Peptide cyclization and cyclodimerization by CuI-mediated azide-alkyne cycloaddition

Article abstract of DOI:10.1021/jo802097m

Head-to-tail cyclodimerization of resin-bound oligopeptides bearing azide and alkyne groups occurs readily by 1,3-dipolar cycloaddition upon treatment with Cu^I. The process was found to be independent of peptide sequence, sensitive to the proximity of the alkyne to the resin, sensitive to solvent composition, facile for a- and β-peptides but not for γ-peptides, and inhibited by the inclusion of tertiary amide linkages. Peptides shorter than hexamers were predominantly converted to cyclic monomers. Oligoglycine and oligo(β-alanine) chains underwent oligomerization by 1,3-dipolar cycloaddition in the absence of a copper catalyst. These results suggest that cyclodimerization depends on the ability of the azido-alkyne peptide to form in-frame hydrogen bonds between chains in order to place the reacting groups in close proximity and lower the entropic penalty for dimerization. The properties of the resin and solvent are crucial, giving rise to a productive balance between swelling and interstrand H-bonding. These findings allow for the design of optimal substrates for triazole-forming ring closure and for the course of the reaction to be controlled by the choice of conditions.

Full text of DOI:10.1021/jo802097m

Peptide Cyclization and Cyclodimerization by CuI-Mediated
Azide-Alkyne Cycloaddition
Reshma Jagasia,† Justin M. Holub,‡ Markus Bollinger,† Kent Kirshenbaum,‡ and
M. G. Finn*,†
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and Department of Chemistry, New York
UniVersity, New York, New York 10003
mgfinn@scripps.edu
ReceiVed September 21, 2008
Head-to-tail cyclodimerization of resin-bound oligopeptides bearing azide and alkyne groups occurs readily
by 1,3-dipolar cycloaddition upon treatment with CuI. The process was found to be independent of peptide
sequence, sensitive to the proximity of the alkyne to the resin, sensitive to solvent composition, facile
for R- and ꢀ-peptides but not for γ-peptides, and inhibited by the inclusion of tertiary amide linkages.
Peptides shorter than hexamers were predominantly converted to cyclic monomers. Oligoglycine and
oligo(ꢀ-alanine) chains underwent oligomerization by 1,3-dipolar cycloaddition in the absence of a copper
catalyst. These results suggest that cyclodimerization depends on the ability of the azido-alkyne peptide
to form in-frame hydrogen bonds between chains in order to place the reacting groups in close proximity
and lower the entropic penalty for dimerization. The properties of the resin and solvent are crucial, giving
rise to a productive balance between swelling and interstrand H-bonding. These findings allow for the
design of optimal substrates for triazole-forming ring closure and for the course of the reaction to be
controlled by the choice of conditions.
Introduction
are often more bioavailable, more stable toward metabolic
degradation, and more selective in receptor binding.4-11 It is
therefore not surprising that peptide sequences are frequently
evaluated for function in both linear and cyclic forms, creating
a need for effective cyclization strategies.
Cyclic peptides and related structures are of longstanding
interest as biologically active compounds because of their ability
to display protein-like epitopes with restricted conformational
flexibility.1-3 Relative to their linear analogues, cyclic peptides
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Sacchi, A.; Longhi, R.; Corti, A. J. Biol. Chem. 2002, 277, 47891–47897.
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5, 251–260.
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B.; Rocchi, R. J. Med. Chem. 2002, 45, 4494–4504.
† The Scripps Research Institute.
‡ New York University.
(1) Davies, J. S. In Cyclic Polymers, 2nd ed.; Semlyen, J. A., Ed.; Kluwer
Academic Publishers: Dordrecht, Netherlands, 2000; pp 85-124.
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2964 J. Org. Chem. 2009, 74, 2964–2974
10.1021/jo802097m CCC: $40.75 2009 American Chemical Society
Published on Web 03/23/2009

Peptide Cyclodimerization
FIGURE 1. On-resin peptide cyclodimerization of two sequences originally reported by Finn et al.26 Amino acids are represented by their single-
letter codes in bold print (X ) propargylglycine).
Peptide cyclization is most often accomplished by the
formation of amide, ester, thioester, disulfide, olefin, or C-C
bonds.12-14 Recently the copper(I)-mediated azide-alkyne
cycloaddition (CuAAC) reaction has been employed as a means
of orthogonal modification15 and cyclization of peptides,16-18
glycopeptides,19 polymers,20 and dendrimers.21,22 In the context
of peptides, this “click reaction”23-25 methodology offers two
important advantages. (1) Azide and alkyne units can be
incorporated into peptide sequences during solid-phase peptide
synthesis (SPPS) without protection, as both of these groups
are stable to typical conditions of peptide synthesis and cleavage.
(2) Azides and alkynes react selectively with each other, but
do so very slowly unless catalyzed, allowing their ligation to
be triggered at will.
serotype that binds several Rv integrins and contains the
canonical Arg-Gly-Asp (RGD) triad. The alkyne, introduced
as a propargylglycine unit, was located near the resin-bound
C-terminus, while the azide group was incorporated as a
5-azidopentanoyl unit at the N-terminus. On-resin cyclization
mediated by cuprous iodide gave high yields of head-to-tail
cyclic dimer rather than the expected cyclic monomer, even for
sequences of substantial length and no obvious conformational,
steric, or torsional constraints.
