A new chiral derivatizing agent for the HPLC separation of α-amino acids on a standard reverse-phase column

Article abstract of DOI:10.1007/s00726-010-0665-5

A new chiral derivatizing agent for α-amino acids is described which leads to diastereomers that can be separated by reverse-phase HPLC with direct detection by a diode array detector. The main advantage of the presented procedure is the fact that an exce

Full text of DOI:10.1007/s00726-010-0665-5

Amino Acids (2011) 40:527–532
DOI 10.1007/s00726-010-0665-5
ORIGINAL ARTICLE
A new chiral derivatizing agent for the HPLC separation
of a-amino acids on a standard reverse-phase column
•
A. F. Kotthaus H.-J. Altenbach
Received: 9 March 2010 / Accepted: 16 June 2010 / Published online: 2 July 2010
Ó Springer-Verlag 2010
Abstract A new chiral derivatizing agent for a-amino
acids is described which leads to diastereomers that can be
separated by reverse-phase HPLC with direct detection by
a diode array detector. The main advantage of the pre-
sented procedure is the fact that an excess of the deriv-
atizing reagent can be employed as the product exhibits an
absorption maximum at 360 nm, while the reagent has its
absorption maximum at 260 nm. Therefore, it is possible to
suppress the reagent signal by a detection wavelength of
400 nm leading to an easy and general method for the
enantioseparation of a mixture of DL-amino acids and the
determination of the enantiomeric purity of a-amino acid as
exemplified by 16 different a-amino acids.
on a reverse-phase column. An ideal reagent should give
good resolvable diastereomers in quantitative yield under
mild conditions in a short reaction time without the for-
mation of troublesome by-products and without an extra
extraction step. It is also advantageous if the derivatives are
better detectable than the substrate due to chromophoric or
fluorophoric properties. Besides, the derivatizing reagent
should be available in both enantiomeric forms to change
the elution order eventually, and it should be easily avail-
¨ ¨
able and stable (Ilisz et al. 2008; Gorog and Gazdag 1994).
A reagent which fulfils a lot of these requirements is the
Marfey reagent A (Bhushan and Bru¨ckner 2004; Bhushan
and Kumar 2009; Bhushan et al. 2009; Fisher et al. 2009;
B’Hymer et al. 2003; Marfey 1984). A special advantage of
this reagent is that the detection is done at 340 nm, so that
the chromatogram is not interfered by impurities which are
in the probe eventually. Often the Marfey reagent is called
a chiral variant of the Sanger reagent B (Sanger 1945).
However, the two compounds considerably differ in their
absorption properties: compound B has a marker function
as it is colorless having an absorption maximum at 232 nm
(Simons 1979) while after reaction with an a-amino acid a
yellow push–pull system is generated exhibiting an
absorption maximum above 330 nm (Sanger 1949). The
Marfey reagent A contains already a push–pull system by
itself thus leading after reaction with an amino acid only to
a small shift in wavelength. Therefore one can follow the
reaction visually only in the case of the Sanger reagent
and, besides, more importantly, the excess of derivatizing
reagent is repressed and furthermore with different elution
conditions or in complex mixtures, respectively, the prod-
uct can be detected by diode array directly.
Keywords Sanger reagent ꢀ Marfey reagent ꢀ
Chiral derivatization agent (CDA) ꢀ Amino acids ꢀ
Enantiomeric purity determination
Introduction
Over the last years, a number of methods have been
described for the detection of specific amino acids in
complex mixtures and the determination of their chiral
purity either by GC, HPLC, TLC, SFC or capillary elec-
trophoresis. For the HPLC detection, indirect method has
been well established that is preparing diastereomers by
derivatizing the a-amino acid with an enantiomerically
pure reagent, which at the same time provides better
detection properties, so that the derivatives can be analyzed
A. F. Kotthaus ꢀ H.-J. Altenbach (&)
In this paper, the synthesis and application of a new
derivatizing agent for a-amino acids is described which
combines the advantages of the Sanger reagent (marker
¨
Department of Chemistry, Bergische Universitat Wuppertal,
Gaußstraße 20, 42199 Wuppertal, Germany
e-mail: altenbach@uni-wuppertal.de
123

