extfieldsofstudy: Medicine Chemistry

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New insight into the dynamical system of αB-crystallin oligomers

α-Crystallin possesses a dynamic quaternary structure mediated by its subunit dynamics. Elucidation of a mechanism of subunit dynamics in homo-oligomers of αB-crystallin was tackled through deuteration-assisted small-angle neutron scattering (DA-SANS) and electrospray ionization (ESI) native mass spectrometry (nMS). The existence of subunit exchange was confirmed with DA-SANS, and monomers liberated from the oligomers were observed with nMS. With increasing temperature, an increase in both the exchange rate and monomer population was observed despite the absence of oligomer collapse. It is proposed that transiently liberated subunits, namely, “traveling subunits,” play a role in subunit exchange. Moreover, we propose that protein function is regulated by these traveling subunits.

Supplementary Text
Small-angle neutron scattering profiles of hydrogenated and deutearted αB-crystallin oligomers at

˚C
The scattering profile of hydrogenated αB-crystallin oligomers (h-αB) coincided with that of deuterated αB-crystallin oligomers (d-αB) in the inverse contrast solvent at 37°C, as shown in Fig. S1. The radii of gyration (R g ) and I(0) of h-αB and d-αB were evaluated from Guinier analysis. The evaluated R g values of h-αB and d-αB were 53.6 ± 1.1 Å and 53.2 ± 1.3 Å, respectively. Here, N in eq. (1) is given by the following equation: where c, N A , and M w are the concentration of protein in weight %, Avogadro's number, and molecular weight, respectively. In addition, the volume of αB-crystallin in solution can be calculated based on the amino acid sequence 1 . Then, M w , which is comparable to the aggregation number of h-αB and d-αB, can be arithmetically calculated from I 0 . The average number of subunits in h-αB and d-αB was calculated to be 26. This result supports that the expected contrasts from h-αB and d-αB were experimentally fulfilled and no structural difference existed between them.

Evaluation of the number of exchangeable subunits in αB-crystallin oligomer at 37 ˚C
We assumed that all subunits in the 26-mer αB-crystallin had an equal probability of exchanging randomly with any other subunit in the other oligomer. Isotopically heterogeneous oligomers comprising hydrogenated and deuterated subunits would be generated with the progress of time. Under the constraint of the number of exchangeable subunits n, we calculated the number of deuterated subunits x in isotopically mixed 26-mers of αB-crystallin and A n in the equilibrium states as follows: Considering the error bar of experimentally obtained A, almost all subunits of the 26-mer αB-crystallin were found to exchange at 37°C (Please refer to Fig. S3).
Detection of αB-crystallin monomer from nMS spectra.
Electrospray ionization (ESI) native mass spectrometry was used for the analysis of oligomeric state of αB-crystallin. The ESI tends to enable proteins highly charged states, which provides multi-valence peaks in mass spectrum even for one protein. In Fig. 3, the peaks of m/z 2240 and 2520 are corresponded to +9 and +8 charged states of αB-crystallin, respectively. From these ion series, the mass was estimated to 20159 Da, which is well consistent with the calculated molecular mass (20146 Da) of αB-crystallin monomer.

Bimolecular collision exchange model.
Firstly, we assumed that the subunit exchange could be described by random collision between two αB-crystallin oligomers (bimolecular collision exchange model), as shown in Fig. S7. Under such an assumption, it is considered that subunit exchange as a function of collision event step (m) can be given by the following equations.
where r, m, N, Δρ, f N correspond to average association number (26 or 40), collision event step, number of deuterated subunits in αB-crystallin oligomer, scattering contrast and fraction of deuterated subunit (N) in S4 αB-crystallin oligomer. In order to reproduce experimentally obtained time dependence of I 0_nor (t), the appropriate collision frequency (k) was selected. The dotted curves in Fig. S8 indicate the best fitted ones for the description of time dependence of I 0_nor (t) at 25, 37 and 48 ºC. It should be noted that the collision frequency could be evaluated from experimentally obtained parameters such as diffusion coefficient (D), sample concentration and so on, assuming the diffusion-limited reaction 2 . Fig. S9 shows the temperature dependence of collision frequency from above-simulation and experiment technique (C_dc). It can be clearly seen that temperature dependence of collision frequency (C_dc) differ from k, suggesting that subunit exchange of αB-crystallin cannot be explained under bi-molecular collision process.


extfieldsofstudy: Chemistry Medicine

text:
Photochemistry of 1,4-Dihydropyridine Derivatives: Diradical Formation, Delocalization and Trapping as a Route to Novel Tricyclic and Tetracyclic Nitrogen Heterocyclic Ring Systems

Irradiation of an acetonitrile solution of 4-aryl-3,5-dibenzoyl-1,4-dihydropyridine derivatives 1a–c and maleimides 2a–c using medium pressure Hg-arc lamp (λ > 290) nm afforded three different cycloadducts 4, 5, 6 in addition to the oxidation products 3. These results indicate that compounds 1a–c undergoes intermolecular cycloaddition reaction through three biradical intermediates and behave photochemically different than those reported previously for the analogous 3,5-diacetyl and 3,5-dicarboxylic acid derivatives. The present work also offers simple access to novel tricyclic and tetracyclic nitrogen heterocyclic ring systems of potential biological and synthetic applications. The structure of the photoproducts was established spectroscopically and by single crystal X-ray crystallography.

Introduction
1,4-Dihydropyridines have historically played a very important role in the synthesis and mechanistic organic chemistry. Derivatives of 1,4-dihydropyridine (DHP) are drugs belonging to a class of pharmaceutical agents known as calcium channel blockers. They are inhibitors of calcium ion penetration inside cells and weaken the contractility of the cardiac muscle. These compounds have been shown to be very effective vasodilators and are useful in the treatment of hypertension, ischemic heart disease, and other cardiovascular disorders [1][2][3][4][5][6]. Among the most important representative of this group of drugs are nifedipine (NPDHP) and felodipine (CPDHP).

Results and Discussion
Several photoirradiation experiments were carried out with 1a and 2a in order to optimize the reaction conditions, involving changes of solvent (CH2Cl2, toluene, CHCl3, and CH3CN), irradiation time, type of lamp (low or medium pressure Hg-arc lamps), the molar ratio of the two reactants (see Supplementary Materials). The optimum reaction conditions was achieved by using acetonitrile as a solvent, 3 h irradiation time, a 400 W medium pressure mercury arc-lamp (λ > 290 nm), a 1:3 molar ratio of 1,4-dihydropyridine 1a with maleimide 2a under a nitrogen atmosphere. These conditions were then applied to all photoreactions and the results are summarized in Table 1. Thus, irradiation of 1,4-dihydropyridines 1a-c with maleimides 2a-c using 400 W medium pressure mercury arc-lamp produced novel fused heterocyclic ring systems 4a-h, 5a-e and 6a, together with the photooxidation products, the 3,5-dibenzoyl-4-aryl-pyridine derivatives 3a-c (Scheme 2, Table 1). In this work, we describe the photochemical behavior of 4-aryl-3,5-dibenzoyl-1,4-dihydropyridine derivatives 1a-c and attempts to trap the possible intermediate diradicals with maleimides 2a-c.

