Dimethylcysteine (DiCys)/o-Phthalaldehyde Derivatization for Chiral Metabolite Analyses: Cross-Comparison of Six Chiral Thiols

Metabolomics profiling using liquid chromatography-mass spectrometry (LC-MS) has become an important tool in biomedical research. However, resolving enantiomers still represents a significant challenge in the metabolomics study of complex samples. Here, we introduced N,N-dimethyl-l-cysteine (dimethylcysteine, DiCys), a chiral thiol, for the o-phthalaldehyde (OPA) derivatization of enantiomeric amine metabolites. We took interest in DiCys because of its potential for multiplex isotope-tagged quantification. Here, we characterized the usefulness of DiCys in reversed-phase LC-MS analyses of chiral metabolites, compared against five commonly used chiral thiols: N-acetyl-l-cysteine (NAC); N-acetyl-d-penicillamine (NAP); isobutyryl-l-cysteine (IBLC); N-(tert-butoxycarbonyl)-l-cysteine methyl ester (NBC); and N-(tert-butylthiocarbamoyl)-l-cysteine ethyl ester (BTCC). DiCys and IBLC showed the best overall performance in terms of chiral separation, fluorescence intensity, and ionization efficiency. For chiral separation of amino acids, DiCys/OPA also outperformed Marfey’s reagents: 1-fluoro-2-4-dinitrophenyl-5-l-valine amide (FDVA) and 1-fluoro-2-4-dinitrophenyl-5-l-alanine amide (FDAA). As proof of principle, we compared DiCys and IBLC for detecting chiral metabolites in aqueous extracts of rice. By LC–MS analyses, both methods detected twenty proteinogenic l-amino acids and seven d-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val), but DiCys showed better analyte separation. We conclude that DiCys/OPA is an excellent amine-derivatization method for enantiomeric metabolite detection in LC-MS analyses.

Initial interests in chiral metabolomics began with D-amino acids, which are physiologically active substances in mammals [16,17]. In fact, D-serine, D-aspartate, D-alanine, and D-cysteine are found in many tissues and body fluids, and several D-amino acids are important neurotransmitters in the brain [18,19]. Enantiomeric amino acids and their derivatives may be useful biomarkers and novel drug candidates; their detection is important in pharmacological research, clinical analysis, agriculture, and food science [20][21][22]. Using isotope tagging, advanced MS instrumentation, and new MS data analysis schemes, it is possible to carry out non-targeted chiral metabolomics profiling and discover novel chiral biomarkers beyond just amino acids [23].
In advanced chiral metabolomics profiling, labeling with heavy isotopes is very important for quantification. To our knowledge, no study has introduced isotope labels via thiol/OPA derivatization. We are particularly interested in developing N,N-dimethyl-Lcysteine (DiCys) with OPA as a potential strategy for isotope tags in chiral metabolomics. DiCys can be easily synthesized in one step from L-cysteine by reductive amination (dimethyl labeling), using formaldehyde (CH2O) and sodium cyanoborohydride (NaBH3CN). The fact that CD2O, 13 CH2O, 13 CD2O and NaBD3CN are commercially available at relatively low costs means that +2, +4, +6, and +8 Da tags can be easily generated via dimethyl labeling [35,36]. Moreover, 13 C-and 15 N-cysteines are also commercially available, which means that up to 10-plex isotope labeling (+0 -+9 Da) is feasible ( Figure S1, supplementary materials).
Due to the potential of DiCys/OPA as a versatile isotope-labeling method, this study sought to understand its performance in standard RP-LC-MS analyses of chiral metabolites. DiCys was evaluated against five commonly used chiral thiols: NAC, NAP, IBLC, NBC, and BTCC. The reaction mechanism of DiCys/OPA with amines is shown in Figure  1a, and the chemical structures of the other thiols are shown in Figure 1b. They were compared based on their fluorescence intensity, separation performance, stability, and ionization efficiency for amino acid enantiomers. DiCys/OPA was also compared against Marfey's reagents, which are commonly used for resolving chiral amino acids. Finally, we compared DiCys against IBLC in identifying D-amino acids in aqueous extracts of rice. Our data suggest that DiCys/OPA is an excellent derivatization method to resolve chiral amines in RP-LC-MS metabolomics profiling.

