Petrorhagiosides A - D, new γ-pyrone derivatives from Petrorhagia saxifraga link

Article abstract of DOI:10.1002/hlca.201200378

Four novel γ-pyrone (=4H-pyran-4-one) metabolites, petrorhagiosides A-D, along with four known analogs, have been isolated from the MeOH extract of Petrorhagia saxifraga, a perennial herbaceous plant typical of Mediterranean vegetation. The structures of the new compounds were established on the basis of extensive spectroscopic analyses including 1D- an 2D-NMR (¹H, ¹H-DQ-COSY, TOCSY, HSQC, CIGAR-HMBC, and HSQC-TOCSY) experiments. Copyright

Full text of DOI:10.1002/hlca.201200378

Helvetica Chimica Acta – Vol. 96 (2013)
1273
Petrorhagiosides A – D, New g-Pyrone Derivatives from Petrorhagia
saxifraga Link
by Brigida DꢀAbrosca, Severina Pacifico, Monica Scognamiglio, Nikolaos Tsafantakis,
Emanuela Pagliari, Pietro Monaco, and Antonio Fiorentino*
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies,
Phytochemistry Laboratory, Second University of Naples, via Vivaldi 43, I-81100 Caserta
(
phone: þ 39-0823-274576; fax: þ 39-0823-274571; e-mail: antonio.fiorentino@unina2.it)
Four novel g-pyrone (¼4H-pyran-4-one) metabolites, petrorhagiosides A – D, along with four
known analogs, have been isolated from the MeOH extract of Petrorhagia saxifraga, a perennial
herbaceous plant typical of Mediterranean vegetation. The structures of the new compounds were
1
1
established on the basis of extensive spectroscopic analyses including 1D- an 2D-NMR ( H, H-DQ-
COSY, TOCSY, HSQC, CIGAR-HMBC, and HSQC-TOCSY) experiments.
Introduction. – Petrorhagia is a small genus of annual and perennial plants,
belonging to the Caryophyllaceae family, mostly native to the Mediterranean region.
The flowers are pink, lilac, or white, and similar to those of the members of the genus
Dianthus.
To the best of our knowledge, the only phytochemical investigations of this genus
has been carried out on Petrorhagia velutina (Guss.) P.W.Ball. et Heywood. A series
of flavonoid C-glycosides from this plant has been reported, and their antiproliferative
activity against human hepatoblastoma cancer cell line HepG2 has been evaluated [1].
From the same species, a new pheophorbide, as well as other nine chlorophyll
derivatives, were isolated. A significant photoinduced phytotoxicity was ascribed to
these compounds [2]. Furthermore, a new dimeric p-coumaroyl glucose, and two
monomeric cis-p-coumaroyl and feruloyl derivatives with radical-scavenging properties
[
3], as well as a new drimane glycoside were reported [4].
This work deals with the phytochemical study of Petrorhagia saxifraga Link, known
as coat flower or saxifrage pink, an eurimediterranean perennial herbaceous species,
typical of woodland and macchia vegetation. The study led to the isolation and
structural characterization of four novel g-pyrone (¼4H-pyran-4-one) glucosides,
named petrorhagiosides A – D.
Results and Discussion. – The phytochemical investigation of the MeOH extract of
Petrorhagia saxifraga led to isolation and identification of eight g-pyrone derivatives,
four of them, 1 – 4, isolated and identified for the first time (Fig. 1).
Compound 1, named petrorhagioside A, had the molecular formula C H O , as
18
26 14
1
deduced from the NMR data and HR-ESI-mass spectrum. The H-NMR spectrum
Table 1) showed, in the downfield region, signals for two H-atoms (d(H) 8.10 (d,
HꢀC(6)) and 6.49 (d, HꢀC(5)); J ¼ 5.7). Moreover, two doublets at d(H) 4.87
(
ꢀ
2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 96 (2013)
Fig. 1. Structures of petrorhagiosides A – D (1 – 4, resp.) and compounds 5 – 8, isolated from P. saxifraga
Table 1. 1H-NMR Data of Petrorhagiosides A – D (1 – 4, resp.) in CD OD. d in ppm, J in Hz. Atom numbering as
3
indicated in Fig. 1.
