You are on page 1of 13

Enzyme assays

Jean-Louis Reymond,* Viviana S. Fluxa` and Noe lie Maillard

Received (in Cambridge, UK) 6th August 2008, Accepted 4th September 2008
First published as an Advance Article on the web 17th October 2008
DOI: 10.1039/b813732c
Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of
enzyme assays have been developed to assist the discovery and optimization of industrial
enzymes, in particular for

white biotechnology where selective enzymes are used with great

success for economically viable, mild and environmentally benign production processes. The
present article highlights the aspects of uorogenic and chromogenic substrates, sensors, and
enzyme ngerprinting, which are our particular areas of interest.
1. Introduction
Enzyme assays are analytical tools to visualize enzyme acti-
vities. In recent years a large variety of enzyme assays have
been developed to assist the discovery and optimization of
industrial enzymes, in particular for

white biotechnology
where selective enzymes are used with great success for
economically viable, mild and environmentally benign produc-
tion processes.
In this context the enzyme assays serve to
screen collections of enzymes available from strain collections,
metagenomic libraries and libraries of mutant enzymes
obtained by random or directed mutagenesis from known
enzymes. This type of screening must be distinguished
from genetic selection experiments, in which a mutation/
selection protocol is set up to allow for the survival of
microorganisms producing an active enzyme, e.g. by comple-
mentation of a biosynthetic pathway in an auxotrophic
bacterial or yeast strain. Both high-throughput screening
and genetic selection are viable protocols for discovering and
improving enzymes.
Important developments of enzyme
assays are also constantly occurring in relation to drug
discovery eorts, medical diagnostics and in the area of
cellular and tissue imaging. Enzyme assays are also used in
enzyme model studies.
The majority of enzyme assays are developed to test isolated
enzymes or enzyme containing samples such as culture suspen-
sions in 96-well microtiter plates or similar parallel liquid
phase systems. The simplest and most practical enzyme assays
are based on synthetic substrates that release a colored or
uorescent product upon reaction or induce a directly detect-
able change in solution such as a precipitation. Many such
substrates are commercially available and often serve as
reference substrates to determine absolute activities of enzyme
samples in Units. Enzyme reactions may also be assayed using
indicators which respond indirectly to product formation or
substrate consumption. The indicator may be as simple as a
pH-indicator and as complex as a functionalized nanoparticle.
Many assays are also based on analytical instruments such
HPLC, GC, MS, NMR or IR spectrometers. These instru-
ments often allow access to reaction parameters not otherwise
accessible such as enantioselectivity and therefore play a
Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, Berne, 3012, Switzerland.
E-mail:; Fax: +41 31 631 80 57;
Tel: +41 31 631 43 25
Jean-Louis Reymond
Jean-Louis Reymond was born
in Switzerland in 1963. He
graduated from the ETH in
Zurich in 1985 and received
his PhD from the University
of Lausanne in 1989. In 1990
he joined the Scripps Research
Institute rst as a post-doctoral
fellow with R. A. Lerner, then
as an assistant professor. In
1997 he moved to the Univer-
sity of Berne, Switzerland, as
an associate professor, and
became a full professor in
1998. His research touches on
three areas of bioorganic chem-
istry: (1) high-throughput screening assays for biocatalysis; (2)
articial protein design with peptide dendrimers; and (3) small
molecule drug discovery.
Viviana Fluxa
Viviana Fluxa was born in
Santiago de Chile in 1980. She
received her MSc in chemistry
from Fribourg University in
2006. While at Fribourg, she
studied diethylstilbestrol deriva-
tives in the laboratory of Chris-
tian Bochet. In March 2006, she
joined the Reymond group at
the University of Berne in the
Department of Chemistry and
Biochemistry. Her current re-
search interests include protease
proling with FRET peptides
and exploring the biological ac-
tivity of cyclic peptides libraries.
34 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009
critical role in biocatalysis for the discovery and optimization
of selective enzymes by directed evolution. Enzyme assays
have been the subject of a recent volume providing an over-
view in the perspective of biocatalysis.
The present article
highlights the aspects of uorogenic and chromogenic sub-
strates, sensors, and enzyme ngerprinting, which are our
particular areas of interest.
2. Chromogenic and uorogenic substrates
Fluorogenic and chromogenic enzyme substrates form the
cornerstone of enzyme assay technology. They incorporate a
chromophore whose absorbency or uorescence properties
change as a result of the enzyme reaction. The key advantage
of these substrates is that the assay is very simple and the
signal produced is directly related to the enzyme-catalyzed
reaction. If the colored or uorescent product is soluble, the
assay is well-suited for microtiterplate based assays. On the
other hand, if the product is insoluble in the reaction media,
the substrate can be used for screening bacterial cultures on
agar plates. Almost all examples to date focus on a small
family of uorophores and chromophores, in particular
umbelliferones, nitrophenols, uoresceins, rhodamines and
BODIPY dyes, all of which are relatively large aromatic
groups which tend to inuence both substrate binding
(stronger binding), catalytic turnover (reactivity may be
lowered or absent compared to non-labeled substrates), and
solubility. Encouraging recent reports by Nau and co-workers
suggest that new, non-aromatic uorophores might provide an
alternative for FRET type assays.
Alternatively various
chemistries can be implemented to separate the enzyme reac-
tive group from the uorophore in a variety of reaction types,
as exemplied with the Clips-O method below.
developments in these directions are still possible and will
mark the future of this type of assays.
2.1 Phenolate and aniline release
Indican is a chromogenic glycosidase substrate belonging to a
family of natural product glycosides found in plants such as
Isatis tinctoria and Polygonum tinctorum, the traditional source
to produce indigo is by fermentation.
Cleavage of the glyco-
sidic bond forms an unstable hydroxyindole intermediate,
which dimerizes oxidatively at air to form indigo as a blue
precipitate. A number of enzyme substrates have been designed
following this natural product example. For instance, numerous
glycosides of uorescent or colored phenols are used to test
glycosidases, e.g. nitrophenyl b-galactoside (1) for detection
of b-galactosidase activity, p-nitrophenyl caproate (2) as a
chromogenic lipase substrate, and the nitrophenyl octyl ether
3 to detect cytochrome P450 activity
(Fig. 1). Note that the
yellow color of nitrophenolate is only visible above pH 7.
Naphthols can be detected indirectly by a secondary reaction
with diazonium salts to form azo-dyes, a principle used in
cytochemistry to test esterase activities in tissue samples with
naphthyl acetate,
and recently adapted to assay aldolase
antibodies in agar plates.
The range of phenol release substrates can be extended using
indirect release mechanisms, as we rst demonstrated with the
alcohol dehydrogenase enzyme substrate 5 (Fig. 2). This chiral
secondary alcohol is oxidized by the enzyme to form the
corresponding ketone 6, which is unstable and undergoes a
b-elimination reaction catalyzed by bovine serum albumin to
produce the blue uorescent umbelliferone anion 7.
signal is only visible above pH 7 where the product exists as a
uorescent anion. The same principle allowed substrates for
aldolase catalytic antibodies
and proline-type catalysts,
and lipases.
A related scheme
involving an intermediate hemiacetal provides various lipase and
esterase substrates,
as wells as a uorogenic substrate for
BaeyerVilliger monoxygenases (BVMO) in the form of the
2-aryloxyketone 8,
via the intermediate lactone 9 which may
be considered as a lactonase-type probe, although it is quite
unstable and spontaneously hydrolyzes in the whole cell
Fig. 1 Chromogenic enzyme substrates releasing nitrophenolate.
Noelie Maillard, born in
Delemont (Switzerland) in
1981, studied chemistry at the
University of Neuchatel, and
complete her diploma thesis
in inorganic chemistry at the
Institute of Chemistry, Univer-
sity of Neuchatel with Prof.
Dr Thomas R. Ward. She
started her PhD in 2006 under
the supervision of professor
Jean-Louis Reymond in the
Department of Chemistry and
Biochemistry at the University
of Bern (Switzerland). Her current research interests include
the development of new methods for the screening of peptide
dendrimer libraries and its application in dierent reactions types.
Noe lie Maillard
This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3446 | 35
conditions used to assay BVMO. Substrate 8 and further analogs
are currently the only available uorogenic substrates for
BVMO and they are readily obtained by alkylation of various
chloroketones with umbelliferone.
The ketone or aldehyde leading to b-elimination may also be
formed by chemical oxidation of a primary 1,2-diol or
1,2-aminoalcohol reaction product in the so-called Clips-Ot
substrates (Fig. 3).
A typical example is epoxide 10, which
provides a highly reliable probe of epoxide hydrolase activity for
screening in microbial cultures.
