Design and synthesis of cell-permeable fluorescent nitrilotriacetic acid derivatives.

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.

Nitrilotriacetic Acid-Functionalized Glucose-Responsive Complex Micelles for the Efficient Encapsulation and Self-Regulated Release of Insulin.

Insulin plays a significant role in diabetes treatment. Although a huge number of insulin-loaded, glucose-responsive nanocarriers have been developed in past decades, most of them showed a lower loading capacity and efficiency due to the weak interaction between insulin and nanocarriers. In this work, a novel insulin-encapsulated glucose-responsive polymeric complex micelle (CM) is devised, showing (i) enhanced insulin-loading efficiency owing to the zinc ions' chelation by nitrilotriacetic acid (NTA) groups of NTA-functioned glycopolymer and the histidine imidazole of insulin, (ii) the glucose-triggered pulse release of insulin, and (iii) long stability under physiological conditions. This CM was fabricated by the self-assembly of block copolymer PEG- b-P(Asp- co-AspPBA) and glycopolymer P(Asp- co-AspGA- co-AspNTA), resulting in complex micelles with a PEG shell and a cross-linked core composed of phenylboronic acid (PBA)/glucose complexations. Notably, the modified nitrilotriacetic acid (NTA) groups of CM could specifically bind insulin via chelated zinc ions, thus enhancing the loading efficacy of insulin compared to that of nonmodified CM. The dynamic PBA/glucose complexation core of CM dissociates under the trigger of high glucose concentration (>2 g/L) while being quite stable in low glucose concentrations (<2 g/L), as demonstrated by the pulse release of insulin in vitro. Finally, in a murine model of type 1 diabetes, NTA-modified complex micelles loading an insulin (NTA-CM-INS) group exhibited a long hypoglycemic effect which is superior to that of free insulin in the PBS (PBS-INS) group and insulin-loaded complex micelles without an NTA modification (CM-INS) group. This long-term effect benefited from Zn(II) chelation by NTA-modified complex micelles and could avoid hypoglycemia caused by the burst release of insulin. Taken together, this constitutes a highly effective way to encapsulate insulin and release insulin via an on-demand manner for blood glucose control in diabetes.

Synthesis and anti-diabetic activity of new N,N-dimethylphenylenediamine-derivatized nitrilotriacetic acid vanadyl complexes.

Vanadium compounds are promising anti-diabetic agents. However, reducing the metal toxicity while keeping/improving the hypoglycemic effect is still a big challenge towards the success of anti-diabetic vanadium drugs. To improve the therapeutic potency using the anti-oxidative strategy, we synthesized new N,N-dimethylphenylenediamine (DMPD)-derivatized nitrilotriacetic acid vanadyl complexes ([VO(dmada)]). The in vitro biological evaluations revealed that the DMPD-derivatized complexes showed improved antioxidant capacity and lowered cytotoxicity on HK-2 cells than bis(maltolato)oxidovanadium (IV) (BMOV). In type II diabetic mice, [VO(p-dmada)] (0.15mmolkg-1/day) exhibited better hypoglycemic effects than BMOV especially on improving glucose tolerance and alleviating the hyperglycemia-induced liver damage. These insulin enhancement effects were associated with increased expression of peroxisome proliferator-activated receptor α and γ (PPARα/γ) in fat, activation of Akt (v-Akt murine thymoma viral oncogene)/PKB (protein kinase-B) in fat and liver, and inactivation of c-Jun NH2-terminal protein kinases (JNK) in liver. Moreover, [VO(p-dmada)] showed no tissue toxicity at the therapeutic dose in diabetic mice and the oral acute toxicity (LD50) was determined to be 1640mgkg-1. Overall, the experimental results indicated that [VO(p-dmada)] can be a potent insulin enhancement agent with improved efficacy-over- toxicity index for further drug development. In addition, the results on brain Tau phosphorylation suggested necessary investigation on the effects of vanadyl complexes on the pathology of the Alzheimer's disease in the future.

Ni(II)-NTA modified poly(ethylene imine) glycopolymers: physicochemical properties and first in vitro study of polyplexes formed with HIV-derived peptides.

