Polyethylene Glycol Linker Research Articles

Magnetoluminescent light switches--dual modality in DNA detection.

The synthesis and properties of two responsive magnetoluminescent iron oxide nanoparticles for dual detection of DNA by MRI and luminescence spectroscopy are presented. These magnetoluminescent agents consist of iron oxide nanoparticles conjugated with metallointercalators via a polyethylene glycol linker. Two metallointercalators were investigated: Ru(bpy')(phen)(dppz), which turns on upon DNA intercalation, and Eu-DOTA-Phen, which turns off. The characteristic light-switch responses of the metallointercalators are not affected by the iron oxide nanoparticles; upon binding to DNA the luminescence of the ruthenium complexes increases by ca. 20-fold, whereas that of the europium complex is >95% quenched. Additionally, the 17-20 nm magnetite cores, having permeable PEG coatings and stable dopamide anchors, render the two constructs efficient responsive contrast agents for MRI with unbound longitudinal and transverse relaxivities of 12.4-9.2 and 135-128 mM(-1)(Fe)s(-1), respectively. Intercalation of the metal complexes in DNA results in the formation of large clusters of nanoparticles with a resultant decrease of both r1 and r2 by 32-63% and 24-38%, respectively. The potential application of these responsive magnetoluminescent assemblies and their reversible catch-and-release properties for the purification of DNA is presented.

Short PEG-Linkers Improve the Performance of Targeted, Activatable Monoclonal Antibody-Indocyanine Green Optical Imaging Probes

The ability to switch optical imaging probes from the quenched (off) to the active state (on) has greatly improved target to background ratios. The optimal activation efficiency of an optical probe depends on complete quenching before activation and complete dequenching after activation. For instance, monoclonal antibody-indocyanine green (mAb-ICG) conjugates, which are promising agents for clinical translation, are normally quenched, but can be activated when bound to a cell surface receptor and internalized. However, the small fraction of commonly used ICG derivative (ICG-Sulfo-OSu) can bind noncovalently to its mAb and is, thus, gradually released from the mAb leading to relatively high background signal especially in the liver and the abdomen. In this study, we re-engineered a mAb-ICG conjugate, (Panitumumab-ICG) using bifunctional ICG derivatives (ICG-PEG4-Sulfo-OSu and ICG-PEG8-Sulfo-OSu) with short polyethylene glycol (PEG) linkers. Higher covalent binding (70-86%) was observed using the bifunctional ICG with short PEG linkers resulting in less in vivo noncovalent dissociation. Panitumumab-ICG conjugates with short PEG linkers were able to detect human epidermal growth factor receptor 1 (EGFR)-positive tumors with high tumor-to-background ratios (15.8 and 6.9 for EGFR positive tumor-to-negative tumor and tumor-to-liver ratios, respectively, at 3 d postinjection).

A Cyclen‐Based Tetraphosphinate Chelator for the Preparation of Radiolabeled Tetrameric Bioconjugates

The cyclen-based tetraphosphinate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene(2-carboxyethyl)phosphinic acid] (DOTPI) comprises four additional carboxylic acid moieties for bioconjugation. The thermodynamic stability constants (logK(ML)) of metal complexes, as determined by potentiometry, were 23.11 for Cu(II), 20.0 for Lu(III), 19.6 for Y(III), and 21.0 for Gd(III). DOTPI was functionalized with four cyclo(Arg-Gly-Asp-D-Phe-Lys) (RGD) peptides through polyethylene glycol (PEG4) linkers. The resulting tetrameric conjugate DOTPI(RGD)4 was radiolabeled with (177)Lu and (64)Cu and showed improved labeling efficiency compared with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The labeled compounds were fully stable in transchelation challenges against trisodium diethylenetriaminepentaacetate (DTPA) and disodium ethylenediaminetetraacetic acid (ETDA), in phosphate buffered saline (PBS), and human plasma. Integrin αvβ3 affinities of the non-radioactive Lu(III) and Cu(II) complexes of DOTPI(RGD)4 were 18 times higher (both IC50 about 70 picomolar) than that of the c(RGDfK) peptide (IC50 = 1.3 nanomolar). Facile access to tetrameric conjugates and the possibility of radiolabeling with therapeutic and diagnostic radionuclides render DOTPI suitable for application in peptide receptor radionuclide imaging (PRRI) and therapy (PRRT).

