A 4-OTBS benzyl-based protective group for carboxylic acids

Article abstract of DOI:10.1016/j.tetlet.2019.05.039

Reported herein is a novel 4-OTBS benzyl-based protective group for carboxylic acids. This protective group can be removed in the presence of TBAF or TFA with high efficiency, which makes it compatible with base-sensitive or acid-sensitive substrates. With this protective group, a near-infrared fluorogenic probe for the detection of γ-glutamyltranspeptidase activities was readily prepared.

Full text of DOI:10.1016/j.tetlet.2019.05.039

Accepted Manuscript
A 4-OTBS Benzyl-based Protective Group for Carboxylic Acids
Zhijun Fang, Yuyao Li, Hexin Xie
PII:
S0040-4039(19)30486-1
https://doi.org/10.1016/j.tetlet.2019.05.039
TETL 50811
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
26 February 2019
17 May 2019
20 May 2019
Please cite this article as: Fang, Z., Li, Y., Xie, H., A 4-OTBS Benzyl-based Protective Group for Carboxylic Acids,
Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.05.039
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Tetrahedron Letters
A 4-OTBS Benzyl-based Protective Group for Carboxylic Acids
Zhijun Fang, Yuyao Li and Hexin Xie*
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Reported herein is a novel 4-OTBS benzyl-based protective group for carboxylic acids.
This protective group can be removed in the presence of TBAF or TFA with high
efficiency, which makes it compatible with base-sensitive or acid-sensitive substrates. With
this protective group, a near-infrared fluorogenic probe for the detection of -
glutamyltranspeptidase activities was readily prepared.
Available online
Keywords:
Protective group
Carboxylic acid
4
-OTBS benzyl
GGT probe
Introduction
carboxylic acids and the removal of benzyl to regenerate carboxylic
acids usually involves hydrogenolysis in the presence of transition
metals (e.g., Pd, Pt). This deprotection strategy, though highly
Carboxylic acid is among the most commonly encountered
functional groups in natural products and biologically interesting
molecules. Due to the high reactivity, carboxylic acids are normally
protected in multiple-step synthesis, mostly in form of ester, such as
allyl, benzyl, tert-butyl, trimethylsilylethyl, and diphenylmethyl
ester. Photolabile protecting groups, for instance, 2,5-
dimethylphenacyl, 1-[2-(2-hydroxyalkyl)phenyl]ethanone,
aminocoumarin, 3-hydroxy -2-naphthalenemethanol, N-alkyl-4-
efficient, may be less compatible with other hydrogenation-sensitive
functional groups, for instance, nitro, carboxybenzyl (Cbz), and
1
1a, 1c
olefin.
To avoid the use of hydrogenolysis and thus make it
orthogonal to hydrogenation-sensitive substrates, we envisaged the
introduction of a caged hydroxyl on the para-position of benzyl
might allow releasing protective group in a different manner: once
the hydroxyl group is uncaged the resulting 4-hydroxyl benzyl ester
2
3
4
5
6
7
8
picolinium (NAP), 2-(1’-hydroxyethyl)-anthraquinone, 3-
4
will undergo a spontaneously 1, 6-elimination to yield free
9
10
aminobenzyl, -carboxy-6-nitroveratryl, and benzoin-derived
carboxylic acid 1 and para-quinone methide (p-QM) under base
conditions (Scheme 1). And the highly active p-QM may be further
attacked by water to form 4-hydroxyl benzyl alcohol (5). As a result,
we may manipulate the deprotection specificity of carboxylic acid by
the selection of proper protective group for 4-hydroxyl group.
1
1
desyl (Dsy), are another type of compelling protective groups for
carboxylic acid because of the unique deprotection strategy. Very
recently, Fang reported the use of dM-Dim for protection of
carboxylic acid, which allowed selectively deprotection under nearly
1
2
neutral oxidative conditions. Almost at the same time, an azulene-
based protection of carboxylic acids was developed by the Harvey
lab, in which, interestingly, the removal of this protection group can
Silyl ethers, such as trimethylsilyl (TMS), triethylsilyl (TES),
tert-butyldimethylsilyl (TBS), and tert-butyldiphenylsilyl (TBDPS),
are a type of well-studied protective groups for hydroxyl. And
stability of these protective groups increases in this order
1
3
be monitored by the change of color. Nevertheless, in spite of
great success of these protection strategies, novel protection group
for carboxylic acids that is orthogonal to other types of protective
groups is still of high demand given the vast variety of chemical
transformations involved carboxylic acids in organic synthesis.
Herein, we wish to report a 4-OTBS benzyl group for the protection
of carboxylic acids, which can be readily introduced and removed in
nearly quantitative yields.
1
a, 14
TBDPS>TBS>TES> TMS.
Proper selection of reaction
conditions allows removal of these protecting groups selectively.
Benzyl is one of the most common protective groups for
*Corresponding authors.
E-mail addresses: xiehexin@ecust.edu.cn (H. Xie)
Scheme 1. Protection of carboxylic acid with caged 4-hydroxyl benzyl.

