N-acylated bacteriohopanehexol-mannosamides from the thermophilic bacterium Alicyclobacillus acidoterrestris

Article abstract of DOI:10.1007/s11745-010-3482-4

Identification of molecular species of various N-acylated bacteriohopanehexol-mannosamides from the thermophilic bacterium Alicyclobacillus acidoterrestris by semipreparative HPLC and by RP-HPLC with ESI is described. We used triple-quadrupole type mass s

Full text of DOI:10.1007/s11745-010-3482-4

Lipids (2011) 46:249–261
DOI 10.1007/s11745-010-3482-4
ORIGINAL ARTICLE
N-Acylated Bacteriohopanehexol-Mannosamides
from the Thermophilic Bacterium Alicyclobacillus acidoterrestris
ˇ
•
•
•
´ˇ
Tomas Rezanka Lucie Siristova Karel Melzoch
Karel Sigler
Received: 20 July 2010 / Accepted: 23 September 2010 / Published online: 21 October 2010
Ó AOCS 2010
Abstract Identification of molecular species of various
N-acylated bacteriohopanehexol-mannosamides from the
thermophilic bacterium Alicyclobacillus acidoterrestris by
semipreparative HPLC and by RP-HPLC with ESI is descri-
bed. We used triple-quadrupole type mass spectrometer, 1H
and 13C NMR for analyzing this complex lipid. CD spectra of
two compounds (model compound—7-deoxy-D-glycero-
D-allo-heptitol obtained by stereospecific synthesis, and an
isolated derivative of hopane) were also measured and the
absolute configuration of both compounds was determined.
On the basis of all the above methods, we identified the full
structure of a new class of bacteriohopanes, represented by
various N-acylated bacteriohopanehexol-mannosamides.
FAME
Fatty acid methyl ester(s)
Gas chromatography–mass
spectrometry
GC–MS
HMBC
Heteronuclear multiple bond
coherence
HMQC
Heteronuclear multiple
quantum coherence
HR-FAB-MS
High resolution fast atom
bombardment mass
spectrometry
LC–MS
Liquid chromatography–mass
spectrometry
LC–MS/ESI SIM
NOE
Selected ion monitoring
Nuclear Overhauser effect
Rotating-frame Overhauser
effect spectroscopy
Keywords Alicyclobacillus acidoterrestris ꢀ
Negative RP-HPLC–ESI–MS/MS ꢀ Circular dichroism ꢀ
N-Acylated bacteriohopanehexol-mannosamides ꢀ
x-Cyclohexyl fatty acids
ROESY
RP-HPLC/MS-ESI
Reversed phase liquid
chromatography–electrospray
ionization mass spectrometry
RP-HPLC–ESI–MS/MS Reversed phase liquid
chromatography–electrospray
ionization tandem mass
Abbreviations
BH
Bacteriohopane
BHpolyols
CD
Bacteriohopanepolyols
Circular dichroism
spectrometry
SPE
Solid phase extraction
Fluoro-N,N,N0,N0-
CID
Collision-induced dissociation
Correlation spectroscopy
Dimethylaminopyridine
TFFH
COSY
DMAP
tetramethylformamidinium
hexafluorophosphate
TLC
Thin layer chromatography
Air volume per volume of
culture medium per minute
ˇ
T. Rezanka (&) ꢀ K. Sigler
VVM
Institute of Microbiology, Academy of Sciences of the Czech
´
Republic, Vıdenska 1083, 142 20 Prague, Czech Republic
e-mail: rezanka@biomed.cas.cz
ˇ
´
Introduction
L. Siristova ꢀ K. Melzoch
Department of Fermentation Chemistry and Bioengineering,
Bacteriohopanepolyols (BHpolyols) are pentacyclic trit-
erpenoids, which are present as membrane constituents in
´
Institute of Chemical Technology Prague, Technicka 5,
166 28 Prague, Czech Republic
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Lipids (2011) 46:249–261
many bacteria [1]. They perform a regulating and rigidi-
fying function in membranes analogous to that of some
sterols in eukaryotes [2–4]. BHpolyol side chains form
many structures, which differ in terms of the number,
position and nature of the functional groups [2, 5]. The side
chain is responsible for the charge and the function of the
molecule [6].
LC-6A pumps (0.5 ml/min), an SCL-6A system controller,
an SPD ultraviolet detector an SIL-1A sample injector and
a C-R3A data processor, with a preparative normal phase
HPLC column Supelcosil LC-Si HPLC column 5 lm
particle size, length 9 I.D. 25 cm 9 21.2 mm with mobile
phase containing hexane–isopropanol-0.05% triethylamine
in water (99.5:0:0.5, v/v/v) to hexane–isopropanol-0.05%
triethylamine in water (20:78:2 v/v/v) for 40 min, a flow
rate of 9.9 ml/min, and monitored by a variable wavelength
detector at 210 nm (HP 1040M Diode Array Detector).
The HPLC equipment consisted of a 1090 Win system,
PV5 ternary pump and automatic injector (HP 1090 series,
Hewlett Packard, USA) and two AscentisÒ Express HILIC
HPLC column 2.7 lm particle size, L 9 I.D. 15 cm 9
2.1 mm (Supelco, Prague) in series were used. This setup
provided us with a high-efficiency column—approximately
30,000 plates/30 cm. LC was performed at a flow rate of
300 ll/min with a linear gradient from the mobile phase
containing methanol/acetonitrile/aqueous 1 mM ammo-
nium acetate (60:20:20, v/v/v) to methanol/acetonitrile/
aqueous 1 mM ammonium acetate (20:60:20, v/v/v) for
40 min. The whole HPLC flow (0.37 ml/min) was intro-
duced into the ESI source without any splitting.
The most commonly occurring BHpolyols in bacteria
contain four functional groups in the side chain, typically
three hydroxyl groups at 32R, 33R, 34S [7, 8] with the C-35
position occupied by either another OH or an amino group
[2].
In numerous strains belonging to various taxonomic
groups (e.g. purple nonsulfur bacteria, methylotrophs, or
cyanobacteria) the bacteriohopane (BH) derivatives are not
present as free polyols but are linked to various polar
moieties like glucosamine or N-acylglucosamine, anhy-
drogalacturonopyranosides, a-amino acids such as trypto-
phan and ornithine, adenosine, cyclitol ethers or other polar
entities the structures of which are still unidentified [2, 9].
