Angewandte
Chemie
from 4c and 4d by removal from the
resin and cleavage of the tert-butyl
ether and trityl thioether; Figure 1).
After treatment at room temper-
ature for two periods of 6 hours, the
estimated peptidyl resin loading was
the same as that observed with DIC/
DMAP (0.47 mmolgÀ1, 64% yield;
see calculation in the Supporting
Information). Moreover, epimeriza-
tion of the cysteine residue linked to
a resin through an ester bond during
peptide elongation was investigated
by the synthesis of d-Cys7-contain-
ing octreotide: no epimerization was
observed (see the Supporting Infor-
mation).
During the synthesis of 1a,
which contains an unfavorable l/d
combination, the risk of diketopi-
perazine formation[36] was analyzed
(see the Supporting Information).
Upon treatment with DMF/piperi-
dine for 1 hour, no release of the
diketopiperazine [d-Leu-l-Trp] was
observed.
Further elongation by the
HBTU/DIEA coupling strategy
yielded the resin-anchored isopepti-
des 5a–c. Cleavage from the resin by
treatment with TFA afforded the free isopeptides 6a–c with
high purity (80–95%). Compounds 6a–c were then subjected
to the O–N acyl-migration reaction. They were dissolved and
stirred in aqueous phosphate buffer at pH 7.4 or in the
organic solvent mixture DMF/piperidine (80:20),[22] and the
intramolecular O–N acyl migration was monitored for each
compound by RP HPLC and LC/MS analysis.
Figure 2. Monitoring of the formation of octreotide (1c). HPLC profiles A) of crude isooctreotide
6c, B) for the conversion of 6c into the corresponding peptide alcohol, and C) of octreotide (1c).
(HPLC conditions: VWR chromolith column; detection at 214 nm; elution: 0–100% B linear
gradient over 3 min at a flow rate of 5 mLminÀ1; solvent A: H2O/0.1% TFA; solvent B: CH3CN/
0.1% TFA).
Experimental Section
3a–c: The Fmoc-b-amino alcohol 2 (3 equiv) was dissolved in a
solution of DBU (2%) in anhydrous DMF, and the resulting solution
was added to the 2-chlorotrityl chloride resin (1.55 mmolgÀ1). The
reaction mixture was stirred at room temperature for 12 h, and the
resin was then removed by filtration and washed with DMF (ꢀ 3),
10% DIEA in MeOH (ꢀ 3), DMF (ꢀ 3), and CH2Cl2 (ꢀ 3).
4a,b and 4c’: DIC (3 equiv) and DMAP (0.3 equiv) were added to
a solution of Fmoc-AA1-OH (6 equiv relative to resin substitution) in
The intramolecular transposition reaction was quantita-
tive in organic solvents at room temperature; it reached
completion within 1 hour to afford the crude compounds 1a,b
with good purity (average, 90%; see the Supporting Informa-
tion). The yields of the recovered peptides were between 80
and 90% on the basis of the calculated resin loading of 4a,b.
For octreotide (1c), the O–N acyl shift and oxidation of 6c
were performed under slightly alkaline conditions at room
temperature in a phosphate buffer, either in the presence of
oxyfold resin (5 equiv)[37] for 6 hour or without the oxidizing
reagent for 2 days. Octreotide (1c) was recovered in high
yield with high purity (Figure 2). Notably, complete conver-
sion of the isopeptide 6c into the corresponding peptide
alcohol occurred prior to the formation of the disulfide bond.
We have reported a simple method for the synthesis of
challenging peptide-alcohol targets from b-amino alcohol
starting materials through the use of an O–N acyl-transfer
reaction. This methodology proved to be efficient for the
loading of the b-amino alcohols onto the resin through their
amino function, and no epimerization was observed when the
Mitsunobu coupling protocol was used for acylation with a
cysteine derivative.
a
mixture of anhydrous CH2Cl2 and DMF (50:50) at 08C
(3 mLmmolÀ1). The resulting mixture was stirred with the b-amino
alcohol functionalized resin for 6 h at room temperature. This
procedure was repeated twice, and then the loading was determined
by Fmoc titration: 4a: 0.46 mmolgÀ1
0.47 mmolgÀ1
; ; 4c’:
4b: 0.42 mmolgÀ1
4c:
A solution of PPh3 (3 equiv) and Fmoc-Cys(Trt)-OH
(3 equiv) in anhydrous THF (20 mL per gram of resin) was added
to the resin. A 40% solution of DEAD in toluene (3 equiv) was then
added, and the mixture was stirred for 6 h at room temperature. This
procedure was repeated twice. The loading was determined by Fmoc
titration: 0.47 mmolgÀ1
.
Elongation and cleavage of the isopeptides were performed by
conventional SPPS (see the Supporting Information).
1c: Isopeptide 6c was dissolved in phosphate buffer (pH ꢀ 7.4),
and the mixture was stirred at room temperature. The reaction was
monitored by HPLC and LC/MS. After 30 min, conversion of the
isopeptide 6c into the corresponding peptide alcohol was observed:
m/z 1021.8 [M+H]+. After 48 h, the filtrate was concentrated under
reduced pressure and lyophilized for analysis by HPLC and LC/MS.
HPLC: tR = 1.21 min (89% purity for the crude compound); LC/MS:
monoisotopic mass calculated for C49H66N10O10S2: 1018.4 Da; found:
Angew. Chem. Int. Ed. 2010, 49, 117 –120
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
119