Immobilization of chiral enzyme inhibitors on solid supports by amide-forming coupling and olefin metathesis

Article abstract of DOI:10.1016/S0040-4020(02)01052-9

The question whether phage display can be used as a selection method in the directed evolution of enantioselective enzymes has not been answered satisfactorily to date. In order to be able to test this in a specific case, namely in the hydrolytic kinetic

Full text of DOI:10.1016/S0040-4020(02)01052-9

Tetrahedron 58 (2002) 8465–8473
Immobilization of chiral enzyme inhibitors on solid supports
by amide-forming coupling and olefin metathesis
a
b
b
Manfred T. Reetz,a Carsten J. Ru¨ggeberg, Melloney J. Droge and Wim J. Quax
,
*
¨
a
¨
¨
Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mulheim/Ruhr, Germany
bDepartment of Pharmaceutical Biology, University Centre for Pharmacy, Antonius Deusinglaan 1, NL-9713 AV Groningen, The Netherlands
Received 19 April 2002; accepted 19 August 2002
Abstract—The question whether phage display can be used as a selection method in the directed evolution of enantioselective enzymes has
not been answered satisfactorily to date. In order to be able to test this in a specific case, namely in the hydrolytic kinetic resolution of the
acetate derived from a,b-isopropylideneglycerol (IPG) catalyzed by the lipase from Bacillus subtilis, suicide enzyme inhibitors anchored on
porous glass or polymer beads were designed and synthesized. These are immobilized phosphonates, which bear a leaving group and also
contain the chiral substrate (D) and (L)-IPG. Modified SIRANw (porous glass) and Tentagelw (polymer) were chosen as carriers, attachment
occurring via amide-forming coupling or Ru-catalyzed olefin metathesis. Initial lipase inhibition studies are also reported. q 2002 Elsevier
Science Ltd. All rights reserved.
1. Introduction
allows the selection of proteins on filamentous phages by
inserting the gene of interest into the phage gene g3p
sequence. The encoded enzyme (‘phage enzyme’) is
displayed on its surface. Consequently, proteins with
desirable binding properties can be selected from a
randomly constructed library of mutants.10,11 In the field
of antibodies new variants have been obtained by affinity
maturation, yielding selections of 1 out of 109.12 Moreover,
phage display has been used recently to select enzyme
variants with improved biophysical properties and/or
enhanced catalytic activities. Specifically, the techniques
of phage display were utilized to select enzymes on suicide
inhibitors,10 transition state analogs13 or substrates anchored
to the phage.10
We have previously shown that the methods of directed
evolution1–4 can be used successfully in the quest to create
enantioselective enzymes for use in organic chemistry.5 The
combination of random mutagenesis and gene-expression
coupled with high-throughput screening for enantioselec-
tivity of thousands of enzyme mutants forms the basis of a
fundamentally new concept in the area of asymmetric
catalysis. Specifically, the lipase from Pseudomonas
aeruginosa, which catalyzes the hydrolytic kinetic reso-
lution of a certain chiral ester with a selectivity factor E of
only 1.1 (wild-type), was evolved into a variant displaying
an E-value of 11.5 By applying recombinant methods such
as DNA shuffling,3 enantioselectivity was recently
increased further to E¼51.6 These efforts involved the
screening of about 40,000 enzyme variants. Since one of the
problems in this new field of asymmetric catalysis concerns
the question of efficient screening-systems for enantioselec-
tivity in a given reaction of interest, a great deal of effort has
gone into developing high-throughput ee-assays.7,8
Although none of these assays are universally applicable,
in optimal cases 1000–20,000 enzyme variants can be
screened per day.
Enantioselectivity in the present context is a particularly
difficult parameter to deal with. Indeed, the idea of applying
the technique of phage display in order identify the most
enantioselective enzyme or group of enzymes in a super-
large library of variants currently does not have an
undisputably sound theoretical basis. It may be argued
that a search based on maximal enantioselective binding is
predestined to lead to the identification of an enzyme
displaying unacceptably low activity (in the worst case a
single turnover only). On the other hand one can speculate
that the process of phage display will reveal enzymes
displaying synthetically sufficient but not overpowering
differentiation between the (R) and (S)-enantiomeric forms
of the chiral substrate under study, while allowing for
acceptable levels of activity. Irrespective of these uncer-
tainties we decided to embark on a long-term project
directed towards testing phage display in identifying
enantioselective ‘hits’ in libraries of millions of enzyme
variants.
A possible alternative to screening is selection by phage
display.9 Potentially it offers a means to ‘evaluate’ millions
of enzyme variants with respect to a given property within a
short time. As originally proposed by Smith9 the method
Keywords: biotransformations; olefin metathesis; asymmetric catalysis;
lipase; phage display.
