Two-step enzymatic modification of solid-supported bergenin in aqueous and organic media

Article abstract of DOI:10.1016/j.tetlet.2009.12.119

The natural flavonoid bergenin was directly immobilized onto carboxylic acid-functionalized controlled pore glass (carboxy-CPG) at 95% yield. Immobilized bergenin was brominated via chloroperoxidase in aqueous solution and then transesterified with vinyl butyrate in diisopropyl ether by subtilisin carslberg (SC) extracted into the organic solvent via ion-pairing. Enzymatic cleavage of 7-bromo-4-butyrylbergenin from carboxy-CPG (9.6% final yield) was accomplished using lipase B (LipB) in an aqueous/organic mixture (90/10 v/v of water/acetonitrile), demonstrating the feasibility of solid-phase biocatalysis of a natural product in aqueous and non-aqueous media.

Full text of DOI:10.1016/j.tetlet.2009.12.119

Tetrahedron Letters 51 (2010) 1220–1225
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Two-step enzymatic modification of solid-supported bergenin in aqueous
and organic media
a
a
a,b
c
a,*
Umar Akbar , Dong-Sik Shin , Elizabeth Schneider , Jonathan S. Dordick , Douglas S. Clark
a
Department of Chemical Engineering, University of California, Berkeley, Berkeley, CA 94720, United States
UCSF/UCB Joint Graduate Group in Bioengineering, Berkeley, CA 94720, United States
Department of Chemical and Biological Engineering, of Biology, and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy,
NY 12180, United States
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The natural flavonoid bergenin was directly immobilized onto carboxylic acid-functionalized controlled
pore glass (carboxy-CPG) at 95% yield. Immobilized bergenin was brominated via chloroperoxidase in
aqueous solution and then transesterified with vinyl butyrate in diisopropyl ether by subtilisin carslberg
Received 12 November 2009
Revised 17 December 2009
Accepted 18 December 2009
Available online 28 December 2009
(SC) extracted into the organic solvent via ion-pairing. Enzymatic cleavage of 7-bromo-4-butyrylbergenin
from carboxy-CPG (9.6% final yield) was accomplished using lipase B (LipB) in an aqueous/organic mix-
ture (90/10 v/v of water/acetonitrile), demonstrating the feasibility of solid-phase biocatalysis of a natu-
ral product in aqueous and non-aqueous media.
Keywords:
Solid Phase
Biocatalysis
Non-aqueous
Enzyme cleavage
Ó 2009 Elsevier Ltd. All rights reserved.
The ability of enzymes to efficiently carry out highly specific
reactions on a broad range of substrates has been exploited in
many diverse applications, such as pharmaceutical development,
leads and other specialized molecules, taking advantage of the
3
1
32
novel synthesis opportunities, tunable specificity, and selectiv-
3
3,34
ity
afforded by such systems.
Successful implementation of this methodology requires two
1
–5
protein engineering, and polymer templating and synthesis.
In
the pharmaceutical industry, biocatalysts have been used to gener-
ate highly focused lead compound libraries. One approach is
termed ‘combinatorial biocatalysis,’ which has been employed in
both aqueous and non-aqueous media as a complement to combi-
natorial chemistry and rational drug design for the optimization of
pharmaceutical compounds.6 Over the past decade, there has
been a concerted effort to combine this concept with solid-phase
chemistry to merge the selectivity of biocatalysis with the high
throughput conferred by solid-phase synthesis.9 Specific areas of
interest include enzymatic modification of immobilized peptides
important elements: enzyme-accessible supports, and for the par-
ticular case of non-aqueous biocatalysis, organic-soluble enzymes.
To satisfy the first requirement, we employed controlled pore glass
(CPG), a material commonly used for enzyme immobilization. CPG
possesses a large, open pore structure (in contrast to supports that
–8
24
require pre-swelling prior to use), is relatively easy to handle,
and, most importantly, provides more favorable access to enzyme
2
4
than many other supports.
