NMR-based characterization of a novel yellow chlorophyll catabolite, Ed-YCC, isolated from Egeria densa

Tetrahedron Letters 55 (2014) 2982–2985

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NMR-based characterization of a novel yellow chlorophyll catabolite, Ed-YCC, isolated from Egeria densa Daigo Wakana a,b, , Hiroki Kato c, , Tadayuki Momose c, Nobuhiro Sasaki c,d, Yoshihiro Ozeki c, Yukihiro Goda b,⇑ a

Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, Ministry of Health, Labour and Welfare, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan c Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan d Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan b

a r t i c l e

i n f o

Article history: Received 9 December 2013 Revised 20 March 2014 Accepted 26 March 2014 Available online 3 April 2014

a b s t r a c t A novel yellow chlorophyll catabolite, Ed-YCC, was isolated from leaves detached from Egeria densa shoots, in which chlorophyll degradation and anthocyanin synthesis were induced in 0.1 M fructose solution under light illumination as a plant senescence process, a model of autumnal leaf coloration. Structure elucidation was accomplished by various NMR techniques including 2D-INADEQUATE. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Chlorophyll Egeria densa Yellow chlorophyll catabolite 2D-INADEQUATE NMR

Autumnal leaf coloration of deciduous tree plants is one of the representative events in plant senescence.1,2 In this process, degradation of chlorophyll occurs by environmental changes such as lower temperature and shorter days, and leaves turn brown, yellow, and red. Reddish coloring of autumn leaves is caused by synthesis and accumulation of anthocyanins coincident with the degradation of chlorophyll.1 Chlorophyll degradation is catalyzed according to its catabolic pathway, which was initially clarified by the discovery of non-fluorescent chlorophyll catabolites (NCCs) in the leaves of barley, Hordeum vulgare, that had been artificially induced into senescence.3 Pheophorbide a,4 red chlorophyll catabolite (RCC)5 and primary fluorescent chlorophyll catabolite (pFCC)6

Abbreviations: ACN, acetonitrile; DPT, distortionless enhancement by polarization transfer; COSY, correlation spectroscopy; ESI, electrospray ionization; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence; HPLC, high-performance liquid chromatography; HR, high-resolution; INADEQUATE, incredible natural abundance double quantum transfer experiment; MS, mass spectrometry; NCC, non-fluorescent chlorophyll catabolite; pFCC, primary fluorescent chlorophyll catabolite; RCC, red chlorophyll catabolite; TFA, trifluoroacetic acid; TOCSY, total correlation spectroscopy; TOF, time-of-flight; YCC, yellow chlorophyll catabolite. ⇑ Corresponding author. Tel.: +81 3 3700 9154; fax: +81 3 3700 9165. E-mail address: [email protected] (Y. Goda).   These authors contributed equally to this work. http://dx.doi.org/10.1016/j.tetlet.2014.03.114 0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

have been identified as important intermediates in the chlorophyll catabolic pathway. After the modification of pFCC with glucose7 or malonic acid,8 modified pFCCs isomerize non-enzymatically to generate their respective NCCs followed by transportation and storage in vacuoles.9 Recently, yellow chlorophyll catabolites (YCCs) were identified in senescent leaves of Cercidiphyllum japonicum10 and Tilia cordata.11 Although the phenomenon of autumnal reddening of leaves of deciduous trees is familiar, the induction mechanisms of this phenomenon have not yet been clarified because this event occurs only once a year and fluctuates according to climatic variations. Attempts have been made to develop model systems in which autumnal reddish leaf coloration is artificially and reproducibly induced over a short time period, at any time and in-house. In our laboratory, we established a system in which autumnal reddish leaf coloration was induced in Egeria densa. When the leaves detached from E. densa shoots were incubated in sugar solution under light illumination, both degradation of chlorophyll and accumulation of anthocyanin, 5-O-methyl cyanidin 3-O-glucoside,12 were simultaneously and reproducibly induced within a week.13 We noticed that, during this process, a remarkable amount of a yellow compound (1, shown in Fig. 1), which was detected by high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC), was produced and accumulated together

