Labile Linker Research Articles

Hydrogels for combination delivery of antineoplastic agents

The systemic delivery of anticancer agents has been widely investigated during the past decade but localized delivery may offer a safer and more effective delivery approach. We have designed and synthesized a novel hydrogel to locally deliver antineoplastic agents, and demonstrate the different types of release that can be achieved from these hydrogels using three model drugs: methotrexate, doxorubicin, and mitoxantrone. Alginate was chemically modified into low molecular weight oligomers and cross-linked with a biodegradable spacer (adipic dihydrazide) to form biodegradable hydrogels. The model antineoplastic agents were loaded into the hydrogel via three different mechanisms. Methotrexate was incorporated within the pores of the hydrogel and was released by diffusion into the surrounding medium. Doxorubicin was covalently attached to the polymer backbone via a hydrolytically labile linker and was released following the chemical hydrolysis of the linker. Mitoxantrone was ionically complexed to the polymer and was released after the dissociation of this complex. These three release mechanisms could potentially be used to deliver a wide selection of antineoplastic agents, based on their chemical structure. This novel delivery system allows for the release of single or combinations of antineoplastic agents, and may find utility in localized antineoplastic agent delivery.

Solid phase synthesis of cyclic peptides by oxidative cyclative cleavage of an aryl hydrazide linker—synthesis of stylostatin 1

The synthesis of cyclic peptides using an oxidation labile aryl hydrazide linker is reported. After oxidation with NBS release from the resin is triggered via an intramolecular cyclative cleavage which proceeds without racemization.

Solid-phase synthesis of oligoesters using a JandaJel™ resin

This communication describes a general method for the solid-phase synthesis of oligomeric esters. A JandaJel™ resin was utilized as the solid support in conjunction with the highly acid labile Rink linker. Ester coupling reactions were performed using buffered reagents and the monomer was protected as its allyl ester. In this manner, ester coupling and deprotection could be performed under essentially neutral conditions, giving rise to products in both excellent yields and purity.

Synthesis and thermal properties of poly(urethane-imide)

The synthesis and thermal properties of thermoplastic poly(urethane-imide) (PUI) resins were studied. Model reaction studies on the reactions of 4,4′-diphenylcarbamatodiphenylmethane and 4,4′-diisocyanatodiphenylmethane with phthalic anhydride were performed. We found that the reaction of anhydrides with urethane groups could take place under certain reaction conditions. According to the model reaction studies, N-2-methyl-pyrrolidone was employed as a solvent, and no catalyst was used in the polymerization. To restrain the side reaction of anhydrides with urethane groups, we adopted a two-step chain-extending procedure in a chain-extending reaction. The inherent viscosity of PUI was 0.83–0.99 dL/g. The prepared polymers not only exhibited improved solubility in organic solvents but also formed flexible films. Thermogravimetric analysis showed that PUI exhibited a two-step thermal weight-loss pattern. The first step of the thermal degradation of PUI was attributed to the thermooxidizing cleavage of weak and labile linkage, such as urethane groups, isopropylidene, and methylene, except for imide rings. The polymer inherent viscosity decreased sharply during the first step of thermal degradation. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 773–781, 2001

Oxidative Modification of Rat Liver 5'-Nucleotidase: The Mechanisms for Protection and Re-Activation

The effect of oxidative stress catalysed by transition metals appears to have a critical relevance for the structure and function not only of membrane lipids but also of integral membrane proteins in a complex lipid-protein assembling, and membrane-dependent function. The integral membrane enzyme 5'-nucleotidase is susceptible to Fe 2+ -ion catalysed oxidative modification, and the extent of enzyme inhibition is in inverse relationship (r = -0.820) with lipid peroxidation (MDA) level. This work is also a comparative study about possible effectiveness of different Fe-ion chelators (deferoxamine, Na-citrate, Na-salicylate, ammonium oxalate and EDTA), antioxidants (GSH, GSH/GSH-Px system, Cu, Zn-SOD and mannitol) and metal cations (Mg 2+ and Mn 2+) to protect or restore Fe 2+ -ion induced 5'-nucleotidase inhibition and to supress Fe 2+ -ion enhanced lipid peroxidation. Among the examined chelators it was only deferoxamine and Na-citrate that exerted a fully protective and reactivating ability; among the antioxidants it was only GSH; among the metal cations it was only Mn 2+. The ability to protect or restore 5'-nucleotidase activity and to diminish chain-induced lipid peroxidation is explicable in terms of: metal-binding ability, capacity of taking iron away from a biological molecule, or ability of transferring the damage to itself. After a short incubation period, the iron associated with enzyme or lipid hydroperoxides could be in a labile coordinative linkage, still able to interact with possible ligands or metal cations.