Several examples of solution-phase Cu-mediated cyclodimer-
ization of peptides and carbohydrates have been reported, all
with azido-alkyne substrates for which monomeric ring closure
is disfavored because of torsional or geometric factors (described
in more detail in the Supporting Information).27-30 Other
investigators have described mixtures of peptide cyclic mono-
mers and dimers as a function of ring strain and concentration,31,32
as well as clean macrocyclization under the influence of
conformational constraints.17,18,33 These observations show that
the CuAAC process is not inherently predisposed toward ring
closure by head-to-tail dimerization. The unexpected and clean
cyclodimerization of the two resin-bound peptides shown in
Figure 1 therefore signaled to us the presence of an unexpected
mechanistic feature. We suggested a sequence-dependent con-
formational bias and/or amide-Cu binding as two possible
contributing factors in our original report.26 We describe here
experimental tests that disprove these hypotheses and suggest
a crucial role for interchain hydrogen bonding. In addition, we
wanted to explore the scope and limitations of the cyclodimer-
ization method, since its products are both unique and potentially
useful. When compared with monocyclic analogues, cyclic
dimers test the contributions of both conformational rigidity and
valency in the binding of peptide motifs to their targets.
We previously described attempts to cyclize two peptides on
resin via the CuAAC reaction (Figure 1).26 The sequences used
in this preliminary study were derived from an adenovirus
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(16) Roice, M.; Johannsen, I.; Meldal, M. QSAR Comb. Sci. 2004, 23, 662–
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Maarseveen, J. H. Org. Lett. 2006, 8, 919–922.
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charjya, A. Tetrahedron Lett. 2006, 47, 2775–2778.
(19) Agren, J. K. M.; Billing, J. F.; Grundberg, H. E.; Nilsson, U. J. Synthesis
2006, 18, 3141–3145.
(20) Grayson, S. M.; Laurent, B. A.; Eugene, D. E. Polym. Mater. Sci. Eng.
2006, 51, 827–828.
(21) Rijkers, D. T. S.; Van Esse, G. W.; Merkx, R.; Brouwer, A. J.; Jacobs,
H. J. F.; Pieters, R. J.; Liskamp, R. M. J. Chem. Commun. 2005, 4581–4583.
(22) Wu, P.; Malkoch, M.; Hunt, J. N.; Vestberg, R.; Kaltgrad, E.; Finn,
M. G.; Fokin, V. V.; Sharpless, K. B.; Hawker, C. J. Chem. Commun. 2005, 46,
5775–5777.
(23) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004–2021.
(24) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3062.
(25) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
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7, 4503–4506.
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(29) Bodine, K. D.; Gin, D. Y.; Gin, M. S. J. Am. Chem. Soc. 2004, 126,
1638–1639.
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(31) Angell, Y.; Burgess, K. J. Org. Chem. 2005, 70, 9595–9598.
(32) Choi, W. J.; Shi, Z.-D.; Worthy, K. M.; Bindu, L.; Karki, R. G.; Nicklaus,
M. C.; Fisher, R. J.; Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2006, 16, 5265–
5269.
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BioChem 2008, 9, 1701–1705.
J. Org. Chem. Vol. 74, No. 8, 2009 2965

Jagasia et al.
TABLE 1. Peptide Sequences To Test the Dependence of
Cyclodimerization on Amino Acid Compositiona
no.
sequenceb
no.
sequenceb
no.
sequenceb
1
2
3
4
5
6
7
8
9
IDASTRLNA
KVASTRLNA
YSMFTRLNA
VEMFTRLNA
RLASFKCLA
TWASFKCLA
HTVMFKCLA
KCVMFKCLA
12 SVAEYRLKA
13 THMFAHLKA
14 SVMFAHLKA
15 IQASAHLKA
16 KVASAHLKA
17 RLRMFWKLF
23 HMFTMAKLF
24 ADFTMAKLF
25 MFAQTRIEF
26 THAQTRIEF
27 KWANTRIEF
28 SVANTRIEF
18 AKRMFWKLF 29 KNASHKIEF
19 SEMAFWKLF 30 IMASHKIEF
MHAQYRLKA 20 GYMAFWKLF 31 VTSFHKIEF
21 KRHDMAKLF 32 KASFHKIEF
11 KWAEYRLKA 22 FVHDMAKLF
10 TLAQYRLKA
a All peptides bear protected side-chain functional groups as listed in
the Experimental Section, and all were cyclodimerized successfully by
treatment with CuI in 4:1 MeCN:DMSO. b Single-letter amino acid
codes represent the resin-bound N-to-C sequence present between azide
and alkyne moieties with the general structure shown above the table.
FIGURE 2. Solution-phase cyclization of an alkyne- and azide-
derivatized peptide.
which would reduce the azide in a linear dimer. To the extent
that our survey of sequence space is representative, it appears
that the CuAAC-mediated peptide cyclodimerization process is
largely insensitiVe to the nature of the amino acids present in
the sequence, keeping in mind that side-chain functional groups
are protected.
Results and Discussion
Sequence Tolerance. The dependence of cyclodimerization
on sequence identity was probed with a set of 32 peptides
synthesized by batch splitting after coupling every second
residue (Table 1). All proteinogenic amino acids were included
except proline; the sequences tested the importance of several
residues in the original sequence that were suspected of playing
a role in the cyclodimerization outcome. Peptides were synthe-
sized on Rink amide MBHA (4-methylbenzhydrylamine) resin
with an initial loading of 0.70 mmol/g, similar to that in our
previous report. As before, alkynes were installed near the
C-terminus, although by N-propargylation rather than in pro-
pargylglycine for synthetic convenience, and azides were
introduced by acylation of the N-terminus. Each linear sequence
was individually cleaved and lyophilized after a standard workup
procedure. All 32 peptides were obtained with good to excellent
purity (>75%, estimated by MALDI mass spectrometry analysis
of the lyophilized crude powders).
