Application of cyanuric chloride-based six new chiral derivatizing reagents having amino acids and amino acid amides as chiral auxiliaries for enantioresolution of proteinogenic amino acids by reversed-phase high-performance liquid chromatography

Article abstract of DOI:10.1007/s00726-011-0832-3

Six dichloro-s-triazine (DCT) reagents having L-Leu, D-Phg, L-Val, L-Met, L-Ala and L-Met-NH₂ as chiral auxiliaries in cyanuric chloride were introduced for enantioseparation of 13 proteinogenic amino acids. Four other DCTs and six monochloro-s

Full text of DOI:10.1007/s00726-011-0832-3

Amino Acids (2012) 42:1371–1378
DOI 10.1007/s00726-011-0832-3
ORIGINAL ARTICLE
Application of cyanuric chloride-based six new chiral derivatizing
reagents having amino acids and amino acid amides as chiral
auxiliaries for enantioresolution of proteinogenic amino acids
by reversed-phase high-performance liquid chromatography
•
Ravi Bhushan Shuchi Dixit
Received: 7 October 2010 / Accepted: 6 January 2011 / Published online: 19 January 2011
ꢀ Springer-Verlag 2011
Abstract Six dichloro-s-triazine (DCT) reagents having
L-Leu, D-Phg, L-Val, L-Met, L-Ala and L-Met-NH2 as chiral
auxiliaries in cyanuric chloride were introduced for enan-
tioseparation of 13 proteinogenic amino acids. Four other
DCTs and six monochloro-s-triazine (MCT) reagents
having amino acid amides as chiral auxiliaries were also
synthesized. These 16 chiral derivatizing reagents (CDRs)
were used for synthesis of diastereomers of all the 13
analytes using microwave irradiation, which were resolved
by reversed-phase high-performance liquid chromatogra-
phy (RP-HPLC) using C18 column and gradient eluting
mixture of aqueous TFA and acetonitrile with UV detec-
tion at 230 nm. It required only 60–90 s for derivatization
using microwave irradiation. Better resolution and lower
retention times were observed for the diastereomers pre-
pared with CDRs having amino acids as chiral auxiliaries
as compared to counterparts prepared with reagents having
amino acid amides as chiral auxiliaries. As the best reso-
lution of all the 13 analytes was observed for their
diastereomers prepared using the DCT reagent having
L-Leu as chiral auxiliary, this CDR was further employed
for derivatization of Lys, Tyr, His and Arg followed by
RP-HPLC analysis of resulting diastereomers. The results
are discussed in light of acid and amide groups of chiral
auxiliaries constituting CDRs, electronegativities of the
atoms of achiral moieties constituting CDRs and hydro-
phobicities of side chains of amino acids constituting
CDRs and analytes.
Keywords Cyanuric chloride Á Amino acids Á Chiral
derivatizing reagents Á Reversed-phase high-performance
liquid chromatographic separation Á Enantioseparation
Introduction
The sequential and controlled substitution of chlorine
atom(s) by nucleophiles in CC (cyanuric chloride; 2,4,6-
trichloro-1,3,5-triazine; s-triazine chloride; trichloro-s-tri-
azine) provides chiral monochloro-s-triazine (MCT) and
dichloro-s-triazine (DCT) reagents. Bru¨ckner and co-
workers synthesized chiral MCT reagents and used them
for enantioseparation of a few selected amino acids by
reversed-phase high-performance liquid chromatography
(RP-HPLC) (Bru¨ckner and Strecker 1992; Bru¨ckner and
Wachsmann 2003a, b). Enantioresolution of carbonyl
compounds using tailor-made chiral derivatizing reagents
(CDRs) based on CC has been reported in literature
(Kempter et al. 2000). CC has also been applied to bind
different amines or amino acids as chiral selectors on solid
supports to prepare a series of chiral stationary phases
(CSPs) for enantioresolution of amino acids and amino
alcohols (Bru¨ckner and Wachsmann 1996; Chen and Lin
1995; Iuliano et al. 1997; Lecci and Iuliano 2005; Li et al.
