Real-time NMR Spectroscopy Research Articles

REACTION KINETICS OF THE BENZYLATION OF ADENINE IN DMSO: REGIO-SELECTIVITY GUIDED BY ENTROPY.

The factors governing the regio-selectivity of the alkylation of adenine have been of interest for many years due to the biological importance of adenine derivatives, however, no reaction kinetic studies have been conducted. Herein, we report the rate constants and activation parameters of the benzylation of adenine under basic conditions in DMSO in the absence and presence of 15-crown-5 ether using real-time 1H NMR spectroscopy. The reaction is second-order for the formation of the N9- and N3-benzyladenine products, with a regio-selectivity factor 2.3 in favour of the N9-adduct. The Gibbs free energy of activation amounts to 87±2 kJ mol-1 for both reactions. The formation of the N9-adduct is more activated by 7 kJ mol-1, but its effect is offset by a less negative activation entropy, demonstrating that the long-contested reason for the regioselectivity in the benzylation of adenine is dominated by compensation of entropy and enthalpy in the transition state. The kinetic parameters obtained in the presence of the 15-crown-5 ether indicate that the crown ether forms a complex with an adenine-sodium ion-pair, increasing the activation barrier. However, the Gibbs free energy in the absence and presence of the crown ether remains constant.

In situ Diazonium Salt Formation and Photochemical Aryl-Aryl Coupling in Continuous Flow Monitored by Inline NMR Spectroscopy.

A novel class of diazonium salts is introduced for the photochemical aryl-aryl coupling to produce (substituted) biphenyls. As common diazonium tetrafluoroborate salts fail, soluble and safe aryl diazonium trifluoroacetates are applied. In this mild synthesis route no catalysts are required to generate an aryl-radical by irradiation with UV-A light (365 nm). This reactive species undergoes direct C-H arylation at an arene, forming the product in reasonable reaction times. With the implementation of a continuous flow setup in a capillary photoreactor 13 different biphenyl derivatives are successfully synthesized. By integrating an inline 19F-NMR benchtop spectrometer, samples are reliably quantified as the fluorine-substituents act as a probe. Here, real-time NMR spectroscopy is a perfect tool to monitor the continuously operated system, which produces fine chemicals of industrial relevance even in a multigram scale.

Monitoring galactolipid digestion and simultaneous changes in lipid-bile salt micellar organization by real-time NMR spectroscopy

The use of Nuclear Magnetic Resonance spectroscopy for studying lipid digestion in vitro most often consists of quantifying lipolysis products after they have been extracted from the reaction medium using organic solvents. However, the current sensitivity level of NMR spectrometers makes possible to avoid the extraction step and continuously quantify the lipids directly in the reaction medium. We used real-time 1H NMR spectroscopy and guinea pig pancreatic lipase-related protein 2 (GPLRP2) as biocatalyst to monitor in situ the lipolysis of monogalactosyl diacylglycerol (MGDG) in the form of mixed micelles with the bile salt sodium taurodeoxycholate (NaTDC). Residual substrate and lipolysis products (monogalactosyl monoacylglycerol (MGMG); monogalactosylglycerol (MGG) and octanoic acid (OA) were simultaneously quantified throughout the reaction thanks to specific proton resonances. Lipolysis was complete with the release of all MGDG fatty acids. These results were confirmed by thin layer chromatography (TLC) and densitometry after lipid extraction at different reaction times. Using diffusion-ordered NMR spectroscopy (DOSY), we could also estimate the diffusion coefficients of all the reaction compounds and deduce the hydrodynamic radius of the lipid aggregates in which they were present. It was shown that MGDG-NaTDC mixed micelles with an initial hydrodynamic radius rH of 7.3 ± 0.5 nm were changed into smaller micelles of NaTDC-MGDG-MGMG of 2.3 ± 0.5 nm in the course of the lipolysis reaction, and finally into NaTDC-OA mixed micelles (rH of 2.9 ± 0.5 nm) and water soluble MGG. These results provide a better understanding of the digestion of galactolipids by PLRP2, a process that leads to the complete micellar solubilisation of their fatty acids and renders their intestinal absorption possible.

