Synthesis of All- L cyclic tetrapeptides using pseudoprolines as removable turn inducers

Article abstract of DOI:10.1021/ol101018w

(Figure Presented) Cyclic tetrapeptides have generated great interest because of their broad-ranging biological properties. In order to synthesize these highly strained 12-membered cyclic compounds, a cyclization strategy using pseudoprolines as removable turn inducers has been developed. The pseudoproline derivatives induce a cisoid amide bond in the linear peptide backbone which facilitates cyclization. After cyclization, the turn inducers can be readily removed to afford cyclic tetrapeptides containing serine or threonine residues.

Full text of DOI:10.1021/ol101018w

ORGANIC
LETTERS
2010
Vol. 12, No. 14
3136-3139
Synthesis of All-L Cyclic Tetrapeptides
Using Pseudoprolines as Removable
Turn Inducers
Kelly A. Fairweather, Nima Sayyadi, Ian J. Luck, Jack K. Clegg, and
Katrina A. Jolliffe*
School of Chemistry, The UniVersity of Sydney, 2006 NSW, Australia
kate.jolliffe@sydney.edu.au
Received May 4, 2010
ABSTRACT
Cyclic tetrapeptides have generated great interest because of their broad-ranging biological properties. In order to synthesize these highly
strained 12-membered cyclic compounds, a cyclization strategy using pseudoprolines as removable turn inducers has been developed. The
pseudoproline derivatives induce a cisoid amide bond in the linear peptide backbone which facilitates cyclization. After cyclization, the turn
inducers can be readily removed to afford cyclic tetrapeptides containing serine or threonine residues.
The significant biological activity displayed by cyclic pep-
tides, together with their potential as biomaterials,1 makes
these compounds of great interest. In particular, cyclic
tetrapeptides are considered to be naturally occurring “privi-
leged structures”2 as they are able to present substituents in
a spatially well-defined manner and display a variety of
biological activities, including histone deacetylase inhibition,3
tyrosinase inhibition,4 antimitogenic activity,5 antitumor
activity,6 and antimalarial acivity.7 However, the synthesis
of head-to-tail cyclic tetrapeptides is generally difficult as a
result of their highly constrained 12-membered ring struc-
ture.8 Cyclization of a linear precursor frequently yields the
cyclic dimer (a cyclic octapeptide), rather than the required
cyclic monomer,9 indicating that the primary hurdle to
cyclization is bringing the N- and C-termini of the peptide
into close spatial proximity. This can be attributed to the
strong π-character of peptide bonds, which tend to adopt a
transoid conformation, resulting in an extended linear peptide
conformation.
(1) Hartgerink, Clark, T. D.; Ghadiri, M. R. Chem.sEur. J. 1998, 4,
1367.
The synthesis of all-L cyclic tetrapeptides is particularly
challenging; almost all of the cyclic tetrapeptides synthesized
to date contain either a mixture of L- and D-amino acids,
(2) Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. ReV. 2003, 103,
893.
(3) Norgren, A. S.; Bu¨ttner, F.; Prabpai, S.; Kongsaeree, P.; Arvidsson,
P. I. J. Org. Chem. 2006, 71, 6814.
(4) Kawagishi, H.; Somoto, A.; Kuranari, J.; Kimura, A.; Chiba, S.
Tetrahedron Lett. 1993, 34, 3439.
(7) Singh, S. B.; Zink, D. L.; Polishook, J. D.; Dombrowski, A. W.;
Darkin-Rattray, S. J.; Schamtz, D. M.; Goetz, M. A. Tetrahedron Lett. 1996,
37, 8077.
(5) (a) Kawai, M.; Jasensky, R. D.; Rich, D. H. J. Am. Chem. Soc. 1983,
105, 4456. (b) Sta¨helin, H.; Trippmacher, A. Eur. J. Cancer 1974, 10, 801.
(c) Pringle, R. B. Plant Physiol. 1970, 46, 45.
(6) Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.; Beppu, T.
J. Biol. Chem. 1993, 268, 22429.
(8) Cavelier-Frontin, F.; Achmad, S.; Verducci, J.; Jacquier, R.; Pe`pe,
G. THEOCHEM 1993, 286, 125.
(9) El Haddadi, M.; Cavelier, F.; Vives, E.; Azmani, A.; Veducci, J.;
Martinez, J. J. Peptide Sci. 2000, 6, 560.
10.1021/ol101018w  2010 American Chemical Society
Published on Web 06/21/2010

