Bioconjugation with Maleimides: A Useful Tool for Chemical Biology

Maleimide chemistry stands out in the bioconjugation toolbox by virtue of its synthetic accessibility, excellent reactivity, and practicability. The second-generation of clinically approved antibody–drug conjugates (ADC) and much of the current ADC pipeline in clinical trials contain the maleimide linkage. However, thiosuccinimide linkages are now known to be less robust than once thought, and ergo, are correlated with suboptimal pharmacodynamics, pharmacokinetics, and safety profiles in some ADC constructs. Rational design of novel generations of maleimides and maleimide-type reagents have been reported to address the shortcomings of classical maleimides, allowing for the formation of robust bioconjugate linkages. This review highlights the main strategies for rational reagent design that have allowed irreversible bioconjugations in cysteines, reversible labelling strategies and disulfide re-bridging.     

Bioconjugation of functionalized fluorescent YVO4:Eu nanocrystals with BSA for immunoassay

Novel fluorescent YVO₄:Eu nanocrystals were ∼15 nm in diameter and had been functionalized with phosphorous polyacrylic acids at the surface during the synthesis, which illuminated their potential in bioassays. In this paper, bovine serum albumin (BSA) was covalently coupled to activated nanocrystals from primary amine groups by EDC and sulfo-NHS chemistry successfully. Reaction condition, including concentration, pH value and separation methods, were carefully chosen and well discussed. Binding site number was determined simply by measuring BSA concentration using optimized microplate bicinchonic acid (BCA) assay and nanocrystals concentration according to their fluorescence emission in the monodisperse bioconjugate suspension. Transmission electron microscopy (TEM) imaging showed the monodispersity of the bioconjugates. Fluorescent characteristics were also retained. Immunoblots verified that BSA was covalently coupled to nanocrystals and remained immunoreactive. Finally, the two-step heterogeneous time-resolved fluoroimmunoassay (TR-FIA) of rabbit anti-BSA polyclonal antibody (PcAb) was operated to further validate the bioconjugates.     

Rapid Formation of Non-canonical Phospholipid Membranes by Chemoselective Amide-Forming Ligations with Hydroxylamines**

There has been increasing interest in methods to generate synthetic lipid membranes as key constituents of artificial cells or to develop new tools for remodeling membranes in living cells. However, the biosynthesis of phospholipids involves elaborate enzymatic pathways that are challenging to reconstitute in vitro. An alternative approach is to use chemical reactions to non-enzymatically generate natural or non-canonical phospholipids de novo. Previous reports have shown that synthetic lipid membranes can be formed in situ using various ligation chemistries, but these methods lack biocompatibility and/or suffer from slow kinetics at physiological pH. Thus, it would be valuable to develop chemoselective strategies for synthesizing phospholipids from water-soluble precursors that are compatible with synthetic or living cells Here, we demonstrate that amide-forming ligations between lipid precursors bearing hydroxylamines and α-ketoacids (KAs) or potassium acyltrifluoroborates (KATs) can be used to prepare non-canonical phospholipids at physiological pH conditions. The generated amide-linked phospholipids spontaneously self-assemble into cell-like micron-sized vesicles similar to natural phospholipid membranes. We show that lipid synthesis using KAT ligation proceeds extremely rapidly, and the high selectivity and biocompatibility of the approach facilitates the in situ synthesis of phospholipids and associated membranes in living cells.     

One-pot synthesis of triazole-linked glycoconjugates

Highly efficient one-pot synthesis of 1,2,3-triazole-linked glycoconjugates was presented involving a Cu(I) catalyzed 1,3-dipolar cycloaddition as the key step. It offers a convenient route to prepare neoglycoconjugates derived from unprotected saccharides or peracetylated saccharides.     

Insertion of Chemical Handles into the Backbone of DNA during Solid-Phase Synthesis by Oxidative Coupling of Amines to Phosphites

Conjugation of molecules or proteins to oligonucleotides can improve their functional and therapeutic capacity. However, such modifications are often limited to the 5′ and 3′ end of oligonucleotides. Herein, we report the development of an inexpensive and simple method that allows for the insertion of chemical handles into the backbone of oligonucleotides. This method is compatible with standardized automated solid-phase oligonucleotide synthesis, and relies on formation of phosphoramidates. A unique phosphoramidite is incorporated into a growing oligonucleotide, and oxidized to the desired phosphoramidate using iodine and an amine of choice. Azides, alkynes, amines, and alkanes have been linked to oligonucleotides via internally positioned phosphoramidates with oxidative coupling yields above 80 %. We show the design of phosphoramidates from secondary amines that specifically hydrolyze to the phosphate only at decreased pH. Finally, we show the synthesis of an antibody-DNA conjugate, where the oligonucleotide can be selectively released in a pH 5.5 buffer.     