*
Corresponding author. Tel.: þ49-208-306-2000; fax: þ49-208-306-2985;
e-mail: reetz@mpi-muelheim.mpg.de
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01052-9

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M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
The first phase of our study entails two problems. Firstly, it
needs to be demonstrated that the enzyme of interest can in
fact be displayed on bacteriophages. Several cases have
been reported.9–11 We ourselves were recently successful in
demonstrating this for the lipase from Bacillus subtilis.14
Secondly, since enantioselectivity is the parameter of
interest, incorporation of appropriate chirality into the
phage display system is necessary. More specifically it
requires the synthesis of a chiral enzyme inhibitor,
preferably a suicide inhibitor, on a solid phase. In this
publication we focus on this synthetic challenge, describing
the preparation of appropriate chiral suicide inhibitors
anchored on porous glass as well as on polymer beads,
designed for a specific enzymatic transformation. Our
efforts originate from the notion that phage display may
possibly be used in the directed evolution of enantioselec-
tive variants of the lipase from B. subtilis,14–16 specifically
in the hydrolytic kinetic resolution of the chiral acetate 1
derived from a,b-isopropylideneglycerol (IPG; 2); the wild-
type enzyme shows a selectivity factor of only E¼2.1 in
slight favor of the alcohol (L)-2.15 This means that the
relevant suicide inhibitor needs to be designed so as to
mimic the geometric parameters of (L)-1.
2. Results and discussion
2.1. Synthesis of immobilized suicide enzyme inhibitors
Our strategy centered around the preparation of solid
materials of the type 3, composed of an insoluble carrier,
an anchor-group, a spacer and a phosphonate moiety which
contains a leaving group as well as the chiral substrate (D) or
(L)-2, depending upon whether a (D) or (L)-selective enzyme
is to be evolved in future work.
In order to put this into practice we first considered the
utilization of the biotin/avidin interaction which is known to
be exceedingly strong. Therefore the biotin-modified
phosphonic acid 5 was synthesized from commercially
available diol 4, hoping to esterify it with formation of the
desired target molecule 6. However, numerous attempts to
affect this seemingly trivial transformation failed.15
Since our initial and most obvious approach failed, we
changed the synthetic strategy. This time the idea was to
anchor the suicide inhibitor on SIRANw (a porous glass) via
an appropriate spacer. Therefore, the activated diastereo-
meric carbonates 8a,b were chosen as the target molecules
because they can be expected to undergo amidation with
amino-modified SIRANw of the type 7 affording the
immobilized enzyme inhibitors 9a,b. During our study a
related strategy was described by Deussen and Borchert in
their efforts to prepare enzyme inhibitors, although in their
case these are linked to biotin via spacers and disulfide
bridges.18
Due to their tetrahedral geometry, bond distances and
charge distribution, phosphonic acid esters are generally
viewed as acceptable mimics of the transition state of
enzyme-catalyzed ester hydrolysis. For example, this idea
has been exploited numerous times in the production of
catalytic antibodies.17 It also forms the basis of our own
strategy. In our case the purpose of the phosphonate is to
function as a suicide inhibitor. This means that it needs to
contain a leaving group, which can be displaced by the
primary alcohol function of serine, which is at the active site
of the lipase. The irreversible formation of a stable
enzyme–phosphonate complex is expected to destroy the
catalytic property of the lipase.
This approach proved to be successful. SIRANw was first
modified chemically with formation of amino-modified
beads 7.
The synthesis of the phosphonates 8a,b was carried out as

M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
8467
to anchor appropriate soluble enzyme inhibitors on solid
carriers. Olefin metathesis reactions, especially when
mediated by Grubbs or Schrock catalysts, have been applied
successful in a wide variety of synthetic problems.19
However, attaching enzyme inhibitors or transition state
analogs to solid carriers by this method has not been
described so far. We hoped to immobilize suicide inhibitors
of the type 17a,b on SIRANw or Tentagelw beads, the latter
being appropriately modified as 18 and 19, respectively
(Scheme 2).
shown in Scheme 1. Compounds 8a and 8b each exist as a
1:1 mixture of diastereomers. Finally, reaction of beads 7
with activated carbonates 8a,b afforded the immobilized
suicide inhibitors 9a,b.
The synthesis of the respective chemically modified beads
18 and 19 as well as the phosphonates 17a,b (each as 1:1
diastereomer mixture) proceeded as planned. Moreover,
olefin metathesis of these building blocks using the Grubbs
catalyst19 was carried out according to Scheme 2, providing
a second set of chiral immobilized suicide inhibitors 20a,b
and 21a,b. It should be noted that materials of the type 9a,b
and 20a,b/21a,b are not easy to characterize.
The primary synthetic goal was therefore reached. Never-
theless, we also took a parallel approach and prepared a
different type of immobilized enzyme inhibitor. In this case
we envisioned that olefin metathesis would provide a means

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M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
Scheme 1. Synthesis of the activated enzyme inhibitors 8a,b.
Scheme 2. Preparation of the carrier-fixed phosphonates 20a,b and 21a,b by olefin metathesis.

M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
8469
Figure 1. Residual enzyme-activity of the lipase from B. subtilis during incubation with the SIRANw-immobilized suicide inhibitor 9a.
2.2. Binding studies of the immobilized suicide enzyme
inhibitors
appears to be too short, not allowing the enzyme to undergo
phosphorylation at the hydroxy-group of serine. Indeed,
X-ray crystallographic investigations of the enzyme with
active site bound phosphonates suggest a minimum distance
of at least six carbon atoms between phosphorus and the
appropriate solid carrier.20 At shorter distances steric
interactions prevent reaction and therefore no deactivation
is observed. An alternative explanation is inefficient
immobilization of the inhibitors. A similar trend was
observed upon comparing 20b with 9b (t1/2¼4.4 min).