BHpolyols have been found in many other structural varia-
tions including double bonds in the ring system or substitu-
tion by methyl groups in ring A. Furthermore, BHpolyols
with five and six functional groups in the side chain, i.e.
BHpolyols with additional one or two OH group(s) at C-30
and/or C-31 position(s) were identified [10–14, 40]. These
structures are restricted to a limited number of organisms or
occur in an explicit group of organisms and can serve as
specific bacterial marker information [2, 5].
The detector was an Applied Biosystems Sciex API
4000 mass spectrometer (Applied Biosystems Sciex,
Ontario, Canada) using electrospray mass spectra. The
ionization mode was negative, the nebulizing gas (N2)
pressure was 345 kPa and the drying gas (N2) flow and
temperature were 9 l/min and 300 °C, respectively. The
electrospray needle was at ground potential, whereas the
capillary tension was held at 4,000 V. The cone voltage
was kept at 250 V. The mass resolution was 0.1 Da and the
peak width was set to 6 s. For an analysis, total ion currents
(full scan) were acquired from 200 to 1,600 Da.
To date there have been only a few hexafunctionalized
side chain structures reported from nature. The basic
structure has five hydroxyl groups and NH2 functionality at
C-35, and has only been observed in Type I methano-
trophic bacteria [14, 15]. Further, BHhexol cyclitol ether
was identified in sediments of two lakes from Antarctica
[16], soils from Northern England [17], and BHhexol in
sediments from the popular Loch Ness (UK) [2].
CID ions mass spectra were acquired by colliding the
Q1 selected precursor ions with Ar gas at a collision target
gas and applying collision energy of 50 eV in Q2. Scan-
ning range of Q3 was m/z 200–1,600 with a step size of
m/z 0.3 and a dwell time of 1 ms. A peak threshold of 0.3%
intensity was applied to the mass spectra. The instrument
was interfaced to a computer running Applied Biosystems
Analyst version 1.4.1 software.
This report is part of our investigation of thermophilic
bacteria, mainly of the genus Alicyclobacillus. We extended
our analysis from glycophospholipids [18], unusual acyl-
phosphatidylglycerols [19], and O-acyl glycosylated cardio-
lipins [20] to hopanoids. The structure of N-acylated
BHhexol-mannosamides was confirmed not only by ESI–MS,
but, after isolation, also by measurement of 1H-, 13C-NMR,
and CD spectra, including synthesis of a model compound.
Gas chromatography–mass spectrometry of FAME was
done on a GC–MS system consisting of Varian 450-GC,
Varian 240-MS ion trap detector with electron impact
ionization, and CombiPal autosampler (CTC, USA). The
sample was injected onto a 25 m 9 0.25 mm 9 0.1 lm
Ultra-1 capillary column (Supelco, Czech Republic) under
a temperature program: 5 min at 50 °C, increasing at
10 °C/min to 320 °C and 15 min at 320 °C. Helium was
the carrier gas at a flow of 0.52 ml/min. All spectra were
scanned within the range m/z 50–600. The spectra was
identified manually, se also Siristova et al. [18].
Experimental
Instrumentation
The liquid chromatograph was the semipreparative Gradi-
ent LC System G-1 (Shimadzu, Kyoto, Japan) with two
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Lipids (2011) 46:249–261
251
The oven temperature for identification of the acetylated
alcohols BH (degradation products) was programmed from
50 to 275 °C at 10 °C/min and further at 5 °C/min to
350 °C and further with 8 min isothermal elution. The
others conditions of GC–MS apparatus were identical.
Saccharides were separated on a single HILIC HPLC
column (see above) by isocratic elution with acetonitrile
and 0.1% acetic acid in water at a ratio of 40:60. They were
identified by comparing their retention times with those of
commercially obtained standards (galactosamine, glucosa-
mine and mannosamine) and single ion monitoring at
m/z 180 ([M ? H]?).
phases. The aqueous phase was aspirated off and the
chloroform phase was washed three times with two parts
1 M KCl each. The resulting chloroform phase was evap-
orated to dryness under reduced pressure.
First, total lipid extracts were applied to Sep-Pak
Cartridge Vac 35 cc (Waters; with 10 g of aminopropyl-
silica-based polar bonded phase), and from the cartridge
were subsequently eluted chloroform–methanol mixture
(98:2) [22] for removing of non-polar lipids, and polar
lipids, but not phospholipids were further eluted by an
acetone. The eluate was reduced in volume and subjected
to normal phase HPLC.
NMR spectra were recorded on a Bruker AMX 500
spectrometer (Bruker Analytik, Karlsruhe, Germany) at
500.1 MHz (1H) and 125.7 MHz (13C). Optical rotations
were measured with a Perkin-Elmer 243 B polarimeter.
The circular dichroism measurement was carried out under
dry N2 on a Jasco-500A spectropolarimeter at 24 °C.
HR-FAB-MS (positive ion mode) were obtained with a
PEG-400 matrix using a VG 7070E-HF spectrometer. All
compounds were purchased from Sigma–Aldrich (Prague,
Czech Republic).
The aliquot of total BHs (5 mg) was stirred for l h at
room temperature with periodic acid (H5IO6; 3 mg) in
tetrahydrofuran/water (3 ml; 8:1 v/v). The vicinal diols
were oxidized to yield aldehyde products. After addition of
water (10 ml) the mixture was extracted with petroleum
ether (3 9 5 ml). The combined extracts were evaporated
and further stirred for l h at room temperature with sodium
borohydride (NaBH4; 1 mg) in ethanol (3 ml) to produce
alcohols. The excess of NaBH4 was destroyed by water and
the mixture was extracted with petroleum ether (3 9 5 ml).
The extracts were evaporated and acetylated, see below.
The total extract was acetylated overnight at room tem-
perature with acetic anhydride/pyridine (30 ml; 1:1 v/v).
The peracetylated BHs were evaporated and further
analyzed.
