Filip Bureš
email: filip.bures@upce.cz

Oldřich Pytela
email: oldrich.pytela@upce.cz

Milan Kivala

François Diederich
email: diederich@org.chem.ethz.ch

Solvatochromism as an efficient tool to study N,N-dimethylamino-and cyano-substituted π-conjugated molecules with an intramolecular charge-transfer absorption

Keywords: solvent effects, solvatochromism, UV/Vis spectroscopy, donoracceptor system, chromophore solvent effects, solvatochromism, UV/Vis spectroscopy, donor-acceptor system

Solvatochromism as an efficient tool to study N,Ndimethylamino-and cyano substituted π-conjugated molecules with an intramolecular charge-transfer absorption

INTRODUCTION

π-Conjugated organic molecules attract much attention due to their prospective application as efficient materials in organic electronics and optoelectronics. [START_REF] Forrest | Organic Electronic and Optoelectronics[END_REF][START_REF] Miller | Materials for Electronics[END_REF] A typical organic charge-transfer chromophore (D-π-A) consists of strong electron donors D (e.g. NR 2 or OR groups), strong electron acceptors A (e.g. NO 2 or CN groups), and a π-conjugated core featuring (hetero)aromatic rings and/or double or triple bonds. [3][4][5] Optical linear and nonlinear properties of such push-pull molecules depend on the polarizability of the electrons localized in π-bonding molecular orbitals. [6] Although the polarizability of a molecule is mainly given by its chemical structure, in particular by the length of the π-conjugated spacer and the electronic nature of the donors and acceptors attached, [7][8][9] it can also be affected by external factors such as strength of radiation, state of matter, and in solution also by the solvent used.

However, a general description of external factors (solvent) affecting optical and nonlinear optical properties is more complex. In our previous investigation we have studied solvent effects on the electronic absorption spectra of donor-substituted 11,11,12,12-tetracyano-9,10anthraquinodimethanes (TCAQs 10-12, Figure 1). [10,11] Optical properties of these model charge-transfer (CT) solvatochromic indicators were investigated in 32 solvents of various nature. The resulting data were evaluated by means of theoretical models and (semi)empirical correlations determining the optical properties related to electron distribution and polarizability. The present work focuses on organic D-π-A molecules with N,N-dimethylanilino (DMA) groups as donors and cyano groups as acceptors featuring various π-conjugated spacers (Figure 1). Whereas the first class of molecules is represented by cyanoethynylethenes 1-3 (CEEs), [12][13][14] the second class consists of three donor-substituted 7,7,8,8-tetracyanoquinodimethane-based molecules 4-6 (TCNQ-based). [15] Molecules 7-9 represent donor-substituted 1,1,4,4-tetracyanobuta-1,3-dienes (TCBDs). [16][17][18] The compounds shown in Figure 1 were chosen according to their different molecular properties and geometries. The chosen set comprises planar (1-3) or nonplanar (others) molecules, centrosymmetric (2) or nearly centrosymmetric (8, 11) compounds, quinoid (4-6) or fused quinoid (10-12) molecules, and molecules bearing CC triple bonds (1-3, 6, and 9) or 1,4-phenylene units as additional π-linkers and simultaneously a different number of DMA and cyano groups.

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A goal of this study is to investigate the relationships between the structure of D-π-A organic molecules and their solvatochromic properties and to find fundamental structural characteristics and solvent properties that can be used for a quantitative description of these relations.

RESULTS AND DISCUSSION

Data and methods

The change in the UV/Vis spectra of compound 5 in the wavelength range from 400 to 900 nm as a function of solvent is illustrated in Figure 2 (for complete spectra see Figures 1SI-45SI in Supporting Information). The absence of possible solute aggregation and concomitant influence on the position of the absorption maxima was verified by measurements of the UV/Vis spectra for 1-9 in dichloromethane (see SI) where the Lambert-Beer law was fully obeyed. The values of the longest-wavelength absorption maxima λ max measured in 32 representative solvents for compounds 1-9 are summarized in Table 1. The λ max values of TCAQs 10-12 were taken from the previous work. [10] All of the λ max values were converted for further evaluation into the wavenumbers ν having energy dimension. The CT character of the longest-wavelength absorption of compounds 1-9 was already confirmed by protonation/neutralization experiments. [10][11][12][13][14][15][16][17][18] _______________________ Figure 2 _______________________ _______________________ (principal component analysis PCA and factor analysis FA), and methods based on multiple linear regression (MLR) showed to be the most suitable.

The algorithm NIPAL [19] in Exner variation [20] was used for the calculation of the latent variables of the native matrix X in PCA. This algorithm involves modification of the coordinate origin for each column of the matrix of manifest variables during the calculation.

The FA factors were calculated from the correlation matrix R, covariance matrix C, and matrix X T X using nonlinear regression according to the basic factor model. Thus, it was possible to efficiently analyze non-standardized data with the same scale but with different origin in this way. Depending on the used method, this calculation provided separate sets of score T and loadings matrixes F, which were further physico-chemically interpreted.

Beside the quantum-chemical quantities used for the description of molecule properties, the structural characteristics expressed as a number of present structural features were also successfully employed. This method is common in QSAR (quantitative structure-activity relationships). With respect to the number of indicators and possible multicollinearity, the maximum number of the explanatory variables was limited to three. Selection of the most suitable explanatory variables in the linear regression was performed by the calculation for all possible combinations of the proposed set of variables according to the minimal residual standard deviation. The regressions with the selected combination of the explanatory variables were tested by regression diagnostic (including t-test, F-test, assessing multicollinearity, residual analysis) for statistic validity. A similar principle was used for the selection of the most suitable explanatory variables characterizing the used solvents.

Exploratory data analysis

Exploratory analysis is a simple and efficient tool for primary data screening. A mutual comparison of the longest-wavelength absorption maxima λ max of the intramolecular CT bands measured for compounds 1-9 in 32 different solvents (Table 1) through the polygon's size and shape is shown in carbonyls and two π-bonds in a mutual interaction. The spectral transitions are given by the local electron transitions on the "enedione" motive (so-called "quinoid transitions"). The substituents in the area of carbonyls or fused aromatic rings (see TCAQ's 10-12) may show their own local electron transitions (e.g. benzoid transitions in the case of benzene rings).

Thus, the interaction of "enedione" and "benzoid" moieties shows CT transitions accompanied by a bathochromic shift of λ max . From the polygon sizes in Figure 3, it is obvious that CT transitions between the "enedione" moiety and the fused benzene rings in molecular structures 10-12 are considerably weaker compared to those of the TCNQ-based molecular structures 4-6. This is presumably due to the electron inter-connection which can change the π-bond character of the "enedione" moiety.

_______________________ Figure 3 _______________________

The TCBD series of solvatochromic indicators 7-9 showed undoubtedly the least polygon's area. The main structural feature of these compounds is the buta-1,3-diene backbone with two appended terminal cyano groups in a non-coplanar arrangement with the donor DMA substituents at the positions 2 and 3 of the butadiene moiety. [17] The CT interaction between both parts is most probably not caused by the number of donors (one DMA in hydrogen bond interaction between the terminal nitrogens of the cyano groups and acidic hydrogens of the HBD solvents or with an interaction between the LUMO of indicator and the lone pairs of the HBA solvents. For the solvent classification see ref. [21] Both of these effects change the LUMO properties of the solvatochromic indicators and, consequently, also the intramolecular interaction and character of the electron transition. Last but not least, the observed "switch off" can be caused by the solvent properties. The molecules of protic solvents are hydrogen bonded to each other and, therefore, the cavity surrounding the solute (indicator) is more rigid. Such a rigid cavity suppresses the effective electron reorganization in the transition from the ground to the excited state, which results in a hypsochromic shift.

This effect can be observed selectively in dependence on the molecular structure of indicators.