unnatural amino acids or at least one Pro residue.9-12 While
there have been several reported syntheses of all-L cyclic
tetrapeptides containing two Pro residues,4,13 most of these
have since been identified to yield the cyclic octapeptides,10
and the most recent example provided the all-L cyclic
tetrapeptide cyclo(Leu-Pro-Leu-Pro) in only 5% yield after
optimization of the cyclization conditions.9 Recently, the use
of a photolabile ring-contraction auxiliary to synthesize a
small library of all-L cyclic tetrapeptides, with yields ranging
from 4 to 29%, has been reported.14,15 However, while this
two-step cyclization/ring contraction procedure provides all-L
cyclic tetrapeptides from linear precursors in which Gly is
the C-terminal amino acid, when a chiral center is present
at the C-terminus significant epimerization is observed during
the cyclization step.
Scheme 1. Cyclization of Linear Tetrapeptides 1a-h
of all-L cyclic tetrapeptides. Both Ser (1a-d) and Thr (1e-h)
derived ΨMe,Me′pro residues were incorporated into the linear
peptides, alternating with “spacer” amino acids (Phe, Ile, Leu,
and Val), and in all cases, a ΨMe,Me′pro residue was
positioned at the C-terminus of the linear precursor.
We have recently reported the use of pseudoprolines
(ΨMe,Me′pro) derived from threonine residues as removable
turn-inducers to facilitate head-to-tail peptide cyclization.16,17
The incorporation of these oxazolidine derivatives into a
linear peptide favors cis-amide conformations at the amide
bonds N-terminal to the ΨR,R′pro residues.18-20 This facili-
tates cyclization by favoring a conformer in which the N-
and C-termini of the peptide are spatially proximate and was
found to provide significantly increased head-to-tail cycliza-
tion yields of hexa- and heptapeptides when compared to
those obtained for analogous peptides with standard Ser/Thr
protecting groups. After cyclization the ΨMe,Me′pro “protect-
ing” groups were readily removed by treatment with acid to
yield cyclic peptides devoid of turn inducers.16,17 To further
demonstrate the utility of this methodology, we now report
the synthesis of a small library of all-L cyclic tetrapeptides
that do not contain turn-inducers, facilitated by the use of
both serine and threonine derived ΨMe,Me′pro residues.
During our previous studies on the cyclization of hexapep-
tides containing alternating Val and Thr residues, we
observed that cyclization yields were significantly improved
if more than one ΨMe,Me′pro turn-inducer was incorporated
into the linear precursor and that the positioning of a
The required linear tetrapeptides 1a-h were prepared
using standard Fmoc solid-phase peptide synthesis (SPPS)
(HBTU/DIPEA couplings) by loading and coupling of the
commercially available dipeptides containing the modified
Ser or Thr residues. The 2-chlorotrityl chloride resin was
used as the solid support to enable cleavage of the linear
peptides from the solid phase, without removal of the side
chain protecting groups; optimized conditions involved
treatment with a mixture of hexafluoro-2-propanol, trifluo-
roethanol, and dichloromethane (1:2:7 v/v/v).
The 1H and 13C NMR spectra of the linear peptides 1a-d
and 1f-h exhibit predominantly a single set of resonances
(>90% peak intensity relative to other resonances as esti-
mated by integration of the 1H spectra), indicating that these
tetrapeptides predominantly adopt a single conformation in
CD3CN solution. In the case of 1e, a major and minor set of
resonances in a 7:3 ratio are clearly observed in both the 1H
and 13C NMR spectra. To identify the structure of the major
tetrapeptide conformers, compound 1c was investigated using
a combination of 1D and 2D NMR experiments. The major
conformer was determined to be that in which the amide
bonds preceding both the ΨMe,Me′pro residues adopt a cisoid
conformation on the basis of the typical cross-peaks observed
by 2D NMR ROESY and NOESY experiments (i.e.,
RHi-1-RHi crosspeaks) (Figure 1) that reflect the spatial
Ψ
Me,Me’pro residue as the C-terminal amino acid prevented
epimerization from occurring during the cyclization reac-
tion.16 We therefore chose the linear tetrapeptides 1a-h
(Scheme 1), which each incorporate two ΨMe,Me′pro residues
to investigate the utility of this methodology for the synthesis
(10) Schmidt, U.; Langner, J. J. Peptide Res. 1997, 49, 67
(11) Bock, V. D.; Percianccante, R.; Jansen, T. P.; Hiemstra, H.; Van
Maarseveen, J. H. Org. Lett. 2006, 8, 919
.
.
(12) Cini, E.; Botta, C. B.; Rodriquez, M.; Taddei, M. Tetrahedron Lett.
2009, 50, 7159–7161.
(13) Aracil, J.-M.; Badre, A.; Fadli, M.; Jeanty, G.; Banaigs, B.;
Francisco, C.; Lafargue, F.; Heitz, A.; Aumelas, A. Tetrahedron Lett. 1991,
32, 2609
.
(14) Horton, D. A.; Bourne, G. T.; Coughlan, J.; Kaiser, S. M.; Jacobs,
C. M.; Jones, A.; Ruhmann, A.; Tuner, J. Y.; Smythe, M. L. Org. Biomol.
Chem. 2008, 6, 1386
.
(15) Meutermans, W. D. F.; Bourne, G. T.; Golding, S. W.; Horton,
D. A.; Campitelli, M. R.; Craik, D.; Scanlon, M.; Smythe, M. L. Org. Lett.
2003, 5, 2711
.
(16) Skropeta, D. S.; Jolliffe, K. A.; Turner, P. J. Org. Chem. 2004, 69,
8804
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(17) Sayyadi, N.; Skropeta, D.; Jolliffe, K. A. Org. Lett. 2005, 7, 5497
(18) Wo¨hr, T.; Wahl, F.; Hefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.;
.
Figure 1. Structure of 1c with the observed NOEs indicated by
double-headed arrows.
Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218
.
(19) Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wo¨hr, T.;
Mutter, M. J. Am. Chem. Soc. 1997, 119, 918
Org. Lett., Vol. 12, No. 14, 2010
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3137

