Design and synthesis of 6-C-alkyl-DMDP type nanomolar inhibitors of ß-galactosidase and ß-glucosidase based on broussonetine S and related derivatives.

Broussonetine S (9), its C-1' and C-10' stereoisomers, and their corresponding enantiomers have been synthesized from enantiomeric arabinose-derived cyclic nitrones, with cross metathesis (CM), epoxidation and Keck asymmetric allylation as key steps. Glycosidase inhibition assays showed that broussonetine S (9) and its C-10' epimer (10'-epi-9) were nanomolar inhibitors of bovine liver ß-galactosidase and ß-glucosidase; while their C-1' stereoisomers were 10-fold less potent towards these enzymes. The glycosidase inhibition results and molecular docking calculations revealed the importance of the configurations of pyrrolidine core and C-1' hydroxyl for inhibition potency and spectra. Together with the docking calculations we previously reported for α-1-C-alkyl-DAB derivatives, we designed and synthesized a series of 6-C-alkyl-DMDP derivatives with very simple alkyl chains. The inhibition potency of these derivatives was enhanced by increasing the length of the side chain, and maintained at nanomolar scale inhibitions of bovine liver ß-glucosidase and ß-galactosidase after the alkyl groups are longer than eight or ten carbons for the (6R)-C-alkyl-DMDP derivatives and their 6S epimers, respectively. Molecular docking calculations indicated that each series of 6-C-alkyl-DMDP derivatives resides in the same active site of ß-glucosidase or ß-galactosidase with basically similar binding conformations, and their C-6 long alkyl chains extend outwards along the hydrophobic groove with similar orientations. The increasing inhibitions of ß-glucosidase and ß-galactosidase with the number of carbon atoms in the side chains may be explained by improved adaptability of longer alkyl chains in the hydrophobic grooves. In addition, the lower ß-glucosidase and ß-galactosidase inhibitions of (6S)-C-alkyl-DMDP derivatives than their C-6 R stereoisomers can be attributed to the misfolding of their alkyl chains and resulted decreased adaptability in the hydrophobic groove. The work reported herein is valuable for design and development of more potent and selective inhibitors of ß-galactosidase and ß-glucosidase, which have potential in treatment of lysosomal storage diseases. Furthermore, part of the 6-C-alkyl-DMDP derivatives and their enantiomers were also tested as potential anti-cancer agents; all the compounds tested were found with moderate cytotoxic effects on MKN45 cells, which would indicate potential applications of these iminosugars in development of novel anticancer agents.

Cellular spermine targets JAK signaling to restrain cytokine-mediated autoimmunity.

Prolonged activation of the type I interferon (IFN-I) pathway leads to autoimmune diseases such as systemic lupus erythematosus (SLE). Metabolic regulation of cytokine signaling is critical for cellular homeostasis. Through metabolomics analyses of IFN-ß-activated macrophages and an IFN-stimulated-response-element reporter screening, we identified spermine as a metabolite brake for Janus kinase (JAK) signaling. Spermine directly bound to the FERM and SH2 domains of JAK1 to impair JAK1-cytokine receptor interaction, thus broadly suppressing JAK1 phosphorylation triggered by cytokines IFN-I, IFN-II, interleukin (IL)-2, and IL-6. Peripheral blood mononuclear cells (PBMCs) from individuals with SLE showing decreased spermine concentrations exhibited enhanced IFN-I and lupus gene signatures. Spermine treatment attenuated autoimmune pathogenesis in SLE and psoriasis mice and reduced IFN-I signaling in monocytes from individuals with SLE. We synthesized a spermine derivative (spermine derivative 1 [SD1]) and showed that it had a potent immunosuppressive function. Our findings reveal spermine as a metabolic checkpoint for cellular homeostasis and a potential immunosuppressive molecule for controlling autoimmune disease.

Perceived rhythmic regularity is greater for song than speech: examining acoustic correlates of rhythmic regularity in speech and song.

