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65d898779138d23161ce552c | 18 | The S-HL of the apoenzyme model comprised of Cys493 and Gly829 residues with adjacent fragments of the main chain (Fig. ). The S-HL of the holoenzyme model was extended by the residue of Gln707 as well as fumarate and toluene with respect to that of apoenzyme. The B-HL comprised of S-HL extended by all residues penetrating 5 Å radius of Gly829 and Cys493 |
65d898779138d23161ce552c | 19 | for the apoenzyme (389 atoms) and 5 Å radius of Gly829 and 3 Å radius Cys493 for plus toluene and fumarate for the holoenzyme (315 atoms). The charge of the S-HL was 0 and -1, and for the B-HL was 1 and 0, respectively. All calculations were conducted for a doublet state due to the presence of a single radical. The geometry of the QM:MM models was optimized at the B3LYP/6-31g(d,p):AMBER using an electronic embedding approach which was followed by vibrational analysis introducing vibrational corrections for stationary points. The transition states of H atom transfer between Cys493 and Gly829 were localized by means of relaxed scans along the reaction coordinate (i.e., d(Cys-S-H … C rad -Gly)) followed by TS optimization using the Berny algorithm. Each stationary state preceding or following a particular TS was optimized individually, with initial geometry derived from the TS. The energy of the final stationary points was corrected with single point calculations using B-HL at B3LYP/6-311g+(2d,2p):AMBER level of theory with electronic embedding and Grimme D3 corrections for disperse interactions . The only exception from this protocol was proS intermediate of the apoenzyme (proS I apo ) for which we were able to calculate energy only at the B3LYP/6-31g(d,p):AMBER level of theory. As a result, the energy for proS I apo was approximated based on the energy difference between proS I apo and proS TS apo calculated at B-HL/B3LYP/6-31g(d,p)/D3:AMBER level of theory. The vibrational corrections were calculated at 303 K and 1 atm. and scaled with 0.9806 factor according to B3LYP/6-31g(d) correction calculated by Scott and Radom and thermal energy correction was added to all electronic energies calculated at the respective level of theory. The corrections for the transfer of the H/D atom were obtained by substitution of the mass of respective protium nuclei with an isovalue of 2, taking into consideration substitution of the transferred hydrogen atom, atom position at Gly829, both of the hydrogen atoms or none. |
65d898779138d23161ce552c | 20 | The transfer of the H atom from Cys to Gly was evaluated for the holoenzyme in either substrates (toluene and fumarate) or product (R-benzylsuccinate) bound state. As a reference, the apoenzyme was examined. For each transition two alternate conformations were evaluated, leading to H atom transfer toward the re or si face of radical Gly. |
65d898779138d23161ce552c | 21 | The steady-state kinetic constant of the deuteration process (Fig. ) was described by a standard scheme assuming a two-step reaction with the first reversible and a second irreversible step. The second step is assumed irreversible due to the H/D exchange of the proton/deuteron with abundant solvent. For such assumptions observed overall kinetic constant of the H/D exchange was calculated according to as: |
65d898779138d23161ce552c | 22 | The C-C bond formation between fumarate and toluene catalyzed by benzylsuccinate synthase is enabled by the interaction of a thiyl radical of Cys493 with the methyl group of the alkylaromatic substrate, which has been taken as starting point for previous studies on the reaction mechanism . However, the reactive thiyl radical first needs to be generated by |
65d898779138d23161ce552c | 23 | transferring an H atom from the sulfhydryl group of Cys493 to the radical Gly829 . This crucial initial process needs to occur in all GRE, but is poorly understood up to date, and almost no approaches have been addressed to obtain computational models. Therefore, we decided to employ QM:MM modeling of this step using BSS as an example. We started our investigation with an enzyme-substrate complex (E:S), where both fumarate and toluene are bound in the active site. The reaction starts with radical Gly829 and the sulfhydryl group of Cys493, which is turned away in the direction of the neighboring residue Gln707. The spin density of the initial glycyl radical is mostly located at the C carbon of Gly (0.9) but is also distributed by resonance along the peptide backbone to the adjacent nitrogen (0.075) and carbonyl oxygen atoms (0.1) of the preceding peptide bond, and with a resulting negative spin density to the carbonyl C atom of Gly829 (-0.16). These values are remarkably similar to previously calculated spin densities of glycyl radicals in crystals of the model compound Nacetylglycine, where the C carbon was attributed to a value of 0.77 . Both residues, Gly829 and Cys493, represent the tips of two turns, which extend as loops from the inner rim to the center of the barrel structure (hereafter called G-loop and C-loop). Both residues are essential contributors to the active site of the enzyme and directly face each other . To A similar transfer can also occur when the H atom is transferred to the si face of the glycyl radical (leading to proS-TS ES ). The position of the Cys, in this case, resembles that in the proR-TS ES , but the G-loop harboring the glycyl radical is bent to present the other face of the Gly. |
65d898779138d23161ce552c | 24 | The C-H and S-H distances at proS-TS ES are still quite short at 1.57 and 1.52 Å, while the S-H-C angle is more bent at 145 o (Fig. , Fig. ). Moreover, the radical density is more localized in proS-TS ES compared to proR-TS ES , as the spin densities are 0.36 at the S atom and 0.66 at C of Gly829. |
65d898779138d23161ce552c | 25 | The H transfer is completed with the formation of the non-radical Gly829 and the thiyl radical at Cys493, which slightly rotates back towards Gln707. The main difference in the structures of both intermediates (I1) is associated with the position of Arg826, which gets into close contact with the G-loop in the proS conformation due to its conformational change. |
65d898779138d23161ce552c | 26 | The analysis of energy barriers calculated for both models by using B-QM and corrected with D3 dispersion and thermal energy corrections (Table ; see Table -S7 for all energies) indicates that the proR-TS ES is energetically more favorable (40.4 kJ/mol) and hence more probable compared to the proS-TS ES (62.2 kJ/mol). However, the difference is not large enough to exclude a reaction via proS-TS ES , rendering both processes kinetically possible. The energy state of intermediate I1 is very similar in both models and turned out to be 32-34 kJ/mol lower (more stable) than that of the substrate-bound E:S state. |
65d898779138d23161ce552c | 27 | Assuming a partial reversibility of the BSS reaction between the product and the product radical, we have investigated the same process for the enzyme:product model (E:P) by modeling the steps of the BSS mechanism in reverse. Thus, the process starts with the bound product in the E:P complex, Gly829 in the radical state (spin density at C 0.91), and Cys493 in the sulfhydryl form with an SH group. The Cys residue of the E:P complex is localized between the G-loop hosting Gly829 and a tightly packed arrangement of the side chains of residues Met494, Leu766, and Gln707. The H transfer occurs in analogy to the E:S complex, involving either an attack on the si (proS) or re (proR) face of the glycyl radical. As expected, the calculated geometries of the transition states are almost identical to those observed for is divided between the C atom of Gly829 and the S atom of Cys493 for both the proR-(0.61 and 0.28, respectively) and the proS-directed reaction (0.68 and 0.32, respectively). The geometric and electronic similarities to the E:S complexes correspond also with the very similar calculated values of the energy barriers between E:P and the respective TS EP , which were 38.8 kJ/mol for proR-TS EP and 61.9 kJ/mol for proS-TS EP . The similarity even extended to the prediction that the first intermediate containing the thiyl radical of Cys493 is energetically more stable (by 33 kJ/mol) than the E:P state with the glycyl radical (Table ). |
65d898779138d23161ce552c | 28 | holoenzyme (E:S or E:P) with either bound substrates (TS ES ) or product (TS EP ) or in the apoenzyme with an empty active site (apo). The electronic energies were corrected with thermal energy calculated for models with protium-only substituted Gly (re-/si-H GlyH), with either enantiomer of mono-deuterated Gly assuming transfer of either its deuterium-(re/si-D GlyH) or protium-substituent (re/si-H GlyD), and with deuterium substituting both H atoms of Gly (re/si D GlyD). For readers' convenience, the barriers associated with the respective reverse processes, i.e. transfer of H/D atoms from Gly to radical Cys are provided in the right column (R/S-H or D transfers). Note that the R-and S-enantiomers of monodeutered Gly are also represented as GlyHD and GlyDH, respectively, to aid comprehension of Fig. . |
65d898779138d23161ce552c | 29 | E:S si E:S 0.0 0.0 0.0 0.0 E:S Strans. Finally, we have also analyzed the same process for the apoenzyme, without substrates or product present in the active site. Such a model represents the situation when water molecules can penetrate the active site despite its hydrophobicity, and the geometry of the enzyme is not influenced by the strong binding of fumarate. Besides the presence of three Furthermore, to reach the si face of the glycyl radical, Cys493 has to attain a higher energy conformation for pushing away the hydrophobic residues of Leu391 and Val709. |
65d898779138d23161ce552c | 30 | The energetics of both processes, when compared to the respective starting points (E apo ) suggest that the proR-TS apo is associated with a higher energy barrier (99.8 kJ/mol) than the proS-TS apo (80.6 kJ/mol) (Table , Table -S9). The latter value however omits an additional required energy input of 86.8 kJ/mol for the conformational shift from proR E apo to proS E apo . |
65d898779138d23161ce552c | 31 | Therefore, this result strongly suggests a high preference for the proR over a proS hydrogen transfer in apo BSS, if it is possible at all. Furthermore, the thiyl radical intermediate (I apo ) exhibits a higher energy than E apo by 7.2 kJ/mol in the case of the proR transfer and by approximately 78 kJ/mol in the case of the proS transfer (although such a high energy of proS I apo seems to be associated with increased size of QM part-see Table ). It should be noted, however, that QM:MM methodology, with mostly frozen coordinates outside of the active site, is not the best tool for precise estimation of energy differences between different conformations of the protein. |
65d898779138d23161ce552c | 32 | This occurs as well in PFL , but not in ARNR, where the hyperfine splitting does not change even after 12 hours of incubation in D2O-based buffers . The observed spectral change for BSS indicates a complete exchange of the remaining protium hydrogen at C of the glycyl radical within the time required for changing the buffer (ca. 1 h) . Since the hydrogen substituents of Gly829 are not acidic enough to spontaneously exchange with water as evident from experiments with model compounds , the process needs to be coupled to hydrogen transfer reactions with residues containing exchangeable hydrogens. The most likely of these reactions are either the GRE activation/deactivation processes or the hydrogen transfer cascades between Gly829 and Cys493 associated with the reaction mechanism. |
