Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation

Despite recent breakthroughs in the structural characterization of G‐protein‐coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double‐cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G‐protein‐coupled Y₂ receptor (Y₂R). Solid‐state NMR measurements of specific isotope‐labeled NPY in complex with in vitro folded Y₂R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C‐terminal α‐helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T³² to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y₂R.     

Solid‐Phase Combinatorial Synthesis and Biological Evaluation of Destruxin E Analogues

The solid‐phase combinatorial synthesis of cyclodepsipeptide destruxin E has been demonstrated. The combinatorial synthesis of cyclization precursors 8 was achieved by using a split and pool method on SynPhase Lanterns. The products were successfully macrolactonized in parallel in the solution phase by using 2‐methyl‐6‐nitrobenzoic anhydride and 4‐(dimethylamino)pyridine N‐oxide to afford macrolactones 9 , and the subsequent formation of an epoxide in the side chain gave 18 member destruxin E analogues 6 . Biological evaluation of analogues 6 indicated that the N‐MeAla residue was crucial to the induction of morphological changes in osteoclast‐like multinuclear cells (OCLs). Based on structure–activity relationships, azido‐containing analogues 15 were then designed for use as a molecular probe. The synthesis and biological evaluation of analogues 15 revealed that 15 b , in which the Ile residue was replaced with a Lys(N₃) residue, induced morphological changes in OCLs at a sufficient concentration, and modification around the Ile residue would be tolerated for attachment of a chemical tag toward the target identification of destruxin E ( 1 ).     

Immunological Evaluation of Co‐Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self‐Adjuvanting Anti‐Breast‐Cancer Vaccine Candidates

Co‐assembling vaccines composed of a lipidated HER2‐derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam₃CSK₄, α‐GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen‐specific immunostimulation properties, observed in reported self‐adjuvanting vaccine candidates, by using self‐assembly and adjuvant‐conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co‐assembly with lipidated CH401, which boosted the anti‐CH401 IgG and IgM production. In particular, α‐GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co‐assembling vaccine design opens the door for efficient and practical self‐adjuvanting vaccine development.     

PET Imaging of the Neuropeptide Y System: A Systematic Review

Neuropeptide Y (NPY) is a vastly studied biological peptide with numerous physiological functions that activate the NPY receptor family (Y₁, Y₂, Y₄ and Y₅). Moreover, these receptors are correlated with the pathophysiology of several diseases such as feeding disorders, anxiety, metabolic diseases, neurodegenerative diseases, some types of cancers and others. In order to deepen the knowledge of NPY receptors’ functions and molecular mechanisms, neuroimaging techniques such as positron emission tomography (PET) have been used. The development of new radiotracers for the different NPY receptors and their subsequent PET studies have led to significant insights into molecular mechanisms involving NPY receptors. This article provides a systematic review of the imaging biomarkers that have been developed as PET tracers in order to study the NPY receptor family.     

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

In spite of the recent success in crystallizing several G‐protein‐coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid‐state NMR spectroscopy. Static ¹⁵N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional ¹H,¹³C order parameter of the CH, CH₂, and CH₃ groups. In directly polarized ¹³C NMR experiments, these order parameters showed astonishingly low values of S_CH=0.55, S=0.33, and S=0.17, which corresponds to segmental amplitudes of approximately 50° in the backbone and approximately 50–60° in the side chain. At physiological temperature, ²H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a very broad spectrum that could stem from the transmembrane α‐helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid‐crystalline membrane at physiological temperature.     

One‐Pot Protection‐Glycosylation Reactions for Synthesis of Lipid II Analogues

(2,6‐Dichloro‐4‐methoxyphenyl)(2,4‐dichlorophenyl)methyl trichloroacetimidate ( 3 ) and its polymer‐supported reagent 4 can be successfully applied to a one‐pot protection‐glycosylation reaction to form the disaccharide derivative 7 d for the synthesis of lipid II analogues. The temporary protecting group or linker at the C‐6 position and N‐Troc protecting group of 7 d can be cleaved simultaneously through a reductive condition. Overall yields of syntheses of lipid II ( 1 ) and neryl‐lipid II N^ε‐dansylthiourea are significantly improved by using the described methods.     

Self‐Assembled BolA‐like Amphiphilic Peptides as Viral‐Mimetic Gene Vectors for Cancer Cell Targeted Gene Delivery

In this study, two types of BolA‐like amphiphilic peptides with dual ligands comprising a tumor‐targeting moiety of RGD sequence and a cell‐penetrating moiety of R8 sequence are designed and synthesized as gene vectors. The BolA‐structural peptide carriers can self‐assemble into spherical nanoparticles with a hydrophilic core and shell, which are similar to the viral capsid and can bind plasmid DNA in an aqueous medium to form viral‐mimetic complexes. It is found that the BolA‐like dual ligands system exhibits significantly enhanced gene expression in both HeLa and 293T cell lines, as compared with poly(ethylenimine) PEI. These BolA‐like amphiphilic peptides are promising in clinical trials of gene therapy.

     

Solid‐Phase Total Synthesis of Yaku'amide B Enabled by Traceless Staudinger Ligation

We report a solid‐phase strategy for total synthesis of the peptidic natural product yaku'amide B ( 1 ), which exhibits antiproliferative activity against various cancer cells. Its linear tridecapeptide sequence bears four β,β‐dialkylated α,β‐dehydroamino acid residues and is capped with an N‐terminal acyl group (NTA) and a C‐terminal amine (CTA). To realize the Fmoc‐based solid‐phase synthesis of this complex structure, we developed new methods for enamide formation, enamide deprotection, and C‐terminal modification. First, traceless Staudinger ligation enabled enamide formation between sterically encumbered alkenyl azides and newly designed phosphinophenol esters. Second, application of Eu(OTf)₃ led to chemoselective removal of the enamide Boc groups without detaching the resin linker. Finally, resin‐cleavage and C‐terminus modification were simultaneously achieved with an ester–amide exchange reaction using CTA and AlMe₃ to deliver 1 in 9.1 % overall yield (24 steps from the resin).     

Staphylococcus aureus Penicillin‐Binding Protein 2 Can Use Depsi‐Lipid II Derived from Vancomycin‐Resistant Strains for Cell Wall Synthesis

Vancomycin‐resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide‐containing modified cell‐wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin‐binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild‐type S. aureus. However, it is unclear whether VRSA processes the depsipeptide‐containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi‐lipid I, a cell‐wall precursor of VRSA. By using this chemistry, we prepared a depsi‐lipid II analogue as substrate for a cell‐free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi‐lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.     

Stereoselective Fluorination Alters the Geometry of a Cyclic Peptide: Exploration of Backbone‐Fluorinated Analogues of Unguisin A

New methods for enhancing the efficiency of peptide cyclization, and for fine‐tuning the conformations of cyclic peptides, are valuable from a drug development perspective. Herein stereoselective fluorination is investigated as a new strategy for achieving these goals. Four vicinal difluorinated analogues of the natural cyclic heptapeptide unguisin A have been efficiently synthesized. The analogues are found to adopt dramatically different secondary structures, controlled by the fluorine stereochemistry.     

Efficient Synthesis of Cryptophycin‐52 and Novel para‐Alkoxymethyl Unit A Analogues

Cryptophycins are a family of highly cytotoxic, cyclic depsipeptides. They display antitumour activity that is largely maintained for multi‐drug‐resistant tumour cells. Cryptophycins are composed of four building blocks (units A–D) that correspond to the respective amino and hydroxy acids. A new synthetic route to unit A allows the selective generation of all four stereogenic centres in a short, efficient and reliable synthesis and contributes to an easier and faster synthesis of cryptophycins. The first two stereogenic centres are introduced by a catalytic asymmetric dihydroxylation, whereas the remaining two stereogenic centres are introduced with substrate control of diastereoselectivity. The stereogenic diol function also serves as the epoxide precursor. The approach was used to synthesise the native unit A building block as well as three para‐alkoxymethyl analogues from which cryptophycin‐52 and three analogous cryptophycins were prepared. Macrocyclisation of the seco‐depsipeptides was based on ring‐closing metathesis.     

