A streamlined workflow for twoplexing of N-linked glycan analysis using light (12C6) and heavy (13C6) isotopologues of 3-aminobenzenesulfonic acid.

Comparative glycosylation analysis of biopharmaceuticals requires the development of methods that deliver the necessary throughput, support structural elucidation and relative quantitation of glycans released from therapeutics. The current study presents the development and applicability assessment of a twoplex approach using light and heavy isotopolouges of 3-aminobenzenesulfonic acid (3-ASA) under wet labeling conditions followed by UHPLC-MS analysis in data dependent acquisition mode. Excellent labelling efficiency, >90%, was achieved for both the light and heavy variants of the reagent. Glycan distributions of two human IgG lots labeled by light and heavy isotopolouges were identical, demonstrating no labeling bias introduced by either of the isotopologues. Peak area distributions of glycan profiles of two human IgG lots were compared to 2-aminobenzamide (2-AB) and RapiFluor-MS protocols. The comparison led to identical results in peak area distribution across the three dyes, but differences in chromatographic selectivity attributed to the different tags. MS1 based relative quantitation was further validated by releasing glycans from the same lot of human IgG, with glycan pools obtained labeled with light and heavy isotopologues separately, followed by mixing and clean-up of the same amount of light and heavy labeled glycan pools. MS analyses of each glycan resulted in a ratio of light and heavy XIC in the range of 0.97 ≤ x ≤ 1.05, demonstrating the method is amenable for the relative quantitation of glycans. Excellent correlation between the relative quantitation data of N-glycans from two human IgG N-glycan pools using the twoplex approach and ratios from peak area distribution calculated from the fluorescent chromatogram was observed (r = 0.986), further corroborating the reliability of the method and its potential applicability in the biopharmaceutical industry. Highly informative HCD-MS2 spectra dominated mostly by Y- and Z-type single and double glycosidic fragment ions facilitate structural interpretation of the oligosaccharides.

Preparation and Evaluation of Potent Pentafluorosulfanyl-Substituted Anti-Tuberculosis Compounds.

The global fight to stop tuberculosis (TB) remains a great challenge, particularly with the increase in drug-resistant strains and a lack of funding to support the development of new treatments. To bolster a precarious drug pipeline, we prepared a focused panel of eight pentafluorosulfanyl (SF5 ) compounds which were screened for their activity against Mycobacterium tuberculosis (Mtb) H37Rv in three different assay conditions and media. All eight compounds had sub-micromolar potency, and four displayed MICs <100 nm. Seven compounds were evaluated against non-replicating and mono-drug-resistant Mtb, and for their ability to inhibit Mtb within the macrophage. The greatest potency was observed against intracellular Mtb (MIC <10 nm for three compounds), which is often the most challenging to target. In general, the SF5 -bearing compounds were very similar to their CF3 counterparts, with the major differences observed being their in vitro ADME properties. Two SF5 -bearing compounds were found to have greater protein binding than their corresponding CF3 counterparts, but were also less metabolized in human microsomes, resulting in longer half-lives.

Photochromism emergence in N-salicylidene p-aminobenzenesulfonate diallylammonium salts.

N-Salicylidene p-aminobenzenesulfonate salts were prepared by in situ condensation of p-aminobenzenesulfonate diallylammonium salt and salicylaldehyde. Modulation of thermo- and photochromism was achieved by varying the alkyl chain length of the diallylammonium counter-cation. A structural-optical properties investigation reveals that both crystal packing and dihedral angle between aromatic rings of the N-salicylidene aniline switch are not sufficient to predict the occurrence of photochromism in the solid state. The available free space around the N-salicylidene p-aminobenzenesulfonate, in addition to the flexibility of the nearby environment, is shown to be of major importance for the cis→trans isomerisation to occur as well as for the stabilisation of the trans-keto form. Emergence of photochromic properties was determined from the diallylhexylammonium cation within the series of investigated counter-cations. High stability is observed for the trans-keto form of one polymorph of N-salicylidene p-aminobenzenesulfonate diallylhexylammonium salt (k = 2.4×10(-7)  s(-1)).

Mono and dinuclear phosphinegold(I) sulfanylcarboxylates: influence of nuclearity and substitution of PPh3 for PEt3 on cytotoxicity.

