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Oligopeptides from Aster tataricus
Pergsmon
0031~!M22@4)EOO3S-Q
Phyrdumisrry.
Vol. 36, No. 4. pp. 945 948, 1594 Ekvin %mc+ Ltd Pnntcd in Great Britain. 003 -9422l94 f7.00 + 0.00
I
OLIGOPEPTIDES DON~LIANG
FROM
CHENG, Yu SHAO,
ASTER
TATARICUS
R. HARTMAN,* E. RODER* and K. ZHAO~
Institute of Organic Chemistry, State Key Laboratory of Applied Organic Chemistry, Lanzhou 730000, P. R. China; * Pharmazeutisches and Physiologishe Chemie Institut, Universit Bonn, D-5300 Bonn, F.R.G.; tI%partment of Chemistry, New York University, New York, NY 10003, U.S.A. (Received 19 October 1993)
Key Word Index--Aster
tataricus;
Compositae; roots; oligopeptides; asterinin A, B and C.
Abstract-Three
novel oligopeptides, asterinin A-C have been isolated from the roots of A. tataricus and their structures elucidated by chemical, enzymatic and spectral methods.
INTRODUmION
We have recently reported the isolation and structures of glycosides from A. tataricus roots [I]. In the course of further studies on the constituents of the above plant, three new oligopeptides, asterinin A, B and C have been obtained. This paper refers to their structures.
RESULTS AND DISCUSSION
Repeated chromatography of the ethyl acetate fraction of 70% ethanol extract of A. taturicus roots on silica gel and Sephadex LH-20 afforded asterinin A (1). B (2) and C (3). Asterinin A (1) gave a positive Ehrlich test and a negative ninhydrin reaction. The FAB-mass spectrum ([M + 1] + ion peaks at m/z 532) and elemental analysis (found: C, 56.45; H, 6.23; N, 13.20%) suggested the molecular formular to be C2SH33Ns0R. It was assumed to be a peptide from the IR absorption at 3296 (NH, OH), 1728 (CO,H), 1651 (amide GO) and UV at 212 nm (1.36 x 104), 267 nm (1.85 x 104). The ‘H and 13CNMR spectra showed signals of five NH protons at 6 12.89, 9.67, 9.34,9.01,8.75 and five carbonyl carbons at 6 162.8,172.8, 170.9, 170.8, 175.5 (Table l), indicating that 1 was a pentapeptide. Amino acid analysis of 1 after acid hydrolysis revealed the presence of L-Ser (1 eq), t_-allo-Thr (1 eq), /I-Phe (/?-amino, /&phenylpropanoic acid) (1 eq), t.-Abu (a-aminobutyric acid) (I eq) and another non-standard amino acid. On enzymatic hydrolysis with z-chymotryp sin in pH 9 buffer solution at 37”, the amino acid compositions were in agreement with those of acid hydrolysis except for the presence of Azs4 Pro (pyrrole-2carboxylic acid) (1 eq). The presence of these amino acid residues was also proved by the ‘H and ’ 3C NMR spectra whose ambiguous assignments were made by the combination of lH-‘HCOSY, 13C-lH COSY and COLOC spectra. The sequence of amino acid residues, A2.4 Pro-Lallo-Thr-t-Ser+Phe-L-Abu was deduced by the correla-
tion between the signals of NH protons and those of carbonyl carbons, and corroborated by a series of fragment peaks at m/z: 438,94,337,195,250,282,103, and429 in the FAB-mass spectrum (Fig. 1). Consequently, the structure of asterinin A was elucidated as A2*4Pro-L-alloThr-L-Ser-/IPhe-L-Abu. Asterinin B (2) had the molecular formula C26H,,N,0, by the FAB-mass spectrum ([M + 11’ at m/z 546) and elemental analysis (found: C, 57.21; H, 6.44, N, 12.87%). The composition of amino acids of 2 was identical with that of 1 on acid and enzymatic hydrolysis. The IR, UV and NMR spectral data of 2 were close to those of 1. However, a three-protons singlet at 63.61 due to a methyl group indicated 2 to be a methyl ester of 1. This proposal was further confirmed by the M, of 2 (14 mass units higher than that of 1). Furthermore, methylation of 1 with diazomethane afforded 2. Therefore, asterinin B was determined to be A2~4Pro-t_-allo-Thr-LSer-/?Phe-L-Abu. Asterinin C (3), C,,H,,N,O,, gave the same molecular formula and was composed of the same amino acids as 2. A detailed analysis of the COSY and COLOC spectra indicated that the positions of Abu and Thr were reversed, compared from those of 2, i.e. the only difference between 3 and 2 was in the amino acid sequence. This difference was corroborated by the fragmentation pattern of the FAB-mass spectrum of 3 (Fig. 1). To further support the sequence of amino acids in 3, ZD-NOESY measurement was performed. Strong NOE correlations were observed between the signals of H-7 and H-8, H-12 and H-13, C-16 and H-17, H-26 and H-27, respectively. The structure of asterinin C was thus assigned as A2.4Prot_-Abu-L-Ser-BPhe-L-allo-ThrOMe.
