"Volatilizable" supports for high-throughput organic synthesis

Article abstract of DOI:10.1021/ja0402396

The concepts and use of "volatilizable" solid supports are presented. Such supports, which are completely removed by volatilization following decomposition, improve the efficiency of the solid-phase synthesis of both individual and mixtures of low-molecul

Full text of DOI:10.1021/ja0402396

Published on Web 05/21/2005
“Volatilizable” Supports for High-Throughput Organic Synthesis
Richard A. Houghten*,† and Yongping Yu*,‡
Torrey Pines Institute for Molecular Studies and Mixture Sciences Inc., 3550 General Atomics Court,
San Diego, California 92121, College of Pharmaceutical Sciences, Zhejiang UniVersity, 353 Yan An Road,
Hangzhou, Zhejiang 310031, P.R. China
Received October 20, 2004; E-mail: houghten@tpims.org; yyu@zju.edu.cn
Scheme 1. Concept of Solid-Phase Organic Synthesis on
“Volatilizable” Support and Linker
Solid-phase synthetic approaches, pioneered by Bruce Merrifield
in 1963,1 in conjunction with combinatorial methods developed over
the past 20 years are highly efficient means for the production of
tens of thousands of individual compounds and/or mixture-based
libraries made up of millions of compounds.2,3 In conjunction with
high-throughput biological screening, combinatorial approaches are
now routinely used in the search for new pharmaceutical lead
compounds. All currently utilized solid-phase organic synthetic
supports require that the desired synthetic compound(s), whether
they are low-molecular weight acyclic or heterocyclic compounds,
peptidomimetics, peptides, or oligonucleotides, etc., must be cleaved
from the support and, as a final step, separated from the spent
support. This final separation step in the synthesis of very large
numbers of compounds, typically exhaustive extraction followed
by filtration or centrifugation, inherently results in reduced yields
and increased costs. We herein report one solution to this problem.
This is exemplified by the solid-phase synthesis on “volatilizable”
solid supports and linkers that can be completely removed by their
ultimate decomposition and complete “volatilization” during the
final cleavage step of the synthetic process. This approach eliminates
the final extraction step and yields solely the desired synthetic
product(s) in the reaction vessel (Scheme 1).
Scheme 2. Selective Synthesis of C- and N-Terminal Protected
Peptides on “Volatilizable” Support and Linker
While many possible fully “volatilizable” solid supports can be
envisioned, silica gel, one of the first solid supports used for the
synthesis of peptides,4 is used herein to illustrate these concepts.
Silica gel is a well characterized, widely available, and inexpensive
solid support that can be functionalized at excellent substitution
levels.5 In the example presented, silica gel was first functionalized
with p-chloromethylphenyltrimethoxysilane in refluxing toluene to
form the desired chloromethylbenzyl-functionalized silica gel 3 (see
Supporting Information). The initial Boc-amino acid was introduced
by the treatment of this functionalized support with the cesium salts
of Boc amino acids in DMF in the presence of KI at 55 °C overnight
to yield the support-bound Boc-amino acid 4 (loading: 0.7 mmol/
g). Selective C-terminal and N-terminal protected peptides were
then synthesized on this “volatilizable” support as shown in Scheme
2. Parallel solid-phase synthesis was carried out using the “teabag”
approach.6 Following removal of the N-terminal Boc group with
50% trifluoroacetic acid in dichloromethane, protected amino acids
4 were sequentially coupled using standard Boc-peptide synthesis
chemistry (Boc/TFA/DIC)7 to afford the resin-bound peptide 5. The
silica gel portion of the benzyl ester-linked peptide 5 was completely
decomposed by treatment with 10% hydrofluoric acid (pH 4.3) for
1 h at room temperature. This yielded the C-terminal benzyl ester-
protected peptide 6, tetrafluorosilane, and water. Following solvent
removal by either rotary evaporation or lyophilization (HF and
tetrafluorosilane are readily trapped and/or decomposed by in-line
traps containing solid CaO), the desired protected peptides were
obtained as the sole products remaining in the reaction vessel in
excellent yields and purities (Table 1, 6a-e). In a separate
experiment, support-bound 6 was N-acylated with phenyacetic acid.
The silica gel was volatilized in this instance by treatment with
anhydrous HF for 1.5 h at 0 °C. Following evaporation of the
anhydrous HF with a gaseous nitrogen stream, the N-terminal
modified peptide 7 was obtained in excellent yield and purity
following lyophilization (Table 1, entries 7a-c). The central feature
of this strategy is the ease in which functionalized silica gel is
decomposed through Si-O-Si and Si-Aryl bond cleavage into
volatile constituents (SiF4 and water) under the influence of aqueous
or anhydrous hydrogen fluoride. Thus, this flexible approach enables
one to rapidly produce either C- and N-terminal protected peptide
† Torrey Pines Institute and Mixture Science.
‡ Zhejiang University and Torrey Pines Institute.
9
8582
J. AM. CHEM. SOC. 2005, 127, 8582-8583
10.1021/ja0402396 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. C-Terminal and N-Terminal Protected Peptides and
Chiral Polyamines Synthesized on “Volatilizable” Supports
in turn, have been used as templates for the diversity oriented
synthesis (DOS) of low-molecular weight acyclic and heterocyclic
compounds.9 We examined the reduction of silica gel-bound
peptides to their corresponding chiral polyamines. The silica gel-
bound L-Tyr-L-Tyr-L-Phe-L-Pro-benzyl amide was reduced with
borane to yield the desire mono N-benzylated chiral pentaamine
11 in excellent yield and purity following volatilizable cleavage
with 10% aqueous hydrofluoric acid and lyophilization (Table 1,
entry 11).
In summary, we have presented here an innovative solid-phase
approach for the synthesis of low-molecular weight acyclic,
heterocyclic compounds, and peptides on functionalized silica gel
as an example illustrating the concept of “volatilizable” solid
supports and linkers. The solid support and linker were completely
removed by their decomposition and ultimate “volatilization” during
the final cleavage step in the synthetic process to yield solely the
desired synthetic product(s) in the final reaction vessel.
product (%)
yielda (%)
6a
6b
6c
6d
6e
7a
7b
7c
10a
10b
11
Ala-Phe-OBzl
Leu-Phe-OBzl
Leu-Ala-Phe-OBzl
Val-Ala-Phe-OBzl
Vla-Leu-Ala-Phe-OBzl
93
95
91
92
94
96
98
92
89
90
81
Phenylacetyl-Val-Ala-OH
Phenylacetyl-Phe-Val-Ala-OH
Phenylacetyl-Asn-Leu-Vla-Ala-OH
Phe-Gly-Arg(Pbf)-Ala-benzyl amide
Ala-Gly-Gly-Arg(Tos)-benzyl amide
reduced (Tyr-Tyr-Phe-Pro-benzyl amide)
a Percent yields are based on the weight of crude material and are relative
to the loading of the first amino acid on silica gel resin.
Scheme 3. “Volatilizable” Solid Support Synthesis of Amide
Protected Peptides and Chiral Polyamines
Acknowledgment. We thank Adel Nefzi and John Ostresh for
their many helpful comments and suggestions. This work was
funded in part by a grant from the National Science Foundation
(R.A.H., CHE 0455072) and the Multiple Sclerosis National
Research Institute (R.A.H., MSRI 04051).
Supporting Information Available: Experimental methods; LC-
MS of representative products. This material is available free of charge
via the Internet at http:/pubs.acs.org.
References
(1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
fragments by the selective treatment of the bound compounds with
either aqueous or anhydrous HF. The majority of commonly used
amino acid side chains, as well as C- and N-terminal protecting
groups, including O-benzyl, benzyloxycarbonyl, Fmoc, tosyl, etc.,
but not Boc, are stable to aqueous HF. This enables fully protected
peptides to be generated for use in the synthesis of larger peptides
and proteins by segment coupling and/or the preparation of libraries
of peptidomimetics.
The automated synthesis of individual compound arrays in a 96-
well format using silica gel as the volatilizable solid support was
also examined. Utilizing an automated centrifuge-based synthesizer,8
Fmoc amino acids were coupled to benzylamine-linked silica gel
support 8 using standard Fmoc peptide chemistry (Fmoc/piperidine/
DIC)7 to yield resin-bound peptide 9 Scheme 3). Following
volatilization with 10% aqueous hydrofluoric acid and lyophiliza-
tion, the desired C-terminal and side-chain-protected peptides were
obtained as the sole products in each well (Table 1, 10 a-b). The
96- or 384-well plates used can serve not only as the reaction vessels
for combinatorial array synthesis but also as “mother” plates for
later storage and/or distribution.
(2) (a) Jung, G., Ed. Combinatorial Chemistry: Synthesis, Analysis, Screening;
Wiley-VCH: Weinheim, 1999. (b) Nicolaou, K. C., Hanko, R., Hartwig,
W., Eds. Handbook of Combinatorial Chemistry; Wiley-VCH: Weinheim,
Germany, 2002 (c) Dolle, R. E. J. Comb. Chem. 2003, 5, 693.
(3) (a) Houghten, R. A.; Pinilla, C.; Appel, J. R.; Blondelle, S.; Dooley, C.
T.; Eichler, J.; Nefzi, A.; Ostresh, J. M. Mixture-Based Comb. Libr. 1999,
3743-3778. (b) Pinilla, C.; Appel, J. R.; Borras, E.; Houghten, R. A.
Nat. Med. 2003, 9, 118.
(4) (a) Parr, W.; Grohmann, K. Tetrahedron Lett. 1971, 2633. (b) Bayer, E.;
Jung, G.; Hala´sz, I.; Sebstian, I. Tetrahedron Lett. 1970, 4503. (c) Parr,
W.; Grohmann, K., Hagele, K. Liebigs Ann. Chem. 1974, 655-666.
(5) (a) Iler, R. K. The Chemistry of Silica; John Wiley & Sons: Ltd.: New
York, 1979. (b) Pesek, J. J., Leigh, I. E., Eds. Chemically Modified
Surfaces; Royal Society of Chemistry: Cambridge, 1994. (c) Pesek, J. J.,
Matyska, M. T., Abuelafiya, R. R., Eds. Chemically Modified Surfaces:
Recent DeVelopments; Royal Society of Chemistry: Cambridge, 1996.
(d) Brook, M. A., Ed. Silicon in Organic, Organometallic, and Polymer
Chemistry; John Wiley & Sons: Ltd.: New York, 2000; Chapter 10. (e)
Tao, T.; Maciel, G. E. J. Am. Chem. Soc. 2000, 122, 3118. (f) Shimada,
T.; Aoki, K.; Shinoda, Y.; Nakamura, T.; Tokunaga, N.; Inagaki, S.;
Hayashi, T. J. Am. Chem. Soc. 2003, 125, 4688.
(6) Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5131.
(7) Stewart, J. M., Young, J. D., Eds. Solid-Phase Peptide Synthesis; Pierce
Chemical Company: Rockford, Illinois, 1984.
(8) Lebl, M. Bioorg. Med. Chem. Lett. 1999, 91, 1305-1310.
(9) (a) Ostresh, J. M.; Husar, G. M.; Blondelle, S. E.; Dorner, B.; Weber, P.
A.; Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11138. (b).
Nefzi, A.; Ostresh, J. M.; Yu, Y.; Houghten, R. A. J. Org. Chem. 2004,
69, 3603.
Chiral polyamines can be obtained by the exhaustive reduction
of support-bound peptides (see ref 9 and citations therein). These,
JA0402396
9
J. AM. CHEM. SOC. VOL. 127, NO. 24, 2005 8583