Advancing HPLC-PDA-HRMS-SPE-NMR Analysis of Coumarins in

Article pubs.acs.org/jnp

Advancing HPLC-PDA-HRMS-SPE-NMR Analysis of Coumarins in Coleonema album by Use of Orthogonal Reversed-Phase C18 and Pentafluorophenyl Separations Rita de Cássia L. Lima,† Simone M. Gramsbergen,† Johannes Van Staden,‡ Anna K. Jag̈ er,† Kenneth T. Kongstad,*,† and Dan Staerk† †

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ‡ Research Center for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa S Supporting Information *

ABSTRACT: A hyphenated procedure involving high-performance liquid chromatography, photodiode array detection, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDAHRMS-SPE-NMR, has proven an effective technique for the identification of compounds in complex matrices. Most HPLCPDA-HRMS-SPE-NMR investigations reported so far have relied on analytical-scale reversed-phase C18 columns for separation. Herein is reported the use of an analytical-scale pentafluorophenyl column as an orthogonal separation method following fractionation of a crude ethyl acetate extract of leaves of Coleonema album on a preparative-scale C18 column. This setup allowed the HPLC-PDA-HRMS-SPE-NMR analysis of 23 coumarins, including six new compounds, 8-O-β-D-glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)-7-methoxycoumarin (4), (Z)-6-(4-β-D-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (6), 6-(4-β-D-glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (8), (Z)-7-(4-β-D-glucopyranosyloxy-3-methylbut-2-en-1-yloxy)coumarin (13), (S)-8-(3-chloro-2hydroxy-3-methylbut-1-yloxy)-7-methoxycoumarin (19), and 7-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)coumarin (20). The use of the pentafluorophenyl column even allowed separation of several regioisomers that are usually difficult to separate using reversed-phase C18 columns. The phytochemical investigation described for C. album in this report demonstrates the potential and wide applicability of HPLC-PDA-HRMS-SPE-NMR for accelerated structural identification of natural products in complex mixtures. reported to occur in the Rutaceae.6,7 Several studies have reported different pharmacological activities for coumarins, such as anticoagulant, respiratory stimulation, vasodilatatory, and diuretic effects.8 Furthermore, coumarins have been reported to have anti-inflammatory activities by acting on different levels of the inflammatory process, such as reducing edema, altering functions of important enzymes such as cyclooxygenases, and preventing generation of free radicals.9 Dereplication of known compounds and full isolation and structural identification of new compounds from complex extracts has traditionally been a laborious and time-consuming task. Initial proof-of-concept experiments with the hyphenation of high-performance liquid chromatography, photodiode array detection, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDA-SPE-NMR,10−12 were applied

Coleonema album (Thunb.) Bartl. & Wendl. (Rutaceae) is a 1 m tall shrub that grows along the coastal area of the Cape Peninsula, South Africa. It is cultivated commonly in gardens and has some use in traditional medicine for its anti-inflammatory properties.1−3 Extracts of C. album have previously been reported to show in vitro inhibition of cyclooxygenase 1 and 2, enzymes intrinsically responsible for inflammatory processes, as well as moderate antimycobacterial activity.1 A crude extract of C. album has also shown antioxidant capacity, with phenolic compounds such as flavonoids and coumarins being the active constituents.3 However, until now, only a few compounds have been identified in C. album, i.e., 7-(3′,3′-dimethylallyloxy)coumarin, (R)-(+)-7-(2′,3′-epoxy-3′-methylbutoxy)coumarin, (R)-(+)-7-(2′,3′-dihydroxy-3′-methylbutoxy)coumarin, (R)(+)-2′,3′-epoxysuberosin, ulopterol, and (R)-(+)-7-methoxy-8(2′,3′-epoxy-3′-methylbutoxy)coumarin.4,5 Coumarins are commonly found in species belonging to the Apiaceae and Rutaceae, and around 200 coumarins have been © 2017 American Chemical Society and American Society of Pharmacognosy

Received: November 7, 2016 Published: March 1, 2017 1020

DOI: 10.1021/acs.jnatprod.6b01020 J. Nat. Prod. 2017, 80, 1020−1027

Journal of Natural Products

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Figure 1. (Bottom) Preparative-scale C18 HPLC-UV chromatogram at 330 nm of the defatted crude extract of C. album leaves. Thirteen fractions were collected as indicated. (Top) Expansions showing base peak chromatograms (blue) and UV chromatograms at 330 nm (red) of selected fractions analyzed by HPLC-PDA-HRMS-SPE-NMR using an orthogonal pentafluorophenyl HPLC column. Peak numbering according to elution order on an analytical-scale C18 HPLC column in a pilot HPLC-PDA-HRMS-SPE-NMR experiment.

mainly for major constituents of organisms. However, the addition of high-resolution mass spectrometry and a cryogenically cooled 1.7 mm probe, i.e., a HPLC-PDA-HRMS-SPEcryoNMR setup, has allowed comprehensive structural analysis of even minor metabolites directly from a crude extract.13 Furthermore, coupling with high-resolution bioassays and ligand fishing,14−16 direct 13C NMR detection,17 and database-assisted structure elucidation18 have facilitated the identification and structural elucidation process of bioactive constituents directly from crude extracts. Hitherto, most studies have been performed using reversed-phase C18 columns. Herein is reported the use of preparative-scale HPLC employing a C18 column for crude extract fractionation, followed by orthogonal HPLCPDA-HRMS-SPE-cryoNMR using an analytical-scale pentafluorophenyl column for analysis of all preparative-scale fractions. This led to identification of 26 compounds from the ethyl acetate extract of leaves of C. album, including six new compounds.

