Expanding the scope of biocatalysis: Oxidative biotransformations on solid-supported substrates

Article abstract of DOI:10.1002/adsc.200800188

Oxidative biocatalytic reactions were performed on solid-supported substrates, thus expanding the repertoire of biotransformations that can be carried out on the solid phase. Various phenylacetic and benzoic acid analogues were attached to controlled pore

Full text of DOI:10.1002/adsc.200800188

FULL PAPERS
DOI: 10.1002/adsc.200800188
Expanding the Scope of Biocatalysis: Oxidative
Biotransformations on Solid-Supported Substrates
Sarah J. Brooks,a,d Lydie Coulombel,a,d Disha Ahuja,a Douglas S. Clark,b
and Jonathan S. Dordicka,c,*
a
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Nanotechnology Center, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA
Fax : (+1)-518-276-2207; e-mail: dordick@rpi.edu
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA
Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA
Both authors contributed equally to the work.
b
c
d
Received: March 31, 2008; Revised: May 20, 2008; Published online: June 23, 2008
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: Oxidative biocatalytic reactions were per- linkage. Controlled pore glass (CPG) modified with
formed on solid-supported substrates, thus expanding a polyethylene glycol (PEG) linker afforded substan-
the repertoire of biotransformations that can be car- tially higher product yields than non-PEGylated
ried out on the solid phase. Various phenylacetic and CPG or non-swellable polymeric resins. This work
benzoic acid analogues were attached to controlled represents the first attempt to combine solid-phase
pore glass beads via an enzyme-cleavable linker. Re- oxidative biotransformations with subsequent pro-
actions catalyzed by peroxidases (soybean and tease-catalyzed cleavage, and serves to further
chloro), tyrosinase, and alcohol oxidase/dehydrogen- expand the use of biocatalysis in synthetic and me-
ase gave a range of products, including oligophenols, dicinal chemistry.
halogenated aromatics, catechols, and aryl aldehydes.
The resulting products were recovered following
cleavage from the beads using a-chymotrypsin to se- Keywords: biotransformations; enzyme catalysis;
lectively hydrolyze a chemically non-labile amide halogenation; oxidation; solid-phase synthesis
Introduction
to the synthesis or modification of complex lead mol-
ecules, including natural products and synthetic com-
Advances in combinatorial chemistry have resulted pounds that contain multiple functional groups. Com-
largely from solid-phase chemistries with high reactiv- plexities arise due to the need for selective attach-
ity and broad specificity.[1] Various solid supports are ment and cleavage sites and chemistries, as well as
now commonly used and generally consist of cross- the need to perform reactions under mild conditions.
linked polymers, which provide a fluid-like environ- For this reason, enzymatic catalysis on solid-support-
ment that aids solution-phase chemistries on the solid ed substrates has been considered.
phase. These materials have facilitated a broad array
Enzymes have a long history of acting on solid sub-
of reactions, including Ugi condensation,[2] Pummerer strates. For example, cellulose degradation[9] (in aque-
cyclizations[3] [2+3]cyclocondensations,[4] Beckmann ous media) and sugar acylation[10] (in organic sol-
rearrangements,[5] cyclization of b-diketones,[6] and vents) involve substrates with limited or no solubility,
Michael additions,[7] among others. As a result, combi- yet biocatalytic reactions are feasible. Biocatalytic
natorial and iterative reactions are possible, including transformations on solid-supported substrates have
multiple reactions in sequence under different reac- been performed since the mid-1990s. Such reactions
tion conditions without the consequences of reagent include oligosaccharide and oligophenol synthesis via
holdover. Product isolation is facilitated by use of glycosyltransferase and peroxidase catalysis, respec-
linkages that provide a labile functionality.[8] Unfortu- tively,[11,12] peptide synthesis[13] and enantioselective
nately, many of the same attributes that make solid- ester hydrolysis using proteases[14] and esterases/lipas-
phase chemistry popular cannot be readily translated es,[15] respectively, and oligonucleotide synthesis via
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RNA ligase.[16] These reactions are either degradative served as substrates for specific oxidative enzymes. A
(e.g., hydrolytic) or are limited to specific oligomer variety of amine-functionalized supports were studied,
formation.
