Contribution of the anomeric effect to the solution and crystal structure of [1S,2S,6S,7s]-1,6-diaza-4,9-dioxa-2,7-dimethoxycarbonylbicyclo[4.4.1]undecane, a condensation product of L-serine methyl ester with formaldehyde.

The reaction of L-serine methyl ester hydrochloride (1) with paraformaldehyde (2) in dichloromethane in the presence of triethylamine afforded a novel compound: [lS,2S,6S,7S]-1,6-diaza-4,9-dioxa-2,7-dimethoxycarbonylbicyclo[4.4.1]undecane (4) as a 2:3 adduct of 1 with 2. 1H and 13C NMR spectroscopy were unable to discriminate between two possible symmetrical structures. The latter was unambiguously proved by X-ray crystallography. The crystal structure established: (i) the existence of two identical seven-membered rings each containing a N-C-O grouping; (ii) the existence of a long C-O-C-N-C-N-C-O-C sequence in which each nitrogen belongs simultaneously to a N-C-O (oxazolidine) and to a N-C-N (aminal) motifs; (iii) the existence of a chair-like conformation for both seven-membered rings; (iv) the antiperiplanar geometry of pN-C-O and consequently the manifestation of a strong anomeric effect in both N-C-O groupings, whereas anomeric effect was virtually absent in the N-C-N sequence, as corroborated by bond distances and bond angles. Chemical shifts, coupling constants and NOE effects confirm that the conformational features of 4 are preserved in solution.

Anomeric effects in non-carbohydrate compounds: conformational differences between the oxazolidine rings of a cis-fused bicyclic system.

Tris(hydroxymethyl)aminomethane (Tris) can react with benzaldehyde (1:2 molar ratio) to produce cis-2,8-diphenyl-5-hydroxymethyl-1-aza-3,7-dioxabicyclo[3.3.0]octa ne, the structure of which has been confirmed by nuclear magnetic resonance spectroscopy and X-ray crystallography. The crystal structure showed that both oxazolidine rings A and B are puckered in opposite directions. Ring A exists in an E3 envelope form with 0-3 noticeably down (0.65 A) the plane of the remaining atoms, whereas ring B adopts the 7E envelope conformation with the 0-7 atom displaced up from the mean reference plane by 0.70 A. Comparison of bond angles and bond distances showed that both oxazolidine rings A and B exhibit cross endo-anomeric effects resulting from electron delocalization over the bond sequence O-3-C-2-N-1-C-8-O-7.

Subcellular distribution of a new fluorinated, biocompatible, non-ionic telomeric carrier: a study in cultured B16 melanoma and rat skin fibroblasts.

Tris-hydroxymethyl-amino-methane telomers bearing a fluorinated end have recently been proposed as potential drug carriers. Using ion microscopy, we have investigated the cell uptake and subcellular distribution of a perfluorinated telomere, called F-TAC, in two cell lines, malignant murine B16 melanoma and normal rat skin fibroblasts. Single layer cell cultures on gold plates were incubated with F-TAC at different concentrations. Ion microscopy using mass spectrometry enabled the detection of Fluorine 19 atoms entering into F-TAC constitution. This microanalytical study showed an elective cytoplasmic localization of the molecule, wherein the distribution is relatively homogeneous. Within same culture and incubation conditions, intercellular variations in F-TAC content were very low. In the malignant line, the intracellular concentration remains practically identical when increasing F-TAC concentration in the culture medium above 0.2 mg/ml, indicating that the uptake phenomenon is saturable. In conclusion, the F-TAC telomer easily crosses the plasma membrane, however, it has difficulties in crossing the nuclear membrane. It is likely that intracellular penetration is essentially due to rapid endocytosis of the telomer.

A new approach to phosphoserine, phosphothreonine and phosphotyrosine synthons and to thiophospho analogs. Stepwise synthesis of mono- and multiphosphorylated phosphopeptides related to src-protein kinase.

Several phosphoserine, phosphothreonine and phosphotyrosine synthons suitable for the stepwise synthesis of phosphopeptides were prepared. Treatment of methylthiomethyl (MTM) esters of either Z-, Boc-, Allocserine and threonine with phosphochloridate in pyridine followed by MgBr2 cleavage of MTM in diethyl ether afforded the title compounds in good yield. Thiophosphoserine and phosphotyrosine synthons were also obtained by the phosphoramidite method using di-(2,2,2-trichloroethyl)-N,N-diisopropylphosphoramidite and MCPBA as oxidizing reagent. Trichloroethyl proved valuable as phosphate protecting group especially in phosphotyrosine derivatives owing to its stability in acidic conditions. These synthons were involved in the liquid-phase synthesis of several phospho and/or thiophosphopeptides related to either src-protein kinase or rat liver pyruvate kinase.

Vesicles and other supramolecular systems from biocompatible synthetic glycolipids with hydrocarbon and/or fluorocarbon chains.

A series of double-tailed hydrocarbon and/or fluorocarbon glycolipids derived from galactose and glucose have been prepared. These compounds were obtained upon opening a lactono- and maltonolactone moiety by the amino group of either a glycine, glycylglycine or lysine residue. The carboxyl terminus of the glycyl and glycylglycine conjugates was further reacted with the appropriate double-tailed amine. In the case of lysine, the lactonamide conjugate was functionalized with a hydrocarbon and/or fluorocarbon fatty amine and acid, respectively. The ability of such glycolipids to disperse in water, the morphology of self-assemblies formed and the stability of the supramolecular structure obtained were shown to depend on the presence or absence and on the nature of the aminoacid spacer. Most of the compounds described were shown by conventional techniques (TEM, Cryo-TEM, LLS, etc.) to produce stable vesicular systems.

Double-tailed perfluoroalkylated glycolipids as components for drug delivery and targeting systems. Preliminary biocompatibility results.

Vesicles are being investigated as drug carriers, especially for enhancing the therapeutic effectiveness of the drug while minimizing its side effects. Drug targeting can be achieved if there is a specific recognition of the vesicle's outer wall by specific cells. With these objectives in mind new glycolipids fitted with fluorinated, hydrogenated or mixed, single and double-tails, containing either a gluco- or a galactopyranose residue in their hydrophilic head, were synthesized and their ability to achieve self-organized supramolecular systems was assessed. Replacement of hydrogen by fluorine in these glycolipids was found to enhance biological tolerance. Thus, a fluorinated single-tailed glycolipid displayed no action on red blood cells at concentrations as high as 50 g/l while its hydrogenated analog was hemolytic at 5 g/l. 100% of survival was obtained one month after intravenous or intraperitoneal injection into mice of isotonic dispersions of single and double-tailed glycolipids at a dose of 500 mg/kg. These glycolipids were innocuous on Namalva cell cultures at a concentration of 0.1 g/l.

Biocompatibility of alkyl and perfluoroalkyl telomeric surfactants derived from THAM.

A preliminary, comparative biological evaluation of two new families of non-ionic telomeric surfactants derived from Tris(hydroxymethyl)acrylaminomethane (THAM) is reported. These trisacryl conjugates, or TAC, were designed with the purpose of improving the stability and biocompatibility of fluorocarbon emulsions to be used in injectable oxygen-delivering systems. Their amphiphilic character arises from the simultaneous presence in the same molecule of several hydrophilic THAM residues and of a hydrophobic tail consisting in either a hydrocarbon (H-TAC family) or a fluorocarbon (F-TAC family) chain. The acute toxicity in mice after intravenous injection is low (LD50 in the 625 to 1250 mg/kg body weight range for the H-TAC compared to 630 to 4500 mg/kg body weight range for F-TAC) and increases with the length of the hydrophobic chain. No hemolytic activity was detected for the F-TAC at concentrations up to 200 g/l, while hemolysis is found for the H-TAC at a concentration of 5 g/l or less and increases with the alkyl chain length. The impact of the new surfactants on the growth and viability of Namalva cell cultures also increases with the length of the hydrophobic chain, with again a better tolerance for the F-TAC. Altogether the fluorinated amphiphiles display better tolerance than their hydrocarbon analogs in spite of their significantly increased surface activity. Both families of compounds appear to have potential as strongly hydrophilic surfactants for biomedical applications.

Telomeric THAM-derived perfluoroalkylated surfactants for fluorocarbon emulsions.

New fluorophilic and hydrophilic, cost efficient telomeric surfactants derived from tris(hydroxymethyl)acrylaminomethane were synthesized in 2 steps in 80% yield with respect to the perfluoroalkylated telogen. They demonstrate better ability to emulsify fluorocarbons than Pluronic f-68. The biological tolerance of these new surfactants is remarkable, the perfluorohexyl derivative was tolerated at doses of 4g/kg bw after i.v. injection in mice. None of the perfluoroalkylated THAM derivatives induces hemolysis of human red blood cells at concentrations up to 200g/l in physiological solutions.

Conformational analysis of the amino termini (5 residues) of human glycophorin AM and AN: differentiation of the structural features of the TN and T antigenic determinants in relation to their specificity.

The N-terminus of glycophorin A, the main transmembrane erythrocyte glycoprotein responsible for the MN blood-group specificity, has been modelled. As the minimum size of the protein recognised by the antiglycophorin A antibodies is the N-terminal glycopentapeptide, attention was focused on the TN and T antigenic determinants of this size in order to determine wether differences in 3D structure exist and how a specific response with different antibodies is induced.

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity.

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.

Solid-phase synthesis of peptides containing phosphoserine using phosphate tert.-butyl protecting group.

In this paper, we report the solid-phase synthesis of peptides containing O-phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino-acids linked to p-alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid-labile side-chain protecting groups (including phosphate residue) were used. Thus, both removal of side-chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.

One- and two-dimensional NMR studies of the N-terminal portion of glycophorin A at 11.7 Tesla.

One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy (at 11.7 Tesla) was used to gain some structural and spectral information about glycophorin AM, glycophorin AM tryptic glycopeptide, a related pentapeptide, and two related monoglycosylated pentapeptides. The protein spectral information suggests that the highly glycosylated N-terminus of glycophorin does not seem to possess a unique tertiary structure. Furthermore, the spectral information provided by the carbohydrate residues also indicates that there is no strong carbohydrate-protein interaction resulting in a unique tertiary structure. This result does not preclude any unique protein-carbohydrate interactions. For the small monoglycosylated pentapeptide containing alpha-D-GalNAc attached to Thr, a unique NOESY cross-peak was observed between the anomeric proton and the beta-proton of Thr. A cross-peak between the beta-proton of Ser and the anomeric proton was not observed for a related monoglycosylated pentapeptide containing alpha-D-GalNAc O-linked to Ser.

Malaria invasion of human erythrocytes. Synthesis of peptides relevant to glycophorin A and evaluation of their inhibitory properties.

The objective of this study was to determine whether a nonglycosylated portion of glycophorin A (GPA), the main erythrocyte membrane glycoprotein, was involved in the process of invasion of red blood cells (RBC) by merozoites of Plasmodium falciparum, a parasite responsible for the most severe form of malaria. A series of peptides covering the sequence 55-76 situated upstream from the intramembraneous hydrophobic region of GPA was synthesized by an active ester coupling strategy and assessed for invasion-blocking capacity by using an in vitro assay system. Tests showed peptide 65-69, Ala-His-His-Phe-Ser, to be a good inhibitor of the invasion of RBC. Results presented here provide a confirmation of the existence of parasite binding sites on the peptide domain of GPA. Furthermore, comparison of inhibitory activity with peptide composition allowed us to rule out any contribution of a toxic parameter related to hydrophobicity as reported earlier.

Blood group antigens: synthesis of Ss antigenic peptides related to human glycophorin B.

The human Ss blood group antigens are located on glycophorin B, a minor human erythrocyte membrane glycoprotein. The structural difference in Ss antigens is determined by a Met/Thr polymorphism at position 29. This report describes the first synthesis of the two peptides carrying the Ss specificities, SS: Asn-Gly-Glu-Met-Gly-Gln-Leu-Val and ss: Asn-Gly-Glu-Thr-Gly-Gln-Leu-Val.

Possible role of the carbohydrate residues on the structure of the N-terminus of glycophorin AM.

Natural-abundance 13C nuclear magnetic resonance (13C-n.m.r.) was used to study the effect of monoglycosylation on the structure and dynamics of a pentapeptide related to the N-terminus of glycophorin AM. The results of this study indicate that a single point of glycosylation, on the pentapeptide, can significantly affect its structure. Moreover, glycosylation of this pentapeptide also affects its dynamic motion in solution. This study further defines the role that the carbohydrate residue plays in determining the structure about the N-terminus of glycophorin AM.

13C n.m.r. study of the structure and the metal ion binding sites of neuropeptides composed of L-Asp and L-Glu.

13C NMR spectral data are presented for a variety of possible neuropeptides composed of L-Asp, Ac-L-Asp, and L-Glu which contain alpha and beta peptide linkages. The data for the various compounds are compared to the data presented for Ac-Asp-Glu, a known neuropeptide, in order to gain structural information about the related compounds. Indications are that for compounds 1 and 5, the cis peptide bond configuration exists due to the interaction of zwitterionic species. This interaction appears to be eliminated when the beta peptide bonds are formed, as in the case of compounds 3 and 7. Spin-lattice relaxation times were used to confirm the structures. Electron-nuclear relaxation rates are also used to elucidate the metal ion binding sites of these species.

Structural, dynamic, and metal-ion binding studies of the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr by 13C-N.m.r. spectroscopy.

13C-N.m.r. spectral data as well as spin-lattice relaxation times (T1 values) are presented for the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr. The binding of Gd3+ to these model compounds containing N-terminal blocking groups and esterified carboxyl groups indicates that the disaccharide contains a rather weak, but unique, binding-site in the vicinity of C-2 of alpha-D-GalNAc (possibly involving N-2', the acetamido carbonyl group, O-3' and/or possibly the glycosidic oxygen atom (O-3)).

Synthesis of the Mg antigenic determinant and peptide analogs related to human glycophorin A.

The Mg antigen is a well known rare mutation of the MN blood group system. The amino-terminal pentapeptide related to human glycophorin AMg, Leu-Ser-Thr-Asn-Glu, as well as pentapeptides representing the peptide backbone of glycophorin AM, AN and AMc and other analogs, were synthesized to serve both as glycosyl transferase acceptors and as artificial antigens. These compounds were obtained by a stepwise peptide coupling strategy in solution.