Intermolecular 1H-1H Two-Dimensional Nuclear Overhauser Enhamncements in the Characterization of a Rationally Designed Chiral Recognition System

Article abstract of DOI:10.1021/jo951255b

Chiral stationary phases (CSPs) for liquid chromatography derived from N-(acyl)proline-3,5-dimethylanilides separate the enantiomers of N-(3,5-dinitrobenzoyl)-α-amino esters and amides with high levels of selectivity.These CSPs have been used to assemble a large body of chromatographic data which indirectly supports tha validity of the mechanistic rationalle originally used in the design of these CSPs.We herein report 1H and 13C chemical shift data obtained when the (S)-enantiomer of chiral solvating agent (CSA) 3, and a soluble analogue of the selector used in CSP (S)-1, acts on each of ther enantiomers of the diemthylamide of N-(3,5-dinitrobenzoyl)leucine, 2.The changes in chemical shift in the mixture of (S)-2 and (S)-3 support the existence of those interactions thought to be essential to chiral recognition in this system.In addition, sugnificant intermolecular NOESY enhancements are observed in this mixture.These NOE data are consistent with the structure expected for the more stable diastereomeric adsorbate formed between (S)-2 and the (S)-proline-derived CSP 1.No intermolecular NOEs are observed for corresponding mixtures of the chiral solvating agent (S)-3 and (R)-2, the enantiomer least retained on (S)-CSP 1.

Full text of DOI:10.1021/jo951255b

J . Org. Chem. 1996, 61, 4769-4774
4769
1
In ter m olecu la r H-1H Tw o-Dim en sion a l Nu clea r Over h a u ser
En h a n cem en ts in th e Ch a r a cter iza tion of a Ra tion a lly Design ed
Ch ir a l Recogn ition System
William H. Pirkle* and Patrick G. Murray†
University of Illinois, School of Chemical Sciences, 600 South Mathews, Urbana, Illinois 61801
David J . Rausch‡ and Stephen T. McKenna§
Department of Chemistry, Illinois Benedictine College, 5700 College Road, Lisle, Illinois 60532, and
Amoco Corporation, Analytical Research and Services Division, P.O. Box 3011, F-9, Naperville, Illinois
60566
Received J uly 13, 1995 (Revised Manuscript Received April 26, 1996X)
Chiral stationary phases (CSPs) for liquid chromatography derived from N-(acyl)proline-3,5-
dimethylanilides separate the enantiomers of N-(3,5-dinitrobenzoyl)-R-amino esters and amides
with high levels of selectivity. These CSPs have been used to assemble a large body of
chromatographic data which indirectly supports the validity of the mechanistic rationale originally
1
used in the design of these CSPs. We herein report H and 13C chemical shift data obtained when
the (S)-enantiomer of chiral solvating agent (CSA) 3, a soluble analogue of the selector used in
CSP (S)-1, acts on each of the enantiomers of the dimethylamide of N-(3,5-dinitrobenzoyl)leucine,
2. The changes in chemical shift in the mixture of (S)-2 and (S)-3 support the existence of those
interactions thought to be essential to chiral recognition in this system. In addition, significant
intermolecular NOESY enhancements are observed in this mixture. These NOE data are consistent
with the structure expected for the more stable diastereomeric adsorbate formed between (S)-2
and the (S)-proline-derived CSP 1. No intermolecular NOEs are observed for corresponding
mixtures of the chiral solvating agent (S)-3 and (R)-2, the enantiomer least retained on (S)-CSP 1.
We recently reported the preparation and evaluation
of a new class of π-basic chiral stationary phases (CSPs)
derived from anilides of N-(acyl)proline.1,2 The a priori
design of these CSPs was based on an understanding of
the factors responsible for enantiodiscrimination in struc-
turally different but mechanistically related systems.3 In
practice, these CSPs exhibit high levels of selectivity in
the liquid chromatographic separation of the enantiomers
of a variety of π-acidic racemates. For example, the
separation factor (R) for the enantiomers of the dimethyl-
amide of N-(3,5-dinitrobenzoyl)leucine, 2, is 30, the
capacity factor for the least retained enantiomer being
0.28.4 Thus, (S)-CSP-1 (Figure 1) displays a strong
affinity for (S)-2, and very little affinity for (R)-2, an ideal
situation from the standpoint of chiral recognition. In
point of fact, separation factors exceeding 80 have been
observed for the enantiomers of selected analytes with
CSP 1.1
The proline-based CSPs were designed to be mecha-
nistically similar to the well-characterized N-(aryl)-R-
amino ester-derived CSPs.5,6 Choosing analyte 2 as a
specific example, the interactions essential to the pref-
F igu r e 1. Structures of the (S)-proline-derived CSP 1 and
N-(3,5-dinitrobenzoyl)leucine dimethylamide (2).
erential retention of (S)-2 on (S)-CSP 1 were expected to
be (1) a face-to-face π-π interaction between the 3,5-
dimethylanilido group of CSP 1 and the 3,5-dinitroben-
zoyl group of (S)-2, (2) a hydrogen bond from the 3,5-
dinitrobenzamide N-H of (S)-2 to the N-(acyl)-carbonyl
oxygen of CSP 1, and (3) a hydrogen bond from the 3,5-
dimethylanilide N-H of CSP 1 to the C-terminal carbonyl
oxygen of (S)-2 (Figure 2).