A comparison of the experimental λ max values measured in methanol with those quantumchemically calculated (ZINDO-CI, methanol) seems to be interesting in this respect. Whereas the experimental λ max values of all indicatores exceeded the calculated ones (e.g. indicator 4calculated 569.2 nm, found 669.5 nm; indicator 8 -calculated 378.4 nm, found 470 nm), the experimental and calculated λ max values for 7 are quite similar (calculated 436.9 nm, found 443 nm).

Solvatochromism-structure relationships for D-π-A molecules

Factor analysis (FA) has been applied to evaluate a relation between solvatochromic indicators characterized by the ν values in different solvents. The FA proved to be more suitable than PCA because the calculated factor loadings do not suffer from unicity of the particular columns. The factor loadings calculated by the decomposition of the correlation matrix R, covariance matrix C, and matrix X T X were significantly different. The performed analysis and several attempts of their interpretation showed that the ν values themselves are crucial and depend on the coordinate origin as well as on the scale. Thus, the factor loadings calculated by the decomposition of the X T X matrix with two factors were used for interpretation of the structure-solvatochromism relationships.

_______________________

_______________________

The calculated f 1 and f 2 loadings are summarized in Table 2. Relationships of points corresponding to the particular indicators 1-12 in the plane of f 1 and f 2 are shown in Figure 4.

It can be clearly seen that indicators 3 and 7 are exceptional, which unambiguously corresponds with the results of the exploratory analysis (see above). In contrast to others, the TCNQ-based indicators 4-6 contain quinoid moieties and, therefore, their λ max are shifted more bathochromically. The near location of 5 and 6 shows, that the acetylene linker in 6 has a small impact on the CT transition in such class of compounds. On the other hand, a replacement of hydrogen in 4 with an additional DMA donor (5) led to a bathochromic shift (see also the polygon expansion in Figure 3). The location of indicators 3 and 7 in the upper part confirm their unique, above-discussed properties.

Interpretation of the factor loadings f 1 and f 2 (Table 2) through the calculated and/or structural characteristics summarized in Table 3 3). In connection with equation ( 1) we can deduce, that the chomophores featuring a low LUMO energy, "suitable π-conjugated enlargers", and rather low permanent dipole moment (less "electron rigid" molecules) will possess the most bathochromically shifted λ max .

In contrast to f 1 , the factor loadings f 2 significantly correlate only with the ν values in protic solvents (R>0.6, positive correlation coefficient). This implies that f 2 describes in particular the influence of such class of solvents (higher f 2 values means also higher ν ).

The anomalous effect of the protic solvents on the λ max of 3 and 7 has been discussed before.

A dependence of the factor loadings f 2 on the structural characteristics of solvatochromic indicators provided a better insight into the problem. Thus, the dependence of the factor loadings f 2 on the structural characteristics provided the strong and statistically valid equation The specific effect of protic and very polar solvents on the longestwavelength absorption maxima λ max accompanied by the data decomposition is significant to such a degree that the structure of the score matrix T is deformed. Hence, the columns corresponding to indicators 3 and 7 were excluded from the matrix X and the procedure was repeated. Now, the first latent variable explains 71.2 %, the second 14.4 %, and the third 10.2 % of the data variability (95.8 % of the explained variability overall). Thus, the first latent variable substantially describes the data variability. The variability described by the first three latent variables is similar to the previous case, however, their structure is different.

Interpretation of the latent variable vectors t 1 and t 2 has been accomplished through the (semi)empirical solvent parameters (f(n 2 ),f(ε r ), V m , and δ H ). [10] The strongest and statistically valid correlation for vector t 1 can be described by equation ( 3)

t
n = 32, s = 5.61 10 -1 , R = 0.850.

A joint interpretation of equations ( 3) and ( 4) implies that dominant solvent effects on the position of λ max are, in descending order, solvent polarizability, solvent cavity size, and solvent polarity. According to the previous discussion, [10] increasing solvent polarizability and polarity shifts the longest-wavelength absorption maxima λ max bathochromically, while the solvent cavity size has the opposite effect (hypsochromic shift).

Solvatochromism-polarizability relationshiphs

First-(α), second-(β), and third-order (γ) polarizabilities of indicators 1-12 calculated by MOPAC2009 are summarized in Table 3. The afore-mentioned structural characteristics and the ν values in all 32 solvents were used for the interpretation of the calculated polarizabilities. A statistically significant dependence has been found only for the first-order polarizability α. This implies that the higher-order polarizabilities are not related to solvatochromism.

A simple and statistically valid interpretation of the first-order polarizability α is given by equation ( 5

EXPERIMENTAL SECTION

The complete synthesis and full spectral characterization of compounds 1-12 has been reported previously. [12][13][14][15][16][17][18] Solvents used for the solvatochromism study were chosen according to their specific difference (acidity, basicity, polarity, polarizability, and their combinations). [21] Solvents were reagent-grade and were purified by methods described in the literature. [10] The measured solution of compounds 1-9 had a concentration of 6×10 -5 mol/L.

Absorbance was in the range from 0.1 to 1.0. If the compound was not completely dissolved, a filtered saturated solution was measured instead. In order to verify the concentration independency of the absorption maxima position and that the Lambert-Beer law was fully obeyed, spectra of indicators 1-9 at different concentrations ranging from 6.45×10 -5 to 6.45×10 -6 M were measured (as shown in SI). Electron absorption spectra were recorded on a Hewlett-Packard 8453 spectrophotometer in the range of wavelengths from 200 to 1100 nm with an accuracy of 0.5 nm. The longest-wavelength absorption maxima were numerically determined from the obtained smoothed dependencies of absorbance on wavelength. Table 1. The longest-wavelength absorption maxima λ max of the intramolecular CT bands measured for compounds 1-9 in dependence on 32 solvents (listed in ascending order according to solvent's N T E values [START_REF] Reichardt | Solvents and Solvent Effects in Organic Chemistry[END_REF] ). [b] 42 51 -142.5 28.5 165.5 159.5 -97 46.5 67

[a] Not measurable. [b] The spectra are listed in ascending order according to

N T E values (see Table 1, main text). The spectra are listed in ascending order according to N T E values (see Table 1, main text). The spectra are listed in ascending order according to N T E values (see Table 1, main text). The spectra are listed in ascending order according to The spectra are listed in ascending order according to N T E values (see Table 1, main text). The spectra are listed in ascending order according to N T E values (see Table 1, main text). The spectra are listed in ascending order according to N T E values (see Table 1, main text). The spectra are listed in ascending order according to N T E values (see Table 1, main text). The spectra are listed in ascending order according to N T E values (see Table 1, main text).

  Figure 1

Figure 3 .

 3 It is obvious that compounds 4, 5, and 6 have the highest λ max values. The cut in the upper part of polygon 4 was caused by the missing triethylamine value. The same solvent was indicated to be exceptional by the rising ray in the right upper part of the polygons 5 and 6 (remarkable also for other compounds). The observed high λ max values for componds 4-6 were most probably caused by the quinoid partial structure (TCNQ) of the molecule. From the spectral point of view, the quinoid motive is a combination of two

7 and two DMAs in 8 and 9 )

 9 but rather by the spatial arrangements and the difference in the energies of the local electron transitions. The polygon's shapes in Figure3evidence the differences between the λ max values given by the various solvent effects. The polygons 3 and 7 have remarkable irregular shape. The histogram of the λ max frequency distribution in dependence on λ max clearly shows bimodal distribution of the λ max values for these two indicators. The group of solvents with the lower λ max values (indicator 3) includes both HBD (hydrogen-bond donor) solvents (alcohols, formamide) and non-HBD solvents (dimethylsulfoxide or N,N-dimethylacetamide -one of the most polar solvents). For indicator 7, this group of solvents is even extended to tetrahydrofuran(!), esters, ketones, nitrocompounds, etc. In other words, the afore-mentioned solvents "switch off" the intramolecular CT transition and the observed λ max values correspond to a local π-π * or n-π * transitions of any molecule part (see also the Supporting Information). We can consider several reasons of such a "switch off" related either to the structure of the compound or to the solvent properties. The first reason is a disproportion of electron donating DMA groups (one) and the number of electron withdrawing cyano groups (three for 3 and four for 7) in contrast to other molecules. The second structural reason is the localization of the well accessible cyano groups, as well as the LUMO, at the molecule terminus. Both of these reasons are related with an intramolecular

( 2 )

 2 f 2 = -(1.16±0.52) 10 -3 -(6.65±1.67) 10 -7 COSMO area + + (1.71±0.55) 10 -3 COSMO area/COSMO volume -(1.51±0.48) 10 -4 i PL , (2) n = 12, s = 3.00 10 -5 , R = 0.941.Equation(2) implies that the factor loadings f 2 , respectively ν , decrease with increasing solvatable surface (COSMO area) of the molecule and decreasing ratio of the solvatable surface to the solvatable volume of the molecule (increasing "sphericity" of the solute). This trend is consistent for planar (i PL = 1) as well as nonplanar (i PL = 0) molecules. Based on these observations we can conclude that an additional effect of the solvent cavity lowering the longest-wavelength absorption maxima λ max takes place in protic solvents, especially for small molecules (Figure4, indicators 3 and 7).

