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Cyclolinopeptides F–I, cyclic peptides from linseed
Phytochemistry 57 (2001) 251±260
www.elsevier.com/locate/phytochem
Cyclolinopeptides F±I, cyclic peptides from linseed Teruki Matsumoto, Akira Shishido, Hiroshi Morita, Hideji Itokawa, Koichi Takeya * School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Received 5 July 2000; received in revised form 23 October 2000
Abstract Four cyclic peptides, cyclolinopeptides F±I, were isolated from seeds of Linum usitatissimum. Their structures were elucidated by extensive 2D NMR spectroscopic methods and by chemical degradation. Further, their immunosuppressive activity is examined. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Linum usitatissimum; Linaceae; Cyclolinopeptide; Cyclic peptide; Immunosuppresive activity
1. Introduction Cyclolinopeptide A (CLA), a cyclic nonapeptide, which exhibits various biological activities (Wieczorek et al., 1991; Naider et al., 1971; Brewster and Bovey, 1971; Tonelli, 1971; Blasio et al., 1989) has been isolated from linseed oil as one of the ®rst isolated natural cyclic peptides (Kaufmann and Tobschirbel, 1959). In a previous paper, we reported on the separation, puri®cation and structure elucidation of cyclolinopeptides B±E having cyclic nona- or octapetides, from the seeds of Linum usitatissimum (Morita et al., 1997, 1999). As part of our investigation on new biologically active cyclic peptides from higher plants (Itokawa et al., 1997), we focused our attention on the isolation of additional cyclic peptides from linseed. In this report, we describe both the isolation and structural elucidation of four new cyclic peptides, cyclolinopeptides F (1)±I (4) by extensive 2D NMR spectroscopic methods and their immunosuppressive activities against mouse splenocytes. 2. Results and discussion Puri®cation of the methanolic extract prepared from the linseed involving Diaion HP-20 column chromatography, using a H2O±MeOH gradient system. The MeOH eluate was next applied to a silica gel column, * Corresponding author. Tel.: +81-426-763007; fax: +81-426771436. E-mail address: [email protected] (K. Takeya).
this being followed by HPLC [ODS] to yield the four new cyclic peptides, cyclolinopetides F±I (CLF±CLI) in yields of 0.0008%, 0.0024%, 0.0002% and 0.00007%, respectively. Cyclolinopeptides F (CLF) and G (CLG) were obtained as white powders. Their molecular formulae were shown to be C55H74N9O10S2 and C56H76N9O10S2, respectively by the quasi-molecular ion peak at m/z 1084.5018 [M+H]+ and at m/z 1098.5138 [M+H]+ in the HR-FABMS. Both IR spectra had absorption peaks which were attributed to amine, amide carbonyl and sulfoxide groups. Since the UV absorption maximum in the neighborhood of 280 nm suggested the presence of a tryptophan residue, the compounds were hydrolyzed with 6 N HCl containing 4% thioglycolic acid. HPLC analysis of each hydrolysate showed that CLF was a cyclic octapeptide consisting of two phenylalanines (Phe), two methionines (Met), proline (Pro), leucine (Leu), valine (Val) and tryptophan (Trp) per mol and CLG was a cyclic octapeptide consisting of two phenylalanines (Phe), two methionines (Met), proline (Pro), leucine (Leu), isoleucine (Ile) and tryptophan (Trp) per mol. However, both methionines in CLF and CLG were present as methionine sulfoxides, because each molecular formula had an additional two oxygen atoms, compared with the cyclic octapeptide presumed from above amino acid degradation and both had IR absorption bands (CLF: 1016 and CLG: 1015 cm 1) indicating sulfoxide groups. In the 1H and 13C NMR spectra (Tables 1 and 2), CLF and CLG had seven amide protons and eight amide carbonyl carbons, respectively, and thus were
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00442-8
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Table 1 1 H and 13C NMR signal assignments for cyclolinopeptide F (1) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO Phe2
" NH CO
4.21 (1H, 2.11 (1H, 1.59 (1H, 1.85 (2H, 3.58 (1H, 3.48 (1H,
t, 7.8) m) m) m) m) m)
4.10 (1H, dt, 6.4, 7.6) 2.80 (2H, m) 7.21 (2H, 7.01 (2H, 7.19 (1H, 8.39 (1H,
m) d, 6.6) m) d, 6.4)
62.04 29.04 24.17 46.89 173.55 56.60 35.10 137.68 127.87 128.39 125.88 170.35
3
Phe
" NH CO Trp4 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.67 (1H, m) 3.44 (1H, m) 2.82 (1H, m) 7.21 (2H, 7.20 (2H, 7.19 (1H, 7.92 (1H,
m) m) m) d, 8.1)
3.99 (1H, m) 3.38 (2H, m) 10.62 (1H, s) 7.15 (1H, d, 7.5) 7.45 (1H, 7.04 (1H, 6.95 (1H, 7.31 (1H,
d, 7.8) t, 7.8) t, 7.8) d, 7.8)
8.06 (1H, d, 6.3)
52.73 36.22 138.09 127.95 128.39 126.06 171.16 55.61 23.42 122.55 110.71 117.63 120.58 117.95 111.04 135.82 127.21
Val
CH3 NH CO Mso6
"CH3 NH CO Leu7
CH3 NH CO Mso8
"CH3 NH CO
4.50 (1H, m) 2.20 (1H, m) 0.94 (3H, d, 6.7) 0.85 (3H, d, 6.7) 7.27 (1H, d, 6.3)
3.98 (1H, m) 2.08 (1H, m) 1.97 (1H, m) 2.74 (2H, m) 2.49 (3H, s) 8.31 (1H, br.s)
56.88 30.92 19.20 17.39 172.52a 54.57 23.23 49.48 38.00 170.74a
3.88 (1H, m)
52.41
1.84 (1H, m) 1.61 (1H, m) 1.62 (1H, m) 0.91 (3H, d, 8.4) 0.85 (3H, d, 8.4) 8.05 (1H, d, 6.5)
37.85 24.30 22.78 20.77 171.07a
4.70 (1H, dt, 6.9, 14.0) 2.39 (1H, m)
50.35 25.60
1.93 (1H, m) 2.78 (2H, m) 2.44 (3H, s) 7.36 (1H, br.s)
50.35 37.85 170.35a
170.74a
Assignments may be interchanged.
obviously octapeptides. However, analysis of the NOE data and 2JH C and 3JH C correlations by HMBC did not permit elucidation of their amino acid sequences as shown in Figs. 1 and 3. Hence, CLF and CLG were chemically transformed to their corresponding linear derivatives (CLF0 and CLG0 ), i.e. by reduction of their methionine sulfoxide using thioglycolic acid and subsequent decomposition with cyanogen bromide. This gave the linear peptides, which displayed an absorption IR bands at 1775 cm 1 attributed to an homoserine lactone. Sequential analyses due to the HMBC correlations indicated by arrows in Figs. 2 and 4 showed that CLF0 and CLG0 were, respectively, the linear peptides
Pro±Phe±Phe±Trp±Val±HomoSer Lactone and as ProPhe±Phe±Trp±Ile±HomoSer Lactone each additional dipeptide was assumed to be Leu7 HomoSer lactone. ROE correlations of CLF were observed between the Mso6 -proton and the Leu7 amide proton, and between the Mso8 -proton and the Pro1 -proton, respectively. Also, ROE correlations of CLG were noted between the Mso8 -proton and Pro1 -proton, and between the Ile5 -proton and the Mso6 amide proton. Consequently, the structure of CLF was identi®ed as cyclo (-Pro±Phe± Phe±Trp±Val±Mso±Leu±Mso-) and CLG as cyclo (Pro±Phe±Phe±Trp±Ile±Mso±Leu±Mso-). The absolute stereochemistries of the component amino acids was
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Table 2 1 H and 13C NMR signal assignments for cyclolinopeptide G (2) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO
4.21 (1H, t, 7.8) 2.09 (1H, m) 1.57 (1H, m) 1.85 (2H, t, 6.6) 3.58 (1H, m) 3.51 (1H, m)
62.03 29.01 24.17 46.90 173.42
2
Phe
" NH CO Phe3
" NH CO Trp4 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.11 (1H, dt, 6.4, 7.5) 2.84 (2H, m) 7.22 (2H, m) 7.02 (2H, m) 7.27 (1H, m) 8.40 (1H, d, 6.4)
4.69 (1H, m) 3.44 (1H, m) 2.79 (1H, m) 7.22 (2H, m) 7.02 (2H, m) 7.06 (1H, m) 7.93 (1H, d, 6.3)
6.95 (1H, m) 3.36 (2H, m) 10.61 (1H, s) 7.18 (1H, d, 6.8) 7.45 (1H, d, 7.8) 7.05 (1H, t, 7.8) 6.95 (1H, t, 7.8) 7.31 (1H, d, 7.8) 8.11 (1H, br.s)
56.59 35.07 137.76 127.84 128.35 125.85 170.36 52.66 36.19 138.09 127.95 128.42 126.01 170.85a 55.65 23.38 122.58 110.65 117.62 120.57 117.94 111.03 135.82 127.20
Ile
CH3 CH3 NH CO Mso6
"CH3 NH CO Leu7
CH3 NH CO Mso8
"CH3 NH CO
4.49 (1H, br.s) 1.91 (1H, m) 1.39 (1H, m) 1.12 (1H, m) 0.92 (3H, d, 6.6) 0.88 (3H, t, 9.2) 7.32 (1H, m)
56.81 37.14 23.84 15.30 11.06 172.30a
4.01 (1H, m) 2.16 (1H, m) 1.97 (1H, m) 2.73 (1H, m) 2.65 (1H, m) 2.49 (3H, s) 8.27 (1H, br.s)
54.36 23.13 49.54 37.83 170.29
3.84 (1H, m) 1.79 (1H, m) 1.62 (1H, m) 1.61 (1H, m) 0.89 (3H, d, 6.1) 0.84 (3H, d, 6.1) 8.01 (1H, m)
4.70 (1H, m) 2.39 (1H, m) 1.95 (1H, m) 2.75 (2H, m) 2.45 (3H, s) 7.36 (1H, m)
52.49 37.83 24.27 22.76 20.83 170.92a 50.35 25.63 50.35 38.05 171.03a
173.17a
Assignments may be interchanged.
