Pair of Stereodynamic Chiral Benzylicaldehyde Probes for

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A Pair of Stereodynamic Chiral Benzylicaldehyde Probes for Determination of Absolute Configuration of Amino Acid Residues in Peptides by Mass Spectrometry Lin Wang, Zhe Jin, Xiayan Wang, Su Zeng, cuirong sun, and Yuanjiang Pan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03804 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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Analytical Chemistry

A Pair of Stereodynamic Chiral Benzylicaldehyde Probes for Determination of Absolute Configuration of Amino Acid Residues in Peptides by Mass Spectrometry Lin Wang,†,‡,§ Zhe Jin,§ Xiayan Wang,‡ Su Zeng,† Cuirong Sun,†,* Yuanjiang Pan§,* †

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China Department of Chemistry and Chemical Engineering, Beijing University of Technology, 100124, Beijing, China § Department of Chemistry, Zhejiang University, Hangzhou 310027, Zhejiang China ‡

ABSTRACT: This paper describes a simple method to determine the absolute configuration of amino acids residues in peptides by mass spectrometry using a newly developed pair of mass-tagged chiral probes without the requirement of reference standards. A pair of benzylicaldehyde probes, 1-(S)-1H in S configuration and 2-(R)-2D in deuterium-labeled R configuration with the ratio of 1:1, were synthesized for in-situ condensation with amino acid residues and transformed into a pair of stereodynamic imine products. The characteristic intensity difference observed in mass spectrometry can be used to determine the absolute configuration and to quantify the enantiomeric composition of chiral amino acid residues. Significant chiral recognition ability was achieved for eighteen natural chiral amino acids and for one β-amino acid by comparing the ion intensity ratio of imine products I[1-(S)-1H -AA]- to I[2-(R)-2D-AA]-. For 16 kinds of amino acids, the L form of the amino acids was more reactive with 1-(S)-1H, while D configuration amino acids preferred to react with 2-(R)-2D. However, for three kinds of amino acid, the opposite result was obtained. The configurations of the residues in the peptides, Phe-Tyr-Ala, D-Phe-Tyr-Ala, Val-Pro-Phe-D-Leu-Met, Val-Pro-Phe-Leu-D-Met, as well as in a natural peptide with unknown chirality were determined by acid hydrolysis followed by the present method. In addition, molecular modeling results illustrate that the recognition process is mainly controlled by kinetic factors. Using the new probes coupled with a mass spectrometry approach avoids time-consuming workup and separation steps. We expect that the probes could be applied as tools to determine the absolute configuration of amino acid residues in proteins in future research.

Chiral molecules have attracted considerable attention from organic and pharmaceutical chemists. Peptides and their basic amino acid units (AA) are especially interesting because they are important for the biological functions and activities. Damino acid-containing peptides share the same sequences with their all-L isomers. But in many cases, the conversion of the amino acid from the L to D form within peptides and proteins induces peculiar folding of the peptides, increases their structural diversity and generally results in modified biological activity. One of the most elegant examples of protein chirality and its relation to functionality was provided by Kent et al.1 They demonstrated that D-HIV protease retained activity, but the enzyme specificity was limited to the substrates and inhibitors of a chirality opposite to the natural L-form. Besides, D-amino acids have been identified in bacterial cell wall,2 cone snails,3- 4 and some mammals.5-6 Isomerized aspartyl residues in β-amyloid peptide Aβ (1-42) at positions 7 and 23 are demonstrated to have relevance in Alzheimer.7 Opioid peptides, such as natural dermorphin containing a D-Ala residue, shows better antimicrobial and analgesic activities than its all L-analog.8 As increasing number of peptides containing D-amino acids are found exhibiting different activities compared to peptides containing L-amino acids, reliable methods that can be used to determine the absolute configuration of amino acid residues in peptides are highly desirable. Sensitive determination of the configurations of amino acid residues in peptides or proteins remains a challenge, as this post-translational modification does not induce any change in molecular mass. A common approach is to hydrolyze the chiral peptides using acid or enzyme followed by liquid chromatography (LC) 9 or capillary electrophoresis (CE)10- 11 by comparing the migration time to the standard. Marfey’s reagent coupled with LC-MS is one of the most extensively used method for the determination of the enantiomeric purity of amino acids, short peptides and drug compounds containing