7-O-β-D-glucopyranoside (5),19 7-(2,3-dihydroxy-3-methylbutyloxy)coumarin (9),4 8-(2,3-dihydroxy-3-methylbutyloxy)-7-methoxycoumarin (12),20 8-(2-hydroxy-3-methylbut-3-en-1-yloxy)7-methoxycoumarin (16),20 6-(2-hydroxy-3-methylbut-3-en-1-yl)7-methoxycoumarin (17),21 6-(2,3-dihydroxy-3-methylbutyl)-7methoxycoumarin (18),5 (E)-3,7-dimethylocta-2,6-dien-1-yl 2-((3-methylbut-2-en-1-yl)amino)benzoate (25),22 and (2E,4E)N-isobutyldeca-2,4-dienamide (26)23 (Figure S2, Supporting Information), by comparison of their NMR data with literature values. Compounds 9 and 18 have previously been isolated from C. album, whereas compounds 12, 16, and 26 are reported for the first time in this species, and compounds 5, 17, and 25 are reported for the first time for the genus Coleonema. In addition, two new coumarins, 6 and 13, for which the structure elucidation is discussed below, were obtained. Preparative-Scale C18 HPLC Combined with Analytical-Scale Pentafluorophenyl HPLC-PDA-HRMS-SPE-cryoNMR Analysis. Due to insufficient separation and/or low

RESULTS AND DISCUSSION Initial Pilot Experiment. In a pilot experiment, a defatted ethyl acetate extract of the leaves of C. album was analyzed by HPLC-PDA-HRMS-SPE-cryoNMR using an analytical-scale octadecylsilica column (C18 column). Fifteen repetitive separations of 10 μL each of a 12.5 mg/mL solution allowed cumulative trapping of 18 peaks (Figure S1, Supporting Information). Using the peak and compound numbering scheme (in italics and bold, respectively) as for the subsequent HPLC-PDA-HRMSSPE-cryoNMR analysis of preparative-scale fractions (Figure 1), the compounds eluted with eight of the trapped peaks were identified as (Z)-6-(4-hydroxy-3-methylbut-2-en-1-yl)coumarin

signal-to-noise of the NMR spectra acquired in the HPLCHRMS-SPE-NMR mode, the identity of the material eluted

■

1021


144.5

147.7

116.0

n.d.

79.0

8

9

10

1′

2′

1022

77.5

62.3

5″

6″

1.19, d (11.2)

3.96, s, OCH3

3.63, ddd (11.5, 6.0, 2.8)

3.55, ddd (11.5, 6.0, 6.0)

3.23, m

3.34, m

3.37, m

3.48, overlapping signal

5.07, d (7.8)

34.3

27.3

112.9

17.3

77.4

71.4

77.0

74.6

103.6

3.71, ddd (11.6, 6.4, 2.8)

62.8 3.56, ddd (11.6, 5.8, 5.8) 62.7

77.0 3.16, ddd (9.3, 5.8, 2.8)

71.5 3.22, ddd (9.3, 8.8, 3.5)

77.7 3.26, dt (3.5, 8.8)

74.6 3.11, ddd (8.8, 7.8, 3.5)

102.2 4.23, d (7.8)

14.2 1.75, s

4.20, d (11.9)

75.0 4.02, d (11.9)

126.4 5.63, t (7.4)

26.9 3.36, d (7.4)

112.0

154.7

102.8

159.3

127.8

129.8

144.7

112.9

3.58, m 3.74, m

3.30, m



3.08, ddd (8.8, 7.8, 2.9)

4.21, d (7.8)

0.97, d (6.5)

3.67, dd (9.5, 6.5)

3.42, dd (9.5, 6.0)

1.76, dd (13.1, 6.5)

1.71, m

1.43, m

2.69, ddd (13.8, 10.1, 5.8)

2.60, ddd (13.8, 10.1, 5.8)

6.74, s

7.33, s

7.73, d (9.5)

6.13, d (9.5)

δH, mult. (J in Hz)

8b

C chemical shift data obtained from direct-detected experiments with 2048 scans using acetonitrile-d3. n.d.: not detected.

a13

OCH3 62.3

77.2

71.0

3″

75.1

2″

4″

26.0

104.1

5′

1″

1.19, d (11.2)


2.96, d (13.6)

2.51, dd (10.4, 13.6)

154.4

102.9 6.76, s

158.3

112.3

129.5 7.30, s

δC 162.3

33.8

154.3

7

7.29, s

144.9 7.73, d (9.5)

113.0 6.13, d (9.5)


75.2

126.7

6

24.9

126.0

5

7.79, d (9.5)

6.29, d (9.5)

δC 161.4

3′

144.8

4


6a,b

4′

115.1

3

δC

160.5

pos.

2

4b

Table 1. NMR Spectroscopic Data (600 MHz) of 4, 6, 8, 13, 19, and 20

δC


δC

57.1

29.7

28.5

71.1

77.6

76.2

114.7

148.6

135.4

155.3

109.8

124.5

144.8

113.9

161.4

3.94, br s, OCH3

1.61, s

1.58, s

3.92, dd (7.9, 2.5)

4.47, dd (10.4, 2.5)

4.03, dd (10.4, 7.9)

7.02, d (8.7)

7.33, d (8.7)

7.78, d (9.6)

6.22, d (9.6)


19b δC

29.7

28.4

77.1

71.1

113.7

156.9

102.4

162.4

113.5

130.0

144.5

113.7

161.6

1.64, s

1.61, s

3.94, ddd (7.8, 5.5, 2.5)

4.41, dd (10.0, 2.5)

4.06, dd (10.0, 7.8)

6.94, overlapped signal

6.94, overlapped signal

7.53, d (9.2)

7.79, d (9.5)

6.21, d (9.5)


20b

C chemical shift data obtained from HSQC and HMBC experiments using acetonitrile-d3.

b13

3.71, ddd (11.5, 6.0, 2.5)

62.6 3.55, ddd (11.5, 5.6, 5.6)

77.0 3.21, ddd (9.5, 5.6, 2.5)

71.4 3.23, ddd (9.5, 8.8, 4.1)

77.5 3.29, dt (8.8, 3.8)

74.5 3.13, ddd (8.8, 7.8, 3.8)

101.9 4.24, d (7.8)

21.6 1.85, s

4.33, d (12.2)

67.3 4.27, d (12.2)

139.1

124.5 5.69, t (6.7)

65.7 4.69, d (6.7)

113.8

157.0

102.6 6.89, overlapping signal

163.1

113.6 6.89, overlapping signal

130.0 7.49, d (9.2)

144.7 7.78, d (9.5)

113.5 6.19, d (9.5)

162.1

13b

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Figure 2. Selected HMBC and NOE correlations used for structure elucidation of 4, 6, 8, 13, 19, and 20 identified in the defatted ethyl acetate extract of the leaves of Coleonema album.

