id
stringlengths 24
24
| idx
int64 0
402
| paragraph
stringlengths 106
17.2k
|
|---|---|---|
60c74788bb8c1a3c3f3daac2 | 15 | properties. An equivalent one-dimensional pore system was constructed for investigating the use of altitudinal rotors (Fig. ) which includes two motors orientated perpendicular to the pore direction, such that the rotation is orientated in the direction of diffusion. The unidirectional rotation for this case is also arranged with the bias torque in the same direction for each rotor. The sampling and simulation details are analogous to that discussed for the azimuthal case. Similar to the system containing azimuthal motors, we observe no discernible influence from the motor rotation in the large pore structures. Moreover, for the small pore structures, where the positions the rotor particles in close proximity there is also the same relative decrease in diffusion. |
60c74788bb8c1a3c3f3daac2 | 16 | The unidirectional motion of molecular rotors is considered imperative for directing movement at the molecular level. Thus, we sought to consider how non-directional motion manifests and effects diffusion in small pore systems, for both the azimuthal and altitudinal rotors. In fact, such a case reassembles the scenario of rotors frequently incorporated in the backbone of porous frameworks but not governed by a stimulus, such as light. To investigate this, we conducted similar simulations as described in the previous two sections, but with the absence of the bias torque. This resulted in non-directional motion of the rotors (Fig. ). Without the application of bias force, the rotation is observed to consecutively oscillate in random directions, clockwise or anti clockwise. The presence and dynamics of the confined LJ liquid is not observed to produce unidirectional or preferred directional rotation of the rotors. |
60c74788bb8c1a3c3f3daac2 | 17 | Non-directional motion of the azimuthal rotor resulted in equivalent diffusivity as the unidirectional motion previously described. Contrastingly, the altitudinal rotor showed enhanced diffusivity, greater than both the considered unidirectional and fixed rotor dynamics (6% greater than the fixed rotor case). This non-directional motion can provide the altitudinal system with the same diffusivity as the azimuthal system. |
60c74788bb8c1a3c3f3daac2 | 18 | As the directionality, and hence cooperativity, of the motors in this small pore structure can effect the transport properties of the confined fluid we examined different combinations of rotation and their effect on the directionality of the diffusion in the small pore, for both orientations of the motor. Please note the frequencies of rotation are aligned, here we only investigate the direction. We considered six different cases: fixed rotors, non-directional rotors and the four combinations of clockwise or anti clockwise rotation. We label the direction of rotation by the sign of the bias torque (Fig. ). The alignment of azimuthal motors with the pore walls shows no distinct direction of diffusivity, with the average displacement close to centre, around 0 𝜎. This indicates equal diffusion in either direction of the one-dimensional pore. Notably, this is not affected by different combinations of rotation by the motors. The altitudinal motors, however, exhibit important dependence on the set rotation of the motors. If the rotors have no rotation (fixed), non-directional rotation or aligned rotation the LJ fluid is observed to show no directional preference. |
60c74788bb8c1a3c3f3daac2 | 19 | Although the magnitude of diffusion is only marginally affected by the arrangement of motors, for example we have not demonstrated on/off switchable diffusion, the induction of a preferential diffusion direction is certainly promising. Notably, the rotors used here are of relative size to the diffusing LJ particles and of equal interaction strength. It is the effect of cooperativity and confinement that is responsible for this significant outcome on the global transport properties. Amplification of these effects are expected to occur if the average rotor-fluid interactions as well as the confinement effects are optimized. An increase in motor density per void volume is certainly also expected to enhance the observed diffusion effects. |
60c74788bb8c1a3c3f3daac2 | 20 | The molecular simulations, described in this study, represents a minimalist and qualitative picture of molecular motors, and the possible consequence of arranging them in confined solid-state. Many approximations are made to describe the motion of rotors in this study. For example, the rotation mechanism is significantly reduced to a two-state motion. Notably, the rotation between the two-state occurs almost instantaneously, when the dihedral potential is switch. In reality molecular motors show complex rotational dynamics with slow and fast movements to produce a full rotation. In future studies, it is possible to use more complex potential energy surfaces and gradual potential changes to more realistically reflect the intricate dynamics of real-world motors. Nevertheless, the potential switching mechanism should provide the foundation to build new atomistic models of molecular machines. |
60c74788bb8c1a3c3f3daac2 | 21 | A key observation from considering the combined dynamics of the rotor surrounded by fluid is the impervious nature of the fluid bulk. The small rotor system is a drop in the ocean compared to the stable dynamics of a fluid. This is clear from the global properties remaining unaffected by rotational dynamics, while only the very local properties, for example the particles in the arc of rotation, show some evidence of change. This observation is in line with recent scepticism about diffusion enhancement by dynamic species in diluted solutions. In turn, this demonstrates the importance of regular, defined arrangement with a maximal high motor density of these machines in confinement. It was only when the fluid was confined in a pore of comparable size to the molecular rotor where the applied rotation began to affect the global transport properties of the fluid. Still, in our initial model we find relatively modest changes to diffusivity. However, by considering the orientation of the motor and the effect of different rotations of rotors we find the greatest change. By combination of altitudinal rotors aligned into the pore and synchronization of the rotational directionality we could produce a turnstile-like system able to direct the diffusion in a specified direction of the pore. This illustrates that random orientation and non-directional rotation of molecular motors in the solid-state are unlikely to produce diffusion enhancement on a larger length scale let alone directed diffusion. Cooperative rotation and specific arrangement of unidirectional rotation are required to produce designed transport phenomena. |
60c74788bb8c1a3c3f3daac2 | 22 | In this work, we have outlined the influence of rotating molecular species and the importance of their orientation, cooperativity and directionality on the transport properties of confined fluids. Our findings help to understand the influence of local dynamics of molecular motors and rotors on the surrounding fluid. This includes, the nature of pore space functionalized with dynamic groups and potential strategies to manipulate transport properties of confined fluids by this dynamic pore space. Although the applied model drastically simplifies real-world molecular motors, it captures the most important aspects being unidirectional rotation and orientation of the rotor. Our results, however, provide helpful indications about the need of high rotational frequencies in addition to the detection of minor changes in diffusivity locally and globally. We envision the guidelines, presented here, to be crucial for the design of stimuliresponsive dynamic materials, capable of manipulating guest transport properties by dynamic molecular machines. However, the currently reported porous materials that include molecular machines or rotors do not meet the specified criteria defined by this investigation. |
60c74788bb8c1a3c3f3daac2 | 23 | As discussed in the introduction, many transport phenomena are supported by activated vibrations of the pore walls, a property neglected in the present work. The combination and alignment of local dynamics from a functional surface, by anchored groups, and global vibrational dynamics of the framework backbone, towards cooperative pore agitation on different length scales, represents an interesting avenue for novel transport phenomena in porous solids and should be further explored. |
676d39296dde43c90862eca2 | 0 | Chlorinated compounds are found in various natural products such as Atpenin A5 (1), Perforenone B (2) or Clionastatin B (3), active pharmaceutical ingredients such as Quinfamide (4) or Chloramphenicol (5), and essential intermediates used in chemical manufacturing (Scheme 1A). Chlorine-based chemistry has been used in the synthesis of around 20% of small molecule drugs and 30% of agrochemical products. Among organochlorides, chloroalkanes serve as important synthetic precursors to access several functional groups, including alcohols, amines and thioethers. The classical methods for synthesizing chloroalkanes involve electrophilic addition of formal [Cl + ] intermediates to an olefin (Scheme 1B, eq. 1), nucleophilic substitution with chlorides [Cl -] (Scheme 1B, eq. 2), and the use of chlorine radicals [Cl•] (Scheme 1B, eq. 3). As they are difficult to control, chlorine radicals have long been considered of lower interest for fine chemical synthesis. Recently, based on important progress in radical chemistry, there has been a renewed focus on this approach. Different transformations have been developed, including radical additions to olefins, hydrogen atom transfer processes onto Csp 3 -H bonds, or oxidative cleavage of C-C bonds. With an emphasis on sustainable chemistry in the past few decades, several photocatalytic protocols have been developed to convert electrophilic or nucleophilic chlorine sources to chlorine radicals. These methods include reductive activation, oxidative activation, and, more recently, ligand-to-metal charge transfer (LMCT) processes for the homolytic cleavage of metal chlorides. Hypervalent iodine reagents (HIRs) have similar properties as heavy metals in organic transformations. In particular, the homolytic fission of the I-Cl bond can generate chlorine radicals in analogy to LMCT processes (Scheme 1B, eq 4). Indeed, the photolysis of HIRs to give chlorine radicals has been described using iodine trichloride ICl3, Willgerodt reagent (6a) and recently chlorinated cyclic HIRs. Among them, 6a stands out as it is more stable and easier to handle than ICl3 and also more atom-economical than cyclic HIRs. 6a has been extensively applied for the electrophilic chlorination of Csp 2 centers such as olefins or aromatic rings (Scheme 1C, eq. 5), as well as for the oxidation of alcohols and thioethers. These pathways are potentially competitive with radical chlorination, suggesting that 6a would not be a good choice to favor this pathway. We hypothesized, however, that irradiation of 6a would lead to rapid homolytic fission of the I-Cl bond, producing chlorine radicals fast enough to suppress electrophilic chlorination pathways. As a result, highly selective radical chlorination of C-H and C-C bonds to form Csp 3 -Cl bonds could be achievable even in the presence of arenes or alcohols (Scheme 1C, eq. 6). Although the use of 6a for chlorination under photochemical conditions was initially introduced by Banks, it has been applied only to the chlorination of steroids by Breslow, Wicha, and co-workers in the 70's and 80's, and the potential of this approach in other chlorination reactions has not been further explored since then. Given that reagent 6a can be easily accessed via the reaction of chloride anions with iodosobenzene, it can potentially be generated in situ using a catalytic amount of iodobenzene in the presence of an external oxidant. Gilmour and Wirth recently reported catalytic iodine(I/III) chlorinations from CsCl or TMSCl, focusing on the electrophilic chlorination of olefins. A radical approach would allow to target not only olefins but also cyclopropanes C-C and alkanes C-H bonds. |
676d39296dde43c90862eca2 | 1 | Herein, we report the use of 6a for the formation of various Csp 3 -Cl bonds including 1,3-, 1,2-and C-H-chlorinated products starting from cyclopropanes, alkenes and alkanes respectively (Scheme 1D). The most efficient protocol was established using a stoichiometric amount of 6a, but preliminary results for a catalytic method were also obtained. Additionally, we introduce a one-pot process to further functionalize benzylic chlorides with aryl, N, O, and S nucleophiles, providing a fast and modular approach to 1,1-diaryl compounds, important building blocks in the synthesis of various pharmacophores. Especially, 1,1diaryl-3-chloropropane compounds were reported as the precursors to synthesize a wide range of commercial drugs such as Prozapine (7a), Fenpiprane (7b), Diisopromine (7c), or Fendiline (7d), and other bioactive compounds via amination of the terminal chloride (Scheme 1E). Scheme 1. A. Natural products and drugs containing Csp We started our investigation by irradiating a mixture of phenylcyclopropane (8a) and chlorobenziodoxolone reagent 6b, which we had previously used as a chlorine radical source in the oxidative activation of cyclopropanes (Scheme 2A). After 4 hours of irradiation with three equivalents of 6b, we observed an 86% NMR yield of the 1,3-dichlorinated product 9a. However, further attempts to improve the yield by increasing the amount of 6b or prolonging the reaction time were unsuccessful. We then examined Willgerodt-type reagents 6a,c-e. Interestingly, a quantitative yield of 9a was formed after 15 minutes of irradiation, and only 1.2 equivalents of 6a were needed to achieve complete conversion. In contrast to electrophilic chlorination, the electronic structure of the aromatic ring did not affect the reactivity of 6, giving 9a quantitatively with either an electron-rich (6c) or electron-poor reagents (6d-e). Further screening of other common solid nitrogen-based chlorinating reagents 6f-h did not deliver the product, demonstrating the unique properties of the Willgerodt-type reagents under photolysis. Further screening of solvents showed that chloroform, acetonitrile, and ethyl acetate could also be used with a slight decrease in yield. The reaction was less efficient in acetone, methanol, water/acetonitrile mixtures or THF. To our delight, the same conditions could be applied for the 1,2 chlorination of vinyl naphthalene 10a and the C-H chlorination of ethyl arene 12a, delivering 76% of 11a and 86% of 13a (Scheme 2B). In the case of 10a and 12a, 1.5 equivalents of 6a were required to reach complete conversion. Performing a control experiment in the dark for the chlorination of 8a for 1 hour resulted in 27% of aromatic chlorination 9a1, 15% of 9a and 54% of recovered cyclopropane 8a. Only 7% of 11a was observed starting from 10a and no product was detected with 12a. These results demonstrated the dominance and higher efficiency of the radical pathway under irradiation. We consistently observed a large amount of iodobenzene ( ) remaining after irradiation of 6a, thereby, we envisioned that a catalytic process could be achieved by adding chlorine salt and an oxidant to re-generate 6a (Scheme 2C). Following a protocol reported by Gilmour and co-workers, we screened reactions in parallel with different oxidants, including Selectfluor, NFSI, oxone, and chlorine salts (LiCl, KCl, CsCl) under visible-light irradiation with 20 mol% iodobenzene (14) as catalyst. We observed the formation of products 9a, 11a, and 13a when using either Selectfluor or oxone as the oxidant. NFSI did not result in product formation for any of the three substrates. We found that the best conditions for C-C chlorination were using CsCl with Selectfluor as the oxidant, yielding 68% of product 9a. Oxone proved to be the best oxidant for chlorination of 10a and 12a, delivering 38% of 11a and 79% of 13a. However, several background reactions were observed and affected the reaction efficiency, such as fluorination in the case of 8a, polymerization of 10a and aromatic chlorination on 12a. Overall, the possibility of performing chlorinations directly from chloride salts under iodine(I/III) photocatalysis had been demonstrated. Nevertheless, the use of stoichiometric 6a still delivered better results without side reactions and required a shorter reaction time than the photocatalytic protocol. Given that 6a is an easily accessible reagent from iodobenzene ( ), we decided to continue to explore the substrate scope using stoichiometric amounts of 6a. Scheme 2. Reaction optimization and control experiments. NMR yield is reported using CH2Br2 as an internal standard. n.r = no reaction. |
676d39296dde43c90862eca2 | 2 | We then investigated the substrate scope of aryl cyclopropanes 8 in the 1,3 chlorination (Scheme 3A). The desired products were obtained cleanly, and no aromatic chlorination was observed regardless of the electronic properties of the phenyl ring (products 9a-h). The isolated yield of 9a was only 80% compared to the quantitative NMR yield due to its instability on column chromatography. We encountered similar issues with products 9b and 9c. Product 9b was even fully hydrolyzed on column chromatography to give the corresponding benzylic alcohol in 51% yield. The reaction tolerated various functionalities, including a bromo (9d), a cyanide (9e), an acetyl (9f), a pinacol boronate (9g), and a naphthyl (9h) groups, with isolated yields ranging from 63% to 91%. A diphenyl-substituted cyclopropane resulted in 79% yield of product 9i. A donor-acceptor cyclopropane with a diester functional group delivered 68% of products 9j. Interestingly, the reaction tolerated a carboxylic acid and a pyridine derivative, giving 91% yield of 9k and 74% yield of 9l. It has been reported that pyridines can substitute chloride on 6a, and carboxylic acids are usually incompatible with reactions involving electrophilic HIRs. The products observed demonstrated that photolysis was fast enough to promote the radical pathway over other reactions. The protocol also allowed the late-stage chlorination of heterocyclic bioactive molecules, yielding chlorinated products 9m derived from the drug Lesinurad in 42% yield and 9n in 59% yield. It is worth mentioning that the thioether in 9m was tolerated, even if it could be sensitive to oxidation. Scheme 3. Substrate scope of chlorination. Yields in bracket are NMR yields with CH2Br2 as an internal standard. Scope on 0.2 mmol scale. 1.5 equiv. of 6a. The chlorinated compound was hydrolyzed on reverse phase chromatography, and the corresponding alcohol was isolated. 2.0 equiv. of 6a. 2.5 equiv. of 6a. 4.0 equiv. of 6a. |
