Generation Of Equivalents Research Articles

Photocatalytic generation of alkyl carbanions from aryl alkenes

AbstractOrganometallic reagents are routinely used as fundamental building blocks in organic chemistry to rapidly diversify molecular fragments via carbanion intermediates. However, the catalytic generation of carbanion equivalents, particularly from sp3-hybridized alkyl scaffolds, remains an underdeveloped goal in chemical synthesis. Here we disclose an approach for the generation of alkyl carbanions via single-electron reduction of aryl alkenes, enabled by multi-photon photoredox catalysis. We demonstrate that photocatalytically induced alkyl carbanions engage in intermolecular C–C bond-forming reactions with carbonyl electrophiles. Central to this method is the controlled formation of an alkene distonic radical anion intermediate that undergoes nucleophilic addition, followed by a kinetically favoured reductive polar crossover to produce a second carbanion available for further diversification. The versatility of this protocol was illustrated by the development of four distinct intermolecular C–C bond-forming reactions with aromatic alkenes (hydroalkoxylation, hydroamidation, aminoalkylation and carboxyaminoalkylation) to generate a range of valuable and complex scaffolds.

Reactions of Platinum Terminal Polyynyl Complexes trans-(C6F5)(p-tol3P)2Pt(C≡C)nH (n = 2-4) and n-BuLi, Generation of Functional Equivalents of Pt(C≡C)nLi Species, and Derivatization with Organic and Inorganic Electrophiles.

Reactions of the title complexes and n-BuLi (1.5 equiv, -45 °C) afford functional equivalents of the deprotonated species trans-(C6F5)(p-tol3P)2Pt(C≡C)nLi (n = 2-4), as assayed by subsequent additions of MeI or Me3SiCl to give trans-(C6F5)(p-tol3P)2Pt(C≡C)nMe (66-52%) or trans-(C6F5)(p-tol3P)2Pt(C≡C)nSiMe3 (63-49%). However, 31P NMR data suggest more complicated mechanistic scenarios, and small amounts of the hydride complex trans-(C6F5)(p-tol3P)2PtH (independently synthesized from the chloride complex, AgClO4, and NaBH4) are detected in most cases. Analogous sequences involving trans-(C6F5)(p-tol3P)2Pt(C≡C)2H and benzyl bromide, D2O, or W(CO)6/Me3O+ BF4- similarly afford products with Pt(C≡C)2Bn, Pt(C≡C)2D, or Pt(C≡C)2C(OCH3)=W(CO)5 linkages. The crystal structures of the tungsten and corresponding SiMe3 adduct, the three Pt(C≡C)nMe species, and hydride complex are determined.

Advances in the design, generation, and application of tissue-engineered myocardial equivalents.

Due to the limited regenerative ability of cardiomyocytes, the disabling irreversible condition of myocardial failure can only be treated with conservative and temporary therapeutic approaches, not able to repair the damage directly, or with organ transplantation. Among the regenerative strategies, intramyocardial cell injection or intravascular cell infusion should attenuate damage to the myocardium and reduce the risk of heart failure. However, these cell delivery-based therapies suffer from significant drawbacks and have a low success rate. Indeed, cardiac tissue engineering efforts are directed to repair, replace, and regenerate native myocardial tissue function. In a regenerative strategy, biomaterials and biomimetic stimuli play a key role in promoting cell adhesion, proliferation, differentiation, and neo-tissue formation. Thus, appropriate biochemical and biophysical cues should be combined with scaffolds emulating extracellular matrix in order to support cell growth and prompt favorable cardiac microenvironment and tissue regeneration. In this review, we provide an overview of recent developments that occurred in the biomimetic design and fabrication of cardiac scaffolds and patches. Furthermore, we sift in vitro and in situ strategies in several preclinical and clinical applications. Finally, we evaluate the possible use of bioengineered cardiac tissue equivalents as in vitro models for disease studies and drug tests.

Hierarchical hydrogel scaffolds with a clustered and oriented structure.

