Table Of ContentSynthesis of a One-Bead One-Compound
Combinatorial Peptide Library
Kit S. Lam and Michal Lebl
1. Introduction
The four general methods to generate and screen a huge combinatorial pep-
tide library +-lo7 peptides) are: biological libraries such as filamentous phage
(I), plasmid (2)) or polysome (3) libraries; the “one-bead one-compound” syn-
thetic combmatonal library method or the “Selectlde process” (4-6); synthetic
peptide library methods that require deconvolution, such as an iterative
approach (7,8), positional scanning (9); orthogonal partition approach (JO), or
recurse deconvolution (II); and synthetic library using affinity column
selection method (12,13).
There are advantages and disadvantages m each of these methods. In gen-
eral, the main advantages of the biological library method are that large pep-
tides can be displayed on a filamentous phage library, and that large protein
folds can be mcorporated into the library. However, the main disadvantage is
that biological libraries, in general, are restricted to all L-amino acids. In con-
trast, the remaining three methods all use synthetic libraries; therefore,
o-amino acids, unnatural ammo acids, nonpeptide components, and small rigid
scaffoldings can all be incorporated into these libraries.
The “one-bead one-compound” library is based on the concept (4,5) that
when a solid-phase split synthesis method (4,8,14) is used, each solid-phase
particle (bead) displays only one peptide entity although there are approx 1013
copies of the same peptide in the same bead. The resulting peptide-bead library
(e.g., lo7 beads) is then screened in parallel using either “on-bead” binding
assays (15) or “solution phase-releasable” assays (16) to identify peptide-beads
with the desired biologic, biochemical, chemical, or physical properties. The
From Methods m Molecular Biology, vol 87 Combmatonal Peptrde Library Protocols
Edlted by S CablIly 0 Humana Press Inc , Totowa, NJ
2 Lam and Lebl
positive peptide-beads are then physically isolated for microsequencing with
an automatic protein sequencer. In this chapter, detailed methods for the syn-
thesis of a random “one-bead one-compound” combinatorial peptide library
will be described. Chapters 2 and 10 give examples of two general screening
methods for such libraries.
2. Materials
2.1. Chemicals
1. Tenta-Gel Resin S-NH, (90-100 pm) resin may be obtained from Rapp Polymere,
Tubmgen, Germany (see Note 1).
2. Fmoc amino acids with standard side chain-protectmg groups, N-hydroxy-
benzotriazole (HOBt), benzotriazolyl-oxy-trisdimethylammo-phosphonmm
hexafluorophosphate (BOP), diisopropylethylamme (DIEA), diisopropyl-
carbodumide (DIC), piperidme, trifluoroacetic acid (TFA), nmhydrm, may be
obtained from many different suppliers, such as Bachem (Torrance, CA), Bio-
science (King of Prussia, PA), Advanced ChemTech (Louisville, KY),
Novabiochem (San Diego, CA), and Peptides International (Louisville, KY)
3. Technical grade solvents such as dimethylformamide (DMF) or dichloromethane
(DCM) may be obtained from many different chemical suppliers HPLC-grade
DMF for the coupling may be obtamed from Burdock and Jackson, Muskegon,
MI. Ethanol, phenol, p-cresole, thioamsole, ethanedithiol, pyndme, and potas-
sium cyanide may be obtained from many different chemical suppliers.
4 0 1 g/mL Nmhydrm in ethanol
5 4 g/mL Phenol m ethanol.
6 10 mM Potassmm cyanide, stock solution.
7. 50% Piperidme m DMF
8. Reagent K: TFAlp-cresolelwaterlthioamsole/ethanedithiol, 82 5*5:5:5*2.5. (v/v/
v/v/v)
9 10% DIEA m DMF.
10 Dimethylsulfoxide (DMSO)/Amsole/TFA, 10:5:85
2.2. Apparatus
1. Polypropylene vials (5-lo-mL) may be purchased from Baxter Scientific Prod-
ucts, McGaw Park, IL. Polyethylene disposable transfer pipets may be purchased
from Elkay Products, Shrewsbury, MA.
2 Motorized rockmg platform.
3 Randomization glass vessel (chromatography column 5-6 x 18 cm) fitted with a
medmm glass smtered frit connected to vacuum and nitrogen via a two-way valve
from below The three positions of the valve are “off,” “vacuum,” or “nitrogen.”
4 Recnculatmg water aspirator or a solvent-resistant vacuum pump with cold trap
5 Nitrogen tank.
One-Bead One-Compound
3. Methods
3.1. Synthesis of a Linear Pentapeptide Library
As indicated earlier, a solid-phase split synthesis method (4,8,14) is used to
generate a random peptide library. The composition and final structure of the
peptide library depends on the number of amino acids (one or more) used m
each coupling cycle and the number of coupling cycles used. The final peptide
library may be linear or cyclic, or have specific secondary structures. For sim-
plicity, the method for the synthesis of a linear pentapeptide library with all 19
eukaryotic amino acids except cysteine is given below:
1. Swell 10 g TentaGel Resin S-NH, beads (- 0 25 mEq/g, see Notes 1 and 2) for at
least 2 h m HPLC-grade DMF with gentle shaking in a silicomzed flask.
2 Wash the beads twice with HPLC-grade DMF in the slllcomzed randomlzatlon
vessel as follows* add 75 mL DMF from the top, gently bubble nitrogen from
below through the smtered glass for 2 min, then remove the DMF by vacuum
from below (see Note 3).
3 Transfer all the beads to a slllcomzed flask in HPLC-grade DMF Then dlstrlbute
the beads into 19 equal allquots. A disposable polyethylene transfer plpet IS
extremely useful m the even distribution of the beads mto each polypropylene
vial (see Note 4).
4 Allow the beads to settle and remove most of the DMF above the settled bead
surface from each polypropylene reaction vial.
5 Add threefold molar excess of each of the 19 Fmoc-protected ammo acids (see
Note 5) and threefold molar excess of HOBt to each reaction vial using a mml-
ma1 volume of HPLC-grade DMF.
6. Add threefold molar excess each of BOP and DIEA to each reaction vial to ml-
tlate the coupling reaction.
7. Cap the reaction vials tightly and rock them gently for 1 h at room temperature
8. To confirm the completion of couplmg reaction, plpet a minute amount of resin
from each reaction vial into small borosilicate glass tubes (6 x 50-mm) and per-
form ninhydrm test (17) as follows:
Wash the minute quantity of resin m the small glass tubes (6 x 50-mm)
sequentially with the following solvents* DMF, t-amyl alcohol
(2-methylbutan-2-ol), acetic acid, t-amyl alcohol, DMF, and ether Add to
each tube one drop of each of the following three reagents, (ninhydrin m etha-
nol (0.1 g/mL), phenol m ethanol (4 g/mL), and potassium cyanide stock
solution diluted 50 times with pyridme. Place the tubes m a heating block at
120°C for 2 min. Observe the color intensity of the beadsu nder a microscope.
To ensure complete couplmg, every bead from the minute quantity of sample
beads should be nmhydrin negative, I e , straw yellow color.
