Localized Chemical Remodeling for Live Cell Imaging of Protein‐Specific Glycoform

Live cell imaging of protein‐specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole‐cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein‐specific glycoform information is reported. The proof‐of‐concept protocol developed for MUC1‐specific terminal galactose/N ‐acetylgalactosamine (Gal/GalNAc) combines affinity binding, off‐on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability.     

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor‐Associated Mucin Glycopeptides by the Human Macrophage Galactose‐Type Lectin

The human macrophage galactose‐type lectin (MGL) is a key physiological receptor for the carcinoma‐associated Tn antigen (GalNAc‐α‐1‐O‐Ser/Thr) in mucins. NMR and modeling‐based data on the molecular recognition features of synthetic Tn‐bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non‐glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca²⁺. NMR data were complemented with molecular dynamics simulations and Corcema‐ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn‐presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH–π contacts involving exclusively the NHAc moiety.     

Metabolic Glycoengineering with N‐Acyl Side Chain Modified Mannosamines

In metabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides. After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates. The feasibility of MGE was first discovered for sialylated glycans, by using N‐acyl‐modified mannosamines as precursor molecules for unnatural sialic acids. Prerequisite is the promiscuity of the enzymes of the Roseman–Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N‐acyl side chain of mannosamine analogues, for example, elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications). Unnatural sialic acids are incorporated into glycoconjugates of cells and organs. MGE has intriguing biological consequences for treated cells (aliphatic MGE) and offers the opportunity to visualize the topography and dynamics of sialylated glycans in vitro, ex vivo, and in vivo (bioorthogonal MGE).     

Increased Affinity of 2′‐O‐(2‐Methoxyethyl)‐Modified Oligonucleotides to RNA through Conjugation of Spermine at Cytidines

Structural modification at the 2′‐O‐position of riboses in oligonucleotide therapeutics is of critical importance for their use as drugs. To date, the methoxyethyl (MOE) substituent is the most important and features in dozens of antisense oligonucleotides that have been tested in clinical trials. Yet, the search for new improved modifications continues in a quest for increased oligonucleotide potency, improved transport in vivo and favorable metabolism. Recently, we described how the conjugation of spermine groups to pyrimidines in oligonucleotides vastly increases their affinity for complementary RNAs through accelerated binding kinetics. Here we describe how spermines can be linked to the exocyclic amino groups of cytidines in MOE‐oligonucleotides employing a straightforward ‘convertible nucleoside approach’ during solid phase synthesis. Singly‐ or doubly‐modified oligonucleotides show greatly enhanced affinity for complementary RNA, with potential for a new generation of MOE‐based oligonucleotide drugs.     

Terminal Alkenes as Versatile Chemical Reporter Groups for Metabolic Oligosaccharide Engineering

The Diels–Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5‐tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate‐linked side chains of varying length terminated by alkene groups and their suitability for labeling cell‐surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N‐butenyloxycarbonylmannosamine, was especially well suited for labeling cell‐surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent.     

Derivatization of sialic acids for stabilization in matrix‐assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2 → 3)‐ and α(2 → 6)‐isomers

Sialylated carbohydrates usually decompose by loss of sialic acid when ionized by matrix‐assisted laser desorption/ionization (MALDI) as the result of the labile carboxylic proton. Stabilization has previously been achieved by forming methyl esters with methyl iodide, a procedure that eliminates the labile proton. In this paper, we describe an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the MALDI spectrum. The sugars were desalted, dissolved in methanol, and treated with 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMT‐MM). After removal of the solvent, the products were transferred directly to the MALDI target and examined from 2,5‐dihydroxybenzoic acid. Small amounts of N‐glycans derived from biological sources benefited from an additional clean‐up stage involving Nafion 117. α(2 → 6)‐Linked sialic acid produced only methyl esters whereas α(2 → 3)‐linked sialic acids were converted into their lactones providing a 32 Da difference in mass. Negative ion collision‐induced decomposition (CID) mass spectra of these neutralized glycans provided information, in many cases, on the antenna of N‐linked glycans to which the variously linked sialic acids were attached. The method was applied to N‐linked glycans released from bovine fetuin and porcine thyroglobulin. Copyright © 2008 John Wiley & Sons, Ltd.     