The substrate is hydrolyzed by
epoxide hydrolases to form the 1,2-diol 11. In the presence of
sodium periodate (NaIO
), the diol is rapidly and quantitatively
oxidized to form aldehyde 12, which undergoes a b-elimination
to form umbelliferone 7 as above. Further examples include
thermally stable lipase, amidase and phosphatase substrates,
an HIV-protease substrate,
and a ceramidase substrate.
Further strategies for indirect phenolate release include intra-
molecular carbamate or carbonate cyclization. The aldolase
Fig. 3 The Clips-Ot epoxide hydrolase substrate.
Fig. 4 A chromogenic substrate for aldolase catalytic antibodies.
Fig. 5 Enzyme assays with direct and indirect release of a uorescent
Fig. 2 Fluorogenic enzyme substrates with indirect release of
36 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009
antibody substrate 13 features an interesting recent example of
this approach (Fig. 4).
This substrate undergoes sequential
retroaldolization, b-elimination and intramolecular carbamate
cyclization to release a catechol which can be made visible by
formation of the insoluble black precipitate 14 in the presence of
Fe(III). Related carbonate and carbamate cyclization are also
used in substrate for epoxide hydrolases
and acylases.
Anilines such as nitrophenyl aniline and rhodamine are
frequently converted to amides to form chromogenic or
uorogenic amidase and protease substrates, as in the protease
probe 15 designed for caspases (Fig. 5).
The aniline can also
be released indirectly, for example via a self-immolative
quinone methide mechanism as in the uorogenic peptide
substrate 16 for the prostate specic antigen (PSA),
or by
spontaneous hydrolysis of a urea following oxidation of a
phenol to a catechol and subsequently an orthoquinone as in
the recently reported tyrosinase substrate 17.
2.2 FRET
Many enzyme assays are based on FRET (Fo rster or Fluores-
cence Resonance Energy Transfer) as detection principle, for
instance to measure hydrolytic reactions separating uorophore
from quencher in the case of proteases,
and in the synthetic direction for fucosyl trans-
Aldehyde 18 features an interesting use of FRET, in
which addition of a nucleophile to the aldehyde such as an
antibody catalyzed aldol addition to form 19 (Fig. 6) removes the
intramolecular quenching eect.
Fluorescence of a label can
also be modulated by proximity eects such as medium eects.
For example, in an aminonitrobenzofurazane-labeled g-cyclo-
dextrin reported as an a-amylase uorogenic substrate, cleavage
of the cyclodextrin ring by the amylase exposes the uorophore
to water and reduces uorescence intensity.
Fluorescent sub-
strates for kinase substrates have been recently reviewed.
In a
recent example, a luminogenic probe was developed for tyrosine
phosphorylation based on a short peptide sequence containing
an iminodiacetate moiety near the site of phosphorylation. In
response to kinase activity, the probe provides a strong lumines-
cence enhancement, resulting from the increased ability of the
probe to bind and sensitize Tb
and Eu
ions upon phos-
Fluorescence modulations by aggregation,
dilution or phase change are also related to FRET substrates.
Thus, lipases can be assayed with 1,3-dioleoyl-2-(4-pyrenyl-
butanoyl)glycerol in the presence of lipoproteins and albumin.
Ester hydrolysis releases the pyrene carboxylate, which then binds
to albumin, resulting in a uorescence increase. The commercially
available FITC-conjugates of casein are used as protease sub-
strates, whereby proteolysis removes the autoquenching and leads
to stronger uorescence of the uorescein chromophores.
Recently reported protease-sensitive nanobers consisting of
aggregated b-sheet forming peptides also rely on dilution-induced
release of autoquenching upon proteolytic cleavage.
2.3 Other approaches
The enzyme reaction may also directly modify the chromophore
itself as a detection mechanism. For example microbial growth
can be monitored by following the activity of nitroreductases
that reduce the nitro group of various 7-nitrocoumarins such as
20 to form the corresponding 7-aminocoumarins 21 as a
uorescent products (Fig. 7).
Peroxidases react with a variety
of aromatic compounds to form colored products, and indoles
are substrates for cytochromes, forming indigo upon secondary
oxidation of the hydroxyindole primary product.
dehydrogenase and aldolases can be screened using 6-methoxy-
naphthaldehyde and related substrates.
The primary amine in
substrate 22 is oxidized by monoamine oxidase for form an
aldehyde, ultimately forming the uorescent indole 23 by con-
densation of with the aniline amino group.
The recent report
of an assay for fatty acid dehydrogenases based on chromo-
phore modication by the enzyme is also of interest.
Luminescent products such as luciferase substrates (biolumines-
or 1,2-dioxetanes (chemiluminescence)
released from
enzyme reactions are also of interest.
Substrate dissolution or product precipitation or crystalliza-
tion may sometimes form the visible signal, without formation
of a colored product. Classically, microbial cultures producing
active lipases will form a clearing zone on agar plates prepared
with tributyrin. Certain polymer degrading enzymes can be
screened by recording dissolution of an insoluble substrate, in
Fig. 7 Fluorogenic substrates with direct modication of the
chromophore upon reaction.
Fig. 6 A FRET probe for following nucleophilic addition reactions
to aldehydes.
This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3446 | 37
particular cellulases.
More sophisticated systems were recently
investigated, such as the formation of hydrogels
dipeptide nanotubes.
In a recent example, incorporating an
ester-containing substrate in a liquid crystal allowed to assay a
lipase through its action on the alignment layer.
2.4 Fluorescence activated cell sorting (FACS)
Fluorescent and/or uorogenic substrates have been used to
directly identify cells expressing active enzymes in liquid culture
based on uorescence-activated cell sorting (FACS), which
provides a particularly meaningful application of such sub-
strates in high-throughput screening.
For example mutants
of the protease OmpA were displayed on the surface of E. Coli
cells and a cell-surface adherent uorogenic substrate for
protease was added to the culture.
Cells expressing an active
protease became uorescent and could be sorted out by FACS,
which allowed discovering mutants with a 30-fold improved
activity. The same technique has been used for sorting micro-
emulsion droplets where an enzyme gene and expression
machinery are compartmentalized together with a uorogenic
substrate system.
Both methods allow to screen very large
numbers of variants (410
). Glycosides labeled with Bodipy
dyes were used to screen sialyl transferases in living cells, relying
on the fact that this substrate but not its sialylated product is
cell permeable. Thus, after washing unreacted substrate, cells
containing an active enzyme retained the uorescent product
and were separated by uorescence activated cell sorting.
high-throughput screening technique for the identication and
isolation of enantioselective enzymes based on FACS has been
developed on the idea of labeling each of the two enantiomers
with a dierent uorescent dye. This method allows to evaluate
cells (and number of clones generated) within a few hours.
3. Indicator assays
A variety of relatively simple chemosensor systems based on
chromogenic or uorogenic reagents can convert a chemical
transformations into a detectable signal, often through a func-
tional group specic reaction or by a separation eect. Bio-
sensors binding to either substrate or product may be used
similarly. Such indicator assays can be used to assay reactions
of specic, unlabeled substrates, which is necessary whenever a
very specic reaction is being optimized. The main drawback of
indicator assays is that they are often sensitive to interferences
(e.g. other events than catalytic turnover may produce a signal,
or turnover may be masked) and may require narrow assay
conditions that render them incompatible with certain enzymes.
In addition the reporter chemistry may be rate limiting, which
prevents their use for kinetic studies. These potential limitations
may be overcome for a high-throughput screening application
by using proper controls and relying on an endpoint measure-
ment rather than on measuring a reaction rate. As long as they
can test authentic substrates, indicator assays will remain one of
the best option for enzyme assays in the future.
3.1 Enzyme-coupled assays
One of the most straightforward methods to render an enzyme-
catalyzed reaction detectable consists in further converting the
reaction product by a second enzyme to form a second product
and so on, until one of these follow-up reactions produces a
detectable signal. The vast majority of enzyme-coupled assays
involve an oxido-reductase, usually an alcohol dehydrogease
(ADH), using NAD or NADP as cofactors, as exemplied
recently in the selection of enantioselective aldolase mutants.
Recent interesting examples include the determination of the
enantioselectivity of lipases and esterases using acetate ester
substrates on the basis of an acetate detection kit.
The assay
was used recently to select a double mutant of Bacillus Subtilisis
esterase with inverted enantioselectivity towards acetylated
tertiary alcohols.
The enantioselectivity of ADH enzymes has
also been used to determine the enantioselectivity of alcohols
formed by the catalyzed addition of diethyl zinc to aldehydes,
for a transition metal catalyzed epoxide opening reactions
the conversion of benzaldehyde and acetyl cyanide to the corres-
ponding acetylated cyanohydrins.
