Selective N-Terminal Acylation of Peptides and Proteins with Tunable Phenol Esters.

Selective modification of peptides and proteins is of foremost importance for the development of biopharmaceuticals and exploring biochemical pathways, as well as other applications. Here, we present a study on the development of a general and easily applicable selective method for N-terminal acylation of biomolecules, applying a new type of phenol esters. Key to the success was the development of highly tunable phenol activators bearing in the ortho-position, sulfonic acid or sulfonamide, acting as a steric shield for hydrolysis, and electron-withdrawing groups in the other ortho- and para-position for controlling the reactivity of the activated phenol esters. A library of heptapeptides, testing all 20 natural amino acids positioned at the N-terminal, were acylated in a selective manner at the N-terminus. The majority showed high conversion and excellent Nα-selectivity. Several biologically relevant biomolecules, including DesB30 insulin and human growth hormone, could also be modified at the N-terminal in a highly selective way, exemplified by either a fluorophore or a fatty acid sidechain. Finally, taking advantage of the possibility to accurately adjust the reactivity of the phenol esters, we present a potential strategy for the construction of dual active biopharmaceuticals through the employment of a bifunctional acylation linker and demonstrate its use in the creation of a GLP-1 insulin analogue, coupled through the lysine residue of GLP-1 and the N-terminal PheB1 amine of DesB30 insulin.

Promoting Selective Generation of Formic Acid from CO2 Using Mn(bpy)(CO)3Br as Electrocatalyst and Triethylamine/Isopropanol as Additives.

Urgent solutions are needed to efficiently convert the greenhouse gas CO2 into higher-value products. In this work, fac-Mn(bpy)(CO)3Br (bpy = 2,2'-bipyridine) is employed as electrocatalyst in reductive CO2 conversion. It is shown that product selectivity can be shifted from CO toward HCOOH using appropriate additives, i.e., Et3N along with iPrOH. A crucial aspect of the strategy is to outrun the dimer-generating parent-child reaction involving fac-Mn(bpy)(CO)3Br and [Mn(bpy)(CO)3]- and instead produce the Mn hydride intermediate. Preferentially, this is done at the first reduction wave to enable formation of HCOOH at an overpotential as low as 260 mV and with faradaic efficiency of 59 ± 1%. The latter may be increased to 71 ± 3% at an overpotential of 560 mV, using 2 M concentrations of both Et3N and iPrOH. The nature of the amine additive is crucial for product selectivity, as the faradaic efficiency for HCOOH formation decreases to 13 ± 4% if Et3N is replaced with Et2NH. The origin of this difference lies in the ability of Et3N/iPrOH to establish an equilibrium solution of isopropyl carbonate and CO2, while with Et2NH/iPrOH, formation of the diethylcarbamic acid is favored. According to density-functional theory calculations, CO2 in the former case can take part favorably in the catalytic cycle, while this is less opportune in the latter case because of the CO2-to-carbamic acid conversion. This work presents a straightforward procedure for electrochemical reduction of CO2 to HCOOH by combining an easily synthesized manganese catalyst with commercially available additives.

Nickel-Mediated Alkoxycarbonylation for Complete Carbon Isotope Replacement.

Many commercial drugs, as well as upcoming pharmaceutically active compounds in the pipeline, display aliphatic carboxylic acids or derivatives thereof as key structural entities. Synthetic methods for rapidly accessing isotopologues of such compounds are highly relevant for undertaking critical pharmacological studies. In this paper, we disclose a direct synthetic route allowing for full carbon isotope replacement via a nickel-mediated alkoxycarbonylation. Employing a nickelII pincer complex ([(N2N)Ni-Cl]) in combination with carbon-13 labeled CO, alkyl iodide, sodium methoxide, photocatalyst, and blue LED light, it was possible to generate the corresponding isotopically labeled aliphatic carboxylates in good yields. Furthermore, the developed methodology was applied to the carbon isotope substitution of several pharmaceutically active compounds, whereby complete carbon-13 labeling was successfully accomplished. It was initially proposed that the carboxylation step would proceed via the in situ formation of a nickellacarboxylate, generated by CO insertion into the Ni-alkoxide bond. However, preliminary mechanistic investigations suggest an alternative pathway involving attack of an open shell species generated from the alkyl halide to a metal ligated CO to generate an acyl NiIII species. Subsequent reductive elimination involving the alkoxide eventually leads to carboxylate formation. An excess of the alkoxide was essential for obtaining a high yield of the product. In general, the presented methodology provides a simple and convenient setup for the synthesis and carbon isotope labeling of aliphatic carboxylates, while providing new insights about the reactivity of the N2N nickel pincer complex applied.

