DNA-binding peptides searched from the solid-phase combinatorial library with the use of the magnetic beads attaching the target duplex DNA

Article abstract of DOI:10.1016/S0968-0896(99)00298-9

We have exhibited successful and rapid screening of DNA-binding peptide ligands from solid-phase library beads with the use of the target DNA- conjugated magnetic beads. The target duplex DNA (3) has a polyether linker between two complementary sequences (T₄A₃G-ether linker-CT₃A₄) and is stable in the duplex form during the selection procedure. Finally, 71 pentapeptide sequences were identified from the solid-phase pentapeptide library. From an analysis of the peptide sequences identified in this study, it has been revealed that peptide ligands contain hydrophobic amino acids as the major component. The synthetic peptides with identified sequences and a combination of the major components have exhibited moderate to high binding affinity to the duplex DNA in competition experiments with ethidium-DNA complexes. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00298-9

Bioorganic & Medicinal Chemistry 8 (2000) 465±473
DNA-Binding Peptides Searched from the Solid-Phase
Combinatorial Library with the Use of the Magnetic Beads
Attaching the Target Duplex DNA
Md. Rowshon Alam, Minoru Maeda and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Received 12 October 1999; accepted 16 November 1999
AbstractÐWe have exhibited successful and rapid screening of DNA-binding peptide ligands from solid-phase library beads with
the use of the target DNA-conjugated magnetic beads. The target duplex DNA (3) has a polyether linker between two com-
plementary sequences (T4A3G-ether linker-CT3A4) and is stable in the duplex form during the selection procedure. Finally, 71
pentapeptide sequences were identi®ed from the solid-phase pentapeptide library. From an analysis of the peptide sequences iden-
ti®ed in this study, it has been revealed that peptide ligands contain hydrophobic amino acids as the major component. The syn-
thetic peptides with identi®ed sequences and a combination of the major components have exhibited moderate to high binding
anity to the duplex DNA in competition experiments with ethidium±DNA complexes. # 2000 Elsevier Science Ltd. All rights
reserved.
Introduction
molecular ligands for DNA-binding. In this paper, we
wish to report the following: (a) random screening of
new peptide ligands from the solid-phase pentapeptide
library by combinatorial technology, and (b) quantitative
analysis of the binding anity of selected pentapeptides
with the duplex.
It is now widely accepted that most diseases contain
genetic disorder. Therefore, development of new thera-
peutic methods for sequence-selective inhibition of
disordered genes is highly desired. A number of
approaches have been attempted with the use of oligo-
nucleotides-based inhibitors, DNA-binding ligands with
small molecular weight such as naturally occurring
substances, etc.1±6 Oligonucleotides and related com-
pounds can recognize a duplex DNA sequence within
the major groove by forming triplexes.7±12 However,
duplex sequences suitable for triplex formation are
limited only to homopurine±hornopyrimidine tracts.
Recently, polyamides as the analogues of natural prod-
ucts have been shown to recognize a DNA sequence
within a minor groove.13±19 Nevertheless, accurate
recognition of a DNA sequence has remained a dicult
problem. In this study, we have attempted to ®nd short
peptide ligands for the target duplex DNA based on
combinatorial technology. Binding motifs of DNA-
binding proteins consist of relatively short peptides, and
their binding properties are originated from the ®xed
conformation of a protein backbone.20 Determination
of peptide ligands by random screening and their struc-
ture analysis would provide useful information on small
Structures of the Library and the Magnetic Beads,
and the Screening Method
The chemical structures of the library and the magnetic
beads are schematically shown in Figure 1, and Figure 2
illustrates the screening procedure. The library beads
were constructed with Merri®eld resin by pool-and-split
synthesis, incorporating one sequence per (one) bead.21
Two units of 6-amionocaproic acid were ®rst incorpor-
ated into the resin as the spacer, followed by elongation
of pentapeptide by conventional solid-phase synthesis
with the use of 20 N-Fmoc-protected L-amino acids to
provide library beads (1) containing 3,200,000 sequences
(205) in total. We have already reported a combinatorial
random screening method with the use of magnetic
beads conjugated with the target molecule and the
library beads.22 In this study, we attempted to apply this
method to search for peptide ligands toward the target
duplex DNA that is attached on the magnetic beads
(2).23,24 The sequence of the target duplex DNA (3)
(T4A3G-ether linker-CT3A4) was chosen as a model
*Corresponding author.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00298-9

466
Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
Figure 1. The structures of the library (1) and the magnetic beads (2).
