Design and synthesis of triglyceride analogue biotinylated suicide inhibitors for directed molecular evolution of lipolytic enzymes

Article abstract of DOI:10.1016/S0960-894X(00)00396-6

The design, synthesis, and inhibition properties of two new triglyceride analogue biotinylated suicide inhibitors (2) and (3) for directed molecular evolution of lipolytic enzymes by phage-display is described. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00396-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2027±2031
Design and Synthesis of Triglyceride Analogue Biotinylated
Suicide Inhibitors for Directed Molecular Evolution of
Lipolytic Enzymes
H.-J. Deussen,* S. Danielsen, J. Breinholt and T. V. Borchert
Â
Protein Discovery, Novo Nordisk A/S, Novo Alle, 2880 Bagsvñrd, Denmark
Received 10 May 2000; revised 30 June 2000; accepted 5 July 2000
AbstractÐThe design, synthesis, and inhibition properties of two new triglyceride analogue biotinylated suicide inhibitors (2) and (3)
for directed molecular evolution of lipolytic enzymes by phage-display is described. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
coated anity support. Cleavage of the disul®de bond
releases these phages displaying the formerly active
enzymes from the support.2b,4 The captured phages with
high anity for the ligand can be enriched, ampli®ed and
further characterized. The evolutionary scheme is repeated
for the necessary number of cycles until the desired
properties have been achieved. So far bifunctional activ-
ity labels for selection of ®lamentous bacteriophages
displaying b-lactamase and penicillin binding proteins,4,5
b-galactosidase,6 and Lipolase1 (a commercially avail-
able Humicola lanuginosa lipase from Novo Nordisk),7
based on the above design, have been used. Lipases (EC
3.1.1.3.) are serine hydrolases and catalyze the hydrolysis
of organic esters as well as esteri®cation. The natural
substrates of lipases are triglycerides of long-chain fatty
acids (fats and oils). The capability of degrading fatty
stain to a mixture of more soluble free fatty acids,
diglycerides, monoglycerides, and glycerol has led to the
common application of lipases, e.g. Lipolase1, in deter-
gents8 and in fat and oil processing.9 Lipases constitute
furthermore the largest group of synthetically useful
enzymes.10
Directed molecular evolution is an attempt to mimic
nature, in producing enzymes with altered properties
(e.g., changes in catalytic properties, stability, speci®city,
or pH pro®le). Large libraries comprising up to 1010
variants can today easily be created by di€erent
methods of random mutagenesis in the laboratory. The
current bottle-neck is the selection and screening of the
library in order to identify individual mutants with the
desired properties. Therefore, the development of
appropriate search systems is of key importance to the
whole process of directed evolution.1 Phage display2 has
become the most developed and commonly used method
in direct coupling of phenotype and genotype of
enzymes: The gene encoding the enzyme is packaged
within a bacteriophage and the enzyme itself is displayed
on the phage surface as a fusion protein. Those phages
displaying the desired enzymes on the surface can be
selected from a phage library by biopanning: Mixtures
of displayed active or inactive enzymes are incubated
with a bifunctional activity label that features a mecha-
nism-based inactivator3 (or suicide inhibitor) of the
target enzyme. The suicide inhibitor is connected to an
anity label (e.g., biotin) through a linker including a
disul®de bond, which can be reductively cleaved. On
incubation of a mixture of phage enzymes, those phages
showing catalytic activity on the inhibitor under the
conditions of the experiment are labeled with the anity
ligand. These phages can be recovered on an streptavidin
Anity Label Design
The structure of the suicide inhibitor of Lipolase1 (1),
which was synthesized earlier according to the design
discussed above,7 is shown in Figure 1. The inhibitor is
of the phosphonate irreversible type, which mimics the
tetrahedral transition state intermediate occuring during
ester hydrolysis. A direct covalent adduct between nucleo-
philic hydroxyl group of active site serine residue in the
catalytic triad and the phosphorus atom of the inhibitor
*Corresponding author. Tel.:+45-4443-7202; fax: +45-4443-4742;
e-mail: hjdu@novo.dk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00396-6

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H.-J. Deussen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2027±2031
Figure 1. Structure of the anity label (1): a 4-nitrophenol activated phosphonate, i.e., a suicide substrate of lipolytic enzymes is connected to a
biotin moiety (anity handle) through a spacer containing a disul®de bridge (reductive cleavage site).7
is formed releasing the nucleofuge 4-nitrophenolate
from the phosphonate.11 The linkage between the
phosphonate and the reductive cleavage site was chosen
to be a C11-alkyl chain in order to ensure a certain
length allowing to ®t into the hydrophobic cleft of lipase
variants leaving the cleavage site outside.12 The leaving
group PNP ensures ecient reactivity towards the active
site together with the necessary stability towards
hydrolysis with water.13 The suicide substrate design of
(1) was chosen on the basis of information about lipase-
substrate interaction at a submolecular level gained
earlier through X-ray crystallography of crystals of
simple organophosphonates conjugated with various
lipases.11b,c,d,12,14 However, the inhibitors used in these
studies showed only little similarity to the natural lipase
substrates, i.e., triacylglycerols. Accordingly the suicide
inhibitor part of (1) only resembles the transition state
intermediate of ester hydrolysis and not the additional
structural features of triacylglycerols, which are important
for speci®c substrate-enzyme interactions apart from the
catalytic triad. The most important rule of screening is
`You get what you screen for', so the ability to screen for
the closest structural analogue of the target compound is
an important advantage. More recently, dicarmoyloxy-
propanol,15 diacylglycerol,16 and dialkylglycerol17 esters
of alkylphosphonic acid have been reported as lipase
inhibitors, containing alkyl chains bound to a glycerol
backbone, thus structurally resembling triacylglycerols.
In order to obtain triglyceride analogue biotinylated
suicide inhibitors useful for the selection of lipases with
improved catalytic activity towards triacyl glycerols, we
synthesized (2) and (3).18 There the phosphonate unit is
attached at the sn-1 position (the most general approach
for 1,3- or unspeci®c lipases) of the triglyceride mimick-
ing moiety (Fig. 2).
The additional glycerol-like moieties providing more
natural lipase±substrate interactions due to the alkyl
chains in the sn-2 and sn-3 positions are thought to
result in a more targeted screening: (2) mimics a short
chain triglyceride with C9 at sn-2 and C10 at sn-3 without,
however, the features of the two ester bonds at those
positions. (3) corresponds to a long-chain (C18N) ana-
logue, where the hydrolyzable ester bonds are replaced
Figure 2. Structures of the triglyceride analogue anity labels (2) and (3): the 4-nitrophenol activated phosphonate is attached at the sn-1 position to
two di€erent glyceride-like analogues. The remaining design (biotin moiety, spacer, disul®de bridge) remained unchanged in comparison to (1).

H.-J. Deussen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2027±2031
2029
by carbamoyl functions.15 As the PNP-phosphonate
ester is the only moiety, which is susceptible towards
nucleophilic attack by the serine residue in the active
site, undesirable side reactions by lipases are avoided.
Hence, improved tools for directed evolution of lipases
towards triglyceride activity have been designed.
these di€erences can only be attributed to the additional
steric requirements of the glycerol-like molecular units
attached to the phosphorus atom, or due to chiral dis-
crimination at one of the stereocenters.17a Accordingly,
the least sterically requiring label (1) has the shortest t-
half, while (3), which is the most bulky, has the longest
t-half. The slow rate of inhibition observed for (2) and
(3) might provide an interesting opportunity for selec-
tion of mutant lipases having acquired increased activity
towards these inhibitors.
Lipase Inhibition
The two anity labels (2) and (3) were tested for their
ability to inactivate Lipolase1 according to method used
previously for (1).19 Like (1) do both new labels irrevers-
ibly inactivate native Lipolase1 in a pseudo-®rst-order
kinetics. However, the half-life of inactivation was found
to be 65 min for (2) and 85 min for (3) (Fig. 3) in com-
parison to the earlier determined 12 min for (1).7 As all
three labels consist of the same type of suicide inhibitor,
Synthesis (Scheme 1)
Ethyl (4-nitrophenyl)[11-([(2,5-dioxotetrahydro-1H-1-pyr-
rolyl)oxy]carbonyloxy)undecyl] phosphonate (4) was
synthesized in a ®ve step synthesis as described ealier.7
(4) was dealkylated by bromotrimethylsilane (a) to give
a trimethylsilyl phosphonate,20 which was transformed to
the PNP-phosphonochloridate by oxaloyl chloride (b).21
The PNP-phosphonochloridate was esteri®ed either with
commercially available racemic 2-decyl-1-tetradecanol (c),
or with (R,S)-1,2-dioctadecylcarbamoyl-glycerol (pre-
pared from octadecyl isocyanate and (R,S)-1-O-benzyl-
glycerol15a) (d) to give 2-decyltetradecyl (4-nitrophenyl)
[11-([(2,5-dioxotetrahydro-1H-1-pyrrolyl)oxy] carbonyl-
oxy)undecyl] phosphonate (5)22 or 2,3-di[(octadecyla-
mino) carbonyl] oxypropyl (4-nitrophenyl) [11-([(2,5-
dioxotetrahydro - 1H - 1 - pyrrolyl) oxy] carbonyloxyun-
decyl] phosphonate (6).23 This reaction sequence of suc-
cessive dealkylation and esteri®cation of the phosphonate
was found superior to the method described by Marguet
et al.13b using a phosphonodichloridate and N-methyl-
imidazole. (5) and (6) were ®nally coupled with biotinyl-
N-e-aminocaproyl-N-cystamine-N0- cystaminammonium
Figure 3. Time dependent inhibition of puri®ed lipolase.19 ^: Control (no
inhibitor); &: anity label/inhibitor (2); ~: anity label/inhibitor (3).
Scheme 1. Synthesis of (2) and (3): (a) 3 equiv Me3SiBr, rt, CH2Cl2, 2 days; (b) 1.5 equiv (COCl)2, 0ꢀC-rt, CH2Cl2, 2h; (c) 1 equiv (R,S)-2-decyl-1-
tetradecanol, 1.2. equiv Et3N, CH2Cl2, rt, 4h, chromatography on silica: CH2Cl2:AcOEt 25:1, (v:v), 57%; (d) 1 equiv (R,S)-1,2-dioctadecylcarba-
moylglycerol, 1.2 equiv Et3N, CH2Cl2, re¯ux, overnight, recryst. from EtOH, 66%; (e) 1 equiv (7), 3 equiv Et3N, DMF, rt, 3h, chromatography on
silica: CH2Cl2:MeOH 13:1, (v:v), 39%; (f) 1 equiv (7), 3 equiv Et3N, DMF, 40oC, 3h, chromatography on silica: CH2Cl2:MeOH 12:1, (v:v), 36%.

2030
H.-J. Deussen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2027±2031
tri¯uoroacetate (7) (prepared in a six step synthesis
according to ref 4d), to give the ®nal biotinylated suicide
inhibitors 2-decyltetradecyl (4-nitrophenyl) (11-[(2-[(2-
[6-(5-[(3aR,4S,6aS)-2-oxoperhydrothieno[3,4-d]imida-
zol-4-yl]pentanoylamino)hexanoyl]aminoethyl)disulfanyl]
ethylamino)carbonyl] oxyundecyl)phosphonate (2)24 (e)
and 2,3-di[(octadecylamino)carbonyl]oxypropyl (4-nitro-
phenyl) 11-[([2-(2-[(6-[5-((3aR,4R,6aS)-2-oxoperhydro-
thieno [3,4-d]imidazol-4-yl) pentanoyl] aminohexanoyl)
amino] ethyldisulfanyl) ethyl]aminocarbonyl)oxy]un-
decylphosphonate (3)25 (f).
11. (a) Kraut. J. Annu. Rev. Biochem. 1977, 46, 331. (b)
Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.; Dod-
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Huge-Jensen, B.; Patkar, S. A.; Thim, L. Nature 1991, 491. (c)
Martinez, C.; Nicolas, A.; van Tilbeurgh, H.; Eglo€, M.-P.;
Cudrey, C.; Verger, R.; Cambillau C. Biochemistry 1994, 33,
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Acknowledgements
We wish to thank Rie Kristine Schjeltved and Mette-
Marie Damborg for excellent technical assistance. We
wish to thank Jacques Fastrez, Allan Svendsen, and
Shamkant Anant Patkar for inspiring discussions.
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(b) Ator, M.A.; Ortiz de Montallano, P. R. In The Enzymes;
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18. As neither the stereochemistry at the phoshorus, nor at the
chiral C-2 of the glycerol-like backbone is important for our
selection purpose, (2) and (3) were prepared as racemic mixtures.
19. Brie¯y, puri®ed recombinant Lipolase1 (100 mg/mL in
100 mM Tris±HCl, 150 mM NaCl, 0.3 mM CaCl2, 0.1 mM
MgCl2, pH 7.5) was mixed with the suicide inhibitors (5 mM in
chloroform) to a ®nal inhibitor concentration of 50 mM). At
t=1, 2, 3 and 4 h, 10 mL samples were withdrawn and added to
990 mL lipase assay bu€er (80 mM PNP-butyrate in 50 mM
Tris±HCl, 0.1% Triton X-100, 10 mM CaCl2, pH 7.5). Release
of 4-nitrophenol was recorded continuously at OD405 on a
Milton Roy 1201 spectrophotometer.
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wetswinkel, S.; Avalle, B.; Fastrez, J. J. Mol. Biol. 2000, 295, 527.
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22. Data for 5: 1H NMR (CDCl3, 400 MHz): 0.88 (t, 6H), 1.2±1.3
(m, ca. 52H), 1.39 (m, 3H), 1.65 (m, 2H), 1.74 (m, 2H), 1.94 (m,
2H), 2.84 (s, 4H), 4.00 (m, 2H), 4.32 (t, 2H), 7.39 (d, 2H), 8.23 (d,
2H); FAB+MS: 851.6 (MH+); mp: 58 ꢀC Anal. C46H79N2O10P:
C 65.08 (calc. 64.92), H 9.48 (9.36), N 3.30 (3.29).
23. Data for 6: 1H NMR (CDCl3, 400 MHz): 0.88 (t, 6H), 1.2±
1.3 (m, ca. 66 H), 1.40 (m, 2H), 1.47 (m, 4H), 1.68 (m, 2H),
1.73 (m, 2H), 1.96 (m, 2H), 2.84 (s, 4H), 3.15 (m, 4H), 4.19 (m,
1H), 4.20 (m, 1H), 4.28 (m, 1H), 4.30 (m, 1H), 4.32 (t, 2H),
4.70 (m, 2H), 5.09 (m, 1H), 7.38 (d, 2H), 8.24 (d, 2H); FAB+MS:
1179.8 (MH+); mp: 67±69 ꢀC. Anal. C63H111N4O14P: C 64.75
(calc. 64.15), H 9.98 (9.48), N 4.46 (4.75).
24. Data for 2: 1H NMR (CDCl3, 400 MHz): 0.89 (t, 6H), 1.2±
1.8 (m, ca. 71H), 1.94 (m, 2H), 2.21 (t, 2H), 2.23 (t, 2H), 2.74

H.-J. Deussen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2027±2031
2031
(d, 1H), 2.83 (t, 2H), 2.85 (t, 2H), 2.93 (dd, 1H), 3.16 (m, 1H),
3.24 (br. q, 2H), 3.49 (br. q, 2H), 3.55 (q, 2H), 3.96 (m, 1H),
4.05 (m, 2H), 4.06 (m, 1H), 4.34 (m, 1H), 4.52 (m, 1H), 5.18
(br. s, 1H), 5.39 (br. t, 1H), 6.05 (br. s, 1H), 6.13 (br. t, 1H),
6.67 (br. t, 1H), 7.38 (d, 2H), 8.23 (d, 2H); FAB+MS: 1227.7
(MH+). Anal. C62H111N6O10PS3: C 61.83 (calc. 60.65), H 9.42
(9.11), N 6.31 (6.85).
2.74 (d, 1H), 2.83 (t, 2H), 2.85 (t, 2H), 2.93 (dd, 1H), 3.15
(m, 4H), 3.17 (m, 1H), 3.24 (br. q, 2H), 3.48 (br. q, 2H),
3.55 (q, 2H), 4.05 (t, 2H), 4.19 (m, 2H), 4.20 (m, 1H), 4.30
(m, 1H), 4.34 (dd, 1H), 4.52 (m, 1H), 4.81 (br. s, 2H), 5.05
(br. s, 1H), 5.10 (m, 1H), 5.41 (br. t, 1H), 5.82 (br. s, 1H),
6.06 (br. t, 1H), 6.67 (br. t, 1H), 7.38 (d, 2H), 8.23 (d,
2H); ESMS: 1555.8 (MH+); mp: 134±135 ꢀC. Anal.
C79H143N8O14PS3: C 61.24 (calc. 60.97), H 9.79 (9.26), N 6.41
(7.20).
25. Data for 3: 1H NMR (CDCl3, 400 MHz): 0.88 (t, 6H),
1.2±1.8 (m, ca. 94H), 1.96 (m, 2H), 2.22 (t, 2H), 2.23 (t, 2H),