2 , 4 - 6 , and 8 ,

 2468 Principal component analysis (PCA) was applied to evaluate solvent effects on the ν values for different D-π-A organic molecules as solvatochromic indicators. The latent variables t (columns of the matrix T) as characteristics of solvent effects were calculated from the native (non-standardized) matrix X. The PCA method has mainly been chosen because the information on solvent effects can be calculated by the methods based on the similarity principle (similar to the structure-solvatochromism relationships of D-π-A molecules). The first latent variable explains 55.7 %, the second 28.7 %, and the third 11.4 % of the data variability (95.8 % of the explained variability overall). It is obvious that, with respect to the experimental accuracy, the three latent variables described the total relevant variability. The first latent variable describes dominantly solvent effects on indicators 3 (communality h 2 = 0.825) and 7 (h 2 = 0.635), while the effect of other indicators is small (h 2 = 0.01 -0.39). The reasons have already been discussed before. The second latent variable particularly comprises solvent effects on indicators 1, while the third variable describes the interaction with indicators 9-12.

  ): α = (9.83±0.85) 10 -30 -(2.46±0.39) 10 -27 ν (1,2-DCE) -(1.32±0.23) 10 -31 n DMA --(2.33±0.84) 10 -31 n CN /n DMA , (5) n = 12, s = 2.67 10 -31 , R = 0.954. The first-order polarizability α increases with increasing λ max in 1,2-dichloroethane (1,2-DCE, polarizable solvent, this also holds true for other similar solvents) and, on the contrary, with the decreasing number of DMA units and the ratio of CN to DMA units. The negative sign of the wavenumber ν (1,2-DCE) implies that more polarizable molecules have the longest-wavelength absorption maxima shifted bathochromically. The signs of the structural characteristics in equation (5) are somewhat surprising. The reason is probably an "electron rigidity" as a result of the D-π-A molecule polarization. Equation (5) can be used to evaluate the polarizability of organic D-π-A molecules in general.

11 CONCLUSIONA

 11 representative data set of the electronic absorption spectra of twelve systematically selected organic D-π-A molecules as solvatochromic indicators measured in 32 solvents allowed the application of a multidimensional statistic analysis and interpretation of the structure-property relationships. Simple structural characteristics based on a number or presence of specific structural features of the molecule showed to be suitable parameters in the correlation analysis. The most important characteristics are the number of DMA and CN groups, their ratio, and eventually the number of triple bond π-linkers. The quantum-chemically calculated molecule parameters such as LUMO energy, permanent dipole moment, COSMO area, and COSMO volume proved to be suitable structural characteristics as well. Whereas the solvent polarizability [(n 2 -1)/(n 2 +2) function] and the solvent cavity size (molar volume V m and Hildebrand δ H ) are the main factors affecting the position of λ max , the solvent polarity (the Born function 1/ε r ) is less important. The correlation dependencies are statistically valid and relatively strong. A relation between the first-order polarizability α, the longest-wavelegth absorption maxima λ max , and the structural features has also been found. The higher-order polarizabilities β and γ are not related to the observed solavtochromism of indicators 1-12.

15 Figure 1 .

 151 Figure 1. Molecular structures of compounds 1-12: CEEs (1-3), TCNQ-based (4-6), TCBDs (7-9), and TCAQs (10-12).

Figure 2 .

 2 Figure 2. UV/Vis spectra of the organic charge-transfer compound 5, measured in the selected solvents (cHx -cyclohexane, TEA -triethylamine, CCl 4 -tetrachloromethane, MePh -toluene, DXN -1,4-dioxane, THF -tetrahydrofuran, EA -ethyl acetate, DCMdichloromethane, DMF -N,N-dimethylformamide, MeOH -methanol).

Figure 3 . 18 Figure 4 .

 3184 Figure 3. The mutual relationships of λ max for compounds 1-12, depicted as polygons (the polygon's rays correspond to the 32 solvents), the λ max histograms for indicators 3 and 7, and the frequency function of normal distribution, N.

  efficient tool to study N,N-dimethylamino-and cyano-substituted π-conjugated molecules with an intramolecular charge-transfer absorptionFilip Bureš, Oldřich Pytela, Milan Kivala, and François Diederich Electronic absorption spectra of twelve systematically chosen charge-transfer D-π-A push-pull systems featuring electron-withdrawing CN groups, electron-donating N(CH 3 ) 2 groups, and various π-conjugated backbones have been measured in 32 solvents. The solvent polarizability and the solvent cavity size proved to be the most crucial factors affecting the position of λ max . The most important characteristics of D-π-A molecules concerning the solvatochromism are the energy of LUMO, the permanent dipole moment, the COSMO area, the COSMO volume, the number of donors/acceptors, and the length of the π-conjugated path.

1 Figure 1SI .

 11SI Figure 1SI. UV/VIS spectra of compound 1 in selected solvents (part 1).

Figure 2SI .

 2SI Figure 2SI. UV/VIS spectra of compound 1 in selected solvents (part 2).

Figure 3SI .

 3SI Figure 3SI. UV/VIS spectra of compound 1 in selected solvents (part 3).

Figure 4SI .

 4SI Figure 4SI. UV/VIS spectra of compound 1 in selected solvents (part 4).

Figure 5SI .

 5SI Figure 5SI. UV/VIS spectra of compound 1 in CH 2 Cl 2 -dependence on concentration.

2 Figure

 2 Figure 6SI. UV/VIS spectra of compound 2 in selected solvents (part 1).

Figure 7SI .

 7SI Figure 7SI. UV/VIS spectra of compound 2 in selected solvents (part 2).

Figure 8SI .

 8SI Figure 8SI. UV/VIS spectra of compound 2 in selected solvents (part 3).

Figure 9SI .

 9SI Figure 9SI. UV/VIS spectra of compound 2 in selected solvents (part 4).

Figure 10SI .

 10SI Figure 10SI. UV/VIS spectra of compound 2 in CH 2 Cl 2 -dependence on concentration.

3 Figure 11SI .

 311SI Figure 11SI. UV/VIS spectra of compound 3 in selected solvents (part 1).

Figure 12SI .

 12SI Figure 12SI. UV/VIS spectra of compound 3 in selected solvents (part 2).

Figure 13SI .

 13SI Figure 13SI. UV/VIS spectra of compound 3 in selected solvents (part 3).

Figure 14SI .

 14SI Figure 14SI. UV/VIS spectra of compound 3 in selected solvents (part 4).

Figure 15SI .

 15SI Figure 15SI. UV/VIS spectra of compound 3 in CH 2 Cl 2 -dependence on concentration.

4 Figure 16SI .

 416SI Figure 16SI. UV/VIS spectra of compound 4 in selected solvents (part 1).

Figure 17SI .

 17SI Figure 17SI. UV/VIS spectra of compound 4 in selected solvents (part 2).

Figure 18SI .

 18SI Figure 18SI. UV/VIS spectra of compound 4 in selected solvents (part 3).

Figure 19SI .