945
EXPERIMENTAL
Mps: uncorr. NMR: ‘H 500 MHz and “C 125 MHz for asterinin A and B, iH 400 MHz and “C 100 MHz for asreinin C.
946
DONGLIANG CHENG er al.
Table
I. ‘H and 13C NMR spectra
Asterinin
A
Asterinin
H I 2 3 4 5 6 7 8 9 10 II I2 13 14
12.89 7.22 6.33 7.27
I5 16 I7 I8 I9 20 21 22 23 24 25 26 27 28
s s .5 s
d (8.8)
9.67 5.23 4. I6 4.49
d (8.4)
dd (8.8, 8.4)
d (6)
ddd (8.4.4.4.4.0) dd (4.4, 10.8) dd (4.0. 10.8)
C
12.89 7.23 6.34 7.28
59.8 69.6 21.4 172.8
dq (8.4, 6)
57.2 62.5
9.34 d (8.4) 6.14 ddd (8.4, 7.6.4.8) 3.16 ddd (7.6,4.8, 12.4)
7.14-7.40
9.01 5.01 I .88 2.09 0.96
H
IO 11 I2 I3 I4 I5
8.76 5.29 2.16 1.92 0.99
m
constants
br s
d (8.8) dd (8.8, 8.4) dq (8.4. 6) d (6)
9.62 5.23 4.20 4.49
d (8.4) ddd (8.4.4.4.4.0) dd (4.4. 10.8)
59.8 69.6 21.4 172.8 57.2 62.5
dd (4.0, 10.8)
170.9
7.14-7.55
51.7 43.8 143.2 127.3 128.8 127.3 128.8 127.3 170.8
m
9.16 d (7.6) 4.76ddd (7.6,6.8,4.8) 54.68 1.72 ddd (7.6, 6.8, 13.6) 25.72 I .85 ddd (4.8, 13.6. 7.2) 0.85 dd (7.6,7.2)
54.7 25.8 10.5 175.5
br s
br s br s br s
d (10.0) m m m I (7.3)
constants
111.1 109.7 122.3 127.3 162.4
data of astertnin
55.4 26.3 10.6 171.7 56.6 62.9 171.1
H I6 I7 I8 I9 20 21 22 23 24 25 26 27 28 29 30 31
54.4 25.4 10.5 173.5 52.1
(J in Hz) in parentheses.
spectral
C
9.39 d (7.6) 5.29 m 4.31 m
Coupling
br s
9.31 d (8.4) 6. I3 ddd (8.4, 7.6.4.8) 3.15 ddd (7.6.4.8, 12.4)
51.7 43.8 143.2 127.3 128.8 127.3 128.8 127.3 170.8
d (7.6) ddd (7.6,4.8, 6.8) ddd (7.6.6.8. 13.6) ddd (4.8, 13.6, 7.2) dd (7.6. 7.2)
2. ‘H and “CNMR
12.80 7.23 6.33 7.23
III.4 109.9 122.6 127.4 162.8
br s
8.84 5.49 4.51 1.53
C
Isolation
C
9.63 d (7.9) 6.10 m 3.17 m
7.14-7.60
m
9.30 d (8.3) 5.12 m 4.44 m 1.44m 3.63 s
(J in Hz) in parentheses.