with the remaining eight peaks in the pilot project could not be established unambiguously. However, as the obtained NMR and MS data suggested the presence of additional glycosylated and chlorinated coumarins, it was decided to perform preparative-scale HPLC separation using a C18 column, followed by HPLC-PDA-HRMS-SPE-cryoNMR analysis of all fractions using an analytical-scale pentafluorophenyl (PFP) column. Preparative-scale HPLC separation led to the collection of 13 fractions (F1−F13), as shown in Figure 1. Subsequent HPLCPDA-HRMS-SPE-cryoNMR analysis led to the identification of six new compounds, i.e., compound 4 from fraction F1, compounds 6 and 8 from fraction F2, compound 13 from fraction F3, and compounds 19 and 20 from fraction F9. The structure elucidation of these will be discussed below. In addition, the HPLC-PDA-HRMS-SPE-cryoNMR analysis led to identification of 7-methoxy-8-O-β-D-glucopyranosylcoumarin (1),24 (2S)-2′-hydroxymarmesin-2′-O-β-D-glucopyranoside (2),25 and (2R)-2′-hydroxymarmesin 2′-O-β-D-glucopyranoside (3)25 in fraction F1, (Z)-6-(4-hydroxy-3-methylbut-2en-1-yl)coumarin 7-O-β-D-glucopyranoside (5)19 and (S)-7-Omethylpeucedanol 3′-O-β-D-glucopyranoside (7)26 in fraction F2, 7-(2,3-dihydroxy-3-methylbutyloxy)coumarin (9), 8-hydroxy-5-methoxy-2H-1-benzopyran-2-one (10),27 5-hydroxy-8methoxy-2H-1-benzopyran-2-one (11),27 and 8-(2,3-dihydroxy3-methylbutyloxy)-7-methoxycoumarin (12)20 in fraction F3, isorhamnetin (14),28 7-(2-hydroxy-3-methylbut-3-en-1-yloxy)coumarin (15),20 8-(2-hydroxy-3-methylbut-3-en-1-yloxy)-7methoxycoumarin (16),20 and 6-(2-hydroxy-3-methylbut-3en-1-yl)-7-methoxycoumarin (17)21 in fraction F6, 6-(2,3dihydroxy-3-methylbutyl)-7-methoxycoumarin (18) 5 and 7-methoxy-6-(3-methyl-2-oxobutyl)coumarin (21)29 in fraction F9, 7-hydroxy-6-(3-methylbut-2-en-1-yl)coumarin (22)30 in fraction F11, 7-methoxy-8-(3-methylbut-2-en-1-yloxy)coumarin (23)20 in fraction F12, and 7-(3-methylbut-2-en-1-yloxy)-coumarin (24),30

(E)-3,7-dimethylocta-2,6-dien-1-yl 2-((3-methylbut-2-en-1-yl)amino)benzoate (25),22 and (2E,4E)-N-isobutyldeca-2,4-dienamide (26)23 in fraction F13. No further compounds were identified in the remaining fractions. This is the first report of compounds 1, 2, 3, 7, 10, 11, 14, 21, and 22 in the genus Coleonema. Compound 24 has previously been isolated from C. album, whereas compounds 15 and 23 are reported for the first time in C. album. Structural Elucidation of New Coumarins. The material eluted as peak 4 (in fraction F1) exhibited a [M + H]+ ion at m/z 457.1703, which suggested the molecular formula C21H28O11 (C21H29O11+, ΔM 0.3 ppm). The 1H NMR spectrum showed a singlet (δ 7.29, H-5) as well as characteristic doublets for H-3 and H-4 of the coumarin skeleton (δ 7.79, d, J = 9.5 Hz, H-4 and δ 6.29, d, J = 9.5 Hz, H-3), which indicated three substituents on the coumarin ring. One of these substituents was identified as a methoxy group (1H: δ 3.96, 3H, s; 13C: δ 62.3), whereas a glucose unit with β-configuration was identified based on the anomeric signal (1H: δ 5.07, d, J = 7.8 Hz, H-1″; 13 C: δ 104.1, C-1″), with the remaining signals for H-2″−H-6″ shown in Table 1. The D-configuration of glucose was established by GC-MS after hydrolysis, reaction with L-cysteine methyl ester hydrochloride, and derivatization with N-methylbis(trifluoroacetamide) (see Experimental Section). The remaining substituent showed a 1H NMR spin system for a CH2−CH−O moiety and two diastereotopic methyl groups with HMBC correlations to the oxygenated methine group and a monooxygenated quaternary carbon atom (Figure 2), in agreement with a 2,3-dihydroxy-3-methylbutyl group. On the basis of NOE correlations from H-4 to H-5 to H-1′a/H-1′b to the methoxy group and to the anomeric proton, as well as the selected HMBC correlations shown in Figure 2, compound 4 was identified as 6-(2,3-dihydroxy-3-methylbut-1yl)-8-O-β-Dglucopyranosyloxy-7-methoxycoumarin. Full assignment of its 1023



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Figure 3. (A) Schematic model for ΔδSR < 0 and ΔδSR > 0 for MTPA esters. (B) ΔδSR values (red) for 19 with negative values on the left-hand side and positive values on the right-hand side. (C) Structure of 19 with absolute configuration of C-2′. 1

