Angewandte

ChemieInternational Edition: DOI: 10.1002/anie.201702931
German Edition:
DOI: 10.1002/ange.201702931

Peptide Synthesis

AJIPHASEU : A Highly Efficient Synthetic Method for One-Pot
Peptide Elongation in the Solution Phase by an Fmoc Strategy
Daisuke Takahashi,* Tatsuji Inomata, and Tatsuya Fukui
Abstract: We previously reported an efficient peptide synthesis
method, AJIPHASEU, that comprises repeated reactions andisolations by precipitation. This method utilizes an anchor
molecule with long-chain alkyl groups as a protecting groupfor the C-terminus. To further improve this method, wedeveloped a one-pot synthesis of a peptide sequence wherein
the synthetic intermediates were isolated by solvent extraction
instead of precipitation. A branched-chain anchor molecule
was used in the new process, significantly enhancing thesolubility of long peptides and the operational efficiency
compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group.
Another prerequisite for this solvent-extraction-based strategy
was the use of thiomalic acid and DBU for Fmoc deprotection,
which facilitates the removal of byproducts, such as the fulvene
adduct.

As the mainstream modalities of drug discovery shift fromsmall molecules to biological entities, an increasing number of
peptide-based drugs and drug candidates that are smaller thanproteins have been developed in recent years.[1] This trend
increases the requirement for improving solid- and liquidphase peptide synthesis (SPPS and LPPS, respectively). SPPS
is mainly used owing to its automation and simple operation.
In contrast, LPPS is advantageous in terms of product purity,
scalability, and cost. However, the physicochemical properties
of intermediate peptides strongly depend on their sequence,and highly lipophilic and severely insoluble peptide intermediates can lead to difficult workups. In particular, thehandling of long-chain hydrophobic or hydrophilic peptides isproblematic in LPPS process development.[2]To address this problem, some LPPS-based methods[3–7]have been reported, such as the methods using PEG, fluorous
molecules, and anchor molecule 1. We have also developed
the AJIPHASEU method, a unique LPPS approach that uses
“anchor” molecules[8, 9] such as fluorene-based 2 or diphenylmethane-based 3 to elongate the sequence by repeatedcoupling/deprotection reactions and isolation by simple

Figure 1. Chemical structures of anchor molecules 1–3.

precipitation (Figure 1); however, this precipitation-based
method requires halogenated solvents, and reducing their use
is important from an economic and environmental perspective.
In addition, further improvement in terms of operational
efficiency with simplified workup steps is required to perform
industrial-scale syntheses on reasonable timescales. In fact,some attempts have been made for one-pot peptide synthesis
where product isolation was replaced by solvent extraction,
but the solubilities of peptide intermediates during extraction
differ significantly depending on their sequences and lengths,
limiting the usefulness of these strategies.[10–13]
Herein, a widely applicable method was examined andused for the one-pot elongation of a peptide where only
solvent extraction was used for the workup procedure.[17] To
establish this method, the following two challenges needed tobe addressed: 1) The peptide intermediates must be fully
soluble in the extraction solvent during elongation, and 2) the
byproducts must be completely removed by aqueous washing
as residual amounts of Fmoc-protected amino acids, coupling
reagents, deprotection reagents, and other byproducts/residues can affect subsequent reactions and generate impurities.
Considering the solubility, long or hydrophobic peptide
intermediates bearing anchor molecule 1–3 are completely
dissolved in halogenated solvents or THF with increased
viscosity, rendering extraction-based processes inefficient.
We hypothesized that increasing the lipophilicity of theanchor molecule would improve the solubility and decrease
the viscosity, and a branched phytyl group was tested. Phytol
was hydrogenated on Pt/C and treated with HBr/H2SO4 toafford dihydrophytylbromide 4 (Figure 2).[14] The phytyl[*] Dr. D. Takahashi, T. Inomata, T. Fukui
Research Institute for Bioscience Products and Fine Chemicals
AJINOMOTO Co., Inc.
1-1 Suzuki-cho, Kawasaki, Kanagawa 210-8681 (Japan)
E-mail: daisuke_takahashi@ajinomoto.com
Supporting information for this article can be found under:
https://doi.org/10.1002/anie.201702931.
T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial License, which permits use,
distribution and reproduction in any medium, provided the original
work is properly cited and is not used for commercial purposes.
Angew. Chem. Int. Ed. 2017, 56, 7803 –7807

Figure 2. Chemical structures of the branched-chain anchor molecules
and related molecules 4–6.

