Novel diphenylmethyl-derived amide protecting group for efficient liquid-phase peptide synthesis: AJIPHASE

Article abstract of DOI:10.1021/ol302002g

An efficient method for the synthesis of peptides bearing an amide at the C-terminal is described. This method involves the attachment of a C-terminal protecting group bearing long aliphatic chains, followed by the repetition of simple reaction and precipitation steps with the combined advantages of liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS). Using this method, a hydrophobic peptide was successfully synthesized in good yield and high purity, which cannot be obtained satisfactorily by SPPS.

Full text of DOI:10.1021/ol302002g

ORGANIC
LETTERS
2012
Vol. 14, No. 17
4514–4517
Novel diphenylmethyl-Derived Amide
Protecting Group for Efficient
Liquid-Phase Peptide Synthesis: AJIPHASE
Daisuke Takahashi,* Tatsuya Yano, and Tatsuya Fukui
Research Institute for Bioscience Products and Fine Chemicals, AJINOMOTO Co.,
Inc., 1730 Hinaga Yokkaichi Mie 510-0885, Japan
daisuke_takahashi@ajinomoto.com
Received July 19, 2012
ABSTRACT
An efficient method for the synthesis of peptides bearing an amide at the C-terminal is described. This method involves the attachment of a
C-terminal protecting group bearing long aliphatic chains, followed by the repetition of simple reaction and precipitation steps with the combined
advantages of liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS). Using this method, a hydrophobic peptide was
successfully synthesized in good yield and high purity, which cannot be obtained satisfactorily by SPPS.
Development of peptide drugs has increasingly ex-
panded in recent years, and the need for peptide synthesis
has increased accordingly.1 The peptide synthesis methods
are roughly divided into liquid-phase peptide synthesis
(LPPS) and solid-phase peptide synthesis (SPPS) techni-
ques, with much of the focus on SPPS, in which a simple
workup procedure is possible. On the other hand, LPPS
furnishes high-purity peptides and is economically
efficient.2 However, by using LPPS, it has been tradition-
ally difficult to synthesize hydrophilic, hydrophobic, and
long-chain peptides, mainly because of solubility issues
and the time-consuming nature of its process development,
which requires the establishment of workup procedures for
each intermediate peptide.
et al. reported an efficient LPPS protocol with a benzyl
alcohol derivative bearing three long alkoxy chains 1.8
Chiba9 and Sunazuka10 also prepared peptides by a similar
protocol (Figure 1). Separately, we have developed and
reported11 a novelfluorene-derivedprotecting group2 that
facilitates the efficient elongation of peptide chains and
involved only simple repeated reaction and precipitation
steps. This approach combines the advantages of both
SPPS and LPPS. The fluorene-type protecting group ex-
hibits an effect similar to that of the chlorotrityl linker used
in SPPS, namely the avoidance of diketopiperazine forma-
tion. The selective detachment of this C-terminal protect-
ing group with the protecting groups of the side chain
intact leads to production of protected peptide acids that
are useful for the convergent synthesis of long-chain
peptides.
However, several efficient LPPS methods have been
developed to overcome these disadvantages.3À7 Tamaki
On the other hand, peptide drugs and peptide drug
candidates are composed of various sequences and struc-
tures, and no efficient method has yet been discovered
for the synthesis of these numerous types of peptides.
(1) Vlieghe, P.; Lisowsli, V.; Martinez, J.; Khrestchatisky, M. Drug
Discovery Today 2010, 15, 40.
(2) Andersson, L.; Blomberg, L.; Flegel, M.; Lepsa, L.; Nilsson, B.;
Verlander, M. Biopolymers 2000, 55, 227.
(3) Carpino, L. A.; Ghassemi, S.; Ionescu, D.; Ismail, M.; Sadat-
Aalaee, D.; Truran, G.; Mansour, E. M.; Siwruk, G. A.; Eynon, J. S.;
Morgan, B. Org. Process Res. Dev. 2003, 7, 28.
(4) Eggen, I. F. Org. Process Res. Dev. 2005, 9, 98.
(5) Pillai, V. N. R.; Mutter, M.; Bayer, E.; Gatfield, I. J. Org. Chem.
1980, 45, 5364.
(8) Tamiaki, H.; Obata, T.; Azefu, Y.; Toma, K. Bull. Chem. Soc.
Jpn. 2001, 74, 733.
(9) Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Bioorg. Med.
Chem. Lett. 2011, 21, 4476.
(6) Isokawa, S.; Kobayashi, N.; Nagano, R.; Narita, M. Bull. Chem.
Soc. Jpn. 1980, 53, 1028.
(7) Mizuno, M.; Miura, T.; Goto, K.; Hosaka, D.; Inazu, T. Chem.
Commun. 2003, 972.
(10) Hirose, T.; Kasai, T.; Akimoto, T.; Endo, A.; Sugawara, A.;
Nagasawa, K.; Shiomi, K.; Omura, S.; Sunazuka, T. Tetrahedron 2011,
67, 6633.
(11) Takahashi, D.; Yamamoto, T. Tetrahedron Lett. 2012, 53, 1936.
r
10.1021/ol302002g
Published on Web 08/24/2012
2012 American Chemical Society

