylic-CH2), 6.00 (1 H, ddt, J 17.3, 10.5 and 5.4, vinylicCH), 7.39 (2 H, d, J 8.0, H-5 and H-3), 7.99 (1 H, d, J 8.1, H-2 and H-6); C (50 MHz,
CDCl3) 30.68 (COCH3), 51.61 (CHOHCH2CO), 65.47 (CO2CH2CH), 69.29 (CHOH),
118.16 (vinylic-CH2), 125.45 (CH-3 and CH-5), 129.28 (C), 129.83 (CH-2 and CH-6),
132.09 (vinylic-CH), 147.81 (C), 165.9 (CO2CH2), 198.2 (CH2COCH3); m/z (EI) 248
(22%, M+) (Found M 248.1055. C14H16O4 requires 248.1049).
Preparation of 4-allyloxybenzaldehyde (45)
4-Allyloxybenzaldehyde was prepared by another member of
the group (J.K, 20/02/1998): H (200 MHz, CDCl3) 4.61 (2 H, dt,
J 5.2 and 1.5, OCH2), 5.34 (1 H, dq, J 10.5 and 1.5, vinylicCH2), 5.44 (1 H, dq, J 17.2 and 1.5, vinylic-CH2), 6.06 (1 H, ddt,
2 3
OHC
1
4
O
6 5
45
C10H10O2
Mol. Wt.: 162.1852
J 17.3, 10.5 and 5.2, vinylic-CH), 7.01 (2 H, d, J 8.8, H-3 and H5), 7.83 (2 H, d, J 8.8, H-2 and H-6), 9.90 (1 H, s, CHO).
Preparation of (4-allyloxyphenyl)dipyrromethane (46)
4-Allyloxybenzaldehyde 45 (2.50 g, 15.4 mmol), pyrrole
(10.3 g, 154 mmol) and trifluoroacetic acid (175 mg, 1.54
3' 2'
O
4'
1'
NH
1
NH
mmol) were refluxed in toluene (50 ml) for 1.5 hours.
Toluene and pyrrole were then removed by conventional
distillation on a water pump.
The crude material was
46
C18H18N2O
Mol. Wt.: 278.3484
submitted to purification by flash column chromatography on silica (Et2O-light
petroleum, 1:4 eluent). The target dipyrromethane 46 was afforded as a brown solid
(2.26 g, 8.12 mmol; 53%): m.p. 132-133 ºC; Rf (1:1, EtOAc-light petroleum) 0.8; H
(200 MHz, CDCl3) 4.52 (2 H, dt, J 5.2 and 5.2, OCH2), 5.29 (1 H, dq, J 10.4 and 1.3,
vinylic-CH2), 5.41 (1 H, dq, J 17.2 and 1.5, vinylic-CH2), 5.43 (1 H, s, H-1), 5.89-5.93
(2 H, m, pyrrole CH), 6.03 (1 H, ddt, J 17.2, 10.5 and 5.2, vinylic-CH), 6.15 (2 H, q, J
129
2.7, pyrrole CH), 6.67-6.71 (2 H, m, pyrrole CH), 6.86 (2 H, d, J 8.7, H-3’ and H-5’),
7.12 (2 H, d, J 8.7, H-2’ and H-6’), 7.91 (2 H, bs, 2  NH); m/z (EI) 277 (14%, [M-H]+)
236 (17%, [M - C3H2]+) (Found M 278.1414. C18H18N2O requires 278.1419).
Preparation of 5,15-bis(allyloxyphenyl)-10,20-diphenylporphyrin (47)
(4-Allyloxyphenyl)dipyrromethane (46; 1.74 g, 6.25 mmol) and benzaldehyde (775
mg, 7.37 mmol) were refluxed in propionic acid (50 ml) for 30 minutes. Propionic acid
was then removed in vacuo and the residue was submitted to a flash column
chromatography on silica (CH2Cl2-light petroleum, 1:1-4:1 gradient). Four porphyrins
were isolated as lustrous purple solids.
5,10,15,20-Tetraphenylporphyrin 27 (52.5 mg, 0.08 mmol; 1.3%) eluted first in 1:1
CH2Cl2-light petroleum: Rf (CHCl3) 0.97.
5-(4-Allyloxyphenyl)-10,15,20-triphenylporphyrin 49 (74.3 mg, 0.11 mmol; 1.8%)
eluted second in 3:2 CH2Cl2-light petroleum: Rf
(CHCl3) 0.94; max (CHCl3)/nm (log ε) 305
2
(4.16), 318 (3.84), 4.19 (5.56), 465 (2.76), 516
(4.10), 534 (3.46), 550 (3.76); H (400 MHz,
CDCl3) –2.67 (2 H, s, 2  NH), 4.72 (2 H, d, J
3'
O 4'
3
2' 4
1'
5
1
N
20
HN
NH
N
15
10
5.3, OCH2), 5.35 (1 H, dd, J 10.5 and 1.5,
vinylic-CH2), 5.52 (1 H, dd, J 17.1 and 1.6,
vinylic-CH2), 6.18 (1 H, ddt, J 17.2, 10.4 and
49
C47H34N4O
Mol. Wt.: 670.7992
5.3, vinylic-CH), 7.20 (2 H, d, J 8.5, H-3’ and H5’), 7.74-7.76 (9 H, m, 6  meta Ph-H and 3  para Ph-H), 8.03 (2 H, d, J 8.5, H-2’ and
H-6’), 8.11-8.16 (6 H, m, 6  ortho Ph-H), 8.74-8.81 (8 H, m, pyrrolic-H); C (100
MHz, CDCl3) 69.14 (OCH2), 112.97 (CH-3’ and CH-5’), 118.00 (vinylic-CH2), 119.96
(meso–C), 120.01 (C), 120.07 (C–10 and C–20), 126.66 (meta Ph-CH), 127.68 (para
Ph-CH), 131.03 (pyrrole CH), 133.36 (vinylic-CH), 134.55 (ortho Ph-CH), 134.70 (C–
1’), 135.58 (CH-2’ and CH-6’), 142.19 (2  Ph–C), 142.21 (1  Ph–C), ~ 147 (C),
158.47 (C–4’); m/z (ESI) 671 (100%, MH+).
130
The target 5,15-bis(4-allyloxyphenyl)-10,20-diphenylporphyrin 47 (157 mg, 0.22
mmol; 3.5%) eluted third with 3:2
CH2Cl2-petroleum: Rf
(CHCl3) 0.89;
max (CHCl3)/nm (log ε) 420 (5.65), 518
2
3'
(5.16), 538 (3.40), 553 (3.88), 577
1'
O 4'
(2.70), 592 (3.61); max (KBr disk)/cm-1
2'
3
4
20
1
N
5
HN
NH
3023, 1718, 1603, 1558, 1504, 1471,
N
15
O
10
1349, 1283, 1239, 1175, 956, 923, 799,
740; H (200 MHz, CDCl3) <0 (2 H, s, 2
47
C50H38N4O2
Mol. Wt.: 726.8625
 NH), 4.72 (4 H, d, J 5.3, 2  OCH2),
5.35 (2 H, dd, J 10.5 and 1.4, 2 
vinylic-CH2), 5.52 (2 H, dd, J 17.2 and 1.5, 2  vinylic-CH2), 6.18 (2 H, ddt, J 17.2,
10.4 and 5.3, 2  vinylic-CH), 7.20 (4 H, d, J 8.5, 2  H-3’ and 2  H-5’), 7.74-7.76 (6
H, m, 4  meta Ph-H and 2  para Ph-H), 8.03 (4 H, d, J 8.5, 2  H-2’ and 2  H-6’),
8.11-8.16 (4 H, m, 4  ortho Ph-H), 8.74-8.81 (8 H, m, pyrrolic-H); C (50 MHz,
CDCl3) 69.04 (OCH2), 112.85 (CH-3’ and CH-5’), 117.94 (vinylic-CH2), 119.84
(meso–C), 126.66 (meta Ph-CH), 127.57 (para Ph-CH), 130.79 (pyrrole CH), 133.26
(vinylic-CH), 134.47 (ortho Ph-CH), 134.47 (C–1’), 135.50 (CH-2’ and CH-6’), 142.35
(2  Ph–C), 158.35 (C–4’); m/z (ESI) 728 (100%, [M + 2H]+); (Found C, 80.87; H,
5.23; N, 7.36%. C50H38N4O2 requires C, 82.62; H, 5.27; N, 7.71%).
5-(4-Allyloxyphenyl)-10,15,20-triphenylporphyrin 50 (63.5 mg, 0.08 mmol; 1.3%)
eluted fourth with 4:1 CH2Cl2-petroleum:
Rf (CHCl3) 0.84; max (CHCl3)/nm (log
2
ε) 421 (5.69), 517 (4.17), 538 (3.39), 555
3'
O 4'
(3.93); H (200 MHz, CDCl3) <0 (2 H, s,
2  NH), 4.72 (6 H, d, J 5.3, 3  OCH2 ),
3
2' 4
1'
5
20
1
N
HN
NH
N
15
O
10
5.35 (3 H, dd, J 10.5 and 1.6, 3  vinylicCH2), 5.52 (3 H, dd, J 17.2 and 1.6, 3 
O
vinylic-CH2), 6.18 (3 H, ddt, J 17.2, 10.4
and 5.2, 3  vinylic-CH), 7.20 (6 H, d, J
50
C53H42N4O3
Mol. Wt.: 782.9257
8.5, 3  H-3’ and 3  H-5’), 7.74-7.76 (3
H, m, 2  meta Ph-H and para Ph-H),
8.03 (6 H, d, J 8.5, 3  H-2’ and 3  H-6’), 8.11-8.16 (2 H, m, 2  ortho Ph-H), 8.74131
8.81 (8 H, m, pyrrolic-H); C (50 MHz, CDCl3) 69.02 (OCH2), 112.85 (CH-3’ and CH5’), 117.94 (vinylic-CH2), 119.72 (meso–C), 126.60 (meta Ph-CH), 127.58 (para PhCH), 130.97 (pyrrole CH), 133.27 (vinylic- CH), 134.50 (ortho Ph-CH), 134.66 (C–1’),
135.51 (CH-2’ and CH-6’), 142.19 (1  Ph–CH), 142.21 (C), 158.35 (C–1’); m/z (ESI)
783 (100%, MH+).
Preparation
of
[5,15-bis(4-allyloxyphenyl)-10,20-diphenylporphyrinato]zinc(II)
(52)
5,15-Bis(allyloxyphenyl)-10,20diphenylporphyrin 47 (75.3 mg, 0.10
2
mmol), zinc chloride (260.8 mg, 1.24
3'
O 4'
mmol) and triethylamine (62.9 mg, 0.63
mmol) were refluxed in CH2Cl2 (20 ml)
3
2' 4
1'
5
1
20
N
N
15
Zn
N
O
N
10
for 2.5 hours. The solvent was removed
under reduced pressure and the crude
52
C50H36N4O2Zn
Mol. Wt.: 790.2366
residue was purified by flash column
chromatography on silica (CHCl3-light
petroleum 3:2 eluent). The metallated porphyrin (52) eluted following residual traces of
the non-metallated starting material (47) but was contaminated with traces of
triethylammonium salt. The latter was removed by treating a CH2Cl2 solution of the
porphyrin with basic resin (Amberlite IRA-67, free base), filtration and evaporation.
The target [5,15-bis(4-allyloxyphenyl)-10,20-diphenylporphyrinato]zinc(II) (52) was
thus obtained as a lustrous purple powder (75.6 mg, 0.09 mmol; 96%): R f (CHCl3) 0.86;
max (CHCl3)/nm (log ε) 424 (5.55), 532 (3.47), 557 (3.85); max (KBr disk)/cm-1 2932,
1715, 1602, 1548, 1503, 1467, 1347, 1283, 1240, 1174, 956, 923, 797, 738; H (200
MHz, CDCl3) 4.72 (4 H, d, J 5.32, 2  OCH2), 5.35 (2 H, dd, J 10.5 and 1.6, 2 
vinylic-CH2), 5.52 (2 H, dd, J 17.2 and 1.6, 2  vinylic-CH2), 6.18 (2 H, ddt, J 17.2,
10.4 and 5.2, 2  vinylic-CH), 7.20(4 H, d, J 8.5, 2  H-3’ and 2  H-5’), 7.74-7.76 (6
H, m, 4  meta Ph-H and 2  para Ph-H), 8.03 (4 H, d, J 8.5, 2  H-2’ and 2  H-6’),
8.11-8.16 (4 H, m, 4  ortho Ph-H), 8.74-8.81 (8 H, m, pyrrolic-H); m/z (ESI) 791
(100%, MH+); (Found C, 75.83; H, 4.51; N, 6.95 %. C50H36N4O2Zn requires C, 75.99;
H, 4.59; N, 7.09%).
