Synthesis, characterization and thermal studies on solid 3-methoxybenzoate of some bivalent transition metal ions

 0  8  7  2017-02-01 13:16:15 Report infringing document Volume 30, número 3, 2005 Synthesis, characterization and thermal studies on solid 3-methoxybenzoate of some bivalent transition metal ions A. C. Vallejo, A. B. Siqueira, E. C. Rodrigues, E.Y. Ionashiro, G. Bannach, M. Ionashiro1 Instituto de Química, UNESP, C. P. 355, CEP 14801 – 970, Araraquara, SP, Brazil. 2 Corresponding author: Abstract: Solid-state M-3-MeO-Bz compounds, where M stands for bivalent Mn, Co, Ni, Cu and Zn and 3-MeO-Bz is 3-methoxybenzoate, have been synthesized. Simultaneous thermogravimetry and differential thermal analysis (TG-DTA), differential scanning calorimetry (DSC), X-ray powder diffractometry, infrared spectroscopy, and chemical analysis were used to characterize and to study the thermal behaviour of these compounds. The results led to information about the composition, dehydration, thermal stability and thermal decomposition of the isolated compounds. Keywords: bivalent transition metals; 3-methoxybenzoate; thermal behaviour. Introduction Several metal-ion salts of benzoic acid, as well as some benzoic acid derivatives, have been investigated in aqueous solutions and in solid state. In aqueous solutions, Yun and co-workers [1] reported the thermodynamics of complexation of lanthanides by some benzoic acid derivatives. Wang and co-workers [2] reported the spectroscopic study of trivalent lanthanides with several carboxylic acids, including benzoic acid. Arnaud and Georges [3] described the influence of pH, surfactant and synergic agent on the luminescent properties of terbium chelates with benzoic acid derivatives. Choppin and co-workers [4] reported the thermodynamic of complexation of lanthanides by benzoic and isophthalic acids. Lam and co-workers [5] described the synthesis, crystal structure and photophysical and magnetic properties of dimeric and polymeric lanthanide complexes with benzoic acid and its derivatives. In the solid state, Wendlandt synthesized and reported the thermal stability and thermal decomposition of thorium salts with several organic acids, including 4-methoxybenzoic acid [6], as well as benzoic and m-hydroxybenzoic acids [7]. Ecl. Quím., São Paulo, 30(3): 7-13, 2005 Glowiack and co-workers [8] reported the reaction of bivalent copper, cobalt and nickel with 3-hidroxy-4-methoxy and 3-methoxy-4hidroxybenzoic acids and a structure for these compounds has been proposed on the basis of spectroscopic and thermogravimetric data. Brzyska and Karasinski [9] described the thermal decomposition of thorium salts of benzoic and 4methoxybenzoic acids in air atmosphere. Ferenc and Walkow-Dziewulska [10] reported the synthesis and characterization of 2,3-dimethoxybenzoates of heavy lanthanides and yttrium by elemental analysis, IR spectroscopy, thermogravimetric studies and Xray diffraction measurements. In the present paper, solid-state compounds of Mn (II), Co (II), Ni (II), Cu (II) and Zn (II) with 3-methoxybenzoate were prepared. The compounds were investigated by means of chemical analysis, X-ray powder diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and simultaneous thermogravimetry-differential thermal analysis (TGDTA). The results allowed us to acquire information concerning these compounds in the solid state, including their thermal stability and thermal decomposition. 7 Experimental The 3-methoxybenzoic acid, 3-CH3O-C6H4CO2H (3-MeO-HBz) 98% was obtained from Acros organics and purified by recrystallization. Thus, aqueous suspension of 3-MeO-HBz was heated near ebullition until total dissolution and cooled to ambient temperature. The crystal in needle form was isolated and after dry kept in a plastic flask. The hydrated basic carbonates of Mn (II), Co (II), Ni (II), Cu (II) and Zn (II) were prepared by adding slowly with continuous stirring satured sodium carbonate solution to the corresponding metal chloride solutions or sulphate for copper until total precipitation of the metal ions. The precipitates were washed with distilled water until elimination of chloride or sulphate ions (qualitative tests with AgNO3/HNO3 solution for chloride ions or BaCl2 solution for sulphate ions) and maintained in an aqueous suspension. Solid state Mn (II), Co (II), Ni (II), Cu (II) and Zn (II) compounds were prepared by mixing the corresponding metal basic carbonates with 3methoxybenzoic acid, in slight excess. The aqueous suspension was heated slowly