Fluorescent organophosphonates as inhibitors of microbial lipases

Article abstract of DOI:10.1016/S0009-3084(03)00085-9

Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were synthesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or nitrobenzoxadiazole as reporter fluorophores covalently bound to the ω-ends of the respective 2-acylamino- and alkoxy- residues. Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, including the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic enzymes but also for discovery of yet unknown lipases/esterases in biological samples.

Full text of DOI:10.1016/S0009-3084(03)00085-9

Chemistry and Physics of Lipids 125 (2003) 103–114
Fluorescent organophosphonates as inhibitors of microbial lipases
Olga V. Oskolkovaa, Robert Safb, Elfriede Zenzmaiera, Albin Hermettera,∗
a
Department of Biochemistry, Graz University of Technology, Petersgasse 12/II, A-8010 Graz, Austria
b
Institute for Chemistry and Technology of Organic Materials, Graz University of Technology, Stremayrgasse 16/I, 8010 Graz, Austria
Received 16 December 2002; received in revised form 12 May 2003; accepted 27 May 2003
Abstract
Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were syn-
thesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or
nitrobenzoxadiazole as reporter fluorophores covalently bound to the ␻-ends of the respective 2-acylamino- and alkoxy- residues.
Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, in-
cluding the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much
better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by
perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas
species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different
lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally
related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic
enzymes but also for discovery of yet unknown lipases/esterases in biological samples.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Microbial lipases; Synthesis of inhibitors; Fluorescent triacylglycerol analog; Covalent inhibition
1. Introduction
and long-chain (C16) saturated fatty acids as sub-
strates (mainly at sn-1 or sn-3 positions) (Rangheard
et al., 1992; Kirk et al., 1992; Ransac et al., 1990;
Rogalska et al., 1993). The mechanism of action of
these enzymes involves the nucleophilic cleavage of
an ester bond by an activated serine which belongs
to the catalytic triad Ser-His-Asp/Glu (Cygler and
Schrag, 1997; Jaerger and Reetz, 1998; Schmid and
Verger, 1998). Lipophilic p-nitrophenyl phosphonate
esters are convenient tools in lipase research, e.g.
for studies of substrate–enzyme interactions on the
molecular level (Ransac et al., 1997) and functional
analysis, e.g. to determine the active enzyme frac-
tion of crude or pure protein preparations (Scholze
Lipolytic enzymes are widely used biocatalysts
in research and industry to achieve chemical reac-
tions with high regio- and stereoselectivity yielding
enantiomeric alcohols or amines (Jaerger and Reetz,
1998; Schmid and Verger, 1998; Schmid et al., 2001;
Koeller and Wong, 2001; Klibanov, 2001). Lipases
(EC 3.1.1.3) catalyze hydrolysis and synthesis of tria-
cylglycerols. All lipases accept esters of medium (C4)
∗
Corresponding author. Tel.: +43-316-873-6457;
fax: +43-316-873-6952.
E-mail address: albin.hermetter@tugraz.at (A. Hermetter).
0009-3084/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0009-3084(03)00085-9

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O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
et al., 1999; Rotticci et al., 2000; Hermetter et al.,
2001). These inhibitors react with the nucleophilic
serine of lipases, thus leading to the formation of
covalent and equimolar lipid–protein complexes that
are stable in aqueous and organic solutions (Rotticci
et al., 2000; Ransac et al., 1997; Bjoerkling et al.,
1994; Zandonella et al., 1999). Such complexes rep-
resent “open” lipase conformations and mimic the
substrate–enzyme interactions in the first (tetrahedral)
transition state. Fluorescent labels in the hydrophobic
tail of the organophosphonates are not only useful
for quantitative analysis of lipases but also for study-
ing lipid–protein interactions in the first transition
state under different environment (solvent) conditions
(Oskolkova and Hermetter, 2002; Zandonella et al.,
1999).
The phosphonates described in the present work
were tested as inhibitors of lipases from Rhizopus
oryzae, Pseudomonas species, and Pseudomonas
cepacia. Comparative experiments showed that the
novel compounds have a very different inhibitory pro-
file towards the individual enzymes. Only one of the
compounds under investigation, namely perylenebu-
tanoylamino phosphonate (XXII), preferably inacti-
vates PCL in comparison to other inhibitors. PSL was
almost quantitatively inhibited by all organophospho-
nates used in this study.
glycerols was performed in our laboratory according
to (Bucher, Ribitsch et al., unpublished data).
Crude Pseudomonas species lipase was purchased
from Nagase Biochemicals Ltd. (Japan), the pure li-
pase from P. cepacia (PCL) was obtained from R.D.
Schmid, University of Stuttgart, Germany. R. oryzae
lipase was kindly provided by F. Spener and L. Haalck,
University of Münster, Germany.
Dichloromethane (Riedel-de Haën) was dried by
refluxing with phosphorus pentoxide (Merck) and
subsequently distilled. Other solvents were of analyt-
ical grade. TLC was carried out on silica gel 60 F254
aluminum sheets (0.2 mm, Merck) using following
mixtures: petroleum ether/ether, 2:1 (v/v) (system
1), chloroform/methanol/acetone, 10:0.2:0.2 (v/v/v)
(system 2), chloroform/methanol/acetone, 10:0.5:0.5
(v/v/v) (system 3), chloroform/methanol, 10:3 (v/v)
(system 4), or ether/tetrahydrofurane, 2:1 (v/v) (sys-
tem 5) as developing solvents. For preparative TLC
purification silica gel 60 aluminum sheets (0.2 mm,
Merck) without fluorescent indicator were used. Com-
pounds were visualized under UV light (360 nm) and
by charring at 120 ◦C after spraying with 50% sulfuric
acid. Phosphorus-containing compounds were visual-
ized on TLC plates by staining with phosphomolybdic
acid (Dittmer and Lester, 1964), or quantitatively de-
termined in solutions by the method of Broekhuyse
(1968). Short column chromatography was performed
on Kieselgel 60 (230–400 mesh, Merck).
2. Experimental procedures
1H NMR spectra were recorded in deuterated sol-
vents at 199.97 MHz, using a Varian Gemini 200 spec-
trometer. Chemical shifts (δ) are given in ppm relative
to tetramethylsilane as a standard.
Standard chemicals were obtained from Merck.
Mercaptoethanol and p-nitrophenol were from Sigma;
methyl phosphonic acid dichloride, n-hexylphosphonic
acid dichloride and methyl suberyl chloride were from
Aldrich. 1-Methylimidazole, perylene, 3-carbometho-
xypropionyl chloride, and N-hydroxysuccinimide
were obtained from Fluka. Succinimidyl 6-[N-(7-ni-
trobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate was
purchased from Molecular Probes, 12-[N-(7-nitro-
benz-2-oxa-1,3-diazol-4-yl)amino]dodecanoic acid
from Lambda Fluoreszenztechnologie GmbH (Graz,
Austria). Activated succinimid esters of fluorescently
labeled acids were synthesized from appropriate fatty
acids and N-hydroxysuccinimide in the presence of
dicyclohexylcarbodiimide by a procedure essentially
described elsewhere (Lapidot et al., 1967). The synthe-
sis of 2-amino-2-deoxy-1(3)-O-trityl-3(1)-O-alkyl-sn-
Positive ion ESI mass-spectra were recorded on
a standard Kratos electrospray ion source that was
fitted to a Kratos Profile HV-4 double-focusing mag-
netic sector instrument (acceleration voltage 2 kV,
m/z-range 25–2400 Da, scan speed 10 s/Dec, resolu-
tion 1700 (10% valley)). The potentials applied to the
ESI source (temperature 50 ◦C, countercurrent flow of
nitrogen 150 ml/min) were +5.98 kV at the spraying
capillary, +3.12 kV at the cylinder, and +2.57 kV at
the endplate. A Harvard Apparatus 22 syringe pump
was used to deliver a constant flow (6 ␮l/min) of dry
methanol containing CsI (250 mg/l). Solutions of the
samples (50 ␮M) in the same solvent were injected
via a 100-␮l sample loop. The m/z values are given
for the most intense peak of any isotope distribution.