132
Attachment of 5,15-bis(4-allyloxyphenyl)-10,20-diphenylporphyrin
Ph
O
Si
O
OMe
S
N
O
HN
NH
N
O
Ph
According to general method A (route 1, Scheme 31):
Porphyrin 47 (300 mg, 0.41 mmol), (3-mercaptopropyl)trimethoxysilane (111 mg,
0.56 mmol) and AIBN (6 mg, 0.02 mmol) were refluxed in acetonitrile (10 ml) for 6
hours. The solvent was then removed in vacuo and the 1H NMR spectrum of the
residue showed that only unreacted starting materials were present. The reaction was
repeated using a larger excess of (3-mercaptopropyl)trimethoxysilane (805 mg, 4.1
mmol) for the same quantity of porphyrin 47. Again, the 1H NMR spectrum of the
crude material showed that only unreacted starting material were present.
According to general method B (route 2, Scheme 31):
Dried silica gel (200 mg) heated with 3-mercaptopropyl)trimethoxysilane (41.2 mg,
0.21 mmol) in dry toluene (5 ml) in a sealed tube at 110 ºC overnight. The resulting
mercaptopropyl-modified silica was collected by filtration and washed according to the
general method: Found C, 2.64; H, 0.83%. The mercaptopropyl-modified silica was
heated with 5,15-bis(4-allyloxyphenyl)-10,20-diphenylporphyrin (145 mg, 0.2 mmol)
in the presence of AIBN (4.0 mg, 0.01 mmol) in dry acetonitrile (5 ml) at 110 ºC in a
sealed tube for 6 hours. The resulting silica was filtered and washed according to the
general method: (Found C, 28.89%; H, 2.43%; N, 2.35%).
Test reaction A: To assess whether the radical initiator is necessary for covalent
attachment of porphyrin 47 to the mercaptopropyl-modified silica.
Dried silica gel (50 mg) was heated with (3-mercaptopropyl)trimethoxysilane (10.3
mg, 0.05 mmol) in dry toluene according to the general method. After filtration and
washing the silica gel was subjected to elemental analysis: Found C, 2.90; H, 0.87%.
The mercaptopropyl-modified silica was then heated for 6 hours with porphyrin 47
(36.0 mg, 0.05 mmol) in dry acetonitrile (1.5 ml) in a sealed tube at 110 ºC in the
absence of AIBN. The recovered silica gel was washed, dried and submitted for
elemental analysis: Found C, 4.86; H, 1.06; N, 0%.
133
Test reaction B: To assess whether the washing method is efficient in removal of
physisorbed material on the surface of the silica.
Dry silica gel (50 mg) was treated with porphyrin 47 (36 mg, 0.05 mmol) in
acetonitrile (1.5 ml) in a sealed tube at 110 ºC overnight. The silica was recovered by
filtration, washed and dried according to the general method and submitted for
elemental analysis: Found C, 0.75; H, 0.43; N, 0.0%.
Attachment of [5,15-bis(4-allyloxyphenyl)-10,20-diphenylporphyrinato]zinc (II)
(52)
Ph
O
N
N
S
Si
O O
Zn
N
O
N
S
S
Ph
OMe
Si OMe
O O
MeO
Si
O O
Dried silica (300 mg) and (3-mercaptopropyl)trimethoxysilane (61.8 mg, 31.5 mmol)
were heated in dry toluene (7 ml) at 110 ºC overnight. The resulting mercaptopropylmodified silica was collected by filtration, washed and dried according to the general
method before being submitted for elemental analysis: Found C, 3.00; H, 0.93%. A
sample (200 mg) of this mercaptopropyl–modified silica was heated with Zn-porphyrin
52 (58.3 mg, 7.45 mol) in the presence of AIBN (3 mg, 11 mol) in acetonitrile (5 ml)
at 110 ºC for 6 hours. The zinc porphyrin-modified silica was then collected by
filtration, washed and dried according to the general method before being submitted for
elemental analysis: Found C, 5.99; H, 1.10; N, 0.17%.
4.3
SURFACE ATTACHMENT VIA MESO-(P-PENTENYLOXYPHENYL GROUPS).
4.3.1 Attachment of 4-(pent-4-enyloxyphenyl)benzaldehyde (53).
Preparation of 4-(pent-4-enyloxy)benzaldehyde (53)
2
4-Hydroxybenzaldehyde (2.05 g, 16.7 mmol), 5 bromoOHC
pent-1-ene (3.00 g, 20.1 mmol) and potassium carbonate
(2.32 g, 16.7 mmol) were boiled in acetone for 24 hours.
134
1
3
4
O
53
C12H14O2
Mol. Wt.: 190.2384
The reaction mixture was then filtered though celite and the filtrate was evaporated.
The residue was then submitted to a flash column chromatography on silica gel (CHCl 3light petroleum 1:1 eluent). The target 4-(pent-4-enyloxy)benzaldehyde (53) was
afforded as a colourless oil (2.95g, 15.5 mmol; 93%): Rf (CHCl3) 0.29; max (neat)/cm-1
3077, 2943, 2738, 1686, 1601, 1577, 1059, 1470, 1428, 1394, 1312, 1258, 1160, 1110,
1014, 916, 857, 833; H (200 MHz, CDCl3) 1.92 (2 H, p, J 6.8, OCH2CH2), 2.25 (2 H,
q, J 7.1, OCH2CH2CH2CH), 4.05 (2 H, t, J 6.5, OCH2), 5.06 (1 H, dq, J 10.0 and 1.3,
olefinic-CH2), 5.26 (1 H, dq, J 17.1 and 1.5, olefinic-CH2), 5.85 (1 H, ddt, J 17.0, 10.2
and J 6.6, olefinic-CH), 6.98 (2 H, d, J 8.7, H-3 and H-5), 7.83 (2 H, d, J 8.8, H-2 and
H-6), 9.89 (1H, s, CHO); C (50 MHz, CDCl3) 28.06 (OCH2CH2CH2CH), 29.87
(OCH2CH2CH2CH), 67.42 (OCH2), 114.64 (CH-3 and CH-5), 115.39 (olefinic-CH2),
129.68 (C–1), 131.90 (CH-2 and CH-6), 137.34 (olefinic-CH), 164.04 (C–4), 190.75
(CHO); m/z (EI) 190 (49%, M+) (Found M 190.1025. C12H14O2 requires 190.0994).
Preparation of 4-{5-[3-(trimethoxysilanyl)propylsulfanyl]pentyloxy}benzaldehyde
(54)
4-(Pent-4-enyloxy)benzaldehyde
2 3
53
OHC
(569
mg,
3.00
mmol),
mercaptopropyl)trimethoxysilane
1
4
O
S
(3-
Si
(OCH 3)3
54
C18H30O5SSi
Mol. Wt.: 386.5793
(587
mg, 3.00 mmol) and AIBN (12.3 mg,
0.07 mmol) were heated in dry acetonitrile (5 ml) at 85ºC for 6 hours under nitrogen.
At the end of the reaction, solvent and unreacted volatile materials were evaporated
under reduced pressure. The target material 54 was afforded as a colourless oil (980
mg, 2.53 mmol; 84% in > 95% purity): Rf (1:1, light petroleum-CHCl3) 0.22; H (200
MHz, CDCl3) 0.61–0.63 (2 H, m, J 8.2, SCH2CH2CH2Si), 1.46-1.69 (8 H, m,
OCH2CH2CH2CH2CH2S,
OCH2CH2CH2CH2CH2S,
OCH2CH2CH2CH2CH2S,
and
SCH2CH2CH2Si), 2.40 (4 H, 2  t overlapping, J 7.2, CH2CH2SCH2CH2), 3.43 (9 H, s,
O(CH3)3), 3.90 (2 H, t, J 6.3, OCH2CH2CH2CH2CH2S), 6.85 (2 H, d, J 8.7, H-3 and H5), 7.68 (2 H, d, J 8.8, H-2 and H-6), 9.7 (1 H, s, CHO).
135
4.3.2 surface attachment with simple molecules carrying pentenyloxy groups.
According to general method A:
(MeO) 3Si
S
CHO
3O
54
OH
OH
O
Si
O
OMe
Method A
S
O
CHO
Dried silica gel (200 mg) was reacted with 4-{5-[3-(trimethoxysilanyl)prop–3–
ylsulfanyl)pent-1-yloxy}benzaldehyde (54; 74.5 mg, 0.21 mmol) in dry toluene (5 ml)
according to the general method.
The resulting silica was collected by filtration,
washed and dried according to the general method and submitted for elemental analysis:
Found C, 7.92; H, 1.28%.
According to general method B:
(MeO)3Si
OH
OH
OHC
SH
21
Method A
O
Si
O
OMe
SH
O
53
Method B
O
Si
O
OMe
S
3
O
CHO
Dried silica gel (200 mg) and (3-mercaptopropyl)trimethoxysilane (41.2 mg, 0.21
mmol) were heated in dry toluene (5 ml) in a sealed tube at 110 ºC overnight. The
resulting mercaptopropyl-modified silica was collected by filtration, washed and dried
according to the general method and submitted for elemental analysis: Found C, 2.88;
H, 0.93%. This mercaptopropyl-modified silica, 4-(pent-4-enyloxy) benzaldehyde (40.0
mg, 0.21 mmol) and AIBN ( 3 mg, 11 mol) were heated in dry acetonitrile (5 ml) in a
sealed tube for 6 hours (110 ºC). The resulting silica was collected by filtration, washed
and dried according to the general method and submitted for elemental analysis: Found
C, 7.53; H, 1.27%.
4.3.3 Surface attachment with porphyrins carrying pentenyloxy groups.
Preparation of [4-(pent-4-enyloxy)phenyl]dipyrromethane (55)
4-(Pent-4-enyloxy)benzaldehyde (53; 2.00 g, 10.5 mmol), pyrrole (3.53 g, 52.6 mmol)
and trifluoroacetic acid (120 mg, 0.11 mmol) were boiled in dry toluene (40 ml) for 1.5
hours under nitrogen. Toluene and pyrrole were then removed by distillation on a water
136
pump and the residue was submitted to a flash column
chromatography (4:1 Et2O-light petroleum eluent).
(Pent-4-enyloxy)phenyl]dipyrromethane
55
2' 3'
[4was
obtained as a brown solid and purified by trituration in
light petroleum (2.42 g, 7.90 mmol; 75%): m.p. 186-
O
1'
4'
6' 5'
NH
1
NH
55
C20H22N2O
Mol. Wt.: 306.4016
187ºC; Rf (4:1, Et2O-light petroleum) 0.38; H (200 MHz,
CDCl3) 1.94 (2 H, p, J 7.0, OCH2CH2CH2CH), 2.29 (2 H, q, J 6.4, OCH2CH2CH2CH),
3.99 (2 H, t, J 6.4, OCH2), 5.07 (1 H, dd, J 10.2 and 1.5, olefinic-CH2), 5.13 (1 H, dd, J
17.2 and 1.5, olefinic-CH2), 5.39 (1 H, s, H-1), 5.89 (1 H, ddt, J 16.8, 10.1 and 6.5,
olefinic-CH), 5.93 (2 H, broad signal, pyrrole CH), 6.19 (2 H, q, J 2.8, pyrrole CH),
6.65–6.70 (2 H, m, J 4.0 and 2.5, pyrrole CH), 6.88 (2 H, d, J 8.6, H-3’ and H-5’),
7.14(2 H, d, J 8.6, H-2’ and H-6’), 7.92 (2H, bs, 2  NH); C (50 MHz, CDCl3) 28.39
(CH2), 31.00 (CH2), 43.04 (CH-1), 61.17 (OCH2), 107.03 (pyrrole CH), 108.25 (pyrrole
CH), 114.50 (CH-3’ and CH-5’), 115.22 (olefinic-CH2), 117.15 (pyrrole CH), 129.34
(CH-2’ and CH-6’), 132.97 (C), 134.13 (C), 137.80 (olefinic-CH), 157.89 (C–4’); m/z
(EI) 306 (100%, M+) (Found M 306.1729. C21H22N2O requires 306.1732).
