Total synthesis of cyclocitropside A and its conversion to cyclocitropsides B and C via asparagine deamidation

Article abstract of DOI:10.1021/ol3023853

The total syntheses of three closely related cyclic peptide natural products, cyclocitropsides A-C, are described. Cyclocitropside A could be readily converted into cyclocitropsides B and C through an asparagine deamidation pathway, indicating that this is a plausible biosynthetic route to these compounds.

Full text of DOI:10.1021/ol3023853

ORGANIC
LETTERS
2012
Vol. 14, No. 19
5110–5113
Total Synthesis of Cyclocitropside A and
Its Conversion to Cyclocitropsides B and
C via Asparagine Deamidation
Robert E. Thompson, Richard J. Payne,* and Katrina A. Jolliffe*
School of Chemistry, The University of Sydney, NSW 2006, Australia
richard.payne@sydney.edu.au; kate.jolliffe@sydney.edu.au
Received August 28, 2012
ABSTRACT
The total syntheses of three closely related cyclic peptide natural products, cyclocitropsides AÀC, are described. Cyclocitropside A could be readily
converted into cyclocitropsides B and C through an asparagine deamidation pathway, indicating that this is a plausible biosynthetic route to these
compounds.
The structrually related cyclic heptapeptides, cyclocitrop-
sides AÀC (1À3, Figure 1), were recently isolated from the
root bark of the African cherry orange Citropsis articulata.1
The three peptides are almost identical in sequence, varying
only in the nature of the amino acid at position five.
Cyclocitropside A contains an asparagine (Asn) residue at
this position, while cyclocitropsides B and C are configura-
tional isomers of each other bearing aspartic acid (Asp) or
isoaspartic acid (isoAsp) residues, respectively. The isolation
of these three closely related structures led to the hypothesis
that cyclocitropsides B and C might be derived from deami-
The deamidation of asparagine residues in peptides and
proteins is one of the most common forms of nonenzy-
matic degradation and appears to be associated with the
aging of peptides and proteins in vivo.2,3 However, to the
best of our knowledge, cyclocitropside C is the first cyclic
peptide natural product reported to contain an isoAsp
residue.4 The deamidation pathway has been well char-
acterized in proteins and model peptides. At neutral or
basic pH, deamidation proceeds via a succinimide inter-
mediate, the rate of formation of which is dependent on the
5
(2) Aswad, D. W.; Paranandi, M. V.; Schurter, B. T. J. Pharm.
Biomed. Anal. 2000, 21, 1129.
dation of Asn in cyclocitropside A and that this might be
catalyzed by the basic guanidino moiety of the side chain of an
arginine residue also present in the cyclic peptide sequence.1
(3) Robinson, A. B.; Rudd, C. J. Curr. Top Cell Regulat. 1974, 8, 247.
(4) Several cyclic peptides containing erythro-β-methyl-D-isoaspartic
acid have been isolated from cyanobacteria. See: Krishnamurthy, T.;
Szafraniec, L.; Hunt, D. F.; Shabanowitz, J.; Yates, J. R., III; Hauer,
C. R.; Carmichael, W. W.; Skulberg, O.; Codd, G. A.; Missler, S. Proc.
Natl. Acad. Sci. U.S.A. 1989, 86, 770.
(1) Lacroix, D.; Prado, S.; Kamoga, D.; Kasenene, J.; Zirah, S.;
Bodo, B. Org. Lett. 2012, 14, 576.
r
10.1021/ol3023853
Published on Web 09/19/2012
2012 American Chemical Society

