Enantiomeric discrimination using NMR and RP-HPLC 441
°
ꢀ1
experiments. The measurements were made using: (a) a
5 mm inverse probe with a z-gradient coil; (b) the GCSTESL18
HR-DOSY sequence; (c) 15 mmol lꢀ1 solutions of host and
0.25 µm). Injector and detector temperatures were 200 C.
°
°
The temperature program was: 90 C, 5 min/3 C min
/
°
180 C, 5 min. Pressure at the head of the column was 0.6 bar.
Optical rotations were measured with a LEP A2 (Carl
Zeiss) polarimeter, equipped with a sodium lamp (0.005
precision), at 20 C and expressed as [˛]2D0 (concentrations in
°
guest at 30.0 š 0.1 C; (d) the pulse width was 4 ms and the
°
spectral width was typically 4200 Hz and the free induction
decays contained 64 K data points; (e) for all experiments,
25 spectra (32 transients each) were collected with gradient
°
grams/100 ml, solvent).
pulse amplitudes ranging from 0.000 685 to 0.003 427 T cmꢀ1
,
Synthesis
where an approximately 100% decrease in the resonance
intensity was achieved at the largest gradient amplitudes.
The baselines of all arrayed spectra were corrected prior
to processing the data. After data acquisition, each FID
was apodized with 3.0 Hz line broadening and Fourier
transformed. The processing program (the DOSY macro in
a Varian instrument) involves the determination of the peak
heights of all signals above a pre-established threshold and
the fitting of the decay curve for each peak to a Gaussian
function. The DOSY macro was run with data transformed
using fn D 64K. Very crowded spectra were processed in
sections due to the limitation of handling only 512 lines at a
time. The results of the DOSY method of analysis are pseudo
two-dimensional spectra with NMR chemical shifts along
Chemicals and reagents
˛-CD (98%), ˇ-CD (98%) and ꢄ-CD (98%) were purchased from
Acros Organics, Merck and Wacker Chemicals respectively. The
permethylated CDs were obtained according to
a procedure
described in the literature.9 The CDs were lyophilized prior to the
preparation of solutions for NMR spectroscopy. Racemic and pure
substrates were synthesized in our laboratory as described below.
All NMR samples (0.6 ml) were prepared in D2O, 99.9% (Aldrich).
(S)-(C)-3-Hydroxy-2,2-dimethylcyclohexan-1-one, (C)-1
The syntheses of (C)-1 and ꢀšꢁ-1 have been previously reported.12
Spectroscopic data of (C)-1: υH (500 MHz; D2O; HOD) 1.04 (3H,
s, Meax), 1.11 (3H, s, Meeq), 1.68 (1H, m, H-50ax), 1.79 (1H, m, H-4a0 x),
1.92 (1H, m, H-50eq), 2.05 (1H, m, H-4e0 q), 2.39 (1H, ddd, J 5.8, 7.6
and 14.8, H-60ax), 2.46 (1H, ddd, J 5.8, 8.0 and 14.8, H-6e0 q), 3.75 (1H,
dd, J 3.4 and 7.4, H-30); υC (125 MHz; D2O; CCl4) 19.02 (Meax), 19.99
(C-50), 22.15 (Meeq), 27.39 (C-40), 36.78 (C-60), 50.87 (C-20), 77.29 (C-30),
221.77 (C-10).
one axis and calculated diffusion coefficients (m2 sꢀ1 ð 10ꢀ10
)
along the other.
(S)-(C)-3-Acetoxy-2,2-dimethylcyclohexan-1-one, (C)-2
The stoichiometry of host–guest complexes was deter-
mined by the continuous variation method.13 The total
concentration of the interacting species in the solution was
kept constant at 15 mmol lꢀ1 and the molar fraction r of the
guest was varied in the range 0.2–0.8. Apparent binding
constants of the enantiomers of (š)ꢀ1, (š)-2 and (š)-3 with
ˇ-CD were calculated on the basis of Scott’s modification of
the Benesi–Hildebrand equation.14
A solution of ꢀCꢁ-1 (30.0 mg, 0.21 mmol) in pyridine (1.0 ml) was
treated with acetic anhydride (1.0 ml). The mixture was stirred at
room temperature for 20 h. Usual work-up and purification yielded
ꢀCꢁ-2 as a viscous oil (34.8 mg, 98% yield).
[˛]D20 D C10.7 (c 1.87 in CHCl3);ꢅmax (film)/cmꢀ1 2952, 1740,
1713, 1374, 1240, 1045, 988; m/z: 184 (MC, 8%), 142 (12), 124 (20), 82
(45), 55 (22), 43 (100); υH (500 MHz; D2O; HOD) 1.02 (3H, s, Meax),
1.17 (3H, s, Meeq), 1.80 (1H, m, H-50ax), 1.86 (1H, m, H-40ax), 1.92 (1H,
m, H-50eq), 2.05 (3H, s, MeAc), 2.11 (1H, m, H-4e0 q), 2.38 (1H, dtd, J
1.2, 5.7 and 15.1, H-60ax); 2.60 (1H, ddd, J 6.1, 9.8 and 15.1, H-6e0 q),
4.94 (1H, dd, J 3.1 and 6.0, H-30); υC (125 MHz; D2O; CCl4) 19.31
(Meax), 20.10 (MeAc), 20.14 (C-50), 22.49 (Meeq), 24.58 (C-40), 36.72
(C-60), 49.13 (C-20), 80.55 (C-30), 173.45 (COAc), 220.34 (C-10).
Enantiomeric excess was determined by chiral GC (97% ee).
The compound (š)-2 was obtained under identical conditions (98%
yield) and presented the same spectral data as the enantiomerically
pure ꢀCꢁ-2.
Chiral chromatography
HPLC chromatograms were obtained with an HP 1090 M
LC system, which consisted of a ternary gradient pump,
a thermostatic compartment and a diode array detector
(DAD). For single wavelength analyses the DAD was
set at 200–210 nm with a bandwidth of 10 nm, whereas
absorbance spectra were recorded from 200 to 400 nm.
The HPLC analyses were carried out using a Waters
Nova Pack ODS (4 µm, 150 mm ð 3.9 mm i.d) column eluted
with chiral mobile phase (15 mmol lꢀ1) ˇ-CD in at a flow
(S, S)-(C)-3-Hydroxy-2-methyl-2-(2-propynyl)
cyclohexan-1-one, (C)-3
The bacterium S. rubidaea CCT5732 was obtained from the Cul-
ture Collection, Fundac¸a˜o de Pesquisa e Tecnologia Andre´ Tosello
(Brazil). The microorganism was grown at 48 h/28 C/120 rpm
°
in culture shaker-flasks in an appropriate medium (nutrient
broth) and cells were harvest by centrifugation. The reduction of
the 2-methyl-2-(2-propynyl)cyclohexane-1,3-dione, was performed
in 125 ml bottles on a rotatory shaker (120 rpm). To bottles
containing pH 7.0 phosphate buffer (25 ml) and washed cells
(350 mg), the dione substrate was added (60 mg, 0.36 mmol).
rate of 0.5 ml minꢀ1 and a column temperature of 30 C.
°
The injection volume was 2.0 µl of a solution with about
10 mg mlꢀ1 of the mixture in water.
°
The mixture was shaken at 28 C and the reaction was mon-
GC-MS analyses were carried out using an HP-5990/5970
system equipped with an HP-5 fused silica capillary
column (30 m ð 0.25 mm ð 0.25 µm); column temperatures
itored by chiral GC. Upon reaching the appropriate conver-
sion degree, the cells were filtered and the product formed
was extracted from the supernatants with ethyl acetate. The
organic layer was dried over Na2SO4, filtered and evaporated
under reduced pressure. The residue (oil) was purified by col-
umn chromatography on silica gel, eluted with hexane, yield-
ing (S, S)-(C)-3-hydroxy-2-methyl-2-(2-propynyl)cyclohexan-1-one,
(C)-3 (18 mg, 27% yield) and (R, S)-(C)-3-hydroxy-2-methyl-2-(2-
propynyl)cyclohexan-1-one, (C)-3a (21 mg, 33% yield).
were programmed from 80 to 290 C at 15 C minꢀ1. GC–MS
°
°
°
°
injector and detector temperatures were 220 C and 285
C
respectively; the flow rate of helium was 1.16 ml minꢀ1. The
injection volume, 0.5 µl of a solution of about 10 mg mlꢀ1
of the mixture in ethyl acetate, was in split mode. The
mass spectra were determined at 70 eV. Scanning speed was
0.84 scan sꢀ1 from m/z 40 to 550.
Data for (C)-3: [˛]D20 D C7.2 (c 6.0 in CHCl3);ꢅmax (film)/cmꢀ1
3450, 3291, 2945, 2114, 1704, 1458, 1125, 995; m/z 166 (MC, 17%),
122 (39), 109 (90), 107 (43), 106 (29), 95 (80), 93 (84), 91 (100),
77 (35), 43 (38); υH (500 MHz; D2O; HOD) 1.25 (3H, s, Meax),
1.78 (1H, m, H-5a0 x), 1.81 (1H, m, H-4a0 x), 1.93 (1H, m, H-5e0 q),
2.10 (1H, m, H-40eq), 2.32 (1H, dtd, J 1.5, 5.4 and 14.6, H-6a0 x),
2.35 (1H, t, J 2.7, H-90), 2.43 (1H, dd, J 2.7 and 17.0, H-70a), 2.54
Chiral GC analyses were carried out using a GC(FID) HP-
5890 chromatograph, equipped with a heptakis-(2,6-methyl-
3-pentyl)-ˇ-cyclodextrin chiral column (25 m ð 0.25 mm ð
Copyright 2002 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2002; 40: 433–442