Conformational Selection in Glycomimetics: Human Galectin‐1 Only Recognizes syn‐Ψ‐Type Conformations of β‐1,3‐Linked Lactose and Its C‐Glycosyl Derivative

The human lectin galectin‐1 (hGal‐1) translates sugar signals, that is, β‐galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound‐state conformations of Galβ‐C‐(1→3)‐Glcβ‐OMe ( 1 ) and its βGal‐(1→3)‐βGlc‐OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)‐NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event. Although free C‐glycoside access four distinct conformers in solution, hGal‐1 recognizes shape of a local minimum of compound 1 , the syn‐Φ/syn‐Ψ conformer, not the structure at global minimum. MD simulations were run to explain, in structural terms, the observed geometry of the complex.     

The α‐Glycosidation of Partially Unprotected N‐Acetyl and N‐Glycolyl Sialyl Donors in the Absence of a Nitrile Solvent Effect

The synthesis of α‐sialosides is one of the most difficult reactions in carbohydrate chemistry and is considered to be both a thermodynamically and kinetically disfavored process. The use of acetonitrile as a solvent is an effective solution for the α‐selective glycosidation of N‐acetyl sialic acids. In this report, we report on the α‐glycosidation of partially unprotected N‐acetyl and N‐glycolyl donors in the absence of a nitrile solvent effect. The 9‐O‐benzyl‐N‐acetylthiosialoside underwent glycosidation in CH₂Cl₂ with a good α‐selectivity. On the other hand, the 4,7,8‐O‐triacetyl‐9‐O‐benzyl‐N‐acetylthiosialoside was converted to β‐sialoside as a major product under the same reaction conditions. The results indicate that the O‐acetyl protection of the sialyl donor was a major factor in reducing the α‐selectivity of sialylation. After tuning of the protecting groups of the hydroxy groups at the 4,7,8 position on the sialyl donor, we found that the 9‐O‐benzyl‐4‐O‐chloroacetyl‐N‐acetylthiosialoside underwent sialylation with excellent α‐selectivity in CH₂Cl₂. To demonstrate the utility of the method, straightforward synthesis of α(2,9) disialosides containing N‐acetyl and/or N‐glycolyl groups was achieved by using the two N‐acetyl and N‐glycolyl sialyl donors.     

Efficient Solid Phase Synthesis of Cleavable Oligodeoxynucleotides Based on a Novel Strategy for the Synthesis of 5′‐S‐(4,4′‐Dimethoxytrityl)‐2′‐deoxy‐5′‐thionucleoside Phosphoramidites

The incorporation of a specific cleavage site into an oligodeoxynucleotide can be achieved by utilizing the four 5′‐S‐(4,4′‐dimethoxytrityl)‐2′‐deoxy‐5′‐thionucleoside 3′‐(2‐cyanoethyl diisopropylphosphoramidites) 5 and 15a – c (Fig. 1). Based on the silver ion assisted cleavage of P? S and C? S bonds, we synthesized oligodeoxynucleotides with an achiral 5′‐phosphorothioate linkage 3′–O–P–S–5′ by the solid‐phase phosphoramidite procedure. The efficient cleavage of these modified oligodeoxynucleotides can be detected by HPLC, PAGE, and surface plasmon resonance (SPR) spectrometry. The liberated 5′‐thiol moiety can be used directly for post‐reaction labeling with appropriately functionalized reporter groups.     

One‐Pot Synthesis of N‐Acetyl‐ and N‐Glycolylneuraminic Acid Capped Trisaccharides and Evaluation of Their Influenza A(H1 N1) Inhibition

Human lung epithelial cells natively offer terminal N‐acetylneuraminic acid (Neu5Ac) α(2→6)‐linked to galactose (Gal) as binding sites for influenza virus hemagglutinin. N‐Glycolylneuraminic acid (Neu5Gc) in place of Neu5Ac is known to affect hemagglutinin binding in other species. Not normally generated by humans, Neu5Gc may find its way to human cells from dietary sources. To compare their influence in influenza virus infection, six trisaccharides with Neu5Ac or Neu5Gc α(2→6) linked to Gal and with different reducing end sugar units were prepared using one‐pot assembly and divergent transformation. The sugar assembly made use of an N‐phthaloyl‐protected sialyl imidate for chemoselective activation and α‐stereoselective coupling with a thiogalactoside. Assessment of cytopathic effect showed that the Neu5Gc‐capped trisaccharides inhibited the viral infection better than their Neu5Ac counterparts.     