up to near ebullition, until total neutralization of the respective basic carbonates. The resulting solutions, except the copper compound, after cooled were maintained in an ice bath to recrystallize the acid in excess and filtered through a Whatman nº 40 filter paper. Thus, the aqueous solutions of the respective metal-3methoxybenzoates were evaporated in a water bath until near dryness and kept in a desiccator over anhydrous calcium chloride. The copper compound, after the neutralization the precipitate was washed with distilled water heated up to near ebullition, filtered through and dried on Whatman nº 40 filter paper, and also kept in desiccator. All attempts to synthesize the iron (II) compound were unsuccessful, due to the oxidation reaction of Fe (II) to Fe (III) during the drying of the compound, even by using nitrogen atmosphere. In the solid-state compounds, metal ions, water and 3-methoxybenzoate contents were determined from TG curves. The metal ions were also determined by complexometry with standard EDTA solution [11, 12] after igniting the compounds of the respective oxides and their dissolution in hydrochloric acid solution. X-ray powder patterns were obtained with 8 a SIEMENS D-5000 X-ray diffractometer using Cu Kα radiation (λ = 1.544 Å) and setting of 40 kV and 20 mA. Infrared spectra for 3-methoxybenzoic acid, sodium 3-methoxybenzoate as well as for its metalion compounds were run on a Nicolet model Impact 400 FT-IR instrument, within the 4000-400 cm-1 range. The solid samples were pressed into KBr pallets. Simultaneous TG-DTA curves were obtained with thermal analysis systems model SDT 2960, from TA instruments. The purge gas was an air flow of 150 mL min-1. A heating rate of 20ºC min1 was adopted with samples weighing about 7 mg. Alumina crucible was used for TG-DTA. The DSC curves were obtained using a Mettler TA-4000 thermal analysis system. The purge gas was flowing air with a flow rate of 150 mL min-1. The heating rate was 20ºC min-1 and the initial sample mass was about 7mg. Aluminium crucible with perforated cover was used for DSC experiment. Results and Discussion The X-ray powder diffraction patterns, Fig. 1 show that the synthesized compound have a crystalline structure, without evidence for formation of an isomorphous series, except for the nickel compound that was obtained in amorphous state. Infrared spectroscopic data on 3methoxybenzoic acid, 3-methoxybenzoate and its compounds with the metal ions considered in this work are shown in table 2. The investigation was focused mainly within the 1700 – 1400 cm-1 range because this region is potentially most informative to assign coordination sites. In sodium 3methoxybenzoate, strong band at 1568 cm-1 and a medium intensity band located at 1402 cm-1 are attributed to the anti-symmetrical and symmetrical frequencies of the carboxylate groups, respectively [13, 14]. The manganese, cobalt, nickel, copper and zinc compounds, presented practically the same symmetrical and anti-symmetrical vibrations of the COO- groups, when compared with the sodium salt, suggesting that the compounds have ionic character. In all the compounds, except for the copper one, the medium intensity band observed within 1682-1693 cm 1 range are attributed to C=O stretch of dimmer structure of the acid, suggest the presence of 3Ecl. Quím., São Paulo, 30(3): 7-13, 2005 methoxybenzoic acid in these compounds. Simultaneous TG-DTA curves of the compounds are shown in Fig. 2. These curves show mass losses in consecutive and/or overlapping steps with partial losses which are characteristic for each compound. Only the copper compound shows an endothermic peak corresponding to the first mass loss ascribed to dehydration. For the other compounds, no endothermic peak compatible with the first mass loss due to the dehydration is observed. Based on the information of infrared spectra and DTA curves, samples of these compounds were heated in a long test glass tube up to the temperature corresponding to the first mass loss and under approximately the same conditions as the TG-DTA curves. In these experiments, sublimation for the nickel compound and sublimation followed by fusion and evaporation for manganese, cobalt and zinc compounds were observed.The sublimated materials were identified as 3-methoxybenzoic acid, by infrared spectra and m.p. = 106 – 108ºC literature; 105 – 108ºC, observed. The presence of 3-MeO-HBz in these compounds must be due to the aggregation phenomenon, indicating that the procedure used to eliminate the