Cultivation, Isolation and Identification
Alicyclobacillus acidoterrestris CCM 4660 (Czech Collec-
tion of Microorganisms, Brno, Czech Republic) was culti-
vated in an alicyclobacillus medium containing (g/l): yeast
extract 6.0, glucose 5.0, CaCl2ꢀ2H2O 0.25, MgSO4ꢀ7H2O
0.5, (NH4)2SO4 0.2, KH2PO4 3.0 and 1 ml/l of trace element
solution (ZnSO4ꢀ7H2O 0.1 g/l, MnCl2ꢀ4H2O 0.03 g/l,
H3BO3 0.3 g/l, CuCl2ꢀ6H2O 0.2 g/l, CaCl2ꢀ2H2O 0.01 g/l,
NiCl2ꢀ6H2O 0.02 g/l, Na2MoO4ꢀ2H2O 0.03 g/l), pH was
adjusted to 4. A volume of 600 ml of inoculum was prepared
in 500-ml round-bottom flasks on a temperature controlled
shaker at 45 °C and 200 rpm. The biomass was cultivated in
a 5-l mechanically stirred fermentor with the aim of
obtaining inoculum for a 50-l fermentor. Inoculum was
cultivated for 12 h under aerobic conditions with aeration
rate 0.8 VVM. The stirrer frequency was 300–400 rpm and
the cultivation temperature was 45 °C.
The strain main cultivation was carried out in a 50-l
mechanically stirred fermentor for 15 h, under aerobic
conditions with an aeration rate of 1 VVM. The stirrer
frequency was 350–400 rpm and the cultivation tempera-
ture was 45 °C. Cells were harvested in the log phase when
the optical density of the culture was maximal. The dry
biomass was 1.49 g/l media.
The fraction of N-acylated BHhexol-glycoside after
semipreparative HPLC was treated for 16 h at 100 °C with
a 10% solution of dry HCl in CH3OH. The reaction mixture
was taken to dryness, suspended in water, the pH was
regulated by 5% NaOH to 9, BHhexol was extracted by
diethyl ether, the water mixture was acidified by 5% HCl to
pH 4, free fatty acids were extracted by CHCl3 and water
phase was after lyophilization was silylated, see above. The
fatty acid methyl esters were prepared by reaction of the
free fatty acids with methanol, see Rezanka et al. [23].
Preparation of 7-Deoxyheptitol
D-glycero-D-allo-Heptose (8a) and D-glycero-D-altro-
heptose (8b) were prepared from KCN and D-allose (6)
according to procedures described previously [24, 25].
Briefly, the pH of an aqueous solution (15 ml) of potassium
cyanide (269 mg, 4.14 mmol) was lowered to 7.2 by
dropwise addition of aqueous acetic acid (3 M) with rapid
stirring at room temperature. An aqueous solution (10 ml)
of D-allose 6 (248 mg, 1.38 mmol) was slowly added, with
addition of acetic acid (3 M) or sodium hydroxide (1 M) as
appropriate to maintain a pH of 7.2–7.5 during addition of
D-allose and subsequent reaction, which was allowed to
proceed for 1.5 h. The reaction mixture was analyzed by
TLC (cellulose sheets, 90:10 acetone–water) to reveal a
The extraction procedure was based on the method of
Bligh and Dyer [21]. The alcohol-water mixture was cooled
and one part chloroform was added and the lipids were
extracted for 30 min. Insoluble material was sedimented by
centrifugation and the supernatant was separated into two
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Lipids (2011) 46:249–261
complete conversion of D-allose (6) (Rf 0.67) into the
aldonitriles (7a and 7b) (Rf 0.8–0.9). The pH of the aldo-
nitrile solution was lowered to 4.7 by dropwise addition of
acetic acid (3 M) and the solution was stored at 4 °C for ca.
1 h. Further, the pH of the solution was again lowered to
4.2 by dropwise addition of acetic acid (3 M). Following
addition of prereduced 5% palladium on barium sulfate
(86 mg = 4.3 mg, 0.04 mmol Pd), the flask was evacuated
three times and flushed with hydrogen. Vigorous stirring
under an atmosphere of hydrogen was then allowed to
proceed at room temperature for ca. 15 h. The reaction
mixture was analyzed by TLC (90:10 acetone–water) to
reveal a complete reduction of the aldonitriles (7a and 7b)
(Rf 0.8–0.9) into the imines (Rf 0.4–0.6). The catalyst was
removed over Celite and the pH of the resultant filtrate was
lowered from pH 4.4 to ca. pH 3.0 by repeated batch-wise
treatment with DowexÒ 50WX2 hydrogen form 200–400
mesh (4 9 4 g). The final filtrate was analyzed by TLC
(95:5 acetone–water) and no imine was detected. The water
was removed by lyophilization to leave the epimeric
mixture of 8a and 8b as a pale yellow oil; the yield of
epimeric sugars was 243 mg (84%) and they were sepa-
rated by semi-preparative TLC.
reddish color, 5 ml of ethylthiol was added and shaking was
continued for 2.5 h. The product was allowed to stand
overnight in the refrigerator. About 5 ml of cold water was
added, the dithioacetal filtered, and washed with cold etha-
nol. The yield, after one recrystallization from ethanol, was
100 mg (63.3%). Very surprisingly, this compound (9) has
1
not yet been described. [a]D ?10.7 (c 0.16, MeOH); H-
NMR (500 MHz, CD3OD) d 4.92 (1H, d, J = 1.6, H-7), 4.93
(1H, dd, J = 1.6, 5.5, H-6), 5.06 (1H, t, J = 5.5, H-5), 5.19
(1H, t, J = 5.5, H-4), 4.71 (1H, t, J = 5.5, H-3), 4.65 (1H,
ddd, J = 5.5, 3.2, 8.2, H-2), 4.52 (1H, dd, H = 11.0, 3.2,
H-1a), 4.42 (1H, dd, J = 11.0, 8.2, H-1b), 2.89 (2H, m,
SCH2), 2.89 (2H, m, SCH2), 1.22 (6H, m, CH2CH3); 13C
NMR (125 MHz, CD3OD) 56.1 (C-7), 74.2 (C-6), 73.3
(C-5), 71.2 (C-4), 74.6 (C-3), 72.8 (C-2), 64.4 (C-1), 64.8
(SCH2), 15.0 (CH2CH3); HR-FAB-MS (m/z): 317.1099
[M ? H]?, calc. for [C11H24O6S2 ? H]? 317.1092.