However, it is not meaningful to compare immobilized
inhibitor 9a with its diastereomeric form 9b in a quantitative
way due to uncertainties in the degree of loading. It needs to
be pointed out that this uncertainty is of no relevance to
future experiments in the selection step of directed evolution,
since only one of the materials will be used (9a or 9b in the
evolution of a (D) or (L)-selective mutant enzyme).
Having prepared different types of immobilized enzyme
inhibitors 9a,b, 20a,b and 21a,b, it was now possible to
carry out binding studies.14 Note that the potential suicide
inhibitors are enantiopure at the stereocenter of the IPG
molecule ((D) or (L)-configuration). We hope that in later
studies this will impart (D) or (L)-inhibitor properties, i.e. be
useful in the selection (D) or (L)-specific enzymes. However,
the materials are ‘racemic’ at the phosphorus atom. Since
separation of the respective diastereomers was not possible,
we used the mixtures in binding studies. In doing so the
residual catalytic activity of the lipase during incubation
with the suicide inhibitors was measured.
Experiments involving the incubation of the lipase from B.
subtilis with the SIRANw-based phosphonate 20a showed
no significant deactivation of the enzyme within a time
range of 5 h. In sharp contrast appreciable deactivation
occurred with the other SIRANw-immobilized suicide
inhibitor 9a within minutes (Fig. 1). The t1/2-value turned
out to be 5.2 min. This pronounced difference in behavior
can be traced to the difference in the length of the respective
spacers. In the case of materials 20a,b, the alkenyl-spacer
Interestingly, the results of the incubation experiments using
the Tentagelw-based inhibitor 21a show that 50% deactiva-
tion requires more than 2 h (Fig. 2). Such lower reactivity
relative to 9a can be explained on the basis of reduced
swelling ability in aqueous medium.15Alternatively, the
orientation of the spacer onto the active site of the lipase
may be unfavorable resulting in a lower reactivity.
Figure 2. Residual enzyme-activity of the lipase from B. subtilis during incubation with the tentagel-based suicide inhibitor 21a.

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M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
3. Conclusion
determined by incubation of the enzyme and the appropriate
phosphonate inhibitor in assay buffer at room temperature.
10 mg of the inhibitor was washed three times and pre-
equilibrated for 10 min using 500 mL assay buffer. Note that
the inhibitor does not dissolve in this solution. Therefore the
reaction mixture was vertically rotated during incubation.
Subsequently, purified lipase (1 mg) was added to the
inhibitor. As control, lipase was also incubated in assay
buffer without the inhibitor for the same time period. During
incubation of the lipase, aliquots of 10 mL were collected to
determine the residual lipase activity using the p-nitro-
phenylcaprylate assay, as described above. No inactivation
of lipase in the controls could be detected. All results are
expressed as mean^SEM. The statistical significance of
differences was tested at a significance level of P,0.05
using a two-tailed Student’s t-test.
We have succeeded in attaching chiral phosphonate-based
enzyme inhibitors covalently to solid supports. In one
approach a soluble chiral phosphonate connected to an
activated carbonate via a spacer was prepared and reacted
with amino-modified SIRANw, resulting in a smooth
coupling reaction. In the second approach to immobilization
a similar chiral enzyme inhibitor connected to an olefinic
moiety via an appropriate alkyl chain was synthesized, in
this case covalent attachment to allyl-modified SIRANw or
Tentagelw occurring by Ru-catalyzed olefin metathesis. We
expect that olefin metathesis as described here will be useful
in the immobilization of other suicide inhibitors and/or
transition state analogs in general, adding to the power of
this olefin-forming process.19
The stage is now set for the implementation of a selection
system based on phage display for the identification of
enantioselective lipase variants from B. subtilis in the model
reaction 1!2. The results of these upcoming studies will
enable us to decide whether the idea of utilizing phage
display in the directed evolution of enantioselective
enzymes is viable, or whether only enantioselective binding
phenomena will be revealed. Irrespective of the outcome,
the synthetic strategies described in the present paper may
be of use in other applications as well.
4.3. Surface modification of SIRANw
A suspension of SIRANw (SIKUG/012/XX/300/A; 100 g)
in 5% nitric acid (200 mL) was heated under reflux for 8 h.
After filtration the glass beads were washed with water
(500 mL) and further purified by dialysis for 48 h. The
resulting SIRANw was then washed with 500 mL of acetone
followed by removal of solvent in vacuo at 1508C. To a
suspension of the dried SIRANw in toluene (300 mL)
allyltrimethoxysilane or 3-aminopropyltrimethoxysilane
(9.6 mmol) was added und heated under reflux for 20 h.
The modified SIRANw was filtered off and washed with
MeOH (750 mL). After a drying period of 8 h in vacuo at
808C the SIRANw was ready to use. n-Aminopropyl-
modified SIRANw 7: elemental analysis: C 0.05, N 0.03.
Allyl-modified SIRANw 18: elemental analysis (%): C 0.03.
4. Experimental
Unless otherwise noted, all starting materials were obtained
from commercial suppliers and were used without further
purification. Solvents were dried according to established
procedures by distillation from an appropriate drying agent
under an inert atmosphere of argon in glassware that has
been flame-dried. Column chromatography was performed
on Merck silica gel 60 (260–400 mesh). Chemical shifts are
quoted in parts per million (ppm, d) using either the solvent
as internal standard (1H NMR, 13C NMR) or phosphoric
acid as external standard (31P NMR). In the case of the
immobilized inhibitors the amount of phosphorus (or
nitrogen) relative to the whole material is very small,
which means that the uncertainty in the elemental analysis
can be large.
