Rapid sequencing of split-and-mix peptide receptor libraries - Identification of binding partners for Val-Val-Ile-Ala in aqueous solution

Article abstract of DOI:10.1002/ejoc.200601004

Split-and-mix peptide libraries have been encoded by capping cleavable, laddered sequences with p-bromobenzoic acid, allowing rapid analysis of the cleaved ladder fragments by mass spectrometry. The encoding methodology has been applied to the synthesis o

Full text of DOI:10.1002/ejoc.200601004

FULL PAPER
DOI: 10.1002/ejoc.200601004
Rapid Sequencing of Split-and-Mix Peptide Receptor Libraries – Identification
of Binding Partners for Val-Val-Ile-Ala in Aqueous Solution
Jon Shepherd,[a] G. John Langley,[a] Julie M. Herniman,[a] and Jeremy D. Kilburn*[a]
Keywords: Peptides / Coding / Sequencing / Combinatorial chemistry / Receptors / Screening
Split-and-mix peptide libraries have been encoded by cap-
ping cleavable, laddered sequences with p-bromobenzoic
acid, allowing rapid analysis of the cleaved ladder fragments
by mass spectrometry. The encoding methodology has been
applied to the synthesis of libraries of peptide receptors and
a library of tripeptides functionalised with a bicyclic guanidi-
nium unit has been successfully screened to identify binding
partners for Val-Val-Ile-Ala, the C-terminal tetrapeptide se-
quence of the amyloid-β protein, Aβ(1–42).
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
Introduction
The combinatorial, split-and-mix approach[1] has been
used by several research groups to prepare combinatorial
libraries which have been successfully screened to identify
novel receptors for small molecules[2] and in particular se-
quence-selective receptors for peptides, in both organic[3]
and aqueous[4,5] solvent systems. In this context we have
used bis(aminoalkyl)guanidiniums as a scaffold to prepare
resin-bound libraries of two-armed “tweezer” receptor
structures 1 (Scheme 1).[4a,4i] Screening experiments with
these libraries involve incubation of a sample of the library
with a dye-labelled guest peptide and selection of “hit” be-
ads, which are strongly coloured as a result of selective
binding of the dye-labelled guest peptide to the receptor on
the particular bead. The “hit” beads are manually selected
and the structure of the receptor on each “hit” bead is iden-
tified. This approach has been successfully used to identify
tweezer receptors of general structure 1 for peptides such
as the bacterial cell wall precursor tripeptide Lys--Ala--
Ala in aqueous media.[4a]
Scheme 1.
thousand receptor structures often lead to many “hit”
(strongly dyed) beads. A limitation of this approach, in our
hands at least, has been that the identification of receptor
structures on these “hit” beads has relied on Edman se-
quencing,[6] either of the peptide arms of the receptor itself,
or of a separately synthesized peptide coding strand. The
Edman sequencing methodology has proved to be both ex-
pensive and time-consuming which has greatly restricted the
number of beads from a screening experiment that can be
conveniently sequenced, and this in turn has restricted the
amount of statistical information about the preferred struc-
ture of potent receptors that can be obtained from a given
screening experiment. Although reasonably strong and
selective receptors have none-the-less been identified, se-
quencing of greater numbers of beads should lead to more
reliable identification of the most potent receptor structure
from a given library. These considerations are even more
relevant if larger libraries incorporating greater diversity
than used to date are to be screened.
These results, and related studies by others,[4] emphasise
the potential of such tweezer structures as strong and selec-
tive peptide receptors even in competitive aqueous solvents,
despite the inherent flexibility of such structures.
An added advantage of this split-and-mix approach has
been that once a library is synthesised, a series of screening
experiments can be carried out very rapidly which in prinic-
iple allows one to probe the binding selectivities of the re-
ceptors as a function of both the structure of the peptide
guest and the solvent used in the screening experiment.
However, screening experiments with libraries of several
With this in mind an alternative sequencing strategy has
been developed which allows rapid, reliable and inexpensive
sequencing of large numbers of beads.[7] Herein we describe
the results of these studies, and the application of the new
sequencing methodology to the screening of a single-armed
[a] School of Chemistry, University of Southampton,
Southampton, SO17 1BJ, UK
Fax: +44-2380-596805
E-mail: jdk1@soton.ac.uk
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receptor library to identify binding partners for Val-Val-Ile- fore cleavage and MS analysis, was used to avoid competi-
Ala, the C-terminal tetrapeptide sequence of the amyloid-β tive binding of a guest peptide carboxylate in screening ex-
protein, Aβ(1–42).
periments.
In preliminary experiments to ascertain the limit of de-
tection of the capped fragments, tentagel resin was deriv-
atised, by sequential coupling of methionine, arginine(Pbf)
and three aminohexanoic acid (Ahx) or phenylalanine (Phe)
moieties. Samples of the resulting resin were coupled with
Results and Discussion
Development of Capping Methodology
A very useful method on the basis of mass spectrometry single amino acids [Gly, Ala, Val, Phe, Leu, Ile, Ser(OtBu),
for the determination of the sequence of bead-based pep- Thr(OtBu), Gln, Asn, Glu(OtBu), Lys(Boc), Pro, Trp and
tides has been described by Youngquist and by Lebl.[8] Dur- Tyr(OtBu)] and then capped with varying amounts (from
ing synthesis of a library, a small percentage of the growing 0–100 mol-%) of p-bromobenzoic acid relative to the resin
peptide chains are capped at each step by an acylating loading and excess DIC/HOBt/DIPEA for 18 h. Single be-
group to generate a family of termination products on the ads were selected from each sample, cleaved using an excess
bead – a “ladder sequence”. Cleavage of an individual bead of CNBr, and the cleavage products were analysed by
and analysis by matrix-assisted laser desorption/ionization MALDI-TOF MS. The resultant data were processed using
time-of-flight mass spectrometry (MALDI-TOF MS), gives data reduction software.[12] It was thus determined that,
the mass of each of the termination products and allows with the inclusion of the arginine in the linker construct,
the full peptide sequence to be read from the N-terminus the capped sequences (as the [M + H]+ adducts) could be
as the mass difference between adjacent peaks equates to detected using as little as 10 mol-% of the p-bromobenzoic
the mass of each consecutive amino acid component. In acid in each capping reaction. The amount of capping in
their original patent[8c] Lebl et al. also suggested using bro- each step is clearly important as each capping reaction
mobenzoic acid as a capping group to generate mass peaks clearly consumes some of the peptide of interest, but using
with a characteristic bromine isotope pattern. The concept only 10 mol-% would in principle allow one to construct a
of introducing a substituent with a readily identifiable isop- suitably encoded nonapeptide, and certainly should satis-
tope pattern was successfully developed by Bradley[9] who factorily allow encoding of shorter peptides. Of course pre-
used an equimolar mixture of AcOH and [D3]AcOH as the paring encoded peptides in this way, even using 10 mol-%
capping reagent, so that the relevant peaks of the ladder capping, means that only a portion of the material on a
sequence are readily identified by the presence of two peaks single bead is represented by the final structure and accord-
of equal intensity at [M + H]+ (for Ac-capped fragments) ingly any screening experiments should take account of
and [M + 3 + H]+ (for the corresponding [D3]Ac-capped contributions from the truncated peptide sequences. How-
fragment). Identification of relevant peaks was greatly facil- ever, in the studies described herein only the full peptide
itated by application of data reduction software which can structure incorporates the crucial guanidinium function-
select peaks with defined isotopic patterns, and as a conse- ality that serves as a carboxylate binding site. As this study
quence greatly increases the effective signal-to-noise ratio of and our earlier studies have shown, the presence of the gua-
the relevant signals in the mass spectra.
nidinium is essential in achieving binding of peptides with
As a coding strategy for our receptor libraries, we de- a free carboxylate terminus. Hence, using the laddered pep-
cided to generate similar ladder sequences using a single tide encoding strategy is particularly suitable for the sorts
molecular identity, p-bromobenzoic acid – as suggested by of libraries we wish to prepare.