Total syntheses of thiocoraline and BE-22179 and assessment of their DNA binding and biological properties.

Full details of the total syntheses of thiocoraline (1) and BE-22179 (2), C(2) symmetric bicyclic octadepsipeptides possessing two pendant 3-hydroxyquinoline chromophores, are described in which their relative and absolute stereochemistry were established. Key elements of the approach include the late-stage introduction of the chromophore, symmetrical tetrapeptide coupling, macrocyclization of the 26-membered octadepsipeptide conducted at the single secondary amide site following disulfide formation, and a convergent assemblage of the tetradepsipeptide with introduction of the labile thiol ester linkage in the final coupling reaction under near racemization free conditions. By virtue of the late-stage introduction of the chromophore and despite the challenges this imposes on the synthesis, this approach provides ready access to a range of key chromophore analogues. Thiocoraline and BE-22179 were shown to bind to DNA by high-affinity bisintercalation analogous to echinomycin, but with little or no perceptible sequence selectivity. Both 1 and 2 were found to exhibit exceptional cytotoxic activity (IC(50) = 200 and 400 pM, respectively, L1210 cell line) comparable to echinomycin and one analogue, which bears the luzopeptin chromophore, was also found to be a potent cytotoxic agent.

The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4 — a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.

Fluorosulfonyl-substituted xanthines as selective irreversible antagonists for the A(1)-adenosine receptor.

FSCPX (1) has been reported to be a potent, selective, and irreversible antagonist for the A(1)-adenosine receptor (AR). To obtain an irreversible A(1)AR antagonist with potentially better stability and to further elucidate the effects of linker structure on the pharmacological characteristics, several new analogues were targeted in which the labile ester linkage of 1 was replaced by more stable functionalities. In particular, alkyl and amide linkers between the xanthine pharmacophore and the reactive 4-fluorosulfonylphenyl group were explored. The data showed that the chemical composition of the linker affects the affinity and apparent irreversible binding to the A(1)AR. Overall, compound 23b appeared to have the most advantageous characteristics as a potential irreversible ligand for the A(1)AR. These include relatively high affinity for the A(1)AR as compared to the A(2A)AR, concentration-dependent and selective apparent irreversible binding to the A(1)AR, and ease of removal of unbound ligand from biological membranes. These properties indicate that 23b has the potential to be a useful tool for further study of the structure and function of the A(1)AR.

ChemInform Abstract: A Novel Synthesis of Oligonucleotide-Peptide Conjugates with a Base-Labile Phosphate Linker Between the Two Components According to the Allyl-Protected Phosphoramidite Strategy.

Abstract An efficient synthesis of base-labile nucleotide–peptide conjugates has been accomplished, in which the two components are directly linked between the terminal hydroxyl of a nucleotide and the hydroxyl of a serine or threonine residue of a peptide by a phosphodiester bond. This synthesis utilizes the phosphoramidite method with allyl for the phosphate linkages and the C-terminal of the peptide and allyloxycarbonyl for the nucleoside bases and the N-terminal of the peptide. In this synthesis, the removal of the allylic protecting groups and the detachment of the products was achieved under non-basic or mild basic conditions to bring about no conspicuous decomposition of the labile phosphate linker, and thus the target conjugates were obtained at a high purity and in high yields.

Solid-phase synthesis of substituted guanidines using a novel acid labile linker.

