RESUMO
Tuberculosis, caused by Mycobacterium tuberculosis, remains a major public health concern, demanding new antibiotics with innovative therapeutic principles due to the emergence of resistant strains. Benzothiazinones (BTZs) have been developed to address this problem. However, an unprecedented in vivo biotransformation of BTZs to hydride-Meisenheimer complexes has recently been discovered. Herein, we present a study of the influence of electron-withdrawing groups on the propensity of HMC formation in whole cells for a series of C-6-substituted BTZs obtained through reductive fluorocarbonylation as a late-stage functionalization key step. Gibbs free energy of reaction and Mulliken charges and Fukui indices on C-5 at quantum mechanics level were found as good indicators of in vitro HMC formation propensity. These results provide a first blueprint for the evaluation of HMC formation in drug development and set the stage for rational pharmacokinetic optimization of BTZs and similar drug candidates.
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Benzothiazinones (BTZs) have widely inspired medicinal chemistry and translational research due to their remarkable antitubercular potency and clinical potential. While most structure-activity relationship campaigns have largely focused on lateral chain modifications and substituents on the BTZ core, scaffold hopping strategies have been rarely investigated previously. In this work, we report the first example of ring expansion of the BTZ core toward benzofuran- and naphthalene-fused thiazinones. In vitro testing showed micromolar activity for both compounds, and molecular docking simulations provided insights into their reduced inhibitory capacity toward the enzymatic target (DprE1). Calculated electrochemical potentials revealed a lower susceptibility to reduction as opposed to BTZ drug candidates, in line with the mechanistic requirement for covalent binding.
Assuntos
Benzofuranos , Mycobacterium tuberculosis , Relação Estrutura-Atividade , Simulação de Acoplamento Molecular , Antituberculosos/farmacologia , Antituberculosos/química , Benzofuranos/farmacologia , Naftalenos/farmacologiaRESUMO
A systematic experimental study was performed to determine laser irradiation conditions for the large-area fabrication of highly regular laser-induced periodic surface structures (HR-LIPSS) on a 220 nm thick Mo film deposited on fused silica. The LIPSS were fabricated by scanning a linearly polarized, spatially Gaussian laser beam at 1030 nm wavelength and 1.4 ps pulse duration over the sample surface at 1 kHz repetition rate. Scanning electron microscope images of the produced structures were analyzed using the criterion of the dispersion of the LIPSS orientation angle (DLOA). Favorable conditions, in terms of laser fluence and beam scanning overlaps, were identified for achieving DLOA values <10∘. To gain insight into the material behavior under these irradiation conditions, a theoretical analysis of the film heating was performed, and surface plasmon polariton excitation is discussed. A possible effect of the film dewetting from the dielectric substrate is deliberated.
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Antigen processing and presentation are the cornerstones of adaptive immunity. B cells cannot generate high-affinity antibodies without T cell help. CD4+ T cells, which provide such help, use antigen-specific receptors that recognize major histocompatibility complex (MHC) molecules in complex with peptide cargo. Similarly, eradication of virus-infected cells often depends on cytotoxic CD8+ T cells, which rely on the recognition of peptide-MHC complexes for their action. The two major classes of glycoproteins entrusted with antigen presentation are the MHC class I and class II molecules, which present antigenic peptides to CD8+ T cells and CD4+ T cells, respectively. This Review describes the essentials of antigen processing and presentation. These pathways are divided into six discrete steps that allow a comparison of the various means by which antigens destined for presentation are acquired and how the source proteins for these antigens are tagged for degradation, destroyed and ultimately displayed as peptides in complex with MHC molecules for T cell recognition.
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Apresentação de Antígeno , Linfócitos T CD8-Positivos , Humanos , Complexo Principal de Histocompatibilidade , Antígenos de Histocompatibilidade Classe I , Antígenos , Peptídeos , Antígenos de Histocompatibilidade Classe IIRESUMO
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.
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Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Pandemias , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , Camelídeos Americanos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias/prevenção & controle , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/genética , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.