7-Deoxy-D-glycero-D-allo-Heptitol, i.e. (2R,3R,4S,5S,
6S)-heptane-1,2,3,4,5,6-hexaol (10). Reductive desulfur-
ization of similar dithioacetals has already been described
[30]. To a solution of the derivative 9 (95 mg, 0.30 mmol)
in dioxane (5 ml) was added Raney Ni (300 mg). The
suspension was heated at 80 °C for 1 h with stirring to give
the derivative 9. Crystallization from absolute ethanol
yielded 43.5 mg (74%) of the desired desoxyheptitol as
clusters of needles, with an m.p. at 124–125 °C and [a]D
?1.7 (c 0.16, MeOH); 1H-NMR (500 MHz, CD3OD) d
3.65 (1 H, dd, J = 10.1, 11.5, 1a-H) and 3.84 (1 H, dd,
J = 3.4, 11.5, 1b-H), 3.77 (1 H, ddd, J = 2.2, 3.4, 10.1, 2-
H), 3.70 (1 H, dd, J = 3.9, 2.2, 3-H), 3.92 (1H, dd,
J = 3.9, 3.4, 4-H), 3.62 (1 H, dd, J = 3.4, 5.2, 5-H), 3.94
(1 H, dq, J = 5.2, 6.5, 6-H), 1.21 (3 H, d, J = 6.5, 7-H);
13C NMR (125 MHz, CD3OD) 19.1 (C-7), 70.4 (C-6), 73.2
(C-5), 71.7 (C-4), 74.2 (C-3), 72.7 (C-2), 64.1 (C-1);
HR-FAB-MS (m/z): 197.1025 [M ? H]?, calc. for
[C7H16O6 ? H]? 197.1025. Again, though it is hard to
believe, this compound has not yet been described.
Aldrich Analtech TLC UniplatesTM with spherical par-
ticles (size 50 lm) of a-cellulose matrix, size 20 cm 9
20 cm, layer thickness 250 lm, fluorescent indicator were
used. The mobile phase was acetone–water (95:5, v/v),
D-glucose being used as standard. Identification under UV
light gave the following Rf values for the compounds:
D-glycero-D-allo-heptose (8a) (0.35), D-glucose (0.51) and
D-glycero-D-altro-heptose (8b) (0.63). The yield of D-gly-
cero-D-allo-heptose (8a) was 106 mg, with [a]2D3 ?12.8
(1 day, c = 0.15), lit. data: [a]2D1 ?14.1 [26], [a]D ?10.7
(24 h) (c 1.6, H2O) [27], [a]D ?7.2 (3 min) ? ?12.7
(24 h) (c 4.6, H2O) [28]; 13C NMR (125 MHz, D2O) d
major: 93.8 (C-1), 73.8, 72.1, 71.3, 71.2, 67.7, 61.8; minor:
92.7 (C-1), 72.0, 71.7, 67.6, 67.1, 66.9, 62.1 [27, 28]; 13C
NMR d 94.4, 72.9, 72.0, 71.9, 74.6, 68.5, 62.6 (the values
for C-1 ? C-7 of b-pyranose), the values for C-1 were
93.4 (a-pyranose), 94.4 (b-pyranose), 96.8 (a-furanose),
and 101.4 (b-furanose) [29]. Our data measured for mixture
of four forms at C-1 were 93.2 (a-pyranose), 94.5
(b-pyranose), 97.0 (a-furanose), and 101.5 (b-furanose)
and the following values are for the major form: 13C NMR
(125 MHz, D2O) d 94.5 (C-1), 73.3 (C-2), 72.5 (C-3), 71.6
(C-4), 73.8 (C-5), 68.4 (C-6), and 62.2 (C-7); HR-FAB-MS
(m/z): 211.0823 [M ? H]?, calc. for [C7H14O7 ? H]?
211.0818.
General Procedure for Bichromophoric Derivatization
Anthroylation
To an ice cooled suspension of the 9-anthracenecarboxylic
acid (0.1 mmol, 22.2 mg) and fluoro-N,N,N0,N0-tetrameth-
ylformamidinium hexafluorophosphate (TFFH, 0.1 mmol,
26.4 mg) in a minimum of CH2Cl2 (ca. 5 ml) was added
Et3N (0.5 mmol, 50.5 mg, 70 ll) resulting in a clear solution
with some heat evolution. After stirring for 30 min at 20 °C
the alcohol (10) (0.1 mmol, 19.6 mg) and a catalytic amount
of DMAP (0.01 mmol, 1.22 mg) were added and the reac-
tion mixture was stirred at 20 °C overnight. Water (5 ml)
was added and the mixture extracted with CH2Cl2 (3 9
3 ml). The combined organic extracts were dried (MgSO4),
D-glycero-D-allo-Heptose diethyl thioacetal = (2S,3R,
4S,5S)-7,7-bis(ethylthio)heptane-1,2,3,4,5,6-hexaol. A mix-
ture of D-glycero-D-allo-heptose (8a) (105 mg) and concen-
trated HCl (5 ml) was shaken for about 10 min to dissolve
most of the sugar and then, as the solution began to assume a
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Lipids (2011) 46:249–261
253
filtered and concentrated in vacuo. The resulting crude
residue was purified by SPE with silica gel (MeOH/CH2Cl2,
1:9) to yield the desired ester (11) [yield 33.6 mg (84%)].
HR-FAB-MS (m/z): 401.1606 [M ? H]?, calc. for [C22
H24O7 ? H]? 401.1600. The eluate was concentrated, the
mixture was separated by normal phase HPLC (SupelcosilTM
LC-Si HPLC Column 5 lm particle size, L 9 I.D.
25 cm 9 10 mm, EtOAc-hexane, 3:7; 2 ml/min; 311 nm
UV detection), and used for the next step, cinnamoylation.
The preparation of the anthroyl derivative of BHhexol (13)
was performed similarly as with (11). From the 9-anthra-
cenecarboxylic acid (1 lmol, 222 lg), TFFH (1 lmol,
264 lg) in CH2Cl2 (200 ll), Et3N (5 lmol, 505 lg, 0.7 ll)
and the alcohol (3) (1 lmol, 578 lg) with catalytic amount of
DMAP (1 lmol, 122 lg) compound 13 was obtained after
purification by SPE with silica gel (MeOH-CH2Cl2, 2:98) in a
yield of 610 lg (78%). HR-FAB-MS (m/z): 783.5199
[M ? H]?, calc. for [C50H70O7 ? H]? 783.5204.
