Gijsbert A. van der Marel

Thioglycosides in sequential glycosylation strategies

This tutorial review surveys the use of thioglycosides in the development of sequential glycosylation methodologies, with a focus on chemoselective, orthogonal and iterative glycosylation strategies reported since the beginning of this century. Both fundamental aspects of glycosidic bond formation and ingenious state-of-the-art methodologies are presented.

Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells *

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam3CysSK4 (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG∼conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam∼conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG∼conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8+ T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

The impact of oxacarbenium ion conformers on the stereochemical outcome of glycosylations

The search for stereoselective glycosylation reactions has occupied synthetic carbohydrate chemists for decades. Traditionally, most attention has been focused on controlling the S(N)2-like substitution of anomeric leaving groups as highlighted by Lemieuxs in situ anomerization protocol and by the discovery of anomeric triflates as reactive intermediates in the stereoselective formation of beta-mannosides. Recently, it has become clear that also S(N)1-like reaction pathways can lead to highly selective glycosylation reactions. This review describes some recent examples of stereoselective glycosylations in which oxacarbenium ions are believed to be at the basis of the selectivity. Special attention is paid to the stereodirecting effect of substituents on a pyranosyl ring with an emphasis on the role of the C-5 carboxylate ester in the condensations of mannuronate ester donors.

Triple Bioorthogonal Ligation Strategy for Simultaneous Labeling of Multiple Enzymatic Activities

Bioorthogonal chemistry plays an important role in chemical biology research by creating the means to carry out selective chemical transformations in complex biological samples. A ligation reaction classifies as being bioorthogonal when it can be performed in a biological sample in a chemoselective manner without any interference with the biological system. Bioorthogonal reactions have been used in cell-surface labeling of glycoproteins and studies of biological processes that involve post-translational modifications. Another area of research that has benefited from bioorthogonal chemistry is two-step activity-based protein profiling (ABPP), where it enables the temporal separation of a reporter group and a chemical probe that is directed to the active site of an enzyme (such a chemical probe is also called activity-based probe, ABP). Two-step ABPP strategies are of particular interest when the presence of a tag interferes with selectivity, affinity, cell-permeability, or bioavailability of the probe. A further advantage of tandem labeling strategies is the option to use different reporter groups depending on the type of experiment and the desired method of analysis while using a single ABP. Several bioorthogonal ligation strategies have been described, and continuing efforts are being made to develop ligations that are more selective and efficient than existing methods. At the same time the high complexity of biological processes often requires the study of multiple targets simultaneously, thereby creating a need for ligation reactions that are orthogonal with respect to each other and can thus be used concurrently in a single experiment. Over the past decade, a number of tandem ligation strategies has been described for use in bioconjugation. The first report of a tandem bioorthogonal ligation in complex biological samples involved a Staudinger–Bertozzi ligation and Diels– Alder cycloaddition procedure, which utilizes mutually orthogonal reagents but suffers from the need to mask free thiol groups prior to the ligation step to avoid nonspecific labeling. More recently, it was reported that a copper-free azide–cyclooctyne cycloaddition can be used concurrently with an inverse-electron-demand Diels–Alder reaction between tetrazine and trans-cyclooctene for the simultaneous labeling of two different receptors on cell surfaces, provided that the proper reagents are carefully selected so that crossreactivity is minimized. Herein, we describe a triple ligation strategy employing the tetrazine ligation, Staudinger–Bertozzi ligation, and copper(I)-catalyzed Huisgen [2+3] cycloaddition (“click” reaction) for the selective and simultaneous labeling of three different enzymatic activities in a single experiment (Scheme 1a). Several examples of two-step ABPP strategies using click chemistry and Staudinger–Bertozzi ligation have been described previously. The tetrazine ligation, however, has thus far not been used for this purpose. Therefore we set out to develop a two-step ABPP strategy in which an ABP is functionalized with norbornene as a ligation handle that can react with a tetrazine reagent conjugated to a reporter group to enable detection and analysis of labeled proteins. As a model system for our studies we selected the 20S proteasome, containing three catalytically active subunits (b1, b2, and b5) that can be targeted by either broad-spectrum or subunit-specific ABPs. We designed two proteasome ABPs that are functionalized with norbornene as a ligation handle: ABP 1 is derived from the pan-reactive proteasome inhibitor epoxomicin, and ABP 2 has a different scaffold based on a b5-subunit-selective proteasome inhibitor (Scheme 1 b). Furthermore, we chose to create a panel of three tetrazine reagents functionalized with different tags, being BodipyTMR (3 a), BodipyFL (3 b), and biotin (3c). Other reagents used herein for two-step labeling of the proteasome by click chemistry and Staudinger ligation are shown in Scheme 1c. The synthesis of all reagents and competition experiments confirming the ability of the ABPs to target all proteolytically active proteasome b subunits (1, 4, 5) or only the b5 subunit (2) in cell extracts and/or in living cells can be found in the Supporting Information. The applicability of the tetrazine ligation for two-step labeling of endogenous proteasome activity was tested by exposing human embryonic kidney (HEK) cell lysates to norbornene-functionalized ABP 1 in a concentration that results in complete proteasome binding followed by ligation with one of the tetrazine reagents 3a–c for one hour at 37 8C. Analysis of labeled proteins by SDS-PAGE using either fluorescent readout or detection by streptavidin Western blotting (Figure 1a and Figure S2 in the Supporting Information) showed that ligation with all three tetrazine reagents results in labeling of the three catalytically active proteasome b subunits in a concentration-dependent manner. In this [*] L. I. Willems, N. Li, Dr. B. I. Florea, M. Ruben, Prof. G. A. van der Marel, Prof. H. S. Overkleeft Leiden Institute of Chemistry and Netherlands Proteomics Centre Gorlaeus Laboratories Einsteinweg 55, 2333 CC Leiden (The Netherlands) E-mail: h.s.overkleeft@chem.leidenuniv.nl

