Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group and their applicability to biomolecule derivatization

Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group (i.e., 1-[4-(6-aminooxyhexamido)benzyl]diethylenetriaminetetraacetic acid lanthanides(III), 6a-d, where Ln(3+) is Eu, Dy, Sm, and Tb) is described. Their applicability to biomolecule derivatization is demonstrated by allowing them to react with a synthetic oligopeptide, a protein, two synthetic drugs, and a steroid. The oligopeptide and protein were linked to 6 after preoxidation of their N-terminal serine residues, while the drugs and the steroid reacted via their ketone functionality. Also some application data is included.     

Targeting of N-(2-hydroxypropyl)methacrylamide copolymers to liver by incorporation of galactose residues

Soluble synthetic polymers have potential as targetable carriers of pharmacological agents. Here we report that incorporation into poly[N-(2-hydroxypropyl)methacrylamide)] of an oligopeptide side-chain terminating in galactose enhanced the polymer's pinocytic uptake from the rat bloodstream by the liver. Within the liver lysosomes enzymic digestion led to the intracellular release of a drug analogue also bound to oligopeptide side-chains of the polymer.     

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

Myelin basic protein (MBP) is a major protein of myelin‐proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12‐mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti‐MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K _d = 0.51–0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10⁻¹ to 2.3 × 10⁻⁴ M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K _d, M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192‐fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins.     

Labeling of proteins and oligopeptides with luminescent lanthanide(III) chelates.

Synthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine-based europium(III) chelate designed for solution phase protein labeling is demonstrated.     

Transport of hepcidin, an iron-regulatory peptide hormone, into retinal pigment epithelial cells via oligopeptide transporters and its relevance to iron homeostasis

Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC₅₀ values in the range of 0.4–1.7 μM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.     

Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

Introduction of lanthanide(III) chelates to oligopeptides on solid phase

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).     

A chitin-oligomer binding peptide obtained by screening of a phage display random peptide library and its affinity modulation corresponding to oxidation–reduction state

Screening of a phage display random 9-mer peptide library, in which cysteine residues were at the both terminals of the 7-mer random region, was performed to obtain an oligopeptide that recognizes a chitin-oligomer. Affinity of the obtained peptide (Cys-Ser-Arg-Thr-Thr-Arg-Thr-Arg-Cys) to chitotriose was modulated by its oxidation–reduction state. Only the oxidized form exhibited specific binding to the target molecule, chitotriose. This is the first report of reversible affinity modulation of a synthetic oligopeptide which can recognize a neutral saccharide.     

The fate of the biomimetic cation triaqua-mu-oxohexapropionatotrichromium(III) in rats

The synthetic biomimetic triaqua-mu-oxohexapropionatotrichromium(III) nitrate when given intravenously to rats lowers fasting blood plasma triglycerides and cholesterol concentrations; thus, the cation has the potential to serve as a therapeutic agent. Its ability to function in vivo presumably is dependent on its ability to mimic the action of the natural, bioactive, chromium-binding oligopeptide chromodulin in stimulating insulin receptor kinase activity. Consequently, the cation should be incorporated into insulin-sensitive cells intact. Thus, the fate of the 51Cr-labeled complex during the first 24 h after injection in tissues, blood, urine, and feces was followed. The complex appears to be readily incorporated into tissues and cells. In hepatocytes, the cation is efficiently transported into microsomes where its concentration reaches a maximum in approximately 2 h.     

Synthesis of protected oligo (γ-methyl-L-glutamates) and study of their conformation in solution and at the air–water interface

Oligopeptides having the general formula N-carbobenzoxy(γ-methyl-L -glutamyl)_n-dimethyl-L -glutamate (with n = 1, 3, 5, 7, 11) were prepared, using both solid-phase synthesis and conventional peptide couplings in solution. The appearance of an ordered structure with increasing chain length was studied in organic solvents and at the air–water interface. The results obtained by thin-layer chromatography were interpreted. All the studies have demonstrated a very similar behaviour of this type of oligopeptide in solution and in monolayers.     

Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species

In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.     

Three complete turns of a 3(10)-helix at atomic resolution: the crystal structure of Z-(Aib)11-OtBu.

The crystal structure of the synthetic protected oligopeptide Z-(Aib)11-OtBu was determined by x-ray crystallography. The undecapeptide folds in a regular 3(10)-helix with nine consecutive 4 --> 1 hydrogen bonds. At present, this is the largest available structure of a homopeptide (including homopeptides consisting of standard amino acids) and also the longest observed regular 3(10)-helix at atomic resolution. Z-(Aib)11-OtBu crystallizes readily from hot ethanol-water mixture and is one of the crystals in which no solvent molecule is co-crystallized. In the crystal head-to-tail hydrogen bonded columns are formed in the [1 0 1] direction. Each helical column is surrounded by six others, whereby two are packed in parallel and four in antiparallel fashion. Helical columns are packed via apolar crystal contacts. The crystal structure of Z-(Aib)11-OtBu is compared with the crystal structures of Z-(Aib)10-OtBu and Z-(Aib)9-OtBu. The similarities and differences are analysed.     

Circular dichroism and conformational analysis of the membrane-modifying peptide -N-t-Boc-(Aib-L-Ala)5-Gly-Ala-Aib-Pro-Ala-Aib-Aib-Glu-(OBzl)-Gln-OMe with respect to alamethicin

The conformational analysis of the CD spectrum is reported for the synthetic and membrane-modifying nonadecapeptide analog of alamethicin N-t-Boc-(Aib-L -Ala)₅-Gly-Ala-Aib-Pro-Ala-Aib-Aib-Glu(OBzl)- Gln-OMe. The CD data are evaluated according to three different methods and are discussed with respect to those obtained from natural alamethicin and suitable models such as N-t-Boc-(Aib-L -Ala)₇-OPOE, fragments of the synthetic nonadecapeptide, and the hexadecapeptide N-t-Boc-(Aib-L -Ala)₅-Pro-Ala-Aib-Aib-Glu(OBzl)-Gln-OMe. The synthetic nonadecapeptide with the longer helical region exhibits membrane activities comparable to those of alamethicin, whereas the hexadecapeptide with the shorter helix is inactive.     

Isolation and Characterization of Low-Molecular-Weight Chromium-binding Substance (LMWCr) from Chicken Liver

Chromodulin (also known as low-molecular-weight chromium-binding substance, LMWCr) is a chromium-binding oligopeptide proposed to play a role in insulin signaling and chromium transport in mammals. This laboratory has isolated and purified this material from a non-mammalian source, an avian. Spectroscopic and physical characterization of the isolated material suggests the material is an oligopeptide with a multinuclear chromium assembly bridged via asparatate and glutamate residues very similar to its mammalian counterparts. The isolated material also possesses a biological activity similar to other LMWCr isolates.     

Coordination modes of N-(2-hydroxyphenyl)methyl-bis-(2-pyridylmethyl)amine with copper (I) and (II) halides

The title ligand, N-(2-hydroxyphenyl)methyl-bis-(2-pyridylmethyl)amine, was prepared via a condensation-reduction synthetic route. The compounds, CuCl(C₁₉H₁₉N₃O) and [CuBr(C₁₉H₁₉N₃O)]⁺Br⁻ · 3H₂O, were readily synthesized from the reaction of CuCl or CuBr₂ and the ligand in acetonitrile. The title copper(I) compound is an O-H ? Cl hydrogen-bonded linear chain of tetrahedrally coordinated copper centers, and the title copper(II) compound exists as two strongly tetragonally distorted dibromide bridged metal cations in a dimer with the phenol hydroxyl groups weakly bound in a trans-fashion to one of the bridging bromides. In the copper(I) complex the phenoxy group acts only as a hydrogen bond donor, whereas in the copper(II) complex it acts both as a ligand and a hydrogen bond donor.     