Hydrogel scaffolds play a critical role in tissue engineering due to their hydrophilic network structure and good biocompatibility. Constructing anisotropic scaffolds geometrically similar to injured tissues is conducive to promoting the generation of tissue and organ equivalents, or to guiding and enhancing the regeneration of injured tissues. In this study, we developed polyvinyl alcohol (PVA)/alginate hierarchical hydrogel scaffolds with a clustered and oriented structure using a method that combines directional freezing and drying under stretching. Our hydrogel scaffolds with an adjustable modulus (50 kPa-20 MPa) can match different types of injured tissues. The clustered and oriented structure successfully guided the alignment and orientation of fibroblasts and chondrocytes. This work provides a new idea for constructing hydrogels with hierarchical and anisotropic microstructures, which have promising applications in tissue regeneration.

Fabrication of Human Skin Equivalents Using Decellularized Extracellular Matrix.

There is a growing demand for in vitro models of human tissues that recapitulate the complex structures and functions found in vivo, and the biomaterials that support these physiologically relevant models are essential underpinning technologies. Here, we present an optimized protocol for generating human skin equivalents (HSEs) using a dermal matrix isolated from decellularized porcine skin. The decellularized extracellular matrix (dECM) contains a complex mixture of fibrillar collagens and matrisomal proteins that mimic native skin and can be produced in large quantities. The procedure for decellularization, digestion, and solubilization of the dECM is described in detail. In addition, we provide instructions for how to construct a three‐dimensional HSE model using the dECM as the dermal support matrix for human keratinocytes and dermal fibroblasts. Recent studies from our laboratory have shown that HSEs generated using porcine dECM display improved epidermal differentiation and stratification compared to existing protocols using type I collagen gels. Thus, dECM‐based biomaterials are a useful tool for replicating human skin physiology in vitro and developing advanced human skin models for therapeutic discovery and testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of decellularized extracellular matrix from porcine skin Basic Protocol 2: Generation of human skin equivalents

Fully Synthetic 3D Fibrous Scaffolds for Stromal Tissues-Replacement of Animal-Derived Scaffold Materials Demonstrated by Multilayered Skin.

The extracellular matrix (ECM) of soft tissues in vivo has remarkable biological and structural properties. Thereby, the ECM provides mechanical stability while it still can be rearranged via cellular remodeling during tissue maturation or healing processes. However, modern synthetic alternatives fail to provide these key features among basic properties. Synthetic matrices are usually completely degraded or are inert regarding cellular remodeling. Based on a refined electrospinning process, a method is developed to generate synthetic scaffolds with highly porous fibrous structures and enhanced fiber-to-fiber distances. Since this approach allows for cell migration, matrix remodeling, and ECM synthesis, the scaffold provides an ideal platform for the generation of soft tissue equivalents. Using this matrix, an electrospun-based multilayered skin equivalent composed of a stratified epidermis, a dermal compartment, and a subcutis is able to be generated without the use of animal matrix components. The extension of classical dense electrospun scaffolds with high porosities and motile fibers generates a fully synthetic and defined alternative to collagen-gel-based tissue models and is a promising system for the construction of tissue equivalents as in vitro models or in vivo implants.

Green oxygen power plants in the brain rescue neuronal activity

SummaryNeuronal activity in the brain depends on mostly aerobic generation of energy equivalents and thus on a constant O2 supply. Oxygenation of the vertebrate brain has been optimized during evolution by species-specific uptake and transport of O2 that originally derives from the phototrophic activity of prokaryotic and eukaryotic organisms in the environment. Here, we employed a concept that exploits transcardial injection and vascular distribution of unicellular green algae or cyanobacteria in the brain of Xenopus laevis tadpoles. Using oxygen measurements in the brain ventricle, we found that these microorganisms robustly produce sizable amounts of O2 upon illumination. In a severe hypoxic environment, when neuronal activity has completely ceased, the photosynthetic O2 reliably provoked a restart and rescue of neuronal activity. In the future, phototrophic microorganisms might provide a novel means to directly increase oxygen levels in the brain in a controlled manner under particular eco-physiological conditions or following pathological impairments.