528
A. F. Kotthaus, H.-J. Altenbach
function) and the Marfey reagent (easy derivatization and
good detection properties) and allowing the quick and easy
analysis of D- and L-amino acids.
times with a little pentane. Drying in vacuum afforded
13 g (18%) (2S,6S,9R)-6-isopropyl-9-methyl-1,4-diazaspi-
ro[4.5]decan-2-one as a colorless solid (the purity is good
enough for the next conversion. Recrystallisation from
cyclohexane is possible and afforded fine needles). To get
better yields, the mother liquor was evaporated and further
product crystallized within a few weeks, which can be
isolated as above. This procedure was repeated several
times, whereby the yield increased over 50%.
NH
O
O
N
O
H2N
F
F
HN
F
TLC: Rf 0.27 (silica 60, 2-propanol/cyclohexane 25:75);
mp 127°C (after recrystallisation). 1H NMR (400 MHz,
CDCl3, 32°C): d = 9.09 (s, 1 H), 3.61 (d, 2JH,H = 16.2 Hz,
O2N
NO2
O2N
NO2
O2N
NO2
A
B
1
2
Marfey reagent
Sanger reagent
1 H,), 3.50 (d, JH,H = 16.2 Hz, 1 H), 2.10–2.01 (m, 1 H),
1.98 (s, 1 H, NH), 1.85–1.76 (m, 2 H), 1.67–1.58 (m, 2 H),
3
1.33–1.17 (m, 3 H), 0.92 (d, JH,H = 7 Hz, 3 H), 0.89
3
(d, JH,H = 6.5 Hz, 3 H), 0.86–0.82 (m, 1 H), 0.78 (d,
Materials and methods
3JH,H = 6.9 Hz, 3 H). 13C NMR (100 MHz, CDCl3, 32°C):
d = 176.5 (C2), 80.0 (C5), 51.9 (C6), 50.5 (C10), 49.6 (C3),
34.4 (C8), 30.0 (C9), 25.3 (C12), 23.9 (C13/14), 22.5 (C7),
22.1 (C11), 17.9 (C13/14). IR (KBr): 3262, 3202, 3097, 2947,
2928, 2908, 2868, 2842, 1708, 1681, 1482, 1459, 1430,
1360, 1340, 1258, 1234, 1157, 991, 925, 884, 831,
723 cm-1. Calcd for C12H22N2O: C, 68.63; H, 10.54; N,
13.32. Found: C, 68.28; H, 10.71; N, 13.25. MS (EI, 70 eV):
m/z (relative intensity) 210 (M?, 36), 195 (29), 167 (14),
153 (33), 125 (100), 111 (22), 98 (61), 55 (27), 41 (47).
NMR spectra were recorded on a Bruker ARX 400 spec-
1
trometer. H and 13C chemical shifts are reported d ppm
relative to the used deutero solvent. Mass spectra were
recorded on a Varian MAT 311A; IR spectra were recorded
on a Nicolet FT-IR spectrometer Avatar 360. Elementary
analyses were obtained with a Perkin-Elmer microele-
mentary analyst 240B. Melting points were recorded
on a Gallenkamp MFB-595 and are uncorrected. UV-
absorbance spectra were recorded on
a Shimadzu
UV-160A. The HPLC system consisted of a Merck/Hitachi
L 7100 pump, a DA L-7455 detector and a MZ Kromasil
RP 18 column (250 9 4.6 mm, 5 lm). For TLC alumi-
num sheets silica gel 60 F254 from Merck were used.
(-)-Menthone, glycinamide, trifluoracetic acid (TFA),
N,N-diisopropylethylamine (DIPEA) and triethylamine
(TEA) were purchased from Aldrich, Acros and Fluka.
2-Fluoro-3,5-dinitrobenzoic acid was synthesised accord-
ing to the procedure of Petersen and Jensen (2001).
Synthesis of (5R,6S,9R)-4-(2-fluoro-3,5-dinitro-
benzoyl)-6-isopropyl-9-methyl-1.4-
diazaspiro[4.5]decan-2-one (1)
1.15 g (5 mmol) 2-fluoro-3,5-dinitrobenzoic acid was sus-
pended in 10 ml dichloromethane. First 0.47 ml (5.5 mmol)
oxalyl chloride was added followed by 40 ll DMF. The
mixture was stirred for 1 h at ambient temperature while an
emission of gas occurred. To the clear solution, 1.05 g
(5 mmol) (5S,6S,9R)-6-isopropyl-9-methyl-1,4-diazaspi-
ro[4.5]decan-2-one dissolved in 20 ml dichloromethane
was added followed by 1.82 ml (11.0 mmol) DIPEA. After
1 h, the reaction mixture was washed two times with brine
and dried over NaSO4. The solvent was removed under
reduced pressure and the crude product was isolated as red
foam. Recrystallization from acetone/water 2:1 afforded the
title compound as a pale yellow solid (yield 71%).
Synthesis of (5S,6S,9R)-6-isopropyl-9-methyl-1,4-
diazaspiro[4.5]decan-2-one (2)
44.2 g of fine pulverized glycinamide hydrochloride was
suspended in 700 ml of absolute methanol. After the
addition of 61.6 g (400 mmol) (-)-menthone and 40.5 g
(55.8 ml, 400 mmol) triethylamine, the reaction mixture
was refluxed for 18 h. The solvent was removed under
reduced pressure and the residue was dissolved in a mix-
ture of water and diethyl ether. The water layer was
extracted three times with diethyl ether. The collected
organic layers were washed with sat. NaHCO3 solution and
brine and dried over Na2SO4. Evaporation under reduced
pressure gave 84 g of a pale yellow oil that crystallized
within 1 week. After addition of 30 ml pentane, the crystal
slurry was transported on a suction filter and washed two
TLC: Rf 0.47 (silica, cyclohexane/ethyl acetate 1:2); mp.
1
158°C dec. H NMR (400 MHz, CDCl3, 32 °C): d = 9.36
(s, 1 H, H4), 9.00 (dd, JH,H = 3.1, JH,F = 6.1 Hz, 1 H,
4
4
4
H21), 8.50 (dd, JH,H = 2.8, JH,F = 4.3 Hz, 1 H, H19),
4
2
3.97 (AB, JH,H = 15.3 Hz, 1 H, H2), 3.91 (AB, JH,H
2
=
3
14.8 Hz, 1 H, H2), 2.70 (dd, JH,H 13.0 and 2.3 Hz, 1 H,
H6), 2.53 (t, 2JH,H = 12.7 Hz, 1 H, H10), 1.89–1.75 (m, 4 H,
2
7,8,10,12), 1.66 (m, 1 H, H9), 1.30 (dq, 1 H, JH,H
H
=
3
3JH,H = JH,H = 13.3 Hz, 3JH,H = 2.9 Hz, H7), 1.12 (dq, 1 H,
123

