Hybrid Implants Based on Calcium-Magnesium Silicate Ceramic Diopside as a Carrier of Recombinant BMP-2 and Demineralized Bone Matrix as a Scaffold: Ectopic Osteogenesis in Intramuscular Implantation in Mice.

High efficiency of hybrid implants based on calcium-magnesium silicate ceramic, diopside, as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously using the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue using in vivo bioreactor principle in the patient's own body, effectiveness of ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At the dose of 7 µg of BMP-2 per implant, dense agglomeration of cells, probably skeletal muscle satellite precursor cells, was observed one week after implantation with areas of intense chondrogenesis, initial stage of indirect osteogenesis, around the implants. After 12 weeks, a dense bone capsule of trabecular structure was formed covered with periosteum and mature bone marrow located in the spaces between the trabeculae. The capsule volume was about 8-10 times the volume of the original implant. There were practically no signs of inflammation and foreign body reaction. Microcomputed tomography data showed significant increase of the relative bone volume, number of trabeculae, and bone tissue density in the group of mice with BMP-2-containing implant in comparison with the group without BMP-2. Considering that DBM can be obtained in practically unlimited quantities with required size and shape, and that BMP-2 is obtained by synthesis in E. coli cells and is relatively inexpensive, further development of the in vivo bioreactor model based on the hybrid implants constructed from BMP-2, diopside, and xenogenic DBM seems promising.

Hybrid Implants Based on Calcium-Magnesium Silicate Ceramics Diopside as a Carrier of Recombinant BMP-2 and Demineralized Bone Matrix as a Scaffold: Dynamics of Reparative Osteogenesis in a Mouse Craniotomy Model.

Calcium-magnesium silicate ceramics, diopside, is a promising material for use in bone plastics, but until now the possibility of its use as a carrier of recombinant bone morphogenetic protein-2 (BMP-2) has not been studied, as well as the features of reparative osteogenesis mediated by the materials based on diopside with BMP-2. Powder of calcium-magnesium silicate ceramics was obtained by solid-state synthesis using biowaste - rice husks and egg shells - as source components. Main phase of the obtained ceramics was diopside. The obtained particles were irregularly shaped with an average size of about 2.3 µm and ~20% porosity; average pore size was about 24 nm, which allowed the material to be classified as mesoporous. Diopside powder adsorbs more than 150 µg of recombinant BMP-2 per milligram, which exceeds binding capacity of hydroxyapatite, a calcium-phosphate ceramic often used in hybrid implants, by more than 3 times. In vitro release kinetics of BMP-2 was characterized by a burst release in the first 2 days and a sustained release of approximately 0.4 to 0.5% of the loaded protein over the following 7 days. In vivo experiments were performed with a mouse model of cranial defects of critical size with implantation of a suspension of diopside powder with/without BMP-2 in hyaluronic acid incorporated into the disks of demineralized bone matrix with 73-90% volume porosity and macropore size from 50 to 650 µm. Dynamics of neoosteogenesis and bone tissue remodeling was investigated histologically at the time points of 12, 21, 48, and 63 days. Diopside particles were evenly spread in the matrix and caused minimal foreign body reaction. In the presence of BMP-2 by the day 63 significant foci of newly formed bone tissue were formed in the implant pores with bone marrow areas, moreover, large areas of demineralized bone matrix in the implant center and maternal bone at the edges were involved in the remodeling. Diopside could be considered as a promising material for introduction into hybrid implants as an effective carrier of BMP-2.

Dynamics of a Molecular Rotor Exhibiting Local Directional Rotational Preference within Each Enantiomer.

Directional internal rotation in molecular systems, generally controlled by chirality, is known to occur in natural and artificial systems driven by light or fueled chemically, but spontaneous directional molecular rotation is believed to be forbidden. We have designed a molecular rotor, whereby ferrocene and triptycene linked by a methylene bridge provide two rotational degrees of freedom. On the basis of experimental observations, in conjunction with computational data, we show that the two different modes of rotation are strongly coupled and the spatial orientation of the bistable ferrocene moiety controls the barrier to its own rotation about the triptycene axis. It is proposed that the barrier to clockwise 120° rotation across each individual triptycene blade is lower in the M-enantiomer and for counterclockwise 120° rotation, it is lower in its P-counterpart. These findings demonstrate the possibility of locally preferred thermal directional intramolecular rotation for each dynamically interconverting enantiomer.

