Letter Cite This: Org. Lett. 2018, 20, 2100−2103
pubs.acs.org/OrgLett
Identification of an Endogenous Organosulfur Metabolite by Interpretation of Mass Spectrometric Data Qibo Zhang,* Lisa A. Ford, Anne M. Evans, and Douglas R. Toal Metabolon, Inc., 617 Davis Drive, Suite 400, Morrisville, North Carolina 27560, United States S Supporting Information *
ABSTRACT: The chemical structure of x11564, a new endogenous organosulfur metabolite, was elucidated by de novo interpretation of mass spectrometric data. The structure was confirmed by comparison to a synthetic standard. Metabolite x11564 is structurally related to intermediates in the methionine salvage pathway.
I
n the biosynthesis of polyamines, S-adenosylmethionine (SAM) is converted to methylthioadenosine (MTA) as a byproduct,1 which can be converted back to methionine in the methionine salvage pathway (MSP) via intermediates that were characterized decades ago.2−4 Here, we report a new organosulfur compound, x11564 (1a), which may be a primary metabolite related to MSP. This metabolite was discovered as a potential biomarker in plasma during an untargeted metabolomic study for more accurate assessment of the glomerular filtration rate. It is well-accepted that de novo interpretation of mass spectrometric data for structure elucidation is more challenging than the interpretation of nuclear magnetic resonance (NMR) spectroscopic data.5−9 Nevertheless, there are compelling cases where correct chemical structures are elegantly elucidated by interpretation of mass spectrometric data.10−14 Here, we describe our strategy using mass spectrometry for the structure elucidation of x11564. All ions in this study were measured with accurate mass, but omitted textually for clarity. On a reversed-phased column, x11564 was retained poorly under basic conditions (1.19 min, Figure S1) while better under acidic conditions (2.53 min, Figure 1a). Due to the low level of x11564 in plasma, urine was tested and found to contain a compound of the same mass eluting at the same retention time (Figure 1b). Co-elution of this compound and x11564 was observed when a mixture of urine and plasma extract was injected (Figure S2). The product ion (MS2) spectrum of the compound in urine (Figure S3) also matched that of x11564 in plasma extract (Figure S4). These results established that x11564 was present in urine and at a much higher concentration, and thus, further analyses were conducted with urine samples. The monoisotopic mass of the deprotonated x11564 was determined to be 177.0227 (Figure 2a; Table 1), which supports a chemical formula of C6H9O4S−. The fine structure of the A + 2 peak confirmed the presence of sulfur with C6H9O434S− at m/z 179.0185 (Figure 2a inset).15 The double bond equivalent (DBE) was calculated to be 2 for the neutral © 2018 American Chemical Society
Figure 1. LC/MS chromatograms for x11564 (2.53 min) in a plasma extract (a) and a diluted urine sample (b), and synthetic cis- and transDMTPAs (2.20 and 2.53 min, respectively) (c).
compound. A collision-induced dissociation (CID) MS2 spectrum of x11564 showed a total of 17 daughter ions (Figures 2c and S3; Table 1). A second MS2 spectrum (Figure S5) by high-energy collision-induced dissociation (HCD) revealed three additional ions (m/z 97, 87, and 74, Table 1). The CID MS3 spectra of the m/z 159, 129, 115, and 85 ions and HCD MS3 spectra of the m/z 159 and 85 ions (Figure S6) detected two more ions (m/z 84 and 67, Table 1) and established the relationships of certain ions. The m/z 57.03 and 84 ions are from m/z 85; the m/z 101 and 85 ions from m/z 129; the m/z 99 and 100 ions from m/z 115; and the m/z 115, 111, 100, 99, 97, 83, 67 ions from m/z 159. Received: February 25, 2018 Published: March 26, 2018 2100
DOI: 10.1021/acs.orglett.8b00664 Org. Lett. 2018, 20, 2100−2103
Letter
Organic Letters
Table 1. Calculated and Measured Mass of x11564 Ionsa measured ion 1d2 1 2d2 2d 2 3d2 3db 3 4d 4 5d2 5d 5 6d2 6d 6 7d 7 8d 8 9d2 9d 9 10d2 10d 10 11d 11 12 13d 13 14 15d2 15d 15 16 17d 17 18d2 18d 18 19 20 21 22d2 22d 22 23
Figure 2. MS spectra for x11564 (a) and deuterated x11564 (b), and MS2 spectra for x11564 (c), deuterated x11564 (d), synthetic transDMTPA (e), and deuterated trans-DMTPA (f).