7‐Iodo‐5‐aza‐7‐deazaguanine ribonucleoside: crystal structure,physical properties,base‐pair stability and functionalization

The positional change of nitrogen‐7 of the RNA constituent guanosine to the bridgehead position‐5 leads to the base‐modified nucleoside 5‐aza‐7‐deazaguanosine. Contrary to guanosine, this molecule cannot form Hoogsteen base pairs and the Watson–Crick proton donor site N3—H becomes a proton‐acceptor site. This causes changes in nucleobase recognition in nucleic acids and has been used to construct stable `all‐purine' DNA and DNA with silver‐mediated base pairs. The present work reports the single‐crystal X‐ray structure of 7‐iodo‐5‐aza‐7‐deazaguanosine, C<sub>10</sub>H<sub>12</sub>IN<sub>5</sub>O<sub>5</sub> ( 1 ). The iodinated nucleoside shows an anti conformation at the glycosylic bond and an N conformation (O4′‐endo) for the ribose moiety, with an antiperiplanar orientation of the 5′‐hydroxy group. Crystal packing is controlled by interactions between nucleobase and sugar moieties. The 7‐iodo substituent forms a contact to oxygen‐2′ of the ribose moiety. Self‐pairing of the nucleobases does not take place. A Hirshfeld surface analysis of 1 highlights the contacts of the nucleobase and sugar moiety (O—H…O and N—H…O). The concept of pK‐value differences to evaluate base‐pair stability was applied to purine–purine base pairing and stable base pairs were predicted for the construction of `all‐purine' RNA. Furthermore, the 7‐iodo substituent of 1 was functionalized with benzofuran to detect motional constraints by fluorescence spectroscopy.     

An Enzymatic N‐Acylation Step Enables the Biocatalytic Synthesis of Unnatural Sialosides

Chitin is one of the most abundant and cheaply available biopolymers in Nature. Chitin has become a valuable starting material for many biotechnological products through manipulation of its N‐acetyl functionality, which can be cleaved under mild conditions using the enzyme family of de‐N‐acetylases. However, the chemoselective enzymatic re‐acylation of glucosamine derivatives, which can introduce new stable functionalities into chitin derivatives, is much less explored. Herein we describe an acylase (CmCDA from Cyclobacterium marinum) that catalyzes the N‐acylation of glycosamine with a range of carboxylic acids under physiological reaction conditions. This biocatalyst closes an important gap in allowing the conversion of chitin into complex glycosides, such as C5‐modified sialosides, through the use of highly selective enzyme cascades.     

Sialoside analogue arrays for rapid identification of high affinity siglec ligands

The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acalpha2-3Gal and Neu5Acalpha-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function.     

Effective Methods for the Synthesis of N‐Methyl β‐Amino Acids from All Twenty Common α‐Amino Acids Using 1,3‐Oxazolidin‐5‐ones and 1,3‐Oxazinan‐6‐ones

N‐Methyl β‐amino acids are generally required for application in the synthesis of potentially bioactive modified peptides and other oligomers. Previous work highlighted the reductive cleavage of 1,3‐oxazolidin‐5‐ones to synthesise N‐methyl α‐amino acids. Starting from α‐amino acids, two approaches were used to prepare the corresponding N‐methyl β‐amino acids. First, α‐amino acids were converted to N‐methyl α‐amino acids by the so‐called ‘1,3‐oxazolidin‐5‐one strategy’, and these were then homologated by the Arndt–Eistert procedure to afford N‐protected N‐methyl β‐amino acids derived from the 20 common α‐amino acids. These compounds were prepared in yields of 23–57% (relative to N‐methyl α‐amino acid). In a second approach, twelve N‐protected α‐amino acids could be directly homologated by the Arndt–Eistert procedure, and the resulting β‐amino acids were converted to the 1,3‐oxazinan‐6‐ones in 30–45% yield. Finally, reductive cleavage afforded the desired N‐methyl β‐amino acids in 41–63% yield. One sterically congested β‐amino acid, 3‐methyl‐3‐aminobutanoic acid, did give a high yield (95%) of the 1,3‐oxazinan‐6‐one ( 65 ), and subsequent reductive cleavage gave the corresponding AIBN‐derived N‐methyl β‐amino acid 61 in 71% yield (Scheme 2). Thus, our protocols allow the ready preparation of all N‐methyl β‐amino acids derived from the 20 proteinogenic α‐amino acids.     

A locked derivative of 8‐aza‐7‐deazaadenosine

The title compound [systematic name: (1S,3S,4R,7S)‐3‐(4‐amino‐1H‐pyrazolo[3,4‐d]pyrimidin‐1‐yl)‐1‐hydroxymethyl‐2,5‐dioxabicyclo[2.2.1]heptan‐7‐ol], C₁₁H₁₃N₅O₄, belongs to a family of nucleosides with modifications in both the sugar and nucleobase moieties: these modifications are known to increase the thermodynamic stability of DNA and RNA duplexes. There are two symmetry‐independent molecules in the asymmetric unit that differ significantly in conformation, and both exhibit a high‐anti conformation about the N‐glycosidic bond, with χ torsion angles of −85.4 (3) and −87.4 (3)°. The sugar C atom attached to the nucleobase N atom is −0.201 (4) and 0.209 (4) Å from the 8‐aza‐7‐deazaadenine skeleton plane in the two molecules. The molecules are assembled into layers via hydrogen bonds and π–π stacking interactions between the modified nucleobases.     

