Synthesis of constrained head-to-tail cyclic tetrapeptides by an imine-induced ring-closing/contraction strategy

Article abstract of DOI:10.1002/anie.201304773

Making heads or tails of it: A strategy involving a head-to-tail imine-captured ring closure followed by ring contraction was used to synthesize otherwise difficult cyclic tetrapeptides. Compared with the direct lactamization process, the estimated activa

Full text of DOI:10.1002/anie.201304773

Angewandte
Chemie
DOI: 10.1002/anie.201304773
Peptide Cyclization
Synthesis of Constrained Head-to-Tail Cyclic Tetrapeptides by an
Imine-Induced Ring-Closing/Contraction Strategy**
Clarence T. T. Wong, Hiu Yung Lam, Tao Song, Guanhua Chen,* and Xuechen Li*
Head-to-tail cyclic peptides exhibit remarkable biological
activities with extraordinary potency and oral-bioavailability
owing to their compacted structures and high stability against
enzymatic degradation and physical denaturation; thus, cyclic
peptides have attracted a lot of attention in the pharmaceut-
[
1,2]
ical industry.
In particular, cyclic peptides with small-sized
rings have a drug-like scaffold that is ideal for therapeutic
1
–3
[3]
Figure 1. Synthesis of cyclic tetrapeptides. R =amino acid side
chains.
development. A range of cyclic tetrapeptides have been
shown to exhibit a wide spectrum of biological activities,
including the cytotoxic agents trapoxin and hirsutide, the
opioid receptor-binding CJ-15208, the antiprotozoal apicidin,
[
4]
and the antifungal rhodopeptins.
The exploration of cyclic peptides for medicinal chemistry
[5,7]
requires efficient synthetic methods.
In principle, lactam-
ization of linear peptides can be achieved by the standard
[6]
dehydration reagent-mediated reactions for coupling pep-
tides. However, peptide cyclization often proceeds more
slowly than a regular intermolecular peptide coupling reac-
tion and, therefore, is accompanied by the competing
[
7]
dimerization and oligomerization reactions. Because of the
constrained geometry of the peptide backbone, small cyclic
peptides (less than six amino acid residues) present a chal-
[
7,8]
lenge for chemical peptide cyclization.
synthesis of cyclic tetrapeptides with rigid 12-membered ring
Particularly, the
[14]
structures rely on: 1) the use of external conformational
assistance and 2) the introduction of internal turn-inducing
[
9]
residues, such as glycine, proline, d-amino acids, tertiary
[8a,10]
[11]
amide,
or pseudoproline. Therefore, synthesis of tetra-
peptides comprising any of the regular l-amino acids without
a pre-organized structure is still considered a challenging, if
[
7,10]
not impossible, task.
Herein, we describe the synthesis of
Scheme 1. Two possible pathways for the proposed tetrapeptide cycli-
zation using serine and threonine ligation.
cyclic tetrapeptides through an intramolecular serine/threo-
[12,13]
nine ligation (Figure 1),
which uses an imine-induced
ring-closure contraction to decrease the activation energy
barrier, thus enabling tetrapeptide cyclization.
We hypothesized that the formation of the head-to-tail
imine (2) with a 16-membered ring structure would be
relatively more favorable than that for a peptide with a 12-
membered ring (Scheme 1). Subsequently, the intramolecular
ring contraction by way of an O!N acyl transfer to afford the
N,O-benzylidene acetal intermediate (4) through intermedi-
ate 3 or 3’ would be made possible by an increase in enthalphy,
resulting from the amide bond formation. Then the resulting
acetal group could be removed to form the cyclic tetrapeptide
with a natural peptidic bond. We were also aware that the four
bonds introduced are not flexible (i.e., the ortho-substituents
on the benzene with a fixed angle), thereby likely not
reducing the strain energy very much during the ring
[
+]
[+]
[+]
[
*] Dr. C. T. T. Wong, H. Y. Lam, Dr. T. Song, Prof. Dr. G. Chen,
Prof. Dr. X. Li
Department of Chemistry, The University of Hong Kong
Pokfulam Road, Hong Kong (P.R. China)
E-mail: ghc@yangtze.hku.hk
[15]
xuechenl@hku.hk
contraction step, which challenges our hypothesis.