A novel acid labile linker for solid-phase synthesis of substituted guanidines has been developed. Its synthetic utility is exemplified by high-yielding pyrazole displacement with structurally and electronically diverse sets of aliphatic and aromatic amines. The final cleavage is achieved by treatment with 95:5 trifluoroacetic acid/water for 1 h. The corresponding guanidines were obtained in high purity (80-95%) and good isolated yields (50-95%). The scope and limitations of this linker were further demonstrated by the solid-phase synthesis of an 880-member library of individual trisubstituted arylguanidines employing pyrazole displacement with a set of 11 anilines and two subsequent Mitsunobu N-alkylations with sets of 10 and 8 alcohols, respectively.

A Novel Synthesis of Oligonucleotide–Peptide Conjugates with a Base-Labile Phosphate Linker between the Two Components According to the Allyl-Protected Phosphoramidite Strategy

An efficient synthesis of base-labile nucleotide–peptide conjugates has been accomplished, in which the two components are directly linked between the terminal hydroxyl of a nucleotide and the hydroxyl of a serine or threonine residue of a peptide by a phosphodiester bond. This synthesis utilizes the phosphoramidite method with allyl for the phosphate linkages and the C-terminal of the peptide and allyloxycarbonyl for the nucleoside bases and the N-terminal of the peptide. In this synthesis, the removal of the allylic protecting groups and the detachment of the products was achieved under non-basic or mild basic conditions to bring about no conspicuous decomposition of the labile phosphate linker, and thus the target conjugates were obtained at a high purity and in high yields.

Enantioselective ester hydrolysis catalyzed by imprinted polymers.

Highly cross-linked network polymers prepared by molecular imprinting catalyzed enantioselectively the hydrolysis of N-tert-butoxycarbonyl phenylalanine-p-nitrophenyl ester (BOCPheONP). The templates were designed to allow incorporation of the key catalytic elements, found in the proteolytic enzyme chymotrypsin, into the polymer active sites. Three model systems were evaluated. These were constructed from a chiral phosphonate analogue of phenylalanine (series A, C) or L-phenylalanine (series B) attached by a labile ester linkage to an imidazole-containing vinyl monomer. Free radical copolymerization of the template with methacrylic acid (MAA) and ethylene glycol dimethacrylate (EDMA) gave a highly cross-linked network polymer. The templates could be liberated from the polymers by hydrolysis, giving catalytically active sites envisaged to contain an enantioselective binding site, a site complementary to a transition state like structure (series A, C), and a hydroxyl, imidazole, and carboxylic acid group at hydrogen bond distance. As predicted, the enantiomer of BOCPheONP complementary to the configuration of the template was preferentially hydrolyzed with D-selectivity for the series A polymers (kD/kL = 1.9) and L-selectivity for the series B polymers (kL/kD = 1.2). The maximum rate enhancement, when compared with a control polymer, prepared using a benzoyl-substituted imidazole monomer as template, was 2.5, and comparing with the imidazole monomer in solution, a maximum rate enhancement of 10 was observed. The catalytic activity was higher for polymers subjected to the nucleophilic treatment. This was explained by a higher site density and flexibility of the polymer matrix caused by this treatment. In a comparison of template rebinding to polymers imprinted with a template containing either a carboxylate (planar ground state structure) or a phosphonate (tetrahedral transition state like structure) functionality, it was observed that imprinted polymers are able to discriminate between a transition state like and a ground state structure for transesterification. However the influence of transition state stabilization on the observed rate enhancements remains obscure. Only at acidic pH's was catalysis observed, whereas at basic pH's the polymers inhibit the reaction. At a later stage, the catalytic activity of the polymers for nonactivated D- and L-phenylalanine ethyl esters was investigated. A rate enhancement of up to 3 was observed when compared to the blank. Most important, however, the polymers imprinted with a D template preferentially hydrolyzed the D-ethyl ester and exhibited saturation kinetics.

