Total synthesis of the antifungal depsipeptide petriellin A

Article abstract of DOI:10.1021/jo201017w

We report the solid-phase total synthesis of the antifungal highly modified cyclic depsipeptide petriellin A. The synthesis confirms earlier reports on the absolute configuration of the natural product. The solid-phase approach resulted in a protected lin

Full text of DOI:10.1021/jo201017w

ARTICLE
pubs.acs.org/joc
Total Synthesis of the Antifungal Depsipeptide Petriellin A
Marianne M. Sleebs, Denis Scanlon, John Karas, Rani Maharani, and Andrew B. Hughes*
Department of Chemistry, La Trobe Institute of Molecular Sciences, La Trobe University, Victoria 3086, Australia
S Supporting Information
b
ABSTRACT: We report the solid-phase total synthesis of the
antifungal highly modified cyclic depsipeptide petriellin A. The
synthesis confirms earlier reports on the absolute configuration
of the natural product. The solid-phase approach resulted in a
protected linear precursor, which was cleaved from the solid
support prior to cyclization and final deprotection. Use of
advanced coupling agents for several hindered amides was a
feature of the synthesis. The natural product was prepared in
overall 5% yield.
’ INTRODUCTION
Discovery and development of novel therapeutics is driven, in
part, by the increase in pathogenic bacteria and fungi resistant
to established drugs. Medicinal chemistry discovery has a focus
on natural products displaying (particularly novel) biological
activity.1,2 These secondary metabolites, in some instances, have
been useful as drugs, while others have served as lead compounds
in drug development. While many different classes of metabolites
have pharmacological importance, our research is directed
toward modified amino acids that include N-methyl R-amino
acids3ꢀ6 and β-amino acids.7ꢀ12 Furthermore, we are interested
in the development of methodologies for the subsequent synth-
esis of bioactive peptides including such modified amino
acids.8,13 In particular, the present research focuses on total
synthesis of the naturally occurring antifungal cyclodepsipeptide,
petriellin A 1 (Figure 1).
Figure 1. Cyclization site for solid-phase synthesis of petriellin A.
can accompany this approach. These difficulties are further
exacerbated in the case of N-methylated peptides by the difficult
coupling of secondary amines to form tertiary amide bonds. Accord-
ingly, in planning the present total synthesis of petriellin A, earlier
difficulties were taken into account and a solid-phase approach
was chosen. Indeed, solid-phase peptide chemistry has difficulties
of its own;17 however, purification and isolation of intermediates
is eliminated from the reaction course. Thus, it was anticipated
that production of the linear precursor of petriellin A would be
achieved in less time, and if difficulties were encountered in
synthesizing the linear precursor or during the cyclization, a new
approach could be investigated in a much shorter time frame.
Furthermore, once the solid-phase technique was optimized and
a total synthesis was achieved, this approach might be applied to
the total synthesis of other similar depsipeptides of interest.
Prior to commencing the petriellin A solid-phase synthesis,
a number of details were considered. In the first instance, only a
Lee and Gloer and co-workers14 isolated petriellin A 1
(Figure 1) from the organic extracts of an antagonistic copro-
philous fungus, Petriella sordida (UAMH 7493). Petriellin A was
found to have activity against Ascobolus furfuraceous (NRRL
6460) and Sordaria fimicola (NRRL 6459).14 Structurally, pet-
riellin A contains one R-D-hydroxycarboxylic acid and 12 L-amino
acids, five of which are N-methylated. At the time of the initial
communication, Lee and Gloer and co-workers14 did not deter-
mine the absolute configuration of N-methylisoleucine and the
two N-methylthreonine residues in the natural product. Clarifi-
cation of the unknown stereochemistry was later communicated
by Aurelio et al.6,15 An attempted total synthesis of petriellin A via
solution-phase chemistry was later reported by Aurelio et al.13
The authors constructed the “northwestern” and “southeastern”
halves of their planned linear precursor. However, the assembly
of the acyclic depsipeptide was unsuccessful.
While solution-phase peptide techniques have been utilized
extensively throughout the history of peptide synthesis and
lead to the production of natural products,16 many difficulties
Received: May 22, 2011
Published: July 08, 2011
r
2011 American Chemical Society
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small number of heavily N-methylated peptides had been
synthesized by solid-phase peptide techniques.19,20,22,23 This
was attributed to the fact that resin-bound secondary amines
have lower reactivities toward activated acids than analogous
reactions in solution.18 Some solid-phase syntheses of N-methy-
lated peptides have been accomplished by utilizing coupling
reagents that have shown success in solution. A successful solid-
phase synthesis of cyclosporins was reported that employed
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hex-
afluorophosphate (HATU) or N,N0-diisopropylcarbodiimide
(DIC)/1-hydroxy-7-azabenzotriazole (HOAt) for the construc-
tion of tertiary amide bonds.19 Other examples achieved N-methyl
amide synthesis by symmetrical anhydride methods.20 However,
both of these approaches required multiple coupling cycles to
obtain adequate coupling yields.
A reliable alternative to overcome the difficulties associated
with the synthesis of N-methylated amide bonds was first
reported by Gilon and co-workers.21 Bis(trichloromethyl)-
carbonate (BTC, or triphosgene) was added to an Fmoc-amino
acid, yielding the corresponding acid chloride. This was imme-
diately added to a peptidyl Rink amide 4-methylbenzhydryla-
mine (MBHA) resin at 50 °C, yielding a tertiary amide bond.
Fmoc-amino acids bearing acid-labile side chains were found
to be stable to these conditions. HPLC analysis established
that the peptides were produced in high yields and purity
without racemization when tetrahydrofuran (THF) was used
as solvent. The absence of racemization was due to the high
reactivity of the acid chloride compared to the slower oxazolone
formation.21 This approach was more versatile than methods
that enlisted the use of premade Fmoc-amino acid chlorides,
as some cannot be synthesized and others are not stable
resulting in, for instance, oxazolone formation. Moreover,
those with acid-labile side-chain protecting groups have a limited
shelf life.
Applying the BTC coupling agent in the solid-phase synthesis
of petriellin A was promising, particularly since total syntheses
of omphalotin A22 and cyclosporin O23 using BTC have been
reported. In the first instance, Jung and co-workers22 conducted
comparative N-methyl couplings on a trityl chloride resin, in
which BTC was compared to dicyclohexylcarbodiimide (DCC),
DIC/HOAt and tetramethylfluoroformamidinium hexafluoro-
phosphate (TFFH). The highly acid-labile trityl chloride resin
was used as a model, rather than the Rink amide resin originally
employed by Gilon and co-workers,21 due to the acid lability of
N-methylated peptides.24,25 In all cases BTC possessed superior
coupling efficiencies; however, the original method21 was found
to be useless for the synthesis of larger peptides on the trityl
chloride resin.21 Under the original BTC-coupling conditions,
premature cleavage of the growing peptide from the highly acid-
labile trityl resin resulted. It was assumed that 2,4,6-collidine
alone was not basic enough to neutralize the hydrogen chloride
formed during the BTC activation and prevent it from cleaving
the peptide from the resin. Jung and co-workers22 eliminated this
problem by pretreating the resin with diisopropylethylamine
(DIPEA) and then adding the preformed Fmoc-amino acid
chloride. The presence of DIPEA accelerated the reaction,
and thus the elevated temperature employed in the original
procedure21 was no longer necessary and byproduct formation
ceased. HPLC analysis confirmed the enantiomeric purity of the
products was unaffected by these modifications.23 It was decided
that the modified BTC method of Jung and co-workers22,23
would be utilized in the present synthesis of petriellin A.
The site chosen for cyclization was dictated by the sequence of
the linear precursor. Tertiary amide bonds generally were
discounted as possible cyclization sites. The junctions between
the C- and N-termini of proline and the two neighboring non-N-
methylated amino acids were two possible sites for cyclization.
One was the amide bond connecting the C-terminus of proline to
the N-terminus of isoleucine; the activated form of proline is
known to resist racemization,26 but the isoleucine side chain is
sterically hindered. The other site was the C-terminus of alanine
and the N-terminus of proline. Although cyclizing at this point
would involve the formation of a tertiary amide bond, proline
behaves more like a primary amine when reacting with activated
acids. The alanineꢀproline junction was chosen as the preferred
cyclization site due to the lower steric hindrance compared to
isoleucine. As a consequence, the linear precursor would begin
from the N-terminus of L-proline and finish at the C-terminus
of alanine.
The final factor considered was the protecting group on
the side chains of the two threonine residues. Conveniently,
Fmoc-N-methyl-O-t-butyl-L-threonine was commercially avail-
able. However, the use of this t-butyl ether derivative would
require establishing a trifluoroacetic acid (TFA) deprotection
method that avoided TFA-induced amidolysis of the cyclization
product. With this in mind, a series of acid stability tests were
conducted on a sample of natural petriellin A isolated from
P. sordida.6 It was established that TFA-induced amidolysis was
avoided, provided reduced temperatures were used during
deprotection of the t-butyl ethers (vide infra).
’ RESULTS AND DISCUSSION
The construction of petriellin A began by loading Fmoc-L-
alanine onto the 2-chlorotrityl chloride resin. After 2 h the
unreacted resin sites were capped with methanol. The resin
was found to have an amino acid loading of 0.52 mmol/1.00 g
of resin. The Fmoc-protected peptidyl resin was treated with
a solution of piperidine/1,5-diazabicyclo[5.4.0]undec-5-ene
(DBU)/N,N-dimethylformamide (DMF) (2:2:96). The pre-
sence of the exposed primary amine was confirmed through a
2,4,6-trinitrobenzenesulfonic acid (TNBS) test on a small sample
of the resin beads.
Fmoc-L-pipecolic acid was the next amino acid in the se-
quence. At the time only DL-pipecolic acid was available, and a
Scheme 1. Commencing Solid-Phase Synthesis of Petriellin
A 1a
a Reagents and conditions: (a) (1) Fmoc-L-Ala-OH, DIPEA, CH2Cl2,
2 h; (2) MeOH; (3) piperidine, DBU, DMF. (b) (1) Fmoc-L-Pip-OH,
HBTU, HOBt, DIPEA, DMF, 5 h; (2) piperidine, DBU, DMF. (c) (1)
Fmoc-L-Ile-OH, HBTU, HOBt, DIPEA, DMF, 24 h; (2) piperidine,
DBU, DMF. (d) (1) Fmoc-L-Pro-OH, HBTU, HOBt, DIPEA, DMF,
24 h; (2) piperidine, DBU, DMF.
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resolution by recrystallization of the tartaric acid salts was
conducted. This purified L-pip-D-(ꢀ)-tartrate salt was treated
with Fmoc-O-succinimide in the presence of base. Fmoc-L-
pipecolic acid was isolated; its data were identical to previously
reported material.27 The protected pipecolic acid residue was
pretreated with O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethylur-
onium hexafluorophosphate (HBTU)/1-hydroxybenzotriazole
(HOBt) in the presence of base and the solution was added to
the resin-bound amino acid. Once this coupling cycle was
complete, the resin was treated with base to afford resin-bound
dipeptide 2 (Scheme 1). The exposed secondary amine group
was verified by a chloranil test. Fmoc-L-isoleucine was next
pretreated with the coupling reagent to form the activated ester
and then it was immediately added to the resin. Base treatment
of the resin followed and revealed the deprotected resin-bound
peptide. Fmoc-L-proline was added to the resin and, again, the
coupling was efficacious with HBTU/HOBt. Subsequent base
treatment yielded the tetrapeptide 3 (Scheme 1).
The resin-bound peptide 3 was treated with a preformed
solution of Fmoc-N-methyl-L-valine HOBt ester (Scheme 2).
Successive basic treatment revealed the resin-bound pentapep-
tide 4. The R-hydroxy-D-phenyllactic acid residue was next in the
sequence. However, this residue was converted to the didepsi-
peptide 9 (Scheme 3) by a Mitsunobu esterification and then
added to the growing peptide. This approach was desirable
because there are few sensitive resin tests for detecting unreacted
resin-bound alcohols. It was initially considered that if the ester
bond was made on the solid support, a sample of the growing
peptide could be cleaved after the solid-phase esterification
and analyzed by electrospray mass spectrometry (ES-MS). This
would give an indication of whether the coupling had been
successful. However, the R-hydroxycarboxylic acid present in
petriellin A is D-configured. A Mitsunobu esterification in solu-
tion gives the product with inversion of configuration at
the alcohol center. This approach allows the solution-phase
synthesis to begin from the less expensive and readily available
L-phenylalanine.
Scheme 2a
Accordingly, L-phenylalanine was diazotized to the corre-
sponding R-acetoxycarboxylic acid28 and subsequently con-
verted to the t-butyl ester derivative29 in 68% yield. The
doubly protected intermediate was subjected to mild base
hydrolysis, giving the alcohol 7 (Scheme 3). Mitsunobu ester-
ification between Fmoc-L-pipecolic acid and the L-phenyllactate
derivative 7 gave the depsipeptide 9 (Scheme 3). Initially, the
esterification was conducted in THF, which resulted in low yields
(40%). However, an improvement resulted when dichloro-
methane was used as the reaction medium (70%). For compara-
tive purposes, D-phenylalanine was also diazotized and then
treated with di-t-butyl dicarbonate to yield a doubly protected
intermediate. Once the acetate group was hydrolyzed, a DCC-
mediated coupling of the D-alcohol 8 and Fmoc-L-pipecolic acid
gave the depsipeptide 9 in 56% yield (Scheme 3). The DCC
coupling gave a reduced yield of the desired product with respect
to the synthesis of the same compound by Mitsunobu esterifica-
tion. Therefore, the economically favorable option is to com-
mence the synthesis of the dipeptide 9 from L-phenylalanine.
The fully protected dipeptide 9 was subjected to a TFA-
induced deprotection to expose the C-terminus (Scheme 3). The
a Reagents and conditions: (a) (1) Fmoc-L-N-MeVal-OH, HBTU,
HOBt, DIPEA, DMF, 18 h; (2) piperidine, DBU, DMF. (b) (1)
Fmoc-L-Pip-D-PheLac-OH, BTC, 2,4,6-collidine, DIPEA, THF, 18 h;
(2) repeat cycle; (3) piperidine, DBU, DMF. (c) (1) Fmoc-N-Me-L-Ile-
OH, BTC, 2,4,6-collidine, DIPEA, THF, 24 h; (2) piperidine,
DBU, DMF.
Scheme 3. Assembly of Depsidipeptide 10a
a Reagents and conditions: (a) (1) NaNO2, CH3CO2H, H2O; (2) (Boc)2O, DMAP, t-BuOH; (3) K2CO3, CH3OH, H2O. (b) Ph3P, DEAD, CH2Cl2.
(c) DCC, DMAP, CH2Cl2. (d) TFA/CH2Cl2 (1:1).
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crude acid 10 was sufficiently clean to use in the subsequent
solid-phase synthesis. Pretreatment of the acid 10 with BTC gave
the acid chloride, which was immediately added to the depro-
tected resin-bound pentapeptide 4 (Scheme 2). Immediately
after an 18 h coupling cycle, a chloranil test indicated that a
mixture of pentapeptide 4 and Fmoc-protected heptapeptide 5
was present. Another overnight BTC-mediated coupling of the
activated depsipeptide 10 was successful in completing the
synthesis of the resin-bound Fmoc-protected heptapeptide 5.
A small sample of the resin was treated with base and the
deprotected heptapeptide 5 was cleaved from the resin. ES-MS
analysis verified that the desired intermediate had formed ([M +
H]+ m/z 784 (100%)) with no deletion products present. The
remaining resin-bound peptide was deprotected and a solution of
the BTC-activated Fmoc-N-Me-L-isoleucine acid chloride was
added. Analysis after 24 h indicated complete coupling had taken
place and that the octapeptidyl resin 6 was present.
Fmoc-N-methyl-O-t-butyl-L-threonine was the next residue in
the sequence of petriellin A. However, prior to committing to the
use of this t-butyl ether derivative, the acid stability of petriellin A
toward a 50% TFA solution was investigated. A sample of natural
petriellin A30 was obtained and analyzed by HPLC to establish
purity. It was then used as a control. A solution of 50% TFA in
dichloromethane was then added to a 1 mg sample of natural
petriellin A for 1 h at room temperature. HPLC analysis of the
TFA-treated sample and the control showed that significant
degradation had occurred in the TFA-exposed petriellin A
sample. Another experiment was conducted whereby a fresh
petriellin A sample was treated with the same TFA solution;
however, the temperature of the mixture was maintained below
0 °C. HPLC analysis showed that no degradation had occurred.
At reduced temperatures it appeared that TFA-induced amido-
lysis was not occurring. Thus, the t-butyl ether protection could
be employed on the threonine residues.
Accordingly, Fmoc-N-methyl-O-t-butyl-L-threonine was pre-
treated to form the acid chloride and the solution was added to
the resin-bound peptide (Scheme 4). A chloranil test indicated
that the nonapeptide was completely converted to the resin-
bound decapeptide 12 and this was further verified by ES-MS
analysis ([M + H]+ m/z 1195 (30%)).
The next residue to be added to the growing peptide was
valine (Scheme 4). In the first instance, a 2-h BTC-mediated
coupling did not result in complete conversion to the undeca-
peptide intermediate 13 (Scheme 4). A second cycle mediated by
DIC/HOAt was necessary for complete conversion. However,
when the complete synthesis of petriellin A was later repeated,
the formation of the undecapeptidyl resin was efficacious after
The peptidyl resin 6 was treated with base, and following this
a solution of Fmoc-N-methyl-L-valine acid chloride was added
(Scheme 4). A sample of the growing peptide was subjected to
ES-MS analysis and the formation of the desired resin bound
nonapeptide 11 was confirmed ([M + H]+ m/z 1024 (50%)).
Scheme 4a
Scheme 5a
a Reagents and conditions: (a) (1) Fmoc-N-Me-L-Val-OH, BTC, 2,4,6-
collidine, DIPEA, THF, 2 h; (2) piperidine, DBU, DMF. (b) (1) Fmoc-
L-Thr(OtBu)-OH, BTC, 2,4,6-collidine, DIPEA, THF, 18 h; (2) piper-
idine, DBU, DMF. (c) (1) Fmoc-L-Val-OH, BTC, 2,4,6-collidine,
DIPEA, THF, 24 h; (2) piperidine, DBU, DMF.
a Reagents and conditions: (a) (1) Fmoc-N-Me-L-Thr(OtBu)-OH,
HBTU, HOBt, DIPEA, DMF, 18 h; (2) piperidine, DBU, DMF. (b)
(1) Fmoc-L-Pro-OH, BTC, 2,4,6-collidine, DIPEA, THF, 18 h; (2)
piperidine, DBU, DMF.
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only one cycle when the first BTC-mediated coupling was left
overnight rather than for 2 h.
Scheme 6. Resin Cleavage, Cyclization, and Deprotection
To Form Petriellin A 1a
The peptidyl resin was deprotected and a sample was sub-
jected to the TNBS test. A dark red color indicated that the
N-terminal protecting group had been removed. The second-to-
last coupling involved another N-methylthreonine derivative.
The amino acid was pretreated with HBTU, forming the HOBt
activated ester, which was immediately added to the peptidyl
resin 13 (Scheme 5). An ES-MS analysis on completion of the
cycle gave a molecular ion peak at m/z 1466 ([M + H]+, 90%) for
the Fmoc-deprotected dodecapeptide 14. The final residue to
complete the linear precursor in the synthesis of petriellin A was
Fmoc-L-proline. This coupling was accomplished by the BTC-
acid chloride method. After an overnight cycle, ES-MS of a small
sample of Fmoc-deprotected material gave a peak at m/z 1577
([M + H]+, 100%), confirming the successful synthesis of the
resin-bound linear cyclization precursor 15.
The remainder of the peptidyl resin was thus deprotected
with a solution of piperidine and DBU in DMF (2:2:96) for
3 min. The resin was washed and dried. Cleavage of the linear
precursor from the resin was effected by a solution of 1.5% TFA
in dichloromethane. Upon addition of the cleavage solution,
the resin beads immediately changed color from yellow to red,
indicating the peptide was free from the resin. The solution was
collected and immediately diluted with excess dichloromethane,
to reduce the concentration of the acid solution. The solution was
carefully removed under reduced pressure at room temperature.
Caution was taken when the peptide was exposed to this acidic
solution due to possible TFA-induced amidolysis observed with
natural petriellin A (vide supra) and other peptides.17,31,32
The crude linear precursor was isolated as an oil. The material
was dissolved in a solution of 50% acetonitrile/Milli-Q water and
the mixture was freeze-dried overnight. A colorless solid was
isolated in a moderate yield (58%, as calculated from the loading
of the first residue). An HPLC analysis was conducted, which
demonstrated that the linear precursor was sufficiently clean for
further manipulations.
Cyclizations of large peptides are generally carried out at high
dilutions. This technique is used to encourage the intramolecular
reaction to take place rather than an intermolecular reaction. In
the current cyclization, approximately 2 mL of solvent was used
for every milligram of the linear precursor. The solvent used is
another important consideration for the cyclization reaction. The
solvent chosen influences the solution-phase conformation the
peptide adopts and consequently directly influences the cycliza-
tion yield. There are no clearly defined parameters one can rely
on in selecting a solvent. Petriellin A is heavily N-methylated and
therefore there is already an increased risk of epimerization.24,25
Dipolar aprotic solvents, such as DMF, were avoided since they
further increase the risk of epimerization. A mixture of dichlor-
omethane and acetonitrile in a ratio of 10:1 was selected, as this
reaction medium has a low dielectric constant, reducing the risk
of epimerization. Acetonitrile was included to aid in solubilizing
the coupling reagents. Upon completion of the coupling reaction,
the solvents were easily removed under reduced pressure.
Initially, trial cyclizationꢀdeprotection reactions were con-
ducted and the products of these reactions were analyzed by
comparative HPLC with a sample of the natural product. The two
reactions, cyclization and deprotection, were conducted sequen-
tially without isolation and analysis of the cyclic t-butyl-protected
petriellin A intermediate. The reason for taking this approach
was to reduce the handling of the intermediate and ultimately
a Reagents and conditions: (a) 1.5% TFA in CH2Cl2. (b) (1) HBTU,
DIPEA, CH3CN/CH2Cl2 (1:9); (2) TFA/CH2Cl2 (1:1), 0 °C.
maximize the overall yield. The trial reactions were conducted on
a 1 mg scale. The linear precursor 16 (Scheme 6) was dissolved in
dichloromethane, and a solution of HBTU in acetonitrile and
dichloromethane was added in the presence of base. After 1 h the
solvent was removed under reduced pressure and the residue that
remained was cooled in an iceꢀsalt bath. In order to effect t-butyl
ether deprotection, a solution of 50% TFA in dichloromethane
was also cooled and then added to the cooled residue. The solu-
tion was left to stir on ice for 30 min and the temperature of the
reaction was maintained below 0 °C. Following this, the solution
was diluted with dichloromethane to a TFA concentration of no
more than 0.5%. The solution was removed in vacuo at room
temperature. The crude residue was analyzed by analytical HPLC.
A peak with a retention time of 16.12 min was confirmed as the
synthetic product after comparison to the retention time of the
natural product. Once the synthetic peptide 1 (Scheme 6) had
been identified, isolation of the desired peak was undertaken on
an analytical scale. Once the purified synthetic peptide was
isolated, analysis by ES-MS further confirmed the total synthesis
of petriellin A.
A control trial cyclizationꢀdeprotection reaction was under-
taken whereby no cooling was applied during TFA treatment.
All other aspects of the trial reaction were identical to those
described above. The crude material isolated was analyzed by
analytical HPLC. As suspected, the peak at 16.12 min for the
synthetic product was the minor component, as opposed to it
being the major component when the TFA deprotection was
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conducted below 0 °C. Furthermore, early-eluting byproduct
peaks dominated the HPLC trace in the room-temperature
experiment, suggesting formation of degradation products re-
sulting from TFA-induced hydrolysis during the uncooled acidic
treatment.
The couplingꢀdeprotection reaction was repeated on a larger
scale according to the first trial reaction described. A small sample
of the crude material was first analyzed by analytical HPLC to
ensure the presence of the synthetic product. After formation
of the synthetic product was verified via analytical HPLC,
purification and isolation by semipreparative HPLC was con-
ducted on the remaining crude material. A major peak was
detected and isolated at 28.4 min. ES-MS analysis confirmed
the component isolated was the desired product ([M + H]+ m/z
1433 (90%), [M + Na] m/z 1454 (90%)). The fractions isolated
were combined and found to have a purity of 87.07% by
analytical HPLC. The overall yield determined from the loading
of the first residue was 5%.
The natural product has been analyzed extensively by NMR.30
1
Therefore, H NMR analyses were conducted on the synthetic
product and compared to the literature. The aromatic region
between 6.8 and 7.6 ppm and the region containing the
R-protons between 4.5 and 6.0 ppm in 1H NMR spectra of the
synthetic product and the natural product were identical
Figure 2. 1H NMR spectrum of synthetic petriellin A 1 in acetone-d6/deuteriochloroform (1:4).
Figure 3. 1H NMR spectrum of natural petriellin A 1 in acetone-d6/deuteriochloroform (1:4).
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(Figures 2 and 3). The spectrum of the synthetic product does,
however, have extra peaks at 1.4 and 3.0 ppm, which were
attributed to solvent peaks from the deuterated solvent used
for the NMR analysis. These comparisons confirmed the total
synthesis of petriellin A by solid-phase chemistry.
1:9). The amino acid solution was added to the resin, followed by the
addition of DIPEA (1.03 mL, 6.0 mmol). The reaction was left to shake
in a reaction vessel equipped with a sintered bottom for 2 h. MeOH
(1.5 mL) was added and the mixture was shaken for a further 30 min.
The resin was filtered and washed with DMF, CH2Cl2 and ether,
successively. The resin was dried in vacuo overnight to yield 1.88 g,
52% (0.52 mmol/1 g) of loaded resin. The synthesis of petriellin A was
performed on 400 mg, 0.21 mmol of preloaded ClTrt resin.
’ CONCLUSIONS
Fmoc Deprotection. Fmoc-peptidyl resin was washed and then
shaken with piperidine/DBU/DMF (2:2:96) for 3 min. The resin was
filtered and washed with the cleavage solution, followed by successive
washing with DMF and CH2Cl2. Completion of the deprotection was
confirmed by a positive TNBS test33 for non-N-methylated amino acids
and by a positive chloranil test34 for N-methylated amino acids.
BTC-Mediated Couplings. To the semidry peptidyl ClTrt resin
(0.21 mmol) that had been previously swollen with CH2Cl2 were added
dry THF (1 mL) and DIPEA (287 μL, 1.6 mmol). In a separate reaction
vessel, BTC (72 mg, 0.24 mmol) was dissolved in dry THF (2.5 mL,
96 mM), followed by addition of the Fmoc-amino acid (0.74 mmol).
sym-Collidine (278 μL, 2.1 mmol) was added to the clear amino acid
solution and a precipitate immediately formed. The amino acid solution
was then added to the solution containing the resin beads and the
reaction was shaken for 4ꢀ24 h. The peptidyl ClTrt resin was washed
successively with DMF and CH2Cl2. Completion of the reaction was
confirmed by a negative TNBS test33 for non-N-methylated residues and
the chloranil test34 for N-methylated residues. In some cases further
confirmation was gained through cleavage of the growing peptide from
a small amount of ClTrt resin, followed by mass spectral analysis.
HBTU/HOBt-Mediated Couplings. To the semidry peptidyl
ClTrt resin (0.21 mmol) that had been previously swollen with CH2Cl2
was added dry DMF (1 mL). In a separate vessel, the Fmoc-amino acid
(0.74 mmol) was dissolved in a solution of HBTU (278 mg, 0.74 mmol)
and HOBt (100 mg, 0.75 mmol) in DMF (3 mL). To this colorless
solution was added DIPEA (287 μL, 1.7 mmol), and the mixture was left
to stir for 5 min, until a yellow solution resulted. This solution was added
to the peptidyl ClTrt resin and the reaction was shaken for 4ꢀ24 h.
Completion of the reaction was confirmed by a negative TNBS test33 for
non-N-methylated residues and the chloranil test34 for N-methylated
residues. In some cases further conformation was gained through
cleavage of the growing peptide from a small amount of ClTrt resin,
followed by mass spectral analysis.
DIC/HOAt-Mediated Couplings. To the semidry peptidyl ClTrt
resin (0.21 mmol) that had been previously swollen with CH2Cl2
was added dry DMF (1 mL). In a separate vessel, the Fmoc-amino acid
(0.63 mmol) and HOAt (86 mg, 0.63 mmol) were dissolved in dry
CH2Cl2/DMF (2.5 mL, 1:1). DIC (98 μL, 0.68 mmol) was added and
the reaction was allowed to stir for 5 min. The amino acid solution was
poured over the peptidyl resin and the reaction was shaken for 4ꢀ24 h.
Completion of the reaction was confirmed by a negative TNBS test33 for
non-N-methylated residues and the chloranil test34 for N-methylated
residues. In some cases further conformation was gained through
cleavage of the growing peptide from a small amount of ClTrt resin,
followed by mass spectral analysis.
Cleavage of Linear Precursor from Resin. The peptidyl ClTrt
resin (0.21 mmol) was treated with a 1.5% solution of TFA in CH2Cl2
(2 mL). Upon this addition, the resin beads immediately turned bright
red in color and the cleavage mixture was filtered. The resin beads were
washed with the cleavage mixture (3 mL ꢁ 2) and then with CH2Cl2
(10 mL ꢁ 2). The solution was reduced in vacuo and the residue was
resuspended in HPLC-grade CH3CN/Milli-Q H2O (1:1). The mixture
was freeze-dried overnight to yield a colorless solid (189 mg, 58%).
A sample (20 μg) of the crude product was subjected to analytical
reverse-phase HPLC on a C18 column (150 mm ꢁ3.2 mm). The crude
product was eluted with a gradient from 0 to 100% buffer B in buffer A
Petriellin A was successfully synthesized by solid-phase pep-
tide synthesis techniques in an overall yield of 5%, as determined
from the loading of the first amino acid. The 2-chlorotrityl
chloride resin was used to synthesize the peptide and the residues
were incorporated as the Fmoc-amino acid derivatives. Solution-
phase synthesis was applied in the production of the depsipeptide
bond contained within petriellin A. The presynthesized depsi-
peptide was successfully incorporated by solid-phase techniques
with no detection of any deletion products. Cleavage of the
linear peptide by 1.5% TFA in dichloromethane did not result
in any detectable TFA-induced amide bond hydrolysis. Most
importantly, cleavage of the t-butyl protecting groups from
the threonine residues in 50% TFA in dichloromethane at room
temperature caused significant loss of the desired synthetic
product. However, when the same TFA treatment was con-
ducted below 0 °C, the yield of synthetic product increased
significantly, as indicated by analytical HPLC. Furthermore, this
reduced-temperature TFA deprotection technique may be useful
in the synthesis of other heavily N-methylated peptides. The
cyclization was conducted at high dilution, and analysis con-
firmed that the linear tridecapeptide intermediate underwent the
desired intramolecular reaction, rather than an undesired inter-
molecular reaction.
’ EXPERIMENTAL SECTION
(R)-N-(9-Fluorenylmethoxycarbonyl)-(S)-pipecolinylphe-
nyllactic Acid 10. Fmoc-L-pipecolic acid (1.4 g, 4.1 mmol), alcohol 7
(1 g, 4.5 mmol), and triphenylphosphine (TPP) (1.4 g, 5.4 mmol) were
dissolved in dry CH2Cl2 (10 mL) under an atmosphere of N2. The
mixture was cooled to 0 °C, and diethyl azodicarboxylate (DEAD)
(840 μL, 5.4 mmol) was added in three portions over 10 min. The
reaction was stirred at 0 °C for 1 h and then at room temperature for 5 h.
After this time the solvent was concentrated in vacuo and the residue was
crystallized several times from diethyl ether. The filtrate was collected
and again concentrated. The crude residue was purified by flash column
chromatography eluted with 20% ethyl acetate/hexane to yield the
depsipeptide 9 as a colorless oil (1.54 g, 69% yield). 1H NMR (300 MHz,
CDCl3) (rotamers) δ 7.77ꢀ7.13 (13H, m), 5.16ꢀ4.85 (1H, m),
4.42ꢀ4.12 (3H, m), 3.13ꢀ3.01 (2H, m), 2.31ꢀ2.17 (2H, m),
1.60ꢀ1.20 (15H, m). 13C NMR (75 MHz, CDCl3) (rotamers) δ
173.1, 170.8, 170.6, 167.9, 167.8, 155.8, 155.0, 143.8, 143.7, 141.0,
136.5, 135.6, 135.4, 129.4, 129.1, 128.1, 128.0, 127.9, 127.4, 126.8, 126.6,
126.4, 124.8, 124.6, 119.7, 82.0, 73.9, 73.6, 71.0, 67.4, 54.3, 54.0, 46.9,
41.6, 41.3, 40.3, 37.0, 27.7, 27.6, 26.8, 26.4, 24.5, 24.3, 20.3, 20.1.
The ester 9 was then dissolved in CH2Cl2 (3 mL) and TFA (3 mL). The
mixture was left to stand for 30 min and then concentrated under
reduced pressure from dichloromethane (10 mL ꢁ 3). Formation of
the acid 10 was confirmed by ES-MS m/z 500.5 ([M + H]+, 90%) and
522.4 ([M + Na], 100%). HRMS m/z for C30H30NO6 [M + H]+ calcd
500.2073, found 500.2059; m/z for C30H29NNaO6 [M + Na]+ calcd
522.1893, found 522.1874.
Loading of Resin. To the 2-chlorotrityl resin (ClTrt) (1.57 g, 1.57
mmol) was added dry CH2Cl2 (10 mL). In a separate vessel, Fmoc-L-
alanine (311 mg, 1.0 mmol) was dissolved in DMF/CH2Cl2 (10 mL,
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dx.doi.org/10.1021/jo201017w |J. Org. Chem. 2011, 76, 6686–6693

The Journal of Organic Chemistry
ARTICLE
(see Supporting Information) over 20 min at a flow rate of 0.8 mL/min
and detected at 254 nm. HPLC retention time was 15.5 min. The
analytical trace showed the crude linear peptide to be sufficiently clean
to proceed to the cyclization without HPLC purification. (Found:
[M + H]+ 1562.0275. C82H137N12O17 requires [M + H]+ 1562.0225).
Cyclization and Deprotection (Synthesis of Petriellin A).
The crude linear peptide (25 mg, 16 μmol) and HBTU (61 mg, 160
μmol) were dissolved in dry CH3CN/CH2Cl2 (1:9) (50 mL, 1 mL/g).
DIPEA (55 μL, 320 μmol) was added and the reaction was left to stir
for 1 h under an atmosphere of nitrogen. The mixture was reduced in
vacuo and the crude residue was cooled in a saltꢀice bath. Meanwhile,
a solution of 50% TFA/dry CH2Cl2 (5 mL) was prepared and cooled in
liquid N2. The cooled TFA mixture was poured onto the cooled crude
peptide and allowed to stir for 30 min. The reaction mixture was diluted
with CH2Cl2 (150 mL) and reduced in vacuo at room temperature. The
crude product was dissolved in HPLC-grade 50% CH3CN/Milli-Q H2O
(20 mL). A sample of this solution (20 μL) and a sample of natural
petriellin A30 were subjected to analytical reverse-phase HPLC on a C18
column (150 mm ꢁ 3.2 mm). The crude product and natural petriellin A
were eluted with a gradient from 0 to 100% buffer B in buffer A over
20 min at a flow rate of 0.8 mL/min and detected at 220 nm. HPLC
(synthetic and natural petriellin A)30 retention time was 16 min. Once
it was confirmed that the synthetic product was in the crude mixture, it
was subjected to semipreparative reverse-phase HPLC on a C18 column
(50 mm ꢁ 21.2 mm). The crude product was eluted with a gradient from
20% to 80% buffer B in buffer A over 30 min at a flow rate of 6 mL/min
and detected at 220 nm. HPLC retention time was 28.4 min. The
appropriate fractions were collected and freeze-dried overnight to yield
a colorless white solid (2.1 mg, 9% from the linear precursor or 5% from
loading of the first residue). Comparative analytical reverse-phase HPLC
between the synthetic product (10 μg) and the natural product30
(10 μg) was conducted on a C18 column (150 mm ꢁ 4.6 mm). The
products were eluted with a gradient from 0 to 100% buffer B in buffer A
over 25 min at a flow rate of 1 mL/min and detected at 220 nm. HPLC
(synthetic and natural30 petriellin A) retention time was 21.5 min.
(Found: [M + H]+ 1431.8813. C74H118N12O16 requires [M + H]+
1431.8867). 1H NMR data were identical to the data previously reported
for the natural product30 (Figures 2 and 3).
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’ ASSOCIATED CONTENT
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S
Supporting Information. General experimental meth-
b
ods and 1H and 13C NMR spectra for compounds 1, 9, and 10.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail tahughes@optusnet.com.au.
’ ACKNOWLEDGMENT
M.M.S. is grateful to the Australian government for the award
of a postgraduate scholarship.
’ REFERENCES
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