a
Table 1. Optimization of deprotection conditions
Scheme 2. Selective deprotection of substituted benzyl esters. 3a or 3a-3 (24
mol) and TsOH (5 mol%) in DCM/MeOH (1:1, 0.42 mL) were stirred at rt
for 45 min, followed by the addition of NaHCO3 (aq., 0.42 mL).
Determined by HPLC analysis.

-
F source
Time
Yield
a
Entry
Substrate
Solvent
b
(
equiv.)
NaF (2)
(NH )HF (1)
Py•HF (2)
Et N•3HF
0.67)
KHF (1)
(h)
(%)
c
1
2
3
3a
3a
3a
PBS:THF = 1:2
PBS:THF = 1:2
PBS:THF = 1:2
2
<5 (14)
c
4
2
2
<5 (12)
a
Table 2. Deprotection of p-OTBS benzyl esters.
2
<5
3
4
3a
PBS:THF = 1:2
2
<5
(
5
6
7
8
9
3a
3a
2
PBS:THF = 1:2
PBS:THF = 1:2
DMF:THF = 1:2
2
2
2
2
2
2
2
2
<5
68
75
74
76
76
81
93
95
99
97
98
b
TBAF (2)
TBAF (2)
TBAF (2)
TBAF (2)
TBAF (2)
TBAF (3)
TBAF (5)
TBAF (8)
TBAF (10)
TBAF (10)
TBAF (10)
Entry
1
Ester
Acid
Yield (%)
95
3a
3a
MeOH:THF = 1:2
1a
3a
MeCN:THF = 1:2
10
11
12
13
14
15
16
3a
THF
THF
THF
THF
THF
THF
THF
3a
2
3
1b
1c
1d
93
98
3a
3a
0.5
0.5
0.5
0.5
3a
3a-1
3a-2
4
98
a
Unless otherwise noted, a mixture of 3 (33 mol) and fluoride reagent in
b
indicated solvent (0.1 mL) were stirred at room temperature. Determined by
RP-HPLC analysis on C18 column. Used NaHCO
c
5
6
7
1e
1f
99
96
97
3
aqueous solution instead
of PBS as co-solvent.
a)
3
a
150
3a + TBAF
1g
1a
1
00
b
c
d
8
1h
1i
93 (97 )
5
0
b
d
9
73 (94 )
0
5
10
15
20
25
30
a
Time (min)
Unless otherwise noted, 3 (0.28 mmol) was treated with TBAF (1.0 M in
b)
b
c
300
200
100
0
THF, 2.8 mL) for 0.5 h at rt. Isolated yield. Mixed with pre-neutralized
d
3
a
TBAF (pH 7) for 0.5 h followed by the treatment of NaHCO
Conducted with a mixture of TFA:DCM (1:1).
3
(aq.).
3
3
a + neutralized TBAF
a + neutralized TBAF + NaHCO
3
1a
Fluoride ion has been reported as efficient TBS-deprotection
1
a
agent. Therefore, a number of fluoride sources, including sodium
fluoride, ammonium hydrogen difluoride, pyridine hydrogen
fluoride, triethylamine trihydrofluoride, potassium bifluoride, and
tetrabutylammonium fluoride (TBAF), were initially examined and a
mixture of phosphate buffered saline (pH = 7.4) and tetrahydrofuran
(THF) was used as solvent (Table 1). To our surprise, the use of
sodium fluoride, ammonium hydrogen difluoride, trimethylamine
trihydrofluoide, and potassium bifluoride, did not lead to the
formation of desired free carboxylic acid 1a but remained basically
unreacted, which might be resulted from the poor solubility of these
fluoride reagents in THF (entries 1, 2, 4, and 5). Replacing PBS with
an aqueous solution of sodium bicarbonate as co-solvent slightly
promoted the deprotection process, affording desired free acid in less
than 15% yield. Pyridine hydrogen fluoride seemed to be more
efficient to remove TBS-protecting group; about 40% of starting
material had been converted to corresponding 4-hydroxyl benzyl
ester (4a) but only trace amount of 1a was detected (entry 3).
Fortunately, TBAF, the typical reagent for TBS-deprotection, was
5
10
15
20
25
30
Time (min)
Figure 1．Deprotection of 3a by TBAF monitored by HPLC. a) HPLC trace
of reaction mixture of 3a and TBAF (red line) in THF for 0.5 h at 310 nm. b)
HPLC trace of reaction mixture of 3a and neutralized TBAF (pH 7) before
magenta line) and after treatment with NaHCO
Supplementary Information for details.
(
3
(aq.) (red line). See
To test the feasibility of our hypothesis, we first investigated the
use of 4-OTBS benzyl as the protective group for carboxylic acids.
Experimentally, we selected 4-OTBS benzyl 1-naphthoic ester (3a)
as model substrate, partially due to its characteristic UV-Vis
absorbance at 310 nm which allows easily monitoring reaction
process with HPLC.

