6-maleimidohexanoic Acid Research Articles

Maleimide grafting onto polysaccharides via mild condition esterification and its impact on their structure.

This study proposes an innovative approach to tailor the properties of two polysaccharides, microcrystalline cellulose (MCC) and potato starch, through chemical modification in dispersion. The methodology involves the grafting of 6-Maleimidohexanoic acid (6-MHA) moieties onto hydroxyl groups of the polysaccharides without dissolving them in order to keep their native structure preserved. To overcome the slow and inefficient reaction between carboxylic acids of 6-MHA and hydroxyl groups of the polysaccharides, a vinyl ester of 6-MHA was synthesized through the transvinylation of 6-MHA acid with vinyl acetate. The resulting 6-MHA ester was employed to introduce a new functionality to polysaccharides' hydroxyl groups via transesterification, catalyzed by potassium carbonate. To enhance the reactivity, the polysaccharides were mercerized prior to modification process. The efficiency of the transesterification reaction between the vinyl ester of 6-MHA and the hydroxyl groups of the polysaccharides was confirmed using Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Thermal behavior analysis was carried out using thermogravimetric analysis (TGA), while changes in crystallinity resulting from the modification were assessed through X-ray diffraction analysis (XRD). Finally, the impact of the modification on the morphology of polysaccharides was examined with environmental scanning electron microscopy (ESEM). Despite changes in microstructure, MCC kept its macrostructure remained morphologically unchanged while the granular structure of starch was damaged. Maleimide grafting onto MCC and starch has the potential to turn them into thermally reversible materials for various applications such as debondable adhesive or coating.

Evidence of Isomerization in the Michael-Type Thiol-Maleimide Addition: Click Reaction between L-Cysteine and 6-Maleimidehexanoic Acid.

The reaction between L-cysteine (Cys) and 6-maleimidohexanoic acid (Mhx) in an aqueous medium at different levels of pH was analyzed via RP-HPLC, finding the presence of two reaction products throughout the evaluated pH range. By means of solid-phase extraction (SPE), it was possible to separate the products and obtain isolated profiles enriched up to 80%. The products were analyzed individually through mass spectrometry, DAD-HPLC, NMR 1H, 13C, and two-dimensional evidence of isomerization between the hydrogen atoms of the α-amino and the thiol group present in the cysteine. Thus, it was concluded that the products obtained corresponded to a mixture of the isomer Cys-S-Mhx, where the adduct is formed by a thioether bond, and the isomer Cys-NH-Mhx, in which the union is driven by the amino group. We consider that the phenomenon of isomerization is an important finding, since it has not previously been reported for this reaction.

Synthesis and New Cytotoxicity Screening Approach of Novel Dirhodium Complexes

Transition metal–based chemotherapeutics have been used to treat a variety of cancers since the 1960s with cisplatin being the most prominent of these drugs used in a clinical setting. Many cancers become chemoresistant to cisplatin, so there is currently a need to synthesize and explore the anticancer abilities of other transition metal complexes. Rhodium, one of the six platinum–group metals, demonstrates cytotoxic effects in cancer cells in the form of dirhodium tetraacetate (Rh2A4). However, Rh2A4 is known to be less cytotoxic than cisplatin in HeLa cervical cancer cells. This research aims to modify Rh2A4 to increase its cytotoxicity to that of cisplatin. One of the acetate ligands of Rh2A4 was selectively replaced with 6‐maleimidohexanoic acid (M) to form a dirhodium triacetate maleimide (Rh2A3M) complex. Maleimides are known to readily react with alkylamines (R‐NH2) under basic conditions to produce amine conjugates. Alkylamines were chosen because of this reactivity and because their presence in a variety of metabolic and physiological processes may aid in the uptake of the dirhodum complex in cancer cells. The Rh2A3M complex was successfully reacted with four alkylamines: benzylamine, cyclohexylamine, ethanolamine, and glucosamine. This reaction was done in a 1:10 molar ratio in a phosphate buffer of pH 7.25 at room temperature for 72 hours to form subsequent Rh2A3M‐NH‐R complexes. The crude reaction mixture was then purified by preparative HPLC and characterized via 1H 1D NMR. A new screening method was developed using MTT reagent to compare the cellular viability of HeLa cervical cancer cells treated with the four crude reaction mixtures and the four HPLC purified Rh2A3M‐NH‐R complexes. Each pure complex had a similar percent cellular viability to its corresponding crude reaction mixture. This procedure will allow for future screens of many Rh2A3M + R‐NH2 crude reaction mixtures to indicate cytotoxicity of Rh2A3M‐NH‐R products without the need for the purification and characterization steps. Out of the four pure complexes, Rh2A3M‐cyclohexylamine was found to be the most cytotoxic at ~20 μM, suggesting it is comparable in cytotoxicity to cisplatin. Current work is focused on the determination of the IC50 value for this complex to definitively compare its cytotoxicity to cisplatin.

