A combinatorial approach to catalytic peptide dendrimers

Article abstract of DOI:10.1002/anie.200460177

Exploring the structural diversity of peptide dendrimers as synthetic protein models: A 65536-membered combinatorial peptide-dendrimer library was prepared by split-and-mix techniques on beads (see picture). The library was screened and revealed peptide dendrimers that catalyze fluorogenic ester hydrolysis and peptide dendrimers that bind to vitamin B₁₂.

Full text of DOI:10.1002/anie.200460177

Communications
Combinatorial Synthesis
A Combinatorial Approach to Catalytic Peptide
Dendrimers**
Anthony Clouet, Tamis Darbre, and
Jean-Louis Reymond*
In de novo protein design one attempts to create artificial
proteins with defined structure and function from first
principles, usually with the help of trial-and-error procedures
[
*] A. Clouet, Priv.-Doz. Dr. T. Darbre, Prof. Dr. J.-L. Reymond
Department of Chemistry and Biochemistry
University of Bern
Freiestrasse 3, 3012 Bern (Switzerland)
Fax: (+41)31-631-8057
E-mail: jean-louis.reymond@ioc.unibe.ch
[
**] This work was financially supported by the University of Bern and
the Swiss National Science Foundation. We thank Mr. Urs Kämpfer
of the Protein Analytical Service of the Department of Chemistry and
Biochemistry, University of Bern, for performing the amino acid
analyses.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
4
612
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DOI: 10.1002/anie.200460177
Angew. Chem. Int. Ed. 2004, 43, 4612 –4615

Angewandte
Chemie
[
1]
that scan a large number of possible amino acid sequences.
problem was solved by taking advantage of the dendritic
Our approach to de novo protein design is based on peptide
dendrimers. Dendrimers are treelike structures that adopt a
globular or disk-shaped structure as a consequence of top-
structure in which amino acids are present in one, two, four, or
eight copies, depending on their placement in the different
branches. As each amino acid was present at most at two
positions in different branches, the sequence could be
unambiguously assigned by quantitative amino acid analysis
[
2]
ology rather than folding. We have shown that peptide
dendrimers that bear histidine residues at the surface catalyze
[
3]
[6]
ester hydrolysis in water. Herein we report a combinatorial
approach to peptide dendrimers based on split-and-mix
synthesis and on-bead screening. The method is exemplified
by the discovery of catalytic and binding peptide dendrimers
in a 65536-membered library.
of the dendrimer.
The peptide-dendrimer library was prepared from a 400-
mg resin batch, which ensured that each sequence would be
present in approximately 15 beads. Aromatic, hydrophobic,
positively charged, negatively charged, and small amino acids
1
4
As peptide dendrimers are synthesized on solid supports,
it should be possible to apply the principles of combinatorial
were distributed evenly among the inner positions A –A and
5
8
the outer positions A –A . Histidine was placed in the
[
4]
6
8
peptide synthesis, which have been exploited successfully
outermost layer (A and A ) as surface placement of this
[
5]
[3]
for peptide-based and small-molecule catalysts. To achieve
this goal, we selected a dendrimeric architecture containing
eight variable positions connected by three successive
branching diamino acid units (Figure 1). Preliminary experi-
residue was known to favor catalysis. The sequence started
with an amino–hexanoyl–glycine spacer to minimize inter-
actions with the solid support. All coupling steps were
quantitative, as indicated by the negative TNBS (2,4,6-
trinitrobenzenesulfonic acid) staining test. Side-chain pro-
tecting groups were removed with trifluoroacetic acid after
cleavage of the Fmoc protecting groups and acetylation of the
last amino acid, resulting in a functional dendrimer library on
beads.
The peptide dendrimer library was screened to find
catalytic peptide dendrimers for an ester-hydrolysis reaction.
Activity screening was realized by soaking the beads in an
aqueous buffered solution of a fluorogenic ester substrate,
followed by decantation and spreading the slurry of beads
onto a Petri dish. Whereas the solvent quickly evaporated at
the glass surface, evaporation inside the beads was slow, so
that each bead functioned as a separate microreactor. The
procedure was successful for screening the hydrolysis of 8-
butyryloxypyrene-1,3,6-trisulfonate,
a known fluorogenic
lipase substrate (Scheme 1). Approximately 60% of all
8
Figure 1. Combinatorial library of 65536 (=4 ) peptide dendrimers
obtained by solid-phase split-and-mix synthesis. * (branching
unit)=(S)-2,3-diaminopropanoic acid. Spacer=NH(CH ) CONH-
2
5
CH CONH-, and NH(CH ) CONHCH CONH after cleavage from the
2
2
5
2
2
8
support for resynthesized dendrimers. The N terminus at position A
is either acetylated (esterolysis studies) or free (vitamin B12 binding
studies). The solid support is tentagel.
