New pulvinic acid and phenylalaninol derivatives from the mushrooms Retiboletus griseus and R. nigerrimus

Article abstract of DOI:10.5560/ZNB.2013-3050

The close genetic relationship of the mushrooms Retiboletus griseus and R. nigerrimus is supported by the occurrence of the orange-yellow retiboletic acid, resp. its methyl ester, in both species. The structures of these highly substituted pulvinic acid derivatives have been elucidated by spectroscopic investigations. R. nigerrimus also contains two N-protocatechuyl-L- phenylalaninol derivatives, nigerrimin A and B, the first reported occurrence of such compounds in nature. Their configuration was established by the synthesis of nigerrimin B.

Full text of DOI:10.5560/ZNB.2013-3050

New Pulvinic Acid and Phenylalaninol Derivatives from the Mushrooms
Retiboletus griseus and R. nigerrimus
Gertraud Grubera, Lydia Kerschensteinera, Ryuji Marumotob†, and Wolfgang Steglicha
a
Department Chemie und Biochemie, Ludwig-Maximilians-Universita¨t, Butenandtstr. 5 – 13,
81377 Mu¨nchen, Germany
Rokko Mycological Laboratory, 53-1, Okuike-minamimachi, Ashiya, Hyogo 659, Japan
b
Reprint requests to Prof. Dr. Wolfgang Steglich. Fax: + 49-89-2180-77756.
E-mail: wos@cup.uni-muenchen.de
Z. Naturforsch. 2013, 68b, 675 – 682 / DOI: 10.5560/ZNB.2013-3050
Received February 14, 2013
Dedicated to Professor Heinrich No¨th on the occasion of his 85th birthday
The close genetic relationship of the mushrooms Retiboletus griseus and R. nigerrimus is supported
by the occurrence of the orange-yellow retiboletic acid, resp. its methyl ester, in both species. The
structures of these highly substituted pulvinic acid derivatives have been elucidated by spectroscopic
investigations. R. nigerrimus also contains two N-protocatechuyl-L-phenylalaninol derivatives, niger-
rimin A and B, the first reported occurrence of such compounds in nature. Their configuration was
established by the synthesis of nigerrimin B.
Key words: Pulvinic Acids, N-(Protocatechuyl)phenylalaninols, Fungi, Taxonomy, Synthesis
Introduction
the fruit body is handled. In an early investigation of
this species, none of the known pulvinic acids could be
Based on DNA sequence data and morphological detected [3]. We now report on the isolation and struc-
criteria, Binder and Bresinsky [1] proposed the new tural elucidation of a penta oxygen-substituted pulvinic
bolete genus Retiboletus, which includes R. retipes, acid, named retiboletic acid (1), from the acetone ex-
R. ornatipes, R. flavoniger, R. griseus, and R. niger- tract of dried fruit bodies. The acid is accompanied by
rimus. Whereas the first three species are characterized its methyl ester 2.
by the occurrence of retipolides [2], the metabolites of
The air-dried mushrooms were powdered and ex-
R. griseus and R. nigerrimus remained unknown. We tracted with acetone-water. The crude extract was
have now carried out a chemotaxonomic survey of the then distributed between dilute HCl and EtOAc, and
latter two species, which resulted in the detection of the dried and concentrated organic phase was inves-
a new pulvinic acid in these fungi, accompanied by two tigated by reversed-phase HPLC. This revealed the
phenylalaninol derivatives in R. nigerrimus.
presence of two orange-yellow pigments, which could
be obtained analytically pure by preparative HPLC.
Their absorption spectra strongly resembled those of
hydroxypulvinic acids, the lead pigments of Boletus
species [4]. On TLC comparison, the more polar pig-
ment exhibited nearly the same Rf values as varie-
gatic acid (3); however, on oxidation with aqueous
Results and Discussion
Isolation and structure elucidation of retiboletic acid
(1) and methyl retiboletate (2)
Retiboletus griseus (Frost) Manfr. Binder & Bresin- NaHCO3/K3[(CN)6], no blue color developed. This
sky is a fairly common mushroom of oak-hickory excluded a catechol moiety as ring A [5].
forests in eastern North America. It is easily recog-
The (–)-ESI mass spectrum of retiboletic acid
nized by its grey appearance and the reticulate stem showed a molecular ion at m/z = 401 [M–H]− cor-
with a yellowish to brown pattern that darkens when responding to the formula C19H13O10. Thus, the new
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G. Gruber et al. · New Pulvinic Acid and Phenylalaninol Derivatives from Retiboletus griseus and R. nigerrimus
5
HO
HO
RO
6
O
1"
O
O
A
2
HO
2"
3"
H
3CO
O
4"
HO
H
2'
B
OH
OH
OH
OH
RO
2C
O2C
5"
6" 6'
OH
1, R = H, retiboletic acid
3, R = H, variegatic acid
2, R = CH3, methyl retiboletate
4, R = CH3, 3-O-methylvariegatic acid
OH
2
HO
HO
HO
HO
RO
6
6
O
O
O
A
2
O
O
O
HO
2'
B
HO
HO
2'
OH
OH
OH
OH
HO2C
HO2C
H3CO2C
6'
6'
OH
OH
5, isogomphidic acid
6, gomphidic acid
7, R = H
8, R = CH3
compound contained one oxygen atom more than var- ment, whereas the corresponding signal for gomphidic
iegatic acid (3) and, in addition, a phenolic O-methyl acid (6) occurs at δH = 7.35 [6, 8]. Structure 1 was con-
group, which appeared as a 3H singlet in the 1H NMR firmed by the 13C NMR spectrum and HMBC experi-
spectrum (δH = 3.84). Four signals in the aromatic ments.
proton region were visible, which could be assigned
The second pigment closely resembled retiboletic
to a 4-hydroxy-3-methoxyphenyl group [ABX system: acid (1) in its H and 13C NMR data, with the ex-
δH = 6.80 (d, J = 8.3 Hz, 5-H), 7.79 (dd, J = 8.3, ception of signals for an additional O-methyl group at
1.9 Hz, 6-H), 8.00 (d, J = 1.9 Hz, 2-H)] and a 3,4,5- δH = 3.90 (δC = 54.1) which exhibited an HMBC cor-
trihydroxyphenyl residue: δH = 6.38 (s, 20/60-H). The relation to the carbonyl group at δC = 172.3. Acetyla-
position of the O-methyl group followed from its corre- tion (Ac2O/cat. DMAP) yielded a tetra acetate; thus,
lation with 2-H in a NOESY experiment and an HMBC the second pigment is methyl retiboletate (2), which is
1
correlation with C-3 (δC = 147.2).
