Further characterization of the antithrombin-binding sequence in heparin

An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin and previously found to have the following predominant structure

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, has been studied further. High-voltage, paper electrophoresis of the ³H-labelled disaccharides obtained by deamination with HNO₂ (pH 1.5) followed by reduction with Na[³H]BH₄ showed 25% of mono-O-sulfated components, in addition to l-iduronic acid(2-O-SO₃)-2,5-anhydro-d-[³H]mannitol(6-O-SO₃). The monosulfated disaccharides were identified by high-pressure, ion-exchange chromatography as l-iduronic acid(2-O-SO₃)-2,5-anhydro-d-[³H]mannitol, l-iduronic acid-2,5-anhydro-d-[³H]mannitol(6-O-SO₃), and d-glucuronic acid-2,5-anhydro-d-[³H]-mannitol(6-O-SO₃). These components originated from the reducing, terminal disaccharide residue (units 7 and 8), as indicated by selective labelling with Na[³H]BH₄. The structural variability within this region suggests that it is not part of the antithrombin-binding sequence. Neither enzymic removal of the non-sulfated l-iduronic acid unit 1 nor N-deacetylation (by hydrazinolysis) at unit 2 had any significant effect on the affinity of the octasaccharide for antithrombin. However, removal of the disaccharide corresponding to units 1 and 2, by selective deamination of the N-deacetylated octasaccharide, yielded a low-affinity hexasaccharide. In addition, a high-affinity deamination product was formed, presumably an octasaccharide containing a 6-sulfated 2-deoxy-2-C-formyl-d-pentofuranosyl unit due to ring contraction in unit 2. These results suggest that the 6-sulfate group in unit 2 may be involved in antithrombin binding. It is concluded that the antithrombin-binding site in heparin is represented by the pentasaccharide sequence extending from unit 2 to unit 6 of the octasaccharide studied.     

Facile Synthesis of <Emphasis Type="Italic">N</Emphasis>-protected Amino Acid Esters Assisted by Microwave Irradiation

A highly efficient and safe methodology for synthesis of various N-protected amino acid ethyl esters have been established in this study. This methodology employs orthoesters as both esterification reagent and solvent for protected amino acids. The reactions were carried out under microwave irradiation in neutral conditions for only 2 min, resulting in highly pure crude products in most cases. This strategy works with a variety of N-protecting groups, such as acid labile protecting group: BOC and tBu; base labile protecting group: Fmoc; hydrogenation labile protecting group: Z and Na/NH₃ labile protecting group: Tos, thus providing facile access to numerous valuable building blocks for solid phase synthesis. Further reduction of the crude protected amino acid ethyl ester by sodium borohydride under mild conditions led to the corresponding protected β-amino alcohols with excellent yield, as demonstrated by three examples.     

Alterations in extracellular calcium concentration differentially influence prostaglandin and thromboxane synthesis in epithelial cells from the toad urinary bladder

The effects of alterations in extracellular calcium concentration on prostaglandin (PGE) and thromboxane (TXB₂) syntheses were studied in isolated epithelial cells from the urinary bladder of the toad, Bufo marinus. In epithelial cells prepared using collagenase, basal iPGE synthesis was greater than iTXB₂ synthesis. Increasing extracellular calcium from zero to 1 mm increased iPGE synthesis and decreased iTXB₂ synthesis equivalently such that total conversion of endogenous arachidonate to these two metabolites was unaltered. Vasopressin stimulated iPGE and iTXB₂ syntheses when the incubation buffer contained 1 mm calcium but had no effect in the presence of 0.4 μm calcium. In contrast, using an EDTA isolation method, basal iPGE and iTXB₂ syntheses were equal in the presence of zero calcium. Increasing extracellular calcium concentration to 1 mm caused a greater enhancement in iTXB₂ synthesis compared to iPGE. Increasing extracellular calcium to 2 mm was associated with a decline in iPGE and iTXB₂ syntheses back to the levels observed with no calcium added to the medium. The effect of increasing the calcium concentration was greater in phosphate than in bicarbonate buffer. In a Tris buffer the effect of altered calcium was almost completely abrogated. These studies demonstrate that the choice of buffer and alterations in extracellular calcium concentration differentially alter basal arachidonic acid metabolism to prostaglandins and thromboxane in isolated toad urinary bladder cells. The results suggest that there may exist several endogenous pools of arachidonic acid which are differentially influenced by calcium. Furthermore, the pool sensitive to vasopressin has an absolute requirement for calcium.     