Preparation of 10, 20-diphenyl-5, 15-bis[4-(pent-4-enyloxy)phenyl]porphyrin (56)
[4-(Pent-4-enyloxy)phenyl]dipyrromethane (55; 2.63 g, 8.60 mmol) and benzaldehyde
(1.10 g, 10.3 mmol) were refluxed in propionic acid (50 ml) for 40 minutes. Propionic
acid was removed under reduced pressure and the residue was submitted to flash
column chromatography on silica gel (CH2Cl2-light petroleum, 1:1-4:1 gradient). Four
porphyrins were isolated as lustrous purple solids.
5,10,15,20-Tetraphenylporphyrin 27 (12 mg, 0.02 mmol; 0.21%) eluted first in 1:1
CH2Cl2-light petroleum: Rf (CHCl3) 0.97.
5-[4-(Pent-4-enyloxy)phenyl]-10,15,20-triphenylporphyrin (15.2 mg, 0.02 mmol;
0.2%) eluted second in 1:1 CH2Cl2-light petroleum: Rf (CHCl3) 0.82; max (CHCl3)/nm
(log ε) 419 (5.54), 461 (2.94), 517 (4.10), 538 (3.34), 552 (3.81), 571 (2.94), 592 (3.59);
H (400 MHz, CDCl3) -2.76 (2 H, s, 2  NH), 2.09 (2 H, p, J 6.9, OCH2CH2), 2.41 (2 H,
q, J 7.2, OCH2CH2CH2CH), 4.26 (2 H, t, J 6.2, OCH2), 5.10 (1 H, dq, J 10.0 and 1.5,
olefinic-CH2), 5.18 (1 H, dq, J 17.2, J 1.7, olefinic-CH2), 5.98 (1 H, ddt, J 17.2, J 10.4, J
6.8, olefinic-CH), 7.25 (2 H, ~d, J 8.8, H-3’ and H-5’), 7.71-7.81 (9 H, m, 6  meta PhH and 3  para Ph-H), 8.10 (2 H, ~d, J 8.8, H-2’ and H-6’), 8.20-8.22 (6 H, m, 6 
137
ortho Ph-H), 8.82-8.91 (8 H, m, pyrrolic-H); C (100
MHz, CDCl3) 28.68 (CH2), 30.30 (CH2), 67.53
2
(OCH2), 112.76 (CH-3’ and CH-5’), 115.33 (olefinicCH2), 119.93 (meso–C), 120.06 (C–10 and C–20),
3'
O
4'
3
2' 4
1'
5
1
N
NH
120.15 (meso–C), 126.67 (meta Ph-CH), 127.68 (para
20
HN
N
15
10
Ph-CH), 131.02 (broad signal, pyrrolic-CH), 134.43
(C–1’), 134.56 (ortho Ph-CH), 135.61 (CH-2’ and
C49H38N4O
Mol. Wt.: 698.8524
CH-6’) 137.93 (olefinic-CH), 142.21 (Ph–C), 142.24
(Ph–C), 158.93 (C–4’); m/z (ESI) 699 (100%, MH+).
The target 10,20-diphenyl-5,15-bis[4-(pent-4-enyloxy)phenyl]porphyrin 56 (154.1
mg, 0.20 mmol; 2.3%) eluted third in 7:3 CH2Cl2light
petroleum:
Rf
(CHCl3)
0.47;
max
(CHCl3)/nm (log ε) 421 (5.71), 453 (3.49), 517
(4.31), 538 (3.79), 552 (4.10), 591 (9.81), 669
(2.59); max (KBr disk)/cm-1 3312, 2923, 1639,
2
3'
4'
O
3
2' 4
1'
5
20
1
N
HN
NH
N
15
O
10
1605, 1505, 1466, 1400, 1350, 1282, 1245, 1175,
966, 802, 737, 701; H (400 MHz, CDCl3) -2.76
(2 H, s, 2  NH), 2.07 (4 H, p, J 7.2, 2 
OCH2CH2CH2CH), 2.40 (4 H, ~ q, J 7.0, 2 
56
C54H46N4O2
Mol. Wt.: 782.9688
OCH2CH2CH2CH), 4.24 (4 H, td, J 6.2 and 1.2, 2 OCH2), 5.09 (2 H, dq, J 10.0, J 1.2,
2  olefinic-CH2), 5.18 (2 H, dq, J 17.2 and 1.2, 2  olefinic-CH2), 5.98 (2 H, ddt, J
17.2, J 10.2, J 6.8, 2  olefinic-CH), 7.25 (4 H, ~ d, J 8.8, 2  H-3’ and 2  H-5’), 7.717.81 (6 H, m, 4  meta Ph-H and 2  para Ph-H) 8.10 (4 H, ~ d, J 8.8, 2  H-2’ and 2 
H-6’), 8.20-8.22 (4 H, m, ortho Ph-H), 8.82-8.92 (8 H, m, pyrrolic-H); C (100 MHz,
CDCl3) 28.67 (CH2), 30.29 (CH2), 67.51 (OCH2), 112.74 (CH-3’ and CH-5’),
115.32(olefinic-CH2), 119.93 (C), 119.97 (C), 120.07 (C), 126.66 (CH, meta Ph-CH),
127.65 (para Ph-CH), 131.06 (broad signal, pyrrolic-CH), 134.44 (C–1’), 134.56 (CH,
ortho Ph-CH), 135.60 (CH-2’ and CH-6’) 137.93 (olefinic-CH), 142.26 (C), 142.29 (C),
158.90 (C–4’); m/z (ESI) 782 (100%, M+); (Found C, 82.56; H 5.99; N, 6.89%.
C54H46N4O2 requires C, 82.84; H, 5.92; N, 7.15%).
138
20-Phenyl-5,10,15-tris[4-(pent-4-enyloxy)phenyl] porphyrin (80.9 mg, 0.09 mmol;
1.15%) eluted fourth in 4:1 CH2Cl2-light
petroleum: Rf (CHCl3) 0.37; max (CHCl3)/nm
(log ε) 422 (5.60), 464 (3.56), 519 (4.22), 538
2
(3.76), 555 (4.06), 626 (3.34), 648 (3.94); H
(400 MHz, CDCl3) -2.76 (2 H, s, 2  NH), 2.07
3'
O
4'
3
2' 4
1'
5
20
1
N
HN
NH
15
N
(6 H, p, J 7.2, 3  OCH2CH2CH2CH), 2.40 (6 H,
O
10
~ q, J 7.0, 3  OCH2CH2CH2CH), 4.24 (6 H, t, J
6.2, 3  OCH2), 5.09 (3 H, dq, J 10.0 and 2.0, 3
O
 olefinic-CH2), 5.18 (3 H, dq, J 16.9 and 3.6, J
C59H54N4O3
Mol. Wt.: 867.0852
1.2, 3  olefinic-CH2), 5.98 (3 H, ddt, J 17.2,
10.2 and 6.8, 3  olefinic-CH), 7.25 (6 H, ~ d, J 8.8, 3  H-3’ and 3  H-5’), 7.71-7.81
(3 H, m, 2  meta Ph-H and para Ph-H) 8.10 (6 H, ~ d, J 8.8, 3  H-2’ and 3  H-6’),
8.21-8.21 (2 H, m, ortho Ph-H), 8.82-8.92 (8 H, m, pyrrolic-H); C (100 MHz, CDCl3)
28.63 (CH2), 30.26 (CH2), 67.43 (OCH2), 112.69 (CH-3’ and CH-5’), 115.29 (olefinicCH2), 119.99 (C), 119.87 (C), 119.99 (C), 126.63 (meta Ph-CH), 127.97 (para Ph-CH),
131.03 (broad signal, pyrrolic-CH), 134.45 (C–1’), 134.49 (CH, ortho Ph-CH), 135.57
(CH-2’ and CH-6’) 137.90 (olefinic-CH), 142.24 (C), 142.29 (C), 158.85 (C–4’); m/z
(ESI) 867 (100%,MH+).
Preparation of {10,20-diphenyl-5,15-bis[4-(pent-4-enyloxy)phenyl]porphyrinato}
zinc(II) (57)
10,20-Diphenyl-5,15-bis[4-(pent-4enyloxy)phenyl] porphyrin 56 (100 mg, 0.12
2
mmol) and zinc acetate dihydrate (280 mg, 1.2
mmol) were refluxed in CHCl3 (5 ml). TLC
monitoring showed that the reaction had reached
completion after 5 minutes. The solvent was then
3'
O 4'
3
2' 4
1'
5
1
20
N
N
Zn
N
N
15
O
10
removed in vacuo and the residue was submitted
to flash column chromatography on silica (CHCl3
eluent). After trituration with light petroleum the
target
compound,
57
C54H44N4O2Zn
Mol. Wt.: 846.3429
{10,20-diphenyl-5,15-bis[4-(pent-4-
enyloxy)phenyl]porphyrinato}zinc(II) 57, was obtained as a lustrous fuchsia powder
139
(92.5 mg, 0.11 mmol; 92%): Rf (CHCl3) 0.46; max (CHCl3)/nm (log ε) 255 (3.84), 372
(3.07), 424 (5.26), 552 (3.75), 575 (2.37), 595 (3.07); H (400 MHz, CDCl3) 2.07 (4 H,
p, J 7.2, 2  OCH2CH2CH2CH), 2.40 (4 H, ~ q, J 7.0, 2  OCH2CH2CH2CH), 4.24 (4
H, td, J 6.2 and 1.2, 2 OCH2), 5.09 (2 H, dq, J 10.0 and 1.2, 2  olefinic-CH2), 5.18 (2
H, dq, J 17.2 and 1.2, 2  olefinic-CH2), 5.98 (2 H, ddt, J 17.2, 10.2 and 6.8, 2 
olefinic-CH), 7.25 (4 H, ~ d, J 8.8, 2  H-3’ and 2  H-5’), 7.71-7.81 (6 H, m, 4  meta
Ph-H and 2  para Ph-H), 8.10 (4 H, ~ d, J 8.8, 2  H-2’ and 2  H-6’), 8.20-8.22 (4 H,
m, ortho Ph-H), 8.82-8.92 (8 H, m, pyrrolic-H); C (100 MHz, CDCl3) 28.68 (CH2),
30.29 (CH2), 67.51 (OCH2), 112.62 (CH-3’ and CH-5’), 115.29 (olefinic-CH2), 120.92
(C), 120.96 (C), 121.01 (C), 126.53 (meta Ph-CH), 127.46 (para Ph-H), 131.86 (
pyrrolic-CH), 132.03 (pyrrolic-CH), 134.42 (C–1’), 135.08 (ortho Ph-CH), 135.40 (CH2’ and CH-6’) 137.95 (olefinic-CH), 142.89 (C), 150.20 (C), 150.55 (C), 158.72 (C–4’);
m/z (ESI) 846 (100%, M+); (Found C, 76.44; H, 5.11, N, 6.47%. C54H44N4O2Zn requires
C, 76.63; H, 5.24; N, 6.62%).
Attachment of 10,20-diphenyl-5,15-bis[4-(pent-4-enyloxy)phenyl]porphyrin (56)
According to general method B:
Ph
O
Si
O
OMe
S
3
N
O
HN
NH
N
O
Ph
Dried silica gel (300 mg) was heated with (3-mercaptopropyl)trimethoxysilane (21;
300 mg, 1.5 mmol) in dry toluene (7 ml) in a sealed tube at 110 ºC overnight. The
resulting mercaptopropyl-modified silica was collected by filtration, washed and dried
according to the general method before being submitted: Found C, 4.05; H, 1.16%.
This mercaptopropyl-modified silica was heated with 10,20-diphenyl-5,15-bis[4-(pent4-nyloxyphenyl)porphyrin (56; 100 mg, 0.13 mmol) in the presence of AIBN (4 mg,
0.01 mmol) in dry acetonitrile (5 ml) at 110 ºC in a sealed tube for 6 hours. The
resulting silica was collected by filtration, washed and dried according to the general
method.