Rhythm is a key feature of music and language, but the way rhythm unfolds within each domain differs. Music induces perception of a beat, a regular repeating pulse spaced by roughly equal durations, whereas speech does not have the same isochronous framework. Although rhythmic regularity is a defining feature of music and language, it is difficult to derive acoustic indices of the differences in rhythmic regularity between domains. The current study examined whether participants could provide subjective ratings of rhythmic regularity for acoustically matched (syllable-, tempo-, and contour-matched) and acoustically unmatched (varying in tempo, syllable number, semantics, and contour) exemplars of speech and song. We used subjective ratings to index the presence or absence of an underlying beat and correlated ratings with stimulus features to identify acoustic metrics of regularity. Experiment 1 highlighted that ratings based on the term "rhythmic regularity" did not result in consistent definitions of regularity across participants, with opposite ratings for participants who adopted a beat-based definition (song greater than speech), a normal-prosody definition (speech greater than song), or an unclear definition (no difference). Experiment 2 defined rhythmic regularity as how easy it would be to tap or clap to the utterances. Participants rated song as easier to clap or tap to than speech for both acoustically matched and unmatched datasets. Subjective regularity ratings from Experiment 2 illustrated that stimuli with longer syllable durations and with less spectral flux were rated as more rhythmically regular across domains. Our findings demonstrate that rhythmic regularity distinguishes speech from song and several key acoustic features can be used to predict listeners' perception of rhythmic regularity within and across domains as well.

Design and synthesis of iso-allo-DNJ and L-isoDALDP derivatives: pursuit of potent and selective inhibitors of α-glucosidase.

A series of iso-allo-DNJ and L-isoDALDP derivatives were synthesized from dithioacetal 16 with sequential and highly diastereoselective Ho and Henry reactions, and aziridinium intermediate-mediated ring rearrangement as key steps. Glycosidase inhibition assay found four of them as selective α-glucosidase inhibitors, and the less substituted compound 30 showed more potent α-glucosidase inhibition (IC50 = 9.3 µM) than the others. Molecular docking study revealed different docking modes of the iso-allo-DNJ and L-isoDALDP derivatives from their parent compounds, and also the similarity of compound 30 to isofagomine.

Design, synthesis and glycosidase inhibition of DAB derivatives with C-4 peptide and dipeptide branches.

A series of DAB-peptide and DAB-dipeptide derivatives were synthesized from D-tartrate-derived nitrone 18. The DAB peptides 16 are derivatives of trans,trans-3,4-dihydroxy-L-proline. Glycosidase inhibition assay found four of them to be weak and selective bovine liver ß-galactosidase inhibitors, and the C-2' methyl substituted compound 23b showed the most potent ß-galactosidase inhibition (IC50 = 0.66 µM). Molecular docking studies revealed different docking modes of compound 23b compared to those of other DAB-peptides, and partial similarity of compound 23b to DGJ.

Nanomolar ß-glucosidase and ß-galactosidase inhibition by enantiomeric α-1-C-alkyl-1,4-dideoxy-1,4-imino-arabinitol derivatives.

A series of α-1-C-alkyl DAB (1,4-dideoxy-1,4-imino-d-arabinitol) and LAB (1,4-dideoxy-1,4-imino-l-arabinitol) derivatives with aryl substituents have been designed as analogues of broussonetine W (12), and assayed as glycosidase inhibitors. While the inhibition spectrum of α-1-C-alkyl DAB derivative 16 showed a good correlation to that of broussonetine W (12), introduction of substituents on the terminal aryl (17a-f) or hydroxyl groups at C-1' position of the alkyl chains (18a-e) decreased their α-glucosidase inhibitions but greatly improved their inhibitions of bovine liver ß-glucosidase and ß-galactosidase. Furthermore, epimerization of C-1' configurations of compounds 18a-e clearly lowered their inhibition potency of bovine liver ß-glucosidase and ß-galactosidase. Notably, some of the α-1-C-alkyl DAB derivatives were also found to have potent human lysosome ß-glucosidase inhibitions. In contrast, enantiomers of compounds 18a-e and 1'-epi-18a-e generally showed increased α-glucosidase inhibitions, but sharply decreased bovine liver ß-glucosidase and ß-galactosidase inhibitions. Molecular docking calculations unveiled the novel two set of binding modes for each series of compounds; introduction of C-1' hydroxyl altered the conformations of the pyrrolidine rings and orientation of their long chains, resulting in improved accommodation in the hydrophobic grooves. The compounds reported herein are very potent ß-glucosidase and ß-galactosidase inhibitions with novel binding mode; and the structure-activity relationship provides guidance for design and development of more pyrrolidine pharmacological chaperones for lysosomal storage diseases.