Results and Discussion
Several photoirradiation experiments were carried out with 1a and 2a in order to optimize the reaction conditions, involving changes of solvent (CH 2 Cl 2 , toluene, CHCl 3, and CH 3 CN), irradiation time, type of lamp (low or medium pressure Hg-arc lamps), the molar ratio of the two reactants (see Supplementary Materials). The optimum reaction conditions was achieved by using acetonitrile as a solvent, 3 h irradiation time, a 400 W medium pressure mercury arc-lamp (λ > 290 nm), a 1:3 molar ratio of 1,4-dihydropyridine 1a with maleimide 2a under a nitrogen atmosphere. These conditions were then applied to all photoreactions and the results are summarized in Table 1. Thus, irradiation of 1,4-dihydropyridines 1a-c with maleimides 2a-c using 400 W medium pressure mercury arc-lamp produced novel fused heterocyclic ring systems 4a-h, 5a-e and 6a, together with the photooxidation products, the 3,5-dibenzoyl-4-aryl-pyridine derivatives 3a-c (Scheme 2, Table 1). The structure of all products has been established based on the 1 H-NMR, 13 C-NMR, HRMS and the X-ray crystal structures of photoproducts 4d, 5a, 5b and 6a (Figure 1). The 1 H-NMR spectra of compounds 5a-e showed interesting small coupling constants (J 0-2) for the vicinal ring protons (C-4, C-5 or C-1, C-29 shown in the X-ray of 5a) consistent with a dihedral angle around 80° as found by the X-ray structure of two of these compounds 5a,b.
(4d) (5a) The structure of all products has been established based on the 1 H-NMR, 13 C-NMR, HRMS and the X-ray crystal structures of photoproducts 4d, 5a, 5b and 6a ( Figure 1). The 1 H-NMR spectra of compounds 5a-e showed interesting small coupling constants (J 0-2) for the vicinal ring protons (C-4, C-5 or C-1, C-29 shown in the X-ray of 5a) consistent with a dihedral angle around 80˝as found by the X-ray structure of two of these compounds 5a,b. The structure of all products has been established based on the 1 H-NMR, 13 C-NMR, HRMS and the X-ray crystal structures of photoproducts 4d, 5a, 5b and 6a ( Figure 1). The 1 H-NMR spectra of compounds 5a-e showed interesting small coupling constants (J 0-2) for the vicinal ring protons (C-4, C-5 or C-1, C-29 shown in the X-ray of 5a) consistent with a dihedral angle around 80° as found by the X-ray structure of two of these compounds 5a,b.
(4d) (5a)  The formation of the different photo-adducts 4a-h, 5a-e, and 6a can be satisfactorily interpreted by the formation of the diradical intermediates A-D. Diradical A, which is formed from the initial excitation of the (n, π*), undergoes a hydrogen shift from the β-carbon to form diradical B, which then undergoes cycloaddition with 2 followed by dehydrogenation to give 4. Diradical A can also resonate to diradicals C, D and E. Trapping of the diradical C with 2a yields 6a, diradical C which is in resonance with D may also go through loss of H2 to give the pyridine derivatives 3a-c. Finally, trapping of diradical E with the appropriate maleimide gives the corresponding interesting tetracyclic system 5 (Scheme 3).  The formation of the different photo-adducts 4a-h, 5a-e, and 6a can be satisfactorily interpreted by the formation of the diradical intermediates A-D. Diradical A, which is formed from the initial excitation of the (n, π*), undergoes a hydrogen shift from the β-carbon to form diradical B, which then undergoes cycloaddition with 2 followed by dehydrogenation to give 4. Diradical A can also resonate to diradicals C, D and E. Trapping of the diradical C with 2a yields 6a, diradical C which is in resonance with D may also go through loss of H 2 to give the pyridine derivatives 3a-c. Finally, trapping of diradical E with the appropriate maleimide gives the corresponding interesting tetracyclic system 5 (Scheme 3). The formation of the different photo-adducts 4a-h, 5a-e, and 6a can be satisfactorily interpreted by the formation of the diradical intermediates A-D. Diradical A, which is formed from the initial excitation of the (n, π*), undergoes a hydrogen shift from the β-carbon to form diradical B, which then undergoes cycloaddition with 2 followed by dehydrogenation to give 4. Diradical A can also resonate to diradicals C, D and E. Trapping of the diradical C with 2a yields 6a, diradical C which is in resonance with D may also go through loss of H2 to give the pyridine derivatives 3a-c. Finally, trapping of diradical E with the appropriate maleimide gives the corresponding interesting tetracyclic system 5 (Scheme 3).  Furthermore irradiation of 1a alone in acetonitrile solution leads to the corresponding oxidized pyridine derivative 3a. Under the same conditions irradiation of diethyl 4-phenyl-1,4 -dihydropyridine-3,5-dicarboxylate (7) produced the cage compound 8 as shown in Scheme 4 and Figure 2. These facts showed different photochemical behavior for compounds 1a-c than those reported for the other dihydropyridine derivatives. The possibility of the formation of compound 6a by [4 + 2] thermal cycloaddition reaction has also been excluded, when attempts to react 1a with 2a at temperature of 180˝C for 1 h, gave only the unreacted starting material.

General Information
Melting points were recorded on a Gallenkamp apparatus (London, United Kingdom) The UV-Vis absorption spectra were scanned by using a Cary 5 instrument (Agilent, Santa Clara, CA, USA) with dry, clean quartz cuvettes of 1.0 cm path length. IR spectra were recorded in KBr disks on JASCO FTIR 6300 spectrophotometer (JASCO, Easton, MD, USA). 1 H-NMR (400 MHz or 600 MHz) and 13 C-NMR (100 MHz or 150 MHz) spectra were recorded on a DPX 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were measured on (GC-MS DFS) (high resolution, high performance, tri-sector GC/MS/MS (Thermo, Bremen, Germany) and by LC-MS using LC-MS DFS (Thermo). with an API-ES/APCI ionization mode. X-Ray single crystals data were performed using a Rapid II (Rigaku, Tokyo, Japan) and X8 Prospector diffractometer (Bruker) An annular reactor model APQ40 (Applied Photo-Physics Ltd., RG 49PA, England, UK) fitted with a 400 W (λ > 290 nm) medium pressure mercury arc-lamp was used for the irradiation. The starting 1,4-dihydropyridine derivatives 1a-c were synthesized as described recently [18]. procedure for the photoreaction of 1,4-dihydropyridines 1a-c with maleimides 2a-c A solution of each of 1a-c (1 mmol) and the appropriate 2a-c (3 mmol) in acetonitrile (100 mL) in a Pyrex tube was purged with nitrogen for 20 min, and then irradiated under N2 atmosphere for 3

General Information
Melting points were recorded on a Gallenkamp apparatus (London, United Kingdom) The UV-Vis absorption spectra were scanned by using a Cary 5 instrument (Agilent, Santa Clara, CA, USA) with dry, clean quartz cuvettes of 1.0 cm path length. IR spectra were recorded in KBr disks on JASCO FTIR 6300 spectrophotometer (JASCO, Easton, MD, USA). 1 H-NMR (400 MHz or 600 MHz) and 13 C-NMR (100 MHz or 150 MHz) spectra were recorded on a DPX 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were measured on (GC-MS DFS) (high resolution, high performance, tri-sector GC/MS/MS (Thermo, Bremen, Germany) and by LC-MS using LC-MS DFS (Thermo). with an API-ES/APCI ionization mode. X-Ray single crystals data were performed using a Rapid II (Rigaku, Tokyo, Japan) and X8 Prospector diffractometer (Bruker) An annular reactor model APQ40 (Applied Photo-Physics Ltd., RG 49PA, England, UK) fitted with a 400 W (λ > 290 nm) medium pressure mercury arc-lamp was used for the irradiation. The starting 1,4-dihydropyridine derivatives 1a-c were synthesized as described recently [18]. procedure for the photoreaction of 1,4-dihydropyridines 1a-c with maleimides 2a-c   A solution of each of 1a-c (1 mmol) and the appropriate 2a-c (3 mmol) in acetonitrile (100 mL)

General Information
Melting points were recorded on a Gallenkamp apparatus (London, United Kingdom) The UV-Vis absorption spectra were scanned by using a Cary 5 instrument (Agilent, Santa Clara, CA, USA) with dry, clean quartz cuvettes of 1.0 cm path length. IR spectra were recorded in KBr disks on JASCO FTIR 6300 spectrophotometer (JASCO, Easton, MD, USA). 1 H-NMR (400 MHz or 600 MHz) and 13 C-NMR (100 MHz or 150 MHz) spectra were recorded on a DPX 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were measured on (GC-MS DFS) (high resolution, high performance, tri-sector GC/MS/MS (Thermo, Bremen, Germany) and by LC-MS using LC-MS DFS (Thermo). with an API-ES/APCI ionization mode. X-Ray single crystals data were performed using a Rapid II (Rigaku, Tokyo, Japan) and X8 Prospector diffractometer (Bruker) An annular reactor model APQ40 (Applied Photo-Physics Ltd., RG 49PA, England, UK) fitted with a 400 W (λ > 290 nm) medium pressure mercury arc-lamp was used for the irradiation. The starting 1,4-dihydropyridine derivatives 1a-c were synthesized as described recently [18].

