Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

The main proteinase (M(pro)) of the severe acute respiratory syndrome (SARS) coronavirus is a principal target for the design of anticoronaviral compounds. Benzotriazole esters have been reported as potent nonpeptidic inhibitors of the enzyme, but their exact mechanism of action remains unclear. Here we present crystal structures of SARS-CoV M(pro), the active-site cysteine of which has been acylated by benzotriazole esters that act as suicide inhibitors. In one of the structures, the thioester product has been hydrolyzed and benzoic acid is observed to bind to the hydrophobic S2 pocket. This structure also features the enzyme with a shortened N-terminal segment ("amputated N finger"). The results further the understanding of the important role of the N finger for catalysis as well as the design of benzotriazole inhibitors with improved specificity.

The synthesis of heterocyclic α‐mercapto acids starting from (RS)‐thiomalic acid using hexafluoroacetone as protecting and activating agent is described. The new compounds are useful building blocks for peptide and depsipeptide modification as well as for drug design.

H e x a f l u o r o a c e t o n e a s P r o t e c t i n g a n d A c t i v a t i n g R e a g e n t Abstract: Methodology for the site-selective functionalization of a-amino alkanedioic acids (Asp, Glu and homologues) using hexafluoroacetone as protecting and activating reagent is described. Via new types of dielectrophiles, alternative approaches to multifunctional non-natural amino acids and some of their conjugates become readily available.Site-selective derivatizations of multifunctional compounds like w-carboxy-a-amino acids (Asp, Glu and homologues) require sophisticated protection/activation concepts. 1,2 Even relatively simple derivatives of aspartic acid like the sweetener aspartame 3 (a-functionalization) and isopeptides 4 (w-functionalization) demand laborious multi-step procedures. Therefore, the development of new methodology is a challenge, where activation of the a-carboxylic group and protection of the adjacent amino group as well as peptide bond formation and deprotection of the amino group are performed as tandem reactions. 5 This challenge can be met by using bidentate protective groups like phosgene, 6 and certain aldehydes and ketones. 7-9 However, on aminolysis of Leuchs anhydrides, formation of oligomers cannot be avoided. During aminolysis of the cyclic anhydride, the amino group of the newly formed dipeptide derivatives are so quickly deblocked, that they compete successfully with the amino acid ester as acyl acceptors. Furthermore, oxazolidinones derived from certain aldehydes like formaldehyde 10 require additional Nprotection and N-deprotection steps.Recently, hexafluoroacetone (HFA) has been successfully applied for simultaneous protection and activation of aamino-, 11 a-hydroxy-, 12 and a-mercapto acids 13 without the need of additional protection and deprotection steps. On reaction of aspartic, malic, and thiomalic acid with HFA the a-functionality and the adjacent carboxy group are simultaneously protected. Concomitantly, the a-carboxy group is activated, while the w-carboxy group remains unaffected and can be derivatized selectively after separate activation. Remarkably, orthogonal protection and w-activation can be achieved in only two steps.We now report on the application of HFA as protecting and activating reagent for w-derivatization of a-amino alkanedioic acids of different chain lengths (four to eight carbon atoms). HFA reacts with a-amino alkanedioic acids 1 in DMSO at room temperature to give 2,2-bis(trifluoromethyl)-1,3-oxazolidin-5-ones (2) in 67-90% yield. A second equivalent of HFA is necessary to trap the water, which is eliminated during lactone formation. 1 H and 19 F NMR control of the progress of the reaction reveals that the five-membered lactone is formed exclusively, but there is some loss during work up, because of the water solubility of compounds 2. HFA hydrate is the only by-product formed. The extraction process with the biphase system CH 2 Cl 2 -H 2 O to remove the hydrate can be avoided when DMF is used as a solvent. For work-up, DMF is distilled off in v...

The SARS coronavirus main proteinase is a prime target for antiviral therapy. In this issue of Chemistry & Biology, Wu et al. describe potent inhibition of the enzyme by benzotriazole esters, which were originally obtained as intermediates in the synthesis of lopinavir derivatives .