determined to be entirely in the l-con®gurations as evidenced by derivatization of the acid hydrolysate with Marfey's reagent (Marfey, 1984), followed by HPLC analyses. Cyclolinopeptides H (CLH) and I (CLI) were isolated as white powders, and their molecular formulae were determined to be C56H76N9O9S2 for CLH by HRFABMS m/z 1082.5201 [M+H]+ and C55H74N9O9S2 for CLI by HR-FABMS m/z 1068.5110 [M+H]+. HPLC of their acid hydrolysates showed that CLH was an octapeptide consisting of two Phe, two Met, Pro, Leu, Ile, Trp per mole and CLI was an octapeptide consisting of two Phe, two Met, Pro, Leu, Val, Trp per
mole. Each amino acid was con®rmed to be in the lcon®guration using Marfey's method. The amino acid components of CLH and CLI were similar to those of CLG and CLF. However, the FAB mass spectra are 16 amu less than those of CLG and CLF. The facts suggested the presence of one methionine and one methionine sulfoxide in CLH and CLI instead of two methionine sulfoxides in CLG and CLF, respectively. According to the above speculation, reductive transformations of methionine sulfoxide in CLG and CLF by thioglycolic acid are demonstrated to give three compounds, respectively. One of them in CLG (or CLF) agreed completely with CLH (or CLI) in comparison of
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T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
Fig. 1. Cyclolinopeptide F (1): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
Fig. 2. Cyclolinopeptide F0 : dashed arrows show HMBC correlations and arrows show some selected NOE relationship.
various physical and spectral data. Consequently, the sequence of amino acids in CLH (or CLI) is basically same to that in CLG (or CLF). The amino acid sequences of CLH and CLI were determined from ROE correlations as shown in Figs. 5 and 6. Consequently, the structures of CLH were identi®ed as cyclo (-Pro± Phe±Phe±Trp±Ile-Mso±Leu±Met-) and CLI as cyclo (-Pro±Phe±Phe±Trp-Val±Met±Leu±Mso-). CLB (cyclo (-Pro±Pro±Phe±Phe±Val±Ile±Met±LeuIle-)) showed an inhibitory eect on mitogen (concanavalin A) induced response of human peripheralblood lymphocytes (IC50: 44 ng/ml), which is comparable to that of cyclosporin A (Morita et al., 1997). Furthermore, CLA (cyclo (-Pro±Pro±Phe±Phe±Leu±Ile±Ile± Leu±Val-)), CLB and CLE (cyclo (-Pro±Leu±Phe±Ile±
Mso±Leu±Val-Phe-)) possessing dierent amino acid sequences showed a moderate inhibitory eect of about the same potency on the mouse lymphocyte proliferation induced by Con. A (IC50: CLA 2.5 mg/ml; CLB 39 mg/ml; CLE 43 mg/ml) (Morita et al., 1999). However, CLC (cyclo (-Pro±Pro±Phe±Phe-Val±Ile±Mso± Leu±Ile-)), CLD (cyclo (-Pro±Phe-Phe±Trp±Ile±Mso± Leu±Leu-)) and CLF±CLI (IC50: >100 mg/ml, respectively) did not show any of the above activities. It is known that the biological activity of CLA is critically dependent on the sequence and conformation (Kessler et al., 1988). The conformational analyses and further conformation±biological activity relationship of CLA± CLI, are currently being investigated in our laboratories.
T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
255
Fig. 3. Cyclolinopeptide G (2): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
Fig. 4. Cyclolinopeptide G0 : dashed arrows show HMBC correlations and arrows show some selected NOE relationship.
3. Experimental 3.1. General Optical rotations were measured on a JASCO DIP-4 spectrometer and [ ]D values are given in 10 1 deg cm2 g 1. The IR and UV spectra were taken on JASCO FT/ IR 620 and Hitachi 557 spectrometers, respectively, and the 1H and 13C NMR spectra were recorded on BruÈker AM-500 spectrometers, with chemical shifts () reported in ppm, the spectra were recorded at 320 . FABMS were taken with a VG Autospec spectrometer. HPLC was carried out using an Inertsil PREP-ODS column
(20 mm i.d.250 mm and 30 mm i.d.250 mm, GL Science Inc.) packed with 10 mm ODS. TLC was conducted on precoated Kieselgel 60 F254 (Art. 5715; Merck) with spots detected by use of the Dragendorff reagent. 3.2. Plant material Seeds (Linum usitatissium, Linaceae) were purchased from Nikka Oil Co., Japan and the oil was removed by manual pressing. The plant was identi®ed by Professor Yutaka Sashida, director of the medicinal garden in Tokyo University of Pharmacy and Life Science. A voucher specimen is deposited in our Laboratory.
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T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
Fig. 5. Cyclolinopeptide H (3): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
Fig. 6. Cyclolinopeptide I (4): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
3.3. Extraction and separation Seeds (L. usitatissium, 30 kg) were manually pressed to remove oil by squeezing, these being extracted with hot MeOH three times to give a MeOH extract (4 kg). The latter was subjected to Diaion HP-20 column chromatography using a H2O±MeOH gradient system (0:1±
1:0), and the fraction eluted with 100% MeOH was further subjected to silica gel column chromatography using a CHCl3±MeOH gradient system (1:0±0:1). A 15% MeOH fraction was subjected to ODS HPLC with 40±60% CH3CN solvent system to give CLF (0.0008%), CLG (0.0024%), CLH (0.0002%) and CLI (0.00007%).
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Table 3 1 H and 13C NMR signal assignments for cyclolinopeptide H (3) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO Phe2
" NH CO Phe3
" NH CO
4.16 2.09 1.66 1.91 3.61 3.56
(1H, (1H, (1H, (2H, (1H, (1H,
t, 7.8) m) m) m) m) m)
4.05 (1H, m) 2.96 (1H, t, 10.3) 2.83 (1H, m) 7.18 6.98 7.22 8.20
(2H, (2H, (1H, (1H,
m) m) m) d, 6.2)
4.58 (1H, m) 3.33 (1H, m) 2.77 (1H, m) 7.18 7.28 7.22 7.88
(2H, (2H, (1H, (1H,
m) m) m) d, 8.8)
61.89 28.75 24.39 46.94 173.17 56.33 35.14 137.78 127.86 128.42 126.00 170.06 53.39 36.47 138.13 127.86 128.60 126.00 170.76a
4
Trp 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.08 (1H, m) 3.33 (2H, m) 10.59 (1H, s) 7.18 (1H, d, 7.6) 7.45 7.04 6.97 7.31
(1H, (1H, (1H, (1H,
d, 7.7) t, 7.7) t, 7.7) d, 7.7)
8.18 (1H, d, 7.5)
55.63 24.31 122.76 110.37 117.65 120.59 117.97 111.04 135.81 127.19
Ile
CH3 CH3 NH CO Mso6
"CH3 NH CO Leu7
CH3 NH CO Met8
"CH3 NH CO
4.34 1.85 1.26 1.11 0.90 0.85 7.45
(1H, m) (1H, m) (1H, m) (1H, m) (3H, d, 7.6) (3H, t, 6.1) (1H, d, 7.7)
57.20 37.01 24.21 15.24 11.05 171.41a
4.03 2.17 1.96 2.69 2.47 8.18
(1H, m) (1H, m) (1H, m) (1H, m) (3H, s) (1H, d, 7.5)
3.78 1.84 1.61 1.58 0.87 0.83 8.06
(1H, m) (1H, m) (1H, m) (1H, m) (3H, d, 6.1) (3H, d, 6.0) (1H, d, 6.1)
54.14 23.20 49.71 37.86 170.76 52.83 37.92 24.31 22.76 20.88 170.06a
4.78 (1H, dt, 3.9, 8.8)
49.57
2.05 1.87 2.48 2.00 7.31
31.69
(1H, m) (1H, m) (2H, m) (3H, s) (1H, d, 8.8)
29.52 14.75 170.91a
171.80a
Assignments may be interchanged.