Stability and Fluorescence of DiCys Derivatives
Some of the most abundant amine-containing metabolites in biological samples are amino acids. L and D amino acid pairs are also among the most important enantiomeric metabolites in terms of biological functions. The charged carboxylate group makes it

Stability and Fluorescence of DiCys Derivatives
Some of the most abundant amine-containing metabolites in biological samples are amino acids. L and D amino acid pairs are also among the most important enantiomeric metabolites in terms of biological functions. The charged carboxylate group makes it somewhat challenging to resolve all 20 proteinogenic amino acids by RP-HPLC. Therefore, we chose amino acids as model metabolites to study DiCys/OPA derivatization.
One of the reported disadvantages of OPA/thiol derivatization is the instability of the product [24,25]. Here, we evaluated the stability of OPA adducts with DiCys and five additional chiral thiols-NAC, NAP, IBLC, NBC, and BTCC. We monitored the fluorescence intensities of OPA/thiol-derivatized amino acids at 4 • C for 60 min ( Figure S2 supplementary materials), and there was no visible sign of fluorophore breakdown, consistent with previous reports [37]. This should therefore be stable enough for routine LC-MS workflows.
We also quantified the fluorescence intensities of five L-amino acids derivatized with six chiral thiols ( Figure 2a) after HPLC separation. Our results indicated that IBLC, NAC, and DiCys derivatives produced stronger fluorescence. In contrast, the NAP and NBC derivatives exhibited very low fluorescence intensities. somewhat challenging to resolve all 20 proteinogenic amino acids by RP-HPLC. Therefore, we chose amino acids as model metabolites to study DiCys/OPA derivatization. One of the reported disadvantages of OPA/thiol derivatization is the instability of the product [24,25]. Here, we evaluated the stability of OPA adducts with DiCys and five additional chiral thiols-NAC, NAP, IBLC, NBC, and BTCC. We monitored the fluorescence intensities of OPA/thiol-derivatized amino acids at 4 °C for 60 min ( Figure S2 supplementary materials), and there was no visible sign of fluorophore breakdown, consistent with previous reports [37]. This should therefore be stable enough for routine LC-MS workflows.
We also quantified the fluorescence intensities of five L-amino acids derivatized with six chiral thiols ( Figure 2a) after HPLC separation. Our results indicated that IBLC, NAC, and DiCys derivatives produced stronger fluorescence. In contrast, the NAP and NBC derivatives exhibited very low fluorescence intensities.

Separation of Enantiomers
When Chernobrrovkin et al. compared NAC, NAP, IBLC, and NMC as chiral thiols for OPA derivatization [33], they found that NAC and NMC provided better chiral resolution than NAP and IBLC. However, the resolution factors may depend on the column;

Separation of Enantiomers
When Chernobrrovkin et al. compared NAC, NAP, IBLC, and NMC as chiral thiols for OPA derivatization [33], they found that NAC and NMC provided better chiral resolution than NAP and IBLC. However, the resolution factors may depend on the column; mobile phase composition; flow rate; and gradient [38,39]. We previously found that optimal elution condition for OPA adducts was around pH 8 instead of the typical acidic condi-tions [40]. Therefore, we conducted RP-LC separation at pH 8 to resolve five enantiomer pairs (Glu, Ser, Ala, Tyr, and Phe) ( Figure 3). The best resolution was obtained with IBLC and DiCys, and the worst was with BTCC (Table S1 supplementary materials). Quantitative conversion to derivatized products for both enantiomers and the lack of racemization were confirmed by MS detection.
Molecules 2021, 26, x FOR PEER REVIEW 4 of 11 mobile phase composition; flow rate; and gradient [38,39]. We previously found that optimal elution condition for OPA adducts was around pH 8 instead of the typical acidic conditions [40]. Therefore, we conducted RP-LC separation at pH 8 to resolve five enantiomer pairs (Glu, Ser, Ala, Tyr, and Phe) (Figure 3). The best resolution was obtained with IBLC and DiCys, and the worst was with BTCC (Table. S1 supplementary materials). Quantitative conversion to derivatized products for both enantiomers and the lack of racemization were confirmed by MS detection.

Ionization Efficiency and MS/MS Properties
Amino acids exhibit low ionization efficiencies in ESI-MS experiments, and OPA derivatization may bring significant enhancements [40]. As shown in Figure 2b, all six thiol adducts have shown 25-100-fold higher ionization efficiencies over non-derivatized amino acids, making them generally useful for ESI-MS detection. In MS analyses, it is easy to identify derivatized amino acid enantiomers in the mass chromatogram based on doublepeak detection via selected ion monitoring. To fragment OPA adducts requires relatively high collision energies: around 20 V [40]. The fragmentation patterns of DiCys/OPA adducts with seven amino acids are shown in Figure S3 supplementary materials, with a neutral loss of the thiol group in all cases.

