Derivatize, Racemize, and Analyze - An Easy and Simple Procedure for Chiral Amino Acid Standard Preparation for Enantioselective Metabolomics

Article abstract of DOI:10.1021/acs.analchem.9b00666

A simple, controllable, and reproducible stereoisomerization (racemization and epimerization) protocol for the preparation of scalemic α-amino acid mixtures from stereoisomerically pure standards was developed. Simply derivatize your amino acids with a racemization tag that incorporates a urea bond on the N-terminus of the target amino acid and incubate at elevated temperatures up to 95 °C for defined time periods until the targeted d-amino acid levels are obtained. The racemization tags investigated were 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), aminophenyl-N-hydroxysuccinimidyl carbamate (AC), and 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a ready-to-use, tailor-made chiral uniformly ¹³C and ¹⁵N labeled [U-¹³C¹⁵N]-amino acid standard with the desired d-amino acid percentage within minutes or hours without sample cleanup. A racemization time of 30 min at 95 °C will lead to a d-amino acid level of 1-5%, while 6 h at 95 °C provides 15-30% d-amino acids. Racemization occurs due to imine formation at the chiral carbon atom bound to the urea-linking group without decomposition of labile amino acids such as Asn, Gln, Trp, Cit, and theanine. For amino acids possessing two chiral centers such as dl-Ile or dl-Thr, only the epimerization of isomers with different stereochemistry at the second chiral center will produce all four possible isobaric enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX(+) column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS analysis. This new racemization method solves the problem of (enantioselective) matrix effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants full MS-compatibility.

Full text of DOI:10.1021/acs.analchem.9b00666

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Article
Derivatize, Racemize and Analyze - an Easy and Simple Procedure for
Chiral Amino Acid Standard Preparation for Enantioselective Metabolomics
Jeannie Horak, and Michael Lämmerhofer
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00666 • Publication Date (Web): 14 May 2019
Downloaded from http://pubs.acs.org on May 14, 2019
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Derivatize, Racemize and Analyze -
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an Easy and Simple Procedure for Chiral Amino Acid Standard
Preparation for Enantioselective Metabolomics
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Jeannie Horak*, Michael Lämmerhofer
Eberhard-Karls-University Tuebingen,
Institute of Pharmaceutical Sciences,
Pharmaceutical (Bio-)Analysis,
Auf der Morgenstelle 8 (Haus B), 72076 Tuebingen, Germany
*Corresponding author: Dr. Jeannie Horak
Tel.: +49 (0)7071 29 74053
Fax.: +49 (0)7071 29 4565
Email: Jeannie.Horak@uni-tuebingen.de
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Abstract
A simple, controllable and reproducible stereoisomerization (racemization and epimerization)
protocol for the preparation of scalemic -amino acid mixtures from stereoisomerically pure
standards was developed. Simply derivatize your amino acids with a racemization-tag that
incorporates a urea bond on the N-terminus of the target amino acid and incubate at
elevated temperatures up to 95 °C for defined time periods until the targeted D-amino acid
levels are obtained.
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The racemization-tags investigated were 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC), aminophenyl-N-hydroxysuccinimidyl carbamate (AC) and 3-aminopyridyl-N-
hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a
ready-to-use, tailor made chiral uniformly 13C and 15N labelled [U-13C15N]-amino acid
standard with the desired D-amino acid percentage within minutes or hours without sample
clean-up. A racemization time of 30 min at 95 °C will lead to a D-amino acid level of 1-5%,
while 6 h at 95 °C provides 15-30% D-amino acids. Racemization occurs due to imine
formation at the chiral carbon atom bound to the urea-linking group without decomposition of
labile amino acids such as Asn, Gln, Trp, Cit and theanine. For amino acids possessing two
chiral centers such as DL-Ile or DL-Thr, only the epimerization of isomers with different
stereochemistry at the second chiral center will produce all four possible isobaric
enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX(+)
column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS
analysis. This new racemization method solves the problem of (enantioselective) matrix
effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants
full MS-compatibility.
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Introduction
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In the continuously expanding field of omics analysis, such as proteomics, glycomics,
lipidomics or metabolomics, high sample throughput and ultra-fast separation are essential,
necessitating reasonably fast and simple sample preparation techniques. Ultra-high
performance liquid chromatography coupled with high resolution mass spectrometry
(UHPLC-HRMS) combines fast analyte separation with high mass resolving power and
highest possible mass-to-charge (m/z) accuracy. The dilute and shoot approach resembles
the fastest sample preparation technique and has hence gained increasing popularity in LC-
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MS, ranging from the analysis of drugs
and metabolites to large molecules such as
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insulin . In this context, the use of isotopically labelled internal standards (IS) to ensure
highest possible accuracy in quantitative analytics is of utmost importance, since matrix
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effects can be adequately compensated. These isotopically labelled compounds share
identical chemical properties, including extraction and ionization efficiency, with their
unlabelled counterparts, the target analytes. In case of the upcoming research field chiral
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metabolomics , which include chiral amino acid metabolomics 8,9, the need for chiral
isotopically labelled IS to compensate for matrix effects, which effect both enantiomers
differently (enantioselective matrix effects) is known. Presently, many chiral metabolomics
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studies still rely on external calibration
or the use of an non-proteinogenic amino acid for
IS such as racemic norleucine 11. The reasons are manifold. Isotopically labelled amino acid
standards mixtures are costly and their cost increase with the number and type of labelled
atoms. Nonetheless, the more expensive [13C] and [15N] labelled IS are preferred over [2H],
since the latter may behave chromatographically different compared to [13C]- and [15N]-
labelled compounds 12,13. Alternatively also isotopically labelled derivatization tags can be
used to produce isotopically labelled IS 14. Since in both cases the natural isotopologue
pattern of organic compounds provide additional m/z peaks with M+1, M+2, M+3 and higher,
the m/z difference between analyte and IS should be at least 2 and preferably higher or even
uniformly [13C] and [15N] labelled ([U-13C15N]). Therefore the application of isotopically
labelled IS, necessitate mass spectrometric detection to determine small m/z differences
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between co-eluting target analyte and IS. In addition, only few isotopically labelled D-amino
acid standards exist and chiral isotopically labelled DL-amino acid standard mixtures
containing all proteinogenic amino acids are not at all available. Even the commercial [U-
13C15N]-L-amino acid metabolomics standard does not contain all proteinogenic L-amino
acids. The amino acids L-Gln, L-Asn and L-Trp are absent, since they are prone to
degradationduring acid 15,16 or base 17,18 catalysed hydrolysis of peptides and proteins applied
for amino acid composition determination. Also, the most common N-terminal amino acid
racemization method involves the formation of a Schiff-base with aromatic aldehydes in the
presence of concentrated acetic acids and elevated temperatures 19,20, which induces
deamidation and degradation of Asn and Gln to Asp and Glu, respectively. Alternatively,
Schiff base formation followed by racemization can also be induced by complex formation
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with an auxiliary ligand and a metal ion such as copper or nickel 22,23. In these cases the
disadvantage lies in the necessity to remove the metal ions prior to mass spectrometric
analysis.Besides that also C-terminal racemization of N-acetyl amino acids via oxazolone
formation and base induced ring-opening was reported in literature 24,25. Furthermore, several
patents illustrate the racemization of N-acyl, N-phenacetyl, N-benzoyl 26-28 and N-phenacetyl-
phosphino amino acid 29 derivatives as well as N-phenacyl-hydroxyl proline 30 in the presence
of the corresponding acid and heat treatment up to 180 °C. The major disadvantage of these
methods is, as previously mentioned, the decomposition of several amino acids during acid
treatment.
The here presented study introduces the mild N-terminal heat induced stereoisomerization
(racemization and epimerization 31) of 6-aminoquinolyl-N-hydroxysuccinimidyl (AQC) tagged
proteinogenic amino acids and some uncommon amino acids without deamidation and
degradation of some metabolically interesting amino acids such as Gln, Asn Trp, Cit and
theanine. Applying this racemization method on DL-Ile or DL-aIle, leads in both cases to a
complete mixture of DL-Ile plus DL-aIle in one standard solution. The same accounts for DL-
Thr and DL-aThr. Furthermore, this procedure allows the preparation of a scalemic DL-[U-
13C15N]-isotopically labelled chiral amino acid mixture containing a defined amount of D-[U-
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13C15N]- amino acids, which can be used as an internal standard for different sample types,
containing varying levels of D-amino acids, to compensate for (enantioselective) matrix
effects.
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EXPERIMENTAL SECTION
Chemicals and standard solutions
All amino acids, L-citrulline (Cit), L-phenylglycine (Phg), ammonium formate, boric acid,
hydrochloric acid, 1,3,5-tri-tert-butyl-benzene (TBB), sodium acetate, sodium hydroxide,
sodium deuteroxide, diethylamine (DEA), dimethyl sulfoxide (DMSO), DMSO-d6, acetonitrile-
d3 (ACN-d3) and 1.5 mL Eppendorf Safe-Lock microcentrifuge tubes were from Sigma Aldrich
(Schnelldorf, Germany). L-Theanine was from TCI Chemicals GmbH (Eschborn, Germany).
Ultra-LC-MS grade ACN ultra-LC-MS methanol (MeOH), formic acid (FA), syringes, 0.22 µm
sterile syringe filters, Mµlti®-lid locks for 1.5 mL microcentrifuge tubes and Parafilm® were
from Carl ROTH (Karlsruhe, Germany). -Phenylalanine and 7-fluoro-4-nitrobenzoxadiazole
(NBD) were from TCI (Zwijndrecht, Belgium). 5-(Dimethylamino) naphthalene-1-sulfonyl-L-
and DL-tryptophan (dansyl, DNS-Trp), N-fluorenylmethoxycarbonyl-L- and DL-phenylalanine
(FMOC-Phe), N-carbobenzyloxy-L and DL-phenylalanine (CBZ-Phe) and N-3,5-
dinitrobenzoyl L and DL-tryptophan (DNB-Trp) were in-house prepared analytical standards.
Ultra-pure water was generated with a Water Purelab Analytics Purification System from
ELGA (Celle, Germany).
6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) was purchased from
Synchem (Felsberg
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Altenburg, Germany). In addition, AQC, aminophenyl-N-
hydroxysuccinimidyl carbamate (AC) and 3-aminopyridyl N-hydroxysuccinimidyl carbamate
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(APC) were in-house prepared as described in literature
yield yellowish off-white crystals.
and re-crystalized in ACN to
Isotopically labelled L-[U-13C15N]-amino acid metabolomics standard mix (2.5 mM in 0.1 M
HCl) was purchased from Euroiso-top GmbH (Saarbrücken, Germany) and does not contain
Asn, Gln and Trp.
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All DL- and L-amino acid stock solutions were prepared at a concentration of 50 mM in 0.1 M
HCl. The single standard solutions were combined to obtain a 2.5 mM L- and DL-[U-12C14N]-
amino acid standard mixtures containing all 20 proteinogenic amino acids in 0.1 M HCl,
respectively. Note that the concentration of each D- and L-enantiomer in the DL-amino acid
standard mixtures was 1.25 mM and 2.5 mM in the L-amino acid standard mixtures.
Separate test mixtures of isobaric amino acids Leu, Ile and aIle as well as Thr, aThr and Hse
were prepared at concentrations of 2.5 mM per solution. If not otherwise stated, all test
solutions were diluted to 0.025 mM prior to derivatization with AQC. Note that for the AQC-
derivatized amino acid solutions, the stated amino acid concentrations are the concentration
of the sample prior to derivatization.
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Sample Preparation
Derivatization with AQC
The AQC reagent solution was prepared at a concentration of 3 mg/mL in HPLC-grade ACN
(AQC: 10.5 mM) and stored at -20 °C prior to use. In general, 10 µL 0.025 mM amino acid
sample solution was added to 80 µL (or 70 µL) 20 mM sodium borate buffer (pH 8.8). After
addition of 10 µL AQC reagent, the reaction solution was immediately heated to 55 °C for 10
min at 800 rpm. Derivatization reactions of standard reference solutions were performed in
1.5 mL Crystal Clear microcentrifuge tubes from Starlab (Hamburg, Germany). Note that for
0.25 mM amino acid standard mixtures, 10 µL sample, 70 µL 0.2 M borate buffer (pH 8.8)
and 20 µL AQC reagent solution were used.
Reagent-type dependent racemization
For comparison two additional urea-group incorporating amino acid tags, aminophenyl-N-
hydroxysuccinimidyl carbamate (AC) 34 and 3-aminopyridyl N-hydroxysuccinimidyl carbamate
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(APC)
were investigated. The 0.25 mM L-[U-12C14N]- and [U-13C15N]-amino acid solution
was derivatized with AC and APC as described for AQC 32,33. The racemization experiment
was performed at 95°C for 18 h, followed by chromatographic evaluation via HPLC-ESI-
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QTOF-MS as described for AQC. Information about the performance of the amino acid tags
FMOC, CBZ, DNB, DNS and NBD during heat treatment is provided in the Supporting
Information.
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Racemization of compounds with two chiral centers
The test solutions 0.25 mM L-Thr, L-aThr, DL-Thr, DL-aThr, L-Ile- L-aIle, DL-Ile and DL-aIle
were derivatized using 20 mM borate buffer and 10 µL AQC, and were racemized for 18 h at
95 °C. For all Ile solutions, a 1:10 dilution to 0.025mM was performed prior to chiral analysis,
while the Thr solutions were injected without prior dilution.
Racemization of asparagine, glutamine, citrulline and theanine
For the racemization experiment, 20 µL of 0.025 mM L-Asn, L-Gln, L-Cit or L-theanine was
added to 160 µL of 20 mM sodium borate buffer (pH 8.5) in 1.5 mL microcentrifuge tubes
followed by addition of 20 µL AQC reagent (3 mg/mL in ACN) and heated to 55°C for 10 min.
After ultrasonication for 1 min and purging with nitrogen for 1 min, the reaction tube was
placed onto the thermoshaker for 15 h at 95°C at 800 rpm.
Note that stereoselective separation of scalemic AQC-DL-Asn and AQC-DL-Gln as well as
their degradation products AQC-DL-Asp and AQC-DL-Glu was performed on a Chiralpak
ZWIX(+) column (150 x 3 mm, 3 µm) using a short gradient elution method 36. Chiral
separation of scalemic AQC-DL-Cit and AQC-DL-theanine was performed on a Chiralpak
ZWIX(+) column (150 x 4 mm, 3 µm) using the dual-gradient elution method. Analysis was
performed with HPLC-ESI-QTOF-MS.
Time and temperature dependent racemization with AQC
A standard solution, containing 0.025 mM L-[U-12C14N]- amino acids and 0.025 mM L-[U-
13C15N]- amino acids was first derivatized with AQC. To ensure a tight closure during the
racemization process, the lid was secured with a small strip of Parafilm® combined with a
Mµlti®-lid lock (Figure S1). Alternatively Eppendorf Safe-Lock microcentrifuge tubes, sealed
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with Parafilm® were used. Amino acid racemization was induced by heat treatment for
defined time periods. The following racemization conditions were investigated, 10 min, 30
min and 1 h at 55 °C, 75 °C and 95 °C. The shaker speed of a Grant-bio PHMT Thermo-
Shaker from Grant Instruments Ltd. (Shepreth, England) was set to 600 rpm. After reaching
room temperature, all samples were centrifuged and analyzed without further treatment.
