Acid-base catalyzed glycosyl donor and then glycosyl acceptor activation with phenylboron difluoride or diphenylboron fluoride permits hydrogen bond mediated intramolecular S(N)2-type glycosidation in generally high anomeric selectivity.

SummaryMalaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double-stranded RNA of cysteine protease genes ( falcipain -1 and -2) in the malaria parasite, Plasmodium falciparum . Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain doublestranded (ds)RNAs are reminiscent of the effects observed upon administering E-64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs ≈ ≈ ≈ ≈ 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence-specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding of RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95Å resolution.Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding. KeywordsRRM; RNA binding domain; Pre-mRNA splicing; mRNA translation; TIA-1; TIAR Post-transcriptional mechanisms for regulating gene expression depend on numerous coordinated interactions among RNA sequences and trans-acting protein factors. The RNA binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions as a posttranscriptional regulator of gene expression by recognizing uridine-rich RNA sites. In the nucleus, TIA-1 regulates alternative splicing of pre-mRNAs, including those encoding fibroblast growth factor receptor-2 (fgfr-2), type II procollagen (col2a1), the cystic fibrosis transmembrane conductance regulator (cftr), and the pro-apoptotic Fas receptor (fas), among others [1;2;3;4;5;6]. In the cytoplasm, TIA-1 regulates mRNA translation [7;8;9;10], and suppresses mRNA translation during environmental stress (reviewed in [11]). The functional importance of TIA-1 is shown by its requirement for viability of DT40 cells [12], and the high rates of embryonic lethality among mice lacking TIA-1 [8;13].*Correspondence E-mail: clara_kielkopf@urmc.rochester.edu; Fax: 585-275-6007. ‡ These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and Methods Protein purificationThe human TIA-1-RRM2 fragment (residues 94-175) was expressed using the pGEX-6p vector (GE Healthcare). Glutathione-S-transferase (GST) fusion proteins were purified by glutathione affinity, and then the TIA-1 fragment was further purified by cation exchange chromatography following removal of the GST tag....

In this study o‐ and m‐xylylene moieties in combination with a triazolylmethyl moiety have been successfully employed as a relatively rigid spacer system in intramolecular glycosylation reactions. Phenyl 3,4,6‐tri‐O‐benzyl‐2‐O‐propargyl‐1‐thio‐D‐glucopyranoside was employed as a donor, which could be readily connected by 1,3‐dipolar cycloaddition (click reaction) to O‐(2‐ or 3‐azidomethylbenzyl)‐protected acceptors to afford, after liberation of the accepting hydroxy groups, the desired donor–spacer–acceptor‐linked intermediates. NIS/TMSOTf‐promoted glycosylation furnished disaccharide‐containing macrocycles. In general, very good results were obtained. The anomeric selectivity is dependent on various factors, the ring size seeming crucial.

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