14 OPTICAL ACTIVITY AND

THE STRUCTURE OF
MACROMOLECULES
F. CIARDELLI
Department of Chemistry and Industrial Chemistry, University of Pisa, Italy and
CNR, Center for Stereordered and Optically Active Macromolecules, Pisa, Italy
O. PIERONI
Department of Chemistry and Industrial Chemistry, University of Pisa, Italy and
CNR, Institute of Biophysics, Pisa, Italy
and
A. FISSI
CNR, Institute of Biophysics, Pisa, Italy

14.1 INTRODUCTION
14.1.1 ORIGIN OF OPTICAL ACTIVITY IN
MACROMOLECULES
As in low molecular weight compounds, optical activity can be observed only in
chiral macromolecules, that is, macromolecules for which all allowed conforma-
tions lack reflection symmetry elements.
The identification of chiral macromolecules differs from that of low molecular
weight molecules because of the substantially linear structure along the chain
backbone. Accordingly, analysis of the symmetry properties has been carried out
on the basis of three different models: (i) the infinite length chain; (ii) the finite
length chain with equal end groups; and (iii) the finite length chain with different
end groups. Point symmetry valid for molecules having definite and 'discrete'
dimensions in all directions can be used only for the last two models, whereas
linear symmetry must be used for the first model, which implies an infinite
dimension [I]. In linear symmetry, in contrast to point symmetry, the new
symmetry operation 'translation' and the new symmetry element 'translation
axis' are introduced. The analysis for aflexiblemacromolecule, which can assume
an extremely large number of conformations, is conveniently carried out on the
most symmetric of these conformations, which is usually the 'planar zigzag'. The
Polymer Spectroscopy. Edited by Allan H. Fawcctt
© 19% John Wiley & Sons Ltd
analysis of the derived Fischer projection of an infinite chain indicates that this is
chiral when a symmetry plane containing the chain, those perpendicular to the
chain and that with translation containing the chain are all lacking [ I ] . For finite
length chains, chirality is guaranteed by lack of symmetry in the plane containing
the chain and the plane perpendicular to the chain at its central point [I].
Thus, in vinyl polymers, only atactic macromolecules can be chiral in the first
model with infinite length chain. The atactic and the syndiotactic macro-
molecules with an even number of monomer residues are chiral in the finite chain
model with identical end groups, whereas all isotactic, syndiotactic and atactic
chains have a chiral structure for the last model with different end groups [1,2].
Even in vinyl polymers consisting of chiral macromolecules, extremely low or
vanishing optical rotation can be predicted when the molecular weight is high,
even if a complete separation of the enantiomeric pair were to be possible. Indeed,
in vinyl polymers every stereogenic carbon atom is flanked by two CH 2 groups,
and its chirality arises only from the different lengths of the two chain sections
attached to it. Thus an appreciable contribution to chiroptical properties is
conceivable only for asymmetric centers close to the chain ends, the concentra-
tion of which decreases with increasing molecular weight. The same holds for
conformational optical activity referred to the presence of secondary structures,
involving the macromolecule as whole or a substantial fraction of it, with
a predominant handedness. This last is not attributable simply to stereogenic
centers (asymmetric centers) with a single absolute configuration in each repeat-
ing unit.
Indeed, the existence of purely conformational optical activity is not a unique
macromolecular requisite, being Well known in low molecular weight atro-
pisomerism. However, in polymers it assumes a very specific characteristic
connected with the occurrence of cooperative effects which allow transmittance
of molecular asymmetry along the chain to very long distances [3].
Most isotactic vinyl polymers assume helical conformations in the crystalline
state [4], but owing to the substantial achirality of the macromolecules both
screw senses are found in the lattice cells in equal amounts. This is even more true
in a melt or in solution, where left-handed and right-handed helical sections
alternate even within the same macromolecular chain. Certainly, an appreciable
optical rotation would be observed in the crystalline state provided that crystalli-
zation occurred under a chiral field inducing a single screw sense helicity in all
chains. Such an optical rotation would promptly be lost on melting or dissolution,
as an immediate equilibration between the two opposite helical senses would occur.
Isotactic macromolecules derived from achiral monomers have no preference
for right- or left-handed screw senses, and the two are perfectly balanced at inter-
and intramolecular level. However, distribution of left- and right-handed helical
secondary structures affects markedly the free energy of the system, alternation of
the two senses in the same chain being favored for entropic reasons [4,5]. If this
last situation takes place, conformational optical activity cannot be obtained
because of intramolecular conformational compensation, which hinders any
isolation of chains with a predominant handedness.
The above considerations are described in fuller detail in previous papers [6,7]
and indicate that macromolecules assuming a single chirality conformation can
show chiroptical properties characteristic of the conformation itself. Moreover, if
chromophores are present in the side chains specific chiroptical properties can
arise from dipole-dipole electronic interactions among these chromophores
disposed along the chirally arranged backbone. This situation is clearly shown in
poly-a-amino acids, in which specific and typical chiroptical properties are
associated with specific and typical conformations (a-helix, /?-structures, random
coil) [8].
In order to make one screw sense largely predominant in a single macro-
molecule, the intramolecular equilibration must be hindered by building very
rigid chains. In the limiting case the chains will form rods having helical structure
with either left- or right-handed helicity. Even if hindering of equilibration can be
considered as a kinetic effect, it cannot be excluded that thermodynamic contri-
butions are involved, particularly when rigidity is due to bulk side chains and
conformational reversals have a very high internal energy [5,9]. In other words,
bulky side chains in a vinyl type structure, for instance, can favor the formation of
longer helical chain sections, as the lower entropy is balanced by the gain in
internal energy due to minimization of the number of conformational reversals.
Indeed these last have, in the case of macromolecules with bulky and branched
side chains, larger internal energy per structural unit than the same unit in the
helical conformation [10]. Which mechanism is actually operating cannot be
established from primary structure as a general rule, an increase in temperature in
both cases favoring the equilibration of the two screw senses within each
macromolecular chain.
Chiroptical properties (molar rotatory power [ $ ] and molar ellipticity [0])
result from a weighted average of the contributions from different conformers, as
shown in the following equations:

where N1 indicates the molar fraction and [O] 1 (or [G]1) indicates the molar
rotatory power (or molar ellipticity) of the i-th conformer. In macromolecules the
molar entities refer to one residue; thus the chiroptical properties are independent
of molecular weight, at least when this is very high.
It has been demonstrated that in isotactic polymers of optically active a-olefins
the molar optical rotation per monomeric residue can be interpreted in terms of
the prevalence of few conformations with very high optical rotation of the same
sign, corresponding to those allowed to the structural unit inserted in an one
screw sense helix [11].
 Moreover, in coisotactic copolymers of optically active a-olefins with viny-
laromatic monomers, it was shown that the aromatic groups in the side chains
give rise to dichroic bands in the spectral region of the n->n* electronic
transitions. In several cases exciton splitting was also observed, correspon-
ding to the strong allowed n-m* electronic transition [12]. This circular-
dichroism (CD) couplet was confirmed to be connected to the dipole-dipole
electronic interaction of transition moments of aromatic moieties disposed
in a mutual chiral geometry with a predominant handedness, such as that of
a one screw sense helix [13,14]. This clear demonstration that the extrinsic
CD bands of side chains are related to main chain conformation indicates
their usefulness as 'conformational probes'. A typical case comprises poly-
peptides with aromatic side chains masking the peptide absorption bands
[15].

14.1.2 OBJECTIVE

With reference to the concepts summarized in the previous section, optical

activity measurements can be particularly effective in providing structural
information on polypeptides with side chains absorbing at a wavelength clearly
distinct from that of the peptide group. In some favorable cases, moreover,
CD spectra can allow the detection of very specific structural features, inclu-
ding non-bonding interactions. On the other hand, the same data can be used
for monitoring even subtle structural changes induced by external factors.
Accordingly, the evaluation of chiroptical properties allows one to follow
crucial conformational changes accompanying such biological phenomena
as substrate binding, macromolecule-macromolecule interactions, and so
on.
In order to substantiate these last considerations, the present paper is devoted
to the description of studies, using mainly CD spectra, concerning poly (L-
glutamic acid)- and poly(L-lysine)-bearing photochromic side chains. Light
irradiation of these polypeptides gives rise to reversible isomerization of the
photoresponsive chromophores attached to the macromolecule's backbone,
which itself can then undergo reversible conformational changes. These may be
accompanied by reversible variations of the polymer's properties, such as viscos-
ity, solubility and so on.
The main objective of this paper is to show that the examination of the
chiroptical properties during the above mentioned phenomena can allow the
correlatation of changes in conformation and properties with the photoresponse
of the chromophores. On the same basis, an interpretation at the molecular level
can be put forward of the reversible variation of viscosity and the solubility. It is
also hoped that these indications may be useful for developing photorecording
devices which can be read using their chiroptical properties.
14.2 CHIROPTICALPROPERTIESOF
PHOTOCHROMIC POLYPEPTIDES
14.2.1 POLYPEPTIDES PHOTORESPONSIVE TO UV LIGHT
14.2.1.1 Azobenzene-containing Polypeptides
Polypeptides sensitive to irradiation with near UV light can be prepared by
introducing photochromic azobenzene units into the side chains of high molecu-
lar weight (Mv = 100000-250000) poly(a-amino acid)s, such as poly(L-glutamic
acid) or poly(L-lysine). Macromolecules having the structures represented in
Figure 14.1, and containing various percentages of azo groups, can be obtained
under various reaction conditions [16,17].
The photoisomerization of azobenzene moieties (Figure 14.2) is the event
responsible for the photochromic behavior of these macromolecules. At room
temperature in the dark all azo groups are in the trans configuration, which is
planar and then fully conjugated. Irradiation produces isomerization to the cis
configuration which, by contrast, is not planar for steric reasons. At the photo-
stationary state, the relative composition of the two isomers depends only on the
incident light. The maximum photoconversion to the cis isomer (85 %) is achieved
by irradiating at 350-370 nm, whereas the maximum yield of the back reaction
from the cis to the trans isomer (80%) is achieved by irradiating at 450 nm. With
a lamp having a power of 100 W, irradiation for 1 or 2 min is enough to achieve
the photostationary state.
By dark adaptation, the metastable cis chromophores decay again to the trans
form. The thermal decay at room temperature in the dark is rather slow for
azo-modified poly(L-glutamic acid) and takes more than 20Oh to restore the
all-trans isomeric composition; for azo-modified poly(L-lysine) the decay in the

