Synthesis and Characterization of Enzymatically Degradable PEG‐Based Peptide‐Containing Hydrogels

Biodegradable hydrogels were synthesized by the click reaction of 4‐arm azido‐terminated PEG differing in molecular weight (2 100 and 8 800) and two alkyne‐terminated peptides: [alkyne]‐GFLGK‐[alkyne] and ([alkyne]‐GFLG)₂K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and microrheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800‐based hydrogels, time‐course analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability.

     

“Click & Seed” Approach to the Biomimetic Modification of Material Surfaces

A simple, versatile, protein‐repulsive, substrate‐independent biomimetic surface modification is presented that is based on the creation of a PEO brush on a polydopamine anchoring layer and its capacity for selective follow‐up modifications with various ligands using a copper‐catalyzed alkyne‐azide cycloaddition reaction. The desired surface concentration of peptide biomimetic ligands can be controlled by adjusting the peptide concentration in the reaction mixture, then measuring the activity of ¹²⁵I‐radiolabeled peptides that are immobilized on the substrates. The performance of the prepared substrates is tested in cell cultures with MEF cells and a human ECC line.

     

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography‐multiple reaction monitoring mass spectrometry (LC‐MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope‐labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC‐MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Polymethine dyes as spectral-fluorescent probes for biomacromolecules

It is known that polymethine dyes (PD) can form complexes with biomacromolecules, in which, as a rule, fluorescence buildup is observed. In addition, PD possess a unique property to form ordered aggregates of different types (dimers, H- and J-aggregates) both in the free state and on biomacromolecules as templates. This creates a basis for application of PD as spectral-fluorescent probes for biomacromolecules, which is a matter of this review. Besides, the review is devoted to studies of noncovalent interactions of PD with biomacromolecules: nucleic acids, proteins, and some others.     

Neutral loss of water from the b ions with histidine at the C-terminus and formation of the c ions involving lysine side chains

Neutral loss of water from the amide bond induced by the His side chain has been reported. The proposed fragmentation pathway is a retro-Ritter reaction catalyzed by the imidazole nitrogen. In our MS/MS study of the neuropeptide GAHKNYLRFamide, we observed that the neutral loss of water from the b(3) ion is abundant. The b(3) ion has a His residue at the C-terminus. As reported previously, in the b ions with His at the C-terminus, the imidazole residue is connected to the carbonyl carbon to form a five-membered ring. Therefore, it is unlikely that the neutral loss of water from the b(3) ion is catalyzed by the imidazole nitrogen. Through MS2 and MS3 studies of a synthetic peptide standard AGHKLL and its chemically labeled and isotope-encoded forms, we discovered that the water loss from the b(3) ion involves the carbonyl group of His, the hydrogen connected to the alpha-carbon of Gly, and the amide hydrogen of His. We also discovered the formation of an unusual c(x) ion in peptides with a Lys or Arg residue at the (x + 1) position of the peptide.     

The effects of L‐ to D‐isomerization and C‐terminus deamidation on the secondary structure of antimicrobial peptide Anoplin in aqueous and membrane mimicking environment

UV resonance Raman spectra of the antimicrobial peptide (AMP) Anoplin (L ‐Anoplin‐NH₂) and two of its derivatives (enantiomer D ‐Anoplin‐NH₂ and C‐terminus deamidated L ‐Anoplin‐OH) were measured in aqueous buffer solution and in membrane‐mimetic environments including 2,2,2‐trifluoro ethanol (TFE), zwitterionic lipid dipalmitoylglycerophosphocholine (DPPC) and anionic lipid dipalmitoylglycerophosphoglycerol (DPPG) vesicle solutions. All three peptides were found to adopt random‐coil/β turn‐like conformation in aqueous solution over the temperature range of 1–60 °C. The conformation was found to become more α‐helical in membrane‐mimetic solutions such as TFE and DPPG but not in DPPC for all Anoplin derivatives. The data demonstrate that Anoplin preferentially binds to the anionic over the zwitterionic model cell membranes. Results also showed that deamidation does not change the conformation of L ‐Ano‐NH₂ very significantly, but does alter membrane rupturing and antimicrobial activities thus confirming that it is the physicochemical properties rather than the peptide conformation that define the mechanism of AMP action. Copyright © 2010 John Wiley & Sons, Ltd.     

Electron predators are hydrogen atom traps. Effects of aryl groups on N—Cα bond dissociations of peptide radicals

Effects of substituted aryl groups on dissociations of peptide aminoketyl radicals were studied computationally for model tetrapeptide intermediates GXD^?G where X was a cysteine residue that was derivatized by S‐(3‐nitrobenzyl), S‐(3‐cyanobenzyl), S‐(3,5‐dicyanobenzyl), S‐(2,3,4,5,6‐pentafluorobenzyl), and S‐benzyl groups. The aminoketyl radical was placed within the Asp amide group. Aminoketyl radicals having the S‐(3‐nitrobenzyl) group were found to undergo spontaneous and highly exothermic migration of the hydroxyl hydrogen atom onto the nitro group in conformers allowing interaction between these groups. Competing reaction channels were investigated for aminoketyl radicals having the S‐(3‐cyanobenzyl) and S‐(3,5‐dicyanobenzyl) groups, e.g. H‐atom migration to the C and N atoms of the C≡N group, migration to the C‐4 position of the phenyl ring, and dissociation of the radical‐activated N? C_α bond between the Asp and Gly residues. RRKM kinetic analysis on the combined B3LYP and ROMP2/6‐311++G(2d,p) potential energy surface indicated > 99% H‐atom transfer to the C≡N group forming a stable iminyl intermediate. The N? C_α bond dissociation was negligible. In contrast, peptides with the S‐(2,3,4,5,6‐pentafluorobenzyl) and S‐benzyl groups showed preferential N? C_α bond dissociation that outcompeted H‐atom migration to the C‐4 position and fluorine substituents in the phenyl ring. These computational results are used to suggest an alternative mechanism for the quenching effect on electron‐based peptide backbone dissociations of benzyl groups with electron‐withdrawing substitutents, as reported recently. Copyright © 2010 John Wiley & Sons, Ltd.     

Croissamide,a proline-rich cyclic peptide with an N-prenylated tryptophan from a marine cyanobacterium Symploca sp.

Croissamide, a proline-rich cyclic peptide that contains an N-prenylated tryptophan, was isolated from a marine cyanobacterium Symploca sp. Its gross structure was determined by spectroscopic analyses, and the absolute configuration was established based on chiral HPLC analyses of acid hydrolysates.