Interaction of the Heparin-Binding Consensus Sequence of β-Amyloid Peptides with Heparin and Heparin-Derived Oligosaccharides.

Alzheimer's disease (AD) is characterized by the presence of amyloid plaques in the AD brain. Comprised primarily of the 40- and 42-residue β-amyloid (Aβ) peptides, there is evidence that the heparan sulfate (HS) of heparan sulfate proteoglycans (HSPGs) plays a role in amyloid plaque formation and stability; however, details of the interaction of Aβ peptides with HS are not known. We have characterized the interaction of heparin and heparin-derived oligosaccharides with a model peptide for the heparin- and HS-binding domain of Aβ peptides (Ac-VHHQKLV-NH2; Aβ(12-18)), with mutants of Aβ(12-18), and with additional histidine-containing peptides.

Design, synthesis, and characterization of heparin-binding peptoids.

A series of N-substituted glycine oligomers (peptoids) of varying length and side chains was synthesized with the aim of producing peptidomimetics that would bind with high affinity to heparin and thereby neutralize its anticoagulant activity. To this end, a library of 29 peptoids was synthesized using solid phase synthesis methodologies. The general design of the peptoids was the repeating trimer sequence N(cationic side chain)-N(alkyl or benzyl side chain)-N(α-chiral side chain), where the monomers are N-substituted glycine residues bearing the indicated side chains.

Determination of the primary structure and carboxyl pK (A)s of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional (1)H NMR.

Determination of the structure of heparin-derived oligosaccharides by (1)H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H(1) protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional (1)H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-](3)-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆(4,5)-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-D: -glucosamine and IdoA(2S) is 2-O-sulfated α-L: -iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra.

Kinetics and equilibria of cis/trans isomerization of secondary amide peptide bonds in linear and cyclic peptides.

The secondary amide peptide bonds that comprise up to one-third of the bonds of peptide or protein backbones can exist as cis and trans isomers, with the trans isomer being highly favored. However, there is little quantitative data on the kinetics and equilibria of cis-trans isomerization of secondary amide peptide bonds due to the difficulty of detecting the very small population of cis isomers. Knowledge of factors that influence the kinetics and equilibria of cis-trans isomerization of secondary amide peptide bonds will contribute to a more complete understanding of the structural and dynamic behavior of the backbones of peptides and unfolded proteins and of complex protein folding kinetics.

Interaction of heparin and heparin-derived oligosaccharides with synthetic peptide analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein.

Background: Although protamine is effective as an antidote of heparin, there is a need to replace protamine due to its side effects. HIP peptide has been reported to neutralize the anticoagulant activity of heparin. The interaction of HIP analog peptides with heparin and heparin-derived oligosaccharides is investigated in this paper.

Oligo(N-aryl glycines): a new twist on structured peptoids.

We explore strategies to enhance conformational ordering of N-substituted glycine peptoid oligomers. Peptoids bearing bulky N-alkyl side chains have previously been studied as important examples of biomimetic "foldamer" compounds, as they exhibit a capacity to populate helical structures featuring repeating cis-amide bonds. Substantial cis/trans amide bond isomerization, however, gives rise to conformational heterogeneity.

Kinetics and equilibria of cis/trans isomerization of backbone amide bonds in peptoids.

The biological activities of N-substituted glycine oligomers (peptoids) have been the subject of extensive research. As compared to peptides, both the cis and trans conformations of the backbone amide bonds of peptoids can be significantly populated. Thus, peptoids are mixtures of configurational isomers, with the number of isomers increasing by a factor of 2 with each additional monomer residue.

Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.

Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH.

Interaction of heparin with two synthetic peptides that neutralize the anticoagulant activity of heparin.

Two synthetic analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (Ac-SRGKAKVKAKVKDQTK-NH2) and the all-d-amino acid version of the same peptide (l-HIPAP and d-HIPAP, respectively) were synthesized, and their efficacy as agents for neutralization of the anticoagulant activity of heparin was assayed. The two analogue peptides were found to be equally effective for neutralization of the anticoagulant activity of heparin, as measured by restoration of the activity of serine protease factor Xa by the Coatest heparin method. The finding that l-HIPAP and d-HIPAP are equally effective suggests that d-amino acid peptides show promise as proteolytically stable therapeutic agents for neutralization of the anticoagulant activity of heparin.

Improved resolution in two-dimensional 1H NMR spectra of peptides by band-selective, homonuclear decoupling during both the evolution and acquisition periods: application to characterization of the binding of peptides by heparin.

Two-dimensional 1H NMR experiments that achieve band-selective, homonuclear decoupling in both the indirectly detected F1 and directly detected F2 dimensions were used to assign the highly overlapped 1H NMR spectrum of the peptide Ac-SRGKARVRAKVKDQTK-NH2, both free in solution and bound to heparin. Band-selective, homonuclear decoupling during the evolution period was achieved using a double pulsed field gradient spin-echo (DPFGSE) with semi-selective shaped pulses; band-selective, homonuclear decoupling during the acquisition period was achieved by time-shared semi-selective shaped pulse decoupling. Regular TOCSY, ROESY and NOESY spectra and TOCSY, ROESY and NOESY spectra measured with band-selective, homonuclear decoupling in the evolution (F1) dimension (BASHD-TOCSY, ROESY and NOESY spectra) and with band-selective, homonuclear decoupling in both the F1 and F2 dimensions (D-(or Double)-BASHD-TOCSY, ROESY and NOESY spectra) are reported and compared for the peptide and its heparin complex.