supported by the molecular ion at m/z = 415 [M–H]−
Due to the strong deshielding effect of the copla- observed in the (–)-ESI mass spectrum. Since work-
nar hydroxybutenolide ring, protons 2 and 6 at ring up and purification were carried out in the absence of
A in pulvinic acids generally appear at distinctly lower methanol, the pigment must be a genuine metabolite.
field than their counterparts 20-H and 60-H in ring B,
Retiboletus nigerrimus (R. Heim) M. Binder &
which is twisted by steric interactions with the neigh- Bresinsky [ = Tylopilus nigerrimus (R. Heim) Hongo
boring carboxyl group [6]. This effect allowed the al- & M. Endo] is a blackish bolete with a purple tint
location of the two phenyl rings in retiboletic acid as and reticulated stipe, which occurs in evergreen, broad-
given in structure 1. Comparison with the correspond- leaved forests in Papua New Guinea, Malaysia, Thai-
ing chemical shifts for 3-O-methylvariegatic acid (4) land, and East Asia. Extracts of dried specimens from
(2-H: δH = 7.84, 6-H: 7.72) [7] and isogomphidic acid Japan contained methyl retiboletate (2) in admixture
(5) (20/60-H: δH = 6.41) [8, 9] showed close agree- with methyl variegatate (7) [7, 10] and methyl 3-O-
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G. Gruber et al. · New Pulvinic Acid and Phenylalaninol Derivatives from Retiboletus griseus and R. nigerrimus
677
5'
an amide moiety (δNH = 7.67, d, J = 8.2 Hz), which is
connected to a methine proton (δH = 3.98, m). 1H,1H-
COSY experiments revealed the coupling of this pro-
ton to a neighboring CH2OH residue (δH = 3.32/3.40,
each dd, J = 10.8/6.1 and 10.8/5.4 Hz, δC = 62.9)
R
R
6'
3
2
O
2"
5"
H 2'
CH2OH
1
HO
HO
N
H
6"
and, in addition, a second methylene group (δH
=
2.57/2.69, each dd, J = 13.9/8.2 and 13.9/5.9 Hz,
δC = 36.1) which connects to the 3,4-dihydroxyphenyl
ring. From this evidence, structure 10 could be pro-
posed for nigerrimin B. It was supported by the 13C
NMR data and HMBC experiments (Fig. 1).
9, R = H, nigerrimin A
10, R = OH, nigerrimin B
In the case of nigerrimin A, the NMR data indi-
cated the same basic structure, however, the signals of
the dihydroxyphenyl group were replaced by those of
a simple phenyl residue. Nigerrimin A is therefore N-
(protocatechuyl)phenylalaninol (9). In order to estab-
lish the absolute configuration of the nigerrimins, the
synthesis of L-nigerrimin B was undertaken.
OH
OH
O
H
HO
HO
N
H
CH2OH
Fig. 1. Selected HMBC correlations of 10.
Synthesis of nigerrimin B
methylvariegatate (8), a known metabolite from Hy-
grophoropsis aurantiaca [7].
The advanced synthesis commenced from 3,4-
The isolation of the highly substituted retiboletic diisopropoxybenzoic acid (13) and O,O-diisopropyl-
acid (1) and its methyl ester (2) from Retiboletus protected L-DOPA methyl ester (16), which were pre-
griseus and ester 2 from R. nigerrimus strongly sup- pared in high yield from ethyl 3,4-dihydroxybenzoate
ports their transfer from Tylopilus to another genus (11) and N-Boc-L-DOPA methyl ester (14), respec-
within the Boletales. The supposed position within tively (Scheme 1). Coupling of compounds 13 and 16
Retiboletus on behalf of molecular data [1] remains using TBTU [11] yielded amide 17, which was re-
to be proved. During our investigations of these two duced with NaBH4 in THF-MeOH [12, 13] to the cor-
species, we were unable to detect any retipolides, the responding alcohol 18. Treatment of the latter with
characteristic metabolites of R. retipes/ornatipes and AlCl3 afforded tetraphenol 10, in every respect iden-
R. flavoniger [2].
tical with L-nigerrimin B. The synthetic compound
showed a similar negative optical rotation ([α]D25
=
Isolation and structure elucidation of nigerrimins
A and B
−53.6) as the natural product ([α]2D5 = −52.2), and
there was close agreement of their CD spectra.
The nigerrimins 9 and 10 are new compounds.
In addition to the pulvinic acids, two colorless In contrast to L-phenylalaninol, there are no reports
metabolites, nigerrimins A and B, were isolated from that 3-(3,4-dihydroxyphenyl)alaninol and its deriva-
R. nigerrimus. Nigerrimin A exhibited a molecular tives have been detected in nature before. Simple N-
peak at m/z = 287 in the EI mass spectrum, corre- benzoyl-L-phenylalaninol has been isolated from cul-
sponding to the formula C16H17NO4, whereas niger- tures of Aspergillus flavipes [14] and Penicillium bre-
rimin B (C16H17NO6) contained two additional oxy- vicompactum [15], as well as from higher plants. Since
gen atoms. Both compounds showed the base peak aqueous solutions of the nigerrimins readily darken on
at m/z = 137 (C7H5O3) and an ion at m/z = 109 exposure to air, the blackish appearance of R. niger-
(C6H5O2), indicating the presence of a dihydroxyben- rimus may be caused by the presence of these com-
1
zoyl residue. In the H NMR spectrum of nigerrimin pounds or of L-DOPA in the fruit bodies. The bio-
B, signals for the 3,4-dihydroxybenzoyl and an addi- logical activity of the polyphenolic compounds 9 and
tional 3,4-dihydroxyphenyl residue were visible. The 10 is unknown and remains to be investigated. In-
carbonyl group of the former (δC = 166.0) is part of terestingly, phenylalanine and 3,4-dihydroxybenzoic
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678
G. Gruber et al. · New Pulvinic Acid and Phenylalaninol Derivatives from Retiboletus griseus and R. nigerrimus
O
O
O
H
H
O
iPrO
iPrO
iPrO
iPrO
a
b
OEt
OH
OEt
93 %
98 %
O
11
14
12
13
OiPr
H
H
O
OiPr
OiPr
c
d
OiPr
O
H
H
97 %
H
CO2
85 %
x H N
Me
CO2
BocHN
Me
CO2
HCl
BocHN
Me
2
15
16
OiPr
OiPr
OiPr
OiPr
O
O
H
CO2
H
C
e
54 %
f
iPrO
iPrO
iPrO
iPrO
+
13 16
N
H
Me
N
H
H
2O
H
73 %
18
10
17
g, 91 %
Scheme 1. Synthesis of L-nigerrimin B (10). Reagents and conditions: a) iPrBr, K2CO3, DMF, 60 ◦C, 3 h; then 20 ◦C, 12 h;
b) NaOH, EtOH-H2O, 95 ◦C, 2 h; c) iPrBr , K2CO3,DMF, 50 ◦C, 12 h; d) 2 N HCl in EtOAc, 20 ◦C, 16 h; e) TBTU, NEt3,
MeCN, 20 ◦C, 36 h; f) NaBH4, THF, MeOH, 20 ◦C, 48 h; g) AlCl3, CH2Cl2, 20 ◦C, 48 h.
