Double Substitution Research Articles

One‐pot Construction of Dibenzooxabicyclo[5.5.0]dodecanes

AbstractUnder the open‐vessel atmosphere conditions, the one‐pot synthesis of bis‐sulfonyl dibenzooxabicyclo[5.5.0]dodecanes was developed by double substitution of 1,3‐dichloroacetone with arenesulfinic acid sodium salts, and C‐ and O‐alkylation of the resulting 1,3‐bis‐sulfonylacetones with 2 equivalents of o‐bis(bromomethyl)arenes. A plausible mechanism is proposed and discussed. This high‐yielding protocol provides an intermolecular substitution and an intramolecular ring‐closure via the formations of three carbon−carbon single (C−C) bonds, two carbon−sulfur single (C−S) bonds, and one carbon−oxygen single (C−O) bond.magnified image

Mg1-xNixFe2-xCrxO4 synthesis via hydrothermal route: Effect of doping on the structural, optical, electrical, magnetic and photocatalytic properties

A pristine MgFe2O4 and a series of Ni and Cr substituted magnesium ferrite NPs have been fabricated via hydrothermal route. The influence of double ions substitution on MgFe2O4 was examined attributed to structural, optical, electrical, magnetic and photocatalytic properties. X-ray diffraction analysis revealed cubic geometry having fd3m space group with an average crystalline size in 25–33 nm. From hysteresis loop saturation polarization (Ps), coercivity and remanence polarization (Pr)was evaluated and found that the value of Ps and Pr were improved on increasing dopants content. VSM analysis confirms the ferromagnetic behavior of Mg1-xNixFe2-xCrxO4. Furthermore, thedielectric loss decreased by increasing frequency as well as the amount of dopant. UV–Vis absorption spectrum exhibited red-shift and band-gap energy reduced i.e., 2.58 to 2.29 eV. PCA study evaluated using crystal violet (CV) dye and Mg1-xNixFe2-xCrxO4 NPs exhibited higher efficiency and 97 % dye degradation was observed for Mg1-xNixFe2-xCrxO4 versus MgFe2O4 (59 %) under sunlight irradiation in 100 min. Moreover, the stability of NPs was confirmed through recycling experiment and Mg1-xNixFe2-xCrxO4 showed promising stability and reusability. Thus, the enhanced photocatalytic activity under solar light revealed its application for remediation of dyes in industrial waste under sunlight irradiation, it will make the process highly feasible.

Water soluble octa-imidazolium zinc phthalocyanine for nucleus/nucleolus cell fluorescence microscopy and photodynamic therapy.

A poly-cationic theranostic macrocycle was developed to perform confocal microscopy imaging and photodynamic therapy studies on a model of melanoma cancer, one of the most aggressive cancer. Hence, an octa-imidazolium zinc phthalocyanine was conveniently synthesized in large amount in three steps in a 44% overall yield: upon double nucleophilic aromatic substitution, cyclo-tetramerization and quaternization reactions. Such an octa-cationic molecule was readily soluble in physiological media, reaching concentrations beyond 1mM. It showed fluorescence properties in aqueous medium (ΦF = 0.31) with no noticeable aggregation, spectroscopy studies showed. In vitro confocal fluorescence microscopy studies carried out on murine melanoma model (B16F10 cells) showed that the fluorophore was mainly located in the cell nucleolus, an organelle of interest for the treatment of cancer. The anticancer photodynamic potential of the octa-cationic photosensitizer could be measured (IC50 = 5.4µM) using the MTS viability assay. Both fluorescence microscopy studies and photodynamic studies demonstrate the octa-cationic molecule is theranostic and could be further developed for future photodynamic diagnosis (PDD) and photodynamic inactivation of micro-organisms (PDI).

