Facile synthesis of peptidyl salicylaldehyde esters and its use in cyclic peptide synthesis

Article abstract of DOI:10.1021/ol402279h

An efficient solid phase synthetic protocol for salicylaldehyde ester peptides is reported. With a Ser or Thr at the N-terminus, these salicylaldehyde ester peptides can be easily converted to Ser/Thr containing cyclic peptides.

Full text of DOI:10.1021/ol402279h

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Facile Synthesis of Peptidyl
Salicylaldehyde Esters and Its Use in
Cyclic Peptide Synthesis
Jun-Feng Zhao,*,† Xiao-Hong Zhang, Ying-Jie Ding, Yong-Sheng Yang, Xiao-Bao Bi,
and Chuan-Fa Liu*
Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang
Technological University, Singapore 637551
cfliu@ntu.edu.sg, zhaojf@hku.hk
Received August 10, 2013
ABSTRACT
An efficient solid phase synthetic protocol for salicylaldehyde ester peptides is reported. With a Ser or Thr at the N-terminus, these salicylaldehyde
ester peptides can be easily converted to Ser/Thr containing cyclic peptides.
Peptide head-to-tail cyclization generates an unusual
class of peptides with a circular backbone structure. With
their interesting structure and richness of pharmacology,
cyclic peptides have always been attractive targets for
synthetic chemistry.1 The classical methods for peptide
macrocyclization, which can be done either in solution or
on resin, involve the activation of the carboxyl group of
appropriately protected linear peptides. However, an in-
herent danger of enthalpic carboxyl activation is racemiza-
tion at the C-terminal residue. The advent of chemo-
selective peptide ligation methods2 has offered new ways
for peptide macrocyclization. These ligation methods fea-
ture a prior capture step that brings the reacting C- and
N-termini into close proximity, allowing spontaneous
peptide bond formation in the subsequent step. With this
entropy-driven activation scheme, the risks of racemiza-
tion are minimized and the use of protection groups is not
needed. In the late 1990s, Tam et al. first applied such
methods in cyclic peptide synthesis.3 Notably, the cysteine-
based nativechemical ligation (NCL)4 was used by them to
synthesize the macrocyclic cystine-knot peptides,3c which
has now become the method of choice for preparing Cys-
rich cyclic peptides. When the thiol-mediated ligation is
followed by a desulfurization step, the method can be
expanded to ligation at non-Cys residues.5 However,
(3) (a) Botti, P.; Pallin, T. D.; Tam, J. P. J. Am. Chem. Soc. 1996, 118,
10018. (b) Zhang, L. S.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363. (c)
Tam, J. P.; Lu, Y. A.; Yu, Q. T. J. Am. Chem. Soc. 1999, 121, 4316.
(4) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science
1994, 266, 776.
(5) (a) Rohde, H.; Seitz, O. Biopolym. (Pept. Sci.) 2010, 94, 397. (b)
Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064. (c) Botti, P.;
Tchertchian, S. Patent WO 2006/133962, 2006. (d) Haase, C.; Rohde, H.;
Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 6807. (e) Chen, J.; Wan, Q.;
Yuan, Y.; Zhu, J.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2008, 47,
8521. (f) Yang, R. L.; Pasunooti, K. K.; Li, F. P.; Liu, X. W.; Liu, C. F.
J. Am. Chem. Soc. 2009, 131, 13592. (g) Ajish Kumar, K. S.; Haj-Yahya,
M.; Olschewski, D.; Lashuel, H. A.; Brik, A. J. Angew. Chem., Int. Ed.
2009, 48, 8090. (h) Yan, L.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123,
523. (i) Thompson, R. E.; Chan, B.; Radom, L.; Jolliffe, K. A.; Payne,
R. J. Angew. Chem., Int. Ed. 2013, 52, 9723. (j) Malins, L R.; Cergol,
K. M.; Payne, R. J. ChemBioChem 2013, 14, 559.
† Department of Chemistry, The University of Hong Kong, Hong Kong.
(1) For reviews on cyclic peptides, see: (a) Clark, R. J.; Craik, D. J.
Biopolymers 2010, 94, 414. (b) White, C. J.; Yudin, A. K. Nat. Chem.
2011, 3, 509. (c) Jiang, S.; Li, Z.; Ding, K.; Roller, P. Curr. Org. Chem.
2008, 12, 1502. (d) Lambert, J. N.; Mitchell, J. P.; Roberts, K. A.