Abstract 2231: Development of bifunctional cross-linking agents to identify intermediate proteasomes.

Abstract Introduction: This research was conducted to develop bifunctional cross-linking agents that can be used to identify intermediate proteasomes. Two well-characterized proteasome subtypes, the constitutive proteasome and immunoproteasome, differ with respect to their distinct sets of catalytic subunits. Additionally, intermediate proteasome subtypes comprised of non-standard mixtures of catalytic subunits have more recently been discovered. These intermediate proteasomes differ from constitutive and immunoproteasomes in their proteolytic activity profiles, suggesting that they have unique cellular functions. In agreement with this, intermediate proteasomes in cancer cell lines were shown to play important roles in the production of several tumor antigens. However, largely due to limitations of methods currently available to distinguish between subtypes, the detailed functions of intermediate proteasomes remain poorly defined. Bifunctional agents capable of cross-linking two different catalytic subunits within a single proteasome complex will facilitate a simple approach to identify distinct proteasome subtypes present within cells. These compounds will serve as important chemical probes for investigating the functions of individual proteasome subtypes in cancer cells. Experimental Procedures: Bifunctional cross-linking agents were synthesized by coupling two broad-spectrum or subunit-selective proteasome inhibitors through hydrocarbon or polyethylene glycol linkers. Positions for derivatization of these inhibitors were selected based on computational modeling, and linker lengths were varied to increase cross-linking efficiency. The ability of the resulting compounds to cross-link two proteasome catalytic subunits in cancer cell lysates was assessed via western blotting. Competition assays with subunit-selective proteasome inhibitors were conducted to verify the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional cross-linking agents were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results revealed the ability of several of these bifunctional agents to cross-link two catalytic subunits within individual proteasome complexes, facilitating compositional analysis of distinct proteasome subtypes. Conclusions: We have successfully developed bifunctional agents capable of cross-linking two catalytic subunits within individual proteasome complexes. These compounds will be utilized in functional studies of intermediate proteasomes present within cancer cells. Citation Format: Kimberly C. Carmony, Lalit Kumar Sharma, Kyung-Bo Kim. Development of bifunctional cross-linking agents to identify intermediate proteasomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2013-2231

Nanomechanics of Lipid Encapsulated Microbubbles with Functional Coatings

Microbubbles (MBs) are increasingly being proposed as delivery vehicles for targeted therapeutics, as well as being contrast agents for ultrasound imaging. MBs formed with a lipid shell are promising candidates due to their biocompatibility and the opportunity for surface functionalization, both for specific targeting of tissues and as a means to tune their mechanical response for localized ultrasound induced destruction in vivo. Herein, we acquired force-deformation data on coated lipid MBs using tip-less microcantilevers in an atomic force microscope. Model lipid MBs were designed to test the effects of adding a functional coating on the outside of the lipid leaflet, including a protein coat (streptavidin) or the addition of quantum dots (Q-dots) as optical reporters. MBs (~3 μm diameter) were repeatedly compressed for deformations up to ~50% to obtain a full bubble response. Addition of a coating increased the initial deformation stiffness related to shell bending ~2-fold for streptavidin and ∼3-fold for Q-dots. The presence of a polyethylene glycol (PEG) linker in between the lipid and functional coating, led to enhanced stiffening at high deformations. The plasticity index has been determined and only those MBs that included the PEG linker showed a force dependent short time-scale (<~1s) plasticity. This study demonstrates modulation of the mechanical response of biocompatible MBs through the addition of functional coatings necessary for rationale design of therapeutic lipid MBs for targeted drug delivery.

