Thiol-Reactive Probes—Chapter 2

Keywords: BBB; Nanoparticles; Drug delivery; Peptides; Solid phase synthesis

Peptide synthesis by prior thiol capture

Richards-Kortum reported a novel approach to conjugating nanoparticles and aptamers []. The aptamer was extended to provide a hybridization site for complementary ONT-coated nanoparticles. GNPs (20 nm) were coated with thiol-modified capture ONTs containing a hexa(ethylene glycol) spacer and a complementary sequence for the aptamer extension, and the extended anti-PSMA aptamer was hybridized to capture ONT-coated GNPs by heating the solution at 70°C for 5 min, followed by incubation at room temperature for 30 min. The resulting aptamer-GNPs showed targeted detection of LNCaP (PSMA+) cells via reflectance imaging. This conjugation strategy has several advantages. First, the negatively charged phosphate groups of the ONTs prevented aggregation of the nanoparticles through electrostatic repulsion, leading to nanoparticle stability, even in high-salt environments. Second, aptamers were easily conjugated to the surfaces of nanoparticles via complementary sequence hybridization with the capture ONTs, which preserved the aptamer integrity and stability during bioconjugation. A PEG spacer between the thiol group and the complementary capture sequence of capture ONT improved the hybridization efficiency by minimizing steric hindrance between the gold surface and the hybridization site. Third, a small number of aptamers was needed for binding to the nanoparticles through the short cheap capture ONTs, unlike thiolated aptamers, in which significant concentrations of aptamers are necessary to coat and stabilize the nanoparticles. Finally, multiplexing capabilities can be obtained by incorporating other types of molecules (targeting, delivery, imaging, or therapeutic agents) into the nanoparticles.

Formation of N-branched oligonucleotides as by-products in solid-phase oligonucleotide synthesis

Cysteine Thiol-Protecting Group - Peptides International

Direct reaction strategies involve nanoparticle surface functionalization with amine, aldehyde, or active hydrogen groups. The functional groups on the nanoparticle surfaces depend on the coating layer applied during the nanoparticle preparation steps. These strategies are particularly suitable for conjugating fluorescence dyes, chelators for nuclear imaging, or drugs. In general, antibody-based or peptide-based targeting moieties that are unmodified prior to conjugation are not natively reactive with these nanoparticles. However, liposomes that include maleimides on their surfaces or GNPs can be used for direct conjugation with thiol group-containing biomolecules, such as antibodies, peptides, or ONTs.

Reversible site-specific tagging of enzymatically synthesized RNAs using aldehyde-hydrazine chemistry and protease-cleavable linkers

Pyridyl disulfide linkers include cleavable disulfide bonds, which facilitates a quantitative evaluation of the reaction efficiency. Jon . calculated the concentration of surface-bound peptide molecules on the nanoparticles by quantifying the released pyridine-2-thione []. In an effort to develop integrin-targeted iron oxide nanoparticles as theranostic agents, amine-modified iron oxide nanoparticles were synthesized, and SPDP was added to convert the primary amine groups on the nanoparticles to sulfhydryl-reactive pyridyldisulfide groups. Conjugation between the thiol group-containing cyclic RGD peptides and the SPDP-activated nanoparticles produced pyridine-2-thione, which was immediately collected by spin filtering (at 100 K). The immobilized cRGD molecules were quantified based on the ultraviolet (UV) absorbance at 343 nm of the collected pyridine-2-thione filtrate, indicating that the average number of conjugated cRGD peptides on each nanoparticle was 0.39 wt%. This linker is useful for enhancing the intracellular gene silencing properties of siRNA. Bhatia . studied the gene-silencing efficacy of siRNA-conjugated QDs using cleavable (sulfo-LC-SPDP) or noncleavable (sulfo-SMCC) cross-linkers []. They immobilized thiol-modified siRNA specific for EGFP to amine-functionalized QDs via sulfo-LC-SPDP or sulfo-SMCC linkers and quantified the EGFP fluorescence intensity. The siRNA attached QDs via the sulfo-LC-SPDP linker provided greater silencing efficiency than those attached via the sulfo-SMCC linker. The cleavable disulfide cross-linker released siRNA from the nanoparticles into the intracellular reducing environment, which affected the interactions between the siRNA and the RNA induced silencing complex (RISC), which is necessary for gene knockdown.

Impurities consist of deletion sequences, incompletely deprotected sequences, truncated sequences and side products of the synthesis process.

combination of solid-phase peptide coupling and in-solution thiol ..

These building blocks were employed in solid-phase PNA synthesis and it was shown that Boc/S- p -methoxybenzyl protection scheme was only satisfactory for the introduction of N-terminal thiol modification while the Fmoc/S-butylthio protected monomer proved to be amenable to elongation.

Facile Preparation of a Thiol-Reactive 18F-Labeling …

9. Kiesewetter DO, Jacobson O, Lang L, Chen X. Automated radiochemical synthesis of [18F]FBEM: A thiol reactive synthon for radiofluorination of peptides and proteins. 2011;69:410-4

Facile Preparation of a Thiol-Reactive 18 F ..

Gold particle bioconjugates are important constructs for cellular imaging [15]. Because of the large scattering cross section of metal particles, individual nanoparticles can be imaged under white-light illumination. This involves the covalent coupling of cysteine in the selected sequence to a particle surface via sulfur-gold bond [16]. This direct coupling affords a simple one-step procedure that produces particles with high surface coverage of peptide. One important aspect of thiol-gold chemistry is that the reaction proceeds at room temperature in aqueous solution.