Preparation and Evaluation of 6-Maleimidohexanoic Acid Grafted Chitosan Nanoparticles as a Novel Carrier for Intranasal Protein Delivery

In this study, 6-maleimidohexanoic acid grafted chitosan nanoparticles (MHA-CS NPs) were prepared and evaluated the efficiency of intranasal protein delivery as compared with well-known chitosan nanoparticles (CS NPs). Fluorescein isothiocyanate labelled with bovine serum albumin (FITC-BSA) was used as a model protein. The results indicated that both CS NPs and MHA-CS NPs were positively charged NPs before and after protein loading. The condition for optimal protein loading was 1:6 mass ratio of protein/NPs at 1 h incubation period. The optimal formulations of CS NPs and MHA-CS NPs were evaluated on porcine mucosa as ex vivo. The mucoadhesive and permeation properties of FITC-BSA loaded MHA-CS NPs showed a greater than FITC-BSA loaded CS NPs and FITC-BSA solution, respectively. These ex vivo studies present the potential of MHA-CS NPs as a novel carrier for intranasal protein delivery that will be a candidate for in vivo study.

Studies about reactive ene-functionalized dextran derivatives for Thiol-ene click reactions

Dextran was applied for the design of biopolymers bearing reactive CC double bond for subsequent “click” reaction with thiols. The polysaccharide was functionalized with maleimido- and norbornene moieties as “ene” compound for thiol-Michael and radical thiol-ene reaction, respectively. The hydroxyl groups of dextran were esterified with 6-maleimidohexanoic acid or 5-norbornene-2-carboxylic acid in an efficient, homogeneous one-step synthesis applying different in-situ activation methods of the carboxylic acids. The water- or organo-soluble dextran derivatives obtained were allowed to react with N-acetyl-L-cysteine to study the reactivity of the ene-functionalized derivatives in thiol-ene “click” reactions dependent on solvent, reaction time, and activation/initiation reagent. The dextran esters and the corresponding N-acetyl-L-cysteine adducts were characterized by means of elemental analysis, NMR-, and FT-IR spectroscopy.

Preparation and Characterization of Albumin Conjugates of a Truncated Peptide YY Analogue for Half-Life Extension

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

Versatile bio-ink for covalent immobilization of chimeric avidin on sol–gel substrates

A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films.

Evaluation of Synthetic Cell-Penetrating Peptides, Pro-Rich Peptide and Octaargine Derivatives, as Adenovirus Vector Carrier

Two cell-penetrating peptides, a Pro-rich peptide derivative, acetyl-(Val-Arg-Leu-Pro-Pro-Pro)(3)-Gly-Cys amide, and an octaarginine derivative, acetyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys amide, were prepared by the solid phase method. Each peptide was coupled to the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, and then conjugated to the Adenovirus vector containing luciferase gene. Peptide-modified Ad, as compared with wild-type Ad, exhibited excellent luciferase activity in B16BL6 cells.

Tailored PEG for rh-G-CSF Analogue Site-Specific Conjugation

A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH(2) was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH(2))(11)-Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH(2))(11)-NH(2) was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal (PEG-C(18)-Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by (1)H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal/PEG-NH(2) molar ratio, %]. Both PEG-C(18)-Mal and the commercial maleimido activated 20 kDa linear PEG (PEG-Mal) were used for conjugation to (17)Cys of recombinant human granulocyte colony stimulating factor (rh-G-CSF). Under denaturing conditions, at pH 7.0, both activated PEGs yielded over 90% protein conjugation. Under native conditions, about 55% and 7% PEGylated protein were obtained with PEG-C(18)-Mal and PEG-Mal, respectively. Circular dichroism analysis showed that the PEGylation does not induce detectable alteration of the protein secondary structure. On the other hand, the PEGylation conditions were found to affect significantly the protein stability. The derivatives obtained either with the two polymers by unfolding/refolding process or with PEG-Mal under native conditions displayed rapid aggregation with half-life ranging from 30 to 90 min. The derivative obtained with PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal in the absence of guanidinium chloride displayed remarkably higher stability with aggregation half-life of about 60 h.

Preparation of a Tat-Related Transporter Peptide for Carrying the Adenovirus Vector into Cells

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, Ac-Tat(48-60)-Gly-Cys-NH(2), having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat(48-60)-Gly-Cys-NH(2) peptide exhibited excellent gene transfer efficacy in B16BL6 cells.