Palladium-Catalysed Coupling Reactions En Route to Molecular Machines: Sterically Hindered Indenyl and Ferrocenyl Anthracenes and Triptycenes, and Biindenyls.

Pd-catalysed Stille and Suzuki cross-couplings were used to prepare 9-(3-indenyl)-, 6, and 9-(2-indenyl)-anthracene, 7; addition of benzyne led to the 9-Indenyl-triptycenes, 8 and 9. In 6, [4 + 2] addition also occurred to the indenyl substituent. Reaction of 6 through 9 with Cr(CO)6 or Re2(CO)10 gave their M(CO)3 derivatives, where the Cr or Re was complexed to a six- or five-membered ring, respectively. In the 9-(2-indenyl)triptycene complexes, slowed rotation of the paddlewheel on the NMR time-scale was apparent in the η5-Re(CO)3 case and, when the η6-Cr(CO)3 was deprotonated, the resulting haptotropic shift of the metal tripod onto the five-membered ring also blocked paddlewheel rotation, thus functioning as an organometallic molecular brake. Suzuki coupling of ferrocenylboronic acid to mono- or dibromoanthracene yielded the ferrocenyl anthracenes en route to the corresponding triptycenes in which stepwise hindered rotations of the ferrocenyl groups behaved like molecular dials. CuCl2-mediated coupling of methyl- and phenyl-indenes yielded their rac and meso 2,2'-biindenyls; surprisingly, however, the apparently sterically crowded rac 2,2'-Bis(9-triptycyl)biindenyl functioned as a freely rotating set of molecular gears. The predicted high rotation barrier in 9-phenylanthracene was experimentally validated via the Pd-catalysed syntheses of di(3-fluorophenyl)anthracene and 9-(1-naphthyl)-10-phenylanthracene.

Mechanisms and Beyond: Elucidation of Fluxional Dynamics by Exchange NMR Spectroscopy.

Detailed mechanistic information is crucial to our understanding of reaction pathways and selectivity. Dynamic exchange NMR techniques, in particular 2D exchange spectroscopy (EXSY) and its modifications, provide indispensable intricate information on the mechanisms of organic and inorganic reactions and other phenomena, for example, the dynamics of interfacial processes. In this Review, key results from exchange NMR studies of small molecules over the last few decades are systemised and discussed. After a brief introduction to the theory, the key types of dynamic processes are identified and fundamental examples given of intra- and intermolecular reactions, which, in turn, could involve, or not, bond-making and bond-breaking events. Following that logic, internal molecular rotation, intramolecular stereomutation and molecular recognition will first be considered because they do not typically involve bond breaking. Then, rearrangements, substitution-type reactions, cyclisations, additions and other processes affecting chemical bonds will be discussed. Finally, interfacial molecular dynamics and unexpected combinations of different types of fluxional processes will also be highlighted. How exchange NMR spectroscopy helps to identify conformational changes, coordination and molecular recognition processes as well as quantify reaction energy barriers and extract detailed mechanistic information by using reaction rate theory in conjunction with computational techniques will be shown.

Synthesis of glycosyl chlorides using catalytic Appel conditions.

The stereoselective synthesis of glycosyl chlorides using catalytic Appel conditions is described. Good yields of α-glycosyl chlorides were obtained using a range of glycosyl hemiacetals, oxalyl chloride and 5 mol% Ph3PO. For 2-deoxysugars treatment of the corresponding hemiacetals with oxalyl chloride without phosphine oxide catalyst also gave good yields of glycosyl chloride. The method is operationaly simple and the 5 mol% phosphine oxide by-product can be removed easily. Alternatively a one-pot, multi-catalyst glycosylation can be carried out to transform the glycosyl hemiacetal directly to a glycoside.

Dynamic Cross-Exchange in Halophosphonium Species: Direct Observation of Stereochemical Inversion in the Course of an SN 2 Process.