amine groups.12 This method has enough reproducibility and accuracy for quantitative analysis. The reaction often takes one to two hours under mild alkaline conditions and needs to be protected from light. Meanwhile, the choice of the stationary phase, internal standard and data interpretation method can cause a lot of variability. Tandem mass spectrometry (MS/MS) can also be used to characterize peptides containing D-amino acids, as peptides with D-amino acid substitutions often display different collision active dissociation behaviors13-16 or electron capture dissociation patterns.17-18 Most MS/MS-based approaches introduce a functional group for the peptide epimers to better discriminate between them. A kinetic method described by Cooks et al, which is based on the dissociation of metal-bound trimeric complex ions of peptide epimers, allows for good discrimination.19-21 Sachon et al. demonstrated that acetylation is an easy protocol for the differentiation of peptide containing a D-residue close to the N-terminus by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF-TOF) mass spectrometry.22 With this method, Sweedler et al successfully distinguished endogenous D-amino acid-containing neuropeptides from individual neurons.23 An alternative strategy is to label the peptides with a chromophore, and then to use radical-directed dissociation (RDD) to produce different backbone dissociation product ions. This method achieves high chiral recognition values and has the advantage of the greatest flexibility in terms of charge state selection.24-26 In addition, the combination of hydrogen/deuterium exchange and tandem MS was also reported to identify the racemization sites in immunoglobulin protein.27 Such MS-based approaches exhibit high sensitivity and good reproducibility. However, they are not suitable for the analysis of compounds with unknown chirality, since standards are required to validate the observed fragmentation pattern. Therefore, it is essential to develop a fast and sensitive MS-based strategy to characterize

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the absolute configuration of individual amino acid residues in unknown peptides without using standards.

Figure 1. Structures of the probes. Chemical derivatization, especially using probes with isotope labeling, has been proven to be an efficient approach for the identification of small chiral molecules in complicated matrixes. 28-30 Herein, a pair of mass-tagged chiral benzylicaldehyde probes, 1-(S)-1H and 2-(R)-2D, was designed for determining the absolute configuration of amino acid residues in peptides, with several structural features shown in Figure 1. The rationale for the design of these two probes is as follows. First, the salicylaldehyde ring (the presence of an adjacent phenol moiety) in the stereodynamic probe can accelerate the condensation reaction between the formyl unit and amino group, facilitating efficient enantiomer recognition.31 Second, the proline unit is an effective skeleton for designing chiral selectors because of its non-rotational chiral center. Third, the formation of an imine results in new stereochemical bias. As we demonstrated below, the pair of 1 H/2D mass-tagged probes is beneficial for the direct determination of the configuration in one mass spectrum when the S and R probes are presented in a 1:1 ratio. For the reaction, a total volume of 500 µL of anhydrous acetonitrile containing 10 µmol of the probes mixture [1-(S)1 H and 2-(R)-2D, 1:1] and 1 µmol of phenylalanine (Phe) was stirred at room temperature. The derivatization process is simple with mild reaction conditions and does not require any special equipment. A typical full scan mass spectrum for the analysis of Phe is depicted in Figure 2. A condensation reaction occurred between Phe and the probes resulting in the loss of one H2O and the appearance of two product ions at m/z 469 and 474, corresponding to the pair of imines. Interestingly, the intensity of the ion at m/z 469 (S, S) was higher than that of the ion at m/z 474 (S, R) for L-Phe (Figure 2B). In contrast, for D-Phe (Figure 2C), the result was the opposite, with a higher intensity observed for m/z 474 (R, R). This finding suggests that the intensity of product ion is related to the absolute configuration of the amino acid and the reaction is controlled by the isomeric differences. It should be noted that the L-Phe tends to produce the light form of the imine(S, S), while D-Phe tends to produce the heavy isotope-labeled form of the imine (R, R). To understand the underlying interactions that cause large differences in chiral selectivity, reaction rate measurements were conducted. For kinetic measurements, 1-(S)-1H and 2(R)-2D probes were allowed to react with either L or D Phe for different time points. Flow injection mass spectra were recorded to obtain the conversion rate by integrating the imine peak areas. The changes in the relative amounts of imine products reflect the different rate constants, which are used to define the configuration of the starting material according to the rate difference. It was found that L-Phe reacts faster with 1-(S)-1H than with 2-(R)-2D by a ratio of 6.5, while D-Phe

