Rapid SABRE Catalyst Scavenging Using Functionalized Silicas

In recent years the NMR hyperpolarisation method signal amplification by reversible exchange (SABRE) has been applied to multiple substrates of potential interest for in vivo investigation. Unfortunately, SABRE commonly requires an iridium-containing catalyst that is unsuitable for biomedical applications. This report utilizes inductively coupled plasma-optical emission spectroscopy (ICP-OES) to investigate the potential use of metal scavengers to remove the iridium catalytic species from the solution. The most sensitive iridium emission line at 224.268 nm was used in the analysis. We report the effects of varying functionality, chain length, and scavenger support identity on iridium scavenging efficiency. The impact of varying the quantity of scavenger utilized is reported for the three scavengers with the highest iridium removed from initial investigations: 3-aminopropyl (S1), 3-(imidazole-1-yl)propyl (S4), and 2-(2-pyridyl) (S5) functionalized silica gels. Exposure of an activated SABRE sample (1.6 mg mL−1 of iridium catalyst) to 10 mg of the most promising scavenger (S5) resulted in <1 ppm of iridium being detectable by ICP-OES after 2 min of exposure. We propose that combining the approach described herein with other recently reported approaches, such as catalyst separated-SABRE (CASH-SABRE), would enable the rapid preparation of a biocompatible SABRE hyperpolarized bolus.

Fam20C in Human Diseases: Emerging Biological Functions and Therapeutic Implications

Fam20C, a typical member of Fam20 family, has been well-known as a Golgi casein kinase, which is closely associated with Raine Syndrome (RS). It can phosphorylate many secreted proteins and multiple substrates, and thereby plays a crucial role in biological functions. More importantly, Fam20C has also been found to enhance the metastasis of several types of human cancers, such as breast cancer, indicating that Fam20C may be a promising therapeutic target. Accordingly, some small-molecule inhibitors of Fam20C have been reported in cancer. Taken together, these inspiring findings would shed new light on exploiting Fam20C as a potential therapeutic target and inhibiting Fam20C with small-molecule compounds would provide a clue on discovery of more candidate small-molecule drugs for fighting with human diseases.

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded scope is a contemporary challenge. Measuring reaction outcomes on mixtures of substrates, called here SUbstrate Multiplexed Screening (SUMS), has long been used to rigorously quantitate enzyme specificity. Despite the potential utility of SUMS to guide engineering of promiscuous enzymes, this approach has not found widespread adoption in biocatalysis. Here, we develop principles of how to design robust SUMS methods that, rather than assess absolute specificity, use heuristic readouts of substrate promiscuity to identify hits for further investigation. This rich information enables engineering of activity for multiple substrates simultaneously and identifies enzyme variants with altered promiscuity, even when overall activity is lower. We demonstrate the effectiveness of SUMS by engineering two enzymes to produce pharmacologically active tryptamines from simple indole precursors in a biocatalytic cascade. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

Comparative Phenotypic, Proteomic, and Phosphoproteomic Analysis Reveals Different Roles of Serine/Threonine Phosphatase and Kinase in the Growth, Cell Division, and Pathogenicity of Streptococcus suis

Eukaryote-like serine/threonine kinases (STKs) and cognate phosphatases (STPs) comprise an important regulatory system in many bacterial pathogens. The complexity of this regulatory system has not been fully understood due to the presence of multiple STKs/STPs in many bacteria and their multiple substrates involved in many different physiological and pathogenetic processes. Streptococci are the best materials for the study due to a single copy of the gene encoding STK and its cognate STP. Although several studies have been done to investigate the roles of STK and STP in zoonotic Streptococcus suis, respectively, few studies were performed on the coordinated regulatory roles of this system. In this study, we carried out a systemic study on STK/STP in S. suis by using a comparative phenotypic, proteomic, and phosphoproteomic analysis. Mouse infection assays revealed that STK played a much more important role in S. suis pathogenesis than STP. The ∆stk and ∆stp∆stk strains, but not ∆stp, showed severe growth retardation. Moreover, both ∆stp and ∆stk strains displayed defects in cell division, but they were abnormal in different ways. The comparative proteomics and phosphoproteomics revealed that deletion of stk or stp had a significant influence on protein expression. Interestingly, more virulence factors were found to be downregulated in ∆stk than ∆stp. In ∆stk strain, a substantial number of the proteins with a reduced phosphorylation level were involved in cell division, energy metabolism, and protein translation. However, only a few proteins showed increased phosphorylation in ∆stp, which also included some proteins related to cell division. Collectively, our results show that both STP and STK are critical regulatory proteins for S. suis and that STK seems to play more important roles in growth, cell division, and pathogenesis.