+
Our first task was to devise an efficient strategy to prepare
the requisite peptide salicylaldehyde (SAL) esters. Owing to
the lability of the O-SAL ester during the process of Fmoc-
based solid-phase peptide synthesis (SPPS), we previously
introduced an Fmoc-SPPS of peptide SAL esters using on-
[
] These authors contributed equally to this work.
[**] This work was supported by the General Research Fund
(
HKU707412P) of the Research Grants Council of Hong Kong.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201304773.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
[
12]
resin phenolysis.
Herein, we developed a Boc-SPPS of
crude cyclization mixture and tried to pinpoint the ratio of the
monomeric product to the dimerized cyclic product.
peptide SAL esters. Towards this goal, we elected to link the
aldehyde group of salicylaldehyde to the resin through an
alkene linker, which could be restored to the aldehyde group
by ozonolysis (Scheme 2).
The obtained peptide SAL esters were dissolved in
a pyridine–acetic acid mixture (molar ratio 1:2) at a concen-
tration of 1 mm. Gratifyingly, these peptide SAL esters
cylclized smoothly and most of the cyclizations were complete
within four hours. The desired monocyclization was observed
as the major reaction product, with ratios of cyclomonomer to
cyclodimer from 3:2 to 9:1. Notably, the cyclization of the
sequence SYIA-SAL ester produced almost exclusively the
cyclomonomer product (Table 1, entry 6). Following acid-
Table 1: Synthesis of a series of cyclic tetrapeptides.
[
b]
Entry
Sequence
Mass [Da]
Monomer:Dimer
[
a]
1
2
3
4
5
6
7
8
9
cyclo-(SAAA)
cyclo-(TALL)
cyclo-(TLLA)
cyclo-(SHIF)
cyclo-(TVVA)
cyclo-(SYIA)
cyclo-(SFIA)
cyclo-(TINA)
cyclo-(TKLA)
404
398
398
484
370
434
418
399
413
4:1
7:3
9:1
3:2
9:1
99:1
9:1
3:2
3:2
Scheme 2. Synthesis of peptide SAL esters using aminomethyl resin
(
sphere). PG=protecting group; THF=tetrahydrofuran; TFA=tri-
fluoroacetic acid; PyBOP=benzotriazol-1-yl-oxytripyrrolidinophospho-
nium hexafluorophosphate; DIEA=N,N-diisopropylethylamine;
DMAP=4-dimethylaminopyridine; DMF=dimethylformamide;
TMSOTf=trifluoromethanesulfonic acid trimethylsilylester.
[a] The cyclized product with N,O-benzylidene acetal was isolated by
reverse-phase HPLC. The cyclic product after acidolysis co-eluted with
the solvent in HPLC. [b] The ratio was determined by analytical LC–MS.
To this end, the hydroxy group of salicylaldehyde was first
protected with an acetate group affording 6, followed by the
Wittig reaction with 7 to form 8. The liberated carboxylic acid
upon TFA treatment was coupled to the aminomethyl resin
with PyBOP, generating 9. The phenol group, after removal of
the acetate group, became amenable to Boc-SPPS. After the
resin-bound peptide (10) was synthesized, TMSOTf/TFA/
thioanisole was used to remove the side-chain protecting
groups if present, while keeping the peptide intact on the
resin. Lastly, the aldehyde was regenerated by ozonolysis and,
at the same time, the peptide SAL ester (11) was released
from the resin (Scheme 2). According to this method, we
prepared and purified, by reverse-phase HPLC, a series of
tetrapeptide SAL esters with an N-terminal serine or
threonine in overall yields of 9–21%, based on resin loading.
