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string | predicted_class
string | confidence
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|---|---|---|
The MFI for IgG staining was calculated by measuring and averaging the IF intensities of 20 randomly selected glomeruli under the same conditions using the image analysis software. For nephrin staining, 20 randomly selected glomeruli were graded semi-quantitatively as follows: 0, severely decreased staining; 1, mildly decreased staining; 2, intense staining. The nephrin IF score was then calculated using the formula: (0 × N0 + 1 × N1 + 2 × N2)/(N0 + N1 + N2), where N is the number of the glomeruli for each staining grade.
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study
| 100.0 |
The cytokine and IgM levels in the sera or culture supernatants, and the levels of serum IgG anti-dsDNA Abs were measured using ELISA kits (IL-4 and IFN-γ: BD Biosciences, San Diego, CA; IgM: Bethyl Laboratories, Inc., Montgomery, TX; IgG anti-dsDNA Abs: Shibayagi Co. Ltd., Gunma, Japan). BUN levels and serum albumin, total protein, and alanine aminotransferase levels were measured using a DRICHEM 3000 V system (Fuji Medical System, Tokyo, Japan). Urinary protein excretion was measured by a clinical laboratory testing company (SRL, Tokyo, Japan).
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study
| 99.94 |
Kidney, liver, and spleen specimens were filtered through a stainless-steel mesh and dissolved. Kidney MNCs were thereafter isolated using a Percoll density gradient (67% and 33%) centrifugation. Liver MNCs were isolated without collagenase, essentially as previously described10.
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study
| 99.94 |
For the identification of whole NKT or iNKT cells, the MNCs were stained with anti-TCR αβ Ab (H57-597, eBioscience, San Diego, CA) and either anti-NK1.1 Ab (PK136, eBioscience) or the α-GalCer-loaded CD1d tetramer (MBL, Nagoya, Japan), respectively. Macrophages were identified by staining MNCs with anti-F4/80 (BM8, eBioscience) and anti-CD11b Abs (M1/70, eBioscience), and their expression of TLR-9 was examined using anti-TLR-9 Ab (J15A7, BD Biosciences). B cells were identified by staining MNCs with anti-B220 Ab (RA3-6B2, eBioscience). Flow cytometry was performed on a Cytomics FC500 instrument (Beckman Coulter, Indianapolis, IN).
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study
| 99.94 |
MNCs (5 × 105 cells) of liver or splenic origin in 200 μL of medium were cultured with either α-GalCer (100 ng/mL), ConA (1 ng/mL, Vector Laboratories, Inc., Burlingame, CA), anti-CD3 Ab (10 μg/mL, eBioscience), CpG-ODN (20 μg/mL, Hycult Biotech, Uden, The Netherlands), or LPS from E. coli 0111:B4 (10 μg/mL, Sigma-Aldrich, St. Louis, MO) in 96-well flat-bottom plates. The culture supernatants were harvested 48 hours after seeding.
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study
| 99.94 |
Data are expressed as the mean ± SEM. Differences between two experimental groups were assessed using the Student’s t-test, and those among more than three experimental groups were assessed by one-way ANOVA with Tukey’s HSD post hoc test. Differences in the proteinuria-free survival time and the survival time of the groups were analyzed using the log-rank test and the Cox proportional hazard model. P-values of < 0.05 were considered statistically significant. All statistical analyses were performed using the JMP software (version 11; SAS Institute Inc., Cary, NC).
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study
| 99.94 |
‘LPS’ describes a variety of cell wall lipopolysaccharides shed by Gram-negative bacteria; also known as ‘endotoxin’, they have been found in various fluids, including whole blood (WB). The ‘concentrations’ are typically assayed using the Limulus amoebocyte lysate assay (e.g. [1–3]). However, although satisfactory in simple matrices, this test is not considered very reliable in blood . Indeed, because it is so hydrophobic, little or no LPS is actually free (unbound), and so it is not even obvious what its ‘concentration’ in blood might mean (see ). Although the quantitative assessment of LPS concentrations in WB can be problematic, its presence in this fluid may have important and clinically relevant effects on the blood microenvironment, and may be central in the treatment of inflammatory conditions [5–8].
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review
| 99.44 |
LPS molecules are potent inflammagens (e.g. [9–11]) and may be both cytotoxic and/or neurotoxic [5,12–15]. They are known to induce the production of a variety of pro-inflammatory cytokines [16–19] that are involved in various apoptotic, programmed necrosis and pyroptotic pathways . Indeed, cytokine production is central to the development of inflammation . A further characteristic of systemic inflammation is a hypercoagulatory state [23–27]. Such hypercoagulability is a common pathology underlying all thrombotic conditions, including ischaemic heart disease, ischaemic stroke and venous thromboembolism . Furthermore, a hypercoagulable state is typically associated with pathological changes in the concentrations of fibrin(ogen) , and in particular an increase in the level of the fibrin degradation product D-dimer is seen as a reliable biomarker for cardiovascular risk .
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review
| 99.8 |
Considering its cytokine-dependent effects, the question then arises as to whether LPS can cause hypercoagulation by acting on the coagulation pathway more directly. One route is via tissue factor (TF) upregulation; TF is related to the cytokine receptor class II family, and is active early in the (extrinsic) coagulation cascade, where it is necessary to initiate thrombin formation from prothrombin . Recently, it was shown that LPS may upregulate TF; 100 ng ml−1 LPS added to healthy cord WB of newborns or the WB of healthy adults induced TF-mediated activation of haemostasis . LPS from Escherichia coli (100 ng ml−1) also activated the coagulation system when added to WB, via a complement- and CD14-dependent upregulation of TF, leading to prothrombin activation and hypercoagulation ; however, this was noted after 2 h, and therefore it was not an acute process . Note that in these studies, the anticoagulant was lepirudin, which prevents thrombin activation such that the effects of thrombin could not be evaluated. In this work, we used citrate as an anticoagulant.
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| 100.0 |
It occurred to us that, in addition to changes in TF expression by LPS, the process might also involve the direct binding of the lipophilic LPS to circulating plasma proteins, particularly fibrinogen, and that this (potentially rapid) binding might also cause pathological changes in the coagulation process. This would be independent of the slower TF activation, and thus an acute and relatively immediate process (figure 1). This indeed turned out to be the case. A preprint has been lodged at bioRxiv . Figure 1.High-level effects of systemic inflammation on the coagulation system and the pathologic effects of LPS when present in blood and how it influences coagulation via a direct or indirect activation. Processes 1A and B are currently known for LPS activity in blood, while process 2 is a newly proposed acute reaction effect of LPS on blood and plasma. (Online version in colour.)
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study
| 99.94 |
High-level effects of systemic inflammation on the coagulation system and the pathologic effects of LPS when present in blood and how it influences coagulation via a direct or indirect activation. Processes 1A and B are currently known for LPS activity in blood, while process 2 is a newly proposed acute reaction effect of LPS on blood and plasma. (Online version in colour.)
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other
| 99.1 |
To investigate our hypothesis that LPS may cause hypercoagulation via an acute and direct binding reaction (by interaction with plasma proteins directly involved in the clotting cascade), we investigated the effect of two LPS preparations from E. coli (viz. O111:B4 and O26:B6). These were added to WB of healthy individuals, to platelet (and cell)-poor plasma (PPP), and to purified fibrinogen.
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| 100.0 |
Although the physiological levels of LPS are said to be 10–15 ng l−1, and little or none of it is free , in our hands the addition of LPS at these concentrations caused immediate coagulation when they were added to WB. Figure 2 shows the effect of 0.2 ng l−1 O111:B4 LPS when added to WB and incubated for 10 min. Dense matted deposits are spontaneously formed; these are not seen in healthy WB. Fibrinogen with added O111:B4 or O26:B6 LPS with just 30 s exposure (no thrombin added) also spontaneously formed matted deposits (results not shown). Figure 2.Effect of O111:B4 LPS (0.2 ng l−1) on whole blood (without thrombin), where dense matted deposits were spontaneously formed, not seen in control whole blood smears.
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| 100.0 |
Figure 3 shows the effects of thrombin on clot formation for a healthy control (figure 3a) and when the PPP was pre-incubated for 10 min with 0.2 ng l−1 O111:B4 LPS (two examples in figure 3b,c). Figure 3d shows the distribution of fibre thicknesses for the 30 individuals, with and without added LPS. The fibre thickness is much more heterogeneous after LPS is added. Clearly, these tiny amounts of LPS are having enormous effects on the clotting process. These kinds of netted structures, which we have also termed ‘dense matted deposits’, were previously seen in inflammatory conditions such as diabetes [37–39], iron overload and stroke [37,40–42]. Typically, healthy fibrin fibre networks form a net where individual fibrin fibres are seen, but with added LPS a matted net develops. There is a significant difference (p < 0.0001) between fibre thickness before and after LPS treatment in the presence of thrombin. Note, though, that the distribution of the fibre thickness in the LPS-treated group varies from very thick to very thin. In some cases, continuous fibre plates are formed, where no individual fibres could be seen or measured. This explains the difference in n between the ‘before’ and ‘after’ treatment (1450 versus 1330 measured fibres). Figure 3.The effect of 0.2 ng l−1 O111:B4 LPS on the morphology of fibrin fibres in the platelet-poor plasma (PPP) of healthy individuals (with added thrombin). (a) Healthy fibres; (b,c) PPP with added LPS. (d) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with added LPS, there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Material and methods.
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study
| 100.0 |
The effect of 0.2 ng l−1 O111:B4 LPS on the morphology of fibrin fibres in the platelet-poor plasma (PPP) of healthy individuals (with added thrombin). (a) Healthy fibres; (b,c) PPP with added LPS. (d) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with added LPS, there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Material and methods.
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study
| 100.0 |
The experiment with the O111:B4 LPS was also repeated with a shorter 30 s exposure time. PPP with LPS and added thrombin showed fibre agglutination starting to happen in only 30 s of LPS exposure. These shorter experiments are to be contrasted with previously reported experiments that showed the much longer-term effect of LPS, involving cytokine production, including increased TF production via monocytes. By adding LPS to PPP (with thrombin), we bypass the possibility that LPS can stimulate TF production via the monocyte route suggested in . To determine if another type of LPS would also cause the changes noted above, we also added E. coli O26:B6 LPS to PPP of five individuals (30 s and 10 min exposure time), followed by addition of thrombin. Scanning electron microscopy (SEM) results showed the same trends as noted with the O111:B4 LPS (results not shown).