65d898779138d23161ce552c | 33 | Biochemical and structural data on PFL and its activating enzyme suggest that the generation of the glycyl radical by the respective activating enzyme is strictly stereospecific for all GRE, and a reversible glycyl radical deactivation process is only known for PFL , but not for any other GRE. Moreover, one of the two conserved Cys residues of PFL has already known to be involved in the H/D exchange of the glycyl radical in PFL . Therefore, the observed H/D-exchange of BSS occurs most likely during the H-transfer reactions between Gly829 and Cys493, which occur in equilibrium in both directions. Because the sulfhydryl hydrogen of Cys493 is exchangeable with solvent, this would lead to H/D exchange at the glycyl radical if the H transfer reaction between Cys and Gly occasionally occurred in the less preferred enantiomeric orientation. Therefore, the observed H/D exchange at C of the glycyl radical can be explained if the energetic barriers of proR-and proS-directed hydrogen transfer from Cys493 to the glycyl radical are not too far apart, making the processes kinetically comparable. |
65d898779138d23161ce552c | 34 | Deuteration of certain positions influences the heights of the energetic barriers of the respective transition states of H/D exchange pathways through primary and secondary kinetic isotope effects (KIE). Therefore, we analyzed all possible scenarios separately, i.e. when the H of the Gly829 radical is exchanged to D, when the D is exchanged back to H, and when Gly829 stays either fully protonated or deuterated, all in both possible geometries via either the re or the si face of the radical Gly (Table , Table , S8). Moreover, we did these calculations with three different forms of BSS: the enzyme in complex with the substrates (E:S), with the products (E:P), or as an apoenzyme with an empty active site cavity (apo). |
65d898779138d23161ce552c | 35 | Fully protonated BSS showed barriers of 40.4 and 72.2 kJ/mol for the forward and reverse reactions of H exchange with Cys493 via the re face of the glycyl radical, but of 62.2 and 95.8 kJ/mol via the si face. Therefore, this hydrogen transfer appears to be highly enantiospecific for the re-face, which proceeds over 5,600 times faster than via the si face and in the reverse process over 12,000 faster for the proR-H transfer compared to proS. |
65d898779138d23161ce552c | 36 | The transfer of deuterium from Cys493 to the re face of the glycyl radical is associated with a barrier of 43.2 kJ/mol (Fig. E:S), representing a slight increase by 2.8 kJ/mol compared to that for H/H transfer, which results in an intrinsic KIE of 3.0. The reverse reaction, i.e. the transfer of a D atom from R-GlyHD back to the thiyl radical, proceeds with a barrier of 77.4 kJ/mol. The predicted intrinsic KIE associated with this transfer is higher than in the forward process (7.9), due to a higher increase of the energy barrier (5.2 kJ/mol) upon replacing protium for deuteron. |
65d898779138d23161ce552c | 37 | If deuterium is transferred from Cys493 to the si face of the glycyl radical, the barrier is at 64.8 kJ/mol, representing an increase of 2.6 kJ/mol compared to the si H/H exchange. The reaction, the backward transfer of a D atom from the S-enantiomer of monodeuterated Gly829, occurs with a barrier of 101.0 kJ/mol, 5.2 kJ/mol more than for the si H/H exchange (Fig. ). |
65d898779138d23161ce552c | 38 | Interestingly, the barriers for both hydrogen transfer reactions from Cys493 to the deuterated glycyl radical turned out to be slightly lower than those for the corresponding H/H exchange reactions due to a secondary kinetic isotope effect: the barriers for protium transfer to the re and si faces are 39.8 and 61 kJ/mol (Fig. ), which is 0.6 and 1.2 kJ/mol lower than the respective values for the H/H transfer and associated with an inverse KIE (0.78 and 0.63, respectively). Similarly, the secondary kinetic isotope effect facilitates also deuteron transfer when the glycyl radical is already deuterated (GlyD . ). Such a process is associated with a barrier of 42.6 kJ/mol for the re-face attack and 61.0 kJ/mol for the si face attack. (Fig. ), resulting in a lower intrinsic KIE with respect to deuteron transfer to a non-deuterated glycyl radical (decrease from 3.0 for GlyH . to 2.24-2.35 for GlyD . ). Thus, it is easier to transfer a second deuteron if the first one is already present at the glycyl radical. |
65d898779138d23161ce552c | 39 | In the reverse process, the hydrogen transfer from R-GlyHD to the thiyl radical occurs with a barrier of 95.6 kJ/mol, which is 0.2 kJ/mol lower than for H/H (KIE 0.9), while the barrier for hydrogen transfer from S-GlyDH is at 72.4 kJ/mol, i.e. 0.2 kJ/mol higher than for the respective H/H exchange (KIE 1.1)(Fig. ). The removal of a deuteron from GlyD2 is significantly slower compared to the process when only protium is involved, and the energy difference of 5.5 kJ/mol results in a KIE of 9 (for values see Table and S6 and Fig. ). |
65d898779138d23161ce552c | 40 | The entire scheme of all possible H and D exchange reactions in BSS is shown in Fig. . The calculated barriers indicate immediately that the conservative exchange reactions resulting in the retention of either H to D in the glycyl radicals are highly favored over those leading to isotope exchange. Judging from the much lower barriers for both the forward and reverse reactions in the case of GlyH . , this process (with deuterated Cys493) occurs mostly via the reface (labeled by blue arrows in Fig. ), whereas in the case of GlyD . , it occurs mostly via the siface (with protiated Cys493, labeled green in Fig. ). To obtain any exchange of H to D (or reverse) in the glycyl radical, some of the less favorable reactions in the other stereochemical orientation have to occur. For exchanging H to D, the more favorable pathway is the si-face directed attack of deuterated Cys493 combined with the transfer of the 'proR'-protium from the S-glycyl intermediate back to the thiyl radical (red arrows in Fig. ). Although the barrier of the first step is significantly higher than that for the re-attack (64.8 vs. 43.2 kJ/mol, indicating a 5,400-fold slower rate), the second step of protium vs. deuterium transfer is highly favored (barriers 72.4 vs. 101 kJ/mol), while the alternative pathway via R-Gly829 contains a prohibitively high barrier at the second step (95.6 kJ/mol). |
65d898779138d23161ce552c | 41 | In conclusion, the very high barrier of removing H from R-GlyHD (95.6 kJ/mol) suggests that deuterium exchange is correlated with the rare si-directed hydrogen transfer events, which produce an S-GlyDH intermediate (Fig. red pathway). The calculated back-transfer of H from S-GlyDH is much faster than that of D (difference of the barriers 28.6 kJ/mol), so an H/D exchange at the glycyl radical will be inevitable if the first step occurs. |
65d898779138d23161ce552c | 42 | First, we compared the calculated rates for the preferential pathway via S-Gly829 (red arrow in Fig. ) with those of the alternate pathway via R-Gly829. It turns out that the value of k S H/D is calculated as 2 s -1 while that of k R H/D equals 2.1*10 -4 s -1 , which means that the pathway proceeds exclusively through S-GlyDH while R-GlyHD is of no consequence. This shows that despite the 5400-fold kinetic preference for the transfer of D via the re face, one of the expected rare D transfers via the si face of the glycyl radical is a prerequisite for the H/D exchange. The same approach can be applied to analyzing the experiment when the deuterated enzyme is incubated in H2O. For the backward exchange of D to H in the glycyl radical, the preferred pathway proceeds via the re-face attack (purple arrows in Fig: ) with a calculated value of k R D/H of 0.29 s -1 while the alternative pathway, starting with the transfer of H to the si face, is associated with a k S D/H value of 2.4*10 -5 s -1 , almost 12,000-fold slower than the process via the re-face. |
65d898779138d23161ce552c | 43 | Comparing the relative rates of preferred pathways for exchanging either H to D or D to H in the glycyl radicals (k S H/D of 2 s -1 vs k R D/H of 0.29 s -1 ), we obtain a ratio of 6.7, suggesting a slightly faster rate of deuterating the glycyl radical in D2O, compared with re-protonation in H2O. |
65d898779138d23161ce552c | 44 | The calculations for the E:P complex indicate differences of only 4 kJ/mol or less from the respective energy barriers of the E:S complex (Tab. 2, S6). Therefore, the pattern of favorable and unfavorable H or D transfer reactions is identical to that of the E:S complex with only minor differences in the ratios of the respective rates. In particular, the difference of the energy barriers for the transfer of D from Cys-SD to GlyH . between the pathways via the reand si-face is 26.4 kJ/mol which translates to a 34,200 faster rate of the former process and a very high enantioselectivity (Table ). As in the case of the E:S complex, the kinetic analysis reveals that the exchange of protium to deuterium in the glycyl radical via S-Gly829 (k S H/D) is almost 11,500 times higher (1.6 s -1 ) than via R-Gly829 (k R H/D = 1.4*10 -4 s -1 ), while in the backward exchange of deuterium to protium, the process via R-GlyHD is preferred (k R D/H = 0.26 s -1 ) compared to that via S-GlyDH (k S D/H = 5.7*10 -6 s -1 ). Similarly, as in the case of the E:S complex, the estimated rate of enzyme deuteration in D2O turns out to be 6.2 times higher than for backward protonation of the deuterated enzyme in H2O. |
65d898779138d23161ce552c | 45 | In the apoenzyme, the situation is seemingly reversed than in the case of the E:S and E:P complexes. Our calculations indicate a preference for H transfer to the si over the re face of the glycyl radical (barriers of 80.6 kJ/mol vs 107 kJ/mol), which would indicate an almost 40,000 faster rate of S-GlyDH formation (Table ). However, we have to remember that those barriers are calculated with respect to the E apo conformational states, which were derived from an internal reaction coordinate scan started at the si or re-oriented TS apo . As a result, the conformational changes enforced during the optimization of the TS may persist at the E apo or I apo states. It turned out that the energy of the proS conformer of E apo is 86.8 kJ/mol higher than that of the proR conformer. Such a situation was not observed in the case of the E:S and E:P models where the energy differences between the respective reference states were minimal. |
65d898779138d23161ce552c | 46 | Therefore, it should be concluded that, based on our calculations, any H/D exchange between Cys493 and Gly829 of the apoenzyme would be highly enantioselective and would not easily exchange the H atom at Gly along the depicted mechanism. The predicted kinetic constants (7.3*10 -7 vs. 1.6*10 -18 s -1 ) show that the favored H/D exchange pathway in apo-BSS proceeds through R-GlyHD, regardless whether the barriers of the proS process need to be increased by 86.8 kJ/mol because the higher energy level of this conformant. However, even the favored H/D exchange pathway in apo-BSS would be 2.67*10 6 -fold slower than that in the E:S or E:P complex. This equals to one predicted exchange per 378 h, practically precluding H/D exchange in the absence of bound substrate or product. |
65d898779138d23161ce552c | 47 | we noticed that the signal changed almost completely from a protium-to a deuteriumcontaining glycyl radical upon changing the solvent from H2O to D2O, but appeared to revert only partially when the buffer was changed back to H2O, resulting in a mixed signal (Fig. ). Aromatoleum toluolicum was used, either as prepared (blue), after exchanging the solvent to D2O (green), and after exchanging it back to H2O (black). The red curve approximates the black spectrum by adding the blue and green spectra at a ratio of 0.44:0.21, which fits well to a 40% loss of radical content in this sample as calculated by spin quantitation. Note that the amplitude-based 68:32 ratio shown here is converted to an 80:20 ratio when analyzed based on the spectrum integrals. |