676d39296dde43c90862eca2 | 3 | We then explored the substrate scope of olefins (Scheme 3B). In general, styrene derivatives having an electron-donating group (products 11a,b) gave lower yields than with an electronwithdrawing group (11c,d), likely due to instability of the products during column chromatography. 1,1-Disubstituted or 1,2disubstituted styrene derivatives delivered 1,2-dichlorination products in high yields (11e: 93%, 11f: 71%, 11g: 80%). Despite having opposite electronic properties, both 11f and 11g can be obtained with good yields, demonstrating the generality of radical chlorination compared to classical electrophilic chlorination. The protocol was also successfully applied to non-activated olefins (products 11h-j). In the case of allyl benzene, an ipso rearrangement was observed, giving a 5:1 mixture of 11h1 and 11h2 in 71% yield. Product 11i was isolated in 79% yield starting from 4-phenyl-1-butene. Although it was reported that 6a can be used as an efficient reagent to oxidize alcohols, the reaction shows good tolerance to both alcohols and amines, yielding 74% of product 11j starting from the drug Oxprenolol. Further exploration of the substrate scope for C-H chlorination revealed excellent yields with 4-bromo ethyl benzene and ethyl benzene (products 13a and 13b) (Scheme 3C). When starting from an aryl cyclobutane, we obtained C-H functionalization instead of C-C cleavage, resulting in cyclobutyl alcohol 13c after isolation on reverse phase column chromatography. We were also able to achieve C-H chlorination of -carbonyl C-H bonds, yielding 93% of isolated product 13d, although four equivalents of 6a were necessary to complete the reaction. Starting from the substrate having both secondary benzylic and -carbonyl C-H bonds, 13e and 13f were isolated as the major products. Only a trace amount of benzylic chlorination was observed from the crude mixture. Interestingly, we obtained product 13g in 64% yield without chlorination of the acetyl group, likely due to the unfavorable formation of a primary radical on the acetyl group. The reaction favored the less steric hindered benzylic C-H bond of Ibuprofen methyl ester, resulting in 75% yield of product 13h. Lastly, the protocol was successful in case of Benzbromarone, giving 43% yield of product 13i. The simplicity of the protocol allowed us to use benzylic chlorides as intermediates for a telescoped functionalization (Scheme 4A). We focused on 1,1-diaryl motifs, which are frequently encountered in bioactive molecules (Scheme 1E). Following modified reported conditions for Friedel-Crafts arylation, we successfully performed a one-pot chlorination/arylation sequence, resulting in 1,1-diaryl-3-chloro scaffolds 15 starting from cyclopropanes 8. The nucleophilic substitution happened at room temperature in the presence of iron(III) chloride and a base, and purification of the chlorinated intermediate was not required. Cyclopropane 8a was converted to 15a in 77% yield after two steps. The reaction was successful for both bromo and boron substituted substrates (products 15b,c). However, the nucleophilic substitution is not efficient with cyclopropanes bearing electron-withdrawing groups, as exemplified by product 15d obtained in 26% only, likely due to the difficulty of forming the benzylic carbocation. Using phenol as a nucleophile exclusively delivered aromatic substitution over O-alkylation, giving a 52% yield of 15f as a para/ortho mixture in a 7:1 ratio. Products 15g and 15h were obtained in 76% and 66% yield, respectively, using 1,3-benzodioxole and benzothiophene as nucleophiles. Arylation product 15i was obtained in 45% yield starting from Indomethacin methyl ester. The drug Nimesulide and Estrone can also be used for the aromatic substitution (products 15j and 15k), demonstrating the possibility for late-stage functionalization of bioactive compounds. We also explored the same reaction conditions with sulfonamides as nucleophiles, obtaining good yields from para-methoxyphenyl sulfonamide (product 15l, 75%) and the drug Celecoxib (15m, 70%). In this case, one equivalent of iron chloride was required to accelerate the reaction. Alcohol and thiol nucleophiles can also be used under the same reaction conditions, giving products 15n-p in 65-74% yield. Additionally, C-H arylation and 1,2 chloro-arylation of styrene derivatives can be achieved, yielding 61% of product 15q and 60% of product 15r. During scope exploration, we often noticed the presence of a byproduct resulting from benzylic arylation with iodobenzene 14. This suggests the possibility of reusing 14 as a substrate in the C-C bond forming step. Since 14 has low reactivity in Friedel-Craftstype arylation, we envisaged the use of a Nickel-catalyzed reductive coupling of 14 with the benzylic chloride intermediate to give a 1,1-diaryl scaffold in an atom-economic manner (Scheme 4B). Following modified reported conditions (see SI for details on optimization), we carried out the reductive coupling directly from the concentrated crude mixture after ring-opening chlorination. After two steps, we successfully obtained chloroarylation products 16a-d with yields ranging from 49% to 61%. This approach is suitable for both electron-poor and electron-rich aromatic partners, making it complementary to the Friedel-Crafts type reaction. We expected that 1,3 chlorinated compounds could also serve as dielectrophilic intermediates (Scheme 4C). A double substitution was conducted with sodium sulfonate and sodium azide, resulting in 57% yield of disulfonate 17a and 63% yield of diazidation product 17b over two steps. Lastly, we used the same method to perform a telescoped C-H benzylic functionalization with azide and morpholine nucleophiles, producing C-H amination products 18a-c (Scheme 4D). All the reactions were conducted without purification of the chlorinated intermediates, demonstrating the practicability of our method. Scheme 4. Two-step protocol for benzylic functionalization. Scope on 0.2 mmol scale. 0.5 equiv. of FeCl3. 1.0 equiv. of FeCl3. |
676d39296dde43c90862eca2 | 4 | In conclusion, we have developed a general protocol for chlorinating cyclopropanes, olefins, and activated C-H bonds using the direct photoexcitation of the Willgerodt reagent (6a). The conditions are mild and the reaction practical to set up, with a wide range of functional groups tolerated. Additionally, we demonstrated that photo-mediated chlorination using Iodine(I/III) catalysis is possible, which serves as a complementary approach to the stoichiometric use of the Willgerodt reagent (6a). Taking advantage of the easily accessible benzylic chlorides, we have developed a one-pot protocol for further substitution with C, N, O, and S nucleophiles, and for repurposing the aryl iodide byproduct in a reductive cross-coupling. Our work provides a practical approach to functionalize cyclopropanes, olefins, and activated C-H bonds via the formation of organo-chlorine intermediates, and we believe it will be, therefore, of interest for accessing useful building blocks in synthetic and medicinal chemistry. |
621ffc5491a2e6fceae2b5ab | 0 | Optical rotation (OR) refers to the phenomenon of rotation of the plane of polarization of impinging linearly polarized light by an optically active sample, e.g., a sample with an enantiomeric excess of a chiral compound. The ubiquity of enantiospecific chiral molecules in biological systems makes the ability to differentiate enantiomers especially relevant. While experimental measurements can provide the sign and magnitude of the rotation, theoretical simulations are necessary to correlate the sign of the rotation to the structure of the molecule. Therefore, the assignment of the absolute configuration of a chiral sample requires a synergistic effort of experiment and theory. |
621ffc5491a2e6fceae2b5ab | 1 | Reliable quantum mechanical (QM) methods based on density functional theory (DFT) and coupled cluster (CC) theory have been developed for the calculation of chiroptical properties, using response theory to evaluate the appropriate OR tensor. However, these techniques are based on approximate solutions of the Schrödinger equation, which affect the accuracy of the calculations and may compli-cate the comparison with experiment. The need to effectively represent electron correlation in the calculation of OR has been well established. While DFT methods present a cost efficient means of accomplishing this, CC methods maintain the allure of being systematically improvable by including higher order excitations. The basis set dependence of OR calculations has been investigated at DFT level. Mach and Crawford examined the basis set dependence of CC with single and double excitations (CCSD) with the LPol basis sets along with the augmented correlation consistent basis sets of Dunning. Srebro et al. also tested the LPol basis sets, showing that they do not outperform aug-cc-pVDZ. This work also provides insight on the potential benefits of using a functional with a long-range correction. Howard et al. investigated the performance of the ORP basis set with CC methods, and Haghdani et al. studied the basis set dependence of CC methods in comparison with DFT for a set of seven molecules. Much work in pursuit of improving accuracy and reducing computational cost of OR calculations has been done through manipulation of the basis set. The work of Cheeseman et al. has demonstrated the importance of diffuse functions for OR calculations. Basis sets specifically for determination of optical properties have been developed. The LPol basis sets of Baranowska and Sadlej 29 augment the sets of Duijneveldt with diffuse and polarization functions. The ORP basis set of Baranowska and Laczkowska was later developed, in which the basis sets of Schäfer et al. were modified with contracted diffuse and polarization functions, resulting in smaller basis sets than the LPol sets. Recently our group proposed two minimal basis sets, augD-3-21G and augT3-3-21G, both of which are formed from augmenting the 3-21G set with diffuse functions from the parent Dunning correlation consistent basis sets optimized for OR calculations, based on an initial study of Wiberg et al. with unoptimized functions. These basis sets were proposed with the intent of maintaining the quality of a large basis set while decreasing the cost of OR calculations. |
621ffc5491a2e6fceae2b5ab | 2 | Another important issue of approximate calculations is that the choice of gauge for the representation of the electric dipole perturbation leads to numerically different results, contrary to a hypothetical exact calculation. Furthermore, in the length gauge (LG) the OR is origin dependent while in the velocity gauge (VG) the OR has a spurious static limit. These are both unphysical artifacts due to the incompleteness of the basis set and of the electron correlation treatment in practical simulations. The origin dependence of LG calculations can be removed in variational methods using London orbitals, also known as gauge including atomic orbitals (GIAOs), at the cost of considerably more complicated equations and computer codes. Additionally, this approach cannot be used with standard CC methods, unless one employs uncommon orbital optimization techniques. Recently, our group has introduced an approach to obtain origin invariant LG results, LG(OI), without the need of GI-AOs so that it can be equally used with variational and non-variational methods. To obtain meaningful results with VG, one needs to calculate the spurious static term explicitly and subtract it out, an approach called modified VG (MVG). Despite all of the work mentioned above, it is not yet clear what choice of gauge, level of theory, and basis set is optimal for OR calculations. The main obstacle to such a determination has been the inability to perform a uniform comparison between choices of gauge and basis sets across variational and non-variational methods due to the unresolved issue of the origin dependence of standard LG calculations (before the development of the LG(OI) approach). Another problem is that a direct comparison with experimental data is difficult for this sensitive molecular property. On the one hand, simulations are not yet able to reliably account for solvation effects, vibrational effects are typically introduced only through approximate perturbative treatments, and proper averaging of multiple conformers of the same enantiomer with opposite OR sign requires very accurate relative energy calculations. On the other hand, experimental measurements of rigid chiral molecules in the gas phase (which would sidestep some of the above simulation limitations, i.e., conformational averaging and solvation effects) are very limited due to the low volatility of these compounds and the overall small signal. In this work, we address a number of these issues by performing a thorough analysis of the basis set dependence in approximate calculations of optical rotation using different choices of gauge. We use an extensive set of chiral molecules (50 at DFT level and 17 at CCSD level) to test how OR calculations approach the complete basis set (CBS) limit with the LG (including GIAOs for DFT), LG(OI), and MVG schemes. The calculations are performed with the aug-cc-pVζZ series up to ζ = Q followed by an extrapolation to the CBS limit, which provide a consistent improvement of the basis set description. We investigate the convergence towards the CBS limit for each choice of gauge within each method, the convergence towards gauge invariance for each method, and a comparison between methods at the CBS limit. Thus, we can draw important conclusions on the effect of basis set and electron correlation incompleteness for a given choice of gauge and model chemistry. We also attempt a limited comparison with experimental data for a subset of 5 rigid molecules for which experimental data in the gas phase are available. |
621ffc5491a2e6fceae2b5ab | 3 | Although this work is based on well-established methodologies, it is useful to briefly review the main equations to compute the optical rotation. The isotropic OR is expressed as specific rotation [α] ω (or [α] λ ) in customary units of deg [dm (g mL)] -1 , where ω is the frequency of the impinging light (and λ is the corresponding wavelength): |
621ffc5491a2e6fceae2b5ab | 4 | where ̵ h is the reduced Planck's constant (J s), N A is Avogadro's number, c is the speed of light (m/s), m e is the mass of an electron (Kg), M is the molecular mass (amu). β is the electric dipole-magnetic dipole polarizability tensor, which is expressed in atomic units together with the frequency ω. The tensor can be written with a sum-over-states (SOS) formula as: |
621ffc5491a2e6fceae2b5ab | 5 | where ψ 0 and ψ j are the ground and jth excited state wavefunctions, respectively, µ is the electric dipole operator, m is the magnetic dipole operator, Greek indices correspond to Cartesian components, and ω j0 is the jth excitation frequency. This expression works in the non-resonant regime 1,64-66 and for wavelengths larger than the average molecular size. In practice, the tensor is evaluated using the linear response formalism (LR), because it does not require the evaluation of excited states. The linear response function, expressed with the notation ⟪X; Y ⟫ ω for two generic operators X and Y , can be evaluated using two representations of the electric dipole operator: |
621ffc5491a2e6fceae2b5ab | 6 | where the summation is over the number of electrons and r and p are the position and momentum operators. The two representations are denoted length gauge (L in Eq. 3) and velocity gauge (V in Eq. 4). The trace of the tensors in the two choices of gauge is equivalent in exact calculations: 14 3-4 lead to the same OR value, i.e., gauge invariance refers to the equality in Eq. 5. This equivalence is also satisfied for approximate methods with variationally optimized orbitals in the CBS limit. However, achieving gauge invariance in approximate variational methods does not guarantee that the OR is numerically the same as that obtained in an exact calculation. For non-variational methods, like in standard approximations of CC theory, and in general with incomplete basis sets, one obtains numerically distinct values of the OR in Eq. 1 with different choices of gauge. |
621ffc5491a2e6fceae2b5ab | 7 | As discussed in the Introduction, approximate calculations with incomplete basis sets lead to an unphysical static limit in the velocity gauge formulation, which is explicitly calculated and subtracted out in the MVG method, although the results are intrinsically origin invariant. Conversely, approximate LG calculations lead to origin-dependent OR values; these can be made origin-independent using GIAOs with variational methods. However, GIAOs are not useful with methods where the orbital response is not included in the LR calculation, as for standard CC methods. Alternatively, origin independence can be achieved with the LG(OI) approach, which works for variational and non-variational methods. The origin dependence of the β tensor in the LG is due to the off-diagonal elements of the mixed length-velocity gauge electric dipole-electric dipole polarizability tensor α (L,V ) , which is non-symmetric for approximate methods. In LG(OI), α (L,V ) is diagonalized with a singular value decomposition (SVD): |
621ffc5491a2e6fceae2b5ab | 8 | This transformation renders the diagonal elements of β, and therefore its trace, origin invariant. Since the α (L,V ) tensor is symmetric in an exact calculation, the LG, LG(OI), and MVG methods become equivalent for a complete treatment of electron correlation and a complete basis set. For variational methods, the choice of gauge [LG(GIAOs), LG(OI), or MVG] becomes immaterial in the CBS limit even with approximate treatments of electron correlation, as demonstrated numerically in section 4. |
621ffc5491a2e6fceae2b5ab | 9 | The OR was computed using standard linear response methods as discussed in section 2. Three choices of gauge were considered: modified velocity gauge (MVG), length gauge (LG), as well as the newly developed origininvariant length gauge [LG(OI)]. LG calculations were performed with gauge-including atomic orbitals (GIAOs) for variational methods (B3LYP, CAM-B3LYP ) and without for CCSD. Thus, CCSD-LG OR calculations are inherently origin dependent. The two density functionals were chosen as representative members that provide reasonable OR values. A test set of 50 molecules with a wide range of molecular sizes and OR magnitudes was compiled from previous OR studies, see Figure . Molecular geometries were also obtained from these sources, as detailed in Tables S1-S50 in the Supporting Information (SI). OR values were calculated for the whole set using DFT methods and for the first 17 molecules in Figure using CCSD because of computational limitations. |