Each resin-bound protected peptide was subjected to cycliza-
tion in 20 mM solutions of cuprous iodide under the convenient
reaction conditions employed in our previous report (20%
DMSO in MeCN, room temperature, 40 h, in screw-cap reaction
vials closed immediately after bubbling with a stream of dry
N2). Successful cyclodimerization was indicated by a clear
transition from a dominant peak of monomer mass to a dominant
peak of dimeric mass in the MALDI mass spectrum of the
lyophilized powders. This result was observed for all of the 32
sequences shown in Table 1, although some were slower to react
than others due to inconsistencies in the exclusion of oxygen,
thereby giving rise to different amounts of active CuI catalyst.
To confirm the expected cyclic nature of the products, most of
the samples were subjected to careful analysis by analytical
reversed-phase (RP) HPLC after cleavage from the resin. In
each case, a significantly shorter retention time was observed
for the major product macrocycle in comparison with corre-
sponding linear monomers. In contrast, linear dimers synthesized
independently showed uniformly longer reversed-phase HPLC
retention times than their monomeric counterparts. Moreover,
the chromatogram and mass spectrum of each product was
unchanged after treatment with excess triphenylphosphine,
Solution-Phase Cyclization. To illuminate the role of the
resin in the peptide cyclodimerization process, solution-phase
reactions were performed with a protected peptide sequence
similar to that in our original studies (Figure 2). Following solid-
phase synthesis, cleavage (without deprotection), and purifica-
tion, structure 33 was subjected to CuI-mediated cycloaddition
in a deoxygenated MeOH/CH2Cl2 mixture at concentrations of
0.8 and 3 mM, to promote an intra- and intermolecular reaction,
respectively.32 After side-chain deprotection, resin cleavage, and
ether precipitation, the samples were analyzed by analytical
HPLC and MALDI mass spectrometry. The major product peak
in both reactions had monomer molecular weight, had signifi-
cantly shorter retention time on RP-HPLC than the linear
deprotected starting material, and was insensitive to reduction
with phosphine, showing that monocyclization to structure 34
was the main pathway in the solution-phase reaction. A small
amount of dimeric product 35 was also observed (approximately
10% and 20% when conducted at 0.8 and 3 mM, respectively,
as determined by HPLC integration) which coeluted with an
authentic sample of cyclic dimer. Therefore, selectiVe cy-
clodimerization of peptides of this length is facilitated by
attachment to a resin. However, a particular resin is not required.
The use of three different polystyrene supports with different
loading levels (chlorotrityl resin at 1.4 mmol/g starting functional
group density, Rink amide MBHA resin at 0.70 mmol/g, and
the PEGylated Rink-modified TentaGel (TGR) resin at 0.26
mmol/g) induced no substantial difference in the course of the
reaction (Supporting Information).
Peptide Length. To evaluate the influence of peptide length
on cyclodimerization, truncated versions of peptide 33 were
prepared. When treated with CuI, resin-bound 36 was converted
cleanly to the cyclic dimer, but the shorter sequence 37 gave
an intractable mixture with no major peak on HPLC and a
MALDI mass spectrum showing both monomer and dimer
masses (Figure 3 and Supporting Information). The shortest
sequence 38 produced a single major peak in the HPLC
chromatogram with monomeric mass and a significantly shorter
2966 J. Org. Chem. Vol. 74, No. 8, 2009

Peptide Cyclodimerization
FIGURE 3. Test of the dependence of sequence length on cyclization. RP-HPLC chromatograms of the crude products. No linear monomers were
observed, which was verified by coinjection of peptides before cyclization (not shown); “x” indicates peak due to a small amount of a chromophore
with a high extinction coefficient.
retention time than the linear starting material; this material was
unaffected by treatment with triphenylphosphine. Accordingly,
this product was assigned as the cyclic monomer of sequence
38. Monomeric cylization of peptides of this length was also
shown to be independent of the sequence (Supporting Informa-
tion). These results indicate that a minimum peptide length of
six residues is required in this case for selectiVe cyclodimer-
ization.
Proximity of Alkyne to Resin. We previously demonstrated
that cyclodimerization is favored by placement of the alkyne
moiety at the C-terminal end of the peptide, which is attached
to the resin polymer.26 This phenomenon was further explored
with sequences 39-43, which provide an increasing number
of glycine spacers between the resin and the alkyne (Figure 4).
Under standard reaction conditions (5 or 20 mM CuI, 4:1 MeCN/
DMSO with 2,6-lutidine), each of the sequences provided cyclic
dimers as the sole detectable products by MALDI mass
spectrometry. However, as the distance of the alkyne to the resin
was increased, a progressive increase in the intensity of
unresolved broad peaks was observed in the analytical RP-HPLC
chromatograms. These side products are presumed to emerge
from peptide degradation, which apparently becomes significant
when cyclodimerization is not optimal (see Supporting Informa-
tion for additional experiments). Thus, cyclodimerization be-
comes less efficient as the alkyne is moVed to peptide positions
farther away from the resin.
Availability of Alkyne. Figure 5 summarizes the outcome
of experiments designed to test whether monocyclization could
be made to occur if either azide or alkyne is made the limiting
functional group. Resin-bound peptides were prepared in which
only 25% of the chains displayed on the resin contained both
groups necessary for triazole formation and 75% lacked either
the azide (44) or the alkyne (45). Upon exposure to standard
CuAAC conditions, no cyclic monomers were observed; in other
words, chains containing both azide and alkyne were somehow
prevented from closing upon themselves, even when the rate
of cyclodimerization was necessarily reduced. Resin 44 provided
only the linear heterodimer and remaining nonazide peptide,
FIGURE 4. Sequences prepared to determine the efficacy of cy-
clodimerization when the distance between alkyne and solid support
(Rink amide MBHA resin) is increased.
whereas resin 45 returned the linear mixed dimer, cyclic dimer,
and both starting peptide sequences. A significant difference
was also noted in the extent of completion for the two reactions.
