Category: Chemistry

Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands

Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2′-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application.(European Journal of Medicinal Chemistry.,Volume 121, 4 October 2016, Pages 132-142.)

Solid-phase synthesis of 5′-triantennary N-acetylgalactosamine conjugated antisense oligonucleotides using phosphoramidite chemistry

A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5′-end of antisense oligonucleotide using phosphoramidite chemistry. Conjugation of the 5′-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5′-triantennary GalNAc cluster conjugated ASOs and helps understand the structure–activity relationship for targeting hepatocytes with oligonucleotide therapeutics.(Bioorganic & Medicinal Chemistry Letters.,Volume 25, Issue 19, 1 October 2015, Pages 4127-4130.)

MMT, Npeoc-protected spermine, a valuable synthon for the solid phase synthesis of oligonucleotide oligospermine conjugates via guanidine linkers

Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea coupling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised in eight steps from spermine. MMT group was used as coupling monitor and resulting oligomeric spermines were conjugated to oligonucleotides. (Bioorganic & Medicinal Chemistry., Volume 19, Issue 6, 15 March 2011, Pages 1972-1977.)

Preparation and characterization of octenyl succinic anhydride modified agarose derivative

Agarose was successfully modified with octenyl succinic anhydride (OSA) and the factors affecting OSA modifying process were studied. The degree of substitution (DS) could be regulated from 0.02 to 0.21 by changing the reaction condition, simultaneously the molecular weight of the OSA-agarose (OSAG) varied from 342 kD to 483 kD. FT-IR spectrum of the OSAG at 1734 cm-1 and 1576 cm-1 revealed characteristic absorption peaks of the ester carbonyl groups (CO) and the carboxylate (RCOO-), respectively. NMR spectrum of the OSAG suggested the main substitution occurred at the C-2 in the d-galactopyranose. The SEM image of agarose showed the porous network structure became dense and the fiber became thin after OSA modification. Compared with original agarose, the prepared OSAG showed novel physical properties including low gelling and melting temperature and high transparency. The remaining gelation ability and newly introduced amphiphilic character anticipate potential application as functional polysaccharide materials in foods. (Food Chem. 2019 May 1;279:30-39.)

Superior anti-neoplastic activities of triacontanol-PEG conjugate: synthesis, characterization and biological evaluations

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Triacontanol (TA, C30H62O), abundantly present in plant cuticle waxes and bee waxes, has been found to display promising anti-neoplastic potentials. As a long chain fatty alcohol, TA possesses limited aqueous solubility, which hinders its medicinal application. To overcome its solubility barrier, a polymer prodrug was synthesized through attaching TA to poly ethylene glycol (PEG), using succinic acid as a linker with bifunctional amide and ester bonds. Anti-neoplastic effects of PEG-TA were assessed in LoVo and MCF7 cells, anti-proliferative and apoptosis-inducing activities were subsequently confirmed in mouse xenograft model. Encouragingly, PEG-TA possessed selective anti-cancer ability. It did not exhibit significant cytotoxicity on normal cells. Mechanistic examination revealed inhibition of NF-κB nuclear translocation, suppression on matrix degradation enzyme and down-regulation of angiogenic signaling might contribute to its anti-malignant effects. Pharmacokinetics clearly indicated PEGylated TA (named as mPEG2K-SA-TA) substantially enhanced TA delivery with increased plasma exposure (19,791 vs. 336.25 ng·mL−1·h−1,p < .001), mean residence time (8.46 vs. 2.95 h, p < .001) and elimination half-life (7.78 vs. 2.57 h, p < .001) compared to those of original TA. Moreover, mPEG2K-SA-TA appeared to be safe in preliminary toxicological assessment. PEGylated TA also emerged as a functional carrier to deliver hydrophobic chemotherapeutic agents, since it readily self-assembled to micelles in aqueous solution with a low critical micelle concentration (CMC, 19.1 µg·mL−1). Conclusively, PEG-TA conjugate displayed superior anti-neoplastic activities and low toxicity, as well as facilitated the delivery of other hydrophobic agents, which appeared to be an innovative strategy for cancer therapy.(Drug Deliv. 2018; 25(1): 1546–1559.)