Mapping CD20 molecules on the lymphoma cell surface using atomic force microscopy

Atomic force microscopy (AFM) was used to locate CD20 molecules on the surface of lymphoma Raji cells. Rituximab (a monoclonal antibody against CD20) molecules were linked onto the AFM tip via a polyethylene glycol (PEG) linker. Raji cells were adsorbed onto glass slides coated with poly-L-lysine. First, the CD20 distribution in a local area of the cell surface was visualized using the AFM lift scan mode. Second, 16 × 16 force curves were obtained from the same cell area to construct the CD20-rituximab binding force map. Finally, free rituximab was added to block the CD20 molecules on the cell surface and the lift phase image and CD20-rituximab force map were obtained again. The experimental results indicated that when the lift height was greater than the length of the PEG linker, no recognition sites were observed in the lift phase image. However, as the lift height decreased to the length of the PEG linker, some recognition sites were observed in the lift phase image and these sites were generally consistent with the pixels in the force map. After blocking, both the recognition sites in the lift phase image and the gray pixels in the binding force map decreased markedly. These results can improve our understanding of the distribution of protein molecules on the cell surface and facilitate further investigations into cellular functions.

Development of highly potent protease‐activated receptor 2 agonistsviasynthetic lipid tethering

Protease-activated receptor-2 (PAR₂) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR₂ is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR₂ is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR₂ agonist (2-aminothiazol-4-yl-LIGRL-NH₂) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC₅₀ values as low as 1.47 nM, representing a ∼200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR₂: EC₅₀ for Ca(2+) response as low as 3.95 nM; EC₅₀ for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC₅₀ of 14.4 pmol. STLs failed to elicit responses in PAR2(-/-) cells at agonist concentrations of >300-fold their EC₅₀ values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR₂ and represent opportunities for drug development at other protease activated receptors and across GPCRs.

Multivalent Protein Binding Kinetics of Variable Lymphocyte Receptors Probed using Dynamic Force Spectroscopy

The binding of an antibody to a target antigen underpins an array of biological warfare agent detection schemes and pharmaceutical therapies. However, current antibody-based detection and treatments suffer from multiple drawbacks, including the lack of an antibody that offers a range of affinities for a target antigen. Here, we measure the binding kinetics of a recently discovered class of antibody derived from jawless vertebrates called Variable Lymphocyte Receptors (VLRs), which can achieve IgG-like binding specificity. We first tether low densities of mono-, di-, and pentavalent VLRs to an atomic force microscope cantilever via polyethylene glycol linkers, and then measure the forces required to rupture their bonds with a substrate tethered anthrax antigen. We find that in all three cases the interactions are load-rate dependent - probed far from equilibrium - over the entire range of load rates for which our apparatus is capable. As expected, binding affinity is enhanced in a noncooperative fashion with increasing VLR valency. Furthermore, for the di- and pentavalent molecules, the at-surface dwell time is varied, with longer dwell times increasing the probability of states in which multiple VLR binding domains of a single VLR protein are complexed with surface bound BclA. Thus, through single molecule force spectroscopy, VLRs with greater valency show increased binding stability and lower dissociation rates, and therefore tunable affinity, while the nature of how the binding state probabilities vary with time provides insight into the intermediate binding state kinetics of this system.

Electrocatalytic effect towards NADH induced by HiPco single-walled carbon nanotubes covalently functionalized by ferrocene derivatives

ABSTRACTThe present work reports the covalent functionalization of single-walled carbon nanotubes (SWCNTs) by ferrocene derivatives with polyethyleneglycol linkers. A very clean initial sample was chosen to avoid any residual catalyst and carbon impurities. Functionalized SWCNTs (f-CNTs) are deposited on the surface of a glassy carbon electrode (GCE) and this modified electrode is used for oxidizing the cofactor NADH (dihydronicotinamide adenine dinucleotide) in the presence of diaphorase. A clear electrocatalytic effect is evidenced, which can only be attributed to the f-CNTs.

