Peptide synthesis 'in water' by a solution-phase method using water-dispersible nanoparticle Boc-amino acid

Article abstract of DOI:10.1002/psc.1367

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in-water solution-phase peptide synthesis method using Boc-amino acids. It is based on a coupling reaction utilizing suspended water-dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity.

Full text of DOI:10.1002/psc.1367

Research Article
Received: 15 July 2011
Revised: 17 January 2011
Accepted: 27 January 2011
Published online in Wiley Online Library: 14 April 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1367
Peptide synthesis ‘in water’ by
a solution-phase method using
water-dispersible nanoparticle
Boc-amino acid
Keiko Hojo,∗ Hideki Ichikawa, Mare Onishi, Yoshinobu Fukumori
and Koichi Kawasaki
Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic
chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in-water
solution-phase peptide synthesis method using Boc-amino acids. It is based on a coupling reaction utilizing suspended water-
c
dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity. Copyright ꢀ
2011 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: nanoparticles; suspension; solution-phase synthesis; peptide synthesis in water; Boc strategy; green chemistry
Introduction
peptide synthesis methods, the most common building blocks are
the t-butyloxycarbonyl (Boc)- and 9-fluorenylmethoxycarbonyl
(Fmoc)-protected amino acids [8,9], which are highly soluble
in organic solvents. These molecules are sparingly soluble in
water and are considered inappropriate for in-water synthesis. We
recently reported a new technique, solid-phase peptide synthesis
in water, which utilizes Fmoc-amino acids converted to water-
dispersible nanoparticles [10,11].
In industrial syntheses, peptide products such as Aspartame
(Asp-Phe-OMe),Leuplin (anLHRHanalogusedforcancertherapy)
(Takeda Pharmaceutical Co. Ltd., Osaka, Japan) and so on are
produced primarily by solution-phase peptide synthesis. The Boc
strategy is well known to be suitable for industrial chemistry and
green chemistry, because only gases are generated, without any
other by-products produced by deprotection of the Boc group.
Gases require less disposal energy than do solid wastes generated
by deprotection of other protecting groups such as the Fmoc
group. Here, we describe an in-water solution-phase peptide
synthesis method in which Boc-amino acids are converted to
water-dispersible nanoparticles.
Since the early 20th century, the use of organic solvents in
chemical reactions has led to a great leap forward in modern
organic chemistry. More stringent rules regulating the use and
disposal of organic solvents are increasingly restricting chemical
synthesis alternatives, and there is an urgent need to develop
non-hazardous alternatives in order to achieve green sustainable
chemistry [1,2]. Owing to the vast importance of peptides in
biological processes, there is an escalating need for synthetic
peptides to be used in a wide variety of applications. However,
the consumption of organic solvent is extremely large in chemical
peptide syntheses because of the multiple condensation steps in
organicsolvents. Theproblemwithorganicsolventsisnotsomuch
their use but the seemingly inherent inefficiencies associated with
the disposal of waste. There is a compelling need for new peptide
synthesis technologies that do not damage the environment.
From the viewpoint of green sustainable chemistry, the best
solvent is no solvent. If a solvent is needed, then water has much
to offer: It is non-toxic, non-inflammable, abundantly available and
inexpensive[3].Seekingamoreenvironmentallybalancedmethod
ofpeptidesynthesis,wefocusedondevelopinganorganicsolvent-
free synthetic method using water, an environmentally friendly
solvent.