65d898779138d23161ce552c | 48 | We quantitated the contributions of the spectra of the protium-and deuterium-containing enzymes on the basis of the integrated original spectra, indicating that 80% of the active BSS molecules had reverted to the protium-state, while 20% still contained the deuterated glycyl radical (Fig. red curve). Since the buffer exchange procedures lasted approximately 1 h in either direction, this observation correlates well with the postulated faster rate of exchanging protium to a deuteron at the glycyl radical than that of the reverse process. In this respect, it is noteworthy that the EPR experiments on BSS or PFL were always performed in cell extracts that contained substrates or products, while EPR on ARNR was performed in the presence of formate but in the absence of nucleoside triphosphates . |
65d898779138d23161ce552c | 49 | The calculated energy barriers for H/D exchange in E:S, E:P and apo BSS indicate that the exchange from protium to deuterium in the glycyl radical occurs at an approximately 7-fold higher rate in E:S and E:P than the backward exchange of the deuterium to the protium. In the apoenzyme, we calculated the inversed situation, with a 4.3-fold slower rate of protium to deuterium exchange compared to the reversed process. However, if the rates of E:S and E:P and apo are compared we find very similar rates for E:S and E:P complexes but the calculated rates for the apoenzyme are more than five orders of magnitude slower. These findings are in qualitative agreement with the results of isotope exchange experiments monitored by EPR spectroscopy. If we assume that the time needed for preparation only allowed for partial exchange of deuterated glycyl radical with protium we would expect full exchange of protium by deuterium because of the higher rate in the case of E:S or E:P complexes. In the case of apo BSS the calculated rate was so low that any H/D exchange would not be observable in reasonable time. |
65d898779138d23161ce552c | 50 | To gain insight into the rate of H/D exchange at the Cyr493 SH group we have conducted two types of isotope-labelled experiments. First, we followed what products were formed in the reaction of fumarate with d8-toluene in H2O, detecting d8-benzylsuccinate and d7benzylsuccinate by MS. If no H/D exchange occurred at the SH group, we would expect only the d8-product to be formed. The rate of formation of the d7-product (with one deuteron from toluene replaced by a proton from the reaction environment) would reveal the rate of SD/SH exchange during the reaction. We indeed detected the simultaneous appearance of both products i.e., d8-and d7-labelled benzylsuccinate. The specific activity of d8-benzylsuccinate formation turned out to be 2. We may assume that the enzyme changes its confirmation upon product release, so the relative energy change and the equilibrium shifts back to that of the glycyl radical state. This may help to safeguard the enzyme from accidental proteolysis, oxygen-dependent cleavage or other side-reactions. |
65d898779138d23161ce552c | 51 | (iii) Forward and backward reactions of the same transition are not equivalent in any of the full enzyme models, due to stabilization of radical cysteine state in holoenzyme. Rather we observe usually very different TS energies for these processes, which may help in guiding the reaction pathway of the overall reaction of BSS. |
65d898779138d23161ce552c | 52 | (iv) All states, apo-BSS, the E:S and E:P complexes, exhibit strong enantioselectivity for the hydrogen transfer process between Gly829 and Cys493. In the substrate-or product-bound states, the re side of the radical Gly829 is more prone to attack by Cys493, but the attack from the si side is still kinetically feasible. |
65d898779138d23161ce552c | 53 | (v) Isotope effects are predicted to be significant for exchanging the protium atoms of the glycyl radical, resulting in an easier H/D exchange process at Gly of the protium by deuterium than vice versa. Combining these calculations with those on the enantioselectivity of the reaction we predicted that BSS should have a low, but detectable isotope exchange activity with the product benzylsuccinate and provided an explanation for the observed H/D exchange of the glycyl radical. |
65d898779138d23161ce552c | 54 | We show experimental support for the predicted H/D transfer reactions in the product-bound state of BSS, since the enzyme catalyzes H/D exchange at the carboxymethyl side chain of the product benzylsuccinate (C3 atom). As expected, this reaction depends on the active, radicalcontaining state of BSS and even proceeds sequentially to exchange both protium atoms of C3 of benzylsuccinate. In addition, we also identified a D/H exchange activity during the BSS reaction with d8-toluene by confirming small amounts of d7-benzylsuccinate as a byproduct of the abundant d8-benzylsuccinate, which confirms H/D exchange at Cys493. |
65d898779138d23161ce552c | 55 | The kinetically favored H-transfer pathways between Cys493 and Gly829 in either model result in the retention of the same H-atom in the glycyl radical, contradicting the experimental evidence. Therefore, an occasional kinetically unfavorable transfer step to the si-side needs to occur, which leads to the observed H/D isotope exchange reactions of the glycyl radical. The same principles applied for the H/D-transfer reaction in substrate-and product-bound enzyme, while in contrast, the calculated rates for apo-BSS suggest that neither H-transfer to nor H/D exchange of the glycyl radical should be observable in a reasonable time frame because of their slow kinetics. |
65d898779138d23161ce552c | 56 | This predicted behavior indeed fits the respective experimental conditions since all EPR experiments with BSS or other FAE to date were conducted in cell extracts , which always contain enough product and leftover substrates to saturate the enzymes (e.g. from the growth of the cells on toluene). This suggests that the observed change of the glycyl radical signal in EPR spectra upon incubation in D2O is facilitated by the binding of either the substrates of the product. This would allow BSS to attain a conformation that exhibits lower H transfer barriers from Cys493 to the glycyl radical and speeds up the H/D exchange process. |
65d898779138d23161ce552c | 57 | Our calculations also indicated that the exchange of H to D in the glycyl radical of BSS (in D2O) occurs significantly faster than the reverse exchange of D back to H (after changing D2O back to H2O). This prediction is consistent with the observed complete exchange of H to D in the glycyl radical within the time needed for sample preparation, while the assumed 6.4-fold slower reverse exchange of D to H was apparently still ongoing after the same preparation time. |
65d898779138d23161ce552c | 58 | Looking at other GRE for which H/D exchange experiments have been reported, the samples of purified and in-vitro-activated PFL contained 2 mM substrate or substrate analog (pyruvate or oxamate) as a necessary component of the activation reaction . Moreover, the covalently bound pyruvate in PFL is attached at Cys418, enabling the presence of bound substrate during the Cys419-mediated H/D exchange at the glycyl radical C atom . In contrast, ARNR was essentially assayed in the apoenzyme state, i.e. with only formate added as a cosubstrate for the activation reaction, but without nucleotide triphosphate, which was correlated with the absence of observable H/D exchange . |
65d898779138d23161ce552c | 59 | In this study, we show by computer modelling that activated BSS containing a radical on Gly829 (1) requires bound substrates or product to enable H-transfer or H/D exchange between Cys493 and the glycyl radical while these reactions are precluded in apo-BSS; retains the H-or D-atoms of the glycyl radicals when acting with the preferred stereospecificity, but (3) is able to initiate H/D exchange into the glycyl radical at a slow rate (as well as even slower reverse D/H exchange) by occasional reactivity in the non-preferential stereospecifity, together with (4) some experimental support for these predictions. The behavior of the enzyme reported here may be valid for all GRE, indicating that substrate or product binding may be a prerequisite for both H-transfer between the active site Gly and Cys residues and the reactions involved in H/D exchange of the glycyl radical. Since BSS has so far been only investigated in the naturally activated state in cell extracts while in-vitro activation is difficult and has only recently been accomplished , a broader data basis with biochemically better accessible GRE will be very helpful to further investigate the alleged requirements proposed in this study. |
65d898779138d23161ce552c | 60 | The Supporting Information contains details on experimental procedures (EPR, LC-MS/MS, MD, QM:MM), RMDS curves from MD simulations, distribution histograms of C-C distances between Gly928 and Cys493, energies and vibrational corrections of all stationary points, differences in energy corrected with thermal corrections calculated at all levels of theory, values of kinetic constant rates and intrinsic kinetic isotope effect, figures of the active site for all stationary points, auxiliary product ion MS spectra of benzylsuccinate standard and details on fragmentation pattern analysis for deuterated benzylsuccinate as well as non-standard AMBER files used for MD simulation (radical Gly, radical Cys, substrates and product) and PDB files of all stationary points. |
63d56c13ae221ab9b240932f | 0 | Chat GPT is a state-of-the-art language model that is developed by OpenAI. It is a deep neural network that has been trained on a large corpus of text, which enables it to generate humanlike responses to a wide range of prompts. Chat GPT utilizes the transformer architecture, which allows it to handle long-term dependencies and context in its language generation. It can be finetuned to specific tasks such as language translation, question answering and text completion, and can generate human-like text for various applications. Chat GPT is a notable example of the advancement in natural language processing and machine learning. Since, it has been trained by up till 2021, it processes the user questions and try to give us good information back on that topic. Hence the information provided by Chat GPT is not novel it will just provide the information uptill 2021. Drug discovery is the process of identifying and developing new medications to treat disease. It is a complex, multi-disciplinary field that involves various steps such as target identification, lead discovery, preclinical development, clinical trials, and regulatory approval. The process typically starts with the identification of a biological target that is involved in a disease process. Researchers then use various techniques such as high-throughput screening, computational chemistry, and structure-based drug design to identify small molecules or biologics that can interact with the target and modulate its activity. These lead compounds are then subjected to preclinical testing in various animal models and cell lines to evaluate their efficacy and safety before moving on to clinical trials in humans. The goal of drug discovery is to identify safe and effective treatments for a wide range of diseases. Computational based drug discovery process if shown in Figure . Drug discovery using computational chemistry is the application of computer-based methods to assist in the discovery of new medications. It involves the use of computer simulations and modeling techniques to predict the properties and interactions of potential drug molecules. These methods can be used to understand the structure and behavior of proteins and other biological targets, and to design and optimize new compounds that can bind to these targets and modulate their activity. Computational chemistry plays a vital role in modern drug discovery by providing a cost-effective and efficient way to screen large numbers of compounds, predict their potential efficacy and toxicity, and identify new lead compounds for further development. It also enables researchers to design new drugs that are more selective and have fewer side effects, as well as to understand the mechanisms of drug action at the molecular level, and to optimize the pharmacokinetics and pharmacodynamics of drugs. Drug discovery is a very difficult and complex process for several reasons: (1) Biological complexity: The human body is an incredibly complex system, and understanding how it works at the molecular level is difficult. Diseases are caused by a variety of factors, and finding a single drug that can treat a disease effectively is challenging; (2) High failure rate: The majority of drugs that enter clinical trials fail to be approved. This is because they are found to be ineffective, or they have unacceptable side effects during testing; (3) Time-consuming and expensive: The drug discovery process is long and costly. It can take up to 15 years and cost billions of dollars to bring a new drug to market; (4) Stringent regulatory requirements: The process of drug development is heavily regulated, and drugs must go through a series of preclinical and clinical trials before they can be approved for use; (5) Unpredictable nature of drug development: Even after a drug has been identified and developed, it's not certain that it will work as expected in human trials, sometimes it can have unexpected side effects; (6) Lack of new targets: Many of the known drug targets have already been exploited, and finding new targets is becoming increasingly difficult. Despite these challenges, researchers continue to work towards developing new and effective drugs. Advances in technology and computational chemistry, as well as a better understanding of disease biology, are helping to make the drug discovery process more efficient and cost-effective. In the current work, we have explored the usage and limitation of Chat GPT in drug discoveries. |
