Four triphosphates of 2'-deoxyuridine that carried the following bioorthogonally reactive groups were synthesized by organic-chemical methods. Two triphosphates with tetrazines and one with a cyclopropene moiety were designed for Diels-Alder reactions with inverse electron demand, and one triphosphate with a tetrazole core was designed for the "photoclick" cycloaddition. These triphosphates were not only successfully applied for oligonucleotide preparation by standard DNA polymerases, including Hemo KlenTaq, Vent, and Deep Vent, but also bypassed for full length primer extension products. Fluorescent labeling of the primer extension products was achieved by fluorophores with reactive counterparts and analyzed by polyacrylamide gel electrophoresis mobility shifts. The tetrazine-oligonucleotide conjugates were reacted with carboxymethylmonobenzocyclooctyne- and bicyclononyne-modified fluorophores. The yield of these postsynthetic reactions could significantly be improved by a more stable but still reactive nicotinic acid-derived tetrazine and by changing the key experimental conditions, mainly the pH of 7.2 and the temperature of 45-55 °C. The cyclopropene-oligonucleotide conjugate could be successfully labeled with a tetrazine-modified rhodamine in very good yields. The "photoclick" cycloaddition between tetrazole-oligonucleotide conjugates and a maleimide-modified dye worked quantitatively. The combination of primer extension, bypass, and bioorthogonal modification works also for double and triple labeling using the cyclopropene-modified 2'-deoxyuridine triphosphate.
Postsynthetic modification of nucleic acids has the advantage that the chemical development of only a few building blocks is necessary, each bearing a chosen reactive functional group that is applicable to its reactive counterpart for a variety of different labeling types. The reactive group is either linked to phosphoramidites for chemical synthesis on solid phase or attached to nucleoside triphosphates for application in primer extension experiments and PCR. Chemoselectivity is required for this strategy, together with bioorthogonality to perform these labelings in living cells or even organisms. Currently, the copper-free reactions include strain-promoted 1,3-dipolar cycloadditions, "photoclick" reactions, Diels-Alder reactions with inverse electron demand, and nucleophilic additions. The majority of these modification strategies show good to excellent reaction kinetics, an important prerequisite for labeling inside cells and in vivo in order to keep the concentrations of the reacting partners as low as possible.
A 13mer DNA duplex containing the artificial 4-aminophthalimide:2,4-diaminopyrimidine (4AP:DAP) base pair in the central position was characterized by optical and NMR spectroscopy. The fluorescence of 4AP in the duplex has a large Stokes shift of Δλ=124 nm and a quantum yield of Φ =24 %. The NMR structure shows that two interstrand hydrogen bonds are formed and confirms the artificial base pairing. In contrast, the 4-N,N-dimethylaminophthalimide moiety prefers the syn conformation in DNA. The fluorescence intensity of this chromophore in DNA is very low and the NMR structure shows no significant interaction with DAP. Primer-extension experiments with DNA polymerases showed that not only is the 4AP C nucleotide incorporated at the desired position opposite DAP in the template, but also that the polymerase is able to progress past this position to give the full-length product. The observed selectivity supports the NMR results.
The
purpose of the present contribution is to illustrate how to
design and grow crystals of POMOFs based on POM-hybrid linkers with
lanthanide ions as nodes. Thus, the MnIII-centered Anderson–Evans
polyoxometalate (Mn-A-E-POM) was functionalized with 4-(((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)methyl)benzoic
acid (H4L) to afford the hybrid inorganic–organic
POM [N(n-C4H9)4]4[(MnMo6O18)(HL)(L)] (1), which in turn reacts with lanthanide salts and yields two three-dimensional
frameworks with the general formulas Ln(DMF)6Ln(DMF)5Ln3(DMF)10[(MnMo6O18)(L)2]3·xDMF (2; Ln = La–Nd) and [Ln(DMF)4(H2O)]2[Ln3(DMF)6][(MnMo6O18)(L)2]3·xDMF (3; Ln = Y, Sm–Lu). The differentiation in
these two families results from the lanthanide contraction. The crystallization
process is crucial for obtaining these two families in a bulk pure
phase. Family 2 can be obtained by stirring, while for
family 3 the less energy demanding layering method proved
to be the most efficient pathway. Notably, the change in the ionic
radii causes a change in space group (from P21 (family 2) to P21/c (family 3); however, the topology
of the frameworks is unaffected.
We present the synthesis and characterization of heterometallic compounds with a very large azide to metal ratio. Their interesting structures give rise to fascinating magnetic properties.
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