3.4. Cyclolinopeptide F (CLF)
3.6. Cyclolinopeptide H (CLH)
Colorless powder, [ ]D 71.4 (c 0.21, MeOH); FABMS m/z 1084.6 [M+H]+; HR-FABMS m/z 1084.5018 [M+H]+, calcd for C55H74N9O10S2, 1084.5000; IR max (KBr) cm 1: 3375, 1657 and 1016; UV lmax (MeOH) nm (log "): 280 (3.65) and 228 (4.02). 1 H and 13C spectra are listed in Table 1.
87.7 (c 0.15, MeOH); Colorless powder, [ ]D FABMS m/z 1081.6 [M+H]+; HR-FABMS m/z 1082.5201 [M+H]+, calcd for C56H76N9O9S2, 1082.5207; IR max (KBr) cm 1: 3422, 1653 and 1029; UV lmax (MeOH) nm (log "): 281 (3.62) and 229 (3.94). 1 H and 13C spectra are listed in Table 3.
3.5. Cyclolinopeptide G (CLG)
3.7. Cyclolinopeptide I (CLI)
Colorless powder, [ ]D 66.6 (c 0.20, MeOH); FABMS m/z 1098.6 [M+H]+; HR-FABMS m/z 1098.5138 [M+H]+, calcd for C56H76N9O10S2, 1098.5157; IR max (KBr) cm 1: 3364, 1657 and 1015; UV lmax (MeOH) nm (log "): 281 (3.65) and 219 (3.96). 1 H and 13C spectra are listed in Table 2.
Colorless powder, [ ]D 60.6 (c 0.20, MeOH); FABMS m/z 1068.2 [M+H]+; HR-FABMS m/z 1068.5110 [M+H]+, calcd for C55H74N9O9S2, 1068.5051; IR max (KBr) cm 1: 3383, 1657 and 1027; UV lmax (MeOH) nm (log "): 281 (3.65) and 228 (4.00). 1 H and 13C spectra are listed in Table 4.
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Table 4 1 H and 13C NMR signal assignments for cyclolinopeptide I (4) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO Phe2
" NH CO Phe3
" NH C=O
4.20 (1H, m) 2.09 (1H, m) 1.61 (1H, m) 1.88 (2H, m) 3.63 (1H, m) 3.52 (1H, m)
4.12 (1H, m) 2.86 (2H, m) 7.22 (2H, m) 7.04 (2H, m) 7.19 (1H, m) 8.38 (1H, d, 7.0)
4.67 (1H, m) 3.41 (1H, m) 2.76 (1H, m) 7.22 (2H, m) 7.26 (2H, m) 7.19 (1H, m) 7.90 (1H, d, 8.3)
62.02 28.97 24.19 46.93 173.38 56.49 35.09 137.76 127.85 128.41 125.87 170.34 52.78 36.97 138.05 127.93 128.41 126.02 171.26a
4
Trp 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.06 (1H, m) 3.36 (2H, m) 10.60 (1H, s) 7.04 (1H, d, 7.3) 7.45 (1H, d, 7.7) 7.18 (1H, m) 6.95 (1H, t, 7.7) 7.32 (1H, d, 7.7) 8.25 (1H, d, 5.5)
55.73 24.19 120.58 110.55 117.62 122.63 117.95 111.04 135.83 127.20
Val
CH3 NH CO Met6
"CH3 NH CO Leu7
CH3 NH CO Mso8
"CH3 NH CO
4.43 (1H, m) 2.14 (1H, m) 0.93 (3H, d, 6.4) 0.84 (3H, d, 6.4) 7.34 (1H, d, 7.1)
4.03 (1H, m) 2.49 (1H, m) 2.39 (1H, m) 2.01 (1H, m) 1.85 (1H, m) 2.02 (3H, s) 8.15 (1H, br.s)
3.83 (1H, m) 1.85 (1H, m) 1.61 (1H, m) 1.55 (1H, m) 0.90 (3H, d, 6.2) 0.86 (3H, d, 6.2) 8.07 (1H, d, 6.2)
57.25 30.64 19.08 17.53 170.74a 54.05 29.46 30.01 14.30 170.94 52.38 37.57 24.32 22.77 20.73 171.03
4.71 (1H, m)
50.29
2.39 (1H, m) 1.94 (1H, m) 2.82 (1H, m) 2.71 (1H, m) 2.46 (3H, s) 7.35 (1H, br.s)
25.61 50.29 37.89 170.43
172.27a
Assignments may be interchanged.
3.8. Absolute con®guration of amino acid A solution of each peptide (1 mg) in 6 N HCl was heated at 110 C for 24 h in a sealed tube. After removal of HCl by repeated evaporation in vacuo, the hydrolysate was dissolved in water and treated with 1-¯uoro2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent) and 1 M NaHCO3 at 35 C for 1 h. After cooling, it was treated with 2 M HCl and then concentrated to dryness. This residue was subjected to HPLC, ¯ow rate 1 ml/ min, detection at 340 nm, solvent: 10±80% MeOH (80 min gradient added on 10 min to be eluted with 100% MeOH)/50 mM triethylamine phosphate (TEAP)
buer (pH 3.2). Retention time (min) of authentic amino acids were as follows: l-Pro (54.8), d-Pro (60.6), l-Met (63.9), d-Met (76.8), l-Val (66.8), d-Val (79.7), l-Trp (67.4), d-Trp (75.8), l-Phe (71.9), d-Phe (84.6), lIle (74.0), d-Ile (86.4), l-Leu (75.5), d-Leu (87.4), lMsn (46.7), d-Msn (51.2), l-Mso (45.4), d-Mso (47.4). 3.9. Reaction of CLF and CLG with thioglycolic acid and cyanogen bromide Solutions of CLF (18.9 mg) and CLG (21.1 mg) in 40% thioglycolic acid (1.0 ml) were individually heated at 50 C for 24 h, respectively. Each reaction mixture
T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
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was subjected to HP-20 column chromatography, then further puri®ed by HPLC, to give two cyclic octapeptides consisting of one methionine sulfoxide and one methionine, and one cyclic octapeptide containing two methionine. The latter was treated with 100 M cyanogen bromide and 70% formic acid (1.0 ml) for 24 h at room temperature. The reaction mixture from CLF (or CLG) was lyophilized and puri®ed by HPLC to give an acyclic homoserine lactonic product CLF0 (3.0 mg) (or CLG0 (5.5 mg)).
(1H, m, ), 4.35 (1H, m, ), 4.22 (1H, m, ), 8.40 (1H, d, 8.0, NH). 13C NMR Pro1 58.59 ( ), 29.46 ( ), 23.28 ( ), 45.61 (), 167.79 (CO); Phe2 54.13 ( ), 37.59 ( ), 137.59 ( ), 127.98 (), 129.17 ("), 126.25 (), 167.79 (CO); Phe3 53.77 ( ), 37.36 ( ), 137.47 ( ), 127.88 (), 129.05 ("), 126.09 (), 170.66 (CO); Trp4 53.17 ( ), 27.88 ( ), 123.54, 109.76, 118.44, 120.73, 118.13, 111.16, 135.97, 127.39, 170.91 (CO); Ile5 56.63 ( ), 36.90 ( ), 24.28 ( ), 15.13 ( ), 10.98 (), 170.77 (CO); HomoSer lactone6 47.71 ( ), 28.63 ( ), 65.24 ( ), 174.93 (CO).
