A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogenous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.
The diastereoselective cyclopropanation of various alkenes with diazoacetate derivatives can be achieved under mechanochemical conditions using metallic silver foil and a stainless-steel vial and ball system. This solvent-free method displays analogous reactivity and selectivity to solution-phase reactions without the need for slow diazoacetate addition or an inert atmosphere. The heterogeneous silver-foil catalyst system is easily recyclable without any appreciable loss of activity or selectivity being observed. The cyclopropanation products were obtained with excellent diastereoselectivities (up to 98:2 d.r.) and in high yields (up to 96 %).
Herein, we describe a copper-free, oxidant-free, solvent-free homocoupling reaction using a palladium catalyst under mechanochemical conditions. We extended the methodology to palladium catalyst on solid support which showed a different reactivity and different product ratios from the non-supported catalyst.
Post-translational modification by covalent attachment of Rub1 (NEDD8),
ubiquitin, SUMO and other small signaling proteins has a profound impact on the
function and fate of cellular proteins. Investigations of the relationship of
these bioactive structures and their functions are limited by analytical methods
that are scarce and tedious. A novel strategy is reported here for analysis of
branched proteins by top-down mass spectrometry and illustrated by application
to four recombinant proteins and one synthetic peptide modified by covalent
bonds with ubiquitin or Rub1. The approach allows an analyte to be recognized as
a branched protein, participating proteins to be identified, the site of
conjugation to be defined, and chemical, native and recombinant modifications to
be characterized. In addition to high resolution provided by the mass
spectrometer, success is based on sample fragmentation by electron transfer
dissociation assisted by collisional activation and software designed for
graphic interpretation adapted for branched proteins. This strategy allows for
the first time structures of unknown two component branched proteins to be
elucidated directly.
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