A novel type of highly negatively charged lipooligosaccharide
from
Pseudomonas stutzeri
OX1 possessing two
4,6-
O
-(1-carboxy)-ethylidene residues in the outer core region
Serena Leone
1
, Viviana Izzo
2
, Alba Silipo
1
, Luisa Sturiale
3
, Domenico Garozzo
3
, Rosa Lanzetta
1
,
Michelangelo Parrilli
1
, Antonio Molinaro
1
and Alberto Di Donato
2
1
Dipartimento di Chimica Organica e Biochimica and
2
Dipartimento di Chimica Biologica, Universita
`degli Studi di Napoli Federico II,
Napoli, Italy;
3
Istituto per la Chimica e la Tecnologia dei Materiali Polimerici – ICTMP – CNR, Catania, Italy
Pseudomonas stutzeri OXI is a Gram-negative microorgan-
ism able to grow in media containing aromatic hydrocar-
bons. A novel lipo-oligosaccharide from P. stutzeri OX1
was isolated and characterized. For the first time, the pres-
ence of two moieties of 4,6-O-(1-carboxy)-ethylidene resi-
dues (pyruvic acid) was identified in a core region; these two
residues were found to possess different absolute configur-
ation. The structure of the oligosaccharide backbone was
determined using either alkaline or acid hydrolysis. Alkaline
treatment, aimed at recovering the complete carbohydrate
backbone, was carried out by mild hydrazinolysis (de-O-
acylation) followed by de-N-acylation using hot KOH. The
lipo-oligosaccharide was also analyzed after acid treatment,
attained by mild hydrolysis with acetic acid, to obtain
information on the nature of the phosphate and acyl groups.
The two resulting oligosaccharides were isolated by gel
permeation chromatography, and investigated by composi-
tional and methylation analyses, by MALDI mass spectro-
metry, and by
1
H-,
31
P- and
13
C-NMR spectroscopy. These
experiments led to the identification of the major oligosac-
charide structure representative of core region-lipid A. All
sugars are
D
-pyranoses and a-linked, if not stated otherwise.
Based on the structure found, the hypothesis can be ad-
vanced that pyruvate residues are used to block elongation
of the oligosaccharide chain. This would lead to a less
hydrophilic cellular surface, indicating an adaptive response
of P. sutzeri OX1 to a hydrocarbon-containing environment.
Keywords:Pseudomonas stutzeri OXI; lipopolysaccharide;
NMR spectroscopy; mass spectrometry; pyruvic acid.
Environmental pollution is recognized worldwide as an
emergency for its negative effects on the biosphere and on
human health. Bioremediation strategies have recently been
devised, based on microbial biotransformations, given the
metabolic potential of selected microorganisms, in partic-
ular by Gram-negative bacteria, and their adaptability to
many different pollutants [1].
Pseudomonas stutzeri OX1 is a Gram-negative bacterium
isolated from the activated sludge of a wastewater treatment
plant, and endowed with unusual metabolic capabilities for
the degradation of aromatic hydrocarbons [2]. In fact, in
contrast with other Pseudomonas strains, this microrganism
is able to grow on a large spectrum of aromatic compounds
including phenol, cresol and dimethylphenol, and on
nonhydroxylated molecules such as toluene and o-xylene,
the most recalcitrant isomer of xylene. Moreover, it is able to
metabolize tetrachloroethylene (PCE), one of the ground-
water pollutants commonly resistant to degradation [3].
Degradation of aromatic hydrocarbons by aerobic bac-
teria comprises an upper pathway, which produces dihy-
droxylated aromatic intermediates by the action of
monooxygenases, and a lower pathway, which processes
these intermediates to molecules that enter the citric acid
cycle [4]. We have recently cloned, expressed and charac-
terized three different enzymatic systems from P.stutzeri
OX1: (a) toluene-o-xylene monooxygenase (ToMO) [5],
endowed with a broad substrate specificity [6] and (b)
phenol hydroxylase (PH) [7], both belonging to the upper
pathway; and (c) catechol 2,3 dioxygenase (C2,3O) (A. di
Donato, unpublished observations), the gatewayenzyme
to the lower pathway. However, chemical toxicity of wastes
can hamper the use of this and other microorganisms in
bioremediation strategies, especially when organic solvents
are present at high concentrations.
