Bioconjugation Chemistry Research Articles

Synthesis of dibromo-triazoles and their amination with hydroxylamine-O-sulphonic acid

N-heterocyclic compounds have a wide range of applications in medicinal chemistry, bio-conjugation and materials chemistry due to their high biological activity, low toxicity and high systemic nature. Dibromo-triazoles and their amino derivatives are important functional materials. 3,5-Dibromo-4H-1,2,4-triazole (1, yield 74%) can be easily synthesised from 1H-1,2,4-triazole (2) using bromine, N-bromosuccinimide or other brominating agents in a high yield. The 3,5-dibromo-4-amino-1,2,4-triazole and 3,5-dibromo-1-amino-1,2,4-triazole (3, yield 21%) can be prepared by reacting compound 1 with hydroxylamine-O-sulphonic acid in weakly alkaline aqueous solutions at reflux temperature. Pure compound 3, a colourless solid, was finally obtained after a column chromatography. 4,5-dibromo-1-amino-1,2,3-triazole and 4,5-dibromo-2-amino-1,2,3-triazole (4, yield 30%) can also be prepared with 1H-1,2,3-triazole (5) as the starting reactant and 4,5-dibromo-1H-1,2,3-triazole (6, yield 78%) as the intermediate. The structures of the compounds above were confirmed using 1H-NMR, 13C-NMR, infrared (IR), LC-MS, XRD and elementary analysis. According to the characterisation results, these compounds have two isomers.

Research spotlight: emergence of EPR effect theory and development of clinical applications for cancer therapy.

Research spotlight: emergence of EPR effect theory and development of clinical applications for cancer therapy.

Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

Rapid kinetic characterization of glycosyl hydrolases based on oxime derivatization and nanostructure-initiator mass spectrometry (NIMS).

Glycoside hydrolases (GHs) are critical to cycling of plant biomass in the environment, digestion of complex polysaccharides by the human gut microbiome, and industrial activities such as deployment of cellulosic biofuels. High-throughput sequencing methods show tremendous sequence diversity among GHs, yet relatively few examples from the over 150,000 unique domain arrangements containing GHs have been functionally characterized. Here, we show how cell-free expression, bioconjugate chemistry, and surface-based mass spectrometry can be used to study glycoside hydrolase reactions with plant biomass. Detection of soluble products is achieved by coupling a unique chemical probe to the reducing end of oligosaccharides in a stable oxime linkage, while the use of (13)C-labeled monosaccharide standards (xylose and glucose) allows quantitation of the derivatized glycans. We apply this oxime-based nanostructure-initiator mass spectrometry (NIMS) method to characterize the functional diversity of GHs secreted by Clostridium thermocellum, a model cellulolytic organism. New reaction specificities are identified, and differences in rates and yields of individual enzymes are demonstrated in reactions with biomass substrates. Numerical analyses of time series data suggests that synergistic combinations of mono- and multifunctional GHs can decrease the complexity of enzymes needed for the hydrolysis of plant biomass during the production of biofuels.

ChemInform Abstract: Chemical Modifications and Bioconjugate Reactions of Nanomaterials for Sensing, Imaging, Drug Delivery and Therapy

AbstractReview: 50 refs.

Highly Colloidally Stable Hyperbranched Polyglycerol Grafted Red Fluorescent Silicon Nanoparticle as Bioimaging Probe

Here we report a surface modification approach for fluorescent silicon nanoparticle that transforms hydrophobic nanoparticle into water-soluble nanoparticle of high colloidal stability. The approach involves ring-opening polymerization of glycidol at the hydroxyl-terminated nanoparticle surface that results in a hyperbranched polyglycerol grafted silicon nanoparticle (Si-HPG). The resultant Si-HPG has 25 nm hydrodynamic diameter, low surface charge, and broad emission in the range of 450-700 nm with a fluorescence quantum yield of 6-9%. The Si-HPG has been transformed into cyclic RGD peptide functionalized nanoprobe using the conventional bioconjugation chemistry and used for specific targeting to αvβ3 integrin overexpressed cervical cancer cells and glioblastoma cells. Result shows that a silicon nanoparticle-based red fluorescent nanoprobe can be developed for in vitro/in vivo bioimaging applications.

Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy

As prepared nanomaterials of metals, semiconductors, polymers and carbon often need surface modifications such as ligand exchange, and chemical and bioconjugate reactions for various biosensor, bioanalytical, bioimaging, drug delivery and therapeutic applications. Such surface modifications help us to control the physico-chemical, toxicological and pharmacological properties of nanomaterials. Furthermore, introduction of various reactive functional groups on the surface of nanomaterials allows us to conjugate a spectrum of contrast agents, antibodies, peptides, ligands, drugs and genes, and construct multifunctional and hybrid nanomaterials for the targeted imaging and treatment of cancers. This tutorial review is intended to provide an introduction to newcomers about how chemical and bioconjugate reactions transform the surface of nanomaterials such as silica nanoparticles, gold nanoparticles, gold quantum clusters, semiconductor quantum dots, carbon nanotubes, fullerene and graphene, and accordingly formulate them for applications such as biosensing, bioimaging, drug and gene delivery, chemotherapy, photodynamic therapy and photothermal therapy. Nonetheless, controversial reports and our growing concerns about toxicity and pharmacokinetics of nanomaterials suggest the need for not only rigorous in vivo experiments in animal models but also novel nanomaterials for practical applications in the clinical settings. Further reading of original and review articles cited herein is necessary to buildup in-depth knowledge about the chemistry, bioconjugate chemistry and biological applications of individual nanomaterials.

Pycup—A Bifunctional, Cage-like Ligand for 64Cu Radiolabeling

In developing targeted probes for positron emission tomography (PET) based on (64)Cu, stable complexation of the radiometal is key, and a flexible handle for bioconjugation is highly advantageous. Here, we present the synthesis and characterization of the chelator pycup and four derivatives. Pycup is a cross-bridged cyclam derivative with a pyridyl donor atom integrated into the cross-bridge resulting in a pentadentate ligand. The pycup platform provides kinetic inertness toward (64)Cu dechelation and offers versatile bioconjugation chemistry. We varied the number and type of additional donor atoms by alkylation of the remaining two secondary amines, providing three model ligands, pycup2A, pycup1A1Bn, and pycup2Bn, in 3-4 synthetic steps from cyclam. All model copper complexes displayed very slow decomplexation in 5 M HCl and 90 °C (t1/2: 1.5 h for pycup1A1Bn, 2.7 h for pycup2A, 20.3 h for pycup2Bn). The single crystal crystal X-ray structure of the [Cu(pycup2Bn)](2+) complex showed that the copper was coordinated in a trigonal, bipyramidal manner. The corresponding radiochemical complexes were at least 94% stable in rat plasma after 24 h. Biodistribution studies conducted in Balb/c mice at 2 h postinjection of (64)Cu labeled pycup2A revealed low residual activity in kidney, liver, and blood pool with predominantly renal clearance observed. Pycup2A was readily conjugated to a fibrin-targeted peptide and labeled with (64)Cu for successful PET imaging of arterial thrombosis in a rat model, demonstrating the utility of our new chelator in vivo.

Gold nanoparticle-based gene delivery: promises and challenges

Gold nanoparticles have emerged as a promising material for biomedical research due to ease of synthesis and highly adjustable optical properties, which can be utilized in the imaging of different diseases. Gold nanoparticles are fabricated by grafting biocompatible polymers and natural or synthethic biomolecules and present a novel avenue for engineering multifunctional smart systems. Many reports on the significant achievements and the bioconjugation chemistry promise to expand the application spectrum of gold nanoparticles. This review summarizes the current state-of-the-art development of functionalized gold nanoparticles for cancer gene therapy.

Carboxyl decorated Fe3O4 nanoparticles for MRI diagnosis and localized hyperthermia

We report the development of carboxyl decorated iron oxide nanoparticles (CIONs) by a facile soft-chemical approach for magnetic resonance imaging (MRI) and hyperthermia applications. These superparamagnetic CIONs (∼10nm) are resistant to protein adsorption under physiological medium and exhibit good colloidal stability, magnetization and cytocompatibility with cell lines. Analysis of the T2-weighted MRI scans of CIONs in water yields a transverse relaxivity (r2) value of 215mM−1s−1. The good colloidal stability and high r2 value make these CIONs as promising candidates for high-efficiency T2 contrast agent in MRI. Further, these biocompatible nanoparticles show excellent self-heating efficacy under external AC magnetic field (AMF). The infrared thermal imaging confirmed the localized heating of CIONs under AMF. Thus, these carboxyl decorated Fe3O4 nanoparticles can be used as a contrast agent in MRI as well as localized heat activated killing of cancer cells. Furthermore, the active functional groups (COOH) present on the surface of Fe3O4 nanoparticles can be accessible for routine conjugation of biomolecules/drugs through well-developed bioconjugation chemistry.