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution

DEVELOPMENT TECHNOLOGY OF SULFITE PULP TCF BLEACHING FOR CHEMICAL PROCESSING

An environmentally friendly chlorine-free technology for producing wood sulfite soluble pulp using only one oxidizing bleaching agent – hydrogen peroxide has been developed: unconventional catalyzed peroxide delignification and hydrogen peroxide bleaching. The conditions of cooking, delignification, extraction and bleaching of spruce sulfite pulp obtained as a result of studies, as well as successful combinations of conditions of all stages made it possible to obtain two types of soluble pulp by one technology: soluble pulp for nitration (for gunpowder) was obtained under a short scheme including a delignification step and hot alkaline extraction; by continuing this scheme with hydrogen peroxide bleaching in two steps, viscose pulp was obtained. The use of this technology will make it possible to implement a double import substitution – in raw materials (wood instead of cotton) and in finished products (pulp for chemical processing - for nitration and viscose pulp, which are currently imported).

No evidence for widespread positive selection on double substitutions within codons in primates and yeasts.

Nucleotide substitutions in protein-coding genes can be divided into synonymous (S) and non-synonymous (N) ones that alter amino acids (including nonsense mutations causing stop codons). The S substitutions are expected to have little effect on function. The N substitutions almost always are affected by strong purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases can modulate the deleterious effect of single N substitutions and, thus, could be subjected to the positive selection. This effect has been demonstrated for mutations in the serine codons, stop codons and double N substitutions in prokaryotes. In all abovementioned cases, a novel technique was applied that allows elucidating the effects of selection on double substitutions considering mutational biases. Here, we applied the same technique to study double N substitutions in eukaryotic lineages of primates and yeast. We identified markedly fewer cases of purifying selection relative to prokaryotes and no evidence of codon double substitutions under positive selection. This is consistent with previous studies of serine codons in primates and yeast. In general, the obtained results strongly suggest that there are major differences between studied pro- and eukaryotes; double substitutions in primates and yeasts largely reflect mutational biases and are not hallmarks of selection. This is especially important in the context of detection of positive selection in codons because it has been suggested that multiple mutations in codons cause false inferences of lineage-specific site positive selection. It is likely that this concern is applicable to previously studied prokaryotes but not to primates and yeasts where markedly fewer double substitutions are affected by positive selection.

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.

Semi-rational protein engineering of a novel ene-reductase from Galdieria sulphuraria for asymmetric reduction of (R)-carvone and ketoisophorone.

Asymmetric reduction of (R)-carvone and ketoisophorone by an engineered ene-reductase from Galdieria sulphuraria (GsOYE) combined with glucose dehydrogenase for NADPH regeneration were studied. A semi-rational protein engineering was used to enhance the activity and selectivity of GsOYE. Upon the sequence alignment and molecular docking results, two amino acid residues at positions 66 and 270 were selected as saturation mutation sites. Finally, a single substitution variant of GsOYE-N270A with complete conversion (100%) and diastereoselectivity (dep >99%) for reduction of (R)-carvone and a double substitution variant GsOYE-Y66P/N270H with improved stereoselectivity for reduction of ketoisophorone were obtained.

Six-Membered Saturated NHC-Stabilized Borenium Cations: Isolation of a Cationic Analogue of Borinic Acid.

The reaction of six-membered saturated NHC [1,3-di(2,6-diisopropylphenyl) tetrahydropyrimidine-2-ylidene; henceforth abbreviated as 6-SIDipp] with PhBCl2 yields a Lewis base adduct, 6-SIDipp·PhBCl2 (1), which readily undergoes nucleophilic substitution reaction with AgNO3, leading to the single (2) and double (3) substitution of both chlorides with ONO2 moieties at the boron atom. The reaction of 1 with 1 equiv of AlCl3 resulted in a borenium cation of composition [6-SIDipp·B(Ph)Cl]+ (4) with AlCl4- as the counteranion. Although borenium cations with different substituents on boron have been reported, a structurally characterized phenylchloroborenium cation remains unknown. Similarly, the reaction of 1 with triflic acid provides the first representative of a new class of borenium cations bearing one hydroxyl and one phenyl group on boron (5), a cationic analogue of borinic acid.

Application of Box and Voronoi CAPs for Metastable Electronic States in Molecular Clusters.

