Bioconjugation Chemistry Research Articles

Strain-Promoted “Click” Chemistry for Terminal Labeling of DNA

1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.

The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.

ChemInform Abstract: Surface Plasmon Resonance Biosensorics in Urine Proteomics

AbstractReview: 45 refs.

Intra- and inter-molecular cross-linking of peptide ions in the gas phase: reagents and conditions.

Intra-molecular and inter-molecular cross-linking of protonated polypeptide ions in the gas phase via ion/ion reactions have been demonstrated using N-hydroxysulfosuccinimide (sulfo-NHS)- based reagent anions. The initial step in the ion/ion reaction involves the formation of a long-lived complex between the peptide and reagent, which is a prerequisite for the covalent bioconjugation chemistry. The sulfonate groups on the NHS rings of the homo-bifunctional cross-linking reagents have high affinity for the protonated sites in the peptide and, therefore, facilitate the long-lived complex formation. In addition to the formation of a long-lived chemical complex, intra-molecular cross-linking also requires two unprotonated primary amine sites within a molecule where the covalent modification takes place. Alternatively, inter-molecular cross-linking demands the availability of one neutral primary amine site in each of the two peptides that are being cross-linked. Nucleophilic displacement of two sulfo-NHS groups by the amine functionalities in the peptide is a signature of the covalent cross-linking chemistry in the gas phase. Upon removal of the two sulfo-NHS groups, two amide bonds are formed between an unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent.

Squaramides: physical properties, synthesis and applications

Squaramides are remarkable four-membered ring systems derived from squaric acid that are able to form up to four hydrogen bonds. A high affinity for hydrogen bonding is driven through a concomitant increase in aromaticity of the ring. This hydrogen bonding and aromatic switching, in combination with structural rigidity, have been exploited in many of the applications of squaramides. Substituted squaramides can be accessed via modular synthesis under relatively mild or aqueous conditions, making them ideal units for bioconjugation and supramolecular chemistry. In this tutorial review the fundamental electronic and structural properties of squaramides are explored to rationalise the geometry, conformation, reactivity and biological activity.

The art of engineering viral nanoparticles.

Viral nanotechnology is an emerging and highly interdisciplinary field in which viral nanoparticles (VNPs) are applied in diverse areas such as electronics, energy and next-generation medical devices. VNPs have been developed as candidates for novel materials, and are often described as "programmable" because they can be modified and functionalized using a number of techniques. In this review, we discuss the concepts and methods that allow VNPs to be engineered, including (i) bioconjugation chemistries, (ii) encapsulation techniques, (iii) mineralization strategies, and (iv) film and hydrogel development. With all these techniques in hand, the potential applications of VNPs are limited only by the imagination.

Quantum Dot DNA Bioconjugates: Attachment Chemistry Strongly Influences the Resulting Composite Architecture

The unique properties provided by hybrid semiconductor quantum dot (QD) bioconjugates continue to stimulate interest for many applications ranging from biosensing to energy harvesting. Understanding both the structure and function of these composite materials is an important component in their development. Here, we compare the architecture that results from using two common self-assembly chemistries to attach DNA to QDs. DNA modified to display either a terminal biotin or an oligohistidine peptidyl sequence was assembled to streptavidin/amphiphilic polymer- or PEG-functionalized QDs, respectively. A series of complementary acceptor dye-labeled DNA were hybridized to different positions on the DNA in each QD configuration and the separation distances between the QD donor and each dye-acceptor probed with Förster resonance energy transfer (FRET). The polyhistidine self-assembly yielded QD-DNA bioconjugates where predicted and experimental separation distances matched reasonably well. Although displaying efficient FRET, data from QD-DNA bioconjugates assembled using biotin-streptavidin chemistry did not match any predicted separation distances. Modeling based upon known QD and DNA structures along with the linkage chemistry and FRET-derived distances was used to simulate each QD-DNA structure and provide insight into the underlying architecture. Although displaying some rotational freedom, the DNA modified with the polyhistidine assembles to the QD with its structure extended out from the QD-PEG surface as predicted. In contrast, the random orientation of streptavidin on the QD surface resulted in DNA with a wide variety of possible orientations relative to the QD which cannot be controlled during assembly. These results suggest that if a particular QD biocomposite structure is desired, for example, random versus oriented, the type of bioconjugation chemistry utilized will be a key influencing factor.

