Effect of fluorine substitution on the agonist specificity of norepinephrine.

Substitution of fluorine for hydrogen in position 2, 5, or 6 of the aromatic ring of norepinephrine markedly alters the alpha- and beta-adrenergic agonist properties of norephinephrine. The 6-fluoro isomer is an beta-adrenergic agonist with virtually no beta agonist activity, while the 2-fluoro isomer is a beta-adrenergic agonist with little alpha activity. The 5-fluoro isomer is equipotent with norepinephrine as an alpha agonist and significantly more potent as a beta agonist. The possible physiochemical basis for these differences is discussed.

A fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy.

Aberrant glycosylation of mucins leads to the exposure of cryptic carbohydrate antigens at the surface of carcinoma cells, which, therefore, represent potent targets for anticancer therapeutic vaccines. To date, the development of immunogens to stimulate immune response to such saccharidic antigens is based on carbohydrate conjugation to carrier proteins. However, these traditional protein conjugates are poorly defined in chemical composition and structure. As an alternative, we synthesized a multiple antigenic O-linked glycopeptide (MAG) carrying the carbohydrate Tn antigen associated with a CD4+ T-cell epitope (MAG:Tn-PV). This fully synthetic immunogen is highly defined in composition and carries a high saccharidic epitope ratio over the entire molecule. The MAG:Tn-PV was able to induce anti-Tn IgG antibodies that recognize human tumor cell lines. A therapeutic immunization protocol performed with this fully synthetic immunogen increased the survival of tumor-bearing mice. Thus, the accurately defined and versatile MAG system represents an efficient strategy to induce carbohydrate-specific antitumor immune responses but may also be applicable to the prevention of infectious diseases, if it is based on bacterial oligosaccharides.

High density O-glycosylation of the MUC2 tandem repeat unit by N-acetylgalactosaminyltransferase-3 in colonic adenocarcinoma extracts.

A synthetic peptide corresponding to the human MUC2 tandem repeat unit was glycosylated in vitro using UDP-GalNAc and extracts of colonic adenocarcinoma and paired normal mucosa, followed by fractionation of the products by reverse phase high-performance liquid chromatography. Several peaks of glycopeptides with different numbers of GalNAc residues attached were detected. It is notable that the adenocarcinoma extract was capable of glycosylating peptides to a much greater extent than was normal mucosa. The levels of mRNA for N-acetylgalactosaminyltransferases-1, -2, and -3 were determined by reverse transcription-PCR. Only N-acetylgalactosaminyltransferase-3 mRNA was expressed at a higher level in the adenocarcinoma than in the normal tissue. When the MUC2 tandem repeat peptide was glycosylated with a mixture of the normal mucosa extract and recombinant N-acetylgalactosaminyltransferase-3, larger amounts of glycopeptides with higher contents of GalNAc residues were produced. The MUC2 tandem repeat peptides glycosylated extensively by recombinant N-acetylgalactosaminyltransferase-1, -2, or -3 were prepared and characterized. Substitution at each Thr residue, as revealed by Edman degradation sequencing, in conjunction with evidence obtained on mass spectrometry indicated a heterogeneous pattern of site-specific glycosylation within the MUC2 tandem repeat. It was found that maximum numbers of 6, 8, and 11 GalNAc residues were incorporated by N-acetylgalactosaminyltransferases-1, -2, and -3, respectively, and that only N-acetylgalactosaminyltransferase-3 could completely glycosylate both consecutive sequences composed of three and five Thr residues in the MUC2 tandem repeat unit. These results suggest that O-glycosylation of the clustered Thr residues is a selective process controlled by N-acetylgalactosaminyltransferase-3 in the synthesis of clustered carbohydrate antigens.

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy.

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

Influence of fluorine substitution on the site of enzymatic O-methylation of fluorinated norepinephrines.

The extent of meta- and para-O-methylation by catechol O-methyltransferase of 2-fluoro-, 5-fluoro-, and 6-fluoronorepinephrine (FNE) at pH 7 and 9 was determined. The rank order of preference for para-O-methylation is 5FNE much greater than NE greater than 6FNE greater than 2FNE. In all cases, increasing the pH to 9 results in an increase in para-O-methylation. Results with 2F- and 5FNE demonstrate the importance of ionization in the methyltransferase reaction when fluorine is situated ortho to one of the phenolic groups. To establish unequivocally the identities of the products, the isomeric, monofluorinated vanillins and isovanillins were synthesized and directly related to the products formed enzymatically from the monofluorinated norepinephrines.

Syntheses and adrenergic agonist properties of ring-fluorinated isoproterenols.