A pair of enzymes was
described that are able to dierentiate the 1,2-hexanediol anti-
podes Lactobacillus ker alcohol dehydrogenase, highly S selective,
and horse liver alcohol dehydrogenase, modestly S selective. This
allows one to obtain simultaneous enantioselectivity readouts on
two distinct substrates for the Co(III)-salen-mediated hydrolytic
kinetic resolution of epoxides.
It has been shown that only
partially enantioselective dehydrogenases are generally sucient
for the ee-determination of chiral alcohols.
The reduction of hydrogen peroxide to water catalyzed by
peroxidases occurs with oxidation of various chromogenic
dyes, such as ABTS.
Horseradish peroxidase (HRP) and
as oxidant was used to detect the naphthol product
formed by hydroxylation of naphthalene by a P450
Turner and co-workers have developed
the reaction to follow the activity of an enantioselective
microbial monoamine oxidase (MAO-N) on amines. The
MAO-N produces hydrogen peroxide as a byproduct, which
is revealed by a peroxidase and 3,3
-diaminobenzidineas a
chromogenic reagent.
In a related experiment, the same
group has recently reported a novel high-throughput screening
method to determine both the rate and the enantioselectivity
of asymmetric ketone reduction by ketoreductases (KRED) in
the presence of an R-selective alcohol oxidase, and optionally
horseradish peroxidase (HRP) and ABTS (Fig. 8).
R-selective KRED induces multiple turnovers producing
Fig. 8 Dual-wavelength spectrophotometric tracking of two chromo-
gens (NADP
and ABTS) for the determination of ketoreductase
(KRED) activity and enantioselectivity.
38 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009
many equivalents of NADP
, or ABTS
if HRP is added to
the assay. By contrast, an S-selective KRED results in only
one turnover of NADPH since no R-alcohol is produced as an
oxidase substrate.
Hydrolytic enzymes such as glycosidases have been used as
secondary enzymes to follow the production of chromogenic
substrates from non-reactive precursors through a primary
enzyme such as a glycosidase, glycosyl transferase, glyco-
Similarly, proteases have been used to track
prolyl cistrans isomerases,
and peptide
Luciferases produce light by oxidation under
consumption of ATP, oxygen and an oxidizable substrate such
as luciferin or an aldehyde and reduced avin. A number of
assays have been reported that use a luciferase as secondary
enzyme to screen a reaction producing one of the luciferase
substrates as a product. The oldest method to quantify
ATP relies on rey luciferase and luciferin.
bacteria have been used to monitor the activity of aldolase
catalytic antibodies releasing nonanal.
The same pheno-
typic screen was recently used to discover new oxido-reductase
enzymes, whereby the substrate spectrum of the bacterial
luciferase was explored and extended.
Firey luciferase
was recently used as the substrate undergoing refolding by
the chaperone Hsp90.
An assay for monoamine oxidases A
and B was recently reported where the phenol-type substrate
for the luciferase is released by b-elimination of a primary
aldehyde oxidation product. For inhibitor assay, it should be
noted that typically 13% of compound libraries inhibit
luciferase activities.
3.2 Functional group selective reagents
Kazlauskas and co-workers have exploited the well-known
fact that ester hydrolysis lowers the pH of the reaction medium
to develop a colorimetric assay to screen lipases and esterases
for enantioselectivity.
pH-indicators have also been used
in the context of solgel encapsulated enzymes.
Recently a
pH-indicator assay was reported for screening glycosyl trans-
ferases based on the acidication induced by glycosyl transfer
from UDP-GalNAc,
and from glycosyl uorides.
tional group selective chromogenic or uorogenic reagents
have been used to detect enzyme activities, including reagents
for amines formed by amidases,
ammonia from nitrile
aldehydes from vinyl ester cleavage
from periodate cleavage of epoxide hydrolysis products,
thiols from thiolactones,
peptides from kinase reactions,
UDP from glycosyl
dimedone from lipase reactions,
and amino
acids from amidases.
For example, amino acids can be
detected by uorescence in real-time using the non-uorescent
Cu(II) complex of calcein 24. The amino acid displaces Cu(II)
from calcein, whereby calcein regains its green uorescence.
This simple assay is suitable to screen acylases, amidases and
proteases, as illustrated for the case of aminopeptidase, for
which no other uorescence assay is known (Fig. 9).
Adrenaline serves as the reporter for detecting for 1,2-diols
(28) and 1,2-aminoalcohols by back-titration of sodium
periodate (Fig. 10). Adrenaline (29) reacts quantitatively
and rapidly with sodium periodate to form the deep red
Fig. 9 A uorescence assay for aminopeptidase using a calcein
Fig. 10 The adrenaline test for enzymes.
This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3446 | 39
adrenochrome (30), a reaction which is does not take place if a
1,2-diol or 1,2-aminoalcohol has already consumed the perio-
date oxidizing agent. This provides a practical end-point assay
for a variety of hydrolytic enzymes.
The adrenaline assay
can be used to screen the enzymatic hydrolysis of epoxides (25)
by epoxide hydrolases, triglycerides such as tributyrin 26 or
various acetate esters
by lipases and the dephosphorylation
of phytic acid (27) by phytases. The adrenaline test for epoxide
hydrolase was recently adapted to an automated format for cell
Sodium periodate also decolorizes certain chromo-
phores and the assay was used to screen epoxide hydrolases
using uorescein as periodate reporter.
In an unusual yet very practical example of indirect sensing
of an enzyme reaction, epoxide hydrolase activity on butane-
oxide was detected in E. coli cultures on agar-plate using
Safranin O.
Oxidation of the 1,2-diol product by E. Coli
modied the membrane potential and lead to accumulation of
the red dye in the colonies producing active enzyme, allowing
for direct selection.
3.3 Bio- and nano-sensors
While the above example apply simple chemical reactivity
principles in the context of enzyme assays, some sensor
systems rely on more sophisticated detection schemes with
biosensors, vesicles and gold nanoparticles, a type of assay
which may be assigned to the nano world. The rst notable
example concerns antibodies for the so-called cat-ELISA
assay developed in the context of catalytic antibody
Further examples ADP selective aptamers
for kinase sensing,
or lectins for testing glycosyl transferases
on microarray displayed substrates.
Vesicles containing
synthetic multifunctional pores (SMPs) have been used for
enzyme assays.
The SMPs are incorporated in the vesicle
membrane and serve as channels for the escape of uorescein,
which results in a uorescence increase since dilution removes
autoquenching. Substrate/product ratios in an enzymatic
reaction can be monitored whenever substrate and product
dierentially modulate the ow of uorescein through the
SMPs. Gold nanoparticles have been used for a variety of
enzyme assays. Solutions of Au(III) (HAuCl
) are reduced by
NAD(P)H, catechols, or thiols, to form colored suspensions of
gold nanoparticles, allowing a colorimetric assay for enzymes
such as lactate dehydrogenase,
acetyl choline esterase
and tyrosinase.
Aggregation of gold nanoparticles in
suspension induces a color change from red to purple grey,
allowing assays for kinases mediated by a biotinavidin
aggregation trigger,
for proteases using a synthetic
peptide with a protease-specic sequence anked by a pair
of S-acetyl cysteine residues,
for alkaline phosphatase
and ATP sensing
using charge induced aggregation of
nanoparticles, and for endonucleases using two sets of gold
nanoparticles coated with complementary single-strand DNA
4. Fingerprinting
The information content of screening assays can be increased
by analyzing multiple substrates simultaneously. The rst
multi-substrate analysis method was the APIZYM system in
the 1960s.
In this method a set of 19 or 32 dierent
enzyme substrates, including chromogenic substrates for
lipases and esterases, aminopeptidases, chymotrypsin, trypsin,
phosphatases, sulfatases and b-galactosidases, is used in a
multi-well format to analyze microbial cultures. The analysis
produces a reactivity pattern indicating which enzymes are
produced by the microorganism. This information serves to
identify the microorganism, and forms the basis of microbial
strain identication in hospitals. Enzyme ngerprinting
proposes to focus the analysis on a single enzyme using a
series of structurally related substrates to characterize its
The data may be used to identify reactive
substrates, or for functional classication of the enzymes.
Generally one analyzes reactivity ngerprints across a series of
similar substrates, such as series of peptides, however it should
be mentioned that certain enzymes may also catalyze reactions
of dierent functional groups, which is called substrate
Fingerprinting with multiple substrates
should be distinguished from the so-called activity-based
protein proling, which is based on labeled suicide enzyme
inhibitors to identify reactive enzymes in protein
The critical problem of enzyme ngerprinting
is to provide a reliable method for producing the data,
and most research eorts are currently still focusing on
this problem. The true potential of ngerprinting will be
realized when reagents will be available for measuring multi-
dimensional datasets reporting enzyme selectivities and
substrate preferences as readily as what is currently possible
with single substrates.
4.1 Parallel assays in microtiter plates
Assays with multiple substrates in microtiterplates have been
mostly used for hydrolase proling.
