Development of Bacteriorhodopsin Analogues and Studies of Charge Separated Excited States in the Photoprocesses of Linear Polyenes


  • This invited paper is part of the Symposium-in-Print: Photobiology in Asia.

*email: (Anil K. Singh)


Development of bacteriorhodopsin (bR) analogues employing chromophore substitution technique for the purpose of characterizing the binding site of bR and generating bR analogues with novel opto-electronic properties for applications as photoactive element in nanotechnical devices are described. Additionally, the photophysical and photochemical properties of variously substituted diarylpolyenes as models of photobiologically relevant linear polyenes are discussed. The role of charge separated dipolar excited states in the photoprocesses of linear polyenes is highlighted.


Nature has created several molecular machines that are powered by photons. These machines work with remarkable specificity and efficiency and are responsible for biological sensory and energy transductions leading to a variety of physiological processes like vision, photosynthesis, photomorphism, phototropism, phototaxis, etc. The photoactive element of these molecular machines is generally a biopigment consisting of a protein bound to a low molecular weight organic chromophore. This biopigment assembly is involved in harvesting and processing the photon energy to power respective biological phenomenon. In many such biological systems, linear polyenes like vitamin A aldehyde (Fig. 1), commonly known as retinal are involved as the photoactive element. Such linear polyene-based photoreceptors are widespread in nature. Retinal-bound photoreceptors like visual pigment rhodopsin (Rh), halobacterial photoreceptors like bacteriorhodopsin (bR), halorhodopsin, sensory rhodopsins I and II, chlamydomonas retinochrome, etc. are some of the well known photoreceptors (1,2). Retinal-bound photoactive proteins are also known to induce phototaxis in fungi (3). More recently, a new light harvesting pigment called xanthorhodopsin from Salinibacter rubber has been discovered (4). Uniquely, xanthorhodopsin binds to two unrelated chromophores: retinal and a carotenoid salinixanthin. A unique protein called photoactive yellow protein (PYP) based on thiocinnamic acid has also been discovered (5). Table 1 lists a few photoresponsive proteins based on retinal and related polyenes. In general, the retinal-bound photoreceptor proteins consist of seven trans-membrane helices connected by interhelical loops and the retinal chromophore is embedded inside the helices, which acts as a light absorption antenna for the protein. Upon absorption of light, the structure of retinal and its surrounding protein gets changed, which ultimately leads to various biological responses. One of the structural changes that is well known to occur in these proteins is the stereo- and regio-specific photo-isomerization of the polyene chromophore.

Figure 1.

 Structure of vitamin A aldehyde, commonally known as retinal. A protein is attached via the aldehyde group of retinal to create a photobiological receptor.

Table 1.   Some biological photoreceptors that contain linear polyenes.
Sl. No.PhotoreceptorChromophoreFunction
2Bacteriorhodopsinall-trans-RetinalLight-driven proton pump
3Halorhodopsinall-trans-RetinalChloride ion pump
4Sensory rhodopsin-Iall-trans-RetinalPhototaxis
5Sensory rhodopsin-IIall-trans-RetinalPhototaxis
7Fungal rhodopsinall-trans-RetinalPhototaxis
8XanthorhodopsinRetinal-carotenoid complexLight-driven proton pump
9Photoactive yellow protein4-Hydroxy-cinnamic acidNegative phototactic response

In recent years, much attention has been focused on the structure and mechanism of functions of Rh and bR (6). While the structure and mechanism of at least one of the proteins viz. bR are known in great detail, the exact nature of the excited state of the chromophore is yet to be completely understood. The photochromic properties of these proteins have also been noted with great interest. Further, a clear knowledge of the structure and mechanism of function of bR together with its easy availability has raised much hope for its application as photoactive element in molecular electronic devices (7). Consequently, recent years have witnessed increased activities towards development of newer analogues of bR having novel opto-electronic properties. Additionally, extensive photophysical and photochemical studies of retinylidene and related chromophores are being undertaken with the aim of developing a clear understanding of the structure and dynamics of the excited states of photobiologically relevant linear polyenes.

We have performed bioorganic studies with a view to prepare new bR analogues by chromophore substitution method for the purpose of further characterizing the bR binding site, and also at the same time developed new bR analogues with novel opto-electronic properties. We have also undertaken extensive photophysical and photochemical studies of model donor-acceptor diarylpolyenes with an objective of examining possible involvement of charge-transfer excited states in the photoprocesses of linear polyenes. In this article, we present some of our efforts in these directions.