2-Fluoro-, 5-fluoro-, and 6-fluoroisoproterenol were synthesized by reduction of the Schiff base formed between the corresponding ring-fluorinated 3,4-bis(benzyloxy)phenethanolamine and acetone, followed by reductive debenzylation in the presence of oxalic acid to yield crystalline neutral oxalates. The apparent beta-adrenergic potencies were determined in the isolated guinea pig atria. 2-Fluoro- and 5-fluoroisoproterenol were equipotent with (+/-)-isoproterenol, while 6-fluoroisoprotenol was virtually inactive. No alpha-adrenergic agonist activity (guinea pig aorta) was shown by any of the fluoroisoproterenols. Displacement of alpha- and beta-specific radioligands from isolated membrane preparations from rat brain by the fluoroisoproterenols were in agreement with the responses of the organ preparations. Thus, the apparent fluorine-induced specificity is due to specificity at the receptor binding site. The effects of fluorine substitution are discussed with regard to the apparent negative influence of the 6-fluoro substituent on the beta-agonist properties of isoproterenol, the lack of any increase in potency due to the 2-fluoro substituent, and the possibility of fluorine-induced changes in the electron density of the aromatic ring as a possible rational for the fluorine-induced specificity of both the fluoroisoproterenols and the fluoronoirepinephrines.

Synthesis and biological properties of 2-, 5-, and 6-fluoronorepinephrines.

2-Fluoro-, 5-fluoro- and 6-fluorodimethoxybenzaldehydes were prepared by photochemical decomposition of the corresponding diazonium fluoroborates. The aldehydes were converted to the cyanohydrin trimethylsilyl ethers, which, in turn, were reduced to the dimethoxyphenethanolamines. Boron tribromide demethylation afforded the racemic ring-fluorinated norepinephrines. An alternate route, using the dibenzyloxyfluoroaldehyde, was also used to prepare 6-fluoronorepinephrine. The fluorine substituent markedly increases the phenolic acidities of these analogues. The biological properties conferred upon norepinephrine by the fluorine substituents in peripheral and central adrenergically responsive systems clearly demonstrate that 2-fluoronorepinephrine is a nearly a pure beta-adrenergic agonist, while 6-fluoronorepinephrine is an alpha-adrenergic agonist. 5-Fluoronorepinephrine retains both beta- and alpha-adrenergic agonist properties. Receptor-binding studies with specific radiolabeled ligands indicate that the specificity conferred by the site of fluorine substituents results from a change in the affinity of these analogues for the alpha- and beta-adrenergic receptors.

Enzymatic synthesis of some O-beta-D-digalactosyl glycopeptides, using beta-D-galactosidase.

Disaccharide-peptide conjugates were obtained in yields of 30-50% from o-nitrophenyl beta-D-galactopyranoside by employing beta-D-galactosidase from E. coli as catalyst. Two series of beta-D-galactosyldipeptides were examined as galactosyl acceptors. They both contain an L-serine residue beta-linked to the anomeric carbon of galactose. In the first series, serine is in the N-terminal position of the dipeptide; in the second series, serine is in the C-terminal position. The second amino acid is L-alanine or glycine. Some of our substrates gave a high yield of beta-(1-->3)-digalactosyldipeptide derivatives and all gave very little of the beta-(1-->6) regioisomer. The conditions and the limitations of the transgalactosylation reaction are discussed.

Synthesis of alpha- and beta-biotinylated T-antigen.

The T-antigen [beta-D-Gal-(1-->3)-D-Ga1NAc] has been linked to biotin through a C6 spacer arm for the detection of a specific 'T-antigen-lectin' complex at the surface and/or on the migration pathway of melanoma cells. When 4,6-di-O-acetyl-2-azido-2-deoxy-3-O-(2,3,4, 6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha- or -beta-D-galactopyranosyl halides were treated with N-benzyloxycarbonyl or N-fluorenylmethoxycarbonyl protected aminohexanols (used as the spacer arm), unusual stereoselectivities were observed for the synthesis of the alpha and beta anomers. The synthesis of the alpha anomer could only be achieved, in reasonable yields, with the Schiff base of aminohexanol.

Unusual lactam formation occurring in the synthesis of a biotinylated T-antigen-serine derivative.

Synthesis of the biotinylated T-antigens, linked to a serine by an alpha (7 alpha) or a beta (7 beta) 2-acetamido-2-deoxy-D-galactoside bond, is described. These derivatives were needed for the detection of a specific endogenous lectin at the surface and/or on the migration pathway of melanoma cells. In the course of the synthesis, an unusual lactam formation was observed with the beta anomer of the azido-disaccharide 5 beta.

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen.

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Synthesis of alpha and beta-glycopyranosyl-serine derivatives by enzymic transglycosylation.

The transglycosylation from raffinose and lactose to Aloc-Ser-OMe is catalyzed respectively by alpha and beta galactosidases. Transglycosylation from cellobiose has been achieved with beta-glucosidase. The simplicity of the enzymatic synthesis, the stereospecificity of the condensations in one-pot reactions and the ease of purification give the method value for large scale preparation of beta-linked derivatives. The protective groups of the serine residue can be cleaved under mild conditions: the ester group has been removed quantitatively by papain catalyzed hydrolysis and the Aloc group by a Pd (0) hydrostannolytic cleavage.

Esterification of 9-fluorenylmethoxycarbonyl-glycosylated serine and cysteine derivatives with an hydroxymethyl resin.

