Improved Synthesis Strategy for N‐Methoxy‐1,3‐oxazinane Nucleic Acids (MOANAs)

Abstract

Dependence of the hydrolysis rate of a series of N-methoxy-2phenyl-1,3-oxazinanes on the Hammett substituent constant of the substituent of the phenyl ring was determined, yielding a reaction constant 1 = À 1.40 � 0.05. Based on this information, 4-(benzoyloxy)benzaldehyde was selected as a protecting group for a new (2R,3S)-4-(methoxyamino)butane-1,2,3-triol phosphoramidite building block. The yield of the preparation of this building block as well as its coupling in automated oligonucleotide synthesis were greatly improved compared to the method reported previously. The 2-[4-(benzoyloxy)phenyl]-1,3-oxazine protection persisted throughout the synthesis of short oligonucleotides but was rapidly removed when the oligonucleotides were released from solid support and dissolved in water.

Watson‐Crick Base Pairing ofN‐Methoxy‐1,3‐Oxazinane (MOANA) Nucleoside Analogues within Double‐Helical DNA

“…While the dissociation rates observed may appear impractically high, we would like to point out that adjusting the pH to physiological (7.40) would cause a rate retardation of nearly two orders of magnitude, effectively “freezing” the equilibrium. Furthermore, we have shown previously [39] that the rate can be controlled in a predictable way by introduction of electron‐withdrawing or ‐donating substituents on the arene moiety of the aldehyde.…”

“…The formation and hydrolysis of N ‐methoxy‐1,3‐oxazinanes is acid‐catalyzed and impractically slow at neutral pH [39] . On the other hand, excessively acidic conditions would prevent hybridization of the oligonucleotide scaffolds into the desired hairpin structures.…”

“…Five hairpin‐forming oligodeoxynucleotides were synthesized by an automated synthesizer, each incorporating the previously reported [39] MOANA building block at position 6 and a variable residue at position 21 (Table 1 ). On completion of chain assembly, the oligonucleotides were deprotected and released from the solid support by conventional ammonolysis.…”

“…The formation and hydrolysis of N ‐methoxy‐1,3‐oxazinanes is acid‐catalyzed and impractically slow at neutral pH. [39] On the other hand, excessively acidic conditions would prevent hybridization of the oligonucleotide scaffolds into the desired hairpin structures. To ensure stability of the hairpins under conditions where incorporation and dissociation of the nucleobase analogues would be reasonably fast, their UV melting profiles were measured at pH 5.5 (100 mM triethylammonium acetate buffer).…”

“…The hairpin oligonucleotide scaffolds ON1a, ON1c, ON1g, ON1t and ON1s (Table 1) were synthesized by an ÄKTA oligopilot plus 10 DNA/RNA synthesizer in 2 μmol scale on CPG support following conventional phosphoramidite strategy. The previously reported [39] 4-(benzoyloxy)benzylidene-protected building block was used for introduction of the (2R The rates of dissociation of the hairpin-aldehyde conjugates were determined by diluting the aforementioned 500 μM stock solutions to 1.0 μM with 200 mM aqueous triethylammonium acetate buffer (pH = 5.5) and analyzing the composition of the solutions thus obtained at appropriate time intervals by RP-HPLC on a Thermo Scientific Hypersil ODS C18 column (250 × 4.6 mm, 5 μm) eluting with a linear gradient of of MeCN in 50 mM aqueous triethylammonium acetate buffer (pH = 7.0), the flow rate being 1.0 mL min À 1 .…”