https://etd.iisc.ac.in/handle/2005/8 Thu, 16 Apr 2026 05:25:15 GMT 2026-04-16T05:25:15Z http://etd.iisc.ac.in:80/bitstream/id/9a7a6f81-27a0-4961-b420-d0687b339bfc/ https://etd.iisc.ac.in/handle/2005/8 https://etd.iisc.ac.in/handle/2005/9348 A-T base sequences: a theoretical and an experimental study Rajagopalan, Malini As a result of the study undertaken, besides predicting the relative stabilities of polynucleotide duplexes containing A–T base pairs, the following conclusions can be drawn (Rajagopalan et al., 1984): a) Base–base interactions depend on both helical parameters and base parameters. b) Hydrogen bonding plays an important role not only in determining stacking patterns but also in defining the pairing scheme. c) Particularly for the D form, the A–T–A–T sequence prefers a right handed structure, whereas the T–A–T–A sequence prefers a left handed structure. These energy calculations highlight the role of base sequence, orientation, and disposition of bases, enabling theoretical predictions of energetically stable structures. Chapter 4 will deal in detail with the structure of the synthetic polynucleotide poly d(A–T) in the D form. The relevance of the energy calculation results to its structure-compared with available X ray data-will be discussed. The results of physicochemical studies on both the impure and pure trimer and the impure dimer have suggested certain criteria important for the interaction of such compounds with DNA. It must be emphasized that purity is essential for quantitatively determining the extent of interaction with various DNAs and for effective comparison with distamycin (Dst). Since the dimer could not be purified, only qualitative studies of its interactions were carried out, and no quantitative data are reported. Work on the dimer-aimed at identifying the reasons for its resistance to purification and possible solutions-is in progress. For the trimer–DNA interaction, only a qualitative analysis was conducted, supporting the idea that compounds resembling A–T specific probes can be synthesized. However, quantitative studies on the interaction of certain trimer analogues of Dst are currently underway in this laboratory (Dasgupta et al., 1987). These compounds resemble Dst, except that either the second or third consecutive methylpyrrole ring is replaced by alanine. The effect of this modification-and the resulting curvature-on binding to natural and synthetic DNAs, in comparison with Dst, has been determined. A few additional analogues of Dst have been synthesized (Hao & Ekambareshwar, 1987) to better understand the role of curvature in Dst analogue–DNA interactions. In these analogues, the N methylpyrrole rings of Dst or netropsin (Nt) have been replaced by meta or para aminobenzoic acid units. These analogues: show specificity for B DNA only, exhibit no affinity for A or Z DNA, display reduced A–T selectivity compared with Dst, and bind via the minor groove. Preliminary antiviral and antitumor studies indicate that these analogues are active against bovine herpes simplex virus and BUK 2 cells. The antibacterial activity of both the dimer and trimer showed interesting results (as mentioned in Chapter 8), even though these preliminary results do not yet establish the comparative strength or specificity of binding of these analogues relative to Dst. Antiviral and antitumor studies on these Dst analogues are ongoing. Thus, it can be concluded that multiple factors contribute to ligand–DNA interactions, influencing both base specificity and binding strength. These factors include: electrostatic attraction, hydrogen bonding, van der Waals radii, curvature, and the chemical structure of the ligand. The design of A–T specific probes using conformational principles is therefore of significant importance. It has been shown that substituting consecutive pyrrole rings of Dst with alanine reduces A–T specificity (Dasgupta et al., 1987), relative to native Dst. A similar loss of A–T specificity was observed in trimer–DNA interactions in the present study. Further investigations are needed to determine whether this reduced specificity arises from chemical substitution, changes in curvature, or a combination of both. These studies are being actively pursued in this laboratory. https://etd.iisc.ac.in/handle/2005/9348 https://etd.iisc.ac.in/handle/2005/7701 Achieving oral bioavailability of therapeutic macrocyclic peptides by bridging backbone chemistry with