An enzyme-labile safety catch linker for synthesis on a soluble polymeric support

Article abstract of DOI:10.1002/1521-3765(20010302)7:5<959::AID-CHEM959>3.0.CO;2-K

The development of new and broadly applicable linker groups which are stable under a variety of reaction conditions and allow release of the desired products from the solid support under very mild conditions is of great interest in organic synthesis and combinatorial chemistry. We describe an enzyme-labile safety-catch linker which releases alcohols and amines through i) enzymatic cleavage of an amino group and ii)subsequent lactam formation. The linker group was investigated on different polymeric supports: TentaGel, PEGA, CPG-beads and the soluble polymer POE-6000. From these linker-polymer conjugates 2-methoxy-5-nitrobenzyl alcohol was released by penicillin G acylase catalysed cleavage of a phenylacetamide and attack of the liberated benzylamine on the neighbouring ester group in ortho position. The model study revealed that only in the case of soluble POE-6000 conjugate high yields for the cleavage could be achieved. In the case of the other solid supports the enzyme does not have access to the interior of the polymer matrix. The application of the POE-6000 linker conjugate was investigated for various esters in Pd⁰-catalysed Heck-Suzuki- and Sonogashira reactions as well as in a Mitsunobu reaction and cycloadditions. These studies proved that the linker is stable under a broad variety of reaction conditions and that the enzymatic method allows for release of the desired product alcohols under extremely mild conditions at pH 7 and 37°C. In addition, the enzymatic reaction proceeds with complete chemoselectivity, that is other esters or amides are not attacked by the biocatalyst. In addition to alcohols amines can also be cleaved by means of the enzyme-initiated two-step process. In these cases the higher stability of amides as compared to esters requires warming to 60°C to induce cyclization and release of the desired product.

Full text of DOI:10.1002/1521-3765(20010302)7:5<959::AID-CHEM959>3.0.CO;2-K

FULL PAPER
An Enzyme-Labile Safety Catch Linker for Synthesis
on a Soluble Polymeric Support
Uwe Grether[a, b] and Herbert Waldmann*[a]
Abstract: The development of new and
broadly applicable linker groups which
are stable under a variety of reaction
conditions and allow release of the
desired products from the solid support
under very mild conditions is of great
interest in organic synthesis and combi-
natorial chemistry. We describe an en-
zyme-labile safety-catch linker which
releases alcohols and amines through
i) enzymatic cleavage of an amino group
and ii) subsequent lactam formation.
The linker group was investigated on
different polymeric supports: TentaGel,
PEGA, CPG-beads and the soluble
polymer POE-6000. From these link-
er± polymer conjugates 2-methoxy-5-ni-
trobenzyl alcohol was released by peni-
cillin G acylase catalysed cleavage of a
phenylacetamide and attack of the li-
berated benzylamine on the neighbour-
ing ester group in ortho position. The
model study revealed that only in the
case of soluble POE-6000 conjugate
high yields for the cleavage could be
achieved. In the case of the other solid
supports the enzyme does not have
access to the interior of the polymer
matrix. The application of the POE-6000
linker conjugate was investigated for
various esters in Pd0-catalysed Heck-,
Suzuki- and Sonogashira reactions as
well as in a Mitsunobu reaction and
cycloadditions. These studies proved
that the linker is stable under a broad
variety of reaction conditions and that
the enzymatic method allows for release
of the desired product alcohols under
extremely mild conditions at pH 7 and
378C. In addition, the enzymatic reac-
tion proceeds with complete chemose-
lectivity, that is other esters or amides
are not attacked by the biocatalyst. In
addition to alcohols amines can also be
cleaved by means of the enzyme-initi-
ated two-step process. In these cases the
higher stability of amides as compared
to esters requires warming to 608C to
induce cyclization and release of the
desired product.
Keywords: combinatorial chemistry
´
enzyme catalysis ´ penicillin G
acylase ´ safety-catch linker ´ solid-
phase synthesis
the demands on the linker groups concerning their stability
under different reaction conditions and selective cleavage
have risen accordingly. Therefore, new and broadly applicable
linkers which are stable under a variety of reaction conditions,
but which at the same time allow release of the synthesized
products under the mildest conditions possible are of great
interest in organic synthesis and combinatorial chemistry.[2]
Enzymatic methods may open up advantageous alternatives
to classical chemical techniques since enzyme-catalysed trans-
formations often proceed under very mild conditions (pH 5 ±
8, 25 ± 378C) and very high chemo-, regio- and stereoselec-
tivity.[3] We now report on the development of a new enzyme-
labile safety catch linker for combinatorial chemistry[4] on a
soluble polymeric support, allowing for the release of the
target compounds in high yields and under neutral conditions.
Introduction
Combinatorial chemistry and parallel synthesis of compound
libraries on polymeric supports are efficient methods for the
generation of new substances with a predetermined profile of
properties.[1] Paramount to the success of this approach is the
availability of suitable polymers and widely applicable linker
groups for the attachment of the desired compounds to the
polymeric carrier. Recent research in this field has focussed
on and achieved the successful transfer of a steadily growing
number of reaction types to synthesis on solid supports, and
[a] Prof. Dr. H. Waldmann, Dr. U. Grether
Max-Planck-Institute für molekulare Physiologie
Abteilung Chemische Biologie, Otto-Hahn-Strasse 11
44227 Dortmund (Germany)
and
Universität Dortmund, Fachbereich 3, Organische Chemie
44221 Dortmund (Germany)
Fax : (‡49)231-133-2499
Results and Discussion
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[b] Dr. U. Grether
In the design of the new linker group a biocatalysed trans-
formation was combined with a subsequent intramolecular
Institut für Organische Chemie der Universität Karlsruhe
Richard-Willstätter-Allee 2, 76128 Karlsruhe (Germany)
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959

H. Waldmann and U. Grether
FULL PAPER
cyclization reaction according to the principle of ªassisted
removalº[5] of the desired target molecules. To this end the
linker embodies a functional group which is recognized and
attacked by the biocatalysts. The enzyme liberates an
intermediate which cyclizes with release of the target
compounds. Furthermore, the linker carries an additional
functional group for attachment to the polymeric support. The
realization of this principle is shown in Scheme 1. The linker
aromatic compound 6 was sub-
jected to a completely regiose-
lective electrophilic amidoalky-
lation with N-hydroxymethyl-
phenylacetic acid amide.[7]
Under the acidic reaction con-
ditions the THP ether was si-
multaneously converted into
MeO
COOMe
HO
5
a)-c)
O
OMe
MeO
O
H
N
HO
the corresponding acetate
7
O
which was then saponified. By
means of this three-step se-
quence linker 8 is accessible in
an overall yield of 67%.
Paramount to the successful
development of the enzyme-
labile linker system is the
choice of an appropriate solid
support. It has to be compatible
with the conditions required for
8
O
X
target molecule
Scheme 2. Synthesis of the
linker group 8. a) THP-
OCH2CH2Br, K2CO3, DMF,
908C, 10 h, 94% (!6);
MeO
O
H
N
O
N
H
O
O
b)
PhCH2C(O)NHCH2OH,
enzyme labile
group
HOAc/H2SO4 20:1, 208C, 10 h,
83% (!7); c) NaOMe, MeOH,
08C, 1 h, 87 %.
X = O, NH, NR
1
penicillin G
acylase
COOH
_
the enzymatic transformation and ideally must give the
enzyme access to all functionalized sites. From earlier work
on the use of biocatalysts on solid supports[4b, 6d] we were
aware of the fact that, in general, polar polymeric supports are
preferable (see also [4d]) and that the polymer must swell well
under the reaction conditions to allow entry of the biocatalyst
into the polymer matrix. For these reasons TentaGel[8] and the
PEGA[9] resin were chosen. In addition, controlled pore glass
(CPG) beads were investigated because they had already
proven to be compatible with the use of the enzyme penicillin
acylase,[6d] that is allowing for quantitative removal of phenyl-
acetamido protecting groups from oligonucleotides assem-
bled on CPG beads. In order to quantify the efficiency of the
enzymatic transformations an assay was established that is
based on the enzyme-initiated release of 2-methoxy-5-nitro-
benzyl alcohol from the corresponding polymer-bound esters
and its determination by means of UV spectroscopy. This
system was established in solution by employing a soluble
non-polymer-bound ester analogue as substrate.
The 2-methoxy-5-nitrobenzyl group was coupled to the
linker by nucleophilic esterification of carboxylic acid 9 with
benzyl bromide 10 (Scheme 3). Subsequent conversion of the
primary alcohol present in product 11 with chloroformic acid
p-nitrophenyl ester 12 or phosgene yielded activated reagents
13 and 14. Treatment of amino-functionalized PEGA-resin or
CPG beads with carbonate 13 resulted in quantitative
acylation of the solid support as judged by means of the
ninhydrin test (see Experimental Section).
Alternatively, methyl ester 8 was first converted into the
corresponding activated carbonate 18 which was used to
acylate TentaGel quantitatively (Scheme 4). In this case
successful loading of the resin was also confirmed by
means of gel phase 13C NMR spectroscopy. Saponification
of the methyl ester 19 and subsequent nucleophilic esterifi-
cation yielded polymer-bound nitrobenzyl ester 21. By
analogy soluble ester 23 was synthesized (Scheme 5). Safety
catch protecting group 22 required for this purpose was
synthesized by analogy to the synthesis of linker group 8
from homoveratric acid and N-hydroxymethyl phenylacetic
acid.[10]
O
X
target molecule
MeO
O
O
NH2
N
H
O
2
MeO
O
O
O
NH
+
HX target molecule
N
O
H
3
4
Scheme 1. Principle of the enzyme-labile safety catch linker.
group is attached as urethane to an amino-functionalized
carrier (!1). It allows for the attachment of, for example,
alkyl halides, alcohols or amines as carboxylic acid esters and
amides and their subsequent conversion to product libraries.
The enzyme-labile functional group, a phenylacetic acid
amide, is remote from the structures to be generated by
combinatorial synthesis. Thereby possible steric or electronic
interactions of the biocatalyst with the synthesis products
which might lead to a reduction of the substrate tolerance of
the enzyme are minimized. The release of the target
molecules proceeds according to the safety catch principle
in an two-step process. In the first step the amidase penicil-
lin G acylase hydrolyses the phenylacetamide with complete
chemo- and regioselectivity and under exceptionally mild
conditions (pH 7.0, room temperature or 378C).[6] Subse-
quently benzylamine 2 thereby generated as activated inter-
mediate cyclizes to polymer-bound lactam 3 and releases the
corresponding target molecule 4.
The linker was synthesized from commercially available
homovanilinic acid methyl ester 5 (Scheme 2). After alkyla-
tion of the phenolic hydroxyl group with THP-protected
bromoethanol in high yield the resulting trisubstituted
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Safety Catch Linker
959±971
O
OH
O
OMe
MeO
O
MeO
O
H
N
H
N
HO
HO
9
O
O
Br
8
a)
MeO
NO2
NO2
a)-d)
10
R
NO2
O
O
OMe
MeO
O
H
O
O
HO
N
OMe
MeO
O
11
H
H
N
O
N
O
O
O
Cl
O
O
O
NO2
Cl
O
Cl
b)
d)
21
12
O
OR
OR
linker
OH
MeO
O
MeO
O
H
H
N
N
=
TentaGel
O
O
Scheme 4. Synthesis of TentaGel-bound 2-methoxy-5-nitrophenyl ester 21.
a) 12, DMAP, CH2Cl2, 08C, 1 h then 208C, 24 h, 48% (!18); b) TentaGel-
NH2, HOBt, DMF, 208C, 10 h, quant. (! 19); c) 0.5m LiOH/CH3OH 1:1,
208C, 2 h, quant. (! 20); d) 10, Cs2CO3, DMF, rt, 81%.
NO2
O
Cl
O
O
13
O
O
14
c)
e)
O
NH2
NH2
POE 6000
CPG, PEGA
O
OH
O
R
NO2
MeO
MeO
MeO
O
H
N
+
H
N
H
N
Br
O
OMe
O
O
O
15:
16:
= CPG
22
10
a)
= PEGA
= POE 6000
17:
NO2
Scheme 3. Synthesis of polymer-bound linker 2-methoxy-5-nitrophenyl
esters 15 ± 17. a) 10, Cs2CO3, H2O/EtOH then DMF, rt, 82%; b) 12, DMAP,
CH2Cl2, rt, 61%; c) CPG-NH2 or PEGA-NH2, HOBt, EtN(iPr)2, DMF,
quant.; d) phosgene, CH2Cl2, rt, quant.; e) POE-NH2, DMAP, HOBt,
CH2Cl2, rt, 92%. HOBt ˆ 1-hydroxybenzotriazole, DMAP ˆ 4-dimethyl-
aminopyridine.
O
O
OMe
MeO
MeO
H
N
O
23
Scheme 5. Synthesis of model ester 23. a) Cs2CO3, DMF, rt, 68%.
In order to determine whether penicillin G acylase accepts
in principle substrates of type 23 this phenylacetamide was
treated with the enzyme at pH 7 in the presence of 20 vol%
acetone and dimethyl-b-cyclodextrin as solubilizing agents.
After 48 h benzyl alcohol 24 and lactam 25 were obtained in
90% and 86% yield, respectively (Scheme 6). In the absence
of the biocatalyst formation of the alcohol could not be
detected, that is non-enzymatic hydrolysis does not occur
under the reaction conditions employed.
Encouraged by this finding the release of alcohol 24 from
resins 15, 16 and 21 was investigated (Scheme 6). In a series of
experiments the pH value (pH 6 ± 8), the reaction temper-
ature (20 ± 378C), the reaction time and the cosolvent were
varied. The amount of released product 24 was quantified by
detecting its absorption at 307 nm (the enzyme does not
absorb at this wavelength). For all polymeric supports the
highest yields were obtained in the presence of 10 vol% of
methanol at pH 7 and 378C. However, disappointingly only
low yields in the cleavage from the different polymeric
NO2
NO2
O
O
CH3O
O
a)
+
OMe
MeO
NH
H
N
RO
OMe
HO
24
RO
O
25: R = CH3
26: R = POE
23: R = Me
90%[a]
1%[b]
TentaGel
21: R =
15: R =
NHCOOCH2CH2
NHCOOCH2CH2
NHCOOCH2CH2
NHCOOCH2CH2
10%[b]
13%[b]
59%[c]
CPG
16: R = PEGA
POE 6000
17: R =
Scheme 6. Enzyme-initiated cleavage of 2-methoxy-5-nitro benzyl alcohol
(24) from different polymeric supports and in solution; a) penicillin G
acylase, conditions; [a] immobilized penicillin G acylase, 20% acetone,
dimethyl-b-cyclodextrin, 378C; [b] penicillin G acylase suspension, 10%
methanol, 378C; [c] penicillin G acylase suspension, 10% methanol, 208C.
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H. Waldmann and U. Grether
FULL PAPER
supports were obtained (Scheme 6). From TentaGel 21 only
1% of the calculated amount could be cleaved. Obviously, in
this case only the outer surface of the bead is accessible to the
biocatalyst. This is in line with observations made by us earlier
in the development of a fragmenting enzyme-labile linker.[4b]
In this case a small lipase that tolerated high amounts of
cosolvent had to be used to obtain preparatively viable results.
Penicillin acylase, however, has a molecular weight of about
80 kDa and appears to be too large to penetrate efficiently
into the interior of the TentaGel beads. Similar results were
obtained by Lebl et al. and Balasubramanian et al.[11] In the
case of PEGA and CPG beads significantly higher yields were
determined (10 ± 13%), although the results obtained with
these commercially available insoluble carriers were still not
satisfying. A possible solution to this problem might be to use
longer spacers for the functionalization of the CPG beads, or
to employ PEGA6000[12] instead of PEGA-1500. This resin
incorporates longer polyethyleneglycol building blocks and
has larger cavities; however, it is not yet commercially
available.
group of the linker was esterified with m-iodobenzylbromide
30 and the polymer-bound aryl iodide 31 generated was then
further transformed in a Heck[15] reaction to a cinnamic acid
ester, a Suzuki[16] reaction to a biphenyl and a Sonogashira[17]
reaction to give an alkine (Scheme 7). NMR spectroscopic
8
a)-c)
29
linker OH
Br
d)
I
30
linker
O
I
31
O
OMe
OtBu
e)
f)
g)
Since the major obstacle for the enzymatic reaction
appeared to be the very limited access of the biocatalyst to
the surface of the insoluble solid supports we envisaged that
the use of a soluble polymer should serve to overcome this
problem. Therefore, a soluble polyethyleneglycol functional-
ized at both termini with an amino group and with an average
molecular mass of 6000 Da (POE 6000) was investigated.[13]
POE 6000 is soluble in numerous organic solvents but can be
precipitated, filtered off, and washed after addition of diethyl
ether, thereby facilitating the separation of surplus reagents
and the side products. It allows for NMR spectroscopic
monitoring of the reactions[14] and, due to its pronounced
solubility in water, for biocatalysed transformations it offers
the advantage that the substrates are accessible to the
biocatalyst in homogeneous phase.