including amino-functionalized controlled pore glass
A gap in technology exists in the use of solid-phase (CPG) and the PEG-based resin TentaGelꢁ (cross-
biocatalysis for general synthetic transformations, and linked polystyrene containing grafted PEG). The sub-
in particular oxidative transformations. Such reactions strates were attached via a linker containing l-phenyl-
include phenolic coupling, as well as hydroxylation, alanine (l-Phe) that enabled hydrolytic release of the
epoxidation, halogenation, and alcohol oxidation, all reaction products from the solid support by a-chymo-
of which represent examples of diverse chemistries trypsin (CT) under ambient conditions. CT-catalyzed
that are difficult to achieve selectively using chemical cleavage allows mild hydrolysis of typically recalci-
catalysis. The current work is focused on expanding trant amide-based linkers.[11,17] Such a strategy was de-
the range of solid-phase biocatalysis through the use sirable to enable structural preservation of com-
of peroxidase, tyrosinase, and alcohol oxidase/dehy- pounds that might be unstable during acid or base
drogenase, all on solid-supported substrates. As a cleavage.[8a]
result, a new repertoire of solid-phase chemistries can
be performed under conditions ultimately amenable
to the synthesis of new chemical entities and the opti- Attachment and Cleavage of Linkers
mization of complex pharmaceutical lead compounds.
In initial studies with CPG, an Fmoc-l-Phe linker was
first attached to the surface of the support. Following
Fmoc removal, the phenylacetic/benzoic acid deriva-
tives p-hydroxyphenylacetic acid (HyPAA), p-
Results and Discussion
Our synthetic strategy involved the attachment of hydroxyACHTREmUGN ethyl-phenylacetic acid (HMPAA), and p-
phenylacetic or benzoic acid derivatives, each with a vinylbenzoic acid (VBA) were coupled to the l-Phe
different functional group at the para position, which moiety (Scheme 1). In the case of PEG-functionalized
Scheme 1. Attachment of solid-supported aromatic acids to CPG and TentaGelꢁ resins. Reagents and conditions: (a) N-Fmoc
amido dPEG4 (3 equiv.), DIC (4 equiv.), HOBt (6 equiv.), DMF, 608C; (b) 3% pyridine in AC2O, r.t.; (c) piperidine (20%, v/
v) in DMF, r.t.; (d) Fmoc-l-phenylalanine (3 equiv.), DIC (4 equiv.), HOBt (6 equiv.), DMF, 608C; (e) 3% pyridine in
AC2O, r.t.; (f) piperidine (20%, v/v) in DMF, r.t.; (g) 4-hydroxyphenylacetic acid or 4-(hydroxymethyl) phenylacetic acid
(3 equiv.), DIC (4 equiv.), HOBt (6 equiv.), DMF, 608C; (h) 4-vinylbenzoic acid (3 equiv.), DIC (4 equiv.), HOBt (6 equiv.),
DMF, 608C. Steps (a)–(c) were not carried out on TentaGelꢁ, the l-Phe-Fmoc was attached directly to the resin.
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Table 1. Enzymatic cleavage of solid-supported substrates from various supports.
Resin or Glass Type Linker Attachment[a] [mmol/g] % Attachment[b]
Substrate Cleavage [%][c]
l-Phe-VBA l-Phe-HyPAA l-Phe-HMPAA
CPG
-
117.0Æ10.0
64.0Æ3.3
80Æ6.8
44Æ2.2
100Æ5.5
0.40Æ0.03
17.0Æ2.2
3.0Æ0.8
3.0Æ0.4
44.0Æ1.4
1.0Æ0.3
5.0Æ0.2
66Æ5.4
-
CPG
PEG4
-
TentaGelꢁ
250.0Æ13.4
[a]
The amount of substrate attached was determined as described in the Results and Discussion.