2005; Lin et al. 1996a, b, c, 2001; Lin and Lin 1994; Lin
and Yang 1993; Oi et al. 1984, 1996). Chiral separation of
racemic mixtures of amino acids by means of micellar
electrokinetic chromatography after derivatization with
3-(4,6-dichloro-1,3,5-triazinylamino)-7-dimethylamino-2-
methylphenazine (DTDP) has also been reported (Ma et al.
2002).
R. Bhushan (&) Á S. Dixit
Certain MCT and DCT reagents have been used as
CDRs for indirect enantioresolution of a-amino acids
(Bhushan and Agarwal 2010; Bhushan and Kumar 2008),
Department of Chemistry, Indian Institute of Technology
Roorkee, Roorkee 247667, India
e-mail: rbushfcy@iitr.ernet.in
123

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R. Bhushan, S. Dixit
(R,S)-baclofen (Bhushan and Dixit 2010a) and (R,S)-mex-
iletine (Bhushan and Dixit 2010b) at this laboratory. The
diastereomers, of compounds possessing amino groups,
prepared with s-triazine reagents show structural similari-
ties with those prepared with Marfey’s reagent (Bhushan
and Martens 2010).
Experimental
Apparatus
The HPLC system consisting of a 10 mL pump head 1000,
manager 5000 degasser, photodiode array detection (PDA)
system 2600, manual injection valve, and Eurochrom
operating software was from Knauer (Berlin, Germany).
Other equipments used were Microwave-Multiwave 3000
(800 W, Perkin-Elmer, Shelton, CT, USA), pH meter
Cyberscan 510 (Singapore, Singapore), Polarimeter P-3002
(Kruss, Hamburg, Germany), Milli-Q system of Millipore
(Bedford, MA, USA), Perkin Elmer 1600 FT-IR spec-
trometer (Boardman, OH, USA), Vario EL III elementar
analyzer, and Shimadzu UV-1601 spectrophotometer
Keeping in view the above cited literature, two new
DCT reagents (having L-Met and L-Met-NH2 as chiral
auxiliaries) were synthesized and used along with four
other DCT reagents (having L-Leu, D-Phg, L-Val, L-Ala as
chiral auxiliaries) for the first time for enantioseparation of
13 proteinogenic amino acids. Besides, four DCT and six
MCT reagents (Bhushan and Kumar 2008) were also used
as CDRs in the present study to compare the separation
efficiencies of the newly introduced CDRs. The DCT
reagent having L-Leu as chiral auxiliary was further
employed for derivatization of Lys, Tyr, His and Arg fol-
lowed by RP-HPLC analysis of the resulting diastereomers.
The separation results are discussed in the light of (a) acid
and amide groups of chiral auxiliaries of CDRs, (b) elec-
tronegativities of the atoms of achiral moieties constituting
CDRs and (c) hydrophobicities of side chains of amino
acids constituting CDRs and analytes. To the best of
authors’ knowledge, this is the first report on microwave
(MW)-assisted synthesis of diastereomers of DL-amino
acids with the aforementioned 16 CDRs (Fig. 1) followed
by their RP-HPLC resolution.
1
(spectra were recorded in acetonitrile). H NMR spectra
were recorded on a Bruker 500 MHz instrument using
dimethyl sulfoxide (DMSO-d6) as deuterated solvent.
Chemicals and reagents
All racemic and chirally pure amino acids, and chirally
pure amino acid amides were obtained from Sigma-Aldrich
(St Louis, MO, USA). All other analytical-grade chemicals
and HPLC solvents were from E. Merck (Mumbai, India).
Double-distilled water purified with a Milli-Q system
(18.2 MX mL) was used throughout.
Fig. 1 Structures of chiral derivatizing reagents (CDR 1–16)
123

Application of cyanuric chloride-based six new CDRs
Preparation of stock solutions
1373
Yield: 92%; color: white; UV (nm, in MeCN): 231
(kmax); IR (KBr): 3,419, 2,972, 1,698, 1,639, 1,620, 1,573,
1,479, 1,376, 1,218, 1,134, 1,046, 814, 618 cm-1; 1H NMR
(DMSO-d6): d 1.96–2.04 (m, 2H, –CH2–), 2.03 (s, 3H, –S–
CH3), 2.46–2.51 (m, 2H, –CH2–S), 4.37–4.43 (m, 1H,
–CH–N), 8.56–8.68 (dd, 1H, –NH). Anal. calcd for
C8H10Cl2N4O2S: C, 32.33%; H, 3.39%; N, 18.85%. Found:
C, 32.34%; H, 3.40%; N, 18.88%.
