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Original molecular vectors that ensure broad flexibility to tune the shape and surface properties of plasmid DNA (pDNA) condensates are reported herein. The prototypic design involves a cyclodextrin (CD) platform bearing a polycationic cluster at the primary face and a doubly linked aromatic module bridging two consecutive monosaccharide units at the secondary face that behaves as a topology-encoding element. Subtle differences at the molecular level then translate into disparate morphologies at the nanoscale, including rods, worms, toroids, globules, ellipsoids, and spheroids. In vitro evaluation of the transfection capabilities revealed marked selectivity differences as a function of nanocomplex morphology. Remarkably high transfection efficiencies were associated with ellipsoidal or spherical shapes with a lamellar internal arrangement of pDNA chains and CD bilayers. Computational studies support that the stability of such supramolecular edifices is directly related to the tendency of the molecular vector to form noncovalent dimers upon DNA templating. Because the stability of the dimers depends on the protonation state of the polycationic clusters, the coaggregates display pH responsiveness, which facilitates endosomal escape and timely DNA release, a key step in successful transfection. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes.

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.

The efficient scalable syntheses of 2-acetamido-1,2-dideoxy-D-galacto-nojirimycin (DGJNAc) and 2-acetamido-1,2-dideoxy-D-gluco-nojirimycin (DNJNAc) from D-glucuronolactone, as well as of their enantiomers from L-glucuronolactone, are reported. The evaluation of both enantiomers of DNJNAc and DGJNAc, along with their N-alkyl derivatives, as glycosidase inhibitors showed that DGJNAc and its N-alkyl derivatives were all inhibitors of ¿-GalNAcase but that none of the epimeric DNJNAc derivatives inhibited this enzyme. In contrast, both DGJNAc and DNJNAc, as well as their alkyl derivatives, were potent inhibitors of ß-GlcNAcases and ß-GalNAcases. Neither of the L-enantiomers showed any significant inhibition of any of the enzymes tested. Correlation of the in vitro inhibition with the cellular data, by using a free oligosaccharide analysis of the lysosomal enzyme inhibition, revealed the following structure¿property relationship: hydrophobic side-chains preferentially promoted the intracellular access of iminosugars to those inhibitors with more-hydrophilic side-chain characteristics.

18 páginas, 11 figuras, 4 esquemas, A molecular-diversity-oriented approach for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) as gene-delivery systems is reported. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic–hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure–activity relationship studies. Gel electrophoresis revealed that paCDs self-assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70–150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL-CL2 and COS-7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen-bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space-oriented dendritic polycationic constructs., This work was supported by the Spanish Ministerio de Innovación y Ciencia (MICINN; contract numbers CTQ2006-15515-C02-01/BQU and CTQ2007-61180/PPQ), the Junta de Andalucía (P06-FQM-01601), the CSIC, the CNRS, and FUSINT (CNR project). A.D.M. and P.B.O. are grateful for predoctoral fellowships (CSIC and MICINN, respectively).

Results obtained over the past decade towards the preparation of multitopic carbohydrate architectures combining the molecular inclusion capabilities of cyclomaltooligosaccharide receptors (cyclodextrins, CDs) and the recognition properties of saccharide ligands towards biological receptors are discussed. The potential of these new sugar-based "intelligent" transporters for site specific delivery of therapeutics is outlined.