B(OH)2
OMe
tBuO
O
linker
O
linker
O
linker
34
O
32
33
h)
h)
h)
OMe
tBuO
O
The required functionalized POE 6000 was synthesized by
treatment of chloroformic acid ester 14 with the amino-
modified polymer as shown in Scheme 3. The resulting
polymer± linker conjugate 17 was then subjected to the
enzymatic reaction, and the desired benzyl alcohol 24 was
obtained in 59% yield after 48 h incubation at pH 7 and 378C
(Scheme 6). This result encouraged us to investigate the use of
the enzyme-labile safety catch linker together with POE 6000
as soluble polymeric support for applications in combinatorial
chemistry.
HO
HO
36 (73%)
HO
37 (94%)
35 (75%)
Scheme 7. Pd0-catalysed reactions on polymer 31 and enzyme-catalysed
release of the coupling products from the polymeric supports. a) phosgene,
CH2Cl2, rt, quant. (! 27); b) POE-NH2, DMAP, HOBt, CH2Cl2, rt, 93%
(! 28); c) LiOH ´ H2O/THF 2:1, rt, 99%; d) 30, Cs2CO3, DMF, 508C, 24 h,
95%; e) [Pd(OAc)2], Bu4NBr, PPh3 DMF/Et3N/H2O 9:1:1, 508C, 20 h,
91%; f) [Pd(PPh3)4], K3PO4, DMF, 808C, 20 h, 93%; g) [Pd(PPh3)2Cl2],
CuI, dioxane/Et3N 2:1, 208C, 24 h, 97%; h) penicillin G acylase, pH 7.0,
10% MeOH, 378C.
The applicability of the polymer± linker conjugate was
investigated for a variety of transformations. First, a simplified
and straightforward procedure for the synthesis of the
conjugate was established. To this end, linker building block
8 was treated with phosgene to give the chloroformic acid
ester 27 in quantitative yield. For the subsequent coupling to
the polymer the solvent simply had to be removed, and the
polymer was then acylated in quantitative yield. Finally, the
methyl ester incorporated into the linker was saponified to
give the desired polymer± linker conjugate 29 (Scheme 7).
In a first series of experiments the suitability of the
polymer± linker conjugate in Pd0-catalysed transformation
that are widely used in organic synthesis and combinatorial
chemistry was investigated. To this end, first the carboxyl
analysis revealed that these transformations proceeded quan-
titatively. Compounds 32 ± 34 were then incubated with
penicillin G acylase at pH 7 and 378C. After 48 h, benzyl
alcohols 35 ± 37 were isolated in high yields and with a purity
of >95% by simple extraction with diethyl ether.
For polymer-bound alkine 34 it was demonstrated that
incubation with the enzyme for 24 h instead of 48 h already
delivers the desired product in preparatively useful yield of
71%, that is if necessary, the time for the enzymatic reaction
can be shortened considerably. In all three cases control
experiments without enzyme were carried out which revealed
that in the absence of the biocatalyst the linker group is
completely stable. To verify that the linker group is cleaved by
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Safety Catch Linker
959±971
enzyme-initiated lactam formation, polymer-bound lactam 26
(Scheme 6) was isolated in 82% yield after the cleavage of
alcohol 35 and characterized spectroscopically (see the
Experimental Section).
In another series of experiments Mitsunobu esterification
and Diels ± Alder reactions were investigated (Scheme 8). For
the Mitsunobu reaction, the polymer was esterified quantita-
tively with 1,4-bis(hydroxymethyl)benzene. The obtained
In a second type of cycloaddition polymer-bound acrylic
acid ester 41 was subjected to a 1,3-dipolar cycloaddition with
phenylnitrile oxide generated in situ from benzaldehyde
oxime by treatment with sodium hypochlorite. Cyclo-
adduct 44 was formed quantitatively,[19] which demonstrates
that the linker group is stable under oxidative conditions
(Scheme 9). After incubation of 44 with penicillin G acylase
O
linker
O
O
linker
OH
d)
41
29
HO
O
OH
HO
O
a)
a)
HO
N
O
linker
O
linker
O
N
O
OH
O
O
linker
O
38
41
O
44
H
N
Scheme 9. 1,3-Dipolar cycloaddition to give the isoxazoline 44. a) 0.7m
NaOCl, THF, 208C, 4 h, 94 %.
e)
b)
O
HO
H
under the conditions detailed above POE-bound lactam 26
was formed quantitatively, however, only traces of the desired
isoxazoline were isolated. Synthesis of reference compound in
organic solution and subsequent exposure to aqueous meth-
anol showed that the unsaturated heterocycle is not stable
under these conditions. Thus, while the enzyme-labile linker
group is stable to the conditions required for formation and
transformation of nitrile oxides, the cycloadducts have to be
converted into more stable compounds before enzyme-
initiated release from the solid support is attempted.
N
O
linker
O
linker
O
O
O
O
42
39
c)
c)
H
N
O
HO
HO
O
O
O
43 (75%)
endo:exo 2.5:1
40 (81%)
In a third series of experiments we investigated whether the
enzymatic method also gives access to target molecules that
are coupled to the linker as amides. For orientation studies
4-pentylaniline was coupled to the POE-derived carboxylic
acid 29 and the resulting polymer 45 was incubated with
penicillin G acylase at pH 7 and room temperature. As ex-
pected the phenylacetamide group of the linker was hydrolyzed
rapidly. However, since amides are considerable more stable
than esters the desired cyclization did not occur at room
temperature. Upon warming to 608C lactam formation pro-
ceeded well and 4-pentylaniline 46 was isolated in 68% yield
(Scheme 10). Similar results were recorded for an aniline
carrying an ester group in the 4-position (see Scheme 10).
With these results in hands 4-iodoaniline 49 was coupled
quantitatively to linker 29 and the polymer fixed aryliodide 50
obtained thereby was subjected to a Suzuki reaction[16] with
4-methoxyphenylboronic acid leading to biphenyl derivative
51 or a Stille reaction[20] to give enol ether 53 (Scheme 11).
Upon treatment with 0.5m hydrochloric acid, enol ether 53
yielded acetophenone 54, thereby proving the acid stability of
the linker. In order to release the coupling products, polymers
51 and 54 were incubated with penicillin G acylase at pH 7.0
and room temperature. As expected under these conditions
the phenylacetamide was hydrolyzed. Upon warming of the
reaction mixture to 608C, the expected lactam was formed
and amines 52 and 55 were released from the polymer.
Scheme 8. Mitsunobu- and Diels ± Alder reaction on polymeric support
and penicillin G acylase mediated cleavage of the target molecules. a) DIC,
DMAP, HOBt, DMF, pyridine, 08C, 1 h, then 208C, 24 h, 92%; b) DEAD,
PPh3, THF/CH2Cl2 1:1, 58C, 2 h, then 208C, 20 h, 86%; c) penicillin G
acylase, pH 7.0, 10% MeOH, 378C, 48 h; d) DIC, DMAP, HOBt, DMF,
pyridine, 08C, 1 h, then 208C, 24 h, 97%; e) THF, 608C, 15 h, 93%.
polymer-bound benzyl alcohol 38 was then treated with
4-acetamidophenol in the presence of the Mitsunobu reagent
to give phenyl ether 39 in quantitative yield. By subsequent
treatment of polymeric substrate 39 with penicillin G acylase,
aryl benzyl ether 40 could be obtained in 81% yield. For the
Diels ± Alder reaction, 4-hydroxybutyl acrylate was coupled
to the linker and the polymer-bound acrylic acid ester 41 was
treated with cyclopentadiene. This reaction was carried out at
608C at ambient pressure, and at 1008C in a closed glass vial
to investigate the pressure and temperature stability of the
polymer-bound linker. According to NMR spectroscopic
analysis cycloaddition product 42 was formed with an endo/
exo ratio of 2.5:1[18] and with quantitative conversion.
Subsequent enzymatic release delivered alcohol 43 in high
yield and purity. The examples shown in Scheme 8 prove quite
impressively the chemoselectivity of penicillin G acylase since
the enzyme attacks only the phenylacetamide unit and not the
ester and amide groups which are also present.
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H. Waldmann and U. Grether
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the products proceeds under particularly mild conditions with
pronounced selectivity and delivers the desired products in
high yield and purity. The linker is stable under different
conditions, even at elevated temperature. Beyond the devol-
opment of a new linker system for combinatorial chemistry
our results prove that enzymes, in general, are valuable
reagents for transformations on soluble polymeric supports.
H
N
O
R
MeO
O
H
H
N
N
O
O
O
45: R = C5H11
47: R = COOEt
a)
Experimental Section
MeO
O
O
NH
General: Melting points were determined in open capillaries using a Büchi
535 apparatus and are uncorrected. 1H and 13C NMR spectra were recorded
on Bruker AC 250, AM 400 or DRX 500 spectrometers at room temper-
ature. The 1H and 13C NMR signals of the POE 6000 linker conjugate 29
always appeared around the same chemical shift (Æ0.2 ppm). Therefore,
these signals were not mentioned in further transformations of the
POE 6000 linker conjugate 29. IR spectra were recorded on a Bruker
IFS 88 spectrometer, UV/Vis spectra on a Perkin ± Elmer Lambda 2 UV/
Vis spectrometer. Mass spectra and high-resolution mass spectra (HRMS)
O
O
N
H
26
+
H2N
R
46: R = C5H11 (68%)
48: R = COOEt (60%)
were measured on
analyses were performed on a Heraeus CHN-Rapid apparatus.
a Finnigan MAT MS70 spectrometer. Elemental
Scheme 10. Enzyme-catalysed release of model anilines from POE 6000.
a) Penicillin G acylase, pH 7.0, 10% MeOH, 208C, 48 h, then 608C, 4 h.
Materials: Solvents were dried by standard methods and stored over
molecular sieves. For column chromatography silica gel (40 ± 60 mm, Baker)
was used. Size-exclusion chromatography was done on Sephadex LH 20
(Pharmacia LKB Biotechnology). Commercially available reagents were
used without further purification. All reactions except for those carried out
with water were performed under argon. Penicillin G acylase (EC 3.5.1.11)
was used as a suspension in phosphate buffer (Fluka) and immobilized on
Eupergit C (Boehringer Mannheim). Amino-functionalized TentaGel and
polyoxyethylene POE 6000 were obtained from Rapp Polymere, Tübingen,
Germany, PEGA (crosslinked 2-acrylamidoprop-1-yl[2-aminopropy-1-yl]-
polyethylene glycol) was acquired from Novabiochem and CPG with an
average pore diameter of 500 Š from Fluka. Bisamino-functionalized
POE 6000 has an average molecular weight of 6000 gmol 1. Average
molecular masses from compounds bound to this support are calculated on
the basis of the average molecular weight of POE 6000.
+
H2N
I
OH
linker
49
29
a)
H
N
I
linker
50
EtO
(HO)2B
linker
OMe
Bu3Sn
b)
d)
Methyl
{3-methoxy-4-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]phenyl}-
acetate (6): THP-protected bromoethanol[21] 14.8 g, 71 mol) was added to
a suspension of homovanillic acid methyl ester (5, 10.8 g, 55 mol) and
potassium carbonate (9.8 g, 71 mol) in DMF (120 mL). After stirring for
16 h at 908C the solvent was evaporated in vacuo. The residue was
redissolved in ethyl acetate (150 mL) and washed with water (2 Â 80 mL).
The organic layer was dried over MgSO4, the solvent was evaporated in
vacuo and the residue was purified by chromatography on silica gel (ethyl
acetate/hexane 1:10 to 1:1 v/v) to yield a colourless oil (16.6 g, 51 mol,
94%). Rf ˆ 0.67 (ethyl acetate/hexane 1:1 v/v); 1H NMR (CDCl3,
400 MHz): d ˆ 6.89 (d, 3J(H,H) ˆ 8.1 Hz, 1H; arom. CH-5), 6.82 ± 6.78
(m, 2H; arom. CH-2/6), 4.70 (t, 3J(H,H) ˆ 3.6 Hz, 1H; CH), 4.20 (t,
3J(H,H) ˆ 4.9 Hz, 2H; COCH2), 4.07 ± 4.03 (m, 1H; CHOCH2ax), 3.90 ±
3.82 (m, 2H; COCH2CH2), 3.85 (s, 3H; OCH3), 3.69 (s, 3H; COOCH3),
OEt
N
OMe
N
H
linker
H
51
53
c)
e)
O
H2N
OMe
N
linker
H
54
52 (60%)
c)
3.55 (s, 2H; CH2C(O)O), 3.53 ± 3.49 (m, 1H; CHOCH2eq), 1.87 ± 1.50 (m,
O
13
ˆ
6H; CH2CH2CH2); C NMR (CDCl3, 100.6 MHz): d ˆ 172.22 (C O),
H2N
149.64 (COCH3), 147.61 (COCH2), 127.03 (CCH2), 121.45 (arom. CH-6),
114.09 (arom. CH-5), 113.11 (arom. CH-2), 98.95 (CH), 68.64 (COCH2),
65.74 (COCH2CH2), 62.09 (CHOCH2), 55.97 (OCH3), 52.01 (COOCH3),
40.73 (CCH2), 30.49 (CHCH2), 25.44 (CHCH2CH2), 19.34
55 (67%)
Scheme 11. Pd0-catalysed synthesis and enzyme-initiated cleavage of the
anilines 52 and 55 from POE 6000. a) EDC, HOBt, DMF, ca. 208C, 12 h,
97%; b) [Pd(PPh3)4], K3PO4, DMF/H2O 10:1, 808C, 20 h, 95%; c) penicil-
lin G acylase, pH 7.0, 10% MeOH, 208C, 48 h, then 608C, 4 h; d) [Pd2dba3],
AsPh3, dioxane 608C, 24 h, 81%; e) 0.5m HCl/THF 1:1, 208C, 4 h, 92%.
ˆ
(CHCH2CH2CH2); IR (KBr, drift): nÄ ˆ 1739 (C O, ester), 1143 (C O),
‡
1036 cm 1 (C O); MS (EI, 70 eV, 508C): m/z (%): 324 (65) [M] , 196 (41),
129 (100); HRMS (70 eV): calcd for C17H24O6: 324.1573, found: 324.1584.]
Methyl (4-[2-(acetyloxy)ethoxy]-5-methoxy-2-{[(phenylacetyl)amino]me-
thyl}phenyl)acetate (7): A precooled solution (08C) of acetic acid (100 mL)
and concentrated sulfuric acid (5 mL) was added to a mixture of N-
hydroxymethyl-2-phenylacetamide[22] (8.1 g, 49 mol) and homovanillic acid
derivative 6 (16.6 g, 51 mol). The solution was stirred for 2 h at 08C and for
12 h at room temperature. Then the reaction mixture was poured into ice
water (200 mL), neutralized with 1m aqueous NaOH at 08C and extracted
with ethyl acetate (5 Â 250 mL). The combined organic layers were dried
over MgSO4 and after evaporation of the solvent in vacuo the product was
Conclusion
We have developed a new enzyme-labile safety catch linker
that allows for the synthesis of different classes of compounds
and the application of various reaction types. The release of
964
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0964 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 5

Safety Catch Linker
959±971
purified by chromatography on silica gel (ethyl acetate/hexane 2:1 v/v) to
yield a white solid (17.5 g, 41 mol, 83%). Rf ˆ 0.51 (ethyl acetate‡1%
acetic acid); m.p. 118 ± 1198C; 1H NMR (CDCl3, 400 MHz): d ˆ 7.34 ± 7.24
(m, 5H; arom. CH), 6.81 (s, 1H; arom CH-6), 6.69 (s, 1H; arom. CH-3),
6.24 (brs, 1H; NH), 4.40 (t, 3J(H,H) ˆ 4.8 Hz, 2H; COCH2), 4.34 (d,
3J(H,H) ˆ 5.6 Hz, 2H; CH2NH), 4.14 (t, 3J(H,H) ˆ 4.8 Hz, 2H;
COCH2CH2), 3.83 (s, 3H; OCH3), 3.64 (s, 3H; COOCH3), 3.59 (s, 2H;
PhCH2), 3.55 (s, 2H; CH2C(O)O), 2.09 (s, 3H; CH3C(O)); 13C NMR
ˆ
ˆ
ˆ
3594 (O H), 1737 (C O, ester), 1645 (C O, amide I), 1522 (C O, amide II;
N O), 1344 (N O), 1028 cm 1 (C O); MS (EI, 70 eV, 2058C): m/z (%): 538
ˆ
ˆ
‡
(0.02) [M] , 355 (51), 237 (79), 183 (100); HRMS (70 eV): calcd for
C28H30N2O9: 538.1951, found: 538.1933; elemental analysis calcd (%) for:
C 62.45, H 5.61, N 5.20; found: C 62.36, H 5.69, N 4.98.