Calculated as a percentage of the measured loading of amines on the resin/beads. All resin/bead types had different
amine loadings: CPG: 146 mmol/g, TentaGel: 250 mmol/g.
Cleavage expressed as a percentage of attached substrate recovered following cleavage with a-chymotrypsin. All values
are the mean of three independent determinations.
[b]
[c]
CPG, a PEG4 amino acid (containing four ethylene access the supported substrate. Thus, PEGA was not
glycol residues with a carboxyl group on one end and suitable for this study, which employed enzymes with
an Fmoc protected amine on the other) was first cou- molecular weights in the range of 37–118 kD. We
pled to the support followed by Fmoc removal and at- therefore employed CPG with a pore size of 500 Š,
tachment of the Fmoc-l-Phe linker. To fully charac- which has been reported to be accessible to higher
terize and quantify the degree of attachment and sub- MW enzymes .[20]
sequent cleavage of the bound substrates, the Kaiser
Low cleavage was obtained with CPG (<5% of the
ninhydrin assay was used to measure disappearance attached substrate in the case of HMPAA) (Table 1).
of amine groups present on the surface and within the While CPG has large pore sizes (500 Š), which are
pores of the beads and enable the facile quantification certainly sufficient for the 25 kD CT molecule, the
of bound PEG, Fmoc-l-Phe, and substrate, respective- limited distance of the substrates (bound to only an l-
ly. To avoid inaccurate results due to the possibility of Phe linker) from the support surface may limit acces-
incomplete reactions during each attachment step, the sibility of the substrate into the active site of CT. The
free amines were acetylated in between each step to added length of the PEG4 moiety (~20 Š including
block the residual amino groups (Scheme 1). Attach- the l-Phe linker), however, improved CT-catalyzed
ment of the three substrates onto TentaGelꢁ contain- substrate cleavage from the CPG for all three sub-
ing the Fmoc-l-Phe linker was 100% efficient strates, with the highest yield obtained for HMPAA
(Table 1). Substrate attachment onto PEG4-CPG was (Table 1). In addition, the PEG moiety may enhance
lower (~45%, Table 1). Yields of the individual at- the effective solubility of the bound substrate in the
tachment steps (Scheme 1) as determined by the dis- aqueous phase within the pores of CPG, thereby re-
appearance of available amino groups were 62% for sulting in greater substrate accessibility to the CT.[22]
the attachment of PEG4 to the CPG followed by Finally, PEG may act as an anti-protein binding sur-
Fmoc deprotection, and 70% for the subsequent at- face,[23,24] thus preventing CT or other enzymes used
tachment of the l-Phe linker to the PEG4.
in this work from binding to the pore surface and
The efficiency of CT-catalyzed cleavage was deter- blocking access of additional enzyme molecules to the
mined by quantifying the cleavage products via LC- bound substrate. PEG4-CPG, therefore, was chosen as
MS in comparison to chemically synthesized stand- the support for subsequent oxidative biotransforma-
ards. In the case of TentaGelꢁ very low cleavage was tion studies.