Solid-phase non-aqueous enzymology presents the additional
requirement that the enzyme, which is typically insoluble in neat
organic solvents, first be made soluble and active in the desired
and other small molecules in aqueous1
0–14
and non-aqueous
15
media, enzymatic screening of combinatorial libraries of immobi-
solvent. Although there are a variety of methods for solubilizing
1
6–21
35–38
lized small molecules,
substrate removal via enzyme-cleav-
enzymes into organic solvents,
based on previous solubiliza-
1
2,22–25
able linkers,
versus solution-phase modifications.
reactions on insoluble, solid-supported substrates have been car-
and preliminary kinetic comparisons of solid
tion studies surfactant ion-pairing via liquid–liquid extraction
15,26–28
30,39
30
To date, enzymatic
(EXT)
and direct solubilization (DS) are particularly promis-
ing. Specific advantages of these methods include ease of prepara-
tion and high activity in either a particular solvent (extraction) or
across a range of solvents exhibiting different polarities (direct
solubilization).
The present work helps to establish a foundation for solid-phase
combinatorial biocatalysis as a drug development tool. In particu-
lar, we describe a means for directly immobilizing potential lead
compounds without the use of a linker, provide the first example
of non-aqueous solid-phase biocatalysis with a molecule other
ried out primarily in aqueous solution.2
9a,b
However, with the re-
cent development of a new protein solubilization method that
has broadened the utility of non-aqueous enzymology,30 we hope
to extend the capabilities of solid-phase biocatalysis to include
non-aqueous modification of solid-supported pharmaceutical
*
Corresponding author. Tel.: +1 510 642 2408; fax: +1 510 643 1228.
E-mail address: clark@berkeley.edu (D.S. Clark).
0
040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.119

U. Akbar et al. / Tetrahedron Letters 51 (2010) 1220–1225
1221
than a peptide, and present a method for regioselectively removing
potential lead compound derivatives from a solid support in non-
aqueous media.
Boc-L-Phe (Table 1). The HATU/DIPEA combination yielded the
highest solution coupling efficiency for the esterification reaction
(71%) and therefore was used to attach bergenin to Phe-CPG. The
Bergenin was directly immobilized onto carboxylic acid-func-
tionalized controlled pore glass (carboxy-CPG) at 95% yield accord-
ing to Scheme 1. Immobilization yields were determined by
measuring the disappearance of bergenin from solution via LC/MS.
To quantify the degree of immobilization at the primary 11-hydro-
xyl versus secondary hydroxyl groups (most likely the 11-hydroxyl
position due to reduced steric hindrance), bergenin was coupled to
maximum loading of bergenin to the Phe-CPG was 34.2 lmol/g
(53% coupling yield), as determined by monitoring the reaction
progress with LC/MS. Removal of attached bergenin was accom-
2 4
plished using H SO , SC, and CT (Table 2). Cleavage with CT gave
a 9.3% cleavage yield, which was two times higher than SC
(4.7%). Although this result shows the feasibility of using a Phe lin-
ker for selective cleavage of bergenin using CT, this yield was lower
than that obtained using solubilized lipase B (lipB) or SC to remove
bergenin directly from carboxy-CPG in organic solvents. Thus, Phe-
CPG was not employed in subsequent solid-phase biocatalytic
reactions.
Solid-supported bergenin was halogenated by chloroperoxidase
(CPO) as the first step of a two-step enzymatic modification
(Scheme 3). When performed for ꢀ20 h, halogenation conversions
of nearly 70% were obtained with solution phase bergenin.
Although it was not expected that conversions for solid-phase
bergenin halogenation would be as high, successful aqueous phase
modifications of immobilized natural products could prove to be
particularly useful for substrates that are poorly soluble in water,
as is typically the case with many potential pharmaceutical lead
compounds.
