Dendrimers and combinatorial chemistry - Tools for fluorescent enhancement in protease assays

Article abstract of DOI:10.1016/j.tet.2004.05.105

FRET based systems are some of the best methods available to detect and monitor proteolytic activity. To enhance fluorescent signals and hence assay sensitivity, two different systems were developed using two different dendrimeric constructs. In the first

Full text of DOI:10.1016/j.tet.2004.05.105

Tetrahedron 60 (2004) 8721–8728
Dendrimers and combinatorial chemistry—tools for fluorescent
enhancement in protease assays
*
´ ´ ´
¨
Michael Ternon, Juan Jose Dıaz-Mochon, Adam Belsom and Mark Bradley
School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
Received 19 November 2003; accepted 4 May 2004
Available online 2 July 2004
Abstract—FRET based systems are some of the best methods available to detect and monitor proteolytic activity. To enhance fluorescent
signals and hence assay sensitivity, two different systems were developed using two different dendrimeric constructs. In the first case, a triple
branched dendrimer bearing three dansyl groups was used to enhance assay sensitivity and showed a significant enhancement of fluorescence
following enzymatic cleavage. In another example, a tris-fluorescein probe, that undergoes self-quenching, was utilized in a combinatorial
library synthesis to map the substrate specificity of proteases.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Combinatorial chemistry, including ‘split and mix’ synthesis,
represents a key method for the identification and character-
isation of new proteases. This methodology has allowed rapid
progress to be made in the determination of the optimal
proteolytic substrate for a protease6 and provides essential
information for the understanding of complex biological
pathways and for the design of inhibitors.7 We herein describe
the use of multilabelled systems to amplify the fluorescent
signal following enzymatic cleavage and their application to
define the substrate specificity of proteases using combina-
torial multi-chromophoric libraries.
Proteases are involved in numerous essential biological
processes, and one of the first steps in understanding any
new protease is the determination of its substrate specificity.
Many assays for proteases are based on fluorescent probes
due to the high sensitivity of this technique and fluorescence
resonance energy transfer (FRET) methods in particular
have been extensively exploited. In the FRET method,
donor and quencher moieties are separated on a peptide
chain to give rise to an internally quenched fluorescent
peptide. This fluorescence is regained following cleavage of
the peptide by a protease and it is this increase in
fluorescence signal that forms the basis of the FRET
assay. Many donor/quencher couples have been described in
the literature with varying solubilities in aqueous or organic
solvents and efficiency of energy transfer.1
2. Results and discussion
2.1. Dendrimers and amplification monomers
A multivalency system bearing a variety of chromophores
should be able to reduce the detection limits of bioassays such
asproteasedetection, orincreasethelengthofsequencingruns
in fluorescence based sequencing. The AB3 type monomers,
firstly developed by Newkome et al.,8 were recently extended
and enhanced in utility by our group9,10 and these multivalent
compoundswereusedasabasistoamplify signalsbycoupling
chromophores to the terminal functional groups.5 Two
different strategies were chosen to monitor enzymatic
cleavage. The first was based on a FRET tri-donor/mono-
quenchersystem1 and used Dansyl as the donor and Dabsyl as
a quencher. The second involved a self-quenched derivative2,
using a single fluorophore (fluorescein) in which the peptidic
part consisted of a combinatorial library of 1000 peptides
(10£10£10) and was used to define protease substrate
specificity (Fig. 1).
Multiplying the number of dyes on an internally quenched
peptide could increase the sensitivity of the assay and
reduce detection levels. Although dendrimers2 bearing
fluorophores have been described in the literature,3 it is
only very recently that research groups have shown their
possible use as a highly sensitive tool for biological assay
amplification.4 Multi-dyes derivatives however can show
two types of behaviour: (a) significant amplification of
fluorescence directly related to the number of chromo-
phores, (b) a self-quenching phenomenon5 for dendrimers
displaying small Stoke’s shift fluorophores.
Keywords: FRET; Enzymatic cleavage; Substrate specificity;
Combinatorial libraries; Self-quenching.