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prefers to react with 2-(R)-2D by a ratio of 5.9 compared to 1(S)-1H (Figure S26 in the Supporting Information). The results demonstrated that the pair of probes has stereodynamic selectivity when reacting with chiral amino acids. Subsequently, 1-(S)-1H probe was allowed to react with D-Phe for 120 min to confirm that the imine product (S, R) was formed. Then probe 2-(R)-2D was added and the imine products were detected at different time points including 0, 10, 20, 30, 40, 50 and 60 min. Chiral recognition ability (CR) was defined as the ratio of ion intensity of two imine products I[11 2 (S)- H -AA] to I[2-(R)- D-AA] . The results showed that the amount of the newly generated (R, R) imine increase quickly and exceeded the (S, R) imine, until the CR remained stable at approximately 0.45. The complimentary reaction was started with probe 2-(R)-2D and 1-(S)-1H was added second. Only a small amount of newly generated (S, R) imine was detected (Figure S27 in the Supporting Information). All the results suggest that the reaction is controlled by kinetic selectivity and that the intensity of the product ion is related to the absolute configuration of the amino acid.

Figure 2. (A) The enantioselective condensation reaction between probes and L-Phe, (B) mass spectra of imines produced from L-Phe with probes, (C) mass spectra of imines produced from D-Phe with probes. Molecular modeling served as a supplementary tool to understand the stoichiometry of the imines and predict the chiral stereodynamic behavior. L-Phe was chosen as the sample to test the chiral selectivity of the pair of probes. The lowest docking energies represent the most favorable conformation. Figure 3 depicts representative conformers of the imine products of L-Phe with 1-(S)-1H and 2-(R)-2D. Although calculation at the B3LYP/6-31 level cannot give accurate binding energy information, it is still possible to obtain the conformations with the lowest energies which are consistent with experimental results. The result shows that both the transition state energy and the product energy for the reaction of 1-(S)-1H and L-Phe are lower than that of the reaction of 2-(R)-2D and L-Phe, which demonstrates a kinetic reaction mechanism. Meanwhile, there is an intramolecular hydrogen bond between the carboxylic acid of Phe and the carbonyl unit of probe in 1-(S)-1H-L-Phe product. This effectively shortens the distance between the carboxylic acid proton Ha and the chiral proton Hb in the probe to 3.86 Å. In the case of imine resulting from the reaction of L-Phe with 2(R)-2D, no intramolecular hydrogen bond was observed, and the distance between proton Ha and Hb was 7.67 Å. Taken together, a kinetically controlled process with a lower-energy

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Analytical Chemistry

transition state and stable product was responsible for the stereodynamic chiral recognition. Next, CR for all chiral amino acids were determined except proline (Pro), which is a secondary amine and cannot react with the probes. The CR values are shown in Table 1. The L and D forms of all the 18 chiral amino acids and a β-amino acid (pregabalin, Pre) could be distinguished based on different CR values. High chiral selectivity was achieved for Valine (Val), Isoleucine (Ile), Leucine (Leu), and Methionine (Met) with 11.1, 10.09, 10.0 and 8.33 for the L configurations and 0.09, 0.1, 0.1, 0.15 for the D forms, respectively. It appears that the CR value increases for amino acids with nonpolar side chains compared to those with polar side chain. This method presented relatively low chiral selectivity for Asp and pregabalin with CR values of 1.35 and 1.28, respectively. Meanwhile, for amino acids in entries 1 to 16, the CR values reflected the preferred formation of homochiral product. This means that when CR > 1, the analyte tended to react towards 1-(S)-1H and it would be in the L configuration, while in the case of CR﹤1, it would be in the D configuration. In contrast, amino acids in entries 17 to 19 tended to form heterochiral products, and their configurations could also be identified by comparing the light and heavy imine intensity peaks in the spectra. In addition to the high selectivity, the probes are also sensitive as the limit detection for analysis of Leu is approximately 10 pg mL-1 using the homemade chip-MS device shown in Figure S31-32 in the Supporting Information.

