Fluoro-labelled sp2-iminoglycolipids with immunomodulatory properties.

The unique electronic properties of the fluorine atom make its strategic incorporation into a bioactive compound a very useful tool in the design of drugs with optimized pharmacological properties. In the field of the carbohydrates, its selective installation at C2 position has proven particularly interesting, some 2-deoxy-2-fluorosugar derivatives being currently in the market. We have now transferred this feature into immunoregulatory glycolipid mimetics that contain a sp2-iminosugar moiety, namely sp2-iminoglycolipids (sp2-IGLs). The synthesis of two epimeric series of 2-deoxy-2-fluoro-sp2-IGLs, structurally related to nojirimycin and mannonojirimycin, has been accomplished by sequential Selectfluor-mediated fluorination and thioglycosidation of sp2-iminoglycals. Exclusively the α-anomer is obtained regardless of the configurational profile of the sp2-IGL (d-gluco or d-manno), highlighting the overwhelming anomeric effect in these prototypes. Notably, the combination of a fluorine atom at C2 and an α-oriented sulfonyl dodecyl lipid moiety in compound 11 led to remarkable anti-proliferative properties, featuring similar GI50 values than the chemotherapy drug Cisplatin against several tumor cell lines and better selectivity. The biochemical data further evidence a strong reduction of the number of tumor cell colonies and apoptosis induction. Mechanistic investigations revealed that this fluoro-sp2-IGL induces the non-canonical activation mode of the mitogen-activated protein kinase signaling pathway, causing p38α autoactivation under an inflammatory context.

A ß-Cyclodextrin-Based Nanoparticle with Very High Transfection Efficiency Unveils siRNA-Activated TLR3 Responses in Human Prostate Cancer Cells.

Synthetic double-stranded small interfering RNAs (siRNAs) mimic interference RNAs (RNAi) and can bind target mRNAs with a high degree of specificity, leading to selective knockdown of the proteins they encode. However, siRNAs are very labile and must be both protected and transported by nanoparticles to be efficiently delivered into cells. In this work, we used a Janus-type polycationic amphiphilic ß-cyclodextrin derivative to efficiently transfect siRNAs targeting mRNAs encoding mitogen-activated protein kinase (p42-MAPK) or Ras homolog enriched in brain (Rheb) into different cancer cell lines as well as astrocytes. We took advantage of this high transfection efficiency to simultaneously knock down p42-MAPK and Rheb to boost docetaxel (DTX)-mediated toxicity in two human prostate cancer cell lines (LNCaP and PC3). We found that double knockdown of p42-MAPK and Rheb increased DTX-toxicity in LNCaP but not in PC3 cells. However, we also observed the same effect when scramble siRNA was used, therefore pointing to an off-target effect. Indeed, we found that the siRNA we used in this work induced toll-like receptor 3 activation, leading to ß-interferon production and caspase activation. We believe that this mechanism could be very useful as a general strategy to elicit an immune response against prostate cancer cells.

Synthesis, self-assembly and anticancer drug encapsulation and delivery properties of cyclodextrin-based giant amphiphiles.

Cyclodextrin-calixarene giant amphiphiles that can self-assemble into nanospheres or nanovesicles have the ability to encapsulate the anticancer hydrophobic drugs docetaxel, temozolomide and combretastatin A-4 with encapsulation efficiencies >80% and deliver them to tumoral cells, enhancing their therapeutic efficacy by 1-3 orders of magnitude. These amphiphiles were modified by inserting a disulfide bridge confering them redox responsiveness. Disassembly of the resulting nanocompounds and cargo release was favored by high glutathione levels mimicking those present in the tumor microenvironment. Anticancer drug-loaded nanoformulations inhibited prostate, breast, glioblastoma, colon or cervix cancer cell lines proliferation with IC50 values markedly below those observed for the free drugs. Cell-cycle analysis indicated a similar mechanism of action for drug-loaded nanocompounds and free drugs. The results strongly suggest that the cyclodextrin-calixarene heterodimer prototype is an excellent scaffold for nanoformulations aimed to deliver anticancer drugs with limited bioavailability due to low solubility to tumoral cells, markedly increasing their effectivity.

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment.

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

Probing the Inhibitor versus Chaperone Properties of sp²-Iminosugars towards Human ß-Glucocerebrosidase: A Picomolar Chaperone for Gaucher Disease.

A series of sp²-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido), the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 ß-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N'-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase). The 1-deoxynojirimycin (DNJ)-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM). At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

Revealing cooperative binding of polycationic cyclodextrins with DNA oligomers by capillary electrophoresis coupled to mass spectrometry.

