Anil Mukund Limaye (Member) for their intellectual input, encouragement, valuable suggestions and comments throughout my research work. Synthesis and studies of the photophysical property of donor-acceptor unnatural tetrazolyl nucleosides are the content of chapter 3.
CHAPTER 1: UNNATURAL NUCLEOSIDE BASE SURROGATES AND THEIR APPLICATIONS: A REVIEW
Several fluorescent DNA base analogs (nucleoside and/or non-nucleoside base surrogates) have been exploited in various applications, such as in DNA analysis, monitoring hybridization events, in delineating the structure and dynamics of DNA, in probing DNA lesions, in the pursuit of light harvesting of DNA-based materials and in other biotechnological and materials science applications. Various photophysical properties, such as FRET, exciplex, and excimer emission, have also been studied with the multichromophoric DNA containing several unnatural fluorescent base analogs that exploit the rigid DNA conformation as the organizing scaffold.
CHAPTER 2: STUDIES ON THE SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF TRIAZOLYL NUCLEOSIDES
Some of the environmentally sensitive fluorescent oligonucleotide probes containing microenvironment-sensitive fluorescence have been used as a molecular signaling device in DNA detection. Analysis of the UV-visible spectra of the mixture and the combined spectra of the individual nucleosides in both dioxane and buffer revealed a possible ground-state charge-transfer complex formation (Figure A2b–c).
CHAPTER 3: STUDIES ON THE SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF TETRAZOLYL NUCLEOSIDES
The high stabilization of the TphenBDo:ΦΦΦΦ duplex represents a remarkable improvement in stability and selectivity over previously reported non-hydrogen-bonded base pairs.
The non-nucleoside base substitute OxoPyS showed significant selectivity for the nucleoside base substitute TPhenBDo in all four natural bases. The mechanism of exciplex formation via FRET that we have demonstrated would further be possible through judicious design and proper positioning of the donor/acceptor pair in the probe, the pair involving a strong intercalative π-π stacking interaction.
Chapter 1: UNNATURAL NUCLEOSIDE BASE SURROGATES AND THEIR APPLICATIONS: A REVIEW
Chapter 2: STUDIES ON THE SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF TRIAZOLYL
Chapter 3: STUDIES ON THE SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF TETRAZOLYL
STUDIES ON THE STABILIZATION OF AN ABASIC SITE PAIRED AGAINST AN UNNATURAL TRIAZOLYL
Stabilization of an abasic site by a non-nucleosidic base surrogate, targeting an abasic site with a nucleosidic base surrogate 270-273.
Introduction
Benner's early research work focused on the development of new base pairs based on hydrogen bonding patterns orthogonal to those in canonical Watson-Crick base pairs.6 Following Benner's work, many researchers contributed to the field of expanding genetic alphabets. New, hydrophobic base pairs have therefore recently been developed, but their use in transcription is still being investigated.
Need for Unnatural Nucleotide Bases
Thus, there is a need to develop conceptually new and novel base analogs that will be recognized by DNA polymerases with high efficiency for both replication and transcription processes.
Design of Unnatural Base Pairs
However, to date, due to the challenging problem of enzymatic replication of such base pairs, very few of these artificial base pairs have been efficiently and selectively replicated. The resulting base pairs are of the following categories: (a) unnatural hydrogen bonding pattern as well as shape complementarity, (b) hydrophobic forces, (c) non-nucleoside base substitution, (d) and even covalent cross-linking.
Artificial Base-Pairs Based on Hydrogen Bonding Interaction
- Purine Base Analogues
- Size Expanded Base Pairs
- Alkynyl Extended Base Pairs
- Four Hydrogen Bonded Base Pair
- Pyridone Based Base-Pairs
- Halogen Bonded Base Pair
These C nucleosides have been shown to form Watson-Crick-like H-bonds (Figure 1.15) just like the natural bases. Minakawa and Matsuda et al.,35 have reported two pairs of extended DNA base pairs capable of forming four hydrogen bonds (Figure 1.16).
A Complete Set of Emissive RNA Alphabet
Later, they have described the use of size-enlarged versions of adenosine (rxA) and uridine (rxU) as a new set of steric probes to investigate the RNAi mechanism. Therefore, they have reported the design and synthesis of isomorphic base analogs, a complete set of ribonucleoside alphabets, consisting of highly emissive purine (thA, . thG) and pyrimidine (thU, thC) analogs (Figure 1.20).