In a second optimized approach, a 0.25 mM [U-12C14N]- and 0.25 mM [U-13C15N]-L-amino
acid standard solution was derivatized with AQC Note that all samples were prepared in
triplicates. The racemization conditions were 1 h, 6 h and 15 h at 95 °C and 600 rpm. After
racemization and reaching room temperature, all samples were centrifuged and additionally
derivatized with AQC (samples (A)), which resembles a 1:10 dilution. Additionally the 15 h
samples were only diluted 1:10 with 0.2 M borate buffer, without additional re-derivatization
with AQC (samples (B)). In a third optimization step to prevent oxidation of methionine, the
amino acid test solution was ultrasonicated for 1 min and purged with nitrogen prior to
stereoisomerization.
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Instrumentation and Methods
HPLC-ESI-QTOF-MS
HPLC-ESI-QTOF-MS measurements were performed on an Agilent 1290 UHPLC system
comprised of a binary pump and a thermostated column oven from Agilent (Waldbronn,
Germany); and a CTC-PAL HTS autosampler from CTC Analytics (Zwingen, Switzerland).
Furthermore, an Agilent 1100 binary pump combined with a 6-port/2-position Valco valve
was used for MS-calibrant delivery. Mass calibration was performed via sodium acetate
clusters (0.1 mg/mL sodium acetate in acetonitrile:water (1:1, v/v)). LC-MS analyses were
performed on a TripleTOF 5600+ instrument from Sciex (Ontario, Canada) with DuoSpray
Ion Source operated in positive electrospray ionization mode. MS results shown in this study
were generated with TOF-MS full scans (survey scans) combined with MS/MS experiments
by information dependent acquisition (IDA) in high sensitivity mode. The IDA inclusion list
comprised the m/z of the precursor ions of all single and double AQC derivatized as well as
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all un-derivatized amino acid compounds. Note that in the present sample set only Lys was
double derivatized with AQC. For the QTOF measurements, the following instrument settings
were used: curtain gas (CUR) 40 psi, ion source gas (nebulizing gas; GS1) 60 psi, heater
gas (drying gas; GS2) 60 psi, ion spray voltage floating (ISVF) 5500 V, source temperature
(TEM) 400 °C, collision energy (CE) 10 V, declustering potential 100 V. The TOF-MS full
scan was set to m/z 30-2000 with an accumulation time of 250 ms, while for the IDA MS/MS
experiments a CE of 46 V, DP of 100 V and an accumulation time of 100 ms were used in
combination with an automated dynamic background subtraction.
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MS Software Information and Data Analysis
The Analyst®TF Software was utilized for data acquisition, while for qualitative HPLC-ESI-
QTOF-MS data analysis the PeakView® and MasterViewTM software and for quantitative
analysis the MultiQuantTM from SCIEX (Ontario, Canada) was used. MultiQuantTM was only
used for peak integration, for which individual pre-defined integration time sections for each
amino acid enantiomer were employed. Note that the same time section width of baseline
noise (closely neighboring the enantiomer peak) was integrated for each amino acid
enantiomer and subtracted from the corresponding enantiomer peak. An overall smoothing
factor of 2 was used prior to peak integration. Due to the use of a dual gradient elution,
changing the mobile phase composition as well as the flow rate, the peak areas of the D- and
L-enantiomers of a racemic mixture are not identical. For the calculation of the percent D-
amino acid value, the D/L ratios of the commercial [U-12C14N]-amino acids was used for
normalization. Since these normalization factors are highly dependent on chromatographic
separation conditions, the analysis of a racemic DL amino acid standard mixture must
accompany each sample measurement sequence. Since a racemic mixture of isotopically
labelled amino acid was not available, the D/L ratios of the [U-13C15N]-amino acids were
assumed to be identical to that of their unlabeled counterparts.
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Chiral Separation of AQC, APC and AC
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Chiral Separation was performed on a Chiralpak ZWIX(+) (150 mm x 4 mm i.d., 3 µm)
column and Chiralpak ZWIX(+) (150 mm x 3 mm i.d., 3 µm) from Chiral Technologies Europe
(Illkirch, France) using the following chromatographic dual gradient elution conditions. Mobile
phase A (MP-A) was 9.4 mM NH4FA and 9.4 mM FA in ACN:MeOH:H2O (75:25:2; (v/v)),
while mobile phase B (MP-B) contained MP-A:9.4 mM NH4FA and 9.4 mM FA in water (1:1,
(v/v)). The gradient consists of the following steps: i) isocratic run: MP-A 0-35 min at 0.2
mL/min; ii) dual gradient mode: gradient 1 (flow rate): 0-45 min 0.2 mL/min, 45-50 min 0.2-
0.5 mL/min and 50-65 min 0.5 mL/min; iii) gradient 2 (hydro-organic): 35-45 min 0% MP-B to
40% MP-B, 45-50 min to 60% B, 50-60 min to 100% B and 60-65 min 100% B, followed by
iv) column re-equilibration to 100% MP A at 0.2 mL/min. Column temperature was kept at 30
°C and sample injection volume were uniformly 2.5 µL. Note that for the NBD-derivatized
sample the same chromatographic condition was used (Figure S3).
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For the short gradient elution method an oven temperature of 30 °C was employed. The
gradient setting was 0-9 min with MP-A containing 2% water in MeOH with 9.4 mM NH4FA
and 9.4 mM FA; and 9-20 min to 100% MP-B containing MP-A:water with 9.4 mM NH4FA
and 9.4 mM FA (1:1, (v/v)), followed by 20-25 min at 100% MP-B and re-equilibration to
100% MP-A. For the Chiralpak ZWIX(+) column (150 x 4 mm, 3µm) a flow rate of 0.7 mL/min
and an injection volume of 2.5 µL was used, while for the ZWIX(+) column (150 x 3 mm,
3µm) a flow rate of 0.45 mL/min and an injection volume of 1 µL was used.
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The reaction of an amino functional compound with N,N′‐disuccinimidyl carbonate (DSC)
leads to an N-hydroxysuccinimidyl carbamate activated reagent 33, such as 6-aminoquinolyl-
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carbamate (AC)
and 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APC) 35. These
reagents are known to easily react with the N-terminus of amino acids or any amine
containing substances, incorporating thereby an aromatic side chain via a urea linking group.
The addition of an aromatic ring system enhances not only the detectability of amino acids,
but provides also an additional hydrophobic and shape selective interaction site for improved
reversed phase and mixed mode liquid chromatographic separation. Since the chiral
stationary phase with zwitterionic (S,S)-trans-2-aminocyclohexanesulfonic acid (S,S-ACHSA)
modified quinine type chiral selector (Chiralpak ZWIX(+)) 37 is actually a mixed mode phase,
the attachment of AQC to amino acids leads also to improved chiral separation of
enantiomers 36,38-40. The formation of the urea linking group between the amino group of the
aromatic tag and the N-terminus of the amino acid provides a close proximity between the
urea group and the chiral center of the amino acid. Upon heating to 95 °C for elevated time
periods racemization (for amino acids with one stereogenic center) and epimerization (for
amino acids with more than one center of chirality) occurs. It can be expected that a heat-
induced movement of delocalized electrons within the electron clouds of the urea link leads
to a reversible and short term transient imine formation at the carbon atom of the chiral
41,42
center (see TOC Graphic) facilitated by keto-enol tautomerism
and charge-transfer
effects 43. The planar imine structure induces the loss of chirality, hence providing an N-
terminal stereointerconversion of the attached amino acid.
