The salts were characterized by the multinuclear NMR and IR spectroscopy as well as X‐ray diffraction. Hirshfeld surface analysis and solid state structures reveal various intermolecular anion‐π and σ‐hole interactions between the corresponding halogenated pyridinium cations and the anion [Al(OTeF5)4]−.The synthesis and the first structural characterization of the halogenated pyridinium salts [C 5F5NH]+ and [C5F4ClNH]+ and the dimeric cations [(C5F5N)2H]+ and [(C5Cl5N)2H]+ as [Al(OTeF5)4]− salts are presented. These cations show noncovalent interactions with the weakly coordinating anion [Al(OTeF5)4]− such as strong fluorine specific interactions as well as σ‐hole and anion‐π interactions.The synthesis and structural characterization of the perhalogenated pyridinium cations [C While non fluorinated pyridine can be protonated by HCl or HBr, pentafluoropyridine can only be protonated by Brønsted superacids such as HF/AsF5 and HF/SbF5.o‐C6H4F2‐H][Al(OTeF5)4] is known for the protonation of weak bases like benzene and white phosphorus, resulting in [C6H7]+ and [P4H]+, respectively.6F6, which can result in anion‐π interactions.4]− and [PF6]−[6] or between perfluoroarenes, like C5F5N, C6F6 and C10F8 with different halide anions.5F5N, C5F4ClN, and C5Cl5N by [o‐C6H4F2‐H][Al(OTeF5)4] (1 a) and studied hydrogen bonding, halogen bonding and anion‐π interactions between pentafluororopyridinium [C5F5NH]+ (2), 4‐chloro‐2,3,5,6‐tetrafluoropyridinium [C5F4ClNH]+ (3), pentachlororopyridinium [C5Cl5NH]+ (4) and the fluorinated WCA [Al(OTeF5)4]− (a).In contrast to pyridine, the perfluorinated pyridine C5F5N<C5F4ClN<C5Cl5N  of the pyridinium cations illustrate that in all cases, their strongest interaction with an anion will be observed via the N−H moiety Figure . Beside 5F5N, C5F4ClN or C5Cl5N with the Brønsted superacid 1 a in ortho‐difluorobenzene (oDFB) °. These moieties are symmetry related by an inversion center. As short fluorine/fluorine (F4⋅⋅⋅F3) contacts of 276.8(21) pm are observed between the two cations we further inspected the fluorine‐specific interaction in an isolated [C5F5NH]+‐dimer by means of atoms in molecules (AIM) analysis. At the bond critical points (CP) between F3 and F4 the electron density is very low (ρCP=0.045 e A−3) and the Laplacian is small and positive (▿2ρCP=0.035 e A−5). Together with a value of the electron localization function (ELF) close to zero (0.01) and a ratio of the absolute potential (|V|) and the kinetic energy density (G) below 1 (0.74), this indicates a non‐shared interaction such as a van‐der‐Waals interaction. An ionic interaction appears to be unlikely for this dimer of two cations.Figure The Hirshfeld surface analysis Figure  reveals 1 a with an excess of C5F5N the salt [(C5F5N)2H][Al(OTeF5)4] (5 a) was obtained. It crystallizes in the monoclinic space group Cc  pm which is about 35 pm shorter than the sum of the van der Waals radii of 310 pm.5 moiety above and below the plane of the pentafluoropyridines are stabilizing the planar arrangement . For 4‐halotetrafluoropyridines, the halogen bond donor strength weakens in the order I, Br, Cl.5)4]− in the solid state.1 a 4] (3 a) crystallizes in the monoclinic space group C2/c . The reaction of two equivalents of pentachloropyridine with 1 a yielded single crystals of [(C5Cl5N)2H][Al(OTeF5)4]⋅C6H4F2 (6 a⋅C6H4F2). The compound crystallizes in the monoclinic space group P21/c  pm which is 20 pm below the sum of the van der Waals radii. Multiple short Fc Figure .5 a the pyridine rings form a pyridinium‐pyridine dimer. The N−N distances in dimers 5 a and 6 a are indistinguishable within the experimental error. Moreover, the two pentachloropyridine rings in 6 are staggered with an angle between the planes of 95.8(1)°, in contrast to the fluorinated analogue 5 which is arranged coplanar  pm which indicates a rather strong interaction.6 . Additionally, a number of halogen interactions are observed beween the chlorine atoms in 2,6‐ and 4‐positions of 6 and a) fluorine atoms (d(Cl6‐F20)=307.47(18) pm, d(Cl3‐F14)=312.3(2) pm, d(Cl15‐F13)=317.93(19) pm), b) oxygen atoms (d(Cl8‐O4)=301.71(18) pm) of the WCA, and c) fluorine atoms of the co‐crystalized oDFB (d(Cl10‐F22)=307.43(19) pm, d(Cl10‐F21)=300.34(19) pm).As for the fluorine analogue r Figure . Single 2 a and 3 a several broad bands for the N−H stretch are observed. The N−H band for 2 a exhibits two maxima at 3104 cm−1 and 2953 cm−1, respectively. The N−H band for 3 a shows three maxima at 3301 cm−1, 3204 cm−1 and 2953 cm−1, respectively. In contrast to the spectra of the monomeric cations the spectra of the pyridinum‐pyridine dimers show very broad bands in the region from 3000 to 1750 cm−1 for 5 a and from 3500 to 2000 cm−1 for 6 a attributed to the N−H stretching vibration. This is well combarable to the reported spectrum of the nonhalogenated pyridinum‐pyridine dimer.In the IR spectra of o‐C6H4F2‐H][Al(OTeF5)4] is able to protonate a series of the very weakly basic perhalogenated pyridines. The cations [C5F5NH]+, [C5F4ClNH]+, [(C5Cl5N)2H]+, and [(C5F5N)2H]+ were structurally characterized for the first time. In the solid state rather strong noncovalent interactions between these electron deficient aromatic cations and the weakly coordinating anion [Al(OTeF5)4]− are observed. Due to the protonation of the halogenated pyridines, their ability to form halogen bonds as well as anion‐π interactions is increased compared to the neutral species.The Brønsted superacid ‐acetone was flame sealed in a glass capillary and the lock oscillator frequency was adjusted to give δ(1H)=7.26 ppm for a CHCl3 sample locked on the capillary. Chemical shifts and coupling constants of strongly coupled spin systems are given as simulated in gNMR.8 Venture diffractometer with a PHOTON 100 CMOS area detector using Mo−Kα radiation. Single crystals were coated with a perfluoroether oil at −25 °C and selected under nitrogen atmosphere. Using Olex2,For detailed experimental data, see electronic Supporting Information. All reactions were carried out under inert conditions using standard Schlenk techniques. Glass vessels were greased with Triboflon III. All solid materials and triethylaluminium (Al(C2 a), 2074488 (for 5 a), 2082692 (for 3 a), 2074489 (for 6 a)2082701 (for  contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition Number (s) 5 a, the 3‐D dnorm surfaces were mapped using a fixed color scale of −0.4727 (red) to 0.5303 (blue), using an isovalue of 0.5. For 2 a, 3 a and 6 a⋅o‐C6H4F2, the 3‐D dnorm surfaces were mapped using a fixed color scale of −0.0964 (red) to 0.9305 (blue), using an isovalue of 0.5. Red contours indicated a contact less than the sum of the van der Waals radii of the respective elements. Blue and white contours indicate that the nearest external atom is at a distance greater or equal to the sum of the van der Waals radii respectively from atomic coordinates. Atoms in Molecules (AIM) analysis of an isolated [C5F5NH]+‐dimer based on calculations at B3LYP‐D3/cc‐pVTZAll calculations were performed using a general‐purpose High‐Performance Computer at ZEDAT (CURTA),5F5NH][Al(OTeF5)4] (2Synthesis of [C a): A sample of Al(C2H5)3  was dissolved in 3 mL of ortho‐difluorobenzene (oDFB). The solution was degassed and HOTeF5  was condensed onto it at −196 °C. The reaction mixture was warmed up to −30 °C. Afterwards a gas bubbler was added and the reaction mixture was stirred for 30 min. A yellow solution was obtained indicating the formation of the corresponding Brønsted superacid. C5F5N  was condensed into a Schlenk tube with a PTFE cap and dissolved in 2 mL of oDFB. This solution was added to reaction vessel via syringe under an argon stream. The gas bubbler was exchanged with a stopper and the mixture was stirred for another 30 min at −30 °C. The pressure in the reaction vessel was reduced and the flask was placed in a −24 °C fridge for crystallization. [C5F5NH][Al(OTeF5)4] was obtained as colourless crystals. 1H NMR : δ=12.1  ppm; 19F NMR : δ=−40.5 , −47.6 , −95.2 , −100.1 , −153.1  ppm; 27Al NMR : δ=49.9 −, d, 22.5 % [Al(OTeF5)3(O125TeF5)]−, 2J=72 Hz; t, 2.5 % [Al(OTeF5)2(O125TeF5)2]−, 2J=69 Hz] ppm. IR : v˜=3103 (w), 2953 (w), 1691 (m), 1610 (m), 1535 (w), 1466 (w), 1325 (w), 1122 (m), 988 (w), 927 , 799 , 684 , 606 , 553  cm−1. 5F5NH][Al(OTeF5)4]Crystal Data for [Al(OTeF5)4] (3Synthesis of [C a): The synthesis was analogous to the one of [C5F5NH][Al(OTeF5)4] using C5F4ClN  that was previously dissolved in 2 mL of oDFB. After the reaction was complete, all volatile parts were removed in vacuo. The flask with the remaining solution was put into a −24 °C fridge for crystallization. [C5F4ClNH][Al(OTeF5)4] was obtained as colourless crystals. 1H NMR : δ=12.2  ppm. 19F NMR : δ=−38.8 , −45.7 , −99.1 , −133.3  ppm. 27Al NMR : δ=50.1 −; d, 22.5 % [Al(OTeF5)3(O125TeF5)]−, 2J=72 Hz; t, 2.5 % [Al(OTeF5)2(O125TeF5)2]−, 2J=73 Hz] ppm. IR : v˜=3301 (w), 3204 (m), 2953 (vw), 1671 (w), 1649 (w), 1570 (m), 1471 (w), 1445 (w), 1305 (m), 928 , 687 , 636 , 633 (m), 618 (m), 553 , 246 (w) cm−1. 5F4ClNH][Al(OTeF5)4]Crystal Data for [Al(OTeFSynthesis of [(C5)4] (5 a): The synthesis was analogous to the one of [C5F5NH][Al(OTeF5)4] using 10 equivalents of C5F5N  that were previously dissolved in 3 mL of oDFB. [(C5F5N)2H][Al(OTeF5)4] was obtained as a colourless crystals. 1H NMR : δ=11.5  ppm; 19F NMR : δ=−38.7 , −45.8 , −89.7 , −132.2 , −161.8  ppm; 27Al NMR : δ=50.1 −, d, 22.5 % [Al(OTeF5)3(O125TeF5)]−, 2J=73 Hz; t, 2.5 % [Al(OTeF5)2(O125TeF5)2]−, 2J=73.2 Hz] ppm. IR : v˜=2323 (w), 1848 (w), 1666 (m), 1610 (m), 1442 (s), 1394 (w), 1307 (m), 1213 (vw), 1104 (m), 1092 (m), 930 , 685 , 641 , 627 (m), 553 , 431 (w) cm−1. 5F5N)2H][Al(OTeF5)4]Crystal Data for [Al(OTeFSynthesis of [(C5)46H4F2 (6]⋅C 6H4F2)a⋅C: The synthesis was analogous to the one of [C5Cl5NH][Al(OTeF5)4] using 2 equivalents of C5Cl5N  that were previously dissolved in 3 mL of oDFB. [(C5Cl5N)2H][Al(OTeF5)4] was obtained as colourless crystals. 1H NMR : δ=17.5  ppm; 19F NMR : δ=−38.6 , −45.7  ppm; 27Al NMR : δ=50.0 −, d, 22.5 % [Al(OTeF5)3(O125TeF5)]−, 2J=73.0 Hz; t, 2.5 % [Al(OTeF5)2(O125TeF5)2]−, 2J=73.0 Hz] ppm. IR : v˜=3555 (m), 3001 (vw), 2953 (vw), 1619 (w), 1541 (w), 1491 (w), 1449 (w), 1403 (w), 1143 (m), 1120 (m), 1088 (m), 1085 (m), 1007 (m), 922 , 859 (m), 693 , 633 (m), 618 (m), 555 , 420 (s) cm−1. 5Cl5N)2H][Al(OTeF5)4]⋅C6H4F2Crystal Data for [(C (M=1599.10 g/mol): monoclinic, space group P21/c (no. 14), a=12.978(3) Å, b=22.529(5) Å, c=14.071(3) Å, β=96.167(8)°, V=4090.4(15) Å3, Z=4, T=100.1 K, μ(MoKα)=3.632 mm−1, Dcalc=2.597 g/cm3, 173293 reflections measured (4.442°≤2Θ≤56.616°), 10174 unique  which were used in all calculations. The final R1 was 0.0208 (I>2σ(I)) and wR2 was 0.0484 .The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The crystal structures and Hirshfeld surface analyses of three salts of 1-(4-nitrophenyl)piperazine with 2-chlorobenzoic acid, 2-bromobenzoic acid and 2-iodobenzoic acid are reported. 