Stock solutions of all racemic and chirally pure amino
acids (100 mM) were prepared in 1 M HCl for derivati-
zation reactions. Solutions of DCT and MCT reagents
(10 mM) were prepared in acetonitrile and dimethyl sulf-
oxide (DMSO), respectively. Stock solutions of NaHCO3
(1 M) and HCl (1 M) were prepared in purified water. All
solutions were filtered through a 0.45 lm filter prior to use.
N-(4-Chloro-6-methoxy-[1,3,5]triazine-2-yl)-L-methionine
amide (CDR 9)
Synthesis of chiral derivatizing reagents
Three sets of CDRs (sets A–C) were synthesized (Fig. 1).
Set A consisting of five DCT reagents having amino acids
as chiral auxiliaries (CDR 1–5) was synthesized by
nucleophilic substitution of one of the chlorine atoms in
CC with amino acids, namely, L-Leu, D-Phg, L-Val, L-Met
and L-Ala, respectively. Set B consisting of five DCT
reagents having amino acid amides as chiral auxiliaries
(CDR 6–10) was synthesized by nucleophilic substitution
of one of the chlorine atoms in CC with amino acid amides,
namely, L-Leu-NH2, D-Phg-NH2, L-Val-NH2, L-Met-NH2
and L-Ala-NH2, respectively.
Yield: 93%; color: white; UV (nm, in MeCN): 232 (kmax);
IR (KBr): 3,422, 2,970, 1,694, 1,635, 1,616, 1,578, 1,475,
1,371, 1,219, 1,136, 1,045, 816, 620 cm-1 1H NMR
;
(DMSO-d6): d 1.99–2.03 (m, 2H, –CH2–), 2.05 (s, 3H, –S–
CH3), 2.47–2.52 (m, 2H, –CH2–S), 4.38–4.41 (m, 1H,
–CH–N), 7.11 (s, 1H, –CONH2), 7.48 (s, 1H, –CONH2),
8.57–8.69 (dd, 1H, –NH). Anal. calcd for C8H11Cl2N5OS:
C, 32.44%; H, 3.74%; N, 23.65%. Found: C, 32.46%; H,
3.75%; N, 23.68%.
MW-assisted synthesis of diastereomers
Set C consisted of six MCT reagents (CDR 11–16) having
amino acid amides as chiral auxiliaries. Five reagents of this
set (CDR 11–15) were synthesized by nucleophilic substi-
tution of one of the chlorine atoms in 6-methoxy derivative
of CC with amino acid amides, namely, L-Leu-NH2, D-Phg-
NH2, L-Val-NH2, L-Met-NH2 and L-Ala-NH2, respectively.
CDR 16 was obtained by substitution of two chlorine atoms
in CC with L-Val-NH2 and L-Phe-NH2, respectively. Syn-
thesis, characterization and determination of enantiomeric
purity of the reagents were carried out as per literature
(Bhushan and Dixit 2010b; Bhushan and Kumar 2008;
Bhushan and Martens 2010). However, representative syn-
thetic procedure for CDR 4 and characterization data for
newly synthesized reagents (CDR 4 and 9) are given below.
The diastereomers of DL-amino acids were synthesized
under MW irradiation. Reaction conditions were investi-
gated using MW irradiation at 75–90% power in a time
range of 50–100 s, one- to fivefold molar ratio of CDRs
and pH range of 8–10 maintained by 1 M NaHCO3. For
quantitative yield, reactions of representative racemic
amino acids, leucine (aliphatic) and phenylalanine (aro-
matic) were optimized with CDR 1 and CDR 11 as rep-
resentatives of DCT and MCT reagents, respectively. The
optimized conditions for derivatization were pH around
8.0, twofold molar excess of CDRs, and MW irradiation of
60 and 90 s at 85% power (of 800 W) using DCT and MCT
reagents, respectively. The diastereomers of all the analytes
were also synthesized by conventional heating for 3 h at
30ꢁC using DCT reagents, and 1 h at 80ꢁC using MCT
reagents, as per literature report (Bhushan and Kumar
2008) for comparison in the present study. A 10 lL volume
of the solution, containing diastereomers, was diluted 10
times with MeCN, and 20 lL of it was injected onto the
column.