3.9.1. CLF0 Colorless powder; MS m/z 778.4 [M+H]+; IR max (KBr) cm 1: 3427, 3293 (NH), 1775 ( -lactone) and 1641 (amide carbonyl). 1H NMR (DMSO-d6, 300 K): Pro1 4.02 (1H, t, 7.4, ), 2.02 (1H, m, ), 1.71 (1H, m, ), 1.79 (2H, m, ), 3.16 (2H, m, ), 9.31 (1H, br.s, NH); Phe2 4.54 (1H, m, ), 2.98 (1H, m, ), 2.68 (1H, dd, 10.3, 14.0, ), 7.21 (2H, m, ), 7.19 (2H, m, "), 7.16 (1H, m, ), 8.63 (1H, d, 8.5, NH); Phe3 4.55 (1H, m, ), 3.00 (1H, m, ), 2.78 (1H, dd, 9.5, 13.9, ), 7.20 (2H, m, ), 7.18 (2H, m, "), 7.15 (1H, m, ), 8.25 (1H, d, 8.2, NH); Trp4 4.68 (1H, dt, 5.4, 7.8, ), 3.15 (1H, m, ), 3.03 (1H, m, ), 10.80 (1H, s, NH), 7.17 (1H, d, 6.6), 7.60 (1H, d, 7.9), 7.05 (1H, t, 7.9), 6.97 (1H, t, 7.9), 7.31 (1H, d, 7.9), 8.22 (1H, d, 7.8); Val5 4.18 (1H, dd, 6.8, 8.9, ), 1.98 (1H, m, ), 0.89 (3H, d, 6.8, ), 0.87 (3H, d, 6.8, ), 7.98 (1H, d, 8.9, NH); HomoSer lactone6 4.60 (1H, m, ), 2.40 (1H, m, ), 2.15 (1H, m, ), 4.35 (1H, m, ), 4.23 (1H, m, ), 8.41 (1H, d, 8.1, NH). 13C NMR Pro1 58.57 ( ), 29.47 ( ), 23.27 ( ), 45.59 (), 167.73 (CO); Phe2 53.79 ( ), 37.36 ( ), 137.48 ( ), 127.88 (), 129.19 ("), 126.09 (), 170.28 (CO); Phe3 54.15 ( ), 37.54 ( ), 137.56 ( ), 127.99 (), 129.19 ("), 126.25 (), 170.71 (CO); Trp4 53.22 ( ), 27.53 ( ), 123.55, 109.77, 118.44, 120.73, 118.14, 111.16, 135.98, 127.38, 171.05 (CO); Val5 57.47 ( ), 30.82 ( ), 19.00 ( ), 18.04 ( ), 170.68 (CO); HomoSer lactone6 47.72 ( ), 27.91 ), 65.22 ( ), 174.95 (CO).
3.10. Biological assay
3.9.2. CLG0 Colorless powder; MS m/z 814.7 [M+H]+; IR max (KBr) cm 1: 3423 (NH), 1775 ( -lactone) and 1640 (amide carbonyl). 1H NMR (DMSO-d6, 300 K): Pro1 4.02 (1H, t, 7.4, ), 2.18 (1H, m, ), 1.68 (1H, m, ), 1.80 (2H, m, ), 3.15 (2H, m, ); Phe2 4.56 (1H, m, ), 2.68 (1H, dd, 10.0, 14.2, ), 2.18 (1H, m, ), 7.21 (2H, m, ), 7.19 (2H, m, "), 7.16 (1H, m, ), 8.61 (1H, d, 8.7, NH); Phe3 4.54 (1H, m, ), 2.96 (1H, m, ), 2.77 (1H, dd, 9.9, 14.0, ), 7.20 (2H, m, ), 7.18 (2H, m, "), 7.15 (1H, m, ), 8.25 (1H, d, 8.3, NH); Trp4 4.67 (1H, ), 3.17 (1H, m, ), 3.03 (1H, m, ), 10.79 (1H, s, NH), 7.18 (1H, d, 7.3), 7.59 (1H, d, 7.8), 7.05 (1H, t, 7.8), 6.97 (1H, t, 7.8), 7.31 (1H, d, 7.8), 8.20 (1H, d, 7.8); Ile5 4.20 (1H, t, 8.8, ), 1.72 (1H, m, ), 1.46 (1H, m, ), 1.11 (1H, m, ), 0.86 (3H, d, 6.9, ), 0.82 (3H, t, 7.4, ), 8.00 (1H, d, 8.8, NH); HomoSer lactone6 4.60 (1H, m, ), 2.38 (1H, m, ), 2.15
The bio-assay method using mouse lymphocytes was as follows. After dislocation of cervical vertebrae of ICR mice aged 5±8 weeks, the spleen was removed and mashed. Then, the cells were suspended in RPMI 1640 medium and passed through a stainless steel mesh. The single cell suspension was washed twice with the medium and ®nally suspended in RPMI 1640 medium containing 10% fetal calf serum, and 10 mg/ml kanamycin to give 3.5106 cells/ml. 200 ml of this suspension was placed in each well of a microtiter plate with 96 ¯atbottom wells. Concanavalin A was added to each well to a ®nal concentration of 2.5 mg/ml. Subsequently, 4 ml of serially diluted EtOH solution of test compound was added to a ®nal concentration of 10±100 000 ng/ml. The plate was incubated for 3 days in 5% CO2/95% air at 37 C. After termination of cell culture, 20 ml of 5 mM 1methoxy PMS and 0.2 mM WST-1 (DOJINDO Laboratories) in phosphate buered saline was added to every well and the plate was incubated again at 37 C in 5% CO2/air for a 4 h. The plate was read on a microplate reader (Corona MT P-32, Corona Co., Japan) at 415 nm. A dose±response curve was plotted for each drug, and the concentration which gave 50% inhibition of cell growth (IC50) was recorded. Acknowledgements The authors thank the Ministry of Education, Science and Culture, Japan, for ®nancial support through Grant-in-Aid for General Scienti®c Research. References Blasio, B.D., Rossi, F., Benedetti, E., Pavone, C., Temussi, P.A., Zanotti, G., Tancredi, T., 1989. Bioactive peptide: solid-state and solution conformation of cyclolinopeptide A. J. Am. Chem. Soc. 111, 9089±9098. Brewster, A.I., Bovey, F.A., 1971. Conformation of cyclolinopeptide A observed by nuclear magnetic resonance spectroscopy. Proc. Natl. Acad. Sci. USA 68, 1199±1202. Itokawa, H., Takeya, K., Hitotsuyanagi, Y., Morita, H., 1997. Macrocyclic peptide alkaloids from plants. The Alkaloids, Vol. 49. Academic Press, New York, pp. 301±387.
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Kaufmann, H.P., Tobschirbel, A., 1959. An oligopeptide from linseed. Chemische Berichte 92, 2805±2809. Kessler, H., Bats, J.W., Griesinger, C., Koll, S., Will, M., Wagner, K., 1988. Peptide conformations. 46. Conformational analysis of a superpotent cytoprotective cyclic somatostatin analog. J. Am. Chem. Soc. 110, 1033±1049. Marfey, P., 1984. Determination of d-amino acids. II. Use of a bifunctional reagent, 1,5-di¯uoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49, 591±596. Morita, H., Shishido, A., Matsumoto, T., Takeya, K., Itokawa, H., Hirano, T., Oka, K., 1997. Cyclic peptides from higher plants. 42. A new immunosuppressive cyclic nonapeptide B from Linum usitatissium. Bioorganic and Medicinal Chemistry Letters 7, 1269±1272.