pharmacokinetics Raj, Nishant Full text embargo up to Dec 11, 2026 Macrocyclic peptides are emerging as very potential candidates for drug development. Due to the advancement in display technologies, macrocycles can target undruggable proteins. These molecules have the appropriate biochemical & therapeutic properties to provide advantages for both small molecules & antibodies. Still, most macrocycles cannot be given orally due to their low gastrointestinal absorption and rapid digestion. Our study shows that site-specific editing of the backbone of these macrocycles can enhance its drug-like properties. We focused on shielding the amide hydrogen bond acceptor (>C = O) & amide hydrogen bond donor (>N-H) to reduce the de-solvation penalty during membrane permeation. Additionally, altering the backbone amide of these macrocycles enhances their metabolic stability. In a proof of concept, we applied our strategy to four different macrocycles with very different physicochemical properties. With our strategy, we were able to develop several orally bioavailable macrocycles. In this process, we explored interesting parameters governing the permeability & compared the influence of hydrogen bond donor & acceptor to de-solvation. Finally, we applied these strategies to bioactive molecules to increase their drug-likeness without altering their bioactivity. Our strategy provides a unique therapeutic niche to macrocycles that can expand its pharmaceutical landscape. https://etd.iisc.ac.in/handle/2005/7701 https://etd.iisc.ac.in/handle/2005/8986 Acyclic peptides as conformational models Balaram Hemalatha Proteins and polypeptides play multi?faceted functional roles in the living organism. Numerous biological processes are controlled by these biomolecules. Hence, there is widespread interest in understanding the structure, function, and action of these molecules. Various techniques such as X?ray diffraction, Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD), Infrared (IR), and Raman spectroscopy have been used to elucidate polypeptide and protein conformation. Studies on synthetic model peptides can provide insight into the conformational preferences of specific peptide sequences and the factors influencing backbone folding. This thesis describes attempts aimed at developing acyclic peptides as conformational models. The thesis is divided into eight chapters. A general introduction to the various regular peptide conformations and their analysis using spectroscopic methods is presented in Chapter I. The importance of introducing conformational constraints using ??aminoisobutyryl (Aib) and/or prolyl residues or disulfide linkages to generate peptide models with reduced conformational flexibility is highlighted. Chapter II describes the synthesis and spectroscopic studies of model ??turn forming peptides having the general sequence Piv–Pro–X–NHMe, where X = Aib, Gly, D?Ala, L?Ala, L?Val, D?Val, L?Leu, D?Leu, and L?Thr. The ??turn conformations were elucidated using Nuclear Overhauser Effects (NOE), CD, and Raman spectroscopy. NOE experiments provided a basis for differentiating Type II ??turns from Type I and Type III ??turns. Using this technique, Type II ??turns were assigned to the dipeptides with X = Aib, Gly, D?Ala, D?Leu, and D?Val. A Type V ??turn structure was observed for peptides containing L?Ala and L?Leu from NOE studies. Solvent?dependent structural changes were monitored using CD. Chapter III describes the synthesis and conformational studies on peptides with the sequence Boc?Aib?X?Pro?NHMe, where X = Pro, L?Ala, D?Ala, Gly, Val, and Leu. These systems were examined as possible models for 5?1 hydrogen bonded structures and for evaluating environmental effects on cis–trans isomerism about X–Pro bonds. Low?temperature ¹H NMR studies on Boc?Aib?Pro?Pro?NHMe in CDCl? provide evidence for the presence of multiple conformational states in this peptide. ¹³C NMR studies indicate the presence of cis–trans isomerism in solution, which is sequence and solvent dependent. The all?trans structure is devoid of intramolecular hydrogen bonds, as indicated by solvent? and temperature?dependent NMR studies. X?ray investigations on Boc?Aib?Pro?Pro?NHMe and Boc?Aib?Leu?Pro?NHMe provide supporting evidence for the NMR interpretation. ¹H NMR studies favour a Type VI ??turn in the cis X?Pro conformers. Chapter IV describes the synthesis and spectroscopic (¹H NMR, CD, and IR) studies on an Aib?containing