Resin 44, which bears alkynes on every peptide chain, gave
complete consumption of the limiting (azide) group, whereas
even extended treatment of 45 with CuI failed to consume all
of the limiting reagent (alkyne). This is consistent with the
expectation that alkynes are converted rapidly to Cu-acetylides,
and that two Cu atoms are required for triazole formation.34-36
(34) Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Angew. Chem., Int. Ed.
2005, 44, 2210–2215.
(35) Ahlquist, M.; Fokin, V. V. Organometallics 2007, 26, 4389–4391.
(36) Rodionov, V. O.; Presolski, S.; D´ıaz, D. D.; Fokin, V. V.; Finn, M. G.
J. Am. Chem. Soc. 2007, 129, 12705–12712.
J. Org. Chem. Vol. 74, No. 8, 2009 2967

Jagasia et al.
FIGURE 5. (Top) Mixed peptide sequences with substoichiometric quantities of azide or alkyne groups. (Bottom) Summary of the results from
exposure to cyclization reaction conditions.
FIGURE 6. Oligomers of ꢀ-Ala, γ-Abu, and Gly tested for on-resin CuAAC cyclization. The observed products of standard CuI treatment are
indicated in parentheses.
Non-Peptide Oligoamides. In an attempt to establish the
structural requirements for cyclodimerization, we prepared resin-
bound oligoamides lacking side chains and with differing
numbers of main-chain methylene spacers and different lengths:
glycine oligomers 46 and 47, ꢀ-alanine oligomers 48-50, and
γ-aminobutyrate oligomers 51 and 52 (Figure 6). Mixed ꢀ-Ala/
γ-Abu sequences 53-54 were also synthesized. Upon exposure
to CuI under standard conditions, the penta(R-peptide) 46 and
penta(ꢀ-peptide) 48 afforded complex product mixtures (linear
and cyclic monomers, dimers, and oligomers), similar to
pentapeptide 37 (Figure 3) used to establish the length depen-
dence of cyclodimerization. Unfortunately, the octapeptide 47
yielded only highly insoluble material (likely due to ꢀ-sheet
aggregation characteristic of long oligoglycines), the composi-
tion of which could not be analyzed. More revealingly,
lengthening the ꢀ-peptide chain by one (49) or three (50) units
gave rise to a change in the product distribution, providing
substantial amounts of cyclic dimers. It is therefore evident that
cyclodimerization does not require the presence of oligopeptide
side chains.
Sequences 49 and 50 also returned significant amounts
(approximately 15% total) of linear oligomers with mass values
ranging from dimer to pentamer (M2-M5). This phenomenon
was investigated with 49 and was found to arise from triazole
formation in the absence of copper with both the resin-bound
protected peptide and the linear deprotected peptide after
cleavage from the resin, when each was stored in the absence
of solvent at room temperature. We have previously observed
2968 J. Org. Chem. Vol. 74, No. 8, 2009

Peptide Cyclodimerization
FIGURE 7. Resin-bound peptoid sequences subjected to standard CuAAC conditions and subsequent cleavage with TFA. All returned cyclic
monomers were in clean fashion, with the exception of 61, which gave a cyclic monomer and oligomers.
such solid-state (or neat oil) oligomerization of other small-
molecule R,ω-azido-alkynes during the course of polymeri-
zation studies.37 This phenomenon has interesting implications
for metal-independent triazole formation38-41 on surfaces or in
the context of materials synthesis.37
alitiesthatenhancestructuraldiversityandbiomedicalrelevance.46,47
We reasoned that the treatment of azido-alkynyl derivatives of
such molecules with CuI would provide novel cyclic structures
and would begin to test our prior hypothesis (now discredited,
see below and Supporting Information) that interactions of
copper with the backbone amide groups are of importance to
cyclodimerization.26 Experiments were performed using the
series of N-benzylated octamers shown in Figure 7, designed
to exhibit various properties of conformational structure and
rigidity.
Homochiral R-substitution on nitrogen confers well-character-
ized helicity to the peptoid architecture with a helical pitch of
three residues.43-45 Sequence 56 thereby places its azide and
alkyne groups in close proximity to each other on the same
side of the helix, while 57 places them a greater distance apart
and on different faces of the helix.33,47 Structures 58 and 59
mimic the alkyne and azide positions of the peptides tested
above. The lack of R-substituents in the latter sequence removes
the steric and torsional interactions between side chains and
hence any predisposition toward helicity, creating an unstruc-
tured, more flexible peptoid strand.48 In all of these cases
(56-59), cyclic monomers were produced exclusively from an
on-resin CuAAC reaction. The cyclic monomers were identified
by mass spectrometry and their substantially different retention
times on analytical RP-HPLC compared with the linear starting
material.
In contrast to the ꢀ-Ala oligomers, oligo(γ-Abu) sequences
51 and 52 were completely converted to cyclic monomers, the
latter being particularly notable because it contains eight
backbone amide bonds in the chain, the same number that gave
reliable cyclodimerization with oligopeptides. Sequences 49 and
51 are nearly the same length but have different numbers and
relative spacings of their amide bonds. To determine which of
these two factors is responsible for selective cyclodimerization,
mixed ꢀ-Ala and γ-Abu sequences 53-55 were prepared, where
the number of amide bonds was conserved (equivalent to 49,
which readily cyclodimerizes) but their spacing along the
oligoamide strand was varied. Upon exposure to CuI, sequences
53-55 all afforded cyclic monomers, suggesting that the spacing
of amide bonds influences the cyclization process, with closer
amide linkages (R- or ꢀ-peptides) faVoring cyclodimer forma-
tion.
