One-pot high-throughput synthesis of β-turn cyclic peptidomimetics-via "volatilizable" supports

Article abstract of DOI:10.1021/jo802583t

A promising method for the high-throughput synthesis of linear C-hydroxyalkylamido peptidomimetics and β-turn cyclic peptidomimetics via "volatilizable" aminoalkyl functionalized silica gels is presented. Boc amino acids and carboxylic acids were coupled

Full text of DOI:10.1021/jo802583t

A were reported to mimic the turn regions of proteins.4
Intramolecular SNAr reaction is often used for the construction
of these macrocycles via formation of an endo alkyl-aryl ether
bond. Solid- and solution-phase synthetic methods have been
investigated to synthesize similar structures. By using the solid-
phase synthesis approaches, an amino acid with protected side-
chain hydroxyl (X)O) or amino (X)N) group was first
anchored on resin. The side-chain amino or hydroxyl group was
used as the nucleophile to conduct the on resin SNAr cycliza-
tion.5 The solution-phase strategies involved intramolecular
SNAr cyclization usually promoted by TBAF or CsF in an
anhydrous organic solvent.6 However, a variable amount of
intermolecular SNAr byproduct was reported by using these
catalysts. To develop a new methodology to these promising
structures, herein we report an efficient “one-pot” synthetic
method, which incorporate the solid-phase synthesis of linear
peptides on “volatilizable” supports7 and cyclization in aqueous
solution.
One-Pot High-Throughput Synthesis of ꢀ-Turn Cyclic
Peptidomimetics via “Volatilizable” Supports
Yangmei Li,‡ Yongping Yu,*,† Marc Giulianotti,‡ and
Richard A. Houghten*,†,‡
College of Pharmacetical Science, Zhejiang UniVersity, Zijin
Campus, Hangzhou 310058, China, and Torrey Pines
Institute for Molecular Studies, 11350 SW Village Parkway,
Port Saint Lucie, Florida 34987
houghten@tpims.org; yyu@zju.edu.cn
ReceiVed NoVember 21, 2008
A promising method for the high-throughput synthesis of
linear C-hydroxyalkylamido peptidomimetics and ꢀ-turn
cyclic peptidomimetics via “volatilizable” aminoalkyl func-
tionalized silica gels is presented. Boc amino acids and
carboxylic acids were coupled on functionalized aminoalkyl
silica gels using a standard DIC/HOBt coupling protocol.
After peptide synthesis, the resin bound peptide was cleaved
using a two-step process to obtain the linear C-hydroxy-
alkylamido peptidomimetics. ꢀ-Turn cyclic peptidomimetics
were generated by intramolecular SNAr cyclization in an
aqueous solution. Both the linear and the cyclic peptidomi-
metics were obtained with good to excellent yields and
purities through a “one-pot” reaction.
Aminopropyl and aminobutyl functionalized silica gels were
synthesized and utilized as the “volatilizable” supports.8 Parallel
solid-phase synthesis was carried out using the “teabag”
approach.9 Boc-amino acids were coupled on the aminoalkyl
silica gel 1 by standard Boc-peptide synthesis chemistry to yield
the resin-bound peptides 2, as shown in Scheme 1. Resin-bound
peptides 2 were N-acylated with carboxyl acids to yield resin-
bound N-acylated peptides 3. The resin bound N-acylated
(3) (a) Maliartchouk, S.; Feng, Y.; Ivanisevic, L.; Debeir, T.; Cuello, A. C.;
Burgess, K.; Saragovi, H. U. Mol. Pharmacol. 2000, 57, 385–391. (b) Bondebjerg,
J.; Xiang, Z.; Bauzo, R.M.; Haskell-Luevano, C.; Meldal, M. J. Am. Chem. Soc.
2002, 124, 11046–11055. (c) Fink, B. E.; Kym, P. R.; Katzzenellenbogen, J. A.