decapeptide, Boc?Aib?Val?Aib?Aib?Val?Val?Val?Aib?Val?Aib?OMe. This sequence was chosen to evaluate the effect of a triplet of L?amino acids on the conformation of Aib?containing oligopeptides. A brief survey of the conformational preferences of Aib?containing polypeptides is also presented. The results obtained for the decapeptide give evidence for the formation of helical conformers in solution, indicating the strong tendency of Aib residues to dictate helical folding even in the presence of a high content of valine residues. Evidence for structural transitions on going from a non?polar to a polar solvent is provided. Studies on acyclic peptide models for intramolecular ??sheet conformation are presented in Chapters V and VI. Sequences designed as models for intramolecular ??sheet conformation were of two kinds: (a) Homovaline peptides having an Aib or Pro residue, or an Aib?Pro segment in the centre of the sequence to nucleate ??turn formation. The sequences synthesized were Boc?(Val)n?X?(Val)m?OMe (n = 2, 3; m = 3; X = Aib, Pro) and Boc?(Val)n?X?Y?(Val)m?OMe (n = 2, 3; m = 3; X?Y = Aib?Pro). ¹H NMR and IR evidence has been provided for the presence of intramolecular ??sheet structures. Solvent?dependent conformational changes have been observed by CD. Evidence is also provided for the population of helical conformations in solvents such as methanol and trimethylphosphate. (b) Acyclic cystine peptides of the type Boc?Cys??????(Val)n?Trp?OMe (n = 1, 2, 3) were synthesized to explore side?chain interactions in antiparallel ??sheet structures promoted by the disulfide moiety. The quenching of tryptophan fluorescence by the proximal disulfide group was used to identify the conformation of the peptides. Reduction studies using sodium borohydride provide evidence for the formation of antiparallel ??sheet conformation about the S–S bond. These results are presented in Chapter VI. Synthesis and spectroscopic (NMR and CD) studies on the fragments (Boc?Asp(OBzl)?Leu?Thr?(Gly)??Val?OBzl and Boc?Leu?Thr?(Gly)??Val?OBzl) and analogs (Boc?Asp(OBzl)?Leu?Thr?(Gly)??Val?OBzl and Boc?Leu?Thr?(Gly)??Val?OBzl) of (Thr?)?speract, a sperm?activating peptide, are described in Chapter VII. ¹H NMR evidence for the presence of intramolecular ??sheets, which are highly aggregated in non?polar solvents, is presented. The results indicate that peptides containing a stretch of three glycine residues aggregate to a much greater extent than peptides with two glycine residues. An appendix to the thesis briefly describes reverse?phase HPIC studies of diastereomeric peptides with the sequence Piv?Pro?X?NHMe, which establish racemization at 1?Pro on activation of pivaloyl?proline. The main findings of this investigation are summarized in Chapter VIII. List of Publications: The type II ??turn conformation of Pivaloyl?L?Prolyl???aminoisobutyryl?Methylamide: Theoretical, Spectroscopic, and X?ray studies. B. V. Venkataram Prasad, Hemalatha Balaram, and P. Balaram. Biopolymers, 21, 1261–1273 (1982). Multiple conformational states of a Pro?Pro peptide: Structural changes on dissolution of single crystals. Hemalatha Balaram, B. V. Venkataram Prasad, and P. Balaram. Biochem. Biophys. Res. Commun., 109, 825–831 (1982). Multiple conformational states of a Pro?Pro peptide: Solid state and solution conformations of Boc?Aib?Pro?Pro?NHMe. Hemalatha Balaram, B. V. Venkataram Prasad, and P. Balaram. J. Am. Chem. Soc., 105, 4065 (1983). Nuclear Overhauser effects and circular dichroism as probes of ??turn conformations in acyclic and cyclic peptides with Pro?X sequences. B. N. N. Rao, A. I. Cumar, H. Balaram, A. Ravi, and P. Balaram. J. Amer. Chem. Soc., 105, 7423–7428 (1983). Peptide models for ??turns: A circular dichroism study. M. Crisma, G. D. Pasman, H. Balaram, and P. Balaram. Int. J. Pept. Protein Res. (1983). Acyclic peptides as conformational models: Crystal structure of Boc?Aib?Leu?Pro?NHMe·2H?O. B. V. V. Prasad, H. Balaram, and P. Balaram. Int. J. Pept. Protein Res. (1984). Side?chain interactions as probes of ??sheet structures in peptides: Fluorescence quenching of tryptophan by proximal cystine residues. Hemalatha Balaram and P. Balaram (Submitted). Acyclic oligopeptide models for ??turns, ??sheets, and helical structures. Hemalatha Balaram, A. Ravi, E. K. S. Vijayakumar, and P. Balaram. Presented at the Eighth American Peptide Symposium, Tucson, Arizona, U.S.A., May 22–27 (1983). https://etd.iisc.ac.in/handle/2005/8986 