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study
| 100.0 |
The reaction, and presumed binding, of the hydrophobic LPS within fibrinogen fibres implies that they contain, or expose, significant hydrophobic elements. Such elements, also common in amyloid-like fibrils , can be stained fluorescently using dyes such as thioflavin T (ThT) . We therefore studied the effect of 0.2 ng l−1 LPS on the ability of the fibrin fibres formed following thrombin treatment of PPP to bind ThT (figure 4). In contrast with the LPS-free controls (figure 4a), there is a very substantial binding of ThT to the fibrin fibres formed in the presence of the LPS (figure 4b). The lipopolysaccharide binding protein (LBP) is a potent binder of LPS, and would therefore be expected to inhibit the amyloidogenic effects on blood clotting observed. Thus, we also studied the effect of 2 ng l−1 LBP and 2 ng l−1 LBP + 0.2 ng l−1 LPS mixture on ThT binding (figure 4c,d) . Figure 4.(a) Control PPP with ThT and thrombin and (b) as panel (a) but pre-incubated with 0.2 ng l−1 LPS; (c) as panel (a) but pre-incubated with 2 ng l−1 LBP; (d) as panel (a) but pre-incubated with 0.2 ng l−1 LPS and 2 ng l−1 LBP. (Online version in colour.)
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study
| 100.0 |
It is worth rehearsing just how big an effect this is in molar terms: fibrinogen (MW 340 kDa) is present at in plasma at concentrations of approximately 2–4 g l−1 (Weisel's authoritative review gives 2.5 g l−1), and its levels are increased during inflammation (see above), while the LPS (MW 10–20 kDa) was added at a concentration of 0.2 ng l−1. We will assume 15 kDa for the MW of LPS and 30 fg LPS per cell. Thus, 0.2 ng l−1 = 13 fM and 2.5 g l−1 fibrinogen approximately 7.35 µM which is a molar ratio of LPS; fibrinogen monomer in the WB of less than 10−8 : 1. As we are here only looking at the terminal stages of clotting, we considered that fibrinogen might be an important mediator of the LPS-induced hypercoagulation. Thus, we also added both LPS types to purified fibrinogen (30 s and 10 min exposure time) with added thrombin. Even the 30 s exposure time changed the fibrin fibres to form fibrils or dense matted deposits without any individual fibres visible (figure 5). Figure 5.(a) Purified fibrinogen with added thrombin but no LPS; (b) purified fibrinogen with added O111:B4 LPS (30 s exposure) and 0.2 ng l−1 LPS; (c) as panel (b) but 10 min exposure.
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study
| 100.0 |
It is also worth rehearsing what 0.2 ng l−1 of LPS means in terms of the bacterial equivalents. Watson and co-workers showed in laboratory cultures that LPS amounted to some 50 fg per cell in a logarithmic growth phase, falling to 29 fg per cell in stationary phase. As remarked previously , this shows at once that LPS contents per cell can be quite variable and that bacteria can shed a considerable amount of LPS at no major harm to themselves. On the basis that 1 mg dry weight of bacteria is about 109 cells, each cell is about 1 pg, so 50 fg LPS per cell equates to about 5% of its dry weight, a reasonable and self-consistent figure for approximate calculations. We shall take the ‘starved’ value of 30 fg per cell. Thus, 0.2 ng l−1 (200 pg l−1) LPS is equivalent to the LPS content of approximately 7 × 103 cells l−1. Most estimates of the dormant blood microbiome (that is derived mainly by dysbiosis from the gut and from the oral cavity as summarized in [5–7]) (some are much greater ) imply values of 103–104 ml, i.e. approximately 1000 times greater. In other words, a bacterial cell need lose only a small amount of its LPS to affect blood clotting in the way we describe here.
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study
| 99.94 |
ITC is a sensitive and convenient method for detecting biomolecular interactions by measuring the heat that is released or absorbed upon binding . Measurements are conducted directly in solution, without modification of immobilization of the interacting species. We used ITC to study potential interactions between human plasma fibrinogen and LPS from E. coli O111:B4. Titration of fibrinogen into LPS resulted in strong endothermic injection heats with a clear sigmoidal saturation curve indicating a direct binding interaction (figure 6a). Assuming molecular weights of 340 kDa for fibrinogen and 20 kDa for monomeric LPS, we determined a binding stoichiometry (n) of approximately 0.135. This is consistent with each fibrinogen monomer binding to a micelle formed from approximately 75 LPS monomers. Reverse titrations were conducted, injecting LPS into plasma concentrations of fibrinogen (3 g l−1 = 8.8 µM). Titration of 2.5 µM LPS into fibrinogen yielded endothermic injection heats greater than those observed for titration of 2.5 µM LPS into buffer alone (figure 6b), again clearly indicating a direct binding of LPS to fibrinogen. Each injection added 125 ng of LPS into the instrument cell, increasing the LPS concentration by approximately 30 nM per injection. Although we expect that LPS binds fibrinogen at sub-nanomolar concentrations, interactions at these concentrations are below the detection limits of the ITC instrument. Figure 6.ITC analysis of the LPS–fibrinogen interaction. (a) Titration of 8.8 μM human plasma fibrinogen (black circles) or buffer (green open circles) into 100 µM of E. coli O111:B4 LPS. (b) Titration of 50 ng µl−1 LPS (2.5 µM) or buffer into 3 µg µl−1 fibrinogen (8.8 µM) or buffer as indicated. Experiments were conducted at 37°C in phosphate buffered saline. (Online version in colour.)
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study
| 100.0 |
ITC analysis of the LPS–fibrinogen interaction. (a) Titration of 8.8 μM human plasma fibrinogen (black circles) or buffer (green open circles) into 100 µM of E. coli O111:B4 LPS. (b) Titration of 50 ng µl−1 LPS (2.5 µM) or buffer into 3 µg µl−1 fibrinogen (8.8 µM) or buffer as indicated. Experiments were conducted at 37°C in phosphate buffered saline. (Online version in colour.)
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study
| 100.0 |
Thromboelastography (TEG®) is a viscoelastic technique for measuring the clotting properties of WB . TEG of WB and PPP was performed. TEG was not performed with purified fibrinogen because the coagulation activator in the TEG is CaCl2 and fibrinogen is only activated by thrombin, not calcium. Figure 7 shows a typical TEG trace from a control WB with and without added LPS, overlaid with lines that explain the parameters extracted by the instrument and the values for those traces. The statistics are given in table 1. Figure 7.TEG overlay from a control whole blood sample with and without added LPS. R, reaction time, first measurable clot formation; K, achievement of clot firmness; angle, kinetics of clot development; MA, maximum clot strength; MRTG, maximum rate of thrombus generation; TMRTG, time to maximum rate of thrombus generation; TTG, final clot strength. (Online version in colour.) Table 1.Demographics of blood from healthy individuals with and without added LPS. Medians, standard deviation and p-values (values lower than 0.05 are indicated in italic) obtained using the Mann–Whitney U-test are shown for iron profiles, and TEG of whole blood and plasma. R, reaction time, first measurable clot formation; K, achievement of clot firmness; angle, kinetics of clot development; MA, maximum clot strength; MRTG, maximum rate of thrombus generation; TMRTG, time to maximum rate of thrombus generation; TTG, final clot strength.variableshealthy individuals (n=30)healthy individuals with added LPS (n=30)p-valueage (years)29.5 (±13.81)gender male15 (50%) female15 (50%)iron profiles iron µM16.9 (±6.18) transferrin g l−12.75 (±0.46) % saturation25.0 (±10.94) serum ferritin ng ml−142.5 (±92.47)fibrin fibre thicknessn = 1450n = 1330 fibre thickness (nm)103 (±40)86 (±82)<0.0001TEG®TEG of whole blood—recalcified with CaCl2 with added O111:B4 LPS (10 min)MRTG (dyn cm−2 s−1)2.61 (±1.13)2.89 (±0.90)0.33TMRTG (min)13.9 (±3.53)9.6 (±3.01)<0.0001TTG (dyn cm−2)615.0 (±179.55)527.9 (±146.65)0.049R (min)8.0 (±1.64)6.2 (±1.77)<0.0001K (min)4.9 (±2.63)4.2 (±1.23)0.07angle (°)49.8 (±5.27)56.2 (±7.02)0.0066MA (mm)55.0 (±8.07)51.3 (±6.90)0.092TEG of platelet-poor plasma—recalcified with CaCl2 with added O111:B4 LPS (10 min)MRTG (dyn cm−2 s−1)3.6 (±4.35)4.2 (±2.13)0.36TMRTG (min)10.6 (±3.22)9.3 (±3.68)0.50TTG (dyn cm−2)203.9 (±137.51)211.6 (±103.67)0.70R (min)8.2 (±2.64)7.1 (±2.70)0.026K (min)4.4 (±3.51)3.8 (±2.42)0.18angle (°)63.2 (±2.70)54.4 (±10.67)0.23MA (mm)28.4 (±8.34)30.1 (±9.27)0.196TEG of platelet-poor plasma—recalcified with CaCl2 with added O111:B4 LPS (30 s)n = 5n = 5MRTG (dyn cm−2 s−1)5.94 (±1.8)8.2 (±2)0.166TMRTG (min)11.58 (±1.2)9 (±1.3)0.0159TTG (dyn cm−2)244.4 (±69.9)290.2 (±66.5)>0.99R (min)9.8 (±1.2)7 (±1.5)0.031K (min)2.8 (±1.6)2 (±0.9)0.119angle (°)63.6 (±6.5)68.8 (±8.2)0.095MA (mm)32.8 (±6.5)36.7 (±5.5)>0.99TEG of platelet-poor plasma—recalcified with CaCl2 with added O26:B6 LPS (30 s)n = 5n = 5MRTG (dyn cm−2 s−1)6.3 (±2.6)6.2 (±3.6)0.70TMRTG (min)11.6 (±2.1)8.9 (±1.5)0.15TTG (dyn cm−2)276.7 (±43.1)230.8 (±202.2)0.69R (min)9.8 (±1.5)6.4 (±1.4)0.05K (min)2.1 (±0.5)2.1 (±1.1)0.88angle (°)64.4 (±4.3)70.8 (±10.4)>0.99MA (mm)35.5 (±3.3)31.5 (±13)0.60
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study
| 100.0 |
TEG overlay from a control whole blood sample with and without added LPS. R, reaction time, first measurable clot formation; K, achievement of clot firmness; angle, kinetics of clot development; MA, maximum clot strength; MRTG, maximum rate of thrombus generation; TMRTG, time to maximum rate of thrombus generation; TTG, final clot strength. (Online version in colour.)