Peptide translocation through the mesoscopic channel: binding kinetics at the single molecule level

Single channel electrophysiological studies have been carried out to elucidate the underlying interactions during the translocation of polypeptides through protein channels. For this we used OmpF from the outer cell membrane of E. coli and arginine-based peptides of different charges, lengths and covalently linked polyethylene glycol as a model system. In order to reveal the fast kinetics of peptide binding, we performed a temperature scan. Together with the voltage-dependent single-channel conductance, we quantify peptide binding and translocation.

Bifunctional inhibition of HIV-1 reverse transcriptase: A first step in designing a bifunctional triphosphate

The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof of concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T–6PEG–TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA.

Flavonoid Dimers as Novel, Potent Antileishmanial Agents

The present study found that synthetic flavonoid dimers with either polyethylene glycol linker or amino ethyleneglycol linker have marked leishmanicidal activity. Compound 39 showed very consistent and promising leishmanicidal activity for both extracellular promastigotes (IC₅₀ ranging from 0.13 to 0.21 μM) and intracellular amastigotes (IC₅₀ = 0.63 μM) irrespective of the drug-sensitivity of parasites. Moreover, compound 39 displayed no toxicity toward macrophage RAW 264.7 cells (IC₅₀ > 100 μM) and primary mouse peritoneal elicited macrophages (IC₅₀ > 88 μM). Its high value of therapeutic index (>140) was better than other highly potent antileishmanials such as amphotericin B (therapeutic index = 119). Compound 39 is therefore a new, safe, and effective antileishmanial candidate compound which is even effective against drug-refractory parasites.

Imaging and Measuring the Molecular Force of Lymphoma Pathological Cells Using Atomic Force Microscopy

Atomic force microscopy (AFM) provides a new technology to visualize the cellular topography and quantify the molecular interactions at nanometer spatial resolution. In this work, AFM was used to image the cellular topography and measure the molecular force of pathological cells from B-cell lymphoma patients. After the fluorescence staining, cancer cells were recognized by their special morphological features and then the detailed topography was visualized by AFM imaging. The AFM images showed that cancer cells were much rougher than healthy cells. CD20 is a surface marker of B cells and rituximab is a monoclonal antibody against CD20. To measure the CD20-rituximab interaction forces, the polyethylene glycol (PEG) linker was used to link rituximab onto the AFM tip and the verification experiments of the functionalized probe indicated that rituximab molecules were successfully linked onto the AFM tip. The CD20-rituximab interaction forces were measured on about 20 pathological cells and the force measurement results indicated the CD20-rituximab binding forces were mainly in the range of 110-120 pN and 130-140 pN. These results can improve our understanding of the topography and molecular force of lymphoma pathological cells.

Cisplatin drug delivery using gold-coated iron oxide nanoparticles for enhanced tumour targeting with external magnetic fields

The platinum-based chemotherapeutic drug cisplatin is highly effective in the treatment of solid tumours, but its use is restricted by poor bioavailability, severe dose-limiting side effects and rapid development of drug resistance. In light of this we have tethered the active component of cisplatin to gold-coated iron oxide nanoparticles to improve its delivery to tumours and increase its efficacy. Iron oxide nanoparticles (FeNPs) were synthesised via a co-precipitation method before gold was reduced onto the surface (Au@FeNPs). Aquated cisplatin was used to attach {Pt(NH3)2} to the nanoparticles by a thiolated polyethylene glycol linker forming the desired product (Pt@Au@FeNP). The nanoparticles were characterised by dynamic light scattering, scanning transmission electron microscopy, UV–Vis spectrophotometry, inductively coupled plasma mass spectrometry and electron probe microanalysis. The nanoparticles increase in size as they are constructed, with the synthesised FeNPs having a diameter of 5–50nm, which increases to 20–80nm for the Au@FeNPs, and to 60–120nm for the Pt@Au@FeNPs. Nanoparticle drug loading was found to be 7.9×10−4moles of platinum per gram of gold. The FeNPs appear to have little inherent cytotoxicity, whereas the Au@FeNPs are as active as cisplatin in the A2780 and A2780/cp70 cancer cell lines. More importantly the Pt@Au@FeNPs are up to 110-fold more cytotoxic than cisplatin. Finally, external magnets were used to demonstrate that the nanoparticles could be accumulated in specific regions and that cell growth inhibition was localised to those areas.

