Molecules 2013, 18 2600

(Figure 1). These compounds were identified by combined spectroscopic methods and comparisons of
NMR data with literature [10,19–21].

Figure 1. Structures of saponins 1–4 from E. ferox.

Compound 1 was isolated as a white amorphous solid, with a molecular formula assigned as
C44H71NO16 on the basis of combined HRESIMS (m/z 892.46222 [M+Na]+) and 13C-NMR data. The
saponin nature of this compound was evidenced in the 1H-NMR spectrum of 1 by the presence of
characteristic terpenoid methyl singlets (δH between 0.6 and 1.5) together with sugar residues
 Table 1.1H (500 MHz) and 13C-NMR (1
1 2

(Figure 1). These compounds were identified by combined spectroscopic m

Position

1a
δH, mult. (J in Hz)
1.86, m
δC, mult. δ
1
37.4, CH2

NMR data with literature [10,19–21]. 1b

2a
1.32, m
2.38, qd (13.5, 2.5)
28.2, CH2
1
2
2b 2.05, m 2
3 3.28 ª 89.8, CH 3
4 50.5, C

Figure 1. Structures of saponins 1–4 from E. ferox

5
6a
1.33, m
1.94, m
53.2, CH 1
1
21.2, CH2
6b 1.66, m 1
7a 2.07, m 2
29.8, CH2
7b 1.93, m 2
8 129.1, C
9 137.3, C
10 38.5, C
11a 2.17, m 2
23.3, CH2
11b 2.08, m 2
12a 2.00, m 1
38.4, CH2
12b 1.43, m 1
13 43.4, C
14 2.11, m 53.3, CH 2
15a 1.63, m 1
24.8, CH2
15b 1.37, m 1
16a 1.92, m 1
29.1, CH2
16b 1.36, m 1
17 1.57, m 52.5, CH 1
18 0.67, s 11.8, CH3 0
19 1.01, s 18.9, CH3 1
20 1.89, m 37.9, CH 1
21 0.93, d (6.8) 12.4, CH3 0
22a 2
3.51, br d (9.5) 73.4, CH
22b 2
23 3.68, dd (9.5, 1.5) 72.2, CH
24 1.56, m 41.0, CH 2
1
24 0.85, d (6.9) 10.0, CH3
25 1.62, m 32.2, CH 2
26 0.95, d (6.6) 21.6, CH3 0
27 0.96, d (6.6) 21.2, CH3 0
28a 3
1.35, s 24.8, CH3
28b 3
29 177.8, C
a
Overlapped wit
 It appears that 1H and 13C-NMR data are fully consistent with a 22R, 23S and 24R configurations
assuming a usual absolute configuration at C-20 (Figure 2). Additionally, we then propose to change
the configurations for ulososides A, C, D and E that were tentatively assigned as 22S, 23R and 24S.

Figure 2. Comparison of NMR data of the aglycon side-chain.

NMR spectra indicated the disaccharide nature of compound 1. For the glycosidic part of the
molecule, two sugar residues were evidenced by the signals corresponding to two anomeric carbons at
 Table 2. 1H (500 MHz) and 13C-NMR (1
Figure 1. Structures of saponins
Position
1 1–4 from E2
δH, mult. (J in Hz) δC, mult. δ
1′ 4.56, d (8.1) 104.8, CH 4
2′ 3.61, dd (8.1, 9.5) 58.0, CH 3
3′ 3.53, t (9.5) 75.5, CH 3
4′ 3.28 ª 72.1, CH 4
5′ 3.49, ddd (9.4, 6.3, 1.6) 77.0, CH 3
6′a 4.11, dd (11.8, 1.8)
70.2, CH2 3
6′b 3.79, dd (11.8, 6.4)
-CO-CH3 1.93, s 23.0, CH3
-CO-CH3 173.5, C
1′′ 4.49, d (7.8) 105.1, CH 5
2′′ 3.26, dd (9.3, 7.8) 74.8, CH 3
3′′ 3.40, t (9.3) 77.5, CH 3
4′′ 3.53, m 73.2, CH 3
5′′a 4
3.78, d (9.6) 76.6, CH
5′′b 3
6′′a
172.5, C
6′′b
1′′′ 4
2′′′ 3
3′′′ 3
4′′′ 3
5′′′ 3
6′′′ 3
a
Overlapped
ules 2013, 18

Figure S1. 1H-NMR spectrum of 1 (500 MHz) in CD3OD.