obtained (Table 1). This is consistent with previous
studies by Kress et al.[14b] who evaluated enzymatic
peptide cleavage from TentaGelꢁ. Low reactivity on Oxidative Biocatalysis on Solid-Supported Substrates
this support was postulated to be due to the poor ac-
cessibility of the enzyme into the pores of the sup- Oxidation reactions of solid-supported substrates
ports. TentaGelꢁ is known to poorly solvate in aque- were catalyzed by
a
diverse set of enzymes
ous solution, which substantially restricts the pore size (Scheme 2, Table 2), which included two peroxidases
and thus provides very little accessibility of the (soybean peroxidase for phenolic coupling and
enzyme to the bound substrate.[18,19] However, other chloro
ACHTREUNG
ACHTREUNG
reported to readily swell in aqueous solution. While oxidation. To facilitate direct comparison of reactivity
CT-catalyzed reactions have been successfully demon- among all enzymes and reactions, in all cases we
strated on PEGA-supported peptide substrates,[20,21] quantified the bound substrate conversion. To that
the resin does not allow enzymes larger than 35 kD to end, substrate conversion was determined by first
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Scheme 2. Enzymatic transformation of solid-supported substrates. Reagents and conditions: All enzymatic reactions were
performed with 50 mg of the appropriate functionalized resin. (a) Citrate buffer (50 mM, pH 5.0), CPO (10 mL, 10,000 U/
mL), 30 mM H2O2 (final concentration, infused at a rate of 0.1 mL/h for 10 h from a 90 mM stock), KCl (20 mM), r.t.; (b)
phosphate buffer (50 mM, pH 6.5), tyrosinase (0.004 mg/mL, 5,300 U/mg), ascorbic acid (5 equiv.), r.t.; (c) phosphate buffer
(100 mM, pH 8.0), SBP (0.2 mg/mL, 100 U/mg), tyramine (50 mM), 4 mM H2O2 (final concentration, infused at a rate of
0.1 mL/h for 10 h from a 20 mM stock), r.t.; (d) MOPS-Tris buffer (100 mM, pH 7.5), AAO (2 mg/mL, 0.1 U/mg), CuSO4
(0.4 mM), 308C; (e) Tricine-KOH buffer (100 mM, pH 9.0), ADH, (0.4 mg/mL, 0.5 U/mg), NADP+ (2 mg/mL), 308C.
Table 2. Solid-supported oxidative biotransformations.
Substrate[a]
Enzyme
Cosubstrate/Cofactor
Reaction
% Conversion[b]
1b R=CH2OH
1b R=CH2OH
1a R=OH
1a R=OH
1a R=OH
1c R=CH=CH2
1c R=CH=CH2
Alcohol dehydrogenase (ADH)
Aryl alcohol oxidase (AAO)
Tyrosinase
Soybean peroxidase (SBP)
Chloroperoxidase (CPO)
Chloroperoxidase (CPO)
Chloroperoxidase (CPO)
NADP+
H2O2
O2
Oxidation
Oxidation
Oxidation
Phenol coupling
Halogenation
Halohydration
Epoxidation
12Æ2.0
23Æ2.2
50Æ1.5
85Æ3.1
100Æ0
100Æ0
30Æ6.0
H2O2
H2O2 + KCl
H2O2 + KBr
H2O2
[a]
Compound nomenclature relates to Scheme 1.
Substrate conversion based on the amount of unreacted substrate (containing an l-Phe moiety). All values are the mean
[b]
of three independent reactions.
cleaving the l-Phe linker with CT followed by quanti- oxidative and CT catalysis versus CT catalysis alone.
fying the amount of unreacted substrate (containing To validate the effectiveness of this approach, we first
an l-Phe moiety) using HPLC. In the absence of CT, evaluated the reactivity of aryl alcohol oxidase
no l-Phe-linker cleavage was observed. In the pres- (AAO, 80 kD) and alcohol dehydrogenase (ADH,
ence of CT alone, only the unreacted substrate (at- 100 kD) catalysis on HMPAA (Scheme 2, a), which
tached to the l-Phe moiety) was observed. This ap- provides an opportunity to measure independently
proach provides an effective conversion based on the the formation of co-products (NADPH for ADH and
comparison of substrate cleaved following combined H2O2 for AAO). Substrate conversions (after 16 h in-
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Figure 1. Soybean peroxidase-catalyzed radical coupling of tyramine to solid-supported HyPAA.
