Exploring the Effect of Conjugation Site and Chemistry on the Immunogenicity of an anti-Group B Streptococcus Glycoconjugate Vaccine Based on GBS67 Pilus Protein and Type V Polysaccharide.

We have recently described a method for tyrosine-ligation of complex glycans that was proven efficient for the site selective coupling of GBS capsular polysaccharides (PSs). Herein, we explored the effect of conjugation of type V polysaccharide onto predetermined lysine or tyrosine residues of the GBS67 pilus protein with the dual role of T-cell carrier for the PS and antigen. For the preparation of a conjugate at predetermined lysine residues of the protein, we investigated a two-step procedure based on microbial Transglutaminase (mTGase) catalyzed insertion of a tag bearing an azide for following copper-free strain-promoted azide-alkyne [3 + 2] cycloaddition (SPAAC) with the polysaccharide. Two glycoconjugates were obtained by tyrosine-ligation through the known SPAAC and a novel thiol-maleimide addition based approach. Controls were prepared by random conjugation of PSV to GBS67 and CRM197, a carrier protein present in many commercial vaccines. Immunological evaluation in mice showed that all the site-directed constructs were able to induce good levels of anti-polysaccharide and anti-protein antibodies inducing osponophagocytic killing of strains expressing individually PSV or GBS67. GBS67 randomly conjugated to PSV showed carrier properties similar to CRM197. Among the tested site-directed conjugates, tyrosine-directed ligation and thiol-malemide addition was elected as the best combination to ensure production of anti-polysaccharide and anti-protein functional antibodies (in vitro opsonophagocytic killing titers) comparable to the controls made by random conjugation, while avoiding anti-linker antibodies. Our findings demonstrate that (i) mTGase based conjugation at lysine residues is an alternative approach for the synthesis of large capsular polysaccharide-protein conjugates; (ii) GBS67 can be used with the dual role of antigen and carrier for PSV; and (iii) thiol-maleimide addition in combination with tyrosine-ligation ensures the production of anti-polysaccharide and anti-protein functional antibodies while maintaining low levels of anti-linker antibodies. Site-specific conjugation methods aid in defining conjugation site and chemistry in carbohydrate-protein conjugates.

Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines.

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.

Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis.

Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drugs vinblastine and vincristine, bisindole alkaloids derived from the dimerization of the terpenoid indole alkaloids vindoline and catharanthine. Full elucidation of the biosynthetic pathways of these compounds is a prerequisite for metabolic engineering efforts that will improve production of these costly molecules. However, despite the medical and commercial importance of these natural products, the biosynthetic pathways remain poorly understood. Here we report the identification and characterization of a C. roseus cDNA encoding an S-adenosyl-L-methionine-dependent N methyltransferase that catalyzes a nitrogen methylation involved in vindoline biosynthesis. Recombinant enzyme produced in Escherichia coli is highly substrate specific, displaying a strict requirement for a 2,3-dihydro bond in the aspidosperma skeleton. The corresponding gene transcript is induced in methyl jasmonate-elicited seedlings, along with the other known vindoline biosynthetic transcripts. Intriguingly, this unique N methyltransferase is most similar at the amino acid level to the plastidic γ-tocopherol C methyltransferases of vitamin E biosynthesis, suggesting an evolutionary link between these two functionally disparate methyltransferases.

Biocatalytic asymmetric formation of tetrahydro-ß-carbolines.

Strictosidine synthase triggers the formation of strictosidine from tryptamine and secologanin, thereby generating a carbon-carbon bond and a new stereogenic center. Strictosidine contains a tetrahydro-ß-carboline moiety - an important N-heterocyclic framework found in a range of natural products and synthetic pharmaceuticals. Stereoselective methods to produce tetrahydro-ß-carboline enantiomers are greatly valued. We report that strictosidine synthase from Ophiorrhiza pumila utilizes a range of simple achiral aldehydes and substituted tryptamines to form highly enantioenriched (ee >98%) tetrahydro-ß-carbolines via a Pictet-Spengler reaction. This is the first example of aldehyde substrate promiscuity in the strictosidine synthase family of enzymes and represents a first step towards developing a general biocatalytic strategy to access chiral tetrahydro-ß-carbolines.

'Novel alkyl side chain sulfone 1alpha,25-dihydroxyvitamin D3 analogs: a comparison of in vitro antiproliferative activities and in vivo calcemic activities'.