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study
| 98.9 |
Demographics of blood from healthy individuals with and without added LPS. Medians, standard deviation and p-values (values lower than 0.05 are indicated in italic) obtained using the Mann–Whitney U-test are shown for iron profiles, and TEG of whole blood and plasma. R, reaction time, first measurable clot formation; K, achievement of clot firmness; angle, kinetics of clot development; MA, maximum clot strength; MRTG, maximum rate of thrombus generation; TMRTG, time to maximum rate of thrombus generation; TTG, final clot strength.
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study
| 100.0 |
TEG analysis of WB (10 min incubation time with O111:B4 LPS) showed that the R, TMRTG and TTG are all significantly decreased (table 1). Changes to R indicate that the clot forms quicker and a decreased TMRTG indicates that the time to maximum thrombus generation is also faster, suggesting a hypercoagulable state. The TTG is also significantly decreased suggesting total thrombus generation, thus implying clot strength is decreased, although the clot forms faster. The SEM fibrin fibre thickness results show areas where no individual fibres are formed; instead, a matted homogeneous layer forms, and there are also areas of fine and short fibres. Here, we suggest that this morphology is probably related to the decreased TTG, where the fibrin structure results in a clot with decreased strength. We have not measured lyses, but a decreased TTG is likely to indicate a hypofibrinolytic nature of the clot. We have previously shown that the same concentration of LPS as used in this paper, when added to naive uncitrated healthy blood but without added CaCl2, also had an effect on coagulation after only 30 s incubation time . Both TMRTG and R of naive WB were also significantly shorter, also showing hypercoagulation, and the TTG was increased, but not significantly increased. However, in this study, the blood was not drawn in citrated tubes and therefore not re-calcified.
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study
| 100.0 |
We also performed similar TEG experiments with PPP (table 1). After 10 min exposure, just the initial clotting time R was changed. The results were not specific for O111:B4 LPS as O26:B6 LPS added to PPP behaved similarly. The decreased R time is indicative of reaction time, and therefore the time to first measurable clot formation is also significantly decreased (as the initiation of the clot starts faster with than without LPS), confirming a hypercoagulable state with added LPS. After 30 s exposure time, both the R and the TMRTG were shorter (in the five patients tested). This confirms our hypothesis that LPS causes (near) instant hypercoagulability. Here also, the results were not specific for O111:B4 LPS as O26:B6 LPS added to PPP also showed a decreased R-time. TTG of PPP was increased (but not significantly increased) after 30 s, as well as 10 min, which compared with results of PPP from patients with Alzheimer's type dementia .
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study
| 100.0 |
Table 1 shows a summary of the various viscoelastic properties (TEG experiments) after LPS has been added to WB and PPP. There are very substantial changes in a number of the clotting parameters. Following these 10 min exposure experiments, we shortened our experimental time to 30 s and we repeated the experiments with five samples, using PPP, where significant changes were still observed. The results were not specific for O111:B4 LPS as O26:B6 LPS. We note that WB with added LPS showed a more pronounced change in relevant viscoelastic parameters than when LPS was added to PPP (table 1).
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study
| 100.0 |
In the introduction, we suggested that LPS might contribute to excessive blood clotting (or an activated coagulation state) via two possible routes: (i) via a direct and acute binding to plasma proteins (e.g. fibrinogen) or (ii) by an indirect or chronic (longer-term) process where it participates in an inflammatory activation via cytokine production. Here, we showed that the first process is indeed possible, using tiny amounts of LPS that amounted in molar terms to less than 10−8 relative to fibrinogen, and demonstrated it by both viscoelastic and ultrastructural methods. We also confirmed that LPS can change the viscoelastic properties of PPP within 30 s of its addition. Furthermore, WB with added LPS, but without thrombin activation, showed spontaneously formed, amyloid-like matted deposits. Purified fibrinogen experiments with O111:B4 LPS and O26:B6 LPS, with and without added thrombin showed a changed ultrastructure, suggesting that LPS indeed binds to the 340 kDa fibrinogen molecule and that the effects of this are visible ultrastructurally.
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study
| 100.0 |
LPS, and especially its lipid A component, is highly lipophilic, and it therefore may be able to bind directly to plasma proteins, in an acute way. This might be one reason underlying the hypercoagulability , as well as a denser clot structure , as seen in various inflammatory diseases. Although we show here that exposure to even tiny amounts of LPS leads to an immediate (acute) change in the coagulability parameters, we recognize that this may happen simultaneously with chronic (longer-term) reactions (figure 1). Fibrinogen molecules are roughly 5×45 nm, and their self-assembly is a remarkable process (some 5800 are involved in generating a fibre of 80–90 nm diameter and 1 µm length). This would explain why the highly substoichiometric binding of LPS can have such considerable effects, especially as observed in WB. Following Anfinsen , it is assumed that most proteins adopt their conformation of lowest free energy. However, this is not true for amyloid fibre formation nor in the case of the autocatalytic conversion of prion protein conformations . At present, the exact mechanisms of action of these small amounts of LPS are not known, although it is indeed simplest to recognize fibrinogen polymerization as a cascade effect, much as occurs for amyloid and prion proteins whose initial conformation is not in fact that of their lowest free energy . Specifically , the ‘normal’ conformational macrostate of such proteins is not in fact that of the lowest free energy, and its transition to the energetically more favourable ‘rogue’ state is thermodynamically favourable but under kinetic control, normally (in terms of transition state theory) with a very high energy barrier ΔG† of maybe 36–38 kcal mol−1 . Indeed, it is now known that quite a number of proteins of a given sequence can exist in at least two highly distinct conformations . Typically the normal (‘benign’) form, as produced initially within the cell, will have a significant α-helical content, but the abnormal (‘rogue’) form, often in the form of an insoluble amyloid, will have a massively increased amount of β-sheet , whether parallel or antiparallel. In the case of blood clotting, we at least know that this is initiated by the thrombin-catalysed loss of fibrinopeptides from fibrinogen monomers (e.g. ). The massive adoption of a β-sheet conformation, as revealed here for the first time by the thioflavin T staining, demonstrates directly that virtually every fibrinogen molecule in the fibrin fibril must have changed its conformation hugely; it is not just a question of static ‘knobs and holes’ as usually depicted. We also showed that LBP, and a mixture of LPS and LBP, shows decreased ThT binding, compared with LPS alone.
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study
| 100.0 |
Previously, we coined the term ‘atopobiotic’ microbes to describe microbes that appear in places other than where they should be, e.g. in the blood, forming a blood microbiome . Here, we suggest that the metabolic and cell membrane products of these atopobiotic microbes correlate with, and may contribute to, the dynamics of a variety of inflammatory diseases [64–67], and that LPS, in addition to (possibly low-grade) long-term inflammation via cytokine production, may lead an acute and direct hypercoagulatory effect by binding to plasma proteins, especially fibrinogen. Specifically, we showed here that, even with very low levels and highly substoichiometric amounts of LPS, a greatly changed fibrin fibre structure is observed. An urgent task now is to uncover the mechanism(s) of this acute and immediate effect, with its remarkable molecular amplification.
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study
| 99.94 |
In total, 30 healthy individuals were included in the study. Exclusion criteria were known inflammatory conditions such as asthma, human immunodeficiency virus (HIV) or tuberculosis, and risk factors associated with metabolic syndrome, smoking, and, if female, being on contraceptive or hormone replacement treatment. Full iron tests were performed, as high serum ferritin and low transferrin levels are acute phase inflammatory protein markers and indicative of inflammation. We included controls only if their iron levels were within normal ranges. WB of the participants was obtained in citrate tubes and either WB or platelet-poor plasma was used in this study for TEG, confocal and SEM experiments.
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study
| 100.0 |
The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762). A final LPS exposure concentration of 0.2 ng l−1 (well below its critical micelle concentration ) was used in all experiments bar as noted for some of the ITC measurements. A final LBP exposure concentration of 2 ng l−1 LBP and a mixture with final exposure concentration of LPS (0.2 ng l−1) and LBP (2 ng l−1), incubated for 10 min with PPP, were also used (only confocal studies).
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study
| 100.0 |
For ITC experiments, a micellar suspension of 10 mg l−1 was vortexed, followed by multiple serial dilutions. The South African National Blood Service (SANBS) supplied human thrombin, which was at a stock concentration of 20 U ml−1 and was made up in a PBS containing 0.2% human serum albumin. In experiments with added thrombin, 5 µl of thrombin was added to 10 µl of PPP or fibrinogen (with and without LPS exposure). Human fibrinogen was purchased from Sigma (F3879–250MG). A working solution of 0.166 mg ml−1 purified fibrinogen was prepared. This concentration was found to be the optimal concentration to form fibrin fibres in the presence of thrombin, similar to that of platelet-rich plasma fibres from healthy individuals . As noted by a referee, LPS is a common laboratory contaminant, and care is needed; however, this was not an issue here as the ‘no-added-LPS’ controls showed.
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study
| 100.0 |
LPS-incubated WB and purified fibrinogen were prepared for SEM without added thrombin (LPS exposure concentration: 0.2 ng l−1). LPS-incubated PPP and purified fibrinogen samples were prepared as above, but with added thrombin to create an extensive fibrin fibre network (also with LPS exposure concentration: 0.2 ng l−1 before addition of thrombin).
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study
| 99.94 |
E. coli O111:B4 LPS and human plasma fibrinogen were purchased from Sigma-Aldrich. Samples were reconstituted in warm phosphate buffered saline and incubated for 1 h at 37°C with shaking. LPS was then sonicated for 1 h at 60°C. Fibrinogen solutions were passed through a 0.2 µm polyethersulfone syringe filter and concentrations were determined by UV absorbance (E1% = 15.1 at 280 nm). Samples were then diluted with buffer to the required concentration and degassed. ITC experiments were performed at 37°C on a MicroCal Auto-iTC200 system (GE Healthcare) in high-gain mode at a reference power of 10 µcal s−1, with an initial 0.5 µl (1 s) injection followed by 15 2.5 µl (5 s) injections with 300 s spacing. For longer titrations, the syringe was refilled and injections continued into the same cell sample. Control runs were performed in which cell samples were titrated with buffer and syringe samples were titrated into buffer, and data from these runs were subtracted from the experimental data as appropriate. Data analysis was performed in Origin, using the supplied software (MicroCal).