Glycosaminoglycan — lipoprotein interactions: 1. Effect of uronic acid composition and charge density of the glycan

Interactions between glycosaminoglycans and lipoproteins have been studied by affinity chromatography of various modified glycans on agarose substituted with low density lipoprotein (LDL). Elution was performed with increasing concentrations of NaCl. The electrostatic attraction between ligand and polyanion generally increased with increasing sulphate content. However, at equal charge density l-iduronic acid-containing glycans displayed higher affinity than D-glucuronic acid-containing ones. Within a population of heparin-related glycosaminoglycans, material containing 1.23 sulphate groups per hexosamine had higher affinity for LDL than did commercial heparin (2.40 sulphate/hexosamine). Decasaccharides or higher oligosaccharides from heparin-related glycans retained affinity only when they contained sulphate groups, while all fragments smaller than decasaccharide did not bind to LDL. Oligosaccharides that contained both sulphated and non-sulphated l-iduronic acid exhibited higher affinity than did fragments (of corresponding size) that contained only sulphated l-iduronic acid. Heparin-related glycans with the highest LDL-affinity contained 55% d-glucuronic acid. 11% non-sulphated l-iduronic acid and 34% l-iduronic acid-O-sulphate of total uronic acid.     

Design and synthesis of a reagent for solid-phase incorporation of the phosphothreonine mimetic (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid (Pmab) into peptides in a bio-reversible phosphonyl-bis-pivaloyloxymethyl (POM) prodrug form

Reported herein are the synthesis and solid-phase peptide incorporation of N-Fmoc-(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid bis-pivaloyloxymethyl phosphoryl ester [Fmoc-Pmab(POM)₂-OH, 2] as a phosphatase-stable phosphothreonine (pThr) mimetic bearing orthogonal protection suitable for the synthesis of Pmab-containing peptides having bio-reversible protection of the phosphonic acid moiety. This represents the first report of a bio-reversibly protected pThr mimetic in a form suitable for facile solid-phase peptide synthesis.     

An improved synthesis of thyrotropin releasing hormone (TRH) and crystallization of the tartrate

An improved synthesis of thyrotropin releasing hormone (TRH), pGlu-His-Pro-NH₂, is reported. Z-pGlu-ONB (N-hydroxy-5-norbornene-2,3-dicarboximide ester) was reacted with H-His-OH to yield a crystalline Z-pGlu-His-OH which was coupled with H-Pro-NH₂ by the $\text{HONB}\text{DCC}$ method to give Z-pGlu-His-Pro-NH₂ as a fine crystal. Hydrogenation of this protected tripeptide yielded pure TRH nearly quantitatively. The optical purity of TRH thus obtained was confirmed by the method L- and D- amino acid oxidase digestion. The crystallization of TRH was achieved as a tartrate, and the properties of the crystalline TRH-tartrate are described.     

Solid-phase synthesis of larger peptides by a new strategy of detachment from the resin

Attachment of the side-chain carboxyl of the protected aspartic or glutamic acid ester to the resin support has been established for the solid-phase synthesis of the asparagine or glutamine peptide. After further elongation of the α-amino deprotected resin-bound peptide ester with protected peptide fragments and the final detachment from the resin support by ammonolysis, the larger peptides containing, or preferably C-terminated with, asparagine or glutamine could be obtained. Thus, the C-peptide of human proinsulin was prepared by coupling to the resin-bound dipeptide derivative, Leu-Glu(OCH₂Ph®)·OtBu, with six fragments consecutively. It was obtained in an overall yield of 36% after detaching from the resin with alcoholic ammonia, followed by mild acidolysis, DEAE cellulose chromatography, and gel filtration. This procedure has now been applied to the synthesis of the C-terminal fragment of the insulin A chain ending in asparagine, and also to the synthesis of the threonine or serine peptide, where the anchorage to the resin was designed by the reaction of the sidechain hydroxyl with succinic anhydride in the presence of 4-dimethylaminopyridine to form the hemiester of succinic acid, which in turn was condensed to the aminomethyl resin by the DCC-HOBt procedure. Model experiments on the synthesis of the Z-Thr(CO-CH₂CH₂CONHCH₂Ph®)·OtBu and Bpoc-Lys(Boc)-Thr(COCH₂CH₂CONHCH₂Ph®)·OtBu, as well as their detachment from the resin by amminolysis or hydrazinolysis, have shown the potential for a milder process in the solid-phase synthesis of larger peptides.     