† Present address: Nalco Chemical Company, Corporate Research
and Development, One Nalco Center, Naperville, IL 60560.
‡ Illinois Benedictine College.
§ Amoco Corporation.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
(1) Pirkle, W. H.; Murray, P. G. J . Chromatogr. 1993, 641, 11-19.
(2) Pirkle, W. H.; Murray, P. G. J . High Resolut. Chromatogr. 1993,
16, 285-288.
(6) Pirkle, W. H.; Deming, K. C.; Burke, J . A. Chirality 1991, 3, 183-
187.
(7) See, for example: (a) Cochran, J . E.; Parrott, T. J .; Whitlock, B.
J .; Whitlock, H. W. J . Am. Chem Soc. 1992, 114, 2269. (b) Hunter, C.
A. J . Chem. Soc., Chem. Commun. 1991, 11, 749-751. (c) Van Doorn,
A. R.; Bos, M.; Harkema, S.; Van Eerden, J .; Verboom, W.; Reinhoudt,
D. N. J . Org. Chem. 1991, 56, 2371-2380. (d) Tanaka, Y.; Kato, Y.;
Aoyama, Y. J . Am. Chem. Soc. 1990, 112, 2807-2808. (e) Stauffer, D.
A.; Daugherty, D. A. Tetrahedron Lett. 1988, 29, 6039-6042. (f)
Cowart, M. D.; Sucholeiki, I.; Bukownik, R. R.; Wilcox, C. S. J . Am.
Chem. Soc. 1988, 110, 6204-6210. (g) Rebek, J .; Nemeth, D. J . Am.
Chem. Soc. 1985, 107, 6738-6739. (h) Zimmerman, S. C.; VanZyl, C.
M.; Hamilton, G. S. J . Am. Chem. Soc. 1989, 11, 1373-1381.
(3) Pirkle, W. H.; Murray, P. G.; Burke, J . A. J . Chromatogr. 1993,
641, 21-29.
(4) R, the chromatographic separation factor, is the ratio of the two
capacity factors (corrected retention times), k′2/k′1, and is related
logarithmically to the difference in free energy between the two
diastereomeric adsorbates formed as each enantiomer interacts with
the CSP.
(5) Pirkle, W. H.; Pochapsky, T. C.; Mahler, G. S.; Corey, D. E.; Reno,
D. S.; Alessi, D. M. J . Org. Chem. 1986, 51, 4991.
S0022-3263(95)01255-2 CCC: $12.00 © 1996 American Chemical Society

4770 J . Org. Chem., Vol. 61, No. 14, 1996
Pirkle et al.
dimensional experiments have advantages in sensitivity
and quantitation of the observed enhancements.
A
disadvantage of one-dimensional experiments, however,
is that they become increasingly more difficult to perform
as spectra become more congested, since they require that
individual resonances be irradiated selectively without
affecting nearby resonances. Although the component
molecules in this study are small, the mixtures have
crowded NMR spectra with areas of significant overlap
between multiplets. Because the need to selectively
irradiate a particular signal is not of concern in the two-
dimensional NOESY experiment, it is possible to observe
NOEs of closely spaced signals in complex aromatic or
aliphatic regions of the spectrum. Therefore, it seemed
likely that NOESY experiments might be useful in
studying chiral recognition systems involving small bi-
molecular complexes. Finally, intermolecular NOEs
observed in the mixture are free from interfering COSY-
type correlations since the two components in the mixture
are not J -coupled to each other.
F igu r e 2. Interactions proposed to account for retention of
the more retained enantiomer, (S)-2, on (S)-CSP 1.
Exp er im en ta l Section
F igu r e 3. Structure of the (S)-proline-derived chiral solvating
agent 3.
All reagents employed were of pharmaceutical or reagent
grade. Elemental analyses were performed by T. McCarthy
and associates of the University of Illinois microanalytical
laboratory. Melting points are uncorrected.
The investigation of molecular recognition phenomena
is facilitated by high-field NMR instruments.7 Of par-
ticular relevance, intermolecular nuclear Overhauser
effect (NOE) experiments aid in the elucidation of
chromatographically-derived chiral recognition mecha-
nisms.8 The nuclear Overhauser effect (or nuclear Over-
hauser enhancement) is manifest as a change in the
intensity of one signal when another nucleus is saturated.
The intensity change results from through space pertur-
bation of the magnetic spin state distribution of nuclei
near the nucleus which has been excited as it subse-
quently undergoes dipole-dipole cross-relaxation.9 Thus,
the utility of the NOE experiments in establishing
conformations, making relative stereochemical assign-
ments, and determining the spacial orientation of specific
groups in a molecule or complex lies in the fact that an
observed NOE indicates proximity of the irradiated and
the observed nuclei in space.
