Synthesis of N-Glycans and Azasugars to Target Disease

<p>In this thesis two aspects of carbohydrate research will be discussed. First, the total synthesis of N-glycans found on allergens that are known to stimulate an allergic immune response and second, the synthesis of iminosugars in an attempt to extend the scope of the PGF-methodology. Asthma affects 235 million people worldwide, with New Zealand ranking among the highest in the world. Although there is a good understanding of how allergens trigger the immune system on a “macroscopic” level, how an allergen’s molecular structure causes such an allergic response remains unknown. Upon close review of carbohydrates present on the allergens that are known to give an allergic T helper (Th 2) immune response, a common structure has been identified. The structure consists of a complex type N-glycan made up of a pentasaccharide core (Man3(GlcNHAc)2), with additional 1,3-linked α-L-fucose and 1,2-linked D-xylose cappings. As part of a structure relationship study this heptasaccharide and structural derivatives thereof have been synthesised. The synthesis of these N-glycans will allow a more detailed study of the role of these defined structures in triggering an allergic immune response. The second part of this thesis focuses on the protecting group free (PGF) synthesis of iminosugars, which are potent glycosidase inhibitors and are currently used in the treatment of a variety of diseases. Synthetic strategies for the synthesis of iminosugars involve the use of protecting groups, which are necessary to block potential competing reactive centres within a molecule during the multistep synthesis. The disadvantage, however, is that the installation of protecting groups introduces additional steps to the total synthesis, which inevitably leads to lower yields and the generation of waste. Within our group, PGF methodologies have been developed which allow for the synthesis of a variety of iminosugars. The work presented in this thesis extends the scope of this methodology for the synthesis of an important class of iminosugars, the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines. For the purpose of introducing an additional hydroxymethyl group, a ketose starting material was required, and therefore an efficient Vasella/reductive amination reaction using ketoses was developed. Additionally, iodocyclisation and carbamate annulation of the intermediate alkenylamines provided successful entry to the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines, exemplified by the efficient 6-step synthesis of 2,5-dideoxy-2,5-imino-L-iditol and 2,5-dideoxy-2,5-imino-D-mannitol (DMDP).</p>

Synthesis of N-Glycans and Azasugars to Target Disease

<p>In this thesis two aspects of carbohydrate research will be discussed. First, the total synthesis of N-glycans found on allergens that are known to stimulate an allergic immune response and second, the synthesis of iminosugars in an attempt to extend the scope of the PGF-methodology. Asthma affects 235 million people worldwide, with New Zealand ranking among the highest in the world. Although there is a good understanding of how allergens trigger the immune system on a “macroscopic” level, how an allergen’s molecular structure causes such an allergic response remains unknown. Upon close review of carbohydrates present on the allergens that are known to give an allergic T helper (Th 2) immune response, a common structure has been identified. The structure consists of a complex type N-glycan made up of a pentasaccharide core (Man3(GlcNHAc)2), with additional 1,3-linked α-L-fucose and 1,2-linked D-xylose cappings. As part of a structure relationship study this heptasaccharide and structural derivatives thereof have been synthesised. The synthesis of these N-glycans will allow a more detailed study of the role of these defined structures in triggering an allergic immune response. The second part of this thesis focuses on the protecting group free (PGF) synthesis of iminosugars, which are potent glycosidase inhibitors and are currently used in the treatment of a variety of diseases. Synthetic strategies for the synthesis of iminosugars involve the use of protecting groups, which are necessary to block potential competing reactive centres within a molecule during the multistep synthesis. The disadvantage, however, is that the installation of protecting groups introduces additional steps to the total synthesis, which inevitably leads to lower yields and the generation of waste. Within our group, PGF methodologies have been developed which allow for the synthesis of a variety of iminosugars. The work presented in this thesis extends the scope of this methodology for the synthesis of an important class of iminosugars, the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines. For the purpose of introducing an additional hydroxymethyl group, a ketose starting material was required, and therefore an efficient Vasella/reductive amination reaction using ketoses was developed. Additionally, iodocyclisation and carbamate annulation of the intermediate alkenylamines provided successful entry to the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines, exemplified by the efficient 6-step synthesis of 2,5-dideoxy-2,5-imino-L-iditol and 2,5-dideoxy-2,5-imino-D-mannitol (DMDP).</p>

Methyl β-lactoside [methyl β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside] monohydrate: a solvomorphism study