O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
105
2.1. 3-(3-Perylenoyl)propanoic acid methyl ester
J 3.4 Hz, Ar), 8.00 (d, 1H, J 8.5 Hz, Ar), 8.21–8.60
(m, 4H, Ar).
To a solution of 3-carbomethoxypropionyl chlo-
ride (51.8 ␮l, 0.415 mmol) in 10 ml dichloromethane,
64.7 mg (0.476 mmol) aluminum chloride were
added in portions under stirring at 0 ◦C. The reac-
tion was kept at this temperature for 1 h and then
100.0 mg (0.396 mmol) perylene were added por-
tionwise. The mixture was kept at 0 ◦C for 1 h and
then overnight at room temperature, poured on about
10 g of ice, acidified with one to three drops of
concentrated hydrochloric acid. The product was ex-
tracted with dichloromethane (6 × 50 ml), washed
with water (2 × 10 ml). The organic layers were
dried over sodium sulfate. After evaporation, the
residue was purified by column chromatography
on silica gel eluting with chloroform to give the
pure product perylenoylpropanoic acid methyl ester
(121.2 mg, 83.5%). Rf 0.16 (system 1). 1H NMR
(CDCl3) (δ, ppm): 2.87 (t, 2H, Ja,b = J2,3 6.5 Hz,
2–CH2–COO–), 3.40 (t, 2H, Ja,b = J2,3 6.5 Hz,
3–CH2–CO–Ar), 3.77 (s, 3H, CH3), 7.47–7.65 (m,
3H, Ar), 7.74 (t, 2H, J 8.6 Hz, Ar), 7.95 (d, 1H, J
8.0 Hz, Ar), 8.14–8.31 (m, 4H, Ar), 8.56 (d, 1H,
J 8.6 Hz, Ar).
2.3. 7-(3-Perylenoyl)heptanoic acid methyl ester
The synthesis and purification of perylenoylhep-
tanoic acid methyl ester was achieved as described for
perylenoyl propanoic acid methyl ester starting from
suberic acid monomethyl ester chloride and perylene.
1
Yield 73.7%. Rf 0.23 (system 1). H NMR (CDCl3)
(δ, ppm): 1.43 (m, 4H, 4-CH2, 5-CH2), 1.67 (m, 2H,
3-CH2), 1.82 (m, 2H, 6-CH2), 2.32 (t, 2H, Ja,b
J2,3 7.4 Hz, 2-CH2), 3.05 (t, 2H, Ja,b = J6,7 7.4 Hz,
7-CH2), 3.69 (s, 3H, CH3), 7.43–7.64 (m, 3H, Ar),
7.74 (t, 2H, J 8.3 Hz, Ar), 7.84 (d, 1H, J 7.9 Hz, Ar),
8.13–8.30 (m, 4H, Ar), 8.48 (d, 1H, J 7.5 Hz, Ar).
=
2.4. 8-(3-Perylenyl)octanoic acid
The synthesis and isolation was accomplished un-
der identical conditions as described for peryleneb-
utanoic acid staring from 7-(3-perylenoyl)heptanoic
acid methyl ester yielding 60.0% perylenyloctanoic
1
acid. Rf 0.54 (system 3). H NMR (CDCl3/CD3OD,
9:1) (δ, ppm): 1.25–1.50 (m, 6H, 3 CH2), 1.59 (m,
2H, 3-CH2), 1.72 (m, 2H, 7-CH2), 2.26 (dt, 2H, Ja,b
1.8 Hz, J2,3 7.5 Hz, 2-CH2), 2.97 (t, 2H, J7,8 7.7 Hz,
8-CH2), 7.28 (d, 1H, J 8.0 Hz, Ar), 7.43 (m, 3H, Ar),
7.59 (d, 1H, J 3.4 Hz, Ar), 7.63 (d, 1H, J 3.4 Hz, Ar),
7.83 (d, 1H, J 8.5 Hz, Ar), 8.10–8.22 (m, 4H, Ar).
2.2. 4-(3-Perylenyl)butanoic acid
To a suspension of 3-perylenoyl propanoic acid
methyl ester (360.2 mg, 0.983 mmol) in 2.0 ml di-
ethylene glycol, 98% hydrazine hydrate (150.6 ␮l,
2.949 mmol) and powdered potassium hydroxide
(275.3 mg, 4.916 mmol) were added. The reaction
mixture was heated to 140 ◦C for 2 h, then to 190 ◦C
and kept over night, cooled to room temperature,
diluted with 20 ml water, acidified with 0.5 ml con-
centrated hydrochloric acid, the precipitate formed
was filtered, washed with water to pH 7.0, dried on
air or washed with acetone, then with a mixture of
chloroform/methanol (8:2, v/v). The filtrate was con-
centrated in vacuo. The crude product was applied
into a silica gel column and eluted with chloroform to
20% methanol in chloroform to give perylenebutanoic
acid (228.9 mg, 68.8%). Rf 0.47 (system 3). 1H NMR
(CDCl3/DMSO-d6, 20:1) (δ, ppm): 1.92 (quin., 2H,
3-CH2), 2.38 (t, 2H, J 7.0 Hz, 2-CH2), 3.03 (t, 2H, J
7.0 Hz, 4-CH2), 7.41 (d, 1H, J 7.8 Hz, Ar), 7.43–7.64
(m, 3H, Ar), 7.76 (d, 1H, J 3.4 Hz, Ar), 7.80 (d, 1H,
2.5. 2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-3-O-
octyl-sn-glycerol (XII)
(A) To a solution of 12.0 mg (0.027 mmol) perylene-
butyric acid succinimide ester (I) in 3 ml THF, 2-
amino-2-deoxy-1-O-trityl-3-O-octyl-sn-glycerol (V)
(12.2 mg, 0.027 mmol) was added. The reaction
mixture was kept at room temperature overnight,
evaporated under a stream of argon. The residue
was applied into a silica gel column and eluted
with chloroform. Fractions containing the product
were collected and the solvent was removed under
reduced pressure. The compound (XI) (20.8 mg)
was obtained with 98.6% yield. Rf 0.81 (system
2). Removal of the trityl blocking group from
2-perylenebutanoylamino-1-O-trityl-3-O-octyl-sn-gly-
cerol (XI) was carried out with boron trifluoride–