H and 13C NMR data is given in Table 1, and selected HMBC and NOE correlations are presented in Figure 2. Due to the limited amounts of 4, attempts to establish the absolute configuration of C-2′ using the Mosher ester method31 were unsuccessful. The material eluted as peak 6 (in fraction F2) showed a [M + H]+ ion at m/z 409.1493, which suggested a molecular formula of C20H24O9 (C20H25O9+, ΔM 0.0 ppm). Two doublets for H-3 (δ 6.13, d, J = 9.5 Hz) and H-4 (δ 7.73, d, J = 9.5 Hz) as well as two singlets for H-5 (δ 7.30) and H-8 (δ 6.76) showed the presence of substituents at C-6 and C-7. Glucosylation at the 4′-position (i.e., the 4-position of the prenyl group) was shown by NOESY and HMBC correlations between the anomeric proton (δ 4.23, d, J = 7.8 Hz) and the downfield-shifted diastereotopic methylene protons (δ 4.02, d, J = 11.9 Hz, H-4′a and δ 4.20, d, J = 11.9 Hz, H-4′b) and their carbon (δ 75.0, C-4′), respectively. NOESY correlations from H-4 to H-5 to H-1′a/H-1′b supported the 4-glucosylated prenyl group being positioned at C-6, leaving a hydroxy group at C-7, in agreement with the upfield shift of H-8 (δ 6.76). Finally, a NOESY correlation between H-2′ and H-5′ confirmed the Z-configuration of the prenyl double bond, and the D-configuration of glucose was established by GC-MS after hydrolysis, reaction with L-cysteine methyl ester hydrochloride, and derivatization with N-methylbis(trifluoroacetamide) (see Experimental Section). Thus, 6 was established as (Z)-6-(4-β-D-glucopyranosyloxy-3methylbut-2-en-1-yl)-7-hydroxycoumarin. Full assignments of the 1H and 13C NMR data are given in Table 1, and selected HMBC and NOE correlations are shown in Figure 2. The material eluted as peak 8 (in fraction F2) showed a [M + H]+ ion at m/z 411.1645, which suggested the molecular formula C20H26O9 (C20H27O9+, ΔM 1.1 ppm). The 1H NMR spectrum showed characteristic signals for H-3 (δ 6.13, d, J = 9.5 Hz) and H-4 (δ 7.73, d, J = 9.5 Hz) as well as the two singlets for H-5 (δ 7.33) and H-8 (δ 6.74), in agreement with the occurrence of substituents at C-6 and C-7. The substituent at C-6 was identified as a 4β-D-glucopyranosyloxy-3-methylbut1-yl group based on a CH2−CH2−CH(CH3)−CH2−O spin system observed in the COSY spectrum as well as a NOESY correlation between H-5 and H-1′ and HMBC correlations from H-5 and H-1′ to C-1′ and C-5, respectively (Figure 2). A glucose unit with β-configuration was identified based on the anomeric signal (1H: δ 4.21, d, J = 7.8 Hz; 13C: δ 103.6) and the remaining signals for H-2″−H-6″ shown in Table 1. Its attachment at C-4′ was demonstrated based on an HMBC correlation from H-1″ to C-4′ (Figure 2). The D-configuration of glucose was established by the GC-MS method mentioned above, but the absolute configuration of C-3′ could not be established due to the limited amount available from the HPLC-PDA-HRMS-SPE-cryoNMR analysis. Thus, 8 was identified as 6-(4-β-D-glucopyranosyloxy-3-methylbut-1-yl)-7hydroxycoumarin. Full assignment of the 1H and 13C NMR

data is given in Table 1, and selected HMBC and NOE correlations are presented in Figure 2. The material eluted as peak 13 (in fraction F3) showed a [M + H]+ ion at m/z 409.1492, which suggested the molecular formula C20H24O9 (C20H25O9+, ΔM 0.3 ppm). A monosubstituted coumarin skeleton was evident based on the characteristic signals for H-3 (δ 6.19, d, J = 9.5 Hz) and H-4 (δ 7.78, d, J = 9.5 Hz) as well as a doublet for H-5 (δ 7.49, d, J = 9.2 Hz) and two upfield-shifted and overlapping signals for H-6 and H-8 (δ 6.89, 2H, overlapped). The single substituent was identified as a 4β-D-glucopyranosyloxy-3-methylbut-2-en-1-yloxy unit. This was based on 1H NMR signals for a 1,4-dioxygenated prenyl group at δ 4.69 (2H, d, J = 6.7 Hz, H-1′), δ 5.69 (1H, t, J = 6.7 Hz, H-2′), δ 4.33 (1H, d, J = 12.4 Hz, H-4′B), δ 4.27 (1H, d, J = 12.4 Hz, H-4′A), and δ 1.85 (3H, s, H5′) as well as a β-glucose unit based on the anomeric signal (1H: δ 4.24, d, J = 7.8 Hz; 13C: δ 101.9) and the remaining 1H and 13C NMR signals for H-2″−H-6″ as shown in Table 1. The D-configuration of the glucose unit was established by the GC-MS method mentioned above. Glucosylation at C-4 of the prenyl group was apparent based on NOESY correlations between H-1″ and H-4′a/H-4′b as well as HMBC correlations from H-1″ and H-4′a/H-4′b to C-4′ and C-1″, respectively. Attachment of the prenyl group at C-7 was established based on a NOESY correlation between H-6/H-8 and H-1′ as well as HMBC correlations from H-6/H-8 and H-1′ to C-1′ and C-7, respectively (Figure 2). Thus, 13 was identified as (Z)-7-(4-β-Dglucopyranosyloxy-3-methylbut-2-en-1-yloxy)coumarin. Full assignments of the 1H and 13C NMR data are shown in Table 1, and selected HMBC and NOE correlations are presented in Figure 2. The material eluted as peaks 19 and 20 (in fraction F9) showed [M + H]+ ions at m/z 313.0836 and 283.0732, which suggested the molecular formulas of C15H17ClO5 (C15H18ClO5+, ΔM 0.4 ppm) and C14H15ClO4 (C14H16ClO4+, ΔM 0.0 ppm), respectively. Both compounds showed isotope patterns of 3:1 and the loss of 36 mass units in the HRESIMS, which supported the presence of a chlorine atom in the molecule. While halogenated natural compounds are known from marine environments and microorganisms, their presence in terrestrial higher plants is less common.32 C. album investigated in this study was collected along the coast with an expected chlorideenriched environment. Moreover, no chlorine-containing solvent was used during extraction and isolation. Compounds 19 and 20 are therefore expected to be biosynthetic products rather than artifacts. 1H NMR signals for a saturated prenyl side chain were observed for both compounds, namely, δ 4.03 (1H, dd, J = 10.4 and 7.9 Hz, H-1′a), δ 4.47 (1H, dd, J = 10.4 and 2.5 Hz, H-1′b), δ 3.92 (1H, dd, 7.9 and 2.5 Hz, H-2′), δ 1.58 (3H, s, H-4′), and δ 1.61 (3H, s, H-5′) for 19, in agreement with a O−CH2−CH(OH)−CHCl(CH3)2 spin system. For compound 19, additional 1H and 13C NMR signals for a methoxy 1024