Esterification of glycosylated serine and cysteine derivatives with a 4-alkoxybenzyl alcohol (Wang) resin is described. The classical methods of ester bond formation (symmetrical anhydride, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate [TBTU]/4-dimethylaminopyridine [DMAP] with or without 1-hydroxybenzotriazole [HOBT], pentafluorophenyl [Pfp] esters gave high percentages of racemization of the glycosylated serine or cysteine residues. To reduce the D-amino acid content, we found that the best results were obtained with the highly efficient MSNT reagent (2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide), which gave a high yield of substitution of the resin and the lowest percentage of racemization. A difference in behavior was observed between the two amino acids. The glycosylated cysteine derivative always gave lower racemization than the analogous glycosylated serine.

Cyclic AMP-generating systems: regional differences in activation by adrenergic receptors in rat brain.

Catecholamine, histamine, and adenosine-mediated accumulations of radioactive cyclic AMP were assessed in adenine-labeled slices from eight rat brain regions. 2-Fluoronorepinephrine, a selective beta-adrenergic agonist, elicited an an accumulation of cyclic AMP in cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, thalamus, hypothalamus, and medulla-pons. In cerebral cortex and most other brain regions, the beta-adrenergic-mediated response appeared to involve primarily beta 1-adrenergic receptors, while in cerebellum, there was a significant involvement of beta 2-adrenergic receptors. 6-Fluoronorepinephrine, a selective alpha-adrenergic agonist, elicited accumulations of cyclic AMP in all regions except cerebellum. Combinations of the two fluoro derivatives afforded in all brain regions an accumulation of cyclic AMP identical with that elicited by norepinephrine. In hypothalamus, the alpha- and beta-adrenergic responses were significantly greater than additive. In cerebral cortex, the alpha-adrenergic receptor-mediated response appeared to involve alpha 1-adrenergic receptors and to be nearly completely dependent on adenosine, while in other brain regions, the dependence of the alpha-adrenergic response on adenosine was less or absent. Combinations of 6-fluoronorepinephrine and histamine had greater than additive effects in cortex and hippocampus. The results indicate that the interactive control of cyclic AMP-generating systems by alpha-adrenergic, beta-adrenergic, adenosine, and histamine receptors differs significantly among rat brain regions.

Effects of ring fluorination on the cardiovascular actions of dopamine and norepinephrine in the dog.

The 2-, 5- and 6-fluoro (F) analogs of dopamine (DA) and (+/-)-norepinephrine (NE) were compared with the respective parent molecules for alpha, beta-1 and beta-2 adrenergic and vascular DA activities in pentobarbital-anesthetized dogs. 2-F and 5-F DA were equipotent while 6-F DA was about 4-fold less active than DA in causing renal vasodilation in phenoxybenzamine pretreated dogs (vascular DA activity). The three analogs were indistinguishable from DA for vasoconstrictor activity in the femoral vascular bed (alpha adrenergic activity). 2-F and 6-F DA were equipotent to DA while 5-F was about 2-fold more active than DA in inotropic activity (beta-1 adrenergic activity). In contrast, F NE analogs showed marked differential activities. 2-F NE resembled isoproterenol in increasing cardiac contractility, lowering diastolic blood pressure and causing vasodilation in the femoral vascular bed. The 5-F analog was the most potent for (beta-1 adrenergic activity and produced biphasic effects on blood pressure and the femoral vascular bed. 6-F NE exerted no inotropic activity in dose 50- to 80-fold higher than the threshold dose of l-NE and caused vasoconstriction. Thus, fluorine substitution in either of the three positions in the benzene ring of DA induced only minor, if any, differences in receptor activation when compared with DA. On the other hand, fluorine substitution in the benzene ring of NE yielded compounds with marked differential receptor activity. These data indicate that the differences between the effects of F substitution on DA and NE analogs must be related to the only structural difference between the two catecholamines, the presence of a beta-hydroxyl group in NE.

Preparation of a multiple antigen glycopeptide (MAG) carrying the Tn antigen. A possible approach to a synthetic carbohydrate vaccine.

The glycosidic tumor-associated Tn antigen was conjugated to a lysine backbone containing a helper T-cell epitope in order to activate immune responses specific for some types of carcinomas. As opposed to traditional protein conjugates, this multiple antigen glycopeptide (MAG) offers the advantages of the lack of immunogenicity of the polylysine core and of accurate chemical definition. The MAG construction was assembled by conventional solid-phase peptide synthesis. The analysis of its antigenicity demonstrated that the Tn antigen on the MAG is recognized by Tn-specific monoclonal antibodies.

RGDS glycosylated peptides as inhibitors of cell-attachment and platelet aggregation.

Glycopeptides derived from the GRGDS sequence were synthesized to study the effect of the sugar residue on the activity of these peptides. The peptides were tested as inhibitors of cell adhesion to fibronectin and of platelet aggregation. The sugar moiety was found to reduce the biological activity of the parent compounds except for the cyclic derivatives P37 and P38 where the inhibition of platelet aggregation was increased. Some interesting differences were observed between the peptides bearing sugar residues with free hydroxyl groups and those with peracetylated sugars.

Anti-tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-Tn glycotope.

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O:-linked to a CD4(+) T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.