Design and Synthesis of Boronic Acid-Modified Nucleotides for Fluorescent Sensing and Cell Imaging

With the rapidly increasing interest in the field of glycomics, which is the comprehensive study of the roles carbohydrates play in a living system, urgent need for developing quick and highly selective carbohydrate sensors is growing. The boronic acid group, with its electron-deficient structure (6 valence electrons with an open shell), acts as a Lewis acid with high intrinsic affinity towards Lewis bases such as fluoride, cyanide and hydroxyl groups. Specifically, formation of a 5or 6membered ring between the boronic acid moiety and a1,2or 1,3-diol in aqueous solution has been fully explored as a strategy of carbohydrate sensor design. Along this line, those binders were termed as “boronolectins” because of their similar functions as lectins. One challenge in developing boronic acid-based carbohydrate sensors is to enhance the discriminating ability among various carbohydrate analytes with diverse building blocks and complex linkage patterns. One approach is using polypeptide or oligonucleotide as a backbone or scaffold with functionalized boronic acid moiety to create a molecular library, and then selecting binders with favorable properties. The work presented here includes three general research parts: synthesis of a naphthalimide-based boronic acid-conjugated thymidine triphosphate (NB-TTP), fluorescence property studies of NB-TTP incorporated DNA (NB-DNA), and cellular imaging studies using NB-TTP: 1) 4-Amino-1,4-naphthalimide (Nap) was chosen as the fluorophore because of its relatively long excitation and emission wavelengths, and stability. The synthesis of naphthalimide-based boronic acid (NB) followed similar route as previously published work. Tethering of boronic acid moiety and TTP was accomplished through Cu(I)-catalyzed azide-alkyne cyclization (CuAAC), known as click chemistry. The synthesized NB-TTP showed fluorescence enhancements at long wavelength (λem: 540 nm) upon sugar addition. 2) NB-TTP was incorporated into DNA through Klenow fragment-catalyzed primer extension reactions. Different DNA sequences were designed with varying number and spacing for NB-TTP incorporation. The preliminary study provided certain insight into several factors that affect the fluorescent properties of different NB -DNA. 3) NB-TTP was added into Hela cell culture medium to study its cell imaging properties. With the observation under fluorescent microscope, it was demonstrated that NB-TTP showed good cell membrane permeability and significant accumulation in cell nucleus. INDEX WORDS: Boronic acid, Fluorescence, DNA, Carbohydrate sensor, Cell imaging DESIGN AND SYNTHESIS OF BORONIC ACID-MODIFIED NUCLEOTIDES FOR FLUORESCENT SENSING AND CELL IMAGING