Figure 2. Schematic procedure of random screeninc, using, the library and magnetic beads.
duplex. The target duplex DNA (3) forms a stable
duplex (Tm=52±54 ꢀC) due to an ether linker as a non-
nucleotide loop, suggesting that the duplex structure is
maintained during the screening process at room tem-
perature. The target duplex DNA (3) was synthesized
bearing an amino-linker at the 50 end, and attached
covalently to the magnetic beads by a reductive amina-
tion procedure.
DMTr-protected amidite precursors of 20-deoxy-
nucleosides of adenosine, thymidine, guanosine, and
cytidine. A polyether spacer as a non-nucleotide loop.
(DMTrO(CH2CH6O),OP(NiPr2)O±CH2CH2CN) was
introduced between the two complementary strands of
DNA.25 At the ®nal step of the synthesis, aminolink2
(TfNH(CH2)6OP(NiPr2)O±CH2CH2CN) was intro-
duced to the 50 end, followed by cleavage with 28%
NH4OH. The crude oligomer was puri®ed by reverse-
phase HPLC to give the aminolink-conjugated target
duplex (3). Melting temperature of the target duplex (3)
ranged from 53 to 56 ꢀC.
A mixture of the library (1) and the magnetic beads (2)
in a bu€er was gently shaken in a plastic tube for 3 h at
room temperature. A strong magnet was placed outside
the wall of the tube. The interacting bead±bead com-
plexes adhered to the inside wall of the tube at the
position of the outer magnet. Interacting beads were
collected, and observed to be attached by a number of
magnetic beads on the surface (Fig. 3). Such library
beads were separated by a ®ne glass capillary under a
microscope, and subjected to a peptide sequencer for
determination of the sequence.
Next, the aminolink-conjugated target duplex (3) was
attached to the magnetic beads (MPG long chain alkyl-
amine, 500A-5 micron) according to the conventional
protocol of reductive amination with the use of glu-
taraldehyde and NaCNBH3 (Fig. 4). A part of the
magnetic beads (2) was hydrolyzed with venom phos-
phodiesterase (VPDE) and bacterial alkaline phospha-
tase (BAP); then the supernatant was analyzed by
reverse-phase HPLC. Four nucleoside components were
observed in the almost expected ratio of A:T:G:C=7:7:
1:1, con®rming successful introduction of the target
duplex (3) on the magnetic beads (Fig. 4).
Synthesis of the magnetic beads connecting duplex DNA
The target duplex DNA (T4A3G-ether linker-CT3A4, 3)
was synthesized by an automated synthesizer using the

Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
467
Table 1. Pentapeptide sequences determined by the random screeninga
EPFSF
GYEAN
IXWHX
IRMYI
NXMMR
DYVHI
SVLGL
TNYDX
NYXEX
FWWII
DIMEX
IDARX
AHNLA
XQGDX
NKYYY
NMWYV
DKXXY
YXVFL
WYHIY
NQYXX
XQXXX
YVVKX
PFRPP
FTGVM
XFNDW
FTNNP
KVIEX
XLYYF
FWGHE
TEHQL
FMYIR
PDADH
ADYLH
DXGIX
AQNRA
FDWXX
MGPFF
IWLXX
NRGII
FYFIS
RLPYM
LQQPL
YLIAN
MQXVY
TGFGW
GFKPF
FLDWW
GVQXV
EYFRV
HXMXX
AQYXI
LDWAA
LQMXR
LFNMX
TFFGX
VTWSX
KGFTD
KTIKA
YFXRT
VQHXY
Figure 3. Microphotograph of complexes between library and mag-
netic beads (Â200 magni®cation).