4.3.1. General procedure for the preparation of phos-
phonates 8a,b. A solution of phosphonate 16 (3.69 g,
16.7 mmol, 20 mL CH2Cl2) was cooled to 08C and
bromotrimethylsilane (6.39 g, 41.8 mmol) was added. The
mixture was stirred for 40 h at room temperature. All
volatile components were removed under reduced pressure
and the orange residue was dissolved in CH2Cl2 (35 mL).
After the addition of oxalyl dichloride (2.43 g, 19.1 mmol)
and DMF (0.1 mL) at 08C the mixture was heated under
reflux for 18 h. All volatile components were then removed
in vacuo and the residue was dissolved in CH2Cl2 (40 mL).
To this solution (D) or (L)-2 (0.84 g, 6.4 mmol) and
triethylamine (0.77 g, 7.6 mmol) was added at 08C. After
2 h at room temperature the solution was concentrated. The
resulting black oil was purified by flash chromatography
eluting with CH2Cl2/EtOAc (3:1). 8a (2.84 g, 71%): 1H
NMR (300.1 MHz, CDCl3): d¼8.23–8.18 (m, 2H), 7.40–
7.36 (m, 2H), 4.31–4.00 (m, 3J(H,H)¼6.7 Hz, 6H), 3.73 (dt,
2J(H,H)¼8.6 Hz, 3J(H,H)¼6.1 Hz, 1H), 2.81 (s, 4H), 2.04–
1.90 (m, 2H), 1.73–1.60 (m, 4H), 1.42–1.21 (m, 20H); 13C
NMR (75.5 MHz, CDCl3): d¼168.5, 155.4, 151.4, 144.4,
125.9, 120.9, 109.8, 74.1 (d, 3J(C,P)¼7 Hz), 71.6, 66.5, 65.8
(d, 4J(C,P)¼5 Hz), 30.3 (d, 3J(C,P)¼17 Hz), 29.3, 29.2,
29.1, 29.0, 28.9, 28.3, 26.7, 25.8 (d, 1J(C,P)¼140 Hz), 25.4
4.1. Enzyme activity
The lipase from B. subtilis was purified as described by
Lesuisse et al.21 Enzymatic activity was determined
spectrophotometrically by the p-nitrophenylcaprylate
assay, since a C8 alkyl chain revealed optimal activity for
the Bacillus lipase.22 A 10 mM solution p-nitrophenyl-
caprylate in methanol was prepared. 0.5 mM p-nitrophenyl-
caprylate was added to 900 mL assay buffer, containing
50 mM phosphate buffer (pH 8), 0.36% Triton X100 (v/v)
and 0.1% gum arabic. The assay buffer was preincubated at
308C. Fifty microliters purified lipase (1 mg lipase) were
added and the absorbance was measured at 410 nm.
2
6
(d, J(C,P)¼7 Hz), 25.1, 22.1 (d, J(C,P)¼2 Hz), 22.0 (d,
6J(C,P)¼2 Hz); 31P NMR (121.5 MHz, CDCl3): d¼32.0,
31.7; IR (neat): n¼3114, 3080, 2931, 2856, 1812, 1790,
˜
4.2. Binding to the suicide inhibitor
1744, 1592, 1523, 1492, 1347, 1257, 1227, 1048, 862 cm21
MS (70 eV): m/z (%): 525 (3), 404 (69), 384 (100), 360 (11),
;
The inactivation of lipase by the phosphonate inhibitors was

M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
8471
263 (32), 217 (37), 139 (10), 69 (14), 55 (27), 41 (13);
elemental analysis calcd (%) for C28H41N2O12P (628.6): C
53.50, H 6.57, N 4.46, P 4.93; found C 53.50, H 6.62, N
4.53, P 4.89. 8b (2.68 g, 67%): 1H NMR (300.1 MHz,
CDCl3): d¼8.19–8.16 (m, 2H), 7.36–7.33 (m, 2H), 4.28–
diluted with ether (200 mL). The organic layer was washed
with water (2£50 mL) and brine (1£50 mL). Drying over
MgSO4, evaporation of the solvent and purification via flash
chromatography eluting with EtOAc/hexane (90:10) gave
1
13 (7.42 g, 91%) as a colorless oil. H NMR (300.1 MHz,
CDCl3): d¼4.52 (dd, 3J(H,H)¼2.8, 4.5 Hz, 1H), 4.11–3.98
3
2
3.97 (m, J(H,H)¼6.6 Hz, 6H), 3.71 (dt, J(H,H)¼8.7 Hz,
3J(H,H)¼6.0 Hz, 1H), 2.79 (s, 4H), 1.99–1.87 (m, 2H),
1.73–1.58 (m, 4H), 1.36–1.23 (m, 20H); 13C NMR
(75.5 MHz, CDCl3): d¼168.7, 155.5, 151.5, 144.5, 125.6,
(m, 4H), 3.98–3.86 (m, 1H), 3.68 (dt, 3J(H,H)¼9.8, 6.8 Hz,
3
1H), 3.48–3.41 (m, 1H), 3.33 (dt, J(H,H)¼9.4, 6.8 Hz,
1H), 1.82–1.47 (m, 12H), 1.30–1.22 (m, 20H); 13C NMR
3
2
120.9, 109.9, 74.1 (d, J(C,P)¼7 Hz), 71.6, 66.5, 65.8 (d,