Scheme 1. Synthesis of Cyclocitropside A
Figure 1. Structures of cyclocitropsides AÀC.
amino acid sequence,5 local peptide structure,2 and a
number of external factors including pH, temperature,
and the dielectric strength of the solvent.6 Hydrolysis of
this intermediate is relatively rapid and can occur at either
the R- or β-carbonyl group to give either isoAsp or Asp,
generally in an approximately 3:1 ratio.7
The unique structure of cyclocitropside C (3) together
with the intriguing possibility that this compound and
cyclocitropside B (2) might bederived from cyclocitropside
A (1) prompted us to undertake the total synthesis of all
three cyclocitropsides and to investigate whether 1 could
be converted into 2 and 3 via biomimetic deamidation of
the 5Asn residue. Inaddition we prepared two analogues of
cyclocitropside A in which the 2Arg residue was replaced
with either Lys or 2-aminoheptanoic acid in order to probe
the contribution of this amino acid residue to the deamida-
tion process. We report here the results of these studies.
We envisaged that a cyclization point at the Gly-5Asx/
isoAsp junction would enable rapid synthesis of the three
required linear peptide precursors 4À6 (Schemes 1 and 2),
which vary only in their N-terminal amino acids, from a
common hexapeptide presursor. This places Gly as the C-
terminal residue which was expected to both facilitate the
cyclization and avoid any C-terminal epimerization during
this reaction. Thus, the resin-bound linear hexapeptide 7was
prepared using standard Fmoc solid-phase peptide synth-
esis (SPPS) techniques with PyBOP/N-methylmorpholine
(NMM) asthe coupling reagent and 2-chlorotritylchloride
resin as the solid support (Scheme 1). This resin-bound
peptide was split into three equal portions to which either
Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, or Fmoc-
Asp(OH)-OtBuwas coupledin ordertoprovideall three of
the required resin-bound linear heptapeptides 4À6. Clea-
vage of the Asn-containing precursor from the resin and
concomitant global deprotection was achieved using an
acidic cocktail of trifluoroacetic acid, water, and triisopro-
pylsilane to afford 4 in crude form following precipitation
from cold ether. Cyclization of 4 was performed using
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
tetrafluoroborate (DMTMM)8 as the coupling reagent at high
dilution (1 mM) in DMF. Following purification of the reac-
tion mixture by RP-HPLC, 1 was obtained in a pleasing 80%
overall yield based on the initial resin loading (Scheme 1).
4
(5) (a) Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7, 787. (b) Clarke,
S. Int. J. Pept. Protein Res. 1987, 30, 808. (c) Robinson, N. E.; Robinson,
Z. W.; Robinson, B. R.; Robinson, A. L.; Robinson, J. A.; Robinson,
M. L.; Robinson, A. B. J. Pept. Res. 2004, 63, 426. (d) Robinson, N. E.;
Robinson, A. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4367. (e) Capasso,
S. J. Pept. Res. 2000, 55, 224. (f) Robinson, N. E.; Robinson, A. B.;
Merrifield, R. B. J. Pept. Res. 2001, 57, 483.
(6) (a) Capasso, S; Kirby, A. J.; Salvadori, S.; Sica, F.; Zagari, A.
J. Chem. Soc. Perkin Trans. 2 1995, 437. (b) Brennan, T. V.; Clarke, S.
Protein Sci. 1993, 2, 331.
(7) Capasso, S.; Di Cerbo, P. J. Pept. Res. 2000, 56, 382.
Org. Lett., Vol. 14, No. 19, 2012
5111

