The first “ready-to-use” benzene-based heterotrifunctional cross-linker for multiple bioconjugation

Abstract

The synthesis and applications of the first water-soluble benzene derivative bearing a set of three different and orthogonal bioconjugatable groups (aminooxy, azido and thiol) are described. The combined use of a 5-amino isophthalic acid scaffold and unusual acid-labile protecting groups for temporarily masking aminooxy and thiol moieties has enabled the development of a highly convergent approach towards the synthesis of such a trivalent bioconjugation platform in good yields. The potential utility of this "ready-to-use" cross-linking reagent for creating complex and fragile tri-component (bio)molecular systems was illustrated through (1) the rapid preparation of a three-colour FRET cascade with valuable spectral properties and (2) the luminescent/fluorescent labelling of peptides and peptide-oligonucleotide conjugates. Thus, such (bio)molecular assemblies were readily obtained via a three-step process or in a "one-pot" manner, both involving oxime ligation, thiol-alkylation (S(N)2 or Michael addition) and copper-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions.

Oxime Ligation: A Chemoselective Click‐Type Reaction for Accessing Multifunctional Biomolecular Constructs

“…Oxime ligation can be used in conjunction with other click‐type reactions for preparing heterotopic multivalent bioconjugates 42–43. Combinations, such as oxime–thiol– CuAAC,43a,c, 44 and oxime–maleimide–CuAAC44 have been reviewed 43b. Dumy and co‐workers recently combined oxime ligation with CuAAC in order to prepare heterotopic multivalent peptide conjugates (45–72 % yield)45 and glycoconjugates 46.…”

A Synthetic Route to 3‐(Heteroaryl)‐7‐hydroxycoumarins Designed for Biosensing Applications

“…On the other hand, a coupling reaction between 3‐(2‐benzimidazolyl)‐7‐hydroxycoumarin 11 and alkyne‐functionalized cystamine 22 37 was carried out under conditions previously optimized for the synthesis of 17 (vide supra), to give, after semipreprative RP‐HPLC, the second molecular partner (i.e., 23 ) for the copper‐catalyzed azide–alkyne 1,3‐dipolar cycloaddition (CuAAC) 38. This latter reaction was achieved using microsized Cu 0 in a mixture of DMSO/H 2 O (2:1, v/v) at 50 °C 39. The resulting disulfide‐based FRET probe 24 was isolated in pure form by semipreparative RP‐HPLC (yield 48 %, purity 98 %).…”

Indolizine-Based Scaffolds as Efficient and Versatile Tools: Application to the Synthesis of Biotin-Tagged Antiangiogenic Drugs

“…and their use for bioconjugations. 3 − 7 So far, there are a few heterocyclic platforms, 8 − 10 and none with intrinsic luminescent properties. In a previous work, we considered the use of pyridinium ylide-alkyne cycloaddition forming indolizine as fluorogenic coupling methodology.…”