proven to be also efficient in the removal of 4-OTBS benzyl,
affording 1a in 68% yield (entry 6).
can promote the hydrolysis of 4-hydroxyl benzyl ester. Therefore,
besides TBS, other hydroxyl-protective groups, such as TMS,
TBDPS, and allyl, could also be used in the protection of carboxylic
acids, and thus renders versatile deprotection selectivities, which is
particularly useful in the synthesis of carboxylic acid-involving
compounds with multiple functional groups. For instance, 4-OTBS
benzyl protected acid (3a) was inert to catalytic amount of p-
toluenesulfonic acid while 4-OTES benzyl acid (3a-3) was labile to
Encouraged these results, we further performed solvent screening
for this deprotection reaction and it turned out this process was
compatible with a number of organic solvents, including N, N-
dimethyl formamide (DMF), methanol, acetonitrile, and THF
(entries 7-9). On the other hand, increasing the amount of TBAF
could further accelerate this reaction (entries 10-14). As shown in
Figure 1a, 10 equivalents of TBAF led to the completion of
deprotection within 0.5 h and exclusively formed free acid 1a (based
on the HPLC analysis of the reaction mixture). Moreover, from the
reaction mixture, the adduct of QM and water, 4-hydroxy benzyl
alcohol (5), was identified by the HPLC (Figure 1S) and LC-MS
analysis, which is in line with our initial hypothesis. Further
investigation revealed that the substituents on the phenyl ring of
benzyl, for example, fluoro (electron withdrawing group, 3a-1) and
methoxyl (electron donating group, 3a-2), had little effect on this
process; all of these substrates gave practically identical results.
15
such condition, yielding 4-OH benzyl ester 4a exclusively. The
addition of sodium bicarbonate aqueous solution led to the formation
of free acid 1a in quantitative yield (Scheme 2 and Figure 3S).
Electron-rich benzyl group (e.g., p-methoxybenzyl) might be
1
a
sensitive to strong acid. To test whether strong acid conditions
could be used as an alternative way to remove the 4-OTBS benzyl
group, we treated 3a with a 1:1 mixture of trifluoroacetic acid (TFA)
and dichloromethane (DCM) at room temperature. HPLC analysis
(Figure 2S) revealed that all of the starting material had been
exclusively converted to 1a within 5 minutes (> 99% yield based on
HPLC), indicating acid condition could be an alternative manner for
the deprotection of 4-OTBS benzyl.
To gain more information on the TBAF-mediated deprotection
process, we incubated 3a and pre-neutralized TBAF (pH = 7) for 30
minutes. HPLC analysis of the reaction mixture indicated that all of
the starting material 3a disappeared, leaving the corresponding 4-
hydroxyl benzyl ester (4a) as the only product (Figure 1b). This ester
was not stable upon addition of sodium bicarbonate aqueous
As a protective group, both the protection and deprotection
process are crucial to the overall synthetic efficiency. We identified
the use of N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydro
chloride (EDCI) as coupling reagent along with 1.1 equivalents of 4-
Dimethylaminopyridine (DMAP) provided optimal yield of 4-OTBS
solution; it underwent a 1,6-elimination and converted to free acid 1a
completely within a short period of time. The successful removal of
1
a, 16
benzyl ester.
Under such reaction conditions, a number of esters,
4
-OTBS benzyl with neutralized TBAF makes it orthogonal to base-
including aromatic (3a-3e) and aliphatic carboxylic esters (3h, 3i), as
well as , -unsaturated carboxylic esters (3f, 3g), were prepared in
excellent yields (94-98%, Scheme 1S).
sensitive functional group.
These results support our initial design that the removal of 4-
OTBS benzyl group is a two-step process and the use of weak base
Having demonstrated our protection strategy for carboxylic acid,
Scheme 2． Preparation of GGT NIR probe 13. (a) 2c, EDCI, DMAP, DCM, 2.5 h, 97%; (b) Piperidine, DMF, 15 min; (c) 4-aminobenzyl alcohol, EDCI, DMF,
o
1
h, 70% from 7; (d) SOCl
2
, DMF, 0 C, 1 h; (e) Changsha NIR dye，KHCO3，18-C-6, KI, DMF, 2 h, 52% from 9; (f) TFA:TIPS:DCM = 45:5:50, 2 h; (g) 12,
CuSO , THPTA, Vc, H O:DMSO = 1:1, 5 h, 41% from 11.
4 2
a)
b)
4
2
0
1
3
13 + GGT
700
750
800
Wavelength (nm)
Figure 2. a) GGT-mediated cleavage of probe 13 and turn-on of fluorescence; b) Fluorescent spectra of probe 13 before (black line) and after incubation with
GGT (red line). λex = 682 nm.

