Matthieu Sollogoub

The first synthesis of substituted azepanes mimicking monosaccharides: a new class of potent glycosidase inhibitors

The synthesis of the first examples of seven-membered ring iminoalditols, molecules displaying an extra hydroxymethyl substituent on their seven-membered ring compared to the previously reported polyhydroxylated azepanes, has been achieved from d-arabinose in 10 steps using RCM of a protected N-allyl-aminohexenitol as a key step. While the (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxy-azepane 10, a seven-membered ring analogue of fagomine, is a weak inhibitor of glycosidases, the (2R,3R,4R,5S,6S)-2-hydroxymethyl-3,4,5,6-tetrahydroxy-azepane 9 selectively inhibits green coffee bean alpha-galactosidase in the low micromolar range (Ki = 2.2 muM) despite a D-gluco relative configuration.

Molecular Basis for Inhibition of GH84 Glycoside Hydrolases by Substituted Azepanes: Conformational Flexibility Enables Probing of Substrate Distortion

Here we report the synthesis of a series of polyhydroxylated 3- and 5-acetamido azepanes and detail the molecular basis of their inhibition of family 84 glycoside hydrolases. These family 84 enzymes include human O-GlcNAcase, an enzyme involved in post-translational processing of intracellular proteins modified by O-linked beta-N-acetylglucosamine residues. Detailed structural analysis of the binding of these azepanes to BtGH84, a bacterial homologue of O-GlcNAcase, highlights their conformational flexibility. Molecular mechanics and molecular dynamics calculations reveal that binding to the enzyme involves significant conformational distortion of these inhibitors from their preferred solution conformations. The binding of these azepanes provides structural insight into substrate distortion that likely occurs along the reaction coordinate followed by O-GlcNAcase during glycoside hydrolysis. This class of inhibitors may prove to be useful probes for evaluating the conformational itineraries of glycosidases and aid the development of more potent and specific glycosidase inhibitors.

Photosensitive surfactants with various hydrophobic tail lengths for the photocontrol of genomic DNA conformation with improved efficiency.

We report the synthesis and characterisation of photosensitive cationic surfactants with various hydrophobic tail lengths. These molecules, called AzoCx, are used as photosensitive nucleic acid binders (pNABs) and are applied to the photocontrol of DNA conformation. All these molecules induce DNA compaction in a photodependent way, originating in the photodependent polarity of their hydrophobic tails. We show that increasing hydrophobicity strongly enhances the compaction efficiencies of these molecules, but reduces the possibility of reversible photocontrol of a DNA conformation. Optimal performance was achieved with AzoC5, which allowed reversible control of DNA conformation with light at a concentration seven times smaller than previously reported.

Multiple Homo- and Hetero-functionalizations of α-Cyclodextrin through Oriented Deprotections

Introduction of one or more functions on a cyclodextrin in a regioselective manner is a complex task. The discovery of a blueprint strategy involving regioselective deprotection reactions of fully protected cyclodextrins, instead of functionalization of native cyclodextrins, allowed us to propose an efficient alternative to reach this goal. In this paper, we have applied previously delineated strategies, based on steric decompression, to duplicate our deprotection reaction, using a combination of mono- or di-deprotections to access cyclodextrins with two, three and five points of attachment for one, two or three different new functions. The patterns of multi-functionalization delineated here are not accessible by any other methods. Indeed, it is the first time ever a cyclodextrin bearing four different groups is synthesized in a completely controlled manner. This work paves the way to the use of cyclodextrins as multi-functional macrocyclic scaffolds, a use they have long been meant for, but lack of reliable functionalization methods hindered this development.

Regiospecific Tandem Azide-Reduction/Deprotection To Afford Versatile Amino Alcohol-Functionalized α-and β-Cyclodextrins