Kinetic studies and predictions on the hydrolysis and aminolysis of esters of 2-S-phosphorylacetates

Background: Heterobifunctional cross-linking agents are useful in both protein science and organic synthesis. Aminolysis of reactive esters in aqueous systems is often used in bioconjugation chemistry, but it must compete against hydrolysis processes. Here we study the kinetics of aminolysis and hydrolysis of 2-S-phosphorylacetate ester intermediates that result from displacement of bromide by a thiophosphate nucleophile from commonly used bromoacetate ester cross-linking agents.Results: We found cross-linking between uridine-5′-monophosphorothioate and D-glucosamine using N-hydroxybenzotriazole and N-hydroxysuccinimde bromoacetates to be ineffective. In order to gain insight into these shortfalls, 2-S-(5′-thiophosphoryluridine)acetic acid esters were prepared using p-nitrophenyl bromoacetate or m-nitrophenyl bromoacetate in combination with uridine-5′-monophosphorothioate. Kinetics of hydrolysis and aminolysis of the resulting p- and m-nitrophenyl 2-S-(5′-thiophosphoryluridine)acetates were determined by monitoring the formation of phenolate ions spectrophotometrically as a function of pH. The p- and m-nitrophenyl 2-S-(5′-thiophosphoryluridine)acetates showed similar reactivity profiles despite the significant difference in the pKaH values of their nitrophenolate leaving groups. Both were more reactive with respect to hydrolysis and aminolysis in comparison to their simple acetate progenitors, but their calculated selectivity towards aminolysis vs hydrolysis, while reasonable, would not lead to clean reactions that do not require purification. Extrapolations of the kinetic data were used to predict leaving group pKa values that could lead to improved selectivity towards aminolysis while retaining reasonable reaction times.Conclusions: Both p- and m-nitrophenyl 2-S-(5′-thiophosphoryluridine)acetates show some selectivity towards aminolysis over hydrolysis, with the m-nitrophenolate system displaying slightly better selectivity. Extrapolation of the data for hydrolysis and aminolysis of these esters suggests that the use of readily accessible trifluoroethyl 2-S-(5′-thiophosphoryluridine)acetate with a leaving group pKaH of 12.4 should afford better selectivity while maintaining reasonable reaction times. Kinetically, p- and m-nitrophenyl 2-S-(5′-thiophosphoryluridine)acetates show similar properties to o-nitrophenyl 2-S-ethylacetate, and show no evidence for intramolecular catalysis of hydrolysis or aminolysis by the phosphoryl groups.

Near-Infrared Quantum Dots as Optical Probes for Tumor Imaging

Molecular imaging plays a key role in personalized medicine, which is the goal and future of patient management. Among the various molecular imaging modalities, optical imaging may be the fastest growing area for bioanalysis, and the major reason is the research on fluorescence semiconductor quantum dots (QDs) and dyes have evolved over the past two decades. The great efforts on the synthesis of QDs with fluorescence emission from UV to near-infrared (NIR) regions speed up the studies of QDs as optical probes for in vitro and in vivo molecular imaging. For in vivo applications, the fluorescent emission wavelength ideally should be in a region of the spectrum where blood and tissue absorb minimally and tissue penetration reach maximally, which is NIR region (typically 700-1000 nm). The goal of this review is to provide readers the basics of NIR-emitting QDs, the bioconjugate chemistry of QDs, and their applications for diagnostic tumor imaging. We will also discuss the benefits, challenges, limitations, perspective, and the future scope of NIR-emitting QDs for tumor imaging applications.