Self-assembled nanoparticles based on chondroitin sulfate-deoxycholic acid conjugates for docetaxel delivery: Effect of degree of substitution of deoxycholic acid

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Hydrophobically-modified polymers based on chondroitin sulfate with different degree of substitution (DS) of deoxycholic acid (DOCA) were developed for docetaxel delivery. Chondroitin sulfate-deoxycholic acid (CSAD) bioconjugates were synthesized via the linker of adipic dihydrazide by amide bond. They were characterized with spherical shape, mean diameter of around 165.2 nm and negative zeta potential (⿿14.87 to ⿿20.53 mV). An increase of DOCA DS reduced size of nanoparticles, while increasing drug loading efficiency. Drug release in vitro showed a triphasic sustained pattern and higher accumulative drug release percentage was observed with increased DS of DOCA on polymer. Self-assemblies with higher DS also had enhanced internalization of nanoparticles and stronger cytotoxicity at the cellular level. The self-assemble nanoparticles demonstrate to be excellent targeting drug delivery systems and the desired therapeutics can be achieved via the alteration of DS.(Colloids and Surfaces B: Biointerfaces. Volume 146, 1 October 2016, Pages 235-244.)

Amphiphilic polysaccharides as building blocks for self-assembled nanosystems: molecular design and application in cancer and inflammatory diseases

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Polysaccharides (PSs) have been extensively studied in healthcare applications; here, we focus our attention on their use as components of nanomaterials in the management of cancer and inflammatory pathologies. Key advantages of PSs are easy availability, general biodegradability and biocompatibility, low or negligible toxicity, often a low immunogenicity and finally an ease of chemical modification. Here, we pay particular attention to the large family of amphiphilic PS derivatives (AMPDs); they are synthesized by modifying hydrophilic PSs with a variety of hydrophobic groups, which allow the constructs to self-assemble into various nanostructures in aqueous solution. This review focuses on AMPD-based self-assembled nanoparticles, from the chemical synthesis of AMPDs, through nanoparticle preparative strategies, to the most recent applications in cancer and inflammation management, including therapeutics, imaging and theranostics. We also offer an overview, which we feel lacks in the current literature, of the relation between the nature of the hydrophilic PSs and that of the hydrophobic components, of linkers, targeting groups and cross-linkers, and of the actual properties and in vivo fate of AMPD-based nanoparticles. Finally, we believe that this comprehensive insight into the possible effects of AMPDs’ structural components on the performance of nanosystems, can provide criteria for a rational and molecular level-based design of AMPDs. (Journal of Controlled Release. Volume 272, 28 February 2018, Pages 114-144.)

‘Dual’ peptidyl-oligonucleotide conjugates: Role of conformational flexibility in catalytic cleavage of RNA

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Traditional therapeutic interventions against abnormal gene expression in disease states at the level of expressed proteins are becoming increasingly difficult due to poor selectivity, off-target effects and associated toxicity. Upstream catalytic targeting of specific RNA sequences offers an alternative platform for drug discovery to achieve more potent and selective treatment through antisense interference with disease-relevant RNAs. We report a novel class of catalytic biomaterials, comprising amphipathic RNA-cleaving peptides placed between two RNA recognition motifs, here demonstrated to target the TΨC loop and 3′- acceptor stem of tRNAPhe. These unique peptidyl-oligonucleotide ‘dual’ conjugates (DCs) were created by phosphoramidate or thiol-maleimide conjugation chemistry of a TΨC-targeting oligonucleotide to the N-terminus of the amphipathic peptide sequence, followed by amide coupling of a 3′-acceptor stem-targeting oligonucleotide to the free C-terminal carboxylic acid functionality of the same peptide. Hybridization of the DCs bearing two spatially-separated recognition motifs with the target tRNAPhe placed the peptide adjacent to a single-stranded RNA region and promoted cleavage within the ‘action radius’ of the catalytic peptide. Up to 100% cleavage of the target tRNAPhe was achieved by the best candidate (i.e. DC6) within 4 h, when conformational flexibility was introduced into the linker regions between the peptide and oligonucleotide components. This study provides the strong position for future development of highly selective RNA-targeting agents that can potentially be used for disease-selective treatment at the level of messenger, micro, and genomic viral RNA. (Biomaterials 112 (2017) 44e61)