 Table 1. Cont.
21 62.7 803 780 641 [M − Hex + Na]+, 627 [M − HexA + Na]+, 465 [M − Hex − HexA + Na]+ AG-4 Hex-HexA f
22 63.5 805 782 643 [M − Hex + Na]+, 467 [M − Hex − HexA + Na]+ AG-7 Hex-HexA f
23 63.9 817 794 641 [M − HexA + Na]+, 479 [M − HexA − Hex + Na]+ AG-8 HexA-Hex c
24 64.5 761 738 629 [M − Pen + Na]+, 467 [M − Pen − Hex + Na]+ AG-7 Pen-Hex-
25 65.8 787 764 655 [M − Pen + Na]+, 479 [M − Pen − HexA + Na]+ AG-8 Pen-HexA-
a
Monosaccharides were identified by residues masses of 162 for hexoses (Hex), 176 for hexuronic acids (HexA), 132 for pentoses (Pen) and 203 for N-acetylhexosamines (HexNAc);
b
Aglycone cores are directly associated with the mass of our previous report as ULO-A: Aglycone from ulososide A (490 Da); URA-A: Aglycone from uraboside A (474 Da); URA-B:
Aglycone from uraboside B (488 Da); and other new aglycones with a specific m/z as AG-1 (506 Da), AG-2 (476 Da), AG-3 (502 Da), AG-4 (472 Da), AG-5 (500 Da), AG-6 (442 Da),
AG-7 (444 Da) and AG-8 (456 Da); c Glycan core could be the same for ulososide A such as O-[β-D-Glucuronopyranosyl-(1→6)-β-D-glucopyranoside]; d Glycan core could be the same for
e f
uraboside A such as O-[β-D-Arabinopyranosyl-(1′′→2′)-(β-D-galactopyranosyl-(1′′′→3′))-β-D-galactopyranoside]; Bidesmoside-like fragmentation; Glycan core could be the same for
uraboside B such as O-[β-D-Galactopyranosyl-(1→2)-β-D-glucopyranosiduronic acid].

Figure 2. Total Ion Chromatogram of saponins from E. ferox acquired by LC-ESI-MS (m/z: 400–1500 Da).
Mar. Drugs 2013, 11 4818