The complex fluxional interconversions between otherwise very similar phosphonium bromides and chlorides R3 PX+ X- (R=Alk, Ar, X=Cl or Br) were studied by NMR techniques. Their energy barriers are typically ca. 11 kcal mol-1 , but rise rapidly as bulky groups are attached to phosphorus, revealing the importance of steric factors. In contrast, electronic effects, as measured by Hammett analysis, are modest (ρ 1.46) but still clearly indicate negative charge flow towards phosphorus in the transition state. Most significantly, detailed analysis of the exchange pathways unequivocally, and for the first time in any such process, shows that nucleophilic attack of the nucleophilic anion on the tetrahedral centre results in inversion of configuration.

Mysterious Decomposition of Alkoxyphosphonium Chlorides: Postulated Involvement of the HCl2 Anion and Its Capture in the Solid State.

P-Alkoxyphosphonium (AP) chlorides were generated by reacting P-chlorophosphonium chlorides with alcohols. Their well-known spontaneous Arbuzov-type collapse leading to phosphine oxides was studied and its rate found to be dependent on a number of factors in an unexpected fashion: it is inversely proportional to the initial concentration and it shows strong dependence on the acidity of the media but is not very sensitive to the presence of base. To explain these observations, we evoke a self-inhibition model with the formation of the less nucleophilic hydrodichloride anion HCl2 in solution. Detailed analysis of the kinetic data yields the association constant (K=3×102 m-1 ) of the putative HCl2 species in chloroform. Experimental observations for the collapse of highly enriched diastereomeric alkoxyphosphonium (DAP) chlorides are fully analogous to the achiral AP also implying the involvement of HCl2 anions. Moreover, crystallisation of a highly enriched DAP salt derived from (-)-menthol furnished, for the first time, crystals of individual (RP )-DAP hydrodichloride as confirmed by X-ray diffractometry. Importantly, the P-configuration and detailed conformation of the DAP moiety is in good agreement with DFT-level computational results. The thermal collapse of (RP )-DAP⋅HCl2 proceeds with complete retention of the P-configuration furnishing the phosphine oxide of exceptional enantiomeric purity.

Two Independent Orthogonal Stereomutations at a Single Asymmetric Center: A Narcissistic Couple.

The energy barriers in our recently discovered Walden-type inversion of chlorophosphonium salts are similar to those for Cope rearrangements of caged cyclic hydrocarbons. Therefore, we have designed a molecular system that integrates the two processes, thereby producing the first embodiment of a chemical species that can undergo two entirely different and independent stereomutation mechanisms at the same nominal asymmetric center. Thus, the energy barrier to the rearrangement of 9-phenyl-9-phosphabarbaralane oxide, which is easily prepared by a new high-yielding synthesis, was found to be roughly 11 kcal mol-1 . This value is in contrast to the parent barbaralane (7.3 kcal mol-1 ) but in good agreement with our computational results for the rearrangement barriers. Crucially, in the corresponding chlorophosphonium derivative, two stereomutations occur simultaneously: a fast Cope rearrangement (barrier ≈12 kcal mol-1 ) and a slow Walden-type inversion of the phosphorus center (barrier ≈21 kcal mol-1 ). The computational model also revealed a relationship between the Cope rearrangement barrier and the bridgehead distance. The phenomenon of two independent and geometrically orthogonal stereomutations at a single asymmetric center provided important general insights into reaction pathway bifurcation, microscopic reversibility, and dynamic stereochemistry. This first example of coexisting alternative mechanisms that involve covalent bonds may encourage the design of new types of dynamic molecular structures.

Hammond Postulate Mirroring Enables Enantiomeric Enrichment of Phosphorus Compounds via Two Thermodynamically Interconnected Sequential Stereoselective Processes.