The self-assembly of l-histidine might be the cause of histidinemia

l-Histidine is an essential amino acid with unique biochemical and physiological properties. Histidinemia is a disease condition caused by the elevated level of l-histidine in our blood. Mutations in the histidase, an enzyme for the breakdown of histidine, is the cause of the rise in histidine concentration. To our knowledge, no research has been done on why a high concentration of histidine causes histidinemia. In this study, we provide a potential explanation why the elevated levels of histidine in the human body causes histidinemia. In this study we have found that l-histidine self-assembled in water to form nano sheet structures at physiological pH and temperature, using 1D 1H NMR spectroscopy, diffusion ordered spectroscopy (DOSY) and scanning electron microscope (SEM) techniques. The kinetics of self-assembly has been studied using real time NMR spectroscopy. We observed that both the aromatic ring and aliphatic part are equally contributing to the self-assembly of l-histidine. The symptoms of histidinemia, neurological deficits and speech delays, are similar to that of the neurodegenerative diseases caused by the self-assembly of peptides and proteins. We speculate that the self-assembly of l-histidine might be the cause of histidinemia.

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors.

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.

Designed for Circularity: Chemically Recyclable and Enzymatically Degradable Biorenewable Schiff Base Polyester-Imines

Bio-based plastics potentially have several positive impacts on the environment; however, in order to make a real difference, they need to have managed and sustainable end of life. This means they should from the start be designed for chemical, mechanical, and/or organic (biological) recycling. Development of energy-efficient and selective chemical recycling processes is a necessary part in reaching truly circular plastic flows. Polyesters are generally well suited for chemical recycling due to the presence of reversible ester bonds. Utilization of dynamic covalent chemistry to include a second, even more easily reversed bond, such as Schiff base (SB, imine bond), could further facilitate chemical recycling, enabling depolymerization back to monomeric products under mild conditions. Here, we present the synthesis of three vanillin-derived SB monomers SBM1, SBM2, and SBM3 and the corresponding polymers SBP3a–b, SBP4a–b, and SBP5a–b. Three different diamines and two potentially bio-sourced diesters were utilized to yield altogether six different polyester-imines with different aliphatic/aromatic contents. All the obtained SB-based polyesters were thermally stable at ∼290–330 °C and had a high char yield during the pyrolysis, which may indicate inherent flame resistance. All the polyesters were amorphous with glass transition temperatures from 36 to 76 °C. The chemical recyclability and hydrolytic degradation of the synthesized polyesters was evaluated by using real-time 1H NMR spectroscopy. Finally, the susceptibility of the synthesized polyester-imines to enzymatic degradation by PETase was demonstrated. The experimental results were further supported by induced-fit docking experiments to theoretically evaluate the potential productive binding of the produced polyester-imines and intermediates thereof to the active site of PETase.

Understanding Self-Assembly of Silica-Precipitating Peptides to Control Silica Particle Morphology.

The most advanced materials are those found in nature. These evolutionary optimized substances provide highest efficiencies, e.g., in harvesting solar energy or providing extreme stability, and are intrinsically biocompatible. However, the mimicry of biological materials is limited to a few successful applications since there is still a lack of the tools to recreate natural materials. Herein, such means are provided based on a peptide library derived from the silaffin protein R5 that enables rational biomimetic materials design. It is now evident that biomaterials do not form via mechanisms observed in vitro. Instead, the material's function and morphology are predetermined by precursors that self-assemble in solution, often from a combination of protein and salts. These assemblies act as templates for biomaterials. The RRIL peptides used here are a small part of the silica-precipitation machinery in diatoms. By connecting RRIL motifs via varying central bi- or trifunctional residues, a library of stereoisomers is generated, which allows characterization of different template structures in the presence of phosphate ions by combining residue-resolved real-time NMR spectroscopy and molecular dynamics (MD) simulations. Understanding these templates in atomistic detail, the morphology of silica particles is controlled via manipulation of the template precursors.

Real-time monitoring of the sialic acid biosynthesis pathway by NMR.

