Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in high yields.     

Changes in Free and Conjugated Indole 3-Acetic Acid and Abscisic Acid in Young Cotton Fruits and Their Abscission Zones in Relation to Fruit Retention during and after Moisture Stress

Experiments were conducted with field-grown cotton (Gossypium hirsutum L.) in 1985 and 1986 to determine effects of water deficit on levels of conjugated indole 3-acetic acid (IAA) and abscisic acid (ABA) in young fruits (bolls) and their abscission zones in relation to boll retention. Tissues were harvested three times during an irrigation cycle in 1985. They were harvested twice during an irrigation cycle and once after irrigation in 1986 to determine extent of recoveries of measured parameters. As reported earlier, the free IAA content of abscission zones decreased with moisture stress. Irrigation caused a partial recovery in free IAA content of abscission zones and caused a partial recovery in rate of boll retention. In contrast to free IAA, conjugated IAA increased with water deficit, both in 3-day-old bolls and in their abscission zones. Bolls contained much more ester IAA than their abscission zones. Some, but not all, of the increase in ester IAA in bolls during moisture stress could have come from a conversion of amide-linked IAA. Amide IAA decreased slightly during stress and increased after irrigation, but the concentration was low relative to ester IAA. Free and conjugated ABA both increased during stress and decreased after irrigation. However, the concentration of conjugated ABA remained relatively high in abscission zones. Ester IAA, being more resistant than free IAA to enzymic destruction during stress, may hasten recovery of fruit retention after relief of stress by providing a source of free IAA in abscission zones to inhibit continued abscission.     

Isolation and Quantitation of beta-d-Glucopyranosyl Abscisate from Leaves of Xanthium and Spinach

From previous work (Zeevaart 1980 Plant Physiol 66: 672-678) Xanthium leaves are known to contain a high level of alkali-hydrolyzable conjugated abscisic acid. This abscisic acid conjugate has been isolated and identified by mass spectrometry, nuclear magnetic resonance, and chemical and enzymic degradation techniques, as the glucosyl ester of abscisic acid, β-d-glucopyranosyl abscisate. The glucosyl ester of abscisic acid was the only abscisic acid conjugate found in Xanthium leaves. It was also isolated from spinach leaves.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

The production of 99mTc-labeled conjugated antibodies using a cyclam-based bifunctional chelating agent

A bifunctional chelating agent (BFCA) based on a macrocyclic amine, cyclam, was used to form specifically ^99mTc labeled rabbit-anti-HSA-antibodies. This study demonstrates the feasibility of forming a highly stable cyclam-based ^99mTc-BFCA with its subsequent conjugation to antibodies.     

Endogenous Auxin and Ethylene in the Lichen Ramalina duriaei

Indole-3-acetic acid (IAA) levels and ethylene evolution rates were measured in a fruticose lichen Ramalina duriaei collected from carob trees growing in northeast Israel. IAA levels were estimated by gas liquid chromatography with electron capture detection of the pentafluorobenzyl ester and also by enzyme-linked immunosorbent assay following methylation. The identity of the isolated IAA was confirmed by gas chromatography-mass spectrometry of both the methyl and the pentafluorobenzyl ester. IAA levels in lichens 1 year after transplanting to an air-polluted urban site were found to be lower than in the control thalli left at a nonpolluted, rural site. The material from the latter contained about 2.5 micrograms per gram fresh weight free IAA and 0.5 microgram per gram fresh weight conjugated IAA, while the urban material contained 0.3 microgram per gram each of free and conjugated IAA. Ethylene production rate was 1.0 nanoliter per gram fresh weight per hour in the material from the rural site and 1.5 nanoliters per gram fresh weight per hour in material from the urban site.     

Two-step enzymatic selective synthesis of water-soluble ketoprofen–saccharide conjugates in organic media

Ketoprofen–saccharide conjugates were synthesized by selectively enzymatic hydrolysis and acylation. Firstly, the (S)-ketoprofen vinyl ester was prepared by enzymatic hydrolysis of (R,S)-ketoprofen vinyl ester. Then enzymatic transesterification of (S)-ketoprofen vinyl ester with a series of saccharides were performed by the catalysis of a commercial protease from Bacillus licheniformis (BLP) in organic medium mixture of pyridine and tert-butanol. The ketoprofen was selectively conjugated onto the primary hydroxyl group of saccharides and with high yield after 72 h. Partition coefficient determination showed that all the products have better water solubility than parent ketoprofen. Chemical hydrolysis experiment indicated that 50% ketoprofen could be release from ketoprofen glucoside and maltoside in aqueous buffer (pH 7.4) within 48 h.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

Quantum dots as strain- and metabolism-specific microbiological labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.     