C-6 fluorinated casuarines as highly potent and selective amyloglucosidase inhibitors: Synthesis and structure-activity relationship study.

A series of C-6 fluorinated casuarine derivatives have been synthesized via organocatalytic stereoselective α-fluorination of iminosugar-based aldehydes or direct nucleophilic fluorination of polyhydroxylated pyrrolizidines. Glycosidase assays against various glycosidases allowed systematic structure-activity relationship (SAR) study using molecular docking calculations. Introduction of fluorine atom(s) at C-6 position removed the trehalase and maltase inhibitory activities of all casuarine derivatives, and greatly increased their specificity towards amyloglucosidase. Inhibition of the fluorinated casuarines depended on the configuration of C-6 fluorine, of which 6-deoxy-6-epi-6-fluoro-casuarine (24) was found approximately 40-fold potent than its parent compound 6-epi-casuarine (2) as a potent and specific inhibitor of amyloglucosidase. Molecular docking calculations showed that replacement of the C-6 hydroxyls by fluorine atom(s) removed the original interactions with trehalase, but helped to reinforce the binding with amyloglucosidase via newly established fluorine related hydrogen bonding or untypical anion-π interactions. To further investigate the quantitative SARs of casuarine derivatives, the CoMFA and CoMSIA models on amyloglucosidase were established, indicating the dominating effect of electrostatic field in amyloglucosidase inhibition. The 3D-QSAR models were validated to be reliable and can be used for further optimization of casuarine-related iminosugars, as well as design and development of anti-diabetic and immunomodulatory drugs.

Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

trans, trans-2-C-Aryl-3,4-dihydroxypyrrolidines as potent and selective ß-glucosidase inhibitors: Pharmacological chaperones for Gaucher disease.

Enantiomeric series of C-4 hydroxymethyl depleted DAB and LAB derivatives (trans, trans-2-C-aryl-3,4-dihydroxypyrrolidines), designed as ß-glucosidase inhibitors by molecular docking calculations, have been synthesized in 2 steps from l- and d-tartaric acid derived enantiomeric cyclic nitrones 29L and 29D, respectively. Both series of C-4 hydroxymethyl depleted DAB and LAB derivatives 28Da-e and 28La-e, which are structurally trans, trans-2-C-aryl-3,4-dihydroxypyrrolidines, were potent and selective human lysosome acid ß-glucosidase (GCase) inhibitors, of which 28Dd and 28Ld with C-4 biphenyls showed the highest potency relative to other compounds of the same series. The work provided a series of pyrrolidine-type potent and selective GCase inhibitors with minimal hydroxyl substitutions and synthetic procedures. Structure-activity relationship study revealed not only the rationality of hydrophobic and aromatic properties of the binding sites in GCase, but also the great potential of pyrrolidine family in development of new GCase inhibitors with minimized undesirable side effects. The results indicate a strategy for the development of drugs for the treatment of related diseases targeting acid ß-glucosidase, such as Gaucher disease and Parkinson's disease.

Diastereoselective Synthesis, Glycosidase Inhibition, and Docking Study of C-7-Fluorinated Casuarine and Australine Derivatives.

C-7-fluorinated derivatives of two important polyhydroxylated pyrrolizidines, casuarine and australine, were synthesized with organocatalytic stereoselective α-fluorination of aldehydes as the key step. The strategy is extensively applicable to some synthetically challenging fluorinated iminosugars and carbohydrates. The docking studies indicated that the potent inhibitions of trehalase and amyloglucosidase by the fluorinated polyhydroxylated pyrrolizidines are due to the interaction modes dominated by fluorine atoms in these iminosugars with the amino acids' residues of the corresponding enzymes. Steady interactions were established between the C-7 fluoride and a hydrophobic pocket in amyloglucosidase by untypical anion-π interactions. These unexpected docking modes and related structure-activity relationship studies emphasize the value of fluorination in the design of polyhydroxylated pyrrolizidine glycosidase inhibitors.