Lebl – as the capping reagent, as a modification to the well
During these preliminary investigations it was discovered
that when partial capping was attempted by simultaneous
described methodology developed by Bradley.[9,10]
The utility of p-bromobenzoic acid was investigated addition of p-bromobenzoic acid and coupling reagents
using tentagel resin (0.2 mmol/g) and standard Fmoc SPPS (DIC/HOBt/DIPEA) to the resin bound peptide some be-
methods for peptide synthesis. Thus, following the pro- ads within a sample of resin were not being capped at all
cedure described by Bradley,[9] methionine, which is selec- whereas other beads were being completely capped, pre-
tively cleaved under conditions of excess CNBr/TFA/ sumably due to inefficient mixing of the capping group
H2O[11] generating the C-terminal homoserine lactone throughout the resin sample in conjunction with a rapid
(Hsl), was used as the linker and several aminohexanoic and efficient coupling reaction. To avoid this problem a
acid, or phenylalanine residues were introduced to provide solution of p-bromobenzoic acid in DMF was equilibrated
sufficient mass such that cleavage products of interest were with the resin sample (for one hour) before addition of
out of range of low-mass noise and matrix ions inherent in coupling reagents (DIC/HOBt/DIPEA) thus allowing an
MALDI-TOF MS analysis. Incorporation of an arginine even distribution of the capping group throughout the resin
unit was also investigated as the readily ionisable guanidine sample.
group increases the sensitivity of the mass spectrometric
To demonstrate the effectiveness of the protocol a tripep-
analysis. In Youngquist’s original development of ladder se- tide (Ile-Ala-Gly) was prepared on the solid phase from the
quences[8] the side-chain guanidine of the arginine used for resin 2, using Fmoc SPPS, with the inclusion of capping
this purpose was unprotected, but here a protected guani- reactions after each cycle of coupling–deprotection, to gen-
dine, Arg(Pbf) or Arg(Mtr), which can be deprotected be- erate the resin 3 (Scheme 2).
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Figure 1. Mass spectra of a sample from a single cleaved bead of 3 a) raw data, b) processed to identify Br-isotope patterns.
MALDI-TOF MS of the single bead cleavage followed Synthesis of Receptor Libraries
by processing with the appropriate software[12] gave easily
interpretable data (Figure 1) and the peptide sequence
could be read off from the spectrum by calculating the mass
differences of the amino acid residues.
With a functioning methodology for generating “ladder
sequences” (and hence “readable” receptor libraries)
achieved, synthesis of two libraries of receptors for carbox-
ylates was undertaken: a single-armed receptor library 7
and a tweezer receptor library 17.
The basic design of the single-armed receptor consists of
a bicyclic guanidinium 6 (which has been developed exten-
sively by de Mendoza[13] and by Schmitdchen[14] as a potent
carboxylate binding site), linked to a randomised peptide
sequence, and attached to Tentagel resin through the methi-
onine linker, and with an arginine residue for greater sensi-
tivity in the mass spectrometry analysis.
In a further demonstration of the methodology, a small
library 4 of 64 tripeptides was synthesised on resin 2 by
split-and-mix synthesis, using the four amino acids Ala,
Val, Ser(OtBu) and Lys(Boc) (Scheme 2). Twenty single be-
ads were selected from the library, cleaved and analysed by
MALDI-TOF MS. The sequence of eighteen of these beads
was readily assigned. The remaining two beads showed no
relevant characteristic signals in the mass spectrum, sug-
gesting either unsuccessful coupling or cleavage reactions.
The resin 5 was thus prepared by sequential coupling of
methionine, arginine(Pbf) and three aminohexanoic acid
moieties on to tentagel resin (Scheme 3). This was followed
by three rounds of split-and-mix synthesis using nine amino
acids [Gly, Ala, Val, Leu, Phe, Asn, Ser(OtBu), Glu(OtBu),
Lys(Boc)], and incorporating a capping reaction with p-bro-
mobenzoic acid (15–20 mol-% of resin loading) to generate
a 729-membered tripeptide library with a capped “ladder”
sequence. The synthesis was completed by attaching the
bicyclic guanidinium 6[15] (DIC/HOBt/DIPEA in DMF)
to generate single-armed receptor library 7. To assess the
“readability” of the library, three beads were picked at
random, deprotected using 50% TFA/DCM, cleaved under
the standard CNBr/TFA/H2O conditions and successfully
sequenced using MALDI-TOF MS.
In order to generate tweezer receptors on the solid-phase
and make use of the new coding methodology we prepared
the functionalised guanidine 12 which, with its amino and
carboxylic acid functionality, could be incorporated into a
Scheme 2. i) Fmoc-Met, DIC, HOBt, DIPEA, DMF, 3 h; ii) 20%
piperidine/DMF, 20 min; iii) Fmoc-Phe, DIC, HOBt, DIPEA,
DMF, 3 h; iv) Fmoc-AA, DIC, HOBt, DIPEA, DMF, 3 h; v) p-
BrC6H4COOH (15–20 mol-%), DIC, HOBt, DIPEA, DMF, 18 h.
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Scheme 3. i) Fmoc-Met, DIC, HOBt, DIPEA, DMF, 3 h; ii) 20% piperidine/DMF, 20 min; iii) Fmoc-Arg(Pbf), DIC, HOBt, DIPEA,
DMF, 3 h; iv) Fmoc-Ahx, DIC, HOBt, DIPEA, DMF, 3 h; v) Fmoc-AA, DIC, HOBt, DIPEA, DMF, 3 h; vi) p-BrC6H4COOH (15–
20 mol-%), DIC, HOBt, DIPEA, DMF, 18 h; vii) 6, DIC, HOBt, DIPEA, DMF, 3 h.
growing peptide chain using standard solid phase peptide
Solid-phase library synthesis began with partial derivatis-
synthesis (SPPS). This approach differs from our previous ation of Tentagel resin (0.47 mmol/g) with Fmoc-Met (ca.
syntheses of tweezer receptors on the solid-phase, which uti- 80 mol-%). A quantitative ninhydrin test[17] indicated a
lised an orthogonally protected bis(amino)guanidine deriv- residual-free NH2 loading of 0.10 mmol/g (ca. 20%). The
ative as the starting scaffold for attachment of two peptidic remaining free sites were treated with excess Boc2O to gen-
arms[4a,4i] (see Scheme 1).
erate the bifunctional resin 13 (Scheme 5). Deprotection
The guanidine 12 was prepared by coupling of the iso- of the methionine residue was followed by coupling of
thiocyanate 9 and an amino ester, prepared by Boc depro- Arg(Mtr), then two aminohexanoic acid moieties giving 14.
tection of the ester 8, to give the thiourea 10 (Scheme 4). The more acid-stable Arg(Mtr) was selected because of its
Alkylation of 10 gave the thiouronium salt which was con- compatability[18] with the several Boc-deprotections that
verted into the trifluoroacetyl-protected guanidine 11 by re- followed.
action with trifluoroacetamide and DBU.[16] Hydrogenation
Resin 14 was then subjected to two cycles of split-and-
of the benzyl ester gave the acid 12. The trifluoroacetyl pro- mix synthesis. Thus the resin was split into eight portions;
tecting group can be easily removed on the solid phase each portion was independently coupled using an Fmoc-
using mild aqueous base.[16]
amino acid [Gly, Ala, Val, Phe, Asn, Glu(OBn), Lys(Cbz),
Thr(OBn)]; Boc-deprotection using dilute TFA; coupling
with the same amino acid as before, but with a Boc rather
than Fmoc protecting group; Fmoc-deprotection; capping
reaction (15% of nominal loading of the Fmoc-deprotected
strand); mixing of the portions to give the resin 15. In this
way, the amino acid incorporated in the Boc-strand is inde-
xed by the same amino acid in the “ladder sequences” gen-
erated in the Fmoc-strand.