The replacement of a t-butyl group with a trifluoromethyl group has profound effects on the biological profile of 1alpha,25-dihydroxyvitamin D(3) sulfone analogs. Investigation of whether the improved biological activities are due to steric and electronic factors of the trifluoromethyl group led to the design, synthesis and biological evaluation of two analogous alkyl sulfone molecules, methyl sulfone (AU-16-ene-25-SO(2)-CH(3)) and isopropyl sulfone (AU-16-ene-25-SO(2)-i-Pr). These alkyl sulfones are sterically comparable to, but electronically very different from a trifluoromethyl group. The syntheses, antiproliferative activities and calcemic activities of these new alkyl sulfones are presented herein. In comparing the in vitro antiproliferative profiles of the new alkyl sulfone 1alpha,25-dihydroxyvitamin D(3) analogs with the trifluoromethylsulfone and an analogous t-butyl sulfone, the activities increase in the following order: CH(3) < t-Bu approximately = i-Pr < CF(3). In contrast to the calcemic t-butyl sulfone, the novel alkyl sulfones and trifluoromethyl sulfone display desirable low calcemic levels.

Malaria-infected mice are cured by oral administration of new artemisinin derivatives.

In four or five chemical steps from the 1,2,4-trioxane artemisinin, a new series of 23 trioxane dimers has been prepared. Eleven of these new trioxane dimers cure malaria-infected mice via oral dosing at 3 x 30 mg/kg. The clinically used trioxane drug sodium artesunate prolonged mouse average survival to 7.2 days with this oral dose regimen. In comparison, animals receiving no drug die typically on day 6-7 postinfection. At only 3 x 10 mg/kg oral dosing, seven dimers prolong the lifetime of malaria-infected mice to days 14-17, more than double the chemotherapeutic effect of sodium artesunate. Ten new trioxane dimers at only a single oral dose of 30 mg/kg prolong mouse average survival to days 8.7-13.7, and this effect is comparable to that of the fully synthetic trioxolane drug development candidate OZ277, which is in phase II clinical trials.

Potent, low-calcemic, selective inhibitors of CYP24 hydroxylase: 24-sulfone analogs of the hormone 1alpha,25-dihydroxyvitamin D3.

The new 24-phenylsulfone 4a, a low-calcemic analog of the natural hormone 1alpha,25-dihydroxyvitamin D(3), is a potent (IC(50) = 28nM) and highly selective inhibitor of the human 24-hydroxylase enzyme CYP24.

Mechanistic advances in plant natural product enzymes.

The biosynthetic pathways of plant natural products offer an abundance of knowledge to scientists in many fields. Synthetic chemists can be inspired by the synthetic strategies that nature uses to construct these compounds. Chemical and biological engineers are working to reprogram these biosynthetic pathways to more efficiently produce valuable products. Finally, biochemists and enzymologists are interested in the detailed mechanisms of the complex transformations involved in the construction of these natural products. Study of biosynthetic enzymes and pathways therefore has a wide-ranging impact. In recent years, many plant biosynthetic pathways have been characterized, particularly the pathways that are responsible for alkaloid biosynthesis. Here we highlight recently studied alkaloid biosynthetic enzymes that catalyze production of numerous complex medicinal compounds, as well as the specifier proteins in glucosinosolate biosynthesis, whose structure and mechanism of action are just beginning to be unraveled.

Unexpected steric effects of "remote" alkyl groups on the rate of conjugate additions to alkyl alpha,beta-ethylenic sulfones, sulfoxides, and esters.

Examination of conjugated ethylenic sulfones, sulfoxides, and esters in Michael-type addition reactions reveals, for the first time, that the size of the heteroatom-attached alkyl group affects the rate of conjugate addition. Molecular modeling strongly suggests that what are generally considered to be "remote" alkyl groups in -CbetaH=CalphaHS(O)n-alkyl systems and -CH2CbetaH=CalphaHCOO-alkyl systems are actually not remote from the beta-carbon atom of the Michael accepting unit. Molecular modeling clearly shows that the alkyl groups in these Michael acceptors shield the beta-carbons in the following order: Eti-Pr>t-Bu.

Antiproliferative, low-calcemic, fluorinated sulfone analogs of 1alpha,25-dihydroxyvitamin D3: chemical synthesis and biological evaluation.

Novel fluorinated sulfone analogs of the hormone 1alpha,25-dihydroxyvitamin D(3) have been designed and synthesized in order to study the biological effects of fluorine incorporation at the terminus of the C,D-ring side chain. Although biologically active 26,27-hexafluorinated 1alpha,25-dihydroxyvitamin D(3) analogs have been synthesized previously, this investigation reports the first successful fluorinated series in which trifluoromethyl sulfone analogs present a favorable biological profile. This study shows that two new analogs featuring incorporation of a synthetically simple single trifluoromethyl sulfone group have significantly increased antiproliferative activity while causing desirably low in vivo calciuria relative to that of calcitriol. Incorporation of additional fluorines, as in a perfluorobutyl analog, results in a loss of antiproliferative activity.