Anti-Lea monoclonal antibody SPM 522 recognizes an extended Lea epitope.

Insights into the differential binding characteristics of anti-Lea and anti-LeaLex monoclonal antibodies (mAbs) provide information to develop LeaLex-based cancer immunotherapeutics while avoiding anti-Lea autoimmune reactions. We characterized the epitope recognized by anti-Lea mAb SPM 522. We synthesized the Lea 6-aminohexyl glycoside and report experimental evidence of a minor conformation in solution. The Lea and three other 6-aminohexyl glycosides were conjugated to BSA and titration experiments with SPM 522 show that: 1. SPM 522 binds to LeaLex better than to Lea; 2. the non-reducing Lea galactosyl residue is essential to binding. Competitive ELISA experiments using a panel of tri- to pentasaccharide fragments of LeaLex as well as Lea analogues indicate that: 1. the Lea ß-d-galactosyl α hydrophobic patch is crucial to binding; 2. the Lea fucosyl residue contributes to binding; 3. the Lexd-galactosyl residue also contributes to binding. These results indicate that anti-Lea mAb SPM 522 recognizes the Lea[1,3]-ß-d-Gal tetrasaccharide. We propose that a major recognition element is the extended hydrophobic surface defined by the Lea-ß-d-Gal residue extending to the α faces of the ß-d-GlcNAc and ß-d-Gal residues.

Synthesis of Tumor-Associated Le(a)Le(x) Hexasaccharides: Instability of a Thiol-Containing Oligosaccharide in Mass Spectrometry and Hypermetalation Detected by ESI FAIMS.

We report the efficient synthesis of three analogues of the tumor-associated carbohydrate antigen Le(a)Le(x). This hexasaccharide was prepared as a soluble inhibitor hexyl glycoside, as a 6-aminohexyl glycoside for conjugation to proteins, and as a 6-thiohexyl glycoside for immobilization to a gold surface. These three analogues were obtained from a common hexasaccharide intermediate and isolated pure following efficient deprotection reactions that involved metal-dissolving conditions. While all other intermediates and analogues gave the expected molecular ions in ESI HRMS, the 6-thiohexyl glycoside final compound gave a complex spectrum in which no signal matched the molecular ion. Using ESI FAIMS HRMS, we were able to prevent ion dissociation reactions and obtained high quality spectral data. The ions detected could be characterized unambiguously from their accurate masses and gave insight into the behavior of the thiohexyl analogue in the gas phase. These results indicate that the 6-thiohexyl glycoside lost water and led to the formation of "hypermetalated" species which we propose are cyclic.

Aggregation of a Tetrasaccharide Acceptor Observed by NMR: Synthesis of Pentasaccharide Fragments of the Le(a)Le(x) Tumor-Associated Hexasaccharide Antigen.

We report the synthesis of a tetrasaccharide and two pentasaccharide fragments of the Le(a)Le(x) tumor-associated carbohydrate antigen α-L-Fuc-(1→4)-[ß-D-Gal-(1→3)]-ß-D-GlcNAc-(1→3)-ß-D-Gal-(1→4)-[α-L-Fuc-(1→3)]-ß-D-GlcNAc-(1→OR). The choice of protecting groups permitted a one-step global deprotection (Na/NH3(l)). The protected chlorohexyl glycoside pentasaccharide was the precursor to the hexyl glycoside, to be used as a soluble inhibitor, and the aminohexyl glycoside analogue, to be conjugated to proteins for surface immobilization and immunization experiments. We observed that a linear tetrasaccharide that contained two N-acetylglucosamine residues and a free OH group gave two distinct sets of (1)H NMR signals when the data were acquired in deuterated chloroform. Data acquisition at variable concentrations and variable temperatures suggests that the second set of NMR signals results from aggregation of the tetrasaccharide driven by the formation of intermolecular H-bonds involving the NHAc. While the formation of intra- and intermolecular H-bonds involving N-acetylgucosamine residues has been reported in non-H-bonding solvents, this is, to our knowledge, the first time that these have lead to the appearance of two distinct sets of signals in the NMR spectra. This aggregation may explain the lack of reactivity observed when an attempt is made to glycosylate such an acceptor using non-H-bonding solvents such as dichloromethane.

Challenging deprotection steps during the synthesis of tetra- and pentasaccharide fragments of the Le(a)Le(x) tumor-associated hexasaccharide antigen.

We report the convergent synthesis of two novel tetrasaccharide and two novel pentasaccharide fragments of the Le(a)Le(x) TACA: the tetrasaccharides contain neither the galactose at the Le(a) nonreducing end nor the fucose at the Le(x) reducing end; the pentasaccharides only lack the galactose residue at the Le(a) nonreducing end. Two of the analogues were prepared as hexyl glycosides to be used in NMR experiments and as soluble inhibitors in binding studies and two as 6-aminohexyl glycosides to be conjugated to carrier proteins. Our strategy relied on stepwise extensions using excess monosaccharide glycosyl donors (trichloroacetimidates and thioglycosides) in sequential glycosylation reactions. The protecting groups were chosen to limit the number of deprotection steps required to obtain the final derivatives. While this strategy ensured that all glycosylation reactions proceeded in very good yields (70-84%), deprotection of the oligosaccharide intermediates was challenging. Global deprotection using Birch metal dissolving conditions did not remove the tert-butyldiphenylsilyl group, which indeed was incompatible with such reaction conditions. Attempts to remove the TBDPS with tetrabutylammonium fluoride was unsuccessful and led to a complex mixture of compounds that could not be separated. The desired hexyl and aminohexyl tetrasaccharides were finally obtained after four- and five-step deprotection sequences, respectively. Deprotection of the pentasaccharide intermediate to give the hexyl and aminohexyl analogues also led to unexpected results. Indeed, during Zemplén deacylation, a chloroacetamide chlorine atom was displaced by methoxide ions leading to the corresponding methoxyacetamide. Once the chloroacetamide was fully reduced to an acetamide the pentasaccharides were obtained in four and five steps, respectively.

Matched and mismatched acceptor/donor pairs in the glycosylation of a trisaccharide diol free at O-3 of two N-acylated glucosamine residues.

Reciprocal donor acceptor selectivity (RDAS) and double diastereodifferentiation are two concepts that have been used to explain how a glycosyl acceptor and donor may be 'matched' or 'mismatched' in glycosylation reactions. We describe here how the α-L-fucosylation and ß-D-galactosylation of a trisaccharide displaying two equatorial OH groups at C-3 of N-acylated glucosamine units led to different regioselectivities or different 'matched' pairs. These results may be explained under the reciprocal donor acceptor selectivity or double diastereodifferentiation concepts. We also suggest a third explanation to these results: the presence of a benzylidene at the nonreducing end acceptor led to rigidity at this residue which resulted into a mismatched pair during ß-D-galactosylation but allowed α-L-fucosylation.

Synthesis of 6-thio pseudo glycolipids and their orientation on a gold slide studied by IRRAS.

We have synthesized four 6-thio pseudo glycolipid analogues and assessed how two of them self-assembled on a gold surface. These structures were designed as candidate tethers molecules to anchor bilayer lipid membranes on gold. 6-Deoxy-6-thiogalactose was chosen to anchor the macromolecule to the gold and define an aqueous zone at the gold surface. A long alkane chain (C-12 or C-18) linked to the anomeric position of the sugar residue was chosen to anchor a bilayer lipid membrane. The linkage between the carbohydrate and the hydrophobic chains is either a glycosidic bond or a 1,4-disubstituted triazole formed by copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) of the propargyl glycoside with azido-dodecane and azido-octadecane. We are expecting that the hydrocarbon chains will orient themselves perpendicular to the gold surface and be incorporated into the first leaflet of the bilayer membrane. We have studied self assembled monolayers of the C-12 aglycone analogues on gold using infrared reflection absorption spectroscopy (IRRAS). We compared the results given by the IRRAS experiments to the IR spectra recorded by attenuated total reflection (ATR) spectroscopy on films of the randomly oriented analogues. Our results demonstrate that the C-12 analogues did bind to gold and did orient themselves perpendicular to the gold slide.