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Proton Transfers on Amphiphilic Molecules. A Nonionic Alkyllipopeptide in Dimethyl Sulfoxide J. Chrisment and J.-J. Delpuech* Laboratoire d’Etude des Solutions Organiques et Colloı¨dales, URA CNRS no. 406, Universite´ Henri Poincare´ sNancy 1, BP 269, 54506 Vandoeuvre-le` s-Nancy Cedex, France Received September 20, 1995. In Final Form: January 15, 1996X The protonation of the peptide bond (CO-NH) in an amphiphilic compound, the decyl N-dodecanoylsarcosylglycyl ester (DSG), has been studied in dimethyl sulfoxide (DMSO) acidified with trifluoromethanesulfonic acid (CF3SO3H). The surface-active properties of DSG (in DMSO) are however not accompanied by extensive micellization, as shown by diffusion NMR measurements combined with viscosity data. Protonation rates show clearly different kinetic laws depending on the concentration range studied (0.010.13 and 0.3-0.4 mol dm-3) and the Z-E stereoisomerism of DSG. At lower concentrations, proton transfers at 25 and 45 °C classically involve the solvated proton DMSO‚‚‚H+: kH(25 °C) ) 7.7 and 5.5 s-1, ∆Hq ) 41.3 and 37.0 kJ mol-1, and ∆Sq ) -83.5 and -100.5 J mol-1 K-1 for the cis and trans isomers, respectively. At higher concentrations and 45 °C, additional mechanisms are required, involving the N- and/or O-protonated peptide as proton carrier.
Introduction Proton transfers on oxygen, nitrogen, and carbon atoms of weak organic acids have been extensively studied since the advent of dynamic NMR methods.1 Aqueous and nonaqueous solutions have been used for this purpose. In this respect, dimethyl sulfoxide (DMSO) is commonly used in NMR studies of peptides and carbohydrates because of its solubilizing properties.2 We have now decided to extend these investigations to amphiphilic molecules dissolved either as monomeric units or as micellar aggregates. The first example reported in this paper concerns a simple sarcosyl-glycine dipeptide doubly protected by long alkyl chains, namely a decyl ester on the carboxylate terminal and a dodecanoyl group on the amino end. In DMSO, aggregation phenomena are expected to be weak on the basis of prior structural investigations on related nonionic alkyl-fluoroalkyl lipopeptides.3 The very first step in these investigations is to compare acid-catalyzed proton transfers on these molecules and a structurally related non-tensioactive compound, the octyl N-acetylsarcosylglycyl ester (ASG) already described in a previous publication.4 Experimental Section Materials. Decyl N-dodecanoylsarcosylglycyl ester (DSG) was synthesized according to procedures worked out by Castro et al.,5 using the BOP reagent, as described in previous publications.4 The trifluoroacetate (TFA) derivative of decyl sarcosylglycyl ester was first synthesized in the same way as the analogous TFA-Sar-Gly-Oct used as an intermediate in the synthesis of ASG. Two acetonitrile solutions were then prepared containing either (a) 1 equiv of BOP reagent, TFA-Sar-Gly-Dec, and diisopropylamine or (b) 1 and 2 equiv, respectively, of lauric acid and diisopropylamine. The two solutions were mixed X
Abstract published in Advance ACS Abstracts, March 15, 1996.
(1) Delpuech, J.-J. In Dynamics of Solutions and Fluid Mixtures by NMR; Delpuech, J.-J., Ed.; John Wiley and Sons: Chichester, 1995; Chapter 3. (2) Craig, L. C.; Cowburn, D.; Bleich, H. Annu. Rev. Biochem. 1975, 44, 477 and references therein. (3) Chrisment, J.; Delpuech, J.-J.; Hamdoune, F.; Ravey, J.-C.; Selve, C.; Ste´be´, M.-J. J. Chem. Soc., Faraday Trans. 1993, 89, 927. (4) Chrisment, J.; Delpuech, J.-J.; Rajerison, W. J. Chim. Phys. 1991, 88, 1757. (5) Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron Lett. 1975, 1219. Castro, B.; Dormoy, J. R.; Dourtoglou, B.; Evin, G.; Selve, C.; Ziegler, J. C. Synthesis 1976, 751. Castro, B.; Evin, G.; Selve, C.; Seyer, R. Synthesis 1977, 413. Selve, C.; Hamdoune, F. Tetrahedron Lett. 1989, 30, 5755.
Scheme 1. cis and trans Isomers of DSG
together and stirred for 3 h at room temperature. After filtration, the solvent was removed by evaporation under reduced pressure. The residue was dissolved into hexane, and DSG was obtained by recrystallization. The purity was controlled by NMR, chromatography, and elemental analysis: yield, 95%; m.p., 71 °C; NMR (0.1 M DSG in DMSO-d6 at 25 °C and 400 MHz), δ 0.85 (t, J ) 7.0 Hz, CH3 (dec and decanoyl)), 1.25 (s, CH2)8 (dodecanoyl) and (CH2)7 (dec)), 1.48 and 1.54 (2m, 2-CH2 (dec)), 2.19 and 2.31 (2t, J ) 7.4 Hz, 2-CH2 (dodecanoyl)), 2.80 and 2.96 (2s, N-CH3), 3.82 and 3.84 (2d, J ) 5.9 Hz, CH2 (Gly)), 3.95 and 3.98 (2s, CH2 (Sar)), 4.03 (t, J ) 7.4 Hz, 1-CH2 (dec)), 8.10 and 8.34 (2t, J ) 5.8 Hz, NH). Solutions. The procedures used to purify DMSO and to prepare DMSO solutions have been described previously.6 Acidic solutions used to promote proton transfers were obtained by addition of CF3SO3H (Fluka, puriss). Hydrogen ion concentrations at equilibrium were taken as the analytical concentrations of added CF3SO3H (0.057-0.57 mol dm-3). DMSO-d6 (Fluka, puriss) was used without further purification. NMR Spectroscopy. 1H-NMR was performed on a Bruker AM-400 spectrometer at 400 MHz. As in the case of ASG, cistrans stereoisomerism about the dodecanoyl N-C partial double bond (Scheme 1) was observed in solution at 25 °C; this results in two series of lines, all of them in the same population ratio cis/trans ) 55/45 all along the temperature range 25-55 °C. Above 55 °C, the two series of lines are progressively coalescing into each other owing to chemical exchange between cis and trans isomers. The kinetics of the rate process was conveniently studied between 55 and 80 °C using 0.1 M solutions of DSG in DMSO-d6 and the coalescence of the sarcosyl cis and trans methylic singlets. Theoretical line shapes were computed according to the matrix formulation of Anderson, Kubo, and Sack, using the program ECHGN.7 Proton transfers were conveniently studied in the temperature range 25-55 °C, where cis-trans isomerization can be neglected. The coalescence of each (cis and trans) glycyl N-methylenic doublet is used to obtain the mean lifetimes τcis and τtrans of each isomeric peptide between two successive proton transfers. Kinetic data were also obtained using theoretical simulations of experimental curves as described in previous (6) Chrisment, J.; Delpuech, J.-J. J. Chem. Soc., Perkin Trans. 2 1977, 407. (7) Martin, M. L.; Delpuech, J.-J.; Martin, G. J. Practical NMR Spectroscopy; Wiley-Heyden: London, 1980; Chapter 8.
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Figure 2. Surface tension of DMSO solutions at 45 °C as a function of the logarithm of DSG concentration, C0. Figure 1. Solubility curve of DSG in DMSO. publications.4,6,8 In the present case, the simulation should be done on the full NMR pattern of the two overlapping doublets. Surface Tension. Surface tension (γ) measurements were performed with a Dognon-Abribat tensiometer, making use of the Wilhelmy plate method, at 45 °C with a waiting time of 10 min. Viscosimetry. The viscosity η ) kdt (d, density; t, running time) of DSG solutions in DMSO was measured at 45 °C with an Ubbelohde microviscosimeter, previously standardized with water (dH2O(45 °C) ) 0.978; ηH2O(45 °C) ) 0.596 cp; t ) 23 s; and, therefore, k ) 0.026 cSt/s). The measurements showed a linear relationship between the viscosity and the analytical concentration C0 of DSG: η ) 1.509C0 + 1.193 with a correlation coefficient of 0.9998. Diffusion Measurements. Diffusion coefficients9,10 were measured in the Laboratoire de Me´thodologie RMN (Pr. D. Canet), using an original RF field gradient method11 and a home-made NMR apparatus operating at 200 MHz. Owing to line separation (see above), diffusion coefficients were obtained for the cis and trans isomers of DSG, individually.
Results and Discussion Surface-Active Properties. DSG is insoluble in water and easily soluble in DMSO. The solubility curve shows a break point (Figure 1) around 40 °C, which is reminiscent of a Krafft point. However such phenomena are generally quoted for ionic surfactants,12-14 although a few nonionic surfactants do exhibit a Krafft point.14 They are considered to result from the fusion of the solvated crystal with the subsequent formation of micelles. We were thus inclined to suspect the existence of micellar solutions of DSG in DMSO. This peculiarity explains why most experiments were performed at 45 °C and not at room temperature, so as to dissolve up to more than 0.5 mol dm-3 DSG. The surface tension γ of DMSO solutions at 45 °C decreases from 39.9 to 31.1 mN m-1 when the concentration C0 of DSG is increased up to 0.5 mol dm-3. This is in sharp contrast with the behavior of DMSO solutions of ASG, where the surface tension remains equal to that of pure DMSO up to the limit of solubility of ASG (ca. 0.3 (8) Chrisment, J.; Delpuech, J.-J.; Rajerison, W.; Selve, C. Tetrahedron 1986, 42, 4743. (9) Lindman, B.; Olsson, V.; So¨derman, O. In Dynamics of Solutions and Fluid Mixtures by NMR; Delpuech, J.-J., Ed.; John Wiley and Sons: Chichester, 1995; Chapter 8. (10) Canet, D.; Decorps, M. In Dynamics of Solutions and Fluid Mixtures by NMR; Delpuech, J.-J., Ed.; John Wiley and Sons: Chichester, 1995; Chapter 7. (11) Canet, D.; Diter, B.; Belmajdoub, A.; Brondeau, J.; Boubel, J.C.; Elbayed, K. J. Magn. Reson. 1989, 81, 1. (12) Lindman, B.; Wennerstro¨n, H. Top. Curr. Chem. 1980, 1, 87. (13) Shinoda, K.; Nakagawa, T.; Tamamuski, B.; Isermura, T. Colloı¨dal Surfactants; Academic Press: New York, 1963; pp 7-9. (14) Kissa, E. Fluorinated Surfactants, Surfactant Science Series; Marcel Dekker: New York, 1993; Vol. 50, pp 205-210.
Figure 3. 1H-NMR spectrum at 400 MHz and 25 °C of a 0.01 M DMSO-d6 solution of DSG, showing the splitting of lines due to cis (b)-trans (bb) stereoisomerism (for assignments, see Experimental Section). NH-CH2 cis and trans doublets (under the arrow) are enlarged in the inset.
mol dm-3). This shows clearly that the presence of an additional C12 alkyl chain in DSG has induced surfaceactive properties. However, the graph of γ vs log C0 (Figure 2) does not display a break at which the cmc would be reached. A smooth curve is observed instead with a slight curvature and an inflection point for C0 ∼ 0.8 mol dm-3. This is generally assigned to a progressive aggregation of surfactant molecules. The decrease of surface tension, by about 8 mN m-1, is less marked than for the analogous alkyl-fluoroalkyl lipopeptide (17 mN m-1) previously studied; this is in line with the reinforced surface-active properties of fluoroalkyl chains compared to alkyl chains. In both cases, these surface-active properties suggest the presence of small aggregates standing in equilibrium with the monomeric compound but cannot by themselves prove their existence. Z-E Stereoisomerism in N-Dodecanoylsarcosylglycyl Ester (DSG). Two series of lines are observed for protons of the amino acid residues (Figure 3) owing to cis-trans isomerism about the N-dodecanoyl partial double bond. The cis isomer was assigned to the more intense absorption in each couple of lines on the same grounds as those given in the case of ASG.4 The cis/trans isomer ratio (55/45) remains constant when the DSG concentration is changed by an order of magnitude or when the temperature is raised from 20 to 55 °C. The latter observation shows that entropic effects are mainly controlling the cis a trans equilibrium, the trans isomer having a higher degree of internal order than the cis isomer, presumably because of a more compact conformation, a view which will also be useful to interpret kinetic data (see below).
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Variable-temperature experiments show the coalescence between the two sets of cis and trans lines. As in the case of ASG, internal rotation was studied between 55 and 80 °C using the coalescence patterns of the two N-methylic singlets (see Experimental Section). Activation parameters were found from an Arrhenius plot of NMR rate constants, allowing us in turn to extrapolate kinetic data to the standard temperature of 25 °C. These data are given below and are compared to those relative to ASG (between parentheses):
k(cisftrans) (25 °C) ) 4.0 × 10-1 (3.6 × 10-2) s-1 ∆Gq ) 75 (81) kJ mol-1; ∆Hq ) 78 (110) kJ mol-1; ∆Sq ) 8.5 (98) J mol-1 K-1 The higher values of ∆Hq and ∆Sq found for ASG as compared to dimethylacetamide15,16 (84 kJ mol-1 and 20 J mol-1 K-1) had been tentatively assigned in a previous publication4 to strongly solvated cis and trans isomers and to a partial desolvation of the transition state, the DMSO molecules being pushed back from the peptide bond in the course of internal rotation. Following the same line of reasoning, the comparison between data relative to ASG and DSG leads us to conclude that DMSO solvation of cis and trans isomers in their ground states is somewhat decreased by substituting an N-dodecanoyl for an N-acetyl chain at one end of the molecule. This could show some steric hindrance to the approach of solvent molecules toward the peptide NH bond (the main site for DMSO solvation), a view which will also be extended below to the approach of reactant molecules. Whatever the explanation may be, internal rotation in DSG is faster by about one order of magnitude than in ASG, this obliged us to restrict further studies of protonation transfers at high DSG concentrations to a relatively narrow temperature window, between 40 °C (for solubility grounds) and 50 °C (to avoid additional line broadening from internal rotation). A temperature of 45 °C was finally selected for these experiments. Aggregation of DSG in DMSO. In a second step, NMR spectroscopy was used to detect an eventual aggregation of monomeric DSG molecules in DMSO. As in the case of alkyl-fluoroalkyl lipopeptides,3 there is a continuous shift of 1H lines as the analytical concentration C0 of DSG is progressively increased from 0.013 to 0.6 mol dm-3 (Figure 4). The NMR shifts may be observed for each isomer individually, using for this purpose the N-methylic protons. Plots of δ vs C0-1 display a marked curvature (Figure 4) which is reminiscent of similar curves relative to alkyl-fluoroalkyl lipopeptides. Chemical shift variations ∆δ ∼ 0.02 ppm however have a much smaller amplitude in the present investigations and could be accounted for as well by some specific solvent effect. Moreover we showed in a previous publication3 that the knowledge of chemical shift/concentration profiles could not be by itself a sufficient proof for the presence of aggregates. In the above publication, quantitative estimations of aggregates had been possible by combining NMR shift data with small-angle neutron-scattering experiments. In the present case, definite evidence for the absence of extensive aggregation phenomena was brought from diffusion NMR measurements. The N-methylic signals were observed in a radio frequency field gradient,11 previously standardized by measuring the self-diffusion of water. Experimental (15) Stewart, W. E.; Siddall, T. H., III. Chem. Rev. 1970, 70, 517. (16) Drakenberg, T.; Dahlquist, K. I.; Forse´n, S. J. Phys. Chem. 1972, 76, 2178.
Figure 4. Chemical shifts δN-CH3(trans) (O) and δN-CH3(cis) (*) (in ppm from internal TMS) as a function of DSG concentration C0 at 45 °C.
results showed a slight decrease of the diffusion coefficients Dcis and Dtrans of both the cis and trans isomers when the concentration of DSG is increased from C0 ) 0.02, in which case Dcis and Dtrans ) 0.363 × 10-5 and 0.355 × 10-5 cm2 s-1, to C0 ) 0.5 mol dm-3, where Dcis and Dtrans ) 0.285 × 10-5 and 0.290 × 10-5 cm2 s-1, at 45 °C. Within experimental uncertainties (5-10%), the observed decrease of Dcis and Dtrans (by a factor of 1.27) is fully accounted for by the subsequent increase of viscosity: η ) 1.22 and 1.95 cp when C0 ) 0.02 and 0.5 mol dm-3, respectively (i.e. by a factor of 1.59). The presence of DSG aggregates is thus undetectable within experimental errors. In conclusion of the above sections, it can be said that DSG displays surface-active properties in DMSO, however without the occurrence of any micellization process. Proton Transfers in Acidic DMSO. NMR exchange rates 1/τex were measured at two temperatures, 25 and 45 °C, and, in each case, for five to seven concentrations of added trifluoromethanesulfonic acid, CH or [H+]. At 25 °C, DSG is slightly soluble in DMSO, and one concentration was only possible in practice, C0 ) 0.01 mol dm-3. At 45 °C, two series of experiments were performed using either low (C0 ) 0.065 and 0.13 mol dm-3) or high (C0 ) 0.32 and 0.41 mol dm-3) concentrations of DSG. The results can visualized on the graphs of Figure 5, where NMR rate constants are plotted against the analytical concentration [H+]. At low concentrations of DSG (C0 ) 0.01-0.13 mol dm-3), the NMR exchange rate is found to be proportional to [H+] and independent of the peptide concentration, hence the presence of single straight lines in plots of Figure 5:
1/τex ) kH[H+]
(1)
where τex is the mean lifetime of one peptidic proton in a substrate molecule.1 This is quite similar to analogous experiments performed with amides and peptides.3,4,6,8 The mechanism
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allowed us to derive the activation parameters reported in Table 2. Relatively high activation enthalpies are obtained for the protonation of the peptide bond, in line with the low basicity of nitrogen as the result of the delocalization of the electron lone pair over the partial N-C double bond. Strongly negative values are also observed for the associate activation entropy. This confirms that the rate-determining step is effectively a protonation of the amide nitrogen with an overall decrease of freedom in the substrate-reactant pair and not a deprotonation of nitrogen, as envisaged in an alternative mechanism involving the deprotonation of an intermediate acetoimidate:21
RC(OH)
+
R′
N
R′ RC(OH)
N
+ H+
H
Figure 5. Plots of NMR exchange rates 1/τ as a function of [H+] in acidic DMSO, for three DSG concentrations C0 and two temperatures: C0 ) 10-2 mol dm-3 (× and *, for the cis and trans isomers, respectively) at 25 °C; C0 ) 6.4 × 10-2 to 1.3 × 10-1 mol dm-3 at 45 °C (b and O); C0 ) 3.2 × 10-1 to 4.1 × 10-1 mol dm-3 at 45 °C (9 and 0).
generally accepted to account for the above rate law, first proposed by Berger et al.,17 involves the protonation of the amide nitrogen according to the following scheme: k1
RCONHR′ + DMSO‚‚‚H+ {\ } RCONH2+R′ + DMSO k d
followed by a fast diffusion-controlled deprotonation, kd ∼ 5 × 109 mol-1 dm3 s-1 at 25 °C in DMSO (for a discussion, see ref 18 and 19). In this view, the protonation rates k1 are consequently twice as fast as the proton exchange rates kH; hence,
k1 ) 2kH
The rate decrease from the cis to the trans isomer arises from entropic effects (∆Sq ) -83.5 and -100.5 J mol-1 K-1, respectively), in line with the above discussion on the temperature-independent cis-trans equilibrium. The freezing of reactants in the transition state is stronger for the trans isomer, owing to the steric hindrance brought about by the neighboring alkanoyl chain. At higher concentrations of DSG (C0 ) 0.3-0.4 mol dm-3), there are substantial deviations from the straight lines drawn at lower concentration in Figure 5. For the cis isomer, the experimental points lie approximately on a straight line with a slope kH ) 28.6 mol-1 dm3 s-1 (at 45 °C). This points to the presence of a concentrationdependent mechanism superimposed on reaction 1 and contributing to kH by a value ∆kH ) 5.3 mol-1 dm3 s-1. A suggestion for this mechanism could be a symmetrical proton transfer between the substrate, again symbolized as RCONHR′, and its conjugate acid RCONH2+R′: k2
The rate constants obtained in this way are summarized in Table 1, where they are compared to those previously obtained with ASG under the same conditions. It can be seen that the rate constants (at 25 °C) are of the same order of magnitude for ASG and DSG, the values for DSG being slightly smaller than those for ASG.4 For both substrates, proton exchange is faster on the cis than on the trans isomer. This shows the importance of the stereochemistry of substituents in the vicinity of the exchanging site. The approach of the solvated proton toward the amide nitrogen in the glycyl residue may be thought to be easier in the cis than in the trans isomer owing to the steric hindrance brought about by the neighboring dodecyl (in trans DSG) or methyl (in trans ASG) substituent as compared to the carbonyl group (in both cis DSG and ASG; see Scheme 1). Another factor is the possibility of internal hydrogen bonding between the peptide hydrogen and the terminal carbonyl of the alkanoyl substituent in the cis isomer, as suggested by the dashed line in Scheme I.20 Application of the Arrhenius or the Eyring rate equation (where the transmission coefficient is taken as unity) (17) Berger, A.; Loewenstein, A.; Meiboom, S. J. Am. Chem. Soc. 1959, 81, 62. (18) Chrisment, J.; Delpuech, J.-J.; Rajerison, W. J. Chim. Phys. 1983, 80, 747. (19) Perrin, C. L.; Dwyer, T. M. Chem. Rev. 1990, 90, 935 and references therein. (20) As pointed out by one reviewer, the relative positions of the NMR signals of these cis (upfield) and trans (downfield) isomers are apparently not consistent with the latter suggestion. However, the simple relationship between intramolecular hydrogen bond and NH downfield shift may be vitiated here by the parallel existence of strong intermolecular hydrogen bonding with DMSO; see ref 4.
RCONHR′ + RCONH2+R′ \ {k } 2
RCONH2+R′ + RCONHR′ The protonation of the observed amide molecule RCONHR′ is fastly followed by the release of either acidic hydrogen in RCONH2+R′ so that protonation rates are again twice as fast as the chemical exchange. Equation 1 is thus completed into
1 1/τex ) (k1[H+] + k2[RCONH2+R′]) or 2 k2C0 1 k1 + [H+] (2) 2 KN
(
)
where
KN ) [RCONHR′][H+]/[RCONH2+R′] is the ionization constant of the N-protonated conjugate acid, assumed to be in negligible concentration with respect to C0. The former proton exchange rate kH ) k1/2 is thus added with a contribution ∆kH ) k2C0/2KN. The corresponding pK value, pKNH2+, has been estimated on the basis of kinetic data obtained at low concentration to lie in the range pK ) -9.5 ( 1.0 for peptides.18 Such symmetrical exchanges are generally very fast, quite close to the diffusion-limited rate kd; this is the case between the two nitrogen centers in ammonium/amine acid-base (21) Martin, R. B.; Hutton, W. C. J. Am. Chem. Soc. 1973, 95, 4752.
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Table 1. Rate Constants for Proton Transfers in Acidic DMSO at Low Concentrationsa T ) 25 °C
T ) 45 °C
compound
isomer
kH (mol-1 dm3 s-1)
k1 (mol-1 dm3 s-1)
kH (mol-1 dm3 s-1)
k1 (mol-1 dm3 s-1)
DSG
cis trans cis trans
7.67c 5.48 10.1 6.0
15.34 10.96 20.3 12.1
23.33 14.96
46.6 29.9
ASGb a
C0 ) 0.01-0.1 mol dm-3. b From ref 4. c Estimated errors: (5%.
Table 2. Activation Parameters for Proton Transfers on DSG at 25 °C ∆Gq b Eq a ∆Hq b ∆Sq b isomer (kJ mol-1) log Aa (kJ mol-1) (kJ mol-1) (J mol-1 K-1) cis trans
43.8 39.5
8.86 7.97
66.2 67.1
41.3 37.0
-83.5 -100.5
a Computed using the Arrhenius equation k ) A exp(-Eq/RT). 1 Computed using the Eyring equation k1 ) (kBT/h) exp(-∆Gq/RT), where ∆Gq ) ∆Hq - T∆Sq.
b
pairs.22 If we use k2 ) kd ∼ 5 × 109 mol-1 dm3 s-1 and C0 ) 0.4 mol dm-3 (cis isomer alone), the additional contribution ∆kH ∼ 5 mol-1 dm3 s-1 may be accounted for by taking KN ∼ 2 × 108 or pKN ) -8.3, a plausible value. An alternative explanation involves the tautomeric O-protonated conjugate acid RC+(OH)NHR′: k3
RCONHR′ + RC+(OH)NHR′ \ {‚} RCONH2+R′ + RCONHR′ (3) with a similar contribution to the exchange rate:
∆kH ) k3C0/2KO where
KO ) [RCONHR′][H+]/[RC+(OH)NHR′] is the ionization constant of the O-protonated amide, still assumed to be in neglible concentration with respect to C0 (see however the next paragraph). Taking pKO ∼ -1 as in amides18 and the above ∆kH and C0 values allows us to compute k3 ∼ 250 mol-1 dm3 s-1, a value somewhat larger than k1 (46.6 mol-1 dm3 s-1) and significantly smaller than k2 (5 × 109 mol-1 dm3 s-1), in line with an increased (or decreased, respectively) acidity of RC+(OH)NHR′ (pKO ∼ -1) compared to that of DMSO (or of RCONH2+R′) (pK ) 0 and ∼ -8, respectively). The two contributions from eqs 2 and 3 are indiscernible on NMR grounds and should be gathered together into a common law:
∆kH ) (k2/KN + k3/KO)C0/2 The behavior of the trans isomer looks much more complicated. The plot of 1/τex in Figure 5 displays a strong curvature, the slope of the tangent being high at the beginning of the curve and then decreasing quickly when [H+] is larger than ca. 0.1 mol dm-3. The initial tangent to the curve has an expected position with respect to the straight line obtained for the cis isomer; the same additional mechanisms (2 and 3) could account for the initial portion of the plot. The second additional phenomenon which decreases the exchange rates at higher (22) Bianchin, B.; Chrisment, J.; Delpuech, J.-J.; Deschamps, M. N.; Nicole, D.; Serratrice, G. In Chemical and Biological Applications of Relaxation Spectrometry; Wyn-Jones, Ed.; Reidel: Dordrecht, Holland, 1975; pp 365-373 and references therein.
acidities of DMSO solutions could be tentatively assigned to O-protonation of the substrate. It is now recognized that O-protonation is much more effective than Nprotonation in amides. O-protonation would make the amide nitrogen site much less basic yet on electrostatic grounds. This means that O-protonation, even if not contributing directly to proton exchanges on nitrogen (see however the discussion in ref 18), may reduce them to zero on the NMR time scale, at least at high concentrations of acid. If we simply consider that a significant fraction λ of the substrate (depending on the exact value of KO) is effectively O-protonated, this reduces the overall exchange rate on the neutral residual amide by a factor 1 - λ; this also decreases the amount of solvated protons DMSO‚‚‚H+ and consequently the contribution of reaction 1. These effects are indeed more important at high concentrations of DSG. The different behaviors of the cis and trans isomers on increasing C0 would be assigned in this view to a slightly increased acidity in the cis (e.g. KO ∼ 10) compared to the trans (e.g. KO ∼ 2) O-protonated isomer. This would result in a preferential O-protonation of the trans isomer at high concentration of DSG, thus accounting for the dramatic deviation to linearity observed in Figure 5 for this isomer. The different acidities assumed for the cis and trans isomers would be assigned again to steric hindrance to the approach of solvated protons toward the alkanoyl carbonyl group (the more basic site among the three carbonyls of DSG on the basis of IR frequency shifts induced by hydrogen bonding) in the cis isomer. Another attractive explanation could have relied on selective aggregation of the trans isomer: it was therefore important to first recognize the absence of extensive micellization for both isomers of this amphiphilic molecule. Conclusion The lipopeptide presently studied in DMSO offers many misleading aspects. A solubility curve with a pseudoKrafft point, NMR chemical shift-concentration profiles, and unusual kinetics of proton transfers are deceptive indications for aggregation phenomena. However, in spite of surface-active properties, diffusion measurements combined with viscosity data clearly show the absence of extensive micellization. The increased solubility of the crystal above 40 °C is presumably related to the fusion of the solvated solid (at ∼40 °C, against 71 °C for the pure solid), simply followed by an easy dissolution of the monomeric surfactant. Proton transfers obey similar kinetic laws for both the monomeric tensioactive molecule (DSG) and its nontensioactive analogue (ASG), at low concentrations. However, owing to the increased solubility of the tensioactive compound presently studied, anomalies in the kinetic laws were observed at high concentrations. This shows the presence of additional pathways for proton transfers, presumably involving the conjugate acids of the substrate on nitrogen and on oxygen (N- and Oprotonation of the amide). LA950786S
