Surajit Sinha

Unambiguous MO capture probe-based detection of nucleic acid sequences using PO ₃ ⁻ mass peaks.

Chemically modified oligonucleotide synthesis has emerged as a major driving force in modern nucleic acid chemistry. Recently, phosphorodiamidate morpholino oligonucleotide (PMO) has gained much interest, for the treatment of rare disease like Duchenne Muscular Dystrophy (DMD), warranting its further exploration. In this vein, the synthesis of carbocyclic analog of PMO, called phosphorodiamidate piperidino oligomers (PPOs) was reported earlier. However, the use of chlorophosphoramidate chemistry for the synthesis of PPOs limited its overall yield and applicability. Herein, we explored the amidite chemistry for the synthesis of tert ‐butyl‐protected (5′‐ ^t Bu)‐piperidino phosphoramidite monomers. Furthermore, their use for the synthesis of full length 19‐mer PPO sequence and diverse chimeric backbones like PPO‐PMO, PPO‐psDNA, and PPO‐DNA chimeras in an automated oligo synthesizer are also investigated. Thermal melting ( T _m ) and circular dichroism (CD) analyses of their duplexes with complementary RNA strand are also reported. Interestingly, we observed a reasonable Tm in our newly reported chimeras which indicated a strategy to deal with the destabilizing effects of PPO for their potential applications in ASOs. CD spectra indicated non‐canonical helical conformations for pure PPO and altered B‐form features in chimeras, underscoring their structural versatility for RNA‐targeted therapeutics.

Morpholino dimer chlorophosphoramidates and phosphoramidites were synthesized with different neutral-linkages (diamidate, urea, carbamate, methylene, glycine) and incorporated in short PMOs, supporting future antisense technology development.

The C-5 position of pyrimidines is a known modification site for tuning the biophysical properties of oligonucleotides and nucleic acid duplexes. Consecutive C-5 phenyl triazole motifs in morpholino oligonucleotides boost duplex stability with RNA.

The clinical importance of iboga alkaloids lies in their efficacy in reversing drug addiction and modulating drug tolerance. However, due to safety concerns, their use is restricted to appropriate medical supervision. These alkaloids often cause severe psychedelic effects due to differential binding to various brain receptors and cardiotoxicity by blocking the human ether-a-go-go-related gene (hERG) potassium channel. To create safer analogs, our group previously synthesized various benzofuran-containing iboga analogs with good opioid binding selectivity and excellent antinociceptive property. However, the present manuscript disclosed a step-economical and cost-effective synthesis of modified ibogaine/ibogamine analogs (C1, C2, C3, & C4) with the bioisosteric replacement of the indole scaffold with a benzofuran moiety and compared their antinociceptive/anxiolytic activity with their natural counterparts. Among the synthesized iboga analogs, the Endo-iboga analogs (C2 & C4) not only exhibited notable anti-inflammatory and oxidative stress-relieving activity but also effectively improved restricted locomotor activity in a formalin-induced acute pain model in mice. C2 & C4 significantly elevated the levels of inhibitory neurotransmitters (GABA and dopamine) and brain-derived neurotrophic factor (BDNF) compared to their respective Exo-counterparts or previously published benzofuran-containing iboga analogs lacking the tetrahydroazepine ring. Among C2 and C4, the latter exhibited superior cytocompatibility in C2C12 cells (IC₅₀ = 235 μM) and showed no adverse effects on rat hearts during in vivo ECG tests, indicated by no significant QTc prolongation. The hERG Fluorescence Polarization assay identified C4 (IC₅₀ = 21.25 ± 4.89 μM) as the compound with the lowest risk for hERG channel blockade as compared to C2 (19.31 ± 4.02 μM). C4 also showed no alteration of the serum potassium level in rats and, moreover, significantly acted as a potent KOR agonist in C6 cells and a MOR antagonist in MCF7 cells. Apart from MOR and KOR, C4 acted as a weak agonist for 5HT2A and a weak antagonist for the σ1 receptor. In silico binding further confirmed its relatively higher binding energies with reduced stability within the receptor active sites. Overall, the development of bioisosteric iboga analogs, particularly C4, demonstrated significant potential for acute pain management without notable cardiotoxicity, representing a potential pharmacophore in pain therapy innovation.

Morpholino-based ASOs with nuclease-resistant backbone and high sequence specificity could modulate the PD-L1/PD-1 axis to overcome T regulatory cell mediated inhibition but poor cell-permeability limits their clinical applications. Here, we develop a guanidinium-modified cell-permeable morpholino specifically targeting PD-L1 in breast cancer. It suppresses PD-L1 expression through effectively mitigating cytokine induced PD-L1 upregulation and enhancing autophagic flux. Notably, the ASO synergistically with durvalumab (anti-PD-L1 antibody) disrupt, hyperglycosylated PD-L1 by mRNA-level interference and also reduced PD-1⁺ T-cell populations. Importantly, systemic and intra-tumoral administration of ASO demonstrated no abnormalities in cardiac tissues which show no off-target toxicity but it reprogrammed the tumor microenvironment by upregulating inducible nitric-oxide synthase and suppressed regulatory cells, collectively enhancing antitumor immune responses. Together, these findings establish morpholino ASO as a promising targeting therapeutic strategy to overcome tumor-induced immunosuppression, opening new avenues for precision treatment in breast-cancer.

In this manuscript, we report the synthesis of 3′-tert-butyl phosphorodiamidite morpholino monomers and demonstrate their straightforward incorporation into DNA analogues using the standard 3′-phosphoramidite synthesis cycle, without requiring additional steps. The couplings of 3′-tert-butyl phosphorodiamidite morpholino monomers in solid-phase synthesis were optimized using different types of activators to achieve the best yield. Thermal melting studies revealed that the Phosphorodiamidate Morpholino (PMO) incorporated DNA analogue was found to have a higher melting temperature with complementary DNA compared to natural DNA duplex. Circular dichroism (CD) analysis confirmed that the chimeric oligonucleotides adopted a B-type helical conformation when hybridized with complementary DNA. The simplicity and versatility of this synthetic approach make a variety of modified oligonucleotides with PMO unit (s) readily accessible and open a new avenue for applications in antisense therapy, diagnostics, and nucleic acid nanotechnology.