Chasing "Zampanalogs": Advancing the Synthesis of the Zampanolide Macrocycle

<p>(-)-Zampanolide (1), a natural product isolated from a marine sponge, is a microtubule-stabilizing agent that exhibits activity in the nanomolar range against various cancer cells, including in P-gp pump overexpressing cells. This attribute makes (-)-zampanolide an interesting target for further investigation. In this work, a new method for a modular and convergent total synthesis of optically pure zampanolide was investigated, which would also allow the generation of “zampanalogs” following the same basic strategy. Their biological activity may then be assessed to allow the elucidation of structure-activity relationships of (-)-zampanolide and its analogs in tubulin binding. The synthetic plan consisted of the modular combination of four major fragments, which would be connected in the late stages of the synthesis and could therefore be easily exchanged to allow the generation of analogs. The C15-C16 bond would be connected via an alkynylation reaction, and a subsequent reductive methylation would install the trisubstituted alkene. The connections at C1 and C3 could be achieved through a Bestmann ylid linchpin reaction, while the macrolactonization would be completed using a ring-closing metathesis to form the C8-C9 alkene. The side chain could be attached at C20 using one of the established aza-aldol methods. The fragments necessary for the formation of the macrocycle were synthesized successfully. The purification strategy throughout the synthetic route was rationalized and provides an improvement with respect to yield and time compared to work previously done in this research group. Alongside these fragments, modified fragments that were originally intended to serve as model systems were synthesized, which could also be used as building blocks in the synthesis of “zampanalogs”. Several methods for a stereoselective alkynylation at C15 were tested. These led to only meager successes, so an approach using a non-stereoselective alkynylation, followed by oxidation and a stereoselective CBS-reduction, was chosen. For the installation of the trisubstituted alkene a reductive methylation with vitride was tested, but this only led to the reduction of the alkyne without methylation. This product may be employed for the synthesis of C17-desmethyl analogs. The reductive methylation at C16-C17 was ultimately achieved using the Gilman reagent in a similar manner to the installation of the C5 methyl group in the C3-C8 fragment. A linchpin strategy with the Bestmann ylid simultaneously formed the connectivity at C1 and C3. This process was successfully performed on multiple substrates arising from the model systems used in the alkynylation and reductive methylation reactions, yielding precursors to the ring-closing metathesis and potentially enabling the synthesis of various analogs. The ring-closing metathesis proved to be difficult in analogs lacking the C17 methyl group and cis-tetrahydropyran ring, and due to this tendency further investigations are necessary. Once the macrocycle has been closed, a global deprotection and oxidation of hydroxy groups is necessary to allow for the installation of the sidechain.</p>

Chasing "Zampanalogs": Advancing the Synthesis of the Zampanolide Macrocycle

<p>(-)-Zampanolide (1), a natural product isolated from a marine sponge, is a microtubule-stabilizing agent that exhibits activity in the nanomolar range against various cancer cells, including in P-gp pump overexpressing cells. This attribute makes (-)-zampanolide an interesting target for further investigation. In this work, a new method for a modular and convergent total synthesis of optically pure zampanolide was investigated, which would also allow the generation of “zampanalogs” following the same basic strategy. Their biological activity may then be assessed to allow the elucidation of structure-activity relationships of (-)-zampanolide and its analogs in tubulin binding. The synthetic plan consisted of the modular combination of four major fragments, which would be connected in the late stages of the synthesis and could therefore be easily exchanged to allow the generation of analogs. The C15-C16 bond would be connected via an alkynylation reaction, and a subsequent reductive methylation would install the trisubstituted alkene. The connections at C1 and C3 could be achieved through a Bestmann ylid linchpin reaction, while the macrolactonization would be completed using a ring-closing metathesis to form the C8-C9 alkene. The side chain could be attached at C20 using one of the established aza-aldol methods. The fragments necessary for the formation of the macrocycle were synthesized successfully. The purification strategy throughout the synthetic route was rationalized and provides an improvement with respect to yield and time compared to work previously done in this research group. Alongside these fragments, modified fragments that were originally intended to serve as model systems were synthesized, which could also be used as building blocks in the synthesis of “zampanalogs”. Several methods for a stereoselective alkynylation at C15 were tested. These led to only meager successes, so an approach using a non-stereoselective alkynylation, followed by oxidation and a stereoselective CBS-reduction, was chosen. For the installation of the trisubstituted alkene a reductive methylation with vitride was tested, but this only led to the reduction of the alkyne without methylation. This product may be employed for the synthesis of C17-desmethyl analogs. The reductive methylation at C16-C17 was ultimately achieved using the Gilman reagent in a similar manner to the installation of the C5 methyl group in the C3-C8 fragment. A linchpin strategy with the Bestmann ylid simultaneously formed the connectivity at C1 and C3. This process was successfully performed on multiple substrates arising from the model systems used in the alkynylation and reductive methylation reactions, yielding precursors to the ring-closing metathesis and potentially enabling the synthesis of various analogs. The ring-closing metathesis proved to be difficult in analogs lacking the C17 methyl group and cis-tetrahydropyran ring, and due to this tendency further investigations are necessary. Once the macrocycle has been closed, a global deprotection and oxidation of hydroxy groups is necessary to allow for the installation of the sidechain.</p>