Tertiary Amides. Peptoids, oligomeric peptides in which the
side chains are placed on the amide nitrogen atom, have attracted
a great deal of interest for their resistance to proteolytic
cleavage,42 fascinatingstructuralandconformationalproperties,43-45
and their ability to display a wide array of chemical function-
Limited N-alkylation of a peptide was also able to abolish
cyclodimerization, as shown in Figure 8. Thus, while the resin-
bound decamer 62 underwent facile cyclodimerization under
standard conditions, N-methylation of a central glycine (63)
induced the formation of significant amounts of both cyclic
monomer and cyclic dimer, along with some trimeric material.
N-Methylation of two flanking positions (Leu and Phe, structure
64) diverted the CuAAC reaction products to mostly cyclic
monomer, along with small amounts of cyclic dimer and
oligomeric materials with masses ranging from M3 to M5. The
complete absence of cyclic dimers from the reactions of peptoids
and the decrease in cyclodimerization upon N-methylation of
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(46) Holub, J. M.; Jang, H. J.; Kirshenbaum, K. Org. Lett. 2007, 9, 3275–
3278.
(47) Jang, H.; Fafarman, A.; Holub, J. M.; Kirshenbaum, K. Org. Lett. 2005,
7, 1951–1954.
(48) Fafarman, A. T.; Borbat, P. P.; Freed, J. H.; Kirshenbaum, K. Chem.
Commun. 2007, 377–379.
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Jagasia et al.
FIGURE 8. Peptides used to test the dependence of backbone amide methylation on cyclodimerization, and their results after applying standard
reaction conditions.
FIGURE 9. IR spectra of protected resin-bound nonapeptide 39 swollen overnight in CH2Cl2 (blue), DMSO (green), or the standard reaction
solvent system of 4:1 MeCN/DMSO (red).
peptides, demonstrate that secondary, not tertiary, amide bonds
are required for cyclodimerization.
stretching band at 1625 cm-1 and an amide N-H absorbance
at 3277 cm-1, both characteristic of strong hydrogen bonding.
Proposed Mechanism of Cyclodimerization. An examina-
tion of the resin-bound protected peptides by IR spectroscopy
revealed an important solvent-dependent change in the state of
hydrogen bonding by the backbone amide groups. Both CH2Cl2
and DMSO swell polystyrene resins, but the latter solvent is
far better at disrupting inter- and intrachain peptide H-bonding.
Figure 9 shows the representative case of resin-bound protected
peptide 39, which in CH2Cl2 exhibited an amide carbonyl
In DMSO, new bands appeared at 1654 and 3461 cm-1
,
consistent with the expected loss of H-bonding due to interac-
tions of the peptide chains with the solvent.49-52 In the standard
reaction solvent system of 4:1 MeCN/DMSO, the IR spectrum
(49) Narita, M.; Honda, S.; Umeyama, H.; Ogura, T. Bull. Chem. Soc. Jpn.
1999, 61, 1201–1206.
(50) Narita, M.; Lee, J.-S.; Hayashi, S.; Hitomi, M. Bull. Chem. Soc. Jpn.
1993, 66, 489–493.
2970 J. Org. Chem. Vol. 74, No. 8, 2009

Peptide Cyclodimerization
TABLE 2. Conditions Found To Promote Dimeric vs Monomeric CuAAC Cyclization of Peptides
dimeric cyclization
competing monomeric cyclization
side-chain groups protected
reliable monomeric cyclization
side-chain groups protected
polystyrene-based resin swelled in 4:1
MeCN:DMSO
six or more amino acids in length
R- or ꢀ-peptides
alkyne group installed close to
polystyrene backbone
side-chain groups protected
polystyrene-based resin swelled in DMSO
or protic solvent, or reaction done in solution
tertiary backbone amide bonds
four or fewer amino acids in length
γ-peptides
matched that in CH2Cl2, showing that this mixture contains an
insufficient amount of DMSO to disrupt peptide H-bonding. We
therefore propose that head-to-tail interchain H-bonding is the
organizing phenomenon responsible for cyclodimerization by
CuAAC.
do not affect the cyclodimerization efficiency, suggesting that
H-bonding in the resin environment is a powerful force.
(4) Cu-mediated cyclodimerization occurs for resin-bound R-
and ꢀ-oligopeptides but not γ-peptides (Figure 6). A potential
antiparallel arrangement for each of these amide oligomers is
shown in Figure 10; head-to-head alignments are somewhat less
favorable and would not be productive in terms of azide-alkyne
ligation. Sufficiently stable interchain interactions could arise
from a combination of H-bonding density and dipolar stabiliza-
tion. Oligo(R-peptides) have the greatest number of H-bonds
per unit length, enough to overcome a less favorable dipolar
arrangement derived from the alternating pattern of H-bond
donors and acceptors.56 Oligo(ꢀ-peptides)57 compensate for the
lesser H-bond density with a favorable H-bond dipolar arrange-
ment.56 Oligo(γ-peptides) have their H-bonds too far apart to
stabilize interchain interactions and do not undergo cyclodimer-
ization.
(5) Cyclic dimers are less efficiently produced when peptides
are inserted between the alkyne and the polystyrene backbone
(Figure 4). We suggest that when H-bonding is maximized in
these extended sequences, alkyne and azide are not placed
optimally for cyclodimerization, as shown at the left of Figure
11. The productive alignment (Figure 11, right) is less favorable
with respect to H-bonding, and so, a poor yield of cyclic dimer
is obtained.
