From sophorose lipids to natural product synthesis

Article abstract of DOI:10.1002/adsc.201101005

Sophorose lipids (SL) are a readily available, scalable source for the disaccharide sophorose: In a four-step sequence the crude product from the fermentation broth was converted to key disaccharide donors 7 that were further utilized in the synthesis of

Full text of DOI:10.1002/adsc.201101005

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DOI: 10.1002/adsc.201101005
From Sophorose Lipids to Natural Product Synthesis
Natalie Hoffmann,a Jçrg Pietruszka,a,* and Christoph Sçffinga
a
Institut fꢀr Bioorganische Chemie der Heinrich-Heine-Universitꢁt Dꢀsseldorf im Forschungszentrum Jꢀlich, Stetternicher
Forst Geb. 15.8, 52426 Jꢀlich, Germany
Fax : (+49)-2461-616196; e-mail: j.pietruszka@fz-juelich.de
Received: December 23, 2011; Published online: April 4, 2012
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201101005.
Abstract: Sophorose lipids (SL) are a readily avail-
able, scalable source for the disaccharide sopho-
rose: In a four-step sequence the crude product
from the fermentation broth was converted to key
disaccharide donors 7 that were further utilized in
the synthesis of natural products Ebracteatoside C,
Zizybeoside I and phenethyl glycoside. While the
aglycones for Zizybeoside I and the phenethyl gly-
coside are commercially available simple primary
alcohols, the synthesis of Ebrateatoside C required
a secondary allylic alcohol (R)-6-hydroxyoct-7-enyl
acetate. The enantiomerically pure secondary alco-
hol was synthesized in two steps using an alcohol
dehydrogenase-catalysed reduction as the key step.
glucose (sophorose) and an (w-1)-hydroxyoleic acid
aglycone. The primary hydroxy groups of sophorose
can be mono- or diacetylated, the oleic acid moiety
can be either in acyclic or lactone form (Figure 1). SL
have been known since 1961, their fermentation from
various renewable resources has been studied exten-
sively[4–12] and the first and only total synthesis of SL
(lactone) was reported by Fꢀrstner et al.[13] Starting
from the lactone form of SL (4) Jones et al.[14] report-
ed the synthesis of a-sophorose and Tulloch et al.[15]
the synthesis of methyl b-sophoroside, respectively.
Due to their amphiphilic structures SL as well as
rhamnolipids,[16] cellobiose lipids and mannosylery-
thritol lipids[17] can act as biosurfactants. Their proper-
ties are of high interest so that SL have even found
industrial applications.[18–21] Therefore considerable ef-
forts have recently been reported on their separation
and extraction.[6,22] Although the sophorose motif can
be found in many different natural products,[1,4] SL
Keywords: alcohol dehydrogenase; asymmetric cat-
alysis; natural product synthesis; sophorose lipids
Nature is commonly accepted to be one of the most
important resources for new active agents and medi-
cal products. In this context sophorose-based natural
products have attracted our attention. Examples are
the successfully isolated and structurally elucidated
sophorose-containing glucosides Ebracteatoside C (1),
Zizybeoside I (2) and the phenethyl glycoside 3 from
semi-mangrove plants of the genus Acanthus, which
are used as anti-inflammatory and anti-hepatitis
agents in traditional Chinese medicine. Ebracteatoside
C (1) and Zizybeoside I (2) were first isolated from
A. ebracteatus from Thailand in 2001. The phenethyl
glycoside 3 was isolated from the same species as well
as from A. ilicifolius from Thailand and Hainan
(China) and A. volubilis.[1,2] The current work focuses
on the synthesis of such natural products.
To avoid a tedious synthesis of the sophorose gly-
cone from glucose or expensive sophorose,[3] we de-
cided to start from sophorose lipids (4) (SL). SL are
glycolipids containing two (1,2)-b-linked molecules of Figure 1. Sophorose-containing natural products.
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Scheme 1. Synthesis of the sophorose glycones. Reaction
conditions: a) NaOMe, MeOH, room temperature; b) Ac2O,
pyr., 08C to room temperature, or BzCl, pyr., 08C to room
temperature; c) H2, Pd/C, EtOAc, room temperature; d)
HBr, AcOH, room temperature.