Non-Hydrogen-Bonded Nucleosides
However, it has been observed that the base pair (1,104) destabilizes the duplex by 15 oC compared to the natural A:T base pair (Figure 1.26). They observed that the duplex DNA containing the bases forms cross-links between the strands upon γ-radiolysis under anaerobic conditions (Figure 1.28).
Non-Nucleosidic Base Surrogates
However, they did not measure the thermodynamic stability of the duplexes containing these non-nucleocytic base surrogates. The phenanthroline derivatives led to the stabilization of the duplex containing the abasic site as the linker length increased.
Applications of Unnatural Nucleobase Analogues
- Applications in Studying the Duplex Stability
- Applications in Studying the Abasic Duplex Stability
- Applications in Studying the Photophysical Processes in Fluorescent Unnatural DNA
It is concluded that stabilization of the basic site by both types of polyaromatic hydrocarbons as non-nucleoside base substitutes results from stacking interactions between polyaromatic residues and base pairs adjacent to the basic site.74 Shimidzu et al. It is observed that the fluorescence emission from the duplex with an inserted G-strand is very strong and clearly distinguishable from the weak fluorescence of the wild-type single-stranded duplex.
Summary and Future Prospect
The field of designing analogs of fluorescent nucleoside bases with high solvofluorochromicity is also flourishing, and much more progress in this direction is expected in the near future. In the near future, much more progress is expected in expanding the genetic alphabet and using it to drive the synthesis of unnatural proteins, with the hope of translating the expanded genetic alphabet into an expanded genetic code that will one day create a synthetic organism with the ability to encode proteins with new physical - chemical properties.
The concept for the design of various types of fluorescent nucleoside base analogs and the application of such fluorescent oligonucleotide probe containing fluorescent nucleoside base analogs may receive special attention in the future of gene detection technology. It is expected that tuning the chromospheres' structure to obtain predictable photophysical property may allow one to create fluorescent nucleoside analogs to be applicable for bioimaging as well as for gene detection technology.
Introduction
In the design of non-hydrogen bonding base pairs, researchers have mainly concentrated on factors such as π-stacking, hydrophobicity, steric shape mimicry, and in few cases the dipole moment, etc., in the stabilization of DNA duplex.14 Furthermore, in order to generate such new and interesting nucleobase pairs that are applicable to various DNA-based applications, several methods have evolved. As for example, Cu(I)-catalyzed azide-alkyne cycloaddition reaction15a has only gained attention in recent years in nucleic acid chemistry for the design of artificial nucleobases with interesting.
Schematic representation of 1,3-dipolar cycloaddition between an azide and an alkyne
Applications of Click Reaction in Nucleic Acids Chemistry
- Click Oligonucleotide Labelling
Most reported protocols involve the use of CuI or the reduction of stable sources of Cu(II), such as CuSO4, with sodium salts or rationing of Cu(II)/Cu(0) species to generate active Cu(I). -catalyst which catalyzes the click reaction. Some important applications of click chemistry in the field of nucleic acids are described below, which include:
Different methods of labeling of oligonucleotides
Nowadays, the CuAAC reaction is used routinely for postsynthetic labeling of oligonucleotide probes suitable for use in DNA analysis. Postsynthetic reaction with two, non-fluorescent 3-azido-7 hydroxycoumarin via click reaction gave the bis-coumaryl-labeled ODN probe (Scheme 2.3).
Post synthetic labelling of bis-propargyl DNA with coumarin derivative via click reaction
Labelling of oligonucleotides with carbohydrates via click chemistry
Oligonucleotide Ligation and Cyclization
Schematic representation of oligonucleotide ligation
Modification of Sugar-Phosphate Backbone with Click Triazole Unit The click chemistry has not only been utilized to modify the natural nucleobases
The search for other alternatives to replace the phosphodiester bond was to eliminate the negative charge in order to improve uptake by cells and increase the biological half-life.26 In this context, few examples have been reported in the literature where 1. Ring 2,3 -triazole has been used as a replacement for phosphodiester bonds. The thermal melting temperatures were found to be 20 oC higher than that of the natural control DNA, which was explained on the basis of the absence of any repulsive interactions between the neutral triazolyl backbone and the anionic phosphate backbone of the natural target .
Modification of Naturally Occurring Bases
Isobe et al.31 reported the design and synthesis of 10-mer triazole-linked analog of DNA (TLDNA) using click chemistry (Scheme 2.8). The chain extension reaction was done using microwave-assisted copper-catalyzed Huisgen cycloaddition and the artificial 10-mer TLDNA thus formed was able to form a stable duplex with the complementary strand of natural DNA.
