Design, synthesis and self-assembly study of guanine analogues and their applications

Abstract

In past few years, the researches on G-quadruplex hydrogels have been increased significantly. Significant efforts have been employed for the development, characterization and applications of G-quadruplex hydrogels. G-quadruplex hydrogels have unique supramolecular structure where four guanosine or guanosine analogues form intermolecular H-bonding to form G-quartet and further several G-quartets build columnar structure by π-π interactions. The G-quadruplex structures are stabilized by central metal ions especially by K+ . The boronate esters mediated low molecular weightbased G-quadruplex hydrogels are dynamic, thixotropic, injectable and highly biocompatible. These unique features make it highly applicable towards different biomedical applications such as cell growth matrix, 3D bioprinting, drug delivery system, and enzymatic reactions. Besides G-quadruplex, guanine derivatives can form G-ribbon structure by intermolecular H-bonding. G-ribbons are highly important in dynamic chemistry and nanotechnology. The main objectives of my present studies are:  To design and synthesize ferrocene functionalized redox-active G-quadruplex hydrogels, study their different properties and explore the injectability, 3D printability and water stability of the hydrogels.  To develop several arylboronate esters mediated dynamic hydrogels and application of suitable hydrogel as 3D bioink.  To explore imino-boronate esters mediated pH responsive, biocompatible, dynamic hydrogel for the delivery of an anticancer drug doxorubicin selectively at lower pH. Delivery of drug selectively at lower pH helps to deliver the drug selectively to cancer cells as the pH of the cancer cells are acidic compare to healthy cells.  To synthesize alkane functionalized guanine derivatives and study their selfassembly behavior from crystal structures and examine their antibacterial properties. 1. Redox-active Dynamic Self-Supporting Thixotropic 3D-Printable G-quadruplex Hydrogels In this work, redox-active dynamic G-quadruplex hydrogel was designed, prepared and studied. The samples were characterized by several spectroscopic and microscopic techniques. Redox-active ferroceneboronic acid and guanosine were used to synthesize the hydrogel. The gelation conditions were optimized by varying pH, and different alkali iv metal ions. Dynamic boronate esters formation between ferroceneboronic acid and guanosine imparted thixotropic, injectable and 3D printability nature of the hydrogel. During preparation of the hydrogel, initially ferrocene moiety of ferroceneboronic acid (FeII) oxidized and formed green ferrocenium center (FeIII) by aerobic oxidation. The hydrophilic, green ferrocenium center (FeIII) reinforced the solubility of the gelators to form homogeneous hydrogel (gel (green)). However, ferrocenium center (FeIII) is 17e center so gel (green) spontaneously converted more stable, robust gel (brown) having stable 18e configuration. Gel (brown) was used for 3D printing and creating different 3D structures. Stability of printed shape and water solubility of gel (brown) was compared with weaker gel (green). 2. Arylboronate Esters Mediated Self-healable and Biocompatible Dynamic Gquadruplex Hydrogels as Promising 3D-bioinks. In this work, several dynamic arylboronate ester mediated hydrogels were prepared by using guanosine with phenylboronic acid (GPBA), 4-methoxyphenyl boronic acid (4- GMPBA) and 4-nitrophenyl boronic acid (4-GNPBA). The samples were characterized by several spectroscopic and microscopic techniques. Thixotropic, self-healable and extrudable nature of the hydrogels was achieved by dynamic boronate esters bond. The gelation conditions were optimized by varying concentrations of the gelators, pH, and different alkali metal ions. The hydrogels were prepared in physiological pH. Among the hydrogels, guanosine and phenylboronic acid hydrogel (GPBA) was found mechanically most stable. After all the mechanical experiments, GPBA was used for 3D bioprinting applications. Different 3D structures were printed with GPBA. Self-healable nature of the gel helped to merge the layers during printing. The hydrogel was mixed with adult human fibroblast cells (HDFs) and printed. The cells were survived during printing as the hydrogel acted as protective agent against printing pressure. The gel was highly biocompatible. Almost 98% of incorporated cells were found live after 24 h of cell culture. 3. PEG Functionalized Stimuli Responsive Self-healable Injectable Dynamic Iminoboronate G‑quadruplex Hydrogel for the Delivery of Doxorubicin In this work, dynamic G-quadruplex hydrogel was engineered by using guanosine, 2- formylphenylboronic acid, and 4-Arm PEG-NH2 (G4PEG). The gelation conditions were optimized by varying concentrations of the gelators, pH, and different alkali metal ions. The formation of imino-boronate bonds during the gelation process, secondary supramolecular G-quadruplex structure and the formation of nanofibrillar morphology v were well examined using several spectroscopic and microscopic techniques. The mechanical strength of the hydrogel was investigated by rheological experiments. The hydrogel was injectable and self-healable due to the dynamic nature of the imonoboronate bonds. The dynamic bonds provided distinct shear-thinning and thixotropic properties to the resulting hydrogel with almost 90% recovery of its mechanical strength after four cycles. The pH responsive behavior of the hydrogel was achieved by pH sensitive imino-boronate bonds. Imino-boronates are unstable at acidic pH. To investigate the biocompatibility of the hydrogel, a wide range of hydrogel concentrations were examined by in vitro cell culture experiments using the MCF-7 cell line. After a biocompatibility test of the hydrogel, the anticancer drug doxorubicin was incorporated inside the gel to analyze the drug release profile at different pHs. The release rate of the loaded drug was observed faster in lower pH (pH 4.8) than in physiological pH (pH 7.4). Different release rate of the drug from the drug loaded hydrogel in different pHs was driven by the pH sensitive imino-boronate bonds. The release profile of the drug was slow, sustain and steady. 4. G-Ribbon Self-assembly of Alkane Functionalized Guanine Derivatives and Study of Their Anti-microbial Activity In this work, three alkane functionalized guanine derivatives (compound 1, compound 2, and compound 3) were synthesized. 6-Chloropurine was reacted separately with 1- bromo-3-methylbutane, 1-bromooctane and 1-bromodecane to synthesize compounds 1a, 2a and 3a respectively. After that, compounds 1a, 2a and 3a were hydrolyzed by 2N HCl to get desired compounds 1, 2 and 3, respectively. Compounds 1 and 2 were formed crystals in ethanol. Crystal structures of these compounds suggest G-ribbon A like selfassembly by intermolecular H-bonding. SEM images of the compounds showed microflower like structures. The SEM morphology was driven by intermolecular H-bonding. The compounds were further examined for their antibacterial activity. Several grampositive and gram-negative bacteria were used for the antibacterial study including Escherichia coli, Bacillus cereus, Salmonella typhi and Staphylococcus aureus. All the bacteria were treated with the compounds in the agar plates. The compounds show well antibacterial activity with gram-positive Staphylococcus aureus.