Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

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1 Supporting Information Copyright Wiley-VC Verlag Gmb & Co. KGaA, 6945 Weinheim, 2008

2 An Enolizable Barbiturate with Adjustable ydrogen-bonding Structure for UV/vis Detection of ucleic Acid Bases and Related Compounds Ina Bolz and Stefan Spange* [a] [a] Chemnitz University of Technology Institute of Chemistry Straße der ationen Chemnitz, Germany

3 Table of contents Materials... 3 Instrumentation Keto-enol-tautomerism and self-aggregation of Solvatochromism of MR titration with the ionic liquid [C 6 -mim] + Cl Determination of the pk A values of... 9 Binding studies of +DAP... Self-association study of the salt +PS... 3 UV/Vis-binding studies with six artificial receptors MR binding studies with five artificial receptors MR Titration with -n-butylthymine BuTy... 7 MR Titration with 2,6-bis(trifluoroacetamido)pyridine TFA MR Titration with 2,6-diacetamidopyridine DAC MR Titration with -n-butylcytosine BuCy MR Titration with 9-ethyladenine EtAd References S-2

4 Materials. Unless otherwise noted, all materials were received from commercial suppliers and used without further purification. All reaction and deuterated solvents were dried over appropriate drying agents and freshly distilled prior to use. Synthesis of the chromophor -n-butyl-5-(4-nitrophenyl)- barbituric acid is described in the literature. [S] 2,6-Diaminopyridine (DAP; Acros rganics, 98%) was purified by recrystallization from hot chloroform after filtration with charcoal. The receptors 2,6-diacetamidopyridine DAC [S2], 2,6-bis(trifluoroacetamido)pyridine TFA [S3], 9-ethyladenine EtAd [S4], -butylcytosine BuCy [S5] and -butylthymine BuTy [S6] were synthesized by literatureknown procedures. 9-Ethylguanine was received from Sigma Aldrich ( 98 %). Instrumentation. The UV/Vis absorption spectra of freshly prepared solutions were obtained by means of an MCS 400 diode-array spectrometer (Carl Zeiss Jena). Multiple regression analysis and the linear curve-fitting were performed with rigin 5.0 statistical program. The MR titration experiments were obtained on a Varian Inova-400 spectrometer. Chemical shifts were reported as δ- values in parts per million (ppm) relative to Si(C 3 ) 4 as relative reference (δ = 0 ppm) and to the solvent as internal reference. Minimization of the sum of squared deviations between the observed experimental data points and those calculated with the proposed model was performed employing the nonlinear least-squares fitting program rigin 5.0 which is based on a modified Levenberg- Marquardt algorithm. A detailed explanation for UV/Vis- and -MR spectroscopic analyses is given in the literature. [S7] S-3

5 Keto-enol-tautomerism and self-aggregation of. 2 C keto 2 enol * * d 6 -dimethyl sulfoxide * d 2 -water * d 4 -methanol d 2 -dichloromethane * * d 3 -nitromethane δ / ppm Figure S. MR spectra of (8.9 mmol/l) in different deuterated solvents (marked with asterisk). The presence of a methine proton (black framed) supports the keto-structure for in nitromethane and dichloromethane. In water a protonation-deprotonation equilibrium is observed. S-4

6 aggregated enol monomeric enol self-aggregation in methanol [] in mmol/l A / a.u. keto enol keto-enol tautomerism in dichloromethane [] in mmol/l λ / nm Figure S2. Concentration-dependent UV/Vis titration of in methanol and dichloromethane. Selfaggregation as well as keto-enol tautomerism of is dependent on the polarity of the solvent. In methanol (E T (30) = 55.4) [S8d] the stacking of the enol form is observed. In the weaker polar solvent dichloromethane (E T (30) = 40.7) [S8d] the non-solvatochromic keto form is preferred. S-5

