There is a high carrying rate of α‐thalassemia in Fujian province. However, there are few large‐scale studies on the correlation between genotype and phenotype in Fujian province. The purpose of this study was to analyze the phenotype and genotype in a cohort of 2923 patients with α‐thalassemia in Fujian province, so as to provide reference data for screening and diagnosis of α‐thalassemia in Fujian province.The genotype of α‐thalassemia was detected by PCR reverse dot blot assay, gap‐PCR, single PCR, nested PCR, and sequencing. Clinical and hematological indices of 2923 patients were collected, and the correlation between genotype and phenotype was analyzed.SEA/αα was the most common genotype, accounting for 64.80%. In addition, rare α‐thalassemia genotypes were detected in Fujian province, including ‐‐THAI/αα (0.41%), HKαα/‐‐SEA (0.03%), and the novel α‐thalassemia gene mutation CD5 (GCC>ACC) (HGVS named HBA1: c.16G>A) (0.03%). Patients with deletional genotypes of α‐thalassemia were found to have higher RBC and lower Hb, MCV, MCH, and HbA2 than patients with non‐deletional genotypes of α‐thalassemia (p < 0.05).Among 10,350 patients, 2923 cases were found with α‐thalassemia, with a detection rate of 28.24%. Among them, ‐‐The clinical phenotype of α‐thalassemia is influenced by molecular mechanisms. HBA1: c.16G>A mutation is a novel mutation that was first reported in Fujian province, which enriches the human hemoglobin mutation spectrum. There were double peaks at nt224, G>A, in HBA1, corresponding to CD5 (GCC>ACC). It was named HBA1: c.16G>A using the Human Genome Variation Society (HGVS) nomenclature. HBA1: c.16G>A is a novel mutation that was first reported in Fujian province. The discovery of this novel mutation in α‐globin gene has enriched the database of hemoglobin variants, and the detailed genetic analysis and clinical symptom description of this mutation will contribute to the further study of hemoglobin function in the future. Its pathogenesis is due to the defect of α‐globin gene; the synthesis of α‐globin peptide chain was partially or completely inhibited, resulting in hereditary hemolytic anemia. It is one of the most common monogenic diseases with the highest incidence in the world and has attracted extensive attention at home and abroad because of its fatal and disabling nature, which can lead to birth death or birth defects.However, there are few large‐scale studies on the correlation between genotype and phenotype in α‐thalassemia patients in Chinese population. In this study, 10,350 patients from Fujian province were analyzed for genotype and phenotype. Such a study may provide more data for genetic counseling and clinical diagnosis in this region.22.1This study was reviewed and approved by the Ethics Review Committee of Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University. Signed informed consent was obtained from all participants following a detailed description of the purpose of the study. All experiments were performed in accordance with relevant guidelines and regulations.2.2From January 2019 to November 2021, peripheral blood samples of patients who underwent thalassemia‐related examinations in outpatients and inpatients of Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University were collected. A total of 10,350 patients  with household registration in Fujian province were screened, including 2923 cases of α‐thalassemia. α‐thalassemia was confirmed by molecular analyses. Information about patients diagnosed with α‐thalassemia was collected, and their genotypes and phenotypes were statistically analyzed. The median age was 27 (8.31) years. The median age was 5.5 (1.30) years for males and 28 (25.31) years for females. Exclusion criteria are as follows: (1) β‐thalassemia; (2) other blood diseases such as iron deficiency anemia and megaloblastic anemia; (3) malignant tumor; (4) systemic immune disorders; and (5) allergic constitution. Peripheral blood samples were collected from selected subjects and anticoagulated with EDTA‐K2. Approximately 2 ml of the anticoagulated blood sample was used for analysis of blood cell parameters on a Sysmex XN‐2000 automatic hematology analyzer , and the hemoglobin components and levels were analyzed using an automated capillary electrophoresis system . Serum ferritin (SF) concentration, as a measure of iron status, was determined by the chemiluminescent microparticle immunoassay (CMIA) .2.3SEA/, ‐α3.7/, and ‐α4.2/) and the mutations  of α‐thalassemia were analyzed by PCR reverse dot blot assay using commercial kits  as described. The 17 common β‐thalassemia was performed using PCR reverse dot blot assay with the thalassemia gene detection kit  following the manufacturer's instructions.DNA was extracted using the DNA Blood Extraction Kit . The deletions (‐‐2.4THAI genotype was tested using gap polymerase chain reaction (gap‐PCR), and HKαα genotype was tested by single PCR and nested PCR, as described previously.‐‐2.5p < 0.05 were considered statistically significant.All data were entered into and managed using Microsoft Excel 2007 . The data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 25 . Associations between hematological indices and the variant genotypes were assessed by a nonparametric Kruskal–Wallis test, presented as median . The results with 33.1Among 10,350 patients, 2923 patients with α‐thalassemia mutation were detected, with a detection rate of 28.24%, including 2666 cases (91.21%) of α deletional genotype, 181 cases (6.19%) of α mutational genotype, 12 cases (0.41%) of α deletion combined with mutation, and 64 cases (2.19%) of α/β complex mutants.SEA/αα (64.80%), ‐α3.7/αα (18.95%), ‐α4.2/αα (4.96%), αQSα/αα (3.63%), αCSα/αα (1.47%), ‐‐SEA/‐α3.7 (1.20%), and αWSα/αα (0.99%). In addition, rare α‐thalassemia genotypes including ‐‐THAI/αα (0.41%), HKαα/‐‐SEA (0.03%), and the novel α‐thalassemia gene mutation CD5 (GCC>ACC) (HGVS named HBA1: c.16G>A) (0.03%) were detected in Fujian province  × 1012/L) (p < 0.05).Conversely, the levels of Hb, MCV, MCH, and HbA2 in patients with deletional genotypes of α‐thalassemia were lower than that in patients with non‐deletional genotypes of α‐thalassemia  g/L vs 119.0  g/L, 67.5  fl vs 76.1  fl, 21.2  pg vs 24.7  pg, and 2.3  % vs 2.5  %) (p < 0.05).  in patients with deletional genotypes of α‐thalassemia was higher than that in patients with non‐deletional genotypes of α‐thalassemia  × 10SEA/αα were found to have higher RBC, HbF, and SF and lower Hb, MCV, MCH, and HbA2 than patients with ‐α3.7/αα (p < 0.05). What is more, patients with ‐‐SEA/αα were found to have higher RBC and lower Hb, MCV, MCH, and HbA2 than patients with ‐α4.2/αα (p < 0.05). Age of ‐‐SEA/αα was lower than that of ‐α3.7/αα and ‐α4.2/αα (p < 0.05) Among the non‐deletional genotypes of α‐thalassemia, patients with αQSα/αα were found to have higher RBC and HbA2 and lower MCV and MCH than patients with αCSα/αα (p < 0.05). Patients with αQSα/αα were found to have lower MCV and MCH than patients with αWSα/αα (p < 0.05). In addition, patients with αCSα/αα were found to have lower HbA2 than patients with αWSα/αα (p < 0.05).  that was suspected to be a α‐thalassemia carrier. Sequencing the full‐length α1‐globin gene and of the full‐length α2‐globin gene of the DNA from the peripheral blood sample was done. The results showed there were double peaks at nt224, G>A, in HBA1, corresponding to CD5 (GCC>ACC). It was named HBA1: c.16G>A using the Human Genome Variation Society (HGVS) nomenclature  with‐‐SEA/αα, 554 cases (18.95%) with ‐α3.7/αα, and 145 cases (4.96%) with ‐α4.2/αα, suggesting that the most common genotype of α ‐thalassemia in this region was ‐‐SEA/αα, which was consistent with the reports from Guangxi, Guangdong, Chongqing, and Hainan.QSα/αα (3.63%), followed by αCSα/αα (1.47%) and αWSα/αα (0.99%), which were the same as those in Chongqing and slightly different from those in Guangxi, Guangdong, and Hainan,SEA/αα was dominant in these provinces, and the frequencies were respectively 57.55%, 52.78%, and 83.87%.There are two types of α‐thalassemia: deletional genotype and non‐deletional genotype. In addition to the most common deletional genotype, rare deletional genotype of α‐thalassemia has been found in different populations.THAI/αα , HKαα/‐‐SEA , and the novel α‐thalassemia gene mutation CD5 (GCC>ACC)  in Fujian Province. ‐‐THAI genotype has been gradually found in the population of southern China, mainly distributed in Guangxi, Guangdong, Taiwan, and Fujian.SEA genotype, the phenotype of ‐‐THAI is manifested as microcytic hypochromic anemia and the Thai‐type homozygote or Thai‐type heterozygotes with SEA‐type α‐thalassemia are manifested as Bart's hydrops fetalis.3.7 fragment and αααanti4.2 fragment formed by recombination and unequal transposition of the homologous X segment of the α‐globin gene cluster. So far, αααanti4.2 fragments cannot be detected through the routine thalassemia diagnostic kit. The genotype of ‐α3.7/αα, HKαα/‐α3.7, and HKαα/αα is entirely the result of ‐α3.7/αα by gap‐PCR.3.7/αα. Therefore, the symptoms of thalassemia associated with HKαα/αα and ‐‐SEA were milder than those of hemoglobin H disease associated with ‐α3.7 and ‐‐SEA, and only mild globin production disorder was observed . If both husband and wife carry HKαα/αα and ‐‐SEA respectively, the fetus can be kept. However, if the HKαα/αα or anti‐HKαα/αα genotypes are incorrectly identified as ‐α3.7/αα, it may lead to incorrect clinical treatment.Three rare α‐thalassemia genotypes were also detected in this study, including ‐‐SEA/αα/βIVS‐2‐654(C→T)/βN (11 cases), ‐α3.7/αα/βIVS‐2‐654(C→T)/βN (11 cases), ‐‐SEA/αα/βCD41‐42(‐CTTT)/βN (11 cases), ‐α3.7/αα/βCD41‐42(‐CTTT)/βN (4 cases), and ‐‐SEA/αα/β−28(A→G)/βN (4 cases). While patients with α‐ and β‐thalassemia have milder symptoms, their offspring are more likely than the general population to develop severe thalassemia, and the long‐term damage is much greater. Therefore, the clinical diagnosis of concurrent α‐ and β‐thalassemia should not be ignored. According to Xiong Fu et al.,In addition, 64 cases (2.19%) with concurrent α‐ and β‐thalassemia were detected in 2923 positive samples. Patients with concurrent α‐ and β‐thalassemia have been reported to suffer from mild anemia due to a reduction in α‐ and β‐globin chain synthesis, which alleviates the imbalance caused by reduced globin chain synthesis and thus reduces the severity of anemia.SEA/αα was lower than that of ‐α3.7/αα and ‐α4.2/αα (p < 0.05); this may be related to the number of inactivated α‐globin genes of ‐‐SEA/αα were more than ‐α3.7/αα and ‐α4.2/αα. Patients with ‐‐SEA/αα were associated with more severe anemia and came to the hospital earlier for diagnosis and treatment. Chen Suqin et al.4.2 or ‐α3.7 variants. Thus, the absence of α‐globin peptide chains or the relative excess of β‐globin peptide chains is more severe in non‐deletional patients. The hematologic appearance of patients with non‐deletional genotype of α‐thalassemia differs from that patients with deletional genotype of α‐thalassemia; anemia is more severe in patients with non‐deletional genotype of α‐thalassemia.p < 0.05).It may be related to the damage of erythrocyte membrane caused by excess β‐chain oxidation and αQSα or αCSα chain oxidation. This results in decreased EPO production and RBC in non‐deletional genotypes of α‐thalassemia.QSα/αα's Hb was lower than ‐α3.7/αα and ‐α4.2/αα, which was partly supported that anemia in the non‐deletional genotype was more severe than in the deletional genotype. Patients with ‐‐SEA/αα were found to have higher RBC, HbF, and SF, it may be related to ‐‐SEA/αα was associated with more γ‐globin mutations, and its RBC synthesis requires more iron, so the RBC, HbF, and SF were higher than ‐α3.7/αα.SEA/αα were lower than ‐α3.7/αα and ‐α4.2/αα in the three common deletional genotypes, suggesting that the severity of anemia depends on the number of inactivated α‐globin genes.There is significant variation in clinical severity among patients with α‐thalassemia, which is indirectly reflected by the span of age at first diagnosis. Age of ‐‐In conclusion, the clinical manifestations and hematologic phenotypes of α ‐thalassemia are related to genotype. The clinical phenotype of α‐thalassemia is influenced by molecular mechanisms. HBA1: c.16G>A is a novel mutation that was first reported in Fujian province. The discovery of this novel mutation in α‐globin gene has enriched the database of hemoglobin variants, and the detailed genetic analysis and clinical symptom description of this mutation will contribute to the further study of hemoglobin function in the future.Yali Pan, Meihuan Chen, Na Lin, Liangpu Xu, and Hailong Huang designed and prepared the study. YanHong Zhang, Min Zhang, and Lingji Chen collected the literature and the data and prepared the study. All authors approved the final study.This work was funded by the National Natural Science Foundation of China (no.81970170), the National Natural Science Foundation of Fujian Province (no.2019J01510), and the Fujian provincial health technology project (no.2018‐1‐21).The authors confirm that they have no competing interests.