Consumption of organic solvents can be radically reduced by
developing alternative methods that utilize water. In conventional
organic syntheses, reactant molecules are routinely dissolved in
solvents to carry out the reaction efficiently. Peptide synthesis can
becarriedoutinwaterviachemicalconversionofprotectedamino
acids to water-soluble forms [4–7]. This procedure, however,
requires an additional conversion step in the synthetic process,
andthisisundesirableintermsofpreparationcost,resourcesaving
and energy conservation. Thus, development of new techniques
other than chemical conversion is a challenging issue. In current
Inrecentyears, utilizationofnanoparticle-basedtechnologyhas
emerged as a strategy to tackle formulation problems associated
withpoorlywater-solubledrugs[12–14].Accordingly,reductionof
drug particle sizes to the nano-scale provides an increased surface
area and allows homogenous mixing of multiple components,
leading to improved in vivo drug performance. When Boc-amino
∗
Correspondence to: Keiko Hojo, Faculty of Pharmaceutical Sciences & Cooper-
ative Research Center of Life Sciences, Kobe Gakuin University, Chou-ku, Kobe
650-8586, Japan. E-mail: hojo@pharm.kobegakuin.ac.jp
Faculty of Pharmaceutical Sciences & Cooperative Research Center of Life
Sciences, Kobe Gakuin University, Chou-ku, Kobe 650-8586, Japan
c
J. Pept. Sci. 2011; 17: 487–492
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

HOJO ET AL.
Figure 1. New physical properties and reactivities acquired by water-dispersible nanoparticulation.
acids are converted to nanoparticles homogeneously dispersed in
water, the specific surface area is increased and water dissolution
speedisimproved.Evenwhenwater-insolubleBoc-aminoacidsare
used, the reaction can be dramatically accelerated. This suggests
that synthesis of peptides in water according to the Boc strategy
is a promising direction to pursue. (Figure 1)
beads were removed by filtration with 60 ml of aqueous 0.4%
Triton X-100 solution. The particle sizes were determined by DLS
analysis and they had the following characteristics:
Boc-Tyr(BrZ)-OH nanoparticles: particle size = 435 118 nm
In-Water Coupling Reaction of Water-Dispersible Boc-Phe-OH
Nanoparticle with H-Leu-NH2 using Water-Soluble Coupling
Reagents
Materials and Methods
Boc-amino acids were purchased from Watanabe Chemical
Industries, Ltd. A planetary ball mill, model pulverisette 7 (Fritsch
GmbH, Markt Einersheim, German), was used for pulverization
to prepare water-dispersible nanoparticles. The size of particles
was determined by a dynamic light scattering (DLS) analysis, using
instrument model LB-500 (Horiba Instruments Inc.). A nanoparticle
image was taken by a scanning electron microscope (SEM), model
JSM-5300LV (JEOL Ltd., Tokyo, Japan). Amino acid ratios in an
acid hydrolysate were determined with a Waters Pico Tag amino
acid analyzer. Reversed phase HPLC was performed using Waters
model 600 equipment with a DISOPAK column and a gradient
system consisting of acetonitrile/water containing 0.05% TFA.
Optical rotations were determined with an automatic polarimeter,
model DIP-360 (Japan Spectroscopic Co., Tokyo, Japan). Mass
spectra were measured with a Kratos MALDI IV mass spectrometer
(Simadzu Co., Kyoto, Japan) using the TOF technique.
H-Leu-NH2·HCl (166 mg, 1.0 mmol) was dissolved in 10 ml
of water, and then water-dispersible Boc-Phe-OH nanoparti-
cles (60 ml, 2.0 mmol) were added. Several coupling meth-
ods using WSCI [1-ethyl-3-(3ꢁ-dimethylaminopropyl)carbodiimide
hydrochloride] (382 mg, 2.0 mmol) and 4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (552 mg,
2.0 mmol) were examined. N-hydroxy-5-norbornene-endo-2,3-
dicarboximide (HONB) (450 mg, 2.0 mmol), N-hydroxysuccinimide
(HOSu) (230 mg, 2.0 mmol) or 3-sulfo-N-hydroxysuccinimide
(sulfo-HOSu) (436 mg, 2.0 mmol) was used as a coupling addi-
tive. N,N-DIEA (348 µl, 2.0 mmol) was used in the WSCI methods,
and NMM (192 µl, 2.0 mmol) was used in the DMTMM method.
After stirring overnight, the reaction mixture was filtered to collect
the precipitates. The precipitates, which contained the crude pep-
tide, were directly applied to analytical HPLC, and the yields and
purities were measured.