*
Corresponding author. Tel.: þ44-23-8059-3598; fax: þ44-23-8059-6766;
e-mail address: mb14@soton.ac.uk
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.05.105

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M. Ternon et al. / Tetrahedron 60 (2004) 8721–8728
The amplification isocyanates 8 and 9 were synthesised in
six steps as shown in Scheme 1.10 Thus, the Michael
addition of 1,1,1-tris(hydroxymethyl)amino-methane onto
acrylonitrile, followed by amino protection (Boc), and
reduction of the nitrile groups with BH3·THF gave 3. This
was reacted with either Dde-OH to give the Dde (2-acetyl-
dimedone) protected amine 4 or the Dansyl donor group to
give 5. Following removal of the Boc protecting group, the
isocyanates 8 and 9 were prepared following the procedure
of Knolker (Scheme 1).11 Monomer 8 was coupled directly to
¨
the resin via the isocyanate and the free amino groups of the
amplification monomer were liberated with hydrazine. The
fully formed tri-fluorophore construct 9 could be coupled
directly to the free amine groups of the resin linked peptide or
used in solution phase synthesis.
Figure 2. Fluorescence spectra of the peptides 10a and 10b (both at
10 mM). Ex¼335 nm, Em¼545 nm (10a) and 560 nm (10b). The tri-
labelled peptide 10a (red) showed a much more significant increase in
fluorescence than the control peptide 10b (blue).
control peptide of 1-donor/1-quencher 10b was also prepared
to compare the differences of fluorescence intensity between
this and the tri-labelled derivative 10a. Peptides 10a and 10b
were precipitated in cold ether, centrifuged and purified by
HPLC before analysis (.95%) (Scheme 2).
2.2. FRET-amplified substrate synthesis:
3-donors/1-quencher
The Dansyl/Dabsyl couple was described by Hartwig as a
‘couple’ to monitor Heck chemistry during catalyst screen-
ing.12 These two dyes are easy to handle in solution or solid
phase and display efficient energy transfer properties.
Jakubke described the peptidic sequence Gly-Pro-Ala-Lys-
Leu-Ala-Ile-Gly as a good substrate for trypsin with a
cleavage site between lysine and leucine.13 Peptides 10a and
10b were constructed on solid phase following an Fmoc-
strategy using a hydroxymethylphenoxyacetic linker
attached to aminomethyl PS resin. The tri-labelled iso-
cyanate9wasgraftedontothe peptidesN-terminusafterafinal
Fmoc deprotection. The first amino acid (Lys(Ddiv)-OH),14 at
the beginning of the peptide sequence, allowed dabsyl
derivatisation after Ddiv-deprotection with hydrazine. A
Enzymatic cleavage was performed on the substrates
(excitation at 335 nm and emission at 545 or 560 nm) as
shown in Figure 1 and Table 1 demonstrating significant
increases in fluorescence in both cases.
Despite the long interchromophore distance, the Dansyl/
Dabsyl couple showed good quenching efficiency, which
was enhanced for the tribranched-labelled peptide (97%). At
equal concentrations (10 mM), the enzymatic cleavage of
tri-branched dansyl derivative 10a (amplification by 28.4)
shows a significant enhancement over the control peptide
10b (amplification by 6.1) and the signal-to-noise ratio
Figure 1. Internally quenched dendritic peptides 1 and 2 which undergo FRET (1) and self-quenching (2) processes.
Scheme 1. Synthesis of the 1!3 C-branched tris-based isocyanate amplification monomers 8 and 9. (i) acrylonitrile, KOH 40%, dioxane; (ii) Boc2O, Et3N; (iii)
BH3·THF, dioxane, 55 8C; (iv) DdeOH, DIPEA for 4 or Dansyl chloride, Et3N for 5; (v) 20% TFA in CH2Cl2; (vi) Boc2O, DMAP.

M. Ternon et al. / Tetrahedron 60 (2004) 8721–8728
8723
Scheme 2. Synthesis of the FRET-amplified substrate 10a and the control peptide 10b. (i) 20% piperidine; (ii) for 10a: monomer 9, DIPEA, DMAP. For 10b:
Dansyl-Cl, NEt3; (iii) 2% hydrazine in DMF; (iv) Dabsyl-Cl, NEt3; (v) TFA/CH2Cl2/TIS 95/3/2.
increased by a factor 4.6 (Fig. 2). Clearly, in this case, the
FRET-amplified substrate was not subjected to a self-
quenching process, due to its long Stoke’s shift.