Resin 15 was again split into eight portions and coupled
with an Fmoc-amino acid (eight amino acids as before).
Boc-deprotection was carried out using dilute TFA and the
acid 12 was coupled to each portion. The resin portions
were then again Boc-deprotected and coupled with the ap-
propriate Boc-amino acid to match the one attached in the
previous Fmoc-coupling. Finally Fmoc-deprotection was
carried out, followed by capping reaction (20% of original
loading of Fmoc-strand), and mixing of the resin portions
to give 16. In this manner the guanidine is incorporated
into only one strand, with a ladder sequence generated
separately.
Scheme 4. i) CF3CO2H, DCM; ii) 9, Et3N, DCM/MeOH, 18 h; iii)
MeI, acetone, 1 h; iv) NH4 PF6 , DCM/MeOH, 18 h; v)
F3CCONH2, DBU, toluene/CHCl3, 2 h; vi) H2, Pd/C, EtOAc, 3 h.
Furthermore, in order to avoid generating truncated se-
quences on each resin bead that contained the guanidine
moiety 12, when implementing the capping strategy, it was
necessary to generate distinct “strands” (receptor-strand
and coding-strands) on each bead, using orthogonal pro-
tecting groups.
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Split-and-Mix Peptide Receptor Libraries
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Scheme 5. i) Fmoc-Met (0.9 equiv.), DMF, DIC, HOBt, DIPEA, 3 h; ii) xs. Boc2O, DIPEA, DMF, 3 h; iii) 20% piperidine/DMF, 20 min;
iv) Fmoc-Arg(Mtr), DIC, HOBt, DIPEA, 3 h; v) Fmoc-Ahx, DIC, HOBt, DIPEA, 3 h; vi) split; vii) Fmoc-AA, DIC, HOBt, DIPEA,
DMF, 3 h; viii) 20% TFA/DCM, 20 min; ix) Boc-AA, DIC, HOBt, DIPEA, DMF, 3 h; x) p-BrC6H4COOH (15 mol-%), DIC, HOBt,
DIPEA, DMF, 18 h; xi) mix; xii) 12, DIC, HOBt, DIPEA, DMF, 3 h; xiii) p-BrC6H4COOH (xs), DIC, HOBt, DIPEA, DMF, 18 h; xiv)
Ac2O, DIPEA, DMF, 3 h; xv) K2CO3, MeOH, DMF, H2O.
A final iteration of split-and-mix synthesis was per- ginine side chain (1  TMSBr in TFA), cleaved (CNBr,
formed similar to the first two iterations: Fmoc-coupling; TFA, H2O) and analysed by MALDI-TOF MS. Successful
Boc-deprotection; Boc-coupling; Fmoc-deprotection as be- sequencing of all five beads indicated successful implemen-
fore, followed by a complete termination of the Fmoc- tation of the synthesis and a suitable diversity in the mem-
strand using excess p-bromobenzoic acid. A final Boc-de- bers of the library.
protection was carried out, the Boc-strand capped using an
excess of acetic anhydride and all resin portions were then
mixed together. Final deprotection of the trifluoroacetyl
Screening Experiments
group was carried out using 0.15  K2CO3 in MeOH/DMF/
H2O[16] to give the 4096-membered library 17.
In order to demonstrate the utility of the new coding
To test the success of the library synthesis, 5 beads were strategy for the rapid sequencing of hit beads from screen-
selected at random, individually treated to deprotect the ar- ing experiments, library 7 has been screened with a peptide
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J. Shepherd, G. J. Langley, J. M. Herniman, J. D. Kilburn
FULL PAPER
guest using the same methodology we have described pre- The system was allowed to equilibrate for at least 24 h be-
viously.[4a,4i] For the peptide guest we used the dye-labelled fore visualisation under a microscope or using a hand-lens.
tetrapeptide Val-Val-Ile-Ala 18, synthesised using standard Additional aliquots of peptide guest could be added to in-
peptide couplings (Figure 2). Val-Val-Ile-Ala is the C-ter- crease the peptide concentration to provide optimal selec-
minal tetrapeptide sequence of the amyloid-β protein, A tivity, as adjudged by the number of highly stained beads
β(1–42). Amyloid-β proteins, consisting of 39–43 amino ac- (typically 0.1–1% of beads) against a background of non-
ids constitute the major proteinaceous component of pro- or lightly stained beads.
tein plaques found in the brains of Alzheimer’s patients.[19]
Control experiments confirmed that selective binding ob-
Inhibition of the self-assembly of amyloid-β proteins, and served was not due to difference in resin composition of
consequent formation of β-amyloid fibrils, provides a individual beads (by incubating dye-labelled guest with un-
potential therapeutic approach for treating Alzheimer’s derivatised resin and noting no selective uptake), and that
disease[20] and a number of small molecules and designed selective binding is not due to selective recognition of the
β-sheet breakers have indeed been reported to inhibit Aβ dye portion (by incubating the library with the dye chromo-
aggregation.[21]
phore alone and noting no selective uptake). Screening the
dye-labelled guest peptide against simple peptide libraries
lacking any guanidinium carboxylate binding site also
showed no selectivity, emphasising the importance of the
interaction between the guest carboxylate and the receptor
guanidinium, and consistent with all of our earlier work on
such receptor libraries.[4a,4i]
Screening experiments were carried out using solvent
compositions of 50% DMSO/buffer and 20% DMSO/
buffer, and with two different buffers: borax (pH = 9.2) and
HEPES (pH = 8.0). The most strongly dyed beads were
manually picked from each screening experiment and se-
quenced using MALDI-TOF MS.
Results from three different screening experiments are
summarised in Table 1, Table 2, and Table 3.
Table 1. Sequencing results for highly-stained beads found in li-
brary screening experiment using library 7 and guest peptide 18 in
50% DMSO/borax buffer (pH 9.2).
Figure 2. Dye-labelled peptide 18 used in screening experiments
with library 7, and dye-lablled peptide 19 used by Schmuck in
screening experiment with library 20.
Bead
AA3
AA2
AA1
1
2
3
4
5
6
7
Glu(OtBu)
Leu
Ala
Val
Val
Leu
Leu
Val
Phe
Val
Gly
Lys(Boc)
Leu
Asn
Val
Leu
Asn
Val
Leu
Val
In particular, Schmuck has described[4g,4h] screening ex-
periments using the dansyl-labelled tetrapeptide Val-Val-Ile-
Ala 19 and a tripeptide receptor library 20 capped with a
guanidinopyrrole.[22] Schmuck successfully identified guani-
dinopyrrole-linked tripeptides, which bound strongly to the
sequence Val-Val-Ile-Ala and subsequently showed that
such guanidinopyrrole-linked tripeptides could inhibit the
formation of β-amyloid fibrils.[21a] Given the similarities be-
tween Schmuck’s library 20 and our own bicyclic guanidi-
nium-linked tripeptide library 7, it was of interest to screen
our library with dye-labelled tetrapeptide Val-Val-Ile-Ala in
order to verify the viability of screening receptor libraries
indexed by “ladder” sequences as well as comparing screen-
ing results with those of Schmuck.