Gene delivery is critical for the development of nucleic acid-based therapies against a range of severe diseases. The conception of non-viral (semi)synthetic vectors with low cytotoxicity and virus-like efficiency is gathering a lot of efforts, but it represents a fantastic challenge still far from accomplishment. Carbohydrate-based scaffolds offer interesting features towards this end, such as easy availability, relatively cheap cost, tuning properties and a good biocompatibility. The lack of analytical methods providing quantitative and qualitative data on their binding properties with oligonucleotides (DNA/RNA), with a minimal time and sample consumption, represents a limitation for these channels. Here, we attempted to fill the gap by hyphenation of capillary electrophoresis with mass spectrometry (CE-MS). This coupling strategy allows discriminating free and complexed DNA oligomers with cationic cyclodextrins (CDs), determining the stoichiometry where the highest observed is always DNAn: n/3(CD), and unambiguously assigning the partners through m/z detection. Very reliable data were obtained with migration time within 5.5 (standard deviation < 0.5%) and 25 min (standard deviation < 1.1%) for UV and MS detection, respectively. Furthermore, varying the nitrogen/phosphorus ratio (N/P), key parameters relating to the thermodynamics e.g. the micro and macroscopic dissociation constants Kd and KD, respectively (both in low µM range) and the Gibbs free energy ΔG (-16.3 to -26.9 kJ mol-1), and also the cooperativity as Hill number (nH between 0.98 and 15.75) of the supramolecular process can be delineated, providing a unique tool for the high throughput screening and selection of efficient gene delivery carriers.

The sp2-iminosugar glycolipid 1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO2-ONJ) as selective anti-inflammatory agent by modulation of hemeoxygenase-1 in Bv.2 microglial cells and retinal explants.

Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp2-iminosugar glycolipid (sp2-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO2-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO2-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO2-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO2-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO2-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO2-ONJ in microglia.

Development of polycationic amphiphilic cyclodextrin nanoparticles for anticancer drug delivery.

Background: Paclitaxel is a potent anticancer drug that is effective against a wide spectrum of cancers. To overcome its bioavailability problems arising from very poor aqueous solubility and tendency to recrystallize upon dilution, paclitaxel is commercially formulated with co-solvents such as Cremophor EL® that are known to cause serious side effects during chemotherapy. Amphiphilic cyclodextrins are favored oligosaccharides as drug delivery systems for anticancer drugs, having the ability to spontaneously form nanoparticles without surfactant or co-solvents. In the past few years, polycationic, amphiphilic cyclodextrins were introduced as effective agents for gene delivery in the form of nanoplexes. In this study, the potential of polycationic, amphiphilic cyclodextrin nanoparticles were evaluated in comparison to non-ionic amphiphilic cyclodextrins and core-shell type cyclodextrin nanoparticles for paclitaxel delivery to breast tumors. Pre-formulation studies were used as a basis for selecting the suitable organic solvent and surfactant concentration for the novel polycationic cyclodextrin nanoparticles. The nanoparticles were then extensively characterized with particle size distribution, polydispersity index, zeta potential, drug loading capacity, in vitro release profiles and cytotoxicity studies. Results: Paclitaxel-loaded cyclodextrin nanoparticles were obtained in the diameter range of 80-125 nm (depending on the nature of the cyclodextrin derivative) where the smallest diameter nanoparticles were obtained with polycationic (PC) ßCDC6. A strong positive charge also helped to increase the loading capacity of the nanoparticles with paclitaxel up to 60%. Interestingly, cyclodextrin nanoparticles were able to stabilize paclitaxel in aqueous solution for 30 days. All blank cyclodextrin nanoparticles were demonstrated to be non-cytotoxic against L929 mouse fibroblast cell line. In addition, paclitaxel-loaded nanoparticles have a significant anticancer effect against MCF-7 human breast cancer cell line as compared with a paclitaxel solution in DMSO. Conclusion: According to the results of this study, both amphiphilic cyclodextrin derivatives provide suitable nanometer-sized drug delivery systems for safe and efficient intravenous paclitaxel delivery for chemotherapy. In the light of these studies, it can be said that amphiphilic cyclodextrin nanoparticles of different surface charge can be considered as a promising alternative for self-assembled nanometer-sized drug carrier systems for safe and efficient chemotherapy.

Molecular determinants for cyclo-oligosaccharide-based nanoparticle-mediated effective siRNA transfection.

AIM: To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector. MATERIALS & METHODS: siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics. RESULTS: The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA. CONCLUSION: The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.

Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry.

Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps.

Cyclodextrin-based facial amphiphiles: assessing the impact of the hydrophilic-lipophilic balance in the self-assembly, DNA complexation and gene delivery capabilities.