Scheme 2. Synthesis of Cyclocitropsides B and C
Figure 2. Analytical HPLC trace of crude reaction mixture of 1
in 200 mM phosphate buffer at pH 7.5 at 100 °C after 0, 240, and
540 min. Peaks corresponding to compounds 1À3 are shown,
as well as linear peptide byproducts (*). Absorbance measured
at 230 nm.
at 100 °C in 200 mM phosphate buffer at pH 7.5. The
reaction was monitored by LCÀMS, by which the starting
material 1 and the deamidated products 2 and 3 were
observed (Figure 2). After 18 h, the starting material had
been completely consumed, and subsequent purification
by RP-HPLC gave 2 and 3 in 15% and 60% yields,
respectively (Scheme 3). Notably, the 1:4 ratio observed
for formation of 2 and 3 is similar to those commonly
observed for Asn deamidation reactions.2,7,10
Robinson and co-workers have shown that the aspar-
agine-leucine junction is one of the least prone dipeptide
junctions to undergo deamidation in peptides or proteins
(the succinimide intermediate can not be formed at the
Asn-Pro junction, so deamidation is not observed for this
dipeptide under the conditions used).5c Local secondary
structure, such as is likely to be found in a small cyclic
peptide, has also been shown to reduce the rate of the
deamidation reaction.11 The surprisingly facile deamida-
tion we observed for 1 under phosphate-buffered condi-
tions led ustoinvestigate the hypothesispresentedby Bodo
and co-workers1 that this process may be catalyzed by the
basic side chain of the arginine residue in the cyclocitrop-
side sequence. In a manner similar to that described above,
we synthesized two unnatural analogues of 1, replacing the
arginine residue with either lysine (9), providing an alter-
nate basic residue, or the unnatural amino acid L-2-ami-
noheptanoic acid(10) toremovethe basic functionalitybut
provide a similar steric environment to that of the native
sequence (Scheme 4, see the Supporting Information for
The synthesis of structural isomers 2 and 3 was achieved
in a similar manner from the resin-bound precursor 7.
Cleavageof these peptidesfromthe resin toliberatethefree
C-terminal carboxylic acid moiety, with protecting groups
intact, was achieved using a mildly acidic solution of 20%
hexafluoro-2-propanol in dichloromethane to give linear
peptides 5 and 6 (Scheme 2), each of which was cyclized
upon treatment with DMTMM as described above. In situ
acidolytic cleavage of the side-chain protecting groups was
conducted using TFA/TIS/H2O to afford 2 and 3 in 72%
and 75% yield (based on the initial resin loading), respec-
tively, after purification by RP-HPLC. All three synthe-
sized cyclic peptides had physical data identical to those
reported for the isolated natural products,1 thus confirm-
ing the structures and identities of these three natural
products.
Having established the structures of 1À3 through total
synthesis, we turned our attention to the biomimetic
synthesis of 2 and 3 via deamidation of 1. However, our
initial attempts to elicit this transformation in 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethane sulfonate (HEPES)
buffer were unsuccessful, leading only to starting material
after 12 h incubation at 100 °C. It is well established that
phosphate buffer and high temperatures promote the
deamidation reaction.3,9 Therefore, in order to effect this
transformation in a reasonable time period, we incubated 1
(10) (a) Athiner, L.; Kindrachuk, J.; Georges, F.; Napper, S. J. Biol.
Chem. 2002, 277, 30502. (b) Reissner, K. J.; Aswad, D. W. Cell. Mol. Life
Sci. 2003, 60, 1281.
(11) (a) Stotz, C. E.; Borchardt, R. T.; Middaugh, C. R.; Siahaan,
T. J.; Vander Velde, D.; Topp, E. M. J. Pept. Res. 2004, 63, 371.
(b) Krogmeier, S. L.; Reddy, D. S.; Vander Velde, D.; Lushington,
G. H.; Siahaan, T. J.; Middaugh, C. R.; Borchardt, R. T.; Topp, E. M.
J. Pharm. Sci. 2005, 94, 2616.
ꢀ
ꢀ
ꢀ
(8) (a) Kaminski, Z. J.; Kolesinska, B.; Kolesinska, J.; Sabatino, G.;
Chelli, M.; Rovero, P.; Blaszczyk, M.; Glowka, M. L.; Papini, A. M.
ꢀ
J. Am. Chem. Soc. 2005, 127, 16912. (b) Fairweather, K. A.; Sayyadi, N.;
Luck, I. J.; Clegg, J. K.; Jolliffe, K. A. Org. Lett. 2010, 12, 3136.
(9) McKerrow, J. H.; Robinson, A. B. Anal. Biochem. 1971, 42, 565.
5112
Org. Lett., Vol. 14, No. 19, 2012

Scheme 3. Deamidation Pathways Leading to 2 and 3 from 1
Scheme 4. Synthesis of Structural Analogues 9 and 10
the deamidation reaction significantly. While the conditions
employed in this study were relatively harsh in order to
facilitate the reaction in a short time frame, it is likely that
the deamidation process could either be an active enzyme-
catalyzed process or a passive process under ambient con-
ditions in the plant root over extended time periods.
In summary, the total syntheses of three cyclic peptide
natural products 1À3 have been completed, providing
confirmation of their structures. In addition, the conver-
sion of 1 to 2 and 3 has been observed under conditions
favoring asparagine deamidation, confirming this as a
plausible biosynthetic pathway for these compounds and
suggesting the existence of other isoAsp-containing cyclic
peptide natural products.
synthetic details). However, incubation of cyclic peptides
1, 9, and 10 at 100 °C in 50 mM phosphate buffer at pH 7.5
over a 30h period revealedlittle tono difference inthe rates
of degradation into their respective deamidated products
as monitored by LCÀMS (see the Supporting Information
for details). Furthermore, no difference was observed in
the rates of deamidation of linear peptide 4 and cyclic
peptide 1 under the same conditions. It appears that the
influence of the Arg residue on deamidation is negligible
under these phosphate-buffered conditions and that the
cyclic peptide structure does not impose a significant
constraint on deamidation. Similar deamidation reactions
have recently been observed in other cyclic peptides,12
indicating that a cyclic peptide structure does not hinder
Acknowledgment. R.E.T. is grateful for the provision of
an Australian Postgraduate Award and a John A. Lamberton
research scholarship.
Supporting Information Available. Experimental pro-
cedures and characterization data. Details of deami-
dation studies including analytical HPLC data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(12) Curnis, F.; Cattaneo, A.; Longhi, R.; Sacchi, A.; Gasparri,
A. M.; Pastorino, F.; Di Matteo, P.; Traversari, C.; Bachi, A.; Ponzoni,
M.; Rizzardi, G.-P.; Corti, A. J. Biol. Chem. 2010, 285, 9114.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 19, 2012
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