7 Solvatochromism of. Table S. UV/Vis absorption maxima (ν ~ max) of the barbiturate in various solvents, the empirical Kamlet- [S8, S9] Taft parameters α, β, π* and the extend of the solvatochromic absorption shift. solvent α β π* ν ~ max [0-3 cm - ],,,3,3,3-hexafluoro-2- propanol (FP) a 2,2,2-trifluoroethanol (TFE) e water a,2-ethanediol methanol ethanol propanol butanol anisole a m-cresol b acetone ethyl acetate γ-butyrolactone acetonitrile benzene toluene chloroform dichloromethane tetrachloromethane c,,2,2-tetrachloroethane diethyl ether b tetrahydrofuran triethylamine b pyridine ,,, -tetramethyl urea (TMU) d formamide methylformamide ,-dimethylformamide dimethyl sulfoxide [C 6 -mim] + BF [C 6 -mim] + Br [C 6 -mim] + Cl Δλ [nm] 93 Δν ~ [cm ] 5232 a excluded for correlation, b compound present in not-solvatochromic higher aggregation or keto-form, c compound insoluble, d highest bathochromic shift, e highest hypsochromic shift S-6

8 Table S2. The solvent-independent correlation coefficients of the solvatochromic compounds using the Kamlet Taft linear solvation energy relationship (excluding FP, 2, anisole). [S8] ν ~ max,0 a b s r SD F n < < < < A ν ~ max [0-3 cm - ] = α -.85 β π* n = 28; r = 0.93, SD = 0.66; F < FP B ν ~ max [0-3 cm - ] = α -.89 β π* n = 25; r = 0.95, SD = 0.47; F < calculated wave number / cm TMU 3 2 anisole TFE water : [C 6 -mim] + BF 4 2: [C 6 -mim] + Br 3: [C 6 -mim] + Cl calculated wave number / cm TMU 2 3 TFE : [C 6 -mim] + BF 4 2: [C 6 -mim] + Br 3: [C 6 -mim] + Cl measured wave number / cm - measured wave number / cm - Figure S3. Comparison of the results of the Kamlet-Taft analysis of in all solvent used (A) and without,,,3,3,3-hexafluoro-2-propanol (FP), water and anisole (B). S-7

9 influence of α and β bathochromic shift R b is negative bathochromic shift a is negative R + - R hypsochromic shift a is positive hypsochromic shift a is positive R R bathochromic shift b is negative ground state + - influence of π first excited state stronger solvatation s is negative Figure S4. Possible solute-solvent interactions. The multiple correlation analyses of the ν ~ max data with the used Kamlet-Taft parameters show a significant influence of the hydrogen bonding acidity α and basicity β of the solvent on ν ~ max. S-8

10 MR titration with the ionic liquid [C 6 -mim] + Cl -. The stock solutions of (5.45 mmol/l) and the ionic liquid -exyl-3-methylimidazolium chloride [C 6 -mim] + Cl (07 mmol/l) were prepared in dried deutero-dichloromethane under Ar-atmosphere. The [C 6 -mim] + Cl -solution was added in 0 μl steps to. Determination of the pk A values of. The UV/Vis spectra of were recorded in aqueous solution (0.95 mmol/l) from 80 to 000 nm. The p was varied by addition of M Cl or M a and was measured with the VARI p-meter (WTW, Weilheim). Three different maxima of the longest wavelength band λ max were found as function of p value: at 378 nm (p <.8), 385 nm (p ) and at 42 nm (p > 2.93). In every spectrum the absorbances at 378 nm (A), 385 nm (A2) and 42 nm (A3) were determined (figure S5). The pk A -values were estimated as the intercept of the plotting log(a2/a) or log (A3/A2) as a function of p according to the enderson- asselbalch equation. [S7a] S-9