Scheme 2. Synthesis of the sophorose products 2 and 3. Re-
action conditions: a) BnOH, Ag2CO3, CH2Cl2, room temper-
ature; b) 0.5M NaOMe, MeOH, room temperature; c)
PhACTHNUGTRNEGNU(CH2)2OH, Ag2CO3, CH2Cl2, room temperature.
have not been utilized as disaccharide source in natu-
ral product synthesis. With a considerable supply of steps). Having confirmed our concept of using sopho-
SL available,[23] we wish to report their application in rose lipids as glycosyl donor source we adapted our
natural product synthesis for the first time.
synthesis by coupling sophorose bromide 7a with 2-
As a first step towards a common building block phenylethanol forming phenethyl glycoside 3 after de-
derived from SL 4, it was essential to obtain a homo- protection in 59% yield.
geneous starting material from the mixture. Hence,
In order to extend the scope of the approach, it was
the crude fermentation broth was treated with sodium planned to utilise the sequence for the synthesis of
methoxide in methanol in order to open the lactone Ebracteatoside C (1). Therefore a route towards the
ring and cleave the acetyl groups thus providing the enantiomerically pure allyl alcohol (R)-8 as aglycone
corresponding methyl ester 5[24,25] (Scheme 1). The was necessary. Based on our previous experience with
challenge of the sophorose glycone synthesis was the similar compounds,[31–34] we envisaged to apply alcohol
selective hydrolysis of the glycosidic linkage between dehydrogenases, and in particular ADH-T (from
the fatty acid and the carbohydrate moiety: direct Thermoanaerobacter species recombinant in Escheri-
acid treatment of the lipid 5 did completely hydrolyse chia coli), for the enantioselective reduction of the
the glycoside, while the known enzymatic degradation corresponding ketone.
of sophorose lipids with a glycosidase would lead to
Treating the commercially available alkyl bromide
the selective cleavage of the disaccharide moiety.[26] 9 with zinc (activated in situ with iodine) delivers the
However, treatment of ester 5 first with acetic anhy- alkylzinc intermediate.[35] Negishi coupling with acryl-
dride furnishing the peracetylated saccharide[27] fol- ic acid chloride provided the required ketone 10
lowed by hydrogenation of the double bound yielded (Scheme 3). The racemic and enantiomerically en-
the deactivated glycoside 6a as a direct precursor for riched reference compounds 8 were obtained after
the final hydrolysis in good yield. Employing HBr so- enantioselective Corey–Bakshi–Shibata reduction[36]
lution, the ultimate step succeeded in furnishing the and Luche conditions[37] (NaBH4/CeCl3), respectively.
anomeric bromide 7a in moderate yield. It should be
As anticipated, the following enzymatic reduction
noted that hydrogenation is only required for conven- of ketone 10 with ADH-T furnished the required sec-
ience during work-up: The conversion minimises the ondary alcohol (R)-8 in good yield and excellent se-
number of side-products during the selective cleavage lectivity (Scheme 4; 70%, ee >99%). Cofactor
of the saccharide.
(NADPH) recycling was readily achieved using iso-
Sophorose bromide 7a was treated under Koenigs– propyl alcohol. Aglycone (R)-8 was thus obtained in
Knorr conditions[28] using benzyl alcohol (BnOH) and enantiomerically pure form in two steps, only.
Ag2CO3 instead of the more toxic Hg(CN)2.[29] The
The ultimate glycosylation with aglycone (R)-8
obtained peracetylated glycoside was subsequently proved to be challenging, since the absence of a direct-
deprotected under Zemplꢃn conditions[30] furnishing ing neighbouring group in the sophorose moiety 7
the known Zizybeoside I (2) (Scheme 2; 51% over 2 rendered anomeric control especially difficult: con-
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From Sophorose Lipids to Natural Product Synthesis
not improve the selectivity in favour of the b-anomer
(Table 1, entries 4–6). Changing the protecting group
from acetate 7a/11a to benzoate 7b/11b (entries 7–9)
did finally result in the selective formation of the en-
riched b-anomer 12b: PdACTHNUTRGENNUG(CH3CN)4ACHTUNGTERN(NUGN BF4)2 proved to
be the promoter of choice (entry 9: a/b 1:3).[41] It is
noteworthy that the corresponding peracetylated tri-
chloroacetimidate 11a showed under identical condi-
tions only some b-product formation (entry 5:
a/b 1.3:1). The natural product Ebracteatoside C (1)
was eventually isolated after hydrolysis of the eight
ester-protecting groups (Scheme 5; 46% b-anomer).
