Synthesis and Bioconjugation of Diene-Modified Oligonucleotides

Abstract

The preparation of diene-modified oligonucleotides as well as their properties and further derivatization are described. Self-complementary oligonucleotides containing a diene moiety in the loop region form stable, hairpin-like secondary structures. These hairpin mimics can be further derivatized via the Diels-Alder reaction. Diene modification in the stem region leads, in contrast, to a marked destabilization of the hairpin structure. No further reduction in stability is observed, however, upon conjugation of the stem-modified derivatives via the Diels-Alder reaction with an N-substituted maleimide dienophile.

Immuno‐Polymeric Nanoparticles by Diels–Alder Chemistry

“…Diels-Alder reactions, which involve a highly selective [4+2] cycloaddition reaction between a diene and a dienophile, [23][24][25] provide a means to overcome the limitations associated with coupling chemically sensitive antibody molecules, particularly in aqueous environments where both the rate and stereoselectivity of Diels-Alder reactions are dramatically increased. [24,25] The great potential of DielsAlder reactions in biotechnology applications has been recognized, [26][27][28][29][30] and here we demonstrate its applicability to conjugate antibodies to nanoparticles for targeted delivery. This simple, one-step reaction without by-products does not require initiators, catalysts, or coupling reagents, is highly efficient and selective under mild conditions, and results in stable products.…”

Synthesis and Evaluation of Modified Oligodeoxynucleotides Containing Diphosphodiester Internucleotide Linkages

“…[1][2][3][4][5] To a great extent, these modifications have focused on replacing the phosphodiester group by phosphodiester mimics, such as phosphorothioate, [6] methylphosphate, [7] phosphoramidates, [8] boranophosphate, [9] alkylphosphotriesters, [10] phosphorodithioate, [11] phosphonomethyl, [12] propynes, [13] diene, [14] phosphonoformate, [15] and other groups. [16] The phosphate-modified ODNs have been used as inhibitors of gene expression, [1] viral enzymes, [2,3] in vitro messenger-RNA translation, [4] and sequence-specific DNA binding proteins.…”

“…For example, fully decoupled 31 P NMR spectra for the thymidine 5-mer analogue (12 a) displayed 10 nonoverlapping peaks corresponding to 10 phosphorus atoms. The synthetic cycle was then used to synthesize modified ODNs with 12 bases (13)(14)(15)(16)(17)(18)(19)(20)(21)(22) To further confirm the chemical structures of the synthesized compounds, two representative modified ODNs, d-(AAAAA) (12 b) and d(CCCCC) (12 d), were also synthesized by using differentially protected nucleoside building blocks (a and b; Scheme 2) that were prepared according to the previously reported procedures. [30][31][32][33][34] The deprotection of 3'-O-tert-butyldimethylsilyl (TBDMS) and 9-fluorenylmethyloxycarbonyl (Fmoc) groups was performed with tetrabutylammonium fluoride (TBAF) in THF and 20 % piperidine in DMF, respectively, when required.…”

“…The free 3'-hydroxy group was subjected to the subsequent diphosphitylation in the presence of an excess amount of [iPr 2 NPOCH 2 CH 2 CN] 2 O (4 equivalents) to yield 3 a-d. 5'-O-coupling and 3'-O-diphosphitylation reactions were repeated n times (n = 0-4, 11) to produce polymerbound diphosphite triesters (5 a-h). Oxidation, removal of the 2-cyanoethoxy group, and cleavage reactions afforded 30 modified ODNs containing diphosphodiester internucleotide linkages with up to 12 bases (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). This strategy offers the advantages of high selectivity for the attachment of a 5'hydroxy group to the polymer-bound-diphosphitylating reagent, use of unprotected nucleosides, and facile isolation of final products.First, ODNs of up to five bases containing diphosphodiester bridges (8-12 a-d) were synthesized and characterized by 1 H, 13 C, and 31 P NMR spectroscopy, high-resolution TOF electrospray mass spectrometry, and quantitative phosphorus analysis.…”

“…For example, fully decoupled 31 P NMR spectra for the thymidine 5-mer analogue (12 a) displayed 10 nonoverlapping peaks corresponding to 10 phosphorus atoms. The synthetic cycle was then used to synthesize modified ODNs with 12 bases (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). The final products were characterized by 1 H and 31 P NMR spectroscopy, MALDI-TOF mass spectrometry, and quantitative phosphorus analysis.…”