Methyl β-lactoside [methyl β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside] monohydrate, C13H24O11·H2O, (I), was obtained via spontaneous transformation of methyl β-lactoside methanol solvate, (II), during air-drying. Cremer–Pople puckering parameters indicate that the β-D-Galp (β-D-galactopyranosyl) and β-D-Glcp (β-D-glucopyranosyl) rings in (I) adopt slightly distorted 4 C 1 chair conformations, with the former distorted towards a boat form (B C1,C4) and the latter towards a twist-boat form (O5 S C2). Puckering parameters for (I) and (II) indicate that the conformation of the βGalp ring is slightly more affected than the βGlcp ring by the solvomorphism. Conformations of the terminal O-glycosidic linkages in (I) and (II) are virtually identical, whereas those of the internal O-glycosidic linkage show torsion-angle changes of 6° in both C—O bonds. The exocyclic hydroxymethyl group in the βGalp residue adopts a gt conformation (C4′ anti to O6′) in both (I) and (II), whereas that in the βGlcp residue adopts a gg (gauche–gauche) conformation (H5 anti to O6) in (II) and a gt (gauche–trans) conformation (C4 anti to O6) in (I). The latter conformational change is critical to the solvomorphism in that it allows water to participate in three hydrogen bonds in (I) as opposed to only two hydrogen bonds in (II), potentially producing a more energetically stable structure for (I) than for (II). Visual inspection of the crystalline lattice of (II) reveals channels in which methanol solvent resides and through which solvent might exchange during solvomorphism. These channels are less apparent in the crystalline lattice of (I).

Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition

Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l–1 glucose–xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.

Inhibition of Aminoglycoside 6′-N-acetyltransferase Type Ib (AAC(6′)-Ib): Structure–Activity Relationship of Substituted Pyrrolidine Pentamine Derivatives as Inhibitors

The aminoglycoside 6′-N-acetyltransferase type Ib (AAC(6′)-Ib) is a common cause of resistance to amikacin and other aminoglycosides in Gram-negatives. Utilization of mixture-based combinatorial libraries and application of the positional scanning strategy identified an inhibitor of AAC(6′)-Ib. This inhibitor’s chemical structure consists of a pyrrolidine pentamine scaffold substituted at four locations (R1, R3, R4, and R5). The substituents are two S-phenyl groups (R1 and R4), an S-hydroxymethyl group (R3), and a 3-phenylbutyl group (R5). Another location, R2, does not have a substitution, but it is named because its stereochemistry was modified in some compounds utilized in this study. Structure–activity relationship (SAR) analysis using derivatives with different functionalities, modified stereochemistry, and truncations was carried out by assessing the effect of the addition of each compound at 8 µM to 16 µg/mL amikacin-containing media and performing checkerboard assays varying the concentrations of the inhibitor analogs and the antibiotic. The results show that: (1) the aromatic functionalities at R1 and R4 are essential, but the stereochemistry is essential only at R4; (2) the stereochemical conformation at R2 is critical; (3) the hydroxyl moiety at R3 as well as stereoconformation are required for full inhibitory activity; (4) the phenyl functionality at R5 is not essential and can be replaced by aliphatic groups; (5) the location of the phenyl group on the butyl carbon chain at R5 is not essential; (6) the length of the aliphatic chain at R5 is not critical; and (7) all truncations of the scaffold resulted in inactive compounds. Molecular docking revealed that all compounds preferentially bind to the kanamycin C binding cavity, and binding affinity correlates with the experimental data for most of the compounds evaluated. The SAR results in this study will serve as the basis for the design of new analogs in an effort to improve their ability to induce phenotypic conversion to susceptibility in amikacin-resistant pathogens.

1-hydroxymethyl Harmine-TGFβSF Inhibitor: Inovasi Terapi Diabetes Melitus Terbaru Melalui Inisiasi Proses Regenerasi Sel β Pankreas pada Penderita DM Tipe 1 dan 2

Background: Type 1 and 2 diabetes mellitus (DM) is a chronic metabolic disease most commonly affects millions of people worldwide. Despite the differences in pathogenesis, both share one thing in common - that is the drastic depletion in the number of pancreatic β cells. Unfortunately, physiological proliferation of β cells has come to a halt starting from the first year of neonatal. To overcome this problem, researchers have been searching for molecules with the ability to induce β cells proliferation. Upon extensive screening, only harmine was proven to be the most potent β cells proliferation inducer. Furthermore, combination of harmine with TGFβSF inhibitor was found to boost harmine’s effectivity even more. Another development was also made to improve harmine’s selectivity by incorporating 1-hydroxymethyl group. Objective: Evaluate the potency of 1-hydroxymethyl harmine-TGFβSF inhibitor as a novel therapy for DM. Method: A systematic literature study was conducted with the database from Pubmed, Google Scholar, ScienceDirect, and Proquest for articles published within 2015-2019. Discussion: This literature review yields result that harmine-TGFβSF inhibitor is proven to induce β cells proliferation up to 18%/day or equal to 18 times the normal cell proliferation rate during embryogenesis. Moreover, incorporating 1-hydroxymethyl group into harmine is proven not only to improve selectivity but also lessen the toxicity, making 1-hydroxymethyl harmine safe as a novel therapy for diabetes. Conclusion: 1-hydroxymethyl harmine-TGFβSF inhibitor display promising potential as a novel therapy for all type of diabetes patients. Keywords: diabetes mellitus, harmine, TGFβSF inhibitor, β cell proliferation Latar Belakang: Diabetes Melitus (DM) tipe 1 maupun tipe 2 merupakan penyakit metabolik kronis yang paling banyak ditemukan di seluruh dunia. Walaupun memiliki proses patogenesis yang berbeda, namun kedua tipe DM ini ternyata memiliki kesamaan, yaitu terjadinya penurunan kuantitas sel β pankreas. Sayangnya, kemampuan regenerasi sel β pankreas manusia telah terhenti semenjak tahun pertama masa neonatal. Untuk menangani permasalahan tersebut, para peneliti menemukan sebuah molekul bernama harmine yang terbukti efektif menginisiasi proses regenerasi sel β pankreas. Selanjutnya, untuk meningkatkan efektifitas dari harmine agar lebih baik lagi, peneliti kemudian mengkombinasikan harmine dengan TGFβSF inhibitor. Sedangkan, untuk meningkatkan selektivitas dari harmine, peneliti menambahkan gugus 1-hidroksimetil pada molekul tersebut. Tujuan: Evaluasi potensi 1-hydroxymethyl harmine-TGFβSF inhibitor sebagai terapi utama bagi semua penderita DM. Metode: Penelitian dilakukan dengan melakukan tinjauan pustaka dari beberapa database jurnal, yakni PubMed, Google Scholar, ScienceDirect dan ProQuest dengan kriteria literatur dipublikasikan dalam kurun waktu 2015-2019. Pembahasan: Studi literatur ini menunjukan bahwa harmine-TGFβSF inhibitor telah terbukti mampu meningkatkan proliferasi sel β pankreas manusia hingga mencapai 18%/hari atau setara dengan 18 kali kecepatan embriogenesis pada sel normal. Selain itu, penambahan gugus 1-hidroksimetil pada harmine juga telah terbukti tidak hanya mampu meningkatkan selektivitas dari molekul tersebut, tetapi juga mampu menurunkan efek toksisitasnya, sehingga aman digunakan sebagai terapi anti-diabetes terbaru. Kesimpulan: 1-hydroxymethyl harmine-TGFβSF inhibitor memiliki potensi yang menjanjikan untuk menjadi terapi baru bagi semua tipe penderita DM. Kata Kunci: diabetes mellitus, harmine, proliferasi sel β, TGFβSF inhibitor

Studies on the Enantioselective Synthesis of E-Ethylidene-bearing Spiro[indolizidine-1,3′-oxindole] Alkaloids

A synthetic route for the enantioselective construction of the tetracyclic spiro[indolizidine-1,3′-oxindole] framework present in a large number of oxindole alkaloids, with a cis H-3/H-15 stereochemistry, a functionalized two-carbon substituent at C-15, and an E-ethylidene substituent at C-20, is reported. The key steps of the synthesis are the generation of the tetracyclic spirooxindole ring system by stereoselective spirocyclization from a tryptophanol-derived oxazolopiperidone lactam, the removal of the hydroxymethyl group, and the stereoselective introduction of the E-ethylidene substituent by acetylation at the α-position of the lactam carbonyl, followed by hydride reduction and elimination. Following this route, the 21-oxo derivative of the enantiomer of the alkaloid 7(S)-geissoschizol oxindole has been prepared.

Development of highly sensitive fluorescent sensor and fluorescent sensor-doped polymer films for trace amounts of water based on photo-induced electron transfer

Anthracene-(aminomethyl)phenylboronic acid pinacol ester (AminoMePhenylBPin) SM-1 having a cyano group as an electron-withdrawing substituent and a hydroxymethyl group has been developed as a highly sensitive PET (photo-induced electron transfer)-type fluorescent...

Visualization of Penetration and Reaction of Aldehyde Tanning Agent in Leather using Fluorescence Technique

Penetration and reaction of tanning agents in leather greatly influence tanning performance and leather quality. Aldehyde tanning agents are widely used in leather manufacture, but their penetration and reaction in leather cannot be detected easily. Considering the fact that aldehyde can form a fluorescent Schiff base with amino groups of collagen, we visualized the distribution and reactivity of typical aldehyde tanning agents in leather during tanning process using a fluorescence microscope. The results showed that glutaraldehyde in leather was easily observed under the fluorescein isothiocyanate (FITC) filter system. The fluorescence intensity of pelt/leather gradually increased with penetrating of glutaraldehyde, basifying and heating, which was consistent with the formation of Schiff base and the increase in Ts of pelt/leather. Oxidized sodium alginate and dialdehyde sodium carboxymethyl cellulose in leathers were also detected using a fluorescence microscope because the aldehyde group of the two tanning agents formed a fluorescent Schiff base with amino groups of collagen. The oxazolidine tanned leather and the tetrakis hydroxymethyl phosphonium tanned leather emitted little fluorescence because they crosslinked collagen with hydroxymethyl group. These results indicate the fluorescent technique is useful to visualize the penetration and reaction of aldehyde tanning agents that crosslink collagen with aldehyde groups.