Covalent organicframeworks (COFs) with a pore size beyond 5 nmare still rarely seen in this emerging field. Besides obvious complicationssuch as the elaborated synthesis of large linkers with sufficientsolubility, more subtle challenges regarding large-pore COF synthesis,including pore occlusion and collapse, prevail. Here we present twoisoreticular series of large-pore imine COFs with pore sizes up to5.8 nm and correlate the interlayer interactions with the structureand thermal behavior of the COFs. By adjusting interlayer interactionsthrough the incorporation of methoxy groups acting as pore-directing“anchors”, different stacking modes can be accessed,resulting in modified stacking polytypes and, hence, effective poresizes. A strong correlation between stacking energy toward highlyordered, nearly eclipsed structures, higher structural integrity duringthermal stress, and a novel, thermally induced phase transition ofstacking modes in COFs was found, which sheds light on viable designstrategies for increased structural control and stability in large-poreCOFs. While micro- andsmall mesoporous COFs are widespread today, large-pore COFs with porediameters exceeding 5 nm are still rare.23 With a large open pore structure, such COFs could host bulky guestmolecules such as biomolecules26 or allow sufficient diffusionof substrates and products in heterogeneous catalysis, making themideal candidates to further widen the scope of applications for COFs.Covalent organic frameworks(COFs) are highly stable, permanentlyporous and crystalline materials assembled from organic building blocksto form defined periodic networks.20 designed thefirst polyimide-based COF with a pore size exceeding 5.0 nm, whichcould absorb the large dye molecule rhodamine B in its channels. RecentlyZhao et al.23 synthesized an ester-linkedCOF spanning 10 phenylene units at one edge. While the polyimide COFcrystallizes—as all other examples known to date—ina slipped configuration, slightly reducing the apparent pore size,the ester-linked COF showed a significantly smaller pore size in dryconditions than anticipated. However, low crystallinity due to thehigh flexibility of the structure precluded a definite structure analysis.Controlling the synthesis and structure of large-pore COFs to maximizetheir pore size seems to impose unique problems compared to COFs withsmaller pores. Besides synthetic challenges such as reduced linkersolubility with increasing length, the observed effect of layer slippingand potential pore collapse upon removal of guests have to be considered.27 For mesoporous systems such as COFs, large interfacialenergies are expected due to their high surface areas, leading toa natural tendency to minimize their free energy by the closure ofthe energetically unfavorable pores.29 For COFs thistendency could be a lateral slipping of the layers to decrease poresize and surface area or ultimately a pore collapse, while both arecounterbalanced by the strong interlayer stacking interactions andrigid linkers, keeping the porous structure intact. The so far generallyobserved slipping of layers within large-pore COFs may be the resultof an imbalance between increasing pore aperture and relatively largerfree volume, while the same “wall-thickness” and thereforesimilar interlayer interactions are maintained. The total free energyof the system may then become unfavorable, and the system is forcedto minimize its free energy by pore collapse or layer slipping insteadof maintaining nearly eclipsed structures. We thus hypothesize thatincreasing the interlayer interactions might help to anchor the layersin nearly eclipsed stacking modes and prevent slipping or even porecollapse. Considering these observations, an in-depth understandingof the factors affecting pore structure and structural stability isnecessary to successfully obtain large-pore COFs without sacrificingcrystallinity and porosity.In 2014, Fang et al.32 In addition, an increasingrepertoire of new linkages or postmodification of linkages was developedto extend the portfolio of synthetic tools to create chemically stablenetworks that withstand harsh chemical conditions.36 However, in terms of thermal stability Evans et al.38 recently showed that TGA analysis is not sufficient to assess thermalstability of the frameworks. They found that the structural integrityand crystallinity of the networks could be compromised by bucklingof the layers under thermal stress at significantly lower temperaturesthan the TGA measurements suggest. Given the high flexibility of largerCOF structures,23 thermal stress can havean even bigger impact on their structural integrity.In recent years a great deal ofattention has been devoted to thetask to increase crystallinity and surface area of COFs, e.g., bymodulating reversibility during the formation process or facilitatingbeneficial stacking interactions, both of which can be instrumentalin the design of large-pore COFs.39 andby adding favorable interlayer hydrogen bonding.40 By varying the amount of methoxy functionalization andthe pore size, a series of six isoreticular, hexagonal COFs with amaximum pore size of 5.8 nm are prepared. Recursive X-ray powder diffraction(XRPD) simulations and refinements, sorption analysis, and densityfunctional theory (DFT) calculations show that increasing the poresize leads to a tendency toward staggered layer arrangements, whichcan be reverted into eclipsed arrangements by increasing the stackinginteraction and anchoring the layers in nearly eclipsed stacking. In situ XRPD and correlating ex situ sorptionanalysis establish a close connection between interlayer interactionsand thermal stability and reveal a novel thermally induced phase transitionfrom eclipsed to staggered conformation at temperatures as low as120 °C. Our observations demonstrate that modulating interlayerinteractions is a viable tool to influence COF stacking and maximizethermal stability and pore sizes in large-pore COF systems.Herein,we reveal the structure-directing influence of interlayerinteractions in two isoreticular series of 2D large-pore COFs. Wepay specific attention to the effect of interlayer interactions onstructure and pore size and investigate the structural integrity oflarge-pore COFs under thermal stress. Methoxy groups are introducedinto the COF structures in meta position to the imine bond to alterand modulate the interlayer interactions by reducing the inherentdipole moment of the imine linkageTo study the influence of interlayer interactions onthe structure,crystallinity, porosity, and thermal stability of large-pore COFs,we selected three phenylphenanthridine-based building blocks, 4,4′-dibenzaldehyde(PP), 4,4′-bis(mPP), and 4,4′-bis(dPP), in combination with 5′-(4-aminophenyl)--4,4″-diamine(TAB) or 1,3,5-tris[4-amino]benzene (TAPB).By the combination of these building blocks, six isoreticular COFs,PP-TAB, mPP-TAB, dPP-TAB, PP-TAPB, mPP-TAPB, and dPP- TAPB, with varyingamounts of methoxy groups—zero to two regarding each iminebond—and differing pore sizes were obtained 1. The de13C cross-polarization magic angle spinning (CP-MAS) NMR spectroscopy,nitrogen sorption analysis at 77 K, and XRPD. FT-IR reveals the fullconsumption of starting materials by the disappearance of typicalamine and aldehyde stretching bands at around 3300 and 1670 cm–1, respectively. Further, the appearance of a new imine-stretchingband around 1620 cm–1 confirms the successful formationof imine linkages for all six COFs . 13C CP-MAS NMR spectra show the typical imine signalat around 160 ppm and reveal that no residual aldehydes are left forall six COFs . The signalsof PP-TAB, PP-TAPB, and mPP-TAPB display significant broadening andoverlapping, indicating higher disorder, compared to the spectra ofmPP-TAB, dPP-TAB, and dPP-TAPB, which show sharper and more definedsignals.The successful conversion of the building blocks into imine-linkedCOFs was established by Fourier transform infrared analysis (FT-IR), 41 or a staggered AB̅-type stacking, wherelayer B̅ is shifted by a certain stacking vector, indicatedby green and light green arrows in XRPD measurements show that allsix COFs of the series are highly crystalline 1a and b.43 using different stacking and faulting scenarios. This simulation shows the transitionof a nearly eclipsed AA̅-type structure into an ordered, staggeredAB̅-type stacking as the most likely scenario. The shift withinthe ab-plane by applying the stacking vector wasincrementally increased to 12 Å. This leads to a decrease ofthe peak intensities of the 010, 1̅20, 020, and 1̅30 reflections,as observed in the measured pattern with low apparent crystallinity.To evaluate this proposed structure model, we performed Rietveld refinementson the collected powder patterns. The layers were modeled as flatlayers without any torsions, as including this would have led to anoverextension of the parameter space. Symmetry-adapted spherical harmonicswere applied to the peak widths in order to compensate the peak broadeningcaused by intra- and interlayer disorder.To gain further insightsinto the different effects of structureand stacking for this isoreticular set of COFs, we performed systematicDIFFaX-like simulations of the XRPD patterns for mPP-TAB as an exemplaryCOF,Figures S9, S11, and S12,respectively) with the nearly eclipsed AA̅-type stacked modelled to a good fit of the measured XRPD pattern and to a satisfyingagreement factor . For PP-TAB, PP-TAPB, and mPP-TAPB , whichexhibit a staggered AB̅-type stacking, a 12-layer supercellhad to be used for refinement to ensure a satisfying fit .Within the refined supercell all layers show a staggered arrangementwith a stacking vector of around 12 Å. The results, summarizedin a and b lattice parametersof the COFs in staggered stacking compared to the isoreticular, eclipsedCOFs, which indicates additional distortion by layer buckling andtwisting, introduced by the additional degrees of freedom in the staggeredarrangement.The refinement ofthe XRPD patterns collected for mPP-TAB, dPP-TAB,and dPP-TAPB . SEM images show a cauliflower-like morphologyfor AB̅-type stacked PP-TAB, PP-TAPB, and mPP-TAPB due to intergrown,curved sheets forming smooth spheres. AA̅-type stacked mPP-TAB,dPP-TAB, and dPP-TAPB show a comparatively rugged morphology exposingcrystal facets, which points to the growth of ordered, plate-likecrystallites.Scanning electron microscopy(SEM) and transmission electron microscopy(TEM) imaging were performed for all samples to gain further insightsinto the morphology of the COF particles .To gain additionalinsights into the structural arrangement of the nearly eclipsed COFs,we performed a computational study to acquire an in-depth understandingof the stacking of phenylphenanthridine units, the role of the methoxygroups, and the imine configurations. We first analyzed the interactionof two isolated molecular building units. We performed DFT calculationsusing TURBOMOLE50 with periodicboundary conditions, the PBE-D347 functional with GTH-pseudopotentials,51 and a TZV2P-GTH52 basis set.In a secondstep, we investigated different arrangements of building units byconstructing cut-out blocks of the COF structure to model single porewalls 3a.All oFor the structures that we identified as the most promising, wethen performed a complete unit-cell treatment, leading to an optimalarrangement of these large-pore COFs 3c.Scheme S5) were initially optimized on a nonperiodic molecular