The Staudinger ligation-a gift to chemical biology

Although the reaction between an azide and a phosphane to form an aza-ylide was discovered by Hermann Staudinger more than 80 years ago and has found widespread application in organic synthesis, its potential as a highly chemoselective ligation method for the preparation of bioconjugates has been recognized only recently. As the two reaction partners are bioorthogonal to almost all functionalities that exist in biological systems and react at room temperature in an aqueous environment, the Staudinger ligation has even found application in the complex environment of living cells. Herein we describe the current state of knowledge on this reaction and its application both for the preparation of bioconjugates and as a ligation method in chemical biology.     

Förster Resonance Energy Transfer Investigations Using Quantum‐Dot Fluorophores

F?rster resonance energy transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited donor fluorophore to a proximal ground-state acceptor fluorophore, is a well-characterized photophysical tool. It is very sensitive to nanometer-scale changes in donor-acceptor separation distance and their relative dipole orientations. It has found a wide range of applications in analytical chemistry, protein conformation studies, and biological assays. Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores with unique optical and spectroscopic properties that could enhance FRET as an analytical tool, due to broad excitation spectra and tunable narrow and symmetric photoemission. Recently, there have been several FRET investigations using luminescent QDs that focused on addressing basic fundamental questions, as well as developing targeted applications with potential use in biology, including sensor design and protein conformation studies. Herein, we provide a critical review of those developments. We discuss some of the basic aspects of FRET applied to QDs as both donors and acceptors, and highlight some of the advantages offered (and limitations encountered) by QDs as energy donors and acceptors compared to conventional dyes. We also review the recent developments made in using QD bioreceptor conjugates to design FRET-based assays.     

Synthesis, electrochemistry and metal binding properties of monosubstituted ferrocenoyl peptides with thioether-containing sidechains

Ferrocenoyl peptides incorporating thioether functionality respond more strongly to mercury(II) than to other heavy metal ions in solution. Compounds reported previously in this context are all 1,1′-disubstituted, and all include two or more sulfur-containing amino acids. To test whether two thioether groups are required for effective mercury binding by these systems, we have prepared a series of singly-substituted ferrocenoyl peptides from ferrocenecarboxylic acid and l-methionine, S-methyl-l-cysteine or S-trityl-l-cysteine, and tested them as electrochemical probes for mercury(II). Nine ferrocenoyl peptides have been synthesised using a Boc-protecting group strategy and HBTU-mediated peptide coupling. The electrochemical properties of these compounds have been determined using cyclic voltammetry, and all show fully reversible one electron oxidation steps. Forward sweep half wave peaks (E_F), reverse sweep half wave peaks (E_R), peak separations (ΔE_P) and half wave potentials (E_1/2) are reported. Changes in the potential of the iron(II)/iron(III) redox couple of the ferrocene core have been used to quantify heavy metal-peptide interactions, revealing that these monotopic systems also respond more strongly to mercury(II) than to zinc(II), cadmium(II), silver(I) and lead(II). NMR experiments to characterise the peptide-mercury interaction implicate the thioether sulfur as the site of mercury binding and indicate 1:1 stoichiometry. The crystal structure of ferrocenoyl-S-methyl-l-cysteine methyl ester is also reported. The greater responsiveness of these systems to mercury(II) makes them interesting leads for the development of biologically inspired sensors for this toxic heavy metal.     

Thermoresponsive Block Copolymer–Protein Conjugates Prepared by Grafting‐from via RAFT Polymerization

Well‐defined “smart” block copolymer–protein conjugates were prepared by two consecutive “grafting‐from” reactions via reversible addition–fragmentation chain transfer (RAFT) polymerization. The initiating portion (R‐group) of the RAFT agent was anchored to a model protein such that the thiocarbonylthio moiety was readily accessible for chain transfer with propagating chains in solution. Well‐defined polymer‐protein conjugates of poly(N‐isopropylacrylamide) (PNIPAM) and bovine serum albumin (BSA) were prepared at room temperature in aqueous media. The retained trithiocarbonate moiety on the free end group of the immobilized polymer allowed the homopolymer conjugate to be extended by polymerization of N,N‐dimethylacrylamide. Polyacrylamide gel electrophoresis, size exclusion chromatography, and NMR spectroscopy confirmed the synthesis of the various conjugates and revealed that the polymerizations were well controlled. As expected, the resulting block copolymer–protein conjugates demonstrated thermoresponsive behavior due to the temperature‐sensitivity of the PNIPAM block, as evidenced by turbidity measurements and dynamic light scattering analysis.

