SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection.

BACKGROUND: Current therapies for recurrent Clostridioides difficile infection do not address the disrupted microbiome, which supports C. difficile spore germination into toxin-producing bacteria. SER-109 is an investigational microbiome therapeutic composed of purified Firmicutes spores for the treatment of recurrent C. difficile infection. METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial in which patients who had had three or more episodes of C. difficile infection (inclusive of the qualifying acute episode) received SER-109 or placebo (four capsules daily for 3 days) after standard-of-care antibiotic treatment. The primary efficacy objective was to show superiority of SER-109 as compared with placebo in reducing the risk of C. difficile infection recurrence up to 8 weeks after treatment. Diagnosis by toxin testing was performed at trial entry, and randomization was stratified according to age and antibiotic agent received. Analyses of safety, microbiome engraftment, and metabolites were also performed. RESULTS: Among the 281 patients screened, 182 were enrolled. The percentage of patients with recurrence of C. difficile infection was 12% in the SER-109 group and 40% in the placebo group (relative risk, 0.32; 95% confidence interval [CI], 0.18 to 0.58; P<0.001 for a relative risk of <1.0; P<0.001 for a relative risk of <0.833). SER-109 led to less frequent recurrence than placebo in analyses stratified according to age stratum (relative risk, 0.24 [95% CI, 0.07 to 0.78] for patients <65 years of age and 0.36 [95% CI, 0.18 to 0.72] for those ≥65 years) and antibiotic received (relative risk, 0.41 [95% CI, 0.22 to 0.79] with vancomycin and 0.09 [95% CI, 0.01 to 0.63] with fidaxomicin). Most adverse events were mild to moderate and were gastrointestinal in nature, with similar numbers in the two groups. SER-109 dose species were detected as early as week 1 and were associated with bile-acid profiles that are known to inhibit C. difficile spore germination. CONCLUSIONS: In patients with symptom resolution of C. difficile infection after treatment with standard-of-care antibiotics, oral administration of SER-109 was superior to placebo in reducing the risk of recurrent infection. The observed safety profile of SER-109 was similar to that of placebo. (Funded by Seres Therapeutics; ECOSPOR III ClinicalTrials.gov number, NCT03183128.).

Epigenetic heterogeneity hotspots in human liver disease progression.

BACKGROUND AND AIMS: Disruption of the epigenome is a hallmark of human disease, including liver cirrhosis and HCC. While genetic heterogeneity is an established effector of pathologic phenotypes, epigenetic heterogeneity is less well understood. Environmental exposures alter the liver-specific DNA methylation landscape and influence the onset of liver cancer. Given that currently available treatments are unable to target frequently mutated genes in HCC, there is an unmet need for novel therapeutics to prevent or reverse liver damage leading to hepatic tumorigenesis, which the epigenome may provide. APPROACH AND RESULTS: We performed genome-wide profiling of DNA methylation, copy number, and gene expression from multiple liver regions from 31 patients with liver disease to examine their crosstalk and define the individual and combinatorial contributions of these processes to liver disease progression. We identified epigenetic heterogeneity hotspots that are conserved across patients. Elevated epigenetic heterogeneity is associated with increased gene expression heterogeneity. Cirrhotic regions comprise 2 distinct cohorts-one exclusively epigenetic, and the other where epigenetic and copy number variations collaborate. Epigenetic heterogeneity hotspots are enriched for genes central to liver function (eg, HNF1A ) and known tumor suppressors (eg, RASSF1A ). These hotspots encompass genes including ACSL1 , ACSL5 , MAT1A , and ELFN1 , which have phenotypic effects in functional screens, supporting their relevance to hepatocarcinogenesis. Moreover, epigenetic heterogeneity hotspots are linked to clinical measures of outcome. CONCLUSIONS: Substantial epigenetic heterogeneity arises early in liver disease development, targeting key pathways in the progression and initiation of both cirrhosis and HCC. Integration of epigenetic and transcriptional heterogeneity unveils putative epigenetic regulators of hepatocarcinogenesis.

Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization.

