Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. (c) 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro

Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr-cis/trans-Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, (RCO)-C-1-pSer-Pro-NHR2, was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with N-15-labeled Pin1 were used to measure K( d )for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.

We report the first experimental evidence for a fluoro-alkene amide isostere participating in n→π* donation, which stabilizes the collagen triple helix. Of the three amide positions in canonical collagen-like peptides, Gly-Pro, Pro-Hyp, and Hyp-Gly, triple helix stability stands to benefit from substitution of only the isomerizable 3° Gly-Pro amide bond with a trans-locked fluoro-alkene. A (Z)-fluoro-alkene isostere of Gly-trans-Pro was synthesized, and its effect on the thermostability of a collagen-like peptide triple helix was measured. The mixture of enantiomers, Boc-Gly-Ψ[(Z)CFC]-L/D-Pro-OH, was synthesized in 8 steps with 27% overall yield, and the Fmoc-Gly-Ψ[(Z)CFC]-L/D-Pro-Hyp-OBn diastereomers were separated. The Gly-Ψ[(Z)CFC]-Pro isostere installed in a collagen-like peptide forms a stable triple helix. By CD, the thermal melting (Tm) value of the fluoro-alkene peptide was +42.2 ± 0.4 °C, and the Tm value of the control peptide was +48.4 ± 0.5 °C, a difference in stability of ΔTm -6.2 °C. Deshielding of the fluorine nucleus in the 19F NMR spectra is evidence of a stabilizing n→π* electronic interaction.

Collagen is the most abundant human protein, with the canonical sequence (Gly-Pro-Hyp)n in its triple helix region. Cis-trans isomerization of the Xaa-Pro amide has made two of these amide bonds the target of alkene replacement: the Gly-Pro and the Pro-Hyp positions. The conformations of Gly-Pro and Pro-Pro (as a Pro-Hyp model) fluoro-, chloro-, and proteo-alkene mimic models were investigated computationally to determine whether these alkenes can stabilize the polyproline type II (PPII) conformation of collagen. Second-order Møller-Plesset (MP2) calculations with various basis sets were used to perform the conformational analyses and locate stationary points. The calculation results predict that fluoro- and chloro-alkene mimics of Gly-Pro and Pro-Pro can participate in n→π* donation to stabilize PPII conformations, yet they are poor n→π* acceptors, shifting the global minima away from PPII conformations. For the proteo-alkene mimics, the lack of significant n→π* interactions and unstable PPII-like geometries explains their known destabilization of the triple helix in collagen-like peptides.

Full integration of green chemistry into the undergraduate curriculum is a necessity to prepare our students for a sustainable future. We discuss the reasons for the need to change the curriculum, the institutions in North America, Europe, and Asia that are leading the way towards integration with classroom resources, and the published textbooks that are currently available for both classroom and laboratory. We plead for more time for hard-pressed college professors to revamp the curriculum, and for these efforts to be valued. We feel compelled by the urgency of this need to implore the chemistry education community to participate in these efforts now.

ON THE COVER Enzyme promiscuity is regarded as a key driving force for the evolution of new enzyme functions, and ancestral sequences reconstruction is a powerful tool to investigate the appearance of enzymatic functions in natural enzyme lineages. In this cover image, some techniques used to characterize an ancestral enzyme are represented—an X-ray crystallography pattern and a phylogenetic tree where the ancestral enzyme structure emerges. The substrates tested in the article are shown as sticks; pyridoxal (vitamin B6) in yellow and hydroxymethyl pyrimidine (vitamin B1 intermediate) in orange. See article by GonzalezOrdenes et al., pp. 842–854.

Angewandte Chemie International EditionVolume 60, Issue 2 p. 517-535 Graphical AbstractFree Access Graphical Abstract: Angew. Chem. Int. Ed. 2/2021 First published: 04 January 2021 https://doi.org/10.1002/anie.202180211AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume60, Issue2January 11, 2021Pages 517-535 RelatedInformation

Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 μM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 μM, and lower affinity Kd2 values ranging from 3.1 to 227 μM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.

Green chemistry as a discipline is gaining increasing attention globally, with environmentally conscious students keen to learn how they can contribute to a safer and more sustainable world. Many universities now offer courses or modules specifically on green chemistry – Green Chemistry: Principles and Case Studies is an essential learning resource for those interested in mastering the subject. Providing a comprehensive overview of the concepts of green chemistry this book engages students with a thorough understanding of what we mean by green chemistry and how it can be put into practice. Structured around the well-known 12 Principles, and firmly grounded in real-world applications and case-studies, this book shows how green chemistry is already being put into practice and prepare them to think about how they can be incorporated into their own work. Targeted at advanced undergraduate and first-year graduate students with a background in general and organic chemistry, it is a useful resource both for students and for teachers looking to develop new courses.