proximity of the RHi-1 and RHi protons in the cisoid form.21
The lack of any observable NOE cross peak between RH2
and RH3 suggests that the remaining amide bond adopts a
transoid configuration.
Further evidence that this “cis,trans,cis” conformer is the
major one adopted by the linear peptides 1a-h was obtained
upon derivitization of 1g to provide the Cbz and Me ester-
protected tetrapeptide 3 (Figure 2). As was observed for
Table 1. Cyclization Yields
yield with yield with
R1, R2
substrate product
Xa (%)
Ya (%)
R1 ) Ile, R2 ) H
R1 ) Leu, R2 ) H
R1 ) Phe, R2 ) H
R1 ) Val, R2 ) H
R1 ) Ile, R2 ) Me
R1 ) Leu, R2 ) Me
R1 ) Phe, R2 ) Me
R1 ) Val, R2 ) Me
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
47
38
45
40
24
27
40
35
73
66
60
60
20
31
57
20
1g
1h
2g
2h
a Yields reported are of RP-HPLC-purified products. X ) FDPP; Y )
DMTMM BF4.
pounds 1b (38%) and 1f (27%) can be directly compared to
that reported previously for H2N-Leu-Pro-Leu-Pro-OH (5%),9
indicating that both the Ser- and Thr-derived ΨMe,Me′pro
residues are more effective turn inducers than Pro itself.
Notably, in contrast to reported cyclizations of Pro containing
tetrapeptides, no products of cyclodimerization were ob-
served in any of the reactions performed in this study. In
1
addition, analysis of the H and 13C NMR spectra of cyclic
peptides 2a-f and 2h indicates that all of these compounds
have C2 symmetry, confirming that the cyclization reactions
occur with no epimerization of the C-terminal amino acid.
While compound 2g does not display the expected C2
symmetry, further reactions on this compound indicate that
this is due to the presence of a single asymmetric conformer
in solution rather than to loss of stereochemical integrity
during the cyclization reaction (see below).
Figure 2. Perspective drawing of the crystal strucutre of 3. Three
phenyl rings are represented with gray balls for clarity.
1
1a-h, the H and 13C NMR spectra of 3 exhibit predomi-
nantly a single set of resonances, indicating that 3 adopts a
single conformation in DMSO-d6 solution. A single-crystal
X-ray diffraction structure determination of 3 indicated that
in the solid state the amide bonds also adopt an alternate
“cis,trans,cis” conformation with torsional angles of 4.0(4)°,
-178(3)°, and 7.1(5)°, respectively, from the N- to C-
terminus.
In attempts to further improve the cyclization yields, a
second series of cyclization experiments were performed
using the same linear tetrapeptides but replacing FDPP with
the newer coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-
2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMM
BF4). This reagent has recently been found to provide good
coupling results (high yields, low epimerization) for a wide
range of amino acid and peptide substrates, including
fragment coupling and cyclization procedures.22 Treatment
of 1a-h with DMTMM BF4, under the same conditions as
described for FDPP above, gave the corresponding cyclic
tetrapeptides 2a-h in 20-73% yield (Table 1). Using this
method the yields for 2e-h, containing Thr-derived
Cyclization of 1a-h was initially performed using pen-
tafluorophenyl diphenylphosphinate (FDPP) as the coupling
reagent, as we have previously found this to give consistently
higher cyclization yields than other standard coupling
reagents (e.g., HATU, PyBOP). Thus, treatment of 1a-h
with FDPP and N,N-diisopropylethylamine (DIPEA) under
high dilution (0.005 M) in DMF for 3 days gave the
corresponding cyclic products 2a-h (Scheme 1) in 24-47%
yield (Table 1). In all cases, the linear precursors containing
Ser-derived ΨMe,Me′pro residues (1a-d) cyclized in higher
yield than the analogous compounds containing Thr-derived
Ψ
Me,Me′pro residues, were similar to those obtained using
FDPP (20-57% using DMTMM BF4, 24-40% using
FDPP). However, significant enhancements in cyclization
yield were observed for 2a-d (60-73% using DMTMM
BF4, 40-47% using FDPP) containing Ser-derived
Ψ
Me,Me′pro residues (1e-h), suggesting that the extra steric
Me,Me′pro residues.
bulk imparted by the additional methyl group at the ꢀ-posi-
tion of the threonine derivatives hinders the cyclization.
However, for both the Ser- and Thr-containing compounds,
the cyclization yields are pleasingly high when compared to
those previously reported for the synthesis of cyclic tet-
rapeptides.9,13 In particular, the cyclization yields for com-
Ψ
Removal of the pseudoproline “protecting” groups from
the tetrapeptides 2a-h was achieved upon treatment with a
standard peptide cleavage cocktail solution of TFA/triiso-
(21) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; John Wiley &
Sons, Inc.: New York, 1986; p 117.
(22) Kamin´ski, Z. J.; Kolesin´ska, B.; Kolesin´ska, J.; Sabatino, G.; Chelli,
M.; Rovero, P.; Blaszczyk, M.; Glo´wka, M. L.; Papini, A. M. J. Am. Chem.
Soc. 2005, 127, 16912.
(20) Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer, G. S.;
Mutter, M. J. Am. Chem. Soc. 1998, 120, 2714.
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Org. Lett., Vol. 12, No. 14, 2010