In the case of solid-supported bergenin, halogenation was car-
ried out under similar conditions to the solution phase reaction,
with the following modifications: the CPO/bergenin and KBr/
bergenin ratios were increased by approximately 70- and 25-fold,
respectively. Under these conditions, a solid-phase halogenation
conversion of 5.9% was obtained, thereby demonstrating the ability
of the CPO to access at least a portion of the solid-supported berge-
nin. Efforts to improve this conversion included the following: (1)
Boc-L-phenylalanine, a model compound containing a carboxylic
acid group mimicking that of carboxy-CPG. Based on LC/MS analysis
of the modified sample, the ratio of primary/secondary hydroxyl
coupling was ꢀ2:1. In addition, the large pore size (500 Å) of car-
boxy-CPG, along with the formation of an enzyme-cleavable ester
bond between bergenin and the support, suggested that enzymatic
cleavage of immobilized bergenin and its potential derivatives
should be possible.
As an alternate immobilization method, bergenin was coupled
to phenylalanine-CPG with the aim of using
a-chymotrypsin (CT)
to selectively cleave bergenin from the support. Previous studies
suggested that CT can be used in solid-phase synthesis to cleave
4
0,25
compounds from a solid support via a phenylalanine linker.
Bergenin was attached to and cleaved from Phe-CPG according to
Scheme 2. For the generation of Phe-CPG, two silanization re-
agents, triethoxysilylpropanal (C3 silane) and triethoxysilyldecanal
(
C10 silane), were used to generate aldehyde groups on CPG and
provide either a C3 or C10 linker between the surface of the CPG
and the aldehyde group. -Phe was then coupled to the aldehyde
CPG. Both 400 Å and 900 Å pore-size CPG were employed in order
L
4
1
to ascertain the effect, if any, of pore size on Phe loading. The
loading of Phe on both C3 and C10 CPG was determined using
elemental analysis by measuring the difference in carbon and
nitrogen levels before and after reaction with Phe.
increasing the H
2 2
O /CPO ratio (4–8-fold), (2) running consecutive
halogenation reactions with complete removal and replenishment
of enzyme solution between reactions (up to six times), and (3) en-
In the case of the C10 linker, no Phe attachment was detected
for either pore-size CPG, possibly due to crowding by the compar-
atively long linker within the smaller (400 Å) pores, and the smal-
ler overall surface area for the larger pore (900 Å) CPG. To preclude
both possible problems, subsequent reactions employed the 400 Å
CPG functionalized by the C3 silane. Attachment of Phe to the
zyme pre-incubation (at 4, 25 °C) in the absence of H
96 h). It was thought that allowing the enzyme to diffuse through
the porous CPG network prior to H addition may enable better
access of CPO to the solid-supported bergenin before enzyme inac-
tivation by H . Of these measures, only the enzyme pre-incuba-
2 2
O (18–24 or
2 2
O
2 2
O
modified CPG resulted in an amino acid loading of 63.9
l
mol/g
tion (at either temperature for ꢀ20 h) afforded any significant
increase in solid-phase halogenation conversion—nearly a three-
fold increase from 5.9% to 17.3% indicating that slow diffusion of
the enzyme into the CPG pores is limiting.
according to elemental analysis. Bergenin coupling onto the
Phe-CPG was carried out using conditions analogous to those used
for the solution phase coupling reaction between bergenin and
Scheme 1. Bergenin immobilization onto carboxy-terminated controlled pore glass (carboxy-CPG).

1
222
U. Akbar et al. / Tetrahedron Letters 51 (2010) 1220–1225
Scheme 2. Immobilization of bergenin on L-phenylalanine-functionalized controlled pore glass (Phe-CPG) and cleavage by a-chymotrypsin.
Table 1
products in both aqueous and non-aqueous media, which could
serve to be a valuable tool for compound generation and lead opti-
mization in drug discovery.
Reaction yields for coupling of bergenin to Boc-L-Phe in solution using various
coupling reagents
Reagent
Yield (%)
Extraction: Native protein (i.e., taken directly from the bottle
without further modification) was dissolved in aqueous buffer
DIC, HOBt, DMAP
DIC, DMAP
HATU, DIPEA
BrOP, DIPEA
21
46
71
1.5
11
(SC: 10 mM bis-Tris Propane with 2 mM CaCl
2
, pH 7.8; LipB:
1
0 mM bis-Tris Propane with 300 mM NaCl, pH 6.0) at 1 mg/mL
and then added to an equivalent volume of 2 mM sodium bis(2-
ethylhexyl) sulfosuccinate (AOT) dissolved in isooctane. The vial
was vortexed for 10 s and placed in an orbital shaker at 30 °C
and 250 rpm for 45 min. The biphasic mixture was then immedi-
ately centrifuged at 2700g for 5 min to separate the two phases.
Following phase separation, the organic phase was removed and
evaporated to dryness by a flowing air stream and the concentra-
tion of the enzyme/surfactant complex in the organic phase was
determined spectrophotometrically by scanning from 220 to
PyBrOP, DIPEA
Table 2
Cleavage yields for bergenin immobilized on Phe-CPG using different reaction
conditions
Reaction conditions
Yield (%)
1
2
2
M H
2
SO
4
(aq)
48.9
4.7
9.4
0 mg/ml SC in aqueous buffer
0 mg/ml CT in aqueous buffer
3
20 nm, recording the peak absorbance at 280 nm, and subtracting
a baseline absorbance reading at 310 nm. The extinction coefficient
for protein solubilized in organic solvent was experimentally
determined to be the same as for protein dissolved in H
2
O.
A major advantage of solid-phase chemistry and biocatalysis
over solution-phase reactions is the ability to perform sequential
reactions without purification of intermediate products from the
reaction mixture. To demonstrate that solid-phase biocatalysis
can be used to carry out sequential reactions on immobilized sub-
strates in multiple solvents, the halogenated bergenin derivative
was subsequently acylated by EXT-SC in diisopropyl ether (IPE)
with 500 mM excess vinyl butyrate. EXT-SC was selected over its
DS counterpart based on its higher solution phase activity (nearly
twofold higher initial rate) in IPE, the solvent in which the highest
overall EXT-SC activity was observed (Table 3). The resulting
product was cleaved from the CPG via concentrated LipB dissolved
in aqueous buffer in both 0% and 10% acetonitrile (ACN) co-solvent
Direct solubilization: Directly solubilized proteins were prepared
3
0
according to the procedure described by Akbar et al. In particular,
a small amount of nano-filtered water (0.25% v/v for SC, 0.70% v/v
for LipB) was added to a flame-dried vial, followed by addition of
2 mM AOT dissolved in isooctane. Native protein was added to
the isooctane/water mixture to yield a final concentration of
1 mg/mL suspended enzyme and the contents were magnetically
stirred for ca. 45 min. After stirring, the vial was centrifuged at
2700g for 5 min and the supernatant was transferred to a separate
vial and evaporated to dryness before being dissolved in the final
reaction solvent.
Bergenin immobilization and cleavage on CPG: In a flame-dried
vial, 30 mM bergenin was dissolved in a 1:1 (v/v) DMF:DCM
solution, followed by addition of 1 equiv 1-hydroxybenzotriazole
(HOBT), 0.2 equiv 4-dimethyl aminopyridine (DMAP), and
0.25 equiv carboxy-CPG. After allowing the resin to settle to the
bottom of the vial, the liquid above the resin was sampled for LC
analysis of initial bergenin concentration and 1 equiv 1,3-diisopro-
pyl carbodiimide (DIC) was added to initiate the immobilization
(
7
(
Table 4), resulting in a ꢀ10% post-cleavage recovery yield of
-bromo-4-butyrylbergenin under optimal cleavage conditions
10% ACN). To our knowledge, this represents the first example
of a two-step aqueous:organic biocatalytic reaction sequence for
modifying a solid-supported natural product. This approach can
likely be expanded to produce libraries of solid-supported natural

U. Akbar et al. / Tetrahedron Letters 51 (2010) 1220–1225
1223
µ
Scheme 3. Two-step enzymatic modification of solid-supported bergenin via halogenation by chloroperoxidase in aqueous solution followed by acylation via EXT-SC in IPE.
Table 3
found in the 2003–2004 Advanced ChemTech combinatorial
chemistry handbook.
4
2
Initial rates of product formation for solution phase bergenin transesterification with
vinyl butyrate catalyzed by directly solubilized (DS), extracted (EXT), and native
subtilisin carlsberg (SC) in isooctane or diisopropyl ether
Bergenin immobilization and cleavage on Phe-CPG: To prepare
aldehyde CPG (CPG-CHO), CPG was added to a solution of 5%
(v/v) triethoxysilylpropanal or triethoxysilyldecanal in chloroform.
The reaction vial was incubated at 50 °C and 250 rpm for 12 h.
After 12 h, the supernatant was filtered out and the CPG was
washed/filtered four times each in excess chloroform and then
MeOH. CPG-CHO was dried under reduced pressure at room tem-
perature prior to further use. For the Phe reaction, 1 equiv CPG-
À1
Enzyme preparation
Reaction solvent
Initial rate (mM min
)
DS-SC
DS-SC
EXT-SC
EXT-SC
Native SC
Native SC
Isooctane
Diisopropyl ether
Isooctane
Diisopropyl ether
Isooctane
Diisopropyl ether
0.088
0.62
0.0035
1.2
0.88
0.36
CHO and 5 equiv
3
L-Phe in water and 5 equiv NaBH (CN) in DMF
(
1:1 water/DMF (v/v)) were mixed together and the vial was incu-
bated at 25 °C and 250 rpm for 12 h. After 12 h, the supernatant
was filtered out and the Phe-CPG was washed/filtered four times
each in excess DMF, DCM and MeOH. Phe-CPG was dried under a
nitrogen stream and stored under reduced pressure at room tem-
perature. Bergenin coupling to the Phe-CPG was carried out using
HATU/DIPEA as coupling reagents. To a vial containing 1 equiv
Phe-CPG, 5 equiv bergenin and 5 equiv DIPEA in a 1:1 DMF/DCM
(v/v) solution was added and incubated at 25 °C and 250 rpm for
12 h. The supernatant was then filtered out and the Bergenin-
Phe-CPG was washed/filtered four times each in excess DMF,
DCM and MeOH. Each reaction step was monitored by the colori-
metric detection of relevant functional groups on the surface of
the CPG, using the reagent Purpald for aldehydes, Malachite Green
for carboxyl acids, and chloranil for secondary amino groups.4
For quantitative analysis of the immobilized phenylalanine and
bergenin on CPG, elemental analysis was performed on CPG after
drying under reduced pressure for 24 h. For solution phase reac-
Table 4
Cleavage yields for multiple products resulting from cleavage reaction of 7-bromo-4-
butyrylbergenin immobilized on carboxy-CPG using either aqueous Lipase B or Lipase
B in 10% ACN co-solvent
Cleavage product
Yield (%) in
aqueous buffer
Yield (%) in 10%
ACN (v/v)
7
7
4
-Bromo-4-butyryl bergenin
-Bromobergenin
-Butyrylbergenin
5.0
10.5
17.7
9.6
7.8
16.6
Total cleavage yield (%)
94.7
48.0
3–47
reaction. Reaction vials were incubated for ꢀ50 h at 25 °C,
1
00 rpm.