621ffc5491a2e6fceae2b5ab | 10 | Basis sets of various sizes were used in order to determine the tendency of OR values to converge for a given method/gauge. Since it is important to obtain a consistent trend towards the basis set limit, 40 the correlation consistent basis set series was used: aug-cc-pVζZ, where ζ = D, T, or Q. OR values were extrapolated to the CBS limit using the two point inverse power extrapolation formula: |
621ffc5491a2e6fceae2b5ab | 11 | where [α] ζ λ is the OR value obtained for the basis set aug-cc-pVζZ, A and n are fitting parameters, and [α] ∞ λ gives the OR value extrapolated to the CBS limit. In order to obtain a two point fitting for this system of equations, n must be fixed. Values of n were set to 3 for double/triple (DT) extrapolations and 5 for triple/quadruple (TQ) extrapolations, following the recommendation of Haghdani et al. The CBS values from the TQ extrapolations are utilized as the reference values in the comparisons, as described below. Basis sets with higher order angular momentum functions would provide results closer to the basis set limit, but we found that quadruple-ζ level was sufficient for the purposes of this study and it allows calculations on fairly large systems in a reasonable amount of time. |
621ffc5491a2e6fceae2b5ab | 12 | The OR values are analyzed to compare convergence towards the CBS limit within each choice of gauge, between gauges within each method, and between methods. The comparison is performed using the signed relative error for each molecule i at a given basis set size, normalized by the total number of molecules in the set (N ): |
621ffc5491a2e6fceae2b5ab | 13 | By changing the OR value used as reference, one can focus on a different type of comparison. For instance, by using the (best estimate) CBS OR value within each gauge for a given method as reference, once can probe the basis set convergence of each choice of gauge. If the CBS OR value for a specific choice of gauge is used as reference, one can test gauge invariance for a given method as a function of the basis set size. Finally, by using the CBS OR value for a specific choice of gauge and method, one can compare differences between methods. For each molecule in the test set, we selected the enantiomer with positive OR (when calculated with the largest basis set) to avoid complications in the interpretation of the sign of ∆ ref i in Eq. 9; thus, ∆ ref i < 0 always indicates an underestimation of the reference value and vice versa. The molecules for which we changed enantiomer compared to the original study are also listed in the SI. The OR signs are consistent for all molecules and basis sets, with a few exceptions with aug-cc-pVDZ that are explicitly discussed in the next section. |
621ffc5491a2e6fceae2b5ab | 14 | The magnitude of the OR across the test set varies between O(1) -O( 3 ) deg [dm (g mL)] -1 . In the plots, each ∆ ref i bar is color coded from blue to red with increasing magnitude of the reference OR value for each molecule. The change in color was restricted to a range of 0 to 100 deg [dm (g mL)] -1 , such that the bars for molecules with OR values exceeding 100 deg [dm (g mL)] -1 are displayed as the same color (red). This is done for ease of comparison as a few of the molecules, in particular the helicenes (45-49 in Figure ), exhibit very large OR values relative to the rest of the set. This approach is slightly altered in the experimental comparisons, where the maximal value for color coding is set to 500 deg [dm (g mL)] -1 to more easily distinguish the molecules in the given set. This color coding visually provides information about any possible correlation between the sign of the error and the magnitude of the OR. Note that, although relative errors tend to be larger for molecules with small-magnitude OR, this is not a major issue for the data and it becomes less significant as the basis set size increases. On the other hand, using a single definition of the error as in Eq. 9 (plus the color coding) allows us to treat the entire test set on the same footing. It also allows us to combine information about the distribution of errors into a single plot. |
621ffc5491a2e6fceae2b5ab | 15 | Calculations used for theoretical comparisons were performed at the sodium D line (589.3 nm) to avoid any potential issue with resonance conditions, whereas those used for the comparison with experiment were performed at 355 nm and 633 nm to match the available experimental values. All calculations were performed using a development version of the GAUSSIAN 74 suite of programs and all raw data is reported in Tables S1-S50 of the SI. |
621ffc5491a2e6fceae2b5ab | 16 | The data in Figure describe the degree to which the OR values converge with increasing basis set size for each choice of gauge and method. In these plots, the reference value used in Eq. 9 is the CBS(TQ) extrapolation for each gauge. For all methods, there is a clear decrease in total error compared to the reference values; the ∆ TQ U values (Eq. 10) with aug-cc-pVQZ are of the order of 0.01 for the DFT methods, panels (a) and (b), and 0.02-0.04 for CCSD, panel (c). Nonetheless, MVG results show a slightly slower convergence compared to the other choices of gauge. |
621ffc5491a2e6fceae2b5ab | 17 | With smaller basis sets, especially ζ = D, the error is driven by the molecules with smallmagnitude OR as expected. In particular, the molecules that have the wrong sign with this basis set (molecule 27 for DFT and 2 for CCSD) account for a large part of the error, e.g., 18% of the total ∆ TQ U with aug-cc-pVDZ and LG(OI) for B3LYP and 44% with CAM-B3LYP. Significant improvement is observed going from ζ = D to T, with ∆ TQ U values going from 0.2-0.3 to 0.04-0.08 for DFT, respectively. However, CBS(DT) extrapolations seem to offer little improvement and even slightly worsen the result in the case of LG. Comparing the convergence across methods, B3LYP shows the fastest convergence with ba-sis set size, but CAM-B3LYP is only slightly worse. Not surprisingly, CCSD shows a more marked basis set dependence than DFT. Nevertheless, ∆ TQ U values with aug-cc-pVQZ only vary between 0.02 for LG(OI) to 0.04 for MVG. ∆ TQ U is always largest with MVG at CCSD level compared to the other choices of gauge; in fact, the MVG error is twice as large as that of LG(OI) for basis sets with ζ = T and Q. The data in Figure also show a rather random distribution of the errors and no particular correlation with the OR magnitude. The ∆ TQ S values (black horizontal lines crossing the bars in the plots) tend to be negative, indicating an overall underestimation of the reference values. However, this is again mostly driven by the molecules with small OR magnitude, especially those with the wrong sign from the ζ = D calculations. |
621ffc5491a2e6fceae2b5ab | 18 | Gauge invariance is tested by selecting the CBS(TQ) extrapolation with LG(OI) as reference for all choices of gauge within a method, and the results are shown in Figure . This choice of reference is somewhat arbitrary, in the sense that any of the gauges could be used. However, LG(OI) was selected because of its slightly faster convergence rate towards the CBS limit compared to the other choices of gauge. Since gauge invariance is expected for variational methods at the CBS limit, it is worth discussing the DFT and CCSD results separately. For the functionals in panels It is apparent from Figure (c) that CCSD does not approach gauge invariance at the CBS limit (the results from Figure indicate that the CBS(TQ) extrapolation is sufficiently close to the CBS limit). Gauge invariance is not expected for standard approximate CC methods unless the treatment of electron correlation is complete, as discussed in section 2 and demonstrated numerically in Ref. 40. Therefore, the difference between choices of gauge at the CBS limit is a measure of the electron correlation incompleteness for a particular level of truncation in the excitation expansion. A value of ∆ TQ/LG(OI) U = 0.26 for the CBS(TQ) extrapolation with the MVG indicates that CCSD is in fact still quite far from convergence in the treatment of electron correlation (at least for this test set). The ∆ TQ/LG(OI) U change for LG by a factor of 3 (from 0.44 with aug-cc-pVDZ to 0.13 at the CBS limit) indicates a considerable decrease in the "origin dependence" for this choice of gauge. In comparison, the corresponding change for MVG is only by a factor of 2 (from 0.59 to 0.26). It can also be inferred from the graph that the MVG tends to give OR values that are larger in magnitude relative to LG(OI), as evidenced by the positive values of ∆ TQ/LG(OI) S from MVG across all basis set sizes. The opposite can be said for LG, which shows a tendency to underestimate the LG(OI) reference OR. |
621ffc5491a2e6fceae2b5ab | 19 | The final comparison is across methods, which can be obtained by using the CBS(TQ) extrapolation with CAM-B3LYP for each gauge as reference for the other methods and basis sets (note that here there is no direct comparison across choices of gauge), see Figure . The results in panel (a) between different functionals indicate that gauge invariance does not lead to the same overall OR values, as the ∆ TQ/CAM-B3LYP U converge to 0.26 at the CBS limit. A reduction in ∆ TQ/CAM-B3LYP U can be seen from the double to triple zeta basis set. However, further increase in basis set size yields little to no improvement. This is due to results from both functionals converging at similar rates and in the same direction to their respective CBS values, such that the difference between OR values remains almost constant as the basis set size is increased. Posi- A comparison with experimental data (used as reference in Eq. 10) is performed for a subset of the molecules: 14, 30, and 41-43, shown in Figure . These compounds were chosen because they are fairly rigid and have a single stable conformer, and experimental data in the gas phase are available. This choice eliminates various difficulties due to solvation effects and averaging over contributions from multiple stable conformers, but it comes at the cost of a limited sample size. Furthermore, contributions from vibrational motions are not included in the calculations. Therefore, these constraints somewhat limit the generality of this comparison. Only DFT methods are used because most of these systems are too large to be treated at CCSD level with large basis sets. |
621ffc5491a2e6fceae2b5ab | 20 | At 355 nm, left panels in Figure , the errors are dominated by molecule 30, which is a notoriously difficult case for simulations. The increase in basis set size does not provide a significant improvement of the results. More informative are the results at 633 nm, shown in the right panels in Figure , where the errors are more evenly distributed. Also in this case, the basis set incompleteness is not the most important source of error; this is the case also for the choice of gauge with smaller basis sets. Overall, CAM-B3LYP shows a smaller ∆ Exp U with each choice of gauge and basis set compared to B3LYP by about 0.05-0.15 units. The smallest ∆ Exp U S values are obtained for both methods with LG(OI) and the CBS(DT) extrapolation, which is likely fortuitous. From this data, it is not possible to determine whether the disagreement with experiment at the CBS limit is due to missing vibrational effects or electron correlation incompleteness in the calculations, although it is likely that both contributions are important. 5 Discussion and Conclusions |
621ffc5491a2e6fceae2b5ab | 21 | In this work, we examine how OR calculations progress towards the CBS limit with different choices of gauge and level of theory. We use an extensive test set of 50 molecules for DFT methods and 17 molecules for CCSD, Dunning basis sets up to quadruple ζ with a two-point inverse power extrapolation to estimate the CBS limit, and three choices of gauge: MVG, LG(OI) and |
621ffc5491a2e6fceae2b5ab | 22 | The plots in Figures show that the TQ extrapolation is essentially at the CBS limit for DFT, with both functionals showing gauge invariance. For CCSD, the convergence towards the CBS limit is slower than for DFT, and it is due to the larger dependence of this level of theory on the basis set size compared to DFT. The CCSD ∆ TQ U values in Figure , up to 0.04, are slightly larger than for DFT, but they are small enough to consider the OR values essentially converged with respect to basis set size. All methods provide an almost perfect consistency in the OR sign with all basis sets, choices of gauge, and molecules. The only exceptions are molecules 27 for DFT and 2 for CCSD, which have a small OR magnitude and the calculations with the smallest basis set give the wrong sign with all choices of gauge. These two molecules are the main cause of the large relative errors with the aug-cc-pVDZ basis set in Figure . The CBS(DT) extrapolation offers little to no improvement over a regular aug-cc-pVTZ calculation for DFT, likely because the double ζ results are of poor quality. On the other hand, the CBS(DT) extrapolation provides significant improvement over the triple ζ results for CCSD-MVG, because this choice of gauge has a slower rate of convergence with basis set size. The slower convergence rate for MVG compared to LG and LG(OI) is consistent across methods (although the differences between gauges are small for DFT). In all cases, the results with the CBS(DT) extrapolation are farther from the CBS limit than those obtained with the aug-cc-pVQZ basis set, compare DT and Q sectors in the plots in Figure . Nev-ertheless, the aug-cc-pVQZ basis set is often prohibitive for production level calculations; therefore, using aug-cc-pVTZ for DFT calculations and the same basis with a CBS(DT) extrapolation for CCSD may provide the best cost/accuracy balance for OR calculations. |
621ffc5491a2e6fceae2b5ab | 23 | CCSD does not reach gauge invariance at the CBS limit, as shown in Figure (c), with a difference of ∆ TQ/LG(OI) U between LG(OI) and MVG equal to 0.26 (i.e., 26%). This indicates that the treatment of electron correlation for this property is not quite at convergence for CCSD, at least for this test set. It would be interesting to see what level of CC expansion (and basis set size) is necessary to reach gauge invariance for these molecules, but these calculations are currently beyond our capabilities. While gauge invariance for the systematically improvable CC methods would correspond to the exact OR values for a molecule (at least in terms of electronic response), gauge invariance provides no such guarantee for approximate density functionals. This is clearly shown in Figure (a), where B3LYP and CAM-B3LYP do not reach the same CBS limit even if they individually reach gauge invariance. A known limitation of current density functionals is that a systematic improvement of electron correlation for a particular functional is not possible, although Autschbach and coworkers have shown that range-separated functionals can provide better optical activity results when the rangeseparation parameter is optimized to reduce the delocalization error. An important question is whether these methods provide OR estimates that become closer to the experimental results as the basis set is increased. This is a difficult question to answer because it is hard to perform a clean comparison with experimental data. In fact, solvation effects, signal averaging over multiple conformers of the same enantiomer, and vibrational contributions to the OR are all factors that significantly affect the final measured value of this property. Theoretical models to account for these effects are still rather approximate and would cloud the comparison. Therefore, we only attempted a limited comparison by focusing on 5 rigid molecules with a single stable conformer for which experimental data in gas phase are available. This comparison still suffers from the lack of vibrational effects in the calculations. With these caveats in mind, the results in Figure indicate that CAM-B3LYP is more accurate than B3LYP for every basis set/gauge choice and the aug-cc-pVTZ basis set is sufficient to obtain good agreement with experiment. The long-range part 77 of CAM-B3LYP allows a more accurate modeling of the outermost parts of the wavefunction, which is especially important for OR calculations. |
621ffc5491a2e6fceae2b5ab | 24 | From all plots, it is evident that the choice of gauge is less important than the choice of basis set and method. However, Figure shows that the LG(OI) results converge faster than MVG to the CBS limit for all methods, especially for CCSD. Considering that LG(OI) is computationally cheaper than MVG and that it is less difficult to implement than LG with GIAOs for DFT, this choice of gauge may be optimal for OR calculations. |