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Synaptotagmin II peptide-bead conjugate for botulinum toxin enrichment and detection in microchannels

This paper reports an enrichment platform for botulinum neurotoxin type B (BoNT/B) that has been realized through the fusion of bioconjugation chemistry and microfluidics. Micrometer-sized magnetic beads were conjugated to a 22mer synthetic peptide derived from the synaptotagmin II (Syt II) neuronal protein that is specific for BoNT/B binding. Exposure to BoNT/B in buffer, whole milk and fruit juices resulted in toxin capture, which was confirmed using immunofluorescence. Peptide-modified beads were integrated into arrayed, polymeric microfluidic channels, and all assay steps, from capture to detection, were performed directly in the microchannels, thereby simplifying assay utility and increasing throughput relative to existing detection methodologies. Our sensitive microscale approach required only 7 μL of intentionally adulterated sample without any pre-processing (i.e. dilution, centrifugation, filtering), and with a “hands-on” time of only 1 h to detect 16.6 pg of BoNT/B in whole milk.

Methods for detection and measurement of hydrogen peroxide inside and outside of cells.

Hydrogen peroxide (H(2)O(2)) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H(2)O(2) requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H(2)O(2) in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3'5'-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H(2)O(2), methods based on dihydro compounds such as 2',7'-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H(2)O(2). Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H(2)O(2)-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H(2)O(2) in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H(2)O(2) can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H(2)O(2), they cannot be used to monitor transient changes in H(2)O(2) concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H(2)O(2) and can be targeted to various compartments of the cell, but they are not selective for H(2)O(2) because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H(2)O(2)-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.

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Citations

Nanotechnology Challenges in Targeted Delivery of Biopharmaceutics

Event Abstract Back to Event Nanotechnology Challenges in Targeted Delivery of Biopharmaceutics Costas Kiparissides1*, Olga Kammona1 and Vassilis Karageorgiou1 1 Aristotle University of Thessaloniki and Chemical Process Engineering Research Institute, Department of Chemical Engineering, Greece New Peptidic/Proteinic (P/P) drugs are being discovered every day and their increased availability offers new ways to treat and prevent diseases. However, the structure, physicochemical properties, stability, pharmacodynamics and pharmacokinetics of these new biopharmaceutics place stringent demands on the way they are delivered into the body. Additionally, P/P drugs do not easily cross mucosal surfaces and biological membranes, are easily denatured or degraded, prone to rapid clearance in the liver and other body tissues, and require precise dosing. Major goals of nanomedicine in terms of controlled delivery of biopharmaceutics, are the maximization of their bioavailability and efficacy, the control of pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity and biorecognition as well as the overcoming of obstacles arising from low solubility, degradation, fast clearance rates, relatively short-lasting biological activity and inability to cross biological barriers. The above goals are expected to be achieved, through the development of targeted delivery systems that can be selectively delivered to specific areas in the human body. However, since P/P drug characteristics differ substantially with respect to chemical composition, molecular size, hydrophilicity, bioavailability, optimum concentration range, etc., the essential characteristics that identify the efficiency of the delivery systems are highly complex. Thus, their development has to be pursued as a multi disciplinary effort, firmly built on extensive experience in polymer science, pharmacochemistry, pharmacology, molecular biology, bioconjugate chemistry and toxicology. Recent advances in the delivery of biopharmaceutics deal with the development of synthetic nanometer sized targeted delivery systems in the form of nanocarriers (e.g., polymeric or hybrid nanoparticles, nanogels, lipid based vesicles, dendrimers) and molecular carriers (e.g., polyelectrolyte complexes, polymer-P/P drug complexes). Targeted delivery systems can have multiple functions, a key one being their ability to recognize specific molecules which can be located either in the membrane of target cells, or in specific compartments within the cell. The carrier-based drug delivery systems can improve the bioavailability and diminish the toxicity of P/P drugs, control their release profile and make alternative administration routes possible. A challenging objective of targeted delivery is the development of novel nanocarriers and molecular carriers for the targeted delivery of biopharmaceutics via oral, nasal and Blood Brain Barrier (BBB) crossing administration routes. Keywords: Nanotechnology, Targeted Drug Delivery, Biopharmaceutics Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Invited speaker Topic: Nanopharmacology / Nanomedicine Citation: Kiparissides C, Kammona O and Karageorgiou V (2010). Nanotechnology Challenges in Targeted Delivery of Biopharmaceutics. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00142 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 Mar 2011; Published Online: 04 Nov 2010. * Correspondence: Dr. Costas Kiparissides, Aristotle University of Thessaloniki and Chemical Process Engineering Research Institute, Department of Chemical Engineering, Thessaloniki, 54124, Greece, cypress@cperi.certh.gr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Costas Kiparissides Olga Kammona Vassilis Karageorgiou Google Costas Kiparissides Olga Kammona Vassilis Karageorgiou Google Scholar Costas Kiparissides Olga Kammona Vassilis Karageorgiou PubMed Costas Kiparissides Olga Kammona Vassilis Karageorgiou Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Advances in Bioconjugation.