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Phenomenex Kinetex PFP column (pentafluorophenyl phase, 150 mm × 4.6 mm i.d., 2.6 μm particle size, 100 Å pore size) at 40 °C. GC-MS analyses were conducted on an Agilent 6890N system equipped with a 5971 A mass spectrometer and an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies). Diethyl ether, ethyl acetate, petroleum ether (bp 40−65 °C), HPLC grade methanol, and absolute ethanol (99.9%) were obtained from VWR - Bie & Berntsen (Søborg, Denmark). Anhydrous pyridine (max. 0.0075% H2O) and 32% formic acid were obtained from Merck (Darmstadt, Germany), and acetonitrile-d3 was from Euriso-top (Saint-Aubin Cedex, France). HPLC grade acetonitrile, dichloromethane, hexane, chloroform-d1, methanol-d4, trifluoroacetic acid, R-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride, S-(+)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride, L-cysteine methyl ester hydrochloride, N-methylbis(trifluoroacetamide) (MBTFA), D-glucose, and L-glucose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Water was prepared by deionization and 0.22 μm membrane filtration using a Millipore system (Billerica, MA, USA). Plant Material. Leaves of Coleonema album were collected in September 2014 along the coast at the town of Hermanus, in KwaZulu-Natal, South Africa, and identified by Professor J. C. Manning at the South African National Biodiversity Institute, Rhodes Drive, Newlands, Cape Town, South Africa. Herbarium specimens are available at the University of KwaZulu-Natal Herbarium (accession number: O Fajinmi 01). Extraction and Preparative-Scale C18-Based HPLC Fractionation. For preparative-scale C18-based HPLC fractionation, 15 g of dried and powdered leaves was extracted with ethyl acetate (2 × 100 mL) by sonication for 2 h. The extracts were filtered, lyophilized under reduced pressure, and defatted by partitioning between 90% methanol (200 mL) and petroleum ether (3 × 100 mL), yielding 380 mg of crude defatted extract. Three consecutive injections of 300 μL each of the crude extract at 165 mg/mL were performed. The aqueous eluent (A) consisted of H2O−MeCN (95:5 v/v), and the organic eluent (B) consisted of MeCN−H2O (95:5, v/v), both acidified with 0.1% formic acid. The following gradient profile was used: 0 min, 20% B; 60 min, 90% B; 70 min, 100% B; and flow rate at 20 mL/min. UV traces were monitored at 280 and 330 nm, and 13 fractions were collected: F1 (31.0 mg); F2 (28.4 mg); F3 (34.7 mg); F4 (25.4 mg); F5 (27.4 mg); F6 (23.0 mg); F7 (21.5 mg); F8 (1.0 mg); F9 (1.8 mg); F10 (4.0 mg); F11 (1.3 mg); F12 (2.3 mg); F13 (11.5 mg). On the basis of dereplication by HPLC-HRMS, fractions F1, F2, F3, F6, F9, F11, F12, and F13 were chosen for analysis by HPLC-PDA-HRMS-SPE-NMR. HPLC-PDA-HRMS-SPE-NMR Analyses. The crude extract was initially submitted to HPLC-PDA-HRMS-SPE-NMR analysis employing 10 consecutive separations with the injection of 8 μL of a 50 mg/mL solution. Separations were performed at 40 °C on a Phenomenex Luna C18(2) column (150 mm × 4.6 mm i.d., 3 μm particle size, 100 Å pore size), maintaining a flow rate of 0.5 mL/min of H2O−MeCN (95:5) (eluent A) and MeCN−H2O (95:5) (eluent B), both acidified with 0.1% formic acid. The elution profile was as follows: 0 min, 20% B; 60 min, 90% B; 70 min, 100% B. HPLC-PDAHRMS-SPE-NMR analyses of fractions F1, F2, F3, F6, F9, F11, F12, and F13 were performed after 10 consecutive separations for each sample at 40 °C using a Phenomenex Kinetex PFP column (pentafluorophenyl phase, 150 mm × 4.6 mm i.d., 2.6 μm particle size, 100 Å pore size; Phenomenex Inc.). The flow rate was maintained at 0.5 mL/min, using H2O−MeOH (95:5) (eluent A) and MeOH−H2O (95:5) (eluent B), both acidified with 0.1% formic acid. Injection volumes, sample concentrations, and elution profiles were as follows: F1, 5 μL, 75 mg/mL, 0 min, 20% B; 20 min, 40% B; 30 min, 100% B; F2, 6 μL, 93 mg/mL, 0 min, 30% B; 20 min, 40% B; 30 min, 50% B, 40 min, 70% B; 43 min, 100% B; F3, 3 μL, 87 mg/mL, 0 min, 35% B; 25 min, 45% B; 27 min, 100% B; F6, 5 μL, 77 mg/mL, 0 min, 60% B; 10 min, 65% B; 20 min, 80% B; 22 min, 100% B; F9, 4 μL, 9 mg/mL, 0 min, 60% B; 15 min, 90% B; 17 min, 100% B; F11, 5 μL, 7 mg/mL, 0 min, 60% B; 15 min, 90% B; 17 min, 100% B; F12, 4 μL, 12 mg/mL, 0 min, 60% B; 15 min, 90% B; 17 min, 100% B; F13, 4 μL, 20 mg/mL, 0 min, 60% B; 15 min, 90% B; 17 min, 100% B. Mass spectra were

group (δ 3.94 and 57.1, respectively) were observed, and based on NOESY correlations from H-3 to H-4 to H-5 to H-6 to OCH3 and to H-1′a/H-1′b, the methoxy group was positioned at C-7 and the saturated prenyl moiety was positioned at C-8. For 20, the saturated prenyl was positioned at C-7 based on NOESY correlations between H-1′a/H-1′b and the two upfield shifted and overlapping signals for H-6 and H-8 (δ 6.94, 2H, m). Thus, 19 and 20 were identified as 8-(3-chloro-2-hydroxy3-methylbut-1-yloxy)-7-methoxycoumarin and 7-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)coumarin, respectively. Full assignments of the 1H and 13C NMR data are given in Table 1, and selected HMBC and NOE correlations are shown in Figure 2. The absolute configuration of C-2′ of 19 was established using the modified Mosher ester method.31 Thus, (R)-(+)-MTPA and (S)-(−)-MTPA esters of 19 was formed by reaction with (S)-(−)-MTPA chloride and (R)-(+)-MTPA chloride, respectively, and differences in 1H NMR chemical shift values, i.e., ΔδSR = δ(S) − δ(R), were calculated (Figure S35 and S36, Supporting Information). Using the model for ΔδSR shown in Figure 3A, the C-4′/C-5′ methyl groups were placed on the right-hand side, and the CH2−O−Ar group was placed on the left-hand side (Figure 3B); thus 19 was assigned the S-configuration (Figure 3C). The Mosher ester method was also attempted with 20, but the very limited amount available did not allow unambiguous assignment of the configuration of C-2′. In conclusion, the use of preparative-scale C18 HPLC fractionation followed by orthogonal analytical-scale pentafluorophenyl-based HPLC-PDA-HRMS-SPE-cryoNMR analysis allowed the detailed investigation of the defatted ethyl acetate extract of leaves of C. album, leading to the characterization of six new coumarins. Whereas the preparative-scale C18 separations were mainly influenced by differences in lipophilicity, the separations on the pentafluorophenyl column were also influenced by the π−π-interactions between the aromatic pentafluorophenyl groups on the stationary phase and the aromatic coumarins.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded at 300 K with a Bruker Avance III 600 MHz spectrometer (1H operating frequency 600.13 MHz) equipped with a cryogenically cooled 1.7 mm TCI probe using methanol-d4 or acetonitrile-d3 (99.8 atom % of deuterium) as solvents. Mass spectra were recorded on a Bruker micrOTOF-Q II mass spectrometer equipped with an ESI interface. Preparative HPLC analyses were performed using an Agilent (Santa Clara, CA, USA) 1100 Series HPLC system comprising a G1361A quaternary pump, a G2260A autosampler, and a G1365 B DAD detector, all controlled by Agilent ChemStation version 3.02 software, equipped with a Phenomenex (Phenomenex Inc., Torrance, CA, USA) Luna C18(2) column (250 mm × 21.2 mm i.d., 5 μm particle size, 100 Å pore size). HPLC-PDA-HRMS-SPE-NMR analyses of the crude extract and fractions of C. album were performed using an Agilent 1260 Series chromatographic HPLC system consisting of a G1311B quaternary pump with built-in degasser, a G1316A thermostated column compartment, a G1315D photodiode-array detector, and a G1329B autosampler connected to the Bruker micrOTOF-Q II mass spectrometer described above and operated via a 1:99 flow splitter, a Knauer Smartline K120 pump for postcolumn dilution, a Spark Holland Prospekt2 SPE unit, a Gilson 215 liquid handler for automated filling of 1.7 mm NMR tubes from the Prospekt2 device, and the above-mentioned Bruker Avance III 600 MHz NMR spectrometer equipped with a cooled SampleJet sample changer. Targeted HPLC isolations were performed on a Agilent 1200 Series HPLC system comprising a G1315A quaternary pump with a G1322A degasser, a G1367C autosampler, and a G1315C DAD detector equipped with a 1025