we next sought to explore the scope of this deprotection process. As
shown in Table 2, TBAF was highly efficient in removal of 4-OTBS
benzyl protection for all aromatic esters with up to 99% yield,
regardless of the electron properties of substituents on the aromatic
rings. Moreover, cinnamic ester 3f and phenylpropioloic ester 3g
were also deprotected smoothly in the presence of TBAF, releasing
corresponding free acids in excellent yields (96% and 97%,
respectively) within 0.5 h. 4-OTBS benzyl N-Fmoc glycine ester
versatile hydroxyl-protective groups to cage 4-hydroxylbenzyl and
thus control the selectivity of carboxylic acid deprotection, which is
particularly useful in the synthesis of multi-functional molecules.
Acknowledgments
This work was financially supported by “Thousand Plan”
Youth Program, the Fundamental Research Funds for the Central
Universities, and East China University of Science &
Technology.
(
3h) seemed to be incompatible with TBAF and led to deprotection
of both 4-OTBS benzyl and N-Fmoc, which is likely due to strong
1
7
basicity of TBAF. Fortunately, the treatment of 3h with neutralized
TBAF helped to prevent the deprotection of Fmoc. Simply workup
with sodium bicarbonate aqueous solution afforded free acid in 93%
yield (entry 8). In the meantime, deprotection of 3h was also
efficient in acid conditions; Fmoc-protected glycine 1h could be
obtained in 97% yield upon incubation 3h with a 1:1 mixture of TFA
and DCM for only 5 minutes in room temperature. Under acid
conditions, the deprotection of 4-OTBS benzyl naphthalene acetic
ester went smoothly, affording desired free acid 1i in 94% yield,
though, to our surprise, the TBAF-mediated deprotection resulted in
only moderate yield (73%).
Supplementary data
1
13
Experimental procedures, characterizations, H NMR and
NMR spectra, HPLC traces are available in the online version.
C
References and notes
Click here to remove instruction text...
1
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8