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| 100.0 |
TEG was used to study the viscoelastic properties of the participants' blood, before and after addition of LPS. WB TEG was performed on day of collection (after 10 min incubation time with LPS—final exposure concentration 0.2 ng l−1 and PPP was stored in 500 µl aliquots in a −70°C freezer. The thawed citrated PPP (with and without LPS—where LPS was added to PPP at a final exposure concentration of 0.2 ng l−1. The incubation time of LPS and PPP was 10 min, as with WB. Standard TEG procedures were followed with addition of CaCl2 to activate the coagulation process as previously described . TEG was also performed on 5 PPP samples, 30 s after adding O111:B4 LPS or O26:B6 LPS.
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| 100.0 |
Thioflavin T (ThT) was added at an exposure concentration of 5 µM to 200 µl of PPP (incubated for 1 min, and protected from light). A second sample was also prepared by adding an exposure concentration of 0.2 ng l−1 LPS (incubate for 10 min, at room temperature) before the addition of ThT. After an incubation time of 1 min and incubation protected from light, 10 µl of the PPP (with and without LPS) was mixed with 5 µl of thrombin (see SEM preparation of extensive fibrin fibres—as described above). To determine if ThT binding will happen in the presence of LBP, 2 ng l−1 LBP was pre-incubated for 10 min with PPP, followed by 1 min ThT exposure, and fibrin fibre preparation by adding thrombin. A mixture of LPS and LBP was also made (final PPP exposure concentration: 0.2 ng l−1 LPS and 2 ng l−1 LBP). PPP was exposed for 10 min to this mixture, followed by a 1 min exposure of ThT and fibrin fibre formation by adding thrombin to mixture-exposed PPP. Samples were viewed under a Zeiss LSM 510 META confocal microscope with a Plan-Apochromat 63×/1.4 Oil DIC objective, excitation was at 488 nm and emission measured at 505–550.
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| 100.0 |
The construction of mechanically responsive materials which exhibit durability, reversibility and good stability is a challenging task (Ramirez et al., 2013 ▸; Panda et al., 2014 ▸). Structural studies on mechanically responsive materials have gained importance due to their wide ranging applications in materials science and medicine (Liu et al., 2014 ▸). Solids which are responsive to external stimuli such as heat, pressure, humidity, light and can convert them efficiently into work are potential candidates for development as smart materials, e.g. artificial muscles, actuators, biomimetic and technomematic materials (Liu et al., 2014 ▸; Lehn, 2002 ▸; Ikeda et al., 2007 ▸; Sagara & Kato, 2009 ▸; Rowan, 2009 ▸; Takashima et al., 2012 ▸; Mather, 2007 ▸; Sato, 2016 ▸).The efficient conversion of light and heat energy into mechanical work by dense and orderly packed crystals was reviewed recently (Naumov et al., 2015 ▸). Mechanical response occurs when the material is heated, or exposed to light/pressure, that causes an increase in the strain inside the crystal lattice. Thermosalient effects of (phenylazophenyl)-Pd-(hexafluoroacetonate) complex was first reported by Etter & Siedle (1983 ▸). Recently, Panda et al. (2014 ▸) revisited the same complex and observed positive and negative thermal expansion and thermosalient effects. Desiraju and co-workers (Ghosh et al., 2015 ▸) reported that thermosalient effects are mainly due to a sharp phase transition and anisotropy in structural parameters. Vittal’s group reported photo actuation in Zn metal complexes due to an increase in strain by sudden expansion during 2 + 2 photo cycloaddition reaction (Medishetty et al., 2014 ▸). Naumov’s group (Sahoo et al., 2013 ▸; Nath et al., 2014 ▸, 2015 ▸) analyzed thermosalient crystals and classified them into three classes. If the molecule does not have any strong hydrogen-bonding donor and acceptor groups, then it is classified as Class I. Class II consists of molecules with hydrogen-bonding groups in a crowded environment, which will not allow strong hydrogen bonding, and Class III molecules have good hydrogen-bonding functional groups which participate in strong hydrogen bonds. There are a few examples of both thermomechanical and photomechanical effects in crystals (Naumov et al., 2015 ▸; Lusi & Bernstein, 2013 ▸; Skoko et al., 2010 ▸; Medishetty et al., 2015 ▸; Mishra et al., 2015 ▸; Takeda & Akutagawa, 2016 ▸; Lieberman et al., 2000 ▸; Brandel et al., 2015 ▸).
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| 99.9 |
Schiff’s bases and their metal complexes find wide applications in biological systems, as well as non-linear optical materials, in electrochemical cells, as corrosion inhibitors, thermo-/photochromic materials and as thermally/chemically resistant flame retardants (Jia & Li, 2015 ▸; Singh et al., 2014 ▸). In the present investigation, we synthesized dichloro (compound A), dibromo (compound B) and diiodo (compound C) derivatives of N-salicylideneaniline (Schiff’s bases) (Scheme 1). The chemical structures (Fig. S1 of the supporting information) and procedures followed for the synthesis of compounds are given in §2. The presence of a strong hydrogen bonding functional group (e.g. the amide group) makes Compound A a Class III category, and the surprising effects of jumping, breaking and sudden blasting upon heating its polymorphic crystals are presented in this paper. To our knowledge, such a phenomenon is unusual and not reported in polymorphs of the same compound (Steiner et al., 1993 ▸; Crawford et al., 2007 ▸; Sahoo et al., 2013 ▸).
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| 99.94 |
For the synthesis of compound A ((E)-4-((3,5-dichloro-2-hydroxybenzylidene)amino)benzamide) two methods were followed: the first is mechanochemical (Zbačnik & Kaitner, 2014 ▸) grinding and the second is a conventional reaction with azeotropic removal of water (Scheme S1) (Safin et al., 2014 ▸).
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| 99.9 |
Method 1: The product was obtained by mechanical grinding of 4-aminobenzamide and 3,5-dichlorosalicylaldehyde in a 1:1 stoichiometric ratio (1 mmol each, 136 mg; 191 mg) by adding 2–3 ml of methanol (drop by drop addition) solvent for 30 min and the resulting product was washed with hexane 3–4 times to remove unwanted by-products. The product salicylideneaniline was finally purified by crystallization from methanol and characterized by 1H-NMR and 13C-NMR, FT–IR (Figs. S24–26) and finally confirmed by single-crystal X-ray diffraction. We followed the same procedure for the synthesis of compound B and compound C and confirmed their structures by 1H-NMR and single-crystal XRD.
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| 100.0 |
Method 2: 4-Aminobenzamide 1 mmol (136.15 mg) was dissolved in anisole (10 ml) in a 100 ml round-bottom flask fitted with a Dean-Stark setup. 1 mmol of 3,5-dichlorosalicylaldehyde (191.01 mg) was added to the solution. The reaction mixture turns to a light yellow color (after 10 min) and it was refluxed for 4 h. The solution was concentrated by removing the solvent and then washed with n-hexane 3–4 times to remove unwanted by-products. Red color crystals were obtained in 80% yield. We followed the same procedure for the synthesis of compound B and compound C and confirmed their structures by 1H-NMR and single-crystal X-ray diffraction.
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| 99.94 |
X-ray reflections for compound A (Form I, Form II and Form III) and compound B were collected on an Oxford Xcalibur Gemini Eos CCD diffractometer at 298 K using Cu Kα radiation (λ = 1.54184 Å) and data reduction was performed using CrysAlisPro (CrysAlis CCD and CrysAlis RED; Oxford Diffraction, 2008 ▸) and OLEX2 (Dolomanov et al., 2009 ▸) to solve and refine the crystal structure. X-ray reflections for compound C were collected on a Bruker D8 Quest CCD diffractometer equipped with a graphite monochromator and Mo Kα fine-focus sealed tube (λ = 0.71073 Å), and high-temperature Form IV (Compound A) was collected on Bruker D8 venture diffractometer at 180°C; reduction was performed using APEXII Software (Bruker, 2000 ▸). Intensities were corrected for absorption using SADABS (Sheldrick, 1997 ▸) and the structure was solved and refined using SHELX97 (Sheldrick, 1997 ▸). All non-hydrogen atoms were refined anisotropically. Hydrogen atoms on hetero atoms were located from difference electron-density maps and all C—H H atoms were fixed geometrically. Hydrogen-bond geometries were determined in PLATON (Spek, 2002 ▸). X-Seed (Barbour, 1999 ▸) was used to prepare packing diagrams. Crystal structures are deposited as part of the supporting information and may be accessed at https://www.ccdc.cam.ac.uk/structures/ (CCDC Nos. 1488992–1488996).
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| 100.0 |
Powder X-ray diffraction was recorded on Bruker D8 Advance diffractometer (Bruker-AXS, Karlsruhe, Germany) using Cu Kα X-radiation (λ = 1.5406 Å) at 40 kV and 30 mA power. X-ray diffraction patterns were collected over the 2θ range 5–40° at a scan rate of 1° min−1.
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| 99.94 |
Differential scanning calorimetry (DSC) was performed on a Mettler Toledo DSC 822e module. Samples were placed in crimped but vented aluminium sample pans. The typical sample size is 3–5 mg; the temperature range was 30–300°C at 20°C min−1. Samples were purged by a stream of nitrogen flowing at 60 ml min−1.
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| 99.6 |
TGA was carried out using Mettler Toledo TGASDTA851e operating with STAR e software to detect solvates and thermal degradation. Accurately weighed (5–15 mg) samples were loaded in alumina crucibles and heated at a rate of 20°C min−1 over a temperature range of 30 to 300°C under a nitrogen purge of 60 ml min−1.
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study
| 99.9 |
Compound A was purified by crystallization from methanol, and after multiple recrystallizations diffraction quality red color crystals were harvested from the same solvent. Further screening in different solvents afforded three polymorphs concomitantly from methanol solvent as well as a fourth high-temperature polymorph. Studies on compounds B and C are ongoing, but with similar efforts at crystallization, we have so far obtained a single-crystal structure only for the bromo and iodo salicylideneanilines (no polymorphism). The subsequent discussion is on the structures and properties of chloro compound A. Polymorphs of A in bulk quantity (several mg up to g) were obtained by different techniques, such as Form I by rotary evaporation, Form II from slurry in methanol, and Form III from acetone at 80°C in a sealed tube (Fig. 1 ▸). The preliminary observations in this study were done on a hot stage microscope (HSM). Upon heating Form I crystals to about 170–180°C, a few crystals were seen to suddenly fly off from the stage (about 2–3 cm height) and they moved outside of the camera zone (Fig. 2 ▸ a). Form II crystals showed sudden blasting (mini explosion, Fig. 2 ▸ b) at 180°C, and Form III crystals behaved similar to those of Form I in that they were flying off suddenly from the hot stage at 180–190°C.