Universal Dark Quencher Based on “Clicked” Spectrally Distinct Azo Dyes

The first synthesis of an heterotrifunctional molecular scaffold derived from the popular DABCYL azo dye quencher has been achieved. The sequential derviatization of this trivalent azobenzene derivative with two other nonfluorescent azo dyes (Black Hole Quencher BHQ-1 and BHQ-3) and through effective reactions from the "bioconjugation chemistry" repertoire has led to an universal dark quencher (UDQ). This "clicked" poly azo dye is able to turn off an array of fluorophores covering the UV/NIR (300-750 nm) spectral range.

Detection of Specific Protein–Protein Interactions in Nanocages by Engineering Bipartite FlAsH Binding Sites

Proteins that form cage-like structures have been of much recent cross-disciplinary interest due to their application to bioconjugate and materials chemistry, their biological functions spanning multiple essential cellular processes, and their complex structure, often defined by highly symmetric protein–protein interactions. Thus, establishing the fundamentals of their formation, through detecting and quantifying important protein–protein interactions, could be crucial to understanding essential cellular machinery, and for further development of protein-based technologies. Herein we describe a method to monitor the assembly of protein cages by detecting specific, oligomerization state dependent, protein–protein interactions. Our strategy relies on engineering protein monomers to include cysteine pairs that are presented proximally if the cage state assembles. These assembled pairs of cysteines act as binding sites for the fluorescent reagent FlAsH, which, once bound, provides a readout for successful oligomerization. As a proof of principle, we applied this technique to the iron storage protein, DNA-binding protein from starved cells from E. coli. Several linker lengths and conformations for the presentation of the cysteine pairs were screened to optimize the engineered binding sites. We confirmed that our designs were successful in both lysates and with purified proteins, and that FlAsH binding was dependent upon cage assembly. Following successful characterization of the assay, its throughput was expanded. A two-dimension matrix of pH and denaturing buffer conditions was screened to optimize nanocage stability. We intend to use this method for the high throughput screening of protein cage libraries and of conditions for the generation of inorganic nanoparticles within the cavity of these and other cage proteins.

Gold Nanoparticle-Conjugated Anti-Oxidized Low-Density Lipoprotein Antibodies for Targeted Lipidomics of Oxidative Stress Biomarkers

Oxidized low-density lipoproteins (OxLDLs), in particular, oxidized phosphatidylcholines (OxPCs), are known to be involved in pathophysiological processes such as cardiovascular diseases and are described as potential biomarkers, for example, for atherosclerosis. In our study, we used the specific affinity of anti-OxLDL antibodies (Abs) conjugated to gold nanoparticles (GNPs) for extraction and enrichment of OxPCs via selective trapping of OxLDLs from plasma combined with the sensitive detection by liquid chromatography/tandem-mass spectrometry (LC-MS/MS). Successful bioconjugation chemistry of Abs via a bifunctional polyethylene glycol (PEG) spacer and protein G linkage, respectively, was controlled by measuring the surface plasmon resonance (SPR) spectra, size, and zeta potentials. Furthermore, the amount of Ab immobilized onto GNP via the PEG linker was determined. With the optimized immobilization chemistry, the ability and potential of the GNP-based extraction procedure was used for the determination of the dissociation constant, K(d), of the OxLDL binding to the GNP-Ab conjugate. Moreover, apparent K(d)'s were determined for individual PCs and their oxidation products using the compound-specific selected reaction monitoring mode, which allows the characterization of the Ab affinity and, thus, assessment of the potential antigenicity of (Ox)PCs bound to OxLDLs. In summary, the application of GNP-based bioanalysis for selective targeting of OxLDLs and the fast and sensitive detection by LC-MS/MS offers new possibilities for targeted lipidomics in lipoproteins as well as for oxidative stress lipid biomarker screening.

ChemInform Abstract: α‐Oxo Aldehyde or Glyoxylyl Group Chemistry in Peptide Bioconjugation

AbstractReview: 272 refs.

Hydrazino-Pictet-Spengler Ligation as a Biocompatible Method for the Generation of Stable Protein Conjugates

Aldehyde- and ketone-functionalized biomolecules have found widespread use in biochemical and biotechnological fields. They are typically conjugated with hydrazide or aminooxy nucleophiles under acidic conditions to yield hydrazone or oxime products that are relatively stable, but susceptible to hydrolysis over time. We introduce a new reaction, the hydrazino-Pictet-Spengler (HIPS) ligation, which has two distinct advantages over hydrazone and oxime ligations. First, the HIPS ligation proceeds quickly near neutral pH, allowing for one-step labeling of aldehyde-functionalized proteins under mild conditions. Second, the HIPS ligation product is very stable (>5 days) in human plasma relative to an oxime-linked conjugate (∼1 day), as demonstrated by monitoring protein-fluorophore conjugates by ELISA. Thus, the HIPS ligation exhibits a combination of product stability and speed near neutral pH that is unparalleled by current carbonyl bioconjugation chemistries.