Scheme 1. Fluorogenic esterolysis reaction of 8-butyryloxypyrene-1,3,6-
trisulfonate catalyzed by peptide dendrimers.
ments showed that the sequence of eleven coupling steps to
form such dendrimers gave good chemical yields for single
sequences when using 2,3-diaminopropanoic acid as the
[
3b]
branching unit.
A split-and-mix library of peptide den-
beads showed low-level fluorescence, among which very few
beads (10 beads/40 mg resin) showed an intense green
fluorescence that indicated product formation (see Support-
ing Information). These active beads were picked, washed
thoroughly with buffer and water, and subjected to amino acid
analysis (Table 1). Two consensus sequences and two original
sequences were synthesized as single sequences and purified
drimers was designed by distributing sixteen proteinogenic
amino acids into four groups of four amino acids each. The
resulting 65536-member library required splitting in four
portions at each variable sequence step, whereby each group
of four amino acids would be used at two positions in different
branches of the dendrimer. The single-bead sequencing
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Communications
Table 1: Peptide dendrimer sequences identified by amino acid analysis
[
6]
of active beads from the combinatorial library and the corresponding
[
a]
consensus sequences.
8
7
6
5
4
3
2
1
Dend.
A
A
A
A
A
A
A
A
Hits for hydrolysis of 8-butyr-
yloxypyrene-1,3,6-trisulfonate
1
2
3
4
5
6
7
8
9
W
H
H
H
E
G
H
H
H
G
H
H
W
E
W
G
G
H
W
W
G
S
L
L
P
R
R
S
S
S
R
S
S
L
P
P
P
R
L
R
P
R
G
H
G
G
G
W
H
G
H
G
H
G
H
G
E
R
S
L
P
S
R
L
R
P
P
L
S
S
R
S
P
R
R
S
R
S
K
Y
Y
K
I
I
F
I
K
I
K
I
V
A
T
T
V
V
A
A
V
V
V
V
A
T
A
V
T
V
V
V
T
I
I
I
I
I
I
F
I
F
I
A
[
b]
D
V
A
V
A
D
V
V
V
V
V
A
D
D
V
D
D
V
D
V
1
0
consensus sequences
Hits for binding to vita-
C11
C12
13
I
I
I
K
Y
Y
K
I
K
I
Y
K
[
c]
min B12
14
Y
Y
Y
I
K
I
1
1
1
1
1
5
6
7
8
9
W
E
G
E
G
E
Figure 2. Hydrolysis of 8-butyryloxypyrene-1,3,6-trisulfonate (Scheme 1)
catalyzed by peptide dendrimers from the combinatorial library. Condi-
tions: substrate (200 mm), dendrimer (5 mm), aqueous 20 mm Bis–Tris
pH 6.0, 258C. The reactions were run in 96-well, polystyrene, half-area
microtiter plates withsubsequent detection on a SpectraMAX fluores-
cence detector with lexc =460 nm, lem =530 nm. Fluorescence was
converted to product concentration by using a calibration curve, which
was linear in the concentration range used.
consensus sequences
C20
C21
Y
I
[
a] Dendrimer structure according to Figure 1. Dendrimers marked bold
were resynthesized by Fmoc-SPPS and purified. [b] All N termini N-
acetylated. Screening conditions: Beads soaked in 20 mm aqueous Bis–
Tris buffer containing 80 mm 8-butyryloxypyrene-1,3,6-trisulfonate, 258C,
3
0 min. Hits are green-fluorescent under UV radiation (356 nm).
[c] N termini are free amine. Screening conditions: 30 min equilibration
in aq. PBS (10 mm phosphate, 160 mm NaCl, pH 7.4) containing 400 mm
cyanocobalamin (vitamin B ), followed by washing with PBS and water.
Hits are bright orange.
1
2
Table 2: Kinetic parameters for esterolytic peptide dendrimers from the
combinatorial library.
