Screening of polymeric supports and enzymes for the development of an endo enzyme cleavable linker

Article abstract of DOI:10.1016/j.tetlet.2010.03.040

Several polymeric supports possessing an ester moiety were prepared and a range of enzymes was investigated to hydrolyse the ester linkage and release a signalling group into solution for applications in immunoassays. Pseudomonas lipases were found to most readily cleave the solution-phase analogue and this observation translated well to the corresponding polymeric supports, where the most effective were PEGA resins and the LPOS support PEG-6000.

Full text of DOI:10.1016/j.tetlet.2010.03.040

Tetrahedron Letters 51 (2010) 2720–2723
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Screening of polymeric supports and enzymes for the development of an endo
enzyme cleavable linker
Ana Chiva a, David E. Williams b, , Alethea B. Tabor a, Helen C. Hailes a,
*
a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H OAJ, UK
b Unipath Ltd, Bedford MK44 3UP, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Several polymeric supports possessing an ester moiety were prepared and a range of enzymes was inves-
tigated to hydrolyse the ester linkage and release a signalling group into solution for applications in
immunoassays. Pseudomonas lipases were found to most readily cleave the solution-phase analogue
and this observation translated well to the corresponding polymeric supports, where the most effective
were PEGA resins and the LPOS support PEG-6000.
Received 14 January 2010
Revised 3 March 2010
Accepted 12 March 2010
Available online 17 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Esterases
Lipases
Enzyme cleavable linkers
Solid-phase reactions
LPOS
Immunoassays are used extensively in medical diagnostics to
detect or measure biological species. Enzymes are widely used as
signal-generating labels in these assays, either to produce a coloured
or fluorescent species from a non-coloured or non-fluorescent
precursor or to generate a reagent that triggers chemiluminescence
or electrochemiluminescence. An alternative approach is to use an
enzyme to release a signal compound into solution for detection or
to cleave the signal compound from a precursor in which the signal
is significantly modified. Despite problems of carrying out analysis
of labelled species including fluorescent analysis on resin beads,1,2
these are potentially extremely powerful, sensitive and general
methods that particularly lend themselves to schemes in which
amplification is achieved by having multiple labels, bound as a
consequence of a single biological recognition event.3 Ferrocene
has been used in a range of carbonylmetallo-immunoassays where
the ability to tailor its redox properties has been of advantage.4 We
have been exploring the use of electrochemicaldetection techniques
in which modified ferrocenes, showing significantly altered electro-
chemistry, are used as the signal compounds.5,6 These ferrocenes
have the potential to be useful in immunoassay schemes in which
the ferrocene is cleaved and released into solution.7 Enzymatic
cleavage of side chains containingthe label, such as ferrocene, would
alter the redox potential of the ferrocene and potentially offer a
simple and general method for using ferrocene reagents in an
enzyme-coupled immunoassay (Scheme 1).
Due to the extensive range of commercially available hydrolytic
enzymes, we have explored enzymatic cleavage of an ester-linked
labelled moiety. In the present model study to identify suitable
polymeric supports, linkers and enzymes, a fluorescent label was
used, but the synthetic methods are easily adaptable to the use
of ferrocenes as the label. The strategy used was initially to estab-
lish solution-phase chemistries using a solid-phase analogue and
then translate this approach onto the solid phase. Enzyme labile
linkers that can be cleaved under mild neutral reaction conditions
A Immunoassay developed by addition of a PEG signal compound: enzyme
(Enz) such as an esterase captured onto a bead bed by immunoreaction
O
O
O
O
R
HO PEG
N
Fc
+
PEG
N
H
Fc
H
E1/2 approx 350
mV Ag/AgCl
Enz
is a bead bed
R is a resin/polymer
B Immunoassay developed by addition of enzyme: bead loaded with
signal compound captured onto a surface by immunoreaction
O
R-Fc
O
O
R-Fc
R-Fc
R-Fc
O
esterase
PEG
O
O
HO
N
H
Fc
* Corresponding author. Tel.: +44 (0)20 7679 4654; fax: +44 (0)20 7679 7463.
