ADPr-Peptide Synthesis.

Synthetic mono-ADPr-peptides are useful for structural, biochemical, and proteomics studies. We describe here a protocol for the preparation of mono-ADPr-peptides based on a fairly standard Fmoc-based solid-phase synthesis. Phosphoribosylated precursor building blocks are introduced into the peptide chain on solid-phase and subsequently converted to ADPr-sites by chemical phosphorylation with adenosine phosphoramidite. Suitably protected phosphoribosylated glutamine, asparagine, and citrulline building blocks described in this protocol allow introduction of ADP-Gln, ADPr-Asn, and ADPr-Cit into peptide chains as demonstrated for three peptides. Trifunctional amino acids, for which base-sensitive side-chain protection is available, can be accommodated in the sequences flanking the ADPr-cites.

Artificial bioconjugates with naturally occurring linkages: the use of phosphodiester.

Artificial orthogonal bond formations such as the alkyne-azide cycloaddition have enabled selective bioconjugations under mild conditions, yet naturally occurring linkages between native functional groups would be more straightforward to elaborate bioconjugates. Herein, we describe the use of a phosphodiester bond as a versatile option to access various bioconjugates. An opposite activation strategy, involving 5'-phosphitylation of the supported oligonucleotides, has allowed several biomolecules that possess an unactivated alcohol to be directly conjugated. It should be noted that there is no need to pre-install artificial functional groups and undesired and unpredictable perturbations possibly caused by bioconjugation can be minimized.

New Synthetic Methods for Phosphate Labeling.

The complexity of phosphorylation pathways and their downstream effects is vast. Synthetic chemistry has been working side by side with biology to develop phosphate labels for biological processes involving phosphorylated compounds. This chapter discusses recently employed methods for the preparation of several phosphate labels. Synthesis of biomolecules and their analogs and other useful or potentially useful phosphate derivatives is discussed.

Microwave-assisted phosphitylation of DNA and RNA nucleosides and their analogs.

Microwave-assisted chemical phosphitylation of novel nucleoside analogs containing a ribulose sugar unit was successful with yields ranging from 50% to 79% using 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite as the phosphitylating reagent. The resultant phosphoramidite products remained intact, with no signs of degradation over extended reaction times (up to 60 min) at an elevated temperature (65°C). When the same microwave-mediated phosphitylating protocols were applied to canonical DNA and RNA nucleoside monomers as substrates, using either 2-cyanoethyl-N,N,-diisopropyl chlorophosphoramidite or 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphane with an activator, 40% to 90% yields of DNA and RNA phosphoramidites were obtained within 10 to 15 min. These results demonstrate that microwave-assisted phosphitylation is an efficient alternative to standard phosphitylating conditions that can be expanded and refined to include a variety of substrates.