Quantum Dots as Strain- and Metabolism-Specific Microbiological Labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.     

Influence of the site of conjugation on the specificity of antibodies to progesterone

In order to determine how the site on the molecule used for conjugation influences the specificty of the resulting antiserum, progesterone was conjugated to bovine serum albumin (BSA) through substituents on the A(C3), B(C6), C(C11), and D(C20) rings for use as a hapten to elicit antibody formation in rabbits. Specificty of the antisera was determined by testing the ability of 24 representative steroids to displace radioactive progesterone in a radioimmunoassay procedure. Progesterone-tyrosine methyl ester (TME) conjugates were radioiodinated and used as the radioactive form of the hormone and radioactivity bound to antibody was separated from free radioactivity by a double antibody procedure. Immunization with progesterone conjugated at C20 resulted in the formation of antibodies which could not distinguish between progesterone and other Δ⁴-3-ketosteroids with structures similar in the A, B, and C ring (namely 17-hydroxyprogesterone, 20α and 20β-hydroxy-4-pregnen-20-one, deoxycorticosterne and testosterone). Immunization with progesterone-3-BSA resulted in the formation of antisera which were fairly specific for progesterone while immunization with progesterone conjugated at the 11 or 6 positions resulted in antisera which were very specific for progesterone. It was concluded that steroid hormones should be conjugated to protein at sites on the B or C ring of the molecule for the production of specific antisera.     

Biomolecule–nanoparticle interactions: Elucidation of the thermodynamics by isothermal titration calorimetry

BackgroundNanomaterials (NMs) are often exposed to a broad range of biomolecules of different abundances. Biomolecule sorption driven by various interfacial forces determines the surface structure and composition of NMs, subsequently governs their functionality and the reactivity of the adsorbed biomolecules. Isothermal titration calorimetry (ITC) is a nondestructive technique that quantifies thermodynamic parameters through in-situ measurement of the heat absorption or release associated with an interaction.Scope of reviewThis review highlights the recent applications of ITC in understanding the thermodynamics of interactions between various nanoparticles (NPs) and biomolecules. Different aspects of a typical ITC experiment that are crucial for obtaining accurate and meaningful data, as well as the strengths, weaknesses, and challenges of ITC applications to NP research were discussed.Major conclusionsITC reveals the driving forces behind biomolecule–NP interactions and the effects of the physicochemical properties of both NPs and biomolecules by quantifying the crucial thermodynamics parameters (e.g., binding stoichiometry, ΔH, ΔS, and ΔG). Complimentary techniques would strengthen the interpretation of ITC results for a more holistic understanding of biomolecule–NP interactions.General significanceThe thermodynamic information revealed by ITC and its complimentary characterizations is important for understanding biomolecule–NP interactions that are fundamental to the biomedical and environmental applications of NMs and their toxicological effects. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

A Feasible Approach to Evaluate the Relative Reactivity of NHS-Ester Activated Group with Primary Amine-Derivatized DNA Analogue and Non-Derivatized Impurity

Synthetic DNA analogues with improved stability are widely used in life science. The 3′and/or 5′ equivalent terminuses are often derivatized by attaching an active group for further modification, but a certain amount of non-derivatized impurity often remains. It is important to know to what extent the impurity would influence further modification. The reaction of an NHS ester with primary amine is one of the most widely used options to modify DNA analogues. In this short communication, a 3′-(NH₂-biotin)-derivatized morpholino DNA analogue (MORF) was utilized as the model derivatized DNA analogue. Inclusion of a biotin concomitant with the primary amine at the 3′-terminus allows for the use of streptavidin to discriminate between the products from the derivatized MORF and non-derivatized MORF impurity. To detect the MORF reaction with NHS ester, S-acetyl NHS-MAG₃ was conjugated to the DNA analogue for labeling with ^99mTc, a widely used nuclide in the clinic. It was found that the non-derivatized MORF also reacted with the S-acetyl NHS-MAG₃. Radiolabeling of the product yielded an equally high labeling efficiency. Nevertheless, streptavidin binding indicated that under the conditions of this investigation, the non-derivatized MORF was five times less reactive than the amine-derivatized MORF.     

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pK_a, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.     