(6) Uncatalyzed azide-alkyne cycloaddition reactions are
extremely slow when the alkyne is not electron-deficient and
so require the reactants to be held in close proximity in order
to occur, even to a small degree, at room temperature.38,39,58
The observation of Cu-free solid-phase oligomerization for
oligo(ꢀ-Ala) species 49 and 50 (and probably oligo(Gly) 47),
but not for sequences containing γ-peptides, therefore supports
the proposed existence of a preorganizing interaction between
peptide chains. We suggest that the slow reaction in the absence
of copper begins to link the head-to-tail oriented chains and
that cyclic dimers are formed when triazole formation is
accelerated under conditions where H-bonding is maintained.
Peptide Cyclization and Cyclodimerization on
Preparative Scale. To demonstrate the capability of the CuAAC
process to produce substantial quantities of a large cyclic
peptide, 240 mg of resin 65 was prepared (Figure 12) and
cleaved to obtain 57 mg of crude linear peptide (approximately
60% of the maximum yield based on the initial functional group
loading of the resin) and 30 mg of purified peptide after
reversed-phase HPLC. Cyclization of a parallel batch under
standard dimerization conditions (20 mL of 4:1 MeCN:DMSO,
Two important corollaries to this hypothesis concern the role
of resin and solvent: (a) the resin provides a relatively
hydrophobic environment that promotes hydrogen bonding,53,54
and (b) the 4:1 MeCN:DMSO solvent mixture solvates (swells)
the resin, allowing CuI access to all of the reactants, without
disrupting H-bonding between pairs of chains. This solvent
system was originally chosen for a very different reason: to
provide the CuI center with what was thought to be advantageous
coordination by the acetonitrile ligand, a notion that later kinetic
studies on solution-phase CuAAC reactions proved to be
erroneous. The crucially balanced role of the solvent was verified
by the CuAAC reaction of resin 62 in pure DMSO (Figure 8),
which is commonly used for solid-phase peptide synthesis due
to its H-bond disrupting ability. Correlating with the observed
change in H-bonding (Figure 9), this change in solvent changed
the outcome of the reaction from cyclodimerization to mono-
meric cyclization, with small amounts of cyclic dimer observed
along with some degraded material. We also note that mono-
cyclization of two azido-alkyne oligopeptides has recently been
reported using the highly polar and protic combination of 20%
H2O in N-methylpyrrolidinone (NMP) as the solvent.55
A summary of the experimental factors favoring cyclodimer-
ization vs monomeric cylization is given in Table 2. The
hypothesis of head-to-tail preorganization by interstrand H-
bonding, along with the above two corollaries, are consistent
with these findings, as follows:
(1) Backbone secondary amide groups in sufficient numbers
are required. When H-bonding cannot occur, as with tertiary
amides (Figures 7 and 8) or is too weak, as with shorter peptides
(Figure 3), monomeric cyclization is favored as each alkyne
finds itself in closest proximity to the azide of its own chain.
(2) Cyclodimerization is a function of main-chain amide
linkages and is independent of the identity of protected peptide
side chains, at least in the series of nonamers tested in Table 1.
(3) Solution-phase cyclization of a protected peptide affords
a predominantly cyclic monomer, whereas on-resin reaction of
the same species gives a cyclic dimer (Figure 2). When
performed in solution (in this case, in MeOH-CH2Cl2),
hydrogen bonding between chains is likely to be disrupted. On
the contrary, variation in resin loadings of 0.26-1.4 mmol/g
(51) Narita, M.; Lee, J.-S.; Hayashi, S.; Yamazaki, Y.; Sugiyama, T. Bull.
Chem. Soc. Jpn. 1993, 66, 500–505.
(52) Narita, M.; Tomotake, Y.; Isokawa, S.; Matsuzawa, T.; Miyauchi, T.
Macromolecules 1984, 17, 1903–1906.
(53) Merrifield, B. Br. Polym. J. 1984, 16, 173–178.
(54) Sherrington, D. C. J. Chem. Soc., Chem. Commun. 1999, 2275–2286.
(55) Goncalves, V.; Gautier, B.; Regazzetti, A.; Coric, P.; Bouaziz, S.;
Garbay, C.; Vidal, M.; Inguimbert, N. Bioorg. Med. Chem. Lett. 2007, 17, 5590–
5594.
(56) Murray, T. J.; Zimmerman, S. C. J. Am. Chem. Soc. 1992, 114, 4010–
4011.
(57) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001, 101,
3219–3232.
(58) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.;
Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 1053–
1057.
J. Org. Chem. Vol. 74, No. 8, 2009 2971

Jagasia et al.
FIGURE 10. (Left) Potential hydrogen patterns formed by head-to-tail alignments of oligo(Gly), oligo(ꢀ-Ala), and oligo(γ-Abu), placing interstrand
alkyne and azide moieties in close proximity to each other. The shaded bar represents the polystyrene chains to which the peptides are attached.
(Right) Proposed CuAAC cyclodimerization pathway directed by head-to-tail H-bonding.
FIGURE 11. Proposed hydrogen-bonded alignments formed by resin 43 (Figure 4).
FIGURE 12. Preparative-scale syntheses of a representative cyclic dimer and monomer.