An efficient method for the synthesis of phenacyl ester‐protected dipeptides using neutral alumina‐supported sodium carbonate ‘Na2CO3/n‐Al2O3’

In the synthesis of dipeptides (Boc‐AA¹‐AA²‐OPac: AA¹ and AA² represent amino acids) protected by phenacyl (Pac) ester, amines and solid bases as the base for the conversion of the trifluoroacetic acid (TFA) salt of the amino component (TFA·H‐AA²‐OPac) into the corresponding free amino component (H‐AA²‐OPac) were examined. The synthesis of a dipeptide (Boc‐Ala‐Gly‐OPac) using amines for the conversion afforded an unsatisfactory yield with by‐products. On the other hand, the use of neutral alumina‐supported Na₂CO₃ (Na₂CO₃/n‐Al₂O₃) as a solid base for the conversion provided the dipeptide in a quantitative yield without by‐products. The application of Na₂CO₃/n‐Al₂O₃ to the synthesis of some dipeptides protected by Pac ester gave the desired peptides in excellent yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Glycosaminoglycan—lipoprotein interactions: 2. Structure of heparin-releted variants with high affinity for low density lipoprotein

A particular heparan sulphate fraction which possessed the largest proportion of high affinity variants for human low density lipoprotein contained almost equal proportions of the repeating units l-iduronosyl(O-sulphate)N-sulphamidoglucosamine and d-glucoronosyl-N-acetylglucosamine. The heparan sulphate was fractionated on lipoprotein-agarose into three populations. Results of periodate oxidation—alkaline elimination indicated that the size of the completely N-sulphated block regions increased with increasing affinity. In contrast, the number of consecutive l-iduronosyl(O-sulphate)-containing repeats decreased with increasing affinity towards lipoprotein. After selective periodate oxidation—alkaline scission of d-glucoronic acid residues only a portion of the heparan sulphate fragments retained high affinity for lipoprotein. This portion consisted of fragments larger than dodecasaccharide which contained both l-iduronic acid-O-sulphate and non-sulphated uronic acid residues (−) 2:1). No affinity or little affinity was displayed by fragments (of comparable size) that contained only sulphated l-iduronic acid residues.     

Synthesis of Cytidylyl(3′→5′)cytidylyl(3′→5′)-(2′)3′-O-[Lα-alanyl]adenosine

Abstract

A new synthesis of protected C-C-A-[L^α-Ala] 14 is reported using a new set of complementary groups such as 2-phenylsulfonylethoxycarbonyl (PSEC) for the protection of exocyclic amino functions, o-chlorophenyl (o-CIPh) for the internucleotide phosphotriester, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDMP) and the 4-monomethoxytrityl (MMTr) for the protection of the ã-amino fuction of the amino acid. 14 could be deprotected in two steps by treatments with 1,1,3,3-tetramethylguanidinium oximate under a dry condition and then by neat trifluoroacetic acid. Treatment with neat trifluoroacetic acid produced a stable salt: [C-C-A-Ala-N^ãH₃+ CF₃CO₂-] and did not promote any internucleotide phosphate migration or degradation of the oligomeric molecule. This salt was considerably more stable than C-C-A-Ala conjugate with a free ã-amino group, and, therefore, it could be easily purified on a silica gel column and was isolated in 82 % yield. This strategy should be useful for the synthesis of longer oligonucleotide-aminoacyl conjugate.     

First total synthesis and antileishmanial activity of (Z)-16-methyl-11-heptadecenoic acid, a new marine fatty acid from the sponge Dragmaxia undata

The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC₅₀ = 165.5 ± 23.4 μM) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC₅₀ = 62.3 ± 0.7 μM.     

Polymer-supported biopolymer synthesis: 3. Preparation of fully and partially protected βh-endorphin segments with a new phenolic poly [N-(2-methoxyethyl)-acrylamide]-based support

A new phenolic peptide resin, which is a bead-form derivative of crosslinked poly[N-(2-methoxyethyl)acrylamide], has been prepared by a suspension polymerization technique. The resin, which is highly expanded in all common peptide solvents, has been used for the solid (gel) phase synthesis of protected acylpeptide hydrazides. Fully protected acylpeptide hydrazide segments corresponding to β_h-endorphin (15–17), β_h-endorphin (6–14) and β_h-endorphin (6–17) have been prepared. Conversion, by selective catalytic transfer hydrogenation, of the fully protected acylpeptide hydrazide segment corresponding to β-_h-endorphin (15–17) to the partially protected form has been explored.     