acid in excess is not efficient and that the acid is more soluble in 3-methoxybenzoate solution and it no recrystallize even an ice bath. Therefore the first mass loss up to 280ºC (Mn), 200ºC (Co) and 230ºC (Ni, Zn), is due to elimination of 3-methoxybenzoic acid. Thus, Table 2 presents the analytical and thermoanalytical (TG) results for the prepared compounds, from which the general formula Cu(L)2.H2O or M(L)2.xHL can be established, where M represents Mn, Co, Ni and Zn, L = 3methoxybenzoate, HL = 3-methoxybenzoic acid and x = 0.19(Mn), 0.28(Co), 0.13(Ni) and 0.081(Zn). Manganese compounds. The TG-DTA and DSC curves are shown in Fig.2a. These curves show mass losses in two steps between 50-180ºC and 180- 500ºC (TG), and endothermic peaks at 180ºC (DTA, DSC), 100ºC (DSC) and exothermic peaks at 360ºC, 430ºC (DTA, DSC), 474ºC (DTA), 500ºC and 520ºC. The first mass loss that occurs through a slow process is attributed to the loss of 0.19 HL (3methoxybenzoic acid) (calcd. = 7.49%; TG = 7.46%). The second step that occurs through a fast process is ascribed to the thermal decomposition of the compounds that occurs in a single step (Calcd. Ecl. Quím., São Paulo, 30(3): 7-13, 2005 = 72,76%; TG =72.92%), with formation of Mn3O4, as final residue (Calcd. 19.75%; TG=19.57%), and confirmed by X-ray powder diffractometry. The endothermic peaks at 100ºC (DSC and 180ºC (DTA, DSC), are due to sublimation of 3-MeO-BP and fusion of the compounds, respectively, and in agreement with the experiment already described, with sample heated in a long test tube. The exothermic peaks at 360ºC, 430º and 475 ºC (DTA, DSC), 500 and 520 ºC (DSC) are attributed to the oxidation of the organic matter. Figure 1. X-ray powder diffraction patterns of the compounds: (a) Mn(L)2.0.19HL, (b) Co(L)2.0.28HL, (c) Ni(L)2.0.13HL, (d) Cu(L) 2 .H 2 O and (e) Zn(L) 2 .0.081HL (L=4methoxybenzoate, HL = 4-methoxybenzoic acid). Cobalt compound. The TG-DTA and DSC curves are shown in Fig. 2b. These curves show mass losses in four consecutives and/or overlapping steps between 80-200ºC, 200-380ºC, 380-480ºC and 900-920ºC (TG) and endothermic peaks at 235ºC(DTA, DSC) and 900ºC(DTA), and exothermic peaks at 480ºC (DTA, DSC) and 490ºC(DSC).The first mass loss, without thermal event observed in the DTA and DSC curves is ascribed to the loss of 9 0.28 HL (3-methoxybenzoic acid) (Calcd. = 10.55%; TG=10.61%). The endothermic peak at 235ºC (235ºC) DTA, DSC is attributed to the fusion of the compound. The second and third steps with losses of 26.13% and 43.77%, corresponding to the single exothermic peak (DTA) or a broad exothermic between 410-550ºC (DSC), are attributed to the thermal decomposition of the compound. The profiles of the TG-DTA curves show that the oxidation of the organic matter occurs with combustion. The total mass loss up to 480ºCis in agreement with the formation of Co3O4 (Calcd. = 80.13%; TG = 80.51%).The last step corresponding to the small endothermic peak is due to the reduction of Co3O4 to CoO (Calcd. =1.32%; TG =1.21%) and is in agreement with literature. [15,16]. X-ray powder patterns of the residue obtained at 1000ºC is coincident with that obtained for Co3O4; this is due to the reoxidation reaction of CoO to Co3O4 which occurs on cooling the former in an air atmosphere at room temperature [16]. Table 1. Spectroscopic data for 3-methoxybenzoic acid (HL), sodium 3-methoxybenzoate (NaL), and compounds with some bivalent metal ionsa. br: broad, m = medium, s = strong. ν(o – H): hydroxyl group stretching frequency. c νsym(COO-) and νas(COO-): symmetrical and anti-symmetrical vibrations of the COO- group, respectively d ν(C=O): stretching frequency of the dimmer structure. a b Table 2. Analytical data for the M(L)2.H2O and ML2.nHL compounds. ∆L = Ligand lost; HL = 3-metoxibenzoic acid. M = metal 10 Ecl. Quím., São Paulo, 30(3): 7-13, 2005 Nickel Compound. The TG-DTA and DSC curves are shown in Fig.2c. The mass loss that occurs between 50 and 200ºC through slow process, without appearing of thermal event in the DTA and DSC curves, is due to the loss of 0.13 HL (3-methoxybenzoic acid) (Calcd. =5.19%; TG = 5.27%). Above this temperature up to 470ºC, the thermal decomposition occurs through a fast process, corresponding to the exothermic peak at 470ºC (DTA, DSC) attributed to the oxidation of the organic matter. The profiles of the TG and DTA curves show that this step the oxidation of the organic matter is followed by combustion. The total mass loss up to 470ºC is in agreement with the formation of