Four different batches were withdrawn of this same sample and were
submitted for elemental analysis. The results are summarised in table C.
140
Batch number
Carbon content
Hydrogen content
Nitrogen content
(% from elemental analysis)
1
2
3
4
21.07
21.39
21.97
21.47
2.14
2.23
2.37
2.16
1.56
1.58
2.12
1.52
Table C
Attachment of [10,20-diphenyl-5,15-bis(4-pent-4-enyloxyphenyl)porphyrinato]zinc
(II)
According to general method B:
Ph
3
O
S
Si OMe
O O
N
N
Zn
N
O
3
N
S
Ph
Si OMe
O O
S
MeO Si
O O
Dried silica gel (500 mg) and (3-mercaptopropyl)-trimethoxysilane (103 mg, 0.52
mmol) were heated in dry toluene (10 ml) at 110ºC overnight.
The resulting
mercaptopropyl-modified silica was collected by filtration, washed and dried according
to the general method. Four different batches were withdrawn of this same sample and
were submitted for elemental analysis. The results are summarised in table D. A
portion of the mercaptopropyl–modified silica gel (200 mg) was heated with zinc
porphyrin 57 (108 mg, 0.13 mmol) in the presence of AIBN (3 mg, 11 mol) in
acetonitrile (5 ml) at 110 ºC for 6 hours. The zinc porphyrin-modified silica was then
collected by filtration, washed and dried according to the general method before being
submitted for elemental analysis: Found C, 5.49%; H, 1.02; N, 0.0%.
141
Batch number
Carbon content
Hydrogen content
(% from elemental analysis)
1
2
3
4
2.80
2.70
2.90
2.91
0.90
0.87
0.90
0.90
Table D
4.3.4 Solid-state NMR studies
Preparation of silica gel
Silica gel was purchased
from Sigma-Aldrich: TLC
OH
single hydroxyl sites
O
standard
grade
without
Si
binder, surface area 500
Si
O
O
Si
OH
O
Si
Si
O
OH
geminal hydroxyl sites
O
Si
O
lattice sites
2
m /g, particle size 2-25
m, pore diameter 60 Å, pore volume 0.75 cm3/g. Silica gel was dried in vacuo (0.1
mmHg, 110 ºC, 24 hours) prior to use: (Found C, 0.05; H, 0.45%); 29Si solid-state NMR
(59.58 MHz, Direct Polarisation) –111.0 (71.1%, lattice sites), -101.2 (26.7%, single
sites), -91.6 (2.2%, geminal sites).
Preparation of phenyldipyrromethane (65)
Benzaldehyde (6.00 g, 56.5 mmol), trifluoroacetic acid (644.2 mg,
NH
0.56 mmol) and pyrrole (51.28 g, 0.76 mol) were stirred at room
1
NH
temperature under nitrogen for 2 hours. Pyrrole was then removed
by conventional distillation and the residue was submitted to a flash
column chromatography on silica (1.5:8.5 Et2O-light petroleum
eluent).
65
C15H14N2
Mol. Wt.: 222.2851
The isolated product was recrystallised from hot Et2O to afford
phenyldipyrromethane60 as a pale yellow solid (65; 5.00 g, 22.5 mmol; 40%): Rf (1:4
Et2O-light petroleum) 0.56; H (200 MHz, CDCl3) 5.46 (1 H, s, H-1), 5.87-5.93 (2 H, m,
pyrrole CH), 6.10 (2 H, q, J 2.9, pyrrole CH), 6.62-6.66 (2 H, m, pyrrole CH), 7.25 (5
H, m, Ph-H), 7.83 (2 H, bs, 2  NH).
142
Preparation of 10, 20-diphenyl-5,15-bis(4-hydroxyphenyl)porphyrin (59)
Phenyldipyrromethane (65; 3.00 g, 13.5 mmol) and 4-hydroxybenzaldehyde (1.65 g,
14.0 mmol) were refluxed in propionic acid (70 ml) for 30 minutes. Propionic acid was
then removed in vacuo and the crude material was submitted to flash column
chromatography on silica gel (CH2Cl2-petroleum 1:19:1 gradient).
Three porphyrins were isolated as
lustrous purple solids.
2
5-(4-Hydroxyphenyl)-10,15,20-triphenylporphyrin
1
3
2' 4
1'
5
3'
HO 4'
(87.2 mg, 0.14 mmol; 1%) eluted first in 4:1 CH2Cl2-
N
20
HN
NH
light petroleum: Rf (CHCl3) 0.57; H (200 MHz,
N
15
10
CDCl3) 7.20 (2 H, d, J 8.0, H-3’ and H-5’), 7.62-7.70
(9 H, m, 6  meta Ph-H and 3  para Ph-H), 8.06 (2
C44H30N4O
Mol. Wt.: 630.7354
H, d, J 8.2, H-2’ and H-6’), 8.12-8.17 (6 H, m, 6 
ortho Ph-H), 8.75-8.82 (8 H, m, pyrrolic-H).
The target 5,15-bis(4-hydroxyphenyl)–10,20-diphenylporphyrin (59) was eluted
along with 5,10,15-Tris(4-hydroxyphenyl)-20-phenylporphyrin (66) in 9:1 CH2Cl2petroleum (339 mg). No further separation could be achieved. Rf (1:9, Et2O-CH2Cl2)
0.49 (59) 0.31 (66); H (200 MHz, CDCl3) 7.20 (1, d, J 8.0, H-3’ and H-5’), 7.62-7.70
(1.9, m, meta Ph-H and para Ph-H), 8.06 (1, d, J 8.15, H-2’ and H-6’), 8.12-8.17 (1, m,
ortho Ph-H), 8.75-8.82 (1.8, m, pyrrolic-H).
2
3'
HO 4'
2'
3
4
1'
5
1
2
20
3'
N
HN
NH
N
HO 4'
15
OH
3
2' 4
1'
5
20
1
N
HN
NH
N
15
10
10
OH
59
C44H30N4O2
Mol. Wt.: 646.7348
66
C44H30N4O3
Mol. Wt.: 662.7342
143
OH
Preparation of 4-bromobutyraldehyde (62)
Ozone gas was bubbled though a solution of 5-bromopent-1-ene
O
(3.00 g, 20.1 mmol) in dry CH2Cl2 (40 ml) at –78 ºC until the
H 1
solution turned a persistant blue due to excess ozone (after 45
minutes). Nitrogen gas was then bubbled through the solution to
3
2
Br
4
62
C4H7BrO
Mol. Wt.: 151.0018
remove the excess ozone. Triphenylphosphine (5.28 g, 20.1 mmol) was added to the
reaction mixture at –78 ºC and the reaction mixture was stirred at that temperature for
another 10 minutes before being allowed to warm to room temperature. Solvent was
evaporated and the residue was submitted to flash chromatography column on silica gel
(1:1 CHCl3-light petroleum eluent). The target aldehyde (62), which was unstable to
aerial oxidation, was obtained as a colourless oil (2.30 g, 15.2 mmol; 77%): Rf (1:1
CHCl3-light petroleum) 0.56; H (200 MHz, CDCl3) 2.16 (2 H, p, J 6.7, CH2-3), 2.66 (2
H, t, J 7.0, CH2-2), 3.44 (2 H, t, J 6.5, CH2-4), 9.81 (1 H, s, CHO); C (50 MHz, CDCl3)
24.83 (CH2-3), 32.70 (CH2-4), 42.01 (CH2-2), 200.70 (CHO).
Preparation of 13C-labelled methyl-triphenylphosphonium iodide (61)
Iodomethane-13C (5.00 g, 35.0 mmol) was added to a solution of
triphenylphosphine (9.17 g, 35.0 mmol) in toluene (70 ml). The
H313C P
reaction mixture was heated at 40 ºC overnight in a sealed flask.
I
The precipitate formed was collected by filtration and thoroughly
61
washed with light petroleum before being dried on an oil pump (0.1
mmHg)
for
24
hours.
13C-Labelled
methyl-
C1813CH18IP
Mol. Wt.: 405.2171
triphenylphosphonium iodide (61) was obtained as a white powder (13.67 g, 33.8
mmol; 98%): H (200 MHz, CDCl3) 3.24 (3 H, dd, J 135.0 and 13.2, 13CH3), 7.65-7.87
(15 H, m, Ph-H).
Preparation of 4-(tert-butyl-dimethylsilanyloxy)butan-1-ol (69)
1,4-Butanediol (6.00 g, 66.0 mmol) was added to a vigorously
1
HO
stirred suspension of sodium hydride (60% dispersion in
mineral oil, 2.66 g, 66.0 mmol) in dry THF (100 ml). The
3
2
O
4
Si
69
C10H24O2Si
Mol. Wt.: 204.3819
reaction mixture was stirred under argon for 45 minutes, at the
end of which time a white precipitate had formed. Tert-butyl-dimethylsilyl chloride
144
(10.0 g, 66 mmol) was added to the reaction mixture in one batch and the mixture was
stirred at room temperature for a further 45 minutes. The reaction mixture was then
diluted in Et2O (400 ml) and successively washed with saturated NaHCO3 solution (2 
50 ml) and brine (2  50 ml ). The organic layer was dried (Na2SO4) and the solvent
was removed under reduced pressure.
The crude material was submitted to flash
column chromatography on silica (3:7 EtOAc-light petroleum eluent). 4-(Tertbutyldimethylsilyloxy)butan-1-ol (69)120 was obtained as a colourless oil (13.40 g,
65.6 mmol; 99%): Rf (3:7 EtOAc-light petroleum) 0.41; max (neat)/cm-1 3350, 2929,
1472, 1445, 1388, 1361, 1255, 118, 1101, 1006, 939, 836, 775, 663; H (200 MHz,
CDCl3) 0.01 (6 H, s, Si(CH3)2), 0.83 (9 H, s, C(CH3)3), 1.56-1.62 (4 H, m, CH2-2 and
CH2-3), 2.52 (1 H, bs, OH), 3.54-3.63 (4 H, m, CH2-1 and CH2-4); C (50 MHz, CDCl3)
– 6.00 (Si(CH3)2), 18.19 (C(CH3)3), 25.79 (C(CH3)3), 29.78 (CH2-2), 30.12 (CH2-3),
62.62 (CH2-1), 63.26 (CH2-4); m/z (EI) 205 (MH+), 147 (36.8 %, [M - C(CH3)3]+);
(Found M 205.1639. C10H25O2Si requires 205.1624).
Preparation of 4-(tert-butyldimethylsilyloxy)butyraldehyde (70)
A solution of dry DMSO (3.50 ml, 49.4 mmol) in dry CH2Cl2
(40 ml) was added dropwise to a solution of oxalyl chloride (2.35
ml, 26.8 mmol) in CH2Cl2 (50 ml) at -78 ºC under argon. The
mixture was left to stir under argon at -78 ºC for 20 minutes.
O
H
1
3
O Si
2
4
70
C10H22O2Si
Mol. Wt.: 202.3660
Then a solution of 4-(tert-butyldimethylsilyloxy)butan-1-ol (69;
5.00 g, 24.5 mmol) in CH2Cl2 (40 ml) was added dropwise to the reaction mixture at -78
ºC. The resulting solution was left to stir at -78 ºC for 25 minutes. Triethylamine (10.3
ml, 73.3 mmol) was then added to the reaction mixture at -78 ºC. Then the reaction
mixture was allowed to warm up to 0 ºC and left to stir for 1 hour. The reaction mixture
was diluted with CH2Cl2 (150 ml) and washed with water (2  50 ml). The aqueous
layer was extracted with further CH2Cl2 (2  50 ml) and the combined organic layers
were dried (Na2SO4). The solvent was removed in vacuo and the residue was submitted
to flash chromatography column on silica (1:4 Et2O-light petroleum eluent). 4-(Tertbutyldimethylsilyloxy)butyraldehyde (70) was obtained as a colourless liquid (3.50 g,
17.3 mmol; 71%). Aldehyde 70 was found to be air sensitive and was therefore stored
in an oxygen-free atmosphere in the refrigerator. Rf (1:4 Et2O-petroleum) 0.62; H (200
MHz, CDCl3) 0.01 (6 H, s, Si(CH3)2), 0.84 (9 H, s, C(CH3)3), 1.82 (2 H, p, J 6.5, CH2145
3), 2.47 (2 H, dt, J 7.0 and 2.1, CH2-2), 3.61 (2 H, t, J 5.9, CH2-4), 9.75 (1 H, s, CHO);
C (50 MHz, CDCl3) – 6.00 (CH3, Si(CH3)2), 18.16 (C(CH3)3), 25.36 (CH2-3), 25.77
(C(CH3)3), 40.66 (CH2-2), 61.94 (CH2-4), 202.51 (CHO); m/z (EI) 201 (MH+), 187
(16%, [M - CH3]+), 145 (58%, [M- C(CH3)]+).