Sialic acids are part of the outermost component of the glycocalyx of all vertebrates; as such, they are fundamental markers in physiological and pathological processes. In this study, we introduce a real-time assay to monitor individual enzymatic steps of sialic acid biosynthesis, either with recombinant enzymes, in particular using UDP-N-acetylglucosamine 2-epimerase (GNE) or N-acetylmannosamine kinase (MNK), or in cytosolic rat liver extract. Using state-of-the-art NMR techniques, we are able to follow the characteristic signal of the N-acetyl methyl group, which displays different chemical shifts for the biosynthesis intermediates UDP-N-acetylglucosamine, N-acetylmannosamine (and its 6-phosphate) and N-acetylneuraminic acid (and its 9-phosphate). Pseudo 2- and 3-D NMR demonstrated that in rat liver cytosolic extract, the phosphorylation reaction of MNK is exclusive for N-acetylmannosamine generated by GNE. Thus, we speculate that phosphorylation of this sugar from other sources (e.g. external application to cells) or N-acetylmannosamine derivatives often applied in metabolic glycoengineering is not conducted by MNK but by a yet unknown sugar kinase. Competition experiments with the most prevalent neutral carbohydrates demonstrated that of these, only N-acetylglucosamine slowed N-acetylmannosamine phosphorylation kinetics, suggesting an N-acetylglucosamine-preferring kinase as the acting enzyme.

Fused Bis(hemi-indigo): Broad-Range Wavelength-Independent Photoswitches.

Wavelength-independent conversion of organic photoswitches in the photostationary state is a rare phenomenon that opens up a way for many practical applications. In this work, three fused bis(hemi-indigo) derivatives with different substitution patterns were synthesized and their photoswitching was investigated by optical spectroscopy, real-time NMR spectroscopy and TD-DFT calculations. We disclosed that the Z-E photoisomerization of the meta-bis(hemi-indigo) derivative was remarkably independent of the irradiation wavelength from UV up to yellow light. The wavelength-independent forward photoswitching together with the inhibited backward photoisomerization, high thermal stability of the photoinduced isomers as well as significant overlap between the photoswitch absorption and the solar spectrum allows to suggest bis(hemi-indigo) derivatives as promising candidates for molecular solar thermal energy storage (MOST) systems.

Real-Time 1H and 31P NMR spectroscopy of the copolymerization of cyclic phosphoesters and trimethylene carbonate reveals transesterification from gradient to random copolymers

Copolymerization is the general approach to combine properties of different polymers in a single material. We prepared the first trimethylene carbonate (TMC) phosphoester (PE) copolymers by statistical copolymerization using either stannous octanoate or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the respective catalyst. Variation of comonomer feed, reaction temperature and solvent, resulted in a library of copolymers with molar masses of up to 83,000 g/mol and moderate molar mass dispersities (<1.8). The monomer consumptions during the copolymerizations were followed by real-time 1H and 31P NMR spectroscopy in solution and in the bulk. As ring-opening copolymerization of cyclic esters often leads to gradient copolymers, also TMC and PE copolymerizations resulted in gradient copolymers, i.e. P(TMC-grad-PE)s, with different gradient strength depending on the copolymerization conditions. If a suitable transesterification catalyst like stannous octanoate was used, randomization of the gradient copolymer occurred. The 31P NMR shift of incorporated phosphoester units was used as a probe to monitor the randomization of the gradient copolymers in real-time by 31P NMR spectroscopy. The P-comonomer acts as an ideal NMR probe for in situ monitoring the changes of the copolymers’ microstructure during the transesterification by following the evolution of diads and triads. This paper focusses on the randomization procedure and gives detailed information about the copolymer structure and the kinetics of the copolymerization. The data give the possibility to understand randomization processes on a molecular scale and to fine-tune properties of polycarbonate-polyphosphoester copolymers in future applications.

Diversification of Metallic Molecules through Derivatization Chemistry of Au25 Nanoclusters.