Morita, H., Shishido, A., Matsumoto, T., Itokawa, H., Takeya, K., 1999. Cyclic peptides from higher plants. 45. Cyclolinopeptides B± E, new cyclic peptides from Linum usitatissium. Tetrahedron 55, 967±976. Naider, F., Benedetti, E., Goodman, M., 1971. Conformation of cyclolinopeptide A by circular dichroism. Proc. Natl Acad. Sci. USA 68, 1195±1198. Tonelli, A.E., 1971. Approximate treatment of the conformational characteristics of a cyclic nonapeptide, cyclolinopeptide A. Proc. Natl. Acad. Sci. USA 68, 1203±1207. Wieczorek, Z., Bengtsson, B., Trojnar, J., Siemion, I.Z., 1991. Immunosuppressive activity of cyclolinopeptide A. Peptide Res. 4, 275± 283.
www.elsevier.com/locate/phytochem
Cyclolinopeptides F±I, cyclic peptides from linseed Teruki Matsumoto, Akira Shishido, Hiroshi Morita, Hideji Itokawa, Koichi Takeya * School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Received 5 July 2000; received in revised form 23 October 2000
Abstract Four cyclic peptides, cyclolinopeptides F±I, were isolated from seeds of Linum usitatissimum. Their structures were elucidated by extensive 2D NMR spectroscopic methods and by chemical degradation. Further, their immunosuppressive activity is examined. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Linum usitatissimum; Linaceae; Cyclolinopeptide; Cyclic peptide; Immunosuppresive activity
1. Introduction Cyclolinopeptide A (CLA), a cyclic nonapeptide, which exhibits various biological activities (Wieczorek et al., 1991; Naider et al., 1971; Brewster and Bovey, 1971; Tonelli, 1971; Blasio et al., 1989) has been isolated from linseed oil as one of the ®rst isolated natural cyclic peptides (Kaufmann and Tobschirbel, 1959). In a previous paper, we reported on the separation, puri®cation and structure elucidation of cyclolinopeptides B±E having cyclic nona- or octapetides, from the seeds of Linum usitatissimum (Morita et al., 1997, 1999). As part of our investigation on new biologically active cyclic peptides from higher plants (Itokawa et al., 1997), we focused our attention on the isolation of additional cyclic peptides from linseed. In this report, we describe both the isolation and structural elucidation of four new cyclic peptides, cyclolinopeptides F (1)±I (4) by extensive 2D NMR spectroscopic methods and their immunosuppressive activities against mouse splenocytes. 2. Results and discussion Puri®cation of the methanolic extract prepared from the linseed involving Diaion HP-20 column chromatography, using a H2O±MeOH gradient system. The MeOH eluate was next applied to a silica gel column, * Corresponding author. Tel.: +81-426-763007; fax: +81-426771436. E-mail address: [email protected] (K. Takeya).
this being followed by HPLC [ODS] to yield the four new cyclic peptides, cyclolinopetides F±I (CLF±CLI) in yields of 0.0008%, 0.0024%, 0.0002% and 0.00007%, respectively. Cyclolinopeptides F (CLF) and G (CLG) were obtained as white powders. Their molecular formulae were shown to be C55H74N9O10S2 and C56H76N9O10S2, respectively by the quasi-molecular ion peak at m/z 1084.5018 [M+H]+ and at m/z 1098.5138 [M+H]+ in the HR-FABMS. Both IR spectra had absorption peaks which were attributed to amine, amide carbonyl and sulfoxide groups. Since the UV absorption maximum in the neighborhood of 280 nm suggested the presence of a tryptophan residue, the compounds were hydrolyzed with 6 N HCl containing 4% thioglycolic acid. HPLC analysis of each hydrolysate showed that CLF was a cyclic octapeptide consisting of two phenylalanines (Phe), two methionines (Met), proline (Pro), leucine (Leu), valine (Val) and tryptophan (Trp) per mol and CLG was a cyclic octapeptide consisting of two phenylalanines (Phe), two methionines (Met), proline (Pro), leucine (Leu), isoleucine (Ile) and tryptophan (Trp) per mol. However, both methionines in CLF and CLG were present as methionine sulfoxides, because each molecular formula had an additional two oxygen atoms, compared with the cyclic octapeptide presumed from above amino acid degradation and both had IR absorption bands (CLF: 1016 and CLG: 1015 cm 1) indicating sulfoxide groups. In the 1H and 13C NMR spectra (Tables 1 and 2), CLF and CLG had seven amide protons and eight amide carbonyl carbons, respectively, and thus were
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00442-8
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T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
Table 1 1 H and 13C NMR signal assignments for cyclolinopeptide F (1) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO Phe2
" NH CO
4.21 (1H, 2.11 (1H, 1.59 (1H, 1.85 (2H, 3.58 (1H, 3.48 (1H,
t, 7.8) m) m) m) m) m)
4.10 (1H, dt, 6.4, 7.6) 2.80 (2H, m) 7.21 (2H, 7.01 (2H, 7.19 (1H, 8.39 (1H,
m) d, 6.6) m) d, 6.4)
62.04 29.04 24.17 46.89 173.55 56.60 35.10 137.68 127.87 128.39 125.88 170.35
3
Phe
" NH CO Trp4 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.67 (1H, m) 3.44 (1H, m) 2.82 (1H, m) 7.21 (2H, 7.20 (2H, 7.19 (1H, 7.92 (1H,
m) m) m) d, 8.1)
3.99 (1H, m) 3.38 (2H, m) 10.62 (1H, s) 7.15 (1H, d, 7.5) 7.45 (1H, 7.04 (1H, 6.95 (1H, 7.31 (1H,
d, 7.8) t, 7.8) t, 7.8) d, 7.8)
8.06 (1H, d, 6.3)
52.73 36.22 138.09 127.95 128.39 126.06 171.16 55.61 23.42 122.55 110.71 117.63 120.58 117.95 111.04 135.82 127.21
Val
CH3 NH CO Mso6
"CH3 NH CO Leu7
CH3 NH CO Mso8
"CH3 NH CO
4.50 (1H, m) 2.20 (1H, m) 0.94 (3H, d, 6.7) 0.85 (3H, d, 6.7) 7.27 (1H, d, 6.3)
3.98 (1H, m) 2.08 (1H, m) 1.97 (1H, m) 2.74 (2H, m) 2.49 (3H, s) 8.31 (1H, br.s)
56.88 30.92 19.20 17.39 172.52a 54.57 23.23 49.48 38.00 170.74a
3.88 (1H, m)
52.41
1.84 (1H, m) 1.61 (1H, m) 1.62 (1H, m) 0.91 (3H, d, 8.4) 0.85 (3H, d, 8.4) 8.05 (1H, d, 6.5)
37.85 24.30 22.78 20.77 171.07a
4.70 (1H, dt, 6.9, 14.0) 2.39 (1H, m)
50.35 25.60
1.93 (1H, m) 2.78 (2H, m) 2.44 (3H, s) 7.36 (1H, br.s)
50.35 37.85 170.35a
170.74a
Assignments may be interchanged.
obviously octapeptides. However, analysis of the NOE data and 2JH C and 3JH C correlations by HMBC did not permit elucidation of their amino acid sequences as shown in Figs. 1 and 3. Hence, CLF and CLG were chemically transformed to their corresponding linear derivatives (CLF0 and CLG0 ), i.e. by reduction of their methionine sulfoxide using thioglycolic acid and subsequent decomposition with cyanogen bromide. This gave the linear peptides, which displayed an absorption IR bands at 1775 cm 1 attributed to an homoserine lactone. Sequential analyses due to the HMBC correlations indicated by arrows in Figs. 2 and 4 showed that CLF0 and CLG0 were, respectively, the linear peptides
Pro±Phe±Phe±Trp±Val±HomoSer Lactone and as ProPhe±Phe±Trp±Ile±HomoSer Lactone each additional dipeptide was assumed to be Leu7 HomoSer lactone. ROE correlations of CLF were observed between the Mso6 -proton and the Leu7 amide proton, and between the Mso8 -proton and the Pro1 -proton, respectively. Also, ROE correlations of CLG were noted between the Mso8 -proton and Pro1 -proton, and between the Ile5 -proton and the Mso6 amide proton. Consequently, the structure of CLF was identi®ed as cyclo (-Pro±Phe± Phe±Trp±Val±Mso±Leu±Mso-) and CLG as cyclo (Pro±Phe±Phe±Trp±Ile±Mso±Leu±Mso-). The absolute stereochemistries of the component amino acids was
T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
253
Table 2 1 H and 13C NMR signal assignments for cyclolinopeptide G (2) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO
4.21 (1H, t, 7.8) 2.09 (1H, m) 1.57 (1H, m) 1.85 (2H, t, 6.6) 3.58 (1H, m) 3.51 (1H, m)
62.03 29.01 24.17 46.90 173.42
2
Phe
" NH CO Phe3
" NH CO Trp4 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.11 (1H, dt, 6.4, 7.5) 2.84 (2H, m) 7.22 (2H, m) 7.02 (2H, m) 7.27 (1H, m) 8.40 (1H, d, 6.4)
4.69 (1H, m) 3.44 (1H, m) 2.79 (1H, m) 7.22 (2H, m) 7.02 (2H, m) 7.06 (1H, m) 7.93 (1H, d, 6.3)
6.95 (1H, m) 3.36 (2H, m) 10.61 (1H, s) 7.18 (1H, d, 6.8) 7.45 (1H, d, 7.8) 7.05 (1H, t, 7.8) 6.95 (1H, t, 7.8) 7.31 (1H, d, 7.8) 8.11 (1H, br.s)
56.59 35.07 137.76 127.84 128.35 125.85 170.36 52.66 36.19 138.09 127.95 128.42 126.01 170.85a 55.65 23.38 122.58 110.65 117.62 120.57 117.94 111.03 135.82 127.20
Ile
CH3 CH3 NH CO Mso6
"CH3 NH CO Leu7
CH3 NH CO Mso8
"CH3 NH CO
4.49 (1H, br.s) 1.91 (1H, m) 1.39 (1H, m) 1.12 (1H, m) 0.92 (3H, d, 6.6) 0.88 (3H, t, 9.2) 7.32 (1H, m)
56.81 37.14 23.84 15.30 11.06 172.30a
4.01 (1H, m) 2.16 (1H, m) 1.97 (1H, m) 2.73 (1H, m) 2.65 (1H, m) 2.49 (3H, s) 8.27 (1H, br.s)
54.36 23.13 49.54 37.83 170.29
3.84 (1H, m) 1.79 (1H, m) 1.62 (1H, m) 1.61 (1H, m) 0.89 (3H, d, 6.1) 0.84 (3H, d, 6.1) 8.01 (1H, m)
4.70 (1H, m) 2.39 (1H, m) 1.95 (1H, m) 2.75 (2H, m) 2.45 (3H, s) 7.36 (1H, m)
52.49 37.83 24.27 22.76 20.83 170.92a 50.35 25.63 50.35 38.05 171.03a
173.17a
Assignments may be interchanged.