 19SI Figure 19SI. UV/VIS spectra of compound 4 in selected solvents (part 4).

Figure 20SI .

 20SI Figure 20SI. UV/VIS spectra of compound 4 in CH 2 Cl 2 -dependence on concentration.

Figure 21SI .

 21SI Figure 21SI. UV/VIS spectra of compound 5 in selected solvents (part 1).

Figure 22SI .

 22SI Figure 22SI. UV/VIS spectra of compound 5 in selected solvents (part 2).

Figure 23SI .

 23SI Figure 23SI. UV/VIS spectra of compound 5 in selected solvents (part 3).

Figure 24SI .

 24SI Figure 24SI. UV/VIS spectra of compound 5 in selected solvents (part 4).

Figure 25SI .

 25SI Figure 25SI. UV/VIS spectra of compound 5 in CH 2 Cl 2 -dependence on concentration.

6 Figure 26SI .

 626SI Figure 26SI. UV/VIS spectra of compound 6 in selected solvents (part 1).

Figure 27SI .

 27SI Figure 27SI. UV/VIS spectra of compound 6 in selected solvents (part 2).

Figure 28SI .

 28SI Figure 28SI. UV/VIS spectra of compound 6 in selected solvents (part 3).

Figure 29SI .

 29SI Figure 29SI. UV/VIS spectra of compound 6 in selected solvents (part 4).

Figure 30SI .

 30SI Figure 30SI. UV/VIS spectra of compound 7 in CH 2 Cl 2 -concentration dependence.

Figure 31SI .

 31SI Figure 31SI. UV/VIS spectra of compound 7 in selected solvents (part 1).

Figure 32SI .

 32SI Figure 32SI. UV/VIS spectra of compound 7 in selected solvents (part 2).

Figure 33SI .

 33SI Figure 33SI. UV/VIS spectra of compound 7 in selected solvents (part 3).

Figure 34SI .

 34SI Figure 34SI. UV/VIS spectra of compound 7 in selected solvents (part 4).

Figure 35SI .

 35SI Figure 35SI. UV/VIS spectra of compound 7 in CH 2 Cl 2 -dependence on concentration.

Figure 36SI .

 36SI Figure 36SI. UV/VIS spectra of compound 8 in selected solvents (part 1).

Figure 37SI .

 37SI Figure 37SI. UV/VIS spectra of compound 8 in selected solvents (part 2).

Figure 38SI .

 38SI Figure 38SI. UV/VIS spectra of compound 8 in selected solvents (part 3).

Figure 39SI .

 39SI Figure 39SI. UV/VIS spectra of compound 8 in selected solvents (part 4).

Figure 40SI .

 40SI Figure 40SI. UV/VIS spectra of compound 8 in CH 2 Cl 2 -dependence on concentration.

Figure 41SI .

 41SI Figure 41SI. UV/VIS spectra of compound 9 in selected solvents (part 1).

Figure 42SI .

 42SI Figure 42SI. UV/VIS spectra of compound 9 in selected solvents (part 2).

Figure 43SI .

 43SI Figure 43SI. UV/VIS spectra of compound 9 in selected solvents (part 3).

Figure 44SI .

 44SI Figure 44SI. UV/VIS spectra of compound 9 in selected solvents (part 4).

Figure 45SI .

 45SI Figure 45SI. UV/VIS spectra of compound 9 in CH 2 Cl 2 -dependence on concentration.

Figure 46SI .

 46SI Figure 46SI. Colour of solutions of compounds 1-9, dissolved in dichloromethane.

Figure 47SI .

 47SI Figure 47SI. Colour of solutions of compounds 1-9, dissolved in N,N-dimethylacetamide.

Table

Table 3 _______________________

 3 The interpretation of the factor loadings f 1 on the molecular structure of indicators was Indicators with the smallest f 1 have simultaneously the highest λ max and vice versa (see Figure

	_______________________
	complicated by the values of 4-6, that were in regression indicated as extremes. Hence, the
	obtained dependencies were represented by a connecting line between two clusters (4-6 and

provided a better insight into the structure-solvatochromism relationships. The following quantum-chemically calculated characteristics were used: (i) E HOMO and E LUMO energies; (ii) permanent dipole moment µ; (iii) COSMO (COnductor-like Screening MOdel) area; (iv) COSMO volume; (v) molecular dimensions MD1, MD2, and MD3, and (vi) their functions MD1×MD2, MD1×MD3, MD2×MD3, MD1×MD2×MD3, MD1/MD2, MD2/MD1, MD1/MD3, and MD3/MD1. As the characteristics describing structural features of the studied molecules were chosen: (i) number of DMA units n DMA ; (ii) number of cyano groups n CN ; (iii) number of triple bonds n T ; (iv) ratio n DMA /n CN ; (v) ratio n CN /n DMA , and (vi) the indicator variables (0 not present, 1 present)presence of quinoid arrangement i CH , planarity i PL , and centrosymmetry i CS .

others) and, therefore, the values of 4-6 were omitted from the further calculations and the new vector was marked as f 1,-456 . The strongest and statistically valid correlation can be described by equation

(1)

Table 2 .

 2 The factor loadings f 1 and f 2 , calculated for compounds 1-12 by nonlinear regression using the two-factor model.

	Indicators 1, 2, 4, 5, 6, 8, and 9 are positively solvatochromic; indicators 3 and 7 are negatively
	solvatochromic.

Table 3 .

 3 The characteristics of compounds 1-12, calculated by the PM6 method (MOPAC2009).

	Indicator	area COSMO	volume COSMO	E HOMO	E LUMO	MD1	MD2	MD3	moment Dipole	10 30 α	10 29 β	10 27 γ
		[Å 2 ]	[Å 3 ]	[eV]	[eV]	[Å]	[Å]	[Å]	[D]	[esu]	[esu]	[esu]
	1	439.39	468.57	-8.36 -1.42 18.6 8.9	1.9	11.2	3.43 2.99 4.61
	2	438.61	468.93	-8.11 -1.45 21.1 6.0	1.7	2.4	3.70 0.27 8.25
	3	303.06	312.56	-8.66 -2.03 13.5 6.1	1.6	11.3	2.17 5.07 2.78
	4	392.90	441.67	-8.76 -2.30 13.3 13.2 5.5	9.9	3.16 1.41 -0.47
	5	497.18	591.40	-8.59 -2.16 13.8 13.3 7.8	12.4	4.23 2.38 1.09
	6	540.82	623.56	-8.57 -2.13 14.6 13.0 8.2	13.1	4.70 4.45 2.96
	7	314.06	347.39	-8.80 -1.96 10.7 7.9	4.9	8.3	2.06 2.63 1.13
	8	418.21	498.43	-8.61 -1.69 14.9 7.7	6.6	8.0	3.16 3.01 2.37
	9	460.68	531.87	-8.57 -1.70 16.5 8.7	7.6	11.1	3.57 4.09 3.50
	10	363.09	421.49	-8.86 -2.19 11.0 7.7	5.6	8.1	2.72 1.82 1.13
	11	406.67	480.57	-8.72 -1.98 13.5 7.9	4.2	7.2	3.15 1.73 1.89
	12	442.15	515.94	-8.41 -2.24 15.3 8.3	6.4	8.4	3.40 3.20 2.58

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Journal of Physical Organic Chemistry

Acknowledgements

This work was supported by the Ministry of Education, Youth and Sport of the Czech Republic (MSM 0021627501) and by the ETH Research Council.

Initial geometries of the compounds 1-12 were calculated by PM3 method (ArgusLab, Ref. [22] ) and subsequently optimized by the PM6 method (MOPAC2009, Ref. [START_REF] Mopac | Stewart Computational Chemistry,version 8.315W[END_REF] ). Employing MOPAC2009, the following characteristics were further calculated: E HOMO and E LUMO energies, dipole moment, COSMO area, COSMO volume, molecular dimensions (MD) 1, 2, and 3, isotropic polarizability α, and average second-and third-order polarizabilities β and γ .