495 mg of resin 62, 5 mM CuI, 30 µL of 2,6-lutidine, sparged
with N2), followed by cleavage and deprotection, returned 52
mg of crude cyclized material (compound 66, 91% of the
amount of the linear peptide obtained without CuAAC treat-
ment) and 32 mg after purification. Thus, the cyclodimerization
process was conducted with little or no loss of material. A third
2972 J. Org. Chem. Vol. 74, No. 8, 2009

Peptide Cyclodimerization
FIGURE 13. Incorporation of alkyne and azide groups during solid-phase peptide synthesis.
parallel batch of resin 65 was cyclized under conditions to
promote monomeric ring closure (20 mL of DMSO + 1 drop
of H2O, 5 mM CuI, 30 µL of 2,6-lutidine, sparged with N2) and
cleaved from the resin to give only 25 mg of crude product,
reflecting a difficulty in precipitating the more soluble cyclic
monomer, and 8.5 mg of monocyclic triazole 67 after HPLC
purification. In each case, HPLC and MALDI analysis showed
the expected species (linear azido-alkyne peptide, cyclic
monomer, or cyclic dimer) as the dominant species in the
unpurified product mixture, and the purified products were
characterized by HPLC, NMR, and high-resolution mass
spectrometry (Supporting Information).
Azides and alkynes are incorporated with ease into peptide
building blocks and remain undisturbed under conditions of
standard protection, deprotection, and coupling steps. The rate
of the reaction is fast, which is crucial to the overall outcome.
However, these features are not unique to CuAAC, and it should
be possible to find other ligation reactions, such as olefin
metathesis,60-62 nitrile imine-olefin cycloaddition,63,64 or thiol-
ene condensation,65 to accomplish the same task. In addition to
such studies, we will continue the development of cyclic
triazole-linked peptides for biological and materials science.
Experimental Section
Conclusions
Peptide Synthesis. All peptides were prepared by Fmoc SPPS
methods using Rink amide MBHA resin with an initial loading of
0.70 mmol/g, unless otherwise noted. Resins were swollen in DMF
for 45 min prior to synthesis. For sequence extension, the Fmoc-
protected amino acid (4 equiv) was activated by treatment with
HBTU or HCTU (3.9 equiv) and N,N-diisopropylethylamine (500
µL per mmol of amino acid) in DMF (4 mL per mmol of amino
acid) for 2 min. This solution was added to the free amine on resin,
and the coupling reaction was allowed to proceed for 15 min (longer
for difficult couplings) with intermittent stirring. After washing with
DMF, Fmoc deprotection was achieved with 20% 4-methylpiperi-
dine in DMF (2 × 7 min). The resin was washed once again, and
the process was repeated for the next amino acid.
The alkyne group was usually incorporated as an N-propargylg-
lycine unit by the method shown in Figure 13. The symmetric
anhydride of 2-bromoacetic acid (5 equiv of anhydride relative to
free amine) was prepared by mixing the acid (10 equiv) with N,N′-
diisopropylcarbodiimide (DIC, 5 equiv) in DMF at room temper-
ature for 15 min. This mixture was then added to the resin and
allowed to react for 1 h with intermittent stirring. The resin was
washed and then treated with propargylamine (20 equiv) in DMF
The CuAAC cyclodimerization process is quite robust for
peptides of six residues or longer, allowing the convenient
synthesis of large bivalent59 cyclic peptides. Furthermore, the
analogous cyclic monomers can be obtained from the same
resin-bound precursor with a simple change in solvent.
The success of peptide cyclodimerization derives from a
fortuitous combination of resin and solvent properties. The
discovery of the beneficial effects of the 4:1 MeCN:DMSO
solvent mixture provides a useful lesson in the properties of
resin-bound peptides or rather reinforces an old one. Merrifield’s
focus on the role of resin in maintaining oligopeptide solubility
as the key enabling feature of solid-phase peptide synthesis53
is perhaps underappreciated today even by some who use
Merrifield’s methods. Our reaction conditions maintain a state
intermediate between the full resin swelling desired for SPPS
and the collapsed aggregates that bedevil peptide-bearing resins
when insufficiently solvated. Furthermore, we failed to initially
recognize interchain hydrogen bonding as the key organizing
event for cyclodimerization, because we assumed that 20%
DMSO was enough to disrupt all H-bonding interactions.
The CuAAC reaction is well suited to joining together of
such noncovalently organized fragments into larger structures.
(60) Clark, T. D.; Ghadiri, M. R. J. Am. Chem. Soc. 1995, 117, 12364–
12365.
(61) Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. J. Am. Chem. Soc. 1996,
118, 9606–9614.
(62) Kazmaier, U.; Hebach, C.; Watzke, A.; Maier, S.; Mues, H.; Huch, V.
Org. Biomol. Chem. 2005, 3, 136–145.
(59) We have verified our earlier report (ref 26) that the synthesis of two
different sequences, A and B, on a resin bead results in the production of AA,
AB, and BB cyclic dimers under standard CuAAC cyclodimerization conditions.
Thus, mixtures of nonsymmetrical cyclic peptides can be prepared by this method.
(63) Song, W.; Wang, Y.; Lin, Q. J. Am. Chem. Soc. 2008, 130, 9654–9655.
(64) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem.,
Int. Ed. 2008, 47, 2832–2835.
(65) Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995–8997.
J. Org. Chem. Vol. 74, No. 8, 2009 2973

Jagasia et al.
for 12-16 h. After washing, coupling of the next residue to the
resultant secondary amine was achieved by preparing the sym-
metrical anhydride of that protected residue (5 equiv of anhydride)
with DIC in DMF at 0 °C for 15 min and coupling this mixture to
the amine for 1 h with occasional stirring. This same coupling
method was used for all instances when acylation of a secondary
amine was required. Where the alkyne was incorporated by coupling
Fmoc-L-propargylglycine, preactivation was carried out by mixing
the protected unnatural amino acid with HOBt and DIC (4 equiv
each) in DMF at 0 °C for 20 min. Azide was incorporated by the
attachment of either 5-azidopentanoic acid or 6-azidohexanoic acid
by the standard peptide coupling procedure.
The following Fmoc-protected amino acids with side-chain
protecting groups were obtained from a commercial supplier and
used as received: Fmoc-γAbu-OH, Fmoc-Ala-OH, Fmoc-ꢀAla-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-
Gly-OH, Fmoc-His(Mtt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and
Fmoc-Val-OH.
Peptide Cyclization. On-resin cyclization was carried out at the
bench with solvents freshly deoxygenated by bubbling with nitrogen
for at least 5 min. A CuI solution (usually 5 mM, but 20 mM was
used in early experiments) was prepared using a solvent mixture
of 20% DMSO in MeCN. Acetonitrile was required for solublizing
the copper salt, while DMSO was crucial for resin swelling and
peptide accessibility. A 4 mL vial was charged with 50 mg of
peptide-loaded resin, to which was added 2 mL of a CuI solution
and 5 µL of 2,6-lutidine. Cyclizations on a larger scale were carried
out in 20 or 40 mL scintillation vials as required. Nitrogen was
gently bubbled through the mixture for 3 min, and the vial was
capped, sealed with parafilm or electrical tape, and gently agitated
on a rotisserie at room temperature for 16 or 40 h. After the reaction,
the resin was washed successively with DMF, MeCN, and water.
The resin was then gently stirred in a saturated aqueous solution
of disodium EDTA (2 × 10 min) to remove residual bound copper,
followed by sequential washing with water, MeCN, CH2Cl2, and
diethyl ether, and drying in a vacuum desiccator.
Peptide Cleavage. Linear and cyclized peptides were cleaved
from the resin with 2% triisopropylsilane (TIS) in trifluoroacetic
acid (TFA, approximately 1 mL of TFA/TIS per g of resin) for
2.5 h. The cleavage mixture (including resin) was mixed with cold
ether to precipitate the peptide and then filtered. The filtrate was
washed with cold ether, and the peptide was extracted from the
residue with H2O/MeCN mixtures (typically 5-20% MeCN in
water, 50% for peptoids) containing 0.1% TFA. The resulting
solution was frozen and lyophilized to afford a white, solid product.
A similar procedure was used for release of fully protected peptides
from the resin, using 4% TFA in CH2Cl2 for 4 h. The peptide was
precipitated from cold ether, filtered, extracted with 20% MeOH
in CH2Cl2, evaporated to dryness, and purified by preparatory HPLC
to give a white powder.
× 150 mm column (5 µm particle size), with detection at 220 nm.
Gradients of 0-35% or 15-50% MeCN in H2O over 20 min (with
constant 0.1% TFA) were used. Preparatory HPLC was performed
at 10 mL/min on a Dynamax SD-1 instrument using a C18 column
(22 × 250 mm, 10 µm particle size) and MeCN/H2O with 0.1%
TFA gradients as needed. MALDI mass spectra were obtained.
Peptoid Synthesis. N-Substituted glycine oligomers (peptoids)
were synthesized on solid-phase support using modified submono-
mer synthesis protocols reported previously by Zuckermann et al.66
Peptoid oligomers were synthesized by manual techniques as well
as automated procedures on a robotic peptide synthesizer with
software program files written in-house. All reactions were
completed at room temperature.
Rink amide resin (100 mg, 0.69 mmol/g) was swollen in 3 mL
of CH2Cl2 for 45 min before Fmoc deprotection. Typically, the resin
was washed with DMF (4 × 2 mL) and CH2Cl2 (3 × 2 mL)
between each synthetic step described below. Stoichiometries are
based on the loading of the starting resin functional groups, that
is, assuming quantitative yield at each coupling step. Fmoc
deprotection was performed by treatment of the resin with 20%
piperidine in DMF (20 min, 15 mL per g of resin). The resin was
washed, and 20 equiv of bromoacetic acid (1.2 M in DMF, 8.5 mL
per g of resin) and 24 equiv DIC (2 mL per g of resin) were added.
The reaction mixture was agitated at room temperature for 20 min.
After another resin wash, 20 equiv of the monomer amine (1.0 M
in DMF, 10 mL per g of resin) was added, and the reaction mixture
was shaken for 20 min at room temperature. Subsequent bromoa-
cylation and amine displacement steps were performed to prepare
peptoids of desired length.
Linear and cyclized peptoid products were cleaved from the solid
support by treatment with 50% TFA in CH2Cl2 (40 mL per g of
resin) for 10 min at room temperature. The cleavage cocktail was
evaporated under nitrogen gas flow, and the products were
resuspended at an approximate concentration of 2 mM in HPLC
solvent (50% acetonitrile in water, with 0.1% TFA) for analysis
by RP-HPLC and electrospray ionization mass spectrometry.
Acknowledgment. This work was supported by The Skaggs
Institute for Chemical Biology, the National Institutes of Health
(CA112075, GM083658, and GM062116), and the National
Science Foundation (CAREER CHE-0645361 to K.K.). The
authors are grateful to Professor Philip Dawson and co-workers
for helpful discussions and advice.
Supporting Information Available: Experimental details and
representative HPLC and MALDI mass spectrometry data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO802097M
Analytical Methods. Peptides were analyzed by reversed-phase
analytical HPLC (RP-HPLC) at a 1.0 mL/min flow rate and a 4.7
(66) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am.
Chem. Soc. 1992, 114, 10646–10647.
2974 J. Org. Chem. Vol. 74, No. 8, 2009