Terminomics methodologies and the completeness of reductive dimethylation: A meta-analysis of publicly available datasets

© 2019 by the authors. Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the labelling was not complete and investigated whether this was a common occurrence. We assessed the completeness of labelling in a selection of existing, publicly available N-terminomics datasets and empirically determined that amine-based labelling chemistry does not achieve complete labelling and potentially has issues with labelling amine groups at sequence-specific residues. This finding led us to conduct a thorough review of the historical literature that showed that this is not an unexpected finding, with numerous publications reporting incomplete labelling. These findings have implications for the quantitation of N-terminal peptides and the biological interpretations of these data

The formation of an apoplastic diffusion barrier in Arabidopsis seeds is regulated by peptide hormone signaling

Diffusion barrier formation is a critical factor in plant development. The most well described diffusion barriers in Arabidopsis are the Casparian strip and the cuticle. They function in the formation of organ boundaries, prevent water and molecule loss, and protect the plant against environmental stresses. The Casparian strip surrounds the root vascular tissue, whereas the cuticle covers aerial plant organs and is formed de novo during seed development. Embryonic cuticle formation is regulated by a peptide hormone signaling pathway, involving the leucine rich repeat receptor like kinases GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) and the subtilisin-like serine protease ABNORMAL LEAF SHAPE 1 (ALE1). Whereas the latter pathway components have been identified in 2001 and 2008, the peptide hormone mediating the signaling has remained elusive. One aim of this work was to identify the missing pathway element. It was hypothesized that the peptide hormone is released from a larger precursor by ALE1 protease activity to trigger cuticle formation via interaction with the GSO receptors. To uncover the unknown element, the signaling pathway for Casparian strip formation, prooved to be a useful lead. Remarkably, Casparian strip and embryonic cuticle formation employ the same receptor (GSO1), and for Casparian strip formation the GSO1 ligands are known to be members of the CASPARIAN STRIP INTEGRITY FACTOR (CIF) protein family (Doblas et al. 2017, Nakayama et al. 2017). Based on its similarity to the mature CIF peptides and on its phenotypic appearance, it was speculated that a seed expressed protein, called TWISTED SEED1 (TWS1), could serve as the sought ALE1 substrate. As it can be challenging to link proteases to their physiological substrates, this work describes methods how to identify protease specific cleavage sites. One of them was applied to test if TWS1 serves as ALE1 substrate. GFP-tagged TWS1 was transiently coexpressed with ALE1 in Nicotiana benthamiana via agroinfiltration. An ALE1-specific TWS1 cleavage product was detected in the protein extract of coinfiltrated leaves. It was identified by pull down via GFP immunoprecipitation, subsequent separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) analysis. Another method, described in this work, is the identification of protease cleavage sites by in-gel reductive dimethylation: cleavage product-containing gel bands are treated with formaldehyde and cyanoborohydride, prior to in-gel tryptic digest, to achieve a dimethylation of N-terminal free amino groups. The chemically modified N-termini can rapidly be identified and assigned to previous cleavage by the protease of interest. With the method described above, it was found that TWS1 is c-terminally cleaved by ALE1. The two amino acids directly flanking the cleavage site were found to be important for ALE1 cleavage site selection, as their substitution caused a loss of ALE1- dependent cleavage. Our cooperation partners demonstrated an interaction of mature TWS1 with the GSO receptors. The binding affinity of mature TWS1 was reduced by a 3 amino acid C-terminal extension, demonstrating the biological relevance of ALE1-mediated TWS1 processing. Like the CIFs, TWS1 contains a DY tyrosine sulfation motive at its N-terminal processing site. The role of tyrosine sulfation in precursor processing is largely unexplored and was addressed in this work by comparing in-vitro cleavage of different sulfated versus nonsulfated TWS1 precursors. SBT1.8 was found to cleave TWS1 at the N-terminal processing site, and cleavage site selection was influenced by the sulfation state of TWS1 P2´ tyrosine. A homology based 3D model of SBT1.8 was created, which suggested that SBT1.8 interacts with the negatively charged sulfate via a positively charged arginine residue (R302). The role of R302 in substrate binding and recognition was confirmed by in-vitro cleavage assays with mutated SBT1.8 versions, in which R302 was replaced. N-terminal TWS1 cleavage was no longer observed when R302 was substituted. Likewise, no N-terminal cleavage was observed for two other seed expressed Arabidopsis subtilases (SBT1.1 and SBT5.4) that feature an arginine at the corresponding position, indicating that the sole presence of R302 is not sufficient for N terminal cleavage site recognition.Die Bildung von Diffusionsbarrieren ist ein entscheidender Faktor in der Entwicklung von Pflanzen. Die am besten beschriebenen Diffusionsbarrieren in Arabidopsis sind der Caspary-Streifen und die Cuticula. Sie dienen der Abgrenzung von Organen, verhindern den Verlust von Wasser und Molekülen und schützen die Pflanze vor umweltbedingtem Stress. Der Caspary-Streifen umgibt das vaskuläre Gewebe der Wurzel, wohingegen die Cuticula oberirdische Pflanzenorgane bedeckt und während der Samenentwicklung de novo gebildet wird. Die Bildung der embryonalen Cuticula wird von einem Peptidhormon Signalweg reguliert, in dem die Leucin-reichen Rezeptor-ähnlichen Kinasen GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) und die Subtilisin-ähnliche Serinprotease ABNORMAL LEAF SHAPE 1 (ALE1) involviert sind. Während letztere bereits 2001 und 2008 identifiziert wurden, blieb das Signal-vermittelnde Peptidhormon schwer zu fassen. Ein Ziel dieser Arbeit war es, dieses letzte Element des Signalwegs zu identifizieren. Es wurde die Hypothese aufgestellt, dass das Peptidhormon von einem größeren Vorläufer durch ALE1 Protease Aktivität freigesetzt wird und durch eine Interaktion mit den GSO Rezeptoren die Bildung der Cuticula auslöst. Um das unbekannte Element aufzudecken erwies sich der Signalweg zur Bildung des Caspary-Streifens als nützlicher Anhaltspunkt. Beachtenswerter Weise wird zur Bildung des Caspary-Streifens und der embryonalen Cuticula der gleiche Rezeptor (GSO1) eingesetzt und es war bekannt, dass die GSO1 Liganden zur Bildung des Caspary-Streifens zur Familie der CASPARIAN STRIP INTEGRITY FACTOR (CIF) Proteine gehören (Doblas et al. 2017, Nakayama et al. 2017). Aufgrund seiner Ähnlichkeit zu den reifen CIF Peptiden und seines loss-of-function Phänotyps wurde spekuliert, dass TWISTED SEED1 (TWS1) als das gesuchte ALE1 Substrat im Samen dienen könnte. Um den oft schwierigen Nachweis einer Verbindung von Proteasen mit ihren physiologischen Substraten zu führen, beschreibt diese Arbeit Methoden zur Identifizierung von Protease-spezifischen Spaltstellen. Eine davon wurde angewendet um zu testen, ob TWS1 als ALE1 Substrat dient. GFP markiertes TWS1 wurde transient mit ALE1 in Nicotiana benthamiana coexprimiert. Im Proteinextrakt der coinfiltrierten Blätter wurde ein ALE1-spezifisches TWS1 Spaltprodukt detektiert. Es wurde durch Pulldown via GFP Immunpräzipitation, anschließende Auftrennung durch Natriumdodecylsulfat-Polyacrylamid-Gelelektrophorese (SDS-PAGE) und massenspektrometrische Analyse identifiziert. Eine weitere in dieser Arbeit beschriebene Methode ist die Identifizierung von Protease-Spaltstellen durch in-Gel reduzierende Dimethylierung: Spaltprodukt enthaltende Gelbanden werden vor tryptischem in-Gel Verdau mit Formaldehyd und Cyanoborhydrid behandelt, um eine Dimethylierung der freien N-terminalen Aminogruppen zu erzielen. Die chemisch modifizierten N-Termini können schnell identifiziert und einer vorhergehenden Spaltung durch die zu untersuchende Protease zugeordnet werden. Mit der oben beschriebenen Methode wurde gezeigt, dass TWS1 C-terminal von ALE1 gespalten wird. Die beiden Aminosäuren, die die Spaltstelle direkt flankieren, sind für die Erkennung wichtig, da deren Austausch zu einem Ausbleiben der ALE1-abhängigen Spaltung führt. Unsere Kooperationspartner haben eine Interaktion von reifem TWS1 mit den GSO Rezeptoren nachgewiesen, wobei die Bindungsaffinität des reifen TWS1 durch eine C-terminale Extension um 3 Aminosäuren reduziert wurde, was die biologische Relevanz der ALE1-vermittelten TWS1 Prozessierung darlegt. Wie die CIFs enthält TWS1 an seiner N-terminalen Prozessierungsstelle ein DY Tyrosin-Sulfatierungs Motiv. Die Rolle von Tyrosin-Sulfatierung in der Prozessierung von Vorläufern ist weitgehend unerforscht und wurde in dieser Arbeit durch das Vergleichen von in-vitro Spaltungen verschiedener sulfatierter mit nicht sulfatierten TWS1 Vorläufern untersucht. Es wurde gezeigt, dass die im Samen exprimierte Protease SBT1.8 TWS1 N-terminal prozessiert und, dass die Selektion der SBT1.8 Spaltstelle durch den Sulfatierungszustand von TWS1 P2´ Tyrosin beeinflusst wird. Ein Homologiemodell von SBT1.8 wies darauf hin, dass ein positiv geladener Argininrest (R302) mit dem negativ geladenen Sulfat interagiert. Die Bedeutung von R302 für Bindung und Erkennung des Substrates wurde durch in-vitro Spaltversuche mit mutierten SBT1.8 Varianten, in denen R302 ersetzt wurde, bestätigt. N-terminale TWS1 Spaltung wurde nach Austausch von R302 nicht mehr beobachtet. Für zwei weitere im Samen exprimierte Arabidopsis Subtilasen (SBT1.1 und SBT5.4) mit Arginin an entsprechender Position wurde auch keine N-terminale Spaltung beobachtet, was darauf hinweist, dass R302 für die Erkennung der N-terminalen Spaltstelle nicht allein verantwortlich ist

유기물의 C1 작용기화 반응 개발: N-메틸화, N-포밀화, 그리고 C(sp3)–H 트리플루오르메틸화 반응

학위논문 (박사) -- 서울대학교 대학원 : 자연과학대학 화학부, 2021. 2. 이홍근.본 논문에서는 유기금속 복합체를 이용한 새로운 C1 작용기화 반응을 개발하였다. 파트 1에서는 메탄올을 C1 원천으로 이용한 촉매적 활성화 방법에 대해 논한다. 메탄올은 다양한 분야에서 활용되는 생분해성 물질로, 경제적이고 지속 가능한 화합물로 주목을 받는다. 1장에서는 메탄올이 유기 합성에 도입되는 대표적인 반응 예시들을 기술한다. 2 장에서는 메탄올과 루테늄 촉매를 이용한 다양한 아민의 단일 N–메틸화 반응에 대해서 기술한다. 메탄올은 탈수소화 반응을 통해 포름알데이드로 활성화가 되고, 메커니즘 연구를 통해서 수소 가스를 이용한 속도론적 반응성 조절이 본 선택성의 핵심임이 제안된다. 같은 루테늄 촉매는 간단한 반응 조건의 조절을 통해서 메탄올이 아민의 N–포밀화, N,N–이중메틸화, N,N-포밀메틸화 반응에도 활용될 수 있는 것을 보였다 (3 장). 파트 2에서는 유기 물질에 C1 트리플루오르메틸 작용기를 도입하여 C(sp3)–CF3 결합을 만드는 반응에 대해서 기술한다. 트리풀루오르메틸 작용화는 의약 개발 등 여러 분야에서의 활용성을 이유로 유기 합성에서 중요성이 대두되고 있다. 4 장에서는 최근의 C(sp3)–CF3 결헙 형성 방법에 대해서 정리한다. C–H 활성화 트리플루오르메틸화는 중요한 해당 작용기를 복잡한 분자에 한번에 도입 할 수 있는 방법을 제시할 수 있어 이상적인 작용기화 반응으로 여겨지고 있다. 5 장에서는 구리–트리플루오르메틸 복합체와 빛을 이용하여 활성화되지 않은 일반적인 알케인의 C(sp3)–H 트리플루오르메틸화 반응에 대해서 기술한다. 본 합성 방법은 도전적인 반응이 간단한 조건에서 가능하도록 하였고, 다양한 생활성 및 복잡한 분자의 작용기화를 통해 그 활용성을 보인다.Two C1 functionalization methods were developed using organometallic complexes. In Part I, catalytic utilizations of methanol as a C1 source are discussed. Methanol is an economical and sustainable chemical because of its wide applicability and biodegradability, which makes it environmentally friendly. Representative reported examples on organic syntheses using methanol were introduced in chapter 1. A selective N-monomethylation of various amines using methanol as the methylating reagent was achieved with ruthenium pincer complex (Chapter 2). Methanol is activated to formaldehyde by the acceptorless dehydrogenation, and kinetic reactivity control by additional hydrogen gas is suggested as the key of controlling the selectivity based on the mechanistic studies. With the same ruthenium catalyst, selective N-formylation, N,N-dimethylation, and N,N-formylmethylation reactions of amines using methanol were realized through the simple tuning of reaction conditions (Chapter 3). Part II describes the introduction of trifluoromethyl C1 group into organic molecules forming a C(sp3)–CF3 bond. Trifluoromethylation is a high demand functionalization in contemporary organic synthesis due to its widespread applicability, especially for drug development. Chapter 4 reviewed the current approaches for the C(sp3)–CF3 bond formation. C–H trifluoromethylation is an ideal method as direct introduction of the important trifluoromethyl group into complex bioactive molecules can be achieved in a single-step. A direct C(sp3)–H trifluoromethylation of unactivated alkanes using a photo-induced bpyCu(CF3)3 (bpy = 2,2’-bipyridine) complex was successfully achieved (Chapter 5). The method enabled the challenging reaction under mild conditions, and was applied for the functionalization of versatile bioactive or complex molecules in a single step.Abstract 1 Table of Contents 3 List of Tables 7 List of Schemes 8 List of Figures 11 Appendix 195 Abstract in Koreans 271 Part I. Selective C–N Functionalizations of amines with Methanol as the C1 Source 13 Chapter 1. Utilization of Methanol as a C1 Source via Transition Metal Catalysis 13 1.1. Introduction 13 1.2. Methanol utilization via oxygen nucleophile 14 1.3. Methanol utilization via transition metal-catalyzed dehydrogenation 17 1.3.1. C–C1 functionalization with methanol 19 1.3.2. N–C1 functionalization with methanol 23 1.4. Methanol utilization via radical pathway 29 1.5. Conclusion 33 1.6. References 34 Chapter 2. Selective Monomethylation of Amines with Methanol as a C1 Source 39 2.1. Introduction 39 2.2. Results and discussion 43 2.2.1. Reaction condition optimization 43 2.2.2. Substrate scope of amines 45 2.2.3. Mechanistic studies 49 2.3. Conclusion 51 2.4. Experimental section 52 2.4.1. General information 52 2.4.2. General reaction procedures 53 2.4.3. Supplementary optimizations 54 2.4.4. Control experiments and byproduct yield 55 2.4.5. Kinetic profiling studies 58 2.4.6. Characterization of newly reported compounds 64 2.5. References 71 Chapter 3. Selective N-Formylation and N-Methylation of Amines Using Methanol as a Sustainable C1 Source 76 3.1. Introduction 76 3.2. Results and discussion 81 3.2.1. Proposed strategy to control the selectivity 81 3.2.2. Reaction condition optimization 83 3.2.3. Substrate scope of amines 88 3.2.4. Mechanistic studies 91 3.3. Conclusion 99 3.4. Experimental section 100 3.4.1. General information 100 3.4.2. General reaction procedures 100 3.4.3. Supplementary optimizations and data 102 3.4.4. Characterization of newly reported compounds 104 3.5. References 107 Part II. Direct C(sp3)–H Trifluoromethylation of Unactivated Alkanes 109 Chapter 4. Development and Application of C(sp3)–CF3 Bond Formation 109 4.1. Introduction 109 4.2. C(sp3)–CF3 bond formation via diverse synthetic methods 110 4.2.1. C(sp3)–CF3 bond formation via substitution of CF3 source 110 4.2.2. C(sp3)–CF3 bond formation via addition of CF3 source 118 4.4. C(sp3)–CF3 bond formation via CH activation 126 4.5. Conclusion 131 4.6. References 132 Chapter 5. Direct C(sp3)–H Trifluoromethylation of Unactivated Alkanes Enabled by Multifunctional Trifluoromethyl Cu Complexes 137 5.1. Introduction 137 5.2. Results and discussion 140 5.2.1. Reaction condition optimization 140 5.2.2. Substrate scope of alkanes 144 5.2.3. Mechanistic studies 149 5.3. Conclusion 158 5.4. Experimental section 159 5.4.1. General information 159 5.4.2. Optimization of reaction condition 160 5.4.3. Cu complex preparation and characterization 163 5.4.4. General procedure for substrate scopes 168 5.4.5. Characterization of newly reported compounds 168 5.4.6. Mechanistic studies 176 5.5. Computational section 187 5.5.1. General considerations 187 5.5.2. Reductive elimination from bisulfate Cu(III) complexes 188 5.5.3. Time-dependent density functional theory calculation 189 5.6. References 190 Appendix Chapter 2 195 Chapter 3 209 Chapter 5 216Docto