10H14N3O2+·C7H4ClO2−, contains whole-ion-disordered cations and anions, which were modeled with two equivalent conformations with occupancies of 0.745 (10)/0.255 (10) and 0.563 (13)/0.437 (13), respectively. The bromobenzoate and iodobenzoate derivatives are isomorphous and crystallize as hemihydrates, viz. C10H14N3O2+·C7H4BrO2−·0.5H2O and C10H14N3O2+·C7H4IO2−·0.5H2O, respectively [the water molecule is disordered over two locations with occupancies of 0.276 (3)/0.223 (3) for the iodobenzoate derivative]. In the extended structures, all three salts feature an R44(12) loop of two anions and two cations linked by N—H⋯O hydrogen bonds.The crystal structures and Hirshfeld surface analyses of three salts of 1-(4-nitrophenyl)piperazine with 2-chlorobenzoic acid, 2-bromobenzoic acid and 2-iodobenzoic acid are reported. The chlorobenzoate salt, C When discussing the conformations of the anion and cation, only the major components will be used. In the chlorobenzoate anion, the carboxylate group is significantly twisted with respect to the 2-chlorophenyl ring with a dihedral angle of 76.7 (4)°, which is likely due to the steric interaction between the ortho-chloro substituent and the carboxylate group. Structures 2 and 3 exhibit similar cation conformations, with equivalent dihedral angles of 65.5 (3) and 67.1 (5)°, respectively. Additionally, in all three structures, the 4-nitrophenyl group occupies an equatorial position in its attachment to the piperazinium ring.Structure ds Fig. 1, which w2 and 3 are isostructural, only 2 will be discussed in detail. This structure consists of a 4-(4-nitrophenyl)piperazin-1-ium cation linked to a 2-bromobenzoate anion by two N—H⋯O hydrogen bonds /0.223 (3) for the iodobenzoate derivative]. Additionally, there is a weak C—H⋯Br interaction accepted by the bromine atom in the 2-bromobezoate anion and a carbon atom in the piperazinium ring, as well as a pair of weak C—H⋯O interactions between adjacent 4-nitrophenyl rings in the 4-(4-nitrophenyl)piperazin-1-ium cation.Since s Figs. 2 and 3 ▸.3.1, which contains both a disordered cation and anion as well as disordered water of solvation, the discussion will focus solely on the major component. The cation forms an x, 1 − y, 1 − z; x, 1 + y, z; 1 − x, 1 − y, 1 − z; see Fig. 4x, −y, −z; Y⋯Cg distance = 3.488 (18) Å; X—Y⋯Cg: 85.8 (12)°].In the packing of 2, two cations and two anions form an et al., 1990x, −y, −z; see Fig. 6x, 1 − y, 1 − z) that form an x, y, z). The phenyl rings in adjacent cations form π–π interactions with a perpendicular distance between centroids of 3.5332 (11) Å . These are all clearly seen in the fingerprint plot generated by CrystalExplorer  that form an Refinement). This species is likely to be involved in hydrogen bonding with an adjacent oxygen atom in the anion  and with the piperazine ring in the cation, forming an x, 1 − y, −z; slippage = 0.379 Å).In the packing of 4.et al., 2014et al., 2022aet al., 2022et al., 2022aRelated structures containing the 4-(4-nitrophenyl)piperazin-1-ium cation include 4-(4-nitrophenyl)piperazin-1-ium chloride monohydrate –(3), a solution of commercially available (from Sigma-Aldrich) 1-(4-nitrophenyl)piperazine  in methanol (10 ml) was mixed with equimolar solutions of the appropriate acids in methanol (10 ml) and ethyl acetate (10 ml), viz., 2-chlorobenzoic acid  for (1), 2-bromobenzoic acid  for (2), and 2-iodobenzoic acid  for (3), The resulting solutions were stirred for 15 minutes at room temperature and allowed to stand at the same temperature. X-ray quality crystals were formed on slow evaporation after one week for all compounds, where ethanol:ethylacetate (1:1) was used for crystallization. The melting points are 439–441 K (1), 443–445 K (2) and 451–453 K (3).For the synthesis of salts (6.Uiso(H) = 1.2Ueq(C) while the N—H hydrogen atoms were refined isotropically. For 1, in which both the cation and the anion exhibit whole-ion disorder, two equivalent conformations were modeled with occupancies of 0.745 (10)/0.255 (10) and 0.563 (13)/ 0.437 (13) respectively. The water hydrogen atoms were refined isotropically with idealized geometries.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S205698902300302X/hb8051sup1.cifCrystal structure: contains datablock(s) 1, 2, 3. DOI: 10.1107/S205698902300302X/hb80511sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S205698902300302X/hb80512sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S205698902300302X/hb80513sup4.hklStructure factors: contains datablock(s) 3. DOI: 2253382, 2253381, 2253380CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


After the introduction of a pyridine‐containing auxiliary, which provided access to challenging junctions (proline or β‐branched amino acids), we herein probe the role of the pyridine‐ring nitrogen. We observed side reactions leading to preliminary auxiliary loss. We describe a new easy to attach β‐mercapto‐β‐(4‐methoxy‐2‐pyridinyl)‐ethyl (MMPyE) auxiliary, which 1) has increased stability; 2) enables NCL at sterically encumbered junctions ; and 3) allows removal under mildly basic (pH 8.5) conditions was introduced. The synthesis of a 120 aa long peptide containing eight MUC5AC tandem repeats via ligation of two 60mers demonstrates the usefulness. Making use of hitherto unexplored NCL to tyrosine, the MMPyE auxiliary provided access to a head‐to‐tail‐cyclized 21‐mer peptide and a His A properly positioned pyridine ring can speed up auxiliary‐mediated native chemical ligation. A methoxy substituent increases auxiliary stability and facilitates rapid syntheses of long and multiphosphorylated multirepeat proteins and of head‐to‐tail cyclized peptides. N‐terminal cysteine for reactions with peptide thioesters. The ligation‐desulfurization involves thiolated amino acids, which mimic the 1,2‐aminothiol structure of cysteine. After ligation, the thiol group is removed by a radical reaction.The native chemical ligation (NCL) reaction is a cornerstone of protein total synthesis.N‐terminus.N‐terminal alkylation reduces the reactivity of the amino group. Exemplary for this problem are the early N‐benzyl type auxiliaries  will become challenging if not impossible at amino acids with larger steric demand than glycine. A solution to the problem became apparent when we, while working on alternative auxiliary scaffolds, became aware of a radical fragmentation reaction (see below) that allows removal of non‐benzyl auxiliaries.8 ethyl (MPyE) scaffold 9 is the first auxiliary enabling ligations at sterically demanding junctions such as Leu‐Arg, Leu‐Val or Pro‐Ala.S,N‐acyl transfer by base catalysis . While shifting the nitrogen from the ortho to the para position should have little effect on its basicity, the intramolecular proton abstraction  should be impossible. For the para isomer of the MPyE auxiliary, we therefore expected a ligation speed comparable to that of the MPE auxiliary 8.The recently introduced MPyE auxiliary enables NCL reactions at ligation junctions inaccessible to previous ligation auxiliaries. For example, we established Ala‐Val, Leu‐Val, Pro‐Ala and Pro‐Arg bonds.17 . Under these conditions, the original ortho isomer of the MPyE auxiliary remained stable. Nevertheless, with quick and careful handling we succeeded in the isolation of product (53 %) and subsequently analyzed reactions of the para‐MPyE peptide with the peptide thioester 19A. The attempted Ala‐Asn ligation was slow . The decrease of ligation rates upon para positioning illustrates the role of the ring nitrogen, which must be in the ortho position to allow acceleration of the S,N‐acyl transfer by intramolecular base catalysis.The aldehyde precursor 7 Figure A requirepara‐MPyE group from peptide 18N prompted us to also critically analyze the stability of the ortho‐MPyE auxiliary. In a published example23LV  and iminium ion 31. The latter would undergo rapid hydrolysis. Alternatively, the ethylpyridine structures may undergo a Hoffman elimination facilitated by anchimeric assistance from a deprotonated thiol group, which would be considerably acidified by pyridine protonation =4.88; pKA (pyridine)=5.17)The considerations on the stability and reactivity of the MPyE auxiliary indicate an important role of the ring nitrogen. We examined methoxy substitution in meta position to the nitrogen. Methoxy substitution decreases the basicity of pyridine nitrogen (pKα‐methyl carbon with lithium diisopropylamide and subjected to a nucleophilic acylation with diethyl carbonate to yield methyl (5‐methoxy‐2‐pyridinyl)acetate (37). Treatment with lithium bis(trimethylsilyl)amide afforded an ester enolate which was converted to the α‐mercapto ester 39 in an electrophilic thiolation. Reduction of 39 with diisobutylaluminium hydride (DIBALH) furnished 40 as a suitable precursor for auxiliary introduction by reductive alkylation. Compound 40 is present in its enole form and shelf‐stable.For the introduction of the MMPyE auxiliary, a suitable precursor was synthesized in 4 steps  in presence of 2 % acetic acid, the peptides were treated with a mixture of TFA/H2O/TIS (95/2.5/2.5 v/v/v) for final deprotection and cleavage of the peptides from resin. The crude peptides were obtained in high purity, indicative of efficient reductive alkylation .We next attached the MMPyE auxiliary to the unprotected N‐terminus of asparagine and valine test peptides Figure B. After 41X with the peptide thioesters 19Z the compounds were dissolved in a mixture prepared by adding 3 vol.% thiophenol to a phosphate buffer containing 6 M guanidinium hydrochloride with an apparent pH 7.5. The ligations proceeded smoothly. Compared to reactions at the non‐substituted MPyE peptide we observed the expected decrease in the reaction rate , we observed a rapid auxiliary cleavage within 3 to 6 h .52 was prepared by microwave‐assisted solid phase peptide synthesis (MW‐SPPS) and used in crude form in the reaction with mercaptopropionate (MPA). The conversion to the MPA ester 53 proceeded smoothly . For an approach to a peptide thioester, we first synthesized the peptide hydrazide by microwave‐assisted solid‐phase synthesis. Dawson's acetoacetone method was applied to establish the peptide thioester 59 without detriment to the three phospho sites .59 and 57 was discontinued by treatment with hydrazine. Though our aim was to develop a method providing rapid access of product without isolation of intermediates, we also analyzed the ligation mixture . We observed an unexpected by‐product resulting from cleavage of the N‐terminal tyrosine from peptide 57. This side reaction was less pronounced in ligations with non‐phosphorylated peptides  and did not occur at all with a test peptide . At current, we cannot explain this unusual side reaction. Other ligations at tyrosine 6 peptide 60 in pure form.Due to our interest in post‐translationally modified multirepeat proteins, we tested the auxiliary in the rapid synthesis of a hexaphosphorylated peptide comprising 6 heptapeptide repeat units from the C‐terminal domain of the large RNA polymerase II subunit.In a further exploration of application scenarios, we studied a cyclopeptide. The chemical synthesis of cyclopeptides is an active area in medicinal peptide chemistry and NCL is powerful method to achieve cyclization of unprotected linear peptide precursors.Previously we have shown that cysteine residues should be protected during auxiliary removal.