Studies of stereo-electronic features of br binding site and development of br analogues

The bR found in the purple membrane (PM) of extreme halophile Halobacterium salinarium is the most extensively studied retinal-containing photoreceptor protein (8). It functions as a light-driven proton pump and facilitates adenosine triphosphate (ATP) synthesis in the bacterium. The three-dimensional structure of bR at atomic level (1.55 Å) and molecular mechanism of functions are known to a great extent (9). It consists of a single polypeptide made up of seven α-helices consisting of 248 amino acids and a chromophore all-trans-retinal is covalently attached to the ε-amino group of Lys-216 via a protonated Schiff base linkage. Illumination of bR with visible light triggers all-trans to 13-cis photo-isomerization of the retinylidene Schiff base chromophore (Fig. 2). In this process, the photoexcited protein relaxes to the initial light-adapted bR (bRLA) state via several intermediates referred as J, K, L, M, N and O. The configurational change in the chromophore induces the conformational changes in bacterioopsin (bOP), the apoprotein of bR. This results in a shifting of proton from the intracellular to extracellular region of the bacterial cell membrane. A membrane ATPase is driven by proton influx from the resulting proton gradient whereby adenosine diphosphate (ADP) is converted to ATP, which is used by the halobacteria for energizing their cellular molecular processes.

Figure 2.

 The phototriggering process in bR involving trans-cis photo-isomerization of retinylidine Schiff base chromophore.

The structural model of bR helps in identifying the geometry of retinal, its binding sites and the proton pathway (9). The state of protonation of the Schiff base linkage has, however, been a subject matter of much discussion and it has been argued that full protonation of Schiff base nitrogen need not be involved to explain the structural and functional characteristics of native protein. These discussions also led to the early suggestions that water molecules play a crucial role in protonation and stabilization of the Schiff base chromophore in retinal-proteins (10,11). Later on many spectroscopic and other studies were undertaken and the role of water molecules in the structure and functions of retinal-bound photoactive proteins was discussed (12–18). Model studies have been very useful in developing a chemical understanding of the role of water molecules in protonation/deprotonation and control of the microenvironment (particularly the pKa) of the retinal Schiff base chromophore at the reaction center of retinal-bound photoactive proteins. The primary proton sources for the protonation of the Schiff base chromophore present in the receptor proteins are rather weak acids (e.g. Asp, Glu). To explain full protonation/deprotonation of the Schiff base nitrogen by rather weak acids in a rather largely hydrophobic microenvironment of the retinal photoreceptors, the role of water molecules was envisaged and mechanisms suggested (19–21). Subsequent model studies of retinal Schiffs bases in reverse micelles and other matrices also demonstrated the involvement of water molecules in the stability and protonation of retinal Schiff base chromophore (22–34). In an interesting model study, the role of formation of a definite angle between the protonated Schiff base linkage and a carboxylate group, which allows for effective interaction with one or more water molecule bridging the two groups, has been discussed (35).

In late 1970s and early 1980s when the structure of bR was not available, valuable information about bR binding site and its functional mechanisms were obtained by bioorganic model studies. Particularly, the characteristic absorption and photochemical properties of bR could be explained in sufficient detail by these studies. One of the methods that proved to be of great value in deriving information about the binding site interactions in bR involved preparation of bR analogues by chromophore analogue substitution method (36,37). In this method, bR was photobleached to remove the natural chromophore (all-trans-retinal) and the resulting apoprotein, the bOP, was incubated with the synthetic analogue of retinal to generate bR analogues. The synthetic retinal analogue was tailored such that when it is bound in retinal binding site in bR, it gives specific information about the chromophore–protein interaction. This technique was successfully used by Nakanishi and his coworkers to provide chemical explanation for the color of bR/Rh, and also to find out the role of polyene chain in the photoreactions of these proteins (36,37). Subsequently, many bR analogues were synthesized and almost the entire reaction center of bR could be mapped in terms of the nature of chromophore–protein interactions.

Thus, over the years, much effort has been directed towards understanding the unique spectral and photochemical properties of bR. A large number of synthetic chromophore analogues have been incorporated into the binding site of bR and the observations of the resulting optical and photochemical properties have been analyzed in terms of chromophore–protein electrostatic and conformationally restrictive interactions. It has been found that the binding site of bR is quite unrestrictive as retinal analogues with substantially bulky groups in the ring portion of retinal can be accommodated in the bR binding site. Several synthetic retinal analogues with modified aliphatic side chain or/and the trimethyl cyclohexenyl ring have been prepared, and employed to study the stereo-electronic features of bR binding site.

Various other models have been proposed in order to explain the chromophore–protein interaction and the wavelength regulation in retinal-binding photoreceptors. These include ring/chain coplanarization (38,39), interaction between the Schiff base proton and its counter ion (40,41), electronic polarization of aromatic residues (42), and hydrogen-bond interaction between the Schiff base and the surrounding negatively charged carboxylic residues (32,33).

It is also believed that the protein matrix behaves like a polarizable medium with a high refractive index (42). Thus, it plays an important role in wavelength regulation in retinal-bound photoreceptors. It has also been suggested that the dipolar nature of the retinylidene chromophore plays a crucial role in such interactions and wavelength regulation of retinal-bound photoreceptors (43). More recently, it has been shown that very large dipolar changes occur in the vertically excited state of retinylidene chromophore formed after light absorption by bR (44). The origin of such large dipole moment changes lies in the protein domain in the vicinity of retinylidene’s β-ionyl ring. It is believed that protein’s tryptophan residues enhance such light-induced dipole in bR. Other groups have also discussed the role of polarizable groups and secondary interactions in the wavelength regulation in retinal-bound photoactive proteins (45–50). Recently, Ebrey and his co-workers (51) have found the wavelength regulation in wild type pigment by site-directed mutation of various amino acid residues that were present near the chromophore. These studies also indicate the role of dipolar interactions in the wavelength regulation of retinal-bound photoreceptors.