Cu(I)-catalyzed ‘click’ reaction between propargylated bases and the azido sugar
Synthesis of 2-(1,2,3-triazolyl)adenosine derivatives 2.49 and 2.51
Synthesis of 6-(1,2,3-triazolyl)adenosine derivative 2.54
Synthesis and biological properties of C-2 triazolyllinosine derivatives were demonstrated by Lakshman et al.35 (Scheme 2.12).
Synthesis of C-2 triazolylinosine derivatives using the Cu(I) catalysed click reaction
Modification of the Nucleobases
Thus, the oligonucleotides containing 1-ethynyl-2-deoxy-β-D-ribofuranose were reacted with a series of azides using the Cu(I)-catalyzed click reaction to prepare ODNs containing artificial triazole nucleobases (Scheme 2.14). Similarly, Ermolat'ev et al.41 (Scheme 2.16) and Broggi et al.42 (Scheme 2.17) reported that the 1,3-cycloaddition can be achieved in high yield within minutes using microwave irradiation.
Synthesis of 1,2,3-Triazolo Nucleoside Analogues by (CuAAC) and (RuAAC)
Background
Furthermore, in many of the examples the triazole moiety has been used as a linker either to connect the fluorophore to the natural bases or as a substitute for phosphodiester linkage. The focus of the synthesis of these triazolyl nucleobases was mainly of synthetic interest or to generate a set of biologically active nucleosides.
Objective
- Synthesis of Triazolyl Donor/Acceptor Nucleosides
- Structural Characterization
- Study of Photophysical Properties
Excitation at absorption maxima of each solvent shows structureless emission at around 333 nm with a quenched appearance of fluorescence as the polarity of the solvent increases (Figure 2.11a-b). The absorption is characterized by a hypsochromic shift (2-5 nm) and hyperchromism as the polarity of the solvent increases.
General Experimental
Mass spectra were recorded using WATERS MS system, Q-tof premier and data analyzed using Mass Lynx 4.1. IR spectra were recorded in KBr or pure on a Perkin Elmer Spectrum an FT-IR spectrometer.
Crystallographic Description and ORTEP Diagram
Synthesis and Characterization
After completion of the reaction, monitored by TLC, the reaction mixture was partitioned between water and EtOAc. The organic layer was washed with water and then brine, dried over Na 2 SO 4 and then concentrated.
General procedure for the synthesis of triazolyl donor/acceptor aromatic nucleosides via “Click” reaction
General Procedure for toluoyl deprotection of triazolyl donor/acceptor aromatic nucleosides
General Procedure for Toluoyl Deprotection of Triazolyl Donor/Acceptor Aromatic Nucleosides. mmol) of compound 2.93α, 144.0 mg (0.448 mmol) of compound 2.104αααα is isolated as a white solid.
Photophysical Studies of the Nucleosides
The time-correlated single photon counting (TCSPC) method was used to calculate the lifetime data. Lifetime data (Global Analysis) were calculated using a software package with range fitting channels.
Introduction
Importance of Tetrazole Molecules
Due to such attractive and important structural features and biological activity, tetrazoles have been successfully used to design various types of commercial drug candidates.
Synthetic Procedures for the Preparation of Tetrazole Compounds
Schematic representation of conversion of cyano moiety to the tetrazole ring
- Application of Tetrazoles as Amino Acid Analogues in Peptidomimetics
- Application of Tetrazole as Nucleosidic Base Analouges and Linkers
- Background
- Objective
- Results and Discussion
- Synthesis of the Tetrazolyl Donor/Acceptor Nucleosides
- Spectral Characterization of Tetrazolyl Nucleosides
- Study of Photophysical Properties
- CONCLUSION
- Experimental Section
- General Experimental Section
- Crystallographic Description
Pedersen et al.25 have reported the synthesis of the thymidine dimers in which the 2,5-disubstituted tetrazole ring substituted the natural phosphodiester bond (Figure 3.5, 3.29). Efforts are therefore being made to develop synthetic methods for natural O- and N-linked glycosyl amino acids and glycopeptides30 as well as for unnatural C-linked analogues31 to be incorporated into peptides. The synthesis of S-linked glycosylamino acids and thioglycopeptides has also received attention in the past.32 Thus, two series of compounds have been prepared, one containing C-galactosyl and C-ribosyl O-tetrazolylserine, while the other contains S-tetrazolylcysteine derivatives (Figure 3.7).