     

A Dinuclear Mercury(II)‐Mediated Base Pair in DNA

The first dinuclear metal‐mediated base pair containing divalent metal ions has been prepared. A combination of the neutral bis(monodentate) purine derivative 1,N⁶‐ethenoadenine (ϵA), which preferentially binds two metal ions with a parallel alignment of the N−M bonds, and the canonical nucleobase thymine (T), which readily deprotonates in the presence of Hg^II and thereby partially compensates the charge accumulation due to the two closely spaced divalent metal ions, yields the dinuclear T‐Hg^II₂‐ϵA base pair. This metal‐mediated base pair stabilizes the DNA oligonucleotide duplex as shown by an increase of 8 °C in its melting temperature. Formation of the base pair was demonstrated by temperature‐dependent UV spectroscopy as well as by titration experiments monitored by UV and CD spectroscopy.     

Making “smart polymers” smarter: Modern concepts to regulate functions in polymer science

This highlight summarizes recent attempts and advances of macromolecular sciences to abstract the biological concept of regulation and import it into synthetic polymer systems. The differences of “responsive switching” exploited in smart polymers and “regulation” present in the biological world of proteins are evident. Therefore, the biomimetic regulation might advance the possibilities of polymer science beyond these of established “smart polymers” and makes precise regulation of functions through signaling events, signal transduction and even complex regulative circuits possible. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1–14, 2010     

Traveling the Vitamin B12 Pathway: Oral Delivery of Protein and Peptide Drugs

Oral routes of administration for therapeutic peptides and proteins face two major barriers: proteolytic degradation in the stomach and an inadequate absorption mechanism for polypeptides within the intestinal lumen. As a result, peptide‐based therapeutics are administered by injection, a painful process associated with lower patient compliance. The development of a means of overcoming these two major obstacles and enabling the successful delivery of peptide therapeutics by the oral route of administration has therefore been the target of extensive scientific endeavor. This Minireview focuses on oral peptide/protein delivery by the dietary uptake pathway for vitamin B₁₂. Recent progress in this field includes the delivery of erythropoietin, granulocyte‐colony‐stimulating factor, luteinizing‐hormone‐releasing hormone, and insulin.     

A Practical Guide on the Synthesis of Metal Chelates for Molecular Imaging and Therapy by Means of Click Chemistry

The copper‐catalyzed cycloaddition of organic azides and alkynes (CuAAC) is one of the most popular reactions for rapid assembly of multifunctional molecular frameworks from commercially available building blocks. It is also attractive for synthesis of conjugates of multidentate chelate ligands (chelators) with molecular targeting vectors, such as peptides or proteins, which serve as precursors for labeling with metal radionuclides or are useful as MRI contrast agents after Gd^III complexation. However, applicability of CuAAC for such purposes is complicated by formation of unwanted copper chelates. The alternative use of copper‐free click chemistry, for example, the strain‐promoted alkyne‐azide cycloaddition (SPAAC) or the Diels–Alder reaction of tetrazines and strained alkenes, entails other specific challenges: Introduction of large, isomerically non‐homogeneous and hydrophobic linker groups affects product homogeneity and can severely change pharmacokinetic profiles. Against this background, this review elucidates scope and applicability of both Cu‐catalyzed and Cu‐free alkyne‐azide cycloadditions pertinent to the elaboration of radiometal chelates and MRI contrast agents, with an emphasis on strategies to tackle the problem of copper complexation during CuAAC.