https://etd.iisc.ac.in/handle/2005/8992 Altered conformations in natural DNA: Studies on supercoiled form V DNA Shouche, Yogesh Shreepad The double helical structure of DNA proposed by Watson and Crick, which led to the development of modern biology, was assumed to be a uniform right-handed helix. However, in recent years, the sequence-dependent conformational flexibility of DNA has been shown to be a rule rather than an exception through both theoretical and X-ray crystallographic studies on oligonucleotides. The discovery of left-handed Z-DNA paved the way for studies on the possible relevance of unusual secondary structures of DNA in biological processes. Applying the topological properties, Weissmann and co-workers first predicted the possible existence of a left-handed structure within Form V DNA of natural sequences. So far, most of the structural studies on left-handed DNA have been done on synthetic oligonucleotides and polynucleotides of alternating purine-pyrimidine sequences, with little focus on naturally occurring sequences. Such a study requires natural DNA with a large amount of altered conformation, extending from right-handed B-DNA to left-handed Z-DNA under the influence of supercoiling. The molecule of choice for the present study is Form V DNA with a linking number of zero. This is an in vitro reconstituted double-stranded circular DNA obtained by the reannealing of two single-stranded circles. Topological constraints require that every right-handed helical turn must be compensated by a left-handed helical turn or a negative supercoiling, since the linking number of Form V DNA is zero. This molecule adopts a negative superhelical density approaching 1.0. Therefore, several sequences are forced to adopt altered structures depending on the nature of the sequences themselves, as well as their neighboring sequences and torsional stress. The questions raised in this thesis concern the altered conformations adopted by natural sequences in Form V DNA and their detection using sequence- and structure-specific probes. The various aspects investigated are: i) Detection of the nature and extent of altered conformations in Form V DNA. ii) Interaction of altered conformations with structure-specific proteins, such as Z-DNA-specific antibody and Z-DNA-binding protein from wheat germ. iii) Effect of Z-DNA-stabilizing and DNA-unwinding agents on the stability of altered structures in Form V DNA. iv) Recognition of sequences present in altered conformation by sequence-specific restriction endonucleases. v) Accessibility of sequences in the altered conformations to methylation by modification methylases. vi) Capacity of sequences in natural DNA to adopt non-B conformations under topological strain. vii) Effect of flanking sequences on structural transitions. viii) Use of sequence-specific interactions to probe altered conformations in DNA and to rank such sequences based on their potential to adopt non-B conformation under the influence of topological unwinding. Chapter 1 provides a brief account of major advances in our understanding of DNA structure. A concise account of the present status of left-handed DNA, various factors controlling B-to-Z structural transitions, and its probable biological role has been given. DNA supercoiling and its role in regulating genetic processes are also discussed. Studies carried out on Form V DNA by other workers are summarized in this chapter. Chapter 2 describes the steps involved in the preparation and purification of Form V DNA from plasmids pFiG, and the monomer and dimer of pBR322. A brief account of various procedures used for the purification of supercoiled DNA, the starting material for Form V, is also provided. Physicochemical and biochemical characterization of Form V DNA is presented in Chapter 3. Using two-dimensional electrophoresis and sedimentation methods, Form V DNA was found to possess high negative superhelical density. Spectroscopic studies indicated that more than 90% of the DNA is double-stranded, of which 35–40% is in left-handed conformation. A significant proportion of the left-handed segment is in the Z-DNA conformation, as indicated by binding of Z-DNA-specific protein and antibody. These left-handed structures in Form V DNA could be stabilized by hexamine cobalt chloride and destabilized by ethidium bromide. To study the effect of altered conformation on sequence recognition by restriction endonucleases, cleavage of Form V DNA