TLQLA
NTIIL
EDVHX
GPHNQ
INGGY
ILPEI
Results
VKPXX
NEIGX
ANREL
KXLXP
Determination of sequences of the selected beads
Table 1 summarizes the 71 sequences of pentapeptides
on the beads selected by the random screening. Un-
identi®ed Ð amino acids were indicated by X, some of
which may be tBu-protected cystein. The name and the
number of amino acids identi®ed at each position of the
pentapeptide sequence are summarized separately in
Figure 5, illustrating only those amino acids identi®ed
in more than ®ve peptides. Phenylalanine (Phe; F) and
asparagine (Asn; N) were determined most frequently at
the N-terminal position. Glutamine (Glu; Q) was the
best amino acid contained in the second residue. Tyr-
osine (Tyr; Y), glycine (Gly; G), tryptophan (Try; W)
and phenylalanine (Phe; F) were determined as the
preferable amino acid for the third amino acid. Iso-
leucine (IIe; I) was favorable at the fourth position, and
tyrosine, isoleucine (IIe; I) and leucine (Leu; L) were
determined frequently at the ®fth residue. Totally, tyr-
osine (Tyr; Y), phenylalanine (Phe; F), isoleucine (IIe; I),
leucine (Leu; L) and asparagine (Asn; N) were deter-
mined most in the pentapeptides. Interestingly, it turned
RWXHX
aX, not identi®ed.
out that hydrophobic amino acids are the most common
components of the pentapeptides that were selected in
this random screening. On the other hand, it should be
noted that no basic or acidic amino acids were deter-
mined as the most frequently appearing amino acids.
Also, some amino acids were completely excluded from
a speci®c position in the pentapeptide sequences, for
example, glutamine in the ®rst residue, serine and ala-
nine in the second, serine and threonine in the third and
glycine in the ®fth position. Therefore, we assumed that
peptides constructed of the most frequently appearing
amino acids at each position might have a high anity
to DNA.
Figure 4. Preparation of the magnetic beadsÐconnecting the target DNA 3, and analysis of enzymatic hydrolysate of the magnetic beads (2).

468
Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
Evaluation of binding anities of pentapeptides by
ethidium displacement assay
inhibition of emission of ethidium bromide does not
re¯ect the binding mode of the competitive ligand.26±32
Thus, the ethidium displacement assay is convenient
when the binding mode of the ligand is not known. In
this study, only selected pentapeptides and model DNA
duplexes (3, A3T3, CA12, CT12) were used for mea-
surement of binding anity (Table 2). The target duplex
(3) and self-complementary 12-mer duplexes (A3T3 and
CA12) consist of a di€erent number of AT pairs at the
center part of the duplex. CT12 has four GC pairs at the
Binding anity of the peptide ligands with the duplex
DNA was estimated by the displacement of DNA-
bound ethidium bromide (ETBr). The emission inten-
sity of ethidium bromide is increased by binding with
duplex DNA, and is inhibited by competition with
another DNA-ligand. The binding of ethidium bromide
with DNA contains several binding modes, and also
Figure 5. The number of amino acids determined at each position of pentapeptides. Only those contained in more than ®ve peptides are shown.
Table 2. Binding anity of peptide ligandsa (C50, mM)
Entry
Peptides
Target duplex (3) KETBr=3.9Â106
A3T3 KETBr=2.5Â106
CA12 KETBr=7.6Â106
CT12 KETBr=2.4Â106
1
2
3
4
5
6
7
8
FQGII
FQWII
FQFII
7.2
24.5
22
35
19
36
9.3
8
24
14.5
27
4.2
34
15.6
17.5
22
33
7.2
9
7.8
11
25
45
19
17.8
11.6
14.5
24.5
23
34.3
2.8
10
12.5
17
FQYII
WATTT
NQGIL
ILPEI
AHNLA
KTIKA
EPFSF
FTGVM
TEHQLb
KGFTD
FQWII
12
11.8
4.9
6
7.5
10
9
9
10
11
12
13
14c
15
10.5
15.7
17.2
37.5
0.2Â106
Ð
12.2
8
14.3
37
20
24
10
1.9Â106
1.4Â106
1.0Â106
Distamycin
Ð
No inhibition
19
aDNA as duplex=ethidium bromide=1.0 mM, 10.0 mM SHE bu€er, pH 5.0, Ex=546 nm, Em=595 nm.
bNo caproic acid unit connected to C-terminal.
cBinding constants (Ks, M 1) were determined directly by titration experiments under the same condition.

Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
469
middle of the duplex. First of all, we synthesized penta-
peptides, FQ-X-II (X=G, W, F, or Y) and NQGIL
which are composed of amino acids that were identi®ed
most frequently at each position. The sequences ILPEI,
AHNLA, KTIKA, EPFSF, FTGVM, TEHQL and
KGFTD were found in the sequences of some DNA or
RNA binding proteins, and were chosen for binding
assay, although it is uncertain whether these sequences
are involved in the binding sites of the proteins. The
sequence of WATTT contains amino acids that were
not found in this assay. All of these peptides except for
TEHQL, contain two units of 6-aminocaproic acid at
the C-terminal.
15). In contrast, FQ-Y-II, FQ-W-II and FQ-F-II with
di€erent amino residue at the third position exhibited
lower anity to DNA compared to FQGII (Table 2,
Entries 2±4). Binding anities of FQWII were obtained
directly by monitoring tryptophan ¯uorescence quench-
ing, and somewhat lower anities to all duplex DNA
than that of ETBr were obtained. Based on the com-
parison with FQWII, FQGII is estimated to have, at
least, comparable binding anity with ETBr. Displace-
ment of only one amino residue from G to W, F or Y
caused a remarkable decrease of binding anity
(Entries 1 versus 2, 3, 4). The sequences WATTT and
NQGIL showed a relatively moderate binding anity
to all duplexes (Entries 5 and 6). Among the other pep-
tides, ILPEI and AHNLA showed a relatively high a-
nity (Entries 7 and 8).
Prior to the ethidium displacement assay, we deter-
mined the binding parameters of ETBr±DNA binding.
Emission intensity of ETBr was measured in the function
of the duplex DNA concentration. Figure 6 illustrates
job plot and titration experiments with ETBr and
the 12-mer duplex (A3T3) at 25 ꢀC, showing a ratio of
The C5O values of FQ-X-II (X=G, W, F, Y) are plotted
against the duplex DNA sequence (Fig. 7). The binding
anity of ethidium bromide (ETBr) is also plotted for
comparison. FQGII exhibited the highest anity to all
sequences nonselectively, similar to ETBr. Change of
one amino acid in FQ-X-II showed little e€ect on the
bindings to CT 12, but induced a di€erent e€ect to the
other DNA sequences. Binding of FQFII or FQYII
became much weaker than that of FQGII toward
CA12; FQWII was the weakest ligand to A3T3; pep-
tides, except for FQGII, showed only low anity to the
target duplex DNA (3). As a result, FQWII showed
selectivity to CA12 and CT12, and FQYII became
selective to A3T3 and CT12.
ethidium:DNA=2:1 with
Ks=2.5Â106 M
a
Binding constants of ETBr to the
other duplexes were also similarly obtained, and are
shown in Table 2.
stability constant of
1 33
.
As binding stoichiometry of the pentapeptides could not
be determined, the binding anities of the peptides were
compared by determining the concentration needed
for 50% decrease of emission intensity of ETBr (C50,
mM) (Table 2). As the tryptophane-containing peptide
(FQWII) is ¯uorescent, the binding parameters could be
obtained directly.34±38 In this case, the intensity of
FQWII was decreased by binding with the DNA. The
job plot and titration experiment con®rmed 1:1 com-
plexation with the binding constants listed at entry 14 in
Table 2.
Discussion
In this study, we have applied combinatorial technology
to search for peptide ligands toward duplex DNA with
the use of the solid-phase library and the magnetic
beads conjugating the target duplex DNA. As a result,
71 pentapeptides were determined, and a clear tendency
was obtained in that only neutral and hydrophobic amino
acids were identi®ed in the selected pentapeptides and no
basic or acidic amino acids were determined (Table 1 and
Fig. 5). Some pentapeptides of the determined sequences
The sequence of FQGII is a simple combination of
amino acids with the highest appearance at each posi-
tion. This peptide showed the lowest C50 values toward
all sequences of DNA, indicating the highest anity
compared to other peptide sequences (Table 2, Entry 1).