Generation of Self-assembled Vascularized Human Skin Equivalents

Generation of Self-assembled Vascularized Human Skin Equivalents

Generation of Self-assembled Vascularized Human Skin Equivalents.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

Generation of Pigmented Skin Grafts from Human Hair Follicles and Dermal Fibroblasts.

Skin equivalents are able to mimic key features of human skin and they can be used for a very broad range of applications, such as fundamental studies of skin biology, disease, and toxicological models, as well as an alternative for animal testing. The high end of their use is in therapy of wound healing and repigmentation and disorders that massively affect individual health as well as quality of life and pose considerable burden to health care systems worldwide. Tissue-engineered skin grafts often originate from invasively obtained cell material (i.e., biopsy). Hereby, an unmet need for noninvasively gained autologous biological starting material has been created. The hair follicle, entirely noninvasively available by plucking, harbors a heterogeneous cell pool, including stem cells with an immense differentiation capacity, hereby representing an attractive source of cells, especially for purposes of regenerative medicine. In this study, we engineered three-dimensional pigmented epidermal and dermoepidermal grafts using human keratinocytes and melanocytes from the outer root sheath of hair follicles combined with dermal fibroblasts. The grafts were generally anatomically correct and functional regarding stratification and formation of epidermal melanin units, as well as extracellular matrix deposition, exhibiting moderate differences to the skin anatomy and function, typical for the in vitro culture. Impact statement The study focuses on generation of tissue-engineered skin equivalents, in particular, as a possible treatment for nonhealing wounds and depigmentation disorders. We developed an in vitro-generated three-dimensional pigmented epidermal and dermoepidermal graft using keratinocytes and melanocytes from the outer root sheath of human hair follicle combined with dermal fibroblasts. The anatomically and functionally correct grafts showed stratification, epidermal melanin units, and extracellular matrix deposition. They present an in vitro base for an autologous, pigmented graft as well as for further personalized in vitro experimental models generated from a noninvasively obtained cell source, addressing the unmet needs of the currently available clinical treatments.

Generation of Epidermal Equivalents from Hair Follicle Melanocytes, Keratinocytes, and Dermal Fibroblasts.

Bench-to-bedside axis of therapeutic product development is currently being oriented towards minimum invasiveness on both ends-not only clinical application but harvesting of the starting biological material as well. This is particularly relevant for Advanced Therapy Medicinal Products and their specific legislative requirements, even more so in skin regeneration. It is precisely the skin equivalents and grafts that benefit from the minimum-to-noninvasive approach to a noteworthy extent, taking in account the sensitive nature of both skin harvesting and grafting.This chapter includes protocols for two separate steps of generating skin equivalent from the cells cultured from hair follicle outer root sheath. The first step is a non-pigmented epidermal equivalent generated from human keratinocytes from the outer root sheath named non-pigmented epidermal graft. The second step consists of co-cultivating human keratinocytes and human melanocytes from the outer root sheath, hereby producing a pigmented epidermal graft.

Synthesis of anti‐Markovnikov Alcohols via Epoxidation and Hydrogenation of Styrenes using Photocatalytically Generated Redox Equivalents

AbstractWe report an efficient method for a formal anti‐Markovnikov addition of water to styrenes through photocatalytic generation of redox equivalents, using a gold‐doped titanium dioxide (Au/TiO2) semiconductor as photocatalyst. Under irradiation with UV−A light, the photocatalytic activity of Au/TiO2 generates in situ under aerobic conditions hydrogen peroxide (H2O2) and under anaerobic conditions dihydrogen (H2). In the presence of oxygen and a Ru catalyst, the epoxidation of different styrene derivatives is achieved. Subsequently, regioselective reductive Pd‐catalyzed epoxide opening in the absence of oxygen gives the corresponding 2‐phenylethanol derivatives.

Direct Synthesis of Dibenzophospholes from Biaryls by Double C-P Bond Formation via Phosphenium Dication Equivalents.

We have developed a new strategy for the generation of phosphenium dication equivalents from readily available phosphinic acids and Tf2O. The in situ-generated dication equivalents can be readily coupled with simple (hetero)biaryls to form the corresponding dibenzophospholes directly. This protocol can also be applied to the concise synthesis of six- and seven-membered phosphacycles as well as the largely π-extended heteroacene derivatives, which are of great interest in the field of organic functional materials.