level(Table S4). Further optimization in a periodiccombination of blocks modeled the restricted in-plane movement ofa multilayer COF pore wall.To investigate the stacking behaviorof the phenylphenanthridineunits, all four possible stacking variants (Table S9) find the s10 variant to be the optimalstacking configuration of the phenylphenanthridine units. Assuminga rotation of the protruding phenyl groups, parallel-displaced orT-stacked configurations .For judgingthe influence of methoxy groups on the imine linkagestacking and orientation, we again initially optimized a nonperiodicmodel to gain insight into the overall properties stemming from thecomponents. The Figure S48).To gain insight in the underlyingelectrostatic interactions, molecularelectrostatic potential maps were calculated for all isolated components.These maps explain the alternating arrangement by the possibilityto arrange differently charged areas in a more favorable manner tominimize repulsion .We could also observe that the difference in interaction energybetween the two orientations is reduced by added methoxy groups. Furthermore,we performed calculations with periodic combinations of blocks tomodel a multilayer COF pore wall. The interaction energies show, inanalogy to the isolated model, alternatingly oriented imine groupsto be energetically favorable, as they minimize repulsion betweenthe layers  and becomes even smaller when methoxy groups are added tothe linking site .However,for the periodic case that restricts the molecules intheir in-plane movement, the difference between alternating and parallelorientations is smaller . Furthermore, we observed that structures with the phenylphenanthridineoriented on different sides become significantly less favorable whenmethoxy groups are added (Table S7), whichis not observed in the methoxy-free case . As a result, the amount of possible orientationscan be expected to be halved in the methoxy case, which indicatesan increase in structural order.This structural effect results from the reduction of the inherentdipole moment of the imine linker by the adjacent methoxy groups,Figure S51, are always energetically favorable compared toalternatingly oriented ones (Table S8).We conclude that the methoxy groups add stability due to their stericinteractions with each other, which is in most cases also indicatedby their apparent out-of-plane orientation .In building blocks with onlyone methoxy group we found that same-side,parallel-oriented methoxy groups, as those shown in Tables S10–S12). It was observedthat systematic out-of-plane orientations of the methoxy groups areenergetically favorable for every utilized method but even more sothe systematic parallel arrangement of the groups . With these results on the configurationthe complete unit cell of eclipsed stacked dPP-TAB and PP-TAB withparallel and alternating imine linkers, as well as s10 and s11 phenylphenanthridineunits, was optimized using the periodic GFN-xTB implementation inCP2K for a broad analysis as well as GPW-DFT calculations for themost probable structures (Table S13).This behavior was further studied by a completeunit cell treatmentof the dPP-TAB COF. Taking into account the influence of the dispersioninteractions, we performed cell optimizations of the whole unit cellfor methoxy groups ordered in parallel and antiparallel arrangements. These differ by almost 2 Å from the smaller latticeparameters found experimentally, probably due to a lack of bucklingof the structure in the DFT calculations. However, if the eclipsedstacking is abandoned by shifting layers toward the actually staggeredAB̅ stacking, increased buckling is observed , and the lattice parameters a and b are reduced to 56.709 Å, matching thevalues found experimentally.For dPP-TAB, the obtained values for the lattice parameters (SBET) of the COFs were characterizedby measuring nitrogen sorption isotherms at 77 K to further confirm our structural models via their pore sizes. SBET of all the COFs was determined in a suitablepressure region (P/P0 = 0.1–0.3), and pore size distributions (PSDs) were calculatedfrom the adsorption branch by quenched solid density functional theory(QSDFT) based on the carbon model for cylindrical pores and are summarizedin 56 All isoreticular COFs are porous with typical type IV isotherms57 and show, except for PP-TAB, a distinct hysteresis to highly porous (1823 m2 g–1 for mPP-TAB). The COFs crystallizing in astaggered conformation show overall significantly lower SBET values compared to the nearly eclipsed staggered COFs.This can be rationalized by an increase in porosity by both well-definedlayer stacking32 and a decrease in effectivepore size through the staggered arrangement.The porosityand Brunauer–Emmett–Tellersurface area . The obtained results summarizedin Table S3 perfectly follow the trendof the obtained values from the adsorption measurements showing theincrease in pore size from 4.05 nm for PP-TAB to 5.90 nm for dPP-TAPBand, thus, further support our interpretation of favored stackingmotifs.For that, we crosschecked the obtained experimental results andour interpretation by using an additional geometric pore analysis.Here we determined the pore diameter by calculating the biggest sphereable to fit through the spanned pore arrangement, by constructinga pore system using the experimentally obtained structures for bothCOF systems using the PP- and dPP-COFs, respectively . An increasing buckling of the COFlayers at higher temperatures was found, leading to amorphizationof the network. In addition, a trend of lower thermal stability withincreasing pore size and pore functionalization was identified. Itwas postulated that larger and more mobile pendant groups, similarto the methoxy groups used here, can already cause significant disruptionof interlayer interactions in 2D COF sheets as thermal energy is added,thus resulting in lower thermal stability. Taking into account thatthermal stability can become a limiting factor in the applicationof COFs,66 insights into this effect, especially atlarge pore sizes, is a key prerequisite.Observing different stacking modes for otherwise isoreticularand chemically extremely similar COF systems, we further investigatedhow these different stacking modes and interlayer interactions influencethe thermal behavior of large-pore COFs. As previously shown by Evanset al.,Figure S7). The XRPD patterns for the in situ heating experimentswere consecutively collected at 30, 80, 100, 120, 140, 160, 180, and200 °C , witha delay period of 4 h prior to each measurement in order to ensureequilibration. The final patterns at 200 °C for all COFs werefurther refined according to the models described above .In TGA measurements, all COFs in ourseries show a typical, high thermal stability of up to 400 °Cbefore any mass loss is detected S7. The Xhk0 reflectionswas observed .These observations stem from additional layer buckling at higher temperaturesas previously described by Evans et al.38 This additionallayer buckling is irreversible and remains upon cooling of the samples.For the staggered AB̅-type stacked COFs, PP-TAB, PP-TAPB,and mPP-TAPB, a minor reduction in crystallinity and peak broadeningas well as a slight shift of the dPP-TAB COF shows almost no change in its reflections upon heatingto 200 °C except for a slight shift of the 002 stacking reflectiontoward lower diffraction angles 4a. This Figure S17). Up to a transition temperature of140 °C for mPP-TAB and 120 °C for dPP-TAPB, both COFs stillbehave comparable to the temperature-stable dPP-TAB. A slight shiftof the 002 stacking reflection indicates thermal expansion, whileno buckling of the layers is evident. At transition temperature, thebasal 002 reflections show a substantial broadening and completelymerge with the background at higher temperatures. Simultaneously,an initial shift of the hk0 reflections and theirbroadening take place. Continuing stepwise further up to 200 °C, the final shift of the 010 reflectionscorresponds to a reduction of the a and b lattice parameters by 2.1 and 3.3 Å, respectively. As for PP-TAB,PP-TAPB, and mPP-TAPB, this shift is a result of temperature-inducedlayer buckling. However, the observed peak broadening that ultimatelyresults in the disappearance of higher angle hk0reflections strongly resembles our simulations for the transitionof a nearly eclipsed stacked AA̅-type structure to an ordered,staggered AB̅-type stacking .mPP-TAB and dPP-TAPB showthe most significant changes upon heatingS17. Up tFigure S29). Reaching the transition temperature, the previously steepuptake at around P/P0 = 0.5 resembling the capillary condensation in 4.8 nm wide poresbegins to flatten out. With increasing temperature, the initiallybroad hysteresis then closes. During the stepwise transition, the SBET almost halves from 1823 m2 g–1 at room temperature to 930 m2 g–1 after activation at 200 °C. The PSD shows a related, harshdrop in the cumulative pore volume of 4.8 nm and the appearance ofa new pore size at 4.0 nm at 140 °C . With higher temperatures, all pores at 4.8 nm disappearafter being transformed into the smaller pores correlating to a staggeredAB̅-type stacking of ht-mPP-TAB. Similar behavior is observedfor dPP-TAPB, with a decrease of the SBET to 920 m2 g–1 and the disappearanceof pores with a 5.8 nm diameter accompanied by a rise of 4.8 nm poresat 120 °C and above .The SBET and PSD of the ht-phases arein good accordance with the values found for isoreticular, alreadyat room temperature staggered, PP-TAB and PP-TAPB COFs.To confirm this observation of a temperature-inducedphase transitionfrom an eclipsed toward a staggered type stacking for mPP-TAB anddPP-TAPB, corresponding sorption experiments were performed. For thispurpose, the samples were consecutively activated under vacuum atthe given temperatures for 4 h before measurement. For mPP-TAPB nomajor changes below the transition temperature of 140 °C occur,in analogy to the XRPD heating experiments . As shown by Sick et al.,67 this process can even at low temperatures sufficientlyrecover interlayer correlation, indicating the near-eclipsed stackingof mPP-TAB and dPP-TAPB is preferred in solution.Whilethe structural change is irreversible and permanent uponcooling, exposing the COFs to solvent followed by supercritical (sc)CO69 or by removal of structurally incorporated solvent70 are known, our heating experiments show a remarkable, temperature-inducedphase transition for COFs due to layer mobility, which was to thebest of our knowledge never observed before. The structural transitionof mPP-TAB and dPP-TAPB from eclipsed into staggered already occursat rather mild temperatures of 140 and 120 °C, respectively,and is reversible upon contact with solvent. The findings show againcorrelation between interlayer interactions and pore size, as forthe room-temperature isoreticular series. While the relatively smaller-sizedpores of dPP-TAB with strong interlayer interactions are unaffectedup to 200 °C, the isoreticular mPP-TAB shows structural integrityonly up to 140 °C, when layermobility gets sufficient to transform it into a staggered structure.With the increased pore size of dPP-TAPB, compared to dPP-TAB, theinterlayer interactions are again not strong enough to prevent layerdisplacement above 120 °C. In our case, the increased interlayerinteractions of pendant methoxy groups, anchoring the layers in nearlyeclipsed stacking, do not follow the general