2-Methoxy-5-nitrobenzyl (5-methoxy-4-(2-{[(4-nitrophenoxy)carbonyl]-
oxy}ethoxy)-2-{[(phenylacetyl)amino]methyl}phenyl)acetate (13): Pre-
cooled CH2Cl2 (08C, 20 mL) was added to a mixture of acetate 11
(518 mg, 0.96 mol), DMAP (123 mg, 1 mol), chloroformic acid-4-nitro-
phenyl ester 12 (203 mg, 1 mol) and 4 Š molecular sieves. The reaction
mixture was stirred for 2 h at 08C and at room temperature overnight. Then
the molecular sieves were filtered off, the solvent was evaporated in vacuo
and the residue was purified by chromatography on silica gel (ethyl acetate/
hexane 1:1 to 2:1 v/v) to yield a colourless oil (414 mg, 0.59 mol, 61%). Rf ˆ
ˆ
ˆ
ˆ
(CDCl3, 100.6 MHz): d ˆ 172.55 (C O), 170.97 (C O), 170.58 (C O),
149.03 (COCH2), 147.27 (COCH3), 134.99 (CCH2C(O)NH), 129.31 (2C;
arom. CH), 128.83 (2C; arom. CH), 127.13 (2C; arom. CH, CCH2C(O)O),
125.65 (CCH2NH), 115.61 (arom. CH-3), 114.25 (arom. CH-6), 67.15
(COCH2), 62.69 (COCH2CH2), 56.03 (OCH3), 52.28 (COOCH3), 43.81
(PhCH2), 41.14 (CH2C(O)O), 37.89 (CH2NH), 20.91 (CH3C(O)); MS (EI,
‡
70 eV, 1458C): m/z (%): 429 (15) [M] , 208 (10), 87 (100); HRMS (70 eV):
1
0.50 (ethyl acetate/hexane 2:1 v/v); H NMR (CDCl3, 250 MHz): d ˆ 8.28
calcd for C23H27NO7: 429.1787, found: 429.1775; elemental analysis calcd
(%) for: C 64.31, H 6.35, N 3.26; found: C 64.34, H 6.49, N 3.24.
(d, 3J(H,H) ˆ 9.0 Hz, 2H; arom. CH), 8.23 (dd, 3J(H,H) ˆ 8.9 Hz,
4J(H,H) ˆ 2.8 Hz, 1H; arom. CH), 8.08 (d, 4J(H,H) ˆ 2.8 Hz, 1H; arom.
Methyl
(4-(2-hydroxyethoxy)-5-methoxy-2-{[(phenylacetyl)amino]me-
3
CH), 7.51 (d, J(H,H) ˆ 9.0 Hz, 2H; arom. CH), 7.32 ± 7.21 (m, 5H; arom.
thyl}phenyl)acetate (8): A 30% sodium methanolate solution in methanol
(10.7 mL, 58.5 mol) was added at 08C dropwise to a solution of acetate 7
(2.5 g, 5.9 mol) in methanol (110 mL). After stirring for 1 h at 08C, the pH
of the solution was adjusted to pH 6 with hydrochloric acid. Methanol was
removed under reduced pressure and the remaining solution was extracted
with ethyl acetate (5 Â 100 mL). The combined organic layers were dried
over MgSO4. Evaporation of the solvent in vacuo yielded a white solid,
which was used without further purification (1.97 g, 5.1 mol, 87%). Rf ˆ
0.21 (ethyl acetate‡0.5% acetic acid); m.p. 1078C; 1H NMR (CD3OD,
400 MHz): d ˆ 7.29 ± 7.19 (m, 5H; arom. CH), 6.81 (s, 1H; arom. CH-6),
6.76 (s, 1H; arom. CH-3), 4.29 (s, 2H; CH2NH), 3.85 ± 3.79 (m, 4H;
CH2CH2OH), 3.78 (s, 3H; OCH3), 3.64 (s, 2H; PhCH2), 3.63 (s, 3H;
COOCH3), 3.49 (s, 2H; CH2C(O)O); 13C NMR (CD3OD, 100.6 MHz): d ˆ
CH), 6.95 (d, 3J(H,H) ˆ 8.9 Hz, 1H; arom. CH), 6.87 (s, 1H; arom. CH-6),
6.76 (s, 1H; arom. CH-3), 6.22 (brs, 1H; NH), 5.14 (s, 2H; CCH2O), 4.63 (t,
3J(H,H) ˆ 4.7 Hz, 2H; COCH2), 4.40 (d, 3J(H,H) ˆ 5.6 Hz, 2H; CH2NH),
4.24 (t, 3J(H,H) ˆ 4.7 Hz, 2H; COCH2CH2), 3.92 (s, 3H; OCH3), 3.83 (s,
3H; OCH3), 3.72 (s, 2H; PhCH2), 3.54 (s, 2H; CH2C(O)O).
General procedure for the synthesis of polymer-bound 2-methoxy-5-
nitrobenzyl esters: Amino-functionalized polymer (47 mmol) was washed
with DMF (5 Â 8 mL) to remove water. Then acetate (13, 100 mg,
0.14 mol), HOBt (38 mg, 0.28 mol) and 4 Š molecular sieves were added
and the mixture was suspended in ice-cold DMF (10 mL). After 30 min
N,N-diisopropylethylamine (12 mL, 70 mmol) was added and the reaction
mixture was shaken 1 h at 08C and 48 h at room temperature afterwards.
The polymer was filtered off and washed with DMF, methanol, ethyl
acetate and CH2Cl2 (3 Â 8 mL each).
General method to determine the loading of the polymer with 2-methoxy-
5-nitrobenzyl alcohol (24): A defined amount of polymer-bound 2-me-
thoxy-5-nitrobenzyl ester was suspended in 0.5% NaOH/THF 1:1 (5 mL)
and shaken for 24 h at room temperature. The polymer was filtered off and
washed with water and THF (3 Â each). The filtrate was adjusted to pH 7
with diluted hydrochloric acid and the solvent evaporated in vacuo. The
residue was redissolved in methanol (10 mL) and the cleaved amount of
2-methoxy-5-nitrobenzyl alcohol (24) was determined through UV spec-
troscopy with a calibration curve (detection at 307 nm).
ˆ
ˆ
173.93 (C O), 173.60 (C O), 149.68 (COCH2), 148.75 (COCH3), 136.96
(CCH2C(O)NH), 130.77 (CCH2C(O)O), 130.07 (2C; arom. CH), 129.59
(2C; arom. CH), 127.90 (arom. CH), 126.57 (CCH2NH), 115.75 (arom. CH-
3), 115.15 (arom. CH-6), 71.64 (COCH2), 61.59 (CH2OH), 56.56 (OCH3),
52.54 (COOCH3), 43.93 (PhCH2), 41.57 (CH2C(O)O), 38.29 (CH2NH); IR
ˆ
ˆ
(KBr, drift): nÄ ˆ 3524 (O H), 3291 (N H), 1731 (C O, ester), 1640 (C O,
amide I), 1522 (C O, amide II), 1101 cm 1 (C O); MS (EI, 70 eV, 1608C):
ˆ
‡
m/z (%): 387 (0.2) [M] , 237 (22), 163 (100); HRMS (70 eV): calcd for
C21H25NO6: 387.1682, found: 387.1700.
Synthesis of CPG- and PEGA-bound 2-methoxy-5-nitrobenzyl esters 15
and 16
General procedure to determine the loading of the polymer by qualitative
ninhydrin test:[23] The polymer (20 mg) was suspended in 1% aqueous
ninhydrin solution and heated for 5 min to 1008C. If the colour of the
polymer turns blue, the loading was not quantitative. If the polymer keeps
its colour the reaction was quantitative.
2-Methoxy-5-nitrobenzyl
(4-(2-hydroxyethoxy)-5-methoxy-2-{[(phenyl-
acetyl)amino]methyl}phenyl)acetate (11): A solution of cesium carbonate
(83 mg, 0.26 mol) in water (10 mL) was added to a solution of acid 9
(191 mg, 0.51 mol) in ethanol (30 mL) and water (5 mL). After evaporation
of the solvent under reduced pressure the residue was dried in vacuo. Then
2-methoxy-5-nitrobenzyl bromide (10, 138 mg, 0.56 mol) was added and
the mixture was dissolved in precooled DMF (08C, 25 mL). The solution
was stirred 1 h at 08C and for another 12 h at room temperature. After
evaporation of the solvent the residue was dissolved in ethyl acetate
(30 mL) and the solution was extracted with water (20 mL). The organic
layer was dried over MgSO4 and the solvent was removed in vacuo.
Chromatography on silica gel (ethyl acetate/hexane 1:10 to ethyl acetate
v/v) yielded a white solid (226 mg, 0.42 mol, 82%). Rf ˆ 0.27 (ethyl
acetate‡0.5% acetic acid); m.p. 108 ± 1098C; 1H NMR (CDCl3,
400 MHz): d ˆ 8.19 (dd, 3J(H,H) ˆ 9.0 Hz, 4J(H,H) ˆ 2.7 Hz, 1H; arom.
CPG-bound 2-methoxy-5-nitrobenzyl ester 15: According to the general
procedure for the synthesis of polymer-bound 2-methoxy-5-nitrobenzyl
esters amino-functionalized CPG (677 mg, 47 mmol, loading 70 mmol per g),
acetate 13 (100 mg, 0.14 mol), HOBt (38 mg, 0.28 0.28 mol) and N,N-
diisopropylethylamine (12 mL, 70 mmol) were stirred in DMF (10 mL).
After washing the polymer was dried in vacuo. Ninhydrin test: quantitative
reaction.
PEGA-bound 2-methoxy-5-nitrobenzyl ester 16: According to the general
procedure for the synthesis of polymer-bound 2-methoxy-5-nitrobenzyl
esters amino-functionalized PEGA (878 mg, 47 mmol, loading 0.4 mol per
g), acetate 13 (100 mg, 0.14 mol), HOBt (38 mg, 0.28 mol) and N,N-
diisopropylethylamine (12 mL, 70 mmol) were stirred in DMF (10 mL).
After washing the polymer was dried in vacuo. Ninhydrintest: quantitative
4
CH), 8.08 (d, J(H,H) ˆ 2.5 Hz, 1H; arom. CH), 7.30 ± 7.20 (m, 5H; arom.
CH), 6.93 (d, 3J(H,H) ˆ 9.1 Hz, 1H; arom. CH), 6.81 (s, 1H; arom. CH-6),
6.72 (s, 1H; arom. CH-3), 6.43 (t, 3J(H,H) ˆ 4.9 Hz, 1H; NH), 5.13 (s, 2H;
CH2OC(O)), 4.36 (d, 3J(H,H) ˆ 5.6 Hz, 2H; CH2NH), 4.00 (t, 3J(H,H) ˆ
ˆ
reaction; IR (PEGA, KBr, drift): nÄ ˆ 1719 (C O, ester, urethane), 1645
1
ˆ
ˆ
3
(C O, amide I), 1521 cm (C O, amide II).
4.5 Hz, 2H; COCH2), 3.91 (s, 3H; OCH3), 3.88 (t, J(H,H) ˆ 4.5 Hz, 2H;
CH2OH), 3.78 (s, 3H; OCH3), 3.70 (s, 2H; PhCH2), 3.52 (s, 2H;
CH2C(O)O), 3.20 (brs, 1H; OH); 13C NMR (CDCl3, 100.6 MHz): d ˆ
Methyl
(5-methoxy-4-(2-{[(4-nitrophenoxy)carbonyl]oxy}ethoxy)-2-
{[(phenylacetyl)amino]methyl}phenyl)acetate (18): Precooled CH2Cl2
(08C, 15 mL) was added to a mixture of alcohol 8 (400 mg, 1.0 mol),
DMAP (126 mg, 1.0 mol), chloroformic acid-4-nitrophenyl ester (12,
208 mg, 1.0 mol) and 4 Š molecular sieves. The reaction mixture was
stirred for 2 h at 08C and then at room temperature overnight. The
molecular sieves were filtered off, the solvent was evaporated in vacuo and
the residue was purified by chromatography on silica gel (ethyl acetate/
hexane 1:1 v/v, Rf ˆ 0.14) to yield a colourless oil (276 mg, 0.5 mol, 48%).
ˆ
ˆ
171.62 (C O), 170.78 (C O), 161.80 (CNO2), 148.95 (COCH2), 147.51
(COCH3), 141.14 (COCH3), 135.03 (CCH2C(O)NH), 129.39 (CCH2O),
129.26 (2C; arom. CH), 128.77 (2C; arom. CH), 127.07 (arom. CH), 125.71
(arom. CH), 125.27 (CCH2C(O)O), 125.17 (CCH2NH), 124.10 (arom. CH),
115.59 (arom. CH), 113.92 (arom. CH-3), 110.09 (arom. CH-6), 70.98
(COCH2), 61.17 (CCH2O), 61.05 (CH2OH), 56.28 (OCH3), 55.90 (OCH3),
43.67 (CH2C(O)O), 41.02 (PhCH2), 38.04 (CH2NH); IR (KBr, drift): nÄ ˆ
Chem. Eur. J. 2001, 7, No. 5
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0965 $ 17.50+.50/0
965

H. Waldmann and U. Grether
FULL PAPER
1H NMR (CDCl3, 400 MHz): d ˆ 8.28 (d, 3J(H,H) ˆ 9.2 Hz, 2H; arom.
(COCH3), 148.50 (COCH3), 148.42 (COCH3), 141.23 (CNO2), 134.98
(CCH2C(O)NH), 129.32 (2C; arom. CH), 129.19 (CCH2O), 128.84 (2C;
arom. CH), 127.14 (2C; arom. CH, CCH2NH), 125.75 (arom. CH), 125.29
(CCH2C(O)O), 124.25 (arom. CH), 113.46 (arom. CH), 112.82 (arom. CH-
3), 110.04 (arom. CH-6), 61.24 (OCH2), 56.25 (OCH3), 55.96 (OCH3), 55.93
(OCH3), 43.85 (CH2C(O)O), 41.25 (PhCH2), 38.10 (CH2NH); IR (KBr,
3
CH), 7.41 (d, J(H,H) ˆ 9.2 Hz, 2H; arom. CH), 7.34 ± 7.25 (m, 5H; arom.
CH), 6.85 (s, 1H; arom. CH-6), 6.71 (s, 1H; arom. CH-3), 6.27 (brs, 1H;
NH), 4.62 (t, 3J(H,H) ˆ 4.5 Hz, 2H; COCH2), 4.35 (d, 3J(H,H) ˆ 5.6 Hz,
2H; CH2NH), 4.24 (t, 3J(H,H) ˆ 4.7 Hz, 2H; COCH2CH2), 3.85 (s, 3H;
OCH3), 3.65 (s, 3H; COOCH3), 3.60 (s, 2H; PhCH2), 3.55 (s, 2H;
13
ˆ
CH2C(O)O); C NMR (CDCl3, 100.6 MHz): d ˆ 172.57 (C O), 170.73
ˆ
ˆ
ˆ
drift): nÄ ˆ 1721 (C O, ester), 1645 (C O, amide I), 1522 (C O, amide II;
ˆ
ˆ
1
(C O), 155.47 (COC(O)), 152.43 (C O carbonate), 149.09 (COCH2),
147.03 (COCH3), 145.42 (CNO2), 134.92 (CCH2C(O)NH), 129.43
(CCH2C(O)O), 129.33 (2C; arom. CH), 128.85 (2C; arom. CH), 127.16
(arom. CH), 126.00 (CCH2NH), 125.31 (2C; arom. CH), 121.80 (2C; arom.
CH), 115.87 (arom. CH-6), 114.33 (arom. CH-3), 67.25 (COCH2), 66.74
(COCH2CH2), 56.05 (OCH3), 52.34 (COOCH3), 43.82 (PhCH2), 41.10
(CH2C(O)O), 37.95 (CH2NH); IR (KBr, drift): nÄ ˆ 3291 (N H), 1764
ˆ
ˆ
N O), 1346 (N O), 1100 cm (C O); UV/Vis (MeOH): lmax (e) ˆ 193
(68400), 204 (64300), 287 (10000), 306 nm (9600 mol 1 dm3 cm 1); MS (EI,
70 eV, 1758C): m/z (%): 508 (6) [M] , 399 (36), 224 (31), 207 (100); HRMS
‡
(70 eV): calcd for C27H28N2O8: 508.1846, found: 508.1828; elemental analysis
calcd (%) for:
N 5.79.