Bligh-Dyer extraction [21] of lyophilized cells and a part of
the total lipids was cleaved by H5IO6 followed by NaBH4
reduction. This releases mainly a mixture of alcohols
arising from the BHpolyol derivatives, identical in GC
retention times and mass spectra with the previously
described alcohols which were used for the identification of
BHpolyols from sediments [31]. Three alcohols were
identified by GC–MS, i.e. bishomohopanol representing
BHtetrols, homohopanol belonging to BHpentols, and the
BHhexol hopanol. The detection of hopanol suggests the
presence of a hopanoid with a hexafunctionalized side-
chain in the total lipid extract.
Further, part of the total lipids was fractionated by
means of cartridges with aminopropyl silica-based polar
bonded phase, which were rinsed in a chloroform–metha-
nol mixture (98:2) [22] for removing non-polar lipids.
Polar lipids, but not phospholipids, were further eluted by
acetone. Polar non-phospholipids (25% of total lipids, i.e.
385 mg) were further separated by normal phase HPLC, as
described by Moreau et al. [22]. Briefly, non-phospholipids
were subjected to semi-preparative normal phase HPLC
with a linear gradient in for 40 min and with detection at
210 nm, as described by Fox et al. [1]. Table 1 gives the
data obtained after normal phase semipreparative HPLC.
The relative amounts of the C32, C31 and C30 alcohols,
i.e. the relative contributions of tetra-, penta-, and hexa-
functionalized side-chains were in a 2:1:1 ratio, which is in
agreement with data from Table 1. This table also sum-
marizes appropriate classes of hopanoids, i.e. tetrols, pen-
tols, and hexols, whereas minority derivatives of BHs
(up to 2% of total lipids) were omitted.
Cinnamoylation
To an ice cooled suspension of the p-methoxycinnamic
acid (712 mg, 4 mmol) and fluoro-N,N,N0,N0-tetramethyl-
formamidinium hexafluorophosphate (TFFH, 5 mmol,
1.32 g) in a CH2Cl2 (35 ml) was added Et3N (25 mmol,
2.52 g, 3.48 ml) resulting in a clear solution with some
heat evolution. After stirring for 30 min at 20 °C the
anthroyl derivative (11) (30 mg, 75 lmol) and a catalytic
amount of DMAP (0.1 mmol, 12.2 mg) were added and the
reaction mixture was stirred at 20 °C overnight. H2O
(10 ml) was added and the mixture extracted with CH2Cl2
(3 9 5 ml). The combined organic extracts were dried
(MgSO4), filtered and concentrated in vacuo. The resulting
crude residue was purified by SPE with silica gel (MeOH/
CH2Cl2, 1:9) to yield (66.6 mg, i.e. 74%) the desired ester
(12). HR-FAB-MS (m/z): 1,201.4221 [M ? H]?, calc. for
[C72H64O17 ? H]? 1,201.4226.
Table 2 gives the data obtained after RP-HPLC from the
fraction of per-acetylated N-acylated BHhexol-glycosides,
Table 1 BH derivatives from A. acidoterrestris after normal phase
semipreparative HPLC
a
%
Compound
The preparation of cinnamoyl derivative of BHhexol
(14) was performed similarly as with the derivative (12).
From the p-methoxycinnamic acid (712 lg, 4 lmol), TFFH
(5 lmol, 1.32 mg) in CH2Cl2 (0.1 ml), Et3N (25 lmol,
2.52 mg, 3.5 ll) and the alcohol (13) (0.77 lmol, 603 lg)
with catalytic amount of DMAP (0.1 lmol, 12 lg), 915 lg
(75%) of compound (14) was obtained after purification by
SPE with silica gel (MeOH-CH2Cl2, 2:98). HR-FAB-MS
(m/z): 1,583.7816 [M ? H]?, calc. for [C100H110O17 ? H]?
1,583.7820.
N-acyls glucosamine BHtetrol
N-acyls-mannosamine-BHpentol
N-acyls-mannosamine-BHhexol
BHtetrol
29.3
5.9
11.2
12.5
8.6
BHpentol
BHhexol
6.7
BHtetrol-glucosamine
BHpentol-mannosamine
BHhexol-mannosamine
4.5
9.4
9.8
BHtetrol-ether
2.1
P
tetrols
P
48.4
23.9
27.7
Results
pentols
P
hexols
A. acidoterrestris was cultivated in a 50-l fermentor as
described previously [20]. The total lipids were obtained by
a
Minority derivatives of BHs (up to 2% of total lipids) were omitted
123

254
Lipids (2011) 46:249–261
m/z 711, 651, 591, 531 and 471 from sequential loss of the
remaining 5 acetylated OH groups from C30 to C34. The
ESI/MS spectrum (Fig. 2) of a proposed acetylated N-acyls
glycoside of BHhexol (2, n = 3) includes all these diag-
nostic ions as well as a further series m/z 579, 519, 459,
399, 339 and 279 from neutral loss of the A and B rings
(i.e. loss of 192 Da) after a loss of one to six of the acet-
ylated hydroxyls [32]. The composite hexafunctionalized
structure 2 is proposed on the basis of observation of the
diagnostic ions m/z 771 (etc.) and ions at m/z 554 (etc.),
indicating the mass of the terminal group, and by com-
parison with spectra reported for the penta- and hexa-
functionalized cyclitol ethers, see also Table 3.
Table 2 Per-acetylated N-acylated BHhexol-glycosides separated by
RP-HPLC and FAs content by GC–MS
a
%
b,c
%
Compound
N-C15-mannosamine-BHhexol
N-C17-mannosamine-BHhexol
N-C19-mannosamine-BHhexol
N-C21-mannosamine-BHhexol
N-C23-mannosamine-BHhexol
N-C25-mannosamine-BHhexol
N-C27-mannosamine-BHhexol
N-C29-mannosamine-BHhexol
2.7
30.8
54.7
5.8
2.94
31.13
54.41
6.11
3.2
2.98
1.7
1.52
0.9
0.76
0.2
0.15
a
Determined by ESI
b
The structure of pentacyclic nucleus including the side
chain was essentially determined by 1H-NMR spectroscopy
using COSY, ROESY, HMQC, and HMBC experiments.