Alternative delivery entities are desirable in immunotherapies in which polyplexes are widely formed by electrostatic interactions to induce cellular uptake processes for bioactive molecules. In our study, biocompatible Ni(II)-nitrilo(triacetic acid)-modified poly(ethylene imine)-maltose (Ni-NTA-DG) is realized and evaluated as complexation agent against His-tagged peptides using fluorescence polarization and dynamic light scattering. The polyplexes are stable until a pH of 6.5-6.0, and also up to 50 mM of imidazole. A first uptake approach shows that polyplexes lead to an increase in peptide uptake in monocyte-derived immature dendritic cells. In summary, Ni-NTA-DG represents a promising (delivery) platform for forthcoming in vitro applications.

A chelating-bond breaking and re-linking technique for rapid re-immobilization of immune micro-sensors.

With high sensitivity and specificity to antigen, immune micro-sensors can be used in rapid detection of pathogenic microbial. This study proposes and develops a method for rapidly regeneration of antibody on a resonant micro-cantilever sensor. A nitrilotriacetic acid (NTA) derivative is synthesized with cystine and bromoacetic acid, then added with 2-mercaptoethanol to prepare a mixed self-assembled monolayer (SAM) on Au (111) surface of the cantilever. Ni²âº ions are thereafter chelated on the mixed SAM to form a breakable and re-linkable chelating-bond layer. Repeatable cycles of antibody immobilization and erasing are experimentally validated with a detectable marker of synthesized biotinylated poly peptides harboring six histidine residues (named as His-Bio). Two distinguished pathogenic microbial, Escherichia. coli O157:H7 and Bacillus Anthracis, are detected with the rapidly regenerated sensor. The E. coli O157:H7 sensor exhibits a three-time repeated detection to the 10³ CFU/ml concentration microbial. Then, an E. coli O157:H7 sensor is eluted with Tris-HCl (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH = 3.0) and rapidly reconstructed into a B. Anthracis sensor by changing the re-immobilized antibody. The cantilever sensor no longer responses to E. coli O157:H7 even in a high concentration of 107 CFU/ml. In contrast, the sensor is experimentally confirmed being resoluble to low concentration B. Anthracis at 10³ spores/ml level. The proposed fast regeneration method is promising in repeatedly or multi-target detection applications of micro/nano immune-sensors, e.g. the resonant micro-cantilevers.

Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays.

Two bisphosphonate adaptors were designed to immobilize histidine-tagged proteins onto glass substrates coated with a zirconium phosphonate monolayer, allowing efficient and oriented immobilization of capture proteins, affitins directed to lysozyme, on a microarray format. These bifunctional adaptors contain two phosphonic acid anchors at one extremity and either one nitrilotriacetic acid (NTA) or two NTA groups at the other. The phosphonate groups provide a stable bond to the zirconium interface by multipoint attachment and allow high density of surface coverage of the linkers as revealed by X-ray photoelectron spectroscopy (XPS). Reversible high-density capture of histidine-tagged proteins is shown by real-time surface plasmon resonance enhanced ellipsometry and in a microarray format using fluorescence detection of AlexaFluor 647-labeled target protein. The detection sensitivity of the microarray for the target protein was below 1 nM, despite the monolayer arrangement of the probes, due to very low background staining, which allows high fluorescent signal-to-noise ratio. The performance of these Ni-NTA-modified zirconium phosphonate coated slides compared favorably to other types of microarray substrates, including slides with a nitrocellulose-based matrix, epoxide slides, and epoxide slides functionalized with Ni-NTA groups. This immobilization strategy has a large potential to fix any histidine-tagged proteins on zirconium or titanium ion surfaces.

A cysteine-based tripodal chelator with a high affinity and selectivity for copper(I).

A C(3)-symmetric ligand containing three converging cysteine chains anchored on a nitrilotriacetic acid moiety has been synthesized. This tripodal pseudopeptide, which provides three soft sulfur donor groups, exhibits a very high affinity for Cu(I) in either a monometallic complex or the cluster species Cu(6)L(3).

Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications.

High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell-penetrating peptides.