E-mail address: h.c.hailes@ucl.ac.uk (H.C. Hailes).
Present address: MacDiarmid Institute for Advanced Materials and Nanotechnol-
ogy, Department of Chemistry, University of Auckland, Private Bag 92019, Auckland,
New Zealand.
O
E1/2 approx 350
mV Ag/AgCl
Scheme 1. Possible electrochemical detection schemes for immunoassays.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.03.040

A. Chiva et al. / Tetrahedron Letters 51 (2010) 2720–2723
2721
have been used in solid-phase peptide and carbohydrate synthe-
sis.8 They have been classified as exo- or endo-linkers which can
be cleaved by exo- or endo-enzymes, respectively.8 Exo-Linkers
have been developed by several groups for applications in carbohy-
drate assembly and combinatorial chemistry.9,10
trichloroacetimidate derivatives using trichloroacetonitrile and
DBU,19 then reacted with 8 and boron trifluoride etherate to give
the coupled resins 10 and 11. PEGA-NH2 1900 g/mol (0.20 mmol/g)
and PEGA-NH2 800 g/mol (0.40 mmol/g) were reacted with the
succinimidyl carbonate of 8 to give resins 12 and 13.
The arrangement of endo-linkers is linear and cleavage normally
results in partial retention of the enzyme recognition group with
the target molecule.8 Several endo-linked systems have also been
described,11,12 including an aminopropyl silica support and ester
Enzymatic hydrolysis of the resins was then performed. The
cleavage to give 7 was monitored by HPLC analysis. The level of re-
sin loading in each case was determined by resin hydrolysis using
1 M NaOH (24 h) and quantitative determination of 7 by HPLC.
Enzymatic cleavage levels were then calculated relative to the total
loading of 8. Resin-conjugate 10 was submitted to enzymatic
hydrolysis with a range of enzymes previously screened against
2. No substantial hydrolysis was observed (Table 2, entry 1) due
to poor diffusion of the enzyme into the resin. For enzyme cleavage
experiments using 11, low hydrolysis yields of up to 6% were
determined (Table 2, entries 2 and 3). Several studies using Tenta-
Gel adducts with enzymes have been reported with variable re-
sults, including a 50% cleavage yield with penicillin amidase
(MW 60 kDa);9 lipase RB001-05 to hydrolyse an acetate group in
73% yield, although penicillin acylase (MW 80 kDa) was used to
cleave a related exo-linker but gave only 1% hydrolysis,10,20 and
using papain (MW 23 kDa) to cleave a resin-bound fluorescently
labelled peptide (MW 23 kDa) where very low yields were ob-
served.21 Our data suggest that the majority of the cleavage with
the TentaGel-conjugate 11 is ‘surface-based’ with poor accessibil-
ity to the enzymes used.1
PEGA resins can swell up to 60-fold the weight of the polymer
in aqueous buffers and allow large proteins to diffuse into the inte-
rior of the polymer, although they are not as easy to handle as the
TentaGel resins.1,21 Resin-conjugates 12 and 13 were used with se-
lected enzymes, predominantly from the family of Pseudomonas,
which proved to work well with the solution-based analogue.
Again regioselective cleavage of the ester to give 7 was observed,
but the levels of hydrolysis were much higher than those for 10
and 11. Lipoprotein from Pseudomonas sp. andPseudomonas cepacia
lipase showed the best catalytic efficiency with both 12 and 13,
and total hydrolysis was achieved using the former with 12 (Table
2, entry 5). Reports in the literature using PEGA in enzyme cleav-
able reactions have highlighted that the higher the molecular
weight of the enzyme, the lower the cleavage. Specifically, near
quantitative yields were achieved with enzymes of MW up to
50 kDa, and above this, reaction yields were normally low.1,20,22
These data agree with our studies where P. cepacia lipase (MW
25 kDa)23 and lipoprotein lipase from Pseudomonas sp., (MW
33 kDa)24 using 12 and 13 gave high hydrolysis yields (Table 2, en-
tries 4, 7 and 5, 8) while the Pseudomonas fluorescens and Aspergil-
lus oryzae lipase with MWs of around 50 kDa24,25 gave lower
cleavage yields (Table 2, entries 6, 9 and 10).
