Synthesis of polyamide oligomers based on 14-amino-3,6,9, 12-tetraoxatetradecanoic acid

A series of oligomers of polyamides based on 14-amino-3,6,9, 12-tetraoxatetradecanoic acid monomers (ATTAn) was synthesized. These materials were designed as monodisperse analogues of poly(ethylene glycol) for use in biomedical applications where reproducible behavior is important. Synthesis of the monomer was evaluated using two routes. For small-scale preparations, tetraethylene glycol (TEG) was monoprotected with dihydropyran, converted to an alkoxide, and alkylated with ethyl bromoacetate. On larger scales, TEG was alkylated directly by treatment with sodium, followed by ethyl bromoacetate. The amine function was introduced by mesylation followed by treatment with sodium azide. Reduction of the azide to amino groups was performed over Pd/C using either hydrogen or formic acid as proton sources. Assembly of the oligomers was accomplished using standard DCC/NHS chemistry and an iterative dimerization sequence after appropriate deprotection of a pair of monomers. The amino group was protected by retaining the azido group as a latent amine. A series of ATTAn oligomers was prepared (n = 1-8). A lipid conjugate of the octamer, ATTA8-DSPE, was synthesized.     

N,N-Diacetylsialyl chloride—a novel readily accessible sialyl donor in reactions with neutral and charged nucleophiles in the absence of a promoter

N,N-Diacetylneuraminic acid glycosyl chloride was prepared for the first time and made to react with various nucleophiles to give the corresponding α-glycosyl phosphate, β-glycosyl dibenzyl phosphate, α-glycosyl azide, α-phenyl thioglycoside and α-glycosyl xanthate in 65-82% yields and high stereoselectivity while its reactions with simple alcohols were not stereoselective. The new sialyl donor made possible the first stereoselective synthesis of sialic acid glycosyl phosphate with α-configuration and highly efficient synthesis of β-configured sialic acid glycosyl dibenzyl phosphate.     

Synthesis of a GM3 ganglioside analogue carrying a phytoceramide moiety by intramolecular glycosylation as a key step

A novel analogue of ganglioside GM3, in which sphingosine was replaced with a phytosphingosine moiety, was synthesized by intramolecular glycosylation as a key step. Glucose, a reducing terminal of the saccharide, and phytoceramide were first tethered by succinic acid and the derivative used for the subsequent glycosidic bond formation. The obtained glycosyl phytoceramide was further glycosylated with the sialyl galactose residue to afford a fully protected GM3 derivative, which was converted into the desired, final compound by using conventional deprotection procedures.     

Synthesis of the Neoglycoconjugates of Phenolic Glycolipid-related Trisaccharides for the Serodiagnosis of Leprosy

The trisaccharide segment of the phenolic glycolipid I of Mycobacterium leprae was synthesized effectively in the form of β-(2-methoxycarbonylethyl)phenyl glycoside by the condensation of β-(2-methoxycarbonylethyl)phenyl 4–0-benzyl-3–0-methyl-α-l-rhamnopyranoside and 2,3-di-O-methyl-4–0-(2,4-di-0-acetyl-3,6-di-0-methyl-/?-d-glucopyranosyl)-α-l-rhamnopyrano-syl chloride in the presence of silver tritiate and 1,1,3,3-tetra-iV-methylurea, and subsequent selective deprotection. This was then coupled to BSA by the acyl azide method, giving NT-P-BSA. The NT-P-BSA showed very high reactivity and specificity to leprosy sera. The trisaccharide-BSA conjugate with a ^-linked rhamnosylrhamnose unit (/TT-P-BSA) was also synthesized.     

Identification of anesthetic binding sites on human serum albumin using a novel etomidate photolabel

We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on alpha1beta2gamma2s GABA(A) receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (beta2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds approximately 75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA(A) receptor or other putative targets.     