Minority fatty acids up to 0.1% of total fatty acids were omitted
Determined by GC–MS
c
1
The H-NMR spectrum (data see Table 4) of acetylated
which had the broadest interval of molecular weights.
A total of 8 peaks were separated by LC–MS/ESI on
narrow-bore columns connected in series. The chromato-
gram is given in Fig. 1.
hopanoids (2) showed signals in the methyl region typical
of bacteriohopane derivatives. Both signals, including the
13C NMR, are in agreement with previously published
papers [7, 14, 33].
Diagnostic fragment ions for hexafunctionalized bacte-
riohopane structures include the ion at m/z 771 resulting
from the loss of the terminal group at C35 (either OH or
any other moiety linked to C35 via oxygen [32]) and
The ROESY spectrum displayed a number of correlation
peaks between methyl groups and ring protons in the 1,3
diaxial relationship. Thus, 24-Me, H-6ax, 25-Me, 26-Me,
and 13-H were shown to be all located on the upper face of
8
m/z 1494
C29
0
16
m/z 1466
C27
0
8
m/z 1438
m/z 1410
m/z 1382
C25
0
8
C23
0
8
C21
0
80
C19
m/z 1354
m/z 1326
m/z 1298
0
80
C17
0
12
C15
0
600
1200
1800
2400
3000
time (s)
Fig. 1 LC–MS/ESI SIM chromatograms of N-acylated BHhexol glycosides from total lipids of A. acidoterrestris after normal phase HPLC;
fraction of N-acyls-mannosamine-BHhexol is shown. The SIM traces depict [M ? H]? masses, see m/z values
123

Lipids (2011) 46:249–261
255
Fig. 2 The tandem quadrupole ESI product-ion spectrum of the [M ? H]? ion of N-C17-mannosamine-BHhexol at m/z 1,326
the molecule, whereas 27-Me, 28-Me, H-5ax, and H-9ax
were all on its lower face. Also the four-bond couplings
(W coupling) of an angular methyl group with a ring proton
in the COSY were observed. These couplings are only
possible when the dihedral angle between the methyl group
and the ring proton is near 180° and these couplings con-
firmed a trans connection of all the rings, hence the ste-
reochemistry of compound 2 was the same as in previously
isolated bacteriohopanoids. This was confirmed by HMBC
correlations between Me groups and carbons of the ring
system. Thanks to this fact the 13C-resonance (see dC
values in Table 5) of the five cycle moiety could be
determined [7, 14, 33].
exchangeable NH proton of the acylamide group as well as
dimensional proton/proton correlation allowed the whole
pattern of the substituent to be established as compatible
with the structure of an acylated sugar.
3
0
0
The large vicinal coupling constants, J3 ,4 = 9.5 Hz
3
and J4 ,5 = 9.5 Hz, and the NOE between H-30 and H-50
indicated that H-30, H-40 and H-50 protons were located at
an axial position. The observation of NOE between H-40
and 20-NH proton suggested that 20-NH was oriented in an
axial position. These data indicate that the monosaccharide
0
0
is a mannosamine in pyranose form; a configuration
1
was determined by a large coupling constant, JC-1 ,H-1
0
0
=
3
0
0
166.1 Hz [34] and small JH-1 -H-2 = 1.8 Hz [35]. This
implies also that the linkage between the BHhexol and the
mannosamine moiety is an O-glycosidic bond.
1
The H-NMR spectrum of the octaacetate of compound
(2) shows that the signals of the protons at C30 to C34 are
nearly identical to those of the corresponding protons of the
synthetic tetraacetate glycoside [33] and/or isolated
pentaacetate of aminoBHtetrol [14] and are easily identi-
fiable. Decoupling experiments performed on the C34
proton permitted an assignment of the signals of the two
C35 protons which are nearly equivalent and appear as two
doublets at 3.68 and 3.98 ppm. The fact that the signals of
the C35 protons as well as the signal of the C35 carbon
atom were shifted in comparison to the signals of the
corresponding atoms of acetylated BHpolyol [9] indicated
that a sugar moiety was located on the oxygen atom at C35.
From the 1H-NMR spectrum eight acetoxy groups are
present: five at C30 to C34 and three on the residue
linked at C35. Decoupling experiments initiated on the
HMBC analysis indicated that 2-NH0 of the mannosa-
mine was esterified by a fatty acyl group by the correlation
peak between H-20 (d 4.63) and C-100 (d 172.8) of the fatty
group and that 3-OH0, 4-OH0, and 6-OH0 of the mannosa-
mine moiety were esterified by acetates by the correlation
peaks between H-30 (d 5.38), H-40 (d 5.06), and H-60 (d 4.30,
4.14) and the other three carbonyl carbons (d 170.7, 170.6,
170.2). The key HMBC are showed in Fig. 3.
The components of N-acylated BHhexol-mannosamide
were also analyzed after hydrolysis (Fig. 4). The hydroly-
zate contained BHhexol as the aglycone moiety and
mannosamine was the only sugar which was detected by
GC–MS in this fraction. Mannosamine had [a]2D5-4.1, com-
pared to the literature data, [a]2D5 -4.2 for D-mannosamine
123

256
Lipids (2011) 46:249–261
Table 4 1H NMR of compound 2 (500 MHz, CDCl3)
Table 3 The main molecular species of N-acylated BHhexol glyco-
side (2) from A. acidoterrestris
No.
1H
Ions
m/z
Abundance
22
23
24
25
26
27
28
29
30
31
32
33
34
2.23 (1H, m)
0.80 (3H, s)
0.83 (3H, s)
0.84 (3H, s)
0.94 (3H, s)
0.96 (3H, s)
0.69 (3H, s)
M ? H
1,326.8
1,266.8
1,206.8
1,146.7
1,134.7
1,086.7
1,026.7
966.7
836.6
776.6
771.5
729.9
716.5
711.5
669.5
651.5
609.5
591.5
579.4
556.4
538.4
549.4
531.4
519.4
494.4
489.4
471.4
459.4
434.4
399.3
374.3
339.3
279.3
100
18
41
32
38
24
19
47
32
21
65
43
49
23
12
42
37
54
28
67
89
19
37
18
32
64
19
61
31
27
42
27
37
M ? H-AcOH
M ? H-2AcOH
M ? H-3AcOH
M ? H-192
M ? H-4AcOH
M ? H-5AcOH
M ? H-6AcOH
M ? H-4AcOH-Acyl
M ? H-5AcOH-Acyl
M ? H-SC
0.92 (3H, d, J22,29 = 6.5)
5.24 (1H, dd, J22,30 = 2.1; J30,31 = 9.0)
5.23 (1H, dd, J30,31 = 9.0; J31,32 = 3.5)
5.26 (1H, dd, J31,32 = 3.5; J32,33 = 6.5)
5.33 (1H, dd, J32,33 = 6.5; J33,34 = 4.2)
M ? H-SC-42
5.10 (1H, ddd, J33,34 = 4.2; J34,35a = 7.2;
J34,35b = 3.8)
M ? H-6AcOH-Acyl
M ? H-SC-AcOH
M ? H-SC-AcOH-42
M ? H-SC-2AcOH
M ? H-SC-2AcOH-42
M ? H-SC-3AcOH
M ? H-SC-192
SC ? 18
35a
35b
10
3.68 (1H, dd, J34,35a = 7.2; J35a,35b = 11.0)
3.98 (1H, dd, J34,35b = 3.8; J35a,35b = 11.0)
0
4.78 (1H, d, J1 ,2 = 1.8)
0
20
0
0
0 0
4.63 (1H, ddd, J1 ,2 = 1.8; J2 3 = 4.8;
0
J2 NH = 9.0)
30
40
50
0
5.38 (1H, dd, J2 ,3 = 4.8; J3 4 = 9.5)
0
0 0
0
0
0
5.06 (1H, t, J3 4 =4 ,5 = 9.5)
0
0
3.96 (1H, ddd, J4 5 = 9.5; J5 6a = 4.5;
0 0
J5 6b = 2.5)
0
0
0
SC
M ? H-SC-3AcOH-42
M ? H-SC-4AcOH
M ? H-SC-AcOH-192
SC-AcOH
6a0
6b0
0
4.14 (1H, dd, J5 6a = 4.5; J6a 6b = 12.5)
0
0
0
0
0
0
4.30 (1H, dd, J5 6a = 2.5; J6a 6b = 12.5)
0
0
6.45 (1H, d, J2 NH = 9.0)
NH
2000
3000
4000-x8
x7
2.14 (2H, t, J = 7.5)
1.66 (2H, m)
M ? H-SC-4AcOH-42
M ? H-SC-5AcOH
M ? H-SC-2AcOH-192
SC-2AcOH
1.23–1.45 (14H, m)
1.14 (2H, m)
Cyclohexyl’s CH
Cyclohexyl’s CH2
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
1.36 (1H, m)
M ? H-SC-3AcOH-192
SC-3AcOH
1.08–1.68 (m)
2.018 (3H, s)
2.023 (3H, s)
2.029 (3H, s)
2.036 (3H, s)
2.036 (3H, s)
2.049 (3H, s)
2.054 (3H, s)
2.054 (3H, s)
M ? H-SC-4AcOH-192
M ? H-SC-5AcOH-192
(2-amino-2-deoxy-D-mannopyranose) [36]. The mixture of
homologous x-cyclohexyl fatty acids, isolated from the
hydrolyzate, was also determined by GC–MS. The fatty
acid composition obtained by GC–MS was nearly identical
with the results obtained from ESI analysis (see Table 2).
Bisseret and Rohmer [37], who synthesized the eight
possible diastereomers of BHtetrols, established the abso-
lute stereochemistry of the side chain on the basis of their
1H-NMR spectra. Since the 32 diastereomers for BHhexols
have not yet been synthesized, we had to use the CD exciton
chirality method [38] that has been applied to sugar polyols
[39] and/or different acyclic polyols such as BHpolyols [40]
with up to five contiguous OH groups, or aminoBHpentols
[41, 42]. These CD studies clearly showed that with flexible
compounds it is preferable to explore the exciton coupling
between two different chromophores such as anthroyl and
cinnamoyl.
Based on the similarity of CD spectra of our compound
(BHhexol, 14) and the previously published aminoBH-
pentol derivative [41], we concluded that our compound is
also a homolog of allo configuration. To verify this
hypothesis, we consequently prepared from a commercially
123

Lipids (2011) 46:249–261
257
Table 5 13C NMR of compound 2 (125 MHz, CDCl3)
available saccharide D-allose (6) appropriate 7-deoxy-D-
glycero-D-allo-heptitol (10) via the D-glycero-D-allo-
heptose (8a) according to Fig. 5. This helped us remove the
obstacle mentioned by Zhou et al. [41], viz. that configu-
rational determination of aminoBHpentol is not straight-
forward due to the lack of reference hexols.
No.
13C
No.
13C
1
40.4t
18.7t
42.2t
33.3s
56.2d
18.7t
33.2t
41.5s
50.5d
37.5s
20.8t
24.0t
49.3d
41.8s
33.7t
22.6t
54.2d
44.5s
41.6t
27.9t
42.9d
38.1d
21.5q
33.3q
15.8q
16.4q
16.5q
15.9q
16.4q
69.8d
70.3d
71.0d
33
71.6d
71.0d
64.5d
100.3d
55.4d
69.3d
71.4d
68.5d
62.1t
2
34
3
35
10
20
30
40
50
60
100
200
4
The original version of the Kiliani-Fischer synthesis was
modified according to several authors [24, 25]. Instead of
conversion of the cyanohydrin (aldonitrile) to a lactone, the
cyanohydrin is reduced with hydrogen (palladium on bar-
ium sulfate) in water to an imine that quickly hydrolyzes at
pH 4.2 to an aldehyde; thus the final sugars are produced in
just two steps rather than three and, moreover, the final
sugars are separated instead of the lactones. We used
semipreparative TLC on cellulose plates and employed a
previously described solvent system (acetone–water 95:5,
v/v) with D-glucose as standard. The compound with an Rf
lower than glucose was D-glycero-D-allo-heptose (8a)
while that with an Rf higher than glucose was D-glycero-D-
altro-heptose (8b), see also paper [25]. The measurement
of 13C-NMR spectra of (8a) showed that, in keeping with
literature data [27], this compound is present as a mixture
of four forms, i.e. a and b pyranoses and a and b furanoses
in a ratio of 15:75:5:5. Experimental part gives only the
values for the most copious form, i.e. b-pyranose.