β‐mercapto‐β‐(2‐pyridinyl)‐ethyl auxiliaries proceed in high rate and enable joining of peptide fragments at junctions, such as the Leu‐Val bond inaccessible to previous ligation auxiliaries. In this work, we probed the role of the ortho‐positioned ring nitrogen. A comparison with the para isomer (β‐mercapto‐β‐(4‐pyridinyl)‐ethyl) scaffold revealed the superior reactivity of the ortho pyridine derivatives. We attribute the high reactivity to the ortho ring nitrogen abstracting a proton from an ammonium ion formed when the amino group intramolecularly attacks the thioester bond. Without deprotonation the S,N‐acyl shift cannot proceed and the reaction stops at the thioester intermediate stage. Our investigations also uncovered a peculiar lability of peptides modified with the β‐mercapto‐β‐(4‐pyridinyl)‐ethyl) group at the N‐terminus. While the para‐pyridine auxiliary‐peptide bond was stable during TFA cleavage, rapid cleavage occurred at moderately acidic pH 2. This reaction only occurs prior to ligation indicating a role of the amino terminus’ lone pair. Though at a slower rate, premature auxiliary loss was also observed with the ortho‐system (β‐mercapto‐β‐(2‐pyridinyl)‐ethyl). Importantly, our studies show that the auxiliary gains stability when a methoxy substituent is added at the 4‐position of the ortho‐pyridine ring (i. e. meta to the ring nitrogen).Native chemical ligation reactions on β‐mercapto‐β‐(4‐methoxy‐2‐pyridinyl)‐ethyl) auxiliary is conveniently attached to the peptide N‐terminus in the last step of solid‐phase synthesis by reductive alkylation. Despite slightly lowered ligation rates, the MMPyE auxiliary is better suited for NCL at challenging junctions than the MPyE auxiliary becausethe increased stability is a significant advantage when reactions such as the selenoester‐NCLs are performed at slightly acidic conditions. Reactions proceed cleanly also when long reaction times are needed in ligations of large peptides. Furthermore, the increased stability enables ligations at elevated temperature. The usefulness of the MMPyE auxiliary was demonstrated in three applications. The synthesis of a 120 aa long peptide containing 8 MUC5AC tandem repeats and two “sticky” coiled coil peptides involved the smooth ligation of two 60mers via a Ser‐Ala junction. Using a hitherto unexplored ligation site in NCL chemistry and demonstrating the potential for the preparation of post‐translationally modified peptides, we applied the MMPyE auxiliary in a Ser‐Tyr ligation for the synthesis of a His6‐tagged hexaphosphorylated peptide spanning 6 tandem repeats of the RNA polymerase II C‐terminal domain. The rapid synthesis of a Phe‐Tyr‐cyclized 21 aa peptides comprising the primary sequence of microcin J25 illustrates an application of the auxiliary in head‐to‐tail cyclization reactions.The new MMPyE  are stable over a wide pH range, 2) enable native chemical ligations at sterically encumbered junctions, 3) allow removal under mildly basic (pH 8.5) conditions and 4) provide options for attaching further handles by means of 4‐alkoxy substituents. The last option offers prospects for the introduction of functional units such as oligoethylene glycol or oligoarginine units that enhance the solubility of unfolded peptide segments.Ethyl 2‐(pyridin‐4‐yl)‐2‐thio)acetate (S4): Under argon atmosphere, lithium hexamethyldisilazide  was added to a stirred solution of ethyl 2‐(pyridin‐4‐yl)acetate  in 75 mL anhydrous THF over the course of 25 min at −78 °C. After 30 min of stirring, thiosulfonate 38  in 40 mL anhydrous THF was added at −78 °C over 100 min. The reaction mixture was allowed to gradually warm to room temperature over the course of 30 min and a saturated solution of NH4Cl was added. The resultant mixture was extracted with EtOAc (3x) and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (EtOAc) to afford desired product  in 82 % yield as a white solid. 1H NMR : δ [ppm]=8.52 , 7.38 , 6.16 , 4.66 , 4.12 , 3.78 , 3.75 , 3.75 ‐ 3.72 , 1.19 . 13C NMR : δ [ppm]=170.94, 161.94, 159.85, 150.77, 147.17, 124.38, 107.02, 91.64, 62.74, 56.44, 56.06, 52.28, 25.19, 14.32.2‐(Pyridin‐4‐yl)‐2‐thio)acetaldehyde (17): Under argon atmosphere ester S4  was dissolved in 5 mL anhydrous DCM and cooled to ‐94 °C. Diisobutylaluminium hydride  was added carefully over the vessel wall over the course of 1 h. The reaction mixture was stirred for an additional 15 min at −94 °C. Subsequently, a mixture of DCM/MeOH  was added via the vessel wall over the course of 30 min at −94 °C. The reaction mixture was stirred at room temperature for 10 min and a saturated solution of potassium sodium tartrate and DCM (20 ml each) was added. The resulting mixture was stirred at room temperature for 1 h and then extracted with DCM (3x). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (EtOAc) to afford the aldehyde 17 (247 mg) as yellow oil. The material was used for reductive alkylation despite impurities from remaining solvents and partial decomposition. 1H NMR : δ [ppm]=9.52 , 8.52 , 7.27 , 6.10 , 4.62 , 3.78 , 3.77 , 3.75 .Ethyl 2‐(5‐methoxypyridin‐2‐yl)acetate (37): nBuLi  was added dropwise to a stirred solution of diisopropylamine  in 20 ml anhydrous THF at −78 °C under argon. The resulting solution was slowly warmed to room temperature and was added dropwise over 1 h to a stirred solution of 5‐methoxy‐2‐methylpyridine  and anhydrous diethylcarbonate  in 26 ml anhydrous THF at −78 °C. After 2 h of reaction, the solution was slowly warmed to room temperature and a saturated solution of NH4Cl was added. The resultant mixture was extracted with EtOAc (3x), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (cyclohexane/EtOAc=6 : 4 v/v) to afford 37  as a light‐yellow oil. 1H NMR : δ [ppm]=8.25 , 7.28–7.20 , 4.18 , 3.86 , 3.82 , 1.26 . 13C NMR : δ [ppm]=170.91, 154.88, 146.23, 136.13, 124.52, 122.22, 61.17, 55.83, 42.59, 14.28. HRMS: m/z=196.0967 (C10H14NO3 (M+H)+, calcd.: 196.0968).Ethyl 2‐(5‐methoxypyridin‐2‐yl)‐2‐thio) acetate (39): Under argon atmosphere, lithium hexamethyldisilazide  was added to a stirred solution of ethyl 2‐(5‐methoxypyridin‐2‐yl)acetate  in 40 mL anhydrous THF over the course of 25 min at −78 °C. After 30 min of stirring, thiosulfonate 38  in 37 mL anhydrous THF was added at −78 °C within 100 min. The reaction mixture was allowed to gradually warm to room temperature over the course of 30 min and a saturated solution of NH4Cl was added. The resultant mixture was extracted with EtOAc (3x) and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (cyclohexane/EtOAc=1 : 1 v/v) to afford 39  as a white solid. 1H NMR : δ [ppm]=8.15 , 7.43 , 7.27 , 6.17 , 4.75 , 4.12 , 3.83 , 3.78 , 3.76 , 1.19 . 13C NMR : δ [ppm]=171.39, 161.69, 159.74, 155.98, 149.63, 137.36, 124.05, 122.02, 107.32, 91.52, 62.23, 56.37, 56.33, 55.95, 54.59, 25.03, 14.33. HRMS: m/z=408.1455 (C20H26NO6S (M+H)+, calcd.: 408.1475).E)‐2‐(5‐methoxypyridin‐2‐yl)‐2‐thio)ethenol (40) was dissolved in 2.1 mL anhydrous DCM and cooled to ‐94 °C. Diisobutylaluminium hydride  was added carefully via the vessel wall over the course of 1 h. The reaction mixture was stirred for an additional 15 min at −94 °C. Subsequently, a mixture of DCM/MeOH  was added via the vessel wall over the course of 30 min at −94 °C. The reaction mixture was stirred at room temperature for 10 min and a saturated solution of potassium sodium tartrate and DCM (20 ml each) was added. The resulting mixture was stirred at room temperature for 1 h and then extracted with DCM (3x). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by recrystallization (MeOH/H2O) to afford the enol 40  as bright yellow crystals. 1H NMR : δ [ppm]=14.83 , 8.07 , 7.79 , 7.45 , 7.15 , 6.11 , 3.86 , 3.76 , 3.70 , 3.61 . 13C NMR : δ [ppm]=162.21, 161.52, 159.95, 154.14, 153.60, 132.01, 124.77, 122.72, 107.40, 102.62, 91.34, 56.68, 56.29, 55.97, 28.49. HRMS: m/z=364.1201 (C18H22NO5S (M+H)+, calcd.: 364.1213).Peptide Purification: The desired peptides were purified by preparative HPLC on a C18‐column with a binary mixture of A  and B  as mobile phase (flow=6.0 mL/min). For model peptides a linear gradient from 3–30 % B was used. For other peptides a linear gradient was used as indicated. All products were isolated as white solids after lyophilization. Yields were determined by measurements of UV absorption as described in chapter 5.3. of the Suppl. Inf. or by weighing.2)2(CO)‐Gly‐NH2 (19A)LYRAA‐S(CH: Thioester 19A was synthesized according to the published procedure.R=1.39 min (3–30 % B in 4 min); m/z=737.5 (C32H53N10O8S (M+H)+, calcd.: 737.4), 369.3 (C32H54N10O8S (M+2H)2+, calcd.: 369.2); C32H52N10O8S (MW=736.4 g mol−1).2)2(CO)‐Gly‐NH2 (19L)LYRAL‐S(CH: Thioester 19L was synthesized according to the published procedure.R=3.57 min (3–35 % B in 6 min); m/z=779.7 (C35H59N10O8S (M+H)+, calcd.: 779.4), 390.4 (C35H60N10O8S (M+2H)2+, calcd.: 390.2); C35H58N10O8S (MW=778.4 g mol−1).LYRAA‐SePh (22A): Selenoester 22A was synthesized according to the published procedure.R=5.31 min (3–30 % B in 6 min); m/z=733.4 (C33H49N8O6Se (M+H)+, calcd.: 733.3), 367.4 (C33H50N8O6Se (M+2H)2+, calcd.: 367.1); C33H48N8O6Se (MW=732.3 g mol−1).LYRAL‐SePh (22L): Selenoester 22L was synthesized according to the published procedure.R=2.31 min (3‐60 % B in 4 min); m/z=775.4 (C36H55N8O6Se (M+H)+, calcd.: 775.3), 388.3 (C36H56N8O6Se (M+2H)2+, calcd.: 388.2); C36H54N8O6Se (MW=774.3 g mol−1).General Procedure for Introduction of MMPyE Auxiliary: The peptidyl‐resin was allowed to swell in MeOH/NMP/AcOH (6 : 4 : 0.02 v/v/v) for 15 min and then treated with a mixture of the enol 40 and NaCNBH3  in MeOH/NMP/AcOH (3 : 1 : 0.02 v/v/v) over night at room temperature . Afterwards the resin was washed with DCM (5x), MeOH (5x) and DCM (5x) and dried under vacuum. Finally, the peptide was deprotected and cleaved of the resin by addition of a mixture of TFA : TIS : H2O . After 18–24 h the cleavage cocktail was collected by filtration and the resin was washed with TFA (3×0.5 mL). The combined filtrates were concentrated and ice cold Et2O (∼8–10‐fold volume) was added. The suspension was centrifuged  and the ether phase was decanted. For HPLC purification, the peptide pellet was dissolved in H2O : ACN : TFA (1 : 1 : 0.001 v/v/v).pMPyE‐NRAEYSGLG (18N): Synthesis of 16N was achieved by microwave‐assisted solid phase peptide synthesis . The auxiliary was introduced according to the published procedure for the MPyE auxiliary2O/TIS (95 : 2.5 : 2.5 v/v/v) for 18 h. After analyzing the resulting fractions with UPLC‐MS, the auxiliary peptide containing solutions were swiftly frozen for lyophilization to avoid decomposition of the product. Yield: 7.69 mg, 53 %. Note: 18N was stored as dry lyophilizate at −80 °C to avoid auxiliary cleavage . UPLC‐MS: tR=2.87 min (3–30 % B in 6 min); m/z=1102.6 (C47H72N15O14S (M+H)+, calcd.: 1102.5), 552.1 (C47H73N15O14S (M+2H)2+, calcd.: 551.8); C47H71N15O14S (MW=1102.2 g mol−1).MMPyE‐NRAEYSGLG (41N): Yield: 6.55 μmol, 66 %. UPLC‐MS: tR=3.71 min (3–30 % B in 6 min); m/z=1132.6 (C48H74N15O15S (M+H)+, calcd.: 1132.5), 566.9 (C48H75N15O15S (M+2H)2+, calcd.: 566.8); C48H73N15O15S (MW=1132.3 g mol−1).MMPyE‐VRAEYSGLG (41V): Yield: 6.15 μmol, 41 %. UPLC‐MS: tR=4.33 min (3–30 % B in 6 min); m/z=1117.7 (C49H77N14O14S (M+H)+, calcd.: 1117.6), 559.4 (C49H78N14O14S (M+2H)2+, calcd.: 559.3); C48H74N14O13S (MW=1117.3 g mol−1).Ligation of Model Peptides with Peptide Thioester: To the ligation buffer  3 vol.% thiophenol was added and the mixture was transferred to a lyophilized mixture of auxiliary peptide (1 equiv) and peptide thioester (1.5 equiv) to a final concentration of 5 mM auxiliary peptide. An argon atmosphere was applied to the reaction vessel. To monitor the progress of the reaction, aliquots were withdrawn from the ligation mixture, quenched with an aqueous solution of 0.1 % TFA or 0.1 % TFA, 2.5 % hydrazine, 30 mM TCEP and analyzed by UPLC‐MS. The progress of the ligation reaction was assessed by integration of the corresponding peak areas. After completion of the ligation, hydrazine and TCEP were added to the ligation solution. Ligated peptides showed partial rearrangement back to the thioester intermediate via N→S acyl transfer due to the acidic conditions used during and shortly after purification. Swift lyophilization after purification minimizes rearrangement. Alternatively, peptide solutions containing high amounts of rearranged thioesters can be treated with a solution of NH4Ac  to a final pH of 5 to retrieve the amide.Ala‐Asn Ligation on pMPyE (20AN): 2 μmol of para‐MPyE peptide 18N and 3 μmol of peptide thioester 19A were allowed to react. The mixture was analyzed via UPLC‐MS after t=5 min and t=24 h : 2 μmol of MMPyE‐peptide 41N and 3 μmol of peptide thioester 19A were allowed to react for 72 h. Yield: 1.02 μmol, 51 %. UPLC‐MS: tR=4.04 min (3–30 % B in 6 min); m/z=854.0 (C75H118N24O20S (M+2H)2+, calcd.: 853.4), 569.9 (C75H118N24O20S (M+3H)3+, calcd.: 569.3); C75H116N24O20S (MW=1705.9 g mol−1).Leu‐Asn Ligation on MMPyE (42LN): 2 μmol of MMPyE‐peptide 41N and 3 μmol of peptide thioester 19L were allowed to react for 72 h. Yield: 1.14 μmol, 57 %. UPLC‐MS: tR = 5.00 min (3–30 % B in 6 min); m/z=875.0 (C78H124N24O20S (M+2H)2+, calcd.: 874.5), 583.9 (C78H125N24O20S (M+3H)3+, calcd.: 583.3); C78H122N24O20S (MW=1748.0 g⋅mol−1).Ala‐Val Ligation on MMPyE (42AV): 2 μmol of MMPyE‐peptide 41V and 3 μmol of peptide thioester 19A were allowed to react for 72 h. Yield: 1.20 μmol, 60 %. UPLC‐MS: tR=6.07 min (3–30 % B in 8 min); m/z=846.7 (C76H121N23O19S (M+2H)2+, calcd.: 845.9), 565.1 (C76H122N23O19S (M+3H)3+, calcd.: 564.3); C76H119N23O19S (MW=1691.0 g mol−1).Leu‐Val Ligation on MMPyE (42LV): 2 μmol of MMPyE‐peptide 41V and 3 μmol of peptide thioester 19L were allowed to react for 120 h. Yield: 0.58 μmol, 29 %. UPLC‐MS: tR=5.86 min (3–35 % B in 6 min); m/z=867.5 (C76H127N23O19S (M+2H)2+, calcd.: 866.9), 578.9 (C76H128N23O19S (M+3H)3+, calcd.: 578.3); C76H125N23O19S (MW=1733.0 g mol−1).Ligation with Model Peptide Selenoester: A lyophilized mixture of MMPyE peptide (1 equiv) and peptide selenoester (2 equiv) was dissolved in the ligation buffer , pH=6.2) to a final concentration of 5 mM auxiliary peptide. An argon atmosphere was applied to the reaction vessel. To monitor the progress of the reaction, aliquots were withdrawn from the ligation mixture, quenched with an aqueous solution of 0.1 % TFA or 0.1 % TFA, 2.5 % hydrazine, 30 mM TCEP and analyzed by UPLC‐MS. The progress of the ligation reaction was assessed by integration of the corresponding peak areas. After completion of the ligation, hydrazine and TCEP were added to the ligation solution.Ala‐Asn Ligation on MMPyE (→42AN) with Selenoester 22A: 1 μmol of MMPyE‐peptide 41N and 2 μmol of peptide selenoester 22A allowed to react for 24 h. Yield: 0.73 μmol, 73 %. UPLC‐MS: tR=4.05 min (3–30 % B in 6 min); m/z=854.4 (C75H118N24O20S (M+2H)2+, calcd.: 853.4), 570.1 (C75H119N24O20S (M+3H)3+, calcd.: 569.3); C75H116N24O20S (MW=1705.9 g mol−1).Leu‐Val Ligation on MMPyE (→42LV) with Selenoester 22A: 0.5 μmol of MMPyE‐peptide 41V and 1.0 μmol of peptide selenoester 22L were allowed to react for 72 h. Yield: 0.24 μmol, 48 %. UPLC‐MS: tR=5.01 min (3–40 % B in 6 min); m/z=867.9 (C76H127N23O19S (M+2H)2+, calcd.: 866.9), 579.1 (C76H128N23O19S (M+3H)3+, calcd.: 578.3); C76H125N23O19S (MW=1733.0 g mol−1).Auxiliary Cleavage from Model Ligation Products: Lyophilized ligation products were dissolved in the auxiliary cleavage mixture  to a final concentration of 1 mM in plastic tubes. The closed lid was punctured to allow supply of oxygen. Note: Hydrazine is a known oxygen scavenger and auxiliary removal does not tolerate the presence of hydrazine.Removal of Auxiliary from Ligation Product AN (51AN): 500 nmol of ligated peptide 42AN was incubated in the cleavage mixture for 3 h. Yield: 250 nmol, 50 %. UPLC‐MS: tR=4.44 min ; m/z=770.7 (C67H108N22O20 (M+2H)2+, calcd.: 770.4), 514.2 (C67H109N22O20 (M+3H)3+, calcd.: 513.9); C67H106N22O20 (MW=1538.8 g mol−1).Removal of Auxiliary from Ligation Product AV (51AV): 500 nmol of ligated peptide 42AV was incubated in the cleavage mixture for 6 h. Yield: 180 nmol, 36 %. UPLC‐MS: tR=3.85 min (then 3–30 % B in 6 min); m/z=763.2 (C68H111N21O19 (M+2H)2+, calcd.: 762.9), 509.2 (C68H112N21O19 (M+3H)3+, calcd.: 508.9); C68H109 N21O19 (MW=1523.8 g mol−1).Removal of Auxiliary from Ligation Product LN (51LN): 260 nmol of ligated peptide 42LN was incubated in the cleavage mixture for 3 h. Yield: 104 nmol, 40 %. UPLC‐MS: tR=5.16 min ; m/z=791.7 (C70H114N22O20 (M+2H)2+, calcd.: 791.4), 528.1 (C70H115N22O20 (M+3H)3+, calcd.: 527.9); C70H112N22O20 (MW=1580.8 g mol−1).Removal of Auxiliary from Ligation Product LV (51LV): 300 nmol of ligated peptide 42AN was incubated in the cleavage mixture for 6 h. Yield: 109 nmol, 36 %. UPLC‐MS: tR=4.14 min (3–30 % B in 6 min); m/z=784.5 (C71H117N21O19 (M+2H)2+, calcd.: 783.9), 523.1 (C71H118N21O19 (M+3H)3+, calcd.: 523.0); C71H115N21O19 (MW=1566.8 g mol−1).4 Thioester EIQALEEENAQLEQENAALEEEIAQLEY‐(APTTSTTS): The peptide was synthesized on a rink amide resin pre‐loaded with Hnb‐(StBu)Cys‐Gly via automated microwave assisted peptide‐synthesis . The peptide resin was treated with H2N−OH ⋅ HCl , washed and deprotected with TFA : phenol : H2O : TIS (88 : 5 : 5 : 2 v/v/v/v) for 2 h. The precipitated crude peptide was shaken at 37 °C in an aqueous buffer  containing 10 vol.% MPA. The reactions was quenched with 10 vol.% TFA. Preparative HPLC: 15–35 % B in 40 min. Yield: 0.43 μmol, 31 %, UPLC: tR=5.95 min (15–35 % B in 8 min). The product was submitted to ligation without further characterization.4‐KIAQLKQKIQALKQENQQLEEENAALEY MMPyE‐(APTTSTTS): Synthesis was performed on a rink amide resin via automated microwave assisted peptide‐synthesis . For reductive alkylation 10 equiv 40 and 30 equiv NaCNBH3 were allowed to react for 24 h. Preparative HPLC: 15–35 % B in 40 min. Yield: 771 nmol, 57 %. UPLC: tR=4.27 min (15–35 % B in 8 min). The product was submitted to the ligation without further characterization.Ligation of 53 and 54 (→55): Peptide thioester 53 (315 nmol) and auxiliary peptide 54 (210 nmol) were dissolved in ligation buffer  and allowed to react for 120 h. Yield: 53 nmol, 25 %. UPLC: tR=5.87 min (15–35 % B in 8 min). The product was submitted to auxiliary cleavage without further characterization.8‐P4 (56)P1‐Muc5AC: Ligation product 55 (18 nmol) was incubated in the cleavage mixture for 4 h. Preparative HPLC: 15–35 % B in 40 min. Yield: 9.5 nmol, 53 %. UPLC: tR=6.6 min (15–35 % B in 8 min); HRMS: m/z=1786.9876 (C522H862N139O214 (M+11H)7+, calcd.: 1786.7282), 1563.5361 (C522H863N139O214 (M+11H)8+, calcd.: 1563.5131), 1389.9659 (C522H864N139O214 (M+11H)9+, calcd.: 1389.9013), 1251.0593 (C522H865N139O214 (M+11H)10+, calcd.: 1251.0119), 1137.4254 (C522H866N139O214 (M+11H)11+, calcd.: 1137.3751), 1042.6994 (C522H867N139O214 (M+12H)12+, calcd.: 1042.6778).Synthesis of phosphorylated MMPyE‐CTD Peptide 57: Synthesis was performed on a Rink amide resin via MW‐SPPS . For reductive alkylation 7 equiv 40 and 16 equiv NaCNBH3 were allowed to react for 18 h. The peptide was cleaved for 6 h with TFA:H2O:phenol:EDT  protecting group to avoid formation of cleavage side‐product). Preparative HPLC: 3–40 % B in 40 min. Yield: 3.46 μmol, 35 %. UPLC‐MS: tR=3.45 min (3–30 % B in 4 min); m/z=1172.4, (C104H148N23O37S (M+3H)3+, calcd.: 1172.5), m/z=783.3, (C104H148N23O37S (M+4H)4+, calcd.: 783), C104H150N23O46P3S (MW=2343.01 g mol−1). UPLC‐MS: tR=2.53 min (3–30 % B in 4 min); m/z=862.0, (C104H150N23O46P3S (M+3H)3+, calcd.: 861.6), C104H150N23O46P3S (MW=2581.9 g mol−1).6‐[(CH2)2O]6CH2CO‐(YpSPTSPS)3 Hydrazide (58)His: The peptide was assembled on a chlorotriyl resin pre‐loaded with Fmoc‐hydrazine via automated solid‐phase peptide synthesis . After introduction of the PEG6‐chain (Fmoc‐HN‐[(CH2)2O]6‐COOH (5 equiv), HATU  and DIPEA (15 equiv) in DMF), capping and coupling of six histidines, the peptide was cleaved with TFA:H2O:phenol:EDT  for 3 h. Preparative HPLC 3–40 % B in 40 min. Yield: 1.86 μmol, 19 %; 58: 2.15 μmol, 22 %. UPLC‐MS (58): tR=2.08 min (3‐50 % B in 4 min); m/z=1197.3, (C147H213N42O58P3 (M+3H)3+, calcd.: 1196.8), 898.8 (C147H210N42O49 (M+4H)4+, calcd.: 897.9), 719.2 (C147H210N42O49 (M+5H)5+, calcd.: 718.5).Formation of Thioester 59, Ligation with 57 and Removal of MMPyE Group (60): Peptide hydrazide 58  was dissolved in H2O containing 200 mM MPAA and 6 M Gdn HCl (pH=3) and acetylacetone  was added. After 1 h reaction at room temperature, the solution was transferred to a mixture of auxiliary peptide 57  in 100 mM TCEP, 200 mM Na2HPO4 and 6 M Gdn HCl (pH=8.5). NaOH (5 M) was added carefully by alternating addition over the vessel cap followed by vortexing to adjust the pH to 6.3. After 120 h, the ligation was quenched by the addition of 5 μl hydrazine solution (81 % in H2O) and 10 μl of TCEP (1 M in H2O) and the resulting mixture was subjected to an Amicon Ultra Mass Filter from Merck Millipore  and washed twice. After lyophilization, the crude peptide mixture was dissolved in 100 μl phosphate buffer  and purified via a His‐Tag kit using Ni2+ Agarose‐NTA beads. The resulting peptide solution was lyophilized and used i without further purification. The peptide mixture obtained after lyophilization was dissolved in 200 μL of the auxiliary removal mixture  for 24 h. Preparative HPLC: 3–40 % B in 40 min. Yield: 18 nmol, 4 % over 3 steps. UPLC‐MS: tR=5.47 min (5‐30 % B in 8 min); m/z=1195.1 (C243H350N62O103P6 (M+5H)5+, calcd.: 1195.1), 996.9 (C243H350N62O103P6 (M+6H)6+, calcd.: 996.0), m/z=854.1 (C243H350N62O103P6 (M+7H)7+, calcd.: 853.9) m/z=745.9 (C243H350N62O103P6 (M+8H)8+, calcd.: 747.3) m/z=664.3 (C243H350N62O103P6 (M+9H)9+, calcd.: 664.4), (C243H350N62O103P6 (MW=5970.3).Synthesis of MMPyE‐peptide 62, Formation of Thioester 63 and Cyclization (64): Microwave assisted peptide‐synthesis was performed on a pre‐loaded chlorotrityl‐tentagel resin . For reductive alkylation 7 equiv of 40 and 16 equiv of NaCNBH3 were allowed to react for 18 h. Approx, 1.0 μmol MMPyE‐peptide 62 was cleaved from the resin with hexafluoroisopropanol/DCM (7 : 3 v/v) and thioester 63 was formed according to the general procedure . The peptide was cleaved for 18 h with TFA, H2O, phenol, thioanisole, EDT (82 : 5 : 5 : 5 : 3 v/v/v/v/v) and incubated in ligation buffer at 37 °C for 2 h. Preparative HPLC: 20–50 % B in 40 min. Yield: 89 nmol, 9 %. UPLC‐MS: tR=4.88 min (20–50 % B in 8 min); m/z=1138.2, (C109H150N24O28S (M+2H)2+, calcd.: 1138.0), 758.9 (C109H151N24O28S (M+3H)3+, calcd.: 759.0), C109H148N24O28S (MW=2274.55 g⋅mol−1).Cyclopeptide 65: 55 nmol of ligation product 64 was incubated in the cleavage mixture for 12 h. Preparative HPLC: 20–50 % B in 40 min. Yield: 24 nmol, 44 %. UPLC‐MS: tR=3.93 min (20–50 % B in 8 min); m/z=1054.42 (C101H141N23O27 (M+2H)2+, calcd.: 1054.02), 703.01 (C101H142N23O27 (M+3H)3+, calcd.: 703.35), C101H139N23O27 (MW=2107.32). MALDI‐MS: m/z=2104 (C101H140N23O27 (M+H)+, calcd.: 2108).More detailed procedures and characterization data is provided in the Supporting Information.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


In the crystal, these components are organized in infinite zigzag chains via intermolecular hydrogen bonds. Weak interactions between the chains lead to a three-dimensional network.The title salt consists of three components, comprising one dibenzylammonium cation, [(C 14H16N+·C6H7AsNO3−·H2O or [(C6H5CH2)2NH2][H2NC6H4As(OH)O2]·H2O, (I), was synthesized by mixing an aqueous solution of (4-aminophenyl)arsonic acid with an ethanolic solution of dibenzylamine at room temperature. Compound I crystallizes in the monoclinic P21/c space group. The three components forming I are linked via N—H⋯O and O—H⋯O intermolecular hydrogen bonds, resulting in the propagation of an infinite zigzag chain. Additional weak interactions between neighbouring chains, such as π–π and N—H⋯O contacts, involving phenyl rings, –NH2 and –As(OH)O3 functions, and H2O, respectively, lead to a three-dimensional network.The title salt, C This compound was employed for pharmaceutical applications, in particular against trypanosomal infection. Subsequently, in the early 20th century, Paul Ehrlich was inspired by this work to develop a new organoarsenic derivative, called Arsphenamine or Salvarsan  has been determined 2NH2][H2NC6H4As(OH)O2]·H2O (I).From a structural point of view, the crystal structure of phenylarsonic acid was first solved in the early 1960s 2NH2]+, one hydrogen (4-aminophenyl)arsonate anion [H2NC6H5As(OH)O2]− and one water molecule of solvation. The three components of I are linked together through intermolecular N—H⋯O and O—H⋯O hydrogen bonds. The As atom of the anion is bonded to three O atoms and one carbon atom of the phenyl ring, describing a slightly distorted tetrahedral geometry . The As—O bonds exhibit two distinct lengths: As—O1 = 1.7267 (10) Å, and As—O2 = 1.6730 (10) Å and As—O3 =1.6699 (10) Å, which can be considered to be identical. The As—O1 distance is consistent with the presence of a hydroxyl group  Å; symmetry code: (iv) −x, −y + 1, −z + 1, Table 1et al., 2002para position to the arsonate function. Both functional groups are contained in the plane of the phenyl ring. The negative charge of [H2NC6H4As(OH)O2]− is compensated by the presence of one dibenzylammonium cation, [(C6H5CH2)2NH2]+, whose NH2+ group is hydrogen bonded to the oxygen atom O3 of the arsonate function . Moreover, the dibenzylammonium cation shows a syn–anti conformation, displaying C—C—N—C torsion angles of 57.65 (16)° and −178.14 (11)°, which are in the range of previous examples of X-ray structures involving [(C6H5CH2)2NH2]+  Å; symmetry code: (v) 1 + x, y, z] completes the composition of salt I. From a spectroscopic point of view, the infrared spectrum of I (ATR mode) highlights ν(As—C) and ν(As—O) absorption bands, which are characteristic of the arsonate function .The asymmetric unit of the title salt, which is depicted in Fig. 13.I:At the supramolecular stage, two levels of organization can be observed in the crystal structure of a-axis direction resulting from the hydrogen-bonding interactions °], with the oxygen atoms O3 of two arsonate moieties  and the O1 oxygen atom of an adjacent –As(OH)O2 function [N2—H2B⋯O1ii = 3.0769 (17) Å] (symmetry codes as in Table 1(ii) The association of chains leading to a three-dimensional network and resulting from a combination of weak interactions Fig. 4. Two typ4.et al., 20166H5As(OH)O2−. To our knowledge, eleven examples including this fragment have already been identified: ammonium 4-nitrophenylarsonate 2NH2]+, 117 hits incorporating such an entity were found in the Cambridge Structural Database.A search of the Cambridge Structural Database 2O] was prepared according to a previous work  and arsenic acid (As(OH)3O). The title salt was obtained by neutralization of an aqueous solution (20 mL) of (4-aminophenyl)arsonic acid  with dibenzylamine ((C6H5CH2)2NH)  dissolved in 20 mL of ethanol. The mixture was stirred for about two h at room temperature (301 K). After three days of slow solvent evaporation, colourless prism-shaped crystals of [(C6H5CH2)2NH2][H2NC6H4As(OH)O2]·H2O , suitable for an X-ray crystallographic analysis, were collected from the solvent (m.p. 393 K). FT–IR : 3447, 3304, 3187, 1595, 1501, 1454, 1096, 923, 878, 825,752, 735, 695. Elemental analysis  – analysis calculated for C20H23N2O3As·0.25H2O (418.83), salt I partially dehydrated: C, 57.35; H, 5.66; N, 6.69; O, 12.41; found: C, 57.82; H, 5.61; N, 6.62; O, 12.37%.All chemicals were purchased from Sigma-Aldrich (Germany) and used without any further purification. (4-Aminophenyl)arsonic acid [H6.Uiso(H) = 1.5Ueq(O). C-bound hydrogen atoms were placed at calculated positions [C—H = 0.95 Å (aromatic) or 0.99 Å (methylene group)] and H atoms of the NH2 and OH terminal groups were placed geometrically  and refined as riding with Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902300837X/dj2065sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698902300837X/dj2065Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902300837X/dj2065Isup3.cmlSupporting information file. DOI: 2297206CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The magnesium cation exhibits a distorted octahedral coordination with two bidentate dimethoxyethane molecules and a dinuclear aluminate anion, coordinated to Mg2+via oxido and hydroxido units. The anion is an oxido-bridged species, [HOAl(hfip)2(μ-O)Al(hfip)3]2–, with one Al3+ cation tetrahedrally coordinated by an oxido (O2–) anion, a hydroxido anion, and two hfip groups, whereas the second Al3+ cation is coordinated by the oxido anion and three hfip groups.Partial hydrolysis of a sample of [Mg(dme) The nearly right angle involves the oxido and hydroxido groups (O—Al1—OH). The anion coordinates to the magnesium cation via hydroxido and oxido units, thus making these ligands μ- and μ3-bridges, respectively, resulting in an Mg1⋯Al1 distance of 2.8074 (3) Å. The angles at the hydroxido and oxido bridges between Al1 and Mg1 are similar , whereas the angles involving μ3-oxido and Al2 are more obtuse . The Mg2+ cation has a distorted octahedral coordination with cis-O—Mg—O angles ranging from 76.40° (2) to 101.00 (2)° and trans-O—Mg—O angles in the range 164.77 (3)–168.38 (3)°. The O—Mg—O bite angles of the dme ligands [76.40 (3) and 77.08 (3)°] and the anion [76.99 (2)°] are nearly identical.The title compound crystallizes in the monoclinic space group ly Fig. 1. In the Å] Fig. 1. The tet2(CF3SO3)2] 2 –1.7444 (10) Å in NMe4[Al(hfip)4] 4] , 2.1146 (12) Å, 77.49 (5)° in  sample 3][Al(hfip)4]2 + 2H2O → [Mg(dme)2{HOAl(hfip)2OAl(hfip)3}] + dme + 3hfipH.[Mg(dme)et al., 2010The crystal data, data collection, and structure refinement details are summarized in Table 210.1107/S2414314623007162/hb4443sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623007162/hb4443Isup2.hklStructure factors: contains datablock(s) I. DOI: 2156683CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The two compounds have a structural overlap r.m.s. deviation of 0.27 Å. The pyran and seven-membered cycloheptene rings in both structures adopt boat and boat-sofa conformations, respectively. Intra- and intermolecular C—H⋯O hydrogen bonds are responsible for the consolidation of the crystal packing of both molecules. In addition to this, weak C—H⋯π interactions are also observed. The intermolecular interactions were quantified and analysed using Hirshfeld surface analysis.The title compounds, C It is the fundamental component of many beneficial alkaloids, including atropine, scopolamine, and cocaine, whose derivatives are important in the treatment of neurological and psychiatric conditions such depression and panic disorder q2 = 1.021 (2) and q3 = 0.391 (2) Å in (I)q2 = 1.053 (2) and q3 = 0.374 (2) Å in (II)q2 = QT = 0.185 (2) Å and φ = 43.9 (1)° [in (I)] and q2 = QT = 0.087 (1) Å and φ = 47.1 (1)° [in (II)]. The cyclohexene ring (C15–C20) has a distorted sofa conformation in both (I)sC(C17) asymmetry parameters  in both compounds has a boat-sofa conformation, with puckering parameters , see Fig. 4In the crystal of (I)via C—H⋯O intermolecular interaction , see Fig. 4In the crystal of (II)if Fig. 5. The dim4.Crystal Explorer 21 et al., 2021To further characterize the intermolecular interactions in the title compound, we carried out a Hirshfeld surface (HS) analysis Compound (II)6.Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698902300275X/zn2026sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S205698902300275X/zn2026Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698902300275X/zn2026IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2251053, 2251052CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The layers are further connected by π–π stacking interactions.In the crystal of the title compound, C—H⋯O hydrogen bonds link the molecules, enclosing  14H11NO3, the dihydroquinoline core deviates slightly from planarity, indicated by the dihedral angle of 1.07 (3)° between the two six-membered rings. In the crystal, layers of molecules almost parallel to the bc plane are formed by C—H⋯O hydrogen bonds. These are joined by π–π stacking interactions. A Hirshfeld surface analysis revealed that the most important contributions to the crystal packing are from H⋯H (36.0%), H⋯C/C⋯H (28.9%) and H⋯O/O⋯H (23.5%) interactions. The evaluation of the electrostatic, dispersion and total energy frameworks indicates that the stabilization is dominated by the dispersion energy contribution. Moreover, the molecular structure optimized by density functional theory (DFT) at the B3LYP/6-311G level is compared with the experimentally determined molecular structure in the solid state. The HOMO–LUMO behaviour was elucidated to determine the energy gap.In the title molecule, C Moreover, a Hirshfeld surface analysis and interaction energy and energy framework calculations were performed. The molecular structure optimized by density functional theory (DFT) at the B3LYP/6-311G level is compared with the experimentally determined molecular structure in the solid state.2.A (C1–C5/N1) and B (C4–C9) rings. Atoms O1, O2, O3, C10, C13 and C14 are −0.1294 (11), 0.1907 (12), −0.2708 (15), 0.0177 (14), −0.0267 (13) and 0.0953 (23) Å from the least-squares plane of the A ring. The O2—C13 [1.3123 (17) Å] and O3—C13 [1.1955 (16) Å] distances in the ester group indicate localized single and double bonds, rather than delocalized bonding arrangements. The O2—C13—O3 bond angle [122.55 (12)°] seems to be slightly increased with respect to that present in a free acid bis(nicotinamide-κN1)zinc(II)  rings  to form a triperiodic network.In the crystal, C—H⋯O hydrogen bonds Table 1 link thene Fig. 2. These l4.CrystalExplorer ] and those delineated into H⋯H, H⋯C/C⋯H, H⋯O/O⋯H, C⋯C, C⋯O/O⋯C, H⋯N/N⋯H, C⋯N/N⋯C and N⋯O/O⋯N contacts –(i), respectively, together with their relative contributions to the Hirshfeld surface. The most important interaction is H⋯H, contributing 36.0% to the overall crystal packing, which is reflected in Fig. 6b) as widely scattered points of high density due to the large hydrogen content of the molecule with the tip at de = di = 1.22 Å. In the absence of C—H⋯π interactions, the pair of characteristic wings resulting in the fingerprint plot delineated into H⋯C/C⋯H contacts [Fig. 6c)] have a 28.9% contribution to the HS, with the tips at de + di = 2.68 Å. The pair of the scattered points of spikes resulting in the fingerprint plot delineated into H⋯O/O⋯H contacts [Fig. 6d)], with a 23.5% contribution to the HS, has an almost symmetric distribution of points, with the tips at de + di = 2.44 Å. The C⋯C contacts [Fig. 6e)] appear as an arrow-shaped distribution of points and have a contribution of 7.0% to the HS with the tip at de = di = 1.69 Å. The tiny spikes of C⋯O/O⋯C contacts [Fig. 6f)], with a 2.5% contribution to the HS, are visible at de + di = 3.58 Å. Finally, the H⋯N/N⋯H [Fig. 6g)], C⋯N/N⋯C [Fig. 6h)] and N⋯O/O⋯N [Fig. 6i)] contacts contribute 1.4, 0.4 and 0.4%, respectively, to the HS.In order to visualize the intermolecular interactions in the crystal of the title compound, a Hirshfeld surface (HS) analysis –(c), respectively.The Hirshfeld surface representations with the function et al., 2015The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions suggest that van der Waals interactions play the major role in the crystal packing  is the sum of electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange–repulsion (Erep) energies , Edis (green cylinders) and Etot (blue cylinders), and are shown in Figs. 8a)–(c). The evaluation of the electrostatic, dispersion and total energy frameworks indicates that in the title compound the stabilization is dominated by the dispersion energy contribution.Using 6.GAUSSIAN09  using the standard B3LYP functional and the 6311G basis set  derived from the conceptual DFT calculations are given in Table 3E = ELUMO − EHOMO] , with layer-by-layer connections approximately parallel to  and tetra-n-butylammonium bromide . The reaction mixture was stirred at room temperature in DMF for 6 h. After removal of the formed salts, the solvent was evaporated under reduced pressure and the residue obtained was dissolved in dichloromethane. The organic phase was dried over Na2SO4 and then concentrated in vacuo. A pure compound was obtained after recrystallization from dichloromethane/hexane (2:3 v/v).To a solution of methyl 2-oxo-1,2-dihydroquinoline-4-carboxylate (4.47 mmol) in 10 ml of dimethylformamide (DMF) were added propargyl bromide (9.83 mmol), K10.1107/S2056989023007557/wm5688sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989023007557/wm5688Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023007557/wm5688Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023007557/wm5688Isup4.cdxSupporting information file. DOI: 2291603CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Gγ- and Aγ-globin genes were examined by DNA analysis and rapid diagnosis of HPFH mutations were developed by PCR-based methods. Twenty subjects with five different mutations were identified including three known mutations, − 202 Aγ (C>T) (n = 3), − 196 Aγ (C>T) (n = 3), and − 158 Aγ (C>T) (n = 12), and two novel mutations, − 117 Aγ (G>C) (n = 1) and − 530 Gγ (A>G) (n = 1). Interaction of the − 117 Aγ (G>C) and Hb E (HBB:c.79G>A) resulted in elevation of Hb F to the level of 13.5%. Two plain heterozygous subjects with − 530 Gγ (A>G) had marginally elevated Hb F with 1.9% and 3.0%, whereas the proband with homozygous − 530 Gγ (A>G) had elevated Hb F of 11.5%. Functional prediction indicated that the − 117 Aγ (G>C) and − 530 Gγ (A>G) mutations dramatically alter the binding of transcription factors to respective γ-globin gene promotors, especially the CCAAT and GATA-1 transcription factors. Diverse heterogeneity of non-deletional HFPH with both known and new mutations, and complex interactions of them with other forms of thalassemia are encountered in Thai population.High Hb F determinants are genetic defects associated with increased expression of hemoglobin F in adult life, classified as deletional and non-deletional forms. We report the first description of non-deletional hereditary persistence of fetal hemoglobin (HFPH) in Thailand. Study was done on 388 subjects suspected of non-deletional HPFH with elevated Hb F expression. Mutations in the  In Thailand, high prevalence of hemoglobinopathies has been documented including 20–30% of α-thalassemia, 3–9% of β-thalassemia, 20–30% of Hb E (HBB:c.79G>A), 1–8% of Hb Constant Spring (HBA2:c.427T>C) and Hb Pakse′ (HBA2:c.429A>T), and other structural Hb variants and high Hb F determinants. These lead to diverse heterogeneity and complex thalassemia syndromes in the country5. Accurate diagnosis of these hemoglobinopathies is important for promoting appropriate management, genetic counseling, and a prevention and control program of thalassemia in the region.The inherited disorders of hemoglobin (Hb) or hemoglobinopathies are the commonest human monogenic diseases, found in about 7% of the world population. These can be broadly classified into 3 groups, structural Hb variants, thalassemia and hereditary persistence of fetal hemoglobin (HPFH)7. Since co-inheritance of these high Hb F determinants in β-thalassemia disease has an ameliorating effect on the disease severity, identification of them in routine practice is important. At the molecular level, they could be classified into deletional and non-deletional high Hb F determinants. While the former involves several large DNA deletions removing δ- and β-globin genes, the latter is usually caused by point mutations on Gγ- or Aγ-globin gene promoters affecting the binding of related transcription factors5.During early childhood, the level of Hb F normally declines to less than 1–2% of total Hb. The level of Hb F is therefore less than 1–2% in normal adult. High Hb F determinants are a group of genetic defects associated with increased expression of Hb F in adult life. The conditions include δβ-thalassemia, γδβ-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). While δβ-thalassemia and γδβ-thalassemia are usually associated with elevated Hb F and hypochromic microcytic red blood cell indices, HPFH is associated with higher Hb F expression with normal red blood cells. Homozygous or compound heterozygous for δβ-thalassemia and β-thalassemia may have clinical phenotype of non-transfusion-dependent thalassemia or transfusion-dependent thalassemia. In contrast, homozygous HPFH or compound heterozygous HPFH and β-thalassemia usually have no clinical symptom. However, these clinical phenotypes are generally overlapped which renders their differentiation in routine setting difficult unless DNA analysis is performed0-thalassemia (12.6 kb deletion) (NG_000007.3:g.64383_76994), Indian deletion-inversion Gγ(Aγδβ)0-thalassemia , Chinese Aγδβ0-thalassemia (78.9 kb deletion) (NG_000007.3:g.48795_127698del), δβ0-thalassemia (11.3 kb deletion) (NG_000007.3:g.60045_71313delinsTACATTAAGAGATACCTTAATG), Siriraj Aγδβ0-thalassemia (~ 118 kb deletion) (AC104389.8:g.52507_165744), Thai deletion-inversion-insertion Aγδβ0-thalassemia  and Vietnamese/SEA HPFH (27 kb deletion) (NG_000007.3:g.64384_76993del)10. However, non-deletional form of HFPH has rarely been described. This study provides for the first time the molecular description of non-deletional HFPH in Thailand including known and novel mutations affecting the CCAAT and GATA-1 transcription factors binding.In Thailand, several deletional forms of δβ-thalassemia and HPFH have been documented. These included HPFH-6 (79.3 kb deletion) (NG_000007.3:g.45595_124872), δβ10. These included subjects with normal β-globin gene but Hb F ≥ 1.0% (n = 81), Hb E heterozygotes with Hb F ≥ 1.0% (n = 287), β-thalassemia carriers with Hb F>10% (n = 6), and homozygous Hb E with Hb F>20% (n = 14). For comparison, normal individuals with Hb F < 1.0% (n = 60) were also analyzed. Hematological data is routinely recorded on standard automated blood cell counter. Hb analysis is performed using automated capillary electrophoresis system  or automated high-performance liquid chromatography (HPLC) .This study was conducted in accordance with the Declaration of Helsinki and ethical approval of this study protocol was obtained from the Institutional Review Board of Khon Kaen University, Thailand (HE652154). Left-over blood specimens of 388 subjects suspected of non-deletional HPFH were selectively recruited at our routine thalassemia diagnostic service at Khon Kaen University, Thailand. They were all negative for deletional forms of high Hb F determinants previously found in ThailandSEA, --THAI, -α3.7, -α4.2, Hb Constant Spring, and Hb Pakse′), β-thalassemia, deletional high Hb F determinants, and Krüppel-like factor 1 (KLF1) mutations found in Thai population were identified by PCR-based methods as described previously14. Gγ- and Aγ-globin promoter mutations were examined by DNA sequencing on ABI PRISM™ 3730 XL analyzer  using primers F35 and γ515, and F229 and γ3516. Sequences of all primers used in this study are listed in the Supplementary Table Common α-thalassemia (--Gγ- globin promoter (C>T) (HBG2:c.− 211C>T) and − 158 Aγ-globin promoter (C>T) (HBG1:c.− 211C>T) was developed as shown in Supplementary Fig. Gγ- and Aγ-globin gene promoters was done using primers γ415 and γ5 (577 bp in length), and F22 & γ5 (639 bp in length), respectively. With this assay, the amplified product was digested to completion with XmnI restriction enzyme (5′-GAANN▼NNTTC-3′) . After digestion, the 577 bp of − 158 Gγ-globin promoter with T allele [XmnI (+)] was digested into two fragments with 402 bp and 175 bp in lengths, and the 639 bp fragment for the − 158 Aγ- globin promoter with T allele [XmnI (+)] was digested into two fragments with 464 bp and 175 bp in lengths. The − 158 Gγ- and Aγ-globin promoters with C allele [XmnI (−)] remain undigested16.The PCR-restriction fragment length polymorphism (PCR–RFLP) assay for detection of − 158 Aγ-globin (C>T) (HBG1:c.− 255C>T)  (HBG1:c.− 249C>T)  (HBG1:c.− 170G>C)  (HBG2:c − 583A>G) , − 196 Aγ-globin (C>T), and − 117 Aγ-globin (G>C) mutations. Similarly, primers (F40 & γ5) were used to produce the 795 bp internal control fragment for − 530 Gγ-globin (A>G) mutation. Specific primer pairs (F38 & F22), (F39 & F22), (G211 & γ5), and (F41 & F40) were used to generate specific fragments of 444, 450, 150, and 276 bps in length for the − 202 Aγ-globin (C>T), − 196 Aγ-globin (C>T), − 117 Aγ-globin (G>C), and − 530 Gγ-globin (A>G), respectively. The PCR mixture (50 μl) contains 50–200 ng genomic DNA, 30 pmol each primer, 200 μM dNTPs and 1 unit Taq DNA polymerase  in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin and 3 mM MgCl2. The amplification reaction was carried out on a T-Personal Thermocycler . A total of 30 cycles after initial heating at 94 °C for 3 min was performed under the following PCR condition: 93 °C 30 s, 62 °C 30 s, and 72 °C for 1 min. The amplified PCR product was separated on 2.0% agarose gel electrophoresis and visualized under UV light after ethidium bromide staining.Allele-specific PCR assays were developed for rapid identification of four non-deletional HPFH mutations including − 202 HincII, Gγ-HindIII, Aγ-HindIII, ψβ-HincII, 3′ψβ-HincII, β-AvaII and 3′β-BamHI were determined using PCR–RFLP assays as described previously17.Seven polymorphic restriction sites on β-globin gene cluster including ε-https://tfbind.hgc.jp/)18 was used to compare the binding affinity of transcription factors to the promoters of γ-globin genes with wild-type sequences and their mutant counterparts including − 117 Aγ (G>A), − 117 Aγ (G>C), − 114 Aγ (C>T), − 530 Gγ (A>G), and − 533 to − 529 (-ATAAG) mutations.The online TFBIND software (Gγ- and Aγ-globin genes was selectively carried out in 177 subjects with Hb F>5.0%. Fourteen subjects (7.9%) with five mutations in the γ-globin gene promoters were identified including three known mutations, − 202 Aγ (C>T) (n = 3), − 196 Aγ (C>T) (n = 3), and − 158 Aγ (C>T) (n = 6), and two novel mutations, − 117 Aγ (G>C) (n = 1) and − 530 Gγ (A>G) (n = 1). All mutations but the − 530 Gγ (A>G) were identified in heterozygotic form. Rapid PCR diagnosis based on PCR–RFLP assays  mutation on PCR–RFLP based assay. No mutation was detected in 60 normal subjects with Hb F < 1.0%. Therefore, a total of 20 of 388 (5.1%) recruited subjects were found to carry HPFH mutations. These included in total − 202 Aγ (C>T) (n = 3), − 196 Aγ (C>T) (n = 3), − 158 Aγ (C>T) (n = 12), − 117 Aγ (G>C) (n = 1) and − 530 Gγ (A>G) (n = 1). As shown in Table Aγ (C>T) HPFH mutation and Hb F 27.1% was a boy (2-yr-old) who was found to be a carrier of β0-thalassemia (β17(AAG>TAG), HBB:c.52A>T) and α+-thalassemia (3.7 kb deletion). This boy was therefore a triple heterozygote for Aγ-HPFH, β0-thalassemia and α+-thalassemia, a hitherto undescribed complex condition. Extended family analysis indicated that the − 158 Aγ (C>T) mutation was detected in trans to the β0-thalassemia gene. He had Hb A2 of 4.3%, still within diagnostic range for a β-thalassemia trait.Among 388 subjects recruited DNA sequencing of +-Thalassemia was detected in 8 of 20 HPFH subjects with relatively decreased Hb F levels as compared to those with the same HPFH mutations but without α-thalassemia. The overall hematological parameters, Hb analysis results as well as α- and β-globin genotypes of these 20 non-deletional HPFH were summarized in Table No mutation in both γ-globin genes was identified in subjects with homozygous Hb E with unusually high Hb F level. Further screening for the KLF1 mutations previously described in Thai population showed that 3 of these 20 subjects with HPFH  carried the KLF1 mutations i.e., H299D (NG_013087.1:g.6869C>G) (n = 1), T334R (NG_013087.1:g.7231C>G) (n = 1) and G176Afs*179 (NG_013087.1:g.6493_6499dupCGGCGCC) (n = 1). αGγ (A>G) novel mutation is located at the GATA-1 transcription factor binding motif of Gγ-globin gene promoter and is identified in homozygotic form, further family analysis was carried out as shown in Fig. Gγ (A>G) mutation. Apparently, heterozygosity for the − 530 Gγ (A>G) mutation is associated with normal hematological features and marginally elevated Hb F (1.9% in the mother and 3.0% in the sister). The father had alternatively reduced Hb F (0.3%) due to a co-inheritance of α+-thalassemia (3.7 kb deletion) since he was a double heterozygote for Gγ-HPFH and α+-thalassemia. In all members, the levels of Hb A2 were within normal range (2.4–2.6%). The proband’s wife (27-year-old) was a patient with Hb E-β+-thalassemia with mild hypochromic microcytic anemia with Hb 10.1 g/dL, MCV 52.7 fL and MCH 18.6 pg. Therefore, they were at risk of having a child with compound Gγ-HPFH/β+-thalassemia or Gγ-HPFH/Hb E syndrome. In addition, screening for KLF1 mutations previously described in Thai population14 yielded negative results in all members. β-Globin gene haplotype analysis on 7 polymorphic restriction sites within β-globin gene cluster as described in the “Gγ (A>G) mutation was linked to the β-globin gene haplotype (- ++-+-+) in this Thai family and pointed to the same origin of this Gγ-HPFH in the father and the mother. Appropriate genetic counselling was provided to the family members.Since the − 530 0-thalassemia and Aγδβ0-thalassemia. At least 7 different deletional forms of HPFH and δβ0-thalassemia have been characterized in Thailand10. In contrast, the molecular basis of non-deletional HPFH in Thailand has not been investigated. This study represents the first extensive study of non-deletional HPFH among Thai population. At present, around 30 non-deletional HFPH mutations on both Gγ- and Aγ-globin genes have been characterized worldwide. These include 15 mutations each on Gγ-globin and Aγ-globin genes 19. We have now reported for the first time in Thai population, five different HPFH mutations found in 20 subjects including four Aγ-HPFH and a Gγ-HPFH. Of these five HPFH mutations, three known mutations were − 202 Aγ (C>T) (n = 3), − 196 Aγ (C>T) (n = 3) and − 158 Aγ (C>T) (n = 12). Two novel mutations namely − 117 Aγ (G>C) and − 530 Gγ (A>G) were unexpectedly detected. As shown in Table In Thailand, we have extensively investigated the molecular basis of deletional form of HPFH and other high Hb F determinants including δβAγ (C>T) and the Italian/Chinese − 196 Aγ (C>T), are located within the G-rich area upstream of the − 195 to − 202 of γ-globin gene. This DNA region is known to be the binding site of a ubiquitous transcription factor Sp1. As compared to the wild-type sequence, these two mutations decreased the binding of Sp1 on gel-shift experiment22. It has also been suggested that mutations in this G-rich sequence disrupt an intramolecular triplex proposed to be the binding site of a repressor, thereby increasing expression of γ-globin gene23. Carriers of the − 202 Aγ (C>T) and − 196 Aγ (C>T) reported previously had Hb F in the ranges of 1.6–3.9% and 12–21%, respectively23. In our study, the three carriers of the − 196 Aγ (C>T) had as expected the average Hb F of 14.3 ± 1.9% (Table Aγ (C>T) demonstrated higher Hb F levels with the average of 9.9 ± 1.6% as compared to the previously reported cases, possibly due to the presence of Hb E and the − 158 Gγ XmnI ( +) in all Thai subjects. In addition, two of them also carried the KLF1 mutations (H299D & T334R) (case no. 2 & 3), known to be associated with increased Hb F expression in Hb E syndrome14.The Black − 202 Aγ (C>T) or the Cretan HPFH was originally described in three unrelated Greek adults with slightly increased Hb F level (2.9–5.1%) and normal hematological parameters. The − 158 Aγ (C>T) mutation has presumably resulted from a gene conversion event24. It is noteworthy that this form of HPFH was the most common one in Thai population, being detected in 12 of 20 Thai non-deletional HPFH subjects in this study (Table Aγ (C>T) mutation on Hb F production can be raised. We found that this mutation is associated in cis with the − 158 Gγ XmnI ( +) polymorphism in Thai population. It has been noted that a 240-kDa activator protein, a member of the CAAT/enhancer-binding proteins family, binds to − 158 to -− 161 nucleotides of γ-globin gene promoter and induces expression of γ-globin gene. This may explain the effect of − 158 Gγ XmnI (+) (C>T) and − 158 Aγ (C>T) to elevated γ-globin gene expression25. While the − 158 Gγ XmnI (+) (C>T) polymorphism is associated with marginally elevated Hb F during erythropoietic stress, the − 158 Aγ (C>T) mutation is associated with higher Hb F expression with average of 7.8 ± 6.8% (Table Aγ-globin gene is more effective in competition with Gγ-globin gene for the Locus Control Region (LCR) in the adult stage25. The combined action of − 158 Aγ (C>T) in cis with − 158 Gγ (C>T) may lead further to higher Hb F production than having − 158 Gγ (C>T) alone24. In addition, study in Chinese and Thai subjects has identified that the − 158 Gγ XmnI (+) (C>T) was linked to the + 25 (G>A) polymorphism of Aγ-globin promoter (rs368698783), the binding motif of Ly-1 antibody reactive (LYAR) transcription factor. This polymorphism decreased the binding efficiency of the repressor of γ-globin genes leading to increased Hb F production26. We found that all Thai subjects with − 158 Aγ (C>T) HPFH mutation also carried the − 158 XmnI Gγ (+) and the + 25 Aγ (G>A) polymorphism. This should explain the higher Hb F production in these Thai subjects  found with the HPFH mutation alone or in combinations several common hemoglobinopathies including Hb E, β-thalassemia, α-thalassemia, and KLF1 mutation (G176Afs*179). The lowest Hb F of 2.1% was found in a heterozygous subject with the − 158 Aγ (C>T) in combination with Hb E and α+-thalassemia (3.7 kb deletion). This is not unexpected since it has been known that co-inheritance of α-thalassemia can lead to the reduced Hb F level in several hemoglobinopathies29. Of interest is the finding of subject with complex interaction of the − 158 Aγ (C>T), α+-thalassemia (3.7 kb deletion), and β0-thalassemia (β17(AAG>TAG)) (Table 2 (4.3%) and as high as 27.1% Hb F 30. This is in contrast with the deletional form of high Hb F determinants in which co-inheritance with β-thalassemia is associated with normal Hb A2 level5. Although diagnosis of a β-thalassemia heterozygote with this complex interaction seen in the case no. 17 is not altered due to elevated Hb A2, an unusually increased Hb F (27.1%) in heterozygous β-thalassemia, as seen in Thai subject, might be a good marker for a co-inheritance of HPFH in β-thalassemia, requiring further investigation. We recommend therefore to investigate all cases of heterozygous β-thalassemia with Hb F higher than 2% for further investigation of a possible co-inheritance of HPFH. This is a very important and useful information at genetic counselling since co-inheritance of HPFH can ameliorate the severity of β-thalassemia disease3. The last case in this group of HPFH with − 158 Aγ (C>T) mutation is an adult male encountered with a homozygous for this mutation who had 7.6% Hb F. He also carried the heterozygosity for KLF1 mutation (p.G176Afs*179), and α+-thalassemia (3.7 kb deletion). Homozygosity for a − 158 XmnI Gγ (+) was not unexpectedly noted due to the linkage of these two mutations. Again, high Hb F with normal level of Hb A2 and other hematological parameters , CCAAT displacement protein (CDP), and an erythroid specific protein NFE132. It has been shown that the Greek/Black/Sardinian − 117 Aγ (G>A) and Japanese − 114 Gγ (C>T) HPFH mutations slightly increased the binding of CP1 and CDP but reduced the binding of NFE1 to the distal CCAAT motif32. A novel mutation at the same region, [− 117 Aγ (G>C)] identified in Thai subject with Hb E heterozygote in this study was associated with 13.5% Hb F. It is conceivable that this Thai − 117 Aγ (G>C) HPFH mutation should behave similar mechanism with that of the Greek/Black/Sardinian − 117 Aγ (G>A) HPFH mutation. Prediction of transcription factors binding to the region using the TFBIND program18 in comparison between the wild-type promoter, − 117 Aγ (G>A) Greek/Black/Sardinian HPFH, − 117 Aγ (G>C) Thai HPFH and − 114 Gγ (C>T) Japanese HPFH was carried out as shown in Table RRCCAATSA) for the CCAAT box as shown in Table Aγ (G>A) with score of 0.922056. The Thai − 117 Aγ (G>C) and the Japanese − 114 Gγ (C>T) had decreased scores of 0.824343 and 0.797797, respectively.Unlike other globin genes which contain only one CCAAT motif, γ-globin gene has duplicated CCAAT sequences. The proximal CCAAT motif is located at -88 nucleotide and the distal one is found at − 115 regions. The proximal CCAAT motif is corresponding to the CCAAT motif of β-globin gene in which mutations in this motif result in reduced β-globin gene expression and β-thalassemia pursue. In contrast, mutations in the distal CCAAT motif of γ-globin gene results in higher γ-globin expression and HPFH phenotypeGγ-globin gene has been proposed as a negative regulatory element based on study in transiently transfected K562 cell33. The Iranian − 567 Gγ (T>G) HPFH mutation (HBG2:c.− 620 T>G) which changed a GATA-1 binding motif to GAGA sequence (AGATAA>AGAGAA) was associated with increased Hb F of 5.9% and 10.2% in two Iranian subjects33. The five bp deletion between − 533 to − 529 (-ATAAG) of Gγ globin gene (HBG2:c.− 582_− 586del ATAAG), located at the GATA-1 binding site was also associated with HPFH phenotype in an Indian family34. Therefore, alteration of GATA-1 and related repressor proteins bindings to the motif should result in up-regulation of γ-globin gene expression. In contrast, the Thai − 530 Gγ (A>G) HPFH mutation which is located at the same region of GATA-1 binding motif (AGATAA>AGATAG) may have less effect on Gγ-globin gene expression since it does not modify the GATA-1 binding site (A/T)GATA(A/G) at this position significantly. Therefore, as shown in Table silico analysis of GATA-1 related transcription factors binding of the Thai − 530 Gγ-HPFH using the online TFBIND program showed less changes on the similarity scores as compared to the wild-type sequence which contrasts with the − 533 to − 529 (-ATAAG) Indian HPFH mutation which reduced binding of all GATA-1 isoforms. As shown in the table, we found that the Thai HPFH mutation had slight increased binding scores of two GATA-1 isoforms and decreased binding scores for another two GATA-1 isoforms. The mechanisms underlying up-regulation of γ-globin gene in these HPFH mutations may be difference. Accordingly, the effect of the − 530 Gγ (A>G) Thai HPFH mutation on the Gγ-globin gene expression could be minimal. This might explain the marginal elevation of Hb F (1.9% and 3.0%) observed in pure heterozygotic form of the mother and the sister of the proband and indicated that two copies of the − 530 Gγ (A>G) mutation in homozygote state is required for dramatically increased in Hb F (11.5%) as seen in the proband  and − 158 XmnI Gγ (−) had normal Hb F level (0.3%) due to a co-inheritance of α+-thalassemia. It is evidenced from the Indian and Thai HPFH families that subjects with combined non-deletional HPFH mutation and β-thalassemia mutation in trans had elevated Hb A2 for β-thalassemia trait i.e., 3.6–3.9% in Indians34 and 4.3% in Thai (Table 25.Nucleotides between − 675 to − 526 of 0-thalassemia, Aγδβ0-thalassemia, and HPFH described before10. Complex interactions between these non-deletional HPFH in both heterozygote and homozygote with other hemoglobinopathies commonly found in the region can lead to various hematological phenotypes and Hb F productions which could render diagnosis difficult. Identification of these non-deletional HPFH using rapid PCR diagnostic assays developed should improve this diagnosis in routine practice in the regions.In summary, three known and two novel HPFH mutations were identified for the first time in Thai population. This result indicates a diverse molecular heterogeneity of non-deletional HPFH in Thai population in addition to the deletional forms of δβSupplementary Information.