Water Soluble Metallo-Phthalocyanines: The Role of the Functional Groups on the Spectral and Photophysical Properties

Strategies are reported that produce symmetrical metal-free and metallo-phthalocyanine dyes, Pc and MPc, respectively, containing various numbers of water solubilizing carboxylic acid groups on their periphery that provide a dual role by also serving as functional groups to covalently link primary amine-containing targets to these dyes. In order to induce water compatibility and to minimize the degree of aggregation, the periphery of the macrocycle was decorated with various numbers of water-solubilizing groups and/or altering the identity of the metal center. The influence of the number of solubilizing groups and metal center on the spectral and photophysical properties were evaluated. MPc dyes containing 4, 8, or 16 carboxylic acid groups exhibited similar absorption and emission maxima (677 and 686 nm, respectively) with the molar absorptivity of the Q-band ∼10⁵ M⁻¹ cm⁻¹. Results indicated that the fluorescence lifetimes and quantum yields varied as a function of the metal center; the degree of carboxylation did not significantly alter these properties in DMSO, but did mediate the solubility and aggregation states when placed in aqueous solvents. The water solubilizing groups could also serve as labeling moieties for targets bearing primary amines. Results showed that the conjugate, produced by covalently linking an MPc to streptavidin through one of its carboxylate groups, generated a red-shift in the emission maximum with a fluorescence lifetime shorter than that of the native MPc dye.     

Novel Heptamethine Cyanine Dyes with Large Stokes’ Shift for Biological Applications in the Near Infrared

A series of novel functionalized, water-soluble, pH-unsensitive, highly photostable heptamethine cyanine dyes (HCDs) has been synthesized. The aim of the synthesis was to obtain novel effective probes for fluorescence detection in the near infrared. Synthesis and characterization of a special HCD with large Stokes’ shift (>100 nm), bioconjugation to IgG and effect of pH upon the new structure are presented.     

Tailored interfaces for biosensors and cell-surface interaction studies via activation and derivatization of polystyrene-block-poly(tert-butyl acrylate) thin films

Thin spin-coated films of polystyrene-block-poly(tert-butyl acrylate) (PS₆₉₀-b-PtBA₁₂₁₀) on various substrates are introduced as versatile, robust reactive platform for the immobilization of (bio)molecules for the fabrication of tailored biointerfaces. The films are characterized by high stability and (bio)reactivity due to the presence of a glassy PS and a reactive PtBA block, respectively. The selective deprotection of the tert-butyl-ester groups in the PtBA skin layer by hydrolysis under acidic conditions, the activation with N-hydroxysuccinimide and the subsequent derivatization with amino functionalized (bio)molecules were investigated. Based on contact angle, FTIR spectroscopy and XPS, fluorescence microscopy and AFM data, it was shown that the (bio)molecules were coupled covalently to the polymer films and that high molecular coverages up to ∼2.4 poly(ethylene glycol) (PEG) molecules per nm² (M_n = 500 g/mol) were obtained. Organic dyes, polyamidoamine dendrimers, polypeptides, proteins and amino end-functionalized DNA were efficiently and homogeneously immobilized on the PS-PtBA platforms. Grafting of ω-amino functionalized PEG afforded surfaces with substantially reduced non-specific adsorption of proteins and DNA. Owing to the glassy nature of PS and the covalent amide linkages, the derivatized films showed excellent stability under a broad range of processing conditions. Finally, the viability of PS₆₉₀-b-PtBA₁₂₁₀ platforms as versatile biointerfaces was demonstrated in DNA hybridization experiments, as well as cell-surface interaction studies using pancreatic cancer and K562 cells.     

Bioconjugation via Hetero-Selective Clamping of Two Different Amines with ortho-Phthalaldehyde

Amino groups are common in both natural and synthetic compounds and offer a very attractive class of endogenous handles for bioconjugation. However, the ability to differentiate two types of amino groups and join them with high hetero-selectivity and efficiency in a complex setting remains elusive. Herein, we report a new method for bioconjugation via one-pot chemoselective clamping of two different amine nucleophiles using a simple ortho-phthalaldehyde (OPA) reagent. Various α-amino acids, aryl amines, and secondary amines can be crosslinked to the ϵ-amino side chain of lysine on peptides or proteins with high efficiency and hetero-selectivity. This method offers a simple and powerful means to crosslink small molecule drugs, imaging probes, peptides, proteins, carbohydrates, and even virus particles without any pre-functionalization.     

Design and synthesis of water-soluble bioconjugatable trans-AB-porphyrins

Three free base porphyrins have been prepared that bear a polar and facially encumbering 2,4,6-tris-(carboxymethoxy)phenyl motif at one meso (5-) position. The only other substituent (15-position) comprises phenyl, formyl, or p-aminophenyl. The porphyrins exhibit solubility in water (or aqueous buffer solutions) at pH ≥7 and concentrations >1 mM at room temperature. The concise syntheses, water solubility, and bioconjugatable handle make these porphyrin constructs suitable for biological applications.