N,O-isopropylidenated threonines as tools for peptide cyclization: Application to the synthesis of mahafacyclin B

Article abstract of DOI:10.1021/ol0522891

(Chemical Equation Presented) The influence of a single N,O-isopropylidenated threonine turn-inducer on the cyclization of a linear heptapeptide precursor to mahafacyclin B has been investigated. Incorporation of an N,O-isopropylidenated threonine more th

Full text of DOI:10.1021/ol0522891

ORGANIC
LETTERS
2005
Vol. 7, No. 24
5497-5499
N,O-Isopropylidenated Threonines as
Tools for Peptide Cyclization:
Application to the Synthesis of
Mahafacyclin B
Nima Sayyadi, Danielle Skropeta, and Katrina A. Jolliffe*
School of Chemistry, The UniVersity of Sydney, 2006 NSW, Australia
jolliffe@chem.usyd.edu.au
Received September 22, 2005
ABSTRACT
The influence of a single N,O-isopropylidenated threonine turn-inducer on the cyclization of a linear heptapeptide precursor to mahafacyclin
B has been investigated. Incorporation of an N,O-isopropylidenated threonine more than doubles the head-to-tail cyclization yield. The N,O-
isopropylidene grouping is then readily disassembled to give the antimalarial cyclic peptide in high yield.
Biologically active cyclic peptides have been isolated from
a variety of sources including plants, bacteria, and marine
organisms. These naturally occurring compounds exhibit a
wide range of biological activities. This, together with the
fact that they generally exhibit improved biological properties
such as metabolic stability and receptor selectivity when
compared to their linear counterparts, has led to much interest
in the synthesis of cyclic peptides.1-3 However, the head-
to-tail cyclization of a linear peptide is often a slow, low-
yielding procedure, accompanied by side reactions such as
cyclodimerization and epimerisation.4,5 We recently reported
on the use of removable turn-inducers, in the form of N,O-
isopropylidenated threonines, to facilitate head-to-tail peptide
cyclization.6 These threonine-derived acetals were recently
introduced as solubilization aids for solid-phase peptide
synthesis.7,8 Their incorporation into linear peptide precursors
was found to substantially increase cyclization yield, com-
pared to cyclization of the same peptides having O-TBS-
protected threonine residues.6 After cyclization, the turn-
inducers were readily removed to give the cyclic peptides
bearing free threonine residues. We now report on the
application of this methodology to the total synthesis of the
naturally occurring cyclic peptide, mahafacyclin B.
Mahafacyclin B (1) is a cyclic heptapeptide [cyclo(Thr-
Phe-Phe-Gly-Phe-Phe-Gly)] with antimalarial activity (IC50
) 2.2 µM), which has been isolated from the latex of
Jatropha mahafalensis (Euphorbiaceae).9 The structure and
conformation of mahafacyclin B were recently elucidated
and the structure confirmed by synthesis. The final step in
the synthesis, namely, cyclization, was reported to occur in
only 30% yield.9 Since mahafacyclin B contains a single
threonine residue, which can be readily N,O-isopropylide-
(1) Davies, J. S. J. Pept. Sci. 2003, 9, 471.
(2) Li, P.; Roller, P. P.; Xu, J. Curr. Org. Chem. 2002, 6, 411.
(3) Lambert, J. N.; Mitchell, J. P.; Roberts, K. D. J. Chem. Soc., Perkin
Trans. 1 2001, 471.
(7) Wo¨hr, T.; Wahl, F.; Hefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.;
Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218.
(4) Kopple, K. D. J. Pharm. Sci. 1972, 61, 1345.
(8) Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wo¨hr, T.; Mutter,
M. J. Am. Chem. Soc. 1997, 119, 918.
(9) Baraguey, C.; Blond, A.; Cavelier, F.; Pousset, J.-L.; Bodo, B.; Auvin-
Guette, C. J. Chem. Soc., Perkin Trans. 1 2001, 2098.
(5) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber, H.;
Carpino, L. A.; Bienert, M. J. Org. Chem. 1996, 61, 8831.
(6) Skropeta, D.; Jolliffe, K. A.; Turner, P. J. Org. Chem. 2004, 69, 8804.
10.1021/ol0522891 CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/29/2005