The urine sample was analyzed again using a deuterated mobile phase, and a new ion with m/z 179.0352 (Figures 2b and S7) was detected as the major species of deuterated x11564, indicating the presence of two deuterons in the molecular ion. The CID MS2 spectrum of the m/z 179 ion (Figures 2d and S8; Table 1) showed 9 doubly deuterated, 14 singly deuterated, and 7 undeuterated fragments, consistent with a mass shift for certain daughter ions of the undeuterated x11564. Detection of the m/z 161 ion was unexpected and unusual, as it would require the loss of H2O from the doubly deuterated parent ion. Fragmentation of the m/z 161 ion generated singly deuterated m/z 112 and doubly deuterated m/ z 117 and 102 ions. Fragmentation of the singly deuterated m/z 160 ion produced singly deuterated m/z 116, 112, 101, and 100, as well as undeuterated m/z 111 (Figure S9). An undeuterated m/z 100 ion was not detected in the CID MS2 spectrum. The MS3 spectra of the m/z 131, 130, 116, 87, and 86 ions (Figure S10) showed one ion each, which were m/z 87, 86, 101, 59, and 58, respectively. The m/z 133 ion appears to be generated from the parent ion by the loss of carbon dioxide, which may also rationalize the formation of the m/z 115 and 85 ions from m/z 159 and 129, respectively, strongly suggesting the presence of a carboxyl
formula
calculated −
C6H7D2O4S C6H9O4S− C6H5D2O3S− C6H6DO3S− C6H7O3S− C5H7D2O2S− C5H8DO2S− C5H9O2S− C5H6DO2S− C5H7O2S− C5H3D2O4− C5H4DO4− C5H5O4− C5H5D2OS− C5H6DOS− C5H7OS− C5H4DOS− C5H5OS− C5H2DO3− C5H3O3− C4H3D2O3− C4H4DO3− C4H5O3− C4H2D2OS•− C4H3DOS•− C4H4OS•− C4H2DOS− C4H3OS− C5H5S− C3H4DOS− C3H5OS− C3H3OS− C4H3D2O2− C4H4DO2− C4H5O2− C4H4O2•− C4H2DO2− C4H3O2− C2HD2O3− C2H2DO3− C2H3O3− C2H2O3•− C2HO3− C4H3O− C3H3D2O− C3H4DO− C3H5O− C2HO2−
179.0353 177.0227 161.0247 160.0184 159.0121 135.0454 134.0392 133.0329 132.0235 131.0172 131.0319 130.0256 129.0193 117.0349 116.0286 115.0223 114.0129 113.0067 112.0150 111.0088 103.0370 102.0307 101.0244 102.0114 101.0051 99.9988 99.9973 98.9910 97.0117 90.0129 89.0067 86.9910 87.0421 86.0358 85.0295 84.0217 84.0201 83.0139 77.0213 76.0150 75.0088 74.0009 72.9931 67.0189 59.0471 58.0409 57.0346 56.9982
H2O
D2 O 179.0352
177.0227 161.0245 160.0183 159.0120 135.0455 134.0391 133.0328 132.0235 131.0172 131.0318 130.0256 129.0193 117.0349 116.0286 115.0223 113.0066 111.0087
114.0129 113.0067 112.0150 111.0088 103.0371 102.0308
101.0245 102.0113 101.0051 99.9989 99.9969 98.9907 97.0118 89.0068 86.9911
90.0131 89.0068 87.0422 86.0359
85.0296 84.0215 83.0140
75.0089 74.0011 72.9933 67.0189
84.0203 83.0140 77.0215 76.0152 75.0089 72.9933 59.0473 58.0410
57.0348 56.9983
56.9984
a
Measured mass within 5 ppm of calculated value. bOne H more than 1d2, maybe from H/D exchange in the collision cell.
