A modification of a conventional technique for the synthesis of hydrazones of racemic carbonyls: Prevention of spontaneous chiral inversion

Article abstract of DOI:10.1039/c5ra19904b

Conventionally hydrazones and other derivatives of carbonyls are synthesized under acidic conditions when spontaneous chiral inversion is a common problem if the carbonyl compound is chiral. A new method has been developed involving solid phase microwave-assisted conditions for the synthesis of 2,4-dinitrophenyl hydrazone(s) of chiral carbonyl compounds wherein there occurred no inversion of configuration. The method provided high yields (91-95%) in short reaction times (4-6 min). The method proposed clearly has synthetic advantages over current practices. The hydrazones were characterized by IR, ¹H NMR and CHN analysis. The hydrazones represent enantiomeric pairs tagged with a strong chromophore rather than diastereomers. The enantiomeric pairs were separated by HPLC using an α₁-acid glycoprotein column and the best resolution of all the analytes was achieved with a mobile phase containing 0.5% 2-propanol in 10 mM citrate phosphate buffer at pH 6.5. The chromatographic peaks clearly showed base line separation with comparable peak areas and thus the results confirmed that there was no spontaneous inversion of configuration during derivatization. The chromatograms corresponding to the products obtained by conventional procedure (from the racemic mixtures of analytes) showed peaks with unequal areas suggesting formation of enantiomers in unequal amounts (i.e., non racemic mixtures) because of spontaneous inversion of the configuration during derivatization.

Full text of DOI:10.1039/c5ra19904b

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A modification of a conventional technique for the
synthesis of hydrazones of racemic carbonyls:
prevention of spontaneous chiral inversion
Cite this: RSC Adv., 2015, 5, 105719
*
Manisha Singh and Ravi Bhushan
Conventionally hydrazones and other derivatives of carbonyls are synthesized under acidic conditions when
spontaneous chiral inversion is a common problem if the carbonyl compound is chiral. A new method has
been developed involving solid phase microwave-assisted conditions for the synthesis of 2,4-dinitrophenyl
hydrazone(s) of chiral carbonyl compounds wherein there occurred no inversion of configuration. The
method provided high yields (91–95%) in short reaction times (4–6 min). The method proposed clearly
has synthetic advantages over current practices. The hydrazones were characterized by IR, 1H NMR and
CHN analysis. The hydrazones represent enantiomeric pairs tagged with a strong chromophore rather
than diastereomers. The enantiomeric pairs were separated by HPLC using an a1-acid glycoprotein
column and the best resolution of all the analytes was achieved with a mobile phase containing 0.5%
2-propanol in 10 mM citrate phosphate buffer at pH 6.5. The chromatographic peaks clearly showed
base line separation with comparable peak areas and thus the results confirmed that there was no
spontaneous inversion of configuration during derivatization. The chromatograms corresponding to the
products obtained by conventional procedure (from the racemic mixtures of analytes) showed peaks
with unequal areas suggesting formation of enantiomers in unequal amounts (i.e., non racemic mixtures)
because of spontaneous inversion of the configuration during derivatization.
Received 26th September 2015
Accepted 1st December 2015
DOI: 10.1039/c5ra19904b
www.rsc.org/advances
enolization in two directions. In saturated molecules, the eno-
late from the less substituted side is favoured in base catalysed
1. Introduction
reaction while in acid the more substituted enol is preferred. It
is the more resonance-stabilized enol or enolate that is always
preferred.
It may not be an exaggeration to state that the carbonyl group is
the centerpiece of organic chemistry. It is not only present itself
in most of the main functional groups with multiple bond from
carbon to heteroatom but it also serves as a model for reactions
of all functions with p-bonds between dissimilar atoms. Besides,
its modes of reaction are simple and are very versatile in terms of
synthetic applications. Compounds having a primary amino
group cause a nucleophilic addition at the electron decient
carbonyl carbon of aldehydes and ketones with a subsequent
condensation step to replace the carbonyl oxygen by nitrogen
(iC]O / iC]N + H2O). Notable among such reagents are
phenyl-, p-nitrophenyl-, and 2,4-dintrophenyl-hydrazines which
are used much more oen and give the corresponding hydra-
zones with most aldehydes and ketones.1 These make excellent
derivatives as sharp melting solids (and having characteristic IR
spectra), useful for characterization of the parent aldehydes or
ketones.