Table 1. Saponins found in Ectyoplasia ferox, with their LC/MS/MS retention times and fragment signatures.
Rt m/z
Cmp + MW Diagnostic ions (m/z) a Aglycone core b Glycan core
(min) [M + Na]
1 45.1 851 828 833, 513 [M − HexA − Hex + Na]+, 495 [M − HexA − Hex − H2O + Na]+, 452 ULO-A Hex-HexA-
2 46.4 851 828 675 [M − HexA + Na]+, 513 [M − HexA − Hex + Na]+, 495 [M − HexA − Hex − H2O + Na]+, 451 ULO-A HexA-Hex- c
3 46.6 985 962 967, 923, 853 [M − Pen + Na]+, 791, 497 [M − Aglycone + Na]+, 479 [M − Aglycone − H2O + Na]+ AG-1 Pen-Hex-Hex d
4 48.0 851 828 833, 801, 513 [M − HexA − Hex + Na]+, 495 [M − HexA − Hex − H2O + Na]+ ULO-A HexA-Hex-
+ + +
5 50.7 837 814 661 [M − HexA + Na] , 675 [M − Hex + Na] , 499 [M − HexA − Hex + Na] , 435 AG-2 HexA, Hex e
6 52.3 716 693 684, 513 [M − HexNAc + Na]+, 495 [M − HexNAc − H2O + Na]+, 451 ULO-A HexNAc-
7 53.0 819 796 687 [M − pen + Na]+, 525 [M − Pen − Hex + Na]+, 479 AG-3 Pen-Hex-R
905 [M − CO2 − H2O + Na]+, 835 [M − Pen + Na]+, 673 [M − Pen − Hex + Na]+,
8 53.6 967 944 + URA-B Pen-Hex-Hex d
497 [M − Aglycone + Na]
9 54.0 951 928 819 [M − Pen + Na]+, 497 [M − Aglycone + Na]+ AG-4 Pen-Hex-Hex d
10 56.1 979 956 497 [M − Aglycone + Na]+ AG-5 Pen-Hex-Hex d
821 [M − Pen + Na]+, 791 [M − Hex + Na]+, 497 [M − Aglycone + Na]+,
11 57.0 953 930 URA-A Pen-Hex-Hex d
479 [M − Aglycone − H2O + Na]+
875 [M − CO2 − H2O + Na]+, 805 [M − Pen + Na]+, 467 [M − Aglycone + Na]+,
12 57.6 937 914 URA-B Pen-Pen-Hex-
449 [M − Aglycone − H2O + Na]+
789 [M − Pen + Na]+, 657 [M − 2 Pen + Na]+, 467 [M − Aglycone + Na]+,
13 58.2 921 898 AG-4 Pen-Pen-Hex-
449 [M − Aglycone − H2O + Na]+
965 [M − Pen + Na]+, 921 [M − HexA + Na]+, 833 [M − 2 Pen + Na]+,
14 58.4 1097 1074 AG-4 Pen-Pen-HexA-Hex-
657 [M − 2 Pen − HexA + Na]+
965 [M − dHex + Na]+, 833 [M − dHex − Pen + Na]+, 657 [M − dHex − Pen − HexA + Na]+,
15 58.6 1111 1088 AG-4 dHex-HexA-Pen-Hex-
495 [M − dHex − Pen − HexA − Hex + Na]+
805 [M − Pen + Na]+, 511 [M − 2 Pen − Hex + Na]+, 493 [M − 2 Pen − Hex − H2O + Na]+,
16 59.7 937 914 URA-B Pen-Pen-Hex-
467 [M − Aglycone + Na]+, 431
17 60.1 965 942 833 [M − Pen + Na]+, 789 [M − Pen − COO + Na]+, 657 [M − Pen − HexA + Na]+ AG-4 Pen-HexA-Hex-
+ + +
18 60.3 849 826 805 [M − CO2 + Na] , 787 [M − COO − H2O + Na] , 449 [M − COO − HexA − Hex − H2O + Na] URA-B Hex-HexA f
19 61.6 803 780 641 [M − Hex + Na]+, 465 [M − Hex − HexA + Na]+ AG-6 Hex-HexA f
20 62.2 805 782 743, 673 [M − Pen + Na]+, 611 [M − HexA − H2O + Na]+ URA-A Pen-HexA e
spectively. Then, the fragmentation of the parent ions could also be initiated by the loss of th
lycone moiety, generating m/z 497 (C3) cations (Figure 3B).

Figure 3. Collision-induced fragmentation pattern for uraboside A (compound 11).

(A)
Mar. Drugs 2013, 11 4821

Figure 3. Cont.

(B)
 (B) compound 6 (ulososide D); (C) compound 8; (D) compound 12; (E) compound 16 and
(F) compound 18 (uraboside B).

Intens. 84. +MS3(851.8->675.7), 46.5-46.9min #1449-#1461

x105 Y1
0.8 675.3 -GlcA
-H2O -Glc
0.6
Z0 Y0
0.4 495.4 513.4

0.2
451.3
0.0
450 500 550 600 650 700 750 800 850 m/z

(A)
Mar. Drugs 2013, 11 4823

Figure 4. Cont.