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D. Wakana et al. / Tetrahedron Letters 55 (2014) 2982–2985 71

Me HO

5

32 31

OH OHC O

*

3

21

A

2

Me

1

H 18 1

20

Me

4

7 6

HN

19

81

8

O

82

O

10 23

HN H C N H D R* 16

15

13 2

18

S*

17 17 1 17 2 17 3

HO

O

13 3

84

85

COOH

11 12

12

14

24

83

9

22

NH 21

B

1

Me

13

13 1

H

O

O

O Me

13 4

Figure 1. Structure of 1 isolated from E. densa.

with chlorophyll degradation and anthocyanin accumulation. Here, we isolated and purified this yellow compound (1) and determined its structure using a JEOL UltraCOOL NMR probe coupled to an ECA-800 spectrometer as 31,32-dihydroxy-82-hydroxymalonyl132-methoxy-carbonyl-4,5,10,15,21,22,23,24-octahydro-4,5-dioxosecophytoporphyrin, which is a novel YCC not previously identified or reported. Shoots of Egeria densa (Planch.) St. John were collected from the middle course of Ochiai River (Tokyo prefecture) in summer 2012. The leaves were detached from the shoots manually and cultured in 0.1 M fructose solution at 25 °C for 7 days under continuous light (20 lmol s 1 m 2) to induce the autumnal reddish leaf coloration.13 The red-colored leaves were dried with paper towels, airdried 15 min using a hair dryer at room temperature, and further dried in a desiccator containing fresh silica gel over several days until extraction of the yellow compound (1). The yellow compound (1) was extracted from 80 g (dry weight) of the red-colored leaves immersed in 4 L of 50% aqueous methanol overnight at room temperature. The extract was filtered and evaporated to approximately 100 mL, and then loaded onto an HP-20 resin (Mitsubishi Chemical Corp., Tokyo, Japan) open column (i.d. 50  80 mm column) equilibrated with H2O. After washing with excess H2O, the compound was eluted with 100% methanol and was visible as a yellow-colored eluate. The eluate was evaporated to approximately 10 mL, and was purified using a medium-pressure liquid chromatograph (Yamazen Flash Liquid Chromatography YFLC-AI-580, Yamazen Corp., Osaka, Japan) equipped with an HP20 ss resin (Mitsubishi Chemical Corp., Tokyo, Japan) column (i.d. 26  100 mm) by the elution program of solvent A [0.1% aqueous trifluoroacetic acid (TFA)] and methanol at 20 mL min 1 as follows: 40% methanol in A for 0–5 min; linear gradient of 40–90% methanol in A for 5–15 min; 90% methanol in A for 15–22 min. The yellow fractions eluted in 90% methanol were collected and diluted with an equal volume of H2O, and then immediately applied to an HP20 resin open column (i.d. 34  60 mm) equilibrated with H2O. Solvent A containing 0.1% TFA was required to achieve better separation on MPLC, but it caused degradation of Ed-YCC under the storage conditions even at 80 °C. Therefore, it should be removed as soon as possible after separation. The HP-20 column was washed with an excess volume of H2O and then the TFA-free yellow compound was eluted with 100% methanol. The eluate was evaporated to approximately 10 mL, and was purified using two tandemjointed Hi-Flash ODS-SM columns (50 lm, i.d. 26  100 mm, Yamazen), by a stepwise elution program of solvent A and methanol at 20 mL min 1 as follows: 40% methanol in A for 0–5 min; 40–60% methanol in A for 5–20 min; 60% methanol in A for 20–27 min. The yellow fractions eluted at 60% methanol were collected and diluted with an equal volume of H2O, and then applied to an HP-20 resin open column (i.d. 14  90 mm) equilibrated with H2O

