Chromatographic determination of the absolute configuration of an acyclic secondary alcohol using difluorodinitrobenzene

Article abstract of DOI:10.1021/ac0003394

A chromatographic determination method was developed for the absolute configuration of an acyclic secondary alcohol using the characteristic functions of 1,5-difluoro-2,4-dinitrobenzene (FFDNB). This method relies on the formation of the fixed favorable c

Full text of DOI:10.1021/ac0003394

Anal. Chem. 2000, 72, 4142-4147
Chromatographic Determination of the Absolute
Configuration of an Acyclic Secondary Alcohol
Using Difluorodinitrobenzene
Ken-ichi Harada,* Youhei Shimizu, Atsuko Kawakami, Makiko Norimoto, and Kiyonaga Fujii
Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan
consist of two processes, the freezing of the conformation of a
resulting diastereomer and the recognition of the desired confor-
mation based on the influence of the NMR anisotropy effect.
However, some of the drawbacks of this method were pointed
out. To the best of our knowledge, no suitable method has been
developed for the first process; therefore, one must always discuss
the conformation of the resulting diastereomer. For example,
Riguera et al. pointed out that MTPA esters are constituted by
three main conformers in close populations and that methoxyphen-
ylacetic acid (MPA) is superior to MTPA based on the results of
extensive conformational studies.2 Although they recommended
a single derivatization method using MPA in combination with
low-temperature measurement, they also needed to discuss the
detailed conformation.3
A chromatographic determination method was developed
for the absolute configuration of an acyclic secondary
alcohol using the characteristic functions of 1 ,5 -difluoro-
2 ,4 -dinitrobenzene (FFDNB). This method relies on the
formation of the fixed favorable conformation of a second-
ary alcohol-DLA (2 ,4 -dinitrophenyl-5 -leucinamide) de-
rivative and its recognition by ODS silica gel. The sec-
ondary alcohol reacted first with FFDNB under mild basic
conditions, and
L-leucinamide or DL-leucinamide was
then introduced into the secondary alcohol-FDNB deriva-
tive. Because the conformations of the resulting alcohol-
DLA derivatives were rigidly fixed by the dinitrobenzene
plane, the absolute configuration at the asymmetric car-
bon of the secondary alcohol tested can be definitely
deduced by the elution behavior of both of the diastere-
omers in the HP LC and/ or LC/ MS. One of the diastere-
omers has the cis (Z)-type arrangement of two more
hydrophobic substituents of the alcohol and leucinamide
moieties, the more hydrophobic side chain and isobutyl
groups, to the plane of the dinitrobenzene, whereas
another diastereomer has the opposite arrangement (trans
(E)-type). Therefore, the cis arrangement interacts more
strongly with the ODS silica gel and has a longer retention
time than that of the trans-type arrangement. This estab-
lished method was successfully applied to various chiral
acyclic secondary alcohols including chloramphenicol and
4 -hydroxyphenyllactic acid. Finally, the limitations of this
method were also examined.
The “advanced Marfey’s method” has been developed to
nonempirically determine the absolute configuration of constituent
amino acids in a peptide using LC/ MS.4,5 This method consists
of Marfey’s method6 as a chromatographic technique for the
separation of amino acids into each enantiomer, the detection of
the amino acid by mass spectrometry, and a procedure for
obtaining the corresponding enantiomer from either the
L
- or
- and
-amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5- -leu-
-FDLA) is due to the difference in their hydrophobicity,
D
-amino acid. In this method, the resolution between the L
D
L
cinamide (
L
which is derived from the cis (Z)-type or trans (E)-type arrange-
ment of two more hydrophobic substituents. Therefore, the
L-amino acid is usually eluted before the corresponding D-amino
acid.7 The separation mechanism for the resolution of both
resulting diastereomers is based on a rigidly fixed conformation
assisted by the intramolecular hydrogen bonding between the
nitro groups and R-amino groups of the amino acid and leucin-
amide moiety. In addition to this method, DL-FDLA derivatization
was introduced for obtaining the corresponding enantiomer on
Although there are several determination methods such as
X-ray analysis, circular dichroism (CD), and NMR spectral
techniques for the determination of the absolute configuration of
secondary alcohols, a modified Mosher’s method has been widely
used for the past decade because of its universality and acces-
sibility.1 This method is based on the derivatization of a compound
to be tested with the two enantiomers of a chiral anisotropic
reagent, methoxytrifluoromethylphenylacetic acid (MTPA) and
comparison of the chemical shifts of the resulting diastereomers.
