Chapter 5 Photoresponsive and Antimicrobial Studies on...
Transcript of Chapter 5 Photoresponsive and Antimicrobial Studies on...
Chapter 5
Photoresponsive and Antimicrobial
Studies on Porphyrin and
Metalloporphyrin- Anchored Linear
and Dendritic Macromolecules
5.1 Introduction
There is much current interest in the study of polymer bound porphyrins because of
their interesting electronic properties and possible use as photosensitizers, catalyst and
complexing agents. Although the synthesis of a wide variety of polymer bound porphyrins
are well developed, relatively few soluble polymer bound porphyrins are studied. Our
interest is to prepare nature friendly and water soluble linear and dendritic polymer
bound metallated as well as metal free porphyrins. Significant changes in the electronic
spectra were seen in polymer bound metal incorporated porphyrins as well as metal free
porphyrins. This chapter gives a critical discussion on the absorption and luminescence
emission properties of porphyrins, metalloporphyrins and their polymer bound analogues.
The potential use of porphyrin bound to hyperbranched polyglycerol as artificial blood
137
substitutes, antimicrobial agents and its photosensitized antimicrobial action are discussed
in detail.
5.2 Tetraphenyl Porphyrin System
The spectral properties of porphyrin complexes have attracted considerable experi-
mental curiosity and theoretical interest because of their vital role in biological processes
such as photosynthesis, respiration and their potential technological applications. In the
UV -visible absorption spectrum, the highly conjugated porphyrin macrocycle showed in-
tense absorption at 400 to 450nm (the B band or soret band) followed by several weaker
absorptions (Q bands) at higher wavelengths from 500 to 650nm (figure 5.1). Peripheral
substituents on the porphyrin ring often cause changes on the intensity and wavelength
of these absorptions. The origin of intensities of the Q and B bands were successfully
explained by Goutermans four-orbital model1,2.
Figure 5.1. UV- visible spectrum of TPP
138
According to four- orbital model, the B and Q bands can be described in terms of
transitions between a pair of top filled orbitals (a1u and a2u) and lowest empty orbitals
(the doubly degenerate eg). The degeneracy of the a1u, a2u, and eg? excited levels leads
to a strong configuration interaction that results in a high-lying state corresponding to B
band and low- lying state corresponding to Q band. The configuration mixing combines
the transition dipoles of the individual one electron transition in such a way that the B
band contains nearly high intensity, while the Q band is weak. The soret band is assigned
as π - π ?type transition from the two highest occupied molecular orbitals (HOMO) a1u
(π) and a2u (π) to the lowest empty doubly degenerate antibonding molecular orbitals
eg?. The schematic representation of porphyrin HOMOs and LUMOs are shown in figure
5.2.
The presence of highly delocalized π electron system in porphyrin macrocycle provides
a variety of advantages for their applications. One of the ways to enhance the use of
porphyrins in optoelectronic devices is to further expand the existing π electron system.
Porphyrins are particularly appealing because of the richness of their properties, and
because expertise is available to modify these useful platform to build more complex
molecular architectures3.
Figure 5.2. Porphyrin HOMOs and LUMOs.
139
5.3 UV-Visible Absorptions of Linear Polymeric Core
Systems Modified with Tetraphenyl Porphyrins
The linear polymeric cores selected for the modification of porphyrin were poly vinyl
alcohol, poly ethylene glycol and polyglycerol poly adipate. All of them possess primary
alcoholic terminal groups which are accessible to common reactions such as esterification,
etherification reactions etc.
5.3.1 Polyvinyl alcohol modified with TPP
Polyvinyl alcohol bearing free OH groups can be bonded to chlorosulphonated por-
phyrins very well, but the resulting polymer is insoluble in almost all the solvents, the
spectral studies become difficult4. The polymer bound TPP was dissolved in DMF and
the absorptions were studied using these solutions.
The TPP have very characteristic electronic spectra having an intense band at 416
nm (soret/B band) and three or four less intense band at 520-650 nm(Q bands).
On chlorosulphonation we have observed a red shift of 23nm for soret band and 11nm
for the Q1 band. The soret band was shifted from 416nm to 439nm and the Q1 band
was shifted from 514 nm to 525 nm. The Q2, Q3 and Q4 bands were also red shifted
from 550nm to 559 nm, 591nm to 601 nm and 648 nm to 656 nm respectively. The lone
pairs on the sulphur atom causes increased electron delocalisation with the porphyrin
macrocycle π -electron framework. The SO2Cl group enhances the π conjugation and the
HOMO-LUMO energy gap is reduced. This effect is reflected in the red shift shown by
these systems.
The polymer bound porphyrin also exhibited the characteristic absorption. The elec-
tronic spectrum was recorded in DMF. The B and Q bands of TPP bound with polyvinyl
alcohol were blue shifted to 414 nm, 513 nm, 547 nm, 590 nm and 644 nm respectively
(figure 5.3).
The planarity of the porphyrin macrocycle is highly sensitive to structural changes. On
attaching to polyvinyl alcohol core, the steric effect causes a great degree of perturbation
140
on the planarity of the porphyrin macrocycle and the π electron framework is seriously
affected by this change. This causes a slight blue shift in the electronic spectral signals. A
shifting of 2nm, 1nm, 3nm, 1nm and 4nm respectively of soret, Q1, Q2, Q3 and Q4 bands
were observed on binding the TPP system on to the PVA core.The blue shift was very
prominent when compared with the absorbances of chlorosulphonated TPP (table 5.1).
The absorption bands of TPP did not show any notable red shift on binding to the PVA
core. However, the water insoluble TPP system became soluble in polar solvents such as
DMF on anchoring to PVA.
PVA is a film forming linear polymer with excellent hydrophilic character. Therefore,
the photosensitivity and photoresponsive character of the hydrophobic TPP π electron
frame work can be made hydrophilic and stable by attaching TPPSO2Cl on to PVA core.
This can be used for various industrial applications due to the excellent photochromic
character of TPP and structural properties of linear PVA core.
Figure 5.3. UV-visible spectra of TPP, TPP SO2Cl and PVA-TPP.
141
Table 5.1. Electronic absorptions of TPP, TPP -SO2Cl and PVA-TPP
System B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
Q3 band
(nm)
Q4 band
(nm)
TPP 416 514 550 591 648
TPP SO2Cl 439 525 559 601 656
PVA-TPP 414 513 547 590 644
5.3.2 Polyethylene glycol modified with TPP
The appending of porphyrin on to the polymers was confirmed by the conspicuous
colour change of the polymer and also by the electronic spectral measurements. Once
covalently bonded to the polymer, porphyrin was not exchangeable under ordinary con-
ditions, and the system was very stable in both polar and non-polar solvents. PEG is a
linear flexible polymer with dipolar character. Because PEG contains periodically spaced
electron rich ether functions along with terminal OH functions, it is highly compatible
with an aqueous medium and ionic species5,6.
The porphyrin bound PEG also exhibited the characteristic absorption properties.
The electronic spectrum was recorded in DMF. The B and the four Q bands were blue
shifted to 411nm, 509 nm, 541 nm, 583 nm and 63 nm respectively on binding to PEG
(figure 5.4).
The PEG end groups were functionalised with TPP. Compared to PVA-TPP system,
the loading of TPP on to the PEG system is low. However the soret and Q bands showed
blue shift due to the structural perturbation caused by the entangled polymer core on
the porphyrin framework. The soret band showed a shifting of 5 nm, from 416 nm to
411 nm on binding to PEG core. The intensity of Q bands were diminished considerably
and the shifting observed were 5nm, 9nm, 8nm and 9nm respectively for Q1, Q2, Q3
and Q4 bands (table 5.2). However, the PEG-TPP system is highly promising due to its
solubility in water and other polar solvents, its film forming properties and its potential
use in medicine and medical diagnosis.
142
Figure 5.4. UV-Vis spectra of TPP, TPP-SO2Cl and PEG-TPP.
Polyethylene glycol is a nature friendly linear polymer, available in a wide range of
molar masses, soluble in water and polar solvents and widely used in medicine, cosmetics
such as moisturisers, creams, gels etc and in coating applications. A typical hydrophobic
system like tetraphenyl porphyrin can be made hydrophilic and water soluble by attach-
ing the suitably made TPP on to PEG. In the present study we could develop a novel
photoactive porphyrin supported on PEG with molar mass 6000. The newly developed
system strongly absorb in the visible region (both B and Q bands). The PEG-TPP sys-
tem is soluble in water and other polar solvents and find many applications in diagnosis,
medicine and industry.
Table 5.2. Electronic absorptions of TPP, TPP-SO2Cl and PEG-TPP
System B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
Q3 band
(nm)
Q4 band
(nm)
TPP 416 514 550 591 648
TPP SO2Cl 439 525 559 601 656
PEG-TPP 411 509 541 583 639
143
5.3.3 Polyglycerol polyol modified with TPP
The porphyrin bound linear polyglycerol polyol adipate is a pink coloured semisolid
soluble in water and its flexible nature made the studies easier. Porphyrin is highly soluble
in CHCl3 but all the polymer bound porphyrins are insoluble in CHCl3. Both the soret
and Q bands are blue shifted but not to a notable extent after binding with the polymer
(figure 5.5).
Figure 5.5. UV-Vis spectra of TPP, TPPSO2Cl and PG-TPP.