J. Chem. Soc., Perkin Trans. 1 2001, 5, 471. (e) Tam, J. P.; Wong, C. T. T.
J. Biol. Chem. 2012, 287, 27020. (f) Davies, J. S. J. Pept. Sci. 2003, 9, 471.
(2) For reviews on peptide ligation methods, see: (a) Tam, J. P.; Yu,
Q.; Miao, Z. Biopolymers 1999, 51, 311. (b) Hahn, M. E.; Muir, T. W.
Trends Biochem. Sci. 2005, 30, 26. (c) Hackenberger, C. P.; Schwarzer,
D. Angew. Chem., Int. Ed. 2008, 47, 10030. (d) Kent, S. B. H. Chem. Soc.
Rev. 2009, 38, 338. (e) Pattabiraman, V. R.; Bode, J. W. Nature 2011,
480, 471.
r
10.1021/ol402279h
XXXX American Chemical Society

except for Ala and Val, which are derived from Cys and
penicillamine respectively, few thiol-modified amino acids
are commercially available, making it synthetically chal-
lenging to use this approach.
Scheme 1. General Scheme for the Synthesis of C-Terminal
Salicylaldehyde (SAL) Ester Peptide
The aldehyde capture ligation by Tam et al. is one of the
earliest ligation methods developed.6 Conceptually similar
to the imine formation-initiated ligation first proposed by
Kemp,7 this method makes use of an N-terminal Cys, Ser
or Thr to react with a C-terminal glycoaldehyde ester
peptide to initially form a relatively stable thiazolidine or
oxazolidine ring. A subsequent intramolecular OꢀN acyl
transfer forms a new peptide bond with a pseudoproline
structure at the junction of the two peptide segments.6
However, the difficulty of regenerating the native amino
acid residue from the pseudoproline structure has limited
the use of this method. Very recently, this problem was
addressed by Li et al. by using an aromatic salicylaldehyde
(SAL) ester in lieu of the glycoaldehyde ester.8 The aux-
iliary benzylidene acetal group on the ligation product can
be easily cleaved by acidic hydrolysis to generate native
Ser/Thr. Since Ser and Thr are much more abundant than
Cys, this ligation method holds good promise in the
practice of protein and cyclic peptide synthesis.9 As ob-
viously seen from Li’s work, a bottleneck in using this
ligation scheme lies in the synthesis of the SAL ester
peptides. Although they have demonstrated that the
C-ter SAL ester can be introduced either though coupling
a protected peptide-COOH with acetal-protected salicy-
laldehyde in solution9a or through phenolysis of resin-
bound peptidyl-benzimidazolinone,9b the limitations of
these methods are that the solution synthesis suffers from
problems of inconvenience and racemization, which would
limit its use to C-ter Gly or Pro, whereas the on-resin
phenolysis method was inefficient and required long reac-
tion time (16 h, as reported). Clearly, solid phase peptide
synthesis (SPPS)10 would be the preferred method for the
synthesis of SAL ester peptides or their immediate pre-
cursors. An efficient SPPS method would greatly promote
the use of Ser/Thr-based ligation. Herein we report a
convenient solid phase synthesis method for SAL-ester
peptides and its application in the head-to-tail cyclization
of Ser or Thr-containing cyclic peptides.9c,d
The difficulty in the direct solid phase synthesis of
peptide-SAL esters is mainly attributed to the liability of
the phenolic ester and the reactivity of the aldehyde group.
Fmoc chemistry is excluded because the phenolic SAL
ester is labile to piperidine used in Fmoc SPPS. We thus
focused our efforts on developing a Boc-based method.
Since most protected forms of aldehyde are acid-sensitive
and would not survive in the strong acidic conditions
repetitively employed in Boc SPPS, we decided to use a
latent group that would be a precursor to the aldehyde
functionality. An ideal aldehyde precursor group must
satisfy several criteria. (1) It should be compatible with
the procedures of Boc SPPS. (2) After SPPS, the aldehyde
group must be generated easily in a highly selective reac-
tion without affecting other functional groups in the
peptide. (3) It should contain a linker functional group
for anchoring to the solid support.
After several attempts, we found that the commercially
available (E)-3-(2-hydroxyphenyl)acrylic acid (i.e., trans-
2-hydroxycinnamic acid) would fulfill these requirements.