N-(4,6-Dichloro-[1,3,5]triazine-2-yl)-L-methionine
(CDR 4)
L-Methionine (746 mg, 5 mmol) was dissolved in 10 mL
of Na2CO3 solution (1 M) and maintained at 0–5ꢁC. Ace-
tone (50 mL) was added to the solution and allowed to
stand for temperature equilibration (20ꢁC). A solution of
CC (922 mg, 5 mmol) in acetone (30 mL) was added with
vigorous stirring. The reaction mixture was then stirred at
20ꢁC for 1 h and water (30 mL) was added and acetone
was removed under reduced pressure. The product began to
crystallize as acetone was removed. The precipitate was
filtered and washed with ice cold water. The filtrate was
extracted with chloroform and evaporated to dryness in
vacuo to give another crop of product.
Literature (Blotny 2006; Ma et al. 2002) reveals that the
substitution pattern of three chlorine atoms of CC can be
controlled by appropriate reaction conditions (temperature,
time, solvent, etc.) and the order of addition of reactants in
the reaction mixture. Substitution of a single chlorine atom
of DCT reagents under optimized derivatization conditions
was confirmed by thin layer chromatography (TLC). The
reaction, e.g. of CDR 1 (that contains L-Leu moiety as
chiral auxiliary) with DL-Val gives the diastereomers of the
123

1374
R. Bhushan, S. Dixit
type, [L-L-] and [L-D-]; the first letter refers to the config-
uration of the chiral auxiliary of the CDR and the second to
that of the analyte. An analysis of Table 1 clearly indicates
that the CDRs used in the present work gain advantages
over several other CDRs in terms of mild derivatization
conditions and excellent stability of derivatives.
182 lg/mL). These studies were also repeated on three
consecutive days to determine inter-day precision. Signal-
to-noise ratio of 3:1 and 10:1 were used for estimating the
detection and quantification limit, respectively.
Results and discussion
HPLC analysis
HPLC analysis
A LiChrospher C18 (250 mm 9 4.6 mm I.D., 5 lm par-
ticle size) column from Merck (Darmstadt, Germany) was
used for HPLC. The successful mobile phases were
(I) eluent A [MeCN(100 mL) ? H2O(900 mL)] and eluent
B [MeCN(800 mL) ? H2O(200 mL)], both containing
0.1% TFA; gradient 100% A to 100% B in 45 min, and (II)
eluent C [MeOH(100 mL) ? H2O(900 mL)] and eluent D
[MeOH(800 mL) ? H2O(200 mL)], both containing 0.1%
TFA; gradient 100% C to 100% D in 45 min, at a flow rate
of 1 mL/min with UV detection at 230 nm. In mobile
phase I linear gradients of eluent B of 0–100, 5–100,
10–100, 15–100 and 20–100% in 45 min were applied to
separate the diastereomeric pairs. Besides, effects of flow
rate (1–1.5 mL/min) and TFA concentration (0.01–0.3%)
were also studied.