The obtained tetrapeptide SAL esters were very stable and
could be stored at room temperature without any decom-
position. This strategy thus provides a reliable and facile
solution to the preparation of peptide SAL esters. Admit-
tedly, ozone is an extremely reactive oxidizing species, which
caused oxidation of some amino acid residues. In our studies,
we observed that Cys, Met, and Trp were affected by this
oxidation.
olysis with TFA/H O, the cyclic tetrapeptides were purified by
2
reverse-phase HPLC and characterized by NMR spectrosco-
py. In comparison, the conventional lactamization conditions
(HATU, DIEA, DMF; DEPBT, DIEA, DMF; PyBOP,
DIEA, DMF) to cyclize TLLA produced none or trace
amounts of the desired cyclic peptides.
The unprotected amino acid residues, including tyrosine,
histidine, asparagine, and lysine, did not cause any apparent
side reactions during the cyclization. In particular, the direct
aminolysis of peptide salicylaldehyde esters, a great concern
of ours, by the N-terminus or the internal amino group (e.g.
lysine) if present, did not occur. This serine/threonine-
mediated cyclization involves two steps. The first step is the
formation of the cyclic acetal intermediate (4 in Scheme 1).
During this step, if the unwanted head-to-side-chain or head-
to-tail cyclization resulting from direct aminolysis were to
occur, the resulting product would have a different molecular
weight and physical properties from the head-to-tail N,O-
benzylidene acetal linked cyclic product, thereby allowing for
easy differentiation and separation. We carefully analyzed the
crude reaction mixture by LC–MS and did not observe any
such undesired products. The second step uses acidolysis to
remove the N,O-benzylidene acetal group, affording the
desired head-to-tail cyclic product with a natural peptidic
bond.
[
17]
To exclude the possibility of a pre-organized conforma-
tion induced by turn-inducing residues, our peptide sequences
were randomly selected, while intentionally avoiding glycine,
proline, or d-amino acids as the turn-inducing element. We
realized that the ring strain inherent in head-to-tail peptide
cyclization, in particular those of small sizes, would favor the
Another important issue was epimerization at the cycli-
zation site. Thus, we synthesized both a SYIA-SAL ester and
the epimeric SYIa-SAL ester to study their cyclization
products. Both cyclizations proceeded well, without epimeri-
zation at the C-terminal alanine site (Figure 2).
[5a,8a,10a]
cyclodimerization reaction.
We carefully analyzed the
2
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Scheme 1 (with R = H). Formation of the intermediate 2
through imine capture is thermodynamically favored (DG)
over the reactant 1 and the product cyclo-(SAAA) by 7.5 kcal
ꢀ1
and 4.7 kcalmol , respectively. Next, formation of oxazoli-
dine intermediate 3 has the enthalpy and free energy changes
ꢀ
1
ꢀ1
of 1.5 kcalmol and ꢀ5.3 kcalmol , respectively, over 1. In
2
contrast, the intermediate 3’ generated by the sp -hybridized
N atom induced acyl transfer is thermodynamically hindered
ꢀ1
with enthalpy and free energy changes of 27.4 kcalmol and
ꢀ1
1
8.7 kcalmol , respectively (Figure S29). Compared to 1, the
formation of intermediate 3 brings the nitrogen atom of the
N-terminus closer and the carbon atom of the C-terminal
carboxylic; their distances in 1 and 3 are 6.94 ꢀ and 3.97 ꢀ,
respectively. This prepares it for the subsequent intramolec-
ular ring contraction by way of an O!N acyl transfer.
The resultant N,O-benzylidene acetal intermediate 4 has
ꢀ
1
the lowest enthalpy and free energy of ꢀ2.3 kcalmol and
ꢀ1
ꢀ
9.3 kcalmol over 1, respectively. This ensures that the
whole reaction stops at the stable intermediate 4 prior to
acidolysis. Although, the ring size decreases from 16-member
to the less-stable 12-member ring during the process of 3!4,
the enthalpy gained from the amide bond formation com-
pensates and surpasses the destabilization caused by the ring
strain. Notably, the process 1!4 is thermodynamically
favorable over the cyclization of tetrapeptides with turn-
inducing residues (APAA, AAN-CH3AA, AaAA) or penta-
peptide (AAAAA), with DG for these ranging from
Figure 2. HPLC analysis of the crude cyclization mixture of linear
peptides SYIA-SAL ester and SYIa-SAL ester. No epimerization at the
cyclization site was observed.