determined to be entirely in the l-con®gurations as evidenced by derivatization of the acid hydrolysate with Marfey's reagent (Marfey, 1984), followed by HPLC analyses. Cyclolinopeptides H (CLH) and I (CLI) were isolated as white powders, and their molecular formulae were determined to be C56H76N9O9S2 for CLH by HRFABMS m/z 1082.5201 [M+H]+ and C55H74N9O9S2 for CLI by HR-FABMS m/z 1068.5110 [M+H]+. HPLC of their acid hydrolysates showed that CLH was an octapeptide consisting of two Phe, two Met, Pro, Leu, Ile, Trp per mole and CLI was an octapeptide consisting of two Phe, two Met, Pro, Leu, Val, Trp per
mole. Each amino acid was con®rmed to be in the lcon®guration using Marfey's method. The amino acid components of CLH and CLI were similar to those of CLG and CLF. However, the FAB mass spectra are 16 amu less than those of CLG and CLF. The facts suggested the presence of one methionine and one methionine sulfoxide in CLH and CLI instead of two methionine sulfoxides in CLG and CLF, respectively. According to the above speculation, reductive transformations of methionine sulfoxide in CLG and CLF by thioglycolic acid are demonstrated to give three compounds, respectively. One of them in CLG (or CLF) agreed completely with CLH (or CLI) in comparison of
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T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
Fig. 1. Cyclolinopeptide F (1): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
Fig. 2. Cyclolinopeptide F0 : dashed arrows show HMBC correlations and arrows show some selected NOE relationship.
various physical and spectral data. Consequently, the sequence of amino acids in CLH (or CLI) is basically same to that in CLG (or CLF). The amino acid sequences of CLH and CLI were determined from ROE correlations as shown in Figs. 5 and 6. Consequently, the structures of CLH were identi®ed as cyclo (-Pro± Phe±Phe±Trp±Ile-Mso±Leu±Met-) and CLI as cyclo (-Pro±Phe±Phe±Trp-Val±Met±Leu±Mso-). CLB (cyclo (-Pro±Pro±Phe±Phe±Val±Ile±Met±LeuIle-)) showed an inhibitory eect on mitogen (concanavalin A) induced response of human peripheralblood lymphocytes (IC50: 44 ng/ml), which is comparable to that of cyclosporin A (Morita et al., 1997). Furthermore, CLA (cyclo (-Pro±Pro±Phe±Phe±Leu±Ile±Ile± Leu±Val-)), CLB and CLE (cyclo (-Pro±Leu±Phe±Ile±
Mso±Leu±Val-Phe-)) possessing dierent amino acid sequences showed a moderate inhibitory eect of about the same potency on the mouse lymphocyte proliferation induced by Con. A (IC50: CLA 2.5 mg/ml; CLB 39 mg/ml; CLE 43 mg/ml) (Morita et al., 1999). However, CLC (cyclo (-Pro±Pro±Phe±Phe-Val±Ile±Mso± Leu±Ile-)), CLD (cyclo (-Pro±Phe-Phe±Trp±Ile±Mso± Leu±Leu-)) and CLF±CLI (IC50: >100 mg/ml, respectively) did not show any of the above activities. It is known that the biological activity of CLA is critically dependent on the sequence and conformation (Kessler et al., 1988). The conformational analyses and further conformation±biological activity relationship of CLA± CLI, are currently being investigated in our laboratories.
T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
255
Fig. 3. Cyclolinopeptide G (2): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
Fig. 4. Cyclolinopeptide G0 : dashed arrows show HMBC correlations and arrows show some selected NOE relationship.
3. Experimental 3.1. General Optical rotations were measured on a JASCO DIP-4 spectrometer and [ ]D values are given in 10 1 deg cm2 g 1. The IR and UV spectra were taken on JASCO FT/ IR 620 and Hitachi 557 spectrometers, respectively, and the 1H and 13C NMR spectra were recorded on BruÈker AM-500 spectrometers, with chemical shifts () reported in ppm, the spectra were recorded at 320 . FABMS were taken with a VG Autospec spectrometer. HPLC was carried out using an Inertsil PREP-ODS column
(20 mm i.d.250 mm and 30 mm i.d.250 mm, GL Science Inc.) packed with 10 mm ODS. TLC was conducted on precoated Kieselgel 60 F254 (Art. 5715; Merck) with spots detected by use of the Dragendorff reagent. 3.2. Plant material Seeds (Linum usitatissium, Linaceae) were purchased from Nikka Oil Co., Japan and the oil was removed by manual pressing. The plant was identi®ed by Professor Yutaka Sashida, director of the medicinal garden in Tokyo University of Pharmacy and Life Science. A voucher specimen is deposited in our Laboratory.
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T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
Fig. 5. Cyclolinopeptide H (3): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
Fig. 6. Cyclolinopeptide I (4): dashed arrows show selected HMBC correlations and arrows show some selected NOE relationship.
3.3. Extraction and separation Seeds (L. usitatissium, 30 kg) were manually pressed to remove oil by squeezing, these being extracted with hot MeOH three times to give a MeOH extract (4 kg). The latter was subjected to Diaion HP-20 column chromatography using a H2O±MeOH gradient system (0:1±
1:0), and the fraction eluted with 100% MeOH was further subjected to silica gel column chromatography using a CHCl3±MeOH gradient system (1:0±0:1). A 15% MeOH fraction was subjected to ODS HPLC with 40±60% CH3CN solvent system to give CLF (0.0008%), CLG (0.0024%), CLH (0.0002%) and CLI (0.00007%).
T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
257
Table 3 1 H and 13C NMR signal assignments for cyclolinopeptide H (3) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO Phe2
" NH CO Phe3
" NH CO
4.16 2.09 1.66 1.91 3.61 3.56
(1H, (1H, (1H, (2H, (1H, (1H,
t, 7.8) m) m) m) m) m)
4.05 (1H, m) 2.96 (1H, t, 10.3) 2.83 (1H, m) 7.18 6.98 7.22 8.20
(2H, (2H, (1H, (1H,
m) m) m) d, 6.2)
4.58 (1H, m) 3.33 (1H, m) 2.77 (1H, m) 7.18 7.28 7.22 7.88
(2H, (2H, (1H, (1H,
m) m) m) d, 8.8)
61.89 28.75 24.39 46.94 173.17 56.33 35.14 137.78 127.86 128.42 126.00 170.06 53.39 36.47 138.13 127.86 128.60 126.00 170.76a
4
Trp 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.08 (1H, m) 3.33 (2H, m) 10.59 (1H, s) 7.18 (1H, d, 7.6) 7.45 7.04 6.97 7.31
(1H, (1H, (1H, (1H,
d, 7.7) t, 7.7) t, 7.7) d, 7.7)
8.18 (1H, d, 7.5)
55.63 24.31 122.76 110.37 117.65 120.59 117.97 111.04 135.81 127.19
Ile
CH3 CH3 NH CO Mso6
"CH3 NH CO Leu7
CH3 NH CO Met8
"CH3 NH CO
4.34 1.85 1.26 1.11 0.90 0.85 7.45
(1H, m) (1H, m) (1H, m) (1H, m) (3H, d, 7.6) (3H, t, 6.1) (1H, d, 7.7)
57.20 37.01 24.21 15.24 11.05 171.41a
4.03 2.17 1.96 2.69 2.47 8.18
(1H, m) (1H, m) (1H, m) (1H, m) (3H, s) (1H, d, 7.5)
3.78 1.84 1.61 1.58 0.87 0.83 8.06
(1H, m) (1H, m) (1H, m) (1H, m) (3H, d, 6.1) (3H, d, 6.0) (1H, d, 6.1)
54.14 23.20 49.71 37.86 170.76 52.83 37.92 24.31 22.76 20.88 170.06a
4.78 (1H, dt, 3.9, 8.8)
49.57
2.05 1.87 2.48 2.00 7.31
31.69
(1H, m) (1H, m) (2H, m) (3H, s) (1H, d, 8.8)
29.52 14.75 170.91a
171.80a
Assignments may be interchanged.