Derivatization is the fine chemistry that can produce chemical compounds from similar precursors and has been widely used in the field of organic synthesis to achieve diversification of molecular properties and functionalities. Ligand-protected metal nanoclusters (NCs) are metallic molecules with a definite molecular formula, well-defined molecular structure, and molecular-like physical and chemical properties. Unlike organic compounds, which have almost infinite species, until now only hundreds of metal NC species have been discovered, and only a few of them have been structurally resolved. Therefore, the diversification of NC species and functions is highly desirable in nanoscience and nanochemistry. As an efficient approach for generating a library of compounds from a given precursor, derivatization chemistry is not only applicable in producing new organic compounds but also a promising strategy for generating new metal NC species with intriguing properties and functions. The key to the derivatization of metal NCs is to design an efficient derivatization reaction suitable for metal NCs and spontaneously realize the customization of this special macromolecule (metallic molecule) at the atomic and molecular level.In this Account, we use the flagship thiolate-protected NC Au25SR18 (SR denotes a thiolate ligand) as a model to illustrate the derivatization chemistry of metal NCs. In the past 3 years we have developed various derivatization reactions of Au25SR18, including isomerization, redox, ligand addition, alloying, and self-assembly reactions. We discuss the mechanisms that govern these reactions to realize precise customization of the NC structure, size, surface, composition, and interactions. It is particularly noteworthy that advanced techniques such as real-time electrospray ionization mass spectrometry and NMR spectroscopy enable us to have an atomic- and molecular-level understanding of the reaction mechanisms, which will further promote our efforts to design derivatization reactions for metal NCs. Through these delicate derivatization reactions, we can produce Au25SR18 derivatives with new physical, chemical, and biological properties, including electronic structures, photoluminescence, surface reactivity, and antimicrobial properties. Finally, we provide our perspectives on the opportunities and challenges of metal NC derivatization.The derivatization chemistry of metal NCs can not only diversify the properties and functions of metal NCs but also help us understand the structure-property relationship and design principles of metal nanomaterials, which will help advance the research frontier of nanoscience toward atomic precision.

NMR Detection Methods for Profiling RAS Nucleotide Cycling.

RAS oncoproteins exhibit a switch-like behavior to drive diverse signaling cascades. In the active GTP-bound state, a conformational change occurs in these enzymes that enables interaction with downstream effectors. Nucleotide-dependent conformational exchange is easily detected with real-time NMR (RT-NMR) spectroscopy. RT-NMR has been firmly established as an effective assay to measure RAS oncoprotein nucleotide exchange and GTP hydrolysis kinetics and can further determine the regulatory activity of guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). It is now possible to multiplex these assays, allowing for the precise monitoring of activation states for mixtures of RAS oncoproteins or other RAS superfamily GTPases. Here, we describe the protocols necessary to express and purify isotopically labeled RAS and detail how to carry out an RT-NMR assay on a singular RAS protein or on a mixture of small GTPases.

Refolding of Cold-Denatured Barstar Induced by Radio-Frequency Heating: A New Method to Study Protein Folding by Real-Time NMR Spectroscopy.

The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio‐frequency power to lossy aqueous samples, refolding of barstar from its cold‐denatured state can be followed by real‐time NMR spectroscopy. Since temperature‐induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real‐time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low‐temperature‐unfolded state, which can potentially induce multiple folding pathways. The high time resolution real‐time 2D‐NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state‐correlated spectroscopy, an alternative folding pathway circumventing the rate‐limiting cis‐trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.

Real-Time NMR Spectroscopy for Studying Metabolism.

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.

Quantitative Isotopomer Rates in Real-Time Metabolism of Cells Determined by NMR Methods.

Tracer-based metabolism is becoming increasingly important for studying metabolic mechanisms in cells. NMR spectroscopy offers several approaches to measure label incorporation in metabolites, including 13 C- and 1 H-detected spectra. The latter are generally more sensitive, but quantification depends on the proton-carbon 1 JCH coupling constant, which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites, and quantification of 1 H-edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer-based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Herein, we compare 13 C-filtered and 13 C-edited methods for quantification and show the applicability of the methods for real-time NMR spectroscopy of cancer-cell metabolism, in which label incorporations are subject to constant flux. We find an approach using a double filter to be most suitable and sufficiently robust to reliably obtain 13 C incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24 h. The proposed method is equally well suited for calculating the level of label incorporation in labelled cell extracts in the context of metabolic flux analysis.