The complex absorbing potential (CAP) approach offers a practical tool for characterization of energies and lifetimes of metastable electronic states, such as temporary anions and core ionized states. Here, we present an implementation of the smooth Voronoi CAP combined with the equation-of-motion coupled cluster with single and double substitutions method for metastable states. The performances of the smooth Voronoi CAP and box CAP are compared for different classes of systems: resonances in isolated molecules and localized and delocalized resonances in molecular clusters. The benchmark calculations show that the Voronoi CAP is generally more robust when applied to molecular clusters, but box CAPs are equally reliable for localized resonances or in the cases when the resonance does not exhibit significant electron density delocalization into the intramolecular region. As such, the choice of the CAP shape and onset should be guided by the character of the metastable states.

Abstract 3627: The genomic landscape of breast cancers from CHEK2 c.1100delC mutation carriers

Abstract Women who carry the germline CHEK2 c.1100delC mutation have a 2.3-fold increased life-time risk to develop breast cancer (BC). Their BCs are mostly of the luminal/ER+ subtype and diagnosed at a younger age than BCs in sporadic patients. BC patients carrying a CHEK2 c.1100delC mutation are at an increased risk of developing a contralateral BC and have a worse survival compared to sporadic BC patients although resistance to either endocrine or chemotherapy does not appear to play a role herein. To unravel the biological mechanism by which CHEK2 c.1100delC exerts its tumorigenic potential, whole genome sequencing was performed of matching normal and primary BC (pBC) DNA from twenty female CHEK2 c.1100delC mutation carriers at Hartwig Medical Foundation (HMF). We compared the somatic pBC genomes of these twenty patients to 560 pBC genomes from the International Cancer Genome Consortium including 30 BRCA1 and 25 BRCA2 mutation carriers and 329 sporadic ER+ pBC patients. The validation cohort consisted of 517 metastatic BC (mBC) genomes from the Center for Personalized Cancer Treatment that were sequenced at HMF and included 24 mBC genomes from CHEK2 c.1100delC mutation carriers. We found that CHEK2 pBC and mBC genomes were dissimilar to pBC and mBC genomes of BRCA1 and BRCA2 mutation carriers and did not show evidence of a homologous recombination deficiency (HRD) phenotype. Instead, CHEK2 pBC and mBC genomes were very similar to genomes of sporadic ER+ pBC and mBC patients in terms of tumor mutational burden, single and double base substitution signatures as well as small insertion/deletion signatures. The size distribution of structural variants (SVs), however, was different for CHEK2 pBCs and mBCs compared with ER+ as well as BRCA1 and BRCA2 pBCs and mBCs. Importantly, the CHEK2 c.1100delC mutation was associated with an increased frequency of larger (> 1Mb) deletions, inversions and tandem duplications (TDs) with peaks at specific sizes. Although CHEK2 mBC genomes more frequently harbored chromothripsis compared with other mBC subtypes, this did not explain the CHEK2-specific SV size distribution pattern. Moreover, increased chromothripsis in CHEK2 mBC genomes was not associated with a higher frequency of somatic TP53 mutations. In fact, we did not observe any somatic TP53 mutations among CHEK2 pBC and mBC genomes. This mutual exclusiveness suggests CHEK2-driven tumorigenesis could act through the TP53 signaling pathway. To conclude, CHEK2 BC genomes do not harbor somatic TP53 mutations and display a unique SV size distribution profile. Moreover, the absence of an HRD phenotype among CHEK2 BC genomes suggests PARP inhibitors will not be effective for treatment of BC in CHEK2 c.1100delC mutation carriers. Increasing knowledge of the mechanisms of CHEK2-driven tumorigenesis may ultimately lead to the development of tailored strategies for treatment and prevention of BC for CHEK2 mutation carriers. Citation Format: Marcel Smid, Marjanka K. Schmidt, Wendy J. Prager - van der Smissen, Kirsten Ruigrok - Ritstier, Sten Cornelissen, Maartje A. Schreurs, J. Margriet Collee, Muriel A. Adank, Maartje J. Hooning, John W. Martens, Antoinette Hollestelle. The genomic landscape of breast cancers from CHEK2 c.1100delC mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3627.