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| 100.0 |
The X-ray crystal structures of Form I and II were solved in triclinic space group . The amide group forms a dimer through N1—H1B⋯O1 [2.00 (4) Å, ∠172 (3)°] hydrogen bonds in a ring motif (Etter et al., 1990 ▸; Bernstein et al., 1995 ▸) in Form I (Fig. 3 ▸). An intramolecular hydrogen bond between the hydroxyl donor and imine nitrogen through the O2—H2A⋯N2 [1.66 (4) Å, ∠149 (4)°] ring makes an motif. The dihedral angle between the two phenyl rings is 20.07 (2)° (Table 1 ▸). The amide dimers extend through C—Cl⋯O [2.961 (3) Å] and N1—H1A⋯O2 [2.25 (4) Å, ∠161 (3)°] interactions in an eight-membered ring motif. The molecular layers are parallel to the crystallographic (1 −1 −2) plane at a distance of 3.4 Å between the molecular layers (Fig. 3 ▸). The crystal structure of Form II (Fig. 4 ▸) has the same amide dimer synthon [N1—H1B⋯O3; 2.06 (1) Å, ∠170 (1)° and N3—H3B⋯O1; 2.06 (3) Å, ∠170.4 (1)°] and N—H⋯O [N3—H3A⋯O2, 2.41 (2) Å, ∠144.3 (2)°; and N1—H1A⋯O4, 2.32 (4) Å, ∠157.4 (1)°] hydrogen bonds as in Form I. The number of symmetry independent molecules is different (Z′ = 1 in Form I and 2 in Form II). The dihedral angles between phenyl ring planes are 11.99 (17)° and 26.52 (17)°. The C—Cl⋯O interactions are 2.983 (2), 3.237 (2) Å. The chain grows parallel to the c-axis and the molecules are arranged in a wavy (corrugated) motif. Form III and IV also crystallized in the space group and with two molecules in the asymmetric unit. Forms III and IV (Figs. 5 ▸ and 6 ▸) have the same type of amide dimers which are described for the above two structures [N1—H1B⋯O3: 2.02 (2) Å, ∠170.7 (3)°; N3—H3B⋯O1: 2.17 (3) Å, ∠160.9 (1)°] and [N1—H1B⋯O3: 2.18 (2) Å, ∠161.2 (2)°; N3—H3A⋯O1: 2.04 Å, ∠170.8 (2)°], which extend via C—Cl⋯O interactions [3.04 (4), Fig. 5 ▸ a; and 3.101 (3) Å, Fig. 6 ▸ a]. The dihedral angle between two phenyl rings of the same molecule in Form III is 13.87 (4) and 23.91 (4)° and in Form IV is 12.72 (2) and 23.49 (2)°, respectively (Table 1 ▸). In Form III the amide dimers are connected by Cl⋯Cl interactions of Type I (Mukherjee et al., 2014 ▸; Desiraju & Parthasarathy, 1989 ▸) [C—Cl⋯Cl–C, 3.466 (2) Å, ∠138.4 (2)°] to form a tetrameric ring motif (Fig. S2). The planar molecular layers are parallel to the crystallographic (101) plane in Form III (Fig. 5 ▸ c). The hydrogen-bond synthons in Forms I, II, III and IV are identical except that the Cl⋯O distances vary in the polymorphs (Table 2 ▸), but these slight differences in halogen-bond distances have a dramatic consequence on the thermal response of compound A crystals (for additional diagrams of Cl⋯Cl interactions and packing in Compound A polymorphs, see Figs. S2–S3), as described in the next section. The crystal structures and hydrogen-bonding interactions for compounds B and C are discussed in the supporting information (Figs. S4–S7 and Table S3).
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| 100.0 |
The powder X-ray diffraction lines of the three polymorphs of compound A are significantly distinct to permit characterization of the bulk material in each case (Fig. S8), as well as to monitor phase transitions. The peaks of Form I appear at 2θ 9.89, 10.84, 13.22, 13.66, 15.66°, for Form II at 7.08, 8.94, 11.58, 12.82, 14.19°, and Form III at 9.21, 12.32, 13.75°.
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study
| 100.0 |
To better understand the events visually observed on the thermal microscope, variable-temperature powder X-ray diffraction (VT-PXRD) and DSC of the three polymorphs were performed. Heating the sample bench of XRD showed transformation to a new polymorph IV at 200°C for all the three polymorphs such as I, II and III. This transient Form IV was characterized by its unique powder XRD lines and in all cases the product on cooling to room temperature was Form III, which is the nearest stable phase (Fig. S9). Thus, a heat–cool cycle exhibited transformation to polymorph III via a new phase IV. In order to observe possible phase changes below ambient temperature (−25°C to 30°C), DSC was carried out on the sample in the range −150°C to 200 °C. Form I showed an endotherm at 147°C and saw tooth-like endotherms (zigzag profile) at 170–185°C due to Form I → IV conversion (confirmed by VT-PXRD experiments, Fig. S9). PXRD of high-temperature Form IV obtained from different polymorphs I–III are compared in Fig. S10. On cooling the same material, Form IV converted to Form III at 0–10°C and on reheating Form III, a small endotherm at 150–160°C was observed, after which it again converted to high-temperature phase Form IV (Form I → IV ↔ III is a solid-to-solid phase transformation); melting occurred at 249°C (Figs. S11 and S12). In the case of Form II, an endotherm was observed at 170°C and upon further heating a second endotherm occurred at 183–185°C, which indicates Form II → IV conversion. Reheating of the same material showed a small endotherm at 150–160°C (Form III → IV) followed by melting at 249°C (Figs. S13 and S14). During heating of Form III, a sawtooth wave DSC profile was observed at 181–190°C, indicating the conversion of Form III to IV. Cooling of the same material showed transformation to Form III, and on reheating small endotherms were observed at 150–162°C (Form III → IV), followed by melting at 251°C (Figs. S15 and S16). All the above experiments were performed multiple times with identical results. We also performed competitive slurry experiments to establish that Form II is more stable (thermodynamically stable state) compared with the remaining two Forms I and III. DSC and VT-PXRD confirmed that Form IV is stable at high temperature (after 180°C) and Forms III and IV are reversible during cooling and heating (III ↔ IV). Thus, the solid-to-solid phase transitions on temperature-modulated powder XRD and DSC exhibit similar transformations under heat–cool conditions. Compounds B and C melt at 274°C and 254 °C, respectively, without any phase transformation (Fig. S17).
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study
| 100.0 |
Compound A showed a mechanical response towards heating but not the Br and I derivatives, even though these structures have the same type of hydrogen bonds and halogen bonding synthons and are three-dimensional isostructural (Xpac analysis; Figs. S18–20). The most probable reason for mechanical response is mainly due to a change in the halogen atom and also the halogen-bond interactions (Table S3). The interaction energy for the C—X⋯O (X = F, Cl, Br and I) bond increases with an increase in polarizability and a decrease of electronegativity for the halogen atom (Politzer et al., 2010 ▸; Riley & Hobza, 2008 ▸; Riley & Merz, 2007 ▸). The combined effects of both factors increases the σ hole for halogens towards a more +ve lobe in the order of I > Br > Cl. The increased electrostatic interaction between the σ hole of the halogen and oxygen lone pair electrons results in strengthening of the halogen bond from C—Cl⋯O through C—Br⋯O to C—I⋯O (Table 3 ▸) (Riley & Merz, 2007 ▸). In effect, the stronger halogen-bonded structures (having near-identical amide N—H⋯O hydrogen bonds) with Br and I atoms make them less responsive to temperature and mechanical stress because the halogen bond is too strong for the heavier halogens to show structural (and property) dynamics, indicating the importance of weaker C—Cl⋯O interactions in exhibiting the mechanical response of molecular crystals and temperature effects.
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study
| 100.0 |
During heating of Form I (Compound A) on the (1 0 1)/(−1 0 −1) faces (Fig. 3 ▸), we observed a mechanical response of the material by jumping (see the video in the supporting information). After heating Form I crystals for 5–10 min at 200°C, it converted to Form IV with a small change in unit-cell volume (ΔV = 20 Å3), after correcting for Z′ being 1 and 2 in Form I and IV, respectively. Similarly Forms II and III also converted to Form IV after heating for 5 min with a much larger change in volume (ΔV = 64, 41 Å3). Examination of the individual cell axes (Table 4 ▸) suggests that there is a decrease in the cell length along the a-axis and increase in the b- and c-direction of the triclinic cells of Forms I and II. Form III crystals also showed jumping (see the video in the supporting information) when heated on the (0 1 1)/(0 −1 −1) faces with a slight increase in cell lengths in a, b and c directions. In contrast, Form II crystals have irregular morphology (Table S2), so we were not able to identify specific faces in which the crystal shows a response to heating. Heating Form II crystals beyond 180°C caused blasting (see the video in the supporting information). To understand the reason behind the different mechanical responses (jumping and blasting) of polymorphic molecular crystals to heating, we compared all four polymorphic structures in terms of variation in conformation, hydrogen-bonding interactions and changes in crystal packing (Table S2). During the transition all hydrogen/halogen bonds were retained in the structure with slight changes in the distances along with slight changes in conformation in the molecule, and significant changes in packing of molecules were observed. Forms I and III molecules are arranged in a layered structure (Figs. 3 ▸ and 5 ▸), whereas in form II symmetry-independent molecules are arranged in a corrugated wave motif (Fig. 4 ▸). We hypothesize that upon heating crystals of Form I and Form III, the heat is transferred uniformly from the face resulting in the transmission of thermal stress largely in a single direction, thereby causing a thermosalient effect of jumping. In Form II, however, due to the corrugated wave-like arrangement of molecules, heat transmission is non-uniform resulting in a sudden blast of the crystal. The most likely explanation for these effects is due to the sudden release of accumulated strain energy during phase transition (Etter & Siedle, 1983 ▸), and anisotropy in the cell parameters. However, such thermosalient effects in molecular crystals is still not fully understood due to the limited number of examples in the literature for structure–property correlation. Thermochromic effects in Compound A polymorphs are described in the supporting information (Fig. S21).