Val
8
9
Val
Val
Glu(OtBu)
Val
Val
Phe
10
11
12
13
14
15
16
17
Leu
Leu
Ala
Phe
Glu(OtBu)
Gly
Leu
Leu
Val
Leu
Val
Val
Ala
Val
Val
Val
Val
Val
Ser(OtBu)
Val
Val
Leu
Leu
Val
In a typical screening experiment, about 8 mg of the
From the screening experiments the high preponderence
resin-bound library 7 (sufficient such that the number of of valine at all three positions (AA1–AA3) and structurally
beads sampled ensures a statistical likelihood of all library very similar leucine at AA2 and AA3, is immediately appar-
members being represented in the screening experiment[23]
)
ent in all screening experiments. Certain receptor structures
was allowed to swell in a mixture of DMSO/buffer and ti- featured several times across all the screening experiments
trated with aliquots of the dye-labelled tetrapeptide 18 (60– and in particular the sequence BiG-Leu-Val-Val (BiG = bi-
300 µ) also in DMSO/buffer. DMSO was used to ensure cyclic guanidinium) was found 4 times, and Val-Leu-Leu
solubilisation of the guest tetrapeptide 18, and the buffer was found 5 times out of a total of 43 beads sequenced.
ensured that the guest was present as the free carboxylate, Overall the sequences BiG-xxx-Val-Val and BiG-xxx-Leu-
and the guanidinium of the receptor remained protonated. Val account for 20 of the 43 beads sequenced. The Glu-
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Table 2. Sequencing results for highly-stained beads found in li- Binding Studies
brary screening experiment using library 7 and guest peptide 18 in
Given the high occurrence of the sequence BiG-Leu-Val-
Val in the screening experiments, this structure was chosen
for further investigation. The resin-bound receptor 21 was
synthesised using standard Fmoc-SPPS procedures
(Scheme 6).
50% DMSO/HEPES buffer (pH 8.0).
Bead
AA3
AA2
AA1
1
2
Val
Val
Leu
Val
Val
Val
Leu
Val
Val
Val
Leu
3
4
5
6
Glu(OtBu)
Glu(OtBu)
Val
Glu(OtBu)
Val
Leu
Leu
Leu
Leu
Val
7
8
9
Lys(Boc)
Val
Leu
Glu(OtBu)
Leu
Leu
Phe
Val
Val
Lys(Boc)
Phe
Val
Val
Val
Leu
Val
Phe
Val
Leu
Val
Val
Val
Glu(OtBu)
Val
Val
Val
Glu(OtBu)
10
11
12
13
14
15
16
Scheme 6. i) Fmoc-Ahx, DIC, HOBt, DIPEA, DMF, 3 h; ii) 20%
piperidine/DMF, 20 min; iii) Fmoc-Val, DIC, HOBt, DIPEA,
DMF, 3 h; iv) Fmoc-Leu, DIC, HOBt, DIPEA, DMF, 3 h; v) 6,
EDC, HOBt, DIPEA, DMAP, DMF, 1 h.
Table 3. Sequencing results for highly-stained beads found in li-
brary screening experiment using library 7 and guest peptide 18 in
20% DMSO/borax buffer (pH 9.2).
AA3
AA2
AA1
The free receptor 22 was synthesised in solution using
Bead
Boc-protected amino acid couplings (Scheme 7).
1
2
3
4
5
6
7
8
9
10
Val
Leu
Leu
Lys(Boc)
Val
Lys(Boc)
Val
Val
–
Val
Lys(Boc)
Leu
Val
Val
Leu
Leu
Val
Leu
Leu
Leu
Val
Ser(OtBu)
Phe
–
Leu
Leu
Glu(OtBu)
[a]
–
Phe
Val
[a] Sequencing was unsuccessful suggesting either unsuccessful
coupling or cleavage reactions.
(OtBu) residue was found at AA3 for 4 out of 16 beads
identified using HEPES buffer, and Lys(Boc) was found at
AA2 for 3 of the 10 beads selected from the screening ex-
periment using the lower proportion (20%) of DMSO.
Overall these results compare very favourably with those
of Schmuck[4g,4h] who found that in his system receptors
featuring the sequences Gua-Val-Val-Val (Gua = guanidi-
nopyrrole) and Gua-Val-Val-Phe were the strongest recep-
Scheme 7. i) CDI, propylamine, THF, 3 h; ii) 20% TFA/DCM, 1 h;
iii) Boc-Val-OH, CDI, Et3N, THF/CMF, 18 h; iv) Boc-Leu-OH,
CDI, Et3N, THF, 18 h; v) 6, EDC, HOBt, DIPEA, DMAP, DMF,
1 h.
The solid-phase bound receptor 21 was used to deter-
tors in MeOH as solvent (Ka ≈ 9000 –1), whereas in water mine an on-bead binding constant with the dye-labelled tet-
(using bis-tris buffer) receptors of the general structure rapeptide 18 by determining the ability of the resin-bound
Lys(Boc)-Lys(Boc)-xxx-Gua were the strongest. With receptor to absorb the peptide guest from solution, using
Schmuck’s system, for example, the receptor Gua-Leu-Lys- previously described methodology.[3c,4a,4h,24] Thus a known
(Boc)-Lys(Boc) bound dansyl-labelled tetrapeptide 19 with mass of resin 21 was incubated with a solution of guest 18
Kass = 2000 –1 (measured using an on-bead binding assay). in an aqueous buffer and the change in the concentration
The analogous sequence BiG-Leu-Lys(Boc)-Lys(Boc) was of the guest in free solution was monitored by UV spec-
identified in the screening experiment with library 7 using troscopy. To account for non-specific absorption of 18 to
HEPES buffer, and again from the screening experiment the polystyrene resin matrix, an equivalent mass of underiv-
with 7 in the more aqueous solvent (20% DMSO), the se- atised tentagel resin was also incubated with 18. This al-
quence Val/Leu-Lys(Boc)-Val/Leu was found for 3 of the 10 lowed the amount of 18 specifically bound by the tweezer
beads sequenced.
receptor to be determined, and hence an association con-
Eur. J. Org. Chem. 2007, 1345–1356
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1351

J. Shepherd, G. J. Langley, J. M. Herniman, J. D. Kilburn
FULL PAPER
Figure 3. Plot of UV absorption (at 500 nm) of peptide 18 vs. concentration of added receptor 22.
stant based on a 1:1 receptor-substrate stoichiometry was tures identified from combinatorial libraries of peptide re-
estimated as Kass = 1440Ϯ440 –1.
ceptors. The methodology has been adapted for the synthe-
Binding of the receptor 22 with the dye-labelled tetrapep- sis of a library of single-armed peptide receptors 7 and a
tide 18 in free solution was investigated using a reverse ti- library of small “tweezer” receptors 17. The synthesis of
tration, monitoring the UV absorption of the dye chromo- the latter library required the tandem synthesis of “ladder
phore of guest 18 during additions of the receptor 22 at sequences” separated from the full receptor. Library 7 was
constant guest concentration, in 50% DMSO/borax solu- successfully screened to identify binding partners for the
tion. Monitoring the absorption at 500 nm showed a signifi- C-terminal tetrapeptide sequence of the amyloid-β protein
cant increase in the absorption as the first equivalent of the (Aβ), with notable structural similarities to similar amyloid-
receptor was added but a subsequent decrease as a second β protein binding partners identified by Schmuck. This cap-
equivalent was added (Figure 3).
ping methodology is now being used for the synthesis of
The data could not be fitted convincingly to simple bind- structurally more complex encoded receptor libraries and
ing models assuming 1:1, 1:2 or 2:1 binding stoichiometries. results from this work will be reported in due course.