51.3 42.7 142.8 127.1 128.7 127.1 128.7 127.1 170.8 59.7 68.3 20.5 173.2 51.7
ojasterinin
A (l),
B (2) and
C (3). Extraction described in ref. [l]. The fraction eluted with CHCl,-MeOH (5: 1) was further purified by CC with C,H,-MeCOC,H,-MeOH (7:2: 1) and prep. TLC to afford 250 mg 2 and 320 mg 3. The fraction eluted with CHCI,-MeOH (2: 1) was further purified by CC with CHCl,-MeOH-H,O (18:6: 1) and Sephadex LH-20 to give 500 mg 1. Asterinin A (1). Amorphous powder (from 70% MeOH), mp 272-274”, [z]n + 38.5” (pyridine; c 0.33). IR rKBr cm ‘: 3296,1728,165 1 and 1546. UV j.“‘&” nm: 2 12 (1.38 x 104) and 267 (1.85 x 104). FAB-MS m/z: 546 [M + l]‘, 438, 429, 337, 282, 250, 195, 103, 94. Analyt. talc. for C,,H,,N,OB (%): C, 56.49; H, 6.21; N, 13.14. Found: C, 56.45; H, 6.23; N, 13.20. ‘H and 13CNMR (Table 1). Asterinin B (2). Amorphous powder (from 70% MeOH), mp 235-237”, [aIn+ 3.1” (pyridine; c 0.57). IR vKBrcm _ ‘: 3304,1749,1651 and 1539. UV i.McOHnm: 209 (1.38 x 104) and 267 (1.81 x 104). FAB-MS m/z: 546 [M + 11’. Analyt. Calc. for C2,HJ5N,0, (%): C, 57.25; H, and
1
br s
3.61 s Coupling
2 3 4 5 6 7 8 9
B
H
111.4 109.9 122.6 127.4 162.8
8.75 5.52 4.51 1.53
A and B (in pyridine-d,)
170.9
29 30 ?I
Table
C br br br br
data of asterinin
fractionation
procedures
were
Oligopeptides from Aster tataricus
947
OH
I
asterinin A R=H asterinin B R=Me OH
I
Me
94
Me
asterinin A
Fig. I. 13C-‘H long range correlations in the COLOC spectra and fragment peaks in FAB-mass spectra of asterinin A and C. 6.42; N, 12.84. Found: C, 57.21; H, 6.44; N, 12.87. ‘H and
13C NMR (Table 1). Asterinin C (3). Amorphous powder, mp 250-252”. [a&,-4.9” (pyridine; c 0.33). IR vmr,cm-‘: 3304, 1749, 1651 and 1539. UV A,,, nm: 209 (1.38 x 104)and 267 (1.82 x 104). FAB-mass m/z: 546 [M + l]‘, 453,413,367,280,
266,179, 133,94. C,,H,,N,O, requires: C, 57.25; H, 6.44; N; 11.00. Found: C, 57.21; H, 6.43; N, 11.03. ‘H and ’ SC NMR (Table 2). Acid hydrolysis o/compounds l-3. Solns of 1-3 (each containing 5 mg peptide) in 6 M HCI were heated at 110 for 2 hr. After cooling, each soln was coned to dryness.
948
DONGLIANGCHENG et al.
Each sample was analysed for amino acids using the amino acid analyser. Enzymatic hydrolysis of compounds 1-3. Compounds 1-3 (each 3.5 mg) were incubated in 0.05 M tris-hydroxymethyl aminomethane (pH 9,5 ml) with a-chymotrypsin at 37” for 7 days. The hydrolysate was analysed for amino acid using the amino acid analyser. Methylation ofcompound 1.A soln of l(5 mg) in MeOH was treated with CH,N2 and 4.5 mg 2 was obtained after recrystallization. Acknowledgements-We
are greatly indebted
to Dr Frau
Peter-Katalinic of Institute of Physiological Chemistry, Bonn University (F. R. G.) for FAB-MS and useful discussions about the elucidation of the two structures. This work was supported by the National Natural Science Foundation of China. REFERENCE
I. Cheng. D. L. and Shao, Y. (1994) Phytochemistry 35, 173.
0031~!M22@4)EOO3S-Q
Phyrdumisrry.