(75.5 MHz, CDCl3): d¼99.2, 68.0, 62.7, 61.7 (d, J(C,P)¼
3
4J(C,P)¼5 Hz), 30.3 (d, J(C,P)¼17 Hz), 29.3, 29.2, 29.1,
7 Hz), 31.2, 31.0 (d, 3J(C,P)¼17 Hz), 30.1, 29.9, 29.9, 29.8,
29.7, 29.4 (d, 4J(C,P)¼1 Hz), 26.6, 26.0 (d, 1J(C,P)¼
140 Hz), 25.9, 22.7 (d, 2J(C,P)¼5 Hz), 20.1, 16.9 (d,
3J(C,P)¼6 Hz); 31P NMR (121.5 MHz, CDCl3): d¼33.2;
1
29.0, 28.9, 28.3, 26.7, 25.8 (d, J(C,P)¼140 Hz), 25.4 (d,
2J(C,P)¼7 Hz), 25.1, 22.1 (d, 6J(C,P)¼2 Hz), 22.0 (d,
6J(C,P)¼2 Hz); 31P NMR (121.5 MHz, CDCl3): d¼31.9,
31.7; IR (neat): n¼3114, 3080, 2931, 2856, 1812, 1789,
IR (neat): n¼2928, 2854, 1247, 1060, 1032 cm21; MS
˜
˜
1744, 1592, 1523, 1492, 1347, 1258, 1226, 1049, 862 cm21
;
(70 eV): m/z (%): 392 (1) [Mþ], 363 (10), 307 (18), 291
(42), 278 (61), 263 (21), 235 (13), 179 (22), 165 (45), 152
(100), 125 (15), 85 (37); elemental analysis calcd (%) for
C20H41O5P (392.5): C 61.20, H 10.53, P 7.89; found C
61.11, H 10.68, P 7.86.
MS (70 eV): m/z (%): 613 (100), 490 (18), 412 (47), 356
(14), 115 (16), 101 (32), 55 (21), 43 (35); elemental analysis
calcd (%) for C28H41N2O12P (628.6): C 53.50, H 6.57, N
4.46, P 4.93; found C 53.38, H 6.52, N 4.49, P 4.85.
4.4. Attachment of phosphonates 8a, b to SIRANw
7
4.4.3. Diethyl (11-hydroxyundecyl)phosphonate (14). A
solution of phosphonate 13 (12.5 g, 31.0 mmol, 120 mL
MeOH) was treated with Amberlite IR-120 (19.0 g). The
resulting mixture was gently shaken at room temperature for
16 h. The ion-exchange resin was filtered off and evapo-
ration of the solvent gave a crude orange product which was
purified by flash chromatography eluting with CH2Cl2/
MeOH (95:5) to yield 14 (8.11 g, 85%) as a pale yellow
solid. 1H NMR (200.1 MHz, CDCl3): d¼4.12–3.97 (m,
4H), 3.58 (t, 3J(H,H)¼6.5 Hz, 2H), 1.86 (broad s, 1H),
1.76–1.24 (m, 3J(H,H)¼7.0 Hz, 26H); 13C NMR
(50.3 MHz, CDCl3): d¼62.9, 61.4 (d, 2J(C,P)¼7 Hz),
32.7, 30.5 (d, 3J(C,P)¼17 Hz), 29.5, 29.4, 29.3, 29.2,
To a suspension of SIRANw 7 (43.3 g, ,0.9 mmol) in
CH3CN (150 mL) 8a or 8b (877 mg, 1.40 mmol) and
triethylamine (236 mg, 2.33 mmol) were added. The
mixture was shaken for 24 h at room temperature. Filtration,
subsequent washings with CH3CN (3£70 mL) and acetone
(3£70 mL) and drying in vacuo overnight provided the
SIRANSw 9a and 9b, respectively. Phosphonate modified
SIRANw 9a: elemental analysis: N 0.03, P 0.55. Phospho-
nate modified SIRANw 9b: elemental analysis: N 0.03, P
0.03.
1
2
4.4.1. 2-[(11-Bromoundecyl)oxy]tetrahydro-2H-pyran
(12). 11-Bromoundecanol (10) (7.45 g, 29.7 mmol, 30 mL
CH2Cl2) was added dropwise to a solution of 3,4-dihydro-
2H-pyran (11) and toluene-4-sulfonic acid (56 mg,
0.3 mmol) in CH2Cl2 (50 mL) at room temperature. The
mixture was stirred overnight. After dilution with ether
(150 mL) the organic layer was washed with saturated
aqueous Na2CO3 (2£80 mL), water (3£80 mL) and brine
(1£80 mL). Drying over MgSO4, concentration and purifi-
cation via flash chromatography eluting with EtOAc/hexane
(10:90) gave 12 (8.46 g, 85%) as a colorless liquid. 1H NMR
(300.1 MHz, CDCl3): d¼4.54 (dd, 3J(H,H)¼2.6, 4.5 Hz,
29.0, 25.7, 25.6 (d, J(C,P)¼140 Hz), 22.3 (d, J(C,P)¼
5 Hz), 16.4 (d, 3J(C,P)¼6 Hz); 31P NMR (81.0 MHz,
CDCl ): d¼33.3; IR (neat): n¼3420, 2927, 2854, 1228,
˜
3
1059, 1029 cm21; MS (70 eV): m/z (%): 308 (1) [Mþ], 363
(10), 290 (18), 278 (38), 165 (50), 152 (100), 138 (27), 125
(41), 55 (30), 41 (25); elemental analysis calcd (%) for
C15H33O4P (308.4): C 58.42, H 10.79, P 10.04; found C
58.42, H 10.74, P 9.92.