11 0,004 exp fit A / a.u log (A 385nm /A 378nm ) 0,002 0,000-0,002-0,004 pk S ()=.93 Lineare Regression für Data4B: Y = A + B * X Parameter Wert Fehler A B R SD P E ,4,6,8 2,0 2,2 2,4 p.0 p= nm λ / nm A / a.u p = ,06 0,04 λ / nm exp fit 2 A / a.u nm log (A 43nm /A 385nm ) 0,02 0,00-0,02-0,04-0,06 pk S (2) = 2.75 Lineare Regression für DataB: Y = A + B * X Parameter Wert Fehler A B R SD P ,3 2,4 2,5 2,6 2,7 2,8 2,9 3,0 3, 3,2 p 405 nm p= λ / nm Figure S5. Set of UV/Vis absorption spectra of under the variation of p value. S-0

12 Binding studies of +DAP. The binding study between the model base DAP and the host was investigated by UV/Vis and MR spectroscopy. UV/Vis titration. The UV/Vis titration was performed using dichloromethane (freshly distilled from Ca 2 ). A typical binding experiment involved the titration of (25 ml, 0.3 mmol/l) where ten aliquots of 0 μl and five aliquots of 20 μl of the DAP stock solution ( mmol/l) were added. The increase in UV/Vis absorption intensity at 4 nm was monitored as a function of guest concentration. The : stoichiometry of the adduct +DAP was confirmed with the method of continuous variations (Job s method, figure S6). [S7a] 2 C 2 DAP complex formation + DAP + 2 proton-transfer reaction - + DAP A 4 nm / a.u mole fraction of Figure S6. Job s plot between and DAP (every stock solution: 0.3 mmol/l) observed by UV/Vis spectroscopy in dichloromethane. The simple acid-base reaction should be favourable. S-

13 MR spectroscopy. MR binding studies were carried out in dried d 6 -dimethyl sulfoxide (residue water concentration < 0.%). In a typical experiment, the MR spectrum of the host solution (, 2.2 mmol/l,.0 ml of solvent) was recorded and then small portions of DAP were added to the MR tube under argon atmosphere. The concentration of DAP was calculated by the comparison of the integrals. After the : stoichiometry was reached, the chemical shifts of the proton of were monitored as a function of DAP concentration for the determination of the association constant K A (figure S7). [S7a] K A for the complex formation of the enolate anion - with DAP was then obtained as a constant using the simple equilibration equation (). + DAP + DAP- + K + DAP A +DAP (equ. ) If we assume that the proton transfer reaction is complete and only a : complex is formed, then the association constant K A is given by K A = ( 0 x x)( DAP 0 x) (equ. 2) where x is the concentration of the formed complex and 0 and DAP 0 are the initial concentrations in mol/l. The observed chemical shift of the enolate proton δ obs is given by δ obs 0 x = 0 δ + x 0 δ - c (equ. 3) where δ is the chemical shift of the proton of the free enolate anion and δ c the chemical shift of the proton of the complexed enolate anion c. The titration curve (equ. 4) represents the relationship between the observed chemical shift of the enolate proton K A : δ obs and the total concentration of DAP DAP 0 with the three unknown parameters δ, δ and c δ obs δ δ = δ + c + DAP ( DAP + + ) ( ) + ( ) + ( ) 0 0 K A DAP K A 2 0 K A K A (equ. 4) S-2

14 Minimization of the sum of squared deviations between the observed experimental data points and those calculated with the proposed model was performed employing the nonlinear least-squares fitting program RIGI which is based on a modified Levenberg-Marquardt algorithm DAP 0,4 0,2 δ obs / ppm 0,0 9,8 9,6 K A = 2.73 ± 0.35 M - experimental values fit [DAP 0 ] / mmol/l Figure S7. bserved chemical shift of the enolate proton of of the total concentration of DAP 0 in dried d 6 -DMS. δ obs ( [ 0 ] = 2.6 mmol/l) as function Self-association study of the salt +PS. UV/Vis titration. The binding study between and the proton sponge PS was performed according to the UV/Vis binding study of and DAP. To the stock solution of (25 ml, 0.3 mmol/l) were added ten aliquots of 0 μl and five aliquots of 20 μl of the PS stock solution ( mmol/l) in dichloromethane (freshly distilled from Ca 2 ). The increase in UV/Vis absorption intensity at 45 nm was monitored as a function of guest concentration [PS 0 ]. Saturation is reached with the : stochiometry which shows that selfaggregation of the enolate anion is neglible in this concentration range. The : stoichiometry of the adduct +PS was confirmed with the method of continuous variations (Job s method, figure S8). [S7a] S-3