63d56c13ae221ab9b240932f | 1 | gave the correct answer. The answer of Chat GPT is as follows: "Zinc is a transition metal with the atomic number 30, it has 30 electrons in its electronic configuration. The electronic configuration of Zn is [Ar] 3d^10 4s^2. Since all the electrons are paired in the orbitals, the multiplicity of Zn is 1 (singlet). This means that all the electrons in the HOMO are paired and the molecule has no unpaired electrons." Therefore, Chat GPT can be used to determine multiplicity of various compounds in quantum mechanics calculations using density functional theory (DFT). |
63d56c13ae221ab9b240932f | 2 | Chat GPT successfully determine the smiles of Tylenol (Acetaminophen), hence it can be used to covert one file format to another. The Chat GPT answer was "The chemical name of Tylenol is Acetaminophen and its molecular formula is C8H9NO2, its Simplified molecular-input line-entry system (SMILES) notation for Tylenol is "CC(=O)NC1=CC=C(C=C1)O". This notation represents the atoms and their connections in the molecule." 3. Generation of input files for gaussian and other software: Chat GPT successfully produces the input file for gaussian software. The input file is shown in Figure . 6. Can give FASTA sequence but cannot compare the FASTA sequence: In this case ChatGPT was unable to suggest the FASTA sequence. However, to suggests that the fasta sequence can be obtained from the UniProt database. |
63d56c13ae221ab9b240932f | 3 | 11. Machine learning and data analysis: it can be integrated into a program or application using its API, which allows developers to input text and receive a response generated by the model. The API can be accessed using programming languages such as Python, JavaScript, and C#. Additionally, the OpenAI team has also released a pre-trained version of the model, which can be fine-tuned for specific tasks using a process called transfer learning. |
63d56c13ae221ab9b240932f | 4 | 1. Identifying and validating new drug targets: ChatGPT can be fine-tuned on a dataset of scientific literature and used to generate summaries of the latest research on a given disease or biological target. This can help researchers quickly identify new potential targets or gain a better understanding of the current state of research in a specific area. |
63d56c13ae221ab9b240932f | 5 | ChatGPT can be fine-tuned on a dataset of drug-related papers and used to generate reports and papers that summarize the current state of research in a specific area. ChatGPT can be fine-tuned on a dataset of known druglike molecules and used to generate new chemical structures with similar properties. This can help researchers identify new lead compounds that have a higher chance of success in pre-clinical and clinical studies. |
63d56c13ae221ab9b240932f | 6 | Although, it has various advantages it cannot perform extensive scientific calculations like computing RMSD, RMSF, PCA, clustering etc. For these complexes work a trained and experience human involvement is required. It's important to note that ChatGPT is just one tool among many that are used in drug discovery, and it is not a substitute for experimental validation and clinical trials. However, by providing a cost-effective and efficient way to process large amounts of data and generate new knowledge, ChatGPT can assist researchers in making more informed decisions and accelerate the drug discovery process. It's important to note that ChatGPT is just one tool among many that are used in drug discovery, and it is not a substitute for experimental validation and clinical trials. However, it can significantly speed up and improve the drug development process by providing a cost-effective and efficient way to process large amounts of data and generate new knowledge. |
63d56c13ae221ab9b240932f | 7 | 1. Reliance on the quality and availability of data: ChatGPT is only as good as the data it has been trained on. If the data is incomplete, biased, or inaccurate, the model's predictions may not be reliable. 2. Lack of experimental validation: ChatGPT can generate predictions and hypotheses, but it cannot perform experiments or measure the properties of compounds. Therefore, the predictions made by the model need to be validated experimentally. 3. Limited understanding of the underlying biology: While ChatGPT can generate humanlike text, it does not understand the underlying biology of the systems it is simulating. Therefore, the predictions made by the model may not always reflect the true complexity of the systems. 4. Limited interpretability: ChatGPT, like other machine learning models, can be difficult to interpret, and it's not always clear how the model arrived at a particular prediction. 5. Limitations in handling uncertainty: ChatGPT is a deterministic model, it cannot account for the uncertainty in the data and predictions. 6. Lack of transparency: ChatGPT is a black box model, it is difficult to understand and explain the internal workings of the model, which can make it difficult to trust the model's predictions. |
63d56c13ae221ab9b240932f | 8 | In conclusion, ChatGPT is a powerful language model that can assist in the field of drug discovery. By processing and generating human-like text, it can help researchers quickly identify new potential targets, gain a better understanding of the current state of research, design new drugs, and optimize the pharmacokinetics and pharmacodynamics of new drugs. The model can be finetuned on specific datasets and used to generate new knowledge that can support decision-making in early-stage drug discovery. However, it's important to note that ChatGPT is just one tool among many that are used in drug discovery, and it is not a substitute for experimental validation and clinical trials. In addition, it failed to perform complex computational calculations like simulating and analyzing the molecular level studies. A major question is any of this is ethical? Or is it asking someone to write your thesis? The type or amount of work ChatGPT can do right now is equal to All the questions I asked ChatGPT is either enhancing what I have already done, or it is giving you starting point where you still need to do more to complete the research. The work done by ChatGPT in this paper in unethical to do and the answers are already available on the internet. However, I would say that ChatGPT make my life easier by giving a starting point. I strongly believe as technology gets better, ChatGPT will be a valuable tool in helping us becoming more efficient in our research. Nevertheless, the use of ChatGPT in drug discovery is a promising area of research, as it has the potential to significantly speed up and improve the drug development process. |
67307a8b7be152b1d09c106e | 0 | Compounds of the group 13 elements boron, aluminium and gallium are routinely employed as Lewis acids in catalysis. The high fluoride-ion affinities of three-coordinate group 13 compounds underpin numerous applications that break and functionalise strong carbon-fluorine bonds. For example, BF3-OEt2 and AlCl3 have been reported as catalysts for the Friedel-Crafts alkylation of arenes with fluoroalkanes. 2 B(C6F5)3 has been shown to catalyse the hydrodefluorination of fluoroalkanes using Et3SiH as a terminal reductant. Similar reactivity has been achieved using aluminium chloride fluoride (ACF), a heterogenous catalyst proposed to contain highly Lewis acid active sites based on aluminium. Aluminium compounds are also competent reagents and catalysts for carbon-heteroatom and carbon-carbon from fluoroalkanes. In the past few years, Frustrated Lewis Pair (FLPs) catalysts based on group 13 compounds have been applied to highly selective catalytic transformations that allow the controlled functionalisation of a single carbonfluorine bond of sp 3 CF3 or sp 2 CF2H groups. |
67307a8b7be152b1d09c106e | 1 | In contrast, the use of nucleophilic group 13 compounds as catalyst for carbon-fluorine bond functionalisation is less common. It might be expected that by increasing the coordination number of a group 13 compound from three to four-coordinate that its Lewis acid behaviour could be tempered and nucleophilic reactivity of the coordinated ligands exposed (Figure ). The switch in electronic behaviour might be expected to expand scope of accessible substrates for catalytic transformations, as Lewis acid catalysts operate primarily on sp 3 C-F bonds, whereas nucleophilic species would be capable of addition to sp 2 C-F bonds. This hypothesis is largely untested. |
67307a8b7be152b1d09c106e | 2 | In the past 10 years, our research group have developed several new reactions that lead to the generation of well-defined four-coordinate aluminium fluoride complexes as products. More recently, we became interested in the use of these complexes as potential catalysts for the functionalisation of carbon-fluorine bonds. Here we show that molecular fluoride complexes of boron, aluminium and gallium are competent catalysts for the thiodefluorination and hydrodefluorination of electron-deficient arenes and alkenes. We report two defined approaches to catalysis, (i) an additive free reaction and (ii) a catalytic protocol that relies on the use of NaBAr F 4 (Ar F = 3,5-C6H3(CF3)2) as an additive. Experimental and computational data is provided to support the formation of nucleophilic four-coordinate complexes as key intermediates, and metal fluoride species as on-cycle species. Furthermore, we suggest that the additive plays a unique role in polarising and activating metal-fluorine intermediates through formation of weak M-F----Na interactions. Our findings complement existing uses of compounds based on main group elements such as tetrabutylammonium difluorophenylsilicate (TBAT), tetrabutylammonium fluoride (TBAF), or CsF as initiators for carbon-fluorine bond functionalisation. |