The carboxylic acid group can be used to couple itself to an
amine-functionalized resin. The phenolic hydroxyl group
can be used to elongate the peptide chain. Most impor-
tantly, ozonolysis of the CdC bond willaffordanaldehyde
group under very mild conditions and with excellent selec-
tivity.
To test the feasibility of our proposal (Scheme 1), we
coupled (E)-3-(2-hydroxyphenyl)acrylic acid to MBHA
resin using PyBOP to prepare (E)-3-(2-hydroxyphenyl)
acrylyl-MBHA 1 (Scheme 1). Ninhydrin test indicated
completion of the coupling reaction in 1 h. Considering
the relative low reactivity of the phenolic hydroxyl group,
the loading of the first protected Boc-amino acid was
run overnight at room temperature. Peptide chain elonga-
tion was effected using standard Boc SPPS protocols
and monitored with ninhydrin test. The peptide was
cleaved from the resin using either TFMSA/TFA or HF
(Supporting Information). MS analysis clearly character-
ized the target peptide with a C-ter ester, 3, formed with
(E)-3-(2-hydroxyphenyl)acrylamide (Scheme 1). HPLC
(6) (a) Liu, C.-F.; Tam, J. P. Proc. Natl. Acad. Sci. U. S. A. 1994, 91,
6584. (b) Liu, C.-F.; Tam, J. P. J. Am. Chem. Soc. 1994, 116, 4149. (c)
Tam, J. P.; Miao, Z. J. Am. Chem. Soc. 1999, 121, 9013.
(7) For reviews, see: (a) Kemp, D. S. Biopolymer 1981, 20, 1793. (b)
Coltart, D. M. Tetrahedron 2000, 56, 3449. For seminal literatures, see:
(c) Kemp, D. S.; Wang, S. W.; Busby, S., III; Hugel, G. J. Am. Chem.
Soc. 1970, 92, 1043. (d) Kemp, D. S.; Vellaccio, J. F. J. Org. Chem. 1975,
40, 3003. (e) Kemp, D. S.; Gratten, J. A.; Reczek, J. J. Org. Chem. 1975,
40, 3465. (f) Kemp, D. S.; Kerkman, D. J.; Leung, S. L.; Hanson, G.
J. Org. Chem. 1981, 46, 490.
(8) Li, X. C.; Lam, H. Y.; Zhang, Y. F.; Chan, C. K. Org. Lett. 2010,
12, 1724.
(9) (a) Lam, H. Y.; Zhang, Y. F.; Liu, H.; Xu, J. C.; Wong, C. T. T.;
Xu, C.; Li, X. C. J. Am. Chem. Soc. 2013, 135, 6272. (b) Zhang, Y. F.;
Xu, C.; Lam, H. Y.; Lee, C. L.; Li, X. C. Proc. Natl. Acad. Sci. U. S. A.
2013, 110, 6657. (c) When we were about to submit our manuscript, a
new paper from the Li group was published online on the synthesis of the
SAL ester peptides, which is very similar to ours: Wong, C. T. T.; Lam,
H. Y.; Song, T.; Chen, G.; Li, X. Angew. Chem., Int. Ed. 2013, DOI:
10.1002/anie.201304773. (d) The SPPS method for the SAL ester peptides
reported herein is also the subject of a patent application by us: Liu, C.-F.;
Zhao, J. US. Pat. Appl. 61/595.853, 2012.
(10) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
Org. Lett., Vol. XX, No. XX, XXXX
B

profiles of the crude products (Supporting Information)
indicated that the purity of the target peptide was in the
range of 50ꢀ90%. Figure 1 shows two typical examples
of synthesized peptide acrylamide-phenolic esters 3. A
quantitative experiment was done on one of the ester pep-
tides, H-SIPLFPI-O-2-PhCHdCHCONH2, and the effi-
ciency of this protocol was shown with a 60% isolated yield
based on the MBHA resin loading (Supporting Information).
This indicates that the phenolic ester linkage between
the C-terminal carboxyl and (E)-3-(2-hydroxyphenyl)-
acrylamide is fully compatible with Boc SPPS.
Table 1. Synthesis of C-Terminal Salicylaldehyde Ester Peptides
via Ozonolysis
entry
peptide sequence
yield (%)a
1
GVGFA
82
85
60
75
89
80
65
60
76
45
56
72
67
55
71
42
2
SGLYGFAG
SGLFGFAG
TGLFGFAG
SGQAG
3
4
5
6
LGFAG
7
Ac-LGFAG
SLSL
8
9
Ac-ASPLFA
LEQKGFS
VGTESFA
SGKAFL
SFLFA
10
11
12
13
14
15
16
TFFGFFG
SIPLFPI
Figure 1. HPLC profiles of the crude peptides after cleavage.