A new chiral derivatizing agent for the HPLC separation
529
3
3
3
2JH,H = JH,H = JH,H = 12.6 Hz, JH,H = 2.5 Hz, H8),
O
3
1.01 (d, 3 H, JH,H = 7.1 Hz, H13/14), 0.99 (d, JH,H
3
=
3
HN
6.1 Hz, 3 H, H11), 0.85 (d, JH,H = 6.6 Hz, 3 H, H13/14).
13C NMR (100 MHz, CDCl3, 32 °C): d = 167.4 (C3),
159.0 (C15), 154.0 (d, JC,F = 274.7 Hz, C17), 143.9
glycinamide*HCl
O
N
1
triethylamine (TEA)
MeOH
H
4
2
(d, JC,F = 4.1 Hz, C20), 137.8 (d, JC,F = 11.2 Hz, C18),
2
3
129.8 (d, JC,F = 21.4 Hz, C16), 128.2 (d, JC,F = 6.1 Hz,
C19), 122.8 (C21), 85.3 (C5), 50.5 (C2), 45.0 (C6), 44.8
(C10), 33.8 (C8), 29.8 (C9), 26.3 (C12), 23.2 (C13/14), 22.6 (C7),
21.9 (C11), 18.1 (C13/14). 19F NMR (376 MHz, CDCl3, 32
50 %
2
Scheme 1 N,N-acetalization of menthone with glycinamide
4
°C): d = –112.07 (dd, JF,H = 4.8 and 5.6 Hz, F17),
–112.16 (13C satelite, JF,C 274.4 Hz, F17). Calcd for
1
shown diastereomer 2 was isolated by crystallization,
whereas the other diastereomer (with equatorial amide
function) does not crystallize. The proposed structure of the
isolated diastereomer 2 was evidenced by X-ray spectros-
copy of a derivate (Kotthaus 2005). Owing to the low costs
of all reagents (including (?)- or (-)-menthone), the
moderate yield of 50% is acceptable.
C19H23FN4O6: C, 54.02; H, 5.49; N, 13.26. Found: C,
54.36; H, 5.19; N, 13.04. MS (EI, 70 eV): m/z (relative
intensity) 422 (M?, 36), 407 (16), 338 (33), 337 (100), 213
(41), 167 (25), 121 (21), 71 (15), 57 (29), 55 (21), 43 (18),
42 (23).
Typical procedure for derivatization of amino acids
To synthesize reagent 1, 2-fluoro-3,5-dinitrobenzoyl
chloride, prepared in situ from the acid 3 with oxalyl chlo-
ride, was stirred for 1 h with 2 and N,N-diisopropylethyl-
amine (DIPEA) as base in dichloromethane (Scheme 2). The
derived product can be isolated easily by crystallization
(yield 71%). For derivatizing operations, we used a 33 mM
solution of reagent 1 in acetonitrile. During our work it could
be shown that this solution, stored at room temperature is
stable for a minimum of 3 months. The UV spectrum from 1
(Fig. 1) exhibits a maximum at 260 nm, typically for such
electron-deficient aromatics.
with 1
50 ll of a 20 mM aqueous amino acid solution containing
1% TEA was transferred to a 2 ml plastic tube and 100 ll
of a 33 mM solution of 1 in acetonitrile were added. The
mixture was diluted with acetonitrile up to a volume of
300 ll. The contents were mixed and heated to 60°C for
30 min. After cooling to room temperature, 10 ll of the
mixture was injected on a HPLC column. Alternatively, the
sample was acidified with 200 ll acetonitrile/1% TFA (aq.)
1:1 before injection.
The marker function of the reagent could be seen after
reaction with amino acids (Scheme 3). Immediately after
the addition, the color of the reaction mixture changes from
colorless to deep yellow, due to a change in the absorption
maximum from 260 to 360 nm for the derived product
(Fig. 1). The procedure to generate HPLC-compatible
samples is very easy and needs no purification step.
Complete conversion is reached after 30 min in acetoni-
trile/1% TEA (aq.) at 60°C. After cooling, the sample was
injected directly onto the column. To separate derivatized
amino acid mixtures, a gradient elution acetonitrile/TFA
0.1% (aq.) from 50:50 to 80:20 within 1 h was used. To
Elution and detection
To separate diastereomers from amino acid mixtures, elu-
tion was done with a linear gradient of acetonitrile/1% TFA
(aq.) from 50:50 to 80:20 during 1 h, flow rate 0.6 ml/min.
To separate diastereomers of one amino acid, elution was
done with acetonitrile/1% TFA (aq.) 70:30, 80:20 or 90:10
depending on the polarity of the amino acids, flow rate
0.8 ml/min. To observe the derivatizing reagent peak, a
detection wavelength of 350 nm was used. To suppress the
reagent signal, the detection was done at 400 nm.
O
O
OH
Results and discussion
HN
F
NO2
NO2
F
N
O
1. (COCl)2
2. 2,DIPEA
The chiral component of reagent 1 was developed originally
as a chiral glycine equivalent 2. Owing to the excellent
diastereoselectivity using 2 as a chiral auxiliary to prepare
amino acid derivatives (Altenbach et al. 1997, 2001), the
N,N-acetal 2 seems to be an optimal compound to introduce
chirality into a derivatizing reagent. 2 is easily synthesized
from (-)-menthone and glycinamide (Scheme 1). The
O2N
NO2
CH2Cl2
71 %
3
1
Scheme 2 Preparation of reagent 1
123