The binding anity of FQGII is higher than that of
distamycin under the same condition (Entries 1 versus
Figure 6. Determination of binding parameters of ethidium bromide (ETBr)-A3T3 complexation: (A) job plot indicates A3T3:ETBr=1:2 com-
plexation: (B) theoretical curve was obtained by assuming A3T3:ETBr=1:2 complexation with Ks=2.5Â106 M
1
.

470
Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
were synthesized and their binding anities were esti-
mated by ethidium displacement assay (Table 2). It was
of great interest that the FQGII, a simple assembly of
the most-frequently-appeared amino acids at each posi-
tion, showed the highest anity to all duplex DNAs
examined here. From these results, a clear conclusion
has been obtained; the hydrophobic nature of small
molecules is one of the important forces for binding
with DNA. This ®nding is also consistent with other
results with hydrophobic compounds.27,28,39 As DNA
and RNA possess a highly negatively charged phos-
phate backbone and the nucleic acid binding sites of
these proteins possess a positively charged amino acid,
it is generally understood that the stabilities of most
protein-nucleic acid complexes receive large contribu-
tions from electrostatic interactions.40±42 Binding
poly(U) of polycationic peptides such as RWRRRR-
NH2 or KWKKKK-NH2 has been investigated, and
binding constants ranging from 103 to 105 were
obtained.43 The peptides identi®ed in this study, includ-
ing FQWII, have one positive charge at the N-free
terminal and one negative charge of the carboxyl group
at the C-terminal, having no net charge. Nevertheless,
these peptides showed comparable or somewhat stron-
ger binding anity to the duplex DNA compared to
the binding anity of the above polycationic peptides.
This comparison again suggests that the hydrophobic
structure plays an important role for binding of small
molecules to duplex DNA. It has recently been reported
that speci®c binding of Hoechst 33258 to the A3T3
duplex is mainly driven by hydrophobic interaction
rather than H-bonding or van der Waals contacts.44
Although we could not obtain general information
about the peptide structure for selective binding to a
speci®c DNA, it is interesting that change of one amino
residue of pentapeptide (FQ-X-II) had a di€erent e€ect
on the DNA binding and produced some selectivity
(Fig. 7). FQGII probably has a ¯exible structure
because of a small glycine residue, and may change its
conformation for binding to duplex DNA, causing
nonselective binding. The conformation of FQ-X-II
may be restricted by a relatively large aromatic residue
of Y, W or F, thus producing some selectivity. More
detailed experiments will be needed for further accurate
analysis of the binding properties of the peptide ligands.
Conclusion
In conclusion, we have exhibited successful and rapid
screening of DNA-binding peptide ligands from library
beads with use of the target DNA-conjugated magnetic
beads. From the analysis of peptide sequences identi®ed
in this study, it has been revealed that peptide ligands
contain hydrophobic amino acids as the major compo-
nent. The synthetic peptides with identi®ed sequences
and a combination of the major components have
exhibited moderate to high binding anity to the
duplex DNA. This study has clearly suggested that
hydrophobic interaction may be a major contributor for
binding of small molecules to duplex DNA.
Experimental
Synthesis of the target duplex DNA (3) conjugated with
aminolink
The target duplex (aminolink-T4A3G-polyether linker-
CT3A4) was synthesized with the automated DNA syn-
thesizer (CyclonePlus DNA synthesizer, MilliGen/Bio-
research) on a CPG resin by the standard phosphoroamidite
chemistry. The amidite precursors of 20-deoxynucleotides
were purchased from Glen Research, and DMTrO(CH2-
CH2O)6±P(Cl)OCH2CH2CN and TfN(CH2)6±P(Cl)OCH2
CH2CN were synthesized according to the literature.