Gold complexes of the type [Au(PEt3)(Hxspa)] were prepared by reacting triethylphosphinegold(I) chloride in ethanol/water (8:1) with the 3-(aryl)-2-sulfanylpropenoic acids H2xspa [x=p=3-phenyl-; f=3-(2-furyl)-; t=3-(2-thienyl)-; py=3-(2-pyridyl); Clp=3-(2-Chlorophenyl)-; -o-mp=3-(2-methoxyphenyl)-; -p-mp=3-(4-methoxyphenyl)-; -o-hp=3-(2-hydroxyphenyl)-; -p-hp=3-(4-hydroxyphenyl)-; -diBr-o-hp=3-(3,5-dibromo-2-hidroxyphenyl-); spa=2-sulfanylpropenoato] or 2-cyclopentylidene-2-sulfanylacetic acid (H2cpa) and KOH in a 1:1:1 mole ratio. The compounds were characterized by IR spectroscopy and FAB mass spectrometry and by (1)H, (13)C and (31)P NMR spectroscopy. The in vitro antitumor activity of these and of the previously described dinuclear [(AuPEt3)2(xspa)] complexes against the HeLa-229, A2780 and A2780cis cell lines was determined and compared with those of the analogous PPh3 complexes. The results show that the substitution of the PPh3 ligand by PEt3 is particularly effective in increasing the cytotoxicity of the dinuclear [(AuPR3)2(xspa)] complexes, giving rise to compounds that are significantly more active than cisplatin against the aforementioned cell lines. In addition, and as a preliminary test for nephrotoxicity, the cytotoxicity of the most active compounds against the normal renal LCC-PK1 cell line was evaluated and compared with that of cisplatin.

Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

Quantitative analysis of the effects of photoswitchable distance constraints on the structure of a globular protein.

Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attachment sites. Here we investigate the effects of varying cross-linker structure on the photocontrol of folding of the Fyn SH3 domain, a well-studied model protein. We develop a theoretical framework based on an explicit-chain model of protein folding, modified to include detailed model linkers, that allows prediction of the effect of a given linker on the free energy of folding of a protein. Using this framework, we were able to quantitatively explain the experimental result that a longer, but somewhat flexible, cross-linker is less destabilizing to the folded state than a shorter more rigid cross-linker. The models also suggest how misfolded states may be generated by cross-linking, providing a rationale for altered dynamics seen in nuclear magnetic resonance analyses of these proteins. The theoretical framework is readily portable to any protein of known folded state structure and thus can be used to guide the design of photoswitchable proteins generally.

Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse.

The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease.

Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.

Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where Cl > Br > I compared to the 4',5'-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

Synthesis of N-o-methylphenyl-N'-(sodium p-aminobenzene-sulfonate) thiourea and its chromogenic reaction with palladium(II).

A new chromogenic reagent, N-o-methylphenyl-N'-(sodium p-aminobenzenesulfonate)thiourea (MSAT), has been synthesized and characterized by elemental analysis, (1)H-NMR, FT-IR and UV-Vis spectra. Based on the absorption spectrum of the colored complex of MSAT with palladium(II), a novel spectrophotometric method for the determination of palladium has been developed. In a pH 4.0 - 5.5 HAc-NaAc buffer solution, palladium(II) reacted with MSAT to form a stable yellow water-soluble complex with an apparent molar absorptivity of epsilon = 2.04 x 10(5) L mol(-1) cm(-1) at the maximum absorption of 318.0 nm. Beer's law was obeyed in the concentration range of 1.2 - 11.8 microg per 25 mL for palladium(II) with a correlation coefficient of 0.9997. The probable interfering ions and their tolerable limits have also been investigated in detail. The proposed method is simple, rapid, and sensitive, and has been applied to the determination of palladium in anode mud and ore samples with satisfactory results.

Antimycobacterial and antifungal isosters of salicylamides.

A set of 40 derivatives of 3-hydroxypicolinic acid and 2-sulfanylbenzoic acid, isosteric to salicylanilides was synthesized. The compounds were evaluated for in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium, Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Trichosporon beigelii, Aspergillus fumigatus, Absidia corymbifera, Trichophyton mentagrophytes and Microsporum gypseum. Structure-activity relationships of antimycobacterial activity and antifungal activity against T. mentagrophytes and M. gypseum were analyzed by the Free-Wilson method. An increase in antimycobacterial activity was observed only for the sulfanylbenzoic acid derivatives, especially those with the benzyl moiety. The antifungal activity was not significant.