530
A. F. Kotthaus, H.-J. Altenbach
Fig. 1 Left UV spectrum of 1;
c = 6.6 9 10-5 mol/l in
acetonitrile, d = 0.5 cm, right
UV spectrum of 1 ? valine,
c = 6.6 9 10-5 mol/l in
aqueous 1% TEA solution
Scheme 3 Derivatization of
amino acids with reagent 1
O
O
HN
R
OH
O
NO2
*
HN
N
O
NO2
NO2
H2N
N
MeCN / 1%TEA (aq.)
HN
NO2
O
F
R
HO
*
5
O
separate only two diastereomers, isocratic conditions from
70:30 to 90:10 were used.
fixed wavelength at 400 nm (illustrated at the right chro-
matogram in Fig. 2) shows only the two diastereomers of
the derivatized amino acid. The area under the two peaks
and of each measured amino acid racemate (Table 1) is
equal (within the margin of error). An equal absorption of
the racemates is necessary if one wants to calculate the
enantiomeric excess of unknown samples.
Figure 2 demonstrates the advantage of the marker
function exemplarily shown for derivatized DL-valine. The
picture at the left shows an integrated chromatogram from
220 to 400 nm. The highest peak represents the excess of
the derivatizing reagent 1. The same sample detected at a
Fig. 2 HPLC chromatogram of
valine derivatized with reagent
1; left integrated chromatogram
from k = 220–400 nm, right
the same sample at
k = 400 nm; MZ Kromasil RP
18, 0.6 ml/min; gradient MeCN/
0.1% TFA 50:50 ? 80:20 in
60 min
123