(S)-N-P iva loylp r olin e. (S)-Proline (Aldrich) (10 g, 0.087
mol) was dissolved in 50 mL of 2 N NaOH, cooled to 0 °C in
an ice-water bath, and stirred magnetically. Trimethyacetyl
chloride (10.7 mL, 0.087 mol) and 40 mL of 2 N NaOH were
added in several alternating portions over the course of 1 h so
as to maintain the temperature at 5-10 °C. The pH was
checked periodically to insure that the solution remained
strongly alkaline. After addition of the acid chloride was
complete, the reaction mixture was allowed to warm to room
temperature and stirred vigorously for 30 min. The basic
solution was extracted with two 75 mL portions of diethyl ether
and then acidified to Congo red with 6 N HCl. The oil which
separated was extracted into two 75 mL portions of diethyl
ether which were combined, dried over anhydrous MgSO4, and
concentrated on a rotary evaporator. The white crystalline
residue, 16.9 g (97.9%), was used without further purifica-
1
tion: mp 130-132 °C; H NMR (200 MHz) (CDCl3) δ 1.30 (s,
9H), 1.95-2.20 (overlapping m, 4H), 3.70 (m, 2H), 4.55 (m,
1H), 11.10 (bs, 1H). Anal. Calcd for C10H17NO3: C, 60.28; H,
8.60; N, 7.03. Found: C, 60.18; H, 8.64; N, 6.96.
In the present study, we have undertaken a detailed
NMR investigation of each enantiomer of 2 with (S)-CSA
3, a soluble analogue of (S)-CSP 1, to improve our
understanding of the structure of the diastereomeric
complexes formed in solution (Figure 3).
Two-dimensional nuclear Overhauser spectroscopy
(NOESY) is extremely useful in studying the conforma-
tions and higher order structures of proteins and other
macromolecules. However, it has been used less fre-
quently in studies of smaller molecules since tumbling
of these molecules is generally sufficiently rapid so as to
place dipole-dipole relaxation into the positive NOE
regime.10 For small molecules, the steady state one-
(S)-N-P iva loylp r olin e 3,5-Dim eth yla n ilid e (3). (S)-N-
Pivaloylproline (2.50 g, 0.013 mol) was dissolved in 40 mL of
dry dichloromethane. 2-Ethoxy-1-(ethoxycarbonyl)-1,2-dihy-
droquinoline (EEDQ) (3.26 g, 0.132 mol) was added, and the
solution was sonicated until homogeneous (about 5 min).
When dissolution was complete, 1.77 g (0.013 mol) of freshly
distilled 3,5-dimethylaniline (Aldrich) was added and the
solution was stirred for 2 h and then poured into a 250 mL
separatory funnel containing 50 mL of dichloromethane. The
dichloromethane solution was washed sequentially with two
75 mL portions of 2 N HCl, two 75 mL portions of 5% NaHCO3,
and 100 mL of water, then dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. Upon addition of ethyl
acetate and hexane, the remaining yellow syrup afforded a
crystalline product determined to be greater than 99.9%
enantiomerically pure by analytical HPLC using an (S)-N-(3,5-
dinitrobenzoyl)leucine-derived CSP (Regis Technologies, Mor-
ton Grove, IL). 3: 1H NMR (500 MHz) (CD2Cl2) δ 1.27 (s, 9H),
1.87 (m, J cis ) 8.1, 10.0, J trans ) 3.8, J gem ) 12.0 Hz, 1H), 1.95
(m, J cis ) 7.1, 5.3, J trans ) 3.8, 3.6, J gem ) 12.5 Hz, 1H), 2.11
(m, J cis ) 10.0, 6.3, J trans ) 3.6, 6.2, J gem ) 12.5 Hz, 1H), 2.26
(s, 6H), 2.30 (m, J cis ) 7.1, J trans ) 3.2, 3.6, J gem ) 12.0 Hz,
1H), 3.67 (ddd, J cis ) 6.3, J trans ) 3.6, J gem ) 10.0 Hz, 1H),
3.74 (ddd, J cis ) 5.3, J trans ) 6.2, J gem ) 10.0 Hz, 1H), 4.72 (dd,
J cis ) 8.1, J trans ) 3.2 Hz, 1H), 6.70 (s, 1H), 7.11 (s, 1H), 8.90
(bs, 1H); 13C NMR (CD2Cl2) δ 178.7, 170.3, 138.9, 138.8, 125.8,
(8) See, for example: (a) Pirkle, W. H.; Pochapsky, T. C. J . Am. Chem
Soc. 1987, 109, 5975-5982. (b) Pirkle, W. H.; Pochapsky, T. C. J . Am.
Chem Soc. 1986, 108, 5627-5628. (c) Uccello-Barretta, G.; Rosini, C.;
Pini, D.; Salvadori, P. J . Am. Chem. Soc. 1990, 112, 2707-2710. (d)
Spisni, A.; Corradini, R.; Marchelli, R.; Dossena, A. J . Org. Chem. 1989,
54, 684-688. (e) Pirkle, W. H., Selness, S. J . Org. Chem. 1995, 60,
3252-3256.
(9) Neuhaus, D.; Williamson, M. P. The Nuclear Overhauser Effect
in Structural and Conformational Analysis; VCH Publishers, Inc.: New
York, 1989.