Enolization causes inversion of conguration at the asym-
metric a-carbon to carbonyl since the enol has no asymmetry.
The most stable conguration at the a-carbon will result from
equilibration by enolization. In addition reactions to carbonyl
carbon, the asymmetry present on the carbon a- to carbonyl
makes the less hindered side of the carbonyl p-orbital more
accessible and may cause spontaneous congurational inver-
sion resulting into enantiomeric mixtures (which are oen not
predictable).
Enantioresolution of chiral aliphatic or alicyclic aldehydes
and ketones by direct approach using liquid chromatography
would require the presence of a suitable chromophore for on-
line detection in UV-visible region. Direct enantioresolution
based on reversible diastereomeric association between solute
enantiomers and chiral stationary phases (CSPs) offers the
advantages of simple chromatographic runs and absence of
kinetic resolution and racemization over indirect method of
chiral separation.2,3 Among the various nucleophilic reagents
containing amino group 2,4-dinitrophenyl hydrazine is the one
that could act as an achiral strong chromophore for carbonyl
compounds for its DNP moiety.
Such derivatizations (including D-exchange and bromina-
tion) proceed via enol formation which is rate determining (and
are generally acid catalyzed). Ketones offer a choice of
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee – 247667,
India. E-mail: rbushfcy@iitr.ernet.in; manishasingh2283949@gmail.com; Fax: +91-
1332-286202; Tel: +91-1332-285795
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carbonyl compound (used either as an enantiomerically pure
sample or a racemic mixture) undergoing derivatization with an
amino group containing nucleophilic reagent has not been
investigated.
1.1. DNP-derivatives
Since the pioneering work of Sanger4 on the preparation of
dinitrophenyl (DNP) derivatives of amino acids their use for
sequence analysis has declined in the modern times. Never-
theless, DNP moiety attracted attention for its application, as
a strong chromophore, for synthesis of several chiral deriva-
tizing reagents (CDRs) which have been used for separation and
detection of diastereomers of a variety of pharmaceutically
important racemic compounds.5–7
1.3. Present work
Taking into account the literature, as noted above, and the
references cited therein the objective of the present report has
been to develop method of synthesis of derivatives (DNP-
hydrazones in the present case) of certain didactic racemic
carbonyl compounds (four ketones and two aldehydes I–VI,
Fig. 1) which would proceed without spontaneous chiral inver-
sion, and to establish ‘racemic’ and ‘non-racemic’ composition
of the products obtained under newly developed method, and
the products obtained by derivatization of the same racemic
carbonyl compound under conventional acidic conditions. To
achieve the objective, (a) solid phase microwave-assisted
conditions were developed for synthesis of 2,4-dinitrophenyl
hydrazone(s) (DNPHz) of certain didactic chiral carbonyl
compounds, and (b) the DNPHz derivatives of racemic carbonyl
compounds were then resolved by chiral HPLC into enantio-
mers using a1-AGP column. The emphasis was not on devel-
oping a method for enantioseparation but it was to verify the
‘racemic’ and ‘non-racemic’ nature of the product. And the
novelty of the present work lies in the above said two aspects.
Literature reports on the importance of carbonyl compounds
in organic synthesis or in pharmaceutical industry or the
methods of their enantioresolution have not been discussed
because the focus of the present paper is to report the aspects
mentioned above.
1.2. Enantioresolution of chiral carbonyl compounds
Literature reveals sporadic reports on enantioresolution of
chiral carbonyl compounds involving application of DNP
moiety or hydrazone derivative. These include indirect enan-
tioresolution using CDRs developed from 1,5-diuoro-2,4-dini-
trobenzene8 wherein the DNP moiety serves as a chromophore
for on-line detection of the corresponding diastereomers. CSP
derived from (S)-1-(6,7-dimethyl-1-naphthyl) isobutylamine was
used for resolution of cyclic and acyclic chiral ketones as their
oxime 3,5-dinitrophenyl carbamates.9 A chiral phosphorylhy-
drazine reagent was used to prepare hydrazone diastereomers
of chiral ketones which were analyzed by 31P NMR and HPLC.10
Some other reports include resolution of chiral cyclic
ketones by direct approach using CSPs based on amylose
tris(3,5-dimethylphenyl carbamates) and cellulose tris(3,5-
dimethylphenyl carbamates),11 cellulose tribenzoate,12 and
b-cyclodextrin.13,14 Enantioresolution of Wieland–Miescher
ketones, their C(5) homologue, and their C(1) dioxolane deriv-
atives has been reported15 using commercially available CSPs
like cellulose tris-(3,5-dimethyl-phenylcarbamate), native
b-cyclodextrin, and acetylated, carboxymethylated and per-
methylated b-cyclodextrins. Direct enantioresolution of Man-
nich ketones has been achieved by using aqueous copper(II)
acetate and L-aspartame16 and, cellulose and cyclodextrin
derivatives17 as chiral mobile phase additives.
2. Experimental
2.1. Apparatus
The HPLC system of Waters (Milford, MA, USA) was used that
Thus, it is evident that till now the scientic issue with consisted of a 515 HPLC pump, a Waters 2489 UV-vis dual
respect to spontaneous congurational inversion of any chiral wavelength detector, high pressure binary gradient pump
Fig. 1 Structures of chiral aldehydes and ketones.
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control module II, a manual injection valve, an empower2
operating soware (build number, 2154). Other equipment
used were Microwave-Multiwave 3000 (800 W, Perkin-Elmer,
Shelton, CT, USA), a1-AGP (L ꢀ I.D. 10 cm ꢀ 4 mm, 5 mm
particle size) column from Chromtech Merck (Darmstadt, Ger-
many), pH meter Cyberscan 510 (Singapore), Polarimeter
2.4. Characterization of the hydrazones
Melting points were determined in open ended glass capillaries
and were uncorrected. The hydrazones were characterized by IR,
1H NMR and CHN analysis; the data is given below.
2,4-DNPHz of (ꢁ)-3-methylcyclohexanone (1). Color: yellow;
ꢂ
mp 103 ꢁ 2 C; UV (lmax, 365 nm, MeOH): 365; IR (KBr): 3308
¨
P-3002 (Kruss, Hamburg, Germany), FT-IR spectrometer 1600
(NH), 1617 (C]N), 1590 (Ar); 1H NMR: d 9.15 (1H, s, ArH),
d 8.31–8.30 (1H, d, ArH), d 8.00–7.98 (1H, d, ArH), d 1.07 (3H, d),
d 2.07 (1H, m), d 1.36 (2H, m), d 1.98 (2H, m), d 1.58 (2H, m),
d 11.2 (1H, s); anal. calcd for C13H16N4O4: C, 53.42%; H, 5.52%;
N, 19.17%. Found: C, 53.22%; H, 5.30%; N, 19.08%.
(Boardman, OH, USA), Vario EL III elementar analyzer, and
Shimadzu UV-1601 spectrophotometer (spectra were recorded
in MeOH). 1H NMR spectra were recorded on a Bruker 500 MHz
instrument using CDCl3 as the solvent.
2,4-DNPHz of (ꢁ)-2-methylcyclopentanone (2). Color: yellow;
mp 105 ꢁ 2 ꢂC; UV (lmax, 363 nm, MeOH); IR (KBr): 3416 (NH),
2.2. Chemicals and reagents
1
1619 (C]N), 1511 (Ar); H NMR: d 10.8 (1H, s), d 9.15 (1H, s,
(ꢁ)-2-Methylcyclopentanone; (ꢁ)-2-methylcyclohexanone; (ꢁ)-3-
methylcyclohexanone, (ꢁ)-3-methyl-2-pentanone, (ꢁ)-2-methyl-
butyraldehyde, (ꢁ)-2-phenylpropionaldehyde and 2,4-dini-
trophenyl hydrazine (2,4-DNPH) were obtained from Sigma-
Aldrich (St. Louis, MO, USA). All other analytical-grade chem-
icals, HPLC grade solvents such as acetonitrile (MeCN) and
silica gel 60 were also from E. Merck (Mumbai, India). Double
distilled water puried (18.2 MU cm3) with Milli-Q system of
Millipore (Bedford, MA, USA) was used throughout.
