Post-synthetic Conjugation Research Articles

Chemistry of Peptide-Oligonucleotide Conjugates: A Review.

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.

Changed reactivity of secondary hydroxy groups in C8-modified adenosine - lessons learned from silylation.

Synthesis of site-specifically modified oligonucleotides has become a major tool for RNA structure and function studies. Reporter groups or specific functional entities are required to be attached at a pre-defined site of the oligomer. An attractive strategy is the incorporation of suitably functionalized building blocks that allow post-synthetic conjugation of the desired moiety. A C8-alkynyl-modified adenosine derivative was synthesized, reviving an old synthetic pathway for iodination of purine nucleobases. Silylation of the C8-alkynyl-modified adenosine revealed unexpected selectivity of the two secondary sugar hydroxy groups, with the 3'-O-isomer being preferentially formed. Optimization of the protection scheme lead to a new and economic route to the desired C8-alkynylated building block and its incorporation in RNA.

Development of functionalized SYBR green II related cyanine dyes for viral RNA detection

Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demand larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts.

Harnessing combinational phototherapy via post-synthetic PpIX conjugation on nanoscale metal-organic frameworks.

Nanomaterial-mediated phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is an effective anticancer intervention that relies on light activation of photoactive nanomaterials localized in tumors. Recently, combinational PDT/PTT offered a practical pathway to relieve resistance of monotherapy, surmount undesirable side effects and provide a synergistic effect to enhance phototherapeutic efficiency. Herein, we report a facile strategy to integrate protoporphyrin IX (PpIX) into nanoscale metal-organic frameworks (NMOFs) and control their photoactive properties for combinational cancer PDT and PTT. With optimized PpIX conjugation, the as-fabricated nanoparticles (nPCU NPs) exhibit (1) improved dispersibility and particle stability, (2) simultaneous generation of reactive oxygen species and heat effectively through activation by a single-wavelength laser of 635 nm, and (3) maintenance of porosity for further application as drug delivery vehicles. Moreover, in vitro investigation of nPCU NPs demonstrates effective cellular uptake, successful endosomal escape and enhanced phototherapeutic efficacy under both normoxic and hypoxic conditions. Therefore, this study developed a novel type of phototherapeutic nanoplatform with optimal properties for applicable cancer phototherapy.

Enzyme-Cleavable Polymeric Micelles for the Intracellular Delivery of Proapoptotic Peptides.

Peptides derived from the third Bcl-2 homology domain (BH3) renormalize apoptotic signaling by antagonizing prosurvival Bcl-2 family members. These potential peptide drugs exhibit therapeutic activities but are limited by barriers including short circulation half-lives and poor penetration into cells. A diblock polymeric micelle carrier for the BIM BH3 peptide was recently described that demonstrated antitumor activity in a B-cell lymphoma xenograft model [Berguig et al., Mol. Ther. 2015, 23, 907-917]. However, the disulfide linkage used to conjugate the BIM peptide was shown to have nonoptimal blood stability. Here we describe a peptide macromonomer composed of BIM capped with a four amino acid cathepsin B substrate (FKFL) that possesses high blood stability and is cleaved to release the drug inside of target cells. Employing RAFT polymerization, the peptide macromonomer was directly integrated into a multifunctional diblock copolymer tailored for peptide delivery. The first polymer block was made as a macro-chain transfer agent (CTA) and composed of a pH-responsive endosomolytic formulation of N,N-diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA). The second polymer block was a copolymer of the peptide and polyethylene glycol methacrylate (PEGMA). PEGMA monomers of two sizes were investigated (300 Da and 950 Da). Protein gel analysis, high performance liquid chromatography, and coupled mass spectrometry (MS) showed that incubation with cathepsin B specifically cleaved the FKFL linker and released active BIM peptide with PEGMA300 but not with PEGMA950. MALDI-TOF MS showed that incubation of the peptide monomers alone in human serum resulted in partial cleavage at the FKFL linker after 12 h. However, formulation of the peptides into polymers protected against serum-mediated peptide degradation. Dynamic light scattering (DLS) demonstrated pH-dependent micelle disassembly (25 nm polymer micelles at pH 7.4 versus 6 nm unimers at pH 6.6), and a red blood cell lysis assay showed a corresponding increase in membrane destabilizing activity (<1% lysis at pH 7.4 versus 95% lysis at pH 6.6). The full carrier-drug system successfully induced apoptosis in SKOV3 ovarian cancer cells in a dose-dependent manner, in comparison to a control polymer containing a scrambled BIM peptide sequence. Mechanistic analysis verified target-dependent activation of caspase 3/7 activity (8.1-fold increase), and positive annexin V staining (72% increase). The increased blood stability of this enzyme-cleavable peptide polymer design, together with the direct polymerization approach that eliminated postsynthetic conjugation steps, suggests that this new carrier design could provide important benefits for intracellular peptide drug delivery.