Preparation of the [1-13C]-5-(tert-butyl-dimethylsilanyloxy)pent-1-ene (71)
n-Butyllithium (6.36 ml, 16 mmol) was added dropwise to a
suspension of
13
C-labelled methyltriphenylphosphonium
2
H213C
1
4
3
iodide (61; 6.36 g, 16.0 mmol) in THF (50 ml) at room
temperature under argon.
The resulting red solution was
O Si
5
71
C1013CH24OSi
Mol. Wt.: 201.3858
stirred at room temperature for 1 hour. The solution was then
transferred
by
canula
to
a
flask
containing
a
solution
of
4-(tert-
butyldimethylsilyloxy)butyraldehyde (70; 3 g, 15.0 mmol) in THF (15 ml) at -78 ºC.
Once the transfer was complete, the reaction mixture was allowed to warm to room
temperature and was left to stir overnight. The reaction mixture was then diluted in
CHCl3 (100 ml) and washed with water (2  30 ml). The organic layer was dried
(Na2SO4) and the solvent was removed under reduced pressure. The residue was
submitted to flash chromatography on silica (0.5:9.5 Et2O-light petroleum eluent).
13C-
labelled 5-(tert-butyldimethylsilyloxy)pent-1-ene (71) was obtained as a colourless oil
(2.00g, 9.93 mmol; 67% in >95% yield): Rf (7:3 petroleum-EtOAc) 0.56; max
(neat)/cm-1 3066, 2929, 2857, 1618, 1387, 1361, 1255, 1101, 1037, 1006, 967, 938, 904,
836, 813, 775, 736; H (400 MHz, CDCl3) 0.01 (6 H, s, Si(CH3)2), 0.87 (9 H, s,
C(CH3)3), 1.60 (2 H, p, J 6.7, CH2-4), 2.12 (2 H, p, J 6.6, CH2-3), 5.60 (2 H, t, J 6.7,
CH2-5), 4.93 (1 H, ddq, J 155, 10.1 and 1.6, olefinic
17.1 and 1.6, olefinic
13
CH2-1), 5.00 (1 H, ddq, J 155,
CH2-1), 5.81 (1 H, ddt, J 17.0, 10.2 and 6.6, CH2-2); C (100
13
MHz, CDCl3) – 6.00 (CH3, Si(CH3)2), 18.35 (C(CH3)3), 25.96 (C(CH3)3), 30.03 (CH23), 32.02 (CH2-4), 62.54 (CH2-5), 114.49 (13CH2-1), 138.55 (d, J 69.4, CH2-2); m/z (EI)
277 (100%, M+) (Found M 201.1654. 13C12C10H24OSi requires 201.1630).
146
Preparation of [5-13C]-labelled pent-4-en-1-ol (72)
13
C-labelled 5-(tert-butyldimethylsilyloxy)pent-1-ene (71; 2.00 g,
10.0 mmol) and TBAF (1M solution in THF, 15.0 ml, 15.0 mmol)
4
2
H213C
5
3
OH
1
72
were refluxed for 2.5 hours. The reaction mixture was diluted in
C413CH10O
CH2Cl2 (25 ml) and washed with brine (2  15 ml). The aqueous
Mol. Wt.: 87.1250
layer was extracted with more CH2Cl2 (10 ml) and the combined organic layers were
dried (Na2SO4). The solvent was removed by distillation under atmospheric pressure
using a Vigreux column.
13C-labelled
pent-4-en-1-ol (72) was obtained as a colourless
liquid (610 mg, 0.70 mmol; 70% in >95% yield): Rf (1:1 CH2Cl2-Et2O) 0.56; max
(neat)/cm-1 3368, 2937, 1741, 1724, 1619, 1143, 1415, 1390, 1372, 1249, 1048, 994,
905, 836, 776, 734; H (200 MHz, CDCl3) 1.60 (2 H, p, J 7.0, CH2-2), 2.06 (2 H, p,
J6.8, CH2-3), 2.06 (1 H, s, OH), 3.59 (2 H, t, J 6.6, CH2-1), 4.91 (1 H, ddq, J 157, 10.1
and 3.2, olefinic
13
CH2-5), 4.98 (1 H, ddq, J 157, 17.1 and 3.2, olefinic 13CH2-5), 5.77
(1 H, dddt, J 17.0, 10.2, 6.9 and 0.95, CH2-4); C (100 MHz, CDCl3) 30.10 (CH2-3),
31.80 (CH2-2), 62.49 (CH2-1), 114.95 (13CH2-5), 138.58 (d, J 69.5, CH–4); m/z (EI) 87
(6%, M+), 70 (36%, [M – OH]+) (Found M 87.0749. 13C12C4H10O requires 87.0765).
Preparation of [5-13C]-labelled trifluoromethanesulfonic acid pent-4-enyl ester (72)
To a solution of 13C-labelled pent-4-en-1-ol (72; 100 mg, 1.15
mmol) and triethylamine (1.51 mg, 1.50 mmol) in dry CH2Cl2
(10 ml) at 0 ºC was added freshly distilled trifluoromethanesulfonic anhydride (220 l, 1.4 mmol). The reaction was left to
4
H213C
5
O
O S CF 3
2
3
1
O
73
C513CH9F3O3S
Mol. Wt.: 219.1877
stir at 0 ºC under argon for 2 hours. The crude mixture was
then washed with cold water (2  5 ml) and then cold 2M aqueous HCl (2  5 ml). The
organic layer was dried (Na2SO4) and the solvent was removed in vacuo affording the
target triflate (73) as a pale yellow liquid (151 mg, 0.69 mmol; 60% in >95% purity): Rf
(CH2Cl2) 0.67; H (200 MHz, CDCl3) 1.84 (2 H, p, J 6.8, CH2-2), 2.12 (2 H, q, J 7.3,
CH2-3), 4.46 (2 H, t, J 6.6, CH2-1), 4.96 (2 H, ddq, J 157, 10.4 and 3.2, olefinic 13CH25), 5.00 (2 H, ddq, J 157, 16.7 and 3.2, olefinic
and 6.6, CH-4).
147
13
CH2-5), 5.77 (1 H, ddt, J 16.8, 10.3
Preparation
of
13C-labelled
5,15-bis[4-(pent-4-enyloxy)phenyl]–10,20-
diphenylporphyrin (58)
A mixture of unknown composition of
5,15-bis(4-hydroxyphenyl)-10,20-
2
diphenylporphyrin (59) and 5,10,15-tris(4-
3'
hydroxyphenyl)–20–phenyl porphyrin (66)
(470
mg),
13
O
4'
3
2' 4
1'
5
20
1
N
HN
NH
N
15
O
C-labelled
10
trifluoromethanesulfonic acid pent–4–enyl
13
H2 C
13
CH 2
ester (73; 156 mg, 0.71 mmol) and
58
triethylamine (123 l, 0.71 mmol) were
C5213C2H46N4O2
Mol. Wt.: 784.9541
heated in CH2Cl2 (15 ml) in a sealed tube at
70 ºC overnight. The solvent was then removed in vacuo and the crude material was
submitted to flash column chromatography on silica gel (9.5:1.5 CH2Cl2-Et2O eluent).
The target labelled porphyrin (58) was afforded as a lustrous purple powder (153 mg,
0.19 mmol): Rf (CHCl3) 0.65; max (CHCl3)/nm (log ε) 421 (5.66), 518 (4.10); max
(KBr disk)/cm-1 2923, 1605, 1558, 1504, 1464, 1441, 1399, 1349, 1281, 1245, 1175,
1107, 1071, 965, 908, 843, 804, 736, 700; H (400 MHz, CDCl3) –2.81 (2 H, s, 2  NH),
2.01 (4 H, p, J 7.2, 2  OCH2CH2CH2CH), 2.33 (4 H, p, J 7.0, 2  OCH2CH2CH2CH),
4.18 (4 H, t, J 6.4, 2  OCH2), 5.02 (2 H, ddd, J 154, 10.1 and 1.2, 2  olefinic-13CH2),
5.11 (2 H, ddd, J 154, 17.1 and 1.2, 2  olefinic-13CH2), 5.90 (2 H, ddt, J 17.0, 10.1 and
6.8, 2  olefinic-CH), 7.19 (4 H, dd, J 8.7 and 1.6, 2  H-3’ and 2  H-5’), 7.64-7.70 (6
H, m, 4  meta Ph-H and 2  para Ph-H), 8.03 (4 H, d, J 8.56, 2  H-2’ and 2  H-6’),
8.13-8.15(4 H, m, 4  ortho Ph-H), 8.75 (4 H, d, J 4.7, pyrrolic-H), 8.81 (4 H, d, J 4.7,
pyrrolic-H); C (100 MHz, CDCl3) 115.32 (CH2,
(Found C, 82.57; H 5.83; N, 6.85%.
13
13
C-1); m/z (ESI) 785 (100%, M+);
C212C52H46N4O2 requires C, 82.63; H, 5.91; N,
7.14%).

The ratio of porphyrins 59 and 66 in the mixture used in this reaction was not determined
148
Preparation
13C-labelled
of
{10,20-diphenyl-5,15-bis(4-pent-4-enyloxyphenyl)
porphyrinato}zinc(II) (74)
13
C-labelled
5,15-bis[4-(pent-4-
enyloxy)phenyl]–10,20-diphenylporphyrin
2 1
(58; 75.0 mg, 95.5 mol) and zinc acetate
3'
O 4'
dihydrate (223 mg, 0.95 mmol) were
3
2' 4
1'
5
20
N
N
Zn
N
refluxed in CHCl3 (5 ml) for 10 minutes.
The solvent was then removed in vacuo and
O
15
10
13
H2 C
13
CH 2
the residue was submitted to flash column
chromatography on silica gel (9:1 CHCl3-
N
74
C5213C2H44N4O2Zn
Mol. Wt.: 848.3282
EtOAc eluent).
The target metallated
porphyrin (74) was obtained as a lustrous fuchsia powder (77 mg, 90.7 mol; 95%): Rf
(CHCl3) 0.75; max (CHCl3)/nm (log ε) 424 (5.21), 552 (3.70), 595 (2.95); H (400
MHz, CDCl3) 2.01 (4 H, p, J 7.2, 2  OCH2CH2CH2CH), 2.33 (4 H, p, J 7.0, 2 
OCH2CH2CH2CH), 4.18 (4 H, t, J 6.4, 2  OCH2), 5.02 (2 H, ddd, J 154, 10.1 and 1.2,
2  olefinic-13CH2), 5.11 (2 H, ddd, J 154, 17.1 and 1.2, 2  olefinic-13CH2), 5.90 (2 H,
ddt, J 17.0, 10.1 and 6.8, 2  olefinic-CH), 7.19 (4 H, dd, J 8.7 and 1.6, 2  H-3’ and 2
 H-5’), 7.64-7.70 (6 H, m, 4  meta Ph-H and 2  para Ph-H) 8.03 (4 H, d, J 8.56, 2 
H-2’ and 2  H-6’), 8.13-8.15 (4 H, m, 4  ortho Ph-H), 8.75 (4 H, d, J 4.7, pyrrolic-H),
8.81 (4 H, d, J 4.7, pyrrolic-H); C (100 MHz, CDCl3) 115.29 (olefinic
13
CH2); m/z
(ESI) 848 (100%, M+).