General procedure for the photoreaction of 1,4-dihydropyridines 1a-c with maleimides 2a-c
A solution of each of 1a-c (1 mmol) and the appropriate 2a-c (3 mmol) in acetonitrile (100 mL) in a Pyrex tube was purged with nitrogen for 20 min, and then irradiated under N 2 atmosphere for 3 h at room temperature using a 400 W (λ > 290 nm) medium pressure mercury arc-lamp. The reaction progress was monitored by TLC. The solvent was removed under reduced pressure and the resulting residue was subject to column chromatography on silica gel (70-230 mesh) using ethyl acetate/petroleum ether (b.p. 60-80˝C) as eluent to give the corresponding photoproducts.


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Effects of the Hydration State on the Mid-Infrared Spectra of Urea and Creatinine in Relation to Urine Analyses

When analyzing solutes by Fourier transform infrared (FT-IR) spectroscopy in attenuated total reflection (ATR) mode, drying of samples onto the ATR crystal surface can greatly increase solute band intensities and, therefore, aid detection of minor components. However, analysis of such spectra is complicated by the existence of alternative partial hydration states of some substances that can significantly alter their infrared signatures. This is illustrated here with urea, which is a dominant component of urine. The effects of hydration state on its infrared spectrum were investigated both by incubation in atmospheres of fixed relative humidities and by recording serial spectra during the drying process. Significant changes of absorption band positions and shapes were observed. Decomposition of the CN antisymmetric stretching (νas) band in all states was possible with four components whose relative intensities varied with hydration state. These correspond to the solution (1468 cm–1) and dry (1464 cm–1) states and two intermediate (1454 cm–1 and 1443 cm–1) forms that arise from specific urea–water and/or urea–urea interactions. Such intermediate forms of other compounds can also be formed, as demonstrated here with creatinine. Recognition of these states and their accommodation in analyses of materials such as dried urine allows more precise decomposition of spectra so that weaker bands of diagnostic interest can be more accurately defined.

Introduction
There is a rapidly increasing literature on applications of vibrational infrared spectroscopy to analyses of complex biological tissues and fluids in order to identify unique signatures of cell types, diseased tissues and diagnostic markers of specific diseases. [1][2][3][4][5][6][7][8][9][10] The mid-infrared (MIR) spectra of such materials are inevitably complex combinations of many overlapping infrared (IR)-active components. Diagnostic features of interest are in general minor components of these spectra whose small IR intensities are usually overlapped by much stronger bands from more dominant components.
Biological samples, such as urine and thin tissue biopsy sections, are often dried before analysis. 4,7 Drying removes the strong, broad absorption bands of water and, in the case of measurements in attenuated total reflection (ATR) mode, increases the IR intensities of all other components by maximizing their concentrations across the IR-active pathlength into which the evanescent wave penetrates. When applying analytical protocols to IR data obtained from dried versus hydrated materials, it is important to take into account the fact that the IR spectra of many materials are different in aqueous solution and the fully dried states. However, a further complication is that some materials can also exhibit additional distinct states during drying whose IR characteristics differ from both the solution and fully dried forms. This can be an important additional consideration when analyzing materials in which residual water levels are not easy to remove in a consistent Urea, with its strong interactions with water, is a particularly clear example of such behavior. Its strong hydrogen bonding propensity results in its well-known chaotropic effects in aqueous solutions. 11 Molecular modeling of its interaction with an increasing number of water molecules suggests that the first four water molecules form the primary hydration sphere and three more form the secondary hydration sphere. [12][13][14] Additional types of urea-water complexes are also predicted. 12 Such urea-bound water molecules have been detected directly in aqueous solutions of urea by polarization-resolved mid-IR pump-probe spectroscopy; 15 bulk water molecules beyond the hydration sphere around urea are not significantly perturbed, 13,[15][16][17] although this has been questioned with nuclear magnetic resonance studies of long range order. 16 At high concentration, urea may also form dimers or higher order structures, 12,13,18,19 although several studies have questioned their existence. 17,20,21 Some of these controversies may arise due to the different nature of the probe between different spectroscopic techniques. For an extensive review see Idrissi. 21 Here, IR spectroscopy is used to demonstrate the different forms of urea and creatinine that can form at different hydration states. Urea and creatinine are two components of urine that are often measured clinically (using non-IR spectroscopic methods) and can be used as markers for urine concentration against which other components can be normalized. Several methods for quantitation of urea and creatinine in wet urine samples have been described that are based on their well-defined solution IR spectra. 6,9 For example, as described here, urea concentrations in aqueous solution or in wet urine can be quantitated by fitting a Gaussian component to its strong n as (antisymmetric C-N stretch) band at 1468 cm -1 . 22 When urea is completely dry this n as (CN) band shifts to 1464 cm -1 . However, at least two additional states of urea with a n as (CN) band at 1454 or 1443 cm -1 can be observed in partially hydrated urea. When whole urine is dried, the solid materials tend to trap these intermediate forms.
Hence, accurate protocols for component decomposition, or for comparison of ''wet'' and ''dried'' biological fluids and tissues, should accommodate such additional states.

Clinical samples
Five urine samples were collected from volunteer healthy donors at University College London and Imperial College London after obtaining informed consent. Urine samples were analyzed fresh. Urine sample composition varies greatly between individuals because it is heavily influenced by factors such as recent diet, hydration, body mass/composition and time of day. The average urea concentration of these samples was 202 mM (standard deviation: 102 mM), the average creatinine concentration was 5 mM (standard deviation: 3.7 mM) and no protein was detectable. These levels are within the expected ranges of healthy subjects.

Data collection
Spectra were recorded with a Bruker IFS/66 S FT-IR spectrometer, fitted with a liquid nitrogen-cooled MCT detector and a KBr beamsplitter. Experiments were run in ATR mode using a 3 mm diameter silicon microprism with ZnSe optics (three reflections; SensIR). Data were recorded at room temperature at 4 cm -1 resolution. Power spectra were computed by Fourier transformation of 1000 (background; clean crystal surface) or 500 (sample) averaged interferograms. Absorbance spectra between 4000-800 cm -1 were then calculated from -log(sample intensities/background intensities). Cited frequencies are accurate to approximately 1 cm -1 .

