Clausenlanins A and B, Two Leucine-Rich Cyclic Nonapeptides from Clausena lansium

Abstract Two new cyclic nonapeptides, named clausenlanins A (1) and B (2), were isolated from the roots and rhizomes of Clausena lansium. Their structures were elucidated as cyclo-(Gly1-l-Leu2-l-Ile3-l-Leu4-l-Leu5-l-Leu6-l-Leu7-l-Leu8-l-Leu9) (1) and cyclo-(Gly1-l-Leu2-l-Val3-l-Leu4-l-Leu5-l-Leu6-l-Leu7-l-Leu8-l-Leu9) (2) respectively on the basis of extensive spectroscopic analysis, particularly 2D NMR spectra taken at the temperature of 338 or 303 K and MS. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-017-0133-y) contains supplementary material, which is available to authorized users.


Introduction
About 30 species of Clausena (Rutaceae) are widely distributed in the world, and 10 of them exist in China. Clausena lansium (Lour.) Skeels is a fruit tree and distributes widely in south of China [1]. Its leaves and roots have been used as a folk herb for the treatment of cough, asthma, dermatological disease, viral hepatitis, and gastrointestinal disease; and its seeds for treating acute and chronic gastrointestinal inflammation, and ulcer [2]. Caryophyllaceae-type cyclopeptides (CPs), carbazole alkaloids, coumarins, amides, and terpenoids have been isolated from C. lansium [3][4][5][6][7][8]. Among them, CPs are formed with the peptide bonds of protein or non-proten aamino acid residues, which are homomonocyclopeptides with mainly five to twelve a-amino acid residues [9]. During this work, two new cyclic nonapeptides, named clausenlanins A (1) and B (2) (Fig. 1), were isolated from the roots and rhizomes of C. lansium. Because the 1 H NMR signals are weak and severely overlapped taken at room temperature, variable temperature NMR experiments were performed [10]. In this paper, their separation and structure elucidation are described.

Results and Discussion
Clausenlanin A (1) was obtained as an amorphous solid. Its molecular formula was shown as C 50 H 91 N 9 O 9 by its negative HRESIMS ([M-H] -, 960.6876, calcd 960.6867), indicating the 10°of unsaturation. The IR spectrum exhibited the absorption bands at 3429 and 1661 cm -1 ascribable to NH and CO groups. The 1 H and 13 C NMR spectra of 1 in C 5 D 5 N (Table 1) displayed the characteristic signals of typical CPs.
The 1 H NMR signals of the amino acid residues of 1, especially the signals of NH and a-H, were severely overlapped taken at room temperature. The significant improvement of the 1 H NMR signals was observed by increasing the temperatures from 243 to 338 K. Finally a well-resolved 1 H NMR spectrum with sharp proton signals ( Fig. 2a) was obtained at 338 K in pyridine-d 5 . Then the assignment of the 1 H NMR signals of the amino acid residues was obtained by analyzing the 1 H-1 H COSY spectrum, particularly amide proton NH and a-H signals. The corresponding 13 C NMR assignments were determined on the basis of the HSQC and HMBC experiments, particularly a-C signals ( Table 1 Fig. 3).  The sequence of the nine amino acid residues in 1 was determined by analyzing the ROESY correlations between the a-H of one amino acid residue and the amide proton NH of the next amino acid residue (Fig. 3). The ROESY correlations of Gly 1 -aH/Leu 2 -NH, Leu 2 -aH/Ile 3 -NH, Ile 3 -aH/Leu 4 -NH, Leu 4 -aH/Leu 5 -NH, Leu 5 -aH/Leu 6 -NH, Leu 6 -aH/Leu 7 -NH, Leu 7 -aH/Lue 8 -NH, Leu 8 -aH/Leu 9 -NH, Leu 9 -aH/Gly 1 -NH indicated that the structure of 1 is cyclo-(Gly 1 -Leu 2 -Ile 3 -Leu 4 -Leu 5 -Leu 6 -Leu 7 -Leu 8 -Leu 9 ). This sequence of 1 was The absolute configuration of the amino acids of 1 was determined using the advanced Marfey's method and LC-MS analysis [11,12]. The results indicated that the absolute configurations of the amino acid residues (Leu and Ile) in 1 were the L-configuration (Table S1; Fig. 3). Therefore the structure of 1 is determined as cyclo-(Gly 1 -L-Leu 2 -L-Ile 3 -L-Leu 4 -L-Leu 5 -L-Leu 6 -L-Leu 7 -L-Leu 8 -L-Leu 9 ).
Clausenlanin B (2) was obtained as an amorphous solid. Its molecular formula was shown as C 49 H 89 N 9 O 9 by its negative HRESIMS ([M-H] -, 946.6723, calcd 946.6710), indicating the 10°of unsaturation. The IR spectrum exhibited the absorption bands at 3430 and 1661 cm -1 ascribable to NH and CO groups. The 1 H and 13 C NMR spectra of 2 in C 5 D 5 N (Table 1) displayed the characteristic signals of typical CPs.
The 1 H NMR signals of the amino acid residues of 2, especially the signals of NH and a-H, were severely overlapped taken at room temperature. The significant improvement of the 1 H NMR signals was observed by increasing the temperatures from 243 to 338 K. Finally a well-resolved 1 H NMR spectrum with sharp proton signals (Fig. 2b) was obtained at 303 K in pyridine-d 5 . After compared all data of 2 with those of 1, the results indicated that 2 and 1 are very similar, and 2 might also be a cyclic nonapeptide too. The only difference is to be replaced the isoleucine residue in 1 by valine residue in 2. The assignment of the 1 H and 13 C NMR signals of the valine residue was obtained by analyzing the HSQC, HMBC and COSY spectra, i.e. d H 4.42 (a-CH), 2.57 (b-CH), 1.18 and 1.19 (2*c-CH 3 ), 9.22 (NH); d C 173.9 (CO), 62.6 (a-CH), 30.6 (b-CH), 20.3 and 20.2 (2*c-CH 3 ). Therefore 2 consisted of seven leucines, one glycine, and one valine (Table 1; Fig. 3).

General Experimental Procedures
Optical rotations were obtained on a Jasco P-1020 polarimeter. IR spectra were measured on a Tensor 27 spectrometer with KBr pellets. UV spectra were obtained using a Shimadzu UV-2401PC spectrophotometer. 1D and 2D NMR spectra were performed on a Bruker AM
The analysis of the L-and D, L-FDLA (mixture of D-and L-FDLA) derivatives was performed using an Waters Sunfire C 18 column (4.6*150 mm, 5 lm) maintained at 30°C. Acetonitrile-0.1% HCOOH/H 2 O was used as the mobile phase under a linear gradient elution mode (acetonitrile, 28-60%, 50 min (compound 1); acetonitrile, 35-60%, 50 min (compound 2)) at a flow rate of 1 mL/ min. A Waters Xevo-TQD mass spectrometer was used for detection in ESImode. The capillary voltage was kept at 2.5 kV, and the ion source at 450°C. Nitrogen gas was used as a sheath gas at 650 L/h. A mass range of m/z 100-2000 was scanned in 0.2 s.