tricyclic ring cycloheptapeptide, tunicyclin A (1) (Figure 1),
was isolated from the titled plant. Herein we describe the
isolation and structural elucidation of 1.
and Ser were observed, respectively (Figure 2).6,7 Further-
more, the carbonyl carbons of Pro, Ser, Glu, Leu, Val, Gly,
Tunicyclin A (1) was isolated as a white solid.5 Its
molecular formula was established as C29H45N7O11 by
positive HR-Q-TOF-MS (m/z [M + Na]+ 690.30764; cacld
690.30747). The 1H NMR spectrum of 1 (Table 1) displayed
1
Table 1. H (600 MHz) and 13C NMR (150 MHz) Data of 1 in
C5D5Na
δH
δC
δH
δC
Pro1
CO
R
Leu4
171.8 CO
169.2
52.6
4.84 (dd, 8.7, 5.7)
2.06 (m)
2.00 (m)
1.48 (m)
1.65 (m)
3.40 (dt, 9.6, 7.2)
3.91 (dt, 9.6, 7.2)
61.7
29.4
R
4.98 (dd, 8.4, 5.4)
Figure 2. Selected 2D NMR correlations for 1.
ꢀa
ꢀ
2.20 (2H, ddd, 8.4, 5.4, 2.4) 45.2
ꢀb
γa
γb
δa
γ
2.00 (m)
0.76 (3H, d, 6.6)
1.08 (3H, d, 6.6)
25.0
23.2
21.7
24.8
δ
δ′
48.2 Val5
and Ser were undoubtedly assigned to δC 171.8, 171.4, 170.6,
169.2, 172.4, 169.6, and 171.7 based on correlations between
carbonyl carbons and R or ꢀ protons of the same amino acid
residues in HMBC experiment, respectively. In addition, the
quaternary carbon at δC 87.6 was determined as the γ carbon
of the Glu residue by HMBC correlations from Glu-RH, ꢀaH,
and ꢀbH to δC 87.6, while the methine at δC 80.4 was
assigned to δ carbon of the Glu residue by HMBC correla-
tions from Glu-ꢀaH and ꢀbH to δC 80.4.
δb
CO
172.4
59.8
26.3
21.0
18.1
Ser2
CO
R
171.4 ꢀ
γ
5.53 (d, 10.8)
2.66 (m)
1.16 (3H, d, 6.0)
1.20 (3H, d, 6.6)
NH 8.29 (d, 7.8)
R
ꢀa
5.08 (ddd, 7.8, 5.4, 3.0) 57.4 γ′
4.49 (dd, 10.8, 5.4)
4.22 (dd, 10.8, 3.0)
62.4 Gly6
CO
ꢀb
169.6
44.2
Glu3
CO
NH 9.19 (dd, 6.6, 5.4)
170.6 Ra 4.82 (dd, 17.1, 6.3)
Rb 3.95 (dd, 17.1, 5.4)
NH 8.52 (d, 8.4)
R
ꢀa
ꢀb
γ
γ-OH 8.52 (s)
δ
δ-OH 8.60 (d, 7.2)
5.46 (ddd, 9.6, 8.4,6.0) 49.6 Ser7
2.97 (dd, 15.0, 9.6)
2.94 (dd, 15.0, 6.0)
40.0 CO
171.7
NH 8.42 (d, 9.6)
R
ꢀa
The peptide sequence and connectivity of amino acid
residues were established by HMBC crosspeaks: Ser2-NH/
CO-Pro1, Glu3-NH/CO-Ser2, Leu4-RH/CO-Glu3, Val5-RH/
CO-Leu4, Gly6-NH/CO-Val5, Ser7-NH/CO-Gly6 (Figure 2).
In conjunction with ROESY correlations of Ser7-RH with
δa and δb protons of Pro1, the backbone of 1 was thus
determined as cyclo-(Pro1-Ser2-Glu3-Leu4-Val5-Gly6-Ser7)
(Figure 2 and 3). Since 1 had 11 degrees of unsaturation,
while seven amino acid residues and the macrocycle simply
accounted for nine degrees of unsaturation, 1 should bear
another two rings, and the γ and δ carbons of the Glu residue
should participate in the cyclization. Further, HMBC cor-
relation of Leu-RH with γ carbon of Glu indicated that γ
carbon of Glu was connected to the amino group of the Leu
residue. Additionally, HMBC correlation of Val-RH with δ
carbon of Glu revealed that δ carbon of Glu was linked to
the amino group of Val residue. The connectivity was also
confirmed by HMBC correlation of Glu3-δH to carbonyl
carbon of Leu4 residue. On the basis of the above evidence,
the planar structure of tunicyclin A (1) was constructed.
The absolute configurations of Pro1, Ser2, Leu4, Val5, and
Ser7 were identified as L (S) on the basis of HPLC-ESI-MS
analysis of the retention times and m/z values of the chiral
derivatives of the amino acid residues in acid hydrolysate
of 1 (see detailed information in the Supporting Informa-
tion).8 Since Val5-RH was in trans configuration to Val5-
ꢀH based on the coupling constant of Val5-RH/Val5-ꢀH (J
87.6
5.56 (dt, 9.6, 6.0)
4.04 (t, 9.6)
3.95 (dd, 9.6, 6.0)
51.3
64.0
5.43 (d, 7.2)
80.4 ꢀb
a All proton signals integrate to 1H, unless otherwise indicated.
six amide NH or hydroxyl proton signals resonating at δH
8.29 (d), 8.42 (d), 8.52 (d), 8.52 (s), 8.60 (d), and 9.19 (dd).
The 13C NMR spectrum (Table 1) exhibited seven amide
carbonyl resonances at δC 169.2, 169.6, 170.6, 171.4, 171.7,
171.8, and 172.4, along with seven R-amino acid carbons
resonating at δC 61.7, 59.8, 57.4, 52.6, 51.3, 49.6, and 44.2.
On the basis of the above data, together with its negative
reaction to ninhydrin, 1 was inferred to be a typical
cycloheptapeptide. Also, the 1D NMR spectra indicated
several side chain groups, including four methyls (due to
two isopropyl groups), four methylenes, one CH2N group,
two CH2OH groups, and two methines. However, one
oxygenated sp3 quaternary carbon (δC 87.6) and one oxygen-
ated sp3 methine (δC 80.4) remained unknown.
Completed assignment for protons and carbons of 1 was
addressed by 2D NMR experiments, including COSY,
TOCSY, HMQC, and HMBC. From H-1H COSY and
1
TOCSY experiments, seven spin coupling systems of Pro,
Ser, Glu (absence of the γ methylene protons), Leu (absence
of the NH proton), Val (absence of the NH proton), Gly,
(5) Tunicyclin A (1): [R]D20 -47.0 (c 0.10, MeOH); IR (KBr)υmax 3338,
2959, 2874, 1653, 1541, 1457, 1056, 706 cm-1. UV (MeOH) λmax 211,
256 nm. Positive ESI-MS m/z: 690 [M + Na]+. Negative ESI-MS m/z: 666
[M - H]-. Positive HR-Q-TOF-MS m/z: 690.30764 [M + Na]+, cacld. for
C29H45N7O11Na, 690.30747.
(6) Wanger, G.; Kumar, A.; Wuthrich, K. Eur. J. Biochem. 1981, 114,
375–384.
(7) Tan, N. H.; Zhou, J. Chem. ReV. 2006, 106, 840–895.
(8) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. I. Anal. Chem.
1997, 69, 5146–5151.
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Org. Lett., Vol. 11, No. 5, 2009