Discovery of selective metal-binding peptoids using 19F encoded combinatorial libraries

Article abstract of DOI:10.1016/S0960-894X(00)00416-9

A method for encoding solid-phase split/mix combinatorial libraries using the chemical shift of synthetic fluoroarenes ('F-codes') has been developed. They have wide chemical shift dispersion and are detectable at the sub-μmol level. ¹⁹F NMR is

Full text of DOI:10.1016/S0960-894X(00)00416-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2115±2118
19
Discovery of Selective Metal-Binding Peptoids Using F Encoded
Combinatorial Libraries
Michael C. Pirrung* and Kaapjoo Park
Department of Chemistry, Levine Science Research Center, Box 90317, Duke University, Durham, NC 27708-0317, USA
Received 19 May 2000; accepted 14 July 2000
AbstractÐA method for encoding solid-phase split/mix combinatorial libraries using the chemical shift of synthetic ¯uoroarenes
(`F-codes') has been developed. They have wide chemical shift dispersion and are detectable at the sub-mmol level. 19F NMR is used
for decoding. Nine ¯uoroarenes bearing linkers for attachment to solid-phase synthesis supports through a photocleavable group
were prepared. A library of 90 N-alkylglycines bearing substituted succinamides was prepared on solid phase from nine amines, in
which the amine is encoded by the ¯uorinated tag, and 10 anhydrides. Metal binding studies followed by decoding identi®ed
unique, speci®c binders of copper(II) and iron(III) with mM KDs. # 2000 Elsevier Science Ltd. All rights reserved.
The power of split-pool synthesis in the preparation of
1-bead/1-compound combinatorial libraries has been
made manifest through a wide range of studies.1 In
specialized cases such as peptides, beads identi®ed as
bearing a library member with a desired property can be
subjected to direct structural analysis.2 More often, a
code molecule is added simultaneously with molecular
building blocks during solid-phase synthesis, and a binary
encoding methodology3 is used to identify the structures
of compounds derived from single beads through an
easily read molecular code. Encoding molecules include
secondary amines,4 isotopes,5 and a,o-diols bearing
electron-de®cient aromatics,6 and decoding methodolo-
gies include ¯uorescence-detected HPLC, mass spectro-
metry, and gas chromatography with electron-capture
detection. These methods require specialized techniques
or instrumentation not broadly available.
background of ¯uorine in the chemical laboratory
environment, making 19F NMR a high-sensitivity ana-
lytical technique. The newly-synthesized molecular tags
are aryl ¯uorides whose chemical shifts are exquisitely
sensitive to substitution. They have an ortho link to the
support that eliminates one large H±F coupling. The
other ortho position is also substituted. The para posi-
tion is available for substitutions that also a€ect the
¯uorine chemical shift.
Our goal was to develop an encoding method using the
most broadly available structural analysis tool, NMR.
Previous discussion of this concept has been sparse,7 but
a related study using high-resolution MAS NMR tech-
niques has recently appeared.8 We chose 19F NMR
because of ¯uorine's large chemical shift dispersion
(ꢀ1000 ppm overall, ꢀ200 ppm in organo¯uorine com-
pounds), so that it should be possible to analyze many
tags that are unambiguously separated by a signi®cant
chemical shift di€erence. There is also essentially no
A series of nine ¯uorinated aromatics (2) bearing a lin-
ker for attachment during solid-phase synthesis was
prepared by ¯uorine-directed metalation (eqs 1 and 2).9
The 19F NMR chemical shift dispersion of this set of
tags (determined at the stage of the alcohols, the state in
which they are read) is >62 d, and the minimum che-
mical shift separation between tags is 4.7 d (Fig. 1). This
synthetic route makes readily accessible up to 16 ¯uori-
nated tags with (calculated10) chemical shift dispersion
of 80 d and minimum separation of 4 d. Tags can be
*Corresponding author. Fax: +1-919-660-1591; e-mail: pirrung@
chem.duke.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00416-9

2116
M. C. Pirrung, K. Park / Bioorg. Med. Chem. Lett. 10 (2000) 2115±2118
Figure 1. 19F NMR spectrum of nine F-codes released by irradiation from encoded synthesis beads.
detected at 376 MHz with 10:1 S/N ratio in a capillary
microcell at a concentration of 5 mM in 13 mL CDCl3
(65 nmol) in a 40 min acquisition. Tags were adapted for
peptoid synthesis11 and photochemical release by alky-
lation with 3, obtained from the Holmes photolinker12
through a Curtius reaction.13
bead), which were bromoacetylated.15 Nine groups of
beads were formed and individual 19F-encoded tags
were added to each as amines (5) (0.7 M in DMSO, rt, 2
days), obtained by TFA treatment of 4. After bromo-
acetylation, the amine diversity components (6) were
added (1 M in DMSO, rt, 12 h) and all groups of beads
(7) were pooled. These beads were split into 10 pools
and an anhydride (8) was added to each pool (DMF, rt,
12 h). The protecting groups were removed from amino
alcohol building blocks by treatment with n-Bu4NF
(1 M, THF, rt, 10 h). Pools (9) were kept separate for
screening.
Bead pools were tested for metal binding by treatment
with Cu(OTf)2 in acetonitrile (rt, 48 h) at concentrations
increasing from 0.1 mM. One pool (No. 6) showed
many sky-blue-colored beads at 4 mM, and no other
pools were colored at concentrations up to 100 mM.16
Ten blue beads were selected from this pool and sub-
jected to irradiation in THF at 350 nm for 12 h. The
released tags were isolated by ®ltration/evaporation and
A peptoid library was designed using a set of nine R1
amines (Chart 1) and ten R2 anhydrides (Chart 2).
Synthesis was performed on 3 g (2.85 mmol) 500 mM
polystyrene benzylamine beads14 (0.95 mmol/g, 83 nmol/
Chart 1.
Chart 2.

M. C. Pirrung, K. Park / Bioorg. Med. Chem. Lett. 10 (2000) 2115±2118
2117
submitted to 19F NMR analysis in the presence of an
internal standard. A single peak was observed (Fig. 2)
corresponding to a single amine. The structure of the
optimum copper-binding library member was thereby
deduced. Determination of its copper-binding proper-
ties in solution required signi®cantly larger quantities of
the compound than were prepared on solid phase. In
order to simplify the synthesis compared to the initial
mono-N-alkylamide peptoid, a dimethylamide peptoid
with the same binding groups was prepared by conven-
tional synthesis.
Screening of this same library for binding to a second
metal ion, iron (as Fe(2-ethylhexanoate)3 in THF), led to
staining of only one pool (No. 10 Ð 3-hydroxyphthalic).
A gradation of intensity permitted the binding beads to be
strati®ed into four groups. Decoding of the most inten-
sely stained group or a frequency-weighted analysis of
the appearance of individual building blocks led to the
same optimum amine. Preparation by conventional
synthesis of the 3-pyridylmethylamine/3-hydroxyphthal-
amide peptoid 11 led to a triethylammonium salt whose
iron binding was evaluated (485 nm). It shows a 31 mM
KD for iron 2-ethylhexanoate.
This study establishes a novel method for decoding of
combinatorial libraries using NMR instrumentation
present in essentially all chemical laboratories and
¯uoroaromatic tags that are resistant to many chemical
reaction conditions. In addition, because this library
incorporates a photolinker to the tags, they are readily
released for NMR analysis in solution, rather than
requiring a solid-phase NMR method. In this applica-
tion, the ¯uoroaromatics perform as analogue codes in
that they directly represent building blocks in the
library. The frequency of appearance of a tag is there-
fore a direct measure of the performance of a building
Compound 10 was obtained as its triethylammonium
salt, which shows a 44 mM KD for copper tri¯ate as
measured by spectrophotometric titration17 at 390 nm.
While the anity of this peptoid for copper might be
slightly modi®ed by the change from the mono-N-alkyl-
amide to a dimethylamide peptoid, the selectivity should
be similar.
Figure 2. 19F NMR spectrum of one F-code released by irradiation from copper-binding encoded synthesis beads.

2118
M. C. Pirrung, K. Park / Bioorg. Med. Chem. Lett. 10 (2000) 2115±2118
block in the assay. This concept is distinguished from
earlier binary (digital) encoding methods, and should be
useful for kinetic studies and the like. The structural
variation leading to selective binding of iron or copper
are non-intuitive (e.g., 2- vs 3-pyridylmethylamine) and
could only be discovered empirically. This method has
been used here in organic solvents. Direct screening for
biological activity would require aqueous solvents,
which should be compatible with this method using
hydrophilic, PEG-grafted support beads.
Navre, M.; DeLuca-Flaherty, C. R. Antimicrob. Agents Che-
mother. 1998, 42, 1447.
5. Geysen, H. M.; Wagner, C. D.; Bodnar, W. M.; Mark-
worth, C. J.; Parke, G. J.; Schoenen, F. J.; Wagner, D. S.;
Kinder, D. S. Chem. Biol. 1996, 3, 679.
6. Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.;
Reader, J. C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still,
W. C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10922. Borch-
ardt, A.; Still, W. C. J. Am. Chem. Soc. 1994, 116, 373.
7. Keifer, P. A. Drug Disc. Today 1997, 2, 468. Keifer, P. A.
Drugs Fut. 1998, 23, 301.
8. Hochlowski, J. E.; Whittern, D. N.; Sowin, T. J. J. Comb.
Chem. 1999, 1, 291.
9. Takagishi, S.; Katsoulos, G.; Schlosser, M. Synlett 1992, 360.
10. Emsley, J. W.; Feeney, J.; Sutcli€e, L. H. High Resolution
NMR Spectroscopy; Pergamon: Oxford, 1966; Vol. 2, p 898.
Fifolt, M. J.; Sojka, S. A.; Wolfe, R. A.; Hojnicki, D. S.;
Bieron, J. F.; Dinan, F. J. J. Org. Chem. 1989, 54, 3019.
11. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos,
W. H. J. Am. Chem. Soc. 1992, 114, 10646. Zuckermann, R.
N.; Go€, D. A. Polym. Prepr. 1994, 35, 975. Simon, R. J.;
Martin, E. J.; Miller, S. M.; Zuckermann, R. N.; Blaney, J.
M.; Moos, W. H. Tech. Protein Chem. V; Crabb, J. W., Ed.;
Academic: San Diego, 1994; p 533. Figliozzi, G. M.; Gold-
smith, R.; Ng, S. C.; Banville, S. C.; Zuckermann, R. N.
Methods Enzymol. 1996, 267, 437.
Acknowledgements
Financial support provided by NIH GM-AI42151. We
thank Nathan Tumey for initial synthetic investigations.
The assistance of L. LaBean in administrative support
of this work is greatly appreciated.
References and Notes
1. Furka, A.; Bennett, W. D. Comb. Chem. High Throughput
Screen 1999, 2, 105.
2. Wu, J.; Ma, Q. N.; Lam, K. S. Biochemistry 1994, 33,
14825.
3. Youngquist, R. S.; Fuentes, G. R.; Lacey, M. P.; Keough,
T. Rapid Commun. Mass Spectrom. 1994, 8, 77. Keough, T.;
Youngquist, R. S.; Lacey, M. P. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 7131.
4. Baldwin, J. J. Mol. Divers. 1996, 2, 81. Chabala, J. C. Curr.
Opin. Biotechnol. 1995, 6, 632. Ni, Z. J.; Maclean, D.; Holmes,
C. P.; Murphy, M. M.; Ruhland, B.; Jacobs, J. W.; Gordon,
E. M.; Gallop, M. A. J. Med. Chem. 1996, 39, 1601. Maclean,
D.; Schullek, J. R.; Murphy, M. M.; Ni, Z. J.; Gordon, E. M.;
Gallop, M. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2805.
Silen, J. L.; Lu, A. T.; Solas, D. W.; Gore, M. A.; Maclean,
D.; Shah, N. H.; Con, J. M.; Bhinderwala, N. S.; Wang, Y.;
Tsutsui, K. T.; Look, G. C.; Campbell, D. A.; Hale, R. L.;
12. Holmes, C. P.; Jones, D. G. J. Org. Chem. 1995, 60, 2318.
13. Ninomiya, K.; Shiori, T.; Yamada, S. Tetrahedron 1974,
30, 2151.
14. Obtained from Rapp Polymere, item Polystyrene AM-
NH2, order No. H500560.02.
15. `Rehearsal' syntheses using beads derivatized with Fmoc-
Rink amide linker, bromoacetic acid, (imidazolyl)propyl-
amine, and succinic anhydride delivered the target peptoid in
quantitative yield and >95% purity after TFA cleavage.
16. Control beads not bearing peptoids were also unstained,
showing that the tags and linker have no intrinsic metal bind-
ing ability.
17. He, Q.-Y.; Mason, A. B.; Woodworth, R. C. Biochem. J.
1996, 318, 145. Yoon, S. S.; Still, W. C. Tetrahedron 1995, 51,
567.