67307a8b7be152b1d09c106e | 3 | Additive-Free Thiodelfuorination of Arenes: A series of four-coordinate group 13 difluoride complexes 1-B, 1-Al and 1-Ga supported by a sterically demanding b-diketiminate ligand was prepared. Initial experiments were conducted to screen a series of conditions for the reaction with pentafluoropyridine with group 13 catalysts using silanes as the terminal reductant. 1-B, 1-Al and 1-Ga were found to be active catalysts for the thiodefluorination of electron-deficient arenes with silicon-based reagents at 10-20 mol% loading, between 60 -160 °C, using a,a,a-trifluorotoluene as a solvent (Figure ). Using Me3Si-SPh as a terminal reagent, pentafluoropyridine could be selectively converted into 2a. A control reaction showed a maximum of 10% conversion for this transformation after 24 h at 100 °C, when carried out in the absence of a catalyst. Focusing on thiodefluorination, a scope of electron-deficient fluorinated aromatics was investigated. A series of substituted perfluoroarenes could be selectively converted into the monosubstituted products 2b-2h. Under more forcing conditions higher levels of substitution of pentafluoropyridine and perfluorotoluene could be achieved (see supporting information for details). Hexafluorobenzene reacted selectively giving 2i the product of 1,4-disubstitution. Similarly, P(C6F5)3 underwent a selective tri-substitution under catalytic conditions to yield 2j which could be crystallographically characterized (Figure ). 2j holds promise as a novel ligand for applications in catalysis. 2-(Perfluorophenyl)pyridine underwent non-selective thioylation to form isomers of 2k, primarily at the 2-and 4-position of the perfluorophenyl ring. This finding suggests that the pyridyl directing group has little control on the reaction arguing against coordination of the substrate to the catalyst expected during Lewis acid catalysts. An electron-deficient poly-fluorinated terphenyl underwent non-selective single and double substitution to form 2l and 2l'. Hexafluoropropene is also reactive under the catalytic conditions, providing a mixture of stereoisomers 2m:2m' from thioylation of the terminal sp 2 C-F bonds along with a side-product 2m" derived from protonation, likely from adventitious water. |
67307a8b7be152b1d09c106e | 4 | Qualitatively, it was found that the catalysts were more active across the series 1-B > 1-Al > 1-Ga. With the heavier group 13 catalysts requiring more forcing conditions and showing more limited scope than the lightest boron analogue. Reaction trends are consistent with a nucleophilic mechanism with regioselectivity determined by the most electrophilic position of the aromatic ring. In contrast to known Lewis acid catalysts based on group 13, these reactions are selective for sp 2 C-F over sp 3 C-F bonds with CF3 groups being tolerated under catalytic conditions. Less electron-deficient arenes, i.e. those with lower fluorine content, do not react under the optimised conditions. |
67307a8b7be152b1d09c106e | 5 | To better understand the role of the catalyst, in two separate experiments, 1-B and 1-Al were reacted with 3 equiv. of Me3Si-SPh. While in the case of 1-B no apparent reaction occurred, 1-Al reacted with Me3Si-SPh after 48h at 100 °C in C6D6 to produce a 3:1 mixture of 3-Al and 4-Al along with Me3Si-F as a side-product (Scheme 1). The latter was readily apparent from a [a] NMR yields measured by 19 F NMR spectroscopy using 1,2-difluorobenzene as an internal standard. Isolated yields in parentheses. Reactions run using 3 equiv. of Me3Si-SPh. were not active catalysts for the reaction of Et3SiH with pentafluoropyridine under the catalytic conditions established for Me3Si-SPh. Following an extensive screen of conditions and additives it was found that 10 mol% NaBAr F could be used as an additive to effectively promote the reaction. |
67307a8b7be152b1d09c106e | 6 | Under the optimised conditions, pentafluoropyridine could be converted into 2,3,5,6tetrafluoropyridine 5a in 99 % using 10 mol% 1-Al + 10 mol% NaBAr F 4 as a pre-catalyst mixture and 10 equiv. of Et3SiH as the terminal reductant. Control reactions using solely 1-Al or NaBAr F 4 led to minimal conversion, similarly NaBPh4 was not an active additive under these conditions. |
67307a8b7be152b1d09c106e | 7 | Qualitatively, 1-Al and 1-Ga appeared to be more active catalysts than 1-B. The scope of hydrodefluorination was investigated. A series of electron-deficient aromatics including nitro, nitrile, ester, and acid functional groups underwent hydrodefluorination with more forcing conditions and higher loadings required for less electron-deficient substrates (5b-5n). Fluorinated alkenes such as perfluorocyclopentene and hexafluoropropene were also reactive and could be transformed into hydrodefluorination products 5o:5o' and 5p:5p' respectively in reasonable yields (Figure ). |
67307a8b7be152b1d09c106e | 8 | A stoichiometric reaction between 1-Al and NaBAr F 4 in fluorobenzene is consistent with an interaction between the two pre-catalyst components (Scheme 2). Hence at 298 K, a 50 mM solution of 1:1 mixture of 1-Al and NaBAr F 4 displayed a defined and sharp F NMR resonance at d = -180.2 ppm. For comparison, 1-Al alone demonstrates a resonance at d = -174.5 ppm at the same concentration. Varying the ratio of NaBAr F 4 : 1-Al from 0.5:1 to 2:1 suggests that the binding event is 1:1 as the maximum Dd » 6ppm is observed at this ratio with only small changes in chemical shift occurring at higher ratios. The reaction between 1-Al and NaBAr F 4 was investigated at nine different temperatures across a 233 -313 K temperature range, with consistent results supporting 1:1 binding at all temperatures (Figure ). No attempts were made to quantify this data as it is likely that the equilibria at play are complicated by the potential for the solvent, fluorobenzene, to act as a competitive ligand for Na + . A similar reaction between a 1:1 mixture of Transition metal fluoride complexes are known to be effective hydrogen bond (and halogen bond donors), examples of coordination of metal fluorides to s-block cations are however, less common. DFT calculations were undertaken to understand the nature of the binding event. |
67307a8b7be152b1d09c106e | 9 | Coordination of NaBAr F 4 to 1-Al was calculated to be exergonic by DG°(298K) = -1.4 kcal mol -1 consistent with a reversible binding event. Binding was calculated to occur through a chelating k 2mode in which Na + interacts with lone-pairs on the two adjacent fluoride ligands of 1-Al. A similar coordination geometry and exergonic reaction DG°(298K) = -2.9 kcal mol -1 was calculated for reaction of NaBAr F 4 with 1-Ga (Figure ). NBO and QTAIM calculations were used to better understand the changes of electronic structure comparing 1-Ga-NaBAr F 4 to 1-Ga. Calculated Ga-F bond lengths in 1-Ga are ca. 0.03 Å shorter than in 1-Ga-NaBAr F 4. The NPA charges on the fluoride ligands become more negative when comparing 1-Ga (-0.67,-0.67) to 1-Ga-NaBAr F 4 (-0.69,-0.70), this is perhaps expected and would be consistent with the binding of Na + effecting a polarisation of the Ga-F bonds. Second-order perturbation analysis within the NBO framework reveals donor-acceptor interactions between 2p LP on F and the empty 3s orbital of Na + . QTAIM calculations return bond paths between Ga and F atoms along with F and Na atoms in 1-Ga-NaBAr F 4. Comparison of the r(bcp) values for bond critical points connecting Ga and F between 1-Ga (0.11 and 0.11 e bohr -3 ) and 1-Ga-NaBAr F 4 (0.10 and 0.11 e bohr -3 ) is consistent with similar electron-density between these atoms on coordination. r(r) and Ñ 2 r(r) values for bond critical points between Na and F atoms in 1-Ga-NaBAr F 4 support the idea that the binding interaction is primarily electrostatic (Figure ). Based on the analysis, it is possible that the increased 2 JH-F coupling constant for 6-Ga-NaBAr F 4 compared to 6-Ga might be due to a lower p-character (and hence increased s-character) for the main group fluoride bond in the Ga-F---Na interaction compared with Ga-F. |
67307a8b7be152b1d09c106e | 10 | A plausible reaction mechanism for the hydrodefluorination of fluoroarenes involves (i) s-bond metathesis between Et3SiH and the group 13 fluoride 1-M forming the group 13 hydride complex and (ii) nucleophilic attack of the hydride on the fluorinated arene by a cSNAr mechanism, liberating the hydrodefluorinated product and regenerating the group 13 fluoride (M= Al, Ga). We have previously shown that a b-diketiminate stabilized aluminum dihydride complex is an effective reagent for the hydrodefluorination of electron-deficient arenes under forcing conditions. Further experiments and calculations were undertaken to shed light on the role of NaBAr after 6 h at 80 °C (Figure ). The expected kinetic product of this reaction 6-Ga was shown to be in equilibrium with 1-Ga and 7-Ga at 25 ºC in fluorobenzene (figure ). DFT calculations support a role of NaBAr F 4 lowering the barrier for metathesis through fluoride coordination (Figure ). |
67307a8b7be152b1d09c106e | 11 | Reaction of 1-Ga-NaBAr F 4 with Et3SiH was calculated to occur through TS-1 (DG ‡ 298K = 28.8 kcal mol -1 ) leading to direct formation of 6-Ga-NaBAr F 4. In the absence of the promoter, 1-Ga was calculated to react with Et3SiH by a higher barrier through TS-1' (DG ‡ 298K = 30.5 kcal mol -1 ) to form 6-Ga. The lower energy of TS-1 relative to TS-1' is explained through Na + playing a role in charge stabilization during the polarisation and breaking of the Ga---F bond. Experimentally it was found that 7-Ga is an effective nucleophile for the hydrodefuorination of pentafluoropyridine at 100 ºC, and that 10 mol% 7-Ga + NaBAr F 4 can be used as a pre-catalyst for the hydrodefluorination of the same substrate with Et3SiH at 160 ºC (Figure ). |
67307a8b7be152b1d09c106e | 12 | In summary, a series of four-coordinate group 13 fluoride complexes (M = B, Al, Ga) are reported as catalysts for the thiodefluorination and hydrodefluorination of poly-and perfluoroarenes using silanes as terminal reagents. The scope of reactivity in the electrophile is broad, provided the aromatic ring is electron deficient. For thiodefluorination, mechanistic studies support a plausible role for group 13 thiolate intermediates which are capable of C-S bond formation through nucleophilic attack on the aromatic ring. Similarly, for hydrodefluorination, group 13 hydrides are likely key catalytic intermediates. In both cases, catalytic turnover requires metathesis (ligand exchange) between the group 13 complex and silicon reagent; a step that requires breaking of strong group 13 metal-fluoride bonds. While in the case of thiodefluorination RS --to-F -metathesis occurs in the absence of an additive, for hydrodefluorination H --to-F -exchange requires the use of NaBAr F 4 as an additive. Stoichiometric experiments and DFT calculations are consistent with NaBAr F 4 playing a role to activate and weaken the group 13 metal-fluoride bond through a M-F---Na + , facilitating metathesis with silane reagents. This additive effect may have important implications for future catalyst design. |
6239e779658bc03404b403df | 0 | All isobutyl phenylcyanoacrylates (IPCA) compounds were synthesized by Knoevenagel condensation of appropriate benzaldehydes with isobutyl cyanoacetate, catalyzed by base, piperidine (Scheme 1). The preparation procedure was essentially the same for all the monomers. In a typical synthesis, equimolar amounts of isobutyl cyanoacetate and an appropriate benzaldehyde were mixed in equimolar ratio in a 20 mL vial. A few drops of piperidine were added with stirring. The reactions was allowed to proceed 48 hrs at r. |
6239e779658bc03404b403df | 1 | Copolymers of the styrene (ST) and the IPCA compounds, P(ST-co-IPCA) were prepared in 25-mL glass screw cap vials at ST/IPCA = 3 (mol) the monomer feed using 0.12 mol/L of ABCN at an overall monomer concentration 2.44 mol/L in 10 mL of toluene. The copolymerization was conducted at 70ºC. After a predetermined time, the mixture was cooled to room temperature, and precipitated dropwise in methanol. The composition of the copolymers was determined based on the nitrogen content. The novel synthesized IPCA compounds copolymerized readily with ST under free-radical conditions (Scheme 2) forming white flaky precipitates when their solutions were poured into methanol. The conversion of the copolymers was kept between 10 and 20% to minimize compositional drift (Table ). |