Abstract 5735: Synthesis and characterization of PEG-ylated porphyrins for targeting the epidermal growth factor receptor in colorectal cancer detection

Abstract ABSTRACT According to the National Cancer Institute, an estimated 102,900 (colon) and 39,670 (rectal) new cases were obtained in 2010. An estimated 51,370 people have died from colon and rectal cancers combined. This makes colorectal cancer (CRC) the second leading cause of cancer-related deaths in the United States. Consequently, the development of an in-vivo imaging agent that is selective for CRC has long been needed. Much of the detection for tumorigenesis relies on the efficacy in detecting small tumors. Current agents are not selective and often stain tissue not associated with the cancer cells making it difficult to delineate between viable and tumorigenic entities. This project introduces a novel macrocycle conjugated to polyethylene glycol linker, which we hypothesize will serve as the template for a selective molecule with high fluorescence yields that greatly increases earlier detection. Our targeted remedy is a porphyrin that is conjugated to a peptide with an affinity for the Epidermal Growth Factor Receptor (EGFR). Porphyrins are characteristically aromatically stable, contain trademark absorption bands in the visible and near-IR range, and have fluorescence quantum yields much above the current fluorophores. This makes the macrocycle optimal for confocal laser endomicroscopy (CLE) agent production. Consequently, we use a polyethylene glycol linker in order to increase water solubility, retain low toxicity, and to achieve high fluorescence quantum yields, as well as high conjugation yields. In this research, we were able to produce both precursors to the porphyrin-peptide conjugate, MesoPOR-(mono)-3PEG and MesoPOR-(di)-3PEG. These molecules were synthesized successfully with the use of peptide conjugation mechanisms. Molecular weights were confirmed using Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). Characterization was performed using 1H Nuclear Magnetic Resonance (1H-NMR) and Ultraviolet-Visible Spectroscopy (UV-Vis) of the intended molecules. The synthesized molecules, MesoPOR-(mono) 3PEG and MesoPOR-(di) 3PEG, will be useful in peptide conjugation that targets EGFR. These peptide ligands will increase selectivity and detect CRC via CLE. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5735. doi:1538-7445.AM2012-5735

Synthetic tethering increases the potency of PAR2 agonists

Protease‐activated receptor‐2 (PAR2) is G protein‐coupled receptor important in a number of protease‐associated pathologies including allergic asthma, cancer and pain. Peptides (SLIGRL‐NH2) that mimic the receptor's native tethered ligand can activate PAR2 without the off‐target effects of proteases. A better understanding of the pathological role of PAR2 is limited by the need for potent, specific agonists. We and others have developed potent PAR2 peptidomimetics (2‐at‐LIGRL‐NH2) based on the trypsin cleavage site and serine substitutions. We hypothesized mimicking the native tethered ligand would increase PAR2 ligand potency. We used palmitate to mimic the natural membrane anchor and a polyethylene glycol (Peg) linker between the anchor and peptidomimetic to make a series of synthetic tethered ligands (STLs; 2‐at‐LIGRLO‐PegN‐Palm; N=1–5). We found 3 Pegs conferred maximal potency; 2‐at‐LIGRL‐Peg3‐Palm induced Ca2+ and MAPK signaling in vitro (EC50 0.4 nM, 12.2 nM) and thermal hyperalgesia in vivo (0.36 nMol), and was specific for PAR2. 2‐at‐LIGRLO‐Peg3‐Palm is 30–1000 times more potent across assays than the best described agonist and specific for PAR2. STLs are a powerful approach to increase ligand potency without sacrificing specificity and may allow for the development of subnanomolar ligands important to understanding the pathologic roles of PAR2. NIH grants NS073664 and T32‐HL007249.