The chromophore analogue substitution method is not only a method for characterizing the chromophore–protein interaction, but also a unique way of generating new bR analogues having desirable absorption or photochemical properties. The unique electro-optical properties of bR render it as a novel photoswitchable material that can find applications as photoactive element in molecular electronic devices. The most interesting application of bR lies in the area of optical information storage and processing technology, and throughout the last decade researchers have shown considerable interest in this area. This has led to more research towards producing bR-based proteins of enhanced performance for opto-electronic applications (7,52–54). It is currently believed that several bR-based molecular electronic technologies will emerge in the near future. Consequently, search for newer bR analogues is still on.

Typical procedure for bR analogue preparation by the chromophore substitution method

Typical procedure for bR analogue preparation employing chromophore substitution method is outlined in Fig. 3. Details of the methods are described in several publications, also including Ref. (55) and (56). Allowing all-trans-retinal with freshly prepared bOP regenerates the PM. For this, to 1 mL suspension of bOP (1.0 × 10−5 M in HEPES buffer), an ethanolic solution of all-trans-retinal (2.0 × 10−5 M) is added at ambient temperature. The final volume of ethanol concentration in the solution is kept below 5% (v/v). Regeneration of retinal bound protein is monitored by UV–vis absorbance at 560 nm. Appearance of absorption maximum at 560 nm indicates that apoprotein, bOP is active after photobleaching process and ready for binding to the synthetic retinal analogue. The bR analogue is prepared by allowing freshly prepared bOP to interact with synthetic retinal analogue. For this, ethanolic solution of the retinal analogue is incubated with bOP and the formation of new bR pigments is monitored over a period of time by UV–vis spectroscopy. The bR pigments formed are washed free of excess chromophore by hexane washings.

Figure 3.

 Isolation of bR/bOP and regeneration of bR and its analogues.

Retinal analogue synthesis

Retinal analogues are synthesized, HPLC purified and fully characterized for their structure and stereochemistry by physicochemical methods. Several methods for retinal analogue synthesis have been worked out. While individual method for synthesis of a particular retinal analogue can be found in the respective literature reference, synthesis of retinoids has been excellently documented in many publications like Ref. (57) and (58). As an example, synthesis (59) of a retinal analogue namely 3-methyl-5-{4-[(E)-2-phenylvinyl]phenyl}penta-2E,4E-dienal is illustrated in Fig. 4.

Figure 4.

 Reagents and conditions: (a) NaH/THF/0°C, (b) LAH/THF/r.t., (c) BaMnO4/CH2Cl2/r.t., (d) DIBAL-H/THF/0°C.

bR Analogues

Spectral and photochemical studies of bR analogues have greatly helped in building a chemical understanding of the nature of interaction between the retinylidene chromophore and its receptor protein, and for this numerous bR analogues have been synthesized and reported. However, it has not been possible to cover all of them in this article. Only a few bR analogues are mentioned here with some emphasis on author’s own work.

Some bR-analogues prepared by different researchers are given in Table 2. We have prepared bR analogues based on iodophenyl (60), anthryl (61), azobenzene (62), diphenylpolyene (59) and indolyl (63) retinal analogues. These are characterized for their absorption behavior, opsin shift, competitive binding, fluorescence emission, light-induced pH activity and chromophore photochemistry. The anthryl bR analogues (bRa-16) show fluorescence in the Vis region. The absorption of azobenzene-based bR analogues (bRa-23–25) has been found to be sensitive to pH, which makes them good pH sensitive probes. Diphenylpolyene retinal analogues (bRa-26, bRa-27) bearing extended conjugated chain bind to bOP and form pigments, whereas 1,2-diphenylethene aldehydes and 1,4-diphenylbutadiene aldehydes lacking conjugated side chain do not form pigments with bOP. This shows that the side chain of polyene system is important in binding of chromophores to the apoprotein to give bR analogues. The bR analogues, bRa-26 and bRa-27 showed smaller opsin shift of 1025 and 656 cm−1, respectively, when compared with a rather larger opsin shift (4870 cm−1) of native bR. This indicates towards drastically modified nature of interactions between the chromophore with the protein residues in comparison with native bR. It has been shown that these chromophores occupy the same binding site as that of the retinal as these chromophores could not be displaced easily by all-trans-retrinal. Very recently, we have prepared bR analogue based on indolic chromophore namely 3-chloro-5-(1H-indol-3-yl)-hexa-2,4-dienal. However, 5-(1-benzenesulfonyl-1H-indol-3-yl)-3-chloro-hexa-2,4-dienal, bearing bulkier substituent, benzene sulfonyl residue does not bind bOP. The new bR analogue (bRa-38) does not exhibit appreciable fluorescence emissions. However, it shows light-induced pH changes (63).