Assuntos
Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/terapia , Monitorização Imunológica/métodos , Neoplasias Cutâneas/terapia , Animais , Anticorpos Monoclonais/metabolismo , Diagnóstico por Imagem , Feminino , Memória Imunológica , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tomografia por Emissão de Pósitrons , Anticorpos de Cadeia Única/metabolismoRESUMO
In vivo imaging has become in recent years an incredible tool to study biological events and has found critical applications in diagnostic medicine. Although a lot of efforts and applications have been achieved using monoclonal antibodies, other types of delivery agents are being developed. Among them, VHHs, antigen binding fragments derived from camelid heavy chain-only antibodies, also known as nanobodies, have particularly attracted attention. Indeed, their stability, fast clearance, good tissue penetration, high solubility, simple cloning and recombinant production make them attractive targeting agents for imaging modalities such as PET, SPECT or Infra-Red. In this review, we discuss the pioneering work that has been carried out using VHHs and summarize the recent developments that have been made using nanobodies for in vivo, non-invasive, imaging.
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Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for â¼120 days, are biocompatible, and are immunologically largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clinical trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiological conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro. This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.
Assuntos
Carbono-Nitrogênio Ligases/química , Membrana Eritrocítica/metabolismo , Peptídeos/química , Anticorpos de Domínio Único/química , Animais , Carbono-Nitrogênio Ligases/genética , Engenharia Celular , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Diabetes Mellitus Experimental/prevenção & controle , Membrana Eritrocítica/química , Transfusão de Eritrócitos , Feminino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Oldenlandia/enzimologia , Proteínas de Plantas/genéticaRESUMO
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells. To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named "Nanoblades") were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for "transfection-free" homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.
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Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/genética , Humanos , TransfecçãoRESUMO
Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in cats with infection in its progressive form. Although there are numerous reports on the occurrence of FeLV in the feline population worldwide, there is a paucity of data in Asia. In this study, we assessed the circulation of FeLV by ELISA and nested PCR in cats from different countries in Southeast Asia (i.e., Thailand, Malaysia, Singapore, Philippines, Indonesia and Vietnam) and Taiwan during 2017-2018. Forty-seven cats were positive to FeLV by antigen or provirus detection, but 32 samples were considered truly positive on the basis of positive molecular testing. Frequency of occurrence of FeLV proviral DNA ranged from 0% (0/43 positive samples) in Indonesia to 18.5% (22/119 positive samples) in Thailand. A statistically significant association (p < 0.05) was found between country of cats origin, age, lifestyle, abnormal oral mucosa, and FeLV molecular positive results. In-depth studies are needed in other countries in Southeast Asia to elucidate the mosaic of knowledge about FeLV epidemiology.
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Doenças do Gato/epidemiologia , Vírus da Leucemia Felina/genética , Animais de Estimação/virologia , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Animais , Sudeste Asiático/epidemiologia , Doenças do Gato/sangue , Doenças do Gato/virologia , Gatos/virologia , DNA Viral/genética , Feminino , Vírus da Leucemia Felina/classificação , Vírus da Leucemia Felina/isolamento & purificação , Masculino , Provírus/genética , Infecções por Retroviridae/sangue , Infecções por Retroviridae/epidemiologia , Fatores de Risco , Taiwan/epidemiologia , Infecções Tumorais por Vírus/epidemiologia , Carga ViralRESUMO
Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.
Assuntos
Anticorpos Monoclonais/imunologia , Bioquímica , Técnicas Citológicas , Anticorpos de Domínio Único/imunologia , Animais , HumanosRESUMO
Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.
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Cisteína Endopeptidases/metabolismo , Engenharia de Proteínas/métodos , Proteínas/química , Motivos de Aminoácidos , Catálise , Cisteína Endopeptidases/genética , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Oldenlandia/enzimologia , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/químicaRESUMO
The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.
Assuntos
Células Epiteliais/virologia , Perfilação da Expressão Gênica/métodos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Sindbis virus/genética , Transcriptoma , Neoplasias do Colo do Útero/virologia , Regiões 5' não Traduzidas , Sítios de Ligação , Células Epiteliais/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Sindbis virus/crescimento & desenvolvimento , Sindbis virus/metabolismo , Sindbis virus/patogenicidade , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Replicação ViralRESUMO
Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.
Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Vetores Genéticos/genética , RNA Guia de Cinetoplastídeos/genética , Ribonucleoproteínas/genética , Animais , Linhagem Celular Tumoral , Reparo do DNA/genética , Embrião de Mamíferos , Fibroblastos , Edição de Genes/economia , Genoma/genética , Células HEK293 , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes Induzidas , Vírus da Leucemia Murina/genética , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Ativação Transcricional/genéticaRESUMO
It is well known that a binary system of nonactive disks that experience driving in opposite directions exhibits jammed, phase separated, disordered, and laning states. In active matter systems, such as a crowd of pedestrians, driving in opposite directions is common and relevant, especially in conditions which are characterized by high pedestrian density and emergency. In such cases, the transition from laning to disordered states may be associated with the onset of a panic state. We simulate a laning system containing active disks that obey run-and-tumble dynamics, and we measure the drift mobility and structure as a function of run length, disk density, and drift force. The activity of each disk can be quantified based on the correlation timescale of the velocity vector. We find that in some cases, increasing the activity can increase the system mobility by breaking up jammed configurations; however, an activity level that is too high can reduce the mobility by increasing the probability of disk-disk collisions. In the laning state, the increase of activity induces a sharp transition to a disordered strongly fluctuating state with reduced mobility. We identify a novel drive-induced clustered laning state that remains stable even at densities below the activity-induced clustering transition of the undriven system. We map out the dynamic phase diagrams highlighting transitions between the different phases as a function of activity, drive, and density.
RESUMO
Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Imunoglobulina G/química , Ligases/química , Proteínas Recombinantes de Fusão/química , Carbono/química , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida , Engenharia de Proteínas/métodosRESUMO
The formation and properties of laser-induced periodic surface structures (LIPSS) were investigated upon fs-laser irradiation of fused silica at different initial substrate temperatures, TS. For substrate heating between room temperature, TRT, and TS = 1200 °C, a continuous wave CO2 laser was used as the radiation source. The surface structures generated in the air environment at normal incidence with five successive fs-laser pulses (pulse duration, τ = 300 fs, laser wavelength, λ = 1025 nm, repetition frequency, frep = 1 kHz) were characterized by using optical microscopy, scanning electron microscopy, and 2D-Fourier transform analysis. The threshold fluence of fused silica was systematically investigated as a function of TS. It was shown that the threshold fluence for the formation of low-spatial frequency LIPSS (LSFL) decreases with increasing TS. The results reveal that the initial spatial period observed at TRT is notably increased by increasing TS, finally leading to the formation of supra-wavelength LIPSS. The findings are discussed in the framework of the electromagnetic interference theory, supplemented with an analysis based on thermo-convective instability occurring in the laser-induced molten layer. Our findings provide qualitative insights into the formation mechanisms of LIPSS, which allow improvements of the control of nanostructure formation to be made for corresponding applications of dielectric materials in the future.
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Interferon-induced transmembrane proteinâ 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5â kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15â mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.
Assuntos
Proteínas de Membrana/síntese química , Proteínas de Ligação a RNA/síntese química , Sequência de Aminoácidos , Técnicas de Química Sintética/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Modelos Moleculares , Fosforilação , Proteínas de Ligação a RNA/químicaRESUMO
Highly regular laser-induced periodic surface structures (HR-LIPSS) have been fabricated on surfaces of Mo, steel alloy and Ti at a record processing speed on large areas and with a record regularity in the obtained sub-wavelength structures. The physical mechanisms governing LIPSS regularity are identified and linked with the decay length (i.e. the mean free path) of the excited surface electromagnetic waves (SEWs). The dispersion of the LIPSS orientation angle well correlates with the SEWs decay length: the shorter this length, the more regular are the LIPSS. A material dependent criterion for obtaining HR-LIPSS is proposed for a large variety of metallic materials. It has been found that decreasing the spot size close to the SEW decay length is a key for covering several cm2 of material surface by HR-LIPSS in a few seconds. Theoretical predictions suggest that reducing the laser wavelength can provide the possibility of HR-LIPSS production on principally any metal. This new achievement in the unprecedented level of control over the laser-induced periodic structure formation makes this laser-writing technology to be flexible, robust and, hence, highly competitive for advanced industrial applications based on surface nanostructuring.
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We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA) ligation. The new synthetic pathway to the enantiopure oxaproline is based on a chiral amine mediated enantioselective conjugate addition of a hydroxylamine to trans-4-oxo-2-butenoate. This route is practical, scalable and economical and provides decagram amounts of material for protein synthesis and conversion to other protected forms of (S)-oxaproline.