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acquired in positive-ion mode, at 200 °C drying temperature, a capillary voltage of 4100 V, a nebulizer pressure of 2.0 bar, and a drying gas flow of 7 L/min. A solution of sodium formate clusters was automatically injected at the beginning of each run to enable internal mass calibration. The HPLC eluate directed to the photodiode array detector was subsequently diluted with Milli-Q water at a flow rate of 1 mL/min. This allowed for cumulative SPE trappings of selected analytes on Hysphere Resin GP cartridges (10 × 2 mm i.d.) using absorption thresholds at 330 nm as well as base peak chromatograms to trigger analyte trapping. After 45 min of drying with pressurized nitrogen gas, analytes were automatically eluted into 1.7 mm o.d. NMR tubes (96-position tube racks) with acetonitrile-d3. Targeted Isolation. For targeted isolation, 12 g of powdered leaves was extracted as described above, yielding 325 mg of defatted ethyl acetate extract. Preparative-scale HPLC fractionation using the method described above yielded F2 (21.4 mg dissolved to 214 mg/mL in MeOH), fraction F3 (29.5 mg dissolved to 295 mg/mL in MeOH), and fraction F9 (4.0 mg dissolved to 23 mg/mL). All fractions were separated using the same chromatographic methods as described for the HPLC-PDA-HRMS-SPE-NMR analyses on an Agilent 1200 Series analytical HPLC with manual fraction collection monitored at 330 nm. This yielded 1.6 mg of 6, 0.7 mg of 8, 2.1 mg of 13, 1.4 mg of 19, and 0.8 mg of 20. NMR Experiments. All NMR spectra were recorded in methanold4 or acetonitrile-d3 at 300 K, and 1H and 13C chemical shifts were referenced to the residual solvent signal of methanol-d4 (δ 3.31 and δ 49.0, respectively) or acetonitrile-d3 (δ 1.94 and 118.26, respectively). 1H NMR spectra were recorded using 30° pulses with a spectral width of 20 ppm, acquisition time 2.72 s, and relaxation delay 1.0 s, collecting 256 FIDs, each consisting of 64 k data points, and Fourier-transformed to 256 k data points with a line broadening factor of 0.1 Hz. Phase-sensitive DQF-COSY and NOESY spectra were recorded using a gradient-based pulse sequence with 20 ppm spectral width and 2 k × 512 data points (processed with forward linear prediction to 1 k data points). A multiplicity-edited HSQC spectrum was acquired with the following parameters: spectral width 20 ppm for 1H and 170 ppm for 13C, 2 k × 256 data points, and 1.0 s relaxation delay. HMBC was acquired using a spectral width of 20 ppm for 1H and 240 ppm for 13C, 2 k × 128 data points, and 1.0 s relaxation delay. Determination of Absolute Configuration. For Mosher ester analyses, each of the analytes was divided into two equal parts and placed in two reaction vials marked as A and B. Then, 16 μL of anhydrous pyridine was added followed by 0.3 mL of anhydrous dichloromethane. To vial A was added 23 μL of R-(−)-MTPA-Cl, whereas to vial B was added 23 μL of S-(+)-MTPA-Cl. The reaction was monitored by TLC analysis with silica gel 60 F254 eluted with hexane−ethyl acetate (4:1) and sprayed with potassium permanganate. The products were isolated by partitioning of the reaction mixtures between diethyl ether (3 mL) and water (1 mL) followed by extraction of the aqueous phase with 3 × 2 mL of diethyl ether. The organic phases were dried, and the remaining residues dissolved in approximately 30 μL of CDCl3 and immediately transferred to 1.7 mm NMR tubes. 1H NMR and 2D NMR (DQF-COSY and HSQC) spectra were acquired. Determination of the absolute configuration of glucose was performed using 0.3 to 1.0 mg of each analyte dissolved in 100 μL of 6 M trifluoroacetic acid. After heating for 1 h at 70 °C, the sample was evaporated to dryness under a stream of N2 gas and co-distillated with 200 μL of absolute ethanol. Subsequently, the sample was dissolved in 1 mL of anhydrous pyridine, and 2 mg of L-cysteine methyl ester hydrochloride was added, whereafter the mixture was heated for 1 h at 65 °C (shaking it every 10 min). The reaction mixture was cooled to room temperature, and 500 μL was reacted with 200 μL of MBTFA for 20 min at 65 °C. The sample was analyzed subsequently by GC-MS with a helium carrier gas flow rate of 1 mL/min and column temperature programmed to 90 °C for 4 min, and thereafter increasing gradually to 200 °C at 3 °C/min and kept at 200 °C for 10 min. The samples were compared to standards of D-glucose and L-glucose.