The ability of an enzyme to recognise a substrate selectively
in a pool of thousands of potential substrates provides vital
information about its mode of action. Split and mix
synthesis makes the synthesis rapid and subsequent screen-
ing of thousands of peptide targets possible.16,17
2.3. FRET-amplified substrate synthesis: self-quenching
system
Here, the determination of the substrate specificity of two
model proteases, papain and trypsin is reported by the
Another approach for assaying protease activity relies on
multiple copies of a single type of fluorophore, giving rise
internally quenched fluorescent peptides. This method
offers a very simple method of ‘internal FRET’ and was
initially described in our group as a method of assaying
proteases but can be applied in variety of biological assays.
The self-quenching process occurs for small Stoke’s shift
dyes, when the emission wavelength and the excitation
wavelength of the fluorophore are close to each other (4(5)-
carboxyfluorescein lex¼495 nm, lem¼520 nm). In effect,
the dendrimer generates a high-local concentration of
fluorophore permitting efficient quenching while peptide
hydrolysis releases multiple copies of the dyes causing a
large increase in emission.
Herein, we show for the first time, that this process of
‘internal quenching’ can be used in a combinatorial
chemistry screening sense, to profile protease specificity.
FRET-based libraries of course have been widely exploited
to investigate the design of the preferred enzyme substrate,15,6
but a single fluorophore approach would simplify the whole
process, the substrates would be chemically much more
accessible, and cleavage could occur anywhere in the
peptide chain.
Scheme 3. Synthesis of the self-quenched substrate 2 (i) 2% hydrazine in
DMF; (ii) (a) Fmoc-amino acid X1 (10 equiv.), HOBt (10 equiv.), DIC
(10 equiv.), (b) 20% piperidine in DMF; (iii) (a) Fmoc-amino acid X2
(5 equiv.), HOBt (5 equiv.), DIC (5 equiv.), (b) 20% piperidine in DMF;
(iv) (a) Fmoc-amino acid X3 (5 equiv.), HOBt (5 equiv.), DIC (5 equiv.),
(b) 20% piperidine in DMF; (v) (a) g-aminobutyric acid (2 equiv.), HOBt
(2 equiv.), DIC (2 equiv.) (b) 20% piperidine in DMF; (vi) 4(5)-
carboxyfluorescein (10 equiv.), HOBt (10 equiv.), DIC (10 equiv.); (vii)
TFA/TIS/CH2Cl2 (95/2/3).
Table 1. Fluorescence intensity of the peptides 10a and 10b before and
after adding trypsin (arbitrary units)
Peptide
Starting
fluorescence
Final
fluorescence
Quenching
efficiency
Amplification
10b
10a
27
16
165
455
84
97
6.1
28.4

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M. Ternon et al. / Tetrahedron 60 (2004) 8721–8728
Figure 3. Screening of the 30 self-quenching sub-libraries against papain.
screening of an internally quenched combinatorial peptide
library. Three tripeptide sub-libraries were synthesised on
the tri-branched resin using the split and mix method
(Scheme 3). Monomer 8 was coupled to the Rink linker
attached to the aminomethyl PS resin. This resin was
deprotected with hydrazine (2% in DMF) and divided into
10 equal sized pools. The X3 position was fixed in the first
library, X2 in the second library and X1 position in the third
library. To minimize the size of the libraries, ten amino
acids with differing properties were chosen (Ala, Arg, Asn,
Asp, Leu, Lys, Phe, Pro, Ser and Tyr). Although quite
simple, logistically it becomes complex as 100 individual
reactions have to be carried out at this split stage. The first
amino acid was coupled following standard Fmoc strategy
but because of the bulk of the dendrimer backbone, the
reactivity of the terminal amine group was affected and
some coupling reactions were performed a second time to
ensure complete reaction. The second and the third amino
acids were attached following the same procedure. The
introduction of a spacer was vital; too long results in a
decrease of the self-quenching efficiency and too small
affects the access of the enzyme to the cleavage site. A 4-
carbon spacer, g-aminobutyric acid, was therefore attached to
the backbone (a previous study showed poor hydrolysis
kinetics for peptides on dendrimers without spacers).
Carboxyfluorescein was finally coupled onto the N-termini
of all the 30 sub-libraries. The multi labelled peptides were
released from the resin with TFA removing, at the same time,
side chain protecting groups. The sub-libraries were dissolved
in buffer and the concentrations of each of the 30 sub-libraries
(each of which contained 100 different tripeptides) were
checked by UV spectroscopy (495 nm) to ensure identical
concentrations of peptides in each assay.