Figure 14.1 Chemical structures of poly(L-glutamic acid) and poly(L-lysine) containing

azobenzene units in the side chains
Figure 14.2 Photochromic behavior of azobenzene-containing poly(L-glutamic acid).
Reproduced by permission of Elsevier Science S.A. from J. Photochem. Photobiol. B: Bioi
1992,12,125-140

dark takes place so slowly that it cannot be observed under normal experimental
conditions. The photochromic cycles are completely reversible and can be
repeated at will, without any apparent fatigue.
As a consequence of the different electronic situations, the two isomers have
markedly different absorption, and the photo-isomerization is accompanied by
strong variations in the spectra (Figure 14.2). In particular, the trans-to-cis
isomerization is revealed by a strong decrease of the intense band at « 350 nm
associated with a n-n* transition and a contemporaneous increase of the band at
450 nm associated with the n-n* transition of the azo-chromophore.

14.2.1.2 Light-induced Conformational Changes

Poly(L-glutamate)s having azobenzene units in the side chains, in organic sol-
vents such as trimethyl phosphate (TMP), show the typical CD curve of the
a-helix structure, with two minima at 208 and 222 nm. Above 250 nm, the
dark-adapted samples exhibit also a couplet of bands centered at 350 nm,
corresponding to the n-n* transition of the azo chromophore in trans configur-
ation. The trans-to-cis photoisomerization completely cancels the side chain CD
bands in the region of 35Onm, but does not modify at all the CD spectra in the
peptide region. This indicates that, in these solvents, light causes the isomeriz-
ation of the azo side chains, but the isomerization does not induce any variation
of the polypeptide main chain.
Figure 143 CD spectra of poly(L-glutamic acid) bearing 36 mol% azobenzene units,
before ( ) and after ( ) irradiation, in aqueous solution at various pH values: A,
pH 4.8; B, pH 6.5; C,pH 8.0

The secondary structure in water depends on the molar content of azobenzene

units and also on the degree of ionization of the unmodified COOH side chains.
Below pH 5, a sample of poly(Glu) bearing 35 mol% of azobenzene units assumes
a /^-structure. Irradiation does not induce any variation of the polypeptide
conformation. At pH values above 7, the polypeptide adopts a random coil
conformation which is again not affected by the photoisomerization of the azo
side chains. However, at pH values of 5-7, irradiation produces a remarkable
decrease of the ordered structure (Figure 14.3). In this range of pH the trans-to-cis
isomerization produces a higher degree of ionization of the unmodified COOH
side chains, thus amplifying the first light effect and causing unfolding of the
polypeptide.
Cationic surfactants are known to affect the conformation of poly(L-glutamic
acid). This suggested to us that it might be possible to combine the isomerization
of the photochromic side chains with the surfactant effect to obtain an amplifica-
tion of the photoresponse. The expectation was realized by irradiating azo-
modified poly(L-glutamic acid) in the presence of dodecylammonium chloride
(DAC) at the critical micelle concentration (c.m.c) [18]. Figure 14.4 shows the CD
spectra of a 20% azo-modified poly(Glu) both in the absence and in the presence
of DAC. In the absence of detergent at pH 7, the polymer is completely in random
coil conformation and not affected at all by irradiation. In the presence of
detergent at the c.m.c, irradiation at 35Onm (trans-to-cis isomerization) induces
Figure 14.4 CD spectra of poly(L-glutamic acid) bearing 20 mol% azobenzene units, at
pH 7.6, before ( ) and after ( ) irradiation: A, in the absence of dodecylammonium
chloride (DAC); B, in the presence of DAC, below the c.m.c; C, in the presence of DAC, at
the c.m.c. Reproduced by permission of Elsevier Science S.A. from J. Photochem. Photo-
bioi B: BioL, 1992,12,125-140