Intens. 118. +MS2(716.8), 52.3-52.4min #1646-#1649

x105 Y0
513.3 -GlcNAc
-H2O
2
717.4

1 Z0

451.4 495.4
0
400 450 500 550 600 650 700 750 m/z

(B)
 400 450 500 550 600 650 700 750 m/z

(B)

Intens. 126. +MS2(968.0), 53.6-53.7min #1691-#1694

x105 -H2O C3
-Aglycone
B3 497.1
6 479.1

4 -Gal
-Ara
2 Y2 Y1
-44–18
673.3 835.3 905.4
0
400 500 600 700 800 900 1000 m/z

(C)
Mar. Drugs 2013, 11 4824

Figure 4. Cont.

Intens. 152. +MS2(937.9), 57.5-57.6min #1826-#1829

x107 C3
467.1
-Aglycone
1.0
0.8
0.6 -H2O
0.4
0.2 B3
449.1
0.0
400 500 600 700 800 900 1000 m/z

(D)
 0.2 3
449.1
0.0
400 500 600 700 800 900 1000 m/z

(D)

Intens. 165. +MS2(937.7), 59.5-59.9min #1895-#1910

x106 467.1C3
-Aglycone

1.00

0.75
Z0 937.5
0.50 493.1 -2Ara-Gal-H2O -Ara

0.25 Y1
Y0
431.1
511.1
-2Ara-Gal 805.4
0.00
500 600 700 800 900 1000 m/z

(E)
 Figure 4. Cont.

Intens. 169.
787.3 +MS2(849.8), 60.3-60.4min #1922-#1925
x105
-44–18
0.8

805.4
0.6 -44

0.4 449.3 851.5

-Gal-GlcA-H2O-COO
0.2 825.2

0.0
400 450 500 550 600 650 700 750 800 850 m/z

(F)
 Table 1. Cont.
21 62.7 803 780 641 [M − Hex + Na]+, 627 [M − HexA + Na]+, 465 [M − Hex − HexA + Na]+ AG-4 Hex-HexA f
22 63.5 805 782 643 [M − Hex + Na]+, 467 [M − Hex − HexA + Na]+ AG-7 Hex-HexA f
23 63.9 817 794 641 [M − HexA + Na]+, 479 [M − HexA − Hex + Na]+ AG-8 HexA-Hex c
24 64.5 761 738 629 [M − Pen + Na]+, 467 [M − Pen − Hex + Na]+ AG-7 Pen-Hex-
25 65.8 787 764 655 [M − Pen + Na]+, 479 [M − Pen − HexA + Na]+ AG-8 Pen-HexA-
a
Monosaccharides were identified by residues masses of 162 for hexoses (Hex), 176 for hexuronic acids (HexA), 132 for pentoses (Pen) and 203 for N-acetylhexosamines (HexNAc);
b
Aglycone cores are directly associated with the mass of our previous report as ULO-A: Aglycone from ulososide A (490 Da); URA-A: Aglycone from uraboside A (474 Da); URA-B:
Aglycone from uraboside B (488 Da); and other new aglycones with a specific m/z as AG-1 (506 Da), AG-2 (476 Da), AG-3 (502 Da), AG-4 (472 Da), AG-5 (500 Da), AG-6 (442 Da),
AG-7 (444 Da) and AG-8 (456 Da); c Glycan core could be the same for ulososide A such as O-[β-D-Glucuronopyranosyl-(1→6)-β-D-glucopyranoside]; d Glycan core could be the same for
e f
uraboside A such as O-[β-D-Arabinopyranosyl-(1′′→2′)-(β-D-galactopyranosyl-(1′′′→3′))-β-D-galactopyranoside]; Bidesmoside-like fragmentation; Glycan core could be the same for
uraboside B such as O-[β-D-Galactopyranosyl-(1→2)-β-D-glucopyranosiduronic acid].