Position
1
2
3
4
5
6
7
6.49 (d, J ¼ 5.7)
8.10 (d, J ¼ 5.7)
4.73 (d, J ¼ 13.5),
6.44 (d, J ¼ 4.9)
8.00 (d, J ¼ 4.9)
2.45 (s)
6.50 (d, J ¼ 5.4)
8.01 (d, J ¼ 5.4)
4.77 (d, J ¼ 14.1),
4.64 (d, J ¼ 14.1)
4.81 (d, J ¼ 7.2)
6.44 (d, J ¼ 4.8)
7.99 (d, J ¼ 4.8)
2.46 (s)
4.59 (d, J ¼ 13.5)
a
1’
2’
3’
4’
5’
6’
4.87 (d, J ¼ 7.2)
4.88 (d, J ¼ 7.5)
4.85 )
b
b
b
b
3.60 )
3.60 )
3.42 )
3.41 )
b
b
b
b
3.63 )
3.62 )
3.30 )
3.30 )
b
b
b
b
3.43 )
3.46 )
3.27 )
3.28 )
b
b
b
b
3.35 )
3.35 )
3.42 )
3.43 )
3.87 (dd, J ¼ 11.1, 2.2), 3.87 (dd, J ¼ 11.1, 2.1), 4.16 (d, J ¼ 11.1),
4.11 (d, J ¼ 11.4),
3
.66 (dd, J ¼ 11.1, 5.7)
3.66 (dd, J ¼ 11.1, 4.5)
4.59 (d, J ¼ 7.8)
3.75 (dd, J ¼ 11.1, 6.6) 3.75 (dd, J ¼ 11.4, 6.3)
1’’
2’’
3’’
4’’
5’’
6’’
4.60 (d, J ¼ 7.0)
4.30 (d, J ¼ 7.8)
4.30 (d, J ¼ 8.0)
b
3.27 (dd, J ¼ 9.3, 7.0)
3.24 )
3.15 (dd J ¼ 8.4, 8.1)
3.15 (dd, J ¼ 8.4, 8.1)
b
b
b
b
3.35 )
3.35 )
3.35 )
3.30 )
b
b
b
b
3.27 )
3.35 )
3.27 )
3.38 )
3.38 )
b
b
b
b
3.42 )
3.43 )
3.42 )
3.83 (dd, J ¼ 11.1, 2.2), 3.83 (dd, J ¼ 11.1, 2.2), 3.86 (d, J ¼ 11.7),
3.85 (d, J ¼ 11.1),
3.66 (dd, J ¼ 11.1, 5.7) 3.66 (dd, J ¼ 11.1, 4.5)
3.65 (dd, J ¼ 11.7, 4.2) 3.65 (dd, J ¼ 11.1, 4.4)
a) Obscured. b) Overlapped signals, reported without designated multiplicity.
(
HꢀC(1’)) and 4.60 (HꢀC(1’’)), and an AB system at d(H) 4.73, 4.59 (2d, J ¼ 13.5,
CH (7)), as well as additional H-atom signals between 3.9 and 3.2 ppm were evident.
The C-NMR spectrum (Table 2) showed 18 signals, identified on the basis of a DEPT
2
13
experiment, as those for three CH and twelve CH groups, and three quaternary C-
2
atoms (d(C) 177.5 (C(4)), 164.0 (C(2)), and 142.6 (C(3)).
The HSQC experiment showed correlations between the H-atoms at d(H) 8.10 (d,
HꢀC(6)) and 6.49 (d, HꢀC(5)) and the C-atoms at d(C) 157.6 (C(6)) and 117.7 (C(5)),
respectively. Furthermore, the AB-system H-atoms correlated with the CH C-atom at
2
d(C) 57.6 (C(7)), while the two doublets at d(H) 4.87 (d, HꢀC(1’)) and 4.60 (d,
HꢀC(1’’)) showed cross-peaks with the C-atoms at d(C) 104.0 (C(1’)) and 105.1

Helvetica Chimica Acta – Vol. 96 (2013)
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Table 2. 13C-NMR Data of Petrorhagiosides A – D (1 – 4) in CD OD. d in ppm. Atom numbering as
3
indicated in Fig. 1.