Exploiting the Substrate Tolerance of Farnesyltransferase for Site‐Selective Protein Derivatization

The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. With the exception of the biotin-functionalized analogue, all the phosphoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diels-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.

Studies on Heterobifunctional Cross-Linking Reagents, 6-Maleimidohexanoic Acid Active Esters

6-maleimidohexanoic acid N-hydroxysuccinimide ester has been used widely for preparation of enzyme immunoconjugates as a unique heterobifunctional cross-linking reagent. Its heterobifunctional reactivity is good, but its ester portion hydrolyzes easily in the presence of water. Several 6-maleimidohexanoic acid active esters (6-maleimidohexanoic acid 4-nitrophenyl ester, 6-maleimidohexanoic acid N-hydroxy-5-norbornene-endo-2,3-dicarboximide ester, and 6-maleimidohexanoic acid pentafluorophenyl ester) were prepared and their reactivity and stability in an aqueous media were tested. Of the synthetic esters, the pentafluorophenyl ester exhibited the highest reactivity and stability in aqueous media.

Design and synthesis of a Tat-related gene transporter: A tool for carrying the adenovirus vector into cells

A Tat-related peptide, acetyl-Gly-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, designed to transport an Adenovirus vector (Ad) into cells, was synthesized. The synthetic peptide was conjugated to Ad, which potentially can act as an efficient carrier of heterologous genes into cells. The Tat-related peptide was synthesized using the solid phase method and then was coupled to the heterofunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. The resulting peptide-succinimidohexanoic acid N-hydroxysuccinimide ester was conjugated to Ad containing the luciferase gene. B16BL6 cells infected with the peptide-conjugated Ad luciferase gene construct exhibit a 50-fold greater luciferase activity than B16BL6 cells infected with wild-type Ad containing the luciferase gene.

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (αvβ3 and αvβ5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro- βAla) 2Lys-PEG- βAla-Cys-NH 2[(Ac-YGGRGDTP βA) 2K-PEG- βAC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP βA) 2K-PEG- βAC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP βA) 2K-PEG- βAC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

Carbohydrate-centered maleimide cluster as a new type of templates for multivalent peptide assembling: synthesis of multivalent HIV-1 gp41 peptides

This paper describes a facile synthesis of carbohydrate-centered maleimide clusters and their application as a new type of templates for multivalent peptide assembling. Simultaneous introduction of multiple maleimide functionalities onto a carbohydrate core was achieved through the reaction of carbohydrate-based polyamines with methoxycarbonylmaleimide or with the N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid. The clustered maleimides placed on the carbohydrate core allow rapid and highly chemoselective ligation with multiple copies of cysteine-containing peptides under virtually neutral conditions at room temperature. This mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. The usefulness of the maleimide clusters as a new type of templates for multivalent peptide synthesis was exemplified by the synthesis of two tetravalent gp41 peptides incorporating the sequence of the potent HIV inhibitor, T20. The synthetic multivalent gp41 peptides are useful as novel immunogens to raise specific antibodies for HIV studies. They are also useful probes for studying HIV membrane fusion mechanisms.

Engineering of a 129-residue tripod protein by chemoselective ligation of proline-II helices

A 129-residue tripod protein was designed, synthesized, and biophysically characterized. This receptor-adhesive modular protein contained three 30-residue proline-II helices linked to a 9-residue proline-II helix through thioether bonds. Coupling of 6-maleimidohexanoic acid succinimido ester to cis- N α-Boc-4-amino-L-proline furnished in 77% yield the maleimido acid cis- N-Boc-4-(6-maleimidohexanamido)-L-proline (Boc-Prm), which was used in the solid-phase synthesis of the linker peptide CH 3-CO-Pro 3-Prm 3-Pro 3-***NH 2. The leg peptide, the 40-residue thiol Gly-Arg-Gly-Asp-Ser-Pro-Gly-Tyr-Gly-Pro 30-Cys-NH2, was also made by solid-phase synthesis. The tripod protein was prepared by Michael addition of the thiol groups of three leg peptides to the three maleimide groups of the linker peptide. By 13C NMR spectrometry, the linker peptide was a proline-II helix, as indicated by the presence of only trans Pro-Pro resonances for its β and γ carbons. By circular dichroic spectroscopy, the model peptide CH 3-CO-Pro 9-NH 2, the linker peptide, the leg peptide, and the tripod protein each contained substantial proline-II helix, as indicated by a strong negative band at 205 nm and a weak positive band at 226 nm. Since the Pro 30 proline-II helix of each leg is about 93 Å long, two Arg-Gly-Asp sites on different legs of the tripod protein could be as much as ≈250 Å apart.