Regioselective functionalizations that confer chirality to large complex molecules, particularly those with a concave shape, are an important goal in the fields of molecular recognition and catalysis. Cyclodextrins (CDs) qualify as interesting chiral hosts in this respect as they have shown excellent artificial enzymatic activity, although they have found only limited applications in asymmetric catalysis. Amino alcohols, or amino acids such as proline, have emerged as powerful chiral tools for enantioselective catalysis. Therefore, the elaboration of a CD scaffold bearing an amino alcohol or an amino acid at a specific position is an attractive goal. However, the synthesis of such compounds requires a judicious functionalization (both regioselective and orthogonal) of two hydroxy groups of the CD. The regiospecific introduction of two different functionalities on the primary rim of CDs is a rapidly emerging area. Although the synthesis of monofunctionalized CDs is a relatively straighforward task, the addition of a second function implies that a regioselective process must take place. Current methodologies rely on the regioselective opening or formation of capped CDs to afford 6-6-imidazolyl-sulfonylor 6-6thioether-ester substituted CDs, respectively, albeit in moderate yields (less than 10% from unfunctionalized CDs). More recently, a selective direct sulfonation of an imidazolyl CD was performed and, among the 20 possible isomeric monosulfonates, the 6-6 isomer was isolated in 13% yield. However, these methods are inadequate for further conversion into amino alcohols because of the nature of the functional groups, which can also require difficult synthetic procedures. We have developed a method that allows the introduction of one, two, or three orthogonally protected alcohol groups on the primary rim of perbenzylated aand b-CDs through successive sterically oriented deprotections induced in high yields by diisobutylaluminum hydride (DIBAL-H). The double deprotection of CDs 1 and 2 that afforded diols 3 and 4, respectively, was shown to occur by a stepwise mechanism in which the two deprotection steps take place sequentially (Scheme 1). This was demonstrated by the deprotection of alcohol 5 to give diol 3. We proposed that protonolysis of the alcohol on 5 and 6 by DIBAL-H affords very hindered aluminum species, leading to CDs 7 and 8, in which a pair of reactive aluminum centers are attached to the glucose units that are furthest apart to maximize their separation, thereby imposing the observed regioselectivity.

Design and synthesis of acetamido tri- and tetra-hydroxyazepanes: Potent and selective β-N-acetylhexosaminidase inhibitors

A series of seven-membered iminosugars bearing an acetamido group beta- or gamma- to the endocyclic nitrogen have been synthesized via simple transformations of previously described polysubstituted azepanes. These tetra- and trihydroxylated acetamido azepanes are ring homologues of 2-acetamido-1,2-dideoxy-glyconojirimycins and 2-acetamido-1-N-iminosugars respectively. Screening of these azepanes towards a range of commercially available glycosidases demonstrated their potential as selective and potent hexosaminidase inhibitors with K(i)s in the submicromolar range. A correlation between the relative configuration of the azepanes and their ability to inactivate hexosaminidases was also observed for the first time for this class of compounds with one notable exception for the most potent compound.

Photocontrol of single-chain DNA conformation in cell-mimicking microcompartments.