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O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
methanol in absolute dichloromethane under proce-
dure elaborated by Hermetter et al. (1989). Rf 0.13
(system 2). Yield 70.0%. (B) The compound (XII) was
achieved by an analogous procedure described above
starting from perylenebutanoic acid succinimide ester
(I) and 2-amino-2-deoxy-3-O-octyl-sn-glycerol (VI)
with 91.0% yield. The products obtained in both cases
were identical.
2.10. 2-(6-NBD-dodecanoyl)amino-2-deoxy-3-O-
hexadecyl-sn-glycerol (XXI)
The synthesis of the long-chain alkyl acylamino
glycerol (XXI) was achieved under identical condi-
tions as described for the compound (XII) (method B)
starting from NBD-dodecanoic acid succinimid ester
(IV) and 2-amino-2-deoxy-3-O-hexadecyl-sn-glycerol
(X). The yield comprised 98.0%.
2.6. 2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-1-O-
octyl-sn-glycerol (XIV)
2.11. 2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-3-
O-octyl-sn-glycero-1-O-(n-hexyl)phosphonic acid
p-nitrophenyl ester (XXII)
The compound (XIV) was obtained from an
equimolar mixture of perylenebutanoic acid succin-
imide ester (I) and 2-amino-2-deoxy-1-O-octyl-3-O-
trityl-sn-glycerol (VII) under identical conditions
described in method (A) for (XII). Overall yield after
two stages was 88.3%.
To a solution of the glycerol derivative (XII)
(20.0 mg, 0.038 mmol), 40 ␮l triethylamine, and 3.5%
tetrazole solution in acetonitrile (7.4 ␮l, 0.004 mmol)
in 2 ml dichloromethane, hexyl phosphonic acid
dichloride (12.9 ␮l, 0.076 mmol) was added at
5 ◦C. After 1 h when the reaction was complete,
p-nitrophenol (10.6 mg, 0.076 mmol) was added and
the reaction mixture was kept overnight at room
temperature. The solvent was removed under a
stream of nitrogen. The product was isolated by
preparative TLC in the enveloping system chloro-
form/methanol/acetone, 10:0.2:0.2 (v/v/v). Yield was
2.7. 2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-1-O-
hexadecyl-sn-glycerol (XVI)
This product was synthesized using the same
procedure as for (XII) (procedure A) starting from
peryleneoctanoic acid succinimide ester (II) and 2-
amino-2-deoxy-1-O-hexadecyl-3-O-trityl-sn-glycerol
(VIII). Yield (after two stages) is 90.0%.
1
13.1 mg (43.3%). H NMR (CDCl3) (δ, ppm): 0.87
(t, 6H, 2CH3), 1.05–2.00 (m, 22H, CH2-alkyl), 2.13
(m, 2H, 2-CH2-acyl), 2.30 (m, 2H, 3-CH2-acyl), 3.05
(m, 2H, CH2-O-alkyl), 3.40 (m, 4H, CH2-O-glycerol,
4-CH2-acyl), 4.25 (m, 3H, CH2-O-P, CH-N), 6.18 (dd,
1H, NH), 7.29-7.40 (m, 3H, Ar), 7.50 (m, 3H, Ar),
7.66 (d, 1H, J 2.4 Hz, Ar), 7.70 (d, 1H, J 2.2 Hz, Ar),
7.89 (dd, 1H, J 3.4 Hz, J 8.1 Hz, Ar), 8.17 (m, 6H, Ar).
2.8. 2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-3-O-
hexadecyl-sn-glycerol (XVIII)
The compound (XVIII) was prepared in 67.9%
yield by the same procedure as described for (XII)
(A), starting from amino glycerol (IX) and peryle-
neoctanoic acid succinimidyl ester (II).
2.12. 2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-1-
O-octyl-sn-glycero-3-O-(n-hexyl)phosphonic acid
p-nitrophenyl ester (XXVI)
2.9. 2-(6-NBD-hexanoyl)amino-2-deoxy-3-O-octyl-
sn-glycerol (XX)
The amino glycerol (XX) was obtained from
NBD-hexanoic acid succinimide ester (III) and
2-amino-2-deoxy-1-O-trityl-3 - O - octyl - sn - glycerol
(V) as described for (XII) (A). Yield 86.0%. Al-
ternatively, the same compound (XX) was prepared
under identical conditions as described for (XII)
(method (B)) but using succinimide ester (III) and
2-amino-2-deoxy-3-O-octyl-sn-glycerol (VI) with a
100% yield. Rf 0.16 (system 2).
To a solution of 2-perylenebutanoylamino-1-O-
octyl-sn-glycerol (XIV) (8.1 mg, 0.016 mmol) and N-
methylimidazole (6.0 ␮l, 0.070 mmol) in 2 ml dichlo-
romethane, n-hexylphosphonic dichloride (10.8 ␮l,
0.063 mmol) was added. The reaction mixture was
kept at room temperature for 3 h. After the reac-
tion was complete, a mixture of p-nitrophenol (10.6
mg, 0.076 mmol) and N-methylimidazole (6.0 ␮l,
0.070 mmol) were added. After 18 h, the solvent was

O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
107
evaporated under a stream of argon. The phospho-
nate (XXVI) was purified by TLC in system 2. Yield
0.7 mg (5.7%).
with water (2 × 20 ml), dried over sodium sulfate,
evaporated under reduced pressure. The product was
purified on a silica gel column eluting with chloro-
form. Rf 0.57 (system 3). Yield 31.7 mg (66.2%).
2.13. 2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-3-
O-hexadecyl-sn-glycero-1-O-(n-hexyl)phosphonic
acid p-nitrophenyl ester (XXIII)
2.18. O-(4-(3-Perylene)butyl)-O-methylphosphonic
acid p-nitrophenyl ester (XXIX)
The phosphonate (XXIII) was prepared under iden-
tical conditions as described for (XXII) with 6.3%
yield and under conditions as for (XXIV) as well
(yield 31.6%) starting from (XVIII).
One-chain phosphonate inhibitor (XXIX) was ob-
tained from perylenebutanol (XXVIII) analogous to
the procedure described for phosphonate (XXVI), but
using methyl phosphonic acid dichloride as phospho-
rylating reagent. Yield 57.6%.
2.14. 2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-1-
O-hexadecyl-sn-glycero-3-O-(n-hexyl)phosphonic
acid p-nitrophenyl ester (XXVII)
2.19. ((6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)
hexanoyl)aminoethanol (XXX)
The synthesis and isolation was accomplished ac-
cording to the procedure described for (XXII) starting
from the amino glycerol analog (XVI). Yield 26.1%.
A mixture of NBD-hexanoic acid succinimidyl es-
ter (III) (12.5 mg, 0.0319 mmol) and 2-aminoethanol
(2 ␮l, 0.0324 mmol) in 2 ml THF was kept for 30 min.
After TLC showed the reaction complete, the solvent
was removed under a stream of argon. The residue
was dissolved in a chloroform/methanol/water mix-
ture (65:25:4, v/v/v) (5 ml), and 1 g of a resin Dowex
50Wx8 (H+-form) was added. After stirring for
30 min, the product (XXX) dissolved was filtered and
the resin was washed with the same solvent system
(3 × 5 ml). The filtrate was evaporated under reduced
pressure. TLC analysis showed single fluorescent spot
with Rf 0.69 (system 4) and no aminoethanol after
spraying with ninhydrine. Yield 10.6 mg (98.3%).
2.15. 2-(NBD-hexanoyl)amino-2-deoxy-3-O-octyl-
sn-glycero-1-O-(n-hexyl)phosphonic acid
p-nitrophenyl ester (XXIV)
The product (XXIV) was obtained from fluorescent
labeled glycerol (XX) under identical conditions as
described for (XXII) with 27.8% yield.
2.16. 2-(NBD-dodecanoyl)amino-2-deoxy-3-O-
hexadecyl-sn-glycero-1-O-(n-hexyl)phosphonic acid
p-nitrophenyl ester (XXV)
2.20. O-((6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)
amino)hexanoyl)aminoethyl-O-(n-hexyl)phosphonic
acid p-nitrophenyl ester (XXXI)
The compound (XXI) was phosphorylated as de-
scribed for the preparation of phosphonate (XXII) us-
ing acylamino O-alkyl glycerol (XXI) as a starting
material. The yield comprised 4.4%.
Using the method described for phosphonate
(XXII), NBD-labeled hexylphosphonate was obtained
starting from the respective alcohol (XXX). The yield
comprised 11.1%.
2.17. 3-Perylenebutanol (XXVIII)
To a solution of perylenebutanoic acid (50.0 mg,
0.148 mmol) in 3.5 ml toluene, Red-Al (0.67 ml,
2.232 mmol) was added carefully. After 1 h, when the
reaction was complete, the mixture was cooled in an
ice bath, and 25% sulfuric acid was added to pH 5 to
decompose the unreacted Red-Al. Then water (30 ml)
was added and the product was extracted with chlo-
roform (5 × 50 ml). The organic phase was washed
2.21. Determination of lipase activity
Inhibition of lipase activity by the water-insoluble
phosphonates was performed in Triton X-100 micelles
(2 mM final concentration) and measured by a contin-
uous fluorescence method according to Duque et al.
(1996) using 3-O-hexadecyl-2-pyrenedecanoyl-1-tri-

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nitrophenylaminododecanoyl-sn-glycerol as a sub-
strate. Lipase activity was determined at 30 ◦C from
the dequenching of pyrene fluorescence at 378 nm (ex-
citation at 342 nm, slit widths 5.0 nm each) (the initial
linear increase in fluorescence intensity after addition
of the lipase was used to measure enzyme activity).
The measurements were performed in ethanol/water
(1:3, v/v) as a solvent.
sized. Perylene and NBD-labels were chosen because
of the longer wavelength absorption and emission
as compared to pyrene-containing inhibitors, which
have been synthesized and characterized previously
(Zandonella et al., 1999).
For the preparation of perylene-labeled lipase
inhibitors, the corresponding fluorescently labeled
perylene fatty acids were needed. Friedel–Crafts
acylation of perylene with appropriate dicarbonic
acid monomethyl ester chlorides gave perylenoyl
propanoic acid methyl ester and perylenoyl heptanoic
acid methyl ester. It is known that perylene is only
acylated at C-3 position under these reaction condi-
2.22. Inhibition experiments
Triton X-100 specially purified for membrane re-
search (Hofmann La Roche) (10 ␮l of a 100 mM stock
solution in chloroform) and the appropriate volume of
a phosphonate solution in chloroform were mixed and
the solvent was removed under a stream of nitrogen.
The residue was dispersed in an 100 ␮M Tris–HCl
buffer (pH 7.40 at 37 ◦C) by vigorous vortexing fol-
lowed by addition of an aliquot of a lipase solution,
giving a total volume of 500 ␮l. Incubations were per-
formed at 4 ◦C. The percentage of inhibition after 16 h
in the reaction mixture was determined by measur-
ing the residual lipase activity of appropriate aliquots
(3 ␮l) as indicated above. Controls were run in par-
allel under identical conditions but in the absence of
inhibitors.
1
tions (Zinke et al., 1940). The H NMR spectra of
the synthesized perylenoyl propanoic and perylenoyl
heptanoic acid esters (protons of the alkyl and aro-
matic moieties) confirmed the indicated structures of
the acylation products. Kizhner–Wolf reduction of the
keto-groups and the concomitant deblocking of the
methyl protective groups gave perylenebutanoic and
peryleneoctanoic acids, respectively. Proton NMR
spectra were in agreement with the ascribed structures.
The synthetic routes leading to the fluores-
cently labeled triacylglycerol analogues are out-
lined in Fig. 1. The respective compounds are
alkylacyl(amino)deoxyglycerophosphonic acids and
alkyloxyphosphonic acid derivatives. Three major
crucial steps are involved in their synthesis that
are: (1) attachment of a fluorescent acyl chain to
1(3)-O-alkyl-2-aminodeoxy-3(1)-O-trityl-sn-glycerol,
(2) detritylation, and (3) phosphorylation of the re-
sultant alkylacyl(amino)deoxyglycerols. In principle,
the acylation of an amino group can be performed
using acid anhydride (Singh and Schmidt, 1989),
acyl-halides (Zimmerman et al., 1988; Dijkman et al.,
1990), mixed carbodiimide (Hammarstroem, 1971),
or mixed anhydrides formed by ethylchloroformate
and a fatty acid (Acquotti et al., 1986). These proce-
dures have several disadvantages especially because
they require a large molar excess of acylating reagents
and long reaction times. The use of acyl chlorides
was not convenient in our case because of the pos-
sible modification of fatty acid derivatives during
acylation. Activated fatty acid esters (for example,
p-nitrophenyl esters) have also successfully been ap-
plied, e.g. for sphingolipid synthesis (Tkaczuk and
Thornton, 1981; Groenberg et al., 1991; Kann et al.,
1991; Shibuya et al., 1992). However, acylation with
2.23. Spectroscopic analyses
A molar absorbance coefficient of 22,000 M−1 cm−1
at 466 nm in methanol or 29,000 M−1 cm−1 at 442 nm
in ethanol for phosphonate inhibitors labeled with
NBD or perylene, respectively was used to determine
the concentration of the fluorescent phosphonates
(measurements were performed on a Hitachi U-3210
spectrophotometer) (Haugland, 1996). Fluorescence
spectra of both perylene and NBD-labelled com-
pounds in ethanol were recorded using a Shimadzu
RF-5301PC spectrofluorophotometer in ethanol.
3. Results and discussion
3.1. Chemical synthesis
Eight novel phosphonic acid esters fluorescently
labeled with perylenebutanoic, perylene octanoic,
NBD-hexanoic or NBD-dodecanoic acid were synthe-

O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
109
Fig. 1. Synthetic routes of fluorescently labeled organophosphonates.
p-nitrophenyl esters of perylenebutanoic and perylene
octanoic acids may give only low yields of acylation
products probably because of the close vicinity of
a bulky fluorophore and the ester group (Oskolkova
et al., 1999). Thus, we used N-hydroxysuccinimide
esters of fluorescently labeled fatty acids to acylate
selectively amino groups in the presence of free hy-
droxy groups with high yields (Julina et al., 1986).
In the present study, this simple method gave high
yields of acylation products (e.g. 91.0% yield of (XII)
when 2-amino-2-deoxy-3-O-octyl-sn-glycerol (VI)
was acylated with perylenebutanoic acid succinimide
ester).
The protective trityl group of the labeled alky-
lacylaminodeoxytritylglycerols was removed under
standard conditions as described for the synthesis of
diradylglycerols (Hermetter et al., 1989) using boron
trifluoride–methanol. The obtained fluorescent alkyla-
cylaminodeoxyglycerols (XI–XVIII) showed fluores-
cence spectra typical for perylene-labeled compounds
(Johanson et al., 1987).
Two further problems are encountered in the syn-
thesis of fluorescent phosphonate inhibitors. Reac-
tion yields of phosphorylation of glycerolipids tend
to be low. Secondly the fluorescently labeled fatty
acids and, as a consequence, the fluorescently labeled
glycerolipid intermediates are expensive and that is
one special reason, why yields should be as high
as possible. The diradylglycerols were reacted with
alkylphosphonic acid dichloride in the presence of
N-methylmorpholine as a base followed by substi-
tution of the second chloro atom at phosphorus by
p-nitrophenol. Although these two steps were car-
ried out consecutively in an one-pot reaction, prod-
uct yields were rather low (5.7% for 2-deoxy-2-(4-
perylenylbutanoyl)amino-1-O-octyl-3-O-hexylphosp-
honate (XXVI) or 6.3% for 2-deoxy-2-(8-perylenylo-
ctanoyl)amino-3-O-hexadecyl-1-O-hexylphosphonate

110
O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
(XXIII)) most likely because of the close vicinity
of the amido group to the phosphoric acid moiety,
which as a consequence, can lead to an intramolecular
cyclic side-product (oxazaphospholane) in consider-
able amounts. Such compounds can in fact be pre-
pared from 2-deoxy-2-amino-glycerophosphocholine
in the presence of phosphoroxytrichloride (Deigner
and Fyrnys, 1992). To avoid formation of such
compounds we used a modification of the phos-
phorylation procedure as described by Zhao and
Landry (1993) for the synthesis of phosphonate es-
ters. This approach used tetrazole as a catalyst and
has already been shown to be successful for the
synthesis of phosphonates from sterically hindered
alcohols such as menthol and testosterone (Zhao and
Landry, 1993) or for the synthesis of organophos-
phonate esters (Rotticci et al., 2000). In our case,
the yields of products were significantly improved
when tetrazole was used as a catalyst (e.g. yields
of 2-deoxy-2-(8-peryleneoctanoyl)amino-3-O-hexa-
decyl-1-O-hexylphosphonate (XXIII) were 6.3% in
the absence of tetrazole and 31.6% in its presence,
Table 1). For the other glycerolipid phosphonate ana-
logues, yields were above 25% if synthesized by the
tetrazole-catalyzed reaction (except of phosphonate
XXV).
Perylenebutanoic acid was first reduced to peryleneb-
utanol (XXVIII) by reaction with Red-Al. Phos-
phorylation of the alcohol (XXVIII) in the absence
of tetrazole gave perylenebutyl methylphosphonate
(XXIX) with high yield (57.6%). NBD-hexanoic acid
succinimide ester reacted with excess aminoethanol
yielding derivative (XXX). Phosphorylation of the lat-
ter compound gave phosphonate (XXXI) with 11.1%
yield.
The proton signals in the 1H NMR-spectrum of
phosphonate (XXII) were consistent with the indi-
cated molecular structure. Since the phosphorylation
procedures for all glycerolipids in this study were
identical, we present here only one proton NMR
spectrum for the latter compound. UV and fluores-
cence spectra of all perylene-labeled organophos-
phorus compounds showed identical λmax which are
consistent with their assumed chemical structures.
The perylene- (XXII, XXIII, XXVI, XXVII, XXIX)
and NBD-phosphonates (XXIV, XXV, XXXI) had
fluorescence maxima at 448 and 533 nm in ethanol
typical for perylene (Haugland, 1996) and NBD con-
taining derivatives (Nicols and Pagano, 1982), respec-
tively. The ratio of fluorescent label and phosphorus
content of all phosphonates was about 1–1.15, addi-
tionally confirming the structures of the compounds
obtained. Moreover, in ESI mass-spectra of the com-
pounds (XXII, XXVI, XXIII, XXVII, XXXI, XXIV,
XXV) only signals corresponding to their molecular
As a second class of lipase inhibitors, phospho-
nates containing single-chain alkoxy groups instead
of glycerolipids were prepared as follows (Fig. 2).
Fig. 2. Synthesis of one-chain perylene- and NBD-inhibitors.

O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
111
Table 1
Comparison of chromatographic characteristics of synthetic fluorescent phosphonates and their yields after phosphorylation
Compounds
Number
Chromatographic
Yield of phosphorylation (%)
behavior (Rf )
Without
catalyst
With tetrazole
as catalyst
O
P
O
NO2
O
C
CH2
HN CH
CH2
O
O
(CH2)3
C6H13
C8H17
XXII
0.33a
0.33a
–
43.3
O
CH2
O
O
C8H17
(CH2)3
C
HN CH
CH2
O
XXVI
5.7
–
O
NO2
P
C6H13
O
O
P
NO2
O
CH2
O
O
(CH2)7
C
HN CH
CH2
C6H13
16H33
XXIII
0.46a
0.46a
6.3
31.6
26.1
C
O
CH O C16H33
2
(CH2)7
C
HN CH
CH
O
XXVII
–
O
NO2
O P
2
C6H13
O
P
(CH2)3
CH2
O
O
NO2
XXIX
XXXI
0.59b
0.22c
57.6
–
CH3
O
O
P
O
NO2
HN (CH2)5
N
C
HN CH2CH2 O
C6H13
–
11.1
O
N
NO2
O
O
P
NO2
O
CH2
HN CH
CH2
O
O
HN (CH2)5
N
C
C6H13
C8H17
XXIV
0.33c
–
–
27.8
O
N
NO2
O
O
O P
NO2
O
CH2
HN CH
CH2
HN (CH2)11
N
C
C6H13
16H33
0.55c
4.4
O
C
XXV
O
N
NO2
Solvents for TLC: (a) chloroform/methanol/acetone, 10:0.2:0.2 (v/v/v); (b) ether/tetrahydrofurane, 2:1 (v/v) or (c) chloro-
form/methanol/acetone, 10:0.5:0.5 (v/v/v).

112
O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
structures were observed (Table 2). Only in the case
of phosphonate (XXIX) it was impossible to obtain a
useful mass-spectrum.
3.2. Interaction of inhibitors with lipolytic enzymes
Di-O-alkylglycero-phosphonates have already been
reported as useful inhibitors of lipase activity (Stadler
et al., 1996; Zandonella et al., 1999). However, it was
still an open question, whether microbial lipases would
react with fluorescent phosphonate inhibitors contain-
ing amide bonds.
Thus, the novel compounds were tested with re-
spect to their ability to inhibit three selected micro-
bial lipases with different substrate preferences. The
lipase from R. oryzae was efficiently inactivated by
the perylene- and NBD-inhibitors (Fig. 3) at 4 ◦C for
16 h in 100 ␮M Tris–HCl buffer at inhibitor and lipase
concentrations of 1.0 and 0.1 mM, respectively.
The two-chain phosphonate (XXII) was a much
more potent inhibitor of the lipase from P. cepacia
than the other inhibitors used (Fig. 3). In contrast, the
Pseudomonas species lipase was quantitatively inacti-
vated by all synthesized organophosphonates (Fig. 3).
Lipases may show very different steric constraints
around and within their active site, and as a conse-
quence, very different substrate and inhibitor prefer-
ences (Pleiss et al., 1998). The lipases ROL, PCL and
PSL, which had been chosen for the inhibition experi-
ments with organophosphonates, are typical examples
for this structural diversity among highly homologous
enzymes. Accordingly, they show different patterns of
reactivity not only towards lipid substrates, but also to
structurally related inhibitors as demonstrated in this
study. Thus, the novel phosphonates might be use-
ful tools not only for analysis and discrimination of
known lipolytic enzymes but also for discovery of yet
unknown lipases/esterases in biological samples.
As an alternative to water-soluble lipase monomers
used in industry, cross-linked enzyme crystals
(CLECs) (Khalaf et al., 1996; Lalonde et al., 1995;
St. Clair and Navia, 1992) have been introduced to
increase enzyme activity (Zelinski and Waldmann,
1997) and stability in organic solvents (Persichetti
et al., 1995). This makes them useful catalysts which
are easy to separate from the reaction mixture and
can be repeatedly used after subsequent filtration and
washing. Preliminary experiments have shown that
Fig. 3. Inhibition of microbial lipases from Rhizopus oryzae
(ROL), Pseudomonas cepacia (PCL), and Pseudomonas species
(PSL). Experimental conditions are described in Section 2. Values
are percentages of inhibition of the indicated lipase in compari-
son to enzyme activity without inhibitor. Inactivation of PCL by
organophosphonates XXVI and XXV was not determined.

O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
113
Table 2
Results of mass-spectrometry analysis of organophosphonates. Mass-spectra were recorded as described in Section 2
Compound number
Formula
Molecular weight
Ions observed
m/ztheor
m/zobserved
XXII
C
47H57N2O7P
792.39
792.39
960.58
960.58
523.15
606.22
748.36
944.58
[C47H57N2O7P·Cs]+
[C47H57N2O7P·Cs]+
925.30
925.30
1093.48
1093.48
925.4
925.3
1093.4
1093.4
XXVI
XXIII
XXVII
XXIX
XXXI
XXIV
XXV
C47H57N2O7P
C59H81N2O7P
[C59
[C59
n.d.
H
H
81N2O7P·Cs]+
C59H81N2O7P
81N2O7P·Cs]+
C31H26NO5P
C26H35N6O9P
C35
C49
[C26
H
35N6O9P·Cs]+
739.13
881.26
1077.48
739.3
881.3
1077.3
H53N6O10
81N6O10
P
P
[C35H53N6O10P·Cs]+
[C49H81N6O10P·Cs]+
H
n.d.: not detectable.
Dijkman, R., Dekker, N., de Haas, G.H., 1990. Competitive
inhibition of lipolytic enzymes. II. Preparation of
‘monoacylamino’ phospholipids. Biochim. Biophys. Acta 1043,
67–74.
Dittmer, J.C., Lester, R.L., 1964. A simple specific spray for the
detection of phospholipids on thin layer chromatography. J.
Lipid Res. 5, 126–127.
Duque, M., Graupner, M., Stütz, H., Wicher, I., Zechner,
R., Paltauf, F., Hermetter, A., 1996. New fluorogenic
triacylglycerol analogs as substrates for the determination and
chiral discrimination of lipase activities. J. Lipid Res. 37, 868–
876.
the novel inhibitors described in this work might
also be useful to characterize the functional quality
of these systems which is otherwise difficult to de-
termine. Work is in progress to characterize activity
properties of CLECs in three dimensions using the
fluorescent inhibitors.
Acknowledgements
Groenberg, L., Ruan, Z.-S., Bittman, R., Slotte, J.P., 1991.
Interaction of cholesterol with synthetic sphingomyelin
derivatives in mixed monolayers. Biochemistry 30, 10746–
10754.
Financial support of this work by the Austrian Sci-
ence Fund (SFB-project F107 to A.H.; FWF-project
13962-CHE to R.S.) is gratefully acknowledged.
We also thank C. Illaszewicz for recording of NMR
spectra.
Hammarstroem, S., 1971. A convenient procedure for the synthesis
of ceramides. J. Lipid Res. 12, 760–765.
Haugland, R.P., 1996. Fluorescent phospholipids, fatty acids and
sterols. In: Spence, M.T.Z. (Ed.), Handbook of Fluorescent
Probes and Research Chemicals. Molecular Probes, Eugene,
p. 293.
References
Hermetter, A., Scholze, H., Stütz, A., Withers, S.G., Wrodnigg,
T.M., 2001. Powerful probes for glycosidases: novel
fluorescently tagged glycosidase inhibitors. Bioorgan. Med.
Chem. Lett. 11, 1339–1342.
Hermetter, A., Stütz, H., Franzmair, R., Paltauf, F., 1989. 1-O-
Trityl-glycero-3-phosphocholine: a new intermediate for the
facile preparation of mixed-acid 1,2-diacylglycerophospho-
cholines. Chem. Phys. Lipids 50, 57–62.
Jaerger, K.-E., Reetz, M., 1998. Microbial lipases form versatile
tools for biotechnology. Trends Biotech. 16, 396–403.
Julina, R., Herzig, T., Bernet, B., Vasella, A., 1986. Enantio-
selective synthesis of d-erythro-sphingosine and of ceramide.
Helv. Chim. Acta 69, 368–373.
Acquotti, D., Sonnino, S., Masserini, M., Casella, L., Fronza,
G., Tettamanti, G., 1986. A new chemical procedure for
the preparation of gangliosides carrying fluorescent or
paramagnetic probes on the lipid moiety. Chem. Phys. Lipids
40, 71–86.
Bjoerkling, F., Dahl, A., Patkar, S., Zundel, M., 1994. Inhibition
of lipases by phosphonates. Biorgan. Med. Chem. 2, 697–705.
Broekhuyse, R.M., 1968. Phospholipids in tissues of the
eye. Isolation, characterization and quantitative analysis by
two-dimensional thin-layer chromatography of diacyl and
vinyl-ether phospholipids. Biochim. Biophys. Acta 260, 449–
459.
Cygler, M., Schrag, J.D., 1997. Structure as basis for understanding
interfacial properties of lipases. Methods Enzymol. 284, 3–27.
Deigner, H.-P., Fyrnys, B., 1992. Rapid synthesis of 2-desoxy-
2-amino-3-phosphocholine-glycerinic-acid-alkylester, 1-alkyl-
1-desoxy- and 1-O-alkyl-2-desoxy-2-amino-sn-glycero-3-phos-
phocholines, -3-phospho-N,N’-dimethylethanolamine and -3-
phospho-Fmoc-serine-methylester. Chem. Phys. Lipids 61,
199–208.
Johanson, L.B.-A., Molotkovsky, J.G., Bergelson, L.D., 1987.
Fluorescence and absorption properties of perylenyl and
perylenoyl probe molecules in solvents and liquid crystals. J.
Am. Chem. Soc. 109, 7374–7381.
Khalaf, N., Govarghan, C.P., Lalonde, J.J., Persichetti, R.A., Wang,
Y.-F., Margolin, A.L., 1996. Cross-linked enzyme crystals as
highly active catalysts in organic solvents. J. Am. Chem. Soc.
118, 5494–5495.

114
O.V. Oskolkova et al. / Chemistry and Physics of Lipids 125 (2003) 103–114
Kann, C.-C., Ruan, Z.-S., Bittman, R., 1991. Interaction of
cholesterol with sphingomyelin in bilayer membranes: evidence
that the hydroxy group of sphingomyelin does not modulate the
rate of cholesterol exchange between vesicles. Biochemistry
30, 7759–7766.
pancreatic lipases, a kinetic study using the monomolecular
film technique. J. Biol. Chem. 265, 20263–20270.
Rogalska, E., Cudrey, C., Ferrato, F., Verger, R., 1993. Stereo-
selective hydrolysis of triglycerides by animal and microbial
lipases. Chirality 5, 24–30.
Kirk, O., Bjoerkling, F., Godtfresden, S.E., Larsen, T.O., 1992.
Fatty acid specificity in lipase-catalyzed synthesis of glucoside
esters. Biocatalysis 6, 127–134.
Rotticci, D., Norin, T., Hult, K., Martinelle, M., 2000. An active-
site titration method for lipases. Biochim. Biophys. Acta 1483,
132–140.
Klibanov, A.M., 2001. Improving enzymes by using them in
organic solvents. Nature 409, 241–246.
Koeller, K.M., Wong, C.-H., 2001. Enzymes for chemical
synthesis. Nature 409, 232–240.
Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M.,
Witholt, B., 2001. Industrial biocatalysis today and tomorrow.
Nature 409, 258–268.
Schmid, R., Verger, R.D., 1998. Lipasen: Grenzflächen-Enzyme
mit attraktiven Anwendungen. Angew. Chem. 110, 1694–1720.
Scholze, H., Stütz, H., Paltauf, F., Hermetter, A., 1999. Fluorescent
inhibitors for qualitative and quantitative analysis of lipolytic
enzymes. Analyt. Biochem. 276, 72–80.
Shibuya, H., Kawashima, K., Narita, N., Ikeda, M., Kitagawa, I.,
1992. Synthesis of two pairs of enantiomeric C18-sphingosines
and a palmitoyl analogue of Gaucher spleen glucocerebroside.
Chem. Pharm. Bull. 40, 1154–1165.
Lalonde, J.J., Govardhan, C., Khalaf, N., Martinez, A.G., Visuri,
K., Margolin, A.L., 1995. Cross-linked crystals of Candida
rugosa lipase: highly efficient catalysts for the resolution of
chiral esters. J. Am. Chem. Soc. 117, 6845–6852.
Lapidot, Y., Rappoport, S., Wolman, Y., 1967. Use of esters of
N-hydroxysuccinimide in the synthesis of N-amino acids. J.
Lipid Res. 8, 142–145.
Nicols, J.W., Pagano, R.E., 1982. Use of resonance energy transfer
to study the kinetics of amphiphile transfer between vesicles.
Biochemistry 21, 1720–1726.
Oskolkova, O.V., Hermetter, A., 2002. Fluorescent inhibitors reveal
solvent-dependent micropolarity in the lipid binding sites of
lipases. Biochim. Biophys. Acta 1597, 60–66.
Oskolkova, O.V., Shvets, V.I., Hermetter, A., Paltauf, F., 1999.
Synthesis and intermembrane transfer of pyrene-labelled
liponucleotides: ceramide phosphothymidines. Chem. Phys.
Lipids 99, 73–86.
Persichetti, R.A., St. Clair, N.L., Griffith, J.P., Navia, M.A.,
Margolin, A.L., 1995. Cross-linked enzyme crystals (CLECs)
of thermolysin in the synthesis of peptides. J. Am. Chem. Soc.
117, 2732–2737.
Pleiss, J., Fischer, M., Schmid, R.D., 1998. Anatomy of lipase
binding sites: the scissile fatty acid binding site. Chem. Phys.
Lipids 93, 67–80.
Singh, N.P., Schmidt, R.R., 1989. Synthesis of
a (4E,8Z)-
sphingadienine moiety containing cerebroside from Tetragonia
tetragonoides with antiulcerogenic activity. J. Carbohydr.
Chem. 8, 199–216.
Stadler, P., Zandonella, G., Haalck, L., Spener, F., Hermetter,
A., Paltauf, F., 1996. Inhibition of microbial lipases with
stereoisomeric triradylglycerol analog phophonates. Biochim.
Biophys. Acta 1304, 229–244.
St. Clair, N.L., Navia, M.A., 1992. Cross-linked enzyme crystals
as robust biocatalysts. J. Am. Chem. Soc. 114, 7314–7316.
Tkaczuk, P., Thornton, E., 1981. Useful synthesis of ery-
thro- and threo-N-oleoyl-d-sphingosines (ceramides) and
galactosylceramides (cerebrosides) from l-serine. J. Org.
Chem. 46, 4393–4398.
Zandonella, G., Stadler, P., Haalck, L., Spener, F., Paltauf, F.,
Hermetter, A., 1999. Interactions of fluorescent triacylglycerol
analogs covalently bound to the active site of a lipase from
Rhizopus oryzae. Eur. J. Biochem. 262, 63–69.
Rangheard, M.S., Langrand, G., Triantaphylides, C., Baratti,
J., 1992. Multi-competitive enzymatic reactions in organic
media:
a
simple test for the determination of lipase
Zelinski, T., Waldmann, H., 1997. Quervernetzte Enzymkristalle
(CLECs)—effiziente und stabile Biokatalysatoren für die
präparative organische Chemie. Angew. Chem. 109, 746–748.
Zhao, K., Landry, D.W., 1993. Tetrazole catalyzed synthesis of
phosphonate esters. Tetrahedron 49, 363–368.
Zimmerman, P., Bommer, R., Bar, T., Schmidt, R.R., 1988.
Azidosphingosine glycosylation in glycosphingolipid synthesis.
J. Carbohydr. Chem. 7, 435–452.
fatty acid specificity. Enzyme Microb. Technol. 14, 966–
974.
Ransac, S., Gargouri, Y., Marguet, F., Buono, G., Berlinger, C.,
Hildebrand, P., Lensfeld, H., Hadvary, P., Verger, R., 1997.
Covalent inactivation of lipases. Methods Enzymol. 286, 190–
231.
Ransac, S., Rogalska, E., Gargouri, Y., Deveer, A.M.T.J., Paltauf,
F., De Haas, G.H., Verger, R., 1990. Stereosectivity of lipases.
I. Hydrolysis of enantiomeric glyceride analogs by gastric and
Zinke, A., Troger, H., Ziegler, E., 1940. Untersuchungen über
Perylen und seine Derivate. Ber. B 75, 1042–1048.