Synthesizing and Modifying Peptides for Chemoselective Ligation and Assembly into Quantum Dot-Peptide Bioconjugates

Abstract

Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

Concentric Förster Resonance Energy Transfer Imaging

“…14−16 The peptides were labeled at their distal terminus with A555 or A647 (Invitrogen, Carlsbad, CA) using a published protocol. 17 These peptides are denoted as Sub ChT (A555) and Sub TRP (A647), with the subscript identifying the target enzyme (ChT, chymotrypsin; TRP, trypsin).…”

Chemotherapeutic Drug‐Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA

“…The content of reactive amines in ligand‐conjugated Amikabeads was used as an indicator of reactive groups left over after conjugation, and was determined using the ninhydrin assay . Drug‐conjugated Amikabeads were collected and washed three times with DMSO in order to remove any unreacted ligands.…”

Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

“…Similar to oligonucleotides, generally, there are two methods for the modification of peptides: 1) SPPS modification, and 2) solution‐phase modification of presynthesized peptides or bioconjugation . Algar et al.…”

“…Algar et al. reported methods for modifying peptides with 4‐formylbenzoyl and 2‐hydrazinonicotinoyl groups . Furthermore, they described the aniline‐catalyzed ligation of both functional groups, as well as subsequent conjugation of the ligated peptides to CdSe/ZnS quantum dots.…”

“…[43] Similar to oligonucleotides,g enerally, there are two methods for the modification of peptides:1 )SPPS modification, and 2) solution-phase modification of presynthesized peptides or bioconjugation. [44] Algar et al reported methods for modifying peptides with 4-formylbenzoyl and 2-hydrazinonicotinoyl groups. [44] Furthermore, they described the aniline-catalyzed ligation of both functional groups,a sw ella ss ubsequent conjugation of the ligated peptides to CdSe/ZnS quantum dots.…”