2.4. Peptide libraries screenings
The 30 sub-libraries were screened against papain, with
excitation at 495 nm (fluorescence at 520 nm). Fluorescent
enhancement was monitored for 20 min after adding the
enzyme (Fig. 3). Library 1 (X3 fixed) showed selective
cleavage for Tyr and Phe. The second library (X2 fixed)
demonstrated a preference for Ala, Arg and Tyr and the
third library (X1 fixed) for small amino acids such as Ser and
Ala. These results were consistent with the literature,18
(with library 1 representing the P2 position, library 2 the P1
position and library 3 the P10 position).
To demonstrate the further potential of the library, the
library was screened against another enzyme, trypsin. The
results of the screening showed a high preference for basic
Figure 4. Screening of the 30 self-quenching sub-libraries against trypsin.

M. Ternon et al. / Tetrahedron 60 (2004) 8721–8728
8725
amino acid residues (Arg and Lys) as cleavage points in all
three sub-libraries (Fig. 4). Trypsin reaches its full
hydrolytic efficiency with positively charged side chain
residues and presumably, this factor is causing bias with
cleavage occurring at all three positions regardless of their
location making it difficult to get reasonable data for the
specific cleavage site a limitation of the method that has
already been observed in other systems.18
naphthalene-1-sulfonic acid]propoxymethyl amide}-
ethyl] carbamic acid tert-butyl ester (5). A solution of 3
(400 mg, 1 mmol), dansyl chloride (891 mg, 3.3 mmol,
1.1 equiv.) and triethylamine (335 mg, 3.3 mmol,
1.1 equiv.) in CH2Cl2 (20 ml) was stirred for 4 days. The
residue was poured into brine (20 ml) and extracted with
CH2Cl2 (2£10 ml). The combined organic layers were
washed with water (20 ml), dried over MgSO4 and the
solvent removed in vacuo. The crude product was purified
by column chromatography (CH2Cl2/MeOH 98:2) to give 5
(524 mg, 48%) as a yellow solid. 1H NMR (300 MHz,
CDCl3) d 1.30 (s, 9H), 1.58 (quint, J¼6.0 Hz, 6H), 2.80 (s,
18H), 2.94 (t, J¼6.0 Hz, 6H), 3.22 (s, 6H), 3.38 (t,
J¼5.5 Hz, 6H), 5.50 (br s, 3H), 7.08 (d, J¼7.0 Hz, 3H),
7.38–7.49 (m, 6H), 8.15 (d, J¼7.5 Hz, 3H), 8.24 (d,
J¼8.5 Hz, 3H), 8.44 (d, J¼8.5 Hz, 3H); 13C NMR
(100 MHz CDCl3) d 27.5, 28.2, 40.7, 44.6, 52.6, 57.5,
69.0, 69.6, 78.7, 114.4, 118.2, 122.4, 127.5, 128.7, 128.9,
129.1, 129.4, 134.2, 151.1, 154.1; IR y 3290, 1716, 1666,
1580; MS (ESþ): 1092 (MþH)þ.
3. Conclusion
A tri-branched amplification monomer has been developed
as a multi-dye carrier to enhance fluorescence signals during
protease assays. Peptides bearing a tri-dansyl derivative,
internally quenched by a dabsyl group in a peptide, showed
a significant increase in fluorescence following hydrolysis.