C-Atom
1
2
3
4
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
C(1’’)
C(2’’)
C(3’’)
C(4’’)
C(5’’)
C(6’’)
164.0
142.6
177.5
117.7
157.6
57.6
104.0
74.7
87.1
69.8
78.1
62.5
105.1
75.4
164.5
143.3
177.1
117.4
157.1
15.8
104.8
74.9
87.5
69.6
78.2
62.4
105.2
75.6
164.0
142.8
177.5
117.8
157.7
57.9
104.6
75.2
77.9
71.5
77.6
69.9
104.7
75.0
164.5
143.3
177.1
117.4
157.1
15.8
104.6
75.2
77.8
71.5
77.6
69.9
104.7
74.9
77.9
71.5
77.8
62.6
78.2
71.6
77.8
62.4
77.9
71.2
77.4
62.7
77.8
71.1
77.5
62.5
13
(
C(1’’)). These signals, as well as the remaining carbinol signals present in the C-NMR
spectrum, established the presence of two sugar moieties.
The CIGAR-HMBC experiment showed correlations between the doublet at d(H)
.10 (HꢀC(6)) and the C-atom signals at d(C) 177.5 (C(4)), 164.0 (C(2)), and 117.7
8
(C(5)); between the H-atom signal at d(H) 6.49 (HꢀC(5)) and the C-atom signals at
d(C) 177.5 (C(4)), 157.6 (C(6)), and 142.6 (C(3)); between the AB-system H-atom
signals and the C-atom signals at d(C) 164.0 (C(2)) and 142.6 (C(3)). These data were
in good agreement with the presence of a pyran-4-one structure with an CH OH group
2
at C(2) (d(C) 160.4) and an OH group at the C(3) (d(C) 142.6). Furthermore, this C-
atom showed correlations, in the HMBC experiment, with the anomeric H-atom (d(H)
4
.87 (HꢀC(1’))).
The C-atoms of both the monosaccharide units were identified on the basis of a
HSQC-TOCSY experiment (Fig. 2). In fact, the signal at d(H) 4.87 (HꢀC(1’))
correlated with five CH signals d(C) 104.0 (C(1’)) (direct correlation), 87.1 (C(3’)), 78.1
(
C(5’)), 74.7 (C(2’)), 69.8 (C(4’)), and with the CH signal at d(C) 62.5 (C(6’)); besides,
2
the signal at d(H) 4.60 (HꢀC(1’’)) correlated with the CH signals at d(C) 105.1 (C(1’’))
1
(
J correlation), 77.9 (C(3’’)), 77.8 (C(5’’)), 75.4 (C(2’’)), 71.5 (C(4’’)), and with the CH2
signal at d(C) 62.6 (C(6’’)) (Fig. 2). The GC/MS analysis of alditol acetate derivatives,
obtained by the hydrolysis of compound 1 with 2n CF COOH (TFA), reduction with
3
NaBH , and acetylation with Ac O in pyridine, suggested only the presence of glucose
4
2
in the molecule. The differences observed in the chemical shifts of the C-atoms
revealed the presence of a disaccharide moiety. The anomeric H-atom signal at d(H)
4
.60 (HꢀC(1’’)) correlated with the C-atom signal at d(C) 87.1 (C(3’)) in the CIGAR-
HMBC experiment. These data agreed with the presence of a glucose moiety bound to
a second sugar unit through its C(3). The coupling constant value (7.2 Hz) of the H-

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Helvetica Chimica Acta – Vol. 96 (2013)
Fig. 2. HSQC-TOCSY Spectrum of compound 1
atom with the signal at d(H) 4.87 (HꢀC(1’)) evidenced a b-configuration at the
anomeric C-atom. A similar value (7.0 Hz), obtained for the second anomeric H-atom
(d(H) 4.60), also evidenced a b-configuration for its anomeric C-atom. The absolute
configuration of glucose was determined by GC/MS after reaction of hydrolyzed
glucosides with l-cysteine methyl ester and acetylation. These data were in good
accordance with a b-d-glucopyranosyl-(1 ! 3)-b-d-glucopyranoside moiety in the
molecule. The remaining hetero- and homocorrelations detected in 2D experiments
confirmed this hypothesis and allowed the structure of 2-(hydroxymethyl)-4-oxo-4H-
pyran-3-yl 3-O-b-d-glucopyranosyl-b-d-glucopyranoside to be assigned to compound 1.
