Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low μM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH₂, exhibited a K_d for hMC4R of 9.1 ± 1.4 μM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-α-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects.     

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining K_d values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited K_d values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The K_i values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the K_i values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.     

Pyrazolyl derivatives as bifunctional chelators for labeling tumor-seeking peptides with the fac-[M(CO)3]+ moiety (M = 99mTc, Re): synthesis, characterization, and biological behavior

Radiolabeling of biologically active molecules with the [(99m)Tc(CO)(3)](+) unit has been of primary interest in recent years. With this in mind, we herein report symmetric (L(1)) and asymmetric (L(2)-L(5)) pyrazolyl-containing chelators that have been evaluated in radiochemical reactions with the synthon [(99m)Tc(H(2)O)(3)(CO)(3)](+) (1a). These reactions yielded the radioactive building blocks [(99m)Tc(CO)(3)(k(3)-L)](+) (L = L(1)-L(5), 2a-6a), which were identified by RP-HPLC. The corresponding Re surrogates (2-6) allowed for macroscopic identification of the radiochemical conjugates. Complexes 2a-6a, with log P(o/w) values ranging from -2.35 to 0.87, were obtained in yields of > or =90% using ligand concentrations in the 10(-5-)10(-4) M range. Challenge studies with cysteine and histidine revealed high stability for all of these radioactive complexes, and biodistribution studies in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal-urinary pathway. Based on the framework of the asymmetric chelators, the novel bifunctional ligands 3,5-Me(2)-pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(6)) and pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(7)) have been synthesized and their coordination chemistry toward (NEt(4))(2)[ReBr(3)(CO)(3)] (1) has been explored. The resulting complexes, fac-[Re(CO)(3)(k(3)-L)]Br (L(6)(7), L(7)(8)), contain tridentate ancillary ligands that are coordinated to the metal center through the pyrazolyl and amine nitrogen atoms, as observed for the other related building blocks. L(6) and L(7) were coupled to a glycylglycine ethyl ester dipeptide, and the resulting functionalized ligands were used to prepare the model complexes fac-[Re(CO)(3)(kappa(3)-3,5-Me(2)-pz(CH(2))(2)N(glygly)(CH(2))(2)NH(2))](+) (9/9a) and fac-[Re(CO)(3)(kappa(3)-pz(CH(2))(2)N(CH(2))(3)(glygly)(CH(2))(2)NH(2))](+) (10/10a) (M = Re, (99m)Tc). These small conjugates have been fully characterized and are reported herein. On the basis of the in vitro/in vivo behavior of the model complexes (2a-6a, 9a, 10a), we chose to evaluate the in vitro/in vivo biological behavior of a new tumor-seeking Bombesin pyrazolyl conjugate, [(L(6))-G-G-G-Q-W-A-V-G-H-L-M-NH(2)], that has been labeled with the [(99m)Tc(CO)(3)](+) metal fragment. Stability, in vitro cell binding assays, and pharmacokinetics studies in normal mice are reported herein.     

Diiron models for the active site of Fe-only hydrogenase with terminal organosulfur ligation: synthesis, structures and electrochemistry

Diiron model complexes (micro-SCH(2)CH(2)CH(2)S)Fe(2)(CO)(5)L with thioether-substitution, L=S(CH(2)CH(3))(2) (2), S(CH(2)CH(3))(CH(2)CH(2)Cl) (3), S(CH(2)CH(3))(C(6)H(5)) (4), or sulfoxide-substitution, L=SO(CH(2)CH(2)CH(3))(2) (5), SO(CH(3))(2) (6), were synthesized as active site analogues of Fe-only hydrogenase. The organosulfur ligands were introduced into the diiron centers via moderately stable intermediates following two routes. The X-ray crystallographic structures of complexes 2-6 show the apical positions of terminal organosulfur ligands. The electrochemical behaviors of the model complexes were investigated, especially for the interesting properties of the derivative of 6 which is proposed to be the first model with weak donor ligand similar to CO.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Squalene-derived flexible linkers for bioactive peptides

A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3+2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-alpha-MSH constructs, presumably due to slow off rates for this high affinity ligand.     