an evident coil-to-helix transition. The variation is completely reversible when

the sample is dark-adapted or irradiated at 450 nm (cis-to-trans isomerization).
Thus, in the presence of DAC micelles, the polypeptide conformation can be
photomodulated by exposure alternately to light or dark, or by irradiating at two
different appropriate wavelengths.
The key factor responsible for the photoinduced variations of conformation is
the affinity of the azo-polymer for the micelles. Such an affinity, in fact, is likely to
be different when the azo side chains are in trans or in cis configuration. When
azo-units are in the planar, apolar, trans form, they dissolve within the hydropho-
bic core of the micelles, forcing the polypeptide chains to assume a coil conforma-
tion. Isomerization of the azo units to the skewed, polar, cis form inhibits
hydrophobic interactions and causes the azo-units to leave the micelles, thus
allowing the polypeptide chains to adopt the a-helix structure (which is favored in
the absence of micelles). In other words, the primary photochemical event is the
trans ^ cis isomerization of the azobenzene units, but the driving force of the
photoresponse should be the different location of the macromolecules relative to
the micelles.
Dark-adapted (all trans azo groups) poly(L-lysine) bearing 43 mol% of azoben-
zene groups, in a medium of hexafluoroisopropanol/water/sodium dodecyl
sulfate, shows a CD spectrum which can be attributed to the presence of a /?-form.
Irradiation at 340 nm causes the disruption of the /!-structure and promotes the
formation of an a-helix (helix content % 50%), as revealed by the appearance of
the typical CD pattern. Upon irradiation at 450 nm, the spectrum reverses again
to the original one. The photoinduced /J;=± helix conformational change is
completely reversible and the two conformations can be obtained by irradiating
alternately at the two different wavelengths.
This photoinduced fi^± helix change can readily be explained on the basis of
the different geometry and hydrophobicity of the trans and cis azobenzene units.
The /J-form is stabilized by hydrophobic interactions among the side chains and
is favored when the azobenzene units have the planar geometry and the high
hydrophobicity of the trans configuration. The interactions are inhibited when
the azo units are isomerized to the skewed cis configuration, and thus the
/^-structure is destabilized and destroyed. The polypeptide chains then adopt the
a-helix form in the helix-supporting solvent hexafluoroisopropanol.
The photochromic behaviour of azobenzene-containing poly(L-lysine) has also
been reported in the monolayer state [19]. When the polypeptide monolayer is
kept at constant area, alternate irradiation with visible and ultraviolet light
produces reversible changes (% 25%) of the surface pressure of the monolayer.

14.2.13 Photostimulated 'Aggregation-Dissaggregation' Effects

CD data provided evidence that azo-modified poly(Glu) containing azobenzene
units can undergo reversible aggregation-disaggregation processes upon expo-
sure to light or dark conditions [20]. Samples stored in the dark or irradiated at
450 nm (azo groups in the trans configuration) show variations of their CD
spectra on aging in a TMP/water solution (Figure 14.5). The time dependence is
characterized by the gradual appearance of an intense side chain CD couplet
together with a progressive distortion of the a-helix pattern, typical of the effects
produced by aggregates of polypeptide chains [21,22].
Irradiation at 361 nm (tran to cis isomerization) produces the full restoration of
the initial CD spectra, indicating dissociation of the aggregates. The spectra
revert again to the distorted ones on irradiating at 450 nm or by dark adaptation,
thus confirming the reversibility of the light-induced effect.
Investigation of azo-modified poly(Glu) containing 85 mol% azobenzene units
in the side chains has provided confirmation of the occurrence of aggregation-
disaggregation processes induced by light, together with the possibility of
photoregulating polymer solubility [23]. This polypeptide, when stored in the
dark, assumes the a-helix structure in hexafluoroisopropanol (HFP). Addition of
a small amount of water (15 vol%) to the HFP solution causes the formation of
aggregates, followed by precipitation of the polymer as a yellow material. The
precipitation is total and quantitative, as can be seen by recording the absorption
spectrum of the filtered colorless liquid.
Complete dissolution of the polymer was obtained by irradiation of the
suspension for a few seconds at 350 nm; irradiation at 450 nm or dark adaptation
of the solution caused the polymer to precipitate. In a HFP/water = 85/15
solvent mixture, therefore, the 'precipitation-dissolution' cycles can be reversibly
Figure 14.5 Poly(L-glutamic acid) bearing 20mol% azobenzene units. CD spectra in
trimethyl phosphate/water = 50/50, recorded at various aging times: (1) freshly prepared
solution; (2) aged 1 day; (3) aged 2 days; (4) aged 3 days. ( ) Dark-adapted samples;
( ) irradiated at 360 nm, at any aging time. Reprinted with permission from [23].
Copyright 1989 American Chemical Society