acid could be detected by GC-MS after trimethylsi- performed under reduced pressure using a rotary evapo-
rator. Analytical TLC: silica gel 60 F254 aluminum foils
(Merck); solvent system A (v/v): toluene-HCO2Et-HCO2H
(10 : 5 : 3); B: toluene-HCO2Et-HCO2H (10 : 10 : 3); C:
hexanes-EtOAc (1 : 2). Column chromatography: silica gel
60, 40 – 63 µm (Merck), Sephadex LH-20 (Pharmacia). An-
alytical and semipreparative HPLC: Waters 510 pump and
autosampler 717+ with photodiode array detector 996, col-
umn heating compartment Techlab K5 and Millenium chro-
matography software; Waters 600 E pump and system
controller with photodiode array detector 990+. Prepara-
tive HPLC separations: Waters-Millipore with gradient con-
troller M 680, two M 590 EF pumps, and U 6 K injector
equipped with a Knauer variable-wavelength monitor with
a super-preparative flow cell; prefiltration of the solutions
through Sep-Pak RP-18 cartridges (Waters). Nucleosil 100
C18 prepacked HPLC columns (Macherey-Nagel) and gra-
dient systems with MeCN-H2O mixtures with up to 0.5%
TFA were used. TBTU [O-(benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium tetrafluoroborate] was purchased from
Sigma-Aldrich.
lylation of the mushroom extract with N-methyl-
N-(trimethylsilyl)trifluoroacetamide (MSTFA). This
is in accord with the known biosynthesis of L-
phenylalaninol from L-phenylalanine in cultures of
Penicillium brevicompactum [15].
In summary, our investigation of Retiboletus griseus
and R. nigerrimus has added four new members to the
list of unique metabolites from this genus [2], which
underlines the special taxonomic position of Retibole-
tus in Boletaceae [1].
Experimental Section
General
Melting points (uncorrected): Reichert Thermovar hot-
stage apparatus. Optical rotations: Perkin-Elmer 241 po-
larimeter. IR: Perkin-Elmer 1420 ratio recording infrared
spectrometer or Bruker FTIR IFS 45 spectrophotometer.
Intensity of the bands: ss (very strong), s (strong), m
(medium), w (weak), sh (shoulder). UV/Vis spectra: Hewlett-
Packard 8452A diode array spectrophotometer. CD spec-
tra: Jobin Yvon CD-6-Dichrograph. NMR: Bruker AMX
Mushrooms
R. griseus was collected in September 1998 in the Lincoln
600, ARX 300, and AC 200 instruments; spectra were Town Forrest, Lincoln/Maynard, and in the Breakhart Reser-
recorded in CDCl3, [D6]acetone, or [D6]DMSO, with the vation, Saugus/Wakefield, both MA (USA) (leg. et det. N.
solvent peak as internal standard. MS: Finnigan MAT 90 Arnold); R. nigerrimus was collected in July and September
and 95 Q spectrometers (direct inlet, 70 eV). All solvents 1996 near Takarazuka/Nakayama, Hyogo (Japan) (leg. et det.
were distilled before use. Evaporation of the solvents was R. Marumoto).
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G. Gruber et al. · New Pulvinic Acid and Phenylalaninol Derivatives from Retiboletus griseus and R. nigerrimus
679
Isolation of retiboletic acid (1) and its methyl ester 2 from
R. griseus
(logε) = 212 (4.24), 258 (4.02), 337 (3.93), 385 (3.74, sh)
nm. – 1H NMR (600 MHz, [D6]acetone): δ = 3.88 (s, 3 H,
3-OMe), 3.90 (s, 3 H, CO2CH3), 6.45 (s, 2 H, 20/60-H), 6.91
(d, J = 8.3 Hz, 1 H, 5-H), 7.65 (dd, J = 8.3, 1.9 Hz, 1 H,
6-H), 7.76 (d, J = 1.9 Hz, 1 H, 2-H). – 13C NMR (150 MHz,
[D6]acetone): δ = 54.1 (CO2CH3), 55.7 (3-OCH3), 104.7 (C-
200), 109.9 (C-20/60), 111.4 (C-2), 115.3 (C-5), 116.4 (C-500),
121.5 (C-6), 121.8 (C-1), 123.7 (C-10), 133.7 (C-40), 145.5 (C-
30/50), 147.3 (C-4), 147.6 (C-3), 153.5 (C-400), 159.1 (C-300),
166.4 (C-100), 172.3 (C-600). – MS ((–)-ESI): m/z (%) = 415
(49) [M–H]−. – HRMS ((–)-ESI): m/z = 415.0633 (calcd.
415.0665 for C20H15O10, [M–H]−).
Air-dried, pulverized fruit bodies of R. griseus (20.9 g)
were extracted with acetone-water (20 : 1, v/v, 4 × 900 mL).
The combined extracts were concentrated at 40 ◦C under
reduced pressure, and the brown residue (1.35 g) was dis-
solved in water with the addition of a few drops of concd.
