Esterified precursors of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; 18) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA; 17,19) ligands bearing a dimethoxytritylated hydroxyl side arm were prepared and immobilized via an ester linkage to long chain alkyl amine derivatized controlled pore glass (LCAA-CPG). Oligonucleotide chains were then assembled on the hydroxyl function and conjugates were released and deprotected by a two-step cleavage with aqueous alkali and ammonia. The 3'-DOTA and 3'-NOTA conjugated oligonucleotides were converted to (68)Ga chelates by a brief treatment with [(68)Ga]Cl(3) at elevated temperature. Applicability of the conjugates for in vivo imaging with positron emission tomography (PET) was verified.

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne–azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs’ outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C 60 -azide scaffold used for the assembly of full-armed SNAs.

The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5'-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5'-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5'-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5'-monophosphate (5'-TMP) is quantitative, while conversion of 1 to 5'-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5'-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.

Synthesis for (68)Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide hyaluronan (HA) tetra- and hexasaccharide conjugates is described. A solid-supported technique is used to introduce NOTA-chelator into the 3'-terminus of oligonucleotides and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/oligonucleotide conjugation. Protecting group manipulation, required for the HA-moieties, is carried out after the SPAAC-conjugation. Positron emission tomography (PET) is used (1) in the whole-body distribution kinetic studies of the conjugates in healthy rats and (2) to show the potential of hyaluronan-induced targeting of oligonucleotides into the infarcted area of rats with myocardial infarction.

The main threshold for the therapeutic applications of nucleotides and oligonucleotides is their ionic structure which implies poor cellular uptake and unfavorable pharmacokinetic parameters. To circumvent these problems, the anionic phosphate moieties may be temporarily masked with enzymolabile protecting groups to form neutral pronucleotides or pro-oligonucleotides. In cells, enzymes cleave the protecting groups and release the parent drug. Several prodrug strategies have been developed, but the kinetics and mechanisms of the deprotection of potential prodrug candidates are still often poorly known. The purpose of the present review is to summarize the current knowledge on the chemical aspects of alternative prodrug strategies at nucleotide and oligonucleotide level.