In conclusion, it was shown that the readily avail-
able crude mixture of sophorose lipids is a convenient
starting material for natural product synthesis. In only
four steps glycosyl donors 7 were obtained and cou-
pled with various alcohols. In particular, allylic alco-
hol (R)-8 proved to be a challenging aglycone: while
its enantioselective synthesis was established via a che-
moenzymatic route, the b-selective glycosylation was
only possible after considerable optimisation. Ulti-
mately three natural sophorosides 1–3 were obtained
by this short sequence.
Scheme 3. Synthesis of ketone 10 and reference compounds.
Reaction conditions: a) Zn/I2, DMF, 808C, then 1 mol%
PdACHTUNGTRENNUNG(PPh3)4, acrylic acid chloride, 608C to room temperature;
b) catecholborane, (S)-CBS-catalyst, toluene, À788C; c)
CeCl3, NaBH4, EtOH, 788C.
Experimental Section
Analytical data and NMR-spectra are presented in the Sup-
porting Information.
Scheme 4. Enzymatic reduction of ketone 10. Reaction con-
ditions: a) TEA buffer (100 mM, pH 7, 1 mM MgCl2),
1 mol% NAD(P)+, 0.5 U/mL ADH-T, 20 vol% i-PrOH.
Procedure for the Enzymatic Reduction
ventional Koenigs–Knorr conditions (Table 1, entry 1)
led preferentially to the undesired a-product 12a. Al-
ternative promoters such as AgOTf (entry 2) and
TMSOTf (entry 3) did only increase the ratio. There-
fore, trichloroacetimidate[38] 11a was synthesised in
two steps from the protected sophorose bromide 7a
(Scheme 5):[39,40] Silver carbonate-promoted hydrolysis
was followed by activation with trichloroacetonitrile
Synthesis of (R)-6-hydroxyoct-7-enyl acetate (R)-8: Ketone
10 (680 mg, 3.69 mmol, 1 equiv.) in 20 mL 2-propanol,
NADP+ (29 mg, 37 mmol, 1 mol%), and ADH-T solution
~50% in glycerol (~62 U) were added to 80 mL TEA buffer
(100 mM, pH 7.0, 1 mmol MgCl2) and stirred at 308C for
20 h. The aqueous solution was extracted with MTBE, the
organic layer dried over MgSO4, filtered, and the solvent re-
moved under reduced pressure. The crude product was puri-
fied by flash column chromatography on silica gel (PE:EE,
80:20) to afford product (R)-8 as a colourless oil; yield:
and 1,8-diazabicyclo
ACHTUNGTRNE[NUNG 5.4.0]undec-7-ene (DBU) provid-
ing the desired a-donor 11a in poor yield (24%).
Again, preliminary experiments with donor 11a did 478 mg (2.60 mmol, 70%).
Table 1. Glycosylation conditions with aglycone (R)-8.
Entry
Donor
Activator (equiv.)
Temperature
a/b[a] ratio
1
2
3
4
5
6
7
8
9
7a
7a
7a
11a
11a
11a
7b
11b
11b
Ag2CO3 (3 equiv.)
AgOTf
TMSOTf (1.5 mol%)
BF3·OEt2 (2 equiv.)
08C to r.t.
08C
À788C
À308C
r.t.
2.4:1
only a
only a
2.4:1
Pd
G
N
1.3:1
TMSOTf (1.5 mol%)
Ag2CO3 (3 equiv.)
AgOtf/base
À308C
only a
only a
only a
1:3
08C
08C
r.t.
Pd
G
N
[a]
Determined by NMR.
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Scheme 5. Synthesis of the Ebracteatoside C. Reaction conditions: a) Ag2CO3, H2O, acetone/CH2Cl2, room temperature; b)
CCl3CN, DBU, 08C; c) TMSOTf (1.5 mol%), (R)-8, À308C, 4 ꢄ MS, CH2Cl2; d) Pd
ACHTUNTGRENGU(N CH3CN)4ACHUTGNTREN(NUGN BF4)2, (R)-8, 4 ꢄ MS, room
temperature; e) 3% KOH in MeOH, room temperature.
b-anomer 1 was separated by flash column chromatography
on silica gel (CH2Cl2:MeOH:H2O=82:12:0.5) affording the
natural product 1 as a colourless oil; yield: 14 mg (30 mmol,
46%).