The enzymatic hydrolysis of 1 with b-glucosidase in acetate buffer at 378 afforded pure
aglycone 7 and glucose, confirming our hypothesis.
Compound 2, named petrorhagioside B, had the molecular formula C H O , as
18
26 13
1
deduced by the HR-ESI-MS and NMR data. The H-NMR spectrum showed two
doublets of an AM spin system at d(H) 8.00 (HꢀC(6)) and 6.44 (HꢀC(5)), two
anomeric doublets at d(H) 4.88 (HꢀC(1’)) and 4.59 (HꢀC(1’’)), besides further
overlapped signals with d values ranging from 3.9 to 3.2 ppm, and a Me singlet at d(H)
13
2
.45. The C-NMR spectrum showed 18 signals, identified by DEPT experiment, as
those of one Me, two CH , and twelve CH groups, and three quaternary C-atoms (d(C)
2
1
77.1 (C(4)), 164.5 (C(2)), and 143.3 (C(3)). The doublet at d(H) 8.00 (HꢀC(6))
correlated, in the CIGAR-HMBC experiment, with the C¼O C-atom signal at d(C)

Helvetica Chimica Acta – Vol. 96 (2013)
1277
1
6
77.1(C(4)), and with those at d(C) 164.5 (C(2)) and 117.4 (C(5)); the doublet at d(H)
.44 (HꢀC(5)) correlated with the signals at d(C) 177.1 (C(4)), 157.1 (C(6)), and 143.3
(C(3)); The Me singlet correlated with the C-atom signals at d(C) 164.5 (C(2)) and
143.3 (C(3)). These data indicated the presence of a 3-hydroxy-2-methylpyran-4-one
bound to a disaccharide. The GC/MS analysis of the alditol acetate derivatives obtained
by the hydrolysis of 2 afforded only glucose. The coupling constant of anomeric H-
1
13
atoms revealed the b-configuration of both glucose moieties. The H- and the C-NMR
data of the glycosidic portions were almost identical to those of compound 1, in
accordance with a b-d-glucopyranosyl-(1 ! 3)-b-d-glucopyranoside moiety in the
molecule. The structure of 2 was, therefore, established as 2-methyl-4-oxo-4H-pyran-3-
yl 3-O-b-d-glucopyranosyl-b-d-glucopyranoside.
Compound 3, named petrorhagioside C, had the molecular formula C H O ,
18
26 14
1
according to the NMR data and the HR-ESI-mass spectrum. The H-NMR spectrum
exhibited an AM spin system at d(H) 8.01 and 6.50, two anomeric H-atom signals at
d(H) 4.81 (HꢀC(1’)) and 4.30 (HꢀC(1’’)), an AB system at d(H) 4.77, 4.64 (2d,
CH (7)), signals of eight CH groups ranging from 3.15 to 3.44 ppm, and two
2
diastereotopic CH signals at d(C) 4.16, 3.75 (CH (6’)) and 3.86, 3.65 (CH (6’’)) bound
2
2
2
to the C-atoms with signals at d(C) 69.9 (C(6’)) and 62.7 (C(6’’)), respectively, as
13
evidenced in the HSQC experiment. The C-NMR spectrum showed 18 signals
assigned to three CH and 12 CH groups, and three quaternary C-atoms. The NMR
2
signals of the aglycone moiety were similar to those of compound 1. Differences were
ascribed to the saccharidic moiety. The GC analysis of the alditol acetate obtained from
compound 3, as described for the previous metabolites, indicated the exclusive
presence of glucose in the molecule. The downfield shifts of the CH signals at d(H)
2
4
.16 and 3.75 (HꢀC(6’)), as well as that of the C-atom at d(C) 69.9 (C(6’)) suggested the
presence of the disaccharide gentiobioside that was confirmed on the basis of
heterocorrelations, evidenced in the CIGAR-HMBC, between the HꢀC(1’’) signal at
d(H) 4.30 and C(6’). The disaccharide was bound to C(3) of the g-pyrone based on the
heterocorrelation, evidenced in the same experiment, between the anomeric H-atom
signal at d(H) 4.81 (HꢀC(1’)) and that of the C-atom at d(C) 142.8 (C(3)).