The failure to obtain clear binding data for receptors iden-
tified from such library screening experiments when trans-
ferred to free solution is a common, if frustrating, occur-
rence, and in part indicates that the environment provided
Experimental Section
General Methods: Commercially available compounds were used
by the solid-phase support can influence the binding selec-
without further purification. Tentagel-S-NH2 resin was used as so-
tivities and potency.[4a] Furthermore, in the case of the re-
lid support and purchased from Rapp Polymere (Germany). Amino
acid derivatives were purchased from NovaBiochem or BAChem.
Peptide synthesis on solid phase was performed in polypropylene
filtration tubes with polyethylene frits on a Visiprep SPE acuum
ceptor-substrate combination described in this work (i.e.
using models for inhibition of amyloid-β peptide self-as-
sembly) it is not surprising that binding studies in free solu-
tion (off-bead) may be complicated by aggregation phe-
nomena, and indeed Schmuck reported[4h] that the simple
tetrapeptide 19 formed insoluble, fibril-like aggregates upon
standing in solution despite its hydrophilic triethylene
glycol chain. None-the-less both the on-bead binding assay
and pronounced changes in UV spectrum observed during
the titration experiment clearly indicate significant interac-
tion between the receptor and the guest tetrapeptide. At-
Manifold from Supelco. The reaction containers were agitated on
a blood tube rotator (Stuart Scientific Blood Tune Rotator SB1).
All coupling reactions were monitored with the Kaiser Ninhydrin
Test.[17] Thin-layer chromatography (TLC) was performed on alu-
minium-backed plates Merck silica gel 60 F254 or Macherey–Nagel
Alugram Sil G/UV254. Silica 60A (particle size 35–70 micron) was
used for column chromatography. Melting points were determined
in open capillary tubes using a Gallenkamp Electrothermal Melt-
ing Point Apparatus, all are uncorrected. Infrared spectra were re-
corded with a Bio-Rad FT-IR spectrometer or a Nicolet 380 FT-
IR spectrometer as neat solids or oils. Proton NMR spectra were
obtained at 300 MHz with a Bruker AC 300 or Bruker AV 300 and
at 400 MHz with a Bruker DPX 400. Carbon NMR spectra were
recorded at 75 MHz with a Bruker AC300 and at 100 MHz with a
Bruker DPX 400. UV Spectra were recorded with a Shimadzu UV-
1601 spectrometer in quartz cells. Low-resolution ESI mass spectra
were recorded with a Micromass Platform II quadrupole mass ana-
1
tempted investigation of this interaction by H NMR was
unsuccessful due to solubility problems with the receptor 22
and peptide 18 at suitable concentrations, again providing
evidence of the likely aggregation of these compounds.
Conclusions
In summary a modified capping methodology has been
developed to facilitate the rapid sequencing of “hit” struc- lyser (Fisons VG platform through a Hewlett Packard 1050 HPLC
1352
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1345–1356

Split-and-Mix Peptide Receptor Libraries
FULL PAPER
system). High-resolution ESI mass spectra were obtained with a
Bruker Apex III FT-ICR mass spectrometer.
LRMS (ESI+): m/z = 489.3 [M + H]+, 511.3 [M + Na]+. HRMS:
(ESI+) calculated for C22H31F3N4O5 [M + H]+ 489.2320, found
489.2317.
Abbrevations: Ahx = 6-aminohexanoyl, Boc = tert-butyloxycar-
bonyl, CDI = carbonyldiimidazole, DBU = 1,8-diazabicyclo[5.4.0]-
undec-7-ene, DIC = N,NЈ-diisopropylcarbodiimide, DIPEA = di-
isopropylethylamine, DMAP = 4-(dimethylamino)pyridine, DMF
= dimethylformamide, Fmoc = 9-fluorenyloxycarbonyl, HOBt =
4-({3-[(tert-Butoxycarbonyl)amino]propylamino}[(2,2,2-trifluoroace-
tyl)imino]methylamino)butanoic Acid (12): Palladium on carbon
(10% by weight, 175 mg) was added to a solution of the carbamate
11 (804 mg, 1.65 mmol) in EtOAc (20 mL) and the mixture stirred
under hydrogen for 3 h. The reaction mixture was filtered through
celite and the residue washed with EtOAc. The combined filtrates
were evaporated to give the acid 12 as a white solid (619.3 mg,
1-hydroxybenzotriazole, Mtr
= 4-methoxy-2,3,6-trimethylben-
zenesulphonyl, Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl, PyBOP = benzotriazol-1-yloxy-tris(pyrrolino)phospho-
nium hexafluorophosphate, SPPS = solid-phase peptide synthesis,
TFA = trifluoroacetic acid, TMS = trimethylsilyl.
94%); m.p. 104–105 °C. IR (cm–1): νmax = 3281 (w), 2960 (w), 1742
˜
(w), 1682 (w), 1637 (s), 1521 (s), 1448 (m), 1389 (m), 1366 (m),
1158 (s), 660 (m). 1H NMR (300 MHz, [D6]DMSO): δ = 12.14 (br.
s, 1 H, COOH), 9.04 (br. s, 1 H, NH), 7.78 and 7.63 (br. s, 1 H,
NH), 6.85 and 6.75 (br. s, 1 H, NH), 3.29 (m, 2 H, NHCH2), 3.19
(m, 2 H, NHCH2), 2.95 (m, 2 H, BocNHCH2), 2.22 (t, J = 7.5 Hz,
2 H, CH2COOH), 1.72 (m, 2 H, CH2CH2CH2), 1.60 (m, 2 H,
CH2CH2CH2), 1.37 (s, 9 H, Boc) ppm. 13C NMR: δ = 174.2 (C),
160.0 (C), 155.9 (C), 120.8 (q, J = 260 Hz, CF3), 77.5 (C), 40.4
(CH2), 37.2 (CH2), 31.1 (CH2), 29.7 (CH2), 28.2 (CH3), 24.6 (CH2),
23.8 (CH2) ppm, COCF3 not visible. LRMS (ESI+): m/z = 303.0
[M + H – COCF3]+, 421.0 [M + Na]+. HRMS: (ESI+) calculated
for C15H25F3N4O5 [M + Na]+ 421.1669, found 421.1665.