Several mechanisms have been described that contribute
to solvent resistance in Gram-negative bacteria, all based on
structural changes in outer and inner membranes [8].
Different short- and long-term responses have been
observed including modifications of the fatty acid and
phospholipid composition of the membrane, extrusion
mechanisms using vesicles, and energy-dependent active
efflux pumps that export toxic organic solvents outside the
cytoplasm [9].
Correspondence to A. Molinaro, Dipartimento di Chimica Organica e
Biochimica, Universita
`di Napoli Federico II, Complesso
Universitario Monte S. Angelo, via Cintia 4, 80126 Napoli, Italy.
Fax: + 39 081 674393, Tel.: + 39 081 674123,
E-mail: molinaro@unina.it
Abbreviations: DEPT, distorsionless enhancement by polarization
transfer; GlcN, 2-amino-2-deoxy-glucose; Hep,
L
-glycero-
D
-
manno-heptose; Kdo, 3-deoxy-
D
-manno-oct-2-ulosonic acid;
LOS, lipooligosaccharide; LPS, lipopolysaccharide.
(Received 1 March 2004, revised 23 April 2004,
accepted 30 April 2004)
Eur. J. Biochem. 271, 2691–2704 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04197.x
Even though lipopolysaccharides (LPSs) are major
components of the outer membrane of Gram-negative
bacteria, little is known about their role and their chemical
modifications under environmental stress [1,9]. It is certain,
however, that LPSs are unique and vital components of
these microorganisms and that they play an important role
in their survival and their interaction with the environment
[10,11]. Smooth-form lipopolysaccharides (S-LPSs) include
three regions, the O-specific polysaccharide (or O-antigen),
the oligosaccharide region (core region) and the lipid part
(lipid A). Conversely, rough (R) form LPSs do not possess
an O-specific polysaccharide and are frequently named lipo-
oligosaccharides (LOSs). LOSs have been found either in
wild-type strains and in mutant strains harboring mutations
in the genes encoding enzymes of the biosynthesis and/or
the transfer of the O-specific polysaccharide [12,13].
The core region from both smooth and rough forms of
enteric bacteria generally includes oligosaccharides built of
up to 11 units [12,13], and consists of two distinct domains:
an inner core, characterized by the presence of 3-deoxy-
D
-
manno-oct-2-ulosonic acid (Kdo) and
L
-glycero-
D
-manno-
heptose (Hep), and an outer core, which contains common
sugars. It is worth noting that the core oligosaccharide of
LOSs has been reported to play a role in the interaction of
the microorganism with the environment [12,13].
In this paper, the structural characterization of the
carbohydrate backbone of the rough form LPS of P. stutzeri
OX1 is reported, obtained by chemical analyses, MALDI-
TOF mass spectrometry and two-dimensional NMR
spectroscopy. This novel oligosaccharide chain was found
to possess unusual structural features, which might be
biologically relevant. Among these is a GalN residue
substituted by two gluco-configured residues, which are
blocked at position O-4 and O-6 by a pyruvate ketal linkage,
a structure peculiar and new to lipopolysaccharide core
regions.
Based on this finding, the hypothesis can be advanced
that the insertion of pyruvate residues at the end of the
oligosaccharide chain blocks its elongation, thereby leading
to a shorter LOS and hence to a less hydrophilic cellular
surface. Moreover, as it has already been proposed [1,9],
these residues may also contribute to the rigidity and
stability of the Gram-negative cell wall by binding cations.
Experimental procedures
Bacterial growth and LPS extraction
Cells were routinely grown on M9-agar plates supplemented
with 10 m
M
malic acid as the sole carbon source, at 27 C.
For growth in liquid medium, 1 mL was inoculated with a
single colony from a fresh plate, and grown for 18 h at 27 C
with constant shaking. This saturated culture was used to
inoculate 100 mL of the same medium and grown at 27 C
until D
600
1. Final growth was started by inoculating the
appropriate volume of the latter culture into 1 L of fresh
medium, to D
600
¼0.02. Cells were grown at 27 C, until
D
600
¼1 was reached and then recovered by centrifugation
at 3000 gfor 15 min at 4 C, washed with an isotonic buffer
and lyophilized. Growth was carried out in M9 salt medium
supplemented with 4 m
M
phenol as the sole carbon and
energy source. Dried cell yield was 0.13 gÆL
)1
.