4.4.4. Preparation of activated carbonate 15. To a
solution of phosphonate 14 (6.02 g, 19.5 mmol) and
triethylamine (5.92 g, 58.5 mmol) in CH3CN (50 mL) was
added di(N-succinimidyl) carbonate (10.0 g, 39.1 mmol).
The resulting mixture was stirred overnight. After dilution
with EtOAc (150 mL) the organic layer was washed with
water (3£80 mL) and dried over MgSO4. The solvent was
then evaporated and the residue was purified by flash
chromatography eluting with EtOAc (100%) to afford 15
(8.28 g, 94%) as a yellow oil. 1H NMR (300.1 MHz,
3
1H), 3.95–3.64 (m, 2H), 3.52–3.29 (m, J(H,H)¼6.8 Hz,
4H), 1.89–1.25 (m, 25H); 13C NMR (75.5 MHz, CDCl3):
d¼98.8, 67.6, 62.3, 34.0, 32.8, 30.7, 29.7, 29.5, 29.4, 28.7,
28.1, 26.2, 25.5, 19.6; IR (neat): n¼2928, 2854, 1034, 645,
˜
563 cm21; MS (70 eV): m/z (%): 335 (2) [Mþ], 261 (1), 151
(1), 135 (1), 115 (3), 101 (8), 85 (100), 69 (7), 56 (16), 41
(11); elemental analysis calcd (%) for C16H31BrO2 (335.3):
C 57.31, H 9.32, Br 23.83; found C 57.42, H 9.26, Br 23.67.
3
CDCl3): d¼4.26 (t, J(H,H)¼6.8 Hz, 2H), 4.24–4.09 (m,
4H), 2.78 (s, 4H), 1.73–1.46 (m, 6H), 1.32–1.22 (m, 20H);
13C NMR (75.5 MHz, CDCl3): d¼169.1, 152.0, 72.0, 61.7
(d, 2J(C,P)¼7 Hz), 30.9 (d, 3J(C,P)¼17 Hz), 29.7, 29.7,
29.6, 29.4, 28.7, 26.0 (d, 1J(C,P)¼140 Hz), 25.8, 25.7, 22.7
(d, 2J(C,P)¼5 Hz), 16.8 (d, 3J(C,P)¼6 Hz); 31P NMR
4.4.2. Diethyl 11-[(2-tetrahydro-2H-pyranyl)oxy]undecyl-
phosphonate (13). Diethyl phosphite (4.84 g, 35.1 mmol,
5 mL NMP) was added dropwise to a ice-cooled suspension
of sodium hydride (0.92 g, 38.3 mmol) in NMP (20 mL).
After 1 h at 08C and 1 h at room temperature the solution
was again cooled to 08C and alkyl bromide 12 (10.69 g,
31.9 mmol, 8 mL NMP) was added slowly. The mixture was
allowed to warm up to room temperature overnight and was
(121.5 MHz, CDCl ): d¼33.5; IR (neat): n¼2923, 2854,
˜
3
1812, 1785, 1740, 1233, 1027 cm21; MS (70 eV): m/z (%):
449 (1) [Mþ], 404 (2), 307 (3), 291 (100), 263 (12), 165
(24), 152 (43), 125 (15), 55 (13); elemental analysis calcd

8472
M. T. Reetz et al. / Tetrahedron 58 (2002) 8465–8473
4
3
(%) for C20H36NO8P (449.5): C 53.44, H 8.07, N 3.12, P
6.89; found C 53.65, H 8.02, N 3.09, P 7.03.
2J(C,P)¼5 Hz), 32.8 (d, J(C,P)¼1 Hz), 29.3 (d, J(C,P)¼
1
2
17 Hz), 25.5 (d, J(C,P)¼143 Hz), 24.9 (d, J(C,P)¼2 Hz),
21.4 (d, J(C,P)¼2 Hz), 21.3 (d, J(C,P)¼2 Hz); 31P NMR
6
6
4.4.5. Preparation of p-nitrophenyl substituted phospho-
nate 16. To a solution of phosphonate 15 (8.00 g,
17.8 mmol, 50 mL CH2Cl2) was added oxalyl dichloride
(4.52 g, 35.6 mmol) and DMF (0.1 mL) at 08C. The
resulting mixture was stirred for 45 min at 08C and was
allowed to warm up to room temperature overnight. All
volatile components were removed under reduced pressure
und the brown residue was dissolved in CH2Cl2 (60 mL).