ArH), d 8.31 (1H, d, ArH), d 8.01–7.99 (1H, d, ArH), d 1.28 (3H, d),
d 2.76 (2H, m), d 2.10 (2H, m), d 2.24 (2H, m), d 2.41 (1H, m);
anal. calcd for C12H14N4O7: C, 51.80%; H, 5.07%; N, 20.13%.
Found: C, 51.62%; H, 4.95%; N, 19.99%.
2,4-DNPHz of (ꢁ)-3-methyl-2-pentanone (3). Color: orange;
ꢂ
mp 98 ꢁ 2 C; UV (lmax, 361 nm, MeOH); IR (KBr): 3290 (NH),
1
1617 (C]N), 1516 (Ar); H NMR: d11.04 (1H, s), d 9.13 (1H, s,
ArH), d 8.31–8.29 (1H, d, ArH), d 7.98 (1H, d, ArH), d 2.51 (1H, q),
d 1.98 (3H, s), d 1.66 (2H, m), d 1.18 (3H, d), d 0.93 (3H, t); anal.
calcd for C12H16N4O4: C, 51.42%; H, 5.75%; N, 19.99%. Found:
C, 51.11%; H, 5.52%; N, 19.46%.
2.3. Synthesis and characterization of dinitrophenyl
hydrazones (2,4-DNPHz)
2,4-DNPHz of (ꢁ)-2-methylcyclohexanone (4). Color: yellow-
brown; mp 112 ꢁ 2 ꢂC; UV (lmax, 364 nm, MeOH); IR (KBr):
3320 (NH), 1621 (C]N), 1586 (Ar); 1H NMR: d 9.14 (1H, s, ArH),
d 8.30–8.29 (1H, d, ArH), d 8.00–7.98 (1H, d, ArH), d 2.00 (1H, m),
d 1.06 (3H, d), d 1.37 (2H, m), d 1.86 (2H, m), d 1.24 (3H, d), d 1.58
(2H, m), d 11.2 (1H, s); anal. calcd for C13H16N4O4: C, 53.42%; H,
5.52%; N, 19.17%. Found: C, 53.2%; H, 5.23%; N, 19.01%.
2,4-DNPHz of (ꢁ)-2-methylbutyraldehyde (5). Color: yellow-
brown; mp 95 ꢁ 2 ꢂC; UV (lmax, 361 nm, MeOH); IR (KBr):
3287 (NH), 1621 (C]N), 1516 (Ar); 1H NMR: d 9.17 (1H, s, ArH),
d 8.30 (1H, d, ArH), d 8.00–7.98 (1H, d, ArH), d 10.9 (1H, s), d 1.21
(6H, d), d 1.48 (2H, m), d 9.87 (1H, d); anal. calcd for
Solid phase microwave-assisted approach. Representative
synthesis of 2,4-DNPHz of (ꢁ)-3-methyl-2-pentanone and char-
acterization data of all the resulting six hydrazones is given
below.
Synthesis of 2,4-DNPHz of (ꢁ)-3-methyl-2-pentanone (3). 2,4-
DNPH (0.019 g; 10 mmol) and (ꢁ)-3-methyl-2-pentanone
(0.010 g; 10 mmol) were dissolved in MeOH (10 mL) followed
by addition of silica gel (6 g) to this solution. Aer about
20 minutes, the solvent was evaporated and the silica gel (on
which the two reactants were adsorbed) was irradiated with MW
in an oven at 500 W for 4 min (with 1 min interval). The MW
irradiated silica gel was then stirred in ethyl acetate (10 mL) for
10 min and then ltered. The residual silica gel was washed
twice with 5 mL ethyl acetate; the combined extract was
concentrated under the stream of nitrogen and was le for
crystallization. The yields were in the range of 91–95%. The
hydrazones obtained, as the product, under experimental
conditions (i) were designated as (1–6). These derivatives were
analysed by chiral HPLC.
