Nerea Ruiz

Construction of N‐Glycan Microarrays by Using Modular Synthesis and On‐Chip Nanoscale Enzymatic Glycosylation

An effective chemoenzymatic strategy is reported that has allowed the construction, for the first time, of a focused microarray of synthetic N-glycans. Based on modular approaches, a variety of N-glycan core structures have been chemically synthesized and covalently immobilized on a glass surface. The printed structures were then enzymatically diversified by the action of three different glycosyltransferases in nanodroplets placed on top of individual spots of the microarray by a printing robot. Conversion was followed by lectin binding specific for the terminal sugars. This enzymatic extension of surface-bound ligands in nanodroplets reduces the amount of precious glycosyltransferases needed by seven orders of magnitude relative to reactions carried out in the solution phase. Moreover, only those ligands that have been shown to be substrates to a specific glycosyltransferase can be individually chosen for elongation on the array. The methodology described here, combining focused modular synthesis and nanoscale on-chip enzymatic elongation, could open the way for the much needed rapid construction of large synthetic glycan arrays.

Organocatalytic Enantioselective Synthesis of Highly Functionalized Polysubstituted Pyrrolidines

The organocatalytic conjugate addition of different aldehydes to beta-nitroacrolein dimethyl acetal, generating the corresponding highly functionalized nitroaldehydes in high yields and with high stereoselectivities, has been studied in detail. These transformations have been achieved by using both readily available starting materials in a 1:1 ratio as well as commercially available catalysts at a 10 mol % catalyst loading. Furthermore, a very short and efficient protocol has been devised for the preparation of highly enantioenriched pyrrolidines containing two or three contiguous stereocenters starting from the obtained Michael adducts. 3,4-Disubstituted pyrrolidines have been obtained in a single step by Zn-mediated chemoselective reduction of the nitro group followed by intramolecular reductive amination, and trisubstituted homoproline derivatives have been prepared by means of an olefination reaction and a cascade process involving chemoselective reduction of the nitro group followed by a fully diastereoselective intramolecular aza- Michael reaction.

MALDI-TOF Mass Spectrometric Analysis of Enzyme Activity and Lectin Trapping on an Array of N-Glycans†