ꢀ1
ꢀ1
To gain insight into the mechanism of cyclization of
tetrapeptide-SAL esters as well as understand the enabling
factors, we performed the molecular modeling studies to
address two critical issues: 1) which pathway 2!3!4 versus
ꢀ5.1 kcalmol to ꢀ6.2 kcalmol (Table S1).
We estimated the activation energies for various cycliza-
tion processes using a previously implemented method (Fig-
[15a]
ure S30).
The calculated activation energies for the imine
2
!3’!4 is favored, and 2) what is the factor that contributes
capture (Figure 3a) and the intramolecular ring contraction
ꢀ
1
ꢀ1
to a lower energy barrier for the tetrapeptide cyclization using
the intramolecular Ser/Thr ligation strategy.
(Figure 3b) are 7.0 kcalmol and 6.7 kcalmol , respectively,
which are similar to those for amide bond formation in the
cyclization of the unsubstituted linear pentapeptide and its
auxiliary-linked equivalents, with values 6.2–7.6 kcal
As shown in Scheme 1, the N,O-benzylidene acetal
intermediate (4) is chemoselectively generated between
a salicylaldehyde and the N-terminal serine or threonine
through imine capture followed by ring formation (5-endo-
ꢀ
1 [15a]
mol .
In contrast, the direct amide bond formation in
the cyclization of both unsubstituted tetrapeptide SAAA
(Figure 3c) and its SAL equivalent (Figure 3d) have sub-
[
16]
trig) by way of an anti-Baldwin cyclization of the a-hydroxy
3
group of Ser/Thr, generating an sp -hybridized N atom (as in
3
) to initiate acyl transfer. Alternatively, it is also likely that
2
an sp -hybridized N atom could induce acyl transfer forming
[
16]
3’, followed by a 5-exo-trig pro-Baldwin cyclization.
Table 2 summarizes the enthalpy changes (DH) and free
energy changes (DG) of cyclization of the tetrapeptide
SAAA-SAL ester using the serine ligation shown in
[
a]
Table 2: Thermodynamic parameters for cyclization of tetrapeptides.
[
b]
ꢀ1
[c]
ꢀ1
Species
DH [kcalmol
]
DG [kcalmol
]
2
3
1.0
1.5
27.4
ꢀ2.3
6.8
ꢀ7.5
ꢀ5.3
18.7
3
4
5
’
ꢀ9.3
ꢀ2.8
ꢀ1
[
a] Change in enthalpy (DH) and free energy (DG) of the cyclization of
Figure 3. Estimated activation energies (kcalmol ) for cyclizations of
(a) SAL ester tetrapeptide SAAA forming a Schiff base; (b) pseudopro-
line structure (3); (c) direct lactamization of tetrapeptide SAAA;
(d) direct aminolysis of the tetrapeptide SAAA-SAL ester.
tetrapeptide SAAA-SAL ester using serine ligation in acetic acid at
2
98.15 K and 1 atm. [b] DH(x)=H(x)+H(H O)ꢀH(1).
2
[c] DG(x)=G(x)+G(H O)ꢀG(1).
2
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
ꢀ
1
ꢀ1
stantially higher values of 14.3 kcalmol and 14.8 kcalmol ,
respectively. This is consistent with the previous observation
that the head-to-tail tetrapeptide cyclization is difficult to
realize using a conventional approach, and indicates that the
high energy barrier in the cyclization can be bypassed by an
imine-induced ring-closing/ring-contraction strategy using
tetrapeptide SAL esters.
d) A. S. Norgren, F. Bꢁttner, S. Prabpai, P. Kongsaeree, P. I.
Arvidsson, J. Org. Chem. 2006, 71, 6814.
5] For reviews, see: a) J. S. Davies, J. Pept. Sci. 2003, 9, 471; b) S.