629c75506057b1651591faad | 0 | Self-assembly of protein monomeric units is of great interest in supramolecular complex design, but owing to the non-covalency of quaternary structural interactions, mimicking their functionality for the synthesis of new protein complexes is demanding. Metal coordination is used as a driving force for the assembly of protein monomers, leading to a degree of multimerization that depends on the metal ion coordination geometry. Different strategies employed the engineering of chelating motifs of natural and unnatural amino acid residues, the incorporation of non-natural ligands onto protein surfaces, and the construction of hybrid coordination motifs, resulting in metal-induced increased stability of the multimer due to higher metal-binding affinity. Proteinprotein interfaces nucleated by metal coordination have led to the formation of 2D and 3D crystalline protein lattices, and novel functional materials. Of particular interest is the formation of metal-bridged dimers because they are considered the precursor of more complex assemblies. Several techniques such as X-Ray crystallography, NMR, and sedimentation velocity have extensively characterized these binding processes. Dipolar electron paramagnetic resonance (EPR) spectroscopy has recently emerged as an excellent complementary tool for studying metal ion-binding equilibria with sub-micromolar sensitivity. The four-pulse DEER (double electron-electron resonance) and the five-pulse RIDME (relaxation-induced dipolar modulation enhancement) experiments (for pulse sequences see ESI section 1.3) allow detection of the weak dipolar interaction between paramagnetic centres, which is characterized by modulation with the dipolar frequency (ωAB) that encodes the inter-spin distance, rAB. The modulation depth (Δ) of these traces (i.e., the amplitude between the signal intensity at 𝑡 = 0 and the time when the signal is entirely damped in the limit of negligible intermolecular decay) informs the number of coupled spins. In previous studies, DEER and RIDME were employed individually for monitoring the metal-templated dimerization of a nitroxide labelled terpyridine-based ligand model system using different divalent metal ions as templates , and proved the feasibility of monitoring binding events at cryogenic temperatures, with the modulation depth informing on the degree of binding at a given metal-to-ligand ratio. Here, DEER and RIDME were employed complementarily for studying the metal-templated dimerization of a protein model system, the B1 immunoglobulin-binding domain of protein G of Streptococcus sp. Group G (GB1). Double-histidine (dHis) motifs are incorporated as artificial metal-binding sites, 26 making this system particularly suitable for this study. Previous works have used this system as a biological model for pulse dipolar electron paramagnetic resonance spectroscopy (PDS) studies, improving the precision and accuracy of distance measurements due to the increased rigidity of Cu II -chelate spin labels through bipedal attachment. The I6R1/K28H/Q32H and I6H/N8H/K28R1 constructs (Figure ) were selected for this work. Only the construct I6R1/K28H/Q32H gave appreciable DEER modulation depth when bound to Cu II and Zn II (see ESI section 2.1), and modulation depth was maximised for the Cu II series. This is not entirely surprising since it is consistent with the finding of metal-induced stabilization of the α-helix motif via histidine residues and its higher affinity towards Cu II than to other metal ions. 30 The DEER experiment measures the intermolecular nitroxide-nitroxide (R1-R1) distances within the metal-templated dimer and therefore provides direct information on the dimer formation. The modulation depth, ∆ 𝑅1-𝑅1 , depends on the dipolar interaction between nitroxide moieties of each GB1 monomer. The RIDME experiment measures the intramolecular metal-nitroxide distances (M-R1) and is a reporter of all metal-bound species. The modulation depth, ∆ 𝑀-𝑅1 provides information about the formation of dimers coordinated around the metal template and fractional saturation of the metal-binding site, the dHis motif. To characterize these binding equilibria, a cooperative binding model was used to fit simultaneously ∆ 𝑅1-𝑅1 and ∆ 𝑀-𝑅1 values. This allows simultaneous characterization of a dissociation constant (KD) for initial metal binding and a cooperativity factor (𝛼) for the metal-templated dimerization event. |
629c75506057b1651591faad | 1 | First, measurements were performed as a nine-point pseudo-titration series (where each datapoint was a discrete sample) for Cu II , in the presence of phosphate buffer (150 mM NaCl, 42.4 mM Na2HPO4 and 7.6 mM KH2PO4, pH 7.4), having been used extensively for previous EPR methodological work involving GB1 and Cu II -NTA. In Figure , the ∆ 𝑀-𝑅1 behaviour was consistent with a reduced apparent binding affinity of Cu II for the dHis motif in phosphate buffer, with <80% occupancy at a metal:protein ratio of 1:1. |
629c75506057b1651591faad | 2 | Indeed, ∆ 𝑀-𝑅1 was systematically lower for the phosphate buffer series than the Tris-HCl buffer series (ESI and vide infra for details). Interestingly, the ∆ 𝑅1-𝑅1 behaviour suggested that in phosphate buffer, metal-templated dimer formation was optimised at a metal:protein ratio of 1:1 and persists even above stoichiometric Cu II concentration. Replicate measurements of the phosphate buffer series (see ESI) reproduced this observation, and the fitted parameters: 𝛼 = 2, and a 𝐾 𝐷 of 2.7 × 10 -5 further supported the positive cooperativity of dimerization. This affinity agrees with reported literature values for Cu II binding to histidine residues on a solvent-exposed α-helix. Being aware of the possible precipitation of Cu II in the presence of phosphate salts, buffers were screened that would retain free Cu II in solution under alkali conditions via CW-EPR measurements (see ESI section 2.4). For this purpose, Tris-HCl buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4) was most suitable, while for comparison the phosphate buffer retained ~15-60% of Cu II in solution when in presence of two equivalents of imidazole or half an equivalent of K28H/Q32H GB1, respectively (see ESI). Leaving a solution of CuCl2 in phosphate buffer to equilibrate led to negligible available Cu II (i.e., subsequent incubation with protein yielded very poor DEER modulation depths). From these observations we hypothesized that the changing availability of metal ions in solution would modify the binding equilibria reflected by the modulation depths of our measurements. |
629c75506057b1651591faad | 3 | Measurements were then performed as an eight-point pseudo-titration series for Cu II in presence of Tris-HCl buffer. Figure shows the background corrected traces for RIDME and DEER measurements of the Tris-HCl buffer series. In Figure the ∆ 𝑀-𝑅1 behaviour indicated a high binding affinity of Cu II for the dHis motif, with >90% occupancy at a metal:protein ratio of 1:1. The ∆ 𝑅1-𝑅1 behaviour suggested that metal-templated dimer formation was optimised at a metal:protein ratio of 4:5, with marginally higher ∆ 𝑅1-𝑅1 than at a metal:protein ratio of 1:2 (see ESI), but was abolished entirely above stoichiometric ratios of Cu II , where either i) significant data cutting was required for processing or ii) the detected echo was free of dipolar modulation. Replicates of metal:protein ratios of 3:5 and 4:5 (see ESI) showed optimised dimer formation at a metal:protein ratio of 3:5, which is consistent with a negative cooperativity mode of templated dimerization (i.e., the initial dHis motif binding event outcompetes the formation of dimer construct), and the global fitting of the 𝐾 𝐷 and 𝛼 parameters support it further. Exploratory simulations validated the robustness of the cooperative binding model (see ESI section 2.5) and indicated a strongly negative cooperativity parameter (𝛼 = 0.15) and a 𝐾 𝐷 of 3.1 × 10 -6 . |
629c75506057b1651591faad | 4 | Despite the imperfect agreement between the fitted 𝐾 𝐷 and 𝛼 parameters to the phosphate series experimental data manifest by Cu II precipitating from solution, bivariate fitting of repeat measurements (see ESI) indicated that the positive cooperativity and reduced initial binding affinity compared to the Tris-HCl buffer series was reproducible. Additionally, scaling experimental Cu II concentration by 0.65 for the phosphate and 0.85 for the Tris-HCl buffer series yielded global RMSD minima upon reprocessing (see ESI). This observation is consistent with the CW-EPR data (see ESI) for the phosphate buffer series that only ~60% of Cu II is retained in the solution. While for the Tris-HCl buffer series, scaling by a factor of 0.85 corresponds to a shift in optimised dimer formation from a metal:protein ratio of 3:5 to 1:2, as expected for negative cooperativity. |
629c75506057b1651591faad | 5 | These observations can be rationalized by the strong negative cooperativity for the Tris-HCl buffer (the metal-templated dimer formation would be disfavoured, i.e., the initial metal-binding event outcompetes the formation of templated dimer) by considering Tris-HCl interacts strongly with Cu II , retaining it in solution. This maximises the effective Cu II concentration able to bind dHis motifs, and since for templated dimer formation, one monomer must have an unoccupied dHis motif, Tris-HCl buffer disfavours this. On the contrary, precipitation of Cu II as Cu3(PO4)2 reduces the effective Cu concentration in solution. Lower Cu II availability reduces the apparent affinity of dHis motifs, fewer dHis motifs are occupied, and metal-templated dimer formation is favoured. |
629c75506057b1651591faad | 6 | Positive cooperativity in metal-templated dimerization is considered to be rare, while negative cooperativity characterizes many systems. The potential utility of the α-helical dHis motif for protein-protein interface nucleation by metal-binding was already well-known; however, the observation that cooperativity of templated dimerization is buffer modulated provides an additional handle for manipulation of the binding equilibrium. We serendipitously observed that in presence of phosphate buffer, Cu II -templated dimerization demonstrated apparent positive cooperativity, while in Tris-HCl buffer, this templated dimerization displayed strongly negative cooperativity behaviour. |
629c75506057b1651591faad | 7 | Additionally, to our knowledge this is the first pulse dipolar EPR methodology to extract cooperativity and 𝐾 𝐷 parameters by global fitting of nitroxide-nitroxide DEER and Cu II -nitroxide RIDME modulation depths. Results also showcase the robustness and accuracy of PDS in monitoring equilibria processes and to detect variations of cooperativity mode while changing buffer conditions (Wort et. al., in preparation). Finally, this methodology can be easily complemented by other PDS measurements (for instance, by Cu II -Cu |
641c5c0a91074bccd0278d77 | 0 | The chiroptical properties of a molecule are fundamentally linked to the interaction between the electromagnetic wave of circularly polarized light and charge in the molecule during its electronic transitions. While the electric transition dipole moment, ⃗ µ, arises from linear charge displacement, the magnetic transition dipole moment, ⃗ m, arises from rotation of charge. Equivalent to the way we envision circularly polarized light as an electric field vector that propagates helically in space, charge needs to be helically displaced in the molecule for the transition to be optically active. Helical topology in the frontier π-orbitals has been suggested as an avenue for designing optically active molecules. This orbital effect has been coined electrohelicity. However, its experimental relevance is limited by the simultaneous presence of both helicities in the electronic structure. Nonetheless, relations to single-molecule conductance, 9,10 reaction selectivity, and optical properties have recently been suggested. Is the electrohelicity effect a fundamental cause for a strong chiroptical response? |
641c5c0a91074bccd0278d77 | 1 | Electrohelicity appears as helical molecular orbitals (MOs) in allene and spiropentadiene when their symmetry is reduced from D 2d to D 2 (or C 2 ). In allene both the degenerate sets of highest occupied MOs (HOMOs) and the lowest unoccupied MOs (LUMOs) are helical (Figure only the HOMOs are helical while the non-degenerate LUMO and LUMO+1 are delocalized due to spiroconjugation (Figure right). These helical frontier MOs are present in several types of linear carbyne-like and spiroconjugated molecules due to the Möbius topology of the π-system. We recently demonstrated a correlation exists between the chiroptical response and the splitting of the near-degenerate helical MO pairs in substituted allenes and longer odd-carbon cumulenes. In this article, we examine the underlying connection between electrohelicity and optical activity in molecules where helical frontier MOs appear. We treat four types of molecules that exhibit electrohelicity: Subsection 3.1 covers allene, 3.2 evencarbon cumulenes, 3.3 polyynes, and 3.4 spiropentadiene. Each of these subsections can in principle be read independently of each other. First, we show that the optical activity of chiral allenes is driven by the helicity of the frontier MOs, and we demonstrate strategies for optimizing the chiroptical response. Then we examine linear and spiroconjugated systems where the helical frontier MOs may not contribute directly to the optical activity. Finally, we discuss the potential implications of these results going forward in the conclusions section. |
641c5c0a91074bccd0278d77 | 2 | Optical activity of electronic transitions in the UV-Vis range is measured by electronic circular dichroism (ECD) as the difference in absorption of left and right circularly-polarized light, ∆ε. Whereas the absorption is theoretically connected to the oscillator strength of the transition, the chiroptical response is proportional to the rotatory strength of the transition, ∆ε ∝ R. In its simplest form, R is given by the product of the lengths of the electric (⃗ µ) and magnetic (⃗ m) transition dipole moment vectors and the angle given by their mutual orientation (θ ) as given in equation 1. |
641c5c0a91074bccd0278d77 | 3 | It follows from this equation that both ⃗ µ and ⃗ m must be non-zero and oriented non-perpendicularly for a transition to be optically active. Often transitions with high R are electrically allowed and therefore driven by large ⃗ µ, which, while enhancing ∆ε, reduces the dissymmetry factor, g = ∆ε/ε, at a given wavelength. Ideally, optically active transitions must be both magnetically and electrically allowed to facilitate both high absolute and relative optical activity as given by ∆ε and g. Electrically allowed transitions have a linear displacement of charge that give rise to ⃗ µ. For a simple electronic transition, e.g., HOMO→LUMO, the transition density is calculated from the direct product of the implicated MOs of the transition, which gives rise to the transition dipole moments. In the π → π * transition of ethene shown in Figure , there is a phase-difference in one end of the molecule. Consequently, charge is depleted in one end and increased in the other end, leading to ⃗ µ pointing along the molecular axis, and the S 0 → S 1 transition of ethene is electric-dipole allowed. Without rotation of charge, ⃗ m is zero. An example of a magnetic-dipole allowed transition is the HOMO→LUMO (n → π * ) transition of formaldehyde shown in Figure . The nodal planes of the HOMO and LUMO have different orientation and the product therefore gives a quadrupole. When the electric quadropole is associated to a rotation of charge it gives rise to the magnetic transition dipole moment. The direction of ⃗ m depends on the direction of the rotation as given by the right-hand rule. The net direction of rotation around the molecular axis is not defined for an achiral species such as formaldehyde, and the direction of ⃗ m in Figure is arbitrarily chosen. This analysis enables us to pinpoint that the S 0 → S 1 transition is optically inactive because it is electric-dipole forbidden. The chiroptical response of a molecule thus depends on the chirality contained in the electronic structure, not specifically the chiral arrangement of atoms in the molecule. There is no direct correspondence between an enantiomer's chiral configuration and the chiroptical sign of its electronic transitions. From the basics of MO theory it is evident that there must be both linear and rotational displacement of charge during a transition if it is to be both electric-and magnetic-dipole allowed. Furthermore, the angle of the dipoles must be as close to (anti-)parallel as possible to optimize the chiroptical response (see equation 1). To achieve this, the transition density must have a helical shape. This is the fundamental theoretical concept that governs electronic circular dichroism and circularly polarized luminescence spectroscopy. The optical properties of molecules are computed using response theory as implemented in time-dependent density functional theory (TD-DFT) in the Gaussian16 code. The rotatory strengths, electric and magnetic transition dipoles associated to each electronic transition are computed directly this way, and we report them in cgs-units to provide scale of the magnitude of the dipole vectors; see SI part 1 for a note on unit conversion. Geometry optimization and single-point computations were carried out using DFT with the ωB97X-D functional and the def2-TZVP basis set as implemented in Gaussian16. Molecules were optimized to the tight criterion using the ultrafine grid setting. To construct torsion profiles, the dihedral angle between end-groups of allene and spiropentadiene structures is changed without further optimization. MOs are plotted with an isosurface value of |
641c5c0a91074bccd0278d77 | 4 | Our goal is to gain insight by assessing the π-MOs and the optical activity of the related ππ * transitions. We initially treat the achiral (D 2d ) allene in quasi-D 2 symmetry in Subsection 3.1. This exercise lets us examine the underlying relation between optical activity and helical MOs without substituents. Furthermore, we gain insight into the limit where allene changes from optically inactive to active when mirror symmetry breaks (cf. ESI Figure when rotation symmetry breaks). |
641c5c0a91074bccd0278d77 | 5 | We proceed by applying this understanding of allene to explore the potential for achieving high optical activity in functionalized systems. Next, in Subsections 3.2 and 3.3 we treat an even-carbon cumulene and a polyyne subjugated to torsion, which have helical MOs similar to those of allene, and we discuss whether the helicity contributes to the chiroptical response. Finally, we treat achiral (D 2d ) spiropentadiene in quasi-D 2 symmetry to assess how the optical activity differs in spiroconjugated systems, which are structurally different from the carbyne-like allenes. |
641c5c0a91074bccd0278d77 | 6 | We start by assessing the parent allene molecule in quasi-D 2 symmetry where only the three rotation axes are preserved. In D 2 symmetry the HOMOs and LUMOs are helical and the four transition densities arising from the direct MO excitations are shown in Figure . Products of MOs of the same helicity give a helical transition density that retains the helicity (P or M). Accordingly, these have notable electric and magnetic transition dipole moments. ⃗ µ is aligned along the z-axis of the molecule (the allenic axis). ⃗ m has a non-zero component along the z-axis and a larger compo-nent pointing in-between the two allenic planes, which results in an 80 o angle between the two transition dipole vectors. As illustrated in Figure , the direction of ⃗ m is in accordance with the right-hand rule considering the helicity of the transition density. The big off-axis component of ⃗ m comes from the linear charge displacement that is characteristic for ππ * transitions; thus the helix pitch is big and the charge does not rotate perfectly around the allenic axis. Only the z-component ( ⃗ m z ) aligns with ⃗ µ (equation 1), which is pointing along the allenic axis. To provide a sense of an upper-bound for the rotatory strength of substituted allenes, we can calculate R for this transition density that is associated to the MO transitions. The z-component of ⃗ m is parallel to ⃗ µ for π P → π * P giving positive R and anti-parallel for |
