Lactosamine Derivatives Research Articles

The Synthesis and Preclinical Investigation of Lactosamine-Based Radiopharmaceuticals for the Detection of Galectin-3-Expressing Melanoma Cells.

Given that galectin-3 (Gal-3) is a β-galactoside-binding lectin promoting tumor growth and metastatis, it could be a valuable target for the treatment of Gal-3-expressing neoplasms. An aromatic group introduced to the C-3' position of lactosamine increased its affinity for Gal-3. Herein, we aimed at developing a radiopharmaceutical for the detection of Gal-3 positive malignancies. To enhance tumor specificity, a heterodimeric radiotracer capable of binding to both Gal-3 and αvβ3 integrin was also synthetized. Arginine-glycine-asparagine (RGD) peptide is the ligand of angiogenesis- and metastasis-associated αvβ3 integrin. Following the synthesis of the chelator-conjugated (2-naphthyl)methylated lactosamine, the obtained compound was applied as a precursor for radiolabeling and was conjugated to the RGD peptide by click reaction as well. Both synthetized precursors were radiolabeled with 68Ga, resulting in high labeling yield (>97). The biological studies were carried out using B16F10 melanoma tumor-bearing C57BL6 mice. High tumor accumulation of both labeled lactosamine derivatives-detected by in vivo PET and ex vivo biodistribution studies-indicated their potential for melanoma detection. However, the heterodimer radiotracer showed high hepatic uptake, while low liver accumulation characterized chelator-conjugated lactosamine, resulting in PET images with excellent contrast. Therefore, this novel carbohydrate-based radiotracer is suitable for the highly selective determination of Gal-3-expressing melanoma cells.

Synthesis of 68Ga-Labeled cNGR-Based Glycopeptides and In Vivo Evaluation by PET Imaging.

Tumor hypoxia induces angiogenesis, which is required for tumor cell survival. The aminopeptidase N receptor (APN/CD13) is an excellent marker of angiogenesis since it is overexpressed in angiogenic blood vessels and in tumor cells. Asparagine-glycine-arginine (NGR) peptide analogs bind selectively to the APN/CD13 recepto, therefore, they are important vector molecules in the development of a PET radiotracer which is capable of detecting APN-rich tumors. To investigate the effect of glycosylation and pegylation on in-vivo efficacy of an NGR-based radiotracer, two 68Ga-labeled radioglycopeptides were synthesized. A lactosamine derivative was applied to glycosylation of the NGR derivative and PEG4 moiety was used for pegylation. The receptor targeting potential and biodistribution of the radiopeptides were evaluated with in vivo PET imaging studies and ex vivo tissue distribution studies using B16-F10 melanoma tumor-bearing mice. According to these studies, all synthesized radiopeptides were capable of detecting APN expression in B16-F10 melanoma tumor. In addition, lower hepatic uptake, higher tumor-to background (T/M) ratio and prolonged circulation time were observed for the novel [68Ga]-10 radiotracer due to pegylation and glycosylation, resulting in more contrasting PET imaging. These in vivo PET imaging results correlated well with the ex vivo tissue distribution data.

Synthesis of Novel, Dual-Targeting 68Ga-NODAGA-LacN-E[c(RGDfK)]2 Glycopeptide as a PET Imaging Agent for Cancer Diagnosis.

Radiolabeled peptides possessing an Arg-Gly-Asp (RGD) motif are widely used radiopharmaceuticals for PET imaging of tumor angiogenesis due to their high affinity and selectivity to αvβ3 integrin. This receptor is overexpressed in tumor and tumor endothelial cells in the case of numerous cancer cell lines, therefore, it is an excellent biomarker for cancer diagnosis. The galectin-3 protein is also highly expressed in tumor cells and N-acetyllactosamine is a well-established ligand of this receptor. We have developed a synthetic method to prepare a lactosamine-containing radiotracer, namely 68Ga-NODAGA-LacN-E[c(RGDfK)]2, for cancer diagnosis. First, a lactosamine derivative with azido-propyl aglycone was synthetized. Then, NODAGA-NHS was attached to the amino group of this lactosamine derivative. The obtained compound was conjugated to an E[c(RGDfK)]2 peptide with a strain-promoted click reaction. We have accomplished the radiolabeling of the synthetized NODAGA-LacN-E[c(RGDfK)]2 precursor with a positron-emitting 68Ga isotope (radiochemical yield of >95%). The purification of the labeled compound with solid-phase extraction resulted in a radiochemical purity of >99%. Subsequently, the octanol–water partition coefficient (log P) of the labeled complex was determined to be −2.58. In addition, the in vitro stability of 68Ga-NODAGA-LacN-E[c(RGDfK)]2 was investigated and it was found that it was stable under the examined conditions.

Novel disaccharide inhibitors for the bacterial galactosyltransferase LgtC: Design, synthesis via Heyns rearrangement, and biochemical evaluation

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. We report a novel inhibitor design for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. Our design is based on the installation of an electrophilic warhead on the LgtC acceptor substrate and targeted at a non-catalytic cysteine residue in the LgtC active site. We have successfully synthesised two prototype inhibitors in four steps from lactulose. The key step in our synthesis is a Heyns rearrangement, during which we observed the formation of a hitherto unknown side product. While both lactosamine derivatives behaved as moderate inhibitors of LgtC, they also retained residual substrate activity. These results suggest that in contrast to our original design, these inhibitors do not act via a covalent mode of action, but are most likely non-covalent inhibitors.

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin‐3 Inhibitor

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3'-mono- and 2,3'-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2'-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7.

Biophysical and structural characterization of mono/di-arylated lactosamine derivatives interaction with human galectin-3

Combination of biophysical and structural techniques allowed characterizing and uncovering the mechanisms underlying increased binding affinity of lactosamine derivatives for galectin 3. In particular, complementing information gathered from X-ray crystallography, native mass spectrometry and isothermal microcalorimetry showed favorable enthalpic contribution of cation-π interaction between lactosamine aryl substitutions and arginine residues from the carbohydrate recognition domain, which resulted in two log increase in compound binding affinity. This incrementing strategy allowed individual contribution of galectin inhibitor moieties to be dissected. Altogether, our results suggest that core and substituents of these saccharide-based inhibitors can be optimized separately, providing valuable tools to study the role of galectins in diseases.

Studies on the synthesis of Lewis-y oligosaccharides

Lewis-y histo-blood group oligosaccharides are tumour-associated antigens prevalent in several different types of cancer, and they may also be secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. The key step in the synthesis of these sterically congested oligosaccharides involves difucosylation of partially protected lactosamine derivatives. Existing methods require either prolonged reaction times or elaborate glycosyl donors to ensure high stereoselectivity. Herein we report an optimised procedure for using a simple thioglycoside donor that leads to the desired products in high yield and excellent stereoselectivity. It is found that initial glycosylation of the 3′-hydroxy group of lactosamine derivatives in dichloromethane solution can inhibit subsequent glycosylation at the 2-position; however, reaction in toluene solution leads to Lewis-y oligosaccharides in high yield.

ChemInform Abstract: Tumor-Associated Antigen Synthesis. Synthesis of the Gal-α-(1 → 3)-Gal-β-(1 → 4)-GlcNAc Epitope. A Specific Determinant for Metastatic Progression?

A versatile synthesis of the Gal-α-(13)-Gal-β-(14)-GlcNAc epitope coupled to an alkyl spacer molecule could be developed. The important building block 3′-allyl-D-lactal 6 was prepared in high yield by the action of dibutyltin oxide and allyl bromide on D-lactal and converted in several steps into the lactosamine derivative 13 with free 3′-OH group. Stereoselective glycosidation with the β-trichloroacetimidate of benzylated D-galactose 18 led to the trisaccharide in high yield. Activation of the protected Gal-α-(13)-Gal-β-(14)-GlcNAc derivative 21 by desilylation and trichloroacetimidate formation followed by glycosidation with ethyl 9-hydroxynonanoate furnished the spacer-bound trisaccharide hapten 24 in excellent yield after simple two-step deprotection. The trisaccharide hapten 24 can easily be used as immunogen after coupling to an immunogenic carrier.

ChemInform Abstract: Synthesis of Lacto-N-neohexaose and Lacto-N-neooctaose Using the Dimethylmaleoyl Moiety as an Amino Protective Group.

The N-DMM-Protected lactosamine derivative 2 was readily transformed into the corresponding glycosyl donor 4 and into acceptor 5. A TMSOTf-catalyzed glycosidation afforded the derived tetrasaccharide 6 which led to glycosyl donor 9. Reaction of 9 with lactose derivative 10 as acceptor gave the desired hexasaccharide 11. Cleavage of all protective groups and N-acetylation afforded the target molecule 1b (lacto-N-neohexaose). Glycosylation of acceptor 10 with donor 4 furnished tetrasaccharide 16 which, employing standard procedures, gave acceptor 18. Glycosylation of 18 with donor 9 furnished, under standard conditions, octasaccharide 19. Cleavage of all protective groups and N-acetylation afforded the target molecule 1c (lacto-N-neooctaose). Both 1b and 1c were obtained in good overall yields.