3.4. Cyclolinopeptide F (CLF)
3.6. Cyclolinopeptide H (CLH)
Colorless powder, [ ]D 71.4 (c 0.21, MeOH); FABMS m/z 1084.6 [M+H]+; HR-FABMS m/z 1084.5018 [M+H]+, calcd for C55H74N9O10S2, 1084.5000; IR max (KBr) cm 1: 3375, 1657 and 1016; UV lmax (MeOH) nm (log "): 280 (3.65) and 228 (4.02). 1 H and 13C spectra are listed in Table 1.
87.7 (c 0.15, MeOH); Colorless powder, [ ]D FABMS m/z 1081.6 [M+H]+; HR-FABMS m/z 1082.5201 [M+H]+, calcd for C56H76N9O9S2, 1082.5207; IR max (KBr) cm 1: 3422, 1653 and 1029; UV lmax (MeOH) nm (log "): 281 (3.62) and 229 (3.94). 1 H and 13C spectra are listed in Table 3.
3.5. Cyclolinopeptide G (CLG)
3.7. Cyclolinopeptide I (CLI)
Colorless powder, [ ]D 66.6 (c 0.20, MeOH); FABMS m/z 1098.6 [M+H]+; HR-FABMS m/z 1098.5138 [M+H]+, calcd for C56H76N9O10S2, 1098.5157; IR max (KBr) cm 1: 3364, 1657 and 1015; UV lmax (MeOH) nm (log "): 281 (3.65) and 219 (3.96). 1 H and 13C spectra are listed in Table 2.
Colorless powder, [ ]D 60.6 (c 0.20, MeOH); FABMS m/z 1068.2 [M+H]+; HR-FABMS m/z 1068.5110 [M+H]+, calcd for C55H74N9O9S2, 1068.5051; IR max (KBr) cm 1: 3383, 1657 and 1027; UV lmax (MeOH) nm (log "): 281 (3.65) and 228 (4.00). 1 H and 13C spectra are listed in Table 4.
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T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
Table 4 1 H and 13C NMR signal assignments for cyclolinopeptide I (4) in DMSO-d6 at 320 K 1
H NMR assignment H [int, mult, J(Hz)]
13
C NMR C
1
Pro
CO Phe2
" NH CO Phe3
" NH C=O
4.20 (1H, m) 2.09 (1H, m) 1.61 (1H, m) 1.88 (2H, m) 3.63 (1H, m) 3.52 (1H, m)
4.12 (1H, m) 2.86 (2H, m) 7.22 (2H, m) 7.04 (2H, m) 7.19 (1H, m) 8.38 (1H, d, 7.0)
4.67 (1H, m) 3.41 (1H, m) 2.76 (1H, m) 7.22 (2H, m) 7.26 (2H, m) 7.19 (1H, m) 7.90 (1H, d, 8.3)
62.02 28.97 24.19 46.93 173.38 56.49 35.09 137.76 127.85 128.41 125.87 170.34 52.78 36.97 138.05 127.93 128.41 126.02 171.26a
4
Trp 1NH 2 3 4 5 6 7 8 9 NH CO a
H [int, mult, J(Hz)]
C
5
4.06 (1H, m) 3.36 (2H, m) 10.60 (1H, s) 7.04 (1H, d, 7.3) 7.45 (1H, d, 7.7) 7.18 (1H, m) 6.95 (1H, t, 7.7) 7.32 (1H, d, 7.7) 8.25 (1H, d, 5.5)
55.73 24.19 120.58 110.55 117.62 122.63 117.95 111.04 135.83 127.20
Val
CH3 NH CO Met6
"CH3 NH CO Leu7
CH3 NH CO Mso8
"CH3 NH CO
4.43 (1H, m) 2.14 (1H, m) 0.93 (3H, d, 6.4) 0.84 (3H, d, 6.4) 7.34 (1H, d, 7.1)
4.03 (1H, m) 2.49 (1H, m) 2.39 (1H, m) 2.01 (1H, m) 1.85 (1H, m) 2.02 (3H, s) 8.15 (1H, br.s)
3.83 (1H, m) 1.85 (1H, m) 1.61 (1H, m) 1.55 (1H, m) 0.90 (3H, d, 6.2) 0.86 (3H, d, 6.2) 8.07 (1H, d, 6.2)
57.25 30.64 19.08 17.53 170.74a 54.05 29.46 30.01 14.30 170.94 52.38 37.57 24.32 22.77 20.73 171.03
4.71 (1H, m)
50.29
2.39 (1H, m) 1.94 (1H, m) 2.82 (1H, m) 2.71 (1H, m) 2.46 (3H, s) 7.35 (1H, br.s)
25.61 50.29 37.89 170.43
172.27a
Assignments may be interchanged.
3.8. Absolute con®guration of amino acid A solution of each peptide (1 mg) in 6 N HCl was heated at 110 C for 24 h in a sealed tube. After removal of HCl by repeated evaporation in vacuo, the hydrolysate was dissolved in water and treated with 1-¯uoro2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent) and 1 M NaHCO3 at 35 C for 1 h. After cooling, it was treated with 2 M HCl and then concentrated to dryness. This residue was subjected to HPLC, ¯ow rate 1 ml/ min, detection at 340 nm, solvent: 10±80% MeOH (80 min gradient added on 10 min to be eluted with 100% MeOH)/50 mM triethylamine phosphate (TEAP)
buer (pH 3.2). Retention time (min) of authentic amino acids were as follows: l-Pro (54.8), d-Pro (60.6), l-Met (63.9), d-Met (76.8), l-Val (66.8), d-Val (79.7), l-Trp (67.4), d-Trp (75.8), l-Phe (71.9), d-Phe (84.6), lIle (74.0), d-Ile (86.4), l-Leu (75.5), d-Leu (87.4), lMsn (46.7), d-Msn (51.2), l-Mso (45.4), d-Mso (47.4). 3.9. Reaction of CLF and CLG with thioglycolic acid and cyanogen bromide Solutions of CLF (18.9 mg) and CLG (21.1 mg) in 40% thioglycolic acid (1.0 ml) were individually heated at 50 C for 24 h, respectively. Each reaction mixture
T. Matsumoto et al. / Phytochemistry 57 (2001) 251±260
259
was subjected to HP-20 column chromatography, then further puri®ed by HPLC, to give two cyclic octapeptides consisting of one methionine sulfoxide and one methionine, and one cyclic octapeptide containing two methionine. The latter was treated with 100 M cyanogen bromide and 70% formic acid (1.0 ml) for 24 h at room temperature. The reaction mixture from CLF (or CLG) was lyophilized and puri®ed by HPLC to give an acyclic homoserine lactonic product CLF0 (3.0 mg) (or CLG0 (5.5 mg)).
(1H, m, ), 4.35 (1H, m, ), 4.22 (1H, m, ), 8.40 (1H, d, 8.0, NH). 13C NMR Pro1 58.59 ( ), 29.46 ( ), 23.28 ( ), 45.61 (), 167.79 (CO); Phe2 54.13 ( ), 37.59 ( ), 137.59 ( ), 127.98 (), 129.17 ("), 126.25 (), 167.79 (CO); Phe3 53.77 ( ), 37.36 ( ), 137.47 ( ), 127.88 (), 129.05 ("), 126.09 (), 170.66 (CO); Trp4 53.17 ( ), 27.88 ( ), 123.54, 109.76, 118.44, 120.73, 118.13, 111.16, 135.97, 127.39, 170.91 (CO); Ile5 56.63 ( ), 36.90 ( ), 24.28 ( ), 15.13 ( ), 10.98 (), 170.77 (CO); HomoSer lactone6 47.71 ( ), 28.63 ( ), 65.24 ( ), 174.93 (CO).