For example,
ngerprinting lipases and esterases with 16 periodate-activated
fatty acid ester substrates in both enantiomeric forms showed,
as expected, that lipases were more active on long-chain
substrates, while esterases preferentially cleaved short-chain
substrates. More surprisingly, a selectivity for intermediate
chain length substrates was apparent as the second principal
component of the observed diversity, an information not
otherwise accessible (Fig. 11).
Using a related series of
water-soluble esters 31ae and 32a/b derived from uorescein,
ngerprinting showed that lipases and esterases dier from
one another by their reactivity in pure aqueous buer vs.
buer containing 20% dimethyl sulfoxide as co-solvent.
Esterases were more active in aqueous environment, while
lipases required the cosolvent for highest activity, as shown by
the color coded pattern (Fig. 12).
Microtiter-plate based assays have also been used to analyze
proteases using multiple peptide substrates to determine the
optimal substrate,
in particular with positional scanning
libraries of millions of uorogenic peptidyl coumarinamides as
series of 20 or 400 dierent substrate mixtures.
4.2 On-bead assays
Meldal and co-workers reported the use of synthetic combi-
natorial libraries of millions of synthetic peptides as FRET
substrates to analyze protease reactivity on solid
40 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009
a very practical method still under further
development today.
One of the diculties in the analysis
is the necessity to introduce a uorescent label on the cleaved
peptide, which may reduce the reactivity of the protease. We
recently reported an assay for on-bead proteolysis of a solid-
supported combinatorial library of N-acetylated non-tagged
octapeptides AcL (Fig. 13).
After proteolysis, the free
N-termini are simply stained by reductive alkylation with a
tagged aldehyde, and the stained beads are analyzed.
4.3 Cocktail ngerprinting
Due to its separating power, chromatographic screening oers
the possibility to assay several dierent substrates simulta-
neously. A single HPLC-analysis returns the activity nger-
print, in which the relative amounts of product formation
dening the reactivity prole or ngerprint, can be precisely
reproduced if the cocktail composition is controlled. We have
demonstrated this principle for a ngerprint analysis of lipases
and esterases using a cocktail of 20 monoacyl-glycerol
and for proteases using a cocktail of ve hexa-
Such characterization tools may prove useful to
identify novel enzymes with unusual selectivities, as well as in
the area of diagnostics. Similarly, the classical APIZYM
substrate palette for microbial characterization can be formu-
lated as a cocktail reagent, allowing 16 dierent enzyme
reactivities to be determined in a single analysis.
A cocktail
of uorescent umbelliferyl glycosides was recently used to
characterize various glycosidases using an HPLC-based
Substrate cocktails can also be analyzed by mass
spectrometry in a variety of setting including enantioselectivity
determination using a mixture of two isotopically labeled
for glycosyl transferase reac-
tion using substrate mixtures
or for protease activity
determination using a mixture of peptide substrates.
4.4 Microarrays
Miniaturization of ngerprinting (and screening) down to
the scale of a few nanoliters per datapoint is possible with
microarrays printed on glass slides. A nanospray system was
used to homogeneously distribute nanodroplets of a solution
Fig. 11 Fingerprint analysis of chain length selectivity of lipases and
Fig. 12 Fingerprint analysis of cosolvent selectivity of lipases and
Fig. 13 On-bead protease proling on TentaGel beads.
This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3446 | 41
containing three uorogenic protease substrates on a micro-
array on which spots of enzyme had been previously printed,
allowing high-throughput screening of enzyme inhibitors.
Depositing uorogenic substrates on poly-lysine-coated glass
slides also allows ecient assays of various enzymes in nano-
Nanodroplets for enzyme assays can be moved
using thermal gradients for mixing.
Fluorogenic substrates
have also been arrayed with covalent attachment to the surface
of glass slides to allow activity proling experiments with
proteases and other hydrolytic enzymes using combinatorial
series of uorogenic coumarin-derived substrates
with lipases using substrate of varying acyl chain length 35af,
relying on chemoselective oxidation of the 1,2-diol product
with sodium periodate followed by reaction with rhodamine
sulfohydrazide to detect conversion (Fig. 14).
Microarrays have also been used for an elegant protease
proling method based on combinatorial libraries of
PNA-encoded dipeptidyl-rhodamine substrates.
important improvement in the study of the biology of phos-
phatases was developed in a microarray. Glass slides having
multiple peptide substrates of Ser/Thr phosphatases
immobilized on them, can be used to simultaneously determine
the preference of the enzymes for the dierent substrates.
Protease proling was also recently reported based on a
multiplexed solution phase assay on microarrays.
arrays were also reported for enantioselectivity, for example to
estimate the optical purity of amino acids after covalent
attachment by reaction with a pseudo-enantiomeric pair of
labels bearing two dierent uorophores based on Horeaus
Mass spectrometry was applied for the creation
of biochips loaded with label-free oligosaccharide arrays, in
order to study glycosyltransferases activities.
It should be mentioned that it is possible to handle low-
volume enzyme assays at the scale of one microliter per
assay using silicagel plates pre-impregnated with a uorogenic
substrate as the reaction medium, which provides a practical
solution for miniaturization that is much simpler than micro-
A robotic arm is used to dispense the enzyme
containing test solutions in a volume of one microliter per
assay, which results in a homogeneously dispersed spot on the
silicagel surface on which the enzyme reacts evenly with the
substrate (Fig. 15).
5. Conclusion
In recent years enzyme assays have greatly advanced in their
scope and in the diversity of detection principles employed. In
the 1990s high-throughput screening of enzyme activity was
perceived as a critical bottleneck in enzyme engineering due to
the advent of random mutagenesis methods for directed evolu-
tion, which multiplied demands for screening by orders of
magnitude. Developments of new screening methods based on
chemistry, biology and instrumentation have followed to rise to
this challenge, in part by reviving and rening older methods.
Fig. 15 High-throughput screening with microtiter reaction on
silicagel plates.
Fig. 14 Periodate-coupled lipase assay on microarrays.
42 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009
For what concerns future developments, the demand for
new enzyme assays remains high in the context of high-
throughput screening in enzyme engineering, in particular
for sensing regio-, stereo- and enantioselectivity. Remarkably,
most of these problems have been in principle solved by
instrument-based assays such as NMR, MS, HPLC and MS,
not reviewed here, but the methods are often complicated and
expensive to implement. Therefore, most application examples
in enzyme engineering continue to use uorogenic and
chromogenic substrates and indicator assays as the main
screening tool, simply because when available such assays
are simple to use and inexpensive. Despite of their apparent
drawbacks in terms of possible artefacts, the most useful
assays seem to be indicator assays that are compatible with
a range of dierent substrates. In particular enzyme-coupled
assays will probably remain high on the list for many years to
come, with assays producing a colored precipitate being the
most useful for high-throughput screening as they can be
applied on agar plates, on paper, or in microtiter plates.
Improvement in ngerprinting reagents should also be
considered in the future to enable high-throughput screening
with a diverse set of substrates simultaneously, which would
allow a much more productive use of enzyme libraries.
This work was supported by the Swiss National Science
Foundation and Prote us SA, Nmes, France.
Notes and references
1 A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts and
B. Witholt, Nature, 2001, 409, 258268.
2 P. Lorenz and H. Zinke, Trends Biotechnol., 2005, 23, 570574.
3 S. Lutz and W. M. Patrick, Curr. Opin. Biotechnol., 2004, 15,
4 T. W. Johannes and H. Zhao, Curr. Opin. Microbiol., 2006, 9,
5 K. L. Tee and U. Schwaneberg, Comb. Chem. High Throughput
Screening, 2007, 10, 197217.
6 S. B. Rubin-Pitel and H. Zhao, Comb. Chem. High Throughput
Screening, 2006, 9, 247257.
7 W. R. Streit and R. A. Schmitz, Curr. Opin. Microbiol., 2004, 7,
8 S. V. Taylor, P. Kast and D. Hilvert, Angew. Chem., Int. Ed.,
2001, 40, 33103335.
9 G. J. Williams, A. S. Nelson and A. Berry, Cell. Mol. Life Sci.,
2004, 61, 30343046.
10 A. Aharoni, A. D. Griths and D. S. Tawk, Curr. Opin. Chem.
Biol., 2005, 9, 210216.
11 M. D. Toscano, K. J. Woycechowsky and D. Hilvert, Angew.
Chem., Int. Ed., 2007, 46, 32123236.
12 H. Lin and V. W. Cornish, Angew. Chem., Int. Ed., 2002, 41,
13 J. Kofoed and J. L. Reymond, Curr. Opin. Chem. Biol., 2005, 9,
14 J. L. Reymond, Enzyme Assays: High-throughput Screening,
Genetic Selection and Fingerprinting, Wiley-VCH, Weiheim, 2005.
15 A. Hennig, D. Roth, T. Enderle and W. M. Nau, ChemBioChem,
2006, 7, 733737.
16 H. Sahoo and W. M. Nau, ChemBioChem, 2007, 8, 567573.
17 F. Badalassi, D. Wahler, G. Klein, P. Crotti and J. L. Reymond,
Angew. Chem., Int. Ed., 2000, 39, 40674070.
18 C. Oberthur, H. Graf and M. Hamburger, Phytochemistry, 2004,
65, 32613268.
19 E. T. Farinas, U. Schwaneberg, A. Glieder and F. H. Arnold,
Adv. Synth. Catal., 2001, 343, 601606.
20 A. Celik, G. A. Roberts, J. H. White, S. K. Chapman,
N. J. Turner and S. L. Flitsch, Chem. Commun., 2006,
21 L. Kass, CRC. Crit. Rev. Clin. Lab. Sci., 1979, 10, 205223.
22 F. Tanaka, L. Kerwin, D. Kubitz, R. A. Lerner and C. F. Barbas,
3rd, Bioorg. Med. Chem. Lett., 2001, 11, 29832986.
23 G. Klein and J. L. Reymond, Bioorg. Med. Chem. Lett., 1998, 8,
24 G. Klein and J. L. Reymond, Helv. Chim. Acta, 1999, 82,
25 N. Jourdain, R. P. Carlon and J. L. Reymond, Tetrahedron Lett.,
1998, 39, 94159418.
26 R. Perez Carlon, N. Jourdain and J. L. Reymond, Chemistry,
2000, 6, 41544162.
27 B. List, C. F. Barbas and R. A. Lerner, Proc. Natl. Acad. Sci.
U. S. A., 1998, 95, 1535115355.
28 J. Kofoed, T. Darbre and J. L. Reymond, Chem. Commun., 2006,
29 J. Kofoed, T. Darbre and J. L. Reymond, Org. Biomol. Chem.,
2006, 4, 32683281.
30 E. Gonzalez-Garcia, V. Helaine, G. Klein, M. Schuermann,
G. A. Sprenger, W. D. Fessner and J. L. Reymond, Chem.Eur.
J., 2003, 9, 893899.
31 A. Sevestre, V. Helaine, G. Guyot, C. Martin and L. Hecquet,
Tetrahedron Lett., 2003, 44, 827830.
32 E. Leroy, N. Bensel and J. L. Reymond, Adv. Synth. Catal., 2003,
345, 859865.
33 N. Bensel, M. T. Reymond and J. L. Reymond, Chemistry, 2001,
7, 46044612.
34 E. Leroy, N. Bensel and J. L. Reymond, Bioorg. Med. Chem.
Lett., 2003, 13, 21052108.
35 Y. Z. Yang, P. Babiak and J. L. Reymond, Helv. Chim. Acta,
2006, 89, 404415.
36 R. Sicard, L. S. Chen, A. J. Marsaioli and J. L. Reymond, Adv.
Synth. Catal., 2005, 347, 10411050.
37 B. Bicalho, L. S. Chen, J. Grognux, J. L. Reymond and
A. J. Marsaioli, J. Braz. Chem. Soc., 2004, 15, 911916.
38 E. Nyfeler, J. Grognux, D. Wahler and J. L. Reymond, Helv.
Chim. Acta, 2003, 86, 29192927.
39 E. M. Gonzalez-Garcia, J. Grognux, D. Wahler and
J. L. Reymond, Helv. Chim. Acta, 2003, 86, 24582470.
40 J. Grognux and J. L. Reymond, ChemBioChem, 2004, 5, 826831.
41 D. Lagarde, H. K. Nguyen, G. Ravot, D. Wahler, J. L. Reymond,
G. Hills, T. Veit and F. Lefevre, Org. Process Res. Dev., 2002, 6,
42 F. Badalassi, H. K. Nguyen, P. Crotti and J. L. Reymond, Helv.
Chim. Acta, 2002, 85, 30903098.
43 C. Bedia, J. Casas, V. Garcia, T. Levade and G. Fabrias,
ChemBioChem, 2007, 8, 642648.
44 M. Shamis, C. F. Barbas and D. Shabat, Bioorg. Med. Chem.
Lett., 2007, 17, 11721175.
45 P. D. Jones, N. M. Wolf, C. Morisseau, P. Whetstone, B. Hock
and B. D. Hammock, Anal. Biochem., 2005, 343, 6675.
46 R. J. Amir and D. Shabat, Chem. Commun., 2004, 16141615.
47 Y. Meyer, J. A. Richard, M. Massonneau, P. Y. Renard and
A. Romieu, Org. Lett., 2008, 10, 15171520.
48 Z. Q. Wang, J. F. Liao and Z. J. Diwu, Bioorg. Med. Chem. Lett.,
2005, 15, 23352338.
49 G. B. Jones, C. F. Crasto, J. E. Mathews, L. F. Xie,
M. O. Mitchell, A. El-Shafey, A. V. DAmico and G. J. Bubley,
Bioorg. Med. Chem., 2006, 14, 418425.
50 J. P. Germanas, S. Wang, A. Miner, W. Hao and J. M. Ready,
Bioorg. Med. Chem. Lett., 2007, 17, 68716875.
51 E. D. Matayoshi, G. T. Wang, G. A. Krat and J. Erickson,
Science, 1990, 247, 954958.
52 E. K. Kainmuller, E. P. Olle and W. Bannwarth, Chem.
Commun., 2005, 54595461.
53 E. K. Kainmuller and W. Bannwarth, Helv. Chim. Acta, 2006, 89,
54 R. Wareld, P. Bardelang, H. Saunders, W. C. Chan, C. Penfold,
R. James and N. R. Thomas, Org. Biomol. Chem., 2006, 4,
55 T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann and
P. Schwille, Proc. Natl. Acad. Sci. U. S. A., 2002, 99,
This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3446 | 43
56 J. H. Wosnick, C. M. Mello and T. M. Swager, J. Am. Chem.
Soc., 2005, 127, 34003405.
57 V. Boyer, S. Fort, T. P. Frandsen, M. Schulein, S. Cottaz and
H. Driguez, Chem.Eur. J., 2002, 8, 13891394.
58 G. Zandonella, L. Haalck, F. Spener, K. Faber, F. Paltauf and
A. Hermetter, Chirality, 1996, 8, 481489.
59 M. Duque, M. Graupner, H. Stutz, I. Wicher, R. Zechner,
F. Paltauf and A. Hermetter, J. Lipid Res., 1996, 37, 868876.
60 Y. Z. Yang, P. Babiak and J. L. Reymond, Org. Biomol. Chem.,
2006, 4, 17461754.
61 T. Maeda and S. Nishimura, Chemistry, 2008, 14, 478487.
62 F. Tanaka, R. Thayumanavan and C. F. Barbas, J. Am. Chem.
Soc., 2003, 125, 85238528.
63 F. Tanaka, N. Mase and C. F. Barbas, J. Am. Chem. Soc., 2004,
126, 36923693.
64 H. M. Guo, M. Minakawa and F. Tanaka, J. Org. Chem., 2008,
73, 39643966.
65 T. Murayama, T. Tanabe, H. Ikeda and A. Ueno, Bioorg. Med.
Chem., 2006, 14, 36913696.
66 D. S. Lawrence and Q. Z. Wang, ChemBioChem, 2007, 8,
67 M. S. Tremblay, M. Lee and D. Sames, Org. Lett., 2008, 10, 58.
68 M. Rosseneu, M. J. Taveirne, H. Caster and J. P. Vanbiervliet,
Eur. J. Biochem., 1985, 152, 195198.
69 S. S. Twining, Anal. Biochem., 1984, 143, 3034.
70 B. Law, R. Weissleder and C. H. Tung, Bioconjugate Chem., 2007,
18, 17011704.
71 A. L. James, J. D. Perry, C. Jay, D. Monget, J. W. Rasburn and
F. K. Gould, Lett. Appl. Microbiol., 2001, 33, 403408.
72 A. Celik, R. E. Speight and N. J. Turner, Chem. Commun., 2005,
73 G. Chen, D. J. Yee, N. G. Gubernator and D. Sames, J. Am.
Chem. Soc., 2005, 127, 45444545.
74 M. K. Froemming and D. Sames, Angew. Chem., Int. Ed., 2006,
45, 637642.
75 W. H. Zhou, M. P. Valley, J. Shultz, E. M. Hawkins, L. Bernad,
T. Good, D. Good, T. L. Riss, D. H. Klaubert and K. V. Wood,
J. Am. Chem. Soc., 2006, 128, 31223123.
76 J. A. Richard, L. Jean, A. Romieu, M. Massonneau, P.
Noack-Fraissignes and P. Y. Renard, Org. Lett., 2007, 9,
77 B. S. Montenecourt and D. E. Eveleigh, Appl. Environ.
Microbiol., 1977, 33, 178183.
78 Z. Yang and B. Xu, Chem. Commun., 2004, 24242425.
79 Z. Yang, P. L. Ho, G. Liang, K. H. Chow, Q. Wang, Y. Cao,
Z. Guo and B. Xu, J. Am. Chem. Soc., 2007, 129, 266267.
80 L. Adler-Abramovich, R. Perry, A. Sagi, E. Gazit and D. Shabat,
ChemBioChem, 2007, 8, 859862.
81 J. Hoogboom, K. Velonia, T. Rasing, A. E. Rowan and
R. J. Nolte, Chem. Commun., 2006, 434435.
82 M. Olsen, B. Iverson and G. Georgiou, Curr. Opin. Biotechnol.,
2000, 11, 331337.
83 M. J. Olsen, J. Gam, B. L. Iverson and G. Georgiou, Methods
Mol. Biol., 2003, 230, 329342.
84 M. J. Olsen, D. Stephens, D. Griths, P. Daugherty,
G. Georgiou and B. L. Iverson, Nat. Biotechnol., 2000, 18,
85 N. Varadarajan, J. Gam, M. J. Olsen, G. Georgiou and
B. L. Iverson, Proc. Natl. Acad. Sci. USA, 2005, 102, 68556860.
86 D. S. Tawk and A. D. Griths, Nat. Biotechnol., 1998, 16,
87 K. Bernath, M. Hai, E. Mastrobattista, A. D. Griths,
S. Magdassi and D. S. Tawk, Anal. Biochem., 2004, 325,
88 O. J. Miller, K. Bernath, J. J. Agresti, G. Amitai, B. T. Kelly,
E. Mastrobattista, V. Taly, S. Magdassi, D. S. Tawk and
A. D. Griths, Nat. Methods, 2006, 3, 561570.
89 A. Aharoni, K. Thieme, C. P. Chiu, S. Buchini, L. L. Lairson,
H. Chen, N. C. Strynadka, W. W. Wakarchuk and S. G. Withers,
Nat. Methods, 2006, 3, 609614.
90 S. Becker, H. Hobenreich, A. Vogel, J. Knorr, S. Wilhelm,
F. Rosenau, K. E. Jaeger, M. T. Reetz and H. Kolmar, Angew.
Chem., Int. Ed., 2008, 47, 50855088.
91 G. J. Williams, S. Domann, A. Nelson and A. Berry, Proc. Natl.
Acad. Sci. U. S. A., 2003, 100, 31433148.
92 T. Woodhall, G. Williams, A. Berry and A. Nelson, Angew.
Chem., Int. Ed., 2005, 44, 21092112.
93 M. Baumann, R. Sturmer and U. T. Bornscheuer, Angew. Chem.,
Int. Ed., 2001, 40, 42014204.
94 S. Bartsch, R. Kourist and U. T. Bornscheuer, Angew. Chem., Int.
Ed., 2008, 47, 15081511.
95 P. Abato and C. T. Seto, J. Am. Chem. Soc., 2001, 123,
96 S. Dey, K. R. Karukurichi, W. J. Shen and D. B. Berkowitz,
J. Am. Chem. Soc., 2005, 127, 86108611.
97 A. Hamberg, S. Lundgren, M. Penhoat, C. Moberg and K. Hult,
J. Am. Chem. Soc., 2006, 128, 22342235.
98 A. Hamberg, S. Lundgren, E. Wingstrand, C. Moberg and
K. Hult, Chem.Eur. J., 2007, 13, 43344341.
99 S. Dey, D. R. Powell, C. Hu and D. B. Berkowitz, Angew. Chem.,
Int. Ed., 2007, 46, 70107014.
100 Z. Li, L. Butikofer and B. Witholt, Angew. Chem., Int. Ed., 2004,
43, 16981702.
101 H. E. Mu ller, J. Microbiol. Methods, 1984, 2, 101102.
102 H. Joo, A. Arisawa, Z. L. Lin and F. H. Arnold, Chem. Biol.,
1999, 6, 699706.
103 H. Joo, Z. L. Lin and F. H. Arnold, Nature, 1999, 399, 670673.
104 M. Alexeeva, A. Enright, M. J. Dawson, M. Mahmoudian and
N. J. Turner, Angew. Chem., Int. Ed., 2002, 41, 31773180.
105 R. Carr, M. Alexeeva, A. Enright, T. S. C. Eve, M. J. Dawson
and N. J. Turner, Angew. Chem., Int. Ed., 2003, 42, 48074810.
106 M. D. Truppo, F. Escalettes and N. J. Turner, Angew. Chem., Int.
Ed., 2008, 47, 26392641.
107 R. A. Dicioccio, C. Piskorz, G. Salamida, J. J. Barlow and
K. L. Matta, Anal. Biochem., 1981, 111, 176183.
108 H. Vankayalapati and G. Singh, Tetrahedron Lett., 1999, 40,
109 D. Indurugalla, J. N. Watson and A. J. Bennet, Org. Biomol.
Chem., 2006, 4, 44534459.
110 C. Mayer, D. L. Jakeman, M. Mah, G. Karjala, L. Gal, R. A.
J. Warren and S. G. Withers, Chem. Biol., 2001, 8, 437443.
111 C. Garciaecheverria, J. L. Kofron, P. Kuzmic, V. Kishore and
D. H. Rich, J. Am. Chem. Soc., 1992, 114, 27582759.
112 J. L. Kofron, P. Kuzmic, V. Kishore, E. Colonbonilla and
D. H. Rich, Biochemistry, 1991, 30, 61276134.
113 J. T. YliKauhaluoma, J. A. Ashley, C. H. L. Lo, J. Coakley,
P. Wirsching and K. D. Janda, J. Am. Chem. Soc., 1996, 118,
114 K. Kupcho, R. Somberg, B. Bulleit and S. A. Goueli, Anal.
Biochem., 2003, 317, 210217.
115 S. M. Rodems, B. D. Hamman, C. Lin, J. Zhao, S. Shah,
D. Heidary, L. Makings, J. H. Stack and B. A. Pollok, Assay
Drug Dev. Technol., 2002, 1, 919.
116 K. T. Nguyen and D. Pei, Methods Mol. Med., 2008, 142,
117 O. Molin, L. Nilsson and S. Ansehn, J. Clin. Microbiol., 1983, 18,
118 H. Shulman, A. Eberhard, C. Eberhard, S. Ulitzur and
E. Keinan, Bioorg. Med. Chem. Lett., 2000, 10, 23532356.
119 G. Krebs, L. Hugonet and J. D. Sutherland, Angew. Chem., Int.
Ed., 2006, 45, 301305.
120 L. Galam, M. K. Hadden, Z. Q. Ma, Q. Z. Ye, B. G. Yun, B. S.
J. Blagg and R. L. Matts, Bioorg. Med. Chem., 2007, 15,
121 D. S. Auld, N. T. Southall, A. Jadhav, R. L. Johnson, D. J. Diller,
A. Simeonov, C. P. Austin and J. Inglese, J. Med. Chem., 2008,
51, 23722386.
122 L. E. Janes and R. J. Kazlauskas, J. Org. Chem., 1997, 62,
123 L. E. Janes, A. C. Lowendahl and R. J. Kazlauskas, Chem.Eur.
J., 1998, 4, 23242331.
124 C. B. Park and D. S. Clark, Biotechnol. Bioeng., 2002, 78,
125 M. Persson and M. M. Palcic, Anal. Biochem., 2008, 378, 17.
126 A. Ben-David, G. Shoham and Y. Shoham, Chem. Biol., 2008, 15,
127 S. J. C. Taylor, R. C. Brown, P. A. Keene and I. N. Taylor,
Bioorg. Med. Chem., 1999, 7, 21632168.
128 E. Henke and U. T. Bornscheuer, Anal. Chem., 2003, 75,
44 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009
129 A. Banerjee, R. Sharma and U. C. Banerjee, Biotechnol. Appl.
Biochem., 2003, 37, 289293.
130 M. Konarzycka-Bessler and U. T. Bornscheuer, Angew. Chem.,
Int. Ed., 2003, 42, 14181420.
131 S. Salahuddin, O. Renaudet and J. L. Reymond, Org. Biomol.
Chem., 2004, 2, 14711475.
132 C. Mateo, A. Archelas and R. Furstoss, Anal. Biochem., 2003,
314, 135141.
133 K. Doderer, S. Lutz-Wahl, B. Hauer and R. D. Schmid, Anal.
Biochem., 2003, 321, 131134.