Bioconjugation is a burgeoning field of research. Novel methods for the mild and site-specific derivatization of proteins, DNA, RNA, and carbohydrates have been developed for applications such as ligand discovery, disease diagnosis, and high-throughput screening. These powerful methods owe their existence to the discovery of chemoselective reactions that enable bioconjugation under physiological conditions-a tremendous achievement of modern organic chemistry. Here, we review recent advances in bioconjugation chemistry. Additionally, we discuss the stability of bioconjugation linkages-an important but often overlooked aspect of the field. We anticipate that this information will help investigators choose optimal linkages for their applications. Moreover, we hope that the noted limitations of existing bioconjugation methods will provide inspiration to modern organic chemists.

金クラスターおよびナノ粒子の無機バイオコンジュゲート

In this review, we focus on the recent development of conjugates between nanoparticles (NPs) and biomolecules such as DNAs, as well as a brief history for inorganic bioconjugate chemistry. NP-biomolecule combination is heterogeneous and thus challenging. This research area was begun about a decade ago as a basic attempt to align AuNPs along DNA. Today, NP–DNA is a current topic whose developments can be applied in diagnosis, new devices, and drug delivery systems. Among these, the nanoscaled patterning of mesoscale NPs is the vital target of current material science, in which the conjugation of gold (Au) NPs and biomolecules is gaining immense importance. The positioning or alignment of NPs on the desired points of highly constructed DNA architectures is an important step for “DNA lithography” that leads to new electronic and optical devices. This review also introduces the current topics in the DNA architecture and its application for AuNP alignment.

Chemical conjugation of biomacromolecules: A mini-review

AbstractBiological studies showed that assembles of biomolecules can dramatically change their physiological effectiveness. Covalent coupling of different types of biomolecules leads to novel biomacromolecules of different properties. Generally, bioconjugate chemistry opens a new dimension in biomedical and biotechnology research. In this review, some important chemical methods of bioconjugates preparation used in the practice are described. Proteins and saccharides modification methods and employment of linkers used to achieve new functionalities are discussed. Common bioconjugation methods are emphasized and novel methods from recent years are described. Except in chemistry, benefits and limits of the studied methods are outlined.

Immobilization of bacteriophages on modified silica particles

Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge – to a maximum – correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops.

Potato Virus X as a Novel Platform for Potential Biomedical Applications

We demonstrate that nanoparticles formed from the rod-shaped plant virus Potato virus X (PVX) can serve as a novel platform for biomedical applications. Bioconjugation protocols including amine modification and "click" chemistry allowed the efficient functionalization of PVX with biotins, dyes, and PEGs. Fluorescent-labeled and PEGylated PVX particles revealed that different fluorescent labels have a profound effect on PVX-cell interactions. Applying bioconjugation chemistries to PVX opens the door for chemical functionalization with targeting and therapeutic molecules.

Research Highlights

Research Highlights