In Vivo Imaging of Mouse Tumors by a Lipidated Cathepsin S Substrate

The synthesis and evaluation of two cathepsin S‐specific probes is described. For long‐term retention of the probe at the target site and a high signal‐to‐noise ratio, we introduced a lipidation approach via the simple attachment of palmitoic acid to the reporter. After cathepsin S‐specific cleavage in cultured cells and in a grafted tumor mouse model, fluorescence increased owing to dequenching and we observed an intracellular accumulation of the fluorescence in the target tissue. The lipidated probe provided a prolonged and strongly fluorescent signal in tumors when compared to the very similar non‐lipidated probe, demonstrating that non‐invasive tumor identification is feasable. The homing principle by probe lipidation might also work for selective administration of cytotoxic compounds to specifically reduce tumor mass.     

Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation

The chemical synthesis of the 184‐residue ferric heme‐binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α‐ketoacid‐hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild‐type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well‐suited for the preparation of hydrophobic protein targets.     

Design,Synthesis, and Application of an Optimized Monofluorinated Aliphatic Label for Peptide Studies by Solid‐State 19F NMR Spectroscopy

A conformationally restricted monofluorinated α‐amino acid, (3‐fluorobicyclo[1.1.1]pentyl)glycine (F‐Bpg), was designed as a label for the structural analysis of membrane‐bound peptides by solid‐state ¹⁹F NMR spectroscopy. The compound was synthesized and validated as a ¹⁹F label for replacing natural aliphatic α‐amino acids. Calculations suggested that F‐Bpg is similar to Leu/Ile in terms of size and lipophilicity. The ¹⁹F NMR label was incorporated into the membrane‐active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.     

Synthesis of 1‐C‐Glycoside‐Linked Lipid II Analogues Toward Bacterial Transglycosylase Inhibition

Preparation of Lipid II analogues containing an enzymatically uncleavable 1‐C‐glycoside linkage between the disaccharide moiety and the pyrophosphate‐ or pyrophosphonate‐lipid moiety is described. The synthesis of a common 1‐C‐vinyl disaccharide intermediate has been developed that allows easy preparation of both an elongated sugar‐phosphate bond and a sugar‐phosphonate moiety, which are coupled with the polyprenyl phosphate to give the desired molecules. Inhibition studies show how a subtle structural modification results in dramatically different potency toward bacterial transglycosylase (TGase), and the results identify Lipid II‐C‐O‐PP (IC₅₀=25 μM ) as a potential TGase inhibitor.     

Synthesis of the Antimicrobial S‐Linked Glycopeptide,Glycocin F

The first total synthesis of glycocin F, a uniquely diglycosylated antimicrobial peptide bearing a rare S‐linked N‐acetylglucosamine (GlcNAc) moiety in addition to an O‐linked GlcNAc, has been accomplished using a native chemical ligation strategy. The synthetic and naturally occurring peptides were compared by HPLC, mass spectrometry, NMR and CD spectroscopy, and their stability towards chymotrypsin digestion and antimicrobial activity were measured. This is the first comprehensive structural and functional comparison of a naturally occurring glycocin with an active synthetic analogue.     

Convenient Access to Fluorescent Probes by Chemoselective Acylation of Hydrazinopeptides: Application to the Synthesis of the First Far‐Red Ligand for Apelin Receptor Imaging

Herein, we develop a convenient method to facilitate the solution‐phase fluorescent labelling of peptides based on the chemoselective acylation of α‐hydrazinopeptides. This approach combines the advantages of using commercially available amine‐reactive dyes and very mild conditions, which are fully compatible with the chemical sensitivity of the dyes. The usefulness of this approach was demonstrated by the labelling of apelin‐13 peptide. Various fluorescent probes were readily synthesized, enabling the rapid optimization of their affinities for the apelin receptor. Thus, the first far‐red fluorescent ligand with sub‐nanomolar affinity for the apelin receptor was characterized and shown to track the receptor efficiently in living cells by fluorescence confocal microscopy.