Figure 3. The stable structure of transition state and the products from L-Phe with 1-(S)-1H and 2-(R)-2D. Calculations were performed at B3LYP/6-31 level.32 Quantitative analysis, especially when one enantiomer comprises only a few percent of a mixture, remains a challenge. The capability of this method for quantitative analysis was investigated for Phe. L/D ratio were chosen at 100:0, 95:5, 90:10, 75:25, 65:35, 50:50, 35:65, 25:75, 10:90, 5:95 and 0:100,. Analysis of each sample required only one measurement of a single MS spectrum. A linear relationship between ee and ln(I[1-(S)-1H-Phe]-/I[2-(R)-2D-Phe]-.) was obtained with a correlation coefficient (r2) of 0.9916. Similar results were obtained for Leu and Val with correlation coefficients of 0.9912 and 0.9874, respectively, indicating that the method has the potential to quantify the relative percentage of the L and D forms in a mixture (Figure S28 in the Supporting Information).

D-Amino acids are promising candidates as biomarkers for several diseases including chronic kidney disease and Alzheimer disease.33- 34 A sensitive and feasible analysis method for determining trace D amino acids from a mixture is essential. In the following step, the probes were employed to determine the absolute configuration of individual amino acids from an amino acid mixture. A mixture containing Ala, Val, Leu, Met, Phe and Trp of “unknown” configuration was analyzed using the above-described method. Each chiral amino acid formed a pair of imine products that could be detected by mass spectrometry. The high abundance ions at m/z 393, 421, 435, 453, 469 and 508, were observed as shown in Figure 4 (A). The CR values suggested that all the six amino acids components were in the L configuration. Mixtures containing 5% D-Val were allowed to react with the probes, and the D-configuration could be clearly identified (Figure S29 in the Supporting Information). Table 1. Chiral selectivity of amino acids with probes. Entry

Amino acid

CR[a]

Amino acid

CR

1

L-Val

11.1

D-Val

0.09

2

L-Ile

10.09

D-Ile

0.10 0.10

3

L-Leu

10.0

D-Leu

4

L-Met

8.33

D-Met

0.15 0.29

5

L-Gln

4.76

D-Gln

6

L-Ala

3.03

D-Ala

0.35

7

L-Lys

3.03

D-Lys

0.31

8

L-Phe

2.86

D-Phe

0.40

9

L-Tyr

2.38

D-Tyr

10

L-Asn

2.22

D-Asn

0.42 0.45

11

L-Glu

2.08

D-Glu

0.55

12

L-Cys

1.85

D-Cys

0.54

13

L-Trp

1.56

D-Trp

0.60 0.65

14

L-Arg

1.53

D-Arg

15

L-Asp

1.35

D-Asp

0.75 0.86

16

L-Pregabalin

1.28

D-Pregabalin

17

L-His

0.31

D-His

3.58

18

L-Ser

0.64

D-Ser

1.70

19

L-Thr

0.71

D-Thr

1.34

[a] CR= I[1-(S)-1H-AA]-/I[2-(R)-2D-AA]-.I represents relative abundance %. The experiments were carried out in triplicate. Each reaction used a 10fold excess of the pair of reagents and the standard deviations for the multiple runs are all less than 8 %.

Next, amino acid residues in peptides were analyzed using this method. As MS has established itself as an indispensable tool for peptide and protein sequence analysis using electron-transfer dissociation and collisional induced dissociation approaches,35-36 we only focused on determining the configuration of the residues in this work. A pair of peptides with the same amino acid sequences but different configurations was analyzed. To determine the configuration of each amino acid residue, the peptide samples were first hydrolyzed with hot 6 M HCl37 and then neutralized with Na2CO3. Hydrolysis products were allowed to react with 10-fold excess of the probes followed by MS analysis. The results for the tripeptides pairs are shown in Figure 4 (B) and (C).