Figure 2. Total Ion Chromatogram of saponins from E. ferox acquired by LC-ESI-MS (m/z: 400–1500 Da).
Mar. Drugs 2013, 11 4818

Table 1. Saponins found in Ectyoplasia ferox, with their LC/MS/MS retention times and fragment signatures.
Rt m/z
Cmp + MW Diagnostic ions (m/z) a Aglycone core b Glycan core
(min) [M + Na]
1 45.1 851 828 833, 513 [M − HexA − Hex + Na]+, 495 [M − HexA − Hex − H2O + Na]+, 452 ULO-A Hex-HexA-
2 46.4 851 828 675 [M − HexA + Na]+, 513 [M − HexA − Hex + Na]+, 495 [M − HexA − Hex − H2O + Na]+, 451 ULO-A HexA-Hex- c
3 46.6 985 962 967, 923, 853 [M − Pen + Na]+, 791, 497 [M − Aglycone + Na]+, 479 [M − Aglycone − H2O + Na]+ AG-1 Pen-Hex-Hex d
4 48.0 851 828 833, 801, 513 [M − HexA − Hex + Na]+, 495 [M − HexA − Hex − H2O + Na]+ ULO-A HexA-Hex-
+ + +
5 50.7 837 814 661 [M − HexA + Na] , 675 [M − Hex + Na] , 499 [M − HexA − Hex + Na] , 435 AG-2 HexA, Hex e
6 52.3 716 693 684, 513 [M − HexNAc + Na]+, 495 [M − HexNAc − H2O + Na]+, 451 ULO-A HexNAc-
7 53.0 819 796 687 [M − pen + Na]+, 525 [M − Pen − Hex + Na]+, 479 AG-3 Pen-Hex-R
905 [M − CO2 − H2O + Na]+, 835 [M − Pen + Na]+, 673 [M − Pen − Hex + Na]+,
8 53.6 967 944 + URA-B Pen-Hex-Hex d
497 [M − Aglycone + Na]
9 54.0 951 928 819 [M − Pen + Na]+, 497 [M − Aglycone + Na]+ AG-4 Pen-Hex-Hex d
10 56.1 979 956 497 [M − Aglycone + Na]+ AG-5 Pen-Hex-Hex d
821 [M − Pen + Na]+, 791 [M − Hex + Na]+, 497 [M − Aglycone + Na]+,
11 57.0 953 930 URA-A Pen-Hex-Hex d
479 [M − Aglycone − H2O + Na]+
875 [M − CO2 − H2O + Na]+, 805 [M − Pen + Na]+, 467 [M − Aglycone + Na]+,
12 57.6 937 914 URA-B Pen-Pen-Hex-
449 [M − Aglycone − H2O + Na]+
789 [M − Pen + Na]+, 657 [M − 2 Pen + Na]+, 467 [M − Aglycone + Na]+,
13 58.2 921 898 AG-4 Pen-Pen-Hex-
449 [M − Aglycone − H2O + Na]+
965 [M − Pen + Na]+, 921 [M − HexA + Na]+, 833 [M − 2 Pen + Na]+,
14 58.4 1097 1074 AG-4 Pen-Pen-HexA-Hex-
657 [M − 2 Pen − HexA + Na]+
965 [M − dHex + Na]+, 833 [M − dHex − Pen + Na]+, 657 [M − dHex − Pen − HexA + Na]+,
15 58.6 1111 1088 AG-4 dHex-HexA-Pen-Hex-
495 [M − dHex − Pen − HexA − Hex + Na]+
805 [M − Pen + Na]+, 511 [M − 2 Pen − Hex + Na]+, 493 [M − 2 Pen − Hex − H2O + Na]+,
16 59.7 937 914 URA-B Pen-Pen-Hex-
467 [M − Aglycone + Na]+, 431
17 60.1 965 942 833 [M − Pen + Na]+, 789 [M − Pen − COO + Na]+, 657 [M − Pen − HexA + Na]+ AG-4 Pen-HexA-Hex-
+ + +
18 60.3 849 826 805 [M − CO2 + Na] , 787 [M − COO − H2O + Na] , 449 [M − COO − HexA − Hex − H2O + Na] URA-B Hex-HexA f
19 61.6 803 780 641 [M − Hex + Na]+, 465 [M − Hex − HexA + Na]+ AG-6 Hex-HexA f
20 62.2 805 782 743, 673 [M − Pen + Na]+, 611 [M − HexA − H2O + Na]+ URA-A Pen-HexA e