[a]
8
C11
C12
by preparative HPLC. Dendrimer 10, which did not contain a
K
M
(mM)
0.065
0.094
2140
1800
39
0.066
0.14
3100
2540
51
0.051
0.054
1200
1300
22
À1
kcat (min
kcat/kuncat
kcat/K /k
Vnet/Vuncat
)
histidine residue, was not active and represented a false
[
7]
positive. The three other dendrimers (8, C11, and C12)
M
2
were catalytically active, and contained histidine at the
8
outermost position A (Figure 2). These dendrimers displayed
[a] Conditions: 50–800 mm 8-butyryloxypyrene-1,3,6-trisulfonate, 5 mm
enzyme-like catalysis for the ester hydrolysis (Table 2) with
dendrimer, 20 mm aqueous Bis–Tris pH 6.0, 258C. The kinetic constants
given are derived from the linear double-reciprocal plots of 1/Vnet versus
multiple turnover and rate enhancement k /k
up to 3000-
cat uncat
fold over background and KM values in the 0.05 mm range. In
terms of apparent catalytic effect Vnet/Vuncat, these dendrimers
are 2–10 times better than those prepared previously by
1
/[S]. Vnet/Vuncat is the apparent rate enhancement observed with S=
200 mm and 2.5 mol% catalytic peptide dendrimer (5 mm). Vnet
VappÀV with Vapp being the apparent hydrolysis rate in the presence
=
uncat
[
3]
rational design.
of dendrimer and Vuncat the hydrolysis rate in buffer alone (kuncat =4.4”
À5
À1
À4
À1
À1
10 min ). k =8.1”10 mm min is the catalytic rate constant for
Activity screening of solid-supported combinatorial libra-
ries for binding affinity can be readily carried out by bead
2
hydrolysis by 4-methyl-imidazole under the same conditions.
[
4a]
staining with a colored ligand. The dendrimer library was
screened for binding to vitamin B , a corrinoid-type chro-
1
2
mophore for which no artificial binding proteins have been
described to date (Figure 3). Vitamin B -containing enzymes
were used in the synthesis, and these beads bound vitamin B12
as observed during screening. Beads carrying a catalytic
sequence were not stained under these conditions. Bead
staining by vitamin B12 was inhibited in the presence of
> 0.5 mm of soluble purified dendrimer C21, but not with the
same amount of the catalytic dendrimer C10, indicating a
relatively weak, yet highly selective, binding interaction.
While aquocobalamin also bound the dendrimers, there was
no binding with cobinamide, which lacks the nucleotide loop,
therefore suggesting that this structural element is involved in
the dendrimer–vitamin B12 interaction (Table 3).
1
2
hold the vitamin by multiple hydrogen bonds to the carbox-
[
8]
amide groups of the corrin ring. Equilibration with vita-
min B12 in aqueous neutral buffer resulted in staining of two
to five beads per experiment (0.01%). The experiment was
repeated twice to give a total of seven colored beads, which
were subjected to amino acid analysis (Table 1). Two original
sequences and two consensus sequence were resynthesized,
cleaved from the resin, and purified by preparative HPLC. In
each case a small portion of noncleavable synthesis beads
4
614
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Chemie
[
1] a) D. A. Moffet, M. H. Hecht, Chem. Rev. 2001, 101, 3191 – 3203;
b) D. N. Bolon, C. A. Voigt, S. L. Mayo, Curr. Opin. Chem. Biol.
002, 6, 125 – 129.
2] a) A. W. Bosman, H. M. Janssen, E. W. Meijer, Chem. Rev. 1999,
2
[
99, 1665 – 1688; b) D. K. Smith, Tetrahedron 2003, 59, 3797 –
3798; c) S. Hecht, J. M. J. FrØchet, Angew. Chem. 2001, 113,
76 – 94; Angew. Chem. Int. Ed. 2001, 40, 74 – 91; d) G. R.
Newkome, C. N. Moorefield, F. Vögtle, Dendritic Molecules:
Concepts, Synthesis, Perspectives, Wiley-VCH, Weinheim, 1996.
3] a) A. Esposito, E. Delort, D. Lagnoux, F. Djojo, J. L. Reymond,
Angew. Chem. 2003, 115, 1419 – 1421; Angew. Chem. Int. Ed.
[
2
003, 42, 1381 – 1383; b) D. Lagnoux, E. Delort, C. Douat-
Casassus, A. Esposito, J. L. Reymond, Chem. Eur. J. 2004, 10,
215 – 1226.
1
[
[
4] a) K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M.
Kazmierski, R. J. Knapp, Nature 1991, 354, 82 – 84; b) R. A.
Houghten, C. Pinilla, C. , S. E. Blondelle, J. R. Appel, C. T.
Dooley, J. H. Cuervo, Nature 1991, 354, 84 – 86.
5] a) A. Berkessel, Curr. Opin. Chem. Biol. 2003, 7, 409 – 419; b) C.
Gennari, U. Piarulli, Chem. Rev. 2003, 103, 3071 – 3100; c) C. A.