Table 2.   Some retinal analogues and the corresponding bR pigments. Thumbnail image of Thumbnail image of

Nakanishi et al. (37,64) prepared bR analogues based on dihydroretinals (e.g.bRa-1) and using their absorption properties they proposed the “External Point Charge”(EPC) model to explain the color of bR. Light-induced proton pump activity studies of these pigment analogues showed that the presence of C5 = C6 double bond and the five methyl groups of retinal is not responsible for the proton pump activity of bR. Similarly, Iwasa and his co-workers (65) synthesized various artificial pigments of bR with retinal analogues having fixed 6-s-cis conformation (e.g.bRa-3, bRa-4). It was found that all the analogues not only formed the bR-analogue pigments but also showed proton pump activity. This indicated that 6-s-trans structure of retinal is not necessary for the proton pump. Liu (66) and Crouch (67) and their co-workers studied the effect of retinal polyene side-chain length on the formation and function of bR analogues. Compounds with six or less carbon atoms in the polyene chain do not form pigment or form pigment very poorly with bOP. Compounds containing at least seven carbons in the chain are found to form reasonably stable pigment with bOP (e.g.bRa-5–8). Thus, the length of the polyene chain plays an important role for binding to bR. Further, the shorter chain length containing analogues do not show photocycle and the proton pumping activity. It has been shown that at least nine carbons are required in a retinal polyene chain for the formation of a stable pigment that shows photocycle and efficient light activated proton pump. Analogue without cyclohexenyl ring (e.g.bRa-6) also forms bR pigment and shows photocycle and efficient proton pumping, which indicates that the cyclohexenyl ring has no role in the physiological function of bR.

Further, Nakanishi and his co-workers (68) studied the proton pumping activity of retinal analogue bearing a blocked 13-ene double bond (e.g.bRa-9). They demonstrated that analogues with blocked 13-ene double bond form bR pigment, but fail to show bR like proton pump activity. Thus, it was concluded that 13-ene double bond plays an important role for the isomerization and the proton pump activity of bR. Recently, Sheves and his co-workers (69) reported similar results, after doing the EPR study of the pigment containing chromophore with non-isomerizable C13 = C14 double bond.

In 1985, Sheves and his co-workers (70) studied the pulsed laser photolysis of artificial bR pigments, which contained retinal analogues with selectively blocked single and double bonds (e.g.bRa-10–12), and suggested that only C13 = C14 double bond undergoes isomerization in the primary photoprocess. Later on, several conformationally rigid retinal analogues such as aryl (67,71) (e.g.bRa-13, bRa-14), napthyl (72) (e.g.bRa-15), anthryl (61) (e.g.bRa-16), etc. were incorporated into bR binding site and based on the observed OS (Table 2), it was concluded that twisted geometry around C-6, C-7 bond has no significant role for binding to bOP. Crouch and her co-workers studied the effect of polyene chain on naphthyl retinals (e.g.bRa-15) and showed that all the analogues form pigment, but only those pigments containing polyene side chain identical to that of retinal show proton pumping activity efficiently.

In 1990, our group (61), and in 1999, Crouch and her co-workers (73) synthesized anthracene (e.g.bRa-16) and pyrylretinal (e.g.bRa-17) analogues and studied their interaction with bOP. Both the analogues formed pigments and showed proton pump activity. It has been concluded that binding site of bR remains unrestricted to the bulky groups present at the ring portion of the retinal.

It has been further observed that a variety of polycyclic aromatic retinal analogues (e.g.bRa-18–22) form pigments with bOP (74). These bR analogues exhibit blue-shifted absorption maximum and a low OS when compared with native bR. This led to the suggestion that steric and electronic effects play a crucial role for binding to bOP.

In 2001, Sheves (75) studied the interaction between bOP and the chromophores which have N,N-dimethylaminoaryl terminal group (e.g.bRa-28–31). They reported that all the analogues bearing single and two double bonds formed bR pigments with a low OS as compared to the native bR. In an interesting work, Liu and his co-workers (76–79) have studied the interaction between a variety of azulene-based chromophores (bRa-32–37) with bOP. They found that analogues that have one bond shortened or one bond lengthened with respect to retinal showed comparable OS. However, analogues which have two bonds shortened exhibited a much smaller OS. Additionally, azulene-based analogues formed highly red-shifted bR pigments. The dramatic red shift was explained in terms of the delocalization involving carbocations.

The unique electro-optical properties of bR render it as a novel photoswitchable material that can find applications as photoactive element in molecular electronic devices. The most interesting application of bR lies in the area of optical information storage and processing technology and throughout the last decade researchers showed considerable interest in this area. This has led to more research towards producing bR-based proteins of enhanced performance for opto-electronic applications. It is currently believed that several bR-based molecular electronic technologies will emerge in the near future. Consequently, search for newer bR analogues is still on. Thus, the chromophore analogue substitution method is not only a method for characterizing the binding site of bR in terms of chromophore–protein interaction, but also a unique way of generating new bR analogues having desirable absorption or photochemical properties.