Controlled rehydration of urea
3 mL of a 50 mM urea solution, prepared in double distilled water, were dried onto the crystal with a gentle stream of dry nitrogen gas at a flow rate of 300 mL/min. This led to loss of liquid water as evident from bands in the 3500-3100 and 1700-1600 cm -1 regions. Drying was continued until the absorbance spectrum was stable in the 1700-1400 cm -1 region. The sample was then allowed to equilibrate for several hours in atmospheres of controlled humidities, imposed by placing different saturated salt solutions (Table 1) in a well within a chamber that enclosed the sample ( Figure S1). Spectra were recorded when the 1700-1400 cm -1 region had stabilized. 23 Serial Dehydration of Urea and Creatinine Solutions and Urine 3 mL of a 50 mM urea solution were dried onto the crystal and rehydrated by placing double distilled water in the humidification chamber described above. Once the spectrum had stabilized, the humidification chamber was removed and a series of spectra (each an average of 10 interferograms; approximately 4 s data acquisition time) were recorded whilst dry nitrogen gas was passed over the sample until the sample had stabilized (typically 50-100 spectra over 3-7 minutes). Rehydrated and dried spectra of 10 mM creatinine solutions were recorded in the same manner.
The same serial dehydration protocol was followed with two urine samples from healthy donors that were diluted Â 4 with distilled water (to ensure that solute band intensities in the dried state did not exceed the instrument limit) before drying a 3 mL aliquot onto the crystal.

Data Processing and Component Fitting
In general, the thickness of the samples was greater than two microns and, therefore, was greater than the depth of penetration (approximately 0.6 microns) and sufficient to fill the entire active volume above the prism surface. Hence, in order to adjust the ATR spectra for the distortion caused by the wavenumber-dependency of the pathlength, 24 ATR data between 1510 and 1410 cm -1 were ramped before fitting of components by multiplying absorbance values by a factor of (wavenumber max /wavenumber) to allow for the inverse relation between effective pathlength and wavenumber. However, because the wavenumber range used for fitting is very narrow (100 cm -1 ), this correction is extremely small and, in practice, does not significantly alter the fitting parameters or the relative areas of the best fits.
Urea spectra were then decomposed using curve fitting analysis with combinations of Gaussian or pseudo-Voigt functions 25-28 using Origin version 8.6 (OriginLab Corporation). A single full width at half-maximum (FWHM) was used to describe the combined Gaussian and Lorentzian components of the pseudo-Voigt function and m u values were assigned to each component to define Lorentzian contributions. A linear baseline was also included, with offset and slope parameters allowed to vary during the fitting. With this protocol, four components were sufficient to fit all spectra at different stages of drying (see below). A similar protocol was used to decompose spectra between 1520 and 1470 cm -1 of rehydrated creatinine during drying using combinations of Lorentzian 25,26 and pseudo-Voigt functions.
Combinations of these same four urea components, together with an additional component from creatinine at 1492 cm -1 , were fitted to the 1510-1410 cm -1 region of spectra of urine at different stages of dehydration, again with baseline offset and slope parameters being allowed to vary during the fitting and with all other parameters fixed according to Tables 2 and 4.
In cases of compounds in dilute solutions where bulk liquid water would have made a significant contribution to the spectral region used (solution spectrum of Figure 1 and Figure 2), the absorbance contributions of bulk water were removed by fractional subtraction of a spectrum of pure liquid water; optimal subtraction was assessed by minimization of the OH stretching modes in the 3400 cm -1 region and the combination band between 2200 and 2000 cm -1 .

Structure and Frequency Calculations
Infrared spectra of urea-water and urea-urea molecular ensembles were modeled. Firstly, nominal molecular models were built by stepwise addition of water or urea using the computer program Facio, version 18.6.2. 29 Structures were then energy minimized using Gaussian 09 Table 3 Predicted frequencies of n as (CN) band of urea in combination with water or other urea molecules. Calculations were performed using Gaussian 09. 29 Where larger urea ensembles were modeled, only the frequencies of those ureas with the highest number of H-bonds to their carbonyl oxygen are listed.

Kinetic Resolution of Multiple Urea Hydration States
As expected with a small molecule that forms multiple hydrogen bonds with water, the majority of bands of urea change their frequencies and relative intensities between the solution and fully dried states. Dehydration resulted in the loss of the broad absorbance bands of liquid water in the 1700-1600 cm -1 and 3700-2900 cm -1 ranges, with concurrent intensification of narrower bands of urea in these regions (Figures 3a and 3b). The rehydrated spectrum of urea is very similar to that of urea in solution and the fully dried spectrum is essentially the same as that of pure, crystalline urea ( Figure S2a) 22,32 . However, the behavior was clearly not a simple transition between these two states. For example, the n as (CN) band in the rehydrated spectrum was centered at 1466 cm -1 , close to its position at 1468 cm -1 in the spectra of urea in solution at physiological concentrations ( Figure S2b). As the sample became dehydrated this peak gradually downshifted, transiently reaching a minimum at 1448 cm -1 before rapidly upshifting to the 1464 cm -1 position that is characteristic of the fully dried state (Figures 3c and 3d).

Stepwise Rehydration of Urea
By controlling the relative humidity of the surrounding atmosphere, it was possible to stabilize the intermediate  partially hydrated forms of urea. Figure 1 summarizes the peak positions of the n as (CN) band after stabilization at different relative humidities. As the relative humidity decreased, the peak (1468 cm -1 in solution) downshifted, settling at 1456 cm -1 at 79% humidity. However, as the relative humidity was further decreased, the peak upshifted towards the fully dried value of 1464 cm -1 .

Component Fitting to Urea Spectra
The n as (CN) normal mode of urea is the only contributor to the 1510-1410 cm -1 region. Combinations of Gaussian and pseudo-Voigt components were fitted to this region of typical spectra obtained during the transition from hydrated to fully dried forms. Before fitting, a correction was made for the ramping of spectral intensities caused by recording in ATR mode (see Methods). Four components (Table 4) were required to adequately fit all spectra in this wavenumber range based on the root mean square error compared to a three or fewer component fit. Fitting with additional components did not improve the error of the fit.  The fitting data with these components are shown in Figure 4. For reference, the best fit of combinations of these same components to this region of urea in solution is shown in Figure 2; this is dominated by the Gaussian component centered at 1468 cm -1 that also dominates the rehydrated spectrum ( Figure 4a). As the rehydrated sample dried, the 1468 cm -1 component diminished and components at 1454 and 1443 cm -1 emerged (Figure 4b). Figure 4 Component fitting to the n as (CN) band of urea during the transition from rehydrated to dry states. Four spectra were selected from the series obtained during drying of rehydrated urea of Figure 1. Times from beginning of the dehydration are indicated on each panel. Before fitting, spectra were corrected for effects of ramping caused by recording in ATR recording mode, followed by optimal fitting of the intensities of the four components of fixed frequencies and bandwidths (Table 4) (Figure 4c) before the band rapidly upshifted to the 1464 cm -1 form that is characteristic of fully dried urea (Figure 4d).

Hydration States of Creatinine
Hydration-dependent variations in absorption band frequencies and intensities occur with other urinary components, though none that were studied were as remarkable as those seen with urea. Creatinine is another major urinary component and has an absorption band at 1492 cm -1 in solution that partly overlaps with the n as (CN) absorption band of urea. Based on previously published work 33,34 and frequency predictions performed in Gaussian 09 of pure creatinine (see Methods), this band can be assigned to a single complex normal mode (n(C ¼ N) (30) n(CN) (27) (NCH) (24)). Spectra of a rehydrated sample during drying were recorded ( Figure 5) and components were fitted to the data between 1520 and 1470 cm -1 after correction for the ramping effect of recording in ATR mode. The 1492 cm -1 band upshifted during dehydration to an intermediate position at 1501 cm -1 (Figure 5c). Continued drying resulted in a slight downshift to 1499 cm -1 , the same position as in dry, crystalline creatinine (not shown). In both solution and rehydrated states, this band can be approximately described by a single Lorentzian component centered at 1492 cm -1 with a FWHM of 24 cm -1 (Figure 6a and Table 2). After drying, or in crystalline creatinine, the band approximates to a single Lorentzian component at 1499 cm -1 with a FWHM of 29 cm -1 (Figure 6d). However, an additional Gaussian component at 1501 cm -1 with a FWHM of 23 cm -1 is required in order to adequately describe spectra in the partially hydrated conditions (Figure 6b). All spectra could be decomposed adequately with combinations of these three components.