A Nickel(II)-Mediated Thiocarbonylation Strategy for Carbon Isotope Labeling of Aliphatic Carboxamides.

A series of pharmaceutically relevant small molecules and biopharmaceuticals bearing aliphatic carboxamides have been successfully labeled with carbon-13. Key to the success of this novel carbon isotope labeling technique is the observation that 13 C-labeled NiII -acyl complexes, formed from a 13 CO insertion step with NiII -alkyl intermediates, rapidly react in less than one minute with 2,2'-dipyridyl disulfide to quantitatively form the corresponding 2-pyridyl thioesters. Either the use of 13 C-SilaCOgen or 13 C-COgen allows for the stoichiometric addition of isotopically labeled carbon monoxide. Subsequent one-pot acylation of a series of structurally diverse amines provides the desired 13 C-labeled carboxamides in good yields. A single electron transfer pathway is proposed between the NiII -acyl complexes and the disulfide providing a reactive NiIII -acyl sulfide intermediate, which rapidly undergoes reductive elimination to the desired thioester. By further optimization of the reaction parameters, reaction times down to only 11 min were identified, opening up the possibility of exploring this chemistry for carbon-11 isotope labeling. Finally, this isotope labeling strategy could be adapted to the synthesis of 13 C-labeled liraglutide and insulin degludec, representing two antidiabetic drugs.

Achieving Near-Unity CO Selectivity for CO2 Electroreduction on an Iron-Decorated Carbon Material.

A straightforward procedure has been developed to prepare a porous carbon material decorated with iron by direct pyrolysis of a mixture of a porous polymer and iron chloride. Characterization of the material with X-ray diffraction, X-ray absorption spectroscopy, and electron microscopy indicates the presence of iron carbide nanoparticles encapsulated inside the carbon matrix, and elemental mapping and cyanide poisoning experiments demonstrate the presence of atomic Fe centers, albeit in trace amounts, which are active sites for electrochemical CO2 reduction. The encapsulated iron carbide nanoparticles are found to boost the catalytic activity of atomic Fe sites in the outer carbon layers, rendering the material highly active and selective for CO2 reduction, although these atomic Fe sites are only present in trace amounts. The target material exhibits near-unity selectivity (98 %) for CO2 -to-CO conversion at a small overpotential (410 mV) in water. Furthermore, the material holds potential for practical application, as a current density over 30 mA cm-2 and a selectivity of 93 % can be achieved in a flow cell.

Direct Access to Isotopically Labeled Aliphatic Ketones Mediated by Nickel(I) Activation.

An extensive range of functionalized aliphatic ketones with good functional-group tolerance has been prepared by a NiI -promoted coupling of either primary or secondary alkyl iodides with NN2 pincer NiII -acyl complexes. The latter were easily accessed from the corresponding NiII -alkyl complexes with stoichiometric CO. This Ni-mediated carbonylative coupling is adaptable to late-stage carbon isotope labeling, as illustrated by the preparation of isotopically labelled pharmaceuticals. Preliminary investigations suggest the intermediacy of carbon-centered radicals.

Access to ß-Ketonitriles through Nickel-Catalyzed Carbonylative Coupling of α-Bromonitriles with Alkylzinc Reagents.

Herein, we report a nickel-catalyzed carbonylative coupling of α-bromonitriles and alkylzinc reagents with near stoichiometric carbon monoxide to give ß-ketonitriles in good yields. The reaction is catalyzed by a readily available and stable nickel(II) pincer complex. The developed protocol tolerates substrates bearing a variety of functional groups, which would be problematic or incompatible with previous synthetic methods. Additionally, we demonstrate the suitability of the method for carbon isotope labeling by the synthesis of 13 C-labeled ß-ketonitriles and their transformation into isotopically labeled heterocycles.