Involvement of charge-transfer excited states in the photoprocesses of photobiologically relevant linear polyenes

The excited state properties of linear polyenes of retinoids series have received a great deal of attention because of their critical role in several photobiological processes. However, the exact nature of the electronic structure and dynamics of the excited state of these chromophores is not clearly understood, despite numerous multidisciplinary investigations. The role of carbon–carbon double bond (>C = C<) photo-isomerization is, however, well recognized. Various mechanistic models including one-bond-flip (80), bicycle-pedal (81) and Hula-twist (82) mechanisms have been proposed for the photo-isomerization process. Based on the results obtained from the photochemical and photophysical studies of model systems like diphenylpolyenes, the role of conformationally and configurationally relaxed intramolecular charge-transfer excited states has been discussed. Many excellent reviews are available on the subject (83–91).

Absorption and fluorescence behavior

Rettig has discussed the charge separation in excited states of push–pull and decoupled systems and emphasized that these models can help elucidate the photophysical behavior of many organic, inorganic and biologically relevant compounds (43). Further, these models can lead to a deeper understanding of basic photobiological processes like vision and photosynthesis. Several other mechanisms have been suggested to explain the photophysical behavior of push–pull and decoupled systems. These include rehybridization intramolecular charge-transfer (RICT) (92), intramolecular charge-transfer (ICT) (93), and wagged intramolecular charge-transfer (WICT) (94) state.

The excited state properties of donor–acceptor diphenylpolyenes [Ar(CH = CH)nAr] have been extensively studied and in such systems the involvement of ICT states has been suggested. Rettig and his co-workers studied the photophysics of selectively bridged and rigid derivatives of N,N-dimethylamino-p-cyano stilbene (Fig. 5) in which the possibility of rotation around single as well as double bonds is restricted (95,96). These rigid compounds show large solvatochromic, red-shifted fluorescence with large Stokes’ shift. In such compounds, the quantum yield of fluorescence (Φf) increases to unity although the anilino group is free to rotate. The results are explained in terms of three kinetic scheme model consisting of planar ICT (highly polar and fluorescent in nature) state, twisted anilino group (A*, highly polar and fluorescent) and the perpendicular phantom excited state (P*, which is formed as a result of twisting of >C = C< double bond, less polar and nonfluorescent). Rulliere and his co-workers (97) studied time-resolved fluorescence of N,N-dimethylamino-p-cyano stilbene and its rigid derivatives and suggested that the dual fluorescence in such systems can be because of the twist over the single bond attached to aryl and donor group (Ar–N). In N,N-dimethylamino-p-cyano stilbene, two excited state specie were reported in polar solvents. However, rigid N,N-dimethylamino-p-cyano stilbenes, where the twisting motion around the anilino group is prevented showed the presence of one excited state specie in polar solvent. The results are explained in terms of four kinetic scheme model consisting of several excited states such as delocalized excited (DE) state, ICT state (highly polar and nearly planar), conformationally relaxed intramolecular charge-transfer excited state (CRICT, highly polar, single bond twisted and fluorescent), which is formed as a result of twisting of the anilino group, and the P*. In such systems, it is found that the lifetime of CRICT state is 2–20 ps depending upon the polarity, viscosity and hydrogen bond ability of the solvent. Also, the rigid N,N-dimethylamino-p-cyanostilbene compounds show large solvatochromic fluorescence with the involvement of highly dipolar excited state. This indicated that internal rotation around the anilino group did not play any role in obtaining the highly fluorescent species in this push–pull stilbene.

Figure 5.

 Structure of N,N-dimethyl-p-cyanostilbene (DCS) and a rigid-DCS compound.

We have observed solvent polarity-dependent dual fluorescence in dimethoxy-cyano-substituted-1,2-diarylethene in which the shorter wavelength emission is the result of locally excited (LE) delocalized planar excited state and the longer wavelength emission is the result of twisted intramolecular charge-transfer (TICT) excited state (98,99).

In nitro-substituted stilbenes, butadienes, hexatrienes and in their stiffen derivatives, a highly polar excited state is believed to be formed upon excitation, which is stabilized more when compared with the perpendicular P* and follows the radiative pathway rather than the P* state pathway leading to photo-isomerization (100–104). These compounds show large solvatochromic fluorescence and exhibit ICT excited state. It is suggested that such properties are not because of their trans–cis photo-isomerization, but because of their excited state, single bond twists across the nitro group, which has been referred to as the TICT state. We have examined the absorption, time-resolved fluorescence and photo-isomerization properties of various nitro-substituted stilbenes and diarylbutadienes (101–103). It is found that all the compounds show solvent-dependent solvatochromic fluorescence and their Stokes’ shifts increase with increasing solvent polarity. It has been suggested that large red-shifted emission in nitro-substituted diphenylbutadine is because of the TICT state. Time-resolved fluorescence studies showed the presence of single excited state specie with very low Φf and the lifetime (τf) of excited state specie increases in polar solvents. Fluorescence studies of nitro-substituted diphenylbutadiene at 77 K and in solid state showed a blue-shifted emission maximum at 77 K as compared with the ambient temperature. In such systems, it is suggested that emission originates from ICT state rather than from the TICT at 77 K because of their restricted twisting motion in rigid medium (102).