Conventional acid catalyzed synthesis. 2,4-DNPH (0.019 g; 10
mmol) was dissolved in 10 mL MeOH, in a small conical ask;
as a representative, solution of (ꢁ)-3-methyl-2-pentanone (0.010
g; 10 mmol) in 10 mL MeOH was added to it. Concentrated
sulphuric acid was then added drop by drop with constant
stirring till pH 4 was obtained. The reaction mixture was
allowed to stand for 10 min. Formation of corresponding
derivative as 2,4-DNPHz occurred during this time. It was
ltered and recrystallized from MeOH. The yields were in the
range of 78–82%. The hydrazones obtained, as the product,
under experimental condition (ii) were analysed by chiral HPLC.
C
11H14N4O4: C, 49.62%; H, 5.30%; N, 21.04%. Found: C,
49.43%; H, 5.21%; N, 20.98%.
2,4-DNPHz of (ꢁ)-2-phenylpropionaldehyde (6). Color:
yellow; mp 98 ꢁ 2 ꢂC; UV (lmax, 362 nm, MeOH); IR (KBr): 3290
(NH), 1617 (C]N), 1518 (Ar); 1H NMR: d 9.30 (1H, s, ArH), d 8.31
(1H, d, ArH), d 8.00 (1H, d, ArH), d 10.8 (1H, s), d 7.28 (2H, m),
d 7.98 (2H, m), d 7.26 (1H, m), d 1.24 (3H, d), d 2.59 (1H, m),
d 10.1 (1H, m); anal. calcd for C15H14N4O4: C, 57.32%; H, 4.49%;
N, 17.83%. Found: C, 57.17%; H, 4.32%; N, 17.44%.
Stock solutions. (i) Solutions of 2,4-DNPHz of each of the six
carbonyl compounds were prepared in 2-propanol at a concen-
tration of 10 mM and then diluted to a nal concentration of
0.1 mM.
(ii) Citrate phosphate buffer was prepared using 0.1 M
solution of citric acid and 0.2 M solution of dibasic sodium
phosphate.18
Chiral HPLC of reaction products. The composition of
mobile phase for achieving enantioresolution was optimized by
using binary mobile phase system consisting of citrate
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phosphate buffer (in the concentration range 5–25 mM, and pH condensation step (replacing the carbonyl oxygen by nitrogen)
3.5–6.5) and 2-propanol (in the range 0.5% to 3.0%) or MeCN (in is shown in Fig. 3.
1
the range 1 to 5%) at a ow rate of 1 mL minꢃ1. Mobile phase
The characterization data based on IR, H NMR and CHN
was ltered through a 0.45 mm lter and degassed by sonication analysis does not differentiate in the enantiomeric ratio of the
and passing nitrogen before use. 20 mL of the sample was products obtained from the two approaches, as the products are
injected onto the column. Detection was at 365 nm.
structurally and chemically the same. In order to investigate the
issue of spontaneous congurational inversion chiral HPLC of
the products obtained under conditions (i) and (ii) was per-
formed; racemic and non-racemic nature of products (enan-
tiomeric composition) was a decisive factor.
3. Results and discussion
The reaction of racemic carbonyl compounds (I–VI) with the
reagent (2,4-DNPH) does not lead to formation of diastereomers
as the reagent is achiral. There occurs ‘tagging’ of enantiomers
with a strong chromophore in the form of DNP moiety of 2,4-
3.1. Determination of racemic and non-racemic nature of
products by chiral HPLC
DNPH. The scheme showing synthesis of derivatives is given in HPLC analysis of the products obtained by approach (ii) showed
Fig. 2. The hydrazones obtained, as the product, under experi- peaks with unequal areas and this observation led to inference
mental condition (i) were designated as (1–6) and are also that the product was non-racemic though the reactant carbonyl
shown in Fig. 2.
compound was racemic in nature. Since the products are
The reaction under condition (ii) requires nearly pH 4 for formed via enol and the enol (formed in rate determining step
maximum rate while basic or highly acidic conditions lower the involving asymmetric carbon a- to carbonyl group) is a planar
rate. In more strongly acid solution (pH < 3.5) the unshared pair moiety it receives H from either side (side ‘a’ or side ‘b’ shown in
of electrons (the nucleophilic site) of N is protonated and is no Fig. 3, depicting mechanism) congurational inversion occurs
more a nucleophile.19 It was interesting to observe that addition and the product hydrazone is a non racemic mixture. It was,
of sulphuric acid (till pH 4 is obtained) to the mixture of 2,4- therefore, contended that spontaneous inversion of congura-
DNPH and the carbonyl compound resulted into higher yield of tion was taking place during derivatization under acidic
the product hydrazone in comparison to an approach in which conditions. A representative chromatogram with unequal areas
sulphuric acid was added at rst to the solution of 2,4-DNPH corresponding to the products of the analyte (III) is shown in
followed by addition of the solution of carbonyl compound.20
Fig. 4.