641c5c0a91074bccd0278d77 | 7 | The helical MO transitions belong to the b 1 irreducible representation and mix by linear combinations into non-degenerate transitions. The resulting transition dipoles into the two B 1 excited states are shown in the right column of Figure ; either ⃗ µ or ⃗ m z cancels out, and the two transitions are optically inactive. The transitions involving MOs of differing helicity also mix in similar fashion (bottom rows of Figure ); those two are totalsymmetric and are electric and magnetic dipole forbidden. We thus see why the allene transitions are optically inactive. It is the linear combination of the two helical MO transitions (P ± M, Figure ) that limits the optical activity of allenic molecules. The D 2d -structure represents a case where the orbital helicity is perfectly cancelled. To increase the optical activity we must split the degenerate helical MOs, so they will not contribute equally to the excited states. This is the principle of electrohelicity-driven optical activity in allene, and we will explore how this is applied in terms of molecular design. |
641c5c0a91074bccd0278d77 | 8 | Axial torsion of allene reduces its symmetry to D 2 and splits the helical MO pairs as the helix either unwinds and become more bonding (lower eigenvalue), or overwinds and becomes more antibonding (higher eigenvalue). Consequently, the two helical components of the transition do not contribute equally when the dihedral angle is changed from 90 o , and the transitions become optically active. This is demonstrated in Figure , where the end-group orientation of allene is changed, and the energies of the two B 1 excited states change accordingly. As the opposing helical components are emphasized in each transition, the rotatory strength increases systematically with differing sign for both transitions. The 2B 1 state becomes more active as it is the one that is electric dipole allowed at 90 o . Torsion of allene is realized in a series of cyclically alkylsubstituted allenes, where the alkyl linker constrains the structure. These have been synthesized and studied for their helical MOs in other contexts. The series of cycloallenes have a perturbed structure where both the allenic bond angle and dihedral angle are distorted. The frontier MOs and electronic transitions of allene are retained in the cycloallenes at the same torsion angle (ESI Figure ). As the helical MOs are split energetically they do not contribute equally to the electronic transitions, and consequently the cycloallenes have improved chiroptical response at more acute torsion angles (Fig. ). This exemplifies how the energetic tuning of the helical frontier MOs of allene can be used to optimize its optical activity. |
641c5c0a91074bccd0278d77 | 9 | Saturated substituents that hyperconjugate into the helical πsystem break the degeneracies of the frontier π-MOs. Breaking the symmetry of allene with simple substituents such as in 1,3-dimethylallene makes the molecule optically active. However, the rotatory strength of the allowed S 0 → S 4 transition is still modest (Table ) because the helical frontier MOs remain near-degenerate. If ethyl substituents are used instead, the rotatory strength comes close to the limit of the pure MO transitions (156•10 -40 erg•esu•cm/G, Figure ). While the substituents themselves may also contribute to increasing the rotatory strength, this enhancement is primarily driven by the perturbation of the allene π-system. As a control, we can place one or two ethyl substituents at the 1-position instead of at the 1,3-positions (ESI Figure ). Despite all molecules having similar low symmetry (Table ), the increase in the rotatory strength is much smaller because the substituent pattern in ethylallene and 1,1-diethylallene does not fully retain the helical frontier MOs and break their degeneracy. This is also evident for 1,3-diethylallene as its optical activity will depend on the conformation of the substituents. We described this mechanism in detail in a previous communication. Table Rotatory strength of S 0 → S 4 transition of substituted allenes |
641c5c0a91074bccd0278d77 | 10 | Another avenue for enhancement of the optical activity of lowsymmetry allenes is through its covalent dimers, bis-allenes. A number of such molecules have been synthesized with different orientations of the allenic units. The frontier MOs of the allene units mix into combinations of the helical MOs, which are thus partially retained (ESI Figure ). The electronic transitions are composed similarly to those of allene. We examine an allene dimer where the allenic units are placed in parallel and rotated relative to each other, and two bis-allenes where the allene moeities have similar orientation (Figure ). The allene dimer is shown at 45 o . The hypothetical Bis-allene1 has an angle of 45 o , while silicon-based Bis-allene2 at 56.7 o was synthesized by Lin et al. All three have D 2 symmetry and we look at the 2B 3 excited state where the large off-axis component in ⃗ m of allene (cf. Figure ) enables high optical activity. The two MO excitations that contribute to the 2B 3 transition of the 45 o allene dimer are shown in Figure . The MOs are in-phase (HOMO-1 and LUMO) and out-of-phase combinations (HOMO and LUMO+1) of helical MOs. As in the optically active transitions of allene, the MO helicites match for the occupied and unoccupied MOs. The direct MO products thus look like those of allene with a clear helical movement of charge in each allenic unit. As the allenic units are rotated 45 o relative to each other, the electric and magnetic dipole moment point out of the screen, towards the viewer. The same transition is shown for Bis-allene1 in Figure . Again, it is visually clear that the helical components of the transition contribute significantly to the optical activity of the transition. The dihedral angle dependence of the rotatory strength of the dimer and the two bis-allenes is plotted in Figure . The rotatory strength of the allene dimer maxes at 642 • 10 -40 erg•esu•cm/G at 56 o orientation of the dimer. The two bis-allenes do not reach this level, but nonetheless the optical activity is significantly improved from monomer allene. The design of optically active allene-based macromolecules may benefit from considering the helical electronic structure of allene. We aim to explore such design rules in a future study. |
641c5c0a91074bccd0278d77 | 11 | Allene is the shortest of the series of odd-carbon cumulenes. The even-carbon series of cumulenes are much more explored due to their increased synthetic availability relative to the odd-carbon series. They have co-planar end-groups in the ground-state structure, and achieves helical frontier MOs as its symmetry is reduced by torsion of the end-groups. As the end-groups of butatriene ([3]cumulene) are rotated out of plane, its two π-systems mix and even at small dihedral angles the MOs achieve visually clear helical character. Shown in Figure , the first two excited states belong to the B 1 irreducible representations and each have clear HOMO→LUMO and HOMO-1→LUMO character. At 0 o , the HOMO→LUMO transition is electric dipole allowed, while the HOMO-1→LUMO is magnetic dipole allowed. Already at 10 o torsion, the HOMO and HOMO-1 have become very helical, while the LUMO only has weak helical character that becomes visually clear at higher dihedral angles (Figure ). The transition density also achieves helical character. Especially in the case of the HOMO-1 →LUMO transition where the MO have same helicity, it is visually clear that the helicity of the MOs lead to a helical rotation of the charge. Both transitions achieve parallel non-zero ⃗ µ and ⃗ m. The dihedral angle dependence of the rotatory strength (Figure ) reveals that both transitions quickly achieve an enhanced chiroptical response as the MOs become helical. We note that the two electronic transitions cross at 17 o torsion in an avoided crossing, and thus both MO transitions contribute significantly to both electronic transi- |
641c5c0a91074bccd0278d77 | 12 | Polyynes are structurally similar to even-carbon cumulenes but are more stable due to the single-triple bond topology, and is thus an oft-used motif for molecular linkers. As in cumulenes, when the end-groups are rotated polyynes become optically active. Some π-orbitals become helical as the symmetry is reduced and the π-systems mix. The mixing is much less significant than in the cumulenes, and at small torsion angles the mixing is not visually clear (Figure ). Although the HOMO and LUMO only achieve weak helicity, there are lower-lying π-MOs that become more helical. The HOMO-LUMO transition of tolane is electric-dipole allowed and becomes optically active with torsion. Just as there is no visually clear helicity in the MOs, there is no significant helicity in the transition density (Figure ). While there is charge rotating around the polyynic wire in both transitions at high torsion angles, it seems that it is primarily the components on the terminal phenyl groups that gives a non-zero ⃗ m. This charge rotation between the phenyl groups increases with higher torsion angle. The rotatory strengths therefore increase until the phenyl groups are almost in perpendicular orientation, as plotted in Figure . |
641c5c0a91074bccd0278d77 | 13 | At high dihedral angles, close to perpendicular orientation, the B 1 transitions are dominated by the HOMO→LUMO and the HOMO-1→LUMO+1 excitations with 79% contribution each to the electronic transitions at 80 o torsion. Though the MOs are helical at near-perpendicular torsion angles, helicity is insignificant in the transition density because the helical components of the MOs are opposite. Through the full range of dihedral angles it is the twisted orientation of the end-groups that is responsible for the magnetic transition dipole. We find that this is also the case for higher-lying excited states of tolane that are optically active; it seems they are all driven by the charge moving between the two terminal phenyl groups and therefore those transitions are thus not of particular interest for this study. The helical MOs of tolane do not contribute to its optical activity. It is yet to be said, if this result is general for all polyynic molecules or specific to tolane. |
641c5c0a91074bccd0278d77 | 14 | Spiropentadiene has D 2d symmetry, and like allene we assess it in quasi-D 2 symmetry. When the symmetry is reduced, the πsystems mix and the occupied π-MOs become helical (Figure ) with a continuous π-nodal plane spanning the entire length of the molecule. As we have discussed in detail in recent work, the (near-)degenerate sets π → π * transitions are separated by symmetry, and the two HOMO→LUMO transitions shown in Figure do not mix. The HOMOs differ in helicity, while the LUMO is a non-helical spiroconjugation MO. Their products show weak linear and circular charge displacement and consequently both ⃗ µ and ⃗ m are relatively small in magnitude, and it is hard to visually identify any helical components in the transition density. ⃗ µ and ⃗ m both point between the two molecular planes. The two transitions are energetically degenerate and the opposite signs of R thus cancel out in an ECD spectrum; consequently the unsubstituted molecule is optically inactive. The same is true for the HOMO→LUMO+1 transitions (ESI Figure ). |
641c5c0a91074bccd0278d77 | 15 | The chiroptical response is enabled by the parallel orientation of the transition dipoles (Figure ). It is the mixing of the πsystems through the formally saturated central spiro-carbon that ensures the electric and magnetic transitions dipole moments are reoriented. If we remove rotation symmetry and treat spiropentadiene as quasi-C 2v , the transition density of the electronic transitions is contained within one of the molecular planes (ESI Figure ). Consequently, spiropentadiene is optically inactive because ⃗ µ and ⃗ m are perpendicularly oriented. |
641c5c0a91074bccd0278d77 | 16 | The mixing of the two π-systems in quasi-D 2 symmetry mediates the optical activity. However, it appears that the rotatory strengths are not mediated by the MO-helicity as such. This is also evident by ⃗ µ and ⃗ m not pointing along the helical axis of the MOs, as they do for the allene and butatriene. Delocalization of the π-systems has been demonstrated to affect the optical properties of spiropentadienes, and this is also the case for the chiroptical response here. Although the optical activity is not driven by electrohelicity, we showed in a previous communication that the helicity manifests in the electron density, which changes in a helical fashion when charge is transferred from helical to nonhelical MOs during the four ππ * transitions. |
641c5c0a91074bccd0278d77 | 17 | Splitting the (near-)degenerate transitions may enhance the ECD response because transitions with opposite rotatory strengths move towards different wavelengths. As as showed for allene, this can be achieved by axial torsion of the molecule, which in particular affects the eigenvalues of the helical MOs. However, as plotted in Figure , torsion also affects the rotatory strengths, which weaken with torsion for many of the transitions. Figure also includes an assessment of a series of hypothetical conformationally-locked cyclic spiropentadienes. Their torsion angles are constrained by the alkyl linkers, and their electronic transitions show reasonable correlation with the constrained nonlinked systems for most of the transitions (ESI Figure ). With the reduction of the rotatory strength, it seems that splitting the near-degenerate electronic transitions has limited potential for enhancing the chiroptical response of spiropentadiene derivates. |
641c5c0a91074bccd0278d77 | 18 | We have assessed the relation between electrohelicity and optical activity by analyzing the MO contributions to the electric and magnetic transition dipole moments. In allene and longer cumulenes there is a fundamental connection between their chiroptical response and helical frontier MOs, which we use for rational design of optically active molecules. However, we also show that the link is not universal, as we find no direct relation between orbital helicity and optical activity in polyynes and spiroconjugated molecules. There is thus a notable variation in the origin of enhanced rotatory strengths in molecules with electrohelicity. These results question if electrohelicity is at all useful as a general concept for understanding and developing novel optically active molecules. When helical π-systems are suggested as an underlying mechanism for a large chiroptical response, it must be supported by careful analysis on a case-by-case basis. Indeed, we found that in allenic systems, such a mechanism involving helical MOs can be used to enable enhancement of optical acitivty using substituents and macrostructures. This insight is to be applied for design of molecules with large chiroptical response. |
62cd82f227b1e463bb36b984 | 0 | Around 10 million people worldwide are living with Parkinson's disease (PD) 1 , a progressive neurodegenerative disorder characterized by both motor (e.g. bradykinesia, resting tremor, postural instability, rigidity) and non-motor (e.g. memory loss, hyposmia) disabilities. Current PD treatment is limited to motor symptoms management with dopamine replacement or by enhancing the activity of the remaining dopaminergic neurons. No known therapy is available that can slow down the progress or prevent the onset of the disease. Furthermore, PD cases are growing at a fast ever speed and are projected to increase to over 17.5 million by 2040 due to the fast-growing aging population . While aging remains to be the major risk factor of PD, >20 genes have been identified to be associated with the onset and progress of PD , suggesting the potential of discovering disease-modifying PD treatments. |
62cd82f227b1e463bb36b984 | 1 | Leucine-rich repeat kinase 2 (LRRK2), encoded by LRRK2 gene, is a large (286 kDa), multi-domain protein that, in addition to its kinase domain, possesses a second enzymatic guanosine triphosphatase (GTPase) domain and several other domains and motifs that are involved in protein-protein interactions . Pathological mutations in the kinase domain and GTPase domain of LRRK2, such as G2019S and R1441C/G/H mutations, can increase the kinase activity of LRRK2 and eventually lead to pathogenic hallmarks associated with PD, such as ciliogenesis inhibition , defective mitophagy and autophagy , and mitochondrial dysfunction . Increased LRRK2 kinase activity, independent of LRRK2 mutations, has also been reported in idiopathic PD patients . Conversely, LRRK2 knockout or pharmacological inhibition of LRRK2 kinase activity are neuroprotective in cellular and in animal models . These observations provide strong rationale for targeting LRRK2 to treat PD. |
62cd82f227b1e463bb36b984 | 2 | Over the past years, several LRRK2 kinase inhibitors have been developed, including LRRK2-IN-1 , HG-10-102-01 , MLi-2 , PF-06447475 , and DNL201 and DNL151 which are the first two LRRK2 kinase inhibitors in clinical trials . However, all these inhibitors are ATP-competitive type 1 kinase inhibitors which preferably bind to the closed active conformation of LRRK2, leading to dephosphorylation of Ser935 and other biomarkers sites, LRRK2 aggregation, and mislocalization to microtubules . These unintended effects may interfere with vesicle trafficking and could underlie undesirable on-target side-effects observed on lungs and kidneys . Alternative LRRK2 targeting strategies, such as G2019S LRRK2 selective inhibitors , LRRK2 Type II inhibitors, LRRK2 dimerization inhibitors , GTPase inhibitors, antisense oligonucleotide , type 2 LRRK2 kinase inhibitors , and LRRK2 proteolysis targeting chimeras (PROTACs) , have therefore been proposed and are under active exploration. |