repeated by irradiation and dark adaptation, or by irradiating at two different

wavelengths.
The dependence of the polymer solubility on the cis/trans composition of the
azobenzene side chains was investigated by performing irradiation experiments
at various wavelengths of the incident light. The considerable amount of photo-
dissolved polymer allowed its determination by evaporating the solutions ob-
tained upon irradiation and weighing the dry residue. The solubility of the
polymer, as a function of the cis/trans ratio of azobenzene side chains, is described
by a sharp sigmoidal curve. The polymer is fully insoluble when more than 60%
of the azo groups is in a trans configuration. By contrast, the maximum amount of
photosolubilization is achieved when 60% of azo groups are in the cis configur-
ation; the solubility then remains unaffected at higher values of cis content [23].
The described photoresponse effects can be well interpreted on the basis of
association among macromolecules through hydrophobic interactions and
stacking of azobenzene side chains. The planar, apolar, trans configuration gives
aggregation and precipitation; when the azo moieties are photoisomerized to the
skewed, polar, cis configuration, interactions and stacking between azo-groups
are inhibited, so that disaggregation of the macromolecules takes place and
polymer dissolution occurs.

14.2.2 PHOTOMODULATION OF POLYPEPTIDE

CONFORMATION BY SUNLIGHT

14.2.2.1 Spiropyran-containing Polypeptides

Azo-modified polypeptides could be considered as models for photoregulated
processes occurring in nature, but the generation of cis and trans photoisomers,
and hence photoregulation, requires artificial sources of ultraviolet light. Ideally,
one would like to have a model system responding to the presence or absence of
sunlight, such as polypeptides bearing spiropyran groups attached to poly(L-
glutamic acid) [24] or poly(L-lysine) [25] (Figure 14.6).
Spiropyran-modified poly(L-glutamate)s in hexafluoroisopropanol (HFP)
exhibit 'reverse photochromism', that is, a photochromic behavior which is

Spiro Spiro

Spiro group

Figure 14.6 Chemical structure of poly(L-glutamic acid) and poly(L-lysine) bearing

spirobenzopyran units in the side chains
 dark
light

Figure 14.7 Structure and reverse photochromic reactions in HFP of poly(L-glutamic

acid) containing spiropyran units in the side chains

opposite to that usually observed in most common organic solvents. Thus, HFP
solutions kept in the dark at room temperature show a yellow-orange color
which is completely bleached upon exposure to visible light and is reversibly
restored in the dark.
NMR data confirm that the photochromism in HFP involves the well-known
interconversion between the colorless closed spiro structure / and the colored
ring-opened merocyanine structure II (Figure 14.7). Accordingly, in the 13C
NMR spectra of the colorless solution the resonances of the geminal methyl
groups appear as two separate peakes, 27.0 and 21.0 ppm, as a consequence of the
presence of the chiral spiro carbon atom. In the colored solution, by contrast, the
two methyl group resonances merge to a single signal at 28.7 ppm, analogously to
that observed for the proton resonances. The spectra of the colored species kept
in the dark do not show nuclear resonances associated with the spiro form,
indicating that in HFP the spiropyran ;=± merocyanine equilibrium is fully shifted
to the right.
The very polar solvent HFP is probably responsible for the reverse photo-
chromism by stabilizing the charged merocyanine species II more than the apolar
spiropyran species I. A protonated open structure III might also be formed
between the zwitterionic species II and HFP, with the solvent functioning as an
acid, as will be described in the following section.
The dark-adapted sample shows a spectrum which displays two absorption
maxima, at 500 and 370 nm, of about the same intensity (Figure 14.8). Irradiation
with visible light (500-550 nm) or exposure to sunlight for a few seconds
completely dispels the absorption in the visible region and gives rise to the
spectrum of the colorless spiro form, characterized by absorption maxima at 355
and 272 nm. In the dark, the original spectrum is progressively restored.
In the colorless indolinospiropyran species, the two halves of the molecule
are topologically independent, so the absorption spectrum consists mainly of
 A