HCl. After extraction of the aqueous solution with EtOAc
(4 × 200 mL), the combined extracts were dried (Na2SO4)
and concentrated. The yellow-brown residue (0.45 g) was
dissolved in a small amount of MeCN-water (1 : 1, v/v),
the solution was filtered through an RP-18 cartridge, and
the pigments were separated by preparative HPLC [Nucleosil
100 C18, 7 µm, 250 × 16 mm; solvent A: H2O-MeCN (9 : 1)
+ 0.5% TFA; solvent B: MeCN; gradient: start 90% A +
10% B, 40 min: 50% A + 50% B, 45 min: 100% B, flow
rate: 6.8 mL min−1, detection at 265 nm). Retention times:
tR (1) = 15.3, tR (2) = 33.4 min. The analytically pure com-
pounds were obtained by semipreparative HPLC [Nucleosil
100 C18, 5 µm, 250 × 10 mm; solvent A: H2O-MeCN (9 : 1)
+ 0.5% TFA; solvent B: MeCN; gradient: start 90% A +
10% B, 40 min: 60% A + 40% B, 45 min: 100% B, flow
rate: 3.0 mL min−1, detection at 265 nm]. Retention times:
tR (1) = 25.4, tR (2) = 24.6 min.
The tetra acetate was formed by stirring ester 2 (0.8 mg)
and a catalytic amount of DMAP in Ac2O (1.5 mL) overnight
at r. t. After addition of a few drops of water, the mixture
was extracted with Et2O, the solvent was removed under re-
duced pressure, and the tetra acetate was dried in vacuo. MS
((–)-ESI): m/z (%) = 583 (79) [M–H]−, 499 (100). – HRMS
((–)-ESI): m/z = 583.1078 (calcd. 583.1088 for C28H23O14
,
[M–H]−).
Isolation of the pulvinic acid derivatives and nigerrimins
from R. nigerrimus
Freeze-dried, pulverized fruit bodies of R. nigerrimus
(68 g) were defatted with petroleum ether (40–60 ◦C) and
extracted with acetone (2 × 600 mL) under an argon at-
mosphere. Concentration of the extracts yielded 2.49 g of
an orange-brown residue. The mushrooms were then ex-
tracted with MeOH (3 × 600 mL), and the combined extracts
were concentrated to yield an orange-brown residue (4.91 g),
which was equilibrated between EtOAc and acidified wa-
ter (HCl, pH = 5). The aqueous phase was extracted with
EtOAc (5 ×); the dark-brown color of the aqueous phase re-
mained. The organic phases were combined, dried (Na2SO4),
and concentrated to yield an orange-brown solid (1.55 g),
which was indistinguishable from the acetone extract by TLC
(solvent system A). The crude extracts were combined, dis-
solved in a small amount of MeOH, and chromatographed on
Sephadex LH-20 with acetone-MeOH (4 : 1). The first frac-
tion contained blue fluorescent compounds, which slowly
turned brown on standing on the column, and the second
fraction contained orange pigments. The last, dark brown,
fraction contained only decomposition products formed dur-
ing the chromatography. From the first two fractions, the
nigerrimins 9 and 10, methyl 3-O-methylvariegatate (8), and
a 1 : 1-mixture of methyl retiboletate (2) and methyl var-
iegatate (7) could be isolated in pure form by preparative
HPLC (Nucleosil 100 C18, 7 µm, 250 × 16 mm; solvent A:
Retiboletic acid, 30,4,40,50-tetrahydroxy-3-methoxypulvinic
acid (1)
3.6 mg (0.017% of dry weight), yellow-brown solid,
m. p. 105 – 110 ◦C. – Rf (TLC) = 0.23 (solvent system A),
orange spot, + K3Fe[(CN)6] red-brown. – tR (analytical
HPLC) = 21.1 min (Nucleosil 100 C18, 5 µm, 250 × 4 mm;
solvent A: H2O-MeCN (9 : 1) + 0.5% TFA; solvent B:
MeCN; gradient: start 100% A, 10 min: 100% A, 50 min:
100% B, 60 min: 100% B, flow rate: 1.0 mL min−1, de-
tection at 265 nm). – UV/Vis (MeOH): λmax (logε) = 212
(3.97), 263 (3.73), 340 (3.28, sh), 394 (3.37) nm. – 1H NMR
(600 MHz, [D6]acetone): δ = 3.84 (s, 3 H), 6.38 (s, 2 H,
20/60-H), 6.80 (d, J = 8.3 Hz, 1 H, 5-H), 7.79 (dd, J = 8.3,
1.9 Hz, 1 H, 6-H), 8.00 (d, J = 1.9 Hz, 1 H, 2-H). – 13C NMR
(150 MHz, [D6]acetone): δ = 55.7 (CH3), 97.8 (C-200), 110.3
(C-2/20/60), 114.7 (C-5), 118.3 (C-500), 119.9 (C-6), 125.0
(C-1), 127.0 (C-10), 132.5 (C-40), 144.8 (C-30/50), 145.0 (C-
4), 147.2 (C-3), 153.3 (C-400), 167.8 (C-300), 168.9 (C-100),
170.0 (C-600). – MS ((–)-ESI): m/z (%) = 401 (33) [M–H]−.
– HRMS ((–)-ESI): m/z = 401.0514 (calcd. 401.0509 for
C19H13O10, [M–H]−).
Methyl retiboletate (2)
1.3 mg (0.006% of dry weight), orange solid, m. p. 140 ◦C H2O-MeCN (9 : 1) + 0.1% TFA; solvent B: MeCN + 0.1%
(decomp.). – Rf (TLC) = 0.24 (solvent system A), orange TFA; gradient: start 100% A, 3 min: 100% A, 57 min: 100%
spot, + K3Fe[(CN)6] red-brown. –tR (analytical HPLC, same B, flow rate: 7 mL min−1). Retention times: tR (10) = 17.3, tR
conditions as for 1) = 26.5 min. – UV/Vis (MeOH): λmax (9) = 27.3, tR (2, 7) = 35.2, tR (8) = 39.1 min.