General Procedure for Preparation of Water-Dispersible
Nanoparticle Boc-Amino Acids
General Procedure for Synthesis of Peptides using
Water-Dispersible Boc-Amino Acid Nanoparticles,
with Boc-Gly-Phe-Leu-NH2 as an Example
An aqueous dispersion of nanoparticulate Boc-amino acids was
prepared by pulverization using a planetary ball mill as follows: A
40-ml agate jar was charged with 1.0-mm diameter pre-cleaned
zirconium oxide beads (80 g), Boc-Phe-OH (530.6 mg, 2.0 mmol),
PEG (average molecular weight 4000 g/mol, 400 mg, 0.1 mmol)
and 20 ml of water. The batch was rolled at 495 rpm for 4 h.
After pulverization, the zirconium oxide beads were removed by
filtration with 40 ml of water. The particle sizes were determined
by DLS analysis and the particles had the following characteristics:
Boc-Phe-Leu-NH2 (674 mg, 1.0 mmol) was treated with 20 ml
of TFA) for 1 h at room temperature. The TFA solution was
concentrated to a residue in vacuo, to which 1 ml of solution
4.0 mol/l HCl in dioxane was added. The residue was triturated
with diethyl ether. The precipitate was collected by filtration
and dried over sodium hydroxide pellets in vacuo. H-Phe-Leu-
NH2·HCl was thus obtained and dissolved in 20 ml of water.
Water-dispersible Boc-Gly-OH nanoparticles (60 ml, 2.0 mmol)
were mixed and then DMTMM (552 mg, 2.0 mmol) and NMM
(196 µl, 2.0 mmol) were added. After stirring at room temperature
overnight, the precipitate was collected by filtration and washed
with water. The residue was dried invacuo to obtain crude peptide,
which was directly applied to analytical HPLC, and the yields and
purities were measured. The yields and characteristics were Boc-
Gly-Phe-Leu-NH2: Yield 82% (calculated from analytical HPLC),
HPLC analytical purity 90%. Crude peptide was crystallized from
Boc-Phe-OH nanoparticles: particle size = 578 48 nm
Boc-Gly-OH nanoparticles: particle size = 712 36 nm
Boc-Tyr(tBu)-OH nanoparticles: particle size = 687 32 nm.
An aqueous dispersion of nanoparticulate Boc-amino acids
havingbenzyl(Bzl)typesidechainprotectiongroupswasprepared
bypulverizationusingaplanetaryballmillasfollows:A40-mlagate
jar was charged with 0.5-mm diameter pre-cleaned zirconium
oxide beads (80 g), Boc-Tyr(Bzl)-OH (494.3 mg, 1.0 mmol) and
20 ml of aqueous 0.4% Triton X-100 solution. The batch was
rolled at 495 rpm for 4 h. After pulverization, the zirconium oxide
ethyl acetate and diethyl ether. mp 155–157 ◦C; [α]D + 55.7
24
(c 1.0 in MeOH); m/z (MALDI-MS) 457.54 ([M+Na]+, C22H34N4NaO5
calculated m/z = 457.52); Boc-Phe-Leu-NH2: white material; mp
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PEPTIDE SYNTHESIS IN WATER
Figure 2. (A) PhotoimageofunprocessedBoc-Phe-OH.(B)SEMimageofunprocessedBoc-Phe-OH.(C) Photoimageofwater-dispersiblenanoparticleBoc-
Phe-OH. (D) SEM image of water-dispersible nanoparticle Boc-Phe-OH. (E) Water-dispersible Boc-Phe-OH nanoparticles showed the Tyndall phenomenon
upon laser irradiation. (F) Water-dispersible nanoparticle Boc-Phe-OH readily passed through a filter paper.
176–178 ◦C; [α]D −20.2 (c 1.0 in MeOH); m/z (MALDI-MS) 400.86
24
Table 1. In-water coupling reaction using water-dispersible
([M+Na]+, C20H31N3NaO4 calculated m/z = 400.47); Boc-Gly-
nanoparticlesa
Gly-Phe-Leu-NH2: white material, mp 137–140 ◦C; [α]D +64.4
24
Entry
Reagent
Additive
Base
Yield (%)b
(c 1.0 in MeOH); m/z (MALDI-MS) 514.27 ([M+Na]+, C24H37N5NaO6
calculated m/z = 514.57); Boc-Tyr(tBu)-Gly-Gly-Phe-Leu-NH2:
1
2
3
4
DMTMM
WSCI
–
NMM
DIEA
DIEA
DIEA
89
25
32
71
24
yellowish material; [α]D +68.6 (c 1.0 in MeOH); m/z (MALDI-MS)
HONB
733.27 ([M+Na]+, C37H54N6NaO8 calculated m/z = 733.85).