Characterization of the selenotrisulfide formed by reaction of selenite with end-capped phytochelatin-2.

The phytochelatins are a family of peptides synthesized by plants in response to exposure to heavy metals and metalloids, including selenium in the form of selenite. The amino acid sequence of the phytochelatin (PC) peptides is (gamma-Glu-Cys)n-Gly, where n typically ranges from 2 to 5. In this paper, the products of the reaction of selenite with an end-capped analogue of PC2, Ac-(gamma-Glu-Cys)2-Gly-NH2, are characterized.

Unexpectedly fast cis/trans isomerization of Xaa-Pro peptide bonds in disulfide-constrained cyclic peptides.

Acyclic dithiol and cyclic disulfide forms of the peptides Ac-Cys-Pro-Xaa-Cys-NH2 (Xaa = Phe, His, Tyr, Gly, and Thr) and Ac-Cys-Gly-Pro-Cys-NH2 and the peptide Ac-Ala-Gly-Pro-Ala-NH2 were synthesized and characterized by mass spectrometry and NMR spectroscopy. Rate constants kct and ktc for cis-to-trans and trans-to-cis isomerization, respectively, across the Cys-Pro or Gly-Pro peptide bonds were determined by magnetization transfer NMR techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. It was found that constraints imposed by the disulfide bond confer an unexpected rate enhancement for cis/trans isomerization, ranging from a factor of 2 to 13.

Characterization of the acid/base and redox chemistry of phytochelatin analogue peptides.

The phytochelatins are a family of polydisperse, thiol-rich peptides that are synthesized by plants in response to exposure to heavy metals. The amino acid sequence of the phytochelatin peptides is (gamma-glutamyl-cysteinyl)n-glycine, where n typically ranges from 2 to 5. In the first phase of a program to characterize the coordination chemistry of the phytochelatins with heavy metals, the phytochelatin analogue peptides acetyl(gamma-glutamyl-cysteinyl)n-glycine amide (Ac-(gamma-Glu-Cys)n-NH2, n = 2-6) have been synthesized by solid-phase peptide synthesis methods and characterized by 1H NMR spectroscopy.

Heparin and heparan sulfate: structure and function.

This review covers the structure and function of heparin and heparan sulfate glycosaminoglycans. Their chemical structures are discussed, including recently developed methods for sequencing picomole to nanomole quantities of heparin- and heparan sulfate-derived oligosaccharides. The biosynthesis of heparin and heparan sulfate is reviewed as it relates to their diverse and varied structures, and their biological activities and functions are discussed.

Convenient synthesis of human calcitonin and its methionine sulfoxide derivative.

The human calcitonin peptide chain was assembled using Fmoc solid-phase peptide synthesis chemistry. The combinations of cleavage Reagent H with trans-[Pt(en)(2)Cl(2)](2+) and Reagents B, K, and R with trans-[Pt(CN)(4)Cl(2)](2-) provide convenient methods for the synthesis of human calcitonin and its methionine sulfoxide derivative; the formation of intramolecular disulfide bonds by the above Pt(IV) oxidants is essentially quantitative.

Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin.

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin.

trans-Dichlorotetracyanoplatinate(IV) as a Reagent for the Rapid and Quantitative Formation of Intramolecular Disulfide Bonds in Peptides.

Oxidation of cysteine thiol groups by trans-dichlorotetracyanoplatinate(IV) to form intramolecular peptide disulfide bonds has been studied for a series of dithiol peptides ranging from 4 to 15 amino acid residues in length. The dithiol peptides are rapidly and quantitatively transformed to their intramolecular disulfide forms by a slight excess of [Pt(CN)(4)Cl(2)](2)(-), as shown by HPLC. Quantitative analyses by HPLC and by spectrophotometric titration confirm a [Pt(IV)]:[dithiol peptide] stoichiometry of 1:1.

Kinetics and Equilibria of the Thiol/Disulfide Exchange Reactions of Somatostatin with Glutathione.

Rate and equilibrium constants are reported for the thiol/disulfide exchange reactions of the peptide hormone somatostatin with glutathione (GSH). GSH reacts with the disulfide bond of somatostatin to form somatostatin-glutathione mixed disulfides (Cys(3)-SH, Cys(14)-SSG and Cys(3)-SSG, Cys(14)-SH), each of which can react with another molecule of GSH to give the reduced dithiol form of somatostatin and GSSG. The mixed disulfides also can undergo intramolecular thiol/disulfide exchange reactions to re-form the disulfide bond of somatostatin or to interconvert to the other mixed disulfide.