Oxime Bond Formation Research Articles

Rapid and Sequential Dual Oxime Ligation Enables De Novo Formation of Functional Synthetic Membranes from Water‐Soluble Precursors

Cell membranes define the boundaries of life and primarily consist of phospholipids. Living organisms assemble phospholipids by enzymatically coupling two hydrophobic tails to a soluble polar head group. Previous studies have taken advantage of micellar assembly to couple single-chain precursors, forming non-canonical phospholipids. However, biomimetic nonenzymatic coupling of two alkyl tails to a polar head-group remains challenging, likely due to the sluggish reaction kinetics of the initial coupling step. Here we demonstrate rapid de novo formation of biomimetic liposomes in water using dual oxime bond formation between two alkyl chains and a phosphocholine head group. Membranes can be generated from non-amphiphilic, water-soluble precursors at physiological conditions using micromolar concentrations of precursors. We demonstrate that functional membrane proteins can be reconstituted into synthetic oxime liposomes from bacterial extracts in the absence of detergent-like molecules.

Rapid and Sequential Dual Oxime Ligation Enables De Novo Formation of Functional Synthetic Membranes from Water-Soluble Precursors.

Cell membranes define the boundaries of life and primarily consist of phospholipids. Living organisms assemble phospholipids by enzymatically coupling two hydrophobic tails to a soluble polar head group. Previous studies have taken advantage of micellar assembly to couple single-chain precursors, forming non-canonical phospholipids. However, biomimetic nonenzymatic coupling of two alkyl tails to a polar head-group remains challenging, likely due to the sluggish reaction kinetics of the initial coupling step. Here we demonstrate rapid de novo formation of biomimetic liposomes in water using dual oxime bond formation between two alkyl chains and a phosphocholine head group. Membranes can be generated from non-amphiphilic, water-soluble precursors at physiological conditions using micromolar concentrations of precursors. We demonstrate that functional membrane proteins can be reconstituted into synthetic oxime liposomes from bacterial extracts in the absence of detergent-like molecules.

PH-Responsive benzaldehyde-functionalized PEG-based polymeric nanoparticles for drug delivery: Effect of preparation method on morphology, dye encapsulation and attachment

Functionalized, pH-responsive and biocompatible polymeric nanoparticles have attracted the interest of many researchers working on novel pharmaceutical formulations. Here, in an effort to develop an efficient drug delivery formulation, all these properties are combined in one polymeric nanoparticle system. More specifically, benzaldehyde-functionalized amphiphilic block copolymers based on PEG-based oligo(ethylene glycol) methacrylate (OEGMA), benzaldehyde-containing para-formyl phenyl methacrylate (pFPMA) and pH-responsive 2-(diisopropyl)aminoethyl methacrylate (DPA) monomers are readily prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. pH-Switch and single emulsion-solvent evaporation post-polymerization processing methods are used to prepare benzaldehyde-functionalized PEGylated pH-responsive nanoparticles with diameters of 180–230 nm. After nanoparticle formation, the benzaldehyde groups on the surface are shown to react with an Alexa Fluor 488 hydroxylamine dye through oxime bond formation, illustrating the potential for these particles to be surface-functionalized with biologically important molecules, such as fluorescent dyes for tracking their intracellular fate or antibodies for targeted therapy. Encapsulation of Nile Red and rhodamine 6G dyes is performed during the post-polymerization processing step for nanoparticle formation. Nanoparticles with a fluorescent cargo were shown to be successfully internalized in both A2780 ovarian cancer and A549 lung epithelial human cells in vitro, further illustrating the potential for these formulations to be used as triggered release therapeutic drug delivery vehicles.

Mechanically tunable, human mesenchymal stem cell viable poly(ethylene glycol)–oxime hydrogels with invariant precursor composition, concentration, and stoichiometry

Hydrogels are used widely for exploratory tissue engineering studies. However, currently, no hydrogel systems have been reported which exhibit a wide range of elastic modulus without changing precursor concentration, identity, or stoichiometry. Herein, ester- and amide-based poly(ethylene glycol) (PEG)–oxime hydrogels with tunable moduli (∼5–30 kPa) were synthesized with identical precursor mass fraction, stoichiometry, and concentration by varying the pH and buffer concentration of the gelation solution, exploiting the kinetics of oxime bond formation. The observed modulus range can be attributed to increasing amounts of network defects in slower forming gels, as confirmed by equilibrium swelling and small-angle neutron scattering (SANS) experiments. Finally, human mesenchymal stem cell (hMSC) viability was confirmed in these materials in a 24-h assay. While this was only an initial demonstration of the potential utility, the controlled variation in defect density and modulus is an important step forward in isolating system variables for hypothesis-driven biological investigations.