Erratum to:Z Gerontol Geriat 202110.1007/s00391-021-02003-5The authors would like to apologize for an error concerning the mCAM-ED and make the following corrections:Page 109, Table 2, mCAM-EDColumn: Screening vs. monitoring/Items:2. If inattention present → MSQ for identifying cognitive impairment, Comprehension subtest of CTD for identifying disorganized thinkingColumn: ScoringModified CAM algorithm: 1a (acute onset) AND 1b (fluctuating course) + 2 + (3 or 4) positive = diagnosed delirium1a OR 1b + 2 + (3 or 4) positive = suspected delirium.Page 111, Legend table 2:SPMSQ: MSQ mental status questionnaire.As a replacement for


Erep) is dominant.In the crystal, hydrogen-bonding interactions between the 2,4,6-triaminopyrimidine cation and the nitrate anions lead to a one-dimensional supramolecular network with weak anionic interactions forming a three-dimensional network. Energy framework analysis showed that of the components of the framework energies, electrostatic repulsion ° between the mean plane of the cation and that defined by both anions. In the crystal, hydrogen-bonding interactions between the 2,4,6-triaminopyrimidine cation and the nitrate anions lead to a one-dimensional supramolecular network with weak anionic interactions forming a three-dimensional network. These interactions were investigated using Hirshfeld surface analysis, which indicates that the most important contributions for the packing arrangement are from O⋯H/H⋯O (53.2%), N⋯H/H⋯N (12.5%) and C⋯H/H⋯C (9.6%) interactions. Energy framework analysis showed that of the components of the framework energies, electrostatic repulsion (Erep) is dominant.The title compound, C The plane of the anion containing N6 is inclined to the mean plane of the cation by 3.25 (6)° while that of the other anion is inclined by 2.84 (6)°. Thus the whole asymmetric unit lies close to a common plane . The ring C—N bond lengths in the cation [C1—N2 = 1.3531 (16) Å and C2—N3 = 1.3267 (16) Å] are only slightly altered from those in the corresponding conjugate base . Pairs of adjacent ribbons are linked by N1—H1B⋯O3, N2—H2⋯O1 and N3—H3A⋯O3 hydrogen bonds  with O3⋯Cg1i = 3.1369 (11) Å, N6⋯Cg1i = 3.4241 (12) Å, N6=O3⋯Cg1i = 92.16 (7)°; O5⋯Cg1ii = 3.0265 (11) Å; N7⋯Cg1ii = 3.5176 (12) Å; N7=O5⋯Cg1ii = 102.62 (7)° ° Fig. 3.4.et al. 2009et al., 2007Crystal Explorer17  through white to blue . Top and bottom views of the surface together with curvedness, and shape-index plots are given in Fig. 4a–d. The red spots symbolize N—H⋯O contacts and C—H⋯O interactions. The fingerprint plots , followed by N⋯H/H⋯N contacts at 12.5%  and C⋯H/H⋯C contacts at 9.6% .The Hirshfeld surface is mapped over ts Fig. 5 give an 2% Fig. 5b, follo5% Fig. 5c and C⋯6% Fig. 5d.5.To synthesize the title compound, 20 mg of 2,4,6-triaminopyrimidine were dissolved in ethanol (10 mL) and the solution stirred for 3 h. A mixture of ethanol (5 mL) and nitric acid (0.5 mL) was taken in a separate round-bottom flask and stirred for 3 h at 333 K. Afterwards, the 2,4,6-triaminopyrimidine solution was added dropwise to the above mixture. The reaction was continued for 4 h at the same temperature. After completion of the reaction, a pale-yellow solution was obtained, which was filtered and kept for slow evaporation at room temperature. After 15 days, pale-yellow crystals were obtained that were suitable for data collection Fig. 6.6.CrystalExplorer 17.5. Electrostatic (Eele), polarization (Epol), dispersion (Edisp), and exchange-repulsion (Erep) are the four energy variables that make up the total intermolecular interaction energy (Etot). Cylinder-shaped energy frameworks represent the relative strengths of interaction energies in individual directions and give the topologies of pair-wise intermolecular interaction energies within the crystal  = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022005333/mw2185sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022005333/mw2185Isup2.hklStructure factors: contains datablock(s) I. DOI: 2173730CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The layers are connected by C—H⋯Cl hydrogen bonds and C—H⋯π(ring) interactions, forming a three-dimensional structure.The methoxy group lies very close to the plane of the phenyl ring while the acetamido group is twisted out of this plane. In the crystal, N—H⋯O and C—H⋯O hydrogen bonds form layers of molecules parallel to the  9H10ClNO2, the methoxy group lies very close to the plane of the phenyl ring while the acetamido group is twisted out of this plane by 28.87 (5)°. In the crystal, a three-dimensional structure is generated by N—H⋯O, C—H⋯O and C—H⋯Cl hydrogen bonds plus C—H⋯π(ring) interactions. A Hirshfeld surface analysis of the intermolecular interactions was performed and indicated that C⋯H/H⋯C interactions make the largest contribution to the surface area (33.4%).In the title molecule, C The Cl1—C1—C2—O1 torsion angle is 52.89 (12)°, illustrating a + synclinal (+ gauche) conformation about the C1—C2 bond. This places atom Cl1 at 1.299 (1) Å from the plane defined by C1, C2, N1 and O1.The methoxy group lies close to the mean plane of the phenyl ring C3–C8, as indicated by the C7—C6—O2—C9 torsion angle of −174.61 (10)° and atom C9 deviating by only 0.065 (1) Å from the mean plane through the C3–C8 ring. In contrast, the acetamido group is rotated out of the above plane with the dihedral angle between the mean plane through the C3–C8 ring and that defined by N1/C2/C1/O1 being 28.87 (5)° Fig. 1. The sum3.1 axes. These chains are linked by C1—H1A⋯O2 hydrogen bonds  yielded 15 hits of which 13 had X = Cl and R = OEt ) (SPh)(NO2) , O⋯H/H⋯O (19.5%) and Cl⋯H/H⋯Cl (20%) interactions, respectively.The analysis was performed with 6.0.047 mol of 4-methoxyaniline were dissolved in 40 mL of pure acetic acid and put in an ice bath. Subsequently, chloroacetyl chloride (0.047 mol) was added portionwise under stirring. At the end of the reaction, a solution of sodium acetate (35 mL) was added and a solid precipitate appeared after 30 min of stirring at room temperature. The resulting solid was filtered and washed with cold water, dried and recrystallized from ethanol to give the title compound as colourless crystals.−1) 3292 (υ N—H amide), 1029 (υ N—C amide), 1660 (υ C=O amide), 3073 (υ C—Harom), 827 (υ C—Cl), 2959 , 1H NMR (DMSO–d6) δ pm: 3.74 ; 4.24 , 6.93–7.5 , 10.23 , 13C NMR (DMSO–d6) δ ppm: 43.48 (CH2), 55.23 (CH3), 131.53 (Carom—N), 155.51 (Carom—O), 113.92–120.92 (Carom), 164.13 (C=O); HRMS (ESI–MS) (m/z) calculated for C9H10ClNO2 199.04, found 199.0105.Yield 80%, m.p. = 398.6–400.3 K, FT–IR  (1.5 for the methyl group). The N-bound H atom was found in a difference-Fourier map and refined with a DFIX 0.91 0.01 instruction and an independent isotropic displacement parameter.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902200576X/vm2264sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698902200576X/vm2264Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902200576X/vm2264Isup3.cmlSupporting information file. DOI: 2175514CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The packing of the title compound features weak C—H⋯O and C—H⋯π interactions. 22H20O3, the dihedral angle between the aromatic rings linked by the methine group is 81.265 (4)° and the ethoxy side chain adopts an extended conformation [C—O—C—C = 177.24 (10)°]. In the crystal, weak C—H⋯π and C—H⋯O interactions link the molecules into sheets.In the title compound, C These link the molecules into sheets lying perpendicular to the c-axis direction °. In the crystal, weak C—H⋯O Table 1 and C—H⋯on Fig. 2.3-Ethoxy-4-hydroxy benzaldehyde (0.20 mmol) and potassium carbonate (0.40 mmol) were mixed in dimethylformamide (25 ml) and stirred for 0.5 h. A solution of diphenylbromomethane (0.2 mmol) in ethanol (20 ml) was added dropwise and the mixture was stirred at 80°C for 24 h. After that, the solution was concentrated under reduced pressure. The cream precipitate of the title compound formed by adding cold water (250 ml) was filtered off and washed several times with cold ethanol. Colourless slabs were recrystallized from the mixed solvents of chloroform and ethanol (1:1).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621003564/hb4378sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314621003564/hb4378Isup2.hklStructure factors: contains datablock(s) I. DOI: 2075001CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Such ICCs are of potential utility, and there arenumerous examples of phenolic compounds that are biologically active,some of which suffer from low aqueous solubility. The propensity toform ICCs sustained by the PhOH···PhO– supramolecular heterosynthon was evaluated through a combinationof Cambridge Structural Database (CSD) mining, structural characterizationof nine novel ICCs, and calculation of interaction energies. Our analysisof these 9 ICCs and the 41 relevant entries archived in the CSD revealedthat phenol groups can reliably form ICCs through charge-assistedPhOH···PhO– interactions. This conclusionis supported by hydrogen-bond strength calculations derived from CrystalExplorerthat reveal the PhOH···PhO– interactionto be around 3 times stronger than the phenol–phenol hydrogenbond. The PhOH···PhO– supramolecularheterosynthon could therefore enable crystal engineering studies ofa large number of phenolic pharmaceutical and nutraceutical compoundswith their conjugate bases.Although crystalengineering strategies are generally well exploredin the context of multicomponent crystals  formed by neutralcoformers , cocrystals comprised of one or moresalts  are understudied. We herein addressthe design, preparation, and structural characterization of ICCs formedby phenolic moieties, a common group in natural products and drugmolecules. Organic and inorganic bases were reacted with the followingphenolic coformers: phenol, resorcinol, phloroglucinol, 4-methoxyphenol,and 4-isopropylphenol. Nine ICCs were crystallized, each of them sustainedby the phenol–phenolate supramolecular heterosynthon (PhOH···PhO CambridgeStructural Database mining and experimental studiesof model compounds reveal that ionic cocrystals sustained by phenol···phenolatehydrogen bonds are formed reliably Etter systematicallyclassified common hydrogen-bonding motifs using “graph set”notation Gda(r), where G is the pattern designator that could be C (chain), R (ring),and D (dimer or other finite set); a represents the number of acceptors; d is the number of donors; and r is thenumber of atoms in the repeat unit.18 Graph settheory remains a valuable tool for researchers to compare structuresto this day. Shortly after, Desiraju introduced the term “supramolecularsynthon” to represent a structural unit or building block incrystal structures.19 Supramolecular synthonswere subclassified by us in 2003 into supramolecular homosynthons,between the same functional groups  and supramolecular heterosynthons and between differentbut complementary functional groups .14 The understandingof supramolecular synthons, their geometries, and their frequencyof occurrence in the Cambridge Structural Database (CSD) is key tothe rational design of novel multicomponent crystal forms such ascocrystals and hydrates.20Certain intermolecular interactions,such as neutral and charge-assistedhydrogen bonds, and coordination bonds, are amenable to crystal engineeringstudies thanks to their relatively high