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G. Gruber et al. · New Pulvinic Acid and Phenylalaninol Derivatives from Retiboletus griseus and R. nigerrimus
Nigerrimin A, (S)-2-(3,4-dihydroxybenzoylamino)-
3-phenylpropanol (9)
3.98 (m, 1 H, 2-H), 6.45 (dd, J = 8.0, 1.9 Hz, 1 H, 60-H),
6.57 (d, J = 8.0 Hz, 1 H, 50-H), 6.61 (d, J = 1.9 Hz, 1 H,
20-H), 6.71 (d, J = 8.2 Hz, 1 H, 500-H), 7.14 (dd, J = 8.2,
2.2 Hz, 1 H, 600-H), 7.22 (d, J = 2.2 Hz, 1 H, 200-H), 7.67 (d,
J = 8.2 Hz, 1 H, NH). – 13C NMR (150 MHz, [D6]DMSO):
δ = 36.1 (C-3), 53.5 (C-2), 62.9 (C-1), 114.8 (C-500), 115.3
(C-200), 115.4 (C-50), 116.6 (C-20), 119.1 (C-600), 119.9 (C-
60), 126.3 (C-100), 130.5 (C-10), 143.4 (C-40), 144.9§ (C-30),
145.0§ (C-300), 148.3 (C-400), 166.0 (C=O) (§ assignments
are interchangeable). – MS (EI): m/z (%) = 319 (6) [M]+,
196 (8) C9H10NO4, 178 (11) C9H8NO3, 166 (17) C9H10O3,
154 (26) C7H8NO3, 137 (100) C7H5O3, 123 (16) C7H7O2,
109 (10) C6H5O2, 81 (11), 77 (6), 44 (4). – HRMS (EI):
m/z = 319.1081 (calcd. 319.1056 for C16H17NO6, [M]+).
133 mg (0.2% of dry weight), colorless solid, m. p.
132 ◦C (decomp.). – [α]D25 = −10.4 (c = 7.6, MeOH). – Rf
(TLC) = 0.40 (solvent system B), spot turns brown on heat-
ing. tR (analytical HPLC) = 18.8 min (Nucleosil 100 C18,
5 µm, 250 × 4 mm; solvent A: H2O-MeCN (9 : 1) + 0.1%
TFA; solvent B: MeCN + 0.1% TFA; gradient: start 100%
A, 60 min: 100% B, 65 min: 100% B, flow rate: 1 mL
min−1). – UV (MeOH): λmax (logε) = 205 (2.94, sh), 256
(2.43), 286 (2.28) nm. – CD (MeOH): λmax (∆ε) = 254
(–1.89), 264 (–2.16), 277 (–1.62), 291 (–2.41) nm. – IR
(KBr): ν = 3400 (ss, br), 2957 (m), 2930 (m), 1710 (m,
sh), 1636 (ss), 1601 (ss), 1546 (s), 1514 (ss), 1453 (s),
1440 (s), 1383 (s), 1360 (s), 1290 (ss), 1251 (s), 1201 (s),
1138 (s), 1114 (s), 1084 (s, sh), 1034 (s), 958 (m), 882 (m,
sh), 822 (w), 784 (m), 760 (m), 722 (m), 702 (m) cm−1. –
1H NMR (600 MHz, [D6]DMSO): δ = 2.75 (dd, J = 13.4,
9.0 Hz, 1 H, 3-HA), 2.91 (dd, J = 13.4, 4.9 Hz, 1 H, 3-HB),
3.35 (dd, J = 10.3, 6.8 Hz, 1 H, 1-HA), 3.44 (dd, J = 10.3,
4.9 Hz, 1 H, 1-HB), 4.08 (m, 1 H, 2-H), 6.71 (d, J = 8.0 Hz,
1 H, 500-H), 7.12 – 7.23 (m, 7 H), 7.79 (d, J = 8.0 Hz, 1
H, NH), 9.04 (br. s, 1 H, OH), 9.39 (br. s, 1 H, OH). –
13C NMR (150 MHz, [D6]DMSO): δ = 36.6 (C-3), 53.2
(C-2), 63.1 (C-1), 114.8 (C-500), 115.3 (C-200), 119.1 (C-
600), 125.9 (C-40), 126.2 (C-100), 128.2§ (C-20/60), 129.2§
(C-30/50), 139.8 (C-10), 144.8 (C-300), 148.3 (C-400), 166.0
(C=O) (§ assignments are interchangeable). – MS (EI): m/z
(%) = 287 (3) [M]+, 256 (4, C15H14NO3), 210 (1), 196 (54,
C9H10NO4), 178 (19, C9H8NO3), 154 (5, C7H8NO3), 137
(100, C7H5O3), 120 (11, C8H10N), 109 (8), 91 (8), 77 (1), 44
(2). – MS (ESI): m/z (%) = 597 (6) [2 M+Na]+, 310 (100)
[M+Na]+. – HRMS (EI): m/z = 287.1157 (calcd. 287.1157
for C16H17NO4, [M]+).
Ethyl 3,4-diisopropoxybenzoate (12)
A stirred mixture of ethyl 3,4-dihydroxybenzoate (11; 1 g,
5.5 mmol), anhydrous K2CO3 (4.94 g, 35.8 mmol), and iso-
propyl bromide (3.1 mL, 33.0 mmol) in DMF (30 mL) was
heated for 3 h at 60 ◦C under an argon atmosphere, and then
stirred for 12 h at 20 ◦C. As some starting material was still
present [determined by TLC comparison (solvent system A,
detection with FeCl3)], the mixture was again treated with
isopropyl bromide (1.55 mL, 16.5 mmol) and heated for 4 h
at 60 ◦C. After cooling to r. t., water was added, and the mix-
ture was acidified to pH = 4 with conc. HCl. Extraction with
EtOAc (3 ×), drying (Na2SO4), and concentration to dryness
under reduced pressure yielded 12 as a colorless oil (1.43 g,
98%). – Rf (TLC) = 0.77 (solvent system A). – 1H NMR
(300 MHz, CDCl3): δ = 1.13 (d, J = 6.2 Hz, 12 H), 1.17 (t,
J = 7.2 Hz, 3 H), 4.14 (q, J = 7.2 Hz, 2 H), 4.33 (m, 2 H),
6.69 (d, J = 8.4 Hz, 1 H), 7.44 (d, J = 2.0 Hz, 1 H), 7.47 (dd,
J = 8.4, 2.0 Hz, 1 H). – 13C NMR (75 MHz, CDCl3): δ =
13.8 (CH3), 21.4 (2 × CH3), 21.6 (2 × CH3), 59.9 (CH2),
70.8 (CH), 71.9 (CH), 114.7 (CH), 118.8 (CH), 122.7 (C),
123.6 (CH), 147.6 (C), 153.0 (C), 165.5 (C=O). – MS (EI):
m/z (%) = 266 (39) [M]+, 224 (13), 182 (100), 167 (9),
154 (67), 137 (94), 109 (7), 97 (2), 42 (11). – HRMS (EI):
m/z = 266.1499 (calcd. 266.1518 for C15H22O4, [M]+).