cubations) to the aldehydic product were 23 and 12% KCl and H2O2 as co-substrates gave complete conver-
for AAO and ADH, respectively, based on the sion to three distinct ring chlorination products, in-
amount of HMPAA bound to CPG (Table 2). Impor- cluding mono- and dichlorinated HyPAA and a mon-
tantly, the AAO-catalyzed formation of H2O2 and the ochlorinated dimer (Scheme 3). This dimeric product
ADH-catalyzed formation of NADPH as measured was intriguing, as its formation could only come
after 16 h matched the HMPAA conversion as deter- about through intermolecular ring coupling through
mined via the HPLC technique. Thus, the HPLC ap- two separate linkages within a single bead. The pres-
proach and the direct product formation (in the case ence of the PEG4 linker likely aided efficient contact
of ADH and AAO catalysis) were consistent.
of adjacent phenolic substrates within the pore to
Tyrosinase (118 kD) catalysis on bound HyPAA re- enable phenolic coupling by CPO. The high yields of
sulted in conversion of ca. 50% of the bound sub- SBP and CPO catalysis were most likely facilitated by
strate (Table 2). In the presence of ascorbic acid to the formation of diffusible products that are able to
reduce the quinone products from the tyrosinase reac- access bound HyPAA: phenoxy radicals for SBP[12]
tion, cleavage resulted in identification of the catechol and HOCl for CPO.[25] These products are able to ef-
products. All three enzymes require direct contact fectively diffuse from the enzymes within the CPG to
with the bound substrate to catalyze the oxidation re- the bound HyPAA (Figure 1 for SBP).
action. Therefore, the pores may restrict access or
hinder mobility of these enzymes to only the most ac- radical-dependent reactions. Therefore, to assess the
cessible PEG4-CPG bound substrate molecules. ability of CPO to catalyze reactions that require
CPO is also capable of catalyzing non-diffusible
Unlike the aforementioned enzymes, peroxidases direct contact with the substrate, epoxidation reac-
do not require direct contact of the enzyme with the tions were performed using VBA in the presence of
bound substrate (Figure 1). As a result, biotransfor- H2O2 (Table 2, Scheme 4,). As shown in Table 2, the
mations involving phenolic coupling [catalyzed by conversion to the epoxide product was 30%. Con-
soybean peroxidase (SBP), 37 kD] and ring halogena- versely, in the presence of KBr, which is converted
tions [catalyzed by chloroperoxidase (CPO), 42 kD] into diffusible HOBr, complete conversion of the
resulted in high conversions (Scheme 2, Table 2) Spe- VBA to the halohydrin product was obtained
cifically, SBP-catalyzed phenolic coupling of tyramine (Table 2, Scheme 4). This result confirmed that a reac-
in the presence of H2O2 resulted in the formation of tion mechanism consisting of a diffusible product can
coupled tyramine residues bound to HyPAA. Cleav- be more efficient on solid phase substrates than a
age with CT provided ca. 85% conversion (Table 2). mechanism that requires direct contact of the sub-
Similarly, CPO-catalyzed chlorination of HyPAA with strate with the enzyme.
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Scheme 3. CPO-catalyzed halogenation of bound HyPAA [putative structures based on LC-MS analysis]. (a) Mono- and di-
chlorination of solid-supported HyPAA. (b) Schematic representing the formation of dimeric chlorinated products within
the pore of a single bead followed by cleavage. The dashed hemispherical line indicates a pore of CPG containing bound
HyPAA groups. Reagents and conditions: All enzymatic reactions were performed with 50 mg of the appropriate functional-
ized resin. (i) Citrate buffer (50 mM, pH 5.0), CPO (10 mL, 10,000 U/mL), 30 mM H2O2 (final concentration, infused at a
rate of 0.1 mL/h for 10 h from a 90 mM stock), KCl (20 mM), r.t.; (ii) ammonium bicarbonate buffer (100 mM, pH 7.8), a-
chymotrypsin (5 mg/mL, 34 U/mg), 308C.