3.9.1. CLF0 Colorless powder; MS m/z 778.4 [M+H]+; IR max (KBr) cm 1: 3427, 3293 (NH), 1775 ( -lactone) and 1641 (amide carbonyl). 1H NMR (DMSO-d6, 300 K): Pro1 4.02 (1H, t, 7.4, ), 2.02 (1H, m, ), 1.71 (1H, m, ), 1.79 (2H, m, ), 3.16 (2H, m, ), 9.31 (1H, br.s, NH); Phe2 4.54 (1H, m, ), 2.98 (1H, m, ), 2.68 (1H, dd, 10.3, 14.0, ), 7.21 (2H, m, ), 7.19 (2H, m, "), 7.16 (1H, m, ), 8.63 (1H, d, 8.5, NH); Phe3 4.55 (1H, m, ), 3.00 (1H, m, ), 2.78 (1H, dd, 9.5, 13.9, ), 7.20 (2H, m, ), 7.18 (2H, m, "), 7.15 (1H, m, ), 8.25 (1H, d, 8.2, NH); Trp4 4.68 (1H, dt, 5.4, 7.8, ), 3.15 (1H, m, ), 3.03 (1H, m, ), 10.80 (1H, s, NH), 7.17 (1H, d, 6.6), 7.60 (1H, d, 7.9), 7.05 (1H, t, 7.9), 6.97 (1H, t, 7.9), 7.31 (1H, d, 7.9), 8.22 (1H, d, 7.8); Val5 4.18 (1H, dd, 6.8, 8.9, ), 1.98 (1H, m, ), 0.89 (3H, d, 6.8, ), 0.87 (3H, d, 6.8, ), 7.98 (1H, d, 8.9, NH); HomoSer lactone6 4.60 (1H, m, ), 2.40 (1H, m, ), 2.15 (1H, m, ), 4.35 (1H, m, ), 4.23 (1H, m, ), 8.41 (1H, d, 8.1, NH). 13C NMR Pro1 58.57 ( ), 29.47 ( ), 23.27 ( ), 45.59 (), 167.73 (CO); Phe2 53.79 ( ), 37.36 ( ), 137.48 ( ), 127.88 (), 129.19 ("), 126.09 (), 170.28 (CO); Phe3 54.15 ( ), 37.54 ( ), 137.56 ( ), 127.99 (), 129.19 ("), 126.25 (), 170.71 (CO); Trp4 53.22 ( ), 27.53 ( ), 123.55, 109.77, 118.44, 120.73, 118.14, 111.16, 135.98, 127.38, 171.05 (CO); Val5 57.47 ( ), 30.82 ( ), 19.00 ( ), 18.04 ( ), 170.68 (CO); HomoSer lactone6 47.72 ( ), 27.91 ), 65.22 ( ), 174.95 (CO).
3.10. Biological assay
3.9.2. CLG0 Colorless powder; MS m/z 814.7 [M+H]+; IR max (KBr) cm 1: 3423 (NH), 1775 ( -lactone) and 1640 (amide carbonyl). 1H NMR (DMSO-d6, 300 K): Pro1 4.02 (1H, t, 7.4, ), 2.18 (1H, m, ), 1.68 (1H, m, ), 1.80 (2H, m, ), 3.15 (2H, m, ); Phe2 4.56 (1H, m, ), 2.68 (1H, dd, 10.0, 14.2, ), 2.18 (1H, m, ), 7.21 (2H, m, ), 7.19 (2H, m, "), 7.16 (1H, m, ), 8.61 (1H, d, 8.7, NH); Phe3 4.54 (1H, m, ), 2.96 (1H, m, ), 2.77 (1H, dd, 9.9, 14.0, ), 7.20 (2H, m, ), 7.18 (2H, m, "), 7.15 (1H, m, ), 8.25 (1H, d, 8.3, NH); Trp4 4.67 (1H, ), 3.17 (1H, m, ), 3.03 (1H, m, ), 10.79 (1H, s, NH), 7.18 (1H, d, 7.3), 7.59 (1H, d, 7.8), 7.05 (1H, t, 7.8), 6.97 (1H, t, 7.8), 7.31 (1H, d, 7.8), 8.20 (1H, d, 7.8); Ile5 4.20 (1H, t, 8.8, ), 1.72 (1H, m, ), 1.46 (1H, m, ), 1.11 (1H, m, ), 0.86 (3H, d, 6.9, ), 0.82 (3H, t, 7.4, ), 8.00 (1H, d, 8.8, NH); HomoSer lactone6 4.60 (1H, m, ), 2.38 (1H, m, ), 2.15
The bio-assay method using mouse lymphocytes was as follows. After dislocation of cervical vertebrae of ICR mice aged 5±8 weeks, the spleen was removed and mashed. Then, the cells were suspended in RPMI 1640 medium and passed through a stainless steel mesh. The single cell suspension was washed twice with the medium and ®nally suspended in RPMI 1640 medium containing 10% fetal calf serum, and 10 mg/ml kanamycin to give 3.5106 cells/ml. 200 ml of this suspension was placed in each well of a microtiter plate with 96 ¯atbottom wells. Concanavalin A was added to each well to a ®nal concentration of 2.5 mg/ml. Subsequently, 4 ml of serially diluted EtOH solution of test compound was added to a ®nal concentration of 10±100 000 ng/ml. The plate was incubated for 3 days in 5% CO2/95% air at 37 C. After termination of cell culture, 20 ml of 5 mM 1methoxy PMS and 0.2 mM WST-1 (DOJINDO Laboratories) in phosphate buered saline was added to every well and the plate was incubated again at 37 C in 5% CO2/air for a 4 h. The plate was read on a microplate reader (Corona MT P-32, Corona Co., Japan) at 415 nm. A dose±response curve was plotted for each drug, and the concentration which gave 50% inhibition of cell growth (IC50) was recorded. Acknowledgements The authors thank the Ministry of Education, Science and Culture, Japan, for ®nancial support through Grant-in-Aid for General Scienti®c Research. References Blasio, B.D., Rossi, F., Benedetti, E., Pavone, C., Temussi, P.A., Zanotti, G., Tancredi, T., 1989. Bioactive peptide: solid-state and solution conformation of cyclolinopeptide A. J. Am. Chem. Soc. 111, 9089±9098. Brewster, A.I., Bovey, F.A., 1971. Conformation of cyclolinopeptide A observed by nuclear magnetic resonance spectroscopy. Proc. Natl. Acad. Sci. USA 68, 1199±1202. Itokawa, H., Takeya, K., Hitotsuyanagi, Y., Morita, H., 1997. Macrocyclic peptide alkaloids from plants. The Alkaloids, Vol. 49. Academic Press, New York, pp. 301±387.
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Kaufmann, H.P., Tobschirbel, A., 1959. An oligopeptide from linseed. Chemische Berichte 92, 2805±2809. Kessler, H., Bats, J.W., Griesinger, C., Koll, S., Will, M., Wagner, K., 1988. Peptide conformations. 46. Conformational analysis of a superpotent cytoprotective cyclic somatostatin analog. J. Am. Chem. Soc. 110, 1033±1049. Marfey, P., 1984. Determination of d-amino acids. II. Use of a bifunctional reagent, 1,5-di¯uoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49, 591±596. Morita, H., Shishido, A., Matsumoto, T., Takeya, K., Itokawa, H., Hirano, T., Oka, K., 1997. Cyclic peptides from higher plants. 42. A new immunosuppressive cyclic nonapeptide B from Linum usitatissium. Bioorganic and Medicinal Chemistry Letters 7, 1269±1272.
Morita, H., Shishido, A., Matsumoto, T., Itokawa, H., Takeya, K., 1999. Cyclic peptides from higher plants. 45. Cyclolinopeptides B± E, new cyclic peptides from Linum usitatissium. Tetrahedron 55, 967±976. Naider, F., Benedetti, E., Goodman, M., 1971. Conformation of cyclolinopeptide A by circular dichroism. Proc. Natl Acad. Sci. USA 68, 1195±1198. Tonelli, A.E., 1971. Approximate treatment of the conformational characteristics of a cyclic nonapeptide, cyclolinopeptide A. Proc. Natl. Acad. Sci. USA 68, 1203±1207. Wieczorek, Z., Bengtsson, B., Trojnar, J., Siemion, I.Z., 1991. Immunosuppressive activity of cyclolinopeptide A. Peptide Res. 4, 275± 283.