It has been well established that the regulation of gene activity is strongly dependent on the higher-order structure of genomic DNA molecules. Several strategies have thus been developed to control the higher-order structure of long DNA molecules. Most of them have been based on the use of chemical compounds that bind to DNA to neutralize its charge, such as polyamines, multivalent metal cations, cationic surfactants, cationic polymers, nanoparticles, or crowding agents such as hydrophilic polymers. Depending on the concentration of these additives, DNA exhibits a folded or unfolded conformation. Nevertheless, with all these strategies, it is impossible to act in a reversible way on the DNA higher-order structure under a constant chemical composition. Moreover, for transfection applications, compacting DNA is an essential step to allow the entry of DNA into the cell. In most cases, however, DNA remains in a compact conformation inside the cell, which can significantly alter the DNA gene expression. Using an external stimulus to control DNA higherorder structure within a cell-sized compartment has thus became an important challenge. On the other hand, motivated by the perspective of DNA vectorization, preparation of artificial cells or biochemical microreactors, many scientists have attempted to encapsulate DNA into cell-like microcompartments, for example, cellsized liposomes or phospholipid-coated microdroplets. Consequently, various successful strategies have been proposed to prepare DNA–liposome complexes or encapsulate DNA inside liposomes. In most cases encapsulated DNA molecules were typically smaller than a few thousands base pairs. However, in nature, genomic DNA molecules can be much larger, up to hundreds of kbp (kilo base pairs). To the best of our knowledge, no method has been proposed to encapsulate efficiently, in a controlled way, and without degradation, DNA molecules that are larger than 1 kbp into cell-sized liposomes. One paper reported the encapsulation of T4 DNA molecules, but the data were not sufficient to draw conclusions about the integrity of encapsulated DNA chains. Another strategy was to encapsulate DNA in a compact state, but DNA molecules remained in their compact state once they were encapsulated. Very recently, Le Ny and Lee made a breakthrough by proposing a system where DNA higher-structure can be controlled by light in a reversible manner. This was achieved by adding to a DNA solution a photosensitive cationic surfactant, azobenzene trimethylammonium bromide surfactant (AzoTAB). The apolar tail of the surfactant contains an azo group, which is mainly in the trans (more hydrophobic) conformation under visible conditions. Under UV illumination (365 nm), the azo group photoisomerizes into the cis (more hydrophilic) conformation. They demonstrated that there exists an AzoTAB concentration range for which DNA is in the compact state under dark/visible conditions but in the unfolded state under UV illumination, that is, DNA higher-order structure can be controlled by light. In this study, the authors mainly characterized the average property of many DNA chains in solution. Here, we characterized the single-chain conformational behavior of long genomic DNA as a function of AzoTAB concentration and time of UV illumination. We established that the transition has a first-order character at the single-chain level. We studied the single-chain unfolding upon UV illumination and evidenced two mechanisms of single-chain DNA unfolding. Then we applied this strategy to unfold genomic DNA molecules that are encapsulated in cell-mimicking microcompartments. To this end, DNA that was compacted by AzoTAB under visible conditions was encapsulated into cell-sized microdroplets that were coated with various phospholipids prior to UV light exposition. We studied the unfolding process of individual DNA molecules inside the microdroplets. We could successfully unfold most of the DNA molecules when the phospholipid was anionic (DOPS phospholipid). We thus demonstrated how an external stimulus, here light, can be used to control the higher-order structure of individual genomic DNA molecules within cell-sized phospholipid-coated microcompartments. By using fluorescence microscopy (FM), we characterized the conformation of individual T4 DNA molecules at a very low DNA concentration (0.1 mm) in Tris–HCl buffer (10 mm, pH 7.4). To the DNA solution, we added azobenzene trimethylammonium bromide (AzoTAB, Figure 1A) at various concentrations under dark conditions (most of AzoTAB molecules are in the trans conformation, that is, more hydrophobic). Depending on AzoTAB concentration, we distinguished three regimes with re[a] M. Geoffroy, Dr. D. Baigl Department of Chemistry, Ecole Normale Sup rieure 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-2402 E-mail : damien.baigl@ens.fr [b] Prof. M. Sollogoub, S. Guieu Institut de Chimie Mol culaire (FR2769) Laboratoire de Chimie Organique (UMR CNRS 7611) UPMC Universit Paris 06 4, Place Jussieu, C. 181, 75005 Paris (France) [c] Dr. A. Yamada, Dr. A. Est vez-Torres, Prof. K. Yoshikawa Department of Physics, Kyoto University Kitashirakawa oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan) [d] Dr. A. Est vez-Torres, Prof. K. Yoshikawa, Dr. D. Baigl Spatio-Temporal Order Project, ICORP JST (Japan Science and Technology Agency) Kyoto University, Kyoto 606-8502 (Japan) A video clip is available as Supporting information on the WWW under http://www.chembiochem.org or from the author.

Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium

Significance Lipofuscin accumulation in the retinal pigment epithelium (RPE) is a hallmark of aging. High lipofuscin levels in the RPE have been associated with retinal degeneration and blindness in Stargardt disease patients and animal models. Currently, there is no treatment to prevent and/or revert lipofuscin-driven retinal degenerative changes. In this study, we report that beta cyclodextrins, cyclic sugars composed of seven glucose units, can bind retinal lipofuscin, prevent its oxidation and remove it from RPE. This study opens an avenue to develop small drugs against, currently untreatable, lipofuscin-associated blinding disorders. Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (β-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of β-CD. Importantly, β-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of β-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of β-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Selection of the biological activity of DNJ neoglycoconjugates through click length variation of the side chain.

A series of neoglycoconjugates derived from deoxynojirimycin has been prepared by click connection with functionalised adamantanes. They have been assayed as glycosidase inhibitors, as inhibitors of the glycoenzymes relevant to the treatment of Gaucher disease, as well as correctors of the defective ion-transport protein involved in cystic fibrosis. We have demonstrated that it is possible to selectively either strongly inhibit ER-α-glucosidases and ceramide glucosyltransferase or restore the activity of CFTR in CF-KM4 cells by varying the length of the alkyl chain linking DNJ and adamantane.

Phenylenediamine catalysis of “click glycosylations” in water: practical and direct access to unprotected neoglycoconjugates

Phenylenediamine-catalyzed click chemistry leads to the efficient, practical, and column-free preparation of neoglycoconjugates from unprotected glucosyl azide, in pure water when aglycon solubility permits.