A new chiral derivatizing agent for the HPLC separation
531
derivatizing reagent or the impurities of a probe which do
not react with the marker 1.
Table 1 Enantioseparation of amino acids after pre column deriva-
tization with reagent 1 on an achiral phase (MZ Kromasil RP 18)
Furthermore, the separation of the formed diastereomers
is very good. Besides DL-valine, we tested 15 other amino
acids which all could be baseline separated easily (Table 1,
S1–S19). We observed that the elution order of DL-amino
acids (derivatized with 1) is normally first the D- than the
L-isomer. The reverse elution order was only observed by
amino acids with very polar side chains like Ser or Asn.
Most separations shown in Table 1 were carried out by
gradient elution MeCN/1% TFA 50:50 to 80:20 in 60 min.
This method was primarily developed to separate amino
acid mixtures. For the analysis of only one enantiomeric
pair one can accelerate the separation time dramatically by
the use of more polar conditions (compare Table 1, S1 with
S14, S2 with S17 and S4 with S12).
Number
DL-Amino acid
kD
kL
a
S1a
S2a
S3b
S4c
S5d
S6d
S7d
S8d
Tle
Phe
Lys
Val
Asn
Thr
Ser
Ala
Met
Abu
Trp
Val
Phg
Tle
Pro
Nor
Phe
Ile
0.20
0.23
0.82
0.99
1.16
1.78
1.87
2.72
2.96
3.60
3.97
4.12
4.16
4.19
4.20
4.45
4.54
5.34
6.07
0.76
0.51
1.26
2.20
0.95
2.91
1.32
4.13
5.79
4.85
5.91
6.81
5.69
8.77
4.36
6.97
7.48
8.12
9.24
3.72
2.23
1.54
2.23
1.23
1.64
1.42
1.51
1.96
1.35
1.49
1.65
1.37
2.09
1.04
1.56
1.65
1.52
1.52
S9d
S10d
S11d
S12d
S13d
S14d
S15d
S16d
S17d
S18d
S19d
Figure 3 shows the separation of an amino acid cocktail.
In less than 45 min 10 different DL-amino acids were
separated after derivatization with 1.
Conclusion
Leu
In summary, the synthesis and application of a new chiral
derivatizing agent for amino acids are reported that con-
joins a lot of positive characteristics: the synthesis of the
reagent is easy, the educts are cheap, both enantiomers are
accessibly due to commercial availability of (?)- and (-)-
menthone, the reagent and its solution are stable over a
long time and derivatizations are fast and need no work up.
The separations of our tested derivatized amino acids on
reverse-phase HPLC were excellent and the derived dia-
stereomers show the same absorption, so one is able to
determine the enantiopurities of amino acids very easily.
Detection at k = 350 nm
a
MeCN/1%TFA 90:10, 0.8 ml/min
b
MeCN/1%TFA 80:20, 0.8 ml/min
c
MeCN/1%TFA 70:30, 0.8 ml/min
d
Gradient MeCN/1%TFA 50:50 ? 80:20 in 60 min, 0.6 ml/min;
k = tr/t0; a = k00/k0
In this way, one can easily determine the enantiomeric
excess of amino acids, with the advantage of getting to see
only the derivatized amino acids and not the excess of the
Fig. 3 Enantioseparation of
10 DL-amino acids after
diastereomer formation with 1
on an achiral HPLC column.
Conditions: phase MZ Kromasil
RP 18; gradient MeCN/0.1%
TFA 50:50 ? 80:20 in 60 min,
0.6 ml/min; detection k = 300–
400 nm (integrated); peaks:
1 L-Asn, 2 D-Asn, 3 L-Ser,
4 D-Ser, 5 D-Met, 6 hydrol.
reagent, 7 D-Trp, 8 D-Val,
9 D-Tle, 10 D-Nor, 11 D-Phe,
12 reagent, 13 L-Met; 14 L-Trp;
15 D-Leu; 16 L-Val; 17 L-Nor;
18 L-Phe; 19 D-Lys; 20 L-Tle;
21 L-Leu; 22 L-Lys
123

532
A. F. Kotthaus, H.-J. Altenbach
with Marfey’s reagent and its four variants. Amino Acids 36:571–
579
Conversion of 3 with other chiral auxiliaries for instance
menthol or prolinamide etc. led to less satisfying results.
´ ´
Fisher G, Szokan G, Galindo E, Tsesarskaia M (2009) HPLC
determination of acidic D-amino acids and their N-methyl
derivatives in biological tissues. Biomed Chromatogr 23:581–
587
¨
Acknowledgments We thank Mr. J. Donecke for technical assis-
tance in the HPLC measurements.
¨ ¨
Gorog S, Gazdag M (1994) Review—enantiomeric derivatization for
biomedical chromatography. J Chromatogr B 659:51–84
´
Ilisz I, Berkecz R, Peter A (2008) Review—application of chiral
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