C 63.76, H 5.55, N 5.51; found: C 63.77, H 5.35,
Enzyme-initiated release of 2-methoxy-5-nitrobenzyl alcohol (24) in the
solution phase: 2-Methoxy-5-nitrobenzyl ester (23, 50 mg, 0.1 mol) and 2,6-
di-O-methyl-b-cyclodextrin (1.96 g, 1.47 mol) were dissolved in acetone
(20 mL). Then sodium phosphate buffer (0.05m, pH 7.0, 80 mL) was added
under ultrasonication and the suspension was incubated with immobilized
penicillin G acylase (100 U) at 378C. After 12 h and 24 h the same amount
of penicillin G acylase was added again and after 48 h the biocatalyst was
filtered off and washed with buffer solution (pH 7.0, 5 Â 5 mL) and acetone
(5 Â 10 mL). Then the solvent was evaporated in vacuo and benzyltriethyl-
ammonium chloride (448 mg, 1.97 mol) was added. The mixture was
redissolved in water (70 mL) and extracted with ethyl acetate (5 Â 50 mL).
The combined organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. The residue was purified by chromatog-
raphy on silica gel (ethyl acetate/hexane 1:4 to ethyl acetate v/v). 24: White
solid (16 mg, 90 mmol, 90%); Rf ˆ 0.45 (ethyl acetate/hexane 1:2 v/v); m.p.
1238C, ref.[24]:123 ± 1258C; 1H NMR (CDCl3, 400 MHz): d ˆ 8.26 (d,
4J(H,H) ˆ 2.8 Hz, 1H; arom. CH-6), 8.19 (dd, 3J(H,H) ˆ 9.0 Hz,
ˆ
ˆ
ˆ
ˆ
(C O, carbonate), 1733 (C O, ester), 1641 (C O, amide I), 1521 (C O,
amide II; N O), 1348 cm (N O); MS (EI, 70 eV, 1908C): m/z (%): 552
(0.4) [M] , 210 (40), 139 (63), 91 (100); HRMS (70 eV): calcd for
1
ˆ
ˆ
‡
C28H28N2O10: 552.1744; found: 552.1758.
TentaGel-bound methyl ester 19: Amino-functionalized TentaGel (500 mg,
0.15 mol, loading 0.29 mmol per g) was washed with CH2Cl2 (3 Â 30 mL) to
remove water. Then the polymer was swollen in CH2Cl2 (20 mL). After
20 min HOBt (118 mg, 0.15 mol), N,N-diisopropylethylamine (49 mL,
0.29 mol) and carbonate 18 (160 mg, 0.29 mol) were added and the
suspension was shaken for 24 h at room temperature. The polymer was
filtered off and washed with CH2Cl2, ethyl acetate, methanol, DMF,
methanol, ethyl acetate and CH2Cl2 (3 Â 15 mL each) and dried in vacuo.
Ninhydrin test: quantitative reaction. Gel-phase 13C NMR (CDCl3,
ˆ
ˆ
ˆ
125.7 MHz): d ˆ 172.49 (C O), 170.79 (C O), 156.46 (C O urethane),
148.85 (COCH2), 147.40 (COCH3), 135.31 (CCH2C(O)NH), 129.54
(CCH2C(O)O), 129.37 (2C; arom. CH), 128.80 (2C; arom. CH), 127.08
(arom. CH), 125.43 (CCH2NH), 115.55 (arom. CH-3), 114.21 (arom. CH-6),
70.6 ± 70.0 (CH2 polymer), 67.81 (COCH2CH2), 63.32 (COCH2), 56.07
(OCH3), 52.27 (COOCH3), 43.71 (PhCH2), 40.99 (CH2C(O)O), 40.87
3
4J(H,H) ˆ 2.8 Hz, 1H; arom. CH-4), 6.93 (d, J(H,H) ˆ 9.0 Hz, 1H; arom.
CH-3), 4.73 (d, 3J(OH,CH2) ˆ 4.1 Hz, 2H; CH2), 3.97 (s, 3H; OCH3), 2.31
(brs, 1H; OH); UV/Vis (MeOH): lmax (e) ˆ 193 (16100), 228 (9900),
307 nm (10200 mol 1 dm3 cm 1); HRMS (70 eV): calcd for C8H9NO4:
183.0531, found: 183.0518; the spectroscopic data are in agreement with
the literature.[24]
ˆ
(NHCH2 polymer), 37.90 (CH2NH); IR (KBr, drift): nÄ ˆ 1719 (C O, ester,
1
ˆ
ˆ
urethane), 1669 (C O, amide I), 1521 cm (C O, amide II).
TentaGel-bound carboxylic acid 20: Polymer-bound methyl ester 19
(100 mg, ca. 30 mmol) was suspended in 0.25m LiOH/ethanol 1:1 (4 mL)
and shaken at room temperature for 4 h. The resin was filtered off and
washed with water/acetic acid 5:1, methanol, ethyl acetate and CH2Cl2 (3 Â
6,7-Dimethoxy-1,4-dihydro-2H-isoquinoline-3-one (25):[10] Colourless oil
(18 mg, 84 mmol, 86%); Rf ˆ 0.09 (ethyl acetate); 1H NMR (CDCl3,
500 MHz): d ˆ 7.60 (brs, 1H; NH), 6.64 (s, 1H; arom. CH), 6.63 (s, 1H;
ˆ
5 mL each) and dried in vacuo. IR (KBr, drift): nÄ ˆ 1717 (C O, carboxylic
3
arom. CH), 4.46 (d, J(H,H) ˆ 1.6 Hz, 2H; CH2NH), 3.88 (s, 3H; OCH3),
1
ˆ
ˆ
acid, urethane), 1669 (C O, amide I), 1521 cm (C O, amide II).
3.87 (s, 3H; OCH3), 3.52 (s, 2H; CH2C(O)); HRMS (70 eV): calcd for
C11H13NO3: 207.0895, found: 207.0883; the spectroscopic data are in
agreement with the literature.[10]
TentaGel-bound 2-methoxy-5-nitrobenzyl ester 21: A mixture of TentaGel-
bound acid 20 (100 mg, 29 mmol), 2-methoxy-5-nitrobenzyl bromide (10,
71 mg, 290 mmol), cesium carbonate (47 mg, 145 mmol) and 4 Š molecular
sieves were suspended in DMF (8 mL). The suspension was shaken 1 h at
08C and another 44 h at room temperature. Then the polymer was filtered
off and washed with DMF, water, methanol, ethyl acetate and CH2Cl2 (3 Â
8 mL each) and dried in vacuo. Loading: 81% (see general description to
determine the loading of the polymer with 2-methoxy-5-nitrobenzyl
General procedure for the enzyme-initiated cleavage of 2-methoxy-5-
nitrobenzyl alcohol (24) from the solid phase: A defined amount of
polymer-bound 2-methoxy-5-nitrobenzyl ester was suspended in aqueous
sodium phosphate buffer (0.05m, pH 7.0) and cosolvent. After 30 min a
suspension of penicillin G acylase (70 mgmL 1, 560 UmL 1) in aqueous
potassium phosphate buffer (0.1m, pH 7.5) was added and the reaction
mixture was shaken at room temperature or 378C respectively. After an
incubation period ranging from 24 to 168 h the polymer was filtered off and
washed repeatedly with methanol (3 Â ), water (4 Â ) and methanol (4 Â ).
The combined filtrates were evaporated under reduced pressure. Then the
residue was redissolved in methanol (10 mL) and the detached amount of
2-methoxy-5-nitrobenzyl alcohol (24) was determined by UV spectroscopy
(detection at 307 nm). The polymer was subsequently washed with ethyl
acetate (3 Â ) and CH2Cl2 (3 Â ), dried in vacuo and investigated by IR
spectroscopy. For detailed reaction conditions and the results of the
enzymatic cleavage of 2-methoxy-5-nitrobenzyl alcohol see Scheme 6.
ˆ
ˆ
alcohol); IR (KBr, drift): nÄ ˆ 1720 (C O, ester, urethane), 1668 (C O,
1
ˆ
amide I), 1519 cm (C O, amide II).
2-Methoxy-5-nitrobenzyl
(4,5-dimethoxy-2-{[(phenylacetyl)amino]me-
thyl}phenyl)acetate (23): Cesium carbonate (237 mg, 0.73 mol) was added
to a solution of acetic acid 22 (500 mg, 1.5 mol) in ethanol (60 mL) and
water (20 mL). After evaporation of the solvent under reduced pressure
the residue was dried in vacuo. Then 2-methoxy-5-nitrobenzyl bromide (10,
394 mg, 1.6 mol) and 4 Š molecular sieves were added and the mixture was
dissolved in precooled DMF (08C, 50 mL). The solution was stirred 2 h at
08C and for another 12 h at room temperature. After evaporation of the
solvent the residue was dissolved in ethyl acetate (100 mL) and washed
with water (50 mL). The organic layer was dried over MgSO4 and the
solvent was removed in vacuo. Chromatography on silica gel (ethyl acetate/
hexane 1:1 v/v) yielded a colourless oil (504 mg, 1.0 mmol, 68%). Rf ˆ 0.27
(ethyl acetate/hexane 2:1 v/v‡0.5% acetic acid); 1H NMR (CDCl3,
500 MHz): d ˆ 8.23 (dd, 3J(H,H) ˆ 9.0 Hz, 4J(H,H) ˆ 2.8 Hz, 1H; arom.
Synthesis of POE 6000-bound 2-methoxy-5-nitrobenzyl ester 17
2-Methoxy-5-nitrobenzyl (4-{2-[(chlorocarbonyl)oxy]ethoxy}-5-methoxy-
2-{[(phenylacetyl)amino]methyl}phenyl)acetate (14): A 1.93m solution of
phosgene in toluene (1.05 mL, 2 mol) was added at 58C to a solution of
acetate 11 (177 mg, 0.33 mol) in CH2Cl2 (25 mL). The reaction mixture was
stirred overnight at room temperature. Evaporation of the solvent in vacuo
yielded a white solid, which was used without further purification (198 mg,
0.33 mol, quant.). Rf ˆ 0.66 (ethyl acetate); 1H NMR (CDCl3, 250 MHz):
4
CH), 8.12 (d, J(H,H) ˆ 2.8 Hz, 1H; arom. CH), 7.32 ± 7.22 (m, 5H; arom.
CH), 6.94 (d, 3J(H,H) ˆ 9.1 Hz, 1H; arom. CH), 6.78 (s, 1H; arom. CH-6),
6.71 (s, 1H; arom. CH-3), 6.18 (brs, 1H; NH), 5.14 (s, 2H; OCH2), 4.40 (d,
3J(H,H) ˆ 5.6 Hz, 2H; CH2NH), 3.92 (s, 3H; OCH3), 3.83 (s, 3H; OCH3),
3
4
d ˆ 8.23 (dd, J(H,H) ˆ 8.8 Hz, J(H,H) ˆ 2.7 Hz, 1H; arom. CH), 8.08 (d,
4J(H,H) ˆ 2.7 Hz, 1H; arom. CH), 7.33 ± 7.22 (m, 5H; arom. CH), 6.94 (d,
3J(H,H) ˆ 8.9 Hz, 1H; arom. CH), 6.84 (s, 1H; arom. CH-6), 6.73 (s, 1H;
3.81 (s, 3H; OCH3), 3.70 (s, 2H; PhCH2), 3.54 (s, 2H; CH2C(O)O);
13
ˆ
ˆ
C NMR (CDCl3, 125.7 MHz): d ˆ 171.76 (C O), 170.60 (C O), 161.78
966
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0966 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 5

Safety Catch Linker
959±971
arom. CH-3), 6.17 (brs, 1H; NH), 5.13 (s, 2H; CCH2O), 4.48 (d, 3J(H,H) ˆ
6.9 Hz, 2H; CH2NH), 4.04 (t, 3J(H,H) ˆ 5.5 Hz, 2H; COCH2CH2), 3.92 (s,
3H; OCH3), 3.90 (t, 3J(H,H) ˆ 5.5 Hz, 2H; COCH2CH2), 3.82 (s, 3H;
OCH3), 3.71 (s, 2H; PhCH2), 3.54 (s, 2H; CH2C(O)O); C29H29ClN2O10
(601.0).
urethane), 4.40 (brs, 2H; COCH2), 4.35 (d, 3J(H,H) ˆ 5.7 Hz, 2H;
CH2NH), 4.15 (brs, 2H; COCH2CH2), 3.83 (s, 3H; OCH3), 3.79 ± 3.51
(m, CH2C(O)O, PhCH2, CH2 polymer), 3.39 (brs, 2H; NHCH2CH2OCH2
polymer), 2.05 (s, 3H; COOCH3); 13C NMR (CDCl3, 125.7 MHz): d ˆ
ˆ
ˆ
ˆ
172.52 (C O), 170.79 (C O), 156.41 (C O urethane), 148.85 (COCH2),
147.41 (COCH3), 135.02 (CCH2C(O)NH), 129.30 (2C; arom. CH), 128.78
(2C; arom. CH), 128.54 (CCH2C(O)O), 127.09 (arom. CH), 125.33
(CCH2NH), 115.60 (arom. CH-3), 114.07 (arom. CH-6), 70.77 ± 70.30
(COCH2CH2, CH2 polymer), 67.79 (COCH2), 55.99 (OCH3), 52.24
(COOCH3), 43.71 (NHCH2 polymer), 42.31 (PhCH2), 41.05 (CH2C(O)O),
POE-bound 2-methoxy-5-nitrobenzyl ester 17: A mixture of acetate (14,
98 mg, 0.16 mol), diamino-functionalized polyoxyethylene POE 6000
(164 mg, 27 mmol), DMAP (37 mg, 0.3 mol) and HOBt (11 mg, 82 mmol)
was dried for 8 h over P2O5 in vacuo. The mixture was dissolved in CH2Cl2
(20 mL), stirred overnight at room temperature and washed with diluted
hydrochloric acid (pH 4, 2 Â 15 mL). The organic layer was dried over
MgSO4. Evaporation of the solvent yielded a white solid, which was
redissolved in CH2Cl2 (2 mL). The polymer was precipitated through slow
addition of ice-cold diethyl ether, filtered off and washed with ice-cold
diethyl ether and ice-cold ethanol (08C). The precipitate was dissolved in
CH2Cl2 (2 mL) and the procedure was repeated to yield a white solid.
Isolated amount of polymer: 177 mg, 25 mmol, 92%. The reaction was
quantitative as determined by the 1H NMR spectrum; Ninhydrin test: also
ˆ
37.90 (CH2NH); IR (KBr, drift): nÄ ˆ 1724 (C O, ester, urethane), 1652
1
ˆ
ˆ
(C O, amide I), 1522 (C O, amide II), 1114 cm (C O); average molec-
1
ular weight: 6827 gmol
.
POE-bound carboxylic acid 29: A solution of the POE-bound methyl ester
28 (430 mg, 63 mmol) in 0.25m LiOH/THF (2:1, 30 mL) was stirred at room
temperature. After 4 h diluted hydrochloric acid was added to adjust to
pH 4. The solution was extracted with CHCl3 (6 Â 30 mL) and the
combined organic layers were dried over MgSO4. The solvent was
evaporated in vacuo to yield a colourless solid, which was used without
further purification. Isolated amount of polymer: 424 mg, 62 mmol, 99%.
The reaction was quantitative as determined by the 1H NMR spectrum.
1H NMR (CDCl3, 500 MHz): d ˆ 7.32 ± 7.21 (m, 5H; arom. CH), 6.84 (s,
1H; arom. CH-6), 6.74 (s, 1H; arom. CH-3), 6.51 (brs, 1H; NH amide),
5.72 (brs, 1H; NH urethane), 4.37 (t, 3J(H,H) ˆ 4.4 Hz, 2H; COCH2), 4.34
(d, 3J(H,H) ˆ 5.7 Hz, 2H; CH2NH), 4.21 (t, 3J(H,H) ˆ 4.5 Hz, 2H;
COCH2CH2), 3.81 (s, 3H; OCH3), 3.78 (t, 3J(H,H) ˆ 4.6 Hz, 2H;
NHCH2CH2 polymer), 3.73 ± 3.49 (m, CH2C(O)O, PhCH2, CH2 polymer),
3.37 (t, 3J(H,H) ˆ 4.8 Hz, 2H; NHCH2CH2OCH2 polymer); 13C NMR
1
3
quantitative: H NMR (CDCl3, 250 MHz): d ˆ 8.22 (dd, J(H,H) ˆ 9.0 Hz,
4J(H,H) ˆ 2.7 Hz, 1H; arom. CH), 8.10 (d,4J(H,H) ˆ 2.7 Hz, 1H; arom.