All the diastereomers were separated by RP-HPLC. The
successful separation conditions, as optimized, were a
linear gradient of eluent B from 0 to 100% in 45 min
(mobile phase I) containing 0.1% TFA at a flow rate of
1 mL/min. MeCN was found to be a better organic modi-
fier in comparison to MeOH as higher resolutions (Rs) and
lower retention times were obtained with the former. The
elution order of diastereomers was confirmed by elution of
diastereomer of a single enantiomer. The L-L-type diaste-
reomers eluted earlier than L-D-counterparts. The D-D-type
of diastereomers eluted earlier than the D-L-type as these
were prepared using CDRs having D-Phg or D-Phg-NH2
moiety as chiral auxiliary (i.e. CDRs 2, 7 and 12). The
appearance of two sets of peaks (i.e. the first set consisting
of two smaller peaks with baseline separation at 19.419 and
20.487 min and the second set consisting of two larger
peaks with baseline separation at 41.888 and 43.064 min)
in the chromatogram obtained from resolution of the dia-
stereomers of Lys prepared with CDR 1 can be attributed to
monosubstituted derivative (as the result of reaction at
a-amino group) and disubstituted derivative (as the result
of reaction at a- and c-amino groups), respectively. The
elution of monosubstituted derivatives followed by disub-
stituted derivatives can be explained on the basis of
Validation procedure
Method validation was performed using diastereomers of
DL-Leu prepared with CDR 1 following ICH guidelines
(ICH 1996). Linearity was established by injecting the
samples in triplicate, containing DL-Leu in the concentra-
tion range of 0.1–200 lg/mL. Intra-day precision was
established by making triplicate injections of five concen-
trations in the above range (i.e. 78, 104, 130, 156 and
Table 1 Comparison of certain derivatization and chromatographic parameters of the diastereomers prepared with different CDRs reported in
literature with the diastereomers prepared with CDR 1 used in the present study
CDR
Label Detection Derivatization
)
Stability of
diastereomers
LOD
Reference
(kmax
(nm)
conditions
´
Peter et al.
(2000a, b)
(S)-N-(4 Nitrophenoxycarbonyl)phenylalanine
methoxyethyl ester (NIFE)
UV
UV
205
250
20 min at RT
30 min at RT
2 weeks
(4ꢁC)
24 h (RT)
1.0 nmol/mL
5.0 ng
2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl
isothiocyanate (GITC)
Kinoshita
et al.
(1981)
4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-
dimethyl-aminosulfonyl)-2,1,3-benzoxadiazole
(DBD-PyNCS)
FL
kexc = 490 20 min at 55ꢁC
kem = 530
24 h (4ꢁC)
0.16–0.75 pmol Jin et al.
(1998)
´
1,3-Diacetoxy-1-(4-nitrophenyl)-2-propyl
isothiocyanate (DANI)
UV
UV
245
230
2 h at 60ꢁC
1 week (4ꢁC) 0.16 nmol/mL Peter et al.
(2000b)
N-(4,6-Dichloro-[1,3,5]-triazine-2-yl)-L-leucine
(CDR 1)
60 s at 85% (800 W)
under MW irradiation
1 month
(5ꢁC)
0.12–0.14
ng/mL
Present
study
RT room temperature
123

Application of cyanuric chloride-based six new CDRs
1375
•
Set C. Diastereomers prepared with the MCT reagent
having L-Leu-NH2 as chiral auxiliary (CDR 11) were
better resolved in comparison to those prepared with
the CDRs 12-15. However, the highest Rs under this
category was observed for the diastereomers prepared
with CDR 16 which has two stereogenic centers (of
L-Val-NH2 and L-Phe-NH2 present as chiral auxilia-
ries). CDRs can be arranged as 16 [ 11 [ 12 [ 13 [
14 [ 15 for the decreasing order of Rs obtained for the
diastereomeric pairs.
increased hydrophobicity of the latter due to presence of
more and bulky hydrophobic groups. Similarly, under the
optimized derivatization conditions, Tyr yielded disubsti-
tuted, and Arg and His yielded monosubstituted derivatives.
The retention factors (k), separation factor (a) and reso-
lution (Rs) of the diastereomers prepared with set A CDRs
are presented in Table 2. Sections of chromatograms
showing some representative separations are shown in
Fig. 2. The main features for the separation of diastereomers
prepared with three sets of CDRs can be summarized as
•
Set A. The highest Rs was observed for the diastereo-
meric pair of DL-Leu prepared with CDR 1 (having
L-Leu as chiral auxiliary). CDRs can be arranged as
1 [ 2 [ 3 [ 4 [ 5 for the decreasing order of Rs
obtained for the diastereomeric pairs.
A comparison of resolution values (Rs) and retention
factors (k2) for the diastereomeric pairs of five amino acids
(Leu, Phe, Met, Thr and Glu as representatives of aliphatic,
aromatic, sulfur containing, basic and acidic amino acids,
respectively) prepared with three reagents (CDR 1, 6 and
11 as representatives of set A, B and C, respectively) is
shown in Fig. 3a and b, respectively. Evaluation of Fig. 3a
indicates that the three sets of CDRs can be arranged as
A [ B [ C for the decreasing order of resolutions for the
diastereomeric pairs. On the other hand, examination of
Fig. 3b reveals that the three sets can be arranged as
•
Set B. The highest Rs was observed for the diastereo-
meric pair of DL-Leu prepared with the CDR having
amide derivative of L-Leu as chiral auxiliary (CDR 6).