light
dark

Figure 14.8 Variation of the absorption spectra as a function of irradiation and dark-
adaptation time for poly(L-glutamic acid) bearing 85mol% spiropyran units in HFP
(c = 5.01 x 10"2 g/1; / = 1 cm); 1, dark-adapted solution; 2, irradiated solution

localized transitions belonging to a particular half of the molecule, rather than

delocalized transitions belonging to the molecule as a whole [26]. The electronic
transition at longer wavelength, which in HFP occurs at 355 nm, has been
assigned to the benzopyran, and the second transition, which in HFP is seen at
272 nm, has been assigned to the indoline portion of the molecule [26]. In the
colored species, the absorption band at 500 nm can be assigned to a n-n*
electronic transition of the extended and conjugated merocyanine chromophore,
and the 370 nm band can be attributed to a charge-transfer transition from the
oxygen atom of the benzopyran ring to the electron-accepting nitro substituent
[27,28].
 light

dark

Figure 14.9 Reverse photochromic reactions of spiropyran salts (see Fig. 14.7)

Considering the acidity of HFP, the band at longer wavelengths should be

assigned to the zwitterionic ring-opened form II (Figure 14.9), whereas the band
at shorter wavelengths might be assigned to the presence of the ring-opened
species III formed between the zwitterionic species and HFP, with this last acting
as an acid (pKa = 9.30) [29] (shown in Figure 14.9). In the polymers, protonation
of the open form by unmodified COOH side chains may also occur [27], even
though this effect cannot play a relevant role in the 85 mol% modified polymer
shown in the figure. The presence of a well-defined isosbestic point should
indicate only two interconverting species. However, the salt of the spiropyran
with trifluoroacetic acid exibits exactly the same isosbestic point (see the follow-
ing section), so that one cannot exclude the presence of both the zwitterionic and
the protonated merocyanine forms.

14.2.2.2 Photomodulation of Conformation

The CD spectra of the dark-adapted samples of poly(Gluj bearing 85 mol%
photochromic units are those of random coil polypeptides. CD bands of small
intensity are also present in the near UV-visible region, in correspondence with
the merocyanine electronic transitions. The solutions bleached after exposure to
visible light display the typical pattern of the a-helix, with the two minima at 222
and 208 nm, thus indicating that the isomerization of the side chains produces
spiralization of the main chain. The back reaction in the dark is accompanied by
a progressive decrease of the helix content and restoration of the original
disordered conformation (Figure 14.10).
The photoinduced helix content can be only approximately estimated on the
basis of the CD spectra. In fact, several polypeptides, all having a-helical
 light
dark

Figure 14.10 Effect of irradiation and dark adaptation on CD spectra of poly(L-glutamic

acid) bearing 85 mol% spiropyran units in HFP at 250C: 1, kept in the dark; 2, exposed to
sunlight; dotted lines are CD spectra recorded during decay in the dark over 8h. Below
250 nm, CD data are expressed in terms of molar ellipticity based on the mean residue
molecular weight; above 250 nm, the molar ellipticity is referred to one spiropyran-
glutamyl residue