Effect of the Substituent Position on the Anionic Copolymerization of Styrene Derivatives: Experimental Results and Density Functional Theory Calculations

In a combined synthetic, kinetic and theoretical study, the living anionic copolymerization of styrene and its ring-methylated derivatives ortho-, meta-, and para-methylstyrene (MS) was examined by real-time 1H NMR spectroscopy in the nonpolar solvents toluene-d8 and cyclohexane-d12 as well as by density functional theory calculations. Based on the NMR kinetics data, reactivity ratios for each comonomer pair were determined by the Kelen–Tudős method and numerical integration of the copolymerization equation (Contour software). The reaction pathway was modeled and followed by density functional theory (DFT) calculations to validate and predict the experimentally derived reactivity ratios. Unexpectedly, two of the three styrene derivatives showed completely different reactivities in copolymerization, governed by the position of the methyl group. While para-MS is considerably less reactive than styrene, resulting in gradient copolymers (rS = 2.62; rpMS = 0.37), ortho-MS showed the opposite behavior and is mo...

Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases determined in parallel

Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg2+ abundance.

Combining Hyperpolarized Real-Time Metabolic Imaging and NMR Spectroscopy To Identify Metabolic Biomarkers in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer that progresses without any symptom, and oftentimes, it is detected at an advanced stage. The lack of prior symptoms and effective treatments have created a knowledge gap in the management of this lethal disease. This issue can be addressed by developing novel noninvasive imaging-based biomarkers in PDAC. We explored in vivo hyperpolarized (HP) 13C MRS of pyruvate to lactate conversion and ex vivo 1H NMR spectroscopy in a panel of well-annotated patient-derived PDAC xenograft (PDXs) model and investigated the correlation between aberrant glycolytic metabolism and aggressiveness of the tumor. Real-time metabolic imaging data demonstrate the immediate intracellular conversion of HP 13C pyruvate to lactate after intravenous injection interrogating upregulated lactate dehydrogenase (LDH) activity in aggressive PDXs. Total ex vivo lactate measurement by 1H NMR spectroscopy showed a direct correlation with in vivo dynamic pyruvate-to-lactate conversion and demonstrated the potential of dynamic metabolic flux as a biomarker of total lactate concentration and aggressiveness of the tumor. Furthermore, the metabolite concentrations were very distinct among all four tumor types analyzed in this study. Overexpression of LDH-A and hypoxia-inducible factor (HIF-1α) plays a significant role in the conversion kinetics of HP pyruvate-to-lactate in tumors. Collectively, these data identified aberrant metabolic characteristics of pancreatic cancer PDXs and could potentially delineate metabolic targets for therapeutic intervention. Metabolic imaging with HP pyruvate and NMR metabolomics may enable identification and classification of aggressive subtypes of patient-derived xenografts. Translation of this real-time metabolic technique to the clinic may have the potential to improve the management of patients at high risk of developing pancreatic diseases.

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

Fast Access to Amphiphilic Multiblock Architectures by the Anionic Copolymerization of Aziridines and Ethylene Oxide.

An ideal system for stimuli-responsive and amphiphilic (block) polymers would be the copolymerization of aziridines with epoxides. However, to date, no copolymerization of these two highly strained three-membered heterocycles had been achieved. Herein, we report the combination of the living oxy- and azaanionic ring-opening polymerization of ethylene oxide (EO) and sulfonamide-activated aziridines. In a single step, well-defined amphiphilic block copolymers are obtained by a one-pot copolymerization. Real-time 1H NMR spectroscopy revealed the highest difference in reactivity ratios ever reported for an anionic copolymerization (with r1 = 265 and r2 = 0.004 for 2-methyl- N-tosylaziridine/EO and r1 = 151 and r2 = 0.013 for 2-methyl- N-mesylaziridine/EO), leading to the formation of block copolymers with monomodal and moderate molecular weight distributions ( Mw/ Mn mostly ≤1.3). The amphiphilic diblock copolymers were used to stabilize emulsions and to prepare polymeric nanoparticles by miniemulsion polymerization, representing a novel class of nonionic and responsive surfactants. In addition, this unique comonomer reactivity of activated-Az/EO allows fast access to multiblock copolymers, and we prepared the first amphiphilic penta- or tetrablock copolymers containing aziridines in only one or two steps, respectively. These examples render the combination of epoxide and aziridine copolymerizations via a powerful strategy for producing sophisticated macromolecular architectures and nanostructures.