and washed with an excess volume of H2O. The yellow compound was collected in 100% methanol without TFA. The eluate was evaporated to approximately 10 mL, and was re-chromatographed using two tandem-jointed Hi-Flash ODS-SM columns as above except that acetonitrile (ACN) was used as the elution solvent instead of methanol at 20 mL min 1 by the following elution program: 20% ACN in A for 0–5 min; linear gradient of 20–40% ACN in A for 5– 20 min; 40% ACN in A for 20–23 min. The yellow fractions eluted in 40% ACN were combined and loaded on a single HP-20 ss resin column (i.d. 20  65 mm) to remove TFA by H2O and methanol as elution solutions following a stepwise program at 20 mL min 1 lacking TFA: 40% methanol in H2O for 0–15 min; 95% methanol in H2O for 15–20 min. The eluted yellow fractions were evaporated and diluted to 3 mL with H2O, and freeze-dried. A yellow powder (1) of 34.0 mg from 80 g (dry weight) of the red leaves was obtained and stored at 80 °C under argon before MS and NMR measurements. Compound 1 was obtained as a yellow amorphous powder, and the HPLC-PDA chromatogram (Supplementary Fig. S-1) of 1 showed that the purity was over 90% calculated from the peak area at UV 409 nm, which was the maximum absorption of 1. The ESI-TOFMS spectrum in positive ion mode showed a peak at m/z 785.2630 which corresponded to C38H42N4O13Na ([M+Na]+, calcd 785.2641); in negative mode, a peak at m/z 761.2650 (C38H41N4O13, [M H] , calcd 761.2665) appeared. Therefore, the molecular formula was confirmed as C38H42N4O13. The ion trap TOF-MS spectrum in positive mode showed the following fragmentations: MS1: m/z 785.2630; MS2: m/z 753.2377; MS3: m/z 709.2474; MS4: m/z 681.2525; and MS5: m/z 621.2312. These results suggested the presence of a methyl ester group, carbonyl group, formyl group, and C2H4O2 fragment which is considered to be a dihydroxyethyl group. The 1H NMR spectrum showed four singlet protons in the lower field (d 9.95, 10.17, 11.30 and 11.34), a formyl proton (d 9.44), an olefinic proton (d 5.98), a methoxy proton (d 3.66), and four methyl protons (d 2.03, 2.03, 2.16, and 2.18) along with thirteen other protons. The 13C NMR and DEPT spectra showed four methyl carbons (d 9.8, 9.3, 9.1, and 8.7), a methoxy carbon (d 52.3), seven methylene carbons (d 64.8, 64.5, 42.2, 35.4, 22.7, 22.4, and 19.3), three methine carbons (d 67.4, 66.3, and 36.1), an Table 1 H, 13C and

1

1

15

N NMR data of 1 in dimethylsulfoxide-d6

Atom

dC (dN)

1 2 21 3 31 32

130.3 144.0 9.8 128.1 67.4 64.8

4 5 6 7 71 8 81 82 83 84 85 9 10

171.5 176.8 128.2 131.6 8.7 117.6 22.7 64.5 167.3 42.2 168.4 136.1 22.4

11 12

133.3 110.0

dH (J in Hz)

2.16s 4.49 (bt, 5.5) 3.47 (dd, 4.6, 10.1) 3.51 (dd, 7.3, 10.1) 9.44s

2.18s 2.63bs 3.94bs 3.28s

3.81 (d, 15) 3.86 (d, 15)

Atom

dC (dN)

121 13 131 132 133 134

9.1 124.6 188.0 66.3 170.0 52.3

14 15 16 17 171 172 173 18 181 19 20 21 22

157.1 36.1 129.8 120.9 19.3 35.4 174.0 122.9 9.3 123.8 98.4 (125.3) (148.9)

23 24

(151.4) (138.4)

dH (J in Hz) 2.03s

3.84s 3.66s

4.83s

2.47m 2.07m, 2.24m

2.03s 5.98s 9.95s 11.34s 11.30s 10.17s

H NMR, 13C NMR and DEPT, HMBC, HMQC and 2D-INADEQUATE (J = 40 Hz and 70 Hz) spectra of 1 in dimethylsulfoxide-d6 are available as Figures S-2 to S-7 in Supplementary Materials.

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A

B Me

HO

OH OHC O

F1

HN

Me

O HO

O

HN

H N

H

F4

O

NH

Me

Me

F3

Me O

HO

COOH

C HMBC

Me

O

O

O Me

HO

O

HN

H N

H

2D-INADEQUATE

O HN

NH

Me

F2 13

OH OHC O

Me O

O

O Me

15 N

Figure 2. Detailed analysis of 2D NMR spectra of 1. (A) 2D-INADEQUATE and selected HMBC correlations. (B)

aldehyde carbon (d 176.8), an sp2 carbon (d 98.4), and 21 quaternary sp2 carbons (Table 1). The 1H NMR spectrum showed four characteristic NH protons (d 11.34, 11.30, 10.17, and 9.95). From the above data, 1 was suggested to be a chlorophyll catabolite that had many quaternary carbons. Therefore we attempted the 2DINADEQUATE (2D Incredible Natural Abundance DoublE QUAntum Transfer Experiment) NMR experiment to elucidate the structure using a JEOL CH UltraCOOL probe. The 2D-INADEQUATE spectrum of 1 at its natural abundance identified four unambiguous fragments (F1–F4) with contiguous carbon skeletons (Fig. 2): F1, C32-C31-C3(C4)-C2-C1-C20-C19-C18-C-17(C171-C172-C173)-C16-C15C132-C133; F2, C131-C13(C14)-C12-C11-C10; F3, C81-C82; and F4, C83-C84-C85. In fragment F1, a 1,2-dihydroxyethyl-4-methyl-2oxo-1H-pyrrole moiety was identified by the HMBC correlations from the 21-NH to C-1, C-2 and C-3, from 21-Me to C-1, C-2 and C-3 (Fig. 2) and two hydroxylcarbon signals at d 64.8 and 67.4 (C-32 and C-31). The 15N-HMBC correlations from 15-H and 20-H to N-24, the HMBC correlations from 181-Me to C-17, C-18 and C-19 and the carboxyl signal at d 174.0 (C-173) suggested the presence of a 4-methyl-1H-pyrrole-3-propanoic acid moiety in fragment F1. Furthermore, the methoxy group was a part of the methyl carboxylate moiety according to the HMBC correlation from 134-Me to C-133 in fragment F1. In fragment F2, a 4-carbonyl-3-methyl1H-pyrrole moiety was confirmed by the HMBC correlations from 23-NH to C-11, C-12, and C-13 and the carbonyl carbon signal at d 188.0. The connectivity of F1 and F2 was confirmed by the HMBC correlations from 132-H to C-131 and from 15-H to C-13 and C-14. The connection of F3 and F4 was deduced by the HMBC correlation from 82-H2 to C-83. The 2D-INADEQUATE correlations of the pyrrole ring of B could not be detected; therefore, we attempted to elucidate the partial structure of ring B by detailed analysis of the HMBC spectrum. The HMBC correlations from 71-Me to C-6, C-7, and C-8, and from 81-H2 existing in the F3 fragment to C-7, C-8, and C-9 suggested the six carbons (C-6, C-7, C-8, C-9, C-71, and C-81) were connected as C6-C7(C71)-C8(C81)-C9. Furthermore, the HMBC correlations from 22-NH to C-7 and C-8 suggested a 1H-pyrrole moiety. The connectivity of this pyrrole ring and the remaining formyl group was elucidated by the HMBC correlation from 5-H to C-7 and the 15N-HMBC correlation from 5-H to N-22. The connectivity of the pyrrole ring B and the F2 fragment was deduced from the HMBC correlations from 10-H2 to C-8 and C-9. The NMR results as described inevitably led to the sequential connectivity from the F1 fragment, via the F2 fragment, the pyrrole ring B and the F3 fragment, to the F4 fragment. Compound 1 has three chiral centers at the C-31, C-132, and C-15 positions. We compared the coupling constant between 132-H and 15-H of Ed-YCC to that of Cj-NCC-29, a chlorophyll breakdown product of which the configuration between 132-H and 15-H was known as trans.14 In

COOH

HMBC

15

N-HMBC correlations.

the literature, the signal of 15-H was described as singlet, while the signals of 132-H and 15-H of 1 were observed as singlet. These data suggested that the relative configuration between 132-H and 15-H was trans. The stereochemistry of C-31 remained unclear. Finally, full assignments of NMR signals as shown in Table 1 and the molecular formula obtained by MS indicated that the structure of compound 1 should be (Z)-3-(2-(2-((6-(3-(2-carboxyethyl)-5((4-(1,2-dihydroxyethyl)-3-methyl-5-oxo-1H-pyrrol-2(5H)-ylidene) methyl)-4-methyl-1H-pyrrol-2-yl)-5-(methoxycarbonyl)-3-methyl4-oxo-1,4,5,6-tetrahydrocyclopenta[b]pyrrol-2-yl)methyl)-5-formyl4-methyl-1H-pyrrol-3-yl)ethoxy)-3-oxopropanoic acid. This compound was designated as Ed-YCC. Author contributions D.W. and H.K. contributed equally to the design of all the experiments from extraction and purification to determination of the molecular structure of yellow compound 1. T.M. prepared the autumnal reddish-colored E. densa leaves. D.W. N.S., Y.O. and Y.G. wrote the paper. Y.O. and Y.G. conducted all experiments. All authors provided comments. Acknowledgements We are grateful to Professor Dr. K. Nagasawa, Mr. K. Iida, and T. Nakagawa, Tokyo University of Agriculture and Technology, for their help in storing the unstable Ed-YCC 1 under argon gas until NMR measurements could be taken. This research was partially supported by a Grant-in-Aid for Exploratory Research (No. 22657012) from the Japan Society for the Promotion of Science (JSPS). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.03. 114. References and notes 1. Ishikura, N. Kumamoto J. Sci. Biol. 1973, 11, 43–50. 2. Keskitalo, J.; Berqquist, G.; Gardeström, P.; Jansson, S. Plant Physiol. 2005, 139, 1635–1648. 3. Kräutler, B.; Jaun, B.; Bortlik, K. H.; Schellenberg, M.; Matile, P. Angew. Chem. Int. Ed. Engl. 1991, 30, 1315–1318. 4. Hörtensteiner, S.; Vicentini, F.; Matile, P. New Phytol. 1995, 129, 237–246. 5. Kräutler, B.; Mühlecker, W.; Anderl, M.; Gerlach, B. Helv. Chim. Acta 1997, 80, 1355–1362. 6. Mühlecker, W.; Ongania, K. H.; Kräutler, B.; Matile, P.; Hörtensteiner, S. Angew. Chem., Int. Ed. 1997, 36, 401–404.

D. Wakana et al. / Tetrahedron Letters 55 (2014) 2982–2985 7. Pruzˇinská, A.; Tanner, G.; Aubry, S.; Anders, I.; Moser, S.; Müller, T.; Ongania, K. H.; Kräutler, B.; Youn, J. Y.; Liljegren, S. J.; Hörtensteiner, S. Plant Physiol. 2005, 139, 52–63. 8. Mühlecker, W.; Kräutler, B.; Ginsburg, S.; Matile, P. Helv. Chim. Acta 1993, 76, 2976–2980. 9. Oberhuber, M.; Berghold, J.; Breuker, K.; Hörtensteiner, S.; Kräutler, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6910–6915.

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10. Moser, S.; Ulrich, M.; Müller, T.; Kräutler, B. Photochem. Photobiol. Sci. 2008, 7, 1577–1581. 11. Scherl, M.; Müller, T.; Kräutler, B. Chem. Biodivers. 2012, 9, 2605–2617. 12. Momose, T.; Abe, K.; Yoshitama, K. Phytochemistry 1977, 16, 1321. 13. Momose, T.; Ozeki, Y. J. Plant. Res. 2013, 126, 859–867. 14. Oberhuber, M.; Berghold, J.; Breuker, K. Angew. Chem., Int. Ed. 2008, 47, 3057– 3061.