Earlier, it has been shown that (1-(4′-cyanophenyl)-4-(4′-nitrophenyl)-buta-1E,3E-diene (101) and 1-(4′-N,N-dimethylphenyl)-4-(4′-nitrophenyl)-buta-1E,3E-diene) (103) show higher red-shifted solvatochromic fluorescence in polar solvents than cyano and methoxy-substituted diphenylbutadienes (98,105,106). The results have been discussed in terms of the twisting of the single bond attached to the nitro group. However, the twist around single bond attached to the donor (amino group) could not be ruled out. In this context, we have very recently examined photophysical and photochemical behavior of variously substituted donor–acceptor 1,4-diphenylbutadienes and ethenyl indoles (Fig. 6) (101,103,105–107). The p-nitro- and p-amino-phenyl-substituted ethenyl indoles can be considered indole derivative of stilbene or 3-arylidene-substituted indole or derivative of diarylbutadiene in which one of the chain double bonds is placed in the indolic ring, thereby restricting the conformational freedom of the single bond connecting the phenyl group to NH2 or NMe2 or ethenyl moieties. Additionally, the indole moiety in such systems can act as a donor and its donor ability can be altered by substituting the –NH hydrogen with an electron releasing (e.g. alkyl) or an electron withdrawing group (e.g. acetyl, sulfonyl).

Figure 6.

 Structure of 1,4-diphenylbutadiene (DPB), its derivatives (1–8) and ethenyl indoles 9–14.

It has been observed that absorption maximum (λabs max) of the substituted diarylbutadienes and ethenyl indoles in general gets moderately red-shifted with increasing solvent polarity, excepting in case of 1,4-diphenylbutadiene and 1. Ethenyl indole 9 shows a maximum red shift (17 nm) from nonpolar solvent heptane to the polar solvent acetonitrile. As far as fluorescence emission is concerned, the λf max of 1,4-diphenylbutadiene is insensitive to the solvent polarity because of the unavailability of donor–acceptor framework. But on increasing solvent polarity, the λf max of nitro-substituted butadienes and ethenyl indoles get highly red-shifted. Hence, for 2, 3, 5 and 7, which contain a strong donor (–NMe2) and a strong acceptor moiety (–CN, –NO2) and for 8–12 (nitro-substituted ethenyl indoles), a redshift of 100–190 nm in λf max is observed. Ethenyl indoles 11 and 12 show dual fluorescence. The large solvatochromic redshift can be explained in terms of involvement of highly polar twisted excited states (101–103,105–107). Thus, the excited state of nitro-substituted compounds strongly interacts with the polar solvent.

Calculation of excited state dipole moment changes (Δμ) using the Lippert-Mataga analysis (105) indicates a large dipole moment of 17 D–26 D for nitro-substituted butadienes (2,3) and ethenyl indoles (9–12). On the other hand, dienes 1 and 5–8 show Δμ of 8–13 D. Such Δμ-values are indicative of a highly polar nature of the excited state, and hence, a strong interaction of these compounds with the solvent molecules is expected.

Thus, the presence of strong donor–acceptor moiety in 9–12 leads to charge separation-induced interaction between the dipolar solute and the polar solvent molecules. This can result in large solvatochromic redshift in the λf max of these compounds in polar solvents. Such solvatochromic redshifts can arise because of ICT from donor to acceptor moiety (NO2). On the other hand, 1,4-diphenylbutadiene, 1, 4 and 13, which are substituted with moderate acceptor/donor or single substituted donor or acceptor groups, the excited state is less polar, and hence moderate solvatochromic shifts of λf max are seen.

The magnitude and the trends of Φf in these compounds are, however, not very clear. The observed variations in Φf may be as a result of the solvent-driven reversal of closely lying Ag and Bu energy states (83). Further, during the formation of P* specie which involves out-of-plane twisting of an ethylene >C = C< chromophore, considerable charge separation can occur (84–87). Hence, appropriate substituents can stabilize the resulting dipolar P* state. This effect can decrease the energy barrier for the formation of P* state facilitating transcis photo-isomerization and reducing Φf. Thus, Φf of these compounds is highly sensitive to solvent polarity and the electronic nature of the substituents.

Further, low temperature fluorescence studies of dienes and ethenes at 77 K in 1 : 1 ethanol–methanol glass matrix indicate a considerable blueshift in their λf max along with a large enhancement in their fluorescence intensity when compared with fluorescence studies at 298 K (101,105). As the torsional motions in the molecule are expected to be considerably restricted at 77 K, limiting the formation of CRICT state, the blueshift in fluorescence maximum is observed. Thus, low temperature fluorescence studies indicate towards the involvement of nonplanar, CRICT excited states in the photoprocesses of nitro-substituted butadienes and ethenyl indoles.

