Synthesis of 6-Alkynylated Purine-Containing DNA via On-Column Sonogashira Coupling and Investigation of Their Base-Pairing Properties

Abstract

Synthetic unnatural base pairs have been proven to be attractive tools for the development of DNA-based biotechnology. Our group has very recently reported on alkynylated purine-pyridazine pairs, which exhibit selective and stable base-pairing via hydrogen bond formation between pseudo-nucleobases in the major groove of duplex DNA. In this study, we attempted to develop an on-column synthesis methodology of oligodeoxynucleotides (ODNs) containing alkynylated purine derivatives to systematically explore the relationship between the structure and the corresponding base-pairing ability. Through Sonogashira coupling of the ethynyl pseudo-nucleobases and CPG-bound ODNs containing 6-iodopurine, we have demonstrated the synthesis of the ODNs containing three ^NPu derivatives (^NPu1, ^NPu2, ^NPu3) as well as three ^OPu derivatives (^OPu1, ^OPu2, ^OPu3). The base-pairing properties of each alkynylated purine derivative revealed that the structures of pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that ^OPu1 bearing 2-pyrimidinone exhibits higher stability to the complementary ^NPz than the original ^OPu, thereby demonstrating the potential of the on-column strategy for convenient screening of the alkynylated purine derivatives with superior pairing ability.

Keywords: DNA; DNA major groove; Sonogashira coupling; hydrogen bonds; modified nucleosides; solid-phase synthesis; unnatural base pair.

Figure 1

(a) Structures of the previously reported alkynylated purine–pyridazine base pairs. (b) A model structure of the duplex DNA incorporating the ^NPu–^OPz pair. The hydrogen bonding between the pseudo-nucleobases is highlighted.

Figure 2

(a) On-column synthesis of ODNs incorporating alkynylated purine derivatives via Sonogashira coupling reactions; CPG: controlled pore glass. (b) Structures of the purine derivatives synthesized in this study.

Scheme 1

Synthesis of the CPG-bound ODNs containing ^IPu at their 5′ terminal.

Scheme 2

Synthesis of the ethynyl pseudo-nucleobases for ^NPu derivatives.

Scheme 3

Synthesis of the ethynyl pseudo-nucleobases for ^OPu derivatives.

Figure 3

(a) On-column Sonogashira coupling reaction using ODN1 containing ^IPu and alkyne 9. (b) RP-HPLC trace of crude ODN1-^NPu1. Conditions: 0.1 M TEAA buffer (pH 7.0) and CH₃CN, with a gradient of 5% to 20% of CH₃CN over 20 min. Flowrate: 1 mL/min, column oven: 50 °C, detection wavelength: 254 nm.

Figure 4

(a) Solid-phase synthesis and deprotection of ODN3-^NPu1. (b) The RP-HPLC trace of crude ODN1-^NPu3. Conditions: 0.1 M TEAA buffer (pH 7.0) and CH₃CN, with a gradient of 9% to 10% of CH₃CN over 30 min; flowrate: 1 mL/min, column oven: 50 °C, detection wavelength: 254 nm.

Figure 5

Circular dichroism (CD) spectra of the duplex DNAs containing (a) ^NPu derivatives and (b) ^OPu derivatives.

Figure 6

(a) ^OPu1 exhibits higher affinity due to the bifacial nature of the 2-pyrimidinone moiety. (b) The pyridazine pseudo-nucleobases of ^NPu2 and ^OPu2 cause static repulsion with the complementary ^NPz and ^OPz.