The dynamic resolution of tertiary phosphines and phosphine oxides was monitored by NMR spectroscopy. It was found that the stereoselectivity is set during the formation of the diastereomeric alkoxyphosphonium salts (DAPS), such that their initial diastereomeric excess (de) limits the final enantiomeric excess (ee) of any phosphorus products derived from them. However, (31)P NMR monitoring of the spontaneous thermal decomposition of the DAPS shows consistent diastereomeric self-enrichment, indicating a higher rate constant for decomposition of the minor diastereomer. This crucial observation was confirmed by reductive trapping of the unreacted enriched DAPS with lithium tri-sec-butylborohydride (commercially distributed as L-Selectride reagent) at different time intervals after the start of reaction, which gives progressively higher ee of the phosphine product with time. It is proposed that the Hammond postulate operates for both formation and decomposition of DAPS intermediate so that the lower rate of formation and faster subsequent collapse of the minor isomer are thermodynamically linked. This kinetic enhancement of kinetic resolution furnishes up to 97% ee product.

A ferrocenyl kaleidoscope: slow interconversion of six diastereo-atropisomers of 2,6-di-tert-butyl-9,10-diferrocenyltriptycene.

The title triptycene, 6, has been isolated as the product of 9,10-cycloaddition of benzyne to 9,10-diferrocenyl-2,6-di-tert-butylanthracene, 5, whose X-ray crystal structure is reported. Each ferrocenyl unit in 6 has access to the same three non-equivalent molecular environments, and their rotations relative to the molecular paddlewheel give rise to six slowly interconverting atropisomers. Their dynamic behaviour in solution is a challenging NMR puzzle that can be successfully solved by taking advantage of the recently described very large diamagnetic anisotropy of the ferrocenyl moiety, together with the C2 symmetry of particular atropisomers. Application of one- and two-dimensional NMR techniques over a range of temperatures together, with a detailed analysis of the homo- and heteronuclear correlations in 6, resulted in unequivocal mapping of the 99 (1)H and 162 (13)C positions in the six interconverting systems. Variable-temperature 2D-EXSY measurements revealed that, while the stability of the atropisomers is almost identical, they are separated by energy barriers which the ferrocenyls must overcome in the course of their interconversions. The heights of two different rotational barriers have been identified and these experimental findings are in good agreement with DFT calculations.

Degenerate nucleophilic substitution in phosphonium salts.

Rates and energy barriers of degenerate halide substitution on tetracoordinate halophosphonium cations have been measured by NMR techniques (VT and EXSY) using a novel experimental design whereby a chiral substituent ((s)Bu) lifts the degeneracy of the resultant salts. Concomitantly, a viable computational approach to the system was developed to gain mechanistic insights into the structure and relative stabilities of the species involved. Both approaches strongly suggest a two-step mechanism of formation of a pentacoordinate dihalophosphorane via backside attack followed by dissociation, resulting in inversion of configuration at phosphorus. The experimentally determined barriers range from <9 kcal mol(-1) to nearly 20 kcal mol(-1), ruling out a mechansm via Berry pseudorotation involving equatorial halides. In all cases studied, epimerization of chlorophosphonium chlorides has a lower energy barrier (by 2 kcal mol(-1)) than the analogous bromo salts. Calculations determined that this was due to the easier accessibility in solution of pentacoordinate dichlorophosphoranes when compared to analogous dibromophosphoranes. In line with the proposed associative mechanism, bulky substituents slow the reaction in the order Me < Et < (i)Pr < (t)Bu. Bulky substituents affect the shape of the reaction energy profile so that the pentacoordinate intermediate is destabilized eventually becoming a transition state. The magnitude of the steric effects is comparable to that of the same substituents on substitution at primary alkyl halides, which can be rationalized by the relatively longer P-C bonds. The reaction displays first-order kinetics due to the prevalence of tight- or solvent-separated ion pairs in solution. Three-dimensional reaction potential energy profiles (More O'Ferrall-Jencks plots) indicated a relatively shallow potential well corresponding to the trigonal bipyramid intermediate flanked by two transition states.

Evaluation of weak interactions in [2]pseudorotaxanes.

Viologens readily thread bis-p-phenylene crown ethers to form [2]pseudorotaxanes. However, the binding of sterically hindered 3,3'-dimethylviologens is very weak. Density functional theory (DFT) calculations indicated that the additional energy cost of "flattening" is substantial, 55 kJ mol(-1), and prevents the formation of a stable host-guest complex. The structures of [2]pseudorotaxanes determined by X-ray crystallography are in good agreement with the NMR characterisation and DFT results. Their association constants and thermodynamic parameters in solution were measured by using a dilution method and, for the first time, by host-guest nuclear Overhauser effect (NOE) correlations. The NOE approach was subsequently applied to study the sterically hindered analogues and it was shown that the binding in 3,3'-dimethyl-N,N-dibenzyl [2]pseudorotaxane is by 8.5 kJ mol(-1) weaker than in its regular analogue. The proposed technique helps to quantify weak interactions in [2]pseudorotaxanes and can be applied to other host-guest complexes.

Turning regioselectivity into stereoselectivity: efficient dual resolution of P-stereogenic phosphine oxides through bifurcation of the reaction pathway of a common intermediate.

Synthetic routes that provide facile access to either enantiomeric form of a target compound are particularly valuable. The crystallization-free dual resolution of phosphine oxides that gives highly enantioenriched materials (up to 94 % ee) in excellent yields is reported. Both enantiomeric oxides have been prepared from a single intermediate, (RP )-alkoxyphosphonium chloride, which is formed in the course of a selective dynamic kinetic resolution using a single enantiomer of menthol as the chiral auxiliary. The origin of the dual stereoselectivity lies in bifurcation of the reaction pathway of this intermediate, which works as a stereochemical railroad switch. Under controlled conditions, Arbuzov-type collapse of this intermediate proceeds through CO bond fission with retention of the configuration at the phosphorus center. Conversely, alkaline hydrolysis of the PO bond leads to the opposite SP  enantiomer.

Rapid synthesis of Pt(0) motors-microscrolls on a nickel surface via H2PtCl6-induced galvanic replacement reaction.

In this study, Pt(0) microscrolls are synthesized on polished Ni via galvanic replacement reaction (GRR). Employing in situ optical microscopy, the dynamic motion of the catalytic microscrolls as micromotors in H2O2 solutions is revealed. This method offers a rapid fabrication of scrolls from diverse noble metals and alloys.

Unexpected rapid P-stereomutation of phosphine oxides catalysed by chlorophosphonium salts.

P-Stereomutation of phosphine oxides is extremely slow. We show that it is catalysed by chlorophosphonium salts (CPS) which can directly be formed in the system in situ. The racemization of phosphine oxides at ambient conditions catalysed by 1 mol% of CPS takes 1-2 hours and can be arrested by additon of a primary alcohol. The process probably proceeds via the development of oxodiphosphonium P-O-P species.

Electron transfer and switching in rigid [2]rotaxanes adsorbed on TiO2 nanoparticles.

Bistable [2]rotaxanes have been attached through a bulky tripodal linker to the surface of titanium dioxide nanoparticles and studied by cyclic voltammetry and spectroelectrochemical methods. The axle component in the [2]rotaxane contains two viologen sites, V(1) and V(2), interconnected by a rigid terphenylene bridge. In their parent dication states, V(1)(2+) and V(2)(2+) can both accommodate a crown ether ring, C, but are not equivalent in terms of their affinity towards C and have different electrochemical reduction potentials. The geometry and size of the tripodal linker help to maintain a perpendicular [2]rotaxane orientation at the surface and to avoid unwanted side-to-side interactions. When the rigid [2]rotaxane or its corresponding axle are adsorbed on a TiO(2) nanoparticle, viologen V(2)(2+) is reduced at significantly more negative potentials (-0.3 V) than in flexible analogues that contain aliphatic bridges between V(1) and V(2). These overpotentials are analysed in terms of electron-transfer rates and a donor-bridge-acceptor (D-B-A) formalism, in which D is the doubly reduced viologen, V(1)(0), adjacent to the TiO(2) surface (TiO(2)-V(1)(0)), B is the terphenylene bridge and A is viologen V(2)(2+). We have also found that, in contrast with earlier findings in solution, no molecular shuttling occurs in rigid [2]rotaxane adsorbed at the surface. The observations were explained by the relative position of the viologen stations within the electrical double layer, screening of V(2)(2+) by the counterions and high capacity of the medium, which reduces the mobility of the crown ether. The results are useful in transposing of solution-based molecular switches to the interface or in the design and understanding of the properties of systems comprising electroactive and/or interlocked molecules adsorbed at the nanostructured TiO(2) surface.