8-O-β-D-Glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)7-methoxycoumarin (4): insufficient material was available to obtain specific rotation; 1H NMR (CD3CN, 600 MHz) δ 7.79 (1H, d, J = 9.5 Hz, H-4), 7.29 (1H, s, H-5), 6.29 (1H, d, J = 9.5 Hz, H-3), 5.07 (1H, d, J = 7.8 Hz, H-1″), 3.96 (3H, s, H-7-OCH3), 3.63 (1H, ddd, J = 11.5, 6.0, 2.5 Hz, H-6″b), 3.55 (1H, ddd, J = 11.5, 6.0, 6.0 Hz, H-6″a), 3.49 (1H, overlapping signal, H-2′), 3.48 (1H, overlapping signal, H-2″), 3.37 (1H, m, H-3″), 3.34 (1H, m, H-4″), 3.23 (1H, m, H-5″), 2.96 (1H, d, J = 13.6 Hz, H-1′b), 2.51 (1H, dd, J = 13.6, 10.4 Hz, H-1′a), 1.19 (6H, d, J = 11.2 Hz, H-4′-CH3, H-5′-CH3); 13C NMR (CD3CN, 150 MHz) δ 160.5 (C-2), 115.1 (C-3), 144.8 (C-4), 126.0 (C-5), 126.7 (C-6), 154.3 (C-7), 144.5 (C-8), 147.7 (C-9), 116.0 (C-10), 79.0 (C-2′), 24.9 (C-4′), 26.0 (C-5′), 104.1 (C-1″), 75.1 (C-2″), 77.2 (C-3″), 71.0 (C-4″), 77.5 (C-5″), 62.3 (C-6″), 62.3 (OCH3); (+) HRESIMS m/z 457.1703 [M + H]+ (calcd for C21H29O11+, 457.1704; ΔM 0.3 ppm). (Z)-6-(4-β- D-Glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (6): yellowish, amorphous solid; insufficient material was available to obtain specific rotation; 1H NMR (CD3CN, 600 MHz) δ 7.73 (1H, d, J = 9.5 Hz, H-4), 7.30 (1H, s, H-5), 6.76 (1H, s, H-8), 6.13 (1H, d, J = 9.5 Hz, H-3), 5.63 (1H, t, J = 7.4 Hz, H-2′), 4.23 (1H, d, J = 7.8 Hz, H-1″), 4.20 (1H, d, J = 11.9 Hz, H-4′b), 4.02 (1H, d, J = 11.9 Hz, H-4′a), 3.71 (1H, ddd, J = 11.6, 6.4, 2.8 Hz, H-6″b), 3.56 (1H, ddd, J = 11.6, 5.8, 5.8 Hz, H-6″a), 3.36 (2H, d, J = 7.4 Hz, H-1′a/ H-1′b), 3.26 (1H, dt, J = 8.8, 3.5 Hz, H-3″), 3.22 (1H, ddd, J = 9.3, 8.8, 3.5 Hz, H-4″), 3.16 (1H, ddd, J = 9.3, 5.8, 2.8 Hz, H-5″), 3.11 (1H, ddd, J = 8.8, 7.8, 3.5 Hz, H-2″), 1.75 (3H, s, H-5′); 13C NMR (CD3CN, 150 MHz) δ 161.4 (C-2), 113.0 (C-3), 144.9 (C-4), 129.5 (C-5), 112.3 (C-6), 158.3 (C-7), 102.9 (C-8), 154.4 (C-9), 112.0 (C-10), 26.9 (C-1′), 126.4 (C-2′), 75.0 (C-4′), 14.2 (C-5′), 102.2 (C-1″), 74.6 (C-2″), 77.7 (C-3″), 71.5 (C-4″), 77.0 (C-5″), 62.8 (C-6″); (+) HRESIMS m/z 409.1493 [M + H]+ (calcd for C20H25O9+, 409.1493; ΔM 0.0 ppm). 6-(4-β-D-Glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (8): yellowish, amorphous solid; insufficient material was available to obtain specific rotation; 1H NMR (CD3CN, 600 MHz) δ 7.73 (1H, d, J = 9.5 Hz, H-4), 7.33 (1H, s, H-5), 6.74 (1H, s, H-8), 6.13 (1H, d, J = 9.5 Hz, H-3), 4.21 (1H, d, J = 7.8 Hz, H-1″), 3.74 (1H, m, H-6″b), 3.67 (1H, dd, J = 9.5, 6.5 Hz, H-4′b), 3.58 (1H, m, H-6″a), 3.42 (1H, dd, J = 9.5, 6.0 Hz, H-4′a), 3.30 (1H, m, H-5″), 3.22 (2H, overlapping signal, H-3″, H-4″), 3.08 (1H, ddd, J = 8.0, 7.8, 2.9 Hz, H-2″), 2.69 (1H, ddd, J = 13.8, 10.1, 5.8 Hz, H-1′b), 2.60 (1H, ddd, J = 13.8, 10.1, 5.8 Hz, H-1′a), 1.76 (1H, dd, J = 13.1, 6.5 Hz, H-3′), 1.71 (1H, m, H2′b), 1.43 (1H, m, H-2′a), 0.97 (3H, d, J = 6.5 Hz, H-5′); 13C NMR (CD3CN, 150 MHz) δ 162.3 (C-2), 112.9 (C-3), 144.7 (C-4), 129.8 (C-5), 127.8 (C-6), 159.3 (C-7), 102.8 (C-8), 154.7 (C-9), 112.9 (C-10), 27.3 (C-1′), 34.3 (C-2′), 33.8 (C-3′), 75.2 (C-4′), 17.3 (C-5′), 103.6 (C-1″), 74.6 (C-2″), 77.0 (C-3″), 71.4 (C-4″), 77.4 (C-5″), 62.7 (C-6″); (+) HRESIMS m/z 411.1645 [M + H]+ (calcd for C20H27O9+, 411.1650; ΔM 1.1 ppm). (Z)-7-(4-β- D -Glucopyranosyloxy-3-methylbut-2-en-1-yloxy)coumarin (13): yellowish, amorphous solid; insufficient material was available to obtain specific rotation; 1H NMR (CD3CN, 600 MHz) δ 7.78 (1H, d, J = 9.5 Hz, H-4), 7.49 (1H, d, J = 9.2 Hz, H-5), 6.89 (2H, m, H-6, H-8), 6.19 (1H, d, J = 9.5 Hz, H-3), 5.69 (1H, t, J = 6.7 Hz, H-2′), 4.69 (2H, d, J = 6.7 Hz, H-1′a/H-1′b), 4.33 (1H, d, J = 12.2 Hz, H-4′b), 4.27 (1H, d, J = 12.2 Hz, H-4′a), 4.24 (1H, d, J = 7.8 Hz, H-1″), 3.71 (1H, ddd, J = 11.5, 6.0, 2.5 Hz, H-6″b), 3.55 (1H, ddd, J = 11.5, 5.6, 5.6 Hz, H-6″a), 3.29 (1H, dt, J = 8.8, 3.8 Hz, H-3″), 3.23 (1H, ddd, J = 9.5, 8.8, 4.1 Hz, H-4″), 3.21 (1H, ddd, J = 9.5, 5.6, 2.5 Hz, H-5″), 3.13 (1H, ddd, J = 8.8, 7.8, 3.8 Hz, H-2″), 1.85 (3H, s, H-5′); 13C NMR (CD3CN, 150 MHz) δ 162.1 (C-2), 113.5 (C-3), 144.7 (C-4), 130.0 (C-5), 113.6 (C-6), 163.1 (C-7), 102.6 (C-8), 157.0 (C-9), 113.8 (C-10), 65.7 (C-1′), 124.5 (C-2″), 139.1 (C-3′), 67.3 (C-4′), 21.6 (C-5′), 101.9 (C-1″), 74.5 (C-2″), 77.5 (C-3″), 71.4 (C-4″), 77.0 (C-5″), 62.6 (C-6″); (+) HRESIMS m/z 409.1492 [M + H]+ (calcd for C20H25O9+, 409.1492; ΔM 0.3 ppm). 8-(3-Chloro-2-hydroxy-3-methylbut-1-yloxy)-7-methoxycoumarin (19): white, amorphous solid; insufficient material was available to obtain specific rotation; 1H NMR (CD3CN, 600 MHz) δ 7.78 (1H, d, 1026