The predicted electronic absorption spectra were calculated by the ZINDO-CI method (10 highest occupied MOs, 10 lowest unoccupied MOs, self consistent reaction field methanol) using the molecular geometry optimized by ArgusLab. [22] Experimental data were examined by exploratory analysis and subsequently treated by principal component analysis PCA, factor analysis FA, and multiple linear regressions MLR. The Program OPstat [START_REF] Opstat | version 6.25[END_REF] was employed for all statistical calculations.


Adam Gryff-Keller

Przemyslaw Szczeciński

Anna Kraska-Dziadecka

Przemysław Szczeciński

P Szczeciński

]cyclohexane-1,3-dione (NTBC) and Its Two Metabolites Isolated from Urine of Patients Suffering from Tyrosinemia Type I

Keywords: NTBC, metabolism, taumtomeris, NMR spectroscopy, DFT calculation NTBC, metabolism, tautomerism, NMR spectroscopy, DFT calculation

    NMR and DFT Calculation Study on Structures of 2-[2nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC) and Its Two Metabolites Isolated from Urine of Patients Suffering from Tyrosinemia Type I.

Introduction

Several natural and synthetic compounds possessing branched β,β'-triketonic structure are inhibitors of 4-hydroxyphenylpyrruvate dioxygenaze (HPPD), an important enzyme on tyrosine biochemical metabolism path. [1][2][3][4][5][6] The first part of this path is common for plants and In plants blocking this enzyme prevents production of some vitally important compounds, whereas in animals blocking tyrosine catabolism does not have such dramatic consequences, so HPPD inhibitors can be widely exploited as herbicides in crops protection.

Search for new and more effective triketonic herbicides has led to invention of 2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC), which appeared to be so selective and effective that it could be used in therapy of patients suffering from tyrosinemia type I, a fatal metabolic disease. [2,4,[6][7][8][9] Children with this genetic error lack an enzyme needed at the end of tyrosine catabolism path, which results in production of succinylacetone, a pathognomonic metabolite damaging liver and kidney. Application of an NTBC-based drug interrupts the biochemical path at the earlier stage which prevents the production of that harmful metabolite. [7][8][9] The mechanism of HPPD action and its inhibition has been widely discussed. [10][11][12][13][14][15][16] The details of molecular structures of the natural substrate of the enzyme and its inhibitors are obviously crucial in such considerations. It seems that the same concerns the inhibitor metabolites, occurence of which has occasionally been reported. [17,18] Recently, it has been found that one such metabolite, 4-hydroxy-2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC-6-OH) appears in the urine of patients treated with nitisinone. [18] In this work, the separation and chemical identification of the second NTBC metabolite, 5-hydroxy-2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC-5-OH), is reported. Apart from the spectroscopic characterisation of NTBC and its metabolites the detailed discussion on their molecular structures and conformational equilibriums, in the light of 1 H and 13 C NMR spectra and the results of DFT calculations, is undertaken.

Results and Discussion

Separation and chemical identification of NTBC metabolites.

In the previous paper we reported the results of our investigations of organic extracts of urine samples from children treated with nitisinone. [18] In 19 F NMR spectra of these samples two major signals of CF 3 groups were always present. At the same time, in 1 H NMR spectra, in the region δ ≈ 8.5 ppm, two characteristic signals were observed for all investigated samples. These signals were assigned to the H3' protons strongly deshielded because of three electron 3 withdrawing groups present in the aromatic ring of NTBC and its derivatives. One compound containing CF 3 group could be separated from the extracts by TLC technique in a small amount. The NMR spectra showed that it was NTBC-6-OH (Fig. 1). Its identification was confirmed by comparison with the sample of the same compound synthesised in vitro. [18] Following the literature suggestions [19,20] it seemed natural to suppose that the second of the compounds containing CF 3 group present in urine extracts was the unchanged NTBC.

Moreover, the comparison of the 1 H NMR spectrum of a urine extract with the spectrum of the same sample recorded after addition of a portion of NTBC seemed to verify positively such a conjecture. In view of presently available data, however, it is clear that we underestimated the possibility that our identification method might be error prone. Namely, In order to check if NMR technique can be applied for medical assays during nitisinone treatment the fluorine NMR spectra of several samples of unprepared urine, originating from four different patients, have been measured. It has been found that also for these much more dilute samples two main CF 3 signals are easily detectable. We have also repeated the measurements of the original urine sample with the addition of NTBC and NTBC-6-OH. The experiments have confirmed the presence of NTBC-6-OH, but, to our surprise, excluded the presence of NTBC, at least in an amount comparable with NTBC-6-OH. Addition of a portion of NTBC to the urine sample caused the new signal to appear downfield with respect to two original signals (Fig. 2). TLC examination of the organic urine extracts has yielded similar results. It was found that when silica gel plates and benzeneacetone mixture (1/1, v/v) as an eluent were used, the experiments performed for all organic urine extracts and their mixtures with the reference compounds revealed the presence of two main spots: one originating from NTBC-6-OH (R f = 0.25) and the second from an unknown compound (R f = 0.15) unequivocally different from NTBC (R f = 0.5). Separation of the compounds giving those two spots was done with the aid of the preparative TLC technique.

The NMR and HRMS spectra confirmed that one of them was NTBC-6-OH, whereas the second one was a new metabolite NTBC-5-OH (Fig. 1). Analogous 6-and 5-hydroxylated derivatives were found in urine of rats and mice treated with 2-(4-methylsulfonyl-2-F o r P e e r R e v i e w 4 nitrobenzoyl)cyclohexane-1,3-dione (mesotrione), a popular herbicide. [17] In this case, however, unchanged mesotrione was also detected in huge amounts.

Having a relatively pure sample of NTBC-5-OH we could compare the 1 H NMR signals of H3' protons for this compound and for NTBC. It appeared that their multiplet patterns were identical and that at comparable concentration the chemical shift difference ∆δ was of the order of 2 Hz only, at B 0 = 11.7 T. Apparently, this was the reason of the erroneous interpretation of this signal made previously.

Tautomerism

So-called triketonic herbicides (leptospermon, mesotrione, sulcotrione, chalcotrione, etc.) as well as three compounds investigated in this work belong to the class of the branched β,β'-diketones. Actually, probably all these compounds exist in organic solutions exclusively as enols stabilised by the formation of strong intramolecular hydrogen bonds. [10,14,21,22] In 2acylcyclohexane-1,3-diones (Fig. 3) the acyl carbonyl and enol group originating from one of the two ring carbonyls form a ring via such a hydrogen bond. In the case of symmetrically substituted cyclohexane ring (NTBC or NTBC-5-OH), this leads to two chemically indistinguishable topomeric structures, while for the unsymmetrical substitution (NTBC-6-OH) the structures involving different oxygen atoms are different. To be specific, such a classification holds only if cyclohexane ring conformation is neglected; otherwise we deal with enantiomeric structures for symmetrical substitution and with diastereomers in the opposite case.

There is firm evidence coming from both 1 H and 13 C NMR that tautomeric transformation leading from one topomer to another is completely hindered in NMR time scale in CDCl 3 solution at room temperature. Already in our previous paper we noticed that several 2-acylcyclohexane-1,3-diones, including NTBC, show in 13 C NMR spectrum six sharp lines for the cyclohexane ring carbons. [14,22] Also the cyclohexane ring protons yield three, rather than two, multiplets in 1 H NMR spectra. The same spectral features are observed in this work for NTBC-5-OH (see Experimental). It is interesting that in the spectra of the other NTBC metabolite only one proton pattern characteristic for six non-equivalent aliphatic protons and one set of six carbon lines for a cyclohexene ring are present (see Experimental). In view of slow exchange limit it proves that only one of two possible structures of this compound exists in the investigated solution. The quantum chemical calculations of the energy of the optimal molecular structures show that 2-acyl-3,6-dihydroxycyclohex-2-enone (acyl = 4-CF 3 -2-NO 2 -C 6 H 3 CO-) is remarkably more stable than the appropriate 2-acyl-3,4dihydroxycyclohex-2-enone (Fig. 3, Table 1). The same conclusion comes out from the analysis of the 13 C NMR chemical shifts (vide infra). It is the reason why we use the abbreviation NTBC-6-OH for this NTBC metabolite. In the discussed system there is another tautomeric transformation in which the hydrogen atom that binds two oxygen atoms moves between two energy minima on the =O … H─O─ ─O─H … O= path. This passage is accompanied by a due electron rearrangement in the chelate ring (Fig. 4). Such a transformation is believed to be extremely fast because of a low energy barrier. [21] As a result, averaged, population-weighted spectra are observed in NMR. This problem was widely discussed in the literature and also in several papers from our laboratory. [10,14,[21][22][23][24][25][26] In ref. [14] it was shown (as opposite to ref. [10] ) that in the case of NTBC the endo-enol structure prevails. The energy calculations (Table 1) and 13 C NMR chemical shift analysis performed in this work (vide infra) confirm this conclusion for NTBC and show that it is valid also for NTBC metabolites.