A new ‘self-quenched’ split and mix peptide library was
designed using fluorescein as the ‘internally quenched dye’,
avoiding the need to use two fluorophores as in traditional
FRET based protease substrates. Using this ‘quenched’ split
0
4.1.2. [2-{3-[5-Dimethylaminonaphthalene-1-sulfonic
acid]propoxy amide}-1,1-bis-{3-[5-dimethylamino-
naphthalene-1-sulfonic acid]propoxymethyl amide}-
ethyl] amine (7). The protected amine 5 (524 mg,
0.48 mmol) was treated with 20% TFA in CH2Cl2 (10 ml)
and stirred for 45 min. The solvent was removed in vacuo
and CH2Cl2 (50 ml) was added to the crude product which
was washed with saturated aqueous NaHCO3 (2£50 ml) and
water (50 ml). The organic layer was dried over Na2SO4 and
the solvent was removed in vacuo to give the amine 7 as a
white solid (470 mg, quantitative yield) which was used
without further purification. 1H NMR (400 MHz, CDCl3) d
1.59 (quint, J¼6.0 Hz, 6H), 2.50 (br s, 2H), 2.80 (s, 18H),
2.96 (t, J¼6.0 Hz, 6H), 3.22 (s, 6H), 3.40 (t, J¼5.5 Hz, 6H),
6.0 (br s, 3H), 7.07 (d, J¼7.0 Hz, 3H), 7.42 (dt, J¼7.5,
4.0 Hz, 6H), 8.15 (d, J¼7.5 Hz, 3H), 8.24 (d, J¼8.5 Hz,
3H), 8.44 (d, J¼8.5 Hz, 3H); 13C NMR (100 MHz CDCl3) d
28.0, 40.3, 44.4, 56.4, 68.7, 70.8, 114.2, 118.1, 122.2, 127.2,
128.2, 128.6, 128.9, 129.1, 134.2, 150.8; IR y 3290, 1662,
1565; MS (ESþ): 992 (MþH)þ.
and mix library, the substrate specificity (P2 to P1 ) of papain
could be rapidly determined by screening the split and mix
sub-libraries.
4. Experimental
4.1. General information
NMR spectra were recorded using Bruker AC 300 or DPX
400 spectrometers operating at 300 or 400 MHz for 1H and
100 MHz for 13C. Chemical shifts are reported on the d
scale in ppm and are referenced to residual non-deuterated
solvent resonances. Electrospray mass spectra were
obtained on a VG Platform single quadripole mass
spectrometer. MALDI spectra were recorded on a Micro-
mass Tofspec 2E reflection matrix assisted laser desorption
ionisation time of flight (MALDI-TOF) mass spectrometer.
IR spectra were obtained on a Biorad FTS 135 spectrometer
with a Golden Gate accessory with neat compounds as oil or
solids. Fluorescent measurements were recorded using a
Perkin Elmer Luminescence Spectrometer LS50B. Com-
mercially available reagents were used without further
purification. THF was freshly distilled under nitrogen from a
solution of sodium and benzophenone. Purifications by
column chromatography were carried out on silica gel 60
(230–400 mesh) purchased from Merck. Analytical HPLC
was performed on a Hewlett Packard HP1100 Chemstation
equipped with a C18 ODS analytical column, (4.6£3 mm i.d.
5 mm, flow rate 0.5 ml min21) eluting with H2O/MeCN/
TFA (90/10/0.1) to H2O/MeCN/TFA (10/90/0.05) over
3 min, detection by UV at 254 nm. Semi-preparative HPLC
was performed on a HP1100 system equipped with a
Phenomenex Prodigy C18 reverse phase column
(250£10.0 mm, flow rate 2.5 ml min21) eluting with water
(0.1% TFA) to MeCN (0.042% TFA) over 20 min. Resin
samples were agitated by spinning on a blood-tube rotor.
Compounds 3, 4 and 8 were synthesized according to
literature procedures.10
4.1.3. [2-{3-[5-Dimethylaminonaphthalene-1-sulfonic
acid]propoxy amide}-1,1-bis-{3-[5-dimethylamino-
naphthalene-1-sulfonic acid]propoxymethyl amide}-
ethyl] isocyanate (9). 7 (470 mg, 0.48 mmol) and DMAP
(61 mg, 0.5 mmol) were dissolved in THF (20 ml) and
stirred at 210 8C for 5 min under nitrogen To this solution
was added dropwise a solution of Boc2O (152 mg,
0.7 mmol) in THF (2 ml). The reaction mixture was stirred
for 90 min and the solvent removed in vacuo. Because of its
instability, the crude product was rapidly used without
further purification for coupling to the peptide. IR y 2253,
1732, 1609. LC-MS (ES2): 1016 (M2H)2.
4.2. Peptide synthesis
Peptide synthesis was carried out using a hydroxylmethyl-
phenoxy-acetic acid linker attached to aminomethyl PS
resin (1.11 mmol g21, 1% DVB, 75–150 mm).
Fmoc-amino acids (3.76 mmol, 2 equiv.) (Fmoc-Lys(Ddiv)-
OH (2.15 g), Fmoc-Gly-OH (1.12 g), Fmoc-Ile-OH
4.1.1. [2-{3-[5-Dimethylaminonaphthalene-1-sulfonic
acid]propoxy amide}-1,1-bis-{3-[5-dimethylamino-

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M. Ternon et al. / Tetrahedron 60 (2004) 8721–8728
(1.33 g), Fmoc-Ala-OH (1.17 g), Fmoc-Leu-OH (1.3 g),
Fmoc-Lys(Boc)-OH (1.76 g), Fmoc-Ala-OH (1.17 g),
Fmoc-Pro-OH (1.27 g), Fmoc-Gly-OH) (1.12 g) and HOBt
(507 mg, 3.76 mmol) were dissolved in CH2Cl2 (10 ml)
(with enough DMF to get complete dissolution) and stirred
at room temperature for 10 min. DIC (0.63 ml, 3.76 mmol)
was then added and the resulting solution stirred for a
further 10 min. The solution was then added to the resin
(approx. 2 g, 1.88 mmol), pre-swollen in CH2Cl2, and the
reaction mixture agitated for 2 h. The solution was then
drained and the resin washed with DMF (£3), MeOH (£3)
and CH2Cl2 (£3). The coupling reactions were monitored by
the qualitative ninhydrin test with the exception of the
coupling onto proline which was monitored by a chloranil
test. A small amount of resin was cleaved with TFA/
CH2Cl2/TIS (95/3/2) ratio and analyzed by analytical
HLPC (254 nm) and mass spectroscopy to check peptide
synthesis.
4.7. TFA cleavage
The resin (approx. 500 mg) was swollen in CH2Cl2 and
treated with TFA/CH2Cl2/TIS (95:3:2) (12 ml) for 45 min.
The solution was drained and the resin was washed with the
cleavage cocktail (2£5 ml) and the mixture solution was
removed in vacuo.
4.8. Purification of peptides 10a and 10b
The crude cleaved peptides 10a and 10b were dissolved in
the minimum amount of cleavage cocktail and added
dropwise to ice-cooled Et2O. The mixture was centrifuged,
the solvent was removed by decantation and the precipitate
was washed with Et2O (£3), before drying in vacuo. The
precipitate was purified by RP-HPLC and lyophilized to
afford the peptides as red solids.
Peptide 10a: Yield: 32%. HPLC (254 nm): 3.98 min.
MALDI-TOF-MS: 2159 (MþH)þ.
4.3. Fmoc-deprotection
To the resin (preswollen in CH2Cl2) was added 20%
piperidine in DMF (15 ml) and the reaction mixture
agitated for 20 min. The solution was then drained and
the resin was washed with DMF (£3), MeOH (£3) and
CH2Cl2 (£3).
Peptide 10b: Yield: 42%. HPLC (254 nm): 3.52 min. LC-
MS: 1374 (MþH)þ.
4.9. Library synthesis
The libraries were carried out using a Rink linker attached to
aminomethyl PS resin (1.11 mmol g21, 1% DVB, 75–
150 mm). Isocyanate 8 was synthesized according to
literature procedures10 and coupled to the resin (5.0 g,
4.15 mmol). The Dde protecting group was cleaved using
5% hydrazine in DMF (20 ml) for 2 h, the resin was drained
and washed using DMF (£3), MeOH (£3), CH2Cl2 (£3) and
Et2O (£3).
4.4. Dansyl chromophores coupling
Peptide 10a. Isocyanate 9 (335 mg, 0.33 mmol), DMAP
(2 mg, 0.02 mmol) and DIPEA (64 mg, 0.33 mmol) were
dissolved in CH2Cl2/DMF (1:1) (4 ml). The resulting
solution was added to the resin (500 mg, 0.22 mmol)
and the mixture was shaken overnight and monitored by
a qualitative ninhydrin test. The solution was then
drained and the resin was washed with DMF (£3),
MeOH (£3) and CH2Cl2 (£3).