Postsynthetic Conjugation of RNA to Carboxylate and Dicarboxylate Molecules

Carboxylates and dicarboxylates are important phosphate mimics. Herein, we present a simple synthetic route for the preparation of RNA carboxylate/dicarboxylate conjugates, starting from suitably protected NH2- and COOH-containing molecules that are coupled to the RNA on the solid support. The key point in our method was the use of trimethylsilylethanol (TMSE-OH) protecting group, which is removed simultaneously with the silyl protecting group on the 2′-OH of the RNA ribose (e.g. t-Butyldimethylsilyl) during the final RNA cleavage/deprotection steps. The usefulness of this method was demonstrated by preparing different RNA-phosphate mimics oligos.

Utilizing the bioorthogonal base-pairing system ofl-DNA to design ideal DNA nanocarriers for enhanced delivery of nucleic acid cargos

DNA nanoconstructs are a potential drug carrier with high biocompatibility. They are promising particularly when therapeutic nucleic acids are the cargos to be delivered, since both the carrier and the cargo are nucleic acids which can be designed, synthesized and assembled in a seamless feature without using post-synthetic conjugation chemistry. However, the unwanted base-paring events between the cargo and the carrier may potentially disturb the desired structure of the cargo-loaded carrier. To address this concern, we propose a DNA nanocarrier composed of L-DNA strands having a bioorthogonal base-paring system. The study presented here provides useful properties of L-DNA as a backbone for the DNA nanocarrier and demonstrates superiority of L-DNA over the natural D-DNA backbone in the delivery of an anti-proliferative aptamer as well as in the construction of the cargo-carrier assembly.

ChemInform Abstract: Synthesis of Oligonucleotide‐Peptide Conjugates for Biomedical and Technological Applications

AbstractReview: 19 refs.

Oligonucleotide-Peptide Conjugates: Solid-Phase Synthesis under Acidic Conditions and Use in ELISA Assays

Here we used solid-phase methods to prepare oligonucleotides carrying fibrin/ filaggrin citrullinated peptides. Post-synthetic conjugation protocols were successfully applied for the synthesis of oligonucleotides carrying small peptides. A stepwise protocol using acid treatment for the final deprotection allowed the preparation of polypyrimidine oligonucleotides carrying longer and arginine-rich peptides. An ELISA-based test using the oligonucleotide-citrullinated peptide conjugates was developed for the detection of anti-citrullinated protein/peptide antibodies in human serum from rheumatoid arthritis patients.

Synthesis of Specifically Modified Oligonucleotides for Application in Structural and Functional Analysis of RNA

Nowadays, RNA synthesis has become an essential tool not only in the field of molecular biology and medicine, but also in areas like molecular diagnostics and material sciences. Beyond synthetic RNAs for antisense, aptamer, ribozyme, and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. This often requires labeling of the RNA with a suitable spectroscopic reporter group. Herein, we describe the synthesis of functionalized monomer building blocks that upon incorporation in RNA allow for selective reaction with a specific reporter or functional entity. In particular, we report on the synthesis of 5′-O-dimethoxytrityl-2′-O-tert-butyldimethylsilyl protected 3′-O-phosphoramidites of nucleosides that carry amino linkers of different lengths and flexibility at the heterocyclic base, their incorporation in a variety of RNAs, and postsynthetic conjugation with fluorescent dyes and nitroxide spin labels. Further, we show the synthesis of a flavine mononucleotide-N-hydroxy-succinimidyl ester and its conjugation to amino functionalized RNA.

