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

Tailoring polyethers for efficient post-polymerization functionalization by cross metathesis

Olefin cross metathesis is reported for the first time to attach small molecules to a range of novel polyethers with a poly(ethylene glycol) backbone and pendent alkene groups, allowing for a loading of up to one compound per monomer unit. These polymers are tailored to prevent the occurrence of self metathesis (reaction of the polymer with itself) by varying the substitution on the pendent alkenes, thus steering their reactivity toward olefin cross metathesis. Efficient functionalization has been observed for a range of coupling partners as a proof of concept for the use of olefin metathesis to graft small and larger molecules to polyethers for drug delivery. This approach also paves the way for the use of olefin cross metathesis as an efficient method to functionalize a wide variety of polymers with pendent olefin groups

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

Improving the aqueous solubility of HCV-E2 glycoprotein epitope mimics by cyclization using polar hinges

In this research we describe the improvement of the water‐solubility of cyclic epitope mimics based on the HCV E2 glycoprotein by incorporation of suitable polar hinges. The poor solubility of epitope mimics based on peptide sequences in the envelope (E2) protein hampered their synthesis and purification and made it very difficult to prepare the molecular constructs for evaluation of their bioactivity. Since changes in the amino acid composition are hardly possible in these epitope mimics in order to increase water‐solubility, a polar cyclization hinge may offer a remedy leading to a significant increase of polarity and therefore water solubility. These polar hinges were applied in the synthesis of better water‐soluble HCV‐E2 epitopes. An azide functionality in the polar hinges allowed attachment of a tetraethylene glycol linker by Cu‐catalyzed azide‐alkyne cyclo‐addition (CuAAC) for a convenient conjugation to ELISA plates in order to evaluate the bio‐activity of the epitope mimics. The immunoassays showed that the use of more polar cyclization hinges still supported anti‐HCV antibody recognition and did not negatively influence their binding. This significantly increased solubility induced by polar hinges should therefore allow for the molecular construction and ultimate evaluation of synthetic vaccine molecules

Synthesis and cellular penetration properties of new phosphonium based cationic amphiphilic peptides

A new category of phosphonium based cationic amphiphilic peptides has been developed and evaluated as potential antimicrobial peptides and cell penetrating peptides. The required building blocks were conveniently accessible from cysteine and could be applied in a solid phase peptide synthesis protocol for incorporation into peptide sequences. Evaluation of the antimicrobial properties and cellular toxicity of these phosphonium based peptides showed that these “soft” cationic side-chain containing peptides have poor antimicrobial properties and most of them were virtually non toxic (on HEK cells tested at 256 and 512 μM) and non-haemolytic (on horse erythrocytes tested at 512 μM), hinting at an interesting potential application as cell penetrating peptides. This possibility was evaluated using fluorescent peptide derivatives and showed that these phosphonium based peptide derivatives were capable of entering HEK cells and depending on the sequence confined to specific cellular areas

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

A New Class of Tunable Acid-Sensitive Linkers for Native Drug Release Based on the Trityl Protecting Group

Core-cross-linked polymeric micelles (CCPMs) are a promising nanoparticle platform due to favorable properties such as their long circulation and tumor disposition exploiting the enhanced permeability and retention (EPR) effect. Sustained release of covalently linked drugs from the hydrophobic core of the CCPM can be achieved by a biodegradable linker that connects the drug and the core. This study investigates the suitability of trityl-based linkers for the design of acid-triggered native active pharmaceutical ingredient (API) release from CCPMs. Trityl linker derivatives with different substituent patterns were synthesized and conjugated to model API compounds such as DMXAA-amine, doxorubicin, and gemcitabine, and their release kinetics were studied. Hereafter, API release from CCPMs based on mPEG-b-pHPMAmLac block copolymers was investigated. Variation of the trityl substitution pattern showed tunability of the API release rate from the trityl-based linker with t1/2 varying from 24 h at pH 7.4, all at 37 °C. A clear difference in release kinetics was found between gemcitabine and doxorubicin, with gemcitabine showing no detectable release for 72 h at pH 5.0 and doxorubicin showing a t1/2 of less than 1 h. Based on these findings, we show that the reaction mechanism of trityl deprotection plays an important role in the API release kinetics. The first step in this mechanism, which is protonation of the trityl-bound amine, is pKa-dependent, which explains the difference in release rate. In conclusion, acid-sensitive and tunable trityl linkers are highly promising for the design of linker-API conjugates and for their use in CCPMs

A new class of tunable acid-sensitive linkers for native drug release based on the trityl protecting group

Core-cross-linked polymeric micelles (CCPMs) are a promising nanoparticle platform due to favorable properties such as their long circulation and tumor disposition exploiting the enhanced permeability and retention (EPR) effect. Sustained release of covalently linked drugs from the hydrophobic core of the CCPM can be achieved by a biodegradable linker that connects the drug and the core. This study investigates the suitability of trityl-based linkers for the design of acid-triggered native active pharmaceutical ingredient (API) release from CCPMs. Trityl linker derivatives with different substituent patterns were synthesized and conjugated to model API compounds such as DMXAA-amine, doxorubicin, and gemcitabine, and their release kinetics were studied. Hereafter, API release from CCPMs based on mPEG-b-pHPMAmLac block copolymers was investigated. Variation of the trityl substitution pattern showed tunability of the API release rate from the trityl-based linker with t1/2 varying from <1.0 to 5.0 h at pH 5.0 and t1/2 from 6.5 to >24 h at pH 7.4, all at 37 °C. A clear difference in release kinetics was found between gemcitabine and doxorubicin, with gemcitabine showing no detectable release for 72 h at pH 5.0 and doxorubicin showing a t1/2 of less than 1 h. Based on these findings, we show that the reaction mechanism of trityl deprotection plays an important role in the API release kinetics. The first step in this mechanism, which is protonation of the trityl-bound amine, is pKa-dependent, which explains the difference in release rate. In conclusion, acid-sensitive and tunable trityl linkers are highly promising for the design of linker–API conjugates and for their use in CCPMs