Peptide asparaginyl ligases (PALs) hold significant potential in protein bioconjugation due to their excellent kinetic properties and broad substrate compatibility. However, realizing their full potential in biocatalytic applications requires precise control of their activity. Inspired by nature, we aimed to compartmentalize a representative PAL, OaAEP1-C247A, within protein containers to create artificial organelles with substrate sorting capability. Two encapsulation approaches were explored using engineered lumazine synthases (AaLS). The initial strategy involved tagging the PAL with a super-positively charged GFP(+36) for encapsulation into the super-negatively charged AaLS-13 variant, but it resulted in undesired truncation of the enzyme. The second approach involved genetic fusion of the OaAEP1-C247A with a circularly permutated AaLS variant (cpAaLS) and its co-production with AaLS-13, which successfully enabled compartmentalization of the PAL within a patch-work protein cage. Although the caged PAL retained its activity, it was significantly reduced compared to the free enzyme (∼30-40-fold), likely caused by issues related to OaAEP1-C247A stability and folding. Nevertheless, these findings demonstrated the feasibility of the AaLS encapsulation approach and encourage further optimization in the design of peptide-ligating artificial organelles in E. coli, aiming for a more effective and stable system for protein modifications.

Deep Parasternal Intercostal Plane Block for Intraoperative Pain Control in Cardiac Surgical Patients for Sternotomy: A Prospective Randomized Controlled Trial.

OBJECTIVES: Sternotomy pain is common after cardiac surgery. The deep parasternal intercostal plane (DPIP) block is a novel technique that provides analgesia to the anterior chest wall. The aim of this study was to investigate the analgesic effect of bilateral DPIP blocks on intraoperative pain control in cardiac surgery. DESIGN: This is a double-blinded, prospective randomized controlled trial (Oct 2020-Dec 2022). SETTINGS: This study was conducted in a single institution, which is an academic university hospital. PARTICIPANTS: Eighty-six elective cardiac surgical patients with median sternotomy were recruited. INTERVENTIONS: Patients were randomly divided into DPIP or control group. Either 20ml 0.25% levobupivacaine or 0.9% normal saline was injected for the DPIP under ultrasound guidance after induction of general anaesthesia. MEASUREMENTS AND MAIN RESULTS: The primary outcome was intraoperative opioids consumption and hemodynamic changes at sternotomy. Secondary outcomes included postoperative morphine consumption, postoperative pain and time to tracheal extubation. Intraoperative opioids requirement was reduced from a median (IQR) intravenous morphine equivalence of 21.4mg (13.8-24.3mg) in control group to 9.5mg (7.3-11.2mg) in the DPIP group (P<0.001). Hemodynamic parameters were more stable in DPIP group at sternotomy, as evidenced by lower percentage increase in systolic, diastolic and mean arterial blood pressure from baseline. No difference was observed in time to tracheal extubation, postoperative morphine consumption, postoperative pain score and spirometry. CONCLUSIONS: Bilateral DPIP block provides effective intraoperative analgesia and opioid-sparing. It may be included as part of the multimodal analgesia for enhanced recovery in cardiac surgery.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Prevalence of Comorbid Factors in Patients With Recurrent Clostridioides difficile Infection in ECOSPOR III, a Randomized Trial of an Oral Microbiota-Based Therapeutic.

BACKGROUND: Although comorbidities are risk factors for recurrent Clostridioides difficile infection (rCDI), many clinical trials exclude patients with medical conditions such as malignancy or immunosuppression. In a phase 3, double-blind, placebo-controlled, randomized trial (ECOSPOR III), fecal microbiota spores, live (VOWST, Seres Therapeutics; hereafter "VOS," formerly SER-109), an oral microbiota therapeutic, significantly reduced the risk of rCDI at week 8. We evaluated the efficacy of VOS compared with placebo in patients with comorbidities and other risk factors for rCDI. METHODS: Adults with rCDI were randomized to receive VOS or placebo (4 capsules daily for 3 days) following standard-of-care antibiotics. In this post hoc analysis, the rate of rCDI through week 8 was assessed in VOS-treated participants compared with placebo for subgroups including (i) Charlson comorbidity index (CCI) score category (0, 1-2, 3-4, ≥5); (ii) baseline creatinine clearance (<30, 30-50, >50 to 80, or >80 mL/minute); (iii) number of CDI episodes, inclusive of the qualifying episode (3 and ≥4); (iv) exposure to non-CDI-targeted antibiotics after dosing; and (v) acid-suppressing medication use at baseline. RESULTS: Of 281 participants screened, 182 were randomized (59.9% female; mean age, 65.5 years). Comorbidities were common with a mean overall baseline age-adjusted CCI score of 4.1 (4.1 in the VOS arm and 4.2 in the placebo arm). Across all subgroups analyzed, VOS-treated participants had a lower relative risk of recurrence compared with placebo. CONCLUSIONS: In this post hoc analysis, VOS reduced the risk of rCDI compared with placebo, regardless of baseline characteristics, concomitant medications, or comorbidities.

D-Peptide and D-Protein Technology: Recent Advances, Challenges, and Opportunities.

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.

Interferon drives HCV scarring of the epigenome and creates targetable vulnerabilities following viral clearance.

BACKGROUND AND AIMS: Chronic HCV infection is a leading etiologic driver of cirrhosis and ultimately HCC. Of the approximately 71 million individuals chronically infected with HCV, 10%-20% are expected to develop severe liver complications in their lifetime. Epigenetic mechanisms including DNA methylation and histone modifications become profoundly disrupted in disease processes including liver disease. APPROACH AND RESULTS: To understand how HCV infection influences the epigenome and whether these events remain as "scars" following cure of chronic HCV infection, we mapped genome-wide DNA methylation, four key regulatory histone modifications (H3K4me3, H3K4me1, H3K27ac, and H3K27me3), and open chromatin in parental and HCV-infected immortalized hepatocytes and the Huh7.5 HCC cell line, along with DNA methylation and gene-expression analyses following elimination of HCV in these models through treatment with interferon-α (IFN-α) or a direct-acting antiviral (DAA). Our data demonstrate that HCV infection profoundly affects the epigenome (particularly enhancers); HCV shares epigenetic targets with interferon-α targets; and an overwhelming majority of epigenetic changes induced by HCV remain as "scars" on the epigenome following viral cure. Similar findings are observed in primary human patient samples cured of chronic HCV infection. Supplementation of IFN-α/DAA antiviral regimens with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine synergizes in reverting aberrant DNA methylation induced by HCV. Finally, both HCV-infected and cured cells displayed a blunted immune response, demonstrating a functional effect of epigenetic scarring. CONCLUSIONS: Integration of epigenetic and transcriptional data elucidate key gene deregulation events driven by HCV infection and how this may underpin the long-term elevated risk for HCC in patients cured of HCV due to epigenome scarring.

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins.

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(II), as a linker for C-to-N SPCL.

Reactions of Tri-tert-Butylphosphatetrahedrane as a Spring-Loaded Phosphinidene Synthon Featuring Nickel-Catalyzed Transfer to Unactivated Alkenes.

Cage-opening reactions of the highly strained tri-tert-butylphosphatetrahedrane (1), shown here to function as a synthon of (tri-tert-butylcyclopropenyl)phosphinidene, are described. Treatment of 1 with a base-stabilized silylene led to the corresponding phosphasilene, which was isolated in 72% yield as a red crystalline solid. Phosphinidene transfer was also observed when 1 (2 equiv) was combined with the Wittig reagent Ph3PCH2 to form a diphosphirane (50% isolated yield). The reaction is proposed to proceed through a generated phosphaalkene intermediate, which was characterized by NMR spectroscopy. In addition, we report on nickel-catalyzed phosphinidene transfer to styrene, ethylene, neohexene, and 1,3-cyclohexadiene; the corresponding phosphiranes were isolated in 51-64% yield. Computational studies suggest the intermediacy of a nickel phosphinidene species. Treatment of the ethylene-derived phosphirane product with triflic acid delivered elimination of [tBu3C3]OTf and formation of a P-H bond, illustrating the ability of the tri-tert-butyl cyclopropenyl group to serve as a protecting group that is removable following phosphinidene transfer.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Introducing N-Heterocyclic Iminophosphoranes (NHIPs): Synthesis by [3 + 2] Cycloaddition of Azophosphines with Alkynes and Reactivity Studies.

Azophosphines (Ar-N═N-PR2) were prepared from N-aryl-N'-(trimethylsilyl)diazenes (Ar-N═N-SiMe3) and R2PCl by Me3SiCl elimination or oxidation of phosphinohydrazines (Ar-NH-NH-PR2) by 2,5-dialkyl-1,4-benzoquinones. Azophosphines underwent 1,3-dipolar cycloaddition with cyclooctyne and dimethylacetylene dicarboxylate to give N-heterocyclic iminophosphoranes (NHIPs), which are structurally similar to cyclic (alkyl)(amino)carbenes. The cycloaddition reaction is compatible with various phosphorus atom substituents including phenyl (NHIP-1,4,6), isopropyl (NHIP-2), cyclohexyl (NHIP-3), and dimethylamino (NHIP-5) groups. The pKBH+ values of the NHIPs in acetonitrile range from 13.13 to 23.14. On the basis of the Huynh electronic parameter, NHIP-1 and NHIP-2 have σ-donor strengths comparable with that of 1,8-diazabicyclo[5.4.0]undec-7-ene. NHIP-1 underwent facile 1,2-addition with pentafluoropyridine to form a rare fluorophosphorane. The treatment of NHIP-1 with triphenylsilane resulted in P-N bond cleavage, accompanied by the reduction of phosphorus(V) to phosphorus(III). A homoleptic, cationic CuI-NHIP-1 complex was also prepared. The potential utility of π-donating NHIPs was demonstrated by the stabilization of a reactive iminoborane (Cl-B≡N-SiMe3). The facile scalable synthesis, tunability of steric demands, and basicity of NHIPs suggest that this new heterocycle class may find a wide range of applications in synthetic chemistry.

Staudinger Reactivity and Click Chemistry of Anthracene (A)-Based Azidophosphine N3PA.

11-Azido-9,10-dihydro-9,10-phosphanoanthracene (N3PA) has been demonstrated recently as a transfer reagent for molecular phosphorus mononitride (PN) because it easily dissociates at room temperature into dinitrogen (N2), PN, and anthracene (A). Here we report further reactivity studies of the N3PA molecule including strain-promoted 1,3-dipolar cycloaddition with cyclooctyne and Staudinger-type reactivity. Calculations at the DLPNO-CCSD(T)/cc-pVTZ//PBE0-D3(BJ)/cc-pVTZ level of theory indicate that the click reaction is faster than the dissociation of N3PA. The Staudinger-type reactivity enabled transfer of the NPA fragment to a base-stabilized silylene. The previously reported intermediate of vanadium trisanilide with an NPA ligand could be isolated in 61% yield and structurally characterized in a single-crystal X-ray diffraction experiment. In line with the previously reported phosphinidene reactivity of the transient vanadium phosphorus mononitride complex, thermolysis or irradiation of the complex leads to A elimination and formation of the corresponding vanadium PN dimer or trimer, respectively.

Frustrated Lewis Pair Stabilized Phosphoryl Nitride (NPO), a Monophosphorus Analogue of Nitrous Oxide (N2O).

Phosphoryl nitride (NPO) is a highly reactive intermediate, and its chemistry has only been explored under matrix isolation conditions so far. Here we report the synthesis of an anthracene (A) and phosphoryl azide based molecule (N3P(O)A) that acts as a molecular synthon of NPO. Experimentally, N3P(O)A dissociates thermally with a first-order kinetic half-life that is associated with an activation enthalpy of ΔH⧧ = 27.5 ± 0.3 kcal mol-1 and an activation entropy of ΔS⧧ = 10.6 ± 0.3 cal mol-1 K-1 that are in good agreement with calculated DLPNO-CCSD(T)/cc-pVTZ//PBE0-D3(BJ)/cc-pVTZ energies. In solution N3P(O)A undergoes Staudinger reactivity with tricyclohexylphosphine (PCy3) and subsequent complexation with tris(pentafluorophenyl)borane (B(C6F5)3, BCF) to form Cy3P-NP(A)O-B(C6F5)3. Anthracene is cleaved off photochemically to form the frustrated Lewis pair (FLP) stabilized NPO complex Cy3P⊕-N═P-O-B⊖(C6F5)3. An intrinsic bond orbital (IBO) analysis suggests that the adduct is zwitterionic, with a positive and negative charge localized on the complexing Cy3P and BCF, respectively.

Dimerization and Cycloaddition Reactions of Transient Tri-tert-butylphosphacyclobutadiene Generated by Lewis Acid Induced Isomerization of Tri-tert-butylphosphatetrahedrane.

Tri-tert-butylphosphatetrahedrane (1) is shown here to act as a synthon of isomeric tri-tert-butylphosphacyclobutadiene in the presence of a Lewis acid or transition-metal complex. When it is combined with a substoichiometric amount of triphenylborane, compound 1 forms a ladderane-type dimer of tri-tert-butylphosphacyclobutadiene in 72% isolated yield. Trapping of a generated intermediate was achieved by repeating the experiment in the presence of excess styrene (20 equiv) or ethylene (1 atm), and the corresponding [4 + 2] cycloadducts of tri-tert-butylphosphacyclobutadiene were isolated in 88% and 74% yields, respectively. The platinum complex (Ph3P)2Pt(C2H4) also reacts with 1 to form an orange η2 complex of tri-tert-butylphosphacyclobutadiene in 80% isolated yield. Additionally, we report a novel method for generating a phosphinidenoid species via fluoride-induced trimethylsilyl fluoride elimination, leading to an improved preparative procedure for 1 (182 mg, 33% isolated yield).