negative influence onthermal stability of other pore functionalization found by Evans etal.64 and, therefore, offers new insights intothe thermal stability of COFs.While phasechanges by interlayer shifting under solvent influencesFigure S18. Several minutes after heating at the transition temperature, adecrease in intensity of the 010, 1̅20, 020, and 1̅30reflections, indicating a shift of the layers toward a staggered AB̅-typestacking, can be observed .In addition, the 010 reflections shift to higher 2θ angles , which is attributed to the developmentof a buckling of the COF layers. These processes continue, even aftermore than 2 h.To monitor the developmentof the observed phase change over time, mPP-TAB and dPP-TAPB wereheated above the transition temperature, to 140 and 120 °C, respectively,and the evolution of the diffraction patterns was monitored whileholding the samples at constant temperatures. As an example, the XRPDpatterns of mPP-TAB are shown in 71 using the previously applied two-layer unit cells with a planarCOF conformation. The almost eclipsed AA̅-type stacked structurewas used for the patterns collected at 30 °C. For the refinementof the patterns collected at higher temperatures, the space groupsymmetry was lowered to P1 and the upper layer wasallowed to shift freely within the ab-plane. Theabsolute value of the shifting vector of the upper lattice plane wasused as a measure of transition from nearly eclipsed toward staggeredAB̅-type stacking. The a and b lattice parameters were constrained and refined as well in orderto get a measure on the development of the COF-layer buckling. Symmetry-adaptedspherical harmonics were applied to the peak widths in order to compensatethe peak broadening caused by intra- and interlayer disorder. Allrefinements were carried out in a serial way, which means that forthe refinement of a pattern collected at a time ti the refined parameters of the precedingpattern collected at ti–1 were used. The parameters were released iteratively. At first, thelattice parameter was refined, and then the layer shift and finallythe peak profile parameters were released. The resulting evolutionof the mean layer offset and the curving of the COF layers of mPP-TABand dPP-TAPB are given in In order to gain further insights into the mechanismof shifting and curving of the layers, the patterns were analyzedby fully weighted Rietveld refinements,a and b lattice parameters indicate that bucklingof the layers does not require any initial shift and shifting of thelayers does not require any initial buckling. The development of thelayer offset . For every displacement we performedgeometry and cell optimizations using GFN-xTB72 as implemented in CP2K, as it showed qualitatively similar behaviorwhen compared with single-point energies for the optimized cells usingGPW-DFT . The obtained energyvalues were then interpolated by a plane-wave expansion with D6 symmetry. The obtained potential energy landscapesshow the distribution in the potential energy landscapes assuminga shift of the A̅-type layer in comparison to the A-type layerby a certain vector as shown in To gain further insightsinto the origin of the phase transition,displacement potential energy landscapes for PP-TAB, mPP-TAB, anddPP-TAB were calculated 6a. Invesshown in S54. For 31 PP-TAB COF shows theenergetic minima consistent with staggered stacking. The mPP-TAB displaysa similar landscape to PP-TAB with the energetic minima predictingstaggered configurations. However, in this case a small energy barrierbetween the nearly eclipsed and staggered configurations was found.These observations are rationalized in a simplified qualitative scheme(For dPP-TAB, with the strongest interlayer interactions,only asmall, clearly favorable energy window corresponding to a nearly eclipsedstacking is found.e scheme6b. PP-TAin situ XRPDmeasurements, shedding light on the two independent processes involved,layer shifting and buckling. Altered porosity and crystallinity forthe high-temperature phases are persistent but were easily reversedby solvent exposure, suggesting solvent-induced changes of the stackingmode to be an additional degree of freedom in influencing the layerstacking besides the observed thermally induced phase transitions.Considering the fact that the synthesis of large-pore COFs with >5nm pore size remains a challenging task in the COF chemistry, we anticipatethat the findings presented herein will aid the rational design ofCOFs with targeted pore size, layer registry, and thermal stabilityfor specific applications.We successfully developed six novel large-pore COFs with a maximumpore size of 5.8 nm, which is among the largest pore sizes reportedfor COFs to date. More importantly, methoxy groups in phenylphenanthridinebuilding blocks were identified to act as anchors that eliminate thetendency toward slipped layer stacking in large-pore COFs, which typicallyleads to notoriously reduced effective pore sizes and surface areas.XRPD results together with theoretical modeling indicate mPP-TAB,dPP-TAB, and dPP-TAPB to adopt a nearly eclipsed stacking mode withmaximized pore sizes, attributed to enhanced interlayer interactionin these COFs, mediated by the methoxy groups. While increasing interlayerinteractions positively influence the thermal stability of the COFs,we further demonstrate, for the first time, a temperature-inducedphase transition to the staggered stacking polytype, enabled by highlayer mobility even at temperatures as low as 120 °C. The slowtransition could be readily followed by


It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains  and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.The scaffold protein Tks4 is a member of the p47 Scaffold proteins have pivotal roles in cellular signaling by providing an assembly platform for various proteins and by specifying the subcellular localizations of the resulting protein complexes ,2. They N-terminal PX domain is often followed by one or more SH3 domains [phox-related organizer superfamily [phox, p47phox, and NOXO1, the PX domain is followed by one or two SH3 domains. These proteins regulate the assembly and activation of NADPH oxidase complexes [phox-related organizer superfamily.In several scaffold proteins, the  domains ,9. This erfamily ,11. In pomplexes . Two recomplexes ,14. The Both Tks4 and Tks5 have roles in cell motility by affecting the formation of podosomes and invadopodia ,15,16. HIt has already been shown that Tks4 has a signal-transmitting role in EGFR signaling ,29. In tphox [N-terminal PX domain is followed by two SH3 domains  [C-terminal part of the molecule (residues 284–390) is unstructured, and it has an important role in the regulation of NADPH oxidase complexes [phox contains one proline-rich region (PRR) that serves as a binding motif for the SH3 domains of p67phox [phox. The AIR is rich in serine and arginine and contains a proline-proline-arginine (“PPR” ) core motif [phox is autoinhibited by two unique intermolecular interactions: the tandem SH3 domain binds to the AIR, and the PX domain interacts with the first SH3 domain [phox and the assembly of the NADPH oxidase complex are inhibited [1 and SH32 in opposing orientations [phox, SH31 and SH32 form a large hydrophobic binding surface that is occupied by the AIR  [1–SH32 connection is also stabilized by hydrogen bonds and hydrophobic interactions [While the structural basis of the activation-induced translocation is not known in Tks4, the molecular structure, interaction partners, and regulation have already been revealed by several studies in the case of p47phox ,33,34. Idomains) . The unsomplexes ,34. p47p p67phox . The regre motif . Based o3 domain ,33,37. Intations . In p47pPPRRSSI) . This inPPRRSSI) . The SH3ractions . The invphox [phox migrates from the cytosol to the plasma membrane, and the released SH31 and SH32 domains bind as a pair to an AIR-like sequence in the cytoplasmic region of p22phox [During assembly of the NADPH oxidase complex, multiple phosphorylation events on Ser residues in the AIR sequence result in the opening of the inhibited conformation of p47phox ,40. Upon p22phox ,34,35,40N-terminal regions of human Tks4 and p47phox clearly shows high sequence similarity between the two proteins  is located adjacent to the PTAM in Tks4  core motif within the AIR in p47 p22phox ,41) can proteins . However in Tks4 A,B. Anot in Tks4 ,40. Ther in Tks4 ,43,44.N-terminal regions of Tks4 and p47phox have long been recognized, whether a similar intramolecular interaction pattern and regulatory mechanism exist in Tks4 has not been investigated. The membrane localization of the protein is regulated by an EGF-dependent mechanism [phox  and protein–protein interaction assays showed that, in the closed conformation, the first two SH3 domains cooperate with each other to bind the PTAM-containing region and the third SH3 domain interacts with the PX domain. PIP strip and liposome co-sedimentation assays identified phosphatidylinositol (3)-phosphate (PtdIns(3)P) as the main lipid binding partner of the PX domain. In the autoinhibited state, PtdIns(3)P binding is blocked. Introduction of a phosphomimicking mutation into the PRR suggests a potential mechanism by which the opening of the autoinhibited state might be regulated in vivo.Here, we report the characterization of the intramolecular interactions within the N-terminal region of Tks4 was expressed and purified as two fragments (2 (residues 1–279) and PRR–SH33 (residues 333–426). Titration of fluorescently labeled PRR–SH33  with PX–SH32 was followed by MST. A relatively strong interaction with a dissociation constant (Kd) of approximately 240 ± 20 nM was observed (2 titrated with PRR–SH33) was also performed (d (300 ± 200 nM) was observed .First, the ragments C: PX–SH3observed A. The reerformed B. In thi3 has already been reported in Tks4 [3 domain with a fragment containing the PX and SH31 domains (PX–SH31) and followed the reaction via MST. While the fragment corresponding to the PX domain alone could be expressed in E. coli cells, the purified protein was found to be unstable and precipitated slowly. By contrast, the PX–SH31 construct showed no stability issues. Although the experiment clearly showed an interaction between PX–SH31 and SH33 (d with high accuracy (Kd > 50 µM). As a control experiment, labelled SH33 domain was titrated with SH31. The SH31 domain alone showed no binding to the SH33 domain  indicates that the association of the PX domain and the third SH3 domain might contribute to the formation of the autoinhibited conformation in full-length Tks4, the much stronger binding of PX–SH32 to PRR–SH33 (Kd ≈ 240 nM) supports the hypothesis that the PRR following the second SH3 domain might fold back and associate with SH31–SH32, as in p47phox [L fragment (residues: 333–370) containing both the PTAM and the -PxxP- motifs was titrated with SH31, SH32, SH33, and SH31–SH32. Titrations were followed based on the change in the intrinsic tryptophan fluorescence emission spectra of the SH3 domains. This approach is generally used for SH3–ligand binding determination when the ligand does not contain a Trp residue [1–SH32 upon the addition of PRRL (1–SH32 fragment with PRRL followed by MST confirmed these results (Kd = 15 ± 3 µM)  .L fragment that is responsible for the interaction with SH31–SH32, a truncated version of PRRL containing only the PTAM (PRRS) was synthesized . Dissociation