N-Alkyl-α-amino acids in Nature and their biocatalytic preparation

PWS would like to acknowledge the European Union for his current funding: “This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No 665919”.N-alkylated-α-amino acids are useful building blocks for the pharmaceutical and fine chemical industries. Enantioselective methods of N-alkylated-α-amino acid synthesis are therefore highly valuable and widely investigated. While there are a variety of chemical methods for their synthesis, they often employ stoichiometric quantities of hazardous reagents such as pyrophoric metal hydrides or genotoxic alkylating agents, whereas biocatalytic routes can provide a greener and cleaner alternative to existing methods. This review highlights the occurrence of the N-alkyl-α-amino acid motif and its role in nature, important applications towards human health and biocatalytic methods of preparation. Several enzyme classes that can be used to access chiral N-alkylated-α-amino acids and their substrate selectivities are detailed.PostprintPeer reviewe

Identification of N-terminal and C-terminal peptides in proteomics

Background: Identifying modifications made to terminal parts of proteins are very useful in understanding diseases and other out of the ordinary biological states. This thesis has focused on developing methods for enriching N-terminal and C-terminal peptides from a complex protein mixture, so that analysis of these samples can give better, more comprehensive and more reproducible results.Materials and methods: This thesis applies a bottom-up proteomics work-flow approach to develop and compare methods for enrichment of N-terminal, using two digestion enzymes, three sample clean-up methods, and two enrichment agents in different combinations. One method for C-terminal enrichment was developed, with basis in the method that gave the best results from N-terminal enrichment.Results and discussion: None of the N-terminal enrichment methods improved the number of terminal peptides compared to the control samples. However, the results suggest that trypsin should be the enzyme of choice when enriching for N-terminal. The method for enrichment of C-terminal peptides was developed with basis in the method that gave the best results for the N-terminal enrichment. This method yielded only one terminal peptide, which is far lower than expected based on existing literature.Conclusion: While none of the methods developed in this thesis improves the number of terminal peptides, it would seem that trypsin should be chosen over chymotrypsin when enriching N-terminal peptides. This result, however, has not been validated and should be investigated further

Synthesis of adjacent-bridged benzo-annelated cyclam and studies of mono- and di-benzo-annelated cyclam derivatives

A new synthetic methodology for the preparation of a novel adjacent-bridged benzoannelated cyclam is presented. This methodology utilizes reductive ring expansion followed by deallylation to give the desired adjacent-bridged benzo-annelated cyclam. This novel ligand is designed to complex metal cations with a trans coordination geometry. Derivatives of this ligand can be prepared by attaching pendant arms on the nitrogens which can aid as coordinating arms and linkers for potential use of this ligand as bifunctional chelator (BFC). Coupling chemistry studies have been carried out in an attempt to increase the lipophilicity of a dibenzo-annelated tetracyclic bisaminal and to functionalize at the para positions to provide a site for bio-conjugation

Heterogeneous PdAg alloy catalyst for selective methylation of aromatic amines with formic acid under an additive-free and solvothermal one-pot condition

The methylation of amines for the synthesis of methylamines and dimethylamines as platform chemicals has been attempted mostly by homogeneous catalysts with acid additives. However, there are scarcely any reports on heterogeneous catalytic methylation reactions except for a routine approach under high temperature and high pressure of CO2 and H-2 gases for extended reaction times. Here we report a heterogeneously catalyzed selective methylation of aromatic amines using reactive and nontoxic formic acid as the only source for the construction of methyl groups, under ambient pressure in an additive-free one-pot reaction condition. Equal proportions of Pd and Ag in the PdAg/Fe3O4/N-rGO catalyst deliver highly selective amine methylation without aromatic ring hydrogenation, as the strained Pd in the alloy is combined with the graphene-derived support, preventing nanoparticle agglomeration and the action of magnetite as a promoter. Both N-methylation and N, N-dimethylation of various substituted aromatic amines were performed with complete conversion and excellent 90-97% selectivity by controlling the reaction times in the range of 10-24 h at 140 degrees C without unwanted aromatic ring hydrogenation. Furthermore, the developed bimetallic catalyst provided high yields (88-91%) of methylation with CO2+H-2 gas under high pressure, which are as good as the results of homogenous catalysts with an acid additive. To the best of our knowledge, our use of this environmentally friendly methodology is the first time that this durable heterogeneous catalyst has readily performed highly selective methylation at ambient pressure, which is attractive for industrial applications.1193Ysciescopu