WSCI
HOSu
WSCI
Sulfo-HOSu
Preparation of Leu-Enkephalinamide (H-Tyr-Gly-Gly-Phe-Leu-
NH2·TFA)
a Reactions were carried out between H-Leu-NH2 and water-dispersible
Boc-Phe-OH nanoparticles.
b The yield of peptides was calculated from analytical HPLC profiles.
The protected pentapeptide Boc-Tyr(tBu)-Gly-Gly-Phe-Leu-NH2
(710 mg, 1.0 ml) was treated with 20 ml of TFA for 1 h at room
temperature. The TFA solution was concentrated to a residue in
vacuo. The residue was purified by preparative HPLC to give an
hydrophobic Bzl type groups are generally the first choice for the
side chain protection step. We also successfully prepared water-
dispersible Boc-Tyr(BrZ)-OH, which has a hydrophobic BrZ side
chain protecting group, as a nanoparticle formulation using the
same wet milling process in the presence of another dispersant,
an aqueous 0.4% Triton X-100 solution. The Boc-Tyr(BrZ)-OH
nanoparticles dispersed in water were found by DLS to have a
mean diameter of 435 118 nm.
24
amorphous powder. The yield was 51%; [α]D + 12.6 (c 1.0 in
H2O); m/z (MALDI-MS) 555.31 ([M+H]+. C28H39N6O6 calculated
m/z = 555.64); amino acid analysis: Tyr, 0.94; Gly; 2.03, Phe, 0.92;
Leu, 1.01 (average recovery: 92%).
Results and Discussion
To evaluate the feasibility of using the water-dispersible Boc-
amino acid nanoparticles as building blocks for solution-phase in-
water peptide synthesis, we examined in-water coupling reactions
between water-dispersible Boc-Phe-OH nanoparticles and Leu-
NH2. The reaction was carried out by several coupling methods
using the water-soluble coupling reagents WSCI [16] and DMTMM
[17,18]. As shown in Table 1, the coupling was more favorable with
DMTMM than with the WSCI combination containing the additives
HONB [19], HOSu [20,21] or sulfo-HOSu [22]. The in-water coupling
reaction using DMTMM in the presence of NMM resulted in good
yields (89%).
Next, solution-phase in-water synthesis of Leu-enkephalina-
mide was carried out using water-dispersible nanoparticles of
Boc-amino acids prepared as described above. Figure 3 shows the
synthetic scheme. DMTMM was used as the coupling reagent. The
peptidewasdeprotectedwithTFAandtheTFAsaltwasexchanged
fortheHClsaltusinga4.0 mol/lHCl-dioxanesolution.Couplingwas
carried out in water by mixing water-dispersible nanoparticles of
Nanoparticles can be produced by dispersion-based processes.
Wet milling is an attrition-based process in which the insoluble
material is dispersed in the aqueous-based surfactant solution,
and the resulting material is converted into water-dispersible
nanoparticles. First, we selected Boc-Phe-OH, which has a
hydrophobic lipophilic aromatic ring in its side chain, to convert
to water-dispersible nanoparticles. In the presence of PEG [15]
as a dispersant, Boc-Phe-OH was ground with zirconia beads in
water using a planetary ball mill for 2 h at room temperature.
The beads were then removed by filtration and water-dispersible
nanoparticles of Boc-Phe-OH were obtained. SEM images of the
nanoparticles are shown in Figure 2: Panels (b) and (d) show
the nanoparticles before and after grinding, respectively. Nano-
or submicron-sized particles were observed. The nanoparticles
dispersed in water were found by DLS to have a mean diameter
of 578 48 nm. The obtained nanoparticles showed the Tyndall
phenomenon upon laser irradiation and readily passed through a
filter paper (ADVANTEC Filter Paper No.2). In the Boc strategy, the
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J. Pept. Sci. 2011; 17: 487–492 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

HOJO ET AL.