Synthesis of photoreactive neoglycolipid probes--instruments for studying membrane lectins

A method for the synthesis of photoaffinity neoglycolipid probes with a highly efficient carbene-generating diazocyclopentadien-2-ylcarbonyl (Dcp) label, which can be radioiodinated under standard oxidation conditions, was developed. The probes are intended for incorporation into the lipid bilayer. They are lipophilic glycoconjugates on the basis of an amphiphilic aglycone built up from a diacylglycerol and a polyethylene glycol spacer (with a polymerization degree of 9-16) bearing the Dcp label at the terminal unit. The location of the label in the aglycone provides the possibility of one-step preparation of a wide range of probes using various carbohydrate synthons. We have synthesized photoaffinity neoglycoconjugates containing the oligosaccharides: sialyl LewisX tetrasaccharide and A trisaccharide, which is specific to some tumor cells. A probe containing an inactive pentaol (aminodeoxyglucitol) was also synthesized to detect nonspecific binding. The Dcp label is bound to the probe molecule by ester bond; its lability under alkaline conditions facilitates the analysis of cross-linked products after photoaffinity labeling. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Synthesis and mutagenicity of the two stereoisomers of an azide metabolite (azidoalanine)

The L- and D-isomers of azidoalanine (azide metabolite) have been chemically synthesized with 60% yield using corresponding N-(tert-butoxycarbonyl)-serine as starting materials. The mutagenic properties of synthesized L-azidoalanine are very similar to those of azide and in vivo synthesized azidoalanine. Synthetic D-azidoalanine shows very low mutagenic activity on Salmonella typhimurium TA1530 strain compared to that of the L-isomer. Thus a stereoselective process is involved in azidoalanine mutagenicity. The data presented in this study suggest that further biochemical activation is required for L-azidoalanine to produce its mutagenic activity.     

Nucleic Acid Related Compounds. LXXXI. Efficient General Synthesis of Purine (Amino,Azido, and Triflate)-Sugar Nucleosides

Abstract

Treatment of 3′,5′-O-(tetraisopropyldisiloxanyl)adenosine and its arabino epimer with trifluoromethanesulfonyl chloride/DMAP gave the 2′-triflates in high yields. Displacements (LiN₃/DMF) and deprotection gave 2′-azido-2′-deoxyadenosine and its arabino epimer which were reduced with Bu₃SnH/AIBN/DMAC/benzene (or Staudinger reduction) to give 2′-amino-2′-deoxyadenosine and its epimer. Oxidation of 2′,5′-bis-O-(tert-butyldimethylsilyl)adenosine, stereoselective reduction, triflation, azide displacement, deprotection, and reduction gave 3′-amino-3′-deoxyadenosine.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Synthesis of deacetylthymosin beta 11 and its effect on the impaired T-lymphocytes of a uremic patient with common variable immunodeficiency

The untetracontapeptide corresponding to the entire amino acid sequence of deacetylthymosin beta 11 was synthesized by assembling six peptide fragments via the azide followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole in trifluoroacetic acid in the presence of dimethylselenide. The synthetic peptide was tested for its effect on the impaired blastogenic response of phytohemagglutinin-stimulated T-lymphocytes of a uremic patient with common variable immunodeficiency. The synthetic peptide had some restoring activity on the impaired blastogenic response of T-lymphocytes in the one patient tested.     

Metabolic activation of the mutagen azide in biological systems

Inorganic azide (N3-) mutagenicity is mediated through a metabolically synthesized organic azide, L-azidoalanine (N3-CH2-CH(-NH2)-COOH). L-Azidoalanine appears to be formed by the action of O-acetylserine (thiol)-Lyase (EC 4.2.99.8) using O-acetylserine and azide as substrates. In both plants and bacteria tested, azide substitutes for the natural substrate sulfide (S2-) in this reaction. Azide (L-azidoalanine) mutagenesis is highly attenuated by a deficiency in the excision of UV-like DNA damage (uvr-). Thus a premutation lesion recognizable by the bacterial excision-repair enzymes must be formed. Mutagenesis appears to proceed from this by 'direct mispairing' pathway. Azide (L-azidoalanine) mutagenicity is highly specific and involves a stereoselective process, but the molecular nature of the specificity has not been determined.     