Alleviating Strain in Organic Molecules by Incorporation of Phosphorus: Synthesis of Triphosphatetrahedrane.

Phosphatetrahedranes (tBuCP)2 and (tBuC)3P were recently reported and represent the first tetrahedranes containing a mixed carbon/phosphorus core. Herein, we report that tetrahydrofuran (THF) solutions of the parent triphosphatetrahedrane HCP3 may be generated in 31% yield (NMR internal standard yield) by combining [Na(THF)3][P3Nb(ODipp)3] (Dipp = 2,6-diisopropylphenyl), INb(ODipp)3(THF), and bromodichloromethane in thawing THF. While HCP3 was found to be stable in dilute THF solutions for extended periods of time, the concentration of the solution at -40 °C led to the formation of a black precipitate, which has been tentatively assigned as a polymerized form of HCP3. HCP3 reacts readily with (dppe)Fe(Cp*)Cl (dppe = 1,2-bis(diphenylphosphino)ethane, Cp*= η5-C5Me5) in the presence of Na[BPh4] to form a purple cationic iron complex of triphosphatetrahedrane (50% yield), which was structurally characterized in a single-crystal X-ray diffraction experiment. Additionally, we present a series of homodesmotic equations analyzed via quantum chemical calculations that suggest triphosphatetrahedrane is the least strained of the mixed C/P phosphatetrahedranes.

An Azophosphine Synthetic Equivalent of Mesitylphosphaazide and Its 1,3-Dipolar Cycloaddition Reactions.

Dibenzo-7-phosphanorbornadiene-substituted diazene MesN2PA (1, where Mes = mesityl, A = anthracene, or C14H10), a synthetic equivalent of mesitylphosphaazide (MesN2P) and anthracene, was synthesized by treatment of [Ph3BPA][Na(OEt2)2] with [MesN2]OTf (OTf = CF3SO3-) in thawing tetrahydrofuran (14% isolated yield). Treatment of 1 with unsaturated molecules cyclooctyne, [Na(dioxane)2.5][OCP] (phosphaethynolate), and Ad-C≡P (Ad = adamantyl) results in the corresponding [3 + 2] phosphaazide-(phospha)alkyne cycloadducts, with concomitant loss of anthracene in 65%, 49%, and 38% isolated yield, respectively. Structural data for the phosphaethynolate cycloadduct ([3][Na(12-crown-4)2]) were obtained in a single-crystal X-ray diffraction study. A diazatriphosphole was generated by combining 1 with P2A2, a thermally activated anthracene-based molecular precursor to diphosphorus (P2). Thermolysis (33-65 °C) of 1 in benzene-d6 leads to anthracene extrusion. This process has a unimolecular kinetic profile and proceeds with activation parameters of ΔH⧧ = 21.6 ± 0.3 kcal/mol and ΔS⧧= -4.9 ± 0.8 cal/(mol K).

Cryo-kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase.

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti-cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo-measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (kHLE /kHHE ) close to unity above 0 °C; however, the enzyme KIE was increased to 1.72±0.15 at -20 °C, indicating that the coupling of protein motions to the chemical step is minimized under optimal conditions but enhanced at non-physiological temperatures. The presented cryogenic approach provides an opportunity to probe the kinetics of mammalian DHFRs, thereby laying the foundation for characterizing their transition state structure.

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency.

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

31P NMR Chemical Shift Tensors: Windows into Ruthenium Phosphinidene Complex Electronic Structures.

A series of octamethylcalix[4]pyrrole/ruthenium phosphinidene complexes (Na2[1=PR]) can be accessed by phosphinidene transfer from the corresponding RPA (A = C14H10, anthracene) compounds (R = tBu, iPr, OEt, NH2, NMe2, NEt2, NiPr2, NA, dimethylpiperidino). Isolation of the tert-butyl and dimethylamino derivatives allowed comparative studies of their 31P nuclear shielding tensors by magic-angle-spinning solid-state nuclear magnetic resonance spectroscopy. Density functional theory and natural chemical shielding analyses reveal the relationship between the 31P chemical shift tensor and the local ruthenium/phosphorus electronic structure. The general trend observed in the 31P isotropic chemical shifts for the ruthenium phosphinidene complexes was controlled by the degree of deshielding in the δ11 principal tensor component, which can be linked to the σRuP/πRuP* energy gap. A "δ22-δ33 crossover" effect for R = tBu was also observed, which was caused by different degrees of deshielding associated with polarizations of the σPR and σPR* natural bond orbitals.