Glycan microarrays are an established platform for the highthroughput screening of substrate specificities of carbohydrate-binding proteins and processing enzymes. The most common detection method, which is based on fluorescently tagged lectins, can only give a measure of binding specificity, as quantification is often compromised by the specific lectin affinity. Therefore, a label-free technique that could overcome these analytical problems is needed for the analysis of array spot compositions. MALDI-TOF-based assays on surface-bound carbohydrates have been reported. Mrksich and co-workers have shown that this technique can be used to study enzyme activity or to trap affinity ligands on biofunctionalized selfassembled monolayers (SAMs) of oligoethylene glycols on gold surfaces. Unfortunately, the covalent attachment of glycans to the monolayer requires high ligand concentrations that are not suitable when working with complex oligosaccharides. More recently, Wong, Siuzdak, and co-workers studied 2,3-sialyltransferase and b-galactosidase activity on fluorous-tagged lactose immobilized on a perfluorinated surface. The difficult preparation of the nanostructured surface and the multistep tagging procedure, however, render this approach less appealing for routine and high-throughput use in on-chip mass spectrometric analysis of enzyme activity. Inspired by the flexible and mobile organization of glycolipids in lipid bilayers, we present herein a novel strategy for the surface-based MALDI-TOF analysis of glycan arrays that is conceptually a fusion of the approaches of Wong, Siuzdak, Mrksich, and their respective co-workers. Glycans are functionalized with a lipid tag (Scheme 1) and noncovalently immobilized on the MALDI plate by insertion into a self-assembled alkylthiolate monolayer. This setup was tested in a series of glycomics applications. The facile preparation of both a hydrophobic MALDI plate and tagged ligands, as well as the general applicability for large oligosaccharides make this method stand out from other surface-based MALDI-TOF approaches. Hydrophobic surfaces have been applied to MALDI-TOF-based proteomics for sample desalting, but, to the best of our knowledge, these surfaces have not been applied to the oriented immobilization of lipid-tagged biomolecules for surface MALDI-TOF analysis. We chose a commercially available gold-coated MALDI sample plate as surface to prepare a hydrophobic selfassembling monolayer of 1-undecanethiol by following a published procedure. Analysis of the monolayer under standard MALDI-TOF conditions showed only peaks corresponding to known matrix ions, while no disulfide or thiolate ions were detected (Figure 1). The hydrophobically tagged carbohydrate ligands 1–10 used in this study were prepared from synthetic N-glycan structures (2–8) by conjugation with stearic acid or from commercial reducing sugars by reductive amination (1, 9, and 10). Glycan arrays (see the Supporting Information) were made by spotting the conjugates 2–10 onto individual wells of the hydrophobic sample plate, drying, and rinsing the plates with water to remove unbound material. MALDI-TOF analysis using 2,4,6trihydroxy-acetyophenone (THAP) as matrix showed strong ion signal intensities with signal-to-noise values of typically 100 or higher for all compounds; these values are comparable to those reported by Siuzdak and co-workers. Ions were detected as sodium adducts with a detection limit of around 5 picomol. The maximum surface capacity for glycan immobilization was determined by deposition of increasing amounts of conjugate 2, washing, and measurement of the signal for 2 normalized to an internal standard. Surface saturation was reached after deposition of around 2 nmol of conjugate 2 per well, which translates to a surface concentration of 0.2 mmolmm . To avoid the unnecessary waste of valuable analytes, glycans were spotted at half the saturation concentration without compromising signal intensity. The stability of the immobilized glycans to repeated washing with aqueous buffers, water, or organic solvents was then determined. Tagged glycans typically resisted 3–5 wash cycles of 1 minute duration without significant reduction of signal intensity, and the intensity of the model glycan 2 was essentially unchanged after 60 seconds of continuous sonication (Figure 1d). However, the glycans were completely removed from the surface [*] Dr. A. Sanchez-Ruiz, Dr. S. Serna, Dr. N. Ruiz, Prof. Dr. M. Martin-Lomas, Dr. N.-C. Reichardt Biofunctional Nanomaterials Unit, CICbiomaGUNE Paseo Miramon 182, 20009 San Sebastian (Spain) Fax: (+ 34)943-005-314 E-mail: nreichardt@cicbiomagune.es

Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

Experimental observations on the regioselectivity of glycosylation of a 4,6-diol system in the β-d-mannopyranosyl unit of a N-glycan pentasaccharide core structure

The regioselectivity of glycosylation of a 4,6-diol system in the β-mannopyranosyl unit of a N-glycan pentasaccharide core structure is found to be strongly dependent on the structure of the glycosyl donor. While glycosylation with a 2-O-acetyl-D-mannopyranosyl trichloroacetimidate and with a d-mannopyranosyl (α1→3) 2-O-acetyl mannopyranosyl trichoroacetimidate regioselectively occurs at the primary OH-6 position, reaction with d-mannopyranosyl (α1→6) mannopyranosyl 2-O-benzoyl, 2-O-acetyl and 2-O-pivaloyl trichloroacetimidate results in approximately 1:1 mixture of regioisomers at primary OH-6 and secondary OH-4 positions.

(S,S)-(+)-pseudoephedrine alpha-iminoglyoxylamide as a chiral glycine cation equivalent: a modular and flexible approach to enantioenriched alpha-amino ketones.

We have studied the ability of an alpha-imino glyoxylamide derived from (S, S)-(+)-pseudoephedrine as a valuable chiral electrophile for the preparation of alpha-amino carbonyl compounds. In this context, the addition of Grignard reagents to the azomethine moiety of this chiral electrophile afforded the expected alpha-amino amide adducts in good yields and diastereoselectivities. Moreover, these adducts have been transformed into enantioenriched alpha-amino ketones by exploiting the ability of pseudoephedrine amides to undergo selective monoaddition to the carbamoyl group with organolithium reagents.