Exhaustive structure-efficacy relationship studies on nonviral gene delivery systems are often hampered by the ill-defined or polydisperse nature of the formulations. Facial amphiphiles based on rigid cage-type molecular scaffolds offer unique possibilities towards these studies. Taking advantage of regioselective functionalization schemes, we have synthesized a library of cationic cyclodextrin (CD) derivatives combining a range of hydrophilic and lipophilic domains. We have scrutinized how the hydrophilic-lipophilic balance (HLB) around the CD scaffold determines their self-assembly capabilities and the DNA binding and release abilities of the corresponding CD : DNA nanocomplexes (CDplexes). These features have been ultimately correlated with their capabilities to deliver a reporter luciferase-encoding pDNA into COS-7 cells. The ensemble of results demonstrates that fine tuning of the HLB is critical to induce compaction of DNA by the CD-based facial amphiphiles into transfection-productive CDplexes.

Tn Antigen Mimics Based on sp(2)-Iminosugars with Affinity for an anti-MUC1 Antibody.

The first examples of amino acid (Ser/Thr)-sp(2)-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp(2)-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp(2)-iminosugar and the peptide aglycone structure (Ser/Thr).

Glycomimetic-based pharmacological chaperones for lysosomal storage disorders: lessons from Gaucher, GM1-gangliosidosis and Fabry diseases.

Lysosomal storage disorders (LSDs) are often caused by mutations that destabilize native folding and impair the trafficking of enzymes, leading to premature endoplasmic reticulum (ER)-associated degradation, deficiencies of specific hydrolytic functions and aberrant storage of metabolites in the lysosomes. Enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) are available for a few of these conditions, but most remain orphan. A main difficulty is that virtually all LSDs involve neurological decline and neither proteins nor the current SRT drugs can cross the blood-brain barrier. Twenty years ago a new therapeutic paradigm better suited for neuropathic LSDs was launched, namely pharmacological chaperone (PC) therapy. PCs are small molecules capable of binding to the mutant protein at the ER, inducing proper folding, restoring trafficking and increasing enzyme activity and substrate processing in the lysosome. In many LSDs the mutated protein is a glycosidase and the accumulated substrate is an oligo- or polysaccharide or a glycoconjugate, e.g. a glycosphingolipid. Although it might appear counterintuitive, substrate analogues (glycomimetics) behaving as competitive glycosidase inhibitors are good candidates to perform PC tasks. The advancements in the knowledge of the molecular basis of LSDs, including enzyme structures, binding modes, trafficking pathways and substrate processing mechanisms, have been put forward to optimize PC selectivity and efficacy. Moreover, the chemical versatility of glycomimetics and the variety of structures at hand allow simultaneous optimization of chaperone and pharmacokinetic properties. In this Feature Article we review the advancements made in this field in the last few years and the future outlook through the lessons taught by three archetypical LSDs: Gaucher disease, GM1-gangliosidosis and Fabry disease.

Molecular basis of 1-deoxygalactonojirimycin arylthiourea binding to human α-galactosidase a: pharmacological chaperoning efficacy on Fabry disease mutants.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene often leading to missense α-galactosidase A (α-Gal A) variants that undergo premature endoplasmic reticulum-associated degradation due to folding defects. We have synthesized and characterized a new family of neutral amphiphilic pharmacological chaperones, namely 1-deoxygalactonojirimycin-arylthioureas (DGJ-ArTs), capable of stabilizing α-Gal A and restoring trafficking. Binding to the enzyme is reinforced by a strong hydrogen bond involving the aryl-N'H thiourea proton and the catalytic aspartic acid acid D231 of α-Gal A, as confirmed by a 2.55 Å resolution cocrystal structure. Selected candidates enhanced α-Gal A activity and ameliorate globotriaosylceramide (Gb3) accumulation and autophagy impairments in FD cell cultures. Moreover, they acted synergistically with the proteostasis regulator 4-phenylbutyric acid, appearing to be promising leads as pharmacological chaperones for FD.

Amphiphilic oligoethyleneimine-ß-cyclodextrin "click" clusters for enhanced DNA delivery.

Monodisperse amphiphilic oligoethyleneimine (OEI)-ß-cyclodextrin (ßCD) clusters have been prepared, and their potential as gene delivery systems has been evaluated in comparison with a nonamphiphilic congener. The general prototype incorporates tetraethyleneimine segments linked to the primary rim of ßCD through either triazolyl or thioureidocysteaminyl connectors. Transfection efficiency data for the corresponding CD:pDNA nanocomplexes (CDplexes) in BNL-CL2 murine hepatocytes evidenced the strong beneficial effect of facial amphiphilicity.

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery.

Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.

Bicyclic (galacto)nojirimycin analogues as glycosidase inhibitors: effect of structural modifications in their pharmacological chaperone potential towards ß-glucocerebrosidase.

A molecular-diversity-oriented approach for the preparation of bicyclic sp(2)-iminosugar glycomimetics related to nojirimycin and galactonojirimycin is reported. The synthetic strategy takes advantage of the ability of endocyclic pseudoamide-type atoms in five-membered cyclic iso(thio)ureas and guanidines to undergo intramolecular nucleophilic addition to the masked carbonyl group of monosaccharides. The stereochemistry of the resulting hemiaminal stereocenter is governed by the anomeric effect, with a large preference for the axial (pseudo-α) orientation. A library of compounds differing in the stereochemistry at the position equivalent to C-4 in monosaccharides (D-gluco and D-galacto), the heterocyclic core (cyclic isourea, isothiourea or guanidine) and the nature of the exocyclic nitrogen substituent (apolar, polar, linear or branched) has been thus prepared and the glycosidase inhibitory activity evaluated against commercial glycosidases. Compounds bearing lipophilic substituents behaved as potent and very selective inhibitors of ß-glucosidases. They further proved to be good competitive inhibitors of the recombinant human ß-glucocerebrosidase (imiglucerase) used in enzyme replacement therapy (ERT) for Gaucher disease. The potential of these compounds as pharmacological chaperones was assessed by measuring their ability to inhibit thermal-induced denaturation of the enzyme in comparison with N-nonyl-1-deoxynojirimycin (NNDNJ). The results indicated that amphiphilic sp(2)-iminosugars within this series are more efficient than NNDNJ at stabilizing ß-glucocerebrosidase and have a strong potential in pharmacological chaperone (PC) and ERT-PC combined therapies.

Comparative studies on lectin-carbohydrate interactions in low and high density homo- and heteroglycoclusters.

A versatile synthetic procedure to construct series of high- and low-density homo- and heteroglycoclusters is reported. The binding properties of these synthetic multivalent glycoconjugates to concanavalin A (Con A), a model lectin, have been assessed by using a range of competitive and non-competitive binding assays including enzyme-linked lectin assays (ELLA), isothermal titration microcalorimetry (ITC) and surface plasmon resonance (SPR). In all cases, highly dense glycoclusters showed a substantial amplification of the lectin-binding strength in comparison with low-density counterparts. Interestingly, highly-dense glycoligand presentations, regardless of their homo- or heteroglycoligand pattern, furnished similar Con A binding properties, supporting the existence of a synergic effect (heterocluster effect) due to secondary interactions of "non-active" structural motifs in the presence of a certain density of "active" glycoligands.

Difructose dianhydrides (DFAs) and DFA-enriched products as functional foods.

This review provides an overview of the current status of the chemistry and biology of di-D-fructose dianhydrides (DFAs) with a focus on their potential as functional foods. The history of this family of cyclic ketodisaccharides has expanded for almost 100 years and offers a paradigmatic example of artificial synthetic molecules that were identified as natural products later on and finally encountered in our own table. Issued from fundamental investigations on the reactivity of carbohydrates in strongly acidic media, DFAs remained laboratory curiosities for decades. Early reports on their isolation from plants raised doubts, until the formation of some DFA representatives by the action of microorganisms on fructans was reported in the middle 1980s. Since then, research on DFAs has run in parallel in the areas of microbiology and carbohydrate chemistry. Evidence of the potential of these compounds as functional food was accumulated from both sides, with the development of biotechnological processes for mass production of selected candidates and of chemical methodologies to prepare DFA-enriched products from sucrose or inulin. In 1994 a decisive discovery in the field took place in the laboratory of Jacques Defaye in Grenoble, France: the presence of DFAs in a commercial sucrose caramel was evidenced in a quite significant 18% mass proportion! The development of an efficient analytical protocol for DFAs and the stereoselective synthesis of individual standards allowed one to demonstrate that DFAs and their glycosylated derivatives (glycosyl-DFAs) are universally formed during caramelization reactions. They are not potential food products; they have actually always been in our daily food. Most important, they seem to exert beneficial effects: they are acariogenic, low-caloric, and promote the growth of beneficial microflora in the gut. Most recent evidence indicates that DFAs can even protect the intestinal tract against agressive agents favor the assimilation of antioxidants, and act as a drug-like food for the treatment of colon ailments such as inflammatory bowel disease (Crohn disease). The development of efficient methodologies for the preparation of DFA-enriched caramels, compatible with the food and agricultural industry regulations, may lead to new natural functional foods and nutraceuticals based on DFAs in the near future.