group in x11564. Loss of methanethiol may yield the m/z 129 and 111 ions from the parent and m/z 159 ions, respectively, indicating that the sulfur atom is not attached to an oxygen atom. The sulfur-containing m/z 100 radical anion may be generated from the m/z 115 ion by loss of a methyl radical, implicating that the molecule contains a methyl group. Therefore, the lost methanethiol is likely from a methylthio group, and the m/z 100 ion is perhaps a thiyl radical. The m/z 2101
DOI: 10.1021/acs.orglett.8b00664 Org. Lett. 2018, 20, 2100−2103
Letter
Organic Letters 75 ion is highly oxygenated with a formula of C2H3O3− and its reasonable candidate structures are basically limited to glycolate species, suggesting the presence of an α-proton and an αhydroxyl group to the carboxyl group. The deuterium exchange experiment shows the incorporation of two deuterons into the parent ion. As the molecule does not appear to have a thiol group, it likely contains two hydroxyl groups. The formation of the m/z 77 ion with two deuterons indicates that the deuterium on the second hydroxyl group somehow also ends up in the glycolate species. A perceived deuterated β-hydroxyl group would allow its deuteron to be captured into the glycolate species via the McLafferty rearrangement.16 The thus far unaccounted two carbon atoms, three hydrogen atoms, and one DBE can only settle in a few possible structures. In consideration of x11564 as a biochemical and the viability to rationalize the formation of other fragments, the most promising structure of x11564 is inferred to be (2R,3R)-2,3dihydroxy-5-methylthio-4-pentenoic acid (DMTPA, 1a or 1b). The absolute configurations of the hydroxyl groups are proposed from the close structural similarity to the 5methylthioribose (MTR) moiety in MTA.1,17 The double bond configuration cannot be predicted from the mass spectrometric data. Possible fragmentation pathways of deprotonated DMTPA (1) are proposed as shown in Scheme 1. McLafferty
generates 3, which yields 4 upon further loss of a hydrogen molecule. Fragment 5 may be formed by elimination of methanethiol. Loss of CO and CO2 from 5 generates 9 and the predominant m/z 85 ion (15), respectively. Fragments 22 and 16 may be formed from 15 after loss of CO and a hydrogen atom, respectively. Dehydration of 1′ yields 2, which loses methanethiol to give 2-furoate (8), followed by elimination of CO and CO2 to generate 17 and the furanide anion (21), respectively. Loss of H2O and CO2 from 1′ generates fragment 6, which loses a hydrogen molecule to give 7, or rearranges to 6′ followed by loss of a methyl radical and acetylene to give 10 and 13, respectively. The radical anion 10 may lose a hydrogen atom to yield thionolactone 11. The epoxide 13 may rearrange and eliminate a hydrogen molecule to yield fragment 14. The fragments from the doubly deuterated DMTPA species (1d2) can simply be rationalized into the pathways proposed for 1 (Scheme 1). Loss of CO2 from 1′d2 yields 3d2, which then loses HD to give 4d. The m/z 160 fragment (2d) may be formed by loss of HOD from 1′d2. Loss of MeSD and MeSH from 2d generates 2-furoates 8 and 8d, respectively, which may lose CO to give 17 and 17d. Loss of CO2 from 2d or HOD from 3d2 may generate 6d, which could lose HD and H2 to give 7 and 7d, respectively. Fragment 6d may also isomerize to 6′d with partial deuterium migration from position 4 to 2. Homolytic cleavage of the methylthio bond in 6′d generates thiyl radical 10d, which may lose a hydrogen atom to yield thionolactone 11d. The species 6′d may also lose acetylene and deuterated acetylene to give fragments 13d and 13, respectively. Four pairs of mono- and dideuterated fragments of 5d/5d2, 9d/9d2, 15d/15d2, and 22d/22d2 may simply be explained by the initial loss of MeSD/MeSH from the doubly deuterated 1′d2. While McLafferty rearrangement of the parent ion 1d2 may generate 18d2, formation of the singly deuterated species 18d is not straightforward. One possible explanation is that 1′d2 reverses back to the open form (1″d2) with the deuterium remaining at position 4, resulting in an exchange between the deuteron on the 2-hydroxyl group and the proton at position 4. McLaffferty rearrangement of 1″d2 then generates 18d. Loss of DH and D2 from 18d and 18d2, respectively, may yield 20, and loss of HOD and D2O from 18d and 18d2 generates 23. This exchanged parent ion 1″d2 may also lose H2O to form the unexpected m/z 161 ion (2d2), which eliminates MeSD to generate linear 8′d or sequentially loses CO2 and a methyl radical to form 10d2 via 6d2. When a urine sample was