4.10. Library 1
The resin was divided into 10 portions (each 30 mg,
60 mmol, 2.0 mmol g21 loading). HOBt (81 mg,
600 mmol) was dissolved in CH2Cl2/DMF (200 mL:
200 mL) along with one of each of the following amino
acids (600 mmol): Fmoc-Pro-OH (202 mg), Fmoc-
Ser(tBu)-OH (230 mg), Fmoc-Asn(Trt)-OH (358 mg),
Fmoc-Asp-(OtBu)-OH (246 mg), Fmoc-Tyr(tBu)-OH
(275 mg), Fmoc-Arg(Pbf)-OH (388 mg), Fmoc-Lys
(Boc)-OH (281 mg), Fmoc-Phe-OH (232 mg), Fmoc-
Ala-OH (186 mg) and Fmoc-Leu-OH (212 mg). DIC
(100 mL, 600 mmol) was added to each mixture. The
resulting solution stirred for a further 10 min and added to
the pre-swollen resins. The 10 resin samples were shaken
overnight. The reaction mixture was drained off and washed
using DMF (£3), MeOH (£3), CH2Cl2 (£3) and Et2O (£3).
The ninhydrin test was still positive for some of pools and
the coupling was repeated under the same conditions as
above until completion of reaction. The resin was then
combined, mixed and Fmoc cleavage was performed as
previously described. The resin was split into 10 portions
and the same procedure was employed in order to couple
each one of the 10 amino acids onto the second and third
positions. Following the coupling of the third amino acid,
the pools of resin were labelled according to their third
position and the Fmoc group was cleaved using 20%
piperidine in DMF.
Peptide 10b. Dansyl chloride (119 mg, 0.44 mmol) and
triethylamine (44 mg, 0.44 mmol) were added to the pre-
swollen resin (500 mg, 0.22 mmol) in CH2Cl2 and the
reaction mixture was agitated overnight. The solution was
then drained and the resin was washed with DMF (£3),
MeOH (£3) and CH2Cl2 (£3). The coupling reaction was
checked by a qualitative ninhydrin test.
4.5. Ddiv deprotection
To the resin (pre-swollen in CH2Cl2) (approx. 500 mg)
was added 2% hydrazine in DMF (5 ml) and the reaction
mixture agitated for 2 h. The solution was then drained and
the resin was washed with DMF (£3), MeOH (£3) and
CH2Cl2 (£3).
4.6. Dabsyl group coupling
Dabsyl chloride (142 mg, 0.44 mmol) and triethylamine
(44 mg, 0.44 mmol) were added to the pre-swollen resin
(approx. 500 mg, 0.22 mmol) in CH2Cl2 and the reaction
mixture was agitated overnight. The solution was then
drained and the resin was washed with DMF (£3), MeOH
(£3) and CH2Cl2 (£3). The coupling reaction was checked
by a qualitative ninhydrin test.

M. Ternon et al. / Tetrahedron 60 (2004) 8721–8728
8727
4.11. Library 2
butyric acid spacer (2 equiv.) in CH2Cl2/DMF along DIC
(2 equiv.) was prepared. To each pool, a tenth of this stock
solution was added and the mixture was shaken overnight.
The solutions were drained off, the resin washed using DMF
(£3), MeOH (£3), CH2Cl2 (£3) and Et2O (£3). Qualitative
ninhydrin tests were negative for all 10 pools. An Fmoc
cleavage was performed in each pool.
The resin was divided into 10 portions (each 100 mg,
200 mmol, 2.0 mmol g21 loading). HOBt (270 mg, 2 mmol)
was dissolved in CH2Cl2/DMF (600 mL: 600 mL) along
with one of each of the following amino acids (2 mmol):
Fmoc-Pro-OH (674 mg), Fmoc-Ser(tBu)-OH (766 mg),
Fmoc-Asn(Trt)-OH (1.20 g), Fmoc-Asp-OtBu (823 mg),
Fmoc-Tyr(tBu)-OH
(918 mg),
Fmoc-Arg(Pbf)-OH
4.14. 4(5)-Carboxyfluorescein coupling
(1.30 g), Fmoc-Lys(Boc)-OH (936 mg), Fmoc-Phe-OH
(774 mg), Fmoc-Ala-OH (622 mg) and Fmoc-Leu-OH
(706 mg). DIC (315 mL, 2 mmol) was added to each
mixture. The resulting solutions were stirred for a further
10 min and added to the pre-swollen resin. The pools of
resin were shaken overnight. The solution was drained off
and the resin washed using DMF (£3), MeOH (£3), CH2Cl2
(£3) and Et2O (£3). The ninhydrin test was still positive for
some of pools and the coupling was repeated as above until
completion of the reaction. The resin was then combined and
mixed and Fmoc cleavage was performed as previously
described. The resin was split into 10 portions. The same
procedure as above was employed in order to couple each of
the amino acid (1 mmol) to each of the portions of resin. The
solutions were drained off, the resin washed using DMF (£3),
MeOH (£3), CH2Cl2 (£3) and Et2O (£3) and dried and 10
Fmoc cleavages performed. Each pool of resin was labelled
accordingtothesecondpositionaminoacidandeachpoolsplit
into 10 further pools (100 in total). The third amino acid was
coupled employing the same procedure (100 mmol) before
recombining back into 10 pools. The Fmoc group was cleaved
using 20% piperidine in DMF.