Jiang, Z. Li, K. Ding, P. P. Roller, Curr. Org. Chem. 2008, 12,
[
1502; c) S. R. Gracia, K. Gaus, N. Sewald, Future Med. Chem.
2009, 1, 1289; d) Y. Hamada, T. Shioiri, Chem. Rev. 2005, 105,
4441; e) J. P. Tam, C. T. T. Wong, J. Biol. Chem. 2012, 287, 27020;
f) J. N. Lambert, J. P. Mitchell, K. D. Roberts, J. Chem. Soc.
Perkin Trans. 1 2001, 471.
In conclusion, we demonstrated the applicability of
salicylaldehyde ester-induced ligation at serine/threonine
sites in cyclizing small peptides. We first developed an
efficient approach to prepare the requisite peptide salicylal-
dehyde ester using Boc-SPPS. Then, several cyclic tetrapep-
tides, which did not contain any turn-inducing amino acid
residue, were synthesized accordingly. On the basis of
molecular-modeling investigations, we concluded that, com-
pared to the conventional head-to-tail lactamization
approach, intramolecular Ser/Thr ligation can bypass the
high energy barrier, which is needed to overcome the
disfavored backbone strain, by a quick and chemoselective
ring-closing capture, followed by ring contraction using an O-
to-N acyl transfer. Finally, after acidolysis, a head-to-tail
amide-bonded cyclic tetrapeptide with natural peptidic bonds
was synthesized. During the cyclization, epimerization at the
ligating C-terminus was not observed. We will extend our
studies to synthesis of cyclic peptides with different sizes using
intramolecular Ser/Thr ligation.
[
[
[
6] A. El-Faham, F. Albericio, Chem. Rev. 2011, 111, 6557.
7] C. J. White, A. K. Yudin, Nat. Chem. 2011, 3, 509.
8] a) M. El Haddadi, F. Cavelier, E. Vivies, A. Azmani, J. Verducci,
J. Martinez, J. Pept. Sci. 2000, 6, 560; b) D. Skropeta, K. A.
Jolliffe, P. Turner, J. Org. Chem. 2004, 69, 8804; c) J. Lꢂcaillon, P.
Gilles, G. Subra, J. Martinez, M. Amblard, Tetrahedron Lett.
2008, 49, 4674; d) J.-S. Zheng, S. Tang, Y. Guo, H.-N. Chang, L.
Liu, ChemBioChem 2012, 13, 542; e) F. Cavelier-Frontin, S.
Achmad, J. Verducci, R. Jacquier, G. Pꢃpe, THEOCHEM 1993,
286, 125; f) M. Malesevic, U. Strijowski, D. Bꢄchle, N. Sewald, J.
Biotechnol. 2004, 112, 73; g) A. Ehrlich, H.-U. Heyne, R. Winter,
M. Beyermann, H. Haber, L. A. Carpino, M. Bienert, J. Org.
Chem. 1996, 61, 8831.
[9] a) D.-F. Tai, Y.-F. Lin, Chem. Commun. 2008, 5598; b) K. Hass,
W. Ponikwar, H. Nçth, W. Beck, Angew. Chem. 1998, 110, 1200;
Angew. Chem. Int. Ed. 1998, 37, 1086.
10] a) U. Schmidt, J. Langner, J. Peptide Res. 1997, 49, 67; b) V. D.
Bock, R. Percianccante, T. P. Jansen, H. Hiemstra, J. H. Van
Maarseveen, Org. Lett. 2006, 8, 919; c) E. Cini, C. B. Botta, M.
Rodriquez, M. Taddei, Tetrahedron Lett. 2009, 50, 7159; d) Y.
Takeuchi, G. R. Marshall, J. Am. Chem. Soc. 1998, 120, 5363;
e) J. Pastuszak, J. H. Gardner, J. Singh, D. H. Rich, J. Org. Chem.
1982, 47, 2982.