While the nitro-substituted diphenylbutadienes (6–8) show single exponential (τf = 0.1 to 0.6 ns) fluorescence decay, the ethenyl indoles (9–14) show multi-exponential fluorescence decay (103,105). This indicates that strong donor and acceptor-substituted conjugated compounds have more than one emitting specie. For ethenyl indoles, two excited species have shorter τf (0–1 ns), whereas the other excited specie (which is believed to be CT state) has a longer τf (1–4 ns). The existence of large solvatochromic fluorescence in ethenyl indoles 9–12 and the presence of multiple fluorescence bands in ethenyl indoles 11 and 12 suggests that these compounds have at least three excited species in their singlet excited manifold. These species can be the LE state, the ICT state and the CRICT state (Fig. 7). In 9 and 10, all three species are found in nonpolar solvent, whereas in medium-polar and polar solvents the CRICT specie is predominantly observed. In nitro-substituted diphenylbutadienes the CRICT state is formed predominantly in all the solvents. On the other hand, 13 and 14 show two excited state specie in solvents of lower dipole moment, indicating towards absence of CRICT state in these compounds.

Figure 7.

 Excited state kinetic scheme applicable to nitro-substituted ethenyl indoles/dienes (LE: locally excited state, planar. ICT: intramolecular charge-transfer excited state. CRICT: conformationally relaxed intramolecular charge-transfer excited state. P*: perpendicular double bond twisted excited state [phantom excited state]).

Photo-isomerization studies

Direct irradiation of 1E,3E dienes 5–8 yields the corresponding 1E,3Z-isomer because of the one-photon-one-bond photo-isomerization. Dienes 5 and 6 (para-substituted dienes) show efficient photo-isomerzation (photo-isomerization quantum yield, ΦPI in cyclohexane ∼ 0.4 and in CH3CN ∼ 0.5) when compared with meta or ortho-nitro-substituted dienes 7 and 8 (ΦPI in cyclohexane ∼ 0.1 and in CH3CN ∼ 0.2). For all the dienes the 1E,3E isomer dominates the photomixture at photostationary state (PSS). In general, however, the ΦPI increases on increasing solvent polarity. This indicates that polar solvents reduce the activation energy barrier for the formation of P* state. The twisting of >C = C< between C3–C4 (near acceptor, CN or NO2) is more preferred. Hence, 1E,3Z isomer is formed predominantly. Further, it is observed that the Φf for these dienes decreases on increasing solvent polarity, which suggests that fluorescence and photo-isomerization processes compete with each other. Hence, singlet excited state can be involved in the photo-isometrization process of these donor–acceptor dienes. However, as the ΦPI are low for all the dienes, the possibility of the involvement of triplet state cannot be ruled out.

Interestingly, as ethenyl indoles 9 and 10 resist photochemical change, ethenyl indoles 11 and 12 undergo photo-isomerization whose efficiency also depended on solvent polarity. As against dienes 5–8, ethenyl indoles 11 and 12 undergo efficient photo-isomerization in nonpolar n-heptane. However, the efficiency of trans–cis isomerization decreases on increasing the solvent polarity. Apparently, the P* state (from where the photo-isomerization occurs) is less polar than the initially excited state. A dipolar initial excited state of these compounds can form, which will get stabilized in polar medium causing a large energy barrier to P* state. Thus, the efficiency of trans–cis photo-isomerization is lower in the polar solvent. Additionally, the rate of reverse photo-isomerization (i.e. cistrans) may be higher.

On the other hand, 13 and 14 on direct irradiation in organic solvents readily show photocyclization reaction yielding a cyclic photoproduct (107). It is suggested that irradiation of these compounds primarily leads to the expected trans–cis photo-isomerization, and the cis isomer then undergoes [6]e electrocyclization yielding dehydrogenated cyclic photoproduct, which subsequently can aromatize to give the novel benzocarbazole nucleus.

Donor–acceptor diarylpolyenes as model to study the wavelength regulation and color tuning in retinal-bound photoactive proteins: Role of polarizable medium and dipolar species

A large range of absorption wavelengths is observed for retinal-bound photoactive proteins. While the primary electrostatic interactions between positively charged nitrogen of the retinylidene Schiff base chromophore and the anionic residues of the receptor protein have long been believed to play a crucial role in regulating the wavelength of these proteins, recent studies involving second harmonic generation (SHG), hyperRayleigh scattering and two photon spectroscopy have shown that the chromophore of bR has a very large light-induced dipole, which is at least 50% larger than that of the retinal chromophore in films or in solution (44,108–113). It has been demonstrated that the SHG of the chromophore is highly influenced by the protein domains and that the tryptophan residues lying in the vicinity of the chromophore act as polarizable groups and enhance the light-induced dipole in bR. Thus, medium-based polarizable groups play an important role in color tuning.

The retinylidene Schiff base chromophore in these photoreceptors is a push–pull donor–acceptor system, whose ground and excited state properties can be influenced by the surrounding medium. It has been shown that the absorption and fluorescence emission properties of some appropriately substituted donor–acceptor diarylethenes/dienes/indolic ethenes are highly dependent on the electronic nature of the substituents and the solvent polarity. Interestingly, these donor–acceptor systems show large alteration between ground and excited state dipole moment. The magnitude of this alteration increases with increasing solvent polarity, indicating towards enhanced interaction between the excited chromophore and the surrounding solvent molecules. Electronic changes between ground and excited state are required for modulative absorption by a chromophore. Thus, the absorption and fluorescence emission properties of these compounds can be used as a model to explain the absorption and excited state properties of retinylidene chromophore and related photobiological receptors.