propylsilane (TIS)/water (95:2.5:2.5 v/v/v). The deprotection
reaction is slow (4 d), but analysis of the reaction mixtures
by LCMS indicated that after this time, complete conversion
to the Ser- and Thr-containing cyclic peptides 4a-h oc-
curred. Cyclic peptides 4a-h were obtained in moderate to
high yield after purification by RP-HPLC (Scheme 2). The
Table 2. Deprotection Yields
R1, R2
substrate
product
yielda (%)
R1 ) Ile, R2 ) H
R1 ) Leu, R2 ) H
R1 ) Phe, R2 ) H
R1 ) Val, R2 ) H
R1 ) Ile, R2 ) Me
R1 ) Leu, R2 ) Me
R1 ) Phe, R2 ) Me
R1 ) Val, R2 ) Me
2a
2b
2c
2d
2e
2f
4a
4b
4c
4d
4e
4f
80
79
quant
39
30
quant
53
50
Scheme 2
.
Removal of the Pseudoproline “Protecting” Groups
2g
2h
4g
4h
a Yields reported are of RP-HPLC-purified products.
tetrapeptides in overall yields of up to 60% from the linear
precursor. This was achieved by employing ΨMe,Me′pro
“protecting” groups to induce linear tetrapeptide conforma-
tions amenable to cyclization. Deprotection of the ΨMe,Me′pro
groups was carried out under standard conditions to afford
cyclic tetrapeptides devoid of turn inducers. Research is
currently underway to extend this methodology to allow the
synthesis of a wider range of cyclic peptides, including those
that lack Ser and Thr residues.
large variation in yields (30% to quant, Table 2) for the
deprotection reaction is attributable to the low solubility of
some of these compounds under the conditions required for
RP-HPLC.
In all cases, the 1H and 13C NMR spectra of the deprotected
cyclic tetrapeptides 4a-h indicate that the compounds have
C2 symmetry, confirming that the two-step cyclization,
deprotection sequence proceeds without epimerization, pro-
viding cyclic tetrapeptides devoid of turn-inducers.
In conclusion, despite their potential in therapeutic and
materials applications the use of cyclic tetrapeptides has been
limited, largely due to the synthetic difficulties enountered
when preparing such highly strained 12-membered cyclic
structures. The present study reports the synthesis of a
number of serine and threonine containing all-L cyclic
Acknowledgment. We thank the Australian Research
Council for financial support and for the award of a Queen
Elizabeth II research fellowship to K.A.J. We thank Samira
Leesch (USyd) for preliminary work.
Supporting Information Available: Experimental pro-
cedures and spectral data for all compounds. X-ray crystal-
lographic data for compound 3. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL101018W
Org. Lett., Vol. 12, No. 14, 2010
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