Compound 4, named petrorhagioside D, had the same molecular formula as 2 as
1
evidenced by the HR-ESI mass spectrum and the NMR data. Its H-NMR spectrum
showed four CH doublets at d(H) 7.99 (HꢀC(6)), 6.44 (HꢀC(5)), 4.85 (HꢀC(1’)), and
4
3
.30 (HꢀC(1’’)), a Me singlet at d(H) 2.46 and further twelve signals between 3.2 and
13
.9 ppm. The C-NMR and DEPT experiments indicated the presence of a Me, two
CH , and twelve CH groups, and three quaternary C-atoms. The NMR data of the
2
aglycone indicated the presence of a 3-hydroxy-2-methylpyran-4-one, while the 1D-
and 2D-NMR experiments were in good accordance with the presence of gentiobioside
in the molecule. The GC/MS analysis of alditol acetate derivatives of the sugars,
obtained after hydrolysis of 4, confirmed the presence of glucose as monosaccharide.
Spectroscopic data of the known metabolites 5 – 8 were identical to those reported
in the literature: glucoside 5 and its aglycone 7 have been reported as constituents of
Pteris inaequalis var. aequata (Miq.) Tagaw [5], while glucoside 6, known as
dianthoside, has been reported as constituent of Dianthus deltoides L. [6] and Derris
trifoliata Lour. [7]. Maltol (8) is a widely occurring metabolite [8] used as a flavor
enhancer [9]. This compound is also reported as complexant to heavy metal ions.

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Helvetica Chimica Acta – Vol. 96 (2013)
Maltol is reported to be an inhibitor of apoptosis of human neuroblastoma cells,
induced by oxidative damage generated by H O [10], and to be a strong enhancer of
2
2
aluminum accumulation in bone and brain [11].
M. S. acknowledges support by the LꢀOr e´ al UNESCO Program ꢁFor Women in Scienceꢀ through the
012 national fellowship ꢂLꢀOr e´ al Italia per le Donne e la Scienzaꢃ.
2
Experimental Part
General. TLC: Kieselgel 60 F254 (Merck), RP-18 F254 (Merck) or cellulose (Merck) plates with 0.2-,
.5-, or 1-mm film thickness. Column chromatography (CC): Kieselgel 60 (70 – 240 mesh, Merck),
0
Amberlite XAD-4 (Fluka), RP-18 (Baker), or Sephadex LH-20 (Pharmacia). Prep. HPLC: Knauer
Smartline 31/40 module on Luna NH (10 mm, 250 ꢁ 10.0 mm i.d., Phenomenex), equipped with Knauer
2
Smartline 1000 pump, UV Knauer Smartline 2500 and RI Knauer Smartline 2300 detectors, and PC
ꢄ
CromGate software. Optical rotation: Perkin-Elmer 343 polarimeter in MeOH. UV Spectra: UV-1700
Shimadzu spectrophotometer in MeOH; l
spectrometer at 300 ( H) and 75 MHz ( C), in CD OD solns., at 258; d in ppm rel. to Me Si as internal
(log e) in nm. NMR Spectra: Varian 300 FT-NMR
max
1
13
3
4
standard, J in Hz. 1H-Detected heteronuclear correlations were measured by gradient Heteronuclear
1
Single-Quantum Coherence (HSQC) optimized for J(H,C) ¼ 140 Hz and by Constant time Inverse-
detection Gradient Accordion Rescaled Heteronuclear Multiple Bond Correlation spectroscopy
n
13
(
3
CIGAR-HMBC) spectra (8 < J(H,C) < 5) with the spectral width of 18000 Hz in f1 ( C) and
1
000 Hz in f 2 ( H). GC/MS: HP 6890 GC instrument equipped with a 5975B VL MSD detector. HR-
ESI-MS: Electrospray hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-
TOF) fitted with a Z-spray electrospray ion source (Waters S.p.A., Manchester, UK); in m/z.