The TPP has aggregation tendency and which when attached to the linear system
like PEG and PG, this tendency may be enhanced due to the entanglements and that
results in spectral shifts and line broadening. Moreover, the planarity and consequently
the electron delocalisation of the π-frame work were perturbed by the steric hindrance
imposed by the polymer backbone on to the porphyrin macrocycle. When compared to
PVA and PEG, the polyglycerol polyadipate system is less entangled so that the structural
144
perturbation caused on the polymer is less and the shifting observed is to a less extent.
The shifting observed in the Q bands were 2nm, 4 nm, 1 nm and 1 nm respectively for
Q1, Q2, Q3 and Q4 bands (table 5.3). The soret band get blue shifted by 2m from 416 of
TPP on binding with PG.
Table 5.3. The comparative study of TPP, TPP-SO2Cl and PG -TPP
System B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
Q3 band
(nm)
Q4 band
(nm)
TPP 416 514 550 591 648
TPP SO2Cl 439 525 559 601 656
PG -TPP 414 512 546 590 647
The absorption phenomenon of PG-TPP are almost comparable to that of TPP. PG-
TPP strongly absorbs in the visible region of UV-visible spectrum. An intense absorption
was noted at 414 nm corresponding to the soret band. The Q bands were also of moder-
ately high intensities. The PG-TPP system is polar and water soluble though TPP is a
hydrophobic system.
5.4 UV-visible Absorptions of TPP Bound Hyper-
branched Polyglycerol
The porphyrin bound hyperbranched polyglycerol possessed wonderful photochemical
properties7,8. It is insoluble in CHCl3 but freely soluble in water and other polar solvents
though TPP shows the opposite behaviour. The UV-visible spectrum shows signals at
472 nm (soret band) and at 563 nm, 600 nm, 640 nm and 694 nm respectively for Q1, Q2,
Q3, and Q4, bands on binding to HPG (table 5.4).
The aggregation tendency of TPP is not found when it is anchored on to hyperbranched
polyglycerol due to the non-entangled structural architecture of HPG. But the aggregation
was very prominent when TPP was bound to linear polymeric cores such as PVA, PEG
and PG. A red shift of 56 nm was observed for the soret band and red shifts in all the Q
145
bands in the electronic spectra of HPG systems confirmed this. The intensity of the Q 4
band was tremendously increased on attaching to HPG backbone (figure 5.6).
Figure 5.6. UV-vis spectra of TPP, TPPSO2Cl and HPG-TPP.
The phenyl ring of TPP is not coplanar with the porphyrin macrocycle. On binding
or coupling with polymers like PVA, PEG or linear polyglycerol systems, the soret band
seems to be slightly blue shifted while the Q bands nearly disappeared but they appear
clearly and red shifted while the TPP is coupled with HPG.
The peaks of the porphyrin in UV-visible region have generally interpreted in terms
of π- π* transition between bonding and antibonding molecular orbitals. The two inter
band transition, Q and B are assigned as π- π* type.
The significant broadening and red shift of 49 nm (Q1), 50 nm, (Q2), 49 nm (Q3), and
46 nm (Q4) bands in HPG-TPP system indicate increased electron delocalisation due to
increased polarity9. It is observed that after coupling with HPG there is increase in the
146
intensity of absorption band as shown in the figure 5.6. This is because, coupling with
the polymer rearranges few of the electric dipoles from opposite in to parallel direction
and consequently the oscillator strengths increased.
Table 5.4. The comparative study of TPP, TPPSO2Cl and HPG -TPP
System B band
(nm)
Q1band
(nm)
Q2 band
(nm)
Q3 band
(nm)
Q4 band
(nm)
TPP 416 514 550 591 648
TPP SO2Cl 439 525 559 601 656
TPP-HPG 472 563 600 640 694
The red shift of TPP moieties after coupling with HPG may be due to the variations
in the electronic charge delocalization within the porphyrin macrocycle assisted by the
highly branched and heavily functionalised hyperbranched polyglycerol core system. It
is well known that any increase in the electron density within a molecular system would
result on an increase in the energy of HOMOs. In TPP the phenyl rings are not coplanar
with the porphyrin macrocycle. Hence such an electric flow from the phenyl rings would
not be possible for the free TPP or MTPPs.
Table 5.5. Comparative study on absorption spectra of TPP bound linear polymers
and HPG
System B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
Q3 band
(nm)
Q4 band
(nm)
TPP 416 514 550 591 648
PVA -TPP 414 513 547 590 644
PEG -TPP 411 509 541 583 639
PG -TPP 414 512 546 590 647
HPG-TPP 472 563 600 640 694
5.4.1 Percentage loading of TPP and UV-visible absorptions
A series of new and novel HPG-TPP system have been developed with varying amounts
of TPP and subjected to spectral measurements. The results showed that all the systems
147
exhibited red shift to almost the same extent with an increase in absorption values (figure
5.7). All of them gave well-resolved spectra indicating the absence of any dipolar interac-
tions in them and proving that porphyrin was well separated on the polymer matrix. The
intensity of both soret and Q bands were enhanced tremendously as a function of loading
of TPP on the HPG core. 100% TPP loaded HPG core shows very intense absorptions
both in the soret and Q regions. The enhanced absorption of HPG-TPP system opens
immense possibilities in photo responsive applications.
Figure 5.7.UV-Vis spectra of 20%, 40%, 60%, 80% and 100% TPP loaded HPG.
148
5.5 Emission Studies on Tetraphenyl Porphyrins and
Polymer Bound Tetraphenyl Porphyrins
Porphyrins are known for their interesting emission properties. Among these, the
fluorescence properties are very unique. Many of the special applications of these systems
are based on such emission properties. When a molecule is excited to an upper electronic
state, it may return to the ground state either by radiationless cascade or by its emission
from lowest excited singlet and triplet states. The nature and quantum yield of such
emissions are determined by the relative rates of various deactivation processes. The
fluorescence yield of most porphyrins is less than 0.2.
Thus the excited state S1 is primarily deactivated by radiationless decay. It appears
fairly certain that the spin forbidden process S1→ T1 is the predominant route for ra-
diationless deactivation of S1 in porphyrins. The fluorescence spectrum of pure TPP is
shown in figure 5.8. A strong emission in the range from 600nm to 800 nm with two
maxima at about 649 nm and 716 nm was observed with an intensity of 34.3 a.u and 24.2
a.u respectively (figure 5.8).
Figure.5.8. Fluorescence spectrum of TPP.
149
5.5.1 Emission spectra of TPP bound polymer systems
The fluorescence spectra were recorded under the excitation wavelength of 400 nm on
a Hitachi F-3000 fluorescence spectrometer. In the entire polymer bound TPP systems,
the positions of the bands are not much shifted, but we could observe blue shifting of
3-5 nm in both the bands in the case of linear polymers like PEG and PG. PEG-TPP
system exhibited an intense emission at 646 nm and a shoulder at 714 nm and the extent
of shifting was by 3nm and 2nm respectively(figure 5.9).
Figure 5. 9. Fluorescence spectrum of PEG-TPP.
When TPP was bound with linear polyglycerol system the intensity of fluorescence
emission was decreased to 0.39 a.u and 0.23 a.u respectively for the bands. The emission
peak of the PG-TPP system appeared blue shifted by 4nm from 649 nm to 645 nm and
from 716 nm to 712 nm compared to that of TPP (figure 5.10).
150
Figure 5.10. Fluorescence spectrum of PG-TPP system.
When TPP was anchored with HPG we have noticed red shifting in the emission and
the peak appeared at 649 nm in TPP was shifted to 652 nm and the peak at 716 nm was
shifted to 718 nm and the extent of red shift was by 7 nm and 2 nm respectively (figure
5.11).
Figure 5.11. Fluorescence spectrum of HPG-TPP system.
151
From the figure 5.11 it is clear that the fluorescence intensity was reduced to 0.91a.u
at 652 nm and 0.51a.u at 718 nm.The emission properties are summarized in table 5.6.
Table 5.6. Summary of emission behaviour of polymer bound TPP
System Emission Wave-
length λ1
Emission Wave-
length λ2
Intensity
I
Intensity
II
TPP 649 716 34.3 24.2
PEG-TPP 646 714 11.1 9.4
PG-TPP 645 712 3.9 2.3
HPG-TPP 652 718 0.91 0.51
On attaching the linear polymeric cores such as PEG and PG, the emission spectra
showed a remarkable decrease in intensity and the emission maxima were blue shifted.
When attached to HPG also the intensity showed a hypochromic shift but the emission
maxima was red shifted. When the fluorophore is in hydrophobic environment the fluores-
cence intensity is enhanced due to the slow radiationless decay. TPP is highly hydrophobic
in nature. So its emission spectra show maximum intensity. Introduction of TPP on to
hydrophilic polymer backbones therefore decrease its hydrophobicity and hence the inten-
sity is decreased. The blue shift in the linear systems may be attributed to the coiling in
these systems. The extent of coiling is less due to the presence of bulky fluorophore and
so the shifting is less. No such coiling is possible in HPG and hece it shows red shift10.
5.6 Electronic Spectra of Metalloporphyrins
Upon metallation the porphyrin ring system deprotonates, forming a dianionic ligand.
The metal ions behave as Lewis acids, accepting lone pairs of electrons from the dian-
ionic porphyrin ligand. Unlike most transition metal complexes, their colour is due to
absorption(s) within the porphyrin ligand involving the excitation of electrons from π to
π * porphyrin ring orbitals. The electronic absorption spectrum of a typical porphyrin
consists of a strong transition to the second excited state (S0 → S2) at about 400 nm
(the Soret or B band) and a weak transition to the first excited state (S0 → S1) at about
152
550 nm (the Q band). Internal conversion from S2 to S1 is rapid so fluorescence is only
detected from S1. The B and the Q bands both arise from π to π * transitions and can
be explained by considering the four frontier orbitals (HOMO and LUMO orbitals). This
is known as the Gouterman four orbital model (figure 5.12).