3
CH), 7.33 ± 7.20 (m, 5H; arom. CH), 6.95 (d, J(H,H) ˆ 9.1 Hz, 1H; arom.
CH), 6.87 (s, 1H; arom. CH-6), 6.71 (s, 1H; arom. CH-3), 6.36 (brs, 1H;
NH amide), 5.67 (brs, 1H; NH urethane), 5.12 (s, 2H; CCH2O), 4.38 (d,
3J(H,H) ˆ 5.6 Hz, 2H; CH2NH), 4.15 (t, 3J(H,H) ˆ 4.5 Hz, 2H;
COCH2CH2), 3.91 (s, 3H; OCH3), 3.90 (t, 3J(H,H) ˆ 4.5 Hz, 2H;
COCH2CH2), 3.82 (s, 3H; OCH3), 3.78 ± 3.48 (m, CH2C(O)O, PhCH2,
CH2 polymer), 3.38 (t, 3J(H,H) ˆ 4.5 Hz, 2H; NHCH2CH2OCH2 polymer);
1
average molecular weight: 7129 gmol
.
ˆ
ˆ
(CDCl3, 125.7 MHz): d ˆ 173.57 (C O carboxylic acid), 171.35 (C O
Enzyme-catalysed cleavage of 2-methoxy-5-nitrobenzyl alcohol (24) from
ˆ
amide), 156.41 (C O urethane), 148.80 (COCH2), 147.27 (COCH3), 135.03
1
POE 6000:
A
suspension of penicillin G acylase (1 mL, 70 mgmL
,
(CCH2C(O)NH), 129.24 (2C; arom. CH), 128.71 (2C; arom. CH), 128.47
(arom. CH), 127.04 (CCH2COOH), 125.74 (CCH2NH), 115.43 (arom. CH-
3), 114.14 (arom. CH-6), 72.57 (NHCH2CH2 polymer), 72.42 ± 69.92
(COCH2CH2, CH2 polymer), 55.97 (OCH3), 43.29 (PhCH2), 41.14 (NHCH2
560 UmL 1) in 0.1m aqueous potassium phosphate buffer (pH 7.5) was
added to a solution of POE-bound 2-methoxy-5-nitrobenzyl ester 17
(10 mg, 2.8 mmol) in methanol (0.5 mL) and 0.2m aqueous sodium
phosphate buffer (pH 7.0, 4.5 mL). The reaction mixture was shaken at
room temperature. After 12, 24 and 36 h the same amount of enzyme was
added again and after 48 h the solvent was removed in vacuo. The residue
was dissolved in ethyl acetate (2 mL), filtered over silica gel, then the
solvent was removed in vacuo. The amount of detached 2-methoxy-5-
nitrobenzyl alcohol 24 was determined according to the general procedure
for the enzyme-initiated cleavage of 2-methoxy-5-nitrobenzyl alcohol from
the solid phase. Yield: 59%.
polymer), 40.76 (CH2COOH), 37.95 (CH2NH); average molecular weight:
1
6799 gmol
.
POE-bound aryl iodide 31: A suspension of polymer-bound carboxylic acid
29 (234 mg, 34 mmol), cesium carbonate (58 mg, 178 mmol), 2-iodo benzyl
bromide (30, 104 mg, 349 mmol) and 4 Š molecular sieves in DMF (10 mL)
was stirred at 508C. After 24 h the molecular sieves were filtered off and
the solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2
(15 mL) and washed with saturated ammonium chloride solution (10 mL).
Then the aqueous layer was extracted with CH2Cl2 (2 Â 15 mL) and the
combined organic layers were dried over MgSO4. After evaporation of the
solvent the residue was dissolved in CH2Cl2 (2 mL). The polymer was
precipitated through slow addition of ice-cold diethyl ether, filtered off and
washed with diethyl ether (08C) and ethanol (08C). The precipitate was
dissolved in CH2Cl2 (2 mL) and the procedure was repeated to yield a white
solid. Isolated amount of polymer: 234 mg, 32 mmol, 95%. The reaction was
quantitative as determined by the 1H NMR spectrum. 1H NMR (CDCl3,
500 MHz): d ˆ 7.66 (d, 3J(H,H) ˆ 7.9 Hz, 1H; arom. CH), 7.62 (s, 1H; arom.
CH), 7.32 ± 7.24 (m, 1H; arom. CH), 7.09 (t, 3J(H,H) ˆ 7.8 Hz, 1H; arom.
Synthesis of the POE-bound carboxylic acid 29
Methyl (4-{2-[(chlorocarbonyl)oxy]ethoxy}-5-methoxy-2-{[(phenylacetyl)-
amino]methyl}phenyl)acetate (27):
A 1.93m solution of phosgene in
toluene (4 mL, 7.74 mol) was added at 58C to a solution of alcohol 8
(500 mg, 1.29 mol) in CH2Cl2 (30 mL). The reaction mixture was stirred
overnight at room temperature. Evaporation of the solvent in vacuo
yielded a white solid, which was used without further purification (580 mg,
1.29 mol, quant.). Rf ˆ 0.92 (ethyl acetate); 1H NMR (CDCl3, 250 MHz):
d ˆ 7.34 ± 7.24 (m, 5H; arom. CH), 6.79 (s, 1H; arom. CH-6), 6.63 (s, 1H;
arom. CH-3), 6.24 (brs, 1H; NH), 4.60 (t, 3J(H,H) ˆ 4.5 Hz, 2H;
COCH2CH2), 4.35 (d, 3J(H,H) ˆ 4.3 Hz, 2H; CH2NH), 4.20 (t, 3J(H,H) ˆ
4.6 Hz, 2H; COCH2), 3.83 (s, 3H; OCH3), 3.64 (s, 3H; COOCH3), 3.63 (s,
2H; PhCH2), 3.58 (s, 2H; CH2C(O)O); HRMS (70 eV): calcd for
C22H24ClNO7: 449.1225, found: 449.1241.
CH), 4.99 (s, 2H; CH2O); 13C NMR (CDCl3, 125.7 MHz): d ˆ 171.75 (C O
ˆ
ester), 137.84 (CCH2O), 137.38 (arom. CH), 136.87 (arom. CH), 128.84
(arom. CH), 125.12 (arom. CH), 94.35 (CI), 65.85 (CH2O); IR (KBr, drift):
1
ˆ
ˆ
nÄ ˆ 1719 (C O, ester, urethane), 1653 (C O, amide I), 1106 cm (C O);
1
average molecular weight: 7231 gmol
.
POE-bound methyl ester 28: A mixture of acetate (27, 1.36 g, 3.0 mol),
diamino-functionalized polyoxyethylene POE 6000 (3.0 g, 0.5 mol),
DMAP (674 mg, 5.5 mol) and HOBt (203 mg, 1.5 mol) was dried for 8 h
over P2O5 in vacuo. The mixture was dissolved in CH2Cl2 (50 mL), stirred
overnight at room temperature and washed with diluted hydrochloric acid
(pH 4, 2 Â 30 mL). The organic layer was dried over MgSO4. Evaporation
of the solvent yielded a white solid, which was redissolved in CH2Cl2
(4 mL). The polymer was precipitated through slow addition of precooled
diethyl ether (08C), filtered off and washed with diethyl ether (08C) and
ethanol (08C). The precipitate was dissolved in CH2Cl2 (4 mL) and the
procedure repeated to yield a white solid. Isolated amount of polymer:
3.19 g, 0.47 mol, 93%. The reaction was quantitative as determined by the
1H NMR spectrum; Ninhydrin test: also quantitative. 1H NMR (CDCl3,
500 MHz): d ˆ 7.34 ± 7.25 (m, 5H; arom. CH), 6.88 (s, 1H; arom. CH-6),
6.68 (s, 1H; arom. CH-3), 6.44 (brs, 1H; NH amide), 5.68 (brs, 1H; NH
POE-bound cinnamic acid tert-butyl ester 32: A solution of polymer-bound
aryl iodide (31, 110 mg, 30 mmol), tetrabutylammonium bromide (10 mg,
30 mmol) and triphenylphosphine (8 mg, 30 mmol) in DMF/H2O/Et3N 9:1:1
(5.5 mL) was degassed for 20 min under ultrasonication. Then acrylic acid
tert-butyl ester (22 mL, 153 mmol) and Pd(OAc)2 (3 mg, 15 mmol) was added
and the solution was stirred at 508C. After 24 h the solvent was evaporated
under reduced pressure, the residue was redissolved in CH2Cl2 (10 mL) and
the insoluble components were filtered off. The volume of the solution was
reduced in vacuo, then the polymer was precipitated through slow addition
of precooled diethyl ether (08C), filtered off and washed with ice-cold
diethyl ether and ice-cold ethanol. The precipitate was dissolved in CH2Cl2
(2 mL) and the procedure was repeated to yield a white solid. Isolated
amount of polymer: 99 mg, 27 mmol, 91%. The reaction was quantitative as
determined by the 1H NMR spectrum. 1H NMR (CDCl3, 500 MHz):
Chem. Eur. J. 2001, 7, No. 5
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0967 $ 17.50+.50/0
967

H. Waldmann and U. Grether
FULL PAPER
3
ˆ
d ˆ 7.56 (d, Jtrans ˆ 16.0 Hz, 1H; CCH CH), 7.46 (m, 2H; arom. CH), 7.36
Enzyme-catalysed release of tert-butyl(2E)-3-[3-(hydroxymethyl)phenyl]-
2-propenoate (35): According to the general procedure for the enzyme-
initiated cleavage of alcohols from POE 6000 cinnamic acid tert-butyl ester
32 (43 mg, 12 mmol) was dissolved in methanol (2 mL) and 0.2m aqueous
sodium phosphate buffer (pH 7.0, 18 mL) and incubated with a suspension
of penicillin G acylase (4 mL, 70 mgmL 1, 560 UmL 1) in 0.1m aqueous
potassium phosphate buffer (pH 7.5). 35: Colourless oil (2.1 mg, 9 mmol,
75%); Rf ˆ 0.2 (ethyl acetate/hexane 1:4 v/v); 1H NMR (CDCl3, 500 MHz):
(t, 3J(H,H) ˆ 7.6 Hz, 1H; arom. CH), 7.32 ± 7.24 (m, 1H; arom. CH), 6.38
(d,3Jtrans ˆ 16.0 Hz, 1H; CCH CH), 5.07 (s, 2H; CH2O), 1.54 (s, 9H;
ˆ
13
ˆ
C(CH3)3); C NMR (CDCl3, 125.7 MHz): d ˆ 171.85 (C O ester), 166.03
ˆ
(CHC(O)O), 142.79 (CCH CH), 136.27 (CCH2O), 135.10 (CCH), 129.65
(arom. CH), 127.90 (arom. CH), 127.65 (arom. CH), 127.10 (arom. CH),
120.92 (CCH CH), 80.64 (C(CH3)3), 66.45 (CH2O), 28.19 (3C; C(CH3)3);
ˆ
ˆ
ˆ
IR (KBr, drift): nÄ ˆ 1718 (C O, ester, urethane), 1669 (C O, amide I), 1638
1
(C C, a,b-unsaturated carbonyl group), 1114 cm (C O); average mo-
d ˆ 7.55 (d, 3Jtrans ˆ 16.0 Hz, 1H; CCH CH), 7.49 (s, 1H; arom. CH), 7.41 ±
ˆ
ˆ
1
3
lecular weight: 7233 gmol
.
7.39 (m, 1H; arom. CH), 7.37 ± 7.33 (m, 2H; arom. CH), 6.36 (d, Jtrans
ˆ
ˆ
16.0 Hz, 1H; CCH CH), 4.69 (s, 2H; CH2), 2.33 (brs, 1H; OH), 1.53 (s,
POE-bound (4'-methoxy[1,1'-biphenyl]-3-yl)methanol 33: A solution of
polymer-bound aryl iodide 31 (200 mg, 56 mmol), 4-methoxyphenylboronic
acid (42 mg, 276 mol) and K3PO4 ´ 3H2O (30 mg, 110 mol) in DMF/H2O 6:1
(14 mL) was degassed for 20 min under ultrasonication. Then [Pd(PPh3)4]
(1 mg, 1 mmol) was added and the reaction mixture was stirred at 808C.
After 4 h the solvent was evaporated in vacuo, the residue was dissolved in
CH2Cl2 (20 mL) and washed with water (15 mL). The organic layer was
dried over MgSO4 and the solvent was removed in vacuo. The residue was
redissolved in CH2Cl2 (2 mL), then the polymer was precipitated through
slow addition of precooled diethyl ether (08C), filtered off and washed with
ice-cold diethyl ether and ice-cold ethanol. The precipitate was dissolved in
CH2Cl2 (2 mL) and the procedure was repeated to yield a white solid.
Isolated amount of polymer: 187 mg, 52 mmol, 93%. The reaction was
quantitative as determined by the 1H NMR spectrum. 1H NMR (CDCl3,
500 MHz): d ˆ 7.50 (d, 3J(H,H) ˆ 7.8 Hz, 1H; arom. CH), 7.45 (d,
3J(H,H) ˆ 8.7 Hz, 2H; arom. CH), 7.44 (s, 1H; arom. CH), 7.39 (t,
3J(H,H) ˆ 7.6 Hz, 1H; arom. CH), 7.23 ± 7.21 (m, 1H; arom. CH), 6.97 (d,
13
ˆ
9H; C(CH3)3); C NMR (CDCl3, 125.7 MHz): d ˆ 166.40 (C O), 143.39
ˆ
(CCH CH), 141.61 (CCH2O), 134.87 (CCH), 128.99 (arom. CH), 128.47
ˆ
(arom. CH), 127.18 (arom. CH), 126.29 (arom. CH), 120.36 (CCH CH),
80.63 (C(CH3)3), 64.80 (CH2), 28.19 (3C; C(CH3)3); IR (KBr, drift): nÄ ˆ
ˆ
ˆ
3434 (O H), 1707 (C O, ester), 1637 (C C, a,b-unsaturated carbonyl),
1153 cm 1 (C O); MS (EI, 70 eV, 458C): m/z (%): 234 (82) [M] , 178 (100),
‡
160 (89), 131 (66); HRMS (70 eV): calcd for C14H18O3: 234.1256, found:
234.1241.
Polymer-bound lactam 26: Isolated amount of polymer: 32 mg, 10 mmol,
82%; 1H NMR (CDCl3, 500 MHz): d ˆ 8.84 (brs, 1H; NH lactam), 6.73 (s,
1H; arom. CH), 6.67 (s, 1H; arom. CH), 5.53 (brs, 1H; NH urethane), 4.55
(brs, 2H; CH2NH), 4.43 (brs, 2H; COCH2), 4.20 (brs, 2H; COCH2CH2),
3.86 (s, 3H; OCH3), 3.78 (t, 3J(H,H) ˆ 4.6 Hz, 2H; NHCH2 polymer),
3.74 ± 3.55 (m, CH2C(O)NH, CH2 polymer), 3.50 (t, 3J(H,H) ˆ 5.1 Hz, 2H;
NHCH2CH2O polymer), 3.37 (t, 3J(H,H) ˆ 4.7 Hz, 2H; NHCH2CH2OCH2
polymer); 13C NMR (CDCl3, 125.7 MHz): d ˆ 173.8 (C O lactam), 157.7
ˆ
3J(H,H) ˆ 8.7 Hz, 2H; arom. CH), 5.13 (s, 2H; CH2O), 3.85 (s, 3H; OCH3);
ˆ
(C O urethane), 149.5 (COCH2), 147.5 (COCH3), 127.6 (CCH2C(O)NH),
13
ˆ
126.0 (CCH2NH), 112.1 (arom. CH), 111.5 (arom. CH), 73.2 (NHCH2CH2
polymer), 72.8 ± 69.8 (CH2 polymer), 68.2 (NHCH2CH2OCH2 polymer),
66.5 (COCH2CH2), 63.2 (COCH2), 56.4 (OCH3), 45.05 (NHCH2 polymer),
C NMR (CDCl3, 125.7 MHz): d ˆ 171.84 (C O ester), 156.36 (COCH3),
141.22 (CCH2O), 136.04 (CH2CCHCC), 132.96 (CH2CCHCC), 129.01
(arom. CH), 128.06 (2C; arom. CH), 127.01 (arom. CH), 126.66 (arom.