Plant Material. Plants of Petrorhagia saxifraga Link (Caryophyllaceae) were collected in June 2009
in Castel Volturno, near Caserta (Campania, Italy), and identified by Dr. Assunta Esposito at the
Department of Scienze della Vita of Second University of Naples (SUN). Avoucher specimen (CE0112)
has been deposited with the Herbarium of the Dipartimento di Scienze della Vita of SUN.
Extraction and Isolation. Dried whole plants (370 g) were extracted with MeOH (5.0 l) for 5 d in the
dark at 48, to obtain 25.5 g of residual material. The MeOH extract was dissolved in dist. H O (2.0 l) and
2
shaken with AcOEt (2.0 l); the aq. fraction was chromatographed on Amberlite XAD-4, eluting with
H O first, to eliminate sugars, peptides, free amino acids, and other small primary metabolites, and then
2
with MeOH. The alcoholic eluate furnished 6.4 g of residual material which was chromatographed on
Sephadex LH-20 (2.5 ꢁ 120 cm) eluting with H O and collecting 20-ml fractions. Frs. 1 – 15 (316 mg)
2
purified by TLC on SiO , eluting with the org. phase of the biphasic soln. constituted by CHCl /MeOH/
2
3
H O 13 :7:2, furnished pure compound 3 (36 mg); Fr. 16 (77 mg) was re-chromatographed (TLC on
2
cellulose) eluting with the upper phase of the biphasic soln. constituted by CHCl /MeOH/H O 13 :7:2, to
3
2
give pure compound 2 (8 mg) and a the second spot which was purified by HPLC (NH , MeCN/MeOH/
2
H O 7:2 :1) to yield compound 4 (49 mg). Frs. 17 – 19 (350 mg) were subjected CC (RP-18; MeOH/H O
2
2
7
:3) to give a fraction of 20-ml volume; Fr. 10 was purified by HPLC (NH , MeCN/MeOH/H O 7:2 :1)
2
2
to give compounds 1 (38 mg) and 3 (36 mg). Fr. 20 (60 mg) purified by flash CC (SiO ; the org. phase of
2
the biphasic soln. constituted by CHCl /MeOH/H O 13 :7:2) furnished pure compounds 5 (124 mg) and
3
2
6
(165 mg). Fr. 26 (51.7 mg) re-chromatographed (TLC on SiO ), eluting with the org. phase of the
2
biphasic soln. constituted by CHCl /MeOH/H O 13 :7:3, gave pure compounds 7 (15 mg) and 8 (10 mg).
3
2
Petrorhagioside A (¼2-(Hydroxymethyl)-4-oxo-4H-pyran-3-yl 3-O-b-d-Glucopyranosyl-b-d-gluco-
2
5
pyranoside; 1). White amorphous powder. [a] ¼ ꢀ13.0 (c ¼ 0.10, MeOH). UV (MeOH): 258 (3.85),
D
1
13
þ
þ
2
02 (4.07). H- and C-NMR: Tables 1 and 2, resp. HR-ESI-MS: 489.3792 ([M þ Na] , C H NaO14
;
18
26
calc. 489.3804).
Petrorhagioside B (¼2-Methyl-4-oxo-4H-pyran-3-yl 3-O-b-d-Glucopyranosyl-b-d-glucopyranoside;
2
5
1
2
). White amorphous powder. [a]
D
¼ ꢀ8.9 (c ¼ 0.13, MeOH). UV (MeOH): 256 (3.47), 202 (3.76). H-
13
þ
þ
and C-NMR: Tables 1 and 2, resp. HR-ESI-MS: 473.3878 ([M þ Na] , C H NaO ; calc. 473.3810).