Figure 5.12 Orbital diagrams showing possible transitions for porphyrins.
According to this theory, the absorption bands in porphyrin systems arise from tran-
sitions between two HOMOs and two LUMOs, and it is the identities of the metal center
and the substituents on the ring that affect the relative energies of these transitions.
Mixing splits these two states in energy, creating a higher energy 1eu state with greater
oscillator strength, giving rise to the soret band, and a lower energy 1eu state with less
oscillator strength, giving rise to the Q-bands.
Metalloporphyrins can be divided into two groups based on their UV-vis and fluores-
cence properties11. Regular metalloporphyrins contain closed-shell metal ions (d 0 or d
10)for example Zn (II) in which the d π (dxz , dyz ) metal-based orbitals are relatively
low in energy. These have very little effect on the porphyrin π to π* energy gap in por-
phyrin electronic spectra (fig. 5.13). Hypsoporphyrins are metalloporphyrins in which
the metals are of dn, n= 6 to 9, having filled d π orbitals. In hypsoporphyrins there is
significant metal d π to porphyrin π* orbital interaction (metal to ligand π- backbonding)
(figure 5.14). This results in an increased porphyrin π to π* energy separation causing
the electronic absorptions to undergo hypsochromic shifts.
153
Figure 5.13. Molecular orbital diagram for metalloporphyrins. Interactions between dπ
and π* occur in hypsoporphyrins.
Figure 5.14. The dπ metal orbital overlap with the π system of the porphyrin ring.
5.7 Electronic Spectra of Metalloporphyrins Bound
to Linear and Hyperbranched Polymers
Metalloporphyrins have similar spectra as that shown by TPP. But on binding with the
polymers, ie, linear as well as hyperbranched polymers, exhibit shifts in their absorption
spectra. We have developed several metalloporphyrins of Cu (II), Zn (II) and Fe (II),
bound with linear polymers such as PVA, PEG and polyglycerol polyol and hyperbranched
polyglycerol, looking forward to carry out their possibility of applying in photoresponsive
applications, artificial blood products and in photodynamic therapy.
154
5.7.1 PVA-MTPP (M=Zn,Cu,Fe) system
The electronic spectra of all the MTPPs are very characteristic, with an intense band
near 415 nm and one or two less intense bands at 520-650 nm. On comparing with
metal free TPP, tetraphenyl porphyrin metallated with Zn (II) did not exhibit shift in
wavelengh while CuTPP and FeTPP exhibited red shift The metal free TPP bound with
PVA gives absorptions at 414 nm corresponding to soret band. The PVA-ZnTPP also
showed corresponding transitions at 415 nm. The soret bands of PVA-CuTPP and PVA-
FeTPP were, however, observed at 419 nm. There were considerable shifts observed in the
Q bands. In Zn (II) the dπ(dxz, dyz) metal-based orbitals are relatively low in energy. So
it has very little effect on π to π* energy gap in porphyrin electronic spectra. The same
trend is observed when ZnTPPs are bound to linear as well as hyperbranched polymers.
The UV-visible spectra of MTPPs (M= Zn (II), Cu (II) and Fe (II)) bound to PVA is
given in figure 5.15 and a comparison of the absorption maxima are given in table 5.7.
Figure 5.15. Comparative study of UV-vis spectra of PVA-MTPP system.
155
Table 5.7. Summary of UV-vis absorptions of PVA-MTPP systems
MTPP-
Polymer
System
B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
PVA -TPP 414 513 547
PVA- ZnTPP 415 514 549
PVA- FeTPP 419 517 554
PVA- CuTPP 419 519 553
5.7.2 PEG-MTPP (M=Zn,Cu,Fe) system
Metallated porphyrins bound with PEG exhibited characteristic transitions in their
UV-visible spectra. Compared to PEG-ZnTPP, PEG- CuTPP and PEG-FeTPP systems
exhibited red shifts. The UV-visible spectra of MTPPs (M= Zn (II), Cu (II) and Fe (II))
bound to PEG are shown in figure 5.16 and the results are summarised in table 5.8.
Figure 5.16. Comparative study of UV-Vis spectra of PEG-MTPP system.
156
Table 5.8. Summary of UV-vis absorptions of PEG-MTPP systems
MTPP-
Polymer
System
B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
PEG -TPP 411 509 541
PEG- ZnTPP 412 512 545
PEG- FeTPP 414 515 554
PEG- CuTPP 416 518 555
5.7.3 PG-MTPP ((M=Zn,Cu,Fe) system
The PG-MTPP systems also exhibited characteristic absorptions from the UV-visible
region. PG-CuTPP and PEG-FeTPP exhibited prominent red shifts compared to PEG-
ZnTPP. The UV-visible spectra of the PEG-MTPP systems are shown in figure 5.17. The
spectral shifts in both soret and Q bands are given in the table 5.9.
Figure 5.17. Comparative study of UV-vis spectra of PG-MTPP system .
157
Significant changes in the electronic spectra (redshifts in both B and Q bands) were seen in
polymer-incorporated MTPPS in comparison with metal free systems. This is explained
in terms of the molecular distortions and associated changes in the metalloporphyrin
orbital overlap and the charge delocalization from the peripheral substituents 12.
Table 5.9.Summary of UV-vis absorptions of PG-MTPP systems
MTPP-
Polymer
System
B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
PG- TPP 414 512 546
PG- ZnTPP 416 512 544
PG- FeTPP 418 516 551
PG- CuTPP 418 518 550
5.7.4 HPG-MTPP (M=Zn,Cu,Fe) system) system
The metallated porphyrin bound to HPG also showed significant shifts in the UV-
visible spectra. The HPG- ZnTPP system showed only small transitions while FeTPP
and CuTPP bound to HPG showed significant red shifts in their Q1and Q2 bands (figure
5.18). The UV-visible spectra of the PEG-MTPP systems are shown in figure 5.18 and
the UV-visible absorption wavelengths of HPG-MTPP systems are given in table 5.10.
The absorption characteristics of the HPG-MTPP systems show superior properties
over PVA-MTPP, PEG-MTPP and PG-MTPP systems (tables 5.7-5.10). The B (soret)
band of the MTPP systems bound to PVA were found to be at 415nm, 419nm and 419nm
for M=Zn, Fe and Cu respectively. The Q1 and Q2 bands of the PVA-MTPP systems
varies from 514 nm to 519 nm and 549 nm to 553 nm respectively. PEG-MTPP systems
showed absorption maxima for soret and Q bands at 416 nm (soret band of PEG-CuTPP)
and 555 nm (Q2 of PEG-CuTPP). The metallated TPP bound to linear polyglycerol
polyadipate showed maximum B-band absorption at 418 nm for PG-CuTPP and Q1 band
maximum at 518 nm and Q2 band maximum at 550 nm. The hyperbranched polyglycerol
anchored with MTPP showed excellent absorption characteristics. The B-bands of these
158
systems observed at 458 nm,475 nm and 476 nm for HPG-ZnTPP, HPG-FeTPP and
HPG-CuTPP respectively. The Q bands also showed remarkable red shift. The Q1 bands
where observed at 557 nm, 574 nm and 566 nm for HPG-ZnTPP, HPG-FeTPP and HPG-
CuTPP systems. The corresponding Q2 band positions where found to be at 587 nm,
609 nm and 603 nm respectively. The absorption intensities of HPG-MTPP systems were
of high value compared to metal analogues. The remarkable red shift and highly intense
absorptions of HPG-MTPP systems make them excellent photoresponsive materials.
Figure 5.18. Comparative study of UV-vis spectra of HPG-MTPP system
(a)HPG-TPP, (b)HPG- ZnTPP, (c)HPG-FeTPP, (d)HPG-CuTPP.
159
Table 5.10. Summary of UV-vis absorptions of HPG-MTPP systems
MTPP-
Polymer
System
B band
(nm)
Q1 band
(nm)
Q2 band
(nm)
HPG- TPP 472 563 600
HPG- ZnTPP 458 557 587
HPG- FeTPP 475 574 609
HPG- CuTPP 476 566 603
5.8 EPR Spectral Studies of Polymer Bound CuTPP
We have made an attempt to confirm the presence of Cu(II) in the polymer by record-
ing the EPR spectra of the compounds CuTPP anchored on to linear and hyperbranched
polymers. The EPR spectra indicate the presence of Cu(II) in all the compounds. The
EPR spectra of Cu (II) complexes were recorded using a Varian E-112 EPR spectropho-
tometer operating at 9.1 GHz with a microwave power of 5mw. The field set is around
3000G and the scan range used was either 2000 or 1000 G. DPPH was used as g marker.
(i) PVA- CuTPP system
The EPR spectrum of PVA- CuTPP system was recorded in DMF at room temper-
ature and the spectrum is given in figure 5.19. The spin Hamiltonian parameters of the
spectrum are A‖ (gauss) 178, A⊥ (gauss) 30, g‖ 2.235 and g⊥ 2.075, characteristic of the
copper porphyrin.
160
Figure 5.19. The EPR spectrum of PVA- CuTPP system.