The duplex was cleaved from the resin with 28% aqu-
eous NH3 and the solution was heated at 55±60 ꢀC for
5±6 h. Then the crude material was puri®ed by HPLC
(Nacalai tesque, Cosmosil 5C 18-MS, solvent: %B=
10!20%/20 min, 100%/30 min, B=CH3CN, A=0.1 M
TEAA) to a€ord the aminolink-conjugated target
duplex 3. The synthesis of the target duplex 3 without
aminolink was synthesized by the same method except
that TfN(CH2)6±P(Cl)OCH2CH2CN was not applied at
the ®nal step.
Aminolink-conjugated target duplex 3: MALDI-TOF
mass: calcd 5397.13, found 5398.63. Target duplex 3
without amino link: MALDI-TOF mass: calcd 5217.98,
found 5217.84. UV melting was measured at 260 nm
with the use of the target duplex 3 (2 mM) in 0.1 M KCl
bu€er at pH 7.0 by raising and lowering the tempera-
ture at 1 ꢀC/min, and melting temperature of 53 ꢀC was
obtained.
Synthesis of the magnetic beads conjugated with amino
link target duplex (2)
Figure 7. Sequence selectivity of pentapeptides. The left axis repre-
sents the C50 (mM), and the right axis represents Ks (M 1) of ethidium
bromide (ETBr).
Magnetic beads (MPG long chain alkylamine, 500 A±
5 mm, 1.2±1.8Â108 particles/30 mg in 1.0 mL) were put

Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
471
into a plastic tube, then the tube was placed on a strong
magnet and the supernatant was taken out. Coupling
bu€er (3.0 mL, 10 mM phosphate, pH 7.5) was added to
the beads, and the whole was mixed well. The beads
were magnetically separated and washed with the same
bu€er (3.0 mLÂ5). A solution of 10% glutaraldehyde
(3.0 mL, in the above bu€er) was added to the above
beads, and the mixture was gently rotated for 2 h. The
solvent was removed and the beads were washed with
the bu€er (3.0 mLÂ5). A solution of the aminolink-
conjugated target duplex DNA (3) (3 mg, 3.0 mL cou-
pling bu€er) was added to the above beads, followed by
the addition of a solution of % NaCNBH3 (300 mL in
coupling bu€er). The reaction mixture was rotated for
11.5 h at room temperature. The beads were washed
with coupling bu€er, followed by treatment of 1% gly-
cine solution (in 3 mL, coupling bu€er) and 1%
NaCNBH3 solution (300 mL in the coupling bu€er) for
3 h at room temperature. The beads were washed with
washing bu€er (10 mM phosphate, pH 7.0, 1.0 mM
NaCl) (3.0 mLÂ5) to give the target-conjugated mag-
netic beads (2), which were stored in a refrigerator
(4 ꢀC). A part of the DNA-bound magnetic beads
(3.0 mg) was treated with venom phosphodiesterase
(VPDE) and bacterial alkaline phosphatase (BAP) in a
bu€er for 2 h at room temperature, then the mixture
was centrifuged (10,000 rpm) to remove the magnetic
beads. The supernatant was analyzed by HPLC (Naca-
lai tesque, Cosmosil packed column, 4.6Â250 mm, 5C
18-MS, solvent: %B=5!20%/20 min, 100%/30 min,
heated at 80 ꢀC for 2 h with occasional shaking. The
beads were collected on a glass ®lter and washed with
DMSO (10 mLÂ3), CH2Cl2 (20 mLÂ5) and dried under
vacuum to give the reacted beads (6.2 g). The beads
(5.36 g) were treated with CF3COOH/CH2Cl2 solution
(4±6 mL) for 30 min at room temperature, and collected
on a glass ®lter, washed with CH2Cl2 (50 mLÂ2) and
dried under vacuum to give the beads (5.14 g). The
above beads (10.0 g, 2.3 mmol) were suspended in N-
methylpyrrolidone (20 mL), followed by addition of
20 mL of 0.5 M DMF solution (10.0 mmol) of HBTU
(2-(1-H-benzotriazol-l-yl-)-1,1,3,3,-tetramethyluronium
hexafuluorophosphate) and HOBt (1-hydroxy-benzo-
triazol hydrate) and diisopropylethylamine (1.8 mL,
10mmol). Into the above mixture was added 6-(t-butoxy-
carbonylamino)caproic acid (1.5 g, 6.9 mmol), and the
mixture was stirred for 1 h at room temperature. The
beads were collected on a glass ®lter, and washed suc-
cessively with N-methylpyrrolidone (50 mLÂ2) and
CH2Cl2 (50 mLÂ2), and dried under vacuum to give the
beads with two units of 6-aminocaproic acid (10.5 g).
The above beads (8.52 g, 5.03 mmol) were treated with
50% TFA-CH2Cl2 (60 mL) for 1 h at room temperature.
The beads were collected on a glass ®lter, and washed
with CH2Cl2 (10 mLÂ5) and dried under vacuum to
give the beads with free amino groups (8.4 g).
Elongation of peptide chain by the pool and split method.
The above beads were divided into 20 vessels, and the
peptide chain elongation was done as follows. The mix-
ture of the above beads (0.55 g), Fmoc-amino acid
(1.0 mmol), diisopropylethylamine (0.35 mL, 2.02 mmol)
and 3.5 mL of 0.5 M DMF solution of HBTU-HOBT
(1.75 mmol) in N-methylpyrrolidone (3.5 mL) were
shaken gently for 0.5 h at room temperature. The
beads were collected on a glass ®lter and successively
washed with N-methylpyrrolidone (5 mLÂ3), methanol
(5 mLÂ3), CH2Cl2 (5 mLÂ3). This washing procedure
was repeated twice and dried under vacuum. At this
stage, the reaction completion was monitored by the
picrate method. When the reaction was not completed,
the same coupling procedure was repeated. The peptide-
elongated beads were treated with 20% piperidine in N-
methylpyrrolidone for 2 h at room temperature, and
were collected on a glass ®lter. The beads were washed
successively with N-methylpyrrolidone (2 mLÂ3) and
methanol (2 mLÂ3). This washing procedure was repeated
twice. The above peptide elongation was done sepa-
rately with using 20 Fmoc-protected amino acid, and
the obtained beads were combined and mixed well, then
divided into 20 vessels again according to Lam's proto-
col.21 Then the same elongation procedure was repeated
to give library beads containing all the pentapeptide
sequences constructed based on the one-bead-one-
sequence concept.
¯ow rate: 1 mL/min, B=CH3CN, A=0.05
M
NH4OOCH, UV=260 nm). The HPLC chromatogram,
is shown in Figure 4, clearly showing the four base
peaks in an almost expected ratio.
Synthesis of the pentapeptide library beads
6-(N-t-Buthytoxycarbonylamino)caproic acid. Di-t-bu-
toxycarbonate (11.8 g, 0.057 mol,) and NaOH (2.2 g,
0.055 mol) was added into a solution of 6-aminocaproic
acid (6.56 g, 0.05 mol) in t-butanol (40 mL), and the
mixture was stirred for 5 h at room temperature. The
reaction mixture was quenched with water (55 mL), and
the whole was extracted with n-hexane (300 mLÂ2). The
aqueous layer was acidi®ed with 10% HCl at pH 2 at
0 ꢀC, and salted out with NaCl, then extracted with
EtOAc (300 mLÂ2). The combined organic layers were
washed with brine (300 mLÂ2) and dried over Mg2SO4,
®ltered and evaporated. The solvent was evaporated to
give a crude oil, which was crystallized from n-hexane to
give colorless ®ne needles (9.68 g, 88%). mp: 32.5±
33.5 ꢀC. IR (cm 1): 3340, 3400±2400, 1710, 1525, 1H
NMR (270 MHz, CDC13/TMS): 10.69 (1H, bs), 4.61
(IH, bs), 3.10±3.08 (2H, m), 2.32 (2H, t, J=7.4 Hz), 1.63
(2H, q, J=7.6 Hz), 1.53±1.29 (4H, m), 1.42 (9H, s),
FABMS (m/z): 463 (2M+H)+, 232 (M+H)+.