Vol. 36, No. 4. pp. 945 948, 1594 Ekvin %mc+ Ltd Pnntcd in Great Britain. 003 -9422l94 f7.00 + 0.00
I
OLIGOPEPTIDES DON~LIANG
FROM
CHENG, Yu SHAO,
ASTER
TATARICUS
R. HARTMAN,* E. RODER* and K. ZHAO~
Institute of Organic Chemistry, State Key Laboratory of Applied Organic Chemistry, Lanzhou 730000, P. R. China; * Pharmazeutisches and Physiologishe Chemie Institut, Universit Bonn, D-5300 Bonn, F.R.G.; tI%partment of Chemistry, New York University, New York, NY 10003, U.S.A. (Received 19 October 1993)
Key Word Index--Aster
tataricus;
Compositae; roots; oligopeptides; asterinin A, B and C.
Abstract-Three
novel oligopeptides, asterinin A-C have been isolated from the roots of A. tataricus and their structures elucidated by chemical, enzymatic and spectral methods.
INTRODUmION
We have recently reported the isolation and structures of glycosides from A. tataricus roots [I]. In the course of further studies on the constituents of the above plant, three new oligopeptides, asterinin A, B and C have been obtained. This paper refers to their structures.
RESULTS AND DISCUSSION
Repeated chromatography of the ethyl acetate fraction of 70% ethanol extract of A. taturicus roots on silica gel and Sephadex LH-20 afforded asterinin A (1). B (2) and C (3). Asterinin A (1) gave a positive Ehrlich test and a negative ninhydrin reaction. The FAB-mass spectrum ([M + 1] + ion peaks at m/z 532) and elemental analysis (found: C, 56.45; H, 6.23; N, 13.20%) suggested the molecular formular to be C2SH33Ns0R. It was assumed to be a peptide from the IR absorption at 3296 (NH, OH), 1728 (CO,H), 1651 (amide GO) and UV at 212 nm (1.36 x 104), 267 nm (1.85 x 104). The ‘H and 13CNMR spectra showed signals of five NH protons at 6 12.89, 9.67, 9.34,9.01,8.75 and five carbonyl carbons at 6 162.8,172.8, 170.9, 170.8, 175.5 (Table l), indicating that 1 was a pentapeptide. Amino acid analysis of 1 after acid hydrolysis revealed the presence of L-Ser (1 eq), t_-allo-Thr (1 eq), /I-Phe (/?-amino, /&phenylpropanoic acid) (1 eq), t.-Abu (a-aminobutyric acid) (I eq) and another non-standard amino acid. On enzymatic hydrolysis with z-chymotryp sin in pH 9 buffer solution at 37”, the amino acid compositions were in agreement with those of acid hydrolysis except for the presence of Azs4 Pro (pyrrole-2carboxylic acid) (1 eq). The presence of these amino acid residues was also proved by the ‘H and ’ 3C NMR spectra whose ambiguous assignments were made by the combination of lH-‘HCOSY, 13C-lH COSY and COLOC spectra. The sequence of amino acid residues, A2.4 Pro-Lallo-Thr-t-Ser+Phe-L-Abu was deduced by the correla-
tion between the signals of NH protons and those of carbonyl carbons, and corroborated by a series of fragment peaks at m/z: 438,94,337,195,250,282,103, and429 in the FAB-mass spectrum (Fig. 1). Consequently, the structure of asterinin A was elucidated as A2*4Pro-L-alloThr-L-Ser-/IPhe-L-Abu. Asterinin B (2) had the molecular formula C26H,,N,0, by the FAB-mass spectrum ([M + 11’ at m/z 546) and elemental analysis (found: C, 57.21; H, 6.44, N, 12.87%). The composition of amino acids of 2 was identical with that of 1 on acid and enzymatic hydrolysis. The IR, UV and NMR spectral data of 2 were close to those of 1. However, a three-protons singlet at 63.61 due to a methyl group indicated 2 to be a methyl ester of 1. This proposal was further confirmed by the M, of 2 (14 mass units higher than that of 1). Furthermore, methylation of 1 with diazomethane afforded 2. Therefore, asterinin B was determined to be A2~4Pro-t_-allo-Thr-LSer-/?Phe-L-Abu. Asterinin C (3), C,,H,,N,O,, gave the same molecular formula and was composed of the same amino acids as 2. A detailed analysis of the COSY and COLOC spectra indicated that the positions of Abu and Thr were reversed, compared from those of 2, i.e. the only difference between 3 and 2 was in the amino acid sequence. This difference was corroborated by the fragmentation pattern of the FAB-mass spectrum of 3 (Fig. 1). To further support the sequence of amino acids in 3, ZD-NOESY measurement was performed. Strong NOE correlations were observed between the signals of H-7 and H-8, H-12 and H-13, C-16 and H-17, H-26 and H-27, respectively. The structure of asterinin C was thus assigned as A2.4Prot_-Abu-L-Ser-BPhe-L-allo-ThrOMe.