This methodology using NMR spectroscopy can be regarded to
(2) Latypov, S. K.; Seco, J. M.; Quinoa´, E.; Riguera, R. J. Org. Chem. 1 9 9 6 , 61,
8569-8577.
(3) Latypov, S. K.; Seco, J. M.; Quinoa´, E.; Riguera, R. J. Am. Chem. Soc. 1 9 9 8 ,
120, 877-882.
(4) Harada, K.-I.; Fujii, K.; Mayumi, T.; Hibino, Y.; Suzuki, M.; Ikai, Y.; Oka, H.
Tetrahedron Lett. 1 9 9 5 , 36, 1515-1518.
(5) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K.-I. Anal. Chem. 1 9 9 7 , 69,
* Corresponding author: (tel) 81-52-832-1781; (fax) 81-52-834-8780;
(e-mail:) kiharada@meijo-u.ac.jp.
(1) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa H. J. Am. Chem. Soc. 1 9 9 1 ,
113, 4092-4096.
5146-5151.
(6) Marfey, P. Carlsberg Res. Commun. 1 9 8 4 , 49, 591-596.
(7) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K.-I. Anal. Chem.
1 9 9 7 , 69, 3346-3352.
4142 Analytical Chemistry, Vol. 72, No. 17, September 1, 2000
10.1021/ac0003394 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/01/2000

the chromatogram,8 and its utility has been successfully extended
from amino acids to primary amines.9
eter. 1H chemical shifts were referenced to the residual methanol-
d4 signal (δ 3.30 ppm) and DMSO-d6 (δ 2.49 ppm). UV spectra
were obtained using a Shimadzu (Kyoto, Japan) UV-2100 or a
model 991J photodiode array detector during HPLC operation
described below.
In a previous paper, we demonstrated that an NMR spectro-
scopic method using the anisotropy effect can determine the
absolute configuration of the R-carbon of a secondary alcohol.10
In this method, a secondary alcohol was derivatized first with 2,4-
dinitro-1,5-difluorobenzene (FFDNB) to yield an alcohol-FDNB
derivative, which led to the desired 2,4-dinitrophenyl-5-phenyl-
ethylamine (DPEA) derivative after derivatization with 1-phenyl-
ethylamine. The resulting conformation of the alcohol-DPEA
derivative was fixed by means of the dinitrobenzene plane and
was quite similar to those of the 2,4-dinitrophenyl-5-leucinamide
(DLA) derivatives of the primary amines including the amino acids
mentioned above. Consequently, the configuration of a secondary
alcohol was effectively determined by this method. These results
strongly indicate that the same methodology can be applied to
the case when HPLC is used as the recognition method instead
of NMR spectroscopy. In this paper, we describe a procedure for
the determination of the configuration of an acyclic secondary
alcohol using HPLC and/ or LC/ MS.
Analysis by HP LC and LC/ MS. HPLC was performed using
a Tosoh (Tokyo, Japan) dual-pump delivery system. Separation
was carried out on a TSK gel ODS-80Ts column (150 × 4.6 mm
i.d., Tosoh) maintained at 40 °C. Acetonitrile-0.01 M trifluoro-
acetic acid (TFA) was used as the mobile phase under a linear
gradient elution mode (acetonitrile, 30-80%, 50 min). The flow
rate was 1 mL/ min with UV detection at 340 nm and 250-500
nm by photodiode array detection. LC/ MS was performed below.