Carbohydrate recognition by Clostridium difficile toxin A

Clostridium difficile TcdA is a large toxin that binds carbohydrates on intestinal epithelial cells. A 2-A resolution cocrystal structure reveals two molecules of alpha-Gal-(1,3)-beta-Gal-(1,4)-beta-GlcNAcO(CH(2))(8)CO(2)CH(3) binding in an extended conformation to TcdA. Residues forming key contacts with the trisaccharides are conserved in all seven putative binding sites in TcdA, suggesting a mode of multivalent binding that may be exploited for the rational design of novel therapeutics.     

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.     

Binding mechanisms of PEGylated ligands reveal multiple effects of the PEG scaffold

A series of synthetic ligands consisting of poly(ethylene glycol) (PEG), capped on one or both ends with the hapten 2,4-dinitrophenyl (DNP), were previously shown to be potent inhibitors of cellular activation in RBL mast cells stimulated by a multivalent antigen [Baird, E. J., Holowka, D., Coates, G. W., and Baird, B. (2003) Biochemistry 42, 12739-12748]. In this study, we systematically investigated the effect of increasing length of the PEG scaffold on the binding of these monovalent and bivalent ligands to anti-DNP IgE in solution. Our analysis reveals evidence for an energetically favorable interaction between two monovalent ligands bound to the same receptor, when the PEG molecular mass exceeds approximately 5 kDa. Additionally, for ligands with much higher molecular masses (>10 kDa PEG), the binding of a single ligand apparently leads to a steric exclusion of the second binding site by the bulky PEG scaffold. These results are further corroborated by data from an alternate fluorescence-based assay that we developed to quantify the capacity of these ligands to displace a small hapten bound to IgE. This new assay monitors the displacement of a small, receptor-bound hapten by a competitive monovalent ligand and thus quantifies the competitive inhibition offered by a monovalent ligand. We also show that, for bivalent ligands, inhibitory capacity is correlated with the capacity to form effective intramolecular cross-links with IgE.     

Synthesis and oxidation of carboxylate-bridged diiron(II) complexes with substrates tethered to primary alkyl amine ligands

The synthesis and crystallographic characterization of a series of diiron(II) complexes with sterically hindered terphenyl carboxylate ligands and alkyl amine donors are presented. The compounds [Fe(2)(mu-O(2)CAr(Tol))(4)(L)(2)] (L=NH(2)(CH(2))(2)SBn (1); NH(2)(CH(2))(3)SMe (2); NH(2)(CH(2))(3)CCH (3)), where (-)O(2)CAr(Tol) is 2,6-di(p-tolyl)benzoate, and [Fe(2)(mu-O(2)CAr(Xyl))(2)(O(2)CAr(Xyl))(2)(L)(2)] (L=NH(2)(CH(2))(3)SMe (4); NH(2)(CH(2))(3)CCH (5)), where (-)O(2)CAr(Xyl) is 2,6-di(3,5-dimethylphenyl)benzoate, were prepared as small molecule mimics of the catalytic sites of carboxylate-bridged non-heme diiron enzymes. The compounds with the (-)O(2)CAr(Tol) carboxylate form tetrabridged structures, but those containing the more sterically demanding (-)O(2)CAr(Xyl) ligand have only two bridging ligands. The ancillary nitrogen ligands in these carboxylate-rich complexes incorporate potential substrates for the reactive metal centers. Their oxygenation chemistry was studied by product analysis of the organic fragments following decomposition. Compound 1 reacts with dioxygen to afford PhCHO in approximately 30% yield, attributed to oxidative dealkylation of the pendant benzyl group. Compound 3 decomposes to form Fe(II)Fe(III) and Fe(III)Fe(IV) mixed-valence species by established bimolecular pathways upon exposure to dioxygen at low temperatures. Upon decomposition, the alkyne-substituted amine ligand was recovered quantitatively. When the (-)O(2)CAr(Tol) carboxylate was replaced by the (-)O(2)CAr(Xyl) ligand in 5, different behavior was observed. The six-coordinate iron(III) complex with one bidentate and two monodentate carboxylate ligands, [Fe(O(2)CAr(Xyl))(3)(NH(2)(CH(2))(3)CCH)(2)] (6), was isolated from the reaction mixture following oxidation.     

Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25 ± 10 Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH₂ was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-_c[Asp-His-2′-D-Nal-Arg-Trp-Lys]-NH₂ and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)₃ units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.     