CDRs can be arranged as 6 [ 7 [ 8 [ 9 [ 10 for the
decreasing order of Rs obtained for the diastereomeric
pairs.
Table 2 Chromatographic data of diastereomers of proteinogenic amino acids prepared with set A CDRs (DCT reagents having amino acids as
chiral auxiliaries)
CDR 1-(L-Leu)a
kL kD Rs
CDR 2-(D-Phg)a
kD kL Rs
CDR 3-(L-Val)a
kL kD Rs
CDR 4-(L-Met)a
kL kD Rs
CDR 5-(L-Ala)a
kL kD Rs
a
a
a
a
a
Leu
Ile
13.14 14.65 13.34 1.12 12.93 14.14 10.62 1.09 11.32 12.46 10.10 1.10 10.60 11.91 9.59 1.12 7.84 8.90 7.78 1.13
12.51 14.02 13.32 1.12 12.43 13.63 10.54 1.09 10.56 12.20 9.88 1.15 10.18 11.44 9.26 1.12 7.48 8.49 7.36 1.13
11.60 12.85 12.09 1.10 11.11 12.21 10.14 1.09 8.85 10.37 9.67 1.17 8.23 9.44 8.89 1.14 5.92 6.87 7.12 1.16
12.51 13.65 10.04 1.09 12.02 13.10 9.56 1.08 10.83 11.91 9.55 1.09 10.05 11.07 7.49 1.10 7.22 8.06 6.18 1.11
11.78 12.67 10.01 1.07 11.16 12.19 9.16 1.09 9.32 10.36 8.93 1.11 8.39 9.35 7.04 1.11 6.10 6.75 4.76 1.10
10.24 11.75 9.43 1.14 10.25 11.21 8.38 1.08 8.31 9.47 7.56 1.14 8.29 9.24 7.00 1.11 5.41 6.00 4.28 1.10
7.52 8.70 8.63 1.15 6.60 7.64 7.60 1.15 5.41 6.51 7.07 1.20 5.29 6.20 6.67 1.17 1.74 2.36 4.15 1.35
6.39 7.50 8.15 1.17 5.86 6.81 6.96 1.16 3.46 4.46 6.36 1.29 2.87 3.70 6.09 1.28 1.43 1.93 3.61 1.34
5.85 6.56 6.27 1.12 5.95 6.53 5.92 1.09 3.56 4.23 4.95 1.18 2.97 3.63 4.87 1.22 1.12 1.44 2.38 1.29
5.96 6.47 3.20 1.08 5.41 5.66 2.20 1.04 3.04 3.51 1.96 1.15 2.58 2.81 1.67 1.08 1.17 1.39 1.50 1.18
8.83 10.39 10.04 1.17 8.70 10.01 9.22 1.15 6.48 7.86 9.03 1.21 5.58 6.86 7.22 1.22 4.32 5.26 5.76 1.21
4.90 5.31 3.51 1.08 4.85 5.19 2.56 1.07 2.69 2.99 2.03 1.11 2.28 2.56 1.87 1.12 2.22 2.53 1.70 1.13
4.77 5.05 1.40 1.05 4.24 4.48 1.35 1.05 3.26 3.47 1.32 1.06 2.37 2.55 1.29 1.07 1.47 1.60 1.20 1.08
Val
Phe
Trp
Met
Ala
Thr
Glu
Asp
Pro
Ser
Asn
His
7.34 8.19 7.00 1.12 NS
NS
NS
NS
Lys(di) 13.96 14.38 3.47 1.03
Arg 8.09 8.76 5.50 1.08
Tyr(di) 14.02 14.80 6.49 1.05
The CDRs are as mentioned in Fig. 1. Chromatographic conditions: column, LiChrospher C18 (250 mm 9 4.6 mm I.D., 5 lm particle size);
eluent, mobile phase (I) consisting eluent A [MeCN(100 mL) ? H2O(900 mL)] and eluent B [MeCN(800 mL) ? H2O(200 mL)], both con-
taining 0.1% TFA; gradient 100% A to 100% B in 45 min; flow rate, 1.0 mL/min; detection, 230 nm; kL and kD, retention factors of
diastereomers of L- and D-amino acid enantiomers, respectively; a, separation factor; Rs, resolution
NS not studied
a
The chiral auxiliaries constituting the respective CDRs are mentioned in parenthesis
123

1376
R. Bhushan, S. Dixit
Fig. 2 Sections of chromatograms showing resolution of diastereo-
mers of representative amino acids prepared with CDR 1. The L-L-type
diastereomers eluted earlier than L-D-counterparts. Chromatographic
conditions: column, LiChrospher C18 (250 mm 9 4.6 mm I.D., 5 lm
[MeCN(100 mL) ? H2O (900 mL)] and eluent B [MeCN(800 mL) ?