Conjugation of oligo-His peptides to magnetic γ-Fe2O3@SiO2 core-shell nanoparticles promotes their access to the cytosol

The endosomal entrapment of functional nanoparticles is a severe limitation to their use for biomedical applications. In the case of magnetic nanoparticles (MNPs), this entrapment leads to poor heating efficiency for magnetic hyperthermia and suppresses the possibility to manipulate them in the cytosol. Current strategies to limit their entrapment are based on their functionalization with cell-penetrating peptides in order to promote their translocation directly across the cell membrane or their endosomal escape. However, these strategies suffer from potential release of free peptides in cell and to the best of our knowledge there is currently a lack of effective methods for the cytosolic delivery of MNPs after incubation with cells. Herein, we report the conjugation of fluorescently labelled cationic peptides to γ-Fe2O3@SiO2 core-shell nanoparticles by click chemistry to improve MNP access to the cytosol. We compare the effect of Arg9 and His4 peptides. On one hand, Arg9 is a classical cell-penetrating peptide, able to enter cells by direct translocation and on the other hand, it has been demonstrated that sequences rich in histidine residues promote endosomal escape, most probably by the proton sponge effect. The methodology developed allows to have a high co-localization of the peptides and core-shell nanoparticles in cells and to attest that the grafting onto nanoparticles of peptides rich in histidine promotes NP access to the cytosol. The endosomal escape was confirmed by a calcein leakage assay and by ultrastructural analysis in transmission electron microscopy. No toxicity of the nanoparticles functionalized with peptides was found. We show that our conjugation strategy is compatible with the addition of multiple substrates and can thus be used for the delivery of cytoplasm-targeted therapeutics.

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded scope is a contemporary challenge. Measuring reaction outcomes on mixtures of substrates, called here SUbstrate Multiplexed Screening (SUMS), has long been used to rigorously quantitate enzyme specificity. Despite the potential utility of SUMS to guide engineering of promiscuous enzymes, this approach has not found widespread adoption in biocatalysis. Here, we develop principles of how to design robust SUMS methods that, rather than assess absolute specificity, use heuristic readouts of substrate promiscuity to identify hits for further investigation. This rich information enables engineering of activity for multiple substrates simultaneously and identifies enzyme variants with altered promiscuity, even when overall activity is lower. We demonstrate the effectiveness of SUMS by engineering two enzymes to produce pharmacologically active tryptamines from simple indole precursors in a biocatalytic cascade. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

A Model for Correcting the Pressure Drop between Two Monoliths

This paper is concerned with the modeling of the pressure drop through monolith honeycombs. Monolith substrates are promising for the intensification of catalytic processes, especially because of their low back-pressure. There have been several improvements in the modeling of monolith reactors during the last decade, most of them focused on a single substrate configuration, while research in multiple substrates in a single reactor is still sparse. One example is the so-called "minor losses", such as those because of the flow entering and leaving a substrate. Both phenomena interact when two monoliths are placed close in series, and the extra losses produced by them may become relevant when relatively short monoliths are used. In this paper, a spatially resolved computational model of monolith channels arranged in series is used to compute the extra pressure drop because of the flow leaving one substrate and entering the next one downstream. Several Reynolds numbers and spacing lengths for the channels between substrates are investigated. According to the results, for close-coupled monoliths, the inlet and outlet effects produce a negligible pressure drop compared to that in a single monolith configuration. This phenomenon can be accounted for by introducing a correction factor. The magnitude of the correction factor depends on the channel’s Reynolds number, diameter, and spacing length. A model for such a factor is proposed. The model accurately predicts the trend and magnitude of the correction factor.