18
26
13

Helvetica Chimica Acta – Vol. 96 (2013)
1279
Petrorhagioside C (¼2-(Hydroxymethyl)-4-oxo-4H-pyran-3-yl 6-O-b-d-Glucopyranosyl-b-d-gluco-
2
5
pyranoside; 3). White amorphous powder. [a] ¼ ꢀ73.5 (c ¼ 0.20, MeOH). UV (MeOH): 258 (4.06),
D
1
13
þ
þ
2
02 (4.36). H- and C-NMR: Tables 1 and 2, resp. HR-ESI-MS: 489.3802 ([M þ Na] , C H NaO
;
18
26
14
calc. 489.3804).
Petrorhagioside D (¼2-Methyl-4-oxo-4H-pyran-3-yl 6-O-b-d-Glucopyranosyl-b-d-glucopyranoside;
2
5
1
4
). White amorphous powder. [a] ¼ ꢀ12.0 (c ¼ 0.02, MeOH). UV (MeOH): 256 (4.14), 202 (4.27). H-
D
1
3
þ
þ
13
and C-NMR: Tables 1 and 2, resp. HR-ESI-MS: 473.3996 ([M þ Na] , C H NaO ; calc. 473.3810).
18
26
Identification of the Sugar Moieties. Compounds 1 – 4 (0.5 mg) were subjected to acid hydrolysis with
n TFA (150 ml). The hydrolysis reaction was conducted at 1208 for 1 h. The mixture was then dried after
2
i
PrOH addition. The sugars were identified by GC analysis of their alditol acetate derivatives and
comparison with eight reference monosaccharides treated under the same conditions. The reduction of
the sugars was achieved by dissolving the sample in MeOH (150 ml) and by adding NaBH (1.0 mg). The
4
soln. was incubated at r.t. for 1 h and then dried under N flow after treatment with glacial AcOH and
2
MeOH. Finally, the obtained alditol was acetylated, in anh. pyridine (200 ml), by addition of Ac O
2
(
200 ml). After 20 min at 1208, 500 ml of H O were added, the product was extracted with CH Cl (500 ml)
2 2 2
in centrifuge (3500 rpm for 5 min), and the org. phase was analyzed by GC/MS.
Enzymatic Hydrolyses of 1 – 4. To a soln. of the pure compound (2 mg) in acetate buffer (0.5m,
pH 5.0, 5 ml), 40 mg of b-glucosidase (b-glucosidase from almonds, Sigma EC 3.2.1.21) were added.
After stirring for 24 h at 378, the mixture was extracted with AcOEt (5 ml ꢁ 2), dried (Na SO ),
2
4
and evaporated in vacuo. The residue was subjected to TLC for aglycone identification. After concen-
tration of the aq. layer, the sugar residue was identified by RI-HPLC by comparison with authentic
standards.
Determination of Absolute Configuration of Monosaccharides of 1 – 4. Compounds 1 – 4 (2 mg each)
were hydrolyzed with 2n TFA (250 ml) at 1208 for 1 h. The mixture was then dried under N flow, and the
2
residue was dissolved in dry pyridine (100 ml). To pyridine solns. of the hydrolysis products 1 – 4, d-
glucose, l-glucose (0.04 mol/l), and 100 ml of pyridine soln. of l-cysteine methyl ester hydrochloride
(
0.06 mol/l) were added, and the mixtures were warmed at 608 for 1 h. Afterwards, Ac O (150 ml) was
2
added, and the mixtures were kept at 1208 for 20 min, then dried under N , redissolved in 500 ml of H O,
2
2
and extracted with CH Cl (500 ml) in a centrifuge (3500 rpm for 5 min). The org. phase was dried under
2
2
N2 flow, dissolved in CH Cl (500 ml), and analyzed by GC/MS. Temp. conditions were as follows:
2
2
injector port, 2508; initial oven temp., 458, then increased linearly to 3008 at 208/min, and held for 25 min.
Sample solns. were injected using the split mode. The retention times were: d-glucose, 14.97 min; and l-
glucose, 15.22 min [12].
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Received July 19, 2012