Nigerrimin B, (S)-2-(3,4-dihydroxybenzoylamino)-3-
(3,4-dihydroxyphenyl)propanol (10)
106 mg (0.16% of dry weight), colorless solid, m. p.
239 ◦C (decomp.). – [α]2D5 = −52.2 (c = 10.6, MeOH).
– Rf (TLC) = 0.20 (solvent system A), 0.16 (solvent sys-
tem B), spot turns brown on air exposure. – tR (analyt-
ical HPLC) = 10.3 min (same conditions as for 9). – UV
3,4-Diisopropoxybenzoic acid (13)
Ester 12 (766 mg, 2.88 mmol) was heated with 10% aque-
(MeOH): λmax (logε) = 206 (3.15, sh), 256 (2.51), 285 ous NaOH (3.8 mL) in 50% aqueous EtOH (100 mL) for
(2.42) nm. – CD (MeOH): λmax (∆ε) = 237 ( – 1.45), 257 2 h with stirring at 95 ◦C. After cooling to r. t., the solution
(–1.93), 277 (–1.36), 289 (–1.61) nm. – IR (KBr): ν = 3414 was diluted with water (100 mL) and washed with CHCl3
(ss, br), 2957 (m), 2928 (m), 1735 (m), 1635 (s, sh), 1603 (100 mL). Then, the aqueous phase was acidified with conc.
(s), 1549 (m), 1514 (s), 1443 (m), 1361 (m), 1289 (s), 1253 HCl to pH = 4 and extracted with EtOAc (3 × 100 mL). The
(s), 1198 (m), 1116 (m), 1085 (w), 1048 (w), 961 (w), 881 combined organic phases were washed with water (150 mL),
(w), 823 (w), 786 (w), 760 (w) cm−1. – 1H NMR (600 MHz, dried (Na2SO4), and concentrated under reduced pressure to
[D6]DMSO): δ = 2.57 (dd, J = 13.9, 8.2 Hz, 1 H, 3-HA), yield 13 (637 mg, 93%) as colorless solid, m. p. 108 ◦C. –
2.69 (dd, J = 13.9, 5.9 Hz, 1 H, 3-HB), 3.32 (dd, J = 10.8, Rf (TLC) = 0.63 (solvent system A). – 1H NMR (200 MHz,
6.1 Hz, 1 H, 1-HA), 3.40 (dd, J = 10.8, 5.4 Hz, 1 H, 1-HB), CDCl3): δ = 1.36 (d, J = 6.0 Hz, 6 H), 1.38 (d, J = 6.0 Hz,
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681
6 H), 4.51 (m, 1 H), 4.62 (m, 1 H), 6.93 (d, J = 8.4 Hz, 1 Methyl (S)-2-(3,4-diisopropoxybenzoylamino)-
H), 7.66 (d, J = 2.0 Hz, 1 H), 7.74 (dd, J = 8.4, 2.0 Hz, 1 3-(3,4-diisopropoxyphenyl)propanoate (17)
H), 12.40 (br. s, 1 H, CO2H). – MS (EI): m/z (%) = 238 (8)
A solution of acid 13 (50 mg, 0.21 mmol) in MeCN
(4 mL) was stirred with hydrochloride 16 (62 mg,
0.19 mmol), triethylamine (0.12 mL, 0.84 mmol), and
TBTU (67.4 mg, 0.21 mmol) for 36 h at 20 ◦C. Then, the
reaction mixture was equilibrated between water (50 mL)
and EtOAc (50 mL); the organic phase was extracted
with water (2 × 50 mL) and then washed with saturated
aqueous KHSO4 (50 mL), aqueous NaHCO3, and water.
After drying (Na2SO4), the solvent was removed, and the
residue was purified by column chromatography on silica
gel (hexanes-EtOAc, 4 : 1) to yield 17 (58.0 mg, 54%) as
a colorless oil. – Rf (TLC) = 0.77 (solvent system A). – UV
(MeCN): λmax (logε) = 204 (4.74), 257 (4.06), 285 (3.84)
nm. – IR (film): ν = 3326 (m, br), 2978 (ss), 2934 (s), 1746
(s), 1640 (s), 1602 (s), 1578 (s), 1537 (s), 1503 (ss), 1467
(s), 1445 (s), 1426 (m), 1384 (s), 1372 (s), 1334 (s), 1269
(ss), 1215 (ss), 1178 (s), 1136 (ss), 1109 (ss), 1026 (w), 985
(s), 951 (m), 885 (w), 852 (m), 752 (s) cm−1. – 1H NMR
(300 MHz, CDCl3): δ = 1.21 – 1.33 (m, 24 H), 3.10 (dd,
J = 14.0, 5.3 Hz, 1 H, 3-HA), 3.16 (dd, J = 14.0, 5.7 Hz, 1
H, 3-HB), 3.73 (s, 3 H, OCH3), 4.37, 4.50 (each, m, 2 H,
2 × iPr-CH), 4.98 (m, 1 H, 2-H), 6.44 (d, J = 7.5 Hz, 1 H,
NH), 6.62 (dd, J = 8.0, 2.0 Hz, 1 H), 6.65 (d, J = 2.0 Hz, 1
H), 6.79 (d, J = 8.0 Hz, 1 H), 6.84 (d, J = 8.4 Hz, 1 H), 7.20
(dd, J = 8.4, 2.0 Hz, 1 H), 7.36 (d, J = 2.0 Hz, 1 H). – 13C
NMR (75 MHz, CDCl3): δ = 22.1, 22.2 (each 4 × CH3),
37.3 (C-3), 52.3 (OCH3), 53.5 (C-2), 71.9 (iPr-CH), 72.1
(2 × iPr-CH), 72.5 (iPr-CH), 116.1 (C-500), 117.1 (C-200),
118.2 (C-50), 119.4 (C-20), 120.5 (C-600), 122.5 (C-60),
126.8 (C-100), 129.2 (C-10), 148.3, 148.7, 149.1 (each C),
152.4 (C-400), 166.3 (CONH), 172.2 (C=O). – MS (EI):
m/z (%) = 515 (16) [M]+, 278 (100), 236 (39), 221 (14),
207 (11), 194 (66), 179 (14), 165 (24), 153 (10), 137 (34),
123 (31), 109 (3). – HRMS (EI): m/z = 515.2875 (calcd.