CH), 126.34 (arom. CH), 114.26 (2C; arom. CH), 63.21 (CH2O), 55.31
(OCH3); average molecular weight: 7191 gmol
ˆ
41.8 (CH2NH), 34.1 (CH2C(O)NH); IR (KBr, drift): nÄ ˆ 1720 (C O,
1
1
ˆ
urethane), 1665 (C O, lactam), 1116 cm (C O); average molecular
.
1
weight: 6529 gmol
.
POE-bound [3-(1-pentynyl)phenyl]methanol 34: 1-Pentine (41 mL,
423 mmol) was added to a solution of polymer-bound aryl iodide 31
(153 mg, 42 mmol) in dioxane/Et3N 2:1 (12 mL) and the solution was
degassed for 15 min under ultrasonication. Then cuprous iodide (2 mg,
11 mmol) and [Pd(PPh3)2Cl2] (3 mg, 4 mmol) were added and the reaction
mixture was stirred at room temperature. After 24 h, CHCl3 (15 mL) was
added and the solution was washed with saturated aqueous ammonium
chloride (10 mL). Then the aqueous phase was washed with CHCl3 (2 Â
15 mL) and the combined organic layers were dried over MgSO4. The
solvent was evaporated under reduced pressure and the residue was
dissolved in CH2Cl2 (2 mL) followed by precipitation of the polymer by
slow addition of ice-cold diethyl ether. The solid was filtered off, washed
with diethyl ether (08C) and ethanol (08C), then the precipitate was
redissolved in CH2Cl2 (2 mL) and the procedure was repeated to yield a
white solid. Isolated amount of polymer: 147 mg, 41 mmol, 97%. The
reaction was quantitative as determined by the 1H NMR spectrum.
1H NMR (CDCl3, 500 MHz): d ˆ 7.35 (t, 4J(H,H) ˆ 1.7 Hz, 1H; arom. CH),
Enzyme-catalysed cleavage of (4'-methoxy[1,1'-biphenyl]-3-yl)methanol
(36): According to the general procedure for the enzyme-initiated cleavage
of alcohols from POE 6000, POE-bound biphenyl 33 (27 mg, 7.6 mmol) was
dissolved in methanol (1 mL) and 0.2m aqueous sodium phosphate buffer
(pH 7.0, 9 mL) and incubated with a suspension of penicillin G acylase
(3 mL, 70 mgmL 1, 560 UmL 1) in 0.1m aqueous potassium phosphate
buffer (pH 7.5). 36:[25] white solid (1.2 mg, 5.5 mmol, 73%); Rf ˆ 0.47 (ethyl
acetate/hexane 1:2 v/v); m.p. 948C, lit.[25] 958C; 1H NMR (CDCl3,
:
500 MHz): d ˆ 7.53 (s, 1H; arom. CH), 7.51 (td, 3J(H,H) ˆ 8.7 Hz,
4J(H,H) ˆ 3.0 Hz, 2H; arom. CH), 7.46 (d, 3J(H,H) ˆ 7.8 Hz, 1H; arom.
CH), 7.38 (t, 3J(H,H) ˆ 7.6 Hz, 1H; arom. CH), 7.27 (d, 3J(H,H) ˆ 6.5 Hz,
1H; arom. CH), 6.95 (td, 3J(H,H) ˆ 8.7 Hz, 4J(H,H) ˆ 3.0 Hz, 2H; arom.
CH), 4.71 (s, 2H; CH2), 3.83 (s, 3H; OCH3), 2.03 (brs, 1H; OH); MS (EI,
‡
‡
70 eV, 608C): m/z (%): 215 (12) [M‡H] , 214 (100) [M] , 199 (12); HRMS
(70 eV): calcd for C14H14O2: 214.0994, found: 214.0985. The spectroscopic
data are in agreement with the literature.[25]
3
7.32 ± 7.24 (m, 2H; arom. CH), 7.18 (d, J(H,H) ˆ 7.6 Hz, 1H; arom. CH),
Enzyme-catalysed cleavage of [3-(1-pentynyl)phenyl]methanol (37): Ac-
cording to the general procedure for the enzyme-initiated cleavage of
alcohols from POE 6000, POE-bound alkine 34 (19 mg, 5.3 mmol) was
dissolved in methanol (1 mL) and 0.2m aqueous sodium phosphate buffer
(pH 7.0, 9 mL) and incubated with a suspension of penicillin G acylase
(2mL, 70 mgmL 1, 560 UmL 1) in 0.1m aqueous potassium phosphate
buffer (pH 7.5). 37: Colourless oil (0.9 mg, 5.0 mmol, 94%); Rf ˆ 0.2 (ethyl
acetate/hexane 1:6 v/v); 1H NMR (CDCl3, 400 MHz): d ˆ 7.38 (s, 1H;
arom. CH), 7.33 ± 7.22 (m, 3H; arom. CH), 4.59 (s, 2H; CH2OH), 2.38 (t,
3J(H,H) ˆ 7.0 Hz, 2 H ; CCH2CH2), 2.29 (s, 1H; OH), 1.62 (sext, 3J(H,H) ˆ
7.4 Hz, 2H; CH2CH3), 1.05 (t, 3J(H,H) ˆ 7.3 Hz, 3H; CH3); 13C NMR
(CDCl3, 100.6 MHz): d ˆ 140.81 (CCH2OH), 130.75 (arom. CH), 130.05
(arom. CH), 128.45 (arom. CH), 126.10 (arom. CH), 124.28 (CCCCH2),
90.51 (CCH2CH2), 80.56 (CCCCH2), 64.88 (CH2OH), 22.21 (CCH2CH2),
21.39 (CH2CH3), 13.60 (CH3); IR (KBr, drift): nÄ ˆ 3310 (O H), 2228
5.02 (s, 2H; CH2O), 2.38 (t, 3J(H,H) ˆ 7.0 Hz, 2H; CH2CH2CH3), 1.63 (sext,
3J(H,H) ˆ 7.2 Hz, 2 H ; CH2CH3), 1.05 (t, 3J(H,H) ˆ 7.4 Hz, 3H; CH3);
13
ˆ
C NMR (CDCl3, 125.7 MHz): d ˆ 171.87 (C O ester), 135.61 (CCH2O),
131.53 (arom. CH), 131.28 (arom. CH), 128.52 (arom. CH), 127.09 (arom.
CH), 124.57 (CCCCH2), 90.95 (CCH2CH2), 80.21 (CCCH2), 63.27 (CH2O),
22.15 (CCH2CH2), 21.37 (CH2CH3), 13.58 (CH3); average molecular
1
weight: 7111 gmol
.
General procedure for the enzyme-initiated cleavage of alcohols from
POE 6000: A defined amount of polymer-bound substrate was dissolved in
methanol and 0.2m aqueous sodium phosphate buffer (pH 7.0). Then a
1
1
suspension of penicillin G acylase (70 mgmL
, 560 UmL ) in 0.1m
aqueous potassium phosphate buffer (pH 7.5) was added and the reaction
mixture was shaken at 378C. The same amount of enzyme was added after
12, 24 and 36 h. After 48 h the reaction mixture was extracted with diethyl
ether (6 Â 20 mL). The combined organic layers were dried over MgSO4,
then the solvent was removed in vacuo to yield the cleaved alcohol. After
that the aqueous phase was extracted with CH2Cl2 (4 Â 20 mL), the
combined organic layers were dried over MgSO4 and the solvent was
evaporated under reduced pressure. The polymer obtained thereby was
dried in vacuo.
‡
(alkine), 1019 cm 1 (C O); MS (EI, 70 eV, 608C): m/z (%): 174 (24) [M] ,
114 (29); HRMS (70 eV): calcd for C12H14O: 174.1045, found: 174.1030.
General procedure for the esterification of the POE-bound carboxylic acid
29: DIC (34 mL, 220 mmol), pyridine (18 mL, 220 mmol) and the respective
alcohol were added at 08C to a solution of polymer-bound carboxylic acid
29 (100 mg, 29 mmol), DMAP (1 mg, 8 mmol) and HOBt (1 mg, 7 mmol) in
968
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0968 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 5

Safety Catch Linker
959±971
3
3
ˆ
DMF (3 mL). After 1 h the ice bath was removed and the reaction mixture
was stirred overnight at room temperature. The solvent was evaporated in
vacuo, the residue redissolved in CH2Cl2 (15 mL) and washed with 0.05m
hydrochloric acid (15 mL). Then the aqueous phase was extracted with
CH2Cl2 (2 Â 15 mL), the combined organic layers were dried over MgSO4
and the solvent was removed under reduced pressure. The residue was
dissolved in CH2Cl2 (2 mL) followed by precipitation of the polymer by
slow addition of ice-cold diethyl ether. The solid was filtered off, washed
with diethyl ether (08C) and ethanol (08C), then the precipitate was
redissolved in CH2Cl2 (2 mL) and the procedure was repeated. The
polymer was dried in vacuo.
500 MHz): d ˆ 6.39 (d, Jtrans ˆ 17.3 Hz, 1H; CH CH2a), 6.10 (dd, Jtrans
ˆ
3
3
ˆ
17.3 Hz, Jcis ˆ 10.4 Hz, 1H; CH CH2), 5.82 (d, Jcis ˆ 10.4 Hz, 1H;
3
ˆ
CH CH2b), 4.19 ± 4.12 (m, 2H; CHC(O)OCH2), 4.08 (t, J(H,H) ˆ 6.8 Hz,
2H; CH2C(O)OCH2), 1.72± 1.64 (m, 4H; OCH2CH2CH2); 13C NMR (CDCl3,
ˆ
ˆ
125.7 MHz): d ˆ 172.6 (C O ester), 164.2 (OC(O)CH), 131.3 (CH CH2),
ˆ
126.9 (CH CH2), 64.1 (CH2C(O)OCH2), 63.1 (CHC(O)OCH2), 25.0
(CH2C(O)OCH2CH2), 25.05 (CHC(O)OCH2CH2); IR (KBr, drift): nÄ ˆ
ˆ
ˆ
ˆ
1721 (C O, ester, urethane), 1666 (C O, amide I), 1519 (C O, amide II),
1116 cm 1 (C O); average molecular weight: 7051 gmol
.
1
POE-bound Diels ± Alder adduct 42
Method A: A solution of polymer-bound acrylic acid ester 41 (44 mg,
13 mmol) in freshly distilled cyclopentadiene/THF 1:1 (2 mL) was heated to
608C. After 24 h the solvent was evaporated in vacuo, the residue was
dissolved in CH2Cl2 (2 mL) followed by precipitation of the polymer by
slow addition of ice-cold diethyl ether. The solid was filtered off, washed
with diethyl ether (08C) and ethanol (08C) and dissolved in CH2Cl2 (2 mL).
The procedure was repeated to yield a white solid. Isolated amount of
polymer: 42 mg, 12 mmol, 93%, endo/exo 2.5:1. The reaction was quanti-
POE-bound benzyl alcohol 38: According to the general procedure
polymer-bound carboxylic acid 29 (100 mg, 29 mmol), DMAP (1 mg,
8 mmol), HOBt (1 mg, 7 mmol), DIC (34 mL, 220 mmol), pyridine (18 mL,
220 mmol) and 4-(hydroxymethyl)-benzyl alcohol (81 mg, 588 mmol) were
allowed to react in DMF (3 mL). White solid; isolated amount of polymer:
94 mg, 27 mmol, 92%. The reaction was quantitative as determined by the
1H NMR spectrum. 1H NMR (CDCl3, 500 MHz): d ˆ 7.34 ± 7.29 (m, 2H;
arom. CH), 7.25 ± 7.23 (m, 2H; arom. CH), 5.06 (s, 2H; OCH2), 4.66 (s, 2H;
1
tative as determined by the H NMR spectrum.
13
ˆ
CH2OH); C NMR (CDCl3, 125.7 MHz): d ˆ 171.71 (C O ester), 141.56
Method B: Polymer-bound acrylic acid ester 41 (29 mg, 8 mmol) and a trace
of hydroquinone were dissolved in freshly distilled cyclopentadiene/
toluene 3:2 (2.5 mL) in a glass vial. The vial was evacuated, sealed and
heated for 24 h in a bomb tube. After cooling the vial was opened, CH2Cl2
(1 mL) was added and the polymer was precipitated by slow addition of
precooled diethyl ether (08C). The solid was filtered off, washed with ice-
cold diethyl ether and ice-cold ethanol. The residue was redissolved in
CH2Cl2 (2 mL) and the procedure was repeated to yield a white solid.
Isolated amount of polymer: 20 mg, 6 mmol, 70%, endo/exo 2.5:1. The
(CCH2OH), 134.57 (CCH2O), 128.28 (2C; arom. CH), 126.97 (2C; arom.
CH), 63.84 (OCH2), 63.12 (CH2OH); IR (KBr, drift): nÄ ˆ 3325 (O H), 1722
ˆ
ˆ
ˆ
(C O, ester, urethane), 1666 (C O, amide I), 1519 (C O, amide II),
1115 cm 1 (C O); average molecular weight: 7039 gmol
.
1
POE-bound benzyl ether 39: A solution of polymer-bound benzyl alcohol
38 (28 mg, 8 mmol), 4-acetamidophenol (12 mg, 79 mmol) and triphenyl-
phosphine (10 mg, 39 mmol) in THF/CH2Cl2 1:1 (600 mL) was stirred at
58C for 15 min in the presence of 4 Š molecular sieves. Then diethyl
azodicarboxylate (6 mL, 39 mmol) was slowly added and the reaction
mixture was stirred 1 h at 08C and 12 h at room temperature. The
molecular sieves were filtered off, the solvent was evaporated in vacuo and
the residue was purified by size-exclusion chromatography (chloroform/
methanol 1:1 v/v) on Sephadex LH 20 to yield a colourless solid. Isolated
amount of polymer: 25 mg, 7 mmol, 86%. The reaction was quantitative as
determined by the 1H NMR spectrum. 1H NMR (CDCl3, 500 MHz): d ˆ
7.62 (brs, 1H; NH), 7.42 (d, 3J(H,H) ˆ 9.0 Hz, 2H; arom. CH), 7.39 (d,
3J(H,H) ˆ 8.0 Hz, 2H; arom. CH), 7.31 ± 7.23 (m, 2H; arom. CH), 6.88 (d,
3J(H,H) ˆ 9.0 Hz, 2H; arom. CH), 5.07 (s, 2H; CH2OC(O)), 5.03 (s, 2H;
reaction was quantitative as determined by the 1H NMR spectrum.
1
ˆ
H NMR (CDCl3, 250 MHz): d ˆ 6.21 ± 6.09 (m, 3H; CHCHCH CH endo,
ˆ
ˆ
CHCHCH CH exo, CHCHCH CH endo), 5.92 ± 5.88 (m, 1H;
CHCHCH CH exo), 4.14 (brs, 2H; CHC(O)OCH2), 3.74 ± 3.50 (m, 2H;
CH2C(O)OCH2), 3.18 (brs, 1H; C(O)CHCH endo), 3.01 (brs, 1H;
C(O)CHCH exo), 2.95 ± 2.86 (m, 2H; C(O)CHCH2CH endo,
C(O)CHCH2CH exo), 2.25 ± 2.23 (m, 1H; C(O)CH), 1.96 ± 1.86 (m, 1H;
C(O)CHCH2a), 1.73 ± 1.64 (m, 4H; C(O)OCH2CH2CH2), 1.61 ± 1.52 (m,
ˆ
1H; C(O)CHCH2b), 1.46 ± 1.21 (m, 2H; C(O)CHCHCH2); average molec-
1
ular weight: 7183 gmol
.
CH2O), 2.14 (s, 3H; CH3); 13C NMR (CDCl3, 125.7 MHz): d ˆ 171.83 (C O
ˆ
ˆ
ester), 168.35 (C O anilide), 155.15 (COCH2C), 135.25 (CCH2OC), 135.06
Enzyme-catalysed cleavage of 4-hydroxybutyl-bicyclo[2.2.1]hept-5-ene-
carboxylate (43): According to the general procedure for the enzyme-
initiated cleavage of alcohols from POE 6000, POE-bound adduct 42
(41 mg, 11 mmol) was dissolved in methanol (3 mL) and 0.2m aqueous
sodium phosphate buffer (pH 7.0, 26 mL) and incubated with a suspension
of penicillin G acylase (5 mL, 70 mgmL 1, 560 UmL 1) in 0.1m aqueous
potassium phosphate buffer (pH 7.5). The crude product was purified by
chromatography on silica gel (ethyl acetate/hexane 1:3 v/v) to yield a
colourless oil (1.8 mg, 9 mol, 75%, endo:exo 2.5:1). Rf ˆ 0.17 (ethyl acetate/
hexane 1:4 v/v); 1H NMR (CDCl3, 250 MHz): d ˆ 6.22 ± 6.15 (m, 1H;
(CCH2OC(O)), 131.73 (CNH), 128.49 (2C; arom. CH), 127.61 (2C; arom.