Ab initio calculations of three models of collagen at positions Pro-Pro-Gly (1), Pro-Gly-Pro (2), and Gly-Pro-Pro (3) were performed to assess the conformational variation of n→π* contributions to the stability of the collagen triple helix. Full conformational analyses by relaxed potential-energy scans of the Ψ dihedral angle of the central residue in models 1, 2, and 3 revealed the presence of several n→π* interactions. In model 2, with Gly as the central residue, both the Φ and Ψ dihedral angles of Gly were scanned. Most minima of each model contained one or two n→π* interactions, with pyramidalization at the π* carbon. We also observed pyramidalization at the n→π* donor amide nitrogens. Minima with hydrogen-bond or non-native n→π* interactions compete with the collagen stabilizing n→π* interactions. The collagen-like n→ re-π* conformation was found as the global minimum only in model 3. The global minimum of 1 had a 5-membered ring hydrogen bond with an additional weak n→ si-π* interaction. The global minimum of 2 was in the extended conformation. We predict that the n→π* interactions found in native collagen, while individually small, cumulatively contribute to the stability of the triple helix conformation of collagen.

The cis vs trans conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the trans conformation, not cis, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation.

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac–pSer–Ψ[(Z)CH = C]–pipecolyl(Pip)–2-(2-naphthyl)ethylamine 1, that mimic L-pSer–D-Pro, D-pSer–L-Pro, and D-pSer–D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH3 debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC50 values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 μM, respectively.

Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.

In eukaryotes, the C‐terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription template through various post‐translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well‐characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. To address this question, we developed chemical compounds that can be used to mimic the cis and trans isomer states of proline residues in the CTD and therefore probe the selectivity of CTD binding proteins that are sensitive for this subtle variation. Our results established that the selectivity of prolyl isomerization state of CTD by phosphatases results in different regulatory effects and can lead to differentiated CTD phosphorylation states and eventually, transcription outcome. In particular, the phosphatases that specially recognize the minor species of cis proline will display enhanced activity upon prolyl isomerase regulation which is essential for accurate transcription termination. The application of such chemical probes can help us understand the molecular mechanism of the interplay between different post‐translational modifications with prolyl isomerization state and how that affects the conformational status of CTD in transcription.

Disinfection by-products (DBPs) arise when natural organic matter in source water reacts with disinfectants used in the water treatment process. Studies have suggested an association between DBPs and birth defects. Neural tube defects (NTDs) in embryos of untreated control mice were first observed in-house in May 2006 and have continued to date. The source of the NTD-inducing agent was previously determined to be a component of drinking water. Tap water samples from a variety of sources were analyzed for trihalomethanes (THMs) to determine if they were causing the malformations. NTDs were observed in CD-1 mice provided with treated and untreated surface water. Occurrence of NTDs varied by water source and treatment regimens. THMs were detected in tap water derived from surface water but not detected in tap water derived from a groundwater source. THMs were absent in untreated river water and laboratory purified waters, yet the percentage of NTDs in untreated river water were similar to the treated water counterpart. These findings indicate that THMs were not the primary cause of NTDs in the mice since the occurrence of NTDs was unrelated to drinking water disinfection.

The CAP2b neuropeptide family plays an important role in the regulation of the processes of diuresis and/or antidiuresis in a variety of insects. While Manse-CAP2b (pELYAFPRV-NH2) and native CAP2bs elicit diuretic activity in a number of species of flies, native CAP2b sequences have been shown to elicit antidiuretic activity in the kissing bug Rhodnius prolixus and the green stink bug Acrosternum hilare, the latter being an important pest of cotton and soybean in the southern United States. Analogs of CAP2b containing either a (Z)-alkene, cis-Pro or an (E)-alkene, trans-Pro isosteric component were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretion by Malpighian tubules isolated from A. hilare. The conformationally constrained trans-Pro analog demonstrated statistically significant antidiuretic activity, whereas the cis-Pro analog failed to elicit activity. The results are consistent with the adoption of a trans orientation for the Pro in CAP2b neuropeptides during interaction with receptors associated with the antidiuretic process in the stink bug. In addition, the results are further consistent with a theory of ligand-receptor coevolution between the CAP2b and pyrokinin/PBAN neuropeptide classes, both members of the '-PRXamide' superfamily. This work further identifies a scaffold with which to design mimetic CAP2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting CAP2b-regulated diuretic/antidiuretic functions.

The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp(2) to sp(3) in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C-H/D and C═O and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp(2) to sp(3) during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.