RNA–peptide conjugate synthesis by inverse-electron demand Diels–Alder reaction

Ameta, Sandeep; Becker, Juliane; Jäschke, Andres

doi:10.1039/c4ob00076e

Cited by 18 publications

(18 citation statements)

References 41 publications

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“…Jäschke et al showed that this polymerase could terminally modify RNA, using norbornene-and BCNmodified guanosine monophosphate (GMP) as the initiator nucleotides. 178,182 A related approach by Royzen et al showed that a cytidine triphosphate (CTP) analogue with a TCO group at the 5-position of the nucleobase is recognized by the T7 RNA polymerase, and therefore is internally and site-specifically incorporated in a RNA strand. 183 Another alternative method for RNA modification uses an engineered methyltransferase that recognises S-adenosyl-L-methionine analogues containing reactive handles and transfers these handles to a RNA of interest (Fig.…”

Section: Alternative Enzymatic Methods or Enzyme Tags For Protein Modmentioning

confidence: 99%

Inverse electron demand Diels–Alder reactions in chemical biology

2017

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The emerging inverse electron demand Diels-Alder (IEDDA) reaction stands out from other bioorthogonal reactions by virtue of its unmatchable kinetics, excellent orthogonality and biocompatibility. With the recent discovery of novel dienophiles and optimal tetrazine coupling partners, attention has now been turned to the use of IEDDA approaches in basic biology, imaging and therapeutics. Here we review this bioorthogonal reaction and its promising applications for live cell and animal studies. We first discuss the key factors that contribute to the fast IEDDA kinetics and describe the most recent advances in the synthesis of tetrazine and dienophile coupling partners. Both coupling partners have been incorporated into proteins for tracking and imaging by use of fluorogenic tetrazines that become strongly fluorescent upon reaction. Selected notable examples of such applications are presented. The exceptional fast kinetics of this catalyst-free reaction, even using low concentrations of coupling partners, make it amenable for in vivo radiolabelling using pretargeting methodologies, which are also discussed. Finally, IEDDA reactions have recently found use in bioorthogonal decaging to activate proteins or drugs in gain-of-function strategies. We conclude by showing applications of the IEDDA reaction in the construction of biomaterials that are used for drug delivery and multimodal imaging, among others. The use and utility of the IEDDA reaction is interdisciplinary and promises to revolutionize chemical biology, radiochemistry and materials science.

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Section: Alternative Enzymatic Methods or Enzyme Tags For Protein Modmentioning

confidence: 99%

Inverse electron demand Diels–Alder reactions in chemical biology

2017

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“…Traditionally the most common post-labelling chemistry has involved coupling dye succinimidyl esters to amino-modified DNA ( 10 , 11 ). More recently the highly efficient copper catalysed alkyne-azide cycloaddition reaction (CuAAC) ( 12 – 16 ), strain-promoted alkyne-azide cycloaddition reaction (SPAAC) ( 17 – 21 ), Diels-Alder (DA) and inverse electron demand Diels-Alder (IEDDA) reaction ( 19 , 22 – 24 ) have been used. Of these reactions, the SPAAC reaction between cyclooctyne and azide is a good choice because it is fast, orthogonal to the functional groups in DNA, it does not require metal catalysts (which can lead to DNA degradation if not carried out with care) and it involves the non-bulky azide group.…”

Section: Introductionmentioning

confidence: 99%

Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

2016

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A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

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“…[17][18][19] In particular, the Diels-Alder reaction with inverse electron-demand (DAR inv ) [20][21][22] between numerous dienophiles [23][24][25] and tetrazines 21,26,27 was found to be a valuable tool for effective bioorthogonal conjugations. [28][29][30] Followed by a retro-Diels-Alder reaction to eliminate nitrogen gas, this so-called tetrazine ligation is characterized by extremely fast kinetics with second-order rate constants up to 2 Â 10 3 M À1 s À1 and has been already used in a number of both in situ and in vivo studies. 25,31,32 In this study we present a modular approach to the conjugation of biomolecules based on the combination of two efficient chemical transformations, oxime ligation and DAR inv .…”

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confidence: 99%

Combination of inverse electron-demand Diels–Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations

et al. 2015

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A modular approach combining inverse electron-demand Diels-Alder coupling (DARinv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.

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RNA–peptide conjugate synthesis by inverse-electron demand Diels–Alder reaction

Cited by 18 publications

References 41 publications

Inverse electron demand Diels–Alder reactions in chemical biology

Inverse electron demand Diels–Alder reactions in chemical biology

Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

Combination of inverse electron-demand Diels–Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations

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