[
Received: June 3, 2013
Published online: && &&, &&&&
[11] K. A. Fairweather, N. Sayyadi, I. J. Luck, J. K. Clegg, K. A.
Jolliffe, Org. Lett. 2010, 12, 3136.
[
12] a) Y. Zhang, C. Xu, H. Y. Lam, C. L. Lee, X. Li, Proc. Natl.
Acad. Sci. USA 2013, 110, 6657; b) X. Li, H. Y. Lam, Y. Zhang,
C. K. Chan, Org. Lett. 2010, 12, 1724.
13] H. Y. Lam, Y. Zhang, H. Liu, J. Xu, C. T. Wong, C. Xu, X. Li, J.
Am. Chem. Soc. 2013, 135, 6272.
14] H. Kawai, R. D. Jasensky, D. H. Rich, J. Am. Chem. Soc. 1983,
Keywords: activation energy · amino acids ·
cyclic tetrapeptides · cyclization
.
[
[
[
[
1] a) D. J. Craik, N. M. Allewell, J. Biol. Chem. 2012, 287, 26999;
b) M. Katsara, T. Tselios, S. Deraos, G. Deraos, M. T. Matsoukas,
E. Lazoura, J. Matsoukas, V. Apostolopoulos, Curr. Med. Chem.
105, 4456.
15] Previous examples using an auxilliary-mediated ring-closure/
ring-contraction strategy with significant epimerization:
a) W. D. F. Meutermans, S. W. Golding, G. T. Bourne, L. P.
Miranda, M. J. Dooley, P. F. Alewood, M. L. Smythe, J. Am.
Chem. Soc. 1999, 121, 9790; b) W. D. F. Meutermans, G. T.
Bourne, S. W. Golding, D. A. Horton, M. R. Campitelli, D.
Craik, M. Scanlon, M. L. Smythe, Org. Lett. 2003, 5, 2711;
c) J. P. A. Rutters, Y. Verdonk, R. de Vries, S. Ingemann, H.
Hiemstra, V. Levacher, J. H. van Maarseveen, Chem. Commun.
2006, 13, 2221.
[
[
2] E. M. Driggers, S. P. Hale, J. Lee, N. K. Terrett, Nat. Rev. Drug
Discovery 2008, 7, 608.
3] a) D. A. Horton, G. T. Bourne, M. L. Smythe, Chem. Rev. 2003,
1
03, 893; b) J. M. Beierle, W. S. Horne, J. H. van Maarseveen, B.
Waser, J. C. Reubi, M. R. Ghadiri, Angew. Chem. 2009, 121,
819; Angew. Chem. Int. Ed. 2009, 48, 4725.
4
[
4] a) M. Kijima, M. Yoshida, K. Sugita, S. Horinouchi, T. Beppu, J.
Biol. Chem. 1993, 268, 22429; b) T. Saito, H. Hirai, Y. J. Kim, Y.
Kojima, Y. Matsunaga, H. Nishida, T. Sakakibara, O. Suga, T.
Sujaku, N. Kojima, J. Antibiot. 2002, 55, 847; c) H. Chiba, H.
Agematu, K. Dobashi, T. Yoshioka, J. Antibiot. 1999, 52, 700;
2012, 48, 8084.
[
[
16] K. Gilmore, I. V. Alabugin, Chem. Rev. 2011, 111, 6513.
17] Arg(Tos) cannot be cleanly deprotected.
4
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Peptide Cyclization
C. T. T. Wong, H. Y. Lam, T. Song,
G. Chen,* X. Li*
&&&& — &&&&
Synthesis of Constrained Head-to-Tail
Cyclic Tetrapeptides by an Imine-Induced
Ring-Closing/Contraction Strategy
Making heads or tails of it: A strategy
involving a head-to-tail imine-captured
ring closure followed by ring contraction
was used to synthesize otherwise difficult
cyclic tetrapeptides. Compared with the
direct lactamization process, the esti-
mated activation energies for the cyclic
imine formation and the ring contraction
ꢀ
1
were lowered by 7.3 and 7.6 kcalmol ,
respectively, which enables cyclization.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!