Critical Evaluation and Rate Constants of Chemoselective Ligation Reactions for Stoichiometric Conjugations in Water

Saito, Fumito; Noda, Hidetoshi; Bode, Jeffrey W.

doi:10.1021/cb5006728

Cited by 153 publications

(189 citation statements)

References 48 publications

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“…This was recently illustrated by Bode and co-workers, who compared the second-order rate constants of several widely used chemo-selective ligation reactions (Figure 10). 140 The kinetic studies were carried out with functionalized heptapeptides, which were reacted with azo dyes featuring the complementary reactive group. The two reactants of each conjugation were used in a 1:1 ratio.…”

Section: Catalysts For Oxime/hydrazone Formationmentioning

confidence: 99%

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

2017

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The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chemistry, biomaterials and hydrogels, dynamic combinatorial chemistry, organic synthesis, and chemical biology. Here we outline chemical developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomolecules are described. The basic chemical properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equilibria in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified especially fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addition, the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A number of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomolecules and biomaterials.

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Section: Catalysts For Oxime/hydrazone Formationmentioning

confidence: 99%

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

2017

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“…24–26 Furthermore, it would be highly beneficial to deploy a tool for routinely increasing peptide/protein concentrations to the mM range in order to generally accelerate chemoselective ligation reactions. 27,28 …”

Section: Introductionmentioning

confidence: 99%

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

Jacobsen

Petersen

et al. 2016

J. Am. Chem. Soc.

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Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a general new tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a hetero-bifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (helping hands) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebolavirus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic L-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of D-GroES, which was inactive in a hetero-chiral mixture with recombinant GroEL—providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.

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“…1-3 An example of a Michael addition commonly used for bioconjugation is the thiol-maleimide reaction. This reaction is considered one of the “click” reactions due to its fast reaction rate and aqueous compatibility.…”

mentioning

confidence: 99%

Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

Chen¹,

Jiang²,

Carroll

et al. 2015

Org. Lett.

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Density functional theory (DFT) was applied to study the thermodynamics and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31G(d) with the SMD solvation model can reliably predict the Gibbs free energy changes (ΔG) of thiol-Michael addition reactions with an error of less than 1 kcal·mol−1 compared with the experimental benchmarks. Taking advantage of this computational model, the first reversible reaction-based fluorescent probe was developed that can monitor the changes in glutathione levels in single living cells.

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Critical Evaluation and Rate Constants of Chemoselective Ligation Reactions for Stoichiometric Conjugations in Water

Cited by 153 publications

References 48 publications

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

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