Long chain arginine esters: A new class of cationic detergents for preparation of hydrophobic ion-paired complexes

The ability of stoichiometric amounts (based on charged groups) of ionic detergents to bind to oppositely charged ionic compounds has been recently reviewed. These hydrophobic ion-paired (HIP) complexes display altered solubility properties. Most of the work to date on HIP compelxes has focused on basic drugs and anionic detergents. It would be extremely useful to extend this approach to acidic compounds, including DNA and RNA. However, most cationic detergents are relatively toxic. It is hypothesized that detergents constructed from naturally occurring or well tolerated components, coupled by labile linkages, will be less toxic and still able to form strong HIP complexes. This study describes the synthesis and characterization of long chain alkyl esters of arginine. This class of cationic detergents, which have not been reported previously, are less cytotoxic than alkyltrimethylammonium detergents, possibly making them more acceptable in drug delivery applications. These arginine esters exhibit detergent-like properties. For example, the dodecyl ester of arginine has a critical micelle concentration of 0.07 mM, while being approximately 5-10 fold less toxic than tetradecyltrimethylammonium bromide. The arginine dodecyl ester forms stable HIP complexes with plasmid DNA. The complex is sufficiently stable to allow some modest level of transfection with Cos-7 cells in a time- and concentration-dependent fashion. This work demonstrates that arginine-based cationic detergents are effective ion-pairing agents, appear to be less toxic than alkyltrimethylammonium compounds, and form stable complexes with DNA.

Surface and bulk modifications to photocrosslinked polyanhydrides to control degradation behavior.

A unique class of surface-eroding polyanhydrides was developed and explored for use in medical applications requiring high-strength biomaterials (e.g., orthopedics). In particular, dimethacrylated anhydride monomers were synthesized that photopolymerize quickly to render densely crosslinked polymer networks that degrade from the surface only by hydrolysis of labile anhydride linkages. Previous research on these materials has shown that the rate of hydrolysis of the degradable linkages is dependent on the hydrophobicity of the network composition. This article demonstrates the versatility in controlling the degradation process and resulting cellular response in these materials through the incorporation of new chemistries and the formation of polymer-polymer composite structures. Specifically, the rate of mass loss was controlled by the addition of hydrophobic linear polymers [e.g., poly(methyl methacrylate)] or monovinyl monomers based on hydrophobic natural components (e.g., cholesterol, steric acid). In addition, a newly established photografting method was used to modify the network surface chemistry with cholesterol- and stearic acid-based polymer grafts to control the degradation front and cellular interactions at the polymer-tissue interface. Finally, a porogen leaching method was used to form porous polyanhydride constructs, which can be subsequently filled with osteoblasts photoencapsulated in a hydrogel, as potential synthetic allograft materials for tissue engineering bone.

Dispersion redox copolymerization of methyl methacrylate with macromonomeric azoinitiator as a macrocrosslinker

The dispersion copolymerization of methyl methacrylate (MMA) with macromonomeric azoinitiator, MIM-400, as a macrocrosslinker initiated by the benzoyl peroxide (BPO)/N,N′dimethylaniline (DMA) coupled in water/ethanol (4/1, v/v) at 20°C was carried out to yield PEG-b-PMMA networks with labile azo linkages in the main chain. Gel fraction, swelling ratios (qv) and the average molecular weights between junction points (Mc) values of the networks were investigated. Maximum gel fraction was obtained using 0.133M MIM-400, 9.2×10−2M BPO, 2.1×10−2M DMA and 2.19M MMA.

Kinetic Comparison of Trifluoroacetic Acid Cleavage Reactions of Resin-Bound Carbamates, Ureas, Secondary Amides, and Sulfonamides from Benzyl-, Benzhydryl-, and Indole-Based Linkers.

The kinetics of cleavage reactions of 16 resin-bound carbamates, ureas, secondary amides, and sulfonamides from four different acid labile linkers including benzyl, benzhydryl, and indole linkers has been investigated. The optimized cleavage conditions are generally milder than those commonly used and reported (e.g., 0.5% TFA as opposed to 5%). Among various linkers studied in this work, the indole linker has been found to be the most acid labile followed by the Rink linker. The rate of cleavage of compounds linked to the resin via various functional groups can be summarized as follows: sulfonamide >carbamate approximately urea > amide. This study shows that cleavages of 16 compounds from four different acid labile linkers have been optimized to much milder conditions in terms of TFA concentration and the reaction time. It also demonstrates that single bead FTIR is an effective tool for optimizing cleavage conditions.