constants were determined via Trp-fluorescence-based titration experiments . However, further extension of PX–SH32 with a long, disordered region in PX–PRR did not affect the Rg. Moreover, the presence of the third SH3 domain in PX–SH33 resulted in a decreased Rg (max) of the constructs followed a similar pattern, although the highest Dmax was observed in the case of PX–PRR  and longest (PX–SH33) fragments  Tks4 fragments were determined via protein–lipid overlay and liposome co-sedimentation assays.PX domains are responsible for the attachment of proteins to membranes by binding phosphoinositide lipids ,9. In p4ractions ,51. Base6–PX–SH31, His6–PX–SH32, His6–PX–PRR, and His6–PX–SH33 were incubated with 15 different phospholipids spotted onto the surface of a nitrocellulose membrane . Following three washing steps, the membranes were incubated with mouse anti-His6 and anti-mouse IgG-HRP antibodies, and bound immunocomplexes were detected via enhanced chemiluminescence (6–PX–SH31 and His6–PX–SH32 exhibited affinity mainly for Ptdlns(3)P (phosphatidylinositol (3)-phosphate) and PA (phosphatidic acid). The longer Tks4 fragments His6–PX–PRR and His6–PX–SH33 showed an affinity for Ptdlns(3)P only P only .6–PX–PRR and His6–PX–SH33, autoinhibition may have reduced lipid binding; however, autoinhibition is a conformational equilibrium between the autoinhibited (closed) and active (opened) states. Even if the active pool of the protein is present in relatively low quantities, it can still interact with the lipid surface and give an observable signal if substantial amplification is applied. Altogether, these results suggest that the main lipid ligand of the PX domain is Ptdlns(3)P, although there might be some cross-reactivity with PA.It is important to note that the PIP strip assay provides only a qualitative estimate of the lipid binding specificity. Quantitative comparisons of lipid binding affinities between different Tks4 fragments are not possible. The extensive signal amplification, which allows the detection of relatively weak protein–lipid interactions, may explain why all of the Tks4 fragments interacted with Ptdlns(3)P in this assay. In His1, PX–SH32, PX–PRR, and PX–SH33) were titrated with liposome solutions. Ptdlns(3)P or PA presented 5% of the total lipid pool, and the final total lipid concentrations were 0 µM, 20 µM, 100 µM, and 500 µM in all titration experiments. Liposome-bound proteins were separated from the free fraction via centrifugation. Protein samples were analyzed via SDS-PAGE and Coomassie staining. Quantitation was performed via densitometry (Neither Ptdlns(3)P nor PA form biological membranes alone in a cell. Moreover, the curvature and various components of a real biological membrane may change the accessibility and the interaction of these lipids with their protein partners. Therefore, we re-tested the binding of our Tks4 fragments to Ptdlns(3)P and PA in liposome co-sedimentation assays. In these experiments, Ptdlns(3)P and PA were mixed with different lipids  to form liposomes. Tks4 fragments at a final concentration of 1 µM (PX–SH3itometry . This ex1 when titrated with liposomes containing Ptdlns(3)P. PX–SH32 clearly showed the same effect. The presence of the PRR in PX–PRR reduced Ptdlns(3)P binding. The addition of the third SH3 domain (in PX–SH33) completely abolished the interaction. None of the Tks4 fragments interacted with PA-containing liposomes (phox [phox).Concentration-dependent depletion of the free (soluble) protein pool was observed in the case of PX–SH3iposomes . These r in PX–SH completees P binding. This small effect size suggests that Thr353 phosphorylation is most likely not the regulatory mechanism responsible for the closed-to-open transition of Tks4 in vivo.Finally, we investigated the hypothesized regulatory role of Thr353 via liposome co-sedimentation assays by using the phosphomimic mutant PX–SH3N-terminal region of Tks4 closely resembles the structure of p47phox, an evolutionarily-related protein in which the same domains and linear motifs exist in the same relative order , most probably due to the presence of the disordered linker regions connecting the PX and SH3 domains. Therefore, we applied multiple protein–protein interaction assays to identify interactions between various Tks4 fragments. Two main interaction sites were identified: (1) between the PTAM and the SH31–SH32 domains; and (2) between the PX domain and the SH33 domain. Interestingly, neither SH31 nor SH32 bound the PTAM alone. The presence of both domains is necessary for a detectable interaction. The regular -PxPPxPP- SH3-binding motif located close to the PTAM  might first be required to disrupt the interaction between the SH31–SH32 domains and the PRR. Such a signal might lead to simultaneous activation of the PX and SH31–SH32 domains, making them accessible to their lipid and protein binding partners, respectively.In addition to autoinhibition, tandem SH3 domains in other members of the p47 in Tks5 . The bin domains ,55. Thesphox, Marcoux et al. applied SAXS and found that the PX domain was located in the proximity of the first SH3 domain [1 domain or whether it is maintained only in close proximity by a structured linker [1–SH32–PRR (containing the PTAM motif) region of Tks4. However, the intramolecular binding of the PTAM to SH31–SH32 can still reduce the ability of the PX domain to bind PtdIns(3)P (see In the case of p473 domain . Howeverd linker . Surpris(3)P see . We hypo(3)P see B,C where3 domains in Tks4 has been observed by Hishida et al. [3 domain showed a weak interaction with the PX domain. This observation suggests that the Tks4 protein has an extra intramolecular connection in addition to those present in p47phox. It should be noted, however, that the apparent Kd values of the separate domains most likely do not reflect the true strength of the interaction when these domains are connected in the intact molecule. In that situation, the two domains remain in close proximity of each other, resulting in high apparent local concentrations and, eventually, a much stronger interaction. Therefore, even if only relatively weak interactions were identified in this work, we can still conclude that these interactions might reflect an effective regulatory mechanism. This conclusion is supported by the fact that PX–SH33 has a folded, compact structure according to our SAXS experiments  concentration-dependent aggregation see . HoweverWhile our protein–lipid overlay assay showed that the PX domain can selectively bind PtdIns(3)P and PA, the liposome co-sedimentation assay confirmed only the interaction with PtdIns(3)P. In this assay, the environment of the lipids  closely resembles that found in the cell membrane. This is a better model system than the PIP strip, where lipids are loaded onto a flat, artificial surface. Therefore, the liposome co-sedimentation assay yields more reliable results that better reflect the in vivo conditions. Interestingly, some studies have reported interactions between the PX domain of Tks4 and other phosphoinositide lipids using dot-blot assays ,18,29,56phox undergoes conformational changes upon phosphorylation of multiple Ser residues  in the PRR. It is believed that these phosphorylation events induce the opening of p47phox [phox [phox, it seems that the opened-to-closed conformational switch is regulated differently. It is possible that the relevant Ser/Thr phosphorylation sites in Tks4 (if any) have not been identified yet. However, it is known that Src tyrosine kinase phosphorylates Tks4 at Tyr25 and Tyr373 . These events are important in the activation (and subsequent membrane translocation) of the molecule, which results in the assembly of a signaling complex  [phox upon activation.We attempted to identify the mechanism by which the closed conformation can be disrupted in Tks4. p47 p47phox ,40,41,62ox [phox ,40,63. Aox [phox . In Tks4ox [phox ,43,44. Tnd EGFR) ,28. It i3 domains lead to a compact packaging of the N-terminal region of Tks4. In this closed conformation, the ability of the PX domain to bind PtdIns(3)P in biological membranes is blocked. Phosphorylation of Thr353 does not lead to the opening of the closed conformation and can only partially restore PtdIns(3)P binding. Further studies are needed to evaluate other potential regulatory mechanisms, such as the phosphorylation of tyrosine residues by Src kinase.In summary, the intramolecular interaction between tandem SH3 domains and the PTAM motif, and the interaction between the PX and SH31: residues 1–210, PX–SH32: residues 1–279, PX–PRR: residues 1–370, PX–SH33: residues 1–426, SH31–SH32: residues 155–279, PRR–SH33: residues 333–426, SH31: residues 155–210, SH32: residues 224–279, SH33: residues 371–426, PRRL: residues 333–370) were amplified by PCR and subcloned into a modified pET vector encoding an N-terminal His-tag followed by a TEV protease recognition site. All constructs were verified by DNA sequencing (Eurofins Genomics). The TEV protease gene cloned into a bacterial expression vector was a kind gift from Dr. László Nyitray. All proteins were expressed in E. coli Rosetta pLysS bacteria (Novagen) and purified as previously described [L and its variants , nickel-affinity chromatography [The expression vector harboring the human Tks4 gene (Uniprot accession number: A1X283) used in our experiments was described previously . DNA seqescribed . In the tography  was follS peptide was chemically synthesized on an automated PSE Peptide Synthesizer  with a standard Fmoc/tBu SPPS strategy. The peptide was purified via RP-HPLC using a ReproSil 300 C18; 5 µm; 250 × 10 mm column (Maisch). The purity and mass of the peptides were verified using a Shimadzu LC-MS 2020 instrument.The PRR1–SH32 (“tandem SH3”) fragment by wild type, mutant, and truncated PRR fragments  was followed by the detection of the intrinsic tryptophan fluorescence signal. Measurements were performed in 384-well GreinerBio microplates (Ref.: 781076) in 50 µL final volumes. Prior to the measurements, protein samples were dialyzed against PBS  supplemented with 0.5 mM TCEP and 0.05% TWEEN 20. Fifteen-step serial 3:4 dilutions of the PRR fragments were prepared and mixed with SH31–SH32 stock solutions to yield a constant final SH31–SH32 concentration of 5 µM for all experiments. The final concentrations of the PRR fragments varied between 1.8 µM and 100 µM. The emission spectra of all samples were recorded between 315–400 nm in 1-nm steps using a PerkinElmer EnSpire Plate Reader. Selective excitation of tryptophane residues was achieved by setting the excitation monochromator to 295 nm. Titrations were followed at 340 nm. The average signal intensity and standard deviation were calculated from three independent experiments. Dose-response data were analyzed by using a simple 1:1 binding equilibrium model.Titration of the SH32 to PRR–SH33 was measured using a MicroCal ITC200 instrument at 25 °C in PBS freshly supplemented with 0.5 mM TCEP. The concentration of PX–SH32 was 20 μM in the measuring cell, and the concentration of the PRR–SH33 domain was 250 μM in the syringe. Data were evaluated by the AFFINImeter ITC software utilizing the “one set of independent binding sites” model [The affinity of PX–SH3s” model .3, SH31–SH32, and SH33 constructs were fluorescently labeled with Nanotemper NT-647 amine-reactive dye  according to the manufacturer’s instructions. The PRR–SH33, SH31–SH32 