The occurrence of a stereoisomerization event of the model compound AQC-phenylglycine
(AQC-Phg) in a variety of different solutions including aqueous buffer and DMSO as well as
in the presence of acid and base catalysis could be verified by a hydrogen-deuterium
exchange at the chiral center using HPLC-ESI-QTOF-MS combined with chiral separation on
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the Chiralpak ZWIX(+) column (Figure S2). Information about the investigated solvent
systems and racemization conditions is provided in the Supporting Information.
< add Figure 1 >
9
A comparison of different N-terminal amino acid tags such as AQC, APC, AC and NBD as
well as protective groups such as DNS, DNB and CBZ in Figure 1 and Figure S3 show that
only those derivatives that carry a urea linking group can induce racemization of the attached
-amino acid. Figure 1 shows the racemization results for AQC-L-[U-13C15N]- Phe, APC-L-
Phe and AC-L-[U-13C15N]-Phe after heat treatment at 95 °C for 18 h. Note that the slight
retention time shift of scalemic AQC-DL-[U-13C15N]-Phe compared to AQC-L-[U-13C15N]-Phe
was due to a too short column equilibration time. The generated D-enantiomers were all
verified via their MS-spectrum. In comparison, Figure S3 summarizes the failed racemization
for DNS-Trp, DNB-Trp, CBZ-Phe and NBD-Arg. In case of FMOC-Phe in Figure S4, the heat
treatment has led to a complete loss of the protective group under the investigated condition.
Note that -amino acids cannot be stereoisomerized by this method, since the keto-enol
tautomerism between urea group and the attached amino acid is disrupted by the additional
methylene group in the -amino acid (Figure S5)..
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In case of amino acids possessing two or more chiral centers, only the chiral center in
position 2 will be epimerized. In order to substantiate this hypothesis, it was necessary to
chromatographically separate the isobaric isomers of Leu and Thr. During method
optimization the L-[U-13C15N]-amino acid standard was used as a chiral peak assignment
standard to determine the L-enantiomers within a single run (Figure S6) 36. Additionally,
single DL-standard solutions of Leu and Thr isomers were analyzed to determine the correct
elution order of all D- and L-isomers. Hereby, the best result was obtained using a dual
gradient elution method, which included a low flow rate of 0.2 mL/min with 2% water in the
mobile phase A at the beginning to separate the DL-Ile and DL-Leu isomers followed by a
gradient elution to a flow rate of 0.5 mL/min with a water content of 52% in the mobile phase
B, which elute all other amino acids. The latter allows the baseline separation of all six Thr
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isomers, namely DL-Thr, DL-aThr and DL-Hse (Figure S7). Note that D-Ile and D-aIle as well
as L-Leu and L-aIle coelute on the ZWIX(+) column.
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< add Figure 2 >
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As shown in Figure 2 and Figure S8, the epimerization of L-Thr, L-aThr, DL-Thr and DL-
aThr as well as L-Ile, L-aIle, DL-Ile and DL-aIle, has led to the generation of the following
new epimers, namely D-aThr, D-Thr, DL-aThr, DL-Thr as well as D-aIle, D-Ile, DL-aIle and
DL-Ile, respectively. Hence, only the epimerization of DL-Thr or DL-aThr as well as DL-Ile or
DL-aIle will ultimately provide the complete set of all 4 possible epimers of Thr and Ile. Note
that the apparent difference in epimerization ratio for isobaric amino acids shown in Figure 2
is due to differences in analyte ionization caused by an enantioselective matrix effect.
< add Figure 3 >
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Another point that needed to be addressed was whether this stereoisomerization method
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was mild enough to racemize deamidation prone amino acids such as Asn
and Gln
,
which are also of metabolic interest. Figure 3a shows the XICs of AQC derivatized DL-Asn
and DL-Gln as well as their deamidation products DL-Asp and DL-Glu. Figure 3b shows the
XICs of L-Asn and L-Gln and their potential degradation products prior to racemization and
Figure 3c shows their XICs after racemization for 15 h at 95 °C. While the XICs of AQC-Asn
and AQC-Gln show additional peaks for their respective D-enantiomers, the corresponding
XICs of their degradation products were absent of AQC-Asp and AQC-Glu.
< add Figure 4 >
To show the applicability of this method for uncommon amino acids, this experiment was
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repeated for theanine
and citrulline (Cit) 48. Figure 4a shows the XICs of AQC
derivatized L-theanine and L-Cit as well as their respective degradation products AQC-Glu
and AQC-ornithine (Orn). Figure 4b shows clearly that theanine and Cit can be racemized
without being decomposed. Note that after the stereoisomerization event all reaction
solutions were additionally derivatized with AQC to counterbalance the possible loss of AQC
during heat treatment and to show that really no Asp, Glu or Orn was produced.
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The following experiment show the racemization result for the other proteinogenic amino
acids as well as their [U-13C15N]-labelled analogs.
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< add Figure 5 >
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Figure 5 shows the XICs of AQC derivatized [U-13C15N]-labelled amino acids as well as the
XICs of Asn, Gln and Trp after stereoisomerization of a 0.025 mM L-[U-12C14N]- amino acid
(bold red) and 0.025 mM L-[U-13C15N]- amino acid mixture (without Asn, Gln and Trp) at 95
°C for 6 h. An overlay of XICs of all labelled and unlabeled amino acids is provided in Figure
S9 and illustrates that the [U-13C15N]-labelled amino acids are racemized to the same extent
as their unlabeled counterparts. For the calculation of retention factors and separation factors
1,3,5-tri-tert-butyl-benzene (TBB) was used as a void volume marker. A comparison with 4-
(dimethylamino)azobenzene showed that TBB was the better choice (Figure S10) 36. Note
that the separation factors are displayed beside the corresponding XICs in Figure 5. In case
of [U-13C15N]-Met ( [AQC-[U-13C15N]-Met + H]+: m/z 326.1202) the displayed peaks at 52.66
min and 59.08 min are not corresponding to AQC-[U-13C15N]-Met, but to isobaric AQC-His
([AQC-His + H]+: m/z 326.1248). Both, AQC-[U-13C15N]-Met and AQC-[U-12C14N]-Met were
oxidized in the presence of oxygen in the reaction tube. Even after re-derivatization no AQC-
[U-13C15N]-Met or AQC-[U-12C14N]-Met was found. Met was oxidized to methionine sulfoxide
([AQC-[U-13C15N]-Met-O + H]+: m/z 342.1151) and methionine sulfone ([AQC-[U-13C15N]-Met-
O2 + H]+: m/z 358.1100). To overcome this problem, the experiment was repeated after
ultasonication and purging of the microcentrifuge tube with nitrogen. The result was a
racemized and intact solution containing AQC-[U-13C15N]-Met and AQC-[U-12C14N]-Met
(Figure S11).
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A potential application product of this simple stereoisomerization process is the generation of
an isotopically labelled chiral metabolite standard mixture for internal standardization, which
includes the chiral peak assignment of the L- as well as the D-enantiomers. The application
36
of AQC-L-[U-13C15N]-amino acids for chiral elution order assignment
was shown for the
isobaric amino acids DL-Ile, DL-Leu and DL-Thr in Figure S6. In addition a chiral DL-[U-
13C15N]-amino acid standard mixture containing all amino acids would also allow a most
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accurate quantification of both enantiomers of every amino acid by counterbalancing an
enantioselective matrix effect induced by sample background matrix or the chromatographic
gradient elution conditions used. Due to the fact that an isotopically labeled internal standard
is subjected to the same matrix effects and ionization effects such as ion suppression or ion
enhancement as the target analyte, it corrects and normalizes quantitative results. A
comparison between an isocratic separation of racemic DL-Phe and AQC-DL-Phe 36 with the
separation of AQC-DL-Phe using a dual elution gradient, shows that under gradient elution
condition the first eluting AQC-D-Phe peak exhibits a much higher and broader peak
compared to its counterpart AQC-L-Phe (Figure S12).