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J = 9.6 Hz, H-4), 7.33 (1H, d, J = 8.7 Hz, H-5), 7.02 (1H, d, J = 8.7 Hz, H-6), 6.22 (1H, d, J = 9.6 Hz, H-3), 4.47 (1H, dd, J = 10.4, 2.5 Hz, H-1′b), 4.03 (1H, dd, J = 10.4, 7.9 Hz, H-1′a), 3.94 (3H, br s, H-7-OCH3), 3.92 (1H, dd, J = 7.9, 2.5 Hz, H-2′), 1.61 (3H, s, H-5′), 1.58 (3H, s, H-4′); 13C NMR (CD3CN, 150 MHz) δ 161.4 (C-2), 113.9 (C-3), 144.8 (C-4), 124.5 (C-5), 109.8 (C-6), 155.3 (C-7), 135.4 (C-8), 148.6 (C-9), 114.7 (C-10), 76.2 (C-1′), 77.6 (C-2′), 71.1 (C-3′), 28.5 (C-4′), 29.7 (C-5′); (+) HRESIMS m/z 313.0836 [M + H]+ (calcd for C15H18ClO5+, 313.0837; ΔM 0.4 ppm), 277.1074 [M + H − Cl]+ (calcd for C15H17O5+, 277.1071; ΔM 0.6 ppm). 7-(3-Chloro-2-hydroxy-3-methylbut-1-yloxy)coumarin (20): white, amorphous solid; insufficient material was available to obtain specific rotation; 1H NMR (CD3CN, 600 MHz) δ 7.79 (1H, d, J = 9.5 Hz, H-4), 7.53 (1H, d, J = 9.2 Hz, H-5), 6.94 (2H, overlapping signal, H-6, H-8), 6.21 (1H, d, J = 9.5 Hz, H-3), 4.41 (1H, dd, J = 10.0, 2.5 Hz, H-1′b), 4.06 (1H, dd, J = 10.0, 7.8 Hz, H-1′a), 3.94 (1H, ddd, J = 7.8, 5.5, 2.5 Hz, H-2′), 1.64 (3H, s, H-5′), 1.61 (3H, s, H-4′); 13C NMR (CD3CN, 150 MHz) δ 161.6 (C-2), 113.7 (C-3), 144.5 (C-4), 130.0 (C-5), 113.5 (C-6), 162.4 (C-7), 102.4 (C-8), 156.9 (C-9), 113.7 (C-10), 71.1 (C-1′), 77.1 (C-2′), 28.4 (C-4′), 29.7 (C-5′); (+) HRESIMS m/z 283.0732 [M + H]+ (calcd for C14H16ClO4+, 283.0732; ΔM 0.0 ppm); 247.0963 [M + H − Cl]+ (calcd for C14H15O4+, 247.0965; ΔM 0.8 ppm).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01020. Table S1 with retention time, HRMS, and 1H NMR data of the material eluted with peaks 1−26, and Figures S1−S36 with trapping event of a pilot HPLC-PDA-HRMS-SPENMR experiment, structures of compounds 1−26, NMR spectra of 4, 6, 8, 13, 19, and 20, and 1H NMR spectra from Mosher ester method with 19 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +45 3533 6411. Fax: +45 3533 6001. E-mail: [email protected]. ORCID

Kenneth T. Kongstad: 0000-0003-4487-7886 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The 600 MHz HPLC-HRMS-SPE-NMR system used in this work was acquired through a grant from “Apotekerfonden af 1991”, The Carlsberg Foundation, and the Danish Agency for Science, Technology and Innovation via the National Research Infrastructure funds. HPLC equipment used for targeted isolation was obtained via a grant from The Carlsberg Foundation. CAPES (Coordenaçaõ de Aperfeiçoamento de ́ Pessoal de Nivel Superior) is acknowledged for financial support of R.L. under protocol BEX 12010-13-8. A. Ö nder is acknowledged for technical assistance with preparative-scale HPLC isolation.

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REFERENCES

(1) Eldeen, I. M. S.; Van Staden, J. S. Afr. J. Bot. 2008, 74, 345−347. (2) Esterhuizen, L. L.; Meyer, R.; Dubery, I. A. Z. Naturforsch., C: J. Biosci. 2006, 61, 489−498. (3) Esterhuizen, L. L.; Meyer, R.; Dubery, I. A. Nat. Prod. Commun. 2006, 1, 367−375. (4) Gray, A. I. Phytochemistry 1981, 20, 1711−1713. 1027


Advancing HPLC-PDA-HRMS-SPE-NMR Analysis of Coumarins in

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