α-Oxo Aldehyde or Glyoxylyl Group Chemistry in Peptide Bioconjugation

Since the 1990s, α-oxo aldehyde or glyoxylic acid chemistry has inspired a vast array of synthetic tools for tailoring peptide or protein structures, for developing peptides endowed with novel physicochemical properties or biological functions, for assembling a large diversity of bioconjugates or hybrid materials, or for designing peptide-based micro or nanosystems. This past decade, important developments have enriched the α-oxo aldehyde synthetic tool box in peptide bioconjugation chemistry and explored novel applications. The aim of this review is to give a large overview of this creative field.

EPR-effect: Utilizing size-dependent Nanoparticle Delivery to Solid Tumors

EPR-effect: Utilizing size-dependent Nanoparticle Delivery to Solid Tumors

Efficient synthesis of diverse heterobifunctionalized clickable oligo(ethylene glycol) linkers: potential applications in bioconjugation and targeted drug delivery

Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible heterobifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (-OMs) group. The -OMs functional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers containing different highly reactive end groups, e.g., iodo, -NH(2), -SH and maleimido, that were orthogonal to the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generating larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG(16) and PEG(24)) by employing a Cu(I)-catalyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in bioconjugation chemistry was demonstrated by attachment of the integrin (α(v)β(3)) receptor targeting peptide, cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The synthetic methodology presented herein is suitable for the large scale production of several novel heterobifunctionalized OEGs from readily available and inexpensive starting materials.

Antibody‐Functionalized Porous Silicon Nanoparticles for Vectorization of Hydrophobic Drugs

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP.

Incorporation Efficiency of 5'-Azido-5'-Deoxyguanosine into 5'-Terminus of RNA for Preparation of Azido-Functionalized RNA

Although the modern biological systems are by large based on DNA genomes and protein enzymes, RNA plays important roles in many fundamental processes in cells, including regulation of protein biosynthesis, RNA splicing, and retroviral replication, with remarkable features. From this point of view, site-specific substitution and derivatization of RNA can provide powerful tools for elucidating RNA structures and functions. The modification of either the 3'and 5'-termini or an internal position of the oligonucleotides with a primary alkylamine group is a widely used method for introducing additional functional groups to the RNA. In particular, several 5'-modifications of RNA molecules such as sulfhydryl modification for functionalizing the 5'-terminus of RNA by a transcription or kinase reaction have been shown to have broad applications in studying RNA structures, mapping RNA-protein interactions, and in vitro selection of catalytic RNAs, since a unique functional group incorporated into the RNA can be subsequently conjugated to the desired molecule by a selective chemical reaction. However, there is still a need to develop coupling chemistry with high stability and yield to modify RNA and other biomolecules. In addition, the coupling functional groups are ideally required to be stable under aqueous reaction conditions, and the coupling reaction should be highly chemoselective. In this regard, Cu-catalyzed azide-alkyne cycloaddition (CuAAC or click chemistry) to form the triazole version of Huisgen’s [2+3] cycloaddition family may be the best choice, because this reaction only occurs between alkynyl and azido functional groups with high yield, and because the resulting 1,2,3-triazoles are stable at aqueous conditions and high temperature. Indeed, the azide group is one of the most utilized bioorthogonal chemical tags for biomolecule-conjugate experiments because of its small size and inertness to most components in a biological environment. We recently reported a two-step synthetic method for 5'azido-5'-deoxyguanosine (azido-G) by adapting literature procedures (Scheme 1) and its efficiency for click chemistry using 6-heptynoyl p-nitroaniline, in consideration that the click chemistry with rapid reaction between the azide and alkyne groups to form a covalent triazole linkage without cross reacting with other functional groups could be used in bioconjugation chemistry. In the present study, we sought enzymatic methods to incorporate azido groups in RNA and measured incorporation efficiency of azido-G into the 5'terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription. This is important in consideration that a terminal azido group is able to be introduced to the 5'termini of RNA molecules, and that azides are unstable under solid-phase synthesis conditions. A 97-mer single-stranded DNA containing a T7 promoter at the 3'-end (5'CAG GAC TGC TCT CAC TCT CAC GCA CCA AGA AGC TGC CAT TGA TCC CGC TGC