515.2869 for C29H41NO7, [M]+). – C29H41NO7 (515.64):
calcd. C 67.55, H 8.01, N 2.72; found C 67.26, H 7.89, N
2.71.
[M]+, 196 (5), 154 (100), 137 (15), 109 (2), 97 (2), 83 (3),
43 (7). – HRMS (EI): m/z = 238.1199 (calcd. 238.1205 for
C13H18O4, [M]+).
Methyl (S)-2-(tert-butoxycarbonylamino)-
3-(3,4-diisopropoxyphenyl)propanoate (15)
To a stirred solution of N-Boc-L-DOPA methyl es-
ter [16 – 18] (14; 2.70 g, 8.67 mmol) in DMF (70 mL)
were added, under an argon atmosphere, anhydrous K2CO3
(7.79 g, 56.36 mmol) and isopropyl bromide (4.9 mL,
52.0 mmol). The suspension was warmed for 12 h at 50 ◦C,
cooled, and filtered, and the filter was washed with EtOAc.
The filtrate was distributed between EtOAc and water; the or-
ganic phase was washed with water (3 ×), dried (Na2SO4),
and concentrated under reduced pressure to yield 15 (3.33 g,
97%) as a colorless oil. – Rf (TLC) = 0.71 (solvent system
C). – 1H NMR (300 MHz, CDCl3): δ = 1.22 (d, J = 6.0 Hz,
6 H), 1.23 (d, J = 6.0 Hz, 6 H), 1.32 (s, 9 H), 2.88 (dd,
J = 13.8, 5.6 Hz, 1 H, 3-HA), 2.93 (dd, J = 13.8, 5.3 Hz, 1 H,
3-HB), 3.60 (s, 3 H, OCH3), 4.34 (m, 2 H, 2 × iPr-CH), 4.44
(m, 1 H, 2-H), 4.99 (d, J = 7.9 Hz, 1 H, NH), 6.56 (dd, J =
8.0, 1.9 Hz, 1 H, 60-H), 6.60 (d, J = 1.9 Hz, 1 H, 20-H), 6.73
(d, J = 8.0 Hz, 1 H, 50-H). – 13C NMR (75 MHz, CDCl3):
δ = 22.1 [2 × CH(CH3)2], 28.1 [C(CH3)3], 37.5 (C-3), 52.0
(OCH3), 54.3 (C-2), 72.0 [2 ×CH(CH3)2], 79.6 [C(CH3)3],
118.0 (C-50), 119.3 (C-20), 122.4 (C-60), 129.2 (C-10), 148.1
(C-30), 148.8 (C-40), 154.9 (CONH), 172.3 (C=O). – EI-MS:
m/z (%) = 395 (16) [M]+, 353 (1), 322 (4), 295 (2), 280 (5),
236 (11), 207 (37), 194 (34), 165 (80), 151 (5), 123 (100), 88
(4), 77 (4), 57 (22), 41 (22). – HRMS (EI): m/z = 395.2313
(calcd. 395.2307 for C21H33NO6).
Methyl (S)-2-amino-3-(3,4-diisopropoxyphenyl)propanoate
hydrochloride (16)
Compound 15 (186 mg, 0.47 mmol) was treated with 2 N
HCl in EtOAc (6 mL), and the solution was stirred for 16 h at
20 ◦C. Concentration of the solution under reduced pressure
yielded 16 (132 mg, 85%) as a colorless solid. – 1H NMR
(300 MHz, CDCl3): δ = 1.24 (m, 12 H), 3.24 (m, 1 H), 3.33
(m, 1 H), 3.60 (s, 3 H), 4.37 (m, 3 H, 2-H, 2 × iPr-CH), 6.80
(S)-2-(3,4-Diisopropoxybenzoylamino)-
3-(3,4-diisopropoxyphenyl)propanol (18)
To ester 17 (87 mg, 0.17 mmol) in anhydrous THF (1 mL)
(m, 3 H), 8.67 (br. s, 3 H, NH+3 ). – 13C NMR (75 MHz, were added NaBH4 (13 mg, 0.34 mmol) and MeOH (1 mL),
CDCl3): δ = 22.1 (2 × CH3), 22.2 (2 × CH3), 35.8 (C-3), and the mixture was stirred for 48 h at r. t. under an argon
52.8 (OCH3), 54.4 (C-2), 72.0 (CH), 72.2 (CH), 118.3 (C- atmosphere. Then, the reaction mixture was cooled to 0 ◦C,
50), 119.5 (C-20), 122.9 (C-60), 127.3 (C-10), 148.5§ (C-30), acidified with 10% aqueous citric acid to pH = 4, and con-
149.0§ (C-40), 169.3 (C-1) (§ assignments are interchange- centrated under reduced pressure. The residue was dissolved
able). – MS (EI): m/z (%) = 295 (8) [M–HCl]+, 253 (2), 236 in water (20 mL) and washed with CH2Cl2 (3 × 25 mL).
(6), 207 (34), 165 (50), 123 (100), 88 (4), 43 (3). – HRMS Then, the combined organic phase was dried (Na2SO4) and
(EI): m/z = 295.1775 (calcd. 295.1783 for C16H25NO4, [M– concentrated under reduced pressure to yield 18 (60 mg,
HCl]+).
73%) as a colorless oil. – Rf (TLC) = 0.37 (solvent system
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682
G. Gruber et al. · New Pulvinic Acid and Phenylalaninol Derivatives from Retiboletus griseus and R. nigerrimus
A). – UV (MeCN): λmax (logε) = 208 (5.43), 259 (4.89), der an argon atmosphere. Then, saturated aqueous ammo-
287 (4.67) nm. – IR (film): ν = 3339 (m, br), 2976 (ss), nium chloride (2 mL) was added, and the reaction mixture
2930 (s), 1631 (m), 1602 (m), 1574 (m), 1538 (m), 1501 (ss), was equilibrated between water (5 mL) and EtOAc (10 mL).