After the addition of p-nitrophenol (2.48 g, 17.8 mmol) the
solution was cooled to 08C and triethylamine (3.60 g,
35.6 mmol) was added. The mixture was stirred for 1 h at
08C and overnight at room temperature. All volatile
components were removed in vacuo and the black
residue was purified by flash chromatography eluting with
CH2Cl2/EtOAc (7:1) to afford 16 (7.05 g, 73%) as a yellow
solid. 1H NMR (300.1 MHz, CDCl3): d¼8.23–8.20 (m,
2H), 7.38–7.32 (m, 2H), 4.28 (t, 3J(H,H)¼6.7 Hz, 2H),
4.25–4.10 (m, 2H), 2.81 (s, 4H), 1.96–1.85 (m, 2H), 1.73–
1.58 (m, 4H), 1.38–1.25 (m, 17H); 13C NMR (75.5 MHz,
CDCl3): d¼168.7, 155.8, 151.6, 144.4, 125.6, 120.9, 71.6,
62.9 (d, 2J(C,P)¼7 Hz), 30.4 (d, 3J(C,P)¼17 Hz), 29.4,
29.3, 29.2, 29.0, 29.0, 28.3, 26.0 (d, 1J(C,P)¼140 Hz), 25.4,
(121.5 MHz, CDCl ): d¼31.7, 31.4; IR (neat): n¼3115,
˜
3
3079, 2987, 2936, 1641, 1592, 1523, 1492, 1347, 1225,
1055, 918, 754 cm21; MS (70 eV): m/z (%): 399 (5) [Mþ],
384 (100), 341 (13), 286 (21), 268 (17), 203 (11), 101 (41),
81 (11), 57 (15), 43 (41); elemental analysis calcd (%) for
C18H26NO7P (399.4): C 54.13, H 6.56, N 3.51, P 7.76;
found C 54.20, H 6.65, N 3.54, P 7.61. 17b (3.47 g, 29%):
1H NMR (300.1 MHz, CDCl3): d¼8.23–8.18 (m, 2H),
7.40–7.33 (m, 2H), 5.81–5.67 (m, 1H), 5.01–4.92 (m, 2H),
2
3
4.31–4.00 (m, 4H), 3.73 (dt, J(H,H)¼8.7 Hz, J(H,H)¼
5.8 Hz, 1H), 2.09–1.91 (m, 4H), 1.77–1.63 (m, 2H), 1.50
(tt, J(H,H)¼7.3 Hz, 2H), 1.39–1.31 (m, 6H); 13C NMR
3
(75.5 MHz, CDCl3): d¼155.4, 144.6, 137.7, 125.7, 121.0,
115.2, 110.1, 73.9 (d, 3J(C,P)¼7 Hz), 66.4, 65.5 (d, 2J(C,P)¼
4
3
5 Hz), 32.8 (d, J(C,P)¼1 Hz), 29.3 (d, J(C,P)¼17 Hz),
25.5 (d, 1J(C,P)¼143 Hz), 24.9 (d, 2J(C,P)¼2 Hz), 21.4
(d, 6J(C,P)¼2 Hz), 21.3 (d, 6J(C,P)¼2 Hz); 31P NMR
(121.5 MHz, CDCl ): d¼31.7, 31.4; IR (neat): n¼3114,
˜
3
3079, 2987, 2937, 1641, 1592, 1523, 1492, 1347, 1225,
1055, 917, 754 cm21; MS (70 eV): m/z (%): 399 (5) [Mþ],
384 (100), 341 (13), 286 (21), 268 (17), 203 (10), 101 (42),
81 (11), 57 (15), 43 (40); elemental analysis calcd (%) for
C18H26NO7P (399.4): C 54.13, H 6.56, N 3.51, P 7.76;
found C 54.18, H 6.47, N 3.58, P 7.80.
2
3
25.3, 22.2 (d, J(C,P)¼5 Hz), 16.3 (d, J(C,P)¼6 Hz); 31P
NMR (121.5 MHz, CDCl ): d¼31.6; IR (KBr): n¼3114,
˜
3
3081, 2929, 2855, 1812, 1789, 1743, 1592, 1522, 1491,
1347, 1258, 1226, 1037, 913, 862 (m) cm21; MS (70 eV):
m/z (%): 525 (3), 404 (69), 384 (100), 360 (11), 263 (32),
217 (37), 139 (10), 69 (14), 55 (27), 41 (13)); elemental
analysis calcd (%) for C24H35N2O10P (542.5): C 53.13, H
6.50, N 5.16, P 5.71; found C 53.03, H 6.53, N 5.23, P 5.79.
4.5. Allyl modified Tentagel HL-OH 19
To a suspension of swollen Tentagel HL-OH (600 mg dry
weight, ,0.24 mmol) in 30 mL of DMSO allyl bromide
(230 mg, 1.90 mmol) and powdered potassium hydroxide
(160 mg, 2.85 mmol) were added. The mixture was shaken
overnight. After filtration and washings with water
(3£30 mL), acetone (3£30 mL), ether (2£30 mL) and
pentane (30 mL) the resulting resin was dried in vacuo to
afford allyl modified tentagel 19 as yellow beads.
4.4.6. General procedure for the synthesis of phospho-
nates 17a,b. A solution of diethyl 6-hexenylphosphonate
(23) (6.61 g, 30.0 mmol) in CH2Cl2 (15 mL) at 08C was
treated with bromotrimethylsilane (11.50 g, 75.1 mmol).