COtab: Expedient and Safe Setup for Pd-Catalyzed Carbonylation Chemistry.

Bench-stable tablets (COtabs) have been developed for the rapid and safe production of carbon monoxide. The tablets can be made in less than 5 min without the use of a glovebox and only require a stock solution of an amine base to liberate a specific quantity of CO in a two-chamber system. The COtabs were tested in five different carbonylation reactions and provided similar yields compared to literature procedures. Finally, a gram-scale reaction was conducted, as well as 13C-isotope labeling of the anticancer drug, olaparib.

Synthesis of Aliphatic Carboxamides Mediated by Nickel NN2 -Pincer Complexes and Adaptation to Carbon-Isotope Labeling.

The development of a nickel-mediated aminocarbonylation utilizing NN2 -pincer Ni-complexes, alkylzinc reagents, stoichiometric carbon monoxide and amines is described for the first time, which can be adapted to late-stage carbon-isotope labeling. This work expands the scope of the highly established palladium-promoted version of the reaction, by allowing carbon-sp3 fragments to take part in the three-component reaction. Finally, the results obtained show a remarkable effect of the pincer ligand for the reductive elimination step with the amine, which is followed by 13 C NMR spectroscopy studies.

Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells.

Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson's disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of ß-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson's disease.

Carbonylative Coupling of Alkyl Zinc Reagents with Benzyl Bromides Catalyzed by a Nickel/NN2 Pincer Ligand Complex.

An efficient catalytic protocol for the three-component assembly of benzyl bromides, carbon monoxide, and alkyl zinc reagents to give benzyl alkyl ketones is described, and represents the first nickel-catalyzed carbonylative coupling of two sp3 -carbon fragments. The method, which relies on the application of nickel complexed with an NN2 -type pincer ligand and a controlled release of CO gas from a solid precursor, works well with a range of benzylic bromides. Mechanistic studies suggest the intermediacy of carbon-centered radicals.

Palladium-Catalyzed Aminocarbonylation in Solid-Phase Peptide Synthesis: A Method for Capping, Cyclization, and Isotope Labeling.

A new synthetic approach for introducing N-capping groups onto peptides attached to a solid support, combining aminocarbonylation under mild conditions using a palladacycle precatalyst and solid-phase peptide synthesis, is reported. The use of a silacarboxylic acid as an in situ CO-releasing molecule allowed the reaction to be performed in a single vial. The method also enables versatile substitution of side chains, side-chain-to-side-chain cyclizations, and selective [13C] acyl labeling of modified peptides.

Application of Methyl Bisphosphine-Ligated Palladium Complexes for Low Pressure N-11 C-Acetylation of Peptides.

A mild and effective method is described for 11 C-labeling of peptides selectively at the N-terminal nitrogen or at internal lysine positions. The presented method relies on the use of specific biphosphine palladium-methyl complexes and their high reactivity towards amino-carbonylation of amine groups in the presence [11 C]carbon monoxide. The protocol facilitates the production of native N-11 C-acetylated peptides, without any structural modifications and has been applied to a selection of bioactive peptides.

Controlled electropolymerisation of a carbazole-functionalised iron porphyrin electrocatalyst for CO2 reduction.

Using a one-step electropolymerisation procedure, CO2 absorbing microporous carbazole-functionalised films of iron porphyrins are prepared in a controlled manner. The electrocatalytic reduction of CO2 for these films is investigated to elucidate their efficiency and the origin of their ultimate degradation.

Chemo- and Regioselective Ethynylation of Tryptophan-Containing Peptides and Proteins.

Ethynylation of various tryptophan-containing peptides and a single model protein was achieved using Waser's reagent, 1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1 H)-one (TIPS-EBX), under gold(I) catalysis. It was demonstrated by NMR that the ethynylation occurred selectively at the C2-position of the indole ring of tryptophan. Further, MS/MS showed that the tryptophan residues could be modified selectively with ethynyl functionalities even when the tryptophan was present as a part of the protein. Finally, the terminal alkyne was used to label a model peptide with a fluorophore by means of copper-catalyzed click chemistry.