Scheme 4. Chloroperoxidase-mediated transformation of solid-supported 4-vinylbenzoic acid. Reagents and conditions: All
enzymatic reactions were performed with 50 mg of the appropriate functionalized resin. (a) Citrate buffer (50 mM, pH 5.0),
CPO (10 mL, 10,000 U/mL), 30 mM H2O2 (final concentration, infused at a rate of 0.1 mL/h for 10 h from a 90 mM stock),
KBr (20 mM), r.t.; (b) citrate buffer (50 mM, pH 5.0), CPO (10 mL, 10,000 U/mL), 30 mM H2O2 (final concentration, infused
at a rate of 0.1 mL/h for 10 h from a 90 mM stock), r.t..
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Interestingly, the halohydrin product expected from General Procedure for the Preparation of CPG-
CPO catalysis in the presence of KBr and H2O2 was Bound-PEG4
not initially observed; rather, the corresponding epox-
ide was generated (Scheme 4). Such epoxide forma-
loading of amine groups on the beads. 1-Hydroxybenzotri-
All reagent equivalents are stated with respect to the stated
tion may have been due to the CT-catalyzed cleavage
conditions at pH 7.8, which promote the well-known
nucleophilic dehalogenation of the halohydrin to
form the epoxide.[26] To eliminate this non-enzymatic
transformation, the CT-catalyzed cleavage reaction
was performed at pH 5 (CT has low but measurable
activity at this pH), which resulted in the formation
of the expected halohydrin as the sole product
(Scheme 4). Hence, in the presence of KBr, CPO cat-
alyzed the expected formation of the HOBr product,
which diffused to the vinyl moiety and, depending on
the cleavage conditions, remained as the halohydrin
or is dehalogenated to the epoxide (Scheme 4). This
synthetic scheme, which is facilitated by retention of
the halohydrin product on the solid phase, did not
lead to the undesired aldehyde product. Hence, such
a chemo-enzymatic strategy may be synthetically
useful in the preparation of complex natural product
epoxides.
AHCTREaUNG zole (HOBt, 6 equiv.), N,N’-diisopropylcarbodiimide (DIC,
4 equiv.), and N-Fmoc amido dPEG4 (3 equiv.) were added
to a suspension of aminopropyl-CPG resin (1 g, 0.146 mmol/
g) in DMF (10 mL). The mixture was shaken at 200 rpm for
16 h at 608C and then filtered. The resin was washed with
DMF (3”) and the procedure was repeated. After the
second cycle, the resin was filtered and then washed with
DMF, MeOH, and acetone (3” each). The unreacted amino
groups of the resin were capped with a solution of 3% (v/v)
pyridine in acetic anhydride (10 mL) at room temperature
overnight. After filtration, the resin was washed with
MeOH and acetone (3” each). Deprotection of the Fmoc
group was carried out in piperidine [10 mL, 20% (v/v) in
DMF] at room temperature for 18 h and filtered. The resin
was washed with DMF, MeOH, and acetone (3” each).
General Procedure for the Preparation of Polymer-
Bound l-Phenylalanine
All reagent equivalents are stated with respect to the stated
loading of amine groups on the beads. HOBt (6 equiv.), DIC
(4 equiv.), and l-Fmoc-Phe (3 equiv.) were added to a sus-
pension of the appropriate amino-functionalized resin
(0.146–0.25 mmol/g) in DMF (10 mL). The mixture was
shaken at 200 rpm for 16 h at 608C. The resin was washed
with DMF (3”) and the procedure was repeated. After the
second cycle, the resin was filtered and then washed with
DMF, MeOH, and acetone (3” each). As described above,
the unreacted amino groups of the resin were capped with a
solution of 3% (v/v) pyridine in acetic anhydride (10 mL) at
room temperature overnight. After filtration and washing,
Fmoc deprotection was achieved as described above.
