J. Blodgett, T. Li / Tetrahedron Letters 45 (2004) 6649–6652
6651
O
O
O
O
O
NH2
O
OH
O
1
2
3
O
C
O
H
N
O
CH
CH2
O
H
N
O
OMe
O
O
5
4
N
HN
O
Scheme 1. Tagged compounds for evaluations.
Table 2. Effectiveness of 4-tert-butylphenyl group as a tag for column
separation using a beta-cyclodextrin column
the potential to be an efficient and general tag for solu-
tion-phase organic synthesis.
Compounds
Retention time (min) Retention time (min)
with mobile phase A with mobile phase B
A model study was then performed to test the practical-
ity of this tag strategy. For this purpose, 4-(tert-but-
yl)benzyl bromide was chosen as the tagging reagent,
as it can be removed cleanly by catalytic hydrogenolysis.
A commercially available amino acid derivative, Fmoc-
Ser(tBu)-OH, was tagged according to the conditions in
Scheme 2. Excess amount (2equiv) of starting material
Fmoc-Ser(tBu)-OH was used in the reaction and the
reaction mixture was evaluated directly by HPLC. It
was found that with mobile phase up to 50% methanol
in 0.5% TFA in H2O, the tagged amino acid is efficiently
retained by the beta-cyclodextrin stationary phase even
when injected in large amounts. Subsequently, a proto-
col involving eluting the column with 40% methanol in
0.5% TFA in H2O until all impurities were eluted, and
then switching to a gradient up to 100% methanol was
employed to elute the tagged Fmoc-Ser(tBu)-OH prod-
uct. An aliquot of reaction mixture that contains
roughly 20mg of reaction product was successfully sep-
arated in one run using just the analytical HPLC column
mentioned earlier by following this protocol with a
recovery yield over 95%. This purification protocol
was then streamlined by changing directly to a pure
methanol mobile phase from 40% methanol in
0.5%TFA/water to elute the tagged product. By follow-
ing this streamlined protocol, tagged Gly 7 was also suc-
cessfully isolated.
Benzyl alcohol
Benzoic acid
4-tert-Butylphenol 91
5
5.6
Broad
85
15
tert-Butyl ester 1
Acid 2
Amine 3
78
70
40
87
81
90
80
Tagged His ester 4 54
Tagged Tyr ester 5 88
90
Mobile phase A: a linear gradient from 0% methanol to 100% meth-
anol in 0.5% TFA/water over 3h. Mobile phase B: a linear gradient
from 0% methanol to 100% methanol in water over 3h. Flow rate:
1mL/min; column size: 25cm · 4.6mm.
are very close to that of 4-tert-butylphenol and very
different from two controlled compounds (benzyl alco-
hol and benzoic acid) with the exceptions of compounds
3 and 4, which bear a basic nitrogen. Upon removing
TFA from the mobile phase, retention times of
compounds 3 and 4 become comparable to that of
4-tert-butylphenol, suggesting that protonation of the
basic nitrogen in these compounds is responsible for
their short retention time with TFA-containing mobile
phase. In this cyclodextrin based chromatographic sys-
tem, the retention mechanism is hydrophobic in nature.
Therefore, a highly hydrophilic functional group such as
ammonium ion could influence the retention time of
these tagged compounds. However, other polar func-
tional groups such as carboxylic acid appear to have lit-
tle impact on the chromatographic retention of these
tagged compounds. For example, the retention times of
tert-butyl ester 1 and free acid 2 are almost identical.
Even for these basic nitrogen-containing compounds 3
and 4, the retention times are still significantly higher
than those of the controlled compounds benzyl alcohol
and benzoic acid. Moreover, for the intended tag appli-
cation, TFA is required only when acidic non-tagged
molecules exist, as they tend to tail in the absence of acid
in the mobile phase. Linkage of the 4-tert-butyphenyl to
the target molecule seems to have little impact on the
retention behavior, as the linkage through a CH2 group
instead of oxygen in compound 3 does not reduce its
retention times when compared to tagged His ester 4.
These results indicate that the 4-tert-butylphenyl has
Another concern with the tagging system is the ease with
which one can remove the tag from the target molecule
after separation. In this particular example, the tag can
be removed conveniently by catalytic hydrogenolysis.
For example, the catalytic hydrogenolysis to recover
O
Ser(OtBu)-Fmoc
(a)
Fmoc-Ser(OtBu)-OH
Boc-Gly-OH
6
O
Gly-Boc
(a)
(b)
7
Scheme 2. Tag attachment and removal. (a) Cs2CO3, 4-(tert-
butyl)benzyl bromide; (b) H2, Pd/C.