Design, synthesis and glycosidase inhibition of C-4 branched LAB and DAB derivatives.

Two series of C-4 alkylated and arylated LAB (1,4-dideoxy-1,4-imino-l-arabinitol) and DAB (1,4-dideoxy-1,4-imino-d-arabinitol) derivatives, synthesized in 6 steps from enantiomeric cyclic nitrones derived from l- and d-tartaric acid, were designed and assayed against various glycosidases. C-4 Branched LAB alkyl and phenyl derivatives 5La-d showed potent α-glucosidase inhibition, particularly against human lysosomal acid α-glucosidase; C-4 DAB derivatives 5Da-d, with small alkyl groups, showed enhanced inhibition of rat intestinal maltase and sucrase. Both enantiomeric C-4 arylated derivatives 5Lf-l and 5Df-l exhibited potent and selective α-glucosidase inhibition; and compound 5Li with a para-electron donating group (EDG) on its C-4 aryl group, showed the most potent rat intestinal sucrase inhibition. Docking studies showed similar hydrogen bonding modes for the iminosugar skeletons of DAB (1) and LAB (2) with ntMGAM,. While C-4 alkylated LAB derivatives showed high similarity in their binding modes with the active site of ntMGAM, binding modes of the DAB derivatives relied on the size of C-4 alkyl groups with methyl and butyl showed the optimum interactions. Furthermore, C-4 arylation improved the interactions of LAB derivatives with enzymes by T-shaped π-π stack with residue Trp-406; for C-4 arylated DAB derivatives, the π-π stack interactions were found with distinct planar distortions caused by EDGs or EWGs on the C-4 aryls. The results reported herein provided insights for the design and development of DAB and LAB related α-glucosidase inhibitors, and may also contribute to the future development of anti-viral, anti-diabetic and anti-Pompe disease drugs.

Total Synthesis of Pseudouridimycin.

Pseudouridimycin (1), a potent antibiotic against both Gram-positive and Gram-negative bacteria including multi-drug-resistant strains with a new mode of action isolated from Streptomyces sp., was synthesized by a convergent strategy from 5'-amino-pseudouridine 5 and N-hydroxy-dipeptide 26 in 23% total yield. The key intermediate 26 was synthesized by hydroxylaminolysis of the nitrone derived from glutamine and subsequent glycylation with glycine chloride. The synthetic method provides an efficient and practical way for the synthesis of N-hydroxylated peptidyl nucleoside.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease.

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

De Novo Synthesis of Orthogonally-Protected C2-Fluoro Digitoxoses and Cymaroses: Development and Application for the Synthesis of Fluorinated Digoxin.

Inspired by Roush's pioneering work on rare sugars, we have developed a scalable, stereoselective, de novo synthesis of orthogonally protected C2-fluoro digitoxose and cymarose, utilizing Sharpless kinetic resolution and organocatalytic fluorination as key steps. The utility of this strategy is demonstrated by the synthesis of a fluorinated analogue of digoxin, which indicates the fluorine on the sugar ring may have a significant impact on biological activity.

Polyglutamine-Specific Gold Nanoparticle Complex Alleviates Mutant Huntingtin-Induced Toxicity.

Huntington's disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs-JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its ß-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs-JLD1, we generated a complex (AuNPs-JLD1-PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs-JLD1-PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.

Stereocomplementary synthesis of casuarine and its 6-epi-, 7-epi-, and 6,7-diepi-stereoisomers.

Four diastereomers belonging to the family of casuarines, including casuarine (1), 6-epi-casuarine (2), 7-epi-casuarine (13) and 6,7-diepi-casuarine (14), have been synthesized from D-arabinose-derived cyclic nitrone 7 and nitrone-derived aldehyde 4 by a stereocomplementary strategy. Glycosidase inhibition comparison showed that 6-epi-casuarine (2) exhibits enhanced inhibition of trehalase (IC50 = 9.7 µM) and 6,7-diepi-casuarine (14) leads to specific inhibition of trehalase.