It was further conrmed by the observation that the products
The problem or the question of spontaneous congurational
inversion in each of the enantiomers (present in the racemic corresponding to analyte (I), i.e., (ꢁ)-3-methylcyclohexanone
mixture of the carbonyls) cannot be overruled when the did not show peaks with unequal areas. It was because in the
synthesis of hydrazones was taking place in acidic liquid molecule (I, Fig. 1) the carbon a- to carbonyl is not asymmetric
medium (ii). As a result the ratio of the two enantiomers is ex- and it is not involved in the formation of enol.
pected to get disturbed resulting into possibly a non-racemic
On the other hand, the chromatograms corresponding to the
mixture of hydrazones of the chiral carbonyls under study. products obtained under solid state microwave-assisted condi-
Congurational inversion would occur only when the asym- tions, approach (i), clearly showed base line separation with
metric carbon (a- to carbonyl function) is involved in the comparable peak areas (as provided by the system soware).
formation of enol. The possible mechanism for enol formation Sections of chromatograms showing baseline resolution of all
and congurational inversion at the asymmetric a-carbon to the six enantiomeric pairs of hydrazones, corresponding to
carbonyl and the formation of hydrazone in the subsequent approach (i), are shown in Fig. 5; a full chromatogram as
Fig. 2 Scheme showing synthesis of hydrazones.
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Fig. 3 The possible mechanism for the formation of hydrazones via enol formation.
Fig. 4 Full chromatogram (as representative) showing resolution of
product (3) obtained by approach (ii). The peak areas were 386 and 691
mAU at retention time 9.09 and 11.32 min, respectively. Peaks with
unequal areas indicate non-racemic nature.
a representative is given as Fig. 6. In approach (i) silica gel
allowed convenient workup. It served as a very efficient adsor-
bent with a large surface area for homogeneous heating and
thus facilitated faster reactions with short reaction times and
higher yields. The catalytic amount of acid could probably have
been provided by the silica gel (having adsorbed water) and the
MWI triggered reaction being very fast provided no opportunity
for spontaneous chiral inversion during derivatization.
It can thus be concluded that each of the products (1–6) was
Fig. 5 Sections of chromatograms showing resolution of enantio-
meric pairs of six 2,4-DNPHz (retention times are in minutes). Column,
a1-AGP (L ꢀ I.D, 10 cm ꢀ 4 mm, 5 mm particle size); mobile phase,
0.5% 2-propanol in 10 mM citrate phosphate buffer at pH 6.5; flow
rate, 1.0 mL minꢃ1; detection, 365 nm.
a racemic mixture of hydrazones (i.e., the tagged enantiomers)
of the corresponding racemic carbonyl compound. Thus, the
chiral HPLC results clearly veried that there was no congu-
rational inversion of the chiral carbonyl compounds when they
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Chromatographic separation data for resolution of the six
pairs of enantiomers (1–6) in the form of 2,4-DNPHz is given in
Table 1. Table 1 shows the values of enantioselectivity, reso-
lution and retention time (in terms of a, Rs and k1) obtained by
using mobile phase, 0.5% 2-propanol in citrate phosphate
buffer (10 mM, pH 6.5). Varying buffer concentration above or
below 10 mM (pH 6.5) resulted in decrease in enantiose-
lectivity (Fig. 7). The enantiomers were not resolved using only
the citrate phosphate buffer (10 mM, pH 6.5) as the mobile
phase; a base line resolution of all the six analytes was
observed aer addition of 2-propanol to it at a level of 0.5%. A
further increment (by a value of 0.5% at a time) in the
concentration of 2-propanol up to 3% caused a decrease in
resolution. 2-Propanol was found to be a better organic
modier in comparison to MeCN as lower enantioselectivity
and resolution (a and Rs) and higher retention time were ob-
tained by using MeCN in the mobile phase. Increment in the
pH of mobile phase (by a value of 0.5 at a time in the range of
3.5 to 6.5) resulted in increase of a, Rs and k1 for all the six pairs
of enantiomeric hydrazones; thus nally pH 6.5 was found to
be the best (Fig. 8).
Fig. 6 Full chromatogram (as representative) showing resolution of
product (3) obtained by approach (i). The peak areas were 402 and
403 mA U at retention time 9.03 and 11.2 min, respectively. Peaks with
equal areas indicate racemic nature.
were derivatized with 2,4-DNPH under the solid state condi-
tions using MWI. The DNP moiety of 2,4-dinitrophenyl
hydrazine serves as a strong chromophore and a suitable
substrate for inclusion phenomenon with the chiral material of
the a1-acid glycoprotein (a1-AGP) column for enantiomeric
resolution.
3.2. Separation mechanism
Over the pH range 3.5 to 6.5, AGP bears a net negative charge.
Electrostatic interactions along with hydrogen bonding play
Table 1 Chromatographic data for direct resolution of six pairs of enantiomers in the form of 2,4-DNPHz of racemic aldehydes and ketonesa
2,4-DNPHz of racemic aldehydes and ketones
(1)
(2)
(3)
(4)
(5)
(6)
k1
a
Rs
k1
a
Rs
k1
a
Rs
k1
a
Rs
k1
a
Rs
k1
a
Rs
5.60
1.42
4.31
5.56
1.56
5.82
5.45
1.44
4.43
5.66
1.46
4.84
5.80
1.48
5.10
5.84
1.49
5.21
a
Chromatographic conditions: column, a1-AGP (L ꢀ I.D. 10 cm ꢀ 4 mm, 5 mm particle size), mobile phase, 0.5% 2-propanol in citrate phosphate
buffer (10 mM, pH 6.5); ow rate, 1.0 mL minꢃ1; detection at 365 nm; k1, retention factor of rst eluting enantiomer; a, separation factor; Rs,
resolution. (1–6) represent 2,4-DNPHz of chiral aldehydes and ketones as mentioned in experimental (Section-2.3). The data presented in the
table are the mean values of three independent experiments. 2,4-DNPHz synthesized by approach (i).
Fig. 7 Effect of buffer concentration (in the range 5–25 mM) in mobile phase consisting 0.5% 2-propanol at pH 6.5, on enantioselectivity.
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Fig. 8 Effect of pH of mobile phase (in the range 3.5–6.5) on enantioselectivity, resolution and retention times (in terms of a, Rs and k1,
respectively) for product (2) using mobile phase 0.5% 2-propanol in 10 mM citrate phosphate buffer at pH 6.5.
important role in the chiral discrimination on an AGP column.21 experimental results conrmed that there was no congurational
Effect of change of pH on enantioresolution of the analytes inversion of any of the chiral carbonyl compounds. The study is
using AGP column (as noted above) can be attributed to the an important step not only for derivatization of enantiomeric
involvement of coulombic interactions between the analytes carbonyls without spontaneous inversion of conguration (during
and the immobilized protein as the overall charge of the protein synthesis) but also for direct enantioresolution of several chiral
and potential conformational changes are pH dependent.22,23 carbonyl compounds via introducing an achiral chromophore.
Lowering of pH from 6.5 to 3.5 caused a decrease in the net
negative charge of the protein that resulted in a reduced elec-
trostatic attraction of cationic dinitrophenyl hydrazones with
the immobilized protein resulting in decrease of retention time,
Acknowledgements
enantioselectivity and resolution (Fig. 8).
The authors are grateful to the University Grants Commission
AGP is also able to bind a variety of hydrophobic compounds
(UGC), New Delhi, for the award of a senior research assis-
due to interactions with an apolar cavity formed by the folding
tantship (to MS).
of the secondary structure of AGP.24 DNP moiety serves as
a strong chromophore and is also a suitable substrate for
inclusion phenomenon with the chiral material of the AGP
References
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sion phenomenon and ionic interactions and/or reversible
changes in the protein conformation are held responsible as the
main factors for enantiomeric separation of the said dini-
trophenyl hydrazones on AGP column.
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