Hydrated Forms of Urea in Dried Urine Samples
The 1510-1410 cm -1 region of spectra of rehydrated human urine samples from healthy donors during drying was recorded. Two representative samples are shown in Figure 7. This region is dominated by the n as (CN) band of urea, together with a smaller contribution from the 1492 cm -1 band of creatinine. Spectra were analyzed by fitting the four components of pure urea (Table 4) together with additional smaller contributions from the possible forms of creatinine (Table 2) and unrestricted linear sloping baselines.
In the rehydrated state, as expected, the urine spectrum is dominated by the 1468 cm -1 form of urea together with a Figure 6 Curve fitting to the 1520 and 1470 cm -1 region of the FT-IR spectrum of creatinine during the transition from rehydrated to dry states. 3 mL of a 10 mM solution of creatinine were firstly dried onto the crystal surface and then rehydrated in a water-saturated atmosphere. Four spectra were selected from the series obtained during drying of this rehydrated sample. Spectra shown were corrected for effects of ramping caused by recording in ATR recording mode, followed by optimal fitting of the intensities of the three components of fixed wavenumbers and bandwidths (Table 2) 10-15% contribution from the 1492 cm -1 form of creatinine (Figure 7a and 7c). This is similar to that observed with fresh, liquid urine ( Figure S3). However, even after drying by extensive exposure to the dry nitrogen flow, the urea components (Figure 7b and 7d) corresponded to those of its partially hydrated (1454 and 1443 cm -1 ) states (Figure 4c), rather than the 1464 cm -1 band of the fully dried form. Because the enhancement of the urea band intensity on drying is much greater than that of creatinine (compare Figures 3c and 5), the creatinine contributions to this region of the dried spectra become much smaller ( 3%). In Figure 7b and 7d, a single component at 1492 cm -1 , corresponding to hydrated creatinine, appears adequate, consistent with water retention in the dried urine samples, though accurate assignment to its different states cannot be made with confidence because of its very low relative intensity.
The retention of intermediate hydration states of urea in dried urine samples is also clearly seen in the 3700-2900 cm -1 region of spectra of dried urine (Figure 8). Whereas it is clear that the broad band of liquid water has been lost, indicating that no major bulk liquid water remained, the remaining urea bands resembled those of the partially hydrated forms, rather than those of the fully dried states (Figure 3b). Hence, again, it is clear that drying had trapped predominantly the intermediate states of urea, rather than the fully dried form.

Factors in Urine Affecting Observed Behavior
The question arises as to what factor(s) control the different behaviors of urea when dried from pure solution versus urine. A ''simulated urine'' solution was prepared containing 115 mM urea, 50 mM NaCl, 29 mM NH 4 Cl, 19 mM KH 2 PO 4 , 10 mM K 2 SO 4 , and 5 mM creatinine, buffered to pH 6 with potassium hydroxide. 35 When a 3 mL sample was dried onto the ATR crystal, the n as (CN) band of urea stabilized around 1445 cm -1 (Figure 9), reproducing the behavior of urea in urine. However, the relative amount of the 1464 cm -1 component formed on drying increased when one of the major components (ammonium chloride, creatinine, or phosphate) was omitted, or if the mixture was diluted substantially before drying a 3 mL sample. In contrast, if one of these components was omitted but a replacement solute (e.g., sodium sulfate) was added to maintain the solute mass, then the urea again failed to form the 1464 cm -1 component. Furthermore, if urine samples were extensively diluted, then a significant fraction of the 1464 cm -1 form of urea appeared on drying. It is therefore concluded that the urinary effect is a non-specific one, caused simply by the mass of dried components (in urine, urea accounts for only 25-50 % of total solutes) creating a surface barrier that prevents complete dehydration of the material close to the crystal surface.

Electronic Structure Modeling and Physical Basis of Intermediates
Changes in the spectra of pure urea during dehydration must arise from changes in urea-water and/or urea-urea interactions. A similar phenomenon has already been reported by Grdadolnik and Maréchal 13 and Jung et al 18 who observed a shift in the urea n as (CN) band in very concentrated solutions; these were assigned to specific urea-urea and/or urea/water interactions in the limited water environments. In aqueous solution, urea interacts directly with four water molecules. [12][13][14] Gaussian 09 30 software was used to calculate the IR spectra of urea alone, in different hydration states and in interactions with other urea molecules. The predicted wavenumber of the n as (CN) band (Table 3) downshifted from 1466 to 1386 cm -1 as the number of urea-bound water molecules decreased from 5 to 0. This downshift was due to the loss of H-bonding interactions, in particular between water hydroxyls and the carbonyl oxygen of urea. 13 In support of this, the most dramatic predicted changes corresponded to the removal of the last two waters which, in our simulations, were those interacting with the carbonyl group.  Conversely, the predicted wavenumber of the n as (CN) band of anhydrous urea upshifted from 1386 to 1445 cm -1 as its carbonyl oxygen made an increasing number of H-bonds with adjacent urea -NH 2 group(s) ( Table 3). Indeed, in an ensemble of urea molecules, multiple n as (CN) frequencies were predicted. However, the highest wavenumber correlated with those ureas with the greatest number of H-bonding interactions of their carbonyl oxygen. Although the precise peak positions predicted during the simulation vary from those observed in the experimental data, these calculations, at least qualitatively, support a proposal that the experimentally observed initial wavenumber downshift of the n as (CN) band on drying is due to dehydration of urea, and the subsequent upshift arises from direct ureaurea interactions as the last waters are removed.

Conclusions
In the context of IR analyses of urinary samples, at least four forms of urea with distinct IR spectra should be considered, with their relative amounts dependent on hydration level. These correspond to the rehydrated form (equivalent to the solution form), a dried form and two additional partially hydrated forms. Additional hydration states of creatinine can also be formed, though the IR differences between them are much weaker than those of urea.
Spectra of dried urine samples show that the partially hydrated states of urea tend to become trapped in the dense matrix of dried urinary components, with a predominance of the lowest wavenumber (1443 cm -1 ) form, preventing direct urea-urea interactions. The retention of such partially hydrated forms with altered IR signatures complicates the analysis of IR spectra of dried samples such as urine, and it is important that they are recognized in diagnostic analyses of dried biological tissues and fluids. However, the limited number of distinct forms can easily be accommodated in decomposition procedures for quantitative analyses, as shown here with the contributions of different states of urea in dried urine samples.


s2fieldsofstudy: Chemistry Biology

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Logarithmic Partition Coefficient Comparison Study and Molecular Weight of Synthesized Prodrugs of Ibuprofen+Paracetamol, Diclofenac Sodium+Paracetamol and Ibuprofen+Diclofenac Sodium

Prodrug is a substance which after administration is metabolized into a pharmacologically active drug. Actually prodrug has least medicinal value in in-vitro/in-vivo but after biotransformation by metabolism in invivo it releases the active medicament. A drug is a substance which is a chemical entity, has definite structural skeleton, obtained by natural or synthetic or semisynthetic source, which can fit on bioreceptor platform having controlling capacity to control over the biochemical malfunction. Every drug is xenobiotic because it is coming from outer source (xeno) and active in biological unit (biotic). Prodrug is the precursor of drug which is made by derivatization of the same to enhance the bioavailability by pharmacokinetics, lipid solubility by partition coefficient and increase the physicochemical and biochemical parameters by pharmacodynamics. Ibuprofen, Diclofenac and Paracetamol have been taken as NSAID (Non-Steroidal Anti Inflammatory Drug) and three prodrugs have been synthesized by reacting with acid chloride of ibuprofen and diclofenac with paracetamol to get prodrug of ester linkage and acid chloride of ibuprofen has been reacted with diclofenac to get prodrug of amide linkage. All three prodrugs showed different logP values and molecular weights according to the solubility parameters and electronegativity: logP profile: Prodrug-C>Prodrug-B>prodrug-A; molecular weight profile: Prodrug-C>Prodrug-B>prodrug-A.