6352afdeecdad55d48e71510 | 0 | Escherichia coli (E. coli) are a group of bacteria, which are a natural part of the intestinal flora of warm-blooded animals, including humans. Most E. coli are non-pathogenic and are essential for the normal function of a healthy intestine . However, there are also pathogenic types of E. coli such as Shiga toxin-producing E. coli (STEC), also called Verocytotoxin-producing E. coli (VTEC). Their virulence is associated mainly with the production of two cytotoxins: stx1 and stx2., whose mode of action involves inhibition of protein synthesis in the target cell . Abdominal cramps, (bloody) diarrhoea, and sometimes fever and vomiting are the most common symptoms of STEC infection but in severe cases infection may lead to the development of haemolytic uremic syndrome (HUS) which is a life-threatening disease . Although the prevalence of STEC infections in The European Union is lower compared with Salmonella spp. and Campylobacter spp , it remains a high risk to public health due to its low infectious dose (1-100 CFU ), its ability for survival and transmission in the agri-food environment, and the high mortality rate (1.2 % for O157 and O104 serotypes and between 0.1%-0.2% for other serotypes )) compared to other food-borne pathogens . Ruminants, especially cattle are considered to be the most important reservoir of STEC and the infections occur from direct or indirect contamination in the agri-food chain . |
6352afdeecdad55d48e71510 | 1 | Detection of generic E. coli, is used as an indicator organism of faecal contamination in potable water and food production , while detection of specific types of pathogenic E.coli, such as STEC, are important to assure a safe agri-food supply . It is also a regulatory requirement for certain foods in some countries, and/or may be required for export market access, e.g., such as red meat entering the USA (USDA, 2020). The Regulation (EC) No 2073/2005 sets the microbiological criteria for certain microorganisms, including general E. coli and STEC, along with the rules for compliance in the food industry. The acceptable limit for E. coli varies based on the food category, for instance the maximum for shellfish is 10 Colony Forming Unit (CFU)/g, for butter and cream made from unpasteurised milk it is 100 CFU/mL, and for minced meat it is 500 CFU/mL. The microbial criteria for STEC are only set for sprouts and they require its absence in 25 g of the product during its whole shelf life. In addition, based on the WHO guidelines for drinking water, no E. coli should be detected in 100 mL of water intended for drinking to consider it safe . Thus, such strict bacteria requirements illustrate the need for a highly sensitive (1-100 CFU/mL) and rapid detection methods which could be applied at each step of food production, to allow for early detection of pathogens in the agri-food chain. |
6352afdeecdad55d48e71510 | 2 | In the past, detection of STEC was mainly based on the somatic, O polysaccharide antigen, and selecting the pathogens of concern was based on epidemiological studies . However, after the biggest STEC outbreak reported in Germany in 2011, it was concluded that the pathogenicity assessment should be focused on molecular detection and all isolates containing any of the Shiga toxin coding gene (stx) may be able to cause severe infection in humans (EFSA Panel on . This conclusion highlighted the need for quick and reliable methods for nucleic acid-based STEC detection in the agri-food chain. Bacterial detection has advanced significantly in recent years and considerable efforts have been focused on the development of genetic-based techniques, i.e., for methods based on the detection of nucleic acids (DNA or RNA). A method which includes a nucleic acid amplification, the polymerase chain reaction (PCR), is the method of choice for STEC . PCR however requires expensive reagents and equipment; highly trained personnel and sample contamination could lead to the amplification of the non-target sequence leading to false-positive results. For these reasons, it is not feasible to apply such an approach to point-of-care (POC) detection on farms or in the food industry . |
6352afdeecdad55d48e71510 | 3 | Biosensors, analytical devices that translate biological responses into a measurable signals, based on nucleic acid detection have the potential for providing high specificity POC detection of microorganisms . They may allow real-time pathogen detection at each step of food production processes and in situ in food plants or in the field during primary production. This review will focus on nucleic acid-based biosensors using electrochemical techniques as a transducer where a biological response is converted into an electrical signal . The key advantages of electrochemical biosensors are their quick response (voltametric measurements can be done in a few seconds), higher sensitivity when compared to colorimetric techniques , and the possibility for use in realtime detection and suitability for miniaturisation . The disadvantages include a limited shelf life and the impact that variable factors, such as temperature, pH, or ionic strength of the sample, have on the signal, which could limit their use in complex food samples. This can be avoided by electrode miniaturisation which increases the signal-to-noise ratio, or by using multiple enzymes to enhance signal per event . This article reviews the advances in nucleic acid-based electrochemical detection of the E. coli and Shiga toxin producing E. coli (STEC) for agri-food applications. Firstly, issues related to probe sequence selection for generic E. coli and STEC detection are discussed, followed by a review of nucleic acid-based electrochemical sensors reported for detection of generic E. coli and STEC since 2015. These are grouped into different categories based on the most used electrode materials (gold, ITO and screen printed), magnetic particles, nanomaterials modified electrodes and finally sensors using isothermal amplification method. Finally, future trends in the development of commercial nucleic acid-based biosensors and their potential application to agri-food samples are discussed. The methods of how this review was conducted are summarised in the Supporting information. |
6352afdeecdad55d48e71510 | 4 | The selection of a suitable nucleic acid probe sequence for specific target recognition is the first step in the development of an electrochemical nucleic acid-based sensor, and have a huge influence on its specificity . This could also affect the affinity of redox molecules, often used in electrochemical detection. For example, methylene blue has a high affinity towards guanine, therefore a higher concentration of guanine bases on the probe DNA, correlates with a higher signal from methylene blue . The probe sequence could also influence the intensity of an electrochemical signal of the sensor. Table highlights the types of nucleic acid and target genes most used in nucleic acid-based sensors for the detection of general E. coli and STEC. The main challenge in nucleic acid-based specific detection of generic E. coli is its close genetic relationship with Shigella spp., another common pathogen that belongs to the family Enterobacteriaceae. It was estimated that their nucleotide similarity is between 80 and 90% and they are usually differentiated based on their biochemical characteristics. This makes the selection of truly unique and specific primer for E. coli detection a real challenge to avoid a non-specific detection of microorganisms not related to faecal contamination . |
6352afdeecdad55d48e71510 | 5 | Most nucleic acid-based sensors for the detection of generic E. coli have targeted recognition based on RNA, more specifically 16S ribosomal RNA (rRNA) region . 16S rRNA is a part of the 30S subunit of prokaryotic ribosome containing a highly conserved region within the same group of microorganisms, due to its low rate of evolution., However, the use of 16S rRNA to differentiate between E. coli and Shigella spp. is not possible due to the >99% sequence similarity . Therefore, the sensors developed based on 16S rRNA detection are not considered specific for E. coli . The electrochemical sensors developed for the detection of generic E. coli based on DNA recognition have typically focused on targeting the uidA gene responsible for the ß-glucuronidase enzyme . This enzyme catalases the breaking of the glycosidic bond and was reported to be specific for E. coli. However, similarly to 16s rRNA, targeting only this gene would not differentiate E. coli from Shigella spp which can also contain it in its genome . |
6352afdeecdad55d48e71510 | 6 | This gene was also employed for the development of an electrochemical sensor by . The lacZ gene, coding for lactose permease, was employed to develop a sensor for the detection of E. coli in the work of . This gene was previously shown to be specific for E. coli by . Another gene of interest which emerged in the recent years for E. coli detection in water samples is ybbW, encoding a putative Allantoin transporter . Based on extensive research on STEC pathogenicity in the last few years, it was concluded that all E. coli strains containing at least one stx gene coding for toxin production may potentially cause human infection and the nucleic acidbased detection should focus on targeting these genes . Several subtypes of each stx gene exist that were categorised into two groups of genes -stx1 and stx2 and having any subtype of stx in a genome or a combination may be pathogenic. Therefore, the sensors developed by based on one of the stx genes are considered to be the most specific for STEC. |
6352afdeecdad55d48e71510 | 7 | Another gene commonly used in the development of STEC-specific DNA sensors is the eaeA gene which codes for intimin production allowing for bacteria attachment to mucous cells . Recent studies, however, concluded that the presence of this gene is not essential to cause a severe illness, showing that not all STEC causing illness outbreaks had this gene present (EFSA Panel on Biological . Less often, other virulence factors were used such as the rfbE gene coding for an O-antigen synthesis for O157 serotype, shown in the work of . An ideal STEC detection technique should, however, focus on all serotypes which potentially cause the infection. Another virulence gene was chosen by . In their sensor, STEC O157 was detected using the z3276 gene which is known for encoding a unique putative fimbrial protein allowing the adhesion to the host cell. This gene is, however, only specific to STEC O157 serotype as well which means it would fail to recognise other, potentially pathogenic STEC strains. |
6352afdeecdad55d48e71510 | 8 | In summary, choosing the right nucleic acid sequence is crucial for selectivity and specificity in bacterial detection. Most electrochemical sensors developed for the detection of E. coli and STEC to date focused on the detection of a single DNA gene or 16S rRNA sequence. Based on the research, using the selected sequences fails to be specific for the detection of E. coli and STEC. |
6352afdeecdad55d48e71510 | 9 | This problem could be solved by including additional genes in the detection assay to confirm the specificity, such as lacY gene in addition to uidA for the detection of E. coli or stx1 and stx2 genes in addition to eaeA gene for STEC. This highlights the need for the development of multiplex sensors which can enable the detection of multiple genes simultaneously. Alternatively, the focus could be placed on the newly identified genes, such as yaiO found to be specific for the detection of generic E. coli. In the following section, the main parameters used for the development of these biosensors are summarised and discussed. |
6352afdeecdad55d48e71510 | 10 | The main DNA probe attachment approaches have already been explained in a great detail in excellent recent reviews published by , , and and therefore, they will not be discussed herein. In this review, we have classified the sensors for general E. coli and STEC detection into different categories based on the electrode materials (gold, ITO, SPE) while the last section summarises the use of magnetic particles. Several strategies have been developed by researchers to improve performance of the DNA sensors, such as integration of nanostructures, or polymers and miniaturisation of the system. |