A stock solution of HOBt (10 equiv.) and 4(5)-carboxy-
fluorescein (10 equiv.) in CH2Cl2/DMF along DIC
(10 equiv.) was prepared. To each pool, a tenth of this
stock solution was added and the mixture was shaken for
24 h. The ninhydrin test was still positive for some of pools
and the coupling was repeated.
4.15. General procedure for library cleavage
The resins (15 mg) were swollen in CH2Cl2 (1 ml) and
treated with TFA/CH2Cl2/TIS (95:3:2) (0.5 ml) mixture for
45 min. The solutions were drained and the resins were
washed with the cleavage cocktail (2£0.5 ml). The residue
was evaporated to dryness and DMSO (1 mL) was added.
These stock solutions were sonicated for 30 min to facilitate
peptide solubilization. All of the pools were diluted by a
factor of 300 in HEPES (50 mM, pH 8) or potassium
phosphate (50 mM, pH 6.8) buffer depending on the enzyme
under investigation and the final concentrations of the
peptide were checked by absorbance intensities at 495 nm
(the absorbance wavelength of the fluorophore) and adjusted
as necessary.
4.12. Library 3
4.16. Enzymatic assays
The resin was divided into 10 portions (each 100 mg,
200 mmol, 2.0 mmol g21 loading). HOBt (270 mg, 2 mmol)
was dissolved in CH2Cl2/DMF (600 mL: 600 mL) along with
each of the following amino acids (2 mmol): Fmoc-Pro-OH
(674 mg), Fmoc-Ser(tBu)-OH (766 mg), Fmoc-Asn(trt)-
OH (1.20 g), Fmoc-Asp-OtBu (823 mg), Fmoc-Tyr(tBu)-OH
(918 mg), Fmoc-Arg(Pbf)-OH (1.30 g), Fmoc-Lys(Boc)-OH
(936 mg), Fmoc-Phe-OH (774 mg), Fmoc-Ala-OH
(622 mg) and Fmoc-Leu-OH (706 mg). DIC (315 mL,
2 mmol) was added to each mixture. The resulting solution
stirred for a further 10 min and added to the pre-swollen
resin. The pools of resin were shaken overnight. The
solutions were drained off and the resin washed using DMF
(£3), MeOH (£3), CH2Cl2 (£3) and Et2O (£3). The
ninhydrin test was still positive for some of pools and
the coupling was repeated as above until completion of the
reaction. The pools of resin were labelled according to the first
positionaminoacidsand eachpoolsplit into10sub-pools. The
second amino acid was coupled following the same procedure
as above. The 10 sub-pools of resin were mixed in accordance
with their labels, Fmoc deprotected and each pool was again
split into 10 sub-pools. The third amino acid was coupled
employing the same procedures as above. The sub-pools were
then recombined to give the 10 pools. The Fmoc group was
cleaved using 20% piperidine in DMF.
4.16.1. Trypsin assays. Enzyme activity was monitored at
25 8C in 50 mM HEPES at pH 8.0, 10 mM CaCl2, 100 mM
NaCl, DMSO (15% for peptides 10a and 10b and less than
1% for the peptide libraries). Reactions were initiated by the
addition of trypsin and monitored fluorimetrically for
20 min with excitation at 335 nm (10a and 10b) or
495 nm (libraries) and emission at 545 nm (10a), 560 nm
(10b) or 520 nm (libraries).
4.16.2. Papain assays. Enzyme activity was monitored at
25 8C in potassium phosphate buffer at pH 6.8, cysteine
(2 mM), EDTA (1 mM) and the pool of peptides. Reactions
were initiated by the addition of papain and monitored
fluorimetrically for 20 min with excitation at 495 nm and
emission at 520 nm.
Acknowledgements
The authors would like to thank the BBSRC (EBS) and the
University of Granada.
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