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| 100.0 |
Hirshfeld surface (Poulsen et al., 2009 ▸) analysis of polymorphs of compound A (Fig. 7 ▸) showed that Form II has a greater contribution from O⋯H and Cl⋯O contacts (14.8 and 2.6%) compared with Form I (12.9 and 2.1%), Form III (11.8 and 1.5%) and Form IV (12.1 and 1.8%, Fig. S22) indicating the stability of Form II compared with the other two forms, which was also supported in competitive slurry experiments.
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| 100.0 |
We have described thermal responses of jumping (Forms I and III) and blasting (Form II) in dichloro N-salicylidene (compound A) polymorphs due to the sudden phase transition at high temperature. The bromo and iodo halogen derivatives did not exhibit any thermal responses. The significance of weaker Cl⋯O interactions in an invariant amide dimer N—H⋯O structural family of polymorphs is the reason ascribed to the mechanical response of chloromolecular crystals. This study demonstrates the utility of hydrogen- and halogen-bonded molecular crystals in exhibiting the thermosalient effect under thermal stress and also presents a rationale for the design of thermoresponsive crystals.
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| 100.0 |
The calculation of spectroscopic phenomena involving molecular vibrations is an example of a successful application of theoretical chemistry to aid the interpretation of experimental observations. The calculation of molecular structure and vibrational spectra was made possible by the pioneering work of Bratoz, Pulay, Pople, Schaefer and others in developing methods for calculating analytical geometric derivatives of the molecular energy.1–5 Second-order geometric derivatives of the energy have since been derived and implemented for a wide range of correlated wave functions,3,5–13 as well as at the level of density functional theory (DFT).14–16 For a detailed historical account we refer to recent reviews of molecular properties in general and molecular force fields in particular.17–19
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review
| 99.9 |
A commonly used approximation in the study of vibrational spectroscopies is the double-harmonic approximation,20 where molecular vibrations are described as harmonic oscillators and where the fundamental properties describing the spectroscopic intensities are determined by the first-order geometric derivative of the polarization property governing the spectroscopic phenomenon under study. This corresponds to a description which obeys the well-known selection rules for e.g. infrared (IR) and Raman spectroscopies,21 where it is the magnitude of the first-order geometric derivative of the molecular electric dipole moment and polarizability, respectively, that determines the spectroscopic intensity associated with the excitation to a singly excited state of a particular normal mode. Although coupled-cluster theory can provide vibrational frequencies of high accuracy,22–28 its computational cost prevents its routine use for larger molecules. For this reason, density-functional theory (DFT) has been gaining increasing popularity in recent years and has been used for calculations of Raman spectra of molecules as large as buckminsterfullerene.29 The calculations have often been done in combination with scaling of the frequencies in order to account for anharmonicities and errors inherent in the exchange–correlation functional used.30,31 A typical computational protocol has been to determine the frequencies of the (harmonic) normal modes by DFT using the B3LYP functional coupled with intensity calculations performed at the HF or DFT level,32–34 the choice of level of theory for the intensity calculations depending on the computational tools available for the calculation of the necessary geometric derivative of the pertinent polarization property.
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study
| 99.94 |
By leaving the double harmonic approximation, it is possible to obtain a more accurate description of both the vibrational frequencies and the spectroscopic intensities.23,35–38 For the latter, the introduction of anharmonic effects will enable both an improved description of the intensities associated with a single excitation of a particular normal mode as well as introduce the leading-order contributions to intensities associated with transitions corresponding to the simultaneous excitation of two or more vibrational quanta, either involving only a single normal mode or several of them, often referred to as overtone and combination bands, respectively.
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| 99.94 |
Calculations of anharmonic contributions for the purpose of correcting vibrational frequencies have regularly been carried out,17,23,39–41 requiring at least third-order geometric derivatives of the molecular energy, from here on referred to as the cubic force constants. We will refer to the corresponding fourth-order derivatives as the quartic force constants. Calculations of the cubic and quartic force constants have previously almost without exception been done using numerical differentiation.23,41–43 The only exception is the analytic calculation of cubic and quartic force constants at the HF level reported by Handy and coworkers.44,45 Recently, we presented an analytic implementation of cubic and quartic force constants at the DFT level46 by the use of a newly developed recursive code47 for the calculation of molecular properties by response theory.48
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| 100.0 |
For the IR and (regular) Raman spectroscopies, programs that allow for the analytic calculation of the required first-order geometric derivatives of the dipole moment and polarizability, respectively, have been available for some time.49–51 The calculation of anharmonic corrections to the intensities in these spectroscopies requires both the development of the necessary vibrational perturbation theory38 to obtain expressions for these corrections and the possibility of calculating second- and third-order geometric polarization property derivatives, as well as the cubic and quartic force constants, that enter into these expressions. Programs that would allow for the analytic calculation of some of these properties are available, but such calculations have mainly been restricted to the HF level of theory, and for some of the properties (and more so if a DFT description is desired), the researcher has had to resort to numerical differentiation. Analytic calculation offers several advantages over numerical methods such as higher attainable accuracy and ease of computation,51 as numerical derivatives are sensitive to the finite perturbation/geometry displacements employed, and this can have significant effects on the results if not managed carefully.52–54 For these reasons, analytic methods are preferred over numerical ones.
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| 94.9 |
In this work, we present the first application of our recursive approach for the analytic calculation of the anharmonic vibrational frequencies and infrared and Raman intensities of methanimine as well as nitromethane and its mono- and di-deuterated isotopomers. Methanimine has been shown to be very sensitive to the numerical differentiation parameters52 and thus provides a good illustration of the advantages of the analytic approach. The nitromethane isotopomers have been selected because experimental spectra display a large number of combination and overtone bands, for which calculation calls for the use of an anharmonic treatment. We remark that anharmonic effects have also been found to contribute appreciably to the spectroscopic intensities for several other molecules.38
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| 100.0 |
The rest of the paper is organized as follows: in Section 2, we outline the theoretical foundation for the analytic calculation of anharmonic corrections to vibrational frequencies and IR and Raman intensities. In Section 3, we provide details about the computational setup used for the calculations on our chosen systems. We present and discuss the results of our calculations in Section 4, and make some concluding remarks in Section 5.
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other
| 99.7 |
We will begin in Section 2.1 by outlining how the high-order molecular properties used in this work can be calculated analytically through the use of our recently developed recursive response code and then in Section 2.2 proceed to show how these properties can be used to determine anharmonic corrections to vibrational frequencies and IR and Raman intensities. Although the general framework has been described previously,46–48 this work is the first report of fifth-order analytic derivatives involving geometrical distortions.
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| 99.94 |
A detailed presentation of the response theory, which in our approach is fundamental for the analytic calculation of the cubic and quartic force fields and the high-order geometric derivatives of the dipole moment and polarizability that are needed in this work, is too long to show here, and we will therefore restrict ourselves to the most salient features. We refer to the original work48 for a more thorough treatment, and to our recent work47 for a description of the recursive implementation used in the present work.
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| 67.1 |
Our analytic scheme uses as a starting point that linear response functions described by perturbations a and b can be formulated as perturbation strength ε i (i = a, b,…) derivatives of a quasienergy Lagrangian gradient, expressed in a density-matrix (D) formulation as48 1where the derivative is evaluated at zero perturbation strength and where higher-order response functions can be found by further differentiation of eqn (1). A tilde is used to represent a quantity considered at an arbitrary perturbation strength, and the absence of a tilde denotes evaluation at zero perturbation strength. The quasienergy Lagrangian L a is given by2where we have introduced the atomic orbital (AO) overlap matrix S as3Sμν = χ̃μ|χ̃ν,where χ̃ is an atomic orbital, and where the energy- and frequency-weighted Fock matrix W is defined as4where the generalized Kohn–Sham Fock matrix is given by5 We also introduced the generalized energy as6 7 In eqn (5)–(7), we introduced the half-time-differentiated overlap matrix T, the one-electron matrix h, the external field operator t and the two-electron matrix G γ with γ-fractional exchange as8 9 10 11and also the exchange–correlation contributions F xc and xc[ρ̃(D)] in addition to a nuclear potential operator h nuc. Here and throughout the paper, atomic units are used unless otherwise stated. Molecular properties characterized by a perturbation tuple abc… can therefore be formulated as derivatives of the quasienergy Lagrangian gradient as12 13 14where we have introduced a short-hand notation for differentiation and tracing by15and16respectively. This theory is sufficient to define any response function using the so-called n + 1 rule formulation,55 where the calculation of a response property of order n + 1 requires the calculation of the density matrix perturbed to order n. However, other formulations placing other conditions on which perturbed density (and Fock) matrices must be calculated are possible.55 Let us represent the idempotency of the density matrix and the time-dependent self-consistent field (TDSCF) conditions as the matrices Y and Z, respectively, so that17Y = DSD – D,and18where the notation19[M]⊖ = M – M†,and20[M]⊕ = M + M†,has been introduced, and where adjungation is defined to happen before time differentiation. It can be shown that the ansatz21λ̃a = [DaSD]⊖,for the multiplier λ̃ a for Y leads to the definition of the multiplier ζ̃ a for Z as22 It is then possible to make a general expression for the quasi-energy Lagrangian for the calculation of response properties as23where the values of k and n in the various terms denote, with minor variations, to which orders perturbed Fock and density matrices must be calculated in order to evaluate this expression: the value of k determines to which order the perturbed matrices must be calculated for perturbation tuples involving perturbation a, whereas the value of n determines the same for perturbation tuples not involving perturbation a. We have that k + n = N – 1, where N is the order of the property considered, and k must be chosen as an integer in the interval k ∈ [0,(N – 1)/2], where (N – 1)/2 is rounded down for even N. In this work, we do not discuss how the necessary perturbed Fock and density matrices can be calculated, as it is described in detail in ref. 48. We remark, however, that since the calculation of high-order properties requires solving linear response equation systems and since this part of the calculation is computationally expensive, a judicious (k,n) rule choice may give a significant reduction in the number of such systems to be solved, both compared to other rule choices and to numerical differentiation schemes. For instance, for the calculation of cubic force constants, the choice (k,n) = (1,1) makes it necessary to solve M systems, where M is the number of geometrical coordinates, whereas (k,n) = (0,2) results in M 2 such systems. Similarly, a scheme where an analytically calculated molecular Hessian is differentiated numerically by nuclear displacements results in the number of such systems being of the order of M 2. Similar savings can be achieved for other properties.