Pragmatic randomised controlled trials in COPD and asthma: how to guide clinical practice.

The use of real-world evidence (RWE) studies, including pragmatic randomised controlled trials (RCTs; randomised RWE studies), to aid the development of treatment guidelines, is gradually becoming a mainstay within clinical practice. RWE is an integral part of patient-driven decision-making and offers important value to add complimentary evidence to traditional RCTs; these provide a more well-rounded view of the benefits to patient-reported outcomes and improve the external validity of a given treatment versus findings from traditional RCTs alone. Discussions in recent scientific workshops explored the importance of pragmatic RCTs in optimising guideline development and patient care in chronic obstructive pulmonary disease (COPD) and asthma. The Salford Lung Study in patients with COPD (NCT01551758) and asthma (NCT01706198) were the world's first prelicence pragmatic RCTs that compared novel investigational treatments with existing COPD and asthma treatments and, more recently (2021), RWE studies have been used by the American Thoracic Society and the US Food and Drug Administration to support the approval of an immunosuppressant drug in patients receiving lung transplants. This highlights the importance of RWE data in supporting clinical guideline development and emphasises the advantages for the use of pragmatic RCTs in guiding clinical practice.

Effect of recombinant BMP-2 and erythropoietin on osteogenic properties of biomimetic PLA/PCL/HA and PHB/HA scaffolds in critical-size cranial defects model.

Osteoplastic materials PLA/PCL/HA and PHB/HA and scaffolds with a highly porous structure based on them with potential applications in regenerative medicine have been obtained by solvent casting with thermopressing and salt leaching for PLA-based samples and solid-state mixing with subsequent thermopressing and salt leaching for PHB-based samples. The scaffolds were characterized by SEM-EDX, DSC, FTIR spectroscopy, mechanical tests in compression, measurement of the contact angle, in vitro studies, including loading by recombinant BMP-2 and EPO and their release kinetics, and in vivo studies on a model of regeneration of critical-sized cranial defects in mice. Biomimetic scaffolds with micropores sizes ranged from 300 to 500 µm and volume porosity of 70% imitate trabecular bone's structure and have increased hydrophilicity to achieve osteoconductive properties. Mechanical characteristics correspond to native trabecular bone. Elastic modulus - key mechanical characteristics of bone implants - showed the values of 0.15 ± 0.04 and 0.18 ± 0.08 GPa for PLA/PCL/HA and PHB/HA scaffolds, respectively. Both materials have high biocompatibility and can be used together with recombinant proteins BMP-2 and EPO. Introduction of BMP-2 leads to induction of new bone formation, introduction of EPO results in increased angiogenesis in the implantation area. The obtained scaffolds with recombinant proteins can be used as bone implants for reconstruction of defects of lightly or non-loaded bones.

Asymmetric addition of Grignard reagents to ketones: culmination of the ligand-mediated methodology allows modular construction of chiral tertiary alcohols.

A new class of biaryl chiral ligands derived from 1,2-diaminocyclohexane (1,2-DACH) has been designed to enable the asymmetric addition of aliphatic and, for the first time, aromatic Grignard reagents to ketones for the preparation of highly enantioenriched tertiary alcohols (up to 95% ee). The newly developed ligands L12 and L12' together with the previously reported L0 and L0' define a set of complementary chiral promoters, which provides access to the modular construction of a broad range of structurally diverse non-racemic tertiary alcohols, bearing challenging quaternary stereocenters. The present advancements bring to completion our asymmetric Grignard methodology by expanding the scope to aromatic organomagnesium reagents, while facilitating its implementation in organic synthesis thanks to improved synthetic routes for the straightforward access to the chiral ligands. The synthetic utility of the method has been demonstrated by the development of a novel and highly enantioselective formal synthesis of the antihistamine API clemastine via intermediate (R)-3a. Exploiting the power of the 3-disconnection approach offered by the Grignard synthesis, (R)-3a is obtained in 94% ee with ligand (R,R)-L12. The work described herein marks the finalization of our ongoing effort towards the establishment of an effective and broadly applicable methodology for the asymmetric Grignard synthesis of chiral tertiary alcohols.