Synthesis and glycosidase inhibition of 5-C-alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives.

5-C-Alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives have been designed and synthesized efficiently from an l-sorbose-derived cyclic nitrone. The DNJ and l-ido-DNJ derivatives with C-5 alkyl chains ranging from methyl to dodecyl were assayed against various glycosidases to study the effect of chain length on enzyme inhibition. Glycosidase inhibition study of DNJ derivatives showed potent and selective inhibitions of α-glucosidase; DNJ derivatives with methyl, pentyl to octyl, undecyl and dodecyl as C-5 branched chains showed significantly improved rat intestinal maltase inhibition. In contrast, most 5-C-alkyl-l-ido-DNJ derivatives were weak or moderate inhibitors of the enzymes tested, with only three compounds found to be potent α-glucosidase inhibitors. Docking studies showed different interaction modes of 5-C-ethyl-DNJ and 5-C-octyl-DNJ with ntMGAM and also different binding modes of 5-C-alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives; the importance of the degree of accommodation of the C-5 substituent in the hydrophobic groove and pocket may account for the variation of glycosidase inhibition in the two series of derivatives. The results reported herein are helpful in the design and development of α-glucosidase inhibitors; this may lead to novel agents for the treatment of viral infection and type II diabetes.

Synthesis of Pyrrolidine Monocyclic Analogues of Pochonicine and Its Stereoisomers: Pursuit ofSimplified Structures and Potent ß-N-Acetylhexosaminidase Inhibition.

Ten pairs of pyrrolidine analogues of pochonicine and its stereoisomers have been synthesized from four enantiomeric pairs of polyhydroxylated cyclic nitrones. Among the ten N-acetylamino pyrrolidine analogues, only compounds with 2,5-dideoxy-2,5-imino-d-mannitol (DMDP) and pochonicine (1) configurations showed potent inhibition of ß-N-acetylhexosaminidases (ß-HexNAcases); while 1-amino analogues lost almost all their inhibitions towards the tested enzymes. The assay results reveal the importance of the N-acetylamino group and the possible right configurations of pyrrolidine ring required for this type of inhibitors.

Synthesis and glycosidase inhibition of N-substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM).

N-Substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM), the pyrrolidine core of swainsonine, have been synthesized efficiently and stereoselectively from d-mannose with 2,3:5,6-di-O-isopropylidene DIM (10) as a key intermediate. These N-substituted derivatives include N-alkylated, N-alkenylated, N-hydroxyalkylated and N-aralkylated DIMs with the carbon number of the alkyl chain ranging from one to nine. The obtained 33 N-substituted DIM derivatives were assayed against various glycosidases, which allowed a systematic evaluation of their glycosidase inhibition profiles. Though N-substitution of DIM decreased their α-mannosidase inhibitory activities, some of the derivatives showed significant inhibition of other glycosidases.

Synthesis and Glycosidase Inhibition of Broussonetine M and Its Analogues.

Cross-metathesis (CM) and Keck asymmetric allylation, which allows access to defined stereochemistry of a remote side chain hydroxyl group, are the key steps in a versatile synthesis of broussonetine M (3) from the d-arabinose-derived cyclic nitrone 14. By a similar strategy, ent-broussonetine M (ent-3) and six other stereoisomers have been synthesized, respectively, starting from l-arabino-nitrone (ent-14), l-lyxo-nitrone (ent-3-epi-14), and l-xylo-nitrone (2-epi-14) in five steps, in 26%-31% overall yield. The natural product broussonetine M (3) and 10'-epi-3 were potent inhibitors of ß-glucosidase (IC50 = 6.3 µM and 0.8 µM, respectively) and ß-galactosidase (IC50 = 2.3 µM and 0.2 µM, respectively); while their enantiomers, ent-3 and ent-10'-epi-3, were selective and potent inhibitors of rice α-glucosidase (IC50 = 1.2 µM and 1.3 µM, respectively) and rat intestinal maltase (IC50 = 0.29 µM and 18 µM, respectively). Both the configuration of the polyhydroxylated pyrrolidine ring and C-10' hydroxyl on the alkyl side chain affect the specificity and potency of glycosidase inhibition.