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residual DIC, diisopropylurea and unreacted EtOC-
Phe-OH. The peptide couplings were carried out using
0.1 M glycine in pH 10 aqueous solution at 21°C for 16
h.15 The products were isolated by repeated rinsing of
the resin with water, followed by acidification of the
aqueous filtrate and extraction into ethyl acetate.
Under these conditions, the HOBt active ester afforded
a small amount of the dipeptide product along with a
greater amount of the direct hydrolysis product, EtOC-
Phe-OH (Table 1). Although HOBt has been used as an
additive in aqueous solution-phase coupling reactions,
the slower reaction rates associated with solid supports
presumably are to blame for the increased levels of
hydrolysis observed with resin 1.
In contrast to the HOBt resin, active esters derived
from both the HOSu resin 2 and the tetrafluorophenol
resin 3 produced the dipeptide as the major product,
with the latter affording a higher ratio of coupling to
hydrolysis. The mass balance in all of these reaction is
around 60–70%. This reflects the effect of the solvent
change on the swelling of the resin. In water, TentaGel
resin only swells to 2/3 the volume observed in THF.
When the recovered resins were treated with t-BuNH2
in THF, the remaining active ester was released as the
t-Bu amide.
Scheme 1. Preparation of TentaGel linked HOBt.
as DMAP must be avoided in this reaction, otherwise
by-products arising from nucleophilic aromatic substi-
tution are observed. The amide 5a was treated with
neat hydrazine to form 6a. Although the reaction
appeared to be quantitative, purification of the product
was extremely difficult. Thus it was treated directly with
aqueous sodium hydroxide to form the 1-hydroxyben-
zotriazole 7 in 94% yield over two steps. Direct applica-
tion of this protocol to TentaGel-NH2 (0.44 mmol/g)
afforded a low loading (<5%) of the final HOBt unit 1
(see below for quantitation procedures). The low yield
was traced to the nucleophilic aromatic substitution
step, as it was observed that TentaGel resins do not
swell significantly in pure hydrazine. The addition of
DMF as co-solvent (3:1 DMF/NH2NH2) in this reac-
tion was a sufficient remedy and using this procedure,
the HOBt resin was obtained in high yield (vide infra).
The remaining resins, HOSu analog (2) and PFP analog
(3), were prepared following minor modifications of
literature procedures. Thus, TentaGel-SH was prepared
by treatment of TentaGel-Br (0.27 mmol/g) with
thiourea followed by hydrolysis.13 The resulting thiol
was allowed to react with N-hydroxymalimide in the
presence of pyridine to form resin 2 in modest yield.6b,14
The PFP analog 3 was prepared in good yield by
acylating TentaGel-NH2 with 4-hydroxy-2,3,5,6-tetra-
fluorobenzoic acid using diisopropylcarbodiimide
(DIC) and pyridine in THF, followed by treatment with
t-BuNH2 to cleave any oligomeric acylation products.6c
Given the higher ratio of coupling to hydrolysis for
resin 3, along with its facile and less costly preparation
relative to 2, we chose to study its properties further. A
pH survey using 0.1 M glycine solution showed that the
efficiency of coupling is highest above pH 9 (Table 2).
Even at pH 8.5, where glycine is 95% N-protonated, the
coupling still proceeds in good yield. Separate experi-
ments established that even under the alkaline condi-
In all three cases, the yield for the synthesis of the
alcohol was quantified by preparing the active ester of
EtOC-Phe-OH using DIC in THF, followed by reaction
with tert-butylamine (10 equiv) in THF to form EtOC-
Phe-NHtBu (Scheme 2). The yield of the amide cou-
pling step was taken as a lower limit for the yield of the
resin preparation, and indicated a 77% yield of 1 (0.34
mmol/g), a 24% yield for 2 (0.065 mmol/g), and a 73%
yield for 3 (0.32 mmol/g).
Scheme 2. Peptide coupling using active esters.
Table 1. Formation of dipeptides in water using of active
esters derived from resins 1–3a
Resin
EtOC-Phe-Gly (10)
EtOC-Phe (11)
1
2
3
30%
51%
63%
41%
14%
3%
The ability of all three resins to effect peptide coupling
in aqueous solution via their active esters was studied.
The active esters of EtOC-Phe-OH were prepared from
all three resins using standard conditions (DIC/THF).
The resins were carefully rinsed to remove all traces of
a All couplings were carried out with 0.1 M glycine at pH 10 in water
at 21°C for 16 h. Yields are relative to the loading of the alcohol as
determined above.