After 48 h at room temperature the solvent and excess of
silane was removed under reduced pressure. The residue
was dissolved in CH2Cl2 (15 mL) and oxalyl dichloride
(11.44 g, 90.1 mmol) as well as DMF (0.1 mL) was added at
08C. The mixture was allowed to warm up to room
temperature and was then heated under reflux for 1 h. All
volatile components were removed in vacuo and the residue
was dissolved in toluene (150 mL). The resulting mixture
was cooled to 08C and treated with p-nitrophenol (3.76 g,
27.0 mmol), triethylamine (3.80 g, 37.6 mmol) and
1H-tetrazole (0.21 g, 3.0 mmol). After 1 h at 08C and 4 h
at room temperature the solution was again cooled to 08C
and (D) or (L)-2 (3.97 g, 30.0 mmol, 15 mL THF) and
triethylamine (3.80 g, 37.6 mmol) were added dropwise.
The mixture was stirred overnight at room temperature.
Filtration, concentration in vacuo and purification via flash
chromatography eluting with EtOAc/hexane (1:1) gave 17a
and 17b, respectively, as yellow oils. 17a (3.86 g, 32%):
1H NMR (300.1 MHz, CDCl3): d¼8.22–8.17 (m, 2H),
7.39–7.34 (m, 2H), 5.80–5.66 (m, 1H), 5.00–4.91 (m, 2H),
4.5.1. General procedure for the preparation of carrier-
fixed phosphonates 20a,b and 21a,b via olefin metathesis.
To a suspension of olefin modified carrier (18 or 19;
,0.2 mmol) in 30 mL of CH2Cl2 phosphonate 17a or 17b
(160 mg, 0.40 mmol) and Grubbs’ catalyst19 (33 mg,
0.04 mmol) was added. The mixture was heated under
reflux for 24 h and filtered off. Subsequent washings with
CH2Cl2 (200 mL), acetone (250 mL) and ether (250 mL)
and drying in vacuo at 408C gave the appropriate carrier-
fixed phosphonates. Tentagel HL fixed phosphonate 21a:
31P NMR (81.0 MHz, CDCl3): d¼32.3; elemental analysis
(%): N 0.11, P 0.19. Tentagel HL fixed phosphonate 21b:
31P NMR (81.0 MHz, CDCl3): d¼32.0; elemental analysis
(%): N 0.11, P 0.14. SIRANw fixed phosphonate 20a:
elemental analysis (%): C 0.02, N 0.01, P 0.05. SIRANw
fixed phosphonate 20b: elemental analysis (%): C 0.02, N
0.01, P 0.01.
4.5.2. Diethyl 6-hexenylphosphonate (23). Diethyl phos-
phite (5.61 g, 40.1 mmol, 20 mL NMP) was added dropwise
to a ice-cooled suspension of sodium hydride (1.06 g,
36.9 mmol) in NMP (30 mL). After 1 h at 08C and 1 h at
room temperature the solution again was cooled to 08C and
6-bromo-hex-1-ene (22) (6.02 g, 36.9 mmol) was added
2
3
4.31–3.99 (m, 4H), 3.72 (dt, J(H,H)¼8.5 Hz, J(H,H)¼
5.8 Hz, 1H), 2.08–1.90 (m, 4H), 1.76–1.62 (m, 2H), 1.49
3
(tt, J(H,H)¼7.3 Hz, 2H), 1.40–1.31 (m, 6H); 13C NMR
(75.5 MHz, CDCl3): d¼155.3, 144.6, 137.7, 125.6, 121.0,
115.1, 110.0, 73.9 (d, 3J(C,P)¼7 Hz), 66.4, 65.5 (d,

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slowly. The mixture was allowed to warm up to room
temperature overnight and diluted with ether (200 mL). The
organic layer was washed with water (2£50 mL) and brine
(1£50 mL). Drying over MgSO4 and evaporation of the
solvent gave 23 (7.42 g, 91%) as a yellow liquid. The
product was used without further purification. 1H NMR
(300.1 MHz, CDCl3): d¼5.76–5.67 (m, 1H), 4.96–4.86 (m,
3
2H), 4.07–3.96 (m, 4H), 1.97 (dt, J(H,H)¼7.1 Hz, 2H)
1.69–1.53 (m, 6H), 1.25 (t, 3J(H,H)¼7.1 Hz, 6H); 13C NMR
´
Mohar, B.; Meunier, S.; Valleix, A.; Renard, P. Y.; Creminon,
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4
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7 Hz), 33.1 (d, J(C,P)¼1 Hz), 29.6 (d, J(C,P)¼17 Hz),
25.4 (d, 1J(C,P)¼141 Hz), 21.8 (d, 2J(C,P)¼5 Hz), 16.3 (d,
3J(C,P)¼6 Hz); 31P NMR (81.0 MHz, CDCl3): d¼32.9; IR
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˜
960 cm21; MS (70 eV): m/z (%): 220 (19) [Mþ], 179 (26),
165 (33), 152 (100), 138 (41), 125 (78), 111 (32), 97 (42), 81
(51), 41 (42), 29 (40); elemental analysis calcd (%) for
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54.18, H 9.78, P 13.53.
Acknowledgements
This work was funded by the European Commission under
proposal number BI04-98-0249. We thank all the partners
for their discussions and contributions leading to the
generation of this project.
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