cleavable linker which was readily hydrolysed using a-chymotryp-
sin.13 Our approach was to use an endo-linker strategy based upon
compound 1 possessing a hydrocarbon chain as a spacer, an ester
linker to be enzymatically cleaved, a diethylene glycol unit to en-
hance the solubility of the signalling molecule when cleaved and
in aqueous solution and a signalling moiety. In this study a fluores-
cent pyrene species was used. To assess the ease of cleavage in
aqueous solution, compound 2 was synthesised possessing a ben-
zyloxy group to mimic the solid support (Scheme 2).
Compound 314 was coupled to Boc-protected 2-(2-aminoeth-
oxy)ethanol 415 using DCC and HOBt. Removal of the Boc group
gave 5 which was then reacted with commercially available N-
hydroxysuccinimide pyrene (6) to give 2 in 95% yield. Compound
7, the corresponding hydrolysis product, was readily prepared by
reacting pyrene butyric acid with 2-(2-aminoethoxy)ethanol and
DCC and HOBt in 65% yield. Several esterases, proteases and lipases
were assessed for the cleavage of analogue 2–7 to enable selection
of a smaller number of hydrolytic enzymes to use with resin ana-
logues. Representative cleavage data after 1 h is summarised in Ta-
ble 1. Several lipases (entries 4, 7, 9, 10 and 11) demonstrated good
levels of hydrolysis and these were explored further in resin-based
reactions.
The choice of the resin solid support was crucial: it must be
compatible with the biocatalyst, swell in the reaction solvent and
enable accessibility of the enzyme to the interior of the resin.16
Several resins were initially selected for the preparation of enzyme
cleavable systems. Polystyrene-based supports were considered,
despite the fact that they are known to shrink in aqueous media,
because the loading level is higher than that in many available res-
ins and they might be useful in exploring non-aqueous-based
chemistries. TentaGel resin, which swells well in aqueous solu-
tions, was investigated, although the polystyrene core can limit
the access of macromolecules for cleavage purposes.16 Polyacryl-
amide resin PEGA has been recognised as highly suitable for use
in enzyme-based chemistries, and was also investigated.17
Initially compound 8 was synthesised for attachment to resins
(Scheme 3). 6-(Tetrahydropyranyloxy)hexanoic acid18 (9) was
coupled to 4 using DCC and HOBt. Deprotection and reaction with 6
gave 8. The Wang (p-benzyloxybenzyl alcohol [HMP] 0.80 mmol/g)
and TentaGel HMP (0.35 mmol/g) resins were converted into their
Two further potential supports were then investigated. PAM-
AMs are commercially available polyamido amine dendrimers with
a high loading capacity, and different generations are available,
reflecting the degree of loading possible.26 They have been used
as a dendrimeric support to synthesise a polysaccharide with the
enzyme cellodextrin phosphorylase.27 Attachment to latex beads
was envisaged to generate multiply-functionalised amine resins
for use in immunoassays. To explore the use of PAMAMs alone in
preliminary studies, a generation 1 PAMAM was coupled to 8 to
yield dendrimer 14 (Scheme 4). Enzymatic cleavage experiments
were performed: however, only lipoprotein lipase from Pseudomo-
nas sp. hydrolysed the ester moiety in low yield (Table 3) and this
approach was not explored further.