and SH33 domains were titrated with PX–SH32, PRRL, and PX–SH31, respectively. All measurements were performed in PBS supplemented with 0.5 mM TCEP and 0.05% TWEEN 20 by using Monolith NT.115 Premium Capillaries (NanoTemper Technologies MO-k025) and a NanoTemper Monolith NT.115 instrument. Sixteen-step serial 2:3 dilutions of PX–SH31 and PRRL were prepared following the addition of the labeled ligand solutions, thus yielding final starting concentrations of 412 μM and 166 μM of PX–SH31 and PRRL in the titrations, respectively. In the case of PX–SH32, sixteen-step serial 1:2 dilutions and a starting concentration of 40 µM were used. The concentrations of the labeled ligands were kept constant (20 nM) in all experiments. All binding assays were performed in triplicate. MST traces were recorded at room temperature. The excitation LED was used at 50% power and the IR laser power was set to 20%. Data evaluation was performed with the MO Affinity Analysis 2.3 software. Dose response curves were analyzed by using the standard Kd Fit Model.The PRR–SH33 constructs, the PRR and two SH3 domains complex were paired together in CORAL runs to treat them as one rigid body. Atomic models used in the CORAL modeling were generated with homology modeling based on the 1UEC (SH3 domain), 1WLP (PRR and SH3 domains complex), and 1GD5 (PX domain) structures using Modeller [SAXS measurements were performed using the BM29 beamline at ESRF or the P12 beamline at EMBL-Hamburg (PETRA). Data were analyzed using the ATSAS 3.0 software package . Each coModeller .1, PX–SH32, PX–PRR, and PX–SH33). PIP strips were purchased from Echelon . All experiments were carried out according to the manufacturer’s instructions. Briefly, the strips were blocked in TBS-T (0.1% v/v TWEEN 20) with 3% fatty acid-free BSA  for 1 h at ambient temperature. The membrane was incubated with 10 ug/mL of the given protein for 1 h at room temperature, and then washed three times over 30 min with gentle agitation. The bound protein was detected via a 1-h incubation with anti-His antibody . Following a washing step, the membranes were incubated with anti-mouse IgG-HRP (Sigma-Aldrich A-9044) for 1 h prior to detection of bound proteins using enhanced chemiluminescence.Overlay assays were carried out using His-tagged fragments of Tks4 ; DOPE ; POPS ; cholesterol (700000); PI ; PA ; and PtdIns(3)P . Control liposome samples were prepared by mixing the following lipids: 13% (mol/mol) POPC, 35% (mol/mol) DOPE, 22% (mol/mol) POPS, 22% (mol/mol) cholesterol, and 8% (mol/mol) PI. The PtdIns(3)P and PA liposomes were prepared similarly, but the concentrations of POPC and cholesterol were reduced to 10% and 20% (mol/mol), respectively, and 5 mol% PtdIns(3)P or 5 mol% PA were added to the lipid mixture. Stock solutions were prepared in methanol at a concentration of 10 mM. Prior to the experiments, the appropriate amounts of lipids were dried under a stream of N1, PX–SH32, PX–PRR, PX–SH33, and PX–SH33T353E) were added to the liposomes to yield a final protein concentration of 1 µM in all cases. Mixtures were incubated on ice for 30 min. Soluble and liposome-bound proteins were separated via centrifugation in a TL-100 ultracentrifuge (Beckman) using a TLA100.3 rotor . The supernatants and pellets were collected and analyzed via SDS-PAGE. Bands were quantitated via densitometry using ImageJ [The freshly prepared liposome solutions were diluted to final total lipid concentrations of 0.01 mM, 0.1 mM, and 0.5 mM in assay buffer (PBS containing 0.1 mM TCEP). Solutions of the Tks4 fragments (PX–SH3g ImageJ .


Recent progress in nanoscale covalent organic frameworks (COFs)-mediated nanomedicines for cancer diagnosis and therapy is comprehensively summarized in this review.Future perspectives and challenges regarding COFs-mediated nanomedicines for diagnosis and therapy are discussed, with particular emphasis on possible clinical translation. Covalent organic frameworks (COFs) as a type of porous and crystalline covalent organic polymer are built up from covalently linked and periodically arranged organic molecules. Their precise assembly, well-defined coordination network, and tunable porosity endow COFs with diverse characteristics such as low density, high crystallinity, porous structure, and large specific-surface area, as well as versatile functions and active sites that can be tuned at molecular and atomic level. These unique properties make them excellent candidate materials for biomedical applications, such as drug delivery, diagnostic imaging, and disease therapy. To realize these functions, the components, dimensions, and guest molecule loading into COFs have a great influence on their performance in various applications. In this review, we first introduce the influence of dimensions, building blocks, and synthetic conditions on the chemical stability, pore structure, and chemical interaction with guest molecules of COFs. Next, the applications of COFs in cancer diagnosis and therapy are summarized. Finally, some challenges for COFs in cancer therapy are noted and the problems to be solved in the future are proposed. Porous nanomaterials, such as mesoporous silica nanomaterials, zeolites, and porous carbon nanomaterials, have showed potential applications in biomedical fields  due to their large surface area, tunable porosity, and easy functionalization. However, these porous nanomaterials are often inert, lacking any chemical, biochemical, or physicochemical activity. In most cases, these chemically inert porous nanomaterials are useful only as carriers of other active species. More recently, however, with the development of “chemistry of the framework” , metal-oTo compensate for these limitations, a library of COFs have been designed and fabricated as alternatives to MOFs. In 2005, Yaghi et al. designed and synthesized the first COF . COFs arImportantly, as a result of continuous research into molecular building blocks, surface functionalization, and fabrication methods, the structure and size of COFs can be continuously adjusted. Moreover, the size of COF can be reduced to nanoscale. Nanoscale COFs possess not only the good crystallization and porous characteristics of traditional COFs, but also the advantages of good dispersion, small volume, and high bioavailability. At the same time, their unique and tailorable nanostructures and properties make COFs ideal candidates for biomedical applications , 28, endThe structures and properties of COFs are determined by the reactive and functional groups of their building blocks, which have been discussed in detail in some other reviews –43. Typi3N4), 2D MOFs, and 2D COFs, 2D materials have been subject to extensive research attention. The “2D” in 2D COFs means that the covalent bonding is in the same plane; only the non-covalent interactions exist between the planes to form a 2D structure  imine condensation reactions  with 2,4,6-tris(4-aminophenyl)-1,3,5triazine (TAPT) [The unique characteristics of COFs, such as their extended π-conjugated structure, endow them with additional functions beyond potential phototherapeutic activity. The π–π interaction between the layers of a COF and the existence of heteroatoms can produce different bonding sites with biomolecules in the body, leading to chemical damage. Reasonable usage of this chemical damage can effectively inhibit and treat cancer. Based on this, Bhaumik et al. prepared a novel triazine-based mesoporous 2D COF (TrzCOF) via the solvothermal polycondensation of 1,3,5-tri(4-formylbiphenyl) benzene [Phe (TAPT) . In vitrBhaumik et al. experimented with a porous and biodegradable COF EDTFP-1 (ethylenedianiline-triformyl phloroglucinol), which was synthesized using a Schiff-base condensation reaction of 4,4′-ethylenedianiline and 2,4,6-triformylphloroglucinol . EDTFP-1Generally, COFs themselves as therapeutic drugs are still in the early stage of research. However, compared with traditional chemotherapeutic drugs, COFs have some highly advanced functions, such as using bond cooperation for biomolecular interaction and triggering protein expression in vivo.Early diagnosis of cancer plays an increasingly important role in improving cancer therapy. The development of early diagnosis technologies for cancer is of great significance for improving the survival rate of patients. As a kind of organic porous material, COFs have an ordered π-conjugated structure, high porosity, low density, good biocompatibility, and stability; these properties make them excellent carriers for in vitro tumor-detection substances, thus allowing for high sensing sensitivity. At the same time, COFs with high porosity can interact with a greater number of guest molecules, which greatly improves the detection limit. These unique properties of COFs greatly improve the performance of tumor detection.–1 with a linear detection range of 0.05–100 pg mL–1 for EGFR. For MCF-7 cells, the p-COF-based aptasensor showed a LOD of 61 cell mL–1 with a linear detection range of 500 × 105 cell mL–1. The excellent selectivity and high sensitivity were mainly attributed to the following points: (1) the high conjugation ability and π-conjugated structure of COFs not only increase the interaction between COFs and biomolecules, but also improve the electrochemical activity; (2) the mesoporous channel of COFs (2.06 nm) was conducive to the fixation and loading of aptamers; and (3) the planar structure of 2D COFs allowed more aptamers to be combined with biomolecules. Therefore, the proposed sensor can act as a new biosensing platform for rapid and sensitive detection of cancer biomarkers and cancer cells.Based on electrochemical measurement, Liu et al. developed a new aptasensor for immobilizing epidermal growth factor receptor (EGFR)-targeting aptamer strands on a porphyrin COF (p-COF), which can sensitively and selectively bind to EGFR to detect human breast cancer MCF-7 cells  into N-butyl-4-amido-1,8naphthalimide (NPH) by improving the activity and stability of Pd NPs, which improved the chromogenic sensitivity  of cancer due to its low cost, ease of operation, and high sensitivity. Moreover, COFs are characterized by chemical adjustability, high porosity, ordered structural integrity, and abundant binding sites. Therefore, COFs are becoming an ideal carrier for anchoring various guest molecules. For instance, Wang et al. developed a water-based stable carboxymethyl cellulose-modified COF hydrogel (Pd NPs/CMC-COF-LZU1) based on the principle of colorimetric determination to facilitate the in situ growth of palladium NPs . This coity Fig. b. SpecifAs a porous material, COFs can effectively load fluorescent dyes to minimize the quenching caused by aggregation. At the same time, COFs’ skeleton can not only protect the dyes for good photochemical stability—thus improving the fluorescent properties of the materials—but it also has excellent anti-interference ability, which can function as an excellent fluorescence sensor.1O2. Therefore, the luminescence from UCCOFs-1 at 800 nm was turned on, which reported in real time the position and dose of 1O2 generation in the body, thus realizing in situ self-reporting PDT.COFs with high surface area and porosity, good electrical conductivity, and special interaction with bioactive molecules have shown great promise in biosensing and bioimaging for cancer diagnosis. For instance, Wang et al. used a lanthanide-doped upconversion nanoparticle (UCNP) core to coat nanoscale COFs (UCCOFs) with various shell thicknesses via core-mediated imine polymerization . The UCCTo increase the high signal-to-noise ratio of imaging and tumor specificity, Liu et al. developed a pH-responsive nanoplatform based on zinc porphyrin COF (ZnCOF), in which the loading rate of zinc porphyrin (ZnPor) was 22.5 wt% . At pH 7To sum up, nanoscale COFs have the following advantages in cancer biosensing and bioimaging: (1) the rich π-conjugation is conducive to sensitive electrochemical signals through π–π stacking interaction with targeting ligands, functional biomolecules, and the like; (2) the optimized particle size for bioavailability can be obtained through COF design; (3) COFs with porous characteristics can fix functional biomolecules such as aptamer inside the material, thereby increasing the adsorption capacity of the probe and its resultant sensing sensitivity; (4) COFs have abundant binding sites and can interact with fluorescent dyes; and (5) COFs constructed from fluorescent molecules can directly have the ability of biological imaging. At the same time, the formation of COF structure can activate the fluorescent activity of the building block.In this review, we outline the recent breakthroughs in fabrication and application of COFs for cancer diagnosis and therapy. Given their versatility and physicochemical properties, COFs have showed unique advantages in cancer diagnosis and therapy, especially in drug delivery, phototherapy (including PDT and PTT), biosensing, and bioimaging. With their high surface area and porous nature, COFs also show unique advantages for the delivery of anti-tumor drugs to desired locations. For PDT and PTT, COFs can act not only as carriers of PSs or PTAs due to their porous structure and high surface area, but also as PSs or PTAs by virtue of their high light absorption cross section, π-conjugated structure, and versatile constructions from photoactive building blocks. COFs can effectively avoid the annihilation of PSs caused by π–π stacking and have good thermal stability, high contact area, and light utilization. These properties also help COFs play a role in bioimaging and diagnosis, and imaging-guided therapy.In spite of these advantages, the study of biomedical applications for COFs is still in the early stages. There are many ongoing challenges and limitations that must be considered and overcome. First, due to the complex structures and arrangements of the pores, it is difficult to precisely control the loading of drugs into and the patterns of release from COFs. In addition, if one more kinds of active molecule  are involved in the system, their location in or on the COFs should be rationally designed. With the abundant active groups acquired during or post-synthesis, the interior capacity and surface area of COFs could be fully utilized via host–guest interaction. Moreover, the structural qualities of COFs, especially their dimensions, have a significant impact on their performance. COFs with different dimensions (2D or 3D) have their respective advantages, and the applications of COFs with other dimensions have rarely been reported. The ingenious and controllable design of COFs’ dimensions is expected to further promote the application of COFs. In terms of possible clinical transformation, the biocompatibility of different nanoscale COFs, especially as regards long-term systemic toxicity, should be a top priority for consideration. Secondly, the large-scale production of nanoscale COFs is still difficult, and further attention is warranted to the study of batch repeatability. Due to the complex molecular arrangement of COFs , the preparation of COFs with stable performance and uniform size distribution in different synthetic batches still poses a great challenge. Finally, possible clinical translation depends on exploring the optimal biocompatible building blocks and achieving the best therapeutic effect through the design of substituents and structures.Overall, compared with other porous nanomaterials, nanoscale COFs have exhibited unique advantages in the field of cancer diagnosis and therapy. Unlike most of other porous nanomaterials, which act only as inactive carriers for drug delivery and realize other function solely through post-synthetic functionalization, COFs themselves have intrinsic electrical, magnetic, and optical properties that could be applied directly for therapeutic purposes. The synergy of drug delivery and other therapeutic modalities, including phototherapy and immunotherapy, could potentially be realized on a single COF-composed nanosystem, which could resolve many of the challenges of cancer therapy, including difficulty in early diagnosis, therapeutic resistance, recurrence, and metastasis.


Spinacia turkestanica, and the selective sweeps involved in domestication of cultivated spinach, S. oleracea, from S. turkestanica. A total 7,065 single nucleotide polymorphisms (SNPs) generated for 16 Spinacia oleracea and 76 S. turkestanica accessions placed the S. oleracea accessions in one group, Q1, and the 76 S. turkestanica accessions, which originated from Central Asia, in two distinct groups, Q2 and Q3. The Q2 group shared greater genetic identity with the S. oleracea accessions, Q1, than the Q3 S. turkestanica group. Likewise, the S. oleracea Q1 group had a smaller Fst (0.008) with the Q2 group than with the Q3 group (Fst = 0.012), and a greater gene flow (Nm = 30.13) with the Q2 group than with the Q3 group (Nm = 21.83). The Q2 accessions originated primarily from Uzbekistan while the Q3 accessions originated mostly from Tajikistan. The Zarafshan Mountain Range appears to have served as a physical barrier that largely separated members of the Q2 and Q3 groups of S. turkestanica. Accessions with admixtures of Q2 and Q3 were collected primarily from lower elevations at the southern end of the Zarafshan Mountain Range in Uzbekistan. Selective sweep regions identified at 32, 49, and 52 Mb on chromosomes 1, 2, and 3, respectively, appear to have played a vital role in the domestication of S. oleracea as they are correlated with important domestication traits, including day length sensitivity for bolting (flowering). High XP-CLR scores at the 52 Mb genomic region of chromosome three suggest that a selective sweep at this region was responsible for early differentiation of S. turkestanica into two groups in Central Asia.Genotype-by-sequencing (GBS) was used to explore the genetic diversity and structure of  Spinacia consists of cultivated spinach (Spinacia oleracea L.) as well as two other species which occur in nature, S. turkestanica Iljin and S. tetrandra ex M. Bieb , and S. turkestanica is distributed across Central Asia  and South Asia (Afghanistan and Pakistan)  . A limitcessions .S. oleracea accessions and accessions of the wild relatives S. turkestanica and S. tetrandra, based on transcriptome sequence analysis, revealed that S. turkestanica is the progenitor of cultivated S. oleracea, with S. tetrandra genetically more distantly related to the other two species (Spinacia species using a limited number of SNP markers (n = 56) for 25 S. turkestanica, 16 S. tetrandra, and 54 S. oleracea accessions.Few genetic diversity studies have been performed on spinach. Diversity analyses were first carried out for spinach accessions using polymerase chain reaction (PCR) assays based on simple sequence repeat (SSR) and target region amplification polymorphism (TRAP) markers to generate genetic fingerprint data . These s species . The samSpinacia accessions may provide further insights into spinach diversity and domestication.Domestication transforms the traits and genomes of crops . During Spinacia species can be sources of many commercially important genetic traits  in S. turkestanica have been transferred successfully into S. oleracea, providing the primary means of controlling this devastating disease  What is the genetic diversity and group structure of Spinacia genome that led to differences among these groups?2) If a group structure exists, what were the selective sweeps in the S. turkestanica selective sweeps were involved in the domestication of spinach?3) Which In 2008, the Centre for Genetic Resources, the Netherlands (CGN) carried out an expedition to collect seed from populations of ral Asia . After tSpinacia spp. was used in this study. Three accessions of S. turkestanica originating from Turkmenistan, another S. turkestanica accession of unknown origin, and 16 S. oleracea accessions were received from the National Plant Germplasm System (NPGS) of the United States Department of Agriculture (USDA). In addition, 72 S. turkestanica accessions collected in Uzbekistan and Tajikistan were obtained from the CGN, Wageningen University and Research (WUR). CGN collected the material under the Standard Material Transfer Agreement (SMTA) of the ITPGRFA. Information on each accession, including the country of origin, collection site , and collection year is provided in A total of 92 accessions of two Spinach plants were grown in RediEarth propagation mix  in a greenhouse at the Washington State University (WSU) Mount Vernon Northwestern Washington Research and Extension Center, with the air temperature set at 22–24°C during the day and 18–20°C by night, and supplemental lighting provided for 10 h/day. Plants were fertigated daily with General Purpose Fertilizer 20-20-20  injected into the irrigation water at a 1:100 ratio for applying a final nitrogen concentration of 200 ppm at each irrigation. A single leaf was harvested from each plant 35 days after planting, frozen immediately in liquid nitrogen, and stored at −80°C.https://www.biotech.wisc.edu/) for sequencing, where DNA quality and integrity were re-evaluated using the Quant-IT PicoGreen fluorescent dye . The GBS method of ApeKI restriction enzyme, as described by Genomic DNA was isolated from the single frozen leaf of each plant using the cetyl trimethylammonium bromide (CTAB) method , after thttp://www.spianchbase.org) using Bowtie two software  for each accession was used to draw a bar plot to visualize clustering of the spinach accessions. True groups were identified as the maximum value of ΔK, based on the rate of change of the natural log probability of the data. Spinacia spp. were assigned to individual groups (Q) based on an assignment value of 75%. Second, the inferred ancestry assessments obtained from Structure were judged based on prior knowledge of the geographic location of the collection site of each accession of both Spinacia spp., provided by the respective gene banks. A total of 7,065 SNPs common to the 76 S. turkestanica and 16 S. oleracea accessions was used to analyze the population structure of the accessions of these two species. To understand better the group structure and clustering pattern of these accessions, principal component analysis (PCA) was completed using PLINK v1.9  and polymorphic information content (PIC) of SNPs were calculated using PowerMarker software V 3.25.  (h), and Shannon’s information index (I) were calculated using GenAlEx 6.3 (Fst), Nei’s unbiased genetic distance (D), and Nei’s unbiased genetic identity (Id) were also estimated using GenAlEx 6.3. Gene flow (Nm) was calculated using the formula Nm = [(1/Fst)-1]/4, according to Spinacia spp., using G-statistics. In total, 999 permutations determined the fixation indices (Fst) at a level of significance of p < 0.001. Nucleotide diversity (π) was calculated using VCFtools v0.1.15 , number of effective alleles (Ne), Nei’s genetic diversity (h) , unbiaseAlEx 6.3 . Analysi v0.1.15  with a 1S. oleracea and S. turkestanica, and for the Q2 and Q3 groups of S. turkestanica (see details in the Results section). Again, for LD analysis between the Q2 and Q3 groups of S. turkestanica, accessions with <0.75 membership assignment (admixed between Q2 and Q3) of the group structure were excluded. As a result, of 76 S. turkestanica accessions, only 63  were used. The physical distance of the LD curve intersecting at the critical value of r2 = 0.20 was used as LD decay for each species and for groups within S. turkestanica. Screening of the spinach genome for selective sweep regions was first performed by comparing allele frequency differentiation between the S. turkestanica and S. oleracea accessions, following the method described by Q2 and Q3, among the S. turkestanica accessions, screening for selective sweep regions associated with the Q2 and Q3 groups of S. turkestanica was carried out by modelling the likelihood of multi-locus allele frequency differentiation between the two groups using XP-CLR v1.0 (https://github.com/hardingnj/xpclr) for each pseudochromosome with 50 Kb sliding window, 10 Kb step size, and setting the maximum number of SNPs in each window to 50 . The adjacent windows (<10 Kb) with high XP-CLR scores (top 1%) were grouped into a single region representing a single selective sweep region. The candidate genes within the selective sweep regions were also identified. XP-CLR analysis was performed using the 76 S. turkestanica accessions as a reference panel and 16 S. oleracea accessions as a query panel to identify selective sweeps. Of the 63 S. turkestanica accessions that remained after filtering for admixture, 33 accessions in Q2 and 30 in Q3 were used as the reference panel in the XP-CLR analysis. Similarly, given the evidence (see Results) that the S. oleracea accessions (Q1) are most closely related to the Q2 group of S. turkestanica, an analysis of selective sweeps was also computed between the 16 Q1 accessions as the query group and 33 Q2 accessions as the reference group.Linkage disequilibrium (LD) was calculated using SNP pairs within a 200 Kb window, and plotted using PopLDdecay3.4.1  for eachCLR v1.0 . XP-CLR Spinacia accessions. After filtering for sequencing adapters, low-quality bases, and de-multiplexing to extract reads matching the sample barcodes, approximately 300.7 million reads were retained with an average of 3.1 million and a median of 3.1 million reads per accession. Using the TASSEL GBS v2 pipeline, 210,968 SNPs were identified across the six chromosomes. After the SNPs were filtered for indels and a MAF <0.05, 105,328 SNPs were retained. Further filtering to keep only bi-allelic SNPs resulted in retention of 103,704 SNPs.Approximately 331.2 million raw reads were generated from the Illumina NovaSeq run for the 92 S. oleracea and S. turkestanica, respectively. Across both species, there were 20.7% missing calls, and the rest of the SNPs were retained for genetic analysis. Among the filtered SNPs, 7,065 SNPs common to all 92 Spinacia accessions that met the filtration criteria were retained. The distribution and density of these SNPs across the six chromosomes of S. turkestanica and S. oleracea are presented in When the SNP dataset was separated into two files, one for each species, with filtering for 20% missing SNP data, 9,456 and 7,629 SNPs were retained for S. oleracea and 76 S. turkestanica accessions ranged from 0.10 (40 SNPs) to 0.50 , with an average of 0.35  was 44.5 among three groups of the two species. The greatest Fst, 0.012, was between groups Q2 and Q3, both of which consisted of accessions of S. turkestanica, followed by 0.011 for group Q1 of S. oleracea and Q3 of S. turkestanica, and then 0.008 between the Q1 and Q2 groups , followed by Q1 and Q3 (0.989), and Q2 and Q3 (0.988). Consequently, the highest Nm was between Q1 and Q2 (30.13), followed by Q1 and Q3 (21.83), and Q2 and Q3 (19.77) (h), unbiased diversity index (uh), Shannon’s Information Index (I) and nucleotide diversity (π) ranged from 1.988 to 1.997, 1.607 to 1.616, 0.347 to 0.351, 0.355 to 0.357, 0.516 to 0.523, and 0.9223 × 10−3 to 0.9371 × 10−3 respectively , followed by Q3  and Q1 . However, the unbiased diversity index (uh) of Q1, which largely comprised S. oleracea accessions, was greater than that of the Q2 and Q3 groups (The AMOVA of the 76  species . The ave2 groups . The gen (19.77) . The meaectively . The mea3 groups .S. oleracea and S. turkestanica accessions revealed three main groups, Q1, Q2, and Q3 , Q2 (34 accessions), and Q3 (30 accessions), with another 17 accessions admixed  collected from Afghanistan, China, Iran, India, Nepal, and Pakistan, as well as nine S. turkestanica accessions admixed with S. oleracea accessions  were collected from Turkmenistan. Interestingly, with few exceptions, group Q2 comprised S. turkestanica accessions originating from Uzbekistan, while a majority of group Q3 comprised S. turkestanica accessions originating from Tajikistan , including sweep regions 1.3, 2.1, 3.2, 3.8, 3.9, 4.1, 5.1, 5.2, 6.1 and 6.2  and the two sub-populations, Q2 (0.9223 × 10−3) and Q3 (0.9304 × 10−3), of wild S. turkestanica relatives was similar. The high nucleotide diversity of the 16 S. oleracea accessions evaluated in this study might reflect the limited sample size (16 accessions) collected from different geographic regions. The nucleotide diversity estimated using GBS-generated SNPs in this study was slightly greater than estimates in −3.The relatively uniform, genome-wide distribution of 7,065 SNP markers identified across all six spinach chromosomes of 16 Spinacia and domestication of cultivated spinach. This study identified 20 signatures of selective sweeps in the Spinacia genome associated with the domestication of S. oleracea from S. turkestanica  and 11.40893 (with 11 SNPs), respectively. Further research is warranted to identify the specific domestication traits within these selective sweep regions in Spinacia.Little information has been published on selective sweeps involved in the evolution of the genus estanica . Howeverestanica . TherefoS. turkestanica groups Q2 and Q3 identified seven genomic regions that may be responsible for differentiation of these two groups. None of these selective sweeps regions coincided with domestication traits, although until present little research has been carried out on these traits in spinach. The selective sweep region 3.3 at the 52 Mb region of chromosome three appears to have had an important role in differentiation of the Q2 and Q3 groups. No genes involved in determining phenotypic traits have yet been associated with this region. A common selective sweep region at the 52 Mb region of chromosome three was also detected between the S. oleracea Q1 and S. turkestanica (Q2 and Q3) groups. This suggests that S. turkestanica was differentiated into groups Q2 and Q3 prior to the domestication of S. oleracea. As the S. oleracea accessions had greater genetic identity (Id = 0.993), less genetic distance (D = 0.007), and more gene flow (Nm = 30.13) with group Q2 accessions compared to group Q3 accessions , it is plausible that most of the S. oleracea accessions evolved from the Q2 group of S. turkestanica.Investigation of selective sweep regions of Q2 is concentrated, and Tajikistan, where group Q3 accessions were collected. Among the three groups, there was least gene flow (Nm = 19.77) between Q2 and Q3. This mountain range probably has served as a physical barrier to gene flow between these two groups for thousands of years. Therefore, we hypothesize that the two groups, Q2 and Q3, of S. turkestanica are in an early stage of allopatric speciation. The Zarafshan mountain range of the Tian Shan orogenic belt loses elevation in the southern part, a region where three accessions were found to have mixed genetic constitutions of the Q2/Q3 groups. This probably points to the occurrence of a hybrid zone between the two groups. Further research is warranted to elaborate on the early speciation of Q2 and Q3 groups of S. turkestanica in Central Asia.The orogenic mountain range of Tien Shan stretches through Central Asia, including Kyrgyzstan, Tajikistan, Uzbekistan, and Turkmenistan . The ZarS. turkestanica and S. oleracea, and to elucidate the origin of cultivated spinach. Three groups were identified among the S. oleracea and S. turkestanica accessions, with the S. oleracea accessions more closely related genetically to the Q2 group of S. turkestanica accessions than the Q3 group of this species. The selective sweep regions identified in the Spinacia genome indicated that S. turkestanica differentiated into the Q2 group (located on the western side of the Zarafshan mountain range) and Q3 group (located on the eastern side of the Zarafshan mountain range) first, followed by domestication of cultivated spinach, with Q2 accessions of S. turkestanica playing a greater role in the domestication of spinach. The selective sweep regions aligned with multiple domestication traits in the 32, 49, and 52 Mb regions of chromosomes 1, 2, and 3, respectively. A hybrid zone between both groups was found at the southern end of the Zarafshan mountain range. The highly polymorphic SNPs identified in this study can be used in future studies, such as genome wide association studies (GWAS) and marker assisted selection for various economically important agronomic traits found in S. turkestanica.High throughput GBS was employed to identify SNPs that were then used to explore the genetic diversity, genetic differentiation, and gene flow among accessions of


Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer. The glycome, the complex glycan repertoire of the cell, is involved in a myriad of cellular events in health and disease ,2,3,4,5.http://www.cazy.org/) Accessed on 24 April 2021 [5 sequences, originating from animal, plant, yeast, and bacterial species, and exhibits sequence heterogeneity except for a portion of the catalytic domain. It is noteworthy that the functional prediction of putative GTs  can be challenging. This is because many closely related sequences reported in the CAZy database, especially those related to polyfunctional GT families, may perform different catalytic functions [GTs are multi-substrate enzymes that transfer the donor’s sugar moiety, mostly an activated nucleotide, to the acceptor molecule, which is a glycan, a protein, or a lipid molecule. In eukaryotes, most GTs are type II transmembrane proteins with a short N-terminal domain, a transmembrane domain followed by a stem region, and a large C-terminal catalytic domain at the luminal side . GTs areril 2021 . This seunctions .There are numerous challenges toward a structural understanding of GTs ,14,15,16Sialyltransferases (STs) and fucosyltransferases (FUTs) are two distinct classes of GT in terms of structural fold architecture, substrate specificity, the nature of their interactions with both donor and acceptor substrates, and catalysis. Intriguingly, the dysregulation of both STs and FUTs results in the altered expression of sialylated and fucosylated epitopes, which are hallmarks of cancer cells ,23,24,25N-Acetylneuraminic acid (Neu5Ac) as well as their linkages, STs are classified into four families: ST3GAL; ST6GAL; ST6GALNAC; and ST8SIA. Each family consists of several STs , small (S), 3rd (III), and very small (VS) ,28,29. HMultiple sequence alignment of STs in vertebrates revealed the presence of family motifs containing four to twenty amino acids specific to each ST family, thus implicating another level of amino acid conservation among STs ,31,35. EN-glycans and are clearly distinguishable from the classical α1,3 FUTs , which utilize short and linear lactosamine-related acceptors [Depending on their linkage specificities and acceptor substrates, 13 human FUTs are organized into four families—α1,2 FUT; α1,3/4 FUT; α1,6 FUT; and O-FUT . The α1,cceptors ,43.x, Ley, Lea, Leb, sLex, and sLea), thus contributing toward the complexity of fucose-containing Lewis epitopes in naturally occurring glycoconjugates [x and sLea) epitopes are selectin ligands that are overexpressed in cancers due to the upregulation of FUT4, FUT5, FUT6, and FUT7 [x/sLea contribute to cancer dissemination and metastasis [O-glycan and selectively fucosylate epidermal-growth-factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs)  and Lewis antigens  , respects (TSRs) .Hydrophobic cluster analysis performed on FUTs from vertebrates, invertebrates, plants, and bacteria revealed conserved peptide sequences termed as FUT motifs—three in the catalytic region common to α1,2 and α1,6 FUTs ,52; two http://www.cazy.org/) Accessed on 24 April 2021. Both STs and FUTs present a complex tissue-, cell-type-, and stage-specific expression pattern, and are expressed as both membrane-bound and soluble proteins [FUT10 gene and investigated their subcellular distributions. The FUT10-319 isoform encodes a soluble protein expressed in human embryos. FUT10-419 and FUT10-479 are reported to be co-localized with calnexin  to the phosphate group of the nucleotide. It is noteworthy that the DxD motif is not a conserved feature of the GT-A fold, since there are examples of enzymes containing this fold that lack this motif [The architecture of GT-A is reminiscent of two tightly associated β/α/β Rossmann domains, the sizes of which vary, leading to the formation of a continuous central β-sheet to create the N-terminal donor and C-terminal acceptor binding regions . Most GTis motif .The GT-B fold, on the other hand, consists of two separate β/α/β Rossman domains, i.e., an N- and a C-terminal domain separated by a large cleft where the active site is located and stabilized by two long C-terminal-helices. The GT-B fold lacks the DxD motif and generally does not require metal ions for catalysis. Donor and acceptor substrates bind to the C- and N-terminal regions of GT-B, respectively .Campylobacter jejuni, belonging to the GT42 CAZy family [SsST3GalI), whose catalytic domain displays a mixed α/β fold with a seven-stranded parallel β-sheet flanked by 12 α-helices. PorcineST3GAL1 exhibits a modest 10% sequence identity with CstII but contains a β-sheet core and a lid-like structure analogous to the CstII fold. Porcine ST3GAL1 is speculated to be a second distinct GT-A variant   from y family . CstII iy family   , which dsST3GAL1 , HsST6GAsST6GAL1 , HsST6GA6GALNAC2 , and HsSsST8SIA3  adopt a sST8SIA3 ,37,40 ansST8SIA3 , i.e., asST8SIA3 , His-367sST8SIA3 , His-316sST3GAL1 , His-370sST6GAL1 , His-3516GALNAC2 , His-346sST8SIA4  , GDP is found in a shallow cavity of the C-terminal domain. In contrast, half of HsTSR1 is embraced by a cleft formed between both domains [HsFUT8 revealed that the GT-B fold contains only one Rossman fold  and revealed another unique property of FUT8 in mammals, which is that it undergoes a conformational change upon binding to GDP [HsFUT8 structure but become ordered upon binding GDP. This transformation leads to the formation of new interactions between loops A and B; in particular, the electrostatic interactions between Asp368 and Arg365 of loop A and Arg441 of loop B are involved , STs/ FUTs can be classified as glycan- or protein-modifying GTs. Glycan-modifying GTs include all ST and FUT subfamilies except the O-FUTs, which are protein-modifying GTs in humans.HsST6Gal1 bound to the Gal2GlcNAc2Man3GlcNAc2-Asn acceptor illustrates that the C6-OH group of the terminal Gal residue from the Gal-β-1,4-GlcNAc moiety of the acceptor is adjacent to the His catalytic base [HsST3Gal1-Gal-β-1,3-GalNAc-oNP binary complex revealed that the plane of the terminal Gal acceptor residue is rotated by 180°, which ultimately positions its C3-OH group adjacent to the catalytic His residue for glycan transfer [HsST3Gal1. In fact, extensive hydrogen bonding stabilizes the complex while taking advantage of the down-facing axial hydroxyl groups of the disaccharide acceptor. These interactions are quite different from the mode of hydrophobic stacking and hydrogen bonding that is common among Gal-specific binding proteins, including HsST6GAL1 [HsST8SIA3-NeuAc-α-2,3-Gal-β-1,4-GlcNAc-6-SO4 showed that the acceptor NeuAc of ST8SIA3 primarily forms hydrogen-bonding interactions by positioning the nucleophilic C8-OH group of NeuAc adjacent to the His catalytic base [HsST6GALNAC2 binary complex, but without an acceptor, revealed minimal sequence and structural similarity in the primary sequence of the loop regions and secondary structural elements involved in acceptor substrate recognition compared to HsST6GAL1, HsST3GAL1, and HsST8SIA3, implying the presence of significant diversity in the acceptor binding region among members of the GT29 family [Glycan-modifying GTs include mammalian GT29 sialyltransferases that employ analogous conserved sugar donors but recognize diverse acceptor substrates. The acceptor binding regions of STs have shown striking differences in their sequence, secondary structure, and position among the members of the GT29 family . The crytic base ,73 ,investigated by Kotzler et al., displays that a hexasaccharide is required as a minimal acceptor structure [HsFUT8 glycan acceptor recognition. Gracia et al. captured a ternary complex of GDP:HsFUT8:A2-Asn and showed that the catalytic domain is connected to the N-terminal coiled-coil domain by interdomain α3; however, the C-terminal SH3 domain is in contact with the catalytic domain by the β10–β11 loop [HsFUT8:A2-Asn ternary complex is aligned with the GDP-Fuc:HsPOFUT2 binary complex, the fucose residue from donor substrates occupies the appropriate position within the active site of FUT8 such that C1 of the Fuc residue is directly aligned for nucleophilic attack by the OH-6 of the GlcNAc-1 of A2-Asn  corroborates their observation that the trisaccharide GlcNAc-β1,2-Man-α1,3-Man moiety is the key determinant for the acceptor recognition of FUT8 [To further understand the preferences of the acceptor site, Jarva et al. solved the ternary structure of FUT8 in both mice and humans. The SH3 domain emerged as contributing toward the evolution of FUT8 as a dimer, which restricts the movement of the SH3 domain and stabilizes the acceptor binding site . Glu373 e enzyme . The str of FUT8  -C3 [1-X-X-(S/T)-C2 or C2-X-X-(S/T)-C3 of TSRs of groups 1 and 2, respectively (where X is any amino acid) [1–C5, C2–C6, and C3–C4, the TSRs of group 2 are arranged as C1–C4, C2–C5, and C3–C6. Interestingly, the binding region of each of these O-FUTs is complementary to the face of the domain of the protein, which interacts with the cleft through multiple hydrogen bonds, especially within the loop with the consensus sequence. This orients the domain to transfer the OH-group of the Ser/Thr acceptor substrate exactly in the correct position to perform the nucleophilic attack on the anomeric C-1 of the Fuc residue of the donor substrate [Protein-modifying GTs target the hydroxyl group of Ser or Thr in proteins. They must first bind to the acceptor protein to orient the respective Ser/Thr hydroxyl nucleophile correctly for the transfer of a sugar moiety from the donor substrate. These include POFUTs, which modify folded cysteine-rich domains . POFUT1 (S/T)-C3 . Howeverno acid) . While dubstrate ,61.HsPOFUTs in complexes with their acceptor substrates are not yet solved; however, the ternary complexes of O-FUTs have been crystallized in mice and C. elegans. The ternary complex of GDP/GDP-Fuc:MmPOFUT1:EGF-LDs revealed multiple points of interaction between POFUT1 and EGF-LDs. However, the core of the interaction involves a conserved preformed cleft on POFUT1 and conserved, sequence-independent structural elements on the fucosylation motif, which is common to all EGF-LDs [MmPOFUT1 is an important feature, which enables the enzyme to accommodate the sequence diversity of its EGF-LD substrates. The structure of CePOFUT2–GDP–HsTSR1 highlighted that the CePOFUT2 binding domain contains large cavities that are filled by an intricate network of water molecules [CePOFUT2 and HsTSR1 that are complemented by many water-mediated interactions. The proactive role of these water molecules is speculated to bestow promiscuity to CePOFUT2 toward dissimilar TSRs, which might be claimed for similar GTs that modify a wide variety of peptide sequences.Experimental structures of  EGF-LDs . The intolecules . The comThe catalytic mechanism is independent of the overall fold of GTs since both inverting and retaining enzymes are common among the GT-A and GT-B superfamilies. Nucleotide-dependent GTs catalyze a glycosyl transfer reaction either by retention or inversion of stereochemistry at the anomeric reaction center of the donor substrate to generate diverse biological glycans with distinct anomeric configurations  Figure A. The orN2 single-displacement reaction mechanism  revealed reduced activities of ST3GAL1, ST3GAL3, and ST6GAL1 in in vitro and cell-based activity analyses . Lith-O-HsFUT3 and HsFUT7, as described in detail by de Vries et al. [HsST8SIA1 and HsST8SIA4 have also been developed with RosettaCM50 using a template alignment generated with Modeller [HsST8SIA4 with its acceptor substrate has been explored with RosettaDock [HsST3GAL1using the crystal structure of porcine ST3GAL [HsFUT8 for donor substrate binding using its crystal structure [HsFUT8 [Molecular modeling of STs and FUTs is challenging since the number of available crystal structures remains limited for humans. Previously, in the absence of crystal structures, homology modeling along with fold recognition or threading techniques were used to predict structures. The first structural model for the mammalian FUT family, FUT4 in mice, was developed using this technique . Furthers et al. . HomologModeller . AdditioettaDock . The SWIe ST3GAL . Strecketructure . In addi [HsFUT8 . Since bSTs and FUTs display conformational plasticity upon substrate binding and catalysis. Notable progress has been made in the last decade in the successful crystallization and molecular modeling of several STs and FUTs, which has opened new avenues to fill gaps in our understanding of their structural architecture, interactions with substrates, and catalytic mechanisms. This progress holds the promise to impact medical and biotechnological development. Despite the remarkable breakthrough in the determination of high-resolution crystal structures of mammalian STs and FUTs in both apo and binary complexes, very few are available as ternary complexes. It is therefore essential to capture donor-enzyme-acceptor ternary complexes to delineate the molecular basis of the catalytic mechanisms of these glycosyltransferases, which are sought-after therapeutic targets in diseases such as cancer.