Chromium-containing biomimetic cation triaqua-mu3-oxo-mu-hexapropionatotrichromium(III) inhibits colorectal tumor formation in rats

The cation [Cr(3)O(propionate)(6)(H(2)O)(3)](+) has recently been found by oral or intravenous administration to increase insulin sensitivity and improve serum lipids in healthy and type 2 diabetic model rats. Serum insulin concentrations and, hence, insulin sensitivity are in part responsible for the relationship between diet and the incidence of colorectal cancer. This strongly suggested that the synthetic cation, which in vitro is able to stimulate insulin receptor tyrosine kinase activity in a fashion similar to the oligopeptide chromodulin, could influence the incidence of colorectal cancer. Consequently, the effects of the cation on the inhibition of colon tumorigenesis induced by 1,2-dimethylhydrazine in male Sprague Dawley rats were examined. Gavage administration of aqueous solutions of the complex (1000 microg Cr/kg body mass daily for 6 months) resulted in significantly decreased colon tumor incidence (P<0.003).     

Antihypertensive Effect of Boysenberry Seed Polyphenols on Spontaneously Hypertensive Rats and Identification of Orally Absorbable Proanthocyanidins with Vasorelaxant Activity

An E. coli expression clone coding for human proinsulin, which was fused to NH₂-terminal β-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH₂ terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)_n, (His)_n, (Trp)_n, and (Ser)_n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH₂-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.     

Expression of the art gene protein of human T-lymphotropic virus type III (HTLV-III/LAV) in bacteria

Human T-lymphotropic virus type III (HTLV-III/LAV or HIV) contains a gene designated art (anti-repressor transactivator). Here, we report the expression of the art gene product in bacteria and show that the 20-kilodalton (kDa) bacterially expressed art protein is recognized by serum of a patient. The bacterially synthesized art protein competed in an immunological reaction with a 20-kDa protein produced in HTLV-III/LAV-infected lymphocytes. Antiserum to a synthetic oligopeptide corresponding to a sequence in the second exon of the art gene also precipitated the 20-kDa protein in HTLV-III/LAV-infected cells. These results demonstrate that the 20-kDa art gene product is expressed in cell lines that produce HTLV-III/LAV virions.     

Binding of tetrakis(pyrazoliumyl)porphyrin and its copper(II) and zinc(II) complexes to poly(dG-dC)2 and poly(dA-dT)2

Interactions of cationic porphyrins bearing five-membered rings at the meso position, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP; M is H₂, Cu^II or Zn^II), with synthetic polynucleotides poly(dG-dC)₂ and poly(dA-dT)₂ have been characterized by viscometric, visible absorption, circular dichroisim and magnetic circular dichroism spectroscopic and melting temperature measurements. Both H₂PzP and CuPzP are intercalated into poly(dG-dC)₂ and are outside-bound to the major groove of poly(dA-dT)₂, while ZnPzP is outside-bound to the minor groove of poly(dA-dT)₂ and surprisingly is intercalated into poly(dG-dC)₂. The binding constants of the porphyrin and poly(dG-dC)₂ and poly(dA-dT)₂ are on the order of 10⁶ M⁻¹ and are comparable to those of other cationic porphyrins so far reported. The process of the binding of the porphyrin to poly(dG-dC)₂ and poly(dA-dT)₂ is exothermic and enthalpically driven for H₂PzP, whereas it is endothermic and entropically driven for CuPzP and ZnPzP. These results have revealed that the kind of the central metal ion of metalloporphyrins influences the characteristics of the binding of the porphyrins to DNA.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix

The archaeon Aeropyrum pernix grows optimally at 90°C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA_Ap) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA_Ap-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA_Ap was about 70% at 90°C using a 1/100 (w/w) OppA_Ap/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA_Ap with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA_Ap as the extracellular component for the uptake of oligopeptides.