Conclusions
In conclusion, we have demonstrated that a range of
enzymatic oxidation reactions can be performed on
solid-supported substrates. In the process, we have es-
tablished that a combination of CPG and a PEG
linker yields relatively high biocatalytic conversions
on the solid phase relative to non-PEGylated CPG or
non-swellable polymeric resins. The PEG4-CPG facili-
tates the use of a diverse set of enzymes and serves to
further expand the feasibility of solid-phase biocataly-
sis as a synthetic strategy. Finally, we have shown that
General Procedure for the Attachment of Substrates
to Resins
the enzymatic cleavage conditions may alter the out- All reagent equivalents are stated with respect to the stated
loading of amine groups on the beads. After attachment of
l-Phe, the substrate (HyPAA, HMPAA, or VBA, 3 equiv.)
was coupled to the resin in the presence of DIC (4 equiv.)
and HOBt (6 equiv.) in DMF (10 mL). The mixture was
shaken at 200 rpm for 16 h at 608C, filtered, and washed
with DMF (3”), and the procedure was repeated. After fil-
tration, the resin was washed with DMF, MeOH and ace-
tone (3” each).
come of a given reaction, in this case a CPO-catalyzed
halohydration reaction, when compared to a corre-
sponding reaction in solution phase.
Experimental Section
Amino-functionalized controlled pore glass (CPG, 146 mmol
NH2/g) was purchased from Biosearch Technologies
(Novato, CA). TentaGelꢁ was acquired from Rapp-Polyme-
re (Tübingen, Germany). N-Fmoc amido dPEG4 acid was
purchased from Quanta Biodesign (Powell, OH). SBP, CPO,
tyrosinase, CT, p-(hydroxymethyl)phenylacetic acid, p-hy-
droxyphenyl acetic acid, and p-vinylbenzoic acid were ob-
tained from Sigma–Aldrich (St. Louis, MO). Fmoc-Phe-OH
was obtained from Fluka (Buchs, Switzerland). Alcohol de-
hydrogenase 102 and aryl alcohol oxidase 102 were obtained
from Codexis (Pasadena, CA). Chemical and solvents were
of the highest grade commercially available and were pur-
chased from Sigma–Aldrich and Acros (Geel, Belgium).
Cleavage of Bound Substrates
Enzymatic hydrolysis of bound substrates from the resins
was performed with CT catalysis. The resin (50 mg) was sus-
pended in ammonium bicarbonate buffer (100 mM, pH 7.8)
in the presence of lyophilized CT (5 mg/mL, 34 U/mg). The
reaction mixture was shaken at 200 rpm for 24 h at 308C. At
the end of the reaction, the mixture was filtered off the
beads by passing the liquid through a membrane filter
(0.2 mm). The cleaved product was analyzed using LC-MS
(Shimadzu LC-MS 2010 AD equipped with a SPD 20AV de-
tector and LC-MS 2010 A mass spectrometer). An Alltech
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(Deerfield, Il, USA) ODS C18 column was used with a gra-
dient elution profile consisting of 0.2% formic acid:CH3CN
95:5 to 10:90 (30 min) then back to 95:5 (10 min) and finally
maintaining for 5 min at a flow rate of 0.2 mL/min. Product
amounts were calculated by using calibration curves of syn-
thesized standard products where available.
of
2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic
acid
(ABTS) and 100 mL of a 6 mg/mL of SBP in a microwell
plate. In the presence of H2O2 produced as a result of AAO
oxidation of bound HMPAA, SBP oxidizes ABTS resulting
in a colored product, which is then measured at 405 nm.
Preparation of l-Phenylalanine Derivatives
Enzymatic Reactions on Solid-Supported Substrates
l-Phenylalanine methyl ester hydrochloride (108 mg,
0.5 mmol) and the phenylacetic/benzoic acid derivative
(0.5 mmol) were dissolved in 6 mL DMF:CH2Cl2 (1:3). The
mixture was sequentially treated at 08C with NaHCO3
(42 mg, 0.5 mmol), HOBt (75 mg, 0.55 mmol), and EDC
(106 mg, 0.55 mmol). The reaction mixture was stirred at
08C for 2 h and allowed to warm to room temperature. Stir-
ring was continued overnight and then the reaction was
quenched with 15 mL H2O followed by extraction of the
aqueous phase with EtOAc (3”10 mL), after which the
combined organic phases were washed with HCl 0.1M (2”
10 mL), H2O (10 mL) and brine (10 mL), dried over MgSO4,
and concentrated under vacuum. The crude product was pu-
rified by column chromatography (CH2Cl2:EtOAc, 6:4).
Hydrolysis of the methyl esters was performed as follows.
To the purified l-phenylalanine methyl ester derivative
(0.4 mmol) was added 1 mL KOH 1M in MeOH. Following
stirring overnight at room temperature, the solvent was
evaporated under reduced pressure, and 10 mL water were
added to the residue. The mixture was extracted with diethyl
ether (2”10 mL) and the organic phase was discarded. The
aqueous phase was acidified with HCl 1M, extracted with
diethyl ether (3”10 mL), dried over MgSO4, and concen-
trated under vacuum. Please see Supporting Information for
spectroscopic data.
Enzymatic reactions were performed with 50 mg of the ap-
propriate functionalized resin in 20-mL vials. For ADH-cat-
alyzed reactions, the resin-bound HMPAA (50 mg) was sus-
pended in 5 mL Tricine-KOH buffer (100 mM, pH 9.0) in
the presence of the enzyme (0.4 mg/mL, 0.5 U/mg) and
NADP+ (2 mg/mL). The mixture was shaken at 200 rpm and
308C for 16 h. For AAO-catalyzed reactions, the resin-
bound HMPAA (50 mg) was suspended in 5 mL MOPS-Tris
buffer (100 mM MOPS, 100 mM Tris titrated to a pH of 7.5)
in the presence of CuSO4 (0.4 mM) and AAO (2 mg/mL,
0.1 U/mg) followed by stirring at room temperature for 16 h.
Tyrosinase-catalyzed reactions were performed in 5 mL
sodium phosphate buffer (50 mM, pH 6.5) with resin-bound
HyPAA (50 mg) in the presence of tyrosinase
(0.004 mgmLÀ1, 5,300 U/mg). In some cases, ascorbic acid
(5 equiv. of substrate concentration) was added to the reac-
tion mixture. The reactions were mixed at room tempera-
ture for 16 h. For SBP-catalyzed carbon-carbon coupling re-
actions, the resin-bound HyPAA (50 mg) was suspended in
5 mL sodium phosphate buffer (100 mM, pH 8.0) containing
SBP (0.2 mg/mL, 100 U/mg) and tyramine (50 mM). The re-
action mixture was stirred at room temperature and 1 mL of
20 mM H2O2 was infused at a rate of 0.1 mL/h for 10 h using
a syringe pump (4 mM H2O2 final concentration). Finally,
for CPO-catalyzed reactions, resin-bound HyPAA or VBA
(50 mg) was suspended in 2 mL sodium citrate buffer
(50 mM, pH 5.0) containing CPO (5 mL/mL, 10,000 U/mL)
and in the presence or absence of KBr (20 mM). The reac-
tion mixture was stirred at room temperature and 1 mL of
90 mM H2O2 was infused at a rate of 0.1 mL/h for 10 h using
a syringe pump (30 mM H2O2 final concentration). At the
end of each enzymatic reaction, the resins were filtered and
washed with H2O and acetone (3” each), dried, and weigh-
ed. The products were then cleaved from the resin as previ-
ously described.
Acknowledgements
This work was supported by the National Institutes of Health
(GM66712).
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Enzymatic reactions were performed with 50 mg of the ap-
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