Yb3+ f−f excitations in NaYbSe2 : Benchmarking embedded-cluster quantum chemical schemes for 4f insulators

$\tilde{S}\!=\!1/2$ triangular-lattice $f$-electron materials define a dynamic research area in condensed matter magnetism. In various Yb 4$f^{13}$ triangular-lattice compounds, for example, spin-liquid ground states seem to be realized. Using {\it ab initio} quantum chemical methods, we here investigate how correlation effects involving the 4$f$ electrons affect the on-site $f$-$f$ excitation spectrum in NaYbSe$_2$. The system is well suited for such a study since unambiguous inelastic neutron scattering data are available for the Yb$^{3+}$ $f$-$f$ transitions. The excitation energies obtained by configuration-interaction calculations with single and double substitutions agree within 3-4 meV with experimental values, which provides a not so expensive alternative to fitting experimental data at the model-Hamiltonian level in order to analyze $f$-center multiplet structures.

Effect of Chiral Purity on Adjuvanticity of Archaeol-Based Glycolipids.

Archaeosomes composed of sulfated lactosyl archaeol (SLA) glycolipids from stereoisomerically pure archaeol (1) are vaccine adjuvants that can boost immunogenicity and vaccine efficacy in preclinical models. Herein, we report a new synthesis of 2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (3) by treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by reductive debenzylation. Three SLA archaeosomes from archaeols of different chiral purities were prepared, and the effect of stereochemistry on their adjuvanticity toward ovalbumin was investigated. It was found that all SLA archaeosomes induced strong humoral and cell-mediated antigen-specific immune responses following immunization of C57BL/6NCrl mice, with no significant differences, irrespective of the chiral purities. The responses were comparable or better than those obtained using mimetics of approved adjuvants. The performance of SLA archaeosomes during immunization and their lack of dependence on the stereochemistry of archaeol points toward a promising, safe, scalable, and economically viable vaccine adjuvant system.

Editors' Selections from Relevant Scientific Publications

Editors' Selections from Relevant Scientific Publications

Octahedral oxide glass network in ambient pressure neodymium titanate

Rare-earth titanates form very fragile liquids that can be made into glasses with useful optical properties. We investigate the atomic structure of 83TiO2-17Nd2O3 glass using pair distribution function (PDF) analysis of X-ray and neutron diffraction with double isotope substitutions for both Ti and Nd. Six total structure factors are analyzed (5 neutron + 1 X-ray) to obtain complementary sensitivities to O and Ti/Nd scattering, and an empirical potential structure refinement (EPSR) provides a structural model consistent with the experimental measurements. Glass density is estimated as 4.72(13) g cm−3, consistent with direct measurements. The EPSR model indicates nearest neighbor interactions for Ti-O at overline{r}_{TiO} = 1.984(11) Å with coordination of n_{TiO} = 5.72(6) and for Nd-O at overline{r}_{NdO} = 2.598(22) Å with coordination of n_{NdO} = 7.70(26), in reasonable agreement with neutron first order difference functions for Ti and Nd. The titanate glass network comprises a mixture of distorted Ti-O5 and Ti-O6 polyhedra connected via 71% corner-sharing and 23% edge-sharing. The O-Ti coordination environments include 15% nonbridging O-Ti1, 51% bridging O-Ti2, and 32% tricluster O-Ti3. This structure is highly unusual for oxide glasses melt-quenched at ambient pressure, as it consists of Ti-Ox predominantly in octahedral (with nearly no tetrahedral) coordination.

Evidence for Non-Essential Salt Bridges in the M-Gates of Mitochondrial Carrier Proteins.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

