Abstract
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 C60-azide scaffold used for the assembly of full-armed SNAs.
Highlights
Spherical nucleic acids (SNAs, introduced originally by the Chad Mirkin laboratory) consist of an appropriate core and densely packed oligonucleotide (ON) chains.[1−7] They share many beneficial properties that overcome some of the major shortcomings perceived for therapeutic ONs: they have efficient free cellular uptake via class A scavenger receptor-mediated endocytosis,[8,9] they have muted innate immune responses and resistance to nuclease degradation, and they are large enough to avoid renal clearance
In our hands, Bingel’s cyclopropanation[18,19] between Buckminster fullerene (C60) and bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)malonate gave a mixture of compounds (1 and 2, ca. 1:1, n/n) with equal molecular masses and similar NMR data (2 with markedly broader resonances, A vs B in Scheme 1)
MS (ESI-TOF) analysis verified that all 12 arms of 1 could be readily functionalized, but reactions with 2 stacked to undecafunctionalized products (Figures S6 and S7). 1H−15N heteronuclear multiple bond correlation (HMBC)
Summary
Spherical nucleic acids (SNAs, introduced originally by the Chad Mirkin laboratory) consist of an appropriate core (gold, silica, liposomes, proteins) and densely packed oligonucleotide (ON) chains.[1−7] They share many beneficial properties that overcome some of the major shortcomings perceived for therapeutic ONs: they have efficient free cellular uptake via class A scavenger receptor-mediated endocytosis (which correlates with the density and chemistry of the component ONs),[8,9] they have muted innate immune responses and resistance to nuclease degradation (due to steric reasons), and they are large enough to avoid renal clearance. We paid extra attention to the homogeneity and authenticity of the initial C60-azide scaffold (1) as it is readily contaminated by a hardly distinguishable azide-C60 [3 + 2] cycloaddition side product that would hamper the assembly, purification, and identification of the target SNAs. Overall, this procedure allows a controlled synthesis scheme in which one of the ON arms of SNAs can be selectively functionalized with labels or other conjugate groups.
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