945
EXPERIMENTAL
Mps: uncorr. NMR: ‘H 500 MHz and “C 125 MHz for asterinin A and B, iH 400 MHz and “C 100 MHz for asreinin C.
946
DONGLIANG CHENG er al.
Table
I. ‘H and 13C NMR spectra
Asterinin
A
Asterinin
H I 2 3 4 5 6 7 8 9 10 II I2 13 14
12.89 7.22 6.33 7.27
I5 16 I7 I8 I9 20 21 22 23 24 25 26 27 28
s s .5 s
d (8.8)
9.67 5.23 4. I6 4.49
d (8.4)
dd (8.8, 8.4)
d (6)
ddd (8.4.4.4.4.0) dd (4.4, 10.8) dd (4.0. 10.8)
C
12.89 7.23 6.34 7.28
59.8 69.6 21.4 172.8
dq (8.4, 6)
57.2 62.5
9.34 d (8.4) 6.14 ddd (8.4, 7.6.4.8) 3.16 ddd (7.6,4.8, 12.4)
7.14-7.40
9.01 5.01 I .88 2.09 0.96
H
IO 11 I2 I3 I4 I5
8.76 5.29 2.16 1.92 0.99
m
constants
br s
d (8.8) dd (8.8, 8.4) dq (8.4. 6) d (6)
9.62 5.23 4.20 4.49
d (8.4) ddd (8.4.4.4.4.0) dd (4.4. 10.8)
59.8 69.6 21.4 172.8 57.2 62.5
dd (4.0, 10.8)
170.9
7.14-7.55
51.7 43.8 143.2 127.3 128.8 127.3 128.8 127.3 170.8
m
9.16 d (7.6) 4.76ddd (7.6,6.8,4.8) 54.68 1.72 ddd (7.6, 6.8, 13.6) 25.72 I .85 ddd (4.8, 13.6. 7.2) 0.85 dd (7.6,7.2)
54.7 25.8 10.5 175.5
br s
br s br s br s
d (10.0) m m m I (7.3)
constants
111.1 109.7 122.3 127.3 162.4
data of astertnin
55.4 26.3 10.6 171.7 56.6 62.9 171.1
H I6 I7 I8 I9 20 21 22 23 24 25 26 27 28 29 30 31
54.4 25.4 10.5 173.5 52.1
(J in Hz) in parentheses.
spectral
C
9.39 d (7.6) 5.29 m 4.31 m
Coupling
br s
9.31 d (8.4) 6. I3 ddd (8.4, 7.6.4.8) 3.15 ddd (7.6.4.8, 12.4)
51.7 43.8 143.2 127.3 128.8 127.3 128.8 127.3 170.8
d (7.6) ddd (7.6,4.8, 6.8) ddd (7.6.6.8. 13.6) ddd (4.8, 13.6, 7.2) dd (7.6. 7.2)
2. ‘H and “CNMR
12.80 7.23 6.33 7.23
III.4 109.9 122.6 127.4 162.8
br s
8.84 5.49 4.51 1.53
C
Isolation
C
9.63 d (7.9) 6.10 m 3.17 m
7.14-7.60
m
9.30 d (8.3) 5.12 m 4.44 m 1.44m 3.63 s
(J in Hz) in parentheses.
51.3 42.7 142.8 127.1 128.7 127.1 128.7 127.1 170.8 59.7 68.3 20.5 173.2 51.7
ojasterinin
A (l),
B (2) and
C (3). Extraction described in ref. [l]. The fraction eluted with CHCl,-MeOH (5: 1) was further purified by CC with C,H,-MeCOC,H,-MeOH (7:2: 1) and prep. TLC to afford 250 mg 2 and 320 mg 3. The fraction eluted with CHCI,-MeOH (2: 1) was further purified by CC with CHCl,-MeOH-H,O (18:6: 1) and Sephadex LH-20 to give 500 mg 1. Asterinin A (1). Amorphous powder (from 70% MeOH), mp 272-274”, [z]n + 38.5” (pyridine; c 0.33). IR rKBr cm ‘: 3296,1728,165 1 and 1546. UV j.“‘&” nm: 2 12 (1.38 x 104) and 267 (1.85 x 104). FAB-MS m/z: 546 [M + l]‘, 438, 429, 337, 282, 250, 195, 103, 94. Analyt. talc. for C,,H,,N,OB (%): C, 56.49; H, 6.21; N, 13.14. Found: C, 56.45; H, 6.23; N, 13.20. ‘H and 13CNMR (Table 1). Asterinin B (2). Amorphous powder (from 70% MeOH), mp 235-237”, [aIn+ 3.1” (pyridine; c 0.57). IR vKBrcm _ ‘: 3304,1749,1651 and 1539. UV i.McOHnm: 209 (1.38 x 104) and 267 (1.81 x 104). FAB-MS m/z: 546 [M + 11’. Analyt. Calc. for C2,HJ5N,0, (%): C, 57.25; H, and
1
br s
3.61 s Coupling
2 3 4 5 6 7 8 9
B
H
111.4 109.9 122.6 127.4 162.8
8.75 5.52 4.51 1.53
A and B (in pyridine-d,)
170.9
29 30 ?I
Table
C br br br br
data of asterinin
fractionation
procedures
were
Oligopeptides from Aster tataricus
947
OH
I
asterinin A R=H asterinin B R=Me OH
I
Me
94
Me
asterinin A
Fig. I. 13C-‘H long range correlations in the COLOC spectra and fragment peaks in FAB-mass spectra of asterinin A and C. 6.42; N, 12.84. Found: C, 57.21; H, 6.44; N, 12.87. ‘H and
13C NMR (Table 1). Asterinin C (3). Amorphous powder, mp 250-252”. [a&,-4.9” (pyridine; c 0.33). IR vmr,cm-‘: 3304, 1749, 1651 and 1539. UV A,,, nm: 209 (1.38 x 104)and 267 (1.82 x 104). FAB-mass m/z: 546 [M + l]‘, 453,413,367,280,
266,179, 133,94. C,,H,,N,O, requires: C, 57.25; H, 6.44; N; 11.00. Found: C, 57.21; H, 6.43; N, 11.03. ‘H and ’ SC NMR (Table 2). Acid hydrolysis o/compounds l-3. Solns of 1-3 (each containing 5 mg peptide) in 6 M HCI were heated at 110 for 2 hr. After cooling, each soln was coned to dryness.
948
DONGLIANGCHENG et al.
Each sample was analysed for amino acids using the amino acid analyser. Enzymatic hydrolysis of compounds 1-3. Compounds 1-3 (each 3.5 mg) were incubated in 0.05 M tris-hydroxymethyl aminomethane (pH 9,5 ml) with a-chymotrypsin at 37” for 7 days. The hydrolysate was analysed for amino acid using the amino acid analyser. Methylation ofcompound 1.A soln of l(5 mg) in MeOH was treated with CH,N2 and 4.5 mg 2 was obtained after recrystallization. Acknowledgements-We
are greatly indebted
to Dr Frau
Peter-Katalinic of Institute of Physiological Chemistry, Bonn University (F. R. G.) for FAB-MS and useful discussions about the elucidation of the two structures. This work was supported by the National Natural Science Foundation of China. REFERENCE
I. Cheng. D. L. and Shao, Y. (1994) Phytochemistry 35, 173.