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 microM) for 1 h displayed approximately 8,400,000 [corrected]NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 microM) and Ni(OAc)(2) (100 microM) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within 4 h at 37 degrees C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell-penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology [corrected]

Electrogeneration of a poly(pyrrole)-NTA chelator film for a reversible oriented immobilization of histidine-tagged proteins.

This contribution reports, for the first time, the synthesis and electropolymerization of a pyrrole N-substituted by a nitrilotriacetic acid acting as a chelating center of Cu2+. A step-by-step approach for protein immobilization was developed via the successive coordination of Cu2+ and histidine-tagged proteins. The self-assembly of histidine-tagged glucose oxidase led to the formation of a close-packed enzyme monolayer at the poly(pyrrole) surface, and the reversibility and reproducibility of this affinity process were demonstrated.

Arginine carrier peptide bearing Ni(II) chelator to promote cellular uptake of histidine-tagged proteins.

Arginine-rich peptide-mediated protein delivery into living cells is a novel technology for controlling cell functions with therapeutic potential. In this report, a novel approach for the intracellular delivery of histidine-tagged proteins was introduced where a Ni(II) chelate of octaarginine peptide bearing nitrilotriacetic acid [R8-NTA-Ni(II)] was used as a membrane-permeable carrier molecule. Significant internalization of histidine-tagged enhanced green fluorescent protein (EGFP) into HeLa cells was observed by confocal microscopic observation in the presence of R8-NTA-Ni(II). Nuclear condensation characteristic in apoptotic cell death was also induced in the cells treated with a histidine-tagged apoptosis-inducing peptide [pro-apoptotic domain peptide (PAD)], indicating that the cargo molecules really went through the membrane to reach the cytosol. The apoptosis-inducing activity of the peptide thus delivered was compared with that of the PAD peptide covalently connected with the octaarginine peptide.

Synthesis of the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) and its use with the IAsys biosensor to study receptor-ligand interactions on model membranes.

This work describes the synthesis and use of the chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). This lipid is readily dispersed in aqueous media, both alone and when mixed with carrier lipids like dimyristoylphosphatidylcholine (DMPC). Fluorescence microscopic examination of membranes deposited from NTA-DTDA-containing liposomes shows that NTA-DTDA mixes uniformly with the carrier lipid, and does not phase separate. NTA-DTDA-membranes deposited onto the sensing surface of IAsys biosensor cuvettes show good stability, permitting use of the biosensor to study protein interactions. Hexahistidine-tagged proteins including recombinant forms of the extracellular regions of murine B7.1 (B7.1-6H) and of the human erythropoietin receptor (EPOR-6H) bind to NTA-DTDA-membranes; the stability of binding is dependent on both protein concentration, and density of NTA-DTDA. Kinetic measurements show that high stability of anchored proteins (t(1/2) approximately 10-20 h, apparent K(d) approximately 1 nM) can be achieved using membranes containing 25 mol% NTA-DTDA, but low levels of bound protein (<200 arc seconds). The system is used to study the interaction of human EPO with the EPOR anchored onto NTA-DTDA-membranes. In addition to the biological applications reported recently, the results show that NTA-DTDA can be a useful reagent in the study of receptor-ligand interactions.

Carboxymethyl-substituted bifunctional chelators: preparation of aryl isothiocyanate derivatives of 3-(carboxymethyl)-3-azapentanedioic acid, 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic++ + acid, and 1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid for use as protein labels.

3-(Carboxymethyl)-3-azapentanedioic acid (NTA), 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid (EGTA), and 1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid (DOTA) structures having a 4-nitrophenyl substituent attached via an alkyl spacer to the methylene carbon atom of one carboxymethyl arm of the chelator were obtained by alkylation of 4-nitrophenylalanine with bromoacetic acid (NTA), by reductive alkylation of 1,8-diamino-3,6-dioxaoctane with (4-nitrophenyl)-pyruvic acid followed by alkylation with bromoacetic acid (EGTA), and by alkylation of the trimethyl ester of 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid with the methyl ester of alpha-bromo-4-(4-nitrophenyl)pentanoic acid and subsequent saponification (DOTA). The nitrophenyl-substituted chelators were converted to the corresponding amines by hydrogenation then reacted with thiophosgene to give the protein-reactive aryl isothiocyanate derivatives.