15 A A / a.u ration of to PS.0 : λ / nm B,2 : stoichiometry,0 A 45 nm / a.u. 0,8 0,6 0,4 0,2 0,0 0,00 0,05 0,0 0,5 0,20 0,25 0,30 [PS 0 ] / mmol/l C A 45 nm / a.u mole fraction of Figure S8. UV/Vis absorption spectra (A) of (0.3 mmol/l, dotted line) with PS ( mmol/l), the corresponding binding curve (B) and Job s plot (C, every stock solution: 0.3 mmol/l) in dichloromethane. S-4

16 Concentration-dependent MR spectroscopy. According to the literature [S9], the salt +PS, which is a : mixture of the enolate anion of and the proton sponge, [S] was concentrated stepwise in the concentration range 33 mmol/l. Each time a MR spectrum was recorded. The measurements were carried out under an argon atmosphere in d 6 -DMS that had been dried over 4 Å molecular sieves (residue water concentration < 0.%). The concentration-dependent shifts of the proton of the enolate anion were fitted with the non-linear least-squares program RIGI similar to the procedure described (figure S9) self-aggregation δ -enolate anion / ppm K A = 2.8 ± M - experimental value fit [+PS] / mmol/l Figure S9. Concentration-dependent MR measurements of the equimolar mixtures of and the proton sponge PS in dried d 6 -DMS. S-5

17 UV/Vis-binding studies with six artificial receptors. The UV/Vis titration was performed using dichloromethane and methanol at a ratio of.00 to 0.04 (both freshly distilled from appropriate drying agents). A typical binding experiment involved the titration of (.0 ml, mol/l) where aliquots of ml of the receptor stock solution ( mol/l) were added. Due to the poor solubility, the BuCy-stock solution was mol/l (added aliquots ml). The stock solution of EtGu was only mol/l, the added aliquots ( ml) correspond to a molar ratio of EtGu from to 4. The solutions were filled up to 5.0 ml. The increase in absorption intensity was monitored as a function of guest concentration. The quantitative determination of the association constant K A is based on the absorbance variation of the host (, ) in the presence of the guest (G, receptor) with certain concentration. [S7] The UV/Vis-control experiments were performed in dichloromethane using the same titration procedure. Figure S0 shows the UV/Vis titration of with the receptor DAC and with EtAd in both solvent systems. A dichloromethane / methanol 0,8 0,7 B dichloromethane 0,8 0,7 0,6 0,6 + DAC,5 A 422 nm / a.u. 0,5 0,4 0,3 0,2,5 A 422 nm / a.u. 0,5 0,4 0,3 0,2 A / a.u.,0 0,5 422 nm 0, 0,0 0,000 0,005 0,00 0,05 0,020 [DAC] / 0-3 M - A / a.u.,0 0,5 422 nm 0, 0,0 0,000 0,005 0,00 0,05 0,020 [DAC] / 0-3 M - DAC 0,0 DAC DAC λ / nm 0,0 DAC λ / nm 0,8 0,8 0,7 0,7 + EtAd,5 A 40 nm / a.u. 0,6 0,5 0,4 0,3 0,2,5 A 40 nm / a.u. 0,6 0,5 0,4 0,3 0,2 A / a.u.,0 0,5 0,0 342 nm 40 nm 0, 0,0 0,000 0,005 0,00 0,05 EtAd [EtAd] / 0-3 M - EtAd λ / nm A / a.u.,0 0,5 0,0 40 nm 0, 0,0 0,000 0,005 0,00 0,05 0,020 EtAd [EtAd] / 0-3 M - EtAd λ / nm Figure S0. UV/Vis absorption spectra of ( mol/l) with the receptors DAC ( mol/l) and EtAd ( mol/l) in dichloromethane/methanol (ratio.00:0.04, A) and in dichloromethane (B). S-6

18 MR binding studies with five artificial receptors. MR titrations were carried out in dried d 2 dichloromethane (residue water concentration < 0.%). In a typical experiment, the MR spectrum of the host solution (, mol/l,.0 ml of solvent) was recorded and then small portions of the receptor were added to the MR tube under argon atmosphere. The concentration of the receptor was calculated by the comparison of the integrals. The chemical shift of the proton of was monitored as a function of the receptor concentration for the determination of the association constant K A. [S7a] In the following sections adequate MR experiments are shown. The MR resonances of the receptor are blue highlighted. The overlapping of the alkyl chains of the chromophor and the receptor are marked with blue dotted lines. -protons are marked with arrows. MR Titration with -n-butylthymine BuTy. [BuTy] 0-3 mol/l δ (Keto) ppm [] = mol/l δ C-5(Keto) ppm C keto - 2 C + keto - + BuTy BuTy [BuTy] / 0-3 mol/l δ / ppm S-7

19 MR Titration with 2,6-bis(trifluoroacetamido)pyridine TFA. [TFA] 0-3 mol/l δ (Keto) ppm [] = mol/l δ C-5(Keto) ppm F 3 C C keto - + TFA CF 3 [TFA] / 0-3 mol/l δ / ppm MR Titration with 2,6-diacetamidopyridine DAC. 2 2 [DAC] 0-3 mol/l δ (Keto) ppm δ C-5(Keto) ppm C keto - + DAC enol DAC [DAC] / 0-3 mol/l [] = mol/l δ / ppm S-8

20 MR Titration with -n-butylcytosine BuCy. [BuCy] 0-3 mol/l δ (Keto) ppm δ C-5(Keto) ppm / * * * * * - * δ(enol) / ppm; [] = mol/l enol - BuCy enol - + BuCy [BuCy] / 0-3 mol/l δ / ppm MR Titration with 9-ethyladenine EtAd. [EtAd] 0-3 mol/l δ (Keto) ppm * complex precipitated; δ C-5(Keto) ppm * * * * Et 2 Et enol 2 - EtAd enol EtAd [EtAd] / 0-3 mol/l [] = mol/l S-9 δ / ppm

21 References S I. Bolz, C. Moon, V. Enkelmann, G. Brunklaus, S. Spange, J. rg. Chem. 2008, 73, S2 K. Kurita, R. L. Williams, J. eterocyclic Chem. 975, 2, S3 E. Beinlinger, A. iemz, V. M. Rotello, J. Am. Chem. Soc. 995, 7, S4 J. S. owick, J. S. Chen, G. oronha, J. Am. Chem. Soc. 993, 5, S5 D. L. elfer II, R. S. osmane,. J. Leonard, J. rg. Chem. 98, 46, S6 A. D. amilton, D. van Engen, J. Am. Chem. Soc. 987, 09, S7 a) K. A. Connors, Binding Constants, J. Wiley & Sons, ew York, 987; b) I. Bolz, C. May, S. Spange, ew J. Chem. 2007, 3, S8 a) M. J. Kamlet, J.-L. M. Abboud, M.. Abraham, R. W. Taft, J. rg. Chem. 983, 48, ; b) Y. Marcus, Chem. Soc. Rev. 993, 22, ; b) A. ehlke, K. ofmann, S. Spange, ew J. Chem. 2006, 30, ; c) R. Lungwitz, M. Friedrich, W. Linert, S. Spange, ew J. Chem. 2008, DI:0.039/b805932b; d) C. Reichardt, Solvents and Solvent Effects in rganic Chemistry, VC, Weinheim, S9 K. S. Schmidt, R. K.. Sigel, D. V. Filippov, G. A. van der Marel, B. Lippert, J. Reedijk, ew J. Chem. 2000, 24, S-20