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Considering that different types of real samples contain different percentages of D-amino
acids, the D-amino acid content within an isotopically labelled internal standard should meet
these requirements. Non-ribosomal lipopeptides, for instance contain not only an
unexpectedly large number of proteinogenic D-amino acids 49, which may even exceed the
racemic 50%-level 50, but also contain many uncommon amino acids 51, such as allo-
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threonine (aThr) 52, homoserine (Hse) 53, allo-isoleucine (aIle) and hydroxylproline (Hyp)
55, just to name a few. On the other hand, physiological fluids may contain trace amounts of
D-amino acids and require isotopically labelled IS with below 1% to 10% D-amino acids 56,57
.
< add Figure 6 >
Therefore, the preparation of scalemic DL-amino acid standards with defined D-amino acid
content was investigated. For this purpose two separate temperature and
stereoisomerization time studies were performed. Figure 6 shows the result for the
racemization of an amino acid standard test solution containing L-[U-13C15N] and L-[U-
12C14N]-amino acids at 55 °C, 75 °C and 95 °C for 10 min, 30 min and 60 min, while Figure 7
illustrates the results for the racemization of the same test solution at 95 °C for 1 h, 6 h and
15 h. In both cases the final concentration was 0.025 mM of scalemic mixtures containing
DL-[U-12C14N ]- amino acid and DL-[U-13C15N]-amino acids.
In general, only a slight stereoisomerization (below 1%) occurred for Val, Thr, Tyr, His, Lys
and Arg after incubation at 55 °C for 30 min. Surprisingly high values were found for [U-
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13C15N]-Ser, reaching above the 1% level. Therefore a derivatization time of 10 min at 55°C
should not be exceeded. Note that under this condition no racemization was observed.
However, significant racemization was only observed for the incubation experiment at 95 °C
for 30 min and 60 min, providing the highest values for Asn, Tyr, Glu, Gln, His and Val, which
went beyond the 5% D-amino acid level with highest values obtained for [U-12C14N]-Asn with
8% and [U-13C15N]-Tyr with 7%. Lowest values were obtained for Pro, which racemized
rather poorly.
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< add Figure 6 >
The follow-up study, employing 95 °C for racemization combined with increased incubation
times up to 15 h was performed in triplicates (n=3). Since a cleavage of the AQC-tag was
observed, all samples were re-derivatized with AQC prior to HPLC-ESI-QTOF-MS analysis
(sample (A)). To evaluate, if there was a general trend, whether the original L-amino acid or
the generated D-amino acid lost their AQC-tag predominantly, the 15 h incubated samples
were only diluted 1:10 with 0.2 M borate buffer without re-derivatization (sample (B)). A
comparison of D-amino acid levels of re-derivatized (A) and diluted (B) samples in Figure 7
showed that an overall larger percentage of L-amino acids had lost their AQC-tags, since the
diluted samples provided higher AQC-D-amino acid levels.
Hence it can be assumed that with increasing incubation time and temperature, more and
more AQC-tags will be cleaved leading to a natural recession in stereoisomerization, since
only AQC-tagged amino acids are isomerized. Furthermore note that the stereoisomerization
results for Met could only be achieved due to ultasonication of the samples after
derivatization with AQC and performing the stereoisomerization reaction in the absence of air
in inert nitrogen atmosphere (Figure S11). The same experiment as shown in Figure 7
performed in the presence of oxygen had led to the absence of AQC-Met as earlier shown in
Figure 5.
In general, an increase in incubation time from 1 h to 6 h and 15 h, increased the
racemization rate of all amino acids by 10% to 15%, with exception of Met, for which 20% D-
Met was found after 6 h of heating. Increasing the treatment time further had only led to the
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complete oxidation of Met. In actual fact, if a chiral [U-13C15N]-amino acid standard solution
with D-amino acid levels below 5% would be favored, 30 min incubation at 95 °C was
sufficient. For 1 h heat treatment at 95 °C, overall racemization rates from 2-10% were
observed, while for D-amino acid levels around 15-25%, which included Met, 6 h incubation
at 95 °C was adequate. An incubation time of 15 h, however, led to D-amino acid values
between 20% and 45%, depending on the amino acid, but this increase went hand in hand
with total loss of Met. Pro however showed maximum D-amino acid values around 3.5% with
little improvement with time. An additional study concerning the influence of sample solute
type, buffer type, as well as molarity and pH-value of the borate buffer on stereoisomerization
rate and amino acid composition will be discussed separately in a follow-up article 58.
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CONCLUSIONS
The here described stereoisomerization method is simple, derivatize your sample with AQC,
which takes 10 min, then ultrasonicate for 1 min, purge with inert gas for 1 min and heat the
sample for 30 min or up to 15 h at 95 °C on a thermoshaker. Stereoisomerization takes place
without loss of Gln, Asn or Trp. The generated D-amino acid levels depend on the
racemization temperature and the incubation time. If a D-amino acid content between 1-5%
is required, a stereoisomerization time of 30 min at 95 °C is sufficient. Increasing the
incubation time to 60 min provides [U-13C15N]-labeled amino acid standard mixtures
containing 2-10% D-amino acids, 6 h and 15 h incubation time, increases the D-amino acid
levels to 15-30% and 20-45%, respectively. Note that a slight racemization of <0.5% was
observed for 30 min to 60 min racemization time at 55 °C. Hence for chiral separation of
AQC-derivatized amino acids, the derivatization condition of 55 °C for 10 min should not be
exceeded in temperature nor derivatization time.
Chiral separation of all proteinogenic amino acids and some uncommon amino acids such as
aIle, aThr, Hse, Cit and theanine has been performed on a chiral zwitterionic Chiralpak
ZWIX(+) column using a dual elution gradient, which included a hydro-organic gradient from
2% to 52% water content and a flow gradient from 0.2 mL/min to 0.5 mL/min. This use of a
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dual gradient for enantiomer separation and fast elution leads unfortunately to
enantioselective matrix effects providing enantiomer peaks with different peak height and
peak areas, which may cause inaccurate results in LC-MS based enantioselective
metabolomics if only an L-enantiomer containing isotopically labelled metabolite solution is
used as an internal standard for the quantification of both L- and D-enantiomers. The use of
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a
chiral uniformly isotopically labelled internal standard would overcome such
enantioselective matrix effect issues in quantitative chiral analysis. The here presented
package of an easy and simple procedure to generate tailor made internal standard with
desired D-amino acids composition of natural and uncommon amino acids with desired D-
amino acid levels, combined with an universal chiral separation protocol for the zwitterionic
Chiralpak ZWIX(+) column, increases flexibility and will in the near future facilitate highly
accurate quantitative chiral amino acid metabolomics studies performed in a simple
derivatize, racemize & analyze approach.
ASSOCIATED CONTENT
Supporting Information
Reaction tubes sealed with Mµlti®-lid locks and Parafilm, stereoisomerization of AQC-L-Phg
in various solvent systems, stereoisomerization conditions tested on DNS, DNB, CBZ, FMOC
and NBD amino acids as well as AQC--D-Phe, chiral separation of isobaric AQC-Thr and
AQC-Ile isomers, XICs of scalemic mixtures of AQC-[U-12C14N]- and AQC-[U-13C15N ]-amino
acids, void volume markers for the Chiralpak ZWIX(+) column, and stereoisomerization of
AQC-Met under oxygen free condition and enantioselective matrix effect under different
elution conditions (PDF).