13C-labelled
Attachment
of
porphyrin
(58)
and
10,20-diphenyl-5,15-bis[4-(pent-4-enyloxy)phenyl]
13C-labelled
{10,20-diphenyl-5,15-bis[4-(pent-4-
enyloxy)phenyl]porphyrinato}zinc(II) (74)
According to general method B:
Dried silica (400 mg) and (3-mercaptopropyl)trimethoxysilane
(82.5 mg, 0.42 mmol) were heated in dry toluene (10 ml) at 110 ºC
overnight. The resulting silica was collected by filtration, washed
and dried according to the general method and submitted for


O
Si
SH

O
OCH 3
O
O Si
O
SH
elemental analysis: Found C, 2.89; H, 0.89%; 29Si solid-state NMR
(59.58 MHz, Direct Polarisation) -111.3 (73%, lattice sites), -101.6 (20%, single
149
hydroxyl sites), -92.1 (0.7%, geminal hydroxyl sites), -56.6 (2.2%, (SiO)3SiCH2), -47.3
(3.3%, (SiO)2(CH3O)SiCH2);
13
C solid-state NMR (75.43 MHz, Direct Polarisation)
10.17 (27%, C-), 27.01 (56%, C- and C-), 48.82 (18 %, SiOCH3).
The two
following reactions were then carried out in parallel.
A portion of the
Ph
mercaptopropylmodified
gel

O Si
O

OMe
silica
(110

*
S
3
N
HN
NH
N
O
mg)
*
O
Ph
was reacted with
the non-metallated poprhyrin (58; 100 mg, 0.31 mmol) in dry acetonitrile (10 ml) in the
presence of AIBN (4 mg, 14 mol) according to the general method. The modified
silica recovered was submitted for elemental analysis: Found C, 9.11; H, 1.44; N,
0.23%;
13
C solid-state NMR (75.43 MHz, Direct Polarisation) 11.8 (4.8%, C-), 22.6
(8.4%), 31.4 (18%, 13CH2SCH2-, C- and C-), 39.4 (16%), 51.5 (31%, 13CH2(SO)CH2and SiOCH3), 113.4 (19%, olefinic 13C), 129.1 (3.3%).
A
second
portion
of
mercaptopropyl-modified
the
Ph
silica
3
*
gel (110 mg) heated with the Zn-
N
O
N
S
O
N
3
*
S


mmol) in acetonitrile (10 ml) in a
N
S
Ph

porphyrin 74 (104 mg, 0.27
Zn
Si OMe
O O
Si OMe
O O
MeO
Si
O O
sealed tube at 110 ºC for 6 hours
in the presence of AIBN (4.0 mg, 14.0 mol). The resulting silica was collected by
filtration, washed and dried according to the general methods: (Found C, 7.84; H, 1.29;
N, 0.21%);
13
C solid-state NMR (75.43 MHz, Direct Polarisation) 11.1 (6.0%, C-),
22.7 (10.7%), 27.7 (5.3%, C-), 32.1 (35%,
13
CH2SCH2–), 48.6 (0.1%), 51.2 (30%,
13
CH2(SO)CH2- and SiOCH3), 113.6 (8.9%, residual olefinic 13C), 130.6 (4.2%).
A
third
portion
of
mercaptopropyl–
Ph
*
N
modified silica (110 mg)
used
for
reactions
heated
the
two
above
was
with
the
Zn-
N
O
SH
N
N
*
O
HS
S
Ph


Zn

Si OMe
O O
porphyrin (74; 50.0 mg,
150
Si OMe
O O
MeO
Si
O O
0.13 mmol) in acetonitrile (2.5 ml) in the absence of AIBN.
The resulting
mercaptopropyl-modified silica was recovered by filtration, washed and dried according
to the general method and submitted for elemental analysis: Found C, 5.52; H, 1.23; N,
0.01%; 13C solid-state NMR (75.43 MHz, Direct Polarisation) 10.53 (C-), 27.09 (C-
and C-), 31.99 (low intensity,
13
CH2SCH2-), 49.23 (SiOCH3), 113.66 (high intensity,
residual olefinic 13C).
4.4
ATTACHMENT OF A PROTOTYPICAL NOS MODEL TO A MERCAPTOPROPYLMODIFIED GOLD SURFACE.
4.4.1 Synthesis of a prototypical NOS model
Preparation of 1-chloro-4-methoxy-2-methylbenzene (9)
4-Chloro-2-methylphenol (50.1 g, 0.351 mol) was dissolved in 1.5 M
aqueous NaOH solution (250 ml) with vigorous stirring at 0 °C.
6
Cl
reaction mixture was heated for an hour at 60-70 °C after which time a
OMe
Me
Dimethyl sulfate (33.5 ml, 0.354 mol) was added dropwise to the
solution, maintaining the temperature at 0 ˚C. After addition, the
5
3
9
C8H9ClO
Mol. Wt.: 156.6092
separate oily phase had formed. The organic material was extracted into CH2Cl2 and
washed with water and then 2M aqueous NaOH solution. The combined organic layers
were then dried (MgSO4) and evaporation of the solvent under reduced pressure
afforded 1-chloro-4-methoxy-2-methylbenzene60 (9; 48.5 g, 0.31 mmol; 88.3%): H
(200 MHz, CDCl3) 2.31 (3 H, s, CH3), 3.75 (3 H, s, OCH3), 6.60 (1 H, dd, J 8.7 and
2.9, H-5), 6.75 (1 H, d, J 3.0, H-3), 7.25 (1 H, d, J 8.7, H-6).
Preparation of 2-bromoethyl-1-chloro-4-methoxybenzene (10)
A two-litre photochemical reaction vessel fitted with a quartz
immersion well was charged with a solution of sodium bromate (136
6
5
Cl
OMe
3
g, 0.81 mol) in water (250 ml).
EtOAc (1.2 l) and 1-chloro-4-
methoxy-2-methylbenzene (9; 97.0 g, 0.68 mol) were added to the
vessel. The two phase-system was vigorously mixed by combined use
Br
10
C8H8BrClO
Mol. Wt.: 235.5052
of a magnetic stirrer and nitrogen gas purge. A solution of sodium
metabisulfite (103 g, 0.54 mol) in water (350 ml) was then added dropwise to the
151
mixture under irradiation from a 400 Watt medium pressure mercury lamp over 5 hours.
The irradiation was stopped and the organic phase was separated and successively
washed with solutions of sodium thiosulfate (4  100 ml) and brine (2  250 ml). The
combined organic layers were dried (Na2SO4) and the solvent was removed in vacuo
yielding an orange oil. Initial crystallisation was carried out by stirring the oil with light
petroleum at -50 C. Recrystallisation from 1:1 light petroleum:CHCl3 afforded 2bromomethyl-1-chloro-4-methoxybenzene (10)144 as a colourless solid (72.0 g, 0.31
mol; 45%): m.p. 43-45 C; Rf (1:9 Et2O/light petroleum) 0.75; H (200 MHz, CDCl3)
3.86 (3 H, s, OCH3), 4.54 (2 H, s, CH2Br), 6.80 (1 H, dd, J 8.7 and 3.0, H-5) 6.96 (1 H,
d, J 3.0, H-3), 7.28 (1 H, d, J 8.7, H-6).
Preparation of (2-chloro-5-methoxyphenylmethyl)triphenyl phosphonium bromide
(11)
3 4
2-Bromomethyl-1-chloro-4-methoxybenzene (10; 13.1 g, 55.6 mmol)
Cl
2
5
and triphenylphosphine (17.5 g, 66.7 mmol) were refluxed in toluene
1 6
(100 ml) for 4 hours. The solid that had formed was collected by
PPh 3
OMe
Br
11
filtration and washed with toluene affording phosphonium salt 11
C26H23BrClOP+
(25.8 g, 51.8 mmol; 94%): Rf (9:1 Et2O-light petroleum) 0.08; m.p.
Mol. Wt.: 497.7907
207-208 ºC; H (200 MHz, CDCl3) 3.56 (3 H, s, OCH3), 5.55 (2 H, d, J 14.3, CH2), 6.74
(1 H, dt, J 8.6 and 2.8, H-4), 7.03 (1 H, d, J 8.6, H-3), 7.20 (1 H, t, J 2.7, H-6), 7.70–
7.78 (15 H, m, Ph-H).
Preparation of 4-[2-(2-chloro-5-methoxyphenyl)ethenyl]benzaldehyde (12)
To
a
vigorously
stirred
suspension
of
(2-chloro-5-
methoxyphenylmethyl)triphenyl phosphonium bromide (11; 24 g,
48 mmol) in dry THF (500 ml) was added LDA (33.0 ml, 66.0
mmol) dropwise to the mixture at room temperature. The reaction
mixture rapidly turned to a dark red solution. After stirring for 1
hour, the ylide solution was transferred via cannula to a vigorously
11
12
Cl
OMe
9
8
6
4
1
7
3
CHO
2
12
C16H13ClO2
Mol. Wt.: 272.7259
stirred solution of terephthalaldehyde (11.0 g, 82.0 mmol) in THF
(100 ml). The dark red brown colour of the ylide dissipated on contact with the
terephthalaldehyde mixture, leaving a yellow solution. The reaction mixture was stirred
152
for a further 2 hours at room temperature after addition was completed. The reaction
mixture was then quenched by addition of water (4 ml) and concentrated under reduced
pressure. The crude mixture containing a mixture of the E and Z stilbenes (2:1) was
subjected to flash column chromatography (1:2:7 EtOAc-CH2Cl2-light petroleum
eluent). A pure mixture of the stilbenes 12 was obtained (Z/E, 2/1; 5.45 g, 20.0 mmol;
42%). Z-stilbene60: H (200 MHz, CDCl3) 3.54 (3 H, s, OCH3), 6.64 (1 H, d, J 3.0, H14), 6.73-6.85 (3 H, m, H-7, H-8 and H-12), 7.31 (1 H, d, J 8.8, H-11), 7.33 (2 H, d, J
8.2, H-3 and H-5), 7.72 (2 H, d, J 8.3, H-2 and H-6), 9.94 (1 H, s, CHO). E-stilbene60 :
H (200 MHz, CDCl3) 3.85 (3 H, s, OCH3 ) 6.82 (1 H, dd, J 8.9 and 3.0, H-12) 7.09 (1
H, d, J 16.3, H-7) 7.2 (1 H, d, J 3.0, H-14), 7.31 (1 H, d, J 8.8, H-11), 7.62 (1 H, d, J
16.4, H-8), 7.69 (2 H, d, J 8.4, H-3 and H-5), 7.89 (2 H, d, J 8.3, H-2 and H-6), 10.00 (1
H, s, CHO).
Preparation of 8-chloro-5-methoxyphenanthrene-3-carboxaldehyde (13)
7 6
A mixture of the Z and E stilbenes (12; 200 mg, 0.73 mmol),
1,2-epoxybutane (3.00 ml, 35.8 mmol) and iodine (188 mg, 0.74
mmol) in petroleum (250 ml) was placed in a photochemical
reactor vessel fitted with a quartz immersion well and irradiated
for 2 hours with a 400 W medium pressure mercury vapour lamp.
Cl
5
OMe
8a
4b
4
9 4a
10a
CHO
10
1 2
13
C16H11ClO2
Mol. Wt.: 270.7100
The reaction mixture completely decolourised and was filtered to
recover the precipitate that had formed during the irradiation. The filtrate was then
evaporated down to half its volume and poured back into the vessel. Fresh starting
materials were added (same quantities) and the reaction mixture was irradiated for
another 2 hours. This procedure was repeated for 4 more batches of 200 mg and a last
one of 100 mg of the stilbenes. The precipitates from the different batches were
combined and recrystallised from 1:1 CHCl3-light petroleum.
The filtrate was
evaporated down, the residue taken up in CHCl3 and washed with sodium bisulfite
solution. After being dried (Na2SO4) the organic layer was evaporated and the residue
was submitted to flash column chromatography (3:47:50 EtOAc-light petroleumCH2Cl2 eluent). Phenanthrene 13 was obtained as a colourless solid (0.872 g, 3.22
mmol; 80%): Rf (1:4 Et2O-light petroleum) 0.43; H (200 MHz, CDCl3) 4.10 ( 3 H, s,
OCH3), 7.03 (1 H, d, J 8.7, H-6), 7.60 (1 H, d, J 8.7, H-7), 7.77 (1 H, d, J 9.1, H-10,)
153
7.89 (1 H, d, J 8.1, H-1), 8.03 (1 H, dd, J 8.2 and 1.5, H-2), 8.27 (1 H, d, J 9.1, H-9),
10.01 (1 H, s, H-4), 10.17 (1H, s, CHO).