conformation, were reported [30] to show significant variations of the maximum

ellipticities when CD spectra were measured in HFP. On the basis of the
literature values [30] of [ G ] 2 2 2 ( - 3 0 0 0 0 — 40000) for 100% a-helix in HFP,
the photoinduced helix content can be evaluated as 90-70%.
The photochromic reaction involves the reversible conversion of the zwit-
terionic merocyanine (sample kept in the dark) to the uncharged spiro form
(sample exposed to light); the isomerization is thus accompanied by large
variations of the electrostatic interactions among the side chains of the polypept-
ides. Intrachain interactions should produce loops in the macromolecules,
whereas intermolecular stacking should produce aggregation phenomena. As
a result, the macromolecules are forced to adopt a disordered structure. When the
sample is exposed to light and merocyanines are converted to the neutral spiro
form, electrostatic interactions are removed and the polypeptide can adopt the
a-helix structure.
When spiropyrans are treated with acids they are converted into 'spiropyran
salts', which exhibit photochromic behavior differing from that of the parent
spiropyran compounds. The gross mechanism proposed is illustrated in Fig-
ure 14.9. In the dark at room temperature, the compounds give colored solutions
due to the presence of the O-protonated merocyanine species III. The open form
is converted by irradiation with visible light to the iV-protonated spiro form IV.
As spiropyrans are fairly strong bases in the open form but very weak bases in the
closed spiro form, the charged species IV can lose a proton and the neutral species
I can be actually formed.
Comparison of Figure 14.9 with Figure 14.7 shows that different photo-
isomers are involved in acidic or non-acidic solution. Therefore we may expect
spiropyran-containing polypeptides to be affected by light in a different way
depending on whether they are irradiated in the absence or in the presence of acid.
Poly(spiropyran-L-glutamate) in HFP solution in the presence of TFA does not
give light-induced conformational changes. Actually, the solutions show the
typical CD spectra of random coil polypeptides both when they are kept in the
dark and when they are exposed to light.
The addition of methanol as a cosolvent induces the coil -> helix conforma-
tional transition, as for other polypeptides having salified side chains [31]. The
most remarkable aspect of this system is that two distinct curves are observed for
the dark-adapted sample and for the irradiated one (Figure 14.11). In particular,
for the polymer containing 85mol% photochromic units, the concentration of
methanol needed to induce the conformational transition is « 10-40% for the
sample kept in the dark and « 5-10% for the sample exposed to light. Therefore,
at any solvent composition in the range between the two curves, exposure to light
produces reversible variations of the helix content (Figure 14.12).
The photochromic reactions schematized in Figure 14.9 and the above dis-
cussed absorption spectra allow us to explain the conformational behavior. In
HFP, in the presence of TFA, the photochromic side chains are protonated by the
acid either when the sample is kept in the dark (photochromic units present as
open species III) or when the sample is exposed to light (photochromic units
present as closed species IV). In both cases the polypeptide is essentially a polyca-
tion, so the repulsive forces among the side chains make the macromolecules
adopt an extended coil conformation, and no photoresponse is observed.
In the presence of methanol (> 10%) the equilibrium between the two colorless
species IV and I (Figure 14.9) is shifted toward the neutral spiro structure I. In
these conditions the 'folding-unfolding' of the macromolecules is photocontrol-
led by the isomerization of the photochromic units. In the dark, they are present
as charged species, so the macromolecules adopt a disordered conformation.
 MeOH,%

Figure 14.11 Variation of ellipticity at 222 nm as a function of methanol concentration

(v/v) for poly(Glu) bearing 85mol% spiropyran units in HFP/MeOH/TFA solvent
system, at 250C: ( ) dark-adapted sample; ( ), irradiated sample

light
dark

Figure 14.12 Effect of irradiation on CD spectra for poly(Glu) containing 85 mol%

spiropyran units at 250C in various HFP/MeOH/TFA solvent mixtures (c = 2.59 x
10"2 g/1; TFA = 1 x 10"3 ml in 2 ml of mixed solvent); MeOH % (v/v): (a) 0-5%; (b) 10%;
(c) 20%; (d) 40%. ( ), dark-adapted; ( ) irradiated samples
 a-helix v a r i a t i o n , %

merocyanine form , %
Figure 14.13 PoIy(GIu) bearing 85 mol% spiropyran units. a-Helix relative variation as
a function of spiropyran/merocyanine isomeric composition of the side chains, in pure
HFP ( ) and HFP/MeOH/TFA = 90/10/5 x 10 " 2 ( ). The a-helix variation in
% was estimated as {[@]V[©]°} x 100, where [0]° and [ 0 ] ' are the ellipticity values
measured at 222 nm at the beginning and at the time t during decay, at 250C