Single-Domain Antibody-Functionalized pH-Responsive Amphiphilic Block Copolymer Nanoparticles for Epidermal Growth Factor Receptor Targeted Cancer Therapy.

Biocompatible antibody-nanoparticle conjugates have attracted interest as anticancer agents due to their potential to selectively target therapeutic agents at disease sites. However, new formulation and conjugation approaches are urgently needed to improve their uniformity for clinical applications. Here, a pH-responsive benzaldehyde-functionalized poly[oligo(ethylene glycol) methacrylate-st-para-formyl phenyl methacrylate]-b-poly[2-(diisopropyl)aminoethyl methacrylate] [P(OEGMA-st-pFPMA)-b-PDPA] block copolymer, prepared by reversible addition-fragmentation chain transfer polymerization, produced PEGylated nanoparticles (pH ∼ 7.4) by a single emulsion-solvent evaporation formulation approach. Efficient site-specific attachment of an aminooxy-functionalized anti-EGFR single-domain antibody (sdAb) on these benzaldehyde-decorated nanoparticles is achieved by oxime bond formation. These nanoconjugates can specifically bind EGFR (modified ELISA) and have enhanced uptake over nonfunctionalized controls in EGFR-positive HeLa cells. Encapsulation of rhodamine 6G dye and its dispersion upon cellular uptake, consistent with nanoparticle stability loss at pH < 5.7, prove their ability to facilitate triggered release in endosomal compartments and highlight their potential for use as next-generation antibody-drug nanoconjugates for therapeutic drug delivery.

Glycosyl Aldehydes: New Scaffolds for the Synthesis of Neoglycoconjugates via Bioorthogonal Oxime Bond Formation

The straightforward preparation of glycosyl neoconjugates by oxime (or hydrazone) bond formation represents a key bioorthogonal tool in chemical biology. However, when this strategy is employed by reacting the reducing end of the glycan moiety, the configuration and the stereochemical information is lost due to partial (or complete) opening of the glycan cyclic hemiacetal and the formation of the corresponding opened tautomers. We have completed the synthesis of a library of glycosyl aldehydes to be used as scaffold for the synthesis of neoglycoconjugates via oxime bond formation. These glycosyl aldehydes constitute a simple and accessible alternative to avoid loss of chiral information when conjugating, by oxime (or hydrazone) bonds, the aldehyde functionality present at the reducing end of natural carbohydrates.

In vivo characterisation of a therapeutically relevant self-assembling 18 F-labelled β-sheet forming peptide and its hydrogel using positron emission tomography.

Positron emission tomography (PET) and fluorescence labelling have been used to assess the pharmacokinetics, biodistribution and eventual fate of a hydrogel‐forming nonapeptide, FEFKFEFKK (F9), in healthy mice, using 18F‐labelled and fluorescein isothiocyanate (FITC)‐labelled F9 analogues. F9 was site‐specifically radiolabelled with 2‐[18F]fluoro‐3‐pyridinecarboxaldehyde ([18F]FPCA) via oxime bond formation. [18F]FPCA‐F9 in vivo fate was evaluated both as a solution, following intravenous administration, and as a hydrogel when subcutaneously injected. The behaviour of FITC‐F9 hydrogel was assessed following subcutaneous injection. [18F]FPCA‐F9 demonstrated high plasma stability and primarily renal excretion; [18F]FPCA‐F9 when in solution and injected into the bloodstream displayed prompt bladder uptake (53.4 ± 16.6 SUV at 20 minutes postinjection) and rapid renal excretion, whereas [18F]FPCA‐F9 hydrogel, formed by co‐assembly of [18F]FPCA‐F9 monomer with unfunctionalised F9 peptide and injected subcutaneously, showed gradual bladder accumulation of hydrogel fragments (3.8 ± 0.4 SUV at 20 minutes postinjection), resulting in slower renal excretion. Gradual disaggregation of the F9 hydrogel from the site of injection was monitored using FITC‐F9 hydrogel in healthy mice (60 ± 3 over 96 hours), indicating a biological half‐life between 1 and 4 days. The in vivo characterisation of F9, both as a gel and a solution, highlights its potential as a biomaterial.

Tuning the Properties of Penetrating Peptides by Oxime Conjugation

We here describe the application of oxime bond formation between a peptide scaffold and different aldehydes to modify the transporting capabilities of penetrating peptides. We show that bonds such as oximes offer a great synthetic advantage for the modification of the properties of model penetrating peptides. We believe that this approach will allow the development of improved intracellular delivery vehicles.

Tandem β-elimination/hetero-michael addition rearrangement of an N-alkylated pyridinium oxime to an O-alkylated pyridine oxime ether: an experimental and computational study.

A novel OH(-)-promoted tandem reaction involving C(β)-N(+)(pyridinium) cleavage and ether C(β)-O(oxime) bond formation in aqueous media has been presented. The study fully elucidates the fascinating reaction behavior of N-benzoylethylpyridinium-4-oxime chloride in aqueous media under mild reaction conditions. The reaction journey begins with the exclusive β-elimination and formation of pyridine-4-oxime and phenyl vinyl ketone and ends with the formation of O-alkylated pyridine oxime ether. A combination of experimental and computational studies enabled the introduction of a new type of rearrangement process that involves a unique tandem reaction sequence. We showed that (E)-O-benzoylethylpyridine-4-oxime is formed in aqueous solution by a base-induced tandem β-elimination/hetero-Michael addition rearrangement of (E)-N-benzoylethylpyridinium-4-oximate, the novel synthetic route to this engaging target class of compounds. The complete mechanistic picture of this rearrangement process was presented and discussed in terms of the E1cb reaction scheme within the rate-limiting β-elimination step.

Synthesis of the tumor associative α-aminooxy disaccharide of the TF antigen and its conjugation to a polysaccharide immune stimulant

The α-aminooxy derivative of the Thomsen-Friedenriech tumor associated carbohydrate antigen has been synthesized in 11 steps utilizing a D-GalN3 acceptor carrying a pre-installed α-N-hydroxysuccinimidyl moiety. The natural α linkage was prepared in high selectivity employing a suitably protected D-GalN3-thioglycoside donor with N-hydroxysuccinimide. With access to α-TF-ONH2, the preparation of the TF-PS A1 vaccine candidate ensued smoothly through oxime bond formation.

A novel approach for the parallel synthesis of glycopeptides by combining solid-phase peptide synthesis and dendrimer-supported enzymatic modifications

Functionalized, high-generation (G7) polyamidoamine (PAMAM) dendrimers are a convenient scaffold for the fully automated enzymatic synthesis of oligosaccharides such as biologically important sialyl Lewis X tetrasaccharide derivatives. In this study, we expanded this strategy to the synthesis of more complicated glycopeptides by assessing the feasibility of G7 PAMAM dendrimer-based polymer supports for attaching glycopeptide intermediates during the subsequent enzymatic modification steps. A monosaccharide-attached glycopeptide containing an N-terminal heterobifunctional linker was prepared by microwave-assisted solid-phase synthesis and was coupled with an aminooxy-functionalized G7 PAMAM dendrimer through oxime bond formation. This reaction proceeded smoothly at pH 4 and afforded the conjugates in 98% yield when 0.2 equivalents of the glycopeptide were combined with 1 equivalent of the aminooxy group of dendrimer. Although modifications using recombinant human β1,4-galactosyltransferase/uridine-5′-diphospho-α-D-galactose disodium salt and recombinant rat α2,3-sialyltransferase/cytidine-5′-monophospho-β-D-N-acetylneuraminic acid disodium salt gave the trisaccharide Neu5Acα2,3Galβ1,4GlcNAc in quantitative yields, treatment with recombinant human α1,3-fucosyltransferase in the presence of excess guanosine 5′-diphospho-β-L-fucose disodium salt did not convert this trisaccharide into the tetrasaccharide sialyl Lewis X tetrasaccharide on the dendrimer. Further optimization studies are required to improve the efficiency of branched-type sugar elongations and product release from polymers by selective peptidases for constructing a high-throughput glycopeptide synthetic system. Toward parallel enzymatic synthesis of biologically and pharmaceutically important glycopeptides, we investigated the feasibility of aminooxy-functionalized G7 PAMAM dendrimer derivative in the enzyme-assisted synthesis of glycopeptides having oligosaccharide side chains. The G7 PAMAM dendrimer was proved to become a nice supporting material that exhibited 99% recovery in a simple centrifugal ultrafiltration (UF) procedure in comparison with the hollow-fiber UF module (86%) used in the high-performance liquid chromatography-based automated glycan synthesizer. An optimization of the protocol was performed for the synthesis of glycopeptides bearing di-, tri- and tetrasaccharide branches.

Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels

Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3′ or 5′ end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3′ end and an electrochemical label at 5′ end.

Quantitative Evaluation of Bioorthogonal Chemistries for Surface Functionalization of Nanoparticles

We present here a highly efficient and chemoselective liposome functionalization method based on oxime bond formation between a hydroxylamine and an aldehyde-modified lipid component. We have conducted a systematic and quantitative comparison of this new approach with other state-of-the-art conjugation reactions in the field. Targeted liposomes that recognize overexpressed receptors or antigens on diseased cells have great potential in therapeutic and diagnostic applications. However, chemical modifications of nanoparticle surfaces by postfunctionalization approaches are less effective than in solution and often not high-yielding. In addition, the conjugation efficiency is often challenging to characterize and therefore not addressed in many reports. We present here an investigation of PEGylated liposomes functionalized with a neuroendocrine tumor targeting peptide (TATE), synthesized with a variety of functionalities that have been used for surface conjugation of nanoparticles. The reaction kinetics and overall yield were quantified by HPLC. Reactions were conducted in solution as well as by postfunctionalization of liposomes in order to study the effects of steric hindrance and possible affinity between the peptide and the liposome surface. These studies demonstrate the importance of choosing the correct chemistry in order to obtain a quantitative surface functionalization of liposomes.

Modular oxime functionalization of well-defined alkoxyamine-containing polymers

Phthalamide-protected O-(4-vinylbenzyl)-hydroxylamine was polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization with good control of the polymer molecular weight and retention of chain end functionality. The resulting polymer was deprotected by cleavage of the phthalamido protecting groups via treatment with hydrazine to reveal the latent side-chain alkoxyamine functionality (R–O–NH2). The alkoxyamine polymer scaffold was coupled with model small molecule aldehydes and ketones via highly efficient “click” oxime bond formation. The ability of the coupling reactions to be conducted at a variety of temperatures, in the presence of oxygen, and without any additional reagents makes this an attractive modular strategy for preparing well-defined polymers with high degrees of functionality.

Combination of Integrin-Binding Peptide and Growth Factor Promotes Cell Adhesion on Electron-Beam-Fabricated Patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.

Engineering Cell Surfaces via Liposome Fusion

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.

Protein nanopatterns by oxime bond formation.

Patterning proteins on the nanoscale is important for applications in biology and medicine. As feature sizes are reduced, it is critical that immobilization strategies provide site-specific attachment of the biomolecules. In this study, oxime chemistry was exploited to conjugate proteins onto nanometer-sized features. Poly(Boc-aminooxy tetra(ethylene glycol) methacrylate) was synthesized by free radical polymerization. The polymer was patterned onto silicon wafers using an electron beam writer. Trifluoroacetic acid removal of the Boc groups provided the desired aminooxy functionality. In this manner, patterns of concentric squares and contiguous bowtie shapes were fabricated with 150-170-nm wide features. Ubiquitin modified at the N-terminus with an α-ketoamide group and N(ε)-levulinyl lysine-modified bovine serum albumin were subsequently conjugated to the polymer nanopatterns. Protein immobilization was confirmed by fluorescence microscopy. Control studies on protected surfaces and using proteins presaturated with O-methoxyamine indicated that attachment occurred via oxime bond formation.

Efficient synthesis of an (aminooxy) acetylated‐somatostatin derivative using (aminooxy)acetic acid as a ‘carbonyl capture’ reagent

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of an (aminooxy)acetylated-somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a 'carbonyl capture' reagent in the final cleavage step. This (aminooxy)acetylated compound was further used for the chemoselective ligation (oxime bond formation) with daunorubicin and 4-fluorobenzaldehyde leading to the formation of conjugates with potential applications in targeted cancer chemotherapy and positron emission tomography.

Dynamic Covalent Diblock Copolymers Prepared from RAFT Generated Aldehyde and Alkoxyamine End-Functionalized Polymers

Aldehyde- or alkoxyamine-containing trithiocarbonate chain transfer agents were prepared and used to mediate the synthesis of a series of polymers end-functionlized with either aldehyde or alkoxyamines functions, utilizing reversible addition−fragmentation polymerization techniques. Aldehyde end-functionalized polymers were shown to link through reversible oxime bond formation with alkoxyamine end-funtionalized polymers forming diblock copolymers. The dynamic nature of the oxime bond linking these polymer blocks together was demonstrated through a simple exchange reaction with a small molecule alkoxyamine. A diblock copolymer prepared from the self-assembly of an aldehyde end-functionalized polyisoprene with an alkoxyamine end-functionalized polystyrene was shown to undergo further hierarchical self-assembly into micellar aggregates in DMF. It was shown that the addition of an excess of a small molecule alkoxyamine triggered the disassembly of these micelles.

One‐Pot Approach to Well‐Defined Biomolecular Assemblies by Orthogonal Chemoselective Ligations

Click-click cyclopeptides: Well-defined biomolecular assemblies are synthesized using orthogonal oxime bond formation and copper(I)-mediated alkyne-azide cycloaddition reactions in a stepwise or in a one-pot approach. To illustrate this strategy, regioselective ligation of biologically relevant peptides onto a cyclopeptidic scaffold was performed.