Scaffolded multimers of hIAPP(20-29) peptide fragments fibrillate faster and lead to different fibrils compared to the free hIAPP(20-29) peptide fragment.

Applying fibril-forming peptides in nanomaterial design is still challenged by the difficulties in understanding and controlling how fibrils form. The present work investigates the influence of motional restriction on peptide fibrillation. We use cyclotriphosphazene and cyclodextrin as templates to make conjugates of the fibril-forming core of human islet amyloid polypeptide. Attachment of the peptide to the templates resulted in multimers containing six peptide fragments at different positions. ThT fluorescence, CD and FTIR spectroscopy, and AFM and TEM imaging reveal that in both conjugates the peptide retained its fibrillating properties and formed fibrils. However, the conjugate fibrils formed more rapidly than the free peptide and were long and thin, as opposed to the thick and twisted morphology of the intact peptide. Thus the motional restrictions introduced by the scaffold modulate the structure of the fibrils but do not impede the actual fibrillation process.

The importance of being capped: Terminal capping of an amyloidogenic peptide affects fibrillation propensity and fibril morphology.

The formation of aggregated fibrillar ß-sheet structures has been proposed to be a generic feature of proteins. Aggregation propensity is highly sequence dependent, and often only part of the protein is incorporated into the fibril core. Therefore, shorter peptide fragments corresponding to the fibril core are attractive fibrillation models. The use of peptide models introduces new termini into the fibrils, yet little attention has been paid to the role these termini may play in fibrillation. Here, we report that terminal modifications of a 10-residue peptide fragment of human islet amyloid polypeptide strongly affect fibrillation kinetics and the resulting fibril morphology. Capping of the N-terminus abolishes fibrillation, while C-terminal capping results in fibrils with a twisted morphology. Peptides with either both termini free or both termini capped form flat fibrils. Molecular dynamics simulations reveal that the N-terminal acetyl cap folds up and interacts with the peptide's hydrophobic side chains, while the uncapped N-terminus in the C-terminally capped version results in twisting of the fibrils due to charge repulsion from the free N-termini. Our results highlight the role of terminal interactions in fibrillation of small peptides and provide molecular insight into the consequences of C-terminal modifications frequently found in peptide hormones in vivo.

Two-chamber hydrogen generation and application: access to pressurized deuterium gas.

Hydrogen and deuterium gas were produced and directly applied in a two-chamber system. These gaseous reagents were generated by the simple reaction of metallic zinc with HCl in water for H2 and DCl in deuterated water for D2. The setup proved efficient in classical Pd-catalyzed reductions of ketones, alkynes, alkenes, etc. in near-quantitative yields. The method was extended to the synthesis and isotope labeling of quinoline and 1,2,3,4-tetrahydroquinoline derivatives. Finally, CX-546 and Olaparib underwent efficient Ir-catalyzed hydrogen isotope exchange reactions.

Generation of stoichiometric ethylene and isotopic derivatives and application in transition-metal-catalyzed vinylation and enyne metathesis.

Ethylene is one of the most important building blocks in industry for the production of polymers and commodity chemicals. (13)C- and D-isotope-labeled ethylenes are also valuable reagents with applications ranging from polymer-structure determination, reaction-mechanism elucidation to the preparation of more complex isotopically labeled compounds. However, these isotopic derivatives are expensive, and are flammable gases, which are difficult to handle. We have developed a method for the controlled generation of ethylene and its isotopic variants including, for the first time, fully isotopically labeled ethylene, from simple alkene precursors by using Ru catalysis. Applying a two-chamber reactor allows both the synthesis of ethylene and its immediate consumption in a chemical transformation permitting reactions to be performed with only stoichiometric amounts of this two carbon olefin. This was demonstrated in the Ni-catalyzed Heck reaction with aryl triflates and benzyl chlorides, as well as Ru-mediated enyne metathesis.

Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells.

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.