4 Lam and Lebl
9 If the couplmg IS mcomplete (some beads remamed purple or brown with nmhy-
drm test), remove the supernatant from those reaction vials and add fresh Fmoc-
protected ammo acids, BOP, DIEA, and HOBt mto the reaction vial for addmonal
coupling
10. If the couplmg 1s complete (beads remained straw yellow color with nmhydrm
test) discard the supernatants of each reaction vial, and transfer and wash all the
beads to the randomtzation vessel with technical grade DMF
11 After all the 19 couplmg reactrons are completed, all the beads are transferred to
the randomizatton vessel Wash the beads (8 times, 2 mm each) with technical
grade DMF
12 Add 75 mL 50% ptpertdme (m DMF) to the randomtzatton vessel to remove the
Fmoc protectmg group After 10 mm, remove the ptpertdme and add 75 mL fresh
50% prpertdme. After another 10 mm, wash the beads 8 times wtth techmcal
grade DMF and twtce with HPLC-grade DMF
13 Distribute the beads mto each of the 19 reaction vials and carry out the next
couplmg reaction as described above
14. After all the randomtzatton steps are completed, remove the Fmoc protectmg
group with prpertdine as described above
15 After thorough washing with technical grade DMF (5X) followed by DCM (3X), add
10 mL of reagent K (18) to the randomrzatron vessel for 3 h at room temperature
16. Wash the deprotected resms thoroughly with DCM (3X), followed by technical
grade DMF (5X), then once with 10% DIEA to neutralize the resin
17. After thorough washing with technical grade DMF, store the bead library m
HPLC-grade DMF at 4°C. Alternatively, the bead library can be washed thor-
oughly with water and stored in 0.1 M HCl or 0.1 Mphosphate buffer with 0 05%
sodmm azide.
3.2. Synthesis of a Cyclic Peptide Library
The synthesis of a cyclic peptide library (disulfide bond formation) is essen-
tially the same as that of the linear library except that Fmoc-Cys (Trt) is added
at the carboxyl as well as amino terminus of the linear random peptide After
deprotectton, add a mixture of DMSO/Anisole/TFA (see Subheading 2.1.,
item 10) into the resin; incubate overnight at room temperature. After thor-
ough washing, store the library at 4°C as described above.
4. Notes
1 We have tested several commerctally available resins for our library synthesis
The two satisfactory resins are TentaGel (polyethylene grafted polystyrene beads)
and Pepsyn gel (polydimethylacrylamtde beads) Overall, the TentaGel 1sp refer-
able as it is nonsticky and mechanically more stable However, unlike Pepsyn
gel, the level of substrtutron of each TentaGel bead is far from uniform Wtth the
advent of combmatorral chemistry, we anticipate newer resins entering the market
m the near future
One-Bead One-Compound 5
2. TentaGel already has a long polyethylene linker and we do not routmely add
additional linker for our library synthesis In contrast, a linker (preferably a
hydrophilic lmker) is necessary for the synthesis of a peptide library with
polydimethylacrylamide beads. We have used Fmoc-P-alanme and/or Fmoc-
aminocaprorc acid as linkers in the past. However, aminocaproic acid is rather
hydrophobic A polyethyleneglycol-based amino acid (Shearwater, Polymers,
Huntsville, AL) is probably preferable.
3 All glass vessels should be sdiconized thoroughly prior to use Besides using
nitrogen bubbling through the randomization vessel to mix and wash the beads,
we have also prepared libraries in hourglass reaction vessels (Peptides Interna-
tional, Louisville, KY), usmg rocking motion to mix the resins.
4. Each polypropylene reaction vial should be engraved with a letter correspondmg
to a specific amino acid to ensure no mix-up during the synthesis
5. We often omit cysteines from the synthesis of linear peptide libraries to avoid the
complication of intracham and/or interchain crosslinking
Acknowledgments
This work was partially supported by NIH grants CA23074 and CA17094.
Kit S. Lam is a scholar of the Leukemia Society of America.
References
1 Scott, J K. and Smith, G. P. (1990) Searchmg for peptide ligands with an epitope
library. Science 249,386-390.
2. Schatz, P. (1993) Use of peptide libraries to map the substrate specificity of a
peptide-modifying enzyme A 13 residue consensus peptide specifies biotinylation
m Escherichia cob Biotechnology 11,1138-l 143.
3. Kawasaki, G. (199 1) Cell-free synthesis and isolation of novel genes and polypep-
tides. PCT International Patent Application W09 l/05058.
4 Lam, K. S., Salmon, S. E , Hersh, E M., Hruby, V. J , Kazmierski, W. M , and
Knapp, R. J. (1991) One-bead, one-peptide: a new type of synthetic peptide library
for identifymg bgand-bmdmg activity. Nature 354,82-84
5 Lebl, M., Krchnak, V., Sepetov, N F., Seligmann, B., Strop, P., Felder, S and
Lam, K S (1995) One-bead-one structure combmatorial libraries. Bzopolymers
37,177-198.
6 Lam, K S , Lebl, M , and Krchnak, V. (1997) The “one-bead-one-compound”
combinatorial library method. Chem. Rev. 97,41 l-448
7 Geysen, H. M , Rodda, S J., and Mason, T J. (1986) A prior-z delmeation of a
peptide which mimics a discontmuous antigenic determinant. Mol. Immunol. 23,
709-715.
8. Houghten, R. A , et al. (199 1) Generation and use of synthetic peptide combmato-
rial libraries for basic research and drug discovery. Nature 354,84-86
9. Dooley, C. T and Houghten, R A (1993) The use of positional scanning syn-
thetic peptide combinatorial libraries for the rapid determination of opioid recep-
tor ligands Life Scl. 56, 1509-1517
6 Lam and Lebl
10. Deprez, B , Willard, X , Bourel, L , Coste, H , Hyafil, F., and Tartar, A (1995)
Orthogonal combmatorial chemical libraries J Am. Chem. Sot. 117,5405-5408
11 Erb, E , Janda, K., and Brenner, S. (1994) Recenstve deconvolutton of combma-
torial chemtcal ltbraries Proc. Nutl Acad. Scz USA 91, 11,422-l 1,425
12. Zuckermann, R. N , Kerr, J. M , Slam, M A , Banvtlle, S. C., and Santa, D V
(1992) Identification of highest-affinity ligands by affinity selection from eqmmo-
lar pepttde mixtures generated by robotic synthesis. Proc Natl. Acad. Scz. USA
89,4505-4509.
13 Songyang, Z., Carraway, K L , Eck, M. J , Harrtson, S C., Feldman, R. A ,
Mohammadi, M , Schlessmger, J , Hubbard, S. R , Smith, D P , Eng, C., Lorenzo,
M. J., Ponder, B. A J , Mayer, B J , and Cantley, L. C (1995) Catalytic spectfrc-
tty of protein-tyrosme kmases 1s crmcal for selecttve stgnallmg Nature 373,
536-539
14. Furka, A., Sebestyen, F., Asgedom, M., and Dtbo, G (1991) General method for
rapid synthesis of multicomponent peptide mixtures. Int J Peptzde Protein Res
37,487+93.
15 Lam, K. S and Lebl, M (1994) Selectide technology-bead bmdmg screening
Methods f&372-380
16 Lebl, M , Krchnak, V , Salmon, S E., and Lam, K. S (1994) Screenmg of com-
pletely random one-bead-one-pepttde libraries for activmes m solution MethodA
f&381-387.
17 Kaiser, E., Colescott, R. L , Bossmger, C D., and Cook, P. I. (1970) Color test for
detection of free terminal ammo groups m the solid-phase synthesis of pepttdes
Anal. Blochem. 34,595-602.
18 King, D. S., Fields, C G , and Fields, G. B. (1990) A cleavage method which
mmtmtzes side reactions followmg Fmoc solid phase pepttde synthesis Znt. J.
Peptlde Protein Res. 36,255-266.
Enzyme-Linked Calorimetric Screening
of a One-Bead One-Compound Combinatorial Library
Kit S. Lam
1. Introduction
In the “one-bead one-compound” combinatorial library method, each bead
displays only one chemical compound although there are approx 1013 copies of
the same compound in and on the same bead (I-3). With an appropriate detec-
tion scheme, compound-beads with specific biological, physical, or chemical
properties can be identified, and physically isolated, and then their chemical
structure can be determined. In biological systems, one important property that
is of interest is the binding property between a ligand and a ligate. The hgate or
acceptor molecule could be an enzyme (4-6)) an antibody (1,7,8), a receptor
(9,10), a structural protein, or even small molecules (II). Furthermore, the
“one-bead one-compound” library method can also be applied to the discovery
of ligands that bind to the whole viral particle, bacteria, or mammalian cell by
screening for compound-beads that bind to intact cells.
When we mix a ligate with an “one-bead one-compound library,” some com-
pound-beads may be coated by the ligate. This interaction can be detected by
either a labeled ligate or a labeled secondary probe that recognizes the ligate.
Common labels are enzyme, fluorescent probe, color dye, or radionuclide.
There are advantages and disadvantages to each of these methods. The choice
of detection scheme depends largely on the nature and availability of specific
labeled ligates. From our experience, enzyme-linked calorimetric assay is prob-
ably the most convenient, economical, and rapid screening method that does
not require any elaborate equipment (12). Methods for the preparation of the
peptide-bead library are detailed in Chapter 7 of this volume. Details on the
enzyme-linked calorimetric screening method will be given in the next
sections,
From Methods m Molecular Bology, vol 87 Combmatonal Pep/de Library Protocols
Edlted by S CablIly 0 Humana Press Inc , Totowa, NJ
7
8 Lam
2. Materials
All the reagents needed are standard enzyme-linked immunosorbent assay
(ELISA) reagents and are readily available from many biochemical and chemi-
cal companies. The following buffers are needed for the screening:
1. PBS-Tween. 8 mM,Na2HP04, 1.5 mMKH2P04, 137mMNaC1,2.7mMKCl,pH
7.2, with 0.1% Tween-20 (v/v)
2 Binding Buffer 16 m&Z Na2HP04, 3 mM KH,PO,, 274 mM NaCl, 5 4 mM KCl,
pH 7 2, with 0.1% Tween-20 (v/v) and 0.1% gelatm (w/v).
3 TBS. 2 5 n&Z Trts-HCl, 13 7 mM NaCl, and 0 27 mM KCl, pH 8 0
4. BCIP/Alkalme phosphatase buffer. 1.65 mg 5-Bromo-4-chloro-3-mdolyl-
phosphate (BCIP) m 10 mL of 0 lMTris-HCl, 0 lMNaC1 with 2.34 mMMgCl,,
pH 8.5-9 0
5 Gelatin. 0 1% in water
6 6M Guamdme HCl, pH 1 0
3. Methods
3.1. Screening with an Enzyme-Linked Ligate
Common enzymes used in ELISA are alkaline phosphatase, horseradish per-
oxrdase, P-galactosrdase, and glucose oxidase. From our experience the alka-
line phosphatase system is more specific and tends to produce the least artifact
when we screen a “one-bead one-compound” library.
1 If ligate-alkaline phosphatase complex is not commercially available, one may
conmgate the ligate to alkaline phosphatase using bifunctional crosslmkmg
reagents Many such reagents are commercially available (e g , Pierce Chemical,
Rockford, IL) and standard coupling procedures are supplied by the manufactur-
ers Before screening a library, one has to make sure that the coqugation method
does not impair the bmdmg property of the ligate This can usually be accom-
plished by an ELISA assay using a 96-well plate coated with a known hgand (see
Note 1)
2 Transfer l-10 mL of the bead-library (200,000 to 2 million beads) to a 50-
lOO-mL polypropylene container Slowly dilute the dimethylformamtde (DMF)
by adding an incremental amount of double-distilled water. Wash the bead-
library thoroughly with double-distilled water m a column (e g., Econo column,
Bio-Rad, Hercules, CA) Coat the bead-library with 0 1% gelatin (w/v) m water
for at least 1 h. Wash the bead-library with PBS-Tween Transfer the library back
into the polypropylene contamer with the bmdmg buffer (see Note 2) Add the
ligate-alkaline phosphatase coqugate into the library with gentle mixing for 1 to
24 h at room temperature (see Note 3).
3. Transfer the bead-library to the column and wash the beads thoroughly with PBS-
Tween. Then wash the bead-library one last time with TBS.
Enzyme-Linked Calorimetric Library Screening 9
Fig. 1. (A) Photomicrograph of a typical enzyme-linked calorimetric bead-library
screen; a positive bead is noted in the middle of the micrograph. (B) Single positive
beads can easily be retrieved with a handheld micropipet under a dissecting
microscope.
4. Transfer and wash the bead-library to lo-20 polystyrene Petri dishes (100 x 20
mm) with the BCIP/alkaline phosphatase buffer (see Notes 4 and 5). More dishes
may be needed if the beads are too crowded and there are too many positive
beads. Let the enzyme-linked color reaction develop for 30 min to 2 h. Stop the
reaction by acidifying the BCIP/alkaline phosphatase buffer with several drops
of 1 M HCl. Figure 1A shows the photomicrograph of a typical bead-library
screen.
5. With the aid of a light box and a micropipet (e.g., Pipetman PlO, Gilson), transfer
the turquoise beads into a small Petri dish. Many colorless beads will also be
transferred during this process.
6. Place the small Petri dish of positive beads under a dissecting microscope and
pipet individual turquoise beads to a small Petri dish of 6 M guanidine-HCI, pH
1 .O (Fig. 1B). At this stage, transfer only the positive beads (see Note 6). After
10 Lam
20-30 mm at room temperature m 6 Mguanidme-HCI, transfer the posmve beads
to a dish of double-dtstrlled water. Then prpet each posrttve bead onto a glass
filter and msert mto the protein sequencer cartrtdge for mtcrosequencmg (see
Notes 7 and 8)
3.2. Screening with an Unlabeled Ligate by Probing
with an Enzyme-Linked Secondary Antibody
1 Prepare the library as m Subheading 3.1., item 2.
2 Add the alkaline phosphatase-linked anti-ligate antibody to the bead library and
incubate m bmdmg buffer for l-2 h at room temperature
3 Wash the bead-library thoroughly with PBS-Tween and finally once with TBS
4. Add BCIP substrate to the library as described m Subheading 3.1., item 4
5 After 30 mm to 2 h, stop the colortmetrrc reaction by adding several drops of 1 M
HCl to each Petri dish. Remove all the color beads from the library over a light
box with a mrcropipet These color beads interact with the secondary antibody
alone and may be discarded.
6 Recycle the remaining colorless library with the following steps: Incubate the
library with 6 M guamdine-HCl, pH 1 .O, 20-30 min, wash 5 times with double-
distilled water, mix the library with DMF for 1 h, wash 5 times wrth double-distilled
water, followed by PBS-Tween
7 Add the unlabeled ligate to the bead-library and incubate l-24 h at room
temperature
8 Wash the bead-library thoroughly with PBS-Tween
9. Add the alkaline phosphatase-linked antrligate antibody to the bead-library and
incubate l-2 h (see Note 3) Then wash the bead-library thoroughly wtth PBS-
Tween and finally once with TBS.
10 Add BCIP substrate to the library as described m Subheading 3.1., item 4. After
30 min to 2 h, stop the colorimetrtc reaction by adding several drops of 1 M HCl
to each Petri dish. Since the library has been prescreened with the secondary
antibody alone, the posrttve beads Identified at this time should be a result of
bmdmg to the ligate and not to the secondary antibody.
11. Isolate those individual posrtive beads for microsequencmg as descrrbed m
Subheading 3.1., items 5,6.
4. Notes
1 For the two-step screening process, instead of using the lrgate/antr-ligate-enzyme
system, one may use a biotmylated-ligate/streptavrdm-enzyme system
2. Most of the methods employed m Western blot or ELISA for lowering the back-
ground can be applied to the screening of the bead-library We routmely add high
salt (2X PBS), 0 1% Tween-20, and 0 1% gelatin to the bmdmg buffer Bovme
serum albumin instead of gelatin has also been used successfully.
3 In order to mmimtze the background and false posmves, the concentration of
ligate, ligate-enzyme conjugate, or antibody-enzyme conjugate used m the
screening should be as dilute as possible Sometrmes lt is advantageous to use a