H2O(200 mL)], both containing 0.1% TFA; gradient 100% A to 100%
B in 45 min; flow rate, 1.0 mL/min; absorbance (detection), 230 nm.
The numbers above the peaks refer to retention times (in min)
particle size); eluent, mobile phase (I) consisting eluent
A
Fig. 3 Comparison of resolution, Rs and retention factor, k2, for
diastereomers of five amino acids (Leu, Phe, Met, Thr and Glu as
representatives of aliphatic, aromatic, sulfur containing, basic and
acidic amino acids, respectively) prepared with a CDR 1 (DCT
reagent having L-Leu), b CDR 6 (DCT reagent having L-Leu-NH2)
and c CDR 11 (MCT reagent having L-Leu-NH2), as chiral auxiliaries
Effect of acid and amide groups of chiral auxiliaries
B [ A [ C for the decreasing order of retention factors
(k2) obtained for the last eluting diastereomer.
In conclusion, CDR 1 was considered to be the best
since the diastereomers (of all the analytes) prepared with it
had the highest resolution (Rs) and among all the diaste-
reomers synthesized in the present study, the highest Rs
was observed for the diastereomeric pair of DL-Leu pre-
pared with it.
The superiority of amino acids as chiral auxiliaries over
amino acid amides was established as among the two sets
of diastereomers prepared with DCT reagents, the diaste-
reomers prepared with CDRs having amino acids as chiral
auxiliaries (set A) were observed to have higher resolutions
and lower retention times (in terms of k) as compared to
123

Application of cyanuric chloride-based six new CDRs
1377
Method validation
those prepared with CDRs having amino acid amides as
chiral auxiliaries (set B). The lower retention times of
diastereomers can be attributed to more polarity of acid
variants compared to their amides counterparts.
Linearity
Method validation was done using diastereomers of DL-Leu
prepared with CDR 1. Calibration graphs [peak area (y) vs.
concentration of enantiomer (x), lg/mL] were plotted for
both diastereomers of DL-Leu prepared with CDR 1 in the
range 0.1–200 lg/mL and linear regression equations were
used to determine slopes and correlation coefficients. A
good linear relationship was obtained over this range. The
regression equations were y = 0.0006x ? 0.0013 (R2 =
0.9981) and y = 0.0009x - 0.0015 (R2 = 0.9977) for the
first- and second-eluted diastereomer, respectively.
The above-said explanation may also hold good for the
observation that the diastereomers of Asparagine and
Aspartic acid prepared with CDR 10 (DCT reagent having
L-Ala-NH2 as chiral auxiliary) were not resolved, while
their diastereomers prepared with DCT reagent having
L-Ala as chiral auxiliary (CDR 5) showed a baseline
separation.
Effect of electronegativities of the atoms of achiral moieties
Chlorine atom and methoxy group present as the achiral
moieties in DCT and MCT reagents, respectively, may be
influencing the retention factors; lower retention factors
were observed for the diastereomers prepared with MCT
reagents (set C) as compared to their DCT counterparts (set
B). Under reversed-phase conditions, chlorine (having
electronegativity 3.0 on the Pauling scale) may have
greater affinity with ODS of the column and oxygen atom
in the methoxy group (having electronegativity value of
3.5) would have greater affinity with water present in the
mobile phase, causing faster elution of diastereomers pre-
pared with MCT reagents as compared to those prepared
with DCT reagents.