Progress Towards a Submonomer Synthesis of Peptide Nucleic Acid

Abstract

We report recent developments in the optimization of a submonomer synthesis of peptide nucleic acid based on the Fukuyama-Mitsunobu reaction. The key steps in the submonomer synthesis are the installation of an appropriately protected 2-aminoethyl group on the α-nitrogen of an amino acid and its subsequent acylation with a protected nucleobase derivative. The aggressive alkylation conditions require a scheme of maximal protection for the nucleobases and that is proposed herein for the pyrimidines.     

Efficient one‐pot synthesis of N‐ethyl amino acids

Mono‐N‐ethylated α‐amino acid esters are obtained in high yields using reductive amination procedures. Formation of imine is achieved by excess of acetaldehyde, followed by removal of acetaldehyde and reduction by NaBH(OAc)₃. The elaborated one‐pot synthesis allows for the efficient synthesis of side‐chain protected amino acid derivatives. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Development of specific inhibitors for heparin-binding proteins based on the cobra cardiotoxin structure: an effective synthetic strategy for rationally modified heparin-like disaccharides and a trisaccharide

Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identified by NMR spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six differentially protected monosaccharide building blocks (three l-iduronic acids and three d-glucosamines) were prepared. These include a l-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-alpha-l-idopyranosyluronate trichloroacetimidate for which a single-crystal X-ray structure was determined to have M(r)=576.79, a=9.3098(11)A alpha=90 degrees , b=10.3967(12)A beta=90 degrees , c=28.026(3)A gamma=90 degrees , V=2712.7(6)A(3), P2(1)2(1)2(1), Z=4, mu=0.71073A, and R=0.0378 for 7586 observed reflections. It shows that the molecular structure of the donor is in the (1)C(4) conformation with significant 1,3-diaxial interactions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the temporary protecting group to protect the hydroxyl for elongation.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Pepsin-catalyzed peptide synthesis

The use of pepsin as a catalyst for the synthesis of peptide bonds was investigated. It is shown that the enzyme enables the preparation of several protected dipeptides and tripeptides containing two adjacent aromatic residues of the type P-Al-Phe-Y, P-PHe-Ar-Y, or P-AR-Phe-Y where P and Y are amino and carboxyl protecting groups, AL is an aliphatic amino acid residue, and Ar is an aromatic, amino acid residue. They yields are in the rang 25–97%. The high yields, combined with the enzyme's stereospecificity, permit the isolation of optically pure enantiomers from racemic mixtures. For example, when Z-DL -Ph-OH is allowed to react with an excess of H-L -Phe-NH₂, the stereoisomer Z-L -Phe-L -Phe-NH₂ is obtained in practically quantitative yield. At the same time, the unreacted, optically pure Z-D -Phe-OH can be recovered (Z = carbobenzyloxy, Phe = phenylalanine). The advantages and disadvantages of the enzymatic coupling procedure as a possible routine method for peptide synthesis are discussed.     

Inhibition of chlorophyll biosynthesis by lead in greeningPisum sativum leaf segments

Supply of 0.01 to 1.0 mM lead acetate to greening pea(Pisum sativum L.) leaf segments either in the absence or in the presence of inorganic nitrogen lowered total chlorophyll (Chl) content. During a time course study, there was not any appreciable effect of Pb²⁺ upto 4 h but thereafter Pb inhibited Chl synthesis. While supply of succinate, cysteine dithiothreitol, 5,5-dithio-bis-2-nitrobenzoic acid and NH₄C1 had no protective action against Pb²⁺ toxicity, and glycine, glutamate 2-oxoglutarate, MgCl₂, KH₂PO₄, CaCl₂, KC1 protected only partially, reduced glutathione (GSH) could completely overcome the inhibition of Chl biosynthesis by the metal. It is suggested that Pb²⁺ interferes with Chl biosynthesis through GSH availability     

Differential alteration of lipoxygenase and cyclooxygenase metabolism by rat peritoneal macrophages induced by endotoxin tolerance

Altered macrophage arachidonic acid metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e. endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT)C₄/D₄ and prostaglandin (PG)E₂ production by tolerant cells was greater than that by non-tolerant controls (p<0.001). However, A23187-stimulated i-6-keto-PGF_1α levels were lower in tolerant macrophages compared to controls. Stimulation of prostaglandin and thromboxane (Tx)B₂ synthesis by endotoxin or glucam was significantly less in tolerant macrophages compaared to controls (p<0.05). iLTC₄/D₄ production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in the non-tolerant cells. Synthesis ofb iLTB₄ by control macrophages was stimulated by endotoxin (p<0.01). These results demonstrate that arachidonic acid metabolism via the lipoxygenase and cyclooxygenase pathways in macrophages is differentially altered by endotoxin tolerance.