Synthesis and Properties of Oligonucleotides with Acylamido Substituents

Abstract

This account recounts how a project, initially designed to generate folded peptidyl-DNA motifs for cellular delivery, led to the discovery of acylamido substituents that increase affinity for target strands and improve mismatch discrimination of hybridization probes. Our syntheses of oligonucleotides with acylamido groups involve phosphoramidites of aminodideoxynucleosides as intermediates and amide-forming reactions on solid support, performed after assembly of the DNA chain. The richly functionalized intermediates invite a host of side reactions. Strategies to suppress these side reactions are discussed. In our quest for oligonucleotides with improved biophysical and biological properties we used late, onsupport combinatorial couplings to generate small chemical libraries subjected to mass-spectrometrically monitored selection experiments. The combinatorial approach was used to identify acylamido substituents at the termini that act as caps for DNA duplexes. Though truly bioavailable DNA derivatives were not found, oligonucleotides with a short dendrimer at the 5¢-terminus and a blocked 3¢-terminus were shown to possess improved enzymatic stability, while retaining high affinity for target strands. The on-support synthesis of acylamido DNA gives access to structurally diverse molecules with potential in biomedical applications, including the use in high-fidelity DNA microarrays. 1 Introduction 2 Synthesis of Acylamido-DNA and the Stability of Its Duplexes 2.1 Design and General Synthetic Aspects 2.2 Common Side Reactions 2.3 Combinatorial Syntheses 2.4 Oligodeoxynucleotide-Peptide Hybrids 2.5 Dendrimer-DNA Hybrids with Increased Nuclease Resistance 3 Oligonucleotides with Improved Duplex-Forming Properties 3.1 General Considerations 3.2 Oligodeoxynucleotides with Directly Linked Acylamido Substituents 3.3 Acylamido Caps with Linkers that Can Be Introduced as Phosphoramidites 3.4

Pyrenylmethyldeoxyadenosine: A 3′‐Cap for Universal DNA Hybridization Probes

“…A side product with a 3'-terminal phosphate group was also detected. [22] Oligonucleotides with the residues of 2-anthraquinoyl carboxylic acid, 1-pyrenyl carboxylic acid, cholic acid, and 3-perylenylbutyric acid were thus prepared. A numbering scheme was employed for the oligonucleotides, in which the number denotes the DNA sequence, the letter immediately following this number indicates what linker is being used, and the last two letters are a short-hand for the stacking moiety (e.g., py for pyrene).…”

A 5′‐Cap for DNA Probes Binding RNA Target Strands

“…The stacking moieties chosen are shown in Scheme 2. Some are known to act as molecular caps for DNA:DNA duplexes, [16] others were selected from commercially available carboxylic acids according to the following criteria: 1) the resulting stacking moiety should contain two or more rings for strong stacking interactions with the terminal nucleobases of the DNA:RNA duplex; 2) the acyl residue should contain heteroA C H T U N G T R E N N U N G atoms to avoid excessive lipophilicity and to allow for favorable dipole-dipole interactions with the duplex terminus; and 3) the functional groups should be compatible with oligonucleotide synthesis. The short-hand names of the resulting cap-bearing strands start with the number of the DNA sequence, followed by a lower-case letter (or letters) for the linker and two upper-case letters for the stacking moiety.…”

“…To install a rigidifying ring structure, we based linkers on l-prolinol. Target strands of the sequences [15][16][17][18] were assembled on supports 19-22, through coupling with phosphoramidite 23, followed by oxidation to give 24-27 (Scheme 5). The Fmoc-terminated chain was then deprotected, acylated with the Fmoc-protected residue of a second amino acid, which was then relieved of its Fmoc group, followed by coupling of the activated form of the carboxylic acid of the stacking moiety (usually the active ester of oxolinic acid).…”

“…Substituents may also be introduced at the termini of oligonucleotides. [15,16] Highly decorated hybridization probes use a combination of 5'-and 3'-caps, as well as substituents in the interior of the sequence. [17] Such probes were introduced to aid the massively parallel detection of diverse DNA target sequences on microarrays by smoothing out the differences in duplex stability between Abstract: Detecting short RNA strands with high fidelity at any of the bases of their sequence, including the termini, can be challenging, since fraying, wobbling, and refolding all compete with canonical base pairing.…”

“…[19] We recently reported a cap for the 3'-terminus of DNA hybridization probes for binding RNA target strands, [20] as well as a number of caps for the 5'-terminus of probes binding DNA target strands. [16] However, no cap for the 5'-terminus of hybridization probes that increases fidelity in the recognition of RNA targets had been identified. Caps commercialized for the 5'-terminus of DNA probes binding DNA tar-A C H T U N G T R E N N U N G gets [19e, 21] did not perform well when binding RNA target strands (see below).…”