Force-controlled equilibria of specific vesicle-substrate adhesion

We have developed "vertical" magnetic tweezers capable of exerting controlled pico and subpico Newton forces. Using this apparatus, we apply a point-like force to a vesicle that is adhered by means of specific bonds between the vesicle-carrying oligosaccharide sialyl LewisX and the surface-grafted E-selectin. An exponential decrease of the bound vesicle area with the decay rate that is insensitive to the force and the composition of the system is observed. We measure an equilibrium under force that is associated with an increased binding in the center of the contact zone. We also show that the determination of the shape is potentially sufficient to determine the number of formed specific bonds.     

Parallel solid-phase synthesis of a model library of 7alpha-alkylamide estradiol derivatives as potential estrogen receptor antagonists.

The C17-THP derivative of 7alpha-(11-azidoundecanyl)-estradiol (4) was synthesized and coupled to an aminomethyl resin via a photolabile o-nitrobenzyl linker. Reduction of the azide by the Staudinger reaction to its corresponding amine followed by acylation using four activated NFmoc protected amino acids gave a first level of diversity. Subsequent deprotection of the Fmoc followed by a second acylation with five activated carboxylic acids produced, after photocleavage, a model library of twenty antiestrogen-related 7alpha-alkylamide estradiol derivatives in acceptable overall yields and very good purities.     

Synthesis of aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides and their invasion to a 19F labeled HIV-1 TAR model

The potential of aminoglycosides to induce RNA-invasion has been demonstrated. For this purpose, aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides have been synthesized entirely on a solid phase. The synthesis includes an automated oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a (19)F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by (19)F NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 2'-O-methyl oligoribonucleotide (5'-CAGGCUCA-3'), has been obtained by the neomycin conjugate. The increased affinity results from a cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first molar equivalent of the conjugate induces hybridization of the second.     

Ceramide-1-phosphate sugars: new types of glycophospholipids

Ceramide-1-phosphate sugars were synthesized by direct glycosyl phosphite/phosphate andO-glycosyl trichloroacetimidate/phosphate exchange reactions, respectively. Thus, ceramide-1-O-phosphoric acid 5 gave with sialyl phosphite1 as sialyl donor directly -linked sialyl phosphate6; deprotection afforded the corresponding glycophospholipid ceramide-1-phosphateN-acetylneuraminate7. Similarly, fromO-glucosyl- andO-galactosyltrichloroacetimidate10 and13 with phosphoric acid derivative5 glycosyl ceramide-1-phosphate sugars12 and15, respectively, were obtained.     

Recent trends in the synthesis of O-glycosides of 2-amino-2-deoxysugars

The discovery of new methods for stereoselective glycoside synthesis and convergent oligosaccharide assembly has been critical for the area of glycosciences. At the heart of this account is the discussion of the approaches for stereoselective synthesis of glycosides of 2-amino-2-deoxysugars that have emerged during the past two decades. The introductory part provides general background information and describes the key features and challenges for the synthesis of this class of compounds. Subsequently, major approaches to the synthesis of 2-amino-2-deoxyglycosides are categorized and discussed. Each subsection elaborates on the introduction (or protection) of the amino functionality, synthesis of glycosyl donors by introduction of a suitable leaving group, and glycosidation. Wherever applicable, the deprotection of a temporary amino group substituent and the conversion onto the natural acetamido functionality is described. The conclusions part evaluates the current standing in the field and provides a perspective for future developments.     