8-chloro-5-methoxy-3-phenanthren–3–carboxylic acid tert–butoxycarbonylmethyl
ester (83) and (8-chloro-5-methoxyphenanthren-3-yl)methanol (84)
Ethanethiol (102 l, 1.38 mmol) was added to a suspension of sodium hydride (60%
dispersion in mineral oil; 62.9 mg, 2.62 mmol) in DMF (15 ml) under nitrogen. After 5
minutes the mixture was transferred via cannula under nitrogen to a dry flask containing
8-chloro-5-methoxyphenanthrene-3-carboxaldehyde (13; 304 mg, 1.12 mmol), washing
the transferring flask with further DMF (5 ml). The mixture was heated at 110 ˚C for
30 minutes. Tert-butylbromoacetate (383 l, 2.59 mmol) was then added to the hot
reaction mixture. After 2 hours the reaction mixture was cooled to room temperature,
and diluted with EtOAc and light petroleum (1:1) to precipitate sodium bromide. The
reaction mixture was filtered and the organic solvent was evaporated to afford a brown
solid residue. This crude material was then submitted to flash column chromatography
(1:1-4:1 CH2Cl2-light petroleum gradient). Two main compounds were isolated:
tert-butylcarboxymethyl
7 6
8-chloro-5-methoxy-3-
phenanthrenecarboxylate (83; 162 mg, 0.40 mmol, 36.7 %):
Rf (1:1 CH2Cl2-light petroleum) 0.17; max (KBr disk)/cm-1
2976, 1769, 1715, 1610.0, 1567, 1525, 1455, 1418, 1392,
1367, 1324, 1222, 1084, 1036, 956, 906, 844, 813; H (400
MHz, CDCl3) 1.61 (9 H, s, C(CH3)3), 4.32 (3 H, s, OCH3),
Cl
5
OMe
8a
4b
4
9 4a
O
10a
10
O
1 2
O
O
83
C22H21ClO5
Mol. Wt.: 400.8518
4.75 (2 H, s, CO2CH2CO2), 7.13 (1 H, d, J 8.7, H-6), 7.65 (1
H, d, J 8.6, H-7), 7.85 (1 H, d, J 9.1, H-10), 7.95 (1 H, d, J 8.3, H-1), 8.25 (1 H, dd, J
8.3, J 1.6, H-2), 8.35 (1 H, d, J 9.1, H-9), 10.5 (1 H, s, H-4); C (100 MHz, CDCl3)
28.11 ((CH3)3), 55.99 (OCH3), 61.83 (CO2CH2CO2), 82.45 (C), 108.9 (CH-6), 122.25
(C), 124.34 (C), 125.43 (CH-9), 126.15 (CH-2), 127.17 (C), 127.48 (CH-7), 128.44
(CH-1), 128.60 (CH-10), 129.35 (C), 131.04 (C), 131.57 (C), 131.95 (C), 157.72 (CH5), 166.60 (CO2), 167.00 (CO2); m/z (FAB) 400 (100%, M+), 401 (29%, MH+), 269
(100%, [M-OCH2CO2C(CH3)3]+) (Found 400.1054. C22H2135ClO5 requires 400.1078).
154
(8-Chloro-5-methoxyphenanthren-3-yl)methanol (84): Rf (1:1
CH2Cl2-light petroleum) 0.4; H (400 MHz, CDCl3) 4.10 (3 H, s,
7 6
Cl
CH3), 5.01 (2 H, s, CH2), 7.04 (1 H, d, J 8.6, H-6), 7.61 (1 H, d, J
8.6, H-7), 7.65 (1 H, d, J 8.2, H-2), 7.83 (1 H, d, J 9.1, H-10), 7.89
5
OMe
8a
4b
4
9 4a
10a
10
OH
1 2
84
C16H13ClO2
Mol. Wt.: 272.7259
(1 H, d, J 8.2, H-1), 8.21 (1 H, d, J 9.2, H-9), 9.62 (1 H, s, H-4); C
(100 MHz, CDCl3) 55.96 (OCH3), 66.34 (CH2OH), 108.29 (CH-
6), 122.13 (C), 122.79 (CH-9), 124.39 (C), 125.73 (CH-7), 126.93 (CH-2 or CH-4),
127.02 (CH-2 or CH-4), 128.44 (CH-1), 128.60 (CH-10), 129.33 (C), 131.20 (C),
132.11 (C), 139.23 (C), 157.77 (CH-5).
Preparation of 8-chloro-5-hydroxyphenanthrene-3-carbaldehyde (15)
To a suspension of sodium hydride (60 % dispersion in mineral
oil; 178 mg, 4.46 mmol) in dry DMF (20 ml) was added
ethanethiol (332 l, 4.48 mmol). After ten minutes, the mixture
was transferred via a cannula under argon to a dry flask containing
8-chloro-5-methoxyphenanthren-3-carboxaldehyde (13; 1.00 g,
7 6
Cl
5
OH
8a
4b
4
9 4a
10a
CHO
10
1 2
15
C15H9ClO2
Mol. Wt.: 256.6835
3.66 mmol) which was dried over P2O5 prior to use. The
transferring flask was washed with further DMF (5 ml). The mixture was then heated at
70 ˚C for 30 minutes and the solution rapidly turned to a dark red colour. Hydrochloric
acid (2M) was then added to the cooled reaction mixture. A yellow precipitate was
formed, collected by filtration and thoroughly washed with water. TLC showed that
some starting material was still present in the crude precipitate. This material was
submitted to flash column chromatography. Unreacted starting material (376 mg, 1.89
mmol; 52%) eluted first with CH2Cl2.
The target compound 8-chloro-5-
hydroxyphenanthren–3–carboxaldehyde (15), eluted with neat acetone was isolated
as a pale yellow powder (420 mg, 1.64 mmol; 45 %): Rf (CH2Cl2) 0.28; m.p. 243-245
ºC; max (KBr disk)/cm-1 3236, 1660, 1610, 1569, 1520, 1383, 1313, 1236, 1211, 1056,
1010, 902, 845, 819, 774, 793, 774; H (200 MHz, C2D6CO) 2.93 (1 H, bs, OH), 7.32 (1
H, d, J 8.4, H-6), 7.66 (1 H, d, J 8.6, H-7), 8.04 (1 H, d, J 8.3, H-10), 8.10 (1 H, d, J 8.3,
H-2), 8.16 (1 H, d, J 8.3, H-1), 8.34 (1 H, d, J 9.2, H-9), 10.21 (1 H, s, H-4), 10.42 (1 H,
s, CHO); C (50 MHz, C2D6CO) 114.59 (CH-6), 120.46 (C), 121.60 (C), 125.11 (CH-9),
125.86 (CH-7), 128.59 (CH-2 or CH-4), 129.08 (CH-2 or CH-4), 129.70 (CH-1), 130.09
(C), 130.71 (C), 133.29 (CH-10), 134.70 (C), 135.99 (C), 156.23 (CHO); m/z (EI) 256
155
(100%, M+) (Found 256.0297. C15H935ClO2 requires 256.0291); (Found C, 69.91; H,
3.43% C15H9ClO2 requires C, 70.19; H, 3.53%).
Preparation of (1-chloro-6-formylphenanthren-4-yloxy)acetic acid tert-butyl ester
(14)
8-Chloro-5-hydroxyphenanthren-3-carboxaldehyde (15; 1.00
O
2 3
Cl
g, 3.90 mmol), tert-butylbromoacetate (0.65 ml, 4.40 mmol)
1
4
8a
4a
5
4b
10
and potassium carbonate (608 mg, 4.40 mmol) were refluxed in
9
O
O
6
8a
CHO
THF (15 ml) for 1 hour. Water (20 ml) was then added to the
8 7
reaction mixture causing the target material to precipitate. The
14
C21H19ClO4
Mol. Wt.: 370.8259
precipitate was collected by filtration and washed thoroughly
with water. Recrystallisation from hot Et2O afforded [(1-chloro-6-formylphenanthren4-yl)oxy]acetic acid tert-butyl ester (14)60 as colourless needles (742 mg, 1.77 mmol;
45%): Rf (1:4 Et2O-light petroleum) 0.51; H (200 MHz, CDCl3) 1.56 (9 H, s, (CH3)3),
4.69 (2 H, s, OCH2), 6.85 (1 H, d, J 8.5, H-3), 7.54 (1 H, d, J 8.5, H-2), 7.75 (1 H, d, J
9.1, H-9), 7.85 (1 H, d, J 8.2, H-8), 8.06 (1 H, dd, J 8.2 and 1.6, H-7), 8.23 (1 H, d, J
9.1, H-10), 10.32 (1 H, s, CHO).
Preparation of 5,15-bis[8-chloro-5-(tert-butoxycarbonylmethoxy)phenanthren-3yl]-10, 20-bis[4-(pent-4-enyloxy)phenyl]porphyrin (76)
Phenanthrene 14 (559 mg, 1.5 mmol) and
dipyrromethane 55 (600 mg, 1.95 mmol)
were refluxed in propionic acid (20 ml) for
30 minutes.
O
Propionic acid was then
removed in vacuo and the residue was
submitted to flash column chromatography
(petroleum-Et2O-CHCl3,
95:4:1-80:18:2
O
Cl
8'
9'
5'
O
2
O
3
4'
3'
4
5
10'
1' 2'
gradient).
O
O
20
1
N
O
HN
NH
The target material was eluted
N
15
10
with 80:18:2 petroleum-Et2O-CHCl3 and as
a lustrous purple powder (46.3 mg, 35.3
mol; 2%): Rf (4:1 petroleum-Et2O) 0.61;
max (CHCl3)/nm (log ε) 254 (5.09), 386
156
O
76
C82H72Cl2N4O8
Mol. Wt.: 1312.3766
Cl
(4.41), 4.26 (5.80), 520 (4.25), 557 (4.02); H (200 MHz, CDCl3) –2.65 (2 H, bs, 2 
NH), 0.78 (18 H, s, 2  (CH3)3), 2.0-2.11 (4 H, m, 2  pentenyloxy chain OCH2CH2),
2.39-2.42 (4 H, m, 2  pentenyloxy chain OCH2CH2CH2), 4.25 (4 H, t, J 6.3, 2 
pentenyloxy chain OCH2), 4.47 (4 H, 2  s, 2  COCH2CO2), 5.07-5.24 (4 H, m, 2 
pentenyloxy chain olefinic CH2), 5.97 (2 H, 2  ddt, J 17.0, 10.3 and 6.2, 2 
pentenyloxy chain olefinic CH), 6.91 (2 H, d, J 8.6, 2  H-6’), 7.26 (2 H, d, J 8.6, 2 
meta Ph-CH), 7.65 (2 H, dd, J 8.6 and 3.4, 2  H-7’), 8.10-8.29 (8 H, m, 2  H-7’, 2 
H-2’ and 4  ortho Ph-CH), 8.46 (2 H, d, J 9.0, 2  H-9’), 8.48 (2 H, d, J 9.2, 2  H10’), 8.91 (8 H, dd, J 9.5 and 5.0, pyrrolic-H), 10.70 (2 H, s, 2  H-4’).
Preparation of {5,15-bis[8-chloro-5-(tert-butoxycarbonylmethoxy)phenanthren-3yl]-10, 20-bis[4-(pent-4enyloxy)phenyl]porphyrinato}iron (III) (85)
Iron insertion into 5,15-bis[8-chloro-5-(tertbutoxycarbonylmethoxy)phenanthren-3-yl]10,20-bis[4-(pent-4enyloxy)phenyl]porphyrin
O
O
(76) was achieved by heating the free-base
porphyrin (15.0 mg, 0.01 mmol) and ferrous
chloride tetrahydrate (7.20 mg, 0.04 mmol) in
acetonitrile (4 ml).
The reaction was
O
O
Cl
8'
9'
10'
5'
O
2
O
3
4' 4
3'
5
1' 2'
20
1
O
N Cl N
Fe
N
N
monitored by uv/visible-spectroscopy and
15
10
appeared to be complete after 3 hours.
Acetonitrile was removed in vacuo and the
residue was submitted to flash column
chromatography
(CHCl3
eluent).
