Click chemistry as a macrocyclization tool in the solid-phase synthesis of small cyclic peptides

Article abstract of DOI:10.1021/ol702228u

Despite the vast number of techniques developed for the cyclization of small peptides, cyclization efficiency remains problematic in peptides that lack turn-promoting structures. Here we demonstrate the utility of click chemistry as a macrocyclization tool in the solid-phase synthesis of cyclic tetra-, penta-, hexa-, and heptapeptides. On-resin cyclization is completed at room temperature within 6 h, resulting in predominantly monomer with small amounts of cyclomultimer byproducts.

Full text of DOI:10.1021/ol702228u

ORGANIC
LETTERS
2007
Vol. 9, No. 24
5011-5014
Click Chemistry as a Macrocyclization
Tool in the Solid-Phase Synthesis of
Small Cyclic Peptides
Rushia A. Turner, Allen G. Oliver, and R. Scott Lokey*
Department of Chemistry and Biochemistry, UniVersity of California at Santa Cruz,
1156 High Street, Santa Cruz, California 95064
lokey@chemistry.ucsc.edu
Received September 11, 2007
ABSTRACT
Despite the vast number of techniques developed for the cyclization of small peptides, cyclization efficiency remains problematic in peptides
that lack turn-promoting structures. Here we demonstrate the utility of click chemistry as a macrocyclization tool in the solid-phase synthesis
of cyclic tetra-, penta-, hexa-, and heptapeptides. On-resin cyclization is completed at room temperature within 6 h, resulting in predominantly
monomer with small amounts of cyclomultimer byproducts.
Although peptides can be potent and selective as ligands in
vitro, poor bioavailability often places severe limits on their
utility as drugs. Cyclization is one approach to solving this
problem, wherein the removal of ionizable C- and N-termini
renders cyclic peptides more permeable and less prone to in
vivo enzymatic degradation than their linear counterparts.1-3
Cyclization can also constrain a bioactive peptide in its active
conformation, thus lowering the entropic cost of binding to
its biological target.4-7 These factors, along with the wealth
of biologically active cyclic peptides from natural sources,
underscore the importance of developing new synthetic
methods for accessing cyclic peptide scaffolds and their
analogs.8-12
Many reagents and techniques have been developed to
facilitate the combinatorial synthesis of cyclic peptide
libraries,13-15 for which the yield-limiting step is generally
the cyclization reaction. Cyclization of tetra-, penta-, and
hexapeptides in the all-L-configuration can be problematic,
particularly in the absence of â-turn promoting structures
such as glycine, proline, or a D-amino acid.16-18 Despite the
wide variety of reagents that promote efficient cyclization
(1) Tugyi, R.; Mezo, G.; Fellinger, E.; Andreu, D.; Hudecz, F. J. Pept.
Sci. 2005, 11, 642-649.
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J. Nat. Prod. 2005, 68, 623-625.
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C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Ghadiri, M. R.; Parr,
T. R., Jr. Antimicrob. Agents Chemother. 2005, 49, 3302-3310.
(13) Seneci, P. Solid-Phase and Combinatorial Technologies; John Wiley
and Sons, Inc.: New York, 2000.
(2) Besser, D.; Muller, B.; Kleinwachter, P.; Greiner, G.; Seyfarth, L.;
Steinmetzer, T.; Arad, O.; Reissmann, S. Pract. Appl. Appl. Chem. 2000,
342, 537-545.
(3) Shibata, K.; Suzawa, T.; Soga, S.; Mizukami, T.; Yamada, K.; Hanai,
N.; Yamasaki, M. Bioorg. Med. Chem. Lett. 2003, 13, 2583-2586.
(4) Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G. Biopolymers
1991, 31, 745-750.
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43, 9140-9150.
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J.; Wang, B.; Borchardt, R. T. J. Pept. Res. 1999, 53, 383-392.
(7) Gudmundsson, O. S.; Vander Velde, D. G.; Jois, S. D.; Bak, A.;
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67.
10.1021/ol702228u CCC: $37.00
© 2007 American Chemical Society
Published on Web 10/23/2007

in general,19-22 there remains a need for general and mild
techniques that are effective in the cyclization of these
difficult sequences.
We examined the utility of the CuI-promoted alkyne/azide
cycloaddition reaction in the macrocyclization of resin-bound
tri-, tetra-, penta-, hexa-, and heptapeptides. We were
interested in the cyclization efficiency of linear peptides that
might present challenging cyclizations under traditional
lactamization conditions. We chose a leucine-rich sequence
that lacked â-turn-promoting structures, such as proline or
glycine, and which contained only L-amino acids. We
envisioned the use of L-propargyl glycine as a C-terminal,
aspartic acid surrogate in a side-chain cycloaddition/macro-
cyclization with an N-terminal L-azido-leucine (Scheme 1).
The triazole moiety has been utilized in â-turn mimetic
peptides23 and as an isostere of the amide bond.24-28 Recently,
a Huisgen-type 1,3-dipolar cycloaddition reaction generat-
ing a 1,4-disubstituted-1,2,3-triazolesthe so-called “click
reaction”swas used as an amide bond surrogate and cy-
clization aid in the solution-phase synthesis of a cyclic
tetrapeptide analogue.29 Previous attempts to synthesize
cyclo[L-Pro-L-Val-L-Pro-L-Tyr] via lactamization proved
unfruitful, despite the presence of two turn-promoting struc-
tures in the sequence.30 The “ring contraction” mechanism
of the CuI-catalyzed alkyne/azide cycloaddition reaction,
together with the increased ring size of the triazole analog,
was proposed to promote cyclization more efficiently than
macrolactamization.31-34 The target tetrapeptide analogue
was achieved with 70% yield by refluxing the linear peptide
with a copper(I) catalyst at 110 °C for 16 h. This solution-
phase macrocyclization demonstrated the proficiency of the
1,3-dipolar cycloaddition reaction under conditions where
lactamization failed, but there have been no reports on the
applicability of the click reaction as a macrocyclization tool
in the solid-phase synthesis of small cyclic peptides, or
its potential for use in the synthesis of cyclic peptide
libraries.
Scheme 1. Click Macrocyclization of Propargyl Glycine
Azido Leucine (Path A) Is an Analogue of Aspartic Acid
Side-Chain Macrocyclization (Path B)
(16) Klose, J.; Ehrlich, A.; Bienert, M. Lett. Pept. Sci. 1998, 5, 129-
131.
(17) El Haddadi, M.; Cavelier, F.; Vives, E.; Azmani, A.; Verducci, J.;
Martinez, J. J. Pept. Sci. 2000, 6, 560-570.
(18) Besser, D.; Olender, R.; Rosenfeld, R.; Arad, O.; Reissmann, S. J.
Pept. Res. 2000, 56, 337-345.
(19) Ehrlich, A.; Heyne, H. U.; Winter, R.; Beyermann, M.; Haber, H.;
Carpino, L. A.; Bienert, M. J. Org. Chem. 1996, 61, 8831-8838.
(20) Spatola, A. F.; Darlak, K.; Romanovskis, P. Tetrahedron Lett. 1996,
37, 591-594.
Since any amino acid with a primary amine can be converted
into the corresponding azido-acid,35 this C-terminal alkyne/
N-terminal azide motif would maximize the potential size
of a cyclic peptide library by allowing the N-terminal position
at which macrocyclization occurs to be a point of diversity
in a parallel split-pool synthesis.
We investigated the general feasibility of this macrocy-
clization technique under the standard solid-phase peptide
synthesis conditions used to generate peptide libraries,
employing the acid labile 2-chlorotrityl chloride resin to
perform a parallel split-split synthesis (Scheme 2). Each
amino acid coupling was verified by the bromophenol blue
test,36 and the final macrocyclization step was carried out at
room temperature using commercially available CuI salts,
sodium ascorbate, DIEA, 2,6-lutidine, and DMF.
(21) Carpino, L. A.; Elfaham, A.; Albericio, F. J. Org. Chem. 1995, 60,
3561-3564.
(22) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang,
C. W.; Lee, Y. S.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mugge, C.;
Wenschuh, H.; Klose, K.; Beyermann, M.; Bienert, M. Angew. Chem., Int.
Ed. 2002, 41, 442-445.
(23) Oh, K.; Guan, Z. Chem. Commun. 2006, 3069-3071.
(24) (a) Brik, A.; Alexandratos, J.; Lin, Y. C.; Elder, J. H.; Olson, A. J.;
Wlodawer, A.; Goodsell, D. S.; Wong, C. H. ChemBioChem 2005, 6, 1167-
1169. (b) Angell, Y.; Burgess, K. J. Org. Chem. 2005, 70, 9595-9598.
(25) Bock, V. D.; Speijer, D.; Hiemstra, H.; van Maarseveen, J. H. Org.
Biomol. Chem. 2007, 5, 971-975.
(26) Hitotsuyanagi, Y.; Motegi, S.; Hasuda, T.; Takeya, K. Org. Lett.
2004, 6, 1111-1114.
(27) Horne, W. S.; Stout, C. D.; Ghadiri, M. R. J. Am. Chem. Soc. 2003,
125, 9372-9376.
(28) Horne, W. S.; Yadav, M. K.; Stout, C. D.; Ghadiri, M. R. J. Am.
Chem. Soc. 2004, 126, 15366-15367.
(29) (a) Huisgen, R. 1,3-Dipolar cycloaddition: introduction, survey,
mechanism. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.;
Wiley: New York, 1984; Vol. 1, pp 1-176. (b) Kolb, H. C.; Sharpless, K.
B. Drug DiscoVery Today 2003, 8, 1128-1137. (c) Bock, V. D.; Perci-
accante, R.; Jansen, T. P.; Hiemstra, H.; van Maarseveen, J. H. Org. Lett.
2006, 8, 919-922.
Initially, we investigated the efficiency of the macrocy-
clization using catalytic CuBr (0.2 equiv). We found that
the long reaction times of 24-72 h were impractical and
fostered a mixture of cyclic and linear products, regardless
of the presence or absence of other additives such as sodium
ascorbate or nitrogenous bases. We were pleased to find that
(30) Schmidt, U.; Langner, J. J. Pept. Res. 1997, 49, 67-73.
(31) (a) Rostovtsev, V. V.; Green; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596-2599. (b) Rodionov, V. O.; Fokin, V. V.;
Finn, M. G. Angew. Chem., Int. Ed. 2005, 44, 2210-2215.
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662-673.
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126, 1638-1639.
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2006, 1, 51-68.
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(36) Kay, C.; Lorthioir, O. E.; Parr, N. J.; Congreve, M.; McKeown, S.
C.; Scicinski, J. J.; Ley, S. V. Biotechnol. Bioeng. 2000, 71, 110-118.
5012
Org. Lett., Vol. 9, No. 24, 2007

Scheme 2. Synthetic Route to On-Resin, Cu(I)-Promoted
Click Macrocyclization
using stoichiometric CuBr (1 equiv) and an excess of sodium
ascorbate reduced the reaction time to 4-6 h and resulted
in a clean mixture of cyclomonomer and cyclodimer
products, and, in some cases, trace amounts of trimer product.
Increasing CuBr to three equivalents had no effect on
cyclization efficiency, reaction time, or product ratios.
Despite having the same molecular weight, in the series
of peptides that we examined, each of the azide-terminated
linear peptides had a longer retention time than did the cyclic
product. We used the evaporative light-scattering detector
(ELSD) trace to determine the optimal reaction time and to
establish the cleanliness of the crude reaction mixture (Figure
1). We determined the cyclomonomer/cyclodimer product
ratios by integrating the peaks from the photodiode array
detector (PDA) trace (Table 1). All peptide products were
purified by preparative HPLC and their identities verified
Figure 1. ELSD traces of crude linear pentapeptide 3 (top), and
crude cyclization mixture (bottom) showing the cyclomonomer 7,
cyclodimer, 12, and trace cyclotrimer.
macrocyclization shows particular potential for the library
synthesis of cyclic penta- and hexapeptides. The pentapeptide
yielded a cyclomonomer/cyclodimer ratio of 5.5:1, with only
trace amounts of trimer product. The click macrocyclization
of the hexapeptide was nearly as efficient, resulting in only
cyclomonomer and cyclodimer products with a ratio of 5.4:
1. With the reputed ease of macrolactamization among
heptapeptides, we were surprised that our heptapeptide
produced a lower, but still useful, ratio of 3.6:1.
An X-ray crystal structure of the pentapeptide cyclomono-
mer revealed an interesting and novel type-I′/-II′ â-turn
structure (Figure 2). Within the macrocycle, the triazole unit
essentially acts as a spacer between the i and i + 3 positions
of a â-turn. Interestingly, the crystal contains two distinct
molecules in the asymmetric unit with virtually identical
backbone conformations, in which one of the molecules has
a type-II′ â-turn, while the other has a type-I′ â-turn. As in
proteins, type-I′ and -II′ turns are also rare in cyclic
peptides.37 A survey of the Cambridge Crystallographic
Database revealed 33 cyclic peptides that contain a type-II′
â-turn, and 12 cyclic peptides with a type-I′ â-turn (out of
a total of ∼570 cyclic peptides in the database). Among
these, most contain proline and/or D-amino acid residues at
1
by H NMR.
Among the peptides examined, the click macrocyclization
seems best suited for peptides 4-6 amino acids in length.
In light of the difficulty associated with lactamization of tri-
and tetrapeptides, we were not surprised to find that the
tripeptide formed almost exclusively cyclodimer product,
with a small amount of cyclotrimer product as well. We were
pleased to find, however, that the click macrocyclization
proceeded smoothly with the tetrapeptide, producing cy-
clomonomer and cyclodimer products with a ratio of 3.5:1,
with only trace amounts of trimer product. The click
Org. Lett., Vol. 9, No. 24, 2007
5013

facilitate the cyclization of linear peptides,38 there remains
a need for mild techniques that enable the cyclization of small
peptides that are inherently difficult due to an absence of
turn-promoting structures. We have demonstrated the useful-
ness of click chemistry in the on-resin cyclization of a series
of leucine rich tetra-, penta-, hexa-, and heptapeptides. The
cyclization is complete within 6 h, utilizing mild conditions
that are orthogonal to the protection groups common to SPPS.
Furthermore, because these mild conditions are unreactive
to the majority of amino acid side chains, it is possible, in
principle, to carry out the cyclization in the absence of side-
chain protection groups.
Table 1. Results of CuI-Promoted Cyclization Reactions
The Fmoc strategy of solid-phase peptide synthesis makes
it possible to generate vast libraries of cyclic peptides for
high throughput screening and lead discovery. The efficiency
of cyclization, however, is highly dependent on the length,
stereochemistry, and sequence of the linear peptide.39 Inef-
ficiencies in the cyclization process can result in byproducts
that compromise yield and purity requirements for library
synthesis. The ease and reliability of on-resin click cycliza-
tion demonstrated here indicates the potential to carry out
the library synthesis in a one-bead-one-compound format
and bioassay directly from the crude products. Deconvolution
of hit compounds would simply entail one additional
purification step to separate cyclomonomer and multimer
products for individual screening.
cmpd
n
monomer:dimera
isolated yieldb (%)
10
6
7
8
9
1
2
3
4
5
0:1
3.5:1
5.5:1
5.4:1
3.6:1
75c
23d
25d
27d
20d
a Ratio by integration of peaks in PDA trace (λ ) 220-240 nm).
b Products isolated by prep HPLC. c Yield of cyclodimer product. d Yield
of cyclomonomer product. The majority of the product is lost on HPLC
purification, as crude yields for all sequences (monomer plus multimers)
were >95%.
Acknowledgment. We gratefully acknowledge the Na-
tional Institutes of Health for funding (CA104569-03). We
also thank Roche (Palo Alto, CA) for donation of the LC/
MS system used in this work. The single-crystal X-ray
diffraction data in this work was obtained on an instrument
supported by the National Science Foundation’s Major
Research Instrumentation (MRI) Program under Grant No.
CHE-0521569.
the i or i + 2 position, while none have standard L-amino
acids at both the i and i + 2 positions. Thus, the triazole
spacer acts to stabilize an unusual turn type and provides
access to type-I′/-II′ turns for “all L” sequences.
Despite the availability of reagents and methods to
Note Added after ASAP Publication. While this manuscript
was in press, a similar on-resin 1,3-dipolar azide-alkyne
cycloaddition reaction was reported in the synthesis of
vascular endothelial growth factor I ligands: Goncalves, V.;
Gautier, B.; Regazzetti, A.; Coric, P.; Bouaziz, S.; Garbay,
C.; Vidal, M.; Inguimbert, N. Bioorg. Med. Chem. Lett. 2007,
17, 5590-5594. The original version was published ASAP
October 23, 2007; the corrected version was published on
November 2, 2007.
Supporting Information Available: Detailed synthetic
procedures; full X-ray coordinates for 7; method for calculat-
1
ing monomer:dimer ratios; H and 13C NMR spectra, LC/
MS traces, and tabulated IR data for all compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL702228U
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Figure 2. Stereoscopic views of the two molecules in the unit
cell from the crystal structure of the pentapeptide 7, exhibiting a
type-I′ â-turn (A) and a type-II′ â-turn (B).
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Org. Lett., Vol. 9, No. 24, 2007