A practical solution-phase synthesis of an antagonistic peptide of TNF-α based on hydrophobic tag strategy

Article abstract of DOI:10.1039/c0cc03090b

A simple acid-resistant hydrophobic tag, which can be removed rapidly in a single-step procedure after overall peptide synthesis, has been developed to accomplish practical solution-phase synthesis of a 15-mer antagonistic peptide of TNF-α (A-TNF-α). Hydrophobically tagged peptides can be separated as precipitates at each step by addition of a polar organic solvent.

Full text of DOI:10.1039/c0cc03090b

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A practical solution-phase synthesis of an antagonistic peptide
of TNF-a based on hydrophobic tag strategyw
Gegen Tana,a Shingo Kitada,a Shuji Fujita,b Yohei Okada,a Shokaku Kima and
Kazuhiro Chiba*a
Received 6th August 2010, Accepted 7th September 2010
DOI: 10.1039/c0cc03090b
A simple acid-resistant hydrophobic tag, which can be removed
rapidly in a single-step procedure after overall peptide synthesis,
has been developed to accomplish practical solution-phase synthesis
of a 15-mer antagonistic peptide of TNF-a (A-TNF-a). Hydro-
phobically tagged peptides can be separated as precipitates at
each step by addition of a polar organic solvent.
intramolecular cyclized by-product, takes place immediately
under the basic conditions required for N-terminal Fmoc
group deprotection (Scheme 2). This troublesome process
can be prevented under acidic conditions because the nucleo-
philicity of the N-terminus should be significantly decreased
by protonation. Therefore, an acid-resistant hydrophobic tag
would allow for the use of both Boc- and Fmoc-chemistry,
extending the potential uses of hydrophobic tag-based peptide
synthesis. We herein describe the development of a new
hydrophobic tag for peptide synthesis that can be applied in
both Boc- and Fmoc-chemistry. Furthermore, hydrophobically
tagged peptides can be effectively separated as precipitates by
addition of a polar organic solvent. The tag can also be removed
in a single step procedure after overall peptide synthesis
(Scheme 3). We chose a peptide possessing a C-terminal prolyl
residue, i.e., antagonist of tumor necrosis factor-a28 (A-TNF-a),
as the model compound to demonstrate our strategy.
Since the pioneering work of Merrifield, peptide synthesis
based on solid-phase techniques has been established to realize
a practical synthesis for both naturally occurring and artificial
peptides, and has also become essential for automated synthesis
and combinatorial chemistry.1–12 The great advantage of solid-
phase synthesis is the facile separation processes, requiring only
filtration. In this context, a wide variety of effective protection/
deprotection methods were developed to elaborate target
sequences. Alternatively, tag-assisted solution-phase synthesis
has also been demonstrated to be an efficient approach,
especially for multi-step reactions.13–21 In this case, high
reaction rate and rapid reaction monitoring are both attained.
The integration of solid-phase separation and solution-phase
synthesis should facilitate highly practical peptide synthesis.
Previously, we developed a solution-phase reaction system
based on a simple hydrophobic tag, 3,4,5-trioctadecyloxy-
benzyl alcohol (HO-TAGa) 1, that could easily be used in
multi-step reactions (Scheme 1).22–27 In particular, 1 can serve
as an effective protecting group for the peptide C-terminus and
as a phase-tag that can selectively be separated from surplus
reagents, including amino acids (AAs) and condensing agents,
in polar reaction solutions. However, C-terminal benzyl
esters are often hydrolyzed under acidic conditions. Thus,
Boc-chemistry could not be applied to this system. This drawback
is crucial when proline is placed at the C-terminus (or next to
the C-terminus), because the formation of diketopiperazines, an
The present work began with the preparation of 2,4-di-
docosyloxybenzyl alcohol (HO-TAGb) 2 and 3,5-didocosyloxy-
benzyl alcohol (HO-TAGc) 3. To evaluate the stability of the
hydrophobic tags 1–3 against the acidic conditions used for
N-terminal Boc group deprotection, N-Boc-leucine was then
introduced to 1–3 using diisopropylcarbodiimide (DIC) in the
presence of a catalytic amount of dimethylaminopyridine
(DMAP) to form hydrophobically tagged N-Boc-leucines
(N-Boc-Leu-O-TAGa–c) 4–6 quantitatively (Scheme 4). When
Scheme 2 Formation of diketopiperazines from N-Fmoc-AA-Pro-O-
TAGa under basic conditions required for N-terminal Fmoc group
deprotection.
Scheme 1 Structures of hydrophobic tags (HO-TAGa–c) 1–3 used in
this study.
a Laboratory of Bio-organic Chemistry,
Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
E-mail: chiba@cc.tuat.ac.jp; Fax: (+81)-42-360-7167;
Tel: (+81)-42-367-5667
b JITSUBO Co. Ltd., 2-24-16 Naka-cho, Koganei, Tokyo 184-0012,
Japan
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c0cc03090b