Fluorous tag method for the simultaneous synthesis of different kinds of glycolipids

A mixture of saccharide primers with partially fluorinated tails, 2-(perfluorooctyl)ethyl 4′- O -(β- d -galactopyranosyl)-β- d -glucopyranoside (Lac H2F8) and 6-(perfluorohexyl)hexyl 2′-acetamido-2′-deoxy-β- d -glucopyranoside (GN H6H6), were introduced to animal cells. The oligosaccharide of Lac H2F8 was elongated by cellular enzymes and gave a GM3-type oligosaccharide. On the other hand, GN H6F6 was galactosylated to afford a lactosamine derivative that was further sialylated. This research confirmed that simultaneous glycosylation processes took place for Lac H2F8 and GN H6F6 primers and that the presence of one did not prevent the glycosylation of the other from proceeding. Each primer was recognized independently and elongated sequentially by cellular enzymes. Significantly, the synthesis of glycolipids from a mixture of these artificial scaffolds did not prevent the synthesis of glycolipids from the natural precursor. The glycosyl transferases recognized both precursors resulting to simultaneous synthesis of glycolipids.

Synthesis of mono-, di- and tri-O-sulfated N-acetyllactosamines in a form suitable for glycochip printing

A convenient and highly efficient technique for the selective sulfation of lactosamine derivatives has been suggested.

Biological Applications of a Chimeric Probe for the Assessment of Galectin-3 Ligands

Beta1-6 branching of N-linked oligosaccharides has been correlated with the progression of different cancers. The leukoagglutinins of Phaseolus vulgaris (L-PHA) have been used to study this pattern of glycosylation whose biological significance is incompletely understood. The animal lectin, galectin-3, also binds to structures recognized by L-PHA. To develop a functional tool for the in situ identification of this pattern of glycosylation, human galectin-3 was fused to bacterial alkaline phosphatase (gal3/AP). Gal3/AP recognized both A and B blood group saccharides (B>A) and lactosamine derivatives. Gal3/AP recognition depended at least in part on the N-linked oligosaccharides of different glycoproteins. The presence and distribution of galectin-3 ligands were analyzed in both murine and human normal and tumor samples. Loss of apical expression of galectin-3 ligands was commonly found in carcinomas. Endothelial and inflammatory cells were enriched in galectin-3 ligands as compared with tumor cells; thus, gal3/AP is a suitable tool for studying tumor microenvironments. Comparative analysis of both gal3/AP and L-PHA binding patterns indicated that although similar, these patterns are not identical. The probe developed was useful for several immunoenzymatic assays and will allow the physiological and clinical significance of the expression pattern of galectin-3 ligands to be established. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Practical Synthesis of Sulfated Analogs of Lactosamine and Sialylated Lactosamine Derivatives#

A series of β‐D‐Gal‐(1 → 4)‐β‐D‐GlcNAc‐octyl, NeuAcα‐(2 → 3)‐β‐D‐Gal‐(1 → 4)‐β‐D‐GlcNAc‐octyl, and their 6‐O‐sulfated and 6′‐O‐sulfated analogs (1–6) were synthesized in a concise manner starting from readily accessible monosaccharide intermediates. The syntheses involved formation of an orthogonally protected disaccharide and a trisaccharide from which all six compounds were derived. #C.D.R.I. Communication no. 6456.

Synthesis of Lacto-N-neohexaose and Lacto-N-neooctaose Using the Dimethylmaleoyl Moiety as an Amino Protective Group

The N-DMM-Protected lactosamine derivative 2 was readily transformed into the corresponding glycosyl donor 4 and into acceptor 5. A TMSOTf-catalyzed glycosidation afforded the derived tetrasaccharide 6 which led to glycosyl donor 9. Reaction of 9 with lactose derivative 10 as acceptor gave the desired hexasaccharide 11. Cleavage of all protective groups and N-acetylation afforded the target molecule 1b (lacto-N-neohexaose). Glycosylation of acceptor 10 with donor 4 furnished tetrasaccharide 16 which, employing standard procedures, gave acceptor 18. Glycosylation of 18 with donor 9 furnished, under standard conditions, octasaccharide 19. Cleavage of all protective groups and N-acetylation afforded the target molecule 1c (lacto-N-neooctaose). Both 1b and 1c were obtained in good overall yields.