position of ester cleavable group
O
O
O
signalling group
1
is a polymeric support
O
N
H
n
m
O
Boc
O
O
HO
OH
N
H
4
Ph
O
O
Ph
O
5
NH2
O
5
5 3
O
a,b
O
pyr
c
6
N O
3
pyr is pyrene
O
O
O
O
O
pyr
Ph
O
O
pyr
HO
N
H
O
N
H
Polyethylene glycol has been successfully used as a soluble
polymeric support in liquid-phase organic synthesis (LPOS).28,29
After reactions, the polymer can be precipitated by the addition
of diethyl ether, which allows ease of separation and purification
and having a solubilised support will enable the enzyme to ap-
3
5
3
7
2
Scheme 2. Cleavable signalling molecule 1, synthesis of solution-phase analogue 2
and structure 7. Reagents and conditions: (a) Et3N, HOBt, DCC, 52%; (b) TFA, 95%; (c)
6, DMAP, 95%.

2722
A. Chiva et al. / Tetrahedron Letters 51 (2010) 2720–2723
Table 1
Selected enzymes used to hydrolyse 2
Entry
Enzymea
Units usedb
Hydrolysisc
1
2
3
Esterase from horse liver
Esterase from hog liver
Subtilisin A
2.6
152
26
+
+
0
4
5
6
Pseudomonas cepacia lipase
Candida antarctica lipase
Candida cylindracea lipase
113
140
14
+++
+
+
7
8
9
10
11
Pseudomonas fluorescens lipase
Lipoprotein lipase from Chromobacterium viscosum
Lipoprotein lipase from Pseudomonas sp. 17,840 U/mg
Lipoprotein lipase from Pseudomonas sp. 282 U/mg
Lipase from Aspergillus oryzae
180
14,160
3568
1410
580
++
++
+++
+++
+++
a
Esterase/protease reactions in 10% CH3CN in 0.1 M phosphate (pH 8) at 25 °C. Lipases used in 50% hexane and 10% CH3CN in 0.1 M phosphate (pH 8) at 37 °C. Initial concn
2 [S]0 1 mM: no background reaction observed. Reaction monitored by TLC analysis.
b
Where incomplete hydrolysis was observed, additional enzyme had a negligible effect on the level of hydrolysis.
No hydrolysis-0; low levels of hydrolysis (ꢀ0 to 30%)-+; moderate levels of hydrolysis but 2 remaining (ꢀ30 to 90%)-++; complete hydrolysis-+++.
c
hydrolyses were observed again with Pseudomonas enzymes (Table
3, entries 2–4). Notably, since the enzyme and support are both in
solution, the molecular weight of the enzyme does not appear to be
a crucial factor and P. fluorescens hydrolysed 16 in a higher yield
than P. cepacia.
Overall, the approach of initially identifying suitable enzymes
from a solution-phase assay then translating this to polymeric sup-
ports proved effective. Several polymeric supports were investigated
for use in the enzymatic assay. Polystyrene-based resins performed
O
a–c
O
O
THPO
HO
O
pyr
8
OH
O
N
H
O
5
9
5
3
d,e
O
O
f,g
O
pyr
10
11
P
O
N
H
5
3
O
O
pyr
P
O
N
5
3
O
O
H
H
N
O
O
pyr
10 P is Wang-O resin
11 P is TentaGel-HMP-O resin
O
N
H
P
5
3
O
12 P is PEGA-NH resin, loading 0.20 mmol/g
13 P is PEGA-NH resin, loading 0.40 mmol/g
O
O
H
N
Scheme 3. Synthesis of 8–13. Reagents and conditions: (a) Et3N, HOBt, DCC, 4, 72%;
(b) TFA, 99%; (c) 6, DMAP, 23%; (d) Wang (HMP) and Tentagel HMP, CCl3CN, DBU,
0 °C; (e) To (d) add 8, BF3ꢁOEt2; (f) N,N0-disuccinimidyl carbonate, Et3N, 74%; (g)
PEGA-NH2 resins.