CH), 121.59 (2C; arom. CH), 115.13 (2C; arom. CH), 70.05 (COCH2), 57.31
ˆ
(CH2OC(O)), 24.28 (CH3); IR (KBr, drift): nÄ ˆ 1722 (C O, ester,
1
ˆ
ˆ
urethane), 1673 (C O, amide I, anilide), 1511 (C O, amide II), 1115 cm
1
(C O); average molecular weight: 7307 gmol
.
Enzyme-catalysed cleavage of N-(4-{[4-hydroxymethyl)benzyl]oxy}phenyl)-
acetamide (40): According to the general procedure for the enzyme-
initiated cleavage of alcohols from POE 6000, POE-bound benzyl phenyl
ether 39 (20 mg, 6 mmol) was dissolved in methanol (3 mL) and 0.2m
aqueous sodium phosphate buffer (pH 7.0, 26 mL) and incubated with a
suspension of penicillin G acylase (3 mL, 70 mgmL 1, 560 UmL 1) in 0.1m
aqueous potassium phosphate buffer (pH 7.5). The crude product was
purified by chromatography on silica gel (ethyl acetate/hexane 1:1 to 2:1
v/v) to yield a colourless oil (1.2 mg, 4.4 mmol, 81%). Rf ˆ 0.15 (ethyl
acetate/hexane 2:1 v/v); 1H NMR (CDCl3, 500 MHz): d ˆ 7.43 ± 7.37 (m,
6H; arom. CH), 7.03 (brs, 1H; NH), 6.92 (d, 3J(H,H) ˆ 8.8 Hz, 2H; arom.
ˆ
ˆ
CHCHCH CH endo), 6.15 ± 6.08 (m, 2H; CHCHCH CH exo,
ˆ
ˆ
CHCHCH CH exo), 5.95 ± 5.90 (m, 1H; CHCHCH CH endo), 4.13 (t,
3J(H,H) ˆ 6.5 Hz, 2H; OCH2 exo), 4.05 (t, 3J(H,H) ˆ 6.5 Hz, 2H; OCH2
endo), 3.69 (brs, 2H; CH2OH), 3.20 (brs, 1H; C(O)CHCH endo), 3.03 (brs,
1H; C(O)CHCH exo), 2.96 ± 2.88 (m, 1H; C(O)CHCH2CH), 2.24 ± 2.21
(m, 1H; C(O)CH), 1.97 ± 1.85 (m, 1H; C(O)CHCH2a), 1.79 ± 1.59 (m, 4H;
CH2CH2CH2OH),
1.55 ± 1.32
(m,
4H;
OH,
C(O)CHCH2b,
ˆ
C(O)CHCHCH2); IR (KBr, film): nÄ ˆ 3434 (O H), 1729 (C O, ester),
CH), 5.05 (s, 2H; OCH2), 4.71 (s, 2H; CH2OH), 2.15 (s, 3H; CH3);
1
13
ˆ
1570 (C C), 1176 (C O), 1046 cm (C O); MS (EI, 70 eV, 408C): m/z
ˆ
C NMR (CDCl3, 125.7 MHz): d ˆ 168.07 (C O), 155.60 (COCH2), 140.68
‡
(%): 210 (0.5) [M] , 138 (11), 66 (100); HRMS (70 eV): calcd for C12H18O3:
(CCH2OH), 136.45 (CCH2OC), 131.20 (CNH), 127.73 (2C; arom. CH),
210.1256, found: 210.1248.
127.25 (2C; arom. CH), 121.84 (2C; arom. CH), 115.27 (2C; arom. CH),
70.06 (OCH2), 65.14 (CH2OH), 24.43 (CH3); IR (KBr, drift): nÄ ˆ 3286 (O H),
POE-bound isoxazoline 44: syn-Benzaldehyde oxime (8 mL, 71 mmol) was
added to a solution of polymer-bound acrylic acid ester 41 (50 mg, 14 mmol)
in THF (500 mL). Then a 0.7m aqueous solution of sodium hypochlorite
(100 mL, 140 mmol) was added to the reaction mixture. After 4 h at room
temperature the solvent was evaporated in vacuo, the residue was
redissolved in CH2Cl2 (10 mL) and washed with water (10 mL). The
aqueous layer was extracted with CH2Cl2 (3 Â 10 mL), the combined
organic layers were dried over MgSO4 and the solvent was evaporated
under reduced pressure. The residue was dissolved in CH2Cl2 (1 mL) and
the polymer was precipitated by slow addition of precooled diethyl ether
(08C). The solid was filtered off, washed with ice-cold diethyl ether and ice-
1
ˆ
ˆ
ˆ
‡
1735 (C O, ester), 1659 (C O, anilide), 1533 (C O, amide II), 1049 cm
‡
(C O); MS (EI, 70 eV): m/z (%): 272 (4) [M‡H] , 271 (36) [M] , 121
(100); HRMS (70 eV): calcd for C16H17NO3: 271.1208, found: 271.1203.
POE-bound acrylic acid derivative 41
According to the general procedure polymer-bound carboxylic acid 29
(100 mg, 29 mmol), DMAP (1 mg, 8 mmol), HOBt (1 mg, 7 mmol), DIC
(34 mL, 220 mmol), pyridine (18 mL, 220 mmol) and 4-hydroxybutylacrylate
(30 mL, 220 mmol) were allowed to react in DMF (3 mL) to yield a white
solid. Isolated amount of polymer: 101 mg, 29 mmol, 97%. The reaction was
quantitative as determined 1H NMR spectrum; 1H NMR (CDCl3,
Chem. Eur. J. 2001, 7, No. 5
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0969 $ 17.50+.50/0
969

H. Waldmann and U. Grether
FULL PAPER
cold ethanol, then the precipitate was redissolved in CH2Cl2 (2 mL) and the
procedure was repeated to yield a white solid. Isolated amount of polymer:
48 mg, 13 mmol, 94%. The reaction was quantitative as determined
1H NMR spectrum. 1H NMR (CDCl3, 500 MHz): d ˆ 7.68 (dd,3J(H,H) ˆ
(3 mL) and 0.2m aqueous sodium phosphate buffer (pH 7.0, 26 mL) and
1
incubated with a suspension of penicillin G acylase (4 mL, 72 mgmL
,
1368 UmL 1) in 0.1m aqueous potassium phosphate buffer (pH 7.5). 46:[26]
Colourless oil (1.5 mg, 9 mmol, 68%); Rf ˆ 0.38 (ethyl acetate/hexane 1:4
v/v); 1H NMR (CDCl3, 250 MHz): d ˆ 6.97 (d, 3J(H,H) ˆ 9.1 Hz, 2H; arom.
CH), 6.61 (d, 3J(H,H) ˆ 9.1 Hz, 2H; arom. CH), 3.47 (brs, 2H; NH2), 2.50
(t, 3J(H,H) ˆ 7.5 Hz, 2H; CCH2), 1.58 (q, 3J(H,H) ˆ 7.5 Hz, 2H;
CCH2CH2), 1.38 ± 1.22 (m, 4H; CH2CH2CH3), 0.90 (t, 3J(H,H) ˆ 7.5 Hz,
3H; CH3). The spectroscopic data are in agreement with the literature.[26]
7.9 Hz, 4J(H,H) ˆ 1.3 Hz, 2H; arom. CH), 7.44 ± 7.40 (m, 3H; arom. CH),
3
5.16 (dd, J(OCH,CH2aC N) ˆ 10.1, 3J(OCH,CH2bC N) ˆ 7.1 Hz, 1H;
ˆ
ˆ
3
3
OCH), 4.20 (t, J(H,H) ˆ 2.4 Hz, 2H; CHC(O)OCH2), 4.12 (t, J(H,H) ˆ
ˆ
4.5 Hz, 2H; CH2C(O)OCH2), 3.73 ± 3.54 (m, 2H; CH2C N), 1.75 ± 1.69
(m, 4H; OCH2CH2CH2); 13C NMR (CDCl3, 125.7 MHz): d ˆ 172.04 (C O
ˆ
ˆ
ester), 170.14 (OC(O)CH), 156.37 (C N), 130.56 (arom. CH), 129.41
Polymer-bound lactam 26: Colourless solid; isolated amount of polymer:
36 mg, 11 mmol, 85%. The reaction was quantitative as determined by the
1H NMR spectrum.
ˆ
(CC N), 128.79 (2C; arom. CH), 126.92 (2C; arom. CH), 78.06 (OCH),
61.70 (CH2C(O)OCH2), 57.30 (CHC(O)OCH2), 38.76 (CH2C N), 25.15
ˆ
(CH2C(O)OCH2CH2), 25.06 (CHC(O)OCH2CH2); IR (KBr, drift): nÄ ˆ
Enzyme-catalysed release of 4-aminobenzoic acid ethyl ester (48): Accord-
ing to the general procedure for the enzyme-initiated cleavage of anilines
from POE 6000, POE-bound 4-aminobenzoic acid ethyl ester 47 (40 mg,
11 mmol) was dissolved in methanol (3 mL) and 0.2m aqueous sodium
phosphate buffer (pH 7.0, 26 mL) and incubated with a suspension of
penicillin G acylase (4 mL, 72 mgmL 1, 1368 UmL 1) in 0.1m aqueous
potassium phosphate buffer (pH 7.5). 48:[27] Colourless oil (1.1 mg, 7 mmol,
60%); Rf ˆ 0.37 (ethyl acetate/hexane 1:4 v/v); 1H NMR (CDCl3,
250 MHz): d ˆ 7.81 (d, 3J(H,H) ˆ 9.1 Hz, 2H; arom. CH), 6.60 (d,
3J(H,H) ˆ 9.1 Hz, 2H; arom. CH), 4.28 (q, 3J(H,H) ˆ 6.4 Hz, 2H; CH2),
4.01 (brs, 2H; NH2), 1.32 (t, 3J(H,H) ˆ 6.4 Hz, 3H; CH3); C9H11NO2
(165.2); The spectroscopic data are in agreement with the literature.[27]
ˆ
ˆ
ˆ
1726 (C O, ester, urethane), 1666 (C O, amide I), 1519 (C O, amide II),
1116 cm 1 (C O); average molecular weight: 7289 gmol
.
1
General procedure for the synthesis of POE-bound anilides: A mixture of
POE-bound carboxylic acid 29, the respective aniline, EDC and HOBt was
dissolved in DMF. The reaction mixture was stirred 3 h at 08C and 9 h at
room temperature. Then the solvent was evaporated in vacuo, the residue
was dissolved in CHCl3 (30 mL) and washed with 0.05n hydrochloric acid
(2 Â 20 mL). The combined aqueous layers were extracted with CHCl3 (3 Â
30 mL) and the combined organic layers were dried over MgSO4. Then the
solvent was removed under reduced pressure, the residue was redissolved
in CH2Cl2 (2 mL) and the polymer was precipitated by slow addition of pre-
cooled diethyl ether (08C). The solid was filtered off and washed with ice-cold
diethyl ether and ice-cold ethanol. The precipitate was dissolved in CH2Cl2
(2 mL) and the procedure was repeated. The polymer was dried in vacuo.
POE-bound 4-iodoaniline 50: According to the general procedure for the
synthesis of POE-bound anilides, POE-bound carboxylic acid 29 (250 mg,
74 mmol), EDC (56 mg, 292 mmol), HOBt (50 mg, 368 mmol) and 4-iodoani-
line (49, 129 mg, 589 mmol) were allowed to react in DMF (5 mL). White
solid; Isolated amount of polymer: 258 mg, 72 mmol, 97%. The reaction
was quantitative as determined by the 1H NMR spectrum. 1H NMR
(CDCl3, 500 MHz): d ˆ 9.25 (brs, 1H; NH), 7.54 (d, 3J(H,H) ˆ 8.6 Hz, 2H;
POE-bound 4-pentylaniline 45: According to the general procedure for the
synthesis of POE-bound anilides, POE-bound carboxylic acid 29 (100 mg,
29 mmol), EDC (22 mg, 116 mmol), HOBt (20 mg, 145 mmol) and 4-pentyl-
aniline (46, 42 mL, 240 mmol) were allowed to react in DMF (5 mL). White
solid; Isolated amount of polymer: 91 mg, 26 mmol, 89%. The reaction was
quantitative as determined by the 1H NMR spectrum. 1H NMR (CDCl3,
500 MHz): d ˆ 8.80 (brs, 1H; NH), 7.47 (d, 3J(H,H) ˆ 8.4 Hz, 2H; arom.
3
arom. CH), 7.40 (d, J(H,H) ˆ 8.6 Hz, 2H; arom. CH); 13C NMR (CDCl3,
ˆ
125.7 MHz): d ˆ 171.98 (C O anilide), 137.55 (2C; arom. CH), 134.56
ˆ
(CNH), 121.56 (2C; arom. CH), 86.78 (CI); IR (KBr, drift): nÄ ˆ 1719 (C O,
3
3
CH), 7.06 (d, J(H,H) ˆ 8.4 Hz, 2H; arom. CH), 2.54 (t, J(H,H) ˆ 7.5 Hz,
2H; CCH2), 1.59 ± 1.53 (m, 2H; CCH2CH2), 1.32 ± 1.25 (m, 4H;
CH2CH2CH3), 0.87 (t, 3J(H,H) ˆ 3.2 Hz, 3H; CH3); 13C NMR (CDCl3,
1
ˆ
ˆ
urethane), 1675 (C O, amide I, anilide), 1521 (C O, amide II), 1114 cm
(C O); average molecular weight: 7201 gmol
1
.
POE-bound biphenyl aniline 51: A solution of polymer-bound 4-iodoani-
line 50 (200 mg, 55 mmol), 4-methoxyphenylboronic acid (42 mg, 277 mmol)
and K3PO4 ´ 3H2O (29 mg, 110 mmol) in DMF/H2O 10:1 (5.5 mL) was
degassed for 20 min under ultrasonication. Then [Pd(PPh3)4] (1.3 mg,
1 mmol) was added and the reaction mixture was stirred at 808C. After 20 h
the solvent was evaporated in vacuo, the residue was redissolved in CH2Cl2
(20 mL) and washed with water (15 mL). The aqueous layer was extracted
with CH2Cl2 (2 Â 20 mL), the combined organic layers were dried over
MgSO4 and the solvent was evaporated in vacuo. The residue was dissolved
in CH2Cl2 (2 mL), the polymer precipitated through slow addition of ice-
cold diethyl ether, filtered off and washed with diethyl ether (08C) and
ethanol (08C). The precipitate was redissolved in CH2Cl2 (2 mL) and the
procedure was repeated to yield a white solid. Isolated amount of polymer:
186 mg, 52 mmol, 95%. The reaction was quantitative as determined by the
ˆ
125.7 MHz): d ˆ 171.24 (C O anilide), 136.17 (CNH), 135.00 (CCH2),
128.56 (2C; arom. CH), 119.54 (2C; arom. CH), 35.27 (CCH2), 31.35
(CH2CH2CH3), 29.63 (CCH2CH2), 22.47 (CH2CH3), 14.02 (CH3); IR (KBr,
ˆ
ˆ
ˆ
drift): nÄ ˆ 1720 (C O, urethane), 1673 (C O, amide I, anilide), 1517 (C O,
1
amide II), 1115 cm 1 (C O); average molecular weight: 7089 gmol
.
POE-bound 4-aminobenzoic acid ethyl ester 47: According to the general
procedure for the synthesis of POE-bound anilides POE-bound carboxylic
acid 29 (126 mg, 37 mmol), DIC (17 mL, 111 mmol), HOBt (19 mg,
139 mmol) and 4-aminobenzoic acid ethyl ester (48, 37 mg, 222 mmol) were
allowed to react in DMF (4 mL). White solid; Isolated amount of polymer:
118 mg, 33 mmol, 90%. The reaction was quantitative as determined by the
1H NMR spectrum. 1H NMR (CDCl3, 500 MHz): d ˆ 7.93 (d, 3J(H,H) ˆ
9.0 Hz, 2H; arom. CH), 7.67 (d, 3J(H,H) ˆ 9.0 Hz, 2H; arom. CH), 7.48
(brs, 1H; NH), 4.41 ± 4.29 (m, 2H; CH2), 1.40 (t, 3J(H,H) ˆ 7.5 Hz, 3H;
1
1H NMR spectrum. H NMR (CDCl3, 500 MHz): d ˆ 8.99 (brs, 1H; NH),
ˆ
ˆ
CH3); IR (KBr, drift): nÄ ˆ 1751 (C O, ester), 1718 (C O, urethane), 1670
7.65 (d, 3J(H,H) ˆ 8.5 Hz, 2H; arom. CH), 7.48 (d, 3J(H,H) ˆ 8.6 Hz, 2H;
(C O, amide I, anilide), 1521 (C O, amide II), 1118 cm 1 (C O); average
ˆ
ˆ
3
3
arom. CH), 7.45 (d, J(H,H) ˆ 8.5 Hz, 2H; arom. CH), 6.95 (d, J(H,H) ˆ
1
molecular weight: 7093 gmol
.
8.8 Hz, 2H; arom. CH), 3.85 (s, 3H; OCH3); 13C NMR (CDCl3,
ˆ
General procedure for the enzyme-initiated cleavage of anilines from
POE 6000: A definite amount of polymer-bound anilide was dissolved in
methanol and 0.05m aqueous sodium phosphate buffer (pH 7.0). Then a
suspension of penicillin G acylase (72 mgmL 1, 1368 UmL 1) in 0.1m
aqueous potassium phosphate buffer (pH 7.5) was added and the reaction
mixture was shaken at 378C. After 12, 24 and 36 h the same amount of
enzyme was added again and after 48 h the solution was heated to 608C for
4 h. The reaction mixture was filtered over celite and the filtrate was
extracted with diethyl ether (4 Â 30 mL). The combined organic layers were
dried over MgSO4, then the solvent was evaporated in vacuo to yield the
detached aniline. After that the aqueous layer was extracted with CH2Cl2
(3 Â 30 mL), the combined organic layers were dried over MgSO4 and the
solvent was removed under reduced pressure. The polymer was dried in vacuo.
125.7 MHz): d ˆ 171.47 (C O anilide), 137.43 (COCH3), 136.25
(NHCCHCHCC), 133.24 (NHCCHCHCC), 133.24 (CNH), 127.75 (2C;
arom. CH), 126.87 (2C; arom. CH), 119.92 (2C; arom. CH), 114.16 (2C;
ˆ
arom. CH), 55.31 (OCH3); IR (KBr, drift): nÄ ˆ 1718 (C O, urethane), 1670
(C O, amide I, anilide), 1521 (C O, amide II), 1109 cm 1 (C O); average
ˆ
ˆ
1
molecular weight: 7161 gmol
.
Enzyme-catalysed release of 4'-methoxy[1,1'-biphenyl]-4-amine (52): Ac-
cording to the general procedure for the enzyme-initiated cleavage of
anilines from POE 6000, POE-bound biphenyl aniline 51 (38 mg, 10 mmol)
was dissolved in methanol (3 mL) and 0.2m aqueous sodium phosphate
buffer (pH 7.0, 26 mL) and incubated with a suspension of penicillin G
1
1
acylase (3 mL, 72 mgmL
, 1368 UmL ) in 0.1m aqueous potassium
phosphate buffer (pH 7.5). 52:[28] Colourless oil (1.2 mg, 6 mmol, 57%);
1
Enzyme-catalysed release of 4-pentylaniline (46): According to the general
prescription for the enzyme-initiated cleavage of anilines from POE 6000,
POE-bound 4-pentylaniline 45 (44 mg, 13 mmol) was dissolved in methanol
Rf ˆ 0.25 (ethyl acetate/hexane 1:4 v/v); H NMR (CDCl3, 250 MHz): d ˆ
7.42 (d, 3J(H,H) ˆ 8.2 Hz, 2H; arom. CH), 7.35 (d, 3J(H,H) ˆ 8.2 Hz, 2H;
3
3
arom. CH), 6.91 (d, J(H,H) ˆ 9.3 Hz, 2H; arom. CH), 6.72 (d, J(H,H) ˆ
970
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0970 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 5

Safety Catch Linker
959±971
[3] Enzyme Catalysis in Organic Synthesis (Eds.: K. Drauz, H. Wald-
mann), VCH, Weinheim, 1995.
9.3 Hz, 2H; arom. CH), 3.73 (s, 3H; OCH3); MS (EI, 70 eV, 508C): m/z
(%): 199 (100) [M] , 184 (59); HRMS (70 eV): calcd for C13H13NO:
‡
[4] For a preliminary publication see: a) U. Grether, H. Waldmann,
Angew. Chem. 2000, 112, 1688 ± 1691; Angew. Chem. Int. Ed. 2000, 39,
1629 ± 1632. Enzyme-labile linker groups for syntheses on solid
supports: b) B. Sauerbrei, V. Jungmann, H. Waldmann, Angew. Chem.
1998, 110, 1187 ± 1190; Angew. Chem. Int. Ed. 1998, 37, 1143 ± 1146 and
references therein; c) G. Böhm, J. Dowden, D. C. Rice, I. Burgess, J.-F.
Pilard, B. Guilbert, A. Haxton, R. C. Hunter, N. J. Turner, S. L. Flitsch,
Tetrahedron Lett. 1998, 39, 3819 ± 3822; d) M. Schuster, P. Wang, J. C.
Paulson, C.-H. Wong, J. Am. Chem. Soc. 1994, 116, 1135 ± 1136. Safety
catch linkers for combinatorial synthesis; see e.g.: e) N. J. Osborn,
J. A. Robinson, Tetrahedron 1993, 49, 2873 ± 2884; f) X.-Y. Xiao, M. P.
Nova, A. W. Czarnik, J. Comb. Chem. 1999, 1, 379 ± 382.
[5] a) I. D. Entwistle, Tetrahedron Lett. 1994, 35, 4103 ± 4106; b) F.
Cubain, Rev. Roum. Chim. 1973, 18, 449 ± 461; c) G. Just, G. Rosebery,
Synth. Commun. 1973, 3, 447 ± 451; d) B. F. Cain, J. Org. Chem. 1976,
41, 2029 ± 2031; e) T. W. Greene, P. G. M. Wuts, Protective Groups in
Organic Synthesis, 3rd ed., Wiley, New York, 1999 and references
therein.
[6] For the use of the PhAc group in the deprotection of peptides,
carbohydrates and nucleosides see: a) H. Waldmann, Liebigs Ann.
Chem. 1988, 1175 ± 1180 and references therein; b) H. Waldmann, A.
Heuser, A. Reidel, Synlett 1994, 65 ± 67; c) M. A. Dineva, B. Galunsky,
V. Kasche, D. D. Petkov, Bioorg. Med. Chem. Lett. 1993, 3, 2781 ±
2784; d) H. Waldmann, A. Reidel, Angew. Chem. 1997, 109, 642 ± 644;
Angew. Chem. Int. Ed. 1997, 36, 647 ± 649.
[7] a) D. Ben-Ishai, J. Sataty, N. Peled, R. Goldshare, Tetrahedron 1987,
43, 439 ± 450; b) H. E. Zaugg, Synthesis 1984, 85 ± 110.
[8] a) W. Rapp, in Combinatorial Peptide and Nonpeptide Libraries (Ed.:
G. Jung), VCH, Weinheim, 1996, 425 ± 464; b) E. Bayer, Angew. Chem.
1991, 103, 117 ± 133; Angew. Chem. Int. Ed. Engl. 1991, 30, 113 ± 129.
[9] M. Meldal, Tetrahedron Lett. 1992, 33, 3077 ± 3080.
199.0997, found: 199.0972. The spectroscopic data are in agreement with
the literature.[28]
POE-bound enol ether 53: A solution of polymer-bound 4-iodoaniline 50
(200 mg, 55 mmol) and triphenylarsine (3 mg, 10 mmol) in dioxane (4 mL)
was degassed for 20 min under ultrasonication. Then tributyl(1-ethoxyvi-
nyl)stannane (52 mL, 156 mmol) and [Pd2(dba)3] ´ CHCl3 (2.6 mg, 2.6 mmol)
were added and the reaction mixture was stirred 24 h at 608C. Then the
solution was decanted to remove a precipitate which formed during the
reaction. The polymer was precipitated through slow addition of ice-cold
diethyl ether, filtered off and washed with diethyl ether (08C) and ethanol
(08C). The precipitate was redissolved in CH2Cl2 (2 mL) and the procedure
was repeated to yield a white solid. Isolated amount of polymer: 74 mg,
21 mmol, 81%. The reaction was quantitative as determined by the 1H NMR
spectrum. 1H NMR (CDCl3, 500 MHz): d ˆ 9.00 (brs, 1H; NH), 7.56 (d,
3J(H,H) ˆ 8.8 Hz, 1H; arom. CH), 7.52 (d, 3J(H,H) ˆ 8.7 Hz, 1H; arom.
CH), 7.31 ± 7.23 (m, 2H; arom. CH), 4.56 (d, 2J(H,H) ˆ 2.3 Hz, 1H;
C CH2a), 4.14 ± 4.13 (m, 1H; C CH2b), 3.90 (q, 3J(H,H) ˆ 7.0 Hz, 2H;
OCH2), 1.41 (t, 3J(H,H) ˆ 6.9 Hz, 3H; CH3); 13C NMR (CDCl3,
ˆ
ˆ
ˆ
ˆ
125.7 MHz): d ˆ 171.34 (C O anilide), 159.49 (C CH2), 135.00 (CNH),
ˆ
125.76 (2C; arom. CH), 119.58 (CC CH2), 119.00 (2C; arom. CH), 81.31
ˆ
ˆ
(C CH2), 63.16 (OCH2), 14.53 (CH3); IR (KBr, drift): nÄ ˆ 1721 (C O,
1
ˆ
ˆ
urethane), 1667 (C O, amide I, anilide), 1515 (C O, amide II), 1116 cm
1
(C O); average molecular weight: 7089 gmol
.
POE-bound acetophenone 54: A solution of polymer-bound enol ether 53
(74 mg, 21 mmol) in 0.5m hydrochloric acid/THF 1:1 (10 mL) was stirred for
4 h at room temperature. Then the pH of the reaction mixture was adjusted
to pH 7 with NaHCO3 solution and the solution was extracted with CH2Cl2
(3 Â 20 mL). The combined organic layers were dried over MgSO4 and the
solvent was evaporated in vacuo to yield a colourless solid. Isolated amount
of polymer: 68 mg, 19 mmol, 92%. The reaction was quantitative as
determined by the 1H NMR spectrum. 1H NMR (CDCl3, 500 MHz): d ˆ
9.57 (brs, 1H; NH), 7.86 (d, 3J(H,H) ˆ 8.7 Hz, 2H; arom. CH), 7.70 (d,
3J(H,H) ˆ 8.7 Hz, 2H; arom. CH), 2.56 (s, 3H; CH3); 13C NMR (CDCl3,
[10] B. Sauerbrei, U. Grether, H. Waldmann, DE 19626762, 1996.
Â
Â
[11] a) J. Vagner, G. Barany, K. S. Lam, V. Krchnak, N. F. Sepetov, J. A.
Ostrem, P. Strop, M. Lebl, Proc. Natl. Acad. Sci. USA 1996, 93, 8194 ±
8199; b) Y. W. Cheung, C. Abell, S. Balasubramanian, J. Am. Chem.
Soc. 1997, 119, 9568 ± 9569.
ˆ
ˆ
125.7 MHz): d ˆ 196.87 (C O ketone), 171.62 (C O anilide), 143.20
(CNH), 132.24 (CC(O)), 127.70 (2C; arom. CH), 118.77 (2C; arom. CH),
ˆ
ˆ
26.30 (CH3); IR (KBr, drift): nÄ ˆ 1712 (C O, urethane, ketone), 1670 (C O,
[12] J. Rademann, M. Grotli, M. Meldal, K. Bock, J. Am. Chem. Soc. 1999,
121, 5459 ± 5466.
[13] a) E. Bayer, M. Mutter, Nature 1972, 237, 512 ± 513; b) D. J. Gravert,
K. D. Janda, Chem. Rev. 1997, 97, 489 ± 509.
[14] J. M. Dust, Z. Fang, J. M. Harris, Macromolecules 1990, 23, 3742 ±
3746.
1
ˆ
amide I, anilide), 1519 (C O, amide II), 1116 cm
(C O); average
1
molecular weight: 7033 gmol
.
Enzyme-catalysed release of 4-aminoacetophenone (55): According to the
general procedure for the enzyme-initiated cleavage of anilines from
POE 6000, POE-bound acetophenone 52 (29 mg, 8 mmol) was dissolved in
[15] a) M. Hiroshige, J. R. Hauske, P. Zhou, Tetrahedron Lett. 1995, 36,
4567 ± 4570; b) T. Jeffery, J.-C. Galland, Tetrahedron Lett. 1994, 35,
4103 ± 4106.
[16] S. R. Piettre, S. Baltzer, Tetrahedron Lett. 1997, 38, 1197 ± 1200.
[17] S. Berteina, S. Wendeborn, W. K.-D. Brill, A. D. Mesmaeker, Synlett
1998, 676 ± 678.
[18] The endo/exo ratio of compound 36 synthesized by a Diels ± Alder
reaction in solution phase was also 2.5:1.
[19] J.-F. Cheng, A. M. M. Mjalli, Tetrahedron Lett. 1998, 39, 939 ± 942.
[20] K. A. Bearer, A. C. Siegmund, K. L. Spear, Tetrahedron Lett. 1996, 37,
1145 ± 1148.
methanol (3 mL) and 0.2m aqueous sodium phosphate buffer (pH 7.0, 26 mL)
1
and incubated with a suspension of penicillin G acylase (3 mL, 72 mgmL
,
1368 UmL 1) in 0.1m aqueous potassium phosphate buffer (pH 7.5). The
crude product was purified by chromatography on silica gel (ethyl acetate/
hexane 1:4 v/v) to yield a colourless oil (0.7 mg, 5 mmol, 67%). 55:[29]
1
Rf ˆ 0.28 (ethyl acetate/hexane 1:4 v/v); H NMR (CDCl3, 250 MHz): d ˆ
3
3
7.81 (d, J(H,H) ˆ 9.1 Hz, 2H; arom. CH), 6.68 (d, J(H,H) ˆ 9.1 Hz, 2H;
arom. CH), 4.13 (brs, 2H; NH2), 2.51 (s, 3H; CH3); C8H9NO (135.2); The
spectroscopic data are in agreement with the literature.[29]
Acknowledgement
[21] A. Anantanarayan, P. J. Dutton, T. M. Fyles, M. J. Pitre, J. Org. Chem.
1986, 51, 752 ± 755.
[22] R. D. Haworth, R. McGillivray, D. H. Peacock, J. Chem. Soc. 1950,
1493 ± 1498.
This research was supported by the BMBF, the BASF AG and the Fonds
der Chemischen Industrie.
[23] E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal. Biochem.
1970, 34, 595 ± 598.
[1] a) F. Balkenhohl, C. von dem Bussche-Hünnefeld, A. Lansky, C.
Zechel, Angew. Chem. 1996, 108, 2436 ± 2487; Angew. Chem. Int. Ed.
Engl. 1996, 35, 2288 ± 2337; b) J. S. Früchtel, G. Jung, Angew. Chem.
1996, 108, 19 ± 46; Angew. Chem. Int. Ed. Engl. 1996, 35, 17 ± 42;
c) L. A. Thompson, J. A. Ellman, Chem. Rev. 1996, 96, 555 ± 600; d) D.
Obrecht, J. M. Villalgordo, Solid-supported Combinatorial and Paral-
lel Synthesis of Small-Molecular-Weight Compound Libraries, Perga-
mon, New York, 1998.
[24] R. F. Collins, M. Davis, J. Chem. Soc. 1966, 873 ± 880.
[25] M. Minssen-Guette, M. Dvolaitzky, J. Jacques, Bull. Soc. Chim. Fr.
1968, 2111 ± 2117.
[26] H. Meier, W. Luettke, Liebigs Ann. Chem. 1981, 7, 1303 ± 1333.
[27] M. Julliard, G. Vernin, J. Metzger, Helv. Chim. Acta 1980, 63, 456 ±
466.
[28] D. Ren, R. A. McClelland, Can. J. Chem. 1998, 76, 78 ± 84.
[29] C.-H. Park, R. S. Givens, J. Am. Chem. Soc. 1997, 119, 2453 ± 2463.
[2] a) I. W. James, Tetrahedron 1999, 55, 4855 ± 4946 and references
therein; b) review: B. J. Backes, J. A. Ellman, Curr. Opin. Chem. Biol.
1997, 1, 86 ± 93.
Received: September 5, 2000 [F2714]
Chem. Eur. J. 2001, 7, No. 5
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0705-0971 $ 17.50+.50/0
971