(A) H-GVGFA-O-2-PhCHdCHCONH2 and (B) H-SIPLFPI-
O-2-PhCHdCHCONH2. The major peak is the desired
product.
SKSIPLFPI
a Isolated yield after HPLC purification on 5ꢀ10 mg reaction scale.
(entry 4, Table 2) prepared from its linear precursor,
H-SGLFGFAG-OPhCHO. The linear peptide was first
dissolved in 2,2,2-trifluoro ethanol (TFE) followed by
addition of pyridine/acetic acid (1:1) mixture, a condition
previously employed for oxazolidine formation in the
earlier work by Tam’s lab.6c To our delight, in 1 h at room
temperature, the cyclization of this peptide proceeded
cleanly to give a bicyclic N,O-benzylidene acetal 5 as the
major product as seen from the HPLC profile of the
reaction mixture (peak 1 in Figure 2, panel A). Remark-
ably, the concentration of the linear peptide used for
cyclization was 10 mM, which is rather high for an
intramolecular reaction, and no significant side products
were observed (Figure 2, panel A). The cyclic product 5
was purified by HPLC and further treated with a cocktail
solution of TFA/H2O/i-Pr3SiH (95: 2.5:2.5) togenerate the
final cyclic peptide 6 with a natural Gly-Ser peptide bond
at the cyclization junction in excellent yield. In a similar
manner, cyclization of H-SGKAFL-OPhCHO also led to a
clean reaction (Figure 2, panel B), and the isolated cyclic
acetal product was subsequently deprotected to afford
cyclo[SGKAFL] with Leu-Ser at the cyclization junction
(entry 1, Table 2). It is worth mentioning that, although
both cyclization and deprotection steps gave high con-
version yield in HPLC, the isolated yield was reduced
significantly (see Table 2) because of attrition on the
small amount (milligram scale) of material during HPLC
purification.
The key step of this strategy is the ozonolysis of the CdC
bond to generate the aldehyde group. We first carried out
ozonolysis using THF as the solvent, followed by addition
of dimethylsulfide as the reductant. Although ozonolysis
on H-GVGFA-OPhCHdCHCONH2 generated the de-
sired peptide-SAL ester 4 in 1ꢀ2 min at room temperature
(Scheme 1), the solubility of most peptides in THF is poor.
In this regard, a miscible water/organic solvent system
would be ideal for small-to-medium sized peptides. It was
reported that miscible water/organic solvent mixtures
could be used as the media for ozonolysis of CdC bond
in small organic compounds.11 Further optimization de-
monstrated that the mixture of acetonitrile and water (5:1)
was the best solvent for ozonolysis of our peptides. Using
this method, 16 different C-terminal SAL ester peptides
were prepared in good to excellent yields (Table 1). Ozone
is a powerful oxidant, and very short reaction time is
needed to oxidize the CdC bond. Under the conditions
used, it did not cause any significant damage to the
peptides under investigation (Table 1). The obtained
SAL ester peptides were purified by reverse-phase HPLC
under normal conditions and characterized by MS
(Supporting Information).
With now an efficient method for solid phase synthesis
of C-terminal SAL ester peptides in hand, we proceeded to
apply it to the synthesis of cyclic peptides. Cyclization was
performed on linear SAL ester peptides with a Ser or Thr
residue at the N-terminus, 4 (Table 2 heading scheme). A
typical example was a cyclic peptide cyclo[SGLFGFAG]
As the aldehyde-mediated cyclization reaction is very
clean, it is actually unnecessary to isolate the cyclic N,O-
benzylidene acetal product 5 before the TFA deprotection
(11) Schiaffo, C. E.; Dussault, P. H. J. Org. Chem. 2008, 73, 4688.
Org. Lett., Vol. XX, No. XX, XXXX
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Table 2. Synthesis of Ser or Thr-Containing Cyclic Peptides
from Unprotected Peptide-O-2-PhCHOa
entry
sequence
yield (%)b
Figure 2. HPLC monitoring of the cyclization reaction after
1 h. (A) Cyclization of H-SGLFGFAG-OPhCHO (peak 1: its
cyclic N,O-benzylidene acetal product 5, m/z [M þ H]þ found
841.32, calcd 841.40). (B) Cyclization of H-SGKAFL-
O-PhCHO (peak 2: its cyclic N,O-benzylidene acetal product
5, m/z found 708.28, calcd 708.38).
1
2
3
4
5
6
7
8
cyclo[SGKAFL]
cyclo[SFLFA]
39
35
63
65
52
20
56
29
cyclo[TGLFGFAG]
cyclo[SGLFGFAG]
cyclo[SGLYGFAG]
cyclo[SLSL]c
cyclo[TFFGFFG]
cyclo[SKSIPLFPI]d
step. The solvent was removed under vacuum, and the
treatment of TFA on the residue yielded the cyclic end
product 6 directly. Mahafacyclin B, a natural cyclic hepta-
peptide with antimalarial activity isolated from the
latex of Jatropha mahafalensis12a (entry 7, Table 2), was
also synthesized using our methodology, and its identity
was verified by MS and NMR analysis (Supporting
Information). A recent total synthesis of mahafacyclin
B12b employing soluble tag-assisted cyclization required
more than 10 solution synthesis and purification steps. In
comparison, only three purification steps were needed in
our case. This clearly demonstrated the convenience and
robustness of our methodology for the synthesis of cyclic
peptides such as mahafacyclin B.
Because of high strains of the small ring structure,
cyclization of H-SLSL-OPhCHO in the synthesis of cyclo-
[SLSL] (entry 6, Table 2) had to be done at very high
dilutions to minimize cyclodimerization, a side reaction
inherently associated with small cyclic peptides.1b Here,
cyclodimerization involved two consecutive ligation steps,
the first intermolecular and the second intramolecular,
forming a symmetric dimer structure. Significant cyclo-
dimerization was observed even at 0.1 mM concentration
of the linear SAL ester precursor (Figure S44, Supporting
Information).
a Unless otherwise indicated, cyclization was performed for 1 h at
10 mM concentration of the linear SAL ester peptide. b Isolated yield for
two steps. c Concentration for cyclization was 0.05 mM. d Cyclization
reaction was performed overnight at 2 mM of the linear precursor.
In summary, a convenient method for the synthesis of
C-terminal salicylaldehyde ester peptides was developed.
The key design of this method is the ozonolysis reaction on
an aldehyde precursor, peptidyl-OPhCHdCHCONH2,
which is easily obtained from Boc SPPS. This method
makes the peptide SAL esters readily available for Ser/
Thr-mediated aldehyde capture ligation.8 It was further
demonstrated that the peptide SAL esters so-prepared can
be easily converted to Ser/Thr-containing cyclic peptides
through SAL capture ligation. A possible limitation of this
method resides in the susceptibility of certain amino acids
such as Cys, Met and Trp to ozonolysis, which would
restrict its application scope. Nevertheless, these amino
acids are of the lowest natural abundance, and our strategy
certainly provides a new and efficient approach to the
synthesis of salicylaldehyde ester peptides. It is simple to
use because no tedious purification and characterization
steps are needed. In addition, all the reagents used in this
protocol are commercially available. Clearly, our method
not only provides a powerful approach to cyclic pep-
tide synthesis but also will spur renewed interest in the
aldehyde-based capture ligation schemes for protein chem-
ical synthesis in the future.
A number of other cyclic peptides ranging from 5 to 9
residues in ring size and with different cyclization junctions
were preparedbyusingour method(Table2). These results
indicate that the cyclization reaction can tolerate a wide
range of Xaa-Ser/Thr junctions, including the sterically
hindered Ile-Ser (entry 8, Table 2), which is consistent
with earlier results reported by Li et al.8,9aꢀ9c As a negative
control, no cyclization occurred with H-LGFAG-OPhCHO
(entry 6, Table 1), which does not have an N-terminal
Ser/Thr.
Acknowledgment. This work is supported by a Tier 2
grant from the Ministry of Education of Singapore.
Supporting Information Available. Experimental pro-
cedures and compound characterization data. This ma-
terial is available free of charge via the Internet at http://
pubs.acs.org.
(12) (a) Baraguey, C.; Blond, A.; Cavelier, F.; Pousset, J. L.; Bodo,
B.; Auvin-Guette, C. J. Chem. Soc., Perkin Trans. 1 2001, 2098. (b)
Fujita, Y.; Fujita, S.; Okada, Y.; Chiba, K. Org. Lett. 2013, 15, 1155.
The authors declare no competing financial interest.
D
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