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Figure 4. The full scan mass spectrum analysis of (A) amino acid mixtures, (B) Phe-Tyr-Ala, (C) D-Phe-Tyr-Ala, (D) Val-Pro-PheD-Leu-Met, (E) Val-Pro-Phe-Leu-D-Met, and (F) Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2. The higher abundant imine products are marked with star style. was of L configuration and another was D, the ratio would be Both spectra displayed similar peaks at m/z 393, 398, 469, 474, approximately 1. The high abundance ion at m/z 469 resulting 485, and 490, and the only difference was the ratio of the peak from Phe and 1-(S)-1H, indicated that Phe is of the L configuration. Another mass-tagged ion pair at m/z 409 and abundances for m/z 469 to 474. When compared with the CRs 414 was the imine products from the reaction of Ser with the of the free amino acids in Table 1, the configurations of Tyr and Ala residues in the two peptides were determined to be Lprobes, with CR ﹤ 1 indicating that the Ser residue is also of configuration. Phe was in the L configuration in Figure 4 (B) the L configuration. In the peptide, Pro is a secondary amine, and the D configuration in Figure 4 (C). which could not be identified with the probes. There is also no To further verify the feasibility of the method, two “unknown” need to identify the chirality of Gly because it is an achiral peptides without L control peptides were tested. For the Valamino acid. Thus, we were able to distinguish the Pro-Phe-D-Leu-Met peptide, the results clearly showed five configuration of each amino acid residue within the peptide on strong peaks at m/z 421, 440, 453 and 469 (Figure 4D). The the basis of the CR value, without comparing to other peptides ratio of ion intensity of 435 to 440 is ﹤1, indicating that the or standards. Leu residue in the peptide is of the D configuration, and the In summary, we have developed a pair of stereodynamic other amino acid residues were all of the L configuration. In benzylicaldehyde probes that exhibit capability not only for the case of Val-Pro-Phe-Leu-D-Met, the ion at m/z 458 was the analysis of chiral amino acid mixtures, but also for the much higher than the ion at m/z 453, indicating that the Met determination of the configurations of amino acid residue in residue is of the D configuration, and the other amino acids chiral peptides based on the ion abundance ratios of imine such as Val, Phe and Leu were all of the L configuration products in a single mass spectrum. The probes produce a pair (Figure 4E). It should be mentioned that Pro does not react of distinct mass spectrometry signals upon condensation with with the pair of probes. Racemization of amino acids must be amino acid residues in peptides which can be correlated to the considered in the process of acid hydrolysis. Acid hydrolysis substrate chirality and quantification of enantiomer. They were of Val-Pro-Phe-D-Leu-Met with 6M HCl of in H2O and 6M of found to work well with both α-amino acids and β-amino acids. DCl in D2O were investigated. If isomerization occurred, a deuterium-labeled amino acid would be produced in 6M of Most importantly, this straightforward strategy is capable of DCl with a molecular mass 1 Da more than that produced in 6 determining the absolute configurations of amino acid residues M of HCl. The results demonstrated that isomerization under in peptides without using standards. In comparison with acidic conditions did not occur and had no influence on the Marfey’s reagent, analysis of residue configurations using the chiral determination (Figure S30 in the Supporting new probes is accurate with minimal sample handlings, and Information). Therefore, each of the configurations of the without time-consuming workup and separation steps. The constituent amino acids can be determined by this practical probes still need to be improved as they may lose some strategy without using standard. racemic site information when used to analyze large proteins Furthermore, a nature peptide dermorphin (Try-D-Ala-Phein complex biological samples. The development of other new Gly-Tyr-Pro-Ser-NH2) containing a D-Ala residue was and versatile probes for detecting the configurations of analyzed using this strategy. Ala is the smallest chiral amino acid, and its chirality is difficult to determine. As described residues in proteins remains an active area of research in our above, D-Ala prefers to react with probe 2-(R)-2D to produce laboratory. the labeled imine, which is 5 mass units larger than the unlabeled imine. As shown in Figure 4 (F), the expected peaks of the derivative from the Ala residue can be clearly detected ASSOCIATED CONTENT at m/z 393 (S, R) and 398 (R, R), and the intensity of the ion at Supporting Information m/z 398 was higher than that of m/z 393, confirming the D The Supporting Information is available free of charge on the configuration. In the meantime, for the Tyr analysis, the peak ACS Publications website. at m/z 485 was higher than the peak at m/z 490, which means that both Tyr residues had L configurations, because if one

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Synthesis and derivative procedure and other supporting figures and tables (PDF).

AUTHOR INFORMATION Corresponding Author * Cuirong Sun; Telephone (86) 571-88208868; Fax (86) 57188208868; E-mail: [email protected] * Yuanjiang Pan; Telephone (86) 571-87951285; Fax (86) 57187951285; E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (81230080, 21472171, 21605002).

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