strength, predictability,ubiquity, and directionality.21 Cocrystals are of interestin part because they can be readily accessible and amenable to crystalengineering. Cocrystals include molecular cocrystals22 (MCCs), which are comprised of two or more nonvolatileneutral molecules (coformers), and ionic cocrystals23 (ICCs), which involve at least one coformer that is a salt.ICCs are necessarily sustained by charge-assisted hydrogen bonds or,if metal ions are involved, coordination bonds.25 ICCs are therefore comprised of at least three components, a cation,an anion, and an additional molecule or salt. Given that MCCs of acompound typically offer a single variable, the coformer, ICCs canexhibit greater diversity in terms of composition and physicochemicalproperties compared to MCCs.4 Additionally,if an ICC is formed in which one coformer is an API and another isan API salt, then this type of ICC provides an opportunity to generatelow-dosage solid forms since the biologically active component(s)represent most of the mass of the ICC. Although ICCs are generallyless studied than MCCs, the first cocrystal involving sodium chlorideand urea was an ICC26 and, to our knowledge,at least five ICCs have been selected and developed for use in marketeddrug products: Depakote 27 in 1983; Lexapro 28 in 2002; Odomzo 29 in 2015; Entresto 30 in 2015; Seglentis (celecoxib and tramadol hydrochloride)31 in 2021.Cocrystalsare solids that are crystalline single-phase materialscomprised of two or more different molecular and/or ionic compoundsgenerally in a stoichiometric ratio that are neither simple saltsnor solvates.2+ and l-ascorbicacid, and inhibit the formation of oxygen free radicals.33 Statistically, ca. 8.7% of approved small-molecule drugs containedin DrugBank Online (Version 5.1.9.)34 arephenolic drugs, and phenol groups are present in ca. 10.1% of single-componentbiologically active compounds archived in the CSD (Scheme S1). However, many such compounds exhibit low aqueoussolubility and would therefore be classified as BCS class II.35 Their efficacy would therefore be hindered andan otherwise promising drug molecule could be rendered unsuitablefor use in an orally delivered drug product.37 Molecular cocrystals are now well known to modulate the physicochemicalproperties of phenolic compounds including solubility,38 bioavailability,39 stability,40 and mechanical properties41 while preserving their inherent biological activity.Phenolic drug molecules and nutraceuticalcompounds are of topicalinterest because they can exhibit biological effects such as antioxidantand anti-inflammatory activity, inhibit lipid peroxidation initiatedin rat brain homogenates by Fe20 carboxylate moieties,42 and aromatic nitrogen bases.43 Conversely,the phenol–phenolate (PhOH···PhO–) interaction is considered to be a strong hydrogen bond. In solution,PhOH···PhO– systems known as homoconjugatedcomplexes readily form as measured by their conjugated constants.44 PhOH···PhO– complexes can have a negative impact on titration experiments andplay an important role in enzyme catalysis.45 Buytendyk et al. reported the PhOH···PhO– interaction energy in the gas phase to be as high as 26–30kcal/mol, 60% of that of the HF···F– interaction, the strongest hydrogen bond known.46 Crystal structures involving PhOH···PhO– interactions archived in the CSD were mainly studiedfor structural insight47 or applicationssuch as organic synthesis (Kolbe–Schmitt synthesis)49 and NLO.50 As such, there remains a lackof systematic crystal engineering studies on ICCs containing the PhOH···PhO– supramolecular heterosynthon.With respect to crystal engineering, phenols offer medium hydrogen-bonddonor strength and are established as being able to form supramolecularheterosynthons with hydrogen-bond acceptors such as chloride anions,– supramolecular heterosynthons. Specifically, phenoland four other substituted phenol derivatives were selected as modelcompounds and reacted with tetraalkylammonium hydroxides and potassiumhydroxide (– and phenol–phenol (PhOH···PhOH)interactions in the obtained ICCs were examined using CrystalExplorer.In this contribution,we address ICC formation involving phenolsthrough a systematic CSD and experimental study that explores PhOH···PhOydroxide 1.In addPHNTMA; phenol·phenolate·potassium, PHNKOH; 1,3,5-benzentriol·1,3,5-benzentriolate·tetramethylammonium, PGNTMA; 1,3,5-benzentriol·1,3,5-benzentriolate·tetraethylammonium, PGNTEA; 4-isopropylphenol·4-isopropylphenolate·potassium, IPPKOH; 4-isopropylphenol·4-isopropylphenolate·tetrapropylammonium, IPPTPA; 4-isopropylphenol·4-isopropylphenolate·tetrabutylammonium, IPPTBA; 4-methoxyphenol·4-methoxyphenolate·tetrabutylammonium, MOPTBA; and resorcinol·resorcinolate·tetramethylammonium, RESTMA.A library of cocrystal formers containing five phenolderivatives,four organic bases, and one inorganic base was chosen for this study1. CocrysAll reagents and chemicals were purchasedfrom Sigma-Aldrich and used without further purification. Single crystalsof ICCs were obtained via slow evaporation of stoichiometric amountsof starting materials in a range of solvents at room temperature andharvested from solution before complete evaporation of their motherliquors had occurred.PHN  andTMA  were dissolved in 10 mL of acetonitrile (MeCN).The solvent was evaporated under vacuum until a viscous liquid wasobtained. Colorless crystals were harvested after exposing the viscousliquid to ambient conditions overnight.PHN  and KOH  were dissolved in 1 mL of methanol (MeOH). Thesolution was slowly evaporated at room temperature, and colorlesscrystals were obtained after 1 day.PGN  and TMA  were dissolved in 1 mL of MeOH. The solutionwas slowly evaporated at room temperature, and colorless crystalswere obtained after 1 day.PGN  andTEA  were dissolved in 1 mL of MeOH. The solutionwas slowly evaporated at room temperature, and colorless crystalswere obtained after several hours.2O) were dissolved in 1 mL of MeOH. Thesolution was slowly evaporated at room temperature, and colorlesscrystals were obtained after 1 day.IPP  and KOH  were dissolved in 1 mL of MeCN. The solutionwas slowly evaporated at room temperature, and colorless crystalswere obtained after 1 day.IPP  and TPA  were slurried in 1 mL of H2O overnight. The undissolved solid was isolated by filtration anddried in an oven. Colorless single crystals were obtained by dissolvingthe solid in MeOH, followed by slow evaporation at room temperature.IPP  andTBA , followed by slow evaporation at room temperature.MOP  and TBA  were slurried in 2 mL of HRES , IPP ,and TMA  were dissolved in 1 mLof MeOH. The solution was slowly evaporated at room temperature, andcolorless crystals were obtained after 1 week.All PXRD data werecollected on an Empyrean diffractometer  with the followingexperimental parameters: Cu Kα radiation (λ = 1.54056Å); 40 kV and 40 mA; scan speed 8°/min; step size 0.05°,2θ = 5–40°.51 Structures were solved using SHELXT and refinedusing SHELXL contained in Olex2.52 Reflectiondata for the nonhydrogen atoms were refined anisotropically. All hydrogenatoms bonded to carbon  were placed geometrically and refined using a ridingmodel with isotropic thermal parameters: Uiso(H) = 1.5Ueq(−CH3), Uiso(H) = 1.2Ueq(−CH), Uiso(H) = 1.2Ueq(−CH2). Hydrogen atoms on water (AFIX 5) and methanol (AFIX 147)were calculated geometrically and refined using a riding model withisotropic thermal parameter: Uiso(H) =1.5Ueq(−OH). The hydrogen atomsof phenolic hydroxyl groups were located from electron density differencemaps and included in the refinement process using a riding model with Uiso(H) = 1.2Ueq(−OH).Single-crystal data are presented in Crystal structureswere determined by single-crystal X-ray diffraction(SCXRD) with either Cu Kα (λ = 1.5418 Å) radiationor Mo Kα (λ = 0.71073 Å) radiation and a Bruker D8Quest fixed-chi diffractometer equipped with a Photon 100 detectorand the nitrogen-flow Oxford Cryosystem attachment. Unit cell determination,data reduction, and absorption correction (multiscan method) wereconducted using the Bruker APEX3 suite with implemented SADABS software.R factor ≤ 0.05, no disorder andsingle-crystal structure only) was conducted to find crystal structuresthat contain PhOH···PhO– interactions.The following parameters were addressed by the search: (1) numberof structures that exhibit phenol–phenolate interactions excludingsingle-component structures, salts, and zwitterions; (2) number ofstructures that form a PhOH···PhOH supramolecular homosynthon;(3) number of structures that contain a phenol group or a phenolateanion with the restriction that only C, H, and O were considered assubstituents on the phenyl ring . The distance distribution of phenol–phenolateand phenol–phenol hydrogen bonds and the bond length distributionof the C–O bond of the phenol or phenolate group were plottedbased on data from Searches 1, 2, and 3. The schematic structuresused in searches 1, 2, and 3 are presented in ACSD survey  level in the CrystalExplorerprogram package followed by geometry optimization carried out usingthe CASTEP module with GGA-type PBE functional contained in MaterialsStudio 8.0. The total interaction energy was partitioned into electronic(E_ele), polarization (E_pol), dispersion (E_dis), and repulsion (E_rel)components.– interactions are present. Of these entries, 69 (44.2%)are single-component compounds with phenol and phenolate moietiesin the same molecule; 30 entries (19.2%) are salts, and 15 (9.6%)are zwitterionic cocrystals or solvates composed of a zwitterion anda neutral molecule; 42 (26.9%) structures meet the definition of anICC that contains a phenol molecule, phenolate anion, and cation.However, of these 42 cocrystals, only one paper54 classifies them as cocrystals (refcode: JICKIS).Our CSD search retrieved 156 entries in which PhOH···PhO– O···O– bond distance determined from 42 ICC structures rangedfrom 2.4195(52)–2.6599(24) Å with an average of 2.528± 0.08Å. A histogram of the O···O– distance distribution for PhOH···PhO– interactions is presented in The PhOH···PhOO–H···Obond distances of PhOH···PhOH interactions determinedfrom search 2 ranged from 2.5218(28)–3.0381(45) Å, averaging2.812 ± 0.103 Å. A histogram of the O–H···Ohydrogen-bond distance distribution for PhOH···PhOHinteractions is presented in – bonds range from 1.2341(27) to 1.3776(95) Å witha mean of 1.307 (0.023) Å. The distribution plots in CSD search3 addressed the C–O bond length in phenols vs phenolates. TheCSD was found to contain 8277 and 192 entries for phenols and phenolates,respectively. –···PhOH; PhOH···PhOH;PhO–···H2O/MeOH; PhOH···H2O/MeOH. Tetramethylammonium (TMA), tetraethylammonium (TEA),tetrapropylammonium (TPA), and tetrabutylammonium (TBA) cations exhibitno strong hydrogen bonds. Rather, coulombic forces occur with phenolateanions. The neutral or ionic nature of phenolic groups in the nineICCs was addressed through proton location56 from difference Fourier map inspection and C–O bond lengths. Table S1 lists the C–O bond lengths inthe neutral (C–OH) and deprotonated (C–O–) moieties.Hydrogen bonds werefound to be present in each of the nine novel ICCs reported herein:PhO– and PhO–···H2O hydrogen bonds. PHNTMA crystallized in the space group P21/c. Theasymmetric unit of PHNTMA contains three phenol molecules, three phenolateanions, three TMA cations, and three water molecules in the ratioof 1:1:1:1 (R1410(28) (– hydrogen bonds withinthe discrete unit exhibit O···O– bonddistances of 2.438(2), 2.434(2), and 2.466(2) Å. At the (100)plane, adjacent discrete units are arranged vertically and interactvia TMA cations. The discrete units align along the a-axis.PHNTMA, IPPTPA, IPPTBA, and MOPTBA formed discreteadducts. PHNTMA and IPPTBA crystallized as hydrates sustained by PhOH···PhO 1:1:1:1 2a. Four 1410(28) 2b. The rP21/c (R42(8) hydrogen-bondedring with each molecule interacting with an extra IPP molecule outsidethe ring to generate a discrete unit (– hydrogen bond with O···O– = 2.528(3) Å. IPP serves as a hydrogen-bond donor and is hydrogen-bondedto a water molecule in each motif. The discrete units are connectedby TBA cations and aligned along the a-axis.IPPTBA crystallized as a 2:1:1:1 ICC of 4-isopropylphenol, 4-isopropylphenolate,TBA, and water in the space group P21/c 2c. Two 4ete unit 2d. The rP1̅ with one (Z′ = 1) and two (Z′ = 2) formula units in the asymmetric unit, respectively.In both structures, two discrete hydrogen-bonding assemblies existin each asymmetric unit, forming two different motifs C32(7) and C21(5) in IPPTPA (C32(7) motifs in MOPTBA Å, 2.562(2) Å, 2.571(3) Å, 2.589(3) Å; O···O– distances in MOPTBA: 2.5920(16) Å, 2.4413(17)Å, 2.4369(17) Å, 2.6111(17) Å). CH···Ointeractions and cations link the discrete units.IPPTPA and MOPTBA both crystallized in space group n IPPTPA 3a andtwn MOPTBA 3b. These48 Herein,single crystals of PHNKOH were grown from MeOH and are isostructuralwith HERCIQ but with a lower unit cell volume (4359.37 vs 4494.18Å3), presumably due to data collection at 150 K vsroom temperature. IPPKOH crystallized in P21/n. Althoughthey adopt different space groups, PHNKOH and IPPKOH both exhibitasymmetric units comprising three phenol molecules, one potassiumcation, and one phenolate anion in a 3:1:1 ratio. Each potassium cationadopts octahedral coordination geometry with five oxygen atoms fromphenol molecules (or phenol moieties of IPP), and the sixth coordinationsite is occupied by the π system of a phenolate anion (S1a). The 1D coordination polymers in PHNKOHand IPPKOH form zigzag chains (S1b) that are supportedby three nonequivalent charge-assisted hydrogen bonds between eachphenolate moiety and three phenol moieties (O···O– in PHNKOH = 2.5276(18) Å, 2.6052(19) Å,2.6315(19) Å; O···O– in IPPKOH= 2.541(2) Å, 2.617(2) Å, 2.546(3) Å).PHNKOH and IPPKOH form one-dimensional (1D) coordinationpolymers.The structure of PHNKOH  and its 2:1:1 variant (CSD Refcode HEQZUY) were previouslyreported as phenol solvates of potassium phenolate from high-resolutionPXRD data by Dinnebier et al.RESTMA isa 1:1:1 ICC of resorcinol, resorcinolate,and TMA  hydrogen-bonded networks. Deprotonated resorcinol serves as ahydrogen-bond donor and acceptor since only one hydroxyl group isdeprotonated. The phenolate moieties of the resorcinolate anion formthree charge-assisted PhOH···PhO– hydrogen bonds. The resorcinolate anions interact with each otherto form a chain supported by PhOH···PhO– hydrogen bonds. These interactions result in perpendicular chainsbridged by resorcinol molecules, resulting in grid-like networks  and one water molecule (PhO–···H2O). Two phenolate moieties hydrogen-bond to phenolic groupsvia PhOH···PhO– hydrogen bonds  ring motif to expandinto a ladder-like plane composed of rails  hydrogen-bonded networks through PhO–···MeOHand PhOH···MeOH hydrogen bonds, respectively  also feature PhOH···PhOHsupramolecular homosynthons. The two most common motifs observed arethose in which a phenolate moiety interacts with two  or three hydroxyl donors . Phenols are generally more acidic than aliphaticalcohols but less acidic than most carboxylic acids. This study presentsa strategy to form ICCs of phenols and their conjugate bases by PhOH···PhO– interactions to drive and sustain crystal packing.It is a well-recognized rule of thumb for salt and cocrystal screeningthat if ΔpKa (ΔpKa = pKa (base)-pKa (acid)) is >3, then a salt is formed and that if ΔpKa is <0, then a cocrystal is favored.57 In this work, ΔpKa is >3 for all bases used, and as such, proton transferwasanticipated even though proton transfer could have been affected byfactors such as solvent and the composition of starting materials.In this study, several solvent systems were used for ICC screening.Except for PHNTMA, only the use of MeOH or H2O as solventafforded high-quality single crystals suitable for SCXRD data collection.Poor quality crystals or gel-like phases were produced using ethanol,2-propanol, or acetone.As detailed above, charge-assisted PhOH···PhO– and PhOH···PhOHinteractions in the nine new ICCs reported herein are tabulated in Table S2 and presented as a scatter plot in Table S2), and the average strengthis presented in Selectedhydrogen bond lengthsfor PhOH···PhO– and PhOH···PhOH hydrogen-bond distancesin the ninenovel ICCs fall in the “red zone” and “greenzone”, respectively. These distance zones are based upon O···O– distance ranges in PhOH···PhO– and PhOH···PhOH hydrogen bonds (Å), respectively,determined from crystal structures archived in the CSD. PhOH···PhO– hydrogen bonds were found to exhibit relatively shortdistances (2.427(2)–2.658(2) Å) and angles nearing 180°,whereas PhOH···PhOH hydrogen-bond distances were longer(2.825(3)–2.6289(19) Å) and angles were more acute (149(5)–177(8)°).Some charge-assisted PhOH···PhO– hydrogenbonds have relatively long O···O– distances or small angle values (2.6315(19) Å, 161(3)°in PHNKOH; 2.617(2) Å, 158(8)° in IPPKOH; 2.726(3) Å,180(7)° in PGNTEA). These parameters are likely affected by thedifferent crystal packing environment in each crystal structure. Nevertheless,as expected, charge-assisted PhOH···PhO– hydrogen bonds were found to be consistently shorter than PhOH···PhOHhydrogen bonds. Further, as presented in – interactions were calculated to offer ca. 3 times the energy ofPhOH···PhOH interactions, with average energies of91.6 ± 8.1 and 28.53 ± 0.67 kJ/mol, respectively. As alsodetailed in – and PhOH···PhOHhydrogen-bond strength. Specifically, for PhOH···PhOHinteractions, the electrostatic contribution comes from dipole···dipoleinteractions, whereas for PhOH···PhO– interactions, the phenolate oxygen and phenolic hydrogen are keyto the electrostatic contribution. The greater electrostatic energiesin PhOH···PhO– can therefore be attributedto coulombic forces. Polarization contributes to total energy forPhOH···PhO–, while it is negligiblefor PhOH···PhOH. A small dispersion contribution wascalculated for all hydrogen bonds. Repulsion forces for PhOH···PhO– were consistently calculated to be greater than forPhOH···PhOH hydrogen bonds. Overall, although repulsiveforces for the charge-assisted hydrogen bonds are relatively high,the increase is more than offset by increases in permanent chargeand electronic and polarization forces. These high intermolecularinteraction energies and desirable hydrogen-bond geometry featuresvalidate that charge-assisted PhOH···PhO– hydrogen bonds are a strong and reliable interaction to enable theformation of ionic cocrystals.As shown by the scatter plot of – supramolecular heterosynthon is understudied in thecontext of crystal engineering of ICCs. To address this issue, ninenovel ICCs involving phenol and phenol-substituted compounds wereformed by reaction with bases and their crystal structures were determinedby SCXRD. Analysis of the resulting crystal structures revealed thatall nine ICCs are sustained by the phenol–phenolate supramolecularheterosynthon with O···O– distancesranging from 2.427(2) to 2.658(2) Å. A computational study validatedthe robustness of the charge-assisted PhOH···PhO– supramolecular heterosynthon vs. the phenol–phenolsupramolecular homosynthon from an energetic perspective. Based onthese results, we consider that the PhOH···PhO– supramolecular heterosynthon is suitable for the reliableformation of ICCs of phenolic compounds, which are prevalent in drugmolecules and nutraceuticals. That phenols form ICCs with their conjugatebases is important from a pharmaceutical perspective as it means thatthe mass of a dosage form can be close to that of the parent phenol.Future studies will focus on pharmaceutical and nutraceutical cocrystals,their physicochemical properties, and polymorphism.Phenolgroups are found in 8.7% of approved small-molecule drugsin the DrugBank database and in 10.1% of bioactive single-componentcompounds deposited in the CSD. Our CSD survey revealed that the PhOH···PhO


The packing of the title compound is consolidated by C—H⋯O interactions. 23H19NO5, the cyano group adopts an axial orientation and the ester group an equatorial orientation. The dihedral angle between the pendant phenyl group and the benzene ring of the fused-ring system is 25.97 (8)°. Intramolecular O—H⋯O and C—H⋯O hydrogen bonds are observed and the packing is consolidated by C—H⋯O and C—H⋯π interactions.In the title compound, C H-benzo[c]chromen-6-ones or 3,4,5,6-dibenzo-α-pyranones) form an important group of biologically active natural products that occur in bacteria, fungi, lichens, higher plants and animal waste °. The Cremer–Pople puckering parameters of the O1/C9/C10/C13–C15 and C14/C15/C17–C20 rings indicate half-chair conformations in each case with puckering amplitudes Q = 0.359 Å; θ = 104.52°; φ = 9.27° and Q = 0.49 Å; θ = 134.17°; φ = 327.35°, respectively. The O atom attached to C17 is stabilized in its enol (hydroxy) form, presumably as a result of forming a strong intramolecular hydrogen bond to O2. The packing is consolidated by weak C—H⋯O hydrogen bonds and C—H⋯π interactions , phenylboronic acid , K3PO4  and Pd(PPh3)4  in degassed 1,4-dioxane (10 mL) was stirred at 100° C for 12 h under nitrogen. After completion of the coupling reaction (TLC), the mixture was cooled to room temperature, diluted with dichloromethane  and decanted. The residue was extracted with DCM (10 mL × 2) twice. The solvent was removed from the combined DCM layers and the residue was subjected to column chromatography on silica gel (100–200 mesh) by using increasing amounts of ethyl acetate in hexane (5% to 15%) as eluent to afford the title compound as a light-yellow solid in 90% yield (84 mg); Rf = 0.4 ; m.p. 155–158° C. A sample suitable for single-crystal X-ray analysis was obtained by recrystallization the 50 mg of the solid from a mixture of 1 mL of distilled chloroform and 0.5 mL of distilled methanol.A mixture of ethyl 10-cyano-7-hydroxy-6-oxo-3-{[(trifluoromethyl)sulfonyl]oxy}-8,9,10,10Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314622001997/hb4401sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314622001997/hb4401Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622001997/hb4401Isup3.cmlSupporting information file. DOI: 2153368CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-isopropylacrylamide) (pNIPAM) and poly(N-tert-butylacrylamide) (pNTBAM) hydrogels were assessed in terms of their chemical differences, mechanical strength, internal architecture, porosity and capacity to support cell viability, migration, and differentiation. pNTBAM polymerized with a Young’s modulus of up to 371 ± 31 kPa compared to the more flexible pNIPAM, 16.5 ± 0.6 kPa. Viability testing revealed biocompatibility of both hydrogels with significantly increased cell numbers observed in pNTBAM (500 ± 95 viable cells/mm2) than in pNIPAM (60 ± 3 viable cells/mm2) (P ≤ 0.05). Mineralization determined through alkaline phosphatase (ALP) activity, calcium ion and annexin A2 markers of mineralization) and osteogenic behaviour (collagen I expression) were supported in both hydrogels, but to a greater extent in pNTBAM. pNTBAM supported significantly elevated levels of chondrogenic markers as evidenced by collagen II and glycosaminoglycan expression in comparison to little or no evidence in pNIPAM (P ≤ 0.05). In conclusion, structurally similar, chemically distinct, acrylamide hydrogels display variable capacities in supporting osteochondral cell behaviours. These systems demonstrate spatial control of cell interaction through simple changes in monomer chemistry. Fine control over chemical presentation during the fabrication of biomaterial implants could lead to greater efficacy and targeted regeneration of semi-complex tissues.Regeneration solutions for the osteochondral interface depth are limited, where multi-material implants have the potential to delaminate affecting the regeneration process and impacting the final integrity of tissue interface. Here we explore regionally mixed hydrogel networks, presenting distinct chemical features to determine their compatibility in supporting osteogenic or chondrogenic cell behaviour and differentiation. Poly( Furthermore, material characteristics influence the cellular capacity to perform biological functions  and N-tert-butylacrylamide (NTBAM) have been described as co-polymers with potential to stimulate tissue regeneration including that of bone and cartilage  was used to link the polymer network. Ammonium persulfate (APS) 10% w/v initiator was used alongside N,N,N′,N′-tetramethylethylenediamine (TMED) 2.6% v/v as an accelerator. NIPAM formed a transparent soft gelatinous structure requiring 2–3 min to form after rapid addition of the initiator. NTBAM was formed in a 1:1 combination of water and methanol heated to 37 °C. pNTBAM hydrogel formation required 10–15 min to completely gelate.Hydrogels were prepared by liquid phase atom transfer radical polymerization (Matyjaszewski Hydrogels were observed using a bench-top Hitachi S4500 scanning electron microscope (SEM) at 5 kV. Hydrogels were frozen at – 20 °C, freeze dried, mounted onto carbon tape, and gold sputter coated. Pore size measurements were performed using Image J, with over 100 features measured per sample and 3 repeat samples measured.Measurement of water contact angle was performed using a “Theta Lite Attension One Attension version 2.4” system. Samples were placed on a glass petri dish and compressed with a cover slip to acquire a flat surface while being dried at 70 °C in an oven. Water droplets, measured at 1 μL, were slowly placed onto test surfaces. Four measures were collected from each sample out of 4 samples.Compressive strengths were measured with the BOSE Electroforce system equipped with a 20 N loading cell and cross head speed of 0.05 mm/s. The samples were cylindrical in shape with dimensions of 4.5–5.0 mm height and 9.4–11.5 mm diameter. The load was applied until strain reached 90%. Compressive strength was determined from the maximum load of the applied stress–strain curve. Four samples of each hydrogel were tested, and an average obtained.L-glutamine. Culture environments were 37 °C in 5% CO2 culture incubators with media changed every 5 days. Primary human osteoblasts (hOB) and primary human chondrocytes (hCH) were obtained commercially from Promo Cell® and cultured under the conditions described above. Routine trypsinization protocols were followed for cell passaging  and immortalized (human telomerase transcriptase (hTERT) transduced) chondrocytes (OK3H)  or chondrogenic induction supplements .Differentiation was induced by addition of either osteogenic induction supplements . The major components of the assay kit are calcein AM and ethidium homodimer-1 reagents. Calcein AM identifies the presence of live cells by detecting intracellular esterase activity and cell membrane integrity. Live/dead staining solution was prepared by mixing both reagents with PBS at the following rates; 1:200 of calcein AM and 1:50 of ethidium homodimer-1 in PBS. The cells viability profile was assessed after 21 days of cell culturing. The hydrogel samples were removed from media and washed 3 times with PBS. Next, these samples were incubated with 1 mL/sample of live/dead staining solution for 30 min at room temperature protected from light. Hydrogels were viewed under an Olympus U-TBI90 confocal microscope to observe the viable (green fluorescence) and the dead (red fluorescence) cells. The viability of cells upon hydrogel samples was identified by calculating the number of live and dead cells per specific regions of each sample. The number of live cells (green) and dead cells (red) were counted over a 1 mm2 area for a maximum of 5 regions of a captured × 4 microscopic images obtained for individual samples and the average was taken. The whole process was carried out using the cell counting tool of Image J software.Viability of cells was examined with the live/deadCell migration was established after 21 days culture within the hydrogels. Hydrogel samples were removed from media, washed 3 times with PBS, and fixed in 10% paraformaldehyde for 30 min at room temperature. Then, each sample was incubated with 1 mL DAPI stain solution for 30 min at room temperature. Samples were then scanned across the z-axis using a 2 µm step size reaching a maximum of 150 slices of sample down from the top layer. The scanned distance for each sample was set to a maximum of 300 µm starting from the surface. Scanned files were processed via Image J software. The depth location was determined by nuclei localisation within the depth of the hydrogel.Primary antibodies for collagens type I, II, and X  confirmed differentiation. Visualization was enabled through appropriate secondary antibodies; Goat Anti-Rabbit IgG H&L, Abcam cat. No. 6718 and 6717, respectively). Hydrogel samples were fixed with 10% formaldehyde for 30 min at room temperature. Samples were blocked with 5% bovine serum albumin (BSA) in PBS for 2–3 h at 4 °C. This was followed by sample incubation with primary antibody solution overnight at 4 °C. The primary solution was prepared by mixing primary antibody  with 5% BSA in PBS at 1:200 ratio. The primary solution was then aspirated, and samples washed 4 times with 1% BSA in PBS solution. Samples were then incubated with secondary antibody (conjugated with either FITC or TRITC) in 5% BSA in PBS solution at 1:200 ratio at 4 °C for 4 h in the dark. Samples were then washed with 1% BSA in PBS followed by additional PBS washes. Nuclear staining was through DAPI stain for 30 min at room temperature followed by PBS washes. Hydrogels were imaged with an Olympus U-TBI90 laser fluorescent confocal microscope.Extraction of calcium minerals from hydrogel samples was performed using 0.5 M HCl solution. Hydrogels were first fixed with 10% paraformaldehyde in phosphate buffer saline (PBS) for 30 min at room temperature. Each hydrogel sample was incubated with 500 µL of HCl extraction solution at room temperature overnight on a rotary shaker. Calcium mineral was assessed using the calcium colorimetric assay kit (Sigma Aldrich MAK022) following manufacturer’s instructions with calcium ions present in samples Morin  detectedGAGs matrix proteins were assessed by dimethylmethylene blue (DMMB) assay . Hydrogels were digested in papain lysis buffer at 60 °C overnight. Cell lysates were then assessed for collagens I, II  and annexin A2  subsequent to the application of the assay protocol specific for each marker. Absorbance was measured with a Synergy II BioTek plate reader.P ≤ 0.05)  Fig. B. pNTBAMP ≤ 0.05). Pore size increased with lowering hydrogel monomeric concentrations for both hydrogels (p ≤ 0.05). For pNIPAM, 0.058 g/mL and 0.04 g/mL concentration resulted in mean pore diameters of 29.9 ± 7.7 µm and 35.7 ± 9 µm, respectively. Similarly, with pNTBAM, 0.058 g/mL and 0.04 g/mL concentrations resulted in mean pore diameters of 16.3 ± 3.6 µmand 21.2 ± 5 µm, respectively   .Osteosarcoma MG63 and chondrogenic OK3H cells seeded onto pNIPAM samples formed distinct aggregates or clusters while conversely, with pNTBAM, they attach and spread out across the hydrogel surface Fig. A, B. ThiP ≤ 0.05) and 0.058 g/mL (44.8 ± 19 pNIPAM µm)  and 0.042 g/mL (pNIPAM) for primary cell experimentation. Further, based on identified properties (stiffer mass and high cellular density) pNTBAM was selected as a putative best choice for support of mineralization while the smaller pores, hydrophobicity, lower cellular density, cluster growth, and flexibility suggested pNIPAM as a putative choice for chondrogenic tissue growth.2) when compared to hOBs (243.6 ± 38.26 cells/ mm2) on pNTBAM (P ≤ 0.05). We again noted that pNIPAM displayed reduced cell numbers (58 ± 7.3 cells/mm2 for hOBs and 65 ± 3.3 cells/mm2) when compared to pNTBAM, . pNTBAM produced 2.09 ± 0.04 versus 0.37 ± 0.04 µg/µL calcium ions whilst pNIPAM produced the lower amount of 1.15 ± 0.04 versus 0.47 ± 0.06 µg/µL calcium ions (P ≤ 0.05). Collagen I levels produced by hOBs were significantly elevated in osteogenic media when compared to control samples  (P ≤ 0.05) . Complementary to increases in collagen I and calcium ion production we noted that ALP activity levels were increased from 0.2 × 103 ± 0.1 × 103 for controls to 3.3 × 103 ± 0.6 × 103 U/µL for pNTBAM osteogenic samples (P ≤ 0.05) which were themselves in turn significantly elevated (P ≤ 0.05) when compared to pNIPAM values of 1.4 × 103 ± 0.2 × 103 U/µL versus 0.3 × 103 ± 0.1 × 103 U/µL in untreated controls (P ≤ 0.05)  Fig. A.Fig. 7OP ≤ 0.05). pNTBAM produced 31.3 × 103 ± 4.7 × 103 in osteogenic compared to 2.7 × 103 ± 1.8 ng/g control, whereas pNIPAM produced 17.2 × 103 ± 2.4 × 103 versus 2.3 × 103 ± 1.2 × 103 ng/g of controls   when compared to pNTBAM (0.52 ± 0.06 µg/µL pNIPAM versus 0.14 ± 0.06 µg/µL for pNTBAM). Both were significantly lower (P ≤ 0.05) than hOB samples in osteogenic media. Hydrogels seeded with hCHs in chondrogenic media displayed increased collagen I levels when compared to controls (pNTBAM (85 ± 23 versus 26 ± 6 ng/g) and pNIPAM (133 ± 33 versus 78 ± 19 ng/g) (P > 0.05)   Fig. A. Howeve Fig. A. P ≤ 0.05). pNIPAMhCHs samples were not significantly different compared to their controls and were significantly lower (P ≤ 0.05) than pNTBAM (1.15 ± 0.49 µg pNIPAM versus 25 ± 0.74 µg for pNTBAM chondrogenic samples).Chondrogenic hCHs samples revealed significant elevation in GAG expression for pNTBAM hydrogels (25 ± 0.7 µg versus 12.26 ± 1.5 µg control) (P ≤ 0.05). pNIPAM displayed no significant changes (224 ± 112 versus 74.8 ± 64.7 ng/g control). pNTBAM, again, showed significant elevation when compared to pNIPAM chondrogenic samples (3105 ± 282 versus 224 ± 112 ng/g for pNIPAM)   (pNIPAM) and poly(N-tert-butylacrylamide) (pNTBAM) hydrogels were produced by polymerization induced phase separation resulting from differing wettablity between monomer and polymer network, a widely applied method in the production of porous polymer scaffolds  with chondrogenic differentiation. We conclude that when taken together, these unique polymer characteristics create a potentially tuneable platform for 3D osteochondral scaffold generation for application in future tissue engineered complex repair situations.