(10) Derome, A. E. Modern NMR Techniques for Chemistry; Perga-
mon Press: New York, 1987.

NOE in Characterization of a Chiral Recognition System
J . Org. Chem., Vol. 61, No. 14, 1996 4771
117.8, 63.0, 48.8, 39.6, 27.7, 26.6, 26.3, 21.4 ppm. Anal. Calcd
for C18H26N2O2: C, 71.49; H, 8.67; N, 9.26. Found: C, 71.32;
H, 8.60; N, 9.24.
Dim eth yla m id e of (R,S)-N-(3,5-Din itr oben zoyl)leu cin e
(2). Racemic N-(3,5-dinitrobenzoyl)leucine, 2.95 g (0.009 mol),
and 2.36 g (0.009 mol) of EEDQ were suspended in 50 mL of
dry tetrahydrofuran, and the solution was stirred vigorously
until dissolution was complete. After 20 min, anhydrous
dimethylamine was bubbled through the homogeneous solu-
tion for 30 s, the reaction vessel was stoppered, and the
mixture was allowed to stand for 4 h. The initially colorless
solution turns purple upon addition of the gaseous amine, then
orange, and finally remains yellow. After 2 h, the mixture
was concentrated on a rotary evaporator, and the residue was
taken up in 50 mL of dichloromethane, poured into a 100 mL
separatory funnel, and washed sequentially with two 25 mL
portions of 2 N HCl and two 25 mL portions of 5% NaHCO3.
The organic layer was removed from the separatory funnel,
dried over anhydrous MgSO4, and concentrated to dryness.
Recrystallization of the remaining material from ethyl acetate-
hexane yielded 2.46 g (77%) of the white crystalline powdery
racemate, 2: mp 205-207 °C; 1H NMR (500 MHz) (CD2Cl2) δ
0.98 (d, J ) 6.5 Hz, 3H), 1.00 (d, J ) 6.5 Hz 3H), 1.49 (m, J )
10.5, 3.5, J gem ) 14.0 Hz, 1 H), 1.83 (m, J ) 4.0, 11.5, J gem
)
14.0 Hz, 1H), 1.86 (m, J ) 6.5, 10.5, 4.0 Hz, 1H), 3.07 (s, 3H),
3.20 (s, 3H), 5.16 (m, J ) 3.5, 11.5, 8.0 Hz, 1H), 8.98 (d, J )
8.0 Hz, 1H), 8.82 (d, J ) 2.0 Hz, 2H), 9.09 (t, J ) 1.0 Hz, 1H);
13C NMR (CD2Cl2) δ 173.5, 162.3, 148.8, 137.6, 127.7, 121.2,
49.6, 41.2, 37.4, 36.2, 25.4, 23.5, 21.5 ppm. Anal. Calcd for
C15H20N4O6: C, 51.13; H, 5.72; N, 15.90. Found: C, 51.18; H,
5.73; N, 15.90.
Racemic 2 (2.0 g) was dissolved in a minimal amount of 1,4-
dioxane and preparatively resolved by MPLC employing a 75
× 2.5 cm column of (S)-N-(1-naphthyl)leucine-derived CSP
bonded to 40 µm silica gel using a mobile phase consisting of
20% 2-propanol in hexane. The (S)-enantiomer of 2 is the more
retained. The NMR spectrum of either enantiomer is es-
sentially identical to that of the racemate.
P r ep a r a tion of NMR Sa m p les. Four NMR samples were
prepared, containing respectively, (S)-N-pivaloylproline 3,5-
dimethylanilide; (S)-N-(3,5-dinitrobenzoyl)leucine dimethyl-
amide; (S)-N-pivaloylproline 3,5-dimethylanilide and (S)-N-
(3,5-dinitrobenzoyl)leucine dimethylamide; and (S)-N-pivaloyl-
proline 3,5-dimethylanilide and (R)-N-(3,5-dinitrobenzoyl)-
leucine dimethylamide. Samples containing the individual
components were initially 0.0505 M in 3 or 2, respectively, and
the mixtures were initially 0.0505 M in each component.
Spectrophotometric grade CD2Cl2 was passed twice through
Laroche basic alumina to remove any hydrogen chloride or
water which might have been present. Each of the samples
initially contained 1 mL of this CD2Cl2 before degassing under
vacuum (10-3 Torr) and sealing after four freeze-thaw degas-
sing cycles. Some concentration of the samples is inevitable
during the degassing procedure, but if all samples are prepared
consecutively under identical conditions, this should occur to
the same extent for all samples.
NMR Exp er im en ts. NOESY experiments were performed
on a Varian Unity 500 spectrometer using the standard
software provided with VNMR version 4.3. The sample
temperature was regulated at 30 or 0 °C. Spinning was not
used. Spectra were acquired in the hypercomplex phase
sensitive mode with 2048 data points in the directly acquired
dimension and 256 real and 256 imaginary FIDs in the second
dimension. The spectral width was 5085 Hz, centered near 5
ppm in both directions. The resulting acquisition time was
201 ms in the f2 dimension and 50 ms in the f1 dimension. A
total of eight scans were acquired for each FID. The relaxation
delay between scans was set to 20 s; this value was chosen on
the basis of a T1 experiment which showed that this was
sufficient time to allow complete relaxation for all protons
other than H13 (T1 ) 5.4 s) and H15 (T1 > 20 s). Several
experiments were performed using a range of mixing times
from 0.25 to 4 s.
F igu r e 4. Numbering scheme for protons and carbon atoms
in (S)-3 and (R)- and (S)-2.
processed with the same Gaussian filter. The spectra were
phased with diagonal peaks and chemical exchange cross-
peaks positive and NOE cross-peaks negative. Volume inte-
gration was performed using standard VNMR “ll2d” software.
Resu lts a n d Discu ssion
For convenience in discussion, the protons on the
proline-derived chiral selector, (S)-3, and the N-(3,5-
dinitrobenzoyl)leucine dimethylamide, 2, are numbered
as shown in Figure 4.
Careful and thorough assignment of each signal in all
four spectra is necessary if one is to accurately interpret
the observed NOE enhancements. All resonances in each
spectrum were unambiguously assigned using 1H and 13C
NMR spectroscopy with various combinations of selective
1H{1H} decoupling, 1H-1H correlation spectroscopy
(COSY), heteronuclear multiple bond correlation (HMBC),
and heteronuclear multiple quantum coherence (HMQC)
spectroscopy and intramolecular NOE data. No substan-
tial change in chemical shifts occurs in either single
component, 2 or (S)-3, as a function of temperature, save
for the dinitrobenzamide N-H resonance, which moves
from δ 8.98 at 30 °C to δ 10.24 at -60 °C. The signals
arising from the diastereotopic protons of the chiral
solvating agent (S)-3 and the analyte 2 are complicated,
particularly in the mixtures where changes in the chemi-
cal shifts occur and multiplets overlap.
The seriously interested reader is urged to assemble
CPK space-filling molecular models (Havard Apparatus,
South Natick, MA) of the more stable (S)-(S) complex
to refer to in following the interpretation of the chemical
shift and NOE data. For a cartoonlike representation
of the more stable diastereomeric complex, the reader is
directed to ref 1.
1H a n d Ch em ica l Sh ift Differ en ces. The chemical
shifts for all protons on 2 and 3 in each of the four
samples at 26 °C are summarized in Table 1.
As the data in Table 1 show, significant changes in
chemical shift occur for many of the protons of (S)-3 and
(S)-2 in the (homochiral) mixture relative to those
Data in the f2 dimension were processed with no zero-filling
and a matched Gaussian filter. Data in the f1 dimension were
extended from 512 to 2048 points using linear prediction and

4772 J . Org. Chem., Vol. 61, No. 14, 1996
Pirkle et al.
Ta ble 1. 1H Ch em ica l Sh ifts for (S)-3, (S)-2, a n d (S)-3
w ith (S)-2 a n d (S)-3 w ith (R)-2 a t 30 °C in CD2Cl2
Rep or ted in p p m Rela tive to TMS
Ta ble 2. 13C Ch em ica l Sh ifts for (S)-3, (S)-2, a n d (S)-3
w ith (S)-2 a n d (S)-3 w ith (R)-2 a t 26 °C in CD2Cl2
Rep or ted in p p m Rela tive to TMS
proton
H1
H4a, H4b
H5a, H5b
H6a, H6b
H7
free
1.27
3.68, 3.74
1.95, 2.11
1.87, 2.30
4.72
(S)-(R) mixture
(S)-(S) mixture
carbon atom
free
(S)-(R) mixture
(S)-(S) mixture
1.28
1.36
3.78
1.92, 2.12
2.02
4.71
C1
C2
C3
C4
27.4
39.6
178.7
48.8
27.4
39.6
178.7
48.8
27.2
39.3
178.0
49.1
3.68, 3.74
1.95, 2.11
1.87, 2.31
4.72
C5
26.3
26.3
26.3
H9
8.90
8.87
9.37
C6
26.6
26.6
28.0
H11
7.11
7.11
6.82
C7
63.0
63.0
63.1
H13
H14
H15
H17
H20
H21
H22a, H22b
H23
H24
6.70
2.26
9.09
8.82
8.98
5.16
1.49, 1.83
1.86
1.00
6.70
2.26
9.03
8.83
8.75
5.16
1.49, 1.80
1.90
1.01
6.45
2.14
8.90
8.86
9.04
5.16
1.44, 1.83
1.91
1.00
C8
170.3
138.9
117.8
138.8
125.8
21.4
121.2
148.8
127.7
137.6
162.3
49.6
41.2
25.4
23.5
21.5
173.5
37.4
36.2
170.3
138.9
117.7
138.8
125.8
21.4
121.2
148.8
127.7
137.7
162.3
49.5
41.4
25.4
23.5
21.5
173.3
37.4
36.1
171.1
139.2
116.6
138.6
125.1
21.2
120.6
148.5
127.9
137.2
162.9
49.7
40.5
25.2
23.7
21.3
173.9
37.5
36.3
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C21
C22
C23
C24
C25
C26
C27
C28
H25
H27
H28
0.99
3.20
3.07
0.98
3.19
3.05
0.98
3.24
3.06
observed for the noncomplexed species. H9 moves down-
field as would be expected if this amide N-H participates
in hydrogen bonding. Dinitrobenzamide H20 is also
thought to be involved significantly in hydrogen bonding,
but shows only a slight downfield chemical shift, most
likely because it is simultaneously shielded (in the
complex) by the 3,5-dimethylanilide ring.11 Protons H11,
H13, H14, and H15 show upfield chemical shifts, sug-
gesting that the two aromatic rings mutually shield each
other, consistent with a face-to-face approach of these
groups during the π-donor/π-acceptor interaction. The
signal of H1, the tert-butyl protons of the N-acyl group
on (S)-3, shows a modest downfield shift in the stable
complex, as these protons are placed in the deshielding
zone of the 3,5-dinitrobenzoyl group during complexation.
The changes in the chemical shifts and coupling
constants of the diastereotopic protons on the proline ring
which attend formation of the stable complex are note-
worthy. While H4b is downfield of H4a in uncomplexed
(S)-3, both resonances are isochronous (δ 3.78 ppm) in
the mixture of (S)-3 and (S)-2. Both H6a and H6b
experience a change in chemical shift, although in
opposite directions, as a result of complexation. These
induced shifts become greater as temperature is reduced
owing to increased interaction of the two components.
Many of the coupling constants of the proline ring protons
change slightly in the mixture, suggesting that a small
conformational change of the ring may accompany com-
plexation.
the noncomplexed species, these shifts may reflect a
change in the extent of self-association stemming from
the slight extent of formation of the heterochiral complex-
(es).
13C Ch em ica l Sh ift Differ en ces. The chemical shifts
for the carbon atoms of 2 and 3 in each of the four
samples are summarized in Table 2.
The resonances for the four carbonyl carbons are of
particular interest. According to the proposed chiral
recognition mechanism, two of the carbonyl carbons (C8
and C19) are in amides which are hydrogen bond donors
(H9 and H20) and two are in amides which serve as
hydrogen bond acceptors (carbonyl oxygens at C3 and
C26). On formation of the more stable complex (the (S)-
(S) mixture), the resonances for C8, C19, and C26 move
downfield, while the resonance for C3 moves upfield.
Intuitively, one expects participation in a hydrogen bond
to cause downfield shifts for the carbons of carbonyls
which serve as hydrogen bond acceptors. Likewise, the
carbonyl carbon of an amide group in which the N-H is
involved in hydrogen bonding should be deshielded and
consequently should also show a downfield shift relative
to the noncomplexed species. Thus, these experimental
observations are consistent with expectation in three of
the four cases. On the basis of the examination of space-
filling molecular models of this complex, it appears as if
C3 may be slightly shielded by the 3,5-dinitrobenzoyl
system, thus accounting for the apparently anomalous
direction of chemical shift.12 Also noteworthy, the rela-
There are no significant chemical shift differences
between the free species and the heterochiral mixture of
(R)-2-(S)-3, save for the two amide proton resonances,
H9 and H20. Since the chemical shifts of these protons
are sensitive to concentration and temperature even as
(11) Experience shows that the chemical shift of this amide hydrogen
(H20) typically increases when it serves as a hydrogen bond donor.
The close approach (2.05 Å) of this hydrogen (H20) to the pivaloyl
carbonyl carbon (O3) in the crystalline 1:1 complex of (S)-2 and (S)-3
indicates that H20 is involved in a hydrogen bond in the solid state.
Because the remaining induced shifts and intermolecular NOEs
indicate that the dominant mode of interaction of (S)-2 and (S)-3 in
solution is similar to that seen in the solid state, we believe this
hydrogen bond to be present in solution. Study of a CPK space-filling
model of this complex leads us to suspect that the absence of the
expected downfield shift of H20 comes about owing to the anisotropic
magnetic environment in the complex. One of the reviewers thought
this explanation “too subjective” on the basis of his own modeling study
and wanted a “more reasonable explanation” of the chemical shift of
H20. We retain our original opinion but can offer no proof of this.
(12) The anilide N-H of uncomplexed 3 is believed to intramolecu-
larly hydrogen bond to the pivaloyl carbonyl oxygen in view of the
chemical shift of this N-H, the observation of but one pivaloyl rotamer,
even at low temperature, and the lack of dependence of the N-H
chemical shift on temperature. Complexation with (S)-2 shifts the
signal of the anilide N-H 0.5 ppm downfield and that of the anilide
carbonyl carbon 0.7 ppm upfield. One of the reviewers points out that
the anomalous upfield shift of C3 may be occasioned by the change
from an intramolecular hydrogen to an intermolecular hydrogen bond.
This seems quite plausible insofar as the complexation could entail
some conformation alteration of the pivalamide group. Indeed, the
changes in coupling constants which occur on complexation indicate
that some conformation change occurs within the proline ring.

NOE in Characterization of a Chiral Recognition System
J . Org. Chem., Vol. 61, No. 14, 1996 4773
Ta ble 3. In tr a m olecu la r 1H-1H NOE En h a n cem en ts
w ith Rela tive Volu m e In tegr a ls Sh ow n in P a r en th eses.
In tegr a ls Ar e Nega tive u n less Oth er w ise In d ica ted (+)a
Ta ble 4. In ter m olecu la r 1H-1H NOE En h a n cem en ts
w ith Rela tive Volu m e In tegr a ls Sh ow n in P a r en th esesa
intermolecular
NOE enhancement
(relative strength)
proton
observed
intramolecular
NOE enhancement
(relative strength)
proton
observed
H1
H22a (s), H22b (m), H24/H25 (m), H17 (m), H20 (w),
H23 (w)
H1
H4a,b (s), H11 (w), H7 (vw)
H4a, H4b
H5a
H5b
H4a,b H1 (s), H5a (m), H6a (w), H6b (w), H7 (vw)
H5a
H5b
H6a
H6b
H7
H5b (vs), H4a,b (m)
H5a (vs), H4a,b (m), H7 (vw)
H6b (s), H7 (m), H4a,b (w)
H6a (s), H7 (m), H4a,b (w), H11 (vw)
H6a (s), H9 (s), H6b (m), H1 (w), H11 (vw), H4a,b (vw),
H5b (vw)
H6a
H27 (vw)
H6b
H17 (vw), H27 (vw)
H7
H9
H11
H13
H20 (w), H22b (w), H27 (vw)
H20 (w), H17 (vw), H27 (vw)
H17 (m), H15 (w), H21 (w), H27 (w)
H17 (w)
H9
H11 (s), H7 (s)
H11
H13
H14
H15
H17
H20
H21
H14 (vs), H9 (s), H13 (m), H1 (w)
H14 (vs), H11 (w)
H13 (vs), H11 (vs)
H14
H15
H15 (s), H17 (s), H27 (w), H28 (w)
H14 (m), H11 (vw)
H17
H20
H14 (s), H1 (m), H11 (m), H13 (vw)
H7 (w), H9 (w), H11 (vw)
H20 (vs), H23 (w), H24/25 (w), H27 (vw), H28 (vw)
H17 (vs), H22b (m), H23 (m), H21 (w), H27 (vw), H28 (vw)
H25a,b (s), H28 (s), H27 (m), H22a (m), H20 (w), H22b (w),
H23 (w)
H21
H22a
H22b
H23
H1 (s)
H7 (vw)
H1 (m)
H22a
H22b
H22b (vs), H21 (m), H24/25 (m), H28 (m), H23 (w), H27 (w)
H22a (vs), H22 (m), H24/25 (m), H23 (w), H21 (vw),
H27 (vw), H28 (vw)
H24/H25
H27
H28
H1 (m)
H11 (vw), H14 (vw)
H11 (m), H14 (m)
H23
H24/25 (s), H20 (m), H22a (w), H17 (vw), H27 (vw),
H28 (vw)
a
H24/25 H21 (s), H22a (s), H23 (s), H22b (m), H27 (w), H28 (w),
H17 (vw)
Legend for relative volume integrals: vvs ) very, very strong
(>10); vs ) very strong (∼5); s ) strong (∼1); m ) medium (∼0.5);
w ) weak (∼0.1); vw ) very weak (∼<0.1).
H27
H28
H28 (vvs, +), H21 (s), H17 (w), H22a (w), H24/25 (w)
H27 (vvs, +), H21 (s), H22a (m), H24/25 (w), H17 (vw)
a
Legend for relative volume integrals: vvs ) very, very strong
(>10); vs ) very strong (∼5); s ) strong (∼1); m ) medium (∼0.5);
w ) weak (∼0.1); vw ) very weak (∼<0.1).
present system indicates that only the trans-rotamer of
(S)-3, the one which places the C3 carbonyl group more
or less eclipsed with the methine hydrogen H7, is
populated in solution to any appreciable extent. Strong
reciprocal NOEs between H1 and H4a and H4b support
this assignment.
In 2, stronger NOEs observed between H21 and one
of the N-methyl groups permit assignment of H27 as the
downfield methyl singlet, since this group is syn to the
methine hydrogen H21 and closer in space than H28. H20
is correlated to H21 which is eclipsed by the carbonyl
oxygen of C19. Interestingly, reciprocal NOEs between
H27 and H28 are positive, since these methyl groups are
exchanging positions, even though slowly relative to the
NMR time scale.
tively large upfield chemical shifts for C11, C13, and C15
in the homochiral mixture are consistent with the shield-
ing caused by a parallel arrangement of the two aromatic
rings. C10 is probably shielded by the dinitrobenzoyl ring
but is simultaneously deshielded since it is attached to
the hydrogen bond donating amide group, C8 and H9.
These offsetting effects apparently result in a small net
downfield shift.
By contrast, changes in the 13C chemical shifts in the
(S)-3-(R)-2 mixture are small to nonexistent.
In tr a m olecu la r NOESY Exp er im en ts. Observed
intramolecular two-dimensional NOE correlations for
protons in 2 and (S)-3 are presented in Table 3.
In ter m olecu la r NOESY Exp er im en ts. Observed
NOE correlations in the more stable (S)-2-(S)-3 complex
are summarized in Table 4.
The intensities of the off-diagonal correlations were
rated on a relative scale (from very, very strong to very
weak) on the basis of the maximum peak volumes
achieved. H24 and H25 are combined in Tables 3 and 4
because even though these methyl groups are distin-
guished as separate resonances in all spectra, they gave
identical cross-correlations in every instance.
In the system under discussion, information obtained
from the intramolecular NOE enhancements is useful in
determining the average (most populated) conformations
of the components, as free and complexed species. In the
chiral solvating agent 3, strong correlations between H9
and H7 and between H7 and H6a suggest that the
Z-rotamer about the anilide bond is preferred and that
H9 is synperiplanar to H7. This is supported by the fact
that H4b is deshielded by the anilide carbonyl oxygen
(C8) and appears downfield relative to H4a. N-(Acyl)-
prolines are known to populate both conformations which
result from rotation about the ring nitrogen to C3 bond.
Both rotamers have been observed by HPLC and by
NMR.12,13 However, all NMR evidence related to the
Many protons in the complex show significant inter-
molecular NOESY enhancements which are consistent
with the originally proposed chiral recognition mecha-
nism. Reciprocal NOEs are abundant between protons
on both aromatic rings, including the methyl protons
H14, indicating the close alignment of these groups
required for effective face-to-face π-π overlap. Both H9
and H11 show correlations to H27 and H28, the two
methyl groups on (S)-2, suggesting that these groups are
in close proximity to the 3,5-dimethylanilide owing to the
hydrogen bond from H9 to the carbonyl oxygen of C26.
This arrangement also places H27 in the vicinity of H6a
and H6b. H9 shows further correlations to H17 and H20,
suggesting that the two amide groups are close neighbors.
H20, in turn, shows correlations to H11 and to H9. There
are reciprocal NOEs between many of the protons on the
isobutyl group of (S)-2 and the tert-butyl group of (S)-3
(H1). This is significant because it verifies approach of
(S)-2 to that face of the selector which presents the C3
(13) (a) J ocobsen, J .; Melander, W.; Vaisnys, G.; Horvath, C. J . Phys.
Chem. 1984, 88, 4536-4542. (b) Gustafsson, S.; Eriksson, B.; Nilsson,
I. J . Chromatogr. 1990, 506, 75-83.
(14) (a) Madison, V.; Schellman, J . Biopolymers 1970, 9, 511-567.
(b) Garner, R.; Watkins, W. B. Chem. Commun. 1969, 386. (c) Maia,
H. L.; Orrell, K. G.; Rydon, H. N. Chem Commun. 1971, 1209-1210.

4774 J . Org. Chem., Vol. 61, No. 14, 1996
Pirkle et al.
carbonyl oxygen for hydrogen bonding to H20. All of the
observed intermolecular NOEs increase in intensity
(become more negative) as the temperature is lowered
because of more extensive complexation.
Intermolecular NOEs are lacking where, according to
the mechanistic hypothesis, there should be little interac-
tion between the components (S)-3 and (S)-2. For
example, no intermolecular correlations are observed to
or from protons H4a, H4b, H5a, or H5b.
Significantly, no intermolecular NOEs are observed in
the (R)-2-3 mixture. While the absence of an observed
NOE should not be taken as evidence that no interactions
occur, this does suggests that, if present, such complex-
ation makes a negligible contribution to the time-
weighted average of all processes affecting these compo-
nents.15
Con clu sion
Two-dimensional nuclear Overhauser effect spectro-
scopy appears to be a worthwhile probe in investigating
the nature of diastereomeric complexes formed in the
course of chiral recognition, particularly if high levels of
enantioselectivity are present in the system under study.
In this instance, these experiments appear to be entirely
consistent with the mechanism initially proposed on the
basis of the examination of space-filling molecular models
and supported by extensive chromatographic data. The
chromatographic separation of enantiomers will continue
to function as an effective screen in the search for new
systems to study by NMR. Likewise, direct NMR evi-
dence for complexation, particularly NOESY-derived
information about the more stable diastereomeric com-
plexes, will become increasingly important in the design
of improved chiral selectors for the chromatographic
separation of enantiomers.
(15) A reviewer suggested that intermolecular interatomic distances
from the accompanying crystallographic study be cited to further
support the conclusions drawn from the NOE study. A complete listing
of these distance is available from author S.W.R. (see following paper
in this issue). The observed NOEs are time-averaged values and are
influenced by the extent of association. For protons in symmetrical
rotors, the major portion of the NOE is presumably generated during
the periods in which the proton(s) in the rapidly spinning group must
closely approach the NOE partner. This is a consequence of the 1/r6
dependence of NOE magnitude upon the distance between the protons
involved. From the lengthy list of intermolecular H-H distances
obtained from the crystallographic study, one notes that strong NOEs
are observed only between those protons which are within about 4 Å
of each other in the solid state complex.
Ack n ow led gm en t. This work was supported by a
research grants from the National Science Foundation
and by funds from Eli Lilly and Glaxo Research Labo-
ratories. The authors thank the Amoco Corporation in
Naperville, IL, for the use of the Varian 500 MHz
spectrometer, and D.J .R. thanks the Amoco Corporation
for providing a sabbatical opportunity.
J O951255B