Conformation of the system

The molecular structure optimizations have been performed for endo-and exo-enols of all three compounds investigated in this work using a DFT-based method. The energy minima have been found for conformations in which five carbons of the cyclohexene ring, C1, C2, C3, C4, C6 and all three carbon-oxygen bonds lie approximately on one plane, whereas C-5 deviates remarkably from it. Moreover, the aromatic ring assumes approximately perpendicular orientation to that plane (Fig. 5), presumably because of steric reasons. There are two such conformations, which will be further addressed as A and B type conformers, dependently on C-5 and -NO 2 being on the same or opposite sides of the main molecular plane. The calculated molecular energy differences (Table 1) between A and B conformers are very small and both of them are expected to be present in the investigated solutions in comparable amounts. Below we will see that the differences between the appropriate shielding constants are also small and so conformation populations cannot be reliably estimated on this basis.

The molecular structure described above actually denotes existence of four different conformers of the investigated compounds. In the case of NTBC they are pairwise enantiomeric: R-A, S-A, R-B and S-B). In the other two cases they are diastereomeric due to the axial or equatorial position of the -OH substituent (ax-A, eq-A, ax-B and eq-B). The Again, neither molecular energies nor 13 C chemical shifts can be a basis for the evaluation of the populations of the axial and equatorial conformers. In this case, however, there is another source of reliable information at our disposal, namely, the vicinal spin-spin coupling constants between cyclohexene ring protons. Tables 2 and3 contain experimental values of 1 H-1 H spinspin coupling constants obtained from the analysis of spectral patterns occurring in aliphatic parts of 1 H NMR spectra of both NTBC metabolites. These tables also contain theoretical coupling constants calculated for the selected conformers of the investigated compounds. In accord with the expectations, the vicinal coupling constants between aliphatic protons are strongly dependent on the appropriate dihedral angles. [27] On the other hand, the coupling constants are almost independent on A B transition and only marginally dependent on endo exo tautomerisation. Comparison of the experimental and theoretical data in Tables 2 and3 shows that in the case of NTBC-6-OH the equatorial conformation highly predominates, whereas NTBC-5-OH is approximately equimolar mixture of eq and ax conformers. It is even possible to estimate conformer populations for NTBC-5-OH expressing measured coupling constants as population-weighted averages of values calculated for the appropriate eq and ax conformers (eq. 1) and assuming that the impact of small amounts of exo-enols present in the solution can be ignored.

J exp = a(p eq J eq,calc + p ax J ax,calc ) + b (1) In this equation a and b are adjustable parameters. The least-squares analysis yields: p eq = 0.43 ± 0.03, p ax = 0.57 ± 0.03, a= 0.988, b= -0.3 Hz. Thus, the result seems quite reasonable, as judged from a and b values and excellent reproduction of the experimental data (Fig. 6), though the error estimations are probably too optimistic. It is also noteworthy that the OH proton signal of NTBC-5-OH is a broadened singlet, whereas in the spectrum of NTBC-6-OH it is a doublet. Presumably, the intramolecular hydrogen bond in -CHOH-C(=O)-fragment in the latter metabolite prevents the proton exchange. The small 3 J(HCOH) value agrees well with the calculated molecular structure in which the dihedral angle between the appropriate C-H and O-H vectors is close to 90º. There is no need to substantiate that 13 C NMR chemical shifts are an efficient source of valuable information on molecular structure. The usefulness of these relatively easily accessible and precise experimental data was enhanced in the last decade with the enhanced access to quantum chemical calculations of NMR parameters. [28][START_REF] Parts | Calculation of NMR and EPR Parameters: theory and applications[END_REF][START_REF] Velde | [END_REF][31] Numerous test calculations showed that the chemical shifts for the i-th carbon, δ i , in a compound existing in the investigated solution in several forms being in dynamic equilibrium (fast in NMR time scale), can be expressed in terms of the calculated shielding constants, σ i,j , and species populations, p j , by the equation:

Analysis of 13 C NMR chemical shifts

δ i = a(p 1 σ i,1 + p 2 σ i,2 + ... -b) (2)
where a and b are compound-independent, adjustable parameters, a being a scaling factor, close to -1, and b -the shielding constant of the (virtual) chemical shift reference. [32][33][34][35] It was pointed out that in order to achieve a good experiment/theory correlation it is desired to use a sufficiently high and possibly the same level of theory in the molecular geometry optimization and NMR parameters calculation. The impact of the solvent should also be taken into account. [33,35] Obviously, all the abundant forms of the investigated compound present in the solution have to be considered. In the light of the above discussion, for NTBC there are four such species: A and B conformers of the endo and exo enols. In the case of NTBC-5-OH the number of species is doubled due to the distinction between axial and equatorial conformers.

Finally, in the case of NTBC-6-OH metabolite, four species have to be considered, when assuming that only the equatorial conformers are present in the investigated solutions.

Additionally, we performed calculations for four structures of the NTBC-4-OH topomers to check whether the chemical shifts could be used to distinguish between the NTBC-4-OH and NTBC-6-OH structures.

The assignment of the signals in the 13 C NMR spectra for all three compounds has been a relatively easy task, when using 2D-HSQC and 2D-HMQC spectra, taking into account Examination of these data shows that A B transformation affects the 13 C shielding constants only marginally, which means that the determination of [A/B] molar ratio would be unreliable, if possible at all. In such a situation we decided to calculate the mean values of the appropriate shielding constants:

σ i,j = 0.5(σ i,j(A) + σ i,j(B) ) (3)
and to use them in correlations. In the case of NTBC-5-OH we are still left with four species:

ax/eq conformers and endo/exo enols. The shielding constants for ax and eq conformers are again not very large and an attempt at determining the appropriate population parameters for the less populated exo-enol seems unrealistic. That is why we decided to average the appropriate shielding constants again. Finally, in the least-squares analysis of the chemical shift data we had 42 carbon chemical shifts and 9 unknowns (p endo and p exo for NTBC, p ax-endo , p eq-endo and p exo for NTBC-5-OH, p eq-endo and p eq-exo for NTBC-6-OH, and a and b parameters.

The results of this analysis are given in Table 4 and in Fig. 7.

The achieved overall fit between experiment and theory (RMSD = 1.87 ppm) is sufficiently good to interpret the determined numerical values of the population parameters. It may be noticed, however, that the fit in the aliphatic parts of the spectra (carbons 1 to 7) is excellent (RMSD = 0.75 ppm), whereas for the CF 3 carbons and the aromatic ring carbons the deviations from the regression line are much larger and very similar for all three compounds (see the results for NTBC-6-OH in Fig. 8). The deviations for CF 3 carbons could be a result of deficiencies of the theoretical method (an impact of electron correlations and ro-vibrational motions), while some test calculations (see Supplementary Materials) suggest that the deviations for the aromatic ring carbons are connected with large amplitude deformations of the equilibrium conformations, such as the rotation about C7-C1' bond, due to ground state internal motions and intermolecular collisions. It is noteworthy that the aromatic parts of the investigated molecules are identical and only marginally influenced by the substitution of the cyclohexane ring in NTBC-5-OH and NTBC-6-OH. This fact is manifested in very small chemical shift variations for aromatic carbons. Thus, almost all information on possible structural changes within the series should be cumulated in the data concerning the aliphatic parts of these compounds. Following this reasoning, we have performed the separate numerical analysis of the 13 The set of simplifying assumptions proposed above seems to be the most reasonable.