Receptor Displacement in the Cell Membrane by Hydrodynamic Force Amplification through Nanoparticles

We introduce an intrinsically multiplexed and easy to implement method to apply an external force to a biomolecule and thus probe its interaction with a second biomolecule or, more generally, its environment (for example, the cell membrane). We take advantage of the hydrodynamic interaction with a controlled fluid flow within a microfluidic channel to apply a force. By labeling the biomolecule with a nanoparticle that acts as a kite and increases the hydrodynamic interaction with the fluid, the drag induced by convection becomes important. We use this approach to track the motion of single membrane receptors, the Clostridium perfringens ε-toxin (CPεT) receptors that are confined in lipid raft platforms, and probe their interaction with the environment. Under external force, we observe displacements over distances up to 10 times the confining domain diameter due to elastic deformation of a barrier and return to the initial position after the flow is stopped. Receptors can also jump over such barriers. Analysis of the receptor motion characteristics before, during, and after a force is applied via the flow indicates that the receptors are displaced together with their confining raft platform. Experiments before and after incubation with latrunculin B reveal that the barriers are part of the actin cytoskeleton and have an average spring constant of 2.5 ± 0.6 pN/μm before vs. 0.6 ± 0.2 pN/μm after partial actin depolymerization. Our data, in combination with our previous work demonstrating that the ε-toxin receptor confinement is not influenced by the cytoskeleton, imply that it is the raft platform and its constituents rather than the receptor itself that encounters and deforms the barriers formed by the actin cytoskeleton.     

Mutagen Formation in the Reaction of Nitrite with the Food Components Analogous to Sorbic Acid

Mutagen formation by the reaction of sodium nitrite with some sorbic acid analogs was investigated by using the microbial mutagenicity tests (rec-assay and Ames test) together with chemical examination using TLC, and it became clear that the conjugated dienoic carbonyl structure is essential for mutagen formation by the reaction with nitrite By a large scale reaction of sodium nitrite with sorbic acid methyl ester, 5-nitro-2,4-hexadienoic acid methyl ester and ethylnitrohc acid were isolated and identified as the mam mutagens The results led to the assumption that a nitro group adjacent to the double bond is an important factor to develop mutagenicity     

The interaction of proteins and cells with affinity ligands covalently coupled to silicon surfaces as monitored by ellipsometry

Two methods for the chemical binding of biomolecules to silicon surfaces are described. The first method utilizes an alkyl silane and a nucleophilic reagent to join the biomolecule to the silicon surface; the second method involves crosslinking with glutaraldehyde in order to couple the biomolecule and albumin molecules, which have first been physically adsorbed. The course of binding to the silicon surface has been followed with the aid of ellipsometry. This optical measuring technique estimates the thicknesses of, e.g., organic layers, by measuring the polarization properties of a light beam before and after reflection at surfaces. The method by which the binding of a biomolecule to its corresponding affinity ligand on silicon wafers can be followed with this technique is reported. The systems studied are concanavalin A-Saccharomyces cerevisiae cells, immunoglobulin G-Staphylococcus aureus cells, and an NAD-analog-lactate dehydrogenase. With ellipsometry it was possible to assess how the incubation time and the concentration of the cells and the biomolecules added influenced the results. It was found that an increasing time of incubation and higher concentration resulted in a more complete coverage of the silicon wafer surfaces.     

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

d-glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 d-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T_50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca²⁺ ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k_cat/K_m) than unmodified trypsin against the substrate N-α-benzoyl-l-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.     

Comparison of two methods of preparing enzyme-antibody conjugates: application of these conjugates for enzyme immunoassay.

Two methods of preparing enzyme-antibody conjugates were evaluated. High yields of conjugate were obtained with both methods. The first procedure utilizes the homobifunctional crosslinking reagent N,N′-o-phenylenedimaleimide. Sulfydryl residues were introduced into second antibodies by reaction with methyl-mercaptobutyrimidate. The modified antibodies were reacted with N,N′-o-phenylenedimaleimide, excess reagent was removed by gel filtration, and the activated antibodies were cross-linked to β-galactosidase. Up to 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of enzyme activity. The second method utilizes the heterobifunctional meta-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antibodies were reacted with MBS, excess reagent was removed by gel filtration and the activated antibodies were crosslinked to β-galactosidase. Typically, approximately 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of both enzyme and antibody activity. Conjugates prepared using these two procedures were used as labels in an immunoassay system and were able to detect approximately 5 to 10 ng of first antibodies. The MBS procedure was simpler to perform, could more easily be adapted to large-scale work, and gave more reproducible results, and the conjugates produced were able to detect slightly lower concentrations of first antibody.