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Abstract
There are several biological chromophores which are utilized in nature as photoreceptors. However, none have aroused as much curiosity among the scientists regarding their structure and mechanism of function as the one found in purple membrane (PM) of Halobacterium halobium. The chromophore of PM called bacteriorhodopsin (BR) is a membrane bound protein which acts as a transmembrane proton pump. BR has received wide interest since it has some similarities to the visual pigment rhodopsin, and because of its intrinsic properties as a very simple light energy transducer. Bacteriorhodopsin exhibits several striking features including its absorption behaviour and proton-pumping ability. Enormous efforts in recent years have been made to clarify the nature of interaction at the BR active site. However a complete and comprehensive understanding of BR structure and function is far from clear. The present investigations have been undertaken to provide chemical clarification to some of these important aspects of BR structure and function. The approach used in the investigation includes a combination of bioorganic models of BR and spectroscopic techniques.
The thesis entitled 'Bioorganic investigations on bacteriorhodopsin' comprises of eight chapters. Chapter 1 contains the general introduction. A brief review of the important features of purple membrane (HI) including the structural and functional aspects of its protein, bacteriorhodopsin (BR) are presented in this chapter.
Chapter 2-4 concerns with the evaluation of active site properties of BR, in terms of its stereo-electronic features. Retinal analogues 3-methyl-5-(9'-anthryl)-2E,4E-pentadienal (60) and 3,7-dimethyl-9- (9 '-anthryl) -2E, 4E, 6E, 8E-nonatetraenal (66) have been used as bioorganic probes to characterize the BR active site in terms of polyene chain length, role of sterically hindered trimethylcyclohexenyl moiety and effect of electronic interaction of opsin bound charges with planar aromatic rings.
Chapter 2 describes the synthesis of the retinal analogues 60 and 66. The chapter begins with a description of the recent methodologies developed for the synthesis of retinal (1b) and related compounds. The compounds 60 and 66 have been synthesized starting from anthracene. The procedures adopted for the synthesis of retinal analogues 60 and 66 are described in schemes 2.1 and 2.8 respectively. The required functional group for the introduction of polyenic chain in anthracene has been introduced at carbon 9 of anthracene by its formylation reaction. The desired extension of 9-anthraldehyde to 3-methyl- 5-(9'-anthryl) -2E,4E,-pentadiena1 (60) has been done by allowing 9-anthraldehyde to react with methyl 4-(diethylphosphono)-3-methyl-2-butenoate (C5-phosphonate 56) in presence of NaH in THF. The required C5 phosphonate in turn has been synthesized starting from 3,3-dimethyi acrylic acid(61 scheme 2.2), Esterification of the acid involved preparation of its acyl chloride followed by in situ reaction with methanol. Allylic bromination of the resulting ester 62 was successfully carried out by Wohler-Ziegler reaction with N-bromosuccinimide. Bromoester 63 was then treated with triethyl phosphite to give the desired C5 phosphonate in isomeric mixture of 2-cis and 2-trans (56a and 56b respectively) in 2:3 ratio. Condensation of 9-anthraldehyde with a cis-trans mixture of the C 5 phosphonate in presence of NaH resulted in the formation of the corresponding ester methyl 3-methyl-5—(9',-anthryl)-2E-4E-pentadienoate (58) as geometrical isomers. The predominant (80 %) 2E, 4E isomer was isolated by a combination of flash column chromatography and crystallization. The yellow coloured crystals were characterized by HPLC e (Rt = 4.4 minutes) and physico chemical data. A satisfactory steteroseleetivity in the formation of trans double bond at C-4 was achieved by using THF in condensation reaction. Careful reduction of the ester (58) with lithium alluminium hydride (LAH) gave the corresponding alcohol which was carefully oxidized with MnO2 to the desired retinal analogue 60 without any loss of stereochemistry. Compound 60 gave satisfactory physico chemical data. A few unsuccessful attempts have been made towards the synthesis of retinal analogue 66. However, further extension of the chain in 60 could only be done when the compound (60) was treated with C5 phosphonate under Horner-Emmons reaction conditions. Subsequent reduction of the ester (T8) followed by oxidation of the alcohol gave the fully extended anthryl retinal analogue 66. The resulting aldehyde was found to be a mixture of geometrical isomers. Retinal analogue 3,7-dimethyl-9-(9,-anthcyl)-2E,4E,6E,8E-nonatetraenal (66), the predominant constituent of the mixture was isolated in configurational pure form by HPLC for further use. These compounds were very sensitive to light, heat and air and required special storage procedure.