In particular, C-terminal amide peptides have been devel-
oped with the aim of improving both their stability and
activity,12 and this type of peptide accounts for a large
number of the current peptide drugs.1 Therefore, an
efficient method for the synthesis of amide peptides is
required. Peptides with an amide at the C-terminus can
be synthesized by an SPPS-based Fmoc strategy using the
4-methylbenzhydrylamine (MBHA) linker, the 5-[3,5-di-
methoxy-4-(Fmoc-aminomethyl)phenoxypentanoic acid
(PAL) linker, and the Rink amide resin, with detachment
of the resin using TFA.13 However, a C-terminal protect-
ing group for efficient synthesis of peptide amides by LPPS
has not yet been developed. Such a C-terminal protecting
group must be able to not only form a stable bond with the
peptide but also be detached by TFA in the final deprotec-
tion step.
high-temperature conditions with a catalytic amount of
methanesulfonic acid to obtain intermediate 6 in quanti-
tative yield. Subsequently, the Fmoc or ethoxycarbonyl
group was deprotected under basic conditions to furnish the
target molecule bis[4-(docosyloxy)phenyl]methylamine (7)
(Dpm-NH2).
Scheme 1. Synthesis, Loading of Fmoc-amino Acid, And
Cleavage for Dpm-OH and Dpm-NH2-Type Protecting Groups
Figure 1. Benzyl alcohol derivative 1 and fluorene-derived pro-
tecting group compounds 2.
In this study, we report the development of a diphenyl-
methyl-derived protecting group at the C-terminal and
demonstrate its use in the efficient synthesis of various
types of terminal amide peptides by an Fmoc strategy with
a simple workup procedure.
Coupling of the Dpm-NH2-type protecting group with
Fmoc-Ala-OH using the typical coupling reagent system
EDC HCl/HOBt was then confirmed, and the coupling
A high-yield syntheticprocessfor the targetedprotecting
group was accomplished (Scheme 1). First, the hydroxy
groups of bis(4-hydroxyphenyl)methanone were treated
with docosylbromide and K2CO3 in DMF to form ketone
3. Second, the ketone was reduced using NaBH4 to obtain
bis[4-(docosyloxy)phenyl]methyl alcohol (Dpm-OH, 4) in
quantitative yield. This Dpm-OH type C-terminal protect-
ing group 4 was then quantitatively coupled with Fmoc-
Ala-NH2 asthefirst amino acidusing a catalyticamount of
methanesulfonic acid to successfully afford 5, suggesting
that the compound 4 is very useful. However, in this Fmoc/
tBu strategy, the acid-labile protecting groups of the side
chain of Fmoc-AA-NH2, such as Fmoc-Lys(Boc)-NH2
and Fmoc-Asp(OtBu)-NH2, could be partially lost during
the loading to the Dpm-OH type protecting group because
this loading step was performed under slightly acidic
conditions. Therefore, the possible routes for the synthesis
of amine 7 from alcohol 4 were investigated. Compound 4
was coupled with Fmoc-NH2 or ethyl carbamate under
3
product 5 was precipitated from polar solvents such as
MeOH or MeCN. As described above, the Dpm-OH-type
protecting group 4 can be synthesized in only two steps
from available reagents and coupled with Fmoc-AA-NH2.
On the other hand, the Dpm-NH2-type protecting group 7
can be coupled with Fmoc-AA-OH with typical coupling
reagents under mild conditions, although two additional
steps are required. Both compounds are widely available
for use as a C-terminal protecting group in the efficient
synthesis of peptides. The Dpm-type protecting group was,
asexpected, readily removed using TFA, and therefore, the
Dpm-type compound can be used asa protecting group for
the C-terminal. These results imply that the protecting
groups 4 and 7 are applicable as a protecting group at the
carboxamide-containing side chain of amino acid residues,
such as Asp/Glu and Asn/Gln. Thus, we confirmed that
the Dpm-type protecting group can be applied to peptide
amide synthesis as part of an efficient LPPS method
involving the repetition of only simple reaction and pre-
cipitation operations. Furthermore, Dpm-type C-terminal
protecting groups that can couple with Fmoc-AA-NH2 or
Fmoc-AA-OH were prepared.
(12) (a) Suwan, S.; Isobe, M.; Yamashita, O.; Minakata, H.; Imai, K.
Biochem. Mol. Biol. 1994, 21, 1001. (b) Imai, K.; Nomura, T.; Katsuzaki,
H.; Komiya, T.; Yamashita, O. Biosci. Biotechnol. Biochem. 1998, 62,
1875.
(13) Williams, P. L.; Albericio, F.; Giralt, E. Chemical Approaches to
the Synthesis of Peptide and Proteins; CRC Press: Boca Raton, 2000; p 45.
Org. Lett., Vol. 14, No. 17, 2012
4515

Using compound 7, the synthesis of the peptide eptifiba-
tide, which is a platelet aggregation inhibitor, was at-
tempted employing this efficient LPPS method. Because
eptifibatide is one of the best selling peptide drugs on the
market, it would be beneficial if this peptide could be
synthesized by our LPPS method.
Figure 3. HPLC analysis. Top: crude peptide 11 (AAQV-
LISELAIA-NH2) synthesized by SPPS after removal from
the resin. Bottom: crude peptide 11 synthesized via the efficient
LPPS using the Dpm-type C-terminal protecting group 7.
It is commonly known that it is difficult to synthesize
hydrophobic sequences of peptides employing the SPPS
approach. Therefore, the synthesis of a hydrophobic
peptide 11 (AAQVLISELAIA-NH2) containing leucine
(Leu), valine (Val), and others as a model peptide by both
SPPS and LPPS was attempted using the Dpm-type
protecting group 7 to compare the two methods. In the
SPPS method, the FastMoc protocol was employed with
preloaded Ala at the C-terminus of a Rink amide resin
(Watanabe Chem. Ind. Ltd.) and 4 equiv of Fmoc-AA-OH
for double coupling. The target peptide was not obtained
as the main product, and some deletion peptides were
observed because of incomplete reactions (Figure 3).
In contrast to the SPPS case, all of the coupling reactions
were completed using only 1.1À1.5 equiv of Fmoc-AA-
OH employing our efficient LPPS method. After the
final deprotection, in which all the protecting groups
of side chains and the Dpm-type C-terminal protecting
group were removed, the crude peptide was successfully
afforded in excellent purity (Figure 3). The yield of the
total synthesis was 83% over 24 steps, indicating a 98.4%
average yield for each amino acid elongation. The advant-
age of this peptide synthesis method can be easily recog-
nized based on a comparison of the HPLC spectra for the
SPPS synthesis and the new efficient LPPS method.
Figure 2. HPLC analysis. Top: crude peptide 9 after final
deprotection. Bottom: crude peptide eptifibatide (10) after S
to S disulfide bridge formation.
First, 7 was dissolved in CHCl3 and coupled with Fmoc-
Cys(Trt)-OH as the first amino acid using EDC HCl/
3
HOBt. The coupling reaction proceeded well with a neg-
ligible amount of racemization, and isolation was achieved
by precipitation with MeOH. Subsequently, the loaded
Fmoc-Cys(Trt)-OH compound was dissolved again in
CHCl3, the Fmoc group was deprotected using DBU/
Et2NH, and the product was precipitated with MeCN.
These steps were repeatedfor the successful synthesis of the
protected 7-mer peptide eptifibatide 8 in good yield (86%)
from 7 (Scheme 2). The fully protected eptifibatide was
thenexposedto the standardcleavagecocktail(TFA/H2O/
triisopropylsilane = 95:2.5:2.5), and the crude linear
eptifibatide 9 was obtained in high yield and in high purity
without any closely related impurities that could affect the
HPLC purification step (Figure 2). The crude peptide 9
was then smoothly cyclized at the disulfide bond between
the mercaptopropionic acid (Mpa) and cysteine (Cys)
residues under basic aqueous conditions, followed by
purification via reversed-phase HPLC. As a result, eptifi-
batide (10) was successfully obtained in high purity. The
quality of synthesized peptide 10 was confirmed with the
comparison of NMR data to the authentic compound
which was purchased from 2A PharmaChem Co.
The reactivity of the coupling and Fmoc deprotection
reactions is remarkably reduced during the synthesis of
hydrophobic peptides via SPPS; thus, each reaction does
not go to completion, and deletion peptides are generated
duringthe elongation of the peptidesequence. To complete
the reactions, heating, large excess of reagents, and ex-
tending reaction times are required. Unfortunately, side
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Org. Lett., Vol. 14, No. 17, 2012

Scheme 2. Elongation of the Eptifibatide Chain Using the Dpm-NH2-Type Protecting Group
reactions, such as racemization and side-chain reactions,
can occur as a result.
the Dpm-type protecting groups 4 and 7, the peptide chain
can be elongated by simple repeated reaction and precipi-
tation steps, and the crude peptide can be obtained using
standard final deprotection conditions with TFA. Good
solubility during a peptide-chain elongation reaction can
be achieved by anchoring a protecting group bearing a
long alkoxy chain at the C-terminus, and the byproducts
can be readily removed by precipitation. In addition, we
demonstrated that a hydrophobic peptide can be synthe-
sized by this efficient method in higher yield and higher
purity than that obtained with SPPS. As a result, this easy-
to-implement and efficient LPPS protocol (AJIPHASE)
was successfully extended to the synthesis of peptide
amides, which account for the majority of current peptide
drugs, and for which a sufficient LPPS method has not yet
been developed to date. The synthesis of bioactive peptides
and long-sequence peptides using the AJIPHASE protocol
is in progress and will be reported elsewhere.
On the other hand, in the case of the conventional
solution-phase approach with Bzl groups, the use of tBu
as the protecting group at the C-terminus results in the
decreased solubility of the protected peptide in the reaction
solvents as the peptide chain is elongated, particularly for
hydrophobic peptides.
However, in the newly developed LPPS method re-
ported here, the long-chain character of the Dpm-type
protecting group promotes the solubility of the growing
peptide in chloroform. In fact, the solubility is increased,
and complete dissolution can be accomplished with slight
heating to only 30À35 °C. Therefore, by employing the
efficient LPPS method using a Dpm-type protecting
group, peptide chain elongation is possible. In addition,
they can be obtained in very high yield and high purity
because the reactivity of each reaction is favorable and side
products are not generated.
Acknowledgment. We thank Mr. Shoichi Kondo and
Mrs. Naori Nakao (AJINOMOTO Co., Inc.) for their
help with the experiments and analysis and Dr. Takashi
Yamamoto (AJINOMOTO Pharmaceuticles Co., Ltd.)
for fruitful discussions and help with preparation of the
manuscript.
Importantly, the bond between the Dpm-type protecting
group and the peptide chain is not cleaved under the
reaction or workup conditions. However, the Dpm-type
C-terminal protecting group is easily cleaved under the
standard final deprotection conditions (TFA) used in
the Fmoc strategy. This behavior is another reason for
obtaining target peptides in high yield and high purity.
Based on an investigation of the efficient LPPS, we
developed Dpm-derived C-terminal protecting groups 4
and 7 that are useful for efficient LPPS. These molecules
were synthesized from benzophenone derivatives in excel-
lent yield and showed sufficient loading efficiency with
both Fmoc-AA-OH and Fmoc-AA-NH2.The bond be-
tween the peptide and the Dpm-type protecting group is
stable during the elongation of the peptide sequence. Using
Supporting Information Available. Synthetic procedure
of diphenylmethyl-derived protecting groups, general
procedure for peptide analysis and synthesis using the
Dpm-type protecting group support compound 7. Com-
parison and examination of model compounds for pro-
tecting group. This material is available free of charge via
the Internet at http://pubs.acs.org
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 17, 2012
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