a,b
8
O
O
pyr
14
O
N
H
5
3
O
PAMAM (g1)
OH
15
O
c,d
O
H O
H
n
O
HO
n
O
e,f
O
O
proach the substrate more easily. They have had limited applica-
tion to date, although penicillin G acylase was used to readily
cleave conjugates in high yields and purities.8,20 It was decided
to use an HMP linker as before with a linear PEG-6000, functional-
ised at both termini. PEG-6000 was converted into the dimesylated
PEG,29 in quantitative yield and then treated with sodium hydride
and 4-hydroxybenzaldehyde and directly reduced to the alcohol
with sodium borohydride to yield 15 in 74% yield.30 The terminal
hydroxy groups were trichloroacetimidated and reacted with 8 to
produce the PEG resin-conjugate 16 (Scheme 4). Good enzymatic
O
5
O
O
HN
O
O
O
n
pyr
O
3
O
5
16
NH
O
pyr
3
Scheme 4. Synthesis of resin-conjugates 14 and 16. Reagents and conditions: (a)
N,N0-disuccinimidyl carbonate, Et3N, 74%; (b) PAMAM (g1); (c) CH3SO2Cl, Et3N,
100%; (d) 4-(HO)–C6H4CHO, NaH, 75 °C, then NaBH4, 74%; (e) CCl3CN, DBU, 0 °C,
37%; (f) 8, BF3ꢁOEt2, 25%.
Table 2
Enzymatic hydrolysis of resin-conjugates 10–13
Entry
Resin conjugate
Enzymea
Units usedb
Hydrolysis (time)
1
2
3
4
10
11
11
12
Pseudomonas cepacia lipase (MW 25 kDa)
Lipoprotein lipase from Pseudomonas sp. (MW 33 kDa) 17,840 U/mg
Pseudomonas cepacia lipase (MW 25 kDa)
600
3920
600
600
1% (4 h)
4% (4 h)
6% (4 h)
Pseudomonas cepacia lipase (MW 25 kDa)
29% (4 h)
41% (16 h)
59% (4 h)
100% (16 h)
1% (4 h)
21% (4 h)
43% (16 h)
78% (72 h)
71% (4 h)
78% (16 h)
4% (4 h)
5
12
Lipoprotein lipase from Pseudomonas sp. (MW 33 kDa) 17,840 U/mg
11,600
6
7
12
13
Pseudomonas fluorescens lipase (MW 50 kDa)
Pseudomonas cepacia lipase (MW 25 kDa)
290
600
8
13
Lipoprotein lipase from Pseudomonas sp. (MW 33 kDa) 17,840 U/mg
11,600
9
10
13
13
Pseudomonas fluorescens lipase (MW 50 kDa)
Lipase from Aspergillus oryzae (MW 51 kDa)
290
180
7% (4 h)
1–4
l
mol-loaded resins 10–13 used.
a
Wang resin reaction: 1% CH3CN, in 0.1 M phosphate (pH 8) at rt. Tentagel resin reactions: 50% hexane in 0.1 M phosphate (pH 8) at 37 °C. PEGA reactions in 0.1 M
phosphate (pH 8) at 37 °C. Reactions monitored by HPLC.
b
Addition of further enzyme gave negligible increase in the level of hydrolysis.

A. Chiva et al. / Tetrahedron Letters 51 (2010) 2720–2723
2723
Table 3
Enzymatic hydrolysis of resin conjugates 14 and 16
Entry
Resin conjugate
Enzymea
Units usedb
Hydrolysis (time)
1
2
3
4
14
16
16
16
Lipoprotein lipase from Pseudomonas sp. (MW 33 kDa)
Pseudomonas cepacia lipase (MW 25 kDa)
Lipoprotein lipase from Pseudomonas sp. (MW 33 kDa)
Pseudomonas fluorescens lipase (MW 50 kDa)
11,620
200
11,600
180
15% (4 h)
56% (4 h)
28% (4 h)
79% (4 h)
1–3
l
mol-loaded resins 14 and 16 used.
a
0.1 M phosphate (pH 8) at 37 °C. Reaction monitored by HPLC.
Addition of further enzyme gave negligible increase in the level of hydrolysis.
b
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