Post-Synthetic Modification of DNA by Inverse-Electron-Demand Diels−Alder Reaction

There is currently a tremendous interest in developing bioorthogonal "click chemistry" methods for the modification of biopolymers. Very recently, inverse-electron-demand Diels-Alder reactions have received attention, but to date they have not been applied to nucleic acids. Here we describe the first example of DNA modification by inverse-electron-demand Diels-Alder reaction. We synthesized four different building blocks for 3'-terminal, 5'-terminal, and internal incorporation of norbornene dienophiles into oligonucleotides. These DNA strands were either directly reacted with suitably derivatized tetrazine dienes or first subjected to enzymatic manipulations. We demonstrate that the inverse-electron-demand Diels-Alder reaction allows efficient site-specific post-synthetic conjugation, often at a 1:1 stoichiometry, without any side reaction. The reaction works in aqueous media at room temperature, and no transition metals are required. Both short chemically synthesized oligonucleotides and long enzymatically amplified DNA strands were successfully conjugated.

Organometallic Activation of a Fluorogen for Templated Nucleic Acid Detection

A nucleic acid detection scheme that employs DNA-mediated delivery of an organomercury activator to unmask a fluorophore is described. The approach relies on adjacent hybridization of two oligonucleotide conjugates containing organomercury and caged rhodamine functionalities. Postsynthetic conjugation of amino-modified DNAs enabled efficient preparation of these probes. Complementary DNA templates yielded fluorescence signals arising from metal-assisted rhodamine uncaging.

Oligonucleotide conjugation to a cell-penetrating (TAT) peptide by Diels–Alder cycloaddition

Modifed oligonucleotides are routinely employed as analytical probes for use in diagnostics, e.g. in the examination of specific RNA sequences for infectious diseases, however, a major limiting factor in oligonucleotide-based diagnostics is poor cellular uptake of naked oligonucleotides. This problem can be overcome by covalent attachment of a so-called 'cell-penetrating peptide' to form an oligonucleotide peptide conjugate. Stepwise solid phase synthesis of such a conjugate is difficult and expensive due to the conflicting chemistries of oligonucleotides and peptides. A simple approach to overcome this is post-synthetic conjugation. Diels-Alder cycloaddition is an attractive methodology for oligonucleotide peptide conjugation; the reaction is fast, chemoselective and the reaction rate is greatly enhanced in aqueous media - ideal conditions for biological moieties. An oligodeoxyribonucleotide sequence has been derivatised with a series of dienes at the 5'-terminus, using a series of unique dienyl-modified phosphoramidites, and investigation into the effect of diene type on the efficiency of conjugation, using Diels-Alder cycloaddition with a maleimido-derivatised cell-penetrating (TAT) peptide, has been performed. This led to the observation that the optimal diene for conjugation was cyclohexadiene, allowing conjugation of oligodeoxyribonucleotides to a cell-penetrating peptide by Diels-Alder cycloaddition for the first time.

Application of 2-Amino-6-Vinylpurine As an Efficient Agent for Conjugation of Oligonucleotides

Attempts have been made to conjugate a variety of molecules with oligonucleotides to achieve useful functions. In this study, we have established a new efficient method for post-synthetic conjugation of oligonucleotides with the use of the 2-amino-6-vinylpurine nucleoside. Amino nucleophiles form the corresponding conjugates under acidic conditions, whereas thiol nucleophiles reacted efficiently under alkaline conditions. Thus, glutathione and HS-Cys-(Arg)8without protecting groups were efficiently conjugated to the 2-amino-6-vinylpurine–bearing ODN under alkaline conditions. The use of 2-amino-6-vinylpurine as an agent for conjugation is advantageous in that it is stable during the reaction and may be applied to conjugation of ODNs with multiple functional molecules.