Enantiomer Identification in Rice Water with DiCys/OPA
To test the usefulness of DiCys/OPA, we analyzed the aqueous extracts of edible rice, otherwise known as rice water. Rice water is the starchy water that remains after soaking or cooking rice, containing vitamins, amino acids, and minerals. It has been used traditionally in the treatment of skin and hair in Asian countries [45][46][47]. Little is known about the composition of amino acid enantiomers in rice water. Therefore, we separately applied DiCys/OPA and IBLC/OPA derivatization to rice water samples. Their RP-HPLC chromatograms are shown in Figure 4, and we observed almost twice as many visible fluorescent peaks with DiCys compared to IBLC. It shows that DiCys is suitable for separating a wide range of naturally occurring amine metabolites.
By MS and MS/MS detection, we could identify all twenty proteinogenic L-amino acids and seven D-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val) in rice water samples using either DiCys or IBLC. Figure 5 shows the integrated ion intensities of individual amino acids. The retention time, precursor ion, and product ion information are listed in Tables S2 and S3 supplementary materials. The ratios between D/L amino acids are shown in Table 1. Interestingly, the highest D/L ratios were found for the two positively charged amino acids, Arg and Lys. Their physiological roles and gustatory effects remain undetermined.  By MS and MS/MS detection, we could identify all twenty proteinogenic L-amino acids and seven D-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val) in rice water samples using either DiCys or IBLC. Figure 5 shows the integrated ion intensities of individual amino acids. The retention time, precursor ion, and product ion information are listed in Tables S2 and S3 supplementary materials. The ratios between D/L amino acids are shown in Table 1. Interestingly, the highest D/L ratios were found for the two positively charged amino acids, Arg and Lys. Their physiological roles and gustatory effects remain undetermined.

Synthesis of N,N-Dimethyl-L-Cysteine
A total of 100 mg of L-cysteine was dissolved in 10 mL dilute HCl (pH 2.5) and mixed with 8.25 mmol of sodium cyanoborohydride (NaBH 3 CN) for 10 min at 4 • C. Then, 8.25 mmol of formaldehyde (37% w/w) was added, stirred for 30 min, and the reaction was monitored by ninhydrin staining on thin-layer chromatography plates. The DiCys product was purified via silica-gel column chromatography using MeOH/CH 2 Cl 2 . DiCys fraction was acidified to pH 2.5 by adding 0.1 N HCl and evaporated to dryness at 60 • C. DiCys powder was dissolved in deionized water and quantified using the Ellman assay. The reaction yield was 87%. HRMS (ESI/Q-TOF) m/z: M = C 5 H 11 NO 2 S, calculated for [M + H] + = 150.0583, found 150.0589.

Rice Water Preparation
Sushi rice samples were purchased from a local grocery store in Taiwan. In total, 50 g of the dried rice was placed in 50 mL of deionized water. After shaking for 30 min, the solution was passed through filter paper. The rice water was lyophilized and dissolved in 250 µL of 0.01% perchloric acid and filtered twice through 0.22 µm nylon filters. Finally, we quantified total amines using a fluorescamine assay [48].

Derivatization Reactions
The following reagents were prepared freshly before use:

Conclusions
We systematically evaluated the suitability of six chiral thiols (DiCys, NAC, NAP, IBLC, NBC, and BTCC) for OPA-assisted separation of amino acid enantiomers. The best separation efficiencies in RP-HPLC were found with DiCys and IBLC. For fluorescence detection, IBLC, NAC, and DiCys gave stronger signals while NAP only gave very weak signals. All six reagents enhanced ionization efficiencies by 25-100 fold, useful for MS detection. Previously, IBLC has been a popular reagent for resolving chiral amino acids [30,49], and our data supported its usefulness. More importantly, our study was the first to introduce DiCys/OPA for enantiomeric separation, and its performance was comparable to IBLC in our tests. DiCys also outperformed Marfey's reagents FDAA and FDVA, which were specially developed for chiral separation purposes. We conclude that DiCys is a highly versatile reagent for resolving enantiomeric amines in chiral metabolomics experiments.
The greatest advantage of DiCys is its potential for multiplex heavy-isotope labeling. Using well-established chemistries [35], heavy isotope versions from +1 Da to +9 Da may be easily synthesized in one step using commercially available reagents. This may provide 10-plex labeling at an affordable cost for high-throughput metabolomics experiments. Combined with fluorogenic detection and excellent chiral separation, DiCys is one of the most versatile amine derivatization reagents currently available.
In real-world metabolomics profiling of rice water, DiCys provided better separation of amine metabolites than IBLC. Both allowed us to detect twenty proteinogenic L-amino acids and identify seven D-amino acids-Ala, Arg, Lys, Phe, Ser, Tyr, and Val. These D-amino acids are primarily associated with sweetness for humans [50,51], suggesting that D-amino acids may be important for the gustatory taste. Moreover, D-amino acids synthesized by gut microbiomes may affect our immune systems [52,53]. How D-amino acids in rice diet may affect our gut microbiome-immune axis will warrant further investigation.
Supplementary Materials: The following are available online: Figures S1-S4 and Tables S1-S3.