Chiral Space Formed by (+)‐(1S)‐1,1′‐Binaphthalene‐2,2′‐diyl Phosphate: Recognition of Aliphatic L‐α‐Amino Acids

(+)‐(1S)‐1,1′‐Binaphthalene‐2,2′‐diyl hydrogen phosphate (bnppa) is one of the useful optical selectors. To disclose the molecular mechanism by which bnppa recognizes aliphatic L ‐α‐amino acids and separates them by fractional crystallization, X‐ray analyses of bnppa and of its salts with L ‐alanine, L ‐valine, L ‐norvaline, and L ‐norleucine have been undertaken. All the amino acids adopt energetically favorable conformations in the crystal structures. The conformations and the packing patterns of bnppa in these crystal structures are very similar. The bnppa molecules are packed in a specific way to form hydrophobic and hydrophilic layers that are well separated. Between bnppa molecules, at the interface of these hydrophobic and hydrophilic layers, a space with chirality is formed. This space, designated as chiral space, recognizes the optically active amino acids. The packing of bnppa is mainly governed by intermolecular CH⋅⋅⋅π interactions between naphthalene moieties. The chiral space is responsible for the molecular recognition by bnppa allowing fractional crystallization of the L ‐α‐amino acids.     

Synthesis of 4‐(5‐aryl‐1,3,4‐oxadiazol‐2‐yl)phenyl‐hydrazine derivatives from 3‐(4‐hydrazinocarbonyl‐phenyl)sydnones

The synthesis of potential fluorescent active 4‐(5‐aryl‐1,3,4‐oxadiazol‐2‐yl)phenylhydrazine derivatives was accomplished in three steps. The key step was the dehydration cyclization of 1,2‐diacylhydrazines to form the 1,3,4‐oxadiazole ring by use of acetic anhydride/perchloric acid mixture as the dehydrating agent. The sydnone moiety served as the masked hydrazines, which could be demasked by HCl for further application. © 2007 Wiley Periodicals, Inc. Heteroatom Chem 18:438–442, 2007; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/hc.20318     

Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens

BACKGROUND: Sialic acid is a component of many tumor-associated oligosaccharide antigens. The repertoire of sialic acids presented by cells can be expanded to include unnatural variants by intercepting the sialic acid biosynthetic pathway with unnatural precursors. We explored whether unnatural cell surface sialosides produced by metabolism can act as neo-antigens and modulate the immunogenicity of cells. RESULTS: Immunization of rabbits with synthetic conjugates of an unnatural sialic acid bound to keyhole limpet hemocyanin produced significant titers of antibodies that were specific for the structurally altered sialic acid. The antibodies recognized cells that were fed the unnatural biosynthetic precursor, and were capable of directing complement-mediated lysis. CONCLUSIONS: Structural alteration of sialic acids replaces a tolerized self-antigen with an antigenic determinant. Incorporation of unnatural sialosides into cell surface glycoconjugates through biosynthetic means can alter the immunoreactivity of cells, providing new possibilities for tumor immunotherapy.     

Syntheses of 4‐(5‐oxo‐1,2,4‐triazol‐3‐yl)‐sydnones and 4‐(4‐arylamino‐5‐oxo‐1,2,4‐triazol‐3‐yl)‐sydnones from Sydnone Derivatives and Their Fragments

4‐(5‐oxo‐1,2,4‐triazol‐3‐yl)‐sydnones 11 and 4‐(4‐arylamino‐5‐oxo‐1,2,4‐triazol‐3‐yl)‐sydnones 13 have been obtained from a‐chloroformylarylhydrazine hydrochloride 2 . Moreover, the intermediates, including 3, 4 , 9 and 10 , in this study are synthetically informative and valuable. It is also noteworthy that three reactants, 1, 2 and sydnonecarbaldehydes, were prepared from sydnone derivatives and their fragments. The oxidative cyclizations of sydnonecarbaldehyde semicarbazones 9 and carbazones 10 with two different oxidizing agents (Cu(ClO₄)₂ and Fe(ClO₄)₃) have been extensively examined. The reaction time and the yields of cyclizations were affected by the substituents of semicarbazones 9 and carbazones 10.