The structure of a copper(II) complex is described. 6H4O2N)2]·2C9H6O6·2H2O, the Cu2+ ion lies on a center of inversion and coordinates with symmetry related pyridine nitrogen and carboxyl oxygen atoms from two pyridine-2-carboxylic acid anions, giving rise to a square-planar coordination geometry. There are weak axial bonds between Cu and an O atom of a symmetry-related trimesic acid moieties [Cu⋯O = 2.837 (2) Å] The Cu⋯O weak interactions and hydrogen bonds stabilize the whole structure.In the title complex, [Cu(C In the axial position, a very weak interaction Cu1⋯O3 [2.837 (2) Å] is observed. Interestingly, the 1,4-bis(3-pyridyl)-2,3-diaza-1,3-butadiene ligand decomposed during the hydrothermal process and is oxidized into pyridine-2-carboxylic acid. According to our earlier research, the occurrence of oxidation may be caused by excess of CuII salt, which may act as an oxidative agent to promote the formation of the carboxyl group  ligand and one crystal water molecule Fig. 1. The Cu23− ligands  are short, there are no π–π interactions because the inter-centroid distance between the two benzene rings is 5.4029 (15) Å, which is much larger than the normal π–π stacking distance of 3.3–3.8 Å. The shortest distance between the two carbon atoms (C1 and C1′) is 3.379 (4) Å. The other C⋯C distances of the two rings are longer than 3.94 Å. In addition, the distance between the centroid of one benzene ring and the C atoms of another is longer than 4.28 Å.In the crystal, C—H⋯O and O—H⋯O hydrogen bonds Table 1 and toge2·2H2O  in 5 mL of distilled H2O was stirred for 10 min in air, and then the mixture was turned into a Parr Teflon-lined stainless steel vessel and heated at 160°C for 60 h. Dark-red crystals suitable for X-ray diffraction were obtained in a yield of 78% (based on CuCl2·2H2O).A mixture of trimesic acid , 1,4-bis(3-pyridyl)-2,3-diaza-1,3-butadiene  and CuClCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621006726/bv4039sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621006726/bv4039Isup3.hklStructure factors: contains datablock(s) I. DOI: 2092720CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures6. Here we identified 2′-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2′-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.Post-transcriptional modifications have critical roles in tRNA stability and function Reversible internal RNA phosphrylation contributes to thermal stability and nuclease resistance of tRNA, and cellular thermotolerance of hyperthermophiles.  Thus far, about 150 types of RNA modification have been reported in various RNA molecules from all domains of life7. In particular, tRNAs contain the widest variety and largest number of modified nucleosides, with 80% of RNA modifications identified in tRNA molecules. Diverse RNA modifications are clustered in the anticodon loop, especially at positions 34 and 37 (refs. 8). These modifications have critical roles in stabilizing and modulating codon–anticodon interactions on the ribosome, ensuring accurate and efficient protein synthesis. Many RNA modifications are also found in the tRNA body composed of the D-loop, TΨC loop (T-loop) and variable loop (V-loop)10  confer conformational rigidity to the tRNA core region by fixing C3′-endo ribose puckering11.Recent advances in epitranscriptomics research have demonstrated the chemical diversity and biological importance of RNA modifications)10 Fig. . These R6. 5-Methyl-2-thiouridine (m5s2U or s2T) is found at position 54 in the T-loop of tRNAs from thermophiles12. m5s2U54 adopts a rigid conformation with C3′-endo ribose puckering, thereby stabilizing the tRNA body in high-temperature environments11. The 2-thiolation level of m5s2U54 increases as the growth temperature rises14. m5s2U54 contributes to the thermotolerance of Thermus thermophilus15. In Pyrococcus furiosus, the relative abundance of N4-acetylcytidine (ac4C) and its 2′-O-methyl derivative (ac4Cm) were markedly increased with rising growth temperature14. ac4C is a prevalent modification that is present in tRNAs, rRNAs and other RNAs in hyperthermophilic archaea16. ac4C favours the C3′-endo form and stabilizes tRNAs18. Loss of ac4C results in a growth defect in Thermococcus kodakarensis at high temperature, contributing to cellular thermotolerance19. In Bacillus stearothermophilus, 2′-O-methylation in tRNAs increases when the growth temperature rises20. Archaeosine (G+) is a unique 7-deazaguanosine derivative found at position 15 in the D-loop of archaeal tRNAs21. On the basis of quantum mechanics calculations, G+15 stabilizes the Levitt base pair with C48 (ref. 22). In line with this, biochemical and genetic studies have shown that G+ confers thermal stability to tRNAs and contributes to thermotolerance23.In thermophilic bacteria and archaea, unique RNA modifications contribute to the thermal adaptation of tRNAsp) in tRNAs, which, to our knowledge, is the first known instance of internal RNA phosphorylation. Biochemical, structural and genetic studies showed that Up47 is a reversible RNA modification and confers thermal stability to tRNA, thereby contributing to cellular thermotolerance.Here we report the identification of 2′-phosphouridine  27 and mapped 13 types of RNA modification at 18 positions in this tRNA molecule  at position 47 of the RNA fragments digested with RNases  modification in class I tRNAs bearing U47 in the V-loop, but not in class II tRNAs with a long V-loop , with a low error value of 4.4 millimass unit , indicating that N324 is a phosphorylated uridine residue. This prediction explains why the N324 nucleoside was not detected in our nucleoside analysis  and biochemical approaches   Fig. .p47 is a thermophile-specific modification found in the tRNA core region, we investigated whether Up47 stabilizes the tertiary structure of tRNA. To this end, we treated S. tokodaii tRNAVal3 with yeast Tpt1p (2′-phosphotransferase) to remove the 2′-phosphate of Up47. We measured the melting temperature (Tm) of S. tokodaii tRNAVal3 with and without Up47  . According to our analysis of Up47 distribution in archaeal species  nucleotide analysis. A pUpm5C dimer was clearly observed in the parental strain (wild type) of T. kodakarensis (KU216), but was absent in the Δtk2051 strain .To identify a gene responsible for Uies Fig. . Among tp47 Fig. . Among tp47 Fig. . We chosp47 Fig. . STK_095ain Fig. . Therefop47 in T. kodakarensis. The ΔarkI strain grew as well as the wild-type strain (KU216) at the nearly optimal temperature of 83 °C  formation, because G+15 thermally stabilizes tRNAs and contributes to cellular thermotolerance19. We confirmed the absence of Up47 and G+15 in tRNAs from the double-knockout strain  and examined in vitro Up47 formation. Up47 was efficiently reconstituted only in the presence of ATP    Fig. , TkArkI The electrostatic surface potential showed a large positive area on one side of the TkArkI structure Fig. . The posTo characterize the conserved residues in TkArkI, we constructed 14 TkArkI mutants in which targeted residues were replaced by alanine Fig. . All mut33. Tpt1/KptA homologues are distributed across all domains of life35  in the presence of NAD+, with the results indicating that the 2′-phosphate of Up47 was efficiently removed , because N. viennensis is a mesophilic archaeon and its ArkI homologue was predicted to have efficient activity in E. coli. The class I tRNA fraction prepared from this strain was subjected to shotgun analysis to detect the Up47 modification. We clearly detected four Up47-containing fragments derived from various E. coli tRNA species -inducible promotor and quantified the peak intensity of each Up47-containing fragment when TkKptA expression was induced by addition of 10 or 100 μM IPTG   and two class II tRNAs (tRNALeu4 and tRNASer3)  Fig.  but was 2+. Especially at high temperatures, RNA is rapidly degraded. Similarly to 2′-O-methylation, the 2′-phosphorylation of Up47 also serves to prevent tRNA degradation. This property partly explains the RNase resistance of tRNA conferred by Up47  with different conformations in the core region  to fill in for the missing G46, forming the non-canonical base triple Ψ13–G22–C9  has a critical role in RNA metabolism and function as a reversible RNA modification45. If Up47 is a reversible modification, it is expected that tRNA function and stability are dynamically regulated by a writer and eraser, raising the possibility of epitranscriptomic regulation of tRNAs in translation. The mechanism closely resembles post-translational modification of proteins. Phosphorylation and dephosphorylation rapidly and dynamically control protein function48. Because tRNA is a stable molecule with a low turnover rate and long lifetime in the cell, it would be reasonable for tRNA function to be regulated by Up47 modification. We found efficient dephosphorylation of Up47 by TkKptA in vitro , T. Yokogawa (Gifu University) and H. Hori (Ehime University), respectively. Sulfolobus acidocaldarius (JCM no. 8929), Saccharolobus solfataricus (JCM no. 8930), Aeropyrum pernix (JCM no. 9820), Pyrobaculum oguniense (JCM no. 10595) and N. viennensis (JCM no. 19564) were obtained from Japan Collection of Microorganisms, RIKEN BRC which is participating in the National BioResource Project of the MEXT, Japan.S. tokodaii and S. acidocaldarius were cultured at 80 °C in JCM medium no. 165 consisting of 1 g l–1 yeast extract, 1 g l–1 casamino acids, 1.3 g l–1 (NH4)2SO4, 0.28 g l–1 KH2PO4, 0.25 g l–1 MgSO4·7H2O, 0.07 g l–1 CaCl2·2H2O, 2.0 mg l–1 FeCl3·6H2O, 1.8 mg l–1 MnCl2·4H2O, 4.5 mg l–1 Na2B4O7·10H2O, 0.22 mg l–1 ZnSO4·7H2O, 0.05 mg l–1 CuCl2·2H2O, 0.03 mg l–1 Na2MoO4·2H2O, 0.03 mg l–1 VOSO4·H2O and 0.01 mg l–1 CoSO4·7H2O (adjusted to pH 2.5 with H2SO4). S. solfataricus was cultured at 80 °C in JCM medium no. 171 consisting of 1 g l–1 yeast extract, 2.5 g l–1 (NH4)2SO4, 3.1 g l–1 KH2PO4, 0.2 g l–1 MgSO4·7H2O, 0.25 g l–1 CaCl2·2H2O, 1.8 mg l–1 MnCl2·4H2O, 4.5 mg l–1 Na2B4O7·10H2O, 0.22 mg l–1 ZnSO4·7H2O, 0.05 mg l–1 CuCl2·2H2O, 0.03 mg l–1 Na2MoO4·2H2O, 0.03 mg l–1 VOSO4·H2O and 0.01 mg l–1 CoSO4·7H2O (adjusted to pH 4.0 with H2SO4). A. pernix was cultured at 90 °C in JCM medium no. 224 consisting of 1 g l–1 yeast extract, 1 g l–1 peptone, 1 g l–1 Na2S2O3·5H2O, 24.0 g l–1 NaCl, 7.0 g l–1 MgSO4·7H2O, 5.3 g l–1 MgCl2·6H2O, 0.7 g l–1 KCl and 0.1 g l–1 CaCl2·2H2O (adjusted to pH 7.0 with NaOH). P. oguniense was cultured at 90 °C in JCM medium no. 165 with addition of 1.0 g l–1 Na2S2O3·5H2O (adjusted to pH 7.25 with NaOH). N. viennensis was cultured at 42 °C in JCM medium no. 1004 consisting of 1 g l–1 NaCl, 0.5 g l–1 KCl, 0.4 g l–1 MgCl2·6H2O, 0.2 g l–1 KH2PO4, 0.1 g l–1 CaCl2·2H2O, 1.0 ml l–1 modified trace element mixture , 1.0 ml l–1 vitamin solution ), 1.0 ml l–1 7.5 mM EDTA·Na·Fe(III) solution (pH 7.0), 2.0 ml l–1 1 M NaHCO3 solution, 10 ml l–1 HEPES solution (238.4 g l–1 HEPES (free acid) and 24 g l–1 NaOH), 1.0 ml l–1 1 M NH4Cl solution and 1.0 ml l–1 1 M sodium pyruvate solution (adjusted to pH 7.6 with NaOH).T. kodakarensis was cultured at 83 °C, 87 °C or 91 °C, in nutrient-rich medium (ASW-YT-S0 or MA-YT-Pyr) or synthetic medium containing amino acids (ASW-AA-S0), under strict anaerobic conditions. ASW-YT-S0 medium contains 0.8× artificial sea water (ASW)50, 10 g l–1 yeast extract, 5.0 g l–1 tryptone, 2.0 g l–1 elemental sulfur and 0.1% (wt/vol) resazurin. MA-YT-Pyr medium contains 30.5 g l–1 Marine Art SF-1 (Osaka Yakken), 10 g l–1 yeast extract, 5.0 g l–1 tryptone, 5.0 g l–1 pyruvate sodium and 0.1% (wt/vol) resazurin. ASW-AA-S0 medium contains 0.8× ASW, 0.5× amino acid solution50, modified Wolfe’s trace minerals 2, 0.01 g l–1 H3BO3 and 0.01 g l–1 NaMoO4·2H2O), 5.0 ml l–1 vitamin mixture51, 2.0 g l–1 elemental sulfur and 0.1% (wt/vol) resazurin. For plate cultivation, 2.0 ml l–1 polysulfide solution  was added instead of elemental sulfur, and the media were solidified with 1.0% Gelrite . When pyrF-negative transformants were selected0, 75% 5-fluoroorotic acid (5-FOA) was added. We used ASW-YT-S0 medium for standard cultivation, MA-YT-Pyr medium for growth comparisons and ASW-AA-S0 medium for construction of the gene knockout strain.N-lauroylsarcosine sodium salt and 1 mM 2-mercaptoethanol) and mixed with an equal volume of water-saturated