One-Step Conversion of NHS Esters to Reagents for Site-Directed Labeling of IgG Antibodies

Abstract

Antibody conjugates are extensively used for diagnostics and therapeutics, and as a tool for molecular biology. To prepare such conjugates N-hydroxysuccinimide (NHS) esters are most often used due to the straightforward experimental procedure and the commercial accessibility of the reagents. Such conjugates are however highly heterogeneous, since only the reactivity of the lysines determines the distribution of labels. This has inspired the development of methods that experimentally are as facile but produce conjugates of higher quality. Herein, we report the development of a reagent that can, in one step, be activated with an NHS ester of choice and subsequently can be directly used for site-directed labeling of antibodies. The reagent can be prepared in three synthetic steps and produces conjugates with similar ease as for NHS esters, however in a site-directed manner. We show that the reagent is quantitatively activated by a variety of NHS esters, and we use these to functionalize IgG1, IgG2, and IgG4 antibodies.

Disintegrate (DIN) Theory Enabling Precision Engineering of Proteins

“…The approach also translates well to the lysine-directed single-site modification of a lysine residue (LDM K–K ) by replacing the epoxide (F H ) with an acylating reagent ( 12b , F K 2 ) . The strategy was further extended to selectively label Lys residues in various therapeutically relevant monoclonal antibodies using p -phenol ester ( 12d ) equipped with a linchpin fragment as the leaving group. , The o -hydroxyl group of the linchpin imine makes it highly inert toward the external nucleophiles. It creates an opportunity to use another aromatic aldehyde (F K 2 ) to create the second imine, which can be captured in the presence of an external nucleophile to deliver a single-site modification.…”

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging

“…Knowing that antibodies target specific and unique cancer cells and deliver their fluorescent 1 or cytotoxic payloads 41 with high accuracy, we have conjugated 17 to the human IgG trastuzumab having four cyclooctynyl harbors (created after literature procedures, shown in Scheme 3), 42 resulting in a potential diagnostical tracer 13 for imaging of Her2+ cells. 43 At first, an activated 44 NHS ester of 11d was prepared with TSTU (18), followed by the smooth acylation of 3azidopropane-1-amine (19), resulting in 17. Then, we were able to label the antibody (12) with the fluorescent dye in a copper-free strain-promoted azide-alkyne click reaction at room temperature.…”

“…Specific fluorescently labeled proteins became effective tools to study the mechanism of complex biological systems. , In tumor diagnostics, dye-labeled antibodies have great importance due to their combined unique selectivity and detectability. ,,,, Nowadays, biorthogonal and click chemistry ( e.g ., azide-alkyne cycloaddition) offers a site-selective and simple labeling method; therefore, most of the fluorescent dyes are equipped with a functional group available for these reactions. − …”

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging

“…The use of specific antibodies to label tumour cells exclusively and with high selectivity is an effective means of accomplishing this, and has led to recent research interest in in vitro and in vivo fluorescence methods based on antibody conjugation. 4–7…”

“…The use of specific antibodies to label tumour cells exclusively and with high selectivity is an effective means of accomplishing this, and has led to recent research interest in in vitro and in vivo fluorescence methods based on antibody conjugation. [4][5][6][7] Cyanine fluorescent probes can be used as sensors to identify small molecules, but are equally suitable complex systems containing proteins and DNA. [8][9][10][11] Cyanines, such as Cy5® and IRDye800, have been used to identify drug binding sites by a linking them to small molecules like cariprazine, 12 or biomolecules such as sugars, peptides, and proteins (Fig.…”