After 50 h, the liquid above the resin was again sampled for LC
analysis and the reaction was quenched by vacuum filtration of
the solvent, followed by four washes/filtrations each with excess
ACN, MeOH, DMF, and DCM. Immobilized substrate-containing
resins were stored under vacuum at room temperature prior to
further use. Additional control experiments were also carried
out to verify the absence of bergenin side reactions and to quan-
tify the amount of bergenin physically adsorbed to the carboxy-
tions, 10 mM bergenin was coupled to 10 mM Boc-L-Phe using
either DIC (1 equiv), HOBt (1 equiv), DMAP (0.2 equiv), or DIC
(1 equiv), DMAP (0.2 equiv), or HATU (1 equiv), DIPEA (2 equiv),
or BrOP (1 equiv), DIPEA (2 equiv), or PyBrOP (1 equiv), DIPEA
(2 equiv). For bergenin cleavage reactions, bergenin immobilized
on the Phe-CPG was added directly to a solution containing either
1 M aqueous sulfuric acid, 20 mg/mL of SC (220 U/mg) in 100 mM
CPG surface. Bergenin was coupled to Boc-
L
-phenylalanine in
solution using 80 mM each of bergenin and Boc-
L
-phenylalanine
K
K
2
HPO
HPO
4
at pH 7.5 or 20 mg/mL of CT (1600 U/mg) in 100 mM
at pH 8.0. For H SO cleavage, the reaction was run at
and the same molar equivalents of all remaining reagents. The
reaction conditions for bergenin immobilization were modified
from conditions for alcohol attachment onto carboxylic acid resins
2
4
2
4
45 °C and 100 rpm for 24 h, followed by quenching via 1 equiv
base. When native enzyme in aqueous buffer was used, the

1
224
U. Akbar et al. / Tetrahedron Letters 51 (2010) 1220–1225
reaction was run at 45 °C and 100 rpm for 48 h (SC) or at 37 °C and
00 rpm for 24 h (CT). Following each cleavage reaction, the filtrate
was analyzed via LC/MS for the presence of bergenin.
cleavage with concentrated LipB (ꢀ75 mg/mL) dissolved in
50 mM potassium phosphate (pH 7.0) with 0–30% ACN co-solvent,
60–70 mg/mL resin was incubated at 45 °C 180 rpm for 20–30 h.
Following completion of each enzymatic cleavage reaction, DMF
was added to the reaction vials to help dissolve the cleaved berge-
nin/derivative (ꢀ20–25% v/v DMF in final mixture), followed by re-
incubation at 45 °C and 180 rpm for 30 min, centrifugation at
2700g for 5 min, and sampling of the supernatant for LC/MS anal-
ysis. When analyzing the LC/MS results, the calculated yields of the
4-butyryl bergenin derivative were multiplied by 1.5 to account for
the fact that approximately 33% of the bergenin initially immobi-
lized had been attached at the 4-hydroxyl position—thereby pre-
cluding formation of the 4-acylbergenin derivative—instead of
the 11-position (i.e., only 2/3 of the total immobilized bergenin
could be acylated at the 4 position).
1
Solid-phase bergenin halogenation and cleavage: 0.5–0.7 mM
immobilized bergenin (50 mg resin/mL, 14.5
was added to a 150 mM citric acid/150 mM potassium phosphate
pH 2.5) aqueous buffer solution containing 150 mM KBr (1.6 mL
total reaction volume). Following addition of 25 L/mL CPO
20.8 kU/mL, 1450 U enz/ mol bergenin), vials were pre-incubated
at either 4 or 25 °C, 180 rpm for 20 h. Reactions were initiated by
continuous infusion of 800 mM H (in buffer) at a flowrate of
L/h (0.4 mmol/h H L CPO) via a syringe pump. Reaction
lmol COOH/g CPG)
(
l
(
l
2 2
O
2
0
l
2 2
O /l
vials—left open to the atmosphere—were incubated at room tem-
perature under orbital shaking at 180 rpm for 18–20 h. If addi-
tional rounds of reaction were desired, the original solution
above the resin was removed, followed by addition of fresh CPO
and KBr dissolved in aqueous buffer and overnight pre-incubation.