INTRODUCTION
Three prodrugs (Prodrug-A, Prodrug-B and Prodrug-C) have been successfully synthesized and have shown different melting points from individual parent drugs (Ibuprofen, Diclofenac sodium and Paracetamol) which indicates the authenticity of fulfillment of prodrug synthesis 1-2 . The synthesized prodrugs were characterized by MP, logP values and IR (Infrared) spectrum for structural authentification. Their solubility parameters also found different from parent drugs. Prodrug is a substance having no medicinal importance but after biotransformation in GIT it releases the parent drug which is able to show the pharmacological activity. [3][4][5] (2,6-dichlorophenyl) amino]phenyl}acetic acetate) is a nonsteroidal anti-inflammatory drug (NSAID) taken or applied to reduce inflammation and as an analgesic reducing pain in certain conditions. logP=4.51, pKa=4.15. Diclofenac sodium m.p.=theoretical (283-285°C), practical (282°C) Paracetamol [C 8 H 9 NO 2 ; MW=151] (N-(4hydroxyphenyl)acetamide), also known as acetaminophen or APAP, is a medication used to treat pain and fever. logP=0.46, pKa=9.38. Paracetamol m.p.=theoretical (169°C), practical (170°C) (Figure 1).
Ibuprofen, Diclofenac and Paracetamol all comes under NSAID and all three are acidic in nature. Ibuprofen and diclofenac both have free carboxylic acid (-COOH) group and paracetamol has free phenolic group (-OH). The idea of formation of prodrug by joining of free carboxylic acid (-COOH) group of ibuprofen and diclofenac with phenolic group (-OH) of paracetamol by converting free carboxylic acid (-COOH) group into acid chloride (-COCl) and conjugated with free phenolic group (-OH) by benzoylation reaction to get the three desired compounds [Prodrug-A and Prodrug-B having ester (-COO-) linkage. Acid chloride (-COCl) of ibuprofen when reacts with imino group (-NH) of diclofenac then it produces Prodrug-C having amide (-CONH-) linkage ( Figure 2). 6

Chemistry
Ibuprofen (2 g, 0.01 m) has been reacted with 5 ml thionyl chloride and refluxed for 30 mins in moisture free environment until all ibuprofen has been dissolved. The reaction mixture was heated on water bath to remove excess thionyl chloride. It was then cooled in ice and paracetamol (1.5 g, 0.01 m) dissolved in methanol was added in it with shaking. Oily droplets separates out which was then mixed with ice water and kept in ice to solidify the droplets into white solid of prodrug-A. It was filtered and %yield and m.p. has been recorded for dried mass. Prodrug-A (logP=4.56, nonpolar) is a combination of ibuprofen (logP=3.97, nonpolar) and paracetamol (logP=0.46, polar) produced a derivative of semipolar unit. MP=162-164°C. Diclofenac sodium (3 g, 0.01 m) has been reacted with 10 ml thionyl chloride and refluxed for 30 mins in moisture free environment until all diclofenac sodium has been dissolved. The reaction mixture was heated on water bath to remove excess thionyl chloride. It was then cooled in ice and paracetamol (1.5 g, 0.01 m) dissolved in methanol was added in it with shaking. Oily droplets separates out which was then mixed with ice water and methanol and kept in ice to solidify the droplets into white solid of prodrug-B. It was filtered and %yield and m.p. has been recorded for dried mass. Prodrug-B (logP=4.90, nonpolar) is a combination of diclofenac sodium (logP=4.51, nonpolar) and paracetamol (logP=0.46, polar). MP=162-165°C, %yield=88.65.
Ibuprofen (2 g, 0.01 m) has been reacted with 5ml thionyl chloride and refluxed for 30mins in moisture free environment until all ibuprofen has been dissolved. The reaction mixture was heated on water bath to remove excess thionyl chloride. Diclofenac sodium (3 g, 0.01 m) was dissolved in acetone and added slowly to this acid chloride mixture with shaking in ice bath to get Prodrug-C after addition of water. Prodrug-C (logP=6.13, nonpolar) is a combination of ibuprofen (logP=3.97, nonpolar) and diclofenac sodium (logP=4.51, nonpolar), MP=162-165°C, %yield=91.32. The beauty of reaction shows that diclofenac sodium which is sodium salt that releases free diclofenac during reaction between thionyl chloride in acid environment to get free -COOH group which then reacts with thionyl [

Future Target
Our future goal is to perform in-vitro biotransformation of these prodrugs by acidic and alkaline hydrolysis of both ester (-COO-) and amide (-CONH-) linkages into free drugs and chromatographically separation of their Rt in HPLC. Study of in-vitro biotransformation of prodrugs of ester and amide linkages of ibuprofen, diclofenac sodium and paracetamol in acidic and alkaline medium will be our next target after this project. Since both ester (-COO-) and amide (-CONH-) linkages are susceptible for hydrolysis in both acidic pH (gastric pH) and basic pH (intestinal pH) to produce parent drug ibuprofen, diclofenac and paracetamol by biotransformation; so it will be implemented as a prodrug which can show prolong action on pain and fever after getting release into free parent drug by biotransformation (Figure 4).
The HPLC (High Performance Liquid Chromatography) study will report the retention time (R t ) and release kinetics of three prodrugs by taking HPLC degradation datas of three samples of prodrugs and individual HPLC datas of parent drugs separately to compare the R t value of release of three drugs from prodrugs.
Prodrug-A (logP=4.56) will release Ibuprofen and Paracetamol, Prodrug-B (logP=4.90) will release Diclofenac sodium and Paracetamol and Prodrug-C (logP=6.13) will release Ibuprofen and Diclofenac. This will be a comparison study of drug release.


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Application of Inelastic Neutron Scattering to the Methanol-to-Gasoline Reaction Over a ZSM-5 Catalyst

Abstract Inelastic neutron scattering (INS) is used to investigate a ZSM-5 catalyst that has been exposed to methanol vapour at elevated temperature. In-line mass spectrometric analysis of the catalyst exit stream confirms methanol-to-gasoline chemistry, whilst ex situ INS measurements detect hydrocarbon species formed in/on the catalyst during methanol conversion. These preliminary studies demonstrate the capability of INS to complement infrared spectroscopic characterisation of the hydrocarbon pool present in/on ZSM-5 during the MTG reaction. Graphical Abstract

Introduction
The conversion of alcohols to hydrocarbons was first introduced in the Mobil methanol-to-gasoline (MTG) process using an HZSM-5 catalyst, commercialised in New Zealand in 1986. Lurgi's methanol to olefins (MTO) process, also using HZSM-5, UOP-Statoil's MTO process using a SAPO-34 catalyst, and Topsoe's improved gasoline synthesis (TIGAS) using a proprietary zeolite catalyst followed. The availability of cheap methanol derived from natural gas was the initial driver for these technologies. More recently, methanol derived from coal has become the source of transport fuels or olefin feedstocks via MTG or MTO type processes. In the future biomass and other renewable resources are likely to provide a ready supply of methanol, e.g. through gasification and subsequent hydrogenation of CO/CO 2 , or via direct oxidation of methane produced by aerobic digestion. MTG and MTO processes therefore provide a route to fuels and chemicals from renewable feedstocks.
The past 30 years have seen numerous investigations of the reaction pathways and mechanisms by which methanol is converted to hydrocarbons over acid zeotype catalysts, as reviewed recently for example in references [1][2][3][4]. Three different components of the reaction pathway can be distinguished: (1) the initial reaction steps in which methanol reacts with acid sites in the zeolite or SAPO catalysts; (2) the formation of hydrocarbon products during steady-state working conditions; and (3) the catalyst deactivation through so-called coke formation. As in any catalytic system, understanding the reaction pathway is essential if optimum product selectivity and catalyst performance are to be achieved.
Most attention in the last 10 years has focussed on the reactions occurring under steady-state working conditions. The so-called 'hydrocarbon pool' mechanism has found widespread support [2,[5][6][7]. In this mechanism, two catalytic cycles operate in parallel: alkenes are methylated and subsequently cracked in one cycle, while aromatics are methylated and subsequently dealkylated in the other. Experimental support for this mechanism has come from, for example, 13 C labelling studies, NMR and UV-VIS identification of polymethyl aromatic species occluded in working catalysts, and post reaction analysis of occluded species liberated from used catalysts by GC-MS. The differences in product distribution found between zeolites with different pore sizes have been rationalised in terms of different contributions from the two different cycles.
The mechanisms by which the hydrocarbon pool is initially formed from methanol are much less clear-cut (and this subject has been comprehensively reviewed in [1]). Infrared spectroscopy has been extensively used to investigate species formed when methanol first contacts the zeolite catalyst, and clear evidence obtained for the formation of reactive methoxy groups from reaction of methanol or dimethylether with Brønsted acid sites [8][9][10][11][12]. After longer reaction times at higher temperatures more complex infrared spectra develop which have been assigned variously to adsorbed methylaromatics and olefinic species [12,13]. Infrared spectroscopy on ZSM-5 is limited to the energy range 1350-4000 cm -1 as a consequence of the intense absorption bands below 1350 cm -1 due to Si-O and Al-O stretching vibrations of the zeolite framework (although Qian et al. [12] were able to observe a small window in the spectrum of SAPO-34 between 800 and 900 cm -1 ). A second limitation of the FTIR technique is that catalysts at a later stage in the reaction path become difficult to observe because of the presence of strongly absorbing species [13]; i.e. the catalysts become dark in colour. One alternative means of accessing the full vibrational spectrum of the catalyst at all stages of the reaction coordinate is to employ the technique of inelastic neutron scattering (INS) [14].
The use of INS to obtain vibrational spectra of molecular systems has been reviewed by Parker et al. [15,16]; highlighting how direct geometry INS spectrometers, e.g. MAPS [17], can provide supplementary and additional information to that achievable with indirect geometry INS spectrometers, e.g. TOSCA [17]; the latter being the more typical instrument of choice for molecular spectroscopy investigations by INS. That work also outlines an increasing trend of using of INS to examine a variety of heterogeneously catalysed reaction systems [15]. For example, following on from the work of Silverwood et al. who used INS to examine alumina-supported nickel catalysts applied to the dry [18-21] and steam [22] reforming of methane, Warringham and co-workers have used a combination of indirect [23] and direct [24][25][26] geometry INS spectrometers to investigate iron based Fischer-Tropsch synthesis (FTS) catalysts. Moreover, Warringham et al. [27] recently reported on sample environment details relevant to the acquisition of INS spectra of heterogeneous catalysts.
Against this background, it is timely to consider whether INS can be applied to look at MTG and/or MTO chemistry over zeolitic materials such as ZSM-5. Given the ability of INS to selectively probe hydrogeneous vibrational modes [15], it is enticing to discover whether the technique can provide new information on the adsorbed hydrocarbon species present at various stages of the methanol to hydrocarbon reaction. Very recently, O'Malley et al. [28] have used a combination of neutron scattering methods matched by ab initio calculations to investigate, amongst other things, the reaction of methanol with ZSM-5 at room temperature. That study showed methanol to be immobilized due to methoxylation, and that the formation of adsorbed methoxy groups is facile over this material at room temperature. In this paper, we present a preliminary report on the INS spectra of a commercial grade ZSM-5 catalyst exposed to a methanol feedstream at 623 K. With mass spectrometry indicating representative MTG chemistry, the reaction was stopped, quenched by rapid cooling to room temperature in flowing helium, and the catalyst sample taken to the INS spectrometer without exposure to air for spectral acquisition. The resulting ex situ spectra unambiguously reveal the presence of hydrocarbon species, which may be associated with a hydrocarbon pool. Whereas O'Malley et al. [28] concentrated on methanol exposure to ZSM-5 at room temperature, this communication describes an investigation performed at reaction temperatures representative of those utilised in MTG unit operations. Thus, INS is being used here to assess how hydrogen is partitioned within the catalyst matrix during conditions that correspond to the commercially relevant active phase of MTG operation. Although further work is necessary to examine spectra at different reaction times and to better correlate catalytic performance with the vibrational spectra, this short communication demonstrates the capability of INS to positively contribute to the understanding of this economically relevant but technically challenging reaction system.

Experimental
A commercial grade ZSM-5 catalyst (ACS Material LLC, USA; SiO 2 /Al 2 O 3 molar ratio, 38:1, specific surface area C250 m 2 g -1 , pore volume C0.25 ml g -1 , pore size *5 Å , bulk density *0.72 kg l -1 ) supplied in the form of column shaped pellets (diameter 2 mm, length 2-10 mm) was employed. Two Inconel reactors [29] were charged with the catalyst pellets. The first reactor was attached to a gas manifold apparatus [27] and the catalyst dried by the following procedure: whilst continually maintaining a flow of helium over the catalyst (1000 sccm, CK Gas, [99.0 %), the sample was heated to 623 K at a heating rate of 10 K min -1 and then maintained at that temperature for 2 h. The heating was stopped and the sample allowed to cool to ambient temperature in a continuous flow of He. The reactor was then isolated and transferred to an argonfilled glove box (MBraun UniLab MB-20-G, [H 2 O] \ 1 ppm, [O 2 ] \ 2 ppm) for loading into an aluminium INS cell that is sealed via an indium wire gasket [17]. The second sample was similarly dried but the dehydration stage was followed by dosing of the catalyst with methanol vapour by means of a bubbler arrangement whilst the catalyst was maintained at 623 K. The eluting stream was analysed by in-line mass spectrometry (Hiden Analytical, HPR-20) with the spectrometer connected to the reactor exit line via a differentially-pumped, heated quartz capillary. The apparatus is equipped with a 'catch-pot' downstream of the catalyst. Material collected there was subsequently analysed by GC-MS. After approximately 6 h on-stream, the methanol flow and heating were stopped, and the sample allowed to cool to ambient temperature in a continuous flow of He. The reactor was then isolated and transferred to an aluminium INS cell using the procedure outlined above. The two samples (dried catalyst and reacted catalyst) were then transported to the INS spectrometer and the spectrum recorded at 20 K.
A series of spectra were originally measured on the MAPS spectrometer using the high resolution A chopper package operating at incident energies of 4840 (frequency = 600 Hz) and 2016 cm -1 (frequency = 400 Hz) respectively. However, the spectrum of the reacted sample exhibited a poor signal:noise ratio, indicating a low degree of hydrogen retention by the catalyst. A previous use of INS by Warringham and co-workers to examine the hydrogenation of propyne over alumina-supported Pd catalysts showed how the enhanced sensitivity of the MER-LIN spectrometer could be used to acquire the vibrational spectra of minority species, albeit at reduced resolution compared to that attainable with the MAPS spectrometer [30]. Consequently, in an attempt to obtain superior spectra, both the dried and reacted samples were also run on the MERLIN spectrometer using the low resolution S chopper package operating at incident energies of 4840 (frequency = 600 Hz) and 2016 cm -1 (frequency = 400 Hz) respectively. Results are presented from both spectrometers.

Reaction Testing
Mass spectrometry showed the reaction system to exhibit an induction period lasting ca. 3 h, followed by operation at approximately steady-state conditions for the remainder of the reaction period investigated (3 h). Figure 1 shows the mass spectrometer profile for the catalyst exit stream after the induction period. The major peaks identified have m/z ratios of: 18 (H 2 O); and 28 (C 2 H 4 ,); 41 (C 3 H 5 ); 55 (C 4 H 7 , C 3 H 3 O); and 91 (C 7 H 7 ). These features indicate the formation of longer-chained hydrocarbons and oxygenates; while the m/z = 91 peak is indicative of methylated benzenes, e.g. toluene, xylenes, etc. the complementary GC-MS analysis of the catch-pot liquid, collected postreaction, shows some unconverted methanol, with smaller amounts of ethanol, alongside species identified as methylated benzenes (ranging from toluene through to hexamethylbenzene). In addition, a small quantity of heavier products, that may be substituted naphthalenes, were also present.
Collectively, these analytical results indicate that after 6 h on-stream the catalyst is operating in a pseudo-steady state regime, whereby methanol is converted to a range of higher molecular weight products that includes C 2 and C 3 olefins, as well as a number of alkylated benzenes. This distribution is typical of previous reports of MTG chemistry over ZSM-5 [1][2][3], although it is recognised that the catalyst is in the early stages of its reaction cycle with, for example, little evidence of a coking stage active over the short reaction period studied. This assumption is consistent with the observation that, post-reaction, the originally white pellets had changed to a mild grey colour; cutting across the pellet column revealed that the grey colouration was only present at the catalyst surface, with the inside of the pellets retaining their original clean white colouration. The presence of retained carbonaceous material on or within the catalyst was confirmed by temperature programmed oxidation (TPO). This indicated the presence of a relatively small quantity of carbonaceous species, however the oxidation temperature ranged from 473 to 973 K, thereby indicating that at least some of the species showed significant complexity and order; beyond that expected for short linear hydrocarbons. TPO results are therefore consistent with the presence of aromatic species in the postreaction sample.

Inelastic Neutron Scattering
INS is inherently a two-dimensional spectroscopy, being able to sample both energy (E, cm -1 ) and momentum transfer (Q, Å -1 ) [15,16]. This attribute is particularly accessible on direct geometry instruments such as MAPS and MERLIN [15,16]. A commonly adopted means of visualising this information is to use ''mitre'' plots [26], where the neutron scattering intensity is plotted as a function of energy (x, y axis) and momentum transfer (Q, x axis). Figure 2 shows the S(Q,x) mitre plots recorded on the MERLIN spectrometer for the clean (left hand frame) and reacted (right hand frame) samples with 4840 cm -1 incident energy. The reacted sample exhibits more intensity than the dried catalyst, indicating retention of hydrogenous species on reaction, consistent with the results of TPO. Further, there is a band of intensity at *3000 cm -1 , in addition to a feature at *3600 cm -1 that is present in both samples. Figures 3 and 4 show the spectra integrated over the low Q range 0 B Q B 12 Å -1 ; this results in the more conventional presentation of INS spectra (a two-  (Fig. 3a) shows a single band centred just below 3700 cm -1 . This is due to O-H stretching vibrations of the zeolite. Infrared spectra of dehydrated HZSM-5 show up to 3 component bands in this region, at around 3740 cm -1 (SiOH associated with external silanol groups), 3660 cm -1 (AlOH associated with extra-framework aluminium species) and 3610 cm -1 (the Brønsted acid, Si(OH)Al) [9]. The INS experiment lacks the spectroscopic resolution to resolve these species. The MERLIN spectrum of the clean zeolite in the region 1800-600 cm -1 (Fig. 4a) shows three bands at *800, 1080 and 1200 cm -1 . This is closely similar to the spectrum of dehydrated HZSM-5 reported in this region by Jobic et al. [31]. These authors assigned a band at 1080 cm -1 to the in-plane deformation of bridging OH groups (the Brønsted acid sites). A shoulder at 1210 cm -1 was attributed to either a combination of the in-plane OH deformation with a low frequency lattice mode, or possibly an in-plane deformation of a second type of hydroxyl group, while a band at 785 cm -1 was assigned to a framework mode coupled with proton motion. In dehydrated HNaY, on the other hand, a shoulder at *860 cm -1 has been assigned to the in plane deformation mode of silanol groups [32].
The MERLIN spectrum of the zeolite catalyst following reaction in methanol shows one additional strong band in the region 2000-4000 cm -1 centred at 3000 cm -1  (Fig. 3b). This is due to C-H stretching vibrations of adsorbed hydrocarbon species, but the spectral resolution of MERLIN does not allow a differentiation between aliphatic (\3000 cm -1 ) and olefinic or aromatic ([3000 cm -1 ) CH groups. The higher resolution of the MAPS spectrum (Fig. 3c) suggests that both are present (a peak at 2960 cm -1 with a higher frequency shoulder at *3080 cm -1 ). In the 600-1800 cm -1 region both the MERLIN (Fig. 4b) and MAPS (Fig. 4c) spectra show new bands at 1380 and 1450 cm -1 .
Together, Figs. 3 and 4 indicate the presence of aliphatic species in/on the catalyst. Bands at 1380 and 1460 cm -1 are typical of the symmetric and antisymmetric deformations of methyl groups. In their infrared study of ZSM-5 exposed to methanol at 370°C Palumbo et al. [13] attributed bands at 1380 and 1465 cm -1 to methyl groups in methyl substituted benzene rings, along with bands between 1500 and 1700 cm -1 due to C=C stretching vibrations of such species. No aromatic C-H stretching modes above 3000 cm -1 were detected in the infrared study of Palumbo et al., which was attributed to the complete methyl substitution of the aromatic rings. The shoulder detected above 3000 cm -1 in the MAPS spectrum (Fig. 3c) indicates however that some sp 2 hybridised C-H bonds, olefinic or aromatic, are present. In INS the intensity is directly proportional to the number of C-H oscillators, not their type [15], whereas in infrared spectroscopy the extinction coefficients can be widely different. Thus, INS is revealing the presence of hydrocarbonaceous species not readily observable by optical spectroscopy.
These preliminary experiments have demonstrated that INS can directly observe hydrocarbon species present in a working methanol to hydrocarbon catalyst. The INS measurements exploit the sensitivity of the technique to hydrogenous species. In comparison with infrared spectroscopy, the technique suffers from lower spectroscopic resolution, but offers several advantages in principle. Analysis can be undertaken directly (as here) on catalyst samples extracted from a working reactor. In particular, there is no restriction on catalyst particle size, or any need to undertake specific sample preparation measures. Furthermore, there is no restriction on the extent of coke formation that can be tolerated before spectra become obscured, and the full spectroscopic range of vibrational frequencies is accessible without obscuration from zeolite framework modes. The catalyst sample analysed here was obtained at a relatively early stage in the reaction profile, but a hydrocarbon pool is clearly already present. We will report later on similar analyses carried out on catalysts reacted for longer periods of time-on-stream, where the advantages over infrared spectroscopy should be even more evident. INS has the potential to be able to correlate catalytic performance with the degree and form of retained surface species. Such information will be valuable in extending our understanding of a role for a hydrocarbon pool in MTG and MTO chemistry.