A structure-function study of ligand recognition by CD22beta

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.     

Cell-specific targeting by heterobivalent ligands

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 ? is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.     

Synthesis of novel heterobimetallic copper(I) hydrazone Schiff base complexes: a comparative study on the effect of heterocyclic hydrazides towards interaction with DNA/protein, free radical scavenging and cytotoxicity

Two new copper(I) hydrazone complexes have been synthesised from bivalent copper precursor [CuCl(2)(PPh(3))(2)] and ferrocene containing bidentate hydrazone ligands HL(1) (1) or HL(2) (2). Based on the elemental analyses and spectroscopic data, the complexes are best formulated as [CuL(1)(PPh(3))(2)] (3) and [CuL(2)(PPh(3))(2)] (4) of the monovalent copper ion. Solid state structures of ligand 2 and its corresponding complex 4 were also determined. The DNA/albumin interactions of all the synthesised compounds were investigated using absorption, emission and synchronous fluorescence studies. Further, antioxidant properties of all the compounds have also been checked against ABTS, O(2)(-) and OH radicals. Additionally, the in vitro cytotoxic activity of compounds 1-4 was assessed using tumour (HeLa, A431) and non-tumour (NIH 3T3) cell lines.     

19F NMR studies of tryptophan/serum albumin binding

19F NMR provides direct measures of the Trp binding avidity of 'fatty acid free' bovine serum albumin when D- and L-6-fluorotryptophan are used as the probes. Both a high and low affinity binding site are present. The addition of octanoate either displaces the ligand from both sites or greatly decreases the affinity such that little binding occurs at 2 mM levels. In the case of L-6-fluorotryptophan separate signals are observed for the high and low affinity binding sites and titrations with competing ligands can be used to establish the relative affinities of ligands at the high affinity site. Binding at this site appears to be hydrophobic and shape specific with L-Phe being a very poor ligand (K(D)[L-Phe]/K(D)[L-Trp]=800) while both GHKalphaNal and GHKW displace L-6-fluorotryptophan from this site. In tripeptides of the general formula GHK[ epsilon NH(CH(2))(n)(CO)W], affinity increases with tether length and binding at the low affinity site is restored. This NMR assay appears well-suited for the discovery of selective binding agents in this and other biorecognition phenomena.     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Interaction of anions with rat liver arginase: specific inhibitory effects of fluoride

The inhibitory effects of anions, such as N(3)(-), NO(2)(-), BO(4)(3-), SCN(-), CH(3)COO(-), SO(4)(2-), ClO(4)(-), H(2)PO(4)(-), CN(-), I(-), Br(-), Cl(-) and F(-), on the hydrolysis of L-arginine (L-Arg) by rat liver arginase (RLA) have been studied. From all these anions, only F(-) exhibited a clear inhibitory effect at the mM level. Inhibition of RLA by F(-) is reversible and uncompetitive towards L-Arg binding with a K(i) value of 1.3+/-0.5 mM at pH 7.4. This effect is dependent on pH as the IC(50) value of F(-) towards RLA increases from 1.2 to 19 mM when increasing the pH from 7 to 10. Another specific inhibitor of RLA, N(omega)-hydroxy-L-nor-arginine (nor-NOHA), that has been recently shown to bind to RLA as a bridging ligand of its (Mn(II))(2) cluster, exhibits some similarities with F(-) in its inhibitory effects (identical pH dependence). It is thus tempting to propose that the inhibitory effects of F(-) could be due to its binding as a bridging ligand of the RLA (Mn(II))(2) cluster. However, further studies are required to determine the modes of interaction of F(-) with RLA.     

Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.     

Synthesis and evaluation of cholecystokinin trimers: A multivalent approach to pancreatic cancer detection and treatment

In the quest for novel tools for early detection and treatment of cancer, we propose the use of multimers targeting overexpressed receptors at the cancer cell surface. Indeed, multimers are prone to create multivalent interactions, more potent and specific than their corresponding monovalent versions, thus enabling the potential for early detection. There is a lack of tools for early detection of pancreatic cancer, one of the deadliest forms of cancer, but CCK2-R overexpression on pancreatic cancer cells makes CCK based multimers potential markers for these cells. In this Letter, we describe the synthesis and evaluation of CCK trimers targeting overexpressed CCK2-R.     

Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.