NiO, as final residue (Calcd. =80.29%; TG = 80.69%) and confirmed by X-ray powder diffractometry. Figure 2. TG-DTA and DSC curves of the compounds: (a) Mn(L)2.0.19HL (m = 6.9788mg), (b) Co(L)2.0.28HL (m = 7.0760mg), (c) Ni(L)2.0.13HL (m = 7.1162 mg), (d) Cu(L)2.H2O ( m = 7.2794 mg) and (e) Zn(L)2.0.081HL (m = 7.1821 mg). L= 3-methoxybenzoate, HL = 3-methoxybenzoate. Ecl. Quím., São Paulo, 30(3): 7-13, 2005 11 Copper compound. The TG-DTA and DSC curves are show in Fig.2d. The mass loss observed between 95 and 130ºC, corresponding to an endothermic peak at 120ºC (DTA), 140ºC (DSC) is due to dehydration with loss of 1 H2O (Calcd. = 4.69%; TG = 4.61%). The anhydrous compound is stable up to 210ºC, and above this temperature the thermal decomposition occurs in two consecutive steps with losses of 48.22% (210-355ºC) and 26.13 % (355-400ºC). These mass losses corresponding to the sharp exothermic peak at 400ºC (DTA) or two exothermics between 290-340ºC and 360ºC (DSC), are attributed to the oxidation followed by combustion of the organic matter. The total mass loss up to 400ºC is in agreement with the formation of CuO as the final residue (Calcd. = 79.28%; TG = 78.96%), which was confirmed by X-ray powder diffractometry. The last mass loss between 1034 and 1050ºC corresponding to the endothermic peak at 1045ºC is attributed to reduction of CuO to Cu2O (Calcd. = 2.08%; TG = 2.0%). Zinc compound. The TG-DTA and DSC curves are shown in Fig. 2e. The mass loss observed between 90 and 240ºC without appearing of thermal event in the DTA curve is due to the loss of 0.081 HL (3-methoxybenzoic acid) Calcd. 3.24%; TG = 3.21%). In this temperature range the DSC curve shows three endothermic peaks at 110ºC, 150ºC and 200ºC, ascribed to sublimation of 3-methoxybenzoic acid, fusion and evaporation respectively. Above 240ºC, the thermal decomposition occurs in three steps with losses of 29.93% (240 -380ºC), 23.64% (380-500º) and 26.02% (500-538%), corresponding to the exothermic peaks at 520 and 538ºC (DTA) and 500oC and exothermic between 540-600 o C (DSC) attributed to oxidation of organic matter. The profiles of the TG and DTA curves show that the last step the oxidation of the organic matter is accompanied by combustion. The total mass loss up to 538ºC is in agreement with the formation of ZnO as final residue (Calcd. = 78.59%; TG = 78.80%) and confirmed by X-ray powder diffractometry. The differences observed concerning the peak temperatures obtained by DTA and DSC, or 12 peaks observed only in the DSC curves are undoubtedly due to the perforated cover used to obtain the DSC curves while the DTA ones are obtained without cover and due to the greater sensibility of the DSC. Conclusions The TG-DTA curves, infrared spectra and chemical analysis, permitted to verify that the binary compounds were obtained, however contaminated with 3-methoxybenzoic acid, except the copper compound. The procedure used to synthesize these compounds permitted to verify that compound little soluble (copper one) was obtained without contamination, and that the contamination occurs during the evaporation of the compounds solutions, due to the aggregation phenomenon. The TG-DTA, also permitted verify that the 3-MeO-HBz is eliminated before thermal decomposition of these compounds, thus binary compounds may be obtained by thermosynthesis. The X-ray powder patterns pointed out that the synthesized compounds have a crystalline structure, except the nickel one, without evidence concerning the formation of isomorphous series. The infrared spectroscopy data suggest that the compounds considered in this work have an ionic character. The TG-DTA and DSC provided previously unreported information about the thermal stability and thermal decomposition of these compounds. Acknowledgements The authors thank FAPESP (Proc. 97/126468), CNPq and CAPES foundations (Brazil) for financial support. Recebido em: 18/03/2005 Aceito em: 29/04/2005 Ecl. Quím., São Paulo, 30(3): 7-13, 2005 A. C. Vallejo, A. B. Siqueira, E. C. Rodrigues, E.Y. Ionashiro, G. Bannach, M. Ionashiro Síntese, caracterização e estudo térmico dos 3-metoxibenzoatos de alguns metais de transição, no estado sólido. __________________________________________________________________________________ Resumo: Compostos M-3-MeO-Bz foram sintetizados no estado sólido, onde M representa os íons bivalentes Mn, Co, Ni, Cu e Zn e 3-MeO-Bz o ânion 3-metoxibenzoato. equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. 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