Deoxyribo‐ and Ribonucleoside H‐Phosphonates

Most methods for preparing H‐phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation prodecures that it compatible with most common protecting groups.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143703/1/cpnc0206.pd

Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According to the H‐Phosphonate Method

Oligonucleotides can be synthesized by condensing a protected nucleoside H‐phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H‐phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone‐modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H‐phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143594/1/cpnc0304.pd

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation

In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context. In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair. The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important

Evaluation of 2'-hydroxyl protection in RNA-synthesis using the H-phosphonate approach.

A number of different protecting groups were compared with respect to their usefulness for protection of 2'-hydroxyl functions during synthesis of oligoribonucleotides using the H-phosphonate approach. The comparison was between the t-butyldimethylsilyl (t-BDMSi), the o-chlorobenzoyl (o-CIBz), the tetrahydropyranyl (THP), the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp), the 1-(2-chloro-4-methylphenyl)-4-methoxypiperidin-4-yl (Ctmp), and the 1-(2-chloroethoxy)ethyl (Cee) protecting groups. All these groups were tested in synthesis of dodecamers, (Up)11U and (Up)11A, using 5'-O-(4-monomethoxytrityl) or (4,4'-dimethoxytrityl) uridine H-phosphonate building blocks carrying the respective 2'-protection. The performance of the t-BDMSi and o-CIBz derivatives were also compared in synthesis of (Up)19U. The most successful syntheses were clearly those where the t-butyldimethylsilyl group was used. The o-chlorobenzoyl group also gave satisfactory results but seems somewhat limited with respect to synthesis of longer oligomers. The results with all tested acetal derivatives (Fpmp, Ctmp, Cee, THP) were much less successful due to some accompanying cleavage of internucleotidic H-phosphonate functions during removal of 5'-O-protection (DMT)

Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According to the H

Oligonucleotides can be synthesized by condensing a protected nucleoside H‐phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H‐phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone‐modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H‐phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143625/1/cpnc0304.pd