134 F. Zocher, M. M. Enzelberger, U. T. Bornscheuer, B. Hauer and
R. D. Schmid, Anal. Chim. Acta, 1999, 391, 345351.
135 H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki and
N. Itoh, Angew. Chem., Int. Ed., 2005, 44, 29222925.
136 O. Khersonsky and D. S. Tawk, ChemBioChem, 2006, 7, 4953.
137 A. Ojida, Y. Mito-oka, M. Inoue and I. Hamachi, J. Am. Chem.
Soc., 2002, 124, 62566258.
138 S. Yamaguchi, L. Yoshimura, T. Kohira, S. Tamaru and
I. Hamachi, J. Am. Chem. Soc., 2005, 127, 1183511841.
139 K. Martin, T. H. Steinberg, L. A. Cooley, K. R. Gee,
J. M. Beechem and W. F. Patton, Proteomics, 2003, 3, 12441255.
140 J. Wongkongkatep, Y. Miyahara, A. Ojida and I. Hamachi,
Angew. Chem., Int. Ed., 2006, 45, 665668.
141 C. E. Humphrey, M. A. M. Easson and N. J. Turner, ChemBioChem,
2004, 5, 11441148.
142 A. L. L. Duchateau, M. G. Hillemans-Crombach, A. van
Duijnhoven, R. Reiss and T. Sonke, Anal. Biochem., 2004, 330,
143 G. Klein, D. Kaufmann, S. Schurch and J. L. Reymond, Chem.
Commun., 2001, 561562.
144 K. E. S. Dean, G. Klein, O. Renaudet and J. L. Reymond, Bioorg.
Med. Chem. Lett., 2003, 13, 16531656.
145 D. Wahler and J. L. Reymond, Angew. Chem., Int. Ed., 2002, 41,
146 J. L. Reymond and V. Fluxa, Nat. Protoc., 2008, 3, 1270.
147 D. Wahler, O. Boujard, F. Lefevre and J. L. Reymond,
Tetrahedron, 2004, 60, 703710.
148 D. Kahakeaw and M. T. Reetz, Chem.Asian J., 2008, 3, 233238.
149 K. Doderer and R. D. Schmid, Biotechnol. Lett., 2004, 26,
150 B. van Loo, J. H. L. Spelberg, J. Kingma, T. Sonke,
M. G. Wubbolts and D. B. Janssen, Chem. Biol., 2004, 11,
151 D. S. Tawk, B. S. Green, R. Chap, M. Sela and Z. Eshhar, Proc.
Natl. Acad. Sci. USA, 1993, 90, 373377.
152 G. Macbeath and D. Hilvert, J. Am. Chem. Soc., 1994, 116,
153 F. Benedetti, F. Berti, M. Flego, M. Resmini and E. Bastiani,
Anal. Biochem., 1998, 256, 6773.
154 P. Geymayer, N. Bahr and J. L. Reymond, Chem.Eur. J., 1999,
5, 10061012.
155 F. Taran, P. Y. Renard, C. Creminon, A. Valleix, Y. Frobert,
P. Pradelles, J. Grassi and C. Mioskowski, Tetrahedron Lett.,
1999, 40, 18871890.
156 F. Taran, P. Y. Renard, C. Creminon, A. Valleix, Y. Frobert,
P. Pradelles, J. Grassi and C. Mioskowski, Tetrahedron Lett.,
1999, 40, 18911894.
157 R. Nutiu, J. M. Y. Yu and Y. F. Li, ChemBioChem, 2004, 5,
158 P. F. Zatta, K. Nyame, M. J. Cormier, S. A. Mattox, P. A. Prieto,
D. F. Smith and R. D. Cummings, Anal. Biochem., 1991, 194,
159 L. S. Khraltsova, M. A. Sablina, T. D. Melikhova, D. H. Joziasse,
H. Kaltner, H. J. Gabius and N. V. Bovin, Anal. Biochem., 2000,
280, 250257.
160 M. C. Bryan, L. V. Lee and C. H. Wong, Bioorg. Med. Chem.
Lett., 2004, 14, 31853188.
161 M. C. Bryan, O. Plettenburg, P. Sears, D. Rabuka, S. Wacowich-
Sgarbi and C. H. Wong, Chem. Biol., 2002, 9, 713720.
162 G. Das, P. Talukdar and S. Matile, Science, 2002, 298,
163 N. Sorde, G. Das and S. Matile, Proc. Natl. Acad. Sci. U. S. A.,
2003, 100, 1196411969.
164 T. Miyatake, M. Nishihara and S. Matile, J. Am. Chem. Soc.,
2006, 128, 1242012421.
165 S. M. Buttereld, D. H. Tran, H. Zhang, G. D. Prestwich and
S. Matile, J. Am. Chem. Soc., 2008, 130, 32703271.
166 H. Tanaka and S. Matile, Chirality, 2008, 20, 307312.
167 Y. Xiao, V. Pavlov, S. Levine, T. Niazov, G. Markovitch and
I. Willner, Angew. Chem., Int. Ed., 2004, 43, 45194522.
168 V. Pavlov, Y. Xiao and I. Willner, Nano Lett., 2005, 5, 649653.
169 R. Baron, M. Zayats and I. Willner, Anal. Chem., 2005, 77,
170 Z. Wang, J. Lee, A. R. Cossins and M. Brust, Anal. Chem., 2005,
77, 57705774.
171 Z. Wang, R. Levy, D. G. Fernig and M. Brust, J. Am. Chem. Soc.,
2006, 128, 22142215.
172 C. Guarise, L. Pasquato, V. De Filippis and P. Scrimin, Proc.
Natl. Acad. Sci. USA, 2006, 103, 39783982.
173 Y. Choi, N. H. Ho and C. H. Tung, Angew. Chem., Int. Ed., 2006,
46, 707709.
174 W. Zhao, W. Chiuman, J. C. Lam, M. A. Brook and Y. Li, Chem.
Commun., 2007, 37293731.
175 X. Xu, M. S. Han and C. A. Mirkin, Angew. Chem., Int. Ed.,
2007, 46, 34683470.
176 J. Buissie` r, A. Fourcard and L. Colobert, C. R. Hebd. Seances
Acad. Sci., Ser. D, 1967, 264, 415417.
177 P. Nardon, D. Monget, M. L. Didierchet and G. Dethe,
Biomedicine, 1976, 24, 183190.
178 M. W. Humble, A. King and I. Phillips, J. Clin. Pathol., 1977, 30,
179 E. Gruner, A. Vongraevenitz and M. Altwegg, J. Microbiol.
Methods, 1992, 16, 101118.
180 P. Garcia-Martos, P. Marin, J. M. Hernandez-Molina, L. Garcia-
Agudo, S. Aou and J. Mira, Mycopathologia, 2001, 150, 14.
181 J. L. Reymond and D. Wahler, ChemBioChem, 2002, 3, 701708.
182 A. Nath and W. M. Atkins, Biochemistry, 2008, 47, 157166.
183 A. Taglieber, H. Hobenreich, J. D. Carballeira, R. J. Mondiere
and M. T. Reetz, Angew. Chem., Int. Ed., 2007, 46, 85978600.
184 Y. S. Liu, M. P. Patricelli and B. F. Cravatt, Proc. Natl. Acad.
Sci. U. S. A., 1999, 96, 1469414699.
185 W. Li, J. L. Blankman and B. F. Cravatt, J. Am. Chem. Soc.,
2007, 129, 95949595.
186 D. Greenbaum, K. F. Medzihradszky, A. Burlingame and
M. Bogyo, Chem. Biol., 2000, 7, 569581.
187 H. Schmidinger, R. Birner-Gruenberger, G. Riesenhuber, R. Saf,
H. Susani-Etzerodt and A. Hermetter, ChemBioChem, 2005, 6,
188 R. Nakai, C. M. Salisbury, H. Rosen and B. F. Cravatt, Bioorg.
Med. Chem., 2008, DOI: 10.1016/j.bmc.2008.03.018.
189 A. Borodovsky, H. Ovaa, W. J. N. Meester, E. S. Venanzi,
M. S. Bogyo, B. G. Hekking, H. L. Ploegh, B. M. Kessler and
H. S. Overkleeft, ChemBioChem, 2005, 6, 287291.
190 D. C. Greenbaum, W. D. Arnold, F. Lu, L. Hayrapetian,
A. Baruch, J. Krumrine, S. Toba, K. Chehade, D. Bromme,
I. D. Kuntz and M. Bogyo, Chem. Biol., 2002, 9, 10851094.
191 M. Bogyo, S. Verhelst, V. Bellingard-Dubouchaud, S. Toba and
D. Greenbaum, Chem. Biol., 2000, 7, 2738.
192 R. Srinivasan, X. Huang, S. L. Ng and S. Q. Yao, ChemBioChem,
2006, 7, 3236.