Affinity Maturation of an Ovarian Cancer‐Targeting Peptide

Ovarian cancer is among the leading causes of cancer deaths in women. The low survival rate is in part due to inadequate means of detection, which emphasizes the need to develop effective screening techniques. Peptide J18 was previously discovered using phage display technology and was found to specifically target and image human ovarian tumors in a SKOV‐3 xenografted mouse model.Here, affinity maturation of J18 was carried out to improve its binding affinity by conducting an alanine scan of the peptide. A cell‐based modified ELISA using SKOV‐3 cells was carried out and dose‐response curves were modelled using non‐linear regression. The results showed that substitution of serine‐5 (S5) and aspartic acid‐6 (D6) with alanine decreased the EC50 value of the original peptide J18 from 45.02 µM (39.15‐52.94 95% confidence interval; CI) to 19.54 (9.70‐38‐51 95% CI) and 6.92 (5.2‐9.80 95% CI), respectively. Next, a double alanine substitution of S5 and D6 (J18‐S5A‐D6A) was investigated to further increase the binding affinity. The EC50 value was determined to be 4.22 (2.16‐16.14 95% CI), thus improving the binding affinity approximately 10‐fold.To ensure that J18‐S5A‐D6A maintained specificity for ovarian cancer cells, the peptide was evaluated against ovarian cancer SKOV‐3, pancreatic cancer Mia‐Paca‐2, prostate cancer LNCaP, and embryonic kidney HEK 293 cells using a modified ELISA and fluorescent microscopy. Both assays showed significantly increased binding of J18‐S5A‐D6A to SKOV‐3 cells compared to the other cell lines. Based on these results, peptide J18‐S5A‐D6A may be used as an improved imaging agent of ovarian cancer.

Substitution mutational signatures in whole-genome-sequenced cancers in the UK population.

Whole-genome sequencing (WGS) permits comprehensive cancer genome analyses, revealing mutational signatures, imprints of DNA damage and repair processes that have arisen in each patient's cancer. We performed mutational signature analyses on 12,222 WGS tumor-normal matched pairs, from patients recruited via the UK National Health Service. We contrasted our results to two independent cancer WGS datasets, the International Cancer Genome Consortium (ICGC) and Hartwig Foundation, involving 18,640 WGS cancers in total. Our analyses add 40 single and 18 double substitution signatures to the current mutational signature tally. Critically, we show for each organ, that cancers have a limited number of 'common' signatures and a long tail of 'rare' signatures. We provide a practical solution for utilizing this concept of common versus rare signatures in future analyses.

Comprehensive Molecular Profiling of Colorectal Cancer With Situs Inversus Totalis by Next-Generation Sequencing.

BackgroundColorectal cancer (CRC) is one of the most prevalent malignances worldwide. However, CRC with situs inversus totalis (SCRC) is extremely rare, and molecular characterization of this disease has never been investigated.MethodsTumor tissue samples from 8 patients with SCRC and 33 CRC patients without situs inversus totalis (NSCRC) were subjected to multigene next-generation sequencing.ResultsThe most frequently mutated genes in SCRC were APC, TP53, CHEK2, MDC1, GNAQ, KRAS, and SMAD4. A high frequency of SCRC tumors had mutations in DNA damage repair genes. Single amino acid substitutions in the DNA damage repair genes caused by continuous double base substitution was identified in the majority of this population. Furthermore, mutational profiles showed notable differences between the SCRC and NSCRC groups. In particular, CHEK2, MDC1, GNAQ, SMAD4, BRCA1, HLA-B, LATS2, and NLRC5 mutations were more frequently observed in SCRC patients. The mutation loci distributions of KRAS in the SCRC cohort differed from that of the NSCRC cohort. Additionally, differences in the targeted genomic profiles and base substitution patterns were observed between the two groups.ConclusionsThese findings comprehensively revealed a molecular characterization of SCRC, which will contribute to the development of personalized therapy and improved clinical management of SCRC patients.

CRISPR/Cas9‐mediated P‐CR domain‐specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar

SummaryCellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high‐value bioproducts. The large transmembrane‐localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para‐crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant‐conserved region (P‐CR) of PalCESA4 subunit, which are involved in the CESA4‐CESA8 heterodimerization. The CRISPR/Cas9‐knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4‐binding affinity resulting in normal roots, 1.5–2‐fold higher plant growth and cell wall regeneration rates, 1.7‐fold thinner cell wall, high hemicellulose content, 37%–67% decrease in cellulose content, high cellulose DP, 25%–37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2‐fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9‐mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose‐based nanomaterial technologies.