Synthesis of N-acetylglucosamine containing Lewis A and Lewis X building blocks based on N-tetrachlorophthaloyl protection—synthesis of Lewis X pentasaccharide

Phenyl 6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-1-thio-β-d-glucopyranoside (5a) and thexyldimethylsilyl 6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-β-d-glucopyranoside (5b) gave with O-(2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl)trichloroacetimidate (8) in the presence of BF3·Et2O as catalyst exclusively lactosamine derivatives 7a and 7b, respectively, in high yields. Ensuing reaction with O-(3, 4-di-O-acetyl-2-O-benzyl-α-l-fucopyranosyl)trichloroacetimidate (9) in the presence of TMSOTf as catalyst afforded LeX trisaccharide intermediates 10a,b. With fucosyl donor 9 and 5a,b as acceptors in the presence of TMSOTf as catalyst glycosylation either at the 3-O or the 4-O was observed, thus leading to mixtures of disaccharides 11a/12a and 11b/12b, respectively; their reaction with 8 furnished LeX trisaccharide intermediates 10a,b and Lea trisaccharide intermediates 14a,b. Transformation of 10b into the corresponding trichloroacetimidate 17 and reaction with lactose acceptor 19 in the presence of Zn(OTf)2 as catalyst gave protected LeX pentasaccahride intermediate 21, which on deprotection led to unprotected LeX pentasaccharide 1.

ChemInform Abstract: Syntheses of Modified Carbohydrates with Glycosidases: Stereo‐ and Regiospecific Syntheses of Lactosamine Derivatives and Related Compounds.

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Syntheses of Modified Carbohydrates with Glycosidases: Stereo- and Regiospecific Syntheses of Lactosamine Derivatives and Related Compounds1

Different lactosamine derivatives, modified in the 2-N- and anomeric positions and suitable as intermediates for synthesis of Lewis-x and related compounds, were prepared with high specificity on a multigram scale directly from lactose, employing different D-glucosamine derivatives as acceptors and the abundant β-D-galactosidase from Bullera singularis as catalyst. Thus, methyl O-β-D-galactopyranosyl-(1→4)-2-azido-2-deoxy-β-D-glucopyranoside, ethyl O-β-D-galactopyranosyl-(1→4)-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside and ethyl O-β-D-galactopyranosyl-(1→4)-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-thio-β-D-glucopyranoside were formed in 20-40 % yield as calculated based on added acceptor. The 2-phthalimido derivative was isolated in crystalline form without chromatography (extraction/crystallization procedure). The trisaccharide derivative ethyl O-β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside was also isolated. The corresponding cellobiosamine derivatives were similarly obtained from cellobiose using the same type of catalyst and acceptors. The β-D-galactosidase from bovine testes was found to catalyze the highly specific formation of the β(1→6)-linked derivative ethyl O-β-D-galactopyranosyl-(1→6)-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside from lactose and ethyl 2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside. Ethyl O-α-L-fucopyranosyl-(1→4)-2-amino-6-O-benzyl-2-deoxy-1-thio-β-D-glucopyranoside, suitable as an intermediate for preparation of Lewis-a and related compounds, was obtained from p-nitrophenyl α-L-fucopyranoside and 2-amino-6-O-benzyl-2-deoxy-1-thio-β-D-glucopyranoside, employing α-L-fucosidase from bovine testes as catalyst. 1. Presented at the XVIIIth International Carbohydrate Symposium, Milan, Italy, July 21-26, 1996.

Synthesis of 3-O-sialyl and 6-O-sulfo derivatives of dimeric N-acetyl lactosamine as specific acceptors for α-l-fucosyltransferases

The stereoselective syntheses of 3-O-sialyl and 6-O-sulfo dimeric lactosamine derivatives 1–4 are accomplished through the use of key glycosyl donors 13 and 15.

A Practical Method for the Synthesis of N‐Acetyl‐D‐lactosamine Derivatives by the Tandem Use of Galactose Oxidase and β‐Galactosidase

A Practical Method for the Synthesis of <i>N</i>‐Acetyl‐<scp>D</scp>‐lactosamine Derivatives by the Tandem Use of Galactose Oxidase and β‐Galactosidase

Synthesis of lactosamine derivatives using β- d-galactosidase from Bacillus circulans

Various lactosamine derivatives that are versatile building blocks for oligosaccharide synthesis were obtained with excellent regioselectivity and moderate to high yields using β- d-galactosidase from Bacillus circulans as the biocatalyst.