Magnetoluminescent Agents for Dual MRI and Time‐Gated Fluorescence Imaging

AbstractThe synthesis and properties of two luminescent iron oxide nanoparticles for dual imaging by MRI and time‐gated confocal microscopy are presented. These magnetoplasmonic agents consist of macrocyclic terbium complexes conjugated onto magnetite nanoparticles by a polyethylene glycol linker. Two macrocyclic terbium complexes were investigated: Tb‐DOTAm‐(Phen)3 and Tb‐DOTAm‐(acetylene)3. Both complexes display the characteristic long luminescence lifetimes of lanthanides (0.8 ms and 1.3 ms, respectively), rendering them ideal for time‐gated luminescence spectroscopy and confocal imaging – a feature currently unexplored in multimodal imaging agents. Additionally, the 5 ± 1 nm magnetite nanoparticles, with their permeable PEG coating and stable dopamide anchor, render the two constructs efficient MRI contrast agents with longitudinal (r1) and transverse (r2) relaxivities of 9.8 mM–1Fe s–1 and 98 mM–1Fe s–1 at 60 MHz and 37 °C.

Controlled Synthesis of Cell-Laden Microgels by Radical-Free Gelation in Droplet Microfluidics

Micrometer-sized hydrogel particles that contain living cells can be fabricated with exquisite control through the use of droplet-based microfluidics and bioinert polymers such as polyethyleneglycol (PEG) and hyperbranched polyglycerol (hPG). However, in existing techniques, the microgel gelation is often achieved through harmful reactions with free radicals. This is detrimental for the viability of the encapsulated cells. To overcome this limitation, we present a technique that combines droplet microfluidic templating with bio-orthogonal thiol-ene click reactions to fabricate monodisperse, cell-laden microgel particles. The gelation of these microgels is achieved via the nucleophilic Michael addition of dithiolated PEG macro-cross-linkers to acrylated hPG building blocks and does not require any initiator. We systematically vary the microgel properties through the use of PEG linkers with different molecular weights along with different concentrations of macromonomers to investigate the influence of these parameters on the viability and proliferation of encapsulated yeast cells. We also demonstrate the encapsulation of mammalian cells including fibroblasts and lymphoblasts.

A general chemical synthesis platform for crosslinking multivalent single chain variable fragments

Multivalent single chain variable fragments (scFv) show increased affinity to tumor-associated antigens compared to monovalent scFv and intact monoclonal antibodies (mAb). Multivalent constructs can be derived from self-associating or covalent scFv with covalent constructs offering improved in vivo and in vitro stability. Covalent attachment of scFv can be achieved using genetically engineered expression vectors that afford scFv with site specific cysteine functionality. Expression vectors for di-scFv-C wherein the cysteine is located in the center of two scFv have also been developed for attaching chemically reactive linkers. In the example illustrated here, the di-scFv-C is derived from a mAb directed against the MUC1 epitope, which is presented on cancer cells. To achieve multivalency, a chemical crosslinking strategy utilizing various azide and multi-alkyne functionalized polyethylene glycol (PEG) linkers was implemented. Conjugation was achieved by attachment of these linkers to the scFv thiol functionality. Chemoselective ligation was employed to covalently link different protein conjugates via copper(I) catalyzed azide alkyne 1,3-dipolar cycloaddition reaction (CuAAC) chemistry. Ligations were achieved in >70% yield using a specific set of linkers as determined by SDS-PAGE and densitometry. ELISA showed increased tumor binding of a tetravalent scFv providing a versatile chemical crosslinking strategy for construction of multivalent and bi-specific immunoconjugates that retain biological activity and have potential application in pre-targeted radioimmunotherapy and imaging.

PEG-tethered guanosine acetal conjugates for the enzymatic synthesis of modified RNA

A short and efficient synthetic route to guanosine monophosphate conjugates connected to a protected aldehyde group via a flexible polydisperse polyethylene glycol linker is described. These conjugates are enzymatically incorporated into RNA transcripts with 52% yield. The resulting aldehyde-modified RNA gave full conversion in the reaction with biotin hydrazide, demonstrating the utility of the method.