Certainly, other approaches are possible and, actually, have been attempted. Nevertheless, our conclusion about small contributions (probably of the order of 0.1 or less) of exo-enols in tautomeric equilibriums for all three compounds seems to be unquestionable. In the case of NTBC-5-OH the axial and equatorial conformers seem to be present in comparable amounts, which is in agreement with the finding based on the values of 1 H-1 H coupling constants.

Finally, the fit between experiment and theory for the correlation of the chemical shift data for the first NTBC metabolite, i.e. 6-hydroxy-2-[(2-nitro-4-trifluoromethyl)benzoyl]cyclohexane-1,3-dione has been examined, assuming for it the NTBC-4-OH or NTBC-6-OH structure. A better fit, especially in the aliphatic part of the spectrum, provides the additional argument towards the letter structure (Fig. 8).

Experimental

Preparation of organic extracts of urine samples was described previously. [18] Separation of NTBC metabolites from those extracts was performed using TLC glass plates 20×20 cm with silica gel 60 F 254 , 0.25 mm and acetone-benzene mixture (1/1, v/v) as eluent.

In these conditions R f values for NTBC-5-OH and NTBC-6-OH were 0.15 and 0.25, respectively. All other compounds present in the urine sample were located at R f larger than 0.5.

NMR measurements were performed using 500 MHz (11.7 T) spectrometer equipped with a temperature controller. 1 H, 13 C and 19 F NMR spectra of the CDCl 3 urine extracts or unprocessed urine samples after addition of ca. 5 % D2O were recorded at 25 ºC using 5 mm NMR tubes. 1 H and 13 C chemical shifts were referenced to the TMS scale using the residual All the theoretical calculations of molecular structures and NMR parameters were performed using Gaussian 03 program [START_REF] Frisch | Gaussian 03, Revision C.02[END_REF] . At both calculation stages the density functional theory (DFT) with PBE1PBE functional [START_REF] Perdew | [END_REF] were used and the influence of the bulk solvent was taken into account using the polarizable continuum model (PCM) of Tomasi et al. [38] , defining the cavity according to the UFF scheme [39] . In the molecular geometry optimizations the 6-31+G* basis set was used, while for calculating NMR parameters the GIAO method and 6-311++G(2d,p) basis set were exploited.

NTBC-5-OH:

The 1 H-1 H coupling constants between cyclohexene ring protons of NTBC-5-OH and NTBC-6-OH were determined by the total lineshape analysis of the appropriate patterns in 1 H NMR spectra using a non-linear least-squares fitting procedure.

The comparison of the experimental 1 H-1 H spin-spin coupling constants and 13 C NMR chemical shifts with the appropriate theoretical values was performed on the basis of eq. 1 and eq. 2, respectively. The unknown parameters occurring in these equations (a, b, p A , p B , ...)

were derived by a non-linear least-squares analysis of the experimental data set. The chemical shift data for the whole series of investigated species were included into one data set. Such an approach is advantageous as the parameters a and b are common for all three compounds, and the total number of unknowns can be reduced. 4). Adam Gryff-Keller* [a] , Przemysław Szczeciński [a] and Anna Kraska-Dziadecka [a] Results of test calculations for 2-nitro-4-trifluoromethylbenzoyl chloride.

F

It was noticed that in the final chemical shifts/shielding constants correlation the fit for aromatic ring carbons (C1'÷C7') was much poorer than that for C1÷C7 and that the deviations from the regression line were very similar for all three investigated compounds. This latter fact was not surprising as the aromatic parts of the investigated molecules were identical. The larger deviations observed for the aromatic ring carbons could be caused by large amplitude deformations of the equilibrium structures, due to the librations about C7-C1' bond. Such movements could be a result of ground state vibrations and intermolecular collisions. We performed test calculations for a model compound, 4-trifluoromethyl-2nitrobenzoyl chloride, starting from its equilibrium geometry, in which NO 2 group was coplanar with and COCl group perpendicular to the aromatic ring. For this geometry the deviations from the δ/σ regression line for aromatic carbons were almost identical to those for NTBC derivatives. On the other hand, the deviations could be almost completely compensated when it was assumed that in reality we deal with 85 % of conformer with NO 2 group forming a 30 deg angle with the aromatic ring plane and 15 % of conformer with perpendicular NO 2 group (Fig S1). The assumed (during molecular geometry optimization) twist of the NO 2 group obviously resulted in a due twist of the COCl group.

  H3' proton and CF 3 fluorine signals of NTBC derivatives are actually broadened, partially resolved multiplets and, simultaneously, the chemical shift differences involved are very small and concentration-dependent. Unfortunately, organic urine extracts contain, apart from NTBC metabolites, many different compounds whose concentrations overwhelmingly exceed the concentration of the compounds of interest. Therefore, most proton NMR signals of NTBC and its metabolites are covered by intense signals of other mixture components.

  conformers demand the aromatic ring rotation and/or cyclohexane ring inversion, both processes expected to be fast in NMR time scale at room temperature.

13 C- 19 F

 1319 spin-spin couplings and characteristic broadening of C2' signal due to the scalar relaxation of the second kind. It is interesting that the assignments alone are insufficient to make the distinction between NTBC-4-OH and NTBC-6-OH structures.Our experience has shown that applying the 6-311++G(2d,p) basis set in NMR parameter calculations usually yields good results. Unfortunately, however, application of the same basis in optimizations of all 20 structures considered would be computationally too demanding. Thus we have decided to use smaller basis in the first computation step. The shielding calculations are collected in Table1S-4S (Supporting Materials).

CHCl 3 1

 31 proton signal (δ = 7.26 ppm) or carbon CDCl 3 signal (δ = 77.0 ppm). Fluorine spectra in CDCl 3 were referenced to CFCl 3 (δ = 0.0 ppm). 2-D 1 H-13 C correlations of the HSQC and HMQC type were recorded using the spectral widths sw = 6000 Hz, sw1=30000 Hz, number of increments, ni = 512, number of scans, ns = 24, and the standard spectrometer software. 1-D NMR spectra were acquired using standard parameter sets adjusting the number of scans to achieve satisfying s/n for the signals of interest (typically sw = 10000, at = 5 s, digital Hz/point, ns = 64 for 1 H NMR signals and sw = 36000, at = 5 s, digital resolution ~ 0.15 Hz/point, ns = 1000 -10000 for 19 F NMR signals).

Fig. 1 .

 1 Fig.1. Chemical formulas of NTBC and its metabolites.

Fig. 2 .

 2 Fig. 2. Fragment of 19 F NMR spectrum of urine sample (5% of D 2 O was added for lock purpose): a. original, b. after NTBC addition, c. after NTBC and NTBC-6-OH addition.

Fig. 3 .

 3 Fig. 3. Topomeric structures resulting from rotation about C2-C7 bond.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Endo-exo tautomerisation.Fig. 5. Definition of conformer types A and B.

Figure 6 .

 6 Figure 6. Reproduction of the experimental 1 H-1 H spin-spin coupling constants for NTBC-5-OH, using Equation 1 and theoretical values of the appropriate coupling constants calculated for axial and equatorial conformers of the endo-enol of this metabolite (p eq = 0.43 ± 0.03, p ax = 0.57 ± 0.03, a= 0.988, b= -0.3 Hz, RMSD= 0.23 Hz).

Figure 7 .

 7 Figure 7. Reproduction of the experimental 13 C NMR chemical shifts for NTBC, NTBC-5-OH and NTBC-6-OH using Equation2and theoretical values of the appropriate magnetic shielding constants calculated for various conformers of the endo-and exo-enols of these compounds (a = -0.966, b= 186.0 ppm, RMSD = 1.87 ppm, population parameters given in Table4).

