An orthogonally protected CycloTriVeratrylene (CTV) as a highly pre-organized molecular scaffold for subsequent ligation of different cyclic peptides towards protein mimics

The synthesis of a semi-orthogonally protected CycloTriVeratrilene (CTV) scaffold derivative as well as the sequential introduction of three different peptide loops onto this molecular scaffold via Cu(I)-catalyzed azide alkyne cycloaddition towards a medium-sized protein mimic is described. This approach for the construction of medium-sized protein mimics is illustrated by the synthesis of a paratope mimic of the monoclonal antibody Infliximab (Remicade®) and provides access to a range of highly pre-organized molecular constructs bearing three different peptide segments. This approach may find wide applications for development of protein-protein interaction disruptors as well as synthetic vaccines

Potential peptidic proteasome inhibitors by incorporation of an electrophilic trap based on amino acid derived α-substituted sulfonyl fluorides

Peptido sulfonyl fluoride derivatives were designed and synthesized containing a substituent on the alpha position (αPSFs) with respect to the sulfonyl fluoride electrophilic trap. The chemical reactivity of these α-substituted amino sulfonyl fluorides was studied and compared with the previously described β-substituted amino sulfonyl fluorides in order to get a deeper insight into the importance of the immediate structural environment of the sulfonyl fluoride moiety. Unfortunately, the poor solubility of the resulting αPSFs precluded a proper evaluation of their biological activity

Proteasome inhibition by new dual warhead containing peptido vinyl sulfonyl fluorides

The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long lasting ties. We have introduced peptido sulfonyl fluorides for this purpose. Tuning the reactivity of this electrophilic trap may be crucial for modulating the biological action. Here we describe incorporation of a vinyl moiety into a peptido sulfonyl fluoride backbone, which should lead to a combined attack of the proteasome active site threonine on the double bond and the sulfonyl fluoride. Although this led to strong proteasome inhibitors, in vitro studies did not unambiguously demonstrate the formation of the proposed seven-membered ring structure. Possibly, formation of a seven-membered covalent adduct with the proteosomal active site threonine can only be achieved within the context of the enzyme. Nevertheless, this dual warhead concept may provide exclusive possibilities for duration and selectivity of proteasome inhibition

Immobilization by surface conjugation of cyclic peptides for effective mimicry of the HCV-envelope E2 protein as a strategy toward synthetic vaccines

Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e. epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functional-ized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate sur-faces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demon-strated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity towards their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key-binding residues by simply changing the amino-acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key-binding residue for binding monoclonal antibody V3.2

Potent and highly selective inhibitors of the proteasome trypsin-like site by incorporation of basic side chain containing amino acid derived sulfonyl fluorides

A unique category of basic side chain containing amino acid derived sulfonyl fluorides (SFs) has been synthesized for incorporation into new proteasome inhibitors targeting the trypsin-like site of the 20S proteasome. Masking the former α-amino functionality of the amino acid starting derivatives as an azido functionality allowed an elegant conversion to the corresponding amino acid derived sulfonyl fluorides. The inclusion of different SFs at the P1 site of a proteasome inhibitor resulted in 14 different peptidosulfonyl fluorides (PSFs) having a high potency and an excellent selectivity for the proteolytic activity of the β2 subunit over that of the β5 subunit. The results of this study strongly indicate that a free N-terminus of PSFs inhibitors is crucial for high selectivity toward the trypsin-like site of the 20S proteasome. Nevertheless, all compounds are slightly more selective for inhibition of the constitutive over the immunoproteasome

Expedient synthesis of a novel asymmetric selectively deprotectable derivative of the ATAC scaffold

An efficient multigram scale synthesis of a new asymmetric triazacyclophane scaffold, the ATAC (Asymmetric-TAC) scaffold, bearing three selectively removable groups is described. This scaffold is slightly more rigid than our frequently used TAC (TriAzaCyclophane) scaffold. The synthesis of the required triamine is very high yielding without difficult steps or purifications and was also applied to a much improved synthesis of our original TAC scaffold. Especially the tedious first reaction step, that is, mono-oNBS-protection of a triamine could be omitted. The rigidity of the triazacyclophane ring in both TAC- and ATAC scaffolds has also been investigated using variable temperature 1H NMR experiments

Synthetic antibody protein mimics of infliximab by molecular scaffolding on novel CycloTriVeratrilene (CTV) derivatives

Syntheses of novel semi-orthogonally protected CycloTriVeratrilene (CTV) analogues with enhanced water solubility, that is 3 and 4, derived from the previously described CTV scaffold derivative 2 are described here. These scaffolds 2–4 enabled a sequential introduction of three different complementarity determining region (CDR) mimics via Cu(I)-catalysed azide–alkyne cycloaddition towards medium-sized protein mimics denoted as “synthetic antibodies”. The highly optimised sequential introduction enabled selective attachment of three different CDR mimics in a one-pot fashion. This approach of obtaining synthetic antibodies, demonstrated by the synthesis of paratope mimics of monoclonal antibody infliximab (Remicade®), provided a facile access to a range of (highly) pre-organised molecules bearing three different (cyclic) peptide segments and may find a wide range of applications in the field of protein–protein interaction disruptors as well as in the development of synthetic vaccines or lectin mimics. The prepared synthetic antibodies were tested for their affinity towards tumour necrosis factor alpha using surface plasmon resonance and synthetic antibodies with micromolar affinities were uncovered

Synthesis of the TACO scaffold as a new selectively deprotectable conformationally restricted triazacyclophane based scaffold

The synthesis of a new triazacyclophane scaffold (TACO scaffold) containing three selectively deprotectable amines is described. The TACO scaffold is conformationally more constrained than our frequently used TAC scaffold, due to introduction of a substituent on the para position of the benzoic acid hinge, which prevents ring flipping and makes it more attractive than the TAC scaffold for preparation of artificial receptor molecules or for mimicking discontinuous epitopes toward protein mimics when more preorganization is required

Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis

The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure