Besides, [Hg(Pip)2]n (2) has been achieved during the attempts to isolate these polymorphs.The selective synthesis of P1A and P1B hasbeen successfully achieved by changing the synthetic conditions. Theformation of each polymorphic form has been ensured by unit cell measurementsand decomposition temperature. The elucidation of their crystal structurerevealed P1A and P1B as polymorphs, whichoriginates from the Hg(II) cores and intermolecular associations,especially pinpointed by Hg···π and π···πinteractions. Density functional theory (DFT) calculations suggestthat P1B, which shows Hg(II) geometries that are furtherfrom ideality, is more stable than P1A by 13 kJ·mol–1 per [Hg(Pip)2]·DMFformula unit, and this larger stability of P1B arisesmainly from metal···π and π···πinteractions between chains. As a result, these structural modificationslead to significant variations of their solid-state photoluminescence.Controlling the formationof the desired product in the appropriatecrystalline form is the fundamental breakthrough of crystal engineering.On that basis, the preferential formation between polymorphic forms,which are referred to as different assemblies achieved by changingthe disposition or arrangement of the forming units within the crystallinestructure, is one of the most challenging topics still to be understood.Herein, we have observed the formation of two concomitant polymorphswith general formula {[Hg(Pip) 2]·DMF}n (P1A and P1B), aswell as their desolvated form 2. Then, both polymorphswere selectively synthesized by temperature or anion-template formation.Their crystal structures revealed distorted pentagonal pyramidal geometriesand show that differences arise from geometry and packing that ledto different solid-state photoluminescence emissions. According toperiodic-DFT calculations, distortions in P1B are counterbalancedleading to a more stable form by Hg(II)···π andπ···π interactions.We have successfully isolated two concomitantpolymorphswith formula {[Hg(Pip) While the roleof the solvent varies from filling voids and partaking in intermolecularinteractions to being coordinated to the metal center,14 temperature is a simple way to control the formationof polymorphic forms. Energetic data of organic polymorphs15 show that usually energy differences fall withinthe range of 0–10 kJ·mol–1, but scarcedata about relative energy calculation of coordination polymers havebeen found.16 Interestingly, these meredifferences in the order of weak interactions are responsible forsignificant modification in the resulting properties. Recent photoluminescencestudies of Au(I) coordination complexes have shown emission-dependentproperties caused by slight structure differences after the absorptionof guest solvent molecules.13 By this token,polymorphism in Cu(I) complexes evinced the impact of such structuraldifferences in the photophysical properties.17Within this frame, slightmodifications, intermediate variations,or major structural changes (polymorphism) throughout the entire crystalstructure are promoted by factors such as time,18 and cocrystals19 of pharmaceutical interest, and despite theknowledge of polymorphs in discrete coordination complexes such asthe archetypal [PtCl2],20 there is a scarce number of structures bearingcoordination polymers reported hitherto comprising Cu(I),23 Ag(I),25 Co(II) andNi(II),29 Cd(II),30 or Pb(II).31 Inthis scenario, free rotation ligands can drive the formation of conformationalpolymorphs.33 Besides, Hg(II) as softer metal comparedto Zn(II) and Cd(II)34 is capable of accommodatingseveral distorted geometries and partake in weak intermolecular interactions.Thus, a combination of them can trigger the assembly of differentarrangements.35Although there is a large amount of polymorphism data aboutcrystallizationof organic salts37 we have combinedHg(OAc)2, 1,3-benzodioxole-5-carboxylic acid  and the free rotational 4,4′-bipyridine ligand. The synthesis performed at 95 °C resultedin two concomitant polymorphs bearing the same zigzag one-dimensional(1D) structure and accommodating guest N,N-dimethylformamide (DMF) molecules with general formula{[Hg(Pip)2]·DMF}n (P1A and P1B). During the attemptsto isolate each polymorphic forms, we recognized the formation ofan additional zigzag 1D coordination polymer with formula {[Hg(Pip)2]}n (2). All of them were characterized by analytical and spectroscopictechniques. In addition, periodic density functional theory (DFT)calculations have been performed to set the relative stabilities ofboth polymorphic forms and rationalize the origin of the differentstability. Finally, these structural differences provoked dramaticvariations in their solid-state photoluminescence.In pursuit of extendingour knowledge on the structure–propertyrelationship of Hg(II) compounds,Hg(II) complexesare toxic, and anymanipulation of the samples has to be carried out into the fume hoodand wearing gloves.2), 1,3-benzodioxole-5-carboxylic acid,4,4′-bipyridine  ligands, methanol (MeOH), N,N-dimethylformamide (DMF), acetic acid(HOAc), and diethyl ether (Et2O) as solvents were purchasedfrom Sigma-Aldrich. The water used in the reactions was Milli-Q water.Deuterated dimethyl sulfoxide-d6 (DMSO-d6) was used for the NMR experiment and was purchasedfrom Eurisotop. All of them were used without further purification.Reactions and manipulation were carried out in a Digitheat-TFT furnace(JP Selecta) using sealed vials under an autogenous pressure of DMFat 95 °C for the synthesis of the mixture (P1A and P1B) and the isolation of either P1A or P1B. Compound 2 was synthesized in DMF at roomtemperature (RT) or both in MeOH and Milli-Q water at RT and 95 °C.Powder X-ray diffraction (PXRD) patterns were measured with a PANalyticalX’Pert PRO MPD θ/θ powder diffractometer of 240mm radius, in a configuration convergent beam with a focalizing mirrorand a transmission geometry with flat samples sandwiched between low-absorbingfilms. A Cu Kα radiation with λ = 1.5418 Å was used(45 kW and 40 mA). All of them were recorded from 2θ = 5 to30° with a step scan of 0.0263° and a measuring time of300 s per step. Thermal decomposition temperature (dT) was measuredon a Stuart Melting Point Apparatus SMP30 with a heating ramp of 2.0°C·min–1 in a temperature range of 20–210°C. Elemental analyses  were carried out on a Euro Vector3100 instrument. Simultaneous thermogravimetric (TG)/differentialthermal analysis (DTA) determinations were performed with a NetzschSTA 409 instrument, using an aluminum oxide powder crucible and anoxide powder as a standard  and heating at 5 °C·min–1 from25 to 350 °C, under nitrogen atmosphere with a flow rate of 80mL·min–1. The Fourier transform infrared-attenuatedtotal reflection (FTIR-ATR) spectra were recorded on a PerkinElmerspectrometer, equipped with a universal attenuated total reflectance(ATR) accessory with diamond window in the range of 4000–500cm–1. 1H, 13C{1H}, and distortionless enhancement by polarization transfer (DEPT)-135NMR spectra were recorded on an NMR-FT Bruker360 MHz spectrometerin DMSO-d6 solution atRT. All chemical shifts (δ) are given in ppm. Solid-state photoluminescencemeasurements were recorded using a Varian Cary Eclipse Fluorescencespectrophotometer between 500 and 660 nm. CIE 1931 chromaticity diagramwas generated using Origin Pro 2019b software.Hg(II)acetate (Hg(OAc)2  was added and the suspension was sonicated for a minute untildissolution. The reaction was sealed and kept under autogenous pressureat 95 °C for 45 min and then allowed to cool down out of thefurnace for 1 h until 25 °C. After cooling, several prism-likecolorless crystals were formed, filtered, and washed twice with 5mL of cold Et2O. Careful inspection of them revealed thepresence of two different crystal sizes but sharing the same crystallinehabit . These single crystals were mechanically sorted for theirX-ray crystal structure elucidation, revealing that the small crystalswere P1A while the big crystals were P1B.DMF (2.5 mL) was placed into a 10 mL vial and heated in a furnaceuntil 95 °C. Once the temperature was reached, 4,4′-bipy and HPip  were introduced.When the solution became transparent, Hg(OAc)P1A and P1B: Yield: 141 mg (66%).dT = 192–196 °C. Anal. Calcd for C29H25N3O9Hg (760.11 g·mol–1): C, 45.82; H, 3.31; N, 5.53. Found: C, 45.56; H, 3.28; N, 5.32%.FTIR-ATR : 3097(w)–3014(w)[ν(CH)]ar, 2985(w)–2784(w) [ν(CH)]al, 1659(m) [ν(C=O)]DMF, 1628(w)–1576(m)[ν(C=C/C=N)], 1542(m) [ν(COO)]as, 1502(m)–1482(m) [ν(C=C/C=N)], 1429(m)[ν(COO)]s, 1418(sh.), 1368(s)–1229(s) [δ(C=C/C=N)],1163(m), 1128(w), 1101(m), 1069(m), 1034(s) [δ(C–H)]ip, 1003(m) [δ(C–H)]ip, 936(m), 920(m),888(m), 866(w), 821(m)–771(s) [δ(C–H)]oop, 721(m), 682(m), 660(m), 639(m), 630(m), 582(m), 551(m), 534(m),503(w). 1H NMR : δ = 2.72 and 2.88 DMF, 6.10 , 6.97 , 7.41 , 7.58 , 7.92, 7.94 DMF, 8.77 .2  and HPip in DMF (3 mL), a solution of 4,4′-bipy in DMF (2 mL) was added dropwise under vigorous stirring. Immediately,a yellowish solid appeared. The reaction was stirred for 1 h. Thesolid obtained was filtered and washed with 10 mL of cold MeOH. Suitablecrystals were obtained by slow diffusion of 5 mL of MeOH into 1 mLof the mother liquors for 5 days. The phase purity of the sample wasconfirmed by PXRD.To a solution ofHg(OAc)2 wasalso achieved at RT and at 95 °C using MeOH or Milli-Q wateras a solvent.The synthesis of compound 26H18N2O8Hg (687.02 g·mol–1): C, 45.45; H, 2.64; N, 4.08. Found: C, 45.38; H,2.38; N, 3.84%. FTIR-ATR : 3107(w)–3045(w)[ν(CH)]ar, 2983(w)–2891(w) [ν(CH)]al, 2792(w), 1631(w), 1606(m) [ν(C=C/C=N)],1574(m), 1541(m) [ν(COO)]as, 1496(m) [ν(C=C/C=N)],1484(m) [ν(C=C/C=N)], 1429(m) [ν(COO)]s, 1418(m), 1367(s)–1230(s) [δ(C=C/C=N)],1160(m), 1106(m), 1094(m), 1067(m), 1034(s) [δ(C–H)]ip, 1008(m) [δ(C–H)]ip, 930(m), 919(m),890(m), 818(m)–765(s) [δ(C–H)]oop,721(m), 681(m), 670(m), 633(m), 586(m), 549(m). 1H NMR: δ = 6.08, 6.95 , 7.38 , 7.56 ,7.89 , 8.74 . 13C{1H} NMR : δ = 169.07 [O2C–C], 150.63 [N–CH–CH], 150.13[O2C–C–(CH)2–C], 147.13 [O2C–C–CH–C], 144.95 [N–(CH)2–C],127.64 [O2C–C], 125.17 [O2C–C–CH–CH], 121.97 [N–CH–CH], 109.63 [O2C–C–CH–C], 107.81 [O2C–C–CH–CH], 101.66 [O–CH2–O].Yield: 165 mg (77%). dT = 201–202 °C.Anal. Calcd forC2  was addedand the mixture was sonicated for 1 min until a yellowish transparentsolution was obtained. Then, the vial was sealed, kept at 95 °Cunder an autogenous pressure for 45 min, and left to cool down slowlyfor 7 h until 25 °C . Suitable crystals of P1A were formed, collected,filtered off, and washed twice with 5 mL of cold Et2O.DMF (5 mL) andHOAc  were introduced into a 10 mL vial,sealed, and heated in a furnace until 95 °C. Once the temperaturewas reached, compound –1): 3098(w)–3012(w) [ν(CH)]ar, 2895(w) [ν(CH)]al, 2780(w) [ν(CH)]al, 1658(s) [ν(C=O)]DMF, 1628(w)–1576(m)[ν(C=C/C=N)], 1542(m) [ν(COO)]as, 1503(m) [ν(C=C/C=N)], 1483(m) [ν(C=C/C=N)],1427(m) [ν(COO)]s, 1368(s)–1229(s) [δ(C=C/C=N)],1163(m), 1101(m), 1069(m), 1035(s) [δ(C–H)]ip, 1003(sh), 935(m), 888(m), 861(w), 820(m)–771(s) [δ(C–H)]oop, 719(m), 680(m), 660(m), 639(m), 629(m), 582(m), 550(m),538(m), 511(m).Yield: 42.8 mg (59%). dT = 192–193 °C. FTIR-ATR .The synthesis of ar, 2894(w) [ν(CH)]al, 2786(w) [ν(CH)]al, 1658(s) [ν(C=O)]DMF, 1628(w)–1578(m)[ν(C=C/C=N)], 1540(m) [ν(COO)]as, 1504(m)–1453(m) [ν(C=C/C=N)], 1428(m)[ν(COO)]s, 1419(m), 1367(s)–1230(s) [δ(C=C/C=N)],1163(m), 1101(m), 1070(m), 1036(s) [δ(C–H)]ip, 1006(sh), 935(m), 920(m), 887(m), 866(w), 819(m)–769(s)[δ(C–H)]oop, 720(m), 680(m), 661(m), 637(m),630(m), 582(m), 554(m), 536(m).Yield: 64.9 mg (73%).dT = 198–199 °C. 3098(w)–3012(w) [ν(CH)]P1A, P1B, and 2 specimens were used for theX-ray crystallographic analysis.The X-ray intensity data were measured on a D8 Venture system equippedwith a multilayer monochromator and a Mo microfocus (λ = 0.71073Å). For P1A, P1B, and 2 the frames were integrated with the Bruker SAINT software package,using a narrow-frame algorithm. For P1A, the integrationof the data using a triclinic unit cell yielded a total of 8231 reflectionsto a maximum θ value of 30.60° (0.70 Å resolution),of which 8231 were independent  and 3761 (45.69%) were greater than 2σ(|F|2). For P1B, the integration ofthe data using a triclinic unit cell yielded a total of 216 059reflections to a maximum θ value of 30.55° (0.70 Åresolution), of which 33 014 were independent  and 26 560 (80.45%) weregreater than 2σ(|F|2). For 2, the integration of the data using a monoclinic unit cellyielded a total of 3368 reflections to a maximum θ value of30.36° (0.70 Å resolution), of which 3368 were independentand 3237 (96.11%) were greater than 2σ(|F|2).Colorless prism-like 38 For P1A, the final anisotropic full-matrixleast-squares refinement on |F|2 with330 variables converged at R1 = 4.58%,for the observed data and wR2 = 12.50%for all data. For P1B, the final anisotropic full-matrixleast-squares refinement on |F|2 with1556 variables converged at R1 = 2.95%,for the observed data and wR2 = 6.80%for all data. For 2, the final anisotropic full-matrixleast-squares refinement on |F|2 with168 variables converged at R1 = 2.18%,for the observed data and wR2 = 5.43%for all data. For P1A, P1B, and 2, the final cell constants and volume are based upon the refinementof the XYZ-centroids of reflections above 20 σ(I). Data were corrected for absorption effects using themultiscan method (SADABS). Crystal data and relevant details of structurerefinement for compounds P1A, P1B, and 2 are reported in P1A are listed in P1B are shown in 2 are listed in The structures were solved and refined using theBruker SHELXTL Software Package (version-2018/3).39 software,which is based on the low continuous-shape measure (CShM) value S..The geometry evaluation of the threecomplexes has been performedusing version 2.1 of SHAPE value S. The corr42 with POV-Ray Package (version 3.7).43 Color codes for molecular graphics: light slate blue (N),suva gray (C), white (H), lavender gray (Hg), and red (O).Complete informationabout the crystal structure and moleculargeometry is available in .cif format and deposited in the CCDC. CCDCnumbers 2101056–2101058 contain the supporting data of this paper. Moleculargraphics were generated using Mercury (version 4.3.1)P1A and P1B were carried out using the VASP code.45 Geometry optimizationswere performed at the PBE-D2 level of theory47 assuming a P1 space group and keeping the unit cell parameters tothose determined by single-crystal X-ray diffraction (SC-XRD). Withthe aim of analyzing the suitability of the D2 Grimme’s empiricalcorrection, we compared the relative stability of P1B and P1A 2]·DMF unit formula) with PBE-D2, PBE-D3and PBE-D*, a modification of the D2 Grimme’s empirical termsuggested to be more suitable for the modeling of molecular crystals.48 Results are reported in Table S1 in the Supporting Information and show that all Grimme’scorrection leads to a preference for the P1B polymorphand the differences between the three methods are small (5.5 kJ·mol–1). Moreover, according to the literature, PBE-D3 isaccurate enough for modeling molecular crystals50 and, indeed, it has been even used to compute reference values forthe validation of less computationally demanding approaches.51 Ionic cores were described with the projectoraugmented wave (PAW) pseudopotentials.53 The valenceelectrons were represented through a plane-wave basis set with a kineticenergy cutoff of 600 eV. Since cell size and the number of Hg(II)units in the cell differs for P1A and P1B, the Brillouin zone was sampled with a different Monkhorst–Pack K-point mesh in each case, i.e., (4 ×4 × 4) and (1 × 1 × 1) for P1A and P1B, respectively.DFT calculations of E) between two metal dimers of vicinal chains in two terms:(i) the energy cost to distort the dimers from the optimal geometryof a hypothetical isolated fragment (EDIS) and (ii) the pure interaction energy (EINT) between the already distorted fragments  were represented with thePople 6-31+G58 and Dunning’s aug-cc-pVDZ59 Gaussian-type basis sets, respectively. TheHg(II) core was taken into account with the pseudopotential associatedwith aug-cc-pVDZ basis set.60These energy partition schemes are standardin computational chemistryand examples where they have been applied with success can be foundin the literature.2, HPip and 4,4′-bipyin DMF as a solvent, three different crystalline products, namely,{[Hg(Pip)2]·DMF}n (P1A and P1B) and[Hg(Pip)2]n (2) have been isolated (From the reaction of Hg(OAc)isolated 1 dependiP1A and P1B was observed when the reaction was performed in DMF at 95 °C.Cooling down of the saturated solution for 1 h resulted in a crystalsmixture of P1A and P1B. The main differencebetween both crystals at first sight is the size, being P1B larger than P1A. Instead, the formation of 2 is achieved at RT in DMF, MeOH, and Milli-Q water or also at 95°C in MeOH and Milli-Q water. The different formation of themixture P1A and P1B or 2 couldrely on the different solubility of the final complexes under thereaction conditions. Compound 2 rapidly precipitatesas a yellow powder when the reaction is performed at RT in DMF, whileit is soluble at 95 °C. We have performed the reaction in a temperaturerange of 25–115 °C, once every 10 °C and the completedissolution and formation of the polymorphs is only observed as of75 °C and rapidly precipitates as temperature drops. Over 105°C, complexes start to decompose. Their low solubility seemsto facilitate the crystallization of different polymorphic intermediates,which suddenly nucleated and grew as temperature dropped.9 Therefore, temperature and thereby solubilityare the key factors to the formation of the polymorphic mixture.The concomitant crystallization of 16 The ability of the linkers to arrange into differentpolymorphic forms relies on directing factors inter alia time, temperature, concentration, solvent, or additional anionsduring the crystallization step.12 Therefore,we modified the synthetic conditions trying to isolate P1A and P1B. These polymorphs were synthesized as singlecrystals by changing concentration, time, and temperature or addingHOAc to identify the formation of the polymorphs. After the initiallyfound concomitant formation of P1A and P1B 2 resulted in the mixture of P1A and P1B, with P1A being the predominant form, we added equivalentamounts of HOAc to incorporate OAc– anions duringthe recrystallization of 2 in DMF at 95 °C. Interestingly,the addition of the OAc– anions drove the formationof P1A , and the phase purity of 2 was confirmed bypowder X-ray diffraction (PXRD) . We also provide the PXRD pattern of P1B, confirmed by unit cell measurements, to ensure the absenceof 2 in the sample . The interconversion between P1B and 2 was followed by PXRD , elemental analysis (EA), FTIR-ATRand P1A and P1B, P1A, P1B, and 2. The FTIR-ATR spectrum of the mixture of P1A and P1B is a combination of the spectra of the isolated products.The absence of bands in all of the FTIR-ATR spectra between 2630 and2518 cm–1, attributable to hydrogen-bonded ν(O–H)HPip and at 1667 cm–1 from ν(C=O)HPip, indicates the deprotonation of the HPip ligand. The correspondingcarboxylate bands appear at 1542 cm–1 (P1A), 1540 cm–1 (P1B), or 1560 cm–1 (2) for νas(COO) andat 1427 cm–1 (P1A), 1428 cm–1 (P1B), or 1430 cm–1 (2) for νs(COO) . The difference between these bands (Δ= νas(COO) – νs(COO)) revealsthe coordination modes of the carboxylate linkers:61 115 cm–1 (P1A), 112 cm–1 (P1B), and 130 cm–1 (2), suggesting bidentate chelate (μ1–η2) coordination mode in the three compounds.In addition, the spectra of P1A and P1B showa characteristic peak at 1658 cm–1 correspondingto the ν(C=O)DMF.6262 Additional bands from the aromaticrings have also been identified.64We have recorded the FTIR-ATR spectra of 1H NMR spectra of P1A and P1B mixtureand 2 have been recorded in DMSO-d6 . In the mixture of P1A and P1B, the presence of DMF is confirmed by signals at 7.94, 2.88, and2.72 ppm. The spectra show aromatic signals from the Pip linkers between7.58 and 6.95 ppm and the −CH2–from the dioxole group at 6.10 ppm (P1A + P1B) and 6.08 ppm (2). The two signals from the 4,4′-bipyappear between 8.77 and 7.89 ppm. The 13C{1H}and DEPT-135 NMR spectra of 2 display the carboxylateband at 169.07 ppm and the −CH2– of the dioxole at 101.66 ppm. The remaining C signals appear between 150.63 and 108.81 ppm. DEPT-135experiment was required to ensure the correct assignation of the carbonatoms from the aromatic rings .The P1A), 46.8 mg (P1B), or 81.2 mg(2). The TG analysis of 2 exhibits a flatprofile without any mass loss up to 187 °C. Besides, its decompositiontemperature is set to 199 °C from the DTA data . The TG analysis of P1A and P1B evidences the loss of a DMF molecule between 50 and 121 °C (P1A)or between 90 and 126 °C (P1B), and no more thermalevents were observed until decomposition, being stable up to 193 °C(P1A) or 199 °C (P1B). From these data,it seems that the release of DMF by heating is favored in P1A with respect to P1B .TG–DTA determinations ofthe complexes were performed using79.9 mg DMF DMF 2. Once tP1A and P1B) crystallize in the triclinic P1̅ space group,whereas 2 crystallizes in the monoclinic C2/c space group. All of them present the same connectivityforming zigzag 1D polymeric structures, in which hexacoordinated Hg(II)centers bearing [HgO4N2] cores are assembled by bridging 4,4′-bipy ligands along the [1̅01](P1A) (P1B) (2) , a seldom reported geometry present ina few complexes and reserved to Cd65 orHg66 when they are coordinated to linkersforcing geometric constrains. The geometric distortions have beenevaluated by the low continuous-shape measure (CShM) value S. The resulting S valuesagree with the better fitting of P1A (S = 5.748) and P1B  with a PPY-6 and the pairing of 2 with a trigonal prismatic geometry . P1A is assembled by planar 4,4′-bipylinkers (Cg(1)–Cg(1); torsion angle, χ = 1.8(11)°)(P1B contains four Hg(II) centers connected by 4,4′-bipyligands oriented with a χ up to 34.4(5)° (Cg(1)–Cg(3),χ = 8.0(5)°; Cg(5)–Cg(6), 3.5(5)°; Cg(2)–Cg(4),34.4(5)°; Cg(7)–Cg(7), 0.1(6)°) (2 and P1A only have a single Cg(1)–Cg(1) χ value of1.8(11)° (P1A) and 0.1(3)° (2).Both polymorphs (1](P1A) 3a, ·DMF formulaunit. The energy difference between the two polymorphs is small, andthis is in agreement with the formation of the two polymorphs andthe possibility of selectively obtaining one of the two species. Apartition energy scheme (P1B. We decomposed the association energy (E) between two metal dimers of vicinal chains  in two terms: (i) distortion of the two dimers withrespect to a hypothetical isolated dimer and (ii) the interactionenergy between the distorted fragments (further details can be foundin the –1). In contrast,the interaction energy between the distorted fragments differs significantly,and it is larger for the vicinal chains of P1B. Consequently,the association between vicinal chains is 21.0 and 36.1 kJ·mol–1 stronger for P1B than for P1A, and this suggests that the thermodynamic preference for P1B originates mainly from metal cation···π andπ···π interactions, rather than the distortionarising from crystal packing. Remarkably, Hg(3) and Hg(4) centers,which present the less conventional coordination environment, showthe smallest distortion energies and the highest interaction energies,thus suggesting that the final geometry around the metal center istuned by several factors.With the aim of getting further insightsinto the relative stabilities of y scheme 7 was perP1A, P1B, and 2 . Under UV excitation of a pulse laser beam at λexc = 335 nm, the samples display the corresponding emissionspectra with emission maxima (λmax-em) locatedat 565 nm (P1A), 591 nm (P1B), and 562 nm(2). The three spectra are composed of unstructured bandssuggesting charge transfer transitions in character (max-em = 401 nm).68 The emission of P1B is considerablyblue-shifted, whereas P1A and 2 presentcloser emission maxima. All of them present moderate Stokes shiftsof 12 152 cm–1 (P1A), 12 930cm–1 (P1B), and 12 057 cm–1 (2). As displayed in CIE 1931 chromaticitydiagrams, both P1A and 2 display yellowemission colors (λmax-em), while P1B is reddish-orange at being irradiated under the selected λexc .A thorough study of the photophysical properties of biphenyl and relatedaromatic ligands69 evinced how molecularstructure affects the shape and wavelength position of the emissionspectrum. The case of biphenyl molecule remarks how the gain or lossof planarity modifies the absorption and emission properties. Sinceits increase is rendered to sharper spectra and better quantum yields,the loss of planarity tends to provoke larger stokes shifts. Noteworthilystrong π···π stacking as well as Hg···πinteractions open relaxation pathways to nonradiative decay processesand quench fluorescence.6 Therefore, theremarkable quenching in the emission of P1A and P1B compared to 2 could be explained by boththe combination of intermolecular Hg···π andπ···π interactions, being the former onlypresent in P1A and P1B. Besides, the aromaticrings in 2 are on the brink of 4.0 Å for an effectiveπ···π interaction  or 3.632 Å (P1B). By the sametoken, the larger stokes shift of P1B could be understoodby considering the 4,4′-bipy torsion angles. Complexes P1A (χ = 1.8(11)°) and 2 (χ= 0.1(3)°)  and 34.4(5)°°) 2 and 6 p34.4(5)°3 with thP1A and P1B), initially found to be concomitantly formed,as well as their desolvated form 2. Interestingly, theseparation between P1A and P1B was achievedby temperature or anion-template-dependent formation. Such controlin polymorphism is scarce, especially when both forms tend to concomitantlycrystallize. The crystal structures of the three compounds have beendeeply analyzed revealing that Hg(II) is able to accommodate severedistortions and access to an uncommon distorted pentagonal pyramidalgeometry. Those differences combined with the conformations of the4,4′-bipy ligands resulted in significant variations of theirphotophysical properties. Besides, distortions in P1B not only modify the emission maxima, but, according to periodic-DFTcalculations, they are counterbalanced leading to a more stable formas a consequence of stronger Hg(II)···π and π···πinteractions. Therefore, this work contributes to the understandingof structure–property relationship in coordination polymersand provides an example of controlling the formation of concomitantpolymorphs.We have successfullyisolated two polymorphic forms (


After this article  was publSpecifically:In Figure 1A in this article, the 0 RBV (μg/ml), 100 IFN-α (IU/ml) panel appears similar to the 40 RBV (μg/ml), 10 IFN-α (IU/ml) panel; the 10 RBV (μg/ml), 0 IFN-α (IU/ml) panel appears similar to the 20 RBV (μg/ml), 10 IFN-α (IU/ml) panel; and the 20 RBV (μg/ml), 0 IFN-α (IU/ml) panel appears similar to the 20 RBV (μg/ml), 1000 IFN-α (IU/ml) panel.In Figure 1D in this article, the 0 RBV (μg/ml), 100 IFN-α (IU/ml) panel appears similar to the 10 RBV (μg/ml), 10 IFN-α (IU/ml) panel; and the 0 RBV (μg/ml), 250 IFN-α (IU/ml) appears similar to the 20 RBV (μg/ml), 250 IFN-α (IU/ml) panel.PLOS ONE Editors issue this Expression of Concern.Follow-up on these issues is ongoing; in the meantime, the In addition, please note that reference 16 in this article .


Therefore, Ham-based chelating agents will produce 99mTc-labeled compounds with a bivalent targeting scaffold. For their universal usage, we developed a novel Ham-based bifunctional chelating agent, “Ham-Mal”, with a maleimide group that can easily conjugate with a thiol group, for to preparing 99mTc-labeled bivalent ligand probes. Ham-Mal was synthesized by a four-step reaction, and then reacted with cysteine or c(RGDfC) to produce Ham-Cys or Ham-RGD. These precursors were reacted with 99mTcO4- for 10 min under room temperature to obtain 99mTc-(Ham-Cys)2 and 99mTc -(Ham-RGD)2. The cellular uptake level of 99mTc-(Ham-RGD)2 by U87MG (high Integrin ɑvβ3 expression) cells was significantly higher than that by PC3 (low Integrin ɑvβ3 expression) cells at 60 min after the incubation, and the uptake was significantly suppressed by pre-treatment for 15 min with excess c(RGDfK) peptide. In the in vivo study with U87MG/PC3 dual xenografted BALB/c-nu mice, the radioactivity of U87MG tumor tissue was significantly higher than that of PC3 tumor tissue at 360 min after the administration of 99mTc-(Ham-RGD)2. These results suggest Ham-Mal may have potential as a bifunctional chelating agent for 99mTc-labeled bivalent ligand probes. Hydroxamamide (Ham) is a thiol-free chelating agent that forms technetium-99m ( To radiolabel 99mTc, many chelating moieties have been developed and used widely4.Technetium-99m  is a thiol-free chelating agent of oxo-technetium(V) [Tc(V)] known to produce 15. To estimate the conditions of the 99mTc radiolabeling reaction, we synthesized “Ham-Cys”, which was acquired by the reaction with Ham-Mal and cysteine, and then performed the 99mTc radiolabeling reaction to produce “99mTc-(Ham-Cys)2” under several reaction conditions. In addition, to evaluate the utility of Ham-Mal as a 99mTc-bifunctional chelating agent, we synthesized “99mTc-(Ham-RGD)2” whose precursor, “Ham-RGD”, was acquired by reacting Ham-Mal with c(RGDfC), a cyclic arginine-glycine-aspartic acid (RGD) peptide with a high affinity to Integrin ɑvβ3, a member of the integrin superfamily of adhesion molecules16.In this study, we designed and synthesized a novel Ham-based bifunctional chelating agent “Ham-Mal” to simplify the development of Ham-based SPECT imaging probes Fig. . As a ch1 and then its maleimide group was protected by furan to produce 2. A Ham group was then introduced into 2 by reacting with hydroxylamine to produce 3. Next, Ham-Mal (4) was acquired by deprotecting the maleimide group of 3, with a total yield of 7.2% from 4-aminobenzonitrile. Ham-Cys and Ham-RGD were acquired by reacting Ham-Mal (4) with cysteine and c(RGDfC), with yields of 23% and 64%, respectively.The synthesis of Ham-Mal, Ham-Cys, and Ham-RGD is outlined in Fig. 99mTc labeling of Ham-Cys was performed by reacting it with 99mTc pertechnetate and tin(II) tartrate hydrate as a reducing agent in a solvent of acetic acid/ethanol (1/4) or phosphate buffer (pH 7.4), with different final concentrations of Ham-Cys (10 or 20 mM) 2 reacted in phosphate buffer were markedly higher than those in acetic acid/ethanol solvent  (Table 99mTc-(Ham-RGD)2 was obtained by reacting Ham-RGD  with 99mTc pertechnetate and tin(II) tartrate hydrate as a reducing agent in the solvent of phosphate buffer (pH 7.4), with a radiochemical yield of a 49.0 ± 4.8% and radiochemical purity of over 95% 2 and 99mTc-(Ham-RGD)2 did not contained the respective precursor   Fig. . The rad/ethanol /4 or pho/ethanol /4 or pho99mTc-(Ham-RGD)2 is expressed as the %dose/mg protein 2 in mouse plasma was evaluated by incubating the probe in murine plasma at 37 °C for 60 and 180 min. The radiochemical purity of 99mTc-(Ham-RGD)2 was 37.3 ± 3.7% (60 min) and 15.7 ± 5.8% (180 min), respectively.The in vitro stability of 99mTc-(Ham-RGD)2 in each organ was expressed as the %ID/g, except for that in the stomach and thyroid, which was expressed as the %ID (Table 99mTc-(Ham-RGD)2 was excreted mainly from the kidneys. The radioactivity of U87MG tumors (high Integrin ɑvβ3 expression) was significantly higher than that of PC3 tumors from 5 min after the administration of 99mTc-(Ham-RGD)2. In addition, the accumulation in U87MG tumors was reduced by blocking with excess c(RGDfK) at 180 min after administration [6.75 ± 1.73%ID/g (non-blocking) vs. 4.00 ± 0.68%ID/g (blocking) (p < 0.01)], but there was no significant difference in accumulation in PC3 tumors between the non-blocking and blocking groups [1.30 ± 0.35%ID/g (non-blocking) vs. 1.29 ± 0.12%ID/g (blocking) (p = 1.00)] 2 ] having a thiol group with Ham-Mal via the thiol-maleimide reaction, and obtained Ham-Cys and Ham-RGD, respectively  and then the appearance of two peaks because it is difficult to synthesize the analogues of the rhenium-Ham complex7. To search for an appropriate 99mTc radiolabeling reaction condition, we first evaluated the reaction of Ham-Cys and 99mTcO4- with different concentrations of Ham-Cys  in different solvents [phosphate buffer (pH 7.4) and acetic acid/ethanol = 1/4]. In our previous study, we used an acetic acid/ethanol mixture as the solvent for 99mTc radiolabeling reactions of compounds containing Ham13 to prevent forming by-products ; however, the radiochemical yields were low  2 in phosphate buffer (pH 7.4) proceeded with radiochemical yields of 68.7 ± 2.4%  and 75.3 ± 4.5% (20 mM)  were observed  Ham-RGD in phosphate buffer (pH 7.4), and acquired 99mTc-(Ham-RGD)2 in a radiochemical yield of a 47% with a radiochemical purity of over 95% 2, and evaluated it as an Integrin ɑvβ3-targeting probe. Integrin ɑvβ3 is a well-known biomolecule that is closely related to the neovasculature and metastasis of cancer18. In addition, c(RGDfC), a targeting ligand of 99mTc-(Ham-RGD)2, is known to have a high affinity to Integrin ɑvβ319. In the in vitro study, the cellular uptake level of 99mTc-(Ham-RGD)2 in U87MG cells (high Integrin αvβ3 expression) was significantly higher than that in PC3 cells (low Integrin ɑvβ3 expression)  2 in U87MG cells was significantly reduced by pretreatment with excess non-radiolabeled cyclic RGD peptide [c(RGDfK)]  2 was taken up specifically by cells with high Integrin ɑvβ3 expression.To confirm that 01) Fig. . In addi99mTc-(Ham-RGD)2 also accumulated highly in U87MG tumors compared with PC3 tumors from 5 min after its i.v. administration (Table 99mTc-(Ham-RGD)2 accumulated specifically in tumors with high Integrin ɑvβ3 expression. We also performed SPECT/CT imaging study of 99mTc-(Ham-RGD)2, and confirmed that 99mTc-(Ham-RGD)2 can selectively visualize U87MG tumors, consistent with the in vivo biodistribution study 2 as a SPECT imaging probe targeting tumors with high Integrin ɑvβ3 expression. In the in vivo biodistribution study, we also observed high radioactivity in the kidneys  is known to accumulate highly22, less radioactivity was observed at 360 min after the administration of 99mTc-(Ham-RGD)2 (Table 99mTc-(Ham-RGD)2 in mouse plasma; however, we found that the radiochemical purity decreased in a time dependent manner, and some unknown hydrophilic metabolites emerged 2 in plasma is the limitation, and further study for redesigning the bifunctional chelate to improve the stability would be needed for use it in in vivo study.In the in vivo biodistribution study, on Table , which sudy Fig. . These rd Figure . Given t24 and isocyanide26, have been reported to form multivalent complexes with 99mTc. These chelates reacted with 99mTc effectively at a low concentration of the precursors, but their reaction temperature was high (100–120 °C). On the other hand, the precursors based on Ham-Mal reacted with 99mTc under moderate conditions (at room temperature), which is suitable for radiolabeling thermally instable compounds such as proteins (e.g. antibody and its fragment). For radiolabeling 99mTc with those thermally instable compounds, some chelates such as hydrazinonicotinamide (HYNIC) have been used28. Given that these chelates form 99mTc-complexes with a metal-to-ligand ratio of 1:1, Ham has an advantage in terms of developing 99mTc-labeled compounds with a bivalent targeting scaffold, so that Ham-Mal would be a promising bifunctional chelate for developing those 99mTc-labeled compounds.To date, several chelating groups, such as D-penicillamine99mTc-chelating agent based on Ham, “Ham-Mal”. 99mTc radiolabeled agents were acquired easily by reacting Ham-Mal and the targeting ligands via thiol-maleimide reaction, and then by the 99mTc-radiolabeling reaction under moderate conditions. The acquired 99mTc radiolabeled agent [99mTc-(Ham-RGD)2 in this study]demonstrated good visualization of specific organs expressing the target biomolecule. This study suggests that Ham-Mal is available for the development of 99mTc-radiolabeled SPECT imaging probes containing bivalent targeting ligands.In this study, we designed and synthesized a novel bifunctional 99mTc-NaTcO4 was purchased from Nihon Medi-Physics Co., Ltd. . W-Prep 2XY  was used for silica gel column chromatography on a Hi Flash silica gel column . 1H NMR and 13C NMR spectra were recorded on a JNM-ECS400  with tetramethylsilane as an internal standard. Coupling constants are reported in Hertz. Multiplicity was defined as singlet (s), doublet (d), or multiplet (m). High-resolution mass spectrometry (HRMS) of fast atom bombardment (FAB) and electrospray ionization (ESI) were carried out with a JEOL JMS-700 (JEOL) and a LCMS-IT-TOF , respectively. High-performance liquid chromatography (HPLC) was performed with a Shimadzu system .All reagents were obtained commercially and used without further purification unless otherwise indicated. 3, and brine. The organic layer was dried over Na2SO4 and the solvent was evaporated under vacuo to afford 3.38 g of 1 (yield: 85%). 1H NMR  δ 7.25 , 7.59 , 7.98 . 13C NMR  δ 109.88, 118.49, 126.86, 133.06, 135.02, 135.88, 169.34.A solution of 4-aminobenzonitrile  and maleic anhydride  in 1,4-dioxane (30 mL) was stirred at 100 °C for 1 h. To the reaction mixture, ammonium persulfate  and dimethyl sulfoxide (DMSO) (2.84 mL) were added and the reaction mixture was heated to 100 °C for 3 h. Thereafter, the reaction mixture was filtered and dioxane was removed under vacuo. The residue was dissolved in ethyl acetate and washed with diluted HCl, saturated aqueous NaHCO1  in acetonitrile (10 mL) was mixed with furan (5 mL) and stirred at 60 °C for 8 h. The solvent was evaporated and ethyl acetate was added, then a solid precipitated out. The material was filtered and washed with ethyl acetate. The filtrated solution was concentrated to afford more of the product, which was also filtered and washed with ethyl acetate. The combined solid portions were dried under high vacuum at room temperature overnight to afford 2.67 g of 2 (yield: 60%). 1H NMR  δ 3.13 , 5.26 , 6.62 , 7.47 , 7.98 . 13C NMR  δ 47.71, 80.91, 110.98, 118.29, 127.55, 133.19, 136.05, 136.72, 175.27. HR-MS  m/z calcd. for C15H11N2O3+, 267.0770; found 267.0768 [M + H]+.A solution of N,N-dimethylformamide (DMF) (5 mL) and H2O (2 mL) was added to a solution of 2  in DMF (10 mL). The reaction mixture was stirred at room temperature for 3 h. Then, H2O was added to the solution and diluted with ethyl acetate. The organic layer was dried over Na2SO4 and the solvent was evaporated under vacuo. The residue was recrystallized with chloroform and dried under high vacuum at room temperature overnight to afford 287 mg of 3 (yield: 19%). 1H NMR  δ 3.08 , 5.24 , 5.88 , 6.60 , 7.19 , 7.74 , 9.74 . 13C NMR  δ 47.51, 80.80, 126.01, 126.46, 132.41, 133.41, 136.66, 150.27, 175.68. HR-MS  m/z calculated for C15H14N3O4+, 300.0984; found 300.0985 [M + H]+.A solution of hydroxylamine hydrochloride  and triethylamine  in 3  in DMF (6 mL) and toluene (18 mL) was stirred at 115 ºC for 2 h. After evaporation of the solvent, the residue was suspended in H2O and then filtered. The solid portion was dried under high vacuum at room temperature overnight to afford 231 mg of 4 (yield: 74%). 1H NMR  δ 5.87 , 7.20 , 7.33 , 7.75 , 9.72 . 13C NMR  δ 125.85, 126.28, 131.94, 132.65, 134.73, 150.29, 169.82. HR-MS  m/z calculated for C11H10N3O3+, 232.0722; found 232.0725 [M + H]+.A solution of 2O (100 µL) was adjusted to pH 7–8 by 0.1 M NaOH and then added to a solution of 4  in DMF (500 µL). The reaction mixture was stirred at room temperature for 1 h. Then, the solvent was evaporated and the residue was purified by RP-HPLC on a Cosmosil 5C18-AR-II column  using the mobile phase [H2O (0.1% TFA)/acetonitrile (0.1% TFA) = 90/10] at a flow rate of 4.0 mL/min to give 3.5 mg of 5 (yield: 23%). 1H NMR  δ 3.07–3.14 , 3.24–3.34 , 4.08–4.18 , 7.39 , 7.70 . ESI-HRMS m/z calculated for C14H17N4O5S+, 353.0920; found, 353.0919 [M + H]+.A solution of L-cysteine  in H2O (200 µL) was added to a solution of 4  in DMF (500 µL). The reaction mixture was stirred at room temperature for 1 h. Then, the solvent was evaporated and the residue was purified by RP-HPLC on a Cosmosil 5C18-AR-II column  using the mobile phase [H2O (0.1% TFA)/acetonitrile (0.1% TFA) = 90/10 (0 min) to 70/30 (30 min)] at a flow rate of 4.0 mL/min to give 1.8 mg of 6 (64% yield). HR-MS  m/z calculated 810.2993, found 810.2988 [M + H]+.A solution of c(RGDfC)  in H5  in an acetic acid/ethanol mixture  or phosphate buffer , 100 μL of 99mTc-NaTcO4 solution (18–30 MBq) and 15 μL of 3 mM tin(II) tartrate hydrate solution in H2O were added. The reaction mixture was incubated at room temperature for 10 min and purified by RP-HPLC on a YMC-Triart C18  using the mobile phase [10 mM phosphate buffer (pH 7.4)/acetonitrile = 100/0 (0 min) to 70/30 (30 min)] at a flow rate of 1.0 mL/min. The two peaks  were collected, mixed, and used as 99mTc-(Ham-Cys)2 in the following study. The radiochemical yield of 99mTc-(Ham-Cys)2 was calculated by dividing the radioactivity of the collected two peaks by the radioactivity of the reaction mixture measured just before the HPLC purification, in accordance with our previous study11. The acquired 99mTc-(Ham-Cys)2 was analyzed by RP-HPLC using the same method described above. The radiochemical purity was calculated by dividing the radioactivity of the two peaks derived from the 99mTc-(Ham-Cys)2 by the total radioactivity acquired from the HPLC chart  in phosphate buffer , 100 μL of 99mTc-NaTcO4 solution (18–33 MBq) and 15 μl of 3 mM tin(II) tartrate hydrate solution in H2O were added. The reaction mixture was incubated at room temperature for 10 min and purified by RP-HPLC on a YMC-Triart C18  using the mobile phase [10 mM phosphate buffer (pH 7.4)/acetonitrile = 80/20 (0 min) to 60/40 (30 min)] at a flow rate of 1.0 mL/min. The two peaks  were collected, mixed, and used as 99mTc-(Ham- RGD)2 in the following study. The radiochemical yield of 99mTc-(Ham-RGD)2 was calculated by dividing the radioactivity of the collected two peaks by the radioactivity of the reaction mixture measured just before the HPLC purification, in accordance with our previous study11. The acquired 99mTc-(Ham-RGD)2 was analyzed by RP-HPLC using the same method described above. The radiochemical purity was calculated by dividing the radioactivity of the two peaks derived from the 99mTc-(Ham- RGD)2 by the total radioactivity acquired from the HPLC chart  or Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% fetal bovine serum, and 100 U/mL of penicillin and streptomycin in a 5% CO2 incubator at 37 ºC. These cells were sub-cultured overnight in 12-well plates (4 × 105 cells/1 mL), and then pre-incubated in DMEM (1 mL) with or without c(RGDfK)  for 30 min at 37 ºC in a humidified atmosphere containing 5% CO2. After the pre-incubation, the medium was exchanged with DMEM containing 99mTc-(Ham-RGD)2  and then incubated for 60 min. After the incubation, the cells were washed twice with phosphate-buffered saline (PBS) (1 mL) and then lysed with 1 M NaOH (0.4 mL). The radioactivity in the lysates was measured by a Wallac WIZARD 2470 gamma counter  and total protein concentrations in samples were measured using the bicinchonic acid method.According to the previously reported study, U87MG human glioblastoma cells  and PC3 human prostate carcinoma cells  were used as Integrin ɑ99mTc-(Ham-RGD)2 in mouse plasma was performed in accordance to our previous study with some modification11. In brief, 99mTc-(Ham-RGD)2 (about 500 kBq) was added to prepared fresh murine plasma (200 μL) collected from ddY mice . After incubating the solution at 37 °C for 60 and 180 min, acetonitrile (200 μL) was added. Subsequently, the solution was centrifuged  and the supernatant was analyzed using RP-HPLC on a YMC-Triart C18  using the mobile phase [10 mM phosphate buffer (pH 7.4)/acetonitrile = 80/20 (0 min) to 60/40 (30 min)] at a flow rate of 1.0 mL/min.The in vitro stability of https://arriveguidelines.org) and the institutional guidelines for animal care of Kyoto University. BALB/c athymic nude mice  supplied by Japan SLC, Inc.  were housed under a 12-h light/12-h dark cycle, and given free access to food and water. PC3 cells [5 × 106 cells/0.1 mL of RPMI1640 and Geltrex  at a ratio of 1:1] were subcutaneously inoculated into the left flank of mice. At 2 weeks after the inoculation of PC3 cells, U87MG cells [5 × 106 cells/0.1 mL of DMEM and Geltrex (Thermo Fisher Scientific) at a ratio of 1:1] were subcutaneously inoculated into the right flank of mice, and a biodistribution study was performed after a 2-week growth period. 99mTc-(Ham-RGD)2  was injected intravenously into the tumor-bearing mice. Mice were sacrificed by decapitation at 5, 60, 180, and 360 min after administration. The tumor, blood, muscle, and other organs were then excised, and their radioactivity and weight were measured. For the blocking study, c(RGDfK) (10 mg/kg weight) dissolved in 0.1 mL of saline was injected intravenously into the tumor-bearing mice. At 15 min after the injection of c(RGDfK), 99mTc-(Ham-RGD)2  was injected intravenously into the mice, which were sacrificed by decapitation at 180 min after administration. The tumor, blood, muscle, and other organs were then excised, and their radioactivity and weight were measured.Animal experiments were approved by the Kyoto University Animal Care Committee, and performed in accordance with ARRIVE guidelines (30. 99mTc-(Ham-RGD)2  was injected intravenously into the tumor-bearing mice prepared by the above method. At 45 and 165 min after administration, the mice were anesthetized with 1.5–2.0% isoflurane for approximately 10 min, and then CT imaging was performed with X-ray sources set to 60 kVp and 360 μA. After CT imaging at 60 and 180 min after the administration of 99mTc-(Ham-RGD)2, SPECT imaging was performed with multi-pinhole collimators (1.0 mm diameter) and the projection data were acquired using a 20% energy window centered at 140 keV for 99mTc, with a 360° circular orbit, 60-s projection time, and 32 projection angles. The acquired SPECT data were reconstructed by a three-dimensional ordered subset expectation maximization (OSEM) algorithm with CT-based attenuation correction and then analyzed using Amide software .SPECT/CT imaging study was performed using a Triumph LabPET12/SPECT4/CT  by the previously reported method with modificationt-test was used to assess the significance of differences. Differences at the 95% confidence level (P < 0.05) were considered significant.Data are presented as the mean ± S.E.M (for the in vitro study) or the mean ± S.D (for the in vivo study). In the in vitro study, statistical analyses were performed using 2-way ANOVA following the Tukey–Kramer test with JMP 14 software . For the in vivo study, the Student’s Supplementary Information.


However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity. Pharmacokinetics of drugs show large interindividual variability, and some drug-metabolizing enzymes and transporters are involved in the variability. Cytochrome P450 (CYP)3A is a major subfamily of metabolic enzymes involved in the metabolism of some drugs in the liver and small intestine . The maiMidazolam has been reported to be useful and considered a standard probe for CYP3A phenotyping , 5. AlthTo overcome these problems, 4β-hydroxycholesterol (4β-OHC) has drawn attention as an endogenous phenotyping probe for CYP3A activity. 4β-OHC is formed by CYP3A4 and CYP3A5 , 12 and et al. . The method was applied to measure plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers and patients with chronic kidney disease (CKD).7  were purchased from Avanti Polar Lipids, Inc (Alabama) and 4α-OHC standard from Toronto Research Chemicals . The standards of 7α-OHC, 7β-OHC, 22(R)-OHC, 22(S)-OHC, 24(S)-OHC, 25-OHC, and 27-OHC, isomers of 4β-OHC and 4α-OHC, were purchased from Sigma-Aldrich (St. Louis). Human serum albumin (HSA) was purchased from FUJIFILM Wako Pure Chemical Corporation . Other reagents  were purchased from Tokyo Chemical Industry Co, Ltd . Other solvents  were of the highest analytical quality and were purchased from FUJIFILM Wako Pure Chemical Corporation.The standards of 4β-OHC and 4β-OHC-DNine healthy volunteers who fasted for at least 8 h before blood sampling were recruited in this study. Blood was collected in tubes containing EDTA anticoagulant, centrifuged, and plasma samples were frozen at −80°C until measurement. Total cholesterol level was measured by enzymatic assay. This study was approved by the Ethics Committee of Meiji Pharmaceutical University . Prior explanations were provided to all volunteers to inform the scientific purpose of the study, and all volunteers gave written informed consent for participation in this study. Plasma obtained from healthy volunteers was used as unadulterated plasma for preparation of quality control (QC) samples and for validation purposes.https://link.springer.com/content/pdf/10.1007%2Fs10157-014-0949-2.pdf, accessed 16 July 2020). Blood samples collected in tubes containing EDTA anticoagulant were centrifuged, and plasma samples were frozen at −80°C until assay. Total cholesterol level was measured by enzymatic assay. This study was approved by the Ethics Committees of Oita University Hospital  and the Ethics Committee of Meiji Pharmaceutical University . Prior explanations were provided to all patients to inform the scientific purpose of the study, and all patients gave written informed consent for participation in this study.Blood samples were collected from 15 patients with stage 3–5 CKD and from 14 patients with stage 5D CKD undergoing hemodialysis in Oita University Hospital. The patients fasted for at least 8 h before blood sampling. CKD stages were classified by estimated glomerular filtration rate according to the evidence-based clinical practice guideline for CKD published by Japanese Society of Nephrology . The stock solutions were stored at −80°C.Independent 4β-OHC and 4α-OHC stock solutions were made separately for the preparation of calibration and QC samples. 4β-OHC and 4α-OHC were weighed in volumetric flasks and dissolved in 2-propanol . The internal standard 4β-OHC-DAll calibrating and QC solutions contained a mixture of 4β-OHC and 4α-OHC. Eight different calibrating solutions containing equal concentrations of 4β-OHC and 4α-OHC at 10, 20, 40, 100, 400, 1000, 4000, and 10,000 ng/ml  were prepared by diluting the respective calibrating stock solutions with 2-propanol. QC solutions were prepared at lower limit of quantification (LLOQ), low (QCA), medium (QCB), and high (QCC) concentrations containing equal concentrations of 4β-OHC and 4α-OHC at 10, 30, 500, and 8000 ng/ml , respectively, by diluting the respective QC stock solutions with 2-propanol.A derivatization solution was prepared by mixing the following reagents: 250 mg of 2-methyl-6-nitrobenzoic anhydride, 75 mg of 4-dimethylamino-pyridine, 200 mg of picolinic acid, 7.5 ml of pyridine, and 1 ml of triethylamine. The derivatization solution was freshly prepared just before plasma sample preparation.7) and 2.5 μl of 2-propanol for volume adjustment. After adding 200 μl of 28% sodium methoxide methanol solution, the mixture was vortex-mixed for 30 s and left at room temperature (15–25°C) for 20 min. Then, 250 μl of water and 1 ml of n-hexane were added, vortex-mixed for 5 min, and centrifuged . The supernatant (700 μl) was transferred to a clean 1.5-ml safe-lock tube and evaporated to dryness under a N2 gas stream at 40°C. The residue was reconstituted with 100 μl of derivatization solution, vortex-mixed for 30 s, and left at room temperature for 30 min. Then, 1 ml of n-hexane was added, vortex-mixed for 5 min, and centrifuged . The supernatant (700 μl) was transferred to a clean 1.5-ml safe-lock tube and evaporated to dryness under a N2 gas stream at 40°C. The residue was reconstituted with 100 μl of acetonitrile, vortex-mixed, and transferred to an autosampler vial.For measurement of 4β-OHC and 4α-OHC concentrations, plasma samples were pretreated as follows. In each 2.0-mL safe-lock tube, 50 μl of subject’s plasma sample was mixed with 50 μl of internal standards solution  with 2.5 μl of calibration solution containing 4β-OHC and 4α-OHC. Each calibration or QC sample was mixed with 50 μl of internal standards solution. Then, these samples were processed as for subjects’ plasma samples.18 column  and a Waters Acquity BEH C18 VanGuard precolumn  were used at 55°C. 0.1% aqueous formic acid (A) and acetonitrile with 0.1% formic acid (B) were used as mobile phase. The gradient started at 80% B (0.5 min). The ratio was changed linearly to 95% B within 5.5 min and maintained for another 4 min. Then, the ratio was returned to 80% B and maintained for another 2.5 min. The flow rate was 0.4 ml/min, and the injection volume was 2 μl.The UHPLC-MS/MS system  consisted of a Nexera X2 LC system and a quadrupole mass spectrometer (LCMS-8040). For chromatographic separation, a Waters Acquity BEH C7 . Multiple reaction monitoring analysis was performed using argon as collision induced dissociation gas. The MS/MS transitions for the di-picolinyl esters and the electrode voltage of Q1 prebias, collision cell Q2, and Q3 prebias were as follows: m/z 613.3→m/z 490.5, −24 V, −13 V, and −16 V, respectively, for 4β-OHC and 4α-OHC and m/z 620.3→m/z 497.6, −24 V, −15 V, and −16 V for 4β-OHC-D7 . The dwehttps://www.fda.gov/media/70858/download, accessed 21 April 2020) as described in our previous studies .Calibration curves for 4β-OHC and 4α-OHC were constructed using the calibration samples and analyte-specific multiple reaction monitoring quantifier transitions. Peak area ratios of each analyte to internal standard were calculated, and weighted linear regression (1/x) was performed for each analytical batch using the LabSolutions LCMS software (Shimadzu). Data are expressed as mean ± SD. Differences between healthy volunteers, stage 3–5 CKD patients, and stage 5D CKD patients were analyzed using one-way ANOVA with Dunnett’s posthoc test or Kruskal-Wallis test with Dunn's posthoc test. A + signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using ESI. The same mass transition (m/z 613.3→m/z 490.5) was monitored for 4β-OHC and 4α-OHC because they are the stereoisomers. The mass transition of internal standard 4β-OHC-D7 (m/z 620.3→m/z 497.6) differed from that of the two analytes by only the mass shift due to isotopic labeling.Intense [M+H]7 for 2% HSA solution (surrogate matrix), LLOQ sample, QCC sample, plasma sample of a healthy volunteer, and plasma sample of a CKD patient. Retention times were approximately 8.3 and 8.8 min after injection for 4β-OHC and 4α-OHC, respectively. In the chromatogram, 4β-OHC and 4α-OHC were separated from the isomers comprising 7α-OHC, 7β-OHC, 22(R)-OHC, 22(S)-OHC, 24(S)-OHC, 25-OHC, and 27-OHC  of the calibration curve for the calibration range of 0.5–500 ng/ml was ≥0.9986. For 4α-OHC, r2 for the same calibration range was 0.9989.For 4β-OHC, the correlation coefficient , whereas no significant difference was detected by posthoc test. There was no significant difference in the ratio of 4β-OHC to total cholesterol among three groups (P = 0.65)  B.Table 3P < 0.0001), and significant differences were also detected by posthoc test (P = 0.0045), and significant differences were also detected by posthoc test   . Posthocet al. (et al. (et al. (In 2001, Bodin et al.  reported (et al.  recently (et al.  did not  (et al. . Thus, i (et al. . Like 4β (et al. . Therefoet al. (et al. (et al. (et al. avoided the derivatization process in plasma sample preparation and monitored sodium adducted ions, causing inadequate sensitivity. Our novel method adopted a short derivatization process using picolinic acid and monitored the proton adducted ions, which increased ionization efficiency and achieved better LLOQ of 0.5 ng/ml for 4α-OHC. Our newly developed method thus has the advantage of having sufficient sensitivity to evaluate plasma 4α-OHC concentrations in different clinical settings, and it seems to be superior to previous reported methods for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma.Three studies have already reported methods of simultaneous measurement of plasma 4β-OHC and 4α-OHC concentrations. Bodin et al.  reported (et al.  reported (et al.  reported (et al. . The metet al. (et al. (We prepared plasma samples by saponification using sodium methoxide, two-step liquid-liquid extraction using n-hexane, and derivatization using picolinic acid. Sodium methoxide was used for saponification of 4β-OHC and 4α-OHC comprised in lipoproteins and was utilized in previous reports , 26, 27.et al.  and Hasa (et al. , 26. ThuValidation results for 4β-OHC and 4α-OHC are within the acceptable ranges according to the recommendations published by the US Food and Drug Administration. High apparent recovery rates and matrix effects suggest effective sample preparation and minimal ion suppression or enhancement. When samples exposed to air were stored under various in vitro conditions, plasma 4β-OHC and 4α-OHC concentrations increased after a transient decrease. However, 4β-OHC and 4α-OHC were stable when stored in safe-lock tubes, suggesting increased stability by avoiding contact with air. The elevation is speculated to be due to autooxidation of cholesterol. However, detailed mechanism of the transient decrease in plasma 4β-OHC and 4α-OHC concentrations is unknown, but other pathways such as additional autooxidation of 4β-OHC and 4α-OHC may be involved. These findings suggest that plasma samples should be stored in rocked tubes to avoid autooxidation of cholesterol to 4β-OHC and 4α-OHC.The validated UHPLC-MS/MS method was used to measure plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers and CKD patients. Plasma 4β-OHC concentrations in this study were similar to previously reported values in healthy volunteers , 23, 27,The 4β-OHC to total cholesterol ratio has been reported to be superior to 4β-OHC alone for CYP3A phenotyping . However7 were different, the matrix effect of the 4α-OHC assay was not corrected by internal standard. However, the variation due to matrix effect was not large in the 4α-OHC assay and thus appeared to have little effect on the results. Second, the rationale for the use of 4β-OHC to 4α-OHC ratio in CYP3A phenotyping is not well established. We showed the potential of 4β-OHC to 4α-OHC ratio for cross-sectional CYP3A phenotyping in healthy volunteers and CKD patients in this study, but the interindividual variability in 4β-OHC to 4α-OHC ratio was large within each group, although a significant difference was observed among three groups (Our method has some limitations. First, because the retention times of 4α-OHC and 4β-OHC-De groups . To estaIn conclusion, simultaneous quantification method for plasma 4β-OHC and 4α-OHC concentrations using UHPLC-MS/MS was developed and validated and was applied to measurement of plasma concentrations in healthy volunteers and CKD patients. Our novel method may contribute to accurate evaluation of in vivo CYP3A activity.The data supporting this study are available in the article, the supplemental data, or available from the corresponding author upon reasonable request.This article contains The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In the crystal, C—H⋯O hydrogen bonds between neighboring quinoxaline rings form chains along the  23H28N2O, the phenyl ring is inclined to the quinoxaline ring system at a dihedral angle of 20.40 (9)°. In the crystal, C—H⋯O interactions between neighbouring molecules form chains along the a-axis direction. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯H (70.6%), H⋯C/C⋯H (15.5%) and H⋯O/O⋯H (4.6%) interactions. The optimized structure calculated using density functional theory at the B3LYP/6–311 G level is compared with the experimentally determined structure in the solid state. The calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy gap is 3.8904 eV. Part of the n-nonyl chain attached to one of the nitrogen atoms of the quinoxaline ring system shows disorder and was refined with a double conformation with occupancies of 0.604 (11) and 0.396 (11).In the title molecule, C The n-nonyl chain of set A (starting from C9) has a ap, ap, ap, +sc, ap, ap, ap conformation, while for set B the conformation can be describes as ap, ap, ap, −sc, ap, −sc, ap.The title compound crystallizes in the triclinic space group it Fig. 1. The molit Fig. 1. The n-nB⋯O1 interactions  resulting in ribbon formation in the a-axis direction. Parallel ribbons show short C9—H9A⋯O1 contacts . The crystal packing shows layers of n-nonyl chains parallel to the (110) plane with layers of rings in between. Despite the presence of aromatic rings, the packing shows no C—H⋯π or π–π interactions [the shortest centroid–centroid distance is 3.8945 (15) Å for rings N1/N2/C1–C3/C8 and C18–C23]. The unit cell contains no residual solvent-accessible voids.The crystal packing is characterized by C9—H9CrystalExplorer program  is dominated by white regions representing contacts equal to the van der Waals separation and shows only one red spot  indicative of a H16B⋯H16Biii contact . A similar observation is made for the minor component  where the tiny red spot represents a H15B⋯H13Bi contact (2.316 Å).The nt Fig. 4b where et al., 2007a and b, while those delineated into H⋯H and H⋯C/C⋯H contacts are illustrated in Fig. 5c–f, respectively, together with their relative contributions to the Hirshfeld surface. The most important interaction is H⋯H, contributing 70.6% (major component) or 70.5% (minor component) to the overall crystal packing, which is reflected in Fig. 5c and d as widely scattered points of high density due to the large hydrogen content of the molecule, with a sharp tip at de = di = 0.87 Å in the case of the major component. The second most important are C—H interactions, contributing 15.5% (major component) or 15.6% (minor component), for which the fingerprint plot  shows characteristic wings with tips at ed + di ≃ 2.80 Å. Other contacts contribute only 4.6% (H⋯O/O⋯H), 4.3% (C⋯C), 2.4% (H⋯N/N⋯H), 2.2% (N⋯C/C⋯N), 0.3% (O⋯O) and 0.1% (O⋯C/C⋯O) to the Hirshfeld surface.The overall two-dimensional fingerprint plots , hardness (η), ionization potential (I), dipole moment (μ), electron affinity (A), electrophilicity (ω) and softness (σ), are collated in Table 3ΔE = ELUMO − EHOMO] of the molecule is 3.8904 eV, and the frontier molecular orbital energies, EHOMO and ELUMO, are −6.1155 and −2.2251 eV, respectively.The structure in the gas phase of the title compound was optimized by means of density functional theory. The density functional theory calculation was performed by the hybrid B3LYP method and the 6–311 G basis-set, which is based on Becke’s model -one fragment yielded multiple matches (180 hits). Of these, three compounds had an alkyl substituent on N1 and a phenyl ring on C2 comparable to (I)et al., 2021et al., 2009et al., 2021n-octyl chain on N1 instead of a n-nonyl chain. The phenyl ring in MAGBIJ is inclined to the quinoxaline ring system by 25.81 (12)°. For BUDMAP, the dihedral angles are 19.3 (1) and 30.4 (1)° for the two molecules present in the asymmetric unit. For ASAZEC, the dihedral angle is 12.90 (4)° and no disorder is observed in the n-octyl chain, which could be the consequence of the data collection being undertaken at 150 (2) K. Despite the similarity to the title compound, ASAZEC crystallizes in space group C2/c and the molecules are linked by C—H⋯π interactions and form stacks in the b-axis direction.A search of the Cambridge Structural Database -one  in dichloromethane (20 ml) were added 1-chlorononane , sodium hydroxide  and a catalytic quantity of tetra-n-butylammonium bromide. The reaction mixture was stirred at room temperature for 24 h. The solution was filtered and the solvent removed under reduced pressure. The residue thus obtained was chromatographed on a silica gel column using a hexane/ethyl acetate 9:1 mixture as eluent. The solid obtained was recrystallized from ethanol to afford colourless crystals (yield: 70%). 1H NMR  δ ppm: 0.89 ; 1.19–1.42 ; 1.65–1.76 ; 4.20 ; 7.22–8.24 ; 13C NMR  δ ppm: 14.12 (CH3); 22.67, 27.11, 27.32, 29.24, 29.36, 29.51, 31.85 (CH2); 42.68 (N—CH2); 113.59, 123.49, 128.05, 129.63, 130.22, 130.28, 130.72 (CHarom); 132.61, 133.42, 136.14, 154.11 (Cq); 154.40 (C=O).To a solution of 3-phenylquinoxalin-2(1Ueq of the parent atoms (1.5 for methyl groups). During the refinement, the difference-Fourier map revealed disorder for atoms C13, C14 and C15 of the nonyl chain and two conformations were refined with distance restraints (1.512 Å) for the C—C bonds involved and RIGU restraints for the nonyl chain C11–C17. At the end of the refinement, the occupancy factors of the two components converged to 0.604 (11) and 0.396 (11) and the final difference-Fourier map showed no residual peaks of chemical significance.Crystal data, data collection and structure refinement details are given in Table 410.1107/S2056989021009737/dj2034sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021009737/dj2034Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021009737/dj2034Isup3.cmlSupporting information file. DOI: 2110486CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their rapid activation and cytotoxic nature make them promising candidates for use in cell-based immunotherapies; however, under certain conditions, they can induce pro-tumour functions. Furthermore, upon transformation, γδ T cells can develop into aggressive lymphomas with a poor prognosis and no curative therapeutic options. Here, we provide a comprehensive summary of our current knowledge on the complex roles of γδ T cells in cancer. We discuss their anti- and pro-tumour functions in both solid and blood cancers, highlighting the key subsets involved and their potential utility in anti-cancer immunotherapy. We also discuss the mechanisms of γδ T-cell transformation, summarising the resulting γδ T-cell leukaemia/lymphoma entities and their genetic and molecular profiles, as well as current and future treatment strategies.γδ T cells are unique players in shaping immune responses, lying at the intersection between innate and adaptive immunity. Unlike conventional αβ T cells, γδ T cells largely populate non-lymphoid peripheral tissues, demonstrating tissue specificity, and they respond to ligands in an MHC-independent manner. γδ T cells display rapid activation and effector functions, with a capacity for cytotoxic anti-tumour responses and production of inflammatory cytokines such as IFN-γ or IL-17. Their rapid cytotoxic nature makes them attractive cells for use in anti-cancer immunotherapies. However, upon transformation, γδ T cells can give rise to highly aggressive lymphomas. These rare malignancies often display poor patient survival, and no curative therapies exist. In this review, we discuss the diverse roles of γδ T cells in immune surveillance and response, with a particular focus on cancer immunity. We summarise the intriguing dichotomy between pro- and anti-tumour functions of γδ T cells in solid and haematological cancers, highlighting the key subsets involved. Finally, we discuss potential drivers of γδ T-cell transformation, summarising the main γδ T-cell lymphoma/leukaemia entities, their clinical features, recent advances in mapping their molecular and genomic landscapes, current treatment strategies and potential future targeting options. The priTCRG) are located on chromosome 13 in mice and chromosome 7 in humans, and genes for the δ TCR (TCRD) are found on chromosome 14 in both mice and humans [With respect to their TCR assembly, unlike αβ T cells that have randomly-paired receptor chains, the γ and δ chain pairing is restricted to only a few common combinations, resulting in distinct γδ T-cell subsets . The gend humans . Betweend humans . As suchd humans . In humad humans  -programmed interleukin-17A (IL-17)-producing γδ T (γδT17) cells ,43 and Tce IL-17 . Three s+ Vγ9Vδ2 cells produce IL-17, are positive for CD27 and CCR6 and express type-3 signature genes such as RORC and IL23R [In contrast to murine γδ T cells, there is no association between TCR Vδ chain usage and the functional subtype of human γδ T cells; Vδ1, Vδ2 and Vδ3 γδ T cells are present most commonly as the IFN-γ-producing γδT1 subtype ,51, whilnd IL23R . Notablynd IL23R .+ γδ T regulatory (Treg)-like cells [Plasticity, or polarisation, is another important aspect of γδ T-cell physiology , resultike cells ). In theke cells . As discke cells ,65,66,67ke cells ,65,68.The dichotomy of γδ T cells in cancer immunity lies in the fact that, in general, the γδT1 effector subtype is associated with anti-tumour roles, whereas γδT17 cells have been extensively shown in murine studies to possess pro-tumour functions. In humans, it has been until recently very difficult to study this subtype due to their rarity, tissue-resident nature and lack of protocols for their in vitro expansion. However, recent advancements in this area have facilitated the study of γδT17 cells and their pro-tumour functions in humans . Herein,γδ T-cell anti-tumour responses can be categorised into: cellular fitness and stress ligand sensing, death receptor engagement, antibody dependent cytotoxicity (ADCC) and execution of T helper functions . These f+ cells by TCR engagement combined with cytokine stimulation  upregulates NKRs, correlating with CD56 expression and potent anti-tumour cytotoxicity [+ T cells can respond strongly to PAgs such as isopentenyl pyrophosphate (IPP), which is a metabolite of the mevalonate or microbial deoxyxylulose phosphate pathway [Interestingly, cellular stress can be perceived differently by the two main human γδ T-cell subtypes. While specific Vδ1 TCR ligands remain elusive, these cells are highly efficient in detecting cellular stress signals  and FAS ligand (FASL) ,103 Fig. Support+ αβ T-cell populations, γδ T cells may support B germinal centre formation [+ T cells were also shown to upregulate CD40, CD86 and HLA-DR, leading to the production of IgM by B cells [Another γδ T-cell mechanism to exert anti-tumour function is through antibody-dependent cellular cytotoxicity (ADCC). Here, a tumour antigen-specific antibody binds to the tumour cell, allowing the Fc segment to be bound by the FcγRIIIA receptor on γδ T cells, inducing γδ T-cell activation and killing of the tumour cell. This mechanism can be exploited to target and kill tumour cells using specific targeting antibodies , which were shown to enhance the cytotoxicity of stimulated/expanded Vγ9Vδ2 cells . γδ T ceormation . A more ormation . For insormation . Interesormation ,108. Sim B cells . Taken t+ T cells [+ T cells can be stimulated to release certain Th1 , Th2 (IL-4) and Th17 (IL-17) cytokines to induce DC maturation [γδ T cells can also act as excellent T helper cells to support other immune cell types involved in anti-cancer immunity. For one, they have the capacity to function as professional APCs , upregul T cells ,114. Ano T cells ,116 via  T cells , and γδ  T cells  via secr T cells ,119  have been proposed by many studies to be relevant prognostic factors; however, there appear to be differences in their anti-tumour functions across different solid cancer types. Notably, their tumour infiltration and cytolytic activity have been linked with a beneficial role for patient outcome in gastric cancer ,122, hep+ T cells, and that patients who had more responsive γδT1 cells upon stimulation had smaller residual tumour burden and increased overall survival (OS), whereas the IFN-γ secretion capacity of αβ T cells was found to have no effect on patient survival [+ TILs displayed cytotoxic capacity towards breast cancer cell lines and positively correlated with increased patient progression free survival (PFS) and OS [+ TILs were also correlated with increased PFS and, interestingly, a positive and significant correlation of tumour-infiltrating TCRα+ cells with Vδ1+ cells was reported, where the authors propose that maximal patient benefit may arise from the synergistic effect of innate-like γδ and adaptive αβ TILs [Foord et al. demonstrated that epithelial ovarian cancer-derived γδ T cells, when stimulated, produced large amounts of IFN-γ but not IL-17 or IL-10 . Furthersurvival . In line) and OS . In thes αβ TILs . In squa αβ TILs . Notably αβ TILs .Interestingly, where the role of γδ T cells in solid tumour immunity can be conflicting, blood cancers in general appear to be more susceptible to γδ T-cell-mediated anti-tumour responses than solid tumours ,138,139.+ versus Vδ2+ cells. A number of studies have demonstrated anti-tumour responses of Vγ9Vδ2+ cells against leukaemia/lymphoma models; Vγ9Vδ2 T cells transplanted into an AML xenograft mouse model were found to localise in close proximity to engrafted leukaemic cells and significantly increase survival [+ cells were also shown to have anti-tumour activity against CML cells in vitro and in vivo; however, this required the pre-treatment of CML cells with zoledronate and the administration of zoledronate plus IL-2 to mice in order to stimulate PAg expression on the tumour cells and maintain Vγ9Vδ2 T-cell activation [+ cells, which was increased to 50% upon pre-treatment of the AML cells with bisphosphonates, while 50% of samples remained resistant [+ T-cell numbers, associated with high IL-4 serum levels and high tumour cell expression of ULBPs, were correlated with stable disease in a 1-year follow-up in patients with low-grade non-Hodgkin B-cell lymphoma [+ T cells reduced tumour growth and dissemination in a CLL model [Notably, there appear to be differences in the efficacy of haematopoietic cancer cell killing by Vδ1survival . Vγ9Vδ2+tivation . Similaresistant . In a clesistant . On the lymphoma . In muriLL model , and redLL model .+ B-cell-depleted bone marrow is now an established therapeutic protocol [+) Vδ1+ cells were in higher proportions compared with their Vδ2+ counterparts [The anti-tumour roles of γδ T cells in haematological cancer are further exemplified in cases of allogenic haematopoietic stem cell transplantation (aHSCT) as a treatment for leukaemia/lymphoma patients. aHSCT of αβ T-cell/CD19protocol ,154,155,protocol . Strikinprotocol . The expprotocol  and AML protocol , and werprotocol . Examinaterparts .The recognition of leukaemia/lymphoma cells by Vγ9Vδ2 T cells is expectedly induced by the recognition of PAgs (often stimulated by treatment with aminobisphosphonates) and is mediated by TCR stimulation and granule exocytosis ,143,156.It is not entirely clear why haematopoietic cancer cells may be intrinsically more susceptible to γδ T-cell recognition and killing compared with solid tumours. Factors such as increased activity of the mevalonate pathway , higher expression of natural cytotoxicity receptor ligands or the inherent nature of various haematopoietic cells as professional APCs to recruit T lymphocytes have been proposed . It coul+ T cells exhibit pronounced inhibitory effects on tumourigenesis and tumour growth in a variety of malignancies and are a current hot topic in cancer immunotherapy efforts [The direct recognition, rapid activation and cytotoxic capacity of γδ T cells towards cancer cells makes them very attractive tools for cancer immunotherapy, as recently extensively reviewed ,163,164. efforts ,165,166. efforts ,165. The efforts ,168,169. efforts ,146, whi efforts ,163,164.Interestingly, as mentioned above, the pro-tumour functions of γδ T cells appear to be mainly relevant in solid tumours. Tumour-promoting functions of γδ T cells are primarily mediated through the direct and indirect actions of γδT17 cells, which can be the result of polarisation programming. Indeed, the presence of high levels of IL-17 has been detected in various cancer types, such as cervical cancer , breast + T cells supressed CD8+ αβ T-cell cytotoxicity and DC function [+ αβ T cells abolished the anti-tumour effect of the CD8+ T cells in a melanoma mouse model [+ γδT17 cells, which in turn secreted large quantities of pro-inflammatory IL-17 and demonstrated the capacity for immunosuppression by inhibiting CD4+ naïve T-cell proliferation in vitro [One mechanism by which γδ T cells have been shown to induce pro-tumour functions is by negatively regulating other cancer-killing immune cells . For exafunction , thus acse model . Supportin vitro .+ T cells was induced by stimulation with a plate-bound anti-Vδ1 antibody, promoting expression of regulatory markers FOXP3, CD25 and CTLA-4, as well as inducing functional suppression of CD4+ T-cell proliferation [+ T cells can be involved in a positive feedback loop, sustaining FOXP3 expression and also leading to production of the anti-inflammatory cytokine IL-10 [In support of their suppressor function, murine γδ T cells stimulated in vitro with TGF-β and IL-15 were shown to express the FOXP3 transcription factor , resultiferation . Additione IL-10 . Importane IL-10 , with th+ γδT17 cells, which were found at higher numbers in colorectal cancer, were capable of enhancing PMN migration, proliferation and survival, and were correlated with colon tumour invasiveness and progression [+ T cells, allowing increased metastatic disease [γδ T cells can also regulate other immune cells to support tumour progression . The intgression . Furthergression . Mechani disease .The promotion of angiogenesis is another key tumour-promoting effect of γδT17 cells in solid cancers  Figure . In miceThe regulation of γδ T cells can play an important role in their overall function in cancer immunity, whereby factors negatively regulating their anti-tumour activity ultimately promote tumour progression. One such example includes the hypoxia-induced immune evasion of solid cancers . HypoxiaMetabolites in the TME also play an important role in regulating the infiltration and function of the different effector subtypes, γδT1 and γδT17, thereby impacting γδ T-cell tumour immune responses. γδT1 cells display a preference for glycolysis, whereas γδT17 cells are dependent on oxidative phosphorylation and have increased lipid uptake . As suchImmune checkpoint receptors act as another important regulatory mechanism for γδ T-cell function. For instance, PD-1 is an immune checkpoint receptor expressed on most T cells, including γδ T cells, which upon ligand/antigen engagement acts as an ‘off switch’ to negatively regulate T-cell function . Its ligSimilarly to the immune checkpoint molecules, expression of the metabolic enzyme indoleamine-2,3-dioxygenase (IDO) in cancer cells can promote an immunosuppressive shift in the TME . Mechani+ T cells, which was mechanistically linked to neutrophil-derived hydrogen peroxide, serine protease and arginase-I activity [Other immune cells can also negatively regulate γδ T cells . For exaactivity . Interesactivity . Therefoactivity .Interestingly, the local microbiota may play a role in regulating the response of γδT17 cells against tumour cells. In a genetically engineered lung cancer model driven by KRAS and p53, the lung microbiota induced the proliferation and activation of γδT17 cells to promote inflammation and tumour cell proliferation, where antibiotics or germ-free conditions suppressed these effects . HoweverThe malignant transformation and outgrowth of γδ T cells seems to be a relatively rare event as most recognised T-cell neoplasms are composed of αβ T cells. Entities arising from γδ T cells include hepatosplenic T-cell lymphoma (HSTL), primary cutaneous γδ T-cell lymphoma (PCGDTL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), enteropathy-associated T-cell lymphoma (EATL), T-cell large granular lymphocytic leukaemia (T-LGLL) and T-ALL . For theHSTL is an aggressive disease that primarily affects younger adults (median age of 34 years), with a higher incidence in males (≈71%) ,211. The+ CTCL patients  [Malignant γδ T cells can also arise in or home to the skin, and PCGDTL constitutes a separate entity among the cutaneous T-cell lymphomas (CTCLs) . PCGDTL ctively) . PCGDTL ctively) ,218.+ EATL have been reported [The intestinal T-cell lymphomas EATL and MEITL are also rare but aggressive diseases . EATL isreported ,222. MEIreported ,224,225.reported ,227,228,reported ). As aggreported ,230.+ CD8+ cytotoxic T cells, where chronic NK-LGLL comprises <10% of cases [+ counterpart (62–114 months median OS for both) [LGLL is a rare lymphoproliferative disorder that can arise from T or NK cell lineages, and unlike the aforementioned diseases, it has a rather indolent course. Nevertheless, the associated cytopenias and autoimmune phenomena make it a highly symptomatic disease. The most frequent form of T-LGLL (≈85% of cases) is derived from αβ TCRof cases . γδ T-LGor both) ,233,234.or both) ,233,235.T-ALL arises from the malignant transformation of immature T cells in the thymus or bone marrow, and it occurs most frequently in children and young adults. Approximately 10–15% of all T-ALL cases express the γδ TCR , which iThe transformation process of a γδ T cell into a malignant clone is, like for other cell types, a complex and multi-factorial process. In contrast to many other types of haematopoietic cells, T-cell oncogenesis involves overcoming the safeguarding mechanism of TCR/MHC niche-mediated cross-clonal control, which is a part of T-cell homeostasis . On the Around 20% of HSTL cases arise in patients with pre-existing immune dysregulatory disorders, such as inflammatory bowel disease (IBD), autoimmune disorders , infections , haematological malignancies (Hodgkin lymphoma) or long-term immunosuppression after organ transplantation ,211,239.+ T-cell clones harbouring somatic mutations linked to cell proliferation [Therefore, at least in mature γδ T cells, the co-occurrence of inflammatory or immune-modulating disorders in a subset of patients with γδ T-cell lymphoma/leukaemia, irrespective of their origin or main site of infiltration, suggests that chronic inflammation and activation signals could be common states driving the transformation process. Of course, these diseases also arise de novo in many cases, but such inflammatory or immunosuppressive environments may accelerate initiating events  to allow damaged cells to persist. Indeed, we previously reported that patients with newly diagnosed rheumatoid arthritis have expanded CD8feration , and alsferation . Notablyferation , highlig+ disease [+ and 20–25% are Vδ2+ [+ disease [+, consistent with their normal distribution in healthy skin [+ and 20–43% Vδ1+ [+ and 10–20% Vδ1+ [The specific γδ T-cell subsets that undergo transformation to drive these diseases vary across the different lymphoma/leukaemia entities. In HSTL patients, 90% of cases are derived from the Vδ1 subset , which i disease . This isare Vδ2+ ,248. A r disease , which i disease , recentlthy skin . The autthy skin . No diffthy skin . In case43% Vδ1+ ,233. Thi20% Vδ1+ , with a 20% Vδ1+ ,246,252.ABCB1, RUNDC3B and PPP1R9A [CDKN2A/B [Irrespective of the sites/tissues in which γδ T-cell transformation takes place, the genetic aberrations that frequently occur across the various γδ T-cell lymphoma/leukaemia entities have distinct commonalities. Interestingly, gains of chromosome 7q are observed in patients from almost all γδ T-cell cancer subtypes . Notably PPP1R9A . VariousCDKN2A/B . In lineCDKN2A/B ,257. ChrCDKN2A/B ,259.SETD2 and the DNA demethylase TET2  peripheral T-cell lymphomas (PTCLs) or B-cell lymphomas are repurposed for use in γδ T-cell lymphoma/leukaemia patients. Newer, more targeted treatment options that are clinically approved are being explored in trials for relapsed/refractory PTCL or as combinations in first-line settings. These trials include patients with γδ T-cell lymphoma/leukaemia, as trials dedicated only to these entities are usually not feasible. The substance classes being tested include folate metabolite inhibitors , oral inhibitors of phosphoinositide-3 kinase  , JAK inhOverall, new therapeutic options are urgently needed for these aggressive γδ T-cell diseases. Given the more recent advances in understanding the molecular profiles and events driving these malignancies  215,251,294, it Recent years have seen a large step forward in both our understanding of the roles of human γδ T cells in cancer immunity, as well as in our understanding of the genetic and molecular basis of aggressive γδ T-cell leukaemia/lymphoma. These new insights will pave the way for considerable advancements in the years ahead, especially regarding the development of Vδ1 T cells as tools for adoptive γδ T-cell immunotherapy for cancer patients. Nevertheless, it will be important to continue to dissect the pro-tumour functions of γδT17 cells, particularly regarding polarisation triggers in the human context and the negative impact this could play on their therapeutic utility. Furthermore, new therapeutic strategies are urgently needed for patients suffering from incurable γδ T-cell leukaemia/lymphoma, and current research focusing on identifying and targeting common dysregulated pathways, as well as the development of new faithful pre-clinical models, should hopefully soon lead to new breakthroughs to help these patients.