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Unnatural amino acid

Georg Wandrey, Joel Wurzel, Kyra Hoffmann, Tobias Ladner, Jochen Büchs, Lorenz Meinel, Tessa Lühmann
BACKGROUND: Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation. RESULTS: We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP)...
2016: Journal of Biological Engineering
Lei Wang
The genetic code can be expanded to include unnatural amino acids (Uaas) by engineering orthogonal components involved in protein translation. To be compatible with live cells, side chains of Uaas have been limited to either chemically inert or bio-orthogonal (i.e., nonreactive toward biomolecules) functionalities. To introduce bioreactivity into live systems, the genetic code has recently been engineered to encode a new class of Uaas, the bioreactive Uaas. These Uaas, after being incorporated into proteins, specifically react with target natural amino acid residues via proximity-enabled bioreactivity, enabling the selective formation of new covalent linkages within and between proteins both in vitro and in live systems...
October 6, 2016: New Biotechnology
Shubhendu Palei, Henning D Mootz
Cyclic peptides are highly desired molecules not only for basic research but also for many biomedical and pharmacological applications. Due to their potentially superior physicochemical properties as compared to their linear counterparts, they are considered as ideal candidates for studying protein-protein interactions, among others. Most of the methods developed in recent years to prepare cyclic peptides focus either on a synthetic or a recombinant route. While the former provides access to diversified, noncanonical peptides, including unnatural and D-amino acid, for example, the latter can harness the power of genetic randomization to generate and select from large peptide libraries...
2017: Methods in Molecular Biology
Shaoquan Lin, Naoya Kumagai, Masakatsu Shibasaki
α,α-Disubstituted α-amino acids have attracted increasing interest due to their potential utility as building blocks of unnatural peptides. Herein we document an enantioselective entry to this class of compounds through the direct catalytic addition of acetonitrile to α-iminoesters bearing an N-thiophosphinoyl group. Chiral N-heterocyclic carbene complexes of [Ir(cod)(OMe)]2 catalytically rendered the catalytic generation of α-cyanocarbanions from acetonitrile in combination with Barton's base, followed by enantioselective addition to the imino carbonyl group, delivering a variety of enantioenriched α-cyanomethylated α,α-disubstituted α-amino acid derivatives...
October 18, 2016: Organic & Biomolecular Chemistry
Meilin Tian, Shixin Ye
Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn(2+) for regulation is unknown...
October 7, 2016: Scientific Reports
Quan Gou, Yu-Wen Yang, Zi-Ning Liu, Jun Qin
The first example of intermolecular amination of unactivated C(sp(3) )-H bonds by cyclic alkylamines mediated by Cu(OAc)2 /O2 is reported. This method avoids the use of benzoyloxyamines as the aminating reagent, which are normally prepared from alkylamines in extra steps. A variety of unnatural β(2, 2) -amino acid analogues are synthesized by this simple and efficient procedure. This approach offers a solution to the previous unmet challenge of C(sp(3) )-H/N-H activation for the formation of C(sp(3) )-N bonds...
September 29, 2016: Chemistry: a European Journal
Yi Yang, Haiping Song, Peng R Chen
Protein-protein interactions (PPIs) play pivotal roles in regulation of many biological processes. Conventional methods are capable of investigating stable and strong interactions within protein complexes, but remain difficult for studying dynamic, transient, and weak PPIs. Herein we review photo-affinity unnatural amino acids that can be site-specifically incorporated into a protein of interest to covalently trap noncovalent PPIs under living conditions. A newly developed cleavable photocrosslinker from our group will also be introduced, which facilitated the prey-bait separation for better enrichment and identification of photocrosslinked PPI complexes...
September 26, 2016: IUBMB Life
Sanggil Kim, Wooseok Ko, Bong Hyun Sung, Sun Chang Kim, Hyun Soo Lee
Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency...
September 15, 2016: Bioorganic & Medicinal Chemistry
Chengcheng Zhang, Jinhe Pan, Kuo-Shyan Lin, Iulia Dude, Joseph Lau, Jutta Zeisler, Helen Merkens, Silvia Jenni, Brigitte Guérin, François Bénard
The neuropeptide Y1 receptor (Y1R) is overexpressed in many human cancers, particularly breast cancer. Due to stability issues, limited success has been achieved for Y1R imaging agents, including full length and truncated neuropeptide Y (NPY) analogues. The goal of this study was to evaluate the possibility of using radiolabeled truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor. Four truncated NPY analogues were synthesized based on the sequence of [Pro(30), Tyr(32), Leu(34)]NPY(28-36), also known as BVD15...
October 3, 2016: Molecular Pharmaceutics
Donghyun Lim, Wan Gi Byun, Ja Young Koo, Hankum Park, Seung Bum Park
MicroRNAs (miRNAs) regulate gene expression by targeting protein-coding transcripts that are involved in various cellular processes. Thus, miRNA biogenesis has been recognized as a novel therapeutic target. Especially, the let-7 miRNA family is well-known for its tumor suppressor functions and is downregulated in many cancer cells. Lin28 protein binds to let-7 miRNA precursors to inhibit their maturation. Herein, we developed a FRET-based, high-throughput screening system to identify small-molecule inhibitors of the Lin28-let-7 interaction...