Upon completion of the reaction, the contents were centrifuged at
Liquid chromatography/mass spectrometry: Bergenin transesteri-
fication reactions were analyzed using LC/MS. Products were re-
solved using a reverse-phase wide-pore C18 column (JT Baker,
2
700g for 5 min, followed by removal of the supernatant. After
4.6 mm  250 mm, 5
acetonitrile (ACN)/H
67/33% ACN/H O, linear gradient) and analyzed on a Hewlett Pack-
l
m particles) with a 0.9 mL/min flow of an
gently drying the vial contents under a flowing air or N stream,
the resin was further dried under vacuum overnight. During opti-
mization of the reaction conditions, enzyme/substrate ratios be-
tween 1000 and 3000 U CPO/
ratios of 0.2–3.3 mmol/h H
times of 20–96 h were also tested. For cleavage via LipB in aqueous
buffer, 40–60 mg/mL resin containing immobilized bergenin and
its derivatives was added to a 50 mM potassium phosphate (pH
7
1
cleaved bergenin/derivatives (ꢀ20–25% v/v DMF in final mixture)
and re-incubation at 45 °C, 180 rpm for 30 min, the vials were cen-
trifuged at 2700g for 5 min, followed by sampling of the superna-
tant for LC/MS analysis.
2
2
O gradient (0–20 min: 0/100% ACN/H O—
2
2
ard (Agilent) 1100 LC/MSD equipped with a diode array detector
(DAD) and an atmospheric pressure electrospray ionization
(API-ES) unit. The quadrupole ion analyzer MS was set to scan from
300 to 500 amu (0.10 amu increments), with a fragmentor setting
l
2
mol bergenin, co-factor/enzymes
per L CPO, and pre-incubation
2
O
l
of 70 V, 3000 V capillary, 35 psig nebulizer pressure, and with N
2
drying gas set to 300 °C and 9.5 L/min. Absorbances were averaged
between 260 and 290 nm (340–380 nm baseline) and were used in
conjunction with positive-ion MS to provide quantitative detection
and unambiguous molecular identification.
.0) solution containing 75 mg/mL LipB and incubated at 45 °C,
80 rpm for ꢀ30–40 h. After addition of DMF to help dissolve the
Acknowledgments
Solution phase bergenin transesterification: 500 mM of bergenin
dissolved in DMF was added to a flame-dried glass vial in a 95:5
v/v ratio of organic solvent (either isooctane or diisopropyl ether)
to DMF, yielding a final bergenin concentration of 25 mM. Solubi-
lized SC and native SC in either isooctane or diisopropyl ether
was added to the reaction so that the final enzyme concentration
was 1.6 mg/ml. Reactions were initiated by adding 500 mM of vi-
nyl butyrate and the contents were incubated at 45 °C and
This work was supported by the National Institutes of Health
GM66712), by the National Science Foundation (BES-0228145)
(
and the Korea Research Foundation Grant funded by the Korean
Government (KRF-2007-357-D00057). The authors thank Prime
Synthesis for custom synthesizing the 12–13 carbon spacer CPG
as well as Peter McDermott, I-Fan Lin, Brandon Gan, and Vincent
Chan for assistance in obtaining both chemical and enzymatic
bergenin cleavage and hydrolysis data.
2
50 rpm. Initial rates of product formation were measured by
removing aliquots from the reaction vials over time and analyzing
them for the presence of product via LC/MS. Prior to LC/MS analysis
aliquots were first quenched by addition of an excess of acetoni-
trile and incubation on ice, followed by centrifugation at 2700g
for 5 min. The resulting samples were then evaporated to dryness
and re-dissolved in acetonitrile for immediate LC/MS analysis.
Solid-phase bergenin transesterification and cleavage: Immobi-
lized bergenin (ꢀ1–3 mM, 100 mg/mL resin) was transesterified
with excess vinyl butyrate (500 mM) using EXT-SC (6 mg/mL) in
IPE. Solubilized enzyme initially prepared in isooctane was trans-
ferred to the reaction vial; the contents were then evaporated to
dryness and re-dissolved in IPE. After incubation at 45 °C and
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