1467 (m), 1424 (w), 1383 (m), 1268 (ss), 1221 (m), 1177 The aqueous phase was washed with EtOAc (2 × 10 mL),
(m), 1108 (ss), 986 (w), 951 (w), 761 (w) cm−1. – 1H NMR and the combined EtOAc phases were again washed with
(300 MHz, CDCl3): δ = 1.23 – 1.31 (m, 24 H), 2.85 (m, 2 water. Then, the solution was dried (Na2SO4), filtered, and
H, 3-H), 3.43 (br. s, 1 H, OH), 3.67 (m, 2 H, 1-H), 4.23 (m, concentrated under reduced pressure to yield nigerrimin B
1 H, 2-H), 4.44 (m, 4 × iPr-CH), 6.40 (d, J = 7.3 Hz, 1 H, (10) (14.9 mg, 91%) as a colorless solid, m. p. 240 – 243 ◦C
NH), 6.73 (dd, J = 8.0, 2.0 Hz, 1 H, 60-H), 6.82 – 6.78 (m, 3 (decomp.), [α]D25 = −53.6 (c = 9.3, MeOH). The synthetic
H, 20/50/500-H), 7.14 (dd, J = 8.4, 2.0 Hz, 1 H, 600-H), 7.31 compound was identical with the natural product by HPLC
(d, J = 2.0 Hz, 1 H, 200-H). – 13C NMR (75 MHz, CDCl3): comparison and exhibited closely similar spectroscopic data
1
δ = 22.1, 22.2 (each 2 × CH3), 22.3 (4 × CH3), 36.7 (C- (UV, IR, H NMR, 13C NMR, MS) and physical properties
3), 53.6 (C-2), 64.9 (C-1), 72.1, 72.3, 72.4, 72.6 (each iPr- (m. p., [α]D, CD).
CH), 116.6 (C-500), 117.4 (C-200), 118.9 (C-50), 119.4 (C-
Acknowledgement
20), 120.4 (C-600), 122.4 (C-60), 127.5 (C-100), 131.1 (C-10),
148.2, 149.0, 149.6 (each C), 152.5 (C-400), 167.8 (CONH).
– MS (EI): m/z (%) = 487 (8) [M]+, 469 (2), 262 (27), 250
(100), 221 (47), 208 (28), 179 (19), 166 (19), 137 (46),
123 (24), 110 (4), 109 (4), 81 (1), 43 (11). – HRMS (EI):
m/z = 487.2933 (calcd. 487.2934 for C28H41NO6, [M]+).
The authors thank the Deutsche Forschungsgemeinschaft
(DFG) and the Fonds der Chemischen Industrie for finan-
cial support. We are grateful to Ms. C. Dubler for NMR
measurements, and Dr. W. Spahl and Mr. R. Seidl for
the mass spectra. Furthermore, we thank Dr. N. Arnold,
Halle/Saale for his help in collecting the mushrooms, and
Ms. C. Gra¨f for technical assistance. Special thanks are due
to Dr. K. Greenfield, Brisbane (Australia), for valuable sug-
Nigerrimin B (10)
To a solution of compound 18 (25 mg, 0.05 mmol) gestions and improving the manuscript and Prof. A. Bresin-
in CH2Cl2 (4 mL) was added anhydrous AlCl3 (49 mg, sky, Regensburg, for his helpful comments regarding the
0.37 mmol), and the mixture was stirred for 48 h at r. t. un- taxonomy.
[1] M. Binder, A. Bresinsky, Feddes Repertorium 2002, [10] P. C. Beaumont, R. L. Edwards, G. C. Elsworthy, J.
113, 30 – 40.
Chem. Soc. C 1968, 2968 – 2974.
[2] K. Justus, R. Herrmann, J.-D. Klamann, G. Gruber, [11] R. Knorr, A. Trzeciak, W. Bannwarth, D. Gillessen,
V. Hellwig, A. Ingerl, K. Polborn, B. Steffan, W. Steg-
lich, Eur. J. Org. Chem. 2007, 5560 – 5572.
[3] A. Bresinsky, H. Besl, Z. Mykol. 1978, 45, 247 – 264.
Tetrahedron Lett. 1989, 30, 1927 – 1930.
[12] K. Soai, H. Oyamada, M. Takase, Bull. Chem. Soc. Jpn.
1984, 57, 2327 – 2328.
[4] Review: M. Gill, W. Steglich, Progr. Chem. Org. Nat. [13] Y. Hamada, M. Shibata, T. Sugiura, S. Kato, T. Shioiri,
Prod. 1987, 51, 32 – 51. J. Org. Chem. 1987, 52, 1252 – 1255.
[5] W. Steglich, W. Furtner, A. Prox, Z. Naturforsch. 1968, [14] A. M. Clark, C. D. Hufford, L. W. Robertson, Lloydia
23b, 1044 – 1050. 1977, 40, 146 – 151.
[6] W. Steglich, H. Besl, K. Zipfel, Z. Naturforsch. 1974, [15] D. L. Doerfler, B. A. Bird, I. M. Campbell, Phytochem-
29b, 96 – 98. istry 1981, 20, 2303 – 2304.
[7] H. Besl, A. Bresinsky, L. Kopanski, W. Steglich, [16] S. N. Banerjee, C. J. Ressler, J. Org. Chem. 1976, 41,
Z. Naturforsch. 1978, 33c, 820 – 825. 3056 – 3058.
[8] D. R. Gedge, G. Pattenden, A. G. Smith, J. Chem. Soc., [17] S. Aubry, S. Pellet-Rostaing, M. Lemaire, Eur. J. Org.
Perkin Trans. 1 1986, 2127 – 2131. Chem. 2007, 5212 – 5225.
[9] G. Pattenden, N. Pegg, A. G. Smith, Tetrahedron Lett. [18] T. Zhou, R. C. Hider, P. Jenner, B. Campbell, C. J.
1986, 27, 403 – 406.
Hobbs, S. Rose, M. Jairaj, K. A. Tayarani-Binazir,
A. Syme, Eur. J. Med. Chem. 2010, 45, 4035 – 4042.
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