Author Contributions
The manuscript was written through the contributions of all authors. All authors have given
approval to the final version of the manuscript.
Notes: The authors declare no competing financial interest.
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REFERENCES
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(1) Kwok, W. H.; Choi, T. L. S.; Kwok, K. Y.; Chan, G. H. M.; Wong, J. K. Y.; Wan, T. S. M.
Doping control analysis of 46 polar drugs in horse plasma and urine using a 'dilute-and-
shoot' ultra high performance liquid chromatography-high resolution mass spectrometry
approach. J. Chromatogr. A 2016, 1451, 41-49.
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(2) Lawson, A. J.; Shipman, K. E.; George, S.; Dasgupta, I. A novel 'dilute-and-shoot' liquid
chromatography-tandem mass spectrometry method for the screening of
antihypertensive drugs in urine. J. Anal. Toxicol. 2016, 40, 17-27.
(3) Rodin, I.; Braun, A.; Stavrianidi, A.; Baygildiev, T.; Shpigun, O.; Oreshkin, D.;
Rybalchenko, I. 'Dilute-and-shoot' RSLC-MS-MS method for fast detection of nerve and
vesicant chemical warfare agent metabolites in urine. J. Anal. Toxicol. 2015, 39, 69-74.
(4) Vanhee, C.; Janvier, S.; Moens, G.; Deconinck, E.; Courselle, P. A simple dilute and
shoot methodology for the identification and quantification of illegal insulin. Journal of
Pharmaceutical Analysis 2016, 6, 326-334.
(5) Stokes, P.; O'Connor, G. Development of a liquid chromatography-mass spectrometry
method for the high-accuracy determination of creatinine in serum. J Chromatogr B
Analyt Technol Biomed Life Sci 2003, 794, 125-136.
(6) Gosetti, F.; Mazzucco, E.; Zampieri, D.; Gennaro, M. C. Signal suppression/enhancement
in high-performance liquid chromatography tandem mass spectrometry. J. Chromatogr.
A 2010, 1217, 3929-3937.
(7) Takayama, T.; Mochizuki, T.; Todoroki, K.; Min, J. Z.; Mizuno, H.; Inoue, K.; Akatsu, H.;
Noge, I.; Toyo'oka, T. A novel approach for LC-MS/MS-based chiral metabolomics
fingerprinting and chiral metabolomics extraction using a pair of enantiomers of chiral
derivatization reagents. Anal. Chim. Acta 2015, 898, 73-84.
(8) Ishii, C.; Akita, T.; Mita, M.; Ide, T.; Hamase, K. Development of an online two-
dimensional high-performance liquid chromatographic system in combination with
tandem mass spectrometric detection for enantiomeric analysis of free amino acids in
human physiological fluid. J. Chromatogr. A 2018, 1570, 91-98.
(9) Kimura, T.; Hamase, K.; Miyoshi, Y.; Yamamoto, R.; Yasuda, K.; Mita, M.; Rakugi, H.;
Hayashi, T.; Isaka, Y. Chiral amino acid metabolomics for novel biomarker screening in
the prognosis of chronic kidney disease. Sci. Rep. 2016, 6, 26137.
(10) Hamase, K.; Miyoshi, Y.; Ueno, K.; Han, H.; Hirano, J.; Morikawa, A.; Mita, M.; Kaneko,
T.; Lindner, W.; Zaitsu, K. Simultaneous determination of hydrophilic amino acid
enantiomers in mammalian tissues and physiological fluids applying a fully automated
micro-two-dimensional high-performance liquid chromatographic concept. J.
Chromatogr. A 2010, 1217, 1056-1062.
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(11) Weatherly, C. A.; Du, S.; Parpia, C.; Santos, P. T.; Hartman, A. L.; Armstrong, D. W. D-
Amino Acid Levels in Perfused Mouse Brain Tissue and Blood: A Comparative Study.
ACS Chem. Neurosci. 2017, 8, 1251-1261.
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(12) Wang, S.; Cyronak, M.; Yang, E. Does a stable isotopically labeled internal standard
always correct analyte response? A matrix effect study on a LC/MS/MS method for the
determination of carvedilol enantiomers in human plasma. J. Pharm. Biomed. Anal.
2007, 43, 701-707.
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(13) Jung, J.; Jeong, K.; Choi, Y.; Kim, S. A.; Kim, H.; Lee, J. W.; Kim, V. N.; Kim, K. P.; Kim,
J.-S. Deuterium-Free, Three-Plexed Peptide Diethylation for Highly Accurate
Quantitative Proteomics. J. Proteome Res. 2019, 18, 1078-1087.
(14) Chen, Y.-T.; Huang, H.-C.; Hsieh, Y.-J.; Fu, S.-H.; Li, L.; Chen, C.-L.; Chu, L. J.; Yu, J.-
S. Targeting amine- and phenol-containing metabolites in urine by dansylation isotope
labeling and liquid chromatography mass spectrometry for evaluation of bladder cancer
biomarkers. J. Food Drug Anal. 2019, 27, 460-474.
(15) Fountoulakis, M.; Lahm, H.-W. Hydrolysis and amino acid composition analysis of
proteins. J. Chromatogr. A 1998, 826, 109-134.
(16) Frank, H.; Woiwode, W.; Nicholson, G.; Bayer, E. Determination of the rate of acid-
catalyzed racemization of protein amino acids. Liebigs Ann. Chem. 1981, 354-365.
(17) Quitain, A. T.; Daimon, H.; Fujie, K.; Katoh, S.; Moriyoshi, T. Microwave-Assisted
Hydrothermal Degradation of Silk Protein to Amino Acids. Ind. Eng. Chem. Res. 2006,
45, 4471-4474.
(18) Ohmori, T.; Mutaguchi, Y.; Doi, K.; Ohshima, T. Effects of alkali or acid treatment on the
isomerization of amino acids. J. Biosci. Bioeng. 2012, 114, 457-459.
(19) Yamada, S.; Hongo, C.; Yoshioka, R.; Chibata, I. Method for the racemization of
optically active amino acids. J. Org. Chem. 1983, 48, 843-846.
(20) Boesten, W. H. J. Process for racemization of optically active amino acid amides. Eur.
Pat. Appl. 442585, 1991.
(21) Gillard, R. D.; Wootton, R. Reactions of some copper(II) complexes of the Schiff bases
of amino-acid esters. J. Chem. Soc. B 1970, 364-371.
(22) Takeda, R.; Kawamura, A.; Kawashima, A.; Sato, T.; Moriwaki, H.; Izawa, K.; Akaji, K.;
Wang, S.; Liu, H.; Acena, J. L.; Soloshonok, V. A. Chemical dynamic kinetic resolution
and S/R interconversion of unprotected α-amino acids. Angew. Chem., Int. Ed. 2014, 53,
12214-12217.
(23) Nian, Y.; Wang, J.; Zhou, S.; Dai, W.; Wang, S.; Moriwaki, H.; Kawashima, A.;
Soloshonok, V. A.; Liu, H. Purely Chemical Approach for Preparation of D-α-Amino
Acids via (S)-to-(R)-Interconversion of Unprotected Tailor-Made α-Amino Acids. J. Org.
Chem. 2016, 81, 3501-3508.
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(24) Matsuo, H.; Fujimoto, Y.; Tatsuno, T. A novel approach to the C-terminal determination
of peptides: selective 2H- and 3H-labelling reaction of C-terminal amino acids through
oxazolone. Tetrahedron Lett 1965, 3465-3472.
