Oligonucleotide Imprinting in Aqueous Environment
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Oligonucleotide Imprinting in Aqueous Environment Dolly Batra and Kenneth J. Shea* Department of Chemistry University of California, Irvine Irvine, CA 92697 INTRODUCTION The development of synthetic receptors that recognize nucleotide bases and their derivatives is an important area of research [1-3]. Applications are envisioned in separation science, biosensors, drug therapy and genetic engineering. Previously in this laboratory, we have developed a molecularly imprinted synthetic receptor for 9-ethyladenine (9-EA). The network polymer has an affinity for adenine and its derivatives with an average association constant (Ka) of 75,000 M–1 in CHCl3 [4]. When a 9-EA imprinted polymer was used as the chromatographic support, adenine eluted at 27 minutes using 92.5/5.0/2.5 CH3CN/H2O/CH3CO2H as the mobile phase, while cytosine, guanine and thymine derivatives all eluted close to the void volume (2.0 min). In addition, imprinted polymers have been made with complementary binding sites for cytosine and guanine [5], as well as other nucleotide base analogues [6]. The extension of these results to construct robust receptors for oligonucleotides requires fundamental changes in imprinting strategies. Most importantly, since oligonucleotides are water soluble, strategies that employ EGDMA/MAA formulations in organic solvents will need to be replaced with those that do not compromise the interactions between template (the oligo) and functional monomer. Initially, the imprinting of a 2'-deoxyadenosine dimer (1) was attempted. Due to the hydrophilicity of a DNA oligomer, it was difficult to find a suitable organic solvent that would solubilize the oligomers without disrupting the template's interaction with the polymer matrix [7]. To combat the solubility problems and to insure the homogeniety of the polymerization solution, we examined various polymer formulations with organic and/or aqueous-based solvents that would dissolve the template without disrupting these key interactions. EXPERIMENTAL DETAILS Synthesis of Template
NH2 N
The first phase of this research involved synthesis of an adenine dimer 1. Since imprinting involves milligram quantities of template molecules, it was decided to synthesize these quantities using a solution phase technique. The synthesis utilized phosphoramidite methodology (Scheme 1) [8]. The coupling of individually protected nucleotides using a phosphoramidite linkage yields phosphite 2. This linkage is later oxidized to the phosphate group, and, following deprotection with TFA, treatment with concentrated NH4OH leads to the desired oligonucleotide (3).
N
N
N
HO O
NH2 N
O HO P O
N
O O OH 1
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N N
Scheme 1 B1
B2 HO
O P N OR
DMTrO
B2
B1 Tetrazole
OBz
OBz
O P O OR
DMTrO
R = CH2CH 2CN
9 B
B
2
B
O
I 2/H2O/Pyr
2
1
B
2
O
1. TFA 2. NH
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