62cd82f227b1e463bb36b984 | 3 | As one of the most promising disease-modifying targets, LRRK2 lies at the nexus of an emerging signaling network of high relevance for understanding and developing treatments for PD . Although three LRRK2 targeting therapies are already in clinical trials, the exact mechanism by which LRRK2 mutations and its kinase activity contribute to the development of PD is still under investigation. Rab GTPases implicated in vesicular trafficking have been identified as bona fide physiological substrates of LRRK2 , but many components involved in the upstream and downstream wiring of LRRK2 signaling pathways are yet to be discovered, and the question remains as to whether LRRK2 kinase inhibitors will have beneficial disease-modifying effects in PD patients. More indepth LRRK2 target validation is therefore warranted. Induced target protein degradation is a paradigm-shifting drug discovery approach. Heterobifunctional degraders (also known as PROTACs) can induce target protein degradation by recruiting an E3 ubiquitin ligase in proximity to the target protein, resulting in the polyubiquitination and subsequent degradation of the target protein by the proteasome . More than 15 PROTAC degraders are in or approaching the clinic currently , against a variety of targets, including hormone receptors (e.g. AR and ER), transcription factor (e.g. STAT3), anti-apoptotic protein (e.g. BCL-XL), kinases (e.g. BTK and IRAK4), and epigenetic proteins (e.g. BRD9). PROTAC is not only an emerging drug discovery modality but also offers new chemical tools for target identification and validation, and for deciphering target biology . For example, PROTAC-mediated degradation can reveal noncatalytic activity of protein kinases . Herein, we report the discovery and characterization of XL01126, a von Hippel-Lindau (VHL)-based, fast, potent, cooperative and selective LRRK2 PROTAC degrader that is also orally bioavailable and blood brain barrier (BBB) permeable. XL01126 qualifies as a chemical probe to study LRRK2 biology, further validate the target as a therapeutic concept in PD, and usher future drug development. |
62cd82f227b1e463bb36b984 | 4 | We began our efforts by designing and synthesizing a small set of PROTACs aiming to maximize sampling of chemical space and target-PROTAC-E3 ternary complex pairing. HG-10-102-01(Figure ), a BBB penetrant type 1 LRRK2 inhibitor, was chosen as the LRRK2 ligand, on the basis of its small molecular size and favorable physicochemical properties . According to homology modeling of HG-10-102-01 with LRRK2, the morpholine ring is pointing towards solvent , suggesting of a suitable exiting vector for PROTAC linkage. We converted the morpholine ring to piperazine to facilitate linker attachment. For the E3 ubiquitin ligases, we decided to recruit Cereblon (CRBN), Cellular Inhibitor of Apoptosis (cIAP), and VHL, which have readily available ligands with known "PROTACable" sites (Figure ). After converting both the warhead and E3 ligase ligands into "PROTACable" intermediates, they were tethered together through linkers and a small library of first-generation compounds containing 12 LRRK2 PROTACs (Figure ) were generated (Schemes S3, S4, and S5). Figure . Screening of the first-generation PROTACs in WT and G2019S LRRK2 MEFs. (A) Representative Western blots monitoring total LRRK2, LRRK2-pSer935, Rab10-pThr73, total Rab10, and Tubulin levels following the treatment of WT and G2019S MEFs with the indicated compounds at 33 nM, 1 µM, or DMSO for 4h. (B) Quantitative analysis of the relative LRRK2 protein and Rab10-pThr73 levels, which are presented as ratios of total LRRK2/Tubulin or Rab10-pThr73/total Rab10, normalized to the DMSO treated sample. Data were obtained from two biological independent experiments. These PROTACs were then biologically evaluated in mouse embryonic fibroblasts (MEFs) by Western blotting. Briefly, MEFs were treated with compounds at 33 nM and 1 µM for 4 h (Figure ) and 24 h (Figure ) separately, and the intracellular level of LRRK2, phosphorylated LRRK2 at Ser935, and phosphorylated Rab10 (pRab10) at Thr73 were determined. Rab10 is one of the bona fide substrates of LRRK2, whose phosphorylation status is directly affected by LRRK2 kinase activity and protein level. Phosphorylation of LRRK2 at Ser935 is a well-studied biomarker site used to assess the efficacy of type 1 LRRK2 inhibitors . HG-10-102-01 based PROTACs can potentially dephosphorylate LRRK2 at Ser935 through both LRRK2 degradation and inhibition. Among the first-generation PROTACs, compounds SD75, SD82, and SD100 (Figure ) degraded 30-70% of G2019S LRRK2 at 1 µM/4h (Figure ) and achieved 70-85% G2019S LRRK2 degradation after 1 µM/24h treatment (Figure ). These three compounds also showed substantial dephosphorylation of LRRK2 and Rab10, with 75-90% pRab10 dephosphorylated after 1 µM/24h treatment in G2019S LRRK2 MEFs (Figure ). A fourth compound, SD13, also looked promising as it degraded 60% G2019S LRRK2 at 33 nM/4h treatment (Figure ) and 68% G2019S LRRK2 at 33 nM/24h (Figure ). However, less G2019S LRRK2 was degraded upon 1µM treatment by SD13, compared to the 33 nM treatments, suggestive of the "hook effect" . Although SD75, SD82, and SD100 showed only moderate LRRK2 degradation, they did not show any sign of the "hook effect" at 1µM concentration. Notably, all three compounds share the same E3 ligase and ligand (VHL, VH101) and exit vector out of the tert-leucine group, suggesting a potential hot-spot of ternary complex formation between VHL and LRRK2. We therefore decided to focus further medicinal chemistry optimization on this chemical series with the goal to further improve the compounds' fitness as LRRK2 degraders. |
62cd82f227b1e463bb36b984 | 5 | Given the modular nature of PROTAC molecules, the structural modification of the second generation of LRRK2 PROTAC degraders focused on modifying the LRRK2 ligand, the linker, and the VHL ligand (Figure and Figure ), separately. To best assess which structural modification would confer the most significant activity improvement, we designed molecular match pairs of SD75, SD82, and SD100 by changing one structural moiety at a time. XL01078B, XL01072, and XL01070B were designed (Figure ) and synthesized (Scheme S3) where the 5-chlorine substitution on the aminopyrimidine ring of HG-10-102-01 was replaced with -CF3 substitution which was reported to improve binding affinity to LRRK2 . XL01119, XL01118 and XL01120 (Figure ) were molecular match pairs of SD75, SD82, and SD100 respectively, by harboring an extra methyl group on the benzylic position of VHL ligand, which was introduced to increase binding affinity to VHL E3 ligase . Fluorine substitution was introduced on the phenyl group of the VHL ligand of XL01123, XL01122, and XL01121 (Figure ) attempting to fine-tune physicochemical properties at a permissible site . The linker length, composition, and rigidity, which can significantly affect the physicochemical and pharmacokinetic properties of PROTACs, as well as their ternary complex formation and activity , were explored as represented by compounds XL01131, XL01140, XL01111, XL01126, XL01134, and XL01076 (Figure ). In an attempt to improve the drug-like properties and reduce molecular size, we designed XL01145, XL01149 and XL01168 (Figure ). These compounds are derived from truncated HG-10-102-01 with the morpholinoamide moiety removed as its absence retains binary binding affinity to LRRK2 . These 18 new compounds were synthesized as outlined in Schemes 1, S1, S2, S3, S6, S7, S8 and S9 and were also screened via Western blotting (Figure and Figure ). Quantitative analysis of the Western blots (Figure and Figure ) revealed that at 33 nM/4h treatment, XL01126 and XL01134 were the most effective optimized compounds that degraded 20-30% of WT LRRK2 and 50-60% of G2019S LRRK2 (Figure ). Accordingly, these two compounds were also the most potent in decreasing pRab10 in both WT and G2019S LRRK2 MEFs, with > 60% pRab10 inhibited in G2019S LRRK2 MEFs at 33 nM/4h (Figure ). In contrast, the first-generation degraders SD75, SD82, and SD100 induced little to no degradation of LRRK2 at 33 nM/4h treatment (Figure ) and showed weak (<40%) degradation at 33 nM/24h treatment (Figure ), at which XL01126 and XL01134 degraded 50-60% of WT LRRK2 and 70-80% of LRRK2 G2019S (Figure ). Most of the compounds exhibited substantial WT LRRK2 and G2019S LRRK2 degradation (30-80%) at 1 µM/4h or 1 µM/24h treatment (Figure and Figure ), leading to potent and almost complete pRab10 inhibition in WT MEFs and G2019S LRRK2 MEFs, respectively. Multiple new compounds, including XL01078B, XL01119, XL01123, XL01131, XL01126 and XL01134, surpassed SD75, SD82 and SD100 in degrading WT LRRK2 and G2019S LRRK2 at 1 µM/4h and 1 µM/24h treatment, suggesting modifications at the warhead (XL01078B), the E3 ligase ligand (XL01119 and XL01123), and the linkers (XL01131, XL01126, and XL01134) can all improve the degraders' fitness to some extent. Nonetheless, the significant improvement exhibited by XL01126 and XL01134, which are isomers of each other, encouraged us to characterize them further. |
62cd82f227b1e463bb36b984 | 6 | To characterize XL01126 and XL01134 and compare them with the top first-generation degrader SD75, a dose dependent degradation assay was carried out in WT and G2019S LRRK2 MEFs (Figure ). SD75 dose-dependently degraded LRRK2 following 24h treatment in WT and G2019S LRRK2 MEFs (Figure ). However, the degradation of LRRK2 was only partial with Dmax reached at 3 µM (Dmax,24h = 51% and 58% for WT and G2019S LRRK2 respectively). Dose-dependent LRRK2 pSer935 and pRab10 dephosphorylation, which account for both LRRK2 inhibition and degradation, were also observed after SD75 treatment, with EC50 = 2270 nM and 379 nM for the dephosphorylation of Rab10 in WT and G2019S LRRK2 MEFs, respectively. XL01134 and XL01126, the top LRRK2 degraders from the second-generation compounds, showed more extensive LRRK2 degradation after a significantly shorter treatment time (4h) when compared to SD75 (Figure and). XL01134 degraded G2019S LRRK2 (DC50, 4h = 7 nM) more potently than WT LRRK2 (DC50, 4h = 32 nM), with maximum LRRK2 degradation reached at 300 nM and Dmax values against WT LRRK2 and G2019S LRRK2 are 59% and 81%, respectively. However, at concentrations above 300 nM, a strong "hook effect" was observed (Figure ). XL01126 also degraded G2019S LRRK2 (DC50, 4h = 14 nM) and WT LRRK2 (DC50, 4h = 32 nM) at nano-molar concentrations, but achieved more complete degradation than XL01134, with Dmax,4h = 82% in WT MEFs and Dmax,4h = 90% in G2019S LRRK2 MEFs, achieved at around 1 µM. Moreover, no "hook-effect" was observed with XL01126 at higher concentrations (Figure ). Due to the potent LRRK2 degradation capabilities, XL01134 and XL01126 resulted in more pronounced pRab10 dephosphorylation (Figure and 5C) than SD75. XL01134, at 4h, showed 30fold more potent pRab10 inhibition than SD75 (at 24h) in both WT MEFs and G2019S LRRK2 MEFs. XL01126 (at 4h) is 40-fold more potent than SD75 (at 24h) in inhibiting Rab10 phosphorylation in WT MEFs, and 25-fold more potent in G2019S LRRK2 MEFs. Figure . Dose dependent LRRK2 degradation, LRRK2 dephosphorylation, and Rab10 dephosphorylation by SD75, XL01134, and XL01126 in WT and G2019S LRRK2 MEFs. Representative Western blots of total LRRK2, LRRK2-pSer935, Rab10-pThr73, total Rab10, and Tubulin levels after treating WT and G2019S LRRK2 MEFs with SD75 (A), XL01134 (B), or XL01126 (C) at the indicated concentrations for indicated time period. The relativeLRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/Tubulin or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO treated samples. The relative total LRRK2 and pRab10 levels were plotted against the compounds concentration and fitted against "non-linear regression, one site-fit LogIC50" in GraphPad to obtain the DC50 and EC50 values. Data were obtained from two to three biological independent experiments. |
62cd82f227b1e463bb36b984 | 7 | To further compare the degradation profiles of XL01134 and XL01126 with that of SD75, a time-dependent degradation assay was performed in MEFs using Western blotting (Figure ). SD75 was shown to degrade WT LRRK2 and G2019S LRRK2 at 1 µM in a time-dependent manner with moderate Dmax (52% for WT LRRK2 and 81% for G2019S LRRK2) and half-lifes (T1/2) against WT LRRK2 (5.1h) and G2019S LRRK2 (1.4h). In contrast, XL01134 and XL01126 degraded LRRK2 at higher rates, achieved higher Dmax values at only 300 nM, a concentration at which SD75 barely degraded LRRK2. Remarkably, XL01126 presented an improved profile (Dmax, WT = 82%, Dmax, G2019S = 92%, T1/2, WT = 1.2h, T1/2, G2019S = 0.6h) when compared to XL01134 (Dmax, WT= 75%, Dmax, G2019S = 82%, T1/2, WT= 2.7h, T1/2, G2019S = 1.4h). With the shortest degradation half-lives and highest degradation percentage, XL01126 emerged as the most efficient and fastest degrader among the three. The timedependent pRab10 dephosphorylation correlates well with the LRRK2 degradation (Figure , 6B and 6C). XL01126 dephosphorylated pRab10 the fastest with T1/2, pRab10 at 0.7h and 0.3h in WT and G2019S LRRK2 MEFs, respectively. This was followed by XL01134, which induced 50% reduction in Rab10 phosphorylation after 2.1h and 0.3h in WT and G2019S LRRK2 MEFs, respectively. In contrast, SD75 exhibited the slowest inhibition of pRab10 (T1/2, pRab10 = 6.7h on WT MEFs and 1.1h on G2019S LRRK2 MEFs). Figure . Time-dependent LRRK2 degradation, LRRK2 dephosphorylation and pRab10 dephosphorylation by SD75, XL01134, and XL01126. Representative Western blots of total LRRK2, LRRK2-pSer935, Rab10-pThr73, Rab10 total, and Tubulin levels after treating the WT and G2019S LRRK2 MEFs with SD75 (A), XL01134 (B), or XL01126 (C) at the indicated concentrations for the indicated period of time. The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/Tubulin or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO treated samples. The relative LRRK2 and pRab10 protein levels were plotted against the treatment time and were fitted against "non-linear regression, one phase decay" in GraphPad to obtain the half-life (T1/2) values. Data were obtained from two independent biological experiments. XL01126 surpassed its warhead and negative PROTAC cis-XL01126 in inhibiting downstream signaling in G2019S LRRK2 MEFs. Representative Western blots of total LRRK2, LRRK2-pSer935, pRab10, Rab10 total, and Tubulin levels following the treatment of G2019S LRRK2 MEFs with HG-10-102-01 (A), XL01126 (A and B), and cis-XL01126 (B) at the indicated concentrations for 4h. The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/Tubulin or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO treated samples. The relative LRRK2 and pRab10 protein levels were plotted against the compounds concentration and fitted against "non-linear regression, one site-fit LogIC50" in GraphPad to obtain the DC50 and EC50 values. Data were obtained from two independent biological experiments. |
62cd82f227b1e463bb36b984 | 8 | The potent and fast degradation of LRRK2 and inhibition of the Rab substrate phosphorylation by XL01126 prompted us to question if our PROTAC could surpass its warhead (HG-10-102-01) in dephosphorylating the substrate of LRRK2 and how much of the substrate dephosphorylation results from the protein degradation. This is of particular relevance for this project because the warhead ligand itself is a strong LRRK2 inhibitor with nanomolar kinase inhibition activities (Figure ) , and is a general challenge with PROTACs against protein kinase. As expected, the warhead HG-10-102-01 did not degrade LRRK2, but potently inhibited LRRK2 phosphorylation and Rab10 phosphorylation (EC50 = 110 nM on G2019S LRRK2 MEFs, EC50 = 214 nM on WT MEFs) (Figure and Figure ). In contrast, XL01126 dose-dependently degraded both WT LRRK2 (Figure ) and G2019S LRRK2 (Figure ). |
62cd82f227b1e463bb36b984 | 9 | Crucially, XL01126, showed around 3-fold more potently inhibited Rab10 phosphorylation in WT MEFs than HG-10-102-01 (Figure ), and 6-fold more potently inhibited Rab10 phosphorylation in G2019S LRRK2 MEFs (Figure ). These observations suggest that converting HG-10-102-01 to a PROTAC degrader not only improves downstream signaling inhibition, but also increases selectivity for G2019S LRRK2 over WT. Cis-XL01126 (Scheme 1), a non-degrading distomer control of XL01126 where the stereochemistry at the hydroxyl group of hydroxyproline is inverted to abrogate VHL binding , showed no degradation of WT LRRK2 (Figure ) and G2019S LRRK2 (Figure ), but inhibited Rab10 phosphorylation at a similar potency as HG-10-102-01 in both WT MEFs (Figure ) and G2019S LRRK2 MEFs (Figure ). However, due to the lack of LRRK2 degradation, cis-XL01126 was around 7-fold less potent than XL01126 in inhibiting Rab10 phosphorylation (116 nM vs 15 nM), further demonstrating the potency boost in downstream functionality achieved from LRRK2 degradation over and above kinase inhibition. Figure . XL01126 degrades LRRK2 in human peripheral blood mononuclear cells (PBMCs) derived from healthy donors, and mouse bone marrow derived macrophages (BMDMs). Representative Western blotting of total LRRK2, LRRK2-pSer935, pRab10, Rab10 total, and GAPDH levels following treating the PBMCs with XL01126 and cis-XL01126 at the indicated concentrations for 4h (A) and 24 h (B). The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/GAPDH or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO treated samples. The relative LRRK2 and pRab10 protein levels were plotted against the compounds concentration and fitted against "non-linear regression, one site-fit LogIC50" in GraphPad to obtain the DC50 and EC50 values. Data points are presented as mean ± SEM from three biological independent replicates. (C) Representative Western blotting of total LRRK2, LRRK2-pSer935, pRab10, Rab10 total, and GAPDH levels following treating the PBMCs with 300 nM of XL01126 and cis-XL01126 for the indicated time periods. The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/ GAPDH or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO treated samples. The relative LRRK2 and pRab10 protein levels were plotted against the treatment time and were fitted against "non-linear regression, one phase decay" in GraphPad to obtain the half-life (T1/2) values. Data points are presented as mean ± SEM from three biological independent replicates. (D) Representative Western blotting of LRRK2 total and Tubulin levels after treating BMDMs with XL01126 and cis-XL01126 for 4h. The relative LRRK2 levels were obtained by quantifying the ratios of total LRRK2/Tubulin, and normalized to the DMSO treated samples. The relative LRRK2 levels were plotted against the compounds concentration and fitted against "non-linear regression, one site-fit LogIC50" in GraphPad to obtain the DC50 values. |
62cd82f227b1e463bb36b984 | 10 | To scope and assess the degradation activity of XL01126 on other LRRK2 mutants and cell lines, dose-dependent degradation assays of XL01126 were carried out in R1441C LRRK2 MEFs (Figure ), bone marrow-derived macrophages (BMDMs), and human peripheral blood mononuclear cells (PBMCs) (Figure ). XL01126 exhibited potent LRRK2 degradation in all these cell types, with significant differentiation observed between XL01126 and cis-XL01126 in terms of Rab10 dephosphorylation (Table , Figure , and Figure ). The fast (T1/2, 300 nM = 2.4h) and potent (DC50, 4h = 72 nM, DC50, 24h = 17 nM) degradation of human LRRK2 in PBMCs suggests the potential of applying XL01126 to additional human cell lines. Testing of XL01126 and cis-XL01126 on SH-SY5Y, a human neuroblastoma cell line widely used as PD cell model , revealed that XL01126 induced 50% or more degradation of LRRK2 after 6h/300 nM or 24h/300 nM treatment (Figure ). |