Of particular interest are the ethenyl indoles wherein λabs max can be altered from 322 (in case of 13) to 418 nm (in case of 10) by putting various electron withdrawing (–SO2C6H5, –COCH3, –NO2) and electron donor (–H, –C2H5) group combinations on the indolic moiety. Compounds 9–12 exhibit highly red-shifted λf max with increasing solvent polarity possibly because of the twist over the single bond attached to the electron withdrawing nitro group. Such a twist leads to dramatic shifts in the fluorescence emission of these compounds in highly polar media, as in the case of 10 where a red shift to 650 nm is observed. These compounds can involve conformationally twisted dipolar excited state in their photoprocesses. The dipolar-excited state of these chromophores gets stabilized in the presence of polar media and due to appropriately positioned donor/acceptor substituents, leading to wavelength shifts.

These studies indicate that a twist over the bond attached to the electron withdrawing group and the Columbic interactions between the dipolar state of the chromophore and the counter ions can play an important role in wavelength tuning. The solvent can cause enhancement of dipole moment of the chromophore after it absorbs light and reaches the LE state. Similar type of interaction(s) can occur in photo-active retinal proteins, wherein dipolar retinylidene chromophore can interact with appropriate nearby receptor protein based polar/polarizable amino acid residues. The solvent in case of donor–acceptor diarylpolyenes and the amino acid residues in case of retinal-bound photoactive proteins can act as polarizable groups. The polarizable groups can influence the redistribution/delocalization of charge in the ground and excited states of the chromophore, and thereby modulate its wavelength. The interactions of chromophore with its surroundings are depicted in Fig. 8. It may, however, be noted that the feasibility of this model is limited as it lacks the presence of medium based full negative charge(s) or a truly polarizable molecular entity, as may be present at the reaction center of rhodopsins. Further studies involving medium with polar/polarizable groups are required.

Figure 8.

 (a) Protonated retinylidene Schiff base chromophore in bR, showing interaction between the dipolar chromophore and the anionic and aromatic amino acid residue of the surrounding receptor protein. (b) An excited dipolar indolic ethene in interaction with the solvent dipoles, leading to wavelength shifts.


Several biological sensory and energy transductions are mediated by protein-based photoreceptor systems containing retinal as the chromophore. Visual pigment Rh and the Halobacterial protein bR are the prime examples of such photoreceptors. Recent years have witnessed feverish efforts to unravel the molecular details of the structure and mechanism of functions of these systems. Consequently, a great deal of information is now available on the structure, function and the excited state properties of these photoreceptors. Bioorganic and photochemical studies of the analogues of rhodopsins, prepared by chromophore substitution technique, have provided valuable information about the nature of interaction between the retinylidene Schiff base chromophore and the surrounding protein residues. Rhodopsins, particularly bR, which functions like a multiple cycle reversible photoswitch is increasingly being considered for application as photoactive component in nanotechnical devices. In this context, attempts are also being made to enhance its performance by various methods.

In order to understand the excited state structure and dynamics of the chromophore of these photoreceptors, donor–acceptor/push–pull diarylpolyenes have been used as model systems. Extensive absorption, fluorescence emission and various solvent polarity parameter correlations of variously substituted diarylethenes and dienes show that the excited state of these compounds can have dipolar structure. It is further noted that these compounds in addition to having the initially formed locally excited state, can have intramolecular charge-transfer states, which can either be planar or twisted. These states are formed depending on the magnitude of solvent polarity and the electronic nature of the substituent. Further, the formation of dipolar specie is not limited to >C = C< twisting, but it can also be formed due to single bond twists as well. Thus, the role of single bond twist in the excited state of push–pull systems like protonated retinylidene Schiff base chromophore in rhodopsins is also important. Such conformationally relaxed structures can be influenced by the surrounding polar/polarizable medium causing alteration between the ground and the excited states, thereby influencing the wavelength of the chromophore. Such interactions can be involved in retinal-bound photoactive proteins, wherein the light-induced dipole of the push–pull system like retinylidene Schiff base chromophore is highly influenced by the nearby polar/polarizable amino acid residues, giving the protein an ability to tune its wavelength. The feasibility of this model based on the observed photophysical properties of some push–pull diarylpolyenes is however limited, as the chosen medium (organic solvents) has neither negative charge nor a truly polarizable molecular entity. Further studies involving medium with polar/polarizable groups, therefore, are required.

Acknowledgements— A.K.S. is grateful to his students/coauthors whose names appear along with him in the references. Financial supports from various funding agencies of the Government of India including the Department of Science and Technology, Department of Atomic Energy (Board of Research in Nuclear Sciences), Council of Scientific and Industrial Research, Ministry of Human Resource Development, University Grants Commission (UGC) and IIT Bombay are gratefully acknowledged. P.K.H. is recipient of a Research Fellowship from the UGC. We also thank the reviewers for their valuable suggestions.