Heptose (8a) was converted first to the diethyl thioacetal
(9), and then, by reductive desulfurization with Raney
nickel, to 7-deoxy-D-glycero-D-allo-heptitol (10). This des-
oxyheptitol was esterified by selective anthroylation of
primary hydroxyl by anthracene-9-carboxylic acid to (2R,
3R, 4S, 5S, 6S)-2,3,4,5,6-pentahydroxyheptyl anthracene-9-
carboxylate (11). To make sure that we had indeed the
appropriate and pure epimer (viz. the separation of heptoses
by TLC after reduction) we used a separation of this com-
pound by means of normal phase HPLC, see also Wiesler
and Nakanishi [43]. They reported that mixtures of sac-
charides (after cyanohydrin synthesis) could be directly
subjected to anthroylation, after which separation of the
fluorescent monoesters was greatly simplified. The
secondary hydroxyls of anthroyl derivative (11) were per-
p-methoxycinnamoylated to an appropriate ‘‘bichromo-
phoric’’ derivative (12) and CD spectrum of this compound
was measured, see Fig. 6. The curve for L-allo (12) is plotted
as a mirror image of the D-series, see also Zhou et al. [39].
On the basis of all above mentioned spectra (including CD
spectrum of synthetic compound 12), we confirmed that the
configuration of the side chain is 30R, 31R, 32R, 33S,
34S (D-glycero-D-allo) and the components of this fraction
areN-acylated-mannosamine-BHhexols whichcontain—a-D-
2-amino-2-deoxymannopyranose esterified by x-cyclohexyl
fatty acids from C-15 to C-29. The correct name of the
glycoside with C-17 fatty acids is 11-cyclohexyl-N-
((2S,3S,4R,5S,6R)-2-((2S,3S,4R,5R,6R,7S)-7-((3S,3aS,5aR,
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
172.8s
34.1t
300
400-x8
x7
24.8t
27.4–30.0t
38.5t
Cyclohexyl’s CH
Cyclohexyl’s CH2
C=O
29.1d
26.6–34.6t
172.8s
170.7s
170.6s
170.5s
170.2s
169.7s
169.5s
169.4s
21.1q
21.1q
20.9q
20.8q
20.7q
20.6q
20.5q
20.4q
C=O
C=O
C=O
C=O
C=O
C=O
C=O
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
R2
3´´
O
OAc
H
1´´
NH
O
AcO
2´
H
AcO
H
1´
H
H
O
H
35
H
(S)
HMBC
34
AcO
R1
Fig. 3 Partial structure of a new compound (2). Only important
HMBC correlations are shown
123

258
Lipids (2011) 46:249–261
NH2
3´´
(
)
n
HO
OH
OH
OH
H
1´´
NH2
O
O
OH
30
OH
OH
HN
O
HO
H
H+; OH-
H
H
HO
12
O
OH
H
(S)
(R)
(R)
(R)
(S)
(S)
H
2´
OH
22
29
33
1´
35
(S)
(R)
OH
5
H
OH
OH
O
(R)
H+; OH-
OH
H
(S)
1 n=1,3,5,7,9,11,13,15
COOH
3
)
(
OH
n
H
Ac2O+Py
4
n=1,3,5,7,9,11,13,15
H+; OH-
OH
OH
OH
H
H
H
OH
(S)
(R)
(R)
OH
3
(R)
(S)
(S)
(S)
(R)
)
(
n
OH
(R)
H
OAc
OAc
OAc
HN
O
(S)
H
H
H
O
OAc
(S)
(S)
(S)
(R)
(R)
(R)
(S)
(S)
9-anthracenecarboxylic acid,
TFFH, Et3N, DMAP
H
(S)
(S)
(R)
OAc
OAc
O
(S)
OAc
(S)
(S)
2 n=1,3,5,7,9,11,13,15
H
H
(S)
OAc
H
OH
OH
OH
H
H
H
(S)
(R)
(R)
(R)
(S)
(S)
(S)
(R)
O
OH
OH
O
(R)
OMe
OMe
OMe
H
(S)
H
13
O
O
O
p-methoxycinnamic acid,
TFFH, Et3N, DMAP
O
O
O
H
H
H
(S)
(R)
(R)
(R)
(S)
(S)
(S)
(R)
O
O
O
O
O
(R)
O
H
(S)
H
MeO
MeO
14
Fig. 4 Structure of 1 and synthesis of appropriate derivatives
5bR,7aS,11aS,11bR,13aR,13bS)-5a,5b,8,8,11a,13b-hexame-
thylicosahydro-1H-cyclopenta[a]chrysen-3-yl)-2,3,4,5,6-
pentahydroxyoctyloxy)-4,5-dihydroxy-6-(hydroxymethyl)
tetrahydro-2H-pyran-3-yl)undecanamide (1).
derivatives, have so far been identified only in sediments
but their configuration has been unknown [2, 16, 17].
Based on the comparison with the synthesized model
compound (12), we determined its absolute configuration
by using CD. Still, the identification of a novel hopanoid
from A. acidoterrestris is not too surprising since only
several tens of bacterial and cyanobacterial genera have
been tested for the presence of hopanoids [16]. Although
the biological significance of our compound is not yet
clear; it is probable that in A. acidoterrestris the long chain
x-cyclohexyl FAs, which are bound by an amidic bond to
Discussion
To the best of our knowledge, this is the first report of
BHhexol from a specific bacterium. Bacteriohopanoids
having hexol in the side chain, including their composite
123

Lipids (2011) 46:249–261
259
123

260
Lipids (2011) 46:249–261
Acknowledgments The research was supported by projects of the
Ministry of Education, Youth and Sports of the Czech Republic (no.
1M06011; GACR P503/10/P182), and by Institutional Research
Concepts AV 0Z 502 0910 and MSM6046137305.
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