Selection procedure of the library beads with the target
conjugated magnetic beads. A suspension of the library
beads (200 mg) was gently rotated for 3 h in a bu€er
containing 100 mM NaCl and 5 mM Na2HPO4 at pH
7.0 at room temperature. The DNA-conjugated mag-
netic beads (5.4 mg in 0.2 mL) were added to the above
suspension, and the mixture was slowly rotated for 3 h
Incorporation of 6-(N-t-buthyloxycarbonylamino)caproic
acid onto the Merri®eld resin. 6-(N-t-Buthyloxy-
carbonylamino)caproic acid (4.6 g, 21 mmol) was added
into a solution of KOtBu (3.5 g, 0.012 mol) in dry
DMSO (50 mL), followed by addition of the Merri®eld
resin (chloride form, 5 g, 4.8 mmol). The mixture was

472
Md. R. Alam et al. / Bioorg. Med. Chem. 8 (2000) 465±473
at room temperature. A strong magnet was placed out-
side the wall of the test tube. The interacting bead±bead
complexes adhered to the magnetic position on the
inside wall of the tube, and were collected. A single
bead±bead complex was separated from the mixture by
a ®ne glass needle under a microscope, and was directly
loaded to a peptide sequencer. The whole operation is
schematically shown in Figure 2.
a solution of DNA by portions into a solution of
FQWII (1.0 mM in 10 mM SHE bu€er). Changes in
emission intensity at 350 nm with excitation wavelength
of 296 nm were analyzed. In the experiments of ethid-
ium-displacement assay, the changes in ¯uorescence
intensity at 595 nm with excitation at 546 nm were
measured by adding a stock solution of the peptide into
a solution of ethidium bromide (1.0 mM) and DNA
(1.0 mM) in 10 mM SHE bu€er at room temperature.
The C50 value represents the concentration needed for
50% decrease of ¯uorescence intensity (Table 2).
Synthesis of pentapeptides. The peptides used in this
study were synthesized by using HMP resin (1.1 mmol/g)
by a standard N-Fmoc chemistry. At ®rst, two units of
6-aminocaproic acid were introduced. Under argon, the
mixture of the HMP resin (3.0 g, 3.3 mmol), 9-¯uor-
enylmethyloxycarbonyl-6-aminocaproic acid (5.63 g,
16.5 mmol), DCC (N,N0-dicyclohexylcarbodiimide)
(3.40 g, 16.5 mmol) and DMAP (4-dimethylaminopyr-
idine) (0.40 g, 3.3 mmol) in N-methylpyrrolidone
(30.0 mL) was shaken gently for 2.5 h at room tempera-
ture. The beads were collected on a glass ®lter and suc-
cessively washed with N-methylpyrrolidone (10.0 mLÂ4),
methanol (10.0 mLÂ4), CH2Cl2 (10.0 mLÂ4) and dried
under vacuum. The above beads were treated with 20%
piperidine in DMF (30.0 mL) for 80 min at room tem-
perature and were collected on a glass ®lter. The same
procedure was done again to give the beads conjugated
with two units of 6-aminocaproic acid. The pentapep-
tides used in this study were synthesized using the above
beads by standard Fmoc chemistry as described for the
synthesis of library beads. The cleavage of the synthe-
sized peptide was done with 50% TFA in CH2Cl2
(4.0 mL) in the presence of 3% triisobutylsilane and 2%
H2O for 1 h at room temperature. The reaction mixture
was ®ltered, and the ®ltrate was diluted with cold
ether (100.0 mL). White precipitates were collected on
a glass ®lter, washed with ether, then dissolved in TFA
(1±1.5 mL). TFA solution was evaporated to dryness
and the crude peptide was puri®ed by HPLC (Nacalai
tesque, Cosmosil, 5C 18-MS, Solvent: %B=35%,
%A=65%, ¯ow rate=10 mL min, UV=254 nm,
B=0.05% TFA in CH3CN, A=0.05% TFA in H2O).
Freeze-drying of the solvents a€orded the desired
peptides as a white powder whose structures were
con®rmed by 1H NMR (500 MHz) and FAB mass
spectrometry.
Acknowledgments
This work was supported by Grant-in-Aid for Scienti®c
Research from the Ministry of Education, Science,
Sports and Culture, Japan (C09672146 to S.S.). Scho-
larship to M. R. A. from the Ministry of Education,
Science, Sports and Culture, Japan is also appreciated.
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