These chains are linked to each other by N—H⋯N hydrogen bonds, N–H⋯π and π–π interactions, forming a three-dimensional network.In the crystal, the molecules are connected by N—H⋯N hydrogen bonds into dimers with an  19H15N5S, the thiophene ring is disordered in a 0.6:0.4 ratio by an approximate 180° rotation of the ring around the C—C bond linking it to the pyridine ring. In the crystal, the molecules are linked by N—H⋯N hydrogen bonds into dimers with an R22(12) motif, forming chains along the b-axis direction. These chains are connected to each other by further N—H⋯N hydrogen bonds, forming a three-dimensional network. Furthermore, N—H⋯π and π–π [centroid–centroid separations = 3.899 (8) and 3.7938 (12) Å] interactions also contribute to the crystal cohesion. A Hirshfeld surface analysis indicated that the most important contributions to the surface contacts are from H⋯H (46.1%), N⋯H/H⋯N (20.4%) and C⋯H/H⋯C (17.4%) interactions.In the title compound, C The dihedral angle between the phenyl (C7–C12) and pyridine (N1/C2–C6) rings is 64.42 (11) °.The pyridine ring (N1/C2–C6) of the title compound Fig. 1 is large3.et al., 1995b-axis direction. These chains are connected to each other by further N—H⋯N hydrogen bonds, forming a three-dimensional network (Tables 1Cg1⋯Cg1i = 3.899 (8) Å; slippage = 1.899 Å; Cg3⋯Cg3ii = 3.7938 (12) Å; slippage = 1.383 Å; symmetry codes: (i) −x, 1 − y, z; (ii) 1 − x, 1 − y, z; Cg1 and Cg3 are the centroids of the major component of the disordered thiophene ring and of the pyridine ring, respectively] also contribute to crystal cohesion . Fig. 8u. Fig. 7a,b. Fig4.et al., 2016et al., 2019et al., 2014aet al., 2014bet al., 2014cThe four related compounds found as a result of the search for ‘2,6-diamino-4-(thiophen-2-yl)pyridine-3,5-dicarbonitrile’ in the Cambridge Structure Database , chains running along the b-axis direction are formed through N—H⋯O interactions between the 1,4-dihydropyridine N atom and one of the O atoms of the ester groups. Neighbouring chains are linked by C—H⋯O and C—H⋯π interactions. In the crystal of WOJCIJ (space group P21/c), inversion dimers linked by pairs of N—H⋯N hydrogen bonds generate P21/n), inversion dimers linked by pairs of N—H⋯Nc (c = cyanide) hydrogen bonds generate Pbca), inversion dimers linked by pairs of N—H⋯Nn (n = nitrile) hydrogen bonds generate In the crystal of MUCLAA (space group 5.To a solution of 2-(thiophen-2-ylmethylene)malononitrile  and malononitrile  in methanol (25 mL), phenylethylamine  was added and the mixture was stirred at room temperature for 48 h. Then 15 mL of methanol were removed from the reaction mixture, which was left overnight. The precipitated crystals were separated by filtration and recrystallized from ethanol/water (1:1) solution .1H NMR : 1.55 ; 5.45 ; 7.21–7.88 ; 13C NMR : 21.69 (CH3), 50.00 (CH—Ar), 79.77 (=Ctert), 80.92 (=Ctert), 116.85 (CN), 116.97 (CN), 127.14 (2CHarom), 127.22 (CHarom), 128.11 (CHthienyl), 128.63 (2CHarom), 130.14 (CHthienyl), 130.75 (CHthienyl), 134.53 (Car), 144.53 (Cthienyl), 152.30 (=Ctert), 158.70 (N=Ctert), 161.38 (=Ctert).6.SHELXL were used for the Uij values of equivalent atom pairs  and DFIX commands were used to restrain the nearest-neighbour and next-nearest-neighbour bond distances in the two disorder components to be equal with a standard deviation of 0.03 Å. All C-bound H atoms were placed in calculated positions (0.95–1.00 Å) and refined as riding with Uiso(H) = 1.2 or 1.5Ueq(C). The N-bound H atoms were located in a difference map and refined with Uiso(H) = 1.2Ueq(N) .Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023003845/vm2282sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989023003845/vm2282Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023003845/vm2282Isup3.cmlSupporting information file. DOI: 2260011CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


These ribbons are connected by N—H⋯O, N—H⋯N hydrogen bonds and van der Waals interactions.In the crystal, molecules are linked by intermolecular N—H⋯O and N—H⋯N hydrogen bonds into ribbons parallel to (022) along the  11H7N5OS, contains two independent molecules (1 and 2). The thiophene ring in molecule 2 is rotationally disordered (flip disorder) by ca 180°  over two sites with the site-occupation factors of 0.9 and 0.1. These two orientations of the thiophene ring in molecule 2 are not equivalent. In the crystal, molecules are linked by intermolecular N—H⋯O and N—H⋯N hydrogen bonds into ribbons parallel to (022) along the a axis. Within the (022) planes, these ribbons are connected by van der Waals interactions and between the (022) planes by N—H⋯O hydrogen bonds. In molecule 1, Hirshfeld surface analysis showed that the most important contributions to the crystal packing are from N⋯H/H⋯N (27.1%), H⋯H (17.6%), C⋯H/H⋯C (13.6%) and O⋯H/H⋯O (9.3%) interactions, while in molecule 2, H⋯H (25.4%) interactions are the most significant contributors to the crystal packing.The asymmetric unit of the title compound, C These two orientations of the thiophene ring in molecule 2 are not equivalent.As seen in Fig. 1Database survey section.In molecule 1, the angle between the thiophene (S1/C8–C11) and pyridine (N1/C2–C6) rings is 50.72 (9)°. In molecule 2, the angle between the two disordered thiophene rings (S2/C19–C22 and S2′/C19/C20′–C22′) is 6.2 (5)°, and they make an angle with the pyridine ring (N6/C13-C17) of 40.3 (1) and 34.8 (5)°, respectively. Molecules 1 and 2 (r.m.s. deviation = 0.126 A) are almost identical and the geometric parameters are normal and comparable to those of related compounds listed in the B⋯N2 and N10—H10A⋯N7 hydrogen bonds, forming S(5) motifs  along the s Table 1. Within s Table 1 and 4 ▸.CrystalExplorer17.5  represent N—H⋯O interaction zones (Table 1b) interactions provide the highest contribution (Table 2c), C⋯H/H⋯C  and O⋯H/H⋯O  contacts are also significant. Table 2dnorm mappings were performed in the range −0.5165 to +1.1535 a.u. The locations of N—H⋯O interactions are shown by bright-red circles on the dnorm surfaces , Table 1c; 25.4%) are the major factor in the crystal packing with N⋯H/H⋯N , O⋯H/H⋯O  and C⋯H/H⋯C  interactions representing the next highest contributions. The percentage contributions of comparatively weaker interactions in molecules 1 and 2 are given in Table 2es Fig. 5a,b reprs Table 1. The fins Table 1 show thas Table 1b intern Table 2, as wouln Table 2c, C⋯H/Hes Fig. 5c,d, Tab⋯N Fig. 6b; 24.3%⋯O Fig. 6e; 11.7%⋯C Fig. 6d; 11.4%4.et al., 2016viz. CSD refcodes BEFFOL , pairs of molecules are linked by pairs of N—H⋯N hydrogen bonds, forming dimers with an II crystallizes in the triclinic space group PIIA and IIB) in the asymmetric unit. In the crystal of II, molecules IIA and IIB are linked by intermolecular N—H⋯N and N—H⋯O hydrogen bonds into layers parallel to (001). These layers are connected along the c-axis direction by weak C—H⋯N contacts. C—H⋯π and C—N⋯π interactions connect adjacent molecules, forming chains along the a-axis direction. In III (space group: Pc), the two molecules in the asymmetric unit are joined together by N—H⋯O hydrogen bonds, forming a dimer with an c-axis direction, which are connected by C—Br⋯π interactions. In IV (space group: Pc), intermolecular N—H⋯N and C—H⋯N hydrogen bonds, as well as N—H⋯π and C—H⋯π interactions, connect the molecules in the crystal, generating a 3D network. In both V (space group: PVI (space group: PVII (space group: P21/n), the crystal structure is stabilized by intermolecular C—H⋯F and C—H⋯π interactions, and in VIII (space group: P21/c), by intermolecular C—H⋯O and C—H⋯π interactions. In IX (space group: Cc), intermolecular N—H⋯N and C—H⋯N hydrogen bonds form molecular sheets parallel to the (110) and (110) planes, crossing each other. Adjacent molecules are further linked by C—H⋯π interactions, which form zigzag chains propagating parallel to [100]. The compound X (space group: Pca21) crystallizes with two independent molecules in the asymmetric unit. In the crystal, the A and B molecules are linked by N—H⋯S, N—H⋯N and C—H⋯S hydrogen bonds, forming a three-dimensional network. In XI (space group: P21/c), molecules are linked into a chain along the b-axis direction via C—H⋯O interactions. In XII (space group: PXIII (space group: P21/c), the molecules form centrosymmetric dimers via N—H⋯S hydrogen bonds.In the crystal of 5.et al., 2020The title compound was synthesized using a recently reported procedure (Babaee 6.Uiso(H) = 1.2Ueq(C). The N-bound H atoms were found in a difference-Fourier map, and refined freely , with Uiso(H) = 1.2Ueq(N). The thiophene ring (S2/C19–C22) in molecule 2 is rotationally disordered (flip disorder) by ca 180°  over two sites with the site-occupation factors of 0.9 and 0.1 (fixed after refinement cycles). A DFIX instruction was applied to constrain the distances in the thiophene rings of disordered molecule 2. For these rings, FLAT and EADP instructions were also used.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023003237/tx2066sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023003237/tx2066Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023003237/tx2066Isup3.cmlSupporting information file. DOI: 2255359CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A co-crystal of quabodepistat and 2,5-dihydroxybenzoic acid was obtained and the crystal structure was solved from microcrystal electron diffraction (MicroED) data. R,4R)-1--3,4-dihydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinolin-2(1H)-one); C21H20ClF3N2O4] and 2,5-dihydroxybenzoic acid  were successfully co-crystallized. Given the small size of the crystals (1 × 0.2 × 0.2 µm) the structure was solved via microcrystal electron diffraction (MicroED). The C—O and C=O bond-length ratio of the carboxylic group in 2,5DHBA is 1.08 (1.34 Å/1.24 Å), suggesting that 2,5DHBA remains protonated. Therefore, the material is a co-crystal rather than a salt. The amide group of quabodepistat participates in a cyclic hydrogen bond with the carboxylic group of the 2,5DHBA. Additional hydrogen bonds involving the quabodepistat amide and hydroxyl groups result in a three-dimensional network.Quabodepistat [(5-{[(3 The angles O21—C16—C17, F30—C28—C23, C6—N7—C8, C9—C10—C11, O1—C14—C15, and C6—C11—C2 also have z-score values greater than 3.Quabodepistat and 2,5DHBA co-crystallize in a 1:1 stoichiometric ratio in the monoclinic system, space group 2 values  exhibit χ3.via hydrogen bonds are observed between quabodepistat and 2,5DHBA. One of the interactions is between a carboxylic group and an amide. As shown in Fig. 1et al., 2020Intermolecular interactions 4.et al., 2016A search for co-crystals with 2,5-dihydroxybenzoic acid (or gentisic acid) in the Cambridge Structural Database . Tetrahydrofuran (THF) and hexane were purchased from FUJIFILM Wako Pure Chemical Corporation . 2,5DHBA was purchased from Tokyo Kasei Kogyo Co., Ltd. . Quabodepistat (5 g) and 2,5DHBA  were dissolved in 100 mL of THF. 250 mL of hexane were added while stirring. Precipitation occurred as soon as hexane was added. The THF/hexane was stirred at room temperature (approximately 298 K) for three days. After filtration, it was dried at room temperature for 24 h, then heated at 383 K for 20 h.6.−Å−2 s−1. Contiguous diffraction frames were collected every 0.5° from each crystal by continuously rotating the sample stage at a goniometer rotation speed of 1° s−1; the sample stage was rotated from −40° to 40° for the first crystal  and from −60° to 60° for the second crystal . The structure was refined kinematically. Refinement with SHELXL was carried out using the scattering factors for electron diffraction  global, I. DOI: Click here for additional data file.10.1107/S2056989023006047/dj2052Isup2.cmlSupporting information file. DOI: https://doi.org/10.5281/zenodo.7156704Deposit the raw data into Zenodo: https://dx.doi.org/10.5517/ccdc.csd.cc2d19zrDeposit the raw data into CCDC: 2205804CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: