Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins

A new protein crosslinking agent, 2,3-dibromopropionyl-N-hydroxysuccinimide ester, has been synthesized and characterized. The potential use of this compound as a temperature-controllable heterobifunctional crosslinking agent has been investigated using model systems and its reactivity compared with that of chlorambucil-N-hydroxysuccinimide ester. The coupling of¹⁴C-labeled phenylethylamine to lysozyme has been used to illustrate the feasibility of the use of this crosslinking agent for the synthesis of immunotoxins.     

Covalent surface chemistry gradients for presenting bioactive peptides

The activation of surfaces by covalent attachment of bioactive moieties is an important strategy for improving the performance of biomedical materials. Such techniques have also been used as tools to study cellular responses to particular chemistries of interest. The creation of gradients of covalently bound chemistries is a logical extension of this technique. Gradient surfaces may permit the rapid screening of a large range of concentrations in a single experiment. In addition, the biological response to the gradient itself may provide new information on receptor requirements and cell signaling. The current work describes a rapid and flexible technique for the covalent addition of bioactive peptide gradients to a surface or gel and a simple fluorescence technique for assaying the gradient. In this technique, bioactive peptides with a terminal cysteine are bound via a heterobifunctional coupling agent to primary amine-containing surfaces and gels. A gradient in the coupling agent is created on the surfaces or gels by varying the residence time of the coupling agent across the surface or gel, thereby controlling the extent of reaction. We demonstrate this technique using poly(l-lysine)-coated glass surfaces and fibrin gels. Once the surface or gel has been activated by the addition of the coupling agent gradient, the bioactive peptide is added. Quantitation of the gradient is achieved by measuring the reaction kinetics of the coupling agent with the surface or gel of interest. This can be done either by fluorescently labeling the coupling agent (in the case of surfaces) or by spectrophotometrically detecting the release of pyridine-2-thione, which is produced when the thiol-reactive portion of the coupling agent reacts. By these methods, we can obtain reasonably precise estimates for the peptide gradients without using expensive spectroscopic or radiolabeling techniques. Validation with changes in fibroblast cell migration behavior across a bioactive peptide gradient illustrates preservation of peptide function as well as the usefulness of this technique.     

A New Phosphorylating Agent,Di(2,2,2-Trifluoroethyl) Trimethylsilyl Phosphite. Its Application in Dna Synthesis by The Phosphotriester Approach

Abstract

A new phosphorylating agent, di(2,2,2-trifluoro-ethyl) trimethylsilyl phosphite, has been prepared and is proved to be a useful agent for the phosphorylation of the 3′-hydroxyl group of deoxyribonucleosides in the absence of coupling agents. The resulting deoxyribonucleoside 3′-(2,2,2-trifluoroethyl) phosphates are key intermediates for the synthesis of deoxyribooligonucleotides by the phospho-triester approach.     

Surface immobilization and entrapping of enzymes on glutaraldehyde crosslinked gelatin particles

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4-d-glucosidase, EC 3.2.1.3), β-d-fructofuranosidase (invertase, EC 3.2.1.26) and β-d-glucoside (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.     

Studies on the preparation of conjugates of leukotriene C4 with proteins for development of an immunoassay for SRS-A (1)

Leukotriene C₄ (LTC₄) has been conjugated with Bovine Serum Albumin (BSA) and Hemocyanin from Giant Keyhole Limpets (KLH) using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. A second LTC₄-KLH conjugate was prepared using a new bifunctional coupling agent, 6- -maleimidohexanoic acid chloride. Conjugates of some representive LTC₄ component parts; - -chlorophenacylglutathione with BSA and of 2,4(E),6,9(Z)-pentadecatetraen-1-ol with BSA were also prepared.     

Studies on the conjugation of leukotriene B4 with proteins for development of a radioimmunoassay for leukotriene B4

Leukotriene B4 (LTB4) (I) has been converted to its N-(3-amino-propyl)amide derivative (III) and to its hydrazide derivative (VII) via LTB4 delta-lactone. The amide (III) was coupled with Bovine Serum Albumin using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. The hydrazide (VII), was coupled with Hemocyanin (Keyhole Limpet) (KLH) using 6-N-maleimidohexanoic acid chloride as coupling agent.     

A general route to D- and L-six-membered nucleoside analogues

A simple synthetic route for novel L-(as well as D-) six-membered nucleosides is described. Particularly, we have provided a general approach to the synthesis of azasugar-based nucleosides, which preparation has been easily achieved starting from the coupling of our three carbon homologating agent 1 with the well known Garner aldehyde 4. Further suitable and stereocontrolled functionalizations of the intermediate 9 will provide, after the base insertion, a wide class of six membered modified azanucleosides to be tested as NRTIs.     

Use of New Phosphonylating and Condensing Agents in The Synthesis of Oligonucleotides Via The H-Phosphonate Approach

Abstract

Bis (1, 1, 1, 3, 3, 3-hexafluoro-2-propyl) phosphonate was most promising as a phosphonylating agent for the preparation of nucleoside-3′-phosphonate units. 1, 3-Dimethyl-2-chloro-imida-zolinium chloride (UWC) as a coupling agent has successfully been used for the internucleotidic H-phosphonate bonds formation via the H-phosphonate approach on a solid support.     

Studies on the conjugation of leukotriene B4 with proteins for development of a radioimmunoassay for leukotriene B4

Leukotriene B₄ (LTB₄) (I) has been converted to its N-(3-aminopropyl)amide derivative (III) and to its hydrazide derivative (VII) via LTB₄ δ-lactone. The amide (III) was coupled with Bovine Serum Albumin using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. The hydrazide (VII), was coupled with Hemocyanin (Keyhole Limpet) (KLH) using 6-N-maleimidohexanoic acid chloride as coupling agent.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

The synthesis of peptides employing 9-fluorenylmethyl chloroformate(Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtainedin good yield (70–95%). Furthermore, both the ¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

Summary The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

New coupling mechanism of the silane coupling agents in the TATB-based PBX

Behaviours of the silane coupling agents in 1,3,5-triamino-2,4,6-trinitrobenzene (TATB)-based polymer bonded explosives (PBXs) were investigated using dissipative particle dynamics simulations. A new and extraordinary coupling mechanism of the silane coupling agent in TATB-based PBXs was revealed, in which the binding between the binders and TATB was improved by making the TATB's affinitive structure units of binders assembling at the interface, whereas the TATB's unaffinitive structure units are bonded together by the silane coupling agent shrinking into the binders. This is quite different from the traditional view, i.e. the coupling agent usually stays at the interface.     

An improved synthesis of the C-linked glucuronide of N-(4-hydroxyphenyl)retinamide

Retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventatives and chemotherapeutics for numerous types of cancer. The C-linked analogue of the O-glucuronide of 4-HPR (4-HPRCG) has been shown to be a more effective agent. The synthetic route to this molecule has been significantly improved by access to a key C-benzyl-glucuronide intermediate through employment of a Suzuki coupling reaction between an exoanomeric methylene sugar and an aryl bromide. Preliminary evidence shows 4-HPRCG has chemotherapeutic activity.     

A method for the detection of urocanase on polyacrylamide gels and its use with crude cell extracts of Pseudomonas.

A method is described for the specific detection of urocanase activity on polyacrylamide gels. It is dependent upon the reduction of nitro blue tetrazolium by the product of the urocanase reaction using phenazine methosulphate as a coupling agent. The method has been characterized using crude cell extracts of Pseudomonas testosteroni and Pseudomonas putida. After growth of the organisms in histidine-succinate medium each extract shows only one band of urocanase activity. The enzymes from the two species have significantly different electrophoretic mobilities.     

Synthesis of hybrid distamycin-cysteine labeled with 99mTc: a model for a novel class of cancer imaging agents

The synthesis of a hybrid constituted by distamycin A and cysteine labeled with the gamma-emitting radionuclide 99mTc to afford the conjugate complex 5 is reported. This new radiopharmaceutical is of potential interest as tumor imaging agent in diagnostic nuclear medicine. The preparation of the hybrid distamycin A-cysteine 4 has been achieved by coupling deformyldistamycin A and Boc-Dmt-OH. Compound 4 was then successfully labeled with 99mTc by reaction with the novel, high-electrophilic, metal-containing fragment [99mTc(N)(PP)]2+ (PP = diphosphine ligand) yielding the 1:1 complex 5.     

Immobilisation of a 5'-Phosphodiesterase from Vegetal Origin by Covalent Binding on Activated Celite

A vegetal enzyme, 5'-phosphodiesterase, has been immobilised by covalent coupling onto activated Celite. This nonmicrobial activity was obtained from barley rootlets, an economical brewer's by-product. 5'-ribonucleotides are selectively cleaved from RNA by a partially purified preparation of the enzyme. These compounds are high added value products used as flavor enhancers in food industries (disodium 5'-inosinate, disodium 5'-guanylate) and have important applications in the pharmaceutical industry (cytidine 5'-phosphate, uridine 5'-phosphate and adenine 5'-phosphate). Glutaraldehyde was used as coupling agent. A concentration of 5 mM of glutaraldehyde was found adequate. Using a charge of 160.8 units μ g carrier_-1 a total of 15% of the activity could be recovered in the carrier. The characterisation of the solid biocatalist is reported.     

Synthesis of a diaminedithiol bifunctional chelating agent for incorporation of technetium-99m into biomolecules

The synthesis of a bifunctional chelating agent (BCA), 1, based on the diaminedithiol (DADT) ligand system, is described. The six-step synthetic sequence has been accomplished in 16% overall yield, affording 1, which contains a thiolactone as a reactive moiety, which permits direct coupling to nucleophiles without the formation of byproducts. The reactivity of 1 toward benzylamine and subsequent labeling of the ligand with technetium-99m has been evaluated as a model for preparation of various bioconjugates. Both coupling and exchange labeling occur in high yield under mild conditions, and competition reactions with diethylenetriaminepentaacetic acid (DTPA) indicate the superior stability of the technetium-99m-DADT complex. Preparation of BCA 1 thus provides a new avenue into technetium-labeled radiopharmaceuticals.     

Combining experiments with multi-cell agent-based modeling to study biological tissue patterning

Agent-based modeling (ABM), also termed ‘Individual-basedmodeling (IBM)’, is a computational approach that simulatesthe interactions of autonomous entities (agents, or individualcells) with each other and their local environment to predicthigher level emergent patterns. A literature-derived rule setgoverns the actions of each individual agent. While this techniquehas been widely used in the ecological and social sciences,it has only recently been applied in biomedical research. Thepurpose of this review is to provide an introduction to ABMas it has been used to study complex multi-cell biological phenomena,underscore the importance of coupling models with experimentalwork, and outline future challenges for the ABM field and itsapplication to biomedical research. We highlight a number ofpublished examples of ABM, focusing on work that has combinedexperimental with ABM analyses and how this pairing producesnew understanding. We conclude with suggestions for moving forwardwith this parallel approach.      

Study of different coupling agents in the conjugation of a V3-based synthetic MAP to carrier proteins.

The conjugation of synthetic peptides to carrier proteins is a widely used method for immunological studies. Different coupling agents have been described to form the conjugate with carrier proteins. In this paper, we demonstrate that the antibody response toward V3-based synthetic MAPs derived from HIV-1, JY1 isolate, conjugated to two different carrier proteins using either m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or beta-maleimidopropionic acid N-hydroxysuccinimide ester (MPS), or succinic anhydride (SA) show different behaviors. An excellent anti-JY1 response without a strong response to the coupling agent is observed in the case of succinic anhydride spacer. In contrast, MBS produces total abrogation of the antibody response with a high response toward the coupling agent.     

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.