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Chemoselective conjugation of proteins and peptides allows the formation of a covalent bond without the need for protective group chemistry to label or assemble macromolecules. Chemical reactions yielding oxime bonds are highly suitable for this purpose. Oxime ligation comprises the reaction of a ketone or aldehyde (1; Scheme 1) with an aminooxy (2) to form a special imine known as an oxime (3). Advantages of this strategy include chemoselectivity, as a ketone is inert to most other reactions, and the mild conditions in which the reaction can be performed. Oxime formation has been thoroughly investigated and was found to proceed in a step-wise manner, depending on pH.[1] Although the reaction proceeds faster in acidic conditions (pH 4-5), oximes will also form at neutral pH. Furthermore, oxime formation can be accelerated by addition of the catalyst aniline, [2] and oxime linkages are stable at neutral pH.[3] Taken together, these properties make the oxime linkage one of the preferred methods for the chemoselective modification of peptides and proteins. [4] Levulinic acid (LA) is one of the most frequently used ketones for oxime formation, and is generally introduced by attachment to a lysine side chain e-NH 2 or the N-terminal NH 2 moiety.[5] The reaction between the protein-LA complex and an aminooxy moiety proceeds well at millimolar concentrations; however, the relatively low quantity and high molecular weight of most proteins result in sub-millimolar protein concentrations and limitations of the oxime reaction. Under these conditions, we and others have found that oxime reaction yields are low because of the formation of a levulinoyl-derived side-product that competes with oxime bond formation.[5a] As oxime ligations are increasingly attractive and provide an orthogonal approach to label proteins, we explored alternative ketone moieties for bioconjugation. Our hypothesis was that the levulinoyl side-product (corresponding to a mass loss of 18 Da) is the result of intramolecular cyclization of LA, thus preventing the LA ketone group from reacting with the aminooxy moiety. The formation of this by-product is mainly seen at low concentrations, because under these conditions the concentration-independent cyclization side-reaction benefits from the slow oxime formation.To study the molecular mechanism underlying levulinoyl cyclization, the pentapeptide LYRAK was synthesized with LA coupled to the lysine e-amine (LYRAK(LA)). Lyophilized LYRAK(LA) was dissolved in water under acidic conditions (pH 4.5) and was left to cyclize spontaneously for 72 h at room temperature. The conversion to cyclized derivatives was monitored by ESI-MS and NMR (both in [D 6 ]DMSO and D 2 O) and structurally characterized by 2D NMR experiments ( Figures S1 and S2). The cyclization of LA was tested at three concentrations and was shown to be concentration independent ( Figure S3).Based on these results, the following reaction mechanism for intramolecular cyclization of the levulinoyl moiety was proposed (Scheme 2). The amide nitrogen in...

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The aminoacyl-tRNA synthetase family of enzymes is the target of many antibacterials and inhibitors of eukaryotic hyperproliferation. In screening analogues of 5'-O-(N-L-aminoacyl)-sulfamoyladenosine containing all 20 proteinogenic amino acids, we found these compounds to have potent immunosuppressive activity. Also, we found that combinations of these compounds inhibited the immune response synergistically. Based on these data, analogues with modifications at the aminoacyl and ribose moieties were designed and evaluated, and several of these showed high immunosuppressive potency, with one compound having an IC50 of 80 nM, when tested in a cellular mixed lymphocyte reaction assay. Apart from showing the potential of aminoacyl-tRNA synthetase inhibitors as immunosuppressants, the current study also provides arguments for careful evaluation of the immunosuppressive activity of developmental antibacterials that target these enzymes.