(ii)PEG- CuTPP system
The EPR spectrum of PEG-CuTPP system recorded in DMF at room temperature
is given in figure 5.20. The spin Hamiltonian parameters of the spectrum are A‖ (gauss)
180, A⊥ (gauss) 20, g ‖ 2.239 and g⊥ 2.080.
Figure 5.20. The EPR spectrum of PEG- CuTPP system.
(iii)PG- CuTPP system
The following figure represents the EPR spectrum of PG- CuTPP system. The spin
Hamiltonian parameters are A‖ (gauss) 173, A⊥ (gauss) 30, g‖ 2.243 and g⊥ 2.047,
indicating the presence of copper incorporated in the porphyrin macrocycle.
161
Figure 5.21. The EPR spectrum of PG- CuTPP system.
(iv) HPG- CuTPP system
The EPR spectrum of HPG- CuTPP system recorded at room temperature showed a
strong signal at high field and the representative of the spectrum is given in figure 5.22.
The spin Hamiltonian parameters of the spectrum are A‖ (gauss) 175, A⊥ (gauss) 30, g‖
2.234 and g⊥ 2.048. all these results give evidence for the incorporation of copper in the
macrocyclic frame work of porphyrin system.
Figure 5.22. The EPR spectrum of HPG- CuTPP system.
162
We could evaluate the spin-Hamiltonian parameters assuming axial symmetry for all
the four systems. The giso and G could be evaluated using the following equations,
giso = g⊥ + g‖ and
The values are given in Table 5.11. Various spin-Hamiltonian parameters were cal-
culated from the spectra using DPPH as the g marker. The trend g‖ > g⊥ > 2.0023
observed for these complexes show that the unpaired electron is localized in the dx2-y2
orbital. All the four compounds gave only broad peaks characteristic of axial symmetry in
the solid state. G> 4.0 indicate that the local tetragonal axes are only slightly misaligned
while for G >4.0 indicate that the misalignment is appreciable. Axial spectra with lowest
g > 2.04 exhibited by all compounds refer to axial symmetry with all the principal axes
aligned parallel, and would be consistent with square planar stereochemistry.
Table 5.11. Summary of Spin Hamiltonian parameters of the polymer bound
Cu(II)TPP systems
compound A ‖ A ⊥
(gauss)
g ‖ g⊥ giso G
PVA- CuTPP 178 30 2.235 2.076 2.131 3.15
PEG-CuTPP 180 20 2.239 2.080 2.133 3.05
PG-CuTPP 173 30 2.243 2.047 2.113 5.30
HPG-CuTPP 175 20 2.234 2.048 2.110 5.04
5.9 Fluorescence Studies of Polymer Bound Metallo-
porphyrins
In order to study the effect of metallation on the emission properties, the emission
spectra of the metallo porphyrin derivatives of linear and hyperbranched polymers were
subjected to luminescent emission studies. We have selected metallated PEG-TPP as a
representative of linear polymeric systems and compared it with hyperbranched polymer.
It was found that the introduction of metal ion increases the fluorescence intensity in both
the cases. This may be attributed to the increased rigidity of the molecule upon metal-
163
lation. The increase in rigidity of the molecule is expected to decrease the intersystem
crossing and hence an increase in fluorescence intensity13.
The fluorescence emission spectra of polymer bound metalloporphyrins were recorded
in DMF at an excitation wavelength of 400 nm. The metalloporphyrins were anchored
on to linear and hyperbranched polymers and the fluorescence emission properties were
investigated. The PEG-ZnTPP, PEG-FeTPP and PEG-CuTPP showed interesting emis-
sion properties. The spectra are given in figure 5.23. The λ max as well as the intensity
values of PEG- ZnTPP, PEG-Fe TPP and PEG-CuTPP are given in table 5.12. PEG-
TPP system gave emission maxima at 646 nm and 714 nm with an intensity of 11.1 a.u
and 9.4 a.u respectively. The corresponding values of PEG- ZnTPP, PEG- FeTPP and
PEG-CuTPP are 645 nm and 707 nm (33.1.u and 21.2 a.u), 648 nm and 715 nm (34.1a.u
and 24.3a.u) and 649 nm and 716 nm (36.8 a.u and 27.7 a.u ) respectively.
Figure 5.23.Emission spectra of (a) PEG- TPP, (b) PEG -ZnTPP, (c) PEG- FeTPP
and (d) PEG- CuTPP.
164
Table 5.12. Summary of emission behaviour of PEG bound MTPP
System Emission Wave-
length λ1
Emission Wave-
length λ2
Intensity
I
Intensity
II
PEG-TPP 646 714 11.1 9.4
PEG-ZnTPP 645 707 34.1 24.3
PEG-FeTPP 648 715 36.8 27.7
PEG-CuTPP 649 716 33.1 21.2
The fluorescence emission studies of HPG-MTPP systems gave promising results.
HPG-TPP system showed emission maxima at 652 nm and 718 nm with an intensity
of 0.91a.u and 0.51a.u respectively. The HPG-CuTPP system showed maximum intensity
of absorption at 649 nm and 716 nm (intensity 33.9a.u and 24.4 a.u ). the spectra are
given in figure 5.24 and the results are summarised in table 5.13.
Figure 5.24. Comparative study of emission spectra of (a) HPG-TPP, (b) HPG
-ZnTPP (c) H PG- FeTPP (d) H PG- CuTPP.
165
Table 5.13. Summary of emission behaviour of HPG bound MTPP
System Emission
Wave-
length
λ1
Emission
Wave-
length
λ2
Intensity
I
Intensity
II
HPG-TPP 652 718 0.91 0.51
HPG-ZnTPP 645 714 4.1 2.2
HPG-FeTPP 646 714 11.1 9.3
HPG-CuTPP 649 716 33.1 21.2
5.10 Solvation Studies
The unique absorption spectra of porphyrin systems have allowed their identifica-
tion and study throughout the biological realm. It is generally observed that metallic
porphyrins exhibit notable variations in spectral properties, which are dependent on the
nature of the solvents. This is attributed to the relative ligation properties of the solvents
used with the metal. Compared to the metalloporphyrins, the free-base porphyrins lack
the ability to co-ordinatively add the ligands. Consequently the metal- free porphyrins
are not expected to show such change in the electronic spectra depending on the solvent,
except due to any possible tendency towards aggregation. But remarkably enough, while
measuring the absorption spectra of TPP-polymer system under study, we found that the
absorption changes were significant enough in solvents. A series of solvents with varying
polarity is used for the solvation studies. The solvents used were DMSO, DMF, methanol,
toluene, hexane and chloroform.. The electronic spectra of the TPP-polymer system in
various solvents were investigated and the λ max values are given in table 5.14.
166
Table 5.14. The λmax(nm) values of TPP and TPP-polymer systems in various
solvents
System Bands DMSO DMF methanol chloroformtoluene
TPP B 439 416 414 414 412
Q1 522 514 512 512 510
Q2 558 550 527 526 526
Q3 603 591 556 550 550
Q4 710 648 641 640 645
PVATPP B 449 414 insoluble insoluble 412
Q1 514 513 insoluble insoluble 506
Q2 548 547 insoluble insoluble 512
Q3 601 590 insoluble insoluble 540
Q4 661 644 insoluble insoluble 582
PEG-TPP B 422 411 408 405 405
Q1 518 509 506 500 501
Q2 535 541 523 518 516
Q3 598 583 578 578 577
Q4 682 639 628 620 620
PG-TPP B 444 414 413 412 405
Q1 518 512 512 501 501
Q2 555 546 548 538 538
Q3 603 590 586 578 577
Q4 691 647 644 640 640
HPG-TPP B 488 472 463 463 460
Q1 582 563 560 558 557
Q2 610 600 597 596 596
Q3 652 640 638 637 637
Q4 710 694 692 687 685
167
The data clearly reveal that all the bands (B, and Q) are red shifted on increasing
the polarity of the solvent. This behavior indicate that the excited state of these com-
pounds are more polar than their ground state and thus, this red shift can be ascribed to
stabilization of the polar excited state as the polarity of the solvent increased i.e., lower
excitation energy is required in DMSO or DMF relative to CHCl3. This confirm the local
excitation nature, i.e. the π- π* character of the bands.
The electronic spectra of polymer-porphyrin system in mixed solvents (DMF and
CHCl3) were also studied. The results also reveal that as the percentage of DMF increases,
the λ max values shifts to the longer wavelength region. This may be due to increased
πconjugation due to increased polarity and the red shift is consistent with the decrease
of HOMO-LUMO gap. This also confirms the π- π* character of the bands. The λmax
values of different TPP polymer systems in various mixed solvents are given in the table
5.15.
5.11 Light Fastening Studies on Polymer TPP Sys-
tems
From the viewpoint of photochemistry and photobiology, interactions of solar radiation
with matter are considered to occur when one photon interacts with one molecule to
produce a photochemically altered molecule or two dissociated molecules. Porphyrin is
highly photosensitive and this macrocyclic ring in chlorophyll acts as the photosensitizer
in photosynthesis.
To study the action of light on TPP and polymer bound TPP we have irradiated the
equimolar solutions of TPP and polymer bound TPP systems under visible radiant ener-
gies and measured the absorbances in definite time intervals. We have noticed significant
changes in the intensity of absorptions for the free TPP as well as the polymer bound
TPP. Compared to other photochromic systems, TPP is very fast towards light but it
also exhibits shifts in the intensity of absorptions (figure 5.25).
168
Table 5.15. The λmax(nm) values of polymer-porphyrin system in mixed solvents
(DMF and CHCl3)
System Bands 0%DMF 20%DMF 40%DMF 60%DMF 80%DMF100%DMF
TPP B 411 412 412 414 414 416
Q1 508 510 510 512 512 514
Q2 526 532 538 546 549 550
Q3 550 559 563 576 583 591
Q4 640 642 642 646 648 648
PVATPP B 406 408 411 414 414 414
Q1 499 502 507 507 507 513
Q2 511 512 512 512 514 547
Q3 532 540 541 544 544 590
Q4 571 568 572 577 578 644
PEG-TPP B 406 408 410 410 410 411
Q1 500 506 508 508 509 509
Q2 520 522 522 524 525 541
Q3 578 578 580 580 582 583
Q4 620 622 627 628 630 639
PG-TPP B 405 410 412 414 414 414
Q1 501 508 508 510 510 512
Q2 538 542 542 544 546 546
Q3 577 588 588 588 590 590
Q4 640 640 643 644 645 647
HPG-TPP B 456 458 461 461 462 472
Q1 557 557 559 560 560 563
Q2 590 591 593 597 600 600
Q3 634 635 635 638 638 640
Q4 685 685 689 691 694 694
169
Figure 5.25. Variation of absorbance of TPP on irradiation.
The curve (a) represents the absorption band of TPP at zero time and the absorbance
intensities in the visible region are B (2.2), Q1 (1.02), Q2 (0.69), Q3 (0.59) and Q4 (0.52).
On irradiation for five hours the intensities were decreased as B (1.83), Q1 (0.84), Q2
(0.36), Q3 (0.24) and Q4 (0. 17). Further irradiation to infinite time did not cause any
change in the spectra.
Table 5.16. Results of light fastening studies on TPP system
time(hrs) B band
(inten-
sity(a.u))
Q1 band
(inten-
sity(a.u))
Q2 band
(inten-
sity(a.u))
Q3 band
(inten-
sity(a.u))
Q4band
(inten-
sity(a.u))
0 2.2 1.02 0.69 0.59 0.52
1 2.04 0.86 0.36 0.24 0.21
2 1.86 0.86 0.36 0.24 0.17
3 1.83 0.84 0.36 0.24 0.17
4.5hr 1.83 0.84 0.36 0.24 0.17
24 1.83 0.84 0.36 .24 0.17
170
On irradiation, chlorosulphonated TPP was found to be more stable than TPP, to-
wards irradiation though there was small regular decrease in the intensity of absorbance
on prolonged irradiation (figure 5.26).
Figure 5.26. Variation of absorbance of TPP SO2Cl on irradiation.
Table 5.17. Results of light fastening studies on TPP-SO2Cl
time(hrs) B band
(inten-
sity(a.u))
Q1band
(inten-
sity(a.u))
Q2band
(inten-
sity(a.u))
Q3band
(inten-
sity(a.u))
Q4band
(inten-
sity(a.u))
0 1.9 0.74 0.48 0.30 0.21
0.5 1.8 0.59 0.38 0.22 0.15
1 1.79 0.54 0.35 .0.19 0.12
2 1.79 0.45 0.29 0.19 0.13
3 1.79 0.41 0.23 0.14 0.08
4 1.79 0.41 0.23 0.14 0.08
5 1.79 0.41 0.23 0.14 0.08
24 1.79 0.41 0.23 0.14 0.08
171
PVA bound TPP was found to be highly stable on irradiation with visible light. No
appreciable change in intensity was noted even after 5 hours of irradiation. The light
fastening behaviour of the TPP macrocycle binding to PVA core is evident from the
results shown in figure 5.27. The intensities observed for PVA-TPP systems were 1.98
and 0.25 for the soret and Q bands respectively.
Figure 5.27. Variation of absorbance of PVA-TPP on irradiation.
Table 5.18. Results of light fastening studies on PVA-TPP system
time(hrs) B band
(inten-
sity(a.u))
Q1band
(inten-
sity(a.u))
Q2band
(inten-
sity(a.u))
Q3band
(inten-
sity(a.u))
Q4band
(inten-
sity(a.u))
0 1.98 0.25 0.16 .12 .08
1 1.76 0.21 0.12 0.09 0.06
2 2.04 0.19 0.10 0.05 0.05
3 1.86 0.19 0.10 0.05 0.05
4 1.83 0.19 0.10 0.05 0.05
4.5 1.83 0.19 0.10 0.05 0.05
24 1.86 0.19 0.10 0.05 0.05
172
When PEG bound TPP was irradiated and measured the absorbance spectra we ob-
served a small decrease in the in the absorbance values during the first one hour. After
that the values remain constant for many hours. In figure 5.28 the band (a) represents
the absorbance at zero time and absorbances were measured for different time intervals.
The spectra do not show any appreciable change in intensity on prolonged irradiation.
This shows the light fastening property of PEG-TPP system and this may be due to the
stabilization of porphyrin macrocycle when it is bound to polymeric core such as PEG.
Figure 5.28. Variation of absorbance of PEG-TPP on irradiation
Figure 5.28. Variation of absorbance of PEG-TPP on irradiation.
173
Table 5.19. Results of light fastening studies on PEG-TPP system
time(hrs) B band
(inten-
sity(a.u))
Q1band
(inten-
sity(a.u))
Q2band
(inten-
sity(a.u))
Q3band
(inten-
sity(a.u))
Q4band
(inten-
sity(a.u))
0 1.20 0.06 0.03 0.01 0.003
1 1.20 0.06 0.03 0.01 0.003
4 1.20 0.06 0.03 0.01 0.003
4.5 1.20 0.19 0.10 0.05 0.05
24 1.20 0.06 0.03 0.01 0.003
Polyglycerol polyol bound TPP was subjected to time controlled irradiation studies
using radiant energy from the visible light. The PG-TPP system showed high stability
and light fastening on prolonged irradiation. The intensity of absorption also shows that
this new system is highly stable even after prolonged exposure to radiant energies. The
results are shown in figure 5.29.
Figure 5.29. Variation of absorbance of PG-TPP on irradiation.
174
Table 5.20. Results of light fastening studies on PG-TPP system
time(hrs) B band
(inten-
sity(a.u))
Q1band
(inten-
sity(a.u))
Q2band
(inten-
sity(a.u))
Q3band
(inten-
sity(a.u))
Q4band
(inten-
sity(a.u))
0 2.11 0.21 0.12 0.07 0.06
1 2.10 0.13 0.06 0.04 0.03
2 2.10 0.21 0.12 0.04 0.04
3 2.10 0.21 0.12 0.04 0.04
4 2.10 0.21 0.12 0.04 0.04
4.5 2.10 0.21 0.12 0.04 0.04
24 2.10 0.21 0.12 0.04 0.04
TPP bound HPG had been irradiated for 7 hours continuously in sunlight and the ab-
sorbances were measured by UV-visible spectroscopy. The absorption values were found
to remain constant and there was no change in λmax as well. This indicated that por-
phyrin macrocycle was highly stabilized when bound to HPG. The intensity of absorption
was appreciably high in this system and the intensity remains unchanged on exposure for
long time. The high loading, non-entangled configuration and optimum spacial rigidity
of the hyperbranched polyglycerol contribute to these properties (figure 5.30)
Figure 5.30. Variation of absorbances of HPG-TPP on irradiation .
175
Table 5.21. Results of light fastening studies on HPG-TPP system
time(hrs) B band
(inten-
sity(a.u))
Q1band
(inten-
sity(a.u))
Q2band
(inten-
sity(a.u))
Q3band
(inten-
sity(a.u))
Q4band
(inten-
sity(a.u))
0 2.12 0.21 0.11 0.07 0.07
1 2.10 0.13 0.06 0.04 0.03
2 2.10 0.13 0.06 0.04 0.03
3 2.10 0.13 0.06 0.04 0.03
4 2.10 0.13 0.06 0.04 0.03
4.5 2.10 0.13 0.06 0.04 0.03
24 2.10 0.13 0.06 0.04 0.03
5.12 Antimicrobial Phototherapy
Phototherapy is the term used to describe treatments, which use light to achieve
their effects14. For example blue light exposure, which is used to treat newborn babies
with neonatal jaundice. In this case blue light absorbed by bilirubin, the yellow pigment
responsible for producing the skin discoloration, and turns it in to a more soluble form,
which is easier to excrete from the body.
Another application of phototherapy, called phototodynamic therapy (PDT), uses a
combination of electromagnetic radiation (usually laser light) and a drug to selectively
target and destroy cancers. Photodynamic therapy matured as a feasible medical technol-
ogy in 19806s at several institutions through out the world for the treatment of carcinomas
and sarcomas15. The German physician, Mayer-Betz performed the first study with pho-
todynamic therapy with porphyrins in humans in 1913. It is also being investigated for
treatment of psoriasis and acne, and is approved for the treatment of muscular degenera-
tion. Mayer-Betz tested the effects of haemato porphyrin-PDT on his own skin. Modern
day versions of it were tested at the Mayo Clinic at Rosewell Park Cancer Center, but
really did not become widespread until Thomas Dougherty16 initiated clinical trials and
formed the International Photodynamic Association in 1986.
176
The mechanism of photodynamic therapy involves the hopefully selective uptake and
retention of a photosensitizer in a tumor, followed by irradiation with light of particu-
lar wavelength, thereby initiating tumor necrosis presumably through the formation of
singlet oxygen17. It is a ternary treatment for cancer involving three key components:
photosensitizer, light and tissue oxygen18.
5.12.1 Photosensitizer
Photosensitizer is a chemical compound that can be excited by light of a specific
wavelength. This excitation uses visible or near infra red light. It is important that the
sensitizer should be easy to administer systemically via injection in to the blood stream.
Water-soluble sensitizer would therefore be expected to be the most useful since the blood
is a water-based system. But the sensitizer must also be able to get in to the cells by
traversing lipid membranes, thus it should ideally also be hydrophobic.
All photosensitizing agents used in PDT have very similar structures and are often
based on naturally occurring molecules including hemoglobin (the substance that makes
blood look red), vitamin B12, and chlorophyll (the chemical used by the plants for pho-
tosynthesis and which gives them green colour). These compounds are all known as
macrocycles and contain nitrogen, oxygen or sulphur atoms locked inside a large hollow
ring. In some cases the ring also contains a metal such as iron or magnesium. The pho-
tosensitizing agent used as the drug in PDT is given to the patient, which accumulates
in the diseased tissue. It is harmless in its inactive form, but when it is excited by light
of the correct wavelength (usually in the form of laser) it produces singlet oxygen, which
is highly toxic and kills the cell (figure 5.31).
Photodynamic therapy currently makes use of a range of agents from plant extracts
to complex synthetic macrocycles, but characteristically, they are all able to accumulate
selectively in the diseased tissue.
177
Figure 5.31. The process of photodynamic therapy. [A drug is given to the patient (1)
which accumulates in the diseased tissue (2) In its inactive form the agent is harmless
(3) but when it is excited by light of the correct wavelength (usually in the form of a
laser) it produces singlet oxygen which is highly toxic (4) and kills the cell]
5.12.2 Porphyrins as photosensitizers
Human tissue transmits light most effectively in red region of the visible spectrum
and hence photosensitizers with strong absorption band in this region (650-800nm) can
be activated to penetrate deeper in to the tissues. Porphyrins are 22π electron systems
whose main aromatic conjugation pathway contains 18π electrons, which explains long
wavelength absorptions and the intense colour associated with them.
Porphyrinoid photosensitizers have more than one absorption band that can be utilized
for tissue depth controlled penetration. Provided that the porphyrin posses an absorption
maximum at a wavelength corresponding to that of the incident laser light, shining light of
highly coloured porphyrin causes excitation to the singlet state (1P*). The singlet-excited
porphyrin can decay back to the ground with release of energy in the form of fluorescence-
enabling identification of tumor tissue. If the singlet excited state lifetime is suitable (and
this is true for many porphyrins) it is possible for the singlet excited state to be converted
in to the triplet-excited state (3P*), which is able to transfer energy to another triplet-
178
excited state. One of the very few molecules with a triplet ground state is dioxygen, which
is found in most cells. Energy transfer therefore takes place to afford highly toxic oxygen
(1O2) from ground state dioxygen (3O2), provided the energy of the molecules are higher
than that of the product (1O2)19,20 . The reason that singlet oxygen is liberated in the
cells is because of simple photophysics. Figure 5.32 shows a simplified Jablonski diagram
showing the photophysics of the sensitization process used in photodynamic therapy.
Figure 5.32. Photophysics of PDT sensitization.
5.13 Phorphyrin Bound Hyperbranched Polyglycerol
for Antimicrobial Phototherapy
5.13.1 Biocompatibility of hyperbranched polyglycerol
Many biologically active compounds are not suitable for therapeutic purposes because
of their poor solubility, limited bioavailability and rapid elimination. More than that,
while the beneficial effects of many drugs arise through their interactions with specific
tissues, their exposure to other cell types frequently lead to other side effects and toxic-
ity. In recent years there has been increasing interest in photoactive dendritic systems.
End group modified dendrimers are under investigation in a variety of applications such
as carriers of drug and other guest molecules. But the major drawback is their mul-
tistep synthesis and therefore hyperbranched polymers selected as an alternative. The
179
hyperbranched polyglycerol synthesized by the anionic ring-opening multibranching poly-
merization (ROMBP) of glycidol consists of an inert polyether backbone with functional
hydroxyl groups at every branch end. This structural feature resembles the well known
poly (ethylene glycol) (PEG) that is accepted for various biomedical applications. Hy-
droxyl terminated dendrimers based on polyether scaffold have been shown to be of low
toxicity. The non-toxic properties make these new polymers very promising candidates
for drug delivery devices.
The hydroxyl groups of HPG are derivatized with chlorosulphonated porphyrin groups.
The porphyrin which is also biocompatible but hydrophobic become water-soluble by
connecting with HPG.
Before considering the HPG-porphyrin system as a photosensitizer we have considered
a number of issues on PDT. It is important that the sensitizer must be easy to administer
systemically (via injection in to the blood stream). Water-soluble sensitizers would there-
fore be expected to be the most useful since the blood is a water-based system. But the
sensitizer must also be able to get in to by traversing lipid membranes. Thus it should
also be hydrophobic. The porphyrin-polyglycerol system is such an amphiphilic molecule.
Another factor is that the sensitizer must also absorb at long wavelength (i.e., at low
energy). This is because the low energy light travels further through tissue than does high-
energy light (which gets scattered). If we want to kill big tumors by generating singlet
oxygen inside them we need to have sensitizer which likewise absorbs low energy light -
one which has absorption peak in the low energy (long wavelength) area of the electronic
absorption spectrum. The absorptions of TPP in the Q band region were strongly red
shifted after coupling it with HPG (figure 5.34).
180
Figure 5.33. Structure of HPG-TPP system
The porphyrinated hyperbranched polyglycerol exhibited intense absorptions in the
visible region of the spectrum (table 5.22).
We have studied the UV-visible absorptions of linear polymer bound porphyrins like
PVA-TPP, PEG-TPP and PG-TPP and compared them with the absorbance of HPG-
TPP system. The HPG-TPP system exhibited significant red shiftin its absorptions for
the soret and all the four Q bands. of 46 nm to a longer wavelength of 694 nm, which is
a primary requirement for an ideal photosensitizer.
181
Figure 5.34. UV-Vis spectra of (a) TPP (b) HPG-TPP (c) PVA- TPP (d) PEG-TPP
and (e) PG-TPP .
Table 5.22. Light absorption by TPP and TPP bound HPG
Bands λ (nm)
(TPP)
εmax
(TPP)
λ (nm)
(HPG-
TPP)
εmax
(HPG-
TPP)
Q1 514 0.35 563 2.4
Q2 550 0.168 600 1.73
Q3 591 0.11 640 1.13
Q4 648 0.103 694 1.008
The linear systems such as PVA-TPP, PEG-TPP and PG- TPP and their metallated
systems do not have absorptions at longer wavelength region and not enough to meet
the benchmark requirements of PDT agents. However HPG-TPP system meets all these
182
requirements and can be used as an efficient system for PDT. The soret band of HPG-
TPP shows signal at 472 nm with an intensity of 2.1. The Q1, Q2, Q3 and Q4 bands were
observed at 563 nm, 600 nm, 640 nm and 694 nm with intensities 2.4, 1.73, 1.13 and 1.008
respectively.
It is extremely important to be able to synthesize pure molecules for the use as pho-
tosensitizer. The porphyrin bound HPG, however can be easily prepared, functionally
modified and purified to equip them with intense light responsive and novel therapeutic
applications.
Simpler and economical therapies that can effectively treat cancer with minimum side
effects are the dire straits of this new millennium. Last century witnessed a renewed surge
in reinventing the medicinal aspects of light in presence of photosensitizers namely, Pho-
todynamic therapy, was found to be exceptionally useful as anticancer treatment protocol.
HPG-porphyrin based PDT can offer a promising treatment protocol for cancers and va-
riety of other diseases for which remedial measures existing are minimal. The realization
that any chromophore that can induce phototoxicity upon illumination leading to selec-
tive destruction of diseased (premalignant, malignant and benign) tissues lead to incessant
possibilities and any step taken in the design and development of such chromophores are
very much appreciated. HPG-TPP system can make wonders in this field.
5.14 Antimicrobial Activity of HPG-TPP System
The science of photodynamic antimicrobial therapy follows similar principles of PDT. It
is argued that the widespread systemic use of antibiotics is a cause of multidrug resistance
and local therapy using photodynamic agents would lessen the risk of such collateral ef-
fects. Localized infections need not be treated with systemic medication if an efficient
alternative is available. In the past decade photosensitized porphyrin derivatives in con-
junction with laser light were adopted for treating several microbial infections on skin.
There were studies on photodynamic action against microorganisms such as Salmonella
typhimurium, or the yeast, Saccharomyces cerevisiae21. Photosensitizers being readily
available and inexpensive should be attractive in the area of low cost topical health
183
care regimens. The natural product porphyrins are effective against a range of anaer-
obic bacteria. Porphyrin sensitizers are more effective against gram-positive bacteria but
gram-negative organisms are more refractory to photodyanamic activity due to their more
complex cell wall22,23. Although several light sources proposed as lasers and xenon arc
lamp etc, antimicrobial therapy uses low power density light rather than lasers. Microbial
photokilling is attained with milliwatts rather than tens or hundreds of watts19. The
power density of a light source is normally given in mW/cm2 where as the light source
describes the energy received (by a wound or a petri dish) and as such can be calculated
as the power density multiplied by the illumination time (in seconds). The use of directed
light against microbial pathogens in situ also causes the problems of the possibility of col-
lateral damage. Such effects can be minimized by keeping a minimum distance between
the light source and petri dish concerned so as to keep the temperature 370C-390C. In
the present studies we have chosen 500W tungsten filament lamp (wave length >600nm)
having a power density 0.28 mW/cm2.
The mechanism of photodynamic microbial killing is same as that of tumor cell necrosis
in PDT. On irradiation the photosensitizer gets excited to the triplet state which trans-
fers its energy to molecular oxygen, and the singlet oxygen formed in situ then reacts
rapidly with its environment-cell wall, nucleic acids, peptides etc. The short half-life of
singlet oxygen again ensures a localized response.18 Haematoporphyrin derivative is the
first preparation used in clinical PDT and has some activity against both bacteria and
viruses24,25. Many porphyrins are benign in the dark but are transferred by sunlight and
produce singlet oxygen, which is toxic to cells. In the present work, the study of photo
toxicity after 4-hr incubation with TPP, MTPPs, HPG-TPP and HPG-MTPPs on both
Staphylococcus aureus and Escherichia coli were carried out in vitro. The different steps
by which the experiment was done is as follows:
1. 18 number of 10ml nutrient broth tubes were prepared and sterilized at 121 0C and
under 15 lbs pressure. Then they are appropriately labeled.
2. A loopful inoculam of Staphylococcus aureus were asceptically transferred in to 9
tubes of the medium kept at room temperature. Similarly a loopful of inoculam
184
of Escherichia coli was also asceptically transferred in to the other 9 tubes of the
medium kept at room temperature.
3. Incubated all the tubes at 37 0C for 4 hours.
4. After incubation, 5 mg of each of the samples to be tested were added in appropri-
ately labeled tubes.
5. Reincubated the culture tubes at 37 0C for further 12 hours.
6. After the incubation period, 100µl aliquot from each broth was transferred to
petridishes and spread plates were prepared. The plates were incubated at 37 0C
for 24 hours.
7. Then the tubes were irradiated using 500W tungsten filament bulb for 2minutes.
8. After irradiation, the tubes were left in atmospheric condition for 30 minutes. After
that 100µl aliquot from each tube is transferred to petridishes and spread plates
were prepared.
9. The tubes were further irradiated for another 3 minutes and spread plates were
again prepared.
10. All the plates were incubated at 370C for 24 hours.
11. After the incubation period, the plates were examined for bacterial growth.
In the present studies we have selected two bacterial cultures, gram positive Staphy-
lococcus aureus and gram negative Escherichia coli. The systems selected to test photo-
senzitivity were TPP, FeTPP, HPG-TPP and HPG-FeTPP. The observations were differ-
ent for different systems and were interesting. The gram-negative E.coli bacterium was
found to be refractory to all the systems but the gram-positive bacteria, Staphylococcus
aureuswere killed by the photo oxidation even though a complete destruction was not
achieved. We have done a comparative study of photosensitizing property of porphyrin,
its metal (Fe) incorporated system and their hyperbranched polyglycerol bound systems.
185
The spread plates prepared using systems before irradiation gave uncountable colonies of
microbes while after irradiation microbial killing was achieved and have obtained count-
able colonies of microbes in the petri dishes. Of these systems FeTPP exhibited a very
significant toxicity. The polymer bound TPP and FeTPP systems were also exhibited
efficient photokilling of the microbes. The photodynamic efficiency of TPP was retained
on binding with HPG. The advantage is that the HPG-TPP as well as the HPG-FeTPP
systems were water soluble and more biocompatible than the polymer free TPP systems.
The comparative study of photodynamic antimicrobial activity of the systems under study
were possible by examining the photographs given below. TPP strongly acted on Staphy-
lococcus aureus and the colonies became countable on irradiation for short period of two
minutes. The colony was reduced to almost 75 percent on irradiation for a time span of
5 minutes (figure 5.35).
Figure 5.35. Staphylococcus aureus colony inoculated with TPP: (a) before irradiation-
uncountable (b) after irradiation for 2 minutes- countable (c) after irradiation for 5
minutes- countable.
186
The action on bacteria, Escherichia coli was not evident and the colony remains
uncountable even after irradiating for 10 minutes. This may be due to its more complex
cell wall compared to gram-positive bacteria23(figure 5.36).
Figure 5.36. Escherichia coli colony inoculated with TPP: (a) before irradiation-
uncountable (b) after irradiation for 10 minutes- Uncountable .
HPG bound metal free TPP also had photodynamic antimicrobial property against
Staphylococcus aureus and this system became superior to TPP because of its hydrophilic-
ity and biocompatibility. The antimicrobial property of HPG-TPP system before and
after irradiation was given in figure 5.37. the entire bacterial colony remain intact before
irradiation of the system with visible radiant energy. On irradiation for two minutes, the
bacterial colony was severely attacked by the newly developed photodynamic antimicro-
bial agent HPG-TPP. This is evident from figure 5.37 (b). On completing irradiation for
5 minutes, more than 75 percent of the bacterial colony was destroyed by the HPG-TPP
photodynamic antimicrobial agent (figure 5.37 (c)).
187
Figure 5.37. Staphylococcus aureus colony inoculated with HPG-TPP : (a) before
irradiation- uncountable (b) after irradiation for 2 minutes- uncountable (c) after
irradiation for 5 minutes- countable) .
When metallated systems were used, against Staphylococcus aureus, the property of
microbial killing was found to be enhanced due to the presence of the heavy metal which
itself inhibits microbial growth. Therefore FeTPP exhibited surprisingly good result when
irradiated for 5 minutes (figure 5.38). The colony becomes countable after 2 minutes of
irradiation and almost 80% of the colony was destroyed on irradation for three minutes
more. It was ensured that the distance between tubes and the bulb were arranged in a
manner that, the temperature within the tubes were not exceed as 37-390C.
188
Figure 5.38. Staphylococcus aureus colony inoculated with FeTPP : (a) before
irradiation- uncountable (b) after irradiation for 2 minutes- countable (c) after
irradiation for 5 minutes- countable.
When FeTPP bound HPG system was used in the antimicrobial phototherapy against
gram positive Staphylococcus aureus, we could notice its significant effect on microbial
killing and the effect was more enhanced than metal free HPG-TPP or simple TPP or
FeTPP system. The bacterial colony almost half on irradiation for two minutes using
visible radiant energy. On further irradiation, the effective photokilling was observed and
further after 5 minutes of irradiation almost 90% of the bacterial colony was photokilled
(figure 5.39).
189
Figure 5.39. Staphylococcus aureus colony inoculated with HPG- FeTPP : (a) before
irradiation- uncountable (b) after irradiation for 2 minutes- countable (c) after
irradiation for 5 minutes- countable.
From the above results it is clear that both porphyrin and HPG bound porphyrin
systems are effective photosensitizers which can be used against gram -positive bacteria
and by increasing the duration of irradiation cidal effect can be achieved.
5.15 Porphyrin Bound Hyperbranched Polyglycerol
as Potential Artificial Blood Product
One of the principal tasks of blood is to transport oxygen throughout the body and
then to release the oxygen to tissues. This is accomplished by the oxygen-carrying pro-
tein haemoglobin, which possess an Fe(II) porphyrin unit at its active site. It is this
Fe(II) porphyrin that is responsible for (reversibly) binding oxygen. The protein serves
to protect and isolate the oxygen binding porphyrin units, as well as helping prevent its
deactivation. Traditional attempts to mimic the reversible oxygen binding properties of
190
heme containing proteins concentrated on constructing simple Fe(II)porphyrins appended
with large groups26.
There are reports on porphyrin bound hyperbranched polyester, which was obtained
by using just one synthetic step, could mimic hemoglobin. The porphyrin cored hy-
perbranched polyester was therefore synthesized by reacting as excess of the branching
monomer, 3-5-diacetoxy benzoic acid with a small amount of tetrakis (4-acetoxy phenyl)
porphyrin under reversible trans esterification. Fe (II) is also inserted using standard con-
ditions. UV-visible spectrophotometric techniques were used to assess the oxygen binding
potential for porphyrin bound hyperbranched polyester.
In the present work the tetrakis Fe (II) porphyrinato sulphonyl chloride was coupled
with hyperbranched polyglycerol and the UV absorption was measured. It exhibited an
absorption maximum at 475 nm in DMF. Oxygen was bubbled through the solution for
1 minute and the absorption maximum was noted and it was found to be get shifted to
486 nm. Nitrogen is bubbled through the solution for 5 minutes, the UV spectrum was
found to be get restored to 475 nm (figure 5.40). By removing oxygen from the solution
the uncomplexation of the Fe (II) species was observed. The experiment was conducted
with the solutions of varying concentrations and the shift observed was consistent.
Figure 5.40. The UV-visible absorptions of HPGFeTPP system. (a) O2 free
HPGFeTPP system and (b)Fe(II)-O2 complex.
191
The oxygen binding capability of the HPG-TPP system was tested in aqueous solutions
of variable pH values. For this, solutions of HPG-TPP system in three different pH were
prepared. The different pH values of the solutions prepared were 4.2, 7.0, and 8.3. All
the solutions are of same concentrations (2x 10−4 millimoles). Through these solutions
N2 was bubbled for five minutes to remove any dissolved O2 and the UV spectra were
taken. At pH 4.2 the soret band was observed at 453 nm (figure 5.41) and at pH 7 and
pH 8.3 the soret band was observed at 430 nm (figure 5.42).
Figure 5.41. The UV-visible spectrum of HPG-Fe TPP system at pH 4.2.
192
Figure 5.42. The UV-visible spectrum of HPG-FeTPP system at pH 7.0 / 8.3.
Oxygen was bubbled through these solutions for half an hour and UV spectra were
taken. No significant shift was observed at pH 4 (figure 5.44) but at pH 7 and at pH 8.3 a
characteristic shift in the soret band was observed by 5nm and 7 nm respectively (figure
5.45 and 5.46). Then N2 was bubbled for 5 minutes and the UV spectra were taken again.
No change was observed at pH 4 but the soret band came to the original position for
solutions at pH 7 and at pH 8.3, that is at 430 nm.
193
Figure 5.43. The UV-visible spectrum of HPG-FeTPP system after O2 binding at pH
4.0.
Figure 5.44. The UV-visible spectrum of HPG-FeTPP system after O2 binding at pH
7.0.
194
From the results it is evident that oxygen-binding capability of HPG-FeTPP system
is appreciably high only at pH 7 or above. When the pH is below 7 it doesnt bind oxygen.
These results approved the efficacy of porphyrin bound HPG as artificial blood product
to reversibly bind oxygen. We came to the conclusion that the solutions at pH 7 and above
are efficient for binding oxygen than solutions at pH below 7. Since the porphyrin bound
HPG was water-soluble and biocompatible it can be used as safe artificial blood product
for human use.
Figure 5.45. The UV-visible spectrum of HPG-FeTPP system after O2 binding at pH
8.3.
In the present work we have synthesized different polymeric derivatives of tetraphenyl
porphyrin and metalloporphyrins. We used both linear and hyperbranched polymeric
systems. The linear systems used were polyvinyl alcohol (PVA), polyethylene glycol
(PEG) and polyglycerol polyadipate (PG). The hyperbranched polymeric system used
was hyperbranched polyglycerol (HPG). All these polymeric systems are water soluble,
nature friendly and having free hydroxyl functional groups in common which can be used
to bound with porphyrin by condensation reaction.
195
Porphyrin on binding with linear polymers like PVA, PEG and polyglycerol polyadi-
pate, exhibited blue shift in the absorption spectra. This is due to the structural per-
turbations caused by the entangled linear polymer core on the porphyrin frame work.
The planarity of the porphyrin macrocycle is highly sensitive to structural changes. On
attaching to linear polymers, the steric effect causes a great degree of perturbation of the
planarity of the porphyrin macrocycle and the p electron framework is seriously affected
by this change. This causes a notable blue shift in the electronic spectral signals. The
TPP has aggregation tendency and which when attached to the linear system like PVA,
PEG and PG, this tendency may be enhanced due to the entanglements and that results
in spectral shifts and line broadening. On attaching to hyperbranched polyglycerol no-
table red shift was observed in the absorption spectrum of porphyrin due to the variations
in the electronic charge delocalization within the porphyrin macrocycle assisted by highly
branched and heavily functionalized HPG core system. Metallation of TPP caused further
red shift in the absorption spectra.
The emission studies of metalloporphyrins bound with linear and HPG polymers re-
vealed several significant and established facts about metallated porphyrins. We noticed
an increased fluorescence yield for FeTPP, ZnTPP and CuTPP bound to linear and hyper-
branched systems. This is due to increased rigidity of TPP structure on metal insertion,
which is expected to decrease the intersystem crossing yields.
Light fastening studies conducted on the various polymer systems proved that the sta-
bility of the TPP was very much increased when anchored to a polymer system. To study
the action of light on TPP and polymer bound TPP we have irradiated the equimolar
solutions of TPP and polymer bound TPP systems under visible radiant energies and
measured the absorbances of definite time intervals. We have noticed significant changes
in the intensity of absorption for the free TPP as well as the polymer bound TPP. Com-
pared to other photochromic systems HPG-TPP is very fast towards light, and it also
exhibits shifts in the intensity of absorption. The red shift and stability towards conti-
nous light exposure make HPG-TPP and its metallo derivatives excellent photoresponsive
systems for photoprobing in medicine and medical diagnosis and in various industrial and
laboratory applications.
196
TPP and MTPP are insoluble in water but when modified with HPG it became sol-
uble. HPG is non toxic and biocompatible and hence the system can find applications
as photosensitizer in photodynamic therapy. A good sensitizer should be able to absorb
light from longer wavelength and should be water soluble. We have studied the UV-visible
absorptions on linear polymer bound porphyrins and compared them with the absorbance
of HPG-TPP system. The HPG-TPP system exhibited significant red shift which is a
primary requirement for an ideal photosensitizer. Since the HPG-TPP system is such an
ambhiphilic molecule it can be used as a photosensitizer.
Photodynamic antimicrobial therapy was done by using this compound and found to be
successful. The antimicrobial studies where carried out against a gram positive bacterium
Stayphylococcus aureus and gram negative bacterium Escherichia coli. The photoinduced
anti microbial activity was studied with TPP, FeTPP, HPG-TPP and HPG-FeTPP. The
gram negative E.coli bacterium was found to be refractory to all the systems but the gram
positive bacterium Stayphylococcus aureus where killed by the photo-oxidation process.
UV-visible spectrometric techniques were used to assess the oxygen binding potential for
porphyrin bound hyperbranched polyester. The oxygen binding capability of HPG-TPP
system was tested in aqueous solutions of variable pH values. From the results it is evident
that oxygen-binding capability of HPG-FeTPP system is appreciable at pH 7 or above.
When the pH is below seven it doesnt bind oxygen. These results approved the efficacy
of porphyrin bound HPG as artificial blood product to reversibly bind oxygen. Since
the porphyrin bound HPG was water soluble and biocompatible it can be used as safe
artificial blood products for human use.
The HPG-TPP system developed in the present work and its metalloderivatives are
excellent systems, which could find many biomedical applications. The very intense ab-
sorptions at longer wavelength region make the system unique in this field. The water
soluble environmental friendly HPG-TPP and its metalloderivatives offer a wide spectrum
of applications in medicine, medical diagnosis, photodynamic therapy and so on.
197
5.16 References
1. Seybold, P.G., and Gouterman, M., J. Mol. Spectrosc., 31, 548 (1971).
2. Gouterman, M., Wagniere, G., and Snyder, H.C., J. Mol. Spectrosc., 11, 108 (1963).
3. http.///www.lasalle.edn/M prushan/Abs and Fluor of TPPH2
4. Batinic H., Spasoojevic, R D., Bindurant, B., Okado-Matsumoto, A., Fridovich, I.,
Vujaskovic, Z., and Dewhirst, M.W., Dalton Trans., 28, 617-624 (2006).
5. Sibrian-Vazquez M., Jensen Yimothy, J., Vicente, M., and Graca, H., J. Photochem.
Photobio., B. Biology, 86, 1011-1344 (2007).
6. Chavez, J.L., Wong, J.L., Jovanovic, A.V., Sinner, E.K., and Duran R.S., Nanobiotec-
nology, IEE proceeding’s 152,73-84 (2005).
7. Olga, Zakhareva, Michael, Grodzicki, Alfred, X. T., Cees Veeger., and Ivonne,
M.C.M. R., Biophysical Chemistry, 73, 189-203 (1998).
8. Marsh, D.F., and Mink, L M., J. Chem.(Ed)., 73, 1181 (1996).
9. Szacilowski, K., Macyk, W., Drzewiecka Matuszek, A., Brindel, M., and Stochel,
G., Chem. Rev., 105, 2647-2694 (2005).
10. Raja, C., Ananthanarayanan, K., and Paramasivam, N., European poly. J., 42,
495-506 (2006).
11. Shiavon, M.D., Iamamoto, Y., Nascimento, O. R., Assis, M. D., J. Molecular catal-
ysis A, 174, 213 (2001).
12. Mikki Vinodu, V., and Padmanabhan, M., J. Polymer Science, Part A: Polymer
Chemistry., 39, 326-334 (2001).
13. Ravikanth, M., Gupta, I., J. Chem. Sci., 117(2), 161-166 (2005).
14. http//www.cancer.org/docroot/ETO/content/ETO/1.3xPhotodynamic Therapy.asp.
198
15. Pushpan, S. K., Venkitaraman, S., Anand, V. G., Sankar, J., Parameswaran, D.,
Ganesan, S., and Chandrasekar, T. K., Curr. Med. Chem. Anticancer Agents, 2,
187-207 (2002).
16. Dougherty, T.J., J. Natl Cancer Inst., 94, 23 (2002).
17. http//www.chemgroups.ucdavis.edu/smith/PDTResearch/PDT html.
18. http//www.photochembgsu.com/applications/therapy.html.
19. Wainwright, M., J. Antimicrobial Chemotherapy, 42, 13-28 ( 1998).
20. Ito, T., J. Photochem. and Photobiol., Reviews, 7, 141-86 (1983).
21. Vaara, M., and Vaara, T., Nature, 303, 526-528 (1983).
22. Jayasankar, L., Macdonald, I. J., and Dougherty, T. J., J. Porphyrins and Phthalo-
cyanins, 5, pp 105-129 (2007).
23. Sham, M, S., Singhal, N., and Rajeswar, P. V., Ind.J.Chem., 40 B., 113-119 (2001).
24. Aneja, H. R., Experiments in Microbiology, New Age International, 69-71(1993).
25. Dubey, R.C., A Textbook of Biotechnology. 4th ed.,S.Chand and Co, Ram Nagar
New Delhi, 732 (2006).
26. http//www.biopolymer.group.shef.ac.uk
27. Collman, J.P., Fu, L., Zingg, A., and Diederich, F., Chem. Comm., 2, 193 (1997).
199