phenol and 1/10 volume of 3 M sodium acetate (pH 5.3). The mixture was shaken for 1 h on ice and mixed with 1/5 volume of chloroform, followed by centrifugation at 8,000g for 10 min at 4 °C. The supernatant was collected and mixed with an equal volume of chloroform, followed by centrifugation at 8,000g for 10 min at 4 °C. Total RNA was obtained from the resultant supernatant by isopropanol precipitation. The total RNA prepared in this manner was separated by 10% denaturing PAGE, followed by staining with SYBR Gold or toluidine blue. The visualized tRNA fraction including class I and class II tRNAs was cut out and eluted from the gel slice with elution buffer (0.3 M sodium acetate (pH 5.3) and 0.1% (wt/vol) SDS), followed by filtration to remove the gel pieces and ethanol precipitation for RNA-MS analysis of the tRNA fraction.For small-scale preparation (~100-ml culture), archaeal cells were resuspended in 3 ml solution D , 0.5% (wt/vol) S. tokodaii, cell pellets (53 g) were resuspended in 530 ml solution D and then mixed with 53 ml of 3 M sodium acetate (pH 5.3) and 425 ml neutralized phenol. The mixture was shaken for 1 h on ice to which 106 ml chloroform/isoamyl alcohol (49:1) was added, followed by centrifugation at 4,500g for 20 min at 4 °C. The supernatant was collected and mixed with 106 ml chloroform/isoamyl alcohol (49:1), followed by centrifugation at 4,500g for 15 min at 4 °C. The aqueous phase was collected and then subjected to isopropanol precipitation. The collected RNA was resuspended in 53 ml water and mixed with 80 ml TriPure Isolation Reagent (Roche), followed by centrifugation at 10,000g for 20 min at 4 °C. The supernatant was collected and mixed with 36 ml chloroform/isoamyl alcohol (49:1), followed by centrifugation at 10,000g for 10 min at 4 °C. The aqueous phase was collected and precipitated with isopropanol. The prepared total RNA (608 mg) was dissolved in 250 ml of buffer consisting of 20 mM HEPES-KOH (pH 7.6), 200 mM NaCl and 1 mM DTT and then loaded on a DEAE Sepharose Fast Flow column (320-ml beads) and fractionated with a gradient of NaCl from 200 to 500 mM. Fractions containing tRNA were collected by isopropanol precipitation.For large-scale preparation of tRNA fractions from 24 or chaplet column chromatography (CCC)52. Approximately 200 absorbance at 260 nm (A260) units of the S. tokodaii tRNA fraction was subjected to RCC. The isolation procedure was carried out as follows: hybridization at 66 °C in 6× NHE buffer (30 mM HEPES-KOH (pH 7.5), 15 mM EDTA (pH 8.0), 1.2 M NaCl, 1 mM DTT), washing at 50 °C with 0.1× NHE buffer (0.5 mM HEPES-KOH (pH 7.5), 0.25 mM EDTA (pH 8.0), 20 mM NaCl, 0.5 mM DTT) and elution at 72 °C with 0.1× NHE buffer. Eluted tRNAs were recovered by ethanol precipitation. Mature and precursor tRNAs were separated by 10% denaturing PAGE and stained with SYBR Gold. Visualized bands of mature and precursor tRNAs were cut out and eluted from the gel slices with elution buffer, followed by filtration to remove the gel pieces and precipitation with ethanol.Isolation of individual tRNAs from thermophilic organisms is extremely difficult owing to their high melting temperatures, which are the consequence of their high G+C content and complex modifications. We thus optimized our original method for RNA isolation by RCCp47, we conducted large-scale isolation of S. tokodaii tRNAVal3 using CCC52. The S. tokodaii tRNA fraction  was subjected to CCC with tandem affinity chaplet columns for tRNAVal3, tRNAIle2 and tRNAPhe. The isolation procedure was carried out as follows: hybridization at 66 °C in 6× NHE buffer, washing separately at 50 °C with 0.1× NHE buffer and elution at 72 °C with 0.1× NHE buffer. The eluted tRNAs were recovered by isopropanol precipitation. The sequences of the DNA probes are shown in Supplementary Table Val3 was further purified by anion exchange chromatography to completely remove tRNAVal2, as described below.To crystalize native tRNA bearing U 2,000 A20 units w1 (Epicentre or Thermo Fisher Scientific) or RNase A (Ambion) and analysed with a linear ion trap–Orbitrap hybrid mass spectrometer  equipped with a custom-made nanospray ion source and a splitless nanoHPLC system  as described previously27. To analyse Ψ sites, tRNA was treated with acrylonitrile to cyanoethylate Ψ53 and subjected to RNA-MS. For dephosphorylation of the Up47-containing fragment . To precisely map tRNA modifications, RNA fragments were decomposed by CID in the instrument. The normalized collision energy of LTQ Orbitrap XL was set to 40%. Mongo Oligo Mass Calculator v2.08  was used for assignment of the product ions in CID spectra.For tRNA fragment analysis by RNA-MS, 30 ng (900 fmol) of the isolated tRNA or 150 ng (4.5 pmol) of tRNA mixture was digested with RNase TVal3 was digested with 0.09 U nuclease P1  in 20 mM ammonium acetate (pH 5.2) at 50 °C for 1 h and mixed with 1/8 volume of 1 M trimethylamine-HCl (TMA-HCl) (pH 7.2) and 0.06 U phosphodiesterase I , followed by incubation at 37 °C for 1 h. To this mixture, 0.08 U BAP was added, and the sample was incubated at 50 °C for 1 h. After that, 9 volumes of acetonitrile were added, followed by LC–MS/MS analysis as described in refs. 54 with some modifications as follows. The samples were chromatographed with a ZIC-cHILIC column  and eluted with 5 mM ammonium acetate (pH 5.3) (solvent A) and acetonitrile (solvent B) at a flow rate of 100 μl min–1 with a multistep linear gradient: 90–50% solvent B for 30 min, 50% solvent B for 10 min, 50–90% solvent B for 5 min and then initialization with 90% solvent B. The chromatographed eluent was directly introduced into the electrospray ionization source of the Q Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Fisher Scientific).For nucleoside analysis, 800 ng (24 pmol) of the isolated tRNA1 in 20 mM ammonium acetate (pH 5.2) at 50 °C for 1 h and then mixed with 9 volumes of acetonitrile for LC–MS. The digests were chromatographed with a ZIC-cHILIC column and analysed by Q Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Fisher Scientific) or LTQ Orbitrap XL (Thermo Fisher Scientific) with a multistep linear gradient: 90–50% solvent B for 30 min, 50% solvent B for 10 min, 50–90% solvent B for 5 min and then initialization with 90% solvent B.For nucleotide analysis, 800 ng (24 pmol) of the tRNA fraction or individual tRNA was digested with 0.09 U nuclease PThe acquired LC–MS data were analysed using Xcalibur 4.1 (Thermo Fisher Scientific) and were visualized with Canvas X .A260 units of the S. tokodaii tRNA fraction was completely digested with nuclease P1. Digests containing pN324m5C dinucleotide were subjected to periodate oxidation with 10 mM NaIO4 for 1 h on ice in the dark. The reaction was stopped by addition of 1 M l-rhamnose and incubation for 30 min. For β-elimination, an equal volume of 2 M lysine-HCl (pH 8.5) was added, and the sample was incubated at 45 °C for 90 min. The product containing pN324p was then subjected to anion exchange chromatography with a Q Sepharose Fast Flow column  equilibrated with 20 mM triethylammonium bicarbonate (TEAB) (pH 8.2). The eluate with 2 M TEAB was collected and dried by evaporation in vacuo. The pellet was dissolved with water and mixed with an equal volume of chloroform, followed by centrifugation at 20,000g for 5 min at 4 °C. The supernatant was recovered and dried again. This process was repeated five times. The resultant digest was mixed with 9 volumes of acetonitrile and subjected to LC–MS/MS using an LCQ-Advantage ion trap mass spectrometer (Thermo Scientific), equipped with an electrospray ionization source and an HP1100 LC system (Agilent Technologies). For LC, the digest was chromatographed with a ZIC-HILIC column  and eluted with 5 mM formic acid (pH 3.4) (solvent A) and acetonitrile (solvent B) at a flow rate of 100 μl min–1 with a multistep gradient: 90–70% solvent B for 25 min, 70–10% solvent B for 15 min, 10% solvent B for 5 min and then initialized with 90% solvent B.Five arkI from T. kodakarensis, Methanocaldococcus fervens, P. oguniense, Aquifex aeolicus, Nautilia profundicola and Leptolyngbya sp. PCC7376 were designed with codons optimized for E. coli expression and synthesized by GENEWIZ or Thermo Fisher Scientific. Each gene was cloned into the pE-SUMO-TEV vector by the SLiCE method55. N. viennensis arkI was PCR amplified from genomic DNA with a set of primers (Supplementary Table Synthetic genes for E. coli BL21(DE3) or Rosetta2(DE3) cells transformed with the pE-SUMO-TEV vector carrying each arkI gene were cultured in 250 ml or 1 l of LB containing 50 μg ml–1 kanamycin and 20 μg ml–1 chloramphenicol when necessary. His6–SUMO-tagged recombinant protein was expressed at 37 °C for 3–4 h by induction with 0.1 or 1 mM IPTG or 2% (wt/vol) lactose when the cells reached OD610 = 0.4–0.6. P. oguniense ArkI was expressed in cells cultured overnight at 18 °C. The collected cells were resuspended in lysis buffer (50 mM HEPES-KOH (pH 8.0), 150 mM KCl, 2 mM MgCl2, 20 mM imidazole, 12% (vol/vol) glycerol, 1 mM 2-mercaptoethanol and 1 mM PMSF) and disrupted by sonication, followed by centrifugation at 15,000g for 15 min at 4 °C. The supernatant was boiled at 60 °C for 20 min  and centrifuged at 15,000g for 15 min at 4 °C. The recombinant protein was affinity captured on an Ni-Sepharose 6 Fast Flow column  and then eluted with lysis buffer containing 300 mM imidazole, followed by gel filtration with a PD-10 column  to remove the imidazole. The recombinant protein for N. viennensis ArkI was purified using a HisTrap column  with a linear gradient of 0–500 mM imidazole, followed by dialysis using a Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific) to remove imidazole. The purified protein was subjected to Ulp1 digestion at 4 °C overnight to cleave the His6–SUMO tag and then passed through a Ni-Sepharose 6 Fast Flow column to remove the tag. Because ArkI homologues from M. fervens (MfArkI) and Leptolyngbya sp. PCC7376 (LeArkI) aggregated following tag removal, His6–SUMO tag-fused proteins of these homologues were used for the phosphorylation assay. Purified protein was quantified by the Bradford method using BSA as a standard.T. kodakarensis ArkI for crystallization, the E. coli BL21(DE3) strain carrying pE-SUMO-TkArkI was cultured in 2 l of LB containing 50 μg ml–1 kanamycin and TkArkI was expressed at 25 °C overnight by induction with 0.1 mM IPTG when the cells reached OD610 = 0.4. The cells were collected and disrupted by sonication in lysis buffer (50 mM HEPES-KOH (pH 8.0), 150 mM KCl, 2 mM MgCl2, 20 mM imidazole, 12% (vol/vol) glycerol, 1 mM 2-mercaptoethanol and 1 mM PMSF). The protein was purified using a HisTrap column with a linear gradient of 20–520 mM imidazole. Fractions containing TkArkI were pooled and subjected to Ulp1 digestion at 4 °C overnight to cleave the tag, followed by passage through a Ni-Sepharose 6 Fast Flow column to remove the tag fragment. The flow-through fraction was filtered through a 0.45-μm PVDF membrane to remove the resin. The protein was further purified by affinity chromatography with a HiTrap Heparin HP column  using a linear gradient of 150–1,150 mM KCl. TkArkI was further purified by size exclusion chromatography using a Superdex 75 10/300 GL column  with buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol and then concentrated to 5.74 mg ml–1 and stored at –80 °C.For large-scale preparation of T. kodakarensis kptA gene was PCR amplified from genomic DNA from T. kodakarensis with the primers listed in Supplementary Table tkkptA. The E. coli Rosetta2(DE3) strain carrying pE-SUMO-TEV-tkkptA was cultured in 1 l LB containing 50 μg ml–1 kanamycin and 20 μg ml–1 chloramphenicol, and TkKptA was expressed at 37 °C for 3 h by induction with 0.1 mM IPTG when the cells reached OD610 = 0.6. The recombinant TkKptA was purified as described above. The gene encoding Tpt1p was PCR amplified from the genomic DNA of S. cerevisiae BY4742 with the set of primers listed in Supplementary Table The p47 by yeast Tpt1p was performed as described33. Individual tRNAs or the tRNA fraction was incubated for 3 h at 30 °C in a reaction mixture (25 μl) consisting of 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA (pH 8.0), 1 mM NAD+, 2.5 mM spermidine, 0.1 mM DTT, 0.9 μM tRNA and 0.1 μg μl–1 