193 A. M. F. Liu, N. A. Somers, R. J. Kazlauskas, T. S. Brush,
F. Zocher, M. M. Enzelberger, U. T. Bornscheuer,
G. P. Horsman, A. Mezzetti, C. Schmidt-Dannert and
R. D. Schmid, Tetrahedron: Asymmetry, 2001, 12, 545556.
194 D. Wahler, F. Badalassi, P. Crotti and J. L. Reymond, Angew.
Chem., Int. Ed., 2001, 40, 44574460.
195 D. Wahler, F. Badalassi, P. Crotti and J. L. Reymond, Chemistry,
2002, 8, 32113228.
196 J. C. Powers, J. L. Asgian, O. D. Ekici and K. E. James, Chem.
Rev., 2002, 102, 46394750.
197 M. Meldal, QSAR Comb. Sci., 2005, 24, 11411148.
198 X. S. Puente, L. M. Sanchez, C. M. Overall and C. Lopez-Otin,
Nat. Rev. Genet., 2003, 4, 544558.
199 P. L. Richardson, Curr. Pharmaceut. Des., 2002, 8, 25592581.
200 D. J. Maly, L. Huang and J. A. Ellman, ChemBioChem, 2002, 3,
201 N. A. Thornberry, T. A. Rano, E. P. Peterson, D. M. Rasper,
T. Timkey, M. Garcia-Calvo, V. M. Houtzager, P. A. Nordstrom,
S. Roy, J. P. Vaillancourt, K. T. Chapman and D. W. Nicholson,
J. Biol. Chem., 1997, 272, 1790717911.
This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3446 | 45
202 J. L. Harris, B. J. Backes, F. Leonetti, S. Mahrus, J. A. Ellman
and C. S. Craik, Proc. Natl. Acad. Sci. USA, 2000, 97, 77547759.
203 J. L. Harris, P. B. Alper, J. Li, M. Rechsteiner and B. J. Backes,
Chem. Biol., 2001, 8, 11311141.
204 T. A. Rano, T. Timkey, E. P. Peterson, J. Rotonda,
D. W. Nicholson, J. W. Becker, K. T. Chapman and
N. A. Thornberry, Chem. Biol., 1997, 4, 149155.
205 C. Pinilla, J. R. Appel, P. Blanc and R. A. Houghten, Biotechniques,
1992, 13, 901905.
206 A. F. Kisselev, M. Garcia-Calvo, H. S. Overkleeft, E. Peterson,
M. W. Pennington, H. L. Ploegh, N. A. Thornberry and
A. L. Goldberg, J. Biol. Chem., 2003, 278, 3586935877.
207 Y. Choe, F. Leonetti, D. C. Greenbaum, F. Lecaille, M. Bogyo,
D. Bromme, J. A. Ellman and C. S. Craik, J. Biol. Chem., 2006,
281, 1282412832.
208 S. S. Cotrin, L. Puzer, W. A. D. Judice, L. Juliano,
A. K. Carmona and M. A. Juliano, Anal. Biochem., 2004, 335,
209 K. Breddam and M. Meldal, Eur. J. Biochem., 1992, 206,
210 M. Meldal, I. Svendsen, K. Breddam and F. I. Auzanneau, Proc.
Natl. Acad. Sci. U. S. A., 1994, 91, 33143318.
211 P. M. St Hilaire, M. Willert, M. A. Juliano, L. Juliano and
M. Meldal, J. Comb. Chem., 1999, 1, 509523.
212 F. M. Alves, I. Y. Hirata, I. E. Gouvea, M. F. M. Alves,
M. Meldal, D. Bromme, L. Juliano and M. A. Juliano,
J. Comb. Chem., 2007, 9, 627634.
213 O. D. Ekici, A. Karla, M. Paetzel, M. O. Lively, D. H. Pei and
R. E. Dalbey, J. Biol. Chem., 2007, 282, 417425.
214 J. Kofoed and J. L. Reymond, Chem. Commun., 2007, 44534455.
215 J. P. Goddard and J. L. Reymond, J. Am. Chem. Soc., 2004, 126,
216 Y. Yongzheng and J. L. Reymond, Mol. Biosyst., 2005, 1, 5763.
217 R. Sicard, J. P. Goddard, M. Mazel, C. Audirin, L. Fourage,
G. Ravot, D. Wahler, F. Lefevre and J. L. Reymond, Adv. Synth.
Catal., 2005, 347, 987996.
218 S. Park and I. Shin, Org. Lett., 2007, 9, 619622.
219 A. Horeau and A. Nouaille, Tetrahedron Lett., 1990, 31,
220 A. Schoofs and A. Horeau, Tetrahedron Lett., 1977, 32593262.
221 J. H. Guo, J. Y. Wu, G. Siuzdak and M. G. Finn, Angew. Chem.,
Int. Ed., 1999, 38, 17551758.
222 M. T. Reetz, M. H. Becker, H. W. Klein and D. Stockigt, Angew.
Chem., Int. Ed., 1999, 38, 17581761.
223 D. X. Zha, A. Eipper and M. T. Reetz, ChemBioChem, 2003, 4,
224 M. T. Reetz, C. Torre, A. Eipper, R. Lohmer, M. Hermes,
B. Brunner, A. Maichele, M. Bocola, M. Arand, A. Cronin,
Y. Genzel, A. Archelas and R. Furstoss, Org. Lett., 2004, 6,
225 G. DeSantis, K. Wong, B. Farwell, K. Chatman, Z. L. Zhu,
G. Tomlinson, H. J. Huang, X. Q. Tan, L. Bibbs, P. Chen,
K. Kretz and M. J. Burk, J. Am. Chem. Soc., 2003, 125,
226 C. J. Zea, S. W. MacDonell and N. L. Pohl, J. Am. Chem. Soc.,
2003, 125, 1366613667.
227 Y. Yu, K. S. Ko, C. J. Zea and N. L. Pohl, Org. Lett., 2004, 6,
228 M. Yang, M. Brazier, R. Edwards and B. G. Davis, ChemBio-
Chem, 2005, 6, 346357.
229 N. Nagahori and S. I. Nishimura, Chem.Eur. J., 2006, 12,
230 M. Yang, G. J. Davies and B. G. Davis, Angew. Chem., Int. Ed.,
2007, 46, 38853888.
231 F. Basile, I. Ferrer, E. T. Furlong and K. J. Voorhees, Anal.
Chem., 2002, 74, 42904293.
232 D. N. Gosalia and S. L. Diamond, Proc. Natl. Acad. Sci. U. S. A.,
2003, 100, 87218726.
233 M. Uttamchandani, X. Huang, G. Y. J. Chen and S. Q. Yao,
Bioorg. Med. Chem. Lett., 2005, 15, 21352139.
234 K. T. Kotz, Y. Gu and G. W. Faris, J. Am. Chem. Soc., 2005, 127,
235 C. M. Salisbury, D. J. Maly and J. A. Ellman, J. Am. Chem. Soc.,
2002, 124, 1486814870.
236 Q. Zhu, M. Uttamchandani, D. B. Li, M. L. Lesaicherre and
S. Q. Yao, Org. Lett., 2003, 5, 12571260.
237 J. Grognux and J. L. Reymond, Mol. Biosyst., 2006, 2,
238 N. Winssinger, R. Damoiseaux, D. C. Tully, B. H. Geierstanger,
K. Burdick and J. L. Harris, Chem. Biol., 2004, 11, 13511360.
239 J. L. Harris and N. Winssinger, Chem.Eur. J., 2005, 11,
240 F. Debaene, L. Mejias, J. L. Harris and N. Winssinger,
Tetrahedron, 2004, 60, 86778690.
241 J. J. Diaz-Mochon, L. Bialy and M. Bradley, Chem. Commun.,
2006, 39843986.
242 H. Sun, C. H. Lu, M. Uttamchandani, Y. Xia, Y. C. Liou and
S. Q. Yao, Angew. Chem., Int. Ed., 2008, 47, 16981702.
243 I. A. Kozlov, P. C. Melnyk, J. P. Hachmann, A. Srinivasan,
M. Shults, C. F. Zhao, J. Musmacker, N. Nelson, D. L. Barker
and M. Lebl, Comb. Chem. High Throughput Screening, 2008, 11,
244 G. A. Korbel, G. Lalic and M. D. Shair, J. Am. Chem. Soc., 2001,
123, 361362.
245 A. Horeau, Tetrahedron Lett., 1961, 506512.
246 A. Horeau, Tetrahedron Lett., 1962, 965969.
247 L. Ban and M. Mrksich, Angew. Chem., Int. Ed., 2008, 47,
248 P. Babiak and J. L. Reymond, Anal. Chem., 2005, 77,
46 | Chem. Commun., 2009, 3446 This journal is c The Royal Society of Chemistry 2009