October 7, 2016: Journal of the American Chemical Society
Lucia Tamborini, Gregorio Cullia, Birgitte Nielsen, Carlo De Micheli, Paola Conti, Andrea Pinto
Homologation of glutamic acid chain together with conformational constraint is a commonly used strategy to achieve selectivity towards different types of glutamate receptors. In the present work, starting from two potent and selective unnatural amino acids previously developed by us, we investigated the effects on the activity/selectivity profile produced by a further increase in the distance between the amino acidic moiety and the distal carboxylate group. Interestingly, the insertion of an aromatic ring as a spacer produced a low micromolar affinity NMDA ligand that might represent a lead for the development of a new class of NMDA antagonists...
September 12, 2016: Bioorganic & Medicinal Chemistry
Kanchana R Ravichandran, Alexander T Taguchi, Yifeng Wei, Cecilia Tommos, Daniel G Nocera, JoAnne Stubbe
Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356•, Y731•, and Y730• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122• and Y356• has not been detected...
October 7, 2016: Journal of the American Chemical Society
Arne H Smits, Annika Borrmann, Mark Roosjen, Jan C M van Hest, Michiel Vermeulen
Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts...
October 17, 2016: ACS Chemical Biology
Taylor May Urquhart, Elisabeth Daub, John Frank Honek
With a mass of ~1.6 x 107 Daltons and composed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, L-azidohomoalanine (Aha), and a suitable Escherichia coliMet auxotroph for phage production, conditions were developed to produce M13 bacteriophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide-alkyne cycloaddition) and capable of azide-selective attachment to 5 nm gold nanoparticles as visualized by transmission electron microscopy...
September 14, 2016: Bioconjugate Chemistry
Jennifer X Qiao, Kenneth J Fraunhoffer, Yi Hsiao, Yi-Xin Li, Chunlei Wang, Tammy C Wang, Michael A Poss
A one-step synthesis of Fmoc-protected aryl/heteroaryl-substituted phenylalanines (Bip derivatives) using the nonaqueous palladium-catalyzed Suzuki-Miyaura cross-coupling (SMC) reaction of Fmoc-protected bromo- or iodophenylalanines is reported. This protocol allows for the direct formation of a variety of unnatural biaryl-containing amino acids in good to excellent yield, which can be readily used in subsequent Fmoc solid-phase peptide synthesis. The synthetic utility of this method is also demonstrated by the SMC reaction of bromophenylalanine-containing tripeptides...
October 7, 2016: Journal of Organic Chemistry
Tessa Lühmann, Valerie Spieler, Vera Werner, Marie-Gabrielle Ludwig, Juliane Fiebig, Thomas Müller, Lorenz Meinel
Driving macrophage (M) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin-4 (IL-4) leverages polarization into the M2-M phenotype but its systemic use is constrained by dose-limiting toxicity. Consequently, we developed IL-4 decorated surfaces aiming at sustained and localized activity. IL-4 muteins were generated by genetic code expansion, replacing Lys 42 by unnatural amino acids (uAA). All muteins had wt-IL-4 comparable cellular performances and binding affinities to the IL4R...
September 5, 2016: Chembiochem: a European Journal of Chemical Biology
Mark Arnold Thomas Blaskovich
Unusual amino acids are fundamental building blocks of modern medicinal chemistry. The combination of readily functionalised amine and carboxyl groups attached to a chiral center group along with one or two potentially diverse side chains provides a unique three-dimensional structure with high degree of functionality. This makes them invaluable as starting materials for syntheses of complex molecules, highly diverse elements for SAR campaigns, integral components of peptidomimetic drugs, and potential drugs on their own...
September 2, 2016: Journal of Medicinal Chemistry
C Hu, J Wang
We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First, we considered the unnatural amino acids and the protein scaffold separately. The scaffold is designed by traditional protein design methods. The unnatural amino acids are inspired by natural structure and organic chemistry methods, and synthesized by either organic chemistry methods or enzymatic conversion...
2016: Methods in Enzymology
R Serfling, I Coin
The site-specific incorporation of unnatural amino acids (Uaas) via genetic code expansion provides a powerful method to introduce synthetic moieties into specific positions of a protein directly in the live cell. The technique, first developed in bacteria, is nowadays widely applicable in mammalian cells. In general, different Uaas are incorporated with different efficiency. By comparing the incorporation efficiency of several Uaas recently designed for bioorthogonal chemistry, we present here a facile dual-fluorescence assay to evaluate relative yields of Uaa incorporation...
2016: Methods in Enzymology
Cathleen Juhl, Sylvia Els-Heindl, Ria Schönauer, Gorden Redlich, Erik Haaf, Frank Wunder, Bernd Riedl, Nils Burkhardt, Annette G Beck-Sickinger, Donald Bierer
The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability...
August 25, 2016: ChemMedChem
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