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NMR Study of the Interaction of Monomeric and Polymeric Chiral Surfactants with (R)- and (S)-1,1′-Binaphthyl-2,2′-diyl Hydrogen Phosphate Joseph K. Rugutt,*,† Eugene Billiot,‡ and Isiah M. Warner§ Department of Chemistry, Massachusetts College of Liberal Arts, 375 Church Street, North Adams, Massachusetts 01247-4100, Louisiana State University, Baton Rouge, Louisiana 70803, and Department of Physical and Life Science, Texas A&M UniversitysCorpus Christi, Corpus Christi, Texas 78412 Received May 4, 1999. In Final Form: November 29, 1999 Chiral discrimination of enantiomers of 1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNDHP) by monomeric chiral surfactants (CS), sodium N-undecylenyl-L-valine-L-leucine and sodium N-undecylenylL-leucine-L-valine , and related polymers is investigated by high-field one (1D)- and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. A general property of the high-resolution 1H NMR spectra of monomeric CS in 90% H2O/10% D2O is the appearance of downfield well-resolved chemical shift signals corresponding to the alpha (RH) protons of valine (ValRH) and leucine (LeuRH) amino acid residues. The remaining skeletal protons resonate in the region 0.5-2.5 ppm, giving rise to an envelope of poorly resolved chemical shifts. The 1H NMR signals of (R)- and (S)-BNDHP were enantiomerically separated into six sets of peaks in the presence of CS. The conformational analysis by means of nuclear Overhauser effect spectroscopy experiments indicates that the CS molecules adopt folded conformations in aqueous solution. The multiple interactions of (S)-BNDHP and CS obtained from intermolecular rotating frame Overhauser effect NMR spectroscopy is direct evidence on the mechanism of chiral recognition in aqueous media.
Introduction The enantiopurity of optically active natural and synthetic products has been the focus of intense research in most branches of chemistry, medicine, biochemistry, the food industry, and agriculture. However, there is one formidable challenge; i.e., currently there is no universal method for obtaining enantiomeric purity. This is especially important to the pharmaceutical industry, as in many cases enantiomers display different physiological effects. Although a number of liquid chromatographic techniques have been developed to separate enantiomers, only a few compounds have been separated.1 The methods for resolution of enantiomers are divided into two major types, direct and indirect.2-5 Capillary electrophoresis (CE) is currently the most powerful method for separation of small quantities of racemic mixtures.6-8 Cyclodextrins (CDs) and chiral metal complexes are the most commonly used chiral selectors in CE.9 Recently, the use of chiral surfactants (CS) for high-efficiency CE enantioresolution has generated a great deal of interest.10 On the basis of chromatographic separation of enanti* To whom correspondence should be addressed. Telephone: (413)-662-5451.Fax: (413)-662-5010.E-mail:
[email protected]. † Massachusetts College of Liberal Arts. ‡ Texas A&M UniversitysCorpus Christi. § Louisiana State University. (1) Camillieri, P.; Thorpe, C. J. J. Chromatogr. 1990, 519, 387-388. (2) Aizoaceae, K.; Terabe, S. Trends Anal. Chem. 1993, 12, 125-130. (3) Lochmu¨ller, C. H.; Souter, R. W. J. Chromatogr. 1975, 113, 283. (4) Boyle, P. H. Q. Rev., Chem. Soc. 1972, 25, 323-341. (5) Purdie, N.; Swallow, K.; Murphy, L. H.; Purdie, R. B. Trends Anal. Chem. Chem. 1990, 9, 136. (6) Novotny, M.; Soini, H.; Stefanson, M. Anal. Chem. 1994, 66, 646A655A. (7) Ward, T. J. Anal. Chem. 1994, 66, 633A-640A. (8) Englehardt, H.; Beck, W.; Kohr, J.; Schmitt, T. Angew. Chem., Int. Ed. Engl. 1993, 32, 629-649. (9) Ishizu, T.; Kintsu, K.; Yamamoto. J. Phys. Chem. B 1999, 103, 8992-8997. (10) Wang, J.; Warner, I. Anal. Chem. 1994, 66, 3773-3776.
omers, a rudimentary formulation of the structural requirements for asymmetric recognition was first proposed by Dalgliesh (1952).11 Clearly, a minimum of three simultaneous interactions must take place between a chiral selector and single enantiomer.12,13 At least one of the interactions must be stereochemically dependent. These chromatographically derived chiral recognition models are currently supported only by indirect evidence. Axially chiral binaphthyls possess diverse biological properties.14 Also, they exhibit atropisomerism15,16 that originates from hindered rotation about the C(1)-C(1′) bond between the two binaphthyl moieties. For example, in 1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNDHP), the phosphate group locks the dihedral angle, θ, about the C(1)-C(1′) axis by bridging the minor groove and therefore improving its enantioselective properties. NMR studies involving the mechanism of chiral recognition in aqueous media are scarce.17a,18-20 1H NMR binding studies in deuterated chloroform (CDCl3) have shown that the efficient enantioselective complexation of cinchona alkaloids occurs at the major groove of the 1,1′-binaphthyl unit. The multiple host-guest hydrogen bonding in addition to aromatic-aromatic interactions represent the (11) Dalgliesh, C. E. J. Chem. Soc. 1952, 47, 3940-3942. (12) Pirkle, W. H.; Finn, J. M. Asymmetric Synth. 1983, 1, 87-224. (13) Pirkle, W. H.; Hoover, D. J. Top. Stereochem. 1982, 13, 263331. (14) Bringmann, G.; Zagst, R.; Schaffer M.; Hallock, Y. F.; Cardellina, J. H., II; Boyd, M. R. Angew. Chem., Int. Ed. Engl. 1993, 32, 11901191. (15) Blascke, G. Angew. Chem., Int. Ed. Engl. 1980, 19, 13-24. (16) Cahn, R. S.; Ingold, S.; Prelog, V. Angew. Chem., Int. Ed. Engl. 1966, 5, 385-415. (17) (a) Chankvetadze, B.; Endresz, G.; Schulte, G.; Bergenthal, D.; Blaschke, G. J. Chromatogr. A 1996, 732, 143-150. (b) Chankvetadze, B.; Endresz, G.; Blaschke, G. Chem. Soc. Rev. 1996, 25, 141-153. (18) Topiol, S. Chirality 1989, 1, 69-72. (19) Endresz, G.; Chankvetadze, B.; Bergenthal, D.; Blaschke, G. J. Chromatogr. A 1996, 732, 133-142. (20) Parker, D. Chem. Rev. 1991, 91, 1441-1457. (21) Lustenberger, P.; Martinborough, E.; Denti, T. M.; Diederich, F. J. Chem. Soc., Perkin Trans. 2 1998, 748-76
10.1021/la990539e CCC: $19.00 © 2000 American Chemical Society Published on Web 02/04/2000
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Figure 1. (a) Structures of monomeric chiral surfactants (CS) 3 and 4. Arrows indicate diagnostic intramolecular NOE interactions. (b) Structures of polymeric chiral surfactants (CS) 5 and 6.
major binding forces in the diastereomeric complexes that form in CDCl3 solution.21 In a search for new types of chiral selectors for enantiomeric separations in CE, we have synthesized amino acid-based CS. These water-soluble CS offer a number of advantages. They can be (1) synthesized in multigram quantities, (2) easily purified, (3) cost-effective, and (4) broadly applicable. The aim of the present paper is 2-fold: (1) elucidation of the solution conformation of CS and (2) relating the observed CE separation efficiency of enantiomers of BNDHP using NMR spectroscopic methods. Experimental Section
The data sizes 512 w in F1 and 1 K in F2, and the data were zero-filled in F1 before subjecting to 2D Fourier transformation to yield a 1 K × 1 K data matrix. The resulting data were then processed using a sine-bell window function in F1 and F2, and the resulting spectra are shown in Figures 5 and 6. Twodimensional nuclear Overhauser effect spectroscopy (NOESY) experiments were performed using the time-proportional-phaseincrement method.25,26 Free induction decays were acquired (64 scans, 4 dummy scans) over 3000 Hz into a 2 K data block for 512 incremental values of the evolution time, t1. The raw data were zero-filled to a 2 K × 2 K matrix and processed with a 1 Hz line-broadening function in both dimensions. The mixing time of 100 ms (for both monomeric and polymeric surfactants) was subjected to a random variation of 5%, and the relaxation delay was 2.0 s. Two-dimensional rotating frame Overhauser effect NMR spectroscopy (ROESY)27,28 spectra were acquired with a mixing time of 125 ms. Solvent suppression was accomplished by use of a presaturation pulse of 2 s. A total of 512 time-domain data points for 256 t1 values of 128 scans each were acquired and then zero-filled to 512 × 512, followed by processing with a 90° shifted sine-squared function in both dimensions.
Sample Preparation. The synthesis of chiral surfactants (CS) (3-6) (Figure 1) was accomplished following the protocol described by Billiot et al.22 Solutions of CS and the (R)- and (S)-BNDHP in H2O/D2O (9:1 v/v) and Tris buffer (chemical shift resonance at 3.53 ppm) were prepared in 5 mm NMR tubes. The concentrations of CS were 100 mM, and the ratio of the [CS]/ [BNDHP] was maintained at 40. Clear solutions were used in order to eliminate the solubility effect. NMR Measurements. The 1H NMR measurements (Figures 2-4; Tables 1 and 2) were performed on a Bruker ARX 300 MHz spectrometer equipped with a Silicon Graphics workstation. Water suppression was accomplished by selective irradiation of the water resonance.23 D2O was used for field-frequency lock, and the observed 1H chemical shifts are reported in parts per million (ppm) relative to an internal standard (sodium 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid salt (TSP, 0 ppm)). 1H-1H correlation spectroscopy (COSY) spectra were recorded using the acquisition parameters:24 recycling delay (D1), 1.5 s; D0 increment, 3 µs; spectral width in F2, 2400 Hz, and in F1, 1200 Hz; temperature, 298 K; number of scans, 64; dummy scans, 4.
1,1′-Binaphthyl Compounds. To date, chirality has become one of the most important and complex topics of separation science.22 The origin of chirality is usually an asymmetric atom, e.g., a carbon atom with four different substituents. Chiral molecules are not superimposable on their mirror images, and their nonsuperimposable images are referred to as enantiomers. Enantiomers have the same physical properties but different optical rotation. A scarcely studied source of chirality involves axially chiral binaphthyl compounds that exhibit atropisomerism.15 It should be noted that several binaphthyl compounds are
(22) Billiot, E.; Macossay, J.; Thibodeaux, S.; Shamsi, S. A.; Warner, I. M. Anal. Chem. 1998, 70, 1375-1381. (23) Hoult, D. I. J. Magn. Reson. 1976, 21, 337-347. (24) Rance, M.; Sørensen O. W.; Bodenhausen, G.; Wagner, R. R.; Ernst, R. R.; Wu¨thrich, K. Biochem. Biophys. Res. Commun. 1983, 117, 479-485.
(25) Bax, A.; Lerner, L. Science 1977, 232, 960-967. (26) Zuiderweg, E. R. P.; Hallenga, K.; Olejniczak, E. D. J. Magn. Reson. 1986, 70, 336-343. (27) Kessler, H.; Gehrke, M.; Griesinger, C. Angew. Chem., Int. Ed. Engl. 1988, 27, 490-536. (28) Bax, A.; Davis, D. J. Magn. Reson. 1985, 63, 207-213.
Results and Discussion
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Figure 2. 1H NMR spectra of UVL (3) (a), solutions of 3 with (S)-BNDHP (b), solutions of 3 with (R)-BNDHP (c), solutions of poly-ULV (6) with (S)-BNDHP (d), solutions of poly-UVL (5) with (R)-BNDHP (e), (S)-BNDHP (f), and (R)-BNDHP (g).
Figure 3. 300 MHz 1H NMR spectrum of sodium N-undecylenyl-L-valine-L-leucine (UVL) (3).
Figure 4. 300 MHz 1H NMR spectrum of sodium N-undecylenyl-L-leucine-L-valine (ULV) (4).
remarkable in many regards: Distinguished by a high anti-HIV and anticytopatic activities, high polarity, high crystallinity, high-symmetry chirality (C2-symmetry), simple spectra, direct attachment of the functional groups to an aromatic system, and the accessibility of both Rand S-enantiomers.14,21 1,1′-Biaryl derivatives possess excellent chiral recognition and induction abilities crucial for obtaining a high degree of enantioselection. Besides, their magnetic screening environment and inclusion in chiral pockets make them particularly suitable by NMR.
Binaphthol (BNOH) is the most famous atropisomeric biaryl compound and provides several bridged derivatives that find myriad applications in varied areas of chemistry, medicine, and pharmacology. In the present paper, we have focused on the R- and S-BNDHP, which satisfy all the above-mentioned properties. Of course the two atropisomers of BNDHP (Figure 7) in the absence of CS have identical 1H and 13C NMR spectra. Chiral Surfactants (CS). Chiral differentiation in cyclodextrin (CD)-guest systems has undoubtedly oc-
Chiral Surfactants Table 1.
1H
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NMR Chemical Shift Changes of Chiral Surfactant (CS) Protons in the Presence and Absence of (S)-1,1′-Binaphthyl-2,2′-diyl Hydrogen Phosphate ((S)-BNDHP) chemical shifts (from DSS; 0 ppm)
surfactant UVL
UVL + (S)-BNDHP poly-UVL
poly-UVL + (S)-BNDHP
spin-spin coupling constants
RCH
βCH
RNH
others
LeuR, 4.26
Leuβ, 1.60
LeuRNH, 7.89
ValR, 4.18
Valβ, 2.11
ValRNH, 7.86
LeuγCH, 1.6 LeuγCH3, 0.92, 0.89 ValγCH3, 0.97, 0.94
LeuR, 4.25
Leuβ, 1.59
LeuRNH, 7.61
ValR, 4.16
Valβ, 4.08
ValRNH, 7.58
LeuR, 4.25
Leuβ, 1.61
LeuRNH, 8.07
ValR, 4.25
Valβ, 2.10
ValRNH, 7.90
LeuR, 4.24 ValR, 4.19
Leuβ, 1.59 Valβ, 2.10
LeuRNH, 7.99 ValRNH, 7.99
3JRβ
LeuγCH, 1.58 LeuγCH3, 0.84, 0.88 ValγCH3, 0.90, 0.92
Val 3Jβγ, 7.2
LeuRβ, 6.8
Leu 3Jγδ, 6.6
ValRβ, 7.9
Val 3Jβγ, 6.8
Val 3Jβγ, 2.9
Leuβ, 1.60 Valβ, 2.10
LeuRNH, 7.56 ValRNH, 7.53
LeuγCH, 1.59 LeuγCH3, 0.88, 0.91 ValγCH3, 0.87, 0.95
ULV + (S)-BNDHP
LeuR, 4.42 ValR, 4.08
Leuβ, 1.68 Valβ, 2.09
LeuRNH, 7.55 ValRNH, 7.52
LeuγCH, 1.61 LeuγCH3, 0.85, 0.88 ValγCH3, 0.88, 0.90 OH 8.16
poly-ULV
LeuR, 4.24 ValR, 2.08
Leuβ, 1.59 Valβ, 2.16
LeuγNH, 7.88 ValRNH, 7.88
LeuγCH, 1.59 LeuγCH3, 0.88 ValγCH3, 0.88
poly-ULV + (S)-BNDHP
LeuR, 4.48 ValR, 4.08
Leuβ, 1.62 Valβ, 2.11
LeuRNH, 7.56 ValRNH, 7.56
LeuγCH, 1.62 LeuγCH3, 0.87-0.91 ValγCH3, 0.87-0.91
a
ValRβ, 7.8
LeuγCH, 1.6 LeuγCH3, 0.88, 0.91 ValγCH3, 0.87, 0.95 OH 8.2
LeuR, 4.46 ValR, 4.09
1H
Leu 3Jγδ, 5.6
LeuγCH, 1.61 LeuγCH3, 0.91-0.95 ValγCH3, 0.91-0.95
ULV
Table 2.
others
LeuRβ, 6.1
LeuRβ, 7.9 ValRβ, 5.6, 2.8
Leu 3Jγδ, 2.9 Val 3Jβγ, 2.9
ValRβ, 2.8, 5.7
Val CH3 7.4
NMR Chemical Changes (∆δ) of Chiral and Amide (rNH) Protons of Chiral Surfactants (CS) in the Presence of (S)-BNDHP
host
LeuRH
ValRH
∆δValRH/∆δLeuRH
LeuRNH
ValRNH
∆δLeuRNH/∆δValRNH
UVL poly-UVL ULV poly-ULV
0.01 0.01 0.04 -0.24
0.02 0.06 0.01 0.01
2 (0.50)a 6 (0.17) 0.25 (4) -0.04 (-24)
0.03 0.08 0.01 0.32
0.28 -0.09 0.001 0.32
0.11(9.33)b -0.89 (-1.13) 10 (0.10) 1.0 (1.0)
The value in parenthesis represents ∆δLeuRH/∆δValRH. b The value in parenthesis represents ∆δLeuRNH/∆δValRNH.
cupied a central position in recent years in view of its importance in separation of pharmaceuticals, agricultural chemicals, and biologically active compounds.6 However, R- and β-CDs cannot discriminate L- and D-enantiomers of certain dipeptide amino acids.8 As part of our ongoing interest in highly efficient CD-type model systems, we have synthesized CS that are soluble in aqueous media. The new CS, above a certain concentration, called the critical micelle concentration (cmc), associate reversibly and cooperatively into large aggregates termed micelles. Studies of micelles in water are more numerous, but micelles have also been studied in many aqueous solvent mixtures and a variety of nonaqueous solvents.8 Generally, the nonpolar tails of the surfactant molecules make up the interior of the micelle, while the polar headgroups, charged or uncharged, are located on the exterior, maintaining contact with the solvent and keeping the micelle in solution. Micellar associations are common in natural biological interactions: lipid-lipid, lipid-protein, carbohydrate-protein, and globular proteins.19 Also, “man made” micelle-drug complexes have been extensively investigated as a method of stabilizing drug molecules and improving their general bioavailability. Interestingly, the pharmacological activities of drugs are based on the biological interaction between the drugs and biopolymers such as enzymes, receptors, tissue proteins, and plasma. Most critically, the chiral molecular recognition generated by these biopolymers is based on the diastereomeric interaction with chiral drugs. The micelle-analyte complex formation appears to be a necessary condition for the enantiomeric distinction process. But the very fast (10-2 to 10-6 s) exchange (monomers T micelles) between
aggregates and bulk water is unfavorable to this association and therefore to the discrimination process.22 We envision that the removal of such equilibrium by polymerization of the micellar structure (monomeric CS) should improve the chiral discrimination process by enhancing the lifetime of micelle-analyte association. Recently, polymerized CS have attracted a great deal of interest as anti-HIV agents.21 In monomeric CS synthesized in our laboratory, the polymerizable vinyl group is part of the hydrophobic chain leading to tail polymerized (T-polymer) products. In T-polymers, the hydrophobic backbone is accommodated in the micellar core and the charged ionic heads are located on the interface in contact with water; thus the T-polymerized CS have structures similar to those of their monomeric counterparts. Polymerization was accomplished according to the method of Billiot et al.22 by a 10 Mrad γ-irradiation of 0.1 M aqueous solutions of monomeric CS in a 0.15 Mrad/h 60Co γ-ray source. The progress of the polymerization was monitored by 1H NMR spectroscopy (in D2O) as a function of the γ-ray dose. Evidence for the structures of polymeric CS was provided by comparison of the 1H NMR spectra of the monomeric CS and the polymerized CS; the disappearance of the peaks due to the vinyl protons (4.6-6.1 ppm) and the broadening of the remaining peaks were the only discernible changes caused by polymerization. A very important question prompted us to undertake detailed NMR studies of CS, that of the arrangement and relative mobility of the various parts of CS molecules in the micelle. We employed NMR spectroscopy to supply useful information regarding this question. 1 H NMR Studies. To establish the connectivities
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Figure 5. 400 MHz 1H-1H COSY map of sodium N-undecylenyl-L-valine-L-leucine (UVL) (3) + (S)-BNDHP in D2O.
between nonexchangeable protons and to identify connectivities involving the exchangeable NH protons in CS, NMR experiments were carried out in D2O and H2O, respectively. 1H NMR measurements were relatively simple to make once the HOD signal had been suppressed by presaturation. Because of a large number of hydrogen atoms in CS, the 1H NMR spectra were severely crowded, especially in the 0.2-2.5 ppm region (Figures 3 and 4). To alleviate this problem, 2D 1H-1H COSY experiments were performed. Unambiguous assignment of chemical shifts of protons in CS and BNDHP was then established by analysis of COSY spectra (Figures 5 and 6). Briefly, a COSY spectrum contains a diagonal, representing the 1D 1H NMR spectrum, and the off-diagonal cross-peaks, which result from through-bond J-coupled protons whose chemical shifts describe the position of the cross-peak. The protons of the methyl groups of Val and Leu in a CS backbone are coupled to a single methine proton. For the polymeric CS, 5 and 6 (data not shown), the line widths of the 1H NMR resonances were largely in excess of 5 Hz and frequently comparable to the value of the three-bond spin-spin coupling constants (3JH-H) of the monomeric CS, 3 and 4 (Table 1). The broadening of peaks is attributable to the large molecular sizes and slow tumbling in solution. To probe the chiral recognition elements of our CS (36) for CE enantiomeric separation of BNDHP, the 1H NMR spectra of (S)-BNDHP were compared with those of its corresponding mixtures in CS (Figure 2a-g). The most interesting spectral region of (S)-BNDHP is 6.0-9.0 ppm in which the four AB-type aromatic protons resonate. The significant broadening of the aromatic resonances in the
Rugutt et al.
Figure 6. 400 MHz 1H-1H COSY map of sodium N-undecylenyl-L-leucine-L-valine (ULV) (4) + (S)-BNDHP in D2O.
Figure 7. Structures of 1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNDHP) enantiomers.
presence of CS indicates that the exchange rates between the free and bound forms of BNDHP (Figure 7) are either intermediate or slow on the NMR time scale. Significant upfield shifts for the aromatic protons of BNDHP occurred
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Figure 9. Hydrogen bond patterns for chiral surfactants (CS).
Figure 8. (a) Important inter-NOEs between ULV (4) and (S)-BNDHP. (b) Important inter-NOEs between UVL (3) and (S)-BNDHP.
as a result of interaction with CS (Figure 2b-e). The extent of the frequency shift was larger and the line widths narrower in the monomeric CS than in the polymeric CS (compare the resonances due to H-8 in spectra b and c and d and e in Figure 2. This implies that the interaction between the enantiomers of BNDHP and CS occurs at the same region but to a different degree.29 In agreement with the inclusion in chiral pocket(s),21 the chemical shift movements of H-5 and H-6 protons of BNDHP were significantly affected upon binding to CS. The upfield chemical shift movement of the H-6 resonance indicates that the major chiral groove (Figure 7) is positioned more toward the micellar hydrophobic chiral pocket of the surfactant (Figure 9). The higher frequency shift of H-4 and H-8 protons in (S)- than in (R)-BNDHP appear to be responsible for strengthened and weakened interactions with CS, respectively. Accordingly, it can be concluded that the mobility of the (S)-BNDHP should be higher than that of the (R)-antipode as a result of stronger interactions with the CS. This result agrees well with that found from CE enantioseparation of BNDHP using chiral CD derivatives and dipeptide CS.17a,b,22 It is worth noting that the 1 H NMR spectra of racemic mixtures of BNDHP in CS (data not shown) were identical to those of the of individual enantiomers in CS. Many of the coupling constants30 of the RH protons, LeuRH, and ValRH of CS changed slightly (Table 1) in the presence of BNDHP, suggesting that small conformational changes may accompany the BNDHP(29) Rugutt, J. K.; Billiot, E.; Warner, I. M. 54th Southwest Regional ACS Meeting, Baton Rouge, LA, November 1-3, 1998. (30) Karplus, M. J. Chem. Phys. 1959, 30, 11-15.
CS complexation.31 Karplus parameters relate the 3JH-H to the dihedral angles (θ) and are very relevant to the confromational analysis of CS. However, for a given 3JH-H value, there are four different θ values. Owing to the flexibility of the amide (C-N) linkages and the hydrophobic side chains, the CS typically adopt several conformations in solution. On the basis of the previous work in chiral discrimination,19 the present work also supports the fact that the differences in enantiomeric 1H NMR signals strongly depend on their position with respect to the chiral centers (RH). As shown in Table 2, the magnitudes of the upfield or downfield chemical shift changes of the RH protons (∆δValRH and ∆δLeuRH), the amide protons (∆δValRNH and ∆δLeuRNH), and their relative ratios may be employed as qualitative indicators of the CS-BNDHP stability and the relative position of BNDHP in the micellar core.32 In addition to the chiral protons (RH), the exchangeable amide protons (NH) protrude from the CS backbone and serve as long-range conformational sensors. Intramolecular NOE Interactions. The method of 2D NOESY is one of the most powerful tools for the study of molecular structure of macromolecules.27,33 In the present studies, the NOESY experiment was employed because it was possible to observe the NOEs of closely spaced resonances34 in the complex aliphatic regions of the CS spectra. The intramolecular NOEs observed in the CS-BNDHP mixture were free from interfering COSYtype correlations since the components in the complex mixture were not J-coupled. Short interproton spatial distances (