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With the recursive program developed by our group,47 it is possible to evaluate eqn (23) for any response property, including the calculation of the required perturbed Fock and density matrices, as long as external routines are available that can provide the necessary (un)perturbed one- and two-electron integral contributions,56–58 exchange–correlation contributions59,60 and perturbed nuclear potential contributions, and solve the response equations61,62 that arise during the evaluation of perturbed Fock and density matrices. More information about the external modules used in this work is given in Section 3. All such modules used in the present work have been parallelized; see e.g. ref. 63.
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Having determined the harmonic vibrational frequencies and normal modes of vibration from the well-established eigenanalysis of the molecular Hessian,20 it is possible to make anharmonic corrections to fundamental vibrational frequencies and frequencies corresponding to combination or overtone excitations of the normal modes by a second-order perturbational approach, where the resulting expressions involve the cubic and quartic force constants and Coriolis vibration–rotation coupling constants. In the VPT2 approach,23,35,36 the corrected fundamental vibrational frequencies ν i1, first overtone frequencies ν i2 and first combination frequencies ν i1j1 are given as, respectively24 25νi2 = 2νi1 + 2Xii, 26νi1j1 = νi1 + νj1 + Xij,where the diagonal and off-diagonal correction terms X ii and X ij are given by27and28where Ω ijk is defined as29Ωijk = (ωi + ωj + ωk)·(–ωi + ωj + ωk)·(ωi – ωj + ωk)·(ωi + ωj – ωk). In the above expressions, ω i denotes a harmonic fundamental frequency, φ ijk and φ ijkl are cubic and quartic force constants, respectively, B α is the rotational constant for axis α, and ζ α ij is a Coriolis coupling constant.
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The method chosen in the present work is the so-called generalized vibrational second-order perturbation (GVPT2) model.38,41 In this method, a VPT2 treatment of the molecular vibrations is used, except for the cases where Fermi resonances are considered to have occurred. In these cases, the terms in the VPT2 treatment that are affected by the Fermi resonance are not included,23 and the affected frequencies are instead resolved in a variational approach.
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Expressions for corrections to spectroscopic intensities can also be identified by a perturbation-theory approach. In a recent work by Bloino and Barone,38 GVPT2 expressions for IR and Raman intensities have been derived. The expression for the IR intensities is30and in classical Raman spectroscopic measurements, the unpolarized (as well as polarized) scattering intensity at a temperature T, related to the Raman cross section, is given by31where32and33where ν i = ω i in the harmonic approximation and is given by eqn (24)–(26) in the anharmonic GVPT2 treatment, ν 0 is the frequency of the incident laser in the Raman experiment, and 0i represents the transition moment of the relevant polarization property from the vibrational ground state to the ith vibrational excited state. In the double-harmonic treatment, these transition moments are determined by first-order geometric derivatives of the polarization property (P 0i1 = ∂P/∂q i), whereas the anharmonic expressions also involve the second- and third-order geometric derivatives of the polarization property and the cubic and quartic force constants. The resulting expressions in the anharmonic case are large and we refer to the work of Bloino and Barone38 where the complete expressions are reported.
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Altogether, the expressions used in the complete VPT2 treatment involve the first-, second-, and third-order geometric derivatives of the molecular electric dipole moment and polarizability in the IR and Raman case, respectively, in addition to the cubic and quartic force constants, meaning that the highest-order property that must be calculated, i.e. the cubic force constants of the frequency-dependent polarizability, is a fifth-order energy derivative. The contributions to this property can be identified from eqn (23) and are shown here in order to demonstrate the complexity involved in the analytic calculations performed in this work and to justify the use of a recursive approach.
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The third-order geometric derivative of the polarizability can be defined from a perturbation tuple (a, b, c, d, e), where perturbations a, b and c correspond to differentiation with respect to geometrical displacements, and d and e to differentiation with respect to a frequency-dependent electric dipole perturbation. Denoting a geometric perturbation as g and the two electric dipole perturbations as f ω and f –ω, where, respectively, each perturbation is associated with a positive or negative frequency ω, eqn (23) takes the form34where the rule choice (k,n) = (2,2) is used because this will give the lowest computational cost. Omitting terms that must be zero straightforwardly or because, in the differentiation carried out, there was lack of dependence on the perturbation operators and, for the sake of brevity, writing contributions that are permutations of identical operators only once, the terms in eqn (34) can be written as35 36 37 38and39where, for example, from eqn (37) is40and where the other differentiated W, Y, and Z terms are of a similar complexity. We consider the length of these expressions, in particular eqn (40), and the corresponding complexity in treating them, as strongly supporting the use of a recursive approach for calculations of the high-order properties required for the GVPT2 treatment, and in a similar manner, automated approaches based on automatic differentiation are needed in order to evaluate the differentiated exchange–correlation energy and kernel .59
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To compute the cubic and quartic force constants and the first-, second- and third-order geometric derivative tensors of the electric dipole moment and of the electric dipole polarizability, the recursive implementation47 of the open-ended response theory framework of Thorvaldsen et al. 48 has been used. This formalism has been implemented in a development version of the Dalton2013 program package.64,65 The linear response solver of Jørgensen et al. 61 has been used for the solution of the response equations. Differentiated one- and two-electron integrals were computed using the Gen1Int56,57 and Cgto-Diff-Eri58,66 programs, respectively, except for some of the lower-order two-electron integral geometric derivatives which were computed using existing functionality in Dalton. The differentiated exchange–correlation (XC) energy and potential contributions up to fifth order needed in the DFT calculations were computed using the XCFun library,59,60 where the integrator XCInt has been used for the integration of the XC contributions. The calculation of the Coriolis coupling constants is not done in a response theory framework, but have been calculated in the manner outlined in ref. 67.
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All calculations have been performed at the DFT level of theory using the B3LYP hybrid functional.68–70 This functional has already been shown to give good results for the calculation of higher-order properties in earlier work.46,71 Dunning's correlation-consistent polarized triple-ζ (cc-pVTZ) basis set72 has been used. The study was conducted for methanimine (CH2NH), and nitromethane (CH3NO2) and its mono- (CH2DNO2) and di-deuterated (CHD2NO2) isotopomers. Two conformations (eclipsed and staggered) have been considered for the non-deuterated isotopomer and four (H-eclipsed, D-eclipsed, H-staggered and D-staggered) for each deuterated isotopomer (cf. Fig. 1).
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For each system, the geometry was optimized and the molecular Hessian and the rotational constants were computed using the Dalton2013 program package.64,65 The other relevant molecular properties were computed at the optimized geometry using the recursive response property implementation, and the Coriolis coupling constants have been implemented in a development version of Dalton2013. The molecular Hessian was then used in a vibrational analysis to find the harmonic vibrational frequencies and to transform the geometric differentiation in the property tensors from a Cartesian basis to a reduced normal coordinate basis73 to calculate anharmonic frequencies and spectral intensities.
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Anharmonic corrections to the fundamental frequencies, as well as first overtones and combination band frequencies were calculated from the cubic and quartic force constants, the rotational constants and the Coriolis coupling constants using a scheme based on vibrational second-order perturbation theory35,36 as described in Section 2.2, where terms found to be affected by Fermi resonances are taken out of the perturbational treatment23 and resolved variationally41 using the GVPT2 model.38
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First-order geometric derivatives of the electric dipole and electric dipole polarizability in the reduced normal coordinate basis were used for the evaluation of the harmonic IR intensities and Raman scattering cross-sections, respectively. Anharmonic corrections to the spectral intensities were calculated by further considering the second and third geometric derivatives of the corresponding properties and the cubic and quartic force constants, in a reduced normal coordinate basis, using the GVPT2 model, resulting in features associated with corrections to the fundamental bands and the appearance of the first overtone and combination bands.
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For methanimine, the cubic and quartic force fields have also been evaluated by numerical differentiation from the molecular Hessians calculated for Cartesian displacements δx of 10–2 and 10–3 Å with Dalton2013 using the expressions41 42where E xixj, E xixjxk and E xixjxkxl represent, respectively, the second-, third- and fourth-order derivatives of the energy with respect to the Cartesian components in superscript, and using convergence thresholds of 10–8 for both the molecular orbital (MO) coefficients and relative to the norm of the perturbed MO coefficients when solving the response equations. The same convergence criteria have been applied to all fully analytic calculations. We remark that the errors in the calculated properties resulting from these strict thresholds are negligible.
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The first, second and third geometry derivatives of the electric dipole moment and polarizability have also been evaluated this way and with the same convergence thresholds, but using the expressions43 44 45where P denotes either the electric dipole moment or the electric polarizability, and P xi, P xixj and P xixjxk represent respectively the first, second and third derivatives with respect to geometry distortions.
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| 87.2 |
The spectral bands have been modeled using Lorentzian functions for the band shape with a 10 cm–1 full width at half maximum. A 1 cm–1 resolution was used to plot all spectra. Raman spectra have been evaluated considering an incident laser wavelength of 514 nm, corresponding to an Ar+ laser at 298.15 K.
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| 99.94 |
In this section, we will illustrate the need for analytic differentiation techniques by calculating the infrared and Raman spectra of methanimine (CH2NH), comparing the analytic approach to the results obtained by numerical differentiation using different step lengths. The sensitivity of methanimine to numerical differentiation parameters52 makes it a suitable system for illustrating the advantages of using an analytic approach.
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In the case of numerical differentiation using a step length of δx = 10–3 Å, the difference in Hessian values between some of the displaced systems was smaller than the numerical precision, thus illustrating one of the problems of this approach. This can be illustrated by the anharmonic correction to the vibrational frequency of the 91 mode which is positive, whereas anharmonic corrections are generally expected to be negative, as is obtained in the analytic approach and when a step length of δx = 10–2 Å is used in the numerical differentiation approach. The anharmonic corrections to the vibrational frequencies of the high-frequency modes appear less sensitive to this problem. The analytic approach does not depend on the energy difference between slightly displaced systems and is therefore free from this source of numerical error.
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Using a step length of δx = 10–2 Å for the numerical differentiation, numerical noise is largely avoided and the anharmonic frequencies are in better agreement with experimental fundamental frequencies. This is also observed for the anharmonic frequencies obtained by analytic differentiation. Nevertheless, the numeric anharmonic corrections are still on average in error by about 10% compared to the analytic corrections, the latter being always larger than the former.