Figure 8 .

 8 Figure 8. Comparison of δ exp /σ calc correlations for NTBC-6-OH assuming 2-acyl-3,4dihydroxycyclohex-2-enone and 2-acyl-3,6-dihydroxycyclohex-2-enone structures (the deviations [ppm] from the regression lines are given).

δδ

  Fig.1

Figure S1 .

 S1 Figure S1. Comparison of δ exp vs. σ calc correlations for the aromatic ring carbons of 4trifluoromethyl-2-nitrobenzoyl chloride assuming equilibrium (a, RMSD = 2.62 ppm) and twisted (b, RMSD = 0,72 ppm) molecular geometries (the deviations [ppm] from the regression lines are given).

  The estimates given in Table4are purely 'statistical errors', having, unfortunately, an unclear meaning in the discussed case, where σ errors are hardly random.

	difficult.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	9

C NMR chemical shifts of C-1 to C-7 carbons and placed its results in Table

4

. It is to be realised that error estimation in the problem in hand is very

Table 1 .

 1 The relative a total scf energies (in kJ/mol) after PCM corrections for various conformers of NTBC and its metabolites as calculated by DFT 6-311++G(2d,p)//PBE1PBE/6-31+G* PCM(CHCl 3 ) method.

	o r
	P
	e
	e r
	R
	e
	v i e
	w

a For NTBC and its metabolites the reference energies are -1268.8867 a.u. and -1344.0593 a.u., respectively.

Table 2 .

 2 The experimental values of spin-spin coupling constants [Hz] for NTBC-6-OH and the values calculated for its selected conformers.

	Coupling constant Experimental eq-endo-A eq-endo-B eq-exo-A
	2 J H4,H4a	-19.4	-19.7	-19.8	-19.2
	2 J H5,H5a	-12.9	-13.2	-13.1	-13.2
	3 J H4a,H5	2.3	1.9	2.0	2.1
	3 J H4,H5	5.4	5.4	5.3	5.4
	3 J H4a,H5a	4.9	4.5	4.4	4.2
	3 J H4,H5a

Table 3 .

 3 The experimental values of spin-spin coupling constants [Hz] for NTBC-5-OH and the values calculated for its selected conformers.

		J H5a,H6	13.1	11.3	11.3	11.2
		3 J H6,OH	1.7	0.8	0.2	1.1
		4 J H4,H6	-	0.3	0.2	0.0
		4 J H4a,H6	-	-1.0	-0.5	-0.6
		OH	O
	H5a
	H4a	H6	Ac
	H5
			OH
	H4

Table 4 .

 4 Results of the analysis of13 C NMR chemical shift data on the basis of eq. 2.

	Compound	Parameter	All data points	Aliphatic carbon
				atoms
	NTBC	p endo	0.84±0.12	0.93±0.05
		p exo	0.16±0.12	0.07 ± 0.05
	NTBC-5-OH	p ax-endo	0.51±0.71	0.36±0.36
		p eq-endo	0.38±0.76	0.64 ± 0.39
		p exo	0.11±0.12	0.01 ± 0.06
	NTBC-6-OH	p endo	0.88±0.13	0.97±0.06
		p exo	0.12±0.13	0.03 ± 0.06
	Total data set	a	-0.966± 0.006	-0.966±0.003
		b [ppm]	186.0±0.8	186.6±0.4
		RMSD [ppm]	1.87	0.75

Table S1 .

 S1 The experimental13 C NMR chemical shifts and magnetic shielding constants calculated for A and B conformers of endo-and exo-enols of NTBC.

	4 5	δ exp -δ calc					a		2'
	-1 1 3 2 2 6 4 0 5	3'	112.65 37.26 31.62 125 19.05	6'	130	70.08 4' 147.86 154.04 135 165.25	71.72 148.96 154.61 140 166.51	70.94 146.72 148.04 145 165.64	150	71.01 147.35 147.62 165.67
	-2							1'		δ exp
	-3
	-4				5'
	-5
	1 2	3' δ exp -δ calc F o r	4' b 1'		2'
	0		125	6'	130 P	135	140	145	150
	-1				5'	e e r		δ exp
	-2							R
								e
									v i e
										w
	Carbon		δ exp			σ endo,A	σ endo,B	σ exo,A	σ exo,B
	7		196.27			-17.66	-16.30	-7.62	-8.33
	3		195.76			-15.56	-15.10	-27.14	-27.13
	1		194.12			-15.05	-12.85	-16.02	-15.95
	2'		145.49			38.96	40.83	37.80	38.68
	1'		139.66			38.05	39.75	44.11	43.83
	4'		132.02			51.64	52.56	50.09	50.31
	5'		130.78			47.67	48.55	49.11	47.37
	6'		127.66			55.37	56.62	55.16	54.05
	7'		122.57			55.99	57.80	56.17	56.18
	3'		121.11			59.86	60.52	57.08	58.66

Table S2 .

 S2 The experimental13 C NMR chemical shifts and magnetic shielding constants calculated for equatorial and axial A and B conformers of endo-and exo-enols of NTBC-5-OH. ,ax,A S,ax,B S,eq,A S,eq,B S,ax,A S,ax,B S,eq,A S,eq,B 7 195.80 -17.32 -17.68 -17.37 -17.38 -6.55 -6.22 -8.09 -8.13 1 192.50 -12.59 -12.45 -12.54 -12.76 -13.67 -14.01 -13.37 -13.45 3 191.72 -11.72 -11.74 -12.62 -12.47 -24.88 -25.03 -23.72 -23.

	Carbon	δ exp	Endo-tautomers			Exo-tautomers
									69
	2'	145.60 39.43	38.81	39.97	38.45	39.57	37.02	39.46	38.29
	1'	139.35 38.23	38.78	38.19	38.54	43.78	44.22	44.27	44.14
	4'	132.33 51.63	50.84	51.26	51.44	50.45	50.43	49.83	50.27
	5'	130.86 47.29	47.12	47.33	48.99	47.49	49.62	47.05	49.27
	6'	127.89 55.18	54.97	55.83	56.03	53.15	55.42	53.15	55.14
	7'	122.58 55.98	56.12	56.10	56.09	56.13	56.23	56.25	56.24
	3'	121.20 59.21	59.38	59.08	58.45	58.70	57.59	58.35	56.93
	2	112.70 69.48	70.64	71.19	70.41	70.01	70.90	73.05	72.37
	5	63.51 119.83 119.36 120.20 120.81 119.60 119.81 120.87 120.97
	6	45.87 142.51 140.26 139.54 139.86 141.65 141.00 139.47 139.08
	4	39.86 145.06 145.29 143.52 143.47 139.33 139.82 137.56 137.36

S

Table S3 .

 S3 The experimental13 C NMR chemical shifts and magnetic shielding constants calculated for equatorial A and B conformens of endo-and exo-enols of NTBC-6-OH.

	Carbon 7 1 3 2'	δ exp 195.86 194.83 195.05 o r σ endo,A -16.63 -15.79 -15.57 P e e r R σ endo,B -17.58 -16.09 -15.18 e v i e 145.68 F w σ exo,A -8.10 -16.38 -25.91	σ exo,B -9.04 -16.73 -25.88

Table S4 .

 S4 The magnetic shielding constants of carbon nuclei calculated for equatorial A and B conformens of endo-and exo-enols of NTBC-4-OH.

	Carbon	σ endo,A	σ endo,B	σ exo,A	σ exo,B
	7	-18.53	-18.61	-7.88	-7.70
	1	-13.71	-13.92	-14.99	-14.85
	3	-13.98	-13.80	-25.09	-25.24
	2'	39.08

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Acknowledgements

This work was financially supported by Warsaw University of Technology.

Supporting information

Results of test calculation for 2-nitro-4-trifluoromethylbenzoyl chloride and carbon chemical shift values for NTBC, NTBC-5-OH and NTBC-6-OH and calculated shielding constants for particular conformers of these compounds.