Scheme 3 General concept of the hydrophobic tag strategy.
Chem. Commun., 2010, 46, 8219–8221 8219
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Scheme 5 N-Boc group deprotection of N-Boc-Arg(Mts)-Pro-O-
TAGc (12).
Scheme 4 Evaluation of the stability of HO-TAGa–c (1–3) against
amount of deprotected product 13 with excellent purity
(Scheme 5). Cleavage of the C-terminal benzyl ester of 12 was
not observed, while no deprotected product 13 was obtained
through N-terminal Fmoc group deprotection of hydro-
phobically tagged N-Fmoc-peptide [N-Fmoc-Arg(Mts)-Pro-
O-TAGc] 14 under basic conditions, and only 3 was recovered
as a precipitate (Scheme 6 and Fig. S4 in supporting information).
Based on these results, repeated peptide elongation with
N-Fmoc-AAs [except the last, N-Boc-Asp(tBu)-OH], deprotec-
tion in solution-phase, and separation by precipitation were
performed using HBTU and HOBt in the presence of DIPEA
to afford hydrophobically tagged N-Boc-A-TNF-a (N-Fmoc-
A-TNF-a-O-TAGc) 15 in 81% isolated yield over 25 steps
(Scheme 7). Finally, deprotection of compound 15 was
achieved under two different types of acidic conditions to give
deprotected compound, A-TNF-a, 16 in 86% isolated yield in
two steps with excellent purity (Scheme 8 and Fig. S6 in
supporting information).
the acidic conditions used for N-terminal Boc group deprotection.
4–6 were subjected to N-terminal Boc group deprotection
conditions, 6 afforded deprotected product 9 quantitatively,
whereas deprotected products 7 and 8 were not obtained.
Hydrolyzed leucine S1 was recovered from the reaction
mixture, along with decomposition products of 1, by acid
treatment of 4 (Scheme S1 in supporting information). On the
other hand, acid treatment of 5 resulted in the quantitative
formation of red-colored resorcinarene S2 (Scheme S2 in
supporting information). These results indicated that the
C-terminal benzyl esters of 4 and 5 were hydrolyzed under
the acidic conditions required for N-terminal Boc group
deprotection, affording the corresponding benzyl cations
stabilized by ortho- and/or para-alkoxy groups, which then
decomposed and cyclized. Meanwhile, hydrolysis of the
C-terminal benzyl ester of 6 was not observed even when 6
was stirred for 3 days under acidic conditions required for
N-terminal Boc group deprotection, clearly suggesting
that 3 functioned as an acid-resistant hydrophobic tag that will
enable the incorporation of Boc-chemistry. It should also be
emphasized that quantitative amounts of both 6 (Fig. S1 in
supporting information) and 9 (Fig. S2 in supporting information)
could be isolated with excellent purities by addition of acetonitrile.
With the acid-resistant hydrophobic tag 3 in hand, we then
turned our attention to the synthesis of A-TNF-a. Initially,
N-Fmoc-proline was introduced to 3 using DIC in the
presence of a catalytic amount of DMAP. After the comple-
tion of the reaction, an excess amount of acetonitrile was
added to the reaction mixture to precipitate hydrophobically
tagged N-Fmoc-proline (N-Fmoc-Pro-O-TAGc) 10 quanti-
tatively (Scheme S3 and Fig. S3 in supporting information).
Compound 10 was then stirred under basic conditions
consisting of 1,3-diazabicyclo[5.4.0]undec-7-ene (DBU) and
piperidine to deprotect the N-terminal Fmoc group, which
was then precipitated by the addition of acetonitrile to give a
quantitative amount of deprotected product 11 (Scheme S4 in
supporting information). Through careful examination, the
Mts group was found to be a suitable protective group for the
arginine side chain. Thus, N-Boc-Arg(Mts)-OH was introduced
to 11 to give hydrophobically tagged N-Boc-dipeptide
[N-Boc-Arg(Mts)-Pro-O-TAGc] 12 quantitatively as a precipitate
by the addition of an excess amount of acetonitrile (Scheme S5
and Fig. S3 in supporting information). It was also found that
the use of O-benzotriazole-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU) and 1-hydroxy-1H-benzotriazole
(HOBt) in the presence of N,N-diisopropylethylamine (DIPEA)
was more effective after the second introduction of an amino
acid. Compound 12 was then stirred under acidic conditions to
deprotect the N-terminal Boc group, which was then precipi-
tated by the addition of acetonitrile to give a quantitative
In conclusion, we have successfully developed a simple
acid-resistant hydrophobic tag, which can rapidly be removed
in a single step procedure after overall peptide synthesis, to
accomplish practical solution-phase synthesis of A-TNF-a.
This strategy could effectively incorporate both Boc- and
Fmoc-chemistry and the products could readily be separated
as precipitates at each step by addition of a polar organic
solvent, which allows for elaboration of target sequences in
peptide synthesis.
Scheme 6 Basic deprotection of N-Fmoc-Arg(Mts)-Pro-O-TAGb
(14).
Scheme 7 Synthesis of N-Boc-A-TNF-a-O-TAGc (15).
Scheme 8 Acidic deprotection of N-Boc-A-TNF-a-O-TAGc (16).
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