62cd82f227b1e463bb36b984 | 11 | As the top two degraders from the second generation, XL01126 and XL01134 are epimers of each other, the only difference being swapped chirality at one of the two tertiary carbons of the cyclohexyl ring in their linkers. This small difference in chemical structure gives rise to very different degradation profiles for the two compounds (Figure and Figure ). These two epimeric PROTACs also exhibited strikingly different binding affinities to VHL as revealed by a fluorescence polarization (FP) displacement binding assay (Figure ) and a VHL target engagement assay (Figure ) . XL01126 has >10-fold weaker binary binding to VHL than XL01134 and also was found to be the weakest LRRK2 binder amongst the compounds tested (Figure ). PROTACs have previously been shown to tolerate weakened binary binding affinities to either their E3 ligase or target protein such that, despite the weak binding, they are able to induce potent protein degradation at a concentration well below the weakened Kd. Conversely, PROTACs made of more potent target ligands do not necessarily guarantee for more potent degraders . These studies together illustrated a now well-established feature with PROTACs, that is the extent of target degradation does not necessarily correlate with the PROTAC's binary binding affinity to E3 ligase or target protein. The ternary binding affinity, cooperativity and stability of the ternary complex, can instead play critically important roles in PROTAC induced protein degradation . To test whether our PROTACs can induce cooperative ternary complex formation and illuminate the relationship between the degradation potency and ternary complex formation, a ternary binding affinity assay and a ternary complex formation assay are warranted. However, we could not implement the mostly commonly used biophysical techniques such as fluorescence polarization and surface plasma resonance for these assays, due to the lack of sufficient recombinant expressed LRRK2 in hand. We therefore turned to endogenously expressed LRRK2 and developed a NanoBRET-based ternary binding affinity assay and ternary complex formation assay in HEK293 cells (Figure ). |
62cd82f227b1e463bb36b984 | 12 | In the NanoBRET-based ternary binding affinity assay, LRRK2-NanoLuc was transiently expressed in HEK293 cells as the BRET donor and LRRK2 tracer which is prepared by conjugating HG-10-102-01 with a fluorophore (BODIPY 576/589 ) (Figure and Scheme S11) was introduced as the acceptor. Titration of PROTAC degraders to the lysed cells and LRRK2 tracer in the presence or absence of recombinant VCB protein (VHL complexed with elongin B-elongin C) gives ternary and binary binding affinities of PROTACs against LRRK2 respectively. Similarly, the ternary complex formation assay also used LRRK2-NanoLuc transiently expressed in HEK293 as the BRET donor, but the acceptor was recombinant VCB protein labeled with BODIPY 576/589 via NHS ester-activated crosslinking reaction. PROTACs that can bridge LRRK2 and VCB together will produce BRET signal (Figure ). In line with the degradation potency, XL01126 induced the most cooperative ternary complex as indicated by its positive cooperativity (α = 5.7) (Figure ) and the highest maximal level of ternary complex formation (Figure ). In contrast, XL01134 induced significantly lower cooperativity (α = 1.4) and SD75 has a negative cooperativity with VHL and LRRK2 (Figure ) Figure . Binding affinities to VHL and LRRK2. (A) Binding affinity of the tested compounds to VHL using FP assay. The indicated compounds were titrated to a solution of VCB protein (10 nM) and JC9 (5 nM) (a FAM labeled probe that binds to VCB) to displace JC9 and the percentage of displacement was plotted against the compounds' concentration and fitted into the "non-linear regression, one site-LogIC50" to obtain the IC50 values, which were used to back calculate the Ki values. NanoBRET target engagement assays of tested compounds to VHL (B) and LRRK2 (C) in permeabilized and live cell modes. The indicated compounds were titrated into HEK293 cells transfected with VHL-NanoLuc (B) or LRRK2-NanoLuc fusion (C) in the presence of VHL tracer (B) or LRRK2 tracer (C). 0.25 µM and 0.5 µM VHL tracer were used for the permeabilized and live mode VHL engagement assay separately. 0.125 µM and 0.5 µM of LRRK2 tracer were used for the permeabilized and live mode LRRK2 engagement assay separately. The Fractional occupancy of the tracers are plotted against the tested compounds' concentrations and fitted into "non-linear regression, one site-LogIC50" to obtain the IC50 values of each compound against both permeabilized and live cells, separately. Data points are presented as mean ± SEM from three independent experiments. (D) The IC50 ratios between permeabilized and live mode target engagements of each compound were used to compare their permeabilities. |
62cd82f227b1e463bb36b984 | 13 | In the NanoBRET-based ternary complex formation assay, SD75, although a less potent degrader than XL01134, induced higher ternary complex than XL01134. However, it should be noted that this assay was carried out in the permeabilized HEK293 cells, and SD75 is likely to induce less intracellular ternary complex formation given its relatively lower permeability comparing to XL01134 (Figure ). The relative permeability (intracellular availability) of each compound was obtained by querying VHL engagement or LRRK2 engagement under live-cell and permeabilized-cell conditions (Figure ). |
62cd82f227b1e463bb36b984 | 14 | To assess the degradation selectivity of XL01126 and identify potential off-targets at the proteome level, we performed unbiased quantitative tandem mass tag (TMT)-based global proteomic profiling in WT MEFs. Over 8000 proteins were quantified in the cell lysate samples from WT MEFs that were treated with 300 nM XL01126, cis-XL01126, or DMSO for 4h (Figure ). The data corroborate a significant chemical knockdown of LRRK2, as validated by Western blotting (Figure ). LRRK1, the closest homologue of LRRK2, and other LRRK2-related proteins such as VPS35 and Rab-specific phosphatase PPM1H remained unaffected. The proteomic data also revealed a small (~30%) depletion in protein levels of phosphodiesterase 6 (PDE6D) (Figure ). PDE6D has a deep hydrophobic ligand-binding pocket, and has been shown to be degradable via PROTACs . Curiously, PDE6D was also found as adventitious off-target degradation of PTK2 PROTACs previously . Inspection of chemical structures highlighted that the PTK2 PROTAC and XL01126 share a similar aminopyrimidine warhead at the target ligand end, a moiety known to be critical to the high binding affinity in PDE6D inhibitor Deltasonamide , suggesting a potential off-target degradation due to adventitious PROTAC binding to PDE6D. Dose-dependent degradation of PDE6D in both WT MEFs and LRRK2 KO MEFs as shown via Western blotting (Figure ) indicated that XL01126 induced PDE6D degradation is LRRK2-independent and excluded it being a downstream consequence of LRRK2 degradation. A study examining the mechanism of LRRK2 degradation demonstrated that degradation by XL01126 is mediated by the ubiquitin-proteasome system as XL01126-induced degradation can be blocked by VHL ligand (VH101), neddylation inhibitor (MLN4924), and proteasome inhibitor (MG132) pretreatments in both WT MEFs (Figure ) and G2019S LRRK2 MEFs (Figure ). However, the LRRK2 dephosphorylation and Rab10 dephosphorylation are not completely rescued by VH101, MLN4924, and MG132 pretreatments owing to the kinase inhibition effect of XL01126 as also evidenced in our kinase inhibition assay (Figure ). |
62cd82f227b1e463bb36b984 | 15 | With a potent, fast and selective LRRK2 degrader in hand, we next established XL01126 cellular functionality in bioassays that report on LRRK2 activity. Mitochondrial dysfunction is one of the pathophysiological hallmarks of PD and can be rescued by mitophagy, a quality control mechanism whereby damaged or unnecessary mitochondria are delivered to lysosomes for degradation through membrane trafficking . It has been shown that increasing mitophagy with inducer agents has the potential as a PD therapy . Previous studies have shown that LRRK2 kinase activity impairs basal mitophagy and that LRRK2 knockout or pharmacological inhibition of LRRK2 with kinase inhibitors was able to rescue the mitophagy level . Utilizing XL01126 as a chemical degrader tool and using cis-XL01126 as a non-degrader, kinase inhibitor control, we found that both XL01126 and cis-XL01126 induced mitophagy level dose-dependently (Figure ) in mito-QC MEFs, a mCherry-GFP-mitochondria reporter cell model developed previously . Although XL01126 and cis-XL01126 act on LRRK2 through different mechanisms, they shared similar potency in inducing mitophagy at 10-100 nM, indicating that mitophagy level is indeed LRRK2 kinasedependent, and that other domains or motifs of LRRK2 are not involved in regulating mitophagy. |
62cd82f227b1e463bb36b984 | 16 | To qualify XL01126 as both a cellular and in vivo suitable degrader probe, and to assess its drug development potential, we next evaluated the physicochemical and ADME properties (Table and Figure ), as well as the in vivo pharmacokinetic profiles of XL01126 (Figure ). Due to the high molecular weight and lipophilicity, XL01126 has low solubility in PBS and moderate solubility in Fed State Simulated Intestinal Fluid (FeSSIF) (Table ), which, however, are all well above its DC50 values (14 -72 nM). The high stability (half-life at 108.29 min) of XL01126 in mouse plasma indicates XL01126 might be suitable for in vivo studies and we reasoned that plasma protein binding may account for its stability as protein binding can decrease the amount of free compound available for enzymatic metabolism. The protein binding also affects the potency of XL01126 in cells as shown by the significant potency shift of XL01126 in MEFs in the presence and absence of 10% fetal bovine serum (FBS) in the culture media (Figure ). To further qualify XL01126 as appropriate for in vivo studies, we assessed its PK profiles in mice (Figure and Table ). Following a single dose of XL01126 via intravenous (IV, 5 mg/Kg), intraperitoneal (IP, 30 mg/Kg), and oral gavage (PO, 30 mg/Kg), the concentrations of XL01126 in plasma, brain tissue, and cerebrospinal fluid (CSF) were determined. XL01126 showed fast absorption in both IP and PO injection with Cmax (7700 ng/mL and 3620 ng/mL for IP and PO separately) reached at 0.25 min and 2h for IP and PO dosing, respectively. High plasma concentrations were achieved in all routes of administration and were maintained at levels way above the DC50 values for XL01126 in the experimental time period. The metabolism of XL01126 seems slow in all administration routes, probably because of high protein binding. Strikingly, XL01126 was also detected in brain tissues and CSF (Figure and 14C), suggesting that XL01126 is capable of penetrating the BBB regardless of its unfavorable in vitro ADME properties and violation of Ro5 and/or RoCNS 76 . To the best of our knowledge, this is the first-time report of a VHL-based PROTAC that is both oral bioavailable (F=15%) and BBB permeable. Further investigation of XL01126 will focus on its in vivo pharmacodynamics and PD-related functional studies. |
62cd82f227b1e463bb36b984 | 17 | Although LRRK2 is a sought-after target for PD, the exact signaling pathways that link LRRK2 with PD pathology are unknown. LRRK2 is a large (286 KD), multi-domain protein that has two enzymatic domains and several other moieties involved in protein-protein interactions. However, LRRK2 kinase inhibitors are the most frequently used, if not the only, pharmacological tools for the study of LRRK2 biology, leaving the GTPase domain and protein-protein interaction domains of LRRK2 underexamined. The LRRK2 degrader that we have developed and characterized in this study offers a new chemical tool for deciphering the biology of LRRK2. |
62cd82f227b1e463bb36b984 | 18 | Employing the target protein degradation strategy to treat neurodegenerative disease can be revolutionary as protein aggregates are among the major pathologies and many attempts to modulate these diseases with conventional small-molecule drugs have not been successful. Significant effort has already been made to target neurodegenerative disease related proteins with either peptide-based or small molecular PROTAC degraders . However, achieving favorable PK profiles with oral bioavailability and BBB penetration have been the major obstacles for central nervous system (CNS)-targeted PROTACs. Amongst the only successes reported to date, Wang et al. developed a tautargeting PROTAC (C004019) that can penetrate the BBB after subcutaneous injection and induce tau protein degradation in the brain . Herein, we disclose the identification of a LRRK2-targeting PROTAC that exhibits remarkable oral bioavailability and BBB penetration. Both CC004019 and XL01126 are VHL-based PROTACs with multiple violations of Ro5 and/or RoCNS. Their capability of penetrating the BBB challenges the Ro5-and RoCNS-based pre-conceptions and dogma and has expanded the chemical space of CNS targeting drugs. Although BBB penetrant, XL01126 showed low concentration in the brain and in CSF, with low brain-to-plasma ratio (< 0.035). Nevertheless, given the substoichiometric/catalytic mechanism of action, which is different from the occupancy-driven mechanism of inhibitors, PROTACs may achieve target protein degradation in the targeted tissue even with low exposure. |
62cd82f227b1e463bb36b984 | 19 | PROTAC is an emerging drug discovery modality, yet the development of an active and efficient degrader is still a laborious and unguided process. Structure-guided PROTAC design is an attractive strategy, but solving the crystal structure of a target protein:PROTAC:E3 ligase ternary complex is a challenging feat. The step-by-step PROTAC development strategy we used here provides an empirical and generalized roadmap for developing PROTACs against LRRK2 and other challenging targets. The ternary binding affinity assay and ternary complex formation assay we developed here successfully circumvented the use of recombinant full-length LRRK2 protein which is challenging to express and purify. These two assays can potentially be applied to PROTAC or molecular glue development for other challenging targets as well. |
62cd82f227b1e463bb36b984 | 20 | Chemicals that are commercially available were purchased from Apollo Scientific, Sigma-Aldrich, Fluorochem, and Enamine and were used without further purification. All solvents use for reactions are anhydrous. LC-MS was carried out on Shimadzu HPLC/MS 2020 equipped with a Hypersil Gold column (1.9 μm 50 × 2.1 mm), photodiode array detector and ESI detector. The samples were eluted with a 3 min gradient of 5-95% acetonitrile in water containing 0.1% formic acid at a flow rate of 0.8 mL/min. Flash column chromatography was performed on Teledyne ISCO Combiflash Companion installed with disposable normal phase RediSep Rf columns (230-400 mesh, 40-63 mm; SiliCycle). Preparative HPLC purification was performed on Gilson Preparative HPLC system equipped with a Waters X-Bridge C18 column (100 mm × 19 mm and 5 μm particle size) using a gradient from 5 to 95% of acetonitrile in water containing 0.1% formic acid over 10 min at a flow rate of 25 mL/min. Compound characterization using NMR was performed either on a Bruker 500 Ultra shield or on a Bruker Ascend 400 spectrometer. The 1 H NMR, C NMR and F NMR reference solvents used are CDCl3 -d1 (δH = 7.26 ppm/δC = 77.16 ppm), CD3OD-d4 (δH = 3.31 ppm/δC = 49.00 ppm) or DMSO-d6 (δH = 2.50 ppm/δC = 39.52 ppm). Signal patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint.), multiplet (m), broad (br), or a combination of the listed splitting patterns. The coupling constants (J) are measured in hertz (Hz). HRMS was performed on a Bruker MicroTOF II focus ESI Mass Spectrometer connected in parallel to a Dionex Ultimate 3000 RSLC system with diode array detector and a Waters XBridge C18 column (50 mm × 2.1, 3.5 µm particle size). All final compounds are >95% pure by HPLC. |
62cd82f227b1e463bb36b984 | 21 | Primary MEFs were generated as described in a previous study . Briefly, the uterine horn was collected from adult female mice at day E12.5 and transferred to a 10 cm tissue culture dish containing cold PBS. Two forceps were used to tear the yolk sacs to isolate each embryo. Forceps were cleaned thoroughly with 70% ethanol between each embryo isolation. The embryos were culled, and a tissue piece was collected in a PCR tube for genotyping. The red tissue of the embryo was removed, and the remainder was minced with a scalpel blade and incubated with 7.5 ml trypsin-EDTA solution for 10 minutes in a 37°C, 5% CO2 tissue culture incubator. The dish was removed from the incubator and checked under a light microscope for single cells. 7.5 ml complete media was added to the trypsinised cells and the cell suspension was transferred to a 15 ml Falcon tube, and centrifuged at 1200 rpm for 5 minutes at room temperature. The trypsin was aspirated, the cell pellet was resuspended in 5 ml fresh complete media, and the cell suspension was plated in a 60 mm tissue culture dish and incubated in a 37°C, 5% CO2 tissue culture incubator. The MEFs at this stage were considered as passage 0 and were passaged and expanded for experimental use once the genotype was confirmed by allelic sequencing and immunoblotting. MEFs were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin supplemented with 1X nonessential amino acids and 1 mM sodium pyruvate. |
62cd82f227b1e463bb36b984 | 22 | Macrophages were cultured in complete media containing DMEM, 10% (v/v) heat inactive FBS, 20% (v/v) L929 preconditioned medium, 2.5% (v/v) HEPES, 2 mM Lglutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, 2% sterile-filtered β-mercaptoethanol, 1X non-essential amino acids and 1 mM sodium pyruvate. Bone marrow isolation and macrophage differentiation was modified from 81 , employing L929 preconditioned medium as the source of M-CSF for differentiation. Briefly, scissors and forceps were used to dissect femurs and tibiae from adult mice, and muscle tissue was carefully removed from bones. Clean femurs and tibiae were placed in a tissue culture dish containing complete media. The ends of each bone were cut with scissors to expose bone marrow. Bone marrow was flushed with a 25-gauge needle attached to a 10 ml syringe containing complete media. Media containing bone marrow was passed through a 70 µm cell strainer and precursor cells were plated on non-tissue culture treated 10 cm bacteriological plates containing 10 ml complete media. This was marked as day 0 of isolation. On day three postisolation, macrophages were topped up with 5 ml fresh complete media. On day seven post-isolation, macrophages were rinsed once with PBS and incubated with versene for 5 minutes in a 37°C 5% CO2 tissue culture incubator. Macrophages were detached with cell scrapers and were centrifuged at 1200 rpm for 5 minutes at room temperature. The versene was aspirated and the remaining cell pellet was resuspended in complete media. The cell suspension was counted, and cells were seeded for experimental analysis in a 6-well format, in tissue culture treated dishes at a final cell density of one million cells per well of a 6-well plate. |
62cd82f227b1e463bb36b984 | 23 | PBMC cells were separated from human blood from healthy volunteer donors following existing protocol 82 and pelleted by centrifugation at 1000 g for 2 min. The supernatant was discarded and the PBMC pellet was resuspended in PBS containing 2% FBS for washing. The suspension was centrifuged at 1000 g for 2min again and the PBMC pellet was resuspended in RPMI-1640 (Gibco) media supplemented with 10% FBS. The cells were then seeded into 6-well plates and treated with testing compounds at indicated concentrations and time period. After treatment, the cells were collected into 2-ml eppendorf tube and centrifuged at 500g for 2 min to pellet the cells, the supernatant was discarded, and the pellet was resuspended in 1 ml PBS and centrifuged at 500 g for 2min again. The PBMC pellet was lysed with 60 µL of lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 1 mM EGTA, sodium orthovanadate, 50 mM NaF, 0.1% (v/v) 2-mercaptoethanol, 10 mM 2-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 µg/ml mycrocystin-LR (Enzo Life Sciences), 270 mM sucrose, 0.5 mM DIFP (Sigma, Cat# D0879) in addition to complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich Cat # 11836170001). DIFP is highly toxic and must be prepared in a fume hood to a stock solution of 0.5 M in isopropanol. The lysed cells were then centrifuged at 1500 g for 15 min at 0 °C. The supernatants were collected for analysis by quantitative immunoblotting. For long term storage, the supernatant was flash frozen and stored at -80°C. Protein concentrations of cell lysates were determined using Pierce™ BCA Protein Assay Kit (ThermoFisher). |
62cd82f227b1e463bb36b984 | 24 | Culturing and passaging of adherent cell lines were carried out using aseptic technique in CL1 or CL2 (for PBMC isolation) biological safety cabinets. All cells were incubated in a 37°C incubator with 5% CO2. Cell lines were regularly tested for mycoplasma contamination. For western blot assay, the cells were seeded in 6-well plates. For immunoprecipitation of LRRK2, SH-SY5Y cells (cultured in DMEM-F12, supplemented with 15% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, 1X non-essential amino acids, and 1 mM sodium pyruvate) were seeded in a 10-cm dish. All cells were treated with the indicated compounds such that the final concentration of DMSO was 0.1%. Following the treatment of cells with compounds at indicated concentrations and time periods, the media was removed and the cells were washed with PBS and lysed in 100 µl ice-cold complete lysis buffer containing 50 mM Tris-HCl pH 7.4, 1 mM EGTA, 10 mM 2-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 270 mM sucrose, supplemented with 1 μg/ml microcystin-LR, 1 mM sodium orthovanadate, complete EDTA-free protease inhibitor cocktail (Roche) and 1% (v/v) Triton X-100. The cells were immediately placed on ice and were scraped and collected into 1.5 ml Eppendorf tubes. Cell lysates were incubated on ice for 10 minutes prior to centrifugation at 15,000 g at 4°C for 15 minutes. The cell pellet was discarded, and supernatant was collected for analysis by quantitative immunoblotting. For long term storage, the supernatant was flash frozen and stored at -80°C. Protein concentrations of cell lysates were determined using the Bradford assay. |
62cd82f227b1e463bb36b984 | 25 | Cell lysates containing a quarter of a volume of 4X NuPAGE LDS sample buffer (NP0007) supplemented with 5% βmercaptoethanol, were heated at 95°C for 5 minutes. 15 to 20 μg of samples were loaded onto pre-cast 4-12% Bis-Tris midi 20W or 26W gels (Thermofisher Scientific, Cat# WG1402BOX or WG1403BOX) and resolved at 130 V for 2 hours with NuPAGE MOPS SDS running buffer (Thermofisher Scientific Cat# NP0001-02). Proteins were electrophoretically transferred onto a 0.45 µm nitrocellulose membrane (GE Healthcare, Amersham Protran Supported 0.45 mm NC) at 90 V for 90 min on ice in transfer buffer (48 mM Tris base and 39 mM glycine supplemented with 20% methanol). The transferred membrane was blocked with 5% (w/v) skim milk powder dissolved in tris-buffered saline with tween (TBS-T) (50 mM Tris base, 150 mM sodium chloride (NaCl), 0.1% (v/v) Tween-20) at room temperature for 1 hour. Membranes were washed three times with TBS-T and were incubated in primary antibody overnight at 4°C. Prior to secondary antibody incubation, membranes were washed three times for 15 minutes with TBS-T. The membranes were incubated with secondary antibody for one hour at room temperature, protected from light. Thereafter, the membranes were washed with TBS-T three times with a 15-minute incubation for each wash, and protein bands were acquired via near-infrared fluorescent detection using the Odyssey CLx imaging system and quantified using Image Studio software. Graphs were generated using Graphpad Prism version 8 software. |
62cd82f227b1e463bb36b984 | 26 | Wildtype MEFs were seeded in 10 cm tissue culture dishes, at a density of two million cells per dish. Cells were treated with 0.1% DMSO, 300 nM XL01126, or 300 nM cis-XL01126 for 4 hours prior to harvest in 400 µl complete lysis buffer, supplemented with 1 μg/ml microcystin-LR, 1 mM sodium orthovanadate, complete EDTA-free protease inhibitor cocktail (Roche) and 1% (v/v) Triton X-100. Cell lysates were incubated on ice for 10 minutes, then underwent three rounds of high energy sonication for 15 cycles (30 seconds on, 30 seconds off) using the Diagenode Bioruptor. Cell lysates were centrifuged at 15,000 g at 4°C for 15 minutes. Cell pellet was discarded, and supernatant was collected for protein quantification using a BCA protein assay kit (Pierce #23225). 100 µg cell lysate was employed for total proteomic analysis. Proteins in cell lysate were reduced with 0.1 M Tris(2-carboxyethyl)phosphine (TCEP) diluted in 300 mM triethylammonium bicarbonate (TEABC) to a final concentration of 10 mM. Samples were incubated on a Thermomixer for 30 minutes at 60°C at 800 rpm then cooled down to room temperature and underwent alkylation with 0.04 M iodoacetamide (IAA) freshly dissolved in water. Samples were then incubated in the dark on a Thermomixer at room temperature for 30 minutes at 800 rpm. Alkylation was quenched with the addition of 0.1 M TCEP dissolved in 300 mM TEABC at a final concentration of 5 mM. Samples were incubated on a Thermomixer at room temperature for 20 minutes at 800 rpm. Sodium dodecyl sulfate (SDS) was added at a final concentration of 5% (w/v) from a 20% (w/v) stock. 12% (v/v) phosphoric acid was then added to a final concentration of 1.2% (v/v). Samples were diluted in 6 times the sample volume of Strap wash buffer containing 90% (v/v) methanol diluted in 100 mM (v/v) TEAB pH 7.1. |
62cd82f227b1e463bb36b984 | 27 | Samples underwent S-trap cleanup to remove detergents and other impurities with S-trap mini columns (PROTIFI Cat# MSPPC02-MINI-80) placed in 2 ml Eppendorfs. The protein mixtures were added to columns and centrifuged briefly (1000 g / 1 minute / RT). Columns were washed with 400 µl S-trap buffer 4 times, centrifuging after each wash at 1000 g / 1 minute / RT. Columns were placed in fresh 2 ml Eppendorfs and 100 µl of 5 µg Trypsin/Lys-C freshly dissolved in 50 mM TEAB, pH 8.5 was added. Columns were centrifuged briefly (200 g / 1 minute / RT) and Trypsin/Lys-C mixture was pipetted back onto the column. 100 µl 50 mM TEAB, pH 8.5 was added directly to the 2 ml Eppendorfs to cover any digested peptides remaining in the tube. The S-trap columns in 2 ml Eppendorfs were incubated at 47°C without shaking for 1.5 hours, then at RT overnight. 80 µl 50 mM TEAB was added to S-trap columns, which were centrifuged, and eluates were collected in new 1.5 ml Eppendorf tubes. 80 µl 0.2% (v/v) formic acid was added to columns, which were centrifuged, and second eluates were pooled with first eluates. 80 µl 50% (v/v) acetonitrile diluted in 0.2% (v/v) formic acid was added to columns, which were centrifuged, and third eluates were pooled with previous eluates. 500 ng digested peptides were set aside to vacuum dry separately to verify that digestion efficiency by calculating the zero and single missed cleavages was >98%. The remaining peptides were divided in half (50 µg peptides each tube) and vacuum dried and stored in -80°C prior to continuation with tandem mass tag (TMT) labeling. |
62cd82f227b1e463bb36b984 | 28 | TMT labeling 800 µg TMT mass tag reagents were dissolved 80 µl 100% (v/v) anhydrous acetonitrile to obtain final concentrations of 10 µg/µl. Resuspended TMT reagents were incubated at RT for 10 minutes, then vortexed and centrifuged briefly (2000 g / 2 minutes / RT). 50 µg lyophilized peptides were resuspended in 50 µl of a mixture containing 42 µl 50 mM TEAB and 8 µl 100% (v/v) anhydrous acetonitrile. Resuspended peptides were sonicated for 10 minutes, then centrifuged at 17,000 g for 10 minutes at RT. Peptides were transferred to fresh protein low-bind 1.5 ml Eppendorf tubes. 20 µl of 10 µg/µl TMT reagent were added to solubilized peptides, vortexed, centrifuged briefly (2,000 g / 1 minute / RT) and incubated on a Thermomixer for 2 hours at 800 rpm at RT. 50 µl of 50 mM TEAB was added to each reaction, followed by vortex, brief centrifugation (2,000 g / 1 minute / RT) and incubation on a Thermomixer at 800 rpm at RT for an additional 10 minutes. 5 µl of each TMT labeled sample was set aside, vacuum dried and injected on MS to confirm that labeling efficiency was >98%. The remaining reactions were stored in -80°C until labeling efficiency was verified. TMT samples were thawed to RT and labeling reactions were quenched with the addition of 5 µl 5% (v/v) hydroxylamine (dissolved in water from a 50% (v/v) stock solution). Samples were incubated on a Thermomixer for 20 minutes at 800 rpm at RT. Quenched TMT labeled samples were pooled, vacuum dried and subjected to High-pH fractionation as described previously , 96 fractions were collected and were concatenated into 48 fraction. Pooled fractions were vacuum dried and stored in -20 freezer until the LC-MS/MS analysis. |
62cd82f227b1e463bb36b984 | 29 | High-pH fractions were solubilized in 60 µl of LC-solution (3% ACN (v/v) and 0.2% Formic acid (v/v) in by placing them on a Thermomixer at room temperature for 30 minutes with an agitation at 1800 rpm. 7 µl of each fraction was transferred into LC-vail inserts for mass spectrometry analysis. LC-MS/MS analysis was carried out on a Thermo Lumos ETD Tribrid mass spectrometer inline with 3000 ultimate RSLC nano-liquid chromatography system. Sample was injected into pre-column (C18, 5µm, 100Ao, 100µ, 2cm Nano-viper column # 164564, Thermo Scientific) at 5 µl/min flow rate and subsequently loaded onto the analytical column (C18, 5µm, 50cm, 100Ao Easy nano spray column # ES903, Thermo Scientific) for the separation of peptides using nano-pump operated at 300 nl/min flow rate. 85 min non-linear gradient was applied (5% Solvent B (80 %ACN v/v in 0.1% Formic acid v/v) to 22% B for 70 min and increased to 35% B for another 10 min for a total of 100 min run time. The eluted peptides were electrosprayed into the mass spectrometer using easy nano source. The data was acquired in a data dependent acquisition (DDA) mode in SPS MS3 (FT-IT-HCD-FT-HCD) method and was acquired using top speed for 2 sec for each duty cycle. The Full MS1 scan was acquired at 120,000 resolution at m/z 200 and analyzed using Ultra high filed Orbitrap mass analyzer in the scan range of 375-1500 m/z. The precursor ions for MS2 were isolated using Quadrupole mass filter at 0.7 Da isolation width and fragmented using normalized 35% Higher-energy collisional dissociation (HCD) of in Ion routing multipole analyzed using Ion trap. Top 10 MS2 fragment ions in a subsequent scan were isolated and fragmented using HCD at 65% normalized collision energy and analyzed using Orbitrap mass analyzer at 50,000 resolution in the scan range of 100-500 m/z. |
62cd82f227b1e463bb36b984 | 30 | Raw MS data of 48 High-pH fractions were searched using MaxQuant search algorithm (Vesion 2.0.3.0) against Uniprot Mouse database (Release version May 20021 containing 25,375 sequences). 10 plex TMT reporter ion MS3 workflow was loaded and used following search parameters. Trypsin as a protease was selected by allowing two missed cleavages, deamidation of Asn and Gln; Oxidation of Met were used as variable modifications and Carbamidomethylation of Cys as a fixed modification. The default mass error tolerance for MS1 and MS2 (4 ppm and 20 ppm) were used. Min of 2 unique+razor peptides were selected for the quantification. The data was filtered for 1% PSM, peptide and protein level FDR. The output protein group .txt files were further processed using the companion Perseus software suite (version 1.6.15.0) . Decoy hits, contaminants, proteins identified by sites and single peptide hits were filtered out. The data was then log2 transformed and T-test was performed between the sample groups and the p-values were corrected using 5% permutation-based FDR to identify the differentially regulated protein groups. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034055. |
62cd82f227b1e463bb36b984 | 31 | FP competitive binding assays were performed following the method described previously . All the measurements were taken on a PHERAstar (BMG LABTECH) plate reader installed with a FP filter that sets excitation and emission wavelengths at 485 nm and 520 nM separately. Each well of 384-well plate (Coring 3575) contains 10 nM VCB protein, 5 nM FAM-lableled HIF-1α peptide (FAM-DEALAHypYIPMDDDFQLRSF, "JC9"), and decreasing concentrations of testing compounds (14 concentrations with 2-fold serial dilution starting from 250 µM) in FP assay buffer (100 mM Bis-Tris propane, 100 mM NaCl, 1 mM TCEP, pH 7) with a final DMSO concentration of 5%. The control wells containing the VCB and JC9 with no compound are set as the maximum signals (zero displacement). And the control wells containing JC9 in the absence of protein are set as the minimum signals. Control values were used to obtain the percentage of displacement which was ploted against Log [Compound]. Average IC50 values were determined for each titration using nonlinear regression analysis with GraphPad Prism (v.9.3.1). The Ki values were back-calculated from the Kd of JC9 (1.5 nM -3.4 nM) and the fitted IC50 values, as described previously . |
62cd82f227b1e463bb36b984 | 32 | For VHL and LRRK2 target engagement experiments in live and permeabilized cells, the HEK293 cells were transfected with VHL-NanoLuc fusion vector (Promega, N275A) or LRRK2-NanoLuc fusion vector (Promega, NV3401) following Promega's protocol and seeded into white 384well plate (Corning3570) at a density of 6000 cells/well. To measure NanoBRET in permeabilized cells, the cells were treated with 50 µg/ ml digitonin (Sigma, D141), 125 nM VHL tracer/125 nM LRRK2 tracer, testing compounds at decreasing concentrations (12 concentrations with 2-fold serial dilution starting from 33 µM), and NanoBRET NanoGlo Substrate (Promega) at concentration recommended by the manufacturer's protocol. In the maximum signal control samples (DMSO control), DMSO was added instead of testing compounds. In the minimum signal control samples (no tracer control), DMSO and tracer dilution buffer were used to replace testing compounds and tracer separately. The filtered luminescence was measured within 10 min following addition of the substrate on a GloMax Discover microplate reader (Promega) or a PHERAstar (BMG LABTECH) plate reader equipped with a 450-nm bandpass filter (donor) and a 600-nm long pass filter (acceptor). To measure NanoBRET in live cells, the cells were treated with 250 nM VHL tracer/500 nM LRRK2 tracer, testing compounds at testing compounds at decreasing concentrations (12 concentrations with 2-fold serial dilution starting from 33 µM) and incubated at 37 °C in an incubator for 2 h. The plates were then cooled down and added with NanoBRET NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega, N2160) before performing the same NanoBRET reading as the permeabilized mode on plate readers. NanoBRET ratio of each well was expressed in milliBRET according to the equation: mBRET = [(signal at 610 nM/signal at 450 nM) -(signal at 610 nMno tracer control/signal at 450 nMno tracer control)]×1000. The fractional occupancy was calculated according to the equation: fractional occupancy = (mBRETtesting compound -mBRETno tracer control)/(mBRETDMSO control -mBRETno tracer control). |
62cd82f227b1e463bb36b984 | 33 | The HEK293 cells were transfected with LRRK2-NanoLuc fusion vector (Promega, NV3401) following Promega's protocol and seeded into white 384-well plate (Corning3570) at a density of 6000 cells/well. The cells were then treated with 50 µg/ml of digitonin, 125 nM of LRRK2 tracer, testing compounds at decreasing concentrations (11 concentrations with 2-fold serial dilution starting from 10 µM) or testing compounds and VCB mix (11 concentrations with 2-fold serial dilution starting from 10 µM compound for the compound. The first 6 concentrations of VCB start from 32 µM with 2-fold dilution, the last 5 concentrations of VCB keep at 1 µM), and NanoBRET NanoGlo Substrate (Promega) at concentration recommended by the manufacturer's protocol. In the maximum signal control samples (DMSO control), DMSO was added instead of testing compounds. In the minimum signal control samples (no tracer control), DMSO and LRRK2 tracer dilution buffer were used to replace testing compounds and LRRK2 tracer separately. The filtered luminescence was measured within 10 min following addition of the substrate on a PHERAstar (BMG LABTECH) plate reader equipped with a 450-nm bandpass filter (donor) and a 600-nm long pass filter (acceptor). The fractional occupancy was calculated according to the equation: fractional occupancy = (mBRETtesting compound -mBRETno tracer control)/(mBRETDMSO control -mBRETno tracer control). |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.