Conjugation Of Small Molecules Research Articles

Cell-surface engineering by a conjugation-and-release approach based on the formation and cleavage of oxime linkages upon mild electrochemical oxidation and reduction.

We report a strategy to rewire cell surfaces for the dynamic control of ligand composition on cell membranes and the modulation of cell-cell interactions to generate three-dimensional (3D) tissue structures applied to stem-cell differentiation, cell-surface tailoring, and tissue engineering. We tailored cell surfaces with bioorthogonal chemical groups on the basis of a liposome-fusion and -delivery method to create dynamic, electroactive, and switchable cell-tissue assemblies through chemistry involving chemoselective conjugation and release. Each step to modify the cell surface: activation, conjugation, release, and regeneration, can be monitored and modulated by noninvasive, label-free analytical techniques. We demonstrate the utility of this methodology by the conjugation and release of small molecules to and from cell surfaces and by the generation of 3D coculture spheroids and multilayered cell tissues that can be programmed to undergo assembly and disassembly on demand.

Unnatural amino acids in novel antibody conjugates.

Antibody-drug conjugates are an important and emerging drug class for the treatment of cancer. Recent evidence strongly suggests that site-specific drug conjugation results in a homogenous population of molecules with more favorable activity and pharmacokinetic properties than randomly conjugated antibodies. Unnatural amino acids (uAAs) can be incorporated in recombinant proteins to enable unique orthogonal chemistries in comparison to the side chains of the natural 20 amino acids. Thus, uAAs present a novel platform for which to create next-generation antibody-drug conjugates. Furthermore, site-specific conjugation through uAAs can also enpower unique small molecule, bispecific, multispecific and other conjugates that could be important constructs for therapeutics, diagnostics and research reagents. Here, we review the progress in uAA incorporation and conjugate construction through both cell-based and -free approaches.

Click modification of multifunctional liposomes bearing hyperbranched polyether chains.

Aiming at controlled modification of liposomal surface structures, we describe a postpreparational approach for surface derivatization of a new type of multifunctional, sterically stabilized liposomes. Application of dual centrifugation (DC) resulted in high encapsulation efficiencies above 50% at very small batch sizes with a total volume of 150 μL, which were conductive to fast and efficient optimization of variegated surface modification reactions. Cholesterol-polymer amphiphiles, including complex hyperbranched polyether structures bearing 1-4 terminal alkynes, were used in DC formulations to provide steric stabilization. The alkyne moieties were explored as anchors for the conjugation of small molecules to the liposomal surface via click chemistry, binding 350-450 fluorophores per liposome as examples for surface active molecules. Using Förster resonance energy transfer (FRET) spectroscopy, the conjugation reaction as well as the uptake of FRET-labeled liposomes by RBE4 cells was monitored, and the distribution of the fluorescent lipids among cellular structures and membranes could be studied. Thus, the combination of clickable hyperbranched amphiphiles and dual centrifugation provides access to well-defined liposomal formulations with a variety of surface moieties.

Supramolecular assemblies of a conjugate of nucleobase, amino acids, and saccharide act as agonists for proliferation of embryonic stem cells and development of zygotes.

The synthetic challenges in glycobiology and glycochemistry hamper the development of glycobiomaterials for biomedicine. Here we report the use of molecular self-assembly to sidestep the laborious synthesis of complex glycans for promoting the proliferation of murine embryonic stem (mES) cells. Our study shows that the supramolecular assemblies of a small molecule conjugate of nucleobase, amino acids, and saccharide, as a de novo glycoconjugate, promote the proliferation of mES cells and the development of zygotes into blastocysts of mouse. Molecular engineering confirms that each motif (i.e., adenine, Arg-Gly-Asp (RGD) domain, and glucosamine) is indispensable for the observed activity of the conjugate. As the first example of using assemblies of the molecular conjugates of multiple fundamental biological building blocks to control cell behaviors, this work illustrates an unprecedented approach to use supramolecular assemblies as multifunctional mimics of glycoconjugates.

An Fc Domain Protein–Small Molecule Conjugate as an Enhanced Immunomodulator

Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A2AR) has been identified as a promising target for immunotherapy, small molecule A2AR agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A2AR agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A2AR interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.

Hyaluronic acid-based drug conjugates: state-of-the-art and perspectives.

Hyaluronan (HA) is biodegradable, highly biocompatible, and contains derivatizable functional groups along its backbone. This relatively simple, non-branched polysaccharide can target specific cell surface receptors making it an attractive polymeric carrier for targeted delivery of therapeutic agents. This article provides an overview of recent developments involving small molecule and bio-macromolecule conjugates of HA as new generation of human therapeutic agents. Several approaches have been developed to prepare conjugates of HA with small molecule drugs, therapeutic peptides, antibodies, and nucleotides. This article discusses such approaches that can modulate the pharmacokinetic and biodistribution of these therapeutic agents so as to appreciate the design criteria for HA based (bio)conjugates or nanoparticles based on their in vitro assay and in vivo pharmacokinetic study.

Targeting HER2 + breast cancer cells: Lysosomal accumulation of anti-HER2 antibodies is influenced by antibody binding site and conjugation to polymeric nanoparticles

Humanized monoclonal antibodies (mAb) against HER2 are being engineered to treat cancer. We utilized phage-display technology to generate a novel anti-HER2 mAb (named 73JIgG) that binds an epitope of HER2 distinct from that of trastuzumab. Although these mAbs bind to the same cell surface receptor, they have different cell distribution profiles. After 3h of incubation, almost 10% of the total 73JIgG reaches the lysosome compared to less than 3% of trastuzumab. Interestingly, 73JIgG disassociates from HER2 whereas trastuzumab remains bound to the receptor. Importantly, HER2 distribution is not affected by the antibody binding epitope, thus negating this mechanism as the reason for the difference in intracellular trafficking of 73JIgG versus trastuzumab. Each of trastuzumab and 73JIgG was chemically-modified with either a small molecule or polymeric nanoparticle to better understand the influence of conjugation on cellular localization. Relative to antibody alone, antibody–nanoparticle conjugates resulted in a higher concentration of antibodies in the lysosome whereas antibody–small molecule conjugates did not affect cell trafficking to the lysosome. Given the importance of lysosomal targeting, these results demonstrate the importance of understanding the influence of the antibody-conjugate on cell trafficking for ultimate optimization of treatment selection.

“Click chemistry” based conjugation of lipophilic curcumin to hydrophilic ε-polylysine for enhanced functionality

This short communication demonstrates an application of “click chemistry” to conjugate small hydrophobic bioactive molecule such as curcumin with water soluble biopolymer such as ε-polylysine to improve the functionality of the biopolymer. ε-Polylysine and curcumin were modified to ε-polylysine azide and monoalkyne curcumin, respectively. The Huisgen cycloaddition (“click chemistry”) between the azide group and monoalkyne group led to formation of a triazole linkage between ε-polylysine and curcumin. The conjugation process was characterized using Fourier transform infrared (FT-IR) spectroscopy. Retention of functional activity of conjugated curcumin was evaluated by comparing the antioxidant activity of conjugates with native ε-polylysine using the oxygen radical absorbance capacity (ORAC) assay. FT-IR spectra showed a significant decrease in the peak intensity of azide group of ε-polylysine (2102cm−1) and a concomitant presence of triazole peak (1459cm−1) after reaction with monoalkyne curcumin, validating that conjugation was successful. ORAC assay showed that the antioxidant activity of ε-polylysine–curcumin conjugate was 3.6 fold higher than control ε-polylysine indicating that conjugated curcumin retained its functional properties. These results highlight the success of “click chemistry” to achieve conjugation of small molecules to polymers.

Ligand-Free Palladium-Mediated Site-Specific Protein Labeling Inside Gram-Negative Bacterial Pathogens

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

One pot conjugation of small molecules to RNA using click chemistry

A convenient, one step conjugation method based on click chemistry has been developed for creating new RNA conjugates. The coupling reaction was performed in RNA 2′-hydroxy deprotection solution, with conjugation and TBDMS-deprotection being accomplished at the same time.

Small molecule fluorophore and copolymer RGD peptide conjugates for ex vivo two-photon fluorescence tumor vasculature imaging

We report the use of small molecule and block copolymer RGD peptide conjugates for deep ex vivo imaging of tumor vasculature in “whole” excised tumors using two-photon fluorescence microscopy (2PFM). The fluorescent probes were administered to mice via tail-vein injection, after which the tumors were excised, fixed, and imaged without further sample preparation. Both RGD conjugates demonstrated specific targeting to tumor blood vessels, and this selectivity imparted excellent contrast in 2PFM micrographs that captured high-resolution 3-D images of the tumor vasculature up to depths of 830 μm in Lewis Lung Carcinoma (LLC) tumors. 2PFM ex vivo fluorescence micrographs clearly revealed tumor vessels, while differences in the sensitivity of tumor vessel imaging were apparent between the small molecule and block copolymer conjugates. Both the small molecule and polymer-based two-photon absorbing probe conjugate are valuable for deep tissue tumor microvasculature imaging.

Clickable PEG conjugate obtained by “clip” photochemistry: Synthesis and characterization by quantitative 19F NMR

In this paper, we describe a grafting methodology associated to a quantitative 19F NMR method (qNMR) for the conjugation of small molecules on a PEG building block aimed at click chemistry applications in the domain of drug delivery systems. Acetylenic PEG (PEG-yne) was first derivatized with a fluorinated benzyl amine (TagF6) by means of photografting of a trifluoromethylphenyl diazirine bifunctional linker (TPD-clip). The amount of TagF6 grafted on PEG-yne was calculated by NMR using an internal standard (trifluoroethanol) and adjusting of the acquisition and processing parameters. NMR is used as a valuable alternative to the complex procedures often employed for the quantification of functionalities on biomaterials. The accuracy of the qNMR methodology was attested by controlling its linearity, the determination of limits of quantification and the percentage of recovery. A good assessment of the TagF6 grafting rates was obtained after taking into account the inherent unspecific adsorption that occurs on materials. This versatile methodology that combines simple chemistry and a common analytical tool was, in a second time, applied to the preparation of a PEG conjugated with a RGD (Arg-Gly-Asp) peptidomimetic in a controlled manner.

Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes.

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.

Crossing borders to bind proteins—a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

Versatile Site-Specific Conjugation of Small Molecules to siRNA Using Click Chemistry

We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5'-alkyne moieties were used for conjugation to the 5'-end of the oligonucleotide. Previously described 2'- and 3'-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3' and 5' termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.

Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen-Pluronic conjugate to the polymerization mixture ( Reddy , S. T. , et al. ( 2007 ) Nat. Biotechnol. 25, 1159 ). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteamine pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented.

Cloning and Comparative Analyses of the Zebrafish Ugt Repertoire Reveal Its Evolutionary Diversity

UDP-glucuronosyltransferases (Ugts) are a supergene family of phase II drug-metabolizing enzymes that catalyze the conjugation of numerous hydrophobic small molecules with the UDP-glucuronic acid, converting them into hydrophilic molecules. Here, we report the identification and cloning of the complete zebrafish Ugt gene repertoire. We found that the zebrafish genome contains 45 Ugt genes that can be divided into three families: Ugt1, Ugt2, and Ugt5. Both Ugt1 and Ugt2 have two unlinked clusters: a and b. The Ugt1a, Ugt1b, Ugt2a, and Ugt2b clusters each contain variable and constant regions, similar to that of the protocadherin (Pcdh), immunoglobulin (Ig), and T-cell receptor (Tcr) clusters. Cloning the full-length coding sequences confirmed that each of the variable exons is separately spliced to the set of constant exons within each zebrafish Ugt cluster. Comparative analyses showed that both a and b clusters of the zebrafish Ugt1 and Ugt2 genes have orthologs in other teleosts, suggesting that they may be resulted from the “fish-specific” whole-genome duplication event. The Ugt5 genes are a novel family of Ugt genes that exist in teleosts and amphibians. Their entire open reading frames are encoded by single large exons. The zebrafish Ugt1, Ugt2, and Ugt5 genes can generate additional transcript diversity through alternative splicing. Based on phylogenetic analyses, we propose that the ancestral tetrapod and teleost Ugt1 clusters contained multiple Ugt1 paralogs. After speciation, these ancestral Ugt1 clusters underwent lineage-specific gene loss and duplication. The ancestral vertebrate Ugt2 cluster also underwent lineage-specific duplication. The intronless Ugt5 open reading frames may be derived from retrotransposition followed by gene duplication. They have been expanded dramatically in teleosts and have become the most abundant Ugt family in these lineages. These findings have interesting implications regarding the molecular evolution of genes with diversified variable exons in vertebrates.

Aliphatic Hyperbranched Polyester: A New Building Block in the Construction of Multifunctional Nanoparticles and Nanocomposites

Herein we report the design and synthesis of multifunctional hyperbranched polyester-based nanoparticles and nanocomposites with properties ranging from magnetic, fluorescence, antioxidant and X-ray contrast. The fabrication of these nanostructures was achieved using a novel aliphatic and biodegradable hyperbranched polyester (HBPE) synthesized from readily available diethyl malonate. The polymer's globular structure with functional surface carboxylic groups and hydrophobic cavities residing in the polymer's interior allows for the formation of multifunctional polymeric nanoparticles, which are able to encapsulate a diversity of hydrophobic cargos. Via simple surface chemistry modifications, the surface carboxylic acid groups were modified to yield nanoparticles with a variety of surface functionalizations, such as amino, azide and propargyl groups, which mediated the conjugation of small molecules. This capability achieved the engineering of the HBPE nanoparticle surface for specific cell internalization studies and the formation of nanoparticle assemblies for the creation of novel nanocomposites that retained, and in some cases enhanced, the properties of the parental nanoparticle building blocks. Considering these results, the HBPE polymer, nanoparticles and composites should be ideal for biomedical, pharmaceutical, nanophotonics applications.

Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers

The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and AMP) and characterized in vitro their interactions with corresponding RNA aptamers. The conjugates preserved specific binding to aptamers while showing several 100-fold increase in fluorescence of the dye (the ‘light-up’ property). In the presence of free small molecules, conjugates can be displaced from aptamers serving also as fluorescent sensors. Our in vitro results provide the proof-of-concept that the small-molecule conjugates with light-up properties can serve as a general approach to label RNA sequences containing aptamers.

Nα‐Fmoc‐Protected ω‐Azido‐ and ω‐Alkynyl‐L‐amino Acids as Building Blocks for the Synthesis of “Clickable” Peptides

AbstractThe growing interest in the 1,4‐disubstituted‐1,2,3‐triazolyl moiety as an amide bond surrogate and its formation through very mild, chemoselective, and bioorthogonal CuI‐catalyzed Huisgen 1,3‐dipolar [3+2] cycloaddition of an alkynyl to an azido function, presented an unmet need for specifically designed α‐amino‐acid‐derived building blocks. Herein we report the synthesis of unnatural homologous series of Nα‐Fmoc‐protected ω‐yne‐ and ω‐azido‐L‐amino acids compatible with the Fmoc/tBu‐based solid‐phase peptide synthesis. These building blocks can be incorporated into pseudopeptides that can serve as precursors of inter‐ and intramolecular click reactions. The homologous Nα‐Fmoc‐ω‐azido‐L‐amino acids were prepared from either the ω‐amino or the ω‐hydroxy precursors by the respective diazo‐transfer reactions and sequential nucleophilic substitutions initially by halide followed by azide. The homologous Nα‐Fmoc‐ω‐yne‐L‐amino acids were prepared by alkylation of a chiral auxiliary, which is a NiII complex of Schiff base derived from glycine and (S)‐2‐(N‐benzylprolyl)aminobenzophenone, with ω‐bromoalkynes. These building blocks will be instrumental for constructing side‐chain modified peptides, interside‐chain peptide chimera, peptide small molecule conjugates, and cyclopeptides, which were cyclized through 1,4‐disubstituted 1,2,3‐triazolyl‐containing side‐chain‐to‐side‐chain bridges. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)