1,2-Amino Alcohols via Cr/Photoredox Dual-Catalyzed Addition of α-Amino Carbanion Equivalents to Carbonyls.

Herein, we report the synthesis of protected 1,2-amino alcohols starting from carbonyl compounds and α-silyl amines. The reaction is enabled by a Cr/photoredox dual catalytic system that allows the in situ generation of α-amino carbanion equivalents which act as nucleophiles. The unique nature of this reaction was demonstrated through the aminoalkylation of ketones and an acyl silane, classes of electrophiles that were previously unreactive toward addition of alkyl-Cr reagents. Overall, this reaction broadens the scope of Cr-mediated carbonyl alkylations and discloses an underexplored retrosynthetic strategy for the synthesis of 1,2-amino alcohols.

Enhanced Time-Invariant Linear Model for the EMI Prediction of Switching Circuits

This article presents an innovative technique for the black-box modeling of the conductive emissions of switching circuits from external observations. The proposed methodology relies on the robust and elegant theory of periodically switched linear systems, providing the user with a tool for the generation of frequency-domain augmented linear time-invariant equivalents. The obtained models offer both a remarkable accuracy for different supply conditions and a deep understanding of the inherent switching mechanisms of this class of devices. The proposed framework also enables the derivation of a simplified enhanced Norton representation which outperforms the classical state-of-the-art models based on linear time-invariant approximations. Model parameters are computed via a blind procedure based only on two measurements. The feasibility and strength of the proposed approach are demonstrated on a dc–dc boost power converter and on a dc electrical motor via the prediction of their conducted disturbances.

Iron-catalyzed α-C-H functionalization of π-bonds: cross-dehydrogenative coupling and mechanistic insights.

The deprotonation of propargylic C–H bonds for subsequent functionalization typically requires stoichiometric metal alkyl or amide reagents. In addition to the undesirable generation of stoichiometric metallic waste, these conditions limit the functional group compatibility and versatility of this functionalization strategy and often result in regioisomeric mixtures. In this article, we report the use of dicarbonyl cyclopentadienyliron(ii) complexes for the generation of propargylic anion equivalents toward the direct electrophilic functionalization of propargylic C–H bonds under mild, catalytic conditions. This technology was applied to the direct conversion of C–H bonds to C–C bonds for the synthesis of several functionalized scaffolds through a one-pot cross dehydrogenative coupling reaction with tetrahydroisoquinoline and related privileged heterocyclic scaffolds. A series of NMR studies and deuterium-labelling experiments indicated that the deprotonation of the propargylic C–H bond was the rate-determining step when a Cp*Fe(CO)2-based catalyst system was employed.

Polarity Inversion Catalysis by the 1,4-Addition of N-Heterocyclic Carbenes

Polarity inversion is the hallmark of N-heterocyclic carbene (NHC) organocatalysis, with the generation and reaction of acyl anion equivalents known for more than 70 years. In contrast, polarity inversion through 1,4-addition of NHCs to conjugate acceptors was first applied in a catalytic reaction in 2006. This sub-field of NHC-organocatalysis has developed steadily over the subsequent years, enabling novel coupling reactions, enantioselective cycloisomerizations, polymerizations, and other reactions. In this review, this emerging area of NHC-organocatalysis is discussed with comprehensive coverage. In addition, notes regarding the use of other Lewis base catalysts for related reactions, and comments regarding NHC selection for this type of catalysis, are provided.

The impact of intercellular communication for the generation of complex multicellular prevascularized tissue equivalents.

In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillary-like structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents.

Inhibition of Anti-Apoptotic Mcl-1 Exerts Anti-Leukemia Activity through Modulation of Leukemia-Stromal Interactions and Metabolic Functions in AML

Inhibition of Anti-Apoptotic Mcl-1 Exerts Anti-Leukemia Activity through Modulation of Leukemia-Stromal Interactions and Metabolic Functions in AML

Unravelling effects of relative humidity on lipid barrier formation in human skin equivalents

Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90–95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH.