The Journal of Organic Chemistry
ARTICLE
(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,
6692
dx.doi.org/10.1021/jo201017w |J. Org. Chem. 2011, 76, 6686–6693