Novel Activity‐Based Probes for Broad‐Spectrum Profiling of Retaining β‐Exoglucosidases In Situ and In Vivo

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases (see picture). These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Dual-Action Lipophilic Iminosugar Improves Glycemic Control in Obese Rodents by Reduction of Visceral Glycosphingolipids and Buffering of Carbohydrate Assimilation

The lipophilic iminosugar N-[5-(adamantan-1-ylmethoxy)pentyl]-1-deoxynojirimycin (2, AMP-DNM) potently controls hyperglycemia in obese rodent models of insulin resistance. The reduction of visceral glycosphingolipids by 2 is thought to underlie its beneficial action. It cannot, however, be excluded that concomitant inhibition of intestinal glycosidases and associated buffering of carbohydrate assimilation add to this. To firmly establish the mode of action of 2, we developed a panel of lipophilic iminosugars varying in configuration at C-4/C-5 and N-substitution of the iminosugar. From these we identified the l-ido derivative of 2, l-ido-AMP-DNM (4), as a selective inhibitor of glycosphingolipid synthesis. Compound 4 lowered visceral glycosphingolipids in ob/ob mice and ZDF rats on a par with 2. In contrast to 2, 4 did not inhibit sucrase activity or sucrose assimilation. Treatment with 4 was significantly less effective in reducing blood glucose and HbA1c. We conclude that the combination of reduction of glycosphingolipids in tissue and buffering of carbohydrate assimilation by 2 produces a superior glucose homeostasis.

Stereodirecting effect of the pyranosyl C-5 substituent in glycosylation reactions.

The stereodirecting effect of the glycosyl C-5 substituent has been investigated in a series of d-pyranosyl thioglycoside donors and related to their preferred positions in the intermediate (3)H(4) and (4)H(3) half-chair oxacarbenium ions. Computational studies showed that an axially positioned C-5 carboxylate ester can stabilize the (3)H(4) half-chair oxacarbenium ion conformer by donating electron density from its carbonyl function into the electron-poor oxacarbenium ion functionality. A similar stabilization can be achieved by a C-5 benzyloxymethyl group, but the magnitude of this stabilization is significantly smaller than for the C-5 carboxylate ester. As a result, the preference of the C-5 benzyloxymethyl to occupy an axial position in the half-chair oxacarbenium ions is much reduced compared to the C-5 carboxylate ester. To minimize steric interactions, a C-5 methyl group prefers to adopt an equatorial position and therefore favors the (4)H(3) half-chair oxacarbenium ion. When all pyranosyl substituents occupy their favored position in one of the two intermediate half-chair oxacarbenium ions, highly stereoselective glycosylations can be achieved as revealed by the excellent beta-selectivity of mannuronate esters and alpha-selectivity of 6-deoxygulosides.

The Stereodirecting Effect of the Glycosyl C5-Carboxylate Ester: Stereoselective Synthesis of β-Mannuronic Acid Alginates

Glycosylations of mannuronate ester donors proceed highly selectively to produce the 1,2-cis-linked products. We here forward a mechanistic rationale for this counterintuitive selectivity, based on the remote stereodirecting effect of the C5-carboxylate ester, which has been demonstrated using pyranosyl uronate ester devoid of ring substituents other than the C5- carboxylate ester. It is postulated that the C5-carboxylate ester prefers to occupy an axial position in the oxacarbenium intermediate, thereby favoring the formation of the (3)H4 half-chair over the (4)H3 conformer. Nucleophilic attack on the (3)H4 half-chair intermediate occurs in a beta-fashion, providing the 1,2-cis-mannuronates with excellent stereoselectivity. The potential of the mannuronate ester donors in the formation of the beta-mannosidic linkage has been capitalized upon in the construction of a mannuronic acid alginate pentamer using a convergent orthogonal glycosylation strategy.

A NOVEL AND FLEXIBLE SYNTHESIS OF PYRANOSE SPIROACETAL DERIVATIVES

Abstract A three-step approach to chiral pyranose [5,4], [5,5], [5,6] and [5,7] unsaturated spiroacetal derivatives from perbenzylated glucopyranolactone 1 is presented. The strategy involves Grignard addition of vinyl or allyl magnesium bromide to 1 to give 2 and 12 , respectively, K-10 mediated glucosidation of different terminal alkenols with 2 and 12 followed by ring-closing metathesis.