A novel acid stable/base labile carbamate linker for N-acyliminium ion reactions on solid support

The development of a novel carbamate linker optimized for solid phase N-acyliminium ion chemistry is reported. Some 2- and 2,4-substituted pyrrolidines were synthesized via addition of several carbon nucleophiles to immobilized N-acyliminium ions. A β-sulfonylethyl carbamate linker appeared especially useful; readily synthesized, stable towards Lewis acids and easily cleavable.

Crosslinked polyanhydrides for use in orthopedic applications: degradation behavior and mechanics.

High-strength, surface-eroding polymers were synthesized from methacrylated anhydride monomers of sebacic acid (MSA) and 1,6-bis(carboxyphenoxy) hexane (MCPH). These multifunctional monomers were photopolymerized using ultraviolet light to produce highly crosslinked polyanhydride networks. Through this approach, the crosslinking density of the resulting polymer network was used to control the final mechanical properties, while the degradation time scale was controlled by the chemical composition of the network. The combined hydrophobicity of the polymer backbone with the hydrolytically labile anhydride linkages led to surface-eroding networks, as confirmed by linear cumulative mass loss profiles as a function of degradation time for crosslinked polymer disks. By copolymerizing varying amounts of MSA and MCPH, the degradation rate of the final network was controlled from 2 days to 1 year. The tensile modulus of crosslinked poly(MSA) (1.4 GPa) was nearly an order of magnitude larger than that of linear poly(sebacic acid). In general, the mechanical properties of the crosslinked polyanhydrides networks were within ranges of those reported for cortical and trabecular bone. However, unlike bulk degrading polyesters such as poly(lactic acid), these surface eroding networks maintained >70% of their tensile modulus with 50% mass degradation.

The preparation of neoglycoconjugates containing inter-saccharide phosphodiester linkages as potential anti-Leishmania vaccines.

The Leishmania express complex glycoconjugates containing phosphosaccharide repeat units at all stages of their life-cycle. One of these molecules, lipophosphoglycan (LPG) has been suggested to be a vaccine candidate. To assess the immunological properties of Leishmania phosphosaccharides, we have prepared neoglycoproteins and neoglycolipids containing synthetic Leishmania phosphosaccharide repeats. The coupling procedure uses the dec-9-enyl spacer of previously synthesised phosphosaccharides for linkage to protein and phospholipid. This alkene moiety is converted by ozonolysis to an aldehyde which is then attached to protein and phospholipid amino groups by reductive amination. The procedure produces neoglycoconjugates in good yield and without compromising the labile phosphodiester linkages within the phosphosaccharide chains.

Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells

Antigen presenting cells (APCs) expressing CD1b mediate the specific T cell recognition of mycobacterial lipid antigens. These lipid antigens require internalization by APCs prior to presentation, but the detailed mechanisms of uptake and intracellular processing are not known. Here we have examined several steps in the presentation of two related classes of CD1b-presented antigens, free and glycosylated mycolates. T cell recognition of glucose monomycolate (GMM) was blocked by agents that fix APC membranes or neutralize the pH of endosomes, indicating a requirement for GMM uptake into an acidic compartment prior to recognition. Different T cell lines responded to free mycolate or GMM without crossreactivity, yet both antigens were taken up by APCs at the same rate. This demonstrated that differential recognition of these antigens resulted from T cell specificity for their hydrophilic caps and that APCs were unable to interconvert these antigens by enzymatic or chemical deglycosylation or glycosylation. APCs were also unable to cleave mycobacterial trehalose dimycolate (TDM) at its most chemically labile linkages to yield antigenic free mycolates or GMM. Our results indicate that these mycolate-containing antigens are resistant to chemical or enzymatic cleavage by APCs, suggesting that molecular trimming is not a universal feature of lipid antigen processing.