7
(25) Dressen, M. H. C. L.; van de Kruijs, B. H. P.; Meuldijk, J.; Vekemans, J. A. J. M.;
Hulshof, L. A. From Batch to Flow Processing: Racemization of N-Acetylamino Acids
under Microwave Heating. Org. Process Res. Dev. 2009, 13, 888-895.
(26) Uzuki, T.; Takahashi, M.; Noda, M.; Komachiya, Y.; Wakamatsu, H. Racemization of
optically active N-acyl amino acids. Ger. Offen. DE 2352579, 1974.
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(27) Jakubke, H. D. N-acyl peptides and their derivatives. Ger. (East) DD 50837, 1966.
(28) Grabley, S. Racemization of optically active amino acids. Ger. Offen. DE. 3334849 A1
19850404., 1985.
(29) Grabley, S. Process for the racemization of optically active amino acids. Germany.
US4638086, Jan. 20, 1987, 1987.
(30) Kuroda, H.; Kubo, S.; Chino, N.; Kimura, T.; Sakakibara, S. Unexpected racemization of
proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino
acids. Int. J. Pept. Protein Res. 1992, 40, 114-118.
(31) Reist, M.; Testa, B.; Carrupt, P.-A.; Jung, M.; Schurig, V. Racemization,
enantiomerization, diastereomerization, and epimerization: their meaning and
pharmacological significance. Chirality 1995, 7, 396-400.
(32) Cohen, S.; Michaud, D. Preparation and use of N-hydroxysuccinimidyl
heterocyclcarbamates. Eur. Pat. Appl. 533200, 1993.
(33) Cohen, S. A.; Michaud, D. P. Synthesis of a fluorescent derivatizing reagent, 6-
aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of
hydrolysate amino acids via high-performance liquid chromatography. Anal. Biochem.
1993, 211, 279-287.
(34) Nimura, N.; Iwaki, K.; Kinoshita, T.; Takeda, K.; Ogura, H. Activated carbamate reagent
as derivatizing agent for amino compounds in high-performance liquid chromatography.
Anal. Chem. 1986, 58, 2372-2375.
(35) Shimbo, K.; Oonuki, T.; Yahashi, A.; Hirayama, K.; Miyano, H. Precolumn derivatization
reagents for high-speed analysis of amines and amino acids in biological fluid using
liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid
Commun. Mass Spectrom. 2009, 23, 1483-1492.
(36) Horak, J.; Lämmerhofer, M. Stereoselective separation of underivatized and 6-
aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatized amino acids using
zwitterionic quinine and quinidine type stationary phases by liquid chromatography-high
resolution mass spectrometry. J Chromatogr A 2019, 1596, 69-78.
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(37) Hoffmann, C. V.; Pell, R.; Laemmerhofer, M.; Lindner, W. Synergistic effects on
enantioselectivity of zwitterionic chiral stationary phases for separations of chiral acids,
bases, and amino acids by HPLC. Anal. Chem. 2008, 80, 8780-8789.
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(38) Rizzi, A. M.; Cladrowa-Runge, S.; Jonsson, H.; Osla, S. Enantiomeric resolution of
derivatized DL-amino acids by high-performance liquid chromatography using a β-
cyclodextrin chiral stationary phase: A comparison between derivatization labels. J.
Chromatogr. A 1995, 710, 287-295.
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(39) Hellinger, R.; Horak, J.; Lindner, W. Enantioseparation of 6-aminoquinolyl-N-
hydroxysuccinimidyl carbamate tagged amino acids and other zwitterionic compounds
on cinchona-based chiral stationary phases. Anal. Bioanal. Chem. 2013, 405, 8105-
8120.
(40) Karakawa, S.; Shimbo, K.; Yamada, N.; Mizukoshi, T.; Miyano, H.; Mita, M.; Lindner, W.;
Hamase, K. Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using
chiral high-performance liquid chromatography-tandem mass spectrometry and its
application to the rat plasma and tissues. J. Pharm. Biomed. Anal. 2015, 115, 123-129.
(41) Piasek, Z.; Urbanski, T. Tautomerism of urea. Tetrahedron Lett. 1962, 723-727.
(42) Thalheim, T.; Wagner, B.; Kuehne, R.; Middendorf, M.; Schueuermann, G. A Branch-
and-Bound Approach for Tautomer Enumeration. Mol. Inf. 2015, 34, 263-275.
(43) West, A. C.; Schmidt, M. W.; Gordon, M. S.; Ruedenberg, K. A Comprehensive Analysis
in Terms of Molecule-Intrinsic, Quasi-Atomic Orbitals. III. The Covalent Bonding
Structure of Urea. J. Phys. Chem. A 2015, 119, 10368-10375.
(44) Jiang, J.; Pavlova, N. N.; Zhang, J. Asparagine, a critical limiting metabolite during
glutamine starvation. Mol. Cell. Oncol. 2018, 5, e1441633/1-e1441633/2.
(45) Marquez, J.; Alonso, F. J.; Mates, J. M.; Segura, J. A.; Martin-Rufian, M.; Campos-
Sandoval, J. A. Glutamine Addiction In Gliomas. Neurochem. Res. 2017, 42, 1735-1746.
(46) Ekborg-Ott, K. H.; Taylor, A.; Armstrong, D. W. Varietal Differences in the Total and
Enantiomeric Composition of Theanine in Tea. J. Agric. Food Chem. 1997, 45, 353-363.
(47) Cheng, S.; Fu, X.; Wang, X.; Liao, Y.; Zeng, L.; Dong, F.; Yang, Z. Studies on the
Biochemical Formation Pathway of the Amino Acid L-Theanine in Tea (Camellia
sinensis) and Other Plants. J. Agric. Food Chem. 2017, 65, 7210-7216.
(48) Papadia, C.; Osowska, S.; Cynober, L.; Forbes, A. Citrulline in health and disease.
Review on human studies. Clin. Nutr. 2018, 37, 1823-1828.
(49) Cava, F.; Lam, H.; de Pedro, M. A.; Waldor, M. K. Emerging knowledge of regulatory
roles of D-amino acids in bacteria. Cell. Mol. Life Sci. 2011, 68, 817-831.
(50) Gross, H.; Loper, J. E. Genomics of secondary metabolite production by Pseudomonas
spp. Nat. Prod. Rep. 2009, 26, 1408-1446.
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(51) Suessmuth, R. D.; Mainz, A. Nonribosomal peptide synthesis-Principles and Prospects.
Angew. Chem., Int. Ed. 2017, 56, 3770-3821.
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(52) De Vleeschouwer, M.; Sinnaeve, D.; Van den Begin, J.; Coenye, T.; Martins, J. C.;
Madder, A. Rapid total synthesis of cyclic lipodepsipeptides as a premise to investigate
their self-assembly and biological activity. Chem. Eur. J. 2014, 20, 7766-7775.
(53) Emanuele, M. C.; Scaloni, A.; Lavermicocca, P.; Jacobellis, N. S.; Camoni, L.; Di
Giorgio, D.; Pucci, P.; Paci, M.; Segre, A.; Ballio, A. Corpeptins, new bioactive
lipodepsipeptides from cultures of Pseudomonas corrugata. FEBS Lett. 1998, 433, 317-
320.
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(54) Horak, J.; Gerhardt, H.; Theiner, J.; Lindner, W. Correlation between amino acid
racemization and processing conditions for various wheat products, oil seed press cakes
and lignin samples. Food Bioprod. Process. 2014, 92, 355-368.