6352afdeecdad55d48e71510 | 11 | Gold is one of the most common electrode material employed in electrochemical sensor development due to its good biocompatibility, high conductivity and stability . Easy and effective chemisorption of thiol modified biomolecules such as nucleic acid onto gold surfaces make them very attractive for several sensing applications. , an example of this method has been reported by . In this study, gold electrodes, which were fabricated on a waste newspaper, were used for immobilisation of thiol-modified probe DNA. The surface changes were investigated via electrochemical impedance spectroscopy (EIS) |
6352afdeecdad55d48e71510 | 12 | and an LOD of 1.6 x 10 -8 M was achieved for this label-free technique. Importantly, the researchers demonstrated the selectivity of the sensor with a PCR product from STEC and Salmonella. reported a sensor for the detection of lacZ gene sequence of E. coli using reporter DNA tagged with methylene blue (MB). As in the previous study, the thiol modified probe DNA was immobilized onto a gold surface and the surface and the DNA hybridisation was measured via alternating current voltammetry (ACV). In this study, an LOD of 3x10 -14 M was obtained and the sensor was validated their using synthetic DNA spiked milk, beer, tap water, and peanut milk, in order to demonstrate the pH and ionic strength dependency of the sensor. The sensor was regenerated six times after hybridisation in milk samples by rinsing it with DI water for 60 seconds which may be a promising result for the continuous monitoring. The real application should, however, be also tested using DNA extracted from bacterial cultures or naturally contaminated samples. |
6352afdeecdad55d48e71510 | 13 | A novel approach for improving the sensitivity of nucleic acid-based sensor was proposed by who immobilized a 3D probe DNA nanostructure onto a gold electrode via thiols, see Figure . (A). The nanostructure comprised of four DNA strands specifically designed to form a 3D structure to avoid surface steric hindrance effect without its overcrowding. The detection was undertaken using amperometry and enzymatically labelled reporter. The developed sensor demonstrated an LOD of 10 -15 M with the synthetic DNA and it was applied to detect PCR amplicons of E. coli genomic DNA to assess its selectivity. An alternative probe DNA attachment approach was used by Li et al 2018 who used poly-adenine (poly-A) blocks instead of thiols. This immobilisation method has been previously reported by other authors offering advantages to the thiolated DNA such as lower cost, faster immobilisation and easier way to control probe density by testing different length of poly-A blocks . A different length of poly-adenine blocks was tested with 30 adenine blocks chosen as most optimal. The enzymatic detection using HRP tagged reporter probe achieved an LOD of 5x10 -15 M. The assay was performed in less than 3 hours, including the PCR amplification. modified the gold electrode with mesoporous silica thin films that offer larger surface area and hydroxyl groups content for biomolecules attachment. The heptylamine-phosphoramidite moiet modified probe, complementary to 16s rRNA sequence of E. coli was immobilised on top of the modified electrode and the detection was done using CV in 5 mM Fe -3-/Fe 4-. This method achieved an LOD of 2.5 x 10 -14 M and tests were done with RNA extracted from the E. coli bacterial culture in around one hour, including the RNA extraction. |
6352afdeecdad55d48e71510 | 14 | Decreasing the surface area of working electrode to micro/nano scale offers several advantages on the electroanalytical performance of the sensor such as enhanced signal-to-noise ratio, increased sensitivity, and small sample volume . The double layer around the micro/nano electrode has a lower capacitance and smaller time constant that allow the signal to be measured more rapidly and with less destruction to the sensor . As the electrode dimensions are smaller than the diffusion layer thickness, the mass transport increases and therefore its sensitivity also increases . Smaller size electrodes can be used to manufacture high-density multiplexed devices which may open up the way for commercialisation . For example, has reported an array consisted of 32 individually addressable gold microelectrodes and their use for |
6352afdeecdad55d48e71510 | 15 | ITO electrodes are also commonly applied for the biosensors development due to their high conductivity, wide electrochemical window and ease of modification . However ITO electrodes have poor electrocatalytic properties that are usually compensated for in biosensors development by their modification with catalytically active metals, such as gold or zinc oxide . Such modification is also necessary for the attachment of biomolecules, such as probe DNA, antibody, or aptamers . For instance, Deshmukh et al. ( ) modified the ITO surface with Aminopropyltrimethoxysilane (APTES) creating amine groups on the surface in order to achieve the immobilisation of amine modified probe DNA via a common crosslinker glutaraldehyde. The label-free detection of z3276 gene from STEC O157:H7 was recorded by using EIS. The authors demonstrated detection of chromosomal DNA and achieved linear range between 6.3x10 -16 -3.2 x 10 -14 M and an LOD 2 CFU/mL in potable water samples in less than one hour also including DNA extraction. Two E. coli strains negative for z3276 gene and Bacillus subtilis were detected using the developed biosensor to evaluate the specificity which is a critical parameter for biosensors. were used to confirm the specificity of the sensor. |
6352afdeecdad55d48e71510 | 16 | Screen printed electrodes (SPE) are types of electrodes fabricated using different types of inks on various substrates such as plastics or ceramics. The major advantage is their low cost which makes them easy to access substrates for the development of disposable sensors, however, their quality can be variable . Carbon is the most used ink for SPE which can then be additionally altered with other metals, polymers, or enzymes, depending on the application. An example for one such application has been reported . Briefly, peptide nucleic acid (PNA) probe was immobilized onto the screen-printed carbon electrode (SPCE) by applying a potential of -0.5 V to working electrode for 300 seconds. It is worth mentioning that PNA is an uncharged synthetic polymer similar to DNA and RNA and it can be applied as an alternative to oligonucleotides. PNA binds the complementary DNA or RNA with high affinity and high specificity which results in more stable complexes compared to natural nucleic acids. |
6352afdeecdad55d48e71510 | 17 | Furthermore PNA is more resistant to high temperature, and the presence of proteases, and nucleases . In this study, the PNA-based biosensor detected a DNA sequence from E. coli by using a sandwich assay with a reporter DNA tagged with latex spheres and biobarcode DNA; see Figure . This barcode DNA was used to attract positively charged silver ions to its negatively charged phosphate groups and afterwards, these silver ions, dissolved with nitric acid, were detected using DPV. Using the sandwich assay platform, the biosensor exhibited an LOD of 5.6x10 -19 M corresponding to 17 CFU/mL and a good discrimination against Salmonella without the need for DNA amplification. The full assay, including DNA extraction, took less than 1.5 hour making it a promising method for POC testing. |
6352afdeecdad55d48e71510 | 18 | Magnetic particles (MPs) in sizes ranging from nanometres to micrometres have gained much attention of researchers to be used in the development of biosensors. Their high surface area allows the immobilisation of the biorecognition molecules at a high density, limiting the background noise. Importantly, they allow to apply sample enrichment and separation in the solution under a magnetic field which makes them perfect candidates for rapid detection of low numbers of pathogens in complex samples . MPs can be either integrated into a transducer or dispersed in a sample and subsequently get attracted using the magnetic field onto the electrode surface (Rocha-Santos, 2014). developed a dual DNA walker strategy for the detection of STEC O157. A DNA walker is defined as a type of nucleic acid nanomachines able to move along a well-designed track comprised of DNA building blocks. These nanomachines are synthesised to mimic the natural DNA walkers like myosin, dynein or kinesin . Subsequently, the modified PCR amplicons were immobilised to streptavidin-magnetic aggregation. In addition, the DNA interacted with positively charged oxidised TMB. As a result of these interactions, the electron transfer and the cathodic current was significantly lower if the target DNA was present in the sample. The authors obtained an LOD of 2x10 -8 M which suggested that this technique was less sensitive compared to other studies summarised in this review. The sensor fabrication was, however, less complex since there was no need for probe immobilisation. |
6352afdeecdad55d48e71510 | 19 | In this review, we presented several electrochemical DNA-based sensors for the detection of general E. coli and STEC were developed since 2015. This review highlights the impact of the electrode material, electrode modification, and the detection technique in the process of the DNA based biosensor development. Sensitive and selective biosensors have been developed by many researchers . Many of these studies have used gold, ITO or carbon electrodes or magnetic nanoparticles. Among them the gold electrodes are the most common used substrates since gold surfaces provide high conductivity, and easy modification with thiol modified probe DNA. The gold electrode surfaces were either directly modified with thiolmodified probe DNA or firstly modified with a mix of polymers and nanomaterials allowing a covalent attachment of probe DNA. These modifications allow high sensitivity of developed biosensors due to increased conductivity, however, they also increase the complexity and preparation time of the biosensors due to additional fabrication steps. Such modifications are difficult to achieve at manufacturing scale with high reproducibility for commercialisation. |
6352afdeecdad55d48e71510 | 20 | Microfabrication and decrease in electrode's size could be an answer to this challenge allowing for large-scale production while achieving high sensitivity. Among the reported literature the microfabricated electrodes were, however, usually modified with at least the nanoparticles for increased conductivity and surface area which that in result remains a limitation for scalability. |
6352afdeecdad55d48e71510 | 21 | Carbon-based electrodes are also a popular choice, and they were found to achieve the highest limit of detection among the collected literature. Carbon electrodes are known to be a cheaper compared to gold meeting the affordability criteria. They, however, were also modified in most cases with a combination of nanomaterials and polymers increasing the complexity of their fabrication. The magnetic nano/microparticles-based sensing methods haven't achieved as low limits of detection compared to the solid-state sensors and typically requires more preparation/ application steps resulting in longer time-to-results. The benefit of MPs that they can be used in high volume, complex samples common in the agri-food industry. Most of the sensors collected in this review are studied in samples between 100 and 200 µL with a maximum of 1.5 mL. The magnetic particles are already used in commercially applied kits for bacteria detection, for instance immunomagnetic separation is a part of ISO protocol for STEC detection (ISO, 2012). Therefore, there is a high potential for their application in the complex samples. Finally, the ITO-based biosensors show good limits of detection, although they typically required complex and laborious modifications which limit their use in the POC detection. |
