1 2 3 4 5 6 7 8 9 10 11 12 13 14 SUPPLEMENTARY INFORMATION Level and activity of D-amino acids in mouse brain tissue and blood Choyce A. Weatherly 1, Siqi Du 1, Curran Parpia 1, Polan T. Santos 2, Adam L. Hartman 2,3, Daniel W. Armstrong 1* 1 Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, 76019, United States 2 Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States 3 Department of Molecular Microbiology and Immunology, Johns Hopkins Blomberg School of Public Health, Baltimore, MD 21205, United States * Corresponding Author: Professor Daniel W. Armstrong, Department of Chemistry and Biochemistry, University of Texas at Arlington, Planetarium Place, Arlington, TX 76019, USA. E-mail: sec4dwa@uta.edu; Fax: 817-272-0619 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
30 31 32 33 34 35 36 Table of Contents Figure-S1 S-3 Figure-S2 S-4 Figure-S3 S-5 Figure-S4 S-6 Table-S1...S-7 37 38 39 40 41 42 43 44 45 46 47 48 Amino acid peaks are identified by comparing the retention time to that of FMOC amino acids standards (shown in Figure-S1) on both first and second dimensions. Representative chromatograms of separations of FMOC-derivatized amino acids in mouse tissues are shown in Figure-S2. Diode-array technology was used to determine the peak purity on the first dimension HPLC (Figure-S3). Peak purity assay was performed using Agilent Chemstation. The UV-Visible spectra acquired during each amino acid peak s elution have a good match for FMOC-amino acid standards. The UV-Visible spectra across each peak are very similar as well. The similarity curve is within the threshold curve limit and the peak purity ratios are below unity (in the green band) confirming peak purity. L- and D-FMOC amino acid peaks from mouse tissues and blood were collected and concentrated, then injected into ESI-triple quadrupole mass spectrometry to confirm their identities. Mass spectrums of Glu and Ser peaks are provided in Figure-S4.
49 Figure 50 51 52 53 Figure-S1. Representative chromatograms of the first and second dimension separations of standard FMOC amino acids. Conditions are described in the experimental section. Serine (ser), aspartic acid (asp), alanine (ala), threonine (thr), leucine (leu), and glutamic acid (glu) are shown with the resolved separation of enantiomers in the second dimension.
54 55 56 57 Figure-S2. Representative chromatograms of the first and second dimension separations of FMOC-derivatized amino acids in mouse tissue. Serine (ser), threonine (thr), valine (val), leucine (leu), are shown with the resolved separation of enantiomers in the second dimension. 58
59 60 A B 61 62 63 Figure S-3 Peak purity analysis of (A) Fmoc-Asp and (B) Fmoc-Glu in mouse cortex tissues using diode-array detector. 64 65 66 67 68 69
70 71 A 72 B 73 74 75 76 77 Figure-S4 Representative mass spectrum of (A) Fmoc-Ser and (B) Fmoc-Glu standards and in mouse tissues. Experiments were performed in full scan mode with m/z from 200 to 800. (A) m/z = 328.2 is [Fmoc-Ser + H] + ;m/z = 346.2 is [Fmoc-Ser + H 2 O+ H] + ; m/z = 350.2 is [Fmoc-Ser + Na] +.(B) m/z = 370.3 is [Fmoc-Glu + H] + ;m/z = 388.2 is [Fmoc-Glu + H 2 O+ H] + ; m/z = 392.2 is [Fmoc-Glu + Na] +. 78 79
80 81 82 83 84 85 86 Table Table-S1. Second dimension chiral chromatography conditions for the separation of FMOC amino acids 1 Amino Acid 2 Mobile Phase 3 Column 4 Leucine 60/400.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Valine 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Serine 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Isoleucine 55/450.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Phenylalanine 55/450.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Alanine 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Glutamic Acid 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Tryptophan 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Threonine 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Methionine 60/400.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Aspartic Acid 70/300.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin Arginine 100/0.1 (w/w%) MeOH/NH TFA 4 4.6 x 100 mm Chirobiotic R Lysine 100/0.1 (w/w%) MeOH/NH TFA 4 4.6 x 100 mm Chirobiotic R Tyrosine 100/0.02 (w/w %) MeOH/NH 4 OAc 4.6 x 100 mm Chirobiotic R
91 92 93 Asparagine 60/400.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin 87 88 Glutamine 60/400.1% TEAA (ph=4.1)/meoh 4.6 x 100 mm SPP Teicoplanin 89 90 their FMOC derivative. 3) Mobile phase constructed by (v/v) 4) SPP is superficially porous particle based column 1) All amino acid derivatives were chromatographically resolved with resolution values 1.5. 2) Amino acids analyzed as