Evans, S. J. Miller, Curr. Opin. Chem. Biol. 2002, 6, 333 – 338;
d) B. Linton, A. D. Hamilton, Curr. Opin. Chem. Biol. 1999, 3,
Figure 3. Structure of vitamin B . In cobinamide the 3’-phospho-
nucleoside is absent and the cobalt center has two cyanide ligands.
1
2
307 – 312; e) K. W. Kunz, M. L. Snapper, A. H. Hoveyda, Curr.
Opin. Chem. Biol. 1999, 3, 313 – 319.
[
a]
Table 3: Interaction between dendrimers and vitamin B12 derivatives.
[6] Single dendrimer-containing resin beads were hydrolyzed with
aqueous HCl (6m) at 1108C for 22 h, and their amino acid
composition was determined quantitatively by HPLC after
derivatization with phenyl isothiocyanate (PITC). Such amino
acid analyses are routine for protein-composition analysis. The
analysis detects as little as 5 pmol per amino acid, which is
sufficient for single-resin-bead analysis, as these contain 50–
200 pmol of dendrimers.
[7] False positives are most likely due to manipulation errors during
bead picking. Sometimes more than one bead is transferred to
the pipette, and the fluorescence staining is diluted when the
beads are washed down the pipette used for picking, which does
not allow staining to be rechecked afterwards.
Dendrimer Vitamin B -cyano Vitamin B -aquo
Cobinamide
1
2
12
4
00 mm 100 mm 400 mm 100 mm 400 mm 100 mm
1
1
C20
C21
C11
9
4
+++
+++
+++
+++
À
+
À
À
À
À
+++
+++
+++
+++
À
++
++
++
++
À
À
À
À
À
À
À
À
À
À
À
[
a] Intensity of bead staining (orange/red coloration) after equilibration
of solid-supported dendrimer withthe given concentration of vitamin B 12
in aqueous phosphate buffer pH 7.4 for 30 min, followed by washing.
[
8] Chemistry and Biochemistry of B12 (Ed.: R. Banerjee), Wiley,
New York, 1999.
[
9] M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J. C. Reader,
G. Asouline, R. Kobayashi, M. H. Wigler, W. C. Still, Proc. Natl.
Acad. Sci. USA 1993, 90, 10922 – 10926.
The experiments above demonstrate a general approach
to functional peptide dendrimers through split-and-mix
combinatorial libraries. Library design and the dendritic
architecture allow single-bead sequencing by quantitative
[
10] a) Enzyme Assays: A Practical Approach (Eds.: R. Eisenthal, M.
Danson) Oxford University Press, Oxford 2002; b) S. Gul, S. K.
Sreedharan, K. Brocklehurst, Enzyme Assays: Essential Data,
Wiley, New York, 1998; c) M. T. Reetz, Angew. Chem. 2001, 113,
[
9]
amino acid analysis, such that bead encoding is not
necessary. Selection for catalysis was realized by soaking the
beads with fluorogenic substrates and spreading on a dry
surface, which allows direct selection for catalytic turnover.
The procedure is, to our knowledge, new and is suitable for
screening reactions with soluble fluorogenic or chromogenic
2
92 – 320; Angew. Chem. Int. Ed. 2001, 40, 284 – 310; d) D.
Wahler, J.-L. Reymond, Curr. Opin. Chem. Biol. 2001, 5, 152 –
58; e) F. Beisson, A. Tiss, C. Riviꢀre, R. Verger, Eur. J. Lipid
1
Sci. Technol. 2000, 133 – 153; f) D. Wahler, J.-L. Reymond, Curr.
Opin. Biotechnol. 2001, 12, 535 – 544.
[
10]
substrates or sensor systems. The method provides a useful
complement to previously reported on-bead screening meth-
[11] a) P. Krattiger, C. McCarthy, A. Pfaltz, H. Wennemers, Angew.
Chem. 2003, 115, 1763 – 1766; Angew. Chem. Int. Ed. 2003, 42,
1722 – 1724; b) I. Lingard, G. Bhalay, M. Bradley, Chem.
Commun. 2003, 2310 – 2311.
[
11]
ods based on the covalent attachment of substrate
or
[
5c]
[5a]
sensor to the beads, the use of precipitating dyes, or IR
[
12] S. J. Taylor, J. P. Morken, Science 1998, 280, 267 – 270.
[
12]
thermography. The exploration of focused libraries should
enable the refinement and improvement of the properties of
the isolated dendrimers, and the exploration of further
problems in binding and catalysis.
Received: March 31, 2004
Keywords: cobalamines · combinatorial chemistry · dendrimers ·
.
enzyme models · peptides
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