6352afdeecdad55d48e71510 | 22 | The number of published articles around electrochemical nucleic acid-based sensors is increasing every year. To visualise it better, the Web of Science database was searched using keywords "DNA or RNA or nucleic acid*" AND "*sensor* or rapid detection or quick detection" as the title AND "bacteria or pathogen*" as the topic. The number of articles found in this search per year since 1980 is shown in Figure . presenting an increase each year. There were fewer than ten articles published in this area per year in the 1980s, around twenty articles a year in the 1990s while the highest numbers were recorded since 2019 with around 160 articles published every year. Even though there is extensive research in this area, there is no commercially available electrochemical nucleic acid-based biosensor for POC detection yet . For the development of a successful POC device, there are several criteria to take in to consideration, so called "ASSURED criteria" such as Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipmentfree and Deliverable to end-users . The performance of a POC should also be comparable to the traditional culture methods . |
6352afdeecdad55d48e71510 | 23 | Following the regulations regarding maximum permitted presence of general E. coli or STEC in different types of food samples, a sensitive sensor for detection of E. coli and STEC should be able to detect <100 CFU/mL. However, a limitation of assessing the suitability of biosensors reported in this review is that an LOD was often expressed by authors in molar concentration due to use of synthetic DNA strands for a proof-of-concept studies. This LOD cannot be directly translated into CFU/ml due to a higher complexity of the sample if a chromosomal DNA was used. However, few authors have studied the developed biosensors with both synthetic and chromosomal DNA. This allows an approximate estimation of the required sensitivity in M concentration for the sensor to detect <100 CFU/mL in bacterial samples. For instance, Widaningrum et al. ( ) detected 5.6x10 -19 M of synthetic DNA and 17 CFU/mL using STEC culture suggesting the LOD would need to reach zM to achieve such high sensitivity. A similar or lower LOD was only demonstrated by and among the selected literature. Several authors reported LOD in aM range which may also be sufficient to detect ~100 CFU/mL. More studies are, however, needed to correlate the LOD in M and CFU/mL. In addition, the use of complex samples, such as food or faeces, was very limited and therefore it is difficult to predict how these systems may perform in POC applications. Typically, DNA extracted from bacterial cultures was only used for validation. The future development of POC nucleic acid based electrochemical sensors should focus on simple ways to prepare the sample or/and incorporate this step into a fully integrated device. As such, development of an easy way for DNA extraction is highly critical key achievement for success. Recycling of the nucleic acid-based biosensors is another crucial factor which clearly may have an impact on their introduction to the market. That means reusing the nucleic acid-based biosensor after dehybridising the target strand and allowing for several uses of a single sensor. Typically, a strong base such as NaOH is used to break the hydrogen bonds in the double-strand DNA. |
6352afdeecdad55d48e71510 | 24 | Alternatively, a high temperature of 100 °C can be applied to achieve the same outcome. However, both ways are not suitable for POC applications because of the need for personal protective equipment (PPE), heating equipment, and a way for disposal of used chemicals. Importantly, reusing a biosensor requires detailed studies on their stability after heat or NaOH treatment conditions. Clearly, the use of nucleic acid-based sensors for continuous monitoring in, for instance water treatment facilities, is now limited. |
6352afdeecdad55d48e71510 | 25 | The detection time of assays presented in this review vary from 1 to 7 hours, depending on the technique used, sample preparation and if any amplification or enrichment methods were used. Since the traditional microbiological methods take a few days for analysis, these biosensors provide an advantage in terms of the analysis period. However, their use in the collected literature was typically limited to pure bacteria culture that only provide a proof-of-concept results. The complex samples such as faeces or food samples may contain low numbers of E. coli or STEC in the presence of other bacteria with high numbers. In that case, the use of an amplification method or enrichment, the common ways to increase the number of target DNA in the sample, may be necessary. This would, however, increase the complexity and time-to-results of the assay. |
6352afdeecdad55d48e71510 | 26 | The major limitation of POC devices developed for STEC from agri-food samples is usually the very low concentration of target bacteria among the existing other species in the same sample It is worth mentioning that these low bacterial counts can cause life-threatening infections. Main approach in order to increase the concentration of the target DNA is including the amplification step. For this purpose, PCR is the most common technique, however, it requires a high precision thermal cycler for temperature control, and long sample preparation, and is prone to non-specific amplifications . These working conditions of PCR limits the application for POC application. The alternative approach which has gained attention in the recent years is isothermal amplification such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), helicase dependent amplification (HAD), or rolling circle amplification (RCA) . The details of each technique have been previously described in the reviews by and . In brief, the isothermal amplification techniques include enzymatic DNA denaturation, which allow to eliminate the high temperature application for denaturation step of PCR. This approach provides an opportunity to use a simple device which can keep the temperature constant for primer annealing and extension. |
6352afdeecdad55d48e71510 | 27 | These isothermal amplification techniques were incorporated in the development of biosensors and state-of-art devices, showing great progress in POC devices development. For instance, Ben Aissa et al. ( ) have reported biotin-streptavidin based approach. Briefly, the target DNA was hybridised with a biotinylated capture probe allowing its attachment to streptavidin-modified magnetic beads. Subsequently, the RCA was undertaken to amplify the target DNA strand and the product of the RCA reaction was labelled with HRP enzyme. The biosensor was constructed onto a SPCE, and the used method was SWV. The enzymatic detection was studied in the presence of hydroquinone as a mediator and H2O2 as a substrate. The biosensor achieved a LOD of 6.7x10 -13 M which corresponded to 10 4.3 CFU/mL in approximately two and a half hours. have applied several amplification strategies to improve the sensitivity of the developed STEC O157:H7 detection method. First, they used the 3D DNA walked strategy followed by RCA to amplify the target DNA and hybridization chain reaction (HCR) to immobilise electrochemical indicators (1, 1′-dicarboxyferrocene (Fc) and doxorubicin (Dox)) to the amplicons allowing for an enhanced electrochemical signal, see Figure . (A). As a result, the biosensor exhibited a very high sensitivity with an LOD of 7 CFU/mL in approximately 2.5 hours including the DNA extraction. |
6352afdeecdad55d48e71510 | 28 | The POC application of this assay may, however, be limited due to its relative complexity. has reported a study of real-time detection of eaeA gene from STEC during the DNA amplification using RPA. The authors fabricated a gold nanogap electrode on silicon wafers, which was able to measure a change in impedance during the DNA amplification. The assay took only 30 minutes, including DNA extraction to achieve an LOD of 7 CFU/mL. These results demonstrate the highly promising step forward success and improvements in the field, however the real-world application of this study requires an easy-to-use approach such as microfluidics. |
6352afdeecdad55d48e71510 | 29 | The miniaturisation of the electrochemical cell onto a single chip and integration with the microfluidics could provide a simple detection platform for target analyte or multiplexed detection of the species with very small sample quantities . Combining such sensing platforms with necessary electronics for the development of an automated device is the overall aim. This enables onsite detection without a fully equipped laboratory environment or highly trained personnel. Microfluidic chips can be designed to couple with several functions for high- |
6352afdeecdad55d48e71510 | 30 | Another promising solution for replacing PCR amplification is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas. CRISPR/CAS is a complex immune system in archaea and bacteria protecting them against foreign virus nucleic acids, which is considered to be the most significant discovery in the recent year in biology . In brief, it is based on the ability of the bacteria to store fragments of foreign DNA in the CRISPR loci as a memory, that together with surrounding repeats, are later used by the Cas enzymes to recognise the invader and inactivate it. CRISPR/Cas system can be classified based on the specific Cas enzymes and the interference mechanism. There are two main classes according to the interference mechanisms and class 2 comprising three different enzyme types (Cas9, Cas12 and Cas13) is most often applied in nucleic acid-based biosensors . For instance, CRISPR/Cas system was recently applied for the detection of whole bacterial cells by and . |
6352afdeecdad55d48e71510 | 31 | The advantage of using the whole-cell detection is the lack of need for DNA extraction, however, the sensor may not be as specific to recognise STEC as a nucleic acid-based detection. In both studies, STEC was recognised using aptamers and the final detection was based on a stem-loop structure probe DNA tagged with MB immobilised onto a gold electrode. In the work of , STEC recognition by a specific aptamer triggered a primer exchange reaction (PER) which initiated the synthesise of ssDNA strands (PER product) leading the activation of Cas12a. The enzyme led to the creaking of the stem-loop structure probe DNA on the electrode and therefore, a current decrease related to MB which was detected by using DPV. The authors developed a highly sensitive sensor with a LOD of 19 CFU/mL, with an analysis time of fewer than three hours. |
6352afdeecdad55d48e71510 | 32 | Chen et al. ( ) achieved even higher sensitivity with a LOD of 10 CFU/mL in less than two hours. In this study, a sandwich-based structure with antibodies modified magnetic beads on one side and aptamers on the other was used for STEC recognition. In the presence of STEC in the sample, the CRISPR/Cas12a complex was triggered leading to hairpin DNA cleavage and effectively signal alteration, detected with DPV as well. Another study by used the CRISPR-Cas12a system coupled with EIS to detect E. coli and S. aureus. In their work, a gold disc electrode was modified with thiolated probe DNA, and the detection principle was schematical presented in Figure . Upon a successful recognition and cleavage of the target DNA by Cas12a, the probe DNA immobilised on the electrode got effectively cleaved by the trans collateral activity of the enzyme. The corresponding change in the electrode's resistance could be subsequently recorded by EIS. Using this system, the authors reported an LOD of 3x10 -9 M. This work is opening the door for further developments of using the novel CRISPR/Cas system for biosensors development. More studies are, however, needed to assess the potential of this novel technology in POC application. |
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