H. Katayama, T. Goto / Tetrahedron Letters 58 (2017) 610–613
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Scheme 1. Synthesis of Fmoc-Cys(Pocam)-OH derivatives. Reaction conditions: (a)
1. Disuccinimidyl carbonate, diisopropylethylamine (DIEA), DMAP, DMF; 2. R-NH2,
DIEA. (b) 1. Formalin, Na2CO3, H2O/1,2-dimethoxyethane; 2. Cysteine hydrochlo-
ride, TFA; 3: Fmoc-OSu, DIEA, 1,2-dimethoxyethane.
electron-donated group on Pocam, we also tried to synthesize iso-
butyl (i-Bu) derivative other than Me-Pocam. The various Cys
(Pocam) derivatives were synthesized essentially according to the
method described previously (Scheme 1).7 Equimolar amounts of
2-hydroxyacetophenone ethylene acetal and disuccinimidyl car-
bonate were mixed, and then alkylamine was added to the reaction
mixture, giving phenacyl N-alkylcarbamate ethylene acetal 1. The
yields of Moe-, Tfe- and i-Bu-derivatives were 81%, 86% and 79%,
respectively, and these values were comparable to that of Me-
derivative (68%) reported previously.7 The products 1 were then
hydroxymethylated with formalin, and introduced to cysteine in
neat TFA. Finally, the amino group of cysteine derivatives was pro-
tected by Fmoc group, giving Moe-, Tfe- and i-Bu-derivatized
Fmoc-Cys(Pocam)-OH 2 in 70%, 55% and 48% yields, respectively.
In order to examine the stability of various Pocam groups under
TFA acidic conditions, these Cys(Pocam) derivatives 2 were sepa-
rately introduced to a peptide by Fmoc-SPPS, and the yields of
Pocam-attached and Pocam-deprotected peptides were deter-
mined after deprotection step with TFA cocktail treatment and
reversed-phase (RP)-HPLC purification. At first, we synthesized
the C-terminal segment of insect growth-blocking peptide (GBP),
GBP(11-25), as a model. GBP was originally isolated from the larval
hemolymph of the host armyworm, Pseudaletia separata, whose
development is halted in the last larval instar stage from parasiti-
zation by the parasitoid wasp, Cotesia hariyai.11–13 Starting from
Fmoc-Gln(Trt)-Wang resin, the peptide chain corresponding to
the GBP sequence was elongated by the ordinary Fmoc-SPPS using
N,N’-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole
(HOBt) as condensation reagents. During SPPS, Fmoc-Lys(N3)-OH
and Fmoc-Cys(Pocam)-OH derivatives were used at Lys20 and
Cys19 positions, respectively. After the chain assembly, crude pep-
tides were cleaved from the solid support, and analyzed by RP-
HPLC. As a result, no significant side reaction on any Pocam groups
was observed during SPPS, and the desired peptides 3 were found
on HPLC chromatograms (Fig. 1).
Figure 1. RP-HPLC elution profiles of GBP(11–25) segments containing various Cys
(Pocam) derivatives after the TFA cocktail treatment at room temperature for 2 h.
(a) Me-Pocam. (b) i-Bu-Pocam. (c) Moe-Pocam. (d) Tfe-Pocam. Column: Mightysil
RP-18 GP (4.6/ Â 150 mm), eluent: 0.1% TFA in aqueous acetonitrile at a flow rate of
1 mL/min.
Table 1
The isolated yields of Pocam-attached (3a-d) and deprotected (4) peptides after TFA
treatment.
Substituent
Protected (3) (%)
Deprotected (4) (%)
Methyl
Isobutyl
2-methoxyethyl
2,2,2-trifluoroethyl
7.8
2.5
25.8
44.9
35.0
37.9
16.4
1.4
Values mean isolated yield of each peptide quantified by amino acid analysis.
isolated yield of 3b was quite low (2.5%). In contrast, the yields
of Tfe- and Moe-Pocam peptides (3c and 3d) were higher than that
of Me-Pocam peptide 3a, indicating that the substituent with EWG
improved the stability of Pocam group. Especially, Tfe-Pocam
group was almost completely stable in TFA solution, and only a
trace amount of Pocam-deprotected peptide 4 was observed
(Fig. 1d). These results were consistent with the observation on
Allocam group reported previously.10
In order to examine the usefulness of Tfe-Pocam group on the
peptide condensation reaction by the thioester method, we synthe-
sized the N-terminal segment of GBP, GBP(1–10) thioester, and
condensed it with the C-terminal segment prepared as described
above. To obtain the peptide thioester, we used N-alkylcysteine
(NAC)-assisted thioesterification reaction.14 Fmoc-(Et)Cys(Trt)-
OH, which was prepared by the previously described method,14,15
was introduced to Rink-Amide MBHA resin. After the removal of
Fmoc group, Fmoc-Gly-OH was condensed using O-(7-azabenzotri-
Pocam-deprotected peptide 4 was also observed on the all chro-
matograms, indicating that all of Pocam groups tested were
cleaved at least in part with exposure to TFA. To evaluate the sta-
bility of Pocam groups in the TFA solution, Pocam-attached peptide
3 and Pocam-deprotected peptide 4 in each crude peptide were
purified with RP-HPLC and quantified by amino acid analysis. The
isolated yield of each peptide was summarized in Table 1. When
i-Bu group was attached to Pocam group, the stability under TFA
acidic conditions was decreased compared to Me-Pocam, and the
azol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HATU) as a condensation reagent. Then, the peptide chain corre-
sponding to the GBP sequence was elongated by the DCC-HOBt
method. After the chain assembly, Fmoc group was removed with