-Glutamyltranspeptidase (GGT) is a cell surface-bound
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protease, which catalyzes hydrolysis of -glutamyl bond of
glutathione, as well other -glutamyl compounds. This enzyme takes
part in cysteine homoeostasis and cellular glutathione, both of which
are closely related to a variety of physiological and pathological
processes, such as diabetes, asthma and cancer. Accurate
visualization activities of GGT is of high importance for disease
diagnosis at early stage. Activatable fluorescent probes, especially
near-infrared (NIR) fluorescent probes, are particularly useful for the
detection of GGT activities in vivo.
GGT probes are mostly derivatives of glutamate, whose
synthesis usually involved protection of free carboxylic acid. Herein,
to further demonstrate the usability of our carboxylic acid protection
approach, we applied it in the synthesis of fully functionalized GGT
NIR-fluorogenic probe 13.
As shown in Scheme 2, the synthesis started from coupling
commercially available protected glutamate 6 and 4-OTBS benzyl
alcohol, resulting 7 in 97% yield. The Fmoc protective group of 7
was then selectively removed by treatment of 10% piperidine in
DMF, followed by coupling with 4-aminebenzyl alcohol to afford 9
in 70% for two steps. These results indicated the 4-OTBS benzyl
protected carboxylic acid is stable enough in 10% of piperidine in
DMF (typical conditions for the deprotection of Fmoc) and thus
makes such protective group compatible with Fmoc protection.
Chlorination of 9 and subsequent nucleophilic substitution by the
alkyne-containing Changsha NIR fluorophore yielded key
intermediate 11 in 52% yield (two steps). The Boc and 4-OTBS
benzyl protective groups were removed simultaneously in the
presence of a mixture of TFA, triisopropylsilane (TIPS) and DCM
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(
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2
0
reaction.
As exhibited in Figure 2, probe 13 is basically non-fluorescent.
However, upon incubation with GGT in PBS, strong fluorescent
signal at 708 nm was detected, demonstrating its potential in
detection of GGT activities. Further application of this probe in
biological system is still under investigation.
In summary, we have developed a novel 4-OTBS benzyl-based
protective group for carboxylic acids. This protective group can be
readily removed in the presence of TBAF or TFA, which makes it
compatible with base-sensitive or acid-sensitive substrates. The
merits of this protective group are highlighted by the excellent
installation and deprotection yields. The usability of this protective
group was further demonstrated by the efficient synthesis of -
glutamyltrans-peptidase NIR probe 13. Moreover, mechanism
studies have confirmed the TBAF-mediated deprotection of 4-OTBS
benzyl ester undergoes 4-hydroxylbenzyl ester intermediate followed
by a 1,6-elimination in base conditions. This allows the use of
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A 4-OTBS Benzyl-based Protective Group
for Carboxylic Acids
Leave this area blank for abstract info.
Zhijun Fang, Yuyao Li and Hexin Xie*

Research highlights



Excellent protection and deprotection
yields.
Compatible with base-sensitive or acid-
sensitive substrates.
High selectivity