subjected to catalytic hydrogenation, LC/MS analysis of the reaction mixture showed the disappearance of x11564 (∼2.5 min) and appearance of the expected hydrogenated x11564 at 3.17 min with m/z 179.0384 (Figure S11). The MS2 spectrum (Figure S12) and MS3 spectra (CID and HCD) of the m/z 131 ion (Figure S13) support the identity of the saturated product (Scheme S1). LC/MS analysis using a deuterated mobile phase revealed that two deuterons were incorporated into the molecular ion with m/z 181.0506 (Figure S14). The MS2 spectrum (Figure S15) and MS3 spectra (Figure S16) of the deuterated species are also consistent with the saturated product (Scheme S2; Table S6). These results provide further evidence for the proposed structure. 2,3-Cyclohexylidene-L-erythruronic acid (24), in which the two hydroxyl groups have the same absolute configurations as those in MTR, was selected as the starting material for the chemical synthesis of DMTPA (Scheme 2). Treatment of 24 with methylthiomethyl triphenyl phosphonium ylide yielded a mixture of protected thioenolethers (25a and 25b),18 which
Scheme 1. Proposed Fragmentation Pathways of x11564
rearrangement (a) may generate fragment 18, which further loses a hydrogen atom, a hydrogen molecule, and H2O to yield 19, glyoxylate (20), and 23, respectively. The m/z 97 ion (12) may be generated from 1 by double dehydration and loss of CO2. Formation of other fragments may be better explained via a tetrahydrofuran intermediate 1′. Loss of CO2 from 1′ 2102
DOI: 10.1021/acs.orglett.8b00664 Org. Lett. 2018, 20, 2100−2103
Letter
Organic Letters
Double bond configuration determination, experimental details, NMR, and LC/MS chromatograms and spectra (PDF)
Scheme 2. Chemical Synthesis of DMTPAs
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
were characterized by 1H, 13C, and 1H−1H COSY NMR spectroscopic (Figures S17−S21) and mass spectrometric (Figures S22 and S23) analyses. When this mixture was treated under mild acidic conditions,19 LC/MS analysis detected two deprotected products eluting at 2.19 and 2.53 min with a molecular ion of m/z 177.0226, consistent with the expected formation of a mixture of E/Z isomers (1a and 1b, Figures 1c and S24). The later eluting product was found to coelute with x11564 (Figure S25), and its CID (Figures 2e and S26) (Figure S27 for the earlier eluting isomer) and HCD MS2 spectra (Figure S28) matched those of x11564 (Figures 2c, S3, and S5) by fragmentation patterns. The CID MS3 spectra of m/z 115, 129, 159, and 85, as well as HCD spectra of m/z 159 and 85 (Figure S29), also matched those of x11564 (Figure S6) remarkably well. The sample was further analyzed using a deuterated mobile phase, and the doubly deuterated molecular ion was found at m/z 179.0351 (Figures S30 and S31). The MS2 spectrum (Figures 2f and S32) of this deuterated product also matched that of deuterated x11564 (Figures 2d and S8), and the MS3 spectra (Figure S33) of m/z 160 and 161 matched those of deuterated x11564 (Figure S9) as well. All these results (Table S9) support the later eluting synthetic DMTPA is the same as x11564. The later eluting DMTPA isomer (1a) was eventually determined to have the trans double bond configuration (see Supporting Information), and thus, x11564 must have the trans double bond configuration. In summary, the chemical structure of x11564 was determined to be trans-DMTPA (1a) by de novo interpretation of mass spectrometric data and subsequent confirmation with a synthetic standard. It would have been difficult to purify this very polar and potentially unstable compound from a complex matrix for NMR analysis. To our knowledge, x11564 represents the first compound with a thioenolether group identified in any human biological samples. At present, it is unknown if x11564 is endogenously synthesized, or results from dietary or other exogenous sources. However, a potential precursor for its biosynthesis is MTA, which is, in turn, formed from SAM during the biosynthesis of polyamines and others.4,20 It is possible that x11564 is generated via an unknown shunt pathway from MTA itself or one of the intermediates in the methionine salvage pathway. At the cost of an essential amino acid, one may conceive that x11564 and/or the shunt pathway plays a necessary biological role.21 It is also possible that the shunt pathway is simply another approach to salvage methionine, in which x11564 is further converted to methionine via transformations hypothetically, including oxidation of the hydroxyl groups, decarboxylation, transamination, and reduction of the olefinic bond.
■
Qibo Zhang: 0000-0002-1497-7707 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank Dr. Xiaoyan (Jennifer) Sun of the Department of Chemistry at North Carolina State University for acquisition and processing of NMR spectra, and Drs. Kay A. Lawton and Meredith V. Brown of Metabolon, Inc. for their suggestions in the preparation of this manuscript.
■
REFERENCES
(1) Avila, M. A.; García-Trevijano, E. R.; Lu, S. C.; Corrales, F. J.; Mato, J. M. Int. J. Biochem. Cell Biol. 2004, 36, 2125. (2) Kang, W.; Hong, S. H.; Lee, H. M.; Kim, N. Y.; Lim, Y. C.; Le, L. T. M.; Lim, B.; Kim, H. C.; Kim, T. Y.; Ashida, H.; Yokota, A.; Hah, S. S.; Chun, K. H.; Jung, Y. K.; Yang, J. K. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, E54. (3) Albers, E. IUBMB Life 2009, 61, 1132. (4) Sauter, M.; Moffatt, B.; Saechao, M. C.; Hell, R.; Wirtz, M. Biochem. J. 2013, 451, 145. (5) Kind, T.; Fiehn, O. Bioanal. Rev. 2010, 2, 23. (6) Dias, D. A.; Jones, O. A. H.; Beale, D. J.; Boughton, B. A.; Benheim, D.; Kouremenos, K. A.; Wolfender, J. L.; Wishart, D. S. Metabolites 2016, 6, 46. (7) Wishart, D. S. Bioanalysis 2011, 3, 1769. (8) Bingol, K.; Bruschweiler-Li, L.; Li, D.; Zhang, B.; Xie, M.; Brüschweiler, R. Bioanalysis 2016, 8, 557. (9) Watson, D. G. Comput. Struct. Biotechnol. J. 2013, 4, e201301005. (10) Kalisiak, J.; Trauger, S. A.; Kalisiak, E.; Morita, H.; Fokin, V. V.; Adams, M. W.; Sharpless, K. B.; Siuzdak, G. J. Am. Chem. Soc. 2009, 131, 378. (11) Schiestl, F. P.; Peakall, R.; Mant, J. G.; Ibarra, F.; Schulz, C.; Franke, S.; Francke, W. Science 2003, 302, 437. (12) Biemann, K. Annu. Rev. Anal. Chem. 2015, 8, 1. (13) Dickschat, J. S. Nat. Prod. Rep. 2014, 31, 838. (14) Zhang, Q.; Ford, L. A.; Evans, A. M.; Toal, D. R. Metabolomics 2017, 13, 92. (15) Solouki, T.; Emmett, M. R.; Guan, S.; Marshall, A. G. Anal. Chem. 1997, 69, 1163. (16) McLafferty, F. W. Anal. Chem. 1959, 31, 82. (17) Riscoe, M. K.; Ferro, A. J. J. Biol. Chem. 1984, 259, 5465. (18) Xu, H.-C.; Campbell, J. M.; Moeller, K. D. J. Org. Chem. 2014, 79, 379. (19) Evans, D. A.; Burch, D. B. Org. Lett. 2001, 3, 503. (20) North, J. A.; Miller, A. R.; Wildenthal, J. A.; Young, S. J.; Tabita, F. R. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E10455. (21) Warlick, B. P.; Evans, B. S.; Erb, T. J.; Ramagopal, U. A.; Sriram, J.; Imker, H. J.; Sauder, J. M.; Bonanno, J. B.; Burley, S. K.; Tabita, F. R.; Almo, S. C.; Sweedler, J. S.; Gerlt, J. A. Biochemistry 2012, 51, 8324.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00664. 2103
DOI: 10.1021/acs.orglett.8b00664 Org. Lett. 2018, 20, 2100−2103