Exposure to light and the consequent photoconversion of the side chains to the
apolar spiro form make the macromolecules adopt the a-helix conformation.
In order to investigate the dependence of the secondary structure on the
isomeric composition of the photochromic side chains, the rate of the helix-to-
coil conversion and the rate of appearance of absorbance at the longer
wavelength in the dark were measured simultaneously. The helix content was
then plotted as a function of the photochromic units present in the merocyanine
form (Figure 14.13). In pure H F P (Figure 14.13, dotted line), the helical structure
starts to break up rapidly as soon as the merocyanine species begin to be formed,
and the helix -* coil conformational change takes place almost entirely following
conversion of % 30% of spiropyran to merocyanine groups. A rather different
behavior is observed in HFP/MeOH/TFA (Figure 14.13, full line): the helix
content decreases slowly with increasing merocyanine percentage, but the helical
structure does not collapse until « 50% of the spiro groups are isomerized to the
merocyanine form.
The different dependence of the helix structure on the percentage of photo-
chromic groups present in the merocyanine form is a confirmation that denatura-
tion of the macromolecules in the dark occurs through different mechanisms in
non-acidic and acidic media. In the former case denaturation should be caused by
stacking and aggregation between zwitterionic merocyanine species II. In the
latter case, denaturation should be caused by repulsive forces among the cationic
side chains III. In both cases, exposure to light removes the electrostatic
interactions between side chains, allowing the formation by the polypeptide
chains.
Also poly(Lys)-containing spirobenzopyran side chains, as well as low molecu-
lar weight model compounds, exhibit intense 'negative' photochromism in HFP
[25]. In the dark the solutions are orange, with two absorption maxima at
«470nm (£mol = 31700) and 370 nm (emol = 32 000). Exposure to sunlight is
accompanied by prompt bleaching, with a shift of the absorption maxima to
353 nm (emol = 11200) and 270-272 nm (emol = 16 200). The original spectrum is
reversibly restored when the illumination is stopped. Decay in the dark at 25 0 C
follows first order kinetics for the model compounds, with a rate constant of
5.7 x 10" 3 min" 1 and a half-life of «122min. For the polymer, the kinetics
deviate slightly from monoexponential and biexponential decay; the time necess-
ary to restore half of the original absorbance is « 80 min.
The analogy of these reversible processes with those observed in spiropyran-
modified poly(Glu) suggests the occurrence of similar photochromic reactions.
Accordingly, HFP stabilizes the colored ionic merocyanine structure, while
irradiation gives the colorless spiro structure.
In pure HFP, the CD spectra are consistent with those of random coil
polypeptide chains, and the photoisomerization reaction does not affect the
polymer conformation at all. Addition of triethylamine to the HFP solution
induces the coil-•helix transition, but the amount of base necessary to induce
the transition is different for the dark-adapted sample (15% v/v) than for
the irradiated one (30% v/v). Thus, at any composition in the range 3-15%
v/v of NEt 3 , exposure to sunlight produces reversible variations of the helix
content. Combination of the effects due to the photochromic behavior with
appropriate amounts of NEt 3 allows modulation of the extent of the photo-
response.
It appears that in pure HFP the conformation for poly(Lys)-containing
spiropyran is determined by the unmodified Lys side chains protonated by the
acid solvent; as a consequence, the polypeptide assumes a coil conformation
which is not affected by the isomerization of the photochromic groups. Addition
of a moderate amount (3-15%) OfNEt3 removes protons from Lys side chains,
whose basicity depends on isomeric composition of the photochromic moieties.
In the range between the transition curves of the dark-adapted and the irradiated
sample, the chain folding ;=± unfolding is then controlled by the isomerization of
the photochromic side chains: when these are in the charged merocyanine form,
the polypeptide chains are in the random coil arrangement, but photoconversion
to the apolar spiropyran form causes the macromolecules to assume a helical
conformation. At NEt 3 contents above 15%, the high concentration of
a NEt 3 • HFP saline complex can probably exert a shielding effect on the charged
side chains, allowing the polypeptide to stay in the helical conformation at any
photoisomeric composition.
The system described provides a well defined example of the combined action
of light and environment on the secondary structure of polypeptides. It can thus
be considered as a macromolecular model resembling the behavior of naturally
occurring photoreceptors [32].

14.2.2.3 Photoinduced Variations of Viscosity

The colored solutions of poly(L-glutamic acid) and poly(L-lysine) containing
spiropyran, when kept in the dark, are characterized by very high values of
viscosity, typical of those displayed by polyelectrolytes. The viscosity decreases
dramatically upon exposure to sunlight and returns to the original value along
with the reappearance of the absorption in the visible region.
In order to correlate viscosity changes with conformational changes, samples
of photochromic polypeptides were exposed to light, then the viscosity and the
CD spectra were measured over time in the dark. Viscosity progressively
increases with the gradual decrease of the helix content for both spiropyran-
containing poly(L-glutamic acid) (Figure 14.14) and poly(L-lysine) (Figure 14.15).
The high viscosity of the solutions in the dark is essentially due to the side chains,
which are charged when macromolecules are in disordered conformation. In
these conditions the polypeptides are able to coordinate many solvent molecules
to give highly solvated an extended macromolecules with a large hydrodynamic
a-helix v a r i a t i o n , %

t i m e , mi n
Figure 14.14 PoIy(GIu) bearing 85mol% spiropyran units. a-Helix content ( ) and
viscosity ( ) variation during decay in the dark at 25 0C. HFP solutions were irradiated,
then dark adapted and monitored over time. The percentage of a-helix variation is
estimated as indicated in Figure 14.13
 a-helix variation,%

t i m e , min
Figure 14.15 Helix content ( ) and viscosity ( ) variation during decay in the dark
for poly(Lys) bearing 46 mol% spiropyran units, in HFP/NEt3 = 94/6

volume, thus exhibiting high values of viscosity. Aggregation phenomena,

through interactions between merocyanine side chains, can also contribute to
viscosity increases. From the figures it appears that viscosity keeps on increasing
even when the a-helix is completely destroyed. In fact, the helix is fully destroyed
by conversions of the spiro to the merocyanine form of « 60%, (Figure 14.13), but
the macromolecules go on expanding until conversion to the merocyanine form
reaches 100%.

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