nated, it appeared an ideal target on which to test our N,O-
acetal-assisted cyclization methodology.
In their previous synthesis of mahafacyclin B, Baraguey
et al. chose the point of cyclization to be between Thr and
Gly to give a linear precursor 2 with Thr as the N-terminal
amino acid and Gly as the C-terminal amino acid (Scheme 1).9
necessary to choose a different cyclization point so that the
Thr residue was not positioned at or near the N-terminus of
the linear peptide precursor. Therefore, we chose to cyclize
between the Phe and Gly residues, which gave us the linear
precursor 3 (Scheme 1), again with Gly as the C-terminal
residue.
The required linear heptapeptide 3 was synthesized by
solution-phase peptide synthesis using a benzyloxycarbonyl
(Cbz)/OMe protection strategy and EDCI (N-(3-dimethy-
laminopropyl)-N′-ethylcarbodiimide hydrochloride) in the
presence of HOBt (1-hydroxybenzotriazole) as the coupling
reagent. The N,O-isopropylidene-containing dipeptide 5 was
prepared in excellent yield from Cbz-Gly-Thr-OMe 4 by
treatment with 2-methoxypropene in the presence of a
catalytic amount of (()-10-camphorsulfonic acid, followed
by hydrolysis of the methyl ester under standard conditions
(Scheme 2). Tripeptide 6 was also prepared using standard
procedures. The two fragments 5 and 6 were then coupled
to give the pentapeptide 7. Sequential addition of the two
phenylalanine residues gave protected heptapeptide 8, and
subsequent deprotection of both the C- and N-termini of the
peptide under standard conditions gave the linear heptapep-
tide 3 which was purified by preparative reversed-phase
HPLC. For the N,O-isopropylidene-containing dipeptide 5
and the methyl ester precursor to 5, a major and minor set
of resonances in a ratio of 85:15 were clearly observed in
Scheme 1. Different Linear Precursors Required for the
Synthesis of Mahafacyclin B
1
the H and 13C NMR spectra.10 The major conformer was
determined to be that with a cisoid amide bond on the basis
of the typical NOE cross-peaks observed by 2D NMR
ROESY and NOESY experiments (i.e., RHi-1 - RHi and
RHi-1 - âHi cross-peaks).11 For the N,O-isopropylidenated
threonine-containing peptides 7, 8, and 3, only a single set
of resonances are observed in the 1H and 13C NMR spectra.
In all cases, these were assigned to be the conformers with
a cisoid amide bond preceding the N,O-isopropylidenated
threonine residue on the basis of the typical NOE cross-peaks
This removed the problem of racemization during the cycli-
zation step. For an N,O-acetal-assisted cyclization, it was
Scheme 2
5498
Org. Lett., Vol. 7, No. 24, 2005

Scheme 3
observed by 2D NMR ROESY and NOESY experiments
tide sequence, we prepared and cyclized the linear heptapep-
tide 10. Heptapeptide 10 has an identical sequence to 3, but
lacks the N,O-isopropylidene, and was readily prepared upon
treatment of 3 with 4 M HCl in dioxane for 10 min. Cycli-
zation of 10 was investigated using conditions identical to
those described above to give the major cyclic peptide pro-
duct, mahafacyclin B (1), in 23% yield. The material obtained
via this route was identical, in all respects, to that obtained
upon cyclization and deprotection of 3. Notably, cyclization
of 10 required more than 72 h to reach completion, as
determined by following both the consumption of the starting
material and the appearance of the product by HPLC, while
the cyclization of 3 was complete in less than 3 h. The much
lower yield and longer reaction time required for cyclization
of 10 compared to 3 indicates that the presence of the N,O-
isopropylidenated threonine residue is assisting 3 to adopt a
conformation amenable to cyclization.
In summary, we have shown that the introduction of a
single N,O-isopropylidenated threonine turn-inducer into a
linear peptide precursor significantly increases the head-to-
tail cyclization yield of that peptide. This method has
successfully been applied to the total synthesis of mahafa-
cyclin B, a naturally occurring cyclic heptapeptide and will
find future application in the synthesis of a wide range of
cyclic peptides.
(i.e., RHi-1 - RHi and RHi-1 - âHi cross-peaks).11
Cyclization of 3 was performed using both pentafluo-
rophenyl diphenylphosphinate (FDPP) and HBTU [(O-
benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate] as coupling reagents under high dilution (0.005
M) in DMF to give the cyclic peptide 9 in 63% and 58%
yield, respectively, twice the yield obtained by Baraguey for
cyclization of the linear precursor 2.9 The 1H NMR spectrum
of 9 indicated two conformers present in a ratio of 83:17.
While overlapping of the signals from different rotamers
makes it difficult to observe the characteristic NOE cross-
peaks for the conformer in which the amide bond preceding
the N,O-isopropylidenated threonine residue is cisoid, it can
be assumed by analogy with the other peptides discussed
here that this is the major conformer. Upon treatment of 9
with trifluoroacetic acid for 24 h, deprotection of the N,O-
isopropylidenated threonine residue gave cyclic peptide 1,
in 90% yield (Scheme 3). This material was found to have
physical and spectral properties identical to those reported
for mahafacyclin B.9
The sequence of a peptide is known to have a strong
influence on cyclization yield.12,13 Therefore, to determine
whether the higher yield obtained from our N,O-isopropyl-
idenated threonine-containing precursor 3 was attributable
to the presence of the turn-inducer, or simply a result of pep-
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.
(10) This ratio is in accordance with those observed for similar Fmoc-
protected dipeptides. Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.;
Fischer, G. S.; Mutter, M. J. Am. Chem. Soc. 1998, 120, 2714.
(11) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; John Wiley &
Sons: New York, 1986; p 117.
Supporting Information Available: Experimental pro-
1
cedures and characterization of all compounds. H and 13C
(12) Brady, S. F.; Varga, S. L.; Freidinger, R. M.; Schwenk, D. A.;
Mendlowski, M.; Holly, F. W.; Veber, D. F. J. Org. Chem. 1979, 44, 3101.
(13) Tang, Y. C.; Xie, H. B.; Tian, G. L.; Ye, Y. H. J. Pept. Res. 2002,
60, 95.
NMR spectra of compounds 1, 3, 4, and 7-10. This material
is available free of charge via the Internet at http://pubs.acs.org.
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