Benzyl
4-[{3-[(tert-Butoxycarbonyl)amino]propylamino}thiocarb-
onyl]amino]butanoate (10): Ester 8 (2.12 g, 7.22 mmol) was stirred
in 20% TFA/DCM (30 mL) for 1 h. The solvents were removed
in vacuo by azeotropic distillation with toluene and the residue
redissolved in 1:1 MeOH/DCM (40 mL). Isothiocyanate 9[16]
(1.56 g, 7.22 mmol) was added, followed by Et3N (5 mL,
36.1 mmol). The mixture was stirred for 18 h and then evaporated
in vacuo. The residue was purified by column chromatography (elu-
ent 30% EtOAc/petroleum ether) to give the thiourea 10 as a yellow
oil (2.22 g, 75%). IR (cm–1): ν
= 3325 (br. w), 2974 (w), 2932
˜
max
(w), 1687 (m), 1548 (m), 1516 (m), 1251 (m), 1163 (s). 1H NMR
(300 MHz, CDCl3): δ = 7.34 (m, 5 H, arom), 6.40 (br. s, 1 H, NH),
5.12 (s, 2 H, CH2Ph), 4.90 (br. s, 1 H, NH), 3.61 (m, 2 H,
General Procedures for Solid-Phase Reactions and Solid-Phase Tests
Resin Preparation and Washing: Prior to reaction, resins were
swollen for 15 min in DCM. After completion of reaction, resins
were drained of the reaction medium and extensively washed with
DCM, DMF, and again with DCM. Each washing consisted of
suspending resin in solvent (≈ 15 mL per g of resin) which was
allowed to stand for one minute before draining and then repeating
the process twice. Any additional/alternate washing is noted in the
appropriate section.
SCNHCH2), 3.41 (m,
BocHNCH2), 2.46 (t, J = 7 Hz, 2 H, CH2COOBn), 1.93 (quin, J
7 Hz, H, CH2CH2CH2); 1.71 (quin, 7 Hz, H,
2 H, SCNHCH2), 3.17 (m, 2 H,
=
2
J
=
2
CH2CH2CH2); 1.42 (s, 9 H, Boc) ppm. 13C NMR (75 MHz,
CDCl3): δ = 181.5 (C), 173.5 (C), 157.1 (C), 135.8 (C), 128.7 (CH),
128.5 (CH), 128.4 (CH), 79.8 (C), 66.8 (CH2), 43.2 (CH2), 41.3
(CH2), 37.3 (CH2), 31.5 (CH2), 29.8 (CH2), 28.5 (CH3), 24.1 (CH2)
ppm. LRMS (ESI+): m/z = 410.2 [M + H]+ 432.2 [M + Na]+.
HRMS: (ESI+) calculated for C19H31N3O4S1 [M + Na]+ 432.1927,
found 432.1926.
Standard Coupling of Amino Acids: Coupling of N-protected amino
acids to an NH2-functionalised resin was carried out according to
the following procedure. Protected amino acid (3 equiv. based on
resin loading) and HOBt (3 equiv.) were dissolved in DMF (≈ 1 mL
per 50 mg of amino acid). DIC (3 equiv.) was added and the mix-
ture added to the pre-swollen resin. Finally excess DIPEA (3–
5 equiv.) was added, the reaction vessel sealed and vented, and the
mixture agitated for at least one hour. The reaction was repeated
as necessary until the resin gave a negative ninhydrin test, and fi-
nally washed according to the general procedure.
Benzyl 4-({3-[(tert-Butoxycarbonyl)amino]propylamino}-[(2,2,2-tri-
fluoroacetyl)imino]methylamino)butanoate
(11):
Iodomethane
(3.4 mL, 54.1 mmol) was added to a solution of the thiourea 10
(2.22 g, 5.41 mmol) in acetone (40 mL). The mixture was stirred
for 1 h and then evaporated in vacuo. The residue was redissolved
in 1:1 MeOH/DCM (40 mL) and ammonium hexafluorophosphate
(1.77 g, 10.8 mmol) was added. The mixture was stirred for 18 h
and then evaporated in vacuo. The residue was taken up in DCM
and washed once with distilled water. The organic phase was dried
with MgSO4 and evaporated to give a white foam. The foam was
dissolved in 4:1 toluene/CHCl3 (50 mL). Trifluoroacetamide
(3.06 g, 27.1 mmol) and DBU (1.6 mL, 10.8 mmol) were added and
the mixture heated at reflux for 1 h. After cooling, the solvents
were removed in vacuo and the residue purified by column
chromatography (20% EtOAc/petroleum ether) to give the guani-
Solid-Phase Fmoc-Deprotection: Pre-swollen resin was treated with
20% piperidine/DMF (≈ 20 mL per g of resin) for 10 min. The
process was repeated before washing as described above.
Solid-Phase Boc-Deprotection: Pre-swollen resin was treated with
20% TFA/DCM (≈ 20 mL per g of resin) for 20 min. The resin was
drained and washed with 50% DIPEA/DMF (≈ 20 mL per g of
resin), before washing as described above.
dine 11 as a yellow oil (1.31 g, 50%). IR (cm–1): ν
= 3333 (br.
˜
max
General Protocol for the Capping Reaction: Pre-swollen resin to be
capped was shaken with a solution of p-BrC6H4COOH in DMF
(for precise quantities see below) for 1 h, before addition of excess
DIC, HOBt and DIPEA (Ͼ10 equiv.) and further shaking for 18 h.
The resin was then drained and washed according to the general
procedure described above.
w), 2979 (w), 1690 (m), 1624 (s), 1517 (m), 1434 (m), 1167 (s), 1137
1
(s). H NMR (300 MHz, CDCl3): δ = 9.56 (br. s, 1 H, NH), 7.34
(m, 5 H, arom), 6.39 (br. s, 1 H, NH), 5.14 (br. s, 1 H, NH) 5.13
(s, 2 H, CH2Ph), 3.52 (m, 2 H, BocNHCH2), 3.26 (m, 2 H,
NHCH2), 3.17 (dt, J = 6, 7 Hz, 2 H, NHCH2), 2.47 (t, J = 7 Hz,
2 H, CH2COOBn), 1.94 (quin, 2 H, CH2CH2CH2), 1.65 (m, 2 H,
CH2CH2CH2), 1.42 (s, 9 H, Boc) ppm. 13C NMR (75 MHz,
CDCl3): δ = 173.3 (C), 166.7 (q, J = 35 Hz, C), 160.8 (C), 157.2
(C), 135.6 (C), 128.7 (CH), 128.5 (CH), 128.3 (CH), 117.1 (q, J =
Synthesis of Resin 2: Tentagel resin (0.2 mmol/g, 642 mg,
0.11 mmol) was pre-swollen using DCM and coupled with Fmoc-
Met-OH (122 mg, 0.33 mmol) using DIC (51 µL, 0.33 mmol),
286 Hz, CF3), 79.7 (C), 66.9 (CH2), 61.1 (CH2), 40.2 (CH2), 37.5 HOBt (44 mg, 0.33 mmol) and DIPEA (57 µL, 0.33 mmol) in
(CH2), 36.6 (CH2), 30.8 (CH2), 28.4 (CH3), 23.8 (CH2) ppm. DMF (5 mL). The resin was washed and gave a negative ninhydrin
Eur. J. Org. Chem. 2007, 1345–1356
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1353

J. Shepherd, G. J. Langley, J. M. Herniman, J. D. Kilburn
FULL PAPER
test. Fmoc-deprotection was followed by three cycles of amino acid
coupling/Fmoc-deprotection using Fmoc-Phe-OH (127 mg,
was coupled with an Fmoc-protected amino acid (22 µmol) and
DIC (10 µL, excess), HOBt (10 mg, excess), and DIPEA (10 µL,
0.33 mmol), DIC (51 µL, 0.33 mmol), HOBt (44 mg, 0.33 mmol) excess) in DMF (2 mL) and one of the following amino acids:
and DIPEA (57 µL, 0.33 mmol) in DMF (5 mL). A final amino
acid coupling using Fmoc-Ahx-OH (116 mg, 0.33 mmol), DIC
(51 µL, 0.33 mmol), HOBt (44 mg, 0.33 mmol) and DIPEA DI-
PEA (57 µL, 0.33 mmol) in DMF (5 mL) was carried out followed
by Fmoc-deprotection. The resin 2 was finally rinsed with Et2O and
dried in vacuo. Quantitative ninhydrin test gave the resin loading as
0.13 mmol/g.
Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Leu-OH,
Fmoc-Phe-OH, Fmoc-Glu(OBn)-OH, Fmoc-Asn-OH, Fmoc-
Ser(OtBu)-OH, Fmoc-Lys(Boc), followed by Fmoc-deprotection.
The resin was then subjected to
a capping reaction using
p-BrC6H4COOH (1.27 mL of 1.15 m solution in DMF,
a
1.47 µmol), DIC (10 µL, excess), HOBt (5 mg, excess) and DIPEA
(10 µL, excess). The resin portions were then combined, suspended
in DCM and shaken for 10 min to ensure thorough mixing. Two
further iterations of the same procedure were carried out (division
into four roughly equal portions; amino acid coupling; Fmoc-de-
protection; capping reaction; mixing) with the same quantities of
reagents as before. The resin was finally coupled with bicyclic gua-
nidinium 6 (55 mg, 108 µmol) and DIC (34 µL, 216 µmol), HOBt
(29 mg, 216 µmol) and DIPEA (38 µL, 216 µmol) in DMF
(10 mL). The resin was then washed according to the general pro-
cedure. A ninhydrin test gave a negative result, indicating that all
remaining free peptide had been capped by the bicyclic guanidi-
nium 6. Resin 7 was rinsed with Et2O and dried in vacuo.
Synthesis of Resin 5: Tentagel resin (0.2 mmol/g, 551 mg, 121 µmol)
was pre-swollen using DCM. Amino acid coupling was carried out
using Fmoc-Met-OH (90 mg, 242 µmol), DIC (57 µL, 363 µmol),
HOBt (49 mg, 363 µmol) and DIPEA (63 µL, 363 µmol) in DMF
(5 mL) followed by Fmoc-deprotection. A second amino acid coup-
ling was carried out using Fmoc-Arg(Pbf)-OH (157 mg, 242 µmol),
DIC (57 µL, 363 µmol), HOBt (49 mg, 363 µmol) and DIPEA
(63 µL, 363 µmol) in DMF (5 mL) followed by Fmoc-deprotection.
Two cycles of amino acid coupling, followed by Fmoc-deprotection
was carried out using Fmoc-Ahx-OH (86 mg, 242 µmol), DIC
(57 µL, 363 µmol), HOBt (49 mg, 363 µmol) and DIPEA (63 µL,
363 µmol) in DMF (5 mL). Resin 5 was finally rinsed with Et2O
and rigorously dried in vacuo. A quantitative ninhydrin test gave
the resin loading as 0.15 mmol/g.
Solid-Phase-Linked Receptor 21: Tentagel resin (119 mg, 0.2 mmol/
g, 23.8 µmol) was pre-swollen according to the general procedure.
Amino acid coupling was carried out using Fmoc-Ahx-OH (17 mg,
47.6 µmol), DIC (11 µL, 71.4 µmol), HOBt (10 mg, 71.4 µmol) and
DIPEA (12 µL, 71.4 µmol) in DMF (3 mL) followed by Fmoc-
deprotection. The resin was subjected to two iterations of amino
acid coupling using Fmoc-Val-OH (24 mg, 71.4 µmol), DIC
(11 µL, 71.4 µmol), HOBt (10 mg, 71.4 µmol) and DIPEA (12 µL,
71.4 µmol) in DMF (3 mL), followed by Fmoc-deprotection. The
resin was subjected to a further amino acid coupling using Fmoc-
Leu-OH (25 mg, 71.4 µmol), DIC (11 µL, 71.4 µmol), HOBt
(10 mg, 71.4 µmol) and DIPEA (12 µL, 71.4 µmol) in DMF (3 mL)
followed by Fmoc-deprotection. Finally, the resin was coupled with
acid 6 (18 mg, 35.7 µmol), DIC (11 µL, 71.4 µmol), HOBt (10 mg,
71.4 µmol) and DIPEA (12 µL, 71.4 µmol) in DMF (3 mL). After
washing the resin gave a negative ninhydrin test. The resin 21 was
rinsed with Et2O and dried in vacuo.
Synthesis of Resin 3: Resin 2 (12 mg, 0.13 mmol/g, 1.5 µmol) was
pre-swollen using DCM. Amino acid coupling using Fmoc-Gly-
OH (1.4 mg, 4.56 µmol), DIC (5 µL, excess), HOBt (5 mg, excess)
and DIPEA (5 µL, excess) in DMF (2 mL) was followed by Fmoc-
deprotection. A capping reaction was then performed using p-
BrC6H4COOH (259 µL, 1.47 m/DMF, 0.38 µmol) and DIC
(5 µL), HOBt (5 mg), DIPEA (5 µL). A second amino acid coup-
ling using Fmoc-Ala-OH (1.4 mg, 4.56 µmol), DIC (5 µL, excess),
HOBt (5 mg, excess) and DIPEA (5 µL, excess) in DMF (2 mL)
was followed by Fmoc-deprotection and again a capping reaction
using p-BrC6H4COOH (259 µL, 1.47 m/DMF, 0.38 µmol) and
DIC (5 µL), HOBt (5 mg), DIPEA (5 µL). A third amino acid
coupling using Fmoc-Ile-OH (1.6 mg, 4.56 µmol), DIC (5 µL, ex-
cess), HOBt (5 mg, excess) and DIPEA (5 µL, excess) in DMF
(2 mL) was followed by Fmoc-deprotection and a capping reaction
using p-BrC6H4COOH (330 µL, 1.15 m/DMF, 0.38 µmol) and
DIC (5 µL), HOBt (5 mg), DIPEA (5 µL). The resin 3 was finally
rinsed in Et2O and dried in vacuo.
Receptor 22: tert-Butyl N-(1S)-3-methyl-1-[{(1S)-2-methyl-1-[{(1S)-
2-methyl-1-[(propylamino)carbonyl]propylamino}carbonyl]-
propylamino}carbonyl]butylcarbamate (62 mg, 0.13 mmol) was
stirred in 20% TFA/DCM (2 mL) for 2 h. The reaction mixture
was rigorously evaporated in vacuo using toluene as an azeotrope.
The residue was redissolved in DMF (3 mL) and acid 12 (67 mg,
0.13 mmol), HOBt (20 mg, 0.14 mmol), DIPEA (45 µL,
0.26 mmol) and DMAP (2 mg) were added followed by EDC
(28 mg, 0.14 mmol). The mixture was stirred for 1 h and then evap-
orated in vacuo. The residue was redissolved in MeOH and a white
solid precipitated on addition of Et2O. The precipitate was recrys-
tallised from MeCN to give the receptor 22 as a white solid (10 mg,
Synthesis of Library 4: Resin 2 (80 mg, 0.13 mmol/g) was divided
into four equal portions (≈ 20 mg each) and pre-swollen using
DCM. Each portion was coupled with an Fmoc-protected amino
acid (7.8 µmol) using DIC (10 µL, excess), HOBt (5 mg, excess) and
DIPEA (10 µL, excess) in DMF (2 mL) and one of the following
amino acids: Fmoc-Ser(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc) followed by Fmoc-deprotection. Each resin sample
was then subjected to a capping reaction using p-BrC6H4COOH
(565 µL of a 1.15 m solution in DMF, 0.65 µmol), DIC (10 µL,
excess), HOBt (5 mg, excess) and DIPEA (10 µL, excess). The resin
portions were then combined, suspended in DCM and shaken for
10 min to ensure thorough mixing. Two further iterations of the
same procedure were carried out (division into four roughly equal
portions; amino acid coupling; washing; Fmoc-deprotection; cap-
ping reaction) with the same quantities of reagents as before. Fi-
nally the resin 4 was rinsed with Et2O and dried in vacuo. 20 single
beads were selected, cleaved and analysed by MALDI-TOF MS.