Accuracy and precision
Replicate HPLC analysis (n = 3) of diastereomers of
DL-Leu (prepared with CDR 1) at five different concen-
tration levels (78, 104, 130, 156 and 182 lg/mL) showed
RSD less than 1.5% in all the cases. RSD for first- and
second-eluted diastereomer varied from 0.40 to 0.86% and
0.47 to 0.75% for intra-day assay precision and 0.64 to
1.40% and 0.75 to 1.30% for inter-day assay precision. The
recovery for first- and second-eluted diastereomer varied
from 98.3 to 98.9% and 98.2 to 98.9% for intra-day assay
and 97.2 to 97.8% and 97.0 to 97.7% for inter-day assay.
LOD was taken as a concentration of the analytes where
S/N was 3 and found to be 0.14 and 0.12 ng/mL for first
and second eluting diastereomers, respectively.
Effect of hydrophobicities of amino acid side chains
Accuracy was determined by investigating the standard
solutions (100 mM) of L-Leu spiked with D-Leu in the range
0.1–1.0%. The results indicate the ability of this method for
the detection of D-Leu in L-Leu up to 0.4% by HPLC.
Bull and Breese (1974) calculated apparent partial specific
volumes of amino acids and constituted their hydropho-
bicity scale; the decreasing order of their hydrophobicity is
Leu (0.842) [ Ile (0.809) [ Val (0.777) [ Phe (0.756) [
Trp (0.731) [ Met (0.709) [ Ala (0.691) [ Thr (0.655) [
Glu (0.632) [ Asp (0.558). The values in parenthesis
represent the apparent partial specific volume. Examination
of chromatographic data reveals that an increment in the
hydrophobicities of the side chains of amino acids consti-
tuting the analytes and the CDRs as well caused longer
retention times and better resolution of resulting
diastereomers.
Conclusion
CDRs based on CC gain advantages over several other
CDRs and many CSPs since these CSPs are not as durable.
The structural features of CC provide a possibility to vary
its hydrophobicity by substituting suitable moieties in it
and make the method quite flexible for resolution of
DL-amino acids in different situations. It further establishes
utility and importance of CC-based CDRs. These made
possible the resolution of diastereomeric pairs of DL-amino
acids, containing acidic, neutral, basic or aromatic side
chains. Besides, CDRs containing opposite enantiomers
(e.g. L-Ala and D-Ala) can be easily prepared in laboratory
with less expenses, while the commercial CDRs are very
costly. Moreover, much less derivatization time using MW
irradiation and improved stability of diastereomers
Separation mechanism
The separation mechanism can be considered to be the
same as proposed in the literature for diastereomers of
certain other analytes (possessing amino group) prepared
with CDRs based on CC (Bhushan and Dixit 2010b;
Bhushan and Kumar 2008). By the same token, the elution
of L–L diastereomer, with the mobile phase, before
L–D diastereomer holds good for the diastereomers inves-
tigated in this study.
´
(Table 1) as compared to other literature reports (Peter
123

1378
R. Bhushan, S. Dixit
Jin D, Nagakura K, Murofushi S, Miyahara T, Toyo’oka T (1998)
Total resolution of 17 DL-amino acids labelled with a fluorescent
chiral reagent R(-)-4-(3-isothiocyanatopyrrolidin-1-y1)-7-(N,N-
dimethylaminosulfonyl-2,1,3-benzoxadiazole, by high-perfor-
mance liquid chromatography. J Chromatogr 822:215–224
et al. 2000a, b; Kinoshita et al. 1981; Jin et al. 1998) were
observed.
Acknowledgments The authors are grateful to the Ministry of
Human Resources Development, Government of India, New Delhi, for
the award of a research assistantship (to SD). Thanks are due to
Alexander von Humboldt Foundation, Bonn, Germany, for donating
Knauer HPLC equipment (to RB). Financial assistance (to RB) from
Council of Scientific and Industrial Research (CSIR), New Delhi, India
(Grant No. 01(2334)/09 EMR-II) is also gratefully acknowledged.
¨
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Lecci C, Iuliano A (2005) Synthesis and evaluation of a new
biselector s-triazine based chiral stationary phase for enantiose-
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