Synthesis of deuterium labeled NMDA receptor inhibitor - 20-Oxo-5β-[9,12,12-(2)H(3)]pregnan-3α-yl-L-glutamyl 1-ester

20-Oxo-5β-[9,12,12-(2)H(3)]pregnan-3α-yl-l-glutamyl 1-ester 11 was synthesized as an internal standard for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5β-pregnan-3α-yl-l-glutamyl 1-ester 18 and its metabolites, in plasma and tissue. 11α-Hydroxy-progesterone (1) was reduced under basic conditions to yield the corresponding 5β-steroid. Protection of the 3- and 20-oxo groups and oxidation of the 11α-hydroxy group was then followed by a deuterium exchange, conducted under basic conditions using deuterated methanol. Next, the carbonyl moiety at C-11 was reduced and the 11α-hydroxyl group removed through utilization of the Barton-McCombie reaction. Subsequent deprotection of the 3- and 20-acetals and stereoselective reduction of the 3-oxo group gave the desired trideuterated pregnanolone (8). This was coupled with protected glutamic acid, which was then deprotected to yield [9,12,12-(2)H(3)]-pregnanolone glutamate (11) with >99% isotopic purity.     

Fluorescent conjugate of sLex-selective bisboronic acid for imaging application

Carbohydrate-based biomarkers such as sialyl Lewis X are known to correlate with cancer formation and progression. By targeting sialyl Lewis X, we have developed a boronolectin-fluorophore conjugate, which was able to selectively label and image xenograft (sc) tumor. This represents the very first example that a small molecule capable of recognizing a carbohydrate biomarker was used for optical imaging application.     

Synthesis of poly(L-Tyr-L-Glu-L-Ala-Gly) and poly(L-Glu-L-Lys-L-Ala) and their circular dichroism spectra in aqueous solution

Two sequential polypeptides, poly(O-benzyl-L -Tyr-γ-benzyl-L -Glu-L -Ala-Gly) and poly(ε-benzyloxycarbonyl-L -Lys-L -Glu-L -Ala), were synthesized, the former by the pentachlorophenyl ester of the tetrapeptide monomer and the latter by the azide of the tripeptide monomer. After deprotection and dialysis, poly(L -Tyr-L -Glu-L -Ala-Gly) was obtained in 71% yield and had a molecular weight of 53,000. The circular dichroism spectra (CD) of the polymer at pH's 7.2, 10.5, and 11.8 and of oligomers and of the monomer at pH 7.2 indicated that the polymer exists in an α-helical conformation. After deprotection, poly(L -Lys-L -Glu-L -Ala) was obtained in 37% yield and had a molecular weight of 3000. The CD spectra of the polymer at pH 7.2 and 2.8, and of the monomer at pH 7.2, indicated that the polymer is in a randomly coiled configuration.     

Purification and characterization of a lymph node sulfotransferase responsible for 6-O-sulfation of the galactose residues in 2'-fucosyllactose and other sialyl LewisX-related sugars

A microsomal galactose-6-O-sulfotransferase (Gal-6-O-Stase) from porcine lymph nodes, able to transfer the sulfate group from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) onto 2'-fucosyllactose (2'-FL) and other sialyl LewisX (sLex)-related sugars, has been purified and characterized. The enzyme was purified to about 35,000-fold by a combination of conventional and affinity chromatographic steps. The purified enzyme preparation exhibited two protein bands at around 80-90 and 170 kDa on 7.5% SDS-PAGE under reducing conditions. Both of these protein bands always comigrated in the gel when peak fractions containing Gal-6-O-Stase activity from the 3',5'-ADP-agarose column were subjected to 6% SDS-PAGE under reducing conditions. These protein bands also showed similar binding patterns to WGA (wheat germ agglutinin), Con A (concanvalin A), and EBA (elderberry agglutinin). Similarly, when the enzyme preparation after the hydroxylapatite step was photolabeled with 8-azido-[32P]-PAPS, both 80-90 and 170 kDa protein bands were labeled in a specific manner. These results suggest a possible association of these two protein bands with the enzyme activity. The carbohydrate substrate specificity of this enzyme suggests that it is well suited to catalyze the sulphonation at the C-6 position of the galactose residues of oligosaccharides that are structurally similar to sLex. Furthermore, a survey of several porcine organs revealed that this enzyme was selectively expressed in lymphoid tissues such as lymph nodes (peripheral and mesenteric) and spleen. These findings suggest that this enzyme may be involved in the assembly of 3'-sialyl-6'-sulfo Lewisx, the major capping group of HEV-ligands for L-selectin.