9%); m.p. 233–235 °C. IR (cm–1): ν
= 2957 (w), 1626 (s), 1543
˜
max
1
(m), 1459 (m), 1310 (m), 1221 (m), 1101 (s), 741 (m), 701 (s). H
NMR (400 MHz, [D6]DMSO): δ = 8.20 (d, J = 8.0 Hz, 1 H, NH),
7.94 (d, J = 9.0 Hz, 1 H, NH), 7.92 (d, J = 8.0 Hz, 1 H, NH), 7.85
(t, J = 5.5 Hz, 1 H, NH), 7.65–7.58 (m, 6 H, 5 CHarom. + NH),
7.50–7.41 (m, 6 H, 5 CHarom. + NH), 4.37 (q, J = 8 Hz, 1 H, Leu
α-CH), 4.16 (dd, J = 8.5, 7.5 Hz, 1 H, α-CH), 4.07 (dd, J = 8.5,
7.5 Hz, 1 H, α-CH), 3.64 (m, 2 H), 3.57 (m, 2 H), 3.42–3.21 (m, 3
H), 3.23 (s, 2 H, SCH2CO), 3.08–2.92 (m, 2 H), 2.80–2.69 (m, 2
H), 2.36–1.85 (m, 4 H), 1.84–1.70 (m, 2 H), 1.59 (m, 1 H), 1.46–
1.34 (m, 4 H), 1.03 (s, 9 H, tBuSi), 0.90–0.79 (m, 21 H, 7CH3) ppm.
Synthesis of Single-Armed Receptor Library 7: Resin 5 (0.09 mmol/
g, 735 mg) was split into nine equal portions (≈ 82 mg each) and 13C NMR (100 MHz, [D6]DMSO): δ = 171.8 (C), 170.5 (C), 170.4,
was pre-swollen according to the general procedure. Each portion
168.7, 150.3, 135.1 (CH), 132.6 (C), 132.4 (C), 130.0 (CH), 128.0
1354
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Split-and-Mix Peptide Receptor Libraries
FULL PAPER
(CH), 126.4 (CH), 124.1 (CH), 118.9 (CH), 109.9 (C), 65.8 (CH2),
57.9 (CH), 57.8 (CH), 51.2 (CH), 49.4 (CH), 47.2 (CH), 44.4 (CH2),
40.8 (CH2), 36.3 (CH2), 34.2 (CH2), 30.5 (CH), 30.1 (CH), 26.6
(CH3), 25.2 (CH2) 24.7 (CH2), 24.1 (CH2), 23.0 (CH2), 22.6 (CH),
22.2 (CH2), 22.1 (CH3), 21.6 (CH3), 19.1 (CH3), 18.8 (CH3), 18.2
(CH3), 18.1 (CH3), 11.3 (CH3) ppm. LRMS (ESI+): m/z = 864.7
[M – Cl]+. HRMS: (ESI+) calculated for C46H74N7O5S1Si1 [M+]
864.5236, found 864.5228.
Acknowledgments
We thank the Commission of the European Union (TMR Network
grant “Enantioselective Separations” ERB FMRX-CT-98-0233)
for financial support and BBSRC for a postgraduate studentship
(J. S.). We also thank Prof. J. de Mendoza, Institute of Chemical
Research of Catalonia (ICIQ), Tarragona, Spain and his research
group for providing us with a sample of the bicyclic guanidinium
derivative 6.
Mass Spectrometric Methods for Single Bead Analysis: Deprotection
and Cleavage of Material from Single Resin Beads: A single resin
bead to be analysed was placed in a glass conical insert (100 µL
volume) using tweezers or a micropipette. The insert was placed
inside an Eppendorf tube. If Arg-deprotection was required, it was
carried out as follows.
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Pbf Deprotection: The bead was treated with 50 µL of 50% TFA/
DCM for 1 h and the solvent was removed in vacuo.
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Mtr Deprotection: The bead was treated with 40 µL of 1  TMSBr
in TFA for 1 h and the reagent mixture removed in vacuo. Cleavage
was performed by treatment of the bead with 20 µL of a solution
of CNBr in 1:1 TFA/H2O (concentration 50–80 mg/mL) and incu-
bation in the dark for 18 h.
Sample Preparation: The cleavage solution was evaporated from the
sample tube in vacuo. 20 µL of either MeCN or 1:1 MeCN/H2O
was then added to the tube. The samples were sonicated for 5 min.
1 µL of solution was spotted onto the MALDI-platen. 1 µL of a
solution of matrix was then applied on top and mixed with the
sample on the platen surface. The spot was allowed to dry under a
stream of warm air before analysis. Matrix was prepared as a satu-
rated solution of α-cyanohydroxycinnamic acid in either acetone
or 1:1 MeCN/H2O for application as above. 1:1 MeCN/H2O gave
consistently better results for more complex mixtures.
MALDI Acquisition and Data Processing: Spectra were acquired
with a Micromass-Tofspec 2E reflectron MALDI-TOF mass spec-
trometer, recording positive ion data at an accelerating voltage of
20 kV and a pulse voltage of 3000 V. Spectra were obtained by
summing multiple laser shots. The following compounds were used
for external calibration; oxybutinin (m/z 358.24 [M + H]+), terfena-
dine (m/z 472.32 [M + H]+), bradykinin (m/z 1060.57 [M + H]+),
substance P (m/z 1347.74 [M + H]+), renin substrate tetradecapep-
tide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser)
(m/z 1758.93 [M + H]+). In the presence of the Arg-linker, species
were generally observed as [M + H]+. In the absence of the Arg-
linker, species were generally observed as [M + Na]+. Data was
acquired and processed using Masslynx v3.2.[12] The cluster analy-
sis function was used with the following parameters; first mass dif-
ference 2.00, first ratio 1.0, mass tolerance 0.05–0.1, ratio tolerance
15–50%, threshold 7–10%.
Screening Experiments: Receptor library resin 7 (≈ 8 mg) was
placed in a flat-bottomed glass dish and pre-swollen in DMSO/
buffer (0.4 mL) for 1 h. A solution of the dye-labelled guest 18 (typ-
ically 60–300 µ) in the same solvent was then added to the mixture
and the system incubated for 24 h. Further aliquots of guest 18
could be added as to the library, followed by further incubation
periods of 24 h, to provide optimal selectivity as adjudged by the
number of highly stained beads against a background of non- or
lightly stained beads. Heavily stained beads were then removed
from the mixture using a micropipette and each individual bead
subjected to cleavage and analysis according to the general pro-
cedure.
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Eur. J. Org. Chem. 2007, 1345–1356
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J. Shepherd, G. J. Langley, J. M. Herniman, J. D. Kilburn
FULL PAPER
[10] During the course of our studies Lam et al. have described the
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Published Online: January 10, 2007
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1345–1356