Antileishmanial dinitroaniline sulfonamides with activity against parasite tubulin.

Novel dinitroaniline sulfonamides based on the herbicide oryzalin 3 were synthesized and evaluated for activity against the parasitic protozoan Leishmania donovani and against leishmanial tubulin, the putative antiparasitic target of oryzalin. A subset of these compounds possess more activity against both Leishmania and the target protein in vitro. Compound 20 displays improved potency against leishmanial tubulin and is 13.4-fold more active against L. donovani axenic amastigotes than oryzalin.

Suramin analogues with a 2-phenylbenzimidazole moiety as partial structure; potential anti HIV- and angiostatic drugs, 2: Sulfanilic acid-, benzenedisulfonic acid-, and naphthalenetrisulfonic acid analogues.

The synthesis of suramin analogues bearing a 2-phenyl-benzimidazole moiety is described. Aminoarene sulfonic acids 2a-e are acylated with 3,4-dinitrobenzoyl chloride 3 yielding the amides 4a-e which are hydrogenated to the corresponding diamines 5a-e. These are treated with 3-nitrobenzaldehyde, yielding the azomethines 7a-e and their isomers 8a-e and 9a-e. Key step in the synthesis of the target compounds 12a-e is the oxidation of the azomethines with oxygen to the benzimidazoles 10a-e. These are hydrogenated to the amines 11a-e reacting with phosgene to yield the symmetric ureas 12a-e. Results of the anti-HIV, cytostatic, and antiangiogenic screening are presented.

Biopharmaceutical evaluation of salicylazosulfanilic acid as a novel colon-targeted prodrug of 5-aminosalicylic acid.

A prodrug of 5-aminosalicylic acid (5-ASA), salicylazosulfanilic acid (SASA), which consists of sulfanilic acid linked to 5-ASA through an azo-linkage was newly synthesized. Biopharmaceutical properties of SASA were evaluated in comparison with those of salicylazosulfapyridine (SASP) in rats. Since SASA is much more hydrophilic than SASP, the absorption of SASA from the small intestine was less in comparison with SASP. When SASA and SASP were incubated with the rat intestinal contents under anaerobic conditions, both compounds were stable in the small-intestinal contents, but were rapidly degraded to 5-ASA in the cecal and the colonic contents. The degradation to 5-ASA by the large-intestinal contents was suppressed by the pretreatment with kanamycin sulfate, suggesting that the bioconversion of SASA is mediated by the intestinal microflora similarly to that of SASP and that SASA is also a prodrug of 5-ASA. After the oral administration, 5-ASA was found neither in the stomach nor in the small intestine in case of both prodrugs. Most of the prodrugs were transferred to the lower intestine where they were degraded to 5-ASA. The recovery of SASA including the metabolites from the gastrointestinal tract at four hours after the oral administration was significantly greater than that of SASP. Accordingly, SASA is free from the liberation of sulfapyridine, the adverse effect moiety of SASP, and less absorbable in the small intestine. Thus, the beneficial characteristics of SASA as an excellent colon-targeted prodrug of 5-ASA were clarified.

Portage transport of sulfanilamide and sulfanilic acid.

Sulfanilic acid, in contrast to sulfanilamide, has poor in vitro antibacterial activity. Paradoxically, it has been shown to be a more effective inhibitor than sulfanilamide of dihydropteroic acid synthase. In order to circumvent the presumed permeability barrier to sulfanilic acid, advantage was taken of the technique of portage transport. Derivatives of the compound were prepared in which it was linked via its primary amino group to the alpha-carbon of glycine residues in di- and tripeptides. L-Alanyl-L-alanyl-L-2-[(4-sulfophenyl)amino]glycine proved to be 207 times more potent than sulfanilic acid and 8 times more active than either sulfanilamide or L-alanyl-L-alanyl-L-2-[[4-(aminosulfonyl)-phenyl]amino]glycine when tested against Escherichia coli. These findings confirm that the weak in vitro activity of sulfanilic acid is due to its limited ability to penetrate the bacterial membrane. They also emphasize the ability of portage transport to reveal therapeutic capability that had been attenuated by poor drug permeation.