The
O
85
C82H70Cl3FeN4O8
Mol. Wt.: 1401.6585
metallated porphyrin (85) eluted following residual traces of the non-metallated starting
material (76). To ensure the axial ligand was Cl, a solution of the porphyrin in CH 2Cl2
was treated with a Amberlite IRA-400 (Cl-) resin. {5,15-Bis[8-chloro-5-(tertbutoxycarbonylmethoxy)phenanthren-3-yl]-10,20-bis[4-(pent-4enyloxy)phenyl]porphyrinato}iron (III) chloride (85) was thus obtained as a dull green
powder (8.00 mg, 5.71 mol; 57 %): Rf (CHCl3) 0.32; max (CHCl3)/nm (log ε) 422
(5.68), 488 (4.02), 515 (3.98).
157
Cl
4.4.2 Modification of a gold wire
A 20 cm long gold wire was divided into 3 segments that were coiled. One segment
was sonicated in DCM for 30 minutes and subsequently dried with N2 gun equipped
with a filter. The clean wire was subsequently reacted with propane-1,3-dithiol (75; 10
mg, 92.3 mol) in DCM at room temperature for 2 hours. The resulting modified wire
was rinsed with DCM to remove any physisorbed material and was then reacted with
chloroiron(III)porphyrin 85 (8.00 mg, 5.71 mol) in boiling acetonitrile in the presence
of AIBN. The resulting modified gold wire was cleaned with DCM to remove any
residual physisorbed porphyrin and was then kept in an oxygen-free atmosphere.
4.5
SYNTHESIS OF NEW SPACER-SUPERSTRUCTURE UNITS FOR THE NOS MODEL.
Preparation of diethyl hydroxymethyl-phosphonate (87)
A
mixture
of
diethyl
phosphite
(8.00
g,
73.5
mmol),
paraformaldehyde (2.36 g, 76.8 mmol) and triethylamine (1.1 ml, 7.71
mmol) was slowly heated to 50 ºC. An exotherm ensued after 5
minutes and the mixture was left to stir for 15 minutes. The crude
O 2
EtO P
OH
EtO 1
87
C5H13O4P
Mol. Wt.: 168.1281
hydroxymethylphosphonic acid diethyl ester (87)86 thus obtained (12.0 g, 71.4 mmol;
97% in > 95% purity) was used for following reactions without further purification: R f
(EtOAc) 0.19; H (200 MHz, CDCl3) 1.22 (6 H, t, J 7.1, 2  ethyl CH3), 3.79 ( 2H, d, J
6.2, PCH2OH), 4.07 (4 H, q, J 7.1, 2  ethyl CH2), 5.01 (1 H, bs, OH); C (50 MHz,
CDCl3) 16.33 (d, J 5.4, 2  CH3CH2), 56.99 (d, J 162, PCH2OH), 62.33 (d, J 6.6, 2 
CH2CH3).
Preparation of diethyl trifluorosulfonyl-methylphosphonate (17)
Hydroxymethylphosphonic acid diethyl ester (87; 2.90 g, 17.2
mol) was added to a vigorously stirred suspension of sodium
hydride (dry powder, 595 mg, 24.8 mmol) in Et2O (25 ml). The
resulting white suspension was stirred overnight at room
O 2
O
O S CF 3
O
17
C6H12F3O6PS
Mol. Wt.: 300.1909
EtO P
EtO 1
temperature. Et2O (25 ml) was added to the suspension which was then transferred via
cannula to a vigorously stirred solution of trifluoromethanesulfonyl chloride (3.00 ml,
28.1 mmol) in Et2O (25 ml) at -78 ºC. The reaction mixture was then allowed to warm
158
to room temperature over the course of 3 hours. The resulting solids were filtered over
celite; the filtrate was taken up in CH2Cl2 (200 ml) and washed with saturated NaHCO3
until TLC of the organic phase showed no starting material was left (5  50 ml). The
organic phase was then dried (Na2SO4) and the solvent was removed in vacuo affording
diethyl trifluorosulfonyl-methylphosphonate (17) as a colourless oil (1.10 g, 3.66 mmol;
21%): Rf(EtOAc) 0.63; H (200 MHz, CDCl3) 1.25 (6 H, t, J 7.1, 2  ethyl CH3), 4.1 (4
H, q, J 7.1, 2  ethyl CH2), 4.54 (2 H, d, J 8.8, CH2P); C (50 MHz, CDCl3) 16.10 (d, J
9.7, 2  ethyl CH3), 63.61 (d, J 8.8, 2  ethyl CH2), 66.30 (d, J 167.3, CH2P), 151.20
(CF3).
Preparation
of
diethyl
[(1-chloro-6-formyl-phenanthren-4-
yl)oxymethyl]phosphonate (19)
Diethyl trifluorosulfonyl-methylphosphonate (17; 199 mg, 0.65
mmol)
was
added
to
a
solution
of
8-chloro-5-
hydroxyphenanthren-3-carboxaldehyde (100 mg, 0.40 mmol)
and diisopropylethylamine (114 l, 0.65 mmol) in dry CH2Cl2
(20 ml) in a sealed tube. The solution was flushed with argon
2' 3'
Cl
1'
4'
10'
9'
O
OEt
O 2 P
OEt
1
5'
6'
CHO
8' 7'
19
C20H20ClO5P
Mol. Wt.: 406.7963
before the tube was sealed and the reaction mixture was heated at
70 ºC overnight. The solvent was then evaporated and the residue submitted to flash
column chromatography (7:3 light petroleum-EtOAc eluent). The target material (19)
was obtained as a light brown solid (31 mg, 7.62 mol; 12%): Rf (EtOAc) 0.35; m.p.
101 ºC; max (KBr)/cm-1 1975, 1675, 1612, 1566, 1522, 1410, 1320, 1299, 1271, 1256,
1207, 1163, 1088, 1026, 962, 842, 820, 778, 704, 658; H (200 MHz, CDCl3) 1.34 (6 H,
t, J 7.1, 2  CH3CH2), 4.30 (4 H, q, J 7.2, 2  CH2CH3), 4.55 (2 H, d, J 10.5, CH2P),
7.15 (1 H, d, J 8.7, H-3’), 7.69 (1 H, d, J 8.6, H-2’), 7.92 (1 H, d, J 9.2, H-9’), 8.00 (1 H,
d, J 8.3, H-8’), 8.15 (1 H, dd, J 8.3 and 1.4, H-7’), 8.39 (1 H, d, J 9.1, H-10’), 10.21 (1
H, s, H-5’), 10.22 (1 H, s, CHO); C (50 MHz, CDCl3) 16.46 (d, J 5.6, 2  CH3CH2),
62.35 (d, J 170, CH2P), 62.97 (d, J 6.3, 2  CH2CH3), 109.38 (CH-3’), 122.17 (C),
123.55 (CH-7’), 125.75 (C), 125.91 (CH-10’), 127.33 (CH-2’), 128.47 (CH-9’), 129.02
(CH-8’), 129.34 (CH-5’), 131.24 (C), 135.10 (C), 136.39 (CH-4’), 193.46 (CHO); m/z
(EI) 406 (2%, M+) (Found M 406.0741; C20H20O5Cl requires 406.0737); (Found C,
58.95; H, 4.75%. C20H20ClO5P requires C, 59.05; H, 4.96%).
159
Preparation of 1-chloro-6-formylphenanthren-4-yl trifluoromethylsulfonate (16)
To
a
solution
of
8-chloro-5-hydroxyphenanthren-3-
2 3
O
O S CF 3
8a
4a
O
5
4b
10
6
8a
CHO
9
8 7
Cl
carboxaldehyde
(15;
300
mg,
1.17
mmol)
and
diisopropylethylamine (264 l, 1.51 mmol) in dry CH2Cl2 (30
ml) at 0ºC was added freshly distilled triflic anhydride (232 l,
1
4
16
C16H8ClF3O4S
Mol. Wt.: 388.7462
1.41 mmol). The reaction was left to stir for 2 hours at 0 ºC and
then at room temperature overnight. The reaction mixture was
then washed with 3M aqueous HCl (3  30 ml). The organic layer was dried (Na2SO4)
and the solvent was removed in vacuo. The crude material was then submitted to a
flash column chromatography on silica (9:1 CHCl3-light petroleum eluent). The yellow
solid obtained was recrystallised from hot Et2O to afford the target material (16; 434
mg, 1.13 mmol; 96%): Rf (CHCl3) 0.66; max (KBr disk)/cm-1 2923, 1703, 1613, 1562,
1520, 1452, 1416, 1298, 1248, 1218, 1137, 984, 865, 829, 774; H (200 MHz, CDCl3)
7.52 (1 H, d, J 8.5, H-3), 7.68 (1 H, d, J 8.6, H-2), 7.85 (1 H, d, J 9.2, H-9), 7.95 (1 H,
d, J 8.3, H-8), 8.10 (1 H, dd, J 8.2 and 1.4, H-7), 8.28 (1 H, d, J 9.2, H-10), 9.51 (1 H, s,
H-5), 10.12 (1 H, s, CHO); C (50 MHz, CDCl3) 121.33 (CH-3), 121.62 (C), 124.57
(C), 125.43 (CH-7), 125.71 (CH-10), 126.88 (C), 127.46 (CH-2), 128.54 (C), 129.67
(CH-9), 129.94 (CH-8), 131.84 (C), 132.72 (CH-5), 133.11 (C), 135.04 (C), 136.75 (C),
145.27 (C-4), 191.9 (CHO); m/z (EI) 388 (49%, M+) (Found M 387.9761;
C16H8O4ClF3S requires 387.9784); (Found C, 50.02; H, 2.11%. C16H8O4ClF3S requires
C, 49.43; H, 2.07%).
Preparation of 2-[(1-chloro-6-formylphenanthren-4-yl)amino]-3-phenylpropionic
acid ethyl ester (20)
A Wheaton round-bottom flask was charged with cesium
and racemic BINAP (14.0 mg, 21.2 mol). A solution of 1chloro-6-formylphenanthren-4-yl trifluoromethylsulfonate (16;
3
2' 3'
carbonate (263 mg, 0.81 mmol), Pd2(dba)3 (5.7 mg, 0.61 mol)
Cl
1'
4'
10'
9'
N 2 CO 2Et
H
1
5'
6'
CHO
8' 7'
120 mg, 0.31 mmol) and L-phenylalanine (240 mg, 1.2 mmol) in
dry toluene (5 ml) was introduced to the flask. Argon was
20
C26H22ClNO3
Mol. Wt.: 431.9105
flushed through the flask before it was sealed and the reaction
mixture was heated at 140 ºC overnight.
The reaction mixture was then directly
transferred onto a silica gel column and chromatographed (9:1 CHCl3-light petroleum
160
eluent).
The target compound 2-[(1-chloro-6-formyl)phenanthren-4-ylamino]-3-
phenylpropionic acid ethyl ester (20), was isolated as a light brown solid (10.1 mg, 2.34
mol; 7.5%): Rf (CHCl3) 0.58; H (200 MHz, CDCl3) 1.17 (3 H, t, J 7.1, ethyl CH3),
3.34 (2 H, dd, J 5.8 and 2.7, H-3), 4.15 (2 H, q, J 7.0, ethyl CH2), 5.10-5.20 (1 H, m, H2), 6.91(1 H, d, J 8.6, H-3’), 7.12-7.35 (5 H, m, Ph-H), 7.56 (1 H, d, J 8.6, H-2’), 7.84
(1 H, d, J 9.1, H-9’), 8.00 (1 H, dd, J 8.3 and 1.4, H-8’), 8.01 (1 H, dd, J 8.3 and 1.3, H7’), 8.31 (1 H, d, J 9.1, H-10’), 9.75 (1 H, s, H-5’), 9.8 (1 H, s, CHO). No further
characterisation was carried out with the small quantity of material obtained.
161
5. References
1 Ignarro, L. J.; Buga, K. S.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G.: Proc. Natl. Acad. Sci. USA, 1987,
84, 9265.
162
2 Palmer, R. M. J.; Ferrige, A. G.; Moncada, S.: Nature, 1987, 327, 524.
3 Ragsdale, R. O.: Development in Inorganic Nitrogen Chemistry, Elsevier, 1973, vol. 2.
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