Dried cells were extracted three times with a mixture of
aqueous 90% phenol/chloroform/petroleum ether (50 mL,
2 : 5 : 8 v/v/v) as described previously [14]. After removal of
the organic solvents under vacuum, the LOS fraction was
precipitated from phenol with water, washed first with
aqueous 80% (v/v) phenol, and then three times with cold
acetone, each time centrifuged as above, and lyophilized
(the yield was 90 mg of LOS, about 4.3% of the dry mass).
Sodium dodecyl sulfate polyacrylamide gel electrophor-
esis (SDS/PAGE) was performed as described previously
[15]. For detection of LPS and LOS, gels were stained with
silver nitrate [15].
Isolation of oligosaccharides
An aliquot of LOS (40 mg) was dissolved in anhydrous
hydrazine (2 mL), stirred at 37 C for 90 min, cooled,
poured into ice-cold acetone (20 mL), and allowed to
precipitate. The precipitate was then centrifuged (3000 g,
30 min, 4 C), washed twice with ice-cold acetone, dried,
dissolved in water and lyophilized (32 mg, 80% of the
LOS). This material was de-N-acylated with 4
M
KOH as
described [16]. Salts were removed using a Sephadex G-10
(Pharmacia) column (50 ·1.5 cm). The resulting oligosac-
charide 1constitutes the complete carbohydrate backbone
of the lipid A-core region (16 mg, 40% of the LOS).
Another aliquot of LOS (40 mg) was hydrolyzed in 1%
(v/v) acetic acid (100 C, 2 h) and the precipitate (lipid A)
was removed by centrifugation (8000 g,30min).The
supernatant was separated by gel-permeation chromatog-
raphy on a P-2 column (85 ·1.5 cm). Two fractions were
obtained, the first contained oligosaccharide 2(28 mg, 70%
of the LOS), whereas the second fraction contained a
mixture of reducing pyranose, furanose, anhydro and
lactone forms of 3-deoxy-
D
-manno-oct-2-ulosonic acid
(3 mg, 7.5% of the LPSs).
General and analytical methods
Determination of Kdo, neutral sugars, carbamoyl analysis,
including the determination of the absolute configuration of
the heptose residues, organic bound phosphate, absolute
configuration of the hexoses, fatty acids and their absolute
configuration, GLC and GLC-MS were all carried out as
described previously [17–21]. For methylation analysis of
Kdo region, LOS was carboxy-methylated with methanolic
HCl (0.1
M
,5min)andthenwithdiazomethanetoimprove
its solubility in dimethyl sulfoxide. Methylation was carried
out as described [22,23]. LOS was hydrolyzed with 2
M
trifluoroacetic acid (100 C, 1 h), carbonyl-reduced with
NaBD
4
, carboxy-methylated as described above, carboxyl-
reduced with NaBD
4
(4 C, 18 h), acetylated and analyzed
by GLC-MS.
Methylation of the complete core region was carried out
as described previously [22–24]. The sample was hydrolyzed
with 4
M
trifluoroacetic acid (100 C, 4 h), carbonyl-reduced
with NaBD
4
, acetylated and analyzed by GLC-MS.
NMR spectroscopy
For structural assignments of oligosaccharides 1and 2,1D
and 2D
1
H-NMR spectra were recorded on a solution of
2692 S. Leone et al.(Eur. J. Biochem. 271)FEBS 2004
5mgin0.6mLofD
2
O, at 55 Corat30C, at pD 14 and
7 (uncorrected values), respectively.
1
H- and
13
C-NMR
experiments were carried out using a Varian Inova 500 or a
Varian Inova 600 instrument, whereas for
31
P-NMR spectra
a Bruker DRX-400 spectrometer was used. Spectra were
calibrated with internal acetone [d
H
2.225, d
C
31.45].
Aqueous 85% phosphoric acid was used as external
reference (0.00 p.p.m.) for
31
P-NMR spectroscopy.
Nuclear Overhauser enhancement spectroscopy
(NOESY) and rotating frame Overhauser enhancement
spectroscopy (ROESY) were measured using data sets
(t
1
·t
2
)of4096·1024 points, and 16 scans were acquired.
A mixing time of 200 ms was used. Double quantum-filtered
phase-sensitive COSY experiments were performed with
0.258 s acquisition time, using data sets of 4096 ·1024
points, and 64 scans were acquired. Total correlation
spectroscopy experiments (TOCSY) were performed with a
spinlock time of 80 ms, using data sets (t
1
·t
2
)of
4096 ·1024 points, and 16 scans were acquired. In all
homonuclear experiments the data matrix was zero-filled in
the F1 dimension to give a matrix of 4096 ·2048 points and
was resolution enhanced in both dimensions by a shifted
sine-bell function before Fourier transformation. Coupling
constants were determined on a first-order basis from 2D
phase sensitive double quantum filtered correlation spectro-
scopy (DQF-COSY) [25,26]. Intensities of NOE signals were
classified as strong, medium and weak using cross-peaks
from intraring proton-proton contacts for calibration.
Heteronuclear single quantum coherence (HSQC) and
heteronuclear multiple bond correlation (HMBC) experi-
ments were measured in the
1
H-detected mode via single
quantum coherence with proton decoupling in the
13
C
domain, using data sets of 2048 ·512 points, and 64 scans
were acquired for each t
1
value. Experiments were carried
out in the phase-sensitive mode according to the method of
States et al. [27]. A 60 ms delay was used for the evolution
of long-range connectivities in the HMBC experiment. In all
heteronuclear experiments the data matrix was extended to
2048 ·1024 points using forward linear prediction extra-
polation [28,29].
MALDI-TOF analysis
MALDI mass spectra were carried out in the negative
polarity in linear or in reflector mode on a Voyager STR
instrument (Applied Biosystems, Framingham, MA, USA)
equipped with a nitrogen laser (k¼337 nm) and provided
with delayed extraction technology. Ions formed by the
pulsed laser beam were accelerated through 24 kV. Each
spectrum is the result of approximately 200 laser shots. A
saturated solution of 2,4,6-trihydroxyacetophenone was
used as the matrix.
Results
Isolation and characterization of the LOS fraction
The LOS fraction was isolated from dried cells by extraction
with phenol/chloroform/petroleum ether, and further puri-
fied with gel permeation chromatography. SDS/PAGE
showed, after silver nitrate gel staining, the presence of fast
migrating species in agreement with their oligosaccharide
nature. Compositional monosaccharide analysis of the LOS
fraction led to the identification of
L
,
D
-Hep,
D
-GalN,
D
-GlcN,
D
-Glc, Kdo (2 : 1.0 : 3.2 : 1.1 : 1.8) and trace
amounts of
L
-Rha. 7-O-Carbamoyl-
L
,
D
-Hep was present in
a stoichiometric ratio with
L
,
D
-Hep. Methylation analysis
showed the presence of terminal Kdo, 6-substituted-HexN,
3-substituted-Hep, 4,5-disubstituted-Kdo, 3,4-disubstituted-
HexN, 4,6-disubstituted-Glc, 4,6-disubstituted-HexN and,
in small amounts, terminal-Rha and 6-substituted-Glc.
In addition, the disaccharide 7-O-carbamoyl-Hep-(1fi3)-
Hep was found.
Fatty acid analysis revealed the presence of typical fatty
acids of pseudomonads LPS [30], i.e. (R)-3-hydroxydodec-
anoic acid [C12:0 (3-OH)], present exclusively in amide
linkage and (R)-3-hydroxydecanoic [C10:0 (3-OH)] (S)-2-
hydroxydecanoic [C12:0 (2-OH)] and dodecanoic acid
(C12:0), present in ester linkage. Moreover, phosphate
colorimetric assays gave positive results.
The LOS fraction was then subjected to both alkaline and
acid degradations and complete structural characterization.
NMR spectroscopy and MALDI-TOF MS spectrometry
of oligosaccharide 1
Oligosaccharide 1was isolated by gel permeation chroma-
tography after complete deacylation of the LOS of
P. stutzeri OX1. The complete structure of fully deacylated
oligosaccharide 1(Fig. 1) was determined by
1
H-,
31
P- and
13
C-NMR spectroscopy. Chemical shifts were assigned
using DQF-COSY, TOCSY, NOESY, ROESY,
1
H,
13
C-
DEPT-HSQC,
1
H,
31
P-HSQC,
1
H,
13
C-HMBC and
1
H,
13
C-
HSQC-TOCSY experiments. Anomeric configurations
were assigned on the basis of
1
Hand
13
C chemical shifts,
of
3
J
H1,H2
values determined from the DQF-COSY experi-
ment (Table 1), and of
1
J
C1,H1
values derived by
1
H,
13
C-
HSQC spectrum recorded without decoupling during
acquisition.
All sugars were present as pyranose rings, as indicated by
1
H- and
13
C-NMR chemical shifts and by the HMBC
spectrum that showed for all residues intraresidual scalar
connectivity between H-1/C-1 and C-5/H-5 atoms (for Kdo
units, between C-2 and H-6). The anomeric region of the
1
H-NMR spectrum (Fig. 2) showed seven major signals in
the region between 5.46 and 4.47 p.p.m. relative to seven
different spin systems (A–G, in order of decreasing chemical
shift), and in addition two AB methylene resonances at high
fields, typical of Kdo residues (I–L). Each spin system was
completely assigned by COSY and TOCSY starting from
anomeric resonances. For Kdo residues Iand Lthe starting
point was the H-3 diastereotopic methylene resonance.
Both spin systems Aand D(5.46 and 5.27 p.p.m.) were
characterized by low
3
J
H1, H2
and
3
J
H2, H3
values, indicative
of two a-manno-configured residues. Moreover, all other
cross peaks within each spin system were assigned in the
TOCSY spectrum from H-2 proton signals, leading to their
identification as two heptoses. Residue Bwas identified as
a-gluco-configured hexosamine on the basis of chemical
shifts and
3
J
H,H
values. Moreover, based on its anomeric
signal at 5.42 p.p.m. present as a double doublet (
3
J
H1,H2
¼
2.9 Hz and
3
J
H1,P
¼8.3 Hz), with one of the couplings due
to a phosphate signal as shown below, it was identified as
GlcN I of the lipid A skeleton. The spin system at
FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2693
Table 1.
1
H,
13
Cand
31
P NMR chemical shifts (p.p.m) of deacylated core-lipid A backbone (oligosaccharide 1) of LOS from P. stutzeri OX1.
Chemical shifts are relative to acetone and external aq. 85% (v/v) phosphoric acid (
1
H, 2.225 p.p.m.;
13
C, 31.45 p.p.m.;
31
P, 0.00 p.p.m. at 55 C).
Residue Nucleus 1 2 3
ax/
45678
A
1
H 5.46 4.39 4.09 4.46 4.39 4.20 3.86/4.08
Hep
13
C 97.8 73.8 76.7 72.0 73.7 69.8 63.9
31
P 1.9 4.0
B
1
H 5.42 2.73 3.64 3.45 4.09 4.30/3.74
GlcN
13
C 95.1 55.9 73.7 70.8 72.0 70.1
31
P 3.0
C
1
H 5.32 3.22 4.06 4.27 4.02 3.71
GalN
13
C 101.3 51.5 78.3 77.5 71.5 61.7
D
1
H 5.27 4.43 4.14 4.05 4.16 4.45 3.75/4.09
Hep
13
C 102.7 69.9 78.2 67.4 71.7 74.2 63.7
31
P 4.3
E
1
H 4.73 3.44 3.71 3.43 3.66 3.93/3.65
Glc
13
C 105.8 75.3 72.9 75.8 67.7 64.6
F
1
H 4.67 2.70 3.54 3.71 3.36 4.04/3.93
GlcN
13
C 104.7 58.2 76.6 77.7 67.2 64.7
G
1
H 4.47 2.67 3.66 3.82 3.47 3.71/3.45
GlcN
13
C 103.5 56.9 73.5 73.4 76.9 63.7
31
P 3.9
I
1
H 1.81/2.07 4.27 4.08 3.69 4.03 3.86/3.70
Kdo
13
C 175.0 101.7 36.1 65.9 67.7 73.0 70.1 63.7
L
1
H 2.00/2.34 4.12 4.24 3.67 3.93 3.86/3.69
Kdo
13
C 175.0 100.9 35.0 71.8 69.5 73.6 70.6 63.9
S-Pyr
1
H 1.48
13
C 175.5 101.9 25.2
R-Pyr
1
H 1.62
13
C 175.8 99.5 17.2
Fig. 1. The structure of oligosaccharide 1 obtained by alkaline hydrolysis of the core region of the LPS of P. stutzeri OX1.
2694 S. Leone et al.(Eur. J. Biochem. 271)FEBS 2004
5.32 p.p.m. (C;
3
J
H1, H2
¼3.6 Hz) was identified as a-GalN
by its J
H,H
values for H-3/H-4 and H-4/H-5, diagnostic of a
galacto configuration (3.4 Hz and less than 1 Hz, respect-
ively). Three spin systems E,Fand G(doublets;
3
J
H1, H2
¼
8.6, 7.8 and 7.7 Hz, respectively) were identified as b-gluco-
configured monosaccharides given their large
3
J
H,H-
values.
A further indication of their bconfiguration was the
observation of NOE contacts in the ROESY spectrum
among H-1, H-3 and H-5, for all E,F,Gresidues.
TheH-3methylenesignalsoftwoa-Kdo residues were
present at 1.82 p.p.m. (H-3ax) and 2.07 p.p.m. (H-3eq)
(residue I), and 2.00 p.p.m. (H-3ax) and 2.34 p.p.m.
(H-3eq) (residue L), respectively. Their aconfiguration
was established on the basis of the chemical shift of their
H-3eq protons and by measurement of the
3
J
H7,H8a
and
3
J
H7,H8b
coupling constants [31,32]. Two methyl singlet
signals were present at higher fields, at 1.48 and 1.62 p.p.m.,
respectively. Each methyl signal was in a 3 : 1 ratio with the
anomeric signals, i.e. in a stoichiometric ratio.
The
13
C-NMR chemical shifts could be assigned by a
DEPT-HSQC experiment, using the assigned
1
H-NMR
spectrum. Seven anomeric carbon resonances were identi-
fied (Table 1), numerous carbon ring signals and four
nitrogen-bearing carbon signals assigned to C-2 of B,C,F
and Gspin systems. Considering the
13
Cchemicalshiftsof
nonsubstituted residues [33], several low-field shifted signals
indicated substitutions at O-3 of residue A, O-6 of residue B,
O-3 and O-4 of residue C, O-3 of residue D,O-4andO-6of
residues Eand F, O-6 of residue G, O-5 and O-4 of residue
L,whereasIwas a terminal residue. In the high field region
of the spectrum two cross peaks at 1.48/25.2 and 1.62/
17.2 p.p.m. were present.
Phosphate substitution was established on the basis of
31
P-NMR spectroscopy. The
31
P-NMR spectrum showed
the presence of five monophosphate monoester signals
(Table 1). The site of substitution was inferred by a
1
H,
31
P-HSQC spectrum that showed correlations of
31
P
signals with H-1 B(GlcN), H-4 Aand H-2 A(Hep I), H-4 G
(GlcN) and H-6 D(Hep II).
The sequence of the monosaccharide residues was
determined using NOE effects of the ROESY (Fig. 3) and
NOESY spectra, and by
1
H,
13
C-HMBC correlations. The
typical lipid A carbohydrate backbone was eventually
assigned on the basis of the NOE signal between H-1 G
and H-6
a,b
B. In the case of Kdo units, which lack the
anomeric proton, the sequence was inferred by NOE
contacts between the methylene-proton H-3
eq
of Kdo L
and H-6 of Kdo I,whereasKdoLwas substituted by
heptose Aas indicated by the NOE effect found between
H-1 Aand H-5 L, and, in addition, between H-5 Aand
H-3
ax
L. All of these NOE contacts were characteristic of
the sequence a-
L
-glycero-
D
-manno-heptose-(1fi5)-[a-D-
Kdo-(2fi4)]-a-D-Kdo [34,35].
Heptose Awas, in turn, substituted at the O-3 position
by heptose D, as demonstrated by the NOE cross peak
between H-1 Dand H-3 A. A disaccharide 7-O-carbamoyl-
Fig. 2.
1
H-NMR spectrum of oligosaccharide 1. The spectrum was recorded under the following conditions: 5 mg of oligosaccharide 1 in 0.6 mL
D
2
O, pD 14 at 30 C.
FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2695