with various restriction enzymes was undertaken (Chapter 4). From studies with restriction endonucleases that have a single site in pBR322, it was found that recognition sequences for enzymes such as Aval, BamHI, and PstI were not accessible to cleavage in Form V DNA, indicating a probable alteration in structure. However, these sites became susceptible to cleavage when the topological constraints were relieved by simultaneous digestion either by topoisomerase or another restriction enzyme. This shows that altered DNA conformation is responsible for resistance to certain restriction endonucleases. Some sites showed partial cleavage. The possible nature of altered conformations adopted at various restriction sites is discussed. In order to probe the altered conformations at regions within Form V DNA, two new methods were developed. Chapter 5 describes one method, which utilizes restriction endonucleases that have more than one site in pBR322. In this case, the nature of structures at various sites for FspI and Narl were assayed by partial cleavage of DNA followed by labeling at a specific site. In Form V as well as in Form I DNA, different sites showed various degrees of reactivity with the enzymes. However, Form V DNA exhibited greater resistance to cleavage compared to Form I DNA. The variable accessibility of sites is discussed in relation to structural alteration and the influence of flanking sequences in modulating such structural transitions. These studies provide strong evidence for the requirement of defined structures, in addition to sequence, for restriction by endonucleases, which can also explain the occurrence of slow sites in natural DNA. A more efficient method for detecting structural alteration is presented in Chapter 6. This method utilizes sequence-specific DNA methylases as probes for B-DNA, exploiting the inability of methylases to methylate DNA adopting altered structures such as Z-DNA or single-stranded DNA. Since the DNA is not cleaved during methylation, the torsional stress of the molecule is maintained during treatment. Thus, the history of the structure at the methylatable site is preserved in terms of methylation level, even when the superhelical strain in Form V DNA is removed by subsequent restriction endonuclease cleavage. The nature and extent of altered conformations present at M.AluI, M.HhaI, and other sites are discussed. Some potential Z-forming alternating purine-pyrimidine sequences of fewer than seven base pairs were not in Z conformation, whereas others did adopt an altered structure, indicating an influence of neighboring sequences. Furthermore, regions of polypurine–polypyrimidine stretches were found to be the second-best candidates to adopt altered conformation. Using the results from restriction endonuclease cleavage and methylation, a hierarchy of altered conformations adopted by DNA sequences in Form V was established, and a map indicating regions with structural alterations was developed. Thus, the combined influence of negative supercoiling and flanking sequences may have a strong effect on the structure adopted by sequences in vivo, thereby playing an important role in the regulation of genetic processes. The work presented in this thesis, along with additional studies carried out by the candidate, has resulted in the following publications: Left-handed DNA in synthetic and topologically constrained Form V DNA and its implications in protein recognition. Shouche, Y.S., Latha, P.K., Ramesh, N., Majumder, K., Mandyan, V., and Samir K. Brahmachari, J. Biosci. 8 (1985) 563–578. Sequences that adopt non-B-DNA conformation in Form V DNA as probed by enzymatic methylation. Samir K. Brahmachari, Shouche, Y.S., Charles R. Cantor, and M. McClelland, J. Mol. Biol. 193 (1987) 201–211. The influence of topological unwinding of DNA on restriction enzyme specificity. Shouche, Y.S., N. Ramesh, and Samir K. Brahmachari, (Submitted to Nucleic Acids Research) Recognition of B- and Z-forms of DNA by E. coli DNA polymerase I. N. Ramesh, Yogesh S. Shouche, and Samir K. Brahmachari, J. Mol. Biol. 190 (1986) 635–638. Stability of left-handed helical structure in pBR322 Form V DNA. Y.S. Shouche and Samir K. Brahmachari, (Manuscript in preparation) Recognition of Z-DNA segments in Form V DNA by Z-DNA-binding protein from wheat germ. Y.S. Shouche, Eugenia T., N. Ramesh, and Samir K. Brahmachari, (Manuscript in preparation) https://etd.iisc.ac.in/handle/2005/8992