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| 99.94 |
Fig. 2 and 3 show, respectively, the calculated infrared and Raman spectra of methanimine for the analytical and numerical approaches. In the calculated infrared spectrum, using a step length of δx = 10–3 Å in the numerical differentiation, not only are the anharmonic corrections to the frequencies in poor agreement with the analytic ones, but so are also the corrections to the intensities, most strikingly so for the low-frequency peaks. In this case, for the IR spectrum, the δx = 10–3 Å numerical differentiation reproduces the analytic anharmonic spectral intensities almost perfectly for the peaks of frequency above 2900 cm–1 but overestimates (in absolute value) drastically the intensity for the other peaks, the lower the frequency the larger the overestimation.
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Numerical (δx = 10–2 Å) and analytic anharmonic corrections to the spectral intensities both go in the same direction for each individual peak, but the magnitude of the corrections differs. The difference in the intensity of the anharmonic corrections to the infrared intensities between the numerical and analytic values varies from 10 to 230% of the analytic correction depending on the peak considered, with the majority of the corrections being in error by 35–85%, the only exceptions being the low-energy modes 81 and 91. However, there is no trend as to whether the numerical corrections under- or overestimate the analytic results. As the anharmonic corrections to the total intensity of the peaks is small, these differences are not easily visible from the spectra plotted in Fig. 2.
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For the Raman spectra, numerical noise does not affect the derivatives of the electronic polarizability when using a step length of δx = 10–3 Å. The numerical anharmonic corrections to the spectral intensities are thus in better agreement with the analytic ones than in the infrared case, but the corrections to the vibrational frequencies remain wrong. Considering the intensities, the numerical spectrum obtained using a step length of δx = 10–3 Å shows differences of less than 10% compared to the analytic spectrum, and is thus in better agreement than the spectrum obtained using a step length of δx = 10–2 Å, where these differences may be as large as 20%. The only exception is the 81 mode, for which both step lengths give corrections that are far from the analytic one. As for the IR spectra, the calculated corrections can be both larger and smaller than the analytic result and whether the corrections are over- or underestimated also depends on the step length. It should also be noted that, depending on the step length used, the ordering of the intensity of the peaks can differ. For example, in the case of δx = 10–2 Å, 11 is slightly more intense than 21, whereas with δx = 10–3 Å, the 21 peak is more intense than 11, in agreement with the analytic differentiation results.
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This example illustrates that even if the use of numerical differentiation can lead to qualitatively sound results, it still depends strongly on the step length used. While methanimine is still a rather small molecule, it could still be expected that these difficulties will be present in larger systems. On this note, we now turn our attention to using the analytic approach to calculate anharmonic vibrational spectra and compare these with available experimental observations.
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| 99.8 |
In Section 4.2.1, we will present and discuss the computed vibrational frequencies, before we in Sections 4.2.2 and 4.2.3 turn to a discussion of the theoretical IR and Raman spectra, respectively, comparing our theoretical results to available experimental data.
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| 83.9 |
All experimental and theoretical studies76–79 on the geometry of nitromethane agree that the barrier (ΔE = 9.6μE h 76) for the rotation of the methyl group around the CN axis is very small, with the staggered conformation being slightly more stable. Our results reproduce quantitatively the barrier height (ΔE B3LYP = 10μE h). Such a low barrier makes it necessary to consider several rotamers when modeling the theoretical spectra, and for this reason all the geometries corresponding to the extrema of the energy along the rotation of the methyl group are considered in this study (cf. Fig. 1). A Boltzmann averaging at room temperature of these rotamers would give a quasi-equal weight for each of the conformers, and for this reason all rotamers will thus be considered of equal weight in the averaging of the spectra from the different rotamers. We note that such a treatment for the low-frequency internal rotation of the methyl group has to be considered approximate, and that this vibration mode probably should be treated by a non-local representation going beyond the normal-mode approximation. For this reason, we will in the following not include this mode in the anharmonic treatment.
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For all rotamers of each isotopomer, the frequency corresponding to the rotation of the methyl group is found to be quite small at the harmonic level and negative at the anharmonic level, which is consistent with what can be expected for a quasi-free rotating methyl group.76,77 The observed spectra should therefore come from the average over all the rotamers. For this study, only the extremum rotamers (staggered and eclipsed) have been considered (cf. Fig. 1), and the system has been treated as having only 14 normal modes (instead of 3N – 6 = 15) by not considering the derivatives with respect to the methyl rotation mode in the anharmonic calculations.
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Using partially deuterated isotopomers lowers the symmetry of the system, thus allowing new rotamers to be spectroscopically active and giving rise to band splittings.77,78 Calculated (harmonic and anharmonic) frequencies for the fundamentals of the non-, mono- and di-deuterated isotopomers of nitromethane are compiled in Tables 2, 3 and 4, respectively. Experimental frequencies77,79–81 are also given for comparison.
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The computed harmonic fundamental frequencies are, in line with previous findings,79 found to be overestimated compared to experiment. Even with anharmonic corrections, the frequencies are in many cases overestimated compared to the experimental data, but lead to a significantly better agreement with experiment. The differences in the calculated vibrational frequencies for the different rotamers are in general very small. Indeed, very similar vibration frequencies are found for the two rotamers of CH3NO2 at both the harmonic and anharmonic level of calculation, the largest difference being 9 cm–1. The calculated vibrational frequencies are also in very good agreement with the experimental assignments of the modes.80–83
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Experimentally, two peaks are assigned in the infrared spectrum to the stretching mode of the C–D bond in CH2DNO2: A strong mode at 2266 cm–1 corresponding to the stretching perpendicular to the plane of the nitro group, and a weak one at 2276 cm–1 corresponding to the stretching parallel to the same plane.79 We find that the maximum frequency for the ν(CD) mode is found for the D-eclipsed geometry in both the harmonic and anharmonic treatment, and the frequency decreases the further away the deuterium atom is from the NO2 plane.
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A similar behaviour is also observed for the stretching of the CH bond in CHD2NO2, with a strong band at 3029 cm–1 corresponding to stretching perpendicular to the plane of the nitro group, and a weak band at 3014 cm–1 corresponding to stretching parallel to the same plane.79 The maximum frequency for the ν(CH) mode is found for the H-eclipsed geometry in both the harmonic and anharmonic treatment, and the frequency then decreases the further away the hydrogen atom is from the NO2 plane, in analogy to the observations for CH2DNO2.
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The infrared spectrum is calculated by summing the calculated spectra of the three rotamers. The harmonic and anharmonic calculated infrared spectra for non-, mono- and di-deuterated isotopomers are shown in Fig. 4–6, respectively. Tables 5–7 show the calculated infrared spectral intensities (before Lorentzian normalization) for the normal modes of the non-, mono- and di-deuterated isotopomers.
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For the three isotopomers considered in this study, the anharmonic corrections do not substantially change the relative intensities of the different fundamental bands below 2000 cm–1. In this region, the main improvements arising from the anharmonic treatment is in the calculated vibrational frequencies, as discussed in the previous section. This observation is in agreement with the findings of Bloino and Barone38 using the GVPT2 approach with numerical calculation of the anharmonic IR spectra for a series of molecules.
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For all three isotopomers, a weak feature appears in the anharmonic spectrum around 2940 cm–1 arising mainly from the combination of the symmetric (mode 4 for all isotopomers) and asymmetric (mode 7 for CH3NO2, mode 6 for CH2DNO2 and mode 5 for CHD2NO2) stretching modes of the NO2 fragment.
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The experimental gas-phase spectrum of CH3NO2 from ref. 84 is reproduced in Fig. 4 for comparison. As already noted, a low-intensity feature around 3000 cm–1, also appearing in the experimental spectrum, is introduced with the anharmonic treatment. The main peak of this feature, at 2955 cm–1, arises mainly from the 4171 combination band and a minor contribution from the 31 fundamental band. Apart from the 11 and 21 fundamental bands (3038 and 3005 cm–1, respectively), another low-intensity combination band, 4181 at 2933 cm–1, appears from the anharmonic treatment. Other low-intensity peaks, also present in the experimental spectrum, appear due to the 41111 combination band at 2471 cm–1 and the 72 overtone band at 2768 cm–1.
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In the CH2DNO2 spectrum, a small shoulder arising from the 101121 combination band of the H-eclipsed conformer and the 91131 combination band from the D-staggered conformer appears on the most intense peak. These two bands are combinations of an angular vibration of the NO2 fragment and an angular motion of the CD fragment. A low-intensity band appears from the anharmonic treatment around 3000 cm–1. The main peak of this band, around 2933 cm–1, corresponds to the 4161 combination band from all four conformers. The rest of the features of this band arise from the 11 and 21 fundamental bands of the four conformers. Other low-intensity peaks appear in the anharmonic spectrum around 2738 cm–1 due to the 62 overtone band of the four conformers and around 2462 cm–1 due to the 41111 combination band from the H-eclipsed and D-staggered conformers.
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In the CHD2NO2 spectrum, the anharmonic treatment gives a modification in the shape of the (broad) band between 1230 and 1280 cm–1, mainly due to the 61 and 71 fundamental bands and from the 122 overtone band. A weak band appears around 1505 cm–1, due to the 101121 and 111121 combination bands from the four conformers. A low-intensity peak, corresponding to the 4151 combination band, appears around 2930 cm–1 from the anharmonic treatment. In addition to the peak at 3020 cm–1, corresponding to the 11 fundamental band of the H-eclipsed conformer, another low-intensity peak appears in the anharmonic spectrum around 2740 cm–1 and is due to the 52 overtone band.
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As done for the IR spectra, the Raman spectra were obtained as the sums of Raman spectra for the individual rotamers. The calculated harmonic and anharmonic Raman spectra for non-, mono- and di-deuterated isotopomers are shown in Fig. 7–9, respectively. Tables 8–10 show the calculated Raman spectral intensities (before Lorentzian normalization) for the normal modes of the non-, mono- and di-deuterated isotopomers.
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The relative intensities of the bands corresponding to CH or CD vibrations compare rather well with the experimental data, as well as the relative intensities of the bands corresponding to the CN and NO2 motions. However, the agreement between theory and experiment for the relative intensities of these two different vibrations is poor. The anharmonic treatment gives slightly better agreement with experiment, though the differences are very small.
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| 99.94 |
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