Chemistries that Facilitate Nanotechnology Kim E. Sapsford,† W. Russ Algar, Lorenzo Berti, Kelly Boeneman Gemmill,‡ Brendan J. Casey,† Eunkeu Oh, Michael H. Stewart, and Igor L. Medintz*,‡ †Division of Biology, Department of Chemistry and Materials Science, Office of Science and Engineering Laboratories, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, United States ‡Center for Bio/Molecular Science and Engineering Code 6900 and Division of Optical Sciences Code 5611, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States College of Science, George Mason University, 4400 University Drive, Fairfax, Virginia 22030, United States Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Sacramento, California 95817, United States Sotera Defense Solutions, Crofton, Maryland 21114, United States

Functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology.

Diels–Alder “click” reactions: recent applications in polymer and material science

Over the past two decades, gene therapy has garnered tremendous attention and is heralded by many as the ultimate cure to treat diseases such as cancer, viral infections, and inherited genetic disorders. However, the therapeutic applications of nucleic acids extend beyond the delivery of double-stranded DNA and subsequent expression of deficient gene products in diseased tissue. Other strategies include antisense oligonucleotides and most notably RNA interference (RNAi). Antisense strategies bear great potential for the treatment of diseases that are caused by misspliced mRNA, and RNAi is a universal and extraordinarily efficient tool to knock down the expression of virtually any gene by specific degradation of the desired target mRNA. However, because of the hurdles associated with effective delivery of nucleic acids across a cell membrane, the initial euphoria surrounding siRNA therapy soon subsided. The ability of oligonucleotides to cross the plasma membrane is hampered by their size and highly negative charge. Viral vectors have long been the gold standard to overcome this barrier, but they are associated with severe immunogenic effects and possible tumorigenesis. Cell-penetrating peptides (CPPs), cationic peptides that can translocate through the cell membrane independent of receptors and can transport cargo including proteins, small organic molecules, nanoparticles, and oligonucleotides, represent a promising class of nonviral delivery vectors. This Account focuses on peptide carrier systems for the cellular delivery of various types of therapeutic nucleic acids with a special emphasis on cell-penetrating peptides. We also emphasize the clinical relevance of this research through examples of promising in vivo studies. Although CPPs are often derived from naturally occurring protein transduction domains, they can also be artificially designed. Because CPPs typically include many positively charged amino acids, those electrostatic interactions facilitate the formation of complexes between the carriers and the oligonucleotides. One drawback of CPP-mediated delivery includes entrapment of the cargo in endosomes because uptake tends to be endocytic: coupling of fatty acids or endosome-disruptive peptides to the CPPs can overcome this problem. CPPs can also lack specificity for a single cell type, which can be addressed through the use of targeting moieties, such as peptide ligands that bind to specific receptors. Researchers have also applied these strategies to cationic carrier systems for nonviral oligonucleotide delivery, such as liposomes or polymers, but CPPs tend to be less cytotoxic than other delivery vehicles.

Peptide Vectors for the Nonviral Delivery of Nucleic Acids

CuI-catalyzed alkyne–azide cycloaddition provides 1,4-disubstituted 1,2,3-triazoles with such efficiency and scope that the transformation has been described as “click” chemistry. An overview of the mechanism of this remarkable reaction is presented as a means to explain the myriad of experimental results, particularly the various methods of catalyst generation, solvent and substrate effects, and choice of base or ligand. Both solution-phase and solid-phase results are comprehensively examined. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

CuI‐Catalyzed Alkyne—Azide “Click” Cycloadditions from a Mechanistic and Synthetic Perspective

Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake mechanisms and pharmacokinetic properties.

https://www.future-science.com/doi/pdf/10.4155/tde.13.47

Oligonucleotide conjugates for therapeutic applications.

Chemical Strategies for the Synthesis of Peptide−Oligonucleotide Conjugates

The use of synthetic oligonucleotides and their mimics to inhibit gene expression by hybridizing with their target sequences has been hindered by their poor cellular uptake and inability to reach the nucleus. Covalent postsynthesis or solid-phase conjugation of peptides to oligonucleotides offers a possible solution to these problems. As feasible chemistry is a prerequisite for biological studies, development of efficient and reproducible approaches for convenient preparation of peptide-oligonucleotide conjugates has become a subject of considerable importance. The present review gives an account of the main synthetic methods available to prepare covalent conjugation of peptides to oligonucleotides.

Chemical Strategies for the Synthesis of Peptide—Oligonucleotide Conjugates

A concise synthesis of oligonucleotide 5'-peptide-conjugates via copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition in aqueous solution is described. Synthesis of reagents was accomplished by on-column derivatization of corresponding peptides and oligonucleotides. This method is well suited for the preparation of peptide-oligonucleotide conjugates containing 1,2,3-triazole linkage between the 5'-position of an oligonucleotide and the N-terminus of a peptide.

Concise Synthesis of Triazole-Linked 5'-Peptide-Oligonucleotide Conjugates by Click Chemistry.

The invention provides a solid-phase synthesis method of a polypeptide structural domain binding DNA. The method comprises the steps as follows: step 1, resin swelling; step 2, removal of fluorenylmethoxycarbonyl protecting groups; step 3, a coupling reaction of amino acid; step 4, polypeptide cutting; step 5, separation and purification; step 6, representation of polypeptide; step 7, interactionof peptide and DNA. By means of the method, the defects of solid-phase synthesis of polypeptide currently are overcome, the coupling efficiency and synthesis yield are increased, and the synthesis yield reaches 60% or above. The five polypeptides P1, P2, P3, P4 and P5 are synthesized by the method; all the synthesized peptides are separated and purified with reversed phase high-performance liquidchromatography, target peptides are represented by mass spectrum, results prove that the synthesized products are corresponding target peptides, and the purity is 95% or above. Besides, the synthesized polypeptides have different structures, and the sequence structure of P3 can provide basic information and support for design of polypeptide medicines taking specific DNA as targets.

Duan Qunpeng

Papers

Chemical Strategies for the Synthesis of Peptide−Oligonucleotide Conjugates

Chemical Strategies for the Synthesis of Peptide—Oligonucleotide Conjugates

Concise Synthesis of Triazole-Linked 5'-Peptide-Oligonucleotide Conjugates by Click Chemistry.

Solid-phase synthesis method of polypeptide structural domain binding DNA