Synthesis and Applications of Oligonucleotide—Carbohydrate Conjugates

AbstractFor Abstract see ChemInform Abstract in Full Text.

Large‐Scale Preparation of Conjugated Oligonucleoside Phosphorothioates by the High‐Efficiency Liquid‐Phase (HELP) Method

A new process for the preparation of large amounts of thioated oligonucleotides in a quasi-classical solution condition is described. This method takes advantage of the use of polyethylene glycol as a soluble, inert support during synthesis. The easy purification of intermediates from a moderate excess of reagents allows very high coupling yields and, consequently, efficient production of the long oligonucleotide sequences required for pharmacological applications. The reaction of properly protected and activated oligonucleotides with high-molecular-weight polyethylene glycols allows a convenient procedure for the postsynthetic conjugation of those biopolymers in solution. The oligonucleotides are modified at the 5' terminus using a liquid-phase procedure with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. The use of N, N'-disuccinimidyl carbonate for the activation of the terminal OH groups of the PEG was preferred.

Preparation of bifunctional silica polymer support for the synthesis of 3'-labeled oligonucleotides

Preparation of a silica polymer support with a linker arm containing protected amino and hydroxy groups is described. 0-Benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBT) was used as an efficient activating reagent at several steps of the polymer synthesis. This bifunctional support is suitable for the preparation of 3'-amino-modified oligonucleotides for post-synthetic conjugation, as well as for the direct solid phase synthesis of 3'-labeled oligonucleotides. The fluorescein-modified silica polymer was synthesized.

Synthesis and Applications of OligonucleotideCarbohydrate Conjugates

Nowadays, oligonucleotide-carbohydrate conjugates are used in antisense biotechnology and in the study of glycosylated DNA functioning in vitro. The application of mono- and disaccharide phosphoramidites, solid-phase supports with immobilized carbohydrates, glycosylated nucleoside phosphoramidites, and postsynthetic conjugation of reactive sugar derivatives with oligonucleotides for preparation of oligonucleotide-carbohydrate conjugates have been systematically studied. The advantages and disadvantages of these approaches are considered. Possible strategies for synthesis of glycoclusters with different topologies conjugated to DNA are discussed. Applications of oligonucleotide-carbohydrate conjugates are highlighted. Studies of interactions of glycosylated oligonucleotides with proteins and effective cell-specific delivery of oligonucleotide-carbohydrate conjugates are discussed.

Postsynthetic conjugation of biopolymers with high molecular mass poly(ethylene glycol): optimization of a solution process tested on synthetic oligonucleotides.

The reaction of oligonucleotides with high molecular weight monomethoxy poly(ethylene glycol)s (MPEGs) has been tested to set up a convenient procedure for the postsynthetic conjugation in solution of biopolymers. A first oligonucleotide was previously modified in 5', using a liquid-phase procedure, with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. Two procedures commonly utilized for the activation of the terminal OH groups of the MPEG were evaluated, that is, the reaction with pNO(2)-phenyl chloroformate and with N,N'-disuccinimidyl carbonate. Both water as well as organic solution conditions were employed and compared. In a second test, a 3'-amino modified, commercial 20-mer was also conjugated in a microscale condition to verify the effect of size and concentration of MPEG on the postsynthetic conjugation of these biopolymers under troublesome synthetic conditions.

Rapid Routes of Synthesis of Oligonucleotide Conjugates from Non-Protected Oligonucleotides and Ligands Possessing Different Nucleophilic or Electrophilic Functional Groups

Optimized methods are described for post-synthetic conjugation of non-protected oligodeoxyribonucleotides to different ligands. Methods for the terminal functionalization of oligonucleotides by amino, sulfhydryl, thiophosphate or carboxyl groups using different chemical reactions and linkers in both organic and aqueous media are described and compared. Experimental conditions for subsequent coupling of ligands containing aliphatic and aromatic amines, aromatic alcohols, carboxylic, sulfiydryl, alkylating, aldehydic and other reactive nucleophilic and electrophilic groups to oligonucleotides were established, including covalent linkage to other oligonucleotides.