Table 2. Yield and purity of in-water synthetic peptidesa
Synthetic peptide
Yield (%)b
Purity (%)b
Boc-Phe-Leu-NH2
89
82
84
86
90
90
92
93
Boc-Gly-Phe-Leu-NH2
Boc-Gly-Gly-Phe-Leu-NH2
Boc-Tyr(tBu)-Gly-Gly-Phe-Leu-NH2
a In-water coupling reactions were carried out with DMTMM. Purity of
peptides was calculated from analytical HPLC profiles.
b Yield and purity of peptides was calculated from analytical HPLC
profiles.
Inthisstudy, water-dispersiblenanoparticlesofBoc-aminoacids
were prepared in a planetary ball mill, and Leu-enkephalinamide
was successfully synthesized in water by Boc chemistry using
these nanoparticles. In addition, coupling reactions mediated
by DMTMM in water containing water-dispersible nanoparticles
were found to progress smoothly, resulting in high yield and
high purity products. Hydrophobic molecules tend to associate
in water. Providing a nano-scale reaction field that promotes
molecular associations might improve the efficiency of these
types of reactions. In the case of water-dispersible nanoparticles
with poor solubility in water, it is likely that the nanoparticles are
homogeneously dispersed in water and recognize each other
by their hydrophobicity. Therefore, it should be possible to
establish a nano-scale, hydrophobic reaction field in aqueous
media to produce high yields of product. Nanoparticles have
novel, interesting physical properties, like much smaller diameters
and much larger specific surface areas, compared to common
particles.
Figure 3. Synthetic scheme of Leu-enkephalinamide.
a Boc-amino acid with an amino component, adding DMTMM and
then stirring the mixture under neutral conditions for 12 h at room
temperature (Figure 4). As nanoparticles readily pass through a
micro-pore filter and the reagent is water-soluble, post-coupling
treatment consists only of removing the unreacted nanoparticles
and reagent-mediated by-products by filtration to obtain the main
product. Figure 5 shows HPLC profiles of the crude main products
obtained after filtration. Each protected peptide fragment showed
a single predominant peak, even without special purification steps
other than filtration, showing that these peptides were obtained
with high purity. Table 2 shows the yield and purity of the crude
product of each coupling reaction. In all reactions, peptides were
obtained with a yield of 82% or higher and with a purity of 90%
or higher, showing the feasibility of efficient in-water coupling
reactions using water-dispersible nanoparticles. After the final
deprotection step, the target product Leu-enkephalinamide was
obtained with a total synthetic yield of 51%. In this study, all
the amino components were in a water-soluble salt form. If the
sparingly soluble amino component was used, a purification step
would have been needed that would include not only filtration
but also some other purification technique(s), e.g. an extraction or
chromatography method.
Ingeneral, mostorganiccompoundsproducedasintermediates
in industrial syntheses are sparingly soluble in water and are thus
not suitable for in-water reactions. However, if a technology
enabling efficient in-water reactions using compounds with poor
water solubility, such as the method described here, is established,
then it could replace standard organic synthesis methods with
more environmentally friendly water-using processes.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research and by an ‘Academic Frontier’ Project for Private
Figure 4. In-water coupling reaction using a water-dispersible nanoparticle Boc-amino acid.
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PEPTIDE SYNTHESIS IN WATER
Figure 5. Analytical HPLC profiles of peptides obtained by in-water synthesis. (A) Boc-Phe-Leu-NH2. (B) Boc-Gly-Phe-Leu-NH2. (C) Boc-Gly-Gly-Phe-Leu-
NH2. (D) Boc-Tyr(tBu)-Gly-Gly-Phe-Leu-NH2. Elution was carried out for over 30 min at a flow rate of 1 ml/min with a linear gradient from 9 : 1 to 3 : 7
mixture of 0.05% aqueous TFA and 0.05% TFA in acetonitrile.
Universities: matching fund subsidy from the Japanese Ministry of
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