J. Am. Chem. Soc. 1998, 120, 4334–4344. (d) Kitagawa, O.; Vander Velde, D.;
Dutta, D.; Morton, M.; Takusagawa, F.; Aube, J. J. Am. Chem. Soc. 1995, 117,
5169–5178. (e) Eguchi, M.; Lee, M. S.; Nakanishi, H.; Stasiak, M.; Lovell, S.;
Kahn, M. J. Am. Chem. Soc. 1999, 121, 12204–12205. (f) Boger, D. L.; Zhou,
J.; Borzilleri, R. M.; Nukui, S.; Castle, S. L. J. Org. Chem. 1997, 62, 2054–
2069. (g) Beugelmans, R.; Singh, P. G.; Bois-Choussy, M.; Chastanet, J.; Zhu,
J. J. Org. Chem. 1994, 59, 5535–5542. (h) Virgilio, A. A.; Schurer, S. C.; Ellman,
J. A. Tetrahedron Lett. 1996, 37, 6961–6964. (i) Zaccaro, M. C.; Lee, H. B.;
Pattarawarapan, M.; Xia, Z.; Caron, A.; L’Heureux, P.-J.; Bengio, Y.; Burgess,
K.; Saragovi, H. U. Chem. Biol. 2005, 120, 1015–1028. (j) Souers, A. J; Virgilio,
A. A.; Rosenquist, S.; Fenuik, W.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121,
1817–1825.
(4) Feng, Y.; Wang, Z.; Jin, S.; Burgess, K. J. Am. Chem. Soc. 1998, 120,
10768–10769.
(5) (a) Feng, Y.; Burgess, K. Chem.sEur. J. 1999, 5, 3261–3272. (b) Kohn,
W. D.; Zhang, L.; Weigel, J. A. Org. Lett. 2001, 3, 971–974.
(6) (a) Boger, D. L.; Borzilleri, R. M.; Nukui, S.; Beresis, R. T. J. Org.
Chem. 1997, 62, 4721–4736. (b) Beugelmans, R.; Bois-Choussy, M. B.; Vergne,
C.; Bouillon, J. P.; Zhu, J. Chem. Commun. 1996, 1029–1030. (c) Laib, T.; Zhu,
J. Tetrahedron Lett. 1998, 29, 283–286.
(7) Houghten, R. A.; Yu, Y. J. Am. Chem. Soc. 2005, 127, 8582–8583.
(8) Li, Y.; Yu, Y.; Giulianotti, M.; Houghten, R. A. Tetrahedron Lett. 2008,
49, 3632–3633.
Protein-protein interactions are known to play a critical role
in many biological processes such as immune responses, protein
enzymeinhibitors,signaltransduction,andapoptosis.Protein-protein
interactions are becoming important targets in medicinal chem-
istry.1 The distinct secondary structural elements such as helices,
sheets, and turns contribute to protein-protein complex inter-
faces. Among these secondary structural motifs, ꢀ-turns have
received the most attention for mediating a myriad of
protein-protein interactions.2 There is an increasing interest in
designing and synthesizing peptidomimetics based on the ꢀ-turn
structure.3 Recently, macrocyclic peptidomimetics of structures
† Zhejiang University
‡ Torrey Pines Institute for Molecular Studies
(1) (a) Betzi, S.; Restouin, A.; Opi, S.; Arold, S. T.; Parrot, I.; Guerlesquin,
F.; Morelli, X.; Collette, Y. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 19256–
19261. (b) Wells, J. A.; McClendon, C. L. Nature. 2007, 450, 1001–1009. (c)
Rush, T. S.; Grant, J. A.; Mosyak, L.; Nicholls, A. J. Med. Chem. 2005, 48,
1489–1495.
(2) Peczuh, M. W.; Hamilton, A. D. Chem. ReV. 2000, 100, 2479–2494.
(9) Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5131–5135.
10.1021/jo802583t CCC: $40.75
Published on Web 01/27/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 2183–2185 2183

SCHEME 1. Synthesis of C-Hydroxyalkylamido and N-Acyl
Peptidomimetics on the “Volatilizable” Aminoalkyl Silica
Gel
SCHEME 2. Synthesis of Cyclic Peptidomimetics
TABLE 1. C-Hydroxyalkylamido and N-Acyl Peptidomimetics
TABLE 2. Cyclic Peptidomimetics
purityc
(%)
yieldd
(%)
puritya yieldb
entry
R1
R2
CH2Ph
n
(%)
(%)
entry
product
4aa
4ba
4ca
4db
4eb
C6H5(CH2)3CO-Leu-NH(CH2)3OH
BnCO-Phe-Gly-Ala-NH(CH2)3OHe
BnCO-Asn-Leu-Phe-Ala-NH(CH2)3OH
BnCO-Phe-Asp-Ala-NH(CH2)4OH
CH3CO-Phe-Phe-Ile-Arg-NH(CH2)4OH
90
85
81
74
84
92
90
77
78
70
6a
6b
6c
6d
6e
6f
CH3
CH3
H
1
1
1
1
1
1
2
2
90
81
88
85
76
82
80
88
92
77
89
88
80
85
78
70
H
CH2CH(CH3)2
Cyo(CHCH2CH2CH2N) CH3
CH2CH2CONH2
CH(CH3)OBzl
CH2Ph
H
CH3
CH3
CH3
a Cleave with 35% aqueous HF at RT for 0.5 h. b Cleave with
anhydrous HF at 0 °C for 2 h. c HPLC purity (in %) was determined by
the peak area at 214 nm. d Yields (in %) are based on the weight of
crude material and are relative to the substitution of the resin (see
Supporting Information). e Bn ) benzyl.
6g
6h
CH(CH3)2
a HPLC purity (in %) was determined by the peak area at 214 nm.
b Yields (in %) are based on the weight of crude material and are
relative to the weight and purity of linear precursors.
peptides 3 were treated with aqueous HF or anhydrous HF (the
side-chain protecting groups of amino acids were removed by
use of anhydrous HF) to decompose the silica gel. Following
the removal of the solvent by lyophilization, H2O2 and DMF
were added to further oxidatively cleave the Si-C bond10
producing the C-hydroxyalkylamido peptidomimetics 4 with
high yields and purities (Table 1).
good-to-excellent purities and yields as shown in Table 2. The
byproducts from the synthesis were mainly generated from
incomplete coupling. The incomplete coupling is most likely
due narrow pores caused by the rigid and nonswelling nature
of silica.
The 20 natural L-amino acids were examined in the synthesis
of the above linear and cyclic peptidomimetics. Protecting
groups such as OBzl, Tos, 4-MeOBzl, and 2-Cl-Z are stable to
oxidative cleavage when aqueous HF is used in the first cleavage
step. Cysteine and methionine were found to be oxidized during
the oxidative cleavage. Byproducts were also generated when
histidine was used in the sequence. Other than noted above,
amino acids were unchanged in the linear peptidomimetics
synthesis.
ꢀ-Turn cyclic peptidomimetics were designed and synthesized
based on the intramolecular SNAr macrocyclization depicted in
Scheme 2. 2-Fluoro-5-nitrobenzoic acid was used as the
N-acylating reagent. After oxidative cleavage, the cyclic precur-
sor 5 was obtained. Following removal of DMF and H2O2 in
vacuum, the precursors 5 were dissolved in 10% aqueous
acetonitrile at a concentration of 0.5 mg/mL. Mixed bed ion-
exchange resins were then added to promote the SNAr macro-
cyclization. To our knowledge this is the first time reporting
the utilizing of mix bed ion-exchange resins to catalyze SNAr
cyclization in aqueous solution. The macrocyclization proceeded
to completion within 12 h at room temperature to yield 14- or
15-membered ꢀ-turn cyclic peptidomimetics depending on the
aminoalkyl functionalized silica gel used. Mixed bed ion-
exchange resins consist of equivalent amounts of strong anion
(OH-) and strong cation (H+) exchange resin. The resins serve
both as a support-bound base to promote the SNAr macrocy-
clization and as a scavenging/desalting agent. No intermolecular
SNAr byproducts were found in solution during the cyclization.
After filtration of the ion-exchange resins and removal of the
solvent, the desired cyclic products were left in the vessel in
Unfortunately, by using this synthetic scheme, no cyclic
petidomimetics containing a carboxyl side chain could be
generated. For example, while peptidomimetics made with
glutamic acid and aspartic acid were stable in oxidative cleavage,
their ester-protected side chains were catalytically hydrolyzed
by the ion-exchange resin. These carboxyl groups then attached
to the ion-exchange resin, resulting in the complete loss of any
cyclic peptidomimetics containing these carboxyl groups.
Larger rings incorporating 3- and 4-amino acid residues
(cyclic peptidomimetics 7 and 8) were also synthesized by this
method. The cyclization took a longer time to complete. The
17/18- and 20/21-membered cyclic peptidomimetics were
obtained with satisfactory purity and yield (data shown in
Supporting Information).
(10) (a) Yang, A.; Li, T. Anal. Chem. 1998, 70, 2827–2830. (b) Stork, G.;
Chan, T. Y.; Breault, G. A. J. Am. Chem. Soc. 1992, 14, 7578–7579. (c) Tamao,
K.; Alita, H.; Iwahara, T.; Kanatanl, R.; Yoshida, J.; Kumada, M. Tetrahedron
1983, 39, 983–990.
Circular dichroism spectra (CD) of compounds 6a were also
recorded in 20% aqueous methanol. Compounds 6a gave an
2184 J. Org. Chem. Vol. 74, No. 5, 2009

MHz, DMSO-d6) δ 21.6, 23.0, 24.3, 27.2, 32.3, 34.6, 34.7, 35.7,
41.0, 50.9, 58.4, 125.7, 128.2, 128.3, 141.8, 171.7, 172.1. MS calcd
for C19H30N2O3, 334.2; (ESI) m/z found, 335.1 (M + H)+.
General Procedure for Cyclic Peptidomimetics (6, 7, and
8). The method for preparation of the linear peptide 5 was the same
as described in the above text. After removal of the solvent by
lyophilization, the product was dissolved in 10% acetonitrile in
water at a concentration of 0.5 mg/mL. Mixed bed ion-exchange
resins were then added to promote the cyclization. The mixture
was shaken overnight to yield the cyclic peptidomimetics 6, 7, and
8.
ellipticity minimum at approximately 203 nm, characteristic of
a type-I ꢀ-turn.
In summary, we present here a novel method to produce
ꢀ-turn cyclic peptidomimetics and C-hydroxyalkyamido linear
peptide analogues on “volatilizable” aminoalkyl functionalized
silica gels. The desired C-hydroxyalkylamido-terminated linear
peptide analogues were obtained by tandem cleavage with
aqueous HF or anhydrous HF and hydrogen peroxide in DMF.
With the use of 2-fluoro-5-nitrobenzoic acid as the N-capping
reagent, ꢀ-turn cyclic peptidomimetics were obtained from the
linear precursor in 10% aqueous acetonitrile through SNAr
cyclization. Mixed bed ion-exchange resins served both as a
base and as a scavenger in the cyclization. 14- and 15-Membered
cyclic peptidomimetics were obtained with high purity through
the “one-pot” reaction. The methodology described is promising
for high-throughput synthesis of C-hydroxyalkylamido linear
peptides and ꢀ-turn cyclic peptidometics.
Cyclic Peptidomimetics (6a). 1H NMR (500 MHz, DMSO-d6)
δ 0.99 (d, 3H, J ) 7.5 Hz), 1.91-1.93 (m, 2H), 2.71 (dd, 1H, J )
13.8, 11.5 Hz), 3.21-3.25 (m, 1H), 3.30 (dd, 1H, J ) 13.9, 3.9
Hz), 3.48-3.51 (m, 1H), 3.87-3.90 (m, 1H), 4.19-4.21 (m, 1H),
4.39-4.41 (m, 1H), 4.48-4.52 (m, 1H), 7.13-7.18 (m, 3H),
7.20-7.23 (m, 2H), 7.30 (dt, 2H, J ) 9.5, 5.5 Hz), 8.05 (d, 1H, J
) 9.5 Hz), 8.27 (d, 1H, J ) 2.8 Hz), 8.36 (dd, 1H, J ) 9, 3 Hz),
8.89 (d, 1H, J ) 4.5 Hz); 13C NMR (125 MHz, DMSO-d6) δ 16.0,
28.2, 36.7, 38.5, 52.1, 52.8, 70.4, 112.8, 123.7, 126.0, 126.1, 127.2,
127.8, 129.2, 138.4, 139.7, 161.4, 165.6, 170.2, 172.2. MS calcd
for C15H18N4O6, 440.2; (ESI) m/z found, 441.1 (M + H)+.
Cyclic Peptidomimetics (7). 1H NMR (500 MHz, DMSO-d6) δ
0.67-0.69 (m, 1H), 0.97-1.03 (m, 1H), 1.32 (d, 3H, J ) 7 Hz),
1.32-1.35 (m, 1H), 2.07-2.11 (m, 1H), 2.71-2.73 (m, 1H),
2.89-2.94 (m, 1H), 3.13 (dd, 1H, J ) 14, 3.5 Hz), 3.27 (dd, 2H,
J ) 14, 5.5 Hz), 4.00-4.08 (m, 3H), 4.24-4.26 (m, 1H), 4.91 (m,
1H), 6.99-7.00 (m, 1H), 7.03-7.07 (m, 4H), 7.10-7.13 (m, 1H),
7.34 (d, 1H, J ) 10 Hz), 8.37 (dd, 1H, J ) 9, 3 Hz), 8.71 (d, 1H,
J ) 7 Hz), 8.75 (dd, 1H, J ) 8, 4.5 Hz), 8.87 (d, 1H, J ) 3 Hz),
9.10 (d, 1H, J ) 2.6 Hz); 13C NMR (125 MHz, DMSO-d6) 16.0,
23.7, 26.5, 37.7, 40.6, 42.4, 50.7, 52.8, 71.1, 114.0, 120.6, 126.3,
126.8, 127.7, 128.5, 130.1, 135.4, 140.8, 160.6, 161.8, 169.3, 171.2,
172.5. MS calcd for C25H29N5O7, 511.2; (ESI) m/z found, 512.3
(M + H)+.
Experimental Section
General Procedure for Synthesis of Linear C-Hydroxy-
alkylamido Peptide (4). A polypropylene mesh packet was sealed
with 100 mg of functionalized silica gel resin 1 (loading 0.2 mmol/
g). After washing with DCM, 5% DIEA in DCM and DCM, the
resin was coupled with N-Boc-protected amino acids using DIC
and HOBt (6 equiv each, 0.1 M) in DMF for 2 h to generate the
desired resin-bound peptides. Boc deprotection was performed using
55% TFA in DCM for 30 min, followed by washing with DCM (3
times). Following neutralization with 5% DIEA in DCM, the resin
was sequentially coupled with Boc-protected amino acids using the
DIC/ HOBt approach for 2 h to yield the resin-bound peptides.
After the terminal Boc group was removed using 55% TFA in DCM
for 30 min, the resin-bound peptide 2 was acylated with a carboxyl
acid using DIC and HOBt (10 equiv each, 0.1 M) in DMF overnight
to yield resin-bound 3. The cleavage of compound 4 was performed
by first adding either 1 mL of 35% aqueous HF at room temperature
for 30 min or with anhydrous HF at 0 °C for 2 h to decompose the
silica, forming tetrafluorosilane and water. Note that when using
anhydrous HF the side chain protecting groups are removed. After
removing the solvent and tetrafluorosilane by lyophilization, DMF
(1 mL) and 30% hydrogen peroxide (1 mL) were added to
oxidatively cleave the Si-C bond at room temperature for 40 h
providing the C-hydroxyalkylamido peptide 4.
Acknowledgment. This work was supported by the National
Science Foundation (RAH CHE 0455072), 1P41GM079590,
1P41GM081261, the State of Florida, Executive Office of the
Governor’s Office of Tourism, Trade, and Economic Develop-
ment, and U54HG03916-MLSCN. Y.Y. would like to thank the
National Science Foundation of China (NSFC20572098) and
Zhejiang Science and Technology Programme (2005c3401).
1
C6H5(CH2)3CO-Leu-NH(CH2)3OH (4a). H NMR (500 MHz,
Supporting Information Available: Experimental detail,
1
compound characterization, and copies of H NMR, 13C NMR
DMSO-d6) δ 0.85 (dd, 6H, J ) 19.8, 6.6 Hz), 1.31-1.45 (m, 2H),
1.50-1.58 (m, 3H), 1.74-1.80 (m, 2H), 2.11-2.16 (m, 2H), 2.53
(t, 2H, J ) 7.5 Hz), 3.04-3.13 (m, 2H), 3.38 (t, 2H, J ) 6.3 Hz),
4.23-4.27 (m, 1H), 7.16-7.18 (m, 3H), 7.26-7.29 (m, 2H), 7.85
(t, 1H, J ) 5.6 Hz), 7.89 (d, 1H, J ) 8.3 Hz); 13C NMR (125
spectra, and LC-MS for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO802583T
J. Org. Chem. Vol. 74, No. 5, 2009 2185