recombinant Tpt1p. The tRNA was extracted by phenol/chloroform treatment and recovered by ethanol precipitation, followed by desalting with Centri-Sep spin columns (Princeton Separations). For crystallization of Tpt1p-treated tRNA, S. tokodaii tRNAVal3 (202.5 μg) was dephosphorylated by yeast Tpt1p in a 200-μl reaction mixture.Removal of the 2′-phosphate of US. tokodaii tRNAVal3 (25 pmol) with or without Up47 was dissolved in degassed buffer consisting of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl and 1 mM MgCl2 and incubated at 80 °C for 5 min, followed by cooling to 25 °C at a rate of 0.1 °C s–1. The samples were placed onto a Type 8 multi-micro UV quartz cell . The hyperchromicity of tRNA was monitored on a UV–visible light spectrophotometer . The gradients were as follows: 25 °C for 30 s, 25–40 °C at 5 °C min–1, 40 °C for 5 min and 40–105 °C at 0.5 °C min–1. The Tm was calculated using Spectra Manager v2 (JASCO). Melting curves were generated using Microsoft Excel.S. tokodaii tRNAVal3 (25 pmol) with or without Up47 was labelled with 32P at the 3′ terminus by ligation with [5′-32P]cytidine 3′,5′-bisphosphate (PerkinElmer). The labelled tRNA was separated on a 7.5% (wt/vol) polyacrylamide gel containing 7 M urea, 1× TBE and 10% (vol/vol) glycerol and was purified by gel extraction. Labelled tRNA was mixed with the S. tokodaii tRNA fraction as a carrier to a concentration of 100,000 counts per minute (c.p.m.) per A260 unit and was precipitated with ethanol. The pellet was dissolved in water to a concentration of 0.1 A260 units per μl. For the RNase degradation assay, the labelled tRNA  was incubated at 65 °C in a reaction mixture consisting of 10 mM HEPES-KOH (pH 7.6), 0.5 mM MgCl2, 100 mM NaCl and 0.1 U μl–1 RNase I (Promega). At time points of 1, 3, 5, 10, 15 and 30 min after starting the reaction, aliquots were taken from the mixture and mixed well with chilled phenol/chloroform/isoamyl alcohol  to stop the reaction, followed by centrifugation at 15,000g for 15 min at 4 °C. The supernatant was collected and treated with an equal volume of chloroform, followed by centrifugation at 15,000g for 5 min at 4 °C. The supernatant was mixed with 2× loading solution  sucrose, 0.05% (wt/vol) xylene cyanol and 0.05% (wt/vol) bromophenol blue) and subjected to 10% denaturing PAGE. The gel was exposed to an imaging plate, and radioactivity was visualized by using an FLA-7000 imaging analyser (Fujifilm). Graphs were generated using Microsoft Excel.S. tokodaii tRNAVal3 (500 μg), isolated as described above, was refolded in annealing buffer (50 mM HEPES-KOH (pH 7.6), 5 mM MgCl2 and 1 mM DTT) by incubation for 5 min at 80 °C and cooling to 25 °C with a rate of 0.1 °C s–1. tRNAVal3 was further purified by anion exchange chromatography using a Mono Q 5/50 GL column  with a linear gradient of 200–1,000 mM NaCl. The major peak was collected, precipitated with isopropanol, dissolved in water and precipitated with ethanol. Tpt1p-treated tRNAVal3 was prepared with the same procedure as described above. The purified tRNA was dissolved in buffer consisting of 10 mM Tris-HCl (pH 7.1) and 5 mM MgCl2 to a concentration of 50 μM. One microlitre of tRNA solution was mixed with 1 μl Natrix 2 no. 32  and 30% (vol/vol) MPD) (Hampton Research) on silicon-coated glass and crystalized by the hanging drop vapor diffusion method at 20 °C.–1 before crystallization. One microlitre of the protein solution was mixed with 0.5 μl reservoir solution, containing 25% (vol/vol) ethylene glycol. TkArkI was crystallized by the hanging drop vapor diffusion method at 20 °C.The concentration of TkArkI was adjusted to 5 mg mlVal3 crystals, the crystals were cryoprotected with a portion of the reservoir solution. For data collection for the native TkArkI crystal, the crystal was cryoprotected with solution containing 25% (vol/vol) ethylene glycol, 2 mM MgCl2 and 1 mM ATP. For data collection for the iodide-derivative TkArkI crystal, the crystal was briefly soaked in and cryoprotected with solution containing 300 mM potassium iodide and 22.5% (vol/vol) ethylene glycol, and the diffraction dataset was collected at a wavelength of 1.5 Å. The datasets were indexed, integrated and scaled using xds56. The initial phase of tRNAVal3 was determined by molecular replacement with Phaser57. The structure of T. thermophilus tRNAVal 58 was used for the model. The initial phase of TkArkI was determined by the SAD method using the anomalous signal of iodide ions. The iodine sites were located by SHELX59, and the initial phase was calculated by Phaser. Subsequent density modification and initial model building were performed with RESOLVE60. The model was further modified with Coot61 and refined with Phenix62. Crystal structures and their electron density maps were visualized using PyMOL, Cuemol or Coot. Torsion angles of the tRNAs were analysed with DSSR software63.The datasets were collected at beamline BL-17A at the Photon Factory at KEK, Japan. For data collection for the tRNA15N]adenosine (10 pmol) and [15N]guanosine (10 pmol) as tracer molecules, followed by addition of 4 volumes of methanol, an equal volume of chloroform and 3 volumes of water and vigorous mixing. The denatured protein was removed by centrifugation at 15,000g for 1 min at 4 °C. The supernatant was dried in vacuo and dissolved in 20 μl water. Half of the extract was analysed by LC–MS. The tracer molecules were prepared by dephosphorylation of [15N]ATP and [15N]GTP as follows: 1,000 pmol each of [15N]ATP (Silantes) and [15N]GTP (Silantes) was treated with 0.04 U alkaline phosphatase  in 20 mM ammonium acetate (pH 8.0) at 60 °C for 30 min. After dephosphorylation, PAP was heat denatured at 95 °C for 5 min.TkArkI purified by affinity chromatography with a HiTrap Heparin HP column  (100 pmol) was mixed with ATP to tRNA similarly to the kinetic studies of TkArkI (see below). tRNA phosphorylation was performed at 70 °C for 15 min in an 8-μl reaction mixture. For PAGE analysis, 4 μl of the reaction mixture was mixed with 4 μl of 2× loading solution, resolved by 10% denaturing PAGE and exposed to an imaging plate to visualize radiolabelled RNA with an FLA-9000 imaging analyser (Fujifilm). The gel image was analysed using Multi Gauge (Fujifilm). Bar graphs with independent plots were prepared with R (R Foundation). For phosphorylation of total RNA, the reaction was performed at 70 °C for 30 min in an 8-μl reaction mixture consisting of 50 mM HEPES-NaOH (pH 7.5), 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 100 μM [γ-32P]ATP , 1.8 μM TkArkI and 50 ng μl–1 total RNA fraction (from the T. kodakarensis ΔarkI strain). Then, 0.5 μl of 50 mM EDTA (pH 8.0) was added, and 4 μl of reaction mixture was mixed with 2× loading solution, resolved by 10% denaturing PAGE and visualized as described above.Up47 by other ArkI homologues was carried out at 70 °C for 30 min in a reaction mixture (30 μl) containing 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 500 μM ATP, 0.05 mg ml–1 BSA (Takara), 1 μM tRNA transcript and 0.5 μM ArkI protein. For NvArkI, the reaction temperature was set to 45 °C. For ArkI homologue from N. profundicola (NpArkI), the reaction was carried out at 50 °C for 60 min. After the reaction, tRNA was prepared as described above. For PAGE analysis, Up47 formation was carried out in a reaction mixture (8 μl) containing 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 100 μM [γ-32P]ATP , 0.1 mg ml–1 BSA (Takara), 0.75 μM recombinant ArkI homologue  and 50 ng μl–1 E. coli total RNA. Then, the reaction mixture was mixed with 2× loading solution, resolved by 10% denaturing PAGE and visualized as described above.Formation of Up47 by TkKptA was carried out at 60 °C for 1 h in a reaction mixture (30 μl) containing 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA (pH 8.0), 1 mM NAD+, 2.5 mM spermidine, 0.1 mM DTT, 0.9 μM T. kodakarensis tRNA fraction and 0.1 μg μl–1 recombinant TkKptA. After the reaction, the tRNA was extracted by acidic phenol/chloroform, desalted on a NAP-5 column  and precipitated with isopropanol. For RNA-MS, the prepared tRNA was desalted by drop dialysis as described above.Dephosphorylation of Up47 formation was quantified by γ-phosphate transfer from [γ-32P]ATP to tRNA. For kinetic measurement of the tRNA substrate, tRNA phosphorylation was performed at 70 °C in a reaction mixture (25 μl) consisting of 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 100 μM [γ-32P]ATP , 0.05 mg ml–1 BSA (Takara), 0.05 μM TkArkI and 0.1–5.0 μM of in vitro-transcribed T. kodakarensis tRNAVal3. For kinetic measurement of the ATP substrate, the ATP concentration was altered from 15.6 to 1,000 μM [γ-32P]ATP  and the tRNA concentration was increased to 1.0 μM. At each time point (2 and 5 min), 8-μl aliquots were taken and mixed with an equal volume of 2× loading solution  bromophenol blue, 0.2% (wt/vol) xylene cyanol and 50 mM EDTA (pH 8.0)) to quench the reaction. Each sample was subjected to 10% denaturing PAGE. The gel was exposed on an imaging plate to measure radiolabelled tRNAs using an FLA-9000 imaging analyser. Kinetic parameters were calculated using Prism 7 (GraphPad).TkArkI-mediated Up47 was quantified by measuring the reduction in radioactivity for tRNA. In vitro-transcribed T. kodakarensis tRNAVal3 was phosphorylated by TkArkI with [γ-32P]ATP as described above and then purified by gel extraction and isopropanol precipitation. In addition, the same tRNA was phosphorylated by TkArkI with unlabelled ATP. By mixing labelled and unlabelled tRNAs, the specific activity of the labelled tRNA was adjusted to 6,250 c.p.m. per pmol in buffer consisting of 50 mM HEPES-KOH (pH 7.6), 5 mM MgCl2 and 1 mM DTT. The labelled tRNA was incubated at 80 °C for 5 min and then cooled at room temperature, followed by isopropanol precipitation. The labelled tRNA was dissolved in water to a concentration of 8 μM . Dephosphorylation of the labelled tRNA by TkKptA was performed at 70 °C in a reaction mixture (30 μl) consisting of 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 1 mM NAD+, 0.05 mg ml–1 BSA (Takara), 1 nM TkKptA and 12.5–800 nM 32P-labelled tRNA. At each time point (2 and 5 min), 8-μl aliquots were spotted on Whatman 3MM filter paper, which was immediately soaked in 5% (wt/vol) trichloroacetic acid. The filter paper was washed three times for 15 min with ice-cold 5% (wt/vol) trichloroacetic acid, rinsed for 5 min with ice-cold ethanol and dried in air. Radioactivity on the filter paper was measured by liquid scintillation counting . Kinetic parameters were calculated using Prism 7.TkKptA-mediated dephosphorylation of UN. viennensis arkI was PCR amplified and cloned into pMW118 (Invitrogen) under the control of the synthetic constitutive J23106 promoter68, followed by insertion of sequences encoding a His6 tag and a 3×Flag tag at the C terminus of the N. viennensis arkI gene, yielding pMW-J23106-nvarkI . The ampicillin resistance cassette (Ampr) was replaced with a chloramphenicol resistance cassette (Camr), yielding pQE-80LC-tkkptA, pQE-80LC-eckptA and pQE-80LC-sctpt1, respectively (Supplementary Table E. coli ΔtrmBΔtapT (Kanr) strain was transformed with pMW-J23106-nvarkI and further transformed with pQE-80LC-tkkptA, pQE-80LC-eckptA or pQE-80LC-sctpt1. The transformants were inoculated in 3 ml LB supplemented with 20 μg ml–1 chloramphenicol, 50 μg ml–1 kanamycin and 100 μg ml–1 ampicillin and cultured at 37 °C until mid-log phase. When the OD610 reached 0.6, IPTG was added to a final concentration of 10 or 100 μM to induce expression of the KptA/Tpt1p homologue and cells were cultured for 3.5 h. A 1.5-ml aliquot of the culture was taken, and the tRNA fraction was extracted and analysed by shotgun RNA-MS as described above. Primers, E. coli strains and plasmids used are listed in Supplementary Tables Chemical structures were drawn with chemical structure drawing tools, including ACD/ChemSketch (ACD/Labs) or ChemDraw (PerkinElmer).Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04677-2.Supplementary InformationThis file contains Supplementary Notes 1–7, Supplementary Figs. 1–10, Supplementary Tables 1–7 and the legend for Supplementary Video 1.Reporting SummaryPeer Review FileSupplementary Video 1S. tokodaii tRNAVal3.Structural switch of the core region in