Procedure for the Synthesis of Trichloroacetimidate
11b
To suspension of bromide 7b (110 mg, 97 mmol) in 1.5 mL
CH2Cl2/acetone/H2O) was added Ag2CO3 (80 mg, 291 mmol,
3 equiv.). The reaction mixture was stirred overnight at
room temperature. Ethyl acetate was then added and the re-
sulting mixture was washed with H2O, 1M HCl, saturated
NaHCO3 solution and saturated NaCl solution. After con-
centration under vacuum the residue was purified by
column chromatography on silica gel (PE:EE=70:30). The
Representative Procedure for the Synthesis of
Zizybeoside I (2) and Phenethyl Glycoside (3)
A
mixture containing the glycosyl donor 7a (147 mg,
210 mmol, 1.0 equiv) in dry dichloromethane (3 mL) was
added to the corresponding acceptor (1.68 mmol, 8 equiv.)
and molecular sieves (150 mg, 3 ꢄ). The reaction mixture
was stirred at room temperature for 1 h and Ag2CO3 (64 mg,
231 mmol, 1.1 equiv.) was added and stirring was continued
for 2 days at room temperature. After filtration through
a pad of Celite the solvent was removed under reduced
pressure. Flash column chromatography on silica gel
(PE:EE=60:40, then 50:50) yielded the respective peracety-
lated disaccharide as a colourless oil.
The disaccharide (55 mmol) in methanol (1.6 mL) was
treated with NaOMe solution (0.16 mL, 0.5M in MeOH)
and stirred for 1 h at room temperature. Ion exchange resin
(5 g, Dowex 650C) was added and stirring was continued for
30 min. After filtration through a pad of Celite the solvent
was removed under reduced pressure yielding the product
as a white solid.
product was obtained as
a white solid; yield: 87 mg
(82 mmol, 84%).
The hemiacetal (350 mg, 327 mmol) was dissolved in
CH2Cl2 (5 mL) and cooled to 08C. Trichloroacetonitrile
(260 mL, 3.27 mmol, 10 equiv.) and 1,8-diazabicyclo-
ACHTUNGTRENNUNG[5.4.0]undec-7-ene DBU (63 mL, 1.3 equiv.) were added, and
the reaction mixture was stirred for 2 h. The solvent was
evaporated to give a crude oil which was purified by column
chromatography (PE:EE=60:40). The product was ob-
tained as a white solid; yield: 365 mg (300 mmol, 92%).
Procedure for the Synthesis of Ebracteatoside C (1)
A mixture containing the glycosyl donor 11b (200 mg,
165 mmol, 1 equiv.), glycosyl acceptor (R)-8 (153 mg,
823 mmol, 5 equiv.), and freshly activated molecular sieves
(100 mg, 4 ꢄ) in dry dichloromethane (4 mL) was stirred
under an atmosphere of dry nitrogen for 1 h. Pd
ACHTUGNRTNE(NUNG CH3CN)4
ACHTUNGTRENNUNG
and stirring was continued at room temperature for 20 h.
After filtration through a pad of Celite the solvent was re-
moved under reduced pressure. Flash column chromatogra-
phy on silica gel (PE:EE=70:30, then 60:40) yielded a mix-
ture of both anomers 12b; yield: 101 mg (82 mmol, 49%).
The anomeric mixture of disaccharides 12b (80 mg,
65 mmol) was dissolved in 3% KOH solution in methanol
(10 mL) and stirred for 2 days at room temperature. Ion ex-
change resin (5 g, Dowex 650C) was added and stirring was
continued for 30 min. After filtration through a pad of
Celite the solvent was removed under reduced pressure. The
Acknowledgements
We gratefully acknowledge the generous support of our proj-
ect by the Bundesministeriums fꢀr Ernꢁhrung, Landwirt-
schaft und Verbraucherschutz (BMELV) via the Fachagentur
Nachwachsende Rohstoffe e.V. (FNR), the BASF Personal
Care and Nutrition GmbH for donations of sophorose lipids
and Julich Chiral Solutions GmbH (now Codexis) for gener-
ous gifts of enzymes. We are indebted to M. Gehsing for per-
forming the enzyme activity tests, Dipl.-Chem. Alexandra
962
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From Sophorose Lipids to Natural Product Synthesis
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