The separation was carried out on a Develosil ODS-HG-5 column
(150 × 2.0 mm i.d., Nomura Chemical, Seto, Japan) maintained
at 40 °C using a HP1050 (Hewlett-Packard, Novi, MI). Acetoni-
trile-water containing 0.01 M TFA was used as the mobile phase
under a linear gradient elution mode at a flow rate of 0.2 mL/
min. The mass spectrometer was a Finnigan TSQ7000 (Finnigan-
Mat, San Jose, CA). All mass spectra were aquired using Q1 as
the scanning quadrupole. The ESI voltage was 4.5 kV with the
auxiliary and sheath gas nitrogen pressure set at 5 units and 70
psi, respectively, and the capillary was heated to 200 °C.
EXPERIMENTAL SECTION
Chemicals.
D- and L-hydroxyphenyllactic acid (Hpla) were
generous gifts from Dr. M. Murakami (Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Japan).
FFDNB, leucinic acid, 1-aminopentane, and 2-aminophenylethane
were purchased from Tokyo Kasei Co., Tokyo, Japan. Triethyl-
amine and chloramphenicol were obtained from Nacalai Tesque,
Kyoto, Japan. Leu and DL-Hpla were purchased from Sigma, St.
Louis, MO. Leucine amide was obtained from Kokusan Chemicals,
Tokyo, Japan. 2-Hydroxybutane and 2-hydroxyhexane were pur-
chased from Aldrich, Milwaukee, WI. 2-Aminobutane, 2-amino-
pentane, 2-aminoheptane, and 1-aminophenylpropane were ob-
tained from Hydrus Chemical, Inc., Japan. 2-Hydroxyheptane,
2-hydroxyoctane, and 1-hydroxyphenylethane were purchased
from AZmax, Kisarazu, Japan. All other reagents and solvents were
of the analytical grade.
P reparation of the DLA Derivative of a Secondary Alcohol.
The secondary alcohol tested was dissolved in dichloromethane
and triethylamine at 40 °C and FFDNB was added to the solution,
which was then allowed to stand for 24 h. The reaction mixture
was evaporated to dryness, and the residue was applied to an ODS
cartridge to remove excess reagent. The cartridge was washed
with 35% acetonitrile (aq) and the desired 2,4-dinitro-5-fluoroben-
zene (FDNB) derivative was eluted with acetonitrile. The eluate
was subjected to preparative TLC using an appropriate solvent
RESULTS AND DISCUSSION
Although a secondary alcohol was directly derivatized with
FDLA, the desired secondary alcohol derivative could not be
prepared. On the basis of the reactivity of FFDNB, the reaction
sequence was changed to obtain the desired secondary alcohol
derivative. That is, the tested secondary alcohol reacted first with
FFDNB to yield quantitatively the secondary alcohol-FDNB
derivative, and then leucinamide was introduced into the resulting
secondary alcohol-FDNB for the recognition (Figure 1). The
derivatization procedure for secondary alcohols was finally opti-
mized as follows: the secondary alcohol reacted with FFDNB and
triethylamine (molar ratio of 1:8:8) in dichloromethane at 40 °C
for 24 h. To remove the excess FFDNB, the reaction mixture was
applied to an ODS silica gel cartridge. The resulting secondary
alcohol-FDNB derivative reacted with leucinamide in 1% triethyl-
amine-acetonitrile at 40 °C for 1 h. After elimination of the excess
reagents, the desired DLA derivatives were subjected to ordinary
HPLC analysis under reversed-phase conditions.
To confirm the conformations of the resulting alcohol-FDNB
and -DLA derivatives, the nuclear Overhauser effect (NOE)
experiments were performed. In the difference NOE spectrum of
the (R)-1-hydroxyphenylethane-FDNB derivative as the typical
chiral secondary alcohol, a strong NOE (12.8%) was observed
between the H-6 of FDNB and the R-proton of the secondary
alcohol. This spectral behavior is consistent with that of (R)-1-
aminophenylethane, in which the strong NOE (11. 5%) was also
observed between the H-6 of FDNB and the R-proton of the
primary amine. These experiments indicated that the R-proton of
the alcohol is spatially located near H-6 of the benzene ring and
the conformations of the alcohol and amine are very similar. The
same results were obtained from the NOE experiment on the DLA
derivatives of (R)-1-hydroxyphenylethane and (R)-1-aminophen-
ylethane. Namely, strong NOEs (∼15%) were observed between
the R-protons of the alcohol or amine tested and leucinamide of
DLA, and the H-6 of the benzene ring (Figure 2), indicating that
system. The FDNB derivative reacted with L-leucinamide in 1%
triethylamine-acetonitrile at 40 °C for 1 h, and the reaction
mixture was evaporated to dryness. The residue was cleaned with
a silica gel cartridge, and the desired DLA derivative was then
subjected to HPLC or LC/ MS.
1
Spectral Measurement. H NMR and NOE difference spectra
were recorded on a JNM-A 400 (JEOL, Tokyo, Japan) spectrom-
(8) Harada, K.-I.; Fujii, K.; Hayashi, K.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron
Lett. 1 9 9 6 , 37, 3001-3004.
(9) Fujii, K.; Shimoya, T.; Ikai, Y.; Oka, H.; Harada, K.-I. Tetrahedron Lett. 1 9 9 8 ,
39, 2579-2582.
(10) Harada, K.-I.; Shimizu, Y.; Kawakami A.; Fujii, K. Tetrahedron Lett. 1 9 9 9 ,
40, 9081-9084.
Analytical Chemistry, Vol. 72, No. 17, September 1, 2000 4143

Figure 1. Derivatization procedure for determination of the absolute configuration of an acyclic secondary alcohol.
Figure 2. (a) 1H NMR and (b) difference NOE spectra of the DLA derivative of (R)-1-hydroxyphenylethane.
the primary amine-DLA has the desired conformation for the
resolution, its spectrum always shows clearly the two absorption
maximums, which are derived from the bridge chromophore
between the nitro groups of the nitrobenzene and the amino
groups of the primary amine and L
-leucinamide.7 However, the
UV spectrum of (R)-1-hydroxyphenylethane-DLA is characterized
by two shoulders at 390 and 320 nm and an absorption maximum
at 290 nm (Figure 4c) and is completely different from that of the
primary amine-DLA. This spectral behavior was similar to that of
the superposition of FDLA (λmax 340 nm, λshoulder 390 nm) (Figure
4b) and (R)-1-hydroxyphenylethane-FDNB (λmax 280 nm) (Figure
4d), indicating that the absorption maximum at 415 nm in the
UV spectrum of the primary amine-DLA is due to the hydrogen
bonding between the nitro group at C-2 of the nitrobenzene and
the amino group of the introduced primary amine. The appearance
of this absorption maximum depends on the presence or absence
of the second hydrogen bonding. Although this hydrogen bonding
is absent in the case of the secondary alcohol-DLA, its conforma-
tion is rigidly fixed as shown in Figure 3, which was confirmed
by the NOE experiment. Because the UV spectrum is readily
available using a photodiode array detector during HPLC analysis,
it is useful to check the desired conformation.
Figure 3. Predominant conformations of the DLA derivatives of (a)
(R)-1-hydroxyphenylethane and (b) (R)-1-aminophenylethane ob-
tained from the NOE experiment.
both of the R-protons are spatially situated near H-6 of the benzene
ring in the alcohol-DLA derivative (Figure 3). In addition, we had
confirmed that this predominant conformation is also formed in
the case of more complicated cyclic alcohols.10
The UV spectral method was very useful for examining the
desired conformation for the resolution of the primary amine
derivatized with FDLA.7 The UV spectrum of (R)-1-aminophenyl-
ethane-DLA shows the characteristic spectral behavior including
the absorption maximums at 415 and 340 nm (Figure 4a). When
4144 Analytical Chemistry, Vol. 72, No. 17, September 1, 2000

groups, relative to the plane of the dinitrobenzene, whereas the
(R)-diastereomer has the opposite arrangement (trans (E)-type)
(Figure 5). Therefore, the cis arrangement interacts more strongly
with the ODS silica gel and has a longer retention time than that
of the trans-type arrangement.7
This method was first applied to leucine and leucinic acid to
examine the chromatographic behavior of the DLA derivative of
amino acid and its corresponding alcohol. We had shown that
the DLA derivative of a hydrophobic neutral amino acid can be
well resolved using this method. Indeed, the peaks of the
derivatives of
respectively, on the chromatogram. Further, the derivatives of
and -leucinic acid were eluted at 19.2 and 26.2 min, respectively,
L- and D-leucine appeared at 19.6 and 26.9 min,
L-
D
on the chromatogram. This chromatographic behavior including
the retention time and the time difference of both isomers of
leucinic acid was closely similar to that of leucine. Subsequently,
several acyclic secondary alcohols were examined together with
the corresponding primary amines. Table 1 shows the elution
order, retention time, and time difference in the HPLC analysis
data of (R)- and (S)-2-hydroxyalkane-DLA and the corresponding
primary amine-DLA derivatives under the reversed-phase condi-
tions. All (R)-diastereomers of the alcohols tested were eluted prior
to the (S)-diastereomers without exception. The resolution became
better with an increase in the length of the alkyl chains, and the
retention time also became longer. This chromatographic behavior
for the alcohols was almost the same as that of the primary amines
as shown in Table 1. These results suggested that both diastere-
omers of the alcohols can be resolved through the common
9
mechanism for the primary amines.7,
The “DL-FDLA derivatization” was developed for obtaining the
corresponding enantiomer from an original chiral compound and
has been applied to primary amines including amino acids.5,8
Namely, this method can form a peak of the corresponding
enantiomer of a chiral compound on the chromatogram using a
simple operation, even if the corresponding enatiomer is not
available. Experimentally, a chiral compound was divided into two
portions and the two portions were separately derivatized with
Figure 4. UV spectra of the DLA derivatives of (a) (R)-1-aminophen-
ylethane and (c) (R)-1-hydroxyphenylethane and (b) FDLA and (d)
the FDNB derivative of (R)-1-hydroxyphenylethane.
As mentioned above, this conformation of the secondary
alcohol derivative was consistent with the case of the primary
amine including amino acids (Figure 3). Therefore, the same
separation mechanism as that of the primary amine can be also
depicted for the secondary-DLA as shown in Figure 5. For
example, in the case of 2-hydroxyalkanes, the (S)-diastereomer
has a cis (Z)-type arrangement of the two more hydrophobic
L-FDLA and DL-FDLA. Both of the derivatives were subjected to
HPLC or LC/ MS analysis, and comparison of the original peaks
with the two peaks can determine the correct absolute configu-
ration. This procedure was slightly modified because the reaction
sequence was changed as mentioned above. After the introduction
substituents of the alcohol and L-leucinamide, the R1, and isobutyl
Figure 5. Separation mechanism for the DLA derivatives of secondary alcohols using HPLC.
Analytical Chemistry, Vol. 72, No. 17, September 1, 2000 4145

Table 1. HPLC Analysis Data of Secondary Alcohol-DLA and the Corresponding Primary Amine-DLA Derivativesa.
x ) OH
tR(R)
x ) NH2
tR(R)
DLA derivatives
elution order
tR(S)
∆tR
elution order
tR(S)
∆tR
2-x-butane
2-x-pentane
2-x-hexane
2-x-heptane
2-x-octane
1-x-phenylethane
1-x-phenylpropane
(R) f (S)
(R) f (S)
(R) f (S)
(R) f (S)
(R) f (S)
(R) f (S)
(R) f (S)
21.5
25.0
28.4
31.7
35.1
23.9
27.2
21.8
25.7
29.5
33.1
36.6
25.9
29.1
0.3
0.7
1.1
1.4
1.5
2.0
1.9
(R) f (S)
(R) f (S)
22.6
26.1
23.1
27.1
0.5
1.0
(R) f (S)
33.2
34.9
1.7
(R) f (S)
(R) f (S)
24.3
27.6
26.5
29.5
2.2
1.9
a
tR, retention time (min); ∆tR, tR(S) - tR(R) (min).
Figure 7. Structure of aeruginopeptin 95A.
Recently, many peptides were isolated from terrestrial cyano-
bacteria12 and classified into several groups, one of which is a
19-membered cyclic depsipeptide possessing a 3-amino-6-hydroxy-
2-piperidone (Ahp) moiety. The structure of a typical compound,
aeruginopeptin 95A, is shown in Figure 7.13 Some compounds in
this group have a Hpla moiety as the N-terminal blocking group.
The established method was applied to the determination of the
absolute configuration of Hpla. Aeruginopeptin 95A was hydro-
lyzed under the usual conditions, and the reaction mixture was
partitioned with diethyl ether and water. The residue from the
organic layer was subjected to the derivatization procedure, and
Figure 6. Mass chromatograms at m/z 1023 [M + TFA - H]- of
the (a) L- and (b) DL-DLA derivatives of chloramphenicol analyzed
using ESI LC/MS in the negative ion mode.
of an (S)-2-hydroxyalkane to FFDNB, the resulting FDNB deriva-
tive was derivatized with
was confirmed that the peak of the
hydroxyalkane is completely identical with that of
L
-leucinamide and DL-leucinamide and it
-DLA derivative of (S)-2-
-derivative of
the resulting L- and DL-DLA derivatives were analyzed under the
LC/ MS conditions. Figure 8 shows the mass chromatograms
monitored at m/ z 769 [M - H]- of both of the derivatives. Because
L
D
(R)-2-hydroxyalkane. This chromatographic behavior was consis-
tent with that of the corresponding amino compound, indicating
that this procedure is applicable to a chiral secondary alcohol.9
To confirm the utility of this method including “DL-FDLA
derivatization”, it was applied to chloramphenicol produced by
Streptomyces venezuelae.11 This antibiotic reacted with FFDNB, and
the resulting FDNB derivative was divided into two portions. Each
the
D
-DLA isomer was eluted prior to the
L-DLA isomer, the
absolute configuration was determined as
coincided with that by Shimizu et al.14 and was also confirmed by
comparison of the derivative of authentic samples.
D
(R). This conclusion
To our knowledge, there is only one method using HPLC, the
modified Horeau method,15 for the determination of the absolute
configuration of a chiral secondary alcohol. This method relies
on the kinetic resolution of racemic 2-phenylbutyric anhydride
by the chiral secondary alcohol. Because the absolute configura-
tion was deduced using the ratio of diastereometric N-[1-(1-
naphthyl)ethyl]-2-phenylbutanamides determined by HPLC, the
results were sometimes ambiguous. Indeed, this method had not
been used; instead, a modified Mosher method was developed
portion was separately derivatized with L- or DL-leucinamide, and
the products were analyzed using LC/ MS. The mass chromto-
grams monitored at m/ z 1023 [M + TFA - H]- are shown in
Figure 6, and the D-DLA derivative was eluted prior to the L-DLA
one. Because the benzene moiety is more hydrophobic than the
side chain, the absolute configuration at C-1 can be determined
as R based on the conformation shown in Figure 5. This result
was consistent with the absolute configuration confirmed previ-
ously.
(12) Namikoshi, M.; Rinehart, K. L. J. Ind. Microbiol. 1 9 9 6 , 17, 373-384.
(13) Harada, K.-I.; Mayumi, T.; Shimada, T.; Suzuki, M.; Kondo, F.; Watanabe,
M. F. Tetrahedron Lett. 1 9 9 3 , 34, 6091-6094.
(14) Tsukamoto, S.; Painuly, P.; Young, K. A.; Yang, X.; Shimizu, Y.; Cornell, L.
J. Am. Chem. Soc. 1 9 9 3 , 115, 11046-11047.
(11) Rebstock, M. C.; Crook, H. M.; Contoulis, J.; Bartz, R. J. Am. Chem. Soc.
1 9 4 9 , 71, 2458-2462.
(15) Svatos, A.; Valterova, I.; Fabryova, A.; Vrkoc, J. Collect. Czech. Chem.
Commun. 1 9 8 9 , 54, 151-159.
4146 Analytical Chemistry, Vol. 72, No. 17, September 1, 2000

amines. Usually, 1 nmol of the DLA derivative can be detected
by this method. Therefore, the method is sensitive and its
procedure is easy to handle. However, there are still three
problems to be resolved in this method at present. Although a
preliminary method was proposed for estimating the hydrophobic-
ity of R1 and R2 (Figure 5),9 no appropriate method has been
established so far. Second, it is difficult to apply this method to a
cyclic secondary alcohol because it is impossible to estimate its
hydrophobicity. For example, the L- and D-DLA derivative of (-)-
menthol could be separated (data not shown), but it was hard to
predict the correct configuration based on our separation mech-
anism. Third, we have to shorten the total analysis time of the
present method because it takes a whole day to do the first
derivatization. The improvement of the derivatization is in progress.
CONCLUSIONS
In the present study, we tried to establish a nonempirical
determination method for the absolute configurations of an acyclic
secondary alcohol using the characteristic functions of FFDNB
in combination with the usual reversed-phase HPLC. This method
relies on the formation of the fixed favorable conformation of a
secondary alcohol-DLA derivative and its recognition by ODS silica
gel. As a result of extensive experiments, it was found that this
method can be successfully applied to an acyclic secondary alcohol
in the same manner as in the case of primary amines. Through
the previous and present studies, we have developed a total system
for the determination of the absolute configuration of secondary
alcohols and primary amines, in which a fixed conformation is
formed using the characteristic functions of FFDNB and the
resulting conformation is recognized by NMR spectroscopy or
HPLC. The methodology is being further extended.
Figure 8. Mass chromatograms at m/z 769 [M - H]- of the (a) L-
and (b) DL-DLA derivatives of Hpla analyzed using ESI LC/MS in the
negative ion mode.
and has been widely used since early 1990.1 To use this method,
we must prepare at least 1 mg of both of the MTPA derivatives of
a secondary alcohol, must assign any 1H signals of the derivative,
and then must calculate the difference in the chemical shift of
each proton in the both isomers. Therefore, this method is not
sensitive and its procedure is time-consuming. Further, this
method does not often give a definite conclusion because of
undesirable conformation. In such a case, a detailed conformation
analysis is required.
In the present study, we developed a chromatographic method
for the determination of the absolute configuration of an acyclic
secondary alcohol as mentioned above. This method is based on
the formation of the fixed favorable conformation of a secondary
alcohol-DLA derivative and its recognition by ODS silica gel, which
can be applied to acyclic secondary alcohols with the aid of mass
spectrometry in the same manner as in the case of primary
ACKNOWLEDGMENT
The authors thank Dr. Kenji Matsuura, Santen Pharmaceutical
Co., for the ESI LC/ MS measurement.
Received for review March 22, 2000. Accepted June 22,
2000.
AC0003394
Analytical Chemistry, Vol. 72, No. 17, September 1, 2000 4147