Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2\'-deoxyuridine 5\'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of ABNO-H-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable 3-fused hexahydropyrrolo-indole conjugates. Similarly modified ABNO-H-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNO-H-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.

While bioorthogonal reactions are routinely employed in living cells and organisms, their application within individual organelles remains limited. In this review, we highlight diverse examples of bioorthogonal reactions used to investigate the roles of biomolecules and biological processes as well as advanced imaging techniques within cellular organelles. These innovations hold great promise for therapeutic interventions in personalized medicine and precision therapies. We also address existing challenges related to the selectivity and trafficking of subcellular dynamics. Organelle-targeted bioorthogonal reactions have the potential to significantly advance our understanding of cellular organization and function, provide new pathways for basic research and clinical applications, and shape the direction of cell biology and medical research.

The desire to create biomolecules modified with functionalities that go beyond nature\'s toolbox has resulted in the development of biocompatible and selective methodologies and reagents, each with different scope and limitations. In this overview, we highlight recent advances in the field of bioconjugation from 2016 to 2023. First, (metal-mediated) protein functionalization by exploiting the specific reactivity of amino acids will be discussed, followed by novel bioorthogonal reagents for bioconjugation of modified biomolecules.

Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.

Oligonucleotide-based therapeutics have attracted attention as an emerging modality that includes the modulation of genes and their binding proteins related to diseases, allowing us to take action on previously undruggable targets. Since the late 2010s, the number of oligonucleotide medicines approved for clinical uses has dramatically increased. Various chemistry-based technologies have been developed to improve the therapeutic properties of oligonucleotides, such as chemical modification, conjugation, and nanoparticle formation, which can increase nuclease resistance, enhance affinity and selectivity to target sites, suppress off-target effects, and improve pharmacokinetic properties. Similar strategies employing modified nucleobases and lipid nanoparticles have been used for developing coronavirus disease 2019 mRNA vaccines. In this review, we provide an overview of the development of chemistry-based technologies aimed at using nucleic acids for developing therapeutics over the past several decades, with a specific emphasis on the structural design and functionality of chemical modification strategies.

Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7-deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiol-containing molecules as well as with cysteine- or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.

Methods that site-selectively attach multivalent carbohydrate moieties to proteins can be used to generate homogeneous glycodendriproteins as synthetic functional mimics of glycoproteins. Here, we study aspects of the scope and limitations of some common bioconjugation techniques that can give access to well-defined glycodendriproteins. A diverse reactive platform was designed via use of thiol-Michael-type additions, thiol-ene reactions, and Cu(I)-mediated azide-alkyne cycloadditions from recombinant proteins containing the non-canonical amino acids dehydroalanine, homoallylglycine, homopropargylglycine, and azidohomoalanine.

Glyoxal-linked 2\'-deoxyuridine 5\'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of 4-azidophenylglyoxal or a Sonogashira reaction of 4-bromophenylglyoxal with 5-ethynyl-dUMP or -dUTP. The triphosphates were used as substrates for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The glyoxal-linked nucleotides reacted with arginine-containing peptides to form stable imizadolone-linked conjugates. This reactive glyoxal modification in DNA was used for efficient bioconjugations and crosslinking with Arg-containing peptides or proteins (e. g., histones) and was found to be more reactive than previously reported 1,3-diketone-linked DNA probes.

All four iodinated 2\'-deoxyribonucleoside triphosphates (dNTPs) derived from 5-iodouracil, 5-iodocytosine, 7-iodo-7-deazaadenine and 7-iodo-7-deazaguanine were prepared and studied as substrates for KOD XL DNA polymerase. All of the nucleotides were readily incorporated by primer extension and by PCR amplification to form DNA containing iodinated nucleobases. Systematic study of the Suzuki-Miyaura cross-coupling reactions with two bulkier arylboronic acids revealed that the 5-iodopyrimidines were more reactive and gave cross-coupling products both in the terminal or internal position in single-stranded oligonucleotides (ssONs) and in the terminal position of double-stranded DNA (dsDNA), whereas the 7-iodo-7-deazapurines were less reactive and gave cross-coupling products only in the terminal position. None of the four iodinated bases reacted in an internal position of dsDNA. These findings are useful for the use of the iodinated nucleobases for post-synthetic modification of DNA with functional groups for various applications.

Linear or branched 1,3-diketone-linked thymidine 5\'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).