T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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group (Phy) was introduced into benzyl- and diphenylmethane-type anchors according to a previously reported
method,[6, 9] and anchors 5 and 6 were obtained (Figure 2;
see also the Supporting Information).
Interestingly, the solubilities of these new branched-chainanchors 5 and 6 in various organic solvents were significantly
better than those of compounds 1 and 3, and allowed for an
efficient extraction-based workup (Table 1). Based on theseresults, compounds 5 and 6 were chosen as the anchor
molecules for examining the new workup procedure. In
addition, the anchor compounds are less soluble in polar
solvents such as MeCN and DMF, which are occasionally usedas solvents in peptide synthesis.
Table 1: Solubility of anchor molecules in extraction solvents [wt %].
Straight chain

Branched chain

Solvent

1

3

5

6

CHCl3
EtOAc
CPME
toluene

9.9
0.2
4.5
–[a]

0.8
0.2
0.01
0.01

> 50
> 50
> 50
> 50

> 50
> 25
> 50
–[a]

[a] No data. CPME = cyclopentyl methyl ether.

To confirm that the use of anchor molecules witha branched alkyl chain enhances the solubility of intermediate
peptides during elongation and leads to less problematic
extractions, we employed compound 6 in the synthesis ofa hydrophobic peptide. This class of peptides is well-known tobe difficult to be synthesized by LPPS owing to their low
solubility in various types of solvents.
Fmoc-Val-OH, Fmoc-Gly-Gly-OH, and Fmoc-Val-OH
were sequentially condensed to form the hydrophobic
sequence Fmoc-Val-Gly-Gly-Val with anchor molecule 1 or
5 at the C-terminus. During the coupling reaction for the forth
residue Fmoc-Val-OH, insoluble matter was observed in thereaction mixture for the intermediate peptide with anchor 1.
Synthesis with the branched-chain anchor 5 led to a completely homogeneous solution. This interesting result indicated that the newly developed anchor with a branched alkyl
chain is useful for the synthesis of hydrophobic peptides
whose intermediates may have low solubility (Figure 3).

ChemieFigure 3. Reaction mixtures of the coupling reaction of Fmoc-Val-OH
with H-Gly-Gly-Val-OAnchor in chloroform with compound 1 or 5 as
the anchor compound, respectively.

Next, we studied the second challenge. After the coupling
reaction, residual amino acids and amines, such as piperidine,
which is conventionally used for the removal of Fmoc groups
and in the formation of fulvene adducts via dibenzofulvene(DBF) derived from Fmoc groups, are a potential cause ofundesirable side reactions. Therefore, it is essential to remove
these byproducts before the next amino acid coupling
reaction. To quench, solubilize, and remove the fulvene
derivatives into an aqueous layer, some reagents were tested
using Fmoc-Leu-OH loaded onto anchor 5 for Fmoc deprotection of Fmoc-Leu-OAnchor 7 into 8 (Table 2).
In the case of diethylenetriamine,[11] unfortunately, separation of the organic layer from the aqueous layer was
troublesome. In fact, standard amine-based Fmoc removal
reagents, including diethylenetriamine and piperidine, did notcompletely capture all DBF; thus a considerable amount of
the fulvene adduct remained intact in the organic layer. Whenwashed with acidified water to remove the base and its adductbyproducts of fulvene, the solution emulsified during theextraction, and the phase separation was insufficient. We
surmise that the acidic aqueous solution that was used for theremoval of solubilized fulvene adducts and residual amines
led to the formation of the emulsion during aqueous washing.
This is presumably due to the protonation of the peptide
N-terminus under acidic conditions, which renders the
peptide amphiphilic, in combination with the long aliphatic
chain of the anchor molecule, which could act as a surfactant.
Accordingly, we then tried basic conditions for aqueous
washing to keep the terminal amine group of the peptide

Table 2: Conversion into the fulvene adduct during Fmoc deprotection of Fmoc-Leu loaded onto 5 and the removal rate in the washing step.

Reagent (equiv)
diethylenetriamine (30)piperidine (5)piperidine (5)Mpa (3)Mpa (3)thiomalic acid (3)cysteine (3)DBU [equiv]

Solvent

DBF/fulvene adduct

0
3
3
6
6
9
6

CHCl3
CHCl3
CPME
CPME
CHCl3
CHCl3
CHCl3

35:65
31:69
8:92
3:97
1:99
1:99
2:98

Washing solvent
HCl aq.
HCl aq.
HCl aq.
Na2CO3 aq.
Na2CO3 aq.
Na2CO3 aq.
Na2CO3 aq.

Layer separability[a]Removal rate [%]

@
@
@
+
+
+
+

48
2
0
100
30
100
8

[a] “@”: Insufficient phase separation; “ + ”: sufficient phase separation. See the Supporting Information for details. Mpa = mercaptopropionic acid.

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deprotonated. To achieve this, mercaptopropionic acid(Mpa), with both carboxylic acid and nucleophilic thiol
moieties, was examined in combination with DBU. FmocLeu loaded onto anchor molecule 5 was treated with Mpa/
DBU, and the resulting solutions were washed with a basic
aqueous solution of sodium carbonate, which resulted in
facile deprotection and the formation of DBF adducts. In thisextraction process, the fulvene adducts were smoothly
removed into the basic aqueous layer with good phaseseparation. Cysteine also showed good results. Finally,
thiomalic acid, with two carboxylic acid moieties, was tested
and showed advantageous properties for transferring thecorresponding fulvene adducts into the basic aqueous layer(Table 2).
Based on the examination above, when Fmoc deprotection is conducted with a compound possessing both thiol andcarboxylate moieties, such as Mpa, thiomalic acid, or Cys, inthe presence of DBU, excess active esters of the amino acids
and dibenzofulvene are converted into acidic species bearing
a carboxylic acid moiety, which are removed by simple
washing with basic aqueous solution. Consequently, the next
condensation can be performed without interference fromthese byproducts. These processes can be performed consecutively, and a practical method for one-pot peptide elongation
without an isolation step has been devised (Scheme 1).

Scheme 1. Novel Fmoc deprotection system for one-pot peptide elongation.

Angewandte

Chemielayer was subjected to the next coupling reaction without
concentration or drying. No impurities derived from thiomalic acid or fulvene were observed in the subsequent
coupling reaction. The fully protected full-length peptide
demonstrated satisfactory solubility in the organic layer, andphase separation of the organic and aqueous layers was
accomplished without any problems. Successive concentration of the organic layer and addition of MeCN afforded thefully protected peptide 10 with an anchor molecule at the
C-terminus. The fully protected compound was then subjected to standard global deprotection conditions (TFA/
triisopropylsilane (TIS)/H2O = 95:2.5:2.5) to afford crude
Degarelix in 89 % purity (Scheme 2 and Figure 4).

Figure 4. HPLC analysis of crude Degarelix. Zorbax Eclipse XDB-C18,
4.6 W 150 mm, 5 mm, 55 8C, 1.2 mL min@1, 17.6 mm aq. Na2HPO4(pH 8.2)/MeCN, gradient: 27–33 % (0–5 min), 33–37 % (5–25 min),
70 % (25–30 min).

We also synthesized a peptide bearing a carboxylic acidterminus. Bivalirudin,[16] with 20 amino acid residues, waselongated using a similar method to that employed for
Degarelix in a one-pot process using compound 5 as theanchor molecule and only solvent extraction for workup(Scheme 3). All reactions and workups proceeded smoothly.
The fully protected 20 residue peptide was completely soluble
in the organic solvent, and phase separation proceededsmoothly in each washing step. As with Degarelix, allintermediate peptides synthesized during elongation were
simply washed and subjected to the next coupling reaction
without isolation, for example, by precipitation as previously
reported.[8, 9] The fully protected product 11 was obtained in

We then studied LPPS with the newly developed anchor
molecule with a branched phytyl group. The first actual
peptide was the anti-ovary-cancer drug Degarelix,[15] whichcontains an amide at the C-terminus. Therefore, the diphenylmethane-type anchor molecule 6 was employed using chloroform as the solvent. Fmoc-protected natural and unnaturalamino acids were used slightly in excess (1.1–1.3 equiv) for
coupling
reactions
with
EDC·HCl/HOBt, and condensation of each residue
proceeded smoothly. Fmoc
deprotection was performedin the presence of 3 equiv ofthiomalic acid and 8 equiv of
DBU. Fmoc was promptly
removed in 1–2 h from eachresidue to form a fulvene
adduct with the scavenger.
Upon washing with an aqueous solution of sodium carbonate, the fulvene adductwas easily removed into theaqueous layer. The organic
Scheme 2. Synthetic scheme for Degarelix elongation.
Angew. Chem. Int. Ed. 2017, 56, 7803 –7807

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Scheme 3. Synthetic scheme for Bivalirudin elongation.

Figure 5. HPLC analysis of crude Bivalirudin. YMC-Pack ODS-A,
4.6 W 150 mm, 5 mm, 40 8C, 1.0 mL min@1, 0.1 % TFA in 5 % THF/waterand 0.1 % TFA in 5 % THF/MeCN, gradient: 20–40 % (0–25 min), 80 %(25–30 min).

73 % yield in this one-pot process (based on 5; Figure 5). The
purity of the crude product after global deprotection was
84 %.
It should be noted that peptide elongation can be
performed in non-halogenated organic solvents such as
cyclopentyl methyl ether (CPME). Only a small amount ofsolvent was added in each elongation cycle, corresponding to
the increasing intermediate molecular weight, and the initialsolvent remained in the vessel throughout the synthesis.
Therefore, solvent consumption is very low, and amounts toapproximately one tenth of that of SPPS. This is an advantage
of our AJIPHASEU method with compounds 5 and 6(Figure 6).

Figure 6. Solvent consumption of various peptide synthesis methods
for 20-mer peptides.

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ChemieConventionally and conveniently, peptides have been prepared by solid-phase
synthesis; however, an easy-to-handle andready-to-scale-up LPPS method for thesynthesis of long and/or hydrophilic peptides in improved purity and yield is stillrequired.
In conclusion, we have developed
a simple, efficient, and widely applicable
peptide synthesis method based on the
branched anchor compounds 5 or 6 incombination with a new Fmoc deprotection
system. This process afforded pure peptides, even for long or hydrophobic peptides, and can be readily scaled up. Solvent
evaporation and dehydration are not necessary during theworkups for the following coupling steps, and only a limited
amount of solvent is added in each reaction. Thus the totalsolvent consumption is significantly lower than for SPPS. We
are confident that these advantages will render the
AJPHASE approach a useful LPPS method even on large
scales.

Acknowledgements
We thank Taisuke Ichimaru for help with analyses and Dr.
Hiroshi Orikabe, Dr. Ken Yamashita, and Dr. Takashi
Yamamoto for fruitful discussions and manuscript preparation.

Conflict of interest
The authors declare no conflict of interest.
Keywords: AJIPHASE · liquid-phase peptide synthesis ·peptides · protecting groups · solvent extraction
How to cite: Angew. Chem. Int. Ed. 2017, 56, 7803 – 7807
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Manuscript received: March 21, 2017
Accepted manuscript online: May 15, 2017
Version of record online: June 1, 2017

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