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Articles Comparison of the Aggregation Behavior of 15 Polymeric and Monomeric Dipeptide Surfactants in Aqueous Solution Fereshteh Haddadian Billiot,† Matthew McCarroll,‡ Eugene J. Billiot,† Joseph K. Rugutt,§ Kevin Morris,⊥ and Isiah M. Warner*,# Department of Physical and Life Science, Texas A&M University-Corpus Christi, Corpus Christi, Texas 78412, Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901, Department of Chemistry, Massachusetts College of Liberal Arts, 375 Church Street, North Adams, Massachusetts 01247-4100, Department of Chemistry, Carthage College, Kenosha, Wisconsin 53140, and Louisiana State University, Baton Rouge, Louisiana 70803 Received July 10, 2001. In Final Form: November 15, 2001 The aggregation numbers of 15 dipeptide surfactants were estimated by use of fluorescence steady-state quenching techniques. Polymerization of the surfactants with γ radiation resulted in molecular micelles with a lower number of repeat units than the corresponding monomer aggregation numbers at the concentration of monomer used for polymerization in this study. In addition, the aggregation numbers of the monomers decreased with increasing size of the N-terminal R group of the dipeptide surfactants examined in this study. The aggregation behavior of the dipeptide surfactants was further investigated using proton NMR (1H NMR) spectroscopy. The proton resonances due to NH and HR were measured above and below the critical micelle concentration of the surfactants. From the differences in proton chemical shifts of the monomeric dipeptide surfactants and aggregation numbers, a model for packing of the monomeric polar head is proposed.
Introduction Polymeric amino acid based surfactants have been used in electrokinetic chromatography (EKC) for several years.1-10 In general, chiral dipeptide surfactants (CDS) have provided better chiral separation when compared to chiral single amino acid surfactants.2 In the case of amino acid based surfactants, the hydrogen-bonding affinity of amide moieties,11,12 as well as the steric interactions of the amino acid side chain,13 appear to play a significant †
Texas A&M University-Corpus Christi. Southern Illinois University. Massachusetts College of Liberal Arts. ⊥ Carthage College. # Louisiana State University. * To whom correspondence should be addressed. ‡ §
(1) Wang, J.; Warner, I. M. Anal. Chem. 1994, 66, 3773. (2) Shamsi, S. A.; Mocossay, J.; Warner, I. M. Anal. Chem. 1997, 69, 2980. (3) Billiot, F. H.; Billiot, E. J.; Warner, I. M. J. Chromatogr. A 2001, 922, 329. (4) Billiot, E.; Warner, I. M. Anal. Chem. 2000, 72, 1740. (5) Billiot, E.; Macossay, J.; Thibodeaux, S.; Shamsi, S. A.; Warner, I. M. Anal. Chem. 1998, 70, 1375. (6) Billiot, E.; Agbaria, R. A.; Thibodeaux, S.; Warner, I. M. Anal. Chem. 1999, 71, 125. (7) Billiot, E.; Thibodeaux, S.; Shamsi, S.; Warner, I. M. Anal. Chem. 1999, 71, 4044. (8) McCarroll, M. E.; Billiot, F. H.; Warner, I. M. J. Am. Chem. Soc. 2001, 123, 3173. (9) Haddadian, F.; Shamsi, S. A.; Warner, I. M. Electrophoresis 1999, 20, 3011. (10) Haddadian, F.; Billiot, E. J.; Shamsi, S. A.; Warner, I. M. J. Chromatogr. A 1999, 858, 219. (11) Pirkle, W. H.; Hyun, M.; Bank, B. J. Chromatogr. 1984, 316, 585. (12) Dobashi, A.; Dobashi, Y.; Kinoshita, K.; Hara, S. Anal. Chem. 1988, 60, 1985.
role in chiral recognition. It should be further noted that in CDS the polar head usually contains two chiral centers and two more hydrogen bond donor/acceptor sites than the corresponding single amino acid surfactants. Several studies reported by Billiot et al. have examined the use of polymeric CDS in chiral separation using EKC.3,7 One study compared the effect of the amino acid order of CDS on chiral separation.5 In another study, the polarity of the polymeric surfactants was measured, and a model was proposed to explain the chiral interaction of this class of polymeric surfactants with binaphthyl derivatives.6 A separate study examined the effect of depth of penetration of the analyte into the micellar core of the polymers on chiral recognition of enantiomers in different charge states.7 Recent studies have provided a comparison of the performance of monomeric and polymeric amino acid based surfactants as chiral pseudo-stationary phases in EKC.3,9 In those studies, polymeric surfactants usually provided better chiral separation than the corresponding monomers. In this paper, we examine and compare the physical properties of several polymeric and monomeric CDS. First, the aggregation numbers of these two kinds of “micelles” were determined using steady-state fluorescence quenching techniques. It should be mentioned that, although it is not strictly correct to refer to polymeric surfactants as “micelles”, for simplicity, this paper will use the term “micelle” to refer to both the aggregated form of the monomeric surfactants and the covalently linked polymeric surfactant. The packing of monomeric CDS was also investigated using 1H NMR. (13) Lin, C.-E.; Li, F.-K.; Lin, C.-H. J. Chromatogr. A 1996, 722.
10.1021/la0110592 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/07/2002
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Billiot et al. Table 1. Aggregation Number of Monomeric Dipeptide Surfactant and Repeating Units of Polymeric Dipeptide Surfactant surfactant monomer polymer surfactant monomer polymer SUGA SUGV SUGL SUAG SUAA SUAV SUAL SUVG
380 140 110 270 358 74 65 62
33 23 23 30 26 24 25 22
SUVA SUVV SUVL SULG SULA SULV SULL
50 62 48 40 42 39 38
19 23 19 21 18 18 19
Fluorescence Measurements. Fluorescence measurements were performed using a SPEX model F2T211 spectrofluorometer equipped with a thermostated cell housing and a thermoelectrically cooled Hamamatsu R928 photomultiplier tube. The aggregation number of the CDS were determined by following the fluorescence quenching method developed by Turro.14 In this study, the quenching of the pyrene by N-acetylpyridinum chloride is measured at λex ) 337 nm and λem ) 392 nm. Using the following expression
ln(I0/I) )
Figure 1. Structure and abbreviation for CDS examined in this study.
Experimental Section Chemicals. Amino acid dipeptides were purchased from Sigma Chemical Co. (St. Louis, MO). N-Hydroxysuccinimide, pyrene, undecylenic acid, and 3-(trimethylsilyl)propionic-2,2,3,3d4 acid sodium salt were obtained from Aldrich Chemical Co. (Milwaukee, WI). The 15 CDS sodium N-undecanoyl-LL-glycylalaninate (SUGA), sodium N-undecanoyl-L-alanylglycinate (SUAG), sodium N-undecanoyl-L-glycylvalinate (SUGV), sodium N-undecanoyl-L-valylglycinate (SUVG), sodium N-undecanoylL-glycylleucinate (SUGL), sodium N-undecanoyl-L-leucylglycinate (SULG), sodium N-undecanoyl-LL-alanylalaninate (SUAA), sodium N-undecanoyl-LL-alanylvalinate (SUAV), sodium Nundecanoyl-LL-alanylleucinate (SUAL), sodium N-undecanoylLL-valylalaninate (SUVA), sodium N-undecanoyl-LL-valylvalinate (SUVV), sodium N-undecanoyl-LL-valylleucinate (SUVL), sodium N-undecanoyl-LL-leucylalaninate (SULA), sodium N-undecanoylLL-leucylvalinate (SULV), and sodium N-undecanoyl-LL-leucylleucinate (SULL) (shown in Figure 1) were synthesized according to a procedure developed in our laboratory.1
N[Q] Cs - cmc
where Cs is the total surfactant concentrations; I0 and I are the relative fluorescence intensity of the pyrene at zero and [Q] concentrations. The aggregation number, N, of the CDS is obtained from the slope of the plot of ln(I0/I) vs [Q]. Prior to examination of the CDS discussed in this study, the aggregation number of sodium dodecyl sulfate (SDS) was determined and compared to literature values. The aggregation number of SDS was determined to be about 60 using this method, which is comparable to values reported by Jobe et al.15 Nuclear Magnetic Resonance Measurements. NMR spectra were recorded on a Bruker ARX 300 MHz spectrometer, and the data were processed with Bruker XWINNMR software operating on a Silicon Graphics Indigo workstation (Silicon Graphics Inc., Bruker Co.). Solutions of CDS at concentrations above and below the cmc were prepared in D2O or in a mixed solvent of 90% H2O and 10% D2O. The D signal of D2O was used for frequency lock, and the intensity of the H2O resonance was suppressed by presaturation.16 Typical one-dimensional 1H NMR spectral acquisition parameters were as follows: data size, 16K; spectral width, 3500 Hz; 90° radio-frequency pulse, with a duration of 7.0 µs; recycling delay between transients, 2.0 s. Adequate signal-to-noise (S/N) ratios in the 1H NMR spectra were achieved after 256 transients. Prior to Fourier transformation, the spectra were multiplied by a Lorentz-Gauss window function and zero-filled to 32 K. Chemical shifts are reported in part per million (ppm) relative to (trimethylsilyl)propionic-2,2,3,3d4 acid sodium salt. Coupling constants (3JH-H) were measured directly from the 1H NMR spectra. Two-dimensional (2D) 1H1H correlation spectroscopy (COSY) experiments were measured with suppression of the water resonance by presaturation. The following acquisition parameters were used: temperature, 298 K; recycling delay, 2.0 s; spectral width in both dimensions, 3500 Hz (11.6 ppm); dummy scans, 4; D0 increments, 3 µs. In all COSY experiments, 2048 data points were acquired in t2, and 64 transients were coadded at each of 256 t1 increments with zerofilling to 2048 points. Gaussian or shifted sinebell apodization was applied in both dimensions.
Aggregation Number of Chiral Dipeptide Surfactants The aggregation numbers of the 15 CDS examined in this study are shown in Table 1. These CDSs have been classified on the basis of aggregation numbers of the monomers. Class I and II surfactants are those having (14) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951. (15) Jobe, D. J.; Reinsborough, V. C.; Wetmore, S. D. Langmuir 1995, 11, 2476. (16) Hoult, D. I. J. Magn. Reson. 1976, 21, 337.
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Figure 2. Comparison of aggregation number of monomeric surfactant.
aggregation numbers above and below 100, respectively. As noted in Table 1, the number of repeat units for the polymers is always lower than the aggregation number of the corresponding monomers. For example, mono-SUAA (in class I) and mono-SULL (in class II) have aggregation numbers of 358 and 38, respectively. However, the number of repeat units for poly-SUAA and poly-SULL was 26 and 19, respectively. This result suggests that, under the conditions used in this study, polymerization results in a change in the size of the “micelle”. Previous studies have shown that the intensity of the radiation source used for polymerization will affect the number of repeat units of the polymers.17 The intensity of the γ radiation source used for the polymerization in this study was about 0.7 krad/h at the point of irradiation. Thus, the smaller “aggregation numbers” of the polymeric CDS are probably a result of the slower polymerization process. It should be mentioned that Paleos et al. have obtained polymers of the same size as the micelles by polymerization of sodium 10-undecenoate with γ radiation of 143 krad/h.18 Other factors that affect aggregation numbers are the size of the polar headgroup of the surfactants as well as ionic repulsive and attractive forces induced by the hydrophobic attraction of the hydrocarbon chain. As indicated in Figure 2, when the size of the C-terminal amino acid is kept constant and size of the N-terminal amino acid is increased, the aggregation number of the monomeric surfactant declines. Note that the aggregation numbers decrease in the series of SUAG (270), SUVG (62), and SULG (40). In addition, the aggregation number of SUGA (380) and SUAA (358) with small polar heads are significantly higher than the aggregation number of
SUVA (50) and SULA (42) with large polar heads. Among the surfactants examined in this study, SULL with the largest polar head had the smallest aggregation number (i.e., 38) and SUGA with the smallest polar head has the largest aggregation number (i.e., 380). It should be mentioned that the aggregation size of the monomers depends more on the size of the N-terminal amino acid than the C-terminal amino acid. No apparent trend was observed in regard to the size of the C-terminal amino acid and the aggregation number. This is possibly due to the electrostatic repulsion among the negatively charged carboxylate groups. The aggregation behavior of these surfactants was further examined using 1H NMR spectroscopy.
(17) Davis, J. E.; Senogles, E. Aust. J. Chem. 1981, 34, 1413. (18) Paleos, C. M.; Malliaris, A. J. Macromol. Sci., Rev. Macromol. Chem. Phys. 1988, C28, 3.
(19) Newcomb, L. F.; Haque, Tasir, S.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 6509. (20) Dobashi, A.; Hamada, M. J. Chromatogr. A 1997, 780, 179.
Packing of Monomeric Chiral Dipeptide Surfactants As with all amino acid based compounds, the amide moieties in CDS are capable of forming strong intermolecular or intramolecular H-bonding. At concentrations below the cmc, these protons hydrogen bond with water, whereas upon micellization, water is largely excluded from the hydrophobic core19,20 and hydrogen bonding between the polar headgroups can then play a major role in determining the conformation and thus the aggregation number of the CDS. As shown in Figure 1, CDS contain two amide moieties, referred to as the C- and N-terminal amide groups. The C-terminal amide is closer to the surface of the micelle. No significant difference was observed in the C-terminal NH proton signal of CDS at concentrations below the cmc where compared to the micellar form. This result could be due to either the mobility of the C-terminal
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Table 2. Proton NMR Chemical Shift for Class II CDS below and above the cmc ((0.01 ppm the Average Standard Deviation) below cmc (1 mM)
SUAV SUAL SUVG SUVA SUVV SUVL SULG SULA SULV a
above cmc (50 mM)
NH (ppm) (3JHz) (θ°)
HR (ppm)
NH (ppm) (3JHz) (θ°)
HR (ppm)
8.22 (6.34) (129) 8.18 (6.48) (129) 8.13 (8.27) (138) 8.10 (8.19) (138) 7.65a 8.10 (8.38) (138) 8.32 (6.53) (129) 8.24 (7.51) (134) 8.29 (7.41) (133)
4.33 4.35 4.20 4.16 4.12 4.10 4.39 4.36 4.39
8.18a 8.10 (7.16) (132) 8.04 (8.60) (140) 8.01a 7.51a 7.99 (8.73) (141) 8.25 (7.45) (133) 8.17 (7.84) (136) 8.21 (7.94) (136)
4.42 4.45 4.27 4.25 4.21 4.20 4.45 4.45 4.48
Extracted from COSY spectra.
amino acid21 or the fact that the C-terminal amino acid interacts with water strongly even in the micellar state. Consequently, no change in chemical shift of the Cterminal amino acid protons were observed in the NMR spectrum. Therefore, in this study, only the amide and HR resonances of the N-terminal amino acids of CDS are discussed. 1H NMR chemical shifts related to change in concentration are due to such factors as hydrogen bonding, structural changes, shielding effects, etc.22,23 These factors are related to the change in intermolecular interactions. For example, the major factor responsible for the changes in chemical shifts of amide protons of the surfactants observed in this study is hydrogen bonding. At low concentration (below the cmc), amides of CDS hydrogen bond strongly with water. By increasing the concentration, these surfactants aggregate. Therefore, the amide protons have less access to water. In the micellar state, amide protons may interact with carbonyl of adjacent amide nitrogens. Therefore, studying chemical shift of the amide protons in different concentrations may reveal the change in intermolecular interaction of the CDS. In this study, 1 H NMR measurements were performed at two concentrations, i.e., above and below the cmc of the CDS. Considering that the cmc’s of the CDS are around 7 mM, 1H NMR was conducted at 1 mM (below the cmc) and 50 mM (above the cmc). The resonance of the N-terminal HR of the class II surfactants were shifted downfield in the micellar state as compared to below the cmc (Table 2). The change in the chemical shift of the HR in the presence of a bulky amino acid is possibly due to the fact that the amino acid side chains (R groups) of CDS in the micellar state tend to aggregate and twist toward the hydrophobic core to avoid exposure to the water. Therefore, reorientation of the side chain upon aggregation causes the bond between the C-HR and the adjacent carbonyl to twist to adopt this new conformation. As noted in Table 2, the vicinal coupling constant (3JH-H) of the N-terminal amino acids are always higher at concentrations above the cmc. The 3JH-H values are related to the torsion angles (θ, see Figure 3) through the Karplus equation.24 The torsion angles below and above the cmc are also reported in Table 2. As indicated in Figure 3, the angle of interest is between the Ha-C-C plane and C-N-Hb plane. Since the magnitude of 3JH-H between Ha and Hb (in Figure 3) varies with the torsion angle, the (21) Janin, J.; Wodak, S.; Levitt, M. Marogret, B. J. Mol. Biol. 1978, 125, 357. (22) Rabenstein, D. L. J. Am. Chem. Soc. 1973, 95, 2797. (23) Ramachandran, G. N.; Sasisekharan, V. Adv. Protein Chem. 1968, 23, 283. (24) Karimi-Nejad, Y.; Schmidt, J. M.; Ruterjan, H.; Schalbe, H.; Griesinger, C. Biochemistry 1994, 33, 5481.
Figure 3. (a) Structure of the N-terminal amino acid. (b) Torsion angle.
greater 3JH-H values reinforce the idea that the R group twists toward the hydrophobic core, which in turn changes the configuration of the surfactant polar head in micellar form. The downfield shift of the HR in micellar states can be attributed to the anisotropic effect of the carbonyl group of the amino acid moieties in the dipeptide backbone. The packing of the monomeric CDS in solution was further investigated by comparing the chemical shift of the amide protons above and below the cmc in 90% H2O and 10% D2O. As shown in Table 2, the N-terminal NH resonances in class II surfactants are shifted upfield in micellar states. There are several plausible explanations for the upfield shift of the N-terminal NH. For example, upon micellization, the R group twists toward the hydrophobic core, and because of steric constraints, the adjacent carbonyl is forced toward the aqueous phase. This conformational change causes the amide proton to twist toward the hydrophobic core, resulting in a decrease in NH-water hydrogen bonding. Therefore, the chemical shift changes of the N-terminal amide in micellar and monomeric states are indicative of the degree of hydrogen bonding in the CDS. Another possible reason for the observed upfield shifts could be that bulky groups do not permit close packing and thus stronger intermolecular hydrogen bonding among the amide groups of the neighboring polar headgroups. Shinitzky et al. have proposed the formation of chiral assemblies of amide planes on the micellar surface of N-stearoylserine by examining the circular dichroism spectrum of this surfactant above and below the cmc.25 Since the polar head of the dipeptide surfactants contains more hydrogen bonding sites than single amino acid based surfactants, it is reasonable to assume that upon micellization stronger hydrogen bonds form among the amides of the polar head of CDS as compared to single amino acid based surfactants. However, bulky polar headgroups may not always allow the formation of strong hydrogen bonds. Examination of the aggregation numbers indicates that, in monomeric surfactants, when a bulky group is located at the N-terminal amino acid of the CDS, lower aggregation numbers are achieved. The unfavorable steric interactions of the bulky side chains prevent the formation of intermolecular hydrogen bonding between the adjacent amide groups. Figure 4a illustrates the proposed conformation of class II CDS. It should be mentioned that no difference in HR signal of class I N-terminal amides were observed. This is possibly (25) Shinitzky, M.; Haimovitz, R. J. Am. Chem. Soc. 1993, 115, 12545.
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intramolecular hydrogen bonds among themselves in micellar form. As mentioned earlier, below the cmc, the amide moiety forms H-bonds with water and above the cmc the amide moieties form H-bonds among themselves. Consequently, no significant difference in the chemical shift of the N-terminal amide hydrogens of the class I surfactants above and below the cmc were observed. Although NMR data are not sufficient to establish the exact conformation for class I surfactants, from the aggregation number of class I surfactants shown in Table 1, it is proposed that class I surfactants form stronger hydrogen bonds among the N-terminal amide and carbonyl of CDS with small polar heads. The model shown in Figure 4b represents the proposed conformation of class I CDS with a small R group in the N-terminal position. This model is also consistent with the model proposed by Shinitzky et al. for the single amino acid surfactant serine.25
Figure 4. Proposed conformation of CDS in solution (a) class II and (b) class I surfactants.
because class I surfactants have small polar headgroups. Therefore, upon micellization, the conformation of the polar head may not change significantly. Because of fast proton exchange of class I N-terminal amide protons, very weak signals were observed for the amide protons of SUAG, SUAA, and SUGA. No significant difference in N-terminal amide chemical shifts were observed above and below the cmc for class I surfactants. As mentioned earlier, class I surfactants have small polar headgroups and large aggregation numbers which suggest that the amide moieties of the class I surfactants form strong
Conclusions The aggregation behavior of the dipeptide surfactants examined in this study appear to depend strongly on the size of the N-terminal amino acid R group than the C-terminal group. Surfactants with smaller polar heads in the N-terminal position form micelles with large aggregation number (i.e.,