(55) Sabiullah, M.; Suleman, M. D.; Rehmatunissa; Ather, M.; Nasreen. Urinary hydroxy-
proline estimation is cheap biochemical marker of bone turnover in post menopausal
women. J. Drug Delivery Ther. 2015, 5, 1-2.
(56) Armstrong, D. W.; Gasper, M.; Lee, S. H.; Zukowski, J.; Ercal, N. D-Amino acid levels in
human physiological fluids. Chirality 1993, 5, 375-378.
(57) Takayama, T.; Mizuno, H.; Toyo'oka, T.; Akatsu, H.; Inoue, K.; Todoroki, K. Isotope
Corrected Chiral and Achiral Nontargeted Metabolomics: An Approach for High Accuracy
and Precision Metabolomics Based on Derivatization and Its Application to
Cerebrospinal Fluid of Patients with Alzheimer's Disease. Anal. Chem. 2019, 91, 4396-
4404.
(58) Horak, J.; Laemmerhofer, Method for the Stereoisomerization of Chiral Compounds.
Eur. Pat. Appl. 18197409, Sept. 28, 2018
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Figure 1. Stereoselective separation of scalemic AQC-DL-[U-13C15N]-Phe, APC-DL-Phe and
AC-DL-[U-13C15N]-Phe on a Chiralpak ZWIX(+) column (150 x 4 mm; 3 µm) using the dual
gradient elution method with HPLC-ESI-QTOF-MS, after racemization at 95 °C for 18 h. Note
that only representative chromatograms are shown, since racemization was performed with
sample mixtures containing L-[U-12C14N]- and L-[U-13C15N]- amino acids.
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Figure 2. Stereoselective separation of scalemic mixtrures of isobaric isomers of a) AQC-Thr
([AQC-Thr + H]+: m/z 290.1135) and b) AQC-Ile ([AQC-Ile + H]+: m/z 302.1499) on a
Chiralpak ZWIX(+) column (150 x 4 mm, 3 µm) using the dual gradient elution method with
HPLC-ESI-QTOF-MS, after epimerization of L-Thr, L-aThr, DL-Thr and DL-aThr as well as L-
Ile, L-aIle, DL-Ile and DL-aIle for 18 h at 95 °C. Note that newly generated isomers are
highlighted in bold red.
Figure 3. XICs of AQC derivatized a) DL-Asn, DL-Asp, DL-Gln and DL-Glu; b) XIC of AQC
derivatized L-Asn and L-Gln before racemization and c) after racemization for 15 hours at 95
°C under nitrogen gas. The XIC traces for Asp and Glu in b) and c) show that no Asp and no
Glu were present before nor after the racemization process. Stereoselective separation was
performed with a Chiralpak ZWIX(+) column (150 x 3 mm, 3 µm) using a short gradient
elution method in combination with HPLC-ESI-QTOF-MS analysis.
Figure 4. XICs of AQC derivatized a) L-theanine and L-Cit as well as their deamidation
products AQC-Glu and AQC-Orn before racemization and b) after racemization for 15 hours
at 95 °C under nitrogen gas. Note that no deamidation products were observed before nor
after the racemization process. Stereoselective separation was performed with a Chiralpak
ZWIX(+) column (150 x 4 mm, 3 µm) using the dual gradient elution method in combination
with HPLC-ESI-QTOF-MS.
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Figure.5. XICs of scalemic AQC-derivatized [U-13C15N]-amino acids (black; without Asn, Gln
and Trp) together with three [U-12C14N]-amino acids, namely the missing amino acids Asn,
Gln and Trp (red; the remaining amino acids are shown in the Supporting Information) after
racemization for 6 h at 95 °C. Separation factors (L-enantiomer/D-enantiomer) are provided
using a void time of 7.041 min. Note that for L-Ile and L-Thr, racemization will provide L-aIle
and L-aThr, hence (L/D) are shown for (L-Ile/D-Ile), (L-Ile/D-aIle), (L-Thr/D-Thr) and
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(L-Thr/D-aThr). Reference chromatograms for the separation of DL-aIle and DL-aThr are
provided in the Supporting Information. Stereoselective separation was performed on
Chiralpak ZWIX(+) (150 x 4 mm, 3 µm) using the dual gradient elution method with HPLC-
ESI-QTOF-MS in positive ionization mode. Gaussian Smoothing Factor 2.5.
Figure6. D-Amino acid content in percent after stereoisomerization of a solution containing
0.025 mM AQC-derivatized a) L-[U-12C14N]-amino acids (20 amino acids) and b) L-[U-
13C15N]-amino acids (17 amino acids without Asn, Gln and Trp) for 10 min, 30 min and 1 h at
55 °C, 75 °C and 95 °C. D-Amino acid results were normalized using the peak areas of the
corresponding enantiomers from a racemic AQC-[U-12C14N]-DL-amino acid mixture.
Stereoselective separation was performed on the Chiralpak ZWIX(+) column (150 x 4 mm, 3
µm) using the dual gradient elution method with HPLC-ESI-QTOF-MS.
Figure 7. D-Amino acid content in percent after stereoisomerization of an AQC-derivatized
amino acid solution containing 0.025 mM of a) L-[U-12C14N]-amino acids (20 amino acids)
and b) L-[U-13C15N]-amino acids (without Asn, Gln and Trp) for 1 h, 6 h and 15 h at 95 °C.
Experiments were performed in triplicates (n=3). All samples were centrifuged after reaching
room temperature, their volumes were adjusted to 100 µL and they were additionally
derivatized with AQC (samples (A)), which resembles a 1:10 dilution. Additionally, the 15 h
samples were only diluted 1:10 with 0.2 M borate buffer, without additional re-derivatization
(samples (B)). Sample (B) showed higher D-amino acid levels compared to sample A, which
illustrates the cleavage of the AQC tag from the L-enantiomer and not from the
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stereoisomerized D-enantiomers. Stereoselective separation was performed on a Chiralpak
ZWIX(+) column (150 x 4 mm, 3 µm) using the dual gradient elution method with HPLC-ESI-
QTOF-MS.
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Figure 1. Stereoselective separation of scalemic AQC-DL-[U-13C N]-Phe, APC-DL-Phe and AC-DL-[U-
13 15
C
N]-Phe on a Chiralpak ZWIX(+) column (150 x 4 mm; 3 µm) using the dual gradient elution method
with HPLC-ESI-QTOF-MS, after racemization at 95 °C for 18 h. Note that only representative chromatograms
12 14
are shown, since racemization was performed with sample mixtures containing L-[U-
C
N]- and L-[U-
13 15
C
N]- amino acids.
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Figure 2. Stereoselective separation of scalemic mixtrures of isobaric isomers of a) AQC-Thr ([AQC-Thr +
H] : m/z 290.1135) and b) AQC-Ile ([AQC-Ile + H]+: m/z 302.1499) on a Chiralpak ZWIX(+) column (150
x 4 mm, 3 µm) using the dual gradient elution method with HPLC-ESI-QTOF-MS, after epimerization of L-
Thr, L-aThr, DL-Thr and DL-aThr as well as L-Ile, L-aIle, DL-Ile and DL-aIle for 18 h at 95 °C. Note that
newly generated isomers are highlighted in bold red.
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Figure 3. XICs of AQC derivatized a) DL-Asn, DL-Asp, DL-Gln and DL-Glu; b) XIC of AQC derivatized L-Asn
and L-Gln before racemization and c) after racemization for 15 hours at 95 °C under nitrogen gas. The XIC